奶牛精子原位杂交蛋白酶K浓度的优化

[目的]确定奶牛精子原位杂交中最佳蛋白酶K浓度。[方法]设置6个蛋白酶K浓度（10、20、30、40、60、80μg/ml）,奶牛精子在37℃消化处理20 min,以精子尾部颈段和中段的存在作为选定最佳蛋白酶K浓度的标准。[结果]30μg/m l蛋白酶K既能有效去除精子蛋白质,又能保持精子的基本形态。[结论]精子原位杂交的最佳蛋白酶K浓度是30μg/ml。     

大额牛冷冻精液技术的研究

本文对大额牛的射精量、精子密度、精子活力等指标进行了测定,同时对大额牛精液的冷冻稀释液、制作程序进行了较为系统的研究。结果得出,大额牛在人工饲养条件下,公牛的射精量为4.92±1.50mL、精子密度为16.79±4.5亿/mL、原精活力0.63±0.078;冷冻精液解冻活力平均可达0.3875±0.0426、冻精解冻后顶体完整率平均52.74%±5.88%,畸形率为12.95%±3.18%,精子复苏率为59.63%±3.67%,其他指标与一般黄牛基本一致。     

哲罗鲑精子冷冻保存

哲罗鲑(Hucho taimen)是分布于额尔齐斯河和黑龙江等水域的冷水性珍稀鱼类,对其精子冷冻保存技术进行研究并建立精子冷冻库,对于哲罗鲑种质保存、人工繁殖发育、苗种培育及种群恢复都具有重要的意义.本研究利用生理盐水、葡萄糖、胎牛血清(fetal bovine serum,FBS)等配制了9种精子稀释液(MPRS,RS,Hanks,ELS,EG1,EG2,Stein,SS-2和D1S),对哲罗鲑精子在稀释液中活力、加入抗冻剂后活力和液氮中冷冻后活力进行检测,发现精子在以上稀释液中均具较高活力(73.00±2.65)％～(96.67±2.08)％,加入二甲基亚砜(dimethyl sulfoxide,DMSO)后活力下降,冷冻后仅在ELS和D15中具有极低活力(1.21±0.00)％和(1.1±0.00)％.进而利用ELS对甲醇、二甲基亚砜浓度进行筛选,发现精子在4％～12％甲醇中平衡20 min后活力仍为(79.00±6.52)％～(88.80±7.89)％,冷冻后仅在6％甲醇中具有相对较高活力(18.33± 10.61)％,但与鲜精活力相比显著下降(P＜0.05).精子在4％～12％ DMSO中平衡5 min后精子活力为(43.75± 11.09)％～(81.67±2.89)％,平衡20 min后精子活力极大下降(3.25±0.30)％～(45.00±5.77)％,冷冻后精子活力极低(1.00±0.00)％～(2.75±1.50)％(P＜0.05).以D15为基础液对葡萄糖浓度进行筛选,发现当葡萄糖浓度为30％(D30)时,冷冻后精子活力相对较高(17.80±2.59)％,但与鲜精相比活力显著下降(P＜0.05).另外对精子在D15、D30、Stein和ELs4种稀释液中短期保存的效果进行比较表明,Stein中精子存活在时间最长(45 h),D30次之(15h).综上所述,分别在ELS和D30中加入6％甲醇冷冻保存哲罗鲑精子具有一定活力,本研究结果为哲罗鲑精子大量冷冻保存和精子库建立等方面研究奠定了基础.     

Effectiveness of in vitro Methods for Predicting in vivo Fertilizing Capacity of Boar Spermatozoa Cryopresemed with 2% or 4% Glycerol1

Contents: Thirteen ejaculates from each of two boars that were greatly different in post-thaw sperm quality were frozen in either 2 or 4% glycerol (final concentration/split-sample). Frozen-thawed sperm were evaluated in vitro, for motility, acrosomal integrity (NAR), plasma membrane integrity (PMI), and for sperm penetration (SPA) of zona-free hamster eggs. To evaluate in vivo fertilizing capacity, 52 sows were inseminated once, 30–34 hours after onset of estrus. Ova were recovered from the reproductive tract 2–4 days following insemination and evaluated for cleavage and sperm binding. The two boars differed significantly for all in vivo and in vitro parameters. Motility and PMI were higher (P < 0.05) for sperm frozen with 4% than 2% glycerol whereas NAR was higher with 2% glycerol. Higher numbers of sperm were bound to eggs in vivo for sperm frozen with 4% than with 2% glycerol (P < 0.05). The fertilization rate for sperm frozen with 4% glycerol was not significantly higher than the fertilization rate for sperm frozen with 2% glycerol (P = 0.12). On the basis of these data, 4% rather than 2% glycerol may be close to optimum when freezing boar semen in straws. However, more extensive in vivo fertility testing is required to confirm this trend. Low correlations were observed between in vitro (and in vivo) evaluation of sperm quality and fertility when the effect of boar and glycerol concentrations was kept constant. Inhalt: Die Brauchbarkeit von in vitro-Methoden für die Voraussage der in vivo-Befruch-tungskapazität von Eberspermatozoen nach Tiefgefrierung mit 2% oder 4% Glycerin Von 2 Ebern, die sich in der Qualität ihres Auftauspermas deutlich unterschieden, wur-den je 13 Ejakulate mit 2% oder 4% Glycerinanted (Endkonzentration — split sample) tiefgefroren. Die aufgetauten Samenproben wurden in vitro nach Motilität, akrosoma-ler Integrität (NAR), Plasmamembran-Integrität (PMI) und im Spermapenetrationstest (SPA) am zonafreien Hamstereiüberprüft. Zur Beurteilung der Befruchtungskapazität in vivo wurden 52 Sauen 30–34 h nach Brunstbeginn einmalig besamt. 2–4 Tage nach der Besamung wurden die Eier aus dem Genitaltrakt gewonnen und auf Teilung und auf Anheftung von Samenzellen an der Zona pellucida beurteilt. Die beiden Eber wa-ren in allen In vitro- und In vivo-Parametern signifikant verschieden. Motilitat und PMI waren besser (p <0,05) für Spermien, die in 4% Glycerin konserviert waren, NAR bes-ser in 2%igem Glycerin. Nach Konservierung in 4% Glycerin waren mehr Spermien an der Eihülle angeheftet als nach 2% Glycerin (p < 0,05). Aber die Befruchtungsraten waren nach 4% und 2% Glycerinkonservierung praktisch nicht verschieden (p = 0,12). Auf der Grundlage dieser Ergebnisse ist zu folgern, daβ die Tiefgefrierkonservierung von Pelletsperma mit 4% Glycerin günstiger ist als mit 2% Glycerin; dieser Trend muβ jedoch in umfangreicheren In vivo-Fertilitätstests noch bestätigt werden. Wenn die Ef-fekte Eber und Glycerinkonzentration konstant gehalten wurden, dann wurden nur niedrige Korrelationen zwischen In vitro- und In vivo-Beurteilung von Spermaqualität und Fruchtbarkeit erhalten.     

梯度离心分离家兔精子试验

用Percoll梯度离心分离家兔精子,收取梯度管上、中、下各层精子给母兔人工授精。上层精子得到10窝共62只仔兔,其中雌性29只(46.8％),雄性33只(53.2％),中层精子得到5窝共31只仔兔,其中雌性16只(51.6％),雄性15只(48.4％);下层精子得到12窝共61只仔兔,其中雌性39只(64.0％),雄性22只(36.0％)。     

番鸭输卵管内贮精腺体的组织学及组织化学的研究

运用微分干涉显微镜对番鸭输卵管内的贮精腺体进行活体观察，并结合组织学和组织化学方法较系统地研究了该腺体的分布、形态及组织化学等特征。结果显示，番鸭贮精腺体分布于子宫－阴道交接处约０．５ｃｍ范围内的固有膜内。腺体呈管状，少数具有分枝，由单层柱状上皮细胞构成，细胞顶部无纤毛，腺体开口呈圆形或狭缝状。腺腔内的精子常聚集于腺体的盲端。对贮精腺体的组织化学研究显示：酸性磷酸酶活性很高，含有脂类、胆固醇、缩醛     

Effect of preovulatory concentrations of estradiol and initiation of standing estrus on uterine pH in beef cows

Previous studies have indicated that initiation of standing estrus within 24h of fixed-time AI influenced pregnancy rates. Furthermore, uterine environment at time of insemination can influence sperm transport. We hypothesized that preovulatory concentrations of estradiol would influence uterine pH at time of insemination. The objective of this study was to determine the influence of elevated preovulatory concentrations of estradiol on uterine pH following a fixed-time AI protocol. Cows were synchronized with the CO-Synch (n=57) protocol, and 29 cows were treated with an injection of estradiol cypionate (ECP; 1mg) 36h before the second injection of GnRH. Cows that exhibited standing estrus or were treated with ECP had increased (P<0.05) concentrations of estradiol compared to cows not in estrus and not administered ECP, respectively. There was an ECP by standing estrus interaction on uterine pH (P=0.01). Control cows that exhibited estrus had a reduced uterine pH (6.72+/-0.10; P=0.05) compared to control cows not exhibiting estrus (7.0+/-0.06). Cows treated with ECP and detected in standing estrus had a greater uterine pH (7.0+/-0.07) compared to control cows in estrus (P=0.02) and ECP cows not in estrus (6.81+/-0.09; P=0.06). The interval between the initiation of standing estrus and when pH was determined also influenced uterine pH. Cows that initiated standing estrus within 4h of pH determination had a lower uterine pH (6.74+/-0.12) compared to cows that initiated estrus 4-8h (7.09+/-0.08; P=0.07) or 8-12h (7.10+/-0.15; P=0.03) after pH determination. In summary, elevated concentrations of estradiol influenced standing estrus but only influenced uterine pH when pH was determined within 4h of the initiation of standing estrus.     

猪颗粒冻精制作试验

对猪颗粒冻精制作工艺进行改革，简化了流程，降低了成本，冻精解冻后精了活率平均为０．３５－０．４０。级水平，受胎率６６．６６％，窝产仔数８．１４头同时对猪精冷冻保存中的谷草转氨酶活性．精子顶体形态变化等生理生化指标进行了测定。     

Effect of Glutathione-Supplemented INRA82 Extender on Miniature Caspian Stallion Sperm Quality during Storage at 5°C

This study was conducted to investigate the effect of glutathione-supplemented INRA82 extender on miniature Caspian stallion sperm quality during storage at 5°C. A total of 12 ejaculates from three stallions (four ejaculates from each stallion) were collected and diluted with INRA82 extender that included different concentrations of glutathione (0 [INRA-G0], 5 [INRA-G5], and 10 mM [INRA-G10]) and stored for 48 hours at 5°C. Sperm motility (computer-assisted sperm analysis), plasma membrane integrity (eosin–nigrosin staining) and functionality (hypo-osmotic swelling test), and malondialdehyde (MDA) level were determined during storage at 5°C. The results showed that the sperm total and progressive motility and plasma membrane integrity and functionality in all extenders were significantly decreased with increasing storage time. However, the MDA level in all extenders was significantly increased with increasing storage time. Also, the results showed that most of the evaluated sperm quality parameters in the present study, with the exception of MDA, were significantly greater in INRA-G5 than in INRA-G0 and INRA-G10 after 24 and 48 hours of storage at 5°C. We have concluded that supplementation of INRA82 with 5 mM glutathione can improve miniature Caspian stallion sperm quality during storage at 5°C by increasing total and progressive motility, plasma membrane integrity and functionality, and decreasing the MDA level compared with INRA-G0 and INRA-G10. More advanced in vitro evaluations and artificial insemination are required to reveal the exact effects of INRA-G5 on miniature Caspian stallion sperm quality and its fertilizing ability.