Ascorbic Acid Improves the Developmental Competence of Porcine Oocytes After Parthenogenetic Activation and Somatic Cell Nuclear Transplantation

In this study, a dose-response assessment was performed to understand the relation between supplementation of media with L-ascorbic acid or vitamin C and porcine oocyte maturation and the in vitro development of parthenotes (PA) and handmade cloned (HMC) embryos. Various concentrations (0, 25, 50 and 100 µg/ml) of vitamin C supplemented in in vitro maturation (IVM) and culture (IVC) media were tested. None of these vitamin C additions affected nuclear maturation of oocytes, yet supplementation at 50 µg/ml led to significantly increased intracellular glutathione (GSH) levels and reduced reactive oxygen species (ROS). When cultured in IVM- and/or IVC-supplemented media, the group supplemented with 50 µg/ml of vitamin C showed improved cleavage rates, blastocyst rates and total cell numbers per blastocyst (P<0.05) compared with other groups (control, 25 µg/ml and 100 µg/ml). In contrast, supplementation with 50 µg/ml vitamin C decreased (P<0.05) the apoptosis index as compared with the groups supplemented with 100 µg/ml. In addition, even with a lower blastocyst rate to start with (37.6 vs. 50.3%, P<0.05), supplementation of HMC embryos with vitamin C ameliorated their blastocyst quality to the extent of PA embryos as indicated by their total cell numbers (61.2 vs. 59.1). Taken together, an optimized concentration of vitamin C supplementation in the medium not only improves blastocyst rates and total cell numbers but also reduces apoptotic indices, whereas overdosages compromise various aspects of the development of parthenotes and cloned porcine embryos.     

Synergistic Effect of Porcine Follicular Fluid and Dibutyryl Cyclic Adenosine Monophosphate on Development of Parthenogenetically Activated Oocytes from Pre‐Pubertal Gilts

This study investigated the effect of porcine follicular fluid (PFF) and dibutyryl cyclic adenosine monophosphate (dbcAMP) during in vitro maturation (IVM) of porcine oocytes on meiotic maturation, fertilization and embryo development, and compared the effect of supplementing the embryo culture media with PFF or foetal bovine serum (FBS) on embryo development. Oocytes from pre‐pubertal gilts were IVM for 44 h, and parthenogenetically activated or in vitro‐fertilized. Embryos were cultured in porcine zygote medium (PZM3) for 7 days. Cleavage and blastocyst rates were evaluated at 48 h and 7 days of culture. The supplementation of the IVM medium with 25% PFF and 1 mm dbcAMP for the first 22 h resulted in more (p < 0.05) embryos developing to the blastocyst stage as compared with the inclusion of dbcAMP alone. The dbcAMP + PFF combination increased (p < 0.05) the average number of nuclei per blastocyst as compared with either of these components alone or in its absence. A synergistic effect of dbcAMP + PFF during IVM was also reflected in the capacity of oocytes to regulate sperm penetration and prevent polyspermy, as twice as many oocytes from the control group were penetrated by more than one sperm as compared with those matured in the presence of both dbcAMP and PFF. The supplementation of PZM3 with 10% FBS from days 5 to 7 of culture significantly improved the total cell quantity in embryos derived either from control or dbcAMP + PFF matured oocytes. There was no effect on the total cell quantity when FBS was replaced by the same concentration of PFF. These studies showed that dbcAMP, PFF and FBS can improve both the quantity (57.3% vs 41.5%) and quality (74.8 vs 33.3 nuclei) of porcine blastocysts derived from oocytes recovered of pre‐pubertal gilts.     

Energy Status Characteristics of Porcine Oocytes During In Vitro Maturation is Influenced by Their Meiotic Competence

The characteristics of energy status in porcine oocytes as related to their meiotic competence and in vitro maturation were studied. Cycling pubertal gilts in the early luteal to early follicular phases of the ovarian cycle were used as oocyte donors. The oocytes recovered from medium (MF) or small follicles (SF) were considered meiotically more or less competent, respectively. A half of oocytes from each category was matured by the standard protocol. The oocytes were examined before or after maturation by confocal microscopy, a bioluminescent cell assay and Western blotting. Four experiments, each in triplicate, were performed to assess both SF and MF oocytes in terms of metabolic units formed by mitochondria and lipids, ATP and lipid consumption and lipid droplets with adipose differentiation‐related protein (ADRP) expression. The proportion of oocytes with metabolic units, the mean ATP content and the number of lipid droplets per oocyte, and the relative number of lipid droplets with ADRP expression were significantly higher in the MF compared to SF oocytes before maturation. On the other hand, after maturation, there was an increase in the proportion of oocytes with metabolic units and the relative number of lipid droplets with ADRP expression in the SF compared to MF oocytes. In conclusion, specific differences in energy characteristics between porcine oocytes with different meiotic competence were found. Meiotically more competent oocytes are more advanced in terms of energy reserves before maturation, while meiotically less competent oocytes are more active in replenishing energy stores during maturation.     

Effects of KDM1A on the Meiotic Maturation and Developmental Potential of Yak Oocytes

The purpose of this study was to explore the effects of KDM1A on the meiotic maturation and developmental potential of yak oocytes. The specific inhibitor GSK-KDM1A of KDM1A was added into in vitro maturation medium of yak oocytes. After 24 h in vitro culture of yak cumulus oocyte complexes (COCs), the expansion of cumulus cells and the extrusion of the first polar body were observed. The expression level of KDM1A in oocyte was measured by immunofluorescence during in vitro culture. The expression levels of Kdm1a, Oct-4, Sox-2 and Nanog in oocytes were detected by RT-qPCR. Then, yak oocytes were fertilized after in vitro culture, and the cleavage rate and blastocyst formation rate were observed, respectively. The results showed that the cumulus cells expansion in GSK-KDM1A groups were significant lower than those in control group (P<0.05) after 24 h culture, and the cumulus cells expansion and the first polar body extrusion rate in 320 nmol·L^-1 group were significantly lower than those in 160 nmol·L^-1 group (P<0.05). During oocyte in vitro maturation, Kdm1a showed dynamic expression profile, and the expression level of Kdm1a in MⅠ-stage was significantly lower than that in GV-stage and MⅡ-stage (P<0.05). The GSK-KDM1A could significantly inhibit the expression of KDM1A protein in oocytes (P<0.05), and the expression of KDM1A in 320 nmol·L^-1 group was significantly lower than that in 160 nmol·L^-1 group (P<0.05). The expression levels of Oct-4 and Sox-2 in GSK-KDM1A group were significantly higher than that in control group (P<0.05), but there was no significant difference in the expression of Nanog (P>0.05). After 24 h culture, the cleavage rates of oocytes in GSK-KDM1A groups were significantly lower than those in control group (P<0.05), while the blastocyst formation rate was not significantly different (P<0.05). In conclusion, KDM1A is involved in regulating the meiotic maturation process of yak oocytes. GSK-KDM1A can effectively inhibit the expression of KDM1A, affect the meiotic maturation and developmental potential of oocytes, which reveal that KDM1A plays an important role in this process.     

KDM1A对牦牛卵母细胞减数分裂成熟及其发育潜能的影响

旨在探讨KDM1A对牦牛卵母细胞减数分裂成熟及其发育潜能的影响。本研究在体外成熟液中添加不同浓度的KDM1A特异性抑制剂GSK-KDM1A,牦牛卵丘-卵母细胞复合体（COCs）体外培养24 h后,观察卵丘细胞的扩展和第一极体的排出情况;利用免疫荧光检测体外培养过程中卵母细胞内KDM1A的表达模式;采用实时荧光定量PCR检测体外培养卵母细胞内Kdm1a、Oct-4、Sox-2以及Nanog的表达水平;体外培养成熟后的牦牛卵母细胞进行体外受精,观察其卵裂率与囊胚形成率。结果显示,体外培养24 h后,GSK-KDM1A组的卵丘细胞扩展程度显著低于对照组（P<0.05）,而320 nmol·L^-1组的卵丘细胞扩展程度和第一极体排出率均显著低于160 nmol·L^-1组（P<0.05）。在卵母细胞体外成熟过程中,Kdm1a呈现动态表达模式,MⅠ期的表达水平显著低于GV和MⅡ期（P<0.05）;添加GSK-KDM1A能显著抑制卵母细胞中KDM1A蛋白的表达（P<0.05）,320 nmol·L^-1组各时间点KDM1A的表达量均显著低于160 nmol·L^-1组（P<0.05）。GSK-KDM1A组卵母细胞内Oct-4与Sox-2的表达水平显著高于对照组（P<0.05）,但Nanog的表达水平无显著差异（P>0.05）。牦牛卵母细胞体外成熟后,GSK-KDM1A组的卵裂率显著低于对照组（P<0.05）,但囊胚形成率无显著变化（P>0.05）。综上表明,KDM1A参与调控牦牛卵母细胞减数分裂成熟过程,GSK-KDM1A能有效抑制KDM1A的表达,影响卵母细胞减数分裂成熟及其发育潜能,揭示KDM1A在此过程中扮演重要角色。     

糖对牦牛卵母细胞体外成熟及其发育能力的影响

试验旨在研究不同种类、不同浓度的糖对牦牛卵母细胞体外成熟和发育能力的影响,进一步探索和优化牦牛卵母细胞培养体系,提高卵母细胞体外成熟和胚胎生产效率。在牦牛卵母细胞成熟液中添加不同浓度（0、5和10 mmol/L）的葡萄糖或蔗糖,培养24 h或预培养2 h后移入无糖培养基中继续培养22 h,统计卵母细胞体外成熟率及体外受精（IVF）后的胚胎卵裂率和囊胚率。结果显示,与对照组（0 mmol/L）相比,5和10 mmol/L葡萄糖组牦牛卵母细胞核成熟率和体外受精胚胎卵裂率均显著提高（P<0.05）,10 mmol/L葡萄糖组的囊胚率最高,且与对照组相比差异显著（P<0.05）。添加10 mmol/L蔗糖可以显著提高牦牛卵母细胞核成熟率（P<0.05）,但胚胎囊胚率与对照组相比差异不显著（P>0.05）。此外,用10 mmol/L葡萄糖预处理牦牛卵母细胞后其核成熟率、胚胎卵裂率和囊胚率最高,且均显著高于对照组（P<0.05）。由此可见,糖对牦牛卵母细胞体外成熟和发育有一定的影响,在成熟过程中添加适当浓度的糖能提高卵母细胞成熟率及体外受精胚胎发育能力。     

Effect of Sugar on in vitro Maturation and Developmental Competence of Yak Oocytes

This study was designed to investigate the effect of different types and different concentrations of sugar on in vitro maturation(IVM) and developmental competence of yak oocytes, for being further research and optimization culture system of yak oocytes for efficient maturity yak oocytes and productivity of embryos. Immature yak oocytes were matured in vitro on culture medium with different concentrations (0,5 and 10 mmol/L) of glucose and sucrose in incubator for 24 h or 2 h pretreament with sugar and 22 h without sugar. Subsequently, then the maturation of oocytes,the cleavage rates and blastocyst formation rates after in vitro fertilization(IVF) were evaluated. The results showed that a medium with 5 and 10 mmol/L glucose IVM could significantly increase the yak oocytes maturation and cleavage (P<0.05), and the highest blastocyst formation rates in 10 mmol/L glucose group was significantly higher than 0 mmol/L glucose (P<0.05).10 mmol/L sucrose could increase significantly the nucleus maturation rates (P<0.05),and there was no significant difference of the blastocyst formation rates after IVF between 0 and 10 mmol/L sucrose (P>0.05). Furthermore, the nucleus maturation rates,IVF cleavage rates and blastocyst formation rates of yak oocytes which pretreated with 10 mmol/L glucose were the highest in these groups, and were higher than 0 mmol/L glucose (P<0.05). It manifested that the appropriate concentration of sugar could improve the quality of yak oocytes and embryos in vitro developmental competence, so it influenced in vitro development of yak oocytes indirectly.     

玻璃化冷冻对牦牛未成熟卵母细胞发育能力及COC转录组的影响

旨在探讨玻璃化冷冻-解冻对牦牛未成熟卵母细胞发育能力及卵丘-卵母细胞复合体（COCs）转录组的影响,为完善牦牛COCs冷冻保存技术提供理论依据。本研究将未经成熟培养的牦牛COCs进行玻璃化冷冻-解冻后分为2组,A组：COCs体外成熟（IVM）后用普通牛精子进行体外受精（IVF）,获得的受精卵在G-1胚胎培养液中培养72 h后转入G-2培养液培养96 h;B组：IVF后,受精卵在G-1培养液培养120 h后转入G-2培养液培养48 h;以未进行冷冻处理的新鲜COCs作为对照组（C组）：IVF后,受精卵在G-1培养液培养72 h后转入G-2培养液培养96 h。对牦牛新鲜COCs（n=3）和玻璃化冷冻-解冻的COCs（n=3）进行扩增、建库和转录组测序（RNA-seq）分析。结果发现,B组的卵裂率、囊胚率显著高于A组（P<0.05）,但A组和B组的卵裂率、囊胚率均显著低于C组（P<0.05）。以|log₂（fold change）|≥ 2,Q<0.05为阈值,牦牛冻融COCs相对于新鲜COCs共筛选出851个差异表达基因（DEGs）,其中上调846个,下调5个。GO分析表明,DEGs主要富集于生物过程、细胞组分和分子功能3大类;KEGG注释结果表明,DEGs富集到258条通路,其中16条通路显著富集（P<0.05）。研究表明,IVF后在G-1培养液中培养120 h可以提高牦牛玻璃化冷冻卵母细胞的后续发育能力;玻璃化冷冻影响牦牛COCs转录组,从而降低卵母细胞的发育潜力。该发现为完善牦牛COCs玻璃化冷冻技术提供了一定的理论基础。     

Effect of Donor Age on the Developmental Competence of Bovine Oocytes Retrieved by Ovum Pick Up

To study the effect of donor age on oocyte developmental competence and steroid profiles, the crossbred cow (Murray Grey × Brahman) in Yunnan province of China were selected and divided into three groups according to its age. The three groups were young cows (n = 12; 12 months old), middle‐aged cows (n = 15; parity: ≤3 calvings; age: 7–8 years old) and old cows (n = 10; parity: ≥8 calvings; age: ≥15 years old). Cumulus–oocyte complexes (COCs) were collected by 10 consecutive ovum pick up (OPU) sessions with a 4‐day interval between each session, followed by in vitro maturation, fertilization and embryo development. Results showed that cleavage rates (CR) and blastocyst rates (BR) were higher in the young cows than those in the middle‐aged and old cows (p < 0.05). CR and BR from COCs of the first and the fourth OPU sessions were lower than those from other sessions in the young cows and the middle‐aged cows (p < 0.05), whereas the similar phenomenon was not observed in the old cows. Plasma concentrations of oestradiol were higher, and plasma concentrations of progesterone were lower before and during OPU sessions in the young cows compared with those in the same period in the middle‐aged cows or the old cows (p < 0.01). In conclusion, donor age of oocytes could affect developmental competence of oocytes recovered by OPU through the action of steroid hormonal balance on follicle development.     

丁内酯-Ⅰ对绵羊卵母细胞体外成熟及发育能力的影响

研究从丁内酯-Ⅰ(BL-Ⅰ)的浓度筛选、作用时间以及作用解除后的成熟培养时间等方面分别进行了试验,以观察其对绵羊卵母细胞核成熟的抑制程度以及对卵母细胞体外成熟及发育能力的影响.结果表明:150μmol/L的BL-Ⅰ能将62.9%的卵母细胞抑制在GV期.BL-Ⅰ抑制8、16 h和24 h后绵羊卵母细胞停滞在GV期的比率差异不显著(P＞0.05);抑制24 h时卵裂率和囊胚率低于对照组,16 h的囊胚率也低于对照组,抑制8 h的卵裂率和囊胚率与对照组差异不显著(P＞0.05).去除抑制后卵母细胞的成熟能力不受影响.由此可得出,丁内酯-Ⅰ能可逆性抑制绵羊卵母细胞的核成熟,但未能提高其发育潜力.     

生殖激素对奶水牛卵母细胞成熟及发育潜能的影响

本试验以屠宰场获取的奶水牛卵巢为试验材料,收集卵母细胞进行体外成熟培养(IVM)、体外受精(IVF)及早期胚胎培养(IVC)。研究激素(FSH、LH、E2、P4)的不同浓度对奶水牛卵母细胞成熟和早期胚胎发育的影响,以期探讨奶水牛卵母细胞成熟和早期胚胎体外培养发育机制,优选不同激素的最佳浓度。结果表明:添加FSH试验组奶水牛颗粒细胞扩散率和卵裂率高于未添加试验组(P<0.05);添加LH试验组奶水牛颗粒细胞扩散率、卵裂率及8-细胞率与未添加试验组比较,差异不明显(P>0.05);17β-E2试验组(1.0μg/mL)的奶水牛颗粒细胞扩散率、卵裂率及8-细胞率高于未添加试验组(P<0.05);添加P4试验各组(0.9μg/mL、1.2μg/mL)的颗粒细胞扩散率明显低于未添加试验组(P<0.01)。     

丁内酯-I对绵羊卵母细胞体外成熟及发育能力的影响

研究从丁内酯-I(BL-I)的浓度筛选、作用时间以及作用解除后的成熟培养时间等方面分别进行了试验,以观察其对绵羊卵母细胞核成熟的抑制程度以及对卵母细胞体外成熟及发育能力的影响。结果表明:150μmol/L的BL-I能将62.9%的卵母细胞抑制在GV期。BL-I抑制8、16 h和24 h后绵羊卵母细胞停滞在GV期的比率差异不显著(P>0.05);抑制24 h时卵裂率和囊胚率低于对照组,16 h的囊胚率也低于对照组,抑制8 h的卵裂率和囊胚率与对照组差异不显著(P>0.05)。去除抑制后卵母细胞的成熟能力不受影响。由此可得出,丁内酯-I能可逆性抑制绵羊卵母细胞的核成熟,但未能提高其发育潜力。     

Maturation Status, Protein Synthesis and Developmental Competence of Oocytes Derived from Lambs and Ewes

The efficacy of oocyte selection for in vitro embryo production depends on the abundance and diameter of follicles, cumulus layers around the oocytes and subsequent fertilization. Application of `ovum pick-up' technique allows us to utilize partially matured oocytes for embryo production even from juvenile subjects. To compare their developmental competence, oocytes derived from lambs and ewes and cultured in maturation medium for up to 26 h were assessed at 2 h intervals by confocal microscopy after chromatin and microtubulin-specific fluorochrome labelling. Lamb oocytes reached second meiotic metaphase (MII) at lower numbers at 24 h (60.0%) and 26 h (28.6%) whereas 85.7% of adult-derived oocytes attained MII status by 24 h of maturation. Radiolabelling of oocyte proteins revealed higher incorporation of [³⁵S-]-methionine and [³⁵S]-cysteine in adult-derived oocytes compared to lamb oocytes. Although the cleavage rate of lamb oocytes was similar to that of ewe oocytes, the proportion reaching blastocyst stage was significantly lower (p < 0.05) in the lamb-derived oocytes. However, blastocysts from both types of oocytes displayed similar cell lineage allocations to inner cell mass and trophectoderm.     

水牛卵母细胞冷冻保存初步研究

①用EFS30、EFS40、EDFS30、EDFS40四种玻璃化冷冻液对MⅡ期水牛卵母细胞进行毒性试验,结果表明:试验组卵母细胞形态正常率与对照组均无显著性差异(P>0.05);对卵母细胞孤雌激活后EDFS30、EDFS40组的卵裂率与对照组(75.28%)及EFS30、EFS40组差异显著(P<0.05);利用4种冷冻保护剂采用OPS法冷冻保存MⅡ期水牛卵母细胞,其中以EDFS40作为冷冻液时,卵母细胞冷冻解冻后孤雌激活卵裂率最高,达31.60%;以EDFS40作为冷冻液,比较了GMP法和OPS法的冷冻效果,结果表明GMP法冷冻效果好于OPS法。②采用不同预处理时间和平衡时间使用细管法常规冷冻G V期卵母细胞,结果表明预处理5 min、平衡15min组的形态正常率和极体排出率相对较高,分别为72.73%、27.27%。     

Undernutrition and Exogenous Melatonin Can Affect the In Vitro Developmental Competence of Ovine Oocytes on a Seasonal Basis

This study evaluated the effects of exogenous melatonin and level of nutrition on oocyte competence, in vitro fertilization (IVF), and early embryonic development in sheep during seasonal anoestrus (SA) and the reproductive season (RS). Adult Rasa Aragonesa ewes were assigned randomly to one of four treatment groups in two experiments based on a 2 × 2 × 2 factorial design. Individuals were treated (+MEL) or not treated (?MEL) with a subcutaneous implant of melatonin for 42 days and then were fed 1.5 (Control, C) or 0.5 (Low, L) times the daily maintenance requirements for 20 days. Ewes were synchronized and mated at oestrus (Day = 0). On Day 5, ovaries were collected and oocytes were used for IVF. Season had a significant (p < 0.01) effect on the number of oocytes recovered (RS: 19.6 ± 1.0; SA: 14.5 ± 1.0) and the number of healthy oocytes (RS: 13.9 ± 0.7; SA: 9.0 ± 0.7). In the RS, neither nutrition nor melatonin had a significant effect on the evaluated oocytes quality parameters although melatonin implants appeared to reduce the number of unhealthy oocytes in the undernourished group (p < 0.05). During SA, in undernourished ewes exogenous melatonin tended to increase the number of healthy (L+MEL: 9.4 ± 1.0, L?MEL: 7.6 ± 1.4; p < 0.1), and significantly improved both cleaved oocytes (L+MEL: 7.0 ± 0.7, L?MEL: 4.1 ± 0.9; p < 0.05) and blastocyst rate (L+MEL: 37.2, L?MEL: 21.9%; p < 0.05). In conclusion, oocyte competence in ewes was affected by season, and melatonin implants appeared to improve developmental competence in the seasonal anoestrous period, particularly in experimentally undernourished ewes.     

玻璃化冷冻对猪MⅡ卵母细胞皮质颗粒分布及其激活后胚胎发育能力的影响

本实验旨在探讨玻璃化冷冻保存对猪MⅡ期卵母细胞皮质颗粒分布和孤雌激活后早期胚胎发育能力的影响。实验将卵母细胞随机分为对照组、毒性实验组和冷冻组。采用EFS40和EDFS40两种玻璃化冷冻液处理,卵母细胞经恢复后对其进行染色,观察皮质颗粒的分布;并对另一部分卵母细胞实施孤雌激活,观察早期胚胎的发育。结果表明:毒性实验组和冷冻组卵母细胞皮质颗粒部分释放、完全释放的比例无显著差异,但均显著高于对照组(P<0.05)。不同毒性处理组和不同冷冻组间对皮质颗粒的分布无显著差异。与毒性实验组相比,冷冻处理组显著降低皮质颗粒在皮质区分布的比例(P<0.05)。EFS40毒性实验组孤雌激活后的存活率、卵裂率、囊胚发育率均显著高于EDFS40毒性实验组(86.6%vs.75.0%)、(61.8%vs.40.7%)、(30.2%vs.23.5%)(P<0.05)。EFS40冷冻组存活率显著高于EDFS40冷冻组,但均显著低于对照组。结果显示,抗冻保护剂处理和玻璃化冷冻均导致猪卵母细胞皮质颗粒释放,与EDFS40相比采用EFS40较适合猪MⅡ期卵母细胞冷冻保存。     

卵泡直径对辽宁绒山羊卵母细胞体外培养的影响

本试验以屠宰场绒山羊卵巢为材料,采用抽吸法收集不同直径卵泡卵母细胞,研究卵泡直径对卵母细胞回收效果和体外成熟、体外受精的影响。结果表明,卵泡直径直接影响卵母细胞的回收效果及随后的体外成熟、体外受精;卵母细胞的回收率和可用卵的比率及成熟率与卵泡直径呈正相关;直径2.1～5 mm卵泡的卵母细胞由于处于生长旺盛期,受精率和卵裂率分别为59.15%和42.50%,显著高于直径5 mm卵泡和≤2 mm卵泡,适合体外胚胎的生产,而直径≤2 mm卵泡的卵母细胞因未发育充分体外发育潜能最差;直径5 mm卵泡的卵母细胞可能因体外培养成熟过度而老化导致受精率和卵裂率降低,缩短其体外成熟时间可能会提高其体外发育潜能。     

马卵母细胞胞质内精子注射后体外发育能力的研究

本研究在非繁殖季节评估卵丘形态(松散型、致密型)、成熟培养体系(TCM 199、NCSU 23)、体外成熟时间(34、38 h)和离子霉素结合6-DMAP激活对马卵母细胞胞质内精子注射(ICSI)后体外发育能力的影响。从屠宰场采集马卵巢,获得的卵母细胞进行体外成熟,然后注射马冷冻解冻精液,统计分裂情况。试验结果表明,①马松散型卵母细胞成熟率显著高于致密型卵母细胞(P<0.05),分别为61.09%和41.24%,但ICSI后36 h分裂率无显著差异(P>0.05),分别为47.34%和44.92%;②两种培养体系对马松散型或致密型卵母细胞成熟率及ICSI后36 h分裂率无显著影响(P>0.05),但相同成熟体系培养松散型卵母细胞成熟率均显著高于致密型卵母细胞(P<0.05),然而ICSI后36 h分裂率差异不显著(P>0.05);③松散型或致密型卵母细胞在TCM 199或NCSU 23中成熟38 h成熟率均高于34 h成熟率,分别为44.43%～68.87%和34.52%～58.90%,松散型卵母细胞在TCM 199体系中成熟34 h、ICSI后激活组或对照组的分裂率显著高于成熟38 h、ICSI后激活组的分裂率(P<0.05),以及致密型卵母细胞在TCM 199体系中成熟34 h、ICSI后激活组的分裂率(P<0.05),而且显著高于松散型卵母细胞在NCSU 23体系中成熟38 h、ICSI后对照组的分裂率(P<0.05);④ICSI后用离子霉素结合6-DMAP激活对马卵母细胞ICSI后36 h分裂无显著影响(P>0.05)。因此,马松散型和致密型卵母细胞的成熟能力存在差异,TCM 199和NCSU 23成熟体系对这2种类型卵母细胞的发育能力无显著影响(P>0.05),马卵母细胞成熟38 h成熟率高于34 h成熟率,TCM 199成熟体系培养松散型卵母细胞34 h进行ICSI后的分裂率最高。离子霉素结合6-DMAP激活对TCM 199或NCSU 23体系成熟马卵母细胞ICSI后的体外发育能力无显著影响(P>0.05)。     

The Effect of Cyclic Adenosine 3', 5' Monophosphate,Methyl Xanthines and Nicotinic Acid on Plasma Nonesterified Fatty Acids in Sheep

The intravenous injection of 3'', 5'' AMP, 10 mg/kg, caused an immediate rise in blood glucose in normal sheep. In sheep where liver glycogen was depleted by pretreatment with 100 mg/kg of nicotinic acid, the blood glucose response was greatly reduced. The glycogenolytic effect of 3'', 5'' AMP was thus easily demonstrated. The injection of 3'', 5'' AMP was followed by an immediate rise in NEFA, which was most pronounced in the pretreated animals. After the initial increase there was a fall in NEFA in both the normal and the pretreated animals. The role of hyperglycemia for the NEFA response to exogenous 3'', 5'' AMP was discussed. It seemed possible that hyperglycemia may modify the initial increase in NEFA. The later fall in NEFA was discussed as a complex response, which may be the result of the action of several hormones, which may be released by 3'', 5'' AMP injection. Nicotinic acid and pyridyl-3-acetic acid showed a potent antilipolytic activity. Nicotinic acid caused a rebound elevation of NEFA. No rebound was observed after pyridyl-3-acetic acid. Pyridyl-3-acetic acid, however, did not change NEFA when administered orally. Pyridyl-2-acetic acid had no antilipolytic effect. Of the methyl xanthines caffeine and theophylline showed about the same lipolytic activity. Theobromine was quite ineffective.     

开放式拉长细管冷冻法对牛卵母细胞体外受精后发育的影响

在常规牛体外受精(IVF)技术的基础上,分别采用开放式拉长细管 (OPS,open pulled straw)法和细管法对未经成熟培养的卵丘卵母细胞(COCs)进行玻璃化冷冻,解冻后再进行体外成熟(IVM)、IVF和早期胚胎的体外培养(IVC)。结果表明,细管组和 OPS组的解冻后 COCs正常率分别为 59.4%±4.3%和 77.9%±4.1%(P<0 01);成熟率分别为48.2%±5.3%和66.0%±5.8%(P<0 01);卵裂率分别为18.5%±2.0%和32.8%±1.4%(P<0 01);8 细胞阶段的成功率分别为14.8%±2.5%和 24.8%±1.5%(P<0 01);桑椹胚发育率分别为 0 和5 3%±1.1%,明显低于未经冷冻的鲜卵组(21.0%±3.8%;P<0 01);囊胚发育率分别为0和4.0%,明显低于鲜卵组(P<0 01)。说明OPS玻璃化冷冻法可以使未经成熟培养的牛 COCs冷冻后获得桑椹胚和囊胚,但桑囊胚发育率仍较低,方法有待改进。