Development of a buffer system for dialysis of bovine spermatozoa before freezing. III. Effect of different inorganic and organic salts on fresh and frozen-thawed semen

THERIOGENOLOGY

DEVEIQEMENTOF A BUFFER SYSTEM FCR DIALYSISOF BOVINE SPERMATOZOABEFORE FREEZING. III. EFFECTOFDIFFEWXW INORGANICAND ORGANIC SALTS CN FRESHANDEROZEN-THAwEDS!mEN M.A. Garcia1 and E.F. Graham Departmentof Animal Science Universityof Minnesota Saint Paul, MN 55108 Received for publication:September 2, 1988 Accepted:February 16, 1989 ABSTRACT Three experimentswere ccmductedto study the effect of inorganic and organic acids m survivalof dialyzed bovine spermatozoa. Ejaculateswere pooled,extended (l:lO),dialvzed (1:50) for 2 h during cooling, and 1 h later they ere frozen in pellets and stored in liquid nitrogen. The pelletswere thawed in aluninm block depressions (peheated at 45°C) and transferredto a test tube at roan temperature as the last icendted. Sperm motility was record& in all samples before freezingand after thawing. The nmber of spermatozoathat passed through the Sephadex colunn was analyzed in all the postthaw samples. No statisticaldifference (p>O.O5)was found between the use of potassiun (ROH) or sodium hydroxide (NaDH)as titrationbases. Howaver, solutionscontainingcalcium (Ca+t)or magnesium 0&g++) provided significantlyless (PO.O5)was found in sperm survivalof the postthawsamples when Cat+ or Mg+t were present. Inorganicsalts of phosphates,carbonatesor chloride providedsignificantly less protectionto the cells than the control extenderswith Na citrate (p
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the motility and oxygen uptake of bull spermatozoais depressedby high concentrationsof @msphate. White (4) indicatedthat phosphateinhibits the oxidationof lactic acid and suggestedthat in #msphate diluents the 02 uptake is lm, and large amounts of lactic acid accumulate. Salisburyet al. (5) developd a sodium citrate extender containing 50% egg yolk, and Kinney and Van de Mark (6) found the optimal Na citrate concentrationto be in a range of 1.55 to 1.95% (W/v). Marsralian seminal plasma is normallyan isotonicand aln-cstneutral fluid containingamong other substancesNa, K, Mg, Ca, Cl and phosphate ions (7,8). Their concentrationvaries between ejaculatesand methods of collection. Their effect cm sperm viabilityhas been studied by a number of researchers. Mann (8) claimed that many of the differencesin metabolicactivitiesand viabilityof spermatozoaof different individuals were due to differencesin chemicalccnqmsitionof seminal plasm in these individuals. White (4) showed a variationin the biological proprties of alkali metals of washed ram and bull semen. Lithiumwas found to be toxic; Na was regardedas neutral;@assium, rubidiumand cessim were beneficial. However,high potassiumconcentrationswere harmful and they depressedmotilitv. These findingsare similar to those of Cragle and Salisbury (9) and Yassen and Foote (101,who reporteda decrease in bull sparm motility,02 uptake and fructoseutilization caused by the increasein the concentrationof sodium and potassiumions. In previousreportswe have shown that dialysismay be used as a tool to improvepostthawquality of bovine spermatozoa(11-14). The use of differentbuffer systems and additionof sugars in the dialysatealso affects the postthawmotility of sparm (15,161. The intent of our researchwas to report the effect of different inorganicand organic salt solutionsand ions on the motility of dialyzed sprmatozoa before and after freezing. MATERIaTSANDMETHODS Ejaculateswere collectedtwice a week with an artificialvagina. Semen samples showing at least 70% motility ware pled and diluted in a 1:lO ratio. Aliquots of extendedsemen ware placed in dialysisbags of 12,000 to 14,000 molecularweight cut off and dialyzed for a period of 2 h during cooling frczn37 to 5°C. The samplesware then transferredto test tubes, and 1 h later they were frozen over dry ice depessions (17) and stored in liquid nitrogenuntil evaluation. Each semen aliquot was extendedand dialyzed against the sm buffer system. The palletswere thawed in preheatedalminum block depressionsas indicatedearlier (11-16). The percentageof motile q.erm was recorded for all samples before freezingand imnediatelyafter thawing. R&thaw sampleswere also analyzedby electroniccount of the nmber of mtile cells that pissed through the Sephadexcolumn (11,18,19). Dialysatesolutionswere preparedas in the previousexperiments (11-16). The canpositionof each dialysatewas 30% PIFEST (11,151+ 35% of each of the solutionslisted in Tables 1 or 2 + 30% isosmoticglucose + 5% glycerol (V/V). The final solutionwas titratedwith isosmotic solutionsof either THIS (Tris (hydroxymathyl) aminamsthane)or PIFES

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(Piperazine-N-N-Bis (2-ethanesulfonicacid)) to pH 7.0 and the final osmotic pessure adjustedto 320-325mOsm/kg before the addition of the cryoprotectant. Semen extender solutionscontained20% egg yolk and the correspondentbuffer system. Experiment1 The inorganicsubstancesstudiedwere Potassiumhydroxide (KOH), Sodium hydroxide (NaOH), Rstassium phosphatemonobasic (KH2FO4), Potassiumphosphatebibasic tK2HOP4),Sodium phosphatebibasic (Na2HFO4),Potassiumchloride (KCl),Sodium chloride (NaCl),Potassium bicarbonate(KHCO3)and S&lium bicarbonate(NaHCO3;Table 1). Sodium citrate was used as the control canpound. Semen aliguots of pooled ejaculates ware extended and dialyzed against the sams buffer system. The experimentwas replicatedsix tires. Sons of the canbinationmaterials precipitatedand could not be preparedas extender systems:therefore, the experimentaldata was analyzed by ANOVA using the General Linear Mcdel (GIM; 20) procedurefor imbslanceddata. Duncan'smultiple range test (21) was used to establishdifferencesbetween the means. The statistical design was 6 (replicates)x 2 (ions)x 6 (inorganic substances). Experiment2 Formate, acetate, citrate, oxalate, succinateand tartrate isosmotic solutionswere pepared as soluble salts of Na, K and, if possible, of Ca and Mg. Calcium salts of citrate,oxalate, succinateand tartrate as well as Mg citrate,oxalate and tartrate precipitatedand could not be used. Tbs dialysatesolutionswsre preparedby the canbinationof the followingpercentagesof isosmoticsolutions: 30% PiperazineN-N-BIS (2-ethanesulfonicacid) tirtratedwith N-IRIS (hydroxymathyl) amino methane URIS) to form PIEEST + 30% glucose t 35% of each of the organic solutionslisted in Table 3. All the dialysatescontained5% glycerol. Semen diluentsware preparedby addition of 20% egg yolk to each of the above dialysates. The data were evaluatedby analysis of varianceusing a 6 (replicates)x 6 (buffers)x 4 (ions) factorialdesign by the GL&Iprocedure for *lanced data (20), followedby Duncan'smultiple range test (21) to canpare the maans obtained. Experiment3 Isosmoticsolutionsof Na and K citrate and Na and K tartratewere by canbining isosmoticsolutionsof prepared. Dialysatesware Farepared 30% PiperazineN-N-BIS (2-ethanesulfonicacid)(PIPES)titratedwith either NaCH or KOH + 30% glucose solution+ 35% of each of the isosmotic solutionslisted in Table 3 W/W. Each dialysatecontained5% glycerol. Extender solutionswere Farepared by the additionof 20% egg yolk to the above solutions. Semen sampleswere handled as in previous experiments. The data was analyzed by ANOVA (22) of a 5 (replicates)x 2

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(titrationbases) x 10 (buffersolutions)factorialdesign, followedby Duncan'smultiple range test (20) to evaluatedifferencesbetweenmeans. FCESULTS

Experiment1 No statisticaldifferences(p>O.O5)in qerm motility of nonfrozen sampleswere found among the KOH, HFO4 or H2P04 extenders (78.3,67.1 and 73.3% motile cells, respectively). Hcwever, spsrm motility in extenderscontainingthe last two substanceswas not significantlydifferent fran the motility in extendersccntainingNaOH (61.6%motile cells). The poorest results wsre obtainedwhen carbonateor Clcontainingsolutionswere used (34.5 or 40.4% motility,respectively). The frozen-thawedsamples showed higher motility of spermatozoain samples containingKOH than all other types of salts used (pO.O5)was found in sperm motility of samples containingKOH, HP04 or H2EO4 when the psrcentageof motile cells after filtrationwas analyzed (39.3, 38.7 and 36.2%, respactively). The number of cells through the colmm in the extendercontainingH2FO4 was not significantlydifferentfran the nmber of cells obtainedwhen NaCfl was used in the extender (33.5%cells). The coorest resultsware obtainedwhen Cl- and IiCO3"-wareincludedin the buffer (29.5 and 25.6% cells through the colmn respectively). The effects of sodium and @assium were studied as main effects. No statisticaldifference (p>O.OS)was observedbetween the ~esence of the two ions when the prcentage of motile cells in nonfrozenand postthawsampleswas analyzed. Hmever, the analysisof the cells that passed through the Sephadexcolunn indicateda si ificantly (pO.O5). Formate,acetate and succinatesolutionsprovidedsignificantlythe least protection (p
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THERIOGENOLOGY motility,respectively)when the percentageof motile cells in the nonfrozen seman smples was evaluated. In the frozen-thawedsamples,the presence ofNa+in the dialysatesprovidedthe highestqermsurvival rate when the percentageof motile oslls ms evaluated (23.5, 18.0, 17.2 Table 1. Effect of sodium and patassim ions and several inorganic buffers cn survival of dialyzed semen Fresh

Frozen thawed

SE%lEIl

Sel?en

Inorganic bufferg

Barcentage motility

Barcentage motility

Na citrateh

71_6a,b,c,f 78.3arb 83.3a 61.6C 72.sa,blc 39.ld 63.3bfc 61.6C 41.6d 41.6d 27.Sd

42.Sarb 50.0a 41.6b

KOH KH2m4 Na2IiFO4 K2Hm4 KC1 NaH2=4 Nash NaHCO3 NaCl KH(303

Z~'c 20:1e 30.0crd ::.P 21:oe 13.6e

Percentagecells filtered in Se@ladexcolmm

49.oa 39.3b 39.3b 38.2b,c :,":$:z: 33.1cpd ;;.O$d 24:Oe 23.5e

KCH= Fotassiumhydroxide,KH2H)q= Botassium-plate monobasic, Na2HW4 = Sodium @ms&ate bibasic,K2HFo4 = Fotassium@msphate bibasic, KC1 = Fotassiunchloride,Na2H!HpD4 = Scdim @m&hate bibasic, NaCH = Scdiunhydroxide,NaRCC3 = Sodium bicarbonate,KC1 = potassium chloride,KHCO3 = Rkassiunbicarbmate. a,b,c,d,e5amesuperscriptsin the same colum indicateno significant difference (DO.05). fN = Six observationspar man. gFina1 canpositionof each dialysatewas 30% PIFEST + 35% of each of the solutionslisted + 30% glucose + 5% glycerol W/V). Each solution hadan osmotic ~essureof 320 to 325n0mAcg. PIPESTwas Wepared by titratinga solution of PiperazineN-N-BIS (2-ethanesulfmic acid)(BIPES)with NlRIS hydroxymathylamcnarethaneURIS) to pH 7.0. hCcntrolsubstance.

and 9.3% motility, respactively). Homver,nodifferencewas foundbetween the use of Na+ or K+in the exteuderswhen the percentageof cells passing through the colum was analyzed (37.0 and 36.2% cells, respactively). SolutionsccntainingCa* and Mg* ware the least effective (28.8 and 26.4% cells, respectively).

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The interactionbetween buffer and ion was statisticallysignificant (RO.05) for the analysisof the percentageof motile cells and the percentageof cells passing through the Se&adex colmm (Table 2). The interactionwas caused by a differentresponseof the semen to the presence of K+ and Na+ in the medium. With tartrate,oxalate, fonaateand acetate an increasein the percentageof cells passingthrough the column was observedwhen Na+ was pesent in the extender. Hcwever,when citrate or succinatewas used, higher values were obtained in the ,presence of K+ containingsolutions. The correspondentNa+ salts were not statisticallydifferentfran each other (DO.O5), except when citrate and for-mate were the solutionsused.

Table 2. Effect of differentsalts of organic acid substrateswhen added to extender and dialysateon seman samples dialyzed before freezing Fresh semen

Organic buffer]

K Citrate Na Tartrate Na Oxalate K Tartrate K Oxalate Na Citrate K Succinate Na Succinate Mg Formate Na Fomate Mg Succinate Ca Formate K Form&e Ca Acetate Na Acetate Mg Acetate K Acetate

Bercentage motility

Frozen-thawed semen

Fercentage motility

Percentagecells filteredin Sephadexcolumn

33.3a 31.3a 32.5a 30.8a 21.2b 30.0a 11.5crd 16.0brcld

49.6a

:;:;?E ,

;;*z'd 31:9d::,f 28.1erfrg 25.7erfrg 24.6frg 21.39 20.99 17.9h

Lg':$d.c 1:.:b,c,d 12:odte 9.gd 1.6e

;;:$1: m;:; mm;:; 34.&d

adwAerftghs~

superscriptsin the same collnn indicateno significant difference(DO.05 ). iN = 6 observationspar mean. jFina1 canpositionof each dialysatewas 30% PIBEST + 35% of each of the solutionslisted + 30% glucose + 5% glycerol (V/v). Each solution had an osmotic messure of 320 to 325 m@m/ka. BIPEST solutionwas preparedby titiationof piprazine-N-N-BIS?2-ethanesulfonic acid)(PIFES)with N-THIS hydroxymethylaminmthaue (THIS)to @I 7.0.

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Experiment 3 No statisticaldifference (p>O.O5)was observedbetween the use of NaOH or KOH as titrationbases when motility of fresh or frozen-thawed samples was analyzed (67.8 and 69.2% motile cells in fresh semen samples and 44.4 and 43.2% in the frozen thawed samples,respectively). Hmever, post-filtrationresults in extenderscontainingNab ware significantly superior (X0.051 to KGfIsolutions (43.4 and 40.1% cells, respectively). The effects of canbinationsof Na and K salts of citrate and tartrate in the buffer and dialysateare illustratedin Table 3. The analysis of the parcentageof motile cells of the fresh semen samples sh& no statisticaldifferencebetween the Na salts of tartrate and citrate and the differentcmbinations employed in extenders5, 6, 7, 8, and 9. The lowest percentagesof motile cells were obtainedwith K salts of tartrateor citrate or their canbinationsin extenders9 and 10. Hmever, no statisticaldifferences(WO.05) ware obtainedbetween the treatmentstien the parcentageof motile cells of frozen-thawed Table 3. Effect of sodium CNa+) and potassium (K+) salts of citric acid, tartaric acid solutionsor their canbinationwhen used 35% of the buffer and dialysate Fresh semen

Extender number 1 2 3 4 5 6 7 8 9 10

as

Frozen thawed seman

Dialysatecontaining Parcentagecells 35% of isosmotic PercentagePercentage filtered in solutionf motility motility Sephadex column K Tartrate MT) K Citrate (KC) Na Tartrate (NaT) r&aCitrate (NaC) 50% NaT-50% KT 50% NaC-50% KC 50% NaT-50% KC 50%NaC-50%KT 50% NaT-50% NaC 50% KT-50% KC

45.0a 43.3a 42.0a 44.1a 45.4a 44.1a 46.2a 40.0a 45.8a 42.0a

44.3a 44.3a 42.3a 41.7a 42.9a 41.9a 41.0a 34.7b 42.0a 42.5a

a,brcrdSamesuperscriptsin the same colmm indicateno significantdifference (p>O.O5). eN = Five observationsper man. fFina1 canpositionof each dialysatewas 30% PIPEST + 35% of each of the solutionslisted + 30% glucose + 5% glycerol W/V). Each solution had an osmotic pessure of 320 to 325 -kg. PIIEST was prepared by titrationof a solutionof piperazineN-N-BIS (2-ethanesulfonic acid)(PIPESlwith N-IRIS hydroxymethylarnincmethane URIS) to @-I 7.0.

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THERIOGENOLOGY samplesware evaluated. The number of cellsthroughthe Se#Wiex colum was significantlylcwer (pCO.05)when the canbinationof 50% of an iscemoticsolutionof citrate and 50% of iscemoticNa tartratewas used as the extender systemas canmredwith all the other buffer canbinationsused. DISCDSSICN The results of our study illustratethat postthawsperm motility in solutionscontaining35% isosmoticNa citrate (V/W in the extenderand dialysatewas statisticallysuprior to any other buffer solution studied. Spam utility of fresh and frozen thati samples was high in potassium@mqbate buffers and was not differentfran motility in samples containingNa citrate. However,the evaluationof the number of cells that passed through the Sephaaexcolmm shd statisticaldifferencesbetween the use of *osphate containingdialysatesand Na citrate. All data obtained for car-ate and chloridesolutionswere significantlylower (pO.O5)was obtained between the use of Na or K salts when sperm motility in fresh samples and nmber of cells passing through the Se@dex colmm wzre analyzed. J&tenderscontainingCa or Mg salts wovidsd the least protection to the cells during freezingand thawing. These results are similar to those of Yassen (231 and Yassen and F'oote(101,who studied the effects of Na, K, Ca and Cl m sperm preservationand reportedthat the use of 20 to 80% of NaCl or KCl, or the use of 10 to 40% CaC12 in TRIS buffer was harmful to the cells. Hmever, it has been found that potassiumreplacments ware twice as harmful. In ccntrastto our results,Yassen and Foote found that the replacementof 25, 50, 75 and 100% of Na citrate buffer with K citrate reduced the freezabilityof the cells. They cmclkied that K, but not Na, is harmful at high levels in the extender. Our experimentsindicatedthatthe use of 35% (V/v) of iscemoticsolutionof K citrate,Na tartrate,Na oxalate,or K oxalate in the extenderand dialysatepovide supariorprotectionto the cells during freezingand thawing than extendersccntainingthe sam mount of Na citrate. Spam motility in the latter was significantlysuprior (X0.051 in fresh and frozen thawad samples than semen extended in solutionscontainingsuccinate,formate or acetate salts (Table 2). Results of our third experimentindicateno difference (p>O.O5) between the use of NaOIior KOH as titrationbases for PIPES, and they indicateno differencesin spsrm postthawmtility or between the number of cells passingthrough the colmm when 9 of the 10 differentcanbinationsof RIFE9 and citrate or tartratesolutionsware used (Table 3). Therefore,it can be concludedthat an effectivesolutionfor use as

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dialysate for extended bovine spermatozoacan be formulatedby canbining 30% (V/V) isosmoticNa salt of PIPES t 30% (V/V) iscsmoticglucose solution + 35% (V/V) of either cne of the canbinationslisted in Table 3 plus 5% glycerol.

1.

Philips, P.H. Preservationof bull semen. J. Biol. Chem. -130: 415-416 (1939).

2. Philips, P.H. and tardy, H.A. A yolk buffer pabulum of bull seman. J. Dairy Sci. -23:339-340(1940). 3. Bishop, M. and Salisbury,G.W. Effect of dilutionwith saline and phosphatesolutionson oxygen uptake of bull semen. Amer. J. Physiol.181:114-118(1955). 4. White, I.G. The effect of sax inorganicions co mamnalian spermatozoa. IIIrd Int. Ccngr. cm Anim. Reprod., Cambridge. pp. 23-25 (1956). 5. Salisbury,G.W., Fuller, H.K. and Willet, E.B. Preservationof bovine sparmatozcain yolk-citrateand field results fran its use. J. Dairy Sci. -24:905 (1941). 6. Kinney, W.C. and Van de Mark, N.L. The effect oE yolk and citrate levels and stage of maturity on the survivalof bull sperm of subzero storage temperatures. J. Dairy Sci. -37:650-652(1954). 7. Maule, J.P. The semen of animals and artificialinsemination. Technical cannunicationN. 54. Cammnwealth Bureau of Animal Breeding and Genetics,Edinburgh,U.K. (1962). 8. Mann, T. The Biochemistryof Semen and the Male Reproductive Tract. Barnes and Noble, Inc., New York, 1964. p. 493. 9. Cragle, R.G. and Salisbury,G.W. Factors influencingmetabolic activity of spermatozoa. IV. RI, osmotic pressureand the cations sodium, potassiumand calcium. J. Dairy Sci. -42:1304-1313(1959). 10. Yassen, A.M. and Foote, R.H. Freezabilityof bovine spermatozoain TRIS-bufferedyolk extenderscontainingdifferentlevels of TRIS, sodium, potassim and calcium ions. J. Dairy Sci. 50:887-892 (1967). 11. Garcia, M.A. Dialysis: A method for Studying and Improvingthe Quality of Frozen Bovine Sparmatozoa. Ph.D. Thesis, Universityof Minnesota,St. Paul, 1984. 12. Garcia, M.A. and Graham, E.F. Effects of low molecular-weight fractions (m) frm milk, egg yolk and seminal plasma on freeza(1987). bility of bovine spermatozoa. Cryobiology-24:429-436

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13.

Garcia, MA. and Graham, E.F. Factors affectingthe removal of the law mlecular weight fraction (IMWF)fran egg yolk and seminal plasma in extendedsemen by dialysis. Effect cm post-thawsperm survival. Cryobiology-24:437-445 (1987).

14.

Garcia, M.A. and Graham, E.F. Dialysisof bovine semen and its effect on fresh and freeze-thawedspermatozoa. Cryobiology-24: 446-454 (1987).

15.

Garcia, M.A. and Graham, E.F. Developmentof a buffer system for dialysis of bovine spermatozoa. I. Effect of zwitterionions on postthawmotility. Theriogenolcqy-31: (1989).

16.

Garcia, M.A. and Graham, E.F. Developnt of a buffer system for dialysis of bovine spermatozoa. II. Effect of sugars and sugar alcohols on pxtthaw motility. Theriogenology31: (1989).

17.

Nagase, H. and Graham, E.F. Pelletedsemen: Canparisonof different extendersand processeson fertilityof bovine spermatozoa. Vth Ccngr. Int. per la RiproduzioneArtificiale,Trento. pp. 387-391 (1964).

18.

Graham, E.F., Schmehl,M.K. and Evensen,B.K. An overviewof column separationof spermatozoa. VIIth Tech. Congr. Artif. Insem. and Reprcd. Madison,WI. pp. 69-73 (1978).

19.

Graham, E.F., Schmehl,M.K., Evensen, B.K. and Nelson, D.S. Viabilityassays for frozen semen. Cryobiology15:242-244(1978).

20.

Goodnight,J.H., Sail, J.P. and Sxle, W.S. SAS User's Guide: Statistics. StatisticalAnalysisSystem. In: Ray, A.A. ted). SAS Institute,Gary, NC. pp. 139-199 (1982). -

21.

Steel, R.G. and Torrie, J.H. Principlesand Proceduresof Statistics. A BicmedicalApproach". New York, 1988. pp. 187-188, 192.

22.

Snedecor,G.W. and Co&ran, W.G. StatisticalMethods. Ima State UniversityPress, .Ams, IA, 1976. pp. 339-369.

23.

Yassen, A.M. Effect of electrolyteconcentration, method of glycerolationand aging on the freezabilityof bull semen extended with TRIS-bufferedegg yolk extender. Diss. Abstr. 2:335b (1967).

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