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Development of a buffer system for dialysis of bovine spermatozoa before freezing. II. Effect of sugars and sugar alcohols on posthaw motility
THERIOGENOLOGY
DEVEUXIWNT OF A BDFFBR SYSTEM FOR DIALYSISOF BWIN!? SPSRMATOZOABEECREFREEZING. II. EFFFCI'OFSUGARS AND SUGAR ALCOHOLSCN RXWTHAWMQl'ILIlY M.A. Garcia1 and E.F. Graham Departsentof Animal Science Universityof Minnesota Saint Paul, MN 55108 Received for publication:September Accepted: February
2, 16,
1988 1989
ABSTRACT !I'cm experimentswere conductedto evaluate the effect of different levels of sugars (glucose,lactose and raffincee)and the effect of those sugars (C3 to C6) or their correspondentsugar alcohols on the dialysis of bovine semen. First, the effect of isosmtic solutionsof glucose, lactose or raffinoseat five differentlevels (0, 25, 50, 75, 95% V/W on sperm utility of semen dialyzed prior to freezingwere studied. These levels were used in extendersand dialysates,and the final voluna was canplemantedwith Picerazine-N-N-BIS (2-ethanesulfonit acid (PIPFS)titrated to @-I7.0 with TRIS (hydroxymthyl)aminomethane (TRIS)to form PIPBST or a 1:l (V/V) canbinationbetween PIPEST and scdiun citrate solutions. In the seccmd expximant, 30% of the buffer VOh_m3 COntained SOlUtimS of sugars (C3 Or c6) or their correspondentsugar alcohol,and the final volune was canpletedwith PIlXST-citratebuffer. Semen aliguotswere extended cl:101and dialyzed. (1:50) for 2 h while cooling fran 37 to 5'C in semipermeabledialysis bags of 12,000 to 14,000 molecularweight cut off. The samples ware frozen in pellets 1 h after dialvsiswas terminated. Spam survivalwas significantlyhigher in PIPEST-citratethan in PIPSST buffer alone (P(O.05). No significantdifference (P>O.O5)was obtained between the use of glucose or lactose or between lactoseand raffinose. High levels of sugar appearedto be detrimentalto spxm motility of fresh and thawed semen samples. Motility of cells extended in buffers containing 30% (V/V) isosmoticsolutionsof glucose,galactose,ribose, xylose, arabinceeor their correspondentsugar alcoholswas significantlyhigher (P
1029
THERIOGENOLOGY intermediarymetabolite (1). Inositol (2), sorbitol Wglucitol,3), Dmnnitol, erythritoland glycerol (4) as wall as sans related enzymes such as sorbitoldehydrogenase(5) have all been identifiedin bovine SeREIl.
The effect of these canpoundson sperm survivalbefore and after freezinghas been studied by many researchers. The addition of mall quantitiesof sane sugars like xylose or fructoseto either yolk-citrate or milk diluentshas been reportedto increasethe Foporticn of sperm that survive freezingand thawing (6). Dilusntscontainingarabincse are reportedto give significantlybetter nutility stxres than those containingfructose;however, fertilitywas unaffectedby a change in sugar levels (7). The additionof 1.25% fructoseto the citrate-yolk-glycerol extender improvedsignificantlythe nonreturnrate at 3 mo fran 56.9 to 66% (8). Also, sucrcee peserved sparm better than lactose,raffincee or a mixture of the three (9). Hajamnann et al. (10) developadthe MinnesotaGC extender that inclmlesaddmitol, galacytol,erythritol,mnnitol, sorbitol,inceitol, dextrose,levulose,citric acid, sodim citrate and a fractionextracted frandriedegg yolk. Caxeption results shcnaadthe extender to be supxior to yolk-citrate. Nagase and Tanizuka (11) found that glycerol and ethyleneglycol gave excellentresults in the pellet and in slow freezing,but very lxxx results with faster freezingmethods, suggesting that the differencein Fotection may be due to differencesin the eutectic points of the sugars used. Tbs authors have previouslyrepxted that postthawsparm survival is improvedby dialysis (12-16),which removes the 1~ mAecul.arweight fraction (W) present in egg yolk and seminal plasma. Ths canposition of the txffer system used for dialysis has also been reportedto influencethe poportion of spermatozoathat best survives freezingand thawing (17). The intent of this poject was to study the effect of differentlevels of mm-, di- or trisaccharides, and of sugar alcohols on fresh and postthawmtility of spermatozoadialyzed pior to freezing.
Bovine ejaculatesware obtainedby artificialvagina fran 12 mature Holsteinbulls. After collection,the samples ware micrcecopicallyevaluated and only sampleswith 60 to 70% motile spermatozoaware used. The ejaculatesme pooledandthe semn aliguotswarediluted (1:lO) in warm exte&ers. The semen was dialyzed (1:50) in semipermeable dialysisbags of 12,000 to 14,000 molecularweight cut off PIWZO) for 2 h while cooling fran 37 to 5°C. Heman was frozen in pellets 1 h after terminationof dialysis (13-161. The pirpxe of dialysiswas to exchange the low molecularwaight fractions WWF (12,000 to 14,000 dalt) wesent in egg yolk and seminal plasma for the differentbuffersugar axnbiuations. The pelletswexe stored in liguid nitrogenuntil evaluation. The palletsware thaw&in aluninumblockde~essions as describedearlier (12,13,17).
1030
MAY 1989 VOL. 31 NO. 5
THERIOGENOLOGY
The effect of the differenttreatmentscn sperm cells was evahated cells before freezingand after thawing. Also, pMZhaw samplesware evaluatedby calculatingthe percentageof cellsthatpassedthroughthe Semdex column by the technigue describedelsewhere (12,13,17,18). by observingthe percentageof mile
Experiment1 The effect of different levels of Dglucose, D-lactoseand Draffinosecn freezabilityof dialyzed bull sgnen was studied. Isosmotic sugar solutions (osmaticpressure 320 to 325 iMsm/kg) of glucose, lactoseand raffinosewere peparedaswall as the buffer solutionsPIFEST preparedby titrationof piperazine-N-N-EJIS(2-ethane sulfonicacid (PIIPES) with N-IRIS (hydroxymsthyl) aminaaathaneYIRIS)to @I 7.0 to form IJIRBT (17) and sodiun citrate (Na citrate). Five levels of each sugar (0, 25, 50, 75 and 95% V/V) were used as Fart_of the extender and dialysate. The dialysatesvlRTewepared by adding 5% V/V glycerol to each sugar solution,and the final voluas was canplemantedwith either one of the two buffers: PIRST or PIIXST-citrateccmbination(1:l v/V; 17). Final separation of extenderswas accanplishedby addition of 20% W/V) egg yolk to each of the 30 differentsugar-buffer-glycerol canbinations. The dialysatesdid not ccotain egg yolk. The data wsre analyzed by analysis of variance (19) of a 6 (replicates)x 2 (buffers)x 3 (sugars)x 5 (levelsof sugar) factorial design, folld by Tukey's multiple range test (20). Experiment2 The ffect of some monosaccharidesor their correspondentsugar alcohols cm survivalof bull semen dialyzed before freezingwas observed. The buffer material used in this experimentuas the 1:l ambination of iscsmoticsolutionsof PIHZST and Na citrate. Five percent of the final volume was glycerol and 30% W/V) was an isosmoticsolution made of the sugars or sugar alcohols (listedin Table 2). Each soluticn was used in extendersand dialysates. The control sample did not contain sugar. Final preparationof each extender also contained20% egg yolk. The semen samples were extended and dialyzedagainst the correspondent dialysateand handled as in the pevious experiment. The data were analyzed by analysis of variance (19) of a 6 (replicates)x 15 (extenders)factorialdesign, and the maans were isolatedby the Tukey's multiple range test (20).
Experiment1 As ccnfirmedin Fevious ex~riments (14,171,the inclusionof 150% V/v) Na citrate solution in the buffer resulted in a significant (X0.05) increase in spsrm rrntilityin the fresh and frozen-thawed samples. The percentagesof nrotiles+erm in ncnfrozensamples and in
MAY 1959 VOL. 31 NO. 5
1031
THERIOGENOLOGY
thawed samples and the percentageof cells that passed through the Sephadexcolunn were 32.2, 20.6 and 26.3% motility in the PIPEST-citrate canbination. No differences(P>O.O5)were obtainedbetween the use of the three typs of sugars when the percentageof motile cells in fresh or thawed semen sampleswere analyzed. The analysisof the cells that pessed through the Sephadexcolumn showed no difference(DO.051 between the samples that had been dialyzed in solutionscontainingglucose or lactose (35.0, 31.9 percentmotility,respectively)and no difference between the use of extenderscontaininglactoseor raffinose (31.4 percent motile cells in the latter). Table 1 illustratesthe effects of the differentlevels of sugars in the extenderson survivalof dialyzed spermatozoa. It can be observed in each of the evaluationmethods that as sugar level was increased,sperm survivaldecreased. The use of a buffer containing95% of an isosmoticsugar solutionand 5% glycerolwas highly detrimentalto the cells. All dependentvariablesshowed significantdifferences(P(O.05) between buffer and level of sugar used (Figure1 a,b,c). Table 1. Effect of sugar levels on survivalof dialyzed semen Fresh Senw7
Percentageisosmtic sugar solutionas plrt of total buffer volma 0
25 50 75 95
Frozen thawed semen
Percentage Parcentage motility rrotility 57.4arf 60.3a 52.0b 31.8c 5.ld
a,b,c,dmame superscriptsin the same calm difference(DO.05). fN = 36 observationsper maan.
37.5arb 38.7a 34.7b 16.6C 1.3d
IQrcentagecells filteredin Sephadexcolmm 49.4a 44.7b 36.7c 24.9d 8.5e
indicateno significant
Experiment2 Table 2 illustratesthe differencesobtainedbetween treatments. Motility of spermatozoain fresh and frozen thawed sampleswas significantly lower (KO.05) in the solutionsthat containederythrose,glyceraldehydeor glycerol ccmparedwith motility in extenderscontaining 30% of any Of the C5 or c6 iSoSmotiCSolutions. Erythrceeand gly-
1032
MAY 1989 VOL. 31 NO. 5
THERIOGENOLOGY
0
R
50 100 0 Percentage of isosmotic sugar solution 3s part of total buffer volume
60
Percentage of isosmotic sugar solution as part of total buffer volume
C
-
---------_ --.
‘\ ‘\
40
-
‘\
‘\
: II : :
20 -
: : :
\
Fiqure
I.
0 50 100 Percentage of isosmotic sugar solution as part cf total buffer volume Effect of different levels of isosmotic sugar solutions included in extender and dislysate. A) Percentage of motile spermatozoa in fresh semen sani~lcs. B) Percentage of motile sperrstozoa in postthaw samples. C) Fcrcencage of cells that passed through the Sephadex column in frozen-thawed snmples. The buffers used ,~erepiperazine-N-X-BIS (Z-ethane sulfonic acid) titrated with TRIS (I’/V)
hydroxymethyl of PIPEST
and
MAY 1989 VOL. 31 NO. 5
aminomethnne sodium citrate.
to
ph
7.0
(PIPEST)
and
~---I
a
1:l
cr’zbination
1033
THERIOGENOLOGY
ceraldehydepreparationswzre toxic to the cell and obstructedsparm motility. Glycerolwas vesent at a high cmcentration aud hence was deletereous,even though 36% of the cells -sad through the Se@mdex colunn. The motilityobserved showed abnormalpatternsdanmstrating glycerol shock. The evaluationof fresh and thawed senen samples showed no significantdifferencesbetween the inclusionof 30% of the isosnotic sugar SOlUtiOnS fran arabitol (C5) up to glucose (C6) in the extending media and dialysate. The postthawevaluationof mtility sM no significantdifferenceswith the inclusionof 30% isosmoticC5 and C6 sugars with the exceptionof D-glucose.
Table 2. Effect of sugars and sugar alcoholson freezabilityof dialyzed bull smen Fresh semen
Typ2 of sugarg D-glucose Sorbitol D-galactose Dulcitol D-ribose Ribitol D-xylose Xylitol D-arabinose Arabitol D-erythrose Erythrol D-glyceraldehyde Glycerol No sugar
Frozen-thawed seam
Fercehtagecells No. carbon IQrcentage Rarcentage filteredin atans motility motility Se&&xcolUnn
$ 2 C5 C5 C5 C5 C5 C4 C4 C3 C3 -
64.2"rbrf 65.0a 61.7a,b
28.4b,c 42.5a
:z'l: . ’ 66.7a 66.7a 65.8arb
:::$?t: 37.5a:b 42.5a 36.6a*b 35.8a,b 40.8a 41.7a
;;:;Z l.Od' 56.7b
l.od
47.5c 42.5c
,:$.b 1:od 22.5c 20.2c
51.2a 48.3a,b 50.8a 42.5b,c*d :;::Z 48.1a:b 48.2arb :;:;Z 23.1e' 44.8a,brc 22.1e 36.4d 39.ocrd
a,bfc,d+Sa superscriptsin the sam colum indicateno significant difference (PO.05). fN=Six observationspsr mean. 930% W/V) of isomotic sugar solution. The remainingbuffer solution was iscmotic RIPEST-citratein a 1:l ambination. RIPESTwas pm-epured by titrationof PiperazineN-N-BIS (2-ethanesulfonicacid) with N-TRIS (hydroxymethyl) aminarethanesolutionto pB 7.0. DISCUSSION Sugar solutionshave been reportedbeneficialfor sperm cells.
1034
MAY 1989VOL. 31 NO. 5
THERIOGENOLOGY Glucose wes chosen in our experimentbecause it is easily metabolizable by spernlatozca (21,221. A beneficialeffect has been reported of milk salts that ocntainmainly lactose 01 bull sperm survival (12,151. Raffincee has also been reportedto ba beneficialfor bull and ram sperm survival (ll). Our results agree with those fran Unal et al. (231, who found no differencebetween sperm responseto lactose or raffinose. Nagase and Graham (24) have also reportedno significantdifferenceson sperm motility when semen was frozen in pelletedform, among extenders ccntainingglucose, lactose,raffinose,stachyceeor their canbination. Haumerstedt(25) dialyzed bovine semen and reportedthat at ccncentrationsexceeding1 x 10-S spann/ml,cellular adenceinetriphospbate (ATP) dropped to very low levels (1 to 3 nMoles par lo'* cells) and the cells became n-tile. He suggestedthatatccncentraticms belowlx 10-8 cells/ml,a matabolizablecanpoundmust be present in the dialysate to maintain cell viability. As the sugar ccocentrationincreased,the proportionof buffer material decreased,which changed the propertiesof the extendingmedia. These cunbinationsdid not best voted the sperm cells fran the changes in @I and osmotic Faressure that occur during cooling and freezing. The differentcunbinationsof PIPEST and sugar were not as effective in maintainingmotility as the acmbination PIFEST-citrate-sugar was. Smnen extended in PI-T-sugar solutions sh& a rapid decline in survivalwhen more than 25% iscemoticsugar solutionswere Feesent. When PIPEST-citrate-sugar solutionsware used, the decline in survival started only after 75% of the extenderwas canposed of an isosnoticsugar solution. In the data obtainedafter filtrationof postthawsamples, sperm motility declined as the sugar levels increased. The analysisof individualvalues of the data obtained after filtrationshawE?dthat higher numbers of notile cells ware obtainedwhen 25% of isosmoticsolutionsof either glucose or lactosewere used. The trisaccharideraffinosewas not as effectiveasthenrx-~o-or the disaccharideused. Sugar alcoholsare sugar molecules in which the aldehydegroup is replaced by an alcohol group. They have been reportedbeneficialfor sperm cell motility (10,111. This smy be due to their structuralsimilarity to the cxyoprotectantglycerol. However,whenglycerolwas used as a sugar alcohol, it wx pesent in very high concentrationand became toxicto sperm. The results in the secondexperimantindicatethatwhen 30% W/V) of isosmoticsolutionsof sugar or sugar alcoholsranging fran G5 t0 c6 WXe used, StatiStiCally SUpriOr motilitywas obtainedand a higher number of cells passed through the Sephadexcolumn. Ths presence of any of these sugar solutionsyielded a statisticallysuperiormotility rate (X0.05) to the absence of sugar in the dialysateor retentate. In suvmary,the results of curtwoexpsrin~nts shwthatsperm survival in PIPSST-citratetmffer is significantlyhigher (X0.05) than in the PIPEST buffer. Nodifferencewas c&air&between the use of gluccse or lactase or between lactose andraffinosewhenthe percentage of motile cells that passed through the sepladexcolumn in pxtthaw samples was analyzed. High levels of sugar in extendingmedia and dialysate peeved not to be beneficialfor sperm motility in fresh
MAY 1989 VOL. 31 NO. 5
THERIOGENOLOGY
or postthawsemen samples. Results of the seccnd exparinmt illustrate that 30% (V/V) of the buffer system for dialysisof spermatozoamy be canposedof ah isosmoticsolutionof glumse, galactose,ribose,xylose, arabinoseor their correspondentsugar alcohols. Motility of spermatozoa in each of these solutionsin fresh or frozen thawed Semen sampleswas significantlysuperior (X0.05) to that of s~rmatozoa extended in samples that did not contain sugar. REFERENCFS
1. Hers, H.G. Le mechanismde la formationdu fructoseseminal et du fructose fcetal. Bicchim.Biophys. Acta. -37:127-138(1960). 2. Hartree,E.F. Inositolin seminal plasma. Bicchem.J. -66:131-134 (1975). 3. King, T-E., Isherwood,F.A. and Mann, T. Sorbitolin semen. AbstractsIVth Int. Cmgr. Biochem.,Vienna, -77 (abstr)(1958). 4. Clark, J.B., Graham, E.F., Lewis, B.A. and Smith, F. D-mahnitol, erythritoland glycerol in bovine semsn. J. Reprcd. Fertil.-13: 189-197 (1967). 5. Georgiev,G.H. Activityof sorbitoldehydrogehasein ram and bull seminal plasma. Int. J. Bicchem.5:21-22 11974). 6. Hafs, H.D. and Elliot, F.I. The effect of nethods of adding egg yolk and monosaccharidesto the survivalof frozen bull spermatozoa. J. Dairy Sci. -38:811-815(1955). 7. ws, C.W. and Martin, I.C.A. The effects of equilibration period and sugar content of the diluehtcnthe survivaland fertility of bull sprmatozoa deep frozen to -79'C. IV Int. Ccngr. Anim. Reprod. and Artif. Insem.,The Hague. pp. 964-967 (1960). 8. Martin, I.C.A. and mns, C.W. Effects of time of equilibration and additionof fructosecm the survivaland fertilityof bull spermatozoadeep frozen to -79Y. J. Repod. Fertil.2:404-410 (1961). of sperm 9. Seglins,A. and Enina, V. Effect of several mnmehts diluent oh bull spermatozoaduring freezing. Vet. Zhivotnovod. pp. 31-35 (19731. 10. Rajamanann,A-H., Graham, E.F. and Smith, F. The effect of a new extender incorporatingdried egg yolk on the fertilityof bovine spsrmatozca. V Cmgr. Intl. pzr la Reprod. Animale e la Fecohd. Artificiale,Trehto, Italy. pp. 392-397 (1964). 11. Nagase,H. andTanizuka,T. Use of @yols as protectahtsagainst freezing injury to lxlllspermatozoa. II. Protectiveeffects of polyols during differentfreezingrates. VI Int. Cmgr. Anim. Reprcd. and Artif. Insem., Paris,France. pp. 1107-1109 (1968).
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MAY 1989 VOL. 31 NO. 5
THERIOGENOLOGY
12.
Garcia, M.A. and Graham, E.F. Effects of lowmolecular-waight fractions (LMWF) fran milk, egg yolk and seminal plasma on freezability of bovine spermatozca. Cryobiology%:429-436 (1987a).
13.
Garcia, M.A. and Graham, E.F. Factors affectingthe removal of low-molecular-weight-fractions (IMWF) fran egg yolk and seminal plasm in extended semen by dialysis: Effect on post-thawsperm survival. Cryobiology-24:437-445(1987b).
14.
Garcia, M.A. and Graham, E.F. Dialysisof bovine semen and its effect on fresh and freeze-thawedspermatozoa. Cryobiology-24: 446-454 (1987c).
15.
Garcia, M.A. Effects of DialyzableDiffusatesfran Milk, Egg Yolk and Seminal Plasma on Freezabilityof Bovine Spermatozoa. MS. Thesis, Universityof Minnesota,1980.
16.
Garcia, M.A. Dialysis: A Method for Studyingand Impoving the Quality of Frozen Bovine Spermatozoa. Ph.D. Thesis, Universityof Minnesota,1984.
17.
Garcia, M.A. and Graham, E.F. Developmentof a buffer system for dialysis of bovine sparmtozoa. I. Effect of zwitterionbuffers. Theriogenolcgy-31: (1989).
18.
Graham, E.F., Vasguez, I.A., SchTlehl, M.K. and Evensen, B.K. An assay of semen quality by use of Sephadex filtration. VIII Int. Cmgr. Anim. Reprod. and Artif. Insem.,Cracm, Poland. pp. 896-899 (1976).
19.
Snedecor,G.W. and Co&ran, W.G. StatisticalMethods. Icwa State UniversityPress, Anrss,1976, pp. 339-369.
20.
Steel, R.G. and Torrie, J.H. Principlesand Proceduresof Statistics. A BianedicalApproach. MCQ-aw Hill, New York, 1980, pp. 187-188,192.
21.
Kraft, L.A., Foley, C.W., Hcwarth, B. and Johnson, A.D. Carbohydratematabolismby washed spermatozoaof various species. J. Dairy Sci. %:786 (1971).
22.
Mann, T. The Biochemistryof Semen and the Male Reproductive Tract. Barnes and Noble, Inc., New York, 1964. p. 493.
23.
Unal, M-B., Berndston,W.E. and Pickett,B.W. Influenceof sugars with glycerol cm pxt-thaw motility of bovine spermatozoain straws. J. Dairy Sci. -61:83-89 (1978).
24.
Nagase, H. and Graham, E.F. Palletedsemen: Canparisonof different extendersand pccesses co fertilityof bovine spermatozoa. V. Int. Ccogr. per la Reprcd. Animale e la Fecond. Artificiale, Trento, Italy, pp. 387-391 (1964).
25.
Hamnerstedt,R. Use of high speed dialysis to prepare bovine sperm for metabolic studies. Biol. of Repmd. g:389-396 (1975).
MAY 1989 VOL. 31 NO. 5
1037
DEVEUXIWNT OF A BDFFBR SYSTEM FOR DIALYSISOF BWIN!? SPSRMATOZOABEECREFREEZING. II. EFFFCI'OFSUGARS AND SUGAR ALCOHOLSCN RXWTHAWMQl'ILIlY M.A. Garcia1 and E.F. Graham Departsentof Animal Science Universityof Minnesota Saint Paul, MN 55108 Received for publication:September Accepted: February
2, 16,
1988 1989
ABSTRACT !I'cm experimentswere conductedto evaluate the effect of different levels of sugars (glucose,lactose and raffincee)and the effect of those sugars (C3 to C6) or their correspondentsugar alcohols on the dialysis of bovine semen. First, the effect of isosmtic solutionsof glucose, lactose or raffinoseat five differentlevels (0, 25, 50, 75, 95% V/W on sperm utility of semen dialyzed prior to freezingwere studied. These levels were used in extendersand dialysates,and the final voluna was canplemantedwith Picerazine-N-N-BIS (2-ethanesulfonit acid (PIPFS)titrated to @-I7.0 with TRIS (hydroxymthyl)aminomethane (TRIS)to form PIPBST or a 1:l (V/V) canbinationbetween PIPEST and scdiun citrate solutions. In the seccmd expximant, 30% of the buffer VOh_m3 COntained SOlUtimS of sugars (C3 Or c6) or their correspondentsugar alcohol,and the final volune was canpletedwith PIlXST-citratebuffer. Semen aliguotswere extended cl:101and dialyzed. (1:50) for 2 h while cooling fran 37 to 5'C in semipermeabledialysis bags of 12,000 to 14,000 molecularweight cut off. The samples ware frozen in pellets 1 h after dialvsiswas terminated. Spam survivalwas significantlyhigher in PIPEST-citratethan in PIPSST buffer alone (P(O.05). No significantdifference (P>O.O5)was obtained between the use of glucose or lactose or between lactoseand raffinose. High levels of sugar appearedto be detrimentalto spxm motility of fresh and thawed semen samples. Motility of cells extended in buffers containing 30% (V/V) isosmoticsolutionsof glucose,galactose,ribose, xylose, arabinceeor their correspondentsugar alcoholswas significantlyhigher (P
1029
THERIOGENOLOGY intermediarymetabolite (1). Inositol (2), sorbitol Wglucitol,3), Dmnnitol, erythritoland glycerol (4) as wall as sans related enzymes such as sorbitoldehydrogenase(5) have all been identifiedin bovine SeREIl.
The effect of these canpoundson sperm survivalbefore and after freezinghas been studied by many researchers. The addition of mall quantitiesof sane sugars like xylose or fructoseto either yolk-citrate or milk diluentshas been reportedto increasethe Foporticn of sperm that survive freezingand thawing (6). Dilusntscontainingarabincse are reportedto give significantlybetter nutility stxres than those containingfructose;however, fertilitywas unaffectedby a change in sugar levels (7). The additionof 1.25% fructoseto the citrate-yolk-glycerol extender improvedsignificantlythe nonreturnrate at 3 mo fran 56.9 to 66% (8). Also, sucrcee peserved sparm better than lactose,raffincee or a mixture of the three (9). Hajamnann et al. (10) developadthe MinnesotaGC extender that inclmlesaddmitol, galacytol,erythritol,mnnitol, sorbitol,inceitol, dextrose,levulose,citric acid, sodim citrate and a fractionextracted frandriedegg yolk. Caxeption results shcnaadthe extender to be supxior to yolk-citrate. Nagase and Tanizuka (11) found that glycerol and ethyleneglycol gave excellentresults in the pellet and in slow freezing,but very lxxx results with faster freezingmethods, suggesting that the differencein Fotection may be due to differencesin the eutectic points of the sugars used. Tbs authors have previouslyrepxted that postthawsparm survival is improvedby dialysis (12-16),which removes the 1~ mAecul.arweight fraction (W) present in egg yolk and seminal plasma. Ths canposition of the txffer system used for dialysis has also been reportedto influencethe poportion of spermatozoathat best survives freezingand thawing (17). The intent of this poject was to study the effect of differentlevels of mm-, di- or trisaccharides, and of sugar alcohols on fresh and postthawmtility of spermatozoadialyzed pior to freezing.
Bovine ejaculatesware obtainedby artificialvagina fran 12 mature Holsteinbulls. After collection,the samples ware micrcecopicallyevaluated and only sampleswith 60 to 70% motile spermatozoaware used. The ejaculatesme pooledandthe semn aliguotswarediluted (1:lO) in warm exte&ers. The semen was dialyzed (1:50) in semipermeable dialysisbags of 12,000 to 14,000 molecularweight cut off PIWZO) for 2 h while cooling fran 37 to 5°C. Heman was frozen in pellets 1 h after terminationof dialysis (13-161. The pirpxe of dialysiswas to exchange the low molecularwaight fractions WWF (12,000 to 14,000 dalt) wesent in egg yolk and seminal plasma for the differentbuffersugar axnbiuations. The pelletswexe stored in liguid nitrogenuntil evaluation. The palletsware thaw&in aluninumblockde~essions as describedearlier (12,13,17).
1030
MAY 1989 VOL. 31 NO. 5
THERIOGENOLOGY
The effect of the differenttreatmentscn sperm cells was evahated cells before freezingand after thawing. Also, pMZhaw samplesware evaluatedby calculatingthe percentageof cellsthatpassedthroughthe Semdex column by the technigue describedelsewhere (12,13,17,18). by observingthe percentageof mile
Experiment1 The effect of different levels of Dglucose, D-lactoseand Draffinosecn freezabilityof dialyzed bull sgnen was studied. Isosmotic sugar solutions (osmaticpressure 320 to 325 iMsm/kg) of glucose, lactoseand raffinosewere peparedaswall as the buffer solutionsPIFEST preparedby titrationof piperazine-N-N-EJIS(2-ethane sulfonicacid (PIIPES) with N-IRIS (hydroxymsthyl) aminaaathaneYIRIS)to @I 7.0 to form IJIRBT (17) and sodiun citrate (Na citrate). Five levels of each sugar (0, 25, 50, 75 and 95% V/V) were used as Fart_of the extender and dialysate. The dialysatesvlRTewepared by adding 5% V/V glycerol to each sugar solution,and the final voluas was canplemantedwith either one of the two buffers: PIRST or PIIXST-citrateccmbination(1:l v/V; 17). Final separation of extenderswas accanplishedby addition of 20% W/V) egg yolk to each of the 30 differentsugar-buffer-glycerol canbinations. The dialysatesdid not ccotain egg yolk. The data wsre analyzed by analysis of variance (19) of a 6 (replicates)x 2 (buffers)x 3 (sugars)x 5 (levelsof sugar) factorial design, folld by Tukey's multiple range test (20). Experiment2 The ffect of some monosaccharidesor their correspondentsugar alcohols cm survivalof bull semen dialyzed before freezingwas observed. The buffer material used in this experimentuas the 1:l ambination of iscsmoticsolutionsof PIHZST and Na citrate. Five percent of the final volume was glycerol and 30% W/V) was an isosmoticsolution made of the sugars or sugar alcohols (listedin Table 2). Each soluticn was used in extendersand dialysates. The control sample did not contain sugar. Final preparationof each extender also contained20% egg yolk. The semen samples were extended and dialyzedagainst the correspondent dialysateand handled as in the pevious experiment. The data were analyzed by analysis of variance (19) of a 6 (replicates)x 15 (extenders)factorialdesign, and the maans were isolatedby the Tukey's multiple range test (20).
Experiment1 As ccnfirmedin Fevious ex~riments (14,171,the inclusionof 150% V/v) Na citrate solution in the buffer resulted in a significant (X0.05) increase in spsrm rrntilityin the fresh and frozen-thawed samples. The percentagesof nrotiles+erm in ncnfrozensamples and in
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THERIOGENOLOGY
thawed samples and the percentageof cells that passed through the Sephadexcolunn were 32.2, 20.6 and 26.3% motility in the PIPEST-citrate canbination. No differences(P>O.O5)were obtainedbetween the use of the three typs of sugars when the percentageof motile cells in fresh or thawed semen sampleswere analyzed. The analysisof the cells that pessed through the Sephadexcolumn showed no difference(DO.051 between the samples that had been dialyzed in solutionscontainingglucose or lactose (35.0, 31.9 percentmotility,respectively)and no difference between the use of extenderscontaininglactoseor raffinose (31.4 percent motile cells in the latter). Table 1 illustratesthe effects of the differentlevels of sugars in the extenderson survivalof dialyzed spermatozoa. It can be observed in each of the evaluationmethods that as sugar level was increased,sperm survivaldecreased. The use of a buffer containing95% of an isosmoticsugar solutionand 5% glycerolwas highly detrimentalto the cells. All dependentvariablesshowed significantdifferences(P(O.05) between buffer and level of sugar used (Figure1 a,b,c). Table 1. Effect of sugar levels on survivalof dialyzed semen Fresh Senw7
Percentageisosmtic sugar solutionas plrt of total buffer volma 0
25 50 75 95
Frozen thawed semen
Percentage Parcentage motility rrotility 57.4arf 60.3a 52.0b 31.8c 5.ld
a,b,c,dmame superscriptsin the same calm difference(DO.05). fN = 36 observationsper maan.
37.5arb 38.7a 34.7b 16.6C 1.3d
IQrcentagecells filteredin Sephadexcolmm 49.4a 44.7b 36.7c 24.9d 8.5e
indicateno significant
Experiment2 Table 2 illustratesthe differencesobtainedbetween treatments. Motility of spermatozoain fresh and frozen thawed sampleswas significantly lower (KO.05) in the solutionsthat containederythrose,glyceraldehydeor glycerol ccmparedwith motility in extenderscontaining 30% of any Of the C5 or c6 iSoSmotiCSolutions. Erythrceeand gly-
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MAY 1989 VOL. 31 NO. 5
THERIOGENOLOGY
0
R
50 100 0 Percentage of isosmotic sugar solution 3s part of total buffer volume
60
Percentage of isosmotic sugar solution as part of total buffer volume
C
-
---------_ --.
‘\ ‘\
40
-
‘\
‘\
: II : :
20 -
: : :
\
Fiqure
I.
0 50 100 Percentage of isosmotic sugar solution as part cf total buffer volume Effect of different levels of isosmotic sugar solutions included in extender and dislysate. A) Percentage of motile spermatozoa in fresh semen sani~lcs. B) Percentage of motile sperrstozoa in postthaw samples. C) Fcrcencage of cells that passed through the Sephadex column in frozen-thawed snmples. The buffers used ,~erepiperazine-N-X-BIS (Z-ethane sulfonic acid) titrated with TRIS (I’/V)
hydroxymethyl of PIPEST
and
MAY 1989 VOL. 31 NO. 5
aminomethnne sodium citrate.
to
ph
7.0
(PIPEST)
and
~---I
a
1:l
cr’zbination
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THERIOGENOLOGY
ceraldehydepreparationswzre toxic to the cell and obstructedsparm motility. Glycerolwas vesent at a high cmcentration aud hence was deletereous,even though 36% of the cells -sad through the Se@mdex colunn. The motilityobserved showed abnormalpatternsdanmstrating glycerol shock. The evaluationof fresh and thawed senen samples showed no significantdifferencesbetween the inclusionof 30% of the isosnotic sugar SOlUtiOnS fran arabitol (C5) up to glucose (C6) in the extending media and dialysate. The postthawevaluationof mtility sM no significantdifferenceswith the inclusionof 30% isosmoticC5 and C6 sugars with the exceptionof D-glucose.
Table 2. Effect of sugars and sugar alcoholson freezabilityof dialyzed bull smen Fresh semen
Typ2 of sugarg D-glucose Sorbitol D-galactose Dulcitol D-ribose Ribitol D-xylose Xylitol D-arabinose Arabitol D-erythrose Erythrol D-glyceraldehyde Glycerol No sugar
Frozen-thawed seam
Fercehtagecells No. carbon IQrcentage Rarcentage filteredin atans motility motility Se&&xcolUnn
$ 2 C5 C5 C5 C5 C5 C4 C4 C3 C3 -
64.2"rbrf 65.0a 61.7a,b
28.4b,c 42.5a
:z'l: . ’ 66.7a 66.7a 65.8arb
:::$?t: 37.5a:b 42.5a 36.6a*b 35.8a,b 40.8a 41.7a
;;:;Z l.Od' 56.7b
l.od
47.5c 42.5c
,:$.b 1:od 22.5c 20.2c
51.2a 48.3a,b 50.8a 42.5b,c*d :;::Z 48.1a:b 48.2arb :;:;Z 23.1e' 44.8a,brc 22.1e 36.4d 39.ocrd
a,bfc,d+Sa superscriptsin the sam colum indicateno significant difference (PO.05). fN=Six observationspsr mean. 930% W/V) of isomotic sugar solution. The remainingbuffer solution was iscmotic RIPEST-citratein a 1:l ambination. RIPESTwas pm-epured by titrationof PiperazineN-N-BIS (2-ethanesulfonicacid) with N-TRIS (hydroxymethyl) aminarethanesolutionto pB 7.0. DISCUSSION Sugar solutionshave been reportedbeneficialfor sperm cells.
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MAY 1989VOL. 31 NO. 5
THERIOGENOLOGY Glucose wes chosen in our experimentbecause it is easily metabolizable by spernlatozca (21,221. A beneficialeffect has been reported of milk salts that ocntainmainly lactose 01 bull sperm survival (12,151. Raffincee has also been reportedto ba beneficialfor bull and ram sperm survival (ll). Our results agree with those fran Unal et al. (231, who found no differencebetween sperm responseto lactose or raffinose. Nagase and Graham (24) have also reportedno significantdifferenceson sperm motility when semen was frozen in pelletedform, among extenders ccntainingglucose, lactose,raffinose,stachyceeor their canbination. Haumerstedt(25) dialyzed bovine semen and reportedthat at ccncentrationsexceeding1 x 10-S spann/ml,cellular adenceinetriphospbate (ATP) dropped to very low levels (1 to 3 nMoles par lo'* cells) and the cells became n-tile. He suggestedthatatccncentraticms belowlx 10-8 cells/ml,a matabolizablecanpoundmust be present in the dialysate to maintain cell viability. As the sugar ccocentrationincreased,the proportionof buffer material decreased,which changed the propertiesof the extendingmedia. These cunbinationsdid not best voted the sperm cells fran the changes in @I and osmotic Faressure that occur during cooling and freezing. The differentcunbinationsof PIPEST and sugar were not as effective in maintainingmotility as the acmbination PIFEST-citrate-sugar was. Smnen extended in PI-T-sugar solutions sh& a rapid decline in survivalwhen more than 25% iscemoticsugar solutionswere Feesent. When PIPEST-citrate-sugar solutionsware used, the decline in survival started only after 75% of the extenderwas canposed of an isosnoticsugar solution. In the data obtainedafter filtrationof postthawsamples, sperm motility declined as the sugar levels increased. The analysisof individualvalues of the data obtained after filtrationshawE?dthat higher numbers of notile cells ware obtainedwhen 25% of isosmoticsolutionsof either glucose or lactosewere used. The trisaccharideraffinosewas not as effectiveasthenrx-~o-or the disaccharideused. Sugar alcoholsare sugar molecules in which the aldehydegroup is replaced by an alcohol group. They have been reportedbeneficialfor sperm cell motility (10,111. This smy be due to their structuralsimilarity to the cxyoprotectantglycerol. However,whenglycerolwas used as a sugar alcohol, it wx pesent in very high concentrationand became toxicto sperm. The results in the secondexperimantindicatethatwhen 30% W/V) of isosmoticsolutionsof sugar or sugar alcoholsranging fran G5 t0 c6 WXe used, StatiStiCally SUpriOr motilitywas obtainedand a higher number of cells passed through the Sephadexcolumn. Ths presence of any of these sugar solutionsyielded a statisticallysuperiormotility rate (X0.05) to the absence of sugar in the dialysateor retentate. In suvmary,the results of curtwoexpsrin~nts shwthatsperm survival in PIPSST-citratetmffer is significantlyhigher (X0.05) than in the PIPEST buffer. Nodifferencewas c&air&between the use of gluccse or lactase or between lactose andraffinosewhenthe percentage of motile cells that passed through the sepladexcolumn in pxtthaw samples was analyzed. High levels of sugar in extendingmedia and dialysate peeved not to be beneficialfor sperm motility in fresh
MAY 1989 VOL. 31 NO. 5
THERIOGENOLOGY
or postthawsemen samples. Results of the seccnd exparinmt illustrate that 30% (V/V) of the buffer system for dialysisof spermatozoamy be canposedof ah isosmoticsolutionof glumse, galactose,ribose,xylose, arabinoseor their correspondentsugar alcohols. Motility of spermatozoa in each of these solutionsin fresh or frozen thawed Semen sampleswas significantlysuperior (X0.05) to that of s~rmatozoa extended in samples that did not contain sugar. REFERENCFS
1. Hers, H.G. Le mechanismde la formationdu fructoseseminal et du fructose fcetal. Bicchim.Biophys. Acta. -37:127-138(1960). 2. Hartree,E.F. Inositolin seminal plasma. Bicchem.J. -66:131-134 (1975). 3. King, T-E., Isherwood,F.A. and Mann, T. Sorbitolin semen. AbstractsIVth Int. Cmgr. Biochem.,Vienna, -77 (abstr)(1958). 4. Clark, J.B., Graham, E.F., Lewis, B.A. and Smith, F. D-mahnitol, erythritoland glycerol in bovine semsn. J. Reprcd. Fertil.-13: 189-197 (1967). 5. Georgiev,G.H. Activityof sorbitoldehydrogehasein ram and bull seminal plasma. Int. J. Bicchem.5:21-22 11974). 6. Hafs, H.D. and Elliot, F.I. The effect of nethods of adding egg yolk and monosaccharidesto the survivalof frozen bull spermatozoa. J. Dairy Sci. -38:811-815(1955). 7. ws, C.W. and Martin, I.C.A. The effects of equilibration period and sugar content of the diluehtcnthe survivaland fertility of bull sprmatozoa deep frozen to -79'C. IV Int. Ccngr. Anim. Reprod. and Artif. Insem.,The Hague. pp. 964-967 (1960). 8. Martin, I.C.A. and mns, C.W. Effects of time of equilibration and additionof fructosecm the survivaland fertilityof bull spermatozoadeep frozen to -79Y. J. Repod. Fertil.2:404-410 (1961). of sperm 9. Seglins,A. and Enina, V. Effect of several mnmehts diluent oh bull spermatozoaduring freezing. Vet. Zhivotnovod. pp. 31-35 (19731. 10. Rajamanann,A-H., Graham, E.F. and Smith, F. The effect of a new extender incorporatingdried egg yolk on the fertilityof bovine spsrmatozca. V Cmgr. Intl. pzr la Reprod. Animale e la Fecohd. Artificiale,Trehto, Italy. pp. 392-397 (1964). 11. Nagase,H. andTanizuka,T. Use of @yols as protectahtsagainst freezing injury to lxlllspermatozoa. II. Protectiveeffects of polyols during differentfreezingrates. VI Int. Cmgr. Anim. Reprcd. and Artif. Insem., Paris,France. pp. 1107-1109 (1968).
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12.
Garcia, M.A. and Graham, E.F. Effects of lowmolecular-waight fractions (LMWF) fran milk, egg yolk and seminal plasma on freezability of bovine spermatozca. Cryobiology%:429-436 (1987a).
13.
Garcia, M.A. and Graham, E.F. Factors affectingthe removal of low-molecular-weight-fractions (IMWF) fran egg yolk and seminal plasm in extended semen by dialysis: Effect on post-thawsperm survival. Cryobiology-24:437-445(1987b).
14.
Garcia, M.A. and Graham, E.F. Dialysisof bovine semen and its effect on fresh and freeze-thawedspermatozoa. Cryobiology-24: 446-454 (1987c).
15.
Garcia, M.A. Effects of DialyzableDiffusatesfran Milk, Egg Yolk and Seminal Plasma on Freezabilityof Bovine Spermatozoa. MS. Thesis, Universityof Minnesota,1980.
16.
Garcia, M.A. Dialysis: A Method for Studyingand Impoving the Quality of Frozen Bovine Spermatozoa. Ph.D. Thesis, Universityof Minnesota,1984.
17.
Garcia, M.A. and Graham, E.F. Developmentof a buffer system for dialysis of bovine sparmtozoa. I. Effect of zwitterionbuffers. Theriogenolcgy-31: (1989).
18.
Graham, E.F., Vasguez, I.A., SchTlehl, M.K. and Evensen, B.K. An assay of semen quality by use of Sephadex filtration. VIII Int. Cmgr. Anim. Reprod. and Artif. Insem.,Cracm, Poland. pp. 896-899 (1976).
19.
Snedecor,G.W. and Co&ran, W.G. StatisticalMethods. Icwa State UniversityPress, Anrss,1976, pp. 339-369.
20.
Steel, R.G. and Torrie, J.H. Principlesand Proceduresof Statistics. A BianedicalApproach. MCQ-aw Hill, New York, 1980, pp. 187-188,192.
21.
Kraft, L.A., Foley, C.W., Hcwarth, B. and Johnson, A.D. Carbohydratematabolismby washed spermatozoaof various species. J. Dairy Sci. %:786 (1971).
22.
Mann, T. The Biochemistryof Semen and the Male Reproductive Tract. Barnes and Noble, Inc., New York, 1964. p. 493.
23.
Unal, M-B., Berndston,W.E. and Pickett,B.W. Influenceof sugars with glycerol cm pxt-thaw motility of bovine spermatozoain straws. J. Dairy Sci. -61:83-89 (1978).
24.
Nagase, H. and Graham, E.F. Palletedsemen: Canparisonof different extendersand pccesses co fertilityof bovine spermatozoa. V. Int. Ccogr. per la Reprcd. Animale e la Fecond. Artificiale, Trento, Italy, pp. 387-391 (1964).
25.
Hamnerstedt,R. Use of high speed dialysis to prepare bovine sperm for metabolic studies. Biol. of Repmd. g:389-396 (1975).
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