发情羊血清、肝素和BSA对绵羊冷冻-解冻精子体外获能的影响

分析了在受精液SOF中添加发情绵羊血清、肝素和牛血清白蛋白(BSA)对绵羊冷冻-解冻精液体外获能和受精的影响。结果表明:(1)使用含发情羊血清浓度为0、5%、10%和20%的受精液,分裂率分别是0、73.79%、73.25%和77.61%。加发情羊血清的3个试验组分裂率差异不显著。而未加血清的试验组分裂率为0,证明发情羊血清促进了绵羊冷冻-解冻精液的体外受精效果。(2)以含5%发情羊血清受精液为基础液,添加0IU/mL、5IU/mL和10IU/mL的肝素,受精后分裂率分别达86.64%、86.63%和75.53%。不加肝素组和5IU/mL肝素组分裂率差异不显著,而10IU/mL高浓度肝素组同其他两组比较,差异极显著(p<0.01)。说明含10IU/mL肝素的受精液,降低了绵羊体外受精后的分裂率。(3)用含5%发情羊血清受精液为基础液,以添加3mg/mLBSA为试验组,不加BSA为对照组,受精后两组分裂率差异不显著。发情绵羊的血清促进绵羊冷冻-解冻精子的体外受精效果。在受精液中添加发情绵羊血清进行绵羊冷冻解冻精子的体外受精,无需添加肝素和BSA。而且添加10IU/ml肝素,降低了受精后的分裂率。建议受精液中添加发情绵羊血清浓度在2%～10%较合适。     

不同浓度肝素对塔里木马鹿精子体外获能效果的影响

为了探讨不同浓度肝素对塔里木马鹿精子体外获能的影响,试验随机取塔里木马鹿冻融精子,添加到SP-TALIP获能液中并添加不同浓度(0,10,20,50,100μg/m L)的肝素,在显微镜下检测0,2,4,6,8小时时的精子活力,试验采用金霉素(CTC)染色法检测精子获能率及顶体反应率等指标,探讨不同浓度肝素对塔里木马鹿精子活力、存活时间、获能率、顶体反应率的影响。结果表明:精子活力随肝素浓度升高而呈下降趋势,4 h后10μg/m L和20μg/m L肝素组精子活力显著高于其他组(P0.05),其中20μg/m L肝素组精子存活时间最长,显著高于其他组(P0.05)。随着培养时间的延长,各试验组精子获能率与对照组相比明显提高,2小时、4小时时20μg/m L肝素组精子获能率显著高于其他组(P0.05);各试验组精子顶体反应率与对照组相比有升高趋势。说明获能液中添加20μg/m L肝素可有效诱导塔里木马鹿精子体外获能。     

延边黄牛精子顶体反应3种检测方法的比较

试验以延边黄牛冷冻精子为材料,用肝素诱导精子体外获能后分别用考马斯亮蓝水溶液、考马斯亮蓝高氯、台盼兰-姬姆萨3种不同方法进行染色,并同时做延边黄牛卵母细胞穿卵试验,以求证精子顶体反应与体外受精率的相关性。     

间情期犬间母细胞体外受精的影响因素

试验对间情期犬卵母细胞体外受精的影响因素进行了系统研究。结果表明①以TALP液作为犬精子获能液，精子回收率(64±10.5)%、顶体反应率(41.5±4.1)%高于TCM199+20%FCS液。②用TCM199+20%FCS作为受精液，卵母细胞单精受精率(18.6%)、原核形成率(10.4%)，高于TALP受精液，而退化率低于TALP受精液(4.9%比19.7%，P＜0.01)。③犬精子与卵母细胞在受精液中共同孵育12h，单精受精率(23.5%)、原核形成率(12.7%)高于共同孵育8、16、20h，而多精受精率(15.7%)、退化率(3.9%)低于共同孵育16、20h。④犬精子为2×106个/mL时，单精受精率(26.0%)、原核形成率(14.6%)，高于0.5×106、1×106、3×106、5×106个/mL组。卵母细胞多精受精率和退化率随精子含量增加而增加。⑤肝素为29mg/L时,卵母细胞单精受精率(24.4%)、原核形成率(12.2%)高于0、10、20、40、50mg/L组。而犬精子获能2h后，顶体反应率和卵母细胞多精受精率随肝素质量浓度增加而增加。     

猪卵母细胞体外受精条件的优化

猪卵母细胞体外受精(in vitro fertilization,IVF)技术为受精和胚胎早期发育机理等基础理论的研究及生产实践提供了重要的研究手段。虽然猪IVF的研究已经取得了一些进展,但其囊胚发育率仍然较低,亟需建立更为稳定、高效的体外受精方法。本试验通过比较精子浓度,精子获能处理,不同精卵共孵育培养体系以及在卵母细胞成熟过程中添加不同激素等影响猪卵母细胞体外受精胚胎发育能力的因素,以求找到最佳的猪卵母细胞体外受精体系。结果显示:在精子浓度为1×10~5~1×10~7/mL,5×10~6/mL的精子浓度显著提高体外受精效率(P<0.05);以茶碱、咖啡及咖啡因联合使用肝素作为获能物质处理精子,发现2.5 mmol/L茶碱处理组体外受精效率显著提高(P<0.05);通过比较不同的受精体系,发现使用40个卵/500μL体系显著提高体外受精效率(P<0.05);在卵母细胞成熟液中添加不同激素组合,发现添加10IU/mL PMSG,10IU/mL HCG和2.5IU/mL FSH激素组合,体外受精效率显著提高(P<0.05)。本试验结果表明,在M199中添加10IU/mL PMSG,10IU/mL HCG和2.5IU/mL FSH体外成熟培养卵母细胞,以5×10~6/mL精子浓度,2.5mmol/L茶碱作为获能物质处理精子,40个卵/500μL共孵育的IVF体系效果最佳,其卵裂率为(61.33±0.77)%,囊胚率为(28.33±1.08)%。本试验将为深入研究猪IVF提供理论参考。     

山羊卵母细胞体外受精方法的研究

优化山羊体外受精条件,为提高体外成熟卵母细胞的受精质量和数量提供理论基础。对肝素、咖啡因、钙离子载体IA三种获能物比较,发现肝素和钙离子载体组受精率分别为56.0%和67.0%,显著高于咖啡因组21.0%(P<0.05)。卵裂率反而是肝素高于钙离子载体和咖啡因组,分别为51.1%、36.3%和19.0%,差异显著(P<0.05);发育到囊胚后,分别为17.8%、11.7%和0,肝素组与钙离子载体组差异不显著。结果表明肝素效果比较稳定。Percoll法处理精子卵裂率高于上浮法,差异显著(59.1%vs42.9%,P<0.05);但是囊胚的发育率分别为11.8%和7.8%,囊胚的平均细胞数分别为116和113,两者差异不显著。     

波尔山羊精子体外获能及穿卵效果的研究

通过不同培养液对精子获能效果、获能后穿卵效果的比较,确定培养液的适宜配方及获能液中肝素的适宜添加浓度。精子用含有肝素的3种培养液进行获能处理,每个处理4个重复。试验设计了5个不同的肝素水平,即0、5、10、15、20μg/mL。于获能处理后4 h检查精子的顶体反应率、活力和活精子率。结果表明,添加15μg/mL和20μg/mL肝素能促进精子的获能效果和获能精子的穿卵效果。     

肝素对体外受精牛卵母细胞发育的影响

肝素是一种氨基葡聚糖,对牛射出体外的精液有诱发精子顶体反应的作用。据 Sei-koh kurosaka 等报道,肝素分别以0.18,45,90μg/ml 加到精子悬浮液中,并保持存活5h,每组精子悬浮液受精卵的分裂率分别为22.0,62.0,56.8,57.0%;肝素以0,18,45,90μg/ml 加到含有体外受精的卵     

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、7.5mmol/L)或亚牛磺酸(0、5、10、20、40μmol/L)的获能处理液中,且每个处理组加入约200μL的精液,在CO_2培养箱里经上游处理45min,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、5.0mmol/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(P0.05),且2.5mmol/L咖啡因组精子活力最高;添加10、20μmol/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(P0.05),且20μmol/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20μmol/L亚牛磺酸采用同样的方法处理,发现20μmol/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2.5mmol/L咖啡因组和对照组(P0.05)。因此,20μmol/L亚牛磺酸可以替代2.5mmol/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。     

精子不同筛选处理方法对体外受精效果的影响

为了比较体外受精所用精子的不同筛选及获能处理方法对肉羊体外受精效果的影响,试验采用离心法和上浮法处理精子。结果表明:试验1组、试验2组中,鲜精组受精率分别为(69.6±4.5)%、(68.3±1.7)%,冻精组受精率分别为(68.4±1.9)%、(67.5±2.1)%,均显著高于对照组鲜精组、冻精组受精率[(35.1±1.6)%、(28.3±2.4)%](P0.05)。试验1组、试验2组中,鲜精组卵裂率分别为(65.6±3.5)%、(64.8±4.2)%,冻精组卵裂率分别为(63.6±4.1)%、(62.9±2.8)%,均显著高于对照组鲜精组、冻精组卵裂率[(33.7±4.1)%、(30.1±3.4)%](P0.05)。试验1组、试验2组中,鲜精组囊胚率分别为(36.8±1.6)%、(32.7±3.3)%,冻精组囊胚率分别为(36.1±3.6)%、(31.1±4.2)%,均显著高于对照组鲜精组、冻精组囊胚率[(14.3±2.5)%、(11.6±4.3)%](P0.05)。精子获能诱导剂肝素组体外受精效果显著好于咖啡因组。     

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.     

不同收集方法及添加半胱氨酸、肝素钠对黄牛卵母细胞体外成熟及发育的影响

试验旨在研究不同卵母细胞收集方法及添加半胱氨酸、肝素钠对黄牛卵母细胞体外成熟及体外受精的影响。以黄牛为研究对象,采用两种方法（抽卵法和割卵法）抽取卵泡中的卵母细胞,比较两种方法获取的卵母细胞成熟率,结果发现,抽卵法获得的卵母细胞成熟率显著高于割卵法（P<0.05）。将获取的卵母细胞分为4组：A组（对照组,只添加基础成熟培养液）、B组（基础成熟培养液+200 μmol/L半胱氨酸）、C组（基础成熟培养液+20 μg/mL肝素钠）、D组（基础成熟培养液+200 μmol/L半胱氨酸+20 μg/mL肝素钠）,结果发现,D组的卵母细胞成熟率显著高于A、B、C组（P<0.05）,B、C组间卵母细胞成熟率无显著差异（P>0.05）,但两组卵母细胞成熟率均显著高于A组（P<0.05）;A组卵母细胞卵裂率均显著低于B、C、D组（P<0.05）,B、C、D组间卵母细胞卵裂率无显著差异（P>0.05）;D组囊胚率显著高于其他3组（P<0.05）。结果表明,抽卵法获得卵母细胞效率显著高于割卵法,肝素钠及半胱氨酸对黄牛卵母细胞体外成熟和体外受精都有促进作用,且同时添加两种物质对体外成熟的效果更佳。     

In vitro capacitation of stallion spermatozoa assessed by the lysophosphatidylcholine-induced acrosome reaction and the penetration rate into in vitro-matured, zona-free mare oocytes

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.     

Effects of bovine serum albumin and trehalose in semen diluents for improvement of frozen-thawed ram spermatozoa

In this study, two following experiments were performed to improve post-thaw motility and viability of frozen-thawed ram spermatozoa. We examined i) the effects of different concentrations of bovine serum albumin (0, 0.3, 1, 5, 10 and 15% BSA) in semen diluents lacking egg yolk and ii) the effects of four semen diluents, fructose (F: control) and trehalose (T) in semen diluents containing egg yolk, 15% BSA in semen diluents without egg yolk (BSA), and modified phosphate buffered saline (m-PBS). Frozen-thawed spermatozoa were examined for progressive sperm motility, viability, morphological abnormality, sperm tail swelling test, and sperm acrosome integrity. In Experiment 1, the rates of sperm motility immediately after thawing (0 h) were significantly (P<0.05) higher in the 10 and 15% BSA groups (55.0 +/- 2.9 and 58.3 +/- 6.7%, respectively) than in the positive control (F) group (41.7 +/- 4.4%). The rate of sperm viability in the negative control (0% BSA) group (80.2 +/- 3.3%) was significantly (P<0.05) lower than in the positive control (F) group (89.8 +/- 1.5%), but when compared with the F group, no significant differences were found among the 0.3, 1, 5, 10 and 15% BSA groups at 0 h. The rates of sperm morphological abnormality of the 10 and 15% BSA groups (6.5 +/- 1.3 and 6.3 +/- 1.1%, respectively) were significantly (P<0.05) lower at 0 h than that in the 1% BSA group (16.3 +/- 5.2%). In Experiment 2, T addition improved (P<0.05) the post-thaw motility compared with the F and BSA groups. Furthermore, at 3 and 6 h, the post-thaw motility of the T group (36.3 +/- 2.4 and 25.0 +/- 2.0%, respectively) was significantly (P<0.05) higher than in the BSA (26.3 +/- 2.4 and 18.8 +/- 1.3%, respectively) and F (28.8 +/- 3.8 and 18.8 +/- 2.4%, respectively) groups. The post-thaw sperm motility and viability in the m-PBS group were significantly (P<0.05) lower than those of the control (F), T, and BSA groups throughout all observation points. These results indicate that 10 and 15% BSA can be substituted for egg-yolk for ram semen diluent and that the addition of trehalose enhances motility and viability of ram spermatozoa after freezing and thawing.     

Effect of relaxin on acrosome reaction and utilization of glucose in boar spermatozoa

Relaxin is a peptide hormone found in seminal plasma that has a physiological influence on sperm motility in some species. There are no reports on the effect of relaxin on acrosome reaction and utilization of glucose in boar spermatozoa. In this study, to investigate the effects of relaxin on sperm motility, acrosome reaction, and incorporation and oxidation of labeled glucose, boar spermatozoa were washed and preincubated for swim-up and then incubated (0-6 h) with 0, 20, or 40 ng/ml relaxin in mTALP medium. The results indicated that the addition of relaxin stimulated sperm motility significantly (P<0.05) during 1-4 h of incubation. The percentage of acrosome-reacted live spermatozoa was higher (P<0.05) when the spermatozoa were treated with 20 or 40 ng/ml relaxin. The rate of incorporation, and oxidation of glucose were also greater (P<0.05) in the spermatozoa incubated with relaxin compared to the control spermatozoa. The rate of incorporation and oxidation of (14)C-glucose were increased in correlation with acrosome reaction up to 4 h of incubation and then decreased in line with the increasing incubation period. In conclusion, the present study demonstrates that relaxin accelerates not only motility but also the acrosome reaction and utilization of glucose in boar spermatozoa.     

用台盼兰—姬姆萨染色检测家畜精子顶导反应的研究

本文探讨了用台盼兰—姬姆萨染色检测家畜精子顶体反应的可行性。用肝素或钙离子载体诱发精子顶体反应。根据染色结果将精子分为四类:a)核后帽部不着色或淡青色,顶体不着色或部分紫红色(有顶体反应活精子);b)核后帽部暗青色.顶体部不着色或部分暗红色(有顶体反应死精子);c)核后帽部不着色或淡青色,顶体部紫红色(无顶体反应活精子);d)核后帽部暗青色,顶体部暗紫红色(无顶体反应死精子)。有顶体反应活精子百分率与仓鼠卵穿透率呈强正相关。从而证明台盼兰—姬姆萨染色是检测家畜精子顶体反应的有效手段,并能预测获能处理后精子的受精能力。     

Hyaluronic Acid as Capacitation Inductor: Metabolic Changes and Membrane‐Associated Adenylate Cyclase Regulation

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.     

绿原酸对猪精液冷冻保存效果的影响

为探究绿原酸对猪精液冷冻保存效果的影响,分别在TCG稀释液中添加不同浓度(15、30、50、80和100 pg/mL)的绿原酸,通过测定冷冻-解冻后精子的活率、顶体完整率、质膜完整率、DNA完整率、超氧化歧化酶(SOD)、过氧化氢酶(CAT)和谷胱甘肽过氧化物酶(GSH-Px)活性来判定对保存效果的影响.结果表明,当添...     

辅酶Q10对绒山羊精液冷冻保存效果的影响

旨在探讨辅酶Q10对绒山羊精液冷冻保存效果的影响。利用添加不同浓度辅酶Q10(4、40、400?滋g/mL)的精液冷冻稀释液对绒山羊精液样本进行冷冻保存,待冷冻精液解冻后,采用流式细胞仪和计算机辅助精液分析系统(CASAS)分别检测不同精液样本的精子活率、质膜完整率、顶体完整率、DNA完整率、线粒体膜电位和细胞内ROS水平。结果表明,当冷冻稀释液中添加浓度为40μg/mL辅酶Q10时,经历冷冻—解冻过程的绒山羊精液样本的精子活率、质膜完整率、顶体完整率均显著高于对照组(P<0.05);在冷冻稀释液中添加浓度为40μg/mL或400μg/mL的辅酶Q10均能显著提高线粒体膜电位并降低细胞内ROS水平(P<0.05)。综上所述,在冷冻稀释液中添加40μg/mL的辅酶Q10能够显著提高绒山羊精子抗氧化能力和冷冻保存效果。     

肝素浓度对辽宁绒山羊精子体外获能的影响

在TALP液中添加不同浓度(25、50、100μg/mL)的肝素对辽宁绒山羊精子进行获能处理,在显微镜下检测处理1、2、3、4、5h后的精子活力,用考马斯亮蓝染色法检测获能处理0.5、1、2、4h后的精子获能状况,探讨肝素浓度对绒山羊精子活力、存活时间、获能率的影响。结果发现,在38.5℃、5%CO2、饱和湿度条件下,精子活力随肝素浓度升高而下降,3 h以后25μg/mL肝素组精子活力显著高于其它肝素组(P<0.05)。添加肝素组获能率显著高于对照组(P<0.05),各肝素组精子获能率差异不显著(P>0.05)。对照组精子存活时间最长,25μg/mL肝素组精子存活时间为10.12 h,显著高于其它两组(P<0.05)。表明25μg/mL肝素处理辽宁绒山羊精子体外获能较为适宜。