• Asian-Australasian Journal of Animal Sciences
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• v.14 no.12
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• pp.1675-1677
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• 2001

The effect of the presence or absence of corpus luteum in the ovaries of slaughtered buffaloes was studied for the oocytes recovery and their subsequent maturation and fertilization in vitro. On an average, 0.41 and 0.67 oocytes per ovary were recovered from ovaries with and without corpus luteum, respectively. Immature oocytes were matured in TCM-199 medium. Significant difference was observed in maturation rate between good (74%) and fair (37%) oocytes. However, there was no significant difference in cleavage rate between the two types. The results of this study show that although the presence of corpus luteum in the ovary at the time of recovery significantly affected availability of total oocytes and in-vitro maturation, but fertilization and cleavage remained unaffected under in vitro conditions.

• Korean Journal of Veterinary Research
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• v.33 no.1
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• pp.179-188
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• 1993

The developmental capacity of bovine oocytes under three different culture systems was investigated in this experiment ; One was culture in TCM199 with bovine oviductal epithelial cells(BOEC) for in vitro culture, another was culture in TCM199 with BOEC for 2 days and then transfer of 4~8cell embryos to rabbit oviduct(RO) and the other was transfer of 1 or 2cell embryos to RO for in vivo culture. And the other concern of this experiment was to investigate the effect of culture period and transfer site on recovery. Immature bovine oocytes were cultured in TCM199 with granulosa cells for 22-24hrs and then fertilized in vitro using frozen-thawed semen treated with BO-caffine and BO-BSA. Fifteen to 18hrs after in vitro fertilization oocytes were cultured in TCM199 with BOEC or transferred to RO for 5 days. The rate of development to the morula or blastocyst was higher in transfer of 1 or 2cell embryos to RO(23.1%) than culture in TCM199 with BOEC(11.7%). But, there was no difference between transfer of 1 or 2cell embryos and transfer of 4~8cell embryos to RO(12.8%). Recovery under different culture periods in RO was significantly higher in 90~95hrs(70.1%) than 122~125hrs(50.9%, p<0.05) and recovery significantly increased when oocytes were transferred deeper in RO(2.5cm>, 47.7% ; 2.5~4.5cm, 63.9% ; 4.5cm<, 77.3%, p<0.05). The results show that transfer of 1 or 2cell embryos to RO is an effective means of supporting the further development of in vitro matured and fertilized bovine oocytes than culture in TCM199 with BOEC or transfer of 4~8cell embryos to RO, and recovery from RO increases when oocytes are transferred deeper and incubated shorter in RO.

• Journal of Embryo Transfer
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• v.15 no.3
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• pp.279-286
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• 2000

This study was conducted to develop an improved method for oocyte pick-up(OPU) with finger-sensibility using ultrasound-guidance from ovarian follicles in Holstein heifers. Oocytes were aspirated from ovarian follicles of clear-outline (>2mm), obscure-outline and invisible($\leq$ 2mm) on ultrasound images with 3 different vacuum pressure(40, 80, 120mmHg). Total number of oocytes recovered/follicles were 309/237(130.4%). 113/80(141.3%) and 107/74(144.6%) with 40, 80 and 120 mmHg of vacuum pressure, respectively. Mean number of oocytes recovered was higher in 2 OPU/week (18.3$\pm$5.3) than 1 OPU/week(14.5$\pm$4.1), but this difference was not statistical1y significant. The recovery rates were not affected by the number of OPU as 135.6%(282 oocytes/208 follicles) in 1~20 OPU, 137.7% (168/122) in 21~40 OPU and 148.4%(92/62) in 41~60 OPU, respectively. The proportions of good oocytes (Grades I) recovered were not significantly different by the number of OPU until 40 OPU(12.4% in 1~20 OPU vs 16.7% in 21~40 OPU). However, a significantly(P<0.05) lower recovery rate resulted from more than 40 OPU compared to less than 40 OPU(7.6%). These results imply that more fertilizable oocytes can be produced from invisible-immature follicles by transvaginal aspiration with finger-sensibility from Holstein heifers.

• Asian-Australasian Journal of Animal Sciences
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• v.20 no.4
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• pp.496-500
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• 2007

Holstein cow ovaries obtained at a slaughterhouse were used to study the influence of the oocyte collection methods (slicing, puncture, aspiration I and II) on recovery efficiency and subsequent in vitro maturation and embryonic development competence of immature oocytes recovered. In the slicing method, the whole ovarian was chopped into small pieces with a surgical blade. In the puncture method, the whole ovarian surface was punctured by 18-g needle. In other 2 aspiration methods, collected oocytes by aspirating from the visible follicles using an 18-g needle attached to a 5 ml syringe (aspiration I) or using a constant negetive pressure (-80 mmHg) with a vacuum pump (aspiration II). The oocytes were classified into 4 classes on the basis of the morphology of cumulus cells and cytoplasmic appearance of oocyte. Slicing ($9.6{\pm}0.4$) and puncture ($9.7{\pm}0.4$)yielded a larger number of oocytes per ovary than other two aspiration methods (aspiration I and II were $5.8{\pm}0.3$and $5.6{\pm}0.4$, respectively) (p<0.05). The number of the highest quality oocytes (grade A) per ovary was significantly higher in slicing ($4.2{\pm}0.2$) and puncture ($4.6{\pm}0.1$) methods than in other methods (aspiration I and II were $1.2{\pm}0.2$ and $1.4{\pm}0.2$, respectively) (p<0.05). The rate of nuclear maturation of the highest and higher quality oocytes (grade A and grade B, respectively) was not affected by the oocytes collection methods. The oocytes collection methods also did not influence subsequent embryonic developmental competence after in vitro fertilization with M II stage oocytes. It is concluded that slicing and puncture methods of the ovaries can be used as an alternative techniques to aspiration by the syringe or vacuum pump.

• Journal of Animal Reproduction and Biotechnology
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• v.34 no.4
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• pp.300-304
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• 2019

This study was conducted to examine the oocyte recovery efficiency through having an OPU session once and twice a week. Also, the oocyte recovery efficiency was examined by using OPU after two and three months of rest period. Six cows were used for oocytes collection and were randomly divided into two groups. In experiment 1, OPU sessions were conducted once and twice a week to collect oocytes. The collected oocytes between once and twice OPU groups were classified into four groups (grade 1, 2, 3 and 4) according to the quality of cumulus cells and ooplasm. Based on the result, the percentage of collected oocytes per aspirated follicle number was similar between once and twice OPU session groups (65.5 ± 1.9 and 68.7 ± 1.4 vs.). However, the percentage of grade 1 oocytes from the twice OPU session group was significantly high compared with that of the once a week OPU session group (25.3 ± 0.9 and 32.5 ± 1.2% vs. once and twice session group, respectively, p < 0.05). In experiment 2, the group with three months of rest period tended to have a high percentage of collected oocyte compared with the group with two months of rest period (64.6 and 70.9% vs. 2 and 3 months rest group, respectively, p = 0.62). The percentage of grade 4 in the group with three months of rest period was significantly low compared with the group with two months of rest period group (27.3 and 36.5% vs. two and three months rest group, respectively, p = 0.05). In conclusion, twice a week OPU session is suitable for collection of high quality oocytes by using OPU, and three months of rest period is needed for the recovery of oocyte quality of a donor cow.

• Reproductive and Developmental Biology
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• v.34 no.3
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• pp.223-227
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• 2010

These study was carried out to investigate the effects of the recovery time, diameter of oocytes on in vitro fertilization or intracytoplasmic sperm injection (ICSI). The in vitro maturation rates to MII stage of oocytes recovered at the inactive, follicular and luteal stages matured for 72 h were $1.4{\pm}0.0%$, $43.4{\pm}3.2%$ and $10.8{\pm}2.7%$, respectively. The fertilization rates of in vitro cultured oocytes recovered from ovaries at the in active, follicular and luteal stages were $0.0{\pm}0.0%$, $15.7{\pm}3.4%$ and $7.6{\pm}3.5%$, respectively. The in vitro maturation rate of oocytes recovered from ovaries at the follicular stage of the reproductive cycle was significantly higher than those at the inactive and luteal stages (p<0.05). The penetration rate determined that the percentages of oocytes with diameters in the < $100\;{\mu}m$, 100 to $100\;{\mu}m$ and 110 to $120\;{\mu}m$ ranges were $17.5{\pm}4.7%$, $43.9{\pm}4.5%$, $21.3{\pm}3.4%$, respectively. The penetration rate of oocytes with diameters between 100 to $100\;{\mu}m$ was significantly higher than that of oocytes whose diameters were < $100\;{\mu}m$ and $110{\sim}120\;{\mu}m$ (p<0.05). The penetration rate of oocytes determined that the percentages of ovaries with diameters between 1 to 5 mm and 6 to 10 mm were $32.9{\pm}3.2%$ and $17.5{\pm}3.7%$, respectively. Thus, the diameters of the ovaries were significantly higher at 1 to 5 mm (p<0.05). A total of 264 oocytes were fixed and stained after co-incubation with sperm, of which 72 had identifiable nuclear material. After in vitro fertilization for 20 hrs, 27.3% of oocytes were penetrated by spermatozoas. Oocytes were fixed and stained after ICSI, of which 38 oocytes contained identifiable nuclear material. After in vitro fertilization and ICSI for 20 hrs, to 27.3% and 67.9% of oocytes were penetrated by spermatozoas. The in vitro fertilization rates by ICSI was significantly higher than that in vitro fertilization method (p<0.05).

• Asian-Australasian Journal of Animal Sciences
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• v.11 no.5
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• pp.491-497
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• 1998

Four types of serum supplements viz. estrus cow serum (ECS), estrus buffalo serum (EBS), pro-estrus buffalo serum (PrBS) and post-estrus buffalo serum (PtBS), added to TCM-199, were evaluated for in vitro maturation and fertilization of buffalo follicular oocytes. The oocytes were recovered from buffalo ovaries after slaughter, using either aspiration or scoring (multiple incisions) method. The recovered oocytes were categorized as A, B and C based on their cumulus investment and ooplasm homogeneity and cultured in four media. The in vitro matured oocytes were inseminated with $1{\times}10^6$ spermatozoa washed in 2.9% sodium citrate solution. The scoring method yielded greater number of morphologically good oocytes than the aspiration method (3.85 vs 1.76 per ovary, p < 0.01). The maturation rates of three categories of oocytes did not differ from one another. The maturation rates of 80.00, 82.08, 78.77 and 66.23%, while the fertilization rates of 54.54, 55.38, 52.80 and 36.76% were recorded for media containing ECS, EBS, PrBS, and PtBS, respectively. The medium containing PtBS gave lower maturation, as well as fertilization, rates than the other three media (p < 0.05). Thus, the scoring method was better than the aspiration method for the recovery of follicular oocytes. The oocytes categorized A, B and C had similar maturation capabilities. The TCM-199 containing buffalo/cow serum collected at pro-estrus or estrus appeared better for in vitro maturation and fertilization of buffalo follicular oocytes than that containing serum collected at post estrus.

• Journal of Embryo Transfer
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• v.13 no.2
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• pp.107-115
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• 1998

The present study was carried out for the comparative study on the collection of bovine follicular oocytes by ultrasound-guided ovum pick-up(OPU) and slaughterhouse-derived (SHD) ovary aspiration and in vitro production of bovine embryos with the follicular oncytes in Korean native cows. Bovine follicular nocytes were observed with a 6.5 MHz convex-array ultrasound transducer designed for intravaginal use and the oocytes were collected with the aspiration equipment attached to the ultrasonograph. Bovine ovaries were collected and transported in phosphate buffered saline from the local slaughterhouse, the follicular oocytes were collected by the aspiration method. The collected follicular oocytes in good quality were matured, fertilized and cultured in the media. The total number of the visible follicles and the recovery rate of follicular oocytes were increased in ultrasonography following follicle stimulating hormone(FSH) treatment in Korean native cows. The mean recovery rate of oocytes was 66.2, 52.8 and 41.7% in the FSH-OPU, non-treatment-OPU and SHD ovaries, respectively. The mean number of recorved oocytes per cow were not significantly(P<0.05) different between the FSH-OPU(14.0$\pm$11.54) and SHD(17.1i6.21) groups, but the numbers in both groups were significantly(P<0.05) higher than the number in the non-treatment-OPU(3.7$\pm$1.57) group. The mean number of usable nocytes in Grade T /11 per ovary was 6.3, 4.8 and 1.3 in the cows of the SHD, FSH-OPU and non-treatment-OPU groups, respectively. The in vitro developmental rate to the blastocyst was not significantly different between the oocytes obtained via OPU(37.1%) and SHD(29.3%). Therefore, the ultrasound-guided OPU technique can be applied to the production of excellent embryos from the high-quality cows, and for the large scale production of in vitro bovine oocytes and embryos, the SHD ovary aspiration method is valuable.

• Journal of Embryo Transfer
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• v.12 no.2
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• pp.195-202
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• 1997

The present study was carried out to evaluate the effect of superovulation treatments on ovarian responses, oocyte recovery rates and grades of collected oocytes using an ultrasound-guided transvaginal approach in Korean native cows. Superovulation in cows was induced with two different regimenes: 1) FSH-decreasing dose(n=8): the cows were received twice per day for three days of the total dose of 400 mg of FSH-p, 2) FSH-single dose(n=9): the cows were administrated a single dose of 400 mg of FSH-p in 25% PVP. The Observation of visible follicles and collection of oocytes were performed 12 hours following the last FSH in FSH-decreasing dose group and 48 hours after the FSH-single dose injection. All visible follicles larger than 6 mm were punctured and aspirated with a 6.5 MHz convex-array ultrasound transducer designed for intravaginal use. The mean number of visible follicles(> 6 mm) was significantly(P<0.05) higher in the FSH-decreasing dose treatment (22.811.9) and FSH-single dose treatment (20.612.0) groups than the non-treatment group(7.0$\pm$8). The mean recovery rate of oocytes was not significantly(P<0.05) different between the treatment and control groups, but the mean number of collected oocytes was significantly(P<0.05) higher in the FSH-decreasing dose treatment( 12.611.5) and FSH-single dose treatment (11.813.6) groups than the non-treatment group(3.7$\pm$0.5). In conclusion, the FSH-single dose treatment at superovulation in cows for ultrasound-guided aspiration might increase the number of aspiratable follicles and the recovery rate of follicular oocytes as the FSH-decreasing dose treatment.

• Korean Journal of Animal Reproduction
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• v.22 no.2
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• pp.119-126
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• 1998

This study was conducted to find out the recovery rate of oocyte according to the different size of follicles from porcine ovaries, and the effect of in vitro maturation of porcine immature oocyte at the different transportation temperature of ovaries from slaughter house. The results obtained were summarized as follows : 1. The number of follicles per ovary was 22.5. The number of A-and B-typed oocytes(type A: cumulus-enclosed oocyte, type-B : corona-enclosed oocyte) per ovary was 2.4. The proportion of A-and B-typed oocytes was 29.6% of the total recovery oocytes. 2. When the immature oocytes were cultured for 36, 40, 44 and 48 h at 5$^{\circ}C$ transportation temperature of ovary, the germinal vesicle breakdown(GVBD) rates of porcine oocytes were 32.5, 28.2, 22.6 and 25.9% respectively. There were no significant differences between all the culture time for GVBD. Especially, most of oocytes were observed to arrest the development beyond germinal vesicle(GV) stage. 3. When the immature oocytes were cultured for 36, 40, 44 and 48 h at $25^{\circ}C$ transportation temperature of ovary, the GVBD rates were 81.0, 90.0, 91.7 and 92.9%, and the maturation (Met-II) rates were 51.2, 78.8, 76.2 and 78.6%, respectively. 4. When the immature oocytes were cultured for 36, 40, 44 and 48 h at 38$^{\circ}C$ transportation temperature of ovary, the GVBD rates were 93.9, 96.5, 96.5 and 95.3%, and the maturation rates were 62.2, 88.4, 84.7 and 86.0%, respectively. 5. The above results showed that the maturation rates of immature oocytes between $25^{\circ}C$ and 38$^{\circ}C$ transportation temperature of ovary did not differ significantly.