Identification of Sperm Subpopulations in Water Buffalo Ejaculates: Changes in Cryopreservation Stages and Bull Variation

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1 Journl of Agriculturl Science nd Technology A 7 (2017) doi: / / D DAVID PUBLISHING Identifiction of Sperm Subpopultions in Wter Bufflo Ejcultes: Chnges in Cryopreservtion Stges nd Bull Vrition Excel Rio Sntos Mylem 1, 2, Mri Elizbeth Del Cruz Leovers 1, Emm Villnuev Venturin 3, Eufrocin Del Peñ Atby 2 nd Edwin Curding Atby 2, 3 1. Deprtment of Biologicl Sciences, College of Arts nd Sciences, Centrl Luzon Stte University, Science City of Muñoz, Nuev Ecij 3120, Philippines 2. Reproductive Biotechnology Unit, Philippine Crbo Center Ntionl Hedqurters nd Genepool, Science City of Muñoz, Nuev Ecij 3120, Philippines 3. Philippine Crbo Center t Centrl Luzon Stte University, Science City of Muñoz, Nuev Ecij 3120, Philippines Abstrct: This study imed to identify nd chrcterize the sperm subpopultions existing in wter bufflo semen using computer ssisted sperm nlyzer (CASA), s well s ssess the effects of cryopreservtion on the sperm subpopultion structure nd evlute bull vribility. The semen of eight Bulgrin Murrh bulls ws collected by four times in n intervl of one week ech. The semen ws cryopreserved following stndrd protocol nd sperm kinemtics ws ssessed. Clustering methods were pplied to individul sperms, forming two significntly different (P < 0.05) subpopultions (P 1 nd P 2 ). Subpopultion P 1 represents those spermtozo tht moved most rpidly nd progressively (46.29%), nd subpopultion P 2 includes spermtozo with reltively low velocity or poorly motile but with high progressiveness (53.41%). There ws decline on the popultion of P 1 sperms from fresh (52.52%), pre-freeze (45.73%) to post-thw (35.17%) stges nd significnt difference on the sperm kinemtics between P 1 nd P 2. A significnt decline in the vlues of distnce, velocity nd mplitude of lterl hed (ALH) prmeters were observed t post-thw stge, while n increse ws observed on trjectory nd bet cross frequency (BCF) kinemtics. Vlues of sperm kinemtics were lso significntly different (P < 0.05) mong ll bulls. The frequency distribution of spermtozo on both subpopultions P 1 nd P 2 ws quite similr for ll bulls in pre-freeze nd post-thw stges, but with significnt (P < 0.05) vribility on fresh stge. Bulls with the highest mintined frequency of P 1 sperms re denoted s good freezer bulls. In sum, kinemtic chrcteriztion of wter bufflo sperm nd clustering into subpopultion enble to identify bulls tht re more resistnt to cryopreservtion nd production of qulity semen for genetic propgtion. Key words: Sperm subpopultions, bufflo semen, sperm kinemtics, cryopreservtion, computer ssisted sperm nlyzer. 1. Introduction The intensified use of rtificil insemintion (AI) in wter buffloes hs put forwrd the interest in improving the quntittive nlysis of semen smples in order to ensure its qulity. In the field, it is essentil to come up with the predictive cpcity of the sperm qulity for potentil fertility of bulls. The prcticl use of the study is to devise method in selecting bulls to become semen donors with known cryosurvivbility Corresponding uthor: Excel Rio Sntos Mylem, M.Sc., reserch field: biology. nd qulity for higher fertilizing cpbility. The rise of the computer ssisted sperm nlyzer (CASA) hs brought n dvntge in determining the semen qulity. Currently, it is the most objective nd detiled method, with improved repetbility nd sensitivity in determining the motility not only of the semen smple, but lso of individul sperms, nd cn even chrcterize them to sperm subpopultions within smple [1]. These sperm subpopultions re bsed on the kinemtics dt, which define the movement of the sperm bsed on its distnce trvelled, velocity, hed

2 Identifiction of Sperm Subpopultions in Wter Bufflo Ejcultes: 185 movement nd trjectory [2, 3]. Sperm subpopultions, commonly devised either by principle component nlysis or clustering, hve been known to hve positive nd significnt correltion on the ejculte qulity nd fertilizing bility in bulls both in vitro nd in vivo [4-6]. The cryopreservtion process hd lso been found out to significntly modify the distribution of spermtozo within subpopultions, nd the mgnitude of the subpopultion in fresh ejcultes ws positively correlted with their resistnce to cryopreservtion. The cryopreservtion process does not only induce loss of sperm vibility, but lso impir the functionlity of the surviving spermtozo, which ccounts for the lower fertilizing cpcity of the frozen-thwed semen [6]. Relizing the effects, the sperm subpopultions re now being used to identify the qulity of the semen nd its potentil fertilizing bility [4]. The chnges during the cryopreservtion process nd individul vritions on the sperms of different mmmls re lso being documented using the subpopultion structure [5, 7-9]. However, very few efforts re being given to wter buffloes. The objective of this reserch ws to identify nd chrcterize the different sperm subpopultions in wter bufflo semen, lso ssess the effects of cryopreservtion on the kinemtic prmeters nd frequency distribution within the different sperm subpopultions, nd finlly to evlute bull vritions on the kinemtic prmeters nd frequency distribution within the different sperm subpopultions structure of fresh, pre-freeze nd frozen-thwed semen. 2. Mterils nd Methods 2.1 Semen Collection Semen ws collected from eight Bulgrin Murrh bulls with ge rnging from 6 to 10 yers old. Four successive collections were performed in one-week intervl using n rtificil vgin nd teser bull. The bulls used in the study were regulr semen donors of the Ntionl Bull Frm, Philippine Crbo Center t Centrl Luzon Stte University, Crrngln, Nuev Ecij, Philippines. 2.2 Cryopreservtion Processing of Semen After semen collection nd initil evlution of the ejcultes (volume, color, ph, sperm concentrtion nd subjective motility grding), smple liquot ws tken nd plced on wter bth for the CASA evlution of fresh semen. The remining semen ws processed for cryopreservtion, following the methods described by Mmud nd Venturin [10] using simple rpid freezer (FHK, FA-1652). A smple liquot ws gin tken during the equilibrtion stge of the semen or before freezing, lso for the CASA evlution for the pre-freeze stge. After 24 h to one week of processing, frozen semen ws thwed in 37 C wter for 15 s for the post-thw qulity ssessment using CASA. 2.3 Sperm Motility Evlution Using CASA The CASA (Hmilton Thorne IVOS II ver. 14) ws used to perform the study. The fresh, pre-freeze nd frozen-thwed semen were evluted using the defult technicl setting for bull semen. The min set of prmeters includes 30 consecutive, digitlized photogrphic imges, which re tken in time lpse of 1 s, which is equivlent to 60 Hz. The evlution procedure ws done using 3 µl of the diluted semen smple with n djusted sperm concentrtion ( /ml), nd loded into the chmbers of the Lej slide for CASA exmintion. Opertion strts by scnning seven rndomly llocted fields for ech smple, recording t lest 100 motile sperms. Semen smples were utomticlly nlyzed by CASA, nd evluted with 11 defults perm kinemtic prmeters, describing the distnce trvelled, velocity, hed movement nd trjectory. For the distnce trvelled, the verge distnce of the smoothed cell pth is mesured s distnce verge pth (DAP), the verge distnce mesured over the

3 186 Identifiction of Sperm Subpopultions in Wter Bufflo Ejcultes: ctul point to point followed by the cell is mesured s distnce curved line (DCL), nd the verge distnce mesured in stright line from the beginning to the end of the trck is the distnce stright line (DSL). For the velocity kinemtics, these re the curviliner velocity (VCL) the verge velocity mesured over the ctul point to point followed by the cell, stright line velocity (VSL) the verge velocity mesured in stright line from the beginning to the end of the trck, nd verge pth velocity (VAP) the verge velocity of the smoothed cell pth. It lso includes the different hed movements, which re the mplitude of lterl hed (ALH) movement the men width of the hed oscilltion s the sperms swim, nd bet cross frequency (BCF) the frequency of sperm hed crossing the verge pth on either direction. Finlly, The trjectory includes linerity (LIN) the rtio of stright line velocity over curviliner velocity, strightness (STR) the rtio of stright line velocity over pth velocity, nd wobble (WOB) the rtio of verge pth velocity over curviliner velocity. Progressive sperm motility is defined by CASA s the percentge of spermtozo with men verge pth velocity bove 50 µm/s nd strightness of 65%. 2.4 Sttisticl Anlysis In determining the subpopultion composition of semen smples using the sperm kinemtics obtined by the CASA nlysis, dt from ll the motile spermtozo during fresh, pre-freeze, post-thw stges collected from the eight bulls were pooled on single dt sheet to represent the whole popultion. Following the nlysis mde by Rencher [11], Wrd s method in hierrchicl clustering, seprte dendrogrms for ll the prmeters ws mde. A multivrite k-mens cluster nlysis ws followed to clssify the number of sperms into reduced number of subpopultions ccording to their ptterns of movement. The clusters mde were bsed on the dendrogrms constructed. The k-mens clustering model used Eucliden distnces computed from the different sperm motion kinemtic prmeters fter normliztion of the dt, so tht the cluster centers re the mens of the observtions ssigned to ech cluster. The popultion of sperms belonging to ech cluster were lso clculted nd presented s reltive frequencies. The effects of cryopreservtion on the different kinemtic prmeters were subjected to nlysis of vrince (ANOVA). Significnt differences for ll the sttisticl tests were set t P = The SSPS sttistics softwre ws used in the study nd with the guidnce of sttisticin. 3. Results 3.1 Motility Chrcteristics of Sperm Subpopultions There re two sperm subpopultions (P 1 nd P 2 ) defined with significntly lrge cluster distnce of There re totl of 82,195 sperms tht re included in the nlysis nd their motility chrcteristics re shown in Tble 1. Subpopultion P 1 represents those spermtozo tht moved most rpidly nd progressively, s indicted by high VAP ( ± µm/s), VSL ( ± µm/s) nd VCL ( ± µm/s) together with high STR (83.13% ± 18.25%), BCF (32.08 ± 9.46 Hz), LIN (49.02% ± 16.06%), WOB (57.92% ± 10.66%) nd moderte ALH (10.32 ± 3.29 µm). It lso hs longer distnces trvelled, s shown by high DAP, DSL nd DCL (54.29 ± 21.48, ± nd ± µm, respectively). Subpopultion P 2 includes spermtozo with reltively low velocity or poorly motile but with high progressiveness, s seen with their low VAP, VSL nd VCL nd high STR, BCF, LIN, WOB nd low ALH. It lso registered the shorter distnces trvelled s indicted by low DAP, DSL nd DCL. The sperm subpopultions formed were significntly different (P < 0.05) from ech other on the distnce nd velocity prmeters, with subpopultion P 1 being the highest. The vlues for hed movement were lso

4 Identifiction of Sperm Subpopultions in Wter Bufflo Ejcultes: 187 Tble 1 Overll sperm kinemtics nd frequency distribution in ech subpopultion in wter bufflo semen (men ± SD). Sperm subpopultions Kinemtic prmeters P 1 P 2 No. of sperms 38,162 (46.29%) 44,033 (53.41%) Distnce trvelled DAP (µm) ± ± b DSL (µm) ± ± b DCL (µm) ± ± b Velocity VAP (µm/s) ± ± b VSL (µm/s) ± ± b VCL (µm/s) ± ± b Hed movements ALH (µm) ± ± 2.85 b BCF (Hz) ± 9.46 b ± Trjectory STR (%) ± ± b LIN (%) ± b ± WOB (%) ± b ± Mens hving different superscripts on ech row re significntly different from ech other t 0.05 significnce level. significntly different (P < 0.05) with ech other, however, there ws higher BCF recorded in subpopultion P 2. In terms of trjectory, LIN nd WOB were significntly higher in subpopultion P 2, while STR ws significntly higher in subpopultion P Frequency Distribution nd Sperm Kinemtics within Subpopultions of Wter Bufflo Semen during Cryopreservtion Tble 2 shows the proportion of spermtozo ssigned in the two subpopultions bsed on their sperm kinemtics cross the cryopreservtion stges. In fresh semen smples, 52.52% belonged to subpopultion P 1 (most rpid nd progressive), nd P 1 ws seen to be significntly decresing (P < 0.05) from 45.73% t pre-freeze to 35.17% t post-thw. This in generl denotes the proportions of spermtozo tht re moving most rpidly nd progressively (P 1 ) during cryopreservtion in declining mnner. Conversely, n incresing percentge of low velocity or poorly motile but with high progressiveness spermtozo ws observed in subpopultion P 2 cross the cryopreservtion stges. A totl of 17.35% of the sperms in the subpopultion P 1 ws lost or ressigned to subpopultion P 2 fter cryopreservtion. Significnt differences (P < 0.05) on the sperm kinemtics between subpopultions cross the cryopreservtion stges were observed. A decrese on the distnce trvelled (DAP nd DCL) by the sperms on both subpopultions from fresh to post-thw stges ws lso observed. However, DSL vlues were lso decresing t pre-freeze stge, but subpopultion P 1 incresed t post-thw stge. A decresing observtion in both subpopultions ws recorded on the velocity prmeters (VAP, VCL nd VSL) from fresh to post-thw stge. Menwhile, n increse in the vlue of ALH ws noticed in both subpopultions t pre-freeze stge, but eventully decresed t post-thw with vlues close with fresh stge (9.30 vs µm nd 4.15 vs µm for P 1 nd P 2, respectively). On the other hnd, BCF decresed t pre-freeze stge but eventully incresed t post-thw with close vlue t fresh stge in both subpopultions (31.99 vs Hz nd vs Hz for P 1 nd P 2, respectively). In

5 188 Identifiction of Sperm Subpopultions in Wter Bufflo Ejcultes: Tble 2 The effect of cryopreservtion on the sperm kinemtics nd frequency distribution in ech subpopultion in wter bufflo semen (mens ± SD). Kinemtic prmeters Sperm subpopultions Fresh stge Pre-freeze stge Post-thw stge P 1 P 2 P 1 P 2 P 1 P 2 No. of sperms 15,114 (52.52%) 13,664 (47.48%) 16,378 (45.73%) 19,435 (54.27%) 6,191 (35.17%) 11,413 (64.83%) Distnce trvelled DAP (µm) ± ± b ± ± b ± ± b DSL (µm) ± ± b ± ± b ± ± b DCL (µm) ± ± b ± ± b ± ± b Velocity VAP (µm/s) ± ± b ± ± b ± ± b VSL (µm/s) ± ± b ± ± b ± ± b VCL (µm/s) ± ± b ± ± b ± ± b Hed movements ALH (µm) 9.42 ± ± 2.73 b ± ± 3.04 b 9.30 ± ± 2.45 b BCF (Hz) ± ± b ± ± b ± ± b Trjectory STR (%) ± ± b ± ± b ± ± b LIN (%) ± ± b ± ± b ± ± b WOB (%) ± ± b ± ± b ± ± b Mens hving different superscripts on ech row re significntly different from ech other t 0.05 significnce level. terms of trjectory, STR, LIN nd WOB decresed t pre-freeze nd eventully incresed t post-thw for both subpopultions. It ws lso observed tht the vlues for LIN nd WOB were significntly higher in subpopultion P 2, unlike for the other sperm kinemtic prmeters wherein the vlues of subpopultion P 1 were lwys higher. 3.3 Frequency Distribution within Subpopultions of Spermtozo of Individul Bulls during Cryopreservtion In fresh stge of the semen smples, the proportion of subpopultion P 1 (most rpid nd progressive) nd subpopultion P 2 (poorly motile or low velocity nd high progressiveness) sperms ws found to be significntly different (P < 0.05) mong bulls, s presented in Fig. 1. In subpopultion P 1, bull 2 hd the significntly highest frequency, while bull 7 ws the lowest. Conversely, bull 7 hd the significntly highest frequency of subpopultion P 2 sperms nd bull 2 hd the lest. During the pre-freeze stge, decline in the percentge of subpopultion P 1 sperms ws observed mong ll the bulls with no significnt difference (P > 0.05) from ech other. On the other hnd, subpopultion P 2 ws significntly different from ech other mong ll bulls with bulls 7 nd 4 hving the highest percentge, respectively. Immeditely fter thwing, the sme decline ws observed in the subpopultion P 1 sperms nd there ws significnt difference mong ll bulls. Overll, bull 8 hd the highest frequency in subpopultion P 1, while bull 7 hd the lest. Menwhile for the subpopultion P 2, ll the bulls were lso significntly comprble with ech other with bulls 7 nd 2 with the highest frequency. The decline percentge t post-thw of subpopultion P 1 sperms ws observed to be highest in bull 1 (46.93%), followed by bull 2 (39.68%) nd bull 3 (37.88%). These bulls could lso be denoted s bd freezer bulls due to their more thn 25% nd lmost 50% decline in the distribution of the most rpid nd progressive sperms. Menwhile, bulls 2 nd 8 could be described/denoted s good freezer bulls

6 Identifiction of Sperm Subpopultions in Wter Bufflo Ejcultes: 189 % spermtozo Fresh bc c b bc bc bc bc bc bc bc bc b c bc Bulls % spermtozo Pre-freeze b bc bc bc b Bulls % spermtozo b b Post-thw 20 b b b b b b b Bulls Fig. 1 Reltive frequency distribution of motile spermtozo in men percentges (n = 8) within subpopultions P 1 (gry) nd P 2 (light gry) between bulls in fresh, pre-freeze nd post-thw semen. Different letters inside the columns indicte significnt differences within subpopultions between bulls t 0.05 significnce level. b b b b with the lowest decline on the percentge of sperms on subpopultion P 1 of 17.57% nd 21.21%, respectively. 3.4 Effect of Individul Vribility on the Sperm Kinemtic Prmeters within the Sperm Subpopultions There ws significnt difference observed on the sperm kinemtics of the two subpopultions mong ll bulls s seen in Fig. 1. In subpopultion P 1, bull 8 hd the significntly (P < 0.05) highest distnce (DAP, DSL nd DCL) nd velocity (VAP, VSL nd VCL) kinemtics, while bull 7 hd the significntly (P < 0.05) lest distnce (DAP: ± vs ± µm/s; DSL: ± vs ± µm/s; DCL: ± vs ± µm/s) nd velocity (VAP: ± vs ± µm/s; VSL: ± vs ± µm/s; VCL: ± vs ± µm/s). Menwhile for the ALH, bull 1 hd the highest (10.18 ± 3.33 µm), while bull 4 hd the lest (8.81 ± 3.37 µm). For the hed movement, bull 8 (34.37 ± 7.90 Hz) nd bull 4 (33.82 ± 9.21 Hz) hd the highest BCF, while bull 2 (30.81 ± 8.02 Hz) nd bull 1 (31.29 ± 8.52 Hz) hd the significntly lest. For the trjectory, bull 4 hd the significntly highest STR of 90.74% ± 9.07%, while bull 2 hd the lest STR of 88.70% ± 8.56%. For the LIN nd WOB, bull 4 hd the highest, while bull 5 hd the lest (LIN: 58.45% ± 15.08% vs % ± 10.79% nd WOB: 63.70% ± 12.46% vs % ± 9.18%, respectively). Significnt differences were lso observed mong bulls on the kinemtic prmeters defining the distnce, velocity, hed movement nd trjectory of subpopultion P 2 sperms. 4. Discussion The results of the present study indicte tht the semen of wter bufflo cn be chrcterized bsed on

7 190 Identifiction of Sperm Subpopultions in Wter Bufflo Ejcultes: their sperm kinemtic prmeters into two subpopultions (P 1 nd P 2 ). This number of sperm subpopultions formed is consistent mong ll eight bulls, either in fresh, pre-freeze or post-thw stges. The differences mong bulls re estblished by the proportion of sperms ssigned to the subpopultion of rpid nd progressive moving sperms (subpopultion P 1 ) t ll stges. The number of sperm subpopultions defined by this study is different from the subpopultions formed in cttle bulls, stllion nd other mmmls with three to four subpopultions [5, 9, 12]. Although the formed subpopultions in this study does not fit with the common finding of three or four well-defined sperm popultions mong mmmlin ejcultes, to our knowledge, the chrcteriztion of sperm subpopultions in fresh or frozen-thwed bufflo semen hs not been previously investigted. The cryopreservtion process hs significnt effects on the frequency distribution of spermtozo within subpopultions. The movement of one spermtozoon to nother subpopultion is due to the chnge in its motility pttern, which is ffected by the entire process from equilibrtion to freezing nd thwing. The shift from subpopultion P 1 to P 2 describes the loss in bility of the sperm to control its semi permebility experiencing sort of flse hyper ctivtion [9]. It cn be seen tht the results on the frequency of spermtozo belonging to subpopultion P 1 were significntly reduced from pre-freeze to post-thw. During freezing, the sperm plsm membrne undergoes phse trnsition from the liquid crystlline phse to the gel phse due to decrese in temperture. This irreversible chnges induced by lipid phse trnsitions during cooling wrming my possibly ffect the movement chrcteristics of spermtozo during semen processing [13]. According to Muiño et l. [9], ejcultes with the highest popultions of rpid nd progressive sperm were lso the most resistnt to cryopreservtion nd showed the best post-thw sperm longevity. The declining percentge of most rpid nd progressive (subpopultion P 1 ) sperms in the study is seemingly cceptble. Although it hs been known tht substntil number (50%) of sperm re dmged during cryopreservtion [14]. In this study, the results of n verge of 35.17% of the most rpid nd progressive sperms subpopultion t post-thw is higher thn tht reported by Muiño et l. [9], who reported only 25.3% kept in cttle bull semen. It cn be then ssumed tht the frozen-thwed wter bufflo semen cn contin good number of most rpid nd progressive sperms cpble for fertiliztion. In this study, individul vrition ws seen mong bulls, especilly on the subpopultion of spermtozo with the most rpid movement nd progressiveness. The chnge in the distribution between the two subpopultions is consistent mong ll bulls s ffected by the cryopreservtion process. There hd been decresing trend on the frequency of the sperms with most rpid nd progressiveness chrcteristics from fresh, pre-freeze to post-thw stge mong ll bulls, in which conversely n increse in the popultion of poorly motile or low velocity but high progressiveness sperms. Bulls in this study with greter proportion of most rpid nd progressiveness sperm subpopultion t the onset still contined higher proportions of it t post-thw. This chrcteristic of the sperms of bull cn be ttributed to its higher cryosurvivbility s compred to others. Severl reserchers suggested tht the sperms belonging to the highest velocity nd progressiveness cn be considered s the sperm with the highest fertilizing potentil [5, 8, 15]. Thus, it is of utmost importnce to determine the different subpopultions of motile spermtozo existing in bull semen smple for better projection of the movement of the sperms nd its possible fertilizing bility. Similr results for the differences on the sperm subpopultion distribution were found in humn [16], dog [8] nd Holstein bulls [9] ejcultes. The study on determining the individul sperm kinemtics could be useful in improving the bufflo

8 Identifiction of Sperm Subpopultions in Wter Bufflo Ejcultes: 191 semen qulity ssessment by detecting subtle chnges on the sperm movement cross cryopreservtion, in which, eventully my ffect its fertilizing function. However, to strengthen justifiction on the fertilizing potentil of the most rpid nd progressive sperms, bulls hving higher proportion of this my be used in vitro or in vivo to see n existing correltion. The findings of this cn be used s predicting formul for the fertilizing rte of bull bsed on the movements of its sperm. This informtion could lso be used to select bulls for continuous use in the rtificil insemintion progrm, or mximize its consumption by lowering the insemintion doses so s to cter more dms to impregnte nd spred its genetics. 5. Conclusions The study on wter bufflo sperm kinemtics indictes two well-defined sperm subpopultions existing in fresh, pre-freeze nd post-thw semen stges. Wter bufflo sperm generlly moves either rpidly nd progressively or slowly nd progressively. The effect of cryopreservtion ws seen in the decline on the frequency of rpidly nd progressively moving sperms of ll bulls. Differences mong bulls re recognized on the distribution of sperm subpopultion with most rpid nd progressive sperms. The greter percentge of most rpidly nd progressively moving sperms mintined t post-thw, indicting good freezbility of the bull. Further studies re needed in determining the importnce of identifiction of sperm subpopultions in wter bufflo nd its use in predicting fertility. Acknowledgments The uthors would like to thnk Ms. Meliss Gzzingn for the sttisticl nlysis of the reserch nd the Philippine Crbo Center for the finncil grnt of the study. References [1] Vincent, P., Underwood, S. L., Dolbec, C., Bouchrd, N., Kroetsch, T., nd Blondin, P Bovine Semen Qulity Control in Artificil Insemintion Centers. Anim. Reprod. 9 (3): [2] Shojei, H., Kroetsch, T., Wilde, R., Blondin, P., Kstelic, J. P., nd Thundthil, J. C Moribund Sperm in Frozen-Thwed Semen, nd Sperm Motion End Points Post-Thw nd Post-Swim-Up, Are Relted to Fertility in Holstein AI Bulls. Theriogenology 77 (5): [3] Sundrrmn, M. N., Klthrn, J., nd Jwhr, K. T. P Computer Assisted Semen Anlysis for Quntifiction of Motion Chrcteristics of Bull Sperm during Cryopreservtion Cycle. Vet. World 5 (12): [4] Ferrz, M. A., Mortó, R., Yeste, M., Arcrons, N., Pen, A. I., Tmrgo, C., Hidlgo, C. O., Muiño, R., nd Mogs, T Evlution of Sperm Subpopultion Structure in Reltion to in Vitro Sperm-Oocyte Interction of Frozen-Thwed Semen from Holstein Bulls. Theriogenology 81 (8): [5] Quintero-Moreno, A., Miró, J., Teres-Rigu, A., nd Rodríguez-Gil, J. E Identifiction of Sperm Subpopultions with Specific Motility Chrcteristics in Stllion Ejcultes. Theriogenology 59 (9): [6] Yániz, J. L., Plcín, I., Vicente-Fiel, S., Sánchez-Ndl, J. A., nd Sntolri, P Sperm Popultion Structure in High nd Low Field Fertility Rms. Anim. Reprod. Sci. 156: [7] Abigr, T., Holt, W. V., Hrrison, R. A., nd Del Brrio, G Sperm Subpopultions in Bor (Sus scrof) nd Gzelle (Gzell dm mhorr) Semen s Reveled by Pttern Anlysis of Computer-Assisted Motility Assessments. Biol. Reprod. 60 (1): [8] Núñez-Mrtínez, I., Morn, J. M., nd Peñ, F. J A Three-Step Sttisticl Procedure to Identify Sperm Kinemtic Subpopultions in Cnine Ejcultes: Chnges fter Cryopreservtion. Reprod. Domest. Anim. 41 (5): [9] Muiño, R., Tmrgo, C., Hidlgo, C. O., nd Peñ, A. I Identifiction of Sperm Subpopultions with Defined Motility Chrcteristics in Ejcultes from Holstein Bulls: Effects of Cryopreservtion nd Between-Bull Vrition. Anim. Reprod. Sci. 109: [10] Mmud, F. V., nd Venturin, E. V Bufflo Semen Collection nd Processing Hndbook. Munoz, Nuev Ecij: PCC-CLSU. [11] Rencher, A. C A Review of Methods of Multivrite Anlysis, Second Edition. Journl of IIE Trnsctions 37 (11): [12] Vázquez, A. J. F., Cedillo, M. J., Quezd, V. J., Rivs, A. C., Morles, E. C. L., Ayl, E. M. E., Hernández, M. J., González, R. A., nd Argón, M. A Effects of Repeted Electroejcultions on Kinemtic Sperm Subpopultions nd Qulity Mrkers of Mexicn Creole

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