---------------------------------------- -------------------------- ,,transcrib]',s ,,notes3 99 ,transcription us$ 9 ?is e-text3 text indicates italics2 text indicates bold-face text2 text indicates sup]script text2 text indicates subscript text4 99 ,lett]$ footnotes 7) an*ors ,a1 ,b1 etc471 expla9+ ! text1 h be5 mov$ to directly bel[ ! p>agraph or table !y ref] to4 ,footnotes ) an*ors #a1 #b1 etc4 ref] to ref]5ces1 t are li/$ t[>ds ! end ( ! text4 99 ,! orig9al text has n be5 modifi$1 except t "s m9or typographical errors h be5 correct$ sil5tly4 ---------------------------------------- -------------------------- ,,pres]vation ,,( ,,bull ,,sem5 ,,at ,,sub-,,z]o ,,temp]atures ,by ,n4 ,l4 ,van,demark ,w4 ,j4 ,mill] ,w4 ,c4 ,k9ney1 ,jr4 ,c>los ,rodriguez ,m4 ,e4 ,fri$man ,bullet9 #fba ,,univ]sity ,,( ,,ill9ois ,,agricultural ,,exp]im5t ,,/ation ,,cont5ts ,page ,,e>ly ,,"w ,,on ,,freez+ ,,sem5 #e ,,selection ,,( ,,sem5 ,,= ,,freez+ #f ,pr$ict+ freezability #f ,freezability ( fir/ & second ejaculates #g ,freezability ( sev]al consecutive ejaculates #g ,freezability ( epididymal sp]m #i ,freezability ( wa%$ sp]m #aj ,,ext5d]s ,,= ,,freez+ ,,bull ,,sem5 #aj ,proportion ( egg yolk 9 f9al dilu5t #aj ,citrate level 9 f9al dilu5t #aa ,/or+ & freez+ dilu5t #ab ,o!r dilu5ts #ac ,,dilution ,,rates #ad ,effect ( fur!r dilution & refreez+ #ad ,,glyc]ol ,,additions #ag ,effect ( glyc]ol on sp]m survival at #e deg4 ,c4 #ag ,glyc]ol levels = freez+ sem5 #ah ,rate1 temp]ature1 & me?od ( add+ glyc]ol #ah ,all[+ sp]m to equilibrate ) glyc]ol #bj ,sug> additions & equilibration "t #bj ,sub/itutes = glyc]ol #bb ,,freez+ ,,rate #bb ,effect ( freez+ rate on sp]m survival #bb ,rate ( cool+ 9 pla/ic & 9 glass #bc ,,/orage ,,temp]ature #bd ,/orage at -#bc deg4 to -#gi deg4 ,c4 #bd ,use ( high] glyc]ol levels & /orage at -#bj deg4 ,c4 #be ,,?aw+ #bf ,comp>ison ( ?aw+ at #e deg4 ,c4 & at #ch deg4 ,c4 #bg ,?aw+ rate 9 pla/ic & 9 glass #bi ,,effects ,,( ,,freez+ ,,proc$ures ,,on ,,metabolic ,,activity ,,( ,,sp]m #cj ,effect ( glyc]ol additions on oxyg5 uptake ( dilut$ sem5 at #cg deg4 ,c4 #cj ,effect ( glyc]ol-plus-catalase on oxyg5 uptake ( dilut$ sem5 #cb ,effect ( freez+ proc$ures on oxyg5 utilization #cc ,effect ( freez+ proc$ures on me?yl5e-blue r$uction "t #cc ,,practical ,,freez+ ,,proc$ure #ce ,collection ( sem5 #ce ,prep>ation ( ext5d] #ce ,dilution aft] collection #cf ,add+ ! glyc]ol #cf ,equilibration #cf ,freez+ #cg ,?aw+ #cg ,,lit]ature ,,cit$ #ch ,,temp]ature ,,conv]sion ,,table #ci ,urbana1 ,ill9ois ,octob]1 #aieg ,publications 9 ! ,bullet9 s]ies report ! results ( inve/igations made or sponsor$ by ! ,exp]im5t ,/ation ,,ackn[l$gm5t ,! au?ors gratefully ackn[l$ge ! f9ancial assi/ance giv5 to ! ,dep>tm5t ( ,dairy ,sci5ce 9 support ( ^! inve/igations by ! ,s\!rn ,ill9ois ,bre$+ ,association ( ,breese1 ,ill9ois1 & ! ,nor!rn ,ill9ois ,bre$+ ,co-op ( ,hamp%ire1 ,ill9ois1 ?r\gh ! ,ill9ois ,dairy ,bre$+ ,f$]ation4 ,,! ,,pres]vation ,,( ,,bull ,,sem5 ,,at ,,sub-,,z]o ,,temp]atures ,by ,n4 ,l4 ,,v&em>k1 ,w4 ,j4 ,,mill]1 ,w4 ,c4 ,,k9ney1 ,,jr41 ,,c>los ,,rodriguez1 & ,m4 ,e4 ,,fri$man,a ,a ,?is publication 0 prep>$ by ,n4 ,l4 ,,v&em>k1 ,pr(essor ( ,dairy ,physiology4 ,rese>* report$ h]e9 0 c>ri$ out coop]atively by ! s5ior au?or & ,w4 ,j4 ,,mill]1 ,w4 ,c4 ,,k9ney1 ,,jr41 ,,c>los ,,rodriguez1 & ,m4 ,e4 ,,fri$man1 =m]ly memb]s ( ! ,dep>tm5t ( ,dairy ,sci5ce4 ,! fir/ ann\ncem5t 9 #aiea ( ! successful impregnation ( a c[ ) bull sem5 t _h be5 froz5 /imulat$ mu* int]e/ & rese>* 9 freez+ z a me?od ( pres]v+ bull sem54 ,rese>* dur+ ! ye>s foll[+ #aiea result$ 9 consid]able progress & success 9 us+ freez+ z a means ( hold+ sem5 = long p]iods ( "t )\t loss ( f]tility4 ,betwe5 #aieb & #aieg1 rese>* on _m aspects ( ! pres]vation ( bull sem5 at sub-z]o temp]atures 0 c>ri$ out 9 ! ,dep>tm5t ( ,dairy ,sci5ce at ! ,univ]sity ( ,ill9ois4 ,_m ( ^! inve/igations h aid$ 9 p]fect+ ! freez+ te*nique t has be5 adapt$ = practical use4 ,"s ( ^! f9d+s h be5 publi%$1 b _m items h gone unreport$ except = g5]al ref]5ces at sci5tific meet+s4 ,x is ! purpose ( ?is bullet9 to br+ toge!r ! results ( sev]al exp]im5ts c>ri$ out 9 connection ) ! freez+ ( bull sem5 & to pres5t a practical freez+ proc$ure bas$ on ! results ( ^! exp]im5ts & f9d+s at o!r in/itutions4 ,p]sons int]e/$ 9 ! developm5t ( ! proc$ures & ! reasons :y c]ta9 /eps are necess>y 9 freez+ sem5 w f9d ! details 9 ! fir/ "p ( ?is bullet94 ,^? int]e/$ only 9 ! freez+ proc$ure may turn to page #ce :]e a practical me?od ( freez+ sem5 is describ$4 ,,e>ly ,,"w ,,on ,,freez+ ,,sem5 ,:ile x has be5 kn[n = a long "t t v>i\s types ( tissues & organisms c )/& freez+ & are ev5 pres]v$ by freez+1 ! fir/ attempts at freez+ sp]m cells 7 made be=e ! turn ( ! c5tury4 ,9 #ahig1 ,dav5port#a;,b f\nd t human sp]m w\ld )/& freez+4 ,= ?irty to =ty ye>s aft] t1 little att5tion 0 paid to freez+ z a possible means ( pres]v+ sem54 ,an excell5t review ( ! e>ly attempts to freeze sp]m has be5 assembl$ by ,polge & ,p>kes4#b ,^! inve/igators also gave a good acc\nt ( _! "w at ! ,briti% ,national ,in/itute ( ,m$ical ,rese>* 9 ,london1 :]e 9 #aidi !y demon/rat$ t glyc]ol w\ld protect f[l sp]m s t x w\ld survive freez+4 ,! next ye> !y f\nd t bull sp]m & ! sp]m ( sev]al o!r species 7 protect$ by glyc]ol dur+ freez+4 ,dur+ ! same ye>1 ,emm5s & ,black%aw#c %[$ t ram & bull sp]m w\ld survive freez+4 ,9 #aiea froz5 sem5 0 us$ to produce a calf 9 ,engl& & a lamb 9 ,au/ralia4 ,b ,^! numb]s ref] to lit]ature citations on page #ch4 ,! highlights 9 ! developm5t ( froz5 sem5 h be5 cov]$ by o!r reviews & reports4 ,int]e/$ p]sons w f9d ! articles ( ,polge & ,p>kes#b & ,smi?#d especially good on ! e>ly hi/ory & !oretical aspects ( freez+ sp]m4 ,lat] progress on ! freez+ proc$ure has be5 review$ & cov]$ 9 a numb] ( detail$ reports4#e1f1g ,_m items n cov]$ 9 ^? articles h be5 assembl$ "h4 ,,selection ,,( ,,sem5 ,,= ,,freez+ ,"o ( ! fir/ consid]ations 9 freez+ sem5 is t ( decid+ :i* sem5 samples are to be froz54 ,s9ce pres]vation ( ! sem5--! ma9t5ance ( ! pot5tial motility & especially ! f]tility ( ! sp]m--is ! prim>y aim1 "s att5tion %\ld be direct$ to ! k9d ( sem5 sample t w )/& freez+4 ,d ! initial *>act]i/ics ( ! sample indicate :e!r ! sp]m w )/& freez+8 ,does maturity ( ! sp]m affect _! freezability8 ,pr$ict+ freezability4 ,e/imates ( sem5 quality 9 ! pa/ h be5 bas$ 9 "p on ! numb]s ( sp]m pres5t 9 a fre% sample & on ! p]c5tage & rate ( motility %[n by ! sp]m4 ,^! *>act]i/ics 7 us$ to det]m9e ! relation%ip betwe5 ! orig9al conc5tration ( sp]m 79 ! fre%1 undilut$ sample71 ! p]c5tage & rate ( sp]m motility 9 ! dilut$ samples j prior to freez+1 & ! p]c5tage & rate ( sp]m motility foll[+ freez+ & ?aw+4 ,f data collect$ be=e & aft] freez+ & ?aw+ #ed ejaculates1 x 0 f\nd t !re 0 n a significant correlation betwe5 ! numb] ( sp]m pres5t 9 ! orig9al sample & ! p]c5t ( motile sp]m pres5t aft] freez+ & ?aw+ 7r #j4jc74 ,a highly significant correlation 7r #j4de7 0 f\nd1 h[ev]1 betwe5 ! p]c5tages be=e freez+ & aft] ?aw+4 ,:ile ?is correlation coeffici5t 0 highly significant1 its magnitude indicates t only ab\t "o fif? ( ! v>iation 9 p]c5tage ( motile sp]m obs]v$ aft] freez+ 0 acc\nt$ = by ! motility ( ! sp]m prior to freez+4 ,freezability ( fir/ & second ejaculates4 ,9 ! e>ly days ( artificial bre$+ 9 ?is c\ntry1 x 0 commonly believ$ t a second ejaculate collect$ a few m9utes aft] ! fir/ result$ 9 a l>g] ejaculate conta9+ m sp]m4 ,) ! developm5t ( ! proc$ure ( /imulat+ sexual excitem5t by re/ra9t prior to collect+ sem51 ?is diff]5ce betwe5 fir/ & second ejaculates has be5 greatly r$uc$4 ,/ill x 0 not$ t second ejaculates frequ5tly )/ood freez+ bett] ?an fir/ ejaculates1 ev5 ?\gh re/ra9t & /imulation ( ! bull occurr$ prior to collection ( ! fir/ ejaculate4 ,dur+ ! c\rse ( a numb] ( exp]im5ts1 x 0 possible to comp>e ! freezability ( #b ejaculates t 7 collect$ a few m9utes ap>t f ! same bull4 ,two consecutive ejaculates 7 obta9$ "o or m times f #bd bulls s t a total ( #eh comp>isons c\ld be made4 ,! mean prefreez+ & po/-?aw+ p]c5tages ( motile sp]m 9 fir/ & second ejaculates are pres5t$ 9 ,table #a4 ,an analysis ( v>iance %[$ t 9 ?is comp>ison ! diff]5ces betwe5 fir/ & second ejaculates 9 sp]m survival dur+ freez+ 7 highly significant4 ,a lat] comp>ison ( #bg fir/ & second ejaculates f #bf bulls did n %[ z great a diff]5ce betwe5 fir/ & second ejaculates 9 _! ability to )/& freez+ 7,table #a74 ,table #a4--;,comp>ison ( ! ,freezability ( ,fir/ & ,second ,ejaculates ,tak5 a ,few ,m9utes ,ap>t ,f ! ,same ,bull ,numb] ,numb] ,prefreez+ ,po/-?aw+ ( ( ,ejaculate motility motility ,survival bulls ejaculates 7p]c5t7 7p]c5t7 7p]c5t7 ---------------------------------------- ----------------------------- #bd #eh #a/ #fj #ci #fe #b;d #fb #de #gd #bf #bg #a/ #fj #cf #fj #b;d #fe #dj #fa ---------------------------------------- ----------------------------- ,freezability ( sev]al consecutive ejaculates4 ,! fact t second ejaculates sometimes )/ood freez+ bett] ?an fir/ ejaculates sugge/$ t ! maturity ( ! sp]m might be a factor affect+ freezability4 ,an opportunity to *eck ?is idea came :5 #bj consecutive ejaculates 7 collect$ f ea* ( #f bulls )9 a #d-;h\r p]iod4 ,! sp]m 9 samples collect$ 9 ?is mann] might be expect$ to be less mature ) ea* additional collection4 ,! results obta9$ 9 freez+ sev]al consecutive ejaculates are %[n 9 ,figure #a z av]ages = ! ejaculates f #f bulls4 ,9 same in/ances1 !re 0 an insuffici5t quantity ( sem5 available to te/ ! freezability4 7,proc$ure3 ,dilut$ to #cj x #ajf sp]m p] ml4 ) #a3a yolk-citrate1 !n cool$ & glyc]olat$ ) an equal volume ( #ad p]c5t glyc]ol 7p]c5t by volume7 9 #b4i p]c5t sodium citrate4 ,f9al sp]m conc5tration1 #ae x #ajf4 ,equilibration "t1 #ae h\rs4 ,freez+ rate1 #b deg4 ,c4 p] m9ute f #e deg4 to -#ai deg4 ,c4 !n #d deg4 ,c4 p] m9ute f -#ai deg4 to -#gi deg4 ,c4 ,held froz5 = #e or m h\rs !n ?aw$ 9 wat] at #e deg4 ,c4 & *eck$ = motility47 ,illu/ration3 ,p]c5t ( motile sp]m be=e & aft] freez+ consecutive ejaculates collect$ )9 a #d-;h\r p]iod f ea* ( #f bulls 7,fig4 #a7 ,9 g5]al1 ! motility be=e freez+ improv$ slightly f ! fir/ to ! f\r? to six? ejaculate & !n decl9$ until ab\t ! #ab? or #ad? ejaculate1 at :i* po9t ! prefreez+ motility seem$ to level (f ?r\gh ! #bj? ejaculate 7,fig4 #a74 ,! p]c5tage ( motile sp]m f\nd aft] freez+ & ?aw+ foll[$ ! same tr5d at an av]age level #aj to #ae p]c5t l[] ?an ! prefreez+ level4 ,z is readily se5 f ! tr5d l9es 9 ,fig4 #a1 ! diff]5ce betwe5 ! prefreez+ motility & ! po/-?aw+ motility increas$ gradually aft] ab\t ! fif? ejaculate4 ,al?\gh ! absolute diff]5ce did n increase greatly1 ! p]c5tage ( survival aft] freez+ dropp$ f #ha p]c5t on ! fir/ #e ejaculates to #bf4e p]c5t on ! la/ #e 7,table #b74 ,table #b4--;,comp>ison ( ! ,freezability ( #bj ,consecutive ,ejaculates ,collect$ ,)9 a #d-;,h\r ,p]iod 7,weight$ av]ages = #f bulls7 ,numb] ,prefreez+ ,po/-?aw+ ,ejaculate ( motility motility ,survival ejaculates 7p]c5t7 7p]c5t7 7p]c5t7 ---------------------------------------- --------------------------- #a/ to #e? #bi #ec4c #dc4b #ha4j #f? to #aj? #bf #dc4h #cj4b #fi4j #aa? to #ae? #bc #bh4f #ad4e #ej4g #af? to #bj? #ai #ah4a #d4h #bf4e ---------------------------------------- --------------------------- ,freezability ( epididymal sp]m4 ,s9ce ! freezability ( bull sem5 seem$ to be bett] 9 second ?an 9 fir/ ejaculates & "s improvem5t 9 freezability 0 evid5t ?r\gh ! fir/ #d to #f ejaculates tak5 consecutively1 ! "q ( :e!r epididymal sp]m w\ld )/& freez+ seem$ to be important4 ,al?\gh :5 #bj collections 7 made1 ! lat] ejaculates no d\bt conta9$ few] mature sp]m1 ! l[]$ freezability c\ld h be5 due to accessory gl& secretions r ?an *anges 9 ! sp]m !mselves4 ,remov+ sp]m directly f ! epididymis w\ld elim9ate any effect t ! accessory gl& secretions c\ld be ex]t+4 ,fur!r1 if epididymal sp]m c\ld be froz51 obta9+ & us+ sem5 f a bull %ortly aft] 8 dea? %\ld be possible4 ,table #c4--;,freezability ( ,sp]m 9 ,samples ,tak5 ,f ! ,epididymides ,prefreez+ ,po/-?aw+ ,bull motility motility ,survival 7p]c5t7 7p]c5t7 7p]c5t7 ---------------------------------------- --------- #a #ej #dj #hj #b #dj #ac #cb #c #fj #ae #be #d #cj #ae #ej #ef #dj #be #fb ,av]age #dd #bb #ej ---------------------------------------- --------- ,to det]m9e :e!r epididymal sp]m w\ld )/& freez+1 ! #ab epididymides 7cauda only7 ( #f slaught]$ bulls 7 flu%$ ) sal9e 7#j4i p]c5t7 & ! sp]m obta9$ 7 froz5 us+ ! same proc$ure z 0 us$ ) ! #bj consecutive ejaculates discuss$ e>li]4 ,av]ages ( ! #b epididymides f ea* bull are giv5 9 ,table #c2 ! samples f bulls #e & #f 7 comb9$4 ,f ! data 9 ,table #c1 x is obvi\s t motile sp]m 7 pres5t aft] freez+ & ?aw+ epididymal samples4 ,x is likely t fur!r exp]i5ce 9 h&l+ epididymal sp]m may lead to improv$ results4 ,us+ froz5 epididymal sp]m f #b bulls1 ,canadian work]s h produc$ confirm$ pregnancies 9 #h out ( #ab c[s4#h ,freezability ( wa%$ sp]m4 ,9 ! laboratory x is frequ5tly desirable to /udy sp]m free ( ! sem9al plasma 9 :i* !y are ejaculat$4 ,sp]m c be sep>at$ f ! sem9al plasma by c5trifugation1 removal ( ! sup]natant plasma1 & resusp5sion 9 a salt solution ( kn[n composition4 ,sometimes x is desirable to repeat ! process4 ,?is t5ds to wa% ! sp]m ) ! salt solution & sp]m h&l$ 9 ?is way are call$ wa%$ sp]m4 ,sp]m cells c5trifug$ ?ree times & wa%$ twice 9 #j4i p]c5t sodium *loride solution )/ood freez+ well :5 f9ally resusp5d$ & froz5 9 yolk-citrate dilu5t4 ,! p]c5tage ( survival 9 ?ree samples subject$ to ?is treatm5t 0 #fj p]c5t4 ,?us x appe>s t ! sem9al plasma itself is n ess5tial = ejaculat$ sp]m to survive ! rigors ( freez+4 ,?is is n surpris+1 s9ce x _h already be5 f\nd t epididymal sp]m1 :i* also are free ( accessory gl& secretions1 c )/& freez+ & ?aw+4 ,,ext5d]s ,,= ,,freez+ ,,bull ,,sem5 ,bo? ( ! ext5d]s t are widely us$ 9 r\t9e /orage ( bull sem5 at #e deg4 ,c4 are us$ = freez+ sem54 ,^! are ! egg yolk-sodium citrate & :ole or skimmilk ext5d]s4 ,mo/ ( ! rese>* ) ext5d]s = freez+ bull sem5 9 ?is laboratory has be5 done ) ! yolk-citrate dilu5ts4 ,proportion ( egg yolk 9 ! f9al dilu5t4 ,"s e>ly exp]i5ces ) a dilu5t consi/+ ( "o "p yolk & "o "p #b4i p]c5t sodium citrate dihydrate 9 di/ill$ wat] %[$ poor sp]m survival foll[+ freez+4 ,! f9al mixture ) ?is dilu5t consi/$ ( ab\t #de p]c5t yolk4 ,9 o!r attempts at add+ glyc]ol 9 ord] to freeze sem51 ! f9al proportion ( yolk 0 dim9i%$ & bett] sp]m survival 0 obta9$4 ,sev]al exp]im5ts 7 c>ri$ out to te/ ! effect ( v>y+ levels ( egg yolk4 ,9 ! fir/ ef=ts to f9d ! optimum level ( egg yolk1 ! level ( yolk 9 ! f9al froz5 mixture 0 v>i$ f ab\t #f to #df p]c5t4 ,^! levels 7 obta9$ by v>y+ ! proportion ( yolk to #b4i p]c5t citrate solution 9 ! orig9al ext5d+ m$ia & also 9 ! m$ia add$ 9 glyc]olat+ ! samples4 ,split portions ( #bj sem5 samples 7 froz5 9 ea* ( ! ext5d] comb9ations indicat$ 9 ,table #d4 ,! mean p]c5tages ( motile sp]m f\nd be=e & aft] freez+ & ?aw+ are %[n also4 ,! highe/ p]c5tages 7 f\nd ) ext5d]s conta9+ #bc & #bd p]c5t yolk4 ,! highe/ p]c5tage ( yolk1 result+ :5 a #a3a 7yolk to citrate7 ext5d] 0 us$ = bo? ext5d+ & glyc]olat+1 prov$ to be mo/ detrim5tal to sp]m survival dur+ freez+4 ,! l[e/ p]c5tage ( yolk us$ 7#f p]c5t7 0 n z effective 9 protect+ sp]m dur+ freez+ z ! int]m$iate levels te/$ 7,fig4 #b74 ,table #d4--;,effect ( ,egg-,yolk ,level 9 ,dilu5t on ,freezability ( ,sem5 7,av]age ( #bj sem5 samples7 ,m$ium ,yolk 9 ,pre- ,po/- ,sur- ---------------------------- f9al freez+ ?aw+ vival ,dilu5t ,ext5d+ ,glyc]olat+,c mix- motility motility 7p]- ------------ ---------------- ture,d 7p]c5t7 7p]c5t7 c5t7 yolk3citrate yolk3citrate 7p]c5t7 ---------------------------------------- --------------------------------- #a #a3a #a3a #de4g #fe #e #h #b #a3a #a3c #cd4i #fd #bg #db #c #a3c #a3a #cc4f #fc #cc #eb #d #a3a #j3a #bd4b #fd #ci #fa #e #a3c #a3c #bb4h #fc #cg #ei #f #a3c #j3a #ab4a #ei #cc #ef #g #a3g #a3g #aa4d #ef #ce #fb #h #a3g #j3a #f4j #eb #bf #ej #i #a3ae #a3ae #e4g #di #be #ea ---------------------------------------- --------------------------------- ,c ,?is mixture includ$ #ad p]c5t glyc]ol4 ,d ,! av]age initial sp]m conc5tration 0 #ijj x #ajf_/ml4 ,suffici5t ext5d] 0 add$ to give #cj x #ajf_/ml4 at ! fir/ ext5sion4 ,?us ! f9al conc5tration 0 #ae x #ajf sp]m_/ml4 aft] glyc]olization4 ,s9ce r l>ge *anges 9 ! p]c5tages ( yolk 7 us$ 9 ?is exp]im5t1 two fur!r trials 7 conduct$ 9 :i* #af1 #bd1 & #cb p]c5t yolk 9 ! f9al mixture 7 comp>$1 ) ! f9al citrate p]c5tages held con/ant4 ,9 ^! te/s1 #af & #bd p]c5t yolk ma9ta9$ sp]m bett] at all citrate levels tri$ ?an #cb p]c5t yolk4 ,! #af p]c5t level 0 slightly bett] at mo/ ( ! levels ( citrate te/$ 7,fig4 #c74 ,citrate level 9 ! f9al dilu5t4 ,! e>ly "w ( ! ,briti% indicat$ t a f9al citrate level ne> #b p]c5t 9 ! dilu5t 0 satisfactory = freez+ bull sp]m4 ,lat]1 9 a p]sonal communication1 ,polge ( ! ,briti% gr\p sugge/$ t a citrate level ( ab\t #b4ce p]c5t might be be/ ) a f9al glyc]ol conc5tration ( #g p]c5t4 ,"s ( ! fir/ attempts 9 ?is laboratory at e/abli%+ ! optimum yolk-to-citrate ratios are %[n 9 ,fig4 #c4 ,9 ^! exp]im5ts1 ! optimum levels ( citrate appe>$ to be l[] ?an anticipat$ f ! ,briti% "w4 ,?us a m complex exp]im5t 0 set up to te/ a wid] range ( citrate levels us+ #af & #bd p]c5t egg yolk 9 ! f9al freez+ mixture4 ,! av]age p]c5tages ( motile sp]m f\nd aft] freez+ #aj sem5 samples at ea* ( ! citrate & yolk levels 9 ?is exp]im5t are %[n also 9 ,fig4 #c4 ,little diff]5ce 9 freezability 0 f\nd betwe5 citrate p]c5tages ( #a4ee & #a4ie4 ,:5 ! rate ( sp]m motility foll[+ freez+ & ?aw+ 0 consid]$ along ) ! p]c5t ( motile sp]m1 a slight advantage 0 f\nd ) #af p]c5t yolk & a citrate conc5tration ( #a4ee p]c5t4 ,illu/ration3 ,p]c5t ( motile sp]m aft] freez+ & ?aw+ sem5 9 dilu5ts conta9+ v>i\s levels ( egg yolk 7,fig4 #b7 ,f ! results ( ^! exp]im5ts1 & f sev]al reports 9 ! lit]ature1#e1f1g1i1aj x appe>s t a dilut+ m$ium result+ 9 a f9al conc5tration ( #af to #be p]c5t yolk & #a4ee to #b4b p]c5t sodium citrate dihydrate is highly satisfactory = freez+4 ,/or+ & freez+ dilu5t4 ,9 "s in/ances x w\ld be advantage\s to h prep>$ dilu5t on h& = use at any "t4 ,! suitability ( /or$ dilu5t 0 te/$ ) a yolk-citrate 7equal p>ts yolk & citrate )\t antibiotics add$7 dilu5t prep>$ & /or$ at #e deg4 ,c4 = #j1 #b1 #e1 #g1 & #i days4 ,sev5 sem5 samples 7 dilut$ & froz5 9 ^! dilu5ts4 ,no diff]5ce 0 not$ 9 ! survival ( sp]m t c\ld be attribut$ to ! age ( ! dilu5t4 ,illu/ration3 ,p]c5t ( motile sp]m aft] freez+ & ?aw+ sem5 9 dilu5ts conta9+ v>i\s levels ( egg yolk & v>i\s p]c5tages ( sodium citrate 7,fig4 #c7 ,9 ano!r trial1 a simil> dilu5t 7#a3a yolk to citrate ) #ajjj units ( p5icill9 & #ejjj units ( /reptomyc97 0 prep>$ & /or$ 9 ! freez] comp>tm5t ( a refrig]ator at -#ae deg4 ,c4 ,^u ?aw+1 x 0 :iti% 9 color & m visc\s ?an fre%ly prep>$ dilu5t4 ,except = ! fact t ! viscosity seem$ to r$uce ! rate ( sp]m motility1 ?is froz5 dilu5t /or$ = #fe days comp>$ favorably ) fre%ly prep>$ dilu5t = freez+ sem54 ,o!r dilu5ts4 ,)\t ! protective action ( egg yolk or milk1 few bull sp]m w survive freez+4 ,sev]al dilu5ts 7 comp>$ on a limit$ scale = freez+ bull sp]m4 ,! results ( ^! trials are compil$ 9 ,table #e4 ,9 ?is trial ! yolk-citrate ext5d] s]v$ be/ 9 ma9ta9+ sp]m motility dur+ freez+4 ,yolk-phosphate & homog5iz$ :ole milk 7 slightly less protective & yolk-sal9e seem$ to furni% ! lea/ protection to sp]m dur+ freez+4 ,a numb] ( inve/igations 9 o!r laboratories h n[ prov5 t milk c be us$ z effectively z ! yolk-citrate dilu5t = freez+ bull sp]m4#f1g ,table #e4--;,comp>ison ( ! ,freezability ( #d ,sem5 ,samples 9 ,diff]5t ,ext5d]s ,dilution ,pre- ,po/- ,motility ,ext5d] rate freez+ ?aw+ ,survival aft] 7sem53 motility motility 7p]c5t7 /orage,e ext5d]7 7p]c5t7 7p]c5t7 7p]c5t7 ---------------------------------------- ------------------------------ ,yolk-citrate #a3a #fj #di #hb #df #a3aj #ec #de #he #cf ,yolk-sal9e #a3a #eg #bi #ea #bh #a3aj #fj #ca #eb #bd ,yolk-phosphate #a3a #ee #cc #fd #be #a3aj #fj #dc #gb #be ,:ole milk #a3a #fj #dj #fg #ce #a3aj #fj #ce #eh #af ---------------------------------------- ------------------------------ ,e ,/or$ at #e deg4 ,c4 = #g h\rs aft] ?aw+4 ,,dilution ,,rates ,! fir/ trials by ! ,briti% at freez+ bull sem5 7 made ) samples conta9+ _m millions ( sp]m cells4 ,9 r\t9e artificial bre$+1 x is common to add ext5d]s to sem5 s t "o millilit] ( dilut$ sem5 may conta9 only #aj million liv+ sp]m cells4 7,?is numb] /ill insures optimal f]tility47 ,frequ5tly ! addition ( #ajj or m p>ts ( ! yolk ext5d] to ea* "p ( ! orig9al sem5 sample is possible )\t r$uc+ ! sp]m numb]s bel[ #aj million p] millilit]4 ,no "o knew if ?is process ( dilution w\ld affect ! resi/ance ( bull sp]m to freez+4 ,! effect ( v>i\s rates ( dilution on ! freezability ( bull sp]m 0 te/$ ) #aj sem5 samples4 ,! results1 pres5t$ 9 ,table #f1 %[ t ! numb]s ( sp]m betwe5 #aj & #ij million p] millilit] did n influ5ce ! p]c5tage ( sp]m t surviv$ freez+4 ,9 a lat] trial x 0 f\nd t sp]m survival 0 slightly bett] at l[] dilution rates ?an 9 ! same samples froz5 foll[+ dilution to #ae million sp]m p] millilit]4 ,h[ev]1 field trials ) froz5 sem5 c>ri$ out by o!rs1 us+ sp]m numb]s z l[ z #ae million p] millilit] ( sem5 insem9at$ or ev5 l[]1 h be5 highly satisfactory4#aa1ab ,dur+ ! e>ly /udies 9 ! ,ill9ois laboratory1 ! effects ( glyc]ol level 7 also te/$4#ac ,^! effects are discuss$ 9 ! section on glyc]ol additions beg9n+ on page #ag4 ,effect ( fur!r dilution & refreez+ aft] ! initial freez+4 ,"u "s circum/ances x might be advantage\s to freeze sem5 ) a high conc5tration ( sp]m cells & !n ext5d x fur!r aft] ?aw+4 ,) su* a proc$ure less /orage space is ne$$ ?an :5 dilution is c>ri$ to ! maximum be=e freez+4 ,two exp]im5ts 7 conduct$ to te/ ! effects ( dilution & /orage at #e deg4 ,c4 & dilution & refreez+ foll[+ an initial freez+ ( conc5trat$ samples4 ,table #f4--;,effect ( ,sp]m ,numb]s & ,glyc]ol ,level 9 ,f9al ,mixture on ,freezability ( ,bull ,sp]m at -#gi deg4 ,c4 7,av]age ( #aj ejaculates7 ,po/-?aw+ motility 7p]c5t7,f ---------------------------------------- ----- ,glyc]ol level ,numb] ( sp]m 7millions_/ml47 7p]c5t7 -------------------------------- #ij #cj #aj ,av]age ---------------------------------------- ------------------------ #e #cf4j #cd4j #cf4j #ce4j #aj #bb4j #bd4j #bc4j #bc4j #ae #c4b #j4i #j4b #a4d ,av]age #bj4c #ai4h #ai4i #bj4j ---------------------------------------- ------------------------ ,f ,mean initial motility ( sp]m be=e freez+ 0 #ee p]c5t4 ,f\r sem5 samples 7 split & ext5d$ at rates ( #a3a 7sem5 to ext5d]7 & #a3aj4 ,^! 7 froz51 !n ?aw$ & halv$4 ,"o half 0 fur!r ext5d$ to a level ( #ae million sp]m p] millilit]2 ! sp]m numb]s 9 ! o!r rema9$ un*ang$4 ,ea* ( ^! halves 0 split aga91 & "o portion ( ea* 0 /or$ at #e deg4 ,c4 = #c to #g h\rs4 ,! o!r two portions 7 refroz54 ,table #g4--;,effect ( ,fur!r ,dilution & ,refreez+ on ,sp]m ,motility ,aft] ! ,initial ,freez+ ( ,bull ,sem5 ,po/-?aw+ motility ,dilution ,pre- ---------------------------------------- ----------- ( freez+ ,aft] ,aft] /orage,g ,aft] refreez+,h sem5 motility fir/ ------------------- -------------------- 7sem53 7p]c5t7 freez+ ,no ,dilut$ ,no ,dilut$ ext5d]7 fur!r to #ae fur!r to #ae dilution million_/ml dilution million_/ml ---------------------------------------- -------------------------------- ,fir/ 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 ,/or$ at #e deg4 ,c4 = #c to #g h\rs aft] fir/ ?aw+4 ,h ,refroz5 foll[+ fir/ ?aw+4 ,table #h4--;,effect ( ,glyc]ol ,level & ,/orage at #e deg4 ,c4 on ,motility ( ,sp]m 9 ,yolk-,citrate ,ext5d] ,sp]m motility ---------------------------------------- ---------------- ,po/- ,aft] /orage at #e deg4 ,c4 ,glyc]ol ?aw+ ---------------------------- ,av]age level #a day #c days #g days 7p]c5t7 --------- --------- --------- --------- --------- p]- rate p]- rate p]- rate p]- rate p]- rate c5t c5t c5t c5t c5t ---------------------------------------- ---------------------------- ,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 ,av]age #ec #b4jc #dg #a4fb #dd #a4ej #cd #a4bb 44 4444 ---------------------------------------- ---------------------------- ,i ,! control diff]$ f ! #j-;glyc]ol treatm5t 9 t no additional citrate or glyc]ol solution 0 add$4 ,a simil> trial 0 c>ri$ out ) sev5 samples2 "o portion 0 dilut$ #a3i2 ! o!r 0 ext5d$ at ! outset to #ae million sp]m p] millilit]4 ,results = bo? te/s are summ>iz$ 9 ,table #g4 ,f ,table #g x c be se5 t refreez+ foll[+ an initial freez+ fur!r r$uc$ ! numb] ( surviv+ sp]m4 ,! second freez+ 0 m detrim5tal to ! portion ( ! samples ext5d$ to #ae million sp]m p] millilit] ?an to ! portion t 0 refroz5 at a high] sp]m conc5tration4 ,! p]c5tage ( motile sp]m rema9$ fairly high 9 ! portions t 7 dilut$ to #ae million sp]m & /or$ at #e deg4 ,c4 ,h[ev]1 9 all cases1 survival 0 be/ 9 ! samples at ! l[] dilution levels4 ,,glyc]ol ,,additions ,:5 ! ,briti% proc$ure = freez+ bull sem5 0 fir/ tri$ 9 ?is c\ntry1 _m ( ! ref9em5ts ( ! te*nique /ill _h n be5 def9$4 ,x 0 kn[n t glyc]ol work$ well 9 protect+ sp]m dur+ freez+4 ,! effects ( glyc]ol on sp]m at #e deg4 ,c41 ! appropriate levels to use 9 freez+1 & ! mann] ( add+ x 7 n well e/abli%$4 ,!re=e1 a numb] ( trials 7 conduct$ 9 an attempt to e/abli% ! be/ proc$ures4 ,effect ( glyc]ol on sp]m survival at #e deg4 ,c4 ,s9ce e>ly "w indicat$ ! ne$ = add+ glyc]ol to dilut$ sem5 9 ord] to protect ! sp]m dur+ freez+1 x 0 consid]$ important to det]m9e ! levels ( glyc]ol t sp]m w\ld tol]ate at #e deg4 ,c4 ,t5 sem5 samples 7 ext5d$ #a3i 7sem5 to dilu5t7 9 a #a3a yolk-citrate dilu5t 7yolk to #b4i p]c5t sodium citrate dihydrate74 ,ea* sample 0 !n split into #f portions & an equal volume ( citrate solution conta9+ glyc]ol 0 add$ sl[ly to ea* to br+ ! glyc]ol 9 ! f9al mixture to #j1 #e1 #aj1 #bj1 or #cj p]c5t 7by volume74 ,^! samples 7 /or$ at #e deg4 ,c4 & exam9$ = motile sp]m aft] #a1 #c1 & #g days4 ,! effects ( glyc]ol levels on ! p]c5tage ( sp]m surviv+ & ! rate 7or spe$7 ( _! =w>d motion 7#j no =w>d motion2 #d extremely rapid progressive motility7 are pres5t$ 9 ,table #h4 ,! p]c5tage ( motile sp]m decreas$ slightly at ! high] levels ( glyc]ol4 ,! mo/ noticeable effect ( ! increase 9 glyc]ol level 0 ! r$uction 9 ! rate ( =w>d motion ( ! sp]m4 ,at ! #cj-p]c5t level1 ! sp]m mov$ sl[ly & c\ld be se5 to rotate z !y mov$ =w>d4 ,"s samples 7 *eck$ aft] sl[ly br++ ! dilu5t up to a level ( #dj p]c5t glyc]ol2 ! sp]m seem$ to be immobiliz$ completely 9 ?is solution4 ,glyc]ol levels = freez+ sem54 ,! ,briti% proc$ure call$ = ! use ( #aj p]c5t glyc]ol 9 ! f9al mixture ( sem5 & ext5d] prior to freez+4 ,yet1 z %[n 9 ,table #f1 9 our laboratory #e p]c5t glyc]ol result$ 9 ! survival ( a high] p]c5tage ( sp]m ?an did #aj or #ae p]c5t4 ,9 ord] to def9e m cle>ly ! optimum glyc]ol level1 sev]al ejaculates ( sem5 7 subsampl$ & portions 7 froz5 aft] ! addition ( yolk-citrate ext5d] & glyc]ol 9 v>y+ quantities4 ,f ,table #i x c be se5 t glyc]ol levels ( #f & #h p]c5t 9 ! f9al mixture result$ 9 maximum sp]m survival dur+ freez+4 ,^! results 7 confirm$ 9 te/s on ! survival ( sp]m at #e deg4 ,c4 /orage = #c days foll[+ freez+ & ?aw+ ) v>y+ glyc]ol levels 7see ,table #aj74 ,! results %[n 9 ,tables #i & #aj 7 confirm$ also 9 lat] exp]im5ts4 ,?irty-six samples 7 subject$ to v>i\s levels ( glyc]ol & no significant diff]5ce 9 freezability 0 f\nd betwe5 #f & #h p]c5t4 ,bas$ on ^! f9d+s1 a glyc]ol level ( #g p]c5t 0 adopt$ = use 9 all exp]im5ts describ$ 9 ?is bullet91 unless o!rwise indicat$4 ,results 9 a numb] ( o!r laboratories h agre$ ) our f9d+s reg>d+ ! use ( approximately #g p]c5t glyc]ol ) ! yolk-citrate dilu5t4#e1f1g1i1aj ,) milk z ! ext5d]1 #aj to #ac p]c5t glyc]ol has be5 pref]r$ by "s4#e1f1g ,table #i4--;,effect ( ,glyc]ol ,level on ,sp]m ,motility ,aft] ,freez+ to -#gi deg4 ,c4 & ,?aw+ ,glyc]ol ,numb] ,pre- ,po/- ,survival level ( freez+ ?aw+ 7p]c5t7 7p]c5t7 samples motility motility 7p]c5t7 7p]c5t7 ---------------------------------------- ------------------ #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 ( ,glyc]ol ,level & ,/orage at #e deg4 ,c4 ,aft] ,?aw+ on ,sp]m ,motility 7,av]age ( #ac ejaculates7 ,sp]m motility 7p]c5t7 ,glyc]ol ---------------------------------- level ,po/- ,aft] /orage at #e deg4 ,c4 7p]c5t7 ?aw+ ----------------------- #a day #c days ---------------------------------------- -------- #d #bi #bb #bj #f #ch #cd #bd #h #db #cc #ag #aj #cc #ah #f ---------------------------------------- -------- ,table #aa4--;,effects ( ,temp]ature1 ,rate ( ,addition ( ,glyc]ol1 & ,equilibration ,"t on ,sp]m ,motility 7,av]age ( #ab ejaculates7 ,temp]ature ,po/-?aw+ motility 7p]c5t7 dur+ ,equilibration ---------------------------------- addition "t ,glyc]ol additions ( glyc]ol 7h\rs7 ---------------------------------- 7 deg4 ,c47 #e #c #a ,av]age ---------------------------------------- --------------------------- #d4e #b #dh #dh #de #dg4d #f #di #ea #dg #dh4h #ah #df #dg #df #df4c ,av]age #dg4h #dh4f #df4j #dg4e #aj4j #b #dd #dc #de #dc4i #f #dh #ej #df #dg4i #ah #dc #df #db #dd4j ,av]age #de4j #df4e #dd4c #de4c #ae4e #b #da #ch #ch #ci4a #f #db #de #dc #dc4f #ah #db #dc #db #db4e ,av]age #db4j #da4h #da4d #da4g ---------------------------------------- --------------------------- ,rate1 temp]ature1 & me?od ( add+ glyc]ol4 ,closely associat$ ) ! "q ( h[ mu* glyc]ol %\ld be add$ is t ( h[ ! additions %\ld be made4 ,orig9ally x 0 believ$ t ! glyc]ol %\ld be add$ 9 /ages s t *anges w\ld occur gradually4 ,h[ev]1 !re w\ld be a sav+ 9 "t if ! entire am\nt c\ld be add$ at once4 ,also1 if ! glyc]ol addition c\ld be made soon aft] ! dilution ) egg yolk-citrate ext5d] at room temp]ature1 "t w\ld be ga9$ 9 process+ ! sem5 = use4 ,s9ce ag+ 9 vitro is kn[n to r$uce ! f]tiliz+ ability ( sp]m1 e ef=t %\ld be made to keep ! process+ "t at a m9imum4 ,! results ( an exp]im5t involv+ ^! items1 along ) t ( h[ mu* "t %\ld be all[$ aft] ! additions be=e freez+ 7equilibration "t71 are pres5t$ 9 ,table #aa4 ,"o c see t sp]m surviv$ freez+ bett] :5 ! dilut$ sem5 0 cool$ to #d4e deg4 ,c4 be=e ! glyc]ol 0 add$4 ,! survival at #aj deg4 & #ae4e deg4 ,c4 0 r$uc$ ) ea* rise 9 temp]ature4 ,?us1 x appe>s t cool+ to refrig]ator temp]ature 7#d-e deg4 ,c47 be=e add+ ! glyc]ol %\ld be a "p ( ! r\t9e proc$ure4 ,a comp>ison ( ! results f add+ ! glyc]ol 9 #e1 #c1 & #a equal portions is giv5 also 9 ,table #aa4 ,little diff]5ce 9 survival dur+ freez+ 0 not$ betwe5 ! ?ree rates ( addition4 ,us+ #c equal additions result$ 9 slightly bett] results1 b ! advantage 0 n /ati/ically significant4 ,:ile little diff]5ce 0 evid5t f add+ ! glyc]ol 9 #c portions z comp>$ to #a1 _m /ill use #c additions 9 ! hope ( obta9+ a slightly bett] sp]m survival4 ,9 fact1 "s h gone to a proc$ure ( add+ ! glyc]ol dropwise ) con/ant g5tle agitation4 ,?is me?od has n be5 te/$ 9 ?is laboratory4 ,all[+ sp]m to equilibrate ) ! glyc]ol4 ,all[+ sp]m to /& 9 ! pres5ce ( glyc]ol is consid]$ by "s to be necess>y 9 ord] t ! glyc]ol p5etrate ! sp]m heads be=e freez+4 ,f ! fir/ successful attempts at freez+ bull sp]m came ! practice ( all[+ #ab to #bj h\rs = ?is process ( equilibration4 ,a long equilibration "t results 9 ag+ ! sp]m4 ,data f a numb] ( s\rces indicate t a drop ( approximately #e p]c5t 9 f]tility 9 ! field occurs ) ea* #bd h\rs ( ag+ 9 ! te/ tube4 ,?us x w\ld seem desirable to r$uce ! equilibration "t to a m9imum comm5surate ) good freezability 9 ord] to r$uce ! effects ( ag+ 7at #e deg4 ,c474 ,results ( attempt+ to r$uce equilibration "t are giv5 9 ,table #aa4 ,at #d4e deg4 ,c41 little v>iation 9 motility foll[+ freez+ & ?aw+ 0 f\nd aft] equilibration times ( #b1 #f1 & #ah h\rs4 ,at ! high] temp]atures ( #aj deg4 & #ae4e deg4 ,c41 ! %orte/ equilibration "t--#b h\rs--0 slightly m detrim5tal ) ! diff]5ces significant at ! #e-p]c5t level at #ae4e deg4 ,c4 ,= all temp]atures comb9$1 #f h\rs 0 significantly bett] ?an #b or #ah h\rs4 ,sug> additions & equilibration "t4 ,e>ly 9 _! exp]i5ces 9 freez+ sem51 ! ,au/ralian work]s f\nd a %ort equilibration "t--#cj m9utes--to be satisfactory if sug>s 7 add$ to ! dilu5t4#e ,?is protective action ( sug>s dur+ ! equilibration p]iod 0 confirm$ 9 our inve/igations4 ,! results ( "o phase ( ?is /udy are %[n 9 ,table #ab4 ,f ^! data x c be se5 t ! pres5ce ( glucose or rhamnose at a level ( #a4be p]c5t improv$ sp]m survival dur+ ! p]iod ( equilibration4 ,9 ano!r trial ^! sug>s & two o!rs1 arab9ose & xylose1 7 te/$ = _! protective action 9 freez+ sem54 ,! p]c5tages ( surviv+ sp]m rema9+ aft] ! v>i\s /eps 9 ! freez+ proc$ure ) & )\t ! pres5ce ( ^! sug>s are %[n 9 ,table #ac4 ,table #ab4--;,effect ( ,add+ ,sug>s to ,yolk-,citrate ,dilu5t on ,sp]m ,motility ,dur+ ,equilibration ,) ,glyc]ol,j ,sp]m motility 7p]c5t7 ------------------------------------- ,/age :5 obs]v$ ,glyc]ol ,glyc]ol ,glyc]ol only & glucose & rhamnose ---------------------------------------- -------------------- ,fre% dilut$ sem5 #ef #ef #ef ,aft] glyc]olization #ed #ed #ed ,aft] equilibration #b h\rs #ea #ec #ec #f h\rs #dh #eb #ec #ab h\rs #df #ej #ea #ah h\rs #dj #df #df ---------------------------------------- -------------------- ,j ,glyc]ol level 9 ! f9al froz5 mixture 0 #g p]c5t4 ,sug>s 7 add$ to a level ( #a4be p]c5t4 ,?ree ( ! sug>s--glucose1 arab9ose1 & rhamnose--protect$ ! sp]m dur+ equilibration & freez+4 ,xylose 0 less effective1 b its addition result$ 9 slightly bett] sp]m survival ?an glyc]ol alone4 ,x 0 f\nd also t ! me?yl5e-blue r$uction "t 7metabolic te/ = sem5 quality7 0 fa/] 9 samples to :i* ! sug>s _h be5 add$--aft] glyc]olization1 aft] equilibration1 & aft] freez+ ! samples4 ,?is is confirm+ evid5ce = ! pres5ce ( m liv+ & actively metaboliz+ sp]m 9 ! portions to :i* sug>s _h be5 add$4 ,table #ac4--;,effect ( ,add+ ,sug>s to ,yolk-,citrate ,dilu5t on ,sp]m ,motility ,dur+ ! ,freez+ ,proc$ures,k 7,av]age ( #aj ejaculates7 ,sp]m motility 7p]c5t7 ---------------------------------------- --------- ,/age ( ,glyc]ol ,glyc]ol ,glyc]ol ,glyc]ol ,glyc]ol obs]vation only & & & & glucose arab9ose xylose rhamnose ---------------------------------------- ------------------------------ ,fre% dilut$ sem5 #fc #fc #fc #fc #fc ,aft] glyc]olization #ed #ee #ed #eg #fj ,aft] #ah h\rs equilibration #ci #dc #dd #ci #df ,aft] freez+ to -#gi deg4 ,c4 & imm$iate ?aw+ #bh #cd #cd #bi #bd ,aft] #d days at -#gi deg4 ,c4 #bc #bf #bf #be #bg ---------------------------------------- ------------------------------- ,k ,glyc]ol level 9 ! f9al froz5 mixture 0 #g p]c5t4 ,sug>s 7 add$ to a level ( #a4be p]c5t4 ,sub/itutes = glyc]ol4 ,s9ce glyc]ol 0 s effective 9 protect+ sp]m dur+ freez+1 _m h assum$ t relat$ comp\nds might be ev5 bett]4 ,sev]al comp\nds1 "s relat$ to glyc]ol & "s n1 h be5 tri$ z sub/itutes = glyc]ol 9 ! freez+ proc$ure4 ,!y include e?yl5e glycol1 propyl5e glycol1 trime?yl5e glycol1 mannitol1 sorbitol1 dextrans1 & sem9al-plasma prote9s4 ,none ( ^! mat]ials has be5 z effective z glyc]ol 9 protect+ sp]m dur+ freez+4 ,9 fact1 sev]al ( ! mat]ials prov$ to be injuri\s to sp]m prior to attempts to freeze ! samples4 ,:ile ! "w 9 our laboratory ) ^! sub/ances z glyc]ol sub/itutes 0 by no means f9ally conclusive1 because ( ! _m possible int]actions ( exp]im5tal conditions1 suffici5t data 7 ga!r$ to lead u to ab&on fur!r /udy until great] promise ( success might be evid5t4 ,,freez+ ,,rate ,effect ( freez+ rate on sp]m survival4 ,reports by "o gr\p ( ,briti% work]s 9 e>ly trials on freez+ bull sem5 indicat$ t ! rate ( cool+ 9 freez+ %\ld n exce$ #b deg4 ,c4 p] m9ute betwe5 #e deg4 & -#ae deg4 ,c41 al?\gh bel[ -#ae deg4 ,c4 ! rate c\ld be fa/]4 ,ano!r gr\p express$ ! view t sem5 c\ld be plung$ into dry ice at -#gi deg4 ,c4 aft] x _h be5 cool$ to -#ae deg4 ,c4 ,to cl>ify ?is "p ( ! freez+ proc$ure1 #aa samples ( sem5 7 subdivid$ & portions ( ea* 7 froz5 at rates ( #j4be deg41 #j4e deg41 #a4j deg41 #b4j deg41 & #d4j deg4 ,c4 drop p] m9ute betwe5 #e deg4 & -#bj deg4 ,c4 & !n twice ^! rates betwe5 -#bj deg4 & -#gi deg4 ,c4 ,vials ( ea* ejaculate at #e deg4 ,c4 7 also plung$ directly into an alcohol ba? at -#gi deg4 ,c4 ,! samples :i* 7 cool$ at ! rates ( #j4be deg41 #j4e deg41 #a4j deg41 #b4j deg41 & #d4j deg4 ,c4 p] m9ute _h ! foll[+ p]c5tages ( motile sp]m aft] ?aw+3 #cj1 #dj1 #df1 #dd1 & #dd4 ,a mean ( #cb p]c5t ( ! sp]m 9 ! samples t 7 plung$ directly into an alcohol ba? at -#gi deg4 ,c4 7 motile aft] ?aw+4 ,!re 7 no /ati/ically significant diff]5ces among ! samples froz5 at #a4j deg41 #b4j deg4 or #d4j deg4 ,c4 p] m9ute4 ,all ( ! o!rs _h significantly l[] survival rates4 ,?us1 x is obvi\s t too sl[ a cool+ rate & plung+ ! samples directly into a -#gi deg4 ,c4 ba? f a temp]ature ( #e deg4 ,c4 cause great] h>m to ! sp]m ?an cool+ at a rate betwe5 #a4j deg4 & #d4j deg4 ,c4 p] m9ute4 ,"s inve/igators h sugge/$ t rapid cool+ bel[ -#bj deg4 ,c4 is n detrim5tal to froz5 sem54 ,?is idea 0 te/$ 9 conjunction ) o!r exp]im5ts4 ,tw5ty-five samples cool$ sl[ly 7#b deg4 ,c4 p] m9ute to -#bh deg4 ,c41 !n #d deg4 ,c4 p] m9ute to -#gi deg4 ,c47 %[$ #fb p]c5t sp]m survival comp>$ ) only #de p]c5t :5 cool$ rapidly bel[ -#bh deg4 ,c4 7#b deg4 ,c4 p] m9ute to -#bh deg4 ,c4 !n plung$ into ba? at -#gi deg4 ,c474 ,?us1 rapid cool+ 0 detrim5tal ev5 aft] ! critical temp]ature range ( #e deg4 ,c4 to -#bj deg4 ,c4 _h be5 pass$4 ,illu/ration3 ,cool+ rates ( dilut$ sem5 samples 9 pla/ic vials & 9 glass ampules 7,fig4 #d7 ,rate ( cool+ 9 pla/ic & 9 glass4 ,pla/ic vials d n conduct ! cold z rapidly z glass ampules d4 ,! temp]ature 9 bo? glass & pla/ic conta9]s t5ds to lag beh9d ! *ange 9 ! ba? 9 :i* !y are imm]s$ z is %[n 9 ,figure #d4 ,temp]atures 9 ! imm]sion ba? 7 record$ 9 a #b-millilit] glass ampule conta9+ #a millilit] dilut$ sem5 & 9 an #h-millilit] pla/ic vial conta9+ #b4e millilit]s ( dilut$ sem54 ,a second pla/ic vial & glass ampule fill$ to capacity ) dilut$ sem5 %[$ a cool+ rate almo/ id5tical to t %[n 9 ,figure #d4 ,x 0 obvi\s f ! comp>ison t samples 9 ! pla/ic vials cool$ sl[] ?an ^? 9 glass & t ! volume ( sem5 7at lea/ ! small volumes us$7 9 ! vials _h little effect on ! rate ( cool+4 ,9 ano!r exp]im5t1 x 0 %[n t ! volume ( dilut$ sem5 9 ! ampule to be froz5 7#j4b1 #a4j or #e4j ml47 _h little or no effect on ! survival ( ! sp]m4 ,,/orage ,,temp]ature ,9 freez+ & /or+ bull sp]m1 an alcohol ba? conta9+ dry ice at a temp]ature ( -#gi deg4 ,c4 has be5 us$ z a cool+ ag5t4 ,9 _m areas1 ! availability ( dry ice is limit$ & ! co/ is r high4 ,me*anical means are available = obta9+ temp]atures z l[ z1 or l[] ?an1 -#gi deg4 ,c4 b = ! mo/ "p !y are exp5sive4 ,if w>m] temp]atures 7 suitable = /or+ froz5 sem51 ! ord9>y deep-freeze1 :i* op]ates at -#ae deg4 to -#be deg4 ,c41 might be us$4 ,/orage at temp]atures f -#bc deg4 to -#gi deg4 ,c4 ,9 te/+ ! effects ( /orage temp]atures on ! survival ( froz5 bull sp]m 79 a dilu5t conta9+ #g p]c5t glyc]ol71 #i ejaculates 7 froz5 & kept at -#bc deg41 -#cg deg41 -#ea deg41 -#fe deg41 & -#gi deg4 ,c4 ,! desir$ temp]atures 7 ma9ta9$ by dropp+ pieces ( dry ice into e?yl alcohol ba?s z ne$$4 ,samples 7 ?aw$ aft] #a h\r1 #a day1 #c days1 & #e days4 ,aft] #a h\r1 ! samples ma9ta9$ at ! v>i\s temp]atures exhibit$ approximately equal motility 7,fig4 #e74 ,illu/ration3 ,effect ( freez+ & /or+ bull sp]m at v>i\s temp]atures on ! sp]m motility at ?aw+ 7av]age ( #i ejaculates7 7,fig4 #e7 ,at ! end ( #a day1 samples /or$ at -#gi deg4 ,c4 exhibit$ approximately ! same motility z did simil> samples /or$ = #a h\r4 ,! samples /or$ at -#fe deg4 ,c4 _h decl9$ slightly 9 motility & ^? ma9ta9$ at -#ea deg4 ,c4 _h only "o-?ird ! motility :i* !y _h display$ at #a h\r4 ,! samples at -#bc deg4 & -#cg deg4 ,c4 exhibit$ practically no motility aft] #a day 9 /orage4 ,aft] #e days1 only #c ( ! #h ejaculates /or$ at -#ea deg4 ,c4 %[$ motility ^u ?aw+4 ,app>5tly detrim5tal *anges take place m rapidly :5 ! samples are /or$ at temp]atures w>m] ?an -#fe deg4 ,c4 ,! nature ( ^! *anges has n be5 det]m9$4 ,reports f o!r laboratories indicate t /orage temp]atures mu* l[] ?an -#gi deg4 ,c4 are j z satisfactory z -#gi deg4 ,c4 ,no te/s ( ! effects ( /orage at -#gi deg4 ,c4 = p]iods long] ?an #ea days h be5 conduct$ 9 ?is laboratory4 ,portions ( #ab ejaculates 7 froz5 & /or$ at -#gi deg4 ,c4 = v>i\s p]iods4 ,"o portion ( ea* ( ^! 0 exam9$ on ! second1 n9?1 #af? & #ea/ day ( /orage4 ,! p]c5t ( motile sp]m & rate ( motility at ea* ( ^! exam9ations 7 z foll[s3 ,day #b #i #af #ea ,p]c5t ( motile sp]m #di #df #dj #ch ,rate ( motility #b4e #b4c #b4b #b4b ,! av]age prefreez+ motility p]c5tage = ! above samples 0 #eh1 ) an av]age rate ( motility ( #b4i4 ,x is app>5t f ^! results t ! loss 9 motility 0 greate/ due to ! initial freez+1 & aft] t ! drop 0 mo/ pron\nc$ dur+ ! fir/ #af days ( /orage4 ,! ,briti% & ! ,au/ralians h bo? report$ ! successful ma9t5ance ( f]tility 9 froz5 sem5 /or$ at -#gi deg4 ,c4 = ov] two ye>s4#e ,use ( high] glyc]ol levels & a -#bj deg4 ,c4 /orage temp]ature4 ,9 #aiec1 a report f ,arkansas sugge/$ t w>m] /orage temp]atures c\ld be us$ if a high p]c5tage ( glyc]ol 7 includ$ 9 ! freez+ mixture4#g ,to te/ ! effectiv5ess ( v>i\s glyc]ol levels on protect+ sp]m /or$ at deep-freeze temp]atures1 glyc]ol levels ( #c4e1 #e4e1 #g4e1 & #i4e p]c5t 7 us$ ) portions ( #d sem5 samples4 ,survival 9 ! portions froz5 & /or$ at -#bj deg4 ,c4 0 poor comp>$ ) ! portions r$uc$ & held at -#gi deg4 ,c4 ,9 a second exp]im5t1 #d samples 7 subdivid$ & froz5 ) a f9al conc5tration ( #g1 #aa1 #ae1 & #ai p]c5t glyc]ol 9 ! sem5-dilu5t mixture4 ,9 ?is trial1 poor results 7 obta9$ at -#bj deg4 ,c4 except t glyc]ol at a level ( #ai p]c5t protect$ ! sp]m m effectively ?an at l[] levels4 ,maximal survival at -#gi deg4 ,c4 0 obta9$ at ! #g-p]c5t glyc]ol level4 ,a f9al trial 0 run1 us+ glyc]ol levels ( #g1 #aa1 #ae1 #ai1 #bc1 #bg1 & #ca p]c5t4 ,! p]c5tages ( motile sp]m pres5t aft] /orage at -#gi deg4 ,c4 & -#bj deg4 ,c4 are %[n 9 ,table #ad4 ,table #ad4--;,effect ( ,glyc]ol ,level & ,/orage ,temp]ature on ,freezability ( ,sem5 7,av]age ( #h ejaculates7 ,/orage ,glyc]ol ,sp]m motility aft] /orage 7p]c5t7 temp]ature level -------------------------------------- 7 deg4,c47 7p]c5t7 #ah h\rs #db h\rs ---------------------------------------- ------------------------ -#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 ---------------------------------------- ------------------------ ,:ile survival 0 fair ov] a %ort p]iod ( "t ) #ai p]c5t glyc]ol at -#bj deg4 ,c41 det]ioration 0 rapid dur+ /orage4 ,aft] #ah h\rs ( /orage1 ! samples at -#bj deg4 ,c4 7#ai p]c5t glyc]ol7 conta9$ only "o half z _m motile sp]m z 7 /ill pres5t 9 ! samples at -#gi deg4 ,c4 7#g p]c5t glyc]ol74 ,aft] #db h\rs ( /orage1 ! be/ samples at -#bj deg4 ,c4 conta9$ only "o-?ird ! numb] ( motile sp]m /ill pres5t 9 ! samples /or$ at -#gi deg4 ,c4 ,^! trials leave little d\bt t "u ! pres5t sy/em ( freez+ & /or+1 /orage at ord9>y deep-freeze temp]atures is f> inf]ior to /orage at dry-ice temp]atures4 ,,?aw+ ,! importance ( c>efully controll$ cool+ & /orage has be5 emphasiz$ 9 ! =ego+ sections4 ,! ne$ = controll+ ?aw+ rates & ! temp]ature ( ?aw+ 0 n cle>ly def9$ 9 ! e>ly "w on freez+ bull sem54 ,! ,briti% us$ a ?aw+ temp]ature ( #dj deg4 ,c41 :i* 0 satisfactory4 ,if !re is a ne$ to hold ! sem5 = a "t aft] ?aw+1 !n a l[] ?aw+ temp]ature might be m desirable s t cool+ aga9 w n be necess>y4 ,comp>ison ( ?aw+ temp]atures ( #e deg4 ,c4 & #ch deg4 ,c4 ,! effects ( ?aw+ at temp]atures ( #ch deg4 7body temp]ature7 & #e deg4 ,c4 7refrig]ator temp]ature7 7 inve/igat$4 ,! fir/ trial involv$ ?aw+ z rapidly z possible by dropp+ glass ampules ( froz5 sem5 into wat] ba?s at ! two temp]atures4 ,! froz5 sem5 samples conta9$ glyc]ol levels ( #d1 #f1 #h1 & #aj p]c5t4 ,! mean p]c5tages ( motile sp]m f\nd aft] ?aw+ ?irte5 dilut$ sem5 samples treat$ 9 ?is mann] are %[n 9 ,figure #f4 ,illu/ration3 ,effect ( glyc]ol p]c5tage & ?aw+ temp]ature on sp]m motility aft] freez+ & ?aw+ 7,fig4 #f7 ,! #e deg4 ,c4 ?aw+ temp]ature result$ 9 a high] p]c5tage ( sp]m survival at all ! glyc]ol levels ?an #ch deg4 ,c41 ) ! diff]5ce 9 favor ( #e deg4 ,c4 becom+ great] z ! glyc]ol level increas$4 ,! reason = ! int]action betwe5 glyc]ol level & ?aw+ temp]ature is n kn[n4 ,x may be t ! pres5ce ( ! high] levels ( glyc]ol at #ch deg4 ,c4 br\ght ab\t h>mful metabolic activity4 ,! diff]5ce 9 survival ( sp]m 9 sem5 ?aw$ at #e deg4 ,c4 & at #ch deg4 ,c4 cont9u$ dur+ /orage at #e deg4 ,c4 7,table #ae74 ,x 0 also evid5t t ! int]action betwe5 glyc]ol level & ?aw+ temp]ature cont9u$ dur+ /orage 7,fig4 #g74 ,illu/ration3 ,effect ( ?aw+ temp]ature on sp]m motility dur+ /orage at #e deg4 ,c4 foll[+ freez+ & ?aw+ 7,fig4 #g7 ,table #ae4--;,effect ( ,glyc]ol ,level1 ,?aw+ ,temp]ature1 & ,/orage at #e deg4 ,c4 ,aft] ,?aw+ on ,sp]m ,motility 7,av]age ( #ac ejaculates7 ,?aw+ ,glyc]ol ,sp]m motility 7p]c5t7 temp]ature level ---------------------------------------- --- 7 deg4 ,c47 7p]c5t7 ,po/- ,aft] /orage at #e deg4 ,c4 ?aw+ ---------------------- ,av]age #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 ,av]age #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 ,av]age #ce4d #bf4h #af4f #bj4f ---------------------------------------- ----------------------------- ,x is obvi\s t motility falls (f rapidly aft] ! sem5 is ?aw$4 ,9 a field trial 9 :i* ! initial int5t 0 to te/ ! effect ( glyc]ol levels on f]tility ( froz5 sem51 ! sem5 0 ?aw$ 9 ! morn+ & us$ dur+ ! same day4 ,survival ( ! sp]m ) #d p]c5t glyc]ol 0 s poor t only a few bre$+s 7 made ) ^! samples4 ,ev5 at #g & #aj p]c5t1 ! f]tility results 7 mu* l[] ?an ) sem5 t _h n be5 subject$ to freez+4 ,at t "t x 0 felt t ?aw+ ! samples & us+ !m ?r\gh\t ! day may h caus$ ! l[ f]tility results4 ,s9ce !n1 a l>ge-scale exp]im5t by ,cornell ,univ]sity inve/igators1 9 coop]ation ) ! ,new ,york ,artificial ,bre$]s' ,coop]ative1 has %[n def9itely t ?aw+ %\ld be delay$ until a few m9utes prior to bre$+4#aa ,if ! sem5 is us$ imm$iately1 a ?aw+ temp]ature ( ei!r #e deg4 or #ch deg4 ,c4 appe>s to be suitable4 ,h[ev]1 !re is less dang] ( cold %ock due to recool+ if #e deg4 ,c is us$4 ,?aw+ rate 9 pla/ic & 9 glass4 ,glass ampules transmit cold or heat m readily ?an pla/ic ones4 ,! temp]ature rise is rapid 9 bo? glass & pla/ic :5 samples are tak5 f ! /orage box at -#gi deg4 ,c4 & plac$ 9 wat] at #e deg4 ,c4 ,h[ev]1 complete ?aw+ occurs m rapidly 9 glass ?an 9 pla/ic ampules4 ,! *anges 9 temp]ature t occurr$ :5 glass & pla/ic ampules 7 ?aw$ 9 a wat] ba? at #e deg4 ,c4 are %[n 9 ,figure #h4 ,! initial temp]ature rise = ! fir/ m9ute or two 0 ab\t ! same1 !n ! rate ( w>m+ 9 ! pla/ic sl[$ & actual melt+ ( ! froz5 sample occurr$ a little ov] a m9ute lat] 9 ! pla/ic ?an x did 9 ! glass4 ,bo? 7 ?aw$ 9 less ?an f\r m9utes4 ,illu/ration3 ,w>m+ rates ( dilut$ sem5 samples 9 pla/ic vials & 9 glass ampules 7,fig4 #h7 ,,effects ,,( ,,freez+ ,,proc$ures ,,on ,,metabolic ,,activity ,,( ,,bull ,,sp]m ,by f9d+ h[ me?ods ( h&l+ affect ! sp]m cells1 "o c sometimes improve ! proc$ures to avoid h>mful effects4 ,"s attempts h be5 made 9 ?is laboratory to det]m9e ! effects ( ! freez+ proc$ures on ! metabolic activity ( bull sp]matozoa4 ,^! inve/igations h be5 limit$ 9 scope1 involv+ ! measurem5t ( oxyg5-consumption & e/imates ( sp]m motility dur+ & aft] incubation at #cg deg4 ,c4 9 a ,w>burg app>atus4 ,effect ( glyc]ol additions on oxyg5 uptake ( dilut$ sem5 at #cg deg4 ,c4 ,! effect ( add+ glyc]ol to dilut$ sem5 on oxyg5 consumption ( ! sp]m 0 te/$ 9 a ,w>burg app>atus1 us+ sem5 dilut$ ) an ext5d] consi/+ ( "o "p egg yolk & "o "p #b4i p]c5t sodium citrate dihydrate4 ,! yolk-citrate ext5d] 0 add$ to ! sem5 at a rate :i* br\ght ! sp]m conc5tration 9 #j4e millilit] to #bjj million to #ejj million4 ,an exact c\nt 0 us$ to calculate ! oxyg5 uptake p] #ajh sp]m p] h\r 7,,zo#b74 ,illu/ration3 ,effect ( glyc]ol additions on oxyg5 consumption ( sp]m at #cg deg4 ,c4 7,fig4 #i7 ,glyc]ol 9 v>i\s p]c5tages 9 #b4i p]c5t sodium citrate dihydrate solution 0 plac$ 9 ! side>m ( ! ,w>burg flasks4 ,! dilut$ sem5 0 held 9 ! ma9 comp>tm5t4 ,aft] a #fj-m9ute prelim9>y run1 9 :i* ! rate ( oxyg5 uptake ( ! sp]m 9 yolk-citrate dilu5t 0 det]m9$1 ! cont5ts ( ! side>m 7 tipp$ into ! ma9 comp>tm5t4 ,! result+ glyc]ol p]c5tages aft] mix+ ! side>m & ma9 comp>tm5t cont5ts 7 #j1 #d1 #h1 & #ab p]c5t4 ,t5 samples ( sem5 7 subsampl$ & ! oxyg5 uptake ( ea* 0 det]m9$ at all f\r levels ( glyc]ol4 ,oxyg5 uptake 0 increas+ly /imulat$ dur+ ! fir/ #bj-m9ute int]val by ea* increase 9 ! am\nt ( glyc]ol add$ 7,fig4 #i74 ,aft] ! fir/ #bj m9utes1 ! rate ( oxyg5 utilization decreas$ at ! two high] levels ( glyc]ol b p]si/$ at #d p]c5t4 ,! rate ( oxyg5 consumption = ! fir/ #bj-m9ute p]iod at ! #d-p]c5t glyc]ol level 0 #acj p]c5t t ( ! control to :i* only sodium citrate _h be5 add$4 ,at #h & #ab p]c5t ! values = ! p]iod 7 #add & #aib p]c5t1 respectively1 ( ! control rate4 ,illu/ration3 ,effect ( glyc]ol & glyc]ol-plus-catalase additions on sp]m motility dur+ incubation at #cg deg4 ,c4 7,fig4 #aj7 ,) ea* increase 9 glyc]ol level1 motility 0 r$uc$ dur+ ! incubation p]iod4 ,?is is %[n 9 ,figure #aj along ) ! effect on motility ( add+ catalase1 :i* is discuss$ 9 ! next section4 ,effect ( glyc]ol-plus-catalase on oxyg5 uptake ( dilut$ sem54 ,c]ta9 bact]ia h be5 %[n to break glyc]ol d[n1 =m+ hydrog5 p]oxide z foll[s3 ,glyc]ol oxyg5 -- lactic acid hydrog5 p]oxide4 ,hydrog5 p]oxide is kn[n to be detrim5tal to sp]m4 ,! addition ( glyc]ol to dilut$ sem5 fir/ increas$ oxyg5 uptake & !n r$uc$ x4 ,s9ce a r$uction 9 sp]m survival foll[$1 "s h>mful action mu/ h tak5 place ) ! addition ( glyc]ol at #cg deg4 ,c4 ,to te/ :e!r ?is action c\ld be due to ! release ( hydrog5 p]oxide z occurs 9 c]ta9 bact]ia1 glyc]ol ) catalase--! enzyme :i* breaks d[n hydrog5 p]oxide--0 add$ to a portion ( #h dilut$ sem5 samples & ! oxyg5 uptake 0 record$4 ,comp>ison ( ! result+ oxyg5 uptake ) glyc]ol & ) glyc]ol plus catalase is %[n 9 ,figure #aa4 ,illu/ration3 ,effect ( additions ( glyc]ol-plus-catalase on oxyg5 consumption ( sp]m at #cg deg4 ,c4 7,fig4 #aa7 ,oxyg5 consumption 0 increas$ by ! pres5ce ( add$ catalase at all glyc]ol levels & 9 ! control4 ,sp]m survival dur+ ! #c-;h\r p]iod at #cg deg4 ,c4 also 0 improv$ by ! pres5ce ( catalase 7,fig4 #aj74 ,h[ev]1 ! g5]al tr5d 9 oxyg5 consumption produc$ by ! addition ( glyc]ol 0 n *ang$ greatly4 ,! high] levels ( glyc]ol /ill /imulat$ oxyg5 uptake dur+ ! fir/ #bj-m9ute p]iod aft] ! additions & !n sl[$ ! rate ( oxyg5 utilization4 ,! rate ( utilization 0 g5]ally high] dur+ ! te/ p]iod 9 ! pres5ce ( catalase ?an )\t add$ catalase4 ,x appe>$ t a "p ( ! h>mful effect ( glyc]ol might be due to ! =mation ( hydrog5 p]oxide4 ,/ill1 ! detrim5tal effects ( ! high] levels ( glyc]ol 7 n completely remov$4 ,table #af4--;,effect ( ,freez+ ,proc$ures on ,oxyg5 ,utilization ( ,bull ,sp]m 9 ,yolk-,citrate ,ext5d] 7,av]age ( #e ejaculates7 ,microlit]s ( oxyg5 ,sem5 sample te/$ utiliz$ p] #ajh sp]m -------------------------- ,fir/ h\r ,second h\r ---------------------------------------- ----------------------------- ,fre% dilut$ sem5 #aj4c #h4a ,fre% dilut$ sem5 glyc]ol tipp$ 9 at end ( fir/ h\r #i4g,l #ab4i,l ,ag$ #bj to #bd h\rs at #e deg4 ,c4 #aa4b #h4c ,ag$ #bj to #bd h\rs at #e deg4 ,c4 glyc]ol tipp$ 9 at end ( fir/ h\r #aa4h,l #ab4i,l ,aft] #bj h\rs equilibration ) glyc]ol #aa4g,l #g4h,l ,aft] freez+ & ?aw+ #i4g #f4c ---------------------------------------- ---------------------------- ,l ,av]age ( #c ejaculates4 ,effect ( freez+ proc$ures on oxyg5 utilization by sp]m4 ,limit$ data h be5 obta9$ on ! effects ( "s ( ! freez+ proc$ures on ! oxyg5 utilization ( bull sp]m4 ,! results obta9$ 9 ^! exp]im5ts confirm$ ! e>li] f9d+s t tipp+ glyc]ol directly into ! dilut$ sem5 at #cg deg4 ,c4 caus$ an increase 9 oxyg5 consumption 7,table #af74 ,all o!r /eps 9 ! freez+ proc$ure _h little effect on oxyg5 consumption by ! sp]m4 ,except :]e glyc]ol 0 add$ dur+ ! det]m9ation1 ! rate ( oxyg5 utilization 0 l[] ! second h\r ?an dur+ ! fir/4 ,! oxyg5 uptake ( sem5 t _h be5 froz5 & ?aw$ seem$ to drop fa/] ?an t ( unfroz5 samples4 ,effect ( freez+ proc$ures on me?yl5e-blue r$uction "t4 ,! me?yl5e-blue r$uction te/ has be5 us$ z a means ( measur+ sem5 quality & is dep5d5t on ! metabolic activity ( ! sp]m4 ,! effects ( v>i\s freez+ proc$ures on ! ability ( samples to decolorize me?yl5e blue 7 det]m9$ ) #aj sem5 samples4 ,sp]m numb]s 7 /&>diz$ to #cjj x #ajf cells p] millilit] & ! "t requir$ = ^! cells to r$uce a #a3dj1jjj solution ( me?yl5e blue 0 det]m9$ on fre%ly dilut$ sem51 aft] ! addition ( glyc]ol1 aft] equilibration1 & aft] freez+ & ?aw+4 ,portions ( ea* dilut$ sample 7 te/$ at ^! /ages ( ! proc$ure ) glyc]ol alone add$ & ) glyc]ol & v>i\s sug>s add$4 ,a m>k$ increase 9 ! "t requir$ = ! sp]m to r$uce me?yl5e blue occurr$ :5 ! glyc]ol 0 add$ 7,table #ag74 ,?is increase 0 greate/ 9 ! portions ) glyc]ol alone & ) glyc]ol & glucose4 ,! "t increase 0 less pron\nc$ 9 ! pres5ce ( ! ?ree p5tose sug>s us$4 ,foll[+ equilibration1 ! samples rega9$ ! ability to r$uce me?yl5e blue at a rate only slightly sl[] ?an :5 !y 7 fre%4 ,freez+ & /orage ( sem5 result$ 9 sl[] r$uction ( ! me?yl5e blue ?an 0 %[n aft] equilibration ) glyc]ol4 ,s9ce freez+ usually kills "s ( ! sp]m1 a sl[+ ( ! r$uction "t aft] freez+ w\ld be expect$4 ,table #ag4--;,effect ( ,freez+ ,proc$ures on ! ,me?yl5e-,blue ,r$uction ,"t ( ,bull ,sem5 ,) & ,)\t ! ,addition ( ,sug>s,m 7,av]age ( #aj ejaculates7 ,me?yl5e-blue r$uction "t 7m9utes7 ---------------------------------------- -------- ,glyc]ol ,glyc]ol ,glyc]ol ,glyc]ol ,glyc]ol only & & & & glucose arab9ose xylose rhamnose ---------------------------------------- ------------------------------ ,fre% sem5 #e4b #e4b #e4b #e4b #e4b ,aft] glyc]olization #bf4d #be4b #ag4c #ad4c #ai4d ,aft] #ah h\rs equilibration #g4d #f4e #f4d #e4c #f4b ,?aw$ imm$iately aft] freez+ #aa4e #aj4e #i4d #i4j #i4d ,?aw$ #dh h\rs aft] freez+ #ad4c #aj4b #aa4c #aj4a #i4e ---------------------------------------- ------------------------------ ,m ,glyc]ol level 9 ! f9al froz5 mixture 0 #g p]c5t4 ,sug>s 7 add$ to a level ( #a4be p]c5t4 ,,practical ,,freez+ ,,proc$ure ,good results usually c be obta9$ 9 freez+ bull sem5 if c>e is tak5 9 collect+1 dilut+ & process+ ! sem54 ,occasionally ! sem5 f c]ta9 bulls w n )/& freez+ well4 ,! reason = ?is is n und]/ood at pres5t4 ,h[ev]1 c>efully foll[+ ! directions & sugge/ions giv5 bel[ w usually produce satisfactory results ) sem5 samples t are ( good quality at ! />t4 ,exp]i5ce 9 ! field has %[n t f]tility results ) froz5 sem5 are usually slightly l[] dur+ ! fir/ few mon?s ?an ) liquid sem5 /or$ at #e deg4 ,c4 7#da deg4 ,f474 ,mo/ units t h work$ ) froz5 sem5 ov] a p]iod ( a few mon?s are able to improve & d get f]tility results z good z1 or bett] ?an1 obta9$ 9 _! liquid sem5 program4 ,collection ( ! sem54 ,9 ord] to obta9 ! be/ possible sem5 = freez+1 c>e & cleanl9ess %\ld be ex]cis$ 9 mak+ ! collection4 ,! artificial vag9a1 & ! glassw>e us$ %\ld be clean & dry4 ,! und]l9e ( ! bull %\ld also be clean & dry4 ,! bull %\ld be re/ra9$ ne> ! teas] c[ = a m9ute or two prior to collection 9 ord] to excite ! fl[ ( secretions prior to ejaculation4 ,all[+ ! bull to m\nt ! teas] once )\t s]v+ ! artificial vag9a is a good practice to use 9 prop]ly /imulat+ ! bull be=e collection ( ! sem54 ,if ! bull has n be5 us$ = ?ree or f\r days1 ! collection ( a second ejaculate = freez+ may be advisable4 ,! second ejaculate seems to )/& freez+ bett] ?an ! fir/ 9 _m in/ances4 ,a clean1 dry artificial vag9a %\ld be us$ = ea* ejaculate collect$4 ,repeat$ collections 9 ! same artificial vag9a may result 9 contam9ation ( ! sem5 ) bact]ia1 lubricat+ jelly & m9ute p>ticles ( dirt4 ,! sem5 sample %\ld be protect$ f contam9ation & f sudd5 temp]ature drops 7cold %ock74 ,prep>ation ( ext5d]4 ,a suitable egg yolk-citrate ext5d] = freez+ bull sem5 c be prep>$ by ! foll[+ proc$ure4 ,"o "p egg yolk 7free ( egg :ite & ! membrane surr\nd+ ! yolk7 is mix$ ) #d p>ts #b4d to #b4i p]c5t sodium citrate dihydrate solution4 ,! citrate is prep>$ ) di/ill$ wat] & !n boil$ or autoclav$4 ,! citrate solution %\ld be cool$ be=e x is mix$ ) ! egg yolk4 ,aft] ! egg & citrate are mix$1 #ajjj units ( p5icill9 & #ajjj micrograms ( /reptomyc9 are add$ p] millilit] ( ext5d]4 ,sulfanilamide %\ld n be add$4 ,?is ext5d] c be prep>$ #ab to #bd h\rs be=e use if x is /or$ at refrig]ator temp]ature4 ,! portion ( ! ext5d] ne$$ = ! orig9al dilution ( ! sem5 %\ld be w>m$ to room temp]ature be=e x is mix$ ) ! sem54 ,dilution aft] collection4 ,z soon z possible aft] collection1 ! sem5 sample %\ld be dilut$ ) ! ext5d]4 ,! ext5d] mu/ be at ! same temp]ature z ! sem5 7room temp]ature7 :5 ! two are mix$ toge!r4 ,at ?is "t ! sem5 c be p>tially dilut$ 7#a "p sem5 to #d p>ts ( ext5d]7 or dilut$ to a sp]m conc5tration twice ! f9al desir$ conc5tration 7lat] 9 add+ ! glyc]ol = freez+1 ! sem5 is dilut$ fur!r ) an equal volume ( glyc]ol conta9+ ext5d]74 ,! dilut$ sem5 is sl[ly cool$ 7#a-a_/b to #b-a_/b h\rs7 to #e deg4 ,c4 7#da deg4 ,f474 ,"s units us+ froz5 sem5 n[ all[ ! sem5 to /& at #e deg4 ,c4 = #e to #f h\rs be=e glyc]olization to all[ ! antibiotics to be m effective aga9/ any vibrio fetus organisms t may be pres5t4 ,?is /ep is tak5 because x has be5 %[n t glyc]ol inhibits ! effectiv5ess ( ! antibiotics4#f ,aft] cool+1 sem5 c be fur!r dilut$ to twice ! desir$ sp]m conc5tration if t 7 n done at ! />t4 7,caution3 ,be sure sem5 & dilu5t are at ! same temp]ature47 ,add+ ! glyc]ol4 ,! glyc]ol solution is prep>$ by add+ #ad volumes ( glyc]ol 7reag5t grade7 to #hf volumes ( yolk-citrate dilu5t 7same z yolk-citrate us$ = orig9al dilution74 ,?is solution may be add$ dropwise ) con/ant g5tle mix+ to ! already dilut$ sem51 or "o-?ird at a "t at #aj-m9ute int]vals ) g5tle mix+ dur+ ea* addition4 ,ei!r me?od %\ld take ab\t #bj to #cj m9utes4 ,! total volume ( glyc]ol-yolk-citrate solution add$ %\ld be equal to ! volume ( ! orig9al dilut$ sem54 ,9 ?is way a conc5tration ( #g p]c5t glyc]ol is obta9$ 9 ! f9al mixture t is to be froz54 ,c>e mu/ be tak5 to keep ! temp]ature at #e deg4 ,c4 7#da deg4 ,f47 dur+ ! "t ! glyc]ol is be+ add$4 7,a cold room is be/ = ma9ta9+ a temp]ature ( #e deg4 ,c41 b ) c>e ! op]ation c be c>ri$ out at room temp]ature by us+ pans ( ice wat] & a refrig]ator47 ,equilibration4 ,! results pres5t$ 9 ?is bullet9 sugge/ t little or no "t ne$ be all[$ aft] ! glyc]ol is add$ be=e freez+4 ,h[ev]1 results obta9$ by o!r work]s %[ improv$ f]tility ) at lea/ #ab h\rs equilibration4 ,"s units gett+ good f]tility results ) froz5 sem5 also are all[+ ! sem5 to /& at #e deg4 ,c4 = #ab to #ah h\rs be=e freez+4 ,aft] ! sem5 has equilibrat$ ) ! glyc]ol1 #a-millilit] portions ( ! mixture are plac$ 9 #a4b- to #b-millilit] vials or ampules :i* are !n seal$4 ,ampul+ c be done ) an automatic syr+e or pipette1 provid$ a l>ge gage ne$le is us$4 ,also1 x is important n to =ce ! fluid mixture rapidly ?r\gh ! syr+e or ! sp]m may be injur$4 ,freez+4 ,! vials or ampules ( dilut$ sem5 are plac$ 9 a ba? ( isopropyl alcohol :i* has be5 cool$ to #e deg4 ,c4 7#da deg4 ,f474 ,?is ba? c be a wide-m\? !rmos bottle or an insulat$ conta9] ( almo/ any sort ) a l>ge op5+ at ! top4 ,! size ne$$ dep5ds on ! numb] ( ampules be+ froz54 ,"s sort ( conv5i5t tray = hold+ ! ampules 9 an ord]ly fa%ion & enabl+ ! samples to be completely subm]g$ is desirable4 ,a few ampules c be kept toge!r easily by plac+ !m 9 a polye?yl5e freez] bag t has _h _m small holes cut 9 x to let ! alcohol ( ! ba? contact ! ampules4 ,! ampules mu/ be completely cov]$ by ! alcohol to insure uni=m cool+4 ,! alcohol ( ! ba? & ! ampules ( sem5 are cool$ by add+ *ipp$ or gr\nd dry ice 9 suffici5t am\nts to l[] ! temp]ature ( ! ba? #b deg4 ,c4 7#c4f deg4 ,f47 p] m9ute f #e deg4 to -#bj deg4 ,c4 ,f -#bj deg4 d[n to -#gi deg4 ,c41 ! rate ( cool+ c be d\bl$ 7#d deg4 ,c4 or #g4b deg4 ,f474 ,electrical equipm5t t regulates ! cool+ rate to ! desir$ temp]atures is available comm]cially1 b ! co/ may be too high = "s small op]ations4 ,! samples %\ld be held at -#gi deg4 ,c4 7-#aaj deg4 ,f47 until !y are ?aw$4 ,?is c be done by us+ an alcohol ba? & dry ice or by special me*anical refrig]at+ equipm5t4 ,at no "t prior to ?aw+ %\ld ! samples be expos$ to w>m] temp]atures4 ,?aw+4 ,! ampules ( froz5 sem5 c be ?aw$ by remov+ !m f ! dry ice /orage box & dropp+ !m into a wat] ba? at #e deg4 ,c4 7#da deg4 ,f474 ,?aw+ temp]atures up to body temp]ature1 #ch deg4 ,c4 7#ajj deg4 ,f471 c be us$ b extreme c>e mu/ !n be tak5 n to pass ! sem5 ?r\gh a cold insem9at+ tube2 = ?is w\ld subject ! sp]m to cold %ock4 ,! sem5 %\ld be us$ = bre$+ )9 a few m9utes aft] ?aw+4 ,,lit]ature ,,cit$ #a ,,dav5port1 ,c4 ,b4 ,effect ( *emical & physical ag5ts ^u protoplasm4 ,macmillan & ,co41 ,new ,york4 #ahig4 #b ,,polge1 ,c41 & ,,p>kes1 ,a4 ,s4 ,possibilities ( long-t]m /orage ( sp]matozoa at l[ temp]atures4 ,anim4 ,bre$+ ,abs4 #bj3a-e4 #aieb4 #c ,,emm5s1 ,c4 ,w41 & ,,black%aw1 ,a4 ,w4 ,! l[ temp]ature /orage ( ram1 bull1 & rabbit sp]matozoa4 ,au/ral4 ,vet4 ,j\r4 #bf3bbf4 #aiej4 #d ,,smi?1 ,,audrey ,w4 ,effects ( l[ temp]atures on liv+ cells & tissues4 ,9 biological applications ( freez+ & dry+4 ,ed4 ,r4 ,j4 ,c4 ,h>ris4 ,academic ,press1 ,inc41 ,new ,york1 #aied4 #e ,,emm5s1 ,c4 ,w41 & ,,black%aw1 ,a4 ,w4 ,artificial insem9ation4 ,physiol4 ,rev4 #cf3bgg-cjf4 #aief4 #f ,proce$+s ( ! ,national ,association ( ,artificial ,bre$]s1 #aiec1 #aied1 & #aiee4 #g ,proce$+s ( ! ,am]ican ,dairy ,sci5ce ,association1 #aiec1 #aied1 & #aiee4 ,publi%$ 9 ! ,june issue ( ! ,j\rnal ( ,dairy ,sci5ce = ea* ye>4 #h ,,b>k]1 ,c4 ,a4 ,v4 ,l[ temp]ature pres]vation ( bov9e epididymal sp]matozoa4 ,canad4 ,j\r4 ,comp4 ,m$4 #ah3cij-cic4 #aied4 #i ,,s>(f1 ,,jack1 & ,,mixn]1 ,j4 ,p4 ,! relation%ip ( egg yolk & glyc]ol cont5t ( dilut]s & glyc]ol equilibration "t to survival ( bull sp]matozoa aft] l[ temp]ature freez+4 ,j\r4 ,dairy ,sci4 #ch3bib-big4 #aiee4 #aj ,,cragle1 ,r ,g41 ,,my]s1 ,r4 ,m41 ,,waugh1 ,r4 ,k41 ,,hunt]1 ,j4 ,s41 & ,,&]son1 ,r4 ,l4 ,! effects ( v>i\s levels ( sodium citrate1 glyc]ol1 & equilibration "t on survival ( bov9e sp]matozoa aft] /orage at -#gi deg4 ,c4 ,j\r4 ,dairy ,sci4 #ch3ejh-ead4 #aiee4 #aa ,,bratton1 ,r4 ,w41 ,,foote1 ,r4 ,h41 & ,,cru!rs1 ,,joan ,c4 ,prelim9>y f]tility results ) froz5 bov9e sp]matozoa4 ,j\r4 ,dairy ,sci4 #ch3dj-df4 #aiee4 #ab ,,hafs1 ,h4 ,d41 & ,,elliott1 ,f4 ,i4 ,! effects ( me?ods ( add+ egg yolk & monosac*>ides on ! survival ( froz5 bull sp]matozoa4 ,j\r4 ,dairy ,sci4 #ch3haa-hae4 #aiee4 #ac ,,mill]1 ,w4 ,j41 & ,,v&em>k1 ,n4 ,l4 ,! influ5ce ( glyc]ol level1 v>i\s temp]ature aspects1 & c]ta9 o!r factors on ! survival ( bull sp]matozoa at sub-z]o temp]atures4 ,j\r4 ,dairy ,sci4 #cg3de-ea4 #aied4 ,,temp]ature ,,conv]sions 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