Evaluation on sperm quality of freshly ejaculated boar semen during in vitro storage under different temperatures

ELSEVIER

EVALUATION ON SPERM QUALITY OF FRESHLY EJACULATED BOAR SEMEN DURING IN VITRO STORAGE UNDER DIFFERENT TEMPERATURES Chun-Xia Zou and Zeng-Ming Yang’ Department of Biotechnology, Northeast Agricultural University Harbin 150030, China Received for publication: August 18, 1999 Accepted: November 3, 1999 ABSTRACT The purposeof this study was to assess the spermquality of fresh ejaculatedboar semen storedunder different temperaturesfor up to 48 h in order to usethe fresh semenefficiently. Spermatozoawere evaluated by 4 methods: Using trypan blue staining, the viability of spermatozoastoredat 39, 20, 15 and 4 “C!for 48 h were 1.6, 46.9, 42.0 and31.O%,respectively. Employing the hypoosmotic swelling test (HOST) showed 1.7%(39 “C), 28.7%(20 “C), 24.1%(15 “C), and 20.1%(4 “C) coiled-tail spermatozoafollowing 48 h storage. With Coomassie blue staining,the ratesof acrosome-intactspermatozoastoredfor 48 h were 4.5%(39 “C), 35.3%(20 “C), 55.7%(15“C) and 22.8%(4 “C). Using fluoresceinisothiocyanate-peanut agglutinin (FITC-PNA), the percentagesof acrosome-intactspermatozoastoredfor 48 h were 4.3%(39 “C), 43.2%(20 “C), 17.3%(15“C) and 14.8%(4 “C), respectively. The cytoplasmic dropletswere found in 18.66% of the spermatozoain fresh semenand were gradually shed during storage. The results of these4 methodswere highly correlatedand could be usedto characterized sperm-cell quality effectively. These findings indicated that both membrane integrity and viability of spermatozoacould be preservedwell during in vitro storageat 20 “C and 15“C for 24 to 48 h. 0 Zoo0 by Elsevier

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Key words: boar,spermatozoa,viability, membraneintegrity, in vitro storage Acknowledgments The authors are indebted to Dr. Michael JK Harper for valuable discussionand critical reading of the manuscript.The authorsthank Yun-Long Li and Kui-Zhong Yue for their technicalhelp. This work wassupportedby ChineseNationalNatural ScienceFoundationNo. 39825120and39730250,andHeilongjiangProvince OutstandingScientistFund. ‘Correspondenceand reprint requests: Zeng-Ming Yang, Department of Biotechnology, Northeast Agricultural University, Harbin 150030, China. Telephone:86-45l-5390846; FAX: 86-451-5303336; E-mail: [email protected] Theriogenology Q 2000 Elsevier

53: 1477-l Science

488,200O Inc.

0093-891WOO/$-see front PII s0093-891x(00)00290-9

matter

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Theriogenology INTRODUCTION

Artificial insemination(AI) in cattle is the reproductive biotechnology which has been widely used for genetic improvement, and most of the semenused for AI has been frozen. Cryopreservationof spermatozoaoffers an effective meansfor long-term storageof important genetic material. This method eliminatesthe difficulty associatedwith transportinganimalsor fresh semenover long distancesor extendedperiods of time. However, current methodsfor cryopreservationof boar spermatozoaare unsatisfactory.To date,the useof cryopreservedboar semen often involves cumbersomeprocessing procedures, and it yields low artificial inseminationdosesper ejaculatedue to low cell survival, resultingin both low fecundity rates and smalllitter sizes(9). The principal advantageof usingunfrozen, liquid semenis that fertility is maintainedeven with low numbersof spermatozoain the inseminate. With fewer than 1 million spermcells per breedingunit, conceptionrateswith liquid semenare similar to those with frozen-thawed semen containing approximately 15 million spermatozoa(27). Liquid storageof boar semenwas superiorto that of frozen storagewith respectto preservingsperm fertility (4). However, the fertility of liquid semenis gradually lost during extendedperiodsat ambient temperature. Therefore, proper assessment of the viability and fertilizing ability of spermatozoais of utmost importancefor choosingthe best way to store semenin vitro and for decidinghow long fresh semencanbe stored. Coomassie blue staining,Hypoosmoticswelling test (HOST) and fluoresceinisothiocyanate-peanutagglutinin (FITC-PNA) have recently been appliedfor assessing the membraneintegrity andacrosomestatusof the spermatozoa(7,8,14,22). The presentstudy was designedto evaluate semenquality during in vitro storageat different temperatures,using4 currently availablemethodsto optimizethe useof freshsemen, MATERIALS AND METHODS SemenPreparation Sperm-richfractions were collected from 1 of 5 crossbredboars(Harbin White x Long White, 1 to 1.5 yr of age) of known reproductivehistory, by the gloved handtechnique.All of the treatmentswere repeatedat least3 timeswith the semensamplesfrom the different boars. The semensamplewere transportedto the laboratoryat 37 “C within 30 min andfiltered through 4 layers of sterile gauze into a prewarmedbeakerto removegel particles. The filtered semen wasrandomlyaliquotedinto 4 sterilebeakersandwasmaintainedat 4 different temperatures:39, 20, 15 and 4 “C, respectively. The semenof eachtemperaturegroup wasthen incubatedfor 0, 1, 2, 4, 8, 12, 24, 36 and48 h, respectively, The time 0 h observationwasmadebeforethe fresh semenwasdivided into 4 temperaturegroups,Boar semenwasstoredat 39, 20, 15 and4 “C for 48 h without any additives before any bacterial contaminationwas detectedunder microscopic examination. SpermMotility To assess motility, 50 uL of the semenfrom eachgroup were mixed with 1 mL of 0.9% NaCl solution. The diluted semenwas examinedfor motile spermcellswith a haemocytometer under X 400 magnification. Sperm motility was assessed by determiningthe percentageof spermatozoashowingany movementof the flagellum. At least200 spermatozoawere counted for eachgroup.

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Analysis of SpermViability by Trypan Blue Staining Spermviability was evaluatedby taking 50 PL semenfrom eachgroup andmixing with 1 mL of 0.4 % trypan blue in 0.9% NaCl and then incubating for 15 min at 37 ‘C. Viable spermatozoaremainedunstainedwhile deadcellswere stainedblue. The percentageof unstained spermatozoawascountedin a sampleof at least200 spermatozoaunderX 400 magnification. HypoosmoticSwelling Test(HOST) The assaywas performed by mixing 50 PL of the semenwith 1 mL of hypoosmotic solutionpreparedby mixing 7.35 g sodiumcitrate 2H,O and 13.51 g fructosein 1 L of distilled H,O, and then incubatingat 37 “C for 30 min (8). Viable spermatozoahad coiled tails after HOST, as observed under phase contrast microscopy. A total of 200 spermatozoawas evaluated for coiled tails by counting in at least 5 different fields under a phase-contrast microscopeat X 400 magnification. l

Coomassie Blue Staining Semenwaswashedtwice in PBS by brief centrifugation(10,000rpm, 20 set) andfixed in 3.7% paraformaldehyde/PBS for 30 min. The sampleswere suspended and spreadon slidesfor air drying. The spermsmearwas stainedin 0.25% Coomassie Blue R250 for 2 min and washed in H,O. The air-dried slideswere mountedand then checkedfor the percentageof acrosomeintact spermatozoain at least200 cells. FITC-PNA Staining The spermsmearpreparedfor Coomassie blue stainingwasfixed in acetoneat -20 “C for 5 min. After nonspecificbinding was blocked with 1% BSA/Hepes-NaCl-Casolution for 30 min, the spermwere incubatedin 5 pg/mL FITC-PNA/Hepes-NaCl-Casolutionfor 30 min (2). The slideswere washedin PBS 3 times and then mountedin 30% glycerin/PBS. The whole acrosomewas visualized with strong green fluorescenceunder a fluorescencemicroscopeand was scored as acrosome-intactsperm cells. The percentageof fluorescent acrosome-intact spermatozoawascountedin at least200 spermcellsunderan Olympusfluorescencemicroscope. CytoplasmicDroplet The percentageof spermatozoawith dropletson the tail was countedon Coomassiebluestained slides at each sampling period during storage in at least 200 sperm cells. The cytoplasmic droplets were easily observedon the tail or neck of a spermatozoonfollowing Coomassie blue stainingundera bright field microscope.

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StatisticalAnalysis The percentagesof spermatozoashowingdifferent staining in individual experiments were comparedwith Time 0 hour valuesusingStudent’st-test. The correlationamongthe abovementionedmethodswasevaluatedby linearregressionanalysis. RESULTS SpermMotility The motility of spermatozoain fresh semenwas 92.Ok3.1%anddeclinedasthe period of storagewasextended(Table 1). In the 39°C storagegroup,motility was higherthan in the other groupsat 4 h, andlower than in the other groupsafter 12 h. Comparedwith the Time 0 h group, the differencesat 2 h and4 h were significant,but the differencesat 8 h were highly significant. No motile spermwere observedafter storageat 39°Cfor 48 h. However,motility of the other 3 groups at 48 h was 25.5%(20 “C), 22.0%(15 “C) and 12.1%(4 “C), respectively. A greater percentageof spermatozoawas motile at 20 “C than at 15 and 4 “C after 4 h of storage.No differencewasobservedat 8 h amongthe 4 groups. Table 1. Percentageof motile spermatozoain boarsemenduring in vitro storageat different temperatures Spermmotility (%) Storage period(hours) 0 1 2 4 8 12 24 36 48

39 “C 92.0*3.1 92.4k5.5 88.5+8.3a 84.3k12.3’ 62.7+10.3b 34.2*4.4b 16.9f4.4b 5.4+2.9b Ob

20 “C

15“C

4 “C

92.Ok3.1 88.7k3.5 81.9f3.9 78.4k5.2’ 70.0k4.3b 60.7,6.3b 42.5+4.7b 33.4k3.6’ 25.5+5.4b

92.0k3.1 84.6f3 .6a 76.9M.8’ 68.7+5.9b 62.5+5.8b 55.6+7.1b 41.7+5.2b 29.1k4.9’ 22.0k4.6b

92.Ok3.1 77.2k4.0b 70.3k6.0b 57.9f4.7b 53.6k6.6b 44.8+7.9b 36.9M.8b 23.4f7.4b 12.1k3.6b

“bThevalue at eachtime point of eachtemperaturegroupwascomparedto the 0 hour value. a: PcO.05; b: P
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SpermViability The spermviability assessed by trypan blue staining is shown in Table 2. There were 93.2+3.1% unstainedspermatozoain fresh semen. Sperm viability in 39 “C group was maintainedat a high level for the first 4 h, then droppedsharply and was only 1.6% by 48 h. The percentageof unstainedspermatozoawashigher in 20 “C group than other groups,although thiswasonly significant at 8 h. Therewasa high percentageof spermatozoaunstainedat 20 “C (46.9%) 15 “C (42.0%) and4 “C (30.9%) by 48 h of storage.Additionally, stainedspermatozoa increasedgradually in these3 groups. In eachtemperaturegroup, there was a sharpdrop in spermviability between2 and 8 of storage. Table2. Percentageof viable spermatozoaduring in vitro storageasassessed by trypan blue staining Trypan blue-unstainedspermatozoa(%> Sperm storage(hours) 0 1 2 4 8 12 24 36 48

39 “C 93.2+3.1 92.8k2.7 89.9f5.2a 88.7+7.0* 70.3+15.4b 44.8k13.0b 28.4+9.6b 7.2k6.2b 1.6fl.lb

20 “C 93.2k3.1 92.9f3.4 91.8f3.9 89.7k3.7’ 86.Ok4.0’ 81.9f9.1b 72.4k2.4b 63.1+4.0b 46.9+6.2b

15°C 93.2k3.1 90.5fl.3 90.4k2.2a 84.4k3.2’ 81.9k2.9b 74.4+9.5b 67.2t7.3b 54.7f5.7b 42.0k10.6b

4 “C 93.2f3.1 82.5k2.2’ 81.1k2.38 78.0k3.5b 76.4+3.2b 67.2f8.4b 52.4k6.7b 40.9f9.1b 30.9+10.0b

“bThevalue at eachtime point of eachtemperaturegroupwascomparedto the 0 hourvalue. a: PcO.05; b: P
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Table3, Percentageof boar spermatozoashowingswellingduring in vitro storageat different temperaturesasassessed by hypoosmoticswellingtest (HOST) Hypoosmoticswellingspermatozoa(%) Storage 2OT 15OC 4°C period (hours) 39-z 59.5rt6.7 59.5k6.7 59.5k6.7 59.5f6.7 0 56.2f5.9 5iOk6.2' 49.0~k6.8~ 1 56.1f3.2 2

52.3k2.9

54.5k6.0'

47.2k6.6b

4 8

43.7+3.3b

48.Ok6.9'

42.8+6.5b

46.5f7.0b 41.6+6.0b

32.1+4.gb

43.2k4.8'

39.2+6.7b

39.0f4.0b

12

27.2+10.0b

41.7+11.2a

36.3+10.4b

33.0k3.0b

24

11.7k3.2b

35.4+12.0a

33.7f12.0b

29.7k4.3b

36

5.2~k22.6~

30.8+14.4b

28.1+8.9b

24.7+6.

48

1.7+0.5b

28.7k11.9b

24.1+8.1b

20.1k5.6b

lb

“bThevalue at eachtime point of eachtemperaturegroupwascomparedwith the 0 hour value a: P 15“C > 4 “C > 39 ‘C. Table4. Percentageof acrosome-intactspermatozoaduringin vitro storageat different temperaturesasassessed by Coomassie blue staining Storage Acrosome-IntactSpermatozoa(%) period (hours) 39°C 2OT 15 “C 4°C 0 98.OkO.9 98.OIkO.9 981OkO.9 98.0k0.9 1 95.6k1.7 96.3k1.9 96.0k1.3 90.2k1.9 89.1k7.2'

95.3k2.4

95:8&1.4

87.3ti.O'

12

4

66.7+13.9b

89.6+3.3"

91.2k2..6a

77.3*3.5b

24

38.9+8.4b

86.4*4.1'

82.5+3.1b

64.3M.5b

36

15.8+8.0b

64.4+2.3b

60.2k5.2b

38.7k3.8b

48

4.5k3.1b

55.3+6.2b

35.7+5.0b

22.8k3.0b

abThe value at eachtime point of eachtemperaturegroupwascomparedwith the 0 hour value. a: PcO.05;b: P
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Changesof AcrosomeStatusAssessed by FITC-PNA Staining The percentageof acrosome-intactspermatozoais shownin Table 5. In the 39 “C group, acrosome-intactspermatozoadecreasedsignificantly by 1 h (68.4%), and there were only 14.8 and4.3% left by 36 and48 h, respectively. Therewasalsoa significant drop by 1 h in the 15’C (80.5%) and4 “C (8 1.2%)groups,but not asgreatasthat at 39 “C. The 20 “C group performed much better than other groups. In this group, 82.3 and 43.2% of the spermatozoaremained acrosome-intactby 24 and 48 h, respectively. However,,there were only 68.8% by 24 h and 17.3%by 48 h at 15“C, and45.5% by 24 h and 14.8%by 48 h at 4 “C. Table5. Percentageof acrosome-intactspermatozoaduring in vitro storageat different temperaturesasassessed by FITC-PNA -___ .Storage Acrosome-IntactSpermatozoa(%) period (hours) 39T 20°C 15 “C 4T 0 94.81kO.8 94.8izO.8 94.8kO.8 94.8kO.8 1 68.4k5.3b 93.2kOo.8 80.5*1.7a 81.2k2.2' 4

54.8k6.8b

92.1+1.0

80.7+0.8*

72.3k22.8b

12

42.5+2.1b

84.5k1.5'

73.2f2.3b

59.5M.3b

24

37.3+3.gb

82.3k2.8"

68.8k2.1b

45.5+5.0b

36

14.8+6.0b

58.5k2.8b

40.7k3.8b

20.8;4.9b

48

4.3fl.lb

43.2k4.0b

17.3M.2b

14.8k4.3b

abThevalue at eachtime point of eachtemperaturegroupwascomparedwith the 0 hour value. a: P
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Table6. Percentageof the spermatozoawith cytoplasmicdropletsduringin vitro storageat different temperatures Spermatozoawith droplets(%) Storage period(hours) 0 1 2 4 8 12 24 36 48

39°C 18.7f1.8 17.3k1.8 16.8f1.6 16.0f1.3 13.6k22.0a 12.3k1.8” 7.1+2.0b 5.7+1.8b 3.7+0.7b

20°C

15°C

18.7rt1.8 17.7’13.2 17.4ti.5 16.7&l.3 14.8+1.9’ 12.9+2.1a 11.6+1.9b 9.6f1.2b 5.0k2.7b

18.7f1.8 11.5+1.8b 11.4k1.9b 9.0*0.5b 8.3+1.7b 7.6+0.6b 4.8+2.0b 4.3kO.8’ 2.8k0.3b

4°C 18.7k1.8 11.9+1.2b 11.3+1.6b 10.9~00.Sb 10.1f1.6b 9.5+1.3b 6.5+1.4b 5.7fl .2b 3.7M.8b

abThevalue at eachtime point of eachtemperaturegroupwascomparedwith the 0 hour value. a: PcO.05; b: P
Trypan blue staining 0.9427

0.9427 0.9532 0.9049 0.8554

0.9283 0.9729 0.9242

HOST Coomassie PNA-FITC procedure blue staining procedure 0.9532 0.9283 0.8733 0.8669

HOST: hypoosmoticswellingtest. PNA-FITC: fluoresceinisothiocyanate-peanut agglutinin.

0.9049 0.9729 0.8733 0.9391

0.8554 0.9242 0.8669 0.9391

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DISCUSSION The spermviability of frozen and extendedboar semenhas been examinedpreviously (11,16,24). In this study, fresh boar semenwas stored for 48 h. Although longer periods of storagewere alsoassessed, contaminationoften occurredafter 48 h. Seminalplasmais credited with having significant anti-bacterialactivity, which is effective on a wide range of organisms andappearsto have a role in preventinginfection of the malereproductivetract (6,23). To use fresh semeneffectively, the quality of spermatozoaduring storageat different temperatureswas examinedand characterizedin the presentstudy. The rate of motile spermatozoain fresh ejaculateswas 92% in this study, which is very similar to the 93.1% reported previously (3). Temperaturealso hasa strong effect on sperm motility. The motility of the spermatozoastoredat 39 “C wasbetterthan that of other groupsin the first 8 h, but it droppednotably thereafterandwasonly 34.2% by 12 h. This may be dueto the high rate of metabolismandrapid energyconsumptionat 39 “C. However, a temperatureof 4 “C alsocauseda rapid decreasein motility at 1 h of storage(from 92.0 to 77.2%), andthis may have beendueto cold shock. Boar spermatozoaareparticularly susceptibleto cold shockwhen cooled below 15 “C. Resistanceto cold shock could be acquiredif boar semenwereaheldat room temperature in seminalplasmafor several hours (21). For the 4 temperaturegroups examinedin this study, 20 and 15 “C were better for maintainingspermmotility than 39 and4 “C. Although motility is an easy way to evaluate spermatozoa,it may not reflect the real viability of the cells. Spermswelling in responseto hypoosmoticconditionsresultsin the appearanceof a coiled tail The HOST procedure was a sensitive, reproducibletest for assessingthe functional integrity of boar spermmembranes after incubationunder hypoosmoticstressconditions,and it may be a useful tool for detecting populationsof less viable spermatozoawhen used in conjunction with another type of membraneintegrity test (26). The HOST score for fresh semenwas 59.5% in our study, which was closeto 61.8% describedby Vazquez et al (26). In bull semen,a 36.2% HOST scorewas recordedwhen spermviability was 87% (5). To date, there are no reportsof HOST findings in boar spermatozoaduring in vitro storage. The HOST scoreat 39 “C wasbetterthan that at 15 and4 “C at 4 h, andwaslower than in the other groups8 h later. Therewere only 11.7%coiledtail spermatozoaat 39 “C by 24 h. The HOST scoresat 15 and 4 “C at 1 h droppedsharply,which may have beenthe result of damageto the sperm membranecaused by low temperatures.In the mouse (lo), the percentageof coiled tail spermatozoawas highly correlated with the percentageof acrosomereactions and zona penetrations(r=O.9). However, the ability of the plasmamembraneof bull spermatozoato swellwithout disruptionin hypotonic solutionsdoesnot alwayscorrelatewith the fertility of the samplesinvestigated,but it representan easymethodof high replication (4). In this study, the HOST scoreof eachgroup waslower thanthe percentageof motile spermcells,which wasalso reportedby Vazquezet al (26). The differencemay be dueto the fact that somespermatozoawith membranedamageremain motile. Functional heterogeneity was demonstratedwith flow cytometry of a spermpopulationwhich includedspermatozoawith a reducedsurvival capacity despitetheir motility (17).

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The feasibility of usingFITC-PNA for studyingthe acrosomereactionin boar spermatozoa has been demonstratedpreviously (25). At the level of fluorescencemicroscopy,the signal representingPNA binding is mainly limited to the acrosomalcap of boar spermatozoa.Thus, FITC-PNA canbe usedasa reliableprobefor detectingacrosomereactionsin boar spermatozoa (7). There were 94.8% acrosome-intactspermatozoain fresh semen. At 39 “C, the numberof acrosome-intactcellswas markedlyreducedto 68.4% by 1 h, 37.3% by 24 h, and 4.3% by 48 h. Both 15 and4 “C temperaturesleadto the lossof acrosomes during storage;however, the sperm acrosomecan be maintainedat a high level during the storageat 20 “C. There were 82.3% acrosome-intactspermatozoaby 24 h and 43.2% by 48 h. Various methodshave been developedto detect the acrosomalstatusof spermatozoa. Coomassieblue staining hasbeen shownto be an easy and reliable methodfor assessing the statusof the spermacrosome(14). Usingthis methodin the presentstudy, 98% of fresh spermatozoawere acrosome-intact. The high temperatureof 39 “C had a harmful effect on the acrosome,resulting in only 38.9% acrosome-intactspermcellsby 24 h. However, the percentageof acrosome-intactspermatozoa at 20, 15 and 4 “C by 24 h of storagewas still 86.4, 82.5 and 64.3%, respectively; after 48 h, there were still 55.3% at 20 “C and 35.7% at 15 “C, but only 4.5% at 39 “C and22.8% at 4 “C. The 20 “C groupwasbetter in keepingthe acrosomeintact than othertemperature.groups, Incubation of bull semenfor 24 h at 4 “C led to an increasein the percentageof spermatozoashowing swelling of the apical ridge (1). The storageof ram semenat room temperaturefor 4 h in the dark has a marked effect on capacitationin a subpopulationof spermatozoa,without changesin their motility or membraneintegrity. Appearanceof the acrosomewas only slightly affected (20). Sincethe increasein the percentageof spermatozoa showingswellingwas numerically similarto the increasein the short-termresponseto A23 187, those spermatozoashowing swelling of the apical ridge were likely to undergothe acrosome reaction, representinga form of capacitation(20). Acrosomalloss may occur becauseof a degenerativeprocessfollowing the death of the spermatozoa(7). Recently,the combineduse of FITC-ConA and Hoechstbis-benzimide33258hasserved asa feasibleand reliable method for the assessment of physiologicalacrosomereactionsof bovine spermatozoa(19). Ejaculated spermatozoastored for 1 or 2 d can penetrate in-vitro matured oocytes in TCM-199 supplementedwith a high concentration of Ca”(8.76 mM) even when no caffeine or preincubationwas employed(15). In anotherstudy,ejaculatedspermatozoafrom semenstored for 18 h at 18 “C could penetratein vitro matured oocytes in modified Tyrode’s medium containing2 mM caffeine and4.72 mM Ca*’ without preincubation.This demonstratedthat boar spermatozoacan be capacitatedwithout preincubationif the fertilization medium contains caffeine and a high concentrationof Ca”(l8). Whether or not boar spermatozoaunderwent capacitationin our study is not clear. The cytoplasm of a spermatidis absorbedby Sertoli cells during spermiation. The remnant of the cytoplasm, known as the cytoplasmic droplet, is located on the neck of the spermatozoon, The dropletscontain many vesicles,tine curved tubulesand various enzymes, but their role in spermfunction is still not clear (12). In boars, the droplets could still be observedin 97% of spermatozoaimmediately after ejaculation; however, only 35% of the spermatozoahaddroplets1 min after ejaculation,with no significantchangesthereafter(13).

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The results of present study indicate a significant correlation among sperm motility, trypan blue staining, HOST, Coomassie blue staining and FITC-PNA methods used to evaluate semen quality. The percentage of spermatozoa shown to be viable by trypan blue staining in each group was greater than that of motile spermatozoa, suggesting that some viable spermatozoa were immotile in semen. Both Coomassie blue staining and FITC-PNA have been used as reliable methods to assess the acrosome status and are highly correlated(t=0.9391). Trypan blue staining was closely correlated with Coomassie blue staining(r=0.9729) and FITCPNA(r=0.9242), suggesting that plasma membrane integrity is related to acrosome membrane integrity. Since all 5 tests were used to assess sperm viability, the close linear correlation among the tests may suggest that any one of them would be an equally effective diagnostic indicator of sperm quality. In conclusion, boar semen may be stored at 20 or 15 “C for 48 h without significant effects on spermatozoal quality. Thus, semen stored for 24 to 48 h at 20 or 15 “C may be useful for AI and IVF, as demonstrated by the high correlations among outcomes with trypan blue staining, HOST, Coomassie blue staining or/and FITC-PNA. REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9 10.

Aalseth EP, Saacke RG. Morphological changes of the acrosome on motile bovine spermatozoa due to storage at 4 “C. J Reprod Fertil 1985; 74:473-478. Ahluwalia B, Farshori P, Jamuar M, Baccetti B, Anderson WA. Specific localizationof lectins in boar and bull spermatozoa. J Submicrosc Cytol Path01 1990; 22: 53-62. Bamba K. Evaluation of acrosomal integrity of boar spermatozoa by bright fieldmicroscopy using an eosin-nigrosin stain. Theriogenology 1988; 29: 1245-125 1. Clarke RN, Johnson LA. Effect of liquid storage and cryopreservation of boarspermatozoa on acrosomal integrity and the penetration of zona-free hamster ova in vitro. Gamete Res 1987; 16:193-204. Correa JR, Zavos PM. The hypoosmotic swelling test: its employment as an assay to evaluate the functional integrity of the frozen-thawed bovine sperm membrane. Theriogenology 1994; 42:351-360. Fair WR, Couch J, Welner N. The purification and assay of the prostate antibacterial factor (PAF). Biochem Med 1973; 8:329-339. Fazeli A, Hage WJ, Cheng F-P, Voorhout WF, Marks A, Bevers MM, Colenbrander B. Acrosome-intact boar spermatozoa initiate binding to the homologous zona pellucida in vitro. Biol Reprod 1997; 56:430-438. Garcia-Lopez 2, Ollero M, Muino-Blanc0 T, Cebrian-Perez JA. A dextran swim-up procedure for separation of highly motile and viable ram spermatozoa from semen plasma. Theriogenology 1996; 46: 141-15 1. Gillmore JA, Liu J, Peter AT, Critser JK. Determination of plasma membrane characteristics of boar spermatozoa and their relevance to cryopreservation. Biol Reprod 1998; 58:28-36. Jeyendran RS, Van der Ven HH, Perez-Pelaez M, Crabo BG, Zaneveld LJ. Development of an assay to assess the functional integrity of the human sperm membrane and its relationship to other semen characteristics. J Reprod Fertil 1984; 70:219-228.

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