Copyright is owned by the Author of the thesis. Permission is given for a copy to be downloaded by an individual for the purpose of research and private study only. The thesis may not be reproduced elsewhere without the permission of the Author. EFFECT OF PROBIOTIC AND LACTOFERRIN -SUPPLEMENTED DIETS ON DAILY GAIN, FEED INTAKE, FEED CONVERSION RATE, MEAN WEEKLY FAECAL SCORES, L YMPHOCYTE TO NEUTROPHIL RATIO, IMMUNITY, GENERAL HEALTH, AND HEMATOLOGICAL PARAMETERS IN WEANLING PIGS SUBJECTED TO AN IMMUNOLOGICAL CHALLENGE A thesis presented in partial fu lfilment of the requirements for the Degree of Master of Science (Animal Science) at Massey University, Palmerston North, New Zealand RICHARD NKAMBA 2007 ABSTRACT Background The digestive system of early weaned pigs is not fully developed and animals can be subjected to a post-weaning check, or lag period, which results in poor feed intake, weight gain, immunity and high diarrhoea cases and mortality. In addition, there is a depression of growth during an immune challenge that results in nutrient intake restriction and redirection to support the immune system. A shift from the unstable flora at weaning into a complex stable one would be achieved by diet manipulation . Different natural products (instead of hazardous antibiotics) are being tested to find their ability in improving pig health and perfom1ance . The aim of this study was therefore to evaluate the effects of dietary supplementation with probiotics and lactoferrin on pigs' growth performance, haematological characteristics and general health. The weaned pigs from four different farms were mixed together upon arrival to place them in an immune challenging environment. Results After 2 1 days post challenge/weaning, average daily feed intake, ADFI (404.64, 426.77, 423 .63 , 378 .48 and 34 1 .48 g/p/d for diet for diet A[control ] , B , C, D (probiotics) and E [lactoferrin] , respectively) was significantly different in pigs that consumed the five diets (p=0.0259) . Pigs that consumed diet B had 5 .47% higher feed intake (p<0.05) than the controls, while those that received diet C consumed 4 .69% more feed than the controls but this feed intake was not significantly different from that of the controls (p>0.05) . The difference in feed intake between pigs in fed diet B and C was also not significant (p>0.05). Animals that were allocated to diet D and diet E (lactoferrin) consumed significantly less feed compared to the control s (p<0.05) . Feed consumption was 6 .47% and 1 5 . 6 1% lower (p<0.05) for pigs fed diet D and E, respectively, compared to the controls. Pigs that received diet D consumed significantly higher amounts of feed (p<0.05) than those fed diet E (lactoferrin). In conclusion, in the first three weeks of life, or at times of stress such as weaning and I or immune challenge, a good probiotic (such as B or C) should produce a faster and more rapid response by increasing I stimulating feed intake so that body weight losses are quickly compensated. Feed intake is a factor that l imit growth in weaned piglets. Weight is gained after the improvement in feed ingestion. Reduction in feed I energy intake also reduces body weight. I f feed (energy) intake is reduced, then, a good diet should stimulate quick repair of the gut and improve intestinal environment architecture and integrity. When feed consumption increases, the levels of digestive enzymes responsible for the breakdown of fats, starches and proteins increase. When feed intake is not increased or increased too late after weaning, bodyweight may not be compensated. 11 DEDICATION I dedicate this thesis to my wife Oli, my sons: Sampa, Bwalya and Mambwe and daughter, Chimwemwe. This is one of the highest achievements for the benefit for all of us. MAY HIS NAME BE BLESSED AND GLORIFIED. lll ACKNOWLEDGEMENT I am grateful to the Ministry of Foreign Affairs and Trade of New Zealand for granting me a New Zealand Aid for International Development (NZAID) scholarship to pursue a postgraduate diploma in science in the Institute ofVeterinary, Animal, and Biomedical Sciences (IV ABS) and a Master of Science degree in Animal Science in the Institute of Food, Nutrition and Human Nutrition (IFNHH) in the Col lege of Sciences at Massey University. Fonterra Farmers Cooperative Limited is thanked for the sponsorship of this research. Sincere thanks and appreciation go to my supervisor, Dr P.H.C. More! for advice, encouragement and help throughout the course of this study and his patience in assisting me to acquire the techniques that are new to me. My thanks and appreciation also go to the technicians at Massey University Farm, Ms Maggie Honeyfield -Ross, Ms Heidi Roesch and Mr. Pereka Kalwin for feed processing, data entries and handl ing of the piglets at the Massey University Pig Biology Unit. IV TABLE OF CONTENTS Abstract Dedication . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . m Acknowledgement . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . IV Table of Contents. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . v List of Tables . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . IX List of Figures . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . x List of Abbreviations . . . .. . . .. .. . . . . .. . . .. .. . .. . .. . .. . XI List of Appendices . . . . . . . . . . . . . . . . xn Introduction . . . . . . . . . . . . . . . . . . . . . . xm CHAPTER 1 . L ITERATURE REVIEW. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 . 1 Probiotics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 .2 1 . 3 1 . 1 . 1 History of Claims . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 . 1 .2 Definition of Probiotics . . . . . . . . . 1 1 . 1 . 3 Revision of Definition . . . . . . . . . . . . . . . . . . . . . . . . 2 1 . 1 .4 Characteristics of effective Probiotics . . . . . . . . . . . . 2 1 . 1 . 5 Development and production of probiotics . . . . . . . . . . . . . . . 3 1 . 1 . 6 Antibiotics and their use . . . . . . . . . . . . . . . . . . . . . . . . 4 1 . 1 . 7 Benefits of antibiotics . . . . . . . . . . . . . . . . . . . . . . . . 4 1 . 1 . 8 Disadvantages and reason for their ban . . . . . . . . . . . . . . . . . 6 1 . 1 .9 Pig growth and appropriate times to use antibiotics or probiotics . . . 7 1 . 1 .9 . 1 The pig growth in general . . . . . . . . . . . . . . . . . . . . . . . . . . . 7 1 . 1 .9 .2 The piglet after weaning . . . . . . . . . . . . 7 1 . 1 .9 . 3 The pig at Weaning . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9 1 . 1 . 1 0 Effect of antibiotic ban . . . . . . . . . . . . . . . . . . . . . . . . 1 1 1 . 1 . 1 1 Alternatives to antibiotics . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 2 1 . 1 . 1 2 Way Forward . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 2 1 . 1 . 1 3 Methods to improve performance after the ban . . . . . . . . . . . . 1 4 1 . 1 . 1 4 Other claims and example of probiotics . . . . . . . . . . . . . . . . . . . . . 1 5 1 . 1 . 1 5 Some requirements for development of probiotics . . . 1 7 1 . 1 . 1 6 Costs and effectiveness of probiotics . . . . . . . . . . . . 1 8 1 . 1 . 1 7 Interactions between probiotics, ingredients and time 1 8 The Immune System Effect of natural products . . . . . . . . 1 . 3 . 1 Effect and mode of action of nonstarch polysaccharide . . . . . . 1 . 3 . 2 Effect of probiotics on blood parameters . . . . . . . . . . . . . . 1 . 3 . 3 1 .3 .4 1 . 3 . 5 . Effect and mode of action o f lactoferrin on performance and general health . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Common methods of preparation I production of l actoferrin . . . The normal or reference values of the leukogram . . . . V 1 9 1 9 1 9 20 2 1 23 23 1 .3 .6 Interpreting the results. . . . . . . . . . . . . . . . . . . . . . . . . . . . . 26 1 .3 .6. 1 Evaluation of leukocytes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 26 1 .3 .6 .2 Differential leukocytes count . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 27 CHAPTER 2 EFFECT OF PROBIOTIC ON AVERAGE DAILY GAIN, FEED INTAKE, FEED CONVERSION RATION, HAEMTOLOGICAL PARAMETERS, IMMUNITY AND GENERAL HEALTHON IMMUNE CHALLENGED WEANLING PIGS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 8 2 . 1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 28 2 .2 Materials and Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 29 2 .2 . 1 Experimental animals . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 28 2 .2 .2 Weaner pens . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 29 2 .3 2 . 3 . 1 2 . 3 .2 2 .4 2 .5 2 .6 2 .7 2 .8 2 .9 Experimental Diets and Feed management . . . . . . . . . . . . . . . . . . . . . Experimental Diets . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Feed Management . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Source and Maintenance of Cultures . . . . . . . . . . . . . . . . . . . . . . . Colony Forming units, Cfu Faecal Scoring Blood Sampling Haematological I Immune Status . . . . . . . . . . . . . . . . . . . . . . . . . . . Statistical Analyses . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 30 30 3 1 32 32 32 33 3 3 34 CHAPTER 3 RESULTS . . . . . . . . . . . . . . . . . . . . . . . . . 36 3 . 1 Effects of mixing pigs from different farms . . . . 3 9 3 .2 Effects of pro biotic- and lactoferrin supplemented diets on animal performance . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 39 3 .2. 1 Week 0 Body Weight (weaning weight) 3 9 3 .2 .2 Week 1 Body Weight 40 3 .2 .3 Week 2 Body Weight 4 1 3 .2 .4 Week 3 Body Weight 4 1 3 .2 .5 Average Daily Gain . . . . . 4 1 3 .2 .6 Average Daily Feed Intake . . . . . . . . . . . . . . . . . . . . . . . . . . . 42 3 .2 .7 Average Feed Conversion Rate . . . . . . . . . . . . . . . . . . . . . . . . . . 43 3 .3 Effects of pro biotic- and lactoferrin-supplemented diets on blood parameters . . . . . . . . . . . . . . . . . . . . . 3 .3 . 1 Whole blood parameters 3 . 3 . 1 . 1 White blood cell s population 3 .3 . 1 . 1 . 1 Absolute White b lood cell VI . . . . . . 44 44 45 4 5 3 . 3 . I . 1 .2 Neutrophil s 0 0 . 0 0 . 0 0 . 0 0 . 0 0 . 0 0 . 0 0 . 0 0 . 0 0 . 0 0 . 0 0 . 0 0 . 49 3 . 3 . 1 . 1 .3 Lymphocytes o o · 0 0 0 0 0 0 o o . 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 50 3 .3 . 1 . I .4 Monocytes 50 3 .3 . 1 . 1 . 5 Eosinophi ls 0 0 . 0 0 . 0 0 . 0 0 . 0 0 . 0 0 . 0 0 . 0 0 . 5 1 3 . 3 . 1 . 1 .6 Basophils 0 0 . 0 0 . 0 0 . 0 0 . 0 0 . 0 0 . 0 0 . 0 0 . 52 3 . 3 . I .2 Erythrocytes or red blood cell population and red cel l indices 5 3 3 . 3 . I .2 . I Erythrocytes or red blood cells 0 0 . 0 0 . 0 0 . 0 0 . 0 0 . 0 0 . 0 0 . 0 0 . 54 3 . 3 . 1 .2 .2 Haemoglobin 0 0 . 0 0 . 0 0 . 0 0 . 0 0 . 0 0 . 0 0 . 0 0 0 0 5 5 3 . 3 . 1 .2.3 Haematocrit 0 0 . 0 0 . 0 0 . 0 0 0 0 0 0 . 5 5 3 .3 . 1 .2 .4 Mean Corpuscular volume 0 0 . 0 0 . 0 0 . 0 0 0 0 0 0 0 . 56 3 . 3 . 1 .2 . 5 Mean Corpuscular Haemoglobin 0 0 . . . 0 0 0 0 57 3 . 3 . I . 2 .6 Mean Corpuscular Haemoglobin Concentration 57 3 .3 . 1 .2 .7 Corpuscular Haemoglobin Concentration Mean 5 8 3 . 3 . I .2 .8 Red cel l Distribution Width 0 0 . 0 0 0 0 0 0 58 3 . 3 .2 Blood Cell Parameter Changes 0 0 . 0 0 . 0 0 . 59 3 . 3 .2 . 1 Absolute White blood cells population changes 59 3 . 3 .2 . 1 . 1 Leukocytes or White blood cel ls 0 0 0 0 0 0 0 59 3 . 3 .2 . 1 .2 Neutrophils 0 0 0 0 0 0 0 0 0 0 0 0 . 0 0 . 0 0 . 0 0 . 0 0 . 0 0 . 0 0 . 0 0 . 0 0 • • • • 0 0 • • 59 3 . 3 .2 . 1 .3 Lymphocytes 0 0 0 0 0 0 0 0 0 0 0 0 . 0 0 . 0 0 . 0 0 . 0 0 . 0 0 . 0 0 . 0 0 . 0 0 0 0 60 3 . 3 .2 . 1 .4 Monocytes 0 0 0 0 0 0 0 0 0 0 0 0 . 0 0 • • 62 3 . 3 .2 . 1 .5 Eosinophils 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 o o . 0 0 . 0 0 0 o o · .63 3 . 3 .2 . 1 .6 Basophils 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 . 0 0 . 0 0 . 0 0 . 0 0 . 0 0 . 0 0 . 0 0 • • 63 3 . 3 .2 .2 Red blood cell population changes 0 0 0 0 0 0 . 0 0 . 0 0 . . . • 0 0 • • 63 3 . 3 .2 .2 . 1 Red blood cell s . . . . . . . . . . 0 0 . . . . . . . 0 0 . . . • 0 0 . . . 63 3 .3 .2 .2 .2 Haemoglobin . 0 0 0 0 0 0 0 0 . . . • 0 0 . 0 0 . 0 0 . 0 0 0 0 • 64 3 . 3 .2 .2 .3 Haematocrit 0 0 . . 0 0 0 0 . 0 0 . 0 0 . 0 0 . . . • 0 0 . . . . 0 0 0 0 0 0 . 65 3 . 3 .2 .2 .4 Mean Corpuscular volume . 0 0 0 0 0 0 0 0 0 0 0 0 . . . . 0 0 . . . 0 0 0 . . . 66 3 . 3 .2 .2 .5 Mean Corpuscular Haemoglobin 0 0 . . . . . o o · 65 3 .3 .2 .2 .6 Mean Corpuscular Haemoglobin Concentration 67 3 .3 .2 .2 .7 Corpuscular Haemoglobin Concentration Mean . . . . . . . . . . 68 3 .3 .2 .2 .8 Red cell Distribution Width . . o o . . .. 0 0 . . . • . . • 0 0 0 0 0 0 69 3 .4 Effects of Probiotic- and lactoferrin-supplemented diets on Mean Weelky Fecal Scores of pigs 0 0 0 0 0 0 . 0 0 . 0 0 . 0 0 . 70 3 . 5 Effects of Probiotic- and lactoferrin supplemeted diets on lymphocyte to neutrophil ratio (stress factor) of pigs 0 0 . 0 0 0 0 0 . . 0 0 0 0 73 3 .6 Effects of Probiotic- and lactoferrin supplemented diets on health parameters of pigs 0 0 0 0 . . . . . 74 CHAPTER 4 DISCUSSION 0 0 . 0 0 . 0 0 . 0 0 . . . . 0 0 . . . . . . . 0 0 . . . . . . . 0 0 . 0 0 0 0 0 0 0 . . . . 0 0 0 0 . 0 0 0 75 Effects o f mixing pigs from different farms to stimulate the immune challenge . . . 7 5 Effects of pro biotic- and lactoferrin-supplemented diets on performance and immunity of pigs . . . 0 0 . 0 0 0 0 0 0 . 0 0 . 0 0 . . . . 0 0 . 0 0 . 0 0 . 0 0 . 0 0 . 0 0 . . . . 0 0 . 0 0 . . . • . . • . . • 75 Vll CHAPTER 5 CONCLUSION CHAPTER 6 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 87 L IST OF REFERENCES . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 92 CHAPTER 7 LIST OF APPENDICES . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 23 Vlll Table Table 1 : Table 2 : Table 3 : Table 4 : Table 5: Table 6 : Table 7 : Table 8 : LIST OF TABLES page Components of the gut mucosal barrier in the piglet . . . . . . . . . . . . . . . . .. . 9 Blood parameters, their abbreviations and units of Measurement . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 24 Accepted physiological values of white and red blood cell parameters in normal and anaemic pigs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 5 Ingredients and diet fomlUlation of experimental diets used in the study to find the effect of d ifferent probiotics and lactoferrin on performance of weaned piglets . .. . . . 31 Least-square means of the effects of pro biotic- and lactoferrin­ supplemented diets on growth performance in weanling pig for average pen liveweight of piglets at the start of the experiment (WkOwt), average pen l ive weight of piglets at the end of week 1 (wk 1wt), average pen l ive weight of piglets at the end ofweek 2 (wk2wt), average pen l ive weight of piglets at the end of week 3 (wk3wt), average daily gain (ADO), average dai ly feed intake (ADFI) and average feed conversion rate (FCR) for the 2 1 day experimental period for each diet with standard error (SE) . . . . . . . . . . . . . . . . . . . . . .. . . . 40 Effects of pro biotic- and Jactoferrin-supplemented diets on blood parameters . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .46 Least square means for effects of pro biotic- and lactoferrin­ supplemented diets on lymphocyte to neutrophil ratio in weanling pigs. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7 1 Least square means for effects of pro biotic- and lactoferrin­ supplemented diets on mean weekly faecal score in weanling pigs. . 73 IX LIST OF FIGURES Page Figure 1 : Effect of non-starch polysaccharide on performance of animals . . . . . . . 1 9 X BASO CH CHCM CH CHOL Cu DNA EOSI ESR HCT HGB LYMPH (LIN ratio MCH MCHC MCV M MONO NEUT ppb RBC RDW SAS TG USDA WBC LIST OF ABBREVIATIONS Basophil cell s Corpuscular haemoglobin constant Corpuscular haemoglobin concentration mean Corpuscular haemoglobin Cholesterol Copper Deoxyribose nucleic acid Eosinophil cells Erythrocyte sedimentation rate Haematocrit level Haemoglobin concentration Lymphocyte cel ls Lymphocyte to neutrophi l ratio Mean cell haemoglobin (or mean erytrocyte haemoglobin content) Mean corpuscular haemoglobin concentration (or mean erythrocyte haemoglobin concentration) Mean cell volume (or mean erythrocyte volume) Molar Monocyte cel ls Neutrophi l cel ls Parts per billion Red blood cel ls Red blood cell distribution width (or erythrocyte distribution width) Statistical Analysis System Triglyceride United States Department of Agriculture White cel ls X I LIST OF APPENDICES Annex 1 : The statistical significance of effects of farm, diet, and their interactions on different parameters in weaned pigs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 23 Xll INTRODUCTION As pointed out by McDonald et al. (2002), World demand for meat on an absolute basis ( i .e al lowing population growth) is predicted to increase rapidly; by 0 .6 per cent per year in developed countries and by 4. 1 per cent per year in developing countries. In general, l ivestock productivity can be increased through good nutrition, improved management and disease control and genetic improvement (Antipas & Weber, 2003 , Blecha & Charley, 1 990). One of the management tools used to reduce disease burden and increase animal performance is antibiotics (Walton, 1 979, 200 1 ; Barnett et al. , 1 989; Cromwell, 2002; Kostantinov et al. 2004). Antibiotics have been used in the animal industry to improve performance (Walton, 200 1 ) . However, public concerns exist about potential risks such as bacterial resistance and al lergenic effects in consumers of animal products. As a result, Sweden and Denmark prohibited the use of prophylactic antibiotics in the mid 1 990's and this was fol lowed by the European Union (Will iams & Heymann, 1 998; Witte, 1 998 ; Yu et al. 2004; Close, 2000; Cullen et al. , 2000; Wenk, 2000; Bamett et al. , 1 989; Jensen, 1 998; Bager et al., 2000; Adj iri-Awere & van Lunen, 2005) . Prohibition of antibiotic use seems reasonable and desirable to consumers (Witte, 1 998; Hiss & Sauerwein, 2003 ; Jensen, 1 998 ; Lopez, 2000). However, the banning of antibiotics may lead to higher stress, mortal ity and increase the number of days to marketing in weaned pigs (Walton, 200 1 ). To increase productivity of sows (and I or farrowing index), piglets are weaned early (at about four weeks or earlier instead of about 8- 1 2 weeks of age) . Early weaned pigs can be stressed by change of feed and of psychological, social environment and dietary stress (Dantzer & Mormede, 1 983 ; Morrow-Tesch & Anderson, 1 994; Lombardi et al. , 2005; Mahan & Lepine, 1 99 1 ; Lalles et al., 2004). Since their digestive system is not ful ly developed, they can encounter a post-weaning check, or growth lag period which is characterised by low feed (energy) intake, diarrhoea and low weight gain loss (Risley et al. , 1 988 ; Barnett et al. , 1 989; Spreeuwenberg et al. , 200 1 ; Kostantinov e t al. 2004; Roth & Kirchgessner, 1 998 ; Aheme e t al. 1 982 ; Mahan & Lepine, 1 99 1 ; Funderburke & Seerley, 1 990; Bosi , 2000). After the ban of antibiotics in Sweden, post-weaning mortality rates increased by 1 .5% and piglets took 6 days longer to reach 25kg target weight for age Xlll (Robertsson & Lundeheim, 1 994 (cited in Adj iri-Awere & van Lunen, 2005)). This evidence shows that lack of antibiotics creates serious negative actions. To correct this, zinc oxide was used and post weaning mortality declined from 1 . 5% (reported above) to 1 %, and time needed to reach the 25kg bodyweight reduced from 6 to 4 . 5 days (Wierup, 1 999, cited in Adj iri-Awere & van Lunen, 2005). Researchers in the animal and feed industry are now trial ling some alternatives and natural products, ranging from chemicals and organic acids to biological products such as probiotics. These products have properties similar to antibiotics, as they improve growth and suppress pathogens (Jensen, 1 998). Antibiotics can be used in known amounts to target certain pathogens. Probiotics, on the other hand, produce variable and unpredictable effects and their mode of action is not ful ly understood. Natural products, such as probiotics and lactoferrin, have been found to improve growth, establish a prophylactic barrier against gastrointestinal disorders. These effects have been greater in young animals and those under stress (Jensen, 1 998). The integrity of the gut mucosa i s a prerequisite to reduce the entry of antigens (Bosi, 2000). The mucosa of the gastrointestinal tract is the first line of contact with pathogens that are ingested. The components of the gut mucosal barrier viz non­ immunological (such as lactoferrin) and immunological are however disturbed at weaning (Bosi , 2000). Reduced feed intake in the weaned pig causes vi l lous atrophy (Bosi, 2000). A lowered supply of milk (Kelly et al., 1 99 1 ) or a restricted ingestion of dry feed (Pluske et al., 1 996) reduce the height of vi llous on the fifth day post wean m g. A good probiotic must not only enhance growth performance but must also be devoid of adverse effects (Bernardeau et al. , 2002). Probiotics such as lactic acid bacteria (LAB) have been incorporated in feeds for many years and have been classified as safe (Fuller, 1 992). These are therefore referred to as "Generally Recognized As Safe": GRAS (Fuller, 1 992 ; Bernardeau et al. , 2002). In addition to probiotics, other natural products such as lactoferrins are said to have an effect on performance and on cellular immune function of ruminants (Wong et al. , 1 997). The aim of this project was to look at the effects of five different diets (a control or basal diet and four supplemented diets) on average dai ly gain, ADG, average daily XlV feed intake, ADFI, feed conversion ratio, FCR, body weight, mean weekly faecal score (MWFS), stress factors (or lymphocyte to neutrophil) (LIN) ratio and general health performance in weanling pigs. Other studies may have looked at different probiotics, environment, species, concentration etc, but not many have looked at using lactoferrin (diet E) in piglet diets. The three probiotics (B, C and D) used in this study are not known (names withheld by the sponsor) except for lactoferrin (a biologically active protein) in diet E and therefore a wide l iterature search has been made in order to match the effects of natural products with the ones used in the current study. XV 1. LITERATURE REVIEW Chapter 1 Probiotics are claimed to have many effects on the host. Among these effects are : improvement of performance, immune system and general health (Lopez, 2000) . In this l iterature review it is therefore important to give brief summaries of what a pro biotic is, and some understanding of effects of probiotics and other natural products on performance of animals. 1.1 Probiotics 1.1.1 History of claims on probiotic use The benefits and use of probiotics such as lactic acid bacteria to cure gastrointestinal ailments was known since time immemorial . In Genesis 1 8 : 8 of the Holy Bible, it can be read that Abraham owed his longevity to the consumption of fermented milk. Similarly, European interest in the gut health benefits of yoghurts containing "beneficial bacteria" began in the early 1 900s, and was indorsed by Metchinkoff and Tissier at the Pasteur Institute (Cummings et al. , 2004). Metchinikoff in 1 907 claimed that consumption of yogurt (Lactobacillus bulgaricus and Streptococcus thermophilus) results in a decline of harmful microorganisms but Lactobacillus bulgaricus were later shown that they did not survive in the gut (Schrezenmeir & de Vrese, 200 1 ). European commercialisation of specific yoghurts based on their gut health was initiated as early as 1 920 by Carasso et al. , as cited in Cummings et al. , 2004. Consumption of this product in Europe is now greater than 1 5 kg capita per year and the amounts are rising (Cummings et al., 2004). 1.1.2 Definition of pro biotic The term probiotic means 'for life ' in Greek language. Lilly and Stil lwell in 1 965 were the first to use the word probiotic to describe "substances secreted by one microorganism which stimulates the growth of another" and thus was different with the term antibiotic ( Lin, 2002) although Komegey & Risley ( 1 996) reported that the administration of "beneficial organisms" to animals started in the 1 920's and the name "probiotics" (in the sense it is used today) was introduced by Parker ( 1 974) when the 1 production of bacterial feed supplements began on a commercial scale and this i s in agreement to Schrezenmeir and de Vrese (2001). 1 . 1 .3 Revision of definition The definition of a pro biotic has had a lot of words added, and subtracted or broadened and narrowed, with some fine tuning and refining. A general definition i s : a pro biotic is a l ive culture of microorganisms, such as lactic acid bacteria (LAB), that exerts a beneficial effect on the host by improving the indigenous microbial balance (Fuller, 1992). Probiotics as applied to human, are defined as live microrganisms which, when administered in adequate numbers, confer a health benefit on the host (FAO/WHO, 2001 (as cited by Casey et al. (2004)). Another definition for probiotics is "living microorganisms, which upon ingestion in certain numbers, exert health benefits beyond inherent basic nutrition" (Guamer and Schaafsma, 1998 (as cited in Adj iri-Awere & van Lunen, 2005)). Some workers have l imited the definition to milk and others on health and nutrition benefit which again included antibiotic but a more improved, revised and broadened definition with respect to host and habitat which does not l imit proposed health effects and excludes nutrition is: 'a probiotic is a preparation of or a product containing viable, defined microorganisms in sufficient numbers, which alter the microflora (by implantation or colonisation) in a compartment of the host and, by that, exert beneficial health effects in the host' (Schrezenmeir & de Vrese, 2001 ) . Furthermore, other workers have found that some probiotics such as lactobacillus cultures sti l l maintain their probiotic effect wether dead or alive (Bemardeau e t al. , 2002; Salminen e t al. , 1999). With these findings, the additional definition proposed by Salminen et al. (1999) is "probiotics are microbial cell preparations or components of microbial cell s that have a beneficial effect on the health and well being of the host". 1 . 1 .4 Characteristics of effective probiotics Probiotics are characterised as living microorganisms. A good probiotic should be able to escape degradation in the gut (Yu et al. , 2004; Adj iri-Awere & van Lunen, 2005), improve growth and feed efficiency and leave no tissue residues or cause mutations (Atherton & Rubbins, 1987; Lopez, 2000; Stavric, 1992). 2 On the other hand, antibiotics are characterised as pure chemical compounds. They are absorbed in the digestive tract. They also improve growth and feed efficiency but they leave residue in tissue and may cause mutation (and resistance) in other microorganisms. The general mode of action of probiotics is that they produce acids, reduce pH and compete with, or discourage growth of pathogenic microorganisms. They posses localised antimicrobial activity and proliferate in the digestive tract and compete for nutrients and space with the pathogenic bacteria whereas antibiotics improve the host ' s performance by blocking living cel l ' s DNA, RNA or protein synthesis and they have a broad spectrum of activity (Lopez, 2000) . Good natural products such as probiotics must not only enhance growth performance but must also not cause adverse side effects (Bernardeau et al. , 2002). Products such as lactic acid bacteria (LAB) have been used in animal feeds for many decades and have been classified as safe (Fuller, 1 992). These products are said to have a "Generally Recognized As Safe": GRAS status (Ful ler, 1 992; Bernardeau et al. , 2002). 1 . 1 .5 Development and production of probiotics Production of probiotics starts with identifying strains that can prol iferate in the gut and be recovered in the same numbers in the faeces of the host. As described by Stavric (992) this starts with the sourcing of isolates. The media for isolation is then identified and this is followed by characterization and storage of isolates. The experimental protocol is then chosen and the method of preparation of defined mixture is estab lished. Selection of isolates from a large number of partial ly identified strains is made and this should match and be representative of all major groups found in the collected bacteria in the gut. These should be able to protect the host from increasing challenge by pathogens. This is then fol lowed by deletion experiments and adheration and determining the mechanisms such as hydrophobic interactions or differences in surface charges, that play a role in the adherence of microorganisms (Gibbons et al. , 1 983 (cited in Stavric, 1 992)). In summary, the prospects for developing a treatment comparable to undefined culture, in efficacy and stability are not promising, despite large numbers of researches conducted . A better understanding of the mode of protection and factors involved would ease the manipulation of caecal flora on a more scientific basis (Stavric, 1 992). Interestingly, dietary condition can affect the development ofprobiotics (Bosi, 2000) and may be, their effect. For example the 3 faecal numbers of Bifidobacteria were higher in pigs fed Bifibobacterium longum with high amylase comstarch than with a low amylase comstarch (Brown et al. , 1 997 as cited in Bosi, 2000)). 1 . 1 .6 Antibiotics and their use Antibiotics and antimicrobials have been used to the furthest extent as additives in the rations of poultry and pigs for more than four decades (Adj iri-Awere & van Lunen, 2005). Antibiotics are substances that can destructively affect bacteria either by interfering with their growth, metabolism or actual ly killing them in situ. Antibiotics are also used to control bacteria that reduce the physiological or metabolic performance of animals. Antibiotics are included to the feed of animals because very large number of them can be given for a particular treatment at the same time, which lessens the trauma of handling that would be required for individual dosing and in the herd situation. It is also important to get all the animals at risk treated in an identical manner with the same concoction at the same time (especially animals housed in large numbers such as pigs and poultry) (Walton, 1 979; 200 I ). Antibiotics are incorporated in food animals for: 1 ) therapy of disease and prevent deaths, 2) preventing the establishment of disease, 3) reducing pain, 4) circumvent secondary bacterial infection, 5) avoid development of an epidemic, 6) establish the gut flora and so enable the animal to cope with periodic changes of dietary ingredients and 7) improve physiological and metabolic performance (Walton, 200 1 ; Nunes & Guggenbuhl . 1 998; Hays, 1 978 ; Stavric, 1 992). 1 . 1 . 7 Benefits of antibiotics The benefits of antibiotic use for humans include : 1 ) less expensive food, 2) food that is more acceptable to the needs of the consumer, such as less fat, 3 ) pigs that are managed free of persistent diseases, 4) more carcass meat with no trace of disease, and 5) shortened growing period to market point and therefore meat wil l be soft. The benefits to the animal are : 1 ) rapid healing of bacterial diseases, 2) lowering of low grade ailments, 3) alleviation of pain, 4) capacity to cope with constantly fluctuating dietary ingredients without the development of intestinal problems and 5) lowering of persistent toxicity by avoiding the growth of intestinal bacteria that produce a range of toxic substances such as ammonia and monoamines (Walton, 200 1 ; Cromwell , 2002; 4 Adjiri-Awere & van Lunen, 2005); Growth promoting effects of antibiotics are not clearly understood, although the increase in growth rate might be as a result of improved gut health, nutrient uti l ization and improved conversion efficiency (Visek, 1978; Adj iri-Awere & van Lunen, 2005). The beneficial effects of subtherapeutic antibiotic use are found to be greatest in herds raised in sub-optimum sanitary conditions (Zimmerman, 1986) and have l ittle effect on healthy animals reared in "exceptional ly clean' conditions (Taylor, 1999; Adj iri-Awere & van Lunen, 2005) . In pig production, during the lactation period, and few days before and after weaning, deaths can range from 10 to 20 % as a result of stress factors (Piloto et al. , 2000 (as cited in Bocourt et al. , 2004)). Piglets weaned earl ier (at 21 to 28 days of age) are exposed to adverse changes in environmental , social and dietary regime, and are often stunted or have retarded l ive weight gain, lowered feed ingestion and are easily predisposed to diarrhoea (Gabert & Sauer, 1994). These factors, among others, can affect the formation of a normal gastrointestinal flora and change its balance (Vandelle, et al. , 1990; Bocourt et al. , 2004), which incite greater incidence of diseases and higher rate of deaths, as well as the reduction in the production levels (Mosson, 2001; Swientek 2003; Bocourt et al., 2004). Newly born and young piglets can easily be exposed to stress, which is, in part, due to their gastrointestinal tract (GIT) being sterile at birth and they do not have sufficient acidification abi lity in their stomach (Vandelle et al. , 1990; Bocourt et al. , 2004). In addition, they are born agammaglobulinemic and the thermoregulating and enzymatic mechanism of their digestive system is poor (Sainsbury, 1993; Bocourt et al. , 2004). Bocourt et al. (2004) reported that mortality in suckling pigs can be as high as 20% and as many as 41.5% of these deaths could be due to diarrheoic diseases. Out of these, 25 .5% are produced by Escherichia coli (Brizuela, 2003 (as cited in Bocourt et al., (2004)). In recent decades, the method to prevent diseases and increase feeding efficiency was the use of antibiotics as feed additives. Hardy (1999) summarised seven reports showing that dietary antimicrobial agents improved digestibility of energy, nitrogen and phoshorus in pigs by 5 .1 , 1. 8 and 3 .4%, respectively. This is also in agreement with Chen et al. (2005) who reported that antibiotics increase growth rate as a result of improved gut health, nutrient util isation 5 and improved feed conversion efficiency, although the mechanisms involved are not fully understood (Adj iri-Awere & van Lunen, 2005). Similarly, other workers have reported the same benefits of growth-promoting antibiotics in animals, including reproductive efficiency (Yu et al., 2004) and decreased excretion of nitrogen (Close, 2000). 1 . 1 .8 Disadvantages of antibiotics and reason for ban Despite their use in low levels (sub-therapeutic) as growth promoters, increasing feed efficiency, reducing mortality and increasing reproductive efficiency, in recent years, their use has much disputed due to the latent risk of other bacteria securing resistance to particular antibiotics and the damaging effects, such as allergies, that this might have on human health (Adj iri-Awere & van Lunen, 2005). These environmental and consumer bones of contention are now very high on the political agenda of many governments, and the lawful use of antimicrobials and hormones in intensively­ housed food animals is coming increasingly into question by sensational istic, welfare, economic and political organisations alike (Walton, 200 1; Adjiri-Awere & van Lunen, 2005). It has also been reported that prolonged inclusion of antibiotics into pig feeds as growth promoters, fai l to produce a benefit in some 20% of cases and probably do not provide an economic return in a further 20% (Rosen, 1992; Stewart & Chesson, 1993). While supporting the use of antibiotics to a small extent (providing they are not used at al l times), Walton (200 1) reported that good management practices (which includes appropriate use of antibiotics and disinfectants) can increase performance, especial ly when outbreaks are foreseen. But he blamed the non-farming public, and often the non-special ist press, who hold onto and disseminate the incorrect opinion that all pigs and poultry are reared from birth on feed containing antibiotics which are also used for the treatment of disease in man and in animals (Walton, 2001). Other workers have reported that, despite increasing performance in some cases, it is also been reported that antibiotics can interfere in the establishment of a normal gastrointestinal flora and alter its balance (Vandelle et al., 1990; Bocourt et al., 2004) which incites greater incidence of diseases and higher death rates, as well as reduction in production levels (Mosson, 2001; Swientek, 2003; Bocourt et al., 2004). Bocourt e t al. , (2004), reported 6 that antibiotics as feed additives have been proved to affect negatively the eubiosis of the gastrointestinal tract. Similar to Adj iri-Awere & van Lunen ( 1 996), they reported that besides, the bacteria become resistant to them and Bocourt et al., (2004) added that, in some cases, there are antimicrobial residue present in meat, milk and other products of animal origin. The Swann Committee Report ( 1 969) (as cited in Awere Adj iri-Awere & van Lunen, 1 996), was the first to recommend that the use of sub-therapeutic levels of antibiotics for growth promotion and disease prophylaxis could increase the risk of bacteria securing resistance to particular antibiotics, and thus negatively impact the medical community's abi lity to combat human bacteria diseases. Since that time, others have reported it repeatedly to emphasize the concern. 1 . 1 .9 Pig growth and appropriate times to use antibiotics or probiotic 1 . 1 .9.1 Pig growth in general Pig growth in general has been reviewed by numerous researchers such as Pearson and Dutson ( 1 99 1 ), Cummings et al. (2004), Lalles et al. (2004). 1 . 1 .9 .2 The piglet after weaning Sources of stress at weaning may be psychological (Funderburke & Seerly, 1 990), nutritional (Funderburke & Seerly, 1 990; Park et al. , 1 986; 1 984; Bamett . , 1 989), environmental (Crenshaw et al. , 1 986; Bamett, 1 989; 1 980; Funderburke & Seerly, 1 990), too early weaning, or low weaning weight (Mahan & Lepine, 1 99 1 ) . Sources of stress at weaning may also be because of too early weaning, low weaning weight and these can interfere with gut development (Crenshaw et al., 1 986; Bamett et al., 1 989; Mahan & Lepine, 1 99 1 ; Dantzer & Mormede, 1 98 1 (cited in Funderburke & Seerly, 1 990); Lalles et al. , 2004; Cummings et al. , 2003). Intestinal alterations often seen post-weaning in piglets include changes in villus/crypt morphology and brush border enzyme activity, and implication of enteric pathogens such as E. coli and rotaviruss (Pluske et al., 1 997; Lalles et al. 2004). 7 The integrity of the gut mucosa is a prerequisite to lower the entry of pathogens (Bosi, 2000). The components of the gut mucosal barrier (non immunological and immunological) are, however, disturbed during stress such as at weaning. Examples of components of the gut mucosa are given in Table I . Lowered feed ingestion in the weaned pig causes villous to reduce in size (Bosi , 2000). A lowered supply of milk (Kel ly et al. , I 99 I ) or a restricted ingestion of dry feed (Pluske et al., I 996) reduce the height of vil lous on the fifth day post weaning. Inadequate feed intake in weaned piglets may result into intestinal inflammation and affect intestinal morphology. In addition, the antigenicity of the diet is another factor (Bosi, 2000). The defense against pathogens is integrated by the endogenous secretion of many antimicrobial components : hydrochloric acid, salivary lysozyme, defensins, lactoferrins, mucous secretion, bile salts (see Table 1 ). Some of these, however, the mechanisms of control of secretion are not adequately known to increase their production by dietary means (Bosi, 2000). As feed intake increases, the levels of digestive enzymes responsible for the breakdown of starches, proteins and fats increase. Therefore, making the animals to consume more feed shortly after weaning is very important (Lombardi et al. , 2005). As reported by Hiss and Sauerwein, 2003 , reduction of pathogen load by �-glucan in the systemic effect medaited by the central nervous system might contribute to increased feed intake . This is also in agreement with Langhans and Hrupka, 1999 (cited in Hiss and Sauerwein, 2003). As reported earlier, during the lactation period and for a few days pre- and post­ weaning, deaths can range from I 0 to 20 % as a result of stress factors (Piloto et al., 2000 (cited in Bocourt et al., 2000)). The cause of diarrhoea may vary, and the impact on health and wel l-being can range from slight discomfort to severe malnutrition and death in humans (Brown, 1 994; Lin, 2000; Tungthanathanich, I 994; Kendiah, I 999) . 8 Table 1 : Components of the gut mucosal barrier in the piglet Non-immunological Mechanical Healthy enterocytes Cell turnover Tight junction Normal motility Chemical Gastric acidity Salivary lysozyme Defensins Lactoferrin Lactoferrin Mucous secretion Bile salts Bacteriological immunological Local Gut-associated lymphoid tissue Intra-epithelial lymphocytes Mesenteric lymph nodes Aggregates in the lamina propria Peyer's patches Secretory lgA: Systemaic endogenous maternal Circulatory lymphocytes Aerobic and anerobic microorganisms Hepatic Kupffer cells Source : Bosi, 2000 p.279 1 . 1 .9.3 The pig at weaning Pig production can be improved by shortening the lactation period hence early weaning (3 to 4 weeks instead of about 8 to 1 2 weeks). But this can also bring some problems. Some of these problems are highlighted below. At weaning there is ( 1 ) reduced lactate (Pluske et al. , 1 997; Lal les et al. 2004). Enteric infections after weaning further depress enzyme activity (Mroz et al., 2003; Lal les et al. 2004) ; (2) reduced absorption (Lalles et al. , 2004). There is also (3) production of intestinal cytokines which are products that occur naturally and are capable of causing inflammations either at a reduced rate or to destroy the malignant cells. Pro­ inflammatory cytokines IL-l�' IL-6 and TNF-a are usually the first harmonizers produced in reaction to tissue damage and are said to influence intestinal epithelial permeability and ion transport (McKay & Baird, 1 999, (cited in Lalles et al. , 2004)). 9 ( 4) Protein and amino acid metabolism - between the time the pig is born and 1 4 days after weaning, the weight of the gastrointestinal tract of the piglet increases from 2 to 6% of body weight (Burrin & Stol l , 2003 ; Lalles et al. , 2004). Briefly after weaning, anorexia is evident and is followed by a reduction in small intestine protein and DNA mass, especially in the area of the small intestines. When anorexia is overcome, feed consumption increases and this is fol lowed by an increase in small intestine weight that exceeds the rate of bodyweight gain, showing the greater important of the intestinal tissues for growth (Seve et al., 1 986, (cited in Lalles et al. , 2004)) . (5) Integration of gut patho-physiological data- there is an interaction between feed intake, body growth and intestinal villus height trans- and paracel lular transport correspond positively, and vi llus height negatively, with CD8+ T lymphocyte density, thus associating inflammation to morphological and functional changes during weaning (Lal les et al. , 2004) and (6) alterations in gut structure and function- low feed intake shortly after weaning in pigs is said to be the leading aetiological factor in gut disorders (Pluske et al. , 1 997; Lalles et al., 2004). Improvement of gut structure, integrity and function would be achieved through use of means that can stimulate postweaning feed intake, such as an adequate diet. Milk products, such as skim milk powder, have a favourable effect on feed intake, growth performance, feed efficiency and health in piglets because of high digestibil ity of proteins and energy (Thacker, 1 999 (cited in Lal les et al. , 2004)). Spray-dried plasma (SDP) has shown some improvement in growth performance as a result of increased feed intake (van Dijk et al. , 2003; Lalles et al. , 2004). This improvement, is not observed in the presence of ingredients of plant origin (Lal les et al. , 2004). Recent studies have proved that spray­ dried plasma is also protective during the first week after weaning when added together with other nutrients into drinking water (Steidinger et al., 2002, (cited in Lalles et al. 2004)), this decreased the incidence and severity of post weaning diarrhoea and intestinal damage. Other studies with SDP, however, did not show improved responses to E. coli challenge (van Dijk et al. 2002; Lalles et al. , 2004) or reported immune over-response and increased intestinal damage fol lowing a lipopolysaccharide challenge (Tochette et al. , 2000; Lalles et al. , 2004) . Weaning, change of diet and use of antibiotics can be stressful in an animal and can change the eubiosis of the gut milieu (Kelly, 1 998). At weaning, piglets have poorly developed non-immune and immune barrier functions. 1 0 As reported earlier, pig production can be improved by shortening the lactation period, i .e early weaning. In summary, the early weaned pigs can be affected by several stresses as explained above, through too early weaning or at low weaning weight (Mahan & Lepine, 1 99 1 , Lalles et al. , 2004). Low feed intake ( <1 00 g/p/d preweaning) may sensitise the pig to antigens in certain feed ingredients (Newby et al. , 1 983; Bamett et al., 1 989). Exposure of sensitized pigs to the dietary antigens at weaning may result in immune responses that damage the l ining of the intestinal tract. The resulting post-weaning scours may be caused by malabsorption and loss of electrolytes, by which, in conjuction with depressed appetite, result in poor performance (Barnett et al. , 1 989). Such dietary hypersensitivity may be transient because pigs recover from this phenomenon in less than 2 weeks postweaning. Longer exposure to, or greater consumption of, the antigenic proteins may induce tolerance to these antigens. The ski l l of the manager aims to prevent young piglets from picking up a variety of diseases during this period and he is aided in this by the use of management aids, including disinfectants and antimicrobials (Walton, 2001 ; Nunes & Guggenbuhl, 1 998). Supplementation of diets with antibiotics to prevent diarrhoea post weaning may result in selection of resistant strains of disease causing bacteria. Therefore, interest in "natural" feed additives, such as probiotics, organic acids and minerals has developed (Gabert & Sauer, 1 994). 1 . 1 . 1 0 Effects of antibiotics ban Livestock development has been rapid because some Governments have given subsidies, both hidden and transparent to develop intensive methods of farming so as to produce cheaper food. Intensively managed animals suffer less from disease due to improved husbandry. But after achieving this goal , the same Governments are now advocating stricter methods of production. Many supermarkets are now confusing management systems by laying down requirements, sometimes umeasonably, for the husbandry and management of the food animal that they intend to purchase from producers and sell through their retail outlets. These requirements focus especially on welfare and reducing the use of in-feed antimicrobial substances, including performance boosters . The supermarkets are trying to ensure that any meat, eggs or milk they sell wil l be produced in a way acceptable to the consumer. Also, they 1 1 consider that because of the controlled methods of production, these foods wil l not contain any antimicrobial substances that would place the consumer at risk thus improving the image of their sales. However, it must be evident that most systems of animal production that incorporate judicial use of antimicrobials can maintain output at a reasonable cost, which can be passed on to the supermarket and thence to the consumer (Walton, 200 1 ). This is similar to Prescott (2000) who estimated that the forbidding of subtherapeutic use of antibiotics in food animals would add $5 to $ 1 0 a year to the cost of food for each American citizen. 1.1. 1 1 Alternatives to antibiotics Since the ban might result in reduced performance, this has driven animal nutritionists and producers to devise natural substitutes , such as probiotics, for commercial pig farms to reduce the problem of higher death rates and reduced growth performance following this restriction (Link et al. , 2005 ; Yu et al, 2004; Adj iri-Awere & van Lunen, 2005). Denmark announced, as of November 2002, a complete withdrawal of growth promoters by 2006 (Fischer-Boel , 2002 as cited in A were Adj iri-Awere & van Lunen, 2005). Despite the current interest in getting rid of subtherapeutic antibiotic use in animal production, there may be a risk that such a lowering or removal would have negative effects on the animal welfare, nutrient uti lisation, manure production and economic sustainability . Close (2000) indicated that in-feed antibiotics for pigs produce a 5- to 1 0-fold return because production efficiency is raised. In the EU two Bacillus products are l icenced for animal use, BioPlus® and Toyocerin®. Toyocerin contains a strain of B. cereus var toyoi that has been deemed safe for animal use because of its fai lure to produce enterotoxins and its failure to transfer antibiotic (Hong, 2005) . 1.1.12 Way forward As reported earlier, consumer groups and the European Union (EU) have decided to forbid all antibiotics used sub-therapeutically in farm animal feed, driving animal nutritionists and producers to devise natural alternatives such as probiotics for 1 2 commercial pig farms to reduce the problem of higher death rates and reduced growth performances fol lowing this block. Recently, studies relating to the health benefits and therapeutic effects of some harmless live microroganisms has become one of the hottest topics in nutritional sciences (Qu, 200 1 ) . Technologies in this area are rapidly developing, based on new biotechnology. Enzymes, yeasts and l ive bacteria and their metabol ites are involved in these investigations. There is also need to develop species resistant to diseases (Tizard, 2000) . Justification for health claims comes mainly from animal studies. These have shown that germ-free (gnotobiotic) animals, or animals whose gut microflora have been perturbed by antibiotics are more susceptible to disease, and resistance can be restored by oral administration of faecal suspension from healthy individuals, a routine taken by the USDA to reduce salmonel losis infection in commercially reared chicks. The organisms used in probiotics are known to produce antimicrobial substances that might affect the colonic microflora balance (Scheinbach, 1 998). On the other hand, probiotics supplementation to piglets to improve growth performance has shown different results, and the precise mode of action through which probiotics exert their positive influence is not ful ly understood, although the mechanisms by which they improve health include: -reduction in the amount of viable microorganisms by- • Production antimicrobial compounds such as acids, peroxides or bacteriocins bactericidal to groups that negatively impact health; • Competition with disease causal organisms • Competition with disease causal organisms for mucosal binding sites; • Competition for substrates -stimulation of the immune system through increase in the concentration of IgG which increases the number of antibodies and macrophage activity (Scheinbach, 1 998 ; Lopez, 2000). 1 3 -modification of the microbial metabolism through increased enzymatic activity (e.g. beta-galactosidase which decreases lactose intolerance) and decrease in enzymatic activity (e.g. beta-glucuronidase and nitroreductase) (Lopez, 2000). Other benefits include reduction of: -lactose intolerance -cholesterol -cancer (Lopez, 2000; Srivanasan, 2005). 1 . 1 . 13 Methods to improve performance after the ban In Sweden, the performance of animals declined after the ban of in-feed antibiotics as stated above. Initially, post-weaning mortality rates in that country worsened I increased by 1 . 5% and days to 25 kg increased by 6 days (Robertsson and Lundeheim, 1 994 (cited in Adj iri-Awere & van Lunen, 2005)) and this is also in agreement with Goransson, 200 1 and Walton, 200 1 who also added that the number of diarrhoea cases doubled. Following the prohibition of in-feed antibiotics in the EU, the effectiveness of some of the available alternatives, such as probiotics (Pollman et al. , 1 980) has evaluated. Enzymes, such as microbial phytase, has been found to release phytate-bound phosphorus in ingredients of plant origin and improve utilisation of phoshorus and amino acids in broiler starter feeds (Ravindran et al. 2006; Patridge and Hazzledine, 1 997). Fermented dairy products, such as yoghurt and fruit juices, (Cummings et al. , 2004); fermentable carbohydrates, such as sugar beet pulp, and fructooligo­ saccharides increase the number of beneficial lactobaci llus bacteria. They also stimulate higher bacterial diversity and more rapid stabil isation of the bacterial community, resulting in improved health but not necessarily growth (Konstantinov et al. , 2003), Mannan oligosaccharides (MOS) inhibit the colonisation of some strains of bacteria in the intestinal tract, such as E. coli. MOS acts as a recptor for E. coli , binding the microbe (Davis et al., 2002). Bacterial adherence results in the alteration of the intestinal micro flora, and may be the mechanism by which MOS improves growth performance in pigs (Davis et al., 2002). Similar prebiotics include dietary fibres (non-starch polysaccharide) such as inulin, that improve gut health, bowel habit 1 4 and satiety, and synbiotics such as bakery, cereal products (Cumming et al. , 2004) Average dai ly gain and feed intake is improved by using dietary �-glucan (Hiss & Saurwein, 2003) and the improvement was higher on farms with low hygienic status (Decuypere et al. , 1 998; Hiss & Sauerwein, 2003). Products such as copper sulphate improves health and growth performance (Davis et al., 2002), due to its antimicrobial effect which improve the gut. Both Cu and mannanologosaccharides (MOS) are bel ieved to alter lymphocyte response in vitro (Davis et al., 2002). However, feeding high levels can result in more Cu in the manure and pose an environmental threat (Davis et al. , 2002) or impair the gut function (Cromwell , 200 1 ; Davis et al. , 2002). MOS can be used in the place of Cu in pig diets to reduced these problems (Davis et al. , 2002). MOS has the abi l ity to attach to mannose binding proteins on the cell surface of some strains of bacteria, thereby preventing these bacteria from colonising the intestinal tract by interfering with the binding of carbohydrate residues on epithelial cell surfaces (Davis et al., 2000). 1 . 1 . 1 4 Other claims and examples of probiotics The digestive tract is most frequently the objective of the functional and health claims and a large market already exists for the gut functional foods worldwide (Lalles et al. , 2004; Cummings et al. , 2004). The most widely used probiotics are lactobacil l i and bifidobacteria that can survive in the intestines. Numerous investigations have been conducted on the beneficial effects on human health for these species (Perdigon et al. , 1 990; Benno et al. , 1 996; Shek, 1 976, (cited for al l in Yu et al. , 2004)). Reports of a culture of Lactobacillus acidophilus actively taking up cholesterol from laboratory media have been validated, and it is known to beneficially modify serum cholesterol levels (De Rodas et al., 1 996 (cited in Yu et al. , 2004 and in Adj iri-Awere & van Lunen, 2005)). In addition, Lactobacillus and Bifidobacterium sp. have been found to be involved in functions that include inhibiting disease causing bacteria, antitumour and anticholesterolaemic activity, positive effects on digestion, and exhilaration of immune system (Piard and Desmazeud, 1 99 1 ; Adachi, 1 992; Wu et al. , 200 1 ; Lin et al. , 2002; Yu et al, 2004; Adj iri-Awere & van Lunen, 2005). Supplementation of 1 5 lactic acid bacteria (LAB) to piglets as been discovered to promote body weight gain, improve feed conversion, boost the populations of bacteria, and lower levels of pathogenic intestinal bacteria. In other studies conducted, it was found that a Lactobacillus acidophilus, L. pentose and Bacillus subtilis mixture could regulate intestinal microbes, boost immune response and lower serum cholesterol (Lin et al. , 2002). LAB do not necessarily need to adhere to or colonise the gastrointestinal tract, as long as they are regularly consumed. Many species and strains of LAB from several genera have been credited with these health benefits, due to their ability to produce different types of antibacterial compounds. They are consumed through many different commercial ly available products (Lin, 2000). General ly a product, depending on the type, can contain one or more of the fol lowing species : Streptococcus thermophilus, Lactococcus lactis, Leuconostoc mesenteroides, Lactobacillus bulgaricus, Lactobacillus casei, Lactobacillus acidophilus, and Lactobacillus reuteri and some Bifidobacterium species. In addition, products can contain other Lactobacillus species, although some are even currently not regarded as a species (such as Lactobacillus caucacicus in some products sold in health feed stores) (Lin, 2000) . There is proof that some pro biotic bacteria can arrest cel l attachment and cell invasion by enterovirulent bacteria. Lactobacilli and bifidobacteria are easily cultured and have a good safety record (Lin, 2000). On the other hand, other studies have not identified any improvement pig in growth performance (Harper et al. , 1 983 ; Yu et al. , 2004), and this is in agreement with Stewart and Chesson, 1 993. Some investigators stil l insist that probiotics cannot replace antibiotics (Walton, 200 1 ; Partridge, 1 99 1 (cited in Stewart & Chesson, 1 993)). Others think that probiotics are more effective in young animals because: 1 . nutrients are more efficiently absorbed because of the thinner small intestinal epithelium; 2. nutrients are spared due to a reduction in competing microorganisms; 3 . microorganisms responsible for subclinical infections are reduced or eliminated; 4. production of growth-depressing toxins or metabolites by the gastrointestinal microbiota is reduced; 5. microbial deconjugation of bile salts is reduced (Jensen, 1 998) . Antibiotics should continue to be used in older animals where probiotics are thought to be less effective and antibiotics should continue to be used when a danger of disease is sensed (Walton, 200 1 ) . 1 6 In other studies, it has been concluded that spores of Bacillus subtilis germinate in the digestive tract (Casula & Cutting, 2002; Link et al. , 2005) . Because the vegetative forms are easily affected by bile salts one may suspect their consequent sporulation or lysis . It is most likely that the spores themselves apply a probiotic effect by acting as stimulators and increasing local cell-mediated immunity (Caruso et al. , 1 993 (cited in Link et al. , 2005)). However in the study by Link et al. , 2005, they did not observe significant differences in either phagocytic activity or polyclonal activation of lymphocytes isolated from the peripheral blood of pigs. In the experiment by Apgar et al. ( 1 993), Streptococcus faecium treatment of weaned pigs had no effect on cell­ mediated immune response and Apgar cited other similar results in the study by Kluber et al. , ( 1 985) . Other investigators observed an increase in macrophage activation in mice fed Lactobaccilus casei and L. bulgaricus. Mice treated with S. thermophilus have also shown to have higher phagocytic activity of peritoneal macrophages and the reticuloendothelial system (Perdigon et al. , 1 987 ; Apgar et al. , 1 993) . Similar research from this station (Clayton, 2002) reported that in addition to vitamin C, the main stimulator of non-heam iron absorption in the diet is meat (Seth & Mahoney, 2000 (cited in Clayton, 2002)) . During digestion, gastric acids denature the proteins before they are split into smaller particles as peptides and free amino acids (Totara, 1 996 (cited in Clayton, 2002)). Kroe et al. ( 1 963) (cited by Clayton (2002)) reported that nine essential amino acids found in meat were capable of promoting the uptake of ferrous iron (and histidine, glutamine, glutamic acid and methionine), whereas cystein, histidine and lysine were able to increase ferric iron uptake. Cysteine, comparable to vitamin C is a reducing agent and helps to transform ferric iron into ferrous form. 1 . 1 . 1 5 Some requirements for development of probiotics The understanding of the structure and operation of the gastrointestinal tract microbial communities and into the activity of the specific microbial species within this ecosystem is important for the development of rational alternatives to in-feed antibiotics, such as probiotics and prebiotics. The wide variety of bacteria in the gut give it a unique environment. The background knowledge on digestion, together with the wide variety of foods offered for consumption today make for great diversity in 1 7 individual patterns of digestive and immune function (Cummings et al., 2004; Scheinbach, 1 998; Stavric, 1 992). Many experiments have been conducted to distinguish microbes in faecal samples. It has been possible to spot gram negative and gram positive bacteria while strict anaerobes have been difficult to identify using conventional means. The new development of ribosomal RNA (rRNA) as a molecular marker, backed by expertise in animal nutrition, and knowledge of elements that dictate the composition of the microbiota and the invasion of pathogenic organisms in the GIT, wil l lead to a better understanding and identification of factors concerning the gut microbiota that affects the ecosystem of the gut at different times, and how these can be manipulated to maintain a normal, working and healthy gut (Lal les et al. , 2004). Many beneficial claims have been made and at this point it is crucial to set the boundary or standards between normal and ill health, the benefits to the host' s health and how to measure aspects of digestion and immunity and interpret the results (Cummings et al. , 2004). 1 . 1 . 1 6 Cost and effectiveness of probiotics Although natural products l ike enzymes from bacteria have been known to improve performance, the method of production can be expensive with low tolerance to heat (Lopez, 2000) . Improvement of production methods wil l increase effectiveness, lower their price and increase the amounts to be used (Lopez, 2000; Scheinbach, 1 998) . It has been observed that some probiotics can be used in low quantities comparable to antibiotics with similar effects (Lopez, 2000). 1 . 1 . 1 7 Interactions between probiotics, ingredients and time As Fugh-Berman (2000) and Chavez et al. (2006) reported herb-drug interactions or, in our our case, probiotic-dietary ingredient interactions are evident and probiotics researchers should advise users about mixing probitics and diet formulations and timing of their use . They need to know whether side effects would arise from using 1 8 these preparations in certain circumstances, such as in combination with drugs or other ingredients (Fugh-Berman (2000) and Chavez et al. (2006). For example, although there is proof of recombinant human lactoferrin (rh-LF) reducing bacteraemia and lowering disease severity scores in infants, oral treatment with rh-LF failed to protect patients with E. coli (Edde et al. , 200 1 ). 1 .2 The immune system Since probiotics are said to stimulate the immune system, it is relevant to know how the immune system operates. The variety of claims for gut health and immune function are found on food on the European market. Generic claims such as ' help keep your body in balance with the probiotics' and ' diets rich in fibre keep your digestive system regular' are common. Further benefits associated with intake of probiotics are widely reported in magazines. The claims made can be broadly categorized into content, functional , enhancement function, reduction of disease risk or disease risk factor and medical claims (Cummings et al. , 2004) . 1 .3 Effect of natural products on pig performance 1 .3 . 1 Effects and mode of action of non-starch polysaccharide (NSP) Various reviews have been made by numerous researchers such as: Pearson and Dutson ( 1 99 1 ), Cumming et al. (2004), Lalles et al. (2004), Lopez (2000), Atherton and Rubbins ( 1 987), Jensen et al. ( 1 998), Jensen & Jensen ( 1 998), to mention but a few. Figure 1 : Assummed mode of action of nonstarch polysaccharides (NSP) and NSP­ hydrolysing enzyme (E) Total effects: Feed intake. performance. nutrient availability. ME, heat production From Simon, 1 998 p 1 1 6 1 9 Growth of indigenous microbiota identified as having beneficial properties on the host, may also be added to the gut. This may be done by the incorporation of compounds in the diet which survive passage through the stomach and small intestine and selectively stimulate selected bacteria in the hind gut. Carbohydrates such as non­ starch polysaccharides (NSP), are the principle energy substrate for large intestinal fermentation in pigs (Jensen et al . , 1 998). NSP and oligosaccharides may pass undegraded to the colon and may influence the composition of microbiota in the large intestines and thus directly affect the total effects (e.g. increase performance by improving feed intake or through decreasing the concentration of ammonia in the hind gut: cage effect (Jensen & Jensen, 1 998). In addition to cage effect, NSP can also stimulate enzymes to improve microflora and reduce digesta viscosity. Microflora can affect either digesta viscosity, or total effects through improved fat digestion or can directly influence these effects. When digestion viscosity is improved it then stimulates intermediate factors which all or separately improve some total end effects (Simon, 1 998). 1 .3.2 Effect of pro biotic on blood parameters Intepretation of leucocyte concentrations in the blood provides insight regarding potential processes that may be occurring in the patient. The leukogram is complete set of numerical data in the leucocyte profile, along with any noted morphological abnormalities. Abnormalities in the leukogram might provide inferences to a pathological process such as inflammation but might not necessari ly establ ish a specific diagnosis. The correct interpretation of leukocyte abnormalities and together with clinical findings, however, may lead to a diagnosis (Thrall et al. , 2004; Tvedten, 1 993 ; Voigt, 2000; Sims, 1 996). B lood examination can be used to detect health status and for production monitoring. It can also be used for early detection of disease and blood disorders (Evans, 1 994) . Exel lent review in the field of interpretation of blood parameters can be obtained from blood interpretation manuals from (electronic) counting machines such as the ADVIA 1 20 and by reaseachers such as Egeli et al. , 1 998 and Thrall et al., 2004) . 20 1 .3 .3 Effect and mode of action of Lactoferrin on performnance and general health of pigs The mucosa of the gut is the first l ine of contact with pathogenic microorganisms that are ingested by the animal and has essential functional role of functional barrier against these agents (Bosi, 2000). Non-immunological and immunological components of the gut mucosal barrier are depicted in Table 1 . Among biological ly active proteins and peptides in milk are glycoproteins and lactoferrins. Proteins and peptides in glycoproteins are lactoferrin, milk mucins (e.g. mannose containing glycoproteins), and adhesion molecules, while those in lactoferrin are lactoferroxins (Zabielski, 1 998). To understand the effect of lactoferrin on animal performance, we have studied the reviews on the chemical and biological properties by Cumberbatch et al. (2000) ; De Wit & Hooydonk ( 1 996); Reiter ( 1 985), Reiter & Perraudin ( 1 99 1 ), lyer & Lonnerdal ( 1 993), Lonnerdal & lyer ( 1 995), Wong et a/. ( 1 997); Lonnerdal (2003), to name but afew. The structure and functions of lactoferrin, as reviewd by the fol lowing researchers such as Lonnerdal & Iyer ( 1 995); Tome & Debbabi ( 1 998); Muri et al. (2005) have also been studied. Lactoferrin is a bioactive protein which makes an important contribution to the host defence system. It eliminates pathogens such as bacteria, viruses and fungi, stimulates and protects cells involved in the host defence mechanisms and controls the cytokine response (Steijns, 200 1 ; Prgomet et al. , 2005). Present commercial bovine lactoferrin products are included in infant formulas, nutritional iron supplements and drinks, fermented milks, chewing gums, immune-enhancing nutraceuticals, cosmetic formulas and pet care supplements (Steijns, 200 1 ) . Bovine lactoferrin is used in cats for the treatment of intractable stomatitis by stimulating the phagocytic activity of neutrophils (Steijns, 200 I ). I t also induces both mucosal and systematic immune response in mice (Steijns, 200 1 ) . Monocytes, neutrophils and macrophages are cel l s of the immune system that ki l l invading pathogens by oxidant reactions. As free iron is often present in the areas of inflammation or infection, these oxidant reactions may be accelerated due to the catalystic effect of iron on free radical production. Lactoferrin wil l bind the free ferric 2 1 iron with high affinity, and thus function as a local antioxidant, protecting the immune cells against the free radicals produced by themselves. Although only the neutrophi ls degranulate and deliver lactoferrin, monocytes and macrophages have lactoferrin receptors on their cel l surface (Tome & Debbabi, 1 998). Protein components of milk have many uses. They provide amino acids which are required for growth and development. They also have more specific functions. The nature of the peptides formed during milk proteins digestion is dependent on the digestive process. The biological properties of milk proteins or milk protein-derived peptides include protein with antimicrobial activity (lactoferrin), peptides which promote nutrient assimilation and peptides with modulatory activity on the physiological function (Tome & Debbabi, 1 998). The role of the different pathways in hypothetical functions, i .e . immune surveil lance, metabolic regulation or gastrointestinal disease, stil l remains unknown. This absorption of large molecules in anti genic and biological ly active quantities are believed in particular to play a role in different physiological and immumunological responses that contribute to humoral tolerance and regulation (Tome & Debbabi, 1 998). Different proteins including lactoferrin, vitamin B 1 2 s binding protein, folate binding protein, P-lactoglobulin and a- lactalbumin, are assumed to interact with either minerals, vitamins or nutrients by a specific mechanism. These interactions may have an effect on absorption of these nutrients (Hanson et al. , 1 994 ; Tome & Debbabi, 1 998). Lactoferrin has been particularly studied for its role as an iron-scavenging protein that could be involved in iron transport, or have an anti-bacterial, anti-inflammatory and immunomodulating properties (lyer & Lonnerdal, 1 993; Tome & Debbabi , 1 998). 22 1 .3.4 Common methods of preparation I production of lactoferrin Lactoferrin and glycoproteins are among biological ly active proteins and peptides in milk and they are claimed to have beneficial effect on the host (Zabielski, 1 998). Bovine lactoferrin may be isolated from fresh skim milk by cation exchange chromatography and gel filtration. Milk at native pH is passed for a short time through S Sepharose fast flow at 4 C and the attached proteins eluted in steps with 0 . 1 , 0.35 , and 1 M Nacl, respectively. The 1 M NaCl fraction containing lactoferrin i s then dialysed and freeze dried. The resulting material is then dissolved in 25 mM sodium phosphate buffer at pH of 6.5 and reapplied to the cation exchanger, which is earl ier equil ibrated in the obove buffer. Lactoferrin is then eluted by appl ication of salt gradient to 1 M NaCl in phosphate buffer and the recovered material dialysed and freeze-dried. Final purification of lactoferrin is achieved by gel filtration through Sephacryl S300 in phosphate buffer and the protein recoverd as a dialysed freeze­ dried powder. Purity of the final product is usual ly greater than 98% as assessed by resource reversed-phase high performace liquid chromatography (HPLC) and mono­ SHPLC (Palmano & Elgar, 2002, cited in Cornish et al. , 2004)). 1 .3.5 The normal or reference values of the leukogram Before making a conclusion concering what is normal or deviates from normal, a number of steps should be followed in order to inteprete the leukograms. Intepretative attention should concentrate only on the absolute values within the differential count. When examining the haematology report, the total leucocyte concentration should be the first thing to look at. The total leukocyte count is not directly interpreted but is only used to calculate absolute differential concentration. If the total count is decreased, the absolute concentration of each type of each type should be examined to determine which are deficient. If the total count is decreased, examine the absolute concentration of each cell type to determine which are present in excess. Even if the total concentration is normal, examine the absolute concentration of each cell type if any abnormalities in distribution are present. Identified abnormalities in the absolute concentration of individual leukocyte types are then interpreted into processes. (Thompson & Forsyth, 2004; Tizard, 2000; Thrall et al. , 2004; Voigt, 2000 and Ege1i et al., 1 998; Comazzi et al. , 2004) . 23 The electronic hematological cell counter is able to provide a differential white cell count, isolating the number of white cell s in each of the blood samples as shown in Table 2 . The acceptable physiological values of the red blood cells parameters in a normal health pig are shown in Table 3 . Table 2 : Blood parameters, their abbreviations and units of measurement White cells Neutrophil cells Lymphocyte cells Monocyte cells Eosinophil cells Basophil cells Red blood cells Haemoglobin concentration Haematocrit level Mean cell volume (or mean erythrocyte volume) Mean cell haemoglobin (or mean erytrocyte haemoglobin content) Mean corpuscular haemoglobin concentration (or mean erythrocyte haemoglobin concentration) Corpuscular haemoglobin constant Corpuscular haemoglobin concentration mean Corpuscular haemoglobin Red blood cell distribution width (or erythrocyte distribution width) Haemoglobin distribution width Platelet Mean packed volume Packed cell volume WBC NEUT LYMPH MONO EOS BASO RBC HGB HCT MCV MCH MCHC CH CHCM CH RDW HOW PLT MPV PCV x 1 09cells!L x 1 09cells/L x 1 09cells/L x 1 09cells!L x I 09cells/L x 1 09cells!L 24 g/L LIL fL p g g/L pg pg % g/L x 1 09 cells/L fL % Table 2: Accepted physiological values of white and red cell parameters in the normal and anaemic pigs (Adapted from Egeli et al. ( 1 998), Clayton (2002), Mi l ler et al. ( 1 96 1 ), Tumbleson & Kalish ( 1 97 1 ), Svoboda et a l . (2004), Ul lrey, 1 959) Blood parameters Abbreviation w1it of measure (Egcli) percent ( Miller ) (Tumblcson (Advia 120) (Svoboda Ullrey Ruakura range of white cell ct al. l 96 1 ) & Kalish, 197 1 ) et al.. 2004) et al., 1959 ma.'is Age (days) Age Age Age Age Age Age (x-x) ( 2 1 -49} (42) (X-x) (7-35) (24-3 1 ) (x-x) nom1al nunnal lowest anemic- healthy White cells WBC x 1 09cells/L 1 0-23 1 00 X X X X 6.92-9.34 5.0-8.0 wbc type Neutrophil cells NEUT x 109cells/L 2.5- 1 0 26.6-56.7 X X X X 1 3 - 1 .9 Lymphocyte cells LYMPH x 109cells/L 7.0- 1 5.5 35. 5-62.0 X X X X 8. 1 -7.3 4.5-13.0 Monocyte cells MONO x 1 09cclls/L 0.32-2.0 1 .6-8.8 X X X X 0.2-0.7 0 .2-2.0 X Eosinophil cells EOS x 1 09cclls/L 0.08- 1 .76 0. 1 -5 .6 X X X X 0.2-0.2 0.5-2.0 Basophi l cdls BASO x 109cel ls/L 0-0.3 0-2.7 0.0 X X X 0.0 -0.2 X Method of calculation Red blood cells RDC x 1 01 2 ct:lls/L 5-8x l 0 1 2 direct X 3. 1 6-5.65 X 4.5 2.29-5.0 5.0-8.0 red cell llQ!< and �arameters Haemoglobin concentration HGB g/L 1 1 0- 1 70 direct 90±0 2 82- 1 08 X 80 40- 98 1 00- 1 60 V) 0-1 Haematocrit level HCT UL 0.37 direct 0.304±0.08 0.26-0.36 X 0.3 0 . 1 2-0.2 0.32-0.50 Mean cell volume MCV fL 50-68 X X 54.6-97.5 X 55 44.2-55.6 50-68 (or mc:m erythrocyte volume) Mean cell haemoglobin MC I-I p g 1 4 .4-20. 1 HGB/RDC X 1 6.2-29.4 X 1 7 X 1 7-2 1 (or mean erytrocyte haemoglobin content) Mean corpuscular haemoglobin concentration MCHC g/L calcu lated as: (or mean crylhrocyte hac:moglobin concenu·0.05) . 39 Table 5 : Least-square means ofthe effects of probiotic-supplemented diets on growth performance in weanling pigs for average pen l ive weight of piglets at the start of the experiment (WkOwt), average pen l ive weight of piglets at the end of week 1 (wk1 wt), average pen l ive weight of piglets at the end of week 2 (wk2wt), average pen live weight of piglets at the end of week 3 (wk3wt), average daily gain (ADG), average daily feed intake (ADFI) and average feed conversion rate (FCR) for the 2 1 day experimental period for each diet with standard error (SE). TRT Parameter A B c D E SE (control) (probiotic B) (probiotic C) (probiotic D) (lactoferrin) Growth Pe1jormance Weaning wt (g) 1 7529.8 7524.3 7452.5 7456.5 7325 . 8 Wk l wt (g) 8303 .05 8423 .65 8269.36 82 1 8 .8 1 80 I 0 .82 1 4 1 .97 Wk2\Vt (g) 1 044 1 .54 I 0490.34 I 0630.63 1 0223 .49 1 0035 .26 249.5 Wk3wt (g) 1 3 866. 1 1 1 4056.9 1 4000.5 1 3480.73 1 322 1 362.4 1 Weight gain Wk l - wkO(g) 773.25 899.35 8 1 6 .86 762 .3 1 685 .02 Wk2 - wk l (g) 2 1 3 8 .45 2066.69 236 1 .27 2004 .68 2024 .45 Wk3 - wk2(g) 3424 .9 1 3566.62 3369.93 3257.24 3 1 85 .73 Wk3-wk0 (g) 6333 . 3 6532.6 6548 6024 .23 5 895.3 A D G (g/d) 305.4 1 3 1 4 .49 3 1 1 . 80 287 .05 274.69 1 7 .26 ADFI (g/pig/d) 404 .64c 426.77d 423 .63cd 378.48b 34 1 .483 1 9 .9 1 FCR 1 .34299 1 .3 5963 1 .4408 1 1 .35278 1 .2574 0 .063 1 actual mean values •. b, c, d values in the same row with different letters are significantly different (p 0.5) between the five groups (mean values : 8303 .05, 8423 .65 , 8269.36, 82 1 8 . 8 1 and 80 1 0.82 for diet groups A, B, C, D and E, respectively. However, on numerical basis, only the pigs in diet B were heavier than the controls while those in diets C, D and E groups were l ighter than the controls . Pigs that consumed diet B were the heaviest while those on diet E were the lightest (Table 5) . 40 3.2.3 Week 2 body weight Bodyweight on day 1 4 was not significantly different (p>0.05) between the five groups (mean values : 1 044 1 . 54, 1 0490.34, 1 0630.63, 1 0223 .49 and 1 0035 .26 for diet groups A, B, C, D and E, respectively. However, pigs in that consumed diets B and C were heavier than the controls whi le those that received diets D and E were l ighter than the controls . Pigs that consumed diet C were now the heaviest while those on diet E were sti l l the l ightest (Table 5) . 3.2.4 Week 3 body weight Bodyweight on day 2 1 was not significantly different between the five groups (mean values : 1 3 866. 1 1 , 1 4056 .9, 1 4000 .5 , 1 3480.73, 1 322 1 for diet groups A, B, C, D and E, respectively. Notwithstanding this, (as in week 2) animals that received diets B and C continued to weighed more compared to those that fed the control diet while those that consumed diets D and E were sti l l numerically l ighter than the controls . The pattern of body weights was similar to week 1 and pigs that were offerd diet B regained the position of heaviest animals while those that consumed diet E maintain the position of lighest animals as in weeks one and two (Table 5) . In summary, animals that received diets B and C were heavier than the control whi le those that consumed diets D and E were l ighter than the controls and remained l ight from week 2 to the end of the experiment. Diets B and C stimulated body weight growth better than the control diet while diets D and E depressed it. 3.2.5 Average daily gain (ADG) Average daily gain is the total weight gained in a given period of time divided by the number of days (measured in g per day). Significance of effects of wkOwt (body weight of pigs at the beginning of the study), diet, run, farm, sex, interaction between farm x diet ADG (average daily gain for the total period is shown in Annex 1 ) . 4 1 Table 5 depicts least-square means of the effects of pro biotic-supplemented diets on growth performance in wean l ing pigs for average pen l ive weight of piglets at the start of the experiment (WkOwt), average pen l ive weight of piglets at the end of week 1 of the experiment ( wk 1 wt ), average pen live weight of piglets at the end of week 2 (wk2wt), average pen liv weight of piglets at the end of week 3 (wk3wt), average daily gain (ADG), average daily feed intake (ADFI) and average feed conversion rate (FCR) for the 2 1 d experimental period for each diet with standard error (SE) . Farm had an influence on ADG of pigs (p = 0.0 1 20) (Annex 1 ). Bodyweight at weaning tendentiously affected ADG of pigs (p=0.08). However, average daily gain was not different (p = 0.4093) for piglets fed control diet (diet A) or probiotic­ supplemented diets B, C, D and E (mean values : 305 .4 1 , 3 1 4.49, 3 1 1 . 80, 287 .05 and 274.69 for diet A, B, C, D and E, respectively) . Replica and sex had no effect on ADG of pigs and the interaction of farm x diet were not detected (p>0.05) . ADG fol lwed the pattern of feed intake and was numerically higher in pigs that consumed diets B and C compared to the controls and lower in pigs that recived diets D and E as wil l be observed below. 3.2.6 Average daily feed intake (ADFI) The average daily feed intake is the total quantity of feed consumed in a given number of days divided by the number of days and number of animals consuming that feed (measured in g per animal per day). The effect of diets on ADFI of pigs is depicted in Table 5 . The significance of effects o f bodyweight at weaning, diet, replica, farm, sex, interaction of farm x diet on ADFI (average dai ly feed intake) are summarised in Annex 1 . There were significant (p = 0.0 1 69) treatment differences for average daily feed intake in piglets fed control or probiotic B, C, D and E (mean values : 404.64, 426.77, 423 .63 , 378 .48 and 34 1 .48 for diet A, B, C, D and E, respectively) . The effect of 42 bodyweight at weaning on ADFI of pigs was significant (p=0.0006) while replication had no effect on ADFI of pigs (p>0.05). Farm and sex tendentiously influenced ADFI for pigs (p=0.08 and p=0.06, respectively). An interaction of farm x diet was not detected (p>0.05) (Annex 1 ) . Feed intake in pigs that received diet B was significantly higher that in pigs that consumed the control diet (p=O.O 1 69) while those in diet C ate more feed compared to the controls, but there was no signicant differences between the two groups (p>0.05) . The pigs that received diets D and E consumed significantly low levels of feed compared to the controls (p<0.05). To summarize, during the 21 day study period, animals that consumed diet B consumed significantly higher amounts of feed compared to the controls (p<0 .05) while those in diet C group also ate more feed than those in the control group although the difference between the two groups was not signi ficant (p>0.05). The pigs that were on diet D and E ate less feed than those given the control diet (p<0.05) [Table 5 ] . Diets B and C stimulated feed (energy) intake better than the control diet while diets D and E depressed it . 3.2. 7 Average feed conversion ratio (AFCR) Feed conversion ratio (FCR) is the ratio of feed eaten in a given period of time to the amount of weight gained over the same period of time. Since it is a ratio of parameters with same units (g) there are no units of measure. The effect of diet on FCR of pigs is summarized in Table 5 . Significance of effects o f bodyweight at weaning, diet, run, farm, sex, interaction between farm x diet on FCR (average feed conversion ratio) over the experimental period of 2 1 days are shown in Annex 1 . There were no treatment effects pigs (p=0. 1 843) on average feed conversion rate for piglets (p = 0.3776). Bodyweight at weaning had no effect on FCR of pigs (mean 43 values : 1 . 34299, 1 .35963, 1 .4408 1 , 1 .3 5278 and 1 .2574 for diet A, B, C, D and E, respectively). Replica and sex did not affect FCR in pigs and the interaction of farm x diet were no significant (p>0.05) (Annex 1 ). FCR was higher in pigs fed diets B, C and D compared to the controls while those in diet E tried to use the less quantity of feed ingested more efficiently compared to the controls (Table 5) . 3 .3 Effect of probiotic on Haematological (blood)parameters 3.3.1 Effect on whole blood parameters Secondly, we investigated the effect of dietary treatment on haematological parameters. The blood samples collected on day 0, 1 4 and 2 1 of the experiment were used to analyse the effect on diet on the blood parameters and final ly how performance and health are affected. To prevent double handling and to reduce stress, the pigs were weighed just before sampling. The fol lowing is a list of parameters that were analysed: White blood cells (WBC), neutrophil cells (NEUT), lymphocyte cel ls (LYMPH), monocyte cel ls (MONO), eosinophil cells (EOS), basophil cell s (BASO), red blood cel ls (RBC), haemoglobin concentration (HOB), haematocrit level (HCT), mean cell volume (or mean erythrocyte volume) (MCV), mean cell haemoglobin (or mean erythrocyte haemoglobin content)(MCH), mean corpuscular haemoglobin concentration (or mean erythrocyte haemoglobin concentration) (MCHC), corpuscular haemoglobin constant (CH), Corpuscular haemoglobin concentration mean (CHCM), corpuscular haemoglobin (CH), red blood cell distribution width (or erythrocyte distribution width)(RDW), haemoglobin distribution width (HDW), platelet (PLT), mean packed volume (MPV), neutrophil (NEUT), lymphocyte (LYMPH), monocyte (MONO), eosinophil (EOSI), and basophil (BASO). Some whole blood parameters varied during the study. Most white cell population and differential counts (NEUT etc ) in b lood of pigs from different groups were within 44 normal range for pigs. Neither the control diet nor probiotic- nor lactoferrin­ supplemeted diets altererd blood parameters (p>0.05) for the 2 1 day experimental , although some numerical differences were observed. WBC parameters showed showed l ittle difference in levels and cellular responses to that of the animals fed the control diet, although they are reported and might be of some use as an indicator of disease or potential benefits (See Table 6 and Annex 1 ). On the other hand, a few Red cell population and differential counts (RBC, HGB, HCT, MCH, etc) in blood of pigs from different groups were noticeably below normal range for pigs of that age group and blood parameters of pigs fed control or pro biotic­ or lactoferrin supplemeted diets did alter (p>0.05) for the 2 1 day experimental and some numerical differences were observed. Although most are reported, it is only clear that RBC, HCT, HGB and MCH were numerically lower in pigs fed diets D and E compared to the controls . It can also be seen that, the animals that consumed diets B and C had numerically higher levels of HCT and RBC than the controls (Table 6 and Annex 1 ). These body iron measures might be of some use as they can affect performance and health in pigs and wil l be discussed in detai l . 3.3. 1 . 1 Effect on white blood cell population parameters For white b lood cel l s we analysed : White blood cel ls (WBC), neutrophil cells (NEUT), lymphocyte cell s (LYMPH), monocyte cel ls (MONO), eosinophil cel ls (EOSI) and basophil cells (BASO). 3.3.1 . 1 . 1 Absolute Leukocyte cells or white blood cells Statistical significance of effects of farm, diet, day and their interactions on white blood cell parameters are presented in Annex 1 . Table 6 depicts the effects of pro biotic-supplemented diets on leukocyte or WBC (white blood cell) components in weanling pigs. The effect of farm on WBC counts in pigs was significant (p=0.0 1 04). Repeated measures of analysis revealed that WBC counts in pigs were unaffected by feeding control or probiotic-supplemented diets (p=0.2609) as there were no statistical ly significant differences between the five diet groups (means values: 1 5 .97, 1 7 .8 1 , 1 6 .46, 1 7 . 35 and 1 6 .3 1 for diet A, B, C, D and E, repectively), although WBC varied 45 (p0.05) were not found . Table 6 : Effects of pro biotic- and lactoferrin-supplementation on whole bood parameters and blood cell parameter changes in weaned piglets Parameter A B c D E SE pvalue (control) (probiotic B) (probiotic B) (probiotic B) (lactoferrin) Effect on whole blood parameters WBC day 0 1 1 . 8 8a 1 2 . 1 a 1 2 . 1 4a 1 2 . 248 1 2 . 02 a 0 . 7 2 0 . 4 2 8 9 (x 1 09 d a y 1 4 1 8 . 1 6b 2 0 . 7 5b 1 8 . 9 2b 2 0 . 2 3b 1 9 . 5b 0 . 7 2 Cel ls/L) d a y 2 1 1 7 . 32 c 2 0 . 5 7b 1 8 . 3 3b 1 9 . 5 8b 1 7 . 4 2c 0 . 72 m e a n va l u e 1 5 . 9 7 1 7 . 8 1 1 6 . 4 6 1 7 . 3 5 1 6 . 3 1 0 . 4 2 Neutrophil (x 1 09 cel ls/L) Day o 4 . 56a 5 . 7 5 a 5. 1 38 5 . 07a 5 . 5 1 a 0 . 5 0 . 2 7 0 8 d a y 1 4 8 . 3 1 c 9 . 4 9c 8 . 6 9b 9 . 67c 9 . 06c 0 . 5 day 2 1 7 . 1 4b 8 . 6 8b 5. 1 38 8 . 92b 7 . 52 b 0 . 5 m e a n v a l u e 6 . 67 7 . 97 7 . 04 7 . 8 9 7 . 36 0 . 2 9 Lymphocytes Day 0 6 . 42a 5 . 458 6 . 1 8 6 . 1 78 5 . 6 5a 0 . 4 0 . 6342 (x 1 09 d a y 1 4 8 . 4 8b 9 . 5 8b 8 . 84b 9 . 06b 8 . 9c 0 . 4 cells/L) day 2 1 9 . 03c 1 0 . 1 9c 9 . 2 b 9 . 2 1 b 8 . 4b 0 . 4 m e a n v a l u e 7 . 97 8 . 4 1 8 . 0 7 8. 1 5 7 . 6 8 0 . 2 3 monocyte (x 1 09 0 . 62a 0 . 52a cells/L) Day 0 0 . 48a 0. 05a 0 . 4 9a 0 . 1 1 0 . 37 1 1 d a y 1 4 0 . 8 1 b 0 . 9 9b 0 . 75b 0 . 8b 1 . 2 8b 0 . 1 1 d a y 2 1 0 . 73ab 1 . 0 5b 0 . 8 8c 0 . 82 b 0 . 74c 0 . 1 1 m e a n v a l u e 0 . 7 2 0 . 8 5 0 . 7 0 . 7 1 0 . 84 0 . 06 Eosinophil D a y 0 0 . 1 9a 0 . 22 a 0 . 1 9a 0 . 24 a 0 . 1 8a 0 . 0 3 0 . 6002 (x 1 09 d a y 1 4 0 . 2 9b 0 . 32b 0 . 34b 0 . 36b 0 . 3 3b 0 . 0 3 cells/L) day 2 1 0 . 3c 0 . 33b 0 . 4c 0 . 33 b 0 . 3 8c 0 . 0 3 46 m e a n 0 . 2 6 0 . 2 9 0 . 3 1 0 . 3 1 0 . 3 0 . 0 2 va l u e Mean Day 0 54 . 1 38 5 4 . 9 9 8 54 . 8 78 5 3 . 8 1 8 5 5 . 268 0 . 76 0 . 93 0 7 Corpuscular 5 2 . 2 4b 5 0 . 9 8b 5 1 . 6 9b 5 0 . 5 b 5 1 . 1 5b Volume (fL) d ay 1 4 0 . 76 day2 1 52 . 8 3b 5 1 . 8c 5 2 . 4 8c 5 0 . 96b 5 1 . 9c 0 . 76 m e a n va l u e 5 2 . 5 9 5 2 . 5 9 53 . 0 1 5 1 . 76 5 2 . 7 9 0 . 4 4 Mean Day 0 1 8 .03 a 1 7 . 79a 1 7 . 9 1 a 1 7 .5a 1 7 . 8a 0 .2 0 . 93 9 corpuscular day 1 4 1 6 .97b 1 6 .42b 1 6 .7 1 b 1 6 .48b 1 6 .5b 0 .2 haemoglobin day 2 1 1 6 . 78b 1 6 .44b 1 6 .68b 1 6 .33b 1 6 .6b 0 .2 (pg) mean value 1 7 . 3 1 7 1 7 1 6 . 8 1 7 0 MCHC (g/L) day 0 32 1 . 5 8 3 2 2 . 6 32 1 . 5 3 2 3 . 2 3 2 0 1 . 2 d a y 1 4 324 . 2 1 32 1 . 5 3 2 3 3 2 5 . 2 32 1 . 3 1 . 2 d a y 2 1 3 1 7 . 44 3 1 3 . 7 3 1 7 . 7 3 1 9. 3 3 1 8 . 1 1 . 2 m e a n va l u e 32 1 . 0 8 3 1 9 . 3 320 . 7 3 2 2 . 6 3 1 9 . 8 2 . 1 CHCM (g/L) d a y 0 31 1 .65' 3 1 2' 309.75' 31 1 09' 309.88' 1 . 4 7 0 . 96 2 5 d a y 1 4 3 16.5b 31 5. 59b 3 1 3 . 9 1 b 3 1 8. 1 3b 3 1 5 . 1 3b 1 . 4 7 d a y 2 1 307.78' 307.05' 306 06' 308.5' 307.75' 1 . 4 7 m e a n va l u e 3 1 1 . 9 8 3 1 1 . 6 3 0 9 . 9 3 1 2 . 6 3 1 0 . 9 0 . 8 5 ROW (%) d a y 0 2 2 . 2 7 2 3 . 4 2 2 3 . 5 2 4 . 0 2 2 3 . 7 5 0 . 3 5 d a y 1 4 2 2 . 3 5 2 4 . 1 3 2 3 . 1 5 2 3 . 2 2 3 . 5 0 . 3 5 d a y 2 1 2 1 . 54 2 3 . 1 2 2 2 2 2 . 3 2 2 . 7 3 0 . 3 5 m e a n v a l u e 22 . 06 2 3 . 56 22 . 8 8 2 3 . 1 8 2 3 . 3 3 0 . 2 Effect o n blood cell parameters changes WBC d 1 4- d 0 6 . 4 1 8 . 52 6 . 7 8 7 . 9 9 7 . 5 0 . 7 1 0 . 2676 ( X 1 09 d 2 1 -d 0 5 . 3 1 8 . 56 6 . 1 9 7 . 34 5 . 39 0 . 7 1 cel ls/L) Trial mean 5 . 86 8 . 56 6 . 49 7 . 66 6 . 44 0 . 5 NEUT, x 1 09 cells/L d 1 4 - d 0 3 . 8 3 . 66 3 . 56 4 . 56 3 . 6 1 0 . 46 d 2 1 -d 0 2 . 53 3 . 0 1 2 . 1 7 3 . 8 8 2 . 0 1 0 . 46 Trial mean 3 . 1 6 3 . 34 2 . 87 4 . 2 2 2 . 8 1 0 . 33 LYMPH, x 1 09 cells/L d 1 4- d 0 2 . 1 2 4 . 1 2 . 6 9 2 . 92 3 . 2 2 0 . 3 8 d 2 1 -d 0 2 . 54 4 . 77 2 . 08 3 . 0 2 2 . 8 5 0 . 3 8 47 Trial mean 3 . 1 6 3 . 34 2 . 8 7 4 . 2 2 2 . 8 1 0 . 3 3 MONO, x 1 09 cells/L d 1 4 - d 0 0 . 1 8 0 . 46 0 . 2 7 0 . 2 9 0 . 7 9 0 . 1 3 d 2 1 -d 0 0 . 1 1 0 . 5 5 0 . 4 1 0 . 3 1 0 . 2 5 0 . 1 3 Trial mean 0 . 1 5 0 . 5 1 0 . 34 0 . 3 0 . 52 0 . 0 9 EOS I , x 1 09 cells/L d 1 4 - d 0 0 . 1 0 . 1 0 . 1 5 0 . 1 2 0 . 1 6 0 . 03 d2 1 -d 0 0 . 1 1 0 . 1 0 . 2 1 0 . 09 0 . 2 0 . 0 3 Trial mean 0 . 1 0 . 1 0 . 1 8 0 . 1 1 0 . 1 8 0 . 02 BASO, x 1 09 cells/L d 1 4 - d 0 0 . 0 1 0 . 07 0 . 0 1 0 . 06 0 . 04 0 . 0 1 d 2 1 -d 0 0 . 0 1 0 . 07 0 . 04 0 . 04 0 . 03 0 . 0 1 Trial mean 0 . 0 1 0 . 07 0 . 02 0 . 05 0 . 04 0 . 0 1 RBC, x 1 06 cells/L d 1 4-d 0 0 . 3 3 0 . 4 3 0 . 36 0 . 44 0 . 54 0 . 08 d2 1 -d 0 0 . 0 8 0 . 2 9 0 . 3 7 0 . 2 0 . 2 4 0 . 08 Trial mean 0 . 2 1 0 . 3 5 0 . 3 7 0 . 32 0 . 3 9 0 . 0 5 HGB (g/L) d 1 4-d 0 -0 . 4 1 - 1 . 82 - 1 . 66 3 . 1 2 -0 . 1 1 . 7 9 d 2 1 -d 0 - 6 . 4 3 -7 . 5 1 - 1 . 7 5 - 1 . 89 - 5 . 3 1 . 7 9 Trial mean - 3 . 4 2 -4 .67 - 1 . 7 0 . 62 - 2 . 7 1 . 26 HCT d 1 4-d0 -0 002 0 .002 -0.004 0 .002 0 .001 0 d2 1 -d0 -0 . 0 1 5 -0.002 -3E-04 -0 008 -0 009 0 Trial mean -0 0086 0.0004 -0 002 -0 003 -0.004 0 MCV (fl) d 1 4-d 0 - 1 . 8 3 -4 . 0 3 - 3 . 1 8 -3 . 3 -4 . 1 0 . 3 8 d2 1 -d 0 - 1 . 3 5 - 3 . 1 6 -2 . 3 9 - 2 . 87 - 3 . 3 0 . 3 8 Trial mean -0 009 0.0004 0 0 0 0.003 MCH (pg) d 1 4 - d 0 - 1 . 0 5 - 1 . 3 5 - 1 . 2 - 1 - 1 . 3 0 . 1 5 d 2 1 -d 0 - 1 . 2 5 - 1 . 3 6 - 1 . 2 3 - 1 . 2 - 1 . 2 0 . 1 5 Trial mean - 1 . 1 5 - 1 . 36 - 1 . 2 1 - 1 . 1 - 1 . 3 0 . 1 1 MCHC (g/L) d 1 4 - d 0 2 . 54 - 0 . 62 1 . 5 2 . 3 1 . 4 8 2 . 1 9 d 2 1 -d 0 - 4 . 0 5 - 9 . 3 6 - 3 . 7 8 -4 . 2 7 - 2 2 . 1 9 Trial mean -0 . 75 -4 . 99 - 1 . 1 4 - 0 . 9 9 - 0 . 3 1 . 55 CHCM (g/L) d 1 4 - d 0 4 . 7 3 3 . 8 1 4 . 1 6 7 . 0 1 5 . 6 3 1 . 33 d 2 1 - d 0 - 3 . 74 - 5 . 1 6 - 3 . 6 9 -2 . 4 8 - 2 . 1 1 . 3 3 Trial mean 0 . 4 9 - 0 . 67 0 . 2 3 2 . 2 7 1 . 7 5 0 . 94 ROW (%) d 1 4 - d 0 -0 . 1 0 . 7 - 0 . 3 5 - 0 . 8 2 - 0 . 2 0 . 3 d 2 1 -d 0 - 0 . 5 5 -0 . 2 9 - 1 . 5 - 1 . 7 1 - 1 0 . 3 Trial mean -0.32 0 . 2 -0 . 9 3 - 1 . 2 7 - 0 . 1 0 . 2 2 a , b c values in the same column with different letters impl ies that from this day to the next the MCV level s are significantly different (p<0.05) . SE standard error 48 3.3 .1 . 1 .2 Neutrophils Statistical significance of effects of farm, diet, day and their interactions on neutrophils cel ls are presented in Annex 1 . The least square means of values for neutrophils (x I 09 cells/L) obtained from each diet treatment are shown in Table 6 . NEUT counts in pigs were affected (pO.OS). As can be observed from in Table 6, the neutrophil cells increased in all diet groups from day 0 to 2 1 with the highest being in pigs fed diet D (from 5 .07 to 8 .92). All diets showed an increase from week 0 to week 2 with diet D showing the highest increase (5 .07 to 9 .67). From day 1 4 to 2 1 , the neutrophi l counts in pigs in all diets groups decl ined with pigs fed diet E declining at a faster rate (from 9.06 to 7.42). The NEUT cell counts of the pigs on day 1 4 and day 2 1 were numerically lower (8 .3 1 and 7 . 1 4, respectively) for the control (diet A) and continued to be low throughout the trial period. When the four treatments under investigation (diet B, C , D and E) were compared, feeding probiotic in diet B resulted in pigs achieving higher neutrophil cel l count ( 9.49 and 8.68 in day 1 4 and day 2 1 , respectively) than the probiotic in diet E. Probiotic supplementation stimulated NEUT in pigs more compared to the control diet. Stimulation was highest in pigs that consumed diet B and least in those given diet C . NEUT varied with time (p<0 .0001 ) . In pigs fed the control diet, NEUT increased from 4 .56 x 1 09 cel ls!L at day 0 (28 days) to 8 .3 1 x 1 09 cells!L at day 1 4 (42 days) . From day 1 4 ( 42 day), the levels decreased to 7 . 1 4 8 .3 1 x 1 09 cells!L at day 2 1 ( 49 days old) (Table 6). 49 3.3. 1 . 1 .3 Lymphocytes Annex 1 shows the statistical significance of effects of farm, diet, day and their interactions on lymphocytes of pigs. Effects of probiotic-supplemented diets on lymphocytes in weanl ing pigs are presented in Table 6 . Lymphocyte counts were affected (pO.OS) . 3.3. 1 . 1 .4 Monocytes Annex 1 depicts the statistical significance of effects of farm, diet, day and their interactions on monocyte cells of pigs. The least square means of values for monocyte ce l l s (x 1 09 cel l s/L) obtained from each diet treatment are presented Table 6. Monocyte cel l numbers in pigs were affected (pO.OS) . 50 3.3. 1 . 1 .5 Eosinophils Annex 1 gives the statistical significance of effects of farm, diet, day and their interactions on eosinophil cells blood parameters of pigs. The least square means of values for eosinophi l cel l s (x 1 09 cel l s/L) obtained from each d iet treatment are presented in Table 6 . Eosinophi l cel l counts in pigs were affected (p0.05). Overal l , trial LSMeans eosinophils counts for piglets fed the control and probiotic­ supplemented diets for the duration of the study were 0 .26, 0.29, 0 .3 1 , 0 .3 1 and 0 .3 for diet A, B, C, D and E, respectively (Table 6). Overall , EOSI increased in pigs in all diet groups from day 0 to 2 1 . Trial means or the least-square means for the increase in EOSI were 0. 1 , 0 . 1 , 0 . 1 8 , 0 . 1 1 and 0. 1 8 for diet A, B, C, D and E, respectively (Table 6) . In our study the eosinophil counts increased. The increase was higher in animals in the diet C group than in control group and lowest in those in diet B group (0 . 1 8 , 0 . 1 and 0 . 1 , respectively). As can be observed from Table 1 8 , the eosinophil cell counts in pigs increased for all diet groups from day 0 to 2 1 . The eosinophil cel ls of pigs that consumed diets A, B, C and E continued to increase throughout the experimental period apart from the eosinophil cell s of pigs fed diet D that increased from day 0 and reached the peak (0. 33 ) on day 1 4 and on day 2 1 it was lower (0. 1 3 x l 09 cells/L). When the four treatments under investigation (diet B, C, D and E) were compared, feeding pro biotic in diet C resulted in pigs achieving higher eosinophil cell count (0.34 and 0 .4) on day 1 4 and 2 1 , respectively) than the probiotic in other diets over the 3 week study. This 5 1 was evidenced by a relatively high increase in number of eosinophil cell s in pigs that consumed diet B for the duration of the study. Probiotic treated diets stimulated eosinophil cellsin pigs more than in the control diet throughout the study period . Stimulation was higher in pigs fed diet C compared to B among the treatment groups . 3.3. 1 . 1 .6 Basophils Annex 1 depicts the statistical significance of effects of farm, diet, day and their interactions on basophil cell s of pigs. The least-square means of values for basophi l cel l s (x 1 09 cel l s/L) obtained from each diet treatment are summarized in Table 6 . Basophi l counts in pigs were affected (p=O .OO 1 2) by time and were lowest on weaning day and an interaction of farm x day (p=0.03 1 5) was detected (Table 6). Treatment means for basophi l ce l l counts did not differ among treatment (diet) groups (p = 0 .4762). The effect of farm on basophi l counts of pigs was not s ignificant (p=0 . 5 1 22) and interactions of fann and diet, farm x d iet x day and diet x day were not found (p>O.OS) . Overall , Diet B and C had higher basophils than control while diet D and E group had lower basophils than control . As can be observed from in Table 20 , from weaning day (day 0) to day 1 4, the basophi l cell counts increased for all diet groups apart from diet C that remained unchanged from weaning day to day 1 4 . (0. 1 and 0 . 1 , respectively). When we compare cell counts from weaning day to day 1 4 and from weaning day to day 2 1 , the cel l counts of all diets are lower from weaning day to day 2 1 except for diet C that had a higher cell count from weaning day to day 2 1 compared from weaning day to day 1 4 (0. 1 and 0 . 1 4, respectively). Cell counts control group were similar in periods between day 1 4 and weaning day and day 2 1 and weaning day (0 . 1 1 and 0. 1 1 , respectively). 52 Suplementation with probiotic B and C stimulated basophil cells more compared to control while stimulation by diet D and E was lower than in the contro l . As can be observed from in Table 6 , the basophil cell counts increased for a l l diet groups from weaning day to 2 1 . The basophil cel ls of pigs fed diet E remained numerically lower throughout the 2 1 d period. Pigs that consumed diet C remained unchanged from day 0 to 1 4 but showed a rapid increase from day 1 4 onwards. Pigs fed the control diet remained unchanged from weaning day to day 1 4 but showed a very small increase from day 1 4 onwards . Pigs that consumed diets D and E showed rapid rise in this cell from weaning day to day 1 4 but showed a decline in BASO from day 1 4 onwards with pigs offered diet D showing a steeper slope of decline compared to those that consumed diets B and E. When the four probiotics under investigation (diet B, C, D and E) are compared, feeding probiotic in diet C resulted in pigs achieving highest basophil cell count (0. 1 and 0 . 1 4 on weaning day and day 2 1 , respectively) than the pigs supplemented with probiotic in diet E. This is evidenced by the highest rise in number of basophil from day 1 4 onwards . 3.3. 1 .2 Erythrocytes or red blood cell populations and red cell indices parameters Red b lood cel ls types analysed included red blood cells (RBC), haemoglobin concentration (HGB), haematocrit level (HCT), mean cell volume (or mean erythrocyte volume) (MCV), mean cell haemoglobin (or mean erythrocyte haemoglobin content)(MCH), mean corpuscular haemoglobin concentration (or mean erythrocyte haemoglobin concentration) (MCHC), corpuscular haemoglobin constant (CH), Corpuscular haemoglobin concentration mean (CHCM), corpuscular haemoglobin (CH), red blood cel l distribution width (or erythrocyte distribution width)(RDW), haemoglobin distribution width (HDW), platelet (PLT), mean packed volume (MPV), neutrophil (NEUT), lymphocyte (LYMPH), monocyte (MONO), eosinophil (EOSI), and basophil (BASO). 53 3.3. 1 .2 . 1 Erythrocytes or red blood cells Statistical significance of effects of farm, diet, day and their interactions on red blood cell parameters are presented in Annex I . Table 6 depicts the effects of pro biotic-supplemented diets on blood components in weanling pigs. RBC counts of pigs were affected (p=0.0008) by time. RBC increased in al l groups from day 0 to day 1 4 (p<0 .05) and decreased in all diet groups from day 1 4 to 2 1 . The decrease was significant (p<0 .05) in all diet groups except in pigs that consumed diet C. They were highest on day 1 4 and reduced from day 1 4 to day 2 1 except in diet C were they increased slightly (by 0. 1 x I 09cells/L). The effect of farm on RBC counts of pigs was significant (p=0.0004). The interactions of farm x diet and farm x day were significant (p=O .O l 03 , and p=0 .0032, respectively) [data not shown] while, interactions of farm x diet x day and diet x day (p>0.05) were not detected (Table 1 2) . Dietary supplementation with control (diet A) and probiotic-supplemented diets had no significant effect (p=0 .4289) on RBC counts of pigs (means values: 5 .93, 5 .99, 5 . 96, 5 . 87 and 5 .75 for diet A, B, C, D and E, respectively) . RBCs carry oxygen from lungs to cells and C02 from cells back to the lungs. High levels of RBCs is an indicator of good health. Pigs were randomly allocated to the five diet groups but by chance those that were in groups D and E had lower RBC levels compared to other groups. This was not expected. As the study progressed, animals that consumed these two diets (D and E) stil l remained with the lowest RBC levels compared to pigs in other diet groups. At the end of the study, pigs fed diets D and E had lower RBC levels compared to the controls while those that consumed diets B and C had higher RBC than the controls . 54 3.3 . 1 .2.2 Haemoglobin concentration Annex I shows the statistical significance of effects of farm, diet, day and their interactions on haemoglobin concentration in pigs. Effects of probiotic-supplemented diets on haemoglobin concentration in weanling pigs are tabled in Table 6 . Farm had a significant effect on HGB of pigs (p0.05 . 3.3.1 .2.3 Haematocrit or packed cell volume or volume of packed red cells Haematocrit (HCT) or packed cell volume (PVC) is the percentage of erythrocytes or RBC in whole blood . Low HCT is an indicator of late stage anaemia (Thral l et al. , 2004; Thompson & Forsyth, 2006; Tvedten, I 993 and Voigt, 2000). Annex I depicts the statistical significance of effects of farm, diet, day and their interactions on haematocrit measures of pigs. Table 6 shows the effects of pro biotic-supplemented diets on haematocrit measures in weanling pigs. Farm had a significant influence on haematocrit measures in pigs (p=0.0002). An interation of farm x day (p=0.0088) was noted for HCT. Nevertheless, no statistical differences (p = 0 .3324) were observed on HCT of pigs by addition of probiotics or lactoferrin on haematocrit between the diet groups (mean values : 0 .3 I 7, 0 .3 1 3 , 0 .3 1 5 , 0 . 302 and 0 .303 for diet A, B, C, D and E, respectively). Interactions of farm x diet, 55 farm x diet x day were not detected (p>0.05). HCT measures were not affected by time (p=0.6 1 8 1 ) (Annex 1 ). HCT or PCV can be used as an indicator of anaemia in animals. High levels indicate high iron levels whereas low is an indicator of i l l health. As reported for RBCs and HGB above, piglets were randomly allotted into the five diet groups but by chance those that were in groups D and E had lower HCT levels compared to other groups . This was, again, not expected. Overall, between weaning day and day 2 1 , pigs that consumed diet D and E had lower levels of HCT compared to the controls. On the other hand, pigs that consumed diets B and C had higher levels of HCT compared to the controls . 3.3 . 1 .2.4 Mean corpuscular volume Erythrocyte volume is the MCV. In other words, MCV is an average volume of a single cell of the red blood cells measured in femtolitres, tL [ 1 tL = 1 o- J s L or 1 11L = 1 09 tL] (Thrall et al . , 2004; Thompson & Forsyth, x ; Tvedten, 1 993 ; Weiser, x ; Bush, x). MCV may be used as a morphological indicator of iron deficiency since this value gives a description of the normality of red blood cell size. Annex 1 shows the statistical significance of effects of farm, diet, day and their interactions on blood parameters of pigs. The effects of probiotic-supplemented diets on blood components in weanling pigs are summarized in Table 6 . MCV of pigs were affected (p=0.0 1 95) by time. MCV decreased (p<0.05) in pigs in al l diet groups from day 0 to 14 . From day 1 4 to 2 1 , MCV increased in pigs in a l l diet groups although the increase was significant (p<0.05) only in pigs that were offered diets B, C and E. Farm had an influence on MCV counts of pigs (p=0.00 1 3) . Trends (p = 0.9307) were observed on MCV of pigs between the diet groups (mean values : 53 .06, 52 .59, 5 3 .0 1 , 5 1 .76 and 52.79 for diet A, B, C, D and E, respectively). Interactions of farm x diet and farm x diet x day, farm x diet x day and diet x farm were not evident (p>0.05) . 56 3.3. 1 .2 .5 Mean corpuscular haemoglobin MCH is calculated by dividing the amount ofHGB (g/L) by the RBC count [x l 0 1 2 cells/L] (Thrall et al. 2004; Thompson & Forsyth, 2006; Tvedten, 1 993) . Annex 1 shows the statistical significance of effects of farm, diet, day and their interactions on blood parameters of pigs. The effects of pro biotic-supplemented diets on MCH in weanling pigs are presented in Table 6. Farm had an influence on MCH of pigs (p<0.000 1 ) . MCH varied over time (p=0 .0 1 23). From day 0 to 1 4, MCH decreased significantly (p<0.05) in pigs in all diet groups. From day 1 4 to 2 1 , MCH further declined (p>0 .05) in pigs that consumed diets A, C and D and increased (p>O.OS) in those that received diet B and E . Statistically no signicant differences (p = 0.9390) in MCH measures were observed in pigs fed control and probiotic-supplemented diets (mean values: 1 7 .26, 1 6 . 88 , 1 7 . 1 , 1 6 .77 and 1 6 .97 for diet A, B, C, D and E, respectively) . There were no significant interactions of farm x diet, farm x day; farm x diet x day and diet x day (p>0.05) . 3.3. 1 .2.6 Mean corpuscular haemoglobin concentration The amount of haemoglobin within erythrocyte (MCHC) is calculated as HGB (g/dL) divided by PCV (ml/1 OOml) x 1 00 = M CH C. Statistical significance of effects of farm, diet, day and their interactions on MCHC of pigs are summarized in Annex 1 . Table 6 depicts the statistical significance of effects of farm, diet, day and their interactions on MCHC of pigs. 57 There was an influence of farm on MCHC of pigs (p=0004). MCHC were not affected by time (p=0.22 1 8) and no significant differences (p = 0.9629) were observed in MCHC between the diet groups. I nteractions of farm x diet, farm x day, farm x diet x day and diet x day were not detected (p>0.05) (Table 6) . 3.3. 1 .2. 7 Corpuscular haemoglobin concentration mean Annex 1 depicts the statistical significance of effects of farm, diet, day and their interactions on corpuscular haemoglobin concentration mean (CHCM) of pigs. Effects of pro biotic-supplemented diets on corpuscular haemoglobin concentration mean in weanling pigs are shown in Table 6. CHCM of pigs was affected (p<0.0 1 49) by time. From day 0 to 1 4, CHCM increased (p<0.05) in pigs in all diet groups. From day 1 4 to 2 1 , CHCM in all the pigs declined (p<0.05). Farm had an influence on CHCM counts in pigs (p0.05). 3.3. 1 .2.8 Red cell distribution width Annex 1 depicts the statistical significance of effects of farm, diet, day and their interactions on red cell distribution width of pigs. The least square means values for red cell distribution width (%) obtained from each diet treatment are presented in Table 6 . Farm had an influence on RDW ofpigs (p < 0.000 1 ) (Annex 1 ) . However, no significant differences (p = 0.7627) were observed by addition of probiotics (B, C, D and E) on RDW of pigs. No interactions of farm x diet and farm x diet x day, diet x 58 day and farm x day were detected (p>0.05) . RDW in pigs did not vary with time (p>0.05). 3.3.2 Blood cell parameter changes Thirdly, we investigated the effect of dietary treatment on blood changes. B lood samples were taken on day weaning day, day 1 4 and 2 1 of the study period. Changes were obtained by subtracting blood counts on weaning day from that of day 1 4 ( d 1 4 - dO) and that of day 2 1 ( d2 1 -d0). 3.3.2 . 1 Absolute white blood cell changes Under white blood cell changes, we analysed WBC, NEUT, L YMP, MONO, EOSI, BASO between day 1 4 and 0 ( d 1 4-0) and day 2 1 and 0 ( d2 1 -0) . 3.3.2. 1 . 1 Leukocyte o r white blood cell changes Statistical significance of effects of farm, diet, day and their interactions on white blood cell changes are presented in Annex 1 . There was an influence of farm on WBC changes of pigs (p=0 .0056), while time had no significant effect on WBC (p=0.2986). LSMeans for WBC changes were not different for piglets fed the control or the probiotic-supplemented diets (p = 0 .2679). Interactions of farm x diet, farm x day, farm x diet x day and diet x day were not observed (p>0.05) . 3.3.2. 1 .2 Neutrophil cell changes The statistical significance of effects of farm, diet, day and their interactions on neutrophil cells are presented in Annex 1 . Least-square means of neutrophil cells of each treatement is shown in Table 6 . Farm had an effect on neutrophi l ce l l changes of pigs (p=0.03 8 1 ) . Neutrophil changes tendentiously varied over time (p=0.06) and were highest on day 1 4 post weaning. However, no significant differences (p=0 .5769) were observed in pigs fed control and pro biotic-supplemented diets on neutrophil cell changes. Interactions of farm x diet, farm x day, diet x day, farm x diet x day were not evident (p>0.05) . 59 From the start of the experiment to the end of week 2 (dO to d 1 4) least-square means of neutrophil cell increase were 3 .8 , 3 .66, 3 .56, 4 .56 and 3 . 6 1 for diet A, B, C, D and E, respectively. From the start of the experiment to the end of week 3 (dO to d2 1 ) least-square means of neutrophil cel l increase were 2 .53 , 3 . 0 1 , 2 . 1 7 , 3 . 8 8 and 2 .0 1 for diet A, B, C, D and E, respectively. Overal l , least-square means of neutrophil cel l changes increased by 3 . 1 6, 3 . 34, 2 . 87, 422 and 2 . 8 1 for diet A , B, C, D and E, respectively. From day 0 to 1 4 , NEUT cells increased in pigs fed control and probiotic­ supplemented diets. However, from day 1 4 to 2 1 , pigs in all diet groups showed areduction in NEUT cells . As can be observed from Table 6 and Annex 1 changes tended to vary with time (p=0.06). Least-significant means for NEUT change during the period from day 0 to 14 was 3 . 8 x 1 09 cell s/L for the control animals. For the period from 0 to 2 1 these NEUT change was 2 .53 x 1 09 cel ls/L. 3.3.2. 1 .2 Lymphocyte cell changes The statistical significance of effects of farm, diet, day and their interactions on lymphocyte cell changes are presented in Annex 1 . Least-square means of lymphocyte cel ls of each treatement is shown in Table 6 . Farm effect had an influence on lymphocyte cells changes of pigs (p0.05) and the interactions of farm x diet, farm x day, diet x day, farm x diet x day were not observed (p>0.05) . 60 From the start of the experiment to the end of week 2 (dO to d 1 4) least-square means of lymphocyte cells increase were 2 . 1 2, 4. 1 , 2 .69, 2 .92 and 3 .22 for diet A, B, C, D and E, respectively The increase in lymphocytes was higher in pigs in the treated groups compared to the controls . Among the treated groups pigs that were fed diet B had the highest increase while those that received diet C had the lowest increase. From the start of the experiment to the end of week 3 (dO to d2 1 ) least-square means of lymphocyte cells increase were 2 . 54, 4 .77, 2 .08, 3 .02 and 2 .85 for diet A, B, C, D and E, respectively. The increase in lymphocytes was higher diet B, D and E groups compared to the control while diet C had a smaller increase compared to control . Among the treated groups diet B had the highest. Overal l , least-square means of lymphocyte cell changes increase were 3 . 1 6 , 3 .34, 2 .87 , 4 .22 and 2 . 8 1 for diet A, B, C , D and E, respectively. From week 0 to week 2 (d 1 4 - dO), lymphocyte cells in pigs receiving diet A had numerically the smallest increase of 2 . 1 2 x 1 09 cells/L while diet B had the highest increase ( 4 . 1 x 1 09 cel ls/L). As seen in Table 6, the LSMeans for the increase in lymphocyte cell during the period from 0 to 2 1 d was highest in diet B(4.77 x 1 09 cel ls) and was lowest in diet C (2.08 x 1 09 cells). Lymphocyte proliferation tended to be higher in pigs that consumed (probiotic- and lactoferrin-) supplemented diets than the control diet and tended to higher in diets B and lower in diets C, D and E (p=0.08). Changes in lymphocytes proliferation tended to be higher in pigs that received diet B . As can be observed from Table 6 and Annex 1 LYMPH changes tended to vary with time (p=0.06). Least-significant means for LYMPH change during the period from 6 1 day 0 to 1 4 was 2 . 1 2 x 1 09 cells/L for the control animals. For the period from 0 to 2 1 these LYMPH change was 2 .54 x 1 09 cell s/L. 3.3.2 . 1 .3 Monocyte cell changes The statistical significance of effects of farm, diet, day and their interactions on monocyte cell changes are depicted in Annex 1 . Least-square means of monocyte cell changes of each treatement is shown in Table 6 . Farm tended to affected monocyte cell changes in pigs (p=0.09) although the monocyte changes were not affected by time (p>O.OS) . No significant differences (p=0. 1 870) were observed in pigs fed control and probiotic­ supplemented diets on monocyte cell changes. Interactions of farm x diet, farm x day, diet x day, farm x diet x day were not detected (p>O.OS) . 3.3.2. 1 .4 Eosinophil cell changes The statistical significance of effects of farm, diet, day and their interactions on eosinophil cell changes are presented in Annex 1 . Least-square means for effects of probiotic-supplemented diets on eosinophil changes in weanling pigs are shown in Table 6 . There was an effect of farm on eosinophil cells changes of pigs (p=0.0008). No significant differences (p=0.3790) were observed in pigs fed control and probiotic­ supplemented diets on eosinophil cel l changes . EOSI changes were not affected by time and interactions of farm x diet, farm x day, diet x day, farm x diet x day were not found (p>O.OS) . 62 3.3.2 .1 .5 Basophil cell changes The statistical significance of effects of farm, diet, day and their interactions on basophil cel l changes are presented in Annex 1 . Least-square means for effects of pro biotic-supplemented diets on basophil changes in weanling pigs are shown in Table 6 . Farm had an effect on basophi l cel l changes in pigs (p=0.0005). However, no significant differences (p=O . l 974) were observed in pigs fed control and probiotic­ supplemented diets on basophil cell changes. Eosinophil cell changes were not affected by time (p>0.05). Interactions of farm x diet, farm x day, diet x day, farm x diet x day were not noted (p>0.05). 3.3.2.2 Erythrocyte or red blood cell changes Under red red blood cell changes, we analysed: red blood cells (RBC), haemoglobin concentration (HGB), haematocrit level (HCT), mean cell volume (or mean erythrocyte volume) (MCV), mean cell haemoglobin (or mean erythrocyte haemoglobin content)(MCH), mean corpuscular haemoglobin concentration (or mean erythrocyte haemoglobin concentration) (MCHC), corpuscular haemoglobin constant (CH), Corpuscular haemoglobin concentration mean (CHCM), corpuscular haemoglobin (CH), red blood cell distribution width (or erythrocyte distribution width)(RDW), haemoglobin distribution width (HDW), platelet (PL T), mean packed volume (MPV) between day 1 4 and 0 ( d 1 4-0) and day 2 1 and 0 ( d2 1 -0). 3.3.2.2 . 1 Erythrocyte or red blood cell changes Statistical significance of effects of farm, diet, day and their interactions on red blood cel l changes are presented in Annex 1 . Effect of diet on RBC is depicted in Table 6. 63 Farm had a significant influence on red blood cells ofpigs (p < 0.00 1 ). LSMeans for red blood cell changes was not different for piglets fed the control or the probiotic­ supplemented diets (p = 0 .879 1 ) and there were no interactions observe (p>0.05) . 3.3.2.2.2 Haemoglobin changes Annex 1 depicts the stati stical significance of effects of farm, diet, day and their interactions on blood HGB of pigs. Effect of diet on HGB is shown in Table 6 . Farm had an influence on haemoglobin of pigs (p < 0.000 1 ) while HGB in pigs did not vary with time (p=O. l 082). LSMeans for haemoglobin changes were not different for piglets fed the control or the probiotic-supplemented diets (p = 0.7947) and interactions of farm x diet, farm x day, diet x day and farm x diet x day were not evident (p>0.05) . Overall, least-square means ofHGB decrease/increase were -3 .42, -4.67, - 1 .7 , 0 .62 and -2.69 g/L for for diet A, B, C, D and E respectively. Pigs in diet D group, however showed an increase of HGB by 0 .62 g. The decrease in HGB was lower in pigs fed diet C, D (increase) and E pro biotic­ supplemented groups compared to the controls while pigs fed diet B had a higher decrease than the controls . Among treatments, diet D pigs had an increase in HGB while those fed diet E had a higher decrease. The decrease in pigs that received diet D was always numerically lower than controls while the decrease in pigs fed diet B was always numenrically higher than control animals . 64 3.3.2.2.3 Haematocrit changes Annex 1 depicts the statistical significance of effects of farm, diet, day and their interactions on blood changes parameters of pigs . Least square means for effects of pro biotic-supplemented diets on haematocrit changes in wean ling pigs are summarized in Table 6 . Farm had an effect on HCT of pigs (p = 0 .000 1 ) whi le HCT did not vary with time (p=0.4088). LSMeans for HCT was not different for piglets fed the control or the probiotic­ supplemented diets (p = 0.9694) and interactions of farm x diet, farm x day, diet x day and farm x diet x day were not noted (p>0.05) . 3.3.2.2.4 Mean corpuscular volume changes Statistical significance of effects of farm, diet, day and their interactions on mean corpuscular volume changes parameters of pigs are presented in Annex 1 . LSMeans for mean corpuscular volume levels was not different for piglets fed the control or the probiotic-supplemented diets (p = 0 .7579) and interactions of farm x diet, farm x day, diet x day and farm x diet x day were not observed (p>0.05) . Farm had no effect on MCV levels of pigs and MCV of pigs did not vary with time (p>0.05). 3.3.2.2.5 Mean corpuscular haemoglobin changes Annex 1 gives the statistical significance of effects of farm, diet, day and their interactions on mean corpuscular haemoglobin of pigs. 65 Farm had a significant effect on MCH of pigs (p=0.0030) while time tended to have an effect on MCH of pigs (p=0.7722) and LS Means for MCH levels was not different for piglets fed the control or the probiotic-supplemented diets (p = 0 .973 5) . Interactions of farm x diet, farm x day, diet x day and farm x diet x day were not observed (p>0.05) . Least square means for mean corpuscular haemoglobin changes are presented in Table 6 . From day 0 to 14 ( d 14 - dO), MCH decreased in a l l diet groups. Pigs receiving diet A had the smallest decrease ( 1 .05). The decrease was highest in diet B group ( 1 . 35 ) . The MCH for al l diets groups reduced during the period from day 0 to 2 1 . The rediction was lowest in pigs fed diet D ( 1 .2) and highest in those in diet B group ( 1 .36). Overall , MCH decrease were 1 . 1 5 , 1 . 36 , 1 .2 1 , 1 . 1 and 1 .25 for diet A, B, C, D and E, respectively. The decrease in MCH is lower in pigs that consumed diet D compared to controls . Decrease in MCH is higher in pigs that received diets B, C and E. Among treatments pigs fed diet C had a lower decrease while those fed diet E had a higher decrease. Decrease in MCH in pigs that consumed diet D was always numerically lower than the controls while pigs fed diet B remained higher than controls throughout the study period. As can be observed from Annex 1 and Table 6, MCH changes did not vary with time (p=0.7722). Least-significant means for MCH change during the period from day 0 to 1 4 was - 1 .05 pg for the control animals . For the period from 0 to 2 1 these MCH change was - 1 .25 pg. 66 3.3.2.2.6 Mean corpuscular haemoglobin concentration changes Annex I gives the statistical significance of effects of farm, diet, day and their interactions on mean corpuscular haemoglobin concentration mean of pigs. Least-square means for mean corpuscular haemoglobin concentration mean changes are presented in Table 6 . MCHC changes in pigs varied over time (p=0.0065). LSMeans for MCHC levels were not different for piglets fed the control or the probiotic-supplemented diets (p = 0 .68 1 4) . Interactions of farm x diet, farm x diet x day, diet x day were not detected and farm had no significant effect on MCHC of pigs (p>0.05) (Annex 1 ) . Overall, Trial means for MCHC reductions were 0 .75 , 4 .99, 1 . 1 4, 0.99 and 0 .25 for diet A, B, C, D and E, respectively. As can be observed from in Table 6 , the levels of MCHC varied with time (p=0.0065) and they increased in pigs that consumed diets A, C, D and E from day 0 and peaked on day 1 4 and then reduced with a higher margin from day 14 to 2 1 to reach a levels lower than at day 0 . However, MCHC in pigs that received diet B decreased by a small margin (0.62) from day 0 to 14 compared to a reduction 9.36 from day 0 to 2 1 . The differences between the decrease/increase in MCHC in pigs between day 0 and 2 1 ( d2 1 -d0) and between day 0 to 1 4 ( d 1 4-dO) was significant and was higher between d2 1 -0 than d 1 4-0 (p<0.05) . As can be observed from Annex 1 and Table 6, MCHC changes varied with time (p=0.0065) . Least-significant means for MCHC change during the period from day 0 to 1 4 was 2 .54 g/L for the control animals. For the period from 0 to 2 1 these MCHC change was -4 .05 g/L. 67 3.3.2.2.7 Corpuscular haemoglobin concentration mean changes Statistical significance of effects of farm, diet, day and their interactions on CHCM changes are presented in Annex 1 . Means for effects of probiotic-supplemented diets on corpuscular haemoglobin concentration and changes in weanling pigs are given in Table 6. CHCM changes for pigs varied over time (p0.05) were not detected. Overall , least-square means of CHCM increased I decrease were +0.49, -0.67, +0.23 , +2 .27 and + 1 .75 for diet A, C, D and E respectively From day 0 to 1 4 ( d 1 4 - dO), CHCM increased in all diet groups. Pigs receiving diet D had the highest increase (7 .0 1 ) and lowest in pigs fed diet B (3 . 8 1 ). The LSmeans of CHCM for all diets groups reduced during the period from day 0 to 2 1 . The reduction was lowest in pigs on diet E (2. 1 3) and highest in pigs in diet B group (5 . 1 6) . As can be observed from in Annex 1 , the levels of CHCM varied with time (p< 0.000 1 ) and they increased for pigs in all diet groups from day 0 and peaked on day 1 4 and then reduced with a higher margin from day 1 4 to 2 1 to reach a levels lower than at day 0 . Pigs fed diet C had numerical ly lower CHCM levels on day 0 and the remained low throughout the study period. 68 As can be observed from Table 6 and Annex 1 , CHCM changes varied over time (p<0.000 1 ) . Least-significant means for WBC changes during the period from day 0 to 1 4 was 4. 73 g/L for the control animals. For the period from 0 to 2 1 these CHCM changes were -3 .74 g/ L. 3.3.2.2.8 Red cell distribution width changes Statistical significance of effects of farm, diet, day and their interactions on RDW changes are presented in Annex I . Least square means for effects of probiotic-supplemented diets on corpuscular haemoglobin concentration mean changes in weanling pigs are presented in Table 6 . LSMeans for RDW levels were not significant different for piglets fed the control or the probiotic-supplemented diets (p = 0.23 59). RDW changes varied over time (p=0.0432) and the decline was greatest during week 2 . Farm had an influence on RDW changes (p0.05) were not detected. From the start of the experiment to the end of week 2 (dO to d 1 4) least-square means of RDW decrease/increase were -0. 1 , +0.7, -0 .35 , -0 .82 and -0.22 for diet A, B, C, D and E, respectively. The increase for the control diet was 800% lower, 250% and 720% lower, and 1 20% higher than diet B, C, D and E, respectively. The decrease was higher in diet C, D and E than contrl while B had and increase. From the start of the experiment to the end ofweek 3 (dO to d2 1 ) least-square means ofRDW changes (decrease) were -0 . 55 , -0.29, - 1 . 5 , - 1 . 7 1 and - l .O l for diet A, B, C, D and E respectively. The decrease in RDW was higher in pigs fed diets C, D and E than control while B was lower than control anmals . 69 Overall least-square means of RDW decreased by -0.32, +0.2, -0.93 , - 1 .27 and -0.062 for diet A, C, D and E respectively but increased by 0 .2 for diet B . As can be observed from in Annex 1 , the levels of RDW varied with time (p=0.0432) and they decreased in pigs that consumed diets A, C, D and E from day 0 and reduced further from day 1 4 to 2 1 to reach levels lower than at day 0. However, pigs that received diet B had an increase in RDW from day 0 to 1 4 and then reduced from day 1 4 to day 2 1 . The differences between the decrease I increase in RDW pigs between day 0 and 2 1 (d2 1 -d0) and between day 0 to 1 4 (d1 4-d0) was significant (p<0.05). RDW decreased in pigs that consumed diet A, C, D and E from day 0 to 1 4 ( d0- 1 4) and further decreased by a higher margin from day 0 to 2 1 (do-d2 1 ) (p<0.05) . Pigs fed diet B had an increase in RD W between dO-d 1 4 and a reduction from d0-d2 1 . As can be observed from Table 6 and Annex 1 , RDW changes varied with time (p=0.0432). Least-significant means for RDW change during the period from day 0 to 1 4 was -0. 1% for the control animals. For the period from 0 to 2 1 these RDW change was -0 .55%. 3.4 Effect of pro biotic- and lactoferrin-supplemented diets on Lymphocyte to neutrophil ratio (stress factor) Forthly, we then investigated the effect of dietary treatment on lymphocyte to neutrophil ratio (Stress factor) of pigs. Blood was collected on weaning day, day 1 4 and 2 1 of the study period.Lymphocyte to neutrophil ratio were determined by dividing the lymphocyte by the neutrophil cell numbers of each pig at samplimg time. Lymphocyte to neutrophi l ratio is a crude indicator of stress). Annex 1 shows the statistical significance of effects of farm, diet, day and their interactions on lymphocyte to neutrophil ratio of pigs. Least square means for effects of pro biotic-supplemented diets on lymphocyte to netrophil ratio in weanling pigs are shown in Table 7 . 70 Table 7 : Effects of pro biotic- and lactoferrin-supplementation on lymphocyte to neutrophil ratio (stress factor) in weaned piglets Parameter A B c D E SE (control) (probiotic B) (probiotic B) (probiotic B) (lactoferrin) Effect on day 0 0 . 8 1 5 1 1 . 1 03 0 . 982 1 . 246 1 . 069 0 . 08 lymphocyte to neutrophil ratio day 1 4 1 . 0657 1 . 0 1 1 1 . 04 1 1 . 422 1 . 1 1 4 0 . 08 day 2 1 0 . 8655 0 . 878 0.87 1 . 3 1 8 0 . 8 9 8 0 . 08 difference day 1 4-day0 0 . 2506 -0.09 0 . 059 0 . 1 76 0 . 044 day2 1 -day0 0.0504 -0.23 -0 . 1 1 0.072 -0. 1 7 Trial mean 0 . 9 1 54 0 . 997 0 . 964 1 . 329 1 . 0 2 7 1 0 . 05 SE standard error Statistical significance of effects of farm, diet, day and their interactions on lymphocyte to neutrophil ratio are presented in Annex 1 . An interaction of farm x diet (p=O .O 1 68) was evident while interactions of farm x day and farm x diet x day and diet x day were not detected (p>0.05) . Farm had a significant effect on lymphocyte to neutrophil ratio of pigs (p=0 .00 1 3) . Least-square means for lymphocyte to neutrophil ratio was not different for piglets fed the control or the probiotic-supplemented diets B, C, D and E (p = 0 . 1 076) . SF were not affected by time (p>0.05) . On weaning day, the least-square means of lymphocyte to neutrophil ratio was 1 .06657 , 1 .0 1 1 3 , 1 .04 1 , 1 .422 and 1 . 1 1 37 for diet A, B, C, D and E, respectively. All diet groups had an increase in lymphocyte to neurophil ratio from weaning day to day 1 4, except diet D group that reduced from 1 . 1 029 to 1 .0 1 3 . Pigs that consumed treated diets had higher SF compared to the control animals. Among treated diets pigs fed diet D had a higher SF and was lower in pigs fed diet C In the last week (week 3) the least-square means of lymphocyte to neutrophil ratio were 0 .8655 , 0 .878, 0 .8703, 1 .3 1 82 and 0.898 1 for diet A, B, C, D and E, respectively. From day 0 to day 21 al l diet groups had a reduction in SF. 7 1 On day 1 4, pigs fed diet B and C had lower SF than the control while SF were higher in pigs fed diets D and E than the controls. Overal l LSmeans of lymphocyte to neutrophil ratio for piglets fed the control and probiotic-supplemented diets for the 2 1 d period were 0.9 1 54, 0 .9974, 0.9644, 1 . 3289 and 1 .027 1 for diet A, B, C, D and E, respectively. Lymphocyte to neutrophil ratio was higher in the treated groups compared to the control . Among treated diets SF was higher in pig that consumed diet D and lower in those that received diet. Although lymphocyte to neutrophil ratio did not vary with time (p>0.05), in pigs fed the control diet, lymphocyte to neutrophil ratio increased from 0 . 82 at day 0 (28 days) to 1 .07 at day 1 4 (42 days). From day 1 4 (42 day), lymphocyte to neutrophil ratio declined 0 .87 at day 2 1 ( 49 days old) (Table 6). On day 7, pigs on pro biotic- and lactoferrin supplemented diets had higher SF compared to the controls . Among the treated groups, pigs fed diet D had a higher SF and was lowest in pigs that received diet C. On day 1 4, pigs fed diets B and C had lower SF than the controls while SF was higher in pigs fed diets D and E than the controls. Among treated diets, pigs that consumed diet C had lower SF values than pigs in diet D. On day 2 1 , pigs in probiotic-added diets had higher SF than the control animals . Among the treated groups, pigs that consumed diet D had higher SF while those fed diet C had lower SF. Diet D stimulated the highest stress factor of all the five diets followed by diet E and B while diets C and A caused the least effective . Although SF in pigs were not affected by time (p>0.05), in pigs fed the control diet, SF increased from 0.8 1 5 1 at day 7 (35 days) to 1 .0657 at day 1 4 (42 days) . From day 1 4 (42 day), SF increased to 0 .8655 at day 2 1 (49 days old) (Table 6). 72 3.5 Effect of probiotic on Mean Weekly Faecal Scores Fifthly, we investigated the effect of dietary treatment on mean weekly faecal score (MWFS) as described earlier (See Materials and Methods, p. 28) . Annex 1 shows the statistical significance of effects of farm, diet, day and their interactions on faecal score of pigs. Least square means for effects of pro biotic-supplemented diets on mean weekly faecal score in weanling pigs are summarized in Table 8 . Table 8 : Effects ofprobiotic- and lactoferrin-supplementation on mean weekly faecal scores in weaned piglets Parameter A B c D E SE (control) (probio1ic B) (probiotic B) (probiotic B) (lactoferrin) mean weekly faecal score day 1 4 2 . 8067 3 . 2 2 1 3 . 2 3 8 2 . 869 3 . 1 06 0 . 1 4 day 2 1 3 . 00 3 3 3 . 1 56 3 . 2 3 7 2 . 956 3 . 1 2 5 0 . 1 4 d ifference d 1 4-d7 - 0 . 66 2 0 . 08 2 - 0 . 4 3 -0 . 1 2 -0 . 0 1 d 2 1 -d 7 -0 .466 0 . 0 1 6 - 0 . 4 3 - 0 . 0 3 0 . 0 0 7 Trial mean 3 . 0 9 2 9 3 . 1 72 3 . 38 2 . 936 3 . 1 1 7 0 . 08 SE standard error Least-sigificant means for weekly fecal scores was not different for piglets fed the control or the probiotic-supplemented diets B, C, D and E (p = 0 . 1 260) . Effects of farm and day on MWFS were not observed (p > 0 .05) . Interactions of farm x diet, farm x day, farm x diet x day and diet x day were not detected (p>0.05) . In week I , the least-square means ofMWFS were 3 .4688, 3 . 1 3 96, 3 .6635 , 2 .9844 and 3 . 1 1 7 for diet A, B, C, D and E respectively. In week 2 the l east-square means ofMWFS were 2 .8067, 3 .22 1 4, 3 .23 84, 2 .8686 and 3 . 1 062 for diet A, B, C, D and E, respectively. 73 In the l ast week (week 3) the least-square means of MWFS were 3 .0033 , 3 . 1 56, 3 .2365, 2 .9557 and 3 . 1 247 1 for diet A, B, C, D and E, respectively without significant differences. The MWFS were higher in diet B, C and E than control and lower in diet D group. Overal l LSmean for MWFS for the 2 1 d period were 3 .0929, 3 . 1 723, 3 .3795, 2 .9362 and 3 . 1 1 65 for pigs receiving probiotic/diet A, B, C, D and E, respectively. Although MWFS in pigs were not affected by time (p>0 .05), in pigs fed the control diet, MWFS decreased from 3 .4688 at day 7 (35 days) to 2 .8067 at day 1 4 (42 days) . From day 1 4 (42 day), MWFS increased to 3 .0033 at day 2 1 (49 days old) (Annex 1 ) . MWFS was numerically lower in pigs that consumed diets D and E compared to the control and higher in those that received diets B and C compared to the contrls. Some pens with higher MWFS also had wet floors indicating some self cleaning mechanisms which is part of physical barrier defense mechanism. 3.6 Effect of pro biotic- and lactoferrin-supplemented diets on Health parameters There were no significant differences due to treatment observed in cumulative diarrhoea cases, other diseases, cull rate and survival rate from 0 to day 2 1 of the experimental period. Control animals as wellas piglets fed probiotic-supplemented diets appearered clinically normal during the whole experiment and no mortality resulted from consuming probiotics . No signs of toxicity were observed in all groups. Piglet mortality was nil in all diet groups . Piglet mortalities after weaning were not positively influenced in the probiotic group. In fact, the piglets that were euthanased were from the pigs that received diets supplemented by probiotic B and D. In our study the probiotic supplemntation did not improve piglet losses. 74 4. DISCUSSION CHAPTER 4 Effect of mixing piglets from different farms The data showed a strong farm effect, that is mixing of piglets at the start of the trial was significant (p<0.05). As possible explanation for this, it can be hypothesized that different hygienic status i .e . different pathogen loads present in different housing and management systems are different from farm to farm and might affect animals responses to different parameters investigated. For example animals ' responses to �­ glucan by Decuypere et al. ( 1 998) using animals from two different farms were different due to hygienic status. ADG was higher in pigs from low hygienic status. Eurell et al. ( 1 992) reported high haptoglobin serum concentrations which are in general elevated during immune challenges and under poor hygienic conditions in pigs. Pigs may also come from areas with some remaining colostral antibodies or earlier individual antigen contact (Hiss and Sauerwein, 2003). It has also been found that piglets from different farms may have diffent number of WBC. This might be as a result of different management. For example WBC wil l show a slower increase if pigs are given one iron injection compared to those given two injections (Johansson et al. , 2005). Effects of pro biotic- and lactoferrin supplementation on physical performance and immunity of weaned pigs To study the effects of probiotics and lactoferrin on the performance and immunity levels of weaned pigs, we mixed different breeds of pigs from four different farms to stimulate the immune chal lenge. This immune challenge model may be considered satisfactory but it would have been better to use a more potent immunological challenge. Stress has been implicated as a factor that stimulates the infection and (in case of swine dysentery) can also be triggered deliberately by the introduction of corticosteroid drugs or starvation of pigs (van Heugten et al. , 1 994a). Although pigs that consumed diets D and E had significantly lowered feed intake, and those that received diet B ate significantly higher amounts than the controls, the body weights at different weighing dates were not significantly reduced by the low intake that resulted 75 from this immunological insult. There were no significant differences in iron levels in pigs on weaning day. Diets could have had another impact on trying to affect iron levels and iron levels were numerically higher in pigs fed diet A, B and C compared to those fed diet D and E. This agreed with the findings of Mahan and Lepine ( 1 99 1 ) who reported that factors such as age, body weight, stress, health status, low feed intake, diet composition, digestive incompetence, and environment at weaning are the main factors of growth check . The fact that no adverse side-effects were observed, the five diets could be tentatively be classified as safe . Good probioticts and other natural alternatives should be safe for inclusion in animal feeds and have both growth promoting effects and GRAS status (Ful ler, 1 992 ; Saxellin et al. , 1 999; Bemardeau et al., 2002). The probiotics and lactoferrin used in the trial were non-pathogenic and safe for animal consumption in addition probiotics B and C had a beneficial effect on growth performance (feed intake) in weaned piglets . Further experiments are, however, warranted. Comparatively, depression of body weight in infected animals was higher [about 1 OOOg (8 .5kg in health compared to 7 . 5kg in sick pigs)] in van Heugten et al. , ' s ( 1 994a) study and was only slightly lowered [by only 200g (8 .5kg in healthy (van Heugten et al.) pigs - 8 .3kg in our controls) on day 7 post challenge] in our study compared to controls in published studies (van Heugten et al. , 1 994a) . The reduction in body weights in other diet groups in our study, compared to the controls in van Heugten et al. , ' s study during the same period, was 1 00, (8 .5kg in healthy van Heugten et al. ' s pigs - 8 .4 in diet B pigs), 200 (8 .5 -8 .3 diet C), 300 (8 .5-8 .2) and 500 (8 . 5-8) for diet B, C, D and E, respectively. Similarly, the body weights were lower than in van Heugten et al. ( 1 994a) because of the beneficial effect of these probiotics . Although we did not have an unchallenged study in the current study, our results can be compared with those reported by van Heugten et al. ( 1 994a). They used feed that was similar in nutritive value and pigs that had similar body weight on day 0 of the challenge (7 .3kg in van Heugten et al. vs 7 .5kg in our current study at 28 days old) . However, the differences in environment and management between the experiments, which are known to affect performance, were excluded. In their study, the body weights in the controls were 7.2kg, 8 . 5kg, 1 0. 8kg and 1 4.0kg at age 28 , 3 5 , 42 and 49 76 days old, respectively. In their study, ADG of controls were 1 90, 320 and 470 g per day at age of 3 5 , 42 and 49 days old, respectively while ADFI was 390, 630 and 900g per pig per day at age of 28 , 3 5 , 42 and 49 days old, respectively. FCR of controls were 2 .0408, 1 . 9608 and 1 . 923 1 at age of 28, 35 , 42 and 49 days old, respectively. With this basic information, we can see that in our study the body weights of pigs were depressed by the immune chal lenge caused by mixing piglets from different farm sources. They had numerically lower iron levels and the body weights in the control group were lowered by 200g, 600g and 1 OOg at age of 35 , 42 and 49 days old, respectively, compared with unchallenged pigs in van Heugten et al. , 's experiment. In our study ADG, ADFI and body weights were all reduced by weaning and the immune challenge compared with the result if animals were not chal lenged. Reduced feed intake is a factor that limits growth in weaned pigs. Weight is gained after the improvement in feed intake (Davies et al., 2002) .The depression in body weight was high in week 1 and even higher in week 2 but the FI , ADG and body weights increased in pigs fed diet B and C ( 1 4.057kg and 1 4 .005kg bodyweight for diet B and C, respectively) to outperform the unchallenged pigs ( 1 4 .0kg) in van Heugten et al . , ' s ( 1 994a) study on day 2 1 post chal lenge. However, those fed diet A, D and E performed poorer compared to controls in van Heugten et al . , ' s study ( 1 3 . 866kg, 1 3 .48 1 kg and 1 322 1 kg body weight for diet A, D and E, respectively). The depression in ADG, FI and body weights in our experiment agrees with those of others (van Heughten et al. , 1 994a, 1 994b; Kegley et al. , 200 1 ; Barnett et al. , 1 989; Pluske et al. , 1 997) and indicates that after weaning I immunological challenge or lower iron levels, animals reduce either ADG, FI, FCR and bodyweight. Animals also attempt to develop a tolerance to the immune challenge and those fed diet B and C in our study mounted a greater immune response by day 2 1 than pigs fed diets A, D and E. On the other hand, animals continuously flooded with a polysaccharide antigen may prevent development of an immune response and pigs wil l quickly surrender to bacterial infection when put under stress or immunological challenge. This is referred to as immunoparalysis and takes place when excess antigen is congested by c irculating antibodies as they are formed (Barnett, 1 983). Furthermore, Marin et al . (2002) reported reduced feed intake and body weights in pigs that received 1 40 and 280 ppb aflatoxin intoxicated feeds compared to healthy 77 controls (0 ppb aflatoxin). The body weights and body weight depression reported by Marin et al. were greater than those reported in our study or by van Heugten et al. ( 1 994a). This work found bodyweight differences between the probiotic­ supplemented and unsupplemented controls and also among supplemented pigs themselves. Differences in body weights of pigs fed diet D and E and controls increased with time. This work concurs with other experiments; weight differences between pigs that consumed 0 ppb or 280 ppb aflatoxin diets (Marin et al., 2002) and also between the unchallenged and infected pigs in van Heugten et al. ( 1 994a) , increased with time as the feeds did not contain any treatments to support immunity against prolonged high levels of the intoxicating agents (immunoparalysis) . Svoboda et al. (2004), also reported decreased body weight in anaemic animals compared to those with adequate HGB in early stages of l ife . It is interesting to note that after the immune challenge in van Heugten et al. ( 1 994a) and Marin et al. (2002), body weight after the immunological challenge remained lower in challenged pigs compared to the controls for the whole duration of these studies, although feed intake was depressed only for a short time immediately after the challenge but improved to outperform the control (in van Heugten et al. , 's ( 1 994a). It is also interesting to note that animals with less stress ( 1 40 ppm aflatoxin and those challenged only once) tried to compensate FI and ADG and body weight (tolerance) while those with higher and continuous stress (of 280 ppb aflatoxin and those chal lenged for second time with l ipopolysaccharide) failed to compensate body weight even though they tried to compensate for FI and ADG (immunoparal ysis). In our study, pigs that received diet D and E had significantly lower feed intake, lower ADG (p>O.OS) and had lower body weights (p>O.OS) for the entire period of the experiment compared with those fed control diet, diet B and C. With these findings we can conclude that probiotics B and C offered a rapid and better immune response, thus an improvement in feed intake which also compensated for higher body weight to outperform the controls . In contrast, pigs that received diets D and E failed to compensate for ADG, FI and body weight due to reduced immunity although they had numerical ly a higher or more efficient FCR (p>O.OS) than pigs fed diet B and C. Lower feed intake might have been worsened by the numerically lower iron levels 78 (low HCT, HGB and RBC). It is interesting to note from van Heugten et al. , ( 1 994a, 1 994b) and Marin et al. , ' s (2002) studies that improved feed intake in immunoparalysed animals does not automatically lead to higher compensatory body weight gain with healthy animals. Reduced ADG, FI, FCR and body weight in pigs have also been documented in many other studies (Klasing et al. , 1 987 ; van Heugten et al. , 1 994 ; Owusu-Asiedu et al., 2002; Coma et al, 1 995 ; Jonasson, 2004; Spurlock et al. , 1 997; Marin et al. , 2002; Odink et al. , 1 990a; Svoboda et al., 2004) caused by change in diet or stress. Important lessons from van Heugten et al. ( 1 994a) are that reduced FI automatical ly results in body weight reduction and even when FI is quickly restored or significantly increased in chal lenged pigs to outperform healthy controls, bodyweight is not automatical ly restored or improved to reach that of healthy pigs . In addition, even though ADG was only slightly (and not significantly) reduced in pigs fed 1 40 ppb aflatoxin compared with controls in Marin et al. ' s (2002) study, body weight was significantly reduced by a wider margin in immunologically insulted pigs compared to controls . Therefore, performance data should be discussed because even those data that are not statistical ly significant (e .g . FI and ADG) can significantly affect other performance parameters (e.g . body weight or body weight gain) . Interestingly, different investigations have reported contradictory results when natural products such as garlic are used. Grela et al. ( 1 998) reported an increase in feed intake when garlic was used in pigs while Corrigan et al. (200 1 ) found that garlic in the diet of nursery pigs decreased feed intake. However, garlic used by Corrigan et al. (200 1 ) was used in combination with a mixture of plant extracts, mixed herb and essential oi ls . Therefore, it is likely that the other herbs and plant extracts may have hidden the strong odour of garlic. This indicates that it is the organoleptic properties of garlic that are to blame for the decrease in feed intake in pigs (Cullen et al. , 2005). S imilarly, in our study, palatability of diet D and E may have been lower than that of diet B and C as pigs preferred the last two diets. As the evidence presented above indicates, the sense of taste is involved in controll ing the selection of food by pigs (Bald win , 1 976) while the influence of olfaction is also recognised (Forbes, 1 995) . In pigs the sense of smell is highly involved in feed intake (Melior, 2000). The active ingredient in garlic 79 (allicin) is a remarkably odoriferous compound (Cavalito & Bailey, 1 994). It can be seen in the investigation by Cavalito and Bailey, 1 994, that the decrease in feed intake was not significant during the finisher period, suggesting that animals may quickly get used to garlic (Cullen et al. , 2005). Similarly, in our study, the initial reduction in FI in al l pigs post challenge and the increase in FI in pigs fed diets B and C, could be due to this reason. Feed intake is a factor limiting growth in weanling pigs. Weight gain accompanies the improvement in feed ingestion. Probiotic may act on the gut to improve performance as observed by an increase in feed intake (Davies et al. , 2002). Ingestion of probiotic D and E in our study, or for l ipopolysaccharide in van Heugten et al. , 's ( 1 994a) study, reduced FI and body weight. However, it is very surprising to note that despite a reduction in feed intake (and digestible energy associated with garl ic inclusion in Cavalito & Bailey' s ( 1 994) experiment), there was no negative effect of garlic on liveweight gain. The inclusion of garlic in the diet at 1 g/kg improved the feed conversion ratio by 9.8% during the grower period and 5 . 7% during the combined grower finisher period, while the inclusion of garlic at 1 0 g/kg improved FCR by 7 .0% during the grower period and 6 .5% during the combined grower-finisher period, when compared to the control diet (Cul len et al., 2005). The improvement in FCR reported above by Cull en et al. (2005), using garlic, and the improvement of both FCR and dai ly gain reported by Grela et al. ( 1 998), using great nettle, garlic and wheat grass mixture, were all much greater than the improvement in FCR or gain noted in studies using antibiotics as growth promoters (Zimmerman, 1 986). Some probiotics have the ability to influence immune response (Bloksma et al. , 1 979). Some beneficial effects of direct-fed-microbials are that they influence imrnunoadjuvant activity (Perdigon et al. , 1 99 1 ) and increase the total amount of intestinal IgA (Tizard, 2000; Lin, 2000) . In contrast, Kluber et al. ( 1 985) , working with weanling pigs, observed no effect ofprobiotic on the cell-mediated immune response. The improvement in feed efficiency by pronutrients I phytogenics such as garlic, might occur for example via ( 1 ) the improvement of gut environment and microbial 80 flora. The explanation for this is traced to the fact that the susceptibility of harmful gram positive bacteria to the antibacterial compounds in garlic is higher than that of the beneficial bacteria in the gut (Rees et al., 1 993) . The desirable bacteria are said to be protected by the presence of garlic as they are less sensitive to its inhibitory effects. Furthermore, garlic containing fluctooligosaccharides, may have a prebiotic effect on gut microflora (Gibson, 200 1 ) ; (2) the improvement in feed efficiency with the inclusion of garlic may be blamed on the lower DE intake of pigs offered the garlic diet compared to those offered the control diet. In finishing pigs, FCR improves with increasing energy intake up to 33 MJ DE/day but becomes less efficient with each increase in DE intake thereafter (Campbel l et al. , 1 985 (cited in Cullen et al. , 2005)). Similar trends in FCR were reported by O 'Doherty and McKeon (2000) when using similar genotypes as in Cullen et al. , ' s (2005) experiment and (3) antimicrobial action of allicin (Ankri & Mirelman, 1 999) may have the ability to prevent microbial fermentation in the gut. In addition garlic has antiviral activity (Ankri & Mirelman, 1 999). The gut and the skeletal musculature in fast growing pigs is derived from a l imited supply of nutrients and are, in effect, antagonists for the accretion of nutrients (Rees et al. , 1 993 ) . V ervaeke et al. ( 1 979) reported that up to 6% of the net energy in pig rations cannot be used for the benefit of the pig due to bacterial uti lisation of glucose in the small intestine. The requirement of amino acids in these bacteria is similar in amount to that of growing pigs (Hays, 1 978). Inclusion of garl ic in feed at I 0 g/kg might stimulate a nutrient sparing and I or economising effect, hence improving FCR (Cullen et al., 2005). Some workers have reported that garlic improved performance (Cullen et al. , 2005 ; Ankri & Mirelman, 1 999; Janz et al., 2007; Horton et al., 1 99 1 ) while others have reported no effect (Reddy et al. , 1 998). These inconsistencies in results have been found in many probiotics and in garlic, and could be due to variable inclusion levels of garlic and in the allicin and alliin concentrations of the garlic used (Cullen et al. , 2005). Several possibilities for why natural products such as probiotics have not been consistently successful include : insufficient bacterial cell numbers; bacteria incapable of surviving and performing their metabolic functions in the gut, which may be influenced the presence of antibiotics in the feed (Turner et al., 200 1 ) ; ageing bacteria 8 1 cultures losing their efficacy over time; and instability of intestinal fermentation (Hil lman, 1 999). The latter can be as a result of overdosing with the probiotic organism such that it exhausts all avai lable nutrients and diminishes other beneficial bacteria as well as pathogenic bacteria. In some cases, the probiotic may replace a colony of harmful bacteria while in others, the pro biotic may replace a colony of beneficial bacteria, thus cancelling out any benefit it may have provided (Ewing & Cole, 1 994). In other studies, it has been seen that some probiotic bacteria, such as Bacillus spp, are not normal components of the indigenous intestinal micro flora, so that those bacteria are hard to establish in the digestive tract, hence no beneficial effect (Jonsson & Conway, 1 992). The use of different methods as a model for in vivo pig inflammatory conditions to investigate immune challenge such as live bacteria (for example enterotoxic Escherichia coli- ETEC) (Owusu-Asiedu et al., 2005 ; Touchette et al. , 2000: Bosi et al. , 2002) or l ipopolysaccharide (LPS) (van Heugten et al. , 1 994a) can yield different results. Nutritional status, diet and low feed intake were the main problems in reduction of performance in our study. When food components, such as lactoferrin, are natural ly injested, they interact with a number of lymphoid cel l s as they move down the gut. Numerous factors can affect these interactions such as sol id or aqueous form of the food component and the mode of exposure (Sfeir et al. , 2004 ; Fugh-Berman, 2000; Chavez et al., 2006). In rats, administration of lactoferrin through injection resulted in higher stimulation of innate and adaptive immune response compare administration through drinking water (Sfeir et al., 2004). In summary, we can conclude that during the 2 1 day period, the effects of pro biotic­ supplementation on ADG, FCR and body weight were not different among diet groups (p>0.05) . By growing fast, pigs fed diets A, B, and C would reach target weight earlier. This can reduce occupation rate thus making housing more efficient, so that more space and time is available for more pigs to be kept (Janz et al., 2007). Low feed intake post weaning I post challenge (or during the time with lower iron levels,) reduced gain resulting in nutritional stress and possibly caused reduced immune resistance and obstruction of antibody formation (Barnett et al. , 1 989). When feed intake is improved after weaning I post challenge, mucin production in the gut improves (Lopez-Pedrosa et al. , 1 998; Lal les et al. , 2004). Mucin contributes to the 82 healthy gut through lubrication, physico-chemical protection and prevention of bacterial adhesion (Forstner & Forstner, 1 994; Lall es et al., 2004; Tizard, 2000) acting as a self cleaning barrier mechanism. This also increases the enzymes necessary for the breakdown of starch, carbohydrares and protein (Lombardi et a!, 2005). During the digestion process, some dietary proteins or polypeptides may escape the luminal hydrolytic process and find their way into the intestinal mucosa in sufficient quantities, where they are, at a later stage, absorbed or incorporated through different mechanism, including both the paracel lular and transcel lular pathways (Tome & Debbabi, 1 998) . This absorption of large molecules in antigenic and biological ly active amounts is presumed to activate different physiological and immunological reactions that lead to oral tolerance and its control (Bahna, 1 985) . Proteins, including lactoferrin, are known to interact with either minerals, vitamins or nutrients by specific mechanisms. These interactions affect incorporation or absorption of these nutrients . Casein phosphopeptides have been found to inhibit the precipitation of calcium phosphate and speed up its absorption in the small intestine (Lee e t al. , 1 980). Lactoferrin is bel ieved to have a part to play in DNA synthesis, proliferation, differentiation and metabolic effects (Tome & Debbabi, 1 998) and is involved in host defence through its bacterial activity (Lonnerdal & Iyer, 1 995). Probiotic B and C might protect proteins and facilitate this system of absorption in pigs, as evidenced by a numerical ly higher gain and protection from immune challenge, although this was not the case for pigs fed lactoferrin-supplemented diet. Different proteins, including lactoferrin, vitamin B 1 2 , protein, folate binding protein, P-lactoglobulin and a­ lactalbumin are assumed to interact with either mineral, vitamin or nutrient s by a specific ( Iyer & Lonnerdal, 1 993 ; Tome & Debbabi, 1 998). The manner in which enzootic diseases affect animals is not wel l understood but more investigations continue to be carried out. Pathogens trigger an immune response specially designed to remove them from the body. In reaction, the immune system activation stimulates a surge of effects, some of which are beneficial to growth, on the animal ' s metabolic system. The type of antigen determines the clinical signs observed in the exposed animal . A non-pathogenic bacteria, a vaccine and I or a natural product may have relatively l ittle or no harmful effects on the animal, whereas exposure to a 83 pathogenic antigen could severely affect the animal ' s performance (Wil liams et al. , 1 997). We found reduced weight gain i n probiotic D - and E-supplemented pigs and this was as a result of reduced feed intake. Similarly, Will iams et al. , 1 997 compared pigs from 6 to 27 kg that differed only in immune system activation and reported significant increases in growth rate and feed efficiency, and only a tendency towards increased feed intake. In addition, van Heugten et al. ( 1 994a), and Pluske et al. ( 1 997) all reported that an antigenic challenge in young pigs suppressed the growth rate, with its greatest impact on feed intake rather than feed efficiency. Metabolic changes linked to infectious diseases can result in decrease in gain and feed efficiency. The fol lowing studies by Klasing et al. ( 1 987), van Heugten et al. ( 1 994a), ( 1 994b), Coma et al. ( 1 995), Jonasson (2004), Spurlock et al. ( 1 997), Marin e/ al. (2002), Odink et al. ( 1 990b) and Svoboda et al. (2004) found decreased weight gain and I or feed intake and I or efficiency of feed util isation in animals that were repeatedly chal lenged with noninfections or infectious (immunological) agents or other stress. The metabolic switch following immune challenge are caused by interleukin 1 ( IL 1 ) and tumor necrosis factor (TNF) produced by stimulatory macrophages (Morrow-Tesch & Anderson, 1 994 ; Marin et al. , 2002 ; Klasing, 1 988 ; Hiss & Sauerwein, 2003) . Nutrients are prevented from reaching intended growing sites (increase in size and volume of cells) and are redirected to the mobil isation of a defence system (Demas et al., 1 997; Hiss & Sauerwein, 2003 ; Beisel, 1 977; van Heugten et al. 1 994a, 1 994b and 1 996; Owusu-Asiedu et al. 2003 ; Spurlock, 1 997; Davis et al. , 2002) . The amount of nutrients being used for the immune system were quickly reversed in pigs that received diets (A,) B and C and this is evidenced by beneficial effects on growth performance originating from increased ADFI which improves ADG. This is consistent with Hiss and Sauerwein (2003) who observed reduced immune function in weaned I challenged pigs that consumed higher amounts of feed supplemented with P-glucan compared to the controls and van Heugten et al. ( 1 994a) who reported reduced weight in l ipopolysaccharide chal lenged piglets . In addition, lowering of interleukins and tumor necrosis factors, which are inhibitors of feed intake, by probiotics, mediated by the central nervous system, might contribute to the increased feed intake (Hiss & Sauerwein, 2003) . Similarly, increase in feed intake in our study could be due to increases as a result of the removal of these inhibitors in pigs fed diet B and C, compared to those fed diet D and E. 84 In our study, feed intake was a factor l imiting growth in young pigs and daily gain and body weight was, therefore, improved with increasing intake (in pigs fed diets B and C). This is consistent with published research ofDavis and coworkers (2002) and Janz e t al. 2007 who reported an increase in gain and feed intake when CuS04 and garlic, respectively were added to feed. Increased feed intake that occurs when natural products such as MOS or copper are added is as a result of reduction of intestinal damage caused by pathogens because of their antimicrobial action. Systematic administration of certain minerals such as copper can improve many functions that they serve in the body. The significance of diet on the immune system was difficult to detect. The explanation could be that piglets were only challenged once or l ightly immunologically challenged and pigs consumed 'adequate' weaning rations with a high protein and energy content. This concurs with Lopez (2000) who reported that in feeding a well formulated diet and I or under good environment, there is no opportunity to further improve nutrients digestibility and the performance of the piglets. The integrity of the gut mucosa is a prerequisite to lower the entry of pathogens (Bosi , 2000). The components of the gut mucosal barrier (non immunological and immunological) are, however, disturbed during stress such as at weaning. Lowered feed ingestion in the weaned pig causes vil lous to reduce in size (Bosi, 2000). A lowered supply of milk (Kelly et al. , 1 99 1 ) or a restricted ingestion of dry feed (Pluske e t al. , 1 996) reduce the height of villous on the fifth day post weaning. Inadequate feed intake in weaned piglets may result into intestinal inflammation and affect intestinal morphology . In addition, the antigenicity of the diet is another factor (Bosi, 2000). The defense against pathogens is integrated by the endogenous secretion of many antimicrobial components such as hydrochloric acid, lactoferrins, mucous secretion. Some of these, however, the mechanisms of control of secretion are not adequately known to increase their production by dietary means (Bosi, 2000) . In our study, piglets did not respond well to the lactoferrin treatment as they were in good hygienic conditions, which would probably result in decreased opportunity for 85 lactoferrin activity. This result i s also consistent with those of other workers . Sarica et al. (2005) reported that wel l nourished healthy chicks do not positively respond to growth-promoters when they are housed under clean conditions and at a moderate stocking density. Future research should be conducted on commercial farms and more potent immune stimulants should be used continuously or at several times during the study. Good feeding or good nutrition involves formulating a feed to meet the requirements of an animal and can therefore stimulate production and improve health. Some nutrients I elements or natural products such as probiotics have the characteristic concentrations and functional forms that should be maintained within narrow limits to maintain a functional and structural integrity of the tissue to safeguard growth and health. Nutrients such as iron (measured as haemoglobin) are important components of tissue (such as blood) and blood components (such as transferrin and ferritin, erythrocytes, leukocytes and monocytes) which play an important role in maintenance of health and a deficiency I excess may result in i l l health (Underwood & Somers, 1 969; Underwood & Mertz, 1 987). Except for ADFI, other parameters studied (ADG, FCR, blood parameters, mean weekly fecal scores (MWFS) and general health) were not significantly different between diet groups, although some numerical diffences were observed. 86 5. CONCLUSION CHAPTER S The present work has researched development of treatments B, C, D and E as alternatives to antibiotics for improving physical performance, general health and immunity in weaned pigs . We have assessed the five diets (A( control), probiotic B, C, D and diet E (lactoferrin)) for their effects on average daily gain, feed conversion, feed intake, mean weekly fecal scores (MWFS), blood parameters, stress factors, and general health. These are essential parameters that can be explored and uti l ised to find effective and safe natural products to be used to reduce diseases and improve the productivity of pigs while at the same time protecting the environment. The findings have shown that weaning a piglet from its mother's milk to a different diet inhibits growth due to hypersensitivity. Stress and some allergenic components contained in the weaned diet can cause scours and prevent growth to some extent. The intestinal microflora is usually stable but is dynamic and can be perturbed by changes in the gut environment (Kelly, 1 998). Growth lag, high mortality, morbidity and diarrhoea have been reported in many studies after weaning I immune challenge (Lalles et al., 2004). Restoration ofthe microbial balance of the intestines is the basic tenent of pro biotic therapy (Kelly, 1 998). Depending on the severity of stress, diet, and animal capacity, the pigs may have a reduced feed intake or feed conversion and may have reduced bodyweight. One of the factors that l imit growth in weaned piglets is feed intake (Davies et al. , 2002). Weight is gained after the improvement in feed ingestion. Pro biotic B may have a beneficial action on the gut to improve performance as observed by an increase in feed intake. As pointed out by Jensen ( 1 998), a good diet wil l produce a stable gut ecosystem that has adequate capacity to resist change as micro niches in the gut ecosystem are well covered and available energy sources are quickly used for the benefit of the host. Immunocompromised animals may fai l to compensate body weight even if feed intake is later improved. On the other hand an animal fed an adequate diet, may develop a tolerance and will compensate for body weight similar to or higher than if they were not stressed. Under these conditions diet B and C were "adequate" diets while D and E were not. A diet or probiotic used in 87 weaned pigs should be able to stimulate feed intake, FCR and I or daily gain and most importantly, compensate for body weight even when pigs are immunological ly challenged. On the other hand when feed intake is reduced, a good diet should improve feed conversion and util isation so that nutrients are better used for growth and lead to bodyweight compensation. Supplementation of pro biotic B and C improved feed intake compared to the control diet. The improvement was only significant in pigs fed diet B. Although, FCR, MWFS, SF and blood characteristics were not significant, some numerical differences and general improvement in pigs were observed between diet groups . ADG followed the pattern of feed intake and was numerically higher in pigs fed diet B and C compared to those that consumed the control diet while pigs that received diets D and E ate less feed compared to the controls . These direct-fed microbials (B and C) can, therefore, be safely used as natural growth promoters and as alternatives to antibiotics in weaned pigs. It is important to report all findings as even non-significant results may significantly affect other performance parameters. Probiotics B, C, D and diet E (lactoferrin) stimulated feed intake performance differently (p<0.05), but there was no significant difference between the diet in the manner in which they affect dai ly gain and feed conversion performance. However they could be further developed and marketed as they can be used safely as feed additives in the place of antibiotics. The mode of action is different and inconsistent between natural products such as probiotics, although several possibilities for why probiotics have not been consistently successful have been proposed. For example, lactoferrin is reported to reduce diarrhoea (Steij ns, 200 1 ) but the difference in mean weekly faecal scores between diet groups was not significant in our study. The failure of pro biotic effect of some products may arise as a result of the environmental state such as a clean environment or a hygienic place in which, the beneficial effect may be minor (Cromwell , 200 1 ; Yu et al. , 2004; Decuypere et al., 1 998; Hiss & Sauerwein, 2003). As earlier stated other consistencies may also be as a result of: insufficient bacterial cell numbers; bacteria incapable of surviving and performing their metabolic functions in the gut, which may be influenced the presence of antibiotics in the feed (Turner et al. , 200 1 ); ageing 88 bacteria cultures losing their efficacy over time; and instabil ity of intestinal fermentation (Hillman, 1 999), to mention but a few. Therefore, having good nutrition, management and disease control are vital important for l ivestock productivity (Walton, 200 1 ) . The performance values of controls in experiments planned to test the effect of probiotics should be low so that the biological variation between the control and treated groups are clear and therefore such studies should be conducted with respect to prevai l ing practical or farm conditions (Jensen, 1 989; Sarica et al. , 2005). Effects are also dependent on diet or component, nutrients, physiological state of animal sick or pregnant, age, concentration, CFU, environment and many other factors and the effects are either local ized at digestive, the systemic, or central level . Al l diets were safe for use in pigs. In addition, some had some benefits which resulted in similar or different effects. The diets should be used at appropriate times depending on the effect that is desired or combined to get a wider or full benefit of two or more benefits e .g . diet B improves weight better in wk 1 whi le diet C improves i t better in wk 2 . At the current concentration levels and method of preparation, (control diet or) diets supplemented with probiotic-B or C can improve FI and ADO and bodyweight and may offer some beneficial effect in gastrointestinal tract from day 0 to 2 1 postweaning I post challenge. As there is only 2% of total blood that flows through the peripheral layer, thi s is not effective for determining diseases (Tizard, 2000; Cummings et al. , 2004). Blood tests cannot be relied upon for detection of immunity I health parameters. When the immune system is impaired clinical symptoms appear, although this is not always the case (Cummings et al. , 2004). For example, pigs can carry disease and can occasional ly shed bacteria without showing clinical symptoms such as diarrhoea or differences in leukocyte population or subpopulations. Therefore, future experiments should collect blood more frequently and include post-mortem examinations to confirm any disease (Jonasson et al. , 2004). Haematological parameters may be used to indicate immunity but should be used together with incidences and severity of infections and growth functions (Cummings et al., 2004). Haptoglobin serum concentrations are generally raised when the immune system is challenged and under poor hygiene and should therefore be included (Hiss & Sauerwein, 2003) . 89 At weaning, scouring in piglets is mainly due to hypersensitivity. The cause of diarrhoea may vary, and the impact on health and wel l -being can range from slight discomfort to severe malnutrition and death in humans (Brown, 1 994; Lin, 2000). After weaning scours are caused by other factors. A good diet stimulates strong chemical barriers, with high gastric acid resulting in low pH (<3 .0) and this improves digestion of nutrients and stimulates iron and calcium absorption. Protein accretion for body weight gain and formation of protective components of the blood is stimulated to support health and body functions. With serious digestion stress, a good selective epithelial mucosa wil l trigger fast movement patterns in the bowels which is l inked to elevate fluid production resulting in higher MWFS . This is an important mechanism of defense by which pathogens are flushed out of the body (self cleaning) (Boudraa et al. , 1 990). Low MWFS is therefore not always a good indicator of gut status as animals may harbour bacteria without showing symptoms. To sum-up, a good probiotic I diet should produce a faster and more rapid response by stimulating feed intake (feed conversion, immune chal lenge and growth and stimulate iron use to alleviate anaemia) in the first three weeks of life or at the times of stress such as weaning and immune chal lenge and enable body weight compensation. A diet should be formulated in such a way that it entirely matches the capacity of the animal ' s haematopoietic system for haemoglobin synthesis and growth. This should also be efficient in producing a higher immune barrier and channel nutrients for growth. A good diet (probiotic or natural cure) should quickly improve feed intake so that pigs ingest sufficient amounts to compensate for body weight loss (such as diet B and C). Feed intake is a factor limiting growth in weanling pigs (Davies et al. , 2002). If feed intake (energy) is reduced, then, an effective diet should improve the feed util isation of the avai lable or ingested nutrients in the animal (e.g. garlic). When feed intake increases, weight gain is also improved. When this is achieved, the less nutrients consumed would be better util ised and the net energy wil l be sufficient to meet requirements for both maintenance and growth and most important, compensate body weight. When feed intake is improved after weaning I post challenge mucin production in the gut improves (Lopez-Pedrosa et al. , 1 998 ; Lalles et al. , 2004; 90 Spreeuwenberg et al., 200 1 ). Mucin contributes to the healthy gut through lubrication, physico-chemical protection and prevention of bacterial adhesion (Forstner & Forstner, 1 994; Lalles et al. , 2004) - self cleaning. As feed intake increases, the levels of digestive enzymes responsible for the breakdown of starches, proteins and fats increase. Therefore making the animals to consume more feed shortly after weaning is very important (Lombardi et al. , 2005). As reported by Hiss and Sauerwein, 2003 and Langham and Hrupka, 1 999, reduction of pathogen load by �-glucan in the systemic effect medaited by the central nervous system might contribute to increased feed intake. A good diet should provide an environment in the gut which favours beneficial bacteria to live longer while exploitative parasites should be quickly purged I flushed out of the alimentary canal - again, self cleaning barrier mechanism. Pathogens may be reduced by using a diet (such as diet B and C) that quickly stimulates fast flow of digesta to reduce the pathogen load (Simon, 1 998). As Fugh-Berman (2000) reported herd-drug interactions or probiotic-ingredient interactions are evident and probiotic reasearchers should advise about mixing probiotics and diet formulations and timing of their use. As pointed out by Bemardeau et al. , 2002 and Ful ler, 1 992, a good probiotic should not only have growth promoting effect, but must also have a general ly recognized as safe (GRAS) status. Further studies are therefore warranted. These should also measure the number of probiotic bacteria both at the start and end of the experiments and know whether these can be retrieved l ive or dead. As pointed out by Bemardeau et al. (2002) and Ful ler ( 1 992), some bacteria can stil l maintain their probiotic effect whether dead or alive. 9 1 6 . LIST O F REFERENCES Abbas, A .K, Lichtman, A.H. & Pober, J .S . ( 1 99 1 ) . Cellular and Molecular Immunology. W.B. Saunders Co. , Philadelphia, USA. Chapters 6 & 9 . Adachi, S . ( 1 992). Lactic acid bacteria and the control of tumours. In : B . J .B . Wood (Editor) . The Lactic Acid Bacteria Adami, A. & Cavazzoni, V. ( 1 999) . Occurrence of selected bacterial group in the faeces of piglets fed with Bacillus coagulans as Probiotics. J Basic Microbial. 39 : 3-9. Adj iri-Awere, A. & van Lunen, T.A. (2005). Subtherapeutic use of antibiotics in pork production: Risks and alternatives. Journal of Animal Science, 85 (2), 1 7- 1 30 Ahmed, F . , & Guadri, S .S . ( 1 985) . Effect of supplementation of essential amino acids on immune response in protein - deficiency rats. Nutr. Rep. Int. 3 1 : 7 1 1 - Ak.kermans, A.D.L. , Konstantinow, S .R . , Zhu, W.Y. , Favier, C.F . & Wil liams, B.A. (2003) . Postnatal development of the intestinal microbiota of the pig. In: Bal l , R.O. (Ed), Proceedings of the 91h International symposium on Digestive physiology in pigs, Banff, AB, Canada. pp. 49-56 Aherne, F.X., Danielson, V. & Nielsen, H .E. ( 1 982). The effect of creep feeding on pre and post waening performance. Acta Agriculture Scandinavica. 32 : 1 55- 1 60 Alverez, S . , de Macias, M.E.N. , de Roux, M.E. de Ruiz, & Holgado, A.P. (x), The oral administration of lactic acid bacteria increase the mucosal intestinal immunity to enteropathogens. J Food Protect. 53 , 404-4 1 Andrews, G.A & Smith, J.E. (2000). In Feldman, B.F. , Zinkl, J. G . , Jain, N.C . (Eds) Schalm 's Vetreinary hematology. 51h Ed . Philadelphia, Lippincott Wi l l iams and Wilkins, pp 1 29- 1 34 Ankiri, S . & Mirelman, D. ( 1 999). Antimicrobial properties of al l icin from garl ic. Microbes and Infection. 1 (2) : 1 25- 1 29 Antipas, C . & Weber, G. (2003). The Role of vitamin nutrition on sow and piglets health. The Pig Journal Proceedings Selection 5 1 : 1 98-2 1 0 Antony, J . , Fyfe, L . & Smith, H . (2005) . Plant active components- a resource for antiparasitic agents. Trends in Parasitology, 2 1 ( 1 0) 462-468 Apgar, G.A. , Kornergey, E.T., Lindemann, M.D. & Wood, C.M. ( 1 993). The effects of feeding various levels of Bifida bacterium globosum A on the performance, gastrointestinal measurements, and immunity of weanling pigs and on the performance and carcass measurements of growing-finishing pigs. J Anim. Sci. 7 1 : 2 1 73-2 1 79 ARC, ( 1 98 1 ). The nutrients requirements of pig's Technical review. Commonwealth Agric. Bureaux, Slough, U.K. Arnold, R.R., Brewer, M. Gauthier, J .J . ( 1 980). Bactericidal activity of human lactoferrin : sensitivity of a variety ofmicroorganisms. Infect Immun 28 : 893-8 Atherton, D . & Robbins, S . ( 1 987). Probiotics- A European perspective. Alltech' s Third Annual Symposium . Biotechnology in the Feed Industry. Lexington, Ky, pp l 67- 1 76. Austyn, J .M. & Wood, K.J. ( 1 993) . Principles of cellular and Molecule Immunology. Oxford University press, UK. Ch. 1 . 92 Avram, N, Maconei, N. , Zabava, R. Voineag, V. ( 1 982). Adverse reactios in piglets inj ected with iron dextran for prevention of anaemia. Rev. Crest Animalelor. 1 1 : p45 Bager, F., Aarestrup, F. M. & Wegener, H. C. (2000). Dealing with antimicrobial resistance-the Danish experience. Can. J Anim. Sci. 80 :223-228 . Bahna, S .L . ( 1 985). Pathogenesis of milk hypersensitivity. Immunology Today 6 : 1 53- 1 54 Baldwin, B.A. ( 1 976). Quantitative studies on taste preference in pigs. Proceedings of the Nutrition Society 3 5 : 69-73 . Barnett, K.L. , Kornegay, E.T., Risley, C.R. , Lindemann, M.D. & Schurig, G.G. ( 1 989). Characterization of creep feed consumption and its subsequent effects on immune response, scouring index and performance of weanling pigs. J Ani m. Sci. 67 :2698-2708. Beard, J.L. (2000). Dietary phosphorus and an inflammatory challenge affect performance and immune function of weaning pigs. Journal of Animal Science 79: Beard, J .L (200 1 ) . Iron biology in immune function, muscle metabol ism and neural functioning. J Nutr. I 3 I : 568-580 Beisel , W.R. ( 1 977). Metabolic and nutritional cosequences of infection. In : H . H . Draper (Ed) Advance in Nutritional research. Vol . 1 . Plenum, New York Bendixen. G. , Bentzen, K. & Clausen, J .E. ( 1 976). Inhibition of human leucocyte migration. In: J .B . Natvig, P. Permann, H. Wigzel (Editors). Lymphocytes. Isolation, Fractionation and Characterisation. Scand. J Immunol., Suppl . 5, 244- 267 Bennett, M. & Davis, J . ( 1 98 1 ) Lactoferrin blinding to human peripheral blood cells: an interaction with a B-enriched population of lymphocytes and a subpopulation of adherent mononuclear cells. Journal of Immunology 127 : 1 2 1 1 - 1 2 1 6 Benno, Y . , He, F . , Hosoda, M. , Hashimoto, H . , Koj ima, T. , Yamazaki, K. , l ino, H . , Mykkanen, H. & Salminen, S. ( 1 996). Effects of Lactobacillus GG yoghurt on human intestinal microecology in Japanese subjects. Nutr. Today, Suppl . 3 1 , 9- 1 1 Ben Mansour, A., Tome, D. , Rautureau, M. , Bisall i , A., & Desjeux, J .-F. ( 1 988) Luminal anti-secretory effects of a b-casomorphin analogue on rabbit i leum treatment with cholera toxin. Paediatric Research. 24:75 1 -755 Berseth, C .L, Lichtenberger, L.M. & Morriss, F .H. ( 1 983). Comparison of the gastrointestinal growth-promoting effects of rat colostrum and mature milk in in new born rats in vivo. Am. J Clin. Nutr. 37 : 52-60 Besong, S . , Jackson, J .A. , Trammwel l , D .S & Akay, V. (200 1 ) . Influence of supplemental chromium on concentration of liver trglyceride, blood metabolites and rumen VF A profile insteers fed moderately high fat diets. Journal of Dairy Science, 84, (7). 1 679- 1 685 . Bernardeau, M. , Vernoux, J-P. & Gueguen, M (2002). Safety and efficacy of probiotic lactobacilli in promoting growth in post weaning Swiss mice. International Journal of Food Microbiology, 77(issue 1 -2) : 1 9-27 . 93 Black, F . , Einarsson, K. , Lid, B .A., Orrhage, K. & Nord C. E. ( 1 99 1 ) : Effect of lactic acid producing bacteria on the human intestine micro flora during amplici ll in treatment. Scandivian Journal of Infectious Diseases. 23 :247-254 B lecha, F. & Charley, B . ( 1 990). Rationale for using immunopotentiators in domestic food animals. In: B lecha, F . ; Charley, B; eds. Immunomodulation in domestic food animals. Academic Press, San Diego, CA. 3-9. Blecha, F. , Pollmann, D .S . , & Nichols, D.A. ( 1 983) . Weaning pigs at an early age decrease cellular immunity. J Anim. Sci. 56:396. Bloksma, N.E. , De Heer, E. , van Dijk, M. & Wil lers, M ( 1 979) . Adjuvaqncity of Lactobacil l i . I . Differential effects of viable and ki l led bacteria. Cl in. Exp. lmmunol. 3 7 :367-375 . Bocourt, R . , Savon, L . , Diaz, J . , Brizuela, M.A. , Serran, P . , Prats, A . & Elias, A . (2004). Effects of the probiotic activity of Lactobacillus rharnnosus on productive and health indicators of piglets. Cuban Journal of Agricultural Science, 3 8 ( 1 ) pp75-79 Bohl , E .H. , Kohmer, E .M. , Saif, L.J., Cross, R.F. , Agness, A.G. & Theil . ( 1 978). J Amer. Vet. Med. Assoc. 1 72:458- Bohmer, B .M. , Kramer, W. & Roth-Maier, D .A. (2006). Dietary probiotic supplementation and resulting effects on performance, health status, and microbial caharacteristics of primiparous sows. Journal of Animal Physiology and Animal Nutrition. 90 :309-3 1 5 . Bosi, P . (2000). Modulation of immune response and barrier function in the piglet gut dietary means. Asian-Aus. J Anim. Sci, vol 1 3 , special issue : 278-293 Bosi, P., Casini, L. , Finamore, A., Cremokolini, C, Merialdi, G., Trevisi, P., Nobil i , F. & Mengeheri, E. (2004). Spray-dried plasm improves growth performance and reduces inflammatory status of weaned pigs challenged with enterocytogenic Escherichia coli K88. J Anim. Sci. 82 : 1 764- 1 772 . Boudraa, G. , Touhami, M. , Pochart, P . , Soltana, R., Mary, J. Y. & Desj eux, J. F. ( 1 990): Effect of feeding yogurt versus milk in chi ldren with persistent diarrhea. Journal of Pediatry & Gastroenterology Nutrition. 1 1 : 509-5 1 2 Bounous, G. , & P.A.L. Kongshavn. 1 978 . Different effect of dietary amino acids on immune reactivity. Immunology 3 5 :257 . Bounous, G. , & P. A. L. Kongshavn. 1 982 . Influence of dietary protein immune system ofmice. J Nutr. 1 1 2 : 1 747. Bounous, G. , & P.A.L. Kongshavn. 1 985 . Differential effect of dietary protein type on the B-cell and T -cel l immune responses in mice. J Nutr. 1 1 5 : 1 403 . Brashears M.M. , Gill iland S .E. , Buck L .M. ( 1 998). Bile salt deconjugation and cholesterol removal from media by lactobacillus casei. J Dairy Sci. 8 1 : 2 1 03 - 2 1 1 0 Brooks, CC. & Davis, J .W. (x) Changes in haematology of the perinatal pig. Brock, J. H. ( 1 993). Iron and immunity. J . Nutr. Immunol . 2 :47- 1 06. Bosi, P . Modulation of immune responses and barrier function in the piglet gut by dietary means. Asian-Aus. J Anim. Sci. 2000. Vol . 1 3 , Special Issue: 278-293 . Brown, K. H . ( 1 994). Dietary management of acute diarrhea disease: contemporary scientific issues. Journal a/Nutrition. 1 24 : 1 455- 1 460. 94 Brown, I .M. , Warhurst, M. , Arcot, 1 . , Playne, R., I llman, 1. & Topping, D .L . ( 1 997). Faecal numbers of Bifidobacteria are higher with a high amylosecomstarch than with a low amylase comstarch. J. Nutr. 1 27 : 1 822- 1 827. Bruner, AB., Joffe, A. , Duggan, A.K, et el.(x). Cl inical hematology - In-clinic analysis, quality control , reference values, and system selection. Veterinary Clinics of North America-Small Animal Practice 26 : Burrin, D .G . , Stol l , B . , Jiang, R . , Chang, X. , Hartmann, B . , Hoist, 1 . 1 . , Greely, G .H . & Reeds, P.J. (2000) . Minimal enteral nutrients requirements for intestinal growth in neonatal piglets: how much is enough? Am. J. Clin.Nutr. 7 1 : 1 603 - 1 6 1 0 Burrin, D . & Stol l , B . (2003). Enhancing intestinal function to improve growth and efficiently, in: Ball R.O. (Ed), proceeding of the 91h International Symposium on Digestive Physiology in Pigs Banff, AB, Canada. pp. 1 2 1 - 1 3 8 Bush, B.M. ( 1 99 1 ). Intepretation of laboratory results for small animal clinicians. (Eds). Blackwel l Scientific Publications. Cai l lard, I. & Tome, D. ( 1 992) Different routes for the transport of x-lactalbumin in rabbit ileum. Journal of nutrition Biochemistry. 3 : 65 3-658 Calhoun , M. L. & Smith, E.M. ( 1 95 8). Hematology And hemopoietic organs. Diseases of Swine. (Ed.) H. W. Dunne, Iowa state college press, Ames. Fraser, A. C . A study of the blood of pigs. Vet. 1 . 94 : 3 . Campbell, R.G, Steele, N.C. , Capetrna, T.J . , McMurtry, J .P . , Solomon, M .B . & Mitchell , A.D. ( 1 989). Interrelationships between sex and exogenous growth hormone administration on performance, body composition and protein and fat accretion of growing pigs. Journal of Animal Science. 67: 1 77- 1 86 Canibe, N. , Hojbert, 0 . , Mikkelsen, L.L & Jensen, B .B . (xx). Improved piglet gut health without the use of antibiotic growth promoters : fermented liquid feed in the diet. Canibe, N. & Jensen, B .B . (2003). Fermented and nonfermented liquid feed to growing pigs: effect on aspects of gastrointestinal ecology and growty performance. Journalof Animal Science. 8 1 . pp20 1 9-203 1 Caruso et al . , 1 993 as cited in Link et al . , 2005). Casey, P.G. , Casey, G.D., Gardner, G.E., Tangney, M. , Stanton, C. , Ross, R.P. , Hi l l , C . & Fitzgerald, G. F. (2004). Isolation and characterisation of anti-Salmonella lactic bacteria from the porcine gastrointestinal tract Casula, G. & Cutting, S .M. (2002). Bacil lus probitics: Spore germination in the gastrointestinal tract. Appl. Environ. Microbial. 68:2344-2352 Cavall ito, C. 1 . & Bailey, J .H. ( 1 994). Allicin, the antibacterial principle of Allium sativum. Isolation physical properties and antibacterial action. Journal of the American Chemical Society 66 : 1 950- 1 95 1 . Chang, K. 1 . , Kil lian, A. , Hazum, E. & Cuatrecas, P . ( 1 99 1 ) and Morphiceptin (NH4- Tyr-pro-CONH2) : a potent specific agonist for morphine (m) receptor. Science. 2 1 2 : 75-77. Chang, X., D . N. Mowat & B.A Mallard. ( 1 994). Supplemental organic and inorganic chromium with niacin for stressed feeders calves. J. Anim. Sci. 72(Suppl . l ) : 1 32 . (Abstract) Chandra, R. K. ( 1 98 1 ). Iron, immunity, and infection: i s there a casual l ink? Food Nutr. Bull. 3 :49-52. Chandrasekhara, N . & Srinivasan, K. ( 1 999). Health benefits of spices. Recent trends in Food Science and technology- Global perspective. Association of Food Scientists and Technologists, Myrore pp725-735 . 95 Chavez, M.L. , Jordan, M.A and Chavez, P.I . (2006). Evidence-based drug-herbal interactions. Life Sciences, 78 ( 1 8) :2 1 46-2 1 57 Chen, Y. J . , Son, K .S . , Cho, J .H. , Kwon, O.S . & and Kim, I .H. (2005a). Effects of dietary Probiotics on growth performance, nutrients digestibility, blood characteristics and fecal noxious gas content in growing pigs . Asian-Aust. J Anim. Sci. 1 8 : 1 464- 1 468 Chen, Y.J . , Kwon, O. S . , Min, B.J . , Shon, K. S . , Cho, J .H. & Kim, I .H. (2005) . The effects of dietary Biotite V supplementation on growth performance, nutrirnt digestibility and fecal noxious gas content Chen, Y.J . , Min, B .J . , Cho, J .H. , Kwon, O.S . , Son, K .S . , Kim, H . . J. & Kim, I .H . (2006). The effects of dietary Bacillus-based probiotics on growth performance, nutrient digestibility, blood characteristics and fecal noxious gas content in finishing pigs. Asian-Australasian Journal of Animal Sciences. 1 9, no.4 : 587-592 Chen, Y. & Johnson, A. G. ( 1 993). In vivo activation of macrophages by prolactin from young and aging mice. International Journal of Immunopharmacology 1 5 :39-45 . Chiang, L.C. , Ng, L .T . , Chiang, M.Y. & Lin, C.C. (2003). Jmmunomodulatory activities of flavonoids, monotepenoids, triterpenoids, iridoid glycosides and phenolic compounds of plantago species. Plant a Med 69: 600-604 Choct, M. & Annison, G. ( 1 992). Anti-nutritive activity effect ofwheat pentosans in boiler chickens: Br. Poult. Sci. 30, 82 1 -834. Chot, M & Annison, G. ( 1 990). Anti-nutritive activity of wheat pen to sans in broiler diets. Br. Poult. Sci. 30 : 82 1 -834. Chowdhury, S .R., Chowdhury, S .D & Smith, T.K. (2002). Effects of garlic on cholesterol metabolism in laying hens. Poultry Science, 8 1 : 1 856- 1 862 Clayton, P. ( 1 998). The effect of probiotics on host mucosal immune responses. MSc Thesis, Massey University, Palmerston North, New Zealand Close, W. H . ( 1 998). Producing pigs without antimicrobial growth promoters. Adv. Pork Prod. 1 1 : 47-56. C lose, W.H. (2000) . Producing pigs without antimicrobial growth promoters. Adv. Pork Prod. 1 1 : 47-56 Clysdale, F.M ( 1 982). The effects of physiological properties of food on chemical status of iron. Nutritional bioavailabli lity of Iron. ( ed) Kies, C, 5 5-84. ACS symposium 203 Coconnier M. H., Bernet M. F . , Demeis S., Chauviere A., Fourniat J. , and Servin A, L ( 1 993): Inhabilition of adhesion of enteroinvasive pathogens to human intestinal Caco-2 cel ls by Lactobacillus acidophilus strain LB decreases bacterial invasion. FEMS Microbiology Letters 1 1 0 :299-306. Cole, D. J. A. ( 1 99 1 ) . The role of the nutritionist in designing feeds for the future. Pages 1 -20 in T. P. Lyons, ed. Biotechnology in the feed industry. Nicholasvi l le, KY Coma, J . , Carrion, D . & Zimmerman, D.R. ( 1 995) . Use ofplasm urea nitrogen as arapid response criterion to determine the lysine requirement of pigs . Journal of Animal Science. 73 :472-48 1 Commazi, S . , Pieralisi, C & Bertazoolo, W. (2004). Haematological and biochemical abnormalities in canine blood: frequency and association in 1 022 samples. Journal ofSrnall Animal Practice, 45 . pp343-349 96 Combs, G.F. & Combs, S .B . ( 1 986). The Role of Selenium in Nutrition. Academic Press, New York Canner, J . & Eckersal l , P .D. ( 1 986). Acute phase response and mastitis in the cow. Res. Vet. Sci. 4 1 : 1 26- Cook, J .D. , Lipschitz, D.A. , Miles, L.E.M. & Finch, C.A. ( 1 974). Serum ferritin as a measure of iron stores in normal subjectes. Am. J Clin. Nutri. 27 :68 1 -687. Cooper, W.C., Good, R.A. & Mariani, T. ( 1 974). Effects of protein insufficiency on immune responsiveness. Am. J Clin. Nutr. 27:647 Corrigan, B.P. , Wolter, B .F . , Ellis, M. & Moreland, S . (200 1 ). Effect of three dietary growth promotions additive on performance of nursery pigs . Journal of Animal Science 79 (Suppl . ) : 1 885 (Abstract). Cornish, J . , Callon, K.E., Naot, D. , Palmano, K.P. , Banovic, T. , Bava, U. , Watson, M . , Lin. J-M., Tong, P.C. , Chen, Q. , Chan, V.A., Reid, H.E. , Fazzalari , N. , Baker, H .M., Baker, E.N. , Haggarty, N.W. , Grey, A.B. & Reid, I .R. (2004). Lactoferrin is a potent regulator of bone cel l activity and increases bone formation in vivo . Endocrinology, vol . 1 45 , no. 9 : 4366-4374. Coste, M. & Tome, D. ( 1 99 1 ) Milk peptides with physiogical activities. I I . Opioid and immunostimulating peptides derived from milk protein. Le Liat. 7 1 :24 1 -24 7 . Coste, M. , Rochet, V . , Leonil, J . , Molle, D. , Bouhallab, S . & Tome, D. ( 1 992a) Identification of C-terminal peptides of bovine B-casein that enhance proliferation of rat lymphocytes . Immunology Letters 3 3 :4 1 -46. Coste, M., Huneau, J . -F . , Mahe, S . & Tome D. ( 1 992b). Interactions of milk derived peptides with the intestinal mucosa. In Dynamic Nutrition Research, Vol. I , ed. A . G. Karger. Base!, Suisse Cox, T.M. & Peters, T.J . , ( 1 980). Cellular mechanisms in the regulation of iron absorption by the human intestine: Studies in patients with iron deficiency before and after treatment. British Journal of Haematology. 44 : 75-86 Crenshaw, T. D., Cook, M. E. , Odle, J. & Martin, R.E. ( 1 986). Effect of nutritional status, ages at weaning and room temperature on the growth and system immune response on weanling pigs. J Anim. Sci. 63 : I 845 Cromwell, G.L. , Cline, T .R. , Crenshaw, J .D. , Crenshaw, T.D., Ewan R.C. , Hamilton, C .R., Lewis , A.J . , Mahan, D.C. , Mil ler, E.R., Pettigrew, J.E, Tribble, L.F. & Veun, T.L. ( 1 993) . The dietary protein and (or) lysine requirements of barrows and gi lts. J. Ani m Sci. 7 I : I 5 I O- I 5 I 9 Cromwell, T.L. (200 I ) . Antimicrobial and promivrobial agents . In: A.J . Lewis, L .L . Southern (Eds) . Swine Nutrition. CRC Press, Boca Raton, FL. Cromwell, G.L. (2002). Why and how antibiotics are used in swine production. Anim. Biotechnol. I 3 : 7-27 Crouch, S . P. , Slater, K. J . & Fletcher, J . ( 1 992). Regulation of cytokine release from mononuclear cells by the iron -blinding protein Lactoferrin. Blood 80(1 ) : 235 -240 Cullen, S.P. , Monahan, F.J . , Callan, J .J . & O'Doherty (2005). The effects of dietary dietary garl ic and rosemary on grower-finisher pig performance and sensory characteristics of pork. Irish Journal of Agricultural and Food Research 44 :57-67 Cumberbatch, M., Dearman, R.J. , Uribe-Luna, S. , Headon, D.R., Ward, P.P. , Coneely, O.M. & Kimber, I (2000). Regulation of epidermal langerhans cel ls migration by lactoferrin. Immunology, I 00:2 I -28 97 Cummings, J .H . , Antoine, J .M. , Azipiroz, F. , Bourdet-Sicard, R. , Brandtzaeg, P. , Calder, P .C. , Gibson, G.R. , Guaner, F . , Isolauri, E. , Pannemans, D . , Shortt, C . , Tuijtelaars, S . & Watzl, B . (2004) . Passclaim-Gut health and immunity. Eur. J Nutr. 43 : 1 1 8- 1 73 , Suppl . 2 Dallman, P .R. ( 1 986). Biochemicalbasis for the manifestations of iron deficiency. Ann. Rev. Ntri. 6, p 1 3 -44 Daniel , H . , Vohwinkel , M. & Rehner, G. ( 1 990). Effect of casein and b-casomorphin on gastriontestines motility in rats. Journal of Nutrition Research, 1 20 :252-257 . Dantzer, R. & Mormede, P. ( 1 98 1 ) . Influence de mode d 'elevage sur le comporment et l ' activite hypophocorticosurrenalienne dup orcaelet. Reprod. Nutr. Develop. 2 1 :66 1 . Dantzer, R & Mormede, P. ( 1 983) . Stress in farm animals : A need for reevaluation. J. Anim. Sci57 :pp6 Davis, M.E. , Maxwell , C .V. , Brown, D.C, de Rodas, B.Z. , Johnson, Z.B, Kegley, E.B. , Hel lwig, D.H. & Dvorak, R.A. (2002). Effects ofmannan oligosaccharides and (or) pharmacological additions of copper sulphate on growth performance and immunocompetence of weanling and growing/finishing pigs. Journal of Animal Science, 80 :2887-2894. Decuypere, N . Dierick, N. & Boddiez, S . ( 1 998). The potentials for immunostimulatory substances (a- 1 ,3/ 1 ,6 glucans) in pig nutrition. J Anim. Feed. Sci.7 : 529- De Lange, C .F .M. 2000. Characterization of the non-starch polysaccharides (x). In : ' Feed Evaluation, Principles and practice' (ed. P.J . Moughan, M.W.A. Verstegen & M.I . Visser-Reyneveld), Wageningen Pers, The Netherlands. Pages 77-92 Demas, G.E. , Cheffer, V . , Talan, M.I . & Nelson, R.J. ( 1 977). Metabolic costs of mounting an antige-stimulated immune response in adult and aged C57BL/6J mice. Am.J Physiol. 273 : 1 63 1 - Demas, G.E. ; Chefer, V . ; Talan, M. I . ; Nelson, R.J. ( 1 997) : Metabolic cost of mounting an antigen stimulated immune response in adult and aged C57BL/6J mice . Am. J. Physiol. 273 , p l 63 1 . De Petrino, C . , Bianchi-Salvadori, B . , Jiri l lo, E. , Baldinell i , L., DiFabio, S . & Vesely, R. ( 1 989). Yoghurt and the immune response. In: yoghurt Nutritional and Health Properties. (Ed.) Chandan, R.C, John Libbey Euro text, London. pp63-67. De Petrino, S . F. , De Jorrat, M.E.B .B . , Meson, 0. & Perdigon, G. ( 1 995) . Protective · abil ity of certain lactic acid bacteria against an infection with Candida Albicans in a mouse immunosuppressant model by corticoid. Food and Agriculture immunology 7 :365-373 Derenzo, E . & Moss, J . (2006). Writing clinical research protocols . (Eds) Elsevier Academic Press, Burlington, USA De Rodas, B. A. , Gill iland, S .E. & Maxwell , C.V. ( 1 996). Hypercholesterolemia action of Lactobacillus acidophilus ATCC 43 1 2 1 and calcium in swine with hypercholesterolemia induced by diet. Journal of Dairy Science. 79:2 1 2 1 -2 1 28 De Simone, C. , Bianchi, Salvadori, B . , Negri, R., Ferrazzi, M. , Baldinell i , L . & Vesely, R. ( 1 986). The adjuvant effect of yoghurt on production of gamma­ interferon by ConA-stimulate human peripheral blood lymphocytes. Nutrition Reports International 3 3 :4 1 9-433 De Simone, C . , Bianchi-Salvadori, B . , Zanzoglu, S . , Cil l i , A. & Lucci, L . ( 1 987). Enhancement of immune response in murine peyers patches by a diet supplemented with yoghurt. Immunopharmacology and Jmmunotoxicology 9 ( 1 ), 87- 1 00. 98 De Simone, C . , Bianchi, Salvadori, B . , Jiril lo, E . , Baldinelli , L . , Bitonti, F . & Vesely, R. ( 1 989) Modulation of immune activities in human and animals y dietary lactic acid bacteria, In: Yoghurt Nutrition and Health Properties . Ed .R.C. Chandan, John Libbey Eurotext, London, pp.20 1 -2 1 3 De Simone, C . , Rosati, E. , Moretti , S . , Bianchi Salvadori, B . , Vesely, R. & Jiri l lo, E . ( 1 99 1 ) . Probiotics and stimulation of the immune response. Eurotext Journal of Clinical Nutrition 45 (suppl.) . 32-34 De Simone, C . , Ciardi, A. & Grassi, A . ( 1 992) Effect of Bifidobacterium bifidum and Lactobacillus acidophilus on gut mucosa and peripheral blood B Lymphocytes. Immunopharmacology and Immunotoxicology. 1 4 : 33 1 -340. De Simone, C . , Vesely, R., B ianchi Sa1vadori, B. & Jirillo, E. ( 1 993) . The role of Probiotics in modulation of the immune system in the man and in the animals . International Journal of Immunotherapy. 9 :23-28 De Vrese, M., Stegelmennn, A., Richter, B., Fenselua, S . , Laue, C . & Shrezenmier, J. (200 1 ) Probiotics - compensation for lactase insufficient. Am. J . Cl in. Nutr. 73 :42 1 S-429S De Wit, J .N. & Hooydonk, A.C .M. ( 1 996). Structure, function of lactoperoxidase in natural antimicrobial systems. Neth. Milk Dairy J. 50: 227-237 . Doomenbal, H . , Tong, A.K. W. & Sather, A.P. ( 1 986). Relationships among serum characteristics and performance and carcass traits in growing pigs. J. Anim. Sci. 62 : 1 675 Dorhoi, A . , Dobream, V . , Zaham, M. &Virag, P. (2006) . Modulatory effects of several herbal extracts on avian peripheral blood cell immune responses. Phytotherapy Research. 20 :352-3 58 . Dtitz, S . S . , Shi, J . , Kielian, T. L . , Gooodband, R . D . , Nelssen, J . L . , Tokach, M. D . , Chengappa, M . M. , Smith, J . E. & B lecha, F. ( 1 995). Influence of dietary beta­ glucan on growth performance, non-specific immunity, and resistance to streptococcus suis infection in weanling pigs. J. Anim. Sci. 73 :334 1 . Dziarski R. ( 1 99 1 ) Demonstration of peptidoglycan blinding sites on lymphocytes and macrophages by photoaffinity cross-linking. Journal o[Biological Chemistry. 266 :47 1 3 -47 1 8 Easter, R.A ( 1 993). Acidification of diets for pigs. In: Recent developments in pig nutrition. 2 (eds) D.J.A. Cole, W.Haresign & P.C. Guamsworthy. Nottingham University Press, Nottingham, U.K. pp 256-266 Eda, S . , Kikugawa, K. & Beppu. M. ( 1 997). Characterization of Lactoferrin-blinding proteins of human macro phages membrane: multiple species of Lactoferrin­ bl inding proteins with polylactosamine-blinding abi l ity. Biological and Pharmaceutical Bulletin 20(2) : 1 27- 1 3 3 Edde, L. , Hipolito, R.B . , Hwang, F .F .Y. , Headon, D .R., Shalwitz, R.A. & Sherman, M.P. (200 1 ) . Am. J. Physiol. Gastrointest. Liver. Physiol. 28 1 :G 1 1 40-G 1 1 50 Egeli , A.K., Framsted, T . , Moberg, H . , and Tverdal Tvedten, A. ( 1 998). Evaluation of piglet blood, uti lising the Technicon H l . Veterinary Cilnical Pathology 27 (4) : 1 23 :228 Eglinton, B . A. , Roberton, D . M. & Cummins, A.G. ( 1 994). Phenotype ofT cells, their soluble receptor levels, and cytokine profile of human breast milk. Immunology and cell Biology. 72 :306-3 1 3 Eicher, S .D, Morril , J .L. , B lecha, F . , Chitko-McKown, C .G. , Anderson, N.V. & Higgins. ( 1 994). Leukocyte functions of young dairy calves fed milk replacers supplemented with vitamin A and E. J. Dairy Sci. 77 : 1 399- 1 407 99 Ellinger, D.K. , Muller, K.L.D. & Glantz, P.J . ( 1 978). Influence of feeding fermented colostrum and Lactobacillus acidophilus on fecal flora and selected blood parameters of young dairy calves. J Dairy Sci. 6 1 (Suppl. 1 ) : 1 26 Elkin, R.G. , Freed, M.B. , Hamakar, B .R. , Zhang, Y & Parsons, C.M. ( 1 996). Condensed tannins are only partially responsible for variations in nutrient digestibility of sorghum grain cultivars. Journal of Agricultural , Food Chemistry. 44 pp.848-853 . Erikson, K.M. , Beard, J .L. & Connor, J .R. ( 1 998). Distribution of brain iron, ferritin, and transferrin in the 28-day-old piglet. Nutritional Biochemistry 9 :276-284 Esteve-Garcia, E., Brufau, J., Perez-Vendrell , A.Miquel, A. , & Duven, K ( 1 997). Biofficacy of enzyme in preparation containing P-glucanase and xylanase activities in boiler diets based on barley or wheat, in combination with flavomycin. Poult. Sci. 76: 1 728- 1 73 7 Evans, R.J. ( 1 994). Porcine Haematology: Reference ranges and the clinical value of haematological examination in the pig. Ewing, W. N. and Cole, D. J . A . ( 1 994). Practical applications ofmicroorganisms in nutrition. Pages 1 1 3 - 1 1 4 in W. N. Ewing and D. J . A. Cole, eds. The living gut. Redwook, Trowbridge, Wilshrine, UK Fairweather-Tait, S .J . ( 1 986). Iron availability - the implication of shrt term regulation . BNF Nutr Bull. 1 1 : 1 74- 1 80 Fairweather-Tait, S.J . , Balmer, S .E. , Scott, P.H., Ninski, M.J. ( 1 987). Lactoferrin and iron absorption in new born infants . Pediatr Res 22:65 1 -4 Fairweather-Tait, S . J . ( 1 995a) . Iron absorption, In: Iron: nutrition and physiological significance, the report of the Brtish Nutrition Foundation task Force. Chapman Hal l . London. United Kingdom. Fairweather-Tait, S .J . ( 1 995b). Iron biochemistry, In: Iron: nutrition and physiological significance, the report of the British Nutrition Foundation Task Force. Chapman Hall . London. Fernandez, C.F . , McGown, K., Shahani, K.M. ( 1 990), Anticarcinogenic and immunological properties of dietary Lactobacilli. J. Food Prot. 5 3 : 704-7 1 Ferralis, R.P. & Diamond, J .M. ( 1 989). Specific regulation of intestinal nutrient transporters by their dietary substrates . Annual Reviews of Physiology. 5 1 : 1 25 - 1 4 1 . Finegold, S .M. , Attebery, H .R. & Sutter, V. L. ( 1 974), Effect of diet on human fecal flora : Comparison of Japanese and America diets. Amer. J Clin. Nutr. 27 : 1 456- 1 469. Fisher-Boel, M. (2002). Danish Minister of food, Agriculture and Fisheries. Opening speech at the International Symposium "beyond Antimicrobial Growth Promoters." Copenhagen, DK [Online] http :/ /www .foulum.dk/ AGPF AP2002/openingspeach.shtmUApril 2006] . Forbes, J .M. ( 1 995) . Learning about food preferences. In: "Voluntary food intake and selection in farm animals", CAB International, Oxon, U.K. p247-276. Forstner, J.F. & Forstner, G.G. (x). Gastrointestinal mucus, in: Johnson L.R (Ed). Physiology of the gastrointestinal tract, Raven Press, New York, USA, 1 994, pp 1 25 5 - 1 283 Fugh-Berman, A. (2000). Herb-drug interactions. The Lancet. 1 3 5 : 1 34- 1 3 8 Fuller, R. ( 1 989) . Probiotics in man and animals. A review. J Appl. Bacterol. 66: 3 65- 378 1 00 Fuller, R. ( 1 992). History and development of probiotics. The Scientific Basis, (Ful ler, R . , Editor, Chapman & Hall Publishers (London) . Funderburke, D .W. & Seerley, R.W. ( 1 990). The effects of postweaning stressors on pig weight change, blood, l iver and digestive tract characteristics. Journal of Animal Science. 68 : 1 55- 1 62 . Franklin, S .T . , Sorenson, C .E . & Hammel, D.C. ( 1 998). Influence of vitamin A supplementation in milk on growth, Health, concentration of vitamins in plasma, and immune parameters of calves. J Dairy Sci. 8 1 :2623-2632 Fraser, A. C. 1 93 8 . A study of the blood ofpigs. Vet. J. 94 :3 . Fraser, D . , Feddes, J . J.R. & Pajor, E.A. ( 1 994). The relationship between creep feeding behaviour of piglets and adaptation to weaning: Effect of diet quality . Canadian Journal of Animal Science. 74 : 1 -6 Friendship, R.M, Lumsden, J.H, McMil lan, I . & Wilson, M.R. ( 1 984). Haematology and biochemistry reference values for Ontario swine. Can. J Camp. Med. 48 : 390- 393 . Gabert, V .M . & Sauer, W.C. ( 1 994) . The effects of supplementing diets for weanling pigs with organic acids. A review. Journal of Animal and Feed Sciences, 3 , 1 994, 73-87 . Gaines, A.M., Carrol l , J .A, Allee, G.L. & Yi, G.F . (2002). Pre- and postweaning performance of pig's inject3ed with dexamethasone at birth. JAnim. Sci. 80 :2255- 2262. Gala, R. R. & Shevach, E. M. ( 1 993a) Influences of prolactin and growth hormone on the activation of dwarf mouse lymphocytes in vivo. Proceeding of the Society for Experimental Biology and Medicine. 205 :224-230 Gala, R. R. & Shevach, E. M. ( 1 993b ) . influence of bromocriptine administration to mother on the development of pupthymocyte and splenocyte subset and on mitogen-induced proliferation in the mouse. Life Science. 5 3 : 1 98 1 - 1 994. Gala, R. R. & Shevach, E. M. ( 1 994) Evidence for the release of a prolactic-like substance by mouse lymphocytes and macrophages. Proceeding of society for Experimental Biology and Medicine. 53 : 1 98 1 - 1 994 Gaudichon, C . , Ross, N. , Mahe, S . , Sick, H . , Bouley, C. & Tome, D. ( 1 994a) Gastric emptying regulates the kinetics of nitrogen absorption from1 5N-label led milk and 1 5N-labelled yogurt in miniature pigs. Journal of Nutrition. 205 : 1 2- 1 9 Gaudichon, C . , Laurent, C. , Mahe, S . , Marks, L . , Tome, D . & Krempf, M . ( 1 994b) Rate of 1 5N-leuncine enrichment and nitrogen characterization from gastro­ duodenal secretion in fasting human. Reproduction Nutrition Development. 205 : 1 970- 1 977 Gaudichon, C., Laurent, C. , Mahe, S. , Ross N. , Benamouzig, R., Luengo, C . , Huneau, J-F. , Sick, H. , Bouley, C. , Rautureau, J. & Tome, D . ( l 995) Exogenous and endogenous nitrogen flow rates and level of protein hydrolysis in the human j ejunum after l 5N-labelled milk and 1 5N-labelled yogurt ingestion. British Journal ofNutrition. 34 :349-3 59 Gaudichon, C . , Mahe, S. , Luengo, C., Laurent C., Meaugeais, M., Krempf, M, & Tome, D . ( 1 996) A 1 5N-jeucine-dilution method to measure endogenous contribution to luminal nitrogen in the human upper jejunum. European journal of Clinical Nutrition. 74:25 1 -260 Gaudichon, C . , Mahe, S . , Luengo, C., Laurent, C. & Meaugeais (x), method to measure endogenous contribution to luminal nitrogen in human upper j ejunum. European Journal ofClinical Nutrition. 50 :26 1 -268 1 0 1 Gehle, M. H . , L. C . Payne, E. R. Peo, Jr. and C. L . , Marsh. ( 1 96 1 ). Interrelationship of growth rate, Hemoglobin dilution, packed cell volume and incidence of anemia in suckling pigs. J Am. Vet. Med.Assn. I 3 8 : 8 1 Gibbons, R. , Etherden, I . & Skobe, Z. ( 1 983) . Association of fimbrae with the hydrophobicity of Streptococcu sanguis FC- 1 andnadherence to salivary pellicles. Infect. Jmmuno. 4 1 :4 1 4-4 1 7 . Gillil and, S .E . & Speck, M.L. ( 1 977a) . Antagonistic action of Lactobacillus acidophilus towards intestinal and food borne pathogens in associative cultures. Journal offood Protection 40 ( 1 2): 820- 823 Gillil and S .E . , Nelson C.R., Maxwell C., 1 995 . Assimilation of cholesterol by Lactobacillus acidophilus. Appl. Environ. Microbio/.49, 377-3 8 1 Goransson, L . (200 I ) . Alternatives to antibiotics-the influence of new feeding strategies for pigs on biology and performance. In: Recent developments in pig nutrition. 3 (eds) J. Wiseman & P.C. Guarnsworthy. Nottingham University Press, Nottingham, U .K. pp 39-50 Graham, P.L. , Mahan, D.C. & Shields, Jr. , R.G ( 1 98 I ). Effects of starter diet and length of feeding regime on performance and digestive enzyme activity of 2-week old weaned pigs. J.Anim. Sci. 53 :299-307. Granick, S . ( 1 946) . Ferritin : IX. Increase of the protein apoferritin in the gastro­ intestines mucosa as a direct response to iron feeding. The function of ferritin in the regulation of iron absorption. J. Bioi. Chem . . I 64 :737 Grela, E.R. , Krusink, R. , & Matras, J . ( 1 998). Efficacy of diets with antibiotics and herb mixture additives in feeding of growing-finishing pigs. Journal of Animal and Feed Sciences, 7 : I 7 1 - 1 75 . Grove, D . S . , Bour, B . , Kacsoh, B . & Mastro, A . M. ( 1 99 1 ) . Effect of neonatal milk­ prolactic deprivation on the ontogeny of the immune system of rat. Endocrine Regulation. 25 : I 1 I - I I 9 . Gwatkin, R.C. & L' Ecuyer, C. ( 1 960) . Rhinitis of swine. IV. Hematology of infected and normal pigs. J Comp. Med. 24 :6 . Halmilton-Miller, J .M.T. ( 1 996), Probiotics-panacea or nostrum? BNF Nutr. Bull. 2 1 : I 99-208 Halpern, G.M., Vruwink, K.G. , Van de Water, J., Keen, C .L. & Gershwin, M .E. ( 1 99 I ) . Influence of long-term yogurt consumption in adults. Internal. J. Jmminotherapy 7, 200-2 I 0 Hamosh, M. ( 1 994 ). Digestion in the premature infant: the effect of human milk. Seminar in Perinatology. 1 8 : 485-494 Hamosh, M. ( 1 997). Should infant formulas be supplemented with bioactive components and conditionally essential nutrients present in human milk? Journal ofNutrition. 1 27 :97 1 S-974S Hampton, D .J. ( 1 986). Attempts to modify changes in the piglet small intestine after weaning. Res. Vet. Sci. 40 :3 1 3- Hansson, L . , Blackberg, I . Edlund, M. , Lundberg, I . , Stromqvist, M. & Herneil, 0 . ( 1 994). Recombination , human, milk bile salt-stimulated l ipase Catalytic activity is retained in the absence of glycosylation and unique proline- rich repeats. Journal of Biological Chemistry.268 : 26,692-26,698 . Hara. H . , Fuj ibayashi, A. & Kiriyama, S . ( 1 992). Pancreatic protease secretion profiles after spontaneous feeding of casein or soybean protein diet in unrestrained conscious rats. Journal of Biochemistry. 3 : 1 76- 1 8 1 Hardy, B . ( 1 999). A world without growth promoters. Concepts Pig Sci. The first annual Turtle Lake Science Conf., Nottingham, U.K. 1 02 Haro A, Lopez-Aiiaga I, Lisbona F, et al (x) . Hematological findings in cats naturally infected with fel ine immunodeficiency virus . Comparative heamatology international Harper A.F . , Kornegay E.T. , Bryantt K.L. , Thomas H.R. , ( 1 983). Efficacy of virginiamycin and a commercially-available lactobacillus probiotics in swine diets. Anim. Feed Sci Tech. 8 :69-76 Hart, E .B. , C. A. Elvehjem, H. Steenbock, A. R. Kummerer, G. Bohstedt & J. M. Far go. ( 1 930). A study of the anemia of young pigs and its pre-vention. J. Nutr. 2 :227 Hartman. D. P . , Holiday, J . W. & Bernton, E. W. ( 1 989). Inhibition of lymphocyte proliferation by antibodies to prolactin. Fraseb Journal. 3 : 2 1 94-2202. Hashimoto, S., Nomoto, K. , Matsuzaki, T. , Yokokura, T. & Mutai, M. ( 1 984) . Oxygen radicle production by peritoneal macrophages and kupffer cel ls elicited with Lactobaci llus casei . Infect. Jmmun.44, 6 1 -67. Hashizume, S . , Kuroda, K. & Murakami, K. ( 1 983) . Identification of Lactoferrin as an essential growth factor for human lymphocyte cell lines in serum-free medium. Biochimica Biophysica Act. 763 :377-382 . Hayashida, K . , Kaneko, T . , Takeuchi, T . , Shimizu, H . , Ando K . & Hrada, E . (2004). Oral administration of lactoferrin inhibits inflammation and nociception in rat adjuvant-induced aththritis. J. Vet. Med. Sci. 66 (2) : 1 49- 1 54 Haydon, K.D. , West, J .W. & McCarter, M.N. ( 1 990). Effect of dietary electrolyte balance on performance and blood parameters of growing-finishing swine in high ambient temperature. J. Anim. Sci . , 68 : 2400-2406 Hays V. W. ( 1 978). The role of antibiotics in efficient livestock production. In : 'Nutrition and Drug Interrelation' , Academic press, New York, pages 545-575 Heird, W.C. , Schwarz, S .M. , & Hansen, I .H . ( 1 984. Colostrum-induced enteric mucosal growth in beagle puppies . . Pediatr Res. 1 8 : 5 1 2-5 . Henschen, A. , Lottspeich, F. Brantl , V. & Techemacher, H. ( 1 979) Novel opioid peptides derived from casein (B-casomorphins) 11. Structure of active component from bovine casein peptone. Hoppe-seyler 's Zeirshrift fur Physiologishe Hill , GM, Cromwell , G .L. , Crenshaw, T.D, Dove, C.R. , Ewan, R.C. , Knabe, D .A. , Lewis, A .J . Libal, D.C. , Mahan, D.C. , Shurson, G.C. , Southern, L .L . & Veun, T.L. (2000). Growth promotion effects and plasma changes from feeding high dietary concentration of zinc and copper to weanling pigs (regional study). Journal of Animal Science. 78 : 1 0 1 0- 1 0 1 6 Hindle VA, Mathij ssen-kamman AA, Stockhofe N , et al . (x) References values of heamatology and blood chemistry parameters in fatting pigs of different bodyweight. Schweizer Archiv Fur Tieheilkunde 1 43 Hiss, S & Sauerwein, H . (2003). Influence of dietary B-glucan on growth performance, lymphocyte proliferation, specific immune response and haptoglobin plasma concentration in pigs. J.Anim. Physiol. A. Anim. Nutrition. 87 :2- 1 1 . Hodge, R.M.W. ( 1 974). Efficiency of food conversation and body composition of the preruminant lamb and the young pig. British Journal of Nutrition 32 : 1 33 - 1 1 26 . Holmgren, N. ( 1 996). Polyarthritis in piglets caused by iron dextran. Bologna, Proc IPVS Congress, p .306 Hong, J. W., Kin, I .H . , Kwon, O.S . , Kim, J .H . , Min, B.J . & Lee, W.B . (2002). Effect of dietary Probiotics supplementation on growing performance and fecal gas emission in nursing and finishing pigs. J. Anim. Sci. Technol. (Kor.) 44 :305-3 1 4 1 03 Hong, H.A. , Due, L.H. & Cutting, S .M.(2005). The use of bacterial spores as probitics .FEMS Microbial Reviews 29, 8 1 3-835 Horton, G.M. J . , Blethen, D .B . & Prasad, B .M. ( 1 99 1 ) . The effect of garlic (Allium sativum) on feed palabil ity of horses and feed consumption, seklected performance and blood parameters in sheep and swine. Can. Anim. Sci : 7 1 :607- 6 1 0 . Horton, G.M. J . , Fennel, M.J . & Prasad, B .M. ( 1 99 1 ) . Effect of garlic (All ium sativum) on performance, carcass composition and blood chemistry changes in broiler chickens. Can. J. Anim. Sci : 7 1 :939-942. Howie, P . W., Forsyth, J. S . Ogaston, S . A. , Clark, A. & Forey, V. C . ( 1 990) Protective effect of breast feeding against infection. British Medical Journal 300: 1 1 - 1 6 Hu, W. L. , Mazuroier, J . , Sawatzki, G. , Montreui l, J, & Spik, G.( 1 988) Lactoferrin receptor of mouse small intestinal brush border. Biochemical Journal 248 :435- 44 l . Inatani, R, Nakatani, N & Fuwa, H . ( 1 983). Antioxidative effe4ct of the constituents of rosemary (Rosmarinus officinahs L.) and their derivatives. Agriculture and Biological Chemistry 47 :52 1 -528 . Iyer, S . & Lonnerdal, B . ( 1 993) Lactoferrin, Lactoferrin receptors and iron metabol ism. European Journal of Clinical Nutrition. 4 7 :232-24 1 Jacobson, M. , Fel lstrom, C. , Lindburg, R., Wallgren, P & Jensen-Waern (2004). Experimental swine dysentery: comparison between infection models Jansman, A.J .M. ( 1 993). Tannins in feedstuffs for simple-stomached animal. Nutrition Research Reviews 6:209-236 Jansman, A, Enting, M.W.A. , Vertsgen, J . , Husman, J .W.O. & van den Berg ( 1 995) . Effects of hul ls of faba beans ( Viciafaba) with a low or high content of condensed tannis on apparent ileal and faecal digestibility and the excretion of endogenous protein in ileal digesta and faeces of pigs. Journal of Animal Science. 73 , 1 1 8- 1 27 Janz, J .A.M. , Morel, P .H.C. , Wilkinson, B.H.P. , & Purchas, R.W. (2007). Preliminary investigation of the effects of low-level dietary inclusion of fragrant essential oils and oleoresins on pig performance and pork quality. Meat Science, 75 :3 50-3 5 5 Jensen, B .B . ( 1 998). The impact of feed additives on microbial ecology of the gut in young pigs. Journal of Animal and feed Sciences, 70:45 -64 Jensen, B .B . & Jensen, M.T. ( 1 998). Microbial production of skatole in the digestive tract of the entire male pigs. In: W.K. Jensen, ( ed). Skatole and Boar taint, p4 1 -76. Jensen, B .B . , Mikkelsen, L. & Christensen, D.N. ( 1 998). Integration of i leum fistulated pigs and in vitro fermentation to quantify the effect of diet on composition of microbial fermentation in the large intestine. In : H . Jorgensen, J .A.Fernadez (eds). Proceeddings ofNJF Seminer No 274 on energy and protein evaluation for the pigs in the Nordic countries. NJF report No. 1 1 9, p 1 06- 1 1 0 . Johansson, A. , Pielberg, G. Anderson, L . & Edfors-Lilja, I . (2005) . Polymorphism at the porcine dorminat white I KIT locus influence coat colour and peripheral blood cell measures. International Society for Animal Genetics, Animal Genetics, 36 :288-296. Jonasson, R. , Johannisson, A . , Jacobson, M. , Fellstorom, C . & Jensen-Waern, M. (2004). Differences in lymphocyte subpopulations and cell counts before and after experimentally induced swine dysentery. Journal of Medical Microbiology, 5 3 :67- 272. 1 04 Jonsson, E. & Conway, P . ( 1 992). Probiotics for pigs. In : (Ed. ) R. Fuller. Probiotics : The Scientific Basis. Chapman and Hall , London. pp. 260-3 1 6 . Kang B.P . B (2000). Hyperlipidermia and type 1 5 '-monodeiodinse activity: regulation by selenium supplementation in rabbits. Ansa] M.P. , Mehta U. Bio. Tr. Elem.Res . 77, 23 1 -239 Kantha et al. ( 1 992). Effects of beta-carotene and retinol repletion on antibody production in vitamin A-deficient rats immunized with pneumonococca1 polysaccharide. Nutr. Res. 1 2 : 1 009- 1 024 Keating K. I . , Caffrey P .B . ( 1 989). Selenium deficiency induced by zinc depreviation in crus. Proc. Nat. Acad. Sci. USA 86, 6436-6440 Kegley, E .B, Spears, J .W. & Auman, S.K. (200 1 ) . Dietary phosphorus and an inflammatory challenge affect performance and immune function of weanling pig. J. Anim. Sci. 79:4 1 3 -4 1 9 Keith M. Erikson, John L . Beard & James R. Connor (x). Welfare and production implication of teeth clipping and iron injection of piglets in outdoor system in Scotland H. C. H. Kemkamp, A. J. Clawson & R. H. Femeyhough. Preventing iron-deficiency anemia in baby pig. Kelly, D. ( 1 998). Probiotics in young and newborn animals. J. Anim. Sci. 7 : 1 5 -23 Kelly, D . , Smyth, J .A. , & McCracken, K.J. ( 1 99 1 ) . Digestive development of early weaned pig. 2 Effect of level of feed intake on digestive enzyme activity during the immediate post-weaning period. Br. J. Nutr. 65 : 1 8 1 - 1 88 . Kendiah, G . ( 1 999). Comparison of the passive prophylactic effect of bovine milk immunoglobulin fed as a bolus or continuously against diarrhoea caused by Escherichia coli K88 using piglets as models . MSc Thesis, Massey University, Palmerston North, New Zealand. Kernkamp, H .C.H, Clawson, A.J. & Femeyhough, R.H. ( 1 959). Preventing iron­ deficiency anemia in baby pig. Kim, I . H . , Hancock, J .D. , Hines, R.H. & Risley, C.R. ( 1 993). Effect of cellulose and bacteria feed addictive on the nutrition value of sorghum grain for finishing pigs. Kansas Agric. Exp. Sta. Rep. Prog. No. 695 : 1 44 . Kimoto H . , Ohmono S . , Okamto T . (2002). Cholesterol removal from media by lactococci . J. Dairy Sci. 85 , 3 1 82-3 1 88 Kin, R & C . L ' Ecuyer. ( 1 960). Rhinitis of swine IV. Hematology of infected and normal pigs. Can. J. Camp. Med. 24 :6 . King, J . W.B. ( 1 97 1 ). The interaction of genotype and environment in pig production. Nottingham Univ. Easter School Agric. Sci . Programs. 1 8 : 2 1 -36 Kirk, J .H . and Bulgin, MS. ( 1 979). Effects of feeding cull domestic onions (Allium cepa) to sheep. Am. J. Vet Res 40, 397-399 Klasing, K. C . &.Austric.R.E. ( 1 984a) . Changes in plasma, Tissue and urinary nitrogen metabolites due to inflammatory challenge. Proc. Soc. Exp. Bioi. Med. 1 76 :276 Klasing, K. C . & Austric. R.E. ( 1 984b ) . Changes in protein synthesis due to an inflammatory chal lenge. Proc. Soc. Exp. Bioi. Med. 1 76:285 Klasing, K . C . , Laurin, D .E, Peng, R.K. & Fry, D.M. ( 1 987). Immunologically mediated growth depression in chicks: Influences of feed intake, Corticosterone and interleukin- 1 . J. Nutr. 1 1 7 : 1 629. Klasing, K. C . ( 1 988) . Nutrition aspect of leukockines. J. Nutr. 1 1 8 : 1 436 Klasing, K. C . & D . M. Barnes. ( 1 988). Decreased amino acid requirement of growth chicks due to immunologic stress. J. Nutr. 1 1 8 : 1 1 58 Klasing, K. C . , & Roura, E. ( 1 99 1 ). Interactions between nutrition and immunity in chickens. Proc. Cornell Nutr. Cof. P94. 1 05 Kleimbeck, S . & McGlone, J.J . ( 1 999). Intensive indoor vs outdoor swine production systems: Genotype and supplemental iron effects on blood haemoglobin and selected immnune measures in young pigs. J Anim. Sci. 2384-2390. Kluber, E. F . , Pollmann, D . S .S . & Blecha, F. ( 1 985) . Effect of feeding Streptococcus faecium to artificially reared pigs on growth, hematology and cell-mediated immunity. Nutr. Rep. Int. 32 :57 Kluge, H. , Broz, J . & Eder, K . (2006). Effects of benzoic acid on growth performance, nutrient digestibility, nitrogen balance, gastrointestinal microflora and paramterters of microbial metabolism in pigs. Journal of Animal Physiology an Animal nutrition 90 :3 1 6-324 . Knol l , J .S . & Rowel l SL. (x). Changes in Hematological and clinicochemical profiles in blood of apparently healthy slaughter healthy slaughter, in relation to the severity of Pathological-Anatomical lesions. Veterinary Quarterly 1 3 Knudsen, K.E. (200 1 ) . The nutritional significance of dietary fibre analysis . Anim. Feed Sci. Technol. 90 :3 -20 Kohno, Y. , Shisaki, K., Mura, T. & Ikawa, S. ( 1 993) Iron-saturated lactoferrin as a comitogenic substance for neonatal rat hepatocytes in primary culture. Acta Paediatrica 83 :650-655 Konstantinow, C.R. , Favier C.F . , Zhu W.Y., Williams, B.A. , Klub, J . , Souffrant W.­ B . , de V os, W.M. , Akkermans, A.D.L. & Smidt H . (2004). Microbial diversity studies of the porcine gastrointestinal ecosystem. Anim.Res.53 :3 1 7-324 Korim P., Bugarsky A . , Juris P., Hadbavhy M., Korimova J . (2003). Protection of experimental animals (in Slovak). Slov. Vet. Cas.28 ( 1 ), I O- I 2 Kornergy, E.T., Tinsley, S .E. & Bryantt, K.L. ( 1 979) . Evaluation of rearing systems of feed flavours for pigs weaned at two or three weeks of age. Journal of Animal Science. 48 :999. Kornegay, E. T. & Risley, C.R. ( 1 996) . Nutrients digestibility of a corm-soybean meal diet as influence by Bacil lus products fed to finishing swing. J. Anim. Sci. 74 :799- 805 . Lalles, J .-P. , Boudry, G . , Favier. C . , Le Floc 'h, N. , Luron, I . , Montagne, L . , Oswald , I .P . , Pie, S . , Pie!, C . & Seve (2004).Gut function and dysfunction in young pigs: microbiology, Anim. Res .53 :30 1 -3 I 6 Langhans, W. & Hrupka, B . ( 1 999). Interleukins and tumor necrosis factor as inhibitors of feed intake. Neuropeptides. 33 (5) :4 1 5 . La Ragione R.M., Casula G. , Cutting S .M. , Woodward M.J (200 1 ). Bacillus subtilis spores competitive exclude E. coli 078:K80 in poultry. Vet. Microbial. 79: 1 33 - I 42 Larson, L.L. , Owen, F .G . , Albright, .J .L. , Appleman, R.D. , Lamb, R.C . , & Muller, L .D ( 1 977). Guidel ines toward more uniformity in measuring and reporting calf experimental data. J Dairy Sci 60:989-99 1 , Langhout, D. J . , Schutte, J .B . , de j ong, J . , Sloetjes, Verstegen, M.W.A. & Tamminga, S. (2000). Effect of viscosity on digestion of nutrients in convectional and germ­ free chicks. Br. J Nutr. 83 :533-540 Lecce, L.G. & King, M .W(x). Role of reo-like virus (rotavirus) in weanling diarrhoea of pigs. Journal of Clinical Microbiology. 8 :454- LeMieux, F. M. , Southern, L.L. & Bidner, T.D. (200 I ) . Effect of a monnan oligosaccharide on the growth of nursery pigs. J Anim. Sci. 79 (suppl . 2) :72 (abstr.) . 1 06 Lessard M. , Yang W.C., Elliot G.S . , Rebar A.H. , Van Vleet J.F., Deslauriers N . , Brisson G.J . , Schultz R .D . ( 1 99 1 ) . Cellular immune responses in pigs fed a vitamin E and selenium-deficient diet. J Anim. Sci. 69. 1 575- 1 5 82 Levy, S.B ( 1 998). The chal lenge of antibiotic resistance. Sci. Am. 278 :46-53 Lewis, S.J . & Freedman, A.R. ( 1 998). Review article : the use of biotherapeutic agents in the prevention and treatment of gastrointestinal diseases. Aliment Pharmacal Ther 1 998; 1 2 :807-822 Lil, D. Y. , Tian, S .J .Z. , Kin, B.J. , Kim, J.H., Kim, K.S. & Kim, Y.Y. (2004) . Effect of continuous feeding of Probiotics on growth performance, nutrient digestibility, blood urea nitrogen and immune response in pigs. J Anim. Sci. Technol. (Kor.) 46 : 39-48 Lim, H. S., Kim, B .H . & Paik, I .K. (2004). Effects of natufermen supplement to the diet on the performance ofweanling pigs. J Anim. Sci. Technol. (Kor.) 46 :98 1 - 988. Lin , C.F. , Gray, J .J . Ashgar, A. , Buckley, D.J . , Booren, A.M & Flega, C .F . ( 1 989). Effects of dietary oils and (a-tocopheral supplementation on lipid composition and stability of broiler meat. Journal of Food Science, 54 : 1 457- 1 460 Lin, H. (2000) . Studies on the protection role of probiotics abd milk calcium on Salmonella typhimurium infection in mice. MSc Thesis, Massey University, Palmerston North, New Zealand Lin, 1 . , Lin, E .C . , Yu, I .T. , Liu, H.T. , Yang, I .S . , Huang, C.H. (2002). Effect of probiotic supplementation on growth performance, serum cholesterol and triglyceride, immune response and fecal bacteria in early weaned piglets. Agri. Assn. China 9 1 , 3 (4) 325-336 Lindemsnn, M.D. , Wood, C.M. , Harper, A.F, Kornegay, E.T. & . Anderson, A (x). Dietary Chromium Picolinate Additions Improve Gain: Feed and Carcass Characteristics in Growing-Fishing Pig and Increase Litter size in Reproduction sows. Department of Animal and Poultry Sciences, Virginia Polytechnic Institute and State University, Black 2406 1 Linder, M.C. Zerounian, N.R. , Moriya, M. & Malpe, R. (2003). Iron and copper homeostasis and intestinal absorption using the CaC02 cell model . Biometals. 1 6 ( 1 ) : 1 45- 1 60 Link, R., Kovac, G. , Pistl , J . , 2005 . A note on probiotics as an alternative for antibiotics in pigs. Journal of Animal and Feed Sciences, 1 4, 5 1 3-5 1 9 Lokaj V. , Oburkova P. , 1 975 . Determination of the tetrazolium-reductase activity of leukocyte. lmunol. Zprav. 6 :42-44 Lombardi, V.R.M. , Fernandez-Nova, L., Etcheverria, 1 . , Seoane, S. & Cacabelos, R. (2005). Studies on immunological , biochemical, haematological and growth regulation by Scomber scombrus fish protein extract supplementation in young pigs. Animal Science Journal. 76: 1 59- 1 70 Lonnerdal , B . & Iyer, S . ( 1 995). Lactoferrin molecular structure and biological function. Ann. Rev. Nutr. 1 5 :93- 1 1 0. Lonnerdal , B . (2003) . Nutritional and psychological significance of human milk proteins . Am. J Clin. Nutr. 77 (suppl .) : 1 537S- 1 547S. Lopez, 1 . (2000). Probiotics in animal nutrition. Asian-Aus. J. Anim. Sci . 1 3 : 1 2-26. Lopez-Pedrosa, J .M. , Torres, M.l. , Fernandez, M . I . , Rios, A & Gill , A. ( 1 998) . Severe malnutrition alters lipids composition and fatty acid profile of the small intestines in newborn piglets, J.Nutr. 1 28 : 224-233 1 07 Loughmil ler, J. (2000). Effects of an acute Salmonella trphimurium or Actinobacillus pleuropneumoniae infection and an evaluation of infrared thermography uses in growing pigs. Ph. D. dissertation, Kansas State University, Manhattan Mahan, D.C. & Seif, L.J. ( 1 983) . Efficacy of vitamin C supplementation for weanling swine. Journal of Animal Science, 56 :63 1 -639. Mahan, D.C. & Lepine, A.J. ( 1 99 1 ) . Effect of pig weaning weight and associated nursery feeding programs on subsequent performance to 1 05kg body weight. Journal of Animal Science. 69: 1 3 70- 1 378 Mahe, S. , Tome, D. , Dunontier, A.-M. & Desj eux, J. -F. ( 1 989) Absorption of intact morphiceptin by DFP-treated rabbits i leum. Peptides 1 0 :45-52. Mahe, S. , Marteau, P . , Huneau, J .-F. , Thruillier, F. & Tome, D. ( 1 993) Intestinal nitrogen and electrolyte movement fol lowing fermented milk ingestion in human. British Journal of Nutrition 7 1 : 1 69- 1 80. Maher, S . , Ross, N. , Benamouzig, R., Davin, L. , Luengo, C . , Gagnon, L . , Gausseres, N . , Rautureau, J. & Tome, D. ( 1 996) Gastrojejunal kinetics and the digestion of �­ lactogoslobulin and casein in the human: influence of the nature and quantity of the protein. American Journal of Clinical Nutrition 63 : 546-552 . Mal in , M. , Suomalainem, H. , Saxelin, M. & Isolauri, E. ( 1 996). Promotion of IgA immune response in patients with Crohn' s disease by oral bacteriotherapy with Lactobacillus GG. Annals. Nutr. Metab. 40: 1 3 7- 1 45 Maner, J . H . , Pond, W.G. & Lowrey, R.S. ( 1 959). Effect and method and level of iron administration on growth, hemoglobin and hematocrit of suckling pigs. J. Animal Sci. l8 : 1 3 73 . Marin, D.E. , Taranu, I . , Bunaciu, R.P. , Pascale, F. , Tudor, D.S , Avram, N. , Sarca, M, Cureu, I . , Criste, R.D. , Suta, V. & Oswald, I .P . (2002) .Changes in performance, blood parameters, numoral and cellula immune responses in weanling piglets exposed to low doses of aflatoxin. Journal of Animal Science . 80, 1 250- 1 257 . Mateo, R . D . , Morrow, J . L. , Da i l ey, J . W . , J i , F . & K im , S . W. (2006) . Use of 5-aminolevulinic acid in swine diet: effect on growth performance, behavioral characteristics and hematological/immune status in nursery pigs. Asian­ Australasian Journal of Animal Sciences 1 9 ( 1 ), 97- 1 0 1 Maxwell , C . V. , Buchanan, D .S . , Owens, F.N. , Gilli land, S .E. , Luce, W.G. & Vencl . , R . ( 1 983 ). Effect of Pro biotic supplement on performance, fecal parameters and digestibil ity in growing-finishing swine. Oklahoma Agric. Exp. Sta. Anim. Sci. Res. Rep. pp. 1 1 4 : 1 57 . Mazurier, J . , Legrand, D . , Hu, W. L. , Montreuil , J . & Spik, G. ( 1 989) Expression of human Lactoferrin receptor in phytohemagglutinin- stimulated human peripheral blood lymphocytes. European Journal of Biochemistry, 1 79:48 1 -497. McAbee, D. D. & Ling, Y. Y. C 1 997) Iron-loading of cultured adult rat hepatocytes reversibility enhances Lactoferrin binding and endocytosis . Journal of Cellular Physiology 1 7 1 :75-86. McCance, R. A & E. M. Widdowson. 1 935 . Phytin in human nutrition. Biochem. J. 29B :2694. McCance, R. A & E. M. Widdowson. 1 944. Activity of the phytase in different cereals and its resistance to dry heat. Nature (Lond.) 1 53 :650 McDonald, P. , Edwards, R.A. , Greenhalgh, J .F.D. & Morgan, C.A. (2002) . Animal nutrition. (eds) Ashford Colour Press Ltd. , Gosport, U.K. Melior, S . (2000). Herbs and spices promote health and growth. Pigs Process 1 6 :27- 30 1 08 Mikkelson, L. L. & Jensen, B . B . ( 1 997). Effect of fermented liquid feed (FLF) on the growth performance and microbial activity in the gastrointestinal tract of weaned piglets. Pages 639-642 in J. P. LaPlace and Fevrier C. Barbeau, (Eds) . Digestive physiology in pigs. INRA, Paris, France. Mil ler, E. R. , Ul lrey, D.E, Ackerman, I . , Schmidt, D .A. , Hoefer, J .A. & Luecke, R.W. ( 1 96 1 a) . Swine hematology from birth to maturity 1 . Serum proteins. J Animal Sci. 20:3 1 -3 5 . Miller, E . R., Ul lrey, D.E . , Ackerman, I . , Schmist. D .A. , Hoefer, J .A. & Luecke, R .W ( 1 96 1 b) . Swine hematology from birth to maturity. I I . Erythrocyte population, size and hemoglobin concentration. J Animal Sci. 20 : 890-897. Miller, E.R. & Ullrey, D.E. ( 1 977). Baby pig anaemia. Pork Ind. Handb. 34 : 1 -4. Miller, E.R, Harmon, B.G., Ul lrey, D.E. , Schmidt, D.A., Luecke, R.W. & Hoefer, J .A. ( 1 962). Antibody absorption, retention and production by the baby pig. J Anim. Sci. 2 1 :309-3 1 4. Mishra, C. & Lambert, 1 . ( 1 996). Production of anti-microbial substances by Probiotics. Asia Pacific. J Clin. Nutr. 5 : 20-24 Mogensen, G. , Salminen, S . , O 'Brien, J . , Ouwehand, A.C. , Holzapfel , W., Shortt, C . , Fonden, R., Miler, G. H . , Donohue, D . , Playne, M.J . , Crittenden, R.G. , Bianchi, Salvador, B . (2002). Inventory of micro organisms with a document history of use in foods. Bulletin of the International Dairy Federation. Moore, A.S . , Gonyou, H.W., Stooky, J .M & McLaren. ( 1 994). Effects of group composition and pen size on behaviour, productivity and immune response of growing pigs. Applied Animal Behavioural Science 40, 1 3 -30 Morrow-Tesch, J & Anderson, G ( 1 994). Immunnological and haematological characterizations of the waisting pig syndrome. Journal of Animal Science, 72 ( 4) : 976- 1 994 Mosmann, T. R. & Coffman, R. L. ( 1 989) Th 1 and Th2 cells: different patterns of Lympokine secretion lead to different properties. Annual Reviews in Immunology 7: 1 45- 1 73 . Mosson, P . (200 1 ) . Probiotics and prebiotics. Pharm. J. 266 : 1 1 8 Muralidhara, K .S . , Sheffeby, G.G. , Ell iker, et al. ( 1 977). Effects of feeding Lactobacilli on the coliform and Lactobaci l l is flora of intestinal tissue and feces from piglets. J. Food Protec. 40288-296 Muri, C . , Schottstedt, T., Hammon, H .M. , Meyer, E. & Blun, 1. W (2005). Haematological, metabolic , and endocrine effects of feeding vitamin A and lactoferrin in neonatal calves. Journal of Dairy Science. 88 : 1 062- 1 077. Mutai, M. , Terashima, T . , Takashi, T. , Tanka, R., Kuroda, A., Ueyama, S . & Matsumoto, K. ( 1 980). Composition for promoting growth of Bifidobacteria and the method for manufacture thereof. · Mroz, Z., Derkker, R.A. , Koopmans, S .J . & Le Huerou-luron, I . (2003) . Performance, functional features of the digestion tract and haematological indices in weaned piglets fed antibiotic free diet and exposed to a viro-bacteria infection, in: Ball R.O. (Ed), Proceeding of the 9th international symposium on digestive physiology in pigs, Banff, AB, Canada, pp 1 80- 1 82 Nader de Macias, M.E. , Apella, M.C. , Romeo, N.C Gonzalez, S.N & Oliver, G ( 1 992). Inhibition of Shigella sonnei by Lactobaci l lus casei and Lactobacillus acidophilus . Journal of Applied Bacterialogy, 73 407-4 1 1 . Nadkarni, K.M. & Nadkarni, A.K. ( 1 976). Indian Material Medica. (eds) Popular Prakashan, Mumbai, India. 1 09 Naylor, A.J . , Choct, M . , Jacques, K.A. (2000). Effects of selenium source and level on performance and meat quality in male broilers . Poultry Sci. 79, Suppl . 1 , 1 1 7 (Abstr.) Neefjes J ., Ndubisi A. & Norburg F. ( 1 993) : cell biology of antigens presentation. Current Opinion in Immunology 5 :27-3 1 . Newby, T.J . Mil ler, B .G . , Stokes, C.R. , & Boume, F.J . ( 1 983) . Hypersensitivity to dietary antigens as predisposing factors in postweaning diarrhoea. Pig Vet. Soc. Proc. I 0 :50- Nichols, B .L . , Henry, J .F . & Putman, M. , ( 1 987). Human lactoferrin stimulates thydimine incorporation into DNA of rat crypt cells . Pediatr Res 2 1 : 563-7 Noh, D .O . , Kim, S . H., Gil l i land, S .E . ( 1 997). Incorporation of cholesterol into the cellular membrane of Lactobacillus acidophilus A TCC 43 1 2 1 . J Dairy Sci. 80, 3 1 07-3 1 1 3 Nonnecke, B .J . , Franklin, T.A. Reihardt. ( 1 993). Retinoid-induced modulation of immunoglobulin M secretion by bovine mononuclear leukocytes in vitro. J Dairy Sci. 76:2 1 75-2 1 83 Nonnecke, B .J . , Franklin, S .T. , Horst, R.L & T.A. Reiharddt. ( 1 994). Invitro modulation of function, proliferation, and phenotype of bovine mononuclear leukocytes by 1 3 -cis retinoic acid. JNutr. immuno. 2 : 39-59 Nordskog, A. W, Comstock, R.E & Winters, L.M. ( 1 944) .The relationship between certain blood components and rate of growth in swine. J Anim. Sci. 3 :422-430). Nunes, C.S. & Guggenbuhl, P . ( 1 998). Evaluation of salinomycin (Biocox (R)) effects on pig performance . Journal of Animal and Feed Sciences. 7: 1 67- 1 70 Nussler A.K and Thomson A. W. ( 1 992) : Immunomodulatory agents in the laboratory and clinic. Parasitology. 1 05 :S5-S23 Odink, J . , Smeets, J .F .M. , Visser, I .J .R. , Sandman & Snijders, J .M.A. ( 1 990a) . Hematological and biochemistry reference values for Ontario swine. Canadian Journal of Comparative Medicine. Odink, J . , Smeets, J .F .M. , Visser, I .J .R. , Sandman & Snijders, J .M.A. ( 1 990b) . Hematological and clinicalchemical profiles of healthy swine and swine with inflammatory processes. J Anim Sci. 68: 1 63 - 1 70 O'Doherty, J .V. & McKeon, M.P.(2000). The use of expel ler copra meal in grower and finisher pig diets. Livestock Production Science 67 :55-65 O'Donovan, P.B., Pickett, R.A., Plumlee, M.P. & Beeson, W.M . (x) . Purdue University, Lafayette, Indiana Iron toxicity in the young pig. Ofek, 1 . , Mirelman, D. & Sharon, N. ( 1 977). Adheerence of Escherichia coli to human mucosal cells mediated by mannose receptors. Nature (Lond) 265 :623-625 Oppenhiermer, S . J . ( 1 989) . Iron and Malaria. Parasitol Today 5:77-9 . Ostrowska, E. , Gaber, N.K. , Sterling, S.J . Tatham, B .G. , Jones, R.B, Eagling, D .R . , Jois Mark. , & Dunshea, F .R . (2004). British Journal of Nutrition. 91 :2 1 1 -2 1 8 . Owusu-Asiedu, A. , Nyachoti, C . M . & Marquardt, R. R. (2002). Response of early­ weaned pigs to spray dried porcine or animal p lasma-based diet supplemented with egg-yolk antibodies against enterotoxic Escherichia coli. J Anim. Sci: 80 :2897-2903 . Pall ister, C.J. ( 1 999). Haematology 1 st Ed. Oxford, Butterworth I Heinmann publishing. Palmano, K.P. & Elgar, D.F . (2002). Detection and quantification of lactoferrin in bovine whey samples by reversed-phase high performabce liquid chromatography on polystyrene-divinylbenzene. J Chomatogr. A 94 7 :307-3 1 1 . 1 1 0 Panda, B & Combs, G.F. ( 1 963) . Impaired antibody production in chicks fed diets low in vitamin A, pantothenic acid or riboflavin. Proc Soc. Exp. Biol. Med. 1 1 3 : 5 30- 534. Pantako, T. 0., Passos. M. , Desrosiers, T & Amiot, J . ( 1 994) [in foreign language] International Dairy Journal vol . 4 (Abstract). Park, H.J . , Lee, J .H. , Song, Y.B. & Park, K.H. ( 1 996). Effects of dietary supplementation of l ipophilic fraction from Panax ginseng on cGMP and cAMP in rat platelets and blood coagulation. Biological and Phamaceutical Bulletin 1 9 ( 1 1 ) pp 1 434- 1 439. Parks, C .W., Grimes, J .L . , Ferket, P.R. & Fairchild, A.S. (2000). The case from mannanoligosccharides in poultry diets. An alternative to growth promotant antibiotics? In Biotechnology in the feed industry. Proceedings of Alltech's 1 61h Annual Symposium (eds) Nottingham University Press. Partridge, K.H & Hazzledine, M. ( 1 997) The influence of feed enzymes on digestion disorders in swine. Proc. 281h Annu. Meeting Am Soc . Swin Pract. , Des Moines, IA. Pp.83- 1 93 Pearson, A. M. & Dutson, T.R. Growth regulation in farm animals ( 1 99 1 ) . (Eds) In : Advances in Meat Research vol 7 . Perdigon, G., Nader, de Macias, M.E. , Alvarez, S . , Oliver, G. & Pesce de Ruiz Holgado, A. ( 1 986a). Effect of a mixture of L. casei and L. acidophilus administration orally on the immune system in mice. Journal of Food Protection. 49:986-989. Perdigon, G., de Macias, M.E. , Alvarez, S., Oliver, G. & de Ruiz Holgado A.P ( 1 986b) Enhancement of immune response in mice fed with S. thermophilus and L . acidophilus. Journal of Diary Science 70 :9 1 9-926 Perdigon, G., Nader de Macrias, M.E. , Alvarez, S . , Ol iver, G. & Pesce de Ruiz Holgado, A. ( 1 987). Enhancement of immune response in mice fed with S. thermophilus and L. acidophilus. Journal of Dairy Science 70, 9 1 9-926 Perdigon, G., Nader de Macias, M. E., Alvarez, S . , Oliver, G., de Ruiz, P . & Holgado, A. ( 1 988) Systemic augmentation of the immune responses in mice by feeding fermented milks with L. casei and L. acidophilus. Immunology. 63 : 1 7-23 Perdigon, G., Nader de Mascias M. E., Alvarez, S., Oliver G., de Ruiz, P . & Holgado, A. ( 1 990) : Prevention of gastrointestinal infection using immunobiological methods with milk fermented with Lactobacillus casei and lactobacillus acidophilus. Journal of Dairy Research 57 :255-264 Perdigon, G., Alvarez, S. , Nader de Macias, M., Roux, M .E. & Pesce de Ruiz Holgado, A. ( 1 990a). The oral administration of lactic acid bacteria increases the mucosal intestinal immunity in response to enter pathogens. Journal of Food Protection 53 (5) :404-4 1 0. Perdigon, G., Alvarez, S . , & Pesce de Ruiz Holgado A. ( 1 99 1 ). Immunoadjuvant activity of oral Lactobacillus casei: influence of dose on the secretory immune response and protective capacity in intestinal infecton. Journal of Dairy Research. 58 : 485-496 Perdigon, G & Alvarez S . , ( 1 992). Probiotics and Immune state. In: Probiotics Ed Fuller R. , Chapman and Hal l , London. 1 46- 1 76 Perdigon, G. , Medici, M. , B ibas Bonet de Jorrat M.E. , Valerde de Buduguer, M . , & Pesce de Ruiz Holgado A. ( 1 993) Immunomodulating effects of lactic acid bacteria on mucosal and tumoural immunity. International Journal of Immunotherapy. 9 :29-52 1 1 1 Perdigon, G., Rachid M. , de Budeguer M.V. , & Valdez K. ( 1 994) . Effect of yogurt feeding on the small and larger intestine associated lymphoid cells in mice. Journal of Dairy Research. 6 1 : 5 53-562 Perdigon G. , Aguero G. , Alvarez S . , Gaudioso de Allori C . , & Pesce de Ruiz Holgado A.( l 995a) . Effect of viable lactobacillus casei feeding on the immunity of the mucosal and microflora in mal-nourished mice. Michwisss. 50 :25 1 -256 Perdigon, G. , Alvarez S. , Rachid M. , Aguero G. , & Gobbato N ( 1 995b) Symposium: pro biotic bacteria for humans: clinical systems for evaluation of effectiveness. Journal of Dairy Science . 78 : 1 597- 1 606 Penston, D .M, Lennard, x. & Jones, lE. ( 1 986) Severe chronic constipation of young women: ' idiopathic slow transit constipation' . Gut 27 :4 1 -48 Piard J .C . & Desmazeaud, M. ( 1 99 1 ). Inhibiting factors produced by lactic acid bacteria. I. Oxygen metabolites and catabolism end-products. La it 7 1 ,525-54 1 Plate! , K. & Srivanasan, K. (2000a) . Stimulatory infuences of selected spices on bile secretion in rats . Nutrition Research 20.pp 1 493- 1 503 . Plate! , K. & Srinivasan, K. (2000). Influence of dietary spices or their active principles on pancreatic digestive enzymes in albino rats. Nahrung, 44 pp42-46. Plumed-Ferrer, C., Kivela, 1 . , Hyvonen, P. & von Wright, A. (2005). Survival , growth and persistence under farm conditions of a Lactobacillus planetarium strain strain inoculated into liquid pig feed. Journal of Applied Microbiology. Issue 4, 99 :85 1 - 860. http ://www.blackwell-synergy.com/doilfull/l O . l l l l /j . l 365- 2672.2005 .02666. downloaded on 3 1 August, 2006 Pluske, J .B. , Wi lliams, I . H. & Aherne, F.X. ( 1 996). Maintenance of vi l lous height and crypt depth in piglets by providing continuous nutrition after weaning. Anim. Sci. 62 : 1 3 1 - 1 44. Pluske, J .B. , Hampson D.J . & Wil l iam I .H . ( 1 997). Factors influencing the structure & functions of the small intestine in the weaned pig: a review, Lives!. Prod. Sci . 5 l ( 1 997) 2 1 5-236. Pollman, D.S . , Danielson, D .M & Peo, E.R. Jr. ( 1 980) Effects of microbial feed additives on performance of starter and growing-finishing pigs. J Anim. Sci. 5 1 : 577-58 1 . Pollman, D.S . , Smith, I .E. , Stephenson, J .S . , Schoneweis, D.A. & Hines, R.H. ( 1 983) . Comparison of gleptoferron with iron dextran for anaemia prevention in young pigs. JAnim. Sci. 56 : 640-644 . Pond, W.G. & Houpt, K.A. ( 1 978). Body fluids hematology, and immunology: The biology of the pig, pp.244-245 . New York: Cornel l University Press Ltd. Poppenga, R.H. (x). Risks associated with herbal remindies. In: Kirk 's Current Veterinary Therapy XII . pp220-226. Prescott, J. F. (2000) . Antimicrobial drugs: Miracle drug or pig feed?. Adv. Pork Prod. 1 1 : 37-45 Preston, C.M. , McCracken, K.J. & Bedford, M.R (200 1 ) . Effect on wheat content, fat source and enzyme supplementation on diet metabolizabil ity and broilers performance. Br. Poult. Sci. 42 :625-632 Prgomet, C., Sarikaya, H . , Bruckmaier, R.M. & Pfaffl, M. W. (2005). Short-term effects on pro-inflammatory cytokine, lactoferrin and CD 1 4 mRNA expression levels in bovine immunoseparated milk and blood cell s treated by LPS. Journal of Veterinary Medicine . 52 :3 1 7-328 . http://www.blackwell-synergy.com/doi/full/l 0 . 1 1 1 1 /j . 1 439-0442.2005 .0074 1 .x downloaded on 3 0th October, 2006). 1 1 2 Qu, S . (200 1 ). Pro biotic Bifidobacterium lactis HNO 1 9 ebhances the resistance and immunity against enteric pathogens. MSc thesis, Massey University. Ragland, D. & Adeola, L . (2002). New Probiotics efficacy demonstrated in University study. [Online] avai lable: http : //www . I n nofeed . eo m. Ramirez, C . G . , Miller, E.R. , Ullrey, D .E & Hoefer, J .H ( 1 963). Swine hematology from birth to maturity. I l l . Blood volume of the nursing pigs. J Animal Sci 22 : 1 068 Ravindran, V., More!, P .C.H . . , Partidge, G.G., Hruby, M. & Sands, J .S . (2006). Influence of an Escherichia coli-derived phytase on nutrient utilisation in broi ler starter fed diets containing varying concentrations of phytic acid. Poultry Science. 8 5 : 82-89. Reddy, N.R., Roth, S .M. , Eigel, W.N. & Pierson, M.D. ( 1 988) . Foods and food ingredients for prevention of diarrhoea! disease in children in developing countries. Journal of food Protection 5 1 :66-7 Rees, As., Lyson, R.J ., Stokes, C.P. & Boume, F. 1. ( 1 989). Antibody production by the pig colon during infection with Treponema hyodysenteriae. Res. Vet. Sci. 47 : 263-269. Rees, L.P. Minney, S .F . , Plummer, N.T., Slater, J .H. & Skyrme, D.A. ( 1 993). A quantitative assessment of the antimicrobial activity of garlic (Allium sativum). World Journal of Microbiology and Biotechnology 9 : 303-307. Reiter, B . ( 1 985) . Protective protein in milk: Biological significance and exploitation. Lysozyme, lactoferrin, l actoperoxidase, xanthineoxidase. Bulletin of International Dairy Federation, 1 95 : Reiter, B & Peraudin, J-P. ( 1 99 1 ) . Lactoperoxidase, biological functions. I : 1 . Everse, K.E. Everse, M.B . Gruisham (eds). Peroxidases in chemistry and biology. Vol . I . Boca Ratan, F.L, USA. C.R.C. Press Inc. , p 1 43 - 1 80 . Ribeiro, G.S, Cardoso, M.R. , Collie, C . , Andrade, Jr. , Borelli , P. , Sommerfelta, I .E . , Santill ian, G, Lopez, C . , Ribicich, M , Franco, A.J . , Sarker, S .A. & Gyr, K. ( 1 992) Non-immunological defence mechanisms of the gut 33 :987-993 Ribeiro, G.S . , Gamica, M.R., Cardoso, M.A., Colli , C . , Andrade, Jr. H .F . & Borel l i , P . (2000). Effects o f dietary iron supplementation on the course of Plasmodium chabaudi malaria in weanling mice. Nutrition Research, 20 : (8): 1 1 93 - 1 1 99 Riley, J .E. ( 1 99 1 ). A review of outdoor pig production, in Manipulating Pig Production IV. p 267-276 Risley, C.R., Komergay, E .T., Bamett, K.L., Lindermann, M.D. &Eigel . W.N. ( 1 988) . Response of early weaned pigs to an Escherichia coli challenges and their absorption of avalbulin or xylose as influenced by creep feeding. Nutr. Rep. Int3 8 : p945 . Richardson, D. ( 1 996). Probiotics & product innovation. Nutrition & Food Science 6 no. 4 :27-33 to go with sheinbach Robert M. Jacobs, John H. Lamsden, Judith A. Taylor. Canine and feline Reference value Robertson , J.A. & Lundeheim, N. ( 1 994). Prohibited use in antibiotics as a feed additive for growth promotion - effects on piglet health and production parameters. Pro c. 1 3th Internatinal Pig Vet. Soc Congr., Bangkok, Thailand Roitt, I . ( 1 99 1 ) . Essential Immunology. (ih ed.). Blackwell Scientific Publications, Oxford, UK. Chapters 1 & 2 . Rosen G.D. , 1 995 . Antibacterial in poultry and p ig nutrition In : R.J. Wallace, A . Chesson (Editors). Biotechnology in animal feeds and animal feeding, VCH Verlagsgsellschaft, Weinheim, pp. 1 43 - 1 72 1 1 3 Roth, F.X. & Kirchgessner, M. ( 1 998). Organic acids as feed aaditives for young: Nutritional and gastrointestinal effects. Journal of Animal and Feed Science, 7 :25- 33 . Row land, I.R. & Tan aka, R . ( 1 993 ) . The effects of transgalactosylated oligosaccharides on gut flora metabolism in rats with human faecal flora. J Appl. Bacterial . 74 :667-674 Roy, C.N & Enns, C.A (2000). Iron homeostasis : new tales from the crypt. B lood 96 ( 1 3 ) : 4020-4027. Rybley, M. E. , Self, H .L, T. Kowalczyk, T & R. H . Grummer, R.H. ( 1 959). The effectiveness of three different methods of iron administration to young pigs. J. Animal Sci. 1 8 :4 1 0 Rydberg, M. E . , Self, H .L. Kowalczyk, T. & Grumrner, R.H. ( 1 959). The effectiveness of three different methods of iron administration to young pigs. J. Animal Sci. 1 8 : 4 1 0 . Sainsbury, D. ( 1 993). Protecting against stress. Probiotics boosts natural resistance. Pigs 9 :37 Salminen, S .J . , Ouwehand, A.S . , Benno, Y. &Lee, Y .K . (2002). Probiotics: how should they be defined? Trends in Food Science and Technology I 0: 1 07- 1 1 0 . Salobir, J . , Bogdanic, C., Pogorelec, R. & Novak, B . ( 1 995). The effect of xylanase and �-glucanase on energy value, apparent nutrition digestibil ity, nitrogen retention and internal viscosity in wheat based broiler diet. Proc . 1 oth Eur. Symp. On Poult. Nutr. 1 5- 1 9 Oct. 1 995, Antalya, Turkey. Pp. 326-327 Sanders M. E. ( 1 994): Lactic acid bacteria as promoters of the human health. In Functional foods. (Ed) I. Goldberg. London: Chapman and Hal l . 294-322 Sanderson, J .H. & Phillips, C.E. ( 1 98 1 ) . An Atlas of laboratory animal haematology. Oxford Science Publications (Ed) . Oxford University Press. New York, USA Sanson, B.F . ( 1 984). The iron requirements of young pigs. Pig Vet. Soc. 1 1 : 67-75 Santos, Jr, A.A., Ferket, P.R., Grimes, J.L. & Edens, F.W. (2004a). Dietary supplementation of endoxylanases and phospholipases for turkeys fed wheat based on growth performance and metabolisable energy of turkey poults. Int. J. Poult. Sci. 3 :20-32. Santos Jr, A .A . , Ferket, P.R., Grimes, J .L. & Edens, F .W. (2004b. Dietary pentosanase supplementation of diets containing different qualities of wheat on growth performance and metabolisation energy of turkey poults. Jnt. J. Poult. Sci. 3 :33-45 Sanyal , A.J. Hirsch, J . I . , Moore, E.W. ( 1 994). Evidence that bile salts are important for iron absorption. Am. J. Physiol . 266 (2 Pt 1 ) : G3 1 8-323 Sarica, S., Ciftci , A., Demir, E. , Kilinc, K & Yildrim, Y. (2005). Use of an antibiotic growth promoters and two herbal natural feed additives witrh and without exogenous enzymes in wheat based broiler diets. South African Journal of Animal Sciences. 3 5 ( 1 ) . SAS Institute, 1 996. SAS User' s Guide: Statistics. SAS Institute, Cary, NC Sato, K . , Saito, H . , Tomioka, H. & Yokokura, T. ( 1 988) . Enhancement ofhost resistance against listreria infection by L. casei: efficacy of cell wal l preparation of L. casei. Microbiology and Immunology 3 2 ( 1 2) : 1 1 89- 1 200 Sato, K., Shinomoto, H . , Taimoto, M., Dosaka, S. & Nakajima, I. ( 1 990) uptake and secretion of human Lactoferrin by B lymphocytes. Agriculture and Biological Chemistry 54, 1 275- 1 279 1 1 4 Sato, R. Shindo, M, Gunshin, H. , Nogushi, T. & Naitom H . ( 1 994) Characterization of Phospopeptides derived from �-casein: an inhibitor of inta-intestine perception of calcium phosphate. Biochimica Biophysica Acta 1 077:4 1 3 -4 1 Savage, D .C . ( 1 977). Microbial ecology of the gastrointestinal tract. Ann. Rev. Microbial. 3 1 : 1 07- 1 3 3 Saxelin, M. , Rautel in, H . , Salminen, S . & Makela, P .H . ( 1 996). Safety and commercial products with viable lactobacillus strains. Jnfectiuos Diseases in Clinical practice 5 : 3 3 1 -3 35 . Scheinbach, S . ( 1 998). Probiotics: Functionality and commercial status. Biotechnology Advances, 1 6 (3) pp 58 1 - 608 Schifrin, E.J . , Domonique, B . , Servin, A.L. , Rochat, F. & Donnet- Hughes, A. ( 1 997). Immune modulation of b lood leukocytes in human buy lactic acid bacteria : criteria for strain selection. American Journal of Clinical Nutrition 66 : 5 1 5s-52oS. Schifrin, E.J . , Rochat, F. , Link-Amster, H . , Aeschlimann, J .M. & Donnet-Hughes, A . ( 1 995). Immunomodulatoin of human blood cell s fol lowing the ingestion of lactic acid bacteria. Journal of Dairy Science 78 (3) :49 1 -497 Schoenherr, W. D . ; Pollmann, D. S . ; Coalson, J. A. , ( 1 994) . Titration of MarcroGard­ S on growth performance of nursery pigs . J Anim. Sci. 72(suppl . 2 ) : 57 Schrezenmeir, J . & de Vrese, M. (200 1 ) . Probiotics, Prebiotics, and synbiotics­ approching a definition. Am. J Clin. Nutr. 73 (Suppl) : 36 1 S-4S Schusdziarra, V., Hollaad, A. , Schick, R., de la Fuente, A. , Brantl, V . & Pfeiffer, E. F. ( 1 983a) Effect of �-casomorphins on somatostatin release by ingested opiate-like substance in dogs. Diahetologia 24: 1 1 3- 1 1 6 . Schusdziarra, V . , Schick, R. De la Fuente, A. Hollaad, A. , Brantl . , V. & Pfeiffer, E .F . ( 1 983a) . Effects of �-casomorphins on some somatostatin release in dogs. Endocrinology 1 1 2 : 1 948- 1 95 1 . Schusdziarra, V. , Hollaad, A. , Schick, R. De la Fuente, Kuer, M. , Maier, V . , Brantl , V. & Pfeiffer, E.F. ( 1 983b) . Modulation of post-prandial insulin release by ingested opiate-like substances in dogs. Diahetologia 24: 1 1 3 - 1 1 6 Sell , S . ( 1 987). Basic Immunology- Immune Mechanisms in health and disease. Elsevier Science Publication Company, Inc . , New York. Chapter 1 0 . Sfeir, R.M., Dubarry, M . , Boyaka, P.N. , Rautureau, M & Tome, D . (2004). The mode of oral bovine lactoferrin administration influences mucosal and systematic response in mice. J Nutr. 1 34:403-409 Si, W., Gong, J . , Tsao, R. , Zhou, T., Yu, H. , Poppe, C . , Johnson, R. & Du, Z. (2006). Antimicrobial activity of essential oils and structurally related synthetic food additives towards selected pathogenic and beneficial gut bacteria. Journal of Applied Microbiology. 1 00:296-305 . Simons, P .C.M. , Versteegh, H .A.J . , Jongbvloed, A .W. , Kemme, P .A. , S lump, P . , Bos, K.D. , Welters, M.G.E. , Beudeker, R.F. & Verschoor, G.J . ( 1 990). Improvement of phosphorus availability by microbial phytase in boilers and pigs. Br. J. Nut. 64 : 525 Sims, L.D ( 1 996). Blood. In : Pathology of the pig-a diagnostic guide. (Ed) Sims, L .D and Glastonbury, J .R. W. Barton, ACT 2600, Australia Called patho in text Sommerfelt, I .E . , Santallan, G. , Lopez, C . , Ribicich, M . & Franco, A.J (x). Effect of dietary iron supplementation on the course of Plasmodium chabaudi Malaria in weanling mice 1 1 5 Spreeuwenberg, M.AM, Verdonk, J .M.A.J . , Gaskins, H .R. & Verstegen, M.W.A. (200 1 ) . Small intestine epithelial barrier function is compromised in pigs with low feed intake at weaning. J Nutr. 1 3 1 : 1 520- 1 527 Spurlock, M.E. , Frank, G.R., Willis, G.M., Kuske, J.L. & Cornelius, S .G . ( 1 997). Effects of dietary energy and immunological challenge on growth performance and immunological variables in growing pigs. J Anim. Sci. 75 : 720-726 Srinivasan, K. (2004a). Spices as nutraceuticals with multi-benefifial health effects. Journal of Herbs and Medicinal Plants, 1 1 : Srinivasan, K. (2005) . Spices as influencers of body metabolism: an overview of three decades of research. Food Research International, 38 ( 1 ) :77-86 Starly, T.S . ( 1 984). Use of fats in diets for growing pigs. In: J. Wiseman (Ed.) . Fats in Animal Nutrition. Butterworts. London Stavric, S. ( 1 992) . Defined cultures and prospects. International Journal of Food Microbiology, 1 5 :245-263 Stavric, S . & Kornegay, E.T. ( 1 995). Microbial probiotic for pigs and poultry. Biotechnology in Animal Feeds and Animal Feeding. (Ed.) R. J. Wallace and A . Chesson. Weinheim: V.C .H . Verlagsgsellschaft mbH. pp. 205-23 1 Steffen, E.K. & Berg, RD. ( 1 983). Relationship between caecal population levels of indigenous bacteria and translocation to the mesenteric lymph nodes. Infection and Immunity 39, 1 252- 1 257 . Steijn, J .M. (200 1 ) . Milk ingredients as nutraceuticals. International Journal of Dairy Technology, 54(3) :8 1 -88 . Sterle, J . A . , Cantley, T . C . Lamberson, W. R . , Lucy, Hametabolism C . , Gerrand, D . E . , Matteri, R . L. & Day, B .N. ( 1 995). Effect of recombinant porcine somatotrophin on placental size, fetal growth, and IGH-I and IGH-II concentrations in pigs. Journal of Animal Science . 73 :2980-2985 Stewart, C.S. & Chesson, A. ( 1 993) . Making sense of probiotics. Pig Veterinary Journal, 3 1 , pp3-33 . Stites, D .P . , Terr, A. I . & Parslow, T.G. ( 1 994). Basic and Clinical Immunology. (81h edn.) . Appleton & Lange, Norwalk, Connecticut, USA. Chapter 4 1 . Stokes, C.R. , Bailey, M. , Haverson, K., Harris, C . , Jones, P., Inman, C . , Pie, S . , Oswald, LP. , Williams, B .A., Akkermans, A.D.L. , Sowa, E . , Rothkotter, H .-J, Miller, B .G. (2004). Postnatal development of intestinal immune system in piglet: implications for the process of weaning. Anim.Res.53 :325-334 Strusinka, D. , Iwanska, S . & Pysera, B . ( 1 998). The effect of digest acid on growth rate and some blood parameters in calves. Journal of Animal and Feed Sciences. 7 :2 1 7-22 1 Stryer, L . ( 1 995). Biochemistry 4th Edition. Ch.30. The integration of metabolism. (Ed). Freeman Publishing, New York. Succi, G . , Sadrucci, A., Tamburini, A. , Adami, A. & Cavazzoni, V. ( 1 995) . Effects of using a new strain of Bacillus coagulans as a probitic on the performance of piglets. Riv. Suinicol. 36, 59-66 Svoboda, M. , Bouda, J . , Drabek, J . & Doubek (2004). Effects of per os lactate supplement on development of haematological profile in early postnatal period. Acta Vet. Brno. 73 :43 1 -436 Swientek, B. (2003) . Beneficial bacteria. Prebiotics and probiotics work in tandem to stimulate a healthy microflora in the gastrointestinal tract. Food product 1 1 6 development. Switala, M., Kolacz, R. , Bodak-Koszalka, E. & Gajewezyk, P. (x). Hematological and biochemical parameters of blood and immune response of runt weaners Switala, M, Kolacz, R, Bodak-koszalka, E, et al . (x). Immunological and hematology response in experimental Toxacara canis-infected pigs Tani , F . , Lio, K., Chiba, H . & Yoshikawa, M. ( 1 990) Isolation and characterization of opioid antagonist peptides derived from human Lactoferrin. Agricultual and Biological Chemistry 54 : 1 803- 1 8 1 0 . Taylor, D. J. ( 1 999). Multiple antibiotics pigs ' medicine. Pig J 43 : 1 70- 1 87 Thacker, P.A ( 1 999). Nutrional requirements of early weaned pigs : a review. Pig News lnfo. 20 pp 1 3N-24N.) Thompson, K. & Forsyth, S . (2006). Pathology II . Study guide and cases for problem­ solving tutorials . Massey University Thornton, R.F. & Shorthose, W.R ( 1 989). Growth promotants, repartitioning agents and pig meat quality . Manipulating pig production I I . Australasian Pig Science Association. Pp3 1 -3 7 Thornton, K . ( 1 990). Outdoor Pig Production. Farming Press, Ipswich, U.K. Thrall, A.M., Baker, D .C . Campbell , T.W., DeNicola, D . , Fettman, M. , Lassen, E .D . , Rebber, A. & Weiser, G. (2004). Veterinary haematology and clinical chemistry (Eds) D.B. Troy and Kerin, R.A. Philadelphia, Pennsylvania, 1 99 1 06, USA. Tizard, I .R. (2000) Veterinary immunology, An introduction (eds). W.B. Saunders Co., Philadelphia. P.A. Tome, D. & Debbabi, H. ( 1 998). Physiological effects of milk protein components. lnt. Dairy Journal. 8 : 3 83-392 Tomioka, H . , Sato, K. & Saito, H . ( 1 990), Effect of oxofloxacin combined with Lactobacillus casei against Mycobacterium fortuitum infection induced in mice. Antimicrob. Ag. Chemother. 34 :632-636 Totara, G.J. & Reynolds-Gtrabowski, S. ( 1 996). The principles of anatomy and physiology 81h edition. Ch. 1 9 . The cardiovascular system: The blood. Harper Collins College Publishers Touchette K.J . , Carrol J .A. , Allee G.L. , Matteri R.L. , Dyer C.J . , Beausang L .A. , Zannell i M. E . (2000) . Effect of spray-dried plasma & lipopolysac-charide exposure on weaned pigs: ! .Effects on the immune axis of weaned pigs, J Anim. Sci . 80494-50 1 Touchette, K.J. , Carrel, J .A. , Allee, G.L. , Matteri, R.L. , Dyer, C.J . , Beausong, L .A. & Zannel l i , M.E. (2002) . Effect of spray-dried plasma and lipopolysaccharide exposure on weaned pigs : Effects on the immune axis of weaned pigs. J Anim. Sci. 80 :494-50 1 Tsuj i, S . Hirata, Y . & Mukai, F . ( 1 990) . Comparison of lactoferrin content of colostrurns between cattle breeds. J Dairy Science. 73 : 1 25- 1 28 Tufano, M.A. , Cipollaro De Lero, G. , Innielo, R. , Galdiero, F. ( 1 99 1 ). Protein A and other surface components of Staphylococcus aureus Stimulate production of IL­ l a, IL-6, TNF-a and IFN-y. European Cytokine Network 2, 3 6 1 -366 1 1 7 Tungthanathanich, P. ( 1 994). The effects of diet and feeding on small intestinal development in piglets during the first 24 hours after birth. Volume 1 text. PhD Thesis, Massey University, Palmerston North, New Zealand. Turner, J .L . , Dritz S .S . & Minton J .E. (2001 ) . Alternatives to conventional antimicrobials in swine diets. ProfAnim. Scientist. 1 7, 2 1 7-226 Turner, J .L . , Dritz., S .S . , Higgins, J .J . & Minton, J .E. (2002). Effects of Ascophyllum nodosum extract on growth performance and immune function of young pigs challenged with Salmonella typhimurium. Journal of Animal Science 80: 1 947- 1 953 . Turner. J .L . Dritz, S . S , Higgins, J .J . , Herkelman, K.L. and Minton, J .E.(2002a) Effects of a Quil laja saponica on growth performance and immune function ofweanling pigs chal lenged with Salmonel la typhimurium. Journal of Animal Science 80 : 1 939- 1 946. Tvedten, H., Scott, M. & Boon, G.D. (x). Interpretation of cytograms and histograms of erythrocytes, leucocytes, and platelets. In Current Veterinary Therapy XIII . P. 3 8 1 Tvedten, H . ( 1 993). Advanced haematology analyzers. Interpretation o f results. Veterinary Clinical Pathology. 22 no.3 : 72-80. Ullrey, D. E. , Miller, E.R, Thompson, O.M., Achern1ann, I .M. , Schmidt, D .A. , Hoefer, J .A. & Luecke, R.W. ( 1 960) . The requirements of the baby pig for orally administered iron. J. Nutr. 70: 1 87 Ullrey, D. E. , Miller, E.R, Brent, B.E, Bradley, B .L. & Hoefer, J .A. ( 1 967). Swine hematology from birth to maturity. IV. Serum calcium, magnesium, sodium, potassium, copper, zinc and inorganic phosphorus. J. Animal Sci. 26 : 1 024 Ullrey, D.E . , Miller, E.R., West, D.A. , Schmidt, R.W., Seerley, R.W., Hoefer, J.A. & Luecke, R. W. ( 1 959) . Oral & Parenteral administration of Iron in the prevention and treatment of baby pig and anemia. J. Anim. Sci. 1 8 :256-263 . Underwood, E.J. & Somers, M. ( 1 969). Australian Journal of Agricultural Research. 20 Underwood, E.J. & Mertz, W. ( 1 987). Trace elements in human and animal nutrition. Vol . l (Ed) Academic Press, Inc. San Diego, California p. 9- 1 4 Usman, A.H. ( 1 999). Bile tolerance, taurocholate deconjugation, and binding of cholesterol by Lactobacillus gasseri strains. J. Dairy Sci. 82 :243-248 Van de Ligt, C.P. A. , Lindermann, M.D. & Cromwell , G.L. (2002). Assessment of chromium tripicolate supplementation and dietary protein level on growth, carcass, and blood criteria in growing pigs. J. Anim. Sci. 80 :24 1 2-24 1 9 Vandelle, M. , Teller, E . & Focant, M . ( 1 990). Probiotics in animal's nutrition : a review. Arch. Amm. 40 : 507- Van Dijk, A.J., Everts, H . , Nabuurs, N.J.A., Margry, R.J .C.F. & Beynen, A.C. (2003) . Growth performance of weaned pigs fed spray-dried animal plasma: review, Livest. Prod. Sci 68. pp 263-274. Van, Heugten, E., Spears, J .W. & Coffey, M.T. ( 1 994a). Effects of dietary protein on performance and immune response in weanling pigs subj ected to an inflammatory challenge. J. Anim. Sci. 72:266 1 -2669 Van, Heugten, E . , Spears, J.W. & Coffey, M.T. ( 1 994b). The effect of dietary protein on performance and immune response in weanling pigs subjected to an inflammatory challenge. Journal of Animal Science 72 : 266 1 -2669. Van, Heugten, E., Coffey , M.T & Spears, J .W. ( 1 996). Effects of immune chall enge, dietary energy density, and source of energy on performance and immunity in weanling pigs. J. Anim. Sci. 74 ( 1 0) :243 1 -2440 1 1 8 Van Kempen, E.J . & Ziij lstra, W.G. ( 1 96 1 ) . Clin. Chim. Act. , 6 :538 . Van Kempen, J , G .M. ( 1 987). Avoid iron deficiency in piglets. Pigs 3 : 1 0- 1 1 . Van Leeuwen, Huisman, J . , Kerkhof, H.M. & Kussendrager, ( 1 998). Small intestinal microbio logical and morphological observations in young calves fed milk replacer enriched with a combination of lactoperoxidase system and lactoferrin . Journal of Animal and Feed Science 7 : 223-228. Visek, W. J . ( 1 978) . The mode of growth promotion by antibiotics. J Anim. Sci. 46: I44 7-I4469 Verstegen, M . W. & Wil liams, B.A. (2002). Alternatives to the use of antibiotics as growth promoters for monogastric animals. Ani m. Biotechnol. l 3 : 1 1 3 - 1 27 . Vervaeke, I .J . , Decuypere, J. A. , Dierick, N.A. & Henderickx, H.K. ( 1 979). Quantitative In-vitro evaluation of energy metabolism influenced by Virginamicin and Spiramyin used as growth promoters in pig nutrition. Journal of Animal Science 49: 846-856 . Voganatsi, A , Panyutich, A , Miyasaki, K.T & Murthy, R.K (200 1 ) Mechanisms of extracellular release of human neutrophi l calprotein. Journal ofleukocyte Biology 70: 1 3 0- 1 34 from http ://www .jleukbio.org Verstegen, M. W. & Williams, B .A. (2002). Alternatives to the use of antibiotics as growth promoters for monogastric animals . Anim. Biotechnol. 1 3 : 1 1 3 - 1 27. Vervaeke, I . J . , Decuypere, J . A. , Dierick, N.A. & Henderickx, H .K. ( 1 979) . Quantitative In-vitro evaluation of energy metabolism influenced by Virginamicin and Spiramyin used as growth promoters in pigs nutrition. Journal of Animal Science 49: 846-856. Voigt, G.L . (2000). Haematological Techniques and concepts for veterinaty technicians (Eds). Iowa State University Press, Ames, Iowa, USA. Waddel l , D . G. , Ullrey, D.E. , Mil ler, E.R., Sprague, J . I , Alexander, E .A. & Hoefer, J .H . ( 1 962). Blood cell population and serum protein concentration in the fetal pig J . Animal Sci. 2 1 : 583 Wahlstrom, R.C. & Juhl, E .W ( 1 960). A comparison of different methods of iron administration on rate of gain and hemoglobin level of the baby pig. J Anim. Sci. 1 9 : 1 83 - 1 8 8 Wannenaccher, R . W. ( 1 977). Key role o f various individual 's amino acids i n the host response to infection. J Clin. Nutr. 30 : 1 269 Walton, J .R. ( 1 979). The place of growth promoters in pig production. Proc. Pig Vet. Soc. 4:pp49. Walton, J .R. (200 1 ) . Benefits of antibiotics in animal feed. In: Recent developments in pig nutrition. 3 (eds) J . Wiseman & P.C. Guamsworthy. Nottingham University Press, Nottingham, U.K. pp 1 1 -37 . Webb, A.J . ( 1 99 1 ) . Genetic programmes to improve litter size in pigs. In Manipulating Pig production I I I . Australasian Pig Science Association, (Ed) E .S . Batterham pp.229-244. Webel , D .M. , Finck, B .N. , Barker, D .H . , Johnson, R.W. ( 1 997). Time course of increased serum cytokines, cortisol, and urea nitrogen in pigs fol lowing intraperitoneal injection of lipopolysaccharides. J Animal Sci. 75 : 1 5 1 4- 1 520 1 1 9 Weiser, M.G. (x) . Haematologic technology for diagnosing anaemias. In Current veterinary therapy XII p. 437 442 Wells, S .M., Kew, S . , Yaqoob, P . , Wallace, F.A. , Calder, P.C. ( 1 999). Dairy glutamine enhances cytokine production by murine macrophages. Nutrition 1 5 : 8 8 1 -884 Wel ls, J .E. Yen, J .T . & Miller, D .N. (2005). Impact of dried skim milk in production diets on lactobacillus and pathogenic bacterial shedding in growing-finishing swine. Journal of Applied Microbiology, 99:400-407. Wenk C. ( 1 998). Environmental effects on nutrient and energy metabolism in pigs. Arch. Tierenahr. 5 1 : 2 1 1 -224 Wenk, C. (2000). Herbs, spices and botanicals : 'old fashioned' or the new feed additives for tomorrow' s feed formulations? Concepts for their successful use. In : 'Boitechnology of the Feed Industry ' . Proceedings of Allteck 1 61h Annual Symposium (ed. T.P. Lyons and K.A. Jacques), pp79-96. Wenk, C. (2000). Recent advantage in animal feed additives such as metabolic modolic modifiers, antimicrobial agents, probiotics, enzymes and highly avail able minerals. Asian-Aust. J Anim. Sci. 1 3 : 86-95 Whang, K.Y. , Kim, S .W., Donovan, S .M. , Mckeith, F.K. & Easter, R.A. (2003). Effect of protein deprivation on subsequent growth performance, gain of body components, and protein requirements in growing pigs. J Anim. Sci. 8 1 : 705-7 1 6 White, L .A. , Newman, M.C. , Cromwell , G.L. Lidemann, M.D. (2002). Brewers dried yeast as a source of manna oligosaccharides for weaning pigs. J.Animal Sci. 80 :26 1 9-2628 Widjaja, W. (2003 ). Use of semi-anaemic piglets to measure iron bioavai labil ity of meat and meat fractions. MSc Thesis, Massey University, Palmerston North, New Zealand. Widowski, T.M. , Curtis, S .E . & Graves, C.N. ( 1 989). The neutrophi l : lymphocyte ration in pigs fed cortisol . Can. J Anim. Sci. 69 : 50 1 -504 Wierup, M. ( 1 999). Changing use patterns in Europe with regulations and educations. Proc. Agriculture ' s role in managing antimicrobial. Resist. Conf. , Toronto, On. Pp. 1 44- 1 47 Will iams D. L . ; Yaeger, R. G. ; Pretus, H . A. ; Browder, I. W.; McNamee, R. B . , & Jones, E. L . ( 1 989) . Immunization against Trypanosome cruzi : adjuvant effect of glucan. Jnt. J. Jmmunopharmacol. I I , 402 Will iams B.A. , Verstegen M.W.A. & Tamminga S (2000). Fermentation in large intestine of single-stomach animals and relationship to animal health, Nutr. Res. Rev. 1 4 :207-227 Wil liams, B .A. Verstegen, M.W.A & Tamminga, S . (200 1 ). Fermentation in the large intestine of single-stomached animals and its relationship to animal health. Nutr. Res. Rev 1 4 :207-227 Wil l iams, N.H. , Stahly, T.S. & Zimmerman, D.R. ( 1 997). Effect of chronic immune system activation on the rate, efficiency and composition growth and lysine needs of pigs from 6 to 27 kg. Journal of Animal Science 75 : 2463-247 1 . Wil l iams, R.J. & Heyamann, D.L . ( 1 998). Containment of antibiotic resistance. Science 279 . 1 1 53 Wintrobe, M.M. , Lee, G.R., Boggs, D .R. , B ithel , T.C., Athens, J .W. & Foerster, J ( 1 975). Clinical Hematology. (Eds) . Henry Kimpton publisher, London 1 20 Witte, W. ( 1 998) : Medical Consequences of Antibiotic use in Agriculture Science 279, 996. Wood, J .D. ( 1 993). Production and processing pratice to meet consumer needs. In Manipulating Pig production IV. PpP1 3 5-247. · Wood, J .D ( 1 993) . Production and processing processing practices to meet consumer needs. In : Manupulating pig production 1 V. Australasian Pig Science Association Wong, C.W. , Seow, H .F . , Husband, A.J . , Regester, G.O. & Watson, D .L . ( 1 997). Effects of purified bovine whey factors on cellular immune functions in ruminants. Vetreinary Immunology and Immunopathology, 56 :85-96 Wong, M.C . ( 1 998). The effect of probiotics on host mucosal immune responses. MSc Thesis, Massey University, Palmerston North, New Zealand. Wu, G. , Meier S .A Knabe D.A ( 1 994). Dietary glutamine supplementation prevents jejuna) atrophy in weaned pigs, J Nutri. 1 26 :2437-2444. Wu, J .F . , Hsu, J .B. , Cheng, C .S . , Hsyu J .N. (200 1 ) . Microbial supplements in pig diets 1 . Growth benefits and cost consideration in the replacement of antibiotics in diets for growing-finishing pigs. Agr. Assn. China 90, 2 ( 1 ) : 1 6-23 Wuryastuti, H .H . , Stowe, D. , Bul l , R.W. & Miller, E .R., 1 993 . Effects of vitamin E and selenium on immune response of peripheral blood, cholostrum, and milk leukocytes of sows. J Anim. Sci. 7 1 :2464-2472 Xuan, Z. N . , Kim, J .D . , Heo, K.N . , lung, H.J . , Lee, J .H. , Han, Y.K. , Kim, Y.Y . & Han, I . H . (200 1 ). Study on the development of a probiotics complex for weaned pigs. Asian-Aust. J Anim. Sci. 1 4 : 1 425- 1 428 Yilmaz, 0. , Celik, S . , Cay, M. ( 1 997). Protective role of intraperiton eal ly administered vitamin E and selenium on the levels of total lipid, total cholesterol , and fatty acid composition of muscle l iver tissues in rats. J Cell. Biochem. 64 :23 3-24 1 Yoon, I. K. & Sem, M.D . ( 1 995). Influence of durect-fed microbials on rumina! fermentation of ruminants : A review. Australas. J Anim. Sci. 8 :533 -555 Yu, I . -T., Ju, C.-C . , Lin, J . , Wu, H . -L . & Yen, H .-T. (2004). Effects of probiotics and selenium combination on the immune and blood cholesterol concentration. Journal of Animal and Feed Sciences, 1 3 :625-634 Young, J . ( 1 997), Development in probiotics and probiotics and symbiotics. A European perspective. Presented at the Annual Meeting of the International Food Technogists. Zabielski, R. ( 1 998). Regulatory peptides in milk, food and in the gastrointestinal lumen of young animals and children. Journal of Animal and Feed Sciences, 7 : 65-78 . Zimecki, M. , Mazurier, J . , Spik, G . & Kapp, J . A. ( 1 996) Lactoferrin inhibits proliferative responses and cytokine production of Th 1 but not Th2 cell lines. Archivum Immunologiae et Therapiae Experimentalis 44, 5 1 -56 Zimmerman, D . R. , Speer, V.C, Hays, V .W. & Catron, D.V. ( 1 959). Inj ection of iron­ dextran and several oral ir on treatments for prevention of iron-deficiency anemia of baby pigs. J Animal Sci. 1 8 : 1 409 Zimmerman, D. R. ( 1 986). Role of subtherapeutic antimicrobials in animal production. Journal of Animal Science 62: (Suppl.3) .6-x 1 2 1 Zioudriou, C. , Streaty, R. A. & Klee, W. A. ( 1 979) Opioid peptides derived from blood food protein : the exorphins. Journal of Biological Chemistry 254: 2446- 2449. Zomborszky-Kovacs, M., Tuboly, S., Biro. , H . , Bardos, L. , Soos, P . , Toth, A . & Tornyos, G. ( 1 998) . The effect of �-carotene and nucleotide base supplementation on haematological biochemical parameters in weaned pigs. Journal of Animal and Feed Sciences 7 :245-25 1 1 22 7 . LIST O F APPENDICES Annexe 1 : The statistical significance of effects of farm, dieta, and their interactions on different parameters in weaned pigs Parameter fam1 Absolute cel ls WBC Neutrophils •• Lymphocytes ••• Monocytes Eosinophil cells ••• Basophils ns RBC ••• Haemoglobin ••• Haematocrit ••• Mean * * Corpuscular Volume Mean corpuscular ••• haemoglobin CHCM • • • RDW • • • Cel l changes WBC • • NEUT • LYMPH • • • MONO ns EOSI • • • BASO ... RBC • • • HGB ••• HCT ... MCV ns d iet" Farm x diet ns ns ns ns ns ns ns ns ns ns ns ns ns ns ns ns ns ns ns ns ns ns ns ns ns ns ns ns ns ns ns ns ns ns ns ns ns ns ns ns ns ns ns ns ns day farm x day diet x day farm x diet x day ... ns ns ns 0.76 ••• ns ns ns 0.62 • • • • • ns ns 0.64 • • • ns ns ns 0.46 ••• ns ns ns 0.59 •• ns ns 0.61 ••• • • ns ns 0.75 ns ns ns 0.75 ns •• ns ns 0.74 ns ns ns 0.8 1 ns ns ns 0.91 ns ns ns 0.88 ns ns ns ns 0.93 ns ns ns ns 0.82 ns ns ns ns 0.84 ns ns ns ns 0.84 ns ns ns ns 0.7 1 ns ns ns ns 0.85 ns ns ns ns 0.87 ns ns ns ns 0.9 ns ns ns ns 0.9 ns ns ns ns 0.92 ns ns ns ns 0.96 1 23 MCH CHCM RDW Mean weekly Faecal Score L:N or stress factor • • • • • • • • ns •• ns ns ns ns ns ns ns ns ns ns ••• ns ns ns ns ns ns ns ns ns ns ns ns ns ns ns ns 0.89 0 .88 0 .87 0.4 0.80 white blood cells (WBC), neutrophil cells (NEUT), lymphocyte cel ls (LYMPH), monocyte cells (MONO), eosinophil cel ls (EOS), basophil cells (BASO), red blood cells (RBC), haemoglobin concentration (HGB), and erythrocyte indices viz., haematocrit level (HCT), mean cel l volume (or mean erythrocyte volume) (MCV), mean cell haemoglobin (or mean erythrocyte haemoglobin content) (MCH), mean corpuscular haemoglobin concentration (or mean erythrocyte haemoglobin concentration) (MCHC), corpuscular haemoglobin constant (CH), Corpuscular haemoglobin concentration mean (CHCM), red blood cel l distribution width (or erythrocyte distribution width)(RDW), haemoglobin distribution width (HDW), platelet (PL T) and mean packed volume (MPV); and lymphocyte to neutrophi l ratio (L;N) or stress factor a d iet A (contro l ) , probiotic 8, C, 0 and lactoferrin (E) R2 R-square ns, * , * * , * * * : not significant, significant at p = 0.05, p=O.O l and p=O.OO l respectively. 1 24