Acrosomal integrity and sperm concentration are two important parameters to assess

the quality of frozen semen doses which in terms validates the fertilizing capacity and conception

rate. The present study was undertaken to evaluate acrosomal integrity by Giemsa’s stain and sperm

concentration of FSS using improved neubauer chamber in Exotic pure Jersey, Crossbred Jersey,

Indigenous Gir cattle and Indigenous Murrah buffalo prior to the field use. The overall values of

Giemsa’s stain were observed as 73.74±0.31, 18.65±0.33 and 7.79±0.25 percent for Intact Acrosome,

Partially Damaged Acrosome and Fully Damaged Acrosome, respectively. Overall values of sperm

concentration were 21.98±0.28 million per straw. The study indicated that there was no significant

difference (P<0.05) among the breeds and the values mostly correlates with the guideline of Minimum

Standard Protocol for Production of bovine semen, 2012 of Govt. of India

Debdulal Sana

Srinibas Das

Subhas Bose

Sumit Chowdhury

Tapas Gupta

Directory of Open Access Journals

101INTRODUCTIONThere is ever increasing demand of frozensemen straws from different parts of the countryfor superior quality germplasm. It is also knownthat a more accurate estimation of fertility ofthe ejaculate is achieved by increasing thenumber of tests performed in the laboratory. Inanimals, capacitation and the subsequentacrosome reaction of spermatozoa are theessentials steps before fertilization andformation of zygote. Acrosomal evaluation, twomethods (double staining and Hoechst/FITC-PSA) are commonly used but producescontradictory result (Risopatron et al., 2001,Kohn et al., 1997). The determination ofacrosomal integrity of spermatozoa by singleGiemsa’s staining technique is an essential toolto evaluate fertilizing capacity of thesemen.thistechnique is very simple, rapid andallows examining the results after many years(Wells and Awa 1970). It binds over an outeracrosomal membrane but does not bind to theacrosome-reacted spermatozoa. Giemsa’s isEVALUATION  OF  FROZEN  SEMEN  BY  A CROSOMALINTEGRITY  AND  SPERM CONCENTRATION - TWO  VITALQUALITY  PARAMETERS  OF  MALE  FERTILITY  IN  BOVINESSumit Chowdhury*,  Srinibas Das,  Tapas Gupta,  Debdulal Sana, Subhas Bose1ABSTRACT: Acrosomal integrity and sperm concentration are two important parameters to assessthe quality of frozen semen doses which in terms validates the fertilizing capacity and conceptionrate. The present study was undertaken to evaluate acrosomal integrity by Giemsa’s stain and spermconcentration of FSS using improved neubauer chamber in Exotic pure Jersey, Crossbred Jersey,Indigenous Gir cattle and Indigenous Murrah buffalo prior to the field use. The overall values ofGiemsa’s stain were observed as 73.74±0.31, 18.65±0.33 and 7.79±0.25 percent for Intact Acrosome,Partially Damaged Acrosome and Fully Damaged Acrosome, respectively. Overall values of spermconcentration were 21.98±0.28 million per straw. The study indicated that there was no significantdifference (P<0.05) among the breeds and the values mostly correlates with the guideline of MinimumStandard Protocol for Production of bovine semen, 2012 of Govt. of  India.KEY  WORDS:  Acrosomal  Integrity, Sperm Concentration, Giemsa’s staining, Active volume.Frozen Semen Bull Station, (Paschim Banga Go-Sampad Bikash Sanstha), Composite State AnimalHusbandry Farm, Salboni, Paschim Medinipur, West Bengal, India,  PIN-721147.1 Chief Executive Officer, Paschim Banga Go-Sampad Bikash Sanstha, Kolkata -700098, India.*Corresponding author:  e-mail : drsumitvo@gmail.comResearch  ArticleExplor  Anim  Med  Res,Vol.4, Issue - 1, 2014, p. 101-107ISSN  2277- 470X (Print), ISSN 2319-247X (Online)Website: www.animalmedicalresearch.org102Exploratory Animal and Medical Research, Vol.4, Issue 1, June, 2014superior over single staining with Trypan blue(Jankovicova et al.,  2008) and Congo red(Kovacs and Foote  1992).Sperm counting is another essential step inthe evaluation of male fertility as the successof artificial insemination depends on theoptimum number of spermatozoa in the frozensemen doses. The World Health Organization(WHO 1992) recommends the use of ahaemocytometer for determining spermconcentration in semen. Photometric estimationis most common method of estimating spermconcentration during processing of semen. Thevalidation of photometric sperm concentrationis generally achieved by improved Neubauerchamber haemocytometer (Mahmoud et al.,1997).  The present study was undertaken tojudge the semen quality with two vital qualitytests i.e. acrosomal integrity and spermconcentration of frozen semen straw in Exoticpure Jersey, Crossbred Jersey, Indigenous Gircattle and indigenous Murrah buffalo.MATERIALS   AND   METHODProduction 0.25 ml French mini FSS:The semen straws were prepared from theExotic pure Jersey, Crossbred Jersey,Indigenous Gir cattle and Indigenous Murrahbuffalo bulls maintained at Frozen Semen BullStation, Salboni, West Bengal.  Twenty samplesof 0.25 ml FSS of different days production overa period of two month were taken into the study.The concentration of neat semen was checkedin photometer (Accucell, IMV, France) to keepthe final concentration of 20 million whenpacked in automated filing and sealing machine(IS-4 machine). Further the straws were kept inLN2 for quality test by Giemsa’s stain foracrosomal integrity and sperm concentration byimproved Neubauer chamber.Acrosomal integrity Test by Giemsa’sstain:The stock Giemsa’s stain was prepared asper manufacturers’ protocol. Firstly, 0.77 gmGiemsa’s powder (MERCK) was measured inweighing balance and saturated for long time(2-3 hrs) with methanol and glycerol mixturepre-warmed at 400C. The methanol-glycerolmixture was prepared by measuring 75 mlabsolute alcohol and 25 ml of glycerol (98%pure). After proper mixing, the stain was filteredwith 0.22 µm sterile Millex (Millipore). Thesolution was kept at 370C in an incubator for7 days in amber color bottle for maturation withintermittent shaking. The stock solution wasready to assess the acrosomal structure ofspermatozoa.The staining procedure was followed theprotocol of Watson, 1975. Firstly, FSS werethawed in 370C for 30 sec and content waspoured into pre-warmed sugar tube at 370C.3 µl of Sorenson Phosphate Buffer (SPB) wasput into clean grease free slide, on that 30 µl offrozen semen was mixed and a smear was drawnFig.1.  Intact Acrosome of Murrah Buffalo.103and air dried. The slides were put into 5%formaldehyde solution for fixing at 370C for 30min (Campbell et al., 1960). The slides wereremoved from the solution, washed in runningtap water and air dried for further processing.The working solution of Giemsa was preparedmixing Giemsa’s stock-3 ml, SPS-2 ml andMilli-Q water- 45 ml in a cup linger and warmedat 370C for 30 min. The smeared slides ofspermatozoa was put into the working solutionand kept at 370C for 2 hrs. The slides wereremoved from the stain and washed in runningtap water and finally air dried. The counting ofintact, partially damaged and fully damagedacrosome was carried out in oil immersionOlympus microscope (BX51) at1000X(10X100) magnification.Particulars Pure Jersey Cross-bred Indigenous Murrah Buffalo Overall (N=80)(N=20) Jersey (N=20)  Gir (N=20) (N=20)Intact Acrosome 73.05 ± 75.15 ±0.71* 73.05 ± 0.48 73.70 ± 0.61 73.74 ± 0.31(%) 0.59PartiallyDamaged 18.60 ± 17.25 ± 0.67 20.00 ± 0.75* 18.75 ± 0.54 18.65 ±0.33Acrosome (%) 0.57Fully Damaged 8.40 ±0.58* 7.70 ± 0.48 7.55 ± 0.48 7.50 ± 0.50 7.79 ± 0.25Acrosome (%)Table 1:  Acrosomal Integrity values.Values depicted as Mean ±SE, * Indicates highest value among the breeds, N= No. of observationsParticulars Yellow for Transparent Orange for Grey for Overallpure Jersey for Cross-bred Indigenous Gir Murrah buffalo (N=80)(N=20) Jersey (N=20) (N=20) (N=20)Packaged 211.90 ± 0.84 212.07 ± 1.04 229.48 ±1.54* 211.49 ± 0.61 216.23 ±1.00volume (ul)Active 207.57 ± 0.70 206.41 ±0.85 218.83 ±1.00* 205.66 ± 0.49 209.62 ± 0.72volume (ul)Wastage 4.33 ±0.44 5.63 ± 0.71 10.65 ±1.59* 5.82 ± 0.41 6.62 ±0.5volume (ul)Table 2 : Straw Volume values.Values depicted as Mean ±SE, * Indicates highest value among the breeds, N= No. of observationsEvaluation  of  frozen  semen  by  a crosomal  integrity  and  sperm concentration -two  vital...104Exploratory Animal and Medical Research, Vol.4, Issue 1, June, 2014Sperm Concentration in 0.25 ml FrenchMini Straw:The sperm concentration was achieved usingImproved Neubauer chambers (China). Thesolution of 0.2% eosin yellow and 0.9% sodiumchloride in 100 ml milli-Q water was used forthis quality test of sperm. Firstly, the thawed-FSS was poured into sugar tube at 370C. Thentwo times dilution of sperm with eosin solutionwere made to give final dilution 1:1000. Then20 µl of diluted semen was charged on aneubauer chamber on both sides with a coverslip on it. Then the chamber was kept in a Petridish in wet environment for 10 min for settlingdown of the sperm. The total 400 small WBCcounting squares were counted for presence ofsperm. Finally, total no of sperm present wascalculated with mathematical formula in thefollowing ways;Sperm concentration/ 0.25 ml straws =AXBX10X 106Where, A = Total no of sperm in upper/lowerchamber, B =Active volume of the semen instraw and 10 is the Multiplication factor.In Minimum Standard Protocol forProduction of Bovine Semen (MSP) of Govt.of India the active volume of semen straw (0.25ml) is given as 0.21 ml. A study was alsoconducted to find out the active volume by thefollowing method.Firstly, the blank straws of different color ofdifferent breeds i.e. orange (Gir), transparent(CBJ), yellow (PJ) and grey (Buffalo) wereweighted in Weighing balance (Sartorius,Germany). The filled straws with semen ofdifferent breeds were also weighted. Thedifference between filled and empty straws wascalculated and packaged volume wasdetermined by multiplying with specific gravityof Tris-citrate-egg-yolk dilutor (specific gravity1.04 gm/dl). Secondly, the active volume in FSSwas calculated. The semen from FSS wasdiscarded with the help of A.I. gun (FrenchMini) cutting the laboratory seal. Then theempty-straw having factory plug and cut portionof laboratory seal was weighted breed wise. Theweight difference between the filled straw andempty-straw was calculated and usable volume/Particulars Pure Jersey Cross-bred Indigenous Murrah Overall (N=80)(N=20) Jersey (N=20) Gir (N=20) Buffalo(N=20)No. of spermatozoa 21.80 ± 0.57 21.57 ± 0.56 22.96 ± 0.56* 21.60 ± 0.55 21.98 ± 0.28as per obtained activevolume (million/FSD)No. of spermatozoa 22.05 ± 0.56 21.95 ± 0.60 22.05 ± 0.56 22.05 ± 0.60 22.02 ± 0.28as per MSP givenactive volume(million/FSD)Table 3:  Sperm Count in frozen semen doses.Values depicted as Mean ±SE, * Indicates highest value among the breeds, N= No. of observations.105active volume was determined multiplying withspecific gravity of dilutor (1.04).The data were analysed using a computerprogramme, SPSS 16.0 ®.RESULT   AND   DISCUSSIONThe aim of the study was focused to evaluateFrozen Semen by acrosomal integrity and spermconcentration test to supply good quality of FSSfor field artificial insemination. Firstly, themorphology of acrosome was tested byGiemsa’s staining methods in all four breeds(Fig. 1). Results revealed that there was nosignificant difference among breeds. Theaverage intact acrosome, partially damagedacrosome and fully damaged acrosome was73.74%, 18.65% and 7.79%, respectively(Table 1). Highest intact acrosome value wasobtained in Crossbred Jersey bulls. The resultswere quite satisfactory in terms of guidelinesfixed in MSP of Govt. of India and such goodmorphology of acrosome of spermatozoaindicated good fertility of the spermatozoa aswell as the efficacy of this simple but accuratemethod of Giemsa’s staining for all four breedsunder study. The acrosomal reaction wasvalidated to the fertility using FITC-PSAtechnique in bovines (Jancovicova et al., 2006).The FITC-PSA labeling technique is less timeconsuming, yolk environment is not limitingfactor, but requires fluorescent microscopy,sperm morphology not cleared, and signal fadeswith time and also have strong poisoning effect.Evaluation of buffalo semen was graded intoacrosome-intact live (AIL), acrosome-intactdead (AID), acrosome-lost live (ALL) andacrosome-lost dead (ALD) with double stainingprocedure Trypan Blue/Giemsa’s stain (Bocciaet al., 2007). The almost same result ofacrosome reaction was achieved by doublestains (37.98%) versus Hoechst/ FITC-PSAstaining (39.33%) in frozen semen (Jancovicovaet al., 2008). It can be concluded that Giemsa’sstain could be used in semen station for rapidscreening of large number of frozen semendoses.Further, the active volume of FSS in allbreeds was determined using mathematicalformulas. Overall active volume was obtainedas 0.2096±0.72 ml (Table 2). In case of FSS ofGir breed, the highest packaged volume as wellas highest wastage- volume was obtained inorange straws (0.2295 ml and 0.0107 mlrespectively). Then the sperm concentration inFSS was calculated in all types of breeds usingobtained active volume and the overallconcentration per frozen semen straw was 21.98and 22.02 million, respectively (Table 3). In allthe cases, recognizable spermatozoa, includingloose heads were counted; other germinal linecells and free tails were ignored. Lineweaveret al. (1970) reported that when 1 ml of freshor frozen semen containing millions of livespermatozoa was placed in mid-cervix inheifers, 4 hours later, 469 or 191 numbers ofspermatozoa were found in the oviducts and1559 or 733 numbers of spermatozoa werefound in uterus, respectively.  We found thatoverall sperm concentration of 21.98±0.28million per dose with effective semen volumeof 0.209±0.72 ml. It was also found that6.62±0.53 ul of semen was absorbed into thefactory plug, reducing the active volumedischarged into the uterus during A.I. The activevolume could be further increased by using nextgeneration filling sealing machine (like MX5of IMV, France) where, only the dilutor will beused to soak the factory plug resulting in zeroabsorption of sperm. Though many researcherscalculated sperm concentration withEvaluation  of  frozen  semen  by  a crosomal  integrity  and  sperm concentration -two  vital...106Exploratory Animal and Medical Research, Vol.4, Issue 1, June, 2014sophisticated techniques i.e. flow cytometry(Takumi et al., 2002) and Computer AssistedSemen Analyzer (CASA) (Coetzee et al., 2001),these instruments also have problems inreproducibility and accuracy, and there isvariation within and between the laboratories(Vantman et al., 1988, Keel et al., 2000). Thesperm counting by improved Neubauer chamberis simple, less cost involvement and also hardlytakes 20-25 min to calculate spermconcentration per sample. We also found thatthere was no significant difference (P<0.05) insperm concentration among all the four breedsof this semen station.Therefore, it can be concluded that bothacrosomal integrity by Giemsa’s stain andsperm concentration using improved neubauerchamber method are most effective semenanalysis techniques to validate male fertility atsemen station level. However, furtherinvestigation requires verifying the semenquality breed wise with calf born data obtainedfrom the field.REFERENCEAnonymous (2012) Minimum Standard Protocolfor Production of Bovine Semen, Govt. of India.World Health Oraganization (1992)  WHOLaboratory Manual for the examination of humansemen and sperm-cervical mucus interaction. 3rdedn.New York, NY: Cambridge University Press.Boccia L, Palo DR, Rosa DE, Attanasio L,Mariotti E, et al. (2007)  Evaluation of buffalo semenby Trypan blue/ Giemsa staining and related fertilityin vitro.  Ital J Anim Sci   6: 739-742.Campbell RC, Hancock JL, Shaw IG (1960)Cytological characteristics and fertilizing capacityof bull spermatozoa.  J Agrie Sci   55: 91-99.Coetzee K, Menkveld R (2001)  Validation of anew disposable counting chamber. ArchivesAndrology   47: 153-156.Jankovicova J, Simon M,  Antalikova D (2006)Methods for evaluation of an acrosome reaction ofbovine spermatozoa.   Actafytotechnicaetzootechnica– Mimoriadnecíslo.  118-119.Jankovicova J, Simon M, Antalikova  J,  HorovskaL (2008)  Acrosomal and viability status of bovinespermatozoa evaluated by two staining methods.Acta Veterinaria Hungarica   56(1): 133-137.Keel BA, Quinn P, Schmidt CFJ (2000)  Resultsof the American Association of Bioanalysts nationalproficiency testing programme in andrology.Hum Reprod.  15:  680-686.Kovacs A,  Forte RH (1992) Viability andacrosome staining of bull, boar and rabbitspermatozoa.  Biotech Histochem   67: 119-124.Kohn FM, Mack SR, Schill WB,  Zaneveld LJD(1997)   Detection of human sperm acrosomereaction: comparison between methods using doublestaining ,Pisumsativum agglutinin, concanavalin A,and transmission microscope.  Hum Reprod   12:714-721.Lineweaver JA, Hafez ESE, Ehlers MH, DicksonWM,  King JR (1970)  Sperm transport in cattle.Cor Vet   60(3): 372.Mahmoud AM, Depoorter B, Piens N, ComhaireFH (1997)  The performance of ten different methodsfor the estimation of sperm concentration.  FertilSteril   68: 340-345.Risopatron J, Pena P, Miska W,  Sanchez R (2001)Evaluation of acrosome reaction in humanspermatozoa: comparison of cytochemical andfluorescence techniques. Andrologia 33:63-67.107Takumi TMD, Hiroshi QMD, Masato FMD,Yukhi H, Sadao KMD (2002)  Automated spermconcentration analysis with a new flow cytometry-based device, S-FCM. Am J Clin  Pathol  117: 401-408.Vantman D, Koukoulis G, Dennison L (1988)Computer assisted semen analysis: evaluation ofmethod and assessment of the influence of the spermconcentration on the linear velocity determination.Fertil Steril   49: 510-515.*Cite this article as:     Chowdhury S,   Das S,   Gupta T,   Sana D,   Bose S (2014)  Evaluation  offrozen  semen  by  a crosomal  integrity  and  sperm concentration -two  vital  quality  parametersof  male  fertility  in  bovines.  Explor Anim Med Res  4(1): 101-107.Evaluation  of  frozen  semen  by  a crosomal  integrity  and  sperm concentration -two  vital...Watson PF (1975)  Use of Giemsa stain to detectchanges in acrosomes of frozen ram spermatozoa.Vet  Rec   97: 12–15.Way AL, Henault MA, Killian GJ (1995)Comparison of four staining method for evaluatingacrosome status and viability of ejaculated andcaudaepididymal spermatozoa.  Theriogenology.43: 131-1361.Wells ME, Awa OA (1970)   New technique forassessing acrosomal characteristics of spermatozoa.J  Dairy Sci    53: 227-232.

EVALUATION OF FROZEN SEMEN BY A CROSOMAL INTEGRITY AND SPERM CONCENTRATION - TWO VITAL QUALITY PARAMETERS OF MALE FERTILITY IN BOVINES

Abstract

Similar works

Full text

Available Versions

Directory of Open Access Journals