牛有腔卵泡卵母细胞体外成熟的研究

试验从屠宰场收集了 4 8头青年黄牛、34头青年水牛的卵巢共 16 4个 ,共回收可用卵母细胞 86 4枚。水牛平均每个卵巢可回收 3.2 2枚可用卵母细胞 ,相当于黄牛 (6 .72枚 )的一半。试验结果表明 :水牛卵巢生长卵泡少 ,卵母细胞产量少、质量差 ;3种采集黄牛卵泡卵母细胞的方法 ,用抽吸加切割法平均从每个卵巢回收的可用卵数 (7.71枚 )都极显著高于抽吸法 (6 .19枚 )和切割法 (6 .4 4枚 ) (P <0 .0 1) ,但是该法手续繁杂 ,适于一次且只能收集少量卵巢时使用 ;在黄牛和水牛卵母细胞成熟培养液中用 0 .3%PVP代替 10 %FBS ,牛卵母细胞的体外成熟率无显著差异 (P >0 .0 5 )。     

影响黄牛小腔卵泡卵母细胞体外受精及早期胚胎发育的因素

从屠宰场收集黄牛卵巢,取皮质深层卵母细胞进行体外成熟、体外受精和早期胚胎体外培养,分析了影响其效果的因素。结果表明,在成熟培养液中添加FSH(10IU/mL)、HCG(20IU/mL)和17β-E2(1mg/L)对卵母细胞受精后早期胚胎发育能力有极显著促进作用;等量牛卵泡液(BFF)与新生牛血清(NCS)对体外受精胚胎发育效果影响不显著,以15?F为宜;颗粒细胞与输卵管上皮细胞均能显著提高卵母细胞体外成熟受精后早期胚胎的发育率,颗粒细胞 输卵管上皮细胞对克服胚胎阻滞现象效果显著。     

有无透明带牛卵母细胞孤雌发育研究

研究探讨了不同浓度的钙离子载体(A23187)结合6-二甲氨基嘌呤(DMAP)或丁内酯Ⅰ(BL-1)激活处理对有或无透明带牛卵母细胞孤雌发育的影响。结果表明：使用A23187浓度从0．625～10．000μmol／L配合2．0mmol／L DMAP孤雌激活牛具透明带卵均能获得良好的卵裂及早期胚胎发育效果；对于去透明带卵来说．使用0．625μmol／L浓度的A23187激活效果最佳。BL-1的激活效果则明显差于DMAP；当采用0．3%PVA取代激活液中的5%血清时，BL-1的激活效率显著提高。     

Recovery of bovine oocytes from small vesicular follicles for in vitro maturation and fertilization

Five dairy and four beef breed, mature cows were used as oocyte donors to develop a system of multiple recovery of oocytes for in vitro maturation and fertilization. The animals were alternately treated with either 20 mg of follicle stimulating hormone (FSH) in four equal intramuscular injections or saline at 12 h intervals starting between days 9 and 13 of the oestrous cycle, and the procedure was repeated at three-week intervals for up to four collections. Eighteen collections resulted in the recovery of 124 oocytes from 181 follicles (69%). No serious side-effects were observed. Recovery was equally successful in both breeds and was not reduced in repeat attempts upon the same animal. Treatment with FSH only marginally increased the recovery rate (p<0.07) and did not affect the number of follicles aspirated (p>0.05), which varied significantly (p<0.05) between cows.From 110 oocytes matured and fertilized in vitro, 70 embryos were recovered after culture in the rabbit oviduct or with trophoblastic vesicles in vitro, of which 30 had cleaved and 5 had progressed to an advanced stage of development. Hormone treatment did not affect zygote development (p>0.05). Four non-surgical transfers of embryos obtained in these studies have resulted in two pregnancies determined ultrasonographically and the birth of a heifer calf. This suggests that the procedure for multiple oocyte recovery is safe and that it can be used sucessfully for obtaining oocytes for in vitro maturation and fertilization.     

牦牛卵巢卵母细胞体外培养成熟条件的建立

研究了不同采集方法和不同培养液对牦牛卵母细胞体外成熟的培养效果。结果:在牦牛乏情期,每个卵巢平均回收卵数为(9.33±4.30),可用卵数为(5.63±4.19)。将牦牛卵丘卵母细胞复合体分别置于5种成熟培养液中培养,成熟率分别为75.56%、71.11%、81.33%、77.33%和77.78%,卵裂率分别为42.22%、33.33%、49.33%、46.67%和42.22%,其中C的成熟液效果最好。来自卵巢表面卵泡的COCs的成熟率(81.33(vs69.33(,P>0.05)高于来自卵巢内卵泡的COCs,卵裂率(49.33%vs34.67%,P<0.05)显著高于来自卵巢内卵泡的COCs。来自明亮卵泡的COCs的成熟率(81.33%vs33.33%,P<0.01)和卵裂率(49.33%vs3.33%,P<0.01)极显著高于来自混浊卵泡的COCs。     

Ghrelin在绵羊卵母细胞体外成熟过程中对BCL-2、BAX的mRNA表达的影响

为了探讨Ghrelin在绵羊卵母细胞体外成熟过程中与BCL-2、BAX的相关性及Ghrelin在绵羊卵母细胞成熟过程中可能的作用机理,试验在绵羊卵母细胞成熟液中添加500 ng/mL Ghrelin,采用实时荧光定量PCR分别在0,8,16,24小时时检测卵母细胞BCL-2、BAX的表达量。结果表明:试验组添加Ghrelin后8,16小时时卵母细胞BCL-2 mRNA相对表达量显著升高(P<0.05),但成熟后16,24小时时较8小时时有下降趋势;下降到24小时时,其表达量与0小时时没有显著变化(P>0.05);8,16小时时试验组与对照组比较差异显著(P<0.05),0,24小时时差异不显著(P>0.05)。试验组添加Ghrelin后8,16,24小时卵母细胞BAX mRNA相对表达量较0小时时下降,且差异显著(P<0.05),但成熟后16,24小时时与8小时时比较有上升趋势;在8,16小时时试验组较对照组BAX mRNA相对表达量均有所下降,且差异有显著性(P<0.05),而在0,24小时时差异不显著(P>0.05)。说明Ghrelin在绵羊卵母细胞成熟过程中与BCL-2和BAX的表达存在相关性,推测Ghrelin在卵母细胞成熟过程中存在积极调控作用。     

CK1 inhibitor affects in vitro maturation and developmental competence of bovine oocytes

The objectives of present study were to evaluate the effect of casein kinase 1 (CK1) inhibition D4476 on in vitro maturation (IVM) and developmental competence of bovine oocytes. The cumulus oocyte complexes (COCs) were cultured in maturation medium with D4476 (0, 2, 5, 10, 20 μM) for 24 hr. After IVM and in vitro fertilization, through expansion average scores of cumulus cells (CCs), oocyte maturation efficiency, cleavage rate and blastocyst rate of zygote, we found 5 μM D4476 could increase the development potential of oocytes. After the COCs were treated with 5 μM D4476, the results of quantitative real‐time PCR analysis, Lichen red staining and PI staining showed that under without affecting germinal vesicle breakdown and nuclear morphology, D4476 could significantly decrease CK1 and upregulate TCF‐4 in oocytes. Furthermore, without influencing the level of Bad and CTSB, D4476 could significantly increase the expression of β‐catenin, TCF‐4, Cx43, MAPK, PTGS‐2, PTX‐3, TGS‐6, Bax and Bcl‐2 in CCs. Western blot analysis revealed that the addition of 5 μM D4476 during the maturation of COCs resulted in a lower level of Cx43 protein at 12 hr and a higher expression of Cx43 protein at 24 hr compared to the group without D4476. These results indicate that adding optimum D4476 (5 μM) to maturation medium is beneficial to maturity efficiency and development competence of bovine oocytes.     

Alpha-lipoic acid improves the maturation and the developmental potential of goat oocytes in vitro

Oxidative stress inevitably occurs during oocyte maturation in vitro. α-lipoic acid (α-LA) has a strong antioxidant capacity, but the effect of α-LA on parthenogenetic activation of oocytes was rarely reported. This study aims to investigate the effect of supplementing α-LA to in vitro maturation medium on the subsequent developmental ability of goat parthenogenetic embryos during oocytes maturation. In the study, the goat cumulus-oocyte complex was divided into the experimental (with 25 μmol/L α-LA) and the control (without α-LA) groups. Oxidase expression was measured using RT-qPCR. After 18–22 hr of maturation, the oocytes were then parthenogenetic activated. The total antioxidant capacity of embryos was measured after 0, 24, 48, 72 and 96 hr of culture. Rates of oocyte maturation and the rates of development for parthenogenetic embryos in the α-LA group were significantly improved by 7.88% (p < .05) and 5.41% (p < .05) compared with those in the control group, respectively. After 24 hr, the difference in total antioxidant capacity was extremely significant in both groups. An evident decrease in the control group and a minor decrease in the α-LA group were observed (p < .01). The ratio of inner cell mass cells to the total cell number of blastocysts in the α-LA group increased compared with that in the control group (p < .05) on day 8. α-LA significantly promoted the expression of SOD and GPX4 of parthenogenetic blastocysts and maturated oocytes. α-LA (25 μmol/L) improved the maturation rate and the developmental competence of the parthenogenetic activation of oocytes, which might be mediated by maintaining the total antioxidant ability of oocytes during the culture period.     

牛卵母细胞体外成熟

卵母细胞体外成熟培养研究主要集中在提高体外成熟卵母细胞支持胚胎发育的能力上。本文主要从成熟机理、卵母细胞的来源、卵母细胞的成熟培养环境及培养时间等方面进行了综述,介绍了目前卵母细胞体外成熟培养的进展情况。     

Meiosis in bovine oocytes matured in vitro and in vivo

Meiosis in bovine oocytes has; been studied after maturation in vitro or in vivo. Oocytes for in vitro maturation were collected from the ovaries of slaughtered cattle without regard to the phase of the estrous cycle while in vivo maturation was studied in oocytes from gonadotrophin-stimulated heifers at times varying between 6 and 36 h after the beginning of behavioural estrus. Oocytes from slaughtered cattle were classified according to their cumulus complex and ooplasm and were cultured for 6, 12, 18, 24, 36 or 48 h in modified Krebs-Ringer bicarbonate buffer before fixation) for cytogenetic analysis. Oocytes from stimulated heifers were aspirated from follicles or flushed from the oviducts, classified according to cumulus and ooplasm, and fixed within 6 h of collection. Nuclear maturation was more rapid in vitro than in vivo. The largest proportion of oocytes reached maturity (Mil) after 12 to 18 h in culture or 30 to 36 h after the onset of behavioural estrus. Oocytes devoid of cumulus cells or showing signs of vacuolation or degeneration had virtually no capacity for nuclear maturation.     

Changes in the amount of proteins, glycogen and lipids in porcine oocytes during in vitro meiotic maturation

Changes in the cytoplasmic inclusions during meiotic maturation were histochemically examined in cultured porcine oocytes. The oocytes contained a small amount of protein and glycogen granules throughout the maturation culture, as well as Sudanophilic lipids composed of small, medium and large droplets. Soon after collection, the amount of Sudanophilic lipid droplets of small and medium size was small and there were 167 ± 11.2 large droplets. After being cultured for 22 h, the number of large lipid droplets decreased remarkably, while the number of small and medium ones increased. There were no differences in the number of Sudanophilic lipid droplets of different sizes between ovulated oocytes and the oocytes cultured for 44 h. The oocytes always contained a large amount of neutral fats and lipoids, but not cholesterols. In the oocytes cultured for 22 h with olomoucine, both the resumption of nuclear maturation and the decrease in the size of the Sudanophilic lipid droplets were inhibited. From the present findings, it appears that the change in the size of the Sudanophilic lipid droplets in the cytoplasm of porcine oocytes is closely related to nuclear maturation.     

Effect of medium additives during liquid storage on developmental competence of in vitro matured bovine oocytes

Our aim was to improve the developmental competence of bovine oocytes during their liquid storage by using additives. In vitro matured oocytes were stored for 20 h at 25°C in HEPES buffered TCM 199 medium (base medium). After storage, in vitro embryo development after in vitro fertilization was compared to those of non‐stored (control) ones. Addition of 10% (v/v) newborn calf serum or 10.27 mmol/L pyruvate alone to the base medium did not improve blastocyst formation rates in stored oocytes; however, their simultaneous addition significantly improved the rate compared with those stored in base medium (P < 0.05). Supplementation of the holding medium with dithiothreitol (DTT) at any concentrations did not improve embryo development from stored oocytes. Although supplementation with cyclosporine A (CsA) significantly reduced apoptosis and membrane damage rates during storage, it did not improve the developmental competence of oocytes. 1,2‐bis(2‐aminophenoxy) ethane N,N,N’,N’‐tetraacetic acid tetrakis‐acetoxymethyl ester and ruthenium red had no effect on oocyte apoptotic rates. Blastocyst formation rates in all stored groups remained significantly lower than that of the control. In conclusion, pyruvate and serum had a synergic effect to moderate the reduction of oocyte quality during storage, whereas mitochondrial membrane pore inhibitor CsA and the antioxidant DTT did not affect their developmental competence.     

In vitro effects of meloxicam on the number,Foxp3 expression,production of selected cytokines,and apoptosis of bovine CD25+CD4+ and CD25-CD4+ cells

The purpose of this study was to evaluate the effect of meloxicam (MEL) on selected immune parameters of bovine CD25^highCD4+, CD25^lowCD4+, and CD25-CD4+ cells. Peripheral blood mononuclear cells (PBMCs) collected from 12-month-old heifers were treated with MEL at a concentration corresponding to the serum level of this medication following administration at the recommended dose (MEL 5 × 10^-6 M) and at a concentration 10 times lower (MEL 5 × 10^-7 M). After 12 and 24 h of incubation with the drug, the percentage of CD25^highCD4+ cells decreased; however, this disturbance was quickly reversed. Furthermore, the absolute number of CD25^highCD4+ cells in the PBMC populations treated with MEL 5 × 10^-6 M for 48 and 168 h was increased. Prolonged (168 h) exposure to the drug increased the percentage of Foxp3+ cells in the CD25^highCD4+ cell subpopulation. The higher dose of MEL was found to significantly increase the percentage of IFN-γ+ cells among the CD25-CD4+ cells. These results indicated that MEL does not exert an immunosuppressive effect by depleting CD4+ cells and suppression of IFN-γ+ production by these cells. Furthermore, IL-10 and TGF-β production was not changed following exposure to MEL.     

牛卵泡卵母细胞的体外成熟和冷冻保存

在简化牛卵母细胞体外成熟的基础上,观察了保护剂、平衡温度、预平衡方法、解冻方法以及冷冻方法对牛卵泡卵母细胞冷冻的影响。结果表明:在体外成熟培养液中添加26.2 mmol/L的NaHCO3和对屠宰场卵巢进行选择更能促进牛卵母细胞的体外成熟;无论是程序冷冻还是玻璃化冷冻,乙二醇(EG)与甘油(GLY)相比更能促进牛卵泡卵母细胞的冷冻效果;在程序冷冻中,冷冻液中添加0.1 mol/L蔗糖比添加0.3mol/L的蔗糖更能促进牛卵泡卵母细胞的冷冻;在玻璃化冷冻中,37℃和25℃的平衡温度比4℃更适合牛卵泡卵母细胞的冷冻;在10%、5%、1%的预平衡浓度之间,5%的预平衡浓度即可达到预平衡的效果;在解冻时,多步脱除保护剂更能保护冷冻的卵母细胞;比较了几种最小样本量(minimum size sample,MSS)玻璃化冷冻方法,解冻成熟培养后的成熟率,拉细毛细玻璃管冷冻法为(41.67±3.19)%,极显著高于未拉细毛细玻璃管冷冻法(glassmicropipette,GMP)的(30.19±1.93)%和固体表面冷冻法(solid surface vitrification,SSV)的(28.33±2.89)%(P<0.01)。     

Chronological changes of bovine follicular oocyte maturation in vitro

Chronological changes of bovine follicular cumulus-oocyte-complexesi were studied after in vitro maturation over a period of 48 h. According to their thickness and compactness of cumulus investments they were classified into 4 groups and cultured in enriched Ham’s F-10 medium with or without human chorionic gonadotrophin (hCG) and estradiolbenzoate (EB) for 0, 6, 12, 18, 21, 24, 27, 30 and 48 h. Representative samples were taken at each time interval for evaluation of nuclear maturation stages, ooplasm quality and size of the peri vitelline space (PVS). The results showed that oocyte nuclear breakdown (ONBD) required 6 to 12 h culture, and the peak of the first polar abstriction occurred at 24 h. The culture period required for ONBD and abstraction of the first polar body were related to the thickness and compactness of cumulus investments with and approximately 6 h delay in heavily compacted complexes. Ooplasm quality evaluation failed to show a clear trend, but the PVS increased in size from 0 h to 30 h and then, retracted again from 30 to 48 h. The overall maturation rate in the presence of hCG and EB was 79.1 %, and a substantial proportion (68.8 %) of nude or partially covered oocytes reached metaphase II stage. In the presence of hCG and EB no block at either metaphase I or at anaphase-telophase I was observed. In the absence of hCG and EB the percentage of oocytes reaching metaphase II was much lower (48.6%) in comparison with oocytes matured in the presence of these hormones (79.1 %). It was concluded a very high proportion of slaughterhouse oocytes could be matured in vitro and that the cumulus investments and addition of certain hormones affected the maturation rate.     

激素和血清对牛卵母细胞体外成熟的影响

卵母细胞的体外成熟直接关系到体外受精胚胎的数量和质量,从而影响到胚胎移植的成功率。本文就促性腺激素、类固醇激素和血清对牛卵母细胞体外成熟的影响,进行了较为详尽的阐述。     

Presence of chlorogenic acid during in vitro maturation protects porcine oocytes from the negative effects of heat stress

Chlorogenic acid (CGA) is known to protect oocytes from oxidative stress. Here we investigated the effects of CGA on porcine oocyte maturation under heat stress and subsequent embryonic development after parthenogenetic activation. For in vitro maturation (IVM) at 41.0°C (hyperthermic condition), supplementation of the maturation medium with 50 μM CGA significantly improved the percentage of matured oocytes and reduced the rate of apoptosis relative to oocytes matured without CGA (p < .05). CGA treatment of oocytes during IVM under hyperthermia tended to increase (p < .1) percentage of blastocyst formation after parthenogenesis and significantly increased (p < .05) the total cell number per blastocyst relative to oocytes matured without CGA. For IVM at 38.5°C (isothermic condition), CGA significantly improved the rate of blastocyst development compared with oocytes matured without CGA (p < .05), but did not affect oocyte maturation, apoptosis rate or the number of cells per embryo. Omission of all antioxidants from the IVM medium significantly reduced the rate of oocyte maturation, but the rate was restored upon addition of CGA. These results demonstrate that CGA is a potent antioxidant that protects porcine oocytes from the negative effects of heat stress, thus reducing the frequency of apoptosis and improving the quality of embryos.     

牛卵泡液对牛卵母细胞体外成熟及受精胚发育力的影响

研究了牛卵母细胞体外成熟液和胚胎培养液中添加不同浓度的牛卵泡液对其体外成熟率和受精胚发育力的影响。结果表明:添加10%牛卵泡液的实验组,卵母细胞的成熟率与血清对照组没有显著差异(P>0.05);添加10%卵泡液的实验组与血清对照组相比,卵裂率和囊胚率没有显著差异,却显著高于添加5%和20%牛卵泡液的实验组(P<0.01),且各实验组囊胚内细胞数差异不大(P>0.05)。因此,用10%的牛卵泡液可以取代成熟液和胚胎培养液中的血清,并可降低实验成本。