THE EFFECTS OF FEES RESTRECTSQR’ AND LIGH? CQHTRGL EN TURKEY E‘tEWQEGCYEVE E’HYQCLGGY Thesis got.- Nu Dogma o§ pk. D. MECHLGAN STATE UNEVEBSXTY Edsei Jorge Biixfier 5.967 'ME.EH:5 v.4¥;-1998\-s‘d “man; ........-‘_' LIBRA R 1" es" I H 4- t . I‘u’ltc' «a: _=- 9-. L ll...o.l.g glatc :2 [larva-,gggty This is to certify that the thesis entitled The Effects of Feed Restriction and Light Control on Turkey Reproductive Physiology presented by Edsel J. Bixler has been accepted towards fulfillment of the requirements for ’ ...——-—.. ’ f, .. I’ .' , ’ M) f, /‘/F --/ degree in / I ' Date 0-169 ABSTRACT THE EFFECTS OF FEED RESTRICTION AND LIGHT CONTROL IN TURKEY REPRODUCTIVE PHYSIOLOGY by Edsel Jorge Bixler Individual seminal volume, sperm counts, spermatocrits, motility scores, testicular weights, ductus deferens weight and sperm reserves were obtained for turkey toms under three different nutritional regimes. Data were also collected on fertility, hatchability, feed consumption, body weight, follicle stimulating hormone content and follicle stimu- lating hormone releasing factor concentration in the hypothalami. The time of oviposition and its relation to the light regime was also studied. Semen volume and sperm counts were increased when feed was with- held twice a week for a period of 24 hours prior to semen collection as compared to gd_1ibitum fed toms and those that had feed withheld 36 hours twice a week prior to semen collection. No significant differences were detected among groups with re— spect to motility scores, testicular weights and ductus deferens weights. An increase in spermatogenesis over that of the §g_libitum fed toms was observed in the testicular homogenates of the toms where the feed was withheld. The follicle stimulating hormone concentration of the pituitary and the follicle stimulating hormone releasing factor found in the Edsel Jorge Bixler hypothalamic extracts were increased in those treatment groups in which the feed was withheld when compared to the §d_libitum fed toms. Feed intake was significantly depressed when the toms were fasted twice a week for periods of 36 hours prior to semen collection when compared to those toms fed §d_libitum or deprived of feed for 24 hours twice a week prior to semen collection. No statistical difference (P > 0.05) was detected between the last two treatment groups. Heavier body weights were recorded for the §g_libitum fed toms when compared to the two treatment groups in which feed was withheld prior to the collection of semen. From the data collected, it appears that restricting the feed intake for 24 hours prior to the collection of semen stimulates sperma- togenesis and produces higher semen volumes. Oviposition time was changed by means of light manipulation. Using a windowless house and a light regime in which the lights came on at 8:00 p.m. and went off at 12 noon, it was possible to obtain approximately ninety percent of all eggs before noon. THE EFFECTS OF FEED RESTRICTION AND LIGHT CONTROL IN TURKEY REPRODUCTIVE PHYSIOLOGY By Edsel Jorge Bixler A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Poultry Science 1967 It is better not to know so much than to know so many things that are not so. Josh Billings ii ACKNOWLEDGEMENTS The author is most grateful to the Rockefeller Foundation who kindly granted him a scholarship which made it possible to attend Michigan State University. He is also grateful to Dr. Robert K. Ringer, Professor of Poultry Physiology for his continuous encouragement, coursework guidance and direction of this research project. Sincere appreciation is expressed to Drs. Cal J. Flegal, Theo H. Coleman, Harold D. Hafs, John H. Wolford, John L. Gill, Joseph Meites and Andres Negro Vilar who in one way or another gave assistance during the development of this thesis. The author also wishes to express his sincere appreciation to Dr. Howard C. Zindel, Chairman of the Department of Poultry Science, and to the people of the State of Michigan for allowing him to use the facilities of Michigan State University. He is also indebted to all those faculty members, fellow graduate students and technicians from whom he received help during the course of this work. Last, but not least, he appreciates the understanding and patience of his wife, Rosa Maria, as well as her continuous encouragement. iii TABLE OF CONTENTS Page I. ACKNOWLEDGEMENTS . . . . . . . . . . . . . . . . . . . . iii II. TABLE OF CONTENTS . . . . . . . . . . . . . . . . . . . . iv III. LIST OF TABLES . . . . . . . . . . . . . . . . . . . . . vi IV. LIST OF FIGURES . . . . . . . . . . . . . . . . . . . . . viii V. LIST OF APPENDICES . . . . . . . . . . . . . . . . . . . ix VI. INTRODUCTION . . . . . . . . . . . . . . . . . . . . . . 1 VII. LITERATURE REVIEW . . . . . . . . . . . . . . . . . . . . 3 A. Effect of Feed Restriction on Semen Output and its Characteristics . . . . . . . . . . . . . . . . . 3 B. Size of Testes and Semen Output . . . . . . . . . . . 10 C. Evaluation of the Sperm Reserves . . . . . . . . . . 11 D. Other Physiological Parameters Affected by Periods of Feed Deprivation . . . . . . . . . . . . . 12 E. Follicle Stimulating Hormone Releasing Factor . . . . 15 F. Effect of Light on Oviposition . . . . . . . . . . . 17 VIII. OBJECTIVES . . . . . . . . . . . . . . . . . . . . . . . 20 IX. MATERIALS AND METHODS . . . . . . . . . . . . . . . . . . 21 A. First Year . . . . . . . . . . . . . . . . . . . . . 27 B. Second Year . . . . . . . . . . . . . . . . . . . . . 28 C. FSH Bioassay . . . . . . . . . . . . . . . . . . . . 40 D. FSH-RF Assay . . . . . . . . . . . . . . . . . . . . 41 iv X. XI. XII. XIII. XIV. RESULTS AND DISCUSSION A. Effect of Feed Restriction During the Breeding Season . 1. Feed consumption 2. Body weight . 3. Semen volume 4. Number of spermatozoa per cubic milliliter S. Spermatocrit values . 6. Motility 7. Fertility and hatchability 8. Blood constituents 9. Organs and glands . 10. The relationship between testicular size and spermatozoal output . . . . . . . . . . . 11. Spermatogenesis and sperm reserves 12. Follicle stimulating hormone content of the pituitaries . 13. Presence and content of a follicle stimulating hormone releasing factor 14. Eschericia coli contamination . B. Effect of Light Pattern on Time of Oviposition 1. First experiment 2. Second experiment . . . . . . . . . . . . . . CONCLUSIONS . . . . . . . SUMMARY . . . . . . . . . . . . LITERATURE CITED APPENDIX Page 44 44 44 47 48 50 51 52 52 53 55 59 61 61 64 66 66 66 68 71 74 76 88 Table 10. 11. 12. 13. 14. LIST OF TABLES Criterium for motility evaluation . Experimental design of feeding patterns for both years . . Composition of the experimental ration Contrasts used for comparing the means Effect of feed being withheld twice weekly on feed consumption . . . . . . . . . . . . . . . . . Effect of feed being withheld twice weekly on body weight . . . . . . . . . . . . . . . Effect of feed being withheld twice weekly prior to semen collection on the volume of semen collected . Effect of feed being withheld twice weekly on spermatozoal concentration . . . . . . . Effect of feed being withheld twice weekly on Spermatocrit values and motility scores . Effects of feed being withheld twice weekly upon fertility and hatchability, expressed as percentage . Average values in percent of blood cells at the end of the last feed deprivation period . Average values of the three different treatments in blood glucose and cholesterol concentration at the end of the experimental period + the standard error of the mean . . . . . . . . . .—L . . . . . . . . . Effect of feed deprivation on certain gland and organ weights given as averages ::mean standard errors . Correlation values between testicular size and spermatozoal output during 1966 and 1967 vi Page 23 27 3O 41 47 49 49 51 52 53 54 55 58 6O Table 15. 16. 17. 18. Effect of feed deprivation upon spermatogenesis and sperm reserves . . . . . . . . . . . . Results obtained on the presence of FRF and its concentration in the hypothalamic extracts of the different treatment groups, represented as rat ovarian weights in milligrams . Effect of light pattern upon oviposition time during the year 1966 Effect of light pattern upon oviposition time during 1967 . vii Page 62 65 67 68 Figure LIST OF FIGURES Schematic diagram of reproductive tract in the abdominal cavity with sites of ligation indicated . Schematic diagram of left side of the reproductive tract indicating the sites of ligation before sectioning into testicle and ductus deferens Microscopic view of a ductus deferens homogenate as seen under phase contract illumination . Microscopic View of a testicular homogenate as seen under the phase contrast illumination Weekly feed consumption per tom during the year, 1966 . . . . . . . . . . . . . Weekly feed consumption per tom during the year, 1967 viii Page 32 34 36 38 45 46 Table II. III. IV. LIST OF APPENDICES Eosin solution for sperm counts . Procedure for cholesterol determinations in blood and adrenal glands Determination of blood sugar Follicle stimulating hormone (FSH) bioassay . ix Page 88 89 91 95 INTRODUCTION The poultry industry has become a highly competitive business and its ever increasing technical and scientific problems have become a continuous challenge for the scientist and husbandman. Breeding highly efficient birds for some desired traits has been done at the expense of some other desirable traits. Loss of these traits has re- sulted in new challenges to other fields of the industry such as physiology, nutrition, etc. For example, development of the Broad Breasted turkey, emphasizing a well developed breast and short legs to obtain meat type conformation, resulted in anatomical disadvantages in the toms. That this type of turkey is widely distributed among commercial farms has made artificial insemination imperative in almost every breeding flock. But, intense use of artificial insemination in commercial operations has created many problems. For example, the majority of the known nutritional re- quirements of breeding turkeys are based on females and little or no information is available for feeding males. There are also problems in collection of clean semen, in semen quality, evaluation techniques and in insemination methods. Field and experimental observations have shown that when both sexes receive the same breeder ration, the females lose weight during the laying season while the males gain weight. Thus, the nutritional requirements of males may differ from those of females during the l 2 breeding season. Therefore, the main purpose of this research was to evaluate, under controlled laboratory conditions, a practice of some turkey producers. In this system, poultrymen remove the feed from toms the night before semen is to be collected. Some claim to obtain larger volumes of semen with less contaminants at the following ejaculation. However, other producers report disastrous results with the same practice. The toms tend to go out of semen production. It is well accepted that obesity is negatively correlated with sexual drive and efficiency in farm animals. The fact that turkey toms become overweight during the breeding season prompted the following study of the effect of feed deprivation before semen collection upon the subsequent production of semen. LITERATURE REVIEW A. Effect of Feed Restriction on Semen Output and its Characteristics Since the development of a technique for artificial insemination in the fowl by Quinn and Burrows (1936) and its application to turkeys by Burrows and Marsden (1938), there has been increasing interest in avian reproduction. The use of artificial insemination in the turkey industry has been accepted as a common practice in the United States because the Broad Breasted type turkey is often physically unable to mate efficiently because of its conformation (Cooper, 1965). Artificial insemination has been shown by various workers to improve fecundity in these turkeys and be a useful tool in turkey breeding (MbCartney, 1951; MacIlraith, 1952). Cooper (1965) stated that the amount of semen which can be col- lected from a tom is dependent upon the influence of nutrition, manage— ment and the normal physiological processes regulating spermatogenesis as well as the training of the male and efficiency of man in obtaining the semen. None of these factors has been extensively studied. The influence of nutrition on semen production or its characteristics in birds has not been thoroughly investigated (Chubb, 1960). This can be appreciated by a review of the National Research Council publication 1345 (1966). Although some commercial turkey breeders follow the practice of removing the feed from the toms prior to semen collection, there 3 4 apparently is no research report on this practice. Flock owners claims are contradictory. Some claim that this practice helped to obtain greater volumes of cleaner semen while others indicated disastrous effects with the toms going out of semen production. 1. Species other than aves —— In species other than aves, several research reports deal with the effect of nutrition upon reproduction. The early paper by Evans and Scott (1922) demonstrated that rats raised on certain diets capable of sustaining growth possessed low fertility in the first generation and were wholly sterile in the second generation. They also showed that the deficiency was nutritional because feeding fresh lettuce, wheat germ or dried alfalfa meal corrected the ailment. Nearly forty years ago, nutritional ”pseudohypophysectomy” was demonstrated by Moore and Samuels (1931) in the rat. This effect could be overcome by administering either testicular hormone or anterior pituitary extracts. These results were corroborated by other researchers (Mulinos and Pomerantz, 1941; Pazos and Huggins, 1945). Rats maintained from four weeks of age to maturity on a high fat diet were found to have underdeveloped accessory reproductive organs (Mann, 1960). Manganese deficiency during the last days of gestation and during lactation in laboratory rats leads to testicular degeneration of the male offspring (Boyer 35.31:» 1942). Comparison of different fasting times in male mice before mating by MCC1ure (1966) showed that fasting up to thirty hours did not affect libido, mating distribution or fertility. However, fasting for 36 to 48 hours decreased libido for a period of 48 hours, but did not affect fertility. 5 Nutrition of boars relative to reproduction has been reviewed by Duncan and Lodge (1960). The published data indicate that feed re- striction from weaning to puberty prevents normal testicular development and results in retarded tubular development and testicular volume. The authors also stated that restriction of feed intake to 50 and 70 per- cent of normal intakes from weaning on,decreased seminal volume without affecting either sperm concentration or fertility. Meduzov and Kudrjavcen (1965) found that boars receiving 0.5 mg of zinc sulphate and manganese salts per kilogram of body weight had markedly better semen quality after 30 to 45 days of administration when compared to boars receiving no trace mineral supplement. The piglets sired by those boars receiving the mineral supplement were heavier at birth than those sired by boars receiving no trace mineral supplement. McCance (1960) reported that pigs suffering severe undernutrition develop large adrenals and have spleens lighter than normal swine when expressed as percentage of body weight. Salamon (1964) showed that in grazing Merino rams a daily supple— mentation of two pounds of a concentrate containing 165 grams of protein and 600 T.D.N. for six weeks prior to the beginning of intensive col- lection of semen improved sperm output. The control rams received a supplementation of one pound per day of grain containing 45 grams of crude protein and 300 grams of T.D.N. during the same period of time. In farm animals, overfattening has been reported to have an ad- verse effect on fertility (Millen, 1962). It has also been shown that the level of nutrition in young Holstein male calves affects the age at onset of puberty (Bratton_e£nal., 1959). These authors showed that young bulls began ejaculations of viable sperm at 37, 43 and 51 weeks 6 of age, when fed on a high, medium and low plane of nutrition, respec— tively. Flipse and Almquist (1961) have shown that overfeeding and, therefore, overweight in dairy bulls produced a decrease in libido and sexual response. However, no effect was demonstrated in total sperm output or extra-gonadal sperm reserves. De Alba (1964) stated that semen output is quite insensible to the plane of nutrition and more so, if the nutritional treatment is of short duration. In humans, obesity may lead to a deficient production of sperma- tozoa. On the other hand, malnutrition may cause sterility and appears to be associated with a reduction in testosterone production (Millen, 1962). P—aminobenzoic acid has occasionally been noted to produce a marked increase in libido and potency in human subjects (Ansbacher, 1944). In some cases of human malnutrition, there is a striking correla- tion between nutritional deficiency and the presence of testicular softening and atrophy associated with diminished libido and potency. These conditions have been reported to be restored to normal by inten- sive vitamin B-complex therapy (Biskind, 1946). Studies carried on with 529 human subjects suffering from chronic malnutrition showed cases of focal fibrosis, basal membrane thickening, decrease of ger— minal epithelium and, therefore, total lack of spermatogenesis. The sertoli cells frequently showed vacuolization, and the interstitial cells were constantly of small size and reduced in numbers. In general, the testes showed typical characteristics of senile retrogression and clinical tests for testosterone production showed a deficient function (Zubiran and Gomez-Mont, 1953). 7 2. Research utilizing poultry -— Regardless of the intensive research that has been carried out in breeding hens with specific nutrients, there are some reports in the literature that cast doubt on the efficacy of the accepted requirements and allowances. As an example of this, Summers et_§1, (1967) reported superior egg production with no difference in fertility and hatchability when meat-type pullets were feed restricted by using wheat bran. These pullets were lighter in weight at the onset of egg production than the full—fed controls and showed better feed conversion for egg production. Lowering the protein content of the feed to ten percent or in- creasing the fiber content to 15 percent in grower feeds produced a delay in sexual maturity with lower body weights in broiler—type pullets, but no differences were detected in egg production, fertility, hatch- ability or mortality (Waldroup EE.§l:a (1966). Feed restriction in broiler pullets has not given consistent results. For instance, feed restriction during the growing period has been reported to decrease subsequent egg production (Davis and Watts, 1955), increase future egg production (Fuller, 1960) and have no effect on subsequent egg produc— tion (Milby and Sherwood, 1953). These findings might be explained by the fact that each researcher used different diets and different methods of feed restriction. Anderson gt 31. (1963) reported that feeding Large White turkey hens at 84 percent of §d_1ibitum consumption of a balanced ration from 12 to 24 weeks of age improved hatchability and feed utilization for egg production during the breeding season. However, similar restriction from 24 to 40 weeks of age had no effect on hatchability. Daniels and Pino (1962) obtained better fertility and hatchability in Broad 8 Breasted Bronze turkeys raised from 8 to 24 weeks of age on clover prairies and supplemented with a mash of white corn and mineral mixture than from those raised on similar conditions but receiving a balanced diet as a supplement. However, sexual maturity was delayed and the birds were significantly lighter in weight at the end of the growing period. Restriction of feed intake from 12 to 28 weeks or from 18 to 28 weeks of age in medium sized white turkeys significantly restricted body weight as well as egg production (Touchburn, 1966). Parker and McSpadden (1943) reported a reduction in turkey semen volume and fer- tilizing capacity when the toms were subjected to a thirty percent restriction in feed intake. On the contrary, Cooper and Barnett (1962) improved the fertility of a naturally mated flock by controlling the weight of the toms by utilizing high fiber diets containing Coastal Bermuda grass. Boone 3£_213 (1967) showed that a six—day starvation period was enough to reduce seminal volume and fertilizing capacity of White Plymouth Rocks. When the starvation was prolonged to 17 days, twenty percent of the males produced no semen. However, severe undernourish- ment of Rhode Island Red cockerels capable of preventing growth beyond 150 to 190 grams at six months of age did not prevent satisfactory reproduction after rehabilitation by ag_libitum feeding (MCCance, 1960). Touchburn (1966) reported that restricting feed consumption from 12 to 24 weeks of age in turkey toms brought about an increase in semen production during the breeding season. Furthermore, the author stated that a separate ration for breeder toms based on the assumptions that toms do not lay eggs and, therefore, their requirements for calcium and protein would logically be lower, had given good results. This tom 9 ration was formulated to furnish 0.9 percent calcium and 15.8 percent protein. In chickens, Arscott et_§1: (1965) showed that diets high in linoleic acid and low in vitamin E affect fertility negatively without affecting seminal volume, hatchability, feed consumption, body weight or testicular weight. Further, increasing the number of inseminations per female did not improve fertility. However, vitamin E supplementation of the diet at a level of 162 mg/Kg of feed prevented the drop in fer— tility. Patrick (1965) fed cocks Se7503 and measured the amount of selenium deposited in the semen and the sperm. Since selenium was present in the semen and sperm, he made the following conclusions: (1) Selenium could be metabolized; (2) selenium could be pooled and retained in the male's reproductive tract and (3) selenium may play a role in the physiology of germ cells. Jones e£_al: (1966) reared cockerels during their growing period under two levels of protein (4.0 and 16.0 percent) and studied the sub— sequent effects on reproduction. Those males that received the lower protein level had also lower body weights and sexual maturity was de- layed for eight weeks. Nevertheless, when the restricted males were placed on a 17.0 percent protein ration at 21 weeks of age, they attained a higher sperm concentration peak which was also maintained for a longer period than the 16.0 percent protein reared controls. The effect of protein level in the breeding ration in relation to fertility in the male chicken has been studied by Arscott and Parker (1963). These authors found that lowering the protein level from 16.9 to 10.7 or 6.9 percent over a 33-week period showed no adverse effect on seminal volume or hatchability. In artificial insemination fertility 10 trials, the 6.9 percent protein level had significantly higher fer- tility (P < 0.55). Several authors have reported that a vitamin A deficiency re- sults in underdeveloped testes, reduction in comb size, increased numbers of abnormal sperms and a decrease in sperm number and motility (Craft Efiigs’ 1926; Burrows and Titus, 1938; Howe_e£ual., 1957). Later work of Rubio and Garcia (1959) showed that a vitamin A deficient diet fed to lO-month—old Single Comb White Leghorn cockerels produced no change in seminal volume. However, severe detrimental effects such as decreased sperm motility, increased numbers of abnormal sperm and decreased sperm concentration resulted in poor fertility. These ad- verse changes were not irreversible and the administration of 30,000 I.U. vitamin A per Kg. of body weight resulted in complete recovery in two weeks. Perek and Snapir (1963) have shown that vitamin C had a bene- ficial effect upon seminal volume and spermatozoa output. Later, Perek (1966) reported results implying that vitamin C might be involved with thyroid metabolism. Since this work is the only one of its kind and was done under Israeli conditions (hot weather implied) further work in this field is required. B. Size of Testes and Semen Output 1. Species other than birds —- Utilizing the exhaustion technique of collecting a large number of ejaculates per week for four consecutive weeks, Edwards (1940) found a correlation of 0.95 in rabbits between testicular weight and the number of spermatozoa in eight consecutive ejaculates. Ortavant (1952) 11 obtained a correlation of 0.80 between the size of ram's testes and the number of spermatozoa therein after electro-ejaculating these males at weekly intervals. Willet and Ohms (1957) reported that the correla- tion between scrotal circumferences EB;§E£E.°E bulls and the weight or volume of the excised testes is 0.94. The correlation between scrotal circumferences and testes spermatozoal production is 0.92 for young bulls and 0.53 with aged bulls when the animals were exhaused with four ejaculations. However, if the bulls were not exhausted, but only maintained in routine service, the correlations were 0.17 and 0.46 for dairy and beef bulls, respectively, with no significant difference be- tween figures, giving a combined correlation of 0.32 (P < 0.01). 2. Research utilizing poultry —- Parker and McSpadden (1943) showed that when cocks were subjected to feed restriction so that the birds lost weight,testicular size and seminal production decreased, however, no correlations were calculated between testicular weight and seminal output. The author explained the decrease in testicular weight as a response of diminished output of gonadotropins by the pituitary due to malnutrition. No report on the correlation between testicular size and sper- matozoal output or production in either chickens or turkeys could be found. C. Evaluation of the Sperm Reserves 1. Species other than birds —- The distribution of sperm in the testes, ductus deferens, and epididymides has been studied in mammals (Amann and Almquist, 1962; Kennelley and Foote, 1964; Kirton §£_§13, 1967). It has also been 12 shown that the frequence of ejaculation in bulls changes this distribu- tion of spermatozoa (Almquist and Amann, 1961). That there is sper- matozoa resorption from the cauda epididymis has been demonstrated by Orgebin—Crist (1964). 2. Research utilizing poultry -- No report could be found of any study of sperm reserves or dis— tribution in turkeys or roosters. Nevertheless, some studies have been done on the spermatogenesis of the duck (Clermont, 1958), the rooster (Kumaran and Turner, 1949) and the turkey (Macartney, 1942). The latter author also showed that left and right testes have the same mitotic activity per gram of tissue. However, it has been reported that in turkeys, the right testicle is larger than the left testicle (Law and Kosin, 1958), this being opposite to the female where the left ovary becomes functional and the right ovary usually does not develop (Nalbandov, 1964). In the domestic fowl, there is an increasing number of degen- erating spermatozoa in the ductus deferens when the frequency of ejaculation is not maximal (Lake, 1966). This observation may indicate that sperm reabsorption could be taking place at this location in fowl which differ from most mammals, in that their epididymidal development is small relative to the ductus deferens. D. Other Physiological Parameters Affected bngeriods of Feed Deprivation It has been reported that in chickens a 24-hour starvation period decreases blood glucose by approximately ten milligrams percent (Golden and Long, 1942). Prolonged fasting periods in adult chickens produced a decline in blood glucose beginning 24 to 36 hours after the fasting 13 period started and an increase in blood glucose of a maximum of 22 milligram percent about six days later (Hazelwood and Lorenz, 1959). Fasting also caused adrenal enlargement in chickens (Sure, 1938). The same was true in a deficiency of thiamine (Beznak, 1923), or in a calcium-vitamin D deprivation (Urist, 1959). The effect of diet has a definite effect on the chicken thyroid as it has been shown that a deficiency of iodine causes thyroid enlargement (Patton, Wilgus, and Harshfield, 1939). Feeding of 10.0 to 14.5 percent fat to chickens produced smaller thyroids than did diets containing only 2.5 percent fat (March and Biely, 1957). However, restricted caloric intake had no effect on thyroid weight even though thyroid function was diminished as was metabolic rate (Premachandra, 1962; Mellen, Hill and Dukes, 1954). Level of cholesterol in blood plasma is directly affected by the thyroid in chickens and its destruction by I131 causes a rise in blood cholesterol. This condition can be reversed by thyroxine admin- istration (Clegg_et_al., 1959). Similar effects of increased choles- terol and lipemia have been observed in ducks (Benoit and Bogdonovitch, 1937) in turkeys (Reineke et_al,, 1946) under hypothyroidism. The observations of the changes in the chemical constituents of the adrenal gland following administration of ACTH led to the develop— ment of new tests for the study of adrenal stimulation either by endogenous or exogenous ACTH (Sayers 35.31:, 1944). In the rat, the decrease in adrenal cholesterol is sensitive enough to serve as an assay technique for corticotropin preparations and to measure the rapidity of adrenal response to stressors. However, these changes do not appear to occur in all species (Elton g£_§l,, 1959). Contradictory to the results of Elton 25 31. (1959), with respect to chickens, Howard l4 and Constable (1958) and Siegle and Beane (1961) produced adrenal cholesterol depletion in chickens following ACTH injection. However, Wolford and Ringer (1962) concluded that adrenal cholesterol content in mature Single Comb White Leghorn hens seemed to be an unreliable test as a stress indicator. Furthermore, no statistical differences were detected in adrenal weight between treatments. The effect of nutrition on the blood corpuscles of the chickens is dependent on the specific nutritional deficiency under observation. For instance, it has been shown that copper or iron deficiencies are capable of producing a decrease in hemoglobin and anemia (Hart, Elvehjem and Kemmerer, 1930). A riboflavin deficiency can produce a significant decrease in lymphocytes and an increase in heterophils (Good e£_§l:, 1953). A folic acid deficient diet caused the development of anemia and decreased numbers of leucocytes of all types in young poults (Gomez and Hogan, 1948). Newcomber (1958) reported that physical restraint in chickens or ACTH or adrenal cortical hormones produced an increase in the numbers of heterophils. Basically, the same results were reported by Wolford and Ringer (1962), except that the latter authors required an extreme stressor such as forty hours of fasting plus an exposure to cold (00 F.) for 15 hours to obtain results similar to those of administering ACTH. Bioassays of turkey pituitary (Witschi, Stanley and Riley, 1937), as well as that of other birds (Leonard, 1937; Burrows and Byerly, 1936) have shown that FSH, LH and Prolactin are present in avian pituitaries. Removal of the anterior pituitary in different avian species has led to a sharp decrease in testicular size. This response plus the fact 15 that gonadotropin hormone preparations contain FSH have proven to be effective in producing reinitiation of spermatogenesis, increased diameter in semineferous tubules and an increase in testicular size, indicate endocrine regulation of the testes (Benoit and Aron, 1934; Nalbandov, Meyer and McShan, 1946; Coombs and Marshall, 1956; Lofts and Marshall, 1956; Chu, 1940). There is abundant experimental evidence in aves that FSH is the gametogenic hormone (Marshall, 1961) and that chronic malnutrition in the chick is capable of inducing nutritional pseudohypophysectomy and, therefore, impaired reproduction (Breneman, 1940 and 1941). E. FRF 1. Species other than birds —— As far as mammalian species are concerned, there is no longer any question as to whether there are neurosecretions of hypothalamic origin, which are usually called "releasing factors”. The existence of these substances and their control upon the secretion of the hor- mones of the anterior lobe of the pituitary, is now well established by many experiments EEHXEXg_and.$E.Vitr° (Guillemin, 1967). This author stated that crude acidic extracts of ventral hypothalamus con— tain substances which stimulate the secretion of pituitary gonadotropins. These stimulating substances have been named according to their function and gonadotropin involved. Therefore, that substance which stimulates the secretion of the pituitary luteinizing hormone (LH) is called LRF for LH—releasing factor. That which stimulates follicle stimulating hormone (FSH) secretion, is named FRF, for FSH—releasing factor. Another l6 substance in these extracts inhibits rather than stimulates the secre— tion of prolactin and it is referred as PIF, for prolactin-inhibiting factor. 2. Research utilizing poultry -- In birds, it has been proposed that environmental factors such as light are capable of causing changes in the activity of specialized hypothalamic cells, the so-called neurosecretory cells (Benoit and Assenmacher, 1955 and 1959). These authors observed neurosecretory material of apparent hypothalamic origin in the hypothalamico-hypophyseal nerve tract of ducks (Assenmacher, 1958; Benoit and Assenmacher, 1959). The same authors have given evidence that sectioning of the portal vessels or sectioning of the hypothalamico-hypophyseal tract in the median eminence without damage to the portal system caused testicular atrophy and lack of gonadal response to light stimulation. Further evidence for the possible role of neurosecretory regulation of gonado— tropins in birds was presented by Oksche_e£flal. (1959). These authors showed that an increase in daily illumination capable of inducing testicular weight in White-crowned sparrows would also produce a de- crease in neurosecretory material in the hypothalamic nuclei and median eminence. These results have been corroborated by Matsui (1966) who classified nine groups of neurosecretory cells in the tree sparrow. However, specific neurosecretory principles or releasing factors in birds have only been shown for prolactin and LH. Kragt and Meites (1964) reported that in pigeons there is a prolactin releasing factor and not a prolactin inhibiting factor as in mammals. Opel and Lepore (1967) have shown the presence of an ovulating releasing factor in the stalk median eminence of the chicken by intrapituitary infustion and 17 premature ovulation. Clark and Fraps (1967) obtained similar results with median eminence extracts infused through a stereotactically placed cannula into the anterior pituitary. F. Effect of Light on Oviposition It is general knowledge among poultrymen that females under natural conditions lay more eggs during the spring when day length is increasing. Furthermore, it has been shown that birds in different parts of the world but at the same latitude show similar seasonal peaks of production (Wheatham, 1933). This effect of light causes gonadotropin release and has been shown to be mediated through the hypothalamus (Benoit and Assenmacher, 1959). The stimulatory effect of light upon the gonads can be appreciated by evaluating the positive effects of light on egg production that have been demonstrated by several authors (Van Albada, 1962; Byerly and Moore, 1941; King, 1959; Palmer, 1966). The time of oviposition in the turkey hen is generally during the hours of the day in which they receive light (Woodward 35.3l3: 1963). However, most of the eggs are laid in the afternoon as observed by several investigators (Stockton and Asmundson, 1950; Resin and Abplanalp, 1951; Woodward E£.El:’ 1963; Wolford 3£_§l:, 1964). How- ever, if the light pattern is modified, there is an effect on the time of oviposition (Albright and Thomson, 1933; Marsden and Fraps, 1960; Davis, 1948; Asmundson and Mbses, 1950). In laying chickens, light has a rather strong influence on oviposition as demonstrated by Warren and Scott (1936) in a series of experiments. These authors found that birds subjected to normal lighting laid during the daytime but if these same birds were subjected by artificial means to continuous lighting, 18 oviposition was distributed uniformly throughout the 24 hours of the day. On the contrary, if the pens were darkened during the day and illuminated during the night, totally shifted their laying time to the illuminated period within four days. When 24 hours of darkness inter- vened, followed by another shift back to normal daytime, change in oviposition time was almost complete within three days after the change had been made. These results demonstrate that under normal cycles of day and night, hens lay during the day. However, if they are illuminated throughout the 24-hour day, the hens will distribute their laying during the 24—hour period. Under reversed conditions of light and darkness, the hens will tend to lay during the illuminated period of artificial daylight. These results have been corroborated by several researchers (Fraps, 1955; Bastian and Zarrow, 1955; Lanson and Sturkie, 1958; Biellier and Ostmann, 1960). The time of oviposition and time of mating undoubtedly affect fertility as indicated by the results obtained by a series of investi- gations reported in the literature. In 1943, Gracewski and Scott showed that better fertility is obtained if the matings take place in the afternoon instead of the morning hours. Moore and Byerly (1942) re— ported that they obtained better fertility when the hens were inseminated after laying and that fertility was lowest when inseminations were made with a hard shell egg in the uterus. wyne gt 31. (1959) also showed that the presence of a hard shell egg in the hen's uterus during in- semination produces a significant drop in fertility. Parker (1945) also obtained better fertility with afternoon inseminations. In turkeys, Parker and Barton (1946) found only a slight decrease in fertility with morning inseminations as compared to afternoon inseminations. 19 The above results showing that the presence of a hard shell egg in the uterus affects fertility negatively, plus the fact that egg laying time has been controlled in other aves (Arrington_gtnal., 1962; Warren and Scott, 1936) encouraged testing the same idea with turkey hens. In addition to the increase in fertility expected if hard shell eggs in utero were to be avoided, it would be to the advantage of the turkey breeders to control the time of oviposition in such a way that he could inseminate at a more convenient time rather than at night as is now generally practiced. OBJECTIVES To study the effect of feed deprivation prior to semen collection from male turkeys upon the semen volume and its characteristics. To study the correlations of testes size and semen output and production. To evaluate the amount of sperm in the testes, epididymi and ductus deferens. To study the feasibility of altering the oviposition time through proper light control so that artificial insemination can be per— formed at a convenient time. 20 MATERIALS AND METHODS General In two successive years, 1966 and 1967, a total of 45 male and 130 female Anderson strain Broad Breasted Large White turkeys were housed under 16 hours of artificial light and eight hours of darkness. The lights came on at 8:00 p.m. and went off at noon from December 15 until the end of the experimental period. This light pattern was de- signed to study the possibility of changing the oviposition time in such a way that a hard shell egg would not be present in the uterus at the time of insemination. The turkeys were June hatched from a com— mercial hatchery and were range-reared during the growing period. All toms were handled and trained for collection of semen for three weeks prior to the beginning of the experiments. During the experimental periods, the semen was collected twice per week on Mondays and Fridays between 7:00 and 9:00 a.m., using the technique described by Burrows and Quinn (1938), with the modification that the semen sample was directly collected in a disposable plastic tuberculin syringe. The use of such a syringe permitted the measuring of the volume of semen produced by a tom to the nearest 1/100 ml. This procedure was followed twice a week and the volumes were recorded for each individual. The individual semen samples were divided into two groups and used alternatively for artificial insemination of hens or for laboratory evaluation. In other words, the semen obtained from 21 22 one—half of the toms in each experimental group was utilized for artificial insemination on Mondays and the other half for laboratory evaluation. On Fridays, the procedure was reversed, the semen samples obtained from those toms whose semen samples were used the previous Monday for laboratory procedures was inseminated and the semen obtained from the other half was taken to the laboratory for evaluation. When the semen sample was utilized for artificial insemination, the hens were inseminated immediately following collection. However, the semen that was used for laboratory evaluation was kept in the syringe or in a small vial and placed in a tray previously filled with crushed ice. The sample was kept in crushed ice for one hour, on the average, before evaluation. The following laboratory tests were made: Spermatozoa motility was determined by examining a hanging drop of semen on a standard size coverslip at room temperature with a light microscope under low-power (10X). The sample was rated from 0 to 5 (Table 1) according to the procedure of Allen and Champion (1955). The motility scoring rates with the highest figure (5) were for samples that showed 80 to 100 percent of the spermatozoa in vigorous movement and the formation of eddies, while the lowest figure (0) was used for no motility at all. Determination of sperm concentration in the collected semen was then made by utilizing two different methods. First, Spermatocrit . 1 . values expre551ng the percentage of spermatozoa in semen volume were Analogous to the usage of ”hematocrit". Spermato referring to the spermatozoa and "crit” originally spelled "krite" meaning to judge. 23 . . , a Table l. Criterium for motility evaluation Scale Characteristics 0 No motility l l — 20% motility, weak oscillatory and undulating movement 2 20 - 40% movement but no waves or eddies formed 3 40 — 60% of the sperm showing progressive motility and vigorous motion slowly moving waves or eddies present 4 6O - 80% sperm motility with waves and eddies of rapid formation and movement 5 80 — 100% of the spermatozoa showing progressive movement and extremely rapid formation of waves and eddies aSlightly modified from Allen and Champion (1958). determined following the procedure of Hickman (1958) with two variants. The first modification was that the capillary tube was sealed with Critoseafi:> instead of by means of heat. The second variant was that in the spinning of the capillary, it was done twice for a period of 15 minutes, totalling thirty minutes at 11,500 rpm instead of 10,000 rpm for ten minutes as used for dairy bulls. The second method of sperm concentration determination consisted of using precisely the same pro— cedure as used in making red blood cell counts. For this purpose an A0 Spencer Bright-Line Hemacytometer was used to determine the sperm concentration per cubic millimeter. For each individual determination, a sample of well—mixed semen was drawn up to the 0.2 mark of the capillary in a red blood cell pipette and then to the 101 mark with a (BUrademark for a vinyl plastic seal manufactured by Biological Research, Inc., St. Louis, Mo. 24 mixture of 1:1 physiological saline and eosin solution (Appendix Table I) used as a diluting fluid. The reason for using such a preparation was to dye the spermatozoa and facilitate their counting. This dilution of l to 500 instead of l to 200 used by most investigators (Wakely, 1949; Burrows and Kosin, 1953; Parker, 1946) was based on the findings of Kosin and Wheeler (1956) who demonstrated that a dilution of 1 to 500 produced more accurate counts and estimates of spermatozoal density. Then, the pipettes were agitated by means of a Clay-Adams Yankee pipette shaker for 45 seconds to make an even mixture. A drop of this fluid was applied to each chamber of the hemacytometer and two microscopic spermatozoal counts were made after allowing the preparation to settle for at least five minutes. The settling period permitted the sperm to fall to the bottom of the counting chamber and, therefore, made more precise counts possible. Two counts such as the one described above were made for each semen sample giving a total of four values. The mean value of these four counts was recorded as the spermatozoa density of that tom's semen. The spermatozoa were counted in five groups of 16 small squares, usually at the center and four corners of the central square millimeter. To avoid counting a spermatozoon twice, those on a line were counted only when on the top or left lines, but not when on the bottom or right lines. The calculations for estimating the number of spermatozoa were done as follows: The number of spermatozoa representing the mean value obtained for a sample was multiplied 500 times since the semen had been diluted 1 to 500 as stated before. This multiplication brought the figure to its original density. Since each chamber consisted of 25 25 large squares and only 5 were counted, a factor five had to be used in multiplying the above figure to obtain the total number of spermatozoa in the whole field. However, the chambers in which the counts were madelneasured onetnillimeter X one millimeter X 0.1 millimeter in depth the figure obtained represents the concentration of sperm in 1/10 of a cubic millimeter. Thus, by multiplying this last figure by a factor of ten, the spermatozoal concentration could be expressed in sperm per cubic millimeter which is the conventional form. In other words, if the mean value of the sample was eighty, the following calculations had to be made: 80 X 500 X 5 X 10 = 4,000,000 spermatozoa per cubic millimeter. All hens were artificially inseminated one week prior to the beginning of the experiment and artificial insemination was continued throughout the experiments as described previously. All eggs were marked with a wax pencil indicating the date, the hen number and time of the day when the egg was collected. The eggs were collected three times a day, just at the onset of light, 12 hours later and just before the lights went off. This system was designed to properly identify the eggs for pedigree purposes and to be able to identify those eggs laid during darkness. For this purpose, each hen had a card where a record was kept of all eggs laid and the time of oviposition as to the light or dark period. All eggs, except those with either soft or broken shells, were incubated weekly in a Jamesway-252 forced-draft incubator which was operated at a temperature of 99.50 to 1000 F. and 70 percent relative humidity. On the 25th day of incubation all eggs were candled for the 26 determination of fertility. Those eggs containing live embryos were transferred and basketed by pedigree for future observations on hatch— ability. All poults were individually weighed and recorded after hatching. The recording of these data was designed to obtain specific information on fertility, hatchability and poult weight as affected by the feed restriction of the sire. All birds were fed a pelleted turkey breeder ration in the fol- lowing manner: The toms were divided and sorted into three groups as described later in more detail for each particular year. The groups were named A, B and C. In both years, group A received feed ad_libitum. In group B, the feed was removed twice a week for periods of 24 hours preceding the collection of the semen. In group C, the feed was removed for periods of 36 hours twice a week previous to semen collection (Table 2). The ration used was calculated to fulfill all known nutrient requirements when fed 3d_libitum to a turkey breeding flock (Table 3). Fresh water was available at all times, except 12 hours before semen collection, with the intention of avoiding overdrinking by the fasted birds. Feed consumption was recorded weekly for the toms, while their body weights were recorded on a monthly basis as well as at the beginning and termination of the experiments. The hens received the same pelleted diet plus oyster shell _ad libitum and fresh water was available to them at all times. All hens were assigned to individual cages that measured 18 inches wide X 24 inches long X 24/26 inches in height, while the toms were housed and kept in conventional floor pens that measured 17.0' X 15' 4" and had cement floors with shavings. 27 The data were analyzed by means of the analysis of variance technique and Duncan's multiple range test for comparisons among treat— ment means (Steel and Torrie, 1960), except as otherwise specified in the particular experiment. A. First Year The 1966 trial was designed to investigate the feasibility of conducting a second study with a larger number of toms. The study was initiated on January 14, 1966 and ended on May 13 of the same year; thus, it had a duration of 17 weeks. A total of 15 males and 70 females were distributed at random in the following manner: The males were divided into three different groups of five toms each to be subjected to the treatments listed above and shown in Table 2. For each tom in group A, four hens were assigned at random for artificial insemination. Groups B and C had five hens per tom and were also assigned at random for the same purpose. Table 2. Experimental design of feeding patterns for both years Group Feeding Program A Full feeding at all times B Feed removed twice a week for 24 hours prior to semen collection C Feed removed twice a week for 36 hours prior to semen collection At the end of the experimental period an extra sample of semen was collected individually from each tom in sterile tuberculin disposable 28 syringes and sent to the Department of Microbiology and Public Health for Escherichia coli counts. After an additional fasting period for treatment groups B and C, blood was drawn from the brachial vein of each bird in all groups for blood smear differentials as well as for blood cholesterol and glucose determinations which were made according to the methods of Zlatkis, §£_§13 (1953) and Folin and Wu (1920), respectively (Appendix Tables II and III). The purpose of making these measurements was to determine if the fasting periods could be regarded as stressors and if decreased feed intake would affect the thyroid. The toms were sacrificed by bleeding and the adrenals, thyroids, testes, crop and rest of the gastrointestinal tract were removed, washed, trimmed and weighed individually on a Roller-Smith or triple-beam balance. The total cholesterol content of the adrenals was also deter- mined according to the method of Zlatkis et 31. (1953) as an indirect measurement of stress. The thyroids were fixed in ten percent formalin and stained by hematoxylin and eosin as described in the Manual of Histologic and Special Techniques (1960) for later histological evalua— tion of the physiological state of the gland. The testes were also prepared for histological observation by means of Allen's fixative (Gray, 1958) and then stained with the periodic acid-Schiff—hematoxylin technique (Biological Stain Commission, 1960). B. Second Year A total of 30 toms and 60 hens were used for this experiment. The hens were assigned to individual cages similar to those described before. The toms were divided into five weight blocks of six birds 29 each. TWO toms per weight block were sorted at random into the three treatment groups and housed in conventional pens similar to those described for the first year. The pens were assigned at random into treatment groups A, B and C (Table 3). Each pen had ten toms which were kept under the conditions of the experiment for a period of 20 weeks, from January 16 to June 5, 1967. In addition to the procedures described previously for both ex- periments, at the end of the experimental period all the toms were sacrificed by decapitation and the pituitaries were carefully removed and frozen at -200 C. A portion of each hypothalamus was removed and placed in a vial, which contained adequate ice cold 0.1N HCl so that each portion of hypothalamus had a ratio of 0.2 milliliters of solution per hypothalamic portion, and stored at —200 C. A similar volume of cerebral cortex was also removed from those toms who belonged to treat- ment group A. The cerebral cortex was also placed in ice cold 0.1N HCl and stored at -200 C. The hypothalamic extracts and cerebral extract were later used for Follicle Stimulating Hormone Releasing Factor (FRF) bioassay as described later in this thesis. The testes were located in the body cavity, then the epididymus- ductus deferens and ductus deferens-cloaca junctions were located and each tightly tied with a thread to avoid sperm movements along the genital tract. Then the entire reproductive tract was dissected free from the surrounding tissues and used for sperm concentration deter— minations. The left testicle and ductus deferens from each tom were used to calculate spermatozoal concentrations in these organs, while the right testicle was fixed and prepared as described previously for the first 30 Table 3. Composition of the experimental ration Ingredient Kg Wheat middlings 50 Ground yellow corn 560 Ground oats 50 Soybean meal, 45% protein 100 Dehydrated alfalfa meal, 17% protein 50 Meat and bone scraps, 50% protein 50 Menhaden fish meal, 60% protein 50 Dried whey 25 Brewers dried yeast 20 Iodized salt 4 Dicalcium phosphate 10 Ground limestone 25 Vitamin and antibiotic mix8 5 Mineral mixb .___1 Total 1000 Calculated analysis: Crude protein, % 17.5 Productive energy, Cal/kg 1980 Crude fat, % 3.6 Crude fiber, % 4.5 Calorie:protein ratio 113 Calcium 2.2 Phosphorus 0.9 aSupplied per kg of ration: Vitamin A, 9,900 USP units; Vitamin D3, 2,248 I.C.U.; Riboflavin, 8.8 mg; D—calcium pantothenate, 22.0 mg; Niacin, 55 mg; Choline chloride, 1,320 mg; Vitamin B12, 26.4 mcg; Vitamin E, 33 units; Menadione sodium bisulfite, 4.4 mg; ethoxyquin, 137.5 mg; Folic acid, 2.2 mg and zinc bacitracin, 55 mg. bSupplies as per kg of ration: Manganous oxide (56% Mn), 95 mg; Zinc oxide (80% Zn), 64 mg; Copper sulpate (25.2% Cu) 33 mg. 31 year experiment for future histological evaluation of spermatogenesis. The procedure used to determine the spermatozoal concentrations in the left testicle and ductus deferens was as follows: After the ductus deferens was tightly tied with a thread at the junction with the epididy— mus and at the entrance to the cloaca, the entire reproductive tract was dissected free of surrounding tissue. Then the ductus deferens was dissected from the testicle at the junction with the epididymus and at the external entrance with the cloaca (Figs. 1 and 2). The left ductus deferens was weighed on a piece of tared wax paper on a Mettler balance to the nearest one hundredth of a gram and minced under refrigerated saline. The two testes were weighed individually exactly as described before for the ductus deferens. Later, the left testicle was dissected free of its tunic, reweighed, minced under refrigerated saline, and placed in fifty milliliters of cold 0.9 percent physiological saline solution. The minced tissues were transferred individually and quanti- tatively to a semimicro Waring blendor. A pinch of Dow antifoam and additional refrigerated saline was added to each individual organ preparation to bring the total volume up to 150 milliliters and the preparation was then homogenized for three minutes. Before the readings were made, the homogenates were diluted further with refrigerated physiological saline to obtain concentrations of spermatozoa, such that 50 to 500 spermatozoa were counted within the grid of the hemacytometer (Figs. 3 and 4). The tissue homogenates were stored for 4 to 48 hours at 5° C. with one or two drops of 100 mg percent merthiolate solution to prevent bacterial destruction of the sperm until the spermatozoal enumeration of 32 Fig. 1. Schematic diagram of reproductive tract in the abdominal cavity with sites of ligation indicated. 33 SITE oF LlGATlON S ITE 0F LIGATION 34 I Fig. 2. Schematic diagram of left side of the reproductive tract indicating the sites of ligation before sectioning into testicle and ductus deferens. 35 Fig. 3. 36 Miscroscopic view of a ductus deferens homogenate as seen under phase contrast illumination (690x). l 1.! 38 Fig. 4. Microscopic view of a testicular homogenate as seen under the phase contrast illumination (690K). 40 each homogenate was estimated by two observers in duplicate, using a phase contrast illumination of the spermatozoa in an A.O. Spencer Bright-Line Hemacytometer. Based upon comparison of their morphology with those in testicular histological sections, spermatids counted in testicular and ductus deferens homogenates were believed to represent only stages 7, 8, 9 and 10 of the cycle of the seminiferous epithelium according to Clermont (1958). C. FSH Bioassay The pituitaries were pooled by treatment groups, weighed and homogenized with normal isotonic saline solution. The solution obtained was used for FSH activity determination. The method used for FSH activity was that of human chorionic gonadotropin augmentation of Steelman and Pohley (1953) as modified by Parlow and Reichert (1963) and described in Appendix Table IV. Each assay consisted of injecting six twenty-day old female rats with the solution from one and one-half tom pituitaries in saline solution at the rate of 0.5 ml twice a day for three consecutive days. The immature female rats were of the Sprague-Dawley strain (Hormone Assay Laboratories, Chicago, 111.), weighing approximately seventy grams each. The rats were maintained in a temperature controlled room (25 :;1° C.) with 14 hours of light and received a commercial diet of Blox pellets (Allied Mills, Chicago, Ill.) which along with water, was available at all times. At the end of the three—day treatment period, the female rats were killed with ether. Their ovaries were removed, trimmed and weighed to the nearest one—tenth of a milligram and an analysis of variance and Duncan's multiple range test were run according to the methods of Steel and Torrie (1960). 41 D. FSH-RF Assay This experiment consisted of a 2 X 4 factorial plan with ortho- gonal contrasts (Steel and Torrie, 1960). The contrasts were planned as follows for mean comparisons. Contrast one consisted of cortical extract versus all others. Contrast two, hypothalamic extract from treatment group A versus hypothalamic extract of treatment groups B and C. And the final contrast number three was hypothalamic extract of treatment group E versus hypothalamic extract in treatment group C (Table 4). Table 4. Contrasts used for comparing the means Contrast Treatment Representation C Cortical extract versus hypothalamic extract of the three treatment groups C2 Hypothalamic extract of treatment group A versus hypothalamic extract of groups B and C C3 Hypothalamic extract of treatment group B versus hypothalamic extract of group C At the end of the experimental period, the toms were decapitated and their hypothalami and anterior pituitaries removed. The anterior pituitaries were assayed for FSH activity as described before in this thesis. The hypothalami were placed in ice cold 0.1N HCl (0.2 ml per hypothalamus) and stored at -20° C. At the time of removing the hypothalami, a similar volume of cerebral cortex was also removed, but only from those toms that had received treatment A. The cortex was treated in exactly the same manner as all the hypothalami used in the 42 experiment. The following week the tissues (hypothalami and cortex) were homogenized separately and by treatment groups in chilled 0.1N HCl and centrifuged at 12,000 g. for forty minutes at 4° C. just prior to use. The supernatant was decanted and placed in a protein-free medium 199 (Difco Laboratories, Detroit, Michigan). The pH was brought up to 7.4 by titration with 1N NaOH solution using a pH meter with glass electrodes. For incubation purposes, adult Sprague-Dawley male rats were decapitated and their pituitaries rapidly removed. The anterior pituitary was separated from the posterior lobe and cut in half. Each half was placed in a different flask containing three milliliters of medium 199. Eight halves were placed in each flask giving a total of four pituitaries per flask. Following this procedure, the flasks were placed for incubation in a Dubnoff metabolic shaker (60 cycles per minute) under constant flow of 95 percent oxygen and five percent carbon dioxide at a temperature of 370 i:0.5° C. for a period of four hours. The pituitaries were preincubated for a period of thirty minutes after which the medium was discarded to avoid any spontaneous release of FSH. The discarded medium was replaced by two milliliters of fresh medium 199 and either one milliliter of hypothalamic or cortical extract per flask. The incubation consisted of four groups with four flasks per group representing the hypothalamic extracts of treatment groups A, B and C as well as the cortical extract of group A. At the end of the incubation period the medium was separated from the anterior pituitaries, frozen and stored at —20° C. until assayed. 43 The FSH content of the medium was bioassayed by means of a four point bioassay with two doses of standard (FSH-NIH-S3) and two doses of the experimental unknown. The difference between doses was two—fold, administering two milliliters of the incubated media per assay rat (Sprague-Dawley strain female rats with an average weight of 60 grams) as a high dose and one milliliter for the low dose. The standard FSH used consisted of 70 and 140 micrograms per rat for the low and high dose, respectively. A total of four rats were used per dose level or a total of 32 rats. the: the the tre the (Ta 196 am be' fe Ca RESULTS AND DISCUSSION A: Effect of Feed Restriction During the Breeding Season 1. Feed consumption -— Feed restriction alone has been shown to influence reproduction; therefore, it would be of interest to know if possible differences are the result of feed restriction, periods of feed withheld or both; hence, the importance of knowing the amount of feed intake per tom during the experiment. The experimental results of the first year showed that feed con- sumption varied little between treatments A (control) and B (24-hr. feed withheld). However, both treatment A and B differed widely from treatment C (36-hr. feed withheld). The same trend was observed during the second year when there were twice as many toms and more observations (Table 5). An analysis of covariance and Dunnett's t-tests (Ostle, 1963; Li, 1964) showed no significant differences between treatments A and B (P > 0.05), but there was a highly significant difference (P < 0.01) between groups A and C for both experimental periods. The trends in feed consumption for each treatment during the first and second year can be observed in Figures 5 and 6, respectively. Apparently a 24-hour feed restriction period twice a week was compensated for by overeating between fasting periods up to a point where there was no real change in total feed intake per tom. However, two 36-hour fasting periods per week were evidently so severe that 44 €63; .. 60> ssh acme—ahn KL... 45 o~ 3 mg 333 : ©~ 2 Va 2 N— S c—a w n e m $99 .235 2:. mew—=0 Son. .8.— comumeacou room 3:33 a em m6 CA m4 9N m.~ 9m Wm 51118180151 46 3.33 AK 9 m. C E m— 3 9 S S 3 m w n o n v N ........ .I o I lllllll m . I. r . ,. .\ c. u \j .7. \ . . I \ \. .\I\ .\ \rv/ x. . 1‘ \ c m. .a .. xx 0 \ l I .‘\ y \ x . a . \ 1t \ w w u \ / .\:. . x \\ .4./ \.ll\V/\\r/\Ilol.\ .\ \ \ VXUHXNITIJ. \A... iiifT... -L ax . xx 89 23> 2E. mutan— th hum aomumanmaou room have? . 6 ea md —.c m4 9N m.~ Wm 9m SlUBJSOUH 47 Table 5. Effect of feed being withheld twice weekly on feed consumptiona’ Time Feed intake per Total feed intake feed tom per week (kg) per tom (kg) withheld Treatment (hrs.) 1966 1967 1966 1967 A O 2.682 2.867 45.594 57.332 B 24 2.773 2.696 47.141 53.918 6 36 1.955** 2.399** 33.235** 47.977** a17 weeks for 1966. b20 weeks for 1967. **(P < 0.01) significantly different from A and B. compensatory overeating could not achieve levels of feed intake similar to those of full—fed toms. If we considered the total number of hours in which the toms were out of feed, and interpret them as follows: Treatment A = 7/7 or ag_libitum feeding; treatment B = 5/7 and treat— ment C = 4/7 ad_libitum feeding. We can conclude from our results that our toms showed a capacity to compensate by overeating if feeding time was restricted by 2/7 (28.6%) that of gd_libitum fed toms. However, the toms could not compensate if restricted to 3/7 (42.9%) §d_libitum fed toms. Based on these results, if actual feed restriction is to be studied, consideration should be made to restrict the toms in excess of that of treatment B. 2. Body weight —- The nutritional status of an individual can be measured to a certain extent by its changes in body weight. Therefore, body weight was measured in the experimental animals as a parameter of the effects 48 of periods of feed restriction upon the physiological state of the body. The differences obtained in body weight do not agree statisti- cally between years as far as treatments A and B are concerned (Table 6). In the first year of the experiment no significant differences (P > 0.05) between treatment groups A and B resulted; however, during the second year a highly significant statistical difference (P < 0.01) was observed between the three groups. Nevertheless, the two—year data agree in that both groups A and B were heavier at the end of the experi- mental period than group C (P < 0.05) and (P< 0.01) during 1966 and 1967, respectively. Possibly these differences were influenced by the original weights of the toms. As shown in Table 6, the weights of the toms at the beginning of the first year were heavier than those in 1967. This suggests that the fasting periods may have affected feed utiliza- tion even though the total intakes were similar for both treatments. Perhaps the amount of feed utilized per tom might have been sufficient in 1966 to maintain body weight, but it was suboptimal in 1967 for maximal growth and gain in body weight. 3. Semen volume —- The main purpose of this experiment was to determine the effects of withholding the feed twice per week upon semen volume. The average semen volume collected per tom during each semen collection of the two experimental periods is summarized in Table 7. The results showed higher volumes of semen for toms of treatment B than for those of treatment groups A and C during both experimental periods. However, in 1966, no statistical significance (P > 0.05) was demonstrable among groups. This was not the case for the 1967 data where the 49 Table 6. Effect of feed being withheld twice weekly on body weight3 Treatment and years A (0 hrs.) B (24 hrs.) C (36 hrs.) 1966 1967 1966 1967 1966 1967 (kg) (kg) (kg) (kg) (kg) (kg) Initial weight 16.180 13.450 16.180 13.500 15.140 13.490 Final weight 16.020 17.360 16.910 16.200 13.420 14.270 Weight difference -0 160 +3.910 +0.73o +2.700** -1.720* +0.780** aStandard error for each final weight mean, (1966) = 1:1.8; + .491. *(P < 0.05) significantly different from A and B. (1967) **(P < 0.01) significantly different from A. Table 7. Effect of feed being withheld twice weekly prior to semen collection on the volume of semen collecteda Yield per collection in m1. Number of Number of Treatment observations 1966 observations 1967 A (0 hrs.) 170 0.33 400 0.33 B (24 hrs.) 170 0.41 400 0.4o** C (36 hrs.) 170 0.33 400 0.32 aStandard error of each mean yield for 1966 =_: 0.010; for 1967 = 0.003. * * (P < 0.01) significantly different from A and C. WI 50 statistical analysis showed a highly significant difference (P < 0.01) favoring treatment group B. Further, the first year numerical values were similar to those obtained for the second year. The agreement of our numerical values in both years strongly indicates that the lack of statistical significance in the first experiment can be attributed to the small number of toms per treatment. 4. Number of spermatozoa per cubic millimeter —- Knowledge of the effect of feed withholding upon seminal volume would be of little value if the effects of such treatment upon the cellular or fluid fractions of semen were not known. Hence, a study was made to determine if there were any effects upon the spermatozoal concentration of the semen obtained under such conditions. The results obtained during the first year varied statistically from those obtained during 1967 (Table 8). The values obtained in 1966 show that treatment group B had nine percent more sperms per cubic milliliter than treatment group A and 12 percent more than treatment group C. However, these figures were not statistically different (P > 0.05) among treatment groups. In 1967, treatment group B had two percent more spermatozoa than treatment group A and twenty percent more than treatment group C. The value obtained for treatment group C in 1967 varied significantly (P < 0.05) from treatment groups B and A. Regardless of the statistical differences, the numerical differences for the three treatments were alike for the two years; treatment group always had the highest concentration and treatment group C the lowest. These results indicate that the observed increase in seminal volume of treatment group B over treatment groups A and C was not due to seminal fluid alone, but was also due to an increase in spermatozoa production. 51 Table 8. Effect of feed being withheld twice weekly on spermatozoal concentration . 3 Number of spermatozoa in thousands per mm _: standard error of the mean Number of Number of Treatment observations 1966 observations 1967 A (0 hrs.) 85 9,700 1:179 200 9,200 ::144 B (24 hrs.) 85 10,600 1:179 200 9,400 1:144 C (36 hrs.) 85 9,300_: 179 200 7,500 i;l44* ”(P < 0.05) significantly different from A and B. 5. Spermatocrit values -- The second evaluation of the semen fractions can be obtained by spermatocrit values. These, therefore, were used as another criteria for semen evaluation. The results obtained by this method of evaluation of spermatozoa concentration agreed with the results obtained in evaluating the number of spermatozoa per cubic millimeter. The spermatocrit values were not statistically different (P > 0.05) between treatments for the first year. However, the results obtained during 1967 showed a significant difference (P < 0.05) among treatment groups (Table 9). Once again treatment B had the highest concentration of spermatozoa, while treatment C had the lowest concentration. Therefore, under our experimental con- ditions, both hemacytometer evaluation and spermatocrit technique re- sulted in higher spermatozoa concentration for treatment group B. These results further corroborate the findings that treatment B stimu- lated seminal volume output by increasing the number of spermatozoa ejaculated as well as possibly increasing the volume of seminal fluid. fc 52 Table 9. Effect of feed being withheld twice weekly upon spermatocrit values and motility scores Percent of spermatozoa MDtility Scores Treatment 1966 1967 1966 1967 A (0 hrs.) 25.1 i .791 20.8 i .496* 4.6 i .114 3.7 i .087 B (24 hrs.) 28.5 i .791 22.6 i .496* 4.4 i .114 3.6 i .087 c (36 hrs.) 29.7 i .791 19.4 i .496* 4.3 i .114 3.4 i .087 *(P < 0.05) A, B and C significantly different from each other. 6. Motility —— Although motility is not a good evaluation of fertilizing capacity in fowl, it is presumably true that immotile spermatozoa are infertile. Hence the importance of measuring the influence of feeding treatment upon motility of the spermatozoa. No statistical differences were detected among treatment groups (P > 0.05) as shown in Table 9. 7. Fertility and hatchability -- The final tests for spermatozoal vitality and fertilizing capacity are detection of egg fertility and hatchability. The effects of feed restriction upon fertility and hatchability showed a repeatable trend from 1966 to 1967 as can be seen in Table 10. The numerical differences showed a tendency of decreased fertility and hatchability as the fasting hours were increased. This trend was re- peatable, but no statistical difference was detected (P > 0.05) among treatment groups. However, if spermatozoal motility is accepted as an indication of fertility, even though no statistical differences were found in fertility and hatchability, the numerical trends tend to 53 agree with the motility results of 1967 showing an adverse effect as the fasting time increased. Table 10. Effects of feed being withheld twice weekly upon fertility and hatchability, expressed as percentagea,b Fertility HatchabilityC Treatment 1966 1967 1966 1967 A (0 hrs.) 91.5 i;2.6 84.1 1:4.1 64.1 1:3.9 58.0 1:4.1 B (24 hrs.) 87.0 1:2.6 82.6 1:4.1 61.2 i:3.9 57.2 1:4.1 C (36 hrs.) 86.7_: 2.6 80.0_: 4.1 60.5 ::3.9 56.7 j:4.l a . Given as treatment means_: standard error of the mean. b 12 hatches. CPercent hatch of fertile eggs. 8. Blood constituents —- In mammals, differential blood cell counts can be used as tests for detecting stress; however, this test in poultry has not been very reliable (Wolford and Ringer, 1962). Fasting the toms was not shown to affect the differential counts (P > 0.05) nor the hematocrit determinations, as shown in Table 11. The lack of response of blood corpuscle to fasting periods indicates that the blood cell producing tissues are either quite refractory to this type of stimulus or else that the stress was not strong enough to produce a response. In the chicken, it has been shown that feed deprivation produces a decrease in blood glucose concentration (Sturkie, 1965). No reports of similar studies with turkeys could be found. 54 .oomH weapon mace meme mums mcoHDQCAEumumo Hmfluoouewwflo HHmo coonm On.m om.o om.o oo.m¢ On.mq om.m¢ U o~.m om.o oq.H om.m¢ oo.mm mm.w¢ m om.~ ow.o om.H ow.m¢ O©.Nm ow.~¢ < mHHLQOmmm mflfizaocHwom mmuxoocoz mmuzoocmemq mawcaoumumm muwwooumamm msouo moofiumm cofium>wudmo meow umma ecu Mo com ecu um mHHmo coofin mo Demo Hod ca modfim> owmum>< .HH mHomH 1101 (\. (‘h II m. d! 55 In the present experiments, the blood glucose concentration did not show a significant change (P > 0.05) among treatment groups when determined after a fasting period. On the other hand, blood cholesterol concentration did show a highly significant increase (P < 0.01) in group C when compared to the other two treatment groups (Table 12). Table 12. Average values of the three different treatments in blood glucose and cholesterol concentration at the end of the experimental period ::the standard error of the mean Cholesterol Glucose Number of Treatment observations mg. % mg. % A 5 160.4 i:10.l 324.2_: 18.3 B 5 139.2_: 10.1 302.8_: 18.3 c 5 214.7 3:. 10.1** 359.5 + 18.3 **(P < 0.01) highly significantly different from A and B. The above results suggest that the turkey tom is quite insensible to blood glucose concentration changes when subjected to fasting periods of 24 to 36 hours. These data do not agree with the results obtained with chickens (Sturkie, 1965) but do agree with those obtained in mammals (Dukes, 1955). 9. Organs and glands —— Since differential blood cell counts are not a very reliable test for stress, total adrenal cholesterol determinations were used for this purpose. The total cholesterol concentration in the adrenals expressed as milligrams per 100 grams of wet weight tissue increased as feed deprivation increased in severity. The values obtained in milligrams In on rn (h 56 per 100 grams of wet adrenals were as follows: Treatment A = 3,697; treatment B = 4,018 and treatment C = 4,130. Since the values were obtained from pooled adrenals, no replication was possible and, there— fore, the data were not analyzed statistically. The amount of choles— terol in the adrenals shows the same pattern as blood cholesterol when related to the treatment received. A possible explanation for the increased amount of cholesterol in the adrenals is an increased synthesis of glucocorticoids as a re- sponse to stress. This increase, and subsequent release, of gluco- corticoids is possibly required by the tom for gluconeogenesis during the fasting periods. If true, this theory helps to explain the absence of a blood glucose concentration difference from that found in chickens. A possible explanation is that the chickens were fasted only once be— fore the glucose measurement while the toms were repeatedly exposed to periods of fasting prior to the glucose determination and, therefore, the possibility of adaptation is suggested by the enlarged adrenals and lighter thyroids. Cholesterol depletion tests have been used for testing adrenal stimulation from either exogenous or endogenous ACTH by several re- searchers (Sayers_g£_al., 1944; Howard and Constable, 1958; Siegle and Beane, 1961). However, these tests have been used with either very acute stressors or direct ACTH injection. In the first of these cases, the general adaptation syndrome might have been carried to the last phase or depletion stage, which was obviously not the case of the experimental toms discussed in this thesis. Evidence for this is that in 1967, both treatment groups made weight gains over the initial weight. 57 With respect to adrenal weight comparisons among treatment groups, both restricted groups had a trend toward heavier adrenal weights, but these differences were not statistically significant (P > 0.05) (Table 13). Since the gastrointestinal tract of the turkey has the capacity of retaining large amounts of ingesta, the thought occurred that, after periodic periods of feed withholding, the toms might overeat prior to such periods and cause an enlargement of the tract. Another possi- bility was a decrease in size of the tract due to a constant restriction of feed. In the present experiment, the gastrointestinal tract showed a tendency to decrease in weight as the fasting regime became more severe; however, no statistical differences (P > 0.05) were shown among treat- ment means (Table 13). No significant differences (P > 0.05) were found with respect to testicular or ductus deferens weights among treatment groups (Table 13). Thyroid weight was significantly depressed in treatment C (P < 0.05) when compared to both treatment groups A and B but there was no significant difference (P > 0.05) in thyroid weight between treat— ment groups A and B (Table 13). Therefore, as far as treatment group B is concerned, the data agree with that reported in the literature, that is, restricted feed intake has no effect on the thyroid weight as far as poultry is concerned (Premachandra, 1962; Mellen e£_§l:, 1954). However, treatment group C did have smaller thyroids and showed choles- teremia which is a sign of hypothyroidism (Reineke g£_gl:, 1946; Benoit and Bogdonovitch, 1937; Clegg e£_§l,, 1959). This sign of hypothyroidism is in agreement with the results obtained by Premachandra 58 .ucmEummuu you mCOwum>ummno OH Roma .ucoeummuu you mcofium>ummno m n oomHm o.H H.~m.m m.N “.m.nm H.N H.o.om m.oH H.0mm N.mm.fl.omm H.Hm H.000 o ogflwma m.NHm.om imfloam msfiflmqm NANHSm fififlao m ©.H H.mN.N m.N H.q.mm H.N H.w.mm m.oH H.Nom N.mN H.¢om H.Hm “.mwm < Amev Amewv Amswv Amev Awev AwEv Boas Roma coma coma seas coma mucmeummue .H.H.u assesses mfimcmun< mcoumwmb mouoso mmummH cmmanfl mmwmum>m mm co>flw muzwwmz cmwuo 6cm Ucmaw cflmuumo mmuouum bumUCmum co cofium>fluamu comm mo uommmm .mH maan 59 (1962) and those of Mellen_e£_a£. (1954) in which restricted caloric intake produced lower metabolic rate and diminished thyroid function. In mammals, Anand (1961) stated that thyroid control and body temperature regulation appeared to be integrated with a third controlling system, that of feed intake regulation. Brobeck (1960) also showed a close relationship between feed intake and both the environmental and body temperature. This triple relationship, temperature-ingesta— thyroid function, raises the possibility of a heat expenditure control through thyroid function regulated by the level of caloric intake. Several authors have supported the idea of a thyroid-temperature rela— tionship by showing that cold exposure increases TSH secretion, while warmth reduces TSH secretion (D'Angelo, 1960 and 1960b; Bottari, 1957; Knigge, 1960; Kuschinsky, 1935). Therefore, it is possible that chronic restriction of feed intake depresses secretion of TSH and thereby pre— vents normal thyroid growth. It has been reported that certain mammals under nutritional stress, have a decreased thyroid function (Stephens, 1940; Reichlin, 1957; D'Angelo, 1951). Thus, under chronic stress the depressed function of the thyroids may be due to nutritional pseudo- hypophysectomy (decreased TSH) with a reduction in weight logically following. 10. The relationship between testicular size and spermatozoal output -- The tests used for evaluating testicular function are tedious and time—consuming. If it could be shown that there is a high positive correlation between testicular weight and spermatozoal output, an easier procedure could be devised. All that would be required for such 60 a test, would be to treat the turkeys, weigh their testes and compare them as measurements of treatment effect. Spermatozoal output per ejaculation of all treatments for the two years as well as testes weight were analyzed and their partial correlations calculated (Table 14). The correlation values were not pooled together by years since treatment group A which was the control group had values statistically significantly different (P < 0.05) be— tween years. This was so even after a possible "outlier" tom was discarded due to extremely small testes and sperm outputs. In this case the correlation value of .970 was lowered to 0.659 but still was statistically significantly different (P < 0.05) from the corresponding value for 1967. Table 14. Correlation values between testicular size and spermatozoal output during 1966 and 1967 Treatment 1966 1967 A 0.97oa** 0.177 B 0.083 0.271 c 0.102 -0.159 aRecalculation of this value due to a possible outlier with extremely small testes and decreased spermatozoal output give a figure of 0.659. **Highly significant difference from zero. All other correlation values were tested against zero and none of the values proved to be statistically (P > 0.05) different from zero. Therefore, there seems to be no significant correlation between 61 testicular weight and spermatozoa output when semen was collected from the toms twice a week. 11. Spermatogenesis and sperm reserves -- Since measurements of semen volume, spermatocrits and sperm counts showed an increase of activity in the testicular tissue of treatment group, B, further studies were performed to determine the total potential of semen production in the different treatment groups per gram of testicle and the amount of semen present in the ductus deferens. Differences among the mean values for number of spermatozoa in' testes and ductus deferens for treatment groups B and C were highly significantly (P < 0.01) greater than that for treatment group A. These differences indicate an increased rate of spermatogenesis when the toms were deprived of feed twice a week prior to semen collection for periods of 24 or 36 hours (Table 15). As reported previously in part D, the number of spermatozoa obtained per collection was lowest for treatment group C. However, in the macerated testes and ductus deferens, the number of spermatozoa from treatment group C was much higher than from treatment group A (P < 0.01); while no significant difference (P > 0.05) was found be— tween treatment groups B and C. A possible explanation is that there was a greater reabsorption of spermatozoa from the excurrent ducts for treatment group C due to the prolonged periods of fast before semen collection. 12. Follicle stimulating hormone content of the pituitaries -- That there was an increase in semen volume and spermatogenesis in treatment group B suggests the possibility that the restriction 62 .mmoHcSU Hamsu osonuHB mmummu oau mzu mo unwfloz HmuOB msu mmEHu maowummu ummfi mo Emuw uma «oncomauoam mo Hones: Hmuoa w .moficsu mow usozuws maowummu uwofl mo ugwflm3 HmBOu mau wcflm: vmuwHSUHmUo .Nx ammuo uwma CH ucsoo Hauoen .movflezvflwflmm modaocw mmumem som.HNH Nmm oqN.¢H 0H 0 mmfi.oNH mom mm~.mH OH m mmo.mk mNN Hmm.w OH < Ummummu o3u msu CH omHoHummu mo Emuw nmcmummmv mduodv mcofium>ummno ucmaummua mONOUmaumam we won mONOUmaumam cfl noncomaumam mo Honesz Mo mCOwHHHE mo muoHHHaz mo maoafifiaz m mo>ummou Sumam cam mHmmcmwOumaummm com: coflum>wummv boom mo uoommm .mH magma 63 periods were acting as pituitary "stimulants” and produced an increase of FSH synthesis which in turn was responsible for the increased Spermatogenic activity of the testes. The FSH content of the pituitaries, expressed as the average of six rat ovarian weights per treatment group was as follows: Treatment group A, 148 :_3.5 mg; treatment group B, 167 ::3.5 mg and tnaatment group C, 167 1:3.5 mg. In preliminary studies, ovarian weights of similar rats treated with similar doses of mature male turkey pituitaries showed ovarian weights extremely close to those obtained in treatment group A. An analysis of variance was performed, followed by Dunnett's t—test for treatment means according to the methods of Steel and Torrie (1960). The results of the statistical analysis show that both treat— ment groups B and C had a significantly higher (P < 0.05) content of FSH in the pituitary than treatment group A. These differences are in accordance with the results observed in the testicular homogenates as reported in the previous section. During the course of the experiment, it was observed that treat- ment group C showed decreased physical activity, with less strutting. Fighting between these toms became practically nil when compared to the other two treatment groups. This suggests a possible decrease in androgen levels which could in turn be a result of decreased LH release. This observation and speculation could possibly explain the difference in results between sperm output of treatment group C and the spermatozoal counts in the testicular homogenates. It has been stated by Turner (1960) that the germ cells require LH or androgen to develop beyond the 64 stage of secondary spermatocytes, but not further with large numbers of secondary spermatocytes being reabsorbed. 13. Presence and content of a follicle stimulating hormone releasing factor (FRF) -- An increase in pituitary FSH does not necessarily reflect an increase in FSH synthesis and release because pituitary content is a function of each. Consequently, further evidence was necessary to demonstrate that the observed increase in FSH was due to increased FSH synthesis as well as to increased FSH release. Due to the fact that there is no evidence for a FRF in turkeys, or for that matter in aves, an experimental design had to be chosen that would not only detect the possible existence of FRF, but also would detect differences in concen- tration of such a releasing factor if there was one. The results suggest that follicle stimulating hormone releasing factor is present in the turkey hypothalamus. The difference in FSH releasing capacity between hypothalamic extracts and cerebral cortex extracts was statistically significant (P < 0.05) as shown in Table 16. Further, analysis of variance also showed a dose response difference (P < 0.01) between the high and low dose of hypothalamic extracts added to incubation media. The results showed that groups B and C had a significantly (P < 0.05) greater concentration of FRF than group A, but there was no significant difference between treatment groups B and C (P > 0.05). The detection of levels of neurosecretory stimu- lating material for FSH corroborates the existence of a FRF in the turkey. The increase in FRF content in the hypothalamic extract of the fasted group supports the findings of increased FSH activity in the 65 .muowuuxm oHEmHmLuoamL mnu Eoum Amo.o V my ucmuomwwb haucmowmwcwwmv .mmmoc 30H bow swan somBumn AHo.o V mv ucmumwuflv maucmowwflcwwm hfinwfimo .0 wow m museum oomeummuu Eoum Amo.o v mv ucmummwwv AHquonwcme n oomuuxo Hmowuuou 0 mo oomuuxm ofiamamnuoamm u mum ao.aa «.0NH ¢.NNH n¢.wm mmmum>m ucmeummuu Hamum>o 0w.mNH m.HHH m.om~ a.mmH m.wo~ Ass NV awam H.ww o.¢u m.¢oH m.ow o.ww AHB HV 304 mmmum>m A.mu£ 0v A.mu: omV A.mu: «NV A.mu: oV mwbma wmumnsocfi meb Hamum>o «nmo 01mm mumm mo own mm Umucmmwuamu .madouw ucoaummuo ucmumMMHw mnu mo muomuuxo owEmHmLuodmn mLu CH c0wumuucmocoo mow new mam mo mocmmmum mzu so wmawmuno muaommm .oH mHQmH 66 anterior pituitary of the same treatment groups as previously reported in this thesis. The increment in spermatogenesis observed in the same groups can, therefore, explain the increase in semen volume observed in treatment group B as a possible result of increased FRF synthesis stimulated by the fasting periods. The increase in FRF may have been responsible for the increased reproductive activity through FSH stimu- lation of spermatogenesis. l4. Escherichia coli contamination Since the toms on treatment groups B and C had been without feed for a period of 24 or 36 hours, the possibility of a decreased amount of feces and, therefore, reduced bacterial contamination of semen was contemplated. For the purpose of checking this possibility Escherichia coli counts were made. Escherichia coli was chosen due to its normal presence in great numbers in the intestines of the turkey. The total number of Escherichia coli counted per milliliter of semen varied tremendously from zero to 112,000,000 organisms with an overall average of 9,917,046 organisms per milliliter. No statistical difference (P > 0.05) or numerical trend was observed among treatment groups. B. Effect of Light Pattern on Time of Oviposition 1. First experiment -- In order to change the time of artificial insemination from that normally used by turkey breeders, it was necessary to alter the light pattern in an attempt to alter oviposition time. Turkey hens were exposed to 15 hours of artificial light per day (8 p.m. until 12 noon). The eggs were collected three times daily, 67 at the time the lights were turned on (8:00 p.m.), 12 hours later (8:00 a.m.) and 15 minutes before the lights went off (11:45 a.m.). The number of eggs gathered per collection was recorded and each egg was marked using a wax pencil with the hen's number and the time of collection. The period of collection and recording of data was January 14 to April 30, 1966. The total number of eggs laid during this experimental period was 3,636 of which 333 were laid during darkness. The distribution of those eggs laid when the lights were off was as follows: January, none; February, 63; March, 153 and April, 117. When expressed as percentages as in Table 17, the data are: January, 0 percent; February, 4.4 percent; March, 11.6 percent and April, 17.6 percent of the total eggs laid in the respective months. The data from this experiment show that there is a trend for an increased number of eggs to be produced during darkness as the season progresses. However, the entireanmnnntof eggs that were laid when the lights were off was only 9.2 percent (Table 17) of the total. Table 17. Effect of light pattern upon oviposition time during the year of 1966 (16 L:8D)a Number of eggs laid Percent of eggs laid Month Dark period Light period Dark period Light period Jan. 0 223 0.0 100.0 Feb. 63 1368 4.4 95.6 Mar. 153 1163 11.6 88.4 Apr. 117 549 17.6 82.4 Total 333 3303 9.2 90.8 3Lights on at 8:00 p.m. and off at 12 noon. 68 The above results indicate that it is possible to reverse the oviposition time in the majority of the hens by light manipulation. 2. Second experiment -— Exactly the same procedure was used as in the first year. The lighting schedule remained the same. The total number of eggs recorded during the 20-week period (January 16 to June 5) was 2,947 out of which 312 were laid during darkness. The distribution of eggs laid during darkness was as follows: January, 2; February, 41; March, 124; April, 92 and May, 54. If we express the number of eggs laid during darkness as a percentage of the total eggs laid during the month, the data are as follows: January, 0.4 percent; February, 6.4 percent; March, 15.0 percent; April, 13.9 percent and May 9.9 percent (Table 18). Table 18. Effect of light pattern upon oviposition time during the year of 1967 (16L:8D)a Number of eggs laid Percent of eggs laid Month Dark period Light period Dark period Light period Jan. 1 273 0.4 99.6 Feb. 41 596 6.4 93.6 Mar. 124 703 15.0 85.0 Apr. 92 569 13.9 86.1 May 54 494 9.9 90.1 Total 312 2635 10.6 89.4 8Lights on at 8 p.m. and off at 12 noon. During this experiment, there was an initial trend of progres— sive increase in the number of eggs laid in the period of darkness and 69 a second trend during April and May of decreasing numbers of eggs pro— duced during darkness. However, the total number of eggs laid during the dark periods only amounted to 10.6 percent of the total. These data confirm those of the first experiment, that is, that the time of oviposition can be controlled by changing the light pattern. The 9.2 and 10.6 percent of eggs laid in darkness do not mean a failure of having successfully changed the oviposition pattern, since it has been reported that even under a regime such as that used commercially, turkey hens laid 5.5 percent of their eggs during darkness (Woodward _e£.al., 1963). The difference in trends on the percentage of eggs laid in darkness between years can be explained as follows: It has been shown that the average interval between successive eggs of a sequence in turkey hens varies from 25 to 27 hours depending on the strain of turkeys (WOlford 3£_§l:, 1964). This sequence time is larger than a 24-hour day and since the turkey ovulates approximately 15 to 30 minutes after oviposition (Wolford_e£nal., 1964), there is a lag time accumulation leading to later ovipositions with each succeeding egg until the end of the sequence. Hence, as the production cycle pro— gresses, there is an increase of later eggs and, therefore, more eggs are laid in darkness. Further, it is common knowledge that after the first weeks of lay a number of turkey hens go out of production for some time and if they do not become broody, they may come back into production. Generally, these hens are the poor producers and will, therefore, have shorter sequences. If what has been reported in the case of chickens by Fraps (1955) is also true for turkeys, the lag time of the terminal oviposition decreases as the sequence length 70 increases from 2 to 7 or 8 eggs and the lag time approaches zero or even may fall below zero as the sequence length increases. This phenomena could explain the reason for two trends during the year of 1967. As the poor producing hens went out of production during April and May, the number of eggs laid during darkness also decreased. CONCLUSIONS Two years of experimental data have indicated that withholding feed prior to semen collection twice a week for periods of 24 hours produced an increase in semen volume without altering feed consumption or body weight gains. However, withholding of feed prior to semen collection twice a week for periods of 36 hours did not increase the amount of semen collected but did depress body weight significantly. The increase in semen volume was not of seminal fluid alone since two evaluations of seminal spermatozoa concentrations (sperm counts and spermatocrits) showed either an increase over the other treatment groups or no statistical difference. Therefore, it can be concluded that, under the experimental conditions described in this thesis, withholding feed twice a week for 24 hours produced an increase in total semen volume. Further, this increase was not of seminal fluid alone which indicates that increased spermatogenesis occurred. Although spermatozoa motility appeared to be decreased as the time feed was withheld increased, the difference was not significant. Both fertility and hatchability decreased after withholding feed, coin- cidental with the motility changes. These differences make the prac— tice of feed withholding prior to semen collection questionable, al- though neither of these characteristics was significantly affected by feeding treatment. Further research on the effects of feed with- holding upon fertility and hatchability are highly desirable. 71 72 The data strongly suggest that the increase in semen volume shown by withholding feed for 24 hours prior to semen collection was due to an increase in follicle stimulating hormone and follicle stimu- lating hormone releasing factor activity. This increased activity seemed to be triggered by the withholding period pe£_§e_and was not due to decreased feed intake. Spermatozoal output at twice-a—week ejaculation and testicular size of the toms were not significantly correlated. Thus, in toms collected twice a week no evaluation of function could be made through the measurement of testicular weight. The total number of spermatozoa in the testes-epididymides structures and ductus deferens were evaluated. The results showed in- creased rates of spermatogenesis in the two groups in which feed was withheld. This trend coincided with similar trends both semen volume and total number of spermatozoa collected in those birds where feed was withheld for 24 hours, but this did not hold for toms where feed was withheld for 36 hours. Possible explanations are: (1) LH activity was not increased parallel to that of FSH; (2) nutritional stress and/or the length of the withholding period pE£_§E_was/were responsible for an increased spermatozoal reabsorption; (3) a combination of (1) and (2). Follicle stimulating hormone releasing factor (FRF) appears to be present in the hypothalamus of the turkey toms and the level of FRF was elevated after feed restriction. This factor had heretofore not been demonstrated in avian species. The oviposition of turkey hens was successfully altered by using artificial light from 8:00 p.m. until noon. About 9 to 10 percent 73 of the eggs were laid between 12 noon and 8:00 p.m. (in darkness), but even under normal artificial lighting some eggs were laid in dark- ness. The laying cycle can be altered sufficiently to permit the breeder to plan the light schedule in such a way that artificial in- semination of the hens can be done at a convenient time. SUMMARY Individual seminal volume, sperm counts, spermatocrits, motility scores, testicular weights, ductus deferens weight and sperm reserves were obtained for turkey toms under three different nutritional regimes. Data were also collected on fertility, hatchability, feed consumption, body weight, follicle stimulating hormone content and follicle stimu— lating hormone releasing factor concentration in the hypothalami. The time of oviposition and its relation to the light regime was also studied. Semen volume and sperm counts were increased when feed was with— held twice a week for a period of 24 hours prior to semen collection as compared to 3d_libitum fed toms and those that had feed withheld 36 hours twice a week prior to semen collection. No significant differences were detected among groups with re- spect to motility scores, testicular weights and ductus deferens weights. An increase in spermatogenesis over that of the ad_1ibitum fed toms was observed in the testicular homogenates of the toms where the feed was withheld. The follicle stimulating hormone concentration of the pituitary and the follicle stimulating hormone releasing factor found in the hypothalamic extracts were increased in those treatment groups in which the feed was withheld when compared to the §d_libitum fed toms. Feed intake was significantly depressed when the toms were fasted twice a week for periods of 36 hours prior to semen collection 74 75 when compared to those toms fed §d_libitum or deprived of feed for 24 hours twice a week prior to semen collection. No statistical difference (P > 0.05) was detected between the last two treatment groups. Heavier body weights were recorded for the ad_libitum fed toms when compared to the two treatment groups in which feed was withheld prior to the collection of semen. From the data collected, it appears that restricting the feed intake for 24 hours prior to the collection of semen stimulates sperma- togenesis and produces higher semen volumes. Oviposition time was changed by means of light manipulation. Using a windowless house and a light regime in which the lights came on at 8:00 p.m. and went off at 12 noon, it was possible to obtain approximately ninety percent of all eggs before noon. LITERATURE CITED Albright, W. P. and R. B. Thomson, 1933. Securing early turkeys by stimulated egg production. Poultry Sci. 12: 124-128. Allen, C. J. and L. R. Champion, 1955. Competitive fertilization in the fowl. Poultry Sci. 34: 1332-1342. Almquist, J. O. and R. P. Amann, 1961. Reproductive capacity of dairy bulls. II. Gonadal and extragonadal sperm reserves as deter— mined by direct counts and depletion trials: dimensions and weight of genitalia. J. Dairy Sci. 44: 1668-1678. Amann, R. P. and J. 0. Almquist, 1962. Reproductive capacity of dairy bulls. VIII. Direct and indirect measurement of testicular sperm production. J. Dairy Sci. 45: 774-781. Anand, B. K., 1961. Nervous regulation of food intake. Physiol. Rev. 41: 677-708. Anderson, D. L., J. R. Smyth, Jr. and R. E. Gleason, 1963. Effect of restricted feeding during the growing period on reproductive performance of large type White turkeys. Poultry Sci. 42: 8-15. Anon., 1960. Staining Procedures. lst Edition. Biological Stain Commission. The Williams and Wilkins Co., Baltimore, Md. Ansbacher, S., 1944. Para-aminobenzoic acid — Experimental and clinical studies. Vitamins and Hormones 2: 215-254. Academic Press, Inc., New York, N. Y. Arrington, L. C., H. Abplanalp and W. O. Wilson, 1962. Experimental modification of the laying pattern in Japanese quail. British Poultry Sci. 3: 105-113. Arscott, G. H. and J. E. Parker, 1963. Dietary protein and fertility of male chickens. J. Nutrition 80: 311-320. Arscott, G. H., J. E. Parker and E. M. Dickinson, 1965. Effect of dietary linoleic acid, vitamin E and ethoxyquin on fertility of male chickens. Jour. Nutrition 87: 63-68. Asmundson, V. S. and B. D. Mbses, 1950. Influence of length of day on reproduction in turkey hens. Poultry Sci. 29: 34-41. 76 77 Assenmacher, I., 1958. Recherches sur le control hypothalamique de la fonction gonadotrope prehypophysaire chez 1e canard. Arch. Anat. Microscop. Mbrphol. Exper. 47: 447-572. Bastian, J. W. and M. X. Zarrow, 1955. A new hypothesis for the asynchronous ovulatory cycle of the domestic hen (Gallus domesticus). Poultry Sci. 34: 776-788. Benoit, J. and I. Assenmacher, 1959. The control by visible radiations of the gonadotrophic activity of the duck hypophysis. Recent. Prog. Hormone Res. 15: 143-164. Benoit, J. and Aron, M., 1934. Sur 1e conditionnement hormonique du developpement testiculaire chez les oiseaux. Ijections d'extrait prehypophysaire chez 1e canard. Remarques sur divers elements d'interpretation des experiences. Influence de l'age. Compt. Rend. Soc. Biol. 116: 215-218. Benoit, J. and Assenmacher, 1955. Le Controle hypothalamique de 1' activite prehypophysaire gonadotrope. J. Physiol. 47: 427-567. Benoit, J. and S. B. Bogdanovitch, 1937. Sue in teneur du sang en acide gras, phosphore lipidique, et cholesterol chez le canard domestique, apres 1'injection d' extraits prehypophysaires et apre's thyroidectomie. Comp. Rend. Soc. Biol. 125: 891. Cited by R. K. Ringer in Avian Physiology, 1965. 2nd Edit. pp. 592—648. Comstock Publishing Associates, Ithaca, N. Y. Beznak, A., 1923. Die Rolle der Nebennieren bei Mangel and vitamin B. Biochem. Z. 141: 1. As cited by P. D. Sturkie, 1965 in Avian Physiology, 2nd edit. pp. 668-696. Biellier, H. V. and O. W. Ostmann, 1960. Effect of varying day lengths on time of oviposition in the domestic fowl. Missouri Agr. Expt. Sta. Res. Bul. 747. Biskind, M. S., 1946. Nutritional therapy of endocrine disturbances. Vitamins and Hormones 4: 147-185. Academic Press Inc., New York, N.Y. Boone, M. A., B. L. Hughes and B. D. Barnett, 1967. Starvation and its effect on semen quality. Poultry Sci. 46: 1235-1236. (Abstract) Bottari, P. M., 1957. The concentration of thyrotropic hormone in the blood of the rabbit under different experimental conditions. Ciba Found. Colloq. Endocrinol. 11: 52—69. Boyer, P. D., J. H. Shaw, and P. H. Phillips, 1942. Studies on manganese deficiency in the rat. J. Biol. Chem. 143: 417-425. 78 Bratton, R., S. D. Musgrave, H. 0. Dunn and R. H. Foote, 1959. Causes and prevention of reproductive failures in dairy cattle. 11. Influence of underfeeding and overfeeding from birth to 80 weeks of age on growth, sexual development and semen production of Holstein bulls. Cornell Agr. Expt. Sta. Bull. No. 940. Breneman, W. R., 1940. Limitation of food consumption as a factor influencing endocrine reactions in the chick. Endocrinol. 26: 1091-1098. Breneman, W. R., 1941. Growth of the endocrine glands and viscera in the chicks. Endocrinol. 28: 946-954. Brobeck, J. R., 1960. Food and Temperature. Recent Progr. Hormone Res. 16: 439-459. Burrows, W. H. and T. C. Byerly, 1936. Studies of prolactin in the fowl pituitary. I. Broody hens compared with laying hens and males. Proc. Soc. Exper. Biol. and Med. 34: 841-844. Burrows, W. T. and I. L. Kosin, 1953. The effects of ambient tempera- ture on production and fertilizing capacity of turkey spermatozoa. Physiol. Zool. 26: 131-146. Burrows, W. H. and S. J. Marsden, 1938. Artificial breeding of turkeys. Poultry Sci. 17: 408—411. Burrows, W. H. and J. P. Quinn, 1935. A method of obtaining spermatozoa from the domestic fowl. Poultry Sci. 14: 251-254. Burrows, W. H. and H. W. Titus, 1938. Vitamin A deficiency and semen production in chickens. Poultry Sci. 17: 224-226. Byerly, T. C. and 0. K. Mbore, 1941. Clutch length in relation to period of illumination in the domestic fowl. Poultry Sci. 20: 387-390. Chu, J. P., 1940. The effects of oestrone and testosterone and of pituitary extracts on the gonads of hypophysectomized pigeons. J. Endocrinol. 2: 21-37. Chubb, L. G., 1960. Nutrition and reproduction in the domestic fowl. Proc. Nutr. Soc., 19: 28-32. Clark, C. E. and R. M. Fraps, 1967. Induction of ovulation in the chicken with median eminence extracts. Poultry Sci. 46: 1245-1246. Clegg, R. E., A. T. Ericson and R. E. Hein, 1959. Distribution of phosphorus in the electrophoretic components of the blood serum of radio-thyroidecrectized chickens. Poultry Sci. 38: 77-80. 79 Clermont, Y., 1958. Structure de l'epithelium seminal et mode de renouvellement des spermatogonies chez 1e canard. Arch. Anat. Microscop. et Morphol. Exp. 47: 47-66. Coombs, C. J. F. and A. J. Marshall, 1956. The effects of hypophy- sectomy on the internal testis rhythm in birds and mammals. J. Endocrinol. 13: 107-111. Cooper, D. M., 1965. Artificial insemination in poultry. World's Poultry Sci. Jour. 21: 12-22. Cooper, J. B. and B. D. Barnett, 1962. Feeding to control body weight to improve fertility of turkey males. Poultry Sci. 41: 1637- 1638. Craft, W. A., C. H. M' Elroy and R. Penquite, 1926. The influence of certain feeds upon the production of spermatozoa by the domestic chicken. Poultry Sci. 5: 187-198. D'Angelo, S. A., 1951. The effect of acute starvation on the thyro— trophic hormone level in the blood of rat and mouse. Endocrinol. 56: 46-58. D'Angelo, S. A., 1960a. Hypothalamus and endocrine junction in per- sistent estrous rats at low environmental temperature. Am. J. Physiol. 199: 701-706. D'Angelo, S. A., 1960b. Adenohypophysial function in the guinea pig at low environmental temperature. Fed. Proc. 19: 51-56. Daniels, R. and J. A. Pino, 1962. Personal communication. Davis, G. T., 1948. The influence of continuous light on reproductive performance in turkeys. Poultry Sci. 27: 658 (Abstract). Davis, W. M. and A. B. Watts, 1955. The effect of ration treatments during various states of the chicken's life upon the subsequent performance of laying hens. Poultry Sci. 34: 1231-1232. De Alba, J., 1964. Reproduccién y genética animal. Instituto Interamericano de ciencias agricolas de la O.E.A. Turrialba, Costa Rica. Dukes, H. H., 1955. The physiology of domestic animals. 7th ed. Comstock Publishing Associates, Ithaca, N. Y. Duncan, D. L. and G. A. Lodge, 1960. Diet in relation to reproduction and the viability of the young. Part III. Pigs. Tech. Comm. No. 21 Commonwealth Bureau of Animal Nutrition. Commonwealth Agricultural Bureaux, Farnham Royal, Bucks, England. 80 Edwards, J., 1940. The effect of unilateral castration on spermato- genesis. Proc. Roy. Soc. 128: 407-411. Elton, R. L., I. G. Zarrow and M. X. Zarrow, 1959. Depletion of adrenal ascorbic acid and cholesterol: A comparative study. Endocrinol. 65: 152-155. Evans, H. M. and K. B. Scott, 1922. On the existence of a hitherto unrecognized dietary factor essential for reproduction. Science 56: 650-651. Flipse, R. J. and J. 0. Almquist, 1961. Effect of total digestible nutrient intake from birth to four years of age on growth and reproductive development and performance of dairy bulls. Jour. Dairy Sci. 44: 905-914. Folin, 0. and H. Wu, 1920. A system of blood analysis Supplement I. A simplified and improved method for determination of sugar. J. Biol. Chem. 41: 367-374. Fraps, R. M., 1955. Egg production and fertility in poultry. In The Physiology of Farm Animals. Editor, J. Hammond. V01. 2, Butterworth's Scientific Publications, London, England. Fuller, H. L., 1960. Restricted feeding of pullets. Proc. Md. Nutr. Conf. 2-4. Goff, S., W. C. Russell and M. W. Taylor, 1953. Hematology of the chick in vitamin deficiencies. I. Riboflavin. Poultry Sci. 32: 54-60. Golden, W. R. C. and C. N. H. Long, 1942. The influence of certain hormones on the carbohydrate levels of the chick. Endocrinol. 30: 675-686. Gomez, B. L. and A. G. Hogan, 1948. Inositol and nicotinic acid in the nutrition of the turkey. J. Nutr. 36: 369-379. Gracewski, J. J. and H. M. Scott, 1943. The influence of time of mating on fertility. Poultry Sci. 22: 264—265. Gray, P., 1958. Handbook of basic microtechnique. 2nd ed. MbGraw- Hill, New York, N. Y. Guillemin, R., 1967. Chemistry and physiology of hypothalamic releasing factors for gonadotropins. Internat. J. Fertil. 12: 359-367. Harper, A. J., and J. E. Parker, 1957. Changes in seasonal egg production of turkeys induced through controlled lighting exposure and force and moulting. Poultry Sci. 36: 967-973. Hart, E. B., C. A. Elvehjem and A. R. Kemmerrer, 1930. Does the practical chick ration need iron and copper additions to insure normal hemoglobin building? Poultry Sci. 9: 91—101. 81 Hazelwood, R. L. and F. W. Lorenz, 1959. Effects of fasting and insulin on carbohydrate metabolism of the domestic fowl. Am. J. Physiol. 197: 47-51. Hickman, C. G., 1958. Spermatocrit values in facilitating the estimation of spermatozoa concentrations. J. Dairy Sci. 41: 318—319. Howard, A. N. and B. J. Constable, 1958. The metabolism of adreno- corticotrophic hormone and ascorbic acid in the chick. Biochem. J. 69: 501-505. Jones, J. E., H. R. Wilson, R. H. Harms, C. F. Simpson and P. W. Waldroup, 1966. Reproduction in male chickens following protein defi- ‘” ciencies during the growing period. Poultry Sci. 45: 1095-1096 (Abstract). Kennelly, J. J. and R. H. Foote, 1964. Sampling boar testes to study spermatogenesis quantitatively and to predict sperm production. J. Anim. Sci. King, D. F., 1959. Artificial light for growing and laying birds. Progress Report Series Number 72. Alabama Polytechnic Institute. 5“‘ Kirton, K. T., C. Desjardins and H. D. Hafs, 1967. Distribution of sperm in male rabbits after various ejaculation frequencies. Anat. Rec. 158: 287—292. Knigge, K. M., 1960. Neuroendocrine mechanisms influencing ACTH and TSH secretion and their role in cold acclimation. Fed. Proc. 19: Suppl. 5: 45-51. Kosin, I. L. and H. Abplanalp, 1951. The pattern of egg laying in turkeys. Poultry Sci. 30: 168—179. Kosin, I. L. and A. Wheeler, 1956. Methods for estimating spermatozoal numbers in turkey semen. Northwest Sci. 30: 41-47. Kragt, C. L. and J. Meites, 1965. Stimulation of pigeon pituitary prolactin release by pigeon hypothalamic extract in vitro. Endocrinol. 76: 1169—1176. Kumaran, J. D. S. and C. W. Turner, 1949. Endocrine activity of the testis of White Plymouth Rocks. Poultry Sci. 28: 636-640. Kuschinsky, G., 1935. Uber den Einflus verchiedener temperatur auf die sekretion des thyreotropen hormons. Arch. Exptl. Pathol. Pharmakol. 179: 726-737. Cited by Reichlin, S. in Neuroendocrinology. V01. 1, Chapt. 12, 1966. Academic Press, New York, N. Y. Lake, P. E., 1966. Physiology and biochemistry of poultry semen. In Advances in reproductive physiology. Acad. Press Inc., Ltd., London. 82 Lanson, R. H. and P. D. Sturkie, 1958. Effect of the length of the dark and light periods on time of oviposition. Poultry Sci. 37: 1219 (Abstract). Law, G. R. and I. L. Kosin, 1958. Seasonal reproductive ability of male domestic turkeys as observed under two ambient temperatures. Poultry Sci. 37: 1034-1047. Leighton, A. T., Jr., and R. N. Shoffner, 1961a. Effect of light regime and age on reproduction of turkeys. I. Effect of 15, 24-hr and restricted light treatment. Poultry Sci. 40: 861— 870. '1 Leighton, A. T., Jr. and R. N. Shoffner, 1961b. Effect of light regime and age on reproduction of turkeys. II. Restricted vs unre- stricted light. Poultry Sci. 40: 871-884. . Leonard, S. L., 1937. Luteinizing hormone in bird hypophysis. Proc. Soc. Exper. Biol. and Med. 37: 566-568. Li, C. C., 1964. Introduction to experimental statistics. lst ed. . McGraw-Hill Book Company, New York, N. Y. Lofts, B. and A. J. Marshall, 1956. The effects of prolactin adminis— tration on the internal rhythm of reproduction in male birds. J. Endocrinol. 13: 101-106. Lowe, J. S., R. A. Morton, N. F. Cunningham and J. Vernon, 1957. Vitamin A deficiency in the domestic fowl. Biochem. J. 67: 215-223. Macartney, E. L., 1942. Diurnal rhythm of mitotic activity in the semineferous tubules of the domestic fowl. Poultry Sci. 21: 130-135. MacIlraith, J. J., 1952. Artificial breeding as a supplement to natural mating in Broad Breasted Bronze turkeys. Poultry Sci. 31: 925 (Abstract). Malmstrom, M. V., 1943. Factors influencing fertility in the domestic fowl. Thesis, Univ. Conn. Cited by L. W. Taylor, 1949. In Fertility and hatchability of chicken and turkey eggs. lst ed. John Wiley and Sons, Inc., New York, N.Y. Mann, T., 1960. Effect of nutrition on androgenic activity and spermatogenesis in mammals. Proc. Nutr. Soc. 19: 15-18. March, B. E. and J. Biely, 1957. The effect of dietary fat level on thyroid activity in the growing chick. Poultry Sci. 36: 1270-1277. 83 Marr, J. E., F. W. Garland, Jr., J. L. Milligan and H. L. Wilcke, 1956. Effect of controlled light during the growing period upon sub- sequent performance of breeder turkeys. Poultry Sci. 35: 1156 (Abstract). Marsden, S. J. and R. M. Fraps, 1960. Effect of light intensity on reproduction in turkeys. Poultry Sci. 39: 1272 (Abstract). Marshall, A. J., 1961. Reproduction. In Biology and Comparative Physiology of Birds. lst Edi. Vol. II, pp. 169—213. Academic Press, New York, N.Y. Matsui, T., 1966. Effect of prolonged daily photoperiods on the hypothalamic neurosecretory system of the tree sparrow (Passer montanus saturatus) Endocrinol. Jap. 13(1): 23-28. MbCance, R. A., 1960. Severe undernutrition in growing and adult animals. 1. Production and general effects. Brit. J. Nutr. 14: 59-73. McCartney, M. G., 1951. The physiology of reproduction in turkeys. I. Effect of method of mating on fertility and hatchability. Poultry Sci. 31: 658-662. MbClure, T. J., 1966. Infertility in mice caused by fasting at about the time of mating. I. Mating behavior and littering rates. J. Reprod. Fert. 12: 243—248. Meduzov, V. S. and A. A. Kudrjavcev, 1965. Effect of zinc and manganese salts on quality of boar semen. Nutr. Abv. and Rev. 35: 534 (Abstract). Mellen, W. J., F. W. Hill and H. H. Dukes, 1954. Studies of the energy level on the basal metabolism of growing chickens. Poultry Sci. 33: 791-798. Milby, T. T. and D. H. Sherwood, 1953. The effect of restricted feeding on growth and subsequent production of pullets. Poultry Sci. 32: 916 (Abstract). Millen, J. W., 1962. The Nutritional Basis of Reproduction. Chas. C. Thomas, Publisher, Springfield, Illinois. Moore, 0. K. and T. C. Byerly, 1942. Relation of time of insemination to percent of fertility. Poultry Sci. 21: 253-255. Moore, C. R. and L. T. Samuels, 1931. The action of testes hormone in correcting changes induced in the rat prostate and seminal vesicles by vitamin B deficiency or partial inanition. Amer. J. of Physiol. 96: 278-288. _ .n- \_m A ‘A-m- 84 Mulinos, M. G. and L. Pomerantz, 1941. The reproductive organs in malnutrition. Effects of chorionic gonadotropin upon atrophic genitalia of underfed male rats. Endocrinol. 29: 267-275. Nalbandov, A. V., 1964. Reproductive Physiology. 2nd ed. W. H. Freeman and Co., San Francisco, Calif. Nalbandov, A. V., R. K. Meyer and W. H. McShan, 1946. Effect of purified gonadotropes on the androgen-secreting ability of testes of hypophysectomized cocks. Endocrinol. 39: 91—104. National Research Council, Publication 1345, 1966. Nutrient Require- ments of poultry. 5th ed., National Academy of Sciences, Washington, D. C. Newcomer, W. S., 1958. Physiologic factors which influence acidophilia induced stressors in the chicken. Am. J. Physiol. 194: 251—254. 0ksche, A., D. F. Laws, F. I. Kamemoto and F. S. Farner, 1959. The hypothalamo—hypophysial neurosecretory system of the White— crowned sparrow, Zonotrichia leucophrys gambellii. Ztschr. Zellforsch, 51: 1-42. Cited by A. Van Tienhoven in Sex and Internal Secretions, V01. II, Chapter 18, 3rd edition. The Williams and Wilkins Co., Baltimore, Md. Opel, H. and P. D. Lepore, 1967. Ovulating hormone-releasing factor in the chicken hypothalamus. Poultry Sci. 46: 1302. Oregbin—Crist, M. C., 1964. Delayed incorporation of thymidine3H in epithelial cells of the ductus epididymis of the rabbit. J. Reprod. Fertil. 8: 259-260. Ortavant, R., 1952. Recherches quantitatives sur la spermatogense et les reserves spermatiques du belier. Proc. Ilnd Inter. Congr. Physiol. and Pathol. Animal Reproduction and Artificial Insemination I. p. 63. Palmer, D. H., 1966. The effects of day-length and light intensity on the performance of meat-type breeder hens. Bull. No. 363, Univ. of Delaware. Parker, J. E., 1945. Relation of time of day of artificial insemination to fertility and hatchability of hen's eggs. Poultry Sci. 24: 314-317. Parker, J. E., 1946. Semen production in Broad Breasted Bronze turkeys. Poultry Sci. 26: 118-121. Parker, J. E., 1965. Semen production of cockerels as related to their subsequent capacity to fertilize hens in flock matings. Poultry Sci. 44: 474-477. 85 Parker, J. E. and O. A. Barton, 1946. Confinement rotation of toms and artificial insemination as related to reproduction in turkeys. N. Dak. Bimo Bul. 9: 5-13. Parker, J. E. and B. J. McSpadden, 1943. Influence of feed restriction on fertility in male domestic fowls. Poultry Sci. 22: 170—177. Parlow, A. F. and L. E. Reichert, Jr., 1963. Species differences in follicle-stimulating hormone as revealed by the slope in the Steelman-Pohley assay. Endocrinol. 73: 740-743. Patrick, H., 1965. Vitamin E, antioxidants, selenium. Feed Age 14: 28-31. Patton, A. R., H. S. Wilgus and G. S. Harshfield, 1939. The production of goiter in chickens. Science 89: 162. Pazos, R., Jr. and C. Huggins, 1945. Effect of androgen on the prostate in starvation. Endocrinol. 36: 416-425. Perek, M., 1966. Fertility in the male in relation to natural and artificial insemination. In Physiology of the domestic fowl. Brit. Egg Marketing Board Symposium. No. 1, pp. 52~60. E. C. Horton and E. C. Amoroso. Oliver and Boyd, Ltd., Edinburgh. Perek, M. and N. Snapir, 1963. Seasonal variations in semen production of different breeds of cocks and the effect of vitamin C feed supplementation upon the semen of White Rocks. Brit. Poultry Sci. 4: 19-26. Premachandra, B. N., 1962. Radioiodine (1131) and thyroid function in the fowl. Proc. XII World's Poultry Congress, pp. 131-135. Quinn, J. P. and W. H. Burrows, 1936. Artificial insemination of fowls. Jour. Hered. 27: 31—37. Reichlin, S., 1957. The effect of dehydration, starvation and of pitressin injections on thyroid activity in the rat. Endocrinol. 60: 470-487. Reineke, E. P., J. A. Davidson, L. F. Wolterink and F. N. Barrett, 1946. The effect of thiouracil on fattening turkeys. Poultry Sci. 25: 410 (Abstract). Rubio Paredes, J. and T. Perez Garcia, 1959. Vitamin A as a factor affecting fertility in cockerels. Poultry Sci. 38: 307. Salamon, S., 1964. The effect of nutritional regimen on the potential semen production of rams. Australian J. Agric. 15: 645-656. Sayers, G., M. A. Sayers, H. L. Lewis and C. N. H. Long, 1944. Effect of adrenotropic hormone on ascorbic acid and cholesterol content of the adrenal. Proc. Soc. Exptl. Biol. Med. 55: 238—239. AX-LY.av-{r .v‘ 3.. 86 Siegel, H. S., 1960. Effect of population density on the pituitary— adrenal cortical axis of cockerels. Poultry Sci. 40: 216-219. Siegel, H. S. and W. L. Beane, 1961. Time responses to single intra— muscular doses of ACTH in chickens. Poultry Sci. 40: 216-219. Silliphant, W. M., 1960. Manual of histologic and special staining technics. 2nd ed. Armed Forces Inst. of Pathol. MCGraw—Hill Book Co., Inc., New York, N. Y. Steel, G. D. and J. H. Torrie, 1960. Principles and Procedures of Statistics. lst edition. McGraw-Hill Book Co., New York, N. Y. Steelman, S. L. and F. M. Pohley, 1953. Assay of the follicle stimu— lating hormone based on the augmentation with human chorionic gonadotropins. Endocrinol. 53: 604-616. Stephens, P. J., 1940. The effect of thyrotropic principle on the anterior pituitary of the thyroid of the undernourished guinea pig. Endocrinol. 26: 485-492. Stockton, K. L. and V. S. Asmundson, 1950. Daily rhythm of egg pro- ' duction in turkeys. Poultry Sci. 29: 477-479. Summers, J. D., W. F. Pepper, S. J. Slinger and J. D. MCConachie, 1967. Feeding meat type pullets and breeders. Poultry Sci. 46: 1158-1164. Sure, B., 1938. Influence of avitaminosis on weights of endocrine glands. Endocrinol. 23: 175-181. Touchburn, S. P., 1966. Feeding the turkey breeder. Turkey Research 1966. Ohio Agr. Res. and Develop. Center, Research Summary 17, Wooster, Ohio. Urist, M. R., 1959. The effects of calcium deprivation upon the blood, adrenal cortex, ovary and skeleton in domestic fowl. Recent Progress in Hormone Res. 15. Academic Press, New York, N. Y. Van Albada, M., 1962. Influencia de los periodos de iluminacion sobre la postura en ciclos de la gallina domestica. In Avicultura Moderna, Prensa Medica Mexicana, Mexico, D. F. Wakely, W. J., 1949. Seasonal variations in the morphology of turkey spermatozoa. Unpublished M.S. thesis, Washington State Univ., Cited by I. L. Kosin and A. W. Wheeler in Methods for estimating spermatozoal numbers in turkey semen. Northwest Science 30: 41-47. Waldroup, W. P., B. L. Damron and R. H. Harms, 1966. The effect of low protein and high fiber grower diets on the performance of broiler pullets. Poultry Sci. 45: 393-402. 87 Warren, D. C. and H. M. Scott, 1936. Influence of light on ovulation in the fowl. J. Exper. Zool. 74: 137*156. Whetham, E. 0., 1933. Factors modifying egg production with specific reference to seasonal changes. Jour. Agric. Sci. 23: 383-418. Wilhelm, L. A., 1965. Personal communication. Willet, E. L. and J. I. Ohms, 1957. Measurement of testicular size and its relation to production of spermatozoa by bulls. J. Dairy Sci. 40: 1559-1569. Wilson, W. 0., A. E. Woodward and H. Abplanalp, 1964. Exogenous regu— lation of oviposition in chickens. Poultry Sci. 43: 1187-1192. Witschi, E., A. J. Stanley and G. M. Riley, 1937. Gonadotrophic hormones of the hypophysis of the turkey. Proc. Soc. Exptl. Biol. and Med. 36: 647-651. Wolford, J. H. and R. K. Ringer, 1962. Adrenal weight, adrenal ascorbic acid, adrenal cholesterol and differential leucocyte counts as physiological indicators of "stressor" agents in laying hens. Poultry Sci. 41: 1521-1529. Wolford, J. H., R. K. Ringer and T. H. Coleman, 1964. Ovulation and egg formation time in the Beltsville Small White turkey. Poultry Sci. 43: 187-189. Wolford, J. H., R. K. Ringer and T. H. Coleman, 1964. Lag time, interval between successive eggs and oviposition time in the turkey. Poultry Sci. 43: 612-615. Wolford, J. H., R. K. Ringer, T. H. Coleman and H. C. Zindel, 1963. Body weight and egg production of turkeys as influenced by lighting regime during the growing period. Mich. Agr. Expt. Sta. Quart. Bul. 45: 506-517. Woodward, A. E., W. 0. Wilson and F. B. Mather, 1963. The egg—laying rhythm of turkeys in cages. Poultry Sci. 42: 1131-1133. wyne, J. W., R. D. Carter, M. G. MbCartney and V. D. Chamberlin, 1959. Relation of time of insemination to fertility in the turkey. Poultry Sci. 38: 828-831. Zlatkis, A., B. Zak and A. J. Boyle, 1953. A new method for the direct determination of cholesterol. J. Lab. Clin. Med. 41: 486-492. Zubiran, S. and F. Gomez-Mont, 1953. Endocrine disturbances in chronic human malnutrition. Vitamins and Hormones 9: 97-132. ‘ m.;‘..li .31 um; Ll}!l ’m‘ “a APPENDIX 88 APPENDIX TABLE I Eosin solution for sperm counts Water 500 milliters 2 per cent eosin solution 25 " 3 II N NaC]. ll 10 It Mixed thoroughly and diluted in a 1:1 proportion with physiological saline for spermatozoal determinations. 89 APPENDIX TABLE II A. Procedures Procedure for cholesterol determination in blood and adrenal glands. Reference: Zlatkis, A., B. Zak and A. J. Boyle, 1953. A new method for the direct determination of cholesterol. J. Lab. Clin. Med. 41: 486-492. Reagents: 1. Standard cholesterol solution (100 mg Z). Dissolve 100 mg of pure, dry and ash-free cholesterol in 100 m1 of 100 percent_Mg£gk glacial acetic acid. 2. Ferric chloride solution (10 mg percent). Dissolve 10 grams of ferric chloride, reagent grade in 100 m1 of 100 percentheggk glacial acetic acid. 3. Color Reagent: Dilute 2.0 ml of the ferric chloride solu- tion to 200 ml with chemically pure concentrated sulfuric acid. To prevent chloride precipitation, add 100 ml of sulfuric acid into volumetric flask. Then add the ferric chloride solution slowly while swirling flask. Procedure: Use triplicate samples A. Blood plasma: 1. Thoroughly mix thawed frozen plasma 2. Pipette 0.1 ml into (Step 3) 90 B. Adrenal tissue: 1. Make a 1.5 gram thawed frozen tissue in 40 m1 of glacial acetic acid. 2. Transfer to 120 m1 Erlenmeyer flask; shake at high speed for 1.5 hrs; filter (Whatman #1) and pipette 0.1 ml into: (Step 3) General Steps: 3. 10 m1 of glacial acetic acid in 20 ml test tube. 4. Add 7 m1 of color reagent and mix immediately. 5. Use plastic stoppers and invert three times with thumb or plastic top.1 6. Let stand for one hour; read in spectrophotometer twenty at 560 millimicrons. 7. Read against a sample of 10 ml glacial acetic acid and seven m1 color reagent. Standard: 1. Use 0.1 ml of standard cholesterol solution and pipette to step #3 of procedures. Calculations: O.D. unknown 100 mg cholesterol 1. X 0.1 ml Std. X = O.D. standard 0.1 ml 100 ml plasma O.D. unknown 60 100 mg cholesterol 2. X 0.1 mg Std. X-——— X = O.D. standard 0.1 gm liver 100 gms wet adrenal 1Caution: tubes become hot. 91 APPENDIX TABLE III A. Procedures - Determination of blood sugar. References: Method of Folin and Wu, 1920. J. Biol. Chem. 41: 367. Reagents: A. 10% sodium tungstate (NaZWo 4 H20) 1. In a 1 liter volumetric flask containing 600 ml of distilled water, dissolve 100 grams reagent grade, sodium tungstate. 2. Dilute to 1 liter with distilled water. B. 2/3 N sulfuric acid (H2804) 1. Into a liter volumetric flask containing about 600 ml of distilled water, add slowly with constant stirring 19 m1 of concentrated sulfuric acid. Dilute to the liter mark with distilled water. Check by titration against a standard sodium hydroxide (NaOH) solution. C. Alkaline copper reagent: 1. Dissolve 40 grams of anhydrous sodium carbonate (Na2C03) in 400 ml of distilled water, and transfer to a 1 liter volumetric flask. Add 7.5 grams of tartaric acid. Add 4.5 grams of crystalline copper sulfate (CuH20°5H20) and dissolve. Mix and dilute to the 1 liter mark with distilled water. On standing for sometime a sediment may appear. If this happens decant or filter through a good quality filter paper. This reagent keeps for a long time. However, it 92 should be checked by transferring 2 m1 of phosphomolybdic acid solution. The blue color of the copper solution should almost completely disappear. Phosphomolybdic acid color reagent: 1. 5. Place 35 grams of molybdic acid and 5 grams of sodium tungstate in a liter beaker. Add 200 m1 of a 10% sodium hydroxide (carbonate free) solution and add 200 ml of distilled water. Boil vigorously for 20 to 40 minutes (until all the ammonia is disposed). Cool, dilute to 350 ml and add 125 ml of 85% (concentrated) phosphoric acid. Dilute to 500 ml with distilled water and mix. Standard glucose solutions: 1. Stock standard (1% solution): a) Dissolve 1 gram of highest purity anhydrous dextrose in 50 ml of saturated benzoic acid solution. b) Make up the volume to 100 ml with saturated benzoic acid solution. c) This solution keeps indefinitely. Dilute standard a) Transfer 2 m1 of the stock 1% glucose standard to a 100 ml volumetric flask. b) Make up the volume with distilled water or saturated benzoic acid solution. This solution stands indefi- nitely and equals 200 mgs percent. 93 3. Saturated benzoic acid solution. a) Dissolve 2.5 grams of benzoic acid crystals in 500 ml of distilled water by boiling. b) Transfer to a 1000 m1 volumetric flask and dilute to a liter rinsing the benzoic acid until the volume has been reached. Procedures for Unknown: 10. ll. Into a Folin-Wu sugar tube place: 2.0 m1 of 1:10 protein free blood filtrate (Filter 42, blue). 2.0 m1 of alkaline copper reagent. Mix by lateral shaking and place into a boiling water bath for 6 minutes. Remove without shaking and place in a large beaker of cold water for 2 to 3 minutes. Add 2.0 ml of phosphomolybdic acid color reagent. Replace in boiling water bath for 5 minutes. Dilute to the 25 m1 mark with distilled water. Mix well by repeated inversions. Allow to stand for 10 to 15 minutes. Transfer a portion of the colored solution into a calorimeter tube. Read in the colorimeter within the next 15 minutes, against a blank tube set at 0. Procedure for blank: Run a parallel determination as described above on 2.0 ml of dis- tilled water in place of the blood filtrate. Transfer a portion of the final solution to a colorimeter tube and set the colorimeter to its 0 reading against this solution. 94 Procedure for standard: Run a determination as described above but use 2.0 m1 of a standard glucose solution instead of the blood filtrate. Read a portion of the final colored solution in the colorimeter against the blank tube set at 0. Calculations (Against the standard): Optical density_of unknown 100 _ Optical density of standard X 0.4 X-6-§-— mg of 51::32e Per 100 m1 of OR Concentration of the standard Reading of the standard X reading of unknown = concentration of the unknown. Calibration Factor: Reading of unknown X Folin-Wu blood sugar factor = mgs % of blood sugar in unknown. The factor is obtained from the reading of the standard solution described above, and calculating the factor from the following formula: Folin-Wu sugar factor = 200 Reading of Sd - reading of blank The calibration factor should be determined in duplicate or triplicate and the average value taken. Once determined, the calibra- tion factor is valid indefinitely within the limits of calibration. 95 APPENDIX TABLE IV A. Procedures - Follicle stimulating hormone (FSH) bioassay. FSH Augmentation — Method of Steelman and Pohley (1953) as modified by Parlow and Reichert (1963). References: 1. Parlow, A. F. and L. E. Reichert, Jr., 1963. Endocrinol. 74: 740. 2. Steelman, S. L. and F. M. Pohley, 1953. Endocrinol. 53: 604. Procedure: 1. The pituitaries are rapidly removed, placed in physiological saline solution and homogenized in a glass homogenizer. 2. The different solutions are brought up to the required volume by adding physiological saline according to the desired concentra— tion of pituitary tissue per milliliter. 3. Doses of three milliliters per rat are prepared by adding physiological saline and 50 I.U. of human chorionic gonadotropin per dose. 4. Inject 0.5 milliliters every 12 hours for three days totalling 3.0 milliliters per rat. 5. 72 hours after the first injection was made, kill the rats and perform a necropsy. 6. Remove the ovaries, dissect free of surrounding tissue and weigh to the nearest one-tenth of a milligram. 7. A FSH preparation of known potency should be run for comparison purposes. 8. Compare the results by appropriate statistical methods. Material required: Female rats 20 to 25 days of age. 96 Note: The use of human chorionic gonadotropin to augment the responses observed with FSH, results in an assay with increased sensitivity and decreases the variation due to luteinizing hormone contamination. 111111111va 058 ll J93 03 111131":er