• Animal Bioscience
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• v.35 no.2
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• pp.177-183
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• 2022

Objective: The present study evaluated the efficiency of embryo development and pregnancy of somatic cell nuclear transfer (SCNT) embryos using different source-matured oocytes in Camelus dromedarius. Methods: Camelus dromedarius embryos were produced by SCNT using in vivo- and in vitro- matured oocytes. In vitro embryo developmental capacity of reconstructed embryos was evaluated. To confirm the efficiency of pregnancy and live birth rates, a total of 72 blastocysts using in vitro- matured oocytes transferred into 45 surrogates and 95 blastocysts using in vivo- matured oocytes were transferred into 62 surrogates by transvaginal method. Results: The collected oocytes derived from ovum pick up showed higher maturation potential into metaphase II oocytes than oocytes from the slaughterhouse. The competence of cleavage, and blastocyst were also significantly higher in in vivo- matured oocytes than in vitro- matured oocytes. After embryo transfer, 11 pregnant and 10 live births were confirmed in in vivo- matured oocytes group, and 2 pregnant and 1 live birth were confirmed in in vitro- matured oocytes group. Furthermore, blastocysts produced by in vivo-matured oocytes resulted in significantly higher early pregnancy and live birth rates than in vitro-matured oocytes. Conclusion: In this study, SCNT embryos using in vivo- and in vitro-matured camel oocytes were successfully developed, and pregnancy was established in recipient camels. We also confirmed that in vivo-matured oocytes improved the development of embryos and the pregnancy capacity using the blastocyst embryo transfer method.

• Journal of Embryo Transfer
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• v.10 no.2
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• pp.105-113
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• 1995

This study was conducted to examine the efficiency of enucleation and blastomere isolation from recipient oocytes and donor embryos, respectively and to determine the effect of oocyte age and electric voltage on the fusion rate and in vitro development of the fused oocytes in rabbit nuclear transplantation. Immature oocytes collected from ovarian follicles were matured in vivo for 12 h in TCM-199 containing FCS and hormones and in vivo matured oocytes were collected 17 to 18 h post-HCG. The fresh and frozen donor embryos of 8- to 16-cell stage were collected from the oviduct of superovulated does. The proportion of successfully enucleated oocytes was greatly lower in in vitro matured oocytes (42.3%) than that (62.7%) in in vivo matured oocytes The level of cytochalasin B for in vivo matured oocytes did not affect the efficiency of enuleation, but 7.5 $\mu$g /mL cytochalasin B for in vitro matured oocytes showed a high enucleation rate significantly. The isolation efficiency of a single blastomere nucleus did not differ between 8- and 16-cell stage embryos. The percentage of single blastomeres isolated from 16-cell stage fresh embryos after 0.5% pronase treatment was greatly higher at 16-min treatment (94.4%) than at 8-min(78. 1%) and the blastomeres(61.5%) isolated from frozen-thawed embryos after 16-min pronase were significantly fewer than those of fresh embryos. The age of recipient oocytes affected nuclear fusion rate. The reconstituted oocytes fused at 24-h age showed slightly higher fusion rate (77.8%) than those (65.0%)fused at 18-h age. The fusion rate of in vitro and in vivo matured oocytes inserted with fresh blastomere did not differ among electric voltages, but the cleavage rate and development to morula-blastocysts of in vitro matured oocytes was more higher under 0.6 kV/cm than under 0.8 to 1.2 kV/cm, while the cleavage rate and development of in vivo matured oocytes was higher under 0.8 to 1.0 kV/cm than under 1.2 kV/cm. The fusion and cleavage rate fol1owing insertion with frozen-thawed blastomere was not different between the in vitro and in vivo matured oocytes and was similar to those from fresh blastomere insertion.

• Journal of Embryo Transfer
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• v.33 no.4
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• pp.281-286
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• 2018

Zona pellucida (ZP), a primarily representative coat of mammalian egg and embryo, has an extremely heterogeneous morphology during different developmental stages. The objective of the present study was to compare the morphological changes of the ZP surface of immature, in vitro and in vivo matured canine oocytes by using scanning electron microscopy (SEM). Canine ovaries were collected from local veterinary hospitals to recover immature oocytes. The ovaries were sliced and the released cumulus oocyte complexes (COCs) were washed with TL-HEPES. The selected COCs were randomly divided into two groups, first group was processed immediately at immature state and the second group was processed 72 h after in vitro maturation, and compared with in vivo derived oocytes. Oocytes were fixed, critical point dried and examined under SEM. The diameters of oocyte and outer holes of the ZP were measured on a total of 249 oocytes; the results were analyzed using One-way ANOVA. Our results showed that, the diameter of immature oocytes significantly differed (p < 0.05) from that of in vivo matured oocytes ($79.60{\pm}0.77{\mu}m$ vs. $101.46{\pm}1.07{\mu}m$, respectively). Similarly, a significant difference (p < 0.05) in the diameters between those of in vitro and in vivo matured oocytes were found ($79.51{\pm}2.36{\mu}m$ vs. $101.46{\pm}1.07{\mu}m$, respectively). Moreover, the diameters of the outer holes of the ZP were significantly (p < 0.05) larger in in vivo matured ($1.48{\pm}0.42{\mu}m$) than in vitro matured for 72 and immature oocytes ($1.10{\pm}0.16$ and $0.43{\pm}0.12{\mu}m$, respectively). Taken together, these data indicates that the ZP surface is related to oocyte maturity in canine.

• Journal of Embryo Transfer
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• v.5 no.1
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• pp.21-27
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• 1990

The technique for maturation of follicular oocyte has been devised to provide such a low cost and in ptentifut number supply of bovine embryo. Some of problems concerning production of bovine embtyo in vitro were discussed in this paper. Bovine follicular oocytes cultured in vitro achieved normal fertilization but cleavage rates to blastocyst were low compared to the oocyte matured in vivo. It has been concluded that a deficient cytoplasmic maturation occurs in the oocytes matured in vitro. These results indicate that the studies for maturation of bovine follicular oocytes in vitro need improvement of culture conditions and to define the characteristics that might be indicative of healthy oocyte.

• Korean Journal of Animal Reproduction
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• v.17 no.2
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• pp.93-101
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• 1993

These experiments were carried out to investigate whether zona hardening affect the efficiency of in vitro fertilization in mouse oocytes. The soluble properties for zona pellucida of oocytes matured in vivo, aged oocytes, and ovarian oocytes matured in vitro have been analyzed with proteolytic enzyme, 3mg/ml of $\alpha$-chymotrypsin. The mean solubility(t50) for the zona of unfertilized oocytes, oocytes not fertilized at the first inseminati and in vitro produced zygotes were 10.1, 20.3 and 32.3min., respectively. The t50 for zona lysis of fertilized oocytes was significantly difference than those observed for unfertilized oocytes and oocytes not fertilized at the first insemination(P<0.01). In addition, the t50 of zona in ovulated oocytes with and without cumulus cells incubated for 0, 3, 6, 9 and 12 hr in vitro, t50 were 13.9, 11.1, 20.7 and 28.0min., and 22.3, 21.0, 30.0 and 33.5min., respectively. In these experiments, the zona pellucida showed a gradual increase in resistance to dissolution by $\alpha$-chyjotrypsin with in vitro aging for more than 6 hrs. This effect was greater in cumulus-free as compared to cumulus-intact oocytes. Finally, in cumulus-intact and cumulus-free ovarian oocytes matured for 0, 5, 10 and 15 hr in vitro the t50 of zona pellucida were 3.0, 10.6, 18.4 and 24.5 min., and 3.0, 14.0, 26.2 and 32.0 min., respectively. Clear differences in solubility between the zona pellucida of oocytes matured in vivo and in vitro. This data were found suggest that under in vitro conditions there is a gradual change in the soluble properties of the zona pellucida, particularly in the absence of the cumulus cells.

• Journal of Veterinary Clinics
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• v.32 no.6
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• pp.536-539
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• 2015

One year old mixed-breed bitch was examined to retrieve in vivo matured oocytes. Laparotomy was performed 72 hr after ovulation determined by serum progesterone concentration, and abnormally enlarged left uterus horn was found. Both ovaries had eight corpus lutea, and a total 16 in vivo matured oocytes having perivitelline space within $25{\mu}m$, polar body, and metaphase II nucleus were recovered by flushing oviducts. This is the first study to confirm in vivo maturation of oocytes from a bitch with hydrometra, which suggests that oocytes recovered from canids with reproductive disease could be valuable sources for assisted reproductive technologies.

• Reproductive and Developmental Biology
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• v.33 no.2
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• pp.85-91
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• 2009

In this study, in vitro maturation system using fetal bovine serum (FBS) or porcine follicular fluid (pFF) was investigated to produce comparable oocytes to those derived from in vivo. Control group of oocytes was cultured in TCM 199 supplemented with 0.1% polyvinyl alcohol (PVA). Other three groups of oocytes were cultured in TCM 199 supplemented with 10% FBS, 10% pFF or 5% FBS + 5% pFF, respectively. After 44 h maturation, oocytes with the first polar body were activated with two electric pulses (DC) of 1.2 kv/cm for 30 ${\mu}sec$. Also, matured oocytes of four groups were reconstructed and fused. Reconstructed embryos were cultured in PZM-3 under 5% $CO_2$ in air at $38.5^{\circ}C$ for 6 days. The oocytes matured in the medium supplemented with FBS or/and pFF showed significantly higher maturation rates (64.0 vs. 73.9 to 85.2%). In PA embryos, cleavage rates (89.7 vs. 77.1 to 86.6%) and blastocysts rates (30.0 vs. 16.2 to 26.2%) were significantly higher in pFF group (p<0.05). In NT embryos, there was no difference among treatments in cleavage rate, but the blastocyst rates (28.5 vs. 15.5 to 24.6%) were significantly higher in pFF group (p<0.05). The apoptosis rate was significantly higher (p<0.05) in the control than other groups (10.8 vs. 4.9 to 8.2% for PA, 3.1 vs. 0.5 to 1.3% for NT). In order to select the comparable oocyte to in vivo oocytes, each group of oocytes was stained with Brilliant cresyl blue (BCB) after 42h maturation. The matured oocytes were separated according to color of cytoplasm; stained group (BCB+) and unstained group (BCB-). The oocytes matured in the presence of FBS or/and pFF showed significantly higher staining rates (70.3 to 72.7 vs. 35.1%) (p<0.05). To verify the fact that the supplementation of FBS or/and pFF can increase the maturation rates, cdc2 kinase activity, the catalytic subunit of MPF, was determined. The cdc2 kinase activity of the oocytes matured in the medium supplemented with FBS or/and pFF was significantly higher than control group (6.7 to 9.3 vs. 3.8). In conclusion, the supplementation of FBS or/and pFF can support in vitro maturation rate of porcine oocytes through the increment of cdc2 kinase activity level in the cytoplasm.

• Asian-Australasian Journal of Animal Sciences
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• v.4 no.3
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• pp.251-254
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• 1991

Immobilized (killed) mouse spermatozoa or sperm head were microinjected into mouse oocytes matured in vivo and cultured for 72h in vitro. When non-capacitated spermatozoon was injected, oocytes that developed to $${\geq_-}$$ 2-cell and $${\geq_-}$$ 4-cell was 27.8 (15/54) and 3.7% (2/54), respectively. When non-capacitated sperm head was injected. development to $${\geq_-}$$ 2-cell and $${\geq_-}$$ 4-cell was 21.3 (16/75) and 8.0% (6/75), respectively. When capacitated spermatozoon was injected, development to $${\geq_-}$$ 2-cell and $${\geq_-}$$ 4-cell was 21.4 (15/70) and 4.3% (3/70), respectively. When capacitated sperm head was injected, development to $${\geq_-}$$ 2-cell and $${\geq_-}$$ 4-cell was 29.9 (35/117) and 10.3% (12/117), respectively. In contrast, none developed beyond 4-cell in the sham-operated group. The results of this study demonstrated that mouse oocytes matured in vivo can undergo normal appearing cleavage to 4-cell stage by dead-sperm injection. Sperm treatment prior to injection did not affect the ability of mouse oocytes to cleave in vitro.

• Korean Journal of Animal Reproduction
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• v.16 no.1
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• pp.39-46
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• 1992

In viro fertilizatin is very important in both human clinical practice and animal breeding. However, the success rate of in vitro fertilization is not high. The purpose of this study ws to determine wheter in the vitro fertilization and culture of porcine oocyte using a hydrogel chamber were possible or not. Hydrogel chambers were made of polymerized 2-hydroxyethyl methacrylate. Matured follicular oocytes in Waymouth's medium and T L Hepes medium, tubal oocytes, and preincubated sperm in M199 medium were treansferred into the lumen of the hydrogel chambers. The chambers containing porcine oocytes and spermatozoa implanted into the mouse peritioneal cavity, and ova were examined after the recovery of the chambers at 84 hours after preservation start. The result was shown that fertilization and culture of porcine oocytes were successfully achieved inside of the hydrogel chamber.

• Korean Journal of Animal Reproduction
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• v.26 no.2
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• pp.133-142
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• 2002

The objective of this study was to examine maturation, fertilization and developmental rate of the in vitro-grown mouse oocytes, and to compare these results with those of oocytes grown and matured in vivo. The preantral follicles isolated from 12-day-old mice were cultured on Transwell-COL membrane inserts. After in vitro growth and maturation, 72.5 % of oocytes grown in vitro produced polar body which can be comparable to in vivo growth (70.5 %). However, the mean oocyte diameter of the in vitro group (69.6$\pm$2.1$\mu$m) was smaller than that of the in vivo group (73.3$\pm$3.0$\mu$m). The fertilization rate was significantly lower (p<0.05) in the in vitro group (76.5%) than in the in vivo group (90.2%), however, there was no difference in the percentage of monospermic and polyspermic oocytes between two groups. The capacities of in vitro grown ova to cleave and develop to blastocyst were (57.8 and 14.4%, respectively) significantly lower (p<0.001) than those of the in vivo counterpart (84.4 and 56.6%, respectively). Moreover, the mean number of cells per blastocyst was significantly lower (p<0.05) in the in vitro group (39.0$\pm$10.8) than in the in vivo group (60.5$\pm$12.5). Live young were produced from transferred 2-cell embryos derived from in vitro-grown and matured oocytes. In conclusion, the results show that in vitro-grown oocytes did not achieve the developmental capacity of in vitro-grown oocytes.