Research Article Computer-Assisted Sperm Analysis of ...Research Article Computer-Assisted Sperm Analysis of Freezable and Nonfreezable Mithun ( Bos frontalis ) Semen P.Perumal, 1

Research ArticleComputer-Assisted Sperm Analysis of Freezable andNonfreezable Mithun (Bos frontalis) Semen

P Perumal1 S K Srivastava2 S K Ghosh2 and K K Baruah3

1 Animal Reproduction Laboratory National Research Centre on Mithun (ICAR) Jharnapani Nagaland 797 106 India2 Animal Reproduction Division Indian Veterinary Research Institute Izatnagar Bareilly Uttar Uttar Pradesh 243 122 India3 Animal Physiology Division National Research Centre on Mithun (ICAR) Jharnapani Nagaland 797 106 India

Correspondence should be addressed to P Perumal perumalponrajgmailcom

Received 27 June 2014 Revised 31 July 2014 Accepted 4 August 2014 Published 18 August 2014

Academic Editor Sami Dridi

Copyright copy 2014 P Perumal et al This is an open access article distributed under the Creative Commons Attribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

The present study was undertaken to assess the motility and velocity parameters of sperm of freezable and nonfreezable ejaculatesby computer-assisted sperm analyser (CASA) such as Hamilton-Thorne Semen Analyser IVOS 11 in mithun semen Fifty ejaculates(twenty-five ejaculates each for freezable and nonfreezable semen ejaculates) were collected from ten matured mithun bulls CASAparameters motility parameters such as forward progressivemotility (FPM) () nonprogressivemotility (NPM) () total motility(TM) () and static sperms (SM) () velocity parameters such as curvilinear velocity (VCL) (120583msec) straight line velocity (VSL)(120583msec) average path velocity (VAP) (120583msec) linearity (LIN) () straightness (STR) () wobble (WOB) () amplitude oflateral head displacement (ALH) (120583m) and beatcross-frequency (BCF) (Hz) weremeasured by CASA analyserThe result revealedthat these parameters varied significantly (119875 lt 005) between the freezable and nonfreezable ejaculates and freezable ejaculateshave significantly (119875 lt 005) higher value than nonfreezable ejaculates It was concluded that most of the CASA parameters weresignificantly lower in nonfreezable ejaculates than in freezable ejaculates in mithun and confirmed that the CASA was effective fora quick and objective analysis of motility and velocity parameters in mithun semen

1 Introduction

Mithun (Bos frontalis) is a semiwild free-range rare bovinespecies present in the North-Eastern Hill (NEH) region ofIndia and is believed to have originated more than 8000years ago from wild Indian gaur (Bos gaurus) [1] The animalhas an important place in the social cultural religious andeconomic life of the tribal population of NEH region As perthe livestock census of India (2007) the mithun populationis decreasing gradually due to lack of suitable breeding bullsincrease in intensive inbreeding practices and lack of suitablebreeding management Greater efforts are required fromall quarters to preserve the mithun population to enhancethe socioeconomic status of this region Since mithuns aresemiwild animal and not fully domesticated natural breedingis practiced in this species with accompanied limitationslike cost and disease transmission Thus use of artificialinsemination for improvement of its pedigree is utmostessential

Etiology for infertility in male animals is varied and com-plex Infertility is diagnosed by history clinical signs physicalexamination of external and internal genitalia assessment oflibido and sexual behaviour semen collection and evaluationand various hormone analyses related to semen production[2] Subjective semen analysis is carried out by conventionalmethod such as assessment of sperm concentration motilityand morphology by phase contrast microscope [3] Howeverthe assessment of semen quality in terms of motility velocityswimming pattern sperm head behaviour and so forth mayhelp in better understanding of the possible sperm functionsemen quality and selection of suitable semen and bullsfor cryopreservation in mithun Various methods are usedto estimate sperm motility and velocity parameters whichhave ranged from very simple techniques such as time-exposure or multiple exposure photomicrographies to thevery sophisticated CASA techniques [4] CASA system yieldsaccurate objective assessment repeatable and reliable resultson different semenparameters such asTM FPManddifferent

Hindawi Publishing CorporationJournal of AnimalsVolume 2014 Article ID 675031 6 pageshttpdxdoiorg1011552014675031

2 Journal of Animals

velocity parameters [5] based on the measurement of indi-vidual sperm cells Recent reports suggested that CASA doesnot only measure the proportion of motile spermatozoa butalso measures other sperm motion parameters derived fromindividual sperm cells and it has more predictive power onfertility potential of semen ejaculates [6] In bovine speciesspecific motion parameters have been positively correlatedwith fertility [7 8] In addition to the use of computerizedtechniques to predict semen fertility CASA also providesa useful tool to study the effects of various in vitro proce-dures on sperm motility as well as the means to study thephenomenon of sperm hyperactivation Spermatozoa FPMalong with certain velocity parameters are essential for thespermatozoa to achieve fertilization Spermatozoa kinematicparameters such as PFM VSL VCL ALH and LIN werepositively correlated with bull fertility [9 10]

A spermatozoon has significantly higher VCL and ALHindicating that there is major bending of the mid piece andlarge amplitude of lateral head displacement This signifiesthe hyperactivation of the spermatozoa Hyperactivation inturn implies high energy state of the spermatozoa which isessential for sperm penetration through cervical mucus zonapellucida fuse with the oocytes and successful fertilization[11] Spermatozoa motility and velocity parameters reflecttheir mitochondrial function and energy status indirectly Inbovine specific motion parameters have been correlated andrelated to fertility of sperm [7 9]

Study of CASA parameters was reported in domesticanimal species such as cattle [10 12] buffalo [13 14] sheep[15] goat [16] boar [17] and dog [18] Further perusal ofliteratures revealed that no information on CASA parametersof freezable and nonfreezable ejaculates in mithun speciesHence the objective of this study was to assess the motilityand velocity parameters measured by CASA to pursuit futuresperm preservation protocols in mithun

2 Material and Methods

21 Experimental Animals Ten apparently healthy mithunbulls of approximately 3 to 5 yrs of age were selected fromthe herd in mithun farm National Research Centre onMithun (ICAR) Jharnapani Nagaland India The averagebody weight of these bulls was 501 kg (493 to 507 kg) at 3ndash5 yrs of age with good body condition (score 5-6) maintainedunder uniform feeding housing and lighting conditionsThe study area lies between 25∘5410158403010158401015840 North latitude and93∘4410158401510158401015840 East longitude and at an altitude range of 250ndash300 mean sea level Each experimental animal was fed in thisexperiment as per the farm schedule These were offered adlibitum drinking water 30 kg mixed jungle forages (184dry matter and 102 crude protein) and 4 kg concentrates(871 dry matter and 145 crude protein) daily fortifiedwith mineral mixture and salt During the study all theexperimental protocols met the Institutional Animal Careand Use Committee regulations

22 Semen Collection Incubation and Evaluation A total of50 ejaculates were collected from ten bulls through rectal

Table 1 Software settings of HTR IVOS 11 used in the study

Parameters ValueChamber type Leja 4Temperature of analysis (∘C) 370Fields acquired 10Frame rate (Hz) 60Number of frames 30Minimum static contrast 35Minimum cell size (pixels) 5Straightness (STR) thresholds () 70VAP cut-off (120583ms) 30Progressive minimum VAP (120583ms) 50VSL cut-off (120583ms) 15Cell intensity 80Magnification 189

massagemethod Brieflymassagewas performed by back andforth hand motion over the ampulla prostate and seminalvesicles and then the urethralis muscles were rhythmicallystroked [19] followed by the gentle milking of ampullae oneby one which resulted in erection and ejaculation Duringcollection the initial transparent secretions were discardedand neat semen drops were collected in a graduated test tubewith the help of a funnel The ejaculates were evaluated andaccepted for evaluation if the following criteriaweremet con-centration gt500 millionmL mass activity gt3+ individualmotility gt70 and total abnormality lt10 Immediatelyafter collection the samples were kept in a water bath at37∘C and evaluated for volume colour consistency massactivity and pH After the preliminary evaluations ejaculateswere subjected to the initial dilution with prewarmed (37∘C)Tris-citrate extender The partially diluted samples were thenbrought to the laboratory in an insulated flask containingwarm water (37∘C) for further processing

23 Sample Selection These ejaculates were split into freez-able and nonfreezable ejaculates based on the postthawmotility [20 21] Ejaculates having postthaw motility 40and above were considered as freezable ejaculates whereasnonfreezable ejaculates were those having postthaw motilityless than 40

231 Computer-Assisted Semen Analysis (CASA) Themotil-ity and velocity parameters were evaluated by HamiltonThorne Sperm Analyser version IVOS 11 (HTM-IVOS Ver-sion 108 Hamilton Thorne Research Beverly MA USA)This CASA system consists of a phase-contrast microscopecamera minitherm heating stage image digitizer and com-puter for saving and analyzing the dataThe software settingsare shown in Table 1

After semen collection the sperm concentration wasfirst estimated using a phase-contrast microscope (NikonEclipse 80i 400 x magnification) 25120583L of semen was dilutedinto 50ndash100120583L of Tris (formulated for bull semen) and 5120583Lof this diluted semen was loaded into a prewarmed dual

Journal of Animals 3

Table 2 Mean (plusmnSE) motility and velocity parameters of freezable and nonfreezable ejaculates of mithun semen

CASA parameters Freezable semen (119899 = 25) Nonfreezable semen (119899 = 25)Forward progressive motility () 5895 plusmn 368a 3817 plusmn 344a

Nonprogressive motility () 1747 plusmn 220b 2023 plusmn 183a

Total motility () 7652 plusmn 329b 5840 plusmn 345a

Static sperms () 2358 plusmn 338a 4160 plusmn 346b

Curvilinear velocity (VCL) (120583msec) 18883 plusmn 465b 13667 plusmn 488a

Straight line velocity (VSL) (120583msec) 8977 plusmn 349b 6063 plusmn 382a

Average path velocity (VAP) (120583msec) 11858 plusmn 372b 8845 plusmn 318a

Linearity (LIN) () 4694 plusmn 153b 4389 plusmn 196a

Straightness (STR) () 7481 plusmn 228b 6776 plusmn 278a

Wobble (WOB) () 6285 plusmn 145 6530 plusmn 198Amplitude of lateral head displacement (ALH) (120583m) 884 plusmn 118b 518 plusmn 135a

Beatcross-frequency (BCF) (Hz) 2823 plusmn 170b 2254 plusmn 163a

Figures with same superscript (a b) do not differ significantly in rows

chamber disposable Leja slide and was allowed to settle onthe minitherm heating stage (38∘C) before the analysis

The following parameters such as percentage of FPMNPM TM SM VAP VSL VCL ALH BCF and LIN weremeasured A minimum of 200 spermatozoa from at leasttwo different drops of each sample were analyzed from eachspecimen The number of objects incorrectly identified asspermatozoa was manually removed and final analysis wasdone for each sample

24 Statistical Analysis The results were analysed statisticallyand expressed as the mean plusmn SEM Significant differencebetween the freezable and nonfreezable semen ejaculateswere estimated with Studentrsquos 119905-test using the SPSSPC com-puter program (version 150 SPSS Chicago IL) Differenceswith values of 119875 lt 005 were considered to be statisticallysignificant after arcsine transformation of percentage data byusing SPSS 15 Correlation between the motility and velocityparameters was established with correlation coefficient beingdone as per Pearsonrsquos method Differences at 119875 lt 005 wereconsidered to be statistically significant

3 Result and Discussion

Thespermmotility and velocity parameterswere evaluated byHamilton Thorne Sperm Analyser and result was presentedin Table 2 The percent of TM and FPM were significantly(119875 lt 005) higher in freezable ejaculates than in nonfreezableejaculates and NPM and SM were significantly higher innonfreezable ejaculates than in freezable ejaculates Similarlyvelocity parameters were significantly (119875 lt 005) higher infreezable ejaculates than in nonfreezable ejaculates inmithunspecies Thus it may enhance the quality of semen by pre-serving efficiently during artificial insemination procedureSimilar report was observed in crossbred cattle [10 22] Balicattle [23] buffalo [13 14] sheep [15] goat [16] boar [17] anddog [18]

The assessment of sperm motility using the conventionalmicroscopical methods is difficult and subjective High vari-ations have been reported for the estimation of motility

parameters of the same ejaculates [24] CASA is an accuratetechnique used for the assessment of themotility and velocityparameters of mithun semen High number of spermatozoacan be analysed individually in a short period of time [5]

Comparedwith the results of report of others the velocityparameters of mithun bulls were highly varied [9] Motilityand velocity parameters are varied with the factors such asage time of collection time between ejaculations energystores of sperm presence of surface acting agents in thecell membrane such as agglutinins and detergents viscosityosmolarity pH temperature ionic concentration of seminalplasma and presence of the mineral elements like Cu ZnMn and hormones prostaglandins and so forth [25]

The sperm mobility phenotype can be attributed tospecific sperm velocity parameters of individual sperm asdetermined by CASA The motion parameters such as VSLLIN and BCF contribute to the overall sperm motilitycharacters in bulls as these were all significantly (119875 lt 005)correlated with sperm mobility In the present experimentvarious types of spermmotility representing VCL VSL VAPLIN STRWOBALH andBCFwere also significantly higherfor the ejaculates classified as freezable compared with thenonfreezable ejaculates The parameter LIN is a measureof linearity and the BCF motion parameter indicates thenumber of times the sperm track crosses the smoothed pathboth of which indicate linear progressionThus spermatozoain freezable ejaculates swim faster and straighter than didspermatozoa in nonfreezable ejaculates This may be biologi-cally significant because the spermmobility phenotype on thebasis of research with whole ejaculates is predictive of fertility[26 27]

Semen samples with high FPM and TM had significantlyhigher positive correlation with velocity parameters in freez-able quality semen (Table 3)The samples with high PFM hadhigher VAP progressive velocity and track speed This wassimilar to the findings of Anil Kumar et al [13] and Perumalet al [10] for path velocity The average path velocity was sig-nificantly and positively correlated with progressive velocitytrack speed andALHThe high positive correlation observedbetween VAP VSL VCL and ALH between VSL and VCL


Table 3 Correlation between the motility and velocity parameters of sperm of freezable semen ejaculates of mithun

FPM NPM TM SM VCL VSL VAP LIN STR WOB ALH BCFFPM 100 minus052 092lowast minus093lowast 075lowast 085lowast 068lowast 068 066lowast minus040 067lowast 088lowast

NPM 100 minus015 015 003 minus005 003 minus014 minus017 016 003 minus008TM 100 minus096lowast 086lowast 096lowast 078lowast 072lowast 068lowast minus039 079lowast 097lowast

SM 100 minus087lowast minus095lowast minus077lowast minus073lowast minus070lowast 038 minus081lowast minus095lowast

VCL 100 087lowast 096lowast 034 033 minus023 096lowast 091lowast

VSL 100 078lowast 061 057 minus033 082lowast 080lowast

VAP 100 024 014 002 098lowast 085lowast

LIN 100 091lowast minus043 025 067lowast

STR 100 minus076lowast 017 062WOB 100 minus002 minus034ALH 100 085lowast

BCF 100lowastCorrelation coefficients were significant 119875 lt 005

Table 4 Correlation between the motility and velocity parameters of sperm of nonfreezable semen ejaculates of mithun

FPM NPM TM SM VCL VSL VAP LIN STR WOB ALH BCFFPM 100 minus038 096lowast minus095lowast 085lowast 090lowast 079lowast 075lowast 090lowast minus082lowast 078lowast 088lowast

NPM 100 minus011 011 003 minus009 minus003 minus025 minus012 minus010 001 minus003TM 100 minus094lowast 092lowast 094lowast 085lowast 073lowast 093lowast minus091lowast 085lowast 094lowast

SM 100 minus090lowast minus095lowast minus086lowast minus075lowast minus091lowast 090lowast minus086lowast minus095lowast

VCL 100 097lowast 098lowast 064lowast 087lowast minus092lowast 097lowast 098lowast

VSL 100 094lowast 082lowast 096lowast minus087lowast 093lowast 096lowast

VAP 100 062 081lowast minus082lowast 098lowast 095lowast

LIN 100 091lowast minus053 061 065STR 100 minus083lowast 081lowast 087lowast

WOB 100 minus085lowast minus091lowast

ALH 100 094lowast


and betweenALHwithVAP VSL andVCL indicated that thevelocity parameters were correlated and interrelated amongthem and with ALH BCF was significantly and positivelycorrelated with ALH Anil Kumar et al [13] and Perumal etal [10] observed a positive correlation similar to the presentstudy between BCF and ALH ALH and BCF representingthe head behaviour of the sperm are highly variable andmean values of these parameters were within the range Theyalso found a highly significant negative correlation betweenSTR and WOB A similar result was observed in the presentstudy In nonfreezable ejaculates of mithun semen there waspositive correlation between the PFM and VCL VSL VAPLIN STR ALH and BCF and negative correlation with SMandWOB Similarly in freezable ejaculates VCL is positivelycorrelatedwithVSL VAP STR ALH and BCF and negativelycorrelatedwithWOB (Table 4) Similar result was reported byAnil Kumar et al [13] and Perumal et al [10]

Spermatozoa FPM along with certain velocity parametersare essential for the spermatozoa to achieve fertilizationSpermatozoa kinematic variables such as FPM VSL VCLALH and LIN were correlated with bull fertility [9 10]A significantly higher VCL and ALH of the spermatozoa

indicate major bending of the mid piece and large amplitudeof lateral head displacement This signifies the hyperacti-vation of the spermatozoa Hyperactivation in turn implieshigh energy state of the spermatozoa which is essential forsperm penetration through cervical mucus and fuse with theoocytes [11] Spermatozoa motility and velocity reflect theirmitochondrial function indirectly In bovine specific motionparameters have been reported to be related to fertility [7 9]but the threshold levels for these motion characteristics havenot yet been established to meet a general consensus

The CASA variable such as linearity or linear motilityis higher indicating that spermatozoa have higher rate offertilization potential in comparison to the total motilitypercentage [28] and semen samples containing such sper-matozoa have higher fertility rates and pregnancy rates afterartificial insemination [8] In bovine artificial inseminationindustry the minimum level of motility percentage requiredis 50 percent [29] and the freezable ejaculates have reachedthat threshold level which shows that cryopreserved mithunsemen was of acceptable quality

Recent findings suggested that assessment of motilespermatozoa in a semen sample may not be considered as


a reliable index for semen evaluationTheobjective and quan-titative measurement of other sperm motion characteristicsderived from observations of individual cells assessed byCASA have been found to be more efficient in predictingsemen samplersquos potential fertility [6] These parameters areprobably important for the progression of spermatozoa intocervical mucus and the penetration of zona pellucida ofoocytes [5] Fertilization rates of human oocytes in vitrohave been shown to correlate positively with sperm velocity[30] In bovine sperm velocity is highly correlated with the59-day nonreturn rate [8] In humans VCL and BCF weresignificantly higher for spermatozoa which penetrated inspermpenetration assay than for those that failed to penetrate[31] In addition to the use of computerized technique topredict semen fertility CASA can be a useful tool to study theeffects of various in vitro procedures on spermmotility as wellas phenomenon of sperm hyperactivation [32] In presentstudy similar observation was reported

The results of present study were varied with otherauthors This may be due to various factors such as semencollection method initial quality of semen method of pro-cessing of semen for CASA time between the collection andanalysis setup of instrument in analysing of sample accuracyof the sample chambers and number of the chambers andfield and sperm examined to provide sufficient statisticalsampling materials analysed [33]

4 Conclusion

It was concluded from the present study that most ofthe sperm motility and velocity parameters of CASA weresignificantly higher in freezable ejaculates in comparison tothe nonfreezable ejaculates of mithun This indicates thatfreezable sperm has structural stability during the freezingand thawing procedure than the nonfreezable sperm thatleads to freezable sperm having higher functional spermstructures to move faster and in the forward directionMoreover CASA system proved its usefulness in routineevaluation of mithun semen especially in frozen semen bank

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

References

[1] F J Simoons ldquoGayal or mithunrdquo in Evolution of DomesticatedAnimals I L Manson Ed pp 34ndash36 Longman London UK1984

[2] S D Johnston M V Root-Kustritz and P N S Olson Canineand Feline Theriogenology WB Saunders Philadelphia PaUSA 2001

[3] M Iguer-ouada and J P Verstegen ldquoEvaluation of the ldquoHamil-ton thorn computer-based automated systemrdquo for dog semenanalysisrdquoTheriogenology vol 55 no 3 pp 733ndash749 2001

[4] S T Mortimer ldquoA critical review of the physiological impor-tance and analysis of sperm movement in mammalsrdquo HumanReproduction Update vol 3 no 5 pp 403ndash439 1997

[5] J Verstegen M Iguer-Ouada and K Onclin ldquoComputerassisted semen analyzers in andrology research and veterinarypracticerdquoTheriogenology vol 57 no 1 pp 149ndash179 2002

[6] D Mortimer Practical Laboratory Andrology Oxford Univer-sity Press New York NY USA 1994

[7] P R Budworth R P Amann and R H Hammerstedt ldquoAmicrocomputer-photographic method for evaluation of motil-ity and velocity of bull spermrdquo Journal of Dairy Science vol 70no 9 pp 1927ndash1936 1987

[8] P B Farrell G A Presicce C C Brocektt and R H FooteldquoQuantification of bull sperm characteristics measured bycomputerndashassisted sperm analysis (CASA) and their relation-ship to fertilityrdquoTheriogenology vol 49 pp 871ndash879 1998

[9] P B Farrell R H Foote M M McArdle V L Trouern-Trend and A L Tardif ldquoMedia and dilution procedures testedto minimize handling effects on human rabbit and bullsperm for computer-assisted sperm analysis (CASA)rdquo Journalof Andrology vol 17 no 3 pp 293ndash300 1996

[10] P Perumal S Selvaraju S Selvakumar et al ldquoEffect of pre-freeze addition of cysteine hydrochloride and reduced glu-tathione in semen of crossbred jersey bulls on spermparametersand conception ratesrdquo Reproduction in Domestic Animals vol46 no 4 pp 636ndash641 2011

[11] R J Aitken M Sutton P Warner and D W RichardsonldquoRelationship between the movement characteristics of humanspermatozoa and their ability to penetrate cervical mucusand zona-free hamster oocytesrdquo Journal of Reproduction andFertility vol 73 no 2 pp 441ndash449 1985

[12] P Perumal S Selvaraju S Selvakumar et al ldquoReduced glu-tathione and cysteine hydrochloride on sperm motility andvelocity parameters of poor crossbred bull semenrdquo InternationalJournal of BiondashResource and StressManagement vol 3 no 2 pp145ndash151 2012

[13] R Anil Kumar M N Sundararaman D V Patel M Iyueand R Kasiraj ldquoCryopreservation of semen as a venture forconservation of wild and endangered toda Buffalo GermplasmrdquoBuffalo Bulletin vol 30 no 3 pp 210ndash218 2011

[14] S Koonjaenak V Chanatinart S Aiumlamai T Pinyopumim-intr and H Rodriguez-Martinez ldquoSeasonal variation in semenquality of swamp buffalo bulls (Bubalus bubalis) in ThailandrdquoAsian Journal of Andrology vol 9 no 1 pp 92ndash101 2007

[15] D Kumar A Joshi and S M K Naqvi ldquoComparative semenevaluation of Malpura and Bharat merino rams by computer-aided sperm analysis technique under semi-arid tropical envi-ronmentrdquo International Journal of Animal and VeterinaryAdvance vol 2 no 1 pp 26ndash30 2010

[16] R Kozdrowski A Dubiel W Bielas and M Dzięcioł ldquoTwoprotocols of cryopreservation of goat semen with the use ofcomputer-assisted semen analysis systemrdquo Acta VeterinariaBrno vol 76 no 4 pp 601ndash604 2007

[17] M L W J Broekhuijse E Sostaric H Feitsma and B MGadella ldquoApplication of computer-assisted semen analysis toexplain variations in pig fertilityrdquo Journal of Animal Science vol90 no 3 pp 779ndash789 2012

[18] A Domosławska S Zdunczyk W Nizanski and T JanowskildquoAssessment of semen quality in infertile dogs using computer-assisted sperm analysis by the Hamilton-Thorne Semen Anal-yserrdquo Bulletin of the Veterinary Institute in Pulawy vol 57 pp429ndash432 2013

[19] C W Palmer S D Amundson L F C Brito C L Waldnerand A D Barth ldquoUse of oxytocin and cloprostenol to facilitate


semen collection by electroejaculation or transrectal massagein bullsrdquo Animal Reproduction Science vol 80 no 3-4 pp 213ndash223 2004

[20] J K Prasad S Kumar GMohanU Shankar and S K AgarwalldquoBiochemical studies pertaining to freezability of cross bred bullsemenrdquo Indian Journal of Veterinary Research vol 8 pp 37ndash402000

[21] A V N Rao and Y V R Rao ldquoSeminal characteristic andfreezability of crossbred bullsrdquo Indian Veterinary Journal vol73 pp 1086ndash1088 1996

[22] P Perumal Cryopreservation of bovine semen with some addi-tives for augmenting fertility [MVSc thesis] Orissa Universityof Agriculture and Technology Bhubaneswar India 2008

[23] K Sarsaifi Y RosninaM Ariff et al ldquoEffect of semen collectionmethods on the quality of pre- and post-thawed bali cattle (Bosjavanicus) spermatozoardquo Reproduction in Domestic Animalsvol 48 no 6 pp 1006ndash1012 2013

[24] D Mortimer M A Shu and R Tan ldquoStandardization andquality control of sperm concentration and sperm motilitycounts in semen analysisrdquo Human Reproduction vol 1 no 5pp 299ndash303 1986

[25] L Blasco ldquoClinical tests of sperm fertilizing abilityrdquo Fertility andSterility vol 41 no 2 pp 177ndash192 1984

[26] D P Froman and A J Feltmann ldquoSperm mobility a quantita-tive trait of the domestic fowl (Gallus domesticus)rdquo Biology ofReproduction vol 58 no 2 pp 379ndash384 1998

[27] D P Froman A J Feltmann M L Rhoads and J D KirbyldquoSperm mobility a primary determinant of fertility in thedomestic fowl (Gallus domesticus)rdquoBiology of Reproduction vol61 no 2 pp 400ndash405 1999

[28] T Cremades J Roca H Rodriguez-Martinez T Abaigar J MVazquez and E A Martinez ldquoKinematic changes during thecryopreservation of boar spermatozoardquo Journal of Andrologyvol 26 no 5 pp 610ndash618 2005

[29] T Hallap M Haard U Jaakma B Larsson and H Rodriguez-Martinez ldquoDoes cleansing of frozen-thawed bull semen beforeassessment provide samples that relate better to potentialfertilityrdquoTheriogenology vol 62 no 3-4 pp 702ndash713 2004

[30] E T Donnelly S E M Lewis J A McNally andWThompsonldquoIn vitro fertilization and pregnancy rates the influence ofspermmotility and morphology on IVF outcomerdquo Fertility andSterility vol 70 no 2 pp 305ndash314 1998

[31] P M Fetterolf and B J Rogers ldquoPrediction of human spermpenetrating ability using computerized motion parametersrdquoMolecular Reproduction and Development vol 27 no 4 pp326ndash331 1990

[32] P B Farrell R H Foote M E Simkin E D Clegg andR J Wall ldquoRelationship of semen quality number of sperminseminated and fertility in rabbitsrdquo Journal of Andrology vol14 no 6 pp 464ndash471 1993

[33] P Farrell L V Trouern-Trend R H Foote and A M DDouglas-Hamilton ldquoRepeatability of measurements on humanrabbit and bull sperm by computer-assisted sperm analysiswhen comparing individual fields and means of 12 fieldsrdquoFertility and Sterility vol 64 no 1 pp 208ndash210 1995

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of


Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology


Molecular Biology International

GenomicsInternational Journal of


The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014


BioinformaticsAdvances in

Marine BiologyJournal of



Signal TransductionJournal of


BioMed Research International

Evolutionary BiologyInternational Journal of



Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014


Genetics Research International


Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational



Enzyme Research



Microbiology


velocity parameters [5] based on the measurement of indi-vidual sperm cells Recent reports suggested that CASA doesnot only measure the proportion of motile spermatozoa butalso measures other sperm motion parameters derived fromindividual sperm cells and it has more predictive power onfertility potential of semen ejaculates [6] In bovine speciesspecific motion parameters have been positively correlatedwith fertility [7 8] In addition to the use of computerizedtechniques to predict semen fertility CASA also providesa useful tool to study the effects of various in vitro proce-dures on sperm motility as well as the means to study thephenomenon of sperm hyperactivation Spermatozoa FPMalong with certain velocity parameters are essential for thespermatozoa to achieve fertilization Spermatozoa kinematicparameters such as PFM VSL VCL ALH and LIN werepositively correlated with bull fertility [9 10]

A spermatozoon has significantly higher VCL and ALHindicating that there is major bending of the mid piece andlarge amplitude of lateral head displacement This signifiesthe hyperactivation of the spermatozoa Hyperactivation inturn implies high energy state of the spermatozoa which isessential for sperm penetration through cervical mucus zonapellucida fuse with the oocytes and successful fertilization[11] Spermatozoa motility and velocity parameters reflecttheir mitochondrial function and energy status indirectly Inbovine specific motion parameters have been correlated andrelated to fertility of sperm [7 9]

Study of CASA parameters was reported in domesticanimal species such as cattle [10 12] buffalo [13 14] sheep[15] goat [16] boar [17] and dog [18] Further perusal ofliteratures revealed that no information on CASA parametersof freezable and nonfreezable ejaculates in mithun speciesHence the objective of this study was to assess the motilityand velocity parameters measured by CASA to pursuit futuresperm preservation protocols in mithun

2 Material and Methods

21 Experimental Animals Ten apparently healthy mithunbulls of approximately 3 to 5 yrs of age were selected fromthe herd in mithun farm National Research Centre onMithun (ICAR) Jharnapani Nagaland India The averagebody weight of these bulls was 501 kg (493 to 507 kg) at 3ndash5 yrs of age with good body condition (score 5-6) maintainedunder uniform feeding housing and lighting conditionsThe study area lies between 25∘5410158403010158401015840 North latitude and93∘4410158401510158401015840 East longitude and at an altitude range of 250ndash300 mean sea level Each experimental animal was fed in thisexperiment as per the farm schedule These were offered adlibitum drinking water 30 kg mixed jungle forages (184dry matter and 102 crude protein) and 4 kg concentrates(871 dry matter and 145 crude protein) daily fortifiedwith mineral mixture and salt During the study all theexperimental protocols met the Institutional Animal Careand Use Committee regulations

22 Semen Collection Incubation and Evaluation A total of50 ejaculates were collected from ten bulls through rectal

Table 1 Software settings of HTR IVOS 11 used in the study

Parameters ValueChamber type Leja 4Temperature of analysis (∘C) 370Fields acquired 10Frame rate (Hz) 60Number of frames 30Minimum static contrast 35Minimum cell size (pixels) 5Straightness (STR) thresholds () 70VAP cut-off (120583ms) 30Progressive minimum VAP (120583ms) 50VSL cut-off (120583ms) 15Cell intensity 80Magnification 189

massagemethod Brieflymassagewas performed by back andforth hand motion over the ampulla prostate and seminalvesicles and then the urethralis muscles were rhythmicallystroked [19] followed by the gentle milking of ampullae oneby one which resulted in erection and ejaculation Duringcollection the initial transparent secretions were discardedand neat semen drops were collected in a graduated test tubewith the help of a funnel The ejaculates were evaluated andaccepted for evaluation if the following criteriaweremet con-centration gt500 millionmL mass activity gt3+ individualmotility gt70 and total abnormality lt10 Immediatelyafter collection the samples were kept in a water bath at37∘C and evaluated for volume colour consistency massactivity and pH After the preliminary evaluations ejaculateswere subjected to the initial dilution with prewarmed (37∘C)Tris-citrate extender The partially diluted samples were thenbrought to the laboratory in an insulated flask containingwarm water (37∘C) for further processing

23 Sample Selection These ejaculates were split into freez-able and nonfreezable ejaculates based on the postthawmotility [20 21] Ejaculates having postthaw motility 40and above were considered as freezable ejaculates whereasnonfreezable ejaculates were those having postthaw motilityless than 40

231 Computer-Assisted Semen Analysis (CASA) Themotil-ity and velocity parameters were evaluated by HamiltonThorne Sperm Analyser version IVOS 11 (HTM-IVOS Ver-sion 108 Hamilton Thorne Research Beverly MA USA)This CASA system consists of a phase-contrast microscopecamera minitherm heating stage image digitizer and com-puter for saving and analyzing the dataThe software settingsare shown in Table 1

After semen collection the sperm concentration wasfirst estimated using a phase-contrast microscope (NikonEclipse 80i 400 x magnification) 25120583L of semen was dilutedinto 50ndash100120583L of Tris (formulated for bull semen) and 5120583Lof this diluted semen was loaded into a prewarmed dual













































ALH 100 094lowast










4 Conclusion




References











































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology













































ALH 100 094lowast










4 Conclusion




References











































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology





















ALH 100 094lowast










4 Conclusion




References











































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology




4 Conclusion




References











































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology
























Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology








Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology

Documents

Research Article Computer-Assisted Sperm Analysis of ...Research Article Computer-Assisted Sperm Analysis of Freezable and Nonfreezable Mithun ( Bos frontalis ) Semen P.Perumal, 1