Comparison of Two Semen Extenders in Terms of In Vitro Development of Bovine Embryos Following IVF

In order to conform with current EC standards with regard to antibiotic cover, the Norwegian Cattle Association is currently investigating the use of Biladyl® as an alternative to the milk-based extender which has been traditionally used in Norway. A study was carried out to investigate the effect of using semen frozen with either milk extender or Biladyl® on the outcome of in vitro fertilization and embryo culture. Semen from 6 Norwegian Red bulls was used. There was a significant difference p<0.05 in terms of cleavage rate between the 2 extenders for 1 bull, 78.2% vs 94.9% for milk and Biladyl® extenders, respectively, and for the overall total of 71.3% vs 76.1% for milk and Biladyl® extenders, respectively. There were no significant differences in terms of blastocyst yield amongst any of the bulls. In conclusion, the results suggest that Biladyl® can be used as a replacement for the traditional milk-based extender without any adverse effects on blastocyst yields following in vitro fertilization.     

荷斯坦牛和西门塔尔牛冻精品质及其体外受精后早期胚胎发育的比较研究

为探讨荷斯坦牛和西门塔尔牛冻精的精液品质及体外受精后胚胎发育能力的差异,利用目测法、低渗膨胀法和考马斯亮蓝染色法评估了荷斯坦牛和西门塔尔牛冻精的活力、质膜完整率和顶体完整率,并比较了二者冻精体外受精后胚胎的卵裂率和囊胚率。结果表明,荷斯坦牛和西门塔尔牛冻精的活力(30.4%和27.2%)、质膜完整率(41.96%和36.22%)和顶体完整率(77.02%和73.02%)均无显著差异(P>0.05),但荷斯坦牛冻精体外受精后的卵裂率(57.5%和48.6%)和囊胚率(30.3%和23.2%)显著高于西门塔尔牛冻精(P<0.05)。提示,不同品种公牛精液体外受精后的发育能力有显著差异(P>0.05)。     

小鼠体外受精及胚胎发育的影响因素

为了完善小鼠体外受精和胚胎的体外培养系统 ,以 M16为基础培养液 ,探讨了葡萄糖、磷酸盐、EDTA、谷氨酰胺及β-巯基乙醇等对小鼠体外受精和胚胎体外培养的影响。结果表明 ,在 M16液中添加 EDTA和 L -谷氨酰胺(m M16液 ) ,小鼠的体外受精率由 2 6 %提高到 5 1%;当在 m M16液中去掉磷酸盐 ,或添加 Hepes,或添加 β-巯基乙醇 ,体外受精率由 5 1%增至 6 2 %～ 74%;m M16液中添加 Hepes,或去掉磷酸盐 ,可阻碍胚胎的体外发育 ,囊胚的扩张率由 80 %降至 49%和 5 8%,而在无糖 m M16液中添加 β-巯基乙醇 ,可显著地提高扩张囊胚率 (91%)。以上结果表明 ,M16液中添加 EDTA、谷氨酰胺和 β-巯基乙醇后 ,适用于小鼠体外受精 ;同时去掉葡萄糖后 ,则适用于 2细胞胚的体外培养。     

Percoll法处理牛精子对体外受精胚胎发育的影响

牛冷冻精液解冻后,在Ｐｅｒｃｏｌｌ梯度液中４００ｇ离心２４ｍｉｎ有效地获得了５７．８７％±８．０％的精子回收率;对照组上游法精子回收率仅为１５．８７％±１．９％（Ｐ〈０．０５）。Ｐｅｒｃｏｌｌ和上游２种方法分离的精子在受精液中８,１８ｈ后的活率,分别为６０％,１０％和７０％,２０％,２种浓度Ｐｅｒｃｏｌｌ法分离的精子对不同成熟处理的卵母细胞体外受精子（ＩＶＦ）卵裂率的影响,精子最终浓度为２×１０＾     

几种冷冻条件对牛体外受精卵发育率的影响

试验比较了乙二醇 (EG)、丙二醇 (PG)、二甲基亚砜 (DMSO)、甘油 (G)和不同的投液氮温度 (- 33℃或 - 4 0℃ )对牛体外受精卵冷冻后发育率的影响。结果 :投液氮温度无论是 - 33℃或 - 4 0℃ ,均以 1.5 mol/ L EG的冷冻效果为最好 ,与 1.5 mol/ L PG相比 ,受精卵的发育率差异显著 (39.7% vs19.2 % ;4 7.8% vs2 4 .7% ,P <0 .0 5 ) ;同 1.5mol/ L DMSO和 1.5 mol/ L G相比 ,受精卵的发育率差异极显著 (39.7% vs 16 .1% or 13.3% ;4 7.8% vs 19.2 % or17.3% ,P <0 .0 1)。 - 4 0℃投液氮 ,受精卵冷冻后的发育率略高于 - 33℃ ,但差异不显著 (P >0 .0 5 )。以 2 5 % EG 2 5 % PG作为细胞外玻璃化溶液对牛体外受精卵进行冷冻 ,冷冻后受精卵的发育率达 5 8.9% ,高于用 1.5 mol/ L EG冷冻在 - 4 0℃投液氮这一处理 (47.8% ) ,但无统计学上差异 (P >0 .0 5 )。然而 ,试验组冷冻后受精卵的发育率均极显著低于对照组 (80 .6 8% ,P <0 .0 1)。结果表明 ,投液氮温度以 - 4 0℃为较好 ,防冻剂以 EG的冷冻效果为最佳。玻璃化冷冻法完全可以应用于冷冻牛体外受精卵     

The Effect of Melatonin on the Secretion of Progesterone in Sheep and on the Development of Ovine Embryos In Vitro

Two experiments were carried out in order to determine whether melatonin can improve secretion of progesterone in vivo, and its effect on embryonic development in vitro. In the first experiment, blood samples were collected from 5 ewes at 15 min intervals for 2 h at 7 and 10 days after withdrawal of progestagen pessaries. The first hour constituted a control period, which ended with an intravenous administration of 3 g/(kg bw)^0.75 melatonin. All the ewes on day 7 and three of the ewes on day 10 showed a progesterone response to melatonin challenge, defined as an increase in the plasma progesterone concentration in at least two consecutive samples during the post-treatment period above the mean+2SD of the values in the pre-treatment period. A paired t-test revealed a significant effect of melatonin on the overall plasma progesterone concentrations before and after the challenge, both on day 7 (pre, 0.61±0.11; post, 0.73±0.13 ng/ml; p<0.01) and day 10 (pre, 1.16±0.19; post, 1.30±0.20 ng/ml; p<0.05). Ninety-one thawed embryos (46 morulae and 45 blastocysts) were used in the second experiment, being cultured with or without 1 g/ml melatonin. If the embryos were blastocysts when the culture started, melatonin increased the percentage that had hatched after 24 h of culture (p<0.01), and there was a lower percentage of degenerated embryos at the end of the incubation period (p<0.05). It may be concluded that melatonin treatment in sheep can increase both fertility and prolificacy by improving luteal function and embryonic survival.     

葡萄糖在牛体外受精及胚胎发育中的影响

在牛精子获能液中添加不同浓度的葡萄糖,应用体外培养技术对牛精子体外获能及早期胚胎发育进行研究,通过观察顶体反应、超激活、活率和早期胚胎发育率,筛选出获能液中最适葡萄糖浓度及其有利于牛早期胚胎发育的最佳浓度.结果表明:葡萄糖是精子获能和维持超激活运动的主要能源物质,其代谢过程中产生的活性氧在牛精子体外获能、受精过程中起重要作用,高浓度(超过9.15 mM)葡萄糖有利于获能的完成;但是对早期胚胎发育不利,对早期胚胎发育来说其最适添加量为6.10 mM,此时的囊胚率最高.     

紫外线照射时间对孤雌激活和体外受精胚胎发育的影响

用核荧光染料 Hoechst3 3 3 42对 5组牛成熟卵母细胞染色后 ,以紫外线 ( UV)分别照射0 ,10 ,2 0 ,3 0 s和 40 s。观察和分析对孤雌激活胚胎和体外受精胚胎卵裂及体外发育的影响。结果表明 ,经 UV照射 0 ,10 ,2 0 ,3 0 s和 40 s的卵母细胞孤雌激活后的卵裂率分别为 80 .8% ,77.9% ,72 % ,61.4%和 45 % ,囊胚发育率分别为 3 9.2 % ,3 6.4% ,19.4% ,14 .5 %和 11.1% ;UV照射 2 0 s以上的囊胚发育率都极显著低于UV未照射和照射 10 s的卵母细胞 ( P <0 .0 1) ,UV照射卵母细胞超过 2 0 s降低了孤雌激活胚胎的体外发育潜力 ;体外受精胚胎中 ,UV照射2 0 s以上与未照射和照射 10 s组相比 ,卵裂率和囊胚发育率显著差异 ( P >0 .0 5 ) ,U V照射卵母细胞超过 2 0 s时 ,显著降低了体外受精胚胎的发育潜力。由此可知 ,卵母细胞经染色后 ,UV照射时间应控制在 2 0 s以内 ,并以照射 10 s时 ,对孤雌激活胚和体外受精胚的体外发育影响最小     

奶牛性控精液品质检测及效果分析

本试验旨在利用精子分析仪对奶牛性控冷冻精液精子的运动性能及体外受精率进行分析。结果表明:性控精液精子活力及顶体完整率、前向性与常规精液相比差异显著(P<0.05);曲线速度、直线速度、平均路径速度、平均移动角度、前向性差异均不显著(P>0.05);体外受精率差异不显著(P>0.05)。结论:奶牛性控精液运动性能及受精率低于常规精液。     

Effect of Antioxidants During Bovine In Vitro Fertilization Procedures on Spermatozoa and Embryo Development

Increased amounts of reactive oxygen species (ROS) during in vitro fertilization (IVF) may cause cytotoxic damage to gametes, whereas small amounts of ROS favour sperm capacitation. The aim of this study was to investigate the effect of antioxidants [50 μ m β-mercaptoethanol (β-ME) and 50 μ m cysteamine (Cyst)] or a pro-oxidant (5 m m buthionine sulfoximine) on the quality and penetrability of spermatozoa into bovine oocytes and on the subsequent embryo development and quality when added during IVF. Sperm quality, evaluated by the integrity of plasma and acrosomal membranes, and mitochondrial function, was diminished (p < 0.05) after 4-h culture in the presence of antioxidants. Oocyte penetration rates were similar between treatments (p > 0.05), but antioxidants adversely affected the normal pronuclear formation rates (p < 0.05). The incidence of polyspermy was high for β-ME (p < 0.05). No differences were observed in cleavage rates between treatments (p > 0.05). However, the developmental rate to the blastocyst stage was adversely affected by Cyst treatment (p < 0.05). The quality of embryos that reached the blastocyst stage, evaluated by total, inner cell mass (ICM) and trophectoderm cell numbers and ICM/total cell ratio was unaffected (p > 0.05) by treatments. The results indicate that ROS play a role in the fertilizing capacity in bovine spermatozoa, as well as in the interaction between the spermatozoa and the oocytes. It can be concluded that supplementation with antioxidants during IVF procedures impairs sperm quality, normal pronuclear formation and embryo development to the blastocyst stage.     

抗氧化剂对家畜胚胎体外发育影响的研究进展

现在研究普遍认为活性氧自由基是导致胚胎体外发育阻滞的主要原因。本文主要综述了不同抗氧化剂对牛胚胎体外发育的影响,并分析其作用机制。     

供体细胞培养处理方法对水牛核移植效果的影响

以经常规培养法 (DMEM 10 % FCS)、血清饥饿法 (DMEM 0 .5 % FCS培养 5～ 10 d)和 Apidicolin- APD结合血清饥饿法 (0 .1mg/ L APD培养 2 4 h,DMEM 0 .5 % FCS培养 1～ 18d)培养处理的水牛卵巢颗粒细胞和水牛成体耳部成纤维细胞作供核 ,分别采取带下注核法和胞质内注核法进行核移植。同一供核细胞各处理组间的核移植胚融合率 (以颗粒细胞作供核 )以及重组胚的囊胚发育率无明显差异 (P>0 .0 5 ) ,但经 APD 0 .5 % FCS培养处理供体细胞核移植后的分裂率显著高于其他组 (P<0 .0 5 )。用 7%乙醇处理的成体耳部成纤维细胞进行核移植 ,其重组胚的分裂率和囊胚发育率与对照组 (不含乙醇 )均无明显差异 (P>0 .0 5 )。结果表明 ,(1)血清饥饿处理水牛供体细胞对其核移植效果没有影响 ;(2 ) DNA合成抑制剂 APD结合血清饥饿培养处理水牛颗粒细胞和成体耳部成纤维细胞 ,可提高其核移植效果 ;(3)乙醇预激活处理水牛成体耳部成纤维细胞 ,对其核移植效果没有影响     

不同浓度CO2对牛卵母细胞体外成熟和受精的影响

对牛卵巢卵泡内卵母细胞在不同浓度的CO2培养箱内体外成熟、体外受精后的卵裂率及胚胎体外发育进行了研究。结果显示,含5%小牛血清(CS)的TCM-199内的卵母细胞在2%CO2条件下培养20h后,达到第二次减数分裂期(MⅡ期)的卵子数明显高于在5%CO2条件下培养的卵母细胞数(P<0.05)。两种浓度的CO2条件下培养的牛卵母细胞体外受精后的卵裂率无明显差异,2%CO2浓度下培养的卵母细胞的囊胚发育率高于5%CO2浓度下培养的卵母细胞。     

添加不同发情时期山羊血清对牛胚胎体外发育的影响

为了提高牛体外胚胎囊胚发育率,添加不同发情时期山羊血清对牛胚胎进行体外培养。分别采集发情周期D0、D2、D4和D7山羊血清（发情当天为D0）,灭活除菌备用。结果表明不同发情时期羊血清对孤紫激活胚胎孵裂率影响差异不显著,囊胚发育率分别为29.5%、32.4%、33.9%和41.3%,D7血清能显著提高囊胚发育率（P＜0.05）,且扩张和孵化胚出现较早。添加D0血清体外受精胚胎卵裂率较高,但对囊胚发育率影响差异不显著。说明孤紫激活和体外受精胚胎发育有差异,D7血清对胚胎后期发育有利。受精胚无血清培养3d后添加D7血清,囊胚发育率为42.9%,是较为理想的培养方法。     

Failure to Remove Bovine Diarrhea Virus (BVDV) from Bull Semen by Swim up and other Separatory Sperm Techniques Associated with in vitro Fertilization

Semen samples, collected from bulls pesistently infected with bovine viral diarrhea virus (BVDV) and containing BVDV (titer 10⁵ - 10⁶TCID₅₀/ml), were subjected to sperm separation procedures (washing, swim up, Percoll gradient, glass wool filtration, glass beads filtration) that are commonly used prior to in vitro fertilization (IVF) to determine if these procedures would yield spermatozoa free from BVDV. The final sperm pellets from frozen and fresh ejaculates were tested for the presence of BVDV by the immunoperoxidase technique; all tests were positive for BVDV in the range of 10³ - 10⁴TCID₅₀/ml). The study shows that when semen containing BVDV at the level of 10⁵ - 10⁶TCID₅₀/ml) is used for IVF, the virus is not completely removed by any of the simple physical methods commonly used to prepare sperm for IVF. Inhalt: Feldversuch das bovine Diarrhoe Virus (BVDV) aus Bullensperma durch Swim up oder andere Trennverfahren in Verbindung mit der In vitro Fertilisation zu entfernen Spermaproben, die von Bullen gewonnen wurden, welche dauerhaft mit dem Virus der bovinen Virus-Diarrhoe (BVDV) infiziert waren und einen Titer zwischen 10⁵ - 10⁶TCID₅₀/ml enthielten, wurden verschiedenen Trennverfahren unterzogen (Waschen, swim up, Percoll gradient, Glaswollenfiltration, Glaskugelfiltration). Diese Verfahren werden üblicherweise für die Vorbereitung zur In vitro Befruchtung verwendet, und es sollte geprüft werden, ob diese Verfahren auch das Sperma von dem BVD-Virus befreien können. Das endgültige Spermapellet von gefrorenen/aufgetauten und frischen Ejakulaten wurde in Gegenwart des BVD-Virus und mit Hilfe der immunoperoxidase Technik getestet. Alle Testergebnisse waren positiv für BVD-Virus in einem Bereich von 10³ - 10⁴ TCID₅₀/ml. Die Studie zeigt, daß Sperma, wenn es 10⁵-10⁶TCID₅₀/ml des Virus enhält, nicht vollständig mit den für die IVF üblichen, einfachen physikalischen Methoden von dem Virus befeit werden kann.     

牛冷冻精液解冻后在不同温度下的存活时间

牛冷冻精液解冻后在3～8℃、室温及37℃条件下保存,分别于2、4、8、12及24小时,观察其活力变化。结果表明:环境温度对精子的存活时间影响很大,活力的下降速度有明显差异。     

季节变化对不同品种种公牛精液品质的影响研究

 选择3~4.5岁的四个品种种公牛25头,观察季节变化对其次均采精量、平均密度、平均活力、精子畸形率、精液利用率及制冻数量的影响并进行比较。结果显示：在四季变化中,除次均采精量要显著低于国外引进品种外,鲁西牛种公牛精液的平均密度、平均活力、精子畸形率、精液利用率及次均制冻数量均表现出一定的优势。因此,鲁西牛作为地方品种受季节变化影响最小,繁殖性能更为稳定。