Effects of BSA and fetal bovine serum in culture medium on development of rat embryos

Rat 1-cell embryos, recovered from naturally mated females, were cultured in a chemically defined medium (mR1ECM) or in mR1ECM supplemented with BSA (4 mg/ml; mR1ECM-BSA) or fetal bovine serum (FBS; 10%, v:v; mR1ECM-FBS) instead of polyvinylalcohol. There was no difference in percentages of embryos that developed to the 2-cell to blastocyst stages between mR1ECM and mR1ECM-BSA, but in mR1ECM-FBS, no development beyond the 2-cell stage was observed. When embryos were transferred to mR1ECM-FBS from mR1ECM after 24 to 64 h of culture, development of embryos to and beyond the 4-cell stage was inhibited. However, when transferred after 80 h of culture, more embryos developed to blastocysts and hatching or hatched blastocysts than in embryos cultured in mR1ECM. When 8-cell embryos and early morulae obtained after 72 and 80 h of culture in mR1ECM, respectively, were cultured in mR1ECM-FBS, a higher proportion of early morulae developed to the blastocyst stage than did 8-cell embryos. When morulae obtained after culture in mR1ECM or mR1ECM-BSA were transferred to recipient females, there was no difference in proportions of fetuses obtained. However, a higher proportion of blastocysts cultured in mR1ECM-FBS developed to fetuses compared with those obtained in mR1ECM. These results indicate that BSA has neither deleterious nor beneficial effects on development of rat 1-cell embryos. In contrast, FBS has deleterious effects on early cleavage of embryos but it promotes more rapid development of morulae to blastocysts, resulting in better quality blastocysts.     

Determination of optimal conditions for parthenogenetic activation and subsequent development of rat oocytes in vitro

The present study was undertaken to determine optimal conditions for parthenogenetic activation and subsequent development of rat oocytes. Oocytes from immature Wistar-Imamichi (WI) and Sprague Dawley (SD) rats were activated by electrical stimulation in combination with 6-dimethylaminopurine (6-DMAP) to assess whether different rat strains display different responses to activation treatment. Since the cleavage rates of activated oocytes were significantly higher in WI than SD strain rats, WI rats were used for the subsequent experiments to determine the effects of post-hCG time, culture duration, different activation protocols (electrical stimulation with 6-DMAP or ionomycin with 6-DMAP) and osmolarity of the activation medium on the activation and subsequent development of WI rat oocytes. For oocytes activated by electrical stimulation combined with 6-DMAP, the percentages of oocytes that were activated and that developed to blastocysts were higher when oocytes were collected at 18-20 h than at any other time points after hCG injection (16, 22-24 h). Culturing for 2-6 h before activation treatment markedly decreased the percentage of activated oocytes that developed to beyond the four-cell stage. There were no differences in the percentages of oocytes with pronuclear formation and subsequent development to the two-cell and blastocyst stages between oocytes that were activated by electrical stimulation or ionomycin, both followed by 6-DMAP treatment. Activation of oocytes by ionomycin and 6-DMAP, both in low osmolarity media (246 mOsM), markedly increased the cleavage rates and percentages of high quality blastocysts (71%). The optimal conditions determined in the present study with simplified activation protocols and high efficiency of activation and subsequent development of WI rat oocytes will be helpful for further research involving nuclear transfer in the rat.     

Defined system for in vitro production of porcine embryos using a single basic medium

We have previously indicated that porcine blastocysts can be produced by in vitro fertilization (IVF) and culture (IVC) in chemically defined porcine gamete medium (PGM) and porcine zygote medium (PZM)-5, respectively, In the present study, the effects of basic media and macromolecular components on in vitro maturation (IVM) were investigated to develop a defined system for in vitro embryo production using a single basic medium through IVM, IVF and IVC. Porcine immature oocytes were matured in porcine oocyte medium (POM) or modified North Carolina State University (mNCSU) 37, which were supplemented with either 10% (v/v) porcine follicular fluid (pFF) or 3 mg/ml polyvinyl alcohol (PVA) as a macromolecular component (designated POM+pFF, POM+PVA, mNCSU37+pFF and mNCSU37+PVA). In the maturation with mNCSU37+PVA, the percentages of oocytes that reached the metaphase II stages were significantly lower than those in the other treatments. Following IVM with the above media, oocytes were treated with an electrical stimulus and cycloheximide for parthenogenetic activation and were cultured in PZM-5 for 5 days. The rates of cleavage and blastocyst formation of parthenogenetic oocytes were significantly lowered for maturation with mNCSU37+PVA compared with the other treatments, while there were no significant differences in the total numbers of cells in blastocysts among the treatments. Following IVF and IVC, the rates of penetration, male pronucleus formation, cleavage and blastocyst formation were significantly lower when oocytes were matured in mNCSU37+PVA than in other maturation media. The normal fertilization rate was significantly higher in POM+PVA compared with the other treatments, although the total number of cells in blastocysts was reduced with the addition of PVA to both POM and mNCSU37 compared with pFF supplementation. These results demonstrate that porcine blastocysts can be produced by the defined system using a single basic medium.     

Donor and recipient rat strains affect full-term development of one-cell zygotes cultured to morulae/blastocysts

The present study was conducted to examine the developmental potential to offspring of rat embryos cultured from 1-cell to morula/blastocyst stage. Pronuclear zygotes from Wistar x Wistar or (SD x DA) x Wistar strains were cultured in modified rat 1-cell embryo culture medium (mR1ECM) for 96 h in 5% CO(2) in air at 37 C. The proportion of the 3-way cross hybrid zygotes developing into morula/blastocyst stage (74%) was higher than that of the Wistar zygotes (66%). Day-5 morulae/blastocysts developed in vitro were transferred into Day-3 or -4 pseudopregnant recipients of Wistar or SD x DA strain. The transfer of cultured embryos resulted in the birth of offspring at 13-59%, while that of non-cultured control blastocysts showed birth rates of 35-65%. The best offspring rate of cultured embryos (59%) was obtained when the hybrid 1-cell zygotes were cultured in mR1ECM medium and transferred into the 2-days earlier uteri of SD x DA recipients. These results suggest that genetic background of recipients as well as donors is a possible factor affecting full-term development of rat morulae/blastocysts derived from 1-cell stage zygotes cultured in vitro.     

Effect of capacitation of stallion sperm with polyvinylalcohol or bovine serum albumin on penetration of bovine zona-free or partially zona-removed equine oocytes

Experiments were conducted to study effects of macromolecules on stallion sperm capacitation and fertilization as determined by penetration of bovine zona-free and equine partially zona-removed oocytes. Stallion sperm were capacitated in TYH medium (modified Krebs-Ringer bicarbonate) supplemented with either 1 mg/mL of polyvinylalcohol (PVA) or 4 mg/mL of BSA. Capacitation was induced with 8 bromoadenosine cyclic monophosphate (8BrcAMP; 0.5 mM) alone or in combination with 0.1 microM of ionomycin. Intraspecies gametes were co-incubated in TYH/PVA or TYH/BSA for 18 to 20 h. For zona-free bovine oocytes, penetration rate (35%) with the combination of 8BrcAMP and ionomycin in PVA-containing medium was higher (P < 0.05) than any treatment in BSA-containing medium (5 to 6%). A similar study was conducted using equine oocytes with partially removed zonae. Sperm capacitated and used for in vitro fertilization (IVF) in PVA-containing medium had higher penetration rates (P < 0.01) than sperm in BSA-containing medium (54 vs. 11%). The effect of equine preovulatory follicular fluid on bovine oocyte penetration was assessed. Bovine oocytes were matured in tissue culture medium-199 with 0, 20, 50, or 100% equine preovulatory follicular fluid, and 1 IU/mL of equine chorionic gonadotropin. Stallion sperm were treated with 8BrcAMP + ionomycin in PVA- or BSA-containing media. The penetration rates of bovine zona-free oocytes by stallion sperm were again higher with PVA (47%) than BSA (18%; P < 0.01). Penetration rates of oocytes matured in 100% follicular fluid were higher (P < 0.05) than for oocytes matured with 0% follicular fluid. The effects of equine follicular fluid and PVA/BSA during sperm capacitation on standard bovine IVF were examined. Culture of bovine oocytes with equine follicular fluid did not affect oocyte maturation or penetration rates after IVF. Bovine sperm capacitated with heparin in PVA-containing medium yielded lower (P < 0.05) fertilization rates than those capacitated in BSA-containing medium when incubated with both zona-intact and zona-free bovine oocytes. In summary, PVA was superior to BSA for ionophore-induced capacitation of equine sperm for penetration of zona-free bovine oocytes or partially zona-removed equine oocytes, but not for standard bovine IVF with bovine sperm. Zona-free bovine oocytes may be useful for assaying in vitro capacitation and fertilization of stallion sperm.     

影响猪ICSI转基因技术效率的主要因素研究

以猪体外成熟卵子和冷冻解冻的死精子为材料,以pEGFP-N1为模式基因,探讨注射台温度、激活后6-DMAP的处理和精子与PEGFP-N1孵育液添加BSA(牛血清白蛋白)对精子胞质内注射(ICSI)转基因效率的影响。结果表明:注射台温度为30℃时的阳性率为40.07%,而38.5℃时为20.97%,差异极显著(P<0.01)。添加BSA的囊胚转基因率为55.56%,对照组为33.33%,差异极显著(P<0.01)。6-DMAP处理组与对照组的转基因率分别为52.53%和26.25%,差异极显著(P<0.01);而且6-DMAP处理组的囊胚率(9.96%)显著高于(P<0.05)对照组(2.30%)。研究表明注射台温度对转基因效率有明显影响,温度高转基因率低;精子与PEGFP-N1孵育液添加BSA对转基因胚胎发育有一定促进和保护作用,有利于提高囊胚转基因率;激活后用6-DMAP处理能提高转基因率和囊胚率。     

Glycosaminoglycans improves early development of zona-free 8-cell rat embryos to blastocysts in a chemically defined medium, but not the pregnancy rate following transfer of the blastocysts

The objective of the present study was to clarify the possible role of the zona pellucida (ZP) in early development of rat embryos and to determine the effect of glycosaminoglycans on the development of ZP-free 8-cell embryos before or after embryo transfer at the blastocyst stage. Eight-cell embryos were divided into three groups comprised of, 1) intact controls, 2) embryos with the ZP was removed with acidic solution and 3) pairs of ZP-free 8-cell embryos aggregated in a small hollow. These embryos were cultured in a chemically defined mR1ECM for 24 h. Developmental ability to the blastocyst stage and mean cell number in the blastocyst was lower in ZP-free embryos than in intact controls. When these blastocysts were transferred, the farrowing rate and efficiency of embryos developed to term were also lower in ZP-free embryos, but not in the aggregated ones. Supplementation with hyaluronan (HA; 63-250 μg/ml) or heparan sulfate proteoglycan (HS; 15 μg/ml) significantly improved blastocyst formation of ZP-free embryos and the cell number in the blastocyst by reducing the incidence of apoptosis. However, there were no beneficial effects of HA or HS on farrowing and newborn rates after transfer of the blastocysts. In conclusion, the ZP plays roles in maintaining successful development of early rat embryos at least from the 8-cell stage not only to the blastocyst stage but also to posttransfer stages. Glycosaminoglycans, such as HA or HS, appear to contribute to successful cleavage during early development to the blastocyst stage but may be insufficient to maintain the posttransfer survival of ZP-free embryos.     

Consequences for the bovine embryo of being derived from a spermatozoon subjected to post-ejaculatory aging and heat shock: development to the blastocyst stage and sex ratio

The objective was to determine whether aging of sperm caused by incubation at normothermic (38.5 C) or heat shock (40 C) temperatures for 4 h prior to oocyte insemination affects sperm motility, fertilizing ability, competence of the resultant embryo to develop to the blastocyst stage and blastocyst sex ratio. In the first experiment, the percent of sperm that were motile was reduced by aging (P<0.001) and the reduction in motility was greater for sperm at 40 C compared to sperm at 38.5 C (P<0.01). In the second experiment, oocytes were inseminated with aged sperm. A smaller percent of oocytes fertilized with sperm aged at either temperature cleaved by Day 3 after insemination than oocytes fertilized with fresh sperm (P<0.05). There was no effect of sperm aging on the percent of oocytes or cleaved embryos that developed to the blastocyst stage. Aging of sperm before fertilization at 38.5 C reduced the percent of blastocysts that were male (P=0.08). In the third experiment, incubation of sperm at 38.5 C or 40 C for 4 h did not reduce fertilizing ability of sperm as determined by pronuclear formation at 18 h post insemination. In conclusion, aging of sperm reduced cleavage rate and the percent of blastocysts that were males but had no effect on the developmental capacity of the embryo. The effect of aging on cleavage rate may represent reduced motility and errors occurring after fertilization and pronuclear formation. Aging at a temperature characteristic of maternal hyperthermia had little additional effect except that polyspermy was reduced. Results indicate that embryo competence for development to the blastocyst stage is independent of sperm damage as a result of aging for 4 h at normothermic or hyperthermic temperatures.     

In vitro culture conditions using chemically defined media for in vitro matured and intracytoplasmically inseminated porcine oocytes

The present study investigated in vitro culture methods [droplet and Well of the Well (WOW)] using semi-defined and defined media [modified porcine zygote medium (mPZM)] and the additional effects of insulin on in vitro matured and intracytoplasmically inseminated porcine oocytes. In Experiment 1, in vitro matured and intracytoplasmically inseminated porcine oocytes were cultured for 6 days in the following four groups: 1) mPZM-3 (containing bovine serum albumin) + droplet (30 mul), 2) mPZM-3 + WOW, 3) mPZM-4 (containing polyvinyl alcohol) + droplet, and 4) mPZM-4+ WOW. The culture media (mPZM-3 and mPZM-4) and methods (droplet and WOW) did not significantly affect the cleavage rate, but the blastocyst rate of the oocytes cultured in mPZM-3 was significantly (P<0.01) higher than that of mPZM-4 (20.1 and 9.4%, respectively). The blastocyst rates as percentages of the cleaved oocytes (51.8 and 16.9%) and the hatched blastocyst rate as percentages of the number of blastocysts (12.3 and 2.2%) were also significantly (P<0.01) higher in mPZM-3 compared with those in mPZM-4. There was significant interaction (P<0.05) between the two main factors; the effects of the culture media and methods on the rate of hatched blasyocysts as percentages of the blastocysts produced and, the hatched blastocyst rate (20.3%) as percentages of the number of blastocysts produced in mPZM-3 were significantly (P<0.05) higher than in the other groups. In Experiment 2, the additional effects of insulin (100 ng/ml) in mPZM-3 and mPZM-4 media was investigated in the WOW culture system. Insulin addition did not improve cleavage, blastocyst formation, or the number of cells in blastocysts. However, as in Experiment 1, mPZM-3 resulted in a significantly higher blastocyst rate as percentages of the cleaved oocytes than mPZM-4 (33.9 and 18.4%). These results indicate that a chemically defined medium (mPZM-4) needs to be improved to provide more suitable culture conditions for in vitro development of in vitro matured and intracytoplasmically inseminated porcine oocytes. However, the WOW system may be a useful IVC method for blastocyst development of in vitro matured porcine oocytes following ICSI when a semi-defined medium (mPZM-3) is used.     

Different factors affect developmental competence and cryotolerance in in vitro produced bovine embryo

The present study was conducted to examine the effects of culture systems and culture media on developmental competence and freezability of bovine embryos obtained by in vitro culture of in vitro matured and fertilized (IVM-IVF) oocytes. No significant difference was observed in the proportions of oocytes developed to blastocysts, the speed at which the oocytes reached the blastocyst stage and the number of cells, when the IVM-IVF oocytes were cultured in CR1aa with or without cumulus cells. Nevertheless, more of the IVM-IVF oocytes cultured either with or without cumulus cells in CR1aa were seen to reach the blastocyst stage much sooner than those cultured with cumulus cells in TCM199 (P<0.05). The proportion of embryos developed to the blastocyst stage by day 7 in CR1aa culture was significantly higher than embryos cultured in TCM199. Viability after frozen-thawed blastocysts were obtained in vitro, was seen in a significantly higher percentage of embryos cultured in TCM199 and developed to the hatched blastocysts than in those cultured in CR1aa (P<0.05). These results indicate that CR1aa was superior to TCM199 for the potential developmental of IVM-IVF oocytes to blastocysts during in vitro culture regardless of co-culture with or without cumulus cells. But the freezability of blastocysts developed in CR1aa was inferior to those developed in TCM199.     

Pig blastocyst development in vitro is affected by amino acids

Pig embryos were removed 5 d after onset of estrus and cultured in modified Krebs-Ringer bicarbonate (mKRB) medium containing bovine serum albumin (BSA; 1 mg/ml). Media in Exp. 1 and 2, but not in Exp. 3, contained 10% heat-inactivated lamb serum (LS). In Exp. 1, embryos were cultured in 1) mKRB + LS or mKRB + LS supplemented with 2) glutamine (2 mM); 3) phenylalanine (.1 mM), methionine (.05 mM) and isoleucine (.2 mM); or 4) phenylalanine, methionine, isoleucine and glutamine. Embryos cultured in media with supplemental phenylalanine, methionine and isoleucine attained larger (P less than .05) volumes during culture and initiated hatching at an increased (P less than .05) frequency compared with embryos cultured without additional amino acids. In contrast, adding glutamine depressed (P less than .05) the maximum volumes observed during culture. In Exp. 2 mKRB + LS was compared with mKRB + LS plus phenylalanine (.1 mM), methionine (.05 mM) and isoleucine (.2 mM); methionine and isoleucine; phenylalanine and isoleucine, or phenylalanine and methionine. Embryos cultured in media supplemented with phenylalanine and methionine attained larger (P less than .05) volumes than embryos cultured in either media without added methionine. Fewer (P less than .05) embryos cultured without methionine initiated hatching (56%) compared with embryos provided methionine (89%). In Exp. 3, we evaluated the addition of glutamine (1 mM) with or without phenylalanine (.1 mM), methionine (.05 mM) and isoleucine (.2 mM) to serum-free mKRB. Adding glutamine alone, but not in combination, increased (P less than .05) blastocyst volumes on d 3 and maximum volume attained during culture compared with embryos cultured in mKRB alone.(ABSTRACT TRUNCATED AT 250 WORDS)     

Consequences for the bovine embryo of being derived from a spermatozoon subjected to oxidative stress

Objective To determine whether oxidative damage of ejaculated frozen–thawed sperm prior to oocyte insemination in vitro affects the competence of the resultant embryo to develop to the blastocyst stage. Method Extended frozen semen from bulls was thawed, subjected to Percoll gradient purification to obtain motile spermatozoa and mixed with medium containing the pro-oxidants menadione or tert-butyl hydroperoxide. After 3 h at 38.5°C, the sperm were washed and used to inseminate oocytes in vitro. Embryo development proceeded until 8 days after insemination. Results Treatment of sperm with 15 or 30 µmol/L menadione reduced the proportions of oocytes that cleaved and those that developed to the blastocyst stage; 30 µmol/L menadione reduced the proportion of cleaved embryos that developed to the blastocyst stage at day 8 after insemination. Oocytes inseminated with sperm treated with 150 or 300 µmol/L tert-butyl hydroperoxide had lower proportions of cleavage and blastocyst development, and the proportion of cleaved embryos becoming blastocysts was also reduced. Conclusion Oxidative damage to ejaculated sperm can compromise the ability of the sperm to cause oocyte cleavage and leads to formation of embryos with reduced competence for development.     

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.     

In vitro fertilization using non-sexed and sexed bovine sperm: sperm concentration, sorter pressure, and bull effects

The objective of these experiments was to study bovine in vitro fertilization (IVF) conditions for blastocyst production using non-sexed sperm (Experiment 1) and sexed sperm (Experiment 2). For Experiment 1, in vitro-matured oocytes (N=707) were allocated to a 2 × 3 × 4 factorial design: time of co-incubation of gametes for fertilization (4 and 18 h), sperm dose (1, 0.33, and 0.11 × 10(6) frozen-thawed sperm/ml, and sperm source (four bulls). Pronuclear status was evaluated for a subset. Experiment 2 (N=2155 oocytes) was a 2 × 3 × 2 × 6 factorial design: sex of sperm (X and Y), sperm dose (1, 0.33, and 0.11 × 10(6) frozen-thawed sperm/ml), and sperm-sorting pressures (40 and 50 psi), replicated with sperm of six bulls. Presumptive zygotes were cultured 60 h in chemically defined medium-1 (CDM-1), and for 114 h in CDM-2. For Experiment 1, pronuclear formation, cleavage and blastocysts rates were greater for 1, and 0.33 × 10(6) than 0.11 × 10(6) sperm/ml (72 and 62 vs 42%; 89 and 81 vs 58%; and 21 and 17 vs 9%, respectively; all p<0.01); polyspermy was greater for 1, than 0.33 and 0.11 × 10(6) sperm concentrations (24 vs 2 and 0%; p<0.01). There were greater main effects (p<0.01) of pronuclear formation (69 vs 48%), polyspermy (13 vs 4%), and cleavage (63 vs 54%), at 18 than at 4 h of co-incubation of gametes (all p<0.01). For Experiment 2, cleavage and blastocyst rates were greater for 1 × 10(6) sperm/ml vs 0.33 and 0.11 (69%, 47%, and 30% cleavage and 30%, 14%, and 8% blastocysts) and 40 vs 50 psi (54% and 44% cleavage and 18% and 15% blastocysts) (p<0.01). A marked bull by fertilization sperm dose interaction was found for cleavage (p<0.05). The main conclusion was that the optimal sperm concentration for cleavage and producing blastocysts via IVF with sexed sperm was considerably higher and more variable among bulls than for unsexed sperm.     

Fertilization and blastocyst development in oocytes obtained from prepubertal and adult pigs

Polyspermic fertilization and embryo quality are important issues for the in vitro production of pig embryos. We hypothesized that oocyte donor (prepubertal gilt vs. sow) affects polyspermy and blastocyst development in vitro and that the sexual maturity of the oocyte donor affects the response to sperm concentration in the fertilization medium. In Exp. 1, oocytes of sows and gilts were mounted and stained 12 h after insemination to provide fertilization data. In Exp. 2, putative embryos were developed in vitro to 144 h post-insemination before mounting. In both experiments, cumulus-oocyte complexes (COC) were collected from ovaries of prepubertal gilts and adult sows. Sperm were added after maturation of COC for 40 to 44 h. Sperm from two boars at 0.5 to 5.0 x 10(6) sperm/mL was used for insemination. More (P < 0.01) monospermic fertilizations were observed in oocytes derived from gilts than for oocytes from sows. There were fewer (P < 0.02) penetrated sperm per fertilized oocyte in oocytes from gilts compared with sows. There were effects of semen donor (boar) on the percentage of monospermic (P < 0.01) and polyspermic (P < 0.002) fertilizations, and on the number of penetrated sperm/fertilized oocyte (P < 0.02). In Exp. 2, cleavage and blastocyst formation was evaluated at 2 and 6 d postinsemination, respectively. More (P < 0.001) blastocysts developed from sow-derived oocytes than from gilt-derived oocytes. More (P < 0.05) total cells per blastocyst were observed in embryos from sow-derived oocytes than from gilt-derived oocytes. Semen donor affected the percentage of oocytes cleaving (P < 0.02), and a boar x sperm concentration interaction affected (P < 0.05) the incidence of blastocyt formation. Results indicate that sexual maturity of the donor is not responsible for the high incidence of polyspermy in porcine in vitro fertilization. However, blastocyst development is improved by the use of oocytes from sows rather than from prepubertal gilts.     

Effects of glutathione treatments during sperm washing and in vitro fertilization on the in vitro early development of embryos of Japanese Black cattle

This study was conducted to examine the effects of adding glutathione (1 mM) to media used for sperm washing and in vitro fertilization (IVF) on the improvement of early development of embryos produced using cryopreserved spermatozoa of the less IVF-competent bull (the one considered unqualified as spermatozoa supplier for the production of bovine blastocysts using IVF). The cryopreserved spermatozoa of this bull were characterized by normal motility and lower ATP content and blastocyst productivity than those of IVF-competent bulls. The addition of glutathione to the sperm washing medium was more effective in improving the productivity of blastocysts and ATP content than the addition of glutathione to the IVF medium or no glutathione addition at all (control). These results suggest that this simple method may be used to improve the potential of cryopreserved spermatozoa of less IVF-competent bulls to fertilize oocytes in vitro and to induce normal embryonic development after fertilization.     

Effect of Chemical or Electrical Activation of Bovine Oocytes on Blastocyst Development and Quality

Activation of in vitro‐matured (IVM) oocytes is essential for successful embryo production following nuclear transfer (NT) or intracytoplasmic sperm injection (ICSI). This study was designed to compare the rates of blastocyst production and embryo quality (as measured by numbers of viable cells) following parthenogenetic activation with electrical pulse or the use of two different calcium ionophores, A23187 (CA) or ionomycin (IO), with or without the addition of bovine serum albumin (BSA). IVM oocytes with a first polar body were randomly allocated to five treatment groups: CA (5 μm CA, 5 min; n = 88), CA + BSA (5 μm CA, 5 min; BSA, 5 min; n = 90), IO (5 μm IO, 5 min; n = 91), IO + BSA (5 μm IO, 5 min; BSA, 5 min; n = 86) and EL (two pulses of 1.5 kV/cm, 20 μs; n = 120). Blastocyst rates were higher (p < 0.05) for CA (54.4%), IO (51.4%) and EL (54.5%) than for IO + BSA (18.3%). Treatment CA + BSA (39.8%) did not differ from the others. There was no difference (p > 0.05) among treatments in total number of cells. However, the percentage of viable cells was reduced in CA (49.9%), CA + BSA (45.8%), IO (64.9%), IO + BSA (50.9%) compared with EL (82.7%). In summary, the addition of BSA to the IO treatment had an adverse effect on blastocyst production rates. Although there was no difference between electrical stimulation and chemical activation on blastocyst production rates, electrical activation resulted in blastocysts with a higher percentage of viable cells.     

Osmolarity- and Stage-Dependent Effects of Glycine on Parthenogenetic Development of Pig Oocytes

The osmolarities of media that are most effective for in vitro culture of mammalian oocytes and embryos are lower than that of oviductal fluid. Oocytes and embryos can survive the high physiological osmolarity in vivo perhaps owing to the presence of amino acids such as glycine, which serve as organic osmolytes in the female reproductive tract. In the present study, the effects of glycine on the parthenogenetic development of pig oocytes were examined in hypotonic or isotonic media. The results showed that culturing oocytes in isotonic media improved the cleavage rates (P<0.01) at 2 days in culture but inhibited any further development beyond cleavage when compared with the hypotonic media. However, addition of 4 mM glycine to the isotonic media resulted in improved blastocyst formation rates compared with that observed in the hypotonic media (P<0.01), and there was no inhibition of development beyond the cleavage stages in oocytes. The beneficial effects of glycine were observed only when oocytes were cultured in isotonic media and glycine was added at day 2 or 3 in culture. The results from the present study indicate that an isotonic medium with glycine is useful for in vitro culture of pig oocytes and that glycine may protect pig oocytes against the detrimental effects of increased osmolarity.     

Full‐Term Development of Rabbit Embryos Produced by ICSI with Sperm Frozen in Liquid Nitrogen without Cryoprotectants

The aim of the present study was to establish the technology of intracytoplasmic sperm injection (ICSI) in rabbit by using the sperm frozen without cryoprotectants. Observation under an electron microscope revealed that the rabbit spermatozoa frozen without cryoprotectants had severe damage especially in the plasma membrane and junction between head and tail. However, after being injected into the oocytes, the sperm frozen without cryoprotectants retained the capability of supporting the cleavage and development of the ICSI oocytes, with no significant difference from that of fresh sperm, although the development of ICSI embryos derived from either frozen sperm or fresh sperm is much lower than that of in vivo‐fertilized zygotes. When additional artificial activation was applied following ICSI, the rates of cleavage and blastocyst formation of ICSI oocytes were significantly increased when compared with the oocytes without additional activation. Yet, the cell numbers in blastocysts were not significantly different between the activation and non‐activation group. After embryo transfer, four offspring were obtained from the oocytes microinjected with the sperm frozen without cryoprotectants. The technology established by this study may facilitate exploring the ICSI‐based transgenic method in rabbit and broaden the application of ICSI technique in related field.