不同化学激活方法和电激活参数对猪体外成熟卵母细胞早期孤雌发育的影响

利用屠宰场猪卵巢卵母细胞,在体外成熟培养44～48 h后,对核成熟卵母细胞进行激活.试验1为不同化学激活方法10% 乙醇 10 mg/L 放线菌酮组、2.5 mmol/L 氯化锶 10 mg/L放线菌酮组、5 μmol/L离子霉素 2.5 mmol/L 6-二甲氨基嘌呤(6-DMAP)组、200 μmol/L 硫柳汞 8 mmol/L二硫苏糖醇组和对照组(不用任何激活剂).试验2为用不同电场强度和脉冲时程进行电激活之后放入胚胎培养液中进行体外培养7 d.结果显示,(1)4种不同化学激活方法处理卵子的1原核形成率、2原核形成率和原核形成率都显著比对照组高(P＜0.05),其中离子霉素 6-DMAP组的2原核形成率和总原核形成率最高,分别为(23.1±3.5)%和(65.2±3.5)%,显著高于其他处理组(P＜0.05);(2)4种不同化学激活方法处理组的囊胚率都比对照组高(P＜0.01),其中离子霉素 6-DMAP组的卵裂率及囊胚率最高,分别为(46.6±18.5)% 和(5.6±4.2)%;(3)本试验所用的4种不同化学激活方法对猪4-细胞孤雌胚SDS-PAGE电泳的蛋白质表达图谱没有显著影响;(4)电场强度为1.7 kV/cm、脉冲时程为50、70 μs时猪体外成熟卵母细胞激活效果最好,卵裂率、囊胚率、囊胚细胞数分别为(77.4±9.7)%、(12.4±3.7)%、(17.6±5.9)%和(75.1±10.6)%、(12.3±2.6)%、(19.1±8.1).以上结果说明本试验所用的4种化学激活方法均能有效激活猪体外成熟卵母细胞,其中离子霉素 6-DMAP的激活效果最理想;在本实验室条件下,采用1.7 kV/cm的电场强度、50～70 μs的脉冲时程均能有效地激活猪体外成熟卵母细胞;本试验所用的4种不同化学激活方法对猪4-细胞孤雌胚SDS-PAGE电泳的蛋白质表达图谱没有显著影响.     

Effect of cycloheximide on in vitro development of electrically activated feline oocytes

This study was conducted to improve parthenogenetic development in vitro of feline oocytes following a combined activation treatment of electrical stimulation and cycloheximide. In vitro matured (IVM) oocytes were stimulated electrically by a DC electrical pulse of 2 kV/cm for 50 micros. The stimulated oocytes were then incubated in MK-1 medium with or without cycloheximide and subsequently cultured in vitro for 6 days. No significant differences were observed between the two groups with respect to the proportions of cleavage, development to the morula stage, and the cell number of blastocysts. However, exposure of electrically stimulated oocytes to cycloheximide significantly increased the rate of development of the stimulated oocytes into the blastocyst stage compared with oocytes stimulated by electrical stimulation alone (31.0% vs 6.7%). The results from the present study suggested that a single electrical stimulation was insufficient to activate the IVM cat oocytes at 24 h of maturation and that exposure to cycloheximide following electrical stimulation improved the efficacy of the parthenogenetic development of domestic cat oocytes.     

Effect of medium change on the development of in vitro matured and fertilized bovine oocytes cultured in medium containing amino acids

Bovine in vitro matured and fertilized oocytes were cultured for 153 hr in groups of 3 or 30 in 30 microl of modified synthetic oviduct fluid medium supplemented with amino acids. The concentration of ammonium in culture medium at 153 hr of culture was significantly decreased by medium change at 72 hr of culture. However, regardless of embryo density, medium change had no beneficial or detrimental effect on the development of bovine embryos. Increase in the development to blastocysts and production of ammonium were observed when embryos were cultured in groups of 30. These results indicated that the ammonium concentration detected in this culture system has a negligible effect on the development of bovine embryos to blastocysts.     

受精液和受精时间对牦牛卵泡卵母细胞体外受精的影响

利用屠宰牦牛卵巢，抽取其表面2～5mm的卵泡内卵母细胞，经体外成熟后分别用BO液和改良Tyrode’S液进行体外受精研究。结果表明，BO液受精6h和改良Tyrode’s液分别受精6h和18h，牦牛体外受精卵的卵裂率差异不显著（分别为52．48％、47．67％和50．00%,P〉0．05）。它们的4细胞发育率分别为75．47％、78．05％和64．10％，8细胞发育率分别为56．60％、56．10％和48．72％，使用改良Tyrode’s液受精18h的发育率最低，与其他2组相比，差异极显著（P〈0．01）；而受精时间同为6h时，2种受精液之间发育率的差异不显著（P〉0．05）。     

Induction of parturition in the sow with a prostaglandin analogue (I.C.I. 80996)

Summary Parturition was induced in 112 gilts and sows on day 111, 112, and 113 of gestation by means of a single intramuscular injection of 175 mcg of a prostaglandin F2α analogue (Cloprostenol, I.C.I. 80996). No side effects were detected immediately after injection and the course of the induced parturition was normal. The interval between injection and parturition was approximately 28 hours. Induction of parturition on day 113 resulted in a significant shortening of this interval as compared with day 111 and 112. The average weights of the piglets at birth and at 5 weeks were within the normal range. The percentage of stillbirths and the loss of piglets up to weaning did not differ significantly between control and experimental groups. The practical applications of induction of parturition are discussed.     

Induction of parturition in the sow with a prostaglandin analogue (I.C.I. 80996)

Summary

Parturition was induced in 112 gilts and sows on day 111, 112, and 113 of gestation by means of a single intramuscular injection of 175 mcg of a prostaglandin F2α analogue (Cloprostenol, I.C.I. 80996). No side effects were detected immediately after injection and the course of the induced parturition was normal. The interval between injection and parturition was approximately 28 hours. Induction of parturition on day 113 resulted in a significant shortening of this interval as compared with day 111 and 112. The average weights of the piglets at birth and at 5 weeks were within the normal range. The percentage of stillbirths and the loss of piglets up to weaning did not differ significantly between control and experimental groups. The practical applications of induction of parturition are discussed.     

Effects of the preservation medium and storage duration of domestic cat ovaries on the maturational and developmental competence of oocytes in vitro

We examined the effectiveness of saline, Euro-Collins solution (EC), and ET-Kyoto solution (ET-K) as preservation media for the cold storage of feline ovaries. Ovaries were maintained in these media at 4°C for 24, 48, or 72 h until oocyte retrieval. The ET-K group exhibited a higher oocyte maturation rate than the saline group after 72 h of storage. Moreover, ET-K could sustain the competence of the feline oocytes to cleave after 48 h, and the morula formation rate of the ET-K group was higher than that of the other groups after 24 and 48 h. Furthermore, the ET-K group exhibited a higher blastocyst formation rate than the other groups after storage for 24 h, and only ET-K retained the developmental competence in blastocysts after 48 h of storage. In addition, regarding the cell numbers of the blastocysts, there was no significant difference among the tested groups. In conclusion, our results indicate that ET-K is a suitable preservation medium for feline ovaries.     

Effect of replacement of pyruvate/lactate in culture medium with glucose on preimplantation development of porcine embryos in vitro

The effect of glucose supplementation at different times in in vitro culture on the developmental competence of in vitro produced (IVP) porcine embryos was examined. In Experiment 1, when IVP embryos were cultured in modified NCSU-37 supplemented with pyruvate and lactate (IVC-pyr/lac) for 0 h, 24 h, 48 h, 72 h, 96 h, or 118 h and subsequently in modified NCSU-37 supplemented with glucose (IVC-glu) until Day 6 (Day 0=day of in vitro fertilization), the rates of blastocyst formation were significantly higher in embryos cultured in IVC-pyr/lac for 24 or 48 h (24.4% and 23.0%, respectively) than in embryos cultured in IVC-pyr/lac for the whole culture period (14.5%). However, there were no significant differences between embryos obtained after the energy source replacement and embryos cultured in IVC-glu for the whole culture period on the rates (15.2%-24.4%, and 16.8% respectively). Replacement of pyruvate/lactate with glucose at 58 h of culture in Experiment 2 significantly enhanced the rate (31.3%) compared to those after replacement at 48 h, 53 h and 63 h of culture (20.6%, 20.8%, and 21.1%, respectively). In conclusion, replacement of pyruvate/lactate with glucose as the energy substrate was optimal at 58 h of culture for the development of porcine embryos to the blastocyst stage.     

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.     

Effect of group culture and embryo-culture conditioned medium on development of bovine embryos

We investigated the effect of group culture on bovine embryo development, and also investigated the effect of embryo-culture conditioned medium on developmental competence of individually cultured bovine embryos. Slaughterhouse-derived bovine oocytes were matured and fertilized in vitro. The presumptive zygotes were cultured individually or cultured in groups of 2 to 5 embryos with a constant culture density (5 mul/embryo). After 7 days of culture, the rates of embryos developed to the blastocyst stage were significantly higher (P < 0.05) in group cultures of more than 3 embryos/drop than for embryo culture of 1 or 2 embryos/drop. These results suggest a beneficial effect of group culture may be exerted by possible growth promoting factors secreted by embryos. In the next experiment, we investigated the effect of timing of fresh medium replacement on the development of embryos cultured in groups. The blastocyst formation rate was lower when culture medium was replaced freshly on days 2-4 after fertilization than on days 5-6. The blastocyst formation rates of single-cultured embryos were significantly (p < 0.05) increased by the addition of conditioned medium derived from multiple-embryo culture. These results indicate that group culture promotes embryo development and that embryo culture-derived conditioned medium is effective for supporting development of single cultured embryos.     

Stage‐specific effects of osmolarity of a culture medium on development of pig oocytes and miniature pig somatic cell nuclear transfer embryos activated by ultrasound treatment

Whether high osmolarity of a culture medium at the early culture stage affects the development of pig oocytes and miniature pig somatic cell nuclear transfer (SCNT) embryos activated by ultrasound was examined. When oocytes were cultured in modified porcine zygote medium‐3 (mPZM‐3) with increased NaCl to 138 mmol/L (mPZM‐3+NaCl; 326 mOsm) or 50 mmol/L sucrose (mPZM‐3+sucrose; 318 mOsm) for the first 2 days and then cultured in normal mPZM‐3 (273 mOsm) for 5 days, the cleavage and blastocyst formation rates were significantly (P < 0.05) higher than those of oocytes cultured in mPZM‐3 for 7 days. The cleavage and blastocyst formation rates of SCNT embryos cultured in mPZM‐3+NaCl for the first 2 days and then cultured in mPZM‐3 for 5 days were also significantly (P < 0.05) higher than those of embryos cultured in mPZM‐3 for 7 days. These results showed that the high osmolarity of a culture medium induced by increasing NaCl concentration during the first 2 days improves the development of pig oocytes and miniature pig SCNT embryos activated by ultrasound.     

体外受精温度、时间及卵子质量对延边黄牛受精卵体外发育的影响

本研究探讨了体外受精温度、时间及卵子质量对延边黄牛卵母细胞体外受精后体外发育的影响。试验结果表明，体外受精温度为39℃、体外受精时间12h时，卵裂率、囊胚发育率及囊胚孵化率最佳；卵子的质量对受精卵体外发育的影响很大。     

两种采卵方法对绵羊卵母细胞体外受精的影响

研究对获取绵羊卵母细胞不同采集方法进行了初步探讨。切剖法平均每个卵巢可采集得到可用卵母细胞数3．95枚(1953枚／494卵巢),显著高于抽吸法的1．96枚(219枚／112卵巢)。抽吸法和切剖法获得的卵母细胞其体外受精后囊胚率分别是20.21％和18．85％,差异不显著。     

Effect of the in vitro maturation medium on equine oocytes: comparison of follicular fluid and oestrous mare serum

The present study evaluated the effect of supplementing the medium used to mature equine oocytes in vitro with oestrous mare serum (EMS) or horse follicular fluid (HFF). To this end, 144 ovaries were obtained from mares aged 16-21 months and transported to the laboratory in Dulbecco's phosphate buffered saline (D-PBS) at 30 degrees C. Oocytes were harvested from the ovaries by slicing, and then selected for in vitro maturation (IVM) according to the number of cumulus cell layers and the characteristics of the cytoplasm. The selected oocytes were washed three times in TCM199 medium plus HEPES (TCM-199H) or in the same medium plus glutamine (TCM-199G), then matured in vitro in six study groups established according to the in vitro maturation (IVM) treatment to see possible interactions between HEPES and glutamine on other supplements: Ten percent EMS was added to two of these media (TCM-199H+EMS and TCM-199G+EMS) and 10% HFF was added to the media in two other groups (TCM-199H+HFF and TCM-199G+HFF). IVM was performed at 38.5 degrees C for 40 h in a controlled atmosphere (5% CO2, 95% relative humidity). The findings indicate that the presence of EMS or HFF in the TCM-199H medium gives rise to the best results in terms of the proportions of oocytes reaching maturity (37.7% and 36.8%, respectively). The values obtained with EMS and HFF were statistically similar to each other but differed from the other treatments. The media containing glutamine led to the highest proportions of degenerated oocytes.     

胰岛素和白血病抑制因子对猪卵母细胞体外成熟和孤雌激活胚胎的影响

为探讨胰岛素(Insulin)和白血病抑制因子(Leukemia inhibit factor,LIF)对猪卵母细胞体外成熟(IVM)和猪孤雌激活胚胎(PAEs)的影响,在卵母细胞体外成熟或者胚胎培养基中添加Insulin和LIF,研究卵裂率和囊胚率的变化。结果:添加了5μg/mL Insulin后猪卵母细胞体外成熟效果显著提高,但成熟后孤雌激活发育能力与非添加组相近;而胚胎培养基中添加Insulin对孤雌胚的卵裂和囊胚的形成也没有明显促进作用;添加1 000 U/mL的LIF后,卵母细胞核成熟率没有明显提高,反而孤雌激活后囊胚率急剧下降,但对卵裂率以及囊胚总细胞数影响不大;在胚胎培养基中添加LIF后,孤雌胚的卵裂和囊胚形成并没有明显的提高。表明:Insulin对卵母细胞体外成熟有益,但是对孤雌胚胎的最佳处理程序还需要摸索;本文所采用的LIF处理对猪卵体外成熟以及孤雌胚胎体外发育没有帮助,还需要进一步研究其他浓度和处理程序对猪卵母细胞体外成熟和孤雌激活胚胎发育能力的影响。     

Breed influences on in vitro development of abattoir-derived bovine oocytes

Background

There is a discrepancy in the reproductive performance between different cattle breeds. Using abattoir-derived ovaries and data base information we studied the effects of breed on in vitro fertilization and early embryo development.

Methods

The in vitro developmental competence of oocytes from cattle (n = 202) of Swedish Red (SR), Swedish Holstein (SH) and mixed beef breeds was compared, retrospectively tracing donors of abattoir-derived ovaries using a combination of the national animal databases and abattoir information. Age was significantly lower and carcass conformation score was higher in the beef breeds than in the dairy breeds.Cumulus oocyte complexes (n = 1351) were aspirated from abattoir-derived ovaries from animals of known breed (visual inspection confirmed through databases), age (databases), and abattoir information. Oocytes were matured, fertilized (frozen semen from two dairy bulls) and cultured according to conventional protocols. On day 8, blastocysts were graded and the number of nuclei determined.

Results

Cleavage rate was not different between the breeds but was significantly different between bulls. The percentage of blastocysts on day 8 was significantly higher when the oocyte donor’s breed was beef or SR than SH. There was no significant difference in blastocyst grades or stages between the breeds, but the number of nuclei in day 8 blastocysts was significantly lower in SH compared to the beef.

Conclusions

The use of abattoir-derived ovaries from animals whose background is traceable can be a valuable tool for research. Using this approach in the present study, oocyte donor breed was seen to affect early embryo development during in vitro embryo production, which may be a contributing factor to the declining fertility in some dairy breeds seen today.     

Effect of maturation culture period of oocytes on the sex ratio of in vitro fertilized bovine embryos

It has been suggested that the maturational stage of oocytes at time of insemination influences the sex ratio of resulting embryos. However, there are very few reports concerning the relationship between the maturation culture period of oocytes and the sex ratio of resulting embryos. The objective of this study was to investigate the effects of in vitro maturation culture period for bovine oocytes on the sex ratio of in vitro produced blastocysts using a novel technique of loop-mediated isothermal amplification (LAMP). Cumulus-oocyte complexes were collected from the ovaries of slaughtered cows, and then matured in vitro for various periods (16, 22, 28, and 34 h). After maturation culture for each period, the oocytes were inseminated with frozen-thawed spermatozoa, and then cultured in vitro. Blastocysts were harvested on Day 7 after insemination, and the sex of the embryos was examined using the LAMP method. The rates of oocytes matured to the metaphase II stage were significantly lower (P < 0.05) in the 16-h maturation group than in the other groups. The proportion of blastocyst formation after insemination was significantly higher (P < 0.05) in the 22-h maturation group than in the other groups. The proportion of male blastocysts increased with the increase in maturation culture period. The proportion of male blastocysts derived from oocytes matured for 34 h was significantly higher (P < 0.05) than from oocytes matured for 16 and 22 h. These results indicate that the sex ratio of in vitro fertilized embryos is apparently influenced by the maturation culture period of the oocytes.     

In vitro fertilization and development of in vitro matured bovine follicular oocytes

Bovine follicular oocytes matured in vitro were fertilized in vitro using epididymal spermatozoa from five different bulls and then cultured to the blastocyst stage in vitro. The fertilization rate, based on one pair of pronuclei and presence of one sperm tail, ranged from 55.2 to 64.3%. Embryo development (cleavage to blastocyst stage) ranged from 21.4 to 31.0% of the cultured ova reaching 8 cells at 3 to 4 d after insemination to 1.3 to 3.7% reaching hatched blastocysts at 9 to 10 d. It is concluded that individual variation among bulls is not a significant factor in fertilization and development rates of bovine follicular oocytes when epididymal spermatozoa are used.     

Parthenogenetic activation and subsequent development of porcine oocytes activated by a combined electric pulse and butyrolactone I treatment

This study was designed to evaluate the parthenogenetic activation of porcine oocytes matured in vitro for a varied period after combined electric pulse (EP; 1500 V/cm, 100 microsec) and Butyrolactone I (BL I). After 36 h of maturation culture, the rates of activated oocytes and oocytes with two pronuclei were significantly lower than those of oocytes cultured for 42 and 48 h after EP. However, when treated by a combined EP and BL I (150 microM), these rates increased to the same level as 42 and 48 h oocytes. When oocytes cultured for 48 h and activated by a combined EP and BL I treatment were subsequently cultured in mNCSU37 medium, the rates of embryos cleaved and developed to the blastocyst stage were significantly higher than those in Whitten's medium. In contrast, when activated oocytes were cultured in mNCSU37 medium under two oxygen environments (5% vs 20% O(2)), there was no difference in the rates of cleavage, blastocyst formation and nuclear numbers per blastocyst. Our results demonstrated that the combined EP and BL I treatment of porcine oocytes matured in vitro is capable of producing high rates of good quality blastocysts when cultured in a suitable in vitro condition.