In vitro fertilisation of in vivo matured porcine oocytes obtained from prepuberal gilts at different time intervals after hCG injection

The goal of the present study was to find out the best interval after hCG injection in PMSG primed prepuberal gilts for retrieval of in vivo matured oocytes for in vitro fertilisation (IVF). Altogether 66 gilts were superovulated with 1500 IU PMSG and 500 IU hCG 72 h later. Ovum pick up was performed endoscopically 24, 28, 32 or 36 h after hCG and a total of 869 cumulus-oocyte-complexes (COCs) were aspirated from 1400 follicles. COCs were tested for quality, and an aliquot was immediately fixed and stained to determine meiotic configuration. The remaining COCs were fertilised in vitro using frozen-thawed epididymal semen. Quality and developmental stage of embryos were tested after IVF, and the number of nuclei was counted. At 24 to 32 h after hCG only few oocytes have entered the second meiotic cycle (18 to 25% vs. 58% at 36 h, p < 0.05). The overall cleavage rate was significantly influenced by insufficient maturation rate at the early collection times (14% at 24 h vs. 49% at 36 h). Additionally, when oocytes were collected 24 to 32 h vs. 36 h the cleavage rate based on mature oocytes was lower (26 vs. 62%, p < 0.05). Once embryonic development has been initiated, the further in vitro development to blastocyst stages did not differ between groups. However, the number of cells was lower at collection times 24 to 32 h as compared to 36 h after hCG (12 to 15 cells vs. 22 cells, p < 0.05). The results indicate that the time of COC collection affects the in vitro developmental competence up to the blastocyst stage and should not be performed earlier than 36 h after hCG treatment.     

Effect of Oocytes in vivo and in vitro on the Developmental Potential of Porcine Somatic Cell Cloned Embryos

To investigate the impact of porcine oocytes in vivo and in vitro maturation (IVM) on the development of porcine somatic cell cloned embryos,the somatic cell cloned embryos cultured in vitro and the sows were treated with hormones to collect mature oocytes in vivo,and the cleavage rate, blastocyst rate and embryo implantation were compared. The results showed that the average number of ovulation in PGC+PMSG+HCG group was significantly higher than that of PGC+HCG,PMSG+HCG and the natural estrus groups (P<0.05). The oocytes collected in vivo could be used for the construction, and the available oocytes rate reached more than 90%,and there was no significant difference among the four groups (P>0.05),which indicated that groups treated by hormone could obtain more available oocytes and the quality of oocytes was not significant different. In vivo and in vitro matured oocytes were used as nuclear transfer embryos of recombinant receptor,the fusion efficiency (80.31% and 79.29%) and cleavage rate (90.40% and 86.51%) were not significant different (P>0.05), but the proportion of in vivo matured oocytes cloned embryos developed into the blastocyst stage was significantly higher (P<0.05). The reconstructed embryos made from in vivo and in vitro matured oocytes were transplanted into surrogate sows (transferred 30 or 60 embryos),10 piglets were born in in vivo maturation of cloned embryo transfer group,while there was no implantation in in vitro maturation of cloned embryo transfer group. The results showed that high quality oocytes obtained by superovulation could significantly increase the blastocyst rate of embryos,reduce the number of embryos transferred and improve the pregnancy rate of surrogate sows.     

Electrical activation enhances pre-implantation embryo development following sperm injection into in vitro matured pig oocytes

The objective of this study was to evaluate the effect of electrical stimulation (EST) on pronuclear formation, chromosomal constitution, and developmental capability among in vitro matured pig oocytes following intracytoplasmic sperm injection (ICSI). After ICSI, the oocytes were randomly distributed and cultured into 3 groups: the EST activated ICSI group, non-activation ICSI group, and in vitro fertilization (IVF) group. The proportion of oocytes in which 2 pronuclei were formed in ICSI groups was significantly higher in the former groups than in the IVF group (96.2 and 93.5 vs. 64.5%, respectively, P<0.05). The cleavage rate was significantly higher in EST activated ICSI group (78.6%) than in the IVF and non-activated ICSI groups (51.8 and 46.0%, respectively, P<0.05), as was the proportion of oocytes that developed to the blastocyst stage at day 7 (18.9 vs. 11.6 and 9.1%, respectively, P<0.05). Diploid blastocysts were observed in 52.4, 63.0, and 65.2% of oocytes in the IVF, activated, and non-activated ICSI groups, respectively. Eight out of 23 gilts (34.8%) were confirmed to be pregnant in activated ICSI groups, but none of these pregnancies were carried to term. These results show that oocyte activation after ICSI is effective in elevating the cleavage rate and blastocyst development, while ensuring normal chromosome composition. Further research is needed to determine the pregnancy maintenance requirements for ICSI-embryos in pigs.     

不同剂量PMSG和hCG对小鼠超排效果的影响研究

选用6周龄雌小鼠48只，随机分为4组，分别用2．5IU、5．0IU、7．5IU、10．0IU的PMSG注射，48h后分别注射hCG2．5IU、5．0IU、7．5IU、10．0IU：次日早晨检栓，60h后冲胚。总见栓率为81．25％（39／48）。冲胚结果：2．5IU、5．0IU、7．5IU和10．0IU剂量组平均每只获胚分别为9．17（110／12）枚、32．67（392／12）枚、16．00（92／12）枚、11．67（140／12）枚，各组超排效果差异极显著（P〈0．01）。结果表明：采用5．0IU PMSG＋5．0IU hCG对小鼠超排效果最好，输卵管分段冲胚法可行性良好。     

体内、外卵母细胞对猪体细胞克隆胚胎发育潜力的影响

为探讨猪体内、外成熟卵母细胞对核移植重组胚胎发育能力的影响,试验通过激素促排获得体内成熟卵母细胞和收集废弃卵巢获取体外成熟的卵母细胞,分别构建核移植重组胚,比较其卵裂率、囊胚率及胚胎移植受孕情况。结果显示,PGC+PMSG+HCG组的平均排卵数（27.8枚/头）显著高于PGC+HCG （12.5枚/头）、PMSG+HCG （13.7枚/头）及自然发情组（11.5枚/头）（P<0.05）,体内收集到的卵母细胞,可用于构建核移植重组胚的可用卵率均达到90%以上,与其他处理组差异不显著（P>0.05）,说明通过激素处理可获得更多的可用卵母细胞,而且卵母细胞的质量没有显著差异;以体内和体外成熟卵母细胞作为核移植受体构建的克隆胚胎,二者的胚胎融合率（80.31%和79.29%）和卵裂率（90.40%和86.51%）差异均不显著（P>0.05）,但来自体内成熟卵母细胞克隆的胚胎发育至囊胚期的比例显著升高（P<0.05）;将体内、外成熟卵母细胞构建的核移植重组胚分别移植代孕母猪,头平均移植30或60枚时,体内成熟卵母构建的克隆胚胎移植出生仔猪10头,而体外培养卵母细胞构建的克隆胚胎均未着床受孕,表明通过激素促排获得的卵母细胞质量更好,能显著提高克隆胚胎的囊胚率,减少胚胎移植数量,提高代孕母猪的怀孕率。     

Optimization of in vitro embryo production and zygote vitrification for the indigenous Vietnamese Ban pig: The effects of different in vitro oocyte maturation systems

The Vietnamese Ban pig is a precious genetic resource that needs to be preserved. In vitro embryo production from in vitro matured (IVM) oocytes is an important tool for the utilization of cryopreserved porcine sperm. The aim of this study was to compare two media for the IVM of Ban pig oocytes. Immature oocytes were subjected to IVM either in a non‐defined (TCM‐199 + pig follicular fluid) or in a defined base medium (POM + epidermal growth factor). At the end of IVM, the oocytes were in vitro fertilized (IVF) with frozen Ban sperm. Ten hours after IVF, the oocytes were either subjected to orcein staining to check fertilization and maturation status or cultured in vitro for 7 days. There was no difference between the two IVM media in terms of percentages of oocyte maturation and blastocyst production. However, the percentage of male pronuclear formation after IVF and the total cell numbers in blastocysts were higher with the defined system. Zygotes obtained by the two IVM systems survived vitrification at similar rates. In conclusion, the two IVM systems were both effective for the production of Ban pig embryos; however, better embryo quality was achieved with the defined one.     

影响辽宁绒山羊母羔胚胎体外生产因素的研究

比较了PMSG+FSH、FSH+PMSG超排方法对超排效果与胚胎发育的影响,EGF对卵母细胞成熟及胚胎发育的影响,以及TALP、SOF受精体系对胚胎发育的影响。结果表明:PMSG+FSH法超排后的卵裂率、囊胚率极显著高于FSH+PMSG法(P<0.01),但超排卵母细胞数差异不显著(P>0.05);成熟液添加20 ng/mL EGF组极体率、囊胚率显著高于对照组(P<0.05),卵裂率差异不显著(P>0.05);SOF受精体系卵裂率显著高于TALP(P<0.05),囊胚率极显著高于TALP(P<0.01)。     

卵丘细胞对猪卵母细胞体外成熟及孤雌胚胎体外发育的影响

为探讨表皮生长因子(epidermal growth factor,EGF)的添加浓度及脱卵丘细胞时间对猪卵母细胞体外成熟及孤雌胚胎体外发育的影响.试验通过在体外成熟液中添加不同浓度(0、10、15、20、30、40 ng/mL)的EGF来研究其对培养44 h的卵母细胞成熟率以及孤雌胚胎发育的影响;在培养开始后的不同时间(18、24、38、44 h)进行脱卵丘细胞处理来研究不同时间脱卵丘处理对培养44 h的卵母细胞成熟率以及孤雌胚胎发育的影响.结果表明,成熟培养基中添加10 ng/mL EGF能显著提高卵母细胞的卵裂率和囊胚率(P <0.05).共培组和独培组卵母细胞培养18 h后脱卵丘细胞成熟率均低于44 h,但差异不显著(P >0.05);共培组卵母细胞培养18 h后脱卵丘细胞的卵裂率和囊胚率显著高于培养44 h(P <0.05);独培组卵母细胞培养18 h后脱卵丘细胞的卵裂率与44 h无显著差异(P >0.05),但囊胚率显著高于培养44 h后脱卵丘细胞(P <0.05).添加10 ng/mL EGF对猪卵母细胞体外成熟及孤雌胚胎体外发育较好;卵母细胞培养18 h后脱卵丘细胞可提高孤雌胚胎早期发育能力.     

Quality and Developmental Ability of Dromedary (Camelus dromedarius) Embryos Obtained by IVM/IVF, In Vivo Matured/IVF or In Vivo Matured/Fertilized Oocytes

The effect of source of cumulus-oocytes-complexes (COCs), maturation and fertilization conditions on developmental competence of dromedary embryos was examined. Thirty-six adult females were superovulated with equine Chorionic Gonadotropin (eCG) injection (3500 IU, IM) and divided in three groups of 12 females each. Group 1 provided 138 COC's collected from follicles >or= 5 mm 10 days after stimulation prior hCG treatment and matured in vitro for 30 h. Group 2 provided 120 in vivo matured oocytes which were aspirated from their follicles 20 h after hCG (3000 IU, IV) given on day 10 follow eCG injection. Group 3 provided 65 in vivo matured/fertilized oocytes. Females in Group 3 received hCG on day 10 following eCG treatment and then were mated 24 h later. Fertilized oocytes were collected from the oviducts of females 48-h post-mating. Quality of the oocytes was assessed after in vitro maturation (IVM), in vitro fertilization (IVF) and in vitro culture (IVC) of COCs. All cultures were performed in three replicates (n = 3) at 38.5 degrees C, under 5% CO(2) and high humidity (>95%). Only COCs with cumulus and homogenous (dark) cytoplasm were used. Nuclear maturation rate for Groups 1 and 2 was determined by epifluorescence microscopy in a sample of COCs (n = 30) denuded, fixed and stained with Hoechst 33342. To study the viability of obtained embryos, hatched blastocysts from each group were transferred to recipients followed by pregnancy diagnosis using ultrasonography at 15, 60 and 90 days. The percentage of COCs reaching metaphase II (MII) after 30 h of maturation was slightly but not significantly higher for in vivo matured oocytes (28/30; 93%) than those in vitro matured (25/30; 84%). The total rate of cleavage (2 cells to blastocyst stage) was not different for the three groups. However, significantly (p < 0.05) more blastocyst and hatched blastocysts were obtained from in vivo matured and in vivo fertilized oocytes (Group 3; 52% and 73%) than from in vitro fertilized oocytes whether they were matured in vitro (Group 1; 35% and 32%) or in vivo (Group 2; 32% and 45%). Pregnancy rates were not significantly different amongst all groups for the three first months following embryo transfer. All pregnancies were lost after day 90 follow transfer except for in vivo matured and in vivo matured/fertilized groups. Only in vivo matured/in vitro fertilized and in vivo matured/fertilized produced embryos continued normal development until term and resulted in the birth of normal and healthy live calves. Six claves (29%; 6/21) were born from Group 3 and one (8%; 1/13) calf was born from Group 2. This study shows that the IVC system used is able to support camel embryo development. However, developmental competence and viability of dromedary embryos may be directly related to the intrinsic quality (cytoplasmic maturation) of oocytes.     

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.     

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。     

Delay in Cleavage of Porcine Embryos after Intracytoplasmic Sperm Injection (ICSI) Shows Poorer Embryonic Development

In pigs, the embryonic developmental ability after intracytoplasmic sperm injection (ICSI) is inferior to that resulting from in vitro fertilization (IVF). We evaluated the timing of cell division up to blastocyst formation on embryonic development after ICSI using either whole sperm (w-ICSI) or the sperm head alone (h-ICSI) and IVF as a control. At 10 h after ICSI or IVF, we selected only zygotes, and each of the zygotes/embryos was evaluated for cleavage every 24 h until 168 h. We then observed a delay in the 1st and 2nd cleavages of h-ICSI embryos and also in blastocoele formation by w-ICSI embryos in comparison with IVF embryos. The rate of blastocyst formation and the quality of blastocysts in both ICSI groups were inferior to those in the IVF group. In conclusion, the delay in cleavage of porcine ICSI embryos shows poorer embryonic development.     

Development of in vitro produced porcine embryos according to serum types as macromolecule

This study was conducted to establish an in vitro maturation (IVM) system by selection of efficient porcine serum during porcine in vitro production. To investigate the efficient porcine serum (PS), different types of PS [newborn pig serum, prepubertal gilt serum (PGS), estrus sow serum, and pregnancy sow serum] were used to supplement IVM media with or without gonadotrophin (GTH) and development rates of parthenogenetic activation (PA) and in vitro fertilization (IVF) embryos were then compared. The maturation rates of the PGS group was significantly higher when GTH was not added. Additionally, during development of PA embryos without GTH, the PGS group showed significantly higher cleavage and blastocyst formation rates. Moreover, the cleavage rates of IVF embryos were significantly higher in the PGS group, with no significant differences in the blastocyst formation. However, when GTH was supplemented into the IVM media, there were no significant differences among the four groups in the cleavage rates, development rates of the blastocyst, and cell number of the blastocyst after PA and IVF. In conclusion, PGS is an efficient macromolecule in porcine IVM, and GTH supplementation of the IVM media is beneficial when PS is used as macromolecule, regardless of its origin.     

猪植入前胚胎体外培养条件的优化

探讨了更换胚胎培养液及添加FBS、高渗透压和不同浓度VE对猪卵母细胞体外受精(IVF)和孤雌激活(PA)胚胎体外发育的影响,进一步优化了猪植入前胚胎体外培养体系。试验一：在第2天、第4天更换新的培养液(换液组),在换液基础上第4天更换为添加10%FBS的培养液(FBS组)。试验二：胚胎分别在0.05 mol/L蔗糖(蔗糖组)和138 mmol/L氯化钠(氯化钠组)的PZM-3(300～320 mOsmol)中培养2 d后移至PZM-3(288 mOsmol)中培养5 d。试验三：在培养液中分别添加50、100和200 μmol/L VE。对照组均在PZM-3(288 mOsmol)中培养7 d。结果表明：试验一,IVF和PA胚胎FBS组囊胚率显著高于对照组和换液组(P<0.05);试验二,IVF胚胎氯化钠组卵裂率、囊胚率均显著高于对照组与蔗糖组(P<0.05);试验三,IVF胚胎添加100 μmol/L VE组囊胚率显著高于对照组(P<0.05)。结果提示,在换液的基础上添加FBS有利于猪IVF和PA胚胎的体外发育;氯化钠调节的高渗透压可以促进猪IVF胚胎的早期发育;添加100 μmol/L VE可以改善猪IVF胚胎的体外发育体系。     

Effects of delipidation and oxygen concentration on in vitro development of porcine embryos

The effects of delipidation and the oxygen (O(2)) concentration in the atmosphere during culture on in vitro development and H(2)O(2) content were investigated in porcine in vivo fertilized embryos and embryos after in vitro maturation and in vitro fertilization (IVM/IVF embryos). There was no significant difference in the developmental rates to the blastocyst stage between the intact and delipidated IVM/IVF embryos. However, the mean number of cells in blastocysts derived from delipidated IVM/IVF embryos (19.8 +/- 0.8 cells) was significantly smaller than that from intact embryos (24.2 +/- 1.2 cells). Although there were no significant differences in the developmental rates to the blastocyst stage of intact and delipidated IVM/IVF embryos between the cultures under 5% O(2) and 20% O(2), the developmental rate of intact IVM/IVF embryos cultured under 5% O(2) (27.1%) was significantly higher than that of the delipidated embryos cultured under 20% O(2) (19.3%). On the other hand, there was no difference in the developmental rate to the blastocyst stage between in vivo fertilized embryos cultured under 5% O(2) and 20% O(2). Hydrogen peroxide (H(2)O(2)), one of the reactive oxygen species (ROS), is thought to cause damage to embryos. The H(2)O(2) content per embryo derived from oocytes cultured under 5% O(2) (in vivo fertilized, 58.0 +/- 2.5 pixels; IVM/IVF, 79.6 +/- 3.2 pixels) was significantly lower than that (in vivo fertilized, 100.2 +/- 3.8 pixels; IVM/IVF, 103.9 +/- 3.2 pixels) under 20% O(2). Furthermore, the level of H(2)O(2) in delipidated IVM/IVF embryos (94.7 +/- 3.9 pixels) was significantly lower than that in intact embryos (103.9 +/- 3.2 pixels) cultured under 20% O(2). The present results indicate that the delipidation of porcine IVM/IVF embryos and reduction of the O(2) concentration decreased the H(2)O(2) level rather than the in vitro developmental rate to the blastocyst stage.     

Effects of ghrelin on in vitro development of porcine in vitro fertilized and parthenogenetic embryos

The objective of the present study was to investigate the effect of addition of ghrelin to in vitro culture medium on preimplantation development of porcine in vitro fertilized and parthenogenetic embryos. In Experiment 1, we sought to compare the in vitro developmental competence of IVF and parthenogenetic embryos. No significant (P<0.05) differences were detected for cleavage rate or blastocyst rate between the in vitro fertilization (IVF)- and parthenogenetic activation-derived embryos. In Experiment 2, parthenogenetic embryos were cultured in Porcine Zygote Medium-3 containing various concentrations of ghrelin. The blastocyst rate was remarkably (P<0.05) increased when 5 ng/ml (PA-5) and 500 ng/ml (PA-500) of ghrelin was added to in vitro culture medium compared with the other groups. Total cell number per blastocyst was slightly promoted in the ghrelin treatment groups compared with the controls. However, the ratio of inner cell mass (ICM) cell number/total cell number was significantly reduced in the PA-50 group compared with the controls (P<0.05). In Experiment 3, we cultured in vitro fertilized embryos in Porcine Zygote Medium-3 supplemented with ghrelin at different dosages. The rate of blastocyst formation was markedly (P<0.05) elevated when 500 ng/ml ghrelin was added to culture medium (IVF-500) compared with the controls. Increased total cell numbers (P<0.05) were observed when in vitro fertilized embryos were cultured in IVF-50 and IVF-500 compared with the controls. However, the ratio of ICM cell number/total cell number was decreased in the ghrelin treatment groups compared with the controls (P<0.05). Taken together, the results suggest that ghrelin can enhance blastocyst formation of porcine in vitro fertilized and parthenogenetic embryos while exerting a negative effect on the structural integrity of the blastocysts.     

ICSI受精卵胞质注射生产转基因猪胚胎的初步研究

试验旨在探讨单精子胞浆内注射（intracytoplasmic sperm injection,ICSI）法生产猪体外受精卵和应用猪ICSI受精卵进行胞质注射生产转基因胚胎的可行性。首先对比了猪体外受精（in vitro fertilization,IVF）受精卵与ICSI受精卵的胚胎发育效率;然后观察了猪ICSI受精卵的双原核形成时间及效率,对精子注射到胞质后6~18 h分6个时间段进行地衣红染色,对比精子进入卵胞质后的状态及原核形成;最后对猪IVF受精卵受精后8~10 h及ICSI受精卵受精后12~14 h进行EGFP-N1质粒（20 ng/μL）胞质注射,观察胚胎发育效率及转基因效率。结果表明,ICSI受精卵的胚胎发育率（卵裂率89.4%和67.9%、囊胚率36.5%和16.1%）显著优于IVF组（P<0.05）,适合用于猪的体外受精卵试验;猪ICSI受精卵双原核在精子注射到卵胞质后12~14 h形成,双原核形成率为54.90%,显著高于其余5个试验组（P<0.05）;ICSI受精卵胞质注射组胚胎卵裂率（86.2%和66.3%）、囊胚率（30.0%和13.6%）及转基因效率（18.5%和0）均显著高于IVF受精卵胞质注射试验组（P<0.05）。本试验结果为采用ICSI受精卵进行胞质注射生产转基因猪的研究奠定了基础。     

水牛卵母细胞和体外受精早期胚胎端粒酶活性测定

为了解水牛卵母细胞和体外受精（IVF）胚胎早期发育过程中端粒酶的活性变化,本研究利用端粒重复序列扩增法（TRAP）进行了水牛未成熟卵母细胞,成熟卵母细胞和2～4细胞,8～16细胞,桑椹胚以及囊胚各阶段的早期胚胎端粒酶活性的测定。依据电泳条带在成像系统下的光密度值,计算端粒酶的相对活性（RTA）。结果发现,未成熟卵母细胞端粒酶活性比成熟卵母细胞高（P〈0.05）,受精后2～4和8～16细胞胚胎端粒酶活性相对较低,桑椹胚端粒酶活性明显升高（P〈0.05）,囊胚阶段达到最高水平。通过对水牛不同发育阶段胚胎细胞数计数及单细胞相对端粒酶活性的分析比较结果显示,卵母细胞的单细胞端粒酶活性最高,囊胚阶段的最低。单细胞端粒酶活性从未成熟卵母细胞到IVF囊胚阶段呈逐渐降低的趋势。这些结果表明,水牛卵母细胞及早期胚胎的端粒酶活性变化与其成熟、发育阻断及全能性的逐步降低有关。     

单宁酸对猪卵母细胞体外成熟及胚胎发育能力的影响

研究旨在探讨单宁酸对猪卵母细胞体外成熟质量及其胚胎发育能力的影响。在猪卵丘卵母细胞复合体（COCs）体外成熟培养液中添加不同浓度（0、1、10、100 μg/mL）单宁酸培养42 h后,检测COCs的扩散程度和卵丘细胞扩散指数,统计COCs的体外成熟率,检测成熟卵母细胞内谷胱甘肽（glutathione,GSH）、活性氧（reactive oxygen species,ROS）和生长分化因子9（growth differentiation factor 9,GDF9）的水平,并统计孤雌激活及体外受精胚胎48和168 h的卵裂率、囊胚率及囊胚总细胞数。结果显示,与对照组相比,10 μg/mL单宁酸组卵丘细胞扩散指数显著提高（P<0.05）,100 μg/mL单宁酸组显著降低（P<0.05）;1和10 μg/mL单宁酸组卵母细胞成熟率差异不显著（P>0.05）,100 μg/mL单宁酸组卵母细胞成熟率显著降低（P<0.05）;1和10 μg/mL单宁酸组GSH和GDF9水平显著提高（P<0.05）,ROS水平显著降低（P<0.05）。孤雌胚胎和体外受精胚胎发育能力结果显示,与对照组相比,各单宁酸组卵裂率差异不显著（P>0.05）,10 μg/mL单宁酸组孤雌胚胎囊胚率及体外受精胚胎囊胚率显著提高（P<0.05）,100 μg/mL单宁酸组孤雌胚胎囊胚细胞数及体外受精胚胎囊胚细胞数均显著低于其他各组（P<0.05）。以上结果表明,10 μg/mL单宁酸可通过提高卵丘细胞扩散能力及GSH和GDF9水平、降低卵母细胞内ROS水平,改善猪卵母细胞成熟质量,提高孤雌胚胎及体外受精胚胎的发育能力。     

Effect of human chorionic gonadotropin on preovulatory luteinizing hormone surge and ovarian hormone secretion in gilts

The influence of varying doses of human chorionic gonadotropin (hCG) on the preovulatory luteinizing hormone (LH) surge, estradiol-17 beta (E2) and progesterone (P4) was studied in synchronized gilts. Altrenogest (AT) was fed (15 mg X head-1 X d-1) to 24 cyclic gilts for 14 d. Pregnant mares serum gonadotropin (PMSG; 750 IU) was given im on the last day of AT feeding. The gilts were then assigned to one of four groups (n = 6): saline (I), 500 IU hCG (II), 1,000 IU hCG (III) and 1,500 IU hCG (IV). Human chorionic gonadotropin or saline was injected im 72 h after PMSG. No differences in ovulation rate or time from last feeding of AT to occurrence of estrus were observed. All gilts in Groups I and II expressed a preovulatory LH surge compared with only four of six and three of six in Groups III and IV, respectively. All groups treated with hCG showed a rapid drop (P less than .01) in plasma levels of E2 11, 17, 23 h after hCG injection when compared with the control group (35 h). The hCG-treated gilts exhibited elevated P4 concentrations 12 h earlier than the control group (3.1 +/- .5, 3.4 +/- .72, 3.1 +/- .10 ng/ml in groups II, III and IV at 60 h post-hCG vs .9 +/- .08 ng/ml in group I; P less than .05). These studies demonstrate that injections of ovulatory doses of hCG (500 to 1,500 IU) had three distinct effects on events concomitant with occurrence of estrus in gilts: decreased secretion of E2 immediately after hCG administration, failure to observe a preovulatory LH surge in some treated animals and earlier production of P4 by newly developed corpora lutea.