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. STUDIES OF NEUROENDOCRINE HECHANISMS INFLUENCING SEASONAL VARIATIONS IN SE?lEN PRODUCTION AND PLASHA HORMONE LEVELS IN RAHS A thesis presented in partial fulfilment of the requirements for the Degree of Doctor of Philosophy at Massey University GRAHAM KEITH BARRELL 1976 Abstract of a thesis presented in partial fulfilment of the re1uirements for the Degree of Doctor of Philosophy STUDIES OF NEUROENDOCRINE NECHANISHS INFLUENCING SEASONAL VARIATIONS IN SEMEN PRODUCTION AND PLASHA HORMONE LEVELS IN RAI1S by Graham Keith Barrell Many workers have shown that sheep are seasonal breeders with peak reproductive activity occurring during the autumn months. The initial experiment in this thesis was designed to ii define the seasonality of reproduction in rams of the N.Z? Romney breed as determined by repeated measurements of semen characterist- ics and of plasma hormone levels. These parameters were studied for 16 months in six N.Z. Romney rams on pasture, with five Merino and four Polled Dorset rams included for comparison. Semen from all three breeds showed relatively regular seas? onal changes in ejaculate volumes and seminal fructose levels with peak values being recorded during March. Likewise, monthly hormone levels VRried in a regular manner with plas?a LH, testost? ?rone and prolactin concentration being elevated during the summer months. Many of the other semen parameters measured showed little tendency for seasonal v?riations, however a change in semen collect? ion technique, from predominantly artificial vagina to entirely electro-ejaculation, may have masked some seasonal changes. All three breeds showed similar seasonal changes in the parameters studied although semen from the Polled Dorsets did not exhibit regular seasonal variations in fructose levels. iii Further experiments were carried out to define the neuroendoc- rine mechanisms which regulate the seasonal reproductive changes in N.Z. Romney rams. Three olfactory bulbectomized rams, three cranial cervical ganglionectomized rams and four rams which had undergone both of these surgical modifications, were grazed to- gether with the rams mentioned above. These surgical treatments disrupted the regular seasonal changes in plasma levels of LH and prolactin , but not, o f testosterone. Spermatozoal concentrations in ejaculates from operated rams were higher than those from un- operated controls, whereas mean fructose concentrations were lower; however the pattern o f seasonal changes in seminal fructose levels was similar in all groups o f rams. Cranial cervical ganglionectomy reduced hydroxyindole-0-methyl transferase activity and cell vol- umes in the pineal glands, so it was concluded that disrupted seasonal patterns of changes in plasma LH and prolactin levels , plus the altered semen production in the surgically treated rams , resulted from modified pineal gland and/or olfacto?y system activity. A preliminary investigation into the role of changes in daily photoperiod as the stimulus for seasonality o f reproduction, was carried out by placing rams in light-controlled rooms at the time of the March equinox. Over the following nine months rams exposed to a phase-reversed annual lighting cycle showed earlier elevations of seminal fructose and plasma testosterone levels than rams on I . either the normal annual or a constant equinoctal lighting regime. ? In all three groups plasma prolactin levels were directly related to the length of daily photoperiod. The findings of the above experiments were extended by a final study in which both pinealectomized and sham-operated rams were exposed to normal or reversed annual lighting cycles. iv Effects of lighting on plasma testosterone and prolactin levels, and on seminal fructose levels, were diminished by pinealectomy. Autopsy data related to gonadal and accessory sex gland function showed significant operations x light ing regimes interactions, which supported the conclusion that in rams pineal gland function mediates endocrine ann gonadal responses to changes in daily photoperiod. Three short-term investigations of hormonal secretion pro? files conducted during the latter experiment, showed that major fluctuations in the release of LH, testosterone, prolactin and cortisol occurred irregularly during the day. A nocturnal elevat- ion of plasma prolactin levels was abolished by pinealectomy. These acute studies tended to confirm the findings of the latter experiment, but in particular they highlighted the pulsatile nature of hormonal secretion. ACKNOWLEDGMENTS I wish to express my gratitude for the help ful advice and generous assistance provided by my chief supervisor , Dr K.R. Lapwood , who also proposed the area of research covered in this thesis. V Further thanks are due to Dr R.M. Greenway for his role as supervisor and provider of encouragement, and to Prof. R.E. Mun? ford for generously providing facilities in the Department o f Physiology and Anatomy and for his major contribution to the statistical analyses o f the experimental data. The financial assistance o f the Veterinary Research Fund o f Massey University is gratefully acknowledged, and the National Institute o f Arthritis, Metabolism and Digestive Diseases, Nation? al Institutes of Health, U.S.A. is acknowledged for provision o f reagents for hormone assays. Dr G.D. Niswender of Colorado State University, U.S.A. is acknowledged for supplying antisera to testosterone and ovine-LH, so also is Dr L.E. Reichert, Jr, Emory University , U.S.A. for donating the highly puri fied ovine prolactin and LH for radioimmunoassay , and Prof. D.S. Flux for donating antisera to bovine prolactin. Dr D.C. Thurley and staff at Wallaceville Animal Research Centre, Upper Hutt, are thanked for performing cortisol assays. I am indebted to Mr M.J. Birtles for carrying out the histo? logical processing and to Mr T.G. Law for the photographs in this thesis. The enthusiastic and unflagging technical assistance o f Mrs H. Carter, Mr R.N. Ward and Mr R.H. Telfer is warmly appreciated , and I wish to thank Mrs D.P. de Wiele, Mrs H.E. Walker and Miss C. Howell for their contrib11tions to the preparation o f this manuscript. vi Finally, I thank my wife Rosalind and daughters, Snrah and Rebecca, for their encouragement, support and perserverance whilst I was engaged in these studies. In these studies,the candidate made the major contribution to the conception and execution o f the experiments. The chief supervisor assisted with the experimental design and in the dev? elopment of surgical techniques and hormone assays. Otherwise, apart from the execution o f cortisol assays and histological processing per formed by others, and the development o f an ovine? LH radioimmunoassay where the candidate's contribution was minimal, all remaining work described in this thesis involved the full participation of the candidate. CHAPTER I: INTRODUCTION TABLE OF CONT?NTS l. SeasonBlity of reuroduction ( 1) General (2) Manifest?tions of reprorluctive seasonality in female animals (a) General (b) Sheep (3) Manifestations of reproductive seasonality in vii PAGE 1 1 2 2 2 male animals 3 (a) General 3 (b) Antlers of deer 3 (c) Gonads and acce?sory sex glands 4 (d) Male libido 5 (e) Semen production 6 (4) Hormonal changes 7 (a) Pituitary and hypothalamic hormone levels 7 (b) Plasma hormone levels 8 (5) Male sexual cycles (6) Male-female reproductive seasonality inter? relationships (7) Summary 10 10 11 2. Factors involved in the seasonality of repro.-:::ction 11 (1) Light (?.) Temperature (3) Nutrition (4) Olfaction (a) General (b) Photoperiodicity of reproduction in m.rymmals (5) Other factors 3. Neuroendocrinolop,y of repronuction in male mammals (1) General 11 11 16 22 25 26 28 29 29 (2) Hyuothalamus 29 (3) Hypothalamic regions involved in gonadotrophin release 30 (4) Extr8hypothalaruic areas involved in gonadotrophin release (5) Mechanisms of GnRH release (6) Negative feedback of steroids (7) Sit es of steroid feedback 30 31 32 32 viii PAGE (8) Hypoth?lamic sites of steroid feedback 33 (9) Role of steroids in hypothalamic differentiation 33 (10) Synthetic GnRH 34 (11) Actions of GnRIT 35 (12) GonAdotrophins (13) Androgens (?) General (b ) FSH anrl LH (c ) Prolactin 4. The role of the pineal gland in mammalian repror3uction 36 36 36 37 3 8 39 (1) General 39 (2) Ef fects o f pinealectomy 39 (3) Pineal indoleamines 40 (4) Pineal peptides 41 (5) Gonadal regulation of pineal function 42 (6) Transport of pin?al principles 42 (7) Afferent nervous pathways 43 (8) Role of the pineal .gland in the seasonality o f reproduction 44 (9) Pineal function after puberty 45 (10) Pineal gland and olfactory function 5. The purpose of th8 present study CHAPTER II: HATERIALS AND HETHODS 45 46 ? 1. Animals 47 2. Animal management procedures (l) On i)asture (2) Indoors 3. Surgical techniques (l ) General surgical procedure (2) Olfactory bulbectomy (3) Cranial cervical ganglionectomy (4) Olfactory bulbectomy/cranial cervical ganglionectomy (5) Pinealectomy (6) Sham-pinealectomy 4. Semen collection 5. Semen appraisal (1 ) Eja culate volume 47 47 47 48 48 49 50 50 50 55 55 55 55 (2) Motility and percentage motile spermatozoa (3) Percenta ges of unstained and morphologically normal ?permatozoa (4) Concentration of soermatozoa per ml semen and ix PAGE 56 56 number of spermatozoa per ejaculate 56 (5) Conc?ntra tion of fructose in semen and seminal plasm?, ann total ejaculate fructose content 56 6. Blood collection 7. Autopsy procedure (1) Histological procedures 61 62 62 (2) Seminal ve?iculer fructose content and concentration 63 64 (3) Epididymal sperm?.tozoal reserves (4) Hydroxyindole-0-methyl transferase a?say 65 8. Hormone assays (1) Reagents (2) LH assay (a ) Discuseion of HIOMT assay method 66 (a ) Radioiodination of ovine-LH with 125r (b) Preparation of precipitating antisera (c) Radioimmunoassay procedure (d) Validation of ov ine-LH radio- 67 67 68 68 69 69 immunoassay 70 (3) Prolactin assay 73 (a ) Labelling o f ovine prolactin with 125r 73 (b) Preparation o f prolactin antiserum 75 (c) Radioimmunoassay procedure 75 (d) Validation of ovine prolactin assay 76 (4) Testosterone assays 76 76 84 87 (a ) Protein-binding assay (b) Radioim?unoas?ay I (c) Radioimmunoa?say II (5) Cortisol assay 9. Experimental design and analysis (1) Analyse? o f variance (2) Missing data (3) Trancform?tions (4) Computations 90 91 91 94 94 95 CHAPTER I II: SEASONALITY OF SEMEN PRODUCTION AND PLASMA HORl'lONE L"SVELS IN NE'.V ZEALAND RONNEY, MERINO AND POLLED DORSET RANS AT PASTURE 1. Introduction 2? Materials and methods (1) Experimental procedure X PAGE 96 96 96 (2) Statistical analyses 97 (a ) Semen data 97 (b) LH and testosterone data 97 (c ) Prolactin data 97 (d ) Rationale for division o f study period into seasons (e ) Breed contrasts 3. Results (1) Semen data (2) Plasma hormone data (a) LH (b) Testosterone (c) Prolactin (3) Autopl'ly data (4) Meteorological data 4 .. Discussion (l ) Seasonal changes in semen production .. (2) Seasonal changes in plasma hormone levels (a) LH e.nd testor;terone (b) Prolactin ( 3) Autopsy data (4) General discussion CHAPTER IV? SEASONALITY OF SEMEN PRODUCTION AND PLASMA HORMONE LEVELS IN RAMS WITH MODIFIED OLFACTORY AND PINEAL FUNCTION l. Introduction 2. Materials and methods (1) Animals and management procedure (2) Data collection (3) Statistical analyses 3 .. Results (1) Semen data (2) Plasma hormone levels (a ) LH (3) Autopsy data (b) TeE"tosterone (c ) Prolactin 97 102 102 102 113 113 113 113 121 121 126 126 130 130 131 133 133 139 140 140 141 141 142 142 152 153 153 153 161 4. Discussion (1) Semen data (2) LH and testosterone (3) Prolactin (4) Autopsy data (5) General discussion CHAPTER V: EFFECTS OF DIFFERENT LIGHTING REGIMES ON SEMEN PRODUCTION AND PLA3MA HORMONE LEVELS IN RAMS 1. Introduction 2. Materials and methods (1) Animals and experimental procedure (2) Statistical analyses 3. Results (1) Semen data (a) Semen data (b) LH and tectosterone (c) Prol9.ctin (d) Treatment contrasts (2) LH and tP-stoderone (3) Prolactin 4. Discussion CHAPTER VI: EFFECTS OF PINEALECTOMY ON SEMEN PRODUCTION AND PLASMA HORMONE LEVELS IN RAMS SUBJECTED TO CONTRASTING LIGHTING REGIMES . 1. Introduction 2. Materials and methods (1) Animals and experimental procedure (2) Statistical analyses 3. Results (1) Semen data (a) Semen and hormone data (b) Treatment contrasts (2) Plasma hormone data (a) LH ( 3) Autopsy da.ta 4. Discussion (b) Testosterone (c) Prolactin (1) Semen production xi PAGE 166 166 167 168 170 172 174 175 175 175 175 180 180 180 180 180 189 189 197 203 203 203 204 204 209 209 209 221 221 225 225 233 236 236 (2) Pla 01) !:: 6 0 0 * 00 ? '-' Q 5 0 4 0 I::Q ? tJ 2 0 LH (ng/ml plasma) F i g u re 2 . 9 Compos i te sta ndard cu rve C mea n?S . E . of seven consecut i ve a s says ) for ov i ne LH rad i o i mmunoa s say . 71 Table 2 . 2 Effec t of d i lution with hypophysectomized sheep plasma , on estimates of LH c oncentration Plasma LH c onc entration (ng/ml ) * Undi luted Di luted 1 : 1 Di luted 1 : 3 5 . 4 5 . 2 4 . 9 4 . 4 4 . 3 3 . 6 4 . 8 - 5 . o 4 . 5 3 . 9 3 . 4 4 . 0 3 . 5 3 . 4 * Each value represents the mean of a triplicate . results of five as says . Di luted 1 : 7 4 . 5 3 . 4 5 . 0 3 . 8 3 . 8 ? 1'\) 73 LH s tandards in the present method were added to tubes containing 200 l-1 1 of hypophysectomized ewe plasma, and then processed through the usual procedure . Als o the data presented in Table 2 . 2 indicated that e ndogenous hormone added to hypophysectomized ewe plasma, was recovered quantitatively. Non-specific binding, e stimated in the absence of unlabelled hormone was always low (less than 1 6;? of the counts bound in the 0 ng/ml s tandard) , and the c ounts obtained were not subtracted from those recorded for the s tandards or samples . Assay sensitivity, defined as the lowest point on the standard curve with a coeffic ient of variation equal to 5? ( Burger, Lee and Rennie , 1 972 ) ranged from 0.04 t o 0.1 1 ng/ml. This range of values corresponded with the lowest plasma LH c oncentrations with 95% fiducial limits which did not overlap zero . Reproducibility of ass ay results was estimated by assaying two plasma samples , four times in each as say, over a number of assays . One s ample with a low Ill concentration (mean 0.55 ng/ml) had a beb?een? assay coefficient of variation (CV) of 22.5% for four ass ays , and a within-assay CV of 1 o. 7%. Another sample (mean 4.1 7 ng/ml ) had a between-assay CV of 1 8.2'}la in twenty ass ays , and a within-as say CV of 8.6%. On the basis of the validation tests performed, it was cons idered that this as say provided reliable estimates of ovine plasma LH levels . ( 3 ) Prolactin Ass5Z Ovine prolactin was measured by a double-antibody radioimmunoassay s imilar to the procedure described above for ovine-LH. (a ) Labe llillji of Ovine Prolactin With 1 25r . Radioiodination of ovine prolactin was carried out by the method of Greenwood, Hunter and Glover ( 1 963 ) with modifications by Fell et al. ( 1 972 ) and Koprowski and Tucker ( 1 971 ) , as described by Langley ( 1 974) . One mCi of iodine-1 25 ( 1 00 m:: i/ml, The Radiochemical Centra , Amersham) was added t o approximately 5 J_Jg of highly purified ovine prolactin ( LER-860-2, c ourtesy of Dr. L.E. Re ichert Jr. , Emory Univers ity, U. S .A. ) in 30 ?1 0.5 M phosphate buffer, pH 7.5. Twenty JJg of chloramine T in 20 ?1 of 0.05 M phosphate buffer, pH 7.5, were added to start the reaction which was allowed to proceed for 60 seconds . The reaction was stopped by the addition of 20 lJ 8 of s odium metabisulphite in 20 lJl of 0 :-05 M phosphate buffer, pH 7.5. Mixing was maintained throughout the period of the reaction and for a further 60 seconds . A tram fer s olution c onsisting of 30 lJl 1 % (w/v) potassium iodide , 0.01 % (w/v) bromophenol blue and 1 6% (w/v) sucrose in 0.05 M phosphate buffer, pH 7.5, plus 30 lJl phosphate buffer pH 7.5, and 30 lJl hypophysectomized ewe plasma was added. The mixture was then transferred quantitatively t o a 0. 7 x 1 8 cm polyacrylamide gel column (Biogel P60, Biorad Laboratories , U. S .A. ) which previously had bee n equilibrated with 1 5 ml of 0 . 02 M barbital buffer, pH 8.6, c ontaining 2?/o ( v/v) aceto?E , and 2 ml of the same buffer containing 5% ? ( w/v ) bovine serum albumin. One ml fractions eluted from the column were collected into 1 ml of PBS-0.1 % gelatin. Aliquots from these fractions were counted for radioactivity and that 74 containing the greatest number of c ounts c orresponding t o radioiodinated prolactin, was further purified on a 1 x 25 cm dextran gel c olumn (Sephadex G1 00, Pharmacia, Swede n) . This column previously had been equilibrated with 25 ml of 0.02 M barbital buffer, pH 8 .6 containing 20}& (v/v) acetone, and 2 ml of the same buffer containing 5% (w/v) bovine serum albumin. Again 1 ml fractions were c ollected into 1 ml of PBS-0.1 % gelatin and aliquots were c ounted for gamma radiation. oP The fraction with the peak level ? radioact ivity was used in the radioimmunoass?. 75 (b) Preparation of Prolac t in Ant iserum. Antiserum ( courtesy Prof. D . S . Flux, Massey University) raised against bovine prolactin (NIH-B1 ) wa3 prepared in rabbits by an initial set of intradermal injections containing a total of 4 mg bovine prolactin in 1 ml of an emul2 ion of Freund' s complete adjuvant and saline . At weekly intervals three further series of injections were made using 2 mg of bovine prolactin in an emulsion of Fround' s incomplete adjuvant and saline. One week after the final injections blood was collected from an ear vein and the anti3er? subsequently collected after clot contraction. ( c ) Radioimmunoas s? Procedure . Polys?rene tubes ( 1 1 x 75 mm) were used for all phases of the radioimmunoass? procedure , and both s tandards and unknown samples were a3 sayed in triplicate . Standard solutions of ovine prolactin (NIH-P-31 1 ) were prepared in 200 ?1 of PBS-3% BSA, to provide a star.dard curve corresponding to a range of plasma c oncentrations from 0 to 256 ng/ml. Hypophysectomized ewe plasma ( 200 ?1) was added to each standard tube , while sample tubes contained 200 ]J l of unkno\vn plasma. Both standard and sample tubes were then adjusted to contain a volume of 500 ]J l by addition of 0.01 M EDTA-PBS-0.1 % gelatin. Two hundred ?1 of rabbit anti ovine prolactin serum, diluted 1 in 8, 000 with 0 .02 M EDTA-PBS containing non-immune rabbit serum (1 in 400) , wa3 added t o each tube prior to incubation at 0 4 C for 24 hours. Radioiodinated prolactin (approximately 50,000 cpm diluted to 1 00 J.l 1 with 0.01 M EDTA-PBS-0.1 % ge latin) was added to each tube and the incub?tion allowed t o proceed for a further 24 hours 0 .. at 4 C . Precipitation of antibo? was completed by centrifuging at 0 1 900 .s, for 30 minutes at 4 C ? The supernatant was removed by aspiration and the precipitate counted for one minute in a Packard Auto-Gamma Scintillation Spectrometer (Model 5285 ) or an LKB-Wallac Ultrogamma counter (Model 1 280) . Non-specific binding and unknown hormone c oncentrations were * After adding 200 ul of the precipitating antiserum (as described for the LH assay) diluted 1 in 8 , the mixture was incubated for 72 hours at 4?c . 76 determined as described for the LH assay. A c omposite standard curve representing the mean value5 from s ix consecutive assays i8 shown in Figure 2 .1 0. (d ) Validation ot_Ovine Prolactin Assay. In general, this ovioo prolactin assay was validated by conducting tests s imilar t o those described for validation o f the LH assay. Specificity of the prolactin antiser?? was indicated by the lack of cross-reaction with the ovine anterior pituitary hormones tested, namely : GH, TSH, LH, FSH and adrenocorticotrophic hormone (ACTH) ( see Figure 2 .1 1 ) . Dilution with hypophyse c t omized ewe plasma did not affect the estimated prolactin content of two plasma samples ( see Table 2 .3 ) . 1 25 . Also non-specific binding of I -prolactl.n was low ( less than 1 2% of the counts bound in the 0 ng/ml standard) and thus ignored in the computation of ass? results . Assay sens itivity ranged from 1 .o t o 2 .0 ng/ml and the reproducibility o f results was checked by ass?ing two samples repeatedly in a nu;nber of ass ays . One wether plasma sample (mean 1 05 .4 ng/ml) displayed a between-assay CV of 6 .4% in seven ass?s , and a within-assay CV of 6 .2%, based on four estimate3 per assay. Another wether plasma sample (mean 42.5 ng/ml) displayed ? a between-assay CV of 1 0.2% in the seven assays , and a within-assay CV of 9.9,%, based on five estimates per assay. Prolactin levels measured in rams by the assay described above were similar to those reported in the literature (Pelletier, 1 973 ; Chamley et al. , 1 974; Forbes et al., 1 975 ) , although both Pe lletier and Forbes et al. reported s ome mean levels higher than 1 50 ng/ml, which exceeded those obtained in the present studies . This assay thus seemed to be reliable and accurate . (4) Testosterone Assgys (a) Protein-binding Ass?. Plasma samples from Experiments 77 8 0 70 10?--?----?----.----.-----.----? 16 32 64 96 128 192 256 PROLACTIN (ng/ml plasma) F i g ure 2 . 1 0 Compos i te sta ndard curve ( mea n?S . E . of s i x consecut i ve a s says ) for ov i ne pro l act i n ra d i o i mmunoassay . 20 M 0 15 )( '"0 c: ::l 0 CO ? a.. 1 0 u 5 1 TSH. FSH 2 LH , ACTH 3 GH 4 PROLACTIN ? ? ? 78 ? e1 ? ?2 ? ...3 1 0 1 00 1000 ng/m l 10,000 100,000 F i g ure 2 . 1 1 Cross-react i on of a nter i or p i tu i ta ry hormones i n the ov i ne pro l act i n rad i o i mmunoassa y . Table 2 . 3 Effec t of di lution with hypophysectomized sheep plasma , on estimates of pro lactin c onc entration of two plasma samples . Plasma prolactin c oncentration (ng/ml )* Sample Number Undi luted Di luted 1 : 1 Di luted 1 : 3 56 56 72 57 66 80 2 80 72 40 85 84 92 73 74 64 * Each value represents the mean of a tripli cate . -'-.) \.0 80 3, 4 and 5 were assayed for testosterone content using a competitive protein-binding assay based on the method described by Anders on ( 1 970) . ( i) Extraction Procedure . Duplicate 1 ml plasma samples were alkalinized with 0.1 ml 1 N NaOH, then extracted in 1 6 x 1 00 mm glass test tubes by vortex mixing for 20 seconds with 5 ml of t oluene- hexane ( 1 :4) . After centrifu ging the tubes to break any emuls ion formed, the aqueous layer was frozen. The a ol vent layer was then decanted into gla?s test tubes and evaporated t o ?ness under air in 0 a water-bath at 45 c . Dried extracts were redi8s olved in 0.6 ml of 0.1 5 M sodium chloride containing 4% (v/v) ethanol ( ethanol-saline) , and stored at 4 ?C overnight. Extraction efficiency was ?' aa judged by the recovery of tritiated testosterone after extraction and redissolution. ( ii ) Ass Cl c 0 '#. en m 70 0 z ? 0 CXl ? c.. 60 u 50 F i g ure 2 . 1 2 82 I I 2 10 2 0 TESTOSTE RONE ( n g/m I p l asm a ) Compos i te sta nda rd cu rve (mea n+S . E . o f 1 2 consecut i ve a ssays ) for testosterone prote i n-b i nd i ng a ssa y . only s light over-estimations of te stos terone concentrations in ram plasma are likely to have occurred. Parallelism of the standard curve with a curve produced by assaying varying quantities of androgen, extracted from varying volumes of unknown plasma, was verified by assaying a plasma sample undiluted ( 1 0. 7?. 8 ng/ml) , diluted 1 : 1 in hypophysectomized ewe plasma (1 0.2 +1 .4 ng/ml) , and diluted 1 : 3 ( 7. 8+2.1 ng/ml) , ( n = 6) . - - Likewise a further plasma sample undiluted ( 2 .4.:!;0.2 ndml) and diluted 1 : 1 in hypophysectomized ewe plasma ( 2 . 5 +0 .2 ng/ml ) , ( n = 6 ) , confirmed parallelism over the useful range of the stande.rd curve . Recovery of standard testosterone added to wether plasma was quantitative ; this result was expected since the s tandards were added to wether plasma and processed in the same manner as the unkno\m samples . Distilled water "blanks" were extracted c.nd assayed by the usual procedure. Also samples of ethanol-saline were processed through the post-extraction s teps as a further set of "blanks" . Results obtained for both sets of blanks were indistinguishable frorr. the zero standard in wether plasma. This agreement of assay results indicated that the assay system detected only those androgens of testicular origin, and also that the wether plasma did not contain dete ctable levels of androgens . Assay sens itivity, defined as the lowest testosterone c oncentration s ignificantly di.fferent from zero (Midgley et al. , 1 971 ) , varied between 0.5 and 0 . 8 ng/ml. Between-assay precis ion was estimated by assaying two plasma samples in each of twelve assays. The between-ns say coefficients of variation (CV) were 1 2.7,% and 22 .5% for samples with mean testos terone concentrations of 1 0.9 n&fml and 1 .o ng/ml, respectively. Within- assay precision was estimated from the variation between duplicates 84 for unknown pla?ma samples over two ranges of ho1?one conce ntration. Within-ass? CV for fifteen samples was 8 .6% in the range 4-7 ng/ml, and 21 .6% in the range 1 -2 ng/ml. Plasma testosterone concentrations determined in t his assay were consistent with published values for testosterone levels in rarr? , which range from 0 to 28 ng/ml (Attal, 1 97o"; Johnson, Des jardins and Ewing, 1 973 ; Katongole , Naftolin and Short, 1 974; Purvis , Illius and Haynes , 1 974; Sanford, Palmer and Howland, 1 974a; Sanford et al. , 1 974]?.; Falvo et al . , 1 975 ; Gomes . and Joyce , 1 975 ; Bremner et al. , 1 976 ; Lee et al. , 1 976 ; Lincoln, 1 976!:) . ( b ) Radioirununoassay I. Plasma s a?ples from Experiment 6 were analysed for testosterone content by a radioimmunoassay based on the method of Smith and Hafs (1 973) . This assay was developed s o that testosterone measurements could be made on smaller volurees of plasma, thereby increas ing the ntwber of replicates that could be performed on any one plasma sample . Als o radioimmunoassays theoretically offered a higher degree of specificity than protein- binding assays . ( i ) Extract1.on Procedure . Triplicate 1 00 lJ 1 plasma samples were alkalinized with one drop of 0.5 N s odium hydroxide , then extracted with 2 ml of toluene-hexane (1 : 2 ) by vortex mixing for 30 seconds in 1 6 x 1 00 mm glass test tube s . After freezing the aqueous layer, the s olvent was decanted into 1 0 x 75 mm glass test tubes ar.d 0 evaporated to dryness under air in a water-bath at 45 c . The walls of the tubes were rinsed with 300 l-.1 1 dichloromethane and the extract again evaporated to dryness . Recovery of tritiated testosterone extracted by this procedure was approximately 9Q%. Aliquots of androgen-free wether plasma, containing appropriate additions of testosterone (Mann Research Laboratories , U. S .A . ) , were extracted in the same manner as the samples to provide a standard curve over the range 0 to 25 ng/ml. ( ii ) Radioimmun0(12SSY Procedure . Two hundred ?1 of testosterone antiserum (Pool 667, raised in rabbits against testos terom-3- (0-carboxymethyl )-oxime-bovine serum albumin, courtesy Dr. G.D. Niswender, Colorado State University, U. S .A. ) diluted 1 in 2, 000 in PBS-0.1 % gelatin c ontaining non-immune rabbit sert? (1 in 200 ) , was added to the bottom of each tube then incubated at room temperature for 30 minutes . Approximately 1 5 , 000 cpm of tritiated testosterone (1 , 2 , 6, 7-3H-testosterone , 84 C?mmole , The Radiochemical Centre , Amersham) in 200 )Jl of PBS-0. 1% gelatin were then added and incubated overnight at 4?C . Free steroid was precipitated by addition of 300 ?1 of dextran-coated charcoal (1% (w/v ) dextran T70, Pharmacia, Sweden, and 0.5% (w/v) charcoal, Darco G6o, Atlas Chemical Industries , U.K. , in distilled water) , incubation at 4 ?C for ten minutes , then centrifuea- tion ( 1 900 ?) for ten minutes at 4?C. One-half ml aliquots of supernatant were transferred to glass scintillation vials, ar? following addition of 8 ml of s c intillation fluid, the radioactivity of the bound testosterone was c ounted in a Packard Tri-Carb Scintillation Spectrometer (Model 3375 ) . Calculation o f testosterone concentrations in samples was performed graphical? as described for the prote in-bi??ing ass?. A composite standard curve showing the mean values from seventeen conse cutive assays i s shown i n Figure 2 .1 3 . ( iii) Validation....2f_ Test osterone Radioimmunoa.ss?Y? Character- isation of the antiserum carried out by Ismail, Niswender and Midgley ( 1 972 ) s howed that 5a-dihydrostestosterone was the only steroid likely to interfere ( 77fo cross reaction) in this radioimmunoassay. However, for reasons already mentioned with respect to the protein-binding assay, this cross-reaction with 5a-dihydrotestosterone would have only 86 9 0 70 6 5 I 60?----?------?------.------.,--------, 0? 5 1 ?0 2?5 5?0 10?0 25?0 TESTOSTERONE (ng/ml plasma) F i g ure 2 . 1 3 Compos i te sta ndard curve ( mea n?S . E . of 1 7 consecut i ve a ssays ) for testosterone ra d i o i mmunoassay I . a minimal effect on the estimat ion of testos terone levels in ram plasma. Paralle lism of the s tandard curve and a curve obtained by as saying varying quanti ties of testosterone extracted from a plasma sample was checked by assaying the sample undiluted (1 0 .0 ng/ml) , or diluted 1 :1 (7 .6 ng/ml) and diluted 1 : 3 ( 6.0 ng/ml) in wether plasma (n = 3 ) . The sens itivity o f the assay was approxin?tely 0.5 ng/ml. This was a cons iderable improvement on that of the protein-binding assay for which it was necessary to extract ten times the v olume of plasma to give a s imilar level of sens itivity. Further evidence for the validity of this radioimmunoassay was obtained from c omparis ons of hormone levels measured in this and the prote in-binding assay. Four samples had values of 0.3,t0.1 , 2 .4,;t0.2, 7.5:.0.5 and 1 o. 7;1J.8 ng/ml in the prote in-binding assay, and 0.4j_0.1 , 2 . 6.;t0.2? 5 . 9;1J.4 and 1 0. 0:!fJ.7 ne/ml in the radioimmunoassay ( n = 6) . 87 Between-assay precision was estimated by repeated assay of plasma samples . Between-assay coeffic ients of variation (CV) were 38 .7.% (mean, 1 . o ng/ml) , 21 .4% (mean, 2 . 6 ng/ml ) , 1 7. 7% (mean, 5 . 9 ng/ml) and 20.0% (mean, 1 0.0 ng/ml) for four samples assayed i n 5 , 7, 6 and 8 assays , respectively. The pooled within-assay CV determined from two estimates per assay of the same samples were 26 . 2%, 39.4%, 1 9.7% and 1 8.9%, respectively. ( c ) Radioimmunoassay II . Plasma samples from Experiment 7 were assayed for testosterone content by a separate radioimmunoassay based on the method described above , but with modifications, including s ome described by Terqui and Thimonier ( 1 974) in their progesterone assay. ( i) Extract ion Procedure . S ingle 500 111 plasma samples were extracted with 9 ml of toluene-hexane ( 1 : 2 ) in 1 6 x 1 25 mm s crew-capped glass culture tubes by shaking for ten minutes in a laborat ory shaker. 88 After f?eezing the aqueous layer, the s olve nt was decanted into 1 6 x 1 00 mm glass test tubes and evaporated to dryness ur?er air in a 0 water-bath at 40 c . The walls o f each tube were rinsed with 1 ml of dichloromethane and the extracts RBain dried under air. Recovery of tritiated testosterone added to plasma prior to extraction was above 95%. ( ii ) Radioimmunoas say Procedure . When dry, the extracts were redissolve d in 500 lJl of ethanol and three 1 00 lJ 1 aliquots transferred to separate 1 1 x 75 mm polystyrene ? test tubes . Testosterone ( Mann Research Laboratories , U. S . A. ) , at appropriate concentrations in 1 00 f.!l ethanol, was als o adde4 to polystyrene tubes to provide a triplicated series of standards, containing 0 to 5, 000 pg testosterone/tube . After evaporating the ethanol, approximately 25, 000 cpm 1 , 2 , 6 , 7-3H-testosterone (? Ci/mmole , The Radiochemical Centre , Amersham) in 200 lJl PBS-0 .1 % ge latin and testosterone antiserum (1 in 2, 000 with 1 in 200 non-immune rabbit serum, as for the previous testosterone radioimmunoassay system) also in 200 lJl PBS-0.1 % gelatin, were added t o each tube . The mixture was incubated at 40?C for 30 minutes and 0 then at 4 C for two hours . The methods of separating free from bound steroids by dextran- coated charcoal, and subsequent counting of radioactivity in 0 .5 ml aliquots of supernatant , were the same as in the previous testosterone radioimmunoassay, except that the counts bound were determined on a Beckman (Model LS-350) liquid s cintillation counter. C omputer estimation of assay results was made by the technique described earlier for the protein hormone radioimmunoassays . A compou ite standard curve showing the mean from twelve conse cutive assays is shown in Figure 2 .1 4. ( iii) Validation of Testosterone Radioimmunoassal? As the antiserum used in this assay was identical to that used in the previous 89 90 80 70 - ? ? ? I > eo = 0 60 * V> c<:l '-" Cl ? 0 CXl 50 ? t> 40 30 ?? 20""' 0 --r-?5--1-r-?0 -----2-r-? 5----5-r?0 ---1- 0 -r? 0 ------,25?0 TESTOSTERONE (ng/ml plasma) F i g ure 2 . 1 4 Compos i te sta ndard c urve ( mea n+S . E . of 1 2 consecut i ve assay s ) f o r testosterone rad i o i mmu noassay I I . 90 testosterone radioimmunoassay, the remarks concerning tho s pecific 1 ty of that assay also apply in this case . Parallelism of the standard curve and curves obtained by assaying varying quantities of plasma, was checked with two sample s , undiluted ( 7.3+0.6, 3 .5,:!!).4 ng/ml ) , diluted 1 : 1 ( 7.5.;!:1 . 7, 3 . 2:f!J.7 ng/ ml ) and diluted 1 : 3 ( 7. 9.;!:1 . 2 , 3. 7:1J .. 4 ng/ml) , respective ly ( n = 6 ) . Assay sens itivity (Burger, Leo and Rennie , 1 972 ) ranged from 0.06 t o 0.1 3 ng/ml which was a c onsideraple improvement o n the two testosterone as says used previously in this thes is . By c omparing results obta iP.ed in this assay with those from the previous assay, it W8.3 shown that the prese nt method provided reliable estimates for plasma testosterone concentration. Three plasma samples which had testosterone concentrations of 0.4:f!J.1 , 2 .6:f!J.2, 5 . 9:f!J.4 ng/ml when assayed by Radioimmunoassay I , were estimated by the present as say t o have testosterone conce ntrat ions of 0.1 9+0 .02, 3 .52 +0.38 and 7.05 +0.29 ng/ml, respective ly ( n = 1 6 ) . - - - Assay precision was evaluated by assaying two samples twice in each of nine assays . Between-assay coefficients of variation (CV ) were 33 .4% and 1 2 .4%, and within-assay CV were 5.8'/o and 1 6 .6%, for samples with mean testosterore concentrations of 0.1 9 and 7.05 ng/ ml, respectively. Therefore it was considered that this testosterone assay was a decided improvement on the two earlier ones, both in terms of sens itivity and precision ( lower CV) , and also because of the greatly increased number of samples which could be assayed each day (65 !!!_ 25 and 30 for the protein-binding assay and first ra4ioimmunoassay , respective ly) . ( 5 ) Cortisol Assaz Plasma cortisol concentrations were determined by Dr. D. C . Thurley at Wallaceville Animal Research Centre , Upper Hutt, using the c ompetitive protein-binding assay of Bassett and H inks ( 1 969) . This assqy was used without modification as outlined below. 9 1 Two hundred ?1 aliquots of ram plasma were deproteinized by precipitation with 400 ?1 ethanol, followed by centrifugation. Duplicate 200 ? 1 aliquots of supernatant were dried and incubated with 500 ?1 of a solution containing cortis ol-binding globulin (CBG) and tritiated corticosterone (1a , 2l-3H-corticosterone ) for 1 5 minutes in a water-bath at 45?C , then cooled in iced water for 20 minutes . Duplicate 200 ? 1 aliquots of s tandard c ortis ol s olutions in ethanol were dried and incubated in the same manner, to prov ide a sta?ndard curve covering the range 0 - 1 0 ng. Steroid-free dog plasma ( approximate ly 0.6% v/v in phosphate buffer) was used as a source of CBG. Separation of protein-bound and free labelled s teroid was achieved by transferring the incubation mixture to a small column of dextran gel (Sephadex G25 , Pharmacia, Sweden) and collecting the initial 1 .5 ml of radioactive eluate into scintillation fluid. C oncentrations of c ortisol in plasma samples were obtained by c omparing the protein-bound radioactiv ity (expressed as a percentage of the protein-bound radioactivity in the 0 ng cortisol star?ard) with the standard curve . According to the authors , the only compour4 likely to interfere in this assay, corticosterone, would contribute little to the values for c ortisol concentration obtained from sheep plasma. 9. EXPERIME.Nl'AL DESIGN AND ANALYSIS Details of the experimental des igns and of the ' analyses of variance used for each experiment are given with the description of the statis tical analyses in the appropriate chapters . ( 1 ) Analyses of Va,ti_?? 92 The analysis of variance applied t o each of the variables measured repeatedly in Experiments 3 , 4 and 5, c ould be represented by the following model : I yi j kl = ? + ai + S j + y jk + ( a B ) i j + ( a y ) i jk + s i j kl Where each individual observation (hormone level ) or mean of several observations (semen character) , made on each animal in each time period or seas on, was assumed to be the sum of a number of parame ters ? - the general populat ion mean, and the deviations due to the following fixed effects ; a . - the ' main effe ct ' of the ith breed or treatment group ( i = 3 ,4) , 1. B . - the 'main effect ' of the j th period or season (j = 1 , 3 , 3 ) , J y'j k - the ' nested effect ' of the k th sampling time within the j th period or sea3on (k = 2, ? ? ? , 7) , (a S) . . - the effe ct of the interact ion of the i th breed or treatment 1. ] group and the j th period or season, I (ayh j k - the effect of the interaction of the i th breed or treatment group and the k th sampling time , within the j th period or seas on, s i j kl - a random error from a distribution with zero ;;:can and homogeneous variance . Likewise , the analys is of variance applied t o each of the variables measured repeatedly in Experiment 6 could be represented by the following model : I Y i j k lm = ? + a i + S j + Y k + 0 k 1 + + ( a Sy ) i j k + ( a o? ikl + ( a S ) . . + ( ay ) ' k + ( By ) ' k 1.] 1. J I I ( S o ) j kl + ( a S o ) i j kl + s i jklm Where each individual observation (hormone leve l) or mean of several observat ions (semen character) , made on each animal in each t ime period, was assumed t o be the sum of a number of parameters ? - the general population mean, and the deviations due t o the following fixed effects ; ai - the ' main effect ' of the i th lighting regime ( i = 2 ) , B . - the ' main effect ' of the j th operation ( j = 2 ) , J Yk - the ' main effect ' of the kth period (k = 2 , 3 ) , 6?1 - the ' nested effect ' of the 1 th sampling time within the kth period ( 1 = 3 ,4, 6 , 7) , 93 (aB1_ j - the e ffect of the interaction of t he i th lighting regime and the j t h operation, (ay1._k - the effect of the interaction of the i th lighting regime and the k th period, ( Sy? k - the effect of the interaction of the jth operation and the k th period, (a Sy) . . k - the e ffect of the interaction of the i th lighting regime 1 J and the j th operation within the kth period, (ao' )ik1 - the effect of the interact ion of the i th lighting regime and th6 1 th sampling time , within the kth period, I (B o j k1 - the effect of the interact ion of the j th operation and the 1 th sampling t ime , within the k th period, (a s o')i j k1 - the e ffect of the interact ion of the i th lighting regime and t he j th operation with t he ' nested effect' of t he l th sampling time , within the k th period, ? ? . k1 - a random error from a distribution with zero mean and 1 J m homogeneous variance . The variance was estimated as the mean square due t o variation between rams within breeds or treatment groups , by samplir? times . A further error variance, betwee n repe ated observations or individual rams within each sampling time , which could be estimated for semen characterswas not included in the analyses of variance . All main effects, the nested effects and the interactions were examined by test ing the s ignificance of contrasts for individual degrees of freedom, based on orthogonal coefficients (C ochran and 94 Cox, 1 960) which were take n from the tables of Fisher and Yates ( 1 963 ) or cons tructed to test specific hypothese s . Individual coefficient:J were then we ighted for differences in : the numbers of rams within breed."! or treatment groups , and subdivis ions within periods or seas ons . C ontrasts were constructed a priori and the number of c omparisons for any e ffect did not, by reason of the ir construction, exceed the number of degrees of freedcm for the correspondiP? mean s quare . Where the c ontrasts were successive terms of a polynomial relationship, the mean s quares for individual degrees of freedom were not calculated beyond the third power. Conseque ntly some of the tables showing summaries of analyses of variance c ontain discrepancies between the number of degrees of freedom available and the number of s ingle degrees of freedom c ontrasts c omputed. In these tables " non s ignificant contrasts" refer only t o the contrasts which were computed. Contrast numbers in the text refer o?ly to contrasts which are summarized in the tables relevant to each particular results section. Levels of s ignificance in all analyses are de noted thus ? ?? ??? p < 0.05 P < 0.01 P< O.C>01 ? (2 ) Missing Dat! Values for miss ing data were calculated, where neces sary by the method described by Cochran and C ox (1 960) . ( 3 ) Transformations Prior to the ir submiss ion as data for statistical analyses, all hormone estimates , except those in Experiments 7.1 , 7.2 and 7.3, were transformed to logarithms using the formula : log hormone c once ntration = 1 00 log1 0 ( x + 1 . 1 ) where x = plasma hormone concentration in ng/ml. This transformation was established on empirical grounds by the finding of a linear relat ionn hip between the e s t imated mean and its standard error for subgroups of hormone data. (4) Computat i ons All calculations of miss ing data, trans formations of hormone data, and analyses of variance were carried out on an IBY. 1 620 computer. 95 The programs used in these analyses included s ome written by the author and others forming part of a statistical analysis package implemented at Massey Univers ity in 1 963 by Dr. F.R .M. C ockrem, and variously modified and exte nded s ince that date by Prof. R.E. :Munford. CHAPI'ER III SE.ASONALITY OF SEMEN PRODtJCTION AND PLASMA HORJ.!ONE LEVELS IN N8W ?ALAND ROMI'EY, MERINO, AND POLLED DORSET RAMS Kr PASTtB.E 1 ? INl'RODUC'l'ION The work described in this chapter was carried out to measure the extent of the seasonal changes in reproductive characteristics of rams under local farming conditions . The N. Z . Romney breed was 96 chosen as this is the predominant breed in New Zealand and, like most breeds of British origin, has a dis tinct breeding seas on. For purposes of comparis on the Merino and Polled Dorset breeds were chos en since these generally are accepted as having extended breeding seasons (Belschner, 1 972 ) . Hafez (1 952 ) reported that Merino and Dorset sheep are capable of breeding all year round in some locations . As pointed out in Chapter I there have been few studies on the seasonal changes of semen characteristics of rams , and even fewer on concomitant changes in plasma levels of reproductive hormones . There have been no reports of any attempt to compare the seasonal changes in semen characteristics and hormone levels, in different breeds of sheep. 2 ? MATERIALS AND METHODS ( 1 ) ExEerimental procedure Animals studied in Experiment 3 were s ix N. Z . Romnoy, five Merino and four Polled Dorset rams, which were maintained under local farming c onditions. Semen collection and appraisal, and blood collection were carried out from February 1 972 until June 1 973 , as described in C hapter II . Blood plasma samples from every fourth week were assayed for concentrations of Uf, testosterone and prolactin. In June 1 973 the ralll8 were slaughtered and subjected to the autopsy procedures described in Chapter II . ( 2 ) Statist ical An??yses ( a) Semen data. The time-course of the experiment was 97 divided into ten equal periods , each period representing one-e ighth of a year. Within-period data for each semen parameter were pooled to provide a s ingle estimate for every ram. The periods were grouped into three seasons , demarcated by the equinoxes as shown in Figure 3 .1 , and the orthogonal coeffic ien:ts used to partition within- and between - seasons effects in the analyses of variance are shown in Table 3 .1 . (b ) LH and Test osterone Data. Seventeen four-weekly LH and testosterone estimates from each ram were grouped into three seasons as shown in Figure 3 .1 , the points of division being the solstices . Orthogonal coefficients used to partition the effects of seasons in the analyses of variance are shown in Table 3 . 2 . ( c ) Prolactin Data. Sixteen monthly prolactin estimates were grouped into seasons in a similar ma??er to the seme n data, with the points of divis ion again being the equinoxes as shown in Figure 3 . 1 . Orthogonal c oe ffic ients used t o partition the effects of seas ons in the analysis of variance are shown in Table 3 .3 . (d ) Rationale Fox- Division of Study Period Into Sea.'3 ons . To perform meaningful c ontrasts with semen da?ta, it was necessary to divide the time-course of the experiment at the equinoxes so that the period in which e jaculates were obtained ? artificial vagina was confined to one season ( Season 1 ) . On the other hand, plasma levels of LH and testosterone were expected to show elevations during the breeding season in the autumn, hence it was c onsidered that divis ion of the study period at the s olstices would provide the most useful grouping of periods for performing contrasts in the case of those ?'H?TER SUMMER T.VINTER SOLSTICE SOLSTICE SOL$TICE EQUINOX EQUINOX EQUINOX 14 . 4 0 PHOTOPERIOD ( h ) 9 . 2 0 SEMEN 1 SEASON 1 2 3 4 PROLACTIN 1 2 3 4 5 6 SEASON 2 SEASON 3 5 6 7 8 9 10 7 8 9 1 0 11 12 1 3 1 4 1 5 16 SEASON 1 LH and TEST ? OSTERONE SEASON 2 SEASON 3 MONTHS 1 2 3 4 5 6 7 8 9 10 1 1 1 2 1 3 1 4 1 5 1 () 1 7 M A M J J A S 0 N D J F M A M J 19 72 1 9 7 3 98 F i g u re 3 . 1 : D i agram to show how the t i me-course of Exper i ments 3 a n d 4 was s ubd i v i ded i nto samp l i ng per i ods a n d seasons for data ana l yses . !t > V\ V\ ,.. m - -< .DI g ? ? V\ =:( -< Tabl e 3 . 1 Orthogonal c oe ffi c i ents* used in partitioning seasonal effects for s emen data from Experiments 3 and 4 . Season 1 Season 2 Season 3 Time Per iods - 1 2 3 4 5 6 7 8 9 1 0 Contrast Seas on 1 - Linear -3 -1 +1 +3 0 0 0 0 0 0 " " - Quadratic +1 -1 -1 +1 0 0 0 0 0 0 " " - Cub ic -1 +3 -3 +1 0 0 0 0 0 0 Seas on 2 - Linear 0 0 0 0 -3 -1 +1 +3 0 0 " 11 - Quadratic 0 0 0 0 +1 -1 -1 +1 0 0 " 11 Cubic 0 0 0 0 -1 +3 -3 +1 0 0 Seas on 3 - Linear 0 0 0 0 0 0 0 0 -1 +1 Season 1 vs Seasons 2 & 3 +3 +3 +3 +3 -2 -2 -2 -2 -2 -2 Seas on 2 vs Season 3 0 0 0 0 +1 +1 +1 +1 -2 -2 *Coeff i c i ents were weighted for unequal group s i z e s prior to the ir use in the analys es of variance . \0 \0 Table 3 . 2 * Orthogonal c oeff i c ients used in partitioning seas onal effe cts for plasma LH and testosterone data from Exper iments 3 and 4 . Season 1 Season 2 Seas on 3 Time Periods - 1 2 3 4 5 6 7 8 9 1 0 1 1 1 2 1 3 1 4 1 5 1 6 Contrast Season 1 - Linear -3 -1 +1 +3 0 0 0 0 0 0 0 0 0 0 0 0 " " - Quadratic +1 -1 - 1 + 1 0 0 0 0 0 0 0 0 0 0 0 0 " 11 - Cubic -1 +3 -3 +1 0 0 0 0 0 0 0 0 0 0 0 0 Season 2 - Linear 0 0 0 0 -5 -3 -1 +1 +3 +5 0 0 0 0 0 0 11 11 - Quadrati c 0 0 0 0 +5 -1 -4 -4 -1 +5 0 0 0 0 0 0 11 11 Cubic 0 0 0 0 -5 +7 +4 -4 -7 +5 0 0 0 0 0 0 Season 3 - Linear 0 0 0 0 0 0 0 0 0 0 -3 -2 -1 0 +1 +2 " 11 - Quadratic 0 0 0 0 0 0 0 0 0 0 +5 0 -3 -4 -3 0 11 11 Cubic 0 0 0 0 0 0 0 0 0 0 -1 +1 +1 0 -1 -1 Seas on 1 vs Seas ons 2 & 3 1 3 1 3 1 3 1 3 -4 -4 -4 -4 -4 -4 -4 -4 -4 -4 -4 -4 Season 3 vs Season 2 0 0 0 0 -7 -7 -7 -7 -7 -7 +6 +6 +6 +6 +6 +6 * Coeff i c i ents were we ighted for unequal group s izes pr ior to their use in the analyses of variance . 1 7 0 0 0 0 0 0 +3 +5 +1 -4 +6 ? 0 0 Table 3 . 3 * Orthogonal c oeffi c ients used in partitioning seasonal effe cts for plasma pro lactin data from Experiments 3 and 4 . Seas on 1 Seas on 2 Season 3 Time Per iods - 1 2 3 4 5 6 7 8 9 1 0 1 1 1 2 1 3 1 4 1 5 1 6 Contrast Season 1 - Linear -5 -3 -1 +1 +3 +5 0 0 0 0 0 0 0 0 0 0 11 11 - Quadrati c +5 -1 -4 -4 -1 +5 0 0 0 0 0 0 0 0 0 0 11 11 Cubic -5 +7 +4 -4 -7 +5 0 0 0 0 0 0 0 0 0 0 Season 2 - Linear 0 0 0 0 0 0 -5 -3 -1 +1 +3 +5 0 0 0 0 " " - Quadratic 0 0 0 0 0 0 +5 -1 -4 -4 -1 +5 0 0 0 0 " " Cubic 0 0 0 0 0 0 -5 +7 +4 -4 -7 +5 0 0 0 0 Season 3 - Linear 0 0 0 0 0 0 0 0 0 0 0 0 -3 -1 +1 +3 11 11 - Quadrati c 0 0 0 0 0 0 0 0 0 0 0 0 +1 -1 -1 +1 " Cubic 0 0 0 0 0 0 0 0 0 0 0 0 -1 +3 -3 +1 Season 1 vs Season 2 +1 . +1 +1 +1 +1 +1 -1 -1 -1 -1 -1 -1 0 0 0 0 Season 3 vs Seas ons 1 & 2 -1 -1 .... 1 -1 -1 -1 -1 -1 -1 -1 -1 -1 +3 +3 +3 +3 *Coeffic i ents were weighted for unequal group s izes prior to the ir use in the analyses of variance . ? 0 ? hormones . The direct relationship between daily photoperiod and plasma prolactin levels reported by Pelletier ( 1 973 ) , provided the basis for division of the study pt?riod at the e quinoxes , s o that seasons of short daily photoperiods could be compared with seaso ns of long daily photoperiods . ( e ) Breed Contr?. Comparis ons between the breeds were made using the orthogonal coefficients shown below, we ighted for disproportionate group numbers . 1 02 Contrast N. Z . Romney Merino Polled Dorse t N. Z . Romrey ? Merino & Polled Dorset +2 -1 -1 Merino E Polled Dorset 0 +1 -1 3 . RESULTS ( 1 ) Semen Data See Tables 3 .4 to 3 .1 0 and Figures 3 . 2 and 3 .3 . The only semen characteristics for which a regular seas onal pattern of change was re corded were e jaculate volume and the three parameters related to seminal fructose levels, all of which reached peak values during the autumn months . Apart from these , motility indices , percentages of motile spermatoz oa and percentages of morphological? normal spermatozoa, declined during March and April, 1 972 , and showed little further change . On the other hand, numbers of spermatozoa per e jaculate declined during the first two months of the study but increased slightly early in the following summer (November, 1 972 ) (Contrasts 1 - 9, Tables 3 . 9 and 3 .1 0 ) . Abrupt changes in mean values for e jaculate volume , concentrat ion of spermatozoa per ml and total e jaculate fructose content occurred between Season 1 and Season 2 , and corresponded with the change in collection method from predominantly artificial vagina to entire? electrical stimulation ( Contrast 8, Tables 3 . 9 and 3.1 0) . Effects of Table 3 . 4 Mean moti l ity indi c e s ( scale 0-4 ) and mean perc entages of mot i l e spermatozoa rec orded from s emen c o l l e c ted :i:n Exper iment 3 . Mot i l i ty index % Mot i l e spermatozoa N . Z . Po l led N . Z . Po l l ed Period Romney Merino Dorset Mean Per iod Romney Merino Dor s et Mean 1 3 . 3 3 . 5 3 . 6 3 . 5 1 6 1 . 3 69 . 9 75 . 0 68 . 7 Season 1 2 2 . 6 3 . 2 2 . 9 2 . 9 Season 1 2 49 . 8 68 . 5 5 5 . 6 58 . 0 3 2 . 2 2 . 8 3 . 0 2 . 7 3 37 . 5 5 3 . 0 57 . 5 49 . 3 4 2 . 2 2 . 7 2 . 2 2 . 4 4 3 9 . 9 49 . 0 46 . 7 4 5 . 2 Mean 2 . 6 3 . 0 2 . 9 2 . 9 Mean 47 . 1 60 . 1 5 8 . 7 5 5 . 3 5 2 . 4 2 . 4 2 . 5 2 . 4 5 4 4 . 9 47 . 7 5 1 . 7 48 . 1 Season 2 6 2 . 2 2 . 3 2 . 4 2 . 3 Season 2 6 40 . 1 47 . 1 4 5 . 4 44 . 2 7 2 . 1 2 . 4 2 . 3 2 . 3 7 43 . 7 54 . 0 48 . 8 48 . 8 8 2 . 2 2 . 7 2 . 2 2 . 4 8 4 5 . 6 53 . 7 40 . 6 46 . 6 Mean 2 . 2 2 . 4 2 . 4 2 . 4 Mean 4 5 . 6 5 0 . 6 46 . 6 46 . 9 Season 3 9 2 . 6 2 . 4 2 . 3 2 . 4 Season 3 9 5 1 . 6 47 . 8 48 . 3 49 . 2 1 0 2 . 0 2 . 3 2 . 5 2 . 3 1 0 47 . 4 42 . 9 5 7 . 8 49 . 4 Mean 2 . 3 2 . 4 2 . 4 2 . 4 Mean 49 . 5 4 5 . 4 5 3 . 1 49 . 3 Overa l l Mean 2 .4 2 . 7 2 . 6 Overal l Mean 46 . 2 5 3 . 4 52 . 7 _, 0 \..>1 Table 3? Mean e jaculate vo lumes and mean total fructose c ontent of e jaculates c o l l ected in Experiment 3 . Ejaculate vo lume (ml ) Total e jaculate fructose c ontent ( mg ) N . z . Po l l ed N . Z . Po l l ed Per iod Romney Mer ino Dorset Mean Per iod Romney Mer ino Dorset Mean 1 0 . 98 0 . 96 0 . 76 0 . 90 1 5 . 53 5 . 6 5 2 . 76 4 ? 65 Season 1 2 0 . 72 0 . 79 0 . 62 0 . 71 Season 1 2 3 . 1 8 2 . 92 2 . 44 2 . 85 3 0 . 54 0 . 73 0 . 6 5 0 . 64 3 1 . 86 2 . 1 9 2 . 68 2 . 24 4 0 . 56 0 ,. 6 1 0 . 47 0 . 5 5 4 1 . 27 1 . 32 2 . 00 1 . 53 Mean 0 .70 0 . 77 0 . 62 0 . 70 Mean 2 ? 96 3 . 02 2 . 47 2 . 8 1 5 1 . 1 0 1 . 20 1 . 00 1 ? 1 0 5 3 . 3 3 3 . 70 1 . 79 2 . 94 Season 2 6 1 . 1 2 0 . 92 1 . 52 1 . 1 9 Season 2 6 4 . 26 3 . 66 4 . 86 4 . 26 7 1 . 1 8 0 . 7 5 1 . 3 1 1 . 08 7 3 . 05 3 . 1 6 1 . 56 2 . 59 8 1 . 5 5 1 . 96 1 ? 5 5 1 . 69 8 1 1 ? 57 20 . 04 3 . 87 1 1 . 83 Mean 1 . 23 1 . 20 1 . 34 1 . 26 Mean 5 . 5 5 7 . 64 3 . 02 5 . 40 Season 3 9 1 . 56 1 . 37 1 . 40 1 . 44 Season 3 9 9 . 65 1 0 . 83 4 . 46 8 . 3 1 1 0 1 . 28 1 . 01 1 ? 1 7 1 . 1 5 1 0 7 . 3 5 4 . 42 3 . 1 1 4 . 96 Mean 1 . 42 1 . 1 9 1 . 28 1 . 29 Mean 8 . 50 7 . 62 3 . 78 6 . 63 Overal l Mean 1 . 06 1 . 03 1 . 04 Overal l Mean .hll 5 . 79 2 . 9 5 ? 0 +=- Table 3 . 6 Mean c oncentrations of fructose in s emen and in s eminal plasma of e jaculates c o l le cted in Experiment 3 . Semina l fructose c oncentration ( mg/ml ) Seminal plasma fructose c oncentrati on ( mg/ml ) . N . z . Po l led N . Z . Po l led Per iod ?Romney Merino Dorset Mean Period Romney Merino Dors et Mean 1 5 . 76 6 . 01 3 . 63 5 . 1 3 1 7 . 26 7 . 85 4 . 92 6 . 68 Season 1 2 4 . 3 1 3 . 98 3 . 49 3 . 93 Season 1 2 5 . 26 5 . 26 4 . 90 5 . 1 4 3 3 . 47 2 . 47 4 . 28 3 . 41 3 4 . 04 3 . 29 4 . 69 4 .0 1 4 2 . 57 2 . 69 3 . 52 2 . 93 4 2 . 99 3 . 64 4 . 66 3 . 76 Mean 4 . 02 3 . 78 3 . 73 3 . 85 Mean 4 . 88 5 . 0 1 4 . 79 4 . 89 5 2 . 68 2 . 65 1 ? 93 2 . 42 5 3 . 00 3 . 23 2 . 09 2 . 77 Seas on 2 6 2 . 90 3 . 57 3 . 1 5 3 . 2 1 Season 2 6 3 . 62 4 . 03 3 . 79 3 . 8 1 7 2 . 26 4 . 44 1 . 46 2 . 72 7 3 . 24 5 . 54 1 . 8 5 3 . 54 8 7 . 24 8 . 69 2 . 64 6 . 1 9 8 8 . 36 1 0 . 04 3 . 04 7 . 1 5 Mean 3 . 77 4 . 83 2 . 29 3 . 6 3 Mean 4 . 5 5 5 . 7 1 2 . 69 4 . 3 1 Season 3 9 6 . 1 2 6 . 6 1 3 . 38 5 . 37 Season 3 9 7 . 1 7 8 . 1 8 3 . 89 6 . 41 1 0 5 . 3 1 3 . 3 6 2 . 5 5 3 .74 1 0 6 . 37 4 . 1 8 3 . 2 1 4 . 59 Mean 5 . 71 4 . 98 2 . 96 4 . 5 5 Mean 6 . 77 6 . 1 8 3 . 5 5 5 . 50 Overal l Mean 4 . 26 4 . 45 3 . 00 Overa l l Mean 5 . 1 3 5 . 52 3 . 70 ? 0 IJl Tabl e 3 . 7 Mean c oncentrations of spermatozoa/ml and mean numbers of spermatozoa/e jaculate in semen c o l lected in Experiment 3 . 9 Spermatozoa/ml ( x1 0 ) Spermatozoa/e jaculate ( x 1 09 ) N . Z . Po l l ed N. Z . Po l led Per iod Romney Mer ino Dors et Mean Per iod Romney Merino Dorset Mean 1 3 . 3 5 3 . 9 1 4 . 0 1 3 . 76 1 3 . 09 3 . 87 3 . 1 9 3 . 38 2 3 . 08 4 . 22 4 . 2 5 3 . 85 Season 1 2 2 . 25 3 . 44 2 . 72 2 .80 Season 1 3 2 . 5 1 3 . 00 3 . 02 2 . 84 3 1 . 30 1 . 84 2 . 5 8 1 . 9 1 4 2 . 5 3 4 . 60 3 . 96 3 . 70 4 1 . 50 2 . 82 2 . 05 2 . 1 2 Mean 2 . 86 3 . 93 3 . 8 1 3 . 53 Mean 2 . 03 2 . 99 2 . 63 2 . 5 5 5 1 . 40 2 . 1 2 1 . 39 1 . 64 5 1 . 84 3 . 03 1 . 64 2 . 1 7 Seas on 2 6 1 . 67 1 . 94 2 . 73 2 . 1 1 Season 2 6 2 . 79 2 . 5 5 4 . 68 3 . 34 7 0 . 86 1 . 1 7 2 . 1 8 1 . 40 7 1 . 27 1 . 28 4 . 26 2 . 27 8 2 . 1 2 2 . 1 0 1 . 66 1 . 96 8 3 . 36 4 . 9 5 3 . 00 3 . 77 Mean .L2J_ 1 . 83 1 . 99 1 . 77 Mean 2 . 3 1 2 . 9 5 3 . 39 2 . 88 Seas on 3 9 2 . 03 2 .7 1 1 . 54 2 . 09 Seas on 3 9 4 . 1 5 4 . 06 2 . 66 3 . 62 1 0 1 . 74 . 1 ? 73 2 . 89 2 . 1 2 1 0 2 . 60 2 . 67 3 . 44 2 . 90 Mean 1 . 88 2 . 22 2 . 2 1 2 . 1 0 Mean 3 . 37 3 . 36 3 . 05 3 . 26 Overa l l Mean 2 . 1 3 2 . 75 2 . 76 Overal l Mean 2 . 42 3 . 05 3 . 02 ? 0 0'\ Table 3 . 8 Mean percentages of unstained and morphological ly normal spermatozoa in s emen c o l le cted in Experiment 3 . % Unstained spermatozoa % Morpho logica l ly normal spermatozoa N . Z . Po l l ed N . Z . Po l l ed Period Romney Merino Dors et . Mean Period Romney Merino Dorset Mean 1 76 . 4 8 1 . 9 85 . 3 8 '1 . 2 1 88 . 0 87 . 8 89 . 4 88 . 4 Season 1 2 62 . 7 77 . 4 77 . 1 72 . 4 Season 1 2 72 . 0 8 1 . 2 85 . 6 79 . 6 3 72 . 4 82 . 5 77 . 3 77 . 4 3 66 . 3 80 . 4 79 . 8 75 . 5 4 70 . 3 8 1 . 3 7 1 . 2 74 . 3 4 67 . 2 8 5 . 6 83 . 5 78 . 8 Mean 70 . 4 80 . 8 77 .7 76 . 3 Mean 73 . 4 83 . 8 84 . 6 80 . 6 5 8 1 . 9 77 . 6 79 . 8 79 . 8 5 70 . 6 75 . 8 84 . 3 76 . 9 Seas on 2 6 73 . 5 72 . 9 78 . 0 74 . 8 Season 2 6 60 . 1 67 . 2 64 . 2 63 . 8 7 88 . 3 77 .7 77 . 9 8 1 . 3 7 76 . 7 6 1 . 0 64 . 1 67 . 3 8 75 . 2 75 . 9 74 . 7 75 . 3 8 53 . 9 64 . 9 57 . 8 5 8 . 9 Mean 79 . 7 76 .0 77 . 6 77 . 8 Mean 6 5 . 3 6 7 . 2 67 . 6 66 . 7 Season 3 9 75 . 7 63 . 4 76 . 5 7 1 . 9 Seas on 3 9 6 8 . 7 57 . 8 67 . 7 64 . 7 1 0 68 . 9 '59 . 1 7 5 . 7 67 . 9 1 0 62 . 0 49 . 0 68 . 9 60 . 0 Mean 72 . 3 6 1 . 2 76 . 1 69 . 9 Mean 6 5 . 4 53 . 4 68 . 3 62 . 4 Overal l Mean 74 . 5 7 5 . 0 77 . 4 Overal l Mean 6 8 . 6 7 1 . 1 74 . 5 ? 0 "">J .Tabl e 3 . 9 Experiment 3 : Summary of Analys es o f Variance for Semen Data . Sourc e of Variation :t-1AIN EFFECTS A . SEASONS Seas on n n 11 " Linear Quadrati c Cubi c Seas on 2 - Linear 11 11 - Quadratic 11 11 - Cubic Seas on 3 - Linear Seas on 1 vs Seas ons 2 & 3 Season 2 vs Season J B . BREEDS N . Z . Romney vs Merino & Po l led Dorset Merino vs Po l l ed Dorset INTERACTION (AxB ) Seas on 1 - Cubic x Contrast 1 0 (Seas ons 1 ? 2 & 3 ) x Contrast 1 1 Seas on 2 - Quadratic x Contrast 1 1 Seas on 3 - Linear x Contrast 1 1 Non s ignificant c ontrasts Res idual Mean Square Contrast No . D . F . 1 2 3 4 5 6 7 8 9 1 0 1 1 1 2 1 3 1 4 1 5 9 1 1 1 1 1 1 1 1 1 2 18 1 1 4 1 20 Vo lume 6 . 78* 0 . 2 5 0 . 1 0 1 4 . 80*** 7 . 50** 4 . 1 8* 4 . 09* 98 . 68*** 0 . 1 5 0 . 1 9 0 . 04 0 . 73 8 . 60** 1 3 . 26?*** 0 . 07 0 . 5 5 0 . 1 4 Moti l ity 2 5 .42*** 0 . 64 0 .4 5 0 . 1 0 0 . 96 0 . 03 0 . 79 34 . 4 5*** 0 . 40 5 . 78* 0 . 05 5 . 1 8* 24 . 45*** 0 . 2 5 0 . 82 0 . 62 0 . 34 Variance Ratios % Moti l e Sperm . /ml Sperm . /ejac . 2 2 . 29*** 0 .74 0 . 01 0 . 03 0 . 1 9 1 . 1 0 0 . 05 1 1 . 92** 0 . 37 7 . 93** o . oo 4 . 08* 2 1 . 1 8*** 0 . 1 2 0 . 68 0 . 6 5 205 . 22 1 ? 01 2 . 23 6 . 49* 0 . 07 0 . 02 3 . 06 0 . 02 89 . 3 1 *** 1 . 60 1 1 . 1 8** 0 . 01 0 . 1 1 1 1 . 02** 4 . 50* 4 . 65* 0 . 34 .l.!1l 4 .73* 0 . 7 1 0 . 46 3 . 37 0 . 2 1 4 . 50* 0 . 88 2 . 27 0 . 66 3 . 70 o . oo 0 . 1 3 3 . 26 1 2 . 79*** 1 ? 2 1 0 . 38 3 . 37 (Key , Semen Parameters : Vo lume = ejaculate vo lume ; Mot i l ity = motil ity index ; % Motile = percentage ? of mot i l e spermatozoa ; Sperm . /ml = c oncentrati on of spermatozoa/ml ; Sperm . /ejac . = number of spermatozoa/ejaculate . ) ? 0 ():) Table 3 . 1 0 Experiment 3 : Summary of Analyses o f Variance for Semen Data . S ourc e of Variation MAIN EFFECTS A . SEASONS Season 1 - Linear 11 11 - Quadratic 11 1 1 - Cubi c Seas on 2 - Linear 1 1 1 1 - Quadrati c 11 11 - Cubi c Season 3 - Linear Seas on 1 vs Seasons 2 & 3 Seas on 2 vs Seas on 3 B ?. BREEDS N . Z . Romney ? Merino & Pol led Dorset Merino vs Po l l ed Dorset INTE??CTION (AxB ) Seas on 1 - Cubi c x Contrast 1 0 ( Seas ons 1 ? 2 & 3 ) x Contrast 1 0 Season 1 - Linear x Contrast 1 1 Season 2 - Linear x Contrast 1 1 ( Seas ons 1 vs 2 & 3 ) x Contra?t 1 1 ( Seas ons 2 vs 3 ) x Contrast 1 1 Non s ignif icant c ontrasts Res idual Mean Square Contrast No . 1 2 3 4 5 6 7 8 9 1 0 1 1 1 2 1 3 1 4 1 5 1 6 1 7 D . F . 9 2 1 8 1 1 1 2 1 20 Fr . Cone . 9 . 82** 0 . 47 0 . 1 1 2 1 . 3 5*** 7 . 89** 4 . 89* 5 . 1 8* o . oo 5 . 47* 2 . 5 6 1 1 . 82*** 0 . 69 2 . 90 4 . 52* 8 . 53** 7 . 40** 0 . 08 1 . 24 3 . 74 Fr . Cont . 6 . 50* 0 . 3 8 0 . 2 1 40 . 93*** 22 . 58*** 1 1 ? 1 1 ** 6 . 80* 29 . 1 1 *** 3 . 0 1 1 . 80 1 5 . 77*** 0 . 1 5 2 . 24 1 . 4 8 20 . 07*** 6 . 59* 0 . 1 4 1 ? 98 ? Variance Rat ios S . P . Fr . % Unstained Cone . 1 4 . 3 3*** 1 . 1 0 0 . 0 1 22 . 70*** 6 . 08* 3 . 8 5 4 . 87* 0 . 78 7 . 0 1 ** 1 . 59 1 3 . 1 8*** o . 50 5 . 09* 4 . 89* 8 . 3 0** 8 . 24** o . 01 1 ? 3 1 4 . 89 2 . 02 1 . 90 1 . 62 0 . 33 o . oo 3 . 1 6 0 . 80 0 . 24 1 0 . 25 ** 1 . 07 0 . 9 1 4 . 08* 77 . 82*** 2 . 46 0 . 3 1 4 . 92* 4 . 66* 0 . 30 1 1 2 . 48 % Normal 4 . 82* 5 . 1 1 * 1 ? 26 8 . 72** 1 ? 38 1 . 53 0 . 37 37 . 26*** 1 . 22 2 . 49 3 . 5 9 1 . 5 1 23 . 69*** o . oo 1 . 03 0 . 02 3 . 00 0 . 72 227 . 1 4 ( Key, Semen Parameters : Fr . Cone . = fructose c onc entration of semen ; Fr . Cont . = total e ja culate fructose c ontent ; S . P . Fr . Cone . = fructo s e c oncentrat ion of s eminal plasma ; % Unstained = perc entage of uns tained spermatozoa ; % Normal = perc entage of morpho logi c a l ly normal spermatozoa ) ..... 0 \.0 5 0 ...... 5 OIQ )( _, UJ f?<( -1 :::) u <( ..., UJ ....... 0 <( 0 N 0 f-<( 5 ? a: UJ a.. (/) I?I /I ?-I- -Y . I--r I?~ N Z ROMNEY PO L L ED DO RSET M A M J J A S 0 N D J F M A M J MO NTHS F i g u re 3 . 2 : Seasona l var i at i ons i n n umbers of s permatozoa/ej ac? u l ate ( mean+S . E . ) i n seme n co l l ected f rom N . Z . Romney , Mer i no a n d Po l l ed Dorset rams , between March 1 972 and J u ne 1 97 3 . 1 1 0 E ........ Cl E UJ U) 0 1-u ::::l 0: u. <{ ? U) <{ _J a... 1 0 5 0 10 N Z R OM N EY I? . I---J"I ME R I N O I /I"'J _J 5 "1? I yi \ <{ z ? UJ U) 0 5 I :I- 0 M A M I-? - J J PO LLED DORS ET I? /I? /A--I I ri A s 0 N D J F M A M J MONTHS F i gure 3 . 3 : Seasona l va r i at i ons i n sem i na l p l asma f ructose con? centrat i on s ( mea n?S . E . ) i n semen co l l ected f rom N .Z . Romney , Mer i no a n d Po l l ed Dorset rams , between Ma rch 1 972 a nd J u ne 1 9 73 . 1 1 1 1 1 2 seasonal changes on semen characteristics during Seas ons 1 and 2 would have been confounded by the change in c olle ction method, however it was likely that seas onal e ffects accounted for the higher mean valuen r e c or? d e d in Season 1 for : motility index, percenta? of motile spermatoz oa and percentage of morphologically normal s permatozoa. In the late autumn months of 1 973 (April, May and June ) , percentages of unstained spermatozoa were lower than in the summer of 1 972-1 973 (Contrast 9, Table 3 .1 0 ) . Semen from N. Z . Romney rams had lower overall mean values for motility index, percenta? of motile spermatoz oa and concentration of spermatozoa per ml than did that from Merino and Polled Dorset rams ( Contrast 1 0, Table 3 . 9 ) . This difference between breeds was especially evident in Seas on 1 , as indicated by the interaction of Seas ons x Breeds , for these parameters (Contrast 1 3 , Table 3 . 9 ) . Similar s ignificant interactions were recorded for e jaculate volumes, and percentages of unstained and morphologically normal spermatoz oa (Contrast 1 3, Tables 3 . 9 and 3 . 1 0 ) . All three measures of seminal fructose levels were higher in semen from Merino rams than in that from Polled Dorsets ( Contrast 1 1 , Table 3 .1 0 ) . This result, i n conjunction with the s ignificant interaction contrasts (Contrasts 1 3, 1 4 and 1 5, Table 3 . 1 0 ) , refle cted the virtual absence of an autumn peak in these para.maters for the semen of Polled Dorsets . During the mid-summer months (December 1 972 , January 1 973 ) the Polled Dorset rams produced semen containing e levated s permatozoal numbers in comparison to that from Merinos (Contrast 1 4, Table 3 . 9 ) . At the same time Merino rams had lower e jaculate volumes than the Polled Dorsets (Contrast 1 4, Table 3 . 9 ) . Of the three breeds , Merinos s howed the greatest decline in percentages of unstained spermatoz oa during the course of the experiment (Contrasts 1 3 and 1 7, 1 1 3 Table 3 .1 0 ) . ( 2 ) Plas?? Hormone Data ( a) L. H. See Tables 3 . 1 1 , 3. 1 2 and Figure. 3 .4. Although changes in plasma LH leve ls were less marked than for testosterone, there was a seas onal pattern with n?imal levels be ing rec orded during the summer months (November to March ) , and lower leve ls during t he midwinter months (April to August ) . Peak mean leve ls \Vera 0.88i9.31 ng LH per ml plasma while midwinter levels ofte n were below the limit of sensitivity of the LH assay ( 0 .04-0.1 1 ng/ml ) . With the orthogonal coefficients used for making contrasts in the Seas ons x Breeds interaction, no s ignificant differe nces were detected. However, the Merinos did appear to have much lower peak plasma LH levels than the N. Z . Romney or Polled Dorset rams . (b) Testos terone . See Tables 3 .1 1 , 3 .1 2 and Figure 3 .5 . All three breeds exhibited a very marked peak of plasma testosterone leve ls during January, February and March, 1 973 , while minimum levels occurred from May t o November in both y8ars . Declining plasma testosterone leve ls from March 1 972 indicated the probable prese nce of a peak in the previous January-February period, s imilar to that recorded in 1 973 . This decline in plasma test osterone leve ls during the autumn of 1 972 appeared to be more pronounced in Merino and Polled Dorset rams than in the N. Z . Romneys . Conseque ntly, in Seas on 1 , mean plasma tes t os terone leve ls were higher in the N. Z . Romneys than in the other two breeds (Seas ons x Breeds interaction contrasts 1 1 and 1 4) . ( c ) Prolactin. See Tables 3 .1 3 , 3 .1 4 and Figure 3 .6 . Plasma prolact in levels showed a well-defined seas onal pattern with ele vated levels during the summer months ( November to April ) and depressed levels during winter (May to September) (C ontrasts 1 -5, 7 ,8 1 0, 1 1 ) . Maximum levels were recorded i n November 1 972 and minimum Tabl e 3 !.ll Mean plasma LH and testosterone c onc entrati ons re c or?ed from rams in Experiment 3 . ( Va lues pre s ented are 1 00 log1 0 ( x + 1 . 1 ) , where x i s hormone c oncentrati on in ng/ml o ) Lute iniz ing Hormone Testosterone N . Z . Po l led N . Z . Po l led Per i od Romney Mer ino Dors et Mean Per i od Romney Merino Dorset Mean 1 . 1 4 . 7 2 1 . 8 1 5 . 3 1 7 . 3 1 77 . 4 68 . 0 5 9 .6 68 . 3 S eason 1 2 1 2 . 6 5 . 0 5 . 2 7 . 6 S eason 1 2 79 . 1 3 5 . 6 24 . 2 46 . 3 3 1 0 . 1 1 4 . 4 6 . 5 1 0 . 3 3 3 0 . 8 2 1 . 3 22 . 8 2 5 . 0 4 7 . 5 4 . 1 4 . 1 5 . 1 4 5 3 . 0 1 5 . 4 3 1 ? 9 3 3 . 4 Mean .l.l.d .!.l...!1. 7 . 8 1 0 . 1 Mean ? 60 . 1 3 5 . 1 34 . 6 3 3 . 4 5 1 3 . 9 7 . 0 1 9 . 1 1 3 . 3 5 45 . 9 22 . 8 56 . 1 4 1 . 6 6 1 5 . 1 1 1 . 6 5 . 0 1 0 . 6 6 46 . 8 1 4 . 9 45 . 4 3 5 . 7 Season 2 7 1 1 ? 5 1 2 . 1 1 0 . 1 1 1 . 2 S eason 2 7 5 4 . 0 49 . 5 3 3 . 1 45 . 5 8 1 3 . 4 1 9 . 8 2 1 . 0 1 8 . 1 8 42 . 6 54 . 5 45 . 9 47 .7 9 23 . 3 1 6 . 0 2 1 . 9 20 . 4 9 44 . 9 57 . 5 70 . 9 57 . 8 1 0 9 . 9 8 . 1 1 5 . 8 1 1 . 3 1 0 37 . 6 43 . 8 58 . 5 46 . 6 Mean 1 4 . 5 1 2 . 4 ? 1 4 . 1 Mean 45 . 3 40 . 5 5 1 . 6 45 . 8 1 1 29 . 0 1 6 . 4 30 . 1 2 5 . 2 1 1 6 5 . 9 1 03 . 6 95 . 8 88 . 4 1 2 24 . 6 1 0 . 6 1 7 . 7 1 7 . 6 1 2 93 . 0 9 0 . 3 1 1 4 . 3 99 . 2 1 3 20 . 0 9 . 3 1 9 . 4 1 6 . 2 1 3 82 . 3 8 5 . 7 87 . 4 8 5 . 1 Season 3 1 4 1 7 . 8 1 1 . 4 1 6 . 5 1 5 . 2 Seas on 3 1 4 77 . 2 87 . 8 1 0 1 . 2 88 .7 1 5 6 . 4 6 . 3 5 . 3 6 . 0 1 5 3 8 . 2 1 3 . 9 3 5 .7 29 o 3 1 6 7 . 1 4 . 1 4 . 1 5 . 1 1 6 30 . 5 1 3 . 8 1 8 . 0 2 0 . 8 1 7 4 . 9 4 . 6 5 . 4 5 . o 1 7 1 8 . 2 20 . 7 8 . 6 1 5 . 8 Mean 1 5 . 7 9 . 0 1 4 . 1 1 2 . 9 Mean 5 7 . 9 59 . 4 6 5 . 9 6 1 . o Overal l Mean 1 4 . 2 1 o . 7 1 3 . 1 Overa l l Mean 5 4 . 0 47 . 0 53 . 5 -- ? ? ? Table 3 . 1 2 :Experiment 3 Summary of Analys es of Variance for LH and Te stosterone Data . Source of Variation MAIN EFFECTS A . SEASONS Season 1 - Linear " " - Quadratic Season 2 - Cubic Season 3 - Linear " " - Quadratic " " - Cubic Season 1 vs Seas ons 2 & 3 Season 3 vs Seas on 2 Non s ignificant c ontrasts B. BREEDS N . Z . Romney vs Merino & Pol led Dors et Merino vs PoTfed Dorset INTERACTION (AxB ) Seas on 1 - Cubic x Contrast 9 Season 2 - Linear x Contrast 9 Seas on 3 - Linear x Contrast 9 ( Seasons 1 ? 2 & 3 ) x Contrast 9 Non s ignificant c ontrasts Res idual Mean Square ?contrast No . 1 2 3 4 5 6 7 8 9 1 0 1 1 1 2 1 3 1 4 1 6 2 32 D . F . 1 1 1 1 3 1 1 1 1 1 8 204 Variance Ratios LH Testosterone 7 . 22** 1 8 . 77*** 0 . 70 5 . 52* 8 . 41 ** 2 . 5 5 42 . 56*** 1 56 . 36*** 0 . 80 8 . 80** 0 . 1 5 1 5 . 00*** 4 . 52* 8 . 3 5** 0 . 66 1 7 . 7 5*** 1 ? 59 1 . 62 2 . 98 1 . 36 1 . 03 0 . 08 2 . 78 9 . 83** 0 . 39 4 . 3 1 * 1 . 62 6 . 5 0* 0 . 07 1 4 . 05** 0 . 85 0 . 73 1 1 2 . 34 6 1 7 . 48 _. _. \Jl 1 ?0 0 ?5 0 -ro 1 ?o E (/) CO c. - E 0?5 ' Cl c I ...J 0 N Z ROM NEY M E R I N O PO LLED DORSET M A M J J A S 0 N D J F M A M J MONTHS F i g u re 3 . 4 : Sea sona l va r i at i ons i n p l a sma LH concentrat i ons ( mea n+S . E . ) recorded f rom N .Z . Romney , Mer i no a nd Po l l ed Dorset rams , between Ma rch 1 972 a n d J une 1 9 73 . 1 1 6 CO E (/) CO 10 5 0 ? 1 0 E -..... Ol c w 5 z 0 a: w I- (/) 0 0 I-(/) w I- 15 Z ROMN EY \yr--r-I--I'-1-r-/ \_1'-I 1?I-l . MER INO \?i POL LE D DO RSET M A M J J A S 0 N D J F M A M J MONTHS F i g u re 3 . 5 : Seasona l va r i at i on s i n p l a sma testosterone concentra t ? i ons ( mea n?S . E . ) recorded f rom N . Z . Romney , Mer i no a n d Po l l e d Dorset rams , between Ma rch 1 972 a n d J une 1 9 73 . 1 1 7 Tabl e 3 . 1 3 Mean plasma pro lactin c onc entrations rec orded from rams in Experiment 3 . (Values pre s ented are 1 00 log1 0 ( x + 1 . 1 ) , where x is hormone c onc entration in ng/ml . ) Period N . Z . Romney Merino Po l led Dors et Mean 1 1 5 . 9 22 1 ? 1 82 . 6 1 0 1 ? 2 2 1 5 5 . 8 1 5 4 . 2 1 54 . 2 1 73 . 7 Season 1 3 42 . 1 1 1 1 ? 6 3 0 . 9 6 1 . 5 4 97 . 3 1 30 . 6 38 . 3 88 . 7 5 1 03 . o 1 3 1 . 2 94 . 6 1 09 . 6 6 92 . 3 1 71 . 9 1 28 . 9 1 3 1 . o Mean 1 01 . 1 1 63 . 4 88 . 2 9 5 . 2 7 1 4 1 ? 4 1 76 . 3 1 38 . 3 1 52 . 0 8 1 43 . 1 1 66 . 6 1 68 . 4 1 5 9 . 4 S eason 2 9 2 1 3 . 3 2 1 2 . 9 2 1 9 . 8 2 1 5 . 2 1 0 1 73 . 2 1 82 . 6 1 99 . 8 1 8 5 . 2 1 1 1 8 5 . 0 1 98 . 8 2 1 2 . 3 1 98 . 7 1 2 1 34 . 7 1 93 . 6 1 80 . 6 1 69 . 6 Mean 1 6 5 . 1 1 88 . 5 1 86 . 5 1 80 . 0 1 3 1 64 . 0 220 . 5 203 . 2 1 9 5 . 9 Season 3 1 4 1 23 . 1 1 70 . 1 1 47 . 4 1 46 . 9 1 5 5 9 . 7 1 57 . 5 63 . 9 93 . 7 1 6 89 . 0 1 5 3 . 4 66 . 7 1 03 . 0 Mean 1 08 . 9 1 75 .4 1 20 . 3 1 34 . 9 Overal l Mean 1 27 . 1 1 75 . 8 1 3 3 . 1 -" -" CO Experiment 3 S ourc e of Variation MAIN EFFECTS A . SEASONS Seas on 1 - Linear " " - Quadrati c 11 11 - Cubic Season 2 - Linear " " - Quadrati c " " - Cubic Season 3 - Linear " 1 1 - Quadrati c " " - Cubic Seas on 1 vs Seas on 2 Seas on 3 vs Seasons 1 & 2 B . BREEDS Tabl e 3 . 1 4 Summary of Analys i s of Varianc e for Pro lactin Data . Contrast No . 2 3 4 5 6 7 8 9 1 0 1 1 D . F . 15 2 1 1 1 l 1 1 1 N . Z . Romney ? Mer ino & Pol led Dors et 1 2 1 3 1 Merino vs Po l led Dorset INTERACTION (AxB ) Seas on 1 - Cubic ? x Contrast 1 2 " " - Linear x Contrast 1 3 ? Season 3 - Linear x Contrast 1 3 ( Season 1 vs Season 2 ) x Contrast 1 3 Non s ignif icant c ontrasts Res idual Mean Square 1 4 1 5 1 6 1 7 1 30 1 1 1 1 1 8 1 92 Var iance Rat i o 5 . 90* 56 . 87*** 5 . 00* 4 . 08* 1 3 . 5 1 *** 0 . 3 1 5 0 . 49*** 7 . 8 1 ** 2 . 03 1 07 . 36*** 5 . 3 5* 27 . 03*** 40 . 79*** 2 1 . 97*** 5 . 75* 5 . 34* 22 . 43*** 0 . 99 1 590 . 23 ? ? \?) 1 20 150 NZ R O M N E Y 100 50 0 ?I eo 150 E (/) ro - Q. - 1oo E ....... I"I-I Ol c ? 50 1-u <( ..J 0 a: 0 a.. 150 M A M J J A S 0 N D J F M A M J MONTHS F i g u re 3 . 6 : Seasona l va r i at i ons i n p l asma p ro l act i n concentrat i ons ( mean+S . E . ) recorded f rom N . Z . Romney , t-.1e r i no a n d Po l l ed Dorset rams , between Ma rch 1 972 a n d J une 1 9 73 . 1 2 1 levels in May 1 972 ( 1 51 . 11-_?1 6 . 6 and 6 ? .3_?1 . 6 ng/ml, respectively ) . Generally, the lowest prolactin levels were recorded from the N. Z . Romneys , and the highest from the Merinos , with the Polled Dorsets intermediate ( overall means 41 .o, 8.3 .1 , and 51 .o ng/ml, respectively ) . In March and April, 1 972, both the N. Z . Romney and Polled Dorset groups had low levels of plasma prolactin in comparison with the Merinos (Contrasts 1 4, 1 5 and 1 7) . Further, in May ?1 973 , the Polled Dorsets displayed a sudden fall in plasma prolactin to near baseline levels , while the Merinos exhibited a more moderate fall (Contrast 1 6 ) . (Note : the N. Z . Romneys displayed a decrease in plasma prolactin levels s imilar to that recorded from the Polled Dorsets , but this decline was not tested separately by the orthoeot?l c ontrasts made in the analysis of variance ) . ( .3 ) Autopsy Data See Tables .3 . 1 5, 3 . 1 6 and .3 .1 7. These data were obtained in June 1 97.3 , after the breeding s eason, when plasma LH and testosterone were at minimum levels . The only s ignificant difference between the breed groups was the recording of lower weights of ampullae from the Merinos than from rams of the other two breeds . (4) Meteorological Data See Figure .3 .7. Both daily photoperiod and temperature displayed similar s inusoidal patterns throughout the year. Total rainfall also showed a seasonal pattern, but in contrast to that for phot?period and temperature , had maximum values during the winter months and minimum values during the summer months . Changes in mean relative humidity ( not shown) were less marked and c orresponded with the changes in total rainfall. Table 3 . 1 5 Data (means* + S .E . ) c o l l e cted f o l l owing autopsy of rams uti l ized in Exper iment 3 . Body Testi cular S eminiferous Epididymal Epididymal Ampul lar Seminal we ights weights tubule we ights spermatozoal we ights ves i cular d iameters res erves we i ghts (Kg ) ( g ) ( p ) ( g ) ( x 1 09 ) ( g ) ( g ) N . Z . Romney 71 . 4_?3 . 4 1 88 . 9_?1 4 . 0 1 58 . 4.? 5 . 5 42 . 6_?2 . 5 36 . 75_?1 3 . 3 5 3 . 73_?0 . 30 7 . 82_?0 . 78 Merino 66 . 4+4 . 6 2 5 2 . 7_?33 . 3 1 60 . 4.? 8 . 8 44 . 7_?3 . 6 23 . 67.? 8 . 37 2 . 47_?0 . 1 1 6 . 39_?0 . 62 Po l l ed Dorset 64 . 5_?5 . 0 264 . 6_?60 . 7 1 63 . 6_?1 4 . 6 50 . 9_?8 . 3 49 . 00_?1 4 . 07 3 . 72+0 . 39 7 . 7 1_?1 . 28 Seminal ves i cular fructose Thyro id Pituitary Pineal Hydroxyindole-0-methyl Pineal c e l l Total c ontent Concentration weights we ights weights transferase activity nuc l ear dens ities ( mg ) (mg/g ) ( g ) ( mg ) ( mg ) (DPM/mg ( DPMjpineal ) (No . /std . grid ) pineal ) N . Z . Romney 36 . 2.? 7 . 5 443 . 2.? 5 5 . 6 5 . 52.? 0 . 53 702 . 2_:!:56 . 3 69 . 1_? 8 . 4 1 2 5 . 0+1 5 . 4 8276+1 076 38 8 . 3+22 . 6 Merino 1 7 . 3.? 8 . 3 245 . 2.? 9 5 . 7 6 . 25.? 0 . 63 837 . 8+58 . 2 8 5 . 5_?1 0 . 1 1 32 . 9_:!:25 . 3 1 0701,:!:1 326 3 5 1 . 7,:!:1 6 . 5 Po l led Dors e t 40 . O.:t 1 5 ? 1 46 1 ? . 9?1 20 . 2 6 . 04,:!: 1 .71 838 . 6?57 . 8 98 . 4,:!:2 5 .7 1 22 . 2?41 . 2 9892,:!:2 543 380 . 2+ 9 . 6 * Where data have been obtained from paired organs , means for each group were based on totals per ram . ..... !\) !\) Tabl e 3 . 1 6 Variance ratios for c ontrasts in the analyses of variance of data pres ented in Tabl e 3 . 1 5 . (D . F . = 1 , 1 2 ) Note : Var iance rat i os re lated to pineal gland data are pre sented separate ly in Tabl e 3 . 1 7 . Contrast Contrast 2 Error Mean Square Contrast 1 Contrast 2 Error Mean Square ? Body we ights 1 . 28 0 . 1 1 89 . 42 Ampul lar weights 3 . 62 8 . 57* 0 . 40 T-e st icular weights 2 . 88 0 . 05 6024 . 83 Semina l ves i cular we ights 0 . 57 1 . 00 3 . 75 Seminiferous tubul e diameters 0 . 1 1 0 . 05 4 1 7 . 58 Semina l ves icular fructose Total c ontent Conc entrat ion 0 . 42 0 . 77 2 . 37 2 . 79 483 . 42 37440 . 83 (Footnote : Contrast 1 Contrast 2 N . Z . Romney vs Merino and Po l led Dors et ; Merino vs Polled Dorset ) . Epididymal we ights 0 . 89 0 . 79 ' 1 05 . 83 Thyroid we ights 0 . 32 0 . 02 4 . 28 Epididymal spermatozoal reserves o . oo 1 . 88 760 .42 Pituitary we ights 3 . 9 1 o . oo 1 69 1 8 . 3 3 ...io 1\) \.!ool Tabl e 3 . 1 7 Variance ratios for c ontrasts in the analyses of variance of pineal gland data pre s ented in Tabl e 3 . 1 5 . ( D . F . = 1 , 1 2 ) Contrast 1 Contrast 2 Error Mean Square ( Fo otnote : Contrast 1 Contrast 2 Pineal we ights 1 . 8 5 0 . 37 1 006 . 67 Hydroxyindole-0-methyl transferase act ivity ( DPM/mg pinea l ) (DPM/pineal ) 0 . 01 1 ? 1 9 0 . 08 0 . 1 2 3357 . 48 1 229029 5 . 1 7 N . Z . Romney vs Merino and Po l led Dorset Merino vs Polled Dors et . ) Pineal c e l l nuc lear dens ities 0 . 9 8 0 . 99 1 8 1 9 . 42 ? 1'\) .::- 14.4 0 0 Q ffi 12.00 0.. 0 b :X: 0.. 9. 2 0 ...... ? 20 ? 0::: ::J ? 0::: 1 0 ? 0.. ::E w .... 0 1 50 ...... ? ? ? 100 ? z ? 0::: ? ? ? 50 0 M A MEAN MAX MEAN MIN M J J A 0 N D J F M A M J 1972 1913 MONTHS F i g u re 3 . 7 : Month l y var i at i on s i n da i l y p hotoper i od ( ca l cu l ated f rom N .Z . Na ut i ca l A l manac , 1 972 ) , a n d temperature a n d tota l ra i n ? ta l I ( recorded a t Massey Un i vers i ty ) , d u r i n g the t i me-course of Expe r i ments 3 a n d 4 . 1 25 4. DISCUSSION ( 1 ) Seas onal Changes in Semen Prod ction Seasonal changes in ram s emen production have been reported 1 26 by a number of workers ( see reviews by Emmens and Robins on, 1 962 ; and Lodge and Salisbury, 1 970) . Although the results have tended to be equivocal, generally higher quality semen has been rec orded during autumn and early winter. Variations between previous reports mostly can be attributed to factors s uch as the particular semen attribute under s tudy, the breed of sheep, and the location of the experiment. In the present experiment a well-defined seas onal pattern was recorded for e jaculate volume and seminal fructose data, these being the s emen parameters which are influenced most by access or,y sex gland function (Mann, 1 964) . This seasonal pattern cons isted of a peak in values for these characteristics during autumn and low levels during spring, which supports earlier reports from studies on rams in California (Cupps et al. , 1 960) and Israel (Amir and Volcani, 1 965 ) . All other semen characteristics studied related to spermatozoal numbers , their activity, or their morphology, and largely were indicative of testicular and epididymal function. These other characteristics did not show aqy evidence of a seas onal pattern, except for numbers of spermatoz oa per e jaculate , which? tended to be elevated during the autumn months . In c ontract to these results, Smyth and Gordon ( 1 967) recorded s ignificant increases during the autumn months in percentages of live spermatozoa, spermatozoa per ml and spermatozoa per e jaculate, for Galway, Suffolk and Wicklow rams , in Ireland. These authors considered that the marked changes in spermatozoal production were related to seasonal changes in le ngth of 1" .?Dnrrq:s t d?light. ? workers in Australia reported that seasonal changes in 1 2 7 semen quality were related to excessive heat and undernutrition, rather than other seas onal factors (Gunn, Sanders and Granger, 1 942 ) . Subsequently other authors have attributed increased frequencies of spermatozoal abnormalities , or impaired spermatogenes is , to high summer temperatures (Hafez, Badreldin and Danvish, 1 955 ; Hiroe et al. , 1 960) . Als o, Moule , Braden and Mattner ( 1 966 ) suggested that a seasonal increase in .the quantity and quality of forage available to grazing Merino rams accounted for an autumnal peak of seminal fructose c oncentrations recorded from these rams . In Experiment 3 no marked seasonal changes in semen characteristics dependent on testicular or epididymal activity were recorded, indicating that ne ither high summer temperatures nor inadequacies in feed supply, had s ignificant effects on production of spermatozoa. On the other hand, the autumnal peak of accessory gland activity, as determined from e jaculate volume and seminal fructose levels, might have bee n related to increased pasture production during the autumn, but more likely resulted from changes in daily photoperiod. T ogether, the present and previous findings, indicate that several environmental factors can influence reproduction in rams , and that the relative importance of each factor can be evaluated only in experiments in which environmental factors can be varied independently, by use of controlled-climate rooms . Hafez, Badreldin and Darwish ( 1 955 ) reviewed a number of studies and concluded that the degree of seas onality in semen characteristics depended on the latitude of the place of st?, a higher degree of seasonality being recorded at higher latitudes. It is thus necessary to consider differences in location when comparing results reported in the literature . Much of the relevant work done in Australia, California and Israel was performed at lower latitudes than the present s tudies , located near Palmerston North ( lat . ?0?21 ' S ) , whereas in the United Kingdom and Europe such work has been carried out at higher latitudes . Amir and Voloani ( 1 965 ) indicated that even for semen characteristics showing a degree of seasonality, the extent of variatioM depended on the breed of rams . For example , no seas onal fluctuations in seminal plasma volume , and in seminal fructose and 1 2 8 citric acid concentrations were recorded from German Mutton Merino, C orriedale and Dorset Horn rams, and contrasted with the marked? seas onal patterns displ?ed by Awass i and Border Leicester rams . In Experiment 3 the N. Z . Romneys had lower values for motility indices and spermatoz oal concentrations than the other two breeds . This may have been related to the deficiency in spermatogenesis shown by rams of a related breed (Romney Marsh) compared with Merino, C orriedale and S outhdown rams , in Brazil (Mies Filho and Ramos , 1 956 ) . The Polled Dorset rams did not display any seas onality i n seminal fructose levels , which may have been related t o their extended breeding seas on, yet the Merinos which have a s imilar breeding pattern had a we ll-defined autumn peak for this semen parameter. A similar pattern of seminal fructose changes in Merino rams was als o reported by Moule , Braden and Mattner ( 1 966 ) . In comparis on t o the Merinos , the Polled Dorsets in the present stuqy showed less distinct fluctuations in e jaculate volume and spermatozoal numbers , which suggested also that the Merinos had a greater tendency for seas onal changes i n semen production than Polled Dorsets . Fertility per se was never tested in this experiment but indices published by Edgar (1 959 ) and Hulet and Ercanbrack ( 1 962 ) have provided s ome guidelines for evaluating semen quality with respect to potential fertility. Spermatozoal concentrations in semen from rams in this experiment were satisfactory or good ( over 1 x 1 09 spermatozo?ml ) according to these guidelines . The rather low values for percentage of motile ( less than 5Q%) and percentage of abnormal spermatoz oa ( les s than 70-80?) probably reflected differences in subjective assessment of these parameters , between this and other studies . 1 29 Subfertility was never expected to occur at any time during the c ourse of Experiment 3. However, changes in potential fertilizing capacity of sperm may have been detectable by use of more stringent in vitro tests of s permatozoal viability. Such tests include spermatozoal motili? after incubation at boqy temperature ( Ludwick, Olds and Carpenter, 1 948; Buckner, Willett and Bayley, 1 965 ) , and spermatozoal viability after freezing ( C olas et al. , 1 972 ) . Abrupt changes in values for e jaculate volume and spermatozoal concentration coincided with the change in semen collection method to e lectro-e jaculat ion, and supported previous evidence that e jaculates collected qy this technique have a greater volume and lower s permatozoal c oncentration than those collected by artificial vagina (Braqy and Gildow, 1 939; Ortavant, Laplaud and Thibault, 1 948; Mattner and Voglmayr, 1 962 ; Salamon and Morrant, 1 963 ; Rathcre , 1 970 ) . An increase i n total e jaculate fructose content als o occurred simultaneously with the change in semen collection method, which supported an earlier observation that ele ctro-e jaculation stimulated the accessor.y glands more than did service into an artificial vagina, and thereby raised seminal fructose c ontent and concentration (Mattner and Voglmeyr, 1 962 ) . In the present experiment no similar change in the concentration of fructose in semen and seminal plasma occurred indicating that the change in total e jaculate fructose content could be accounted for by the increased e jaculate volume . Because the change in semen collection method was confounded with seasonal changes, it was not possible to attribute the concomitant fall in indices of motility and percentages of motile and morphologically normal spermatozoa, t o collection method alone, if at all. Other workers have reported higher indices of motility or percentages of motile spermatozoa in semen c ollected by artificial vagina than in that obtained by electro-e jaculation (Brady am 1 30 Gildow, 1 9.39; Mattner and Voglmayr, 1 962 ; Salamon and Morrant, 1 963 ) , however it has bee n pointed out that the changes in spermatozoal concentration influence the assessment of spermatozoal motility (Mattner and Vog?r, 1 962 ) . Therefore it is likely that the major effect of electrical stimulation is the product ion of an increased volume of accessory gland secre tion compared with that found in artificial vagina e jaculates . ( 2 ) Seasonal C hanges in Plasma Hormone Levels (a ) LH and Testosterone. E levated levels of plasma LH in rams during the autumn months have been reported by a small number of workers (Pelletier, 1 971 ; John.'ion, Des jardirl3 and Ewing, 1 97.3 ) . In Experiment .3 peak LH levels were recorded in December (near midsummer) which c orresponded with a recent French report of a peak during June (Hochereau-de Reviers, Loir and Pelletie?, 1 976 ) . other workers have failed to demonstrate seasonal changes in plasma LH levels in rams (Katongole, Naftolin and Short, 1 974; Sanford, Palmer and How land, 1 9742) . Autumnal peaks in plasma testosterone concentrations in rams have bee n reported by Attal ( 1 970 ) , Johnson, Des jardins and Ewing ( 1 973 ) , Katongole , Naftolin and Short ( 1 974 ) , and Sanford, Palmer and Howland (1 97?) . ? However, in Experiment 3 pronounced seasonal changes in testosterone levels were diapl?ed by all three breeds , with peak concentrations occurring during January, February and March; this result was more in agreement with the pattern described by Gomes and Joyce (1 975 ) , who recorded peak plas? testosterone levels from S outhdown, Shropshire and Targhee rams during the midsummer months. 1 31 Although a seas onal pattern of LH secretion was rec orded in the present study, the period of elevated plasma levels lasted from September t o April. This period encompassed the period of elevated testosterom concentratiom , but the peak of LH secretion occurred approximate? one month earlier than the highest testosterone peak. Moreover, the period of elevated plasma tes.tosterone concentrations ( January, February and March) in Experiment 3 preceded, by approximate? one month, the corresponding August-November testosterone peaks described in the literature .(Attal, 1 970 ; Katongole , Naftolin and Short, 1 974; Sanford, Palmer and Howland, 1 974?) . August? November in the Northern Hemisphere corresponds to February-May in the S outhern Hemisphere , yet in the present stu? plasma testosterone levels had returned to baseline concentrations by May, in agreement with the corresponding results of Gomes and Joyce ( 1 975 ) in Ohio, u. s .A. There was less evidence for a seas onal peak of plasma LH levels in the case of Merino rams . Such differences bet'.veen breeds in the present study could account for the disparities betwee n results in previous papers . However, these differences undoubtedly have been c ontributed t o by differences in localities , and by the small numbers of rams (2-5 per breed) s tudied in each paper. The as sociation in timing of peak levels of secretion of LH and testosterone supported the general view that LH stimulates the production of androgens by the teste s . This topic and the roles of feedback control and seasonal effects will be discUssed later ( See 4, General Discuss ion) . (b) Prolactin. Elevated plasma prolactin levels during the summer months were re corded from rams in Experiment 3 and s uggested a seas onal pattern s imilar to that described for goats ( Buttle , 1 974) and bulls ( Schams and Reinhardt, 1 974) . In each case the animals 1 32 studied were subjected to seasonal changes in both daily photoperiod and temperature , s o the re lative importance of these environ?ental factors c ould not be determined. Pelletier ( 1 973 ) sho?;ved that there was a direct relat ionship 'between the le ngth of the daily photoperiod and plasma prolactin levels in rams and wethers subjected to artificial lighting regimes with c ontrasting seas onal cycles . It thus seems clear that daylight is the major factor c ontrolling plasma prolactin levels in rams . Als o, Hart ( 1 975 ) showed that the provision of additional lighting to goats during the autumn months , prevented the seasonal declir? in prolactin response to milking. In view of the influence of the stress of venepuncture in causine elevated prolactin levels in goats (Hart, 1 973 ) and c ows (Johke, 1 969; Raud et al? , 1 971 ) , i t is c onceivable that the plasma levels obtained in this study were elevated above normal. However the present results , and those of Pelletier ( 1 973 ) , Buttle ( 1 974) and Schams and Reinhardt ( 1 974 ) , were all based on plasma samples obtained by venepuncture . Seas onal or lighting e ffects on plasma prolactin levels were therefore of s t?ficient magnitude to be detectable even though sampling procedures may have e levated prolactin levels . Als o, very low prolactin levels were measured in plasma samples c ollected during winter months suggesting that the e levations caused by venepuncture stress were of minor importance, at leest at that time of the year. Furthermore , when taking s ingle blood samples at weekly, or less-frequent intervals , it c ould be assumed that the influence of stress during sampling was constant for all groups throughout each experiment. Nevertheles s it would have been more desirable t o sample blood remotely by meana of indwelling venous cannulae , t o eliminate the possibility that aqy differences betwee n groups of rams merely represented difference s in susceptibility to stress . Although Merino rams had higher mean plasma prolactin levels, 1 33 they displayed s im ilar seasonal pa t t erns o f prolactin secretion t o the N . Z . Romney and Polled Dorset rams , so the s i gnificant be ? tween-breeds contrast s may have been of doubt f?l importanc e . ( 3 ) Autopsy Data The negative results as r egards any c onsistent patt ern o f breed differences i n the autopsy data was i n ac cord w i t h the lack of ma j or dif ferences in plasma hormone levels or semen character - i s t ics . Als o , the data were c ollected at a t ime when hormonal and s eminal parame t ers were at the ir minimum values . This result does not exclude the poss ibility that bre ed differences in these data c ould have o ccurred during the br e ed ing season . The l ower we ights of ampullae from the Mer inos were of dubious imp ortance . (4) General Dis cuss ion No d ire c t a t tempt was made in th is experiment to inves t igate the interrela t ionships between the var ious parame ters s tudied . It was poss ibl e , however , to make informal c omparisons o f the t iming of changes in s ome var iables . The relat ionships between the s easonal pat terns of plasma LH and t es tos t erone , and seminal plasma fruc t ose c oncentrations , are illustrated in Figure 3 . 8 . This graphic c orn- par ison indicated that the highes t plasma LH levels pre ceded those of testost erone by approximately one month . I n turn , t h e increase in testosterone l evels oc curred about one month earl ier than the corresponding elevations in seminal plasma fructose concentrations . The delay between peak plasma LH levels and the c onsequent peaks in plasma t es tosterone was unexpectedly long , s ince t es tost erone levels usually rose within twenty t o forty minutes of an LH surge in rams during a 24 -hour sampling period ( Katongole , Naftol in and Short , (1J E C/l (1J Q. E ........ Ol c I __j UJ -z ro o E a: C/l UJ ? r- o. (f) O E r- , (j) Ol ??-=- E ........ Ol E UJ (f) 0 r? u ? a: u.. <( ? (f) <( __j c... __j <( z ? UJ 1 .0 0.5 0 10 5 0 10 5 ? 04---?--?--?--?--?--?--?--?--,---.--.---.---.----.--? M A M J J A S 0 N D J F M A M J MO NTHS F i gu re 3 . 8 : I nterre l at i on s h i ps between seasona l va r i at i on s i n p l a sma LH a n d testosterone, a n d i n sem i na l p l a sma f ructose concentrat i on s , for N . Z . Romney ( ) , Mer i no ( - - - - - ) a n d Fb l l ed Dorset ( -------- ) rams , between Ma rch 1 972 a n d J une 1 9 73 . 1 35 1 974; Sanford et al . , 1 974l1) . . Also, after Gnllli administration to r?, plasma testosterone levels rose at the s ame time as plasma LH (Galloway et al. , 1 974 ) , or within fifty minutes Bremner et al. , 1 976 ; Lee et al. , 1 976 ) . However, the fact that hormone concentrations were determined on plasma samples collected at four? we ekly intervals in Experiment .3 , meant that precise statements about their time-relationships could not be made . Furthermore , the present study was an investigation of changes in mean hormone levels throughout the year, not testosterone responses to individual pulse s of LH secretion. So, the results presented here indicated that there was a seasonal change in the degree of testicular response to LH stimulation, as well as a seasonal change in LH output . The change in LH output itself might have reflected seas onal changes in hypothalamic or pituitary responsiveness to testosterone feedback, this responsiveness be ing influenced by other factors , s uch as daily photoperiod or pineal gland activity. Seas onal changes in testicular respoMiveness to LH have bee n described in the male goat by Racey, Rowe and Chesworth (1 975 ) , who suggested that these chan?s were the result of the long-term effects of the earlier rise in plasma LH levels . It is possible that a certain duration of stimulation of the testes by raised levels of circtuating LH is required to proliferate steroidogenic cells . Thereafter the testosterone response to LH ? become progressively greater, and testosterone production may c ontinue at elevated levels , even with reduced LH stimulation. It has been suggested that the frequency of pulses of LH se cretion m? vary through the year (Katongole, Naftolin and Short, 1 974), and could alter the degree of testicular stimulation by affecting the mean LH concentration without necessarily changing the maximal plasma content. Further experiments will have to be performed to investigate the various aspects of s easonal changes in the controlling me chanisms 1 36 for LH and testosterone release in ram3. Such experiments include the study of seasonal changes in : the LH and testosterone responses to GnRH injections ; the testosterone secretory resporu.Jes to LH injections ; and the feedback effects of testosterone injections on LH output. IncreaDed levels of fructose in the seminal plasma of castrated rama have been reported during daily (Moule , Braden and Mattner, 1 966 ) or alternate dai? (Knight, 1 973 ) treatment with testosterone injections . An e ight to eleven day del? from commencement of treatment to seminal plasma fructose response was noted by Moule , Braden and Mattner (1 966 ) , and a similar delay can be seen on inspection of Kni&ht ' s ( 1 973 ) results. Likewise the r? in Experiment 3 did not show an immediate rise in seminal fructose levels following the seasonal increase in plasma testostero}')..e levels ? Also, seminal plasma fructose levels remained elevated after plasma testosterone concentrations had declined. These results indicate that the responses of ram accessory sex glands to androgen stimulation require a certain amount of time to reach their full secretory potential, this time probably being needed to allow full development of active secretory cells . Once maximum secretory activity has been attained, high levels of fructose output may be maintained even by reduced levels of androgens. In summary, Experiment 3 showed that the seas0}')41 pattern of changes which characterize the breeding seas on in rams took the? form of an elevated output of LH, followed by a subsequent peak in secretion of andro?ns by the testes . This led, in turn, tc increased secretory_ activity of the accessory sex glands and als o (according to Ahmed, 1 955) to increased sexual activity. In field experiments of the kind described in this chapter it is difficult to ascertain which environmental factors were responsible for the seasonal patterns recorded for semen production and plasma hormone levels . Variations in daily phot operiod, temperature , rainfall or nutrition could not be controlled independent?, and hence could not be ruled out as poss ible 1 37 regulators of seas onality in sheep. However several authors , in discus s ion of similar field experiments , have implicated photoperiod as the major environmental factor influencing seasonal reproductive changes in rams ( Smyth and Gordon, 1 967; Pelletier, 1 971 ; KHtongo1e , Naftolin and Short, 1 974; Purvls , . Illius and Haynes , 1 974; Gomes and Joyce , 1 975 ) . Further support for this v iew has been obtained from studies in which environmental factors were controlled s o that the effects of photoperiod could be studied independently ( Yeates , 1 949 ; Fowler, 1 961 ; Ortavant, Maule on and Thibault, 1 964; Pelletier, 1 971 ; Jackson and Williams, 1 973 ; Pelletier and Ortavant , 1 975??) . The need t o establish the role of photoperiod in producing s ome of the results obtained in the present experiment made it obligato? to perform experiments described later in this thesis, in which rams were subjected to controlled environments . Evidence of marked seasonality, shown by the Merino and Polled Dorset rams particularly with re gard to plasma testosterone levels , was not anticipated as these rams had been expected to be less seas onal than the N.Z . Romney breed (Hafez . 1 952 ) . It s eemed, from the data obtained during this study, that rams of all three breeds probably were capable of reproduction throughout the whole year. Nevertheless , a marked seas onal pattern was exhibited by these rams with regard t o plasma hormone levels a nd accessory sex gland function, a nd this pattern might have been reflected by subtle variations in the relative levels of fertility, which were not detectable in Experiment 3 . It might therefore have been worthwhile to investigate ram fertility e ither in vivo by inseminating ewes , or by use of in vitro viability 1 38 tests on semen, in the present experiment . Merinos and Polled Dorsets lacked seasonal variatior? in plasma LH and seminal fructose levels , respectively, which indicated that important breed differences in seasonality m? have occurred. Although the link between plasma prolactin levels and reproduction in rams may be tenuous ( see Chapter I ) , the markedly seas onal pattern of prolactin secretion indicated that if prolactin was gonadotrophic in rams , it could provide a means by which changes in daily phot operiod could modif.y reproductive activity. Plasma prolactin levels appeared to be more dire ctly influenced by photo? period than those of LH and testosterone. Thus prolactin release could be influenced by photoperiodic stimuli via the pineal gland, an organ which some authors feel warrants further investigation regarding its possible involvement in reproductive rhythms in manunals (Reiter, 1 974!_) . A sharp drop occurred in 1H levels in plasma collected on 4 December 1 972 , with a subsequent and less severe fall in testosterone levels . This was contrary t o the general seas onal pattern and thus warrants some explanation. In an attempt t o e lucidate this matter, plasma samples collected both one week prior to and one week following this occasion were assqyed; the results revealed that this decline was spread over a few weeks am was not just an is olated event on one particular day. Approximately seven weeks after the decline in plasma LH levels , semen samples displayed a transient drop in quality, which together with the hormone data, suggested that a severe depression of reproductive system function had occurred in early December 1 972 . No satisfactory explanation can be put forward to account for this event, but it was not considered t o be a normal feature of the pattern of seas onal changes in reproductive character? istics which formed the major findings of this experiment . 1 39 CHAPI'ER IV SEASONALITY OF SEMEN PRODUCTION AND PLASMA HORMONE LEVELS IN RAMS WITH MODIFIED OLFACTORY AND PINEAL FUNCTION 1 ? INl'RODUCTION The work described in this chapter was a preliminary investigation of the neuroendocrine mechanisms mediating the seasonal changes in semen characteristics and hormone levels observed in the previous chapter. The two systems inves tigated were the pineal gland and the olfacto?J system. Both of these systems have been described in Chapter I as possible modifiers of reproductive and e ndocrine activity in rodents , although little research has been published on their fur.ctions in domestic animals . Pinealectomy in ewes failed t o have any effects on the incidence of oestrus or ovulation, or LH levels (Roche et al. , 1 970?) . On the other hand, C ardinali , Nagle and Roaner (1 974?) reported a s ignificant relationship between pineal gland activity and stage of the oestrous cycle in ewe s . N o other r?ports have appeared in the literature re lating pineal gland function to reproduction in sheep. Likewise the role of the olfactory system, on seas onal changes in semen quality and hormone leve ls , haa not been established for sheep. The classical approach to such a study would have bee n to observe the e ffects of surgical removal of the organs. However, difficulties were encountered in initial attempts at surgical removal of the pineal gland from rams , s o instead, the cranial cervical ganglia were removed. These ganglia are part of an afferent nervous pathway to the pineal gland in rats and hamsters , and the ir removal 1 40 abolishes the responses of the pineal gland to environmental darkness (Wurtman, Axelrod and Fis cher, 1 9?; Eichler and Moore , 1 971 ) . It was assumed that if cranial cervical ganglionectomy produced any changes in the parameters under study in this experiment, these probably could be attributed to altered pineal gland function. Similarly, the olfactory bulbs of rams were removed to investigate the poss ibility that the resultant impairment of olfactory function ? alter the normal seas onal changes in semen characteristics and blood hormone levels . 2. MATERIALS AND METHODS ( 1 ) Animals and Mana??nt Procedure Adult rams of the N.Z . Romney breed were allocated randonuy to a control group (s :!..x rams ) , or to three surgically treated groups (four rams each) . Rams in the three treated groups were : olfactory bulbeotomized (BulbX) , cranial cervical ganglionectomized (GanglionX) , or olfactory bulbectomi7.?d and cranial cervical ganglionectomized (Bulbx/GanglionX) . Th? control group ( C ontrols ) in this experiment was the group of N. Z . Romney rams studied in Experiment 3 and the two experiments described in this and the previoua chapter were run concurrently, the one group of control N. Z . Romney rams providing data for both studies . All the rams grazed together as one flock, under the management procedures described in Chapter II . The surgical techniques for olfactory bulbectomy and superior cervical ganglionectomy were described in Chapter II. The general health of the operated rams appeared to be identical to that of the Controls yet, one BulbX ram and one GanglionX ram died from unknown causes in the early s tages of the experiment. Acy data obtained from these rams were excluded from the results . There was no postmortem evide nce that these deaths resulted from the surgical procedures, or their after-effects . ( 2 ) Data C ollection Semen and blood plasma samples , as well as autopsy data were collected in Experiment 4 in the same manner as described for Experiment 3 . ( 3 ) Stat istical Analyses Effects of seasons on semen characteristics and plasma hormone levels were studied using the orthogonal coeffic ients de?cribed in Experiment 3 ( see Figure 3 .1 , and Tables 3 .1 , 3 .2 and 3 .3 ) . Comparis ons between the effects of the surgical treatments were performed using the orthogonal coefficients shown below, we ighted for disproportionate numbers of rams in each group. Contrast C ontrols ? BulbX , GanglionX, & BulbX/ GanglionX BulbX & GanglionX ? BulbX/GanglionX BulbX ? GanglionX Controls Bulb X +3 -1 0 +1 0 +1 GanglionX Bulb? GanglionX -1 -1 +1 -2 -1 0 However, in the case of autopsy data relating to the pineal gland (pineal gland weight, cell nuclear density, and hydroxyindole-o-methyl transferase activity ) the following set of orthogonal coefficients was used, again weighted for disproportionate group size : Contrast Controls GanglionX, Bulb? -1 GanglionX vs C ontrols, BulbX - GanglionX vs Bulb? 0 GanglionX - Controls !:!. BulbX +1 BulbX -1 0 -1 GanglionX Bulb? GanglionX +1 +1 +1 -1 0 0 1 42 This latter set of orthogonal coefficie nts was specifical? devised t o investigate the assumption made in the introduction, that cranial cervical ganglionecto? would alter pineal gland function in rams . 3 . RESULTS ( 1 ) Semen Data See Tables 4.1 to 4.7 and Figures 4.1 and 4.2 . (? - Olfactory bulbectomized ram3 appeared t o show normal behavioural responses , with no impairment of ability to serve the artificial vagina. ) Semen from all groups of rams tended to display s imilar changes in characteristics to those rec orded from the N. Z . Romney rams in Experiment 3. Again e jaculate volume and fructose levels displayed a seasonal increase during autumn, while motility, percentage mot ile spermatozoa and percentage morphologically normal spermatozoa declined in the first s ix months and never regained their March 1 972 leve ls . Estimates of spermatozoal numbers i n semen aisplayed erratic changes , total numbers of spermatozoa per e jaculate being particularly variable , showing a double peak during the summer months (November 1. 972 to April 1 973 ) and rising again in Jur..e 1 973 . Apart from a slight fall in May 1 972 , percentages of unstained spermatozoa showed little evidence of change throughout the experiment ( Contrasts 1 -7, Table 4.6 and Contrasts 1 -9, Table 4.7 ) . Significant results in Contrast 6 (Table 4. 6 ) for e jaculate volume, spermatozoa per ml, spermatozoa per e jaculate , and in C ontrast 8 (Table 4.7 ) for total e jaculate fructose c ontent, could be attributed to changing the semen collection method from artificial vagina t o e lectro-e jaculation. On the other hand the s ignificant results for motility indices , and percentages of motile and Tabl e 4 . 1 ?Mean mot i l ity indices ( scale 0-4 ) and mean perc entages of mot i le spermatozoa rec orded from s emen c o l l e cted in Experiment 4 . Moti l ity index % Moti l e spermatozoa BulbX/ BulbX/ Period Controls BulbX Gangl i onX Gangl ionX Mean Per i od Controls BulbX Gangl i onX Gangl i onX Mean ?1 3 . 3 3 . 2 3 . 2 3 . 4 3 . 3 1 6 1 . 3 6 5 . 3 6 3 . 1 69 . 8 64 . 9 Season 1 2 2 . 6 2 . 7 3 . 1 2 . 6 2 . 8 Season 1 2 49 . 8 5 1 ? 9 6 1 . 4 47 . 1 52 . 6 3 2 . 2 2 . 4 2 . 8 3 . 1 2 . 6 3 37 . 5 40 . 0 5 4 . 2 57 . 5 47 . 3 4 2 . 2 2 . 3 2 . 5 2 . 0 2 . 2 4 39 . 9 42 . 5 43 . 9 36 . 7 40 . 8 Mean 2 . 6 2 . 6 2 . 9 2 . 8 2 . 7 Mean 47 . 1 49 . 9 5 5 . 6 5 2 . 8 5 1 . 4 5 2 . 4 2 . 1 2 . 3 2 . 3 2 . 3 5 44 . 9 42 . 5 43 . 6 44 . 6 43 . 9 Seas on 2 6 2 . 2 . 2 . 0 2 . 1 2 . 3 2 . 2 Seas on 2 6 40 . 1 32 . 8 3 2 . 8 44 . 6 37 . 6 7 2 . 1 2 . 6 1 . 8 2 . 4 2 . 2 7 43 . 7 50 . 0 45 .8 5 0 . 6 47 . 5 8 2 . 2 1 ? 8 2 . 0 2 . 9 2 . 2 8 4 5 . 6 3 5 . 8 32 . 7 62 . 7 44 . 2 Mean 2 . 2 2 . 1 2 . 1 2 . 5 2 . 2 Mean 43 . 6 40 . 3 38 . 7 5 0 . 6 43 . 3 Seas on 3 9 2 . 6 1 . 9 2 . 7 2 . 7 2 . 5 Seas on 3 9 5 1 . 6 3 8 . 4 5 9 . 2 53 . 7 50 . 7 1 0 2 . 0 2 . 3 2 . 5 2 . 8 2 . 4 1 0 47 . 4 49 . 4 52 . 5 57 . 2 5 1 . 6 Mean 2 . 3 2 . 1 2 . 6 2 . 8 2 . 4 Mean 49 . 5 43 . 9 5 5 . 8 5 5 . 4 5 1 . 2 Overal l Mean 2 . 4 2 . 3 2 . 5 2 . 6 Overal l Mean 4 6 . 2 44 . 9 48 . 9 52 . 4 ? .::- VJ Table 4 . 2 Mean e jaculate vo lumes and mean total fructose c ontent of e jaculates c o l lected in Experiment 4 . Ejaculate vo lume ( ml ) Tota l ejaculate fructose c ontent ( mg ) BulbX/ BulbX/ Per i od Contro l s BulbX Gangl i onX GanglionX Mean Period Controls BulbX Gangli onX Gangl ionX Mean 1 0 . 98 1 . 1 7 0 . 86 0 . 92 0 . 98 1 5 . 5 3 5 . 5 1 4 . 62 5 . 70 5 . 34 Season 1 2 0 . 72 1 ? 26 0 . 6 1 0 . 98 0 . 89 Seas on 1 2 3 . 1 8 4 . 1 4 1 . 86 4 . 48 3 . 42 3 0 . 54 1 . 05 0 . 57 0 . 60 0 . 69 3 1 . 86 1 . 83 1 . 26 1 . 1 4 1 . 52 4 0 . 56 0 . 68 0 . 64 0 . 93 0 .70 4 1 . 27 0 . 74 0 . 93 2 . 5 3 1 . 37 Mean 0 . 70 1 . 04 0 . 67 0 . 85 0 . 8 1 Mean 2 . 96 3 . 05 2 . 1 6 3 . 46 2 . 9 1 5 1 . 1 0 0 . 93 1 . 37 1 . 22 1 ? 1 6 5 3 . 3 3 1 . 34 1 . 9 1 2 . 37 2 . 24 Seas on 2 6 1 ? 1 2 1 . 08 1 ? 1 9 1 . so 1 . 3 0 Season 2 6 4 . 26 1 . 82 2 . 00 4 . 54 3 . 1 6 7 1 . 1 8 1 . 23 1 . 27 1 . 56 1 . 3 1 7 3 . 05 5 . 03 2 . 1 5 4 . 3 5 3 . 64 8 1 . 5 5 1 . 54 1 . 88 1 . 68 1 . 66 8 1 1 ? 57 7 . 75 6 . 36 1 2 . 6 1 9 . 57 Mean 1 . 2 3 hl2 1 . 42 1 . 56 1 ? 3 5 Mean 5 . 5 5 3 . 98 3 . 1 0 5 . 96 4 . 6 5 Seas on 3 9 1 . 56 0 . 94 . 1 . 1 7 1 . 47 1 ? 28 Seas on 3 9 9 . 6 5 5 . 88 5 . 96 7 . 04 7 . 1 3 1 0 1 . 28 1 . 78 1 . 4 1 1 ? 56 1 . 5 1 1 0 7 . 3 5 4 . 26 4 . 0 1 4 . 84 5 . 1 2 Mean 1 .42 1 . 36 1 . 29 .L2l 1 . 39 Mean 8 . 50 5 . 07 4 . 98 5 . 94 6 . 1 2 Overal l Mean 1 . 06 1 . 1 7 1 . 1 0 1 . 27 Overal l Mean 5 . 1 1 3 . 83 3 . 1 1 4 . 96 ? -l=" -l=" Table 4 . 3 Mean c onc entrati ons of fructose in semen and in s eminal plasma of e jaculates c o l lected in Experiment 4 . Seminal fructose c onc entration (mg/ml ) Seminal plasma fructose c ouc entration (mg/ml ) BulbX/ BulbX/ Per i od Controls BulbX Gangl i onX Gangl i onX Mean Period Controls BulbX Gang l i onX Gangl ionX Mean 1 5 . 76 4 . 20 5 . 63 5 . 97 5 . 39 1 7 . 26 5 . 75 7 . 28 6 . 94 6 . 8 1 Seas on 1 2 4 . 3 1 3 . 64 2 . 98 4 . 3 5 3 . 82 Seas on 1 2 5 . 26 4 . 99 3 . 85 5 . 42 4 . 88 3 3 . 47 1 . 42 2 . 1 9 1 . 3 5 2 . 1 1 3 4 . 04 1 . 81 2 . 59 1 ? 58 2 , 5 1 4 2 . 57 1 ? 1 6 1 . 6 1 2 . 02 1 . 84 4 2 . 99 1 . 42 2 . 3 3 2 . 9 5 2 . 42 Mean 4 . 02 2 . 60 3 . 1 0 3 . 42 3 . 29 Mean 4 . 88 3 . 49 4 . 0 1 4 . 2 2 4 . 1 5 5 2 . 68 1 . 38 1 . 43 1 . 9 1 1 . 85 5 3 . 00 1 . 62 1 . 80 2 . 1 4 2 . 1 4 Season 2 6 2 . 90 1 . 45 1 ? 57 2 . 4 1 2 . 08 Seas on 2 6 3 . 62 1 ? 83 1 . 96 3 . 1 6 2 . 64 7 2 . 26 2 . 1 9 1 . 76 2 . 92 2 . 28 7 3 . 24 6 . 1 9 2 . 04 3 . 1 9 3 . 66 8 7 . 24 5 . 27 3 . 89 7 . 46 5 . 96 8 8 . 36 6 . 40 4 . 48 9 . 1 5 7 . 1 0 Mean 3 . 77 2 . 57 2 . 1 6 3 . 67 3 . 04 Mean 4 . 5 5 4 . 01 2 . 57 4 . 4 1 3 . 88 Season 3 9 6 . 1 2 4 . 84 5 . 6 5 4 . 56 5 . 29 Seas on 3 9 7 . 1 7 5 . 4 1 6 . 63 6 . 1 6 6 . 34 1 0 5 . 3 1 2 o 64 3 . 34 3 . 1 5 3 . 6 1 1 0 6 . 37 3 . 44 4 . 1 3 4 . 38 4 . 58 Mean 5 . 71 3 . 74 4 . 49 3 . 85 4 . 45 Mean 6 . 77 4 . 42 5 . 38 5 . 27 5 . 46 Overa l l Mean 4 . 26 2 . 82 3 . 0 1 3 . 6 1 Overa l l Mean 5 . 1 3 3 . 89 3 . 71 4 . 5 1 ? ? \J1 Table 4 . 4 Mean c onc entrations o f spermatozoa/ml and mean numbers o f spermatoz oa/e j aculate i n s emen c o l lected in Exper iment 4 . 9 Spermatoz oa/ml ( x1 0 ) Spermatozoa/e jaculate ( x1 09 ) BulbX/ BulbX/ Per i od Controls BulbX Gangl ionX Gangl i onX Mean Period Contro l s BulbX Gangl i onX Gangl i onX Mean 1 3 . 3 5 3 . 00 3 . 84 2 . 47 3 . 1 6 1 3 . 09 3 . 26 3 . 30 2 . 2 1 2 . 96 Seas on 1 2 3 . 08 4 . 1 9 3 . 54 3 . 1 5 3 . 49 Season 1 2 2 . 2 5 5 . 5 5 2 . 23 3 . 02 3 . 26 3 2 . 5 1 2 . 94 2 . 40 2 . 70 2 . 64 3 1 ? 30 3 . 39 1 . 38 1 . 54 1 . 90 4 2 . 53 2 . 83 4 . 77 2 . 57 3 . 1 8 4 1 . 5 0 2 . 0 1 3 . 24 3 . 77 2 . 63 Mean 2 . 86 3 . 24 3 . 63 2 . 72 3 . 1 1 Mean 2 . 03 3 . 5 5 2 . 5 3 2 . 63 2 . 68 5 1 . 40 1 . 7 5 2 . 47 1 . 42 1 . 76 5 1 . 84 1 . 84 3 . 64 2 . 46 2 . 44 Season 2 6 1 . 67 1 . 6 5 2 . 9 5 3 . 5 2 2 . 45 Season 2 6 2 . 79 2 .64 3 . 9 1 7 . 53 4 . 22 7 0 . 86 0 . 68 2 . 1 6 1 . 29 1 . 2 5 7 1 . 27 0 . 80 3 . 20 2 . 05 1 . 83 8 2 . 1 2 2 .6 0 1 . 92 2 . 84 2 . 37 8 3 . 36 4 . 94 5 . 29 . 4 . 80 4 . 60 Mean h2.l. 1 . 67 2 . 37 2 . 26 1 ? 9 5 Mean 2 . 3 1 2 . 5 5 4 . 0 1 4 . 2 1 3 . 27 Seas on 3 9 2 . 03 . 1 ? 52 2 . 39 3 . 9 5 2 . 47 Seas on 3 9 4 . 1 5 1 . 43 3 . 3 5 5 . 96 3 . 72 1 0 1 . 74 3 . 72 2 . 86 4 . 27 3 . 1 5 1 0 2 . 60 7 . 64 4 . 03 7 . 1 2 5 . 3 5 Mean 1 . 88 2 . 62 2 . 62 4 . 1 1 2 . 8 1 Mean 3 . 37 4 . 5 3 3 . 69 6 . 54 4 . 53 Overal l Mean 2 . 1 3 2 . 49 2 . 93 2 ., 82 Overal l Mean 2 . 42 3 . 3 5 3 . 36 4 . 05 --" ? 0'\ Table 4 . 5 Mean percentages o f unstained and morpho logically normal spermatozoa in s emen c o l lected in Experiment 4 . % Unstained spermatozoa % Morpho logically normal spermatozoa BulbX/ BulbX Period Controls BulbX Gangl ionX Gangl i onX Mean Period Contro ls BulbX Gangl i onX Gangli onX He an 1 76 . 4 76 . 6 75 . 2 89 . 0 79 . 3 1 88 . 0 86 . 5 74 . 8 8 5 .6 83 .7 Seas on 1 2 62 . 7 6 5 . 7 7 5 . 6 60 . 6 66 . 2 ?eas on 1 2 72 . 0 75 . 1 64 . 9 7 5 . 4 7 1 . 8 3 72 . 4 68 . 0 73 . 3 8 1 ? 7 73 . 8 3 66 . 3 77 . 0 67 . 7 78 . 0 72 . 2 4 70 . 3 78 . 7 70 . 8 67 . 3 7 1 . 8 4 67 . 2 8 1 . 8 72 . 6 80 . 0 7 5 . 4 Mean 70 .4 72 . 2 73 . 7 74 . 6 72 . 8 Mean 73 . 4 80 . 1 70 . 0 79 . 8 75 . 8 5 81 . 9 66 . 8 68 . 1 77 . 5 73 . 6 5 70 . 6 77 . 5 60 . 0 7 1 .7 70 . 0 Season 2 6 73 . 5 66 . 9 73 . 4 79 . 4 73 . 3 Seas on 2 6 60 . 1 63 . 0 47 . 8 67 . 0 59 . 5 7 88 . 3 77 . 5 69 . 3 72 . 0 76 . 8 7 76 . 7 63 . 5 44 . 8 5 4 , 2 59 . 8 8 7 5 . 2 67 . 5 5 1 . 8 78 . 0 68 . 1 8 53 . 9 48 . 8 2 5 . 7 54 . 5 45 . 7 Mean 79 . 7 69 . 7 6 5 . 6 76 . 7 73 . 0 He an 6 5 . 3 63 . 2 44 . 6 6 1 . 8 58 . 8 Season 3 9 7 5 . 7 66 . 5 79 . 6 73 . 2 73 . 8 Seas on 3 9 68 . 7 55 . 2 66 . 7 5 8 . 5 . 62 . 3 1 0 68 . 9 67 . 4 70 . 5 6 5 . 2 68 . 0 1 0 62 . 0 60 . 4 60 . 2 5 5 . 4 59 . 5 Mean 72 . 3 67 . 0 75 . 1 69 . 2 70 . 9 Mean 65 . 4 57 . 8 63 . 4 57 . 0 60 . 9 Overa l l Mean 74 . 5 70 . 2 70 . 8 74 . 4 Overa l l Mean 68 . 6 68 . 9 58 . 5 68 .0? ? ? -.._) Table 4 . 6 Experiment 4 ? : Summary of Analyse s of Var ianc e for Semen Data . Source of Variat i on Contrast No . D . F . Var iance Rat ios 2. MAIN EFFECTS A . SEASONS Season 1 - Linear " 1 1 - Cubi c Season 2 - Linear " " - Cubi c Seas on 3 - Linear Season 1 vs Seas ons 2 & 3 Seas on 2 vs Seas on 3 Non s ignificant c ontrasts B . SURGICAL TREATMENTS I C ? BX, GX, BX/GX BX, GX ? BX/GX BX VS GX INTERACTION (AxB ) Sea s on 3 - Linear x Contrast 8 ( Seas ons 1 vs 2 & 3 ) x Contrast 8 Seas on 1 - Cubic x Contrast 9 Seas on 2 - Quadratic x Contrast 9 11 1 1 - Cubi c x "Contrast 9 ( S eas ons 1 vs 2 & 3 ) x Contrast 9 ( Seasons 2 vs 3 ) x Contrast 9 Sea s on 2 - Linear x Contrast 1 0 Non s ignificant c ontrasts Res idual Mean Square 1 2 3 4 5 6 7 8 9 1 0 1 1 1 2 1 3 1 4 1 5 1 6 1.7 1 8 9 3 27 1 1 1 1 1 1 2 1 1 1 1 1 9 1 20 Volume 6 . 3 5 * 0 . 1 1 1 1 . 74*** 1 . 1 3 2 . 47 74 . 44*** 0 . 30 1 ? 1 1 3 . 5 3 3 . 45 0 . 38 5 . 44* 0 . 1 5 0 . 0 1 3 . 52 0 . 70 0 . 64 0 . 1 1 0 . 04 0 . 68 0 . 1 5 Notil ity 32 . 97*** 0 . 92 0 . 03 0 . 08 0 . 03 . 26 . 98*** 2 . 80 0 . 1 8 1 . 74 5 . 1 3* 1 . 5 1 4 . 1 5* 1 . 06 4 . 54* 0 . 5 9 0 . 33 4 . 48* o . oo 0 . 1 4 0 . 78 0 . 2 5 % Moti l e 26 . 5 6*** 0 . 03 0 . 38 2 . 73 0 . 37 6 . 1 5* 6 . 52* 0 . 84 0 . 92 5 . 47* 1 . 63 0 . 9 1 1 . 41 3 . 90 0 . 56 1 . 47 3 . 1 2 0 . 4 5 0 . 04 0 . 56 1 73 . 1 9 ( Key 1 , Surgical Treatments : C = Contro ls ; BX = BulbX ; GX = GanglionX ; BX/GX = BulbX/Gangl i onX ) Sperm . /ml Sperm . /e jac . 4 . 33* 4 . 1 6* 1 . 70 1 . 62 3 .79 9 . 02** 5 . 93* 0 . 03 9 . 80** 0 . 1 0 1 . 62 3 . 1 4 4 . 62* 0 . 00 1 2 . 72*** 0 . 07 4 . 2 5* 7 . 0 1 ** 5 . 5 8* 0 . 77 1 ? 30 1 ? 1 3 2 . 76 2 . 60 1 3 . 27*** 4 . 94* 8 . 27** 6 . 28* 0 . 49 9 . 87** 2 . 57 o . oo 7 .73** 0 . 6 1 0 . 02 2 . 99 4 . 05* 3 . 98* 1 . 63 0 . 1 3 0 . 79 4 . 76 ( Key 2 , Semen Parameters : Vo lume = e jaculate volume ; Moti l ity = mot i l ity index ; % Moti le = perc entage of motile spermatozoa ; Sperm . /ml = c oncentration of spermatozoa/ml ; Sperm . /ejac . = number of spermatoz oa/e jaculate ) -lo -+=- CO Experiment 4 : Source or Var iation MAIN EFFECTS A . SEASONS Seas on 1 - Linear t l t t - Quadratic '' 11 - Cubic Season 2 - Linear t l t l - Quadratic tl tl - Cubic Season 3 - Linear Season 1 ? Seasons 2 & 3 Seas on 2 vs Season 3 B . SURGICAL TREATMENTS I! C ? BX , GX , BX/GX BX, GX ? BX/GX BX vs GX INTERACTION (AxB ) ( Seas ons 1 ? 2 & 3 ) . x C ontrast 1 0 Seas on 1 - Cubi c x C ontrast 1 1 Non s ignificant c ontrasts Res idual Mean Square Tabl e 4 .1, Summary of Analyses of Var ianc e for Semen Data . C ontrast No . D . F . Variance Ratios 1_ Fr . Cone . Fr . Cont . S .P . Fr . % Unstained Cone . 9 1 1 3 1 . 22*** 20 . 1 9*** 3 7 . 6 1 *** 1 ? 5 1 2 1 1 ? 23 1 . oo 2 . 08 4 . 8 5* 3 1 0 . 30 0 . 08 0 . 94 4 . 27* 4 1 3 1 . 1 6*** 51 . 24*** 36 . 1 4*** 1 . 08 5 1 1 3 . 36*** 1 6 . 49*** 8 . 97** 1 ? 93 6 1 2 . 36 4 . 63* 1 . 04 1 . 30 7 1 6 . 33* 5 . 02* 5 . 1 4* 0 . 5 5 8 1 0 . 27 2 1 . 07*** 0 . 23 0 . 06 9 1 1 0 . 49** 6 . 26* 1 0 . 9 5** 0 . 94 3 1 0 1 1 1 . 9 1 *** 7 . 07** 8 . 68** 1 ? 91 1 1 1 3 . 79 9 . 41 ** 3 . 08 2 . 98 1 2 1 o . 3 3 0 . 46 o . oo 0 . 03 27 1 3 1 0 . 09 4 . 1 7* 0 . 03 6 . 98** 1 4 1 0 . 09 0 0 2 1 0 . 22 5 o 59* 2 5 0 . 43 0 . 54 0 . 5 5 0 . 82 1 20 3 . 73 7 . 1 2 5 . 00 1 2 1 . 27 ( Key 1 , Surgical Treatments : C = Contro ls ; BX = BulbX ; GX = Gangl i onX ; BX/GX = BulbX/Gangl ionX ) % Normal 1 . 88 4 . 45* 0 . 07 1 7 . 59*** o . o6 0 . 8 5 o . oo 42 . 60*** 0 . 3 3 1 . 40 1 ? 9 5 7 . 97** 3 . 26 0 . 1 3 0 . 45 237 e42 (Key 2 , Semen Parameters : Fr . Cone . = fructose c onc entration of s emen ; Fr . Cont . = total e jaculate fructose c ontent ; S . P . Fr . Cone . = fructose c onc entrati on of s emina l plasma ; % Unstained = perc entage of unstained spermatozoa ; % Normal = perc entage of morpho l ogica l ly normal spermatozoa ) ? +? ? 5 - 0 ? )o( - w ? 5 ..J ;:) (.) ? I 0 <.D XNOI1DNVD/XH'lfiH : XNOI'lDNVD } I 0 N I J I I 0 I . I I I S'lO"M I 0 0 0 N I 0 0 0 CD I 0 0 0 V J I ONV'lD 'lVtiNid/WdO 1 65 .. lf) "D c 0 CO L +- Q) c 0 0 CO Q) E c 0 L Q_ '+- ......._ L lf) Q_ "D 0 c CO lf) CO Q) "D Q) ro lf) Q) lf) c Q) L Q_ Q_ X c Q) lf) > +- 0 >-> +- ?- +- > u ?- ro +- u Q) CO lf) ro Q) L E Q) >- '+- N lf) c c w ro L +- lf) E >- ro ..c: L +- Q) X E c I 0 0 I Q) Q) - c 0 ro I I "D (9 c ......._ . X "D >- ..0 c X ro 0 ::J L ()) Q) "D >- "D ..c: c ro ro Q) c c ro X Q) c Q_ L 0 Q) E Q) ......._ 1.0 c L ro Q_ """ (9 0 Q) .. lf) L X ro ::J ..0 Q) "D ::J c LL ()) ro I 0 to determine aqy e ffect of ganglionecto? on the numbers of axons in the pineal glands . 4. DISCUSSI ON 1 66 The seasonal changes in s emen characteristics and plasma hormone levels recorded from the control N. Z . Romney rams have been discussed in Chapter III and will not be discussed again. This discuss ion will be concerned only with results which arose from the surgical tre atments . ( 1 ) Semen Data Although the surgical treatments altered some semen parameters in comparis on to the control group, it was difficult t o categorize such results as being caused by altered pineal gland function, or altered olfactory function. This difficulty arose because res ults obtained from the BulbX/GanglionX group did not appear to be different from the C ontrols by an amount equivalent to the sum of the effects of the two operations singly. Further, it would even have been poss ible to conclude , in respect of seminal fructose levels , that the effects of the double-operative treatment nearly cancelled out the effects of the two s ingle operations , giving values nearer those of the Controls . There have been no reports in the literature on the effects of these operations on semen quality, however Whitten ( 1 956 ) reported that olfactory bulbectomy reduced the volume of secretions from the accessory glands of mice . Reiter ( 1 973?) pointed out that pinealectomy in lower mammals had been notoriously unreliable in producing gonadal responses . He mentioned that anosmia had been designated as a "potent- iating factorn which sensitized the neural-gonadal axis of rats t o the influence of the pineal gland. This conclus ion was based on the finding that anosmia exaggerated the nypertrophY of the gonads and accessory sex organs which followed blinding of male rats (Reiter, 1 67 Kle in and Donofrio, 1 969 ) , and the hypertrophy of the gonads of similar? treated female rats (Reiter and Ellison, 1 970) . The semen data obtained i n the prese nt stuqy did not enable aqy conclusive comment t o be made on such statements . ( 2 ) LH and Testosterone Previo\.13 workers havo not been able to demonstrate ariY effects of olfactory bulbectomy ( Maule on and Signoret , 1 964) or pinealecto? (Roche et al. , 1 970?) on reproductive hormone levels in the ewe . In Experiment 4 all three surgical treatments caused a reduction in the regular seasonal changes in plasma LH and testosterone concentratiollil, and e levated mean LH levels , compared to those recorded from the unoperated control rams . These differences in the seasor?l pattern of plasma LH and testosterone secretion were most evident during the winter months . Cranial cervical ganglionectomy reversed the st)?ulatory effects of blinding or darkness on pineal gland activity in rats (Wurtman, Axelrod and Fi?cher, 1 964 ) and golden hamsters (Eichler arrl Moore , 1 971 ) ? Consequently, because pineal act ivity caU3ed gonadal degeneration in these specie s (Hoffman and Re iter, 1 965 ; Reiter, 1 968 ) , ganglionectomized rams in the present study might have been expected to displqy higher gonadotrophin levels than the c ontrol rams , only during periods of reduced daily photoperiod ( increased darkness ) . In view of the present paucity of knowled@e of olfactory influences on reproduct ion in domestic animals , it is difficult to acc ount for the effects of olfactory bulbectomy, which tended to be s imilar to the results of ganglionectomy. Blask am Reiter ( 1 975 ) found that both blinding and anosmia ( singly or combined) significantly lowered plasma 1H levels in female rats . Their results vrere attributed to impaired release of LH, because the c ombined treatments e levated pituitary levels of this hormone. As the effects of these 1 68 surgical treatments were reversed by pinealecto!I{Y', the authors c oncluded that the antigonadotrophio capacity of the pineal gland was potentiated by the two operations . A s imilar conclus ion could not be applied to the effects of olfacto? bulbecto? in Experiment 4, because this operation tended to elevate plasma LH levels . Plasma LH results from the present experiment indicated that the pineal gland in rams may have had an antigonadotrophic function which was inhibited by cranial cervical ganglionectomy or olfactory bulbectomy. Although plasma testosterone data would have been expected to support this conclusion with e levated levels i n surBical? treated rams , only non-significant trends were observed, and the contrasts tested in the analysis of variance of testosterone data did not reveal any notable differences between the surgically treated and control groups of rams. One significant difference ( between the rate of decline of plasma testosterone levels during March t o June 1 972 , for the single operation groups versus the double-operated group) was of doubtful importance because of its low leve l of signifi? cance , and the absence of' a similar effect in the same period of 1 973 . In summary, the seas onal patterns of secretion of reproductive hormones were diminished by cranial cervical ganglionectorey ( by modifying pineal activity) or olfactory bulbecto!I{Y' (by unknown actions e ither on pineal function, or on hypothalamic or pituitary function, directly) . ( 3 ) Prolactin Ronnekleiv and McCann ( 1 975 ) reported that olfactory bulbectonzy? or cranial cervical ganglionecto!I{Y' lowered serum prolactin levels in male rats . They concluded that the pineal gland, or its principles , normal? stimulated the re lease of prolactin from untreated c ontrol animals, and that this stimulation was induced by olfactory and visual 1 69 input to the pineal gland. Unfortunately, all the ir blood aamples were obtained during ether anaesthesia, s o the effects of this stress on prolactin re lease may have been confounded with th8 surgical treatments. As pointed out by the authors , this objection is particularly valid since the e ffe ct of ether stress may have been entirely dependent on the ability o f a n aniw.al to smell the anaes thetic, and this alone might ac c o u nt for the lower prolactin leve ls in anosmic rats. In the present experi:nent olfactory bulbecto?ey did not cause a? change in prolactin levels . However cranial cervical ganglionecto?ey induced an elevation of plasma prolactin levels and an almost complete loss of the annual seasonal rhythmic fluctuations in levels of this hormone . It is presumed that the very s imilar results obtair?d from rams which had bee n olfactory bulbectomized as we ll as ganglionectomized, was the result of cranial cervical ganglionectol'Il'J alone . Goats which had bee n cranial cervical ganglionectomized in the winter experienced an elevation of the ir plasw? prolactin levels before unoperated control animals and had s ignificantly higher levels for three succes sive months ; however both groups of goats had s imilar midsummer levels (H. Buttle , pers onal c ommunication) . Although group size was small ( n = 3 ) , these preliminary observations from goats were similar to those obtained from rams in Experiment 4. If the pineal gland is an important regulator of prolactin release in rams , as it is presumed t o be in rats and hamsters (Re iter, 1 974?) , the n its major role could be as an inhibitor of prolactin secretion during winter, because pineal activity would be greatest during the period of shorte ned daily photoperiods (Re iter, 1 974?) . Although it c ontrasts with the earlier mentioned conclusions of Ronnekle iv and McCann ( 1 975 ) , the latter hypothes is was supported by the elevated plasma prolactin levels recorded during winter from 1 70 cranial cervical ganglionectomized rams . Further, this result indicated that an intact sympathetic nervoU3 system normally provides the neural pathways by which the pineal gland receives stimuli necessary for its inhibito? influence over prolactin release . Autopsy data which showed that cranial cervical ganglionecto? reduced pineal activity, provided further support for the above 1?pothesis . ThU3 the results from ganglionectomized rams in the present stuqy implicated the pineal gland as having a major role in regulating the seas onal changes in plasma prolactin leve ls recorded in Experiment 3 1 and in the photoperiodic control of prolactin release in rams reported by Felletier ( 1 973 ) . (4) Autopsy Data C omparison of the results for the semen data with those obtained from autopsy material, helped to clarify the nature of gonadal responses to the surgical treatments . Towards the end of the experiment and at autopsy, operated rams, in particular those which had undergone both operations (BulbX/GanglionX ) , tende? to have higher values for indices of spermatozoal production than the control rams . These parameters included spermatozoa per e jaculate , spermatozoal c.once ntration and motility in semen, testicular and epidiqymal weights , and seminiferous tubule diameters . On the other hand, untreated rams had higher mean values for variables assoc iated with access ory gland function, for example : seminal fructose levels , seminal vesicular we ights , and seminal vesicular fructose contents and concentrations . Evidence of a higher level of spermato@enic activity i? surgical? treated rams, together with the higher output of LH in these animals , supported the view that the operations stimulated gonadal activity by increasing gonadotrophin secretion. This finding also was consistent with the theor.y that cranial cervical ganglionecto? reduced the antigonadotrophio activity of the pineal gland. The 1 7 1 effects of olfactory bulbectoll\Y were unclear, except when combined with cranial cervical ganglione cto?, in which case it appeared t o ampli? som of the effects of the latter treatment . Reduced accessory gland function in the operated rams c orre sponded with lowered seminal ves icular we ights and secre tion volumes recorded from anosmic male mice by Whitten ( 1 956 ) . Furthermore Sorrentino, Re iter and Schalch ( 1 971 ) suggested that olfactory bulbecto? and cranial cervical ganglionecto? interfered with the nutritional status and growth hormone secretion in rat s . Such effects c ould reduce accessory gland activity. Plasma testosterone data obtained from rams in Experiment 4 did not shol'l variations which c ould be used in explaining the different levels of accessory sex gland activity caused by the surgical treatments . Reduced HIOMr activity of pineal glanfu3 from rams which had been cranial cervical ganglionectomized provided a check on the e fficie ncy of the surgery, as well as confirming the assumption that this operation would alter pineal function. Pineal cell nuclear density provided an index of ce llular activity as it is reduced when pineal cells are active and have large cytoplasmic volumes , hence lower nuclear densities (Roth, Wurtman and Altschule , 1 962 ) . The e ffect of ganglionecto? on pineal activity als o was indicated by the lower pineal weights in the ganglionectomized rams, however the reduction in we ights was not statistically significant. With the limitations in the histological staining technique available , it was not poss ible t o assess whether changes i n density o f pineal axons occurred as a result of ganglionecto?. It seeiD3 reasonable to conclude that s ince removal of the cranial cervical ganglia reduced pineal activity then, in the ram as in the rat and golden haiDBter, these ganglia are an integral part of the affere nt nerve supp? to the pineal gland. ( 5 ) General D iscussion Cranial cervical sympathectomy may have had effects on structures other than the pineal gland and these must be cons idered. The cranial cervical ganglia are involved in the sympathetic 1 72 autonomic control of blood vessels in the head, including those supplying the pituitary gland and the rest of the brain. Removal of these ganglia has bee n reported t o lead to a fall in cerebral blood volume in mice (Edvinss on, Owman and West, 1 971 ) . Thus it may be poss ible to ascribe all the endocr?ne co??e quences of ganglionector? to alterations in rate of pituitary blood flow . Consequently, definitive statements about the role of the pineal gland in regulating reproduction should be made only after observing the effects of pinealectomy. The s ignificant results which could be attributed to olfactory bulbecto? did not provide much ins ight into the poss ible role of the olfactory system i n the seasonality of reproduction. Disruption of the seas onal changes of plasma LH, and to a lesser exte nt , of plasma testosterone levels s hown by the BulbX group, lent support to the possibility that olfaction could play a part in controlling the onset of the breeding seas on. Furthermore , as destruction of the olfactory bulbs would have disrupted nervous connections from the vomeronasal organ (Alberta, 1 974; Powers and Winans? 1 975 ) , it is not possible to discount the idea that the alteration in seasonal changes of plasma LH and testosterone secretion resulted indirectly from the loss of the vomeronasal component of olfaction. However, the hormone and semen data was obtained from only three rams , s o aey conclusions must be treated with caution. Nevertheles s this experiment established that e nvironmental factors c ould influence the reproductive systems of rams via the ir pineal glands and olfactory systems. The importance of olfactory 1 73 stimuli in seas onal reproductive changes could not be ascertain(?d, nor was it possible to ascertain the mechanisms by which such stimuli c ould modi? reproduction. However on the other hand photoperiodic s timuli, which are known to control annual photoperiodic cycles in domestic animals ( Ortavant, Maule on and Thibault , 1 964) , may exert their influence on reproduction in rams by reducing pineal activity during the summer months . This e ffect of dai? photoperiod probab? is manifested by the increased se cretion of gonadotrophins at this time of the year, as was seen for plasma LH leve ls recorded in Experiment 3 . A s imilar explanation may have accounted for the changes in plasma prolactin levels i n unoperated animals , s o it would have been informative to have measured FSH as well. Unfortunately, a s atisfactory assay for this hormone was not available . 1 74 CHAPI'ER V EFFECTS OF DI FFERE NI' LIGHTING REGIMES ON SEMEN PRODUCTION' AND PLASMA HORMONE LB."'VELS IN RAMS 1 ? IN.rRODUCTION The work described in this chapter was a prelimi? attempt t o determine the impor-tance of daily photoperiod as an e nvironme ntal factor influencing the se ao onal changes in plas ma hormone leve ls and seo::e n production, describe d i n Chapter III . Similar studie s me ntioned in C hapter I have demonstrated that seasonal changes in reproductive characteristics of ram3 c an be simulated, or s ubs tantially modified, by exposing them t o appropriate artificial lighting regime s . However, none o f these studies have involved N. Z. Romney rams , nor have there bee n aqy previous attempts t o combine hormonal and semen production studie s . Some of the experime nts described i n the lite?cture have s imulated the natural annual cycle of photoperiod changes . The role of these has bee n studied by altering the phase of the cycle , usual? in simple phase reversal/non-reversal experiments (Fowler, 1 961 ; Moule , Braden and Mattner, 1 966 ) . On the other hand, " phase-shorte ning" experiments, in which change s in daily phot operiod e quivale nt to the annual cycle were completed in six months , have bee n used t o investigate photoperiodic e ffects on ram seme n product ion (Colas et al. , 1 972 ; Jackson and WilliaiD8 , 1 973 ) and plasma prolactin and LH leve ls ( Pe lletier, 1 973 ; Pe lletier and Ortavant , 1 975??) . I n this experiment the natural seas onal cycle of c hange s in d?light were simulated and their e ffects on reproduction in rams c ompared with the effe cts of a reversed cycle , or a non-changing 1 75 photoperiodic regime . The latter regime was included aa a check on the persistence of res idual r?thms in reproduct ive parame ters , in the absence of changes in photic stimulation. 2 . MATERIALS AND METHODS (1 ) Animals and Experime ntal Procedure Twe lve adult N. Z . Romney rams were allocated at random to three experimental groups , each group being sub jected to a different lighting regime . C omme ncing at the autumn equinox 1 973 , one group continued on the normal seas onal decline in daylight (Normal ) , another experienced an increasing photoperiodic regime (Reversed) , whilst the third group remained on a constant 1 2 hr light : 1 2 hr dark (Even) regime . Information regarding the annual changes in daylight was obtained from the New Zealand Nautical Almanac ( 1 972 ) using the sunrise and s unset tables for Wanganui ( latitude 27 minutes N. of Palmerston North) . Daily lighting was taken as the tiJOO between sunrise and s unset ; this varied in a s inusoidal manner from 9 h 2:1 min minimum to 1 4- h 40 min maximum. No allowance was made for twilight . Collection of blood samples in Experiment 5 c ommenced in March 1 973 and continued until December that year. A programme o f two -weekly semen collections commenced at the Jum solstice and continued until the end of the experiment at the December solstice , 1 973 . experiment autops ies were not performed. (2 ) Statistical Analyses In this ?? In each case below, c ontrasts were selected in an attempt to provide the most meaningful evaluation of treatment effects . (a) Semen Data. Semen data were pooled to give a s ingle mean estimate for each variable for every ram, on e ach of the s ix months between the June and December s olstices ( see Figure 5 .1 ) . Orthogonal 1 76 SOLSTICE SOLSTICE EQUINOX PHOTOPERIOD ( h ) SEMEN 1 PERIOD 1 LH AND 1 TESTOSTERONE PROLACTIN 1 ?10NTHS M 2 2 A Key : N - Normal E - Even R - Reversed 3 4 5 PERIOD 1 3 M J 4 EQUINOX 2 3 4 5 6 PERIOD 2 6 7 8 9 1 0 PERIOD 2 5 6 7 8 9 J A s 0 N D 19 7 3 F i g u re 5 . 1 : D i agram show i ng the cyc l i c cha nges i n da i l y p hotoper i od for ? the I i g ht i ng reg i mes used i n Exper i ment 5 , a n d the subd i v i s i on of t he t i me-cou rse of the exper i ment for data a na l y ses . Table 5 . 1 Orthogona l c oeffic ients used in partitioning t ime period effects f or semen data in Experiment 5 . Sampl ing t ime - 1 2 3 4 5 6 Contrast Linear -5 -3 -1 +1 +3 +5 Quadratic +5 -1 -4 -4 -1 +5 Cubi c -5 +7 +4 -4 -7 +5 ? ---J ---J Table 5 . 2 Orthogonal c oeff i c ients used in partitioning t ime period effects for plasma LH and testosterone data i n Experiment 5 . Period 1 Period 2 Sampl ing t ime - 1 2 3 4 5 6 7 8 9 1 0 Contrast Per i od 1 - Linear -3 -1 +1 +3 0 0 0 0 0 0 11 11 - Quadratic +1 -1 -1 +1 0 0 0 0 0 0 11 11 Cubic -1 +3 -3 +1 . 0 0 0 0 0 0 Per i od 2 - Linear 0 0 0 0 -5 -3 -1 +1 +3 +5 11 11 - Quadratic 0 0 0 0 +5 -1 -4 -4 -1 +5 11 11 Cubic 0 0 0 0 -5 +7 +4 -4 -7 +5 Period 1 vs Per i od 2 +3 +3 +3 +3 -2 -2 -2 -2 -2 -2 ? --,:) CO Table 5 . 3 Orthog?nal c oeff i c i ents used in partitioning time period effects for plasma pro lactin data in Experiment 5 . Per iod 1 Period 2 Sampl ing t ime - 1 2 3 4 5 6 7 8 9 Contras t Per iod 1 - Linear -5 -3 -1 +1 +3 +5 0 0 0 " " - Quadratic +5 -1 -4 -4 -1 +5 0 0 0 " " - Cubic -5 +7 +4 -4 -7 +5 0 0 0 Per iod 2 - Linear 0 0 0 0 0 0 -1 0 +1 " " - Quadratic 0 0 0 0 0 0 +1 -2 +1 Per i od 1 vs Per i od 2 +1 +1 +1 +1 +1 +1 -2 -2 -2 ? --.) \.0 1 80 c oefficients used to partition time period effects in the analyses of variance are shown in Table 5.1 . (b ) LH and Testosterone . The ten four-weekly assay results for mean plasma LH and testosterone c once ntrations were groupe d into two periods , with the June solstice as the dividing point (see Figure 5 . 1 ) ? Orthogonal coefficients used to partition time period effects in the analyse s of variance are shown in Table 5 . 2 . ( c ) Prolactin. The nine monthly mean estimates of plasma prolactin c once ntration were grouped into two periods , with the dividing point at the September equinox ( see Figure 5.1 ) . Orthogonal c oefficients used to partit ion t ime period effects in the analysis of variance are s hown in Table 5 . 3 . ( a) Treatment Contrasts. In the analyses of variance for all semen and hormonal data, c omparisons of the effectB of the three lighting regimes were performed us ing the orthogonal coefficients shown below : Lighting Regiroo C ontrast Normal vs Reversed - Even vs Normal and Reversed 3 . RESULTS ( 1 ) Semen Data Normal +1 -1 See Tables 5.4 t o 5 .1 0 and Figure 5.2 . Even Reversed 0 -1 +2 -1 With the exceptions of e jaculate volume and peroenta? of unstained spermatozoa, all semen characteristics exhibited a general increase in quality over the period of study. The Reversed lighting group contributed most to these increases , and in fact displ?ed a sharp rise in e jaculate volume at the e nd of the experiment. Over the whole period of the experiment, the Even lighting 1 8 1 Table 5 . 4 Mean moti l ity ind i c e s ( s ca l e 0-4 ) and mean perc entages of mot i le s permatozoa r e c orded from e j acu late s c o l lected in Experiment 5 . Mot i l ity index Lighting Regime Sampl ing Normal Reversed Even Mean t ?me 1 2 . 1 1 . 4 1 ? 2 1 . 6 2 2 . 3 1 . 7 1 ? 1 1 . 7 3 1 . 7 1 . 3 0 . 9 1 . 3 4 3 . 2 2 . 3 2 . 2 2 . 6 5 3 . 6 3 . 1 2 . 1 2 . 9 6 2 . 9 2 . 7 2 . 3 2 . 6 Mean 2 . 6 b..1. 1 ? 6 % Mot i l e spermatozoa Lighting Reg ime Sampl ing Normal Reversed Even Mean t ime 1 41 . 2 34 . 4 24 . 4 3 3 . 3 2 5 1 ? 9 28 . 8 20 . 8 3 3 . 8 3 32 . 5 28 . 1 1 8 . 8 26 . 5 4 67 . 8 45 . 0 48 . 1 5 3 .6 5 74 . 4 56 . 9 45 .0 58 . 8 6 5 5 . 2 6 1 . 2 50 . 2 5 5 . 5 Mean 53 . 8 42 . 4 34 . 6 Tab l e 5 . 5 Mean e j aculate vo lumes and mean tota l fructose c ontents o f e j aculates c o l l e c ted in Exper iment 5 . Ejaculate volume ( ml ) Light ing Regime 1 82 Sampl ing Normal Reve rsed Even Mean t ime 1 . 67 1 . 36 0 .75 1 . 26 2 0 . 85 0 .75 1 ? 1 5 0 . 92 3 0 . 62 0 . 64 0 . 99 0 . 75 4 1 . 22 0 . 98 0 . 79 1 . 00 5 0 . 94 1 . 06 0 . 79 0 . 93 6 0 . 9 5 1 . 5 1 0 . 75 1 . 07 Mean 1 . 04 1 . 05 0 . 87 Tota l e jaculate fructose content ( mg ) Lighting Regime Sampl i ng Normal Reversed Even Mean t ime 1 3 . 48 1 . 48 1 . 1 2 2 . 03 2 1 .01 0 . 87 2 . 5 1 1 . 46 3 0 . 65 0 . 77 0 . 85 0 . 76 4 0 . 96 2 . 2 1 0 . 5 1 1 . 23 5 1 . 07 6 . 1 5 o . 5 1 2 . 58 6 1 . 5 1 6 . 88 0 . 3 5 2 . 92 Mean 1 . 45 3 . 06 0 . 97 Tab l e 5 . 6 Mean c oncentrat i ons o f fructose in s emen and in semina l p lasma o f e jacu lates c o l l e c t ed i n Exper iment 5 . Semina l fructose c onc entrat ion (mg/ml ) Lighting Regime Sampl ing Normal Revers ed Even Mean time 1 . 72 1 . 20 1 . 02 1 . 3 1 2 1 . 1 7 0 . 9 8 1 . 99 1 . 3 8 3 0 . 77 1 ? 0 1 1 ? 05 0 . 9 4 4 0 . 74 1 ? 54 0 . 58 0 . 9 5 5 1 ? 1 2 5 . 09 0 . 68 2 . 30 6 1 ? 74 4 . 42 0 . 5 3 2 . 2 3 Mean 1 ? 2 1 2 . 37 0 . 98 Semina l plasma fruc tose c onc entration (mg/m l ) Light ing Regime Sampl ing Normal Reversed Even Mean time 1 2 . 2 2 1 . 25 1 . 06 1 . 5 1 2 1 . 3 2 1 . 04 2 . 02 1 . 46 3 0 . 89 1 . 03 0 .74 0 . 89 4 0 . 9 3 2 . 07 0 . 87 1 . 29 5 1 . 4 1 6 , 28 0 . 76 2 . 8 1 6 2 . 1 0 5 . 72 0 . 6 1 2 . 8 1 Ne an 1 . 48 5.90 1 . 0 1 1 84 Tab l e 5 . 7 Mean c onc entrations o f s permatozoa/m! and mean numbers of s permatozoa/ e jaculate in semen c o l l e c ted in Exper iment 5 . Spermatozoa/m! ( x 1 09 ) Lighting Regime Sampling Normal Reversed Even Mean t ime 1 1 . 74 0 . 63 1 . 20 1 . 1 9 2 1 . 6 5 0 . 9 5 1 . 23 1 . 28 3 1 . 47 0 . 87 0 . 44 0 . 93 4 2 . 89 2 . 65 2 . 94 2 . 83 5 3 . 1 1 2 . 6 5 3 . 1 7 2 . 98 6 2 . 90 3 . 22 3 . 42 3 . 1 8 Mean 2 . 30 1 . 83 2 . 07 Spermatozoa/e jaculate ( x1 09 ) Lighting Regime Sampling Normal Revers ed Even Mean time 1 4 . 1 1 0 . 8 5 1 .70 2 . 22 2 1 . 69 0 . 60 2 . 1 4 1 . 48 3 1 ? 5 2 0 . 60 0 . 43 o . 8 5 4 4 . 29 2 .74 2 . 44 3 . 1 6 5 3 . 05 3 . 05 3 . 70 3 . 26 6 2 . 96 5 . 78 3 . 64 4 . 1 3 Mean 2 . 94 2 . 27 2 . 34 1 85 Tab l e 5 . 8 Mean perc entages of uns ta ined and morpho l ogi ca l ly norma l spermato zoa in s emen c o l lec ted in Experiment 5 . % Uns tained spermatozoa Lighting Regime Sampl ing Normal Re versed Even Mean time 1 5 5 . 0 45 . 2 30 . 9 43 .7 2 69 . 5 52 . 1 48 . 4 56 . 7 3 3 4 . 0 3 5 . 0 24 . 2 3 1 . 1 4 3 1 ? 5 5 1 . 0 43 . 6 42 . 0 5 5 5 . 1 6 4 . 6 5 6 . 8 5 8 . 8 6 5 6 . 6 56 . 1 5 7 . 2 56 . 6 Mean 5 0 . 3 5 0 . 7 43 . 5 % Morphologica l ly normal spermatozoa Lighting Regime Sampl ing Normal Reversed Even Mean time 1 50 . 4 29 . 8 38 . 2 3 9 . 5 2 56 . 8 3 5 . 4 37 . 5 43 . 2 3 47 . 6 50 . 0 22 . 8 40 . 1 4 79 . 4 49 . 2 5 7 . 6 62 . 1 5 73 . 9 72 . 6 60 . 5 69 . 0 6 57 . 5 8 1 . 0 60 .4 66 . 3 Mean 60 . 9 5 3 . 0 46 . 2 Table 5 . 9 Exper iment 5 : Summary of Analys es of Var iance . for Semen Data . Source of Variat i on Contrast No . D . F . Varianc e Ratios Volume Moti l ity % Moti l e Sperm . /ml Sperm . /e jac . MAIN EFFECTS A . LIGHTING REGIMES 2 Normal vs Reversed 1 1 0 . 0 1 3 . 23 4 . 24* 1 . 20 0 . 6 1 Even vs Normal and Reversed 2 1 1 .89 6 . 25* 5 . 96* o . oo 0 . 1 3 B . TIME PERIODS 5 Linear 3 1 0 . 29 1 3 . 32*** 1 1 . 64** 1 8 . 1 7*** 5 . 4 1 * Quadrati c 4 1 3 . 3 2 0 . 00 o . oo 0 . 24 1 . 79 Cubic 5 1 0 . 7 5 2 . 60 2 . 28 2 . 08 1 . 04 INTERACTION (AxB ) 1 0 Contrast 2 x C ontrast 4 6 1 4 . 1 4* 0 . 1 7 0 . 1 5 0 . 24 0 . 01 Non s ignificant c ontrasts 5 1 . 26 0 . 08 0 . 47 0 . 24 0 . 83 Res idual Mean Square 54 0 . 30 1 . 43 6 30 .7 5 2 . 67 9 . 33 ( Key , Semen Parameters : Vo lume = e jaculate volume ; Mot i l ity = motil ity index ; % Motile = perc entage of mot i le spermatozoa ; Sperm . /ml = c oncentration of spermatozoa/ml ; Sperm . /e jac . = number of spermatozoa/e jaculate ) _.. CO 0'. Tab l e 5 . 1 0 'Experiment 5 : Summary of Analys es of Var iance for Semen Data . Source of Variat i on Contrast No . D . F . Variance Ratios Fr . Conc . Fr . Cont . S .P . Fr . % ? Unstained % Normal Cone . MAIN EFFECTS A . LIGHTING REGIMES 2 Normal v? Reversed 1 1 8 . 46** 6 . 23* 9 . 67** 0 . 09 1 . 82 Even vs Normal and Reversed 2 1 6 . 1 7* 5 . 36* 9 . 64?* 2 . 68 3 . 23 B . TIME PERIODS 5 Linear 3 1 5 . 02* 2 . 3 5 8 . 52** 2 .73 1 2 . 4 1 *** Quadrati c 4 1 3 . 02 4 . 64* 4 . 1 6* 2 . 24 0 . 03 Cubic 5 1 0 . 09 0 . 36 0 . 5 1 0 . 09 1 . 20 INTERACTION (AxB ) 1 0 Contrast 1 x Contrast 3 6 1 1 3 . 5 3*** 1 6 . 68*** 1 8 . 90*** 1 . 86 2 . 96 Contrast 2 x Contras t 3 7 1 9 . 48** 5 . 88* 1 0 . 1 3** 2 . 1 6 0 . 02 Non s ignificant c ontrasts 4 1 . 20 0 . 98 1 . 1 5 0 . 50 0 . 56 Res idual Mean Square 54 1 . 80 4 . 94 2 . 38 382 . 38 720 . 50 (Key , Semen Parameters : Fr . Cone . = fruc tose c onc entration of s emen ; Fr . Cont . = t ota l e j aculate fructose c ontent ; S . P . Fr . C one . = fructose c onc entrati on of s eminal plasma ; % Unstained = perc entage of uns tained spermatozoa ; ? % Normal = percentage of morphologically normal spermatozoa ) ? 1 88 l s ' - + ..c: N 2;' 1 4.40 0 ..... ? ? 1 2 .0 0 E 0 E-< 0 R ::t: p.. NORMAL 3 0 EVEN 2 0 REVERSED 6 5 4 3 2 r/ T ___ J J A s 0 N D MONTHS F i g u re 5 . 2 : Month l y va r i at i ons i n sem i na l p l a sma f ructose concen? t rat i ons ( mea n+S . E . ) i n semen co l l ected f rom rams subj ected to Norma l ( N ) , Even ( E ) or Reversed C R ) I i g h t i ng reg i mes . Da i l y p hotoper i od changes a re i I l ustrated a n d the t i m i ng of the eq u i nox ( E 1 ) a n d so l st i ce C S ' ) i nd i cated . 1 89 group had the lowest mean spermatozoal motility and percenta? of motile spermatozoa, while the Reversed group had a mean percenta? of motile spermatozoa intermediate between the other two groups ( Contrasts 1 and 2 ) . The most notable result was the e levation in seminal fructose levels recorded from the Reversed lighting ? group during the last two months of the experiment ( Contrasts 6 and 7) . Although the ra.II13 on Even lighting tended to display change s similar to the Normal lighting group for most semen characteristics, the ir seminal fructose levels fell over the latter part of the study (Contrast 7 ) . ( 2 ) LH and Testosterone - See Tables 5 .1 1 , 5 . 1 2 and Figures 5.3 and 5 .4. The period from March to June 1 973 was characterised by a decline in plasma levels of LH and testosterone . On the other hand, during the second half of the year there was little overall chan@S in plasma LH concentrations , while an increase in plasma testosterone content was almost entirely due to the result from the rams subje cted to Reversed lighting (Contrasts 5, 8 and 1 1 ) . These rams als o had _higher plasma LH levels in the final s ix months of the year than did the Normal lighting group (Contrast 1 0) . During October, the Even lighting group displayed a sharp peak in mean plasma LH concentrations ( Contrast 1 2 ) . ( 3 ) Prolactin See Tables 5.1 3 , 5 .1 4 and Figure 5.5. During the March-September period the Reversed lighting group were subjected to a summer lighting regime and displayed an e levation of plasma prolactin levels approximately in phase with the change in photoperiod. A s imilar e levation of prolactin levels , in phase with Sampl ing time 1 Peri od 1 2 3 4 Mean 5 6 Period 2 7 8 9 1 0 Mean Overal l Mean Tabl e 5 . 1 1 Mean plasma LH and testosterone c onc entrations rec orded from rams in Experiment 5 . (Va lues pre s ented are 1 00 l og1 0 ( X + 1 . 1 ) , where x is hormone c onc entration in ng/ml . ) Luteiniz ing Hormone Testosterone Lighting Regime Lighting Regime Normal Rev1!rs ed Even Mean Sampl ing Normal Reversed Even time 28 . 5 1 2 . 4 1 4 . 2 1 8 . 4 1 60 . 2 37 . 1 1 8 .7 4 . 1 6 . 5 1 1 . 9 7 . 5 Period 1 2 2 1 . 9 5 . 9 5 . 1 1 5 . 4 5 . 1 5 . 1 8 . 5 3 2 1 . 3 9 . 5 7 . 5 1 0 . 1 7 . 1 1 4 . 2 1 0 . 5 4 1 2 . 1 12 . 1 1 1 . 7 1 4 . 5 7 . 8 ? 1 1 ? 2 Mean 28 . 9 1 6 . 1 1 0 . 7 1 2 . 9 7 . 1 1 2 . 1 1 0 . 7 5 28 . 1 5 . 9 1 4 . 9 1 3 . 8 1 5 . 2 7 . 9 1 2 . 3 6 1 5 . 2 1 6 . 3 4 . 1 9 . 4 1 3 . 0 1 5 . 7 1 2 . 7 Per i od 2 7 5 . 1 5 1 . 7 27 . 2 7 . 5 1 5 . 8 3 4 . 2 1 9 . 2 8 1 7 . 1 63 . 3 3 2 . 4 8 . 7 1 2 . 5 8 . 6 9 . 9 9 20 . 2 68 . 5 8 . 2 1 6 . 5 20 . 4 1 3 . 7 1 6 . 9 1 0 30 . 5 77 . 7 2 1 . 8 .lL2 1 4 . 0 1 5 . 4 1 3 . 6 Mean 1 9 . 4 47 . 2 1 8 . 1 1 2 . 7 .!.l..!2. 1 3 . 8 Overal l Mean 23 . 2 34 . 8 1 5 . 2 Mean 3 8 . 7 1 1 ? 0 1 2 . 8 1 2 . o 1 8 . 6 1 6 . 3 1 1 ? 9 28 . 0 37 . 6 32 . 3 43 . 3 28 . 2 _. ? 0 Experiment 5 Source of Variati on MAIN EFFECTS A . LIGHTING REGIMES Normal vs Reversed Even vs Normal and Reversed B. TIME PERIODS Per iod 1 - Linear " " - Quadrati c Per i od 2 - Linear " " - Quadratic Per iods 1 vs 2 Non s ignificant c ontrasts INTERACTION (AxB ) Contras t 1 x Contrast 5 " " x Contras t 6 " " x Contrast 7 Contrast 2 x Contrast 5 1 1 11 x Contrast 6 Non s ignificant c ontrasts Res idual Mean Square Tab l e 5 . 1 2 Summary of Analyses of Var iance for LH and Testosterone Data . C ontrast No . .D . F . Var ianc e Rat ios LH Testosterone 2 1 1 0 . 38 5 . 7 1 * 2 1 0 . 84 1 1 . 07** 9 3 1 3 . 97* 8 . 1 1 ** 4 1 6 . 1 9* 4 . 78* 5 1 1 . 87 1 5 . 60*** 6 1 0 . 23 0 . 02 7 1 1 . 83 5 . 63* 2 0 . 42 0 . 5 5 1 8 8 1 1 ? 23 1 4 . 59*** 9 1 1 ? 1 2 4 . 47* 1 0 1 5 . 1 6* 1 7 . 2 1 *** 1 1 1 o . oo 4 . 3 5* 1 2 1 4 . 79* 0 . 53 9 1 ? 5 1 0 . 49 90 84 . 63 465 . 69 -? \0 __.. NORMAL I ?I EVEN I- -I- _..,_--I-0 2 ? 5 REVERSED MONTHS F i g u re 5 . 3 : Month l y va r i at i ons i n p l a sma LH concentrat i ons ( mea n +S . E . ) recor de d f rom rams s ubjected to Norma l ( N ) , Even ( E ) or Reversed ( R ) I i ght i ng reg i mes . Da i l y photoper i od changes a re i I l ustrated a n d the t i m i ng of the equ i noxes ( E 1 ) a n d so l st i ces ( S 1 ) i n d i cated . 1 93 E ' s' E' I s - ? ? ? ? ? .._, 1 4.40 N Cl 0 ..... ? w 1 2 .00 E p.. 0 E-< 0 :r:: 9.20 R p.. NORMAL 5 EVEN REVERSED I-V --I 0?--?-=?e:==?=-?----??r?--?--?--??? A M J J A s 0 N 0 MONTHS F i g u re 5 . 4 : Month l y va r i at i ons i n p l a sma testosterone concentrat? i on s ( mea n+S . E . } recorded f rom rams s ubjected to Norma l ( N ) , Even ( E ) or Reversed ( R ) l i g ht i ng reg i mes . Da i l y p hotoper i od cha nges a re i I l ustrate d and the t i m i ng of the eq u i noxes ( E 1 ) a n d so l st i ces ( S 1 ) i nd i cate d . Tab le 5 . 1 3 Mean plasma pro lactin c oncentrations rec orded from rams in Experiment 5 . (Values presented are 1 00 log1 0 (x + 1 . 1 ) , where x is hormone c oncentration in ng/ml . ) Lighting Regime Sampl ing Normal Reversed Even Mean t ime 1 60 . 0 98 . 4 1 05 . 8 88 . 1 2 8 1 ? 1 1 40 . 1 90 . 3 1 03 . 8 Period 1 3 1 1 4 . 1 1 5 1 . 6 1 29 . 3 1 3 1 ? 7 4 1 1 8 . 7 1 8 1 . 4 1 34 . 7 1 44 . 9 5 1 1 3 . 6 1 75 . 0 1 29 . 5 1 3 9 . 4 6 1 22 . 7 1 26 . 7 1 42 . 2 1 30 . 5 Mean 1 0 1 ? 7 1 4 5 . 5 1 22 . 0 1 23 . 1 7 1 57 . 1 1 1 2 . 0 1 57 . o 1 42 . 0 Period 2 8 1 54 . 4 93 . 5 1 44 . 2 1 30 . 7 9 1 67 . 4 1 04 . 3 1 24 . 4 1 32 . 0 Mean 1 59 . 6 1 03 . 3 1 4 1 ? 9 1 34 . 9 Overal l Mean 1 2 1 . o 1 3 1 . 4 1 28 . 6 ? \!) -+='" Experiment 5 Source of Variati on MAIN EFFECTS A . LIGHTING REGIMES Normal vs Reversed Even vs Normal and Reversed B . TIME PERIODS Period 1 - Linear " " .:. Quadrati c Periods 1 vs 2 Non s ignifi cant c ontrasts INTERACTION (AxB ) Contrast 1 x Contrast 5 Contrast 2 x Contr?st 4 Non s ignif icant c ontrasts Res idual Mean Square Tabl e 5 . 1 4 Summary of Analys i s of Var iance for Prolactin Data . Contrast No . 1 2 3 4 5 6 7 D . F . 2 1 8 t 1 1 1 3 1 6 1 1 1 0 8 1 Variance Ratio 2 . 84 0 . 29 27 . 9 1 *** 1 3 . 84*** 4 . 98* 0 . 73 5 9 . 67*** 5 . 1 0* 0 . 86 670 . 58 ? \.!) \J1 - .s:::: '-' ? 1 4.40 - 1:1:: ? ? 1 2.00 E-< 0 s: 9.20 50 2 5 75 50 2 5 0 S ' t I- A M J 1 96 E ' t N E R EVEN REVERSED il:. J s N 0 MONTHS F i g u re 5 . 5 : Month l y va r i at i ons i n p l asma p ro l act i n concentrat i ons ( mean+S . E . ) recorded f rom rams s u bjected to Norma l ( N ) , Even ( E ) or Reversed ( R ) I i g ht i ng reg i mes . Da i l y p hotoper i od cha nges a re i 1 1 - ? ustrated a n d the t i m i ng of the eq u i nox ( E 1 ) a n d so l st i ces ( S ' ) i n d i cated . 1 97 increasing photoperiod, was recorded from the rams on the Normal lighting regime during the Oct ober-December period. The above two results, in which plasma prolactin c oncentration increased with le ngthening photoperiod, e ntirely acc ounted for th? very highly s ignificant c omponent (Contras t 6 ) of the Lighting Regimes x Periods interaction. Compared t o the range of fluctuations in plasma prolactin concentrations recorded from the rams on the Normal and Reversed lighting regimes , o nly relatively minor changes were recorded from those on the Even lighting regime . 4. DISCUSSION The results obtained in this experiment indicated that, in the absence of changes in other e nvironmental factors , alterations in lighting influenced neuroendocrine mechanisms in N. Z . Romney rams . This finding supported the hypothesis that changes in daily photoperiod were the major s timulus for seasonal changes in semen characteristics and plasma hormone levels in rams at pasture. Plasma LH c oncentrations recorded in this experiment were s o low that detection of s ignificant differences betwee n treatment groups was unlikely. This failure to s how a? e ffect of photoperiod on plasma LH levels , similar t o those reported by Felletier and Ortavant ( 1975a) , - large? can be accounted for by the differences in concentrations of this hormone obtained in the two laboratories . While the mean levels for plasma LH from rams in the French study ranged from 1 .5 t o 4.0 nf!/' ml, those in the present experiment rare? exceeded 0.5 nf!/'ml. Bioass? comparis ons have indicated that this differenc? probab? c ould not be attributed to the different standards used in each assay : the LH-M1 standard use d by Felletier and Ortavant had 1 .5 times the biological act ivity of 1?H-LH-S1 , whereas the NIH-LH-S1 8 used in this laboratory was equivale nt to 1 .03 time s the NIH-lli-S1 standard. Nevertheless , it is possible that a di?ference in immunore activity of the assay standards , which was not reflected as a difference in biological activity, or s ome other dif?erence between the assays , accounted for the different levels of lJ{ recorded in this and the French work. Alternatively, the low lJ{ le?;els of the N. Z. Romney rams could have reflected e ither a breed difference , or there may have been a diurnal pattern of secretion, s o that low leve ls of LH always were recorded from blood samples c ol le cted at 1 0. 00 h ( however, see C hapter VII) . Although there was little evide nce for aqy se as onal cha? in plasma LH levels , the rise in plasma testosterone concentration rec orded during the final four months of the experirrent from the rams on the Reversed lighting regime , demonstrated that photoperiodic influences could alter gonadal funct ion. Since changes in testosterone production would be expected to refle ct altered LH secretion, the difference in pattern of results for these two hormones in Experiment 5 was surprising. This disparity may have re flected deficiencies in the LH ass?, or it was possible that t he frequency of pulses of LH output had altered, without necessarily changing mean plasma leve ls at the particular t ime of the day when sample s were collected (Katongole , Na?olin and Short , 1 974) . The photoperiodic i?luence on gonadal testosteror? production was confirmed by the simultane ous increase in seminal fructose levels and in e jaculate volumes rec orded from the Reversed lighting group. Only one previous report has indicated that seas onal changes in seminal fructose levels c ould be s imulated by manipulating the photo? period (Jacks on and Williams, 1 973 ) . In an Australian study no seas onal pattern of seminal fructose leve ls was observed from rams on normal or reversed lighting regimes (Moule , Braden and Mattner, 1 966 ) . 1 99 This latter result caused the authors to c onclude that seas onal changes in seminal fructose levels in graz ing rams wero attributable to nutritional factors ; in the p1?sent work, the diet was of constant composition throughout the experiment . However, ur.der some extensive Australian grazing conditions, nutrition may have an influe nce on seminal fructose levels . For rams on Reversed lighting the de l? between the longest daily photoperiod and the increase in plasma testosterore levels was longer than that recorded from rams at pasture (Experiment 3 ) . Jacks on and Williams ( 1 973 ) indicated that a trans ient phase occurs during adaptation of rams t o an imposed light rhythm, so that gonadal responses do not bec ome n in phase" with the new rhythm until after a few photoperiodic cycles . Presumably such an adaptation period is required for establishment of new " in phase" rhythms of LH secretion, and results from refract oriness at pituitary, hypothalamic, or other leve ls :i.n the central nervous system. Other studies on ram semen have shown that photoperiod reversal can reverse the seasonal changes in parameters related t o spermatogenesis ( Ortavant, 1 956 ; Ortavant and Thibault, 1 956 ; Fowler, 1 961 ; Colas et al. , 1 972 ; Jackson and Williams , 1 973 ) ; no such response for these parameters was seen in the present stuqy nor in seroon f'rom grazing N. Z. Romney r?!3 (Experiment 3 ) . This apparent conflict of results could have resulted from breed dif'ferences . Alternatively, it may indicate that spermato?nic responses to photoperiodic rnythms are significant o? when using rhythms with greater (8 h ) amplitude , and a shorter ( 6 months ) cycle length, than that used in the current experiment. Results of experiments on the e ffects of different liehting regimes on epidiqymal spermatozoal reserves ( Ortavant, Yaule on and Thibault , 1 964 ) led to the authors making two important c onclus ions 200 firstly, the e ffects of lighting reversal were more pronounced with a six-monthly cycle than with the natural lightine changes , and secondly, the e ffects were more pronounced in the second cycle than in the first . In Experiment 5 the natural changes in photoperiod which occur at this locality were simulated, thus the results of this experiment were more comparable to those obtained from rams at pasture in Experiments 3 and 4, than to findings from many of the studies reported in the literature. One point of departure from natural d?light changes was the absence of allowance for twilight in the present experiment. At Palmerston North twilight provided grazing animals with a slightly variable period of additio1?l lighting throu?1out the year (about 30 minutes per day ) , but the amplitude of the annual photoperiodic cycle was not significantly different from that used in this experiment ( civil twilight = 32 minutes midsummer and 26 minutes midwinter, N. Z . Aeronautical Information Publication, Ministry of Transport ) . Thus , at this latitude twilight would not account for differences between results rec orded from grazing rams and from those housed indoors . However at higher latitudes twilight becomes progressively longer and has a greater seas onal variation so that it could represent a significant factor in the annual photoperiodic c hanges. The second point rBised by Ortavant, Mauleon and Thibault ( 1 964) exposes the major limitation of the present experiment ; this was the fact that the experiment did not continue for more than one cycle of lighting changes. At the design stages of this preliminary experiment, it was not envisaged that light rhythm reversal would take s o long to produce its effects . Consequently the experiment was c oncluded at the December solstice . It is now obvious that extension of the experiment for a few months may have shown whether the elevated plasma testosterone and seminal fructose levels in the Reversed liv1ting group did in fact decline , while those for the Normal lighting group continued to rise . Furthermore , if the experiment had been continued for a year or two, it is posnible that data for other semen characteristics and LH may have shown seas onal changes in phase with the photoperiodic cycle . 201 No s ignificant changes in plasma testosterone or seminal fructose c oncentrations were re corded from the rams on Eve n lighting indicating that this group resembled the Normal lighting eroup in its seas onal pattern. . This result was consistent with the c onclus ion of Ortavant ( 1 961 ) , who claimed that although ten to twelve hours of daylight represented the optimum for reproduction in rams , this only applied when daily photoperiod was being progressive? decreased. Rams on Normal lighting did not display major changes in semen production, or plasma LH or testosterone levels during the course of this study. As s imilar results were rec orded from rams on Even lighting, no conclusion could be reached regarding the existence of res idual reproductive rhythms in the latte r group. Res idual rhythms of semen production by rams were reported by Jackson and Willia!D3 ( 1 973 ) , but more than one photoperiodic cycle was requ ired t o demonstrate their existence . Even with the short time-course of this experiment it was possible t o reach definite conc lusions about prolactin se cretion in rams : all groups of rams showed changes in plasma prolactin levels which were in phase with the lighting regimes . Thus this experiment confirmed the finding of Felletier ( 1 973 ) that prolactin secretion in ra.Ill3 was directly related t o the length of the photoperiod. Also, the relatively c or?tant plasma prolactin levels of rams on Even lighting suggested that there was no inherent seasonal rhythm of prolactin release. 202 In some species , it has bee n shown that the pinnal gland has an important role in the regulation of hormone release by lighting changes (Reiter, 1 974a ) . It remains to be established whether the seasonal prolactin responses to changing photoperiods are regulated by the pineal gland in the ovine species , and in turn, whether the changes in prolactin secretion have a? s ignificant dire ct or indirect role in re gulating ovine reproductive seas onality. 203 C H.API'SR VI EFFECTS OF PINEALECTOMY ON SEMSN FRODUCTION AND PLASMA HORMO? LEVELS IN RAMS SUBJECTED TO CONI'RASTING LIGHI'If-.TG REGIMES 1 . INTRODUCTION Experiment 6 was undertake n to study the effects of pineal? ectoii\Y on seme n production and plasma hormone leve ls in rams . In Experiment 4, cranial cervical ganglionectoii\Y produced chan@3s in a number. of parameters ass ociated with reproductive activity, and als o altered pineal gland function. This result indicated that the pineal gland may have an important role in inducing seasonality of reproduction in rams . Thus it was necessary to remove the pineal gland t o allow dire ct investigation of this possibility. Reiter ( 1 973?) noted that pinealectomy was a notoriously unreliable means of demonstrating pineal gland influe nces on the reproductive system. He suggested that ofte n this wa;:s due to the use of inappropriate lighting regimes , which by virtue of their exce s sive photoperiod le ngths , effective ly " pinealectomized" experimental animals whether they had bee n surgically pinealectomized or not . The present experiment was des igned t o overc ome this shortcoming both pinealectomized and c ontrol rams were submitted to normal or reversed lighting regimes, in an attempt to evaluate the poss ible role of the pineal gland in mediating the reproductive responses t o photoperiod reversal, which were recorded in Chapter V. 2 . MATERIALS AND METHODS ( 1 ) Animals and Experimental Procedure Sixteen adult N.Z . Romney rams were allocated at random to the 204 pinealecto? ( PX) or sham-operation (Controls ) s urgical treatme nts des cribed in Chapter II. Surgery was carried out between six and ore weeks immediate? prior to the c ommencement of the experime nt , by which tire all rams appeared to have rec overed from surgery and were in good health. Post-mortem macroscopic examination of formalin-fixed brains verified the completeness of the pinealecto? operations, but histological examination of paramedian brain sections revealed minute remnants of pineal s talk in a few pinealectomized rams . These remnants were not c onsidered to be of s ufficient importance to warrant exclusion of the data obtained from a? of the rams c oncerned. Brains from s ham-operated rams were examined in a like manner and appeared normal in every respe ct . See Figures 6 . 1 and 6 . 2 . Commencing at the September e quinox 1 974, one light-c ontrolled room ( containing e ight rams : four pinealectomized and four sham? operated) was submitted to the normal seas onal rhythm of photoperiodic chan? (Normal) , whilst another room ( also containing four pineale ct? omized and four s ham-operated rams ) was subjected to a reversed pattern of photoperiodic changes (Reversed) . The amplitude of the photoperiodic cycle was identical to that used in Experiment 5 , with a minimum photoperiod of 9 hours and 20 minutes and a maximum of 1 4 hours and 40 minutes . These lighting treatments were continued until the e nd of the experiment in May 1 975. (2 ) Statistical Analyses (a ) Semen and Hormone Data. For each semen parameter, data were pooled to provide a monthly mean estimate for each ram. The e ight complete months of the experiment were divided into two periods , demarcated by the December solstice , as shown in Figure 6 .3 . Orthogonal coefficie nts used t o partition time period e ffects for semen data are F i g u re 6 . 1 F i g u re 6 . 2 M i dsa g i tta l sect i on of b ra i n f rom sham-operated ram s how i ng i ntact p i nea l g l a n d ( p ) . So l och rome cya n i n a n d c resy l ? f a st v i o l et sta i n . M i dsag i tta l sect i on o f b ra i n f rom p i nea l ectom i zed ram s how i ng remnant of p i nea l sta l k ( r ) . So l och rome cyan i n a nd cresy l fa st v i o l et sta i n . ( Note : I n F i g u res 6 . 1 a n d 6 . 2 t he rostra ! end of the b ra i n i s to the l eft of each f i gu re . ) 205 EQUINOX PHOTOPERIOD (h ) PERIOD l SOLSTICE EQUINOX PERIOD 2 PERIOD 3 HORMONES l 2 3 4 5 6 7 8 9 10 l l ?1 2 l 3 14 15 16 1 7 lR PERIOD l SEMEN l 2 MONTHS s 0 N KEY : R - Reversed N - Normal 1974 3 D PERIOD 2 4 5 6 7 8 J F M A M 1975 F i g u re 6 . 3 : D i ag ram s how i ng the cyc l i c changes i n da i l y p hotoper i od for the I i g ht i ng reg i mes used i n Exper i ment 6 , and the s u b d i v i s i on of t he t i me-course of the exper i ment for data a na l yses . 206 Table 6 . 1 Orthogonal c oeff i c i ents used in partitioning t ime per iod effects f or s emen data in Experiment 6 . Sampl ing t ime Contrast Per iod 1 - Linear 1 1 1 1 Quadratic Per i od 2 - Linear 1 1 1 1 1 1 - Quadratic 1 1 - Cubic Period 1 vs Period 2 Peri od 1 - 1 2 -1 0 +1 -2 0 0 0 0 0 0 +5 +5 Period 2 3 4 5 6 7 +1 0 0 0 0 +1 0 0 0 0 0 -2 -1 0 +1 0 +2 -1 -2 -1 0 -1 +2 0 -2 +5 -3 -3 -3 -3 8 0 0 +2 +1 +1 -3 f\) 0 ? Tabl e 6 . 2 Orthogonal c oeffic ients used in partiti oning time period effects for plasma hormone data in Experiment 6 . Per i od 1 Per i od 2 Period 3 Sampl ing t ime - 1 2 3 4 5 6 7 8 9 1 0 1 1 1 2 1 3 1 4 1 5 1 6 1 7 Contrast Period 1 - Linear -3 -2 -1 0 +1 +2 +3 0 0 0 0 0 0 0 0 0 0 11 11 - Quadrati c +5 0 -3 -4 -3 0 +5 0 0 0 0 0 0 0 0 0 0 " 11 - Cubic - 1 +1 +1 0 -1 -1 +1 0 0 0 0 0 0 0 0 0 0 Period 2 - Linear 0 0 0 0 0 0 0 -3 -2 -1 0 +1 +2 +3 0 0 0 11 11 - Quadratic 0 0 0 0 0 0 0 +5 0 -3 -4 -3 0 +5 0 0 0 11 11 - Cubic 0 0 0 0 0 0 0 -1 +1 +1 0 -1 -1 +1 0 0 0 Period 3 - Linear 0 0 0 0 0 0 0 0 0 0 0 0 0 0 -3 -1 +1 1 1 11 - Quadratic 0 0 0 0 0 0 0 0 0 0 0 0 0 0 +1 -1 -1 11 11 Cubic 0 0 0 0 0 0 0 0 0 0 0 0 0 0 -1 +3 -3 Peri od 1 vs Per iod 2 +1 +1 +1 + 1 +1 +1 +1 -1 -1 -1 -1 -1 -1 -1 0 0 0 Period 3 vs Periods 1 & 2 -2 -2 -2 -2 -2 -2 -2 -2 -2 -2 -2 -2 -2 -2 +7 +7 +7 1 8 0 0 0 0 0 0 +3 +1 +1 0 +7 f\J 0 CO 209 shown in Table 6 .1 . For each hormone the 1 8 two-weekly estimates of plasma hormooo c oncentrations were grouped into three period.s , demarcated by the December s olstice and March e quinox, as shown in Figure 6 . 3 . Orthogonal coefficients used to partition time period effects for hormone data are s hown in Table 6 .2 . Results from Experiment 5 indicated that these orthogonal c ontrasts should have provided the most meaningful evaluation of photoperiodic e ffects . (b ) Treatment Contrasts . .Treatment effects of lighting regimes , operations , and their interaction were examined using the orthogonal coefficients shown below. Contrast Normal Lighti? Controls Pinealectomized Reversed _&i&hti? C ontrols Pinealectomized Main Effects Lighting Regimes Oper.::?.tions Interaction Lighting Regime s x +1 +1 Operations ?1-'i 3 . RESULTS ( 1 ) Se ?re n Data +1 -1 -1 -1 +1 -1 See Tables 6 .3 to 6 .9 and Figures 6 .4 to 6 .7 . -1 -1 +1 Semen from sham-operated rams had higher values for spermatoz oal motility and percentage of motile spermatozoa than sexoo n from pineal- e et omize d rams . Als o pinealectomized rams on Reversed lighting produced e jaculates with low motility indices and low . peroenta?s of motile spermatozoa during the final five months of the experiment (Contrasts 1 , 2 , 3 , 4 and 6 ) . In the period from the December solstice till the end of the experiment , mean e jaculate volumes tended to increase (C ontrast 3) and were higher than in the previous three months ; 2 1 0 Tab l e 6 . 3 Mean moti l ity i nd i c es ( s ca l e 0-4 ) and mean pe r c entages o f mot i l e s pe rmatozoa r e c o rded from e jacu lates c o l l e c ted in Experiment 6 . Moti l ity index Normal Lighting Revers ed Light ing Sampl ing C ontrols PX Controls PX Mean time 1 2 . 9 2 . 8 3 . 0 2 . 9 2 . 9 Period 1 2 3 . 1 3 . 0 3 . 1 3 . 2 3 . 1 3 3 . 1 2 . 8 3 . 3 2 . 7 2 . 9 Mean 3 . 0 2 . 9 3 . 1 2 . 9 3 . o 4 3 . 0 3 . 0 2 . 9 2 . 4 2 . 8 5 2 . 9 3 . 2 2 . 5 2 . 3 2 . 7 Per i od 2 6 3 . 6 2 . 8 3 . 1 2 . 7 3 . 0 7 3 . 0 2 . 9 3 . 0 2 . 0 2 . 7 8 2 . 6 2 . 9 2 . 7 2 . 1 2 . 5 Mean 3 . 0 3 . 0 2 . 8 2 . 3 2 . 7 Overal l Mean 3 . 0 2 . 9 3 . 0 2 . 5 % Mot i l e Spermatozoa Normal Lighting Reversed Lighting Sampl ing Contro ls PX Controls PX Mean t ime 1 58 . 3 5 5 .6 5 8 . 3 57 . 5 5 7 . 4 Per i od 1 . 2 63 . 8 60 . 6 63 . 8 59 . 4 6 1 . 9 3 60 . 6 57 . 5 65 . 0 5 1 ? 9 58 .7 Mean 60 . 9 57 . 9 62 . 4 56 . 3 5 9 . 3 4 59 . 4 58 . 1 60 . 0 50 . 6 57 .0 5 5 3 . 8 59 . 4 5 2 . 6 45 . ? 1 52 .7 Per i od 2 6 67 . 5 54 .4 58 . 1 44 . 4 56 . 1 7 6 1 . 3 54 .4 56 . 3 4 1 . 9 5 3 . 4 8 5 0 . 0 52 . 5 46 ? 9 3 8 . 8 47 . 0 Mean 58 . 3 5 5 . 8 5 4 . 8 44 . 2 5 3 . 2 Overall Mean 59 . 3 56 . 6 57 . 6 48 .7 ( Key : Controls = Sham-operated ; PX = Pinealectomized ) 2 1 1 Tab l e 6 . 4 Mean e jaculate volumes and mean total fructo s e contents o f ejaculates c o l lected in Exper iment 6 . E jaculate vo lume ( m l ) Normal Lighting Reversed Lighting Sampl ing Contro l s PX C ontro l s PX time 1 1 . 45 1 . 24 1 ? 41 1 . 42 Per i od 1 2 1 . 1 1 1 . 04 0 . 95 1 . 1 1 3 1 . 08 1 ? 1 6 1 . 43 1 . 33 Mean 1 . 2 1 1 ? 1 4 1 ? 26 1 . 28 4 1 ? 1 4 1 . 73 0 . 95 1 . 46 5 1 . 1 9 1 . 66 1 . 30 2 . 06 Per i od 2 6 1 . 03 2 . 03 1 ? 96 1 . 88 7 1 . 09 2 . 60 1 . 70 1 . 89 8 1 . 50 2 . 20 1 . 8 1 1 . 73 Mean ? 2 . 04 1 ? 54 1 . 80 Overal l Mean ? 1 . 70 ., . 43 1 . 6 1 Tota l e jaculate fruc t o s e c ontent ( mg ) Norma l Light ing Reversed Lighting Sampl ing Contro l s PX Controls PX time 1 0 . 67 1 . 1 4 0 . 47 1 . 69 Period 2 0 . 64 0 . 7 5 0 . 47 1 . 32 3 0 . 93 0 . 9 5 3 . 1 9 0 . 8 5 Mean 0 . 75 0 . 94 1 . 38 1 ? 29 4 1 . 87 1 . 83 3 . 4 1 1 . 1 4 5 2 . 34 3 . 02 5 . 76 5 . 23 Per i od 2 6 2 . 4 1 5 . 73 6 . 50 9 . 00 7 3 . 64 7 . 39 3 . 08 8 . 57 8 8 . 9 1 1 o . 77 2 . 1 4 6 . 07 Mean 3 . 84 5 .7 5 4 . 1 8 6 . 00 Overal l Mean 2 . 67 3 . 94 3 . 1 2 4 . 23 (Key : Control s = Sham-operated ; PX = Pinea lectomized ) He an 1 . 38 1 . 05 1 . 2 5 1 ? 2 2 1 . 3 2 1 . 5 5 1 ? 72 1 . 82 1 ? 81 1 . 64 Mean 0 . 99 0 . 80 1 . 48 1 . 09 2 . 06 4 . 08 5 . 9 1 5 . 67 6 . 97 4 . 94 2 1 2 Tab l e 6 . 5 Mean c oncentrat ions o f fruct o s e in s emen n.nd in s emina l plasma of e j aculates c o l l e c ted in Exper iment 6 . S emina l fructos e concentrat i on ( mg/ml ) Normal Lighting Reversed Light ing Sampl ing Contro l s p? Contro l s PX Mean t ime 1 0 . 48 1 . 0 5 0 . 37 1 . 1 8 0 . 77 Per i od 1 2 0 . 47 o . 11? 0 . 48 1 ? 01 0 . 68 3 0 . 87 0 . 7 5 1 . 39 0 . 76 0 . 94 Mean 0 . 6 1 0 . 86 0 . 75 0 . 98 0 . 80 4 1 . 1 2 0 . 90 1 . 9 5 1 . 08 1 . 26 5 1 . 38 2 . 04 3 . 59 2 . 72 2 . 43 Per i od 2 6 2 . 1 7 2 . 7 1 2 . 32 4 . 3 1 2 . 88 7 2 . 84 2 . 64 2 . 1 6 4 . 3 1 2 . 9 9 8 5 . 76 4 . 62 1 . 07 3 . 90 3 . 84 Mean 2 . 65 2 . 58 2 . 22 2 . 26 2 . 68 Overal l Mean 1 . 88 1 . 93 1 . 66 2 . 40 Seminal plasma fructose c onc entrat i on ( mg/ml ) 'Norma l Lighting Reversed Lighti ng Sampl ing C ontrols PX Controls PX Mean t ime 1 0 . 62 1 . 37 0 . 46 1 . 3 8 0 . 96 Per iod 1 2 0 . 62 0 . 9 5 o . 5 1 1 . 23 0 . 83 3 1 . 1 7 0 . 9 4 1 . 72 0 . 87 1 . 1 8 Mean 0 . 8 1 1 .09 0 . 9 0 1 . 1 6 0 . 99 4 1 .42 1 . 1 5 2 . 27 1 . 23 1 . 52 5 1 . 85 2 . 64 4 . 3 5 ' 3 . 28 3 . 03 Per i od 2 6 2 . 63 3 . 3 2 3 . 37 4 . 90 3 . 5 6 7 3 . 1 5 3 . 48 2 . 5 3 5 . 0 1 3 . 5 4 8 6 .7 1 5 . 49 1 . 40 4 . 27 4 . 47 Mean 3 . 1 5 3 . 22 2 . 78 3 . 74 3 . 22 Overa l l Mean 2 , 27 2 . 4 1 2 . 07 2 . 77 (Key : Contro ls = Sham-operated ; PX = Pinea lec tomi zed ) Tab l e 6 . 6 Mean c onc entrat i ons of s permatozoa/m! and mean numbers of spermatozoa/e ja?ulate in s emen c o l le c ted in Experiment 6 . Spermatozoa/m! ( x 1 09 ) Normal Lighting Rever s ed Light ing Samp l i ng Controls PX Controls PX t ime 1 3 . 80 2 . 9 5 5 . 04 3 . 28 Per i od 1 2 3 . 25 2 . 60 3 . 2 2 3 . 1 1 3 3 . 48 3 . 20 4 . 1 0 2 . 7 1 Mean 3 . 5 1 2 . 92 4 . 1 2 3 . 03 4 3 . 42 3 . 3 6 3 . 27 3 . 32 5 3 . 1 0 3 . 69 3 . 06 2 . 9 1 Per i od 2 6 2 . 85 2 . 84 4 . 48 2 . 33 7 2 . 47 3 . 4 1 3 . 88 2 . 39 8 2 . 3 1 2 . 5 5 4 . 69 2 . 0 1 Mean 2 . 83 3 . 1 7 3 . 88 2 . 59 Overa l l Mean 3 . 08 3 . 07 3 . 96 2 . 75 Spermatoz oa/ejaculate ( x1 09 ) Normal Lighting Rever s ed Lighting Sampling Controls PX Contro ls PX t ime 1 6 . 1 6 4 . 3 1 7 . 01 4 . 96 Per i od 1 2 3 . 93 2 . 57 3 . 05 3 . 69 3 4 . 54 3 .73 5 . 9 5 4 . 03 Mean 4 . 88 3 . 53 5 . 34 4 . 22 4 3 . 9 5 5 . 9 5 3 . 26 5 . 2 5 5 4 . 20 6 . 22 4 . 5 6 5 . 92 Per i od 2 6 3 .6 1 6 . 60 1 0 . 05 4 . 6 1 7 3 . 49 9 . 66 6 . 9 1 5 . 05 8 3 . 63 6 .. 00 8 . 66 3 . 75 Mean 3 .77 6 . 88 6 . 69 4 . 92 Overa l l Mean 4 . 1 8 5 . 62 6 . 1 8 4 . 65 ( Key : Contro ls = Sham-operated ; PX = Pineal ectomi z ed ) 21 3 -Mean 3 . 77 3 . 04 3 . 37 3 . 39 3 . 34 3 . 1 9 3 . 1 3 3 . 04 2 . 89 3 . 1 2 He an 5 .6 1 3 . 3 1 4 . 5 6 4 . 49 4 . 60 5 . 2 2 6 . 22 6 . 28 5 . 5 1 5 . 57 2 1 4 Tab l e 6 . 7 Mean perc entages of uns tained and morpho l ogica l ly normal spermatozoa in s emen c o l l e c ted in Experiment 6 . % Uns tained spermatozoa Normal Lighting Reversed Lighting Sampl ing Contro ls PX Contro ls PX Mean t ime 1 62 . 6 58 . 8 5 3 . 0 56 . 8 57 . 8 Per i od 1 2 65 . 9 64 .0' 60 .4 58 . 5 62 . 2 3 67 . 9 72 . 3 67 . 5 59 . 6 66 . 8 Mean 65 . 5 65 . 0 60 . 3 58 . 3 62 . 3 4 64 . 3 75 . 5 62 . 5 64 . 5 66 .7 5 70. 3 79 . 1 70 . 6 73 . 4 73 . 3 Per i od 2 6 78 . 9 79 . 6 65 . 1 71 . 3 73 . 7 7 77 . 0 76 . 4 63 . 6 63 . 0 70 . 0 8 7 1 . 0 72 . 6 62 . 5 66 . 9 68 . 2 Mean 72 '. 3 76 . 6 64 . 9 67 .8 70 . 4 Overall Mean 69 . 7 72 . 3 63 . 2 64 . 2 % Morpho logica l ly normal -? s permatozoa Normal Lighting Reversed Light ing Sampl ing Control s PX Contro l s PX Nea!l t ime 1 82 . 0 68 . 9 70 .7 71 . 5 73 . 3 Period ? 1 2 73 . 3 76 . 4 72 . 5 78 . 1 75 . 1 3 77 . 3 74 . 5 69 . 3 ' 73 . 4 73 . 6 Mean 77 . 5 73 . 3 70 . 8 74 . 3 74 .0 4 75 . 6 69 . 4 67 . 9 70 . 2 70 . 8 5 80 . 1 75 . 8 68 . 3 60 . 3 71 . 1 Per iod 2 6 77 . 6 6 1 . 6 72 . 9 6 5 . 9 69 . 5 7 77 . 0 6 5 . 5 72 . 9 67 . 6 70 .7 8 8 1 . 4 77 . 0 68 . 9 7 1 . 5 74 . 7 Mean 78 . 3 69 . 9 70 . 2 67 . 1 71 . 4 Overal l Mean 78 . 0 7 1 . 1 70 . 4 6 9 . 9 ( Key : Contro l s = Sham-operated ; PX = Pinealectomized ) Source of Variat i on MAIN EFFECTS A . LIGHTING REGIMES B . OPERATIONS C . TIME PERIODS Per i od 2 - Linear Per i ods 1 vs 2 Non s ignificant c ontrasts INTERACTIONS A X B A x C A X ( Periods 1 vs 2 ) Non s ignificant c ontrasts B X c B x ( Peri ods 1 ? 2 ) Non s ignificant c ontrasts A X B X c A x B x Per iod 2 - Linear A x B x ( Periods 1 ? 2 ) Non s ignificant c ontrasts Res idual Mean Square Table 6 . 8 Experiment 6 : Summary of Analyse s of Variance for Semen Data . Contrast No . 1 2 3 4 5 6 7 8 9 D . F . 7 7 7 4 1 1 5 1 5 7 1 4 96 Vo lume 0 . 48 1 0 . 84** 7 . 3 3** 1 5 . 3 8*** 0 . 89 2 . 72 0 . 02 0 . 68 7 . 41 ** 0 . 24 2 . 72 2 . 6 1 0 . 66 0 . 34 Mot i l i ty 3 . 56 5 . 3 8* 0 . 97 3 . 1 3 0 . 60 2 . 07 4 . 67* 0 . 05 0 . 2 9 0 . 65 0 . 43 0 . 69 0 . 98 0 . 42 Variance Rat i os % Moti l e 5 . 1 4* 7 . 66** 4 . 1 7* 7 . 80** 0 . 87 2 . 1 3 2 . 9 5 0 . 1 4 0 . 23 0 . 42 o . oo 0 . 3 1 0 . 1 9 1 42 . 80 Sperm . /ml 1 . 70 8 . 09** 1 . 2 1 1 . 56 0 . 71 7 . 76** 0 . 08 0 . 5 5 0 . 69 0 . 70 4 . 07* 1 . 63 0 . 3 1 1 . 48 Sperm . /ejac . 0 . 84 0 . 01 1 . 33 3 . 48 1 . 5 2 7 . 1 0** 0 . 0 1 0 . 26 2 . 73 0 . 56 4 .85* 4 . 9 5* 0 . 48 9 . 9 1 ( Key , Semen Parameters : Vo lume = e j acu:Late vo lume ; Moti l ity = mot i l ity index ; % Moti le = perc entage of mot i le spermatozoa ; Sperm ./ml = c oncentrati on of spermatozoa/ml ; Sperm . /ejac . = number of spermatozoa/e j aculate ) !\) ? \Jl Tabl e 6 . 9 Exper iment 6 : Summary of Analyses of Var ianc e for Semen Data . S ourc e of Variat i on MAIN EFFECTS A . LIGHTING REGIMES B . OPERATIONS C . TIME PERIODS Per i od 1 - Linear Per i od 2 - Linear 11 " - Quadrati c Per iods 1 vs 2 Non s ignificant c ontrasts INTERACTIONS A X B A x C A x Per i od 2 - Linear 11 11 1 1 - Quadrati c Non s ignifi cant c ontrasts B X c Non s ignificant c ontrasts A X B X c A x B x Per iod 2 - Linear Non s ignifi cant c ontrasts Res idual Mean Square Contras t No . 1 2 3 4 5 6 7 8 9 1 0 :O . F . 1 7 1 1 7 1 2 1 1 4 7 6 7 1 5 96 Fr . Cone . 0 . 1 3 1 ? 22 0 . 06 1 2 . 70*** 0 . 26 2 5 . 86*** 0 . 7 1 0 . 94 5 . 3 1 * 4 . 58* 0 . 05 0 . 46 4 . 2 1 * 0 . 2 1 4 . 1 0 Fr . Cont . 0 . 22 2 . 34 0 . 1 0 1 0 . 8 5** 0 . 73 23 . 09*** 0 . 90 0 . 01 4 . 54* 6 . 63* 0 . 04 1 . 06 0 . 67 0 . 88 1 9 . 24 Var ianc e Ratios S .P . Fr . Cone . 0 . 03 0 . 98 0 . 07 1 1 . 44** 0 . 5 8 26 . 05*** 0 . 70 0 . 42 5 . 0 1 * 4 . 89* 0 . 05 o . 59 3 . 27 0 . 1 8 5 .75 % Unstained 1 2 . 89*** 0 . 69 5 . 1 8* 0 . 02 4 . 57?* 1 5 . 2 3*** 0 . 57 0 . 08 0 . 73 0 . 46 0 . 3 5 0 . 36 0 . 46 0 . 34 1 2 5 . 62 % Normal 4 . 70* 3 . 29 o . oo 0 . 66 0 . 86 1 ? 52 1 ? 25 2 . 34 0 . 0 1 0 . 20 0 .97 0 . 92 0 . 03 0 . 1 9 1 36 . 08 ( Key , Semen Parameters : Fr . Cone . = fructose c onc entrati on of s emen ; Fr . Cont . = total e jaculate fruc tos e c ontent ; S . P . Fr . Cone . = fructose c onc entration of s emina l plasma ; % Unstained = perc entage of unstained spermatozoa ; % Normal = perc entage of morphological ly normal spermatozoa ) 1\) ? ? -0 1 4.40 0 0 C2 ? 1 2 . 0 0 ? 0 E-< g 9. 2 0 ? 8 6 ,-.. 0\ 0 :>< 2 '-' D-1 ? < .....l :::> u < .... D-1 10 ---< 0 N 0 8 ? ? ? D-1 6 ? r/l 4 2 E s E + + + SHAM-OPERATED PINEALECTO MIZED r?/r/r I MONTHS F i g u re 6 . 4 : Month l y va r i at i ons i n n umbers of s permatoza/ej acu l ate ( mea n?S . E . ) i n semen co l l ected f rom sham-ope rated a n d p i nea l ectom i zed rams s ubj ected to Norma l I i ght i n g reg i me . Da i l y p hotoper i od ch a nges a re i I l u strated a n d the t i m i ng of the eq u i noxes ( E ) a n d so l st i ce ( S ) i n d i cated . 2 1 7 6 14.40 0 0 ....... ? ? ? 0 E-o 55 9,20 A.. 1 0 8 6 ..-, 4 0\ 0 - >< '-' IJ.l 2 E-o < ? :::> u 0 < ...... IJ.l -< 0 8 N 0 E-o < 6 ? ? IJ.l A.. Cf.l 4 2 s 0 N s ? D J MONTHS E ? SHAM-OPERATED PINEALECfOMIZED F M A M F i g u re 6 . 5 : Month l y va r i at i ons i n n umbers of spermatoza/ejacu l ate ( mea n?S . E . ) i n semen co l l ected f rom sham-operated and p i nea l ectom i zed rams s ubjected to the Reversed I i g ht i ng reg i me . Da i l y p hotoper i od changes a re i l l ustrated a nd the t i m i ng of the eq u i noxes ( E ) a n d so I st i ce ( S ) i n d i cated . 21 8 - ..t:: Q' I4 A-O 0 -? ? 12.00 ? ? 9.20 6 5 4 3 2 0 5 4 3 2 E + s 0 N s + 0 J MONTHS E t SHAM-OPERATED PINEALECTOMIZED F M A M F i g u re 6 . 6 : Month l y va r i at i on s i n sem i na l p l asma f ructose concen? trat i on s ( mean+S . E . ) i n semen co l l ected f rom s ham-operated a n d p i nea l ectom i zed rams s u bj ected to the Norma l I i g ht i ng reg i me . Da i l y p hotoper i od changes a re i I l ustrated a n d the t i m i ng of the equ i noxes ( E ) and so l st i ce ( S ) i nd i cated . 2 1 9 E - ? 0 1 4.40 Cl 0 ..... ? l:lJ 1 2.00 A.. 0 ? 0 ::r:: 9.2 0 A.. 4 3 - 2 - s - en s '-" l:lJ I Cl) 0 ? 0 g ? ? < 5 ::s Cl) < ....:l 4 A.. ....:l < z ..... 3 ::s l:lJ Cl) 2 s y 0 N s ? / 0 J MONTHS 220 E ? SHAM-OPERATED ? "' ? PINEALECTOMIZED /[ l'"- F M F i g u re 6 . 7 : Month l y v ar i at i on s i n sem i na l p l a sma f ructose concen? trat i ons ( mean+S . E . ) i n semen co l l ected f rom sh am-operated a n d p i nea l ectom i ze d rams s u bj ected to the Reversed I i g ht i ng reg i me . Oa i l y photoper i od changes a re i I l u strated a nd the t i m i ng of the eq u i noxes ( E ) and so l st i ce ( $ ) i nd i cated . 22 1 this trend was most marked for pinealectomized ram3 (C ontrasts 4 and 7 ) . Overall, sham-operated rams had lower e jaculate volumes than pineal? ectomized rams (Contrast 2 ) . All three measures of seminal fructose leve ls displayed higher values during the last five months of the study. Although e jaculates from the rams on Reversed lighting showed peak fructose values duri ng this period and declining levels towards the end of the experiment, the fructose content of semen from rams on the Normal lighting regime c ontinued to increase until the e nd of the experiment (C ontrasts 4, 6 and 8 ) . Pinealectoiey" had no overall e ffe ct on seminal fructose levels , however the decline in seminal fructose conce ntrations in e jaculates from rams under Reversed lighting was s ignificantly less marked in the PX rams than in the Controls ( Contrasts 8, 9 and 1 0) . E jaculates from sham-operated rams had higher mean concentrations of spermatozoa than those from pinealectomized rams (Contrast 2 ) . This differe nce was largely attributable to results from rams on Reversed lighting ; the opposite trend was seen in res ults from rams on Normal lighting during the final five months of the experiment, both for spermatozoal concentration and for total numbers of spermatozoa per e jaculate (Contrasts 5, 8 and 9 ) . Rams under Normal lighting produced semen with higher overall percentages of unstained and morphologically normal spermatozoa than did rams under Reversed lighting. The mean percentage of unstained spermatozoa, calculated from all four groups of rams , rose steadi? from September 1 974, until February 1 975, then showed a s light decline (Contrasts 1 , 3, 5 and 6 ) . ( 2 ) Plasma Hormone Data (a ) 1tl? See Tables 6 . 1 0, 6 .1 3 and Figures 6 . 8 and 6 . 9. All four groups of rams displayed peaks of LH secretion during 222 Tab l e 6 . 1 0 Mean plasma LH c onc entrat i ons r e corded from rams ?i n Experiment 6 . ( Va lues pre s ented are 1 00 log 1 0 (x + 1 . 1 ) , where x is hormone c onc entration in ng/ml . ) Normal Light ing Reversed Lighting Sampl ing Controls PX Controls PX Mean t ime 1 1 6 . 8 1 4 . 3 1 2 . 2 1 3 . 0 1 4 . 1 2 1 8 . 4 1 8 . 6 1 1 . 1 1 6 . 9 1 6 . 2 3 1 6 . 8 1 7 . 1 1 3 . 6 1 6 . 9 1 6 . 1 Per i od 1 4 2 5 . 1 26 . 3 1 6 . 6 23 . 8 22 . 9 5 1 4 . 9 1 6 . 3 1 2 . 7 1 4 . 6 1 4 . 6 6 1 5 . 9 1 5 . 2 20 . 6 2 0 . 6 1 8 . 1 7 1 2 . 0 1 3 . 7 1 1 . 6 1 1 . 7 1 2 . 2 Mean 1 7 . 1 1 7 . 4 1 4 . 1 1 6 . 8 1 6 . 3 8 1 7 . 9 1 6 . 2 1 8 . 5 1 0 . 8 1 5 . 8 9 8 . 9 1 2 . 2 1 2 . 3 1 3 . 0 1 1 ? 6 1 0 9 . 4 9 . 5 8 . 8 1 2 . 9 1 0 . ?1 Per i od 2 1 1 9 . 3 1 0 . 9 9 . 6 1 5 . 6 1 1 . 3 1 2 1 7 . 9 9 . 1 8 . 9 1 I ? 1 1 1 . 7 1 3 1 5 . 0 9 . 1 8 . 3 1 7 . 2 1 2 . 4 1 4 2 1 ? 1 7 . 3 5 . 8 9 . 7 1 1 . 0 Mean 1 4 .2 1 0 . 6 1 0 . 3 1 2 . 9 1 2 . o 1 5 1 4 . 5 1 0 . 2 6 . 1 1 0 . 8 1 0 . 4 Per iod 3 1 6 1 0 . 9 9 . 8 7 . 3 7 . 3 8 . 8 1 7 1 2 . 5 8 . 4 5 . 8 1 4 . 5 1 0 . 3 1 8 1 1 . 4 8 . 9 7 . 8 7 . 0 8 . 8 Mean 1 2 . 3 9 . 3 6 .7 9 . 9 9 . 6 Overal l Mean 1 4 .9 1 3 . 0 .J...hQ 1 3 .7 (Key : Contro l s = Sham-operated rams ; PX g Pinealec tomized rams ) o 14AO 0 - ? l:..t.:l t5 1 2.0 0 f-0 ::r:: t:l. 9. 20 0 ? 6 0 ? 4 0 ?2 0?6 E t s 0 N 223 s E t + SHAM-OPERATED PINEALECfOMIZED 0 J F M A M MONTHS F i g u re 6 . 8 : Fortn i ght l y va r i at i on s i n p l a sma LH concentrat i on s ( mea n?S . E . ) recorded f rom sham-operated a n d p i nea l ectom i zed rams s ubjected to the Norma l I i ght i ng reg i me . Da i l y p hotoper i od changes a re i I l u s t rate d and the t i m i ng o f the eq u i noxes ( E ) a n d so l st i ce ( S ) i n d i ea te d ? 224 E s E ,-... + + * ..c:: '-' 0 1440 0 -? IJ.l C; l 2.00 E-< 0 :I: ? 9.20 I SHAM-OPERATED 0 ? 6 0 ? 4 I-r/frYY) 0 ? 2 \-I-I-r'-x-YI?I ,-... 0 "' s Cl) "' 0. .... s -bO PINEALECTOMIZED . ?:: '-' :I: 0 ? 6 ....l 0 ?4 0?2 0 s 0 N D J F M A M MONTHS F i g ure 6 . 9 : Fortn i g ht l y va r i at i ons i n p l a sma LH concentrat i ons C mean+S . E . ) recorded f rom sham-operated a n d p i nea l ectom i zed rams subjected to the Reversed I i ght i ng reg i me . Da i l y p hotoper i od changes a re i I l u strated a n d t he t i m i ng of the equ i noxes ( E ) a n d so l st i ce C S ) i n d i cated . 225 the period from September t o December (Period 1 ) , followed by lower mean levels during the final five months (Contrasts 6 and 7 ) . Rams under the Normal lighting re gime had higher mean plasma LH levels than those sub jected to the Reversed lighting re gime . This res ult , and the s ignificant Lighting Re gimes x Operations interaction, arose from the lower mean plasma LH levels recorded from the sham-operated rams s ubjected to the Reversed lighting regime (C ontra..sts 1 and 8 ) . From December 1 974 t o March 1 975 (Period 2 ) , plasma LH leve ls of s ham-operated rams under Normal lighting displayed rising values whilst those of sham-operated rams under Reversed lighting declined, as also did those of pinealectomized rams under Normal lighting (C ontrast 20) . 6 . 1 1 . (b ) Testosterone . See Tables 6 .1 1 , 6 . 1 3 and Figures 6. 1 0 and Regardless of lighting regime , sham-operated rams showed a we ll? defined peak of plasma testosterone leve ls at around the time of the s hortest daily photoperiod. On the other hand, plasma from pineal- ectomized rams showed no c onsistent pattern of change in testosterone levels . The above statements accounted for all the significant interaction c ontrasts ( Contrasts 8, 1 0, 1 4 and 20) , while the fact that s ham? operated rams on both lighting regimes did not have elevated plasma testosterone levels during the first four months gave rise t o the highly significant between-periods c ontrast ( Contra..st 6 ) . ( c ) Prolactin. See Tables 6 .1 2 , 6 .1 3 and Figures 6 .1 2 and 6. 1 3 . Substantial fluctuations in plasma prolactin levels were recorded from all groups of rams in this experiment . Generally these changes were such that mean prolactin levels were related directly t o the photoperiods t o which the animals were exposed. However, the fluctuations in plasma prolactin c oncentration were not as marked in pinealectomized ra1113 as they were in sham-operated rams . Also the PX 226 Tabl e 6 . 1 1 Mean plasma testosterone c onc entrati ons recorded from rams in Exper iment 6 . (Values pres ented are 1 00 log1 0 (x + 1 . 1 ) , ''here x is hormone c oncentrat i on in ng/ml . ) Normal Light ing Revers ed Light ing Samp l ing Contro l s PX Controls PX Mean time 1 20 . 9 3 3 . 6 22 .7 33 . 8 27 .7 2 2 1 . 6 20 . 1 1 6 . 4 2 8 . 7 2 1 . 7 3 34 . 8 3 2 . 8 1 5 . 3 2 9 . 8 28 . 2 Per i od 1 4 40 . 8 5 2 . 0 1 2 . 8 52 . 5 3 9 . 5 5 56 . 9 3 3 . 2 29 . 5 25 . 2 3 6 . 2 6 22 . 0 2 6 . 6 57 . 0 42 .9 37 . 1 7 34 . 9 1 9 . 5 34 . 6 30 . 5 2 9 . 9 Mean 3 3 . 1 3 1 . 1 26 . 9 34 . 8 3 1 . 5 8 47 . 8 3 6 . 8 88 . 6 43 . 8 5 4 . 2 9 33 . 1 48 . 1 78 . 3 5 4 . 3 5 3 . 4 1 0 3 7 . 2 40 . 4 50 . 5 32 . 5 40 . 1 . Per i od 2 1 1 3 1 . 1 46 . 5 48 . 6 5 5 . 5 45 . 4 1 2 64 . 4 41 . 8 5 5 . 3 5 4 . 8 54 . 1 1 3 76 . 4 56 . 8 3 3 . 5 68 . 1 5 8 . 7 1 4 82 . 4 23 . 4 1 9 . 0 50 .7 43 . 9 Mean 5 3 . 2 42 . 0 5 3 . 4 5 1 . 4 5 0 . 0 1 5 82 .7 3 5 . 9 2 8 . 0 40 . 3 46 .7 Per i od 3 1 6 57 . 0 40 . 8 24 . 8 3 1 . 2 3 8 .4 1 7 69 . 6 3 1 . 8 2 0 . 3 29 . 2 37 . 7 1 8 3 2 .7 27 . 3 3 2 . 4 27 .7 3 0 . 0 Mean 60 . 5 3 3 . 9 26 . 4 32 . 1 3 8 . 2 Overal l Mean 47 . 0 3 6 . 0 37 . 1 40 . 6 (Key : Contro l s = Sham-operated rams ; PX = Pinea l e c tomiz ed rams ) E s E t t t ,.-_ ..c:: 2;'14.40 0 -? ? 1 2.00 0 f-< 0 g: 9. 20 SHAM-OPERATED I I I ,.-_ ? s "" ? -0.. - s / -bll I YI" c= .._, J- ? z ?? 0 ? ? f-< ? 0 f-< PINEALECfOMIZED ? ? f-< 3 2 !' Ivl /ki'} 0 s 0 N D J F M A M MONTHS F i g u re 6 . 1 0 : Fortn i ght l y var i at i on s i n p l a sma testosterone con? centrat i ons C mean?S . E . ) recorded f rom s h am-operated a n d p i nea l ? ectom i zed rams su bjected to the Norma l I i ght i ng reg i me . Da i l y p hotoper i od cha nges a re i I l ustrated a n d the t i m i ng of the equ i n? oxes C E ) a n d so l st i ce C S ) i n d i cated . 227 . 228 E s E - ? ? ? .s:: '-' ? 1 4.40 -? (.1.J Cl 1 2 .00 E-< g ? 9.2 0 10)..- SHAM-OPERATED 4 - 3 c.s e {/) c.s -0.. 2 e -bO c: '-' ? I'i--r-( z 0 ? ? 0 E-< tl.l PINEALECTOMIZED 0 E-< tl.l 5 (.1.J E-< 4 3 2 s 0 N 0 J F M A M MONTHS F i gu re 6 . I I : Fortn i ght l y va r i at i ons i n p l asma testosterone con? centrat i ons ( mean?S . E . ) recorded f rom s ham-operated a n d p i nea l ? ectom i zed rams subjected to the Reversed I i ght i ng reg i me . Da i l y photoper i od changes a re i I l u strated and the t i m i ng of the eq u i noxes ( E ) and so l st i ce ( S ) i n d i cated . 229 Tab l e 6 . 1 2 Mean plasma pro lac t in c onc entrat i ons r e c o rded from rams i n Exper i ment 6 . ( Va lues pre s ented are 1 00 l og 1 0 ( x + 1 . 1 ) , wher e x i s hormone c onc entrat i on in ng/ml . ) Normal Lighting Reversed Light ing Sampl i ng Contro l s PX Contro l s PX Mean t ime 1 1 73 . 2 1 88 . 0 1 6 5 . 4 1 33 . 1 1 64 . 9 2 1 6 5 . 0 1 57 . 8 1 52 .6 1 57 . 5 1 58 . 2 3 1 73 . 8 1 5 5 . 1 92 o 5 1 45 . 4 1 4 1 . 7 Per i od 4 1 8 1 .2 1 62 o0 90 . 4 1 54 . 7 1 47 . 1 5 1 87 . 5 1 74 . 5 1 06 . 1 1 5 1 .6 1 54 . 9 6 1 89 . 9 1 73 . 7 9 9 . 7 1 2 5 . 5 1 47 . 2 7 1 90 . 6 1 49 . 8 1 02 . 1 1 38 . 7 1 45 . 3 Mean 1 80 . 2 1 65 . 8 1 1 5 . 5 1 43 . 8 1 5 1 . 3 -- 8 1 75 . 2 1 5 5 . 5 53 . 6 1 28 0 6 1 28 . 2 9 1 57 . 1 1 45 . 2 9 6 o7 1 6 1 . 8 1 40 . 2 1 0 1 72 .6 1 56 . 9 1 0 1 . o 1 48 . 5 1 44 . 7 Per i od 2 1 1 1 37 .o 1 63 . 3 1 1 0 . 6 1 57 . 6 1 42 . 1 1 2 1 77 .4 1 89 . 4 1 9 5 . 2 20 1 . 1 1 90 . 8 1 3 1 1 4 . 4 1 67 . 2 1 38 . 6 1 77 . 1 1 49 . 3 1 4 1 04 .7 1 78 . 1 1 69 . 0 209 .7 1 6 5 .4 Mean 1 48 . 3 1 65 . 1 1 23 . 5 1 69 . 2 1 5 1 ? 5 1 5 60 . 1 1 6 1 . 4 1 66 . 2 1 9 5 . 5 1 45 . 8 Per iod 3 1 6 48 . 0 1 66 . 1 1 92 . 8 1 82 . 0 1 47 . 2 1 7 1 28 . 3 1 83 . 5 1 78 . 6 1 68 . 6 1 64 . 7 1 8 1 1 5 . 1 1 63 . 1 1 88 . 9 1 76 . 5 1 60 . 9 Mean 87 .9 1 68 . 5 1 8 1 .6 1 80 .6 1 54 . 6 Overal l Mean 1 47 . 3 1 66 . 1 1 3 3 . 3 1 6 1 ? 9 (Key : Contro ls = Sham-operated rams ; PX = Pinea l ectomi z ed rams ) ? 14.40 .s:::. 0 0 a: ? 12.00 0 f-0 :r: 0.. 9.20 0 E en 100 ? 50 a. E ....... go c z f- 0 <( __. 0 0 100 a::: a.. 50 230 E s E ? t ? ?J?r I S HAM - OPERAT E D ?-/r-1 0?--?----?----?-----r-----.-----.-----.-----..-----.- s 0 N 0 J M O N T H S F M A M F i g u re 6 . 1 2 : Fortn i ght l y v ar i at i on s i n p l asma p ro l a ct i n concentrat? i on s ( mea n?S . E . ) recorded f rom sham-operated a n d p i nea l ectom i zed rams subj ected to t he Norma l I i ght i ng reg i me . Da i l y photoper i od cha nges a re i l l ustrated and the t i m i ng of the equ i noxes ( E ) a n d so l st i ce ( S ) i n d i cated . Cl 0 0:: w 11..12.0 0 ? 0 I a.. 9.20 1 00 -;; 50 E ., 0 0. E ....... Cl 5 ? 150 ? <..> .s of hormone secretion, without changing the mean plasma concentration at the morning sampling time used in Experiment 6 . The only evidence that pinealectomy might alter diurnal hormonal se cretion patterr? in sheep was that of Thurley, Gibb and Russe ll ( 1 975 ) who presented data which indicated that the diurnal cortis ol rnythm tended to be abolished in pinealectomized ewes . No reports have been published on the e ffects of different lighting regimes on the plasma hormone profiles of rams . Thus Experiments 7.1 , 7 .2 , and 7.3 were d?signed to examine whether it was possible to detect any effects of pinealectomy or daily photoperiod on the patterns of reproductive hormone secretion in rams, in a series of experiments in which plasma samples were c ollected at fre quent intervals for exte nded periods of time. 2 . MATERIALS AND METHODS ( 1 ) Animals and Experirental Procedure In Experiment 7.1 the experimental animals used were the rams on 247 the Normal lighting regime of Experiment 6. Four rams were s ampled on November 20, 1 974, and the remaining four rams were sampled on December 4, 1 974. On each occasion two pinealectomized and two sham- operated rams were sampled and blood was c ollected via indwelling venous cannulae ( see Chapter II) eve? twenty minutes for twenty-s ix hours, commencing at o6.oo h. Occasional blockages in the cannulae resulted in a few samples not being collected. In Experiment 7.2 all sixteen rams in Experiment 6 were sampled on March 3, 1 975, blood be ing colle cted by venepuncture at 30 minute intervals for four hours c orr?nc- ing at 1 3 .00 h. A s imilar procedure was adopted for Experiment 7.3, which was performed on May 20, 1 975. However, in this study venepuncture blood samples were collected at 30 minute intervals for six hours c ommencing at 1 0.30 h. As disturbance to the rams was minimal, and no changes were made t o the ir lighting and feeding routine, it was considered that these acute studies did not interfere with the results of Experiment 6. ( 2 ) Hormone Ass a?r Procedure The particular interest in circadian hormone secretion profiles , which has appeared i n the field of reproductive endocrinology was a stimulus to measure a number of different hormones in the plasma samples obtained from Experiment 7.1 LH, prolactin, testosterone and cortisol were ass?ed as described in Chapter II. LH . and prolactin orLcy- were measured in samples from Experiments 7.2 and 7 .3 . (3 ) Statistical Analyses Estimated values for the few miss ing samples were calculated as the mean of the preceding and subsequent samples. Hormone concentrations were transformed to their logarithm using the relationship : logarithm of x = 1 0 lo? 0( x + 1 . 1 ) where, x is hormom conce ntration in ng/ml plasma. 248 For each ram, tran.sfornl:!d values were progress ively 3Wnmed in numerical order of sampling, to form a series of cumulative totalB . The gradient of this cumulative distribution against time, which was tested for linearity by ana?s is of variance , was used as a variable in between-treatment contrasts . It was c onsidered that this gradient provided a parameter which reflected hormone output more comprehens- ively than the arithmetic mean of the hormone concentrations . Between- group c omparis ons of the gradients of the cumulative hormone distributions in the first s tudy were perforrred by Student ' s ' t ' test. In Experiments 7.2 and 7.3 treatme nt effects of lighting re gimes , operations , and the ir interaction were examined using the orthogonal coefficients shown below : C ontrast Normal Lighting Sham? Operated Lighting Regimes ? Operations +1 Lighting Regimes +i x Operations Pineal- e ctomized +1 -1 -1 Reversed Lighting Sham- Pineal- Operated e ctomized -1 -1 +1 -1 -1 +1 Studies on goats have s hmvn that venepuncture initially elevates prolactin leve ls which, in 3Ubse quent samples , return to normal as the animals become accustomed t o this stress (Hart, 1 973 ) . On this basis, the prolactin data for the first samples collected by venepuncture in Experiments 7 .2 and 7.3 were excluded from the statistical analyses. 3. RESULTS Experiment 7.1 was desigmd as a study of the normal patterns of secretion of LH, testosterone , prolactin and cortis ol, and also to examine the e ffects of pinealeoto? on these secretor,y patterns. All e ight rams exhibited a highly irregular pattern of plasma LH secretion. (See Figures 7.1 and 7.2 ) . These pulses occasional? 2 0 0 E ., 0 0.. E - 0> c: J: 4 _, 2 4 2 08.00 12.00 Lights off 16.00 20.00 24.00 TIME ( h ) 59 50 08.00 F i g u re 7 . I : LH secret i on patterns reco rded f rom p l asma samp l es co l l ected f rom fou r sham-operated rams , samp l ed at 20 m i n u te i n terva l s for 26 hours . 249 -0 E lit 0 a.. e - 0> c :X: _, 8 6 4 2 0 4 2 0 6 4 2 0 4 2 Lights on Lights off 63 51 ... ... ... , _.. , - ?,_ 47 08.00 12.00 16.00 20.00 TIME ( h ) F i gure 7 . 2 : LH secret i on patterns recorded f rom p l a sma samp l es co l l ected f rom fou r p i nea l ectom i zed rams , samp l ed at 20 m i n ute i n terva l s for 26 ho urs . 250 ? I _J cu E Ul ro 0 ? 0 . . . . . ? ? J i l l i llU - ? rJ J J I I ! 1 ll r?r1V f? rr r trJil JJH !tl1ll 1 ; ' s I ope = 3 . 1 1 8 ? f ;-ff 1 1 1 .:':. o . o6J l -f'f I , . . I l{? , . 11 tlfft!ttfttH t r.Jff11 ' , ' ' ' ' ' ' ' r r , JJ-f-H4-1i;\! (t1 l . . rJ 1 111 ' t ?' n i l t l l i t? l ope = I . 776 . {-i-1"' 11 p?? .:':. 0 . 023 ??rr - . . . -r-rrtf ?? . . . h am-operated ?/..A"-'1 ? . s ? l n= 4 1 08 . 00 1 2 . 00 ? I ? ??????????????????c ? 1 6 . 00 20 . 00 24 . 00 04 . 00 08 . 00 T i me ( h ) F i g ure 7 . 3 : 26-Hou r cumu l at i ve LH l eve l s ( Mean s ? S . E . ) show i ng poo l ed cumu l at i ve reg ress i on I i nes . rv \J1 .... 252 raised plasma LH conoe ntrations from bare ly detectable leve l.s to more than six ng/ml in the next twe nty-minute s ample . There wa? no circadian pattern of distribution of the pulses . ?inealectomized rams appeared to produce more LH peaks , with higher peak hormone levels , than did the sham-operated rams . Student ' s ' t ' te s t c onfirmed that the s lopes of the cumulative LH distributions for the pinealectom? ized rams were greater than those for the s ham-operated animals (3 . 1 1 8 ? 1 . 776 , p < 0.05 ) . (See Figure 7.3 and Table 7.1 ) . Plasma testosterone levels (Figures 7.4 and 7.5 ) displayed a pulsatile pattern of secretion ve? s imilar to that of LH. Major peaks of tes tosterone c oncentration corresponded with pulses of LH release and each elevation in testosterone levels usually occurred twenty to forty minutes following an LH pulse . Testosterone peaks usually exceeded 5 ng/ml. Again, as with LH, there was no indication of a circadian pattern of testosterone secretion. Pinealectomized rams had an output of testosterone which was higher than that of s ham? operated rams , howeve:.t ... comparison of the s lopes of the cumulative distributions indicated that this difference just failed t o reach s tatistical s ignificance (5 . 1 41 ? 4.00l?, p = 0.052 ) . ( Se e Table 7.1 ) . In all rams plasma prolactin levels fluctuated markedly through? out the twenty-six hour sampling period (Figures 7.6 and 7.7 ) . ?lthough the slopes of the cumulative prolactin distributions were not s ignificantly different for the two groups (Table 7.1 ) ,? a distinct circadian pattern was displayed by the s ham-operated rams , but not by the pinealectomized rams . The circadian pattern recorded from c ontrol animals took the form of an e levation of plasma prolactin c oncentrations, c ommencing in the ear? afternoon and reaching maximum values ( in excess of 1 00 ng/ml) approximately between 20.00 h and 22 .00 h; s ubsequently prolactin levels fell relat ively rapidly to reach low levels at about 24.00 h. Lights on lights off 253 10 59 nl 5 E _IV (/) nl Q. E 0 ......... 0'1 c: w z 10 0 48 a: w ..... en 0 ..... 5 en w ..... 0 10 50 5 08. 00 12 .00 16.00 20.00 24.00 04.00 T I M E ( h ) F i g u re 7 . 4 : Testosterone secret i on patterns recorded f rom p l a sma samp l es co l l ected f rom fou r s ham-operated rams , samp l ed at 20 m i n ute i nterva l s for 26 hou rs . ?l E (/) ?l Q. E ........ C) c::: w z 0 a: w t-(/) 0 t-(/) w t- 5 0 10 5 ..._ , .... , , , , 0 10 5 0 10 08.00 . Lights on 12.00 L ights off 51 - - "'""- 54 47 16.00 20.0.0 24 .00 04.00 08.00 T l M E (h) 254 F i gure 7 . 5 : Testosterone secret i on p atterns recorded f rom p l a sma samp l es co l l ected f rom fou r p i nea l ectom i zed rams , samp l ed at 20 m i n ute i nterva l s for 26 hours . 150 50 0 150 - 0 100 E . Ill 0 a. e 50 ....... 0) c z 0 ? ? 150 ..... ? c.. 100 50 0 150 100 50 08.00 Lights on Lights off 12.00 16.00 20.00 24.00 TIME (h) 59 04.00 08.00 F i g u re 7 . 6 : Pro l act i n secret i o n p atterns recorded f rom p l asma samp l es co l l ected f rom fou r s ham-operated rams , samp l ed at 20. m i nute i nterva l s for 26 hours . 255 Lights on Lights off 100 63 50 0 150 0 100 E Ill 0 Q_ 51 E 50 - ??vi' -0) ..... --........ c z ..... 0 ? ..... ? 100 0.. 50 ' ? ? 0 100 50 08.00 12.00 16.00 20.00 24.00 04.00 08.00 TIME (h) F i g u re 7 . 7 : Pro l act i n secret i on patterns reco rded f rom p l a sma samp l es co l l ected f rom fou r p i nea l ectom i zed rams , samp l ed at 20 m i nute i n te rva l s for 26 ho urs . 256 30 20 0 25 20 ? 10 11) 0 1i .e. 0 0) -5 40 _, 0 (/) ? 30 0 u 20 10 0 Lights on Lights off 50 16.00 20.00 24.00 04.00 08.00 TIME (h) F i g u re 7 . 8 : Cort i so l secret i on patterns recorded f rom p l a sma samp l es co l l ected f rom four s ham-operated rams , samp l ed at 20 m i n u te i nterva l s for 26 hou rs . 257 20 10 0 30 I, I \ / \ I \ I \ --- 20 I \ 0 I E ell ...2 Q. 1 0 E ' 0> c 0 ...__ _. 0 20 V) t= 0::: 0 u 10 0 4?1 40? 20 10 \ Lights on Lights off 63 51 54 16.00 20.00 24.00 04.00 08.00 TIME ( h ) F i g u re 7 . 9 : Cort i so l secret i on patterns recorded f rom p l a sma samp l es co l l ected f rom fou r p i nea l ectom i ze d rams , samp l ed at 20 m i n ute i nterva l s for 26 hours . 258 Table 7 . 1 Gradi ents of Cumulative LH, Testosterone , Prolactin and Cort i s o l Distr ibuti ons Rec orded from Plasma Samples Co l le cted over 26 hours from Individual Sham-operated or Pinealectomiz ed Rams . Log LH Log Testost erone Log Pro lact in ? Log Cort i s o l 4 . 045 5 . 439 1 3 . 66 27 . 32 Pinealectomized 3 . 3 88 4 . 003 1 7 . 29 36 . 3 5 2 , 808 5 . 009 1 5 . 26 1 9 . 47 2 . 230 6 . 1 1 4 1 4 . 2 5 22 . 40 Mean+S .E . 3 . 1 1 8+0 . 067 5 ? 1 4 1,:?:0 ? 442 1 5 . 1 2,:?:0 . 1 5 26 . 39+1 . 1 0 1 . 689 3 . 9 3 5 1 7 . 67 22 . 27 Sham-operated 1 . 48 1 3 . 609 1 5 . 91 23 .97 1 . 989 4 . 3 1 2 1 7 . 1 8 3 1 ? 1 1 1 . 944 4 . 1 5 8 1 6 . 26 3 0 . 92 Mean+S . E . 1 . 776,:?:0 . 023 4 . 004,:?:0 . 1 52 1 6 . 76+0 . 1 0 27 . 07+0 .7 1 .16 3 . 298* 2 . 432+ -0 . 1 26 -0 . 1 56 ( + p = 0 . 05 2 ) 1\) \Jl ....0 260 All rams exhibited a c ortisol secretion pattern which appe&-cd as a se quence of peaks, most of which had a duration of approximately ore hour, and maximum values of about 7 ng/ml. (See Figur"es 7. 8 and 7.9 ) . However, much larger penk.'3 ( s ome higher than 30 ng/ml ) were rec orded, particularly between 07. 00 h and 1 4. 00 h. J?eaks of this type were evident in three of the four sham-operated rams ( Nos . 48, 58 and 59 ) , and, to a lesser extent, in two of the pinealectomized ram3 ( Nos . 47 and 51 ) . There were, however, no s ignificant differences betwee n the s lopes of the cumulative cortis ol distributions for the two groups of rams (Table 7.1 ) . Two o f the raffi3 (Nos . 47 and 50) displ?ed a large c ortis ol peak at about 22.30 h. This might have been a stress effect, s ince these two rams were in adjacent crates . Experiment 7 . 2 was carried out with several aims i n mind firstly to determine whether the effects of pinealect o? on LH secretion, which were revealed in Experiment 7.1 , c ould be detected in a shorter term experiment ; secondly to see whether these effects of pinealecto? were apparent later in the year; and thirdly to investigate possible effects of lighting regimes on LH and prolactin secretion. Very few peaks of LH secretion were recorded from any of the rams and, in the case of pinealectomized rams under Normal lighting, there were no peaks at all. ( See Figure 7.1 0 ) . In the analysis of variance of LH data, none of the treatment contrasts were s ignificant ,, (Tables 7.2 and 7.3 ) . Individual rams showed quite c ons iderable variat ions i n plasma prolactin levels, and nine out of the sixteen had elevated levels at the initial sampling. presumab? as a result of the venepuncture sampling procedure . Pinealectomized rams under both lighting regimes had a higher output of prolactin than did sham-operated rams ( p < 0.05 ) . (See Figure 7.1 1 and Tables 7.2 and 7.3 ) . Lighting regimes had no 4 NORMAL LIGHTING SHAM-OPERATED PINEALECfOMIZED ,.-... C':l s "' C':l -0. - s -CO c:: '-" 5 4 REVERSED LIGHTING SHAM-OPERATED PINEALECTOMIZED 2 o ??--????--???-. 1 7 .00 13.00 15 .00 TIME (h) F i g u re 7 . 1 0 : ( Exper i ment 7 . 2 ) LH l eve l s ( mean+S . E . ) i n s ham-operated and p i nea l ectom i zed rams recorded f rom p l a sma samp l es co l l ected at 30 m i n ute i nterva l s for a four hou r per i od on Ma rch 5 , 1 975 . 261 17 .00 - (1j s r/) (1j -0.. s -eo 1: '-' z -E-< u < ....:l 0 ? ? NORMAL LIGHTING SHAM-OPERATED PINEALECTOMIZED 15 10 REVERSED LIGHTING SHAM-OPERATED PINEALECTOMIZED 10 5 1 /I'i-vY ?'i-r J?, ---r--r---r---1 ? 13 .00 15 .00 1 7 .00 TIME (h) 15 .00 1 7 .00 F i g u re 7 . 1 1 : ( Exper i ment 7 . 2 ) Pro l act i n l eve l s C mea n.?_S . E . ) i n s ham? ope rated a n d p i nea l ectom i zed rams recorded f rom p l asma samp l es co l l ected at 30 m i n ute i nterva l s for a four hour per i od on Ma rch 5 , 1 975 . 262 263 Tab l e 7 . 2 Grad i ents of Cumulat ive LH and Pro lact in Di s t r i but ions Re c o rded f rom Plasma Samp l e s C o l l e cted o ver 4 hours from Individual Sham-operated or Pineal cctomized Rams on Contrast ing Ligh t i ng Reg imes . Log LH Log Pro lactin Norma l Lighting Regime 2 . 1 02 1 4 . 27 Sham-operated 2 . 294 1 4 . 70 1 . 452 1 1 . 40 1 . 1 1 5 1 5 . 1 7 Nean+S . E . 1 . 74 1,:t0 . 276 1 3 o89,:t0 . 85 1 . 04 1 1 9 . 4 1 Pineal e ctom i zed 1 . 090 1 7 . 00 1 . 3 1 3 20 . 3 5 ?J . 769 1 9 . 06 Mean+S . E . 1 . 3 03,:!0 . 1 66 1 8 ? 9 6 .?0 . 7 1 Revers ed Lighting Regime 0 . 89 5 1 6 . 48 Sham-operated 1 . 979 1 4 . 2 5 0 . 83 ?1 1 2 . 70 2 . 722 1 8 . 00 Mean+S . E . 1 . 620,:t0 .448 1 5 . 36,:t1 . 1 7 2 . 1 80 1 7 . 42 Pineal e ctomized 0 . 900 1 7 . 75 0 . 83 4 20 . 50 3 . 504 1 7 . 9 1 Mean+S . E . 1 . 85 4,:!0 . 6 3 1 1 8 . 39_?0 . 7 1 Table 7 . 3 Summary o f Analyses of Variance for LH and Pro lactin Data Pres ented in Tabl e 7 . 2 . Source of Variation D . F . Variance Ratios LH Pro lactin Lighting Regimes 0 . 26 0 . 27 Operations 0 . 06 2 1 . 20*** Lighting Regimes x Operations 0 . 64 1 . 3 3 Res idual Mean Square 1 2 0 . 70 3 . 1 0 C\) 0'\ ? 265 significant e ffect on mean plasma prolactin levels . As the LH data for plasma samples c ollected ovor a four hour period in Experiment 7.2 was inconclU3ive , a further s hort-term s tudy (Experiment 7 .3 ) was undertaken to examine the effects of pinealecto? and lighting regimes on LH and prolactin production rates . In this experiment, however, the sampling period was extended t o s ix hours . Plasma LH c oncentrations from individual rams again indicated a pulsatile re lease of this hormone with most atrlmals displ?ing one or more peaks of LH secretion. However, the sham-operated rama on the Normal lighting regime were an exception in that the ir plasma LH levels changed little during the study ( Figure 7 . 1 2 ) . Analysis of variance of the gradients of the cumulative LH distributions revealed that the rams on Reversed lighting had a higher mean leve l of LH output than those on normal lighting ( p < 0.05 ) . ( See Tables 7.4 and 7 .5 ) . Pinealectomized rams tended t o have higher mean levels o f LH production that did the sham-operated animals , but in the analysis of variance this difference failed to reach s ignificance (p = 0.052 ) . In ?Aperiment 7.3 (Figure 7. 1 3 ) individual rams again had markedly variable plasma levels of prolactin, and thirteen of the sixteen rams had e levated levels at the first sampling. Analysis of variance of the gradients of the cumulative prolactin - distributions (Tables 7.4 and 7.5 ) indicated that the output of prolactin was higher in pineale ctomized rams than in sham-operated rama, whilst both groups subje cted to Reversed lighting apparently had higher prolactin production rates than those under Normal lighting. However, inspection of Figure 7.1 3 and the cumulative distribution data (Table 7 .4) indicated that both of these s ignificant results c ould be misleading as they occurred virtually entirely as a result of the low rate of prolact in output from the sham-operated rams on the Normal 266 NORMAL LIGHTING 4 SHAM-OPERATED PINEALECTOMIZED 2 REVERSED LIGHTING 4 SHAM-OPERATED PINEALECTOMIZED 2 0+-r-r-r-???--?-.?,.? I I i I I I 1 0 .00 1 2 .00 1 4.0 0 16 .00 14 .00 16.00 TIME (h) F i g u re 7 . 1 2 : < Exper i ment 7 . 3 ) LH l eve l s ( mean?S . E . ) i n s h am-operated a n d p i nea l ectom i zed rams recorded f rom p l a sma samp l es co l l ected at 30 m i n ute i nterva l s for a s i x hou r per i od on May 20 , 1 975 . 5 10 5 NORMAL LIGHTING SHAM-OPERATED PINEALECTOMIZED REVERSED LIGHTING SHAM-OPERATED PINEALECTOMIZED 1 2 . 00 14 .00 16 . 00 TIME (h) 1 2 .00 14.00 16 .00 F i g u re 7 . 1 3 : ( Expe r i ment 7 . 3 ) Pro l act i n l eve l s ( mean?S . E . ) i n s h am? operated and p i nea l ectom i zed rams recorded f rom p l a sma samp l es co l l ? ected at 30 m i n ute i nterva l s for a s i x hour pe r i od on May 20 , 1 975 . 267 268 Table 7 . 4 Gradi ents of Cumulat ive LH and Pro lac tin Distributions Rec orded from Plasma S amples C o l l e c ted over 6 hours from I nd ividua l Sham-operated or Pinealectomized Rams on Contras t ing Light ing Regimes . Log LH Log Pro lactin Normal Lighting Regime 1 . 1 79 1 6 . 5 5 Sham-operated 0 . 622 1 8 . 9 5 1 . 22 5 1 7 . 3 8 0 . 736 1 5 .8 5 Mean+S . E . 0 . 940_?0 . 1 5 3 1 7 . 1 8.?0 .67 2 . 8 1 5 24 . 1 2 Pinealectomiz ed 0 . 97 1 2 1 . 04 1 . 8 5 1 26 . 3 5 1 . 644 21 . 66 Mean+S . E . 1 . 820_?0 . 38 1 23 . 29+"1 . 22 Revers ed Lighting Regime 1 . 564 2 5 . 37 Sham-operated 2 . 763 2 5 . 0 1 1 . 9 5 6 2 5 . 44 1 . 2 3 5 2 3 . 83 Mean+S . E . 1 . 880_?0 . 329 2 4 . 9 1_?0 . 37 1 . 707 2 1 . 69 Pineal ectomiz ed 1 . 838 2 5 . 54 2 .797 2 5 . 69 3 . 227 2 2 .7 5 Mean+S . E . 2 . 392_?0 . 369 2 3 . 92_?1 . 00 Table 7 . 5 Summary o f Analyses of Var iance for LH and Pro lactin Data Presented in Table 7 . 4 . Source o f Variation D .F . Variance Ratios LH Pro lactin Lighting Regimes 5 . 53* 22 .74*** Operations 4 . 69+ 8 . 52* Lighting Regimes x Operations 1 0 . 30 1 6 . 44** Res idual Mean Square 1 2 0 . 4 1 3 . 07 ( + p = 0 . 052) ---- (\) 0'\ \.{) lighting regime . This conclusion was c onfirrood by the highly significant Lighting Regimes x Operations interaction. 4. DISCUSSION ( 1 ) LH - Hie;hly pulsatile patterns of LH release in rams have been 270 des cribed in several earlier reports (Bolt, 1 971 ; Katongole , Naftolin Short . 1 974; Sanford et al. , 1 974?; Falvo et al. , 1 975 ) but the pnysiological s ig1uficance of this type of secretory pattern is not clear. It is poss ible that LH responsive tissues re quire a digital rather than an analogue control, such that they respond to hormone pulse freque ncy rather t han to absolute c oncentrations of the gonadotrophin. On the other hand, the rapid increase in peripheral concentrations of LH which follows such pulses, could represent a means for momentarily saturating binding sites on recaptor tissues, thereby obviating the need for constant production of large amounts of the hormone . Inspect ion of the records of LH output for individual rams (Figures 7.1 and 7.2 ) revealed that most of the secretory pulses ?caused concentrations to rise very steeply but to declim in an exponential fashion. A similar pattern was seen in the data presented by Sanford et al. ( 1 974?) who took blood samples at twenty minute intervals also. This finding suggested that the anterior pituitary releases LH in pulses and that between each pulse ,. little or no further secretion takes place . It was estimated that ma? of the higher peaks in the present study fell to fifty per cent of their peak value in approximately thirty minutes . Similar or slightly lower figures could be estimated by inspection of the data of Sanford et al. ( 1 974?) . This half-lifo of approximate ly thirty minutes is s imilar t o that reported by Butler et al. ( 1 972 ) and by Geschwind ( 1 972 ) for ovino LH in ewes . The fact that this figure was fairly constant for secretory peaks with a wide range of heights gave further support t o Geschwind' s ( 1 972 ) concept that a basal level of LH secretion does not occur between secretory phases . A half-life of this duration indicates that the sampling interval of twe nty minutes used in the present 271 study would enable detection of every LH pulse during the observat ion period, even if not allovdng measurement of absolute peak values . AB noted in the previous reports mentioned above , there was no evidence for a circadian pattern of LH release in the present study. It is interesting to note that no circadian rhythm occurred in the output of a hormone which shows annual secretory fluctuations (Chapter III and Hochereau-de Reviers, Loir and Felletier ( 1 976 )) which presumably are caused Qy seas onal changes in daily ?hotoperiod. Steeper s lopes for the cumulative lli distributions in pineal- ectomized rams indicated that these rams had a greater output of LH than the sham-operated animals . This result was in accord with the wide ly accepted be lief that the pineal gland has an antigo??dotrophic role (Kappers, 1 969; Reiter, 1 973b ) . - Previous workers have demonstrated antigonadotrophic properties of sheep pineal glands in in vitro syste?ms ( Citharel et al. , 1 972 ) , but this is the first report which has presented direct evidence that the sheep pineal gland probab? has a true antigonadotrophic function. A similar tendency for the pinealeotomized rams to have e levated LH levels waa recorded in Experizoont 7 . 3 . However, in the analysis of variance this result just failed to reach s ignificance , probab? because the sham-operated rams on the Reversed lighting regime were subje cted to a long daily photoperiod, which in turn caused a stimulation of LH secretion such as recorded in Experiment 3 . 272 Little importance is attached to the fact that in Experime nt 7.2 mean LH levels were not elevated in the pinealectomized rams . This is a result which may be interpreted as contradictory to those obtai.ned in the other experiments in this chapter. More likely, however, it was a spurious result which arose due to the blood sampling period be ing t oo short and coinciding with a tiroo in which the pinealectomized rams experienced virtually nooo of the randomly occurring spikes of LH secretion. Also it was poss ible that seasonal changes modified between? operation differences to the extent that the effects of pinealecto? were apparent only at particular tirres of the year. The significant Lighting Regimes x Operations interaction seen in Experiment 7.3 indicated that pinealectomized rams continued t o display seas onal changes in LH secretion, even i n the absence o f a pineal gland. This finding may have resulted from the presence of residual seasonal rhythm3 s imilar to those which Herbert (1 972 ) claimed persisted in ferrets until the sec ond year after pinealecto?. The effects of the two different lighting regimes were not examined in Experirrent 7.1 , while in Experiment 7.2 lighting regimes had no effect on LH secretion. Again this latter result may mere? have reflected the short period of blood sampling. More pertinent was the fact that the time of sampling was close to the equinox in both regimes, at which time little or no difference in .LH output was expected. For example in an earlier study (Experiment 3 ) rams on pasture displayed a seas onal pattern of plasma LH levels which indicated that highest LH levels were ass ociated with the longest daily photoperiods . This e ffect of photoperiod was confirmed in Experiment 7.3 in which the LH output of rams on Reversed lighting \vas higher than for those on Normal lighting. However these results c ontrast sharply with those of Felletier and Ortavant ( 1 975?) and Lincoln (1 976?) , in which highest plasma LH levels were assoc iated 273 with de creasing daily photoperiods . This disparity in results undoubtedly was attributable mainly to the photoperiodic cycles used by Pelletier and Ortavant ( 6 months cycle length, 8 h amplitude ) and Lincoln ( abrupt change in daily photoperiod from 1 8 h to 8 h) , which were vastly different fro:n those used in Experirrent 6 . In Experiments 5 and 6 lighting regimes had little effect on plasma LH levels ; this result may reflect the inade quacy of the blood s ampling regimes used in those particular experiments . C ollectively, the results of the experiments described in this and earlier chapters can be interpreted as emphasizing the fact that experiments involving c ollection of single plasma samples or multiple samples over a short period of time ( e . g. Experiments 7.2 and 7.3 ) , will not reveal the fUll extent of the e ffects of pinealecto? or lighting regimes, on LH secretion patterns. These e ffects probably will only be e lucidated by experiments which incorporate : plasma sampling regimes similar to that used in Experime nt 7.1 ; with samplings of this type at monthly intervals throughout the year; and which utilize animals which have been exposed to their various lighting regimes for an extended period of time prior to commencing sample collection, in order that res idual seasonal rhythms m? be abolished. ( 2 ) Testosterore Fluctuations in plasma testosterone levels recorded throughout twenty-s ix hours from rams in Experiment 7.1 resembled s imilar patterns of secretion reported by earlier workers (Katongole , Naftolin and Short, 1 974; Purvis, Illius and Haynes , 1 974; Sanford et al. , 1 974?) Furthermore , elevations in plasma testosterone following LH pulses in these rams were consistent with the results of previous studies in which both LH and testosterone were measured (Katongole , Naftolin and Short, 1 974; Sanford et al. , 1 974? . The twenty to forty minute interval between LH pulses and the subsequent peak in plasma 2 74 testosterone levels recorded in those two papers and in the present s tudies, confirred similar results obtained by Bremner et al. ( 1 976 ) and Lee et al. ( 1 976 ) following GnRH administration to rams . On the other hand Gallow? et al. (1 974) recorded a s imultane ous e levation i n plasma levels of both hormones foll?Ning GnRH injection, however this unusual result ? have resulted from the large doses of releasing hormone used by these workers in comparison to the studies reported above. None of the research mentioned above , including the present experiments, revealed a? evidence for the presence of a circadian pattern of testosterone secretion, nor did other studies in v1hich ram plasma was s ampled at les s frequent intervals (Attal, 1 970 ; Falvo et al. , 1 975 ) . I n view of the absence of an LH rhythm o f this type , this result was anticipated. The testosterone output of pinealectomized rams was almost s ignificantly higher than that of s ham-operated rams, a result which undoubtedly was related directly to the differe nce in LH output between the two groups . A separate experiment would be required to determine whether these differences were due to an effect of pineal gland activity on LH re lease per se, or on the sens itivity of LH release to testosterone feedback. The fact that fortnightly plasma testosterone levels recorded during November and December (Experiment 6 ) did not reveal any differences between the two groups of rams (o . 68 .? 0.23 ? 0.94 .! 0.33 ng/ml; t1 4 = 1 . 690, n. s . ) , again indicated the need to perform a number of studies s imilar t o Experiment 7.1 , at various times throughout the year. (3 ) Prolactin Sham-operated rams in Experimnt 7.1 displayed c ons istent nocturnal increases in plasma prolactin c oncentrations . Similar 275 nocturnal e levations have been recorded by Forbes et al. ( 1 975 ) from castrated male lambs, and by Davis and Borger (1 974) from ovariect- omi zed ewe s . In contrast, other workers have failed to detect c ircadian patterns of prolactin release in entire rams (Chamley et al. , 1 974) and vasectomized ra?? (Davis and Borger, 1 973 ; S . L. Davis , pers onal communication) . However Chamley e t al. ( 1 971 .. ) , measured prolactin levels in " pooled" two-hourly samples , s o they c ould have failed to detect short peaks of prolactin re lease ; als o presumably they had no control over poss ible ? degradation or loss of immunoreactivity of the hormone during each two-hour collection period. Also Davis and Borger ( 1 973 ) may have c onducted their s tudy at a time of the year when circadian patterns of prolactin release did not occur; more over blood sampling was performed only two weeks after placement of the animals in metabolism crates , so the ram3 may not have become accustomed to the ir surroundings whe n the experiments were carried out . The nocturnal rise in plasma prolactin leve ls shown by sham? operated rams in Experiment 7.1 c ould not be attributed t o feeding or stress ? Although its onset was during the early afternoon, it appeared to increase sharply at about the time that the lights were switched off ( 1 9.30 h) . The apparent relationship between the rise in prolactin leve ls and the chc:.ng::: from light t o dark.ness may have involved an alteration in pineal gland activity in response t o the change in intensity of photic stimulation of the eye s . Although the possibility of pineal gland involvement in this mechanism is spe c ulat ive, the fact that this nocturnal e levation of plasma prolactin leve ls appeared to be abolished in pinealectomized rams provided s trong support for the idea. It is possible that the prolactin secretion patterns of the two groups of rams might have bee n proven to be s ignificantly 276 differe nt if a more appropriate statis tical test had been applied. The technique of fitt ing a linear regre ssion to the cumulative output probably was inadequate for dete ction of the type of nocturnal prolactin peak displayed by the sham-operated rams . This short? c oming may have been ove rcome by fitt ing polynomial expressions t o the logarithms of the hormone c once ntrations , or by performing non? parametric analyses , although this latter technique usually re quires larger numbers of differe nt observat ions t o show significant differences . As the data has been inspected, it would be inappropriate t o apply further tests of s ignificance in the analys is of these data. In Experire nt 6, the pineal gland was s hown to be involved in the prolactin response to changing daily photoperiod, as the respo??e was diminished in pineale ctomized rams . The poss ible involveme nt of the pineal gland in the prolactin response t o diurnal light-dark changes provided evidence that the pineal gland influences the effe cts of both short term (daily ) and long term ( seasonal ) changes in photo? period on neuroendocrine mechanisms in rams . There did not appear to be any ass ociation between the plasma LH and prolact in leve ls rec orded in Experiment 7.1 . Likewise , similar disso c iations betwee n LH and prolactin secretion patterns have bee n observed in ewes (Butler et al. , 1 972 ; Fell et al. , 1 972 ) and in men (McNeilly et al. , 1 974) . Als o, peaks of prolactin re lease in bulls did not coincide with serum tes tosterone pe aks ( Smith et al. , 1 973 ) . Thus the reproductive significance , if any, of a nocturnal peak of prolactin secretion in rams is difficult t o assess . The effects of stress , including stress of venepuncture, on prolactin release in ruminants have been reported by a number of authors (Johke , 1 969 ; Raud, Kidqy and Odell, 1 971 ; Butler et al. , 1 972 ; Hart, 1 973 ) . In all cases , stress e levated plasma prolactin 277 leve ls . Temporary elevation of plasma prolactin leve ls was shown by maey of the rams studied in Experime nts 7.2 and 7.3 and indicated that caution must be exercised whe n comparing the results of experiments in which blood was c olle cted by venepuncture with those in which indwe lling cannulae were used . Also, it must be re c ognised that differences in plasma prolactin leve ls between groups of rams studied in this thesis (except Experiment 7. 1 ) may refle ct differences in response to the stress of venepuncture , rather than differe nces in the hormone leve ls in undis turbed animals . However, stress e ffects could not have accounted for differences in se as onal or daily patterns o f prolactin secretion between the various groups of rams , as s uch patterns were dependent on re lative changes in hormone leve ls rather than on differences in absolute concentrations. Nevertheless , it is probably better to stuqy prolactin leve ls in s heep which have been cannulated on the day be fore s ampling ( Forbes e t al. , 1 975 ) . I n Experiment 7.2 , higher prolactin output o f pirealectomized rams under both lighting regimes refle cted the general pattern of differences betwe en the groups shown in early March in Experim::mt 6. In that experiment pinealectomized rams maintained re lative ly high and c onstant plasma prolactin levels throughout the whole nine months . On the other hand sham-operated rams under Normal lighting showed diminishing plasma prolactin conce ntrat ions and those on the Reversed lighting regime had increas ing leve ls of this hormone . These previous observations account for the prese nt results from s ham-operated rams , including the low prolactin leve ls recorded from those on the Normal lighting treatment. Results from Experirrent 7.3 c onfirzred those of Pelletier ( 1 973 ) and those of Experiment 6 which showed that plasma prolactin leve ls were reduced in rams exposed to shortened daily photoperiods . This conc lusion was indicated by the lowered prolactin output recorded 278 from sham-operated rams on Normal lighting in May. No s imilar depression of prolactin output occurred in pineale ctomized rams on Normal lighting; this result provided further evidence that the pineal gland influences the e ffects of seas onal changes in photoperiod on prolactin secretion by rams. A longer sampling period would have bee n re quired to determine whether the suggestion of an afternoon rise in plasma prolactin levels in pinealectomized rams resembled that recorded from sham-operated rams over a twe nty-six hour period (Experiment 7.1 ) . A c ircadian pattern of prolactin secretion by pinealectomized rams would be difficult to explain, as the evide nce from Experiments 6, 7. 1 and 7.2 indicated that seasonal, and probably circadian, patterns of prolactin secretion we re abolished in pineal? ectomized rams , because the pineal gland diminished endocrine responses t o changing light stimuli. A change in susceptibility of pineal- ectomized rams to stress of venepuncture could explain a rise in plasma prolactin leve ls , but there was no indicat ion of a similar change in Experime nt 7.2 . Although it is probable that the afternoon prolactin elevation was a random event , a longer-duration study us ing indwe lling cannulae for blood sampling would be necessary to clarify this point. (4) Cortisol It is assumed that the pulsatile pattern of c ortis ol se cretion recorded in Experiment 7.1 reflected the pattern of ACTH secretion by the anterior pituitary. The indication of a diurnal pattern of cortis ol secretion, with e levated leve ls during the morning daylight hours , c onfirmed s imilar findings in intact ( McNatty, C ashmere and Young, 1 972 ) and ovariectomized ewes (Butler et al. , 1 972 ) . A slightly different diurnal pattern in rams was reported by Holley, Beckman and Evans (1 975 ) , who found om peak at 1 6 . 00 h and a second smaller peak at approximate ly 04.00 h. The se authors s uggested that 279 the 1 6 . 00 h peak may have resulted from the animals being fed at that t ime each day. Disturbance of the rams during feeding and cleaning operations may have contributed to the morning peak of cortisol levels seen in the present experiment. A s imilar explanation c ould not account for the results of McN.:ttty, C ashmere and Young ( 1 972 ) , whose animals were fed ad libitum; however these authors did not state when c leaning operations were performed. Two of the four pinealectomized rams (Nos . 54 and 63 ) showed c ortisol secretion patterns which indicated that pinealectomy abolished the normal diurnal pattern of secretion. A s imilar, but also inconclusive trend was recorded from ewes by Thurley, Gibb and Russell ( 1 975 ) . Although it has been reported that the pineal gland may suppresn adrenal c ortical function in rats (Defronzo and Roth, 1 972 ) and mice (Dickson and Hasty, 1 972 ) , there is insufficient evide nce available to support aey suggestion of a link between pineal gland and adrenal cortex function in sheep. Coghlan e t al. ( 1 960 ) found that pil".ealectozey did not appear to alter electrolyte balance in two salt? depleted ewes , which indicated that the pineal gland at least did not influence mineralocortic oid secretion by the adrenal c ortex. Further studies , similar t o Experiment 7.1 , would be required to investigate poss ible pineal-adrenal cortex links in detail, but such experiments would be of only peripheral interest to the _ s tudy of reproductive endocrinology. ( 5 ) General Discussion Hormone secretion profiles studied in the experiments described in this chapter have demonstrated that the anterior pituit? of rams releases hormones in a highly pulsatile manner. It j_s difficult to sa:y whether this reflects : a dynamic and highly variable pattern of control from the hypothalamus ; a c onstant reverberation between hormone secretion and negative feedback; o r the c ombined effects of 280 both type3 of c ontrol. The independent fluctuations in plasma leve ls of LH, prolactin, and c ort isol argue strongly for indepe ndent mechanisma regulating the release of these hormones, and probably ACTH as we ll. More information about the c ircadian patterna of secretion of these hormones and the poss ible influence of the pineal gland on such patterns , would undoubted? have been obtained by performing twenty-six hour sampling studies s imilar to Experiment 7.1 at monthly intervals . Such a sampling procedure would have amplified the findings of Experiment 6, in which pinealectomized and s ham-operated rams on c ontrasting lighting regimes were housed for a period of nine months . Ma? of the results which mere ly indicated possible patterns of hormone secretion, or showed non-s ignificant trends , would have bee n firmly established by a more intensive study. In spite of the s hortc omings in experimental technique outlir..ad above , results from Experiments 7.2 and 7.3 indicated that seas onal changes in the secretion patterns of LH and prolactin in rams did occur. A s imilar observation, based on plasma LH data from rams was made by Katongole , Naftolin and Short ( 1 974 ) who suggested that it was the fre quency of discharge , rather than the peak horiLone leve 1, that altered during the course of the year. This statement was confirmed recently by Lincoln (1 976a ) who described an increased - frequency of plasma LH peaks recorded in S oay rams during the period of testicular activity. In experiments des igned to investigate seas onal c hanges in hormone production by measuring hormone levels in single weekly or monthly blood samples , the need to sample at the same time of the day is firmly es tablished by the probable existence of regular c ircadian secretion patterns, particularly in the case of prolactin and cortisol. The present group of acute studies has als o highlighted the fact that studies based on s ingle blood samples each week or month do not provide the complete picture about seasonal effe cts of hormone production. C HAPI'ER VIII GE??RAL DISCUSSION A?? C ONCLUSIONS The experiments which have been described in this thesis were de signed to establish basic information about the degree of seasonal variations in reproductive parameters , particularly in New Zealand Romney rams . In addition to providing data on seas onality of semen 28 1 production, i t was hoped to obtain c omparable data on plasma leve ls of the hormones which ? have influenced the reproductive tract. Final:!J', as a result of c onducting experiments which involved surgical modification of the nervous system, and artificial manipulat ion of the environment, an attempt was made to e lucidate the neuroendocrine and e nvironmental mechanisms modulating the seasonality of reproduction in N.Z . Romney rams. Experiments 3 , 4 and 5 did not produce such 'definitive results as expected, large ly due t o small numbers of animals , insuffic ient frequency of s ampling, and the relative ly brief duration of the studie s . These deficienc ies arose large ly because of the dearth of previous information on these characteristics for the N. Z . Romney ram, and also because the experiments were tine-sequenced in such a manner that it was not possible to utilize data from the earlier two studies when des igning Experiment 5. Neverthe less the initial three experiments did provide information which was valuable for the des ign of Experiments 6, 7.1 , 7. 2 and 7. 3. , In the latter experinents important observations were made on the role of daily photoperiod as the principal stimulus for seasonal fluctuations in reproduction of rams, and indicated that the pineal gland was a vital mediator of these reproductive effects of photic stimuli. 1 . SEASONALITY OF REPRODUCTION IN GRAZING RAMS 2 82 Pelle t i er ( 1971) point ed out that s eas onally rhythmic changes in plasma LH leve ls of rams were more pronounc e d when they were sub? j e c t e d t o a s ix-monthly photoper iodic cycle rather than the normal twelve month cycl e . This obs ervat ion by Pelle t ier may have ac count ed for the relat ively small number o f s i gnif icant changes in plasma LH levels recorded in Experiments 5 and 6 in which the annual cycle of daily photoper iodic change was simulat e d . Furthermore , the photoper i od i c s t imulat ion of LH s e cret ion in rams reported by L incoln ( 1976?) may have been partly the resul t of natural seas onal changes , on t o which a shortened ( 32 week ) phot oper iodic cycle was super impose d . However in Exper iment 3 , grazing rams displayed a seas onal pat t ern of changes in plasma LH levels which r eached peak values during m i dsummer . Although a summer p eak o f LH s e cret ion might allow a p er iod for conditioning gonadal t issue pri or to the bre ed ing s eason , this result was not expected s ince peaks of LH s e cr e t ion dur ing autumn were reported by Pelle t i er ( 1971 ) , whilst other workers had not f ound any signifi cant seas onal changes in plasma LH concentrat ions ( Sanford , Palmer and H owland , 1974?) . On the o ther hand , Hochereau-de Reviers , Loir and .Pellet ier ( 1976 ) also have described e levated plasma LH levels in rams during midsummer months . S everal points ment ic?ed in Chapt er VI I , about th e need f or an increased plasma sampling frequenc y , may par tially ?ave a c count ed f or di fferenc es in results between the pr esent and previous s tudi es , par t icularly the lack of s i gnifi cant s easonal e ffects in the work o f Sanford , Palmer and Howland ( 1974?) . The low LH values ob? tained throughout th e present experiments raised some doubts concern? ing the accuracy of abs olute hormone conc entrat i ons obta ined wi th the present LH assay . However , the appropriate assay validat ion tests were adequa t e t o dispel such doubts , an d furthermore , there was no reason to suspect inaccuracies in the patterna of changing LH secretion obtained when using this ass?. 283 In contrast to the LH results , plasma testosterone and prolactin leve ls followed clearly de fined annual cycles in rams of all three breeds . Regardless of any spermatogenic effects of testosterone , the annual cycle of change s in secretion of this hormone undoubtedly is of major importance to the seas or.ality of reproduction in rams through its influence on libido. Difficulties in obtaining semen when using an artificial vagina during the winter months were e ncountered in Experiments 3 and 4, as well as by S?h and Gordon ( 1 967) . It was assumed that this observation represented a seas onal depression of libido related to low plasma testosterone conce ntrat ions. Possible implications of the seas onal pattern of prolactin se cretion are discussed later (Section 3 ) . I n Experiment 3 , N. Z . Romney rams displayed marked seas o.nal fluctuations in seminal fructose levels as we ll as in plasma levels of LH, testosterone and prolactin. Polled Dorset and Merino rams also displayed annual changes in these parameters, which were s imilar to those recorded from N. Z . Romneys, except that the seminal fructose levels of Polled Dorset rams and plasma LH levels of Werino rams , did not follow such clearly-defined patterns . Autumnal peaks o f seminal fructose production in all three breeds of rams confirmed s imilar patterns reported from other breeds ( Hiroe ? ? ? 1 960; Amir and Volcani, . 1 965 ) . D istinct seas onal patterns of plasma hormone and seminal fructose leve ls in Merino and Polled Dorset rams did not reflect the less well-defined breeding seasons of ewe s of these breeds reported by Hafez (1 952 ) . Together, these results may have indicated c onsiderable sex differences in reproductive seas onality, or the fact that seas onal changes in plasma hormone leve ls do not necessarily have to c oinc ide 284 with changes in other reproductive characteristics . Although fructose levels in semen showed annual rhyth!Il3 in this study, other se Ire n parameters tended to be more variable and did not display autumnal pea.lal. This differe nce from results o f previo\.13 reports in the literature ( see review by Lodge ar? Salisbury, 1 970 ) may have resulted from the relative ly mild seasonal changes in climatic conditions in New Zealand. The e nforced change in semen colle ction te chnique during Experiments 3 and 4 also may have masked seas onal changes in s ome seminal parameters .. A more likely explanation was that at Palmerston North (latitude 40?21 ' S ) the annual changes in daily photoperiod were less marked than at the higher latitudes where much of the other reported research has taken place . Ma? seminal parameters studied in this experiment are not highly c orrelated with fertility (Wiggins , Terrill and Emik, 1 953 ; Hulet a?d Ercanbrack, 1 962 ; Hulet, Foote and Blackwe ll, 1 964) , so it was not poss ible to comment on seas onal changes in relative fertility of the semen. In view of this shortcoming, investigation of semen with respect t o fertility may have been a worthwhile exercise , if not fraught with enormous practical difficulties . Stress tests such as viability after incubation at bo? temperature (Ludwick, Olds and Carpenter, 1 948; Buckner, Willett and Bayley, 1 954) , or after freezing (Colas et al. , 1 972 ) , provide more a?ate indirect methods for assessing the fertilizing capacity of semen, and may have bee n useful additions to the present semen studies . Surgical modification of olfactory and pineal gland functions in Experiment 4 did not alter the seasonal changes in plasma hormone levels or semen production of rams t o any great extent . These treatments did, however, provide s ome insight into which neural structures might have mediated the normal responses to environmental changes . Removal of the olfactor,y bulbs altered the regular seasonal cycles of changes in plasrr? LH and testosterone c once ntrations , s o it was pos sible that olfactory stimuli provided some regulatory influence over hormonal secretion patterns . Olfactory cues c ould have been derived from 285 pheromones produced by ewes , or other rams , during the breeding season. Evidence that pheromones produced by rams stimulated oestrous activity in ewe s at the onset of the breeding seas on has bee n presented by Schinckel ( 1 954) and Morgan, Arnold and Lindsay (1 972 ) . It has been suggested that in s ome male ungulates s uch olfactory functions involve the vomeronasal organ (Estes , 1 972 ) . In the present studies the function of this organ was not studied directly, but olfactory bulbecto? m? have modified vomeronasal function (Alberts , 1 974; Powers and Wina?, 1 975 ) . Significant e ffects o f cranial cervical ganglionecto? on pineal ce ll volumes and RIO?? activity, indicated that this surgical procedure modified pineal gland activity. C onsequently the disrupt? ion of regular se asonal changes in LH and testosterone secretion patterns in ganglionectomized rams in Experiment 4 was attributed to altered pineal function. A major difficulty arose from the finding that seminal fructose levels continued to display regular seasonal changes in all surgically treated groups of rams in Experiment 4 (Figure 4.2 ) . Since seminal fructose levels were expected t o reflect changes in plasma testosterone c oncentration, regular vatterns of seminal fructose output from these rams were difficult to explain. However, inspection of the annual fluctuations in seminal fruct ose , plasma testosterom and plasma LH levels recorded in Experi.mnt 4 (Figures 4.2, 4.3 and 4.4) revealed that normal seas onal rhythms were disrupted most of all in the case of LH, to a lesser extent for testosterone, and least for seminal fructose . A te ntative qypothesia could have been that the pineal gland an4/or olfactory system exerted direct influences on LH release , s o that parameters 286 further removed from these direct influences were more likely to retain res idual seasonal rhythms . Alternative?, it must be c oncluded that acces sory sex gland function can be regulated by factors for which seas onality of act ivity was not abolished by e ither surgical treatment . The recent deve lopment of a radioimmunoassay for plasma melatonin leve ls in sheep (Rollag and Niswender, 1 976 ) will allow iwTestigation of the e ffects of ganglionectomy and olfactory bulbe cto? on a ? seas or?l pattern of secretion of this c ompound. With respe ct to plasma prolactin data and much of the autopsy data, it was noted that the untreated and olfactory bulbectomized groups were similar to each other; also there were few major differences betwe en the ganglionectomized and bulbe ctomizeq/gar?lion? ectomized groups . A8 a c onsequence , it was concluded that the changes shown by the double-operated groups resulted principally from the ganglionecto? operation, and there was no synergism between olfactory and pineal influences on these parameters . Likewise, there was no reas on to suspect that e ither surgical treatment reversed or nullified any e ffe cts of the other, a possibility that was suggested by work on blinded anosmic rats (Blask and Reiter, 1 975 ) . Moule , Braden and Mattner ( 1 966 ) ascribed seasonal c hanges in seminal fructose concentrations recorded from rams on pasture s olely t o changes in the quality and quantity o f the forage available . However, that stu4y was c onducted in Australia where severe nutrit ional and temperature stresses can be expected; also seasonal c hanges in dail? photoperiod would have been smaller because of the lower latitude at which their experiment was performed. In Experiments ? 3 and 4 rams were never underfed, while adverse effe cts of temperature on the ir reproductive systems could be ruled out because extreme temperatures did not occur (Figure 3.7 ) . Nevertheles s temperature may be an important e nvirorur.ental factor in other regions of the world (VanDemark 287 and Free, 1 970) . A major hypothesis in this thesis was that the most important e nvironmental factor contributing t o the seasonal pattern of reproduction in ra?s was daily photoperiod. It has been shown in Experiment 4 that the pineal gland might have been involved in mediating the influence of changing daily photoperiod on reproduction in rams ; this influence presumably was caused by optic st imuli which reached the pineal glarn by w? of the cranial cervical ganglia. Since the pineal gland of ganglionectomized rama could not r?rce ive photoperiodic changes via this neural pathway, ganglionectorr;y should have produced results e quivalent to pinealectomy. Furthermore blinding would be expected to generate results s imilcu? to those rec orded following ganglionecto?, since blinding would abolish optio activity. As these assumptions have not been explored, future fie ld experiments with blinded and/or hooded rams, as well as pinealectomized rams , would appear to be jus tified. 2 . EFFECTS OF LIGHl'ING REGIMES ON REPRODUCTION I N RAMS In the ir review of the photoperiodic control of gonadal and ,hypophyseal act ivity in domestic animals , Ortavant, Maule on arn Thibault (1 964 ) deduced a set of four hypotheses : 1 . An optimal photoperiod t?or gonadal stimulation existed, this being ten to eleven hours per day for most rams. 2. Continued exposure t o the optimal photoperiod did not sustain gonadal activity indefinite ly, which suggested the existence of a " refract ory period" which varied according to the species or breed, and to the preceding photoperiodic treatnent. 3. Light facilitated the release of gonadotrophins whereas darkness favoured the ir synthesis . 4. Sensitivity to photoperiodic stimulation varied between 288 breeds and between individual animals , as well as between specie s . These deductions of Ortavant, Mauleon and Thibault ( 1 964) were based on the literature available at that time as well as on their own studies with rams and ewe s . The ir studies had included observations of : spermatogenic activity in testicular tissue , te sticular and epididYmal we ights, and pituitary gonadotrophin measurements . Further to the above hypotheses is the question of whether seas onal patterns of reproductive activity are controlled by decreas ing or increasing daily photoperiods . Yeates ( 1 949 ) c oncluded that the autumnal peak of breeding activity in s heep was induced by the period of increasing daily photoperiods during the previous spring. In c ontrast, more recent evide nce has shown that increased spermatogenic activity in rams was a direct response to decreasing daily photoperiods (Ortavant and Thibault, 1 956 ; . Ortavant , 1 961 ) . The findings o f Fe lletier and Ortavant ( 1 975?) showed that de creasing daily photoperiods were stimulatory to LH re lease in rams , whereas increasing phcto?iods were inhibitory or less stimulatory; this result corresponded with that of a previous st? by Pelletier (1 971 ) . Likewise , an abrupt decrease in daily photoperiod produced high plasma leve ls in Soay rams (Lincoln, 1 976?) . There was not always such a close relationship between decreasing daily photoperiods and plasma LH levels in the experiments des cribed in this thesis . For instance , in rams on pasture (ExperiiOO nt 3 ) , maximal plasma LH levels were rec orded during midsummer rather than in the autumn period, yet for rams on artificial lighting regimes (Experiments 5 and 6 ) plasma LH data did not provide c onclusive evidence of any relationship with photoperiod. Part of the difficulty in reconciling the present results with those of other workers may have arisen from differences in breeds , latitudes , LH ass?s, or the photoperiodic cycles to which the animals were subjected. Beak levels of testonterone secretion were ass ociated with the late summer-early autumn months in rams on pasture (Experimant 3 ) , and with decreasing and short daily photoperiods in Experiments 5 and 6. These results for plasma testosterone levels gave substance to conclusions reached by Pelletier ( 1 971 ) arid Fellet ier and Ortavant ( 1 975a) , which were based on plasma LH levels of rams subjected t o sLx-monthly photoperiodic cycle s . In addition, the e ffects of lighting regimes on testes and acces s ory sex glands recorded in Experiment 6 confirmed the findings of Ortavant and Thibault ( 1 956 ) . It is interes ting to speculate about whe ther or not the French workers would have reached the s ame conclusions had they used twe lve-monthly instead of s ix-monthly photoperiodic cycle s . This group of authors apparent? has never published plasma testosterore data from rams submitted to artificial lighting regimes , although Felletier and Ortavant ( 1 975k) have des cribed the e ffects of tes tosterone propionate injections on pituitary and plasma LH levels in s uch rams . Their res ults shovred that the negative feedback of testosterone on LH re lease was less effe ctive during short daily photoperiods ( 8 h light ) than during long daily photoperiods ( 1 6 h light ) . Also they pointed out that alteration in sensitivity to andro@Bn feedback might be more important in natural ( 1 2-monthly) lighting cycles , s ince in the latter case light may be a less effective modifier of LH re lease beca ??e of its reduced rate of chan@B compared to that in a s ix-monthly cycle . A model for the c ontrol of gonadotrophin secretion by light and gonadal steroid negative feedback was proposed by Hoffmann ( 1 973 ) . The major c omponent of this model was a hypothetical comparator which c ontrolled releas ing hormone secretion in response to changes in gonadal s teroid feedback, plus st imuli from another source which in turn depended on photic information. Two possibilitie s for the 290 control of gonadotrophin release arose from the moda l proposed by Hoffmann. One such possibility required re leasing hormone output t o alter directly under the influence of light-derived stimuli and steriod levels . The other poss ible mechanis!Il3 involved an alteration in eypothalamic sensitivity to steroid feedback in response to changes in photic stimuli. Although Pelletier and Ortavant (1 968) found that hypothalamic LH-releas ing activity was higher in rams on s ixteen hours light per day than in those on e ight hours per day, t his result did not distinguish be tween impaired secretion of the releasing hormone ( causing a build up in eypothalamic levels ) or its increased synthesis and release , under long dai? photoperiods . However alterations in releas ing hormone secretion are consistent with the mechanisms proposed by Hoffmann. Another nypothetical model to account for the influence of photoperiod on the c ontrol of gonadotrophin secretion in rams was postulated by Felletier and Ortavant (1 975b ) . This particular model embodie d the proposals of Hoffmann and the authors made the f'ollovring statement about the influence of the photoperiod : " de creas ing light photoperiod has two effects 1 . stimulation of gonadotrophin release 2 . lowering of the intensity of the negative feedback of testicular androgens . Conversely, increas ing light photoperiod 1 . is inhibito? or at least less stimulator,y to the LH release , 2. increases the negative feedback effect of androgens on hypothalamo-hypopeyseal activity." These proposals of Felletier and Ortavant are compatible with the results obtained in the present project . In fact t he work described in this thesis extend? the ir hypotheses by providing positive evidence that the pineal gland is involved in the photo? periodic regulation of gonadotrophin secretion ( see later for discussion) . 291 In Experiments 5 and 6 the parameters affected most by phot operiod reversal were : plasma testosterone and prolactin levels , se minal fructose levels , testicular we ights, epididymal we ights, se minal ves icular we ights, epidi?al spermatozoalreserves , and the c oncentration and total conte nt of fructose in the seminal ves icles . S ignificant effects of lighting regimes on the parameters listed above indicated that gonadal activity was regulated by daily photo? period; no s ignificant influences of lighting regimes were recorded for plasma LH levels , other semen characteristics, or the remaining autopsy data, largely because of the high degree of variability in these parameters , allied with the limited numbers of rams per group. Although Experiments 5 and 6 did not c lari? whether the e ffects of photoperiod on gonadal activity were mediated by changes in LH secretion patterns, French workers have recorded LH secretion patter? which would support such a c onclusion (Felletier, 1 971 ; Pelletier and Ortavant, 1 975?) . It is now clear that s ince LH is secreted in a highly pulsatile manner, groups of experimental animals must be larger if s ignificant differences in the patterns of secretion of this hormone are to be detected. Nevertheless the poss ibility remains that gonadotrophins other than LH may have been involved in photoperiodic e ffects on gonadal act ivity. Results of research, mainly carried out with rats, has shown that FSH may not have any significant funct ion in the control of spermatogenesis in adult animals ( Ortavant, Courot and de Reviers , 1 969 ; Courot , Ortavant and de Reviers , 1 971 ) . However, as it is not known to what extent this statement about FSH can be applied to rams, 292 the possibility that this hormone c ould be involved in responses to altered dai? photoperiod can not be eliminated. A radioimmunoass? for ovine FSH was not available during the c ourse of tre present studie s and as yet workers at other laboratories have not published information on the FSH se cretion patterns of ra.Ill3 exposed to different lighting regimes. (In fact Linc oln ( 1 976,2) recently . presented a paper which indicated that both plasma levels and frequency of e pisodic release of FSH in Soay rams were stimulated by decreas ing daily photoperiods , and suppresse d by reversed lighting changes. ) In addition prolactin mqy function as a gonadotrophin in ra.Ill3 , and the present results have s hown that plasma concentrations of this hormone in rams were ve? respons ive to daily photoperiod . Programmes involving active immunization o f rDms against specific protein hormones such as LH, FSH, or prolactin, or the use of long? acting specific antagonists t o the s ecretion of these hormones , should provide useful data relating to the ir respective role s in seas onal reproduct ive changes . Long-term field studie s with rams s ub je cted t o such treatments would thus be useful adjuncts t o the experiments in this the s is . 3 . REPRODUCTIVE FUNCTIONS OF PROLACTIN IN RAMS Seasonal changes in plasma levels of LH and prolactin rec orded in Experiment 3 indicated that b oth hormones were secreted maximal? during midsummer, which suggested that their release was c ontrolled on a seas onal basis by the same e nvironmental factor, name ly daily photo? period. French studies with rams subjected to artificial lighting regimes showed that although se cretion of both prolactin and LH in ram3 was influenced by photoperiod, plasma prolactin levels were highest in rams subjected to long daily photoperiods (Felletier, 1 973 ) whilst LH levels were highest in rams subje cted to short or decreasing daily 293 photoperiod.3 (Pelle tier, 1 971 ; Thlletier and Ortavant, 1 975_!) . This appare nt relationship between the timing of the maximal rates of secretion of prolactin and LH was supported in Experiroo nts 5 and 6 by the timing of plasma prolactin and testosterone pe rucs . However, plasma LH res ults in these two experiments were inconc lusive , while results from Experirrent 3 indicated that maximal rates of LH secretion occurred approximately one month before the highest peak of testosterone secretion. In Experiment 6, sham-operated rams subjected to the Normal lighting regime had the highest values for parameters ass ociated with gonadal and accessory sex gland act ivity at autopsy eve n though these ram3 had the lowest plasma prolactin levels . None of the above findings excluded the possibility that prolactin may have been involved in reproductive seasonality in rams as a " conditioning" hormone. Evidence from comparative studie s of the physiology of prolact in has s hown that this hormone is involved in a multitude of e ndocrire systems and also has a function in various e ndocrire tissue s " c onditioning" their respons iveress to their specific trophic hormones (Nicol l , 1 973 ) . The elevated plasma prolactin leve ls recorded during exposure of rams t o long daily photoperiods in the experiments in this thesis may have conditiornd the ir reproductive organs to the influence of LH, so that peaks of reproductive organ activity c ould follow during the period of decreasing daily photo? periods. In other words, prior exposure of reproductive tissue s to high levels of prolactin m? have be e n a pre-requisite for optimal responses t o gonadotrophic s timulation, even though the exposure to raised plasma prolactin leve ls m? have pre ceded plasma te stosterone and seminal fructose peaks by approximately two and three months , respectively. Since the period of elevated plasma prolactin levels recorded from rams on pasture (Experiment 3 ) overlapped that of plasma LH, 294 the poss ibility that these two hormones act syr?rgistically can not be overlooked. Evide nce for such synergism between prolactin and LH, in male rats has been summarized in Chapter I. 4. PINEAL GLAND FUNCTION AND REPRODUCTION IN RAMS Semen from grazing cranial cervical ganglionectomized rams studied in Experirrent l.,. exhibited much greater fluctuat ions in sperm? atozoal n?nbers , motility indices and perce ntages of unstained sperm? atozoa than that from c ontrol rams . Als o, in the ganglionectomized rams , the normal seas onal patterns of change in plasma LH and prolactin levels were disrupted. At autopsy, testicular and epididymal we ights of gangliorectomized rams were e levated, while seminal fructose levels were below those of the c ontrol rams. As discussed earlier, these e ffects of ganglionecto? probably c ould be attributed to altered pineal gland function. Likewise , much of the plasma LH, testosterone and prolactin data from Experiments 6, 7.1 , 7 .2 and 7.3, revealed s ignificant effe cts of pineale cto?, and s ignificant Lighting Regimes x Operations interactions . These s ignificant findings provided direct evidence that the pineal g?and did influence reproductive function in rams, and als o that this gland was an important regulator of prolactin secretion. Because cranial cervical ganglionectomized rams were not studied under identical c onditions to those used in the later experiments involving pinealectomized rams , definitive statements about the effectiveness of ganglionecto? in paralleling the e ffects of pineal? e ctoll\Y can not be made , even though cranial cervical ganglionectoll\Y was effective in depres sing pineal e nzyme act ivity and cell volumes. Support for the c onclusion that the pimal gland is re quired for the expression of seas onal patterns of reproductive activity c ould undoubtedly be provided by studying grazing pinealectomized rams over 295 a period of ooo or more years . If such an experiment also included ganglionectomized rams, s o?e of the questions raised by the prenent work should be answered. Fighting amongst rams which had undergoro surgical removal of the pineal gland would have to be avoided by methods such as individual paddock grazing or tethering. Most reports of the effects of the pineal gland on reproductive function in rats , golden hamsters , and ferrets have favoured the c oncept that this gland is antigonadotrophic (Reiter, 1 973?) . C onsequently the onset of breeding ? activity in these animals must result from inhibition of pineal gland act ivity, which supposedly occurs during increased light exposure. However in an experiment utilizing long versus s hort daily phot operiods , and pineale ct omized versus sham-operated female hamsters , s ignificant Light ing Regimes x Operat ions interactions indicated that the pineal gland also could be pro gonadotrophic (Hoffma.nn and Re iter, 1 966 ) . A progonadotrophic function of the pineal gland of rams was suggested by data from Experiment 6 , since in rams subjected to the normal lighting regime, parameters related to gonadal and accessory sex gland activity were higher in sha?operated than pinealectomized rams . Research on pineal function in sheep has been very limited, but evidence from other species has indicated the possible existence of several mechanisms of action. As the pineal gland c ontains high levels of melatonin and serotonin, these and other closely related indoleamines are cor.sidered to be the most likely mediators of. pineal antigonadotrophic actions (Fraschini, C ollu and Martini, 1 971 ) . Serotonin suppressed the spontaneous LH release in castrated rams (Riggs and Malven, 1 974) . In ewes melatonin preve nted the post? castration rise in plasma LH levels (Roche et al. , 1 970b ) , while both melatonin and serotonin blocked the pre-ovulatory peak of LH secretion and prolonged oestrous cycles (Domanski et al. , 1 975 ) . In 296 their report, Domanski et al., described inhibition of LH release in ewes with les ions of the anterior hypothalamic area (AHA), which indicated that the inhibitor,y actions of melatonin and serotonin occurred in the region of the medial basal hypothalamus. This cor?lusion was based on the fact that these lesions blocked the normal inhibitor.y influences of the AHA over GnRH secretion, so the effects of the indoleamines must have occurred nearer to the s ite of GnRH secretion. Antigonadotrophic peptides from sheep pineal glands have been studied using in vitro systems with pituitar.y tissue from rodents (Moszkowska et al. , 1 974; Ebels, 1 9 75); as yet comparable research with ovire pituitary tissue has not been reported. C onsidering the high antigonadotrophic potency of s ome of these pineal peptides, they must be regarded as potential regulators of reproductive seas onality in rams, and s o merit further study. Such research could include immunologic investigations , using antisera raised against these peptides conjugated to proteins. Since no potentially progonadotrophic substances had previously been isolated from the pineal gland, the f'inding of high levels of immunoass?able GnRH in sheep pineal glands (\fhite et al, 1 974) has provided at least one possible explanation for the progonadotrophic property of this organ. Another possible mechanism of action of pineai principles could involve direct effects on the gonads themselves. For example, in rat testes Liu and Kinson ( 1 973) described inhibitory effects of melatonin on the production of testosterone, and of serotonin on spermatogensis . These findings indicated the need for investigation of pineal-eonadal interrelationships in rams. Evidence of gonadal steroid regulation of pineal function has been presented in Chapter I . This evidence was c onfined to studies with rodents because of the 297 paucity of s imilar studies in large domestic animals . Results from Experiment 6 showed that the pineal gland altered the e ndocrine statu.s of rams? probably entirely due t o its own responses t o changes in daily photoperiod. This finding was not surprising s ince the pineal gland has been described as a neuro? e ndocrine transducer? instrumental in produc ing endocrine respon..'ies to photic stimuli (Axelrod, 1 974) . Possible interactions between photoperiod, the pineal gla??? and olfactor,y function can not, however, be overlooked cons idering the disrupted seasonal patterns of hormone secretion recorded from o lfactor,y bulbectomized and cranial cervical gangliorectomized rams, and also those subjected to both surgical treatments (Experiment 4 ) . Interactions between lighting and olfaction i n male rats were suggested by the fact that both blinding and olfactory bulbectonw were I'6 quired to produce re gres sion of their reproductive organs (Reiter et al . , 1 971 ) . In grazing rams ;:;.lthough e levation of plasma LH levels preceded the rise in plasma testosterone levels, LH output remained high during the period of peak plasma testosterone secretion (Figure 3 . 7 ) . As te stosterone inhibited the release of LH by a negative feedback effe ct (Pellet ier? 1 970 ) , the occurrence of high levels of both hormones at the same t ime of the year re quires discussion. Felletier and Ortavant ( 1 975?) considered that decreasing daily photo? periods lowered the sensitivity of the hypothalamo-hypophyseal system to the negative feedback of androgens, so this change in sensitivity could account for the c ontinued release of LH whe n plasma androgen levels were high. It is poss ible that the change in sensitivity to androgen feedback resulted from the production of pineal substances which altered hypothalamic function. The fact that the activity of the pineal gland could be modified directly by changes in daily photoperiod supported this hypothesis . Acute studies need to be carried out to study the effect of testosterone injections on LH secretion in rams in the presence or absence of the pineal gland or s ore of its principle s . Provided that the testosteror? dose used produced physiological leve ls of this hormone , such studies c ould confirm or refute this suggested role of the pineal gland, and thus help establish the c ontrol mechanism for the seas onality of reproduction in rams . 298 The existence of complex physiological interrelationships between neural structures and endocrine glands c onfirmed the c on..cept that the hypothalamus rece ives a wide variety of inputs and integrates these for the final expression of its regulation of anterior pituitary function. The individual role of acy input to the hypothalamus is difficult to discern because mammalian e ndocrine systems show a remarkable degree of con:pensation. This point was illustrated by the fact that in the present studies eve n though s ome of the surgical treatments had marked effects on the patterns of hormone secre?ion, there was no evidence of a clear impairment of reproductive function. Thus further experiments will be required t o e laborate the exact importance of the pineal gland and the olfactory system in the regulation of reproduction in rams . 5 ? POSSIBLE APPLICATIONS OF THE FRESENr FINDINGS Knowledge of the photoperiodicity of reproduction in ewes has been used to increase reproductive rates by allowing n greater frequency of lambings (William8, 1 970; Ducker and Bowman, 1 972 ; Newton and Betts , 1 972 ) . However the application of such knovrledge must not be confined to ewes since the problems ass oc iated with deep? freezing ram sexren have meant that high quality seme n may not be 299 available throughout the year. Exposure to artificially decreased daylight improved libido in rams in Queensland, Australia, and thw assisted out of seas on semen collection for artificial insemination programmes ( S . J. Miller, personal c ow?unication) . However, the general applicability of photoperiodicity in sheep is severely limited because of the impracticability of manipulating daily photoperiod for large numbers of s he ep. Surgical removal of pineal glands from rams might alter seas onal bree ding patterns and produce a relat ively constant high level of reproductive activity throughout the year. Technical difficulties involved in this procedure would prohibit its general use , although cranial cervical ganglionectomy or chemical sympathectomy (for example , using 6-hydroxy dopamine ) , might provide alternative means of altering pineal function. On the other hand s??tained production of high quality semen may not be possible , s ince Ortavant ( 1 961 ) has s hown that maximal spermatogenesis is maintained only for a limited period, and it is likely that the unknown limiting factors involved would operate in the same manner in pinealectomized rams . More likely, expanded knowledge of neuroendocrine control of reproduction in s heep will ass ist the development of pharmacological techniques which have practical agricultural benefits . For example, the use of steroids , prostaglandins and gonadotrophins : in such techniques as : induction of parturition or lactation, regression of corpora lutea, c ontraception, ovarian stimulation and superovulation, and treatment of infertility and impotence , has followed fundamental scientific studies on the endocrine control of reproduction. In the prese nt case, the demonstration that the pineal gland of rams can exert a controlling influence over reproductive function, paves the w? for further investigations t o determine the chemical nature of the substances involved. Such research c ould lead to the discovery of pharmacological age nts which improve prese nt day techniques of manipulating ovine reproduction. 300 3 0 1 .1\hJN;d , S . I . ( 1 9 5 .5 ) . 'J'he effe c t or tc ::d? 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( l 9 G4 ) . r?. ?c l ?J to11i 11 ::) ?:- :1tl1 ?.:: :--_; j_ 1 i ?1 the :-- i_ :,lc .. :? l ??: J . n rl?? : c :c _:"j?? c c c f l i _7l1 t F.l.e J i:.:! ?? e(1 b?r t.::. c 3? '? :;:..J. t 1?: ..; t ic r?:-:: 1?vous ?"j : r :--J t \3 : :: . S c i e nce , I\ . Y . J ! .-t3, .l) 2 {) -lJ)O . ?;;urL1Dn , H . J . , pineal . i\ :-??2 1 1'") 0:.1 , J . (:_ I??e \\? Ycr .. ?c c?r?.J :-:cl lJ , I. o !;.l or: D . ? . ( 1 9 63 ) . 'l'llc J'. ca.:J c': ic Fre .J s . Yea te s , ?: . ?r . r . ( 1 9l.J-9 ) . The br c e j_j_ nfi? seo. s on of she c ::; rli th p8 r ti cu la :c :L'??ferenc e t o i t s nccUf:L c a t ion by o. r t i fi c i o l rr.e?nG u s ir..G; l ight . J . s c;r i. c . Sc i . 22_, l-L?3 .