STUELY 05 THYRG?§£$T°E§H $2.5m»! ON 3251*. SEEK N "Mes-is far has Baggage 05 M, S MECHiCxAM STkfi U‘i‘z‘WéRSE'FY' kasr N. Kunzru 196:3 LIBRAR Y Michigan State University ABSTRACT STUDY OF THYROPROTEIN THERAPY 0N RAM SEMEN by Omkar N. Kunzru ' Five su5pected infertile rams from privately owned sheep breeding farms, including one from the Michigan State University Sheep Breeding Farm, were used in the study. The two control, known fertile rams, were selected from the Michigan State University Sheep Breeding Farm. Volume, concentration and total number of sperm produced were evaluated. Respira- tion rate of the sperm was also measured on a multi-unit constant-pressure respirometer. Starting on May 2i, oral treatment of .2 gm of thyroprotein (Pro- tamone) was given daily to the suspected infertile rams. No statistically significant difference was found between the fertile and infertile rams, in volume and total sperm ejaculated. Concentration was found to be statis- tically significantly higher in the fertile group than the infertile group. The sperm from the ejaculates collected during the treatment period consumed more oxygen than from the ejaculates of the pretreatment period. The treatment appeared to have some favorable effect on the respiration rate of the sperm. On July ll, the thyroprotein dosage was increased to .# gm daily. Volume of the semen was not found to be statistically significantly different between the control and the treated group. Concentration and total number of Sperm ejaculated during this period were highly significantly higher in the control rams than the treated rams. No respiration studies could be made during the higher level of thyro- protein feeding. Omkar N. Kunzru Study on the ribonucleic acid (RNA) and desoxyribose nucleic acid (DNA) content of the sperm collected from the fertile and infertile rams was also started but the experiment had to be abandoned before any relevant data could be collected. STUDY OF THYROPROTEIN THERAPY 0N RAM SEMEN BY Omkar N. Kunzru A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of MASTER OF SCIENCE Department of Animal Husbandry I963 ACKNOWLEDGMENTS The author desires to express his deepest appreciation, with grati- tude, to his Major Professor, Dr. L. H. Blakeslee of the Department of Animal Husbandry for his considerate guidance, supervision, counsel and helpful suggestions so graciously extended by him. His constant interest has been of great value and encouragement during the course of this study. Sincere thanks are acknowledged to Dr. E. P. Reineke of the Depart- ment of Physiology for his genuine interest, constant guidance and help during the study. Grateful appreciation is extended to Dr. w. T. Magee of the Depart- ment of Animal Husbandry for his guidance in the statistical analysis and the constructive suggestions he made from time to time. His ever willing- ness to offer assistance and inspiration will always be remembered. The author wishes to thank the Michigan State University Department of Animal Husbandry and the Endorcrine Research Unit for the facilities provided to carry out this research. In addition, he wants to thank Bill Heiss and Fred Howe for their help in handling the sheep. Sincere thanks to my friend Mr. A. S. Summanwar for his help in typing and arranging the manuscript. In addition, the author is deeply indebted to Mrs. Kay Butcher for her help and the excellent job in typing the final copy of this thesis. TABLE OF CONTENTS Introduction ........................................... Review of Literature ................................... Experimental Procedure ........ . .................. ...... Results and Discussion ................................. Summary and Conclusions ............................ .... Bibliography .............................. . ............ Appendix Tables ........................................ 2] 24 27 Table I. Table 2. LIST OF TABLES Averages of volume, concentration and total number of sperm per ejaculate produced during the period of April 9 to May l7 ..................... Analysis of variance for volume, concentration and total number of sperm per ejaculate during the period of April 9 to May l7 ..................... Table 3. Averages of volume, concentration and total number of sperm per ejaculate produced during the period of May 2# to July 9 ...................... Table A. Analysis of variance for volume, concentration Table 5. Table 6. Table 7. Table 8. Table 9. and total number of sperm during the period of May 2A to July 9 ........ ...... ..... . ................ Averages of volume, concentration and total number of sperm per ejaculate produced during the period of July l2 to August 2 ................... Analysis of variance for volume, concentration and total number of sperm per ejaculate during the period of July l2 to August 2..... .............. Average pl. of oxygen consumed/billion Sperm/hour for the pretreatment period and treatment period.... Analysis of variance for oxygen consumption/billion sperm/hour of the pretreatment period and the treatment period .................................... Analysis of variance of the change in the respira- tion rate of sperm (pl. of oxygen/billion sperm/hour) due to .2 gm. of Protamone fed daily to the infer- tile rams ......................... . ................. Page 12 IA 17 I7 20 2] 2i INTRODUCTION In the past quarter of a century many studies have been done on the effect of high temperatures on the semen production of rams and other male farm animals. The effect of both high atmospheric temperature and artificial insulation on the scrotum on different traits of the semen has been studied. The importance of the thyroid gland in the normal production of semen in rams has long been established. Thyroxine has also been tried as a “cure'l for the so-called IIsummer sterilityll of rams, a phenomenon in which the thyroid gland reduces its secretion of thyroxine because of the increased atmospheric temperatures resulting in part, in the inferior quality of semen produced during these hot months. Originally this research was designed to study three things: I. To compare the volume, concentration and the total number of sperm pro- duced in known fertile and suspected infertile rams. II. To study the effect of thyroprotein-feeding through the summer months on the semen production of the suspected infertile rams. III. To compare the respiration rate of the sperm in the semen of known fertile and suspected infertile rams and to see the effect of feed- ing thyroprotein on it. However, for the reasons explained further in these pages experiment III had to be terminated before all the data could be collected relative to number III. Comparison of ribonucleic acid (RNA) and desoxyribose nucleic acid (DNA) contents in the sperm of the fertile and infertile rams was also started but the experiment had to be discontinued before any relevant data could be collected, because very frequently all of the infertile rams did not ejaculate half a billion sperm.+ +Personal communication from Dr. Manuel Gordon, formerly of the Department of Dairy, Michigan State University, East Lansing. REVIEW OF LITERATURE Smelser (l93h) compared thyroidectomized rats with control litter- mate rats and found that the thyroidectomized rats produced fewer sperm than the controls. 0n autopsy it was found that the treated rats had smaller accessory reproductory organs than the controls. Phillips and McKenzie (l93h) showed that the scrotum of the ram functions very much as a thermostat and maintains the temperature of the testes constant and considerably lower than that of the body cavity. That an artificial insulation on the scrotum has a detrimental effect on the semen production, was also noted. Low temperatures (lowest 6-8°C.) did not have any harmful effects on the testes of rams. An inferior quality of semen was produced by rams during late win- ter when McKenzie and Berliner (1937) kept them in a room artificially kept at high temperature. Berliner and Warbritton (I937) studied seasonal variation in rams and found summer as the lowest season. Semen production fell in thyroid- ectomized rams but injections of h mg. of thyroxine improved the semen toward normal. Green (l9h0) found that the general quality of semen decreases slightly but steadily from January to May, becoming quite in- ferior in June and July, presenting an increased number of the abnormal sperm. But from July to October a very rapid readjustment towards higher quality is underway. After October all sperm types become relatively stationary in their proportional relationship. At Cornell, Phillips g£_§l; (I9A3) during their study of seasonal variation in the semen of sheep and goats, found relatively little dif- ference in volume between the seasons. Strangely enough, the concentration was found lowest in the fall instead of summer and highest in winter. -2- -3- No deleterious effect on ram semen was found by Accame e£_§l; (I944) when the rams were subjected to reduced barometric pressure. The effect of synthetic thyroprotein on sterility in bulls was studied by Reineke (I946). A definite improvement in the libido of bulls with some indication of improved fertility was observed. Number of ser- vices per conception was also reduced. Bogart and Mayer (I946) administered thyroxine to rams subcutaneously and later orally. They found that it did not prevent the gradual decline in the motility and did not have any effect on the volume. The concentra- tion and/or total sperm ejaculated were, however, stabilized at a higher level than that during the pretreatment period. Increase in the percent- age of abnormal sperms produced was also prevented. Shaffner (I948) found that thyroprotein fed to mature cocks reduced their fertility. Volume and concentration, however, were not affected. Eaton §£_al; (I948) made the comparison of feeding two different levels of thyroprotein on the quality of semen produced in rams. Results did not indicate that feeding thyroprotein was beneficial to rams since semen concentration, percent normal sperm and motility was higher in the untreated groups than either of the treated groups. Volume was, however, higher in the higher level (2 gm.) of thyroprotein than the lower level (I gm.). Huston and Wheeler (I949) fed the synthetic thyroprotein to mature cocks. They found that the volume of semen from the treated birds was significantly lower than from the untreated. The concentration and total number of Sperm ejaculated were higher in the treated ones, although not statistically significant. -4- Black g£_§l;_(l950) did not find any increase in the number of total sperm produced by feeding thyroprotein to rams. Volume was increased but it was not significantly different. Concentration was affected to a very small extent. Casady giggl;_(l953) tested the effect of high ambient temperature on spermatozoa of dairy bulls kept in mechanically controlled chambers. They concluded that under chamber conditions spermatogenesis in young dairy bulls may be impaired when the animals are kept continuously exposed to temperature exceeding 85° F. for 5 weeks. Wilwerth‘gt_al; (I954) fed thiouracil to male chickens. The treated birds exhibited a trend toward decreasing volume. When these animals were fed thyroprotein it increased the volume. A higher level of thyro- protein increased the concentration also without effecting the volume. A very high level significantly decreased the concentration and volume. Henneman, Reineke and Griffin (I955) found that in sheep the thyroid se- cretion rate was lowest during July. Dutt and Hamm (I955) exposed rams to artificially maintained high temperatures of 90° C. during winter. They found that the semen quality for unsheared rams in the hot room was inferior but there was very little effect on the sheared rams in the same room. Eight weeks after the test period, there was no significant difference between the treated and un- treated groups. El-sheikh and Casida (I955) working with rabbits concluded that increased ambient temperature may effect the male fertility without bringing about any apparent depression of sperm motility. The ratio of -5- fertilized to unfertilized ova in test females was lower for the treated animals. Dutt and Bush (I955) and Dutt and Simpson (I957) found that keeping the rams in a cooled room the temperature of which was artificially maintained between 45°-48° F., improved their semen characteristics in motility and percent abnormal cells significantly but volume and concen- tration were not significantly effected. Foote 23.21; (I957) concluded that in high temperatures, shearing is more effective in preventing a rise in testes temperature when humidity is low than when humidity is high. Simpson §£_gl; (I959) found that rams which had free access to an air-conditioned room had better semen characteristics in volume, concen- tration, percent motile sperm and percent abnormal sperm than those which did not. Recently Brooks and Ross (I962) in a series of experiments found the same results as Dutt and Bush (I955) and Simpson et al. (I959) except that in their experiment volume was higher in the heated room than in the cooled room. Wilson g£_§l; (I959) concluded that rams kept in a chamber in which both temperature and light conditions were simulated to October conditions had a higher fertility rate than others, including those in which only temperature conditions were simulated to October conditions. Whiteman and Brown (I959) cooled rams during the day and found that cooled rams made more matings and produced more lambs per mating than the controls, although it was found not statistically significant. Brooks, Ross and Pipes (I960) found that the thyroid secretion rate decreases with increasing temperatures. The correlation coefficient was found to be -.363. -6- Brooks and Ross (I962) found that thyroxine given at the rate of .4 mg/IOO lb. bodyweight was detrimental to semen quality at high en- vironmental temperatures. In a cooled room the thyroxine therapy had little effect except that sperm concentration was higher in the treated rams. The .3 mg/lOO lb. bodyweight treatment did not have any appreci- able effect on the quality of semen in the heated room but in the cooled room the treated rams had somewhat superior semen than the untreated, in motility, percent normal sperm and sperm concentration. When thyroxine therapy was reduced .2 mg/IOO lb. bodyweight in the heated room there were no significant differences but the treated rams had higher sperm concentration and higher percent of normal sperm while the untreated rams had a higher volume, higher motility and a higher percent of live sperm. In the cooled room, the semen from untreated rams was of a higher quality in all indices. It was suggested that there is a lowering of semen quality associated with thyroxine therapy at higher environmental temperatures. Gray (I928) working with the sea urchin spermatozoa, found that the oxygen consumption per spermatozoa is inversely related to the number of sperm present. The same results have also been observed on ram and boar spermatozoa by Winchester (l94l) and on unaltered human semen by Ross, Miller and Kurzrok (l94l). Bishop g£_gl; (I954) diluted the whole bull semen four times with egg yolk phosphate and still obtained the same results. Henle and little (I942), however, concluded that with epididymal bull spermatozoa in Ringer's solution, maximal oxygen uptake was at the approximate concentration of 5 x l08 spermatozoa/ml. -7- Bishop (I942) measured the oxygen consumption of fox sperm and con- cluded that the oxygen uptake is directly related to the number of sperm cells. Walton and Edwards (I938) reported on the use of respiration rate of the spermatozoa as a criterion of quality of bull semen. They found that bulls whose sperm exhibited higher re5piration rates required fewer services per conception. Windstosser (I935) found that the respiration rate of semen diluted 1:200 with saline solution, was higher than in whole semen, but in both the series the spermatozoa were immotile throughout the experiment. Sergin (I939) found that the respiration of spermatozoa is lower than most of the other tissues of the body. He also found that the higher the concentration of the semen the higher was the oxygen consump- tion. Thus ram semen which is more concentrated than the semen of the boar or stallion absorbs more oxygen than semen from the other Species. Ely, Herman and Winchester (I942) thought as a result of their ex- periments that the spermatozoa exhibiting a greater activity may live longer than those of lower activity. Bishop and Salisbury (I955) measured the oxygen uptake of both un- diluted and seminal plasma diluted bull semen. The oxygen uptake per cell was found to be inversely related to the concentration of the sample subjected to the experiment. Mohri and Horiuchi (l96l) found that the presence or absence of carbon dioxide influences the respiration rate of sea-urchin spermatozoa. EXPERIMENTAL PROCEDURE Seven rams were used in the experiment (2 known fertile and 5 sus- pected infertile). The 2 known fertile rams were selected from the Michigan State University Sheep Breeding Farm, on the basis of their previous performance, in the number of ewes settled by them. The infer- tile rams were also randomly selected from the farms of breeders who were running private sheep breeding enterprises. Only one ram was taken from each farm, the ram settling the lowest number of ewes being selected. The infertile rams were not all available for the same length of time, thus the data presented here and used in the analysis is from the date all seven rams were available for the experiment, to the date the first ram was taken away. All the rams were kept in the indoor pens and hand fed. The hay and grain given was of the same quality for each ram. Semen samples were collected twice every week at 3-5 day intervals with an electro- ejaculator. If the volume was not enough in the first ejaculate, another collection was tried immediately and the ejaculate collected in the same tube. Graduated tubes were used throughout the experiment. The collec- tion tube was kept in a water jacket at a temperature of approximately 43° C. Immediately after the collection was completed the tube was securely corked, marked and transferred to a thermos flask in which fro- zen water in a can had been stored for about I5-20 minutes. The arti- ficial vagina was washed and dried properly between the collections from different rams. After all seven collections were made the samples were taken to the laboratory immediately. Volume was recorded directly from the tube and concentration of sperm was counted on a haemocytometer slide. The staining fluid used had the following composition: -3- 50 ml of H20 2 ml of 2% Eosin l ml of 3% Sodium Chloride Unfortunately, at the beginning of the experiment only three traits of the semen, namely, volume, Sperm concentration and total sperm ejacu- late were considered. Later motility ratings in approximate percentages were also given to the samples. Respiration studies were made on ejaculations taken prior to July 5. After July 5 no determinations could be made for respiration rate because of one of these three reasons: a. either the samples did not contain any sperm, b, the concentration per cc. was so low that .2 cc. of that sample would have had very few sperm, or c. no semen sample was given by the animal. The respiration study was made on the multiple-unit constant-pres- sure micro-respirometer devised by Reineke (l96l), using .2 cc. samples of semen. While analyzing the data, if no sample was obtained a zero was entered for that animal in all the three traits, namely volume, concen- tration and total sperm ejaculated, because it was thought that the animal did not have anything to offer. Whenever a urine sample was given it was omitted from calculation because it was difficult to know whether the reason was that he did not have anything to give or that this was due to the poor technique on the part of the collector. In either case, there usually was an erection of the penis. When a semen sample was given but no Sperm were found in it, the volume was recorded and counted in the calculations. A zero was entered in the other two traits. RESULTS AND DISCUSSION The averages of the pretreatment period of the three semen traits studied in this experiment are tabulated in table I, the analysis of variance for those traits is shown in table 2. No significant differ- ence (P<.05) in volume was found between the fertile and infertile rams, although a significant difference (P<.05) was found between the rams within the groups. The mean square for between rams/groups was used in all cases to test the significance of the mean square between groups. This was done because of the assumption that these rams were just samples of rams from two populations, fertile and infertile, and what we are really interested in testing is the difference between these two populations. No significant difference (P<.05) was found in the Sperm concentra- tion of the fertile and infertile rams. The Sperm concentration between the rams within the groups was also not statistically significant (table 2). In the total number of sperm produced per ejaculate, again, no significant difference (P<.05) was found between the groups but there was a significant difference (P<.05) between the rams within the groups (table 2). Since no significant difference was found in any of the three semen traits considered, between the fertile and the infertile rams, it can safely be said that the reason why these rams were suspected to be in- fertile is something other than these. Since these rams were selected on their previous fertility performance records there is a possibility that the semen of these rams is inferior in some traits other than these three. It is unfortunate that the motility, percent normal sperm -10- -1]- and percent live sperm were not taken into consideration from the very beginning of the experiment. The poor fertility record could also be because of lack of libido in some of the rams. Since there was no way of determining the libido in these infertile rams, it cannot be ruled out. On the other hand, the two control rams were selected from the Michigan State University Sheep Breeding Farm on their previous record and were known to be fertile.' From table I it can be seen that the total number of sperm produced by either of the two fertile rams was considerably higher than 4 of the infertile rams. Ram No. 7 was higher than the two fertile rams. But there was no significant difference between the two groups. Table l. AVERAGES OF VOLUME, CONCENTRATION AND TOTAL NUMBER OF SPERM PER EJACULATE PRODUCED DURING THE PERIOD OF APRIL 9 TO MAY 17. Trait Fertile Infertile Ram l Ram 2 Ram 3 Ram 4 Ram 5 Ram 6’ Ram 7 Numbera l0 ll 9 l0 l0 7 IO Volume I.39 0.67 0.54 0.66 0.47 0.57 1.17 (cc.) Concentration l.l6 l.7l l.25 l.l5 l.l3 0.69 l.30 (billion/cc.) Total number I.56 l.l8 0.71 0.73 0.54 0.39 I.69 of sperm ejaculated (billion) aNumber of observations. -12- Table 2. ANALYSIS OF VARIANCE FOR VOLUME (A), CONCENTRATION (B) AND TOTAL NUMBER OF SPERM (C) PER EJACULATE PRODUCED DURING THE PERIOD OF APRIL 9 TO MAY 17.3 Source Mean squares for traits df A B C Between groups I l.48 l.46 3.88 Between rams/groups 5 l.l6* 0.69 2.l2* Between ejaculates/rams 60 O.l5 0.34 0.56 *P<.05. From May 24 to July 9, .2 gm of Protamone was given orally in a gelatin capsule to the infertile rams. The averages of the three traits for this period are tabulated in table 3. The analysis of vari- ance for the three traits is reported in table 4. No significant difference in volume (P<.05) was found between the two groups. The rams within the groups were, however, significantly different (P<.05). Table 4. Total number of sperm produced by the two groups was also found not to be statistically significant (P<.05). The rams within the groups were also not significantly different (P<.05). Table 4. In the analysis of the pretreatment data no significant difference was found to exist between the two groups of fertile and infertile rams in volume, concentration or total sperm produced. However, all the in- fertile rams were kept in the Hsuspected infertile'l group, because of their previous poor fertility performances. By the time this experiment was started, moderately high temperatures had already set in, in this area, which must have effected the semen production. Since all the rams, fertile and infertile, were fed the same ration and housed together -13- there were no known environmental differences between the two groups. In other words the deterioration in the semen quality because of the high temperatures should have been the same for both the groups. Still no significant difference was found in the volume, although average vol- ume produced by 3 of the infertile rams was higher than their volume of the pretreatment time and the other 2 were almost the same. Both the fertile rams also produced a little more volume, on an average, in this period than in the pretreatment period, which as far as the know- ledge of the author goes, is unaccountable even on the basis of any previous work. Since no Significant difference was found between the groups it will be safe to say that .2 gm. of thyroprotein did not have any great effect on the semen volume of the infertile rams. This agrees with the results of Bogart and Mayer (I946), Shaffner (I948), Wilwerth g£_al; (I954) and Brooks and Ross (I962) but does not seem to be in line with the results that Eaton §£_§l;_(l948), Warwick g£;§l; (I948) and Black g£_§l; (I950) obtained in their experiments. But no detrimental effect was apparent on the volume as was found by Huston and Wheeler (I949) working with cock semen. The sperm concentration was statistically different between the fertile and infertile groups. On the average all the seven rams pro- duced semen of lower concentration than their respective concentra- tions of the pretreatment period. Since in the pretreatment period there was no statistical difference between the two groups, the only conclusion that can be drawn from the available data is that the thyro- protein did not have any effect on the concentration of the sperm. -14- Table 3. AVERAGES OF VOLUME, CONCENTRATION AND TOTAL NUMBER OF SPERM PER EJACULATE PRODUCED DURING THE PERIOD OF MAY 24 TO JULY 9. Trait Fertile Infertilea Ram l Ram 2 Ram 3 Ram 4 Ram 5 Ram 6 Ram 7 Numberb 14 14 14 14 12 14 14 Volume I.54 0.7] 0.90 0.53 0.60 0.89 1.00 (cc.) Concentration l.l4 l.40 0.93 0.8l 0.46 0.54 l.03 (billion/cc.) Total number 2.0] l.l3 0.94 0.58 0.35 0.55 l.3l of Sperm ejaculated _(billion) aRams received .2 gm. protamone daily. bNumber of observations. Table 4. ANALYSIS OF VARIANCE FOR VOLUME (A), CONCENTRATION (B), AND TOTAL NUMBER OF SPERM (C) PER EJACULATE PRODUCED DURING THE PERIOD OF MAY 24 TO JULY 9.a Source Mean squares for traits df A B 0 Between groups I 2.l6 5.10% 12.89 Between rams/groups 5 l.40* 0.74 2.62 Between ejaculates/rams 87 0.34 0.36 l.48 aInfertile rams given .2 gm. of protamone daily. *P<.05. Thyroprotein did not have any effect on the volume of the semen of the treated rams. Bogart and Mayer (I946) had concluded on the same kind of results in their experiment that this was due to the fact that a . -15- thyroprotein did not effect the interstitial activity of the testes. However, their conclusion that thyroprotein affected the spermatogenic activity was not substantiated in this experiment because of the fact that sperm concentration and total number of sperm were not affected by thyroprotein feeding. But one difference that very definitely exists between the two experiments is that their experiments were on the known fertile or supposedly fertile rams, whereas, this study was on suspected infertile rams. On July II, the thyroprotein dose was increased to .4 gm. daily. In the analysis of variance and in the averages only six rams are used because on July 20, Ram No. 7 a Michigan State University Sheep Breeding Farm ram was removed from the experiment and was put on pasture. After about 3 weeks, a sample was collected from him by the same method but he did not ejaculate any sperm. From the day he was put on pasture the treatment was also discontinued. Since the ram was taken away in the middle of the experiment, it was decided to omit him from all analyses. The averages of the three traits of the semen for this period are tabulated in table 5 and the analysis of variance in table 6. There was no statistically significant difference (P<.05) in volume between the two groups (table 6). It seems that although the difference in the volumes was not significant, this higher level of thyroprotein had some undesirable effect on the rams. When we compare table 5 with table 3, it appears that the treatment did not have a desirable effect on the rams. This conclusion was made by comparing the performance of the infertile rams for this period with their performance of previous -l6- periods. The fertile (control) rams had also shown some decline in their semen characteristics but since the same rams had been kept as controls for all the periods, a relative comparison between the performance of the infertile rams for different periods was made. The relative decline in the semen of the fertile rams was also kept in view, although there was one infertile ram (No. 4) whose average volume for this period was higher than the pretreatment and the period during which .2 gm. of thyro- protein was given. Ram No. 3 produced a higher volume than in the pre- treatment period but was lower than during the lower level period. All the other rams produced their lowest volume of the experiment in this period. Sperm concentration was highly significantly (P<.Ol) higher in the fertile rams than in the infertile rams (table 6). The total number of Sperm produced in the control group was also highly significant (P<.0l) over the treated group (table 6). There was no difference within the groups in both the traits. As can be seen from table 5, this higher level of thyroprotein was harmful on the spermatogenic activity of the testes. These results are in accordance with the results of Warwick et al. (I948) who had fed a lower level but of a highly potent thyro- protein; Wilwerth g£_al; (I954) who found that .08% and .I6% of prota- mone were detrimental to cock semen in both volume and concentration; and Brook and Ross (I962) who concluded that there appeared to be a lowering of semen quality associated with thyroxine therapy at higher environmental temperatures. The averages of the oxygen consumption/billion Sperm/hour of the pretreatment period and the period in which .2 gm. of thyroprotein was -17- Table 5. AVERAGES OF VOLUME, CONCENTRATION AND TOTAL NUMBER OF SPERM PER EJACULATE PRODUCED DURING THE PERIOD OF JULY l2 TO AUGUST 2. Trait Fertile Infertilea Ram l Ram 2 Ram 3 Ram 4 Ram 5 Ram 6 Numberb 7 7 7 6 7 7 Volume 0.76 0.4I 0.6I I.I2 0.24 0.33 (cc.) Concentration 0.044 0.047 0.02l 0.0l7 0.00l 0.006 (billion/cc.) Total number 0.029 0.03l 0.0I3 0.0l3 0.00I 0.003 of sperm ejaculated ,(billion) aRams received .4 gm. of protamone daily. bNumber of observations. Table 6. ANALYSIS OF VARIANCE FOR VOLUME (A), CONCENTRATION (B) AND TOTAL NUMBER OF SPERM (C) PER EJACULATE PRODUCED DURING THE PERIOD OF JULY 12 TO AUGUST 2.a Source Mean squares for traits df A B C Between groups I 0 0.0l09** 0.00483** Between rams/groups 4 0.84 0.0005 0.0002l Between ejaculates/ram 35 0.45 0.0006 0.00044 aInfertile rams given .4 gm. of protamone daily. **P<.0I. uyam’J -]8.. given orally are tabulated in table 7. The analysis of variance is tabulated in table 8. The between ejaculates/rams mean square was used to test the between group mean square for the pretreatment period in table 8 since the only reason that the between rams/groups can be smaller is sampling variation. As can be seen in the pretreatment period table 7, the average oxygen consumption of both the known fertile rams was higher than six of the suspected infertile rams. One infertile ram had a higher average than the fertile ones. The range in the oxygen consumption for any one ejaculate in the fertile rams was from 35.90 ul/billion/hour to l9l.8l ul/billion/hour. For the infertile rams it was 29.llpl/billion/hour to l24.00 ul/billion/hour. The analysis of the available data showed that there was no signi- ficant difference between the fertile and infertile rams, though the 'F' value approached significance (P<.05). In the treatment period although the average oxygen consumption of the infertile rams was higher, or almost equal, to that of the fertile rams, still there was no statistically Significant difference between them. This may be because of a bigger range in both the groups. The range for the fertile rams was 40.33 ul/billion/hour to 2l0.00 pl/billion/ hour and for the infertile rams 20.7l ul/billion/hour to 506.00 ul/billion/ hour. On an average the sperm in the semen of all the infertile rams con- sumed more oxygen in the treatment period than they did in the pretreat- ment period. By looking at table 7 we can see that the semen of all the -]9- treated rams consumed relatively more oxygen than the semen from the fertile rams. Semen from Ram No. l consumed less oxygen in this period than during the pretreatment period, whereas the semen from Ram No. 2 consumed a little more oxygen than it did in the control period. By making a relative comparison between the performances of the fertile and the infertile rams it appears that the thyroprotein did have an effect on the respiration rate of the spermatozoa, although it was not statistically significant as demonstrated by table 8. A statistical comparison was also made on the difference in the average respiration rate between the pretreatment and the treatment periods (table 9). Although there seems to be a difference in the aver— age change in the respiration rate of the fertile and the infertile rams, after feeding the thyroprotein to the infertile rams, it was not found to be statistically significantly different (P<.05). This may be be- cause of the few degrees of freedom and also because of a large varia- tion between the individual rams from the pretreatment to the treatment period. From tables I and 3 it was seen that the concentration for the same period (May 24 to July 9) was significantly higher in the untreated rams. In other words the concentration of sperms was higher in the pretreatment period in the infertile rams than it was during the treatment period. On the contrary the oxygen consumption was higher during the treatment period than the pretreatment period. This agrees with the result of Gray (I928), Rothschild (I950), Winchester (l94l), Ross, Miller and Kurzrok (l94l) and Windstosser (I935) although it does not agree with some of the other workers' results, that higher the concentration, lower the oxygen consumption. -20- .mco_um>comno mo LobEJc oz“ 305m momozucocma c_ mot:m_ma .>__mo ocOEmuoLa .Em N. co>_m mEmL o__ucomc_m mm.m~ mm.m~ mm.mm_ _m.nm .m.:o_ Nk.m_ _:.~- ma-¢e _m.~k Axe JM.N__AmV om.m_~ Ase mm.mo_A~v _m.om_A~v ~m.mo_A:v mw.ow Ame mp oo.m:AmV _s.ww AMV ks._m AMV 04.“: Ame oo.m: Ame om.mw ASV m~.mm QAMV an N Ema m Ema m Ema # Ema m Ema N Ema _ Emm o__ucowc_ o__ucom oo_com m.A¢ev ao_¢ma pzmzexo no ._1 mo<¢m><..~ o_nmp -2]- Table 8. ANALYSIS OF VARIANCE FOR OXYGEN CONSUMPTION/BILLION SPERM/HOUR OF THE PRETREATMENT PERIOD (PP) AND THE TREATMENT PERIOD (TP).a Source df Mean squares PP TP PP TP Between groups I l 5l69 9628 Between rams/groups 5 5 783 ll808 Between ejaculates/rams 2l 28 l257 l0480 aInfertile rams given .2 gm. of protamone daily. Table 9. ANALYSIS OF VARIANCE OF THE CHANGE IN THE RESPIRATION RATE OF SPERM (pl. of oxygen/billion sperm/hour) DUE TO .2 GM. OF PROTAMONE FED DAILY TO THE INFERTILE RAMS. Source df Mean squares Between groups I 6243.80 Between rams/groups 5 2902.l9 SUMMARY AND CONCLUSIONS Five suspected infertile rams were selected from the nearby private- ly owned sheep farms, including one from the Michigan State University Sheep Breeding Farm. Two known fertile rams were selected randomly from the University flock. All the rams were kept in indoor pens, in the same barn and hand-fed the same ration. Samples were collected, by electro-ejaculator in an artificial vagina, twice a week at an interval of 3-5 days. Volume, concentration and total sperm ejaculated were evaluated. -22- Starting on May 2], oral treatment of .2 gm of thyroprotein (Prota- mone) was given daily in a gelatin capsule. No statistically significant difference was found to exist between the fertile and infertile groups in volume and total sperm ejaculated. Concentration was found to be statis- tically significantly higher in the fertile ones over the infertile during this period. On July II, the thyroprotein dosage was increased to .4 gm daily. Vol- ume was not found to be statistically different but concentration and total number of sperm produced were highly significantly higher (P<.Ol) in the control rams as compared to the treated rams. Respiration rate of the sperm was also measured on the multiple-unit constant-pressure respirometer devised by Reineke (l96l), using .2 ml of the sample, before and after the oral treatment with .2 gm of protamone. In both the groups the sperm from the ejaculates collected during the treat- ment period consumed more oxygen than the ejaculates of the pretreatment per- iod. The treatment appeared to have a favorable effect on the respiration rate of the sperm, although it was not statistically Significantly different. The most important semen characteristic of the three, studied in this study, is the total number of Sperm ejaculated, because it takes into consideration both the other traits which were studied, namely volume and concentration. Judging from this point of view, out of the 5 _infertile rams, there were only two rams, Ram No. 3 and Ram No. 6 which at the .2 gm level ejaculated more sperm than their pretreatment performance. They also produced a relatively larger volume during this 'period as compared to their pretreatment period. The higher number of Sperm ejaculated could have been because of this larger volume rather than as an effect of the thyroxine therapy. Ram No. 5 also produced a -23- little higher volume but the concentration was lower than during the pretreatment period. The highest number of sperm in the period, in the infertile group, were ejaculated by Ram No. 7, although it was lower than the pretreatment performance. The only thing that can be concluded from the available data is that at least three of the five infertile rams, Ram No. 4, 5 and 7 were not infertile because of any thyroxine deficiency. They could be infertile because of some other factor or a combination of factors. Ram No. 7 could be suspected for being one of those in which one of the factors could be thyroxine deficiency because this was the only ram in which the decline in the semen quality was not as acute as in others during the Summer months. The thyroxine therapy could counter- act only that factor but the semen quality still declined because of the other factor or factors involved. Ram No. 3 and 6 could be suspected of suffering from thyroxine deficiency. They did show some improvement as far as the total number of sperm is concerned on feeding the thyroprotein at the .2 gm level. It would also be safe to conclude that the .4 gm. level did not have any desirable effect on these three semen characteristics. On an average, the samples from the infertile ram consumed more oxygen in the treatment period (not statistically significant) but it is hard to say whether this was because of the lower concentration of sperm present in the samples or because of the effect of thyroxine therapy. -24- BIBLIOGRAPHY Accame, F., Luis Monge and J. C. Miller. I944. Effect of reduced baro- metric pressure on ram semen. J. Animal Sci. 3:43l. Berliner, V. and V. Warbritton. I937. The pituitary and thyroid in relation to sperm production in rams. Proc. Am. Soc. Animal Prod. pp.l37. Bishop, 0. W. I942. Oxygen consumption of fox sperm. Biol. Bull. 83(3):353. Bishop, M. W. H., R. C. Campbell, J. L. Hancock and A. Walton. I954. Semen characteristics and fertility in bulls. J. Agr. Sci. 44:227. Bishop, M. W. H. and G. W. Salisbury. I955. Effect of sperm concentra- tion on the oxygen uptake of bull semen. Am. J. Physiol. l80:l07. Black, W. G., A. L. Schick, A. L. Pope and L. E. Cassida. I950. Some effects of testosterone propionate and thyroprotein in rams. J. Animal Sci. 9:l86. Bogart, R. and D. T. Mayer. I946. Environmental temperature and thyroid gland involvement in lowered fertility in rams. Mo. Agr. Exp. Sta. Res. Bull. 402. Brooks, J. R. and C. V. Ross. I962. Effect of ambient temperature and thyroxine therapy on semen quality of rams. J. Animal Sci. 2l:700. Brooks, J. R., C. V. Ross and G. Pipes. I960. Effects of environmental temperature on thyroid secretion of rams. J. Animal Sci. l9:13l7. Casady, R. B., R. M. Myers and J. E. Legates. I953. The effects of ex- posure to high ambient temperature on Spermatogenesis in dairy bulls. J. Dairy Sci. 36:l4. Dutt, R. H. and L. F. Bush. I955. The effect of low environmental temperature on initiation of the breeding season and fertility in sheep. J. Animal Sci. l4:885. Dutt, R. H. and P. T. Hamm. I955. Effect of exposure to high environ- mental temperature and shearing on semen production of rams in winter. J. Animal Sci. l4:l245. Dutt, R. H. and E. C. Simpson. I957. Environmental temperature and fertility of Southdown rams early in the breeding season. J. Animal Sci. l6:l36. Eaton, O. N., R. G. Schott, V. L. Simmons and A. H. Frank. I948. The effects of feeding thyroprotein on semen characteristics of rams. J. Animal Sci. 7:449. -25- El-sheikh, A. S. and L. E. Casida. I955. Motility and fertility of spermatozoa as affected by increased ambient temperature. J. Animal Sci. l4:ll46. Ely, R. E., H. A. Herman and C. F. Winchester. I942. Studies of respir- ation rate of dairy bull spermatozoa. Mo. Agr. Exp. Sta. Res. Bull. 353. Foote, W. C., A. L. Pope, R. E. Nichols and L. E. Casida. l957. The effect of variations in ambient temperature and humidity on rectal and testes temperatures of sheared and unsheared rams. J. Animal Sci. l6:l44. Gray, J. I928. The effect of dilution on the activity of spermatozoa. J. Exp. Biol. 5:337. Green, W. W. I940. Seasonal trends of sperm cell types in sheep. Proc. Am. Soc. An. Prod., pp.207. Henle, G. and C. A. little. I942. Studies of the metabolism of bovine epididymal spermatozoa. Am. J. Physiol. I36:70. Henneman, H. A., E. P. Reineke and S. A. Griffin. I955. The thyroid secretion rate of sheep as effected by season, age, breed pregnancy and lactation. J. Animal Sci. l4:4l9. Hulet, C. V. and S. K. Ercanbrack. I962. Fertility index for rams. J. Animal Sci. 2l:489. Huston, T. N. and R. S. Wheeler. I949. Effect of synthetic thyroprotein on seasonal variation in volume and concentration of cock semen. Poultry Sci. 28:262. McKenzie, F. F. and V. Berliner. I937. The reproductive capacity of rams. Mo. Agr. Exp. Sta. Res. Bull. 265. Mohri, H. and K. Horiuchi. l96l. Studies on respiration of sea urchin spermatozoa. III. Respiratory quotient. J. Exp. Biol. 38:249. Phillips, R. W. and F. F. McKenzie. I934. The thermo-regulatory func- tion and mechanism of scrotum. Mo. Agr. Exp. Sta. Res. Bull. 217. Phillips, R. W., R. G. Schott, O. N. Eaton and V. L. Simmons. l943. Seasonal variation in the semen of sheep and goats. Cornell Vet. 33:227. Reineke, E. P. I946. The effect of synthetic thyroprotein on sterility in bulls. The problem of fertility. Proc. Conf. on Fertility, pp.233, Princeton Univ. Press, Princeton. -26- Reineke, E. P. I96I. A new multiple-unit constant-pressure micro- respirometer. J. Appl. Physiol. l6:944. Ross, V., E. G. Miller, Jr. and R. Kurzrok. I94l. Metabolism of human sperm. Endocrin. 28:885. Shaffner, C. S. I948. The influence of thyroprotein feeding on semen quality. Poultry Sci. 27:527. Shergin, N. P. I937. Respiration of sperm of farm animals. An. Breed. Abst. 7:I8l. Simpson, E. C., H. C. Rice, 0. G. Steele and R. H. Dutt. I959. Summer semen characteristics of rams having free access to a plastic air- conditioned room. J. Animal Sci. l8:ll57. Smelser, G. K. I934. Effects of thyroidectomy on testicular function. Anat. Rec. 60:53. Walton, A. and J. Edwards. I938. Criteria of fertility in bull-l. The exhaustion test. Proc. Am. Soc. Animal Prod. 3I:254. Warwick, E. J., C. E. Childs, A. E. Flower and W. E. Ham. I948. Ram semen production and characteristics as influenced by administra- tion of thyroprotein. J. Animal Sci. 7:I98. Whiteman, J. V. and K. I. Brown. I959. Effects of delayed shearing of ewes and daytime cooling of rams on the spring breeding performance. J. Animal Sci. l8:392. Wilson, R. L., W. C. Godley and V. Hurst. I959. Some factors affecting breeding performance and early Iambing in sheep. J. Animal Sci. l8:ll57. Wilwerth, A. M., C. Martinez-Campos and E. P. Reineke. I954. Influ- ence of thyroid status on volume and concentration of cock semen. Poultry Sci. 33:729. Winchester, C. F. and F. F. McKenzie. l94l. Influence of cell concen- tration on respiration rate. Proc. Soc. Exp. Biol. Med. 48:648. Windstosser, K. I935. Respiration of mammalian spermatozoa. Animal Breed. Abst. 3:339. APPENDIX TABLE A KNOWN FERTILE Ram #I - MSU 0029 - Columbia (2 years) Sheared April 26 and September 7 Date Volume Concentration Total No. Motility Atmospheric (c.c.) per c. c. of sperm (percent) temperature (billion) in degrees ejaculate Fahrenheit (billion)_ (average) January 8 0.7 l.50 l.05 20.0 ” l7 I.0 l.80 l.80 15.0 February l7 0.9 l.50 l.35 25.5 March 25 0.7 l.60 I.I2 38.5 April 9 0.8 1.40 1.12 45.0 ” l3 2.5 I.I9 2.975 40.5 ” I8 l.2 l.20 l.44 45.5 ” 23 No sample (urine) ” 27 2.I I.0I 2.I2I 7I.5 ” 30 I.2 l.5 l.8 65.5 May 2 1.7 0.56 0.952 51.0 “ 5 I.6 l.20 I.92 62.5 ” 8 1.5 l.27 I.905 47.5 ” I2 l.0 0.98 0.98 54.5 ” l7 0.3 l.24 0.372 75.0 ” 24 2.0 l.86 3.72 68.0 “ 27 3.I l.0I 3.I3I 53.5 ” 3I I.0 I.IZ l.l2 74.0 June 4 0.8 l.08 0.864 67.5 “ 8 l.2 l.80 2.I6 65.0 ” II l.3 l.09 I.4I7 72.0 ” I4 l.5 0.32 0.48 66.0 ” I8 I.2 0.97 I.I64 76.0 ” 2I I.2 l.87 2.244 65.5 ” 25 3.0 2.I8 6.54 50 70.5 ” 28 2.7 l.42 3.834 70 70.0 July 2 l.5 0.69 l.035 ‘ 55 67.5 ” 5 0.8 0.52 0.4l6 50 7I.5 ” 9 No sperms ” l2 l.8 0.04 0.072 30 74.0 ” I6 0.8 0.08 0.064 25 66.0 ” I9 0.4 0.08 0.032 30 69.5 ” 23 0.3 0.04 0.0I2 I0 66.0 ” 26 0.4 0.02 0.008 I0 62.0 ” 30 0.3 0.05 0.0I5 IO 72.5 August 2 l.3 No sperms 60,5 ” 7 0.8 No sperms 73,0 ” I5 0.6 0.3I 0.I86 I5 65.5 ” 23 0.3 0.2l 0.063 20 7I.0 “ 29 0.4 0.2l 0.084 40 74.0 September 5 0.6 0.20 0.I20 40 63.5 ” I2 0.3 0.02 0.006 40 7I.O ” I9 l.5 I.73 2.59 50 54.5 ” 26 2.l l.37 2.877 55 53.0 October 4 3.I 2.28 7.068 50 62.5 ” I0 2.3 3.I2 7.I76 65 55.5 ” I7 3.0 2.72 8.]60 60 5l.5 -97- APPENDIX TABLE B KNOWN FERTILE Ram #2 - MSU 444 - Shropshire (2 years) Sheared April 26 and September 7 Date Volume Concentration Total No. Motility Atmospheric (c.c.) per c.c. of Sperm (percent) temperature (billion) in degrees ejaculate Fahrenheit (billion) (average) April 9 0.7 1.19 0.833 45.0 ” I3 I.0 2.03 2.03 40.5 “ I8 0.8 0.98 0.784 45.5 ” 23 I.0 2.83 2.83 50.5 ” 27 0.4 l.02 0.408 7I.5 ” 30 0.5 I.II 0.555 65.5 May 2 0.4 3.38 1.352 51.0 “ 5 0.6 2.02 l.2l2 62.5 ” 8 0.8 I.90 l.52 47.5 ” I2 0.6 I.8I l.086 54.5 ” I7 0.6 0.57 0.342 75.0 ” 24 I.I 2.98 3.278 68.0 ” 27 I.0 l.82 l.82 53.5 .. 31 0.4 2.40 0.96 7‘1-5 June 4 0.5 2.0I l.005 67.5 ” 8 0.3 I.98 0.594 65.0 ” II I.I I.II l.22l 72.0 ” l4 I.I l.57 I.727 66.0 “ l8 l.4 l.2l l.694, 76.0 ” 2I 0.7 l.05 0.735 65.5 ” 25 I.0 I.98 I.98 60 70.5 ” 28 0.8 0.98 0.784 70 70.0 July 2 No sample 67.5 ” 5 0. 0.44 0.044 50 7I.5 ” 9 0.5 0.08 0.04 35 69.5 ” I2 0.I 0.0I 0.00I 20 74.0 ” l6 No sample 66.0 ” I9 0.3 0.04 0.0I2 20 69.5 ” 23 0.2 No sperms ” 26 0.9 0.I0 0.090 25 62.0 ” 30 0.4 0.II 0.044 30 72.5 August 2 1.0 0.07 0.070 20 60.5 ” 7 0.6 0.09 0.054 45 73.0 ” I5 0.3 0.06 0.0I8 40 65.5 ” 23 0.2 0.I2 0.024 45 7l.0 ” 29 0.2 0.I8 0.036 40 74.0 September 5 0.4 0.07 0.028 50 63.5 ” I2 0.3 0.2l 0.070 60 7I.O Sold by MSU Sheep Farm -28- APPENDIX TABLE C SUSPECTED INFERTILE Ram #3 - Taylor 362 - Columbia (3gyears) Sheared April 26 Date Volume Concentration Total No. Motility Atmospheric (c.c.) per c. c. of sperm (percent) temperature (billion) in degrees ejaculate Fahrenheit ,(billion) (average) January 8 0.7 l.30 0.9I 20.0 ” I7 0.4 l.20 0.48 I5.0 February I7 0.6 l.00 0.60 25.5 March 25 0.4 l.40 0.56 38.5 April 9 1.1 1.61 1.771 45.0 ” I3 0.5 0.90 0.45 40.5 ” l8 0.4 I.72 0.688 45.5 ” 23 0.6 I.62 0.972 50.5 ” 27 0.4 l.50 0.60 7I.5 ” 30 0.8 l.02 0.8l6 65.5 May 2 No sample (urine) 5l.0 ” 5 0.4 0.98 0.392 62.5 ” 8 No sample (urine) 47.5 ” I2 0.3 l.0I 0.303 54.5 ” I7 0.4 0.9I 0.364 75.0 May 2l Protamone started .2 gm “ 24 2.7 l.58 4.266 68.0 ” 27 I.I I.IO l.2I 53.5 ” 3I 0.6 0.72 0.432 74.0 June 4 0.3 l.02 0.306 67.5 “ 8 0.8 l.53 l.224 65.0 “ ll l.2 0.96 I.I52 72.0 ” I4 l.3 0.80 l.04 66.0 ” I8 0.9 0.62 0.558 76.0 ” 2l 0.5 l.75 0.875 65.5 ” 25 0.5 l.23 0.6I5 40 70.5 ” 28 0.5 0.32 0.I6 55 70.0 July 2 l.0 0.86 0.86 50 67.5 “ 5 0.9 0.49 0.44I 45 7I.5 ” 9 0.3 0.0I 0.003 25 69.5 July ll Protamone .4 gm ” I2 I.6 0.02 0.032 25 74.0 ” l6 0.7 0.02 0.0I4 I5 66.0 .. 19 0.7 0.02 0.014 20 69.5 ” 23 0.3 0.0I 0.003 I0 66.0 ” 26 0.2 0.02 0.004 5 62.0 ” 30 0.5 0.02 0.0I I0 72.5 August 2 0.3 0.04 0.0I2 l0 60.5 ” 7 0.6 No sperms 73.0 ” I5 0.4 0.07 0.028 20 65.5 ” 23 No sample 7l.0 Taken away by the owner. -29- APPENDIX TABLE 0 SUSPECTED INFERTILE Ram #4 - Frank 443 - Corriedale (5 years)eSheared.Ap[il 26 and September l7 Date Volume Concentration Total No. Motility Atmospheric (c.c.) per c. c. of sperm (percent) temperature (billion) in degrees ejaculate Fahrenheit (billion) (average) January 8 0.3 l.00 0.300 20.0 " I7 0.4 l.30 0.520 I5.0 February I7 0.8 I.IO 0.880 25.5 March 25 0.5 l.30 0.650 38.5 April 9 0.8 l.0l 0.808 45.0 “ l3 2.0 l.l3 2.26 40.5 “ l8 0.6 No sperms 45.5 ” 23 0.3 I.70 0.5I0 50.5 “ 27 0.2 l.34 0.268 7I.5 ” 30 0.6 l.06 0.636 65.5 May 2 0.6 2.57 l.542 ' 5I.0 ” 5 0.4 l.20 0.480 62.5 ” 8 0.6 0.66 0.396 47.5 “ I2 No sample (urine) 54.5 ” I7 0.5 0.86 0.430 75.0 May 2l Protamone started .2 gm ” 24 l.8 I.62 2.9I6 68.0 “ 27 0.6 0.9l 0.546 53.5 ” 3l 0.7 0.93 0.65I 74.5 June 4 0.2 O.7I 0.I42 67.5 “ 8 0.3 0.83 0.249 65.0 “ II 0.5 I.0l 0.505 72.0 “ I4 0.9 l.54 l.386 66.0 ” I8 0.5 0.43 0.2I5 76.0 ” 2l No sample (urine) 65.5 “ 25 0.I l.l3 0.Il3 50 70.5 ” 28 0.4 0.4l 0.I64 40 70.0 July 2 No sample (urine) 67.5 ” 5 0.I 0.23 0.023 40 7I.5 ” 9 0.2 0.02 0.004 25 69.5 July II Protamone .4 gm ” I2 I.I No sperms 74.0 ” I6 0.6 0.04 0.024 I5 66.0 ” I9 4.0 0.0l 0.040 I0 69.5 ” 23 No sample (urine) 66.0 ” 26 0.3 0.03 0.009 5 62.0 ” 30 0.2 0.02 0.004 I5 72.5 August 2 0.5 No sperms 60.5 ” 7 0.I No sperms 73.0 " l5 No sample 65.5 “ 23 0.4 No sperms 7l.0 ” 29 0.3 0.Il 0.033 I5 74.0 September 5 1.0 0.11 0.11 10 63.5 ” l2 0.I No sperms 7l.0 “ I9 0.3 0.04 0.0l2 20 54.5 Taken away by the owner in the afternoon of September l9 an- APPENDIX TABLE F SUSPECTED INFERTILE Ram #6 - Bortel - Dorset (Westhoven) - (5 years) Sheared September 7 Date Volume Concentration Total No. Motility Atmospheric (c.c.) per c. c. of Sperm (percent) temperature (billion) in degrees ejaculate Fahrenheit ,_(billion) ,_(average) April 9 0.6 0.98 0.588 45.0 ” I3 No sample (urine) 40.5 ” l8 No sample (urine) 45.5 ” 23 0.4 0.72 0.288 50.5 ” 27 0.5 0.64 0.320 7I.5 ” .30 0.6 0.56 0.336 65.5 May 2 No sample (urine) 5I.0 ” 5 No sample (urine) 62.5 ” 8 0.7 0.7l 0.497 47.5 ” I2 0.4 0.69 0.276 54.5 “ I7 0.8 0.5I 0.408 75.0 May 2l Protamone started .2 gm ” 24 2.0 l.3l 2.620 68.0 ” 27 l.2 I.IO l.320 53.5 ” 3I 0.7 0.82 0.574 74.0 June 4 0.3 0.67 0.20I 67.5 “ 8 0.4 0.72 0.288 65.0 ” ll l.2 0.22 0.264 72.0 “ I4 0.6 0.59 0.354 66.0 “ I8 0.6 0.70 0.420 76.0 ” ZI I.7 0.26 0.442 65.5 ” 25 I.0 0.3I 0.3I0 IO 70.5 “ 28 0.6 0.35 0.2l0 IS 70.0 July 2 l.5 0.44 0.66 . I0 67.5 ” 5 0.3 0.06 0.0I8 5 7I.5 “ 9 0.4 No sperms 69.5 July ll Protamone .4 gm ” I2 0.5 No Sperms 74.0 ” I6 0.5 0.04 0.020 5 66.0 " l9 0.3 No sperms 69.5 “ 23 No sample 66.0 ” 26 0.4 No sperms 62.0 ” 30 No sample 72.5 August 2 0.6 No sperms 60.5 H 7 No sample 73.0 ” l5 No sample 65.5 “ 23 No sample 71.0 ” 29 No sample 74.0 September 5 No sample 63.5 ” l2 0.5 No sperms 7I.0 ” I9 No sample 54.5 September 22 Last dose of protamone “ 26 0.3 0.28 0.084 l5 53.0 October 4 I.7 0.39 0.663 l5 62.5 ” I0 l.4 0.52 0.728 I0 55.5 ” l7 l.0 0.47 0.470 I0 5I.5 -32- APPENDIX TABLE G SUSPECTED INFERTILE Ram #7 - MSU 237874 - Shropshire (3 years) Sheared April 26 Date Volume Concentration Total No. Motility Atmospheric (c.c.) per c.c. of sperm (percent) temperature (billion) in degrees ejaculate Fahrenheit _(billion)_ (average) March 25 0.6 I.90 I.I40 38.5 April 9 1.4 1.34 1.876 45.0 ” I3 0.9 l.50 l.350 40.5 ” I8 l.0 l.42 l.420 45.5 ” 23 l.5 I.75 2.625 50.5 “ 27 l.2 l.ll l.332 7I.5 .. 30 2.1 2.34 4.914 65.5 May 2 No sample (urine) 5I.0 ” 5 I.I I.72 l.892 62.5 ” 8 I.0 0.42 0.420 47.5 ” I2 0.8 0.8I 0.648 54.5 ” I7 0.7 0.58 0.406 75.0 May 2] Protamone started .2 gm “ 24 2.6 2.88 7.488 68.0 ” 27 l.2 I.IO l.320 53.5 ” 3l l.0 0.92 0.920 74.0 June 4 0.5 0.82 0.4I0 67.5 ” 8 0.6 I.IZ 0.672 65.0 ” II 0.4 l.02 0.408 72.0 ” I4 I.O 0.83 0.830 66.0 ” l8 I.I l.45 I.595 76.0 ” 2I l.4 0.56 0.784 65.5 ” 25 l.4 2.04 2.856 55 70.5 ” 28 0.5 0.7l 0.355 40 70.0 July 2 0.7 0.53 0.37I 45 67.5 “ 5 0.9 0.38 0.342 50 7I.5 “ 9 0.7 No sperms 69.5 July ll Protamone .4 gm ” I2 0.4 No sperms 74.0 ” I6 0.5 0.02 0.0I IO 66.0 ” I9 No sample 69.5 ll 23 ” 26 Taken away and put on pasture - treatment discontinued II 30 August 2 H 7 0.9 No sperms ' 73-0 Taken away again put on pasture. Tried to collect a sample on a non-cyclic ewe but refusyed to mount. Later on tried to collect a sample by an electro- ejaculator but did not give any sample. -33- .003}. U FE Oil-ELY 4351'.) .J' :2 1 IIIIIIII Ill 3 II III 451184 illlllllllllli