---------------------------------------- -------------------------- ,,transcriber',s ,,notes3 99 ,transcription used in this e-text3 text indicates italics2 text indicates bold-face text2 text indicates superscript text2 text indicates subscript text4 99 ,lettered footnotes 7with anchors ,a1 ,b1 etc471 explaining the text1 have been moved to directly below the paragraph or table they refer to4 ,footnotes with anchors #a1 #b1 etc4 refer to references1 that are listed towards the end of the text4 99 ,the original text has not been modified1 except that some minor typographical errors have been corrected silently4 ---------------------------------------- -------------------------- ,,preservation ,,of ,,bull ,,semen ,,at ,,sub-,,zero ,,temperatures ,by ,n4 ,l4 ,van,demark ,w4 ,j4 ,miller ,w4 ,c4 ,kinney1 ,jr4 ,carlos ,rodriguez ,m4 ,e4 ,friedman ,bulletin #fba ,,university ,,of ,,illinois ,,agricultural ,,experiment ,,station ,,contents ,page ,,early ,,work ,,on ,,freezing ,,semen #e ,,selection ,,of ,,semen ,,for ,,freezing #f ,predicting freezability #f ,freezability of first and second ejaculates #g ,freezability of several consecutive ejaculates #g ,freezability of epididymal sperm #i ,freezability of washed sperm #aj ,,extenders ,,for ,,freezing ,,bull ,,semen #aj ,proportion of egg yolk in final diluent #aj ,citrate level in final diluent #aa ,storing and freezing diluent #ab ,other diluents #ac ,,dilution ,,rates #ad ,effect of further dilution and refreezing #ad ,,glycerol ,,additions #ag ,effect of glycerol on sperm survival at #e deg4 ,c4 #ag ,glycerol levels for freezing semen #ah ,rate1 temperature1 and method of adding glycerol #ah ,allowing sperm to equilibrate with glycerol #bj ,sugar additions and equilibration time #bj ,substitutes for glycerol #bb ,,freezing ,,rate #bb ,effect of freezing rate on sperm survival #bb ,rate of cooling in plastic and in glass #bc ,,storage ,,temperature #bd ,storage at -#bc deg4 to -#gi deg4 ,c4 #bd ,use of higher glycerol levels and storage at -#bj deg4 ,c4 #be ,,thawing #bf ,comparison of thawing at #e deg4 ,c4 and at #ch deg4 ,c4 #bg ,thawing rate in plastic and in glass #bi ,,effects ,,of ,,freezing ,,procedures ,,on ,,metabolic ,,activity ,,of ,,sperm #cj ,effect of glycerol additions on oxygen uptake of diluted semen at #cg deg4 ,c4 #cj ,effect of glycerol-plus-catalase on oxygen uptake of diluted semen #cb ,effect of freezing procedures on oxygen utilization #cc ,effect of freezing procedures on methylene-blue reduction time #cc ,,practical ,,freezing ,,procedure #ce ,collection of semen #ce ,preparation of extender #ce ,dilution after collection #cf ,adding the glycerol #cf ,equilibration #cf ,freezing #cg ,thawing #cg ,,literature ,,cited #ch ,,temperature ,,conversion ,,table #ci ,urbana1 ,illinois ,october1 #aieg ,publications in the ,bulletin series report the results of investigations made or sponsored by the ,experiment ,station ,,acknowledgment ,the authors gratefully acknowledge the financial assistance given to the ,department of ,dairy ,science in support of these investigations by the ,southern ,illinois ,breeding ,association of ,breese1 ,illinois1 and the ,northern ,illinois ,breeding ,co-op of ,hampshire1 ,illinois1 through the ,illinois ,dairy ,breeding ,federation4 ,,the ,,preservation ,,of ,,bull ,,semen ,,at ,,sub-,,zero ,,temperatures ,by ,n4 ,l4 ,,vandemark1 ,w4 ,j4 ,,miller1 ,w4 ,c4 ,,kinney1 ,,jr41 ,,carlos ,,rodriguez1 and ,m4 ,e4 ,,friedman,a ,a ,this publication was prepared by ,n4 ,l4 ,,vandemark1 ,professor of ,dairy ,physiology4 ,research reported herein was carried out cooperatively by the senior author and ,w4 ,j4 ,,miller1 ,w4 ,c4 ,,kinney1 ,,jr41 ,,carlos ,,rodriguez1 and ,m4 ,e4 ,,friedman1 formerly members of the ,department of ,dairy ,science4 ,the first announcement in #aiea of the successful impregnation of a cow with bull semen that had been frozen stimulated much interest and research in freezing as a method of preserving bull semen4 ,research during the years following #aiea resulted in considerable progress and success in using freezing as a means of holding semen for long periods of time without loss of fertility4 ,between #aieb and #aieg1 research on many aspects of the preservation of bull semen at sub-zero temperatures was carried out in the ,department of ,dairy ,science at the ,university of ,illinois4 ,many of these investigations have aided in perfecting the freezing technique that has been adapted for practical use4 ,some of these findings have been published1 but many items have gone unreported except for general references at scientific meetings4 ,it is the purpose of this bulletin to bring together the results of several experiments carried out in connection with the freezing of bull semen and to present a practical freezing procedure based on the results of these experiments and findings at other institutions4 ,persons interested in the development of the procedures and the reasons why certain steps are necessary in freezing semen will find the details in the first part of this bulletin4 ,those interested only in the freezing procedure may turn to page #ce where a practical method of freezing semen is described4 ,,early ,,work ,,on ,,freezing ,,semen ,while it has been known for a long time that various types of tissues and organisms can withstand freezing and are even preserved by freezing1 the first attempts at freezing sperm cells were made before the turn of the century4 ,in #ahig1 ,davenport#a;,b found that human sperm would withstand freezing4 ,for thirty to forty years after that1 little attention was paid to freezing as a possible means of preserving semen4 ,an excellent review of the early attempts to freeze sperm has been assembled by ,polge and ,parkes4#b ,these investigators also gave a good account of their work at the ,british ,national ,institute of ,medical ,research in ,london1 where in #aidi they demonstrated that glycerol would protect fowl sperm so that it would survive freezing4 ,the next year they found that bull sperm and the sperm of several other species were protected by glycerol during freezing4 ,during the same year1 ,emmens and ,blackshaw#c showed that ram and bull sperm would survive freezing4 ,in #aiea frozen semen was used to produce a calf in ,england and a lamb in ,australia4 ,b ,these numbers refer to literature citations on page #ch4 ,the highlights in the development of frozen semen have been covered by other reviews and reports4 ,interested persons will find the articles of ,polge and ,parkes#b and ,smith#d especially good on the early history and theoretical aspects of freezing sperm4 ,later progress on the freezing procedure has been reviewed and covered in a number of detailed reports4#e1f1g ,many items not covered in those articles have been assembled here4 ,,selection ,,of ,,semen ,,for ,,freezing ,one of the first considerations in freezing semen is that of deciding which semen samples are to be frozen4 ,since preservation of the semen--the maintenance of the potential motility and especially the fertility of the sperm--is the primary aim1 some attention should be directed to the kind of semen sample that will withstand freezing4 ,do the initial characteristics of the sample indicate whether the sperm will withstand freezing8 ,does maturity of the sperm affect their freezability8 ,predicting freezability4 ,estimates of semen quality in the past have been based in part on the numbers of sperm present in a fresh sample and on the percentage and rate of motility shown by the sperm4 ,these characteristics were used to determine the relationship between the original concentration of sperm 7in the fresh1 undiluted sample71 the percentage and rate of sperm motility in the diluted samples just prior to freezing1 and the percentage and rate of sperm motility following freezing and thawing4 ,from data collected before and after freezing and thawing #ed ejaculates1 it was found that there was not a significant correlation between the number of sperm present in the original sample and the percent of motile sperm present after freezing and thawing 7r #j4jc74 ,a highly significant correlation 7r #j4de7 was found1 however1 between the percentages before freezing and after thawing4 ,while this correlation coefficient was highly significant1 its magnitude indicates that only about one fifth of the variation in percentage of motile sperm observed after freezing was accounted for by the motility of the sperm prior to freezing4 ,freezability of first and second ejaculates4 ,in the early days of artificial breeding in this country1 it was commonly believed that a second ejaculate collected a few minutes after the first resulted in a larger ejaculate containing more sperm4 ,with the development of the procedure of stimulating sexual excitement by restraint prior to collecting semen1 this difference between first and second ejaculates has been greatly reduced4 ,still it was noted that second ejaculates frequently withstood freezing better than first ejaculates1 even though restraint and stimulation of the bull occurred prior to collection of the first ejaculate4 ,during the course of a number of experiments1 it was possible to compare the freezability of #b ejaculates that were collected a few minutes apart from the same bull4 ,two consecutive ejaculates were obtained one or more times from #bd bulls so that a total of #eh comparisons could be made4 ,the mean prefreezing and post-thawing percentages of motile sperm in first and second ejaculates are presented in ,table #a4 ,an analysis of variance showed that in this comparison the differences between first and second ejaculates in sperm survival during freezing were highly significant4 ,a later comparison of #bg first and second ejaculates from #bf bulls did not show as great a difference between first and second ejaculates in their ability to withstand freezing 7,table #a74 ,table #a4--;,comparison of the ,freezability of ,first and ,second ,ejaculates ,taken a ,few ,minutes ,apart ,from the ,same ,bull ,number ,number ,prefreezing ,post-thawing of of ,ejaculate motility motility ,survival bulls ejaculates 7percent7 7percent7 7percent7 ---------------------------------------- ----------------------------- #bd #eh #ast #fj #ci #fe #b;d #fb #de #gd #bf #bg #ast #fj #cf #fj #b;d #fe #dj #fa ---------------------------------------- ----------------------------- ,freezability of several consecutive ejaculates4 ,the fact that second ejaculates sometimes withstood freezing better than first ejaculates suggested that the maturity of the sperm might be a factor affecting freezability4 ,an opportunity to check this idea came when #bj consecutive ejaculates were collected from each of #f bulls within a #d-;hour period4 ,the sperm in samples collected in this manner might be expected to be less mature with each additional collection4 ,the results obtained in freezing several consecutive ejaculates are shown in ,figure #a as averages for the ejaculates from #f bulls4 ,in same instances1 there was an insufficient quantity of semen available to test the freezability4 7,procedure3 ,diluted to #cj x #ajf sperm per ml4 with #a3a yolk-citrate1 then cooled and glycerolated with an equal volume of #ad percent glycerol 7percent by volume7 in #b4i percent sodium citrate4 ,final sperm concentration1 #ae x #ajf4 ,equilibration time1 #ae hours4 ,freezing rate1 #b deg4 ,c4 per minute from #e deg4 to -#ai deg4 ,c4 then #d deg4 ,c4 per minute from -#ai deg4 to -#gi deg4 ,c4 ,held frozen for #e or more hours then thawed in water at #e deg4 ,c4 and checked for motility47 ,illustration3 ,percent of motile sperm before and after freezing consecutive ejaculates collected within a #d-;hour period from each of #f bulls 7,fig4 #a7 ,in general1 the motility before freezing improved slightly from the first to the fourth to sixth ejaculate and then declined until about the #abth or #adth ejaculate1 at which point the prefreezing motility seemed to level off through the #bjth ejaculate 7,fig4 #a74 ,the percentage of motile sperm found after freezing and thawing followed the same trend at an average level #aj to #ae percent lower than the prefreezing level4 ,as is readily seen from the trend lines in ,fig4 #a1 the difference between the prefreezing motility and the post-thawing motility increased gradually after about the fifth ejaculate4 ,although the absolute difference did not increase greatly1 the percentage of survival after freezing dropped from #ha percent on the first #e ejaculates to #bf4e percent on the last #e 7,table #b74 ,table #b4--;,comparison of the ,freezability of #bj ,consecutive ,ejaculates ,collected ,within a #d-;,hour ,period 7,weighted averages for #f bulls7 ,number ,prefreezing ,post-thawing ,ejaculate of motility motility ,survival ejaculates 7percent7 7percent7 7percent7 ---------------------------------------- --------------------------- #ast to #eth #bi #ec4c #dc4b #ha4j #fth to #ajth #bf #dc4h #cj4b #fi4j #aath to #aeth #bc #bh4f #ad4e #ej4g #afth to #bjth #ai #ah4a #d4h #bf4e ---------------------------------------- --------------------------- ,freezability of epididymal sperm4 ,since the freezability of bull semen seemed to be better in second than in first ejaculates and some improvement in freezability was evident through the first #d to #f ejaculates taken consecutively1 the question of whether epididymal sperm would withstand freezing seemed to be important4 ,although when #bj collections were made1 the later ejaculates no doubt contained fewer mature sperm1 the lowered freezability could have been due to accessory gland secretions rather than changes in the sperm themselves4 ,removing sperm directly from the epididymis would eliminate any effect that the accessory gland secretions could be exerting4 ,further1 if epididymal sperm could be frozen1 obtaining and using semen from a bull shortly after his death should be possible4 ,table #c4--;,freezability of ,sperm in ,samples ,taken ,from the ,epididymides ,prefreezing ,post-thawing ,bull motility motility ,survival 7percent7 7percent7 7percent7 ---------------------------------------- --------- #a #ej #dj #hj #b #dj #ac #cb #c #fj #ae #be #d #cj #ae #ej #ef #dj #be #fb ,average #dd #bb #ej ---------------------------------------- --------- ,to determine whether epididymal sperm would withstand freezing1 the #ab epididymides 7cauda only7 of #f slaughtered bulls were flushed with saline 7#j4i percent7 and the sperm obtained were frozen using the same procedure as was used with the #bj consecutive ejaculates discussed earlier4 ,averages of the #b epididymides from each bull are given in ,table #c2 the samples from bulls #e and #f were combined4 ,from the data in ,table #c1 it is obvious that motile sperm were present after freezing and thawing epididymal samples4 ,it is likely that further experience in handling epididymal sperm may lead to improved results4 ,using frozen epididymal sperm from #b bulls1 ,canadian workers have produced confirmed pregnancies in #h out of #ab cows4#h ,freezability of washed sperm4 ,in the laboratory it is frequently desirable to study sperm free of the seminal plasma in which they are ejaculated4 ,sperm can be separated from the seminal plasma by centrifugation1 removal of the supernatant plasma1 and resuspension in a salt solution of known composition4 ,sometimes it is desirable to repeat the process4 ,this tends to wash the sperm with the salt solution and sperm handled in this way are called washed sperm4 ,sperm cells centrifuged three times and washed twice in #j4i percent sodium chloride solution withstood freezing well when finally resuspended and frozen in yolk-citrate diluent4 ,the percentage of survival in three samples subjected to this treatment was #fj percent4 ,thus it appears that the seminal plasma itself is not essential for ejaculated sperm to survive the rigors of freezing4 ,this is not surprising1 since it had already been found that epididymal sperm1 which also are free of accessory gland secretions1 can withstand freezing and thawing4 ,,extenders ,,for ,,freezing ,,bull ,,semen ,both of the extenders that are widely used in routine storage of bull semen at #e deg4 ,c4 are used for freezing semen4 ,these are the egg yolk-sodium citrate and whole or skimmilk extenders4 ,most of the research with extenders for freezing bull semen in this laboratory has been done with the yolk-citrate diluents4 ,proportion of egg yolk in the final diluent4 ,some early experiences with a diluent consisting of one part yolk and one part #b4i percent sodium citrate dihydrate in distilled water showed poor sperm survival following freezing4 ,the final mixture with this diluent consisted of about #de percent yolk4 ,in other attempts at adding glycerol in order to freeze semen1 the final proportion of yolk was diminished and better sperm survival was obtained4 ,several experiments were carried out to test the effect of varying levels of egg yolk4 ,in the first efforts to find the optimum level of egg yolk1 the level of yolk in the final frozen mixture was varied from about #f to #df percent4 ,these levels were obtained by varying the proportion of yolk to #b4i percent citrate solution in the original extending media and also in the media added in glycerolating the samples4 ,split portions of #bj semen samples were frozen in each of the extender combinations indicated in ,table #d4 ,the mean percentages of motile sperm found before and after freezing and thawing are shown also4 ,the highest percentages were found with extenders containing #bc and #bd percent yolk4 ,the highest percentage of yolk1 resulting when a #a3a 7yolk to citrate7 extender was used for both extending and glycerolating1 proved to be most detrimental to sperm survival during freezing4 ,the lowest percentage of yolk used 7#f percent7 was not as effective in protecting sperm during freezing as the intermediate levels tested 7,fig4 #b74 ,table #d4--;,effect of ,egg-,yolk ,level in ,diluent on ,freezability of ,semen 7,average of #bj semen samples7 ,medium ,yolk in ,pre- ,post- ,sur- ---------------------------- final freezing thawing vival ,diluent ,extending ,glycerolating,c mix- motility motility 7per- ------------ ---------------- ture,d 7percent7 7percent7 cent7 yolk3citrate yolk3citrate 7percent7 ---------------------------------------- --------------------------------- #a #a3a #a3a #de4g #fe #e #h #b #a3a #a3c #cd4i #fd #bg #db #c #a3c #a3a #cc4f #fc #cc #eb #d #a3a #j3a #bd4b #fd #ci #fa #e #a3c #a3c #bb4h #fc #cg #ei #f #a3c #j3a #ab4a #ei #cc #ef #g #a3g #a3g #aa4d #ef #ce #fb #h #a3g #j3a #f4j #eb #bf #ej #i #a3ae #a3ae #e4g #di #be #ea ---------------------------------------- --------------------------------- ,c ,this mixture included #ad percent glycerol4 ,d ,the average initial sperm concentration was #ijj x #ajf_/ml4 ,sufficient extender was added to give #cj x #ajf_/ml4 at the first extension4 ,thus the final concentration was #ae x #ajf sperm_/ml4 after glycerolization4 ,since rather large changes in the percentages of yolk were used in this experiment1 two further trials were conducted in which #af1 #bd1 and #cb percent yolk in the final mixture were compared1 with the final citrate percentages held constant4 ,in these tests1 #af and #bd percent yolk maintained sperm better at all citrate levels tried than #cb percent yolk4 ,the #af percent level was slightly better at most of the levels of citrate tested 7,fig4 #c74 ,citrate level in the final diluent4 ,the early work of the ,british indicated that a final citrate level near #b percent in the diluent was satisfactory for freezing bull sperm4 ,later1 in a personal communication1 ,polge of the ,british group suggested that a citrate level of about #b4ce percent might be best with a final glycerol concentration of #g percent4 ,some of the first attempts in this laboratory at establishing the optimum yolk-to-citrate ratios are shown in ,fig4 #c4 ,in these experiments1 the optimum levels of citrate appeared to be lower than anticipated from the ,british work4 ,thus a more complex experiment was set up to test a wider range of citrate levels using #af and #bd percent egg yolk in the final freezing mixture4 ,the average percentages of motile sperm found after freezing #aj semen samples at each of the citrate and yolk levels in this experiment are shown also in ,fig4 #c4 ,little difference in freezability was found between citrate percentages of #a4ee and #a4ie4 ,when the rate of sperm motility following freezing and thawing was considered along with the percent of motile sperm1 a slight advantage was found with #af percent yolk and a citrate concentration of #a4ee percent4 ,illustration3 ,percent of motile sperm after freezing and thawing semen in diluents containing various levels of egg yolk 7,fig4 #b7 ,from the results of these experiments1 and from several reports in the literature1#e1f1g1i1aj it appears that a diluting medium resulting in a final concentration of #af to #be percent yolk and #a4ee to #b4b percent sodium citrate dihydrate is highly satisfactory for freezing4 ,storing and freezing diluent4 ,in some instances it would be advantageous to have prepared diluent on hand for use at any time4 ,the suitability of stored diluent was tested with a yolk-citrate 7equal parts yolk and citrate without antibiotics added7 diluent prepared and stored at #e deg4 ,c4 for #j1 #b1 #e1 #g1 and #i days4 ,seven semen samples were diluted and frozen in these diluents4 ,no difference was noted in the survival of sperm that could be attributed to the age of the diluent4 ,illustration3 ,percent of motile sperm after freezing and thawing semen in diluents containing various levels of egg yolk and various percentages of sodium citrate 7,fig4 #c7 ,in another trial1 a similar diluent 7#a3a yolk to citrate with #ajjj units of penicillin and #ejjj units of streptomycin7 was prepared and stored in the freezer compartment of a refrigerator at -#ae deg4 ,c4 ,upon thawing1 it was whitish in color and more viscous than freshly prepared diluent4 ,except for the fact that the viscosity seemed to reduce the rate of sperm motility1 this frozen diluent stored for #fe days compared favorably with freshly prepared diluent for freezing semen4 ,other diluents4 ,without the protective action of egg yolk or milk1 few bull sperm will survive freezing4 ,several diluents were compared on a limited scale for freezing bull sperm4 ,the results of these trials are compiled in ,table #e4 ,in this trial the yolk-citrate extender served best in maintaining sperm motility during freezing4 ,yolk-phosphate and homogenized whole milk were slightly less protective and yolk-saline seemed to furnish the least protection to sperm during freezing4 ,a number of investigations in other laboratories have now proven that milk can be used as effectively as the yolk-citrate diluent for freezing bull sperm4#f1g ,table #e4--;,comparison of the ,freezability of #d ,semen ,samples in ,different ,extenders ,dilution ,pre- ,post- ,motility ,extender rate freezing thawing ,survival after 7semen3 motility motility 7percent7 storage,e extender7 7percent7 7percent7 7percent7 ---------------------------------------- ------------------------------ ,yolk-citrate #a3a #fj #di #hb #df #a3aj #ec #de #he #cf ,yolk-saline #a3a #eg #bi #ea #bh #a3aj #fj #ca #eb #bd ,yolk-phosphate #a3a #ee #cc #fd #be #a3aj #fj #dc #gb #be ,whole milk #a3a #fj #dj #fg #ce #a3aj #fj #ce #eh #af ---------------------------------------- ------------------------------ ,e ,stored at #e deg4 ,c4 for #g hours after thawing4 ,,dilution ,,rates ,the first trials by the ,british at freezing bull semen were made with samples containing many millions of sperm cells4 ,in routine artificial breeding1 it is common to add extenders to semen so that one milliliter of diluted semen may contain only #aj million living sperm cells4 7,this number still insures optimal fertility47 ,frequently the addition of #ajj or more parts of the yolk extender to each part of the original semen sample is possible without reducing the sperm numbers below #aj million per milliliter4 ,no one knew if this process of dilution would affect the resistance of bull sperm to freezing4 ,the effect of various rates of dilution on the freezability of bull sperm was tested with #aj semen samples4 ,the results1 presented in ,table #f1 show that the numbers of sperm between #aj and #ij million per milliliter did not influence the percentage of sperm that survived freezing4 ,in a later trial it was found that sperm survival was slightly better at lower dilution rates than in the same samples frozen following dilution to #ae million sperm per milliliter4 ,however1 field trials with frozen semen carried out by others1 using sperm numbers as low as #ae million per milliliter of semen inseminated or even lower1 have been highly satisfactory4#aa1ab ,during the early studies in the ,illinois laboratory1 the effects of glycerol level were also tested4#ac ,these effects are discussed in the section on glycerol additions beginning on page #ag4 ,effect of further dilution and refreezing after the initial freezing4 ,under some circumstances it might be advantageous to freeze semen with a high concentration of sperm cells and then extend it further after thawing4 ,with such a procedure less storage space is needed than when dilution is carried to the maximum before freezing4 ,two experiments were conducted to test the effects of dilution and storage at #e deg4 ,c4 and dilution and refreezing following an initial freezing of concentrated samples4 ,table #f4--;,effect of ,sperm ,numbers and ,glycerol ,level in ,final ,mixture on ,freezability of ,bull ,sperm at -#gi deg4 ,c4 7,average of #aj ejaculates7 ,post-thawing motility 7percent7,f ---------------------------------------- ----- ,glycerol level ,number of sperm 7millions_/ml47 7percent7 -------------------------------- #ij #cj #aj ,average ---------------------------------------- ------------------------ #e #cf4j #cd4j #cf4j #ce4j #aj #bb4j #bd4j #bc4j #bc4j #ae #c4b #j4i #j4b #a4d ,average #bj4c #ai4h #ai4i #bj4j ---------------------------------------- ------------------------ ,f ,mean initial motility of sperm before freezing was #ee percent4 ,four semen samples were split and extended at rates of #a3a 7semen to extender7 and #a3aj4 ,these were frozen1 then thawed and halved4 ,one half was further extended to a level of #ae million sperm per milliliter2 the sperm numbers in the other remained unchanged4 ,each of these halves was split again1 and one portion of each was stored at #e deg4 ,c4 for #c to #g hours4 ,the other two portions were refrozen4 ,table #g4--;,effect of ,further ,dilution and ,refreezing on ,sperm ,motility ,after the ,initial ,freezing of ,bull ,semen ,post-thawing motility ,dilution ,pre- ---------------------------------------- ----------- of freezing ,after ,after storage,g ,after refreezing,h semen motility first ------------------- -------------------- 7semen3 7percent7 freezing ,no ,diluted ,no ,diluted extender7 further to #ae further to #ae dilution million_/ml dilution million_/ml ---------------------------------------- -------------------------------- ,first trial3 #d samples #a3a #fj #di #df #cd #ca #f #a3aj #ec #de #cf #cj #be #e ,second trial3 #g samples #a3i #fg #dg #da #ce #bh #aa #ae million_/ml #fg #cj #cb 44 #ah 44 ---------------------------------------- -------------------------------- ,g ,stored at #e deg4 ,c4 for #c to #g hours after first thawing4 ,h ,refrozen following first thawing4 ,table #h4--;,effect of ,glycerol ,level and ,storage at #e deg4 ,c4 on ,motility of ,sperm in ,yolk-,citrate ,extender ,sperm motility ---------------------------------------- ---------------- ,post- ,after storage at #e deg4 ,c4 ,glycerol thawing ---------------------------- ,average level #a day #c days #g days 7percent7 --------- --------- --------- --------- --------- per- rate per- rate per- rate per- rate per- rate cent cent cent cent cent ---------------------------------------- ---------------------------- ,control,i #ef #b4e #ee #a4i #df #a4h #ch #a4d #dh #a4ij #j #ed #b4d #dd #a4i #df #a4h #cf #a4d #de #a4hg #e #eb #b4b #ej #a4i #df #a4g #cb #a4d #de #a4hj #aj #eb #b4c #df #a4h #db #a4g #bh #a4f #db #a4he #bj #eb #b4a #ej #a4g #dd #a4f #ch #a4a #df #a4fb #cj #ej #j4g #dd #j4e #db #j4d #cj #j4d #db #j4ea ,average #ec #b4jc #dg #a4fb #dd #a4ej #cd #a4bb 44 4444 ---------------------------------------- ---------------------------- ,i ,the control differed from the #j-;glycerol treatment in that no additional citrate or glycerol solution was added4 ,a similar trial was carried out with seven samples2 one portion was diluted #a3i2 the other was extended at the outset to #ae million sperm per milliliter4 ,results for both tests are summarized in ,table #g4 ,from ,table #g it can be seen that refreezing following an initial freezing further reduced the number of surviving sperm4 ,the second freezing was more detrimental to the portion of the samples extended to #ae million sperm per milliliter than to the portion that was refrozen at a higher sperm concentration4 ,the percentage of motile sperm remained fairly high in the portions that were diluted to #ae million sperm and stored at #e deg4 ,c4 ,however1 in all cases1 survival was best in the samples at the lower dilution levels4 ,,glycerol ,,additions ,when the ,british procedure for freezing bull semen was first tried in this country1 many of the refinements of the technique still had not been defined4 ,it was known that glycerol worked well in protecting sperm during freezing4 ,the effects of glycerol on sperm at #e deg4 ,c41 the appropriate levels to use in freezing1 and the manner of adding it were not well established4 ,therefore1 a number of trials were conducted in an attempt to establish the best procedures4 ,effect of glycerol on sperm survival at #e deg4 ,c4 ,since early work indicated the need for adding glycerol to diluted semen in order to protect the sperm during freezing1 it was considered important to determine the levels of glycerol that sperm would tolerate at #e deg4 ,c4 ,ten semen samples were extended #a3i 7semen to diluent7 in a #a3a yolk-citrate diluent 7yolk to #b4i percent sodium citrate dihydrate74 ,each sample was then split into #f portions and an equal volume of citrate solution containing glycerol was added slowly to each to bring the glycerol in the final mixture to #j1 #e1 #aj1 #bj1 or #cj percent 7by volume74 ,these samples were stored at #e deg4 ,c4 and examined for motile sperm after #a1 #c1 and #g days4 ,the effects of glycerol levels on the percentage of sperm surviving and the rate 7or speed7 of their forward motion 7#j no forward motion2 #d extremely rapid progressive motility7 are presented in ,table #h4 ,the percentage of motile sperm decreased slightly at the higher levels of glycerol4 ,the most noticeable effect of the increase in glycerol level was the reduction in the rate of forward motion of the sperm4 ,at the #cj-percent level1 the sperm moved slowly and could be seen to rotate as they moved forward4 ,some samples were checked after slowly bringing the diluent up to a level of #dj percent glycerol2 the sperm seemed to be immobilized completely in this solution4 ,glycerol levels for freezing semen4 ,the ,british procedure called for the use of #aj percent glycerol in the final mixture of semen and extender prior to freezing4 ,yet1 as shown in ,table #f1 in our laboratory #e percent glycerol resulted in the survival of a higher percentage of sperm than did #aj or #ae percent4 ,in order to define more clearly the optimum glycerol level1 several ejaculates of semen were subsampled and portions were frozen after the addition of yolk-citrate extender and glycerol in varying quantities4 ,from ,table #i it can be seen that glycerol levels of #f and #h percent in the final mixture resulted in maximum sperm survival during freezing4 ,these results were confirmed in tests on the survival of sperm at #e deg4 ,c4 storage for #c days following freezing and thawing with varying glycerol levels 7see ,table #aj74 ,the results shown in ,tables #i and #aj were confirmed also in later experiments4 ,thirty-six samples were subjected to various levels of glycerol and no significant difference in freezability was found between #f and #h percent4 ,based on these findings1 a glycerol level of #g percent was adopted for use in all experiments described in this bulletin1 unless otherwise indicated4 ,results in a number of other laboratories have agreed with our findings regarding the use of approximately #g percent glycerol with the yolk-citrate diluent4#e1f1g1i1aj ,with milk as the extender1 #aj to #ac percent glycerol has been preferred by some4#e1f1g ,table #i4--;,effect of ,glycerol ,level on ,sperm ,motility ,after ,freezing to -#gi deg4 ,c4 and ,thawing ,glycerol ,number ,pre- ,post- ,survival level of freezing thawing 7percent7 7percent7 samples motility motility 7percent7 7percent7 ---------------------------------------- ------------------ #b #aj #ec #b #d #d #ai #ee #bi #ec #f #ai #ee #cd #fb #h #ai #ee #ce #fd #aj #ai #ee #bd #dd #ab #aj #ec #ac #be ---------------------------------------- ------------------ ,table #aj4--;,effect of ,glycerol ,level and ,storage at #e deg4 ,c4 ,after ,thawing on ,sperm ,motility 7,average of #ac ejaculates7 ,sperm motility 7percent7 ,glycerol ---------------------------------- level ,post- ,after storage at #e deg4 ,c4 7percent7 thawing ----------------------- #a day #c days ---------------------------------------- -------- #d #bi #bb #bj #f #ch #cd #bd #h #db #cc #ag #aj #cc #ah #f ---------------------------------------- -------- ,table #aa4--;,effects of ,temperature1 ,rate of ,addition of ,glycerol1 and ,equilibration ,time on ,sperm ,motility 7,average of #ab ejaculates7 ,temperature ,post-thawing motility 7percent7 during ,equilibration ---------------------------------- addition time ,glycerol additions of glycerol 7hours7 ---------------------------------- 7 deg4 ,c47 #e #c #a ,average ---------------------------------------- --------------------------- #d4e #b #dh #dh #de #dg4d #f #di #ea #dg #dh4h #ah #df #dg #df #df4c ,average #dg4h #dh4f #df4j #dg4e #aj4j #b #dd #dc #de #dc4i #f #dh #ej #df #dg4i #ah #dc #df #db #dd4j ,average #de4j #df4e #dd4c #de4c #ae4e #b #da #ch #ch #ci4a #f #db #de #dc #dc4f #ah #db #dc #db #db4e ,average #db4j #da4h #da4d #da4g ---------------------------------------- --------------------------- ,rate1 temperature1 and method of adding glycerol4 ,closely associated with the question of how much glycerol should be added is that of how the additions should be made4 ,originally it was believed that the glycerol should be added in stages so that changes would occur gradually4 ,however1 there would be a saving in time if the entire amount could be added at once4 ,also1 if the glycerol addition could be made soon after the dilution with egg yolk-citrate extender at room temperature1 time would be gained in processing the semen for use4 ,since aging in vitro is known to reduce the fertilizing ability of sperm1 every effort should be made to keep the processing time at a minimum4 ,the results of an experiment involving these items1 along with that of how much time should be allowed after the additions before freezing 7equilibration time71 are presented in ,table #aa4 ,one can see that sperm survived freezing better when the diluted semen was cooled to #d4e deg4 ,c4 before the glycerol was added4 ,the survival at #aj deg4 and #ae4e deg4 ,c4 was reduced with each rise in temperature4 ,thus1 it appears that cooling to refrigerator temperature 7#d-e deg4 ,c47 before adding the glycerol should be a part of the routine procedure4 ,a comparison of the results from adding the glycerol in #e1 #c1 and #a equal portions is given also in ,table #aa4 ,little difference in survival during freezing was noted between the three rates of addition4 ,using #c equal additions resulted in slightly better results1 but the advantage was not statistically significant4 ,while little difference was evident from adding the glycerol in #c portions as compared to #a1 many still use #c additions in the hope of obtaining a slightly better sperm survival4 ,in fact1 some have gone to a procedure of adding the glycerol dropwise with constant gentle agitation4 ,this method has not been tested in this laboratory4 ,allowing sperm to equilibrate with the glycerol4 ,allowing sperm to stand in the presence of glycerol is considered by some to be necessary in order that the glycerol penetrate the sperm heads before freezing4 ,from the first successful attempts at freezing bull sperm came the practice of allowing #ab to #bj hours for this process of equilibration4 ,a long equilibration time results in aging the sperm4 ,data from a number of sources indicate that a drop of approximately #e percent in fertility in the field occurs with each #bd hours of aging in the test tube4 ,thus it would seem desirable to reduce the equilibration time to a minimum commensurate with good freezability in order to reduce the effects of aging 7at #e deg4 ,c474 ,results of attempting to reduce equilibration time are given in ,table #aa4 ,at #d4e deg4 ,c41 little variation in motility following freezing and thawing was found after equilibration times of #b1 #f1 and #ah hours4 ,at the higher temperatures of #aj deg4 and #ae4e deg4 ,c41 the shortest equilibration time--#b hours--was slightly more detrimental with the differences significant at the #e-percent level at #ae4e deg4 ,c4 ,for all temperatures combined1 #f hours was significantly better than #b or #ah hours4 ,sugar additions and equilibration time4 ,early in their experiences in freezing semen1 the ,australian workers found a short equilibration time--#cj minutes--to be satisfactory if sugars were added to the diluent4#e ,this protective action of sugars during the equilibration period was confirmed in our investigations4 ,the results of one phase of this study are shown in ,table #ab4 ,from these data it can be seen that the presence of glucose or rhamnose at a level of #a4be percent improved sperm survival during the period of equilibration4 ,in another trial these sugars and two others1 arabinose and xylose1 were tested for their protective action in freezing semen4 ,the percentages of surviving sperm remaining after the various steps in the freezing procedure with and without the presence of these sugars are shown in ,table #ac4 ,table #ab4--;,effect of ,adding ,sugars to ,yolk-,citrate ,diluent on ,sperm ,motility ,during ,equilibration ,with ,glycerol,j ,sperm motility 7percent7 ------------------------------------- ,stage when observed ,glycerol ,glycerol ,glycerol only and glucose and rhamnose ---------------------------------------- -------------------- ,fresh diluted semen #ef #ef #ef ,after glycerolization #ed #ed #ed ,after equilibration #b hours #ea #ec #ec #f hours #dh #eb #ec #ab hours #df #ej #ea #ah hours #dj #df #df ---------------------------------------- -------------------- ,j ,glycerol level in the final frozen mixture was #g percent4 ,sugars were added to a level of #a4be percent4 ,three of the sugars--glucose1 arabinose1 and rhamnose--protected the sperm during equilibration and freezing4 ,xylose was less effective1 but its addition resulted in slightly better sperm survival than glycerol alone4 ,it was found also that the methylene-blue reduction time 7metabolic test for semen quality7 was faster in samples to which the sugars had been added--after glycerolization1 after equilibration1 and after freezing the samples4 ,this is confirming evidence for the presence of more living and actively metabolizing sperm in the portions to which sugars had been added4 ,table #ac4--;,effect of ,adding ,sugars to ,yolk-,citrate ,diluent on ,sperm ,motility ,during the ,freezing ,procedures,k 7,average of #aj ejaculates7 ,sperm motility 7percent7 ---------------------------------------- --------- ,stage of ,glycerol ,glycerol ,glycerol ,glycerol ,glycerol observation only and and and and glucose arabinose xylose rhamnose ---------------------------------------- ------------------------------ ,fresh diluted semen #fc #fc #fc #fc #fc ,after glycerolization #ed #ee #ed #eg #fj ,after #ah hours equilibration #ci #dc #dd #ci #df ,after freezing to -#gi deg4 ,c4 and immediate thawing #bh #cd #cd #bi #bd ,after #d days at -#gi deg4 ,c4 #bc #bf #bf #be #bg ---------------------------------------- ------------------------------- ,k ,glycerol level in the final frozen mixture was #g percent4 ,sugars were added to a level of #a4be percent4 ,substitutes for glycerol4 ,since glycerol was so effective in protecting sperm during freezing1 many have assumed that related compounds might be even better4 ,several compounds1 some related to glycerol and some not1 have been tried as substitutes for glycerol in the freezing procedure4 ,they include ethylene glycol1 propylene glycol1 trimethylene glycol1 mannitol1 sorbitol1 dextrans1 and seminal-plasma proteins4 ,none of these materials has been as effective as glycerol in protecting sperm during freezing4 ,in fact1 several of the materials proved to be injurious to sperm prior to attempts to freeze the samples4 ,while the work in our laboratory with these substances as glycerol substitutes was by no means finally conclusive1 because of the many possible interactions of experimental conditions1 sufficient data were gathered to lead us to abandon further study until greater promise of success might be evident4 ,,freezing ,,rate ,effect of freezing rate on sperm survival4 ,reports by one group of ,british workers in early trials on freezing bull semen indicated that the rate of cooling in freezing should not exceed #b deg4 ,c4 per minute between #e deg4 and -#ae deg4 ,c41 although below -#ae deg4 ,c4 the rate could be faster4 ,another group expressed the view that semen could be plunged into dry ice at -#gi deg4 ,c4 after it had been cooled to -#ae deg4 ,c4 ,to clarify this part of the freezing procedure1 #aa samples of semen were subdivided and portions of each were frozen at rates of #j4be deg41 #j4e deg41 #a4j deg41 #b4j deg41 and #d4j deg4 ,c4 drop per minute between #e deg4 and -#bj deg4 ,c4 and then twice these rates between -#bj deg4 and -#gi deg4 ,c4 ,vials of each ejaculate at #e deg4 ,c4 were also plunged directly into an alcohol bath at -#gi deg4 ,c4 ,the samples which were cooled at the rates of #j4be deg41 #j4e deg41 #a4j deg41 #b4j deg41 and #d4j deg4 ,c4 per minute had the following percentages of motile sperm after thawing3 #cj1 #dj1 #df1 #dd1 and #dd4 ,a mean of #cb percent of the sperm in the samples that were plunged directly into an alcohol bath at -#gi deg4 ,c4 were motile after thawing4 ,there were no statistically significant differences among the samples frozen at #a4j deg41 #b4j deg4 or #d4j deg4 ,c4 per minute4 ,all of the others had significantly lower survival rates4 ,thus1 it is obvious that too slow a cooling rate and plunging the samples directly into a -#gi deg4 ,c4 bath from a temperature of #e deg4 ,c4 cause greater harm to the sperm than cooling at a rate between #a4j deg4 and #d4j deg4 ,c4 per minute4 ,some investigators have suggested that rapid cooling below -#bj deg4 ,c4 is not detrimental to frozen semen4 ,this idea was tested in conjunction with other experiments4 ,twenty-five samples cooled slowly 7#b deg4 ,c4 per minute to -#bh deg4 ,c41 then #d deg4 ,c4 per minute to -#gi deg4 ,c47 showed #fb percent sperm survival compared with only #de percent when cooled rapidly below -#bh deg4 ,c4 7#b deg4 ,c4 per minute to -#bh deg4 ,c4 then plunged into bath at -#gi deg4 ,c474 ,thus1 rapid cooling was detrimental even after the critical temperature range of #e deg4 ,c4 to -#bj deg4 ,c4 had been passed4 ,illustration3 ,cooling rates of diluted semen samples in plastic vials and in glass ampules 7,fig4 #d7 ,rate of cooling in plastic and in glass4 ,plastic vials do not conduct the cold as rapidly as glass ampules do4 ,the temperature in both glass and plastic containers tends to lag behind the change in the bath in which they are immersed as is shown in ,figure #d4 ,temperatures in the immersion bath were recorded in a #b-milliliter glass ampule containing #a milliliter diluted semen and in an #h-milliliter plastic vial containing #b4e milliliters of diluted semen4 ,a second plastic vial and glass ampule filled to capacity with diluted semen showed a cooling rate almost identical to that shown in ,figure #d4 ,it was obvious from the comparison that samples in the plastic vials cooled slower than those in glass and that the volume of semen 7at least the small volumes used7 in the vials had little effect on the rate of cooling4 ,in another experiment1 it was shown that the volume of diluted semen in the ampule to be frozen 7#j4b1 #a4j or #e4j ml47 had little or no effect on the survival of the sperm4 ,,storage ,,temperature ,in freezing and storing bull sperm1 an alcohol bath containing dry ice at a temperature of -#gi deg4 ,c4 has been used as a cooling agent4 ,in many areas1 the availability of dry ice is limited and the cost is rather high4 ,mechanical means are available for obtaining temperatures as low as1 or lower than1 -#gi deg4 ,c4 but for the most part they are expensive4 ,if warmer temperatures were suitable for storing frozen semen1 the ordinary deep-freeze1 which operates at -#ae deg4 to -#be deg4 ,c41 might be used4 ,storage at temperatures from -#bc deg4 to -#gi deg4 ,c4 ,in testing the effects of storage temperatures on the survival of frozen bull sperm 7in a diluent containing #g percent glycerol71 #i ejaculates were frozen and kept at -#bc deg41 -#cg deg41 -#ea deg41 -#fe deg41 and -#gi deg4 ,c4 ,the desired temperatures were maintained by dropping pieces of dry ice into ethyl alcohol baths as needed4 ,samples were thawed after #a hour1 #a day1 #c days1 and #e days4 ,after #a hour1 the samples maintained at the various temperatures exhibited approximately equal motility 7,fig4 #e74 ,illustration3 ,effect of freezing and storing bull sperm at various temperatures on the sperm motility at thawing 7average of #i ejaculates7 7,fig4 #e7 ,at the end of #a day1 samples stored at -#gi deg4 ,c4 exhibited approximately the same motility as did similar samples stored for #a hour4 ,the samples stored at -#fe deg4 ,c4 had declined slightly in motility and those maintained at -#ea deg4 ,c4 had only one-third the motility which they had displayed at #a hour4 ,the samples at -#bc deg4 and -#cg deg4 ,c4 exhibited practically no motility after #a day in storage4 ,after #e days1 only #c of the #h ejaculates stored at -#ea deg4 ,c4 showed motility upon thawing4 ,apparently detrimental changes take place more rapidly when the samples are stored at temperatures warmer than -#fe deg4 ,c4 ,the nature of these changes has not been determined4 ,reports from other laboratories indicate that storage temperatures much lower than -#gi deg4 ,c4 are just as satisfactory as -#gi deg4 ,c4 ,no tests of the effects of storage at -#gi deg4 ,c4 for periods longer than #ea days have been conducted in this laboratory4 ,portions of #ab ejaculates were frozen and stored at -#gi deg4 ,c4 for various periods4 ,one portion of each of these was examined on the second1 ninth1 #afth and #east day of storage4 ,the percent of motile sperm and rate of motility at each of these examinations were as follows3 ,day #b #i #af #ea ,percent of motile sperm #di #df #dj #ch ,rate of motility #b4e #b4c #b4b #b4b ,the average prefreezing motility percentage for the above samples was #eh1 with an average rate of motility of #b4i4 ,it is apparent from these results that the loss in motility was greatest due to the initial freezing1 and after that the drop was most pronounced during the first #af days of storage4 ,the ,british and the ,australians have both reported the successful maintenance of fertility in frozen semen stored at -#gi deg4 ,c4 for over two years4#e ,use of higher glycerol levels and a -#bj deg4 ,c4 storage temperature4 ,in #aiec1 a report from ,arkansas suggested that warmer storage temperatures could be used if a high percentage of glycerol were included in the freezing mixture4#g ,to test the effectiveness of various glycerol levels on protecting sperm stored at deep-freeze temperatures1 glycerol levels of #c4e1 #e4e1 #g4e1 and #i4e percent were used with portions of #d semen samples4 ,survival in the portions frozen and stored at -#bj deg4 ,c4 was poor compared with the portions reduced and held at -#gi deg4 ,c4 ,in a second experiment1 #d samples were subdivided and frozen with a final concentration of #g1 #aa1 #ae1 and #ai percent glycerol in the semen-diluent mixture4 ,in this trial1 poor results were obtained at -#bj deg4 ,c4 except that glycerol at a level of #ai percent protected the sperm more effectively than at lower levels4 ,maximal survival at -#gi deg4 ,c4 was obtained at the #g-percent glycerol level4 ,a final trial was run1 using glycerol levels of #g1 #aa1 #ae1 #ai1 #bc1 #bg1 and #ca percent4 ,the percentages of motile sperm present after storage at -#gi deg4 ,c4 and -#bj deg4 ,c4 are shown in ,table #ad4 ,table #ad4--;,effect of ,glycerol ,level and ,storage ,temperature on ,freezability of ,semen 7,average of #h ejaculates7 ,storage ,glycerol ,sperm motility after storage 7percent7 temperature level -------------------------------------- 7 deg4,c47 7percent7 #ah hours #db hours ---------------------------------------- ------------------------ -#gi #g #fa #fa -#bj #g #b #a #aa #c #a #ae #ad #aj #ai #cj #bb #bc #bi #ai #bg #be #ah #ca #ba #ab ---------------------------------------- ------------------------ ,while survival was fair over a short period of time with #ai percent glycerol at -#bj deg4 ,c41 deterioration was rapid during storage4 ,after #ah hours of storage1 the samples at -#bj deg4 ,c4 7#ai percent glycerol7 contained only one half as many motile sperm as were still present in the samples at -#gi deg4 ,c4 7#g percent glycerol74 ,after #db hours of storage1 the best samples at -#bj deg4 ,c4 contained only one-third the number of motile sperm still present in the samples stored at -#gi deg4 ,c4 ,these trials leave little doubt that under the present system of freezing and storing1 storage at ordinary deep-freeze temperatures is far inferior to storage at dry-ice temperatures4 ,,thawing ,the importance of carefully controlled cooling and storage has been emphasized in the foregoing sections4 ,the need for controlling thawing rates and the temperature of thawing was not clearly defined in the early work on freezing bull semen4 ,the ,british used a thawing temperature of #dj deg4 ,c41 which was satisfactory4 ,if there is a need to hold the semen for a time after thawing1 then a lower thawing temperature might be more desirable so that cooling again will not be necessary4 ,comparison of thawing temperatures of #e deg4 ,c4 and #ch deg4 ,c4 ,the effects of thawing at temperatures of #ch deg4 7body temperature7 and #e deg4 ,c4 7refrigerator temperature7 were investigated4 ,the first trial involved thawing as rapidly as possible by dropping glass ampules of frozen semen into water baths at the two temperatures4 ,the frozen semen samples contained glycerol levels of #d1 #f1 #h1 and #aj percent4 ,the mean percentages of motile sperm found after thawing thirteen diluted semen samples treated in this manner are shown in ,figure #f4 ,illustration3 ,effect of glycerol percentage and thawing temperature on sperm motility after freezing and thawing 7,fig4 #f7 ,the #e deg4 ,c4 thawing temperature resulted in a higher percentage of sperm survival at all the glycerol levels than #ch deg4 ,c41 with the difference in favor of #e deg4 ,c4 becoming greater as the glycerol level increased4 ,the reason for the interaction between glycerol level and thawing temperature is not known4 ,it may be that the presence of the higher levels of glycerol at #ch deg4 ,c4 brought about harmful metabolic activity4 ,the difference in survival of sperm in semen thawed at #e deg4 ,c4 and at #ch deg4 ,c4 continued during storage at #e deg4 ,c4 7,table #ae74 ,it was also evident that the interaction between glycerol level and thawing temperature continued during storage 7,fig4 #g74 ,illustration3 ,effect of thawing temperature on sperm motility during storage at #e deg4 ,c4 following freezing and thawing 7,fig4 #g7 ,table #ae4--;,effect of ,glycerol ,level1 ,thawing ,temperature1 and ,storage at #e deg4 ,c4 ,after ,thawing on ,sperm ,motility 7,average of #ac ejaculates7 ,thawing ,glycerol ,sperm motility 7percent7 temperature level ---------------------------------------- --- 7 deg4 ,c47 7percent7 ,post- ,after storage at #e deg4 ,c4 thawing ---------------------- ,average #a day #c days ---------------------------------------- ----------------------------- #ch #d #bh4e #ag4c #e4a #ag4j #f #ca4e #bb4d #i4b #ba4a #h #cc4a #ae4j #d4f #ag4f #aj #ai4e #c4f #j4h #h4j ,average #bh4b #ad4f #d4i #ab4b #e #d #bi4b #ba4g #ai4h #bc4i #f #cg4g #cc4h #bc4e #ca4g #h #da4e #cc4a #ag4c #cj4f #aj #cc4a #ah4e #f4j #ai4b ,average #ce4d #bf4h #af4f #bj4f ---------------------------------------- ----------------------------- ,it is obvious that motility falls off rapidly after the semen is thawed4 ,in a field trial in which the initial intent was to test the effect of glycerol levels on fertility of frozen semen1 the semen was thawed in the morning and used during the same day4 ,survival of the sperm with #d percent glycerol was so poor that only a few breedings were made with these samples4 ,even at #g and #aj percent1 the fertility results were much lower than with semen that had not been subjected to freezing4 ,at that time it was felt that thawing the samples and using them throughout the day may have caused the low fertility results4 ,since then1 a large-scale experiment by ,cornell ,university investigators1 in cooperation with the ,new ,york ,artificial ,breeders' ,cooperative1 has shown definitely that thawing should be delayed until a few minutes prior to breeding4#aa ,if the semen is used immediately1 a thawing temperature of either #e deg4 or #ch deg4 ,c4 appears to be suitable4 ,however1 there is less danger of cold shock due to recooling if #e deg4 ,c is used4 ,thawing rate in plastic and in glass4 ,glass ampules transmit cold or heat more readily than plastic ones4 ,the temperature rise is rapid in both glass and plastic when samples are taken from the storage box at -#gi deg4 ,c4 and placed in water at #e deg4 ,c4 ,however1 complete thawing occurs more rapidly in glass than in plastic ampules4 ,the changes in temperature that occurred when glass and plastic ampules were thawed in a water bath at #e deg4 ,c4 are shown in ,figure #h4 ,the initial temperature rise for the first minute or two was about the same1 then the rate of warming in the plastic slowed and actual melting of the frozen sample occurred a little over a minute later in the plastic than it did in the glass4 ,both were thawed in less than four minutes4 ,illustration3 ,warming rates of diluted semen samples in plastic vials and in glass ampules 7,fig4 #h7 ,,effects ,,of ,,freezing ,,procedures ,,on ,,metabolic ,,activity ,,of ,,bull ,,sperm ,by finding how methods of handling affect the sperm cells1 one can sometimes improve the procedures to avoid harmful effects4 ,some attempts have been made in this laboratory to determine the effects of the freezing procedures on the metabolic activity of bull spermatozoa4 ,these investigations have been limited in scope1 involving the measurement of oxygen-consumption and estimates of sperm motility during and after incubation at #cg deg4 ,c4 in a ,warburg apparatus4 ,effect of glycerol additions on oxygen uptake of diluted semen at #cg deg4 ,c4 ,the effect of adding glycerol to diluted semen on oxygen consumption of the sperm was tested in a ,warburg apparatus1 using semen diluted with an extender consisting of one part egg yolk and one part #b4i percent sodium citrate dihydrate4 ,the yolk-citrate extender was added to the semen at a rate which brought the sperm concentration in #j4e milliliter to #bjj million to #ejj million4 ,an exact count was used to calculate the oxygen uptake per #ajh sperm per hour 7,,zo#b74 ,illustration3 ,effect of glycerol additions on oxygen consumption of sperm at #cg deg4 ,c4 7,fig4 #i7 ,glycerol in various percentages in #b4i percent sodium citrate dihydrate solution was placed in the sidearm of the ,warburg flasks4 ,the diluted semen was held in the main compartment4 ,after a #fj-minute preliminary run1 in which the rate of oxygen uptake of the sperm in yolk-citrate diluent was determined1 the contents of the sidearm were tipped into the main compartment4 ,the resulting glycerol percentages after mixing the sidearm and main compartment contents were #j1 #d1 #h1 and #ab percent4 ,ten samples of semen were subsampled and the oxygen uptake of each was determined at all four levels of glycerol4 ,oxygen uptake was increasingly stimulated during the first #bj-minute interval by each increase in the amount of glycerol added 7,fig4 #i74 ,after the first #bj minutes1 the rate of oxygen utilization decreased at the two higher levels of glycerol but persisted at #d percent4 ,the rate of oxygen consumption for the first #bj-minute period at the #d-percent glycerol level was #acj percent that of the control to which only sodium citrate had been added4 ,at #h and #ab percent the values for the period were #add and #aib percent1 respectively1 of the control rate4 ,illustration3 ,effect of glycerol and glycerol-plus-catalase additions on sperm motility during incubation at #cg deg4 ,c4 7,fig4 #aj7 ,with each increase in glycerol level1 motility was reduced during the incubation period4 ,this is shown in ,figure #aj along with the effect on motility of adding catalase1 which is discussed in the next section4 ,effect of glycerol-plus-catalase on oxygen uptake of diluted semen4 ,certain bacteria have been shown to break glycerol down1 forming hydrogen peroxide as follows3 ,glycerol oxygen -- lactic acid hydrogen peroxide4 ,hydrogen peroxide is known to be detrimental to sperm4 ,the addition of glycerol to diluted semen first increased oxygen uptake and then reduced it4 ,since a reduction in sperm survival followed1 some harmful action must have taken place with the addition of glycerol at #cg deg4 ,c4 ,to test whether this action could be due to the release of hydrogen peroxide as occurs in certain bacteria1 glycerol with catalase--the enzyme which breaks down hydrogen peroxide--was added to a portion of #h diluted semen samples and the oxygen uptake was recorded4 ,comparison of the resulting oxygen uptake with glycerol and with glycerol plus catalase is shown in ,figure #aa4 ,illustration3 ,effect of additions of glycerol-plus-catalase on oxygen consumption of sperm at #cg deg4 ,c4 7,fig4 #aa7 ,oxygen consumption was increased by the presence of added catalase at all glycerol levels and in the control4 ,sperm survival during the #c-;hour period at #cg deg4 ,c4 also was improved by the presence of catalase 7,fig4 #aj74 ,however1 the general trend in oxygen consumption produced by the addition of glycerol was not changed greatly4 ,the higher levels of glycerol still stimulated oxygen uptake during the first #bj-minute period after the additions and then slowed the rate of oxygen utilization4 ,the rate of utilization was generally higher during the test period in the presence of catalase than without added catalase4 ,it appeared that a part of the harmful effect of glycerol might be due to the formation of hydrogen peroxide4 ,still1 the detrimental effects of the higher levels of glycerol were not completely removed4 ,table #af4--;,effect of ,freezing ,procedures on ,oxygen ,utilization of ,bull ,sperm in ,yolk-,citrate ,extender 7,average of #e ejaculates7 ,microliters of oxygen ,semen sample tested utilized per #ajh sperm -------------------------- ,first hour ,second hour ---------------------------------------- ----------------------------- ,fresh diluted semen #aj4c #h4a ,fresh diluted semen glycerol tipped in at end of first hour #i4g,l #ab4i,l ,aged #bj to #bd hours at #e deg4 ,c4 #aa4b #h4c ,aged #bj to #bd hours at #e deg4 ,c4 glycerol tipped in at end of first hour #aa4h,l #ab4i,l ,after #bj hours equilibration with glycerol #aa4g,l #g4h,l ,after freezing and thawing #i4g #f4c ---------------------------------------- ---------------------------- ,l ,average of #c ejaculates4 ,effect of freezing procedures on oxygen utilization by sperm4 ,limited data have been obtained on the effects of some of the freezing procedures on the oxygen utilization of bull sperm4 ,the results obtained in these experiments confirmed the earlier findings that tipping glycerol directly into the diluted semen at #cg deg4 ,c4 caused an increase in oxygen consumption 7,table #af74 ,all other steps in the freezing procedure had little effect on oxygen consumption by the sperm4 ,except where glycerol was added during the determination1 the rate of oxygen utilization was lower the second hour than during the first4 ,the oxygen uptake of semen that had been frozen and thawed seemed to drop faster than that of unfrozen samples4 ,effect of freezing procedures on methylene-blue reduction time4 ,the methylene-blue reduction test has been used as a means of measuring semen quality and is dependent on the metabolic activity of the sperm4 ,the effects of various freezing procedures on the ability of samples to decolorize methylene blue were determined with #aj semen samples4 ,sperm numbers were standardized to #cjj x #ajf cells per milliliter and the time required for these cells to reduce a #a3dj1jjj solution of methylene blue was determined on freshly diluted semen1 after the addition of glycerol1 after equilibration1 and after freezing and thawing4 ,portions of each diluted sample were tested at these stages of the procedure with glycerol alone added and with glycerol and various sugars added4 ,a marked increase in the time required for the sperm to reduce methylene blue occurred when the glycerol was added 7,table #ag74 ,this increase was greatest in the portions with glycerol alone and with glycerol and glucose4 ,the time increase was less pronounced in the presence of the three pentose sugars used4 ,following equilibration1 the samples regained the ability to reduce methylene blue at a rate only slightly slower than when they were fresh4 ,freezing and storage of semen resulted in slower reduction of the methylene blue than was shown after equilibration with glycerol4 ,since freezing usually kills some of the sperm1 a slowing of the reduction time after freezing would be expected4 ,table #ag4--;,effect of ,freezing ,procedures on the ,methylene-,blue ,reduction ,time of ,bull ,semen ,with and ,without the ,addition of ,sugars,m 7,average of #aj ejaculates7 ,methylene-blue reduction time 7minutes7 ---------------------------------------- -------- ,glycerol ,glycerol ,glycerol ,glycerol ,glycerol only and and and and glucose arabinose xylose rhamnose ---------------------------------------- ------------------------------ ,fresh semen #e4b #e4b #e4b #e4b #e4b ,after glycerolization #bf4d #be4b #ag4c #ad4c #ai4d ,after #ah hours equilibration #g4d #f4e #f4d #e4c #f4b ,thawed immediately after freezing #aa4e #aj4e #i4d #i4j #i4d ,thawed #dh hours after freezing #ad4c #aj4b #aa4c #aj4a #i4e ---------------------------------------- ------------------------------ ,m ,glycerol level in the final frozen mixture was #g percent4 ,sugars were added to a level of #a4be percent4 ,,practical ,,freezing ,,procedure ,good results usually can be obtained in freezing bull semen if care is taken in collecting1 diluting and processing the semen4 ,occasionally the semen from certain bulls will not withstand freezing well4 ,the reason for this is not understood at present4 ,however1 carefully following the directions and suggestions given below will usually produce satisfactory results with semen samples that are of good quality at the start4 ,experience in the field has shown that fertility results with frozen semen are usually slightly lower during the first few months than with liquid semen stored at #e deg4 ,c4 7#da deg4 ,f474 ,most units that have worked with frozen semen over a period of a few months are able to improve and do get fertility results as good as1 or better than1 obtained in their liquid semen program4 ,collection of the semen4 ,in order to obtain the best possible semen for freezing1 care and cleanliness should be exercised in making the collection4 ,the artificial vagina1 and the glassware used should be clean and dry4 ,the underline of the bull should also be clean and dry4 ,the bull should be restrained near the teaser cow for a minute or two prior to collection in order to excite the flow of secretions prior to ejaculation4 ,allowing the bull to mount the teaser once without serving the artificial vagina is a good practice to use in properly stimulating the bull before collection of the semen4 ,if the bull has not been used for three or four days1 the collection of a second ejaculate for freezing may be advisable4 ,the second ejaculate seems to withstand freezing better than the first in many instances4 ,a clean1 dry artificial vagina should be used for each ejaculate collected4 ,repeated collections in the same artificial vagina may result in contamination of the semen with bacteria1 lubricating jelly and minute particles of dirt4 ,the semen sample should be protected from contamination and from sudden temperature drops 7cold shock74 ,preparation of extender4 ,a suitable egg yolk-citrate extender for freezing bull semen can be prepared by the following procedure4 ,one part egg yolk 7free of egg white and the membrane surrounding the yolk7 is mixed with #d parts #b4d to #b4i percent sodium citrate dihydrate solution4 ,the citrate is prepared with distilled water and then boiled or autoclaved4 ,the citrate solution should be cooled before it is mixed with the egg yolk4 ,after the egg and citrate are mixed1 #ajjj units of penicillin and #ajjj micrograms of streptomycin are added per milliliter of extender4 ,sulfanilamide should not be added4 ,this extender can be prepared #ab to #bd hours before use if it is stored at refrigerator temperature4 ,the portion of the extender needed for the original dilution of the semen should be warmed to room temperature before it is mixed with the semen4 ,dilution after collection4 ,as soon as possible after collection1 the semen sample should be diluted with the extender4 ,the extender must be at the same temperature as the semen 7room temperature7 when the two are mixed together4 ,at this time the semen can be partially diluted 7#a part semen to #d parts of extender7 or diluted to a sperm concentration twice the final desired concentration 7later in adding the glycerol for freezing1 the semen is diluted further with an equal volume of glycerol containing extender74 ,the diluted semen is slowly cooled 7#a-a_/b to #b-a_/b hours7 to #e deg4 ,c4 7#da deg4 ,f474 ,some units using frozen semen now allow the semen to stand at #e deg4 ,c4 for #e to #f hours before glycerolization to allow the antibiotics to be more effective against any vibrio fetus organisms that may be present4 ,this step is taken because it has been shown that glycerol inhibits the effectiveness of the antibiotics4#f ,after cooling1 semen can be further diluted to twice the desired sperm concentration if that were not done at the start4 7,caution3 ,be sure semen and diluent are at the same temperature47 ,adding the glycerol4 ,the glycerol solution is prepared by adding #ad volumes of glycerol 7reagent grade7 to #hf volumes of yolk-citrate diluent 7same as yolk-citrate used for original dilution74 ,this solution may be added dropwise with constant gentle mixing to the already diluted semen1 or one-third at a time at #aj-minute intervals with gentle mixing during each addition4 ,either method should take about #bj to #cj minutes4 ,the total volume of glycerol-yolk-citrate solution added should be equal to the volume of the original diluted semen4 ,in this way a concentration of #g percent glycerol is obtained in the final mixture that is to be frozen4 ,care must be taken to keep the temperature at #e deg4 ,c4 7#da deg4 ,f47 during the time the glycerol is being added4 7,a cold room is best for maintaining a temperature of #e deg4 ,c41 but with care the operation can be carried out at room temperature by using pans of ice water and a refrigerator47 ,equilibration4 ,the results presented in this bulletin suggest that little or no time need be allowed after the glycerol is added before freezing4 ,however1 results obtained by other workers show improved fertility with at least #ab hours equilibration4 ,some units getting good fertility results with frozen semen also are allowing the semen to stand at #e deg4 ,c4 for #ab to #ah hours before freezing4 ,after the semen has equilibrated with the glycerol1 #a-milliliter portions of the mixture are placed in #a4b- to #b-milliliter vials or ampules which are then sealed4 ,ampuling can be done with an automatic syringe or pipette1 provided a large gage needle is used4 ,also1 it is important not to force the fluid mixture rapidly through the syringe or the sperm may be injured4 ,freezing4 ,the vials or ampules of diluted semen are placed in a bath of isopropyl alcohol which has been cooled to #e deg4 ,c4 7#da deg4 ,f474 ,this bath can be a wide-mouth thermos bottle or an insulated container of almost any sort with a large opening at the top4 ,the size needed depends on the number of ampules being frozen4 ,some sort of convenient tray for holding the ampules in an orderly fashion and enabling the samples to be completely submerged is desirable4 ,a few ampules can be kept together easily by placing them in a polyethylene freezer bag that has had many small holes cut in it to let the alcohol of the bath contact the ampules4 ,the ampules must be completely covered by the alcohol to insure uniform cooling4 ,the alcohol of the bath and the ampules of semen are cooled by adding chipped or ground dry ice in sufficient amounts to lower the temperature of the bath #b deg4 ,c4 7#c4f deg4 ,f47 per minute from #e deg4 to -#bj deg4 ,c4 ,from -#bj deg4 down to -#gi deg4 ,c41 the rate of cooling can be doubled 7#d deg4 ,c4 or #g4b deg4 ,f474 ,electrical equipment that regulates the cooling rate to the desired temperatures is available commercially1 but the cost may be too high for some small operations4 ,the samples should be held at -#gi deg4 ,c4 7-#aaj deg4 ,f47 until they are thawed4 ,this can be done by using an alcohol bath and dry ice or by special mechanical refrigerating equipment4 ,at no time prior to thawing should the samples be exposed to warmer temperatures4 ,thawing4 ,the ampules of frozen semen can be thawed by removing them from the dry ice storage box and dropping them into a water bath at #e deg4 ,c4 7#da deg4 ,f474 ,thawing temperatures up to body temperature1 #ch deg4 ,c4 7#ajj deg4 ,f471 can be used but extreme care must then be taken not to pass the semen through a cold inseminating tube2 for this would subject the sperm to cold shock4 ,the semen should be used for breeding within a few minutes after thawing4 ,,literature ,,cited #a ,,davenport1 ,c4 ,b4 ,effect of chemical and physical agents upon protoplasm4 ,macmillan and ,co41 ,new ,york4 #ahig4 #b ,,polge1 ,c41 and ,,parkes1 ,a4 ,s4 ,possibilities of long-term storage of spermatozoa at low temperatures4 ,anim4 ,breeding ,abs4 #bj3a-e4 #aieb4 #c ,,emmens1 ,c4 ,w41 and ,,blackshaw1 ,a4 ,w4 ,the low temperature storage of ram1 bull1 and rabbit spermatozoa4 ,austral4 ,vet4 ,jour4 #bf3bbf4 #aiej4 #d ,,smith1 ,,audrey ,w4 ,effects of low temperatures on living cells and tissues4 ,in biological applications of freezing and drying4 ,ed4 ,r4 ,j4 ,c4 ,harris4 ,academic ,press1 ,inc41 ,new ,york1 #aied4 #e ,,emmens1 ,c4 ,w41 and ,,blackshaw1 ,a4 ,w4 ,artificial insemination4 ,physiol4 ,rev4 #cf3bgg-cjf4 #aief4 #f ,proceedings of the ,national ,association of ,artificial ,breeders1 #aiec1 #aied1 and #aiee4 #g ,proceedings of the ,american ,dairy ,science ,association1 #aiec1 #aied1 and #aiee4 ,published in the ,june issue of the ,journal of ,dairy ,science for each year4 #h ,,barker1 ,c4 ,a4 ,v4 ,low temperature preservation of bovine epididymal spermatozoa4 ,canad4 ,jour4 ,comp4 ,med4 #ah3cij-cic4 #aied4 #i ,,saroff1 ,,jack1 and ,,mixner1 ,j4 ,p4 ,the relationship of egg yolk and glycerol content of diluters and glycerol equilibration time to survival of bull spermatozoa after low temperature freezing4 ,jour4 ,dairy ,sci4 #ch3bib-big4 #aiee4 #aj ,,cragle1 ,r ,g41 ,,myers1 ,r4 ,m41 ,,waugh1 ,r4 ,k41 ,,hunter1 ,j4 ,s41 and ,,anderson1 ,r4 ,l4 ,the effects of various levels of sodium citrate1 glycerol1 and equilibration time on survival of bovine spermatozoa after storage at -#gi deg4 ,c4 ,jour4 ,dairy ,sci4 #ch3ejh-ead4 #aiee4 #aa ,,bratton1 ,r4 ,w41 ,,foote1 ,r4 ,h41 and ,,cruthers1 ,,joan ,c4 ,preliminary fertility results with frozen bovine spermatozoa4 ,jour4 ,dairy ,sci4 #ch3dj-df4 #aiee4 #ab ,,hafs1 ,h4 ,d41 and ,,elliott1 ,f4 ,i4 ,the effects of methods of adding egg yolk and monosaccharides on the survival of frozen bull spermatozoa4 ,jour4 ,dairy ,sci4 #ch3haa-hae4 #aiee4 #ac ,,miller1 ,w4 ,j41 and ,,vandemark1 ,n4 ,l4 ,the influence of glycerol level1 various temperature aspects1 and certain other factors on the survival of bull spermatozoa at sub-zero temperatures4 ,jour4 ,dairy ,sci4 #cg3de-ea4 #aied4 ,,temperature ,,conversions deg4,c4 deg4,f4 #ch #ajj #ce #ie #cj #hf #be #gg #bj #fh #ae #ei #aj #ej #e #da #j #cb -#e #bc -#aj #ad -#ae #e -#ah #j -#bj -#d -#be -#ac -#cj -#bb -#ce -#ca -#dj -#dj -#de -#di -#ej -#eh -#ee -#fg -#fj -#gf -#fe -#he -#gj -#id -#ge -#ajc -#gi -#aaj