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. .. THE EFFECT OF CONDENSED TANNIN UPON THE PROTEIN NUTRITIONAL VALUE OF SOL VENT EXTRACTED COTTONSEED MEAL FOR RUMINANT AND MONOGASTRIC ANIMALS A Thesis Presented in Partial Fulfilment of the Requirements for the Degree of Doctor of Philosophy in Animal Science at Massey University FENG YU 1995 DECLARATION The studies presented in this thesis were completed by the author whilst a postgraduate student in the Department of Animal Science, Massey University, Palmerston North, New Zealand. This is all my own work and the views presented are mine alone. Any assistance received is acknowledged in the thesis. All references cited are included in the bibliography. I certify that the substance of the thesis has not already been submitted for any degree and is not being currently submitted' for any other degree. I certify that to the best of my knowledge any help received in preparing this thesis, and all sources used, have.been acknowledged in this thesis. . FengYu PhD candidate � Professor T N Barry Chief Supervisor /t;;k!� ;'/ Dr W C McNabb Co-supervisor October 1995 Professor P J Moughan Co-supervisor t!tvIL . Dr G F Wilson Co-supervisor ABSTRACT (Feng Yu, 1 995 The Effect of Condensed Tannin upon the Protein Nutritional Value of Solvent Extracted Cottonseed Mealfor Ruminant and Monogastric Animals. Ph D thesis, Department of Animal Science, Massey University, Palmerston North, New Zealand) A series of indoor experiments were conducted at Massey University and AgResearch Grasslands, Palmerston North, New Zealand, to study the effect of cottonseed condensed tannin (cr) upon the nutritional value of solvent extracted cottonseed meal (CSM) for ruminant and monogastric livestock. Ruminant nutrition experiments were conducted using samples suspended in situ in the rumen of fistulated sheep and by incubating samples with rumen fluid in vitro, to study effects upon solubility and degradability of cottonseed proteins. Monogastric nutrition experiments were done initially with laboratory rats as a model for production animals such as the pig, and then with pigs. In all cases half of the animals were supplemented with polyethylene glycol (PEG; MW 3500). PEG specifically binds and inactivates cr and can be used to deduce the effects of CT by comparing control animals (CT acting) with PEG supplemented animals (CT inactivated). 1 . Experimental varieties of cottonseed and of industrial CSM were analysed for extractable and bound cr and free gossypol, crude protein, oil and fibre. CT was present in the hulls of all varieties, with higher concentrations recorded for high tannin and glandless selections (55 g kg- I and 58 g kg- I DM) than for the multiple host plant resistant and high gossypol selections (38 g kg- I DM). cr was present in trace amounts in the kernels of high tannin selections, but was not detected in the kernels of all other selections. On average for the hulls of all varieties, approximately 22, 60 and 18% of total CT was present in the extractable, protein-bound and fibre-bound forms, respectively. Free gossypol was mainly found in the kernels, with negligible amounts being found in the hulls of the experimental varieties. Kernels of high gossypol selections contained higher concentrations of free gossypol ( 18 g kg-I DM) than kernels of multiple host plant resistant, high tannin and commercial selections ( 10-1 2 g kg-l DM), with free gossypol concentration being very low (0.8 g kg- I DM) in the kernels of glandless cottonseed. A negative correlation (r = -0.50, P 10,(00) fraction affords an estimate of endogenous loss. Inclusion of hulls in the EHC based diet increased ileal flow of total N ( 1 387 vs. 1623 mg kg-1 dry matter intake; p8.0 (Jones and Mangan 1977). Hagerman and Butler (1978) reported that precipitation was greatest at a pH within ± 1 unit of the isoelectric points of protein. The CT composition and their molecular size or weight also influences the protein precipitating properties of cr. Bate­ Smith (1973) reported that the minimum molecular weight for effective protein precipitation is about 350 Da. Horigome et al. (1988) showed that for a given cr, the protein-precipitating capacity increased with the increasing degree of polymerisation. Proteins having open, loose conformation, high molecular weight and high contents of proline and other hydrophobic amino acids have a high affinity for cr (Asquith and Butler 1 986). cr can also react with carbohydrates (or polysaccharides), however, the reactions are less well understood than with protein. McLeod (1974) showed that the precipitating capacity of cr with protein was stronger than with carbohydrates. Observation showed a similar pattern of affinity to those noted for proteins, in particular the affinity of polyphenols for sites and environments which permit the hydrophobic interactions to develop (Haslam 1989). Polyethylene glycol (pEG) can .specifically combine with cr to form cr -PEG complexes. The bonds in these complexes are stronger than the bonds in cr -protein complexes. Thus PEG can be used to displace protein from the cr -protein complexes (Jones and Mangan 1 977 ; Barry and Manley 1986). Therefore, effects of cr can be quantified by comparing controls (cr acting) with PEG treatments (cr inactivated). This reaction provides a unique way to study the effect of cr without affecting other nutrients in the diet (Oh et al. 1980; Waghom et al. 1987a; Barry 1 989; McNabb et al. 1993 ; Wang 1 995). 1. s. 2. 3 Analytical Methods for Tannin Detection 3 1 A major problem in studying tannins is the tendency of phenols to oxidise during sample preparation and extraction (Gundagni et al. 1949; Craft 196 1 ). Chopping or macerating the fresh plant material or wilting in the field was found to reduce the extractable CT content in forage (Lyford et al. 1967). Drying forage samples in a forced draught oven can also cause oxidative changes to tannins (Goldstein and Swain 1 963). Suitable reducing agents have been shown to increase yields of tannins when added to the extraction solvents (Khanna et al. 1 968). In some species of legumes such as sainfoin the level of tannin extraction can be improved by fine milling ( 100 mesh) the leaves (Bate-Smith 1 973b). Various methods are available for quantification of polyphenolic compounds. The methods are based on their chemical properties, for example, Folin-Ciocalteu, Folin­ Denis, Prussian Blue, ferric ammonium citrate, and formaldehyde-HCI methods are used to determine total phenols. The vanillin-HCI and butanol-HCI methods are specific to cr and were developed to measure extractable cr. Colorimetric methods have been discussed at length by Deshpande et al. ( 1986). Lately, attention has been focused on quantification of tannins based on their operational property viz. binding/precipitation of proteins because the nutritional, physiological and ecological roles of tannins are attributed to the complexing of tannins with proteins (Deshpande et al. 1 986; Mole and Waterman 1 987a). Other methods employing nuclear magnetic resonance (NMR), near infra-red (NIR) and HPLC (Makkar 1989; Haro et al. 1 989; Okuda et al. 1989) are less frequently used because of high cost and the complexities involved. Different chemical methods measure different classes of phenolic compounds. Therefore, these methods besides providing information on levels of tannins in a sample also help in elucidating their structure. Different methods require different tannins as standards and the values obtained for the sample are expressed relative to the standard. Two types of standards are used: absolute standard, obtained by purifying the standard from the plant of interest; and relative standard, commercially available tannins. The use of absolute standard, although desirable, is not always practicable, due to laborious methods of obtaining well characterised tannins (Hagerman and Butler 1989). The relative standard, such as tan.nic acid for methods based on the oxidation-reduction principle and protein precipitation, catechin for vanillin-HCI assay and quebracho tannin for butanol-HCI method, has been widely used (Makkar 1993). However, various impurities are present in the sample of tannic acid and quebracho tannins (Hagerman and Butler 1 989). The commercially available purified and well characterised cr standards are very important in polyphenolic research. Use of such standards ensures reproducibility within and between laboratories and allows meaningful comparison of values obtained by different laboratories. 32 Methods generally used by our laboratory are the vanillin-Hel assay and a modification of the butanol-He I colorimetric procedure (Porter et al. 1986), as described by Terrill et al. ( 1992a). In the latter method, a technique for separation of total CT into extractable, protein-bound and fibre-bound fractions was developed, and the butanol­ Hel procedure was also modified to use aqueous extracts of these three fractions. Developments or modifications include a more rapid procedure for obtaining extractable CT, the use of different standard curves for extractable and bound CT, the use of aqueous plant extracts and an evaluation of the effects of boiling time. The fractionation of cr into extractable, protein- and fibre-bound cr is important for some kinds of studies, particularly for studies involving protein concentrate meals, since the cr in protein concentrate meals, unlike CT in forage, is mainly in the protein- and fibre-bound forms (see Table 1 . 6). This procedure has been used to measure contents of extractable and bound cr in many samples of different feedstuffs (Terrill et al. 1992a; Wang et al. 1994), with the standard being CT extracted from Lotus pedunculatus. Table 1 . 6 Extractable and bound condensed tannin contents (g kg-l DM) of a range of forages and protein concentrate meals, determined by the butanol-He I procedure. �QnQens� Illonins Extract- Protein Fibre Total able bound bound Reference Legumes Canary clover 83.0 54.0 6.0 143.0 Terrill et al. (1992a) Big trefoil 61.0 14.0 1 .0 76.0 Sulla 33.0 9.0 3.0 45.0 Birdsfoot trefoil 27. 1 6. 1 1 .8 35.0 Wang et al. ( 1994) Grasses Perennial ryegrass l . l 0 0 l . l Terrill et al. (1992a) Yorkshire fog 1 . 1 0.3 0.4 1 .8 Protein concentrate meals Cottonseed 2.1 10.0 3.9 16.0 Rapeseed 0.7 3.7 1 .5 5.9 So�a bean 1 .0 0 0 1 .0 1. 5. 2. 4 Effects of Tannins on Animal Nutrition Numerous studies have been conducted on the effect of tannins in feedstuffs on animal performance. Some of these have been carried out with isolated tannins from feedstuffs or with "standards" of commercial tannins, such as tannic acids, which were thought to 33 be representative of tannins in a number of feedstuffs . Most studies, however, were carried out with raw or fractionated feedstuffs (e .g. hulls of legume seeds) of the same plant species and forages containing different levels of tannins as analysed by one of the available analytical methods . In these studies the effects or differences found were fully or partly related to the differences in tannin level in the experimental diets. 1. 5. 2. 4. 1 Monogastric Animals Ikrior and Fetuga (1984), Balogun et al. (1990) and Terrill et al. (1992a) have detected cr, as a further group of secondary compounds present in commercially produced CSM. cr are likely to further reduce protein nutritional value in diets for monogastric animals (Huisman et al. 1990; Longstaff and McNab 1991; Jansman 1993). Table 1. 7 summarises the nutritional effects of tannins in several feedstuffs on the performance of monogastric animals , and upon nitrogen, amino acid and energy digestibility in these species. Some general observations presented in Table 1. 7 are: i) it has not been conclusively demonstrated that tannins will reduce feed intake in monogastric animal species; ii) tannins in diets generally will reduce weight gain and impair feed conversion efficiency in growing animals; iii) tannins reduce the apparent digestibility of protein, amino acids and, to a lesser extent , energy. The large number of variables that tend to modify the harmful effects of tannins limits the usefulness of direct comparison among the different studies. Tannins are known to have a bitter or astringent taste and may reduce palatability and hence feed intake. In contrast , it has been suggested that a slightly astringent taste increases the palatability of feed and stimulates feed intake (Gupta and Haslam 1980). The physical basis for astringency ·may be that tannins bind and perhaps precipitate salivary proteins and inactivate enzymes (Harborne 1976). This would reduce the lubricating property of saliva, give the mouth a feeling of dryness and affect the animals ' ability to swallow the food (Mole 1989). A second , more direct way by which tannins affect feed palatability may be that tannins directly bind to taste receptors (Mole 1989). Glick and J oslyn (1970) and Vohra et al. (1966) showed a reduction in feed intake in rats and chickens due to the supplementa tion of tannic acid. In contrast , an increased feed. intake was found in chicks fed sal seed, a seed that contains high levels of HT (Zambade et al. 1979). The opposite results , however , were found by Ahmed et al. (1991). The level and type of tannins as well as differences among animal species may explain the contrasting results with respect to the effect of tannins on feed intake. In natural ecosystems there is clear evidence that different animal species select feeds of vegetable origin on the basis of their tannin contents and that the normal or accepted Table 1 . 7 The examples of tannins and their nutritional effects in monogastric species Source Poultry: Sorghum Sorghum Faba bean Tannin level (glkg) 0/19.2 D 0.8/19.1/28.3 D 010.611 .7 S Effect) ADG FC DCn FI FC (% changes) -9.0 +20 -44.4 no effect no effect References Dale et al. ( 1980) Mitaru et al. ( 1985) Jansman et al. (1993b) 34 Faba bean low( l )lhigh(1 ) trDCn - 1 8 (Max) Martin-Tanguy et al. (1977) Pigs: Sorghum Sorghum Faba bean Faba bean Rats: Sorghum Carob tannins Faba bean Faba bean low(2)lhigh(2) 1 .011 .0/36/38 S low(2)lhigh(2) 0.8119. 1128.3 low(2)lhigh(2) 1011511 7 S 10w( 1 )lhigh(3) 0.611 .211 .5 S 5.0n.0/13 S 0114. 1119.9 D lowe 1 )lhigh( 1 ) ADG FI FC il.DCd il.DCn OCd DCn -5.4 +5.9 - 14.6 +0.5 -6.9 +7.4 -10.6 ADG (23-60 kg) -7.5 FC +9.7 ADG (60-103 kg) -6.4 FC +6.7 il.DCn il.DCaa DCn FI DE DCn OCnfe ADG FI FC DCn FI DCaa trDCn NPU -13.2 - 10.7 -6.8 no effect -4.0 -15.4 -3.0 -22.9 -4.7 +23.3 - 1 1 .8 -46.3 -33.3 -3. 1 - 1 1 .8 Myer & Gorbet (1985) Mitaru et al. ( 1984) Grosjean & Castaing ( 1984) Jansman et al. (1993c) Muindi & Thornke ( 1981 ) Tamir & Alumot ( 1969) Jansman et ai. ( 1993a) Ford & Hewitt (1979) 1 Difference between the value for the high-tannin group(s) and the control or low-tannin group(s). D or S : level in diet or source il.DCd : apparent ileal DM digestibility (units) ADG : average daily gain il.DCn : apparent ileal N digestibility (units) FI : feed intake OCd : apparent faecal DM digestibility (units) FC : feed conversion efficiency OCnfe : apparent faecal N-free extract digestibility (units) DE : digestible energy (units) DCn : apparent faecal N digestibility (units) NPU : net protein utilisation DCaa : apparent faecal amino acid digestibility (units) tr.DCn : true faecal N digestibility (units) tannin level in the diets of animals in their natural environment differs among species (Mole 1989). 35 Dietary tannins reduced apparent protein and amino acid digestibilities (Ford and Wewitt 1979, Jansman et al. 1 993a, 1993c). Also a reduced energy digestion in pigs (Duee et al. 1979) and in poultry (Hersted 1 979) has been observed. The dietary tannins also affect vitamin and mineral metabolism (Salunkhe et al. 1 990). However, these effects seem to be less important than the effects on protein digestion. The reduced apparent protein digestibility of tannin-rich feeds may be explained either by a direct binding of tannins to dietary protein, by a reduced activity of protein-degrading enzymes (Longstaff and McNab 1991 ; Griffiths 1979, 1 98 1 ), or by increased secretion of endogenous proteins (digestive enzymes, mucus or mucosal cells; Mangan 1 988; Marquardt 1989). Griffiths ( 1 98 1 ) determined the activity of digestive enzymes in intestinal contents of rats fed diets containing hulls of high- and low-tannin varieties of faba bean. Activities of trypsin, chymotrypsin and (X-amylase were reduced in animals fed the high-tannin diet. Tannin-containing extracts from rapeseed (Yaper and Clandinin 1972), green gram and ripe carobs (Tamir and Alumot 1969), chickpeas and pigeon peas (Singh 1 984) have also been found to impair the in vitro activity of digestive enzymes. Griffiths and Moseley ( 1 980) suggests that dietary tannins may also increase pancreatic secretion of digestive enzymes, and in some animal species tannins may stimulate pancreatic secretion in a manner analogous to that of protease inhibitors from legume seeds (Liener 1 989). This could explain why dietary tannins in some cases increase activities of lipase in intestinal digesta. 1. 5. 2. 4. 2 Ruminant Animals Tannins are present in many forages and fodder tree leaves, agricultural by-products and several agricultural wastes, where they often provide a natural protective system against attack by micro-organisms, insects or against being eaten by ruminants. High concentration of CT in plant material restricted the voluntary intake and reduced both body gain and wool growth rates of sheep (pritchard et al. 1988, 1 992). Barry ( 1 985) found oral supplementation of PEG to lambs grazing L pedunculatus (76-90 g extractable cr kg-I DM) increased live weight gain and wool growth by 4 1 -6 1 g d-I and 23 mg ( 100 cm2)-I , respectively, indicating that these levels of CT were limiting animal production. Barry et al. ( 1986a) and Barry ( 1989) suggested that for the forage diets, levels of extractable cr in the range 20-40 g kg-I DM may be beneficial in terms of dietary 36 protein utilisation. Studies (Ulyatt et al. 1977; John and Lancashire 198 1 ) showed that animal performance was improved by feeding low CT -containing forages compared with other non CT -containing forages, such as red clover, lucerne and perennial ryegrass. However, these results cannot be considered as solely due to cr in the diets, since the forages differed in several other aspects. Terrill et al. ( 1992b) using oral PEG supplement to the grazing sheep showed that the action of cr in sulla (total CT 40-50 g kg-1 0M) increased live weight gain and wool growth. They, however, suggested that PEG supplement might not completely render all CT inert, and the animal response to the CT might not be fully expressed. Barry and Manley ( 1984) established a significant linear relationship between dietary cr concentration and duodenal non-ammonia nitrogen (NAN) flow in sheep fed forage diets (Fig 1 . 7). NAN flow out of the rumen per unit N eaten increased as forage extractable CT concentration increased, and it became unity at the value of 40 g cr kg-I OM (i.e. undegraded dietary N+microbial N flowing at the duodenum equalled the N eaten). Waghom et al. ( 1 987a, 1994) showed that the abomasal amino acid flow was increased significantly by CT, particularly for essential amino acids, when sheep were fed Lotus species (Table 1 . 8). McNabb et al. ( 1993) found that the cr in L. pedunculatus (extractable cr 55 g kg-1 0M) reduced the proteolysis of forage sulphur amino acids in the rumen. Unlike the effect of CT upon rumen protein degradation, there was no clear relationship between the apparent post-ruminal digestibility ofN and CT concentration. However, there do apPear to be differences between concentrations of cr upon the apparent post-ruminal digestion of essential amino acids. The low concentration of cr in L comiculatus (22 g extractable cr kg-1 0M) increased the apparent absorption (proportion of intake) of essential amino acids in the small intestine, whereas the high concentration of cr in L pedunculatus (55 g extractable CT kg-1 0M) decreased the apparent digestibility of essential amino acids (excluding methionine and cysteine; Waghorn et al. 1 987a, 1 994; Table 1 . 8) in the small intestine and resulted in no increase in net absorption. The effect of CT on post-ruminal N digestion may be a product of . both concentration and sources of CT. The depression of apparent amino acid digestibility in the small intestine of sheep fed L pedunculatus may be due to slow release of amino acids from the CT:protein complex, which will affect the true digestibility, and/or ; increase endogenous protein losses. The mechanism of CT -carbohydrate formation is similar to that of CT -protein, but CT react more strongly with protein than with carbohydrate and compared with cr­ protein, the CT -carbohydrate complex is less stable (McLeod 1974). In forage materials, usually only a small amount of CT binds with carbohydrate either before or after animal 37 chewing (ferrilJ et al. 1 992a). Therefore it is reasonable that the interaction between CT and carbohydrate has a lower influence on the digestion of carbohydrate. However, this situation may not apply to some protein concentrate meals since the majority of CT in those meals is in bound forms, either with protein or with fibre (fable 1. 6). Barry and Manley ( 1984) found that the high CT concentration ( 106 g kg- l DM) in L. pedullculatus reduced readily fermentable carbohydrate, structural carbohydrate and for lignin digestibility in the rumen, but this was compensatedrPy increased digestibility in the intestine (Table 1. 9). In contrast, the low CT concentration (22 g kg-I DM) in L. comiculatus had no effect on the digestion of both water soluble carbohydrate and structural carbohydrate (Ulyatt and Egan 1979; Waghom et al. 1987b). II> .lo:: co C z -;;; e � 0 - z < z ::; c; C> "C 0 :::l 0 1 ·4 1 ·3 1 ·2 1 ' 1 1 -0 O·g o-a 0-7 0'6. • o 1 20 Fig. 1 . 7 Duodenal non-ammonia nitrogen (NAN) flow per unit total N intake as a function of forage condensed tannin (CT) concentration in sheep fed on Lotus species. 0, high extractable CT ( l 06g kg-I DM) Lotus pedunculatus; e, low extractable CT (46g kg-I DM) Lotus pedunculatus; A. high extractable CT (14.5 g kg-I DM) Lotus comiculatus; .A, low extractable CT (2.5 g kg-I DM) Lotus comi�ulatus (John & Lancashire 1981 ); (!I, short ratetion ryegrass; 0, perennial ryegrass; . , white clover (MacRae & Ulyatt 1974) and x, sainfoin (Ulyatt & Egan 1979; Source: Barry and Manley 1984) Table 1 . 8 The effect of condensed tannins (Cf) upon the digestion of essential amino acids in sheep fed fresh Lotus comiculatus (22 g extractable cr kg- 1 0M) and Lotus pedunculatus (55 g extractable Cf kg-1 0M; Sources: Waghom et al. 1987a, 1 994) L. comi{;;.ulatus L. rJedunc.111gtu� Control PEG Control PEG 38 Amino acid intake (g dol ) 98.9 98.9 103.2 1 16.8 Abomasal flow (g d-l) 84.7 55.5 12 1 . 1 105.6 Proportion of intake 0.86 0.56 1 . 1 7 0.90 Apparent absorption from small intestine (g dol ) 58.8 36.2 8 1 .4 83.5 Proportion of intake 0.59 0.37 0.79 0.72 Proportion of abomasal flow 0.67 0.67 0.67 0.79 Table 1 . 9 Ruminal and post-ruminal digestion of readily fennentable carbohydrate (RFC) and structural carbohydrate in sheep fed Lotus pedunculatus differing in extractable condensed tannin (Cf) content (Source: Barry and Manley 1984) Low-Cf Lotus. (46 g kg-l..DM} High-Cf Lotu� (106 g kg- l DM) RFC Hemi- Cellulose RFC Hemi- Cellulose cellulose cellulose Apparent digestibility Proportion of intake 0.95 0.73 0.78 0.93 0.56 0.63 Ruminal digestion Proportion of intake 0.80(0.93) 1 0.44(0.58) 0.69(0.69) 0.78(0.93) 0.21 (0.42) 0.53(0.54) Proportion of total digestion 0.84 0.61 0.89 0.83 0.38 0.85 Post-ruminal digestion ProE2rtion of intake 0. 15(0.06) 0.28(0. 15) 0.09(0.09) 0.16(0.06) 0.35(0. 14) 0 . 10(0.09) I Number in parentheses are predicted from the equation of Ulyatt & Egan (1979); derived with non­ tannin-containing fresh forages. Compared to the effect of cr in forages, the effect of CT in protein supplement meals on animal production is less understood. Lambs fed tannin-treated or untreated soya bean meal for a 1 6 day preliminary period gained 217 and 1 17 g d-I , respectively (Driedger and Hatfield 1972). The N retention was 7.5 and 10.6 g d- I for untreated and treated meal. However, the effect of the treatment on stability of the pellets may have been responsible' for some of the beneficial effects of tannin. Balance studies with sheep fed skim milk powder treated with chestnut tannin showed a small decrease in digestible N (68.2 vs 72.0%), but a significant improvement in N retention (23.6 vs 1 6. 1 %; Oelort­ Laval et al. 1 972). ZeIter et al. ( 1970) reported that treatment of peanut, soya bean, linseed, rapeseed and sunflower seed meal, dried skim milk and casein with aqueous solutions of chestnut tannin prevented their degradation by rumen microorganisms. 39 Ehoche et al. (1983) compared cottonseed cake treated with Bagaruwa tannin at O. 50 and 100 g kg-I . They found that all levels of tannin treatment of cottonseed cake reduced ammonia accumulation in the rumen. Average daily gains were increased for the 50 g kg-I level, but decreased for lOOg kg-I group. The treated cake reduced N digestibility by 3% units in the 100g kg-I tannin group, and N retention was improved by 50g kg-I but not lOOg kg-I level of tannin treatment. 1. 5. 2. 5 Animal Defensive Response towards Dietary Tannins A number of herbivorous species consume tannin-rich feedstuffs as a part of their natural diet, without showing severe toxic or otherwise detrimental effects. The effect of tannins is minimised by physiological (Bemays et al. 1989) or behavioural (Roy and Bergeron 1990) adaptations that neutralise dietary tannin. The major physiological adaptation in mammals that has been identified so far is production of salivary tannin-binding proteins (Mehansho et al. 1987b; Austin et al. 1989; Robbins et al. 1991; Hagerman and Robbins 1993). When rodents such as rats are fed tannin-rich diets, they show an initial loss of body weight, and then the animals start to gain weight again (Glick and Joslyn 1970b; Mehansho et al. 1983). The latter authors found that in the adapted animals the parotid glands had undergone dramatic hypertrophy, accompanied with an increase in production of a series of proline-rich proteins (PRPs). It was subsequently shown that these proteins have a very high binding affinity for tannins, being ten times higher than the affinity of BSA (Butler et al. 1982). It is assumed that the secreted PRPs in animals receiving a tannin-rich diet act as binding agents for tannins, thereby preventing other harmful and antinutritional effects {Butler et al. 1986). The PRP response due to dietary tannins was also found in mice (Mehansho et al. 1985), but not in the hamster (Mehansho et al. 1987a). The mechanism of. PRP .induction by dietary tannins is most likely mediated via �­ receptors, but the exact mechanism is unknown (Butler et al. 1986; Jansman 1993). Other functions of these proteins have been described (Bennick 1982) or suggested (Mole et al. 1990). Besides the adaptive mechanism of the parotid glands of rodents towards dietary tannins, no information is available on adaptive mechanisms in other simple-stomached species, including those important farm animals, such as pigs and poUltry. The high tannin diets of many browsing ruminants may be associated with active defences against plant tannins. Increased secretion of salivary proteins that bind and, 40 thereby, neutralise tannins may also be one defence used by browsing ruminants. Provenza and Malechek ( 1 984) suggested that salivary or plant protein consumed by goats might bind with as much as 50% of the dietary tannins during ingestion. Robbins et al. ( 1987) conducted a study to measure binding capacities of saliva in ruminants with different feeding habits, and parotid salivary gland size has been measured in several ruminants (Hofmann 1973; Kay et al. 1980). Parotid salivary glands (g kg-l body weight) are three times larger in browsing ruminants (Le. goat and deer) than in domestic ruminants (Le. sheep and cattle). Those authors suggested that size of different parotid salivary glands are best explained by the range in tannin intake and the requisite production of salivary proteins. The production of glycoproteins for lubrication (Clifford 1 986) and proline-rich salivary proteins to bind dietary tannins, the rapid disintegration of leaf cell walls and passage from the rumen (Spalinger et al. 1986), and the higher rumen fermentation rate (Hungate 1959; Prins and Geelen 1 97 1 ) in browsing ruminants are essential for tree and shrub leaves to be useful food for animals. 1. S. 3 Other Antinutritional Factors 1. S. 3. 1 Cyclopropenoid Fatty Acids The cyclopropenoid fatty acids (CPFA's) are acids structurally relat�d to oleic acid that contain a cyclopropane ring at the site of the double bond. Two of these, malvalic acid and sterculic acid, occur naturally as esters in plants of the Malvales order, which includes all of the Gossypeae (Frank 1987). CH /\ 2 CH3(CH2hC=C(CH2)n COOH n=6, malvalic acid n=7, sterculic acid The CPF A content varies significantly with cultivar and with location (Cherry et al. 1 978). and is much higher in immature seed. The level of CPF A's is highest in G. arboreum species and lowest in G. barbadense species. Examination of individual seeds shows that 75% of the CPFA is located in the axial tissue. almost half of it in the radicle tip. The axis and radicle tip together account for only 5% of the weight of the kernel (Fisher and Cherry 1983). The ratio of malvalic to sterculic acid in cottonseed is usually about 2.50-2.75: 1 . and is independent of location in the seed. Malvalic acid is not as physiologically active as sterculic acid (Jones 1 98 1 ). Cottonseed also contains dihydromalvalic and dihydrosterculic acids. the immediate precursors of malvalic and 41 sterculic acid in the biosynthetic pathway from oleic acid (Bianchini et al. 1 98 1 ), but these are of no concern since the saturated acids are physiologically inactive whether as denaturation inhibitors or cocarcinogens (Jones 198 1 ). The level of the CPFA's ranges from 2 1 to 1 53 ppm (0.02 1 -0. 153 g kg-I) in CSM and from 5 to 1 5 g kg- I in crude cottonseed oil (Levi et al. 1967; Martinez et al. 1970). The more oil left in the meal, the higher the CPF A content. The CPF A content of CSM can be reduced to 5- 10 ppm by successive extraction with solvent mixtures such as acetone-hexane-water. Hexane alone is less efficient (Reilich et al. 1968). Chemical inactivation with anhydrous sulphur dioxide reduces the CPF A content of CSM by over 90%; organic acid and sultbydryl compounds are only partially effective and require the use of solvents (Reilich et al. 1969). In practice, the CPFA content is minimised by processing only mature seed and by extracting the oil as thoroughly as possible. The CPF A's were of little concern other than for their ability to discolour egg whites when cottonseed oil was added to the di�ts of laying hens, but it was later discovered that both compounds are carcinogenic to rainbow trout, particularly, when combined with aflatoxin BI (Anon 1973). An excellent review of the biological effects of various cyclopropenoid compounds is available (Phelps et al. 1965), so only general comments in regard to the effects of these compounds on animal production will be made here. Although it is possible to demonstrate adverse effects of such compounds on the growth rate of broilers, it requires extremely high levels which would not nonnally be encountered from CSM per se (Waldroup 198 1 ). However, the. CPFA's may cause detrimental effects in laying hens, including disturbed lipid metabolism and release of iron from yolk, through their effects on membrane permeability. These acids are also inhibitors of desaturase enzymes, which if protected from rumen hydrogenation in lactating cows, increase the proportion of stearic acid at the expense of oleic acid (Cook et al. 1976). However, these workers showed that the effect of these acids on stearic acid was considerably less if they were unprotected in the rumen, which suggests that during feeding of whole cottonseed most of the CPFA's are probably saturated. The CPF A's can be carried into the food chain by ingestion of animal products that have been fed with them. An appreciable portion of the ingested acids is deposited in the egg yolks of hens and turkeys, and in the body fat of laying hens and dairy cattle (Anon 1973). 1. 5. 3. 2 Pbytate Phytate, the salt of phytic acid, is a cyclic compound (inosited) containing six phosphate radicals. The concentration of the phytic acid in CSM varies between 29-43 g kg-I depending on different process methods (Smith 1970; Vix et al. 197 1 ; Gardner et al. 1976). 42 Physiological significance of phytate lies in the fact that it readily chelates with di­ and tri-valent metal ions such as calcium, magnesium, zinc, and iron to form poorly soluble compounds that are not readily absorbec:i from the intestines (Huisman 1 989). Thus, phytate has generally been regarded as an antinutritional factor which interferes with bioavailability of minerals from plant sources (Reddy et al. 1982; Forbes and Erdman 1983). It has been shown that high dietary calcium accentuates the effect of phytate on zinc bioavailability. The formation of Zn-Ca-phytate complexes in the upper intestinal tract of monogastric animals is believed to be a major mechanism by which phytate reduces zinc bioavailability. The bioavailability of zinc can be best predicted by the expression: phytate-calciumlzinc molar ratio (Fordyce et al. 1987). Kumar and Kapoor ( 1 983) reported that rats receiving diets containing the lowest phytate:zinc molar ratio ( 1 3.7: 1 ) had the highest protein efficiency ratio 1 .97 while rats with the highest phytate:zinc molar ratio (38.5: 1 ) had the lowest protein efficiency ratio. Although the ability of phytate to interfere with the availability of minerals accounts for its major antinutritional effect, phytate has also been shown to interact with basic residues of proteins. Bailey ( 1948) reported that phytic acid is responsible for the decreased solubility of cottonseed protein at acid pH. It is not surprising, therefore, that phytate inhibits a number of digestive enzymes such as pepsin, pancreatin, and a-amylase (Liener 1 989; Savage 1989). Inhibition may also result from the chelation of calcium ions which are essential for the activity of trypsin and a-amylase, or possibly to an interaction with the substrates for these enzymes. Phytic acid is not removed from cottonseed meal by solvent extraction or air classification (Wozenski and Woodburn 1975), but tends to concentrate in the non­ storage protein isolate on fractionation (Berardi et al. 1 969; Lawhon 1975). The phytate content can be reduced by taking advantage of the endogenous enzyme, phytase, which accompanies phytate in separate compartments of the plant tissue, or by providing an exogenous source of the enzyme from microbial sources (Liener 1987). Thus the phytate content can be greatly reduced by simply allowing aqueous suspensions of cottonseed meal to undergo autolysis under appropriate conditions of time, temperature, and pH. 1. S. 3. 3 Aflatoxin Aflatoxin contamination of cottonseed is uncommon, and where it does occur the level is usually very low. Nevertheless, high levels of aflatoxin B I are occasionally found in seeds from bolls that have been infected with A. flavus (Marsh et al. 1 969). Aflatoxin, , whose presence is readily detected by its bright greenish-yellow fluorescence (Marsh and Simpson 1 984), tends to be carried with the protein when contaminated cottonseed is 43 processed into meal, concentrates and isolates (Stoloff et al. 1976). It is insoluble in hexane but can be extracted from cottonseed meal by polar solvents such as aqueous 2- propanol (Rayner et al. 1977). The aflatoxins can be inactivated by treating the contaminated cottonseed meal with ammonia at elevated temperature and pressure (Gardner et al. 197 1 ). The effect of this treatment on the aflatoxin and the protein quality of the meal have been studied (Lee \ and Cucullu 1978; Conkerton et al. 1980). Although the treatment is not approved for human use, fears that the products of ammoniation might be mutagenic have been allayed (Lawlor et al. 1985). 1. 5. 3. 4 Allergens Cottonseed, in common with other oilseeds, contains a number of allergens that are capable of inducing allergic responses in hypersensitive people (Frank 1987). Most of these allergens are water-soluble and are readily isolated from solvent-extracted CSM by extraction with water (Bailey 1948). The most potent cottonseed allergen, CS- l A, comprising 13.8 g kg-I of the meal? is identical to a cottonseed 2S protein as judged by gel-electrophoretic pattern, amino acid composition and immuno-cross-reactivity (Spies et al. 195 1 ; Y oule and Huang 1979). Another type of allergen, designated 2CS, is present in the water-insoluble globulin fraction of cottonseed. Little is known about this allergen (Frank 1 987). The water soluble antigens found in cotton bract or textile mill dust implicated in the byssinosis of cotton mill workers are not present in defatted CSM or hulls. 1. 6 EFFECT OF HEAT AND SOLVENT EXTRACTION ON THE NUTRITIVE VALUE OF COTTONSEED MEAL Table 1 . 1 0 presents the nutrient composition of CSM is produced by the three primary processes (screw press, prepress solvent extraction and direct solvent extraction). They were derived from a comprehensive study involving more than 1 ,300 individual analyses (Jones 198 1 ). Screw pressed CSM is relatively high in residual lipid and is low in free gossypol and has a low protein quality for non-ruminant animals. The high heat and pressure conditions of screw pressing seem to decease the availability of some of the lysine (Table 1 . 1 0). Pr:epress solvent meals are low in residual lipid and free gossypol and have moderate to high protein quality. The direct solvent meals have a high protein quality, : have moderate · . residual oil, and high free gossypol. In a comparison of different processing methods (Luo et al. 1994), screw pressing yielded a lysine availability for chicks of 0.53, prepressing yielded a lysine availability of 0.76 and direct solvent meal yielded a lysine availability of 0.83. Table 1 . 1 0 Mean nutrient composition of cottonseed meal by process (on an "as fed" basis; Sources: Smith 1970; Jones 1 981 ) Screw Prepress Direct Composition Press Solvent Solvent Dry matter g kg-l 914 899 904 Ash g kg-l 62 64 64 Crude fibre g kg-l 135 13 6 124 Ether extract g kg-l 37.2 5.8 15 1 Crude protein g kg-l 4 10 414 4 14 N-solubility 1 g kg-l , 368 544 694 EAF lysine2 mg 16mg N-l 23.6 30.2 34.8 Gossypol Free g kg-l 0.4 0.5 3.0 Bound g kg-l 9.8 10.8 7.4 Total g kg-l 10.2 1 1 .3 10.4 1 Nitrogen soluble in 0.02 N NaOH. 2 Epsilon amino free lysine "available lysine" (Rao et aI. 1963). 44 In the processed meal, the remaining free gossypol plus that which is bound, equals the total gossypol. Therefore, the total gossypol content of CSM is not affected by the process used in oil extraction_ Free and total gossypol contents for different meals are given in Table 1 . 10. The degree of binding is also critical due to the importance of available lysine, especially when the meal is to be fed to monogastric animals. This creates the trade off in CSM where more bound gossypol results in a lower level of the already marginal lysine. It is unknown at this time if some of the bound gossypol, is released in the gut of the animal (Martin 1990) and this is a fertile area for further research. For ruminants, the value of dietary protein is influenced by the proportion of amino acids bypassing the rumen without being degraded to ammonia (Chalupa 1 984; Preston and Leng 1 987). The rumen degradabilities of CSM protein vary, resulting in different bypass characteristics_ Screw press meals usually have a low rate of ruminal solubility, hence one would expect that greater amounts of dietary protein in this type of meal are presented to the intestine for digestion and assimilation by the animal_ Goetsch and Owens ( 1 985) compared the ruminal degradation of CSM protein from different commercial processing methods (screw press, SP; prepress solvent, PP and direct solvent extraction, OS). They found that the rate of in situ N disappearance tended to be greater for OS than for PP and SP meals. In the cow trial, ruminal degradation of supplemental N was lowest for the SP meal (Table 1 . 1 1). Rumen degradation of CSM N did not differ significantly with processing method in the trial with steers, though trends were 45 similar to those found in the cow trial. In both trials, organic matter (OM)and starch digestion were decreased in the rumen and increased post-ruminally with SP as compared with PP and DS. They concluded that the processing method of CSM altered site of protein and OM digestion and that protein degradation will vary with experimental conditions. Table 1 . 1 1 Ruminal degradation of nitrogen (N) and digestibilities of different commercial processed cottonseed meal (CSM) fed to lactating cows and steers (Source: Goetsch and Owens 1985) �QttQn:i�d m�1 Screw Prepress Direct press solvent solvent N solubility (% ofN; in 0. 15 N NaCl) 1 5 29 27 N disappearance (in situ) 4 to 1 2 h (%Ih of 4-h residue) 1 .0 2.0 2.0 1 2 to 20 h (%Ih of 12-h residue) 2.0 1 .0 2.6 In the study with cows: Ruminal degradation of supplemental N (% of intake) 43 (57)1 65 (62) 65 (64) Microbial efficiency (g microbial Nlkg OM fermented) 23 ( 16) 19 ( 14) 22 ( 16) Organic matter digestibility (%) Ruminal 35 (54) 45 (58) 43 (57) Post-ruminal 20 ( 1 5) 1 3 ( 1 1 ) 8 ( 1 1 ) Starch digestibility (%) Ruminal 5 1 (78) 67 (80) 69 (80) Post-ruminal 28 ( 15) 29 ( 1 2) 45 (9) 1 Numbers presented in parentheses are results obtained from the study with steers. Table 1 . 12 presents bioavailable energy values for the various commercially available cottonseed meals. The level of oil in a meal greatly influences the energy level. As noted, screw pressed meals are relatively high in residual lipids, while prepress solvent meals are low and direct solvent meals are intennediate. 1. 7 THE EFFICIENCY OF UTILISATION OF COTTONSEED MEAL FOR ANIMAL PRODUCTION CSM has long been a popular and economic protein concentrate for animal feeding. A relatively low cost has been the prime reason why this by-product of the cottonseed oil extraction industry has been used in feeds for many classes of animals. However, CSM has some natural limiting factors that must be considered for its safe use, particularly for nonruminant animals. Chief among these factors are protein level and quality, fibre level, gossypol and condensed tannin contents (Stem and Ziemer 1 993; Terrill et al. 1 992a; 46 Batterham 1 992; Martin 1 990). In this section, the efficiency of utilisation of CSM in the diets of different animal species is reviewed. The latest recommendations for safe levels of gossypol and CPEA's in the diets are also reviewed. Table 1 . 12 Energy values of commercially processed cottonseed meal (MJ kg- I ; on an "as fed" basis; Source: Jones 198 1) Species and type of meal _______ --!=E""'ne,...r.o.gy1-v ..... a..,lu..,.ei ______ _ Poultry: Screw press Prepress solvent Direct solvent Pig: Screw press Prepress solvent Cattle: DE ME ME-N 1 1 . 1 10.9 10.3 9.8 9.5 9.0 9 . 1 NEp Screw press 13.3 9.8 6.8 Prepress solvent 13 .4 9.8 7.5 Direct solvent 1 3.6 10.4 6 . 1 Sheep: Screw press 13.9 10.3 7.4 Prepress solvent 14.4 1 1 . 1 7.6 Direct solvent 14.3 1 1 . 1 7.2 1MB 9.4 10.2 i DE, digestible energy; ME, metabolizable Energy; ME-N, metabolizable Energy corrected for nitrogen retention; NEp, net energy for production; TME, true metabolizable Energy by the method of Sibbald ( 1976). 1. 7. 1 Cottonseed Meal in Pig Diets CSM can replace a portion of the soya bean meal in pig diets and maintain equal performance, if the nutrient content and presence of free gossypol in CSM are considered in diet formulation (Tanksley and Knabe 198 1 ). Too many times the popular conception of CSM is based on feeding trials conducted many years ago in which CSM, often of poor quality, was the only source of supplemental protein. Many early workers, however, have clearly demonstrated that excellent pig performance, often superior to soya bean meal alone, has been obtained when limited amounts of CSM were fed in combination with other sources of high quality protein in practical growing-finishing diets (Sewell et al. 1955; Haines et al. 1955; Hale and Lyman 1957; Hale and Lyman 1 96 1 ; Tanksley and Lyman 1966, Hintz and Heitman 1967; Tanksley 1 969). The protein quality of CSM for pigs is limited by low levels of several essential amino acids, particularly lysine. Protein quality is further lowered if the CSM is processed at high temperatures, which promotes a reaction between free gossypol and free amino groups in the protein to form an indigestible complex; lysine, because of its 47 free epsilon-amino group, is primarily affected. The lower digestibility (Tanksley et al. 198 1 ) and availability (Batterham et al. 1990) of lysine in processed CSM, compared with soya bean meal, has been reported. Supplementation of cereal grain-CSM based grower-finisher pig diets with synthetic lysine has consistently improved pig performance ( Aguirre et al. 1960; Bell and Larsen 1963; Noland et al. 1968; LaRue et al. 1985, 1987; Ikurior and Fetuga 1988 and Batterham et al. 1 990). Tanksley and Knabe ( 198 1 ) have shown that lysine was the least digestible essential amino acid in processed CSM (Table 1 . 1 3). They found that the digestibilities of the essential amino acids isoleucine, leucine, methionine, threonine, tryptophan and valine were also lower when compared with soya bean meal. Batterham (1 992) discussed the availability and ileal digestibility of some amino acids in CSM for growing pigs and summarised that for lysine, threonine, methionine and tryptophan, heating processed CSM induced changes which depressed ileal digestibility slightly but appeared to result in a substantial proportion of these amino acids being absorbed in inefficiently utilised forrn(s). In contrast, the branched chain amino acids, isoleucine, leucine and valine, appeared less susceptible to the effects of heat. Thus, for these amino acids, reduced ileal digestibility appeared to be the main cause of reduced availability. Table 1 . 1 3 Apparent digestibility values for N and essential amino acids in cottonseed meals and soya bean meal as determined at the terminal ileum of growing-finishing pigs (Source: Tanksley and Knabe 198 1 ) Direct solvent Nitrogen 0.73 Lysine 0.62 Arginine 0.88 Histidine 0.80 Isoleucine 0.68 Leucine 0.70 Methionine 0.71 Phenylalanine 0.81 Threonine 0.62 Tryptophan 0.74 Valine 0.7 1 CQtlQnlieed m�1 Screw press 0.75 0.64 0.90 0.8 1 0.70 0.73 0.66 0.82 0.65 0.68 0.71 Glandless 0.86 0.87 0.96 0.92 0.84 0.85 0.83 0.90 0.79 0.83 0.85 Soya bean meal 0:8 1 0.86 0.90 0.87 0.82 0.8 1 .088 0.85 0.75 0.80 0.80 The digestibility of lysine in a laboratory-processed glandless CSM (0.87) equalled that of soya bean meal, and digestibilities of other essential amino acids were similar or higher to soya bean meal. With supplemental lysine, glandless CSM can replace about one-half of the soya bean meal protein in com-based diets without reducing pig performance (LaRue et al. 1 987). 48 Commercial CSM contains 1 20-1 30 g crude fibre kg-1 0M, which lowers its digestible energy content for pigs. Husby and Kroening ( 1971 ) found an inverse relationship between . crude fibre content and digestible energy values for screw press and prepress solvent CSM. Higher dietary crude fibre content on CSM in pig diets may slightly depress feed efficiency, but the effect should be miniJ}1al. Substitution of CSM for soya bean meal in a grower �iet will increase dietary fibre content by only 1 % (Tanksley and Knabe 198 1 ). LaRue et al. ( 1985) compared energy values in glandless CSM and soya bean meal using growing and finishing swine and 'found that digestible and metabolizable energy for glandless CSM �ere significantly (P8.0, the cr-protein complexes dissociate and protein is released from the complexes. Reactivity increases with increasing polymerisation of the cr. Proteins with open, loose conformations, high molecular weights and high contents of proline and other hydrophobic amino acids have a high affinity for cr. 59 1. 9. 7 With monogastric animals, the presence of extractable cr in the diet has been shown to reduce the efficiency of protein digestion. Extractable CT depresses apparent amino acid digestibility due to a direct binding of cr to dietary proteins, and/or a reduced activity of protein-degrading enzymes, and/or increased secretion of endogenous proteins. However, the effects of bound cr upon protein digestion in the small intestine are unknown. Therefore, the effect of bound cr in CSM upon amino acid digestion and absorption in the small intestine needs to be studied. 1. 9. 8 With ruminants fed forages, low concentrations of extractable cr (20-40 g kg-l DM) improve the efficiency of protein digestion and increase amino acid absorption from the small intestine. However, high concentrations of extractable cr depress feed intake, rumen fibre digestion and apparent amino acid digestibility in the small intestine. Studies are necessary to investigate the effects of bound CT in CSM upon solubility and degradability of both protein and fibre in the rumen. 1. 9. 9 Different manufacturing conditions during processing may be needed to produce CSM that has optimum nutritive value for ruminant and monogastric animals. For CSM produced as a protein supplement for monogastric animals high temperatures and pressures during processing should be avoided. CSM produced for ruminants should use controlled heat to reduce the rumen degradation of protein. Optimum processing conditions for producing the best quality CSM for each species needs to be precisely defined and any interaction between heat (including the amount of heat required), the concentration of cr required and any interaction between cr and heat need to be established. Research is needed in these areas. The research reported in this thesis concentrated upon the effect of cr in CSM for both ruminant and monogastric livestock, with particular reference to effects upon protein digestion. 60 1. 10 METHODOLOGY FOR STUDYING PROTEIN DIGESTION 1.10. 1 Measurement of Amino Acid Digestibility in Monogastric Animals The commonly used procedure for determining amino acid digestibility in pigs has been the faecal index method (Kuiken and Lyman 1948). While the overall apparent digestibility measurement is not technically difficult, there are basic objections to this approach because of the presence of undigested and unabsorbed endogenous protein in the faeces, and possible microbial alterations of undigested and unabsorbed endogenous and exogenous N residues, in the large intestine. As a result, the amino acid composition of faeces in pigs fed diets that differ widely in amino acid composition and digestibility is rather similar (Mason et aI. 1976). From many studies, involving the determination of ileal and faecal N and amino acid digestibilities in pigs, some general findings have emerged, indicating the superior predictive accuracy of ileal digestibility values. Zebrowska ( 1978) highlighted the significance of differences between faecal and ileal estimates of the apparent digestibility of N and amino acids in feedstuffs used in practical dietary formulation. Some studies indicate close correlation between the apparent ileal digestibility of amino acids and animal performance. Moreover, apparent ileal digestibility coefficients have been shown to be sensitive in detecting small differences in protein digestibility due to the processing of foods (Sauer and Ozimek 1986; Knabe et al. 1989) compared with faecal estimates. Tanksley and Knabe ( 1984) concluded that ileal digestibility values offer great potential for increasing the precision of diet formulation for the growing pig. The traditional faecal measurement of amino acid digestibility is inadequate, and the ileal measurement should be used to determine amino acid digestibility. Considerable work has been done with ileal digesta collection methods in pigs, such as the ileo-ileo and ileo-caecal re-entrant cannulation procedures, ileo-rectal anastomosis, nylon bag technique, post-valvular ileo-colic fistulation or ileo-colic post valve procedure and the postvalve T -caesium technique. These methods mainly involve the surgical implantation of cannulas. The use of cannulated pigs for the routine determination of amino acid digestibility is costly, labour intensive and time consuming. Although these methods may be appropriate for research purposes, they are unlikely to be appropriate for routine use in feed evaluation. The method used for the assessment of ileal amino acid digestibility in the studies reported in this thesis involved collecting digesta from the ileum under anaesthesia before sacrifice of the animal (Moughan and Smith 1987; van Barneveld et al. 1 99 1 ) . Digestibility was measured with Cn03 as an indigestible marker given with the test feedstuff. This method involves minimal disruption of normal digestive function. Digestibility data obtained using this technique coupled with a frequent feeding regime were not necessarily any more variable than those obtained from cannulated animals (Moughan 1991 ). In using this technique there is shedding of mucosal cells into the intestinal lumen at death. Therefore digesta samples have to be obtained by avoiding mucosal cells. 6 1 Accepting that amino acid digestibility should be based on measurement made at the terminal ileum of monogastric animals, it needs to be recognised that ileal digesta are derived both from dietary and endogenous sources. Endogenous amino acid loss is used to correct apparent digestibility coefficients to true values. True amino acid digestibility has the advantage over apparent digestibility in that it is a fundamental property of a feed ingredient regardless of the dietary conditions under which the ingredient is fed. For a given amino acid, apparent digestibility increases exponentially with increasing ingested feed because endogenous excretion, as a percent of total excretion, decreases proportionally (Sauer et al. 1980; Furuya and Kaji 1 989; Keith and Bell 199 1 ). Using true rather than apparent digestibility allows raw materials to be accurately compared, even if they are ingested in different quantities. The argument as to whether apparent or true digestibility values are preferred for practical dietary formulation is inextricably linked to the approaches adopted in estimating amino acid requirement for growth. In the formulation of diets for pigs, it is assumed that the supply of digestible amino acid in a mixture of feedstuffs is equal to the sum of the supply based on the digestibility values determined for single ingredients. For feedstuffs with a lower level in one amino acid, their apparent ileal digestibility would be reduced by the influence of the endogenous ileal contribution (Furuya and Kaji 1 989). As true ileal amino acid digestibility is corrected for the endogenous ileal amino acid, true rather than apparent digestibility values would be expected to be more additive (favemer et al. 198 1 ; Furuya and Kaji 199 1 ) . However, the difficulty of accurately determining endogenous excretion has prompted many researchers to recommend the use of apparent amino acid digestibility values for the formulation of diets (Austic 1 983; Sauer et al. 1983). Various approaches to the estimation of endogenous amino acids have been employed, such as the protein-free method, regression method, homoarginine procedure, radioactive isotope or tracer techniques and peptide alimentation method. The peptide alimentation method used in this thesis allowed investigation of the effect of dietary cr on endo-genous ileal amino acid flow in monogastric animals. In this method the animal is fed a semi-synthetic diet containing enzymically hydrolysed 62 casein (EHC) as its sole nitrogen source. Deal digesta are collected and the nitrogenous fraction separated physically using large volume disposable ultrafiltration devices. The high molecular weight (MW> 1 0,000 Da) fraction resulting from the ultrafiltration provides a measure of endogenous amino acid flow. If some of the dietary amino acids and small peptides are not absorbed, they will be removed in the low molecular weight fraction. In addition to the unabsorbed dietary amino acids and peptides, the low molecular weight fraction will contain some non-protein N, and endogenous free amino acids and small peptides. The latter if present, are expected to be at a low concentration (Butts et al. 199 1 ). Nevertheless, their removal in the low molecular weight fraction may lead to some underestimation of the actual endogenous loss of amino acids. For the above reasons, ileal N and amino acid digestibilities were used in this thesis in studies involving effects of cottonseed cr upon protein digestion by . monogastric animals. Apparent ileal digestibility was determined in all monogastric experiments, whilst true ileal digestibility and endogenous protein loss were determined in some experiments. All monogastric experiments used the laboratory rat and in the final experiment the rat was compared with the pig for studying the effects of cottonseed cr upon protein digestion. 1. 10. 2 Measurement of Protein Solubilization and Degradation in the Rumen The value of a protein fed to ruminants is influenced substantially by the extent to which it is digested in the rumen. The digestion of dietary protein in the rumen can be attributed to the combined processes of solubilization and degradation. Solubilization is the release of protein from plant cells into the fluid phase of the rumen following chewing and is an important prerequisite for degradation (Hungate 1966), whilst degradation is the catabolism of protein by microbial proteases resulting in the release of amino acid and NH3. Between 20 and 100% of the protein in many diets based on high protein forages, protein meals and grains may be soluble. Protein concentrate meals such as CSM are of most value in ruminant nutrition if the rumen degradability is low (ie undegradable protein as a proportion of crude protein is high). It has been proposed that the solubility of protein-N in buffer solution can be used as an index of degradability of protein meals in the rumen (Preston and Leng 1987). However, soluble proteins such as serum albumin, ovalbumin, chloroplast protein extract and soluble proteins from soya bean meal and rapeseed meal have variable resistance to degradation in the rumen (Mahadevan et al. 1980). There are several techniques (both laboratory and animal) that have been used to estimate rumen protein solubility and degradability, which include the in vitro total N solubility, in vitro enzymatic techniques, in vitro rumen inoculum, in situ techniques and the in vitro incubation with rumen fluid, followed by fractionation of individual proteins using sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and their quantification using imaging densitometry. 63 Many solvents varying in complexity have been employed for the purpose of determining in vitro soluble N. Solubility of total N in vitro is dependent on the chemical characteristics of the buffer, ionic strength, temperature, and pH (Waldo and Goering 1979). Differences between laboratories were due to differences in extraction time, degree of agitation, feed particle size and solvent to feed ratio. In 'vitro N solubility is highly correlated to short-term ( l to 3 h) rumen digestion of protein (1-0.80-0.97; Crawford et al. 1978; Madsen and Hvelplund 1985), not to extent of rumen digestion (Nocek 1988). Digestion systems utilising proteolytic enzymes offer several advantages over live microbial cultures for measuring in vitro rumen protein digestion (low cost, time reduction, less contamination of feed residue, no cannulated animal required) . However, enzymatic specificity of commercial enzyme preparations in relation to rumen proteolytic activity may be different and becomes an important factor. Several studies have employed single enzyme systems of non-bacterial origin including pepsin, trypsin, papain and pronase (Pion et al. 1983). Other studies have utilised broad specificity fungal and bacterial enzyme sources (poos-Floyd et al. 1 985) or cell coat protease from mixed rumen isolates (Russell et al. 198 1 ) to estimate rumen protein degradability. Several studies have found good correlation (1-0.61 -0.94) between rumen protein degradability using single enzyme incubation techniques and in vivo techniques (Pion et al. 1983; Poncet et al. 1 983; Krishnamoorthy et al. 1983). In general, proteolytic enzyme systems have potential for estimating rumen protein digestion, and may be more suitable for measuring relative differences between feedstuffs than providing absolute values. However, potential sources of variation include buffer pH and composition, duration of incubation, enzyme saturation conditions and incubation temperature. Several researchers (Little et al. 1963; Broderick 1978, 1982; Broderick et al. 1 980; Raab et al. 1983) used ammonia release to predict protein digestion in vitro. Broderick ( 1982) showed negative ammonia release values for sorghum grain and corn after 2 h of incubation. Negative ammonia release occurs when feed contains a considerable amount of fermentable carbohydrate. Protein degradation will be underestimated because of the ammonia used for microbial growth (Broderick 1 978; Chamberlain and Thomas 1979). Broderick ( 1978) refined the procedure by adding 64 hydrazine sulfate to inhibit uptake of amino acids and ammonia by bacteria. This procedure requires analysis of the incubation material for both ammonia and total amino acids to calculate degradation. Although this procedure has been applied to typical feedstuffs (Broderick 1984), interpretation of results may limit its usefulness with many feeds. Raab et al. ( 1983) and Menke et al. ( 1979) developed an approach whereby protein degradation was based on production of ammonia and gas when increments of starch were provided for microbial synthesis. The procedure integrates the relationship between fermentation of carbohydrates and microbial protein synthesis for determination of NH3N incorporated into microbial protein. A potential concern is the type of carbohydrate source (fermentability) used in relation to protein source (degree of protection) with regard to gas production (Nocek 1988). The suspension of feed materials in the rumen (e.g. in situ technique, in sacco technique, artificial fibre bag technique) allows intimate contact of the test feed with the rumen environment. There is no better way to simulate the rumen environment within a given feeding regimen (temperature, pH, buffer substrate, enzymes), although in the rumen environment, the feed is not subjected to the total rumen experience: i.e., mastication, rumination and passage. This technique has been used since late 1970s or early 1980s (Mehrez and 0rskov 1977; 0rskov and McDonald 1979) and is the basis for predicting rumen protein digestion in several feeding systems (Chalupa 1975; NRC 1985; Waldo and Glenn 1 984). The in situ protein digestion in the rumen is estimated using the following equation (0rskov and McDonald 1979) Y = A+B ( 1 -e-ct) where Y represents percent protein digestion at time (t) in hours spent in the rumen. The constants A, B and C represent, respectively, the instantly soluble fraction (A), the proportion digested in time t (B) and the digestion rate of the 'B' fraction (C). Potential digestibility is calculated as A+B. Predicted digestibility (P) is estimated from the equation of 0rskov and McDonald ( 1 979) P =A + [BC/(C+k)] ( 1 ) (2) where k is the rumen particulate fractional outflow rate. Predicted d igesti­ bility thus gives an estimate of rumen digestibility at a specified rumen out­ flow rate. However, its increased popularity has also subjected the in situ technique to extensive evaluation and criticism with regard to the many inherent factors that influence digestion (Le., bag porosity, sample size, feed particle size). In addition, bacteria can enter artificial fibre bags suspended in the rumen and contaminate the undigested residue with microbial protein. Unless undigested residues are corrected for bacterial contamination, rumen protein digestion will be under-estimated (Chalupa and Sniffen 1994). 65 The loss of total N from synthetic-fibre bags using the in situ technique has gained wide acceptance as an index of protein degradability in the rumen. However, Spencer et al. ( 1988) reported that individual proteins (albumins) of pea seed were relatively resistant to rumen degradation, despite the almost complete loss (Le. solubilization) of total pea seed N from synthetic-fibre bags suspended in the rumen. Therefore, rates of dietary protein solubilization and degradation in the rumen may not be similar and the loss or solubilization of total N from synthetic-fibre bags suspended in the rumen may not always be a good index of dietary protein degradation in the rumen. Protein degradation in the rumen, either in vitro or in vivo has been successfully studied by identification of individual proteins using SDS-PAGE (Nugent and Mangan 198 1 ; Spencer et al. 1988; Romagnolo et al. 1990; McNabb et al. 1994) and measuring their rates of breakdown. Therefore, in this thesis protein solubility is defined as the rate of disappearance of total N from samples suspended in the rumen of sheep using the polyester bag technique (Mehrez and 0rskov 1977). Protein degradability is defined as the rate of disappearance of individual proteins during in vitro incubation with rumen fluid, with identification of individual proteins using SDS-PAGE. Cottonseed contains three major types of proteins in approximately equal amounts, having sedimentation values of 2S, 5S and 9S. Protein bodies isolated from .cottonseed also contain these three major proteins in similar proportions. The 5S and 9S proteins are typical globulin storage proteins, whilst the 2S proteins are albumins and are also storage protein as judged by their amino acid composition, developmental properties and high amount in the seed (You Ie and Huang 1979). The 2S proteins are distinct from the 5S and 9S proteins in solubility, amino acid composition, sedimentation values, and SDS gel electrophoretic patterns. Storage proteins comprise about 70% of the total protein in cottonseed kernels, and are located within protein bodies (aleurone grains; Lui and Altschul 1967). The two principal globulin storage proteins (a-globulin and �-globulin) have been identified in cottonseed kernel, and have molecular weights of 52,000 ancJ 48,000 Da. They have similar solubilities, ultraviolet absorption spectra, and contain similar proportions of amino acid residues (Dure and ChI an 198 1 ). 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Zebrowska T 1 978 Determination of available amino acids in feedstuffs for monogastrics. Feedstuffs 50(53) 1 5- 17, 43-44. 92 ZeIter S Z, Leroy F, Tissier J P 1970 frotein des proteines alimentaires contre la desamination bacterienne dans Ie rumen (Protection of feed proteins against bacterial deamination in the rumen). Ann Bioi Anim Biochem Biophy 10 1 1 - 122. Zirkle S M, Line Y C, Gwazdanskas F C, Canseco R S 1 988 Effects of gossypol on bovine embryo development during the preimplantation period. Theriogenology 30 575-582. Chapter 2 CONDENSED TANNIN AND GOSSYPOL CONCENTRATIONS IN COTTONSEED AND IN PROCESSED COTTONSEED MEAL This Chapter has been published in Journal of the Science of Food and Agriculture 63: 7- 15 ( 1993). Reproduced by permission of Society of Chemical Industry, London, UK. 94 2. 1 ABSTRACT Experimental varieties of cottonseed and of industrial cottonseed meal (CSM) were analysed for extractable and bound condensed tannin (CT) and free gossypol. CT was present in the hulls of all varieties, with higher concentrations recorded for high tannin and glandless selections (55 and 58 g kg-1 0M) than for the multiple host plant resistant and high gossypol selections (38 g kg-1 0M). CT was present in trace amounts in the kernels (meats) of high tannin selections, but was not detected in the kernels of all other selections. Industrial CSM contained 8-1 5 g kg-I CT, due to contamination of meats with hull components. On average for the hulls of all varieties, approximately 22, 60 and 1 8 % of total cr was present in the extractable, protein-bound and fibre-bound forms, respectively. Total cr content in the hulls was positively correlated with the lignin content of kernels (r=O.67, P-(I) III 0 0.4 co C) C) .. u. 0.0 '--_---' __ --'-__ -'-__ -'-_-.....-.A....-_---' __ --'-__ --' 1 .0 1 .5 2 .0 2 .5 3.0 Total CT in hulls (mg/seed) Fig 2. 3 The correlation between free gossypol in the kernels and total cr in the hulls of experimental cottonseed varieties. 0, multiple host plant resistance; 0, high gossypol; �, high tannin; ., commercial or Australian bred cultivars; ., glandless cottonseed. r= -0.50; n= 16; p<0.05. 2. 4. 3 Crude Protein, Oil and Fibre The crude protein (CP), oil, neutral detergent fibre (NDF), acid detergent fibre (ADF) and lignin contents of the kernels of cottonseed are shown in Table 2. 4. The CP and oil contents of the kernel of the DP 1 6 glandless were 2% lower and 5% higher, respectively, than the mean levels (346 g kg-I and 348 g kg-I) for the rest of the varieties. Kernels of the three multiple host plant resistant varieties had notably lower 1 04 contents of NDF, ADF and lignin than kernels of the other varieties. Also, kernels from the high tannin varieties tended to have higher lignin levels than kernels from the other varieties. There were very few differences in the CP, oil, NDF, ADF and lignin contents of the hulls from the experimental cottonseeds (Table 2. 5). In all varieties hull vs seed weight was similar (Table 2. 5). Table 2. 5 The content) (g kg-I DM) of crude protein, oil, neutral detergent fibre (NDF), acid detergent fibre (ADF) and lignin in the hulls of cottonseed of experimental varieties Plant selection % of seed Crude Oil NDF ADF Lignin criteria weight I!rotein Multiple host plant resistant: MHR IO 374 42 1 6 884 608 230 MHR 1 1 376 42 1 5 877 606 254 MHR 1 7 361 38 14 903 624 236 High gossypol: HG 063 379 36 1 3 899 6 1 8 232 HG 065 365 35 9 906 636 247 HG 660 358 37 I I 9 1 0 634 238 High tannin: HT-35-5- 1 Smooth 380 3 1 1 2 894 6 1 3 250 HT-35-5 - l Hirsute 350 35 1 2 888 6 1 8 247 HT-35- l 4-3 392 35 1 5 878 596 234 CS 38 1 0 347 39 1 1 882 635 245 Commercial or Australian-bred cultivars: DP 90 312 35 1 1 901 590 2 1 6 Sicala 3-3 374 38 9 904 603 23 1 Siokra 1 4 354 34 9 89 1 6 1 8 232 N74-720- 1 99B 370 3 8 -1 5 885 6 1 3 208 OGFLine 8 364 36 1 2 882 604 229 Glandless: DP 1 6 379 38 1 2 872 625 238 ) Mean of duplicate determinations. Industrial CSM from Narrabri had a greater fibre content than that from Brisbane (Table 2. 4). NDF, ADF and lignin of Narrabri CSM were 14%, 50% and 7% higher than those of Brisbane CSM. Industrial cottonseed meat contained higher fibre compared with the kernels from experimental varieties. The oil content, however, of industrial cottonseed meat was 2% lower than the average level of the experimental cottonseed varieties. 2. 5 DISCUSSION 1 05 The present results demonstrate that the method of Terrill et al. ( 1992) for measuring extractable and bound CT in forages can be extended to cottonseeds and cottonseed products. A complete recovery of added purified CT ( 106%) was found. Lower absorbances for purified cotton cr made up in SDS solution compared with water were found in the present study, similar to the results of Terrill et al. (1992) for CT extracted from the forages Lotus pedunculatus and Hedysarum coronarium. This can be corrected for by using the aqueous standard curve for measuring "free" CT and the SDS standard curve for measuring protein-bound and fibre-bound CT. The low amounts of cotton cr detected in the initial acetone:water:diethyl ether extract and the high amounts of CT detected as protein-bound and fibre-bound CT show that a bound CT method is essential to reliably measure cr in cottonseed and cottonseed products. CT in cottonseed was estimated using cr extracted from cotton leaf as a standard, and it is possible that degree of polymerization may differ in CT from the two sources. However, as the butanollHCI reaction involves hydrolysis of CT to monomer units and formation of anthocyanidin from a portion of these, it is unlikely that differences in chain length would significantly influence the results, as found by Porter et al. ( 1986). Repeatability of measurements between samplings and between duplicates was acceptable. Whilst samples of the 16 experimental cottonseeds were not replicated, the precision of the method suggests that differences found between selection lines (varieties) are probably real. Greater variation in CT detected in the protein-bound and especially the fibre-bound steps with industrial CSM is probably due to cr only being present in hulls, and represents sampling variation from a mixture of meats and hulls. The cottonseed kernels contained negligible amounts of cr, with almost all of the CT being found in the hulls. Kernels from the high tannin varieties tended to be high in lignin (also defined as a secondary compound), which can be explained by lignin and cr being produced by related biochemical pathways (Figure 2. 2; Barry 1 989). However, no significant correlation between total cr and lignin contents in the cottonseed hulls was found in this study. Gossypol, CT and lignin probably evolved in cotton as chemical defences against insect attack. From the results of the present study it seems that selection for high levels of CT or gossypol has led to corresponding reductions in the other, with high and low (ie glandless) gossypol selection producing seed hulls low and high in CT, respectively (Figure 2. 3). 1 06 Fitt et al. ( 1992) showed reduced larval growth on the leaves of both high tannin and high gossypol selections, with the latter showing most promise, and recommended this factor be incorporated into selection programmes for Australian cotton. Selection for high tannin to control insects poses no animal nutrition problems because CT only occurs in the hull and manufacturing processes can be devised to remove most of this component. However, as gossypol occurs in the kernels, as well as in leaves and other plant parts, selection for high gossypol in insect control programmes will probably cause additional nutritional problems, especially for monogastric species. Recent studies have shown that erythrocyte fragility in feeder lambs (Calhoun et al. 1 990) and heifers (Gray et al. 1 990) was increased by feeding moderate amounts of cottonseed products containing free gossypol. Smalley and Bicknell ( 1 982) reported case studies of gossypol toxicosis in mature dairy cattle fed relatively high amounts of ammoniated whole cottonseed. These findings in ruminants indicate that ingestion of free gossypol at high levels may overwhelm ruminal detoxification and hence result in the absorption of quantities of free gossypol that may be potentially toxic. More than 95% of total gossypol in dehulled cottonseed kernels was free gossypol (Pons and Eaves 1 967; Ikurior and Fetuga 1 984). The free gossypol contained in the cottonseed kernels becomes bound to other seed components andlor is destroyed during seed processing (lkurior and Fetuga 1 984). There was little difference among the protein contents of the kernels, whereas the kernel of glandless cottonseed contained a higher concentration of oil than the kernels from the other varieties, which was similar to the findings of Lawhon et al. ( 1977). Kernels of the three multiple host plant resistant varieties had notably lower contents of total fibre and lignin than kernels made from the other varieties, and this should result in improved digestion in monogastric animals. However, these selections have shown very little resistance to insect egg laying and larval damage under Australian conditions (Fitt et al. 1 992). Industrial CSM produced at both the Narrabri and Brisbane plants contained CT, probably due to contamination of the kernels with parts of the hulls under large scale . manufacturing. The higher concentrations of CT in CSM from the Narrabri mill is probably due to the practice of leaving some hulls with the kernels, to facilitate passage through the extruders prior to solvent extraction of the remaining oil. According to the CT concentrations, CSM produced at Narrabri and Brisbane contained approximately 300 and 1 50 g kg-l of hulls, respectively. The tannin to protein ratio in Narrabri and Brisbane CSM was 0.04 and 0.02, respectively. Terrill et al. ( 1992) also found 0.04 g CT per 100 g crude protein in cottonseed meals. The small amounts of CT detected in the industrial cottonseed meats from Narrabri indicates that approximately 5% of the original hulls were left in the meats after decortication. 1 07 CT may be a further contributing factor to the reduced true amino acid digestibility in CSM diets for monogastrics (Batterham et al. 1990; Beech et al. 1 99 1 ) and to the high degree of rumen "by-pass" of CSM protein in ruminant diets (Lee et al. 1 987). 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University of New England, Annidale, NSW, Australia. Chapter 3 EFFECT OF BOUND CONDENSED TANNIN FROM COTTONSEED UPON IN SITU PROTEIN SOLUBILITY AND DRY MA TIER DIGESTION IN THE RUMEN This Chapter has been accepted for publication in Journal of the Science of Food and Agriculture 69 (In press; 1995). Reproduced by pennission of Society of Chemical Industry, London, UK. 1 12 3. 1 ABSTRACT The effect of adding cottonseed hulls upon the solubility of protein in unheated solvent extracted cottonseed kernels was studied using both in vitro incubation in mineral buffer and the in situ polyester bag technique. The latter technique was also used to study effects on rumen dry matter (OM) digestion. Effects attributable to condensed tannin (cr) were assessed by making measurements in the presence and absence of polyethylene glycol (PEG; MW 3,500), which binds and inactivates CT. Cottonseed hulls contained 5 1 g cr kg-1 0M, with 56 and 20% of the total cr being bound to protein and fibre, respectively; no cr was detected in kernel. Hulls and extracted kernel contained 33 and 509 g protein kg- 1 0M, and 887 and 289 g fibre kg-1 0M. In the absence of hulls, 42% of the total nitrogen (N) in cottonseed kernel was soluble in mineral buffer in vitro, whilst potential in situ N solubility and predicted rumen N solubility (corrected for rumen outflow rate) were 99 and 86% respectively. Addition of hulls linearly reduced both in vitro N solubility and potential in situ N solubility, with 1 00% hulls addition reducing potential N solubility and predicted rumen N solubility to 94 and 79% respectively. PEG addition had no effect upon the protein solubility of kernels, but increased N solubility in mixtures of hulls and kernels in vitro but not in situ. Two mg PEG mg-l total cr was shown to reverse the effect of CT in reducing in. vitro protein solubility. Potential in .situ OM digestion and predicted rumen OM digestion (corrected for rumen outflow) were substantially lower for cottonseed hulls (4 1 and 33%) than for kernels (99 and 88%). Increasing the addition of hulls to kernels lowered the rumen OM digestion of mixtures in a quadratic manner, with increasing rate of hulls causing progressively smaller depressions. Addition of PEG had no effect upon the digestion of kernel OM, but increased potential OM digestibility and predicted rumen OM digestion of hulls to 47 and 40% respectively, and also produced an increase in mixtures of hulls and kernels. It was concluded that the high protein solubility of unheated solvent extracted cottonseed kernels can be linearly reduced by the addition of cottonseed hulls, with the magnitude of the reduction being small, and that the presence of bound cr in hulls substantially depressed fibre digestion by rumen micro-organisms. It is doubt full that cr plays a significant role in the reduction of rumen protein solubility produced by cottonseed hulls. 1 13 3. 2 INTRODUCTION Solvent extracted cottonseed meal (CSM) has long been recognized as a protein supplement for ruminant and monogastric livestock. Free gossypol, a polyphenolic binaphthyl aldehyde contained in the seed pigment glands, is considered one of the major anti-nutritional factors (ANFs) in CSM. In CSM the concentration of free gossypol is influenced by the method of oil extraction, with high temperatures and pressures causing "binding" of gossypol with the £-amino group of lysine, thus reducing free gossypol concentration (<0.2 g kg-l DM; Berardi and Goldblatt 1980). Toxicity symptoms attributed to gossypol have been reported primarily in non-ruminants. Ruminants appear to be less susceptible to gossypol toxicity; apparently due to their ability to detoxify gossypol by binding with soluble proteins present in the rumen fluid (Reiser and Fu 1 962), except for high intake of free gossypol, which can overwhelm this protective system, leading to toxicoses in adult ruminants (Lindsey et al. 1 980; Risco et al. 1 993). Recent investigations have found another group of plant secondary compounds, condensed tannins (CT), present in commercially produced CSM (Balogun et al. 1990; Terrill et al. 1 992; Yu et al. 1993). These have been shown to originate from cottonseed hulls, where they occur mainly bound to protein and fibre, whilst cottonseed kernel does not contain cr (Yu et al. 1993). cr are recognized as being an ANF in monogastric animal diets (Huisman et al. 1990), but in ruminants low concentrations may improve the efficiency of protein digestion by forming hydrogen bonded complexes with proteins (Barry 1989). The protein-cr complexes are stable and insoluble at rumen pH (5.5-7.0) but dissociate and release protein below pH 3.0 (Jones and Mangan 1 977). Waghom et al. ( 1 987) showed that the action of cr in the forage of Lotus comiculatus increased the absorption of essential amino acids from the small intestine of sheep by 62%. Barry ( 1989) concluded that low concentrations ( 10-30 g kg-I DM) of CT in forage diets are likely to increase amino acid absorption from the small intestine, but that higher concentrations reduced both feed intake and rumen fibre digestion. There is less information available on the effects of CT on rumen solubility/degradability of protein concentrate meals, particularly for oil-seed meals. Objectives of the present study were to determine the effect of cr in cottonseed hulls upon the in vitro and in situ solubility of total nitrogen (N) and two individual proteins (52,000 and 48,000 Da molecular weight) in unheated solvent extracted cottonseed kernel, and upon the in situ dry matter (OM) digestion of cottonseed hulls and mixtures of kernels and hulls. The 52 and 48 kDa proteins are two principal storage proteins, and the storage proteins comprise about 70% of the total protein in cottonseed kernel (Dure and Chlan 198 1 ). 1 14 The loss of DM and total N from polyester bags immersed in rumen fluid in vitro and in situ has had wide acceptance as an index of DM and N degradability in the rumen (Mehrez and 0rskov 1977; Crooker et al. 1978; Kempton 1 98 1 ). However, Stem and Satter ( 1984) compared protein solubility in a mineral buffer solution with in situ rumen protein degradation, measured as disappearance from dacron-fibre bags, and obtained a correlation coefficient of only 0.26. Spencer et al. ( 1988) reported that individual pea seed proteins were relatively resistant to rumen degradation, despite the almost complete loss of total pea seed-N from synthetic-fibre bags suspended in rumen fluid in vitro. Protein solubilization is an important prerequisite for rumen degradation (Hungate 1 966), however the rates of solubilization and degradation of protein in the rumen are not always similar (McNabb 1990). Therefore, in this paper, protein solubility is defined as the extractability of total N in mineral buffer solution (pH 7.0) in vitro, and also as the rate of disappearance of total N and individual storage proteins from samples suspended in the rumen of sheep using the polyester bag technique (Mehrez aild 0rskov 1977). Protein degradability is defined as the rate of disappearance of individual storage proteins during in vitro incubation with rumen fluid, with identification of individual proteins using sodium-dodecyl-sulphate polyacrylamide gel electrophoresis (SDS-PAGE). The effect of hulls and CT upon the degradability of cottonseed kernel proteins is described by Yu et al. ( 1 995a). Loss of DM from polyester-fibre bags is defined as in situ rumen DM digestion. 3. 3 MATERIALS AND METHODS 3. 3. 1 Experimental Design Three experiments were conducted to determine the effect of Cf in cottonseed hulls upon the in vitro and in situ protein solubility and in situ DM digestion in cottonseed kernels. All investigations were done with seeds of Gossypium hirsutum, var. Siokra L22, as supplied by Cotton Seed Distributors Ltd, Wee Waa, NSW, Australia. The effects of Cf, in the present study. were assessed by making measurements in the presence and absence of polyethylene glycol (PEG, molecular weight (MW) 3.500, Union Carbide, Danbury, cr, USA). PEG binds strongly to CT and can be used to displace protein from the Cf-protein complexes (Jones and Mangan 1977; Barry and Manley 1 986). Therefore, effects of cr can be quantified by comparing controls (CT acting) with PEG treatments (CT inactivated). 1 15 In Experiment 1 the effect of CT in cottonseed hulls upon the in vitro solubility of total N in cottonseed kernel was detennined. Experiment 2 investigated the in situ solubility of total N and of two individual storage proteins in cottonseed kernels; and the in situ rumen OM digestion in cottonseed kernel and mixtures of kernel and hulls. Experiment 3 determined the effect of bound CT upon the in situ rumen OM digestion of cottonseed hulls. 3. 3. 2 Preparation of Cottonseed Kernel and Hulls Delinted whole cottonseed was cracked by passage through a Crushing-Mill (AB Thorell & Persson, Uppsala, Sweden), and then separated into kernels and hulls using an air-blow technique, at the Seed Technology Centre, Massey University. Separated kernels were freeze-dried for 2 days, and then ground to pass through a 2 mm diameter sieve. Ground kernels were extracted in 1 kg batches by continuous stirring with pure hexane ( 1 :2 w/v) for 6 h at ambient temperature, according to the method of Pons and Eaves ( 1 967). Mter displacement washing with hexane (5 x 1 ,000 ml), using vacuum filtration, the extracted meal was air-dried, and ground to pass through a I mm sieve. Gossypol was then extracted, with minimal removal of protein using the following conditions: acetone in water (70:30, w/w, as the solvent); temperature 300C; extracting time 30 min; solvent to kernel ratio 2: 1 ; displacement washing two times. The resulting meal from the second extraction was air-dried again at ambient temperature. Separated hulls were ground to pass through a 1 mm diameter sieve. Fully extracted cottonseed kernel and ground hulls were stored at -200C until used. The chemical composition of cottonseed kernel before and after extraction and of cottonseed hulls is shown in Table 3. 1 . Total CT in ground hulls comprised 24% extractable CT, 56% protein-bound CT and 20% fibre-bound CT. 3. 3. 3 ExperUnent l In Experiment 1 a, the effect of different concentrations of PEG ( 1 , 2, 3 , 4 and 5 mg mg-I total en upon total N solubility in a mixture of 1 g solvent extracted cottonseed kernel plus 200% cottonseed hulls (ie 2 g) was determined. The mixture contained: 1 94 g kg-1 0M of crude protein; 34 and 8.3 g kg-1 0M of total and extractable CT, respectively. Experiment 1 b investigated the effect of CT in cottonseed hulls upon in vitro total N solubility of cottonseed kernel. . The treatments comprised 1 g of solvent extracted cottonseed kernel plus 0, 25, 50, 100 and 200% of ground cottonseed hulls, 1 1 6 with or without 2 mg PEG mg-l total CT. The crude protein and CT contents in each treatment are shown in Table 3. 1 . Table 3 . 1 The chemical composition 1 (g kg- l DM) of cottonseed kernel, hulls and mixtures used in the experiments Dry Crude Oil NDF ADF Lignin Free Total matter protein gossypol cr Cottonseed kernel: untreated 938 360 387 1 98 63 37 1 2.6 0 extractedb 909 509 138 289 137 5 2 0.95 0 Cottonseed hulls 900 33 9 887 734 1 53 0.23 5 1 .0 Extracted kernelblhull mixtures: +25% hulls 9 1 4 420 0.87 1 0.2 +50% hulls 907 354 0.76 1 7.0 +100% hulls 908 269 0.58 25.5 + 1 50 % hulls 905 223 0.47 30.6 +200% hulls 904 194 0.42 34.0 I Mean of duplicate determinations. 2 Hexane+acetone Phosphate buffer (pH 7.0) was prepared by mixing 195 ml of 0. 1 5 M NaH2P04 with 305 ml 0. 15 M Na2HP04 and making up to I litre with distilled water. Freshly pr�pared phosphate buffer (50 ml, pH 7.0), maintained at 390C, was added to groups of volumetric flasks (250 ml); 1 g of unheated solvent extracted cottonseed kernel, and the required amounts of ground cottonseed hulls and PEG were then added. Flasks were fitted with Bunsen valves and incubated in a shaking water bath (90 rpm) at 390C for 2 hours. The mixture was centrifuged at 27,000 x g for 15 min and total N content determined on 10 ml of the clear supernatant solution. All treatments were done in triplicate. 3. 3. 4 Experiments 2 and 3 3. 3. 4. 1 Animals and Diet In Experiments 2 and 3, two groups each of six adult male castrated sheep (mean liveweight 59 kg. SE 1 .9), fitted with a rumen cannula (55 mm ID) were maintained on a basal diet of 900 g d-l of meadow hay (20 g kg-l N, 890 g kg-I DM) supplemented with 250 g d-I of lucerne chaff (30 g kg-I N. 860 g kg-I DM). offered at hourly intervals. from overhead belt-feeders, for at least 2 weeks before the experiments commenced. Water was provided and also a multimineral salt block (Summit®, NZ) was freely available. All sheep were drenched with an anthelmintic to control internal parasites ( 12 ml Ivomec; Merck Sharp and Dohme (NZ) Ltd) and were treated for lice ( 10 ml Wipeout; Coopers Animal Health (NZ) Ltd) before the experiments commenced. 1 17 In both Experiments, one group of six sheep (PEG sheep) received an intraruminal infusion of PEG at a rate of 25 g d-l (in 240 ml water) in all experimental periods, whilst the remaining group of six sheep (Control sheep) received an intraruminal infusion of the same volume of water. There was a two day rest period between the two experiments. 3. 3. 4. 2 Experimental Procedures In Experiment 2, the rate of in situ DM digestion and N solubility of test feeds was measured using the polyester bag method of Mehrez and 0rskov ( 1 977). Test feeds incubated in the bags were unheated solvent extracted cottonseed kernel mixed with 0, 25, 50, 100, 1 50 and 200% of ground cottonseed hulls. The experiment was run over six consecutive periods, each of 2 days, with the six test feeds rotated among the six sheep in each group (Control sheep and PEG sheep), according to a paired 6 x 6 Latin square design. Control sheep comprised one Latin square and PEG sheep comprised a second Latin square. Six polyester bags (Estal-mono, 47 Jlm pore size, Swiss Screens, Sydney, Australia) measuring 7x14 cm internally and having rounded comers, each containing a marble (approximately 5 g) to ensure that the bags would not float in the rumen, plus approximately 5 g of air dried test feed, were lowered into the rumen of each sheep. Bags were removed from the rumen after 4, 8, 12, 24, 36 and 48 h incubation and were rinsed with tap water until the rinse fluid was clear. Bags with their contents were then dried in a forced-draught oven at 600C for 48 h, cooled in a desiccator and weighed. Loss in dry weight was reported as in situ DM digestibility. Dried residue of the test feeds in the bags was then analyse<;l for total N. DM and N disappearance in the rumen due to simple solubilization was estimated by soaking control bags in water in a shaking water bath (90 rpm) at 390C for 2 h (Kempton 1981 ) . This represented initial solubility (1=0). In Experiment 3, six Control and six PEG-infused sheep were used in one time . period to determine the in situ DM digestion of cottonseed hulls. Six polyester bags each containing a marble and approximately 3 g of hulls were suspended in the rumen of each sheep. Bags were removed from the rumen after 4, 8, 1 2, 24, 36 and 48 h incubation. The DM disappearance of hulls at (t=O) was determined, and the residues from the polyester bags were washed and dried as described for Experiment 2. 1 1 8 3. 3. 5 Sample Analysis CT content was detennined using the method of Terrill et al. ( 1992), as described by Yu et al. ( 1993). Extractable CT was extracted using a mixture of acetonelwater/diethyl ether (4.7:2.0:3.3 v/v), followed by extraction of protein-bound CT using boiling sodium dodecyl sulphate containing 2-mercaptoethanol in 10 mM TrislChloride, adjusted to pH 8.0 with HCI (SDS solution). Fibre-bound cr was detennined by boiling the residue remaining from protein extraction with butanol-Hel and SDS solution. CT concentration in each fraction was then detennined by the butanol-HCI procedure (Porter et al. 1986). Free gossypol was detennined using method Ba 7-58 of the American Oil Chemists Society (AOCS 1 975). Total nitrogen (N) was detennined by the Kjeldahl procedure, and crude protein calculated as total N x 6.25. Neutral detergent fibre (NDF), acid detergent fibre (AOF) and lignin were detennined using the method of Robertson and Van Soest ( 198 1). Dry matter was detennined by drying at lOOOC for 24 hours and oil content was determined by extraction with petroleum ether (Boiling point 40-6()OC) for 8 hours. In Experiment 2, the polyester bag residues remaining after total N analysis were pooled for each treatment at each time point (n=6 per treatment), for detennination of in situ solubility of the 52 kDa and 48 kDa proteins in cottonseed kernel using the SDS-PAGE method of McNabb et al. ( 1 994), as described by Yu et al. (1 995a). Between 100 and 1600 J.1g DM samples containing approximately 8 J.1g of protein from polyester-fibre bag residue were loaded in each well, and the electrophoresis was carried out for approximately 3 h at 80 V. The mini-gels ( 1 .0 x 75 x 100 mm) consisted of a stacking gel approximately 22 mm high, layered over a separating gel. After SDS-PAGE, the gels were washed and then total soluble protein was visualised by staining with Fast Green FCF (0. 1 % Fast Green FCF; 40% methanol; 10% acetic acid) for 30 min. The gels were destained in 10% methanol; 7.5% acetic acid. The bands of the 48 kDa and 52 kDa proteins on the developed gels were scanned and quantified using imaging densitometry (Bio-Rad, Model GS-670 Imaging Densitometer, USA). 3. 3. 6 Calculation of Data and Statistical Analysis Data from Experiment 1 were subjected to a one-way analysis of variance. Significant differences between the treatment groups were determined by the least significant difference (LSD) test (Steel and Torrie 1980). 1 19 In Experiment 2, the in situ DM digestion and N solubility rate in the rumen was calculated using the following equation (0rskov and McDonald 1 979). ( 1 ) where Y represents percent DM digestion at time (t) in hours spent in the rumen. The constants A, B and C represent, respectively, the instantly soluble fraction (A), the proportion digested in time t (B) and the digestion rate of the 'B' fraction (C). The constants A, B, C for each animal were calculated by using NLIN (non-linear regression) procedures (SAS 1985). Potential digestibility was calculated asA+B. Predicted rumen digestibility (P) was calculated from the equation of 0rskov and McDonald ( 1 979). P =A + [BC/(C+k)] (2) where k is the rumen particulate dry matter fractional outflow rate, determined with sheep fed a similar lucerne hay to that used in the present investigation (0.033 h-l ; Domingue et al. 1991 ). The same procedures were used to quantify N solubility, with rumen fractional outflow rate being assumed to be 0.046 h- l (0rskov and McDonald 1979). The significance of differences between means for C, A+B and P was established using GLM (General Linear Models) procedures (SAS 1985), with the factors examined being level of hulls, PEG, animals, and the PEG x hulls and PEG x time interactions. If the hulls or the hulls x PEG interaction was significant (p<0.05), then the hulls and hulls x PEG effects were partitioned into linear and quadratic effects and their interactions with PEG. In Experiment 3, the in situ DM digestion rate was calculated from the equation of Mead and Curnow (1983). Y = (A+B)/[ I+e(A-ct)] (3) where Y represents DM digestion at time (t) in hours spent in the rumen, and the constants A, B and C have the same meaning as that described for equation ( 1 ). Predicted rumen digestibility (P) was also calculated using equation (2). The significance of differences between treatments for the constants C, A + B, and P was tested using one way analysis of variance. 3. 4 RESULTS 3. 4. 1 Experbnent l 1 20 In a mixture of 1 g cottonseed kernel with 200% hulls, 23% of total N was soluble in mineral buffer in the absence of PEG. This was increased to 30% at 2 mg PEG mg-l CT and further addition of PEG did not result in further increases in soluble N, showing that two mg PEG mg- 1 total CT was required to prevent or reverse binding of CT in hulls to cottonseed protein. In the absence of hulls, 42% of the total N in cottonseed kernel was soluble in mineral buffer (Figure 3. 1 ). This progressively declined (p<0.05) as increasing quantities of hulls were added to the kernel. The addition of PEG increased N solubility (p<0.05) at each level of hull addition, although values did not increase to the same level as found for pure cottonseed kernel. In PEG treated flasks, N solubility still declined with increasing levels of hull addition. 45 35 25 * • 1 5 �- -�----�----�--�----�----�--�----� o 50 1 00 150 200 g Hulls per 1 00 g cottonseed kernel Fig 3. 1 Experiment lb. The effect of adding cottonseed hulls and polyethylene glycol (pEG; MW 3,500) upon the in vitro nitrogen solubility of solvent extracted cottonseed kernel in phosphate mineral buffer (pH 7.0). Each point is the mean of 3 flasks per treatment. PEG was added at 2 mg mg-l of total CT. ., cottonseed kernel + hulls; 0, cottonseed kernel + hulls + PEG; I, standard error; *, p�0.05. 1 2 1 3. 4. 2 Experiment 2 Increasing rate of hull addition to cottonseed kernel (Table 3. 2) had no effect upon the in situ solubilization rate (constant C; 0. 1 7% h-1 ) of total N, but linearly decreased potential N solubility (constant A +B; pO.05; *** p<0.00 1 . Solubilization rate C (% h- IL + 0. 1 7 0. 1 6 0. 1 9 0. 1 5 0. 1 5 · 0. 1 8 0. 1 6 0. 1 9 0. 1 6 0. 1 8 0. 1 6 0. 1 9 0.0 1 9 . NS NS NS NS Potential Predicted solubility solubility A+B (%) P (%) + + 99 99 86 87 95 97 84 82 94 94 82 81 95 93 78 80 89 90 76 76 88 90 75 74 1 .2 1 .0 *** *** NS NS NS NS NS NS Solubilization of the 52 and 48 kDa cottonseed kernel proteins was variable during the initial 12 h incubation in the rumen, but stable values were attained between 24 and 48 h of incubation, with the mean value over this period being referred to as maximum solubility. Averaged over all six treatment groups, approximately 61 and 70% of the 52 and 48 kDa proteins were instantly soluble in water (ie disappeared at 0 h of incubation), and 97 and 98% were solubilized during the initial 8 h of incubation, indicating that these storage proteins in cottonseed kernel were highly soluble in the rumen. Addition of hull and PEG had no effect upon the in situ maximum solubility of both the 52 and 48 kDa proteins (Table 3. 3). 1 22 Increasing rates of hull addition to cottonseed kernel progressively depressed in situ DM digestion rate (constant C), potential DM digestibility (constant A+B) and predicted rumen DM digestibility (P; pO.05). Table 3. 3 Experiment 2. Effect of adding cottonseed hulls upon the in situ maximum solubility 1 of the 52 and 48 kDa proteins in extracted cottonseed kernel in the rumen of sheep intra-ruminally infused with water or water containing polyethylene glycol (PEG; MW 3,350) 52 kDa 48 kDa protein protein solubilit� (%) soluQilit� (%) PEG + + Cottonseed kernel 99.2 95.0 99.6 98.5 Kernel+25% hulls 97.3 99.9 97.6 99.8 Kernel +50% hulls 99.9 99.4 99.9 99.7 Kernel+ 1 00% hulls 98.3 99.7 97.2 99.5 Kernel+ 1 50% hulls 99.8 99.8 99.9 99.6 Kernel+200% hulls 99.7 99.4 99.7 99.6 1 Average of three values for solubility measured at 24, 36 and 48 hours of incubation. Samples were pooled from sheep on each treatment at each sampling time, to give one value at each time for each treatment. 3. 4. 3 Experiment 3 In situ DM digestion (%) of cottonseed hulls in the rumen of sheep is shown in Figure 3. 2. The rate of in situ DM digestion was very slow for 12 h, and then progressively increased to 48 h. Although the in situ DM soluble component ( constant A) was not affected by PEG (Table 3. 5), the in situ insoluble component (constant B; po.05; * p<0.05; ** p<0.01 ; *** pO.05; * p. 60 ..... -.- .l:I as "0 as 40 ... 01) u Q 20 8 0 0 4 8 1 2 1 6 20 24 Incubation time (hours) Fig. 4. 3 Experiment 2, 3 and 4. Repeatibility of degradation of the 48-kDa protein from cottonseed kernels only (A) and kernels'+ 100% hulls (B) during the in vitro incubation with rumen fluid. Protein degradation (Y;%) expressed as a percentage of that present at t=O, was fitted to the equation (0rskov and McDonald 1979): Y=A + B ( 1 - e-Ct). Rumen fluid was collected from sheep fed on 800 g d-l meadow hay and 125 g d-l cottonseed kernel. Fifteen ml of strained rumen fluid, 60 ml of artificial saliva, 150 mg cellobiose and 1 g of cottonseed kernel were incubated at 390C for 24 h. • Experiment 2; ... Experiment 3; • Experiment 4. Each point is the mean of duplicate determinations. 143 Adding cottonseed hulls significantly reduced potential degradability and degradability after 8 h of both the 52 and 48 kDa proteins (p0.05 ; * p<0.05; ** p0.05 ; *** p0.05; * p<0.05; ** p 10,000) fraction affords an estimate of endogenous loss. Inclusion of CSH in the EHC based diet increased ileal flow of total nitrogen ( 1 387 vs 1 623 mg kg- l dry matter intake; p<0.05), increased ileal flow of total amino acids (23%; p 10,000 Da) from peptides and free amino acids (MW<1O,OOO Da). The high molecular weight fraction (retentate; MW> 10,000 Da) was added back to the precipitate. The total precipitate plus retentate and diet samples were subsequently freeze-dried, finely ground and stored at -200c for the determination of nitrogen, chromium, and amino acid concentrations. The diets and ileal digesta were analysed in duplicate for total nitrogen using the Kjeldahl procedure. The chromium contents of duplicate 15 mg samples of ileal digesta and each diet were detennined by the method of Costigan and EIIis ( 1987). The extractable (ie. free) and bound CT contents of the diets were determined using the method of TerriII et al. ( 1992), and the amino acid compositions of duplicate 5-7 mg samples were determined using high performance liquid chromatography (HPLC, Waters Associates, USA) as described by Yu et al. ( 1995b). Free amino acid molecular weights were used to calculate the weights of amino acids. 5. 3. 3 Calculation and Statistical Analysis Endogenous flows of amino acids at the terminal ileum relating to the ingestion of 1 g of freeze dry matter (FDM) were calculated using the following equation: Amino acid (AA) flow* AA concentration in diet chromium concentration ileal digesta X ileal chromium concentration * All11nits were mg g-1 FUM. The endogenous ileal amino acid flows were determined based on the amino acid contents of the precipitate plus high molecular weight fraction (MW> 1 0,000 Da) following centrifugation and ultrafiltration. A linear statistical model, which included terms for hulls, PEG and hulls x PEG, was fitted to the data for each amino acid singly, and reduction in sums of squares was used to determine levels of significance (SAS 1985). 1 59 5. 4 RESULTS The rats consumed the experimental diets readily and remained healthy throughout the study. The overall mean (±SE) daily food intake (days 10- 1 3) was 1 1(±OA) g. Faeces were not detected in the gastric contents at slaughter indicating that coprophagy had not occurred at least on the last day of study. Adding 50g kg- l hulls to the EHC based diet led to an increase in the determined endogenous ileal flow of total nitrogen (N; p<0.05), total essential amino acids (pO.05). For three of the individual amino acids (threonine, proline and aspartic acid) the differences approached statistical significance (pO.05). 5. 5 DISCUSSION The peptide alimentationldigesta ultrafiltration method allowed investigation of the effect of CSH and CT on endogenous ileal amino acid flow in the growing rat. In this method the animal is fed a semi-synthetic diet containing EHC as its sole nitrogen source. Ileal digesta are collected and the nitrogenous fraction separated physically using large volume disposable ultrafiltration devices. The high molecular weight (MW> 1 0,000 Oa) fraction resulting from the ultraflltration provides a measure of endogenous amino acid flow. If some of the dietary amino acids and small peptides are not absorbed, they will be removed in the low molecular weight fraction (MWl O.OOO). 2 NS. no significant; *, p<0.05; **. p<0.01 . 3 HxP, hulls x PEG interaction. The present estimates of endogenous amino acid flow for the rat given an EHC based diet (0 g kg-I CSH) were slightly higher than these reported by other workers (Butts et al. 1 99 1 ; Donkoh 1993). The inclusion of CSH in the EHC based diet increased ileal N and amino acid flows, indicating that either fibre and, or cr from CSH had an effect on the endogenous ileal loss of amino acids in the rat. However, the protein in the CSH (35 g kg-I ; Yu et al. 1995b) could have caused part of the increase in 161 the estimate of endogenous ileal amino acid loss. If it is assumed that the CSH protein was completely indigestible, then it can be shown that the CSH protein could possibly have contributed a 10% increase in the ileal excretion ofN. Ileal N excretion increased some 23% with CSH addition, so it appears that there was a small effect of the CSH on endogenous protein loss. Given that there was no response to the dietary addition of PEG, cr in the cottonseed hulls did not appear to influence endogenous protein or amino acid flow at the terminal ileum of the rat. Jansman ( 1993) fed pigs diets containing faba bean hulls with low and high concentrations of cr, and found that either dietary fibre and, or cr in faba bean led to a decreased true digestibility of dietary protein and increased excretion of endogenously secreted proteins. Shahkhalili et al. ( 1990) suggested that diets rich in polyphenols from varying' sources influence faecal N excretion in the rat. The rats in the present study initially lost body weight, but after a week had started to gain in weight (data not included). A similar observation was made by Glick and Joslyn ( 1970) who fed rats different types of tannin, including tannic acid, and by Mehansho et al. ( 1983) who gave rats a high-tannin sorghum. The consumption of diets containing tannin was shown to specifically increase the size of the parotid glands in the rat and the synthesis and secretion of proline-rich proteins (PRPs; Mehansho et al. 1992). Tannin-induced PRPs were shown to have a very high binding affinity for tannins (Mehansho et al. 1983). The binding of tannins to both dietary and endogenous proteins has been used to explain the reduced apparent digestibility of protein in tannin containing diets. However, evidence for dietary cr increasing endogenous ileal amino acid loss was not found in the present study. Overall, inclusion of CSH in the EHC based diet led to an increase in the ileal excretion of amino acids. There may have been some effect of the CSH fibre component on endogenous ileal amino acid loss, but the cottonseed cr did not appear to influence this loss. Based on the present result, the presence of some hulls in commercial cottonseed meal will partly contribute to the reported low apparent ileal amino acid digestibility coefficients for CSM protein. However, this cannot be explained by an effect of the cr content of the hulls on endogenous ileal amino acid loss. 5. 6 REFERENCES Batterham E S, Anderson L M, Baigent R D, Darnell R E, Taverner M R 1990 A comparison of the availability and ileal digestibility of lysine in cottonseed and soya-bean meals for grower/finisher pigs. Br J Nutr 64 663-677. 1 62 Butts C A, Moughan P J, Smith W C 1991 Endogenous amino acid flow at the terminal ileum of the rat determined under conditions of peptide alimentation. J Sci Food Agri 55 1 75- 1 87. Costigan P, Ellis K J 1987 Analysis of faecal chromium derived from controlled release marker devices. NZJ Techno 3 89-92. Donkoh A 1993 Amino acid digestibility in meat and bone meal for the growing pig: the development of a digestibility assay based on the laboratory rat. PhD thesis, Massey University, Palmerston North, New Zealand. Glick Z, Joslyn M A 1970 Effect of tannic acid and related compounds on the absorption and utilization of proteins in rats. J Nutr 100 51 6-520. Huisman J 1989 Antinutritional factors (ANFs) in the nutrition of monogastric farm animals. In: Nutrition and Digestive Physiology in monogastric farm animals. Ed. Weerden E J, Huisman. Pudoc Wageningen, the Netherlands. pp I 7-34 . Jansman A J M 1993 Tannins in faba bean (Viciafaba L.) - anti nutritional properties in monogastric animals. PhD thesis, Wageningen Agricultural University, Wageningen, the Netherlands. Jones W T, Mangan J L 1977 Complexes of the condensed tannins of sainfoin (Onobrychis viciifolia Scop.) with fraction 1 leaf protein and with submaxillary mucoprotein, and their reversal by polyethylene glycol and pH. J Sci Food Agri 28 1 26- 1 36. Longstaff M, McNab J M 1991 The inhibitory effects of hull polysaccharides and tannins of field beans (Vicia faba L.) on the digestion of amino acids, starch and lipid and on digestive enzyme activities in young chicks. Br J Nutr 65 199-2 16. Mangan J L 1988 Nutritional effects of tannins in animal feeds. Nutr Res Rev 1 209- 23 1 . Mehansho H, Hagerman A, Clemens S, Butler L, RogIer J, Carlson D M 1983 Modulation of proline-rich protein biosynthesis in rat parotid glands by sorghums with high tannin levels. Proc Nati Acad Sci 80 3948-3952. Mehansho H, Asquith T N, Butler L G, Rogier J C, Carlson D M 1992 Tannin­ mediated induction of proline-rich protein synthesis. J Agri Food Chem 40 93- 97. Moughan.P J, Darragh A J, Smith W C, Butts C A 1990 Perchloric and trichloroacetic acids as precipitants of protein in endogenous ileal digesta from the rat. J Sci Food Agri 52 1 3-2 1 . Statistical Analysis System (SAS) 1 985 SAS User's Guide, Version 5 Edition, Cary, NC: SAS Institute Inc. USA. Shahkhalili Y, Finot P A, Hurrell R, Fern E 1990 Effects of foods rich in polyphenols on nitrogen excretion in rats. J Nutr 120 346-352. 163 Terrill T H, Rowan A M, Douglas G B, Barry T N 1 992 Determination of extractable and bound condensed tannin concentrations in forage plants, protein concentrate meals and cereal grains. J Sci Food Agri 58 32 1 -329. Yu F, Barry T N, Moughan P J, Wilson G F 1993 Condensed tannin and gossypol concentrations in cottonseed and in processed cottonseed meal. J Sci Food Agri 63 7- 15. Yu F, McNabb W C, Barry T N, Waghorn G C 1995a Effect of condensed tannin in cottonseed hulls upon the in vitro degradation of cottonseed kernel proteins by rumen micro-organisms. J Sci Food Agri 69 223-234. Yu F, Moughan P J, Barry T N 1995b The effect of cottonseed condensed tannin on the ileal digestibility of amino acids in casein and cottonseed kernel. Br J Nutr (In press). Chapter 6 THE EFFECT OF COTTONSEED CONDENSED TANNINS ON THE ILEAL DIGESTIBILITY OF AMINO ACIDS IN CASEIN AND COTTONSEED KERNEL This Chapter has been accepted for publication in the British Journal of Nutrition (In press; 1 995). Reproduced by pennission of the Nutrition Society. London. UK. 1 65 6. 1 ABSTRACT The effect of adding cottonseed hulls to casein and cottonseed kernel based diets on the apparent and true ileal digestibility of nitrogen (N) and amino acids, and the proportion of this effect accounted for by condensed tannin (CT), was detennined using the growing rat. Sixty rats were allocated randomly to ten semi-synthetic diets, containing either casein (4 diets) or purified unheated solvent-extracted cottonseed kernel (6 diets) as the sole protein sources, with chromic oxide added as an indigestible marker. Two of the casein diets contained no hulls whilst the remaining two diets contained 70 g cottonseed hulls kg- I . Two of the cottonseed kernel based diets contained no hulls, with two containing 23 g hulls kg-I and the remaining two containing 46 g hulls kg-I . For each pair of diets, polyethylene glycol (PEG) was either included or excluded. The effect of CT was quantified by comparing control rats (-PEG; CT acting) with PEG supplemented rats (+PEG; cr inactivated) at each level of dietary hulls. The rats were given their respective experimental diet for 14 days. Each rat was given the food ad libitum for 1 0 min, hourly from 0800 to 1 800 h . On day 14, samples of digesta were collected at death from the terminal 1 5 cm of ileum at 7 hours from the first meal. Apparent and true ileal digestibilities were calculated for dry matter (DM), N and the individual amino acids. The principal finding was that the inclusion of hulls depressed the apparent and true ileal digestibility of N arid amino acids, but with the response differing between diets. With the casein based diet the mean apparent and true ileal amino acid digestibilities were significantly depressed from 0.89 and 0.96 to 0.85 and 0.92, respectively, by the inclusion of 70 g hulls kg- I in the diet, and addition of PEG then restored these to 0.89 and 0.95. All of the depression could be explained by the CT content of the hulls. However, with the cottonseed kernel based diet the response fell into three categories. The apparent and true ileal digestibilities of the essential amino acids cystine and methionine were not affected by hull addition, ileal digestibilities of leucine, isoleucine, lysine, threonine and valine were markedly depressed by hull addition with approximately 50% of the depression being explained by CT, whilst the ileal digestibilities of histidine, arginine and phenylalanine were depressed by hull addition but little or none of this effect could be explained by cr. Thus the effect of hulls on protein digestion clearly differed with source of protein. With the cottonseed kernel based diet it seems that components of the hulls other than cr also depressed the apparent and true ileal digestibility of N and amino acids. The identity of these components is unknown. 1 66 6. 2 INTRODUCTION Cottonseed meal (CSM) is an important source of protein for monogastric fann animals (Lusas and Jividen 1987), but is regarded as being of variable quality and generally of low amino acid availability (Batterham et al. 1990; Batterham 1992). The nutritional value of CSM is influenced by the processing conditions applied to cottonseed during oil extraction, and by the presence of anti-nutritional factors (ANFs) and non-starch polysaccharides (Frank 1 987; Huisman et al. 1 990; Yu et al. 1 993). Gossypol, a naturally-occurring polyphenolic substance in cottonseed, reacts with the epsilon-amino group of lysine during heating of the seed to form insoluble, indigestible complexes (Lyman et al. 1 959; Oamaty and Hudson 1979; Berardi and Goldblatt 1 980; Ikurior and Fetuga 1 988). Other components of the seed, such as asparagine and glutamine (Varnish and Carpenter 1 975), raffinose and phospholipids (Martinez et al. 1967) have been found to interact during seed processing, leading to inter- and intra-molecular cross-linkages, which reduce protein digestibility by obstructing enzymic attachment (Varnish and Carpenter 1 975). - Recent studies have found that condensed tannins (CT) are present in commercially produced CSM in significant concentrations (8 to 16 g kg- 1 0M; Balogun et al. 1 990; Terrill et al. 1992; Yu et al. 1 993). CT occur in cottonseed hulls (32-65 g kg-1 DM) mainly bound to protein and fibre, but are absent from cottonseed kernel (Yu et al. 1 993). Condensed tannins are polyphenolic compounds, capable of precipitating proteins from aqueous solutions, and have been shown to have anti-nutritional effects in non-ruminant animals (Huisman et al. 1 990; Helsper et al. 1993). Specifically, they are known to increase faecal excretion of nutrients, particularly amino acids, thus reducing apparent nutrient digestibility (Mangan 1 988; Salunkhe et al. 1 990). Moreover, in vitro and in vivo studies have demonstrated that CT can inhibit the activity of digestive enzymes (Longstaff and McNab 1 99 1 ; Jansman 1993) due to the formation of tannin­ enzyme complexes which are biologically inactive (Griffiths 1979; Griffiths and Moseley 1 980). In the rat, sorghum and faba bean CT are known to cause hypertrophy of the parotid glands, accompanied by an increased secretion of proline-rich proteins (Mehansho et al. 1983, 1 992; Jansman 1 993). CT may cause damage to the gut mucosa (Mitjavila et al. 1 977; van Leeuwen et al. 1993), and may also cause an increased loss of mucin in the faeces (Sell et al. 1 985). There is no information on the effects of bound CT in cottonseed hulls on nutrient digestibility in monogastric animals. The objective of the present study was to determine the effect of cr in cottonseed hulls on the apparent and true ileal digestibility of amino acids in casein and in unheated solvent-extracted cottonseed kernel fed to the growing rat. Dietary polyethylene glycol (pEG) addition was used in the present work, 1 67 to allow an effect of the CT, consequent upon an increase in the level of dietary inclusion of cottonseed hulls, to be distinguished from an effect of the increased fibre. PEG binds strongly to CT and can be used to completely displace protein from the cr -protein complexes (Jones and Mangan 1 977; Barry and Manley 1986). Work was also conducted to demonstrate that adding PEG to the diet had no effect on protein digestion in the absence of cr. 6. 3 MATERIALS AND METHODS 6. 3. 1 Preparation of Cottonseed Kernel and Hulls Delinted whole cottonseed (var. Siokra L22) supplied by Cotton Seed Distributors Ltd, Wee Waa, NSW, Australia was cracked using a Crushing-Mill (AB Thorell and Persson, Uppsala, Sweden), and separated into kernels and hulls using air-flow, at the Seed Technology Centre, Massey University, with final manual separation. The separated kernels were freeze-dried for 48 h, ground to pass a 2 mm diameter sieve, and the oil and gossypol were extracted using hexane and then acetone in water (70:30 w/w) using a modification of the Pons and Eaves ( 1 967) procedure, as described by Yu et al. ( 1 995a). Finally, extracted cottonseed kernel and hulls were then re-ground to pass through a 1 mm diameter sieve and were stored at -200c. The chemical composition of the unheated solvent-extracted cottonseed kernel and hulls are shown in Table 6. 1 . 6. 3. 2 Animals and Diets Male and female Sprague-Dawley rats, which had been weaned at 4 weeks of age, were reared on a high quality diet at the Small Animal Production Unit, Massey University. The animals were kept individually in raised stainless steel cages with wire mesh floors, at 20±2OC and with a 1 2 h light/dark cycle. Ten semi-synthetic diets were formulated based on com starch, and containing either casein or purified unheated solvent-extracted cottonseed kernel as the sole protein source (Table 6. 2). The diets contained graded levels of cottonseed hulls, and chromic oxide was added, as an indigestible marker compound, to all diets. At each level of dietary hulls, PEG (molecular weight (MW) 3500, Union Carbide, Danbury, CT, USA) was either included or excluded. Thus, the effect of CT can be quantified by comparing control rats (-PEG; CT acting) with PEG-supplemented rats (Cf inactivated) at each level of dietary hulls. The PEG was added at a minimum ratio of 2 mg mg-1 total cr to maximise the displacement of protein from the Cf-protein complexes (Yu et al. 1 995b). 1 68 Table 6 . 1 Chemical compositions 1 (g kg-1 DM) of the unheated solvent-extracted cottonseed kernel and hulls Cottonseed Cottonseed kernel hulls Dry matter (g kg-I) 909 91 1 Crude protein 537 35 Oil 1 38 10 Neutral detergent fibre 89 886 Acid detergent fibre 37 624 Lignin 27 209 Free gossypol 0.8 0.2 Condensed tannin: Extractable 0 13 Protein-bound 0 29 Fibre-bound 0 10 Total (calculated) 0 52 Essential amino acid Arginine 64 1 .0 Histidine 1 6 0.5 Isoleucine 22 0.9 Leucine 43 1 .4 Lysine 30 I . l Phenylalanine 39 0.9 Threonine 21 0.9 Valine 30 I . l Total (calculated) 265 7.8 NQn-����ntiaI aminQ a�iQ Alanine 26 1 .3 Aspartic acid 7 6.9 Glutamic acid 101 2.9 Glycine 24 1 .0 Proline 23 1 .2 Serine 24 1 .6 Tyrosine 19 1 . 1 Total (calculated) 224 16.0 1 Mean of duplicate determinations. 169 Table 6. 2 Ingredient (g kg-I air dry weight) and chemical compositions of the casein and unheated solvent-extracted cottonseed kernel based diets Qi�I �a�ein �ottQIl��eQ kernel Cottonseed hulls (g kg-I) � --..ZL _0_ --2L 46 PEG I + + + + + Ingredient Casein 160 160 160 160 Cottonseed kernel 290 290 290 290 290 290 Cottonseed hulls 70 70 23 23 46 46 PEG I 8 8 5 2.5 5 Maize starch 599 591 529 521 469 464 446 443.5 423 4 18 Sucrose 100 100 100 100 100 100 100 100 100 100 Maize oil 50 50 50 50 50 50 50 50 50 50 Cellulose2 35 35 35 35 35 35 35 35 35 35 Mineral/vitamin premix3 15 15 15 15 15 1 5 1 5 1 5 1 5 15 Sodium chloride 5 5 5 5 5 5 5 5 5 5 Magnesium sulphate 2 2 2 2 2 2 2 2 2 2 Potassium carbonate 4 4 4 4 4 4 4 4 4 4 Oicalcium phosphate 24 24 24 24 24 24 24 24 24 24 Chromic oxide 6 6 6 6 6 6 6 6 6 6 Mytrient cQlllent {OM l:!��i�}4 OM5 983 982 977 984 973 971 971 975 966 975 OM5 95 1 945 950 949 924 927 926 922 927 93 1 Crude protein 150 156 162 156 160 160 163 161 169 165 Oil 17 20 20 15 94 94 91 94 93 91 NDP 61 61 26 26 46 46 67 67 ADP 43 43 1 1 1 1 25 25 39 39 Lignin 14 14 8 8 1 3 1 3 1 8 1 8 Gross Energy (MJ kg-I) 1 8 1 8 1 8 1 8 18 19 19 19 19 19 Free gossypol (mg kg-I) 174 1 82 175 1 89 1 84 1 86 Condensed tannin (g kg- I) Total 0 0 3.6 3.6 0 0 1 .2 1 .2 2.4 2.4 Free 0 0 0.91 0.9 1 0 0 0.30 0.30 0.60 0.60 . I Polyethylene glycol, MW 3,500. 2 Avicel, Asahi Chemical Industry Company Ltd, Tokyo, Japan. 3 Rat Pellet Premix 9327, Technik Products, Auckland. New Zealand. Supplied the following per kg diet: 10,000 IU Vitamin A; 1 ,500 IU Vitamin 03; 30 IU Vitamin E; 1 mg Vitamin K; 1 mg Thiamine (B 1); 4 mg Riboflavin (B2); 3 mg Pyridoxine (B6); 0.02 mg Vitamin B 12; 1 5 mg Pantothenic acid; I mg Folic acid; 25 mg Niacin; 125 mg Antioxidant; 250 mg Choline; 100 mg Manganese; 35 mg Iron; 1 0 mg Copper; 60 mg Zinc; 1 mg Cobalt; 0.15 mg Selenium. 4 Means of duplicate determinations. 5 OM, dry matter; OM, organic matter; NDF. neutral detergent fibre; ADF, acid detergent fibre. 1 70 6. 3. 3 Expernnental Procedure Sixty rats (mean±SE bodyweight, 1 76±4.5 g) were assigned randomly to the ten experimental diets (Table 2), such that there were three males and three females per diet. The animals were initially fed a casein-based diet (approximately 100 g crude protein kg- 1 ) for 2 days and were then given the experimental diets for a further 1 4 days. The diets were offered in stainless steel feeders fitted with anti-spill devices similar to those described by Thomsen ( 1 98 1 ). The rats were trained to consume their experimental diet between 0800 and 1 800 h, with the feeder being placed in the cage for 10 min at hourly intervals. The training was achieved within 7 days, and feed intakes were recorded after each 10 min feeding. Fresh water was freely available. On day 1 4, the rats were asphyxiated in carbon dioxide gas and decapitated (immediately ceasing all neural stimulation to the gut) at 7 hours from the start of feeding. The abdomen was opened by an incision along the mid-ventral line and the skin and musculature were folded back to expose the viscera. The final 1 5 cm of the ileum was immediately dissected from the body, and the intestinal surface cleaned using absorbent tissue paper, taking care not to apply pressure to the intestine. The digesta were slowly flushed out into plastic bags with distilled water from a plastic syringe. The digesta from each animal were kept separate and packed in ice immediately after collection. . Ileal digesta and samples of the test diets were subsequently freeze-dried, finely ground and stored at -200c for the determination of nitrogen, chromium, and total amino acids. The stomach contents were inspected for signs of faecal contamination resulting from coprophagy. 6. 3. 4 Chemical Analysis The diets and ileal digesta were analysed in duplicate for total nitrogen using the Kjeldahl procedure, and crude protein calculated as total N x 6.25. The chromium contents of duplicate 1 5 mg samples of ileal digesta and each diet were determined by the method of Costigan and Ellis ( 1987). The CT contents of the diets were determined using the method of Terrill et al. ( 1992). Free gossypol in the diets was estimated by the method Ba 7-58 of AOCS ( 1 975). The NDF, ADF and lignin contents were determined by the method of Robertson and van Soest ( 198 1 ). The crude ash, crude oil .and gross energy contents of the feeds were analysed according to conventional methods (AOAC 1 975). The amount of freeze-dried matter (FDM) collected from the tenninal ileum of each rat was detennined after freeze drying the samples for 3 days. 17 1 Amino acid composition was detennined on 5-7 mg samples using high perfonnance liquid chromatography (HPLC, Waters Associates, USA), using a reverse phase column and the Pico.Tag analytical method (Cohen et al. 1 989). Duplicate samples were hydrolysed in 500 pi of 6 M HCI with I % added phenol, for 24 hours at 1 10± 1 °C in glass tubes sealed under vacuum. For the detennination of methionine and cystine in the samples obtained from the cottonseed kernel based diets, separate duplicate samples were oxidised with 90: 10 (v/v) of perfonnic acid:H202 prior to hydrolysis. Methionine and cystine in the samples obtained from the casein based diet, and tryptophan, which was partly destroyed during acid hydrolysis, were not detennined. The amino acids were detected by the fluorescence of their phenylisothiocyanate (PITC) derivatives using a programmable multi-wavelength detector (Waters 490E, USA). Free amino acid molecular weights were used to calculate the weights of amino acids. 6. 3. 5 Data Analysis Apparent and true amino acid digestibility coefficients were calculated using the following equations: Apparent amino acid Dietary AA intake - ileal AA output (AA) digestibility* Dietary AA intake True amino acid (AA) Dietary AA intake - (ileal .AA output - endogenous AA output) digestibility * Dietary AA intake Amino acid (AA) output* * All units were mg g-1 FDM. AA concentration in ileal digesta x diet total chromium ileal total chromium Endogenous amino acid flows used for calculating true amino acid digestibility coefficients in the present study were obtained in a separate but related study (Yu et al. 1995c), in which endogenous ileal amino acid flows were determined in rats given diets containing graded levels of cottonseed hulls. The enzymically hydrolysed casein (EHC) ultrafiltration method (Moughan et al. 1990; Butts et al. 199 1 ) was used in the latter work to determine endogenous amino acid losses. Data from the present study were subjected to ANOV A. A linear statistical model, which included tenns for hulls, PEG and hulls x PEG, was initially fitted to the digestibility data for each amino acid singly, and reduction in sums of squares was used to detennine levels of significance. Relevant comparisons between treatment means were made using orthogonal contrasts (Snedecor and Cochran 1982). Where a cause and effect trend in the data was expected (e.g. ileal digestibility as a function of hull addition), the data (n= 18) were subjected to a simple linear regression and slopes were tested for statistical significance from zero. 6. 4 RESULTS 1 72 The overall mean (±SE) liveweight for the rats at the end of the study was 197±8.4 g. Mean food intakes for the rats on day 13 of the study are given in Table 3. Food intake was within the normal range for the 200 g body weight rat (NRC 1978). Daily food intake tended to be lower with the casein based diet in comparison with the cottonseed kernel based diet, and dietary PEG addition did not affect food intake on either diet (Table 6. 3). On the last day of study, the rats had high food intakes over the first two hourly meals and then consumed generally even sized meals for the remainder of the feeding period (Table 6. 3). The latter was important to ensure an even flow of digesta at the terminal ileum. Faeces were not detected in the gastric contents at slaughter indicating that coprophagy had not occurred at least on the last day of study. Table 6. 3 Mean food intakes1 of the growing rats on day 13 and hourly meal intakes for the last day (day 14) of study Diet Food intake Hourly rneal intakes on day 142 on day 1 3 (g) (g) 2 3 4 5 6 7 Total Casein: -PE

I< * 0.91 0.94 0.004 NS *** ** 0.92 0.95 0.004 >I< ** *'" 0.93 0.96 0.004 NS *** *** 0.78 0.83 0.0 1 0 * ** ** 0.84 0.88 0.008 NS ** ** 0.8 1 0.86 0.009 ** ** ** 0.80 0.85 0.0 1 0 NS ** ** 0.82 0.88 0.007 N S *** *** 0.89 0.67 0.92 0.74 0.004 0.0 1 5 NS NS *** *** * * True digestibility o 70 Overall Significance) + + SE . PNH HNP PWH 0.94 0.94 0.90 0.93 0.004 NS *** ** 0.98 0.98 0.95 0.97 0.004 NS *** ** 0.98 0.98 0.93 0.97 0.005 N S *** *** 0.93 0.98 0.98 0.98 0.9 1 0.95 0.95 0.94 0.94 0.93 0.98 0.99 0.99 0.93 0.96 0.96 0.95 0.95 0.88 0.9 1 0.008 0.95 0.97 0.003 0.95 0.98 0.004 0.96 0.98 0.003 0.89 0.92 0.008 0.9 1 0.93 0.007 0.90 0.94 0.007 0.90 0.94 0.007 0.90 0.93 0.008 0.98 0.98 0.95 0.97 0.003 0.88 0.89 0.82 0.86 NS *** ** NS *** * * NS *** *** NS *** ** NS * * NS ** * NS *** ** NS **'" * * NS ** * *** *>1<* *** *>1< Tyrosine 0.96 0.96 0.93 0.96 0.004 NS ** *** 0.99 0.99 0.97 0.98 0.0 10 0.005 NS NS NS * NS 1 Fractions of cottonseed hulls in the diet. 2 Polyethylene glycol, MW 3,500. 3 PNH: PEG+ vs PEG-, 0 g kg-I hulls; HNP: 0 g kg- I vs 70 g kg- I hulls without PEG; PWH: PEG+ vs PEG-, 70 g kg- I hul ls; . NS, non significant; *, p<0.05; ** , pO.05) between heat treatment and PEG for apparent amino acid digestibility, except for threonine and tyrosine. The significant heatxPEG interactions for threonine (p<0.05) and tyrosine (pO.05). The mean increase in apparent ileal digestibility for the 14 amino acids, due to the addition of PEG ( 1 2 g kg- l DM) to the CSM diet, was 2% units. The species x PEG interaction was not statistically significant, except for the non-essential amino acid, glycine. The commercial CSM had a low apparent ileal amino acid digestibility overall. Table 8. 5 Mean (n=6) apparent digestibility of dry matter, total nitrogen and amino acids determined at the terminal ileum of the growing rat and pig given a commercial cottonseed meal based diet in Experiment 2 Dry matter Nitrogen Essential aminQ acir;! Arginine Histidine Isoleucine Leucine Lysine Phenylalanine Threonine Valine NOD-����Dtial aminQ a�iQ Alanine Aspartic acid Glutamic acid Glycine Serine Tyrosine PEG_2 0.70 0.69 0.85 0.87 0.57 0.62 0.50 0.76 0.50 0.63 0.60 0.70 0.79 0.54 0.61 0.68 CottQnseed meal diet Rat Pig PEG+2 PEG- 0.70 0.63 0.7 1 0.66 0.87 0.85 0.87 0.77 0.62 0.61 0.65 0.64 0.52 0.58 0.77 0.75 0.53 0.56 0.66 0.64 0.63 0.60 0.7 1 0.70 0.8 1 0.76 0.62 0.60 0.64 0.63 0.69 0.67 PEG+ 0.66 0.70 0.85 0.76 0.64 0.66 0.59 0.78 0.58 0.67 0.63 0.74 0.78 0.62 0.66 0.70 Overall Level of significan�e I SE Species PEG SXP 0.01 1 *** NS NS 0.010 0.08 ** NS 0.006 * NS NS 0.012 *** NS NS 0.012 * ** NS 0.01 1 NS * NS 0.013 *** NS NS 0.010 NS 0.09 NS 0.009 *** * NS 0.010 NS * NS 0.012 NS * NS 0.010 NS * NS 0.007 *** * NS 0.01 1 ** *** ** 0.010 * ** NS 0.008 NS * NS 1 SxP, interaction between species and PEG; NS, non significant; *, p