Ca2+ Cascade and Meiotic Resumption of the Caprine Primary Oocyte

In this study, we investigated the fluctuations of concentration of intracytoplasmic free Ca(2+) during in vitro maturation of caprine primary oocytes and its role in meiotic resumption. Oocytes that were extracted from caprine ovaries were cultured and allowed to mature in vitro to determine their developmental stages including germinal vesicle (GV), germinal vesicle breakdown (GVBD), metaphase of the first meiotic division (MI) and metaphase of the second meiotic division (MII). Intracytoplasmic free Ca(2+) turnovers of caprine oocytes at these different developmental stages were measured using the calcium fluorescent probe Fura-2/AM (C(44)H(47)N(3)O(24)) to investigate the dynamics of cytosolic free Ca(2+) during in vitro maturation of oocytes and the role of Ca(2+) in inducing the initiation of meiotic resumption of oocytes. Moreover, the oocytes were cultured in Ca(2+) culture medium and Ca(2+)-free culture medium to examine the effect of extracellular Ca(2+) on the oocyte maturation. The results indicated that Ca(2+) concentrations at GV, GVBD, MI and MII stages were 78.06, 147.41, 126.97 and 97.73 nmol/l, respectively, and that 86.30% of oocytes remained at the GV stage and no oocyte developed to MII in Ca(2+)-free culture medium, and 1.1% of oocytes stayed at the GV stage and 83.5% of oocytes developed to MII in Ca(2+) culture medium. These results suggest that the occurrence of GVBD and cell cycle progression to MI and MII stages are closely related to Ca(2+), and that extracellular Ca(2+) performs a specific function for the initiation of meiotic resumption in caprine oocytes.     

蛋白激酶C在哺乳动物卵母细胞第1次减数分裂成熟过程中的调节作用

蛋白激酶C（PKC）是一个广泛分布在真核细胞中的丝氨酸/苏氨酸蛋白激酶家族。它在卵母细胞的生发泡破裂（GVBD）、染色体凝集、MⅠ期纺锤体组装和第一极体排放等过程中起着重要的调节作用。PKC的活性变化调节着GVBD的发生，GVBD标志着第1次减数分裂的启动。PKC活性在卵母细胞成熟过程中逐渐升高，在第1次减数分裂中/后期转变时活性下降，使卵母细胞得以释放出第一极体，至此卵母细胞完成第1次减数分裂进入第2次减数分裂。作者就PKC在卵母细胞第1次减数分裂成熟过程中的作用综述如下。     

催产素在奶牛生产中的应用

从催产素的作用机理入手,总结了催产素在奶牛助产、预防和治疗胎衣不下、治疗子宫迟缓、治疗子宫内膜炎和子宫积脓、催乳、治疗持久性黄体不孕症、提高受胎率等方面的应用情况和注意事项。     

延边黄牛卵母细胞去核方法的研究

用改进的Willaden去核法与McGrath-Solter去核方法对延边黄牛卵母细胞进行去核操作,比较其去核操作效率。结果表明,本实验改进的willaden去核法——挤压去核法,无论在去核操作时间(1．0 min／枚,P<0．05)上,还是在去核效率(94%,P<0．01)上都显著地高于McGrath-Solter去核法(1．4 min／枚、72%)。表明改进的Willaden去核方法是一种适用于延边黄牛卵母细胞操作的快速、简单、实用的去核方法。     

卵丘细胞在卵母细胞体外成熟培养过程中的作用

介绍了卵丘细胞在卵母细胞体外成熟培养过程中的作用,主要从卵丘细胞与卵母细胞的结构与功能联系,卵丘扩展对卵母细胞体外成熟的影响及卵丘细胞层数对卵母细胞体外成熟的影响三个方面加以阐述。     

The Adverse Effect of Phytoestrogens on the Synthesis and Secretion of Ovarian Oxytocin in Cattle

The current investigations were undertaken to study the mechanism of the adverse effect of phytoestrogens on the function of bovine granulosa (follicles >1< cm in diameter) and luteal cells from day 1–5, 6–10, 11–15, 16–19 of the oestrous cycle. The cells were incubated with genistein, daidzein or coumestrol (each at the dose of 1 × 10^?6 m ). The viability and secretion of estradiol (E2), progesterone (P4) and oxytocin (OT) were measured after 72 h of incubation. Moreover, the expression of mRNA for neurophysin‐I/OT (NP‐I/OT; precursor of OT) and peptidyl‐glycine‐α‐amidating monooxygenase (PGA, an enzyme responsible for post‐translational OT synthesis) was determined after 8 h of treatment. None of the phytoestrogens used affected the viability of cells except for coumestrol. The increased secretion of E2 and P4 was only obtained by coumestrol (p < 0.05) from granulosa cells from follicles <1 cm in diameter and decreased from luteal cells on days 11–15 of the oestrous cycle, respectively. All three phytoestrogens stimulated (p < 0.05) OT secretion from granulosa and luteal cells in all stages of the oestrous cycle and the expression of NP‐I/OT mRNA in the both types of cells. The expression of mRNA for PGA was stimulated (p < 0.05) by daidzein and coumestrol in granulosa cells, and by genistein and coumestrol in luteal cells. In conclusion, our results demonstrate that these phytoestrogens can impair the ovary function in cattle by adversely affecting the synthesis of OT in follicles and in corpus luteum. However, their influence on the ovarian steroids secretion was less evident.     

The Importance of Having Zinc During In Vitro Maturation of Cattle Cumulus–Oocyte Complex: Role of Cumulus Cells

The aim of this study was to investigate the influence of zinc (Zn) on the health of cumulus–oocyte complex (COC) during in vitro maturation (IVM). Experiments were designed to evaluate the effect of Zn added to IVM medium on: DNA integrity, apoptosis, cumulus expansion and superoxide dismutase (SOD) activity of cumulus cells (CC). Also, role of CC on Zn transport during IVM was evaluated on oocyte developmental capacity. DNA damage and early apoptosis were higher in CC matured with 0 μg/ml Zn compared with 0.7, 1.1 and 1.5 μg/ml Zn (p < 0.05). Cumulus expansion did not show differences in COC matured with or without Zn supplementation (p > 0.05). Superoxide dismutase activity was higher in COC matured with 1.5 μg/ml Zn than with 0 μg/ml Zn (p < 0.05). Cleavage and blastocyst rates were recorded after IVM in three maturation systems: intact COCs, denuded oocytes with cumulus cells monolayer (DO + CC) and denuded oocytes (DO). Cleavage rates were similar when COC, DO + CC or DO were matured with 1.5 μg/ml Zn compared with control group (p > 0.05). Blastocyst rates were significantly higher in COC than in DO + CC and DO with the addition of 1.5 μg/ml Zn during IVM (p < 0.01). Blastocyst quality was enhanced in COC and DO + CC compared with DO when Zn was added to IVM medium (p < 0.001). The results of this study indicate that Zn supplementation to IVM medium (i) decreased DNA damage and apoptosis in CC; (ii) increased SOD activity in CC; (iii) did not modify cumulus expansion and cleavage rates after in vitro fertilization; (iv) improved subsequent embryo development up to blastocyst stage; and (v) enhanced blastocyst quality when CC were present either in intact COC or in coculture during IVM.     

代谢激素及营养对牛卵泡发育的影响

营养与牛繁殖的关系十分复杂，它影响到繁殖过程的诸多方面。该文仅局限于讨论其对卵泡发育的影响，即代谢激素(GH，IGF—1，INS)及短期增加日粮摄入对青年母牛卵泡发育的影响。并讨论营养与代谢激素，卵泡发育的相互关系及生产中的一些实际问题，如高产奶牛繁殖率下降，超排反应不稳定等问题。     

Cilostazol Improves Developmental Competence of Pig Oocytes by Increasing Intraoocyte Cyclic Adenosine Monophosphate Level and Delaying Meiotic Resumption

Cilostazol (CLZ) is a cyclic adenosine monophosphate (cAMP) modulator that influences the steady state of the meiotic stage. This study was conducted to determine the effects of CLZ treatment during in vitro maturation (IVM) on developmental competence of pig oocytes. Immature oocytes were exposed to 0 (control), 0.5, 2 and 4 μm CLZ during the first 22 h of IVM. Nuclear maturation, intraoocyte glutathione content and embryo cleavage after parthenogenesis (PA) and somatic cell nuclear transfer (SCNT) were not influenced by CLZ at any concentrations. However, 4 μm CLZ significantly (p < 0.05) improved blastocyst formation after PA (52.1% vs 38.7–46.0%) and SCNT relative to other concentrations (40.8% vs 25.0–30.7%). The mean cell numbers of SCNT blastocysts were significantly increased by 4 μm CLZ compared to the control (42.6 cells vs 35.3 cells/blastocyst). CLZ treatment significantly increased the intraoocyte cAMP level and effectively arrested oocytes at the germinal vesicle (GV) and GV break down stages compared to the control (74.5% vs 45.4%). Our results demonstrated that improved developmental competence of PA and SCNT pig embryos occurred via better synchronization of nuclear and cytoplasmic maturation induced by increased cAMP and delayed meiotic resumption after CLZ treatment.     

卵母细胞的激活

哺乳动物卵母细胞人工激活的方法、机理及激活时间进行总结，目前使用卵母细胞去核前人工激活的方法较多，去核后移核前激活的次之，移核后激活的较少。     

Assessment of Meiotic Spindle Configuration and Post‐Warming Bovine Oocyte Viability Using Polarized Light Microscopy

The objectives of this study were to assess the efficiency of polarized light microscopy (PLM) in detecting microtubule‐polymerized protein in in vitro‐matured bovine oocytes; to examine its effects on oocyte developmental competence; and to assess the meiotic spindle of in vitro‐matured oocytes after vitrification/warming and further assessment of oocyte developmental competence. In the first experiment, the presence of microtubule‐polymerized protein (MPP) was confirmed as a positive PLM signal detected in 99.1% of analysed oocytes (n = 115), which strongly correlated (r = 1; p < 0.0001) with the presence of MPP as confirmed by immunostaining. In the second experiment, oocytes (n = 651) were exposed or not (controls) to PLM for 10 min and then fertilized and cultured in vitro. Oocytes exposed to PLM did not significantly differ from controls with regard to cleavage, total blastocyst and expanded blastocyst rates and cell numbers. In the third experiment, meiotic spindles were detected in 145 of 182 oocytes (79.6%) following vitrification and warming. Interestingly, after parthenogenetic activation and in vitro culture, oocytes that displayed a positive PLM signal PLM(+) differed significantly from PLM(?) in cleavage and Day 8 blastocyst rates. These results suggest that polarized light microscopy is an efficient system to detect microtubule‐polymerized protein in in vitro‐matured bovine oocytes and does not exert detrimental effects on bovine oocyte developmental competence. Moreover, PLM could be used as a tool to assess post‐warming viability in vitrified bovine oocytes.     

Role of Eicosanoids in the Regulation of the Periparturient Period in Cattle

Contents: In this review, the role of eicosanoids in regulation of parturition and the postpartum period was described with special emphasis on the bovine species. The metabolism of arachidonic acid and the production of eicosanoids during the peripartum period was discussed. Prostaglandin E₂ and F_2α (PGE₂, PGF_2α) play an important role in mechanisms controlling parturition. They are involved in luteolysis, uterine contractions and dilation of the cervix. Eicosanoids also seem to influence the loosening processes of the fetal membranes. However, in the literature, conflicting results were found. Many investigations suggested that retained placental membranes could be related to low PGF_2α production and/ or an imbalance of arachidonic acid metabolism in the uterus. The possible role of the lipoxygenase pathway metabolite 15-hydroxy-eicosatetraenoic acid (15-HETE) in expulsion of the fetal membranes was also discussed. As far as the postpartum period is concerned, a relationship between postpartum PGF_2α release and the involution of the uterus was found. Cows with undisturbed uterine involution had higher PGF_2α production than cows with delayed involution. In contrast to the positive effect of PGF_2α on uterine involution, PGE₂ seems to delay the involution processes. Further experiments are necessary in order to study the function of eicosanoids in mechanisms regulating parturition, release of the fetal placental membranes and involution of the uterus.     

Maternal Liver Damage Delays Meiotic Resumption in Bovine Oocytes through Impairment of Signalling Cascades Originated from Low p38MAPK Activity in Cumulus Cells

The main objective of the present study is to investigate the molecular mechanism underlying the delay in progression of nuclear maturation in oocytes derived from cows with damaged livers (DL cows), which was previously reported. In present study, delayed progression of nuclear maturation of oocytes derived from DL cows relative to oocytes derived from cows with healthy livers (HL cows) was accompanied by low maturation promoting factor (MPF) activity (0.43 fold, p < 0.05). When cumulus cells were removed from cumulus‐oocyte complexes and the denuded oocytes were cultured, there was no difference in the progression of nuclear maturation between the two liver conditions. In addition, gap junctional communication (GJC) between the oocyte and cumulus cells was higher in DL cows than in HL cows at 3 and 7 h of in vitro maturation (IVM) (p < 0.05). Supplementation of IVM medium with epidermal growth factor (EGF) increased the ratio of germinal vesicle breakdown (GVBD) of oocytes derived from DL cows to the level seen in oocytes derived from HL cows. Additionally, the level of p38MAPK phosphorylation at 0 h of IVM was significantly lower in cumulus cells derived from DL cows than in cumulus cells derived from HL cows (HL cows, 53.5%; DL cows, 28.9%; p < 0.05). Thus, a low level of p38MAPK phosphorylation in cumulus cells induced slow GJC closure between oocyte and cumulus cells, which resulted in slow meiotic maturation of oocytes derived from DL cows.     

功能性复合营养舔砖对育肥期肉牛生产性能的影响

本试验的目的在于研究育肥期肉牛补饲添加有枸杞多糖、复合酶制剂、过瘤胃氨基酸、单宁酸等添加剂的功能性复合营养舔砖,分析对其生产性能及经济效益的影响。选择西门塔尔杂种牛80头,按体重随机分成2个处理组,每组40头,对照组使用常规日粮,试验组在常规日粮的基础上补饲功能性复合营养舔砖,自由舔食,预饲期15d,正试期90d。结果表明:1）试验观察 ：适口性较好,试验初始,大部分牛啃食,采食一段时间后舔砖采食量相对恒定,同时表明营养舔砖具有一定的营养调节功能,试验育肥肉牛毛色、精神状态均比对照组牛群有所改善;2）只均日增重 ：试验组高于对照组10.78%（P＜0.05）;3）料肉比 ：试验组低于对照组10.63%（P＜0.05）;4）只均毛利润：试验组高于对照组26.54%。