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1.
Split fractions of 25 ejaculated semen samples and spermatozoa from 5 caudae epididymides were used to study the effect of different levels of caffeine on the motility and fructolytic activity. During the first hour of incubation at 37°C, addition of caffeine to unwashed ejaculated buffalo sperm significantly increased the percentage of motility and amount of fructose utilized. In presence of 2–8 mM caffeine, sperm maintained their initial motility at least 2 hours at 37°C. Maximal stimulation of fructolytic activity was obtained with 2 mM whereas the minimal stimulation was found with higher concentration of 8–10 mM caffeine. Compared to ejaculated spermatozoa, epididymal sperm appeared to be more influenced by caffeine. Fructolytic activity was stimulated at least 1.5 times in the presence of 2 mM caffeine. Nearly during all incubation periods, the amounts of fructose utilized by caffeine-treated sperm were greater than in control samples. Data on epididymal sperm motility demonstrated that caffeine (2–10 mM) significantly stimulated and maintained the initial motility at least 3 hours at 37°C .  相似文献   

2.
This study was designed to apply the method of discontinuous Percoll gradients for sex preselection in bovine semen by using a current developed molecular technique, fluorescence in situ hybridization (FISH). In addition, we attempted to amplify the level of enrichment of X- or Y-bearing spermatozoa by treating for activating sperm motility performance with 10 mM caffeine. Bovine spermatozoa were fractionated on Percoll gradients into two major subpopulations of motile spermatozoa (bottom fraction) and weak motile spermatozoa (top fraction). The percentage of Y-bearing spermatozoa in the top fraction (52.9%) slightly exceeded and that in the bottom fraction (44.3%) decreased significantly (P<0.001) compared with the theoretical ratio (50:50). Washing sperm with BO medium affected a deviation between the two sex populations, whereas semen activated with caffeine showed no difference in the percentage of X- and Y-bearing spermatozoa in both fractions compared with the theoretical ratio (50:50). These results show that the proportion of X- and Y-bearing bovine spermatozoa can deviate after discontinuous Percoll gradients, although the proportion of X- and Y-bearing bovine spermatozoa was affected by sperm motility of the sample applied.  相似文献   

3.
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.  相似文献   

4.
To evaluate effects of different concentrations of pentoxifylline, as phosphodiesterase inhibitor, on quality of motility, capacitation and acrosome reaction, Ejaculated spermatozoa were collected from crossbred dogs. The sperm were incubated at concentrations of 0.1, 1, 10 and 100 mM pentoxifylline for 2 h. Conventional assessment was also made on the percentage of motility and quality of motility of spermatozoa; values were expressed as sperm motility index (SMI). Capacitation and acrosome reaction were also evaluated by chlortetracycline fluorescence staining. SMI as quality index of sperm was significantly increased in concentrations of 10 and 100 mM pentoxifylline during 1 and 2 h compared to control. The number of capacitated or acrosome reacted spermatozoa significantly (P < 0.05) were higher than controls at high concentrations of pentoxifylline (10 and 100 mM) during 1 and 2 h. In conclusion, high concentration of pentoxifylline is able to induce capacitation and acrosome reaction and improves quality of motility in canine ejaculated spermatozoa.  相似文献   

5.
Sperm culture media used for in vitro fertilization (IVF) procedures are important factors concerning the viability, motility and acrosomal integrity of spermatozoa. The aim of this study was to investigate the effects of three different sperm diluting media, tissue culture medium (TCM‐199), sperm culture medium (Sp‐TALP) and human tubular fluid (HTF) supplemented with varying concentrations of bovine serum albumin (1, 4 and 6%) or polyvinyl alcohol (0.8%) on the acrosomal integrity, motility and viability of canine spermatozoa. Ejaculates collected from four dogs were diluted in all media and spermatozoa were separated from seminal plasma by the swim‐up technique. Sperm progressive motility was assessed using a phase contrast microscope. Viability and acrosomal integrity were evaluated using a dual stain technique (Giemsa–Trypan blue). The results demonstrated that the number of live canine spermatozoa was similar in culture media supplemented or not supplemented with macromolecules. A minimal concentration of albumin (1%) in the three media showed similar effects on vitality, motility and acrosomal integrity, as had higher concentrations (4 and 6%). The percentage of acrosome‐intact spermatozoa was markedly higher after HTF (94.1%) than after TCM‐199 (70.1%) or Sp‐TALP (71.0%) without supplementation. It is concluded that serum bovine albumin, irrespective of the concentration, preserved sperm viability and function, and HTF is the most suitable medium for preserving the acrosome in canine spermatozoa prepared for in vitro manipulation through short incubation.  相似文献   

6.
A study was conducted to determine the optimum dosage of the exogenous cholesterol-loaded cyclodextrins (CLC) to get maximum cryoprotection for bubaline spermatozoa. In the present study, 120 × 106 spermatozoa were incubated in 2, 3 and 4 mg of CLC as grouped as Gr II, III and IV, respectively, and sperm progressive motility, intracellular Ca2+, capacitation status by protein tyrosine phosphorylation (PTP) assay and zona binding per cent (ZBP) and cleavage rate (CR) of the cryopreserved buffalo spermatozoa by in vitro fertility assay were assessed in comparison with an untreated control group (Gr I). Results revealed that there was a significant (p < .05) linear decrease in percentage of sperm population with higher intracellular Ca2+ and percentage of sperm population with medium or high capacitated by PTP in CLC treated from 2 to 3 mg and then increased to 4 mg/120 × 106 spermatozoa whereas sperm progressive motility, percentage of sperm population with low capacitated, ZBP and CR were increased significantly (p < .05) in sperm population treated from 2 to 3 mg CLC and then decreased to 4 mg/120 × 106 spermatozoa. The study has clearly indicated that CLC at 3 mg/120 × 106 spermatozoa has maximum beneficial effects in protection of sperm progressive motility, membrane fluidity (low intracellular Ca2+); prevention of cryocapacitation (low capacitation pattern in immunolocalization) and enhancement of in vitro ZBP and CR. Post-thaw motility of the CLC-treated sperm has shown positively significant (p < .05) correlation with sperm population with low intracellular Ca2+, low capacitated sperm population, ZBP and CR, whereas it was negatively (p < .05) correlated with sperm population with high intracellular Ca2+, medium or high capacitated sperm. The present study has revealed for the first time that incubation of spermatozoa with CLC of higher dose (>3 mg/120 × 106 spermatozoa) had adverse effects on sperm cryopreservation, although incubation of sperm with 3 mg/120 million prior to processing had minimised the freezing–thawing-associated damages in bubaline species.  相似文献   

7.
Frozen-thawed sexed semen from six bulls (Holstein) was used for studying their efficiency in an in vitro fertilization (IVF)-programme and to compare their ultrastructure with in vitro produced bovine blastocysts produced with non-sorted sperm. Progressive motility of sorted spermatozoa, their IVF rate, development of produced blastocysts and the ultrastructure of the blastocysts were analysed. The cleavage rates of sexed sperm of bulls (groups S1, S2 and S4) were significantly lower than that of unsorted control sperm (P < 0.01). Blastocyst development at day 7 of the sexed semen groups varied between 3.5% and 28.8% versus 33.6% for non-sexed semen. The individual blastocyst yield with sexed semen of group S5 (28.8%) was similar to the mean blastocyst production of the non-sexed control spermatozoa (C, 33.6%; P > 0.05). The remaining five sexed sperm groups resulted in significantly lower developmental rates of blastocysts on day 7 (S1, 4.9%; S2, 0%; S3, 0%, S4, 3.5%; S6, 25.8%, P < 0.01). Group S2 showed microbiological contamination in 50% (four of eight) and S3 in 100% of the experiments (eight of eight). Progressive motility of sexed sperm was significantly lower than that of unsorted sperm (S1, 48 +/- 12.0%; S2, 41 +/- 11.9%; S3, 39.0 +/- 9.9%; S4, 42 +/- 4.6%; P < 0.01; S5, 72 +/- 7.1% and S6, 64 +/- 9.3; P < 0.05 versus C 82 +/- 4.6%). The percentage of progressive motile spermatozoa showed a good correlation with the developmental capacity of blastocysts (r(2): >0.70), the regression parameter was significant (P < 0.01). Furthermore, with a straw containing 10 x 10(6) sexed spermatozoa significantly lower number oocytes was fertilized than with the same concentration of non-sexed sperm (P < 0.01). Our results demonstrate that the suitability of sperm sorting for in vitro fertilization (IVF) is lower than no sexed sperm. Our ultrastructural studies showed that blastocysts produced with flow-cytometrically sex-sorted spermatozoa possessed deviations in the number and structure of organelles like mitochondria, rough endoplasmic reticulum (ER) and nuclear envelope. These morphological alterations may be responsible for compromised development that observed in embryos produced with sex-sorted spermatozoa. Thus, we conclude that sperm sex sorting can markedly affect the efficiency of an IVF-programme.  相似文献   

8.
The present study aimed to establish an efficient system for bovine embryo production by in vitro fertilization (IVF) that can achieve stable normal fertilization and blastocyst developmental rates in any bull without optimization of the sperm concentration in IVF medium. We examined the effects of a PHE mixture (20 μM D-penicillamine, 10 μM hypotaurine and 1 μM epinephrine), theophylline (2.5 mM), and sperm concentration (1, 2 or 5 × 106 cells/ml) on fertilization and blastocyst developmental rates. High cleavage rates (78.3 to 92.4%) and blastocyst developmental rates (31.9 to 62.0%) at day 7 were obtained in the presence of PHE and theophylline in IVF medium with a sperm concentration of 2 × 106 cells/ml using sperm from 9 bulls. In addition, the synergistic effect of PHE and theophylline on normal fertilization (2 pronuclei) was clarified at 12 h after IVF with a sperm concentration of 1 × 106 cells/ml. Moreover, high linearity, high flagellar beat cross frequency, and low amplitude of lateral head of motile sperm were found by computer-assisted sperm analysis. In conclusion, the combination of the PHE mixture and theophylline synergistically accelerates sperm motility and sperm penetration of bovine oocytes. Theophylline activates sperm motility with increasing intracellular cAMP. However, PHE prevents an excessive increase of cAMP and maintains sperm motility without hyperactivation. When the combination of PHE and theophylline is added to IVF medium at a sperm concentration of 2 × 106 cells/ml, we can achieve stable normal fertilization and blastocyst development in any bull.  相似文献   

9.
Despite normal eucrasia, mating desire and semen quality, sire bulls sometimes have spermatozoa with poor freezing tolerance. This study assessed effects of the addition of linoleic acid albumin (LAA) and long-term (LT) equilibrium to frozen semen on their sperm freezing tolerance. Immediately after collection using an artificial vagina and a breeding mount, semen was diluted with yolk citrate buffer; then, it was cooled slowly to 4°C during more than 5 h. Equilibrium treatment at 4°C was applied using the same extender supplemented with glycerol. Semen of bull A, with low sperm freezing tolerance, was treated with 1 mg/ml of LAA added to the first extender. The equilibrium treatment at 4°C was prolonged to 30 h. Significantly higher motility rates were obtained for the LT + LAA-treated sperm before and after freezing-thawing. However, for semen of bulls B and C with normal sperm freezing tolerance, the LT + LAA treatment barely exhibited a small effect on the motility rate. Almost no difference was found among bulls A, B and C in the motility rates of LT + LAA-treated sperm after freezing-thawing. No difference of fertility was apparent on LT + LAA-treated frozen sperm in comparison with normal sperm in embryonic collection and in vitro fertilization. It was not an aberration of fertility in vivo or in vitro. In addition, the conception rate of artificial insemination did not have a difference, and a normal calf was obtained. Results show that addition of LAA to an extender for frozen bovine spermatozoa and 30 h of low-temperature equilibrium might improve the motility of freezing-thawing spermatozoa with poor freezability. Sperm exhibited normal fertilization capability and ontogenic capability.  相似文献   

10.
Evaluation of glucose as a cryoprotectant for boar semen   总被引：2，自引：0，他引：2  
Fertility parameters of boar spermatozoa were evaluated in vitro, after freeze-thawing the semen in three different extenders containing permeable and non-permeable cryoprotectants: A (111.0 mM Tris, 31.4 mM citric acid, 185.0 mM glucose, 20 per cent egg yolk, 3 per cent glycerol and 100 iu/ml penicillin G); B (200 mM Tris; 70.8 mM citric acid, 55.5 mM glucose, 20 per cent egg yolk, three per cent glycerol and 100 iu/ml penicillin G); C (200 mM Tris, 70.8 mM citric acid, 55.5 mM fructose, 20 per cent egg yolk, 3 per cent glycerol and 100 iu/ml penicillin G). The freeze-thawing techniques were the same for each extender. Eight ejaculates from four boars were obtained; the sperm-rich fraction of each ejaculate was extended in each of the three media at a final concentration of 400 x 106 sperm/ml, loaded into 0.5 ml straws and frozen at a rate of 30 degrees C/minute to -196 degrees C. The straws were thawed at 60 degrees C for eight seconds. Sperm motility, acrosomal integrity and in vitro sperm penetration through the zona pellucida of gilt oocytes matured in vitro were evaluated. The motility of unfrozen spermatozoa was 93.1 per cent compared with 60.7 per cent, 48.2 per cent and 35 per cent for sperm frozen in extenders A, B and C respectively; these values were all significantly different (P<0.05). There was no significant decline in sperm motility after incubation for 30 minutes in extender A, but there were significant decreases in sperm motility after 30 minutes of incubation in B and C. The percentage acrosomal integrities were 97.2 per cent for the control and 45.5 per cent, 30.3 per cent and 16.8 per cent for the frozen-thawed spermatozoa in extenders A, B and C respectively. The results of the in vitro penetration assay were 80.7 per cent when using control spermatozoa, and 42.2 per cent, 18.4 per cent and 3.3 per cent when using frozen-thawed spermatozoa in extenders A, B and C respectively  相似文献   

11.
The aim of the present study was to improve the penetration during in vitro fertilization (IVF) of a frozen lot of epididymal sperm with a notoriously low fertilization ability of a Ban boar which is a native Vietnamese breed by optimizing different parameters of the IVF system. In Experiment 1, we determined that Pig‐fertilization medium was superior medium to Tyrode's albumin lactate pyruvate‐polyvinyl alcohol medium for IVF and defined the optimum the sperm concentration (1 × 106 sperm/ml). In Experiment 2, we clarified that partial removal of cumulus cells from cumulus‐oocyte complexes by hyaluronidase treatment before IVF enhances sperm penetration, whereas complete cumulus removal reduces penetration. Finally, in Experiment 3 the elevation of concentration of caffeine in Pig‐fertilization medium from 2 to 5 mmol/L and the prolongation of the co‐culture of gametes from 3 to 5 hr significantly increased the total penetration rate from 15.2% to over 50%. In conclusion, the combination of partial oocyte denudation, an elevated caffeine concentration in Pig‐fertilization medium and an extended interval of IVF with using an optimized sperm concentration was a potent way to improve the fertilization results for a frozen epididymal Ban sperm lot with low fertility.  相似文献   

12.
The purpose of this study was to evaluate the ability of various chemicals to induce capacitation of stallion spermatozoa using 2 different assay systems. In Experiment 1, freshly ejaculated spermatozoa were treated for 0, 3 and 6 h with 10 μ g/ml heparin, 0.5 mM hypotaurine or 5 mM caffeine, or were incubated for 0, 3 and 6 h following 1 min exposure to 0.1 μ M ionophore A23187. The acrosome reaction (AR) in the capacitated spermatozoa was induced by 15 min challenge with 100 μ g/ml lysophosphatidylcholine (LPC). In the BO/BSA-control medium (Brackett and Oliphant medium with 0.3% BSA), mean percentage of AR spermatozoa at 0 h was 30%, and the AR rates increased to 40 and 48% after 3 and 6 h incubation, respectively. There was no significant further increase of the AR rates in the spermatozoa treated with heparin (50% at 6 h) and hypotaurine (58% at 6 h) when compared to the control. Caffeine had a beneficial effect on inducing sperm capacitation after 3 and 6 h incubation (AR rates; 61 and 66%, respectively, P<0.01). Immediately after ionophore A23187 treatment, the AR rate increased to 56%, and reached 68 and 67% after 3 and 6 h incubation, respectively (P<0.01). Spermatozoal motility at any time points did not differ between control and any chemical treatment groups, except one treatment (ionophore; 3 h group).In Experiment 2, frozen-thawed spermatozoa were treated with 4 different chemicals as described above. Aliquot of spermatozoa was added to a microdrop of BO/BSA medium in which 6 to 10 in vitro-matured, zona-free mare oocytes were placed, and the oocytes were fixed and stained 20 h after insemination. The penetration rate by BO/BSA-treated spermatozoa was 76%, which was comparable to the results with heparin (73%), hypotaurine (78%) and caffeine (58%). In contrast, treatment of spermatozoa with ionophore A23187 gave a significantly lower penetration rate (30%) than the control value. Surprisingly these two experiments had different conclusions in assessing capacitation of stallion spermatozoa.  相似文献   

13.
Egg yolk is a common component to sperm refrigeration for most of the deer species, the role of which is to protect sperm membranes against cold shock. In addition, there have been many studies of conservation of ejaculated semen from stags, but few have been reported for epididymal spermatozoa. This work was designed to investigate the combined effects of cooling rates (slow: 0.23 degrees C/min vs rapid: 4.2 degrees C/min) from room temperature to 5 degrees C, and egg-yolk concentration (0, 5 or 20%) in the extender on the survival of Iberian red deer epididymal spermatozoa refrigerated at 5 degrees C. Heterospermic sperm samples were diluted to a final sperm concentration approximately 400x10(6) sperm/ml with a Tris-citrate-fructose (TCF)-egg-yolk diluent. Sperm quality was in vitro judged by microscopic assessments of individual sperm motility [sperm motility index (SMI)], and of plasma membrane (hypo-osmotic swelling test) and acrosome (NAR) integrities. Our results first showed that the presence of egg yolk in the extender significantly improves (p=0.01) the viability and sperm motility after sperm dilution. In addition, acrosome and plasma membrane integrities post-refrigeration did not differ significantly between cooling procedures; however, the SMI differed significantly between cooling procedures (slow: 46.6% vs rapid: 50.0%; p=0.01). Our results also showed that sperm quality was significantly (p<0.01) affected by the combined effects of egg-yolk concentration and cooling procedure, being rapid cooling with 20% of egg yolk the most suitable combination for epididymal sperm refrigeration. In conclusion, egg-yolk improved red deer epididymal spermatozoa characteristics after dilution. Rapid cooling protocol using TCF with 20% egg-yolk significantly improved sperm motility of red deer epididymal spermatozoa after cooling.  相似文献   

14.
In the present study, we investigated the effect of various carbohydrates on the ability of bovine spermatozoa to bind to the bovine oviduct epithelial cells (OECs). We also examined the fertilization competence and motility of spermatozoa that bind to OECs in the presence of carbohydrates. Frozen-thawed spermatozoa were incubated with OECs, with and without various carbohydrates. The sperms were then divided into two fractions: OEC-binding sperms (B-sperm) and non-OEC binding sperms (NB-sperm). The fertilization rate, ability to bind the zona pellucida, and membrane integrity of the spermatozoa as determined using a hypo-osmotic-swelling test (HOST) were lower in NB-sperm than in the unseparated spermatozoa (control). The motility of the B-sperm was maintained for a longer time than that of the control spermatozoa. The addition of N -acetyl- d -glucosamine (GlcNAc, 5 m m ) to the sperm-OEC mixture increased the number of B-sperm. D -mannose (5 m m ) and D -fucose (5 m m ) had no effect on the number of B-sperm. The motility of B-sperm, which bound to OECs in the presence of GlcNAc, however, was not maintained. When either OECs or the spermatozoa were treated with GlcNAc prior to sperm-OEC co-incubation, only sperm-side treatment enhanced sperm-OEC binding, but B-sperm motility was not maintained. The motility of spermatozoa incubated with GlcNAc was lower than that of controls. These results indicate that GlcNAc enhances sperm binding to OECs, probably via sperm surface modification, but does not promote increased sperm survival.  相似文献   

15.
Porcine relaxin is a peptide hormone belonging to the insulin super family that has a variety of biological functions. The present experiment was designed to investigate the effects of relaxin on sperm function and on in vitro fertilization (IVF) of porcine oocytes. Porcine spermatozoa were washed, swum-up, and incubated for 1-4 h in mTALP medium supplemented with 0, 20 or 50 ng/ml porcine relaxin. Motility was determined by observing the type of forward movement of the spermatozoa, and acrosome status was evaluated by applying the triple staining technique. Immature oocytes were aspirated from antral follicles and matured in IVM medium (modified NCSU-37). Matured oocytes were co-cultured with spermatozoa in IVF medium (mTALP) supplemented with 0, 5, 10, 15 or 20 ng/ml relaxin. After 6 h of sperm-oocyte co-incubation, putative zygotes were cultured for 18 h in oocyte culture medium NCSU-37 and then assessed for the rates of monospermy, polyspermy, and male pronucleus formation after acetic orcein staining. Relaxin improved (P<0.05) sperm motility and increased the percentage of acrosome-reacted live spermatozoa during 1-4 h of incubation, although viability was not significantly improved. Significantly (P<0.05) the highest percentage of monospermic (31.7%) and lowest percentage of polyspermic (16.5%) fertilization was achieved from the sperm-oocyte co-culture group treated with 20 ng/ml relaxin as compared to other groups. The percentage of male pronucleus formation was significantly (P<0.05) greater in the 20 ng/ml relaxin-treated sperm-oocyte co-culture group than in the other groups. These results indicate that supplementation with relaxin is capable of improving sperm function and fertilization of porcine oocytes in vitro.  相似文献   

16.
海藻糖对猪精液冷冻保存效果的影响   总被引：8，自引：0，他引：8  
在传统的Tris-柠檬酸-葡萄糖稀释液基础上，分别添加25％、50％、75％、100％的海藻糖，研究不同浓度海藻糖对猪精液冷冻后精子质量的影响。结果表明，海藻糖相对于对照TCG稀释液能够显著改善和提高猪精液的冷冻效果，其最佳添加浓度为25％，冷冻-解冻后猪精子活力、活率、线粒体活性、质膜完整性以及顶体完整率均显著提高（P〈0．05），分别达到41．38％、46．34％、44．56％、43．51％和64．09％。海藻糖可以明显抑制精子获能，获能处理前精子获能率仅为3．68％，而获能处理后达到41．82％，有利于促进精子获能。精液稀释液中甘油的适宜添加浓度为2％，海藻糖只有与甘油共同作用，才能在冷冻-解冻过程更加有效地保护精子。猪精子活力、活率、线粒体活性、质膜完整率、顶体完整率等之间存在极显著的正相关关系（P〈0．01），而与获能处理前精子的获能率存在显著的负相关关系（P〈0．05）。  相似文献   

17.
The aim of this research was to study the effect of hyaluronic acid on bovine cryopreserved spermatozoa compared with heparin as regards the variation of capacitation induction, cellular oxidative metabolism and intracellular signal induced by membrane‐associated adenylate cyclase to propose hyaluronic acid as a capacitation inductor. Heparin or hyaluronic acid and lysophosphatidylcholine were used to induce sperm capacitation and acrosome reaction, respectively. 2′,5′‐dideoxyadenosine was used as a membrane‐associated adenylate cyclase inhibitor. The highest percentages of capacitated spermatozoa and live spermatozoa with acrosome integrity were obtained by incubating sperm for 60 min using 1000 μg/ml hyaluronic acid. In these conditions, capacitation induced by hyaluronic acid was lower compared with heparin; nonetheless both glycosaminoglycans promote intracellular changes that allow true acrosome reaction in vitro induced by lysophosphatidylcholine in bovine spermatozoa. Oxygen consumption in heparin‐capacitated spermatozoa was significantly higher than in hyaluronic acid‐treated spermatozoa. With all treatments, mitochondrial coupling was observed when a specific uncoupler of the respiratory chain was added. The inhibition of membrane‐associated adenylate cyclase significantly blocked capacitation induction produced by hyaluronic acid, maintaining a basal sperm oxygen uptake in contrast to heparin effect in which both sperm parameters were inhibited, suggesting that the membrane‐associated adenylate cyclase activation is involved in the intracellular signal mechanisms induced by both capacitation inductors, but only regulates mitochondrial oxidative phosphorylation in heparin‐capacitated spermatozoa.  相似文献   

18.
The effect of Mycoplasma bovis (Donetta strain) on the ability of bull spermatozoa to interact with zona pellucida-free hamster oocytes was studied in an in vitro assay. Ejaculates of semen from a fertile Holstein bull were used fresh on the day of collection (unextended semen) as well as diluted with egg yolk-citrate and used the following day (extended semen). The addition of M. bovis to both unextended and extended semen at a mycoplasma to sperm cell ratio of 10:1 significantly reduced sperm penetration rates and the mean number of sperm per penetrated egg. Similarly, the ability of spermatozoa to form pronuclei and the activation of penetrated oocytes were adversely affected by M. bovis. No apparent effect on sperm motility was detected. When M. bovis was added to the oocytes, there was a marked reduction in the sperm penetration rates and fertilization processes suggesting that the organism affects certain oocyte function(s). The results indicate that the presence of M. bovis in semen or in the female reproductive tract may affect fertilization. Moreover, the in vitro assay with hamster oocytes was found to be useful for demonstrating the effects of contaminating microbial agents on bovine fertilization processes.  相似文献   

19.
Stallion spermatozoa maintain high fertilizing capacity if cooled to 5 degrees C and inseminated within 24 h. However, if spermatozoa are stored for 48 h, fertilizing capacity declines. Therefore, multiple shipments of semen are often required to inseminate mares that remain in estrus for days. Therefore, experiments were designed to determine if adding antioxidants to stallion spermatozoa stored at 5 degrees C for 48 h could maintain motility and fertilizing ability. In the first experiment stallion spermatozoa were incubated in a skim milk (SM) or a skim milk-egg yolk medium in combination with 10 mM pyruvate, 5 mM xanthurenic acid separately or in combination for up to 48 h at 5 degrees C. Spermatozoa incubated in SM for 48 h exhibited higher percentages of motile sperm (57%) than did sperm incubated in skim milk-egg yolk (34%); antioxidant treatment had little effect. In the second experiment, spermatozoa were incubated in SM containing 0, 1, 2, or 5 mM pyruvate. After 24 h of incubation at 5 degrees C, sperm incubated with 1, 2, or 5 mM pyruvate exhibited higher percentages of progressively motile spermatozoa (45%) than control exhibited (26%; P < 0.05). After 48 h, percentages of progressively motile spermatozoa were similar (27, 19, and 30 vs 14, respectively; P > 0.05). However, when incubated at 5 degrees C for 48 h and then incubated an additional 4 h at 25 degrees C, samples containing pyruvate exhibited higher percentages of motile (63 to 80%) and progressively motile (36 to 42%) sperm than did sperm in SM alone (28 and 5%, respectively; P < 0.05). The third experiment attempted to determine the optimal pyruvate concentration to maintain spermatozoal motility. Spermatozoa incubated with 0, 2, 3.5, or 5 mM pyruvate for 48 h at 5 degrees C and then an additional 4 h at 25 degrees C, exhibited similar percentages of progressively motile cells (31, 35, and 28%, respectively) that were higher than control (11%, P < 0.05). The last experiment evaluated the fertilizing potential of cooled spermatozoa. Embryos were recovered from 35, 20, and 30% of mares inseminated with spermatozoa that had been incubated at 5 degrees C, for 24 h in SM, or for 48 h in SM or SM + 2 mM pyruvate, respectively (P > 0.05). These studies indicate that 2 mM pyruvate in SM was beneficial in maintaining spermatozoal motility in 48 h-stored sperm and, although not significant, seemed to help maintain the fertility of 48 h-cooled spermatozoa.  相似文献   

20.
Bull spermatozoa samples contain variable portion of motile and normal morphology spermatozoa along with spermatozoa incapable of fertilization due to their pathologic changes. As semen quality is influenced by biochemical and morphological characteristics of all spermatozoa, the aim of the study was to separate spermatozoa in discontinuous iodixanol density gradient solution and to determine their cholesterol, phospholipid, triacylglycerol and lipid peroxide concentrations and creatine kinase activity. The study was performed in winter and included seven Simmental bulls aged 1.5-3.5 years. Semen samples were collected by use of artificial vagina. Upon evaluation of semen quality (volume, concentration and progressive sperm motility), the samples were centrifuged in iodixanol density solution to obtain two sperm fractions. The two fractions included sperms with progressive motility greater than 90% and less than 20%, respectively. A statistically significantly higher lipid peroxide concentration was determined in sperm fraction with <20% progressive motility. Different sperm subpopulations can be obtained by separating bull spermatozoa in different iodixanol density gradient solutions, while monitoring their biochemical properties can help assess the sperm quality.  相似文献   

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