Production of offspring from one-day-old oocytes stored at room temperature

To determine the feasibility of preserving oocytes without freezing, we stored mouse oocytes in several media at different temperatures for one day. Confocal microscopy of the metaphase-II spindle in these stored oocytes revealed gross abnormalities in both the spindle and the arrangement of chromosomes. The abnormal spindles could not be rescued by transplanting the aged spindle-chromosome complex into a fresh enucleated oocyte. A diploid parthenogenetic development showed that some of the oocytes stored at room temperature could still develop into blastocysts (10-57%). However, oocytes stored in a refrigerator (5%) or incubator (0%) lost the potential almost entirely. Fertilization of room-temperature-preserved oocytes with fresh spermatozoa by ICSI or IVF resulted in, respectively, 4 and 10%, full-term births. These results suggest that when oocytes are stored at room temperature for one day, most have irreversible damage not only to their cytoplasm but also to the spindle. However, since at least a few percent of stored oocytes retained the potential for full-term development, it may be possible to overcome these problems and develop a simple method for preserving mammalian oocytes without freezing.     

Maturation competence of swamp buffalo oocytes obtained by ovum pick-up and from slaughterhouse ovaries

This study was designed with the final goal of improving in vitro embryo production in the Thai swamp buffalo (Bubalus bubalis carabensis). Oocytes were collected by ovum pick-up (OPU) from six non-lactating multiparous swamp buffalo twice per week for 10 consecutive sessions followed by once-weekly collection for 10 consecutive sessions without hormone stimulation. In addition, oocytes were collected from slaughterhouse ovaries that were classified as follows: ovaries from non-pregnant cows with a visible corpus luteum (NPCL); pregnant cows with a corpus luteum (P); and non-pregnant cows without a corpus luteum (NP). Follicles in each group of ovaries were categorized as small (2-4 mm), medium-sized (5-8 mm) or large follicles (≥ 9 mm). The quality of the oocytes was assessed by their capacity to undergo in vitro maturation. The total number of observed follicles per session (all sizes combined) was larger in the once-weekly OPU group compared with the twice-weekly OPU group. In particular, the numbers of small and large follicles were higher in the once-weekly OPU group (5.2 ± 0.7 and 0.9 ± 0.2, respectively) than in the twice-weekly OPU group (3.9 ± 0.5 and 0.5 ± 0.1). The number of medium-sized follicles did not differ between the groups. The percentages of oocytes with an abnormal spindle morphology were not different between oocytes from the twice-weekly (30.0%) and the once-weekly (28.6%) OPU groups. A higher percentage of oocytes obtained in vitro (49.5%) exhibited nuclear abnormalities compared with those obtained in vivo (≤34.8%) after in vitro maturation. In conclusion, oocytes can be successfully collected by OPU in the swamp buffalo, without hormonal pretreatment, and per week more good-quality oocytes can be collected by twice-weekly OPU. In addition, oocytes collected from slaughterhouse ovaries can be used with the reproductive status of the cow having no influence on the maturation competence of oocytes.     

Effect of exposure duration of ovaries and oocytes at ambient temperature on parthenogenetic development of porcine follicular oocytes

This study was conducted to evaluate the effects of exposing porcine ovaries to 30-33 C during transportation for 4 h and subsequently room temperature (25 C) for 6 h of storage on in vitro maturation (IVM) and subsequent parthenogenetic development of oocytes collected from the ovaries. After IVM, oocytes having a tight oopalsm membrane and no signs of degeneration were exposed to Dulbecco's phosphate-buffered saline (DPBS) with 7% ethanol (v/v) for 7 min to induce parthenogenetic activation. Moreover, we also determined whether exposure of the collected oocytes to room temperature for 1, 2 and 4 h in DPBS or porcine follicular fluid (pFF) affected parthenogenetic development. When porcine ovaries were stored after transportation, oocytes collected from the stored ovaries showed a significantly higher rate of degeneration after 65 h of IVM (58.4%) and a significantly lower rate of cleavage after parthenogenetic activation (40.1%) than oocytes collected from ovaries immediately after transportation (38.9% and 47.4%, respectively). However, there was no significant difference in developmental rates to the morula and blastocyst stages between these two groups (14.4% and 14.3%, respectively). The duration of preservation, 1, 2, and 4 h, of oocytes in DPBS did not affect parthenogenetic development. In contrast, when preserved for 4 h in pFF, the developmental rates of the oocytes were significantly decreased. This suggested that some factor(s) in follicular fluid affects the developmental rate of oocytes with the passage of time in ambient conditions. These results suggest that even after 6 h storage of ovaries, oocytes having normal morphology after IVM have the same rate of parthenogenetic development as oocytes collected from ovaries just after 4 h of transportation, except for a lower cleavage rate, and that exposure of oocytes to room temperature for 4 h in DPBS does not affect their parthenogenetic developmental competence.     

Developmental competence of heat stressed oocytes from Holstein and Limousine cows matured in vitro

The negative effects of heat stress on dairy cattle's fertility have been extensively studied, but the relevant knowledge for beef cattle is rather limited. The aims of this study were to investigate the effects of HS during in vitro maturation on the developmental potential of oocytes derived from Limousine and Holstein cows and to estimate the effect of the differential gene expression of important genes in oocytes, cumulus cells and blastocysts in the growth competence between the breeds. In seven replicates, cumulus oocyte complexes from Holstein and Limousine cows were matured for 24 hr at 39°C (controls C; Hol_39, Lim_39) or at 41°C from hour 2 to hour 8 of IVM (treated T; Hol_41, Lim_41), fertilized, and presumptive zygotes were cultured for 9 days at 39°C. Cleavage and embryo formation rates were evaluated 48 hr post-insemination and on days 7, 8 and 9, respectively. From all groups, subsets of cumulus cells, oocytes and blastocysts were analysed for the relative expression of genes related to metabolism, stress, apoptosis and placentation. No difference was detected in cleavage rate or in blastocyst formation rate among the control groups. In both breeds, heat stress reduced blastocyst yield, but at all days the suppression was higher in Limousines. In Holsteins, altered gene expression was detected in cumulus cells (G6PD, GLUT1) and blastocysts (PLAC8), while in Limousines, differences were found in oocytes (G6PD, HSP90AA1), in cumulus cells (CPT1B, HSP90AA1, SOD2) and blastocysts (DNMT, HSP90AA1, SOD2). It appears that Holstein COCs are more tolerant than Limousine COCs, possibly due to compulsory, production driven selection.     

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.     

Oocyte transport: Developmental competence of bovine oocytes arrested at germinal vesicle stage by cycloheximide under air

The effects of the medium (TCM 199 or SOFaa) and temperature (20 or 39 C) during meiotic arrest by cycloheximide (CHX) under air on the developmental competence of bovine oocytes after in vitro maturation (IVM) and fertilization (IVF) were investigated. Oocytes were maintained in meiotic arrest by 10 microg/ml CHX in a 50-microl droplet of 25-mM HEPES-buffered TCM 199 (H199) at 39 C or synthetic oviduct fluid (HSOFaa) at 20 or 39 C in air for 24 h. After release from the arrest, the oocytes was matured and fertilized in vitro and their developmental competence was examined. The developmental rate of oocytes arrested in HSOFaa at 20 C to the blastocyst stage was similar to that of non-arrested oocytes but was significantly higher (P<0.05) than that of oocytes arrested at 39 C in H199 or in HSOFaa. In consideration of oocyte transport conditions, we also investigated the meiotic arrest of oocytes maintained in a 0.25-ml straw by CHX individually with 10 microl HSOFaa or as a group (40-50 oocytes) with 170-200 microl HSOFaa at 20 C in air for 24 h. After release from meiotic arrest, the developmental competence of these oocytes was assessed similarly. The developmental rate of oocytes treated with CHX individually was similar to that of those treated with CHX in 50-microl droplet of HSOFaa at 20 C. However, the developmental rate of oocytes treated with CHX as a group was lower than that of oocytes treated with CHX in a 50-microl droplet. Five blastocysts developed from oocytes maintained in meiotic arrest in a plastic straw were transferred to five recipient heifers. Consequently, three recipients became pregnant and 2 calves were delivered. The results of the present study indicate that bovine oocytes treated with CHX in HSOFaa at 20 C under air retain the same developmental competence as non-arrested oocytes.     

Developmental competence and glutathione content of maternally heat-stressed mouse oocytes and zygotes

The loss of developmental competence and the glutathione (GSH) content of maternally heat‐stressed mouse oocytes and zygotes were determined. In experiment 1, zygotes were collected from female mice that were heat‐stressed at 35°C for 10 h after hCG injection (oocyte maturation stage), or for 12 h on Day 1 of pregnancy (zygote stage), followed by in vitro culture. To minimize the effects of heat stress on the fertilization process, heat‐stressed oocytes that were fertilized in vitro were also included in this experiment. In experiment 2, heat‐stressed oocytes and zygotes were assayed for GSH content. The application of heat stress to the oocytes resulted in a significant decrease in the percentage of zygotes that developed to morulae or blastocysts, both for naturally fertilized oocytes (56.9% for heat‐stressed vs 85.4% for control) or in vitro‐fertilized oocytes (54.5%vs 73.6%). In the heat‐stressed zygotes, the disruption of embryonic development was more drastic (24.3%vs 90.3%), with the majority of zygotes being arrested at the two‐cell stage. In contrast, the GSH content decreased significantly in heat‐stressed zygotes, but not in heat‐stressed oocytes. These results demonstrate that the loss of developmental competence of early embryos is associated with a decrease in the GSH content of maternally heat‐stressed zygotes, but not of maternally heat‐stressed oocytes.     

绵羊卵巢卵母细胞体外成熟培养的研究

在体外受精过程中,绵羊卵巢卵母细胞体外成熟情况在很大程度上决定着卵母细胞的体外受精率和胚胎发育率.影响卵母细胞成熟的因素很多,主要包括卵泡大小、卵巢的发育阶段、培养基、激素生长因子等.试验从屠宰场采集屠宰后的绵羊卵巢,抽取卵巢表面卵泡中的卵母细胞,选择一级和二级卵母细胞进行体外成熟培养.试验证明,绵羊卵巢卵母细胞体外成熟培养时间以24h为佳,FCS浓度以20%最适宜.试验还发现无菌条件、温度、湿度以及在体外对卵母细胞的操作时间对试验的结果都有很大的影响.通过试验初步探明了适宜绵羊卵母细胞体外成熟的一些基本条件,为全面掌握绵羊卵母细胞体外成熟培养以至整个体外受精技术的完善和规范提供了有益的资料.     

In vitro embryo production from oocytes from ovaries of a single slaughtered cow

This communication describes the in vitro maturation and in vitro fertilization of bovine follicular oocytes. Further development of the embryos was achieved by using a granulosa cell culture system. The in vitro development of oocytes to morula/blastocyst stages obtained from individual cows was compared to the results of pooled simultaneous cultured oocytes and to our over-all results of this method. While there were no statistical differences in the developmental rates between these three groups (individual cows: 28.1%, simultaneous pool: 34.0%, over-all results: 32.7%) marked differences were found between the 22 animals investigated separately. These results indicate that there were great individual variabilities due to the oocyte population comparable to the variations in ovarian response to superovulation.     

Developmental competence of in vitro matured ovine oocytes vitrified in solutions with different concentrations of trehalose

This study aimed to determine the optimum concentration of trehalose in solutions used for vitrification of in vitro matured (IVM) ovine oocytes. IVM oocytes were randomly divided into four experimental (vitrified) and one control (fresh) groups. Experimental groups were treated with different concentrations (0.0, 0.25, 0.5 and 1.0 M) of trehalose. After warming, some viable oocytes were exposed to 0.25% pronase to test zona pellucida hardening, whereas the others were fertilized and cultured in vitro for 8 days to evaluate their developmental competence. Blastocysts quality was assessed by differential staining and TUNEL test. Survival and developmental rates of oocytes vitrified in the presence of 0.5 M trehalose were significantly higher than those of the other vitrified groups. Furthermore, there was a significant difference between fresh and vitrified groups in total blastocyst rate. Analysis of blastocysts quality also revealed a significant difference between the group treated with 0.5 M trehalose and other groups in terms of apoptotic index. Furthermore,zona pellucida digestion time period was longer in trehalose‐free (0.0 M) group compared to other groups. In conclusion, we found that IVM ovine oocytes vitrified in solutions containing 0.5 M trehalose are fertilization‐competent and are able to produce good‐quality blastocysts with an apoptotic index comparable to that of the fresh oocytes. Therefore, 0.5 M may be considered the optimum concentration of trehalose to be used in solutions prepared for vitrification of oocytes.     

Piezo-assisted in vitro fertilization of mouse oocytes with spermatozoa retrieved from epididymides stored at 4 degree C

This study was performed to investigate the effect of partial zona pellucida incision by piezo micromanipulation (ZIP) on the in vitro fertilizing ability of stored mouse spermatozoa. The storage conditions were optimized by storing the mouse epididymides at 4 C in mineral oil or in the mouse body for up to 4 days after death, and the retrieved spermatozoa were used to fertilize fresh oocytes. No significant difference was observed in fertilization rates between the treatments when epididymides were stored for up to 2 days, but the fertilization rates in mineral oil were higher (P<0.05) than those in the mouse body at 3 (41.4 vs. 16.2%) and 4 days (26.0 vs. 15.8%). Spermatozoa retrieved from epididymides stored in mineral oil were then used to fertilize fresh and vitrified oocytes with or without ZIP treatment. The fertilization rates of the ZIP fresh oocytes were higher than those of the zona-intact oocytes at each time point (1 to 4 days). After ZIP, the fertilization rates of spermatozoa stored for 1 and 2 days (91.2 and 86.6%, respectively) were similar (P>0.05) to that of fresh spermatozoa (91.9%). In regard to vitrified oocytes, the fertilization rates of zona-intact and ZIP oocytes using fresh spermatozoa were 46.7 and 84.7%, while the fertilization rates of vitrified ZIP oocytes using spermatozoa stored for 1 to 4 days ranged from 49.3 to 79.6%. When 2-cell embryos derived from ZIP fresh and vitrified oocytes inseminated with 2 day-stored spermatozoa were transferred into recipient females, 47.9 and 15.0% of the embryos developed to term, respectively. These results indicate that storing mouse epididymides at 4 C in mineral oil is more suitable than storage in the mouse body and that the ZIP technique improves the in vitro fertilizing ability of stored mouse spermatozoa in fresh oocytes and significantly increases the fertilization rate of vitrified oocytes with fresh spermatozoa.     

保存卵巢生理盐水温度的下降对猪卵母细胞体外成熟的影响

本试验研究了在37～38.5℃生理盐水中保存猪卵巢,卵巢运回实验室后生理盐水温度对猪卵母细胞体外成熟的影响。37～38.5℃的生理盐水中保存猪卵巢,运回实验室后,当温度下降为31℃、32℃、33℃、34℃及36℃时卵巢上抽取卵母细胞的成熟率分别为24.86±7.31%、21.34±5.12%、28.32±7.49%、21.14±4.82%和17.70±2.27%,淘汰率分别为53.42±12.10%、48.05±21.04%、43.93±12.77%、58.19±6.82%和62.06±12.03%,各组间差异不显著。结果表明:在37～38.5℃的生理盐水下保存猪卵巢,运回实验室后,当温度下降到31～36℃时对卵母细胞的成熟没有显著影响。     

The quality of cryopreserved sperm collected from feline caudal epididymides stored at room temperature

On the assumption that animals of wild feline species died in the field, caudal epididymal sperm were cryopreserved following storage of the feline epididymides at 20°C for 0-24 hr, and their qualities were observed. Compared to the qualities at 0 hr, no significant differences were noted following 12 hr of storage at 20°C. On comparison of the qualities between caudal sperm cryopreserved after 24 hr storage at 4°C and after 12 hr at 20°C followed by 12 hr storage at 4°C, no significant differences were noted. These findings suggest that the cryopreserved sperm collected from epididymides of dead animals might be useful for artificial insemination if cryopreservation was performed within 12 hr exposure to ambient temperature.     

In vitro development of equine oocytes from preserved ovaries after intracytoplasmic sperm injection

In this study, we evaluated the meiotic competence of equine oocytes from ovaries preserved for one day. We also investigated fertilization, cleavage rate, developmental competence and freezability of equine embryos after intracytoplasmic sperm injection (ICSI). After collection from ovaries, the oocytes were classified into two groups comprised of those having compact cumulus layers (Cp) or those having expanded cumulus layers (Ex). Oocytes with a first polar body were subjected to fertilization by ICSI using frozen-thawed stallion spermatozoa and were then cultured in CR1aa medium. The rates of metaphase II-stage oocytes, normal fertilization and cleavage were not significantly different between the two oocyte categories (38.5, 70.0 and 48.7% for CP and 43.5, 60.0 and 58.8% for Ex, respectively). However, the blastocyst development rate of Ex was significantly (P<0.05) higher than that of Cp (25.5 vs. 7.7%). Three Cp-derived and 12 Ex-derived early blastocysts were cryopreserved using the slow cooling protocol, and all of them developed to hatching blastocysts after thawing. These results suggest that equine oocytes fertilized by ICSI can develop to the preimplantation stage in culture conditions similar to those used in the bovine. Furthermore, the Ex oocytes had higher developmental competence than the Cp oocytes, and the in vitro-produced blastocysts had high viability after freezing and thawing.     

Pregnancies attained after collection and transfer of oocytes from ovaries of five euthanatized mares

After euthanasia, ovaries were removed from 5 horses and shipped to a laboratory where 46 oocytes were collected. The oocytes were cultured for 24 to 30 hours, and 36 oocytes were transferred to 10 recipient mares via flank laparotomies. Recipient mares were inseminated with semen from various stallions. Sixteen days after transfer, 4 of the recipients were pregnant with at least 1 embryonic vesicle. Embryonic death occurred in 3 recipients, whereas a healthy live foal was born from 1 recipient. Ovaries from valuable mares can be a source of viable oocytes after death of the mare. For shipping to a laboratory, fluctuations in temperature should be minimized and the ovaries should not be chilled.     

The quality and maturation of bitch oocytes recovered from ovaries by the slicing method

Oocytes were recovered from bitch ovaries at various stages of the estrous cycle by the slicing method. The proportion of Grade A oocytes (darkly pigmented and surrounded in part, or whole, by dense layers of cumulus cells) were counted. Only Grade A oocytes were cultured in TCM199 supplemented with 5% fetal calf serum for evaluation of meiotic competence. There were no significant differences in the total number of oocytes or the proportion of Grade A oocytes that were recovered from bitches at various stages of the estrous cycle. Only 11% of the oocytes reached metaphase II (MII) at 72 hr after initiation of maturation culture. However, the proportions of oocytes reaching MII did not increase with culturing for up to 120 hr.     

Developmental competence of frozen-thawed blastocysts from fair-quality bovine embryos cultured with beta-mercaptoethanol

Two-hundred-and-thirty-one fair-quality embryos at the compacted morula stage collected from 89 superovulated cows were cultured in TCM199 or Brinster's BMOC-3 medium with or without 100 microM beta-mercaptoethanol (beta-ME). After 24 h culture, a total of 142 fair-quality embryos developed to the blastocyst stage, of which 106 were subsequently frozen with 1.8 M ethylene glycol. The mean cell number and development rates of frozen-thawed blastocysts from the fair-quality embryos cultured in TCM199 containing beta-ME were higher than those of the fair-quality embryos directly frozen without culture. The pregnancy rates obtained with frozen blastocysts from fair-quality embryos tended to be lower than those of non-cultured fresh fair-quality embryos and cultured fresh blastocysts. These results indicate that the inclusion of beta-ME in pre-freezing culture media improve the development of frozen-thawed blastocysts from fair-quality embryos, but not the pregnancy rate.