THE FERTILIZING CAPACITY OF FROZEN RAM SEMEN USING VARIOUS DILUENTS AND ADJUVANTS BY Neal Lloyd First AN ABSTRACT Submitted to the School for Advanced Graduate Studies of Michigan State University of Agriculture and Applied Science in partial fulfillment of the requirements for the degree of Doctor of Philosophy Department of Animal Husbandry Approved X/ 1959 s _____ ProQuest Number: 10008623 All rights reserved INFORMATION TO ALL USERS The quality o f this reproduction is dependent upon the quality of the copy submitted. In the unlikely event that the author did not send a complete manuscript and there are missing pages, these will be noted. Also, if material had to be removed, a note will indicate the deletion. uest. ProQuest 10008623 Published by ProQuest LLC (2016). Copyright of the Dissertation is held by the Author. All rights reserved. This work is protected against unauthorized copying under Title 17, United States Code Microform Edition © ProQuest LLC. ProQuest LLC. 789 East Eisenhower Parkway P.O. Box 1346 Ann Arbor, Ml 4 8 1 0 6 - 1346 ABSTRACT THE FERTILIZING CAPACITY OF FROZEN RAM SEMEN USING VARIOUS DILUENTS AND ADJUVANTS Neal L. First Motility determinations on six thawed frozen milk diluted ejacu lates of ram semen indicated that, motility means for glycerol levels of 6, 6.5, 7* 7»5> 8 8.5 percent were significantly greater than the motility means for 5 or 5*5 percent glycerol. No significant difference In spem survival after freezing in a milk diluter occurred among added egg yolk levels of 0, 3* 6, 12 and 2^ percent, although the average ram sperm survival after freezing tended to decrease as yolk levels increased. The number of sperm which survived freezing in glycerol levels of six and nine percent was highly significant over the 12 or 15 percent level. A highly significant interaction occurred between glycerol levels and egg yolk levels. The motility of both frozen and unfrozen semen incubated for eight hours at 5° C. was significantly greater than at 39° C. No significant difference existed between the motility of thawed frozen semen or unfrozen semen after eight or 120 hour incubation at 39° C. or 5° 8. Further, no significant difference occurred between thawed frozen semen equilibrated for one or 18 hours before freezing. Thawed frozen and unfrozen samples averaged 57 percent motility after 2*f0 hours of 5° C. incubation. None of the constituents in a milk-arabinose-glycerol diluter or a yolk-citrate-arabinose-glycerol diluter significantly depressed the - 2 - motility of ram sperm after incubation for eight hours at 39° C. Milk and yolk citrate diluted semen samples had significantly greater motility after eight hours of 39° incubation than un diluted ram semen. The frozen milk diluted ejaculates of two rams were periodically examined for motility throughout l^f- months of -79° C. storage. The greatest motility decline occurred during the first two months of storage with very little decline thereafter. In 1956, two of 12 ewes lambed when cervically inseminated with 500 million sperm in one ml. of thawed frozen milk diluted ram semen. Whereas, seven of 11 lambed when cervically inseminated with 600 million sperm in .2 ml. of undiluted semen. In 1957.» three of 10 ewes lambed when cervically inseminated with 213 million sperm in one ml. of thawed frozen milk plus egg yolk diluted ram semen and six of 10 ewes lambed when cervically inseminated with 600 million sperm in .2 ml. of undiluted ram semen. In 1958> none of b^ ewes lambed when cervically inseminated with 100 million sperm in .9 or one ml. of thawed frozen ram semen even though the average motility one week after freezing was 70 percent. Milk, yolk-citrate and a Bussian-Ukranian diluter were used to dilute the semen before freezing. An analysis of conception rates from b6 ewes cervically insemin ated with unfrozen semen from eight rams indicated no significant difference among glycerolated; milk, yolk-citrate or XJkranian diluters. - 3 - Insemination of milk diluted ram semen resulted in a k6 *7 percent lambing rate; whereas, the insemination of yolk-citrate or Ukranian diluted ram semen resulted in 21 and 18 percent lambing rates re spectively. All was stored less than two days at 5° C* No significant conception rate difference occurred between in semination of 100 million ram sperm in .5 or one ml. of undiluted semen. Thirty-two ewe lambs were each cervically inseminated with 150 million sperm in one ml. of thawed frozen semen from one of two rams. No significant difference in conception rate occurred between ewes inseminated once during estrus or each 12 hours during estrus. In each case 12.5 percent of the ewes inseminated conceived. There was no significant conception rate difference between ewes mated to a vasectomized ram before Insemination and ewes which were inseminated without prior vasectomized mating. percent of the ewes inseminated conceived. In each case, 12.5 TEE FERTILIZING CAPACITY OF FROZEN RAM SEMEN USING VARIOUS DILUENTS AND ADJUVANTS BY Neal Lloyd First A THESIS Submitted to the School for Advanced Graduate Studies of Michigan State University of Agriculture and Applied Science in partial fulfillment of the requirements for the degree of Doctor of Philosophy Department of Animal Husbandry 1959 Neal Lloyd First candidate for the degree of Doctor of Philosophy Final examination: Dissertation: August 10, 1959 > 1:30 p.m., 103 Anthony Hall The Fertilizing Capacity of Frozen Ram Semen Using Various Diluents and Adjuvants Outline of Studies: Major subject: Minor subject: Animal Husbandry Physiology Anatomy Biographical Items: Born - October 8, 1930^ Ionia, Michigan Undergraduate Studies, Michigan State College, 19^8-1952 Graduate Studies, M. S. - Michigan State University, 195^-1957 Ph.D. - Michigan State University, 1957-1959 Experience: ' Member United States Army Signal Corps, 1952-195^ Member of American Society of Animal Production American Dairy Science Association Farmhouse Fraternity Alpha Zeta Sigma Xi ACKNOWLEDGEMENTS Th© author greatly appreciates the guidance and encouragement of Dr. H. A. Henneman as a major professor and sincere friend. Without whose inspiration this thesis would not have been possible. Th© assistance of Dr. Afif Seving© has been sincerely appreciated and a grateful thanks is extended to Dr. W. T. Mage© for his helpful advice and assistance with the statistical analysis. A note of thanks is due Dr. M. Lois Calhoun, Dr. J. A. Williams, Dr. J. E. Nellor, Dr. H. A. Hafs, Dr. D. E. Ullrey, Dr. W. T. Magee and Dr. R. H. Nelson who critically reviewed this manuscript and made many helpful suggestions. Sincere graditude is expressed to Dr. R. H. Nelson, Dr. D. E. Ullrey, th© Michigan State University Animal Husbandry Department and th© Michigan Artificial Breeders Cooperative for the facilities which made this research possible. Th© author wishes to thank th© shepard, Lee Bell and also Larry Cotton and Gary Seevers for their assistance in handling the sheep. The excellent typing and preparation of this manuscript by Amanda Kay Butcher is gratefully appreciated. To his wife, Edith, the author extends his deepest appreciation for her enduring patience and continued encouragement throughout this study. TABLE OF CONTENTS Page I. INTRODUCTION............................. ...... 1 II. OBJECTIVES ..................................... 2 III. REVIEW OF LITERATURE ........................ A. Storage of Frozen Ram Spermatozoa.... 3 3 B . Glycerol Requirement for Freezing Ram Semen •••. • 9 C . Ram Semen Extenders ......... 11 D. pH Electrolytes and Osmotic Pressure .......... 21 E. Spermatozoa Death After Freezing and Thawing .... 23 F . Equilibration Time and Temperature ..... G. The Storage Potential of Frozen S e m e n . 27 31 E, Sperm Numbers Necessary for Satisfactory Concep tion ..................... 33 I. Volume of Semen Inseminated ........... 36 J. Cervical versus Vaginal Insemination.... .... 37 K, When to Inseminate the Ewe in Reference to the Beginning of Estrus and Sperm Life in the Ewe ... 39 L. Insemination or Mating Frequency During one Estrus ..................... 43 M. Vasectomized Mating 46 IV. MATERIALS AND METHODS V. RESULTS AND DISCUSSION ..... ......................... 48 .................... 5h A. The Glycerol Requirement for Freezing Ram Semen . 54 B. The Addition of Egg Yolk to a Milk Diluter and its Effect on Glycerol Requirement ............ 58 C. Frozen and Unfrozen Semen Equilibrated with Glycerol for One and 18 Hours and Incubated at 39° and 5° C. 64 D. 71 Extender Toxicity at 39° C ..................... TABLE OF CONTENTS (continued) Page E, Storage Potentialof Frozen Earn S e m e n ..... F. Breeding Trials ...... ..... ••...... VI. SUMMARY AND CONCLUSIONS...... VII. BIBLIOGRAPHY............... VIII. APPENDIX........................................ 77 80 104 109 126 LIST OF TABLES Page 1. Ejaculates Used in Glycerol Level Experiment ..... 54 2. A test of Interaction Between Glycerol Level and Ejacu lates ..... 55 3. Variance Due to Glycerol Levels ...................... 56 4. Percent Ram Sperm Motility After Freezing in Various Levels of Glycerol ................ 56 5* A test of Interaction Between Rams and Glycerol Levels . 57 6. Ejaculates Used in Yolk Glycerol Experiment ....... 60 7- Average Percent Survival of Sperm Frozen in a Milk Dil uter with Combinations of Egg Yolk and Glycerol....... 60 8. Analysis of Variance on Combinations of Yolk andGlycerol 6l 9. Variance in the Motility of Frozen and Unfrozen Ram Semen Incubated Eight Hours at 39° and- 5° C. and Equili brated with Glycerol for On© and l8 Hours............. 68 10. Variance in the Motility of Frozen and Unfrozen Ram Semen Incubated 120 Hours at 5° C. and Equilibrated with Glycerol for One and 18 Hours.................... 68 11. Ejaculates Used in Studying Extender Toxicity at 39° C. 71 12. Variance in the Extender Toxicity Study 73 ....... 13. Percent Eight Hour Survival of Ram Sperm Diluted in Con stituents of Two Diluters and Incubation at 5° aud39° C. 7^*14. Student Range Test of Treatment Means ............... 75 15. Ejaculates Used to Determine the Storage Potential of Frozen Ram S e m e n ......... 77 1 6 . 1956 Breeding T r i a l 80 ..... 17. 1957 Breeding Trial ................................. 8l 18. Frozen Ram Semen Used in Experiment A .......... 84 19* Unfrozen Ram Semen Used in Experiment A .............. 84 LIST OF TABLES (continued) Page 20. Ewes Conceiving from Insemination with Unfrozen Semen .. 87 21. Analysis of Variance Among Ewes Conceiving from Unfrozen S e m e n ........ 88 22. Conception After Insemination of Ram Semen of Various Ages and Diluted in Three Different Diluters ......... 89 23. The Ram Semen Used in Experiment B ........... 91 24. Ewes Conceiving from Frozen Semen Inseminated In Experi ment B ... ............ 92 25. Summary of Breeding Trials ........«•«..«............ 96 LIST OF FIGUEES Figure 1 Motility Decline of Unfrozen and Frozen Ram Semen Equilibrated for One or 18 Hours at 5° C. 2 Motility Decline of Unfrozen and Frozen Earn Semen Equilibrated for One or 18 Hours at 39 ° C. 3 Earn Sperm Motility Decline at -79° C* Storage LIST OF APPENDICES Appendix A B C D Page Percent Motile Ram Sperm after Freezing in Various Levels of Glycerol 127 Percent Survival of Ram Sperm Frozen In Milk Diluter with Combinations of Egg Yolk and Glycerol 128 Percent Motility of Frozen and Unfrozen Ram Semen Incubated Eight Hours at 39 ° and 5° C. and Equilibrated with Glycerol for One or 18 Hours 129 Percent Eight Hour Survival of Ram Sperm Diluted in Constituents of Two Diluters and Incubation at 5° and 39° C. 130 Percent Motility of Ram Sperm Frozen for Various Lengths of Time 131 - 1 - I. INTRODUCTION Within a comparatively short period of time artificial insemina tion and the use of frozen semen has brought about a revolution in dairy cattle breeding practices. Considerable work has been carried on vith bovine semen to improve methods, materials and techniques until the use of frozen semen and artificial insemination has become an efficient operation in widescale use. Research with ram semen has been limited probably due to the lack of economic reasons for the practical use of artificial insemination. However, with the possibilities of synchronized estrus, the advent of progeny testing to locate valuable sires and the increased use of production testing it is possible that sheepmen will be taking a new look at the use of artificial insemination. To further facilitate this possibility the perfection of a tech nique for freezing and storing ram semen would be of utmost importance. It would make possible the collection of semen at the time of year when it is of highest quality, extend the influence of outstanding sires, and improve conception rates for those breeders desiring early lambs. A limited amount of research has been conducted with frozen ram semen. Enough was done to learn that the techniques used successfully for bull semen were not directly applicable to ram semen. Therefore, this study was undertaken in an attempt to find techniques which would result in frozen ram semen with high fertilizing capacity at the time of insemination. II. OBJECTIVES To develop improved techniques and materials for the low temper ature storage of ram spermatozoa which will result in highly motile semen samples after freezing. To fertility test the insemination of frozen ram semen and to develop insemination procedures which will improve conception from the insemination of unfrozen and frozen ram semen. - 3 - III. REVIEW OF LITERATURE A. Storage of Frozen Ram Spermatozoa Ram sperm were first frozen "by Emmens and Blackshaw (1950) wherein semen samples were diluted one to one and frozen "by the slow rate of Smith and Polge (1950). A sodium citrate phosphate buffer of pH 7*1 was the base around which diluters were formed. Diluters containing only various levels of glycerol were not successful, therefore, combin ations of glycerol and sugars were tried. It was concluded that 7*5 to 10 percent glycerol and 1 .2 5 percent arabinose, rhamnose, or xylose in the citrate phosphate buffer resulted in the greatest ram sperm survival after freezing. Some of the other substances tried as diluter constituents were: N propyl alcohol, iso propyl alcohol, ethylene glycol, monomethyl ether, trimethylene glycol, propylene glycol, hexylene glycol, erythritol, adonitol, sorbitol, dulcitol, glyceraldehyde, glucose, levulose, mannose, galactose, saccharose, trehalose, raffinose, urea and inositol. Smirnov (19 51 ) reported that eight of 19 ewes inseminated with ram semen which was stored at -78 ° C., conceived and produced 12 lambs. Blackshaw and Emmens (1953) observed that ram semen initially dil uted with 50 percent egg yolk phosphate citrate gave consistenly higher survival after freezing than without egg yolk. glycerol was found to be necessary. Equilibration with Frozen ram semen of high motility after freezing resulted in a lambing rate of only 10 percent. Glycerol and arabinose could be added to ram semen at temperatures up to 15° C. without reducing sperm survival after freezing. White et al. (195*0 observed that hull and ram spermatozoa thawed quickly after slow freezing to -79° C. in a phosphate fructose diluent, showed little respiratory or glycolytic activity and were immotile. A large number of bull and ram sperm survived freezing in a diluent containing 7*5 percent glycerol and 1 .2 5 percent arabinose. These cells, however, had little metabolic activity and became immotile, particularly at 37° C. Emmens and Blackshaw (1955) inseminated ewes with unfrozen and frozen semen that was undiluted as well as diluted one to four. volume inseminated varied between and .8 ml. The A 5^ percent con ception rate was obtained from unfrozen undiluted semen; 31 percent from semen diluted with sodium citrate; 25 percent from semen diluted in 50 percent egg yolk, 7*5 percent glycerol, 1 .2 5 percent arabinose and citrate; three percent from semen frozen in citrate, glycerol and arabinose diluter. Four percent of the ewes inseminated with non equilibrated semen frozen in an egg yolk citrate glycerol arabinose diluter conceived and five percent conceived after insemin ation with semen equilibrated 18 hours in the same diluter. Blackshaw (1955b) studied the effect of several sugars on the motility revival of bull and ram semen after freezing to -79 ° C. The sugars were arabinose, dulcitol, galactose, fructose, mannose and sucrose. These sugars were added to the diluter conventionally used in other studies from that laboratory. The addition of arabinose or fructose resulted in the greatest survival of ram sperm. - 5 - Emmens and Blackshaw (1955) found that ram semen may he mixed with the glycerol containing diluent at temperatures up to 15 ° C. without harmful effects on revival after freezing. Egg yolk or lecithin addition to the Australian low temperature diluent re sulted in greater sperm survival after freezing than with a lipopro tein or a milk diluter. A greater number of ram sperm revived from the frozen state when thawed at ^0° C. than at 9° C . Graca (1955) cervically inseminated 1 J6 ewes with .2 ml. of yolk citrate diluted thawed frozen ram semen. whs diluted 1:5 or 1:10 before insemination. The thawed semen The conception rate, based on non-returns, was 31 *2 percent and was 16 percent higher in the 1 :5 dilution group, Szumowski et al. (1956) reported that a reconstituted powdered skimmilk diluter was superior to liquid milk or the common egg yolk diluters (Cambridge, Ukranian or Australian) for freezing ram semen. Motility of thawed sperm was 60 percent with over 70 percent survival after freezing. There was no difference in survival between samples of ram semen frozen in six to 16 percent glycerol. However, the length of glycerol equilibration did affect ram sperm survival. At least four hours of equilibration were necessary and the optimum appeared to be between six and 14 hours. Ho great loss of motility occurred after storage at -79° C. for four weeks. Thirty-nine ejaculates from one ram were studied. First, et al. (1957) used 37 split ejaculates of ram spermatozoa to determine the effect of the following factors on ram sperm survival - 6 - after freezing and storage at -79° C.: (l) levels of glycerol; (2) the addition of L(-) arahinose; (3) a milk extender and (!*) a yolk phosphate citrate extender containing various levels of egg yolk. The survival of frozen ram sperm in a milk diluter contain ing 0, 2, h, 6, 8, 10, 12 or 15 percent glycerol was curvilinear with the greatest survival at six and eight percent. The addition of I .2 5 percent arahinose significantly improved sperm survival in hoth milk and egg yolk-phosphate-citrate extenders. There was no significant difference between egg yolk levels of Mt-, 3^ or 27 per cent, however, the survival from all three was significantly higher than that of an extender containing 23 percent egg yolk. Markovic (1956) observed that the after-thawing spermatozoan survival rate of ram semen diluted in yolk-arabinose before freez ing was 60 percent provided that six to l4 hours were allowed for equilibration. Glucose and arabinose increased spermatozoan sur vival. Galkin (195^-) inseminated 500 ewes with the semen of eight rams which was diluted in yolk-glucose-citrate plus glycerine. The highest lambing rate (86 percent) was that of ewes inseminated with fresh semen. It was 69 percent when the semen was stored for two days at 0° C., 30 percent when stored for five days at 0° C., percent when stored for five days at -8° C., and ^3 percent when stored for 10 days at -8° C. The semen of the last two groups was thawed to 18-22° C. before using. - 7 - Morozov (1957) diluted ram semen in a hypertonic solution of glucose, yolk citrate and glycerine, placed it in -waxed capsules and stored it at -20° to -20.5° C. the spermatozoa were motile* After thawing 60 percent of Spermatozoa were still viable after storage for 12 to 13 days. Kuznetsov (1 9 56 ), in reviewing Russian research, mentioned that at 0° C. storage the spermatozoa of the ram retain motility but lose fertilizing power rapidly. At 0° C. storage a gradual swelling of the thin membrane of the sperm head took place. At -79° C. storage, 40 to 50 percent of the frozen sperm regained motility after thawing. With ram semen frozen 20 days, 19*3 per cent of 303 ewes inseminated conceived. In another trial, 33*5 percent of 512 ewes conceived from ram semen frozen for 30 to 50 days. In the previous work sperm were diluted l:k or less. In a discussion following this paper Professor Emmens stated that 15 to 20 percent of 2,000 ewes conceived after insemination with frozen semen in Australia. Lopyrin and Loginova (1958) reported that the semen of Merino rams was diluted in an isotonic solution to which 7.5 percent glycerol was added before freezing to -78° or to -I830 C; on thaw ing, spermatozoa!, motility was low and survival was poor. When a hypertonic glucose-yolk-citrate diluent plus 7*5 percent glycerine was used spermatozoan motility on thawing averaged Ml- to bG percent. Fewer spermatozoa had penetrated the cranial portion of the neck of - 8 - the uterus six to seven hours after insemination -with thawed frozen semen than in ewes inseminated with fresh semen. Of 36 ewes inseminated two to three times in one estrus with thawed semen^ 32 returned in heat. The four ewes conceiving produced a total of five lambs. Hill et al. (1959) found that fewer ram sperm survived freez ing in a yolk citrate extender containing 14 percent glycerol than in 3*5j 5«25 or 7 percent glycerol. ing rate of 2° C per minute from They found that a slow freez C. to -20° C., U° C. per minute from -20° C. to -50° C. and 10° C. per minute from -50° C. to -79° C. resulted in greater sperm recovery after freezing than a faster rate. - 9 - B. Glycerol Requirement for Freezing Ram Semen* First et al. (1957) have shown a curvilinear ram sperm sur vival after freezing in glycerol levels of 0, 2, k, 6, 8, 10, 12 and 15 percent. However, no significant difference existed Between six and eight percent. Hill et al. (1959) found greater survival after freezing yolk citrate diluted ram sperm in 3*5, 5 *25 or 7 percent glycerol than from ram sperm frozen in 14 percent glycerol. There was a significant ram times glycerol interraction of semen stored for 10 days. Emmens and Blackshaw (1950) concluded that 7*5 to 10 per cent glycerol in a yolk phosphate citrate arahinose diluter re sulted in the greatest ram sperm survival after freezing. White et al. (195^-), Blackshaw (I955u,b), Emmens and Blackshaw (l955), Lopyrin and Loginova (1958) have frozen ram semen in 7*5 percent glycerol. The Russian-Ukranian diluter used by Smirnov (1951), Szumowski et al. (1 958 ) and referred to later in this paper contained approxi mately 6 .5 percent glycerol. Szumowski et al. (195&) observed no difference in survival after freezing ram sperm in six to 16 percent glycerol. Glycerol levels for freezing bull semen have been a controver sial issue. Research concerning this matter was reviewed by Emmens and Blackshaw (1956 ). Amann and Almquist (1957) and have shown that *A11 glycerol levels referred to are on a volume per volume basis. - 10 - glycerol level depends on the solid content of a skimmllk diluter. Saroff and Mixner (1955) found that the glycerol level used in a yolk citrate diluter was dependent upon the amount of egg yolk in the diluter. Kuznetsov (195^) reported that high yolk levels decreased survival and fertilizing capacity of ram semen. Polge (1 956 ) found that hoar sperm could not tolerate ten per cent glycerol in the diluting media. Polge (1951) found that glycerol adequately protected fowl sperm during freezing, however, conception did not occur unless the glycerol was slowly removed before insemination. Embryonic mortality was greatly increased when the inseminated semen contained glycerol. - 11 - C. Ram Semen Extenders Ram semen extenders can be divided into two broad groups; (l) those which are useful for dilution and immediate insemination of fresh warm ejaculates and (2) diluters which allow cooling and storage of the ejaculate. Anderson (19*1-5) stated that one must consider osmotic pres sure, pH, buffering capacity and toxicity when preparing a mammalian semen diluter. Diluters Used for Immediate Insemination Echenique et al. (19*H) after studying several well-known dilut ing media, found that the most suitable diluent was the seminal fluid of a vasectomized ram or bull. Physiological saline has long been used with semen that is diluted and inseminated immediately after collection. However, Milovanov (193*0 mentioned that salts such as chlorides should not be used in diluting fluids. Physiological salt solution is stated to have a destructive action on the sperm lipid capsule. The K, Ca and Mg ions were also reported to have similar effects on the capsule. However, a certain amount of salt ions are necessary for normal sperm irritability. Milovanov proposed the use of colloidal diluters. Emmens and Swyer (19*1-8) found that the harmful effects of physiolt ogical saline dilution were due to high dilution rates rather than due to HaCl toxicity per se. - 12 - Rao and Hart (19^-8) found 0,9 percent sodium chloride had a stimulating effect on hull spermatozoa hut that cells in highly diluted semen died within two hours after dilution. Cheng, Casida and Barrett (19^9) reported that motility of hull sperm was greater in 0.9 percent sodium chloride hut that the use of sodium citrate permitted greater dilution before motility ceased. Emmens and Swyer (19^8), Blackshaw and Emmens (1951) ancl Blackshaw (19 53 ) have found that sodium chloride in isotonic diluents is non-toxic to spermatozoa except at an. alkaline pH. Blackshaw (1953b) also reported that .005 M potassium chloride partially restored the motility of ram sperm washed four times. It did not however, prevent head agglutination. tion prevented sperm head agglutination. Seminal plasma addi He suggested that the protective action of substances such as lecithin is a physical pro tection of the cell surface instead of a chemical effect. Storage Diluents The common storage diluents for the most part contain singly or in combination gelatin, egg yolk, milk, skimmilk and/or sugars in sodium citrate, glycine, sodium phosphate or tartrate buffer. Anderson (19^-5) has reviewed diluters conventionally used. Graziette (19^2) observed that ram semen diluted with sodium potassium phosphate, sodium sulphate, glucose and ram testis serum retained motility longer on storage at 2° to 5° C. than did undiluted semen. The addition of gelatin to the diluter resulted in a rapid loss of motility. - Aslanjan (1950) 13 - inseminated ^,709 eweswith ram semen in yolk citrate and stored at 0° C. for six to 168 hours. three percent of the ewes conceived. to inseminationhad diluted Ninety- The age of the semen prior no effect on conception rates. Dauzier et al.(195 ^*) tested several diluters and found that sperm motility was preserved the longest in a diluter consisting of three parts of three percent sodium citrate solution added to one part egg yolk. Motility was not preserved as long in milk diluents and was the poorest in a salt diluent. In a breeding trial conception rate was low with the citrate yolk diluent 1101088 it was used within two hours of collection when a rate of 66.6 percent was obtained. The highest conception (8 3 .3 percent) was obtained after the insemination of p°p stored ram semen diluted in a sodium citrate,magnesium sulphate, calcium acetate diluter, Koger (1951) has studied the motility of ram sperm diluted In several diluters at various dilutions and under 5° C. storage. The diluters studied were physiological saline, Phillips phosphate buffer and 2.5 percent sodium citrate. singly and in combination with egg yolk. varied with different diluents. These diluters were tested The effect of dilution In diluters without egg yolk, there was a negative correlation between rate of dilution and dura tion of sperm motility. This was apparently due to excessive meta bolic activity immediately following a high rate of dilution. response was the most pronounced in physiological saline. The This addition of egg yolk to citrate and phosphate buffers improved storage in these diluters and the detrimental effects of high dilution were largely overcome. Egg yolk apparently had no bene ficial effect when added to saline or to whole semen. Although saline was the least desirable for storage it resulted in the highest motility immediately after dilution. The most satisfactory diluter for ram semen storage contained 75 parts of 2.9 percent sodium citrate solution and 25 parts egg yolk. When ram semen diluted in this diluter and in a phosphate ©gg yolk diluter, each stored from zero to five days, were used in a breeding trial, fertility dropped noticeably during the first 2k hours of storage and declined sharply with further stor age. A higher percentage of pregnancies occurred from sperm dilu ted in seminal fluids than diluted in phosphate egg yolk. Hyaluron idase addition did not reduce the number of sperm required for a satisfactory conception rate. Sakala (1957) studied the effect of 19 recommended diluents on semen character. The best results were obtained with a diluter composed of 25 percent yolk, sodium citrate and glucose or fructose In recent years several authors have investigated milk dil uents . Thacker and Almquist (1953) found that homogenized or skimmilk must be heated to at least 92 ° C . before it was used as a bovine semen diluter. - 15 - Thacker et al. (195*0 discovered that the toxic factor in unboiled milk was in the albumin fraction and that casein and protein free milk is usable. Boyd et al. (195*0 found, in vitro, that bull spermatozoa survived better in milk treated with thioglycolic acid (a sulphydryl containing compound) than in heated pasteurized milk. In essence this was confirmed by Almquist (195*0, Almquist et al. (195*0 > Flipse et al. (195*0 and by Johnson et al. (1955)• These authors found heated homogenized or pasteurized milk, or one mg. per ml. of cysteine hydrochloride (another sulphydryl compount) added to pasteurized skimmed milk to be as suitable as egg yolk citrate in terms of bull sperm motility and fertility. The sulphydryl compounds lowered the oxidation reduction potential of the milk and inactivated lacterin, the anti-streptococcal and supposedly spermicidal substance present. Istvan (1 956 ) inseminated 535 owes with ram semen diluted in boiled cows milk, ewes with semen diluted in yolk citrate glucose and 257 owes with undiluted ram semen. The percentages of ewes lambing after one insemination were 75*5j> 6 3 *2 and 6 3 .5 respectively. In the following year 8 3 .0 percent of 2 ,6 5 2 ewes inseminated with milk diluted ram semen lambed and 8 3 *1 percent of 638 ewes inseminated with undiluted semen lambed. Dauzier (195^) reported that conception rates obtained with ram semen after storage for 12 to 2b hours at 2° to *f° C. varied -16- from six to 25 percent when the semen was stored undiluted, or diluted in citrate, egg yolk citrate or egg yolk phosphate diluents. When it was stored in skimmed cows milk, heated to 95° C. for ten minutes, conception rate after 12 hours storage was 51 percent. Following the addition of antibiotics, it was 65 percent. Goat semen stored in skimmilk for periods under eight hours resulted in a conception rate of 6k percent. Fillimon et al. (1956) found that when ram semen was diluted in milk and cooled to 0° C., 80 percent of the spermatozoa were viable after storage for kQ hours and 55 percent were viable after 72 hours. Fifty-nine percent of 155 ewes inseminated conceived, as compared to 5 2 .1 percent of 56 ewes inseminated with undiluted semen. When gluco-phosphate plus gelatin diluent was used, 73*9 percent of 23 ewes inseminated conceived versus 71.**- percent of 21 ewes inseminated with undiluted ram semen. Mihailov (1957) reported that ram semen diluted in boiled cows milk was used to inseminate ^,700 ewes at six insemination stations. The conception rate varied between 83 and 97 percent at the differ ent stations. Hill et al. (195&) stored the semen of four rams for 15 days at 5° C. in reconstituted skimmilk and egg yolk sodium citrate diluters. Sperm survival was higher in the reconstituted skimmilk, however, no fertility tests were made. ' Toshioka et al. (1951) conducted experiments on the storage of semen and on the insemination of sheep and goats with stored -17semen. The effects of two kinds of diluents were examined. One was prepared hy mixing two parts by volume of 2 percent boric acid with one part of 1 percent sodium bicarbonate. added to the semen in a ratio of 1:3. RH solution. This was The other diluent was called It was prepared by mixing equal volumes of 0.3 percent sodium sulphamerazine and 0.2 percent homosulphamine. This was dissolved in five percent sodium citrate and the solution was added to the semen in a 1:1 ratio. Both diluents, but especially EH solution, Improved the keep ing quality of the semen. Insemination with semen to which either of these diluents had been added resulted in higher conception rates than insemination with •untreated semen. The percentages are as follows: untreated semen, 56 ,k percent; semen treated by boric acid and sodium bicarbonate, 6 5 .3 percent; semen treated by RH solution, 6 5 .1 percent. The length of time for which the semen retained fertility was: untreated semen, 111-120 hours; semen treated with boric acid and sodium bicarbonate, lMj- hours; semen treated with RH solution, 168 hours. Toshioka and Koike (19 56 ) studied the storage and fertility properties of five semen diluters. These diluters being (l) a glucose sodium citrate diluter, (2) egg yolk sodium citrate, (3 ) the RH diluter previously mentioned and (^) a glucose-RH diluter containing two volumes RH and one volume of egg yolk. The yolk-RH diluter maintained high sperm motility the longest. After 120 hours storage at 2° to 7° C., the diluters -18- were ranked from highest to lowest sperm motility as follows: (l) yolk-RH solution, (2) yolk citrate, (3 ) glucose-RH solution, (!}•) RH solution, (5 ) glucose citrate and (6) undiluted semen. The conception rate for the first *1-8 hours of storage after insemination with yolk-RH diluted semen was as high as from fresh undiluted semen. Four conceptions occurred when the semen was preserved as long as 1 0 -1 3 days. No significant difference could he found, in the influence upon spermatozoa, between heated goat milk and RH solution, when both were used as the diluents of gelatinized semen. A low electrolyte glycine buffer was studied by Ahmed (1955)Ram semen was stored at *f° C. in a standard citrate-egg yolk diluent, which served as a control, and in various glycine dilu ents . Replacing citrate buffer by five percent glycine considerably improved survival over that of other concentrations of glycine and the citrate buffer control. Higher survival in five percent glycine- egg yolk buffer was obtained with a semen dilution ratio of 1 :2 0 than with a ratio of 1:^0. The addition of fructose to the glycine buffer did not improve survival. A 50 percent concentration of egg yolk in a four percent glycine buffered egg yolk diluent was reduced to 3 3 .3 percent egg yolk without affecting either motility or sur vival and to 25 percent egg yolk with no effect on survival but with a slight reduction in motility. -19- a-*-» (1956 ) found the motility and survival of egg yolk glycine diluted sperm stored at h° C. to he greater than the sperm diluted with egg yolk citrate. Aamdal and Hogset (1955) inseminated ewes with ram semen dil uted 1:6 with three percent citrate 50 percent yolk and three percent glycine 50 percent yolk diluter. More ewes conceived from the yolk citrate diluted semen. Kuznetsov (1956 ) from work done in the U.S.S.P., stated that the best diluting medium for ram semen was a three component synthetic medium composed of 90 ml. of isotonic sodium citrate plus 10 ml. isotonic glucose, fructose or glycocole and 20 ml. of egg yolk. When the quantity of egg yolk was increased the survival of spermatozoa and fertilization of ewes decreased. Pozdnjakov (1958) found yolk glucose citrate diluent with anti biotics superior to the same diluent without antibiotics. Pauzier et al. (195*0 observed that ram sperm motility was maintained longer in a yolk citrate diluent than in yolk phosphate, yolk bicarbonate glucose, skimmilk, whole milk or salt diluents. Twenty-five and 50 percent egg yolk maintained sperm motility longer than five percent. Preservation of motility was not affected by rate of cooling or dilution. The fastest rate was 1.5° C. per minute. The conception rate was low (15-7 percent) from I+38 ewes cervically inseminated with one ml. of yolk citrate diluted semen which had been diluted 30 to *^0 times. Seventy-three percent of 21 -20- ewes conceived when inseminated with undiluted ram semen and 83 per cent of natural mated control ewes conceived. A further study in which semen was diluted and inseminated within two hours after col lection, resulted in the following conception data: 66.6 percent of 21 ewes bred with 33 percent yolk citrate diluted semen conceived, 53.3 percent conceived from a diluent of five percent yolk in a sodium citrate buffer, 50 percent from Milovonov diluter, 8 3 .3 per cent from a salt diluter of sodium citrate, magnesium sulphate and calcium acetate and 6*l-.l percent conceived after insemination with milk diluted semen. It was suggested that egg yolk had an unfavorable action against the fertilizing ability of ram sperm and that ram sperm motility was not necessarily indicative of fertility. -21- D. pH, Electrolytes and Osmotic Pressure Emmens (l9*J-7> 19 *<-8 ) using rabbit sperm, concluded that motility was not significantly affected by changes in the proportion of sodium chloride and glucose in buffered diluents of the same osmotic pressure, except above a pH of 9* Blackshaw and Emmens (1951) reported that ram and bull sperma tozoa exhibited optimum motility at pH 7* Hypertonic solutions were less harmful than hypotonic media, at all pH levels. The slight adverse effect of hypertonicity could be diminished by par tial replacement of sodium chloride with glucose at alkaline pH levels * Salisbury and Kinney (1957) have found that pH markedly in fluences aerobic fructolysis as pH increases fructolysis rate in creases and more lactic acid is produced in excess of fructose utili zation. Anderson (19^5)* after reviewing Russian work, stated that a certain concentration of electrolytes was necessary for normal sperm irritability, but that too high concentrations over stimulated spermatozoa, which rapidly lost motility and died. Oxygen consumption by spermatozoa was stimulated by a diluent of 0.9 percent sodium chloride. The rate of motility and the liv- ability were higher and greater than motility and livability of sperm in a phosphate diluent, so long as the oxidation of lactic acid proceeded (Bishop and Salisbury, 1955) • When aeration was -22- incomplete, lactic acid accumulated to toxic levels, pH of the media dropped rapidly and the cells died (Salisbury, 1957)* According to Mann (195*0 > appreciable quantities of potassium, magnesium and calcium occur in mammalian semen. Winters et al. (1938) and Milovanov (193*0 found calcium necessary in diluents for ram spermatozoa. However, Lardy et al. (19*^5) t Lardy and Phillips (19*^3) and Blackshaw (1953&) found that calcium depressed the viability of ram and bull spermatozoa. According to Lardy and Phillips (19*^3) magnesium improved the motility and glycolysis of washed bull spermatozoa. The importance of potassium for the normal functioning of ram and bull spermatozoa has been demonstrated by Lardy and Phillips (19*K3) > White (l953ajljc an& 1956) and Blackshaw (I953ajb). The motility of mammalian spermatozoa was decreased by high dilution even if the diluent was an isotonic one containing a glycolysable sugar (Emmens and Swyer, 19*+8; Blackshaw, 1953a> and White, 1953c, 195**-.). It was postulated that the dilution of salts was responsible for this reduced sperm motility after dilution. The osmotic pressure of the semen of several species, in terms of freezing point depression, has been summarized by Mann (195*0 • The data expressed in centigrade temperature depression were: man *5 5 -*58 °; bull .5*J— *73°j ram .55 -*70 °; stallion .58 -.62 °; and boar .5 9 -.63 0. -23- E. Spermatozoa Death After Freezing and Thawing The studies of Davenport (18 9 7 ), as reported by Davenport (1938 ), Jabnel (1938), Shettles (19*1-0), Hoagland and Pincus (19*1-2) and Parkes (19 ^5 ) illustrated that human spermatozoa were exceptional in that a small portion could survive freezing. Emmens and Blackshaw (1950) resuspended human, bull, ram and rabbit sperm in each respective seminal plasma. The human sperm survived freezing irrespective of the plasma species it was suspended in, indicating the survival prop erty was with the cell itself. Attempts to revive frozen spermatozoa of other species met with little success until Polge, Smith and Parkes (191^9 ) found that glycerol exerted a protective action on cells dur ing freezing. The studies of Polge (1957)} Smith and Polge (1 9 50 ), Emmens and Blackshaw (1950), Szumowski (195*0 > Roy (1955) and Polge (1 9 56 ) indicate that considerable species difference appears to exist as to the tolerance of sperm cells and red blood cells to gly cerol. For most species studied the critical temperature during freezing seems to be between -15° and -20° C. (Polge 1957)* The literature suggests (Polge, 1957; Walton, 1957; Sherman, 1 959; and Parkes, 1956 b) that more than one factor is responsible for sperm cell death during freezing. Luyet and Gehenio (l9 *+0 ) have reviewed the factors responsible for life and death of cells at low temperatures. They postulated that cells were killed during freezing by intra-cellular ice forma tion and subsequent protoplasmic alterations. -2k- Luseva and Cook (1953) found that glycerol modified the rate of ice crystal growth, membrane permeability and related phenomena and partially controlled the rate of freezing. Smith, Polge, and Smiles (1951) observed a fern like pattern of channels in semen frozen in the presence of glycerol. However, glycerol did not prevent crystal formation in the media. The work of Sherman (1957) suggested that extra-cellular mech anical injury from ice formation on freezing and thawing did not cause sperm death. There is some doubt as to whether crystalization is the primary factor causing sperm death during freezing. Lovelock (1953a, b, c) found that destruction of red blood cells during freezing and thawing was caused by an increased concentration of salts which resulted from the conversion of water to ice. In the presence of glycerol the rise in electrolyte concentration below the freezing point of the fluid was sufficiently reduced to abolish this effect. Glycerol failed to exert any protective action when it was prevented from entering the blood cell by the action of copper ions. The experiments of Lovelock and Polge (195*0 have shown a rela tionship between the degree of sperm cell damage caused by freezing at various temperatures below zero and that caused by exposing the spermatozoa to high concentrations of salts at 0° C. The glycerol requirement for freezing sperm of various species and for freezing various cells depended on the salt concentrations of the cells normal environment. Observations of Lovelock (1953b), (195*<-c ) indicated that the high concentration of salt produced by ice formation did cause dispersion of lipids and liproproteins from the red cell membrane which was modified by the presence of glycerol. Lovelock (l95**-b) found that red blood cells which ordinarily did not suffer from thermal shock, did succumb to thermal shock in the presence of excess salt. There are, according to Polge (1957), apparently two major pro blems in freezing semen. The first problem being to freeze the semen fast enough between -15 and -25° C. such that exposure to high salt concentration is reduced. The second problem is to freeze the semen slow enough or with sufficient protection to prevent the occurrence of temperature shock. The studies of Easley, Mayer and Bogart (19*1-2); Walton (1951); Blackshaw (195*0; suk! Kamschmidt et al. (1953) have illustrated that egg yolk, and particularly its lipid and lipoprotein constituent protect smperatozoa against the damaging effects of temperature shock Stallion and boar sperm are the most susceptible to cold shock and ram sperm are more resistent to cold shock than bull sperm (Mayer 1955) • Electron photomicrographs taken by Walton (1957b) indicated that a galecapitis and cell surface destruction took place on cold shocked sperm and sperm dying natural death. The mechanism of the temperature shock protective action of sub stances such as egg yolk and lecithin is not known. Blackshaw and Salisbury (1957) suggested that this protection was due to a surface property which maintained the integrity and function of the cell -26membrane. Their research has proven, that cold shock of whole semen causes a loss of potassium and ether-alcohol extractable phosphorus from the cells into the plasma, and an uptake of sodium and calcium by the cells. Sherman (1959) has shown that some sperm, although motile after freezing and thawing, were injured or weakened by the ordeal. This injury occurred during freezing or thawing and not as a consequence of extender toxicity. Recent studies have proven that glycerol was metabolized by bull spermatozoa (Odell et al. 1956, White et al. 195*4- ami White 1957) and by ram spermatozoa, (Mann and White, 1956, 1957)* Glycerol metabolism by ram and bull sperm is primarily aerobic with a sparing effect on fructolysis (Mann and White 1957 and White 1957) * The rate of glycerol utilization by bull sperm (Odell et al. 1959a) varies with the type of diluent employed. Odell et al. (1959b) have shown that the sugars arabinose and fructose have a sparing effect on glycerol utilization by bull sperm. Lactic acid was not produced from arabinose metabolism whereas large quantities of lactic acid were produced by aerobic meta bolism of fructose. White et al. (195*4-) also could find no lactic acid formed when bull and ram spemn metabolized arabinose. The studies of Elliott et al. (195*4-) > Emmens and Blackshaw (1950)^ Blackshaw (I955a)> Odell and Almquist (1957), VanDemark et al. (1957) and First et al. (1957) indicated that adding various monosaccharides to diluted, glycerolated bull or ram sperm increased the percentage of spermatozoa surviving freezing to -79° C. or below. Shaffner et al. (19*1-1) found that levulose partially protected fowl sperm during quick freezing. -27- F. Equilibration Time and Temperature Considerable discussion has centered around the necessity of equilibrating semen with glycerol for several hours before freezing. The temperature at which this equilibration should take place has also been questioned. The idea of glycerol equilibration period for bull semen began with the original work of Polge and Rowson (1952a) which indicated optimum recovery and fertility after freezing were obtained with a 15 to 20 hour glycerol equilibration. Holt (1953) and Stower (1953) reported greatest recovery with equilibration of 20 to 2k hours. Rill at al. (1959) found a .5 hour equilibration period to be superior to an 18 hour equilibration period, in terms of ram sperm recovery after freezing in a yolk citrate extender. Szumowski et al. (1956 ) found at least four hours of glycerol equilibration necessary for freezing ram semen. The optimum equili bration period was reported to be between six and 1*J- hours. Graham et al. (1957) reported a 6 7 .8 percent first service non return rate from 1,012 cows bred with semen that was equilibrated with glycerol 12 hours before freezing, 6 5 .2 percent non-return from 1 ,0 2 6 first services with semen equilibrated for eight hours and 6 3 .^ percent non-return from 996 first services to bull semen equilibrated for eight hours. significant. The four and 12 hour differences were -28- Williams (195*4-) 9 however, reported a higher percentage of non return when cows were inseminated with semen equilibrated six hours as compared to that equilibrated 18 hours. Saroff and Mixner (1955) stated that as glycerol equilibra tion time was increased from two to 18 hours there was a progres sive increase in sperm survival on thawing. Cragle et al. (1955)> using a three dimensional central composite design, found the opti mum percent citrate, percent glycerol and equilibration time to be 2.9 percent, 7*6 percent and 1*1-.9 hours respectively. Odell and Almquist (195*4-) found no significant differences in bull sperm survival among glycerol equilibration periods of 0.5* *4and 18 hours after freezing as skimmilk diluted bull semen. Odell and Hurst (1956) compared glycerol equilibration time of 0.5 and 18 hours for semen diluted in heated skimmilk and found a significant difference in favor of the shorter time. Odell and Almquist (1957) found that in skimmilk diluent, pre freezing glycerol equilibration periods of 30 minutes and four hours resulted in spermatozoan survival equal to or better than 18 hours. They state that equilibration time was not related to the level of glycerol present in the diluent. The stepwise addition of glycerol resulted in slightly greater post-thaw motility than if the glycerol were added in one portion at 5° C. They found that 1.25 percent fructose added to the skimmilk diluent resulted in a significant increase in freezability of bull -29- spermatozoa. However, the addition of this sugar had no effect on the equilibration period or level of glycerol needed for optimum freezability. Blackshaw (l955a) reported that equilibration of bull spermatozoa with glycerol for 18 hours before freezing does not increase survival after freezing. Blackshaw (1955b) reported that bull and ram semen may be mixed with the glycerol containing diluent at temperatures up to 15° C. without harmful effects on revival after freezing. VahDemark et al. (1957) studied glycerol equilibration time and equilibration temperature. At an equilibration temperature of 4.5° C., little variation in motility was found after freezing and thawing, among semen equilibrated at 2, 6 and 18 hours under equilibration temperatures of 10° C. and 15*5° C. The shortest equilibration time, (two hours) was slightly more detrimental, with the differences at 15*5° C. being significant. For all temperatures considered, six hours was significantly better than two or 18 hours. Their data also supports the findings of Emmens and Blackshaw (1950)> Blackshaw (1955a), Odell and Almquist (1957) and Amann et al. (1957) who reported the addition of simple sugars such as glucose rhamnose, arabinose and fructose does improve sperm survival after freezing. Emmens and Blackshaw (1950), Blackshaw (1955a) and First et al. (1957) have shown that 1 .2 5 percent arabinose combined with glycerol before freez ing results in a very marked increase in ram sperm survival with yolk phosphate citrate and milk diluters. -30- Odell et al'. (1956b) have recently shown that fructose and arabinose have a sparing effect on the utilization of glycerol-lC1^ by bull spermatozoa. In summarizing the research data pertaining to equilibration time where some aspect of motility after freezing has been used as a criterium, the work of Polge and Rowson (1952a), Holt (1953), Stower (1953) and Saroff and Mixner (1955) support an equilibration period of 18 hours or more. The reports of Cragle et al. (1955), VanDemark (1957) and Szumowski et al. (1956 ) favor equilibration of six to 15 hours. The research of Odell and Almquist (195*0 and Odell and Hurst (1 956 ), Odell and Almquist (1957), Blackshaw (1955a) and Hill et al. (1959) indicate no more than one hour of equilibration with glycerol.is necessary. In breeding trials, Graham et al. (1957), found bull semen equilibration of less than one hour the most satis factory and Williams (195*4-) found six hours equilibration resulted in a higher 60 to 9° day non-return rate than 18 hours equilibration. The studies of Polge (1957) indicate a possible explanation for the differences in equilibration results reported by different authors. Bull, ram, stallion and boar semen exhibited greater resistance to j temperatures between -1 5 and -25 ° C. after 18 hours equilibration when compared with one hour equilibration. These facts suggested that dif ferences in equilibration effects on sperm survival may not be noticed unless sperm are frozen very slowly. -31- G. The Storage Potential of Frozen Semen The decrease in motility and decreased fertilizing capacity of hull sperm after prolonged frozen storage has been investigated by several workers. Bratton et al. (1957) reported non-return rates of 73*2 percent of 1 ,1 5 1 first services to frozen semen stored one week and 6 9 .8 per cent for 1,09*4- first services to frozen semen stored 17 weeks at -79 ° C. This difference was not significant. Mixner and Wiggin (1957) found that a first service non-return rate of 6 6 .7 percent occurred with frozen bull semen stored for seven and l*f- days at -79° G. After six and 12 months storage the non return rates were 70*1 and 6 5 .7 percent respectively. Graham et al. (1958) found no significant differences in non return rates from frozen semen used after one, two, three and four weeks of storage at -79° C. The first service non-return rates were 6 3 .6 percent after one week, 6 2 .3 percent after two weeks, 6k ,6 percent after three weeks and 67*1 percent after four weeks storage. VariDemark et al. (1957) found that bull semen stored at -79° C. exhibited the following motility: k9 percent after two days, k6 per cent after nine days, *1-0 percent after 16 days and 38 percent after 51 days storage. Hill et al. (1959) found a significant decline in motility re covery between ram semen frozen two days and 10 days at -79° C. -32- Szumowski et al. (1956) observed no decline in motility of ram sperm stored four weeks at -79° C. Ludwick (1958) observed that frozen bull semen declined more rapidly in motility after thawing than unfrozen semen of the same age. Etgen et al. (1957) and Polge and Rowson (1952b) have even observed a slight increase in percent motile or live cells with increasing storage age at -79° C. Etgen et al. (1957) explain this on the basis that there is (l) a necessary sperm adjustment period after freezing, (2) disintegration of sperm during the first weeks of storage or (3 ) personal bias of the evaluator. -33- H. Sperm Numbers Necessary for Satisfactory Conception Kuznecov (193*4-) recommended the insemination of 50 million or more sperm in the cervix of the ewe. Milovanov (193*4-) reported that at least 500 million ram sperm were essential for vaginal inseminat ion. The data of Hahihullin (1937) indicated that lambing percent ages were higher for 1 :1 and 1 :2 dilutions than 1 :3 dilution or un diluted semen and that 1 :1 dilution resulted in the highest lambing percentage. volume. All the above treatments were inseminated in a .1 ml. He recommended the total sperm numbers inseminated should be not less than one billion. Habibullin (1938) found that after 2k hours storage a dilution of 1 :2 maintained fertilizing ability at 6k percent, whereas sperm diluted 1 :1 showed a marked drop and if diluted 1 :3 fertility dropped to nearly the level of undiluted sperm (36 percent). Lukin and Eremeev (1938) estimated that to ensure 90 percent fertilization the number of undiluted ram sperm inseminated should be 1 .1 billion or if diluted 506 million. Anderson (19*4-1) reported that when *4-62 ewes were inseminated with undiluted ram semen, or semen diluted 1 :3 , 1 :8 or l:l6 ; con ception was much lower following 1 :1 6 dilution than it was for the other treatments (*4-2 to *46 percent). Keast and Morley (19*4-9) concluded from their work that although the highest conceptions were obtained with insemination of 350 million - 3^ - ram sperm in 0.1 ml. volume, reasonably satisfactory results were obtained with a concentration as low as 50 million ram sperm per 0.1 ml. insemination. Carbonero (1955) reported the optimum dilution rate, using a physiological sub-alkaline UaCl and NaoH diluter, to be one part ram semen to five parts of diluter. After reviewing the literature, Terrill (1952), recommended that 50 million or more sperm should be inseminated into the cervix in not more than 0.2 ml. of fluid carrier. Barretto and Mies Fihlo (19U0, however, achieved moderately successful results from as low as five million sperm per insemination. Panyseva (19 ^-0 ) reported a direct correlation between the number of sperm inseminated and conception rate (r = .8 5 ). Kbger (1951 ) found a linear relation between number of sperm inseminated and conception rate. Fifty million or more fresh sperm per insemination were required for a con ception rate of more than 50 percent. A preliminary study was con ducted with sperm diluted in egg yolk citrate, stored at 5° 0. and used from zero through five days. The insemination of ram sperm at concentrations of k20, 105 and 26 million sperm per ml. resulted in pregnancy rates of 35-2, 2 6 .5 &ud 17.1 percent respectively. When only fresh diluted semen of less than one day of age was con sidered, the pregnancy rates were 6 7 , 50 and 33 percent. In a second experiment ram semen diluted in egg yolk citrate was inseminated without storage and the following sperm concentration conception relationships were recorded: -35- Million Sperm/insemination Percent Conception Hate 420.0 210.0 105.0 62.5 6 5 .6 5 0 .0 52.5 35.0 17.5 46.7 33-3 21.4 8.8 2 5 .8 A similar relationship existed between sperm numbers per insemination and conception when either physiological saline or seminal fluids were used as the diluter. However, the corresponding conception rates were all higher when sperm were diluted in ram seminal fluids. These differences were not significant. Kuznetsov (1956)> reported on work in Russia, stated that only 30 to 90 million ram sperm from a normal ejaculate penetrate the cervix and that sperm in the vagina perish within three to six hours after mating. These investigations have shown that spermatozoa leave the cervix in small portions, moving into the uterus and oviducts. The sperm in the uterus retained activity for up to 12 hours. The cervix contained seven to 12 million sperm, 15 to 20 percent of which were motile 30 to 48 hours after Insemination. It was suggested that the cervix acts as a store for sperm allowing a periodic release of motile sperm throughout estrus. -36- I. Volume of Semen Inseminated Smirnov et al. (1939) concluded that the insemination of 0.3 ml. of undiluted ram semen in gelatin or paper capsules resulted in conception rates equal to that of 0.1 to 0 .1 5 nil. of undiluted semen inseminated with a syringe in the cervix. The conception rates from these treatments were between 7^- and 75 percent. Sinclair (1957)* using fresh undiluted ram semen, obtained no difference in conception between cervically inseminated doses of 0.1, 0.2, 0.3 or 0.^ ml. Malikov (1957) cervically inseminated ewes with .05 ml. of undiluted semen and with .1 and .05 ml. of semen diluted in yolk citrate or in boiled cows milk. Seventy-three percent of the ewes inseminated with .1 ml. of milk, 66 percent of those inseminated with .05 ml. milk and 66 percent and 53 percent respectively of the ewes inseminated with one ml. and .05 ml* of yolk citrate diluted ram semen conceived. Kbger (1 9 5 1 ) has achieved 50 percent conception from vaginal inseminations of one ml. of ram semen. -37- J. Cervical versus Vaginal Insemination The difference in sperm numbers necessary for vaginal insem ination (Milovanov 193*0 and cervical insemination (Kuznecov 193*0 would suggest that cervical insemination might be more effective and efficient. Anderson (1937) considered vaginal inseminations responsible for low fertility in a group of ewes wherein cervical inseminations were attempted. Kelly et al. (19 *1-2 ) found no differ ence in lambing percentage whether semen was deposited in the cervix, vaginal folds or forynx of the vagina. Keast and Morley (19*1-9) reported that of 30 cases wherein semen was deposited in the vagina, because the cervix could not be located, only four ewes conceived. These four were from treat ments wherein conception after cervical insemination was from 37 to 69 percent. Koger (1951) inseminated one-half of 201 ewes in the cervix with 0.2 ml. of diluted semen. The other one-half were inseminated in the vagina with one ml. of diluted semen containing the same total number of sperm. There was no difference in the pregnancies obtained between the two methods, cervical insemination resulting in *1-8 .5 per cent pregnancies compared to 50 percent from the vaginal inseminations. Dauzier et al. (195*0 found no difference in conception rate between cervical or vaginal insemination. Mies Filho and De Almieda Ramos (1955) after inseminating 872 ewes with fresh semen, found a significant conception difference in favor of vaginal as opposed to cervical insemination. -38- Dun (1955) studied the reproductive tracts of 112 slaughtered Merino ewes and concluded that an experienced inseminator would he able to deposit semen in the cervical opening in 100 percent of young maiden ewes and 70 percent of older ewes. -39- K, When to Inseminate the Ewe in Deference to the Beginning of Estrus and Sperm Life in the Ewe The optimum time for insemination should depend mainly on the life of sperm in the reproductive tract of theewe, the time of ovula tion and the life of the ova. Quinlan et al. (1932, 1933) found that in Merino sheep the majority of ram sperm inseminated were non-motile after12 hours. Live sperm were found in the cervix of the ewe up to 48hours after mating. Green and Winters (1935) stated that sperm did not live more than 24 hours in the genital tract of the ewe. ram sperm reached the Palovceva et al. Kelly (1937) found threshold of infertility 34 (1938) estimated hoursafter mating. the average duration of sperm survival in the ewe to he 34 to 36 hours and in some cases as much as 40 to 50 hours. Lopyrin and Loginova (1939) found that sperm maintained motility for 32 hours in the tipper portion of the genital tract of ewes in heat. The data of Green (1947) indicated that ram sperm may retain their ability to fertilize the ovum up to approximately two days after insemination, Quinlan et al. (1932) mated ewes at intervals after the begin ning of estrus and found a constant conception rate up to 30 hours, after which time a definite drop in conception occurred. Kardymovic et al. (1935) found the optimum time to breed was 18 to 25 hours after the onset of estrus. -40- Warbritton et al. (1937) observed that of three insemination periods tried, the most desirable was 12 hours before ovulation. This was approximately ten to 18 hours after the onset of estrus. Anderson (l94l) reported that insemination from 12 to 30 hours after the onset of estrus resulted in the best conception. Kelly et al. (1942) reported that there was no difference in the rate of lambing between ewes still in estrus 16 to 24 hours after insemination and those out of estrus. Carbonerro (1955)? after inseminating 8,470 ewes, concluded that the best time for insemination was 12 to 18 hours after the beginning of estrus, when the vaginal mucus was turbid. Sinclair (1957) found no difference in conception rates among ewes inseminated, 0-20, 1 6 -2 8 and 0-28 hours after the beginning of estrus. Further information was gained on this matter from another trial in which the stage of estrus was determined by the condition of the vaginal mucus. Clear mucus was associated with early estrus, clear and copious mucus with middle estrus, cloudy mucus with late estrus and creamy mucus with very late estrus. It was concluded that insemination very late in estrus, a period char acterized by the presence of creamy mucus in the vagina, was likely to result in a lowered rate of conception. Concerning the time of ovulation in reference to estrus, McKenzie and Terrill (1937) found that ovulation in the ewe generally takes place near the end of estrus and that inseminations made after the end of estrus are less likely to be successful than those before the end of estrus. -4l- Palovceva et al. (1938) reported that in the sheep fertilization occurred not earlier than two to four hours after ovulation and ovulation occurred 30 hours or more after the onset of estrus. Lopyrin and Loginova (1939) found that the average time of ovulation was 30 to 32 hours after the onset of estrus. Larrea (1944) reported that Corriedale ewes remained in estrus for 28 to 30 hours. Schott and Phillips (1941) found a time interval of 20 minutes was usually sufficient for sperm to reach the upper part of the Fallopian tube after a normal service and that this was not influenced by the stage of estrus or by ovulation. 20 to 36 hours after the onset of estrus. were equally active. Ovulation usually occurred The right and left ovaries Of several breeds studied Corriedale and Dorset ewes tended to remain in estrus after ovulation. Paloveeva (1940) observed definite uterine movements associated with estrus in the ewe and that simultaneous contraction of longi tudinal and relaxation of circular muscles of the uterine neck assist in opening the cervical canal. Dauzier (1955&^) found that uterine contraction is the essen tial factor in uterine migration of sperm and that spermatozoan mi gration in the Fallopian tube was primarily due to sperm motility. However, the studies of VanDemark and Moeller (1951) indicated that bull sperm travel the Fallopian tube of the cow at a much greater rate than can be accounted for by their own propulsion. -42- Braden (1 9 53 ) observed that in rabbits the tubal uterine junction acts as a barrier against sperm passage and estimated that one of every 160 sperm in the upper uterus enters the Fallo pian tube. He postulated that sperm reaching the distal half of the uterus within one hour after mating may be transported by uterine activity while those reaching this location two to three hours later do so by virtue of their own motility. Woyes et al. (19 5 8 ) have shown in rabbits, that only motile sperm pass the cervix. Other recent papers concerning this subject are those of Black and Asdell (1958) and Adams (1956) • The work of Chang (1951, 1955) and of Braden and Austin (1953), indicated that a sperm conditioning or capacitation period of several hours in the Fallopian tube was necessary before fertilization. Austin and Bishop (1958) suggest that capacitation involves a loosening of the acrosome from the sperm head. It was postulated that the loosened acrosome released an enzyme which enabled the sperm to pass though the zona pellucida of the ova. -1*3- L. Insemination or Mating Frequency During One Estrus Quinlan et al. (1932) found that "breeding eves twice during one estrus resulted in a slightly greater percentage of pregnancies than breeding once. However, this difference was not significant. Milovanov (193^0 reported that results of repeated insemina tion have been excellent in some cases and negative in others. Milovanov (193&) a-s cited by Anderson (19*1-5) reported that some Russian sheep stations had a large increase in lambing percentages when the ewes were inseminated several times. Peregan (1936 ) as cited by Anderson (19**-5)j inseminated 56 Karakul ewes twice at an interval of 20 to 30 hours, this resulted in a 171 percent lambing rate whereas the average for the flock inseminated once was 115 percent. Avramov (1937) inseminated 106 ewes twice at an interval of eight to 12 hours or 20 to 2k hours. When the interval between inseminations was 2*4- hours, the lambing rate of Tigai ewes was raised by 11.7 percent and of Chusbeka ewes by 1^.1 percent. Gavrilov (1937) reported that an eight hour inverval between inseminations gavesomewhat better results than a 2k hour interval and suggested thatthe second insemination should be carried out not later than 12 hours after the first. Kirillov(1938) as cited by Anderson (19*1-5) jconcluded that all ewes should be tested for estrus twice a day, morning and evening, and only those with an estrus period of more than 2*1- hours should be inseminated twice (**7 percent of the ewes were in estrus less than 2k hours). -M*- ^hree or four inseminations did not raise the rate of lambing of ewes with estrus periods longer than !§■ to two days. Lopyrin and Loginova (1939) observed from multiple insemina tions of 2 ,5 8 2 ewes that there was little difference in conception between ewes inseminated once and ewes given two or three insemina tions at 16 hour intervals. Dauzier et al. (195*0 after inseminating **38 ewes found no difference between insemination once or several times during one estrus. Anderson (19*4-1) studied *4-62 ewes and reported that two to four inseminations in one estrus period gave better results than a single one. A single service was reported to give better results than a single insemination. Larrea (I9 M O reported on the results of 523 single and 25 double inseminations in 19*4-3 and 703 single and 23 double insemina tions in 191**1-. The percent lambs born was 69 percent from one and 95 percent from two inseminations in 19*4-3> with 7*4- percent from one and 11** percent from two inseminations in 19****. Glembockii and Vasiljev (I9M*) inseminated Precoce ewes within the first 2** hours of estrus, with the semen of Precoce rams, and if estrus lasted more than 2*f hours ewes were inseminated a second time, with the semen of Karakul rams. The percent lamb crop for 2*4-6 ewes inseminated once was 13*4-.5 percent. The lambs born to 153 out of 190 ewes inseminated twice were of the Precoce type (8 0 .5 percent). lambing percent for these ewes was 135*3 percent, indicating that The -45- 8 0 *5 percent of these ewes conceived from the first insemination. The insemination after 24 hours was effective for the remaining 37 of the 190 ewes that delivered nine Precoce and 52 Karakul x Precoce lambs for a 164.0 percent lamb crop. The nine Precoce were the result of a protracted heat and probably twins to a crossbred. Gutierrez (1948), after analyzing the results from insemination of 38,000 ewes in Uruguay, reported that two inseminations per estrus increased conception rates by 10 percent. Aamdal and Hogset (1955) reported that 40 days after insemina tion 22 of 64 ewes inseminated once with yolk citrate diluted ram semen were pregnant and four of eight ewes were pregnant after insem inating twice. None of 19 ewes became pregnant from one insemination but five of 10 were pregnant after two inseminations with ram semen diluted in yolk glycine. The data of Lopyrin et al. (1957) from 1,000 double insemina tions indicated that 80.2 percent of the ewes conceived when the two inseminations were eight hours apart and 72 percent conceived when the inseminations were 24 hours apart. The lambing percentages were 1 2 7 .8 percent from the eight hour interval and 124.4 percent from the 24 hour interval. -46- M. Vasectomized Mating The question of what effect a vasectomized check ram may have on conception from subsequent inseminations has been considered by Dauzier et al. (195*0 • They found no difference in conception when ewes were and were not mated to a vasectomized ram immediately prior to insemination. Echenique et al. (1941) found the seminal fluids of vasectomized rams to be a more satisfactory diluent than several common diluents tested. The semen of vasectomized bulls was equally effective in diluting ram semen. Larrea (1944) also found the most satisfactory diluent to be the ejaculate of a vasectomized ram. Vasectomized rams have been used to induce earlier breeding in ewes going from anestrus to estrus. Coleman (1950) found with 180 ewes that having vasectomized Merino rams running with one of two groups of ewes for three weeks before mating resulted in earlier lambing and a higher lambing per cent than in the control group. Coleman (1951a, b) used the same ewes previously reported but reversed the ewes to each treatment. Vasectomized rams were run with the experimental groups for 26 days prior to a six week mating period. During the first two weeks of lambing 59 lambs were born in the teased group versus 29 in the unteased group. The total number of lambs produced was also higher in the teased group. -1*7- Radford and Watson (1957) observed that estrus occurred approxi mately one week earlier in anestrus ewes run with vasectomized rams before the breeding season. In at least 80 percent of the ewes estrus was accompanied by massive desquamation of the vaginal epithelium and it was preceded by one or more periods of massive desquamat ion. Pitkjanen (1958 a/b) used uterine fistulas to discover that sperm reached the tubo uterine junction of the sow much more rapdily if natural mating occurred than when the sows were artificially in seminated. sented. Evidence of hastened ovulation due to mating was pre Ninety-five percent of the sows natural mated had ovulated 39 hours after the beginning of estrus whereas 58 percent of the unmated sows had ovulated by that time. Marion et al. (1950) observed that sterile copulation in the cow caused ovulation to occur significantly earlier than in unmated controls. This suggested an earlier release of gonadotrophin as a result of increased oxytocin secretion. This matter was discussed by Hansel (1958) and Hansel et al. (1958). IV. MATERIALS AND METHODS General Procedures Collections: Ram semen was collected with either an artificial vagina or by means of electrical stimulation using a Production Products model 2500 stimulator. collection. Rams were restrained on their side for electrical All semen was ejaculated into graduated test tubes pro tected by a 39° G. water jacket for both types of collection. When the artificial vagina was used rams were excited by either a ewe in natural estrus or a ewe in which estrus had been induced with subcutaneous injection of two to three mg. estradiol cylopentol propionate. The rams were allowed one or two false mounts with a four to five minute restraining period before each collection. It was observed that volume of the ejaculate was increased by this excitation procedure. Dilution: All samples were diluted Initially after collection and then again after cooling to 5° G. Split ejaculates were needed for most of the experiments, therefore, in order to prevent temperature shock, all initial dilutions were performed immediately after collection in a 35° G. room. ator. The semen was cooled slowly to 5° G. in a refriger Samples were then transferred to a 5° C, walk-in cooler where the second dilution occurred. When glycerol and arabinose were to be added, they were contained entirely in the second dilution fraction. The second fraction was added to the initially diluted semen in three equal portions at five minute intervals. Unless otherwise - ^9“ specified all sauries ■were equilibrated approximately 1 ^ hours before freezing. Freezing: All samples were frozen as a one ml, volume in ff^rost T" heat-sealed plastic ampoules. Freezing was accomplished in the laboratory of the Michigan Artificial Breeders Cooperative. The samples to be frozen were suspended in a bath of ethyl alcohol at a temperature of 5° C . The temperature of the bath was further lowered by adding small pieces of dry ice to the alcohol so that it dropped 1 ° per minute from 5° to -10 ° C . then 2 ° per minute from -10 ° to -18 ° C ., per minute from -18 ° to -35 ° C . and 5 ° per minute from -35° to -75° C. The temperature drop was con trolled by manually following a graph of this rate with a poten tiometer which recorded the temperature of the alcohol bath and of the semen every b.5 seconds. After freezing, the samples were stored under dry ice and isopropyl alcohol in a "Head Styrofoam" semen storage chest. The storage temperature varied between -77° aud -8 l° C. Thawing Procedure: All samples were thawed in 6 ° to 8 ° C. water. were performed as soon as the semen was liquid. Inseminations For motility studies all samples were held at 5 ° C. °ne hour then transferred to a 39 ° C. incubation room and held there for 15 minutes before microscopic examination. -50- Microscopic Examination Procedures: Slides were prepared in the 39° C. incubation room by adding a small loop of semen to a drop of saline and then cover slipping. The slides were examined on a heated microscope stage at approximately 1j-0° C. Sperm cells were counted at 210x magnifica tion using a Bausch and Lomb binocular phase contrast microscope. Two slides were counted per ampoule of semen and at least two ampoules were examined for each recorded observation in the experi ments reported. The counting procedure consisted of: (l) diluting initially in the warm room with saline so that 10 to 30 cells were visable in the microscope field at 210x magnification. 100 cells were counted per slide. A total of approximately The number of cells exhibiting forward progressive motility, those motile but not progressively motile and non-motile cells were all recorded using a differential counter. The percent motile cells and the percent progressively motile cells were determined from these counts. Motility examination of semen after collection and dilution was performed in the same manner. Concentration determinations were obtained from the freshly collected undiluted ejaculate according to the procedure of Laing (1955)* More recently the spermatocrit method of Hickman (1958) was used since it was much more rapid. -51- Materials and Their Preparation Glycerol: Glucose: Arabinose: Urea: Cevco U.S.P. grade glycerol Nutritional Biochemicals Anhydrous Dextrose B.L. arabinose from Nutritional Bio chemicals Merck reagent grade urea Egg Yolk: Egg yolk was obtained by washing the intact yolk in distilled water and blotting dry. The yolk membrane was ruptured and the yolk poured out. Only eggs of less than one week of age were used. Sodium citrate: A 2.7 percent (dry formula weight) solution of sodium citrate was maintained for foimulating the yolk citrate extender. The sodium citrate was of the formula ^ 305 ^ 0 7 .2 H2 O and U.S.P. grade from Mallinckrodt Chemical. Milk Extender: The milk extender was prepared in two parts. Part A - The homogenized pasteurized milk used as an extender was obtained from Michigan Artificial Breeders Cooperative after it had been heated to 208° F . for ten minutes. The average solid content was 1 2 .0 5 percent before heating and 12.19 percent after heating. ficant difference. This incidentally was a signi The fat content of the milk averaged 3-^ percent before heating and 3*^5 percent after heating. milk was less than four days old when used. The Five hundred oxford units penicillin and 500 micrograms of dihydro streptomycin were added per ml. -52- Part B - of the milk extender consisted of 1 ^ ml. of glycerol and 2 .2 5 grams arabinose raised to 100 ml. volume with the milk described previously. Egg Yolk Citrate Extender: The egg yolk citrate extender used for freezing was prepared in two parts: Part A - which was used for the initial dilution, consisted of 25 ml. egg yolk added to 75 ml. of a 2 .7 percent sodium citrate solution. Part B - consisted of 25 ml. egg yolk, 2.25 gm. arabinose and 1 ^ ml. of glycerol all raised to a 100 ml. volume with 2 .7 per cent sodium citrate. Parts A and B were mixed in equal portions after the semen was initially added to A and cooled to 5° C. Three mixings at five minute intervals were used. Bussian-Ukranian Extender: The formula for the Russian-Ukranian yolk-glucose-urea extender was obtained from Jack Judy of Ohio State University. Two separate dilutions were necessary. The initial dilution was made using 50 ml. distilled water, 10 ml. egg yolk, l.lj- gm. sodium citrate and 0 A gm. glucose. The glycerolized fraction of the diluter was as follows: 8 ml. of % sodium citrate solution 2 ml. of 2$ urea solution 2 ml. of egg yolk 1 .8 ml. of glycerol Equal volumes of each of the two fractions were used. Glyceroli- zation was accomplished by adding the glycerolized fraction in -53- three equal volumes at five minute intervals, under refriger ation at all times. -54- V. RESULTS AM) DISCUSSION A. The Glycerol Requirement for Freezing Ram Semen* This study ■was initiated to more precisely determine the amount of glycerol required for the greatest motility of ram sperm after freezing. Three ejaculates were collected and processed on each of two Hampshire rams according to the previously described procedure. Data pertaining to these ejaculates is presented in Table 1. Table 1. Ram Ejaculates Used in Glycerol Level Experiment__________ Ejaculate Pope Rayl Volume (ml.) Concentration Percent (billion/ml.) Progressive Motility Percent Motile Cells 1 1 .0 3*8 60 80 2 1 .0 4.8 54 70 3 1 .0 3*4 44 80 1 0 .8 4.4 45 70 2 1.5 4.2 33 58 3 0 .8 2.7 45 75 Each ejaculate was split into treatment groups of 5.5, 6 , 6 .5 , 7 3 7 *5 * 8 8 *5 percent glycerol. The samples were diluted 1:2 0 0 and frozen. Approximately three vials from each ejaculate were examined for motility and progressive motility, 10 months after the semen was frozen. Samples were examined *A11 glycerol levels referred to here are a volume per volume basis. -55- according to procedures previously described. constituted one observation. Each vial examined The data may be seen in Appendix Table A. A preliminary analysis indicated no significant interaction between ejaculates and glycerol levels as reported in Table 2 . Table 2. A Test of Interaction Between Glycerol Levels and Ejaculates Source df ss ms Glycerol level x ejaculate 35 3,3k2.8 95.5 108 8 ,7 3 6 .8 80.9 Error E 1 .1 8 The mean square of interaction of glycerol level x ejaculate con tains some of the main effects of glycerol level and ejaculate. How ever, since this mean square was not significantly larger than the error mean square, the mean square for interaction alone could not be significant. Least square equations were set up tomeasure the effect of glycerol levels, rams and days. This type of analysis was considered necessary because a disproportionate number of vials were available on each treatment. The procedure for setting up and solving the equations was given by Snedecor (195& section 12.17). The estimates of the main effect were obtained with the aid of the electronic computer ’MISTIC1 using program M-13. An analysis of the treatment effects (Table 3) indicated a signi ficant difference among glycerol levels (F<-05)• -56- Table 3* Variance Due to Glycerol Levels Source df Glycerol levels Error ss F ms 7 1 ,8 5 3 .6 1 264.80 136 1 6 ,9 8 7 .6 8 124.91 2 .12* *Significant (P<.05) A student range test indicated that the motility means for glycerol levels of 6 , 6 .5 ., 7 , 7 *5 ., 8 and 8 .5 percent were significantly greater than the motility means for 5 or 5*5 percent glycerol (P<*05). The mean motility after freezing of the 8*5 percent level was signifi cantly greater than the mean motility of five percent glycerol (P{.01) • The mean percent motility for each glycerol level is shown in Table if. Table if* Percent Pam Sperm Motility after Freezing in Various Levels of Glycerol Glycerol level Percent Motility after Freezing % 5 .5$ €$ 6 .55i 3 6 .05 * 39.12* In . 76 11-5.17 7$ b2.73 7-5% b2.35 8# 8.5# ^5 .1 8 it-7.o6 *Significantly less motility than other treatments (P<.05) When this experiment was initiated it was anticipated that the optimum level of glycerol would be somewhere between the extremes of five percent and 8*5 percent. However, these data indicate the optimum was at least 8.5 percent or higher. The results of First et al* (1957)^ Hill et al* (1 959 ) and the experiment in this paper concerning yolk glycerol combinations, indicated that Glycerol levels of 10 to 15 percent were definitely harmful to ram sperm. -57- The results of Szumowski et al. (1956 ), however, indicate no harmful effect from glycerol levels as high as 16 percent. Hill et al. (1959) found an interaction beteween rams and glycerol levels. No interaction was noted between rams and glycerol levels as indicated by the F value of 1.18 in Table 5 (P<.05). However, this is not to say that such an interaction might not be expected with more rams or different rams. Ample evidence for the reality of such treatment sire interactions have been established for frozen bull semen by White et al. (195*0, Odell and Hurst (195^) and Hendrikse and Joling (1957)• Table 5» A Test of Interaction Between Bams and Glycerol Levels______ Source Glycerol Level x Bams Error df ss 7 1,107 158.14 136 18,291 134.49 ms F 1 .1 8 In drawing a conclusion'from research to date, as to the glycerol level to be used in freezing ram semen, it would seem that satisfactory motility or survival after freezing can be obtained with levels from six to nine percent and that the precise optimum level is as yet undetermined. -58- B. The Addition of Egg Yolk to a Milk Diluter and its Effect on ; Glycerol Requirements___________________ This study was initiated to find out if added phospholipid from egg yolk would improve temperature shock protection provided "by a heated whole milk diluter and subsequently improve freezing survival • The data of Sikes and Merilan (1958 ) indicated that the motility of unfrozen sperm was maintained longer in a skimmilk diluter if 10 percent egg yolk were added, Hendrikse and Joling (19 57 ) observed that the motility of bull sperm was maintained better in skimmilk with five percent egg yolk added than with 10 or 15 per cent, Fourteen thousand inseminations indicated that a higher con ception was obtained from the yolk plus milk diluent after storage than from milk diluent without added egg yolk. This study was also designed to determine what combination of egg yolk and glycerol might be the most effective in terms of ram sperm survival after freezing. This was considered necessary, since Amann and Almquist (1957)> reported that increased solids in a skimmilk diluter increased the glycerol need of frozen bull semen. The egg yolk addition obviously would increase the total solid con tent of the milk diluter. Also, Saroff and Mixner (1955) found that as the level of egg yolk in a yolk citrate extender was increased, correspondingly higher levels of glycerol were required for greater sperm survival. An experiment was designed to study the effect of the addition of egg yolk to a milk diluter on motility and glycerol requirements -59- using the pooled ejaculates of two Hampshire rams. The pooled ejaculates were initially split into six egg yolk groups. The semen of group one was diluted with the milk diluter previously mentioned. Each subsequent group was diluted with the same diluter with egg yolk added. The diluter for group two contained three per cent egg yolk; group three, six percent egg yolk; group four, 12 percent egg yolk and group five, 2k percent egg yolk. All collections, handling, freezing and examination procedures were those previously mentioned except that the warm diluted semen was purposely cooled 3 ° C. per minute to 5 ° C. and the freezing rate was 3° C. per minute to -15° C. and 5° C. per minute to -75° C. These modificiations were an attempt to insure that some temper ature shock might occur. At 5° each group was further split into four sub groups of 6 , 9, 12 and 15 percent glycerol. The final dilution rate was 1:200 and samples were equilibrated one hour before freezing. The semen quality and concentration for each trial are reported in Table 6 . Frozen samples were thawed at 5° C., warmed and examined. Two slides per vial were used for one recorded observation. The data are expressed as percent survival one week after freezing. The data pertaining to the two trials are in Appendix Table B. -6o- Table 6 . Ejaculates Used in the Yolk Glycerol Experiment Earn Volume (ml.) Concent ration (billion/ml.) Progressive Motility ») .8 3.2 3.0 70 65 80 80 3.1 67 80 2 .8 3.1 53 ^7 68 2.95 50 70 Trial I Pope Eayl .7 Pooled ejaculate Average .75 Trial II Pope Eayl .7 .2 Pooled ejaculate Average mk-5 Motile Cells (« 72 The average percent sperm survival from the combination of egg yolk and glycerol in the milk diluter are presented in Table 7. Table 7* Average Percent Survival of Speim Frozen in a Milk Diluter with Combinations of Egg Yolk and Glycerol 0 3 6 12 2k 6 71 65 71 55 62 65 ** 9 85 66 77 67 36 56** 12 h9 30 25 15 7 25 * 15 1 2 0 0 1 Average 51 iA 3^ 26 Percent Yolk Average Percent Glycerol 2 in **The mean is significantly greater than the two lowest means *The mean is significantly greater than the lowest mean(P<.05) -6l- The nature of the variance in this experiment is illustrated in Table 8 * Table 8 , Analysis of Variance on Combinations of Yolk and Glycerol Source df Total 79 8 2 ,1 1 2 .8 Yolk levels k Glycerol levels Sum of Squares Mean Square F 5 ,8 7 2 .7 1 ,^6 8 .2 0 1.77 3 6 0 ,9 9 8 .3 2 0 ,3 32.80 2^5** Yolk x Glycerol Int. 12 9 ,9 7 7 .0 831.^8 9.32** Trials 1 1 .5 Error 59 5 ,2 6 2 .5 1.5 0 .2 8 9 .1 9 **Highly significant (P^.0 1 ) The F value of 2 k indicates that a highly significant differ ence existed between the glycerol levels. A student range test was applied to test differences among the glycerol level means, using the highly significant interaction mean square as the error mean square. The means which were significantly different are shown in Table 7* Significantly more sperm survived freezing with glycerol levels of 6 , 9 and 12 percent than survived when frozen in 15 percent glycerol (P^.0 5 ). The means for either six or nine percent glycerol were signifi cantly greater than the sperm survival at 12 or 15 percent glycerol (PC.Ol) . The apparent reduction in freezing survival of ram sperm due to high levels of glycerol support the findings of First et al. (1957) -62- and Hill et al. (1959) • The rapid reduction in ram sperm survival “with glycerol levels above 10 percent certainly need further investi gation, It is recognized that bull sperm tolerate glycerol levels much higher than 10 percent even though the optimum for most bull semen diluters Is between seven and 12 percent (Smith and Polge, 1950> Polge and Rowson, 1953 > Emmens and Blackshaw, 1956 ). Glycerol has been shown to have a damaging effect on fertility of fowl sperm (Polge, 1951) "but this was prevented if the glycerol was removed by dialysis before insemination, Polge (195 6 ) observed that boar sperm do not tolerate glycerol levels as high as ten per cent, Polge and Rowson (1952a) found that glycerol does not reduce fertility of bull semen. This has been supported by the practical field application of glycerol in freezing bull semen with subsequent successful conceptions reported in studies around the world. However, this does not mean that glycerol may not reduce fertility when added to the semen of other species. The highly significant interaction of glycerol and egg yolk shown in Table 8 when the data of six and 15 percent glycerol are considered was the result of relatively little change in sperm survival as yolk increased. While survival definitely decreased as yolk in creased when the percent glycerol was either nine or 1 2 . These data do not support the reports of Amann and Almquist (1957) and Saroff and Mixner (1955)> wherein higher levels of solids -63- or of egg yolk increase the glycerol required for best freezing survival of bull sperm* It is in agreement with Hendrikse and Joling (1957) "Who found greater motility of bull sperm exhibited by lower levels of egg yolk as compared with 10 to 15 percent. The analysis of variance shown in Table 8 also indicates that no significant difference occurred among egg yolk levels. The lack of significance is partially caused by the great amount of varia tion due to a glycerol yolk interaction. Sikes and Merilan (1958) &ad shown unfrozen sperm were main tained longer by the addition of egg yolk to a skimmilk diluter. The data of this experiment while not significant, suggest a depressing effect on ram sperm survival as egg yolk is increased in the milk diluter. -61j~ _ C. Frozen and Unfrozen Ram Semen Equilibrated -with Glycerol for One and 18 Hours and Incubated at 39° smd 5° Centigrade___________________ This experiment was designed to answer the following questions: (1) would thawed frozen semen lose motility and die more rapidly than unfrozen when "both were stored at the conventional 5° C. temperature? (2) could such a difference be expected to occur at 39° C., the approximate temperature to which semen would be exposed after insem ination in the ewe? (3 ) was an 18 hour glycerol equilibration period necessary before freezing the semen? or (^) would the additional time involved in the 18 hour glycerol equilibration actually reduce the expected life of frozen or unfrozen sperm at either or both of the incubation temperatures? In order that these questions might be answered, a 2x2x2 factorial experiment was utilized. It consisted of two replications of eight treatments each entailing all of the possible combinations of the following factors: fresh vs. frozen semen, one hour vs 18 hours equili bration time and incubation temperatures of 39° vs. 5° C. The semen used for each trial was obtained by pooling ejaculates of two Hampshire rams. The pooled ejaculates for trial one had a concentration of 3 *7 billion cells per ml., 65 percent of these cells were progressively motile and 85 percent were motile. The semen used for trial two had a concentration of 3 .3 billion cells per ml. with 63 percent of these cells progressively motile and 77 percent motile. A complete split ejaculate technique was utilized in dividing and diluting the various treatments. -65- The final dilution rate of all samples was 1:300. The samples were examined at 0, 2, k , 8, 16, '$2 , 6k , 96 and 120 hours after freezing. The zero time started immediately after the frozen samples were thawed. after freezing. Frozen samples were thawed immediately The procedure was the same for unfrozen samples except that they were held at 5° C. The results of this experiment are shown in Appendix Table C. Figures 1 and 2 illustrate the motility decline of ram sperm of each treatment as time progressed. Semen samples incubated at 39° C. lost motility rapidly and were nearly immotile at the-end of eight hours. Because the eight hour examination was the last period when sperm in all treatments exhibited motility, the eight hour examination values were used in determining differences bet ween the treatments. Semen samples incubated at 5° 0. were again analyzed after 120 hours. The F tables for these two analysis are shown in Tables 9 a^d 10 . For semen incubated eight hours, the F value of 37*922 for variance due to treatments was highly significant which indicates that at least one of the main effects or interactions differed from zero. The only significant main effect was B, the effect of 39° C. incubation. The F value of 257.9 was highly significant. The F test at 120 hours where only the 5° C. incubated samples were considered, indicated a highly significant difference between the two trials but no difference among treatments. -66- o CM Figure 1. Motility Decline of Unfrozen and Frozen Ram Semen Equilibrated for One or 18 Hours at 5° C. r-H VO ON a o •H cd Pi 5 r—I a1 © CO Pi 2 O -=tVO CO ao H FJ o EH Pi © CH cd lf\ -P ■o§ f5 (0 2 Pi ir\ O O ir\ © N O Pi £4 O CM W on vo rH CO -=dCM O o < o O CO o c— o VO o ITN tausds Q lTq.o^ O -=}- Pi ■cs d •H -P •H fctO o < o on -67- Figure 2. 80 * Motility Decline of Unfrozen and Frozen Ram Semen Equilibrated for One or 18 lours at 39° C. * . Sperm 60 Percent Motile 70 50 * x ko 30 20 10 0 Hours B X X XAB BC ......... ABC — — — — • 2 h 6 8 10 Unfrozen 39° C. incubation one hr. equilibration Frozen 39° C . incubation one hour equilibration Unfrozen 39° C. incubation 18 hours equilibration Frozen 39° C. incubation 18 hours equilibration -68Table 9 . Variance in the Motility of Frozen and Unfrozen Ram Semen Incubated Eight Hours at 39° and 5° C. and Equilibrated _________ with Glycerol for One or 18 Hours_______________________ Source df ss Total 15 1 8 ,1 1 0 .9 F ms Replicate 1 5 2 .5 2 52.52 Treatment T 17,594.42 2,513.48 0.79 37.92** A 1 1 6 2 .5 6 1 6 2 .5 6 B 1 17,095.56 17,095.56 AB 1 45.56 45.56 0 .6 9 C 1 1 5 0 .0 6 15 0 .0 6 2 .2 6 AC 1 14.06 14 .06 0 .2 1 BC 1 1 2 6 .5 6 1 2 6 .5 6 1.91 ABC 1 0 .0 6 0 .0 6 0 .0 0 463.96 6 6 .2 8 Error 7 2.45 257.93** **Highly significant (P<.01) Table 10. Variance in the Motility of Frozen and Unfrozen Ram Semen Incubated 120 Hours at 5 ° C. and Equilibrated with Glycerol for One or 18 Hours ' ss Source df Total 7 4 ,9 8 2 .0 Trials 1 4 ,5 1 2 .5 Treatment s 3 231 ms ^,512.5 77.0 A 1 1 8 0 .5 180.5 C 1 0 .5 0.5 AC 1 5 0 .0 5 0 .0 2 3 8 .5 79.5 Error 3 **Highly significant (P<.Ol) F 56.760** O .968 2.27 0 .1 O .63 -69- It can, therefore, he concluded that on the basis of this experiment significantly more frozen and unfrozen sperm remained motile after eight hours storage at 5° C. than at 39° C. There ■was no significant difference in motility between thawed frozen semen and unfrozen semen of the same age after both were stored at 39 ° C • or 5 ° C, for eight hours• No interactions occurred between equilibration time, eight hour storage temperatures and unfrozen versus frozen semen. The lack of significance due to the main effect A at 120 hours indicates that there was no significant difference between the motil ities of thawed frozen semen and unfrozen semen from the same ejacu late. There was no significant difference between glycerol equilibration periods of one or 18 hours before freezing as indicated by the AC interaction effect and main effect A. It can be noted from the data that a motility of 58 percent was maintained by one hour equilibrated frozen samples of trial two which had been thawed for 2^0 hours. Frozen and unfrozen samples equilibrated for 18 hours each had an average of 57 percent motile cells after 2^0 hours incubation at 5 ° C. From these data it would seem that frozen ram semen could not be expected to lose motility more rapidly after thawing than unfrozen semen. This fact appears to be true at the 39° C., simulated uterine temperature, as well as at 5 ° C. storage. It was evident that ram sperm lost motility more rapidly at 39 o C. than at 5° C. -70- The studies of Anderson (19^1), Kelly (19 ^-2 ), Carbonerro (1955) and Sinclair (1957) indicated that the majority of ewes bred 12 hours after the beginning of estrus conceived. The studies of McKenzie and Terrill (1937) > Polovceva et al. (1938) and Lopyrin and Loginova (1939) indicated that ovulation occurred approxi mately 30 hours after the onset of estrus. It seems evident that ram sperm must live longer than eight hours in the female tract. The fact that a high percent of motile cells remained in frozen and unfrozen semen after 10 days of 5 ° 0 . storage with a glycerolated milk diluter suggested that long time storage of unfrozen ram sperm may be possible. The data suggest that one hour glycerol equilibration was as satisfactory as 18 hour equilibration. This is in agreement with the ram semen data of Hill et al. (1959) and the bull semen equili bration data of Odell and Almquist (1957), Blackshaw (1955a) and Graham et al. (1957)* The ram semen data of Szumowski et al. (195&) and the bull semen data of Polge (1952a), Stower (1953), and Saroff and Mixner (1955) do not agree with these results. “71- D. Extender Toxicity at 39° C. After observing the rapid motility decline of ram sperm at 39° 0 ., "the question arose as to a possible extender toxicity at 39° 0. which was not evident at 5° 0* The experiment designed to test the possibility of such an extender toxicity at warm temperatures consisted of one ejaculate each from three Hampshire rams, split equally among eight treatments with incubation at 5 ° or 39° C. Motility determinations were made at zero and eight hours after the beginning of incubation. Data pertaining to the quality of the three ejaculates are presented in Table 1 1 . Earn Volume (ml.) Concent rat ion (billion/ml.) Progressive Motility m Motile Cells (*) Pope .8 2 .8 75 86 Rayl .6 2 .2 78 90 Pope lamb .6 2 .0 85 92 Average .67 2.5 79 89 Collection, cooling and handling procedures were those previously described. Each ejaculate was initially split three ways. The three groups being ram semen diluted with milk, yolk citrate and undiluted. -72- At 5° C., second dilution fractions were added to the milk diluted semen and the treatments were: milk only, milk plus 7 percent glycerol, milk plus li}- percent glycerol, milk plus 1 .2 5 percent arabinose and milk plus 7 percent glycerol and 1 .2 5 per cent arabinose. The yolk citrate diluted semen was split into two treatment groups; yolk citrate and yolk citrate plus 7 percent glycerol and 1 .2 5 percent arabinose. Semen samples from each treatment were then divided and incu bated at 5° or 39° C. ginning of incubation* Zero time for all diluted samples was the be Zero time for the undiluted samples was determined when they entered 39 ° C. incubation immediately after collection. All samples were diluted 1:250. Two slides on two vials of each ejaculate for each treatment were examined at zero hours and again after eight hours incubation at the two temperatures. The average of the values for the two vials was used as an observation. The data pertaining to this experiment are presented in Appendix Table D. -73- The criterium used to test differences in this experiment was percent eight hour survival. An analysis of the variance in these data is shown in Table 12 . Table 12. Variance in the Extender Toxicity Study Source df Total kj 55,117 Treatments 7 2 2 ,9 0 6 3,272.3 4.02* Conditions (5 ° versus 39 ° C.) 1 2,552 2 ,5 5 2 .0 3.13 Treatment x conditions 7 3 ,6 0 6 515.0 0.63 32 26,053 814.2 Error ss F ms *Highly significant Table 13 presents the average eight hour percent survival for each treatment and incubation temperature. The F value for treatments of 4.02 indicates that a highly signi ficant difference occurred among treatment means. A student range test was used to determine the significance of the difference between the means. -7*1— Table 13* Percent Eight Hour Survival of Earn Sperm Diluted in Constituents of Two Diluters and Incubated at 5° C. or 39° C. Treatment 39° C. Incubation conditions 5 ° c. Average Undiluted ejaculate 28 0 llj-.O Milk hj 62 5*w5 Milk plus Tfo glycerol k2 66 5^.0 Milk plus 1 31 59 1*5. 0 Milk plus 1,25$ arabinose 39 53 1*6 . 0 Milk plus 7$ glycerol plus 1 .25 $ arabinose 31 62 1*6.5 Yolk citrate 68 88 78.0 Yolk citrate plus Tf° glycerol plus 1 .25 # arabinose 85 99 92.0 Average glycerol k6.l* 6 1 .1 The student range test of treatment means show in Table \k, indicate that significantly more diluted sperm survived incubation for eight hours than did undiluted sperm in the incubated undiluted ejaculate (p<*05) • The greatest survival following incubation was achieved by ram sperm diluted in yolk citrate plus glycerol and arabinose. -75- © I K Id rH “ P dO H © p •H rpH»
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