咖啡因和肝素、钙离子载体对家畜冷冻精子体外获能的协同作用

本文报道了用咖啡因与肝素、钙离子载体(I-A)协同处理诱导马、驴、猪冷冻精子体外获能的研究结果.咖啡因与肝素、I-A均有协同作用.在肝素或I-A处理中添加咖啡因,不仅能提高穿透率,而且能促进卵内雄原核的形成和发育.精子先经洗涤,再用咖啡因与肝素或I-A协同处理,可得到良好的获能效果.     

Effect of density gradient composition on in vitro maturation of stallion sperm

The present study was conducted to determine the influence of density gradient composition on in vitro capacitation of stallion spermatozoa. In Experiment I spermatozoa were isolated on either 90% Percoll (P), 90% arabinogalactan (AG), or 12% BSA gradients and then challenged with 1 μM A23187 (15, 150, and 270 min) and heat-solubilized equine zonae pellucidae (270 min, sEZP, 2 ZP/μl). The P gradient enhanced the percentage of progressively motile spermatozoa (PMS) more (P=.0001) than AG or BSA gradients immediately post-processing, but was not sustained throughout the culture period. The viability for P-separated spermatozoa was higher (P=.01) than that of BSA or AG-separated spermatozoa. Gradient composition had no effect (P=.68) on the percentage of live, acrosome reacted spermatozoa (PAR), before and following ionophore and sEZP challenge. In Experiment II, the number of spermatozoa penetrating the ZP of saltstored oocytes was not influenced (P=.35) by gradient composition; however, the number of spermatozoa bound per oocyte was higher (P=.02) for P-separated spermatozoa than for AG or BSA-separated spermatozoa. These data suggest that isolation on a P density gradient may enhance in vitro capacitation of stallion spermatozoa.     

Capacitation of Frozen Thawed Buffalo Bull (Bubalus bubalis) Spermatozoa with Higher Heparin Concentrations

The present study was conducted to examine the effect of high heparin concentration on capacitation of buffalo spermatozoa with a short incubation time. Frozen thawed spermatozoa from three buffalo bulls were pooled and treated with either 50, 100 or 200 microg/ml heparin for 30 min. Capacitation was evaluated by acrosome reaction of spermatozoa and in vitro fertilization rate (per cent cleavage rate, per cent cleavage index). Acrosome reaction was induced in heparin treated spermatozoa with calcium ionophore A23187 and staining was carried out with Coomassie G-250 to evaluate the response as compared with control (0 heparin + calcium ionophore). Significantly higher percentage of acrosome reaction (AR) spermatozoa was noted after heparin treatment (36.8-48.2%) as compared with control (8.1% ; p < 0.05) but differences among the three heparin concentrations were non-significant. However, a significantly higher in vitro fertilization rate was recorded in spermatozoa capacitated by 50 and 100 microg/ml heparin (80.4 and 75.9% cleavage rate, respectively) as compared with 200 microg/ml heparin (47.2% cleavage rate; p < 0.001). It is concluded that buffalo spermatozoa capacitated with 50-100 microg/ml heparin had significantly higher ability to improve in vitro fertilization rate in buffalo.     

In vitro induction of the acrosome reaction in ovine spermatozoa by calcium ionophore A23187

The relationship between concentration of calcium ionophore A23187 and incubation time upon the proportion of spermatozoa undergoing acrosome reaction (AR) in vitro was investigated in rams from a commercial artificial insemination (AI) program. Two ejaculates were collected by artificial vagina from each of nine rams of three breeds (Finn Dorset, Charolais and Suffolk) aged 8-36 months. Each ejaculate was diluted in a skimmed milk extender. Spermatozoa were thereafter incubated for 45 or 60 min in modified Tyrode's medium (TALP) which contained either zero, 0.1 or 1.0 microM/l A23187. After fixing in 10% formaldehyde, the number of spermatozoa that had undergone AR was determined by phase contrast microscopy. In pre-incubation samples, 21.3 +/- 3.3% of spermatozoa had undergone AR. Percentages of acrosome reacted spermatozoa were significantly (P < 0.001) increased after incubation with A23187. After incubation with 0.1 microM/l A23187 for 45 and 60 min there were 22.4 +/- 3.0% and 31.7 +/- 4.3% acrosome reacted spermatozoa, respectively. After incubation with 1.0 microM/l A23187 for 45 and 60 min there were 46.2 +/- 6.5% and 53.8 +/- 5.9% acrosome reacted spermatozoa, whilst corresponding numbers in control samples were 17.0 +/- 2.7% and 22.3 +/- 4.2%. There was also a significant (P < 0.001) effect of individual animals upon the responses to different concentrations of A23187. These findings indicate that (i) A23187 can be used to assess the AR of ovine spermatozoa in vitro and (ii) there are effects of individual animals upon the proportion of spermatozoa undergoing AR.     

In vivo capacitation and acrosome reaction of bovine sperm in estrous and diestrous cows

A staining procedure which enables distinction between spermatozoa possessing a true and false acrosome reaction (AR) was utilized to assess the incidence of capacitation and the true AR of bull spermatozoa recovered from the uterine horns of estrous and diestrous cows. Results show that at 3 and 6 h post-insemination, approximately 14.5 and 31.5%, respectively, of the live spermatozoa recovered had undergone a true AR in the uterus of estrous cows. An increasing percentage of those spermatozoa recovered from estrous cows with time were categorized as undergoing a false AR. This suggests that spermatozoa underwent capacitation, a true AR, then died prior to fixation and staining, therefore being grouped as false acrosome-reacted. Few spermatozoa were observed to have undergone a true AR in diestrous cows. It is apparent from this study that individual spermatozoa undergo capacitation and a true AR at different times during incubation in utero in estrous cows.     

钙离子载体及咖啡因对绵羊精子获能的影响

通过添加不同浓度(0.05、0.1、0.2、0.5、1、5、10μmol/L)的钙离子载体(A23187,IA)及2 mmol/L咖啡因,探讨不同作用时间其对绵羊精子体外获能的影响。结果发现,IA浓度越高,精子的体外获能率越高,顶体反应率越低,活力越低,死精子就越多;而延长作用时间对获能、顶体反应的影响不明显。IA和咖啡因共同作用后,咖啡因能够促进IA诱导顶体反应的发生。建议用IA诱导绵羊精子获能时,其浓度以0.05~0.2μmol/L为宜,作用时间1 min。     

The Effect of 2-Hydroxypropyl-β-Cyclodextrin on Post-Thaw Parameters of Cryopreserved Jack and Stallion Semen

Cyclodextrins improve post-thaw viability and motility of semen as well as mediate cholesterol efflux and subsequent acrosome reaction in spermatozoa from several species. The objectives of this study were: (a) to assess the effect of prefreeze addition of 60 mM hydroxypropyl-β-cyclodextrin (β-CD) on post-thaw viability and motility of jack and stallion semen cryopreserved in ethylene glycol-based freezing extenders containing 5% or 20% (v/v) egg yolk (LEY and HEY, respectively), and (b) to evaluate the ability of 1 μM calcium ionophore A23187 and/or 60 mM β-CD to induce acrosome reaction in thawed jack and stallion spermatozoa. Post-thaw motility of spermatozoa cryopreserved in HEY was higher (P < .05) for jack but lower (P < .05) for stallion spermatozoa when compared with LEY. Jack and stallion spermatozoa both exhibited higher (P < .05) motility when cryopreserved in 60 mM β-CD than without β-CD. Curvilinear velocity was faster (P < .05) for jack and stallion spermatozoa cryopreserved in LEY than in HEY. A treatment × time interaction affected (P < .05) the proportion of spermatozoa that underwent acrosome reaction. Post-thaw incubation of jack and stallion spermatozoa with β-CD for 90 minutes induced acrosome reaction in 85% and 22% of viable sperm cells, respectively; however, only 32% of jack and 8% of stallion spermatozoa incubated with calcium ionophore underwent acrosome reaction. This study is the first to evaluate the effect of β-CD (not loaded with cholesterol) on jack semen cryopreservation, and results reveal that β-CD may be a useful tool to enhance semen cryopreservation and to induce post-thaw acrosome reaction in jack spermatozoa.     

Single Layer Centrifugation of Stallion Spermatozoa through Androcoll™‐E does not Adversely Affect their Capacitation‐Like Status,as Measured by CTC Staining

This study was designed to evaluate the effect of single layer centrifugation (SLC) and subsequent cold storage on stallion sperm capacitation‐like status and acrosome reaction. Three stallions were included in the study, with three ejaculates per stallion. The samples were examined 4, 24 and 72 h after collection, extension and SLC, with storage at 6°C. Sperm capacitation‐like status was investigated using the fluorescent dye chlortetracycline (CTC). There was no difference in capacitation‐like status between colloid‐selected and non‐selected spermatozoa. Sperm motility decreased significantly during cold storage, whereas the proportion of apparently capacitated spermatozoa increased. There was no change in the proportion of acrosome‐reacted spermatozoa. In conclusion, SLC through Androcoll?‐E does not adversely affect the capacitation‐like status of stallion spermatozoa, although it did increase with time during cold storage.     

Hyperactivation and acrosome reaction in vitro in spermatozoa ejaculated by cryptorchid dogs after orchiopexy.

Orchiopexy of the cryptorchid (CR) testis and castration of the scrotal testis were performed in three unilaterally CR beagles at six months of age. Induction rates for ejaculated sperm hyperactivation (HA) and the acrosome reaction (AR) in vitro in these orchiopexied dogs were compared with five those in normal beagles one year later. Canine spermatozoa were incubated for 9 hr at 38 degrees C under 5% CO2 in air in canine capacitation medium at a concentration of 30 x 10(6) sperm/ml. HA was observed using high-speed videomicrography. The AR spermatozoa were evaluated by the triple stain technique. As a result, there was no significant difference between 'the CR dogs after orchiopexy' (CDO) and the normal dogs (ND) with respect to sperm motility just after ejaculation. However, sperm motility of CDO decreased markedly during incubation. There was a significant difference in sperm motility between CDO (Mean +/- SD; 47 +/- 12%) and ND (80 +/- 9%) after three hours of incubation (p less than 0.01). No significant difference was observed between CDO and ND with respect to the HA rate of motile spermatozoa throughout the incubation period. The peak of HA rate was found in both CDO (58 +/- 5%) and ND (61 +/- 16%) after seven hours of incubation. The AR rate of spermatozoa in CDO was lower than that in ND after six hours of incubation. The AR rate of CDO (26 +/- 4%) was significantly lower than ND (46 +/- 5%) after eight hours of incubation (p less than 0.01). It is assumed that there might be relation between a rapid decrease of motility and low AR rate in spermatozoa of CDO during incubation.     

水牛附睾尾精子的体外受精

采用肝素诱导获能 ,比较了 TAL P液和 BO液处理水牛附睾尾精子进行体外受精和细管冷冻精液体外受精的效果。结果表明 ,用 BO液和 TAL P液分别处理水牛附睾尾精子 ,受精后的卵裂率分别为 4 6 .6 7%和 5 3.73% ,发育率分别为 2 1.6 7%和 2 6 .87% ,其受精效果差异不显著。综合 2种方法 ,水牛附睾尾精子的受精率为 5 7.14 % ,受精后的卵裂率为 5 0 .39% ;发育率相对于培养卵为 2 4 .4 1% ,相对于卵裂卵为 4 8.4 4 % ;与细管冷冻精液 (5 6 .0 0 % ,5 4 .31% ,2 6 .72 % ,4 9.2 1% )相比 ,差异不显著。形态学观察还表明 ,用保温干储的方法可获得活率好、存活时间长的附睾尾精子。试验结果说明水牛附睾尾精子用于体外受精可以得到与细管冷冻精液相当的效果。     

Effect of capacitation of stallion sperm with polyvinylalcohol or bovine serum albumin on penetration of bovine zona-free or partially zona-removed equine oocytes

Experiments were conducted to study effects of macromolecules on stallion sperm capacitation and fertilization as determined by penetration of bovine zona-free and equine partially zona-removed oocytes. Stallion sperm were capacitated in TYH medium (modified Krebs-Ringer bicarbonate) supplemented with either 1 mg/mL of polyvinylalcohol (PVA) or 4 mg/mL of BSA. Capacitation was induced with 8 bromoadenosine cyclic monophosphate (8BrcAMP; 0.5 mM) alone or in combination with 0.1 microM of ionomycin. Intraspecies gametes were co-incubated in TYH/PVA or TYH/BSA for 18 to 20 h. For zona-free bovine oocytes, penetration rate (35%) with the combination of 8BrcAMP and ionomycin in PVA-containing medium was higher (P < 0.05) than any treatment in BSA-containing medium (5 to 6%). A similar study was conducted using equine oocytes with partially removed zonae. Sperm capacitated and used for in vitro fertilization (IVF) in PVA-containing medium had higher penetration rates (P < 0.01) than sperm in BSA-containing medium (54 vs. 11%). The effect of equine preovulatory follicular fluid on bovine oocyte penetration was assessed. Bovine oocytes were matured in tissue culture medium-199 with 0, 20, 50, or 100% equine preovulatory follicular fluid, and 1 IU/mL of equine chorionic gonadotropin. Stallion sperm were treated with 8BrcAMP + ionomycin in PVA- or BSA-containing media. The penetration rates of bovine zona-free oocytes by stallion sperm were again higher with PVA (47%) than BSA (18%; P < 0.01). Penetration rates of oocytes matured in 100% follicular fluid were higher (P < 0.05) than for oocytes matured with 0% follicular fluid. The effects of equine follicular fluid and PVA/BSA during sperm capacitation on standard bovine IVF were examined. Culture of bovine oocytes with equine follicular fluid did not affect oocyte maturation or penetration rates after IVF. Bovine sperm capacitated with heparin in PVA-containing medium yielded lower (P < 0.05) fertilization rates than those capacitated in BSA-containing medium when incubated with both zona-intact and zona-free bovine oocytes. In summary, PVA was superior to BSA for ionophore-induced capacitation of equine sperm for penetration of zona-free bovine oocytes or partially zona-removed equine oocytes, but not for standard bovine IVF with bovine sperm. Zona-free bovine oocytes may be useful for assaying in vitro capacitation and fertilization of stallion sperm.     

添加咖啡因、亚牛磺酸对牛精子功能的影响

为研究获能液中添加咖啡因和亚牛磺酸对牛精子功能的影响,本研究将荷斯坦牛冻精解冻后分别添加在含不同浓度咖啡因（0、2.5、5.0、7.5 mmol/L）或亚牛磺酸（0、5、10、20、40 μmol/L）的获能处理液中,且每个处理组加入约200 μL的精液,在CO₂培养箱里经上游处理45 min,以评估咖啡因和亚牛磺酸对牛精子活力、顶体及质膜完整性的影响,进而探讨在获能液中亚牛磺酸替代咖啡因的效果。结果显示,添加2.5、5.0 mmol/L咖啡因组经上游法获能处理后的牛精子活力和顶体完整率均显著高于对照组（P<0.05）,且2.5 mmol/L咖啡因组精子活力最高;添加10、20 μmol/L亚牛磺酸组经上游法获能处理后的牛精子活力、顶体完整率和质膜完整率均显著高于对照组（P<0.05）,且20 μmol/L亚牛磺酸组的精子功能参数值最高;将筛选的最佳浓度2.5 mmol/L咖啡因和20 μmol/L亚牛磺酸采用同样的方法处理,发现20 μmol/L亚牛磺酸组的精子顶体完整率和质膜完整率均显著高于2.5 mmol/L咖啡因组和对照组（P<0.05）。因此,20 μmol/L亚牛磺酸可以替代2.5 mmol/L咖啡因用于体外受精体系中的精子获能处理,有助于提高精子功能参数。     

肝素、咖啡因、丙酮酸钠、BSA对延边黄牛卵母细胞体外受精效果的影响

试验为延边黄牛体外胚胎产业化生产奠定基础,进行了肝素、咖啡因、丙酮酸钠、BSA对延边黄牛体外受精效果的影响研究。结果表明,以改良BO液为基础液,添加20 μg/mL肝素时,精子顶体反应率和受精率分别为82.52%和75.34%,高于添加20 μg/mL咖啡因组(P<0.05)和添加10 μg /mL肝素与10 μg/mL咖啡因协同作用组(P>0.05);结合使用肝素(20 μg/mL),添加0.0020 g/mL丙酮酸钠时,精子顶体反应率和受精率分别为78.13%和71.57%, 高于添加0.0015 g/mL和0.0025 g/mL丙酮酸钠组(P<0.05);结合使用肝素(20 μg/mL),添加0.006 g/mL BSA时,精子顶体反应率和受精率分别为80.78%和71.44%,高于添加0.001 g/mL和0.003 g/mL BSA组(P<0.05)。因此,添加适当浓度的肝素、丙酮酸钠和BSA可显著提高延边黄牛精子体外获能效果及体外受精率。     

Relationship between Sperm Response to Glycosaminoglycans in vitro and Non-return Rates of Swedish Dairy AI Bulls

In this study, the relations between fertility (56‐day non‐return rates, 56‐day NRR) after artificial insemination (AI) and bull sperm characteristics post‐thaw, after swim‐up and after co‐incubation with heparin (Hep) and hyaluronan (HA), respectively, were determined, attempting to determine if such a procedure could be of value to evaluate the potential fertilizing ability of frozen‐thawed AI bull spermatozoa. Spermatozoa from 20 semen batches derived from 20 Swedish Red and White AI bulls ranging widely in their field fertility after AI (55–79% 56‐day NRRs) were evaluated with regards to post‐thaw motility, membrane integrity, and migration through a simple swim‐up procedure. Sperm viability and capacitation status were evaluated by two different vital staining procedures and chlortetracycline hydrochloride staining. Sperm motility and membrane integrity post‐thaw (e.g. indicators of sperm viability) were significantly correlated (r = 0.53, p < 0.05 and r = 0.59, p < 0.01, respectively) with fertility. Heparin (5 µg/ml) significantly (p lt; 0.001) increased the frequencies of capacitation and acrosome‐reaction (AR) among swim‐up separated spermatozoa, whereas HA at a concentration of 50 ng/ml did not have any significant capacitating effect. The incidences of capacitated or AR‐spermatozoa following Hep‐treatment were not correlated with fertility. On the other hand, the percentage of viable spermatozoa was significantly (p < 0.001) lower in Hep‐treated samples than in control and HA‐treated samples and was significantly (r = 0.49, p < 0.05) correlated with fertility after AI (56‐day NRR). The results indicate that the percentage of viable spermatozoa after swim‐up separation and heparin‐exposure from a selected population of AI bulls were significantly and positively related to the AI fertility of the donors and thus could be used as a parameter to determine the fertilizing ability of frozen—thawed AI bull spermatozoa.     

Effect of Exogenous Progesterone on Post-thaw Capacitation and Acrosome Reaction of Bovine Spermatozoa

The aim of this work was to study the effect of progesterone (P4) on capacitation and acrosome reaction (AR) of post-thaw bovine spermatozoa in vitro. Spermatozoa were incubated (0-180 min) in capacitation medium supplemented with 0, 0.1, 1.0 and 10.0 microg/ml of P4. At different time intervals aliquots were taken to determine sperm plasma membrane lipid destabilization, or capacitation (AR induced by lysophosphatidylcholine) in spermatozoa. The second experiment aimed to study the effects of P4, as potential inducer of AR in heparin-capacitated spermatozoa. The acrosomal status and viability of spermatozoa were evaluated under an epifluorescence microscope using Ethidium homodimer/peanut agglutinin fluorescein isothiocyanate staining method. Plasma membrane scrambling in spermatozoa was assessed by a flow cytometer, using merocyanine staining. The results show that P4 at the concentrations used had no negative effects on sperm viability. Progesterone significantly enhanced sperm capacitation (p < 0.001), but had no effect on plasma membrane lipid stability (p > 0.05) and did not significantly increase the AR of heparin-capacitated spermatozoa (p > 0.05). Progesterone displayed its effects in a dose-dependent manner with a maximum effect of 10 microg/ml P4 at 180 min of incubation. The results demonstrate that in cryopreserved bovine semen, P4 acts as capacitating, but not as an AR-inducing agent.     

Comparative Immunolocalization of Heat Shock Proteins (Hsp)-60, -70, -90 in Boar, Stallion, Dog and Cat Spermatozoa

Heat shock proteins (Hsp)-60, -70 and -90 are important testis chaperones that fulfil several functions during sperm cell maturation. In post-meiotic cells, their expression may change or may be undetectable and in some species it may be evident in mature spermatozoa. The aims of this study were to verify whether Hsp60, -70 and -90 are present in the sperm, and to compare their localization in boar, stallion, cat and dog spermatozoa by immunofluorescence. Hsp-60 immunoreactivity was detected in sperm midpiece in all the species examined. In stallion sperm, Hsp70 signal was localized in the sub-equatorial band, whereas immunoreactivity was evident on the neck of dog spermatozoa and on both neck and sub-equatorial region of cat spermatozoa. In agreement with our previous observations, a triangular fluorescent signal in the equatorial segment of fresh boar sperm was detected. Hsp90 immunoreactivity was present in different portions of sperm tail: in the midpiece of both boar and cat spermatozoa and in the neck and throughout the tail in dog and stallion spermatozoa, respectively. When capacitation and acrosome reaction were induced in boar, stallion and dog spermatozoa, no changes in both Hsp60 and -90 were recorded by either Western blot or immunofluorescence. After induction of acrosome reaction, a Hsp70 redistribution in boar spermatozoa and an increased percentage of stallion spermatozoa showing the post-acrosomal signal were observed although no changes were recorded by Western blot; in dog spermatozoa, no changes in Hsp70 were found by Western blot and immunofluorescence after capacitation and acrosome reaction.     

钙离子浓度对绵羊精子获能的影响

为探讨钙离子浓度对绵羊精子获能的影响,将绵羊精子在不同钙离子浓度的绵羊输卵管合成液（SOF液）中培养,并用钙离子载体A23187进行诱导。结果发现,不同钙离子浓度对绵羊精子的影响差别不明显,经A23187诱导后,1.7 mmol/L组和2 mmol/L组的顶体反应率（AR）与其他组有显著差异（P<0.05）,1.7 mmol/L浓度中,绵羊精子存活时间为16 h,2 mmol/L浓度中的存活时间为10 h,因此认为绵羊精子获能的适宜钙离子浓度为1.7 mmol/L,且获能过程中钙离子的存在是必要的。     

利用CTC染色进行猪精子不同体外获能方法的比较

为了探讨不同方法对猪精子体外获能的影响,本试验以猪的新鲜精液为研究对象,分别经肝素上游法、钙离子载体诱导法和咖啡因诱导法进行精子不同获能时间的处理,并利用金霉素荧光染色法（chlortetracycline,CTC）对其获能效果进行检验。结果表明,采用肝素上游法孵育40 min处理后,精子的获能比例显著高于其他各处理组（P<0.05）,说明肝素上游法可以较好地诱发猪精子在体外获能。     

In vitro Capacitation and Acrosome Reaction of Dog Spermatozoa can be Feasibly Attained in a Defined Medium Without Glucose

Incubation of dog spermatozoa in a medium without glucose and in the presence of lactate and pyruvate (l-CCM) for 4 h at 38.5 degrees C in a 5% CO(2) atmosphere induced in vitro capacitation of these cells. This was verified after the combined specific capacitation-like changes in percentages of viability and altered acrosomes, motility characteristics, sperm location of reactivity against Pisum sativum, Arachis hypogaea and Helix pomatia lectins and the tyrosine phosphorylation pattern. Furthermore, a feasible acrosome reaction (AR) was induced when spermatozoa incubated in l-CCM for 4 h were further co-incubated for 1 h with canine oocytes. This was demonstrated by AR-like changes in percentages of viability, altered acrosomes, motility characteristics and sperm location of reactivity against P. sativum, A. hypogaea and H. pomatia lectins. All these results clearly indicate that in vitro capacitation, and subsequent AR, can be feasibly achieved without the presence of sugars. This ability can be related to the specific characteristics of energy-metabolism regulation reported in dog spermatozoa.     

Fertility of stallion spermatozoa isolated on albumin gradients

Fertility of stallion semen extended with bovine serum albumin (BSA) sucrose extender or cream-gelatin extender was compared. Pregnancy rates were 95% of 47 mares and 86% of 46 mares inseminated with BSA-sucrose and creamgelatin extended semen, respectively. Foaling rates and cycles per conception were not significantly different between treatment groups.Semen from 5 mature stallions was used in an attempt to isolate a population of highly motile spermatozoa. Immediately following collection, samples were evaluated for motility and forward movement. Seven to 10 ml of semen, extended 1:1 with BSA-sucrose extender, were pipetted onto BSA medium separation columns. After 1 hour incubation at 37°C, the top, middle and bottom layers were separately withdrawn from each separation column and pooled, respectively. A marked decrease (P<.001) was noted in the mean motility of spermatozoa in the top layer (35%) as compared to the mean pre-incubation motility (59%). Spermatozoa from middle and bottom layers were significantly (P<.001) more motile (70.6 and 87%) than those from the top layer and pre-incubation samples. Rate of forward movement (RFM) of spermatozoa in lower fractions was higher (P<.001) than RFM of spermatozoa in the top layer. Concentration of spermatozoa decreased (P<.001) as the concentration of BSA in the medium increased.