175 DETECTION OF LIPID GRANULES ON IVF BLASTOCYSTS MATURED IN APO-TRANSFERRIN IN KOREAN NATIVE COWS (HANWOO)

2007 ◽  
Vol 19 (1) ◽  
pp. 204
Author(s):  
S. H. Choi ◽  
S. R. Cho ◽  
M. H. Han ◽  
H. J. Kim ◽  
C. Y. Choe ◽  
...  
Keyword(s):  
In Vitro Maturation ◽  
Blastocyst Stage ◽  
Blastocyst Formation ◽  
Granule Formation ◽  
Vitro Fertilization ◽  
Sudan Black B ◽  
Chi Squared ◽  
Maturation Rate ◽  
Lipid Granules

Apo-transferrin (apo-Tf) is found in mammalian sera and plays a role as an anti-oxidant in media. The objective was to investigate the effects of apo-Tf on in vitro maturation and development and to examine the characteristics of blastocysts derived from IVF production in the presence of apo-Tf in Korean native cows (Hanwoo). Ovaries were collected from a slaughterhouse and cumulus–oocyte complexes (COCs) were taken from 2–6-mm antral follicles. The collected COCs were washed 3 times with 0.1 M PVA-TCM-199 and matured in 10 �g mL-1 apo-Tf with TCM-199 at 39�C, 5% CO2, 95% air for 24 h. Matured COCs were fertilized with frozen–thawed Korean native cattle semen treated with BO medium containing 5 mM caffeine and 1 �g mL-1 heparin for 8 h, and developed to the blastocyst stage in 5% FBS, 0.3% BSA in TCM-199, and IVMD (IFP, Japan). To detect the lipid granules, blastocysts were fixed with 10% formalin-PBS for 2 h and then 50% ethanol for 2 min. Staining of blastocysts was performed with 1% Sudan Black B in 70% ethanol for 2 min. IVM and IVF were replicated 3 times. The results of blastocyst formation were analyzed by chi-squared test. The maturation rate was 92.6% at Met II in 10 �g mL-1 apo-Tf. The blastocyst formation was 22.5%, 38.4%, and 34.8% in 5% FBS (control), 0.3% BSA in TCM-199 (P < 0.05), and IVMD (P < 0.05), respectively. The lipid granules in blastocysts from the 5% FBS-TCM-199 group were larger than those in the 0.3% BSA-TCM-199 and IVMD groups. The results suggest that apo-Tf is an important factor for in vitro maturation and that FBS in culture medium affects lipid granule formation on bovine blastocysts derived from in vitro fertilization.

154 COMPARISON OF DIFFERENT CULTURE MEDIA AND INCUBATION METHODS ON CULTURING MURINE EMBRYOS IN VITRO USING STRAW AS A RECEPTACLE

2017 ◽  
Vol 29 (1) ◽  
pp. 185
Author(s):  
L. R. Madzhie ◽  
M. A. Raseona ◽  
L. P. Nethenzheni ◽  
O. Ajao ◽  
M. L. Mphaphathi ◽  
...  
Keyword(s):  
Developmental Stages ◽  
Culture Media ◽  
Uv Light ◽  
Fertilization Rate ◽  
Blastocyst Stage ◽  
Blastocyst Formation ◽  
Vitro Fertilization ◽  
Stage Of Development ◽  
Murine Embryos

In vitro fertilization in the straw system might increase the efficiency of fertilization and the quality of blastocyst formation as compared with micro-drops-IVF systems. The aim of the study was to in vitro fertilize mouse oocytes and culture the resulting zygotes in bi-gas incubator and in a goat vagina and compare the in vitro embryo developmental stages in TCM-199 and Ham’s F10 culture media until the blastocyst-stage of development. F1 generations (Balb C × C57) were used to harvest oocytes and spermatozoa. The fresh sperm were capacitated in different incubation methods (bi-gas incubator and in the vagina of a goat). A volume of 2–4 µL of Ham’s F10 containing capacitated sperm (~8 × 106 per mL) were placed into Ham’s F10 fertilization drops under the oil, containing 10 oocytes and penicillamine, hypotaurine, and epinephrine for enhancing sperm motility and penetration of oocytes. The same procedure was used with the TCM-199 medium and IVF drops without oil (both TCM-199 and Ham’s F10) for straw filling. The presumptive embryos in Ham’s F10 and TCM-199 were divided into different groups: first group were cultured in micro-drops, second group the embryos were aspirated in semen straws and placed in the incubator (incubator straws) for culture, and other straws were covered with a sponge and inserted in the vagina of a goat (vaginal straws) for culture. The resulted blastocysts were stained using Hoechst 33528 solution and blastomeres were counted on a fluorescent UV light inverted microscope at 400× magnification (Nikon Eclipse TI, Narishige Co., Ltd., Amityville, NY, USA). The results were analysed by 2 × 2 factorial designs and Student’s t-test was used to separate the mean. There was no statistical difference (P > 0.05) between the media and incubators on the stage of murine embryo development. The overall fertilization rate was 94 to 99%. The incubator straws with Ham’s F10 (80.5%) had the highest rate of embryos that reached the blastocyst stage, followed by incubator straws with TCM-199 (77.0%), and vaginal straws with Ham’s F10 (60.0%) had the lowest rate of embryos that reached the blastocyst stage. The overall mean number of blastomeres in the blastocyst stage of the embryos ranged from 85 ± 9 to 90 ± 9 cells in all receptacles and incubators. It was concluded that the fertilization and culturing of murine embryos are possible in straws incubated in a bi-gas incubator and in the goat vagina as an alternative method of fertilizing oocytes and culturing murine embryos. In addition, Ham’s F10 and TCM-199 can both be used to fertilize oocytes and culture murine embryos until blastocyst formation embryo in vitro, incubated in a bi-gas incubator or in the vagina.

scholarly journals Effect of follicle-stimulating hormone on Bligon goat oocyte maturation and embryonic development post in vitro fertilization

2020 ◽  
Vol 13 (11) ◽  
pp. 2443-2446
Author(s):  
Diah Tri Widayati ◽  
Mulyoto Pangestu
Keyword(s):  
In Vitro Fertilization ◽  
Embryonic Development ◽  
Oocyte Maturation ◽  
In Vitro Maturation ◽  
Follicle Stimulating Hormone ◽  
Fertilization In Vitro ◽  
Vitro Fertilization ◽  
Maturation Rate ◽  
Stimulating Hormone

Background and Aim: Bligon goat is a crossbreed between Etawah and Kacang goat. This crossbreed goat is mostly reared by small farmers. In vitro maturation allows female goat (does) contributes toward reproduction despite the fact that the animal has been slaughtered. The aim of this study was to determine the in vitro maturation rate of Bligon goat oocytes supplemented with follicle-stimulating hormone (FSH), and their ability for further embryonic development after in vitro fertilization. Materials and Methods: Experiment was conducted at the Laboratory of Animal Physiology and Reproduction, Faculty of Animal Science, Universitas Gadjah Mada, Yogyakarta, using Bligon goat ovaries obtained from local slaughterhouse around Yogyakarta. One thousand five hundred cumulus-oocyte complexes were matured for 24 h in tissue culture medium 199 supplemented with 50 IU/L FSH or without FSH (control). First, matured oocytes were evaluated its morphology based on the expansion of cumulus cells and PB1 extrusion. Next, 600 oocytes were then stained with 1% aceto-orcein to examine maturation based on changes in the configuration of chromosomes and nuclear membrane breakdown. Oocytes were considered mature when they reached metaphase II. To prove the ability of mature oocytes to develop into embryos, 900 oocytes were processed for fertilization in vitro. The data were analyzed using analysis of variance. Results: The results indicated that FSH supplementation significantly increased oocyte maturation rate (65.21±7.26 vs. 43.25±6.23%) as indicated by extrusion of PB1 and homologous chromosome pairing and lined in the equator. The rate of degeneration was lower in the FSH-supplemented medium (3.21±0.25 vs. 10.17±3.15%). The blastocyst stage of oocyte developed embryos was reached by 12.43±2.15% and 22.28±4.86% of the control and treatment groups, respectively. Conclusion: FSH supplementation significantly improves oocyte maturation and yields mature oocytes for future embryo development in vitro.

scholarly journals 205.The effect of FSH concentration during IVM and gamete co-incubation length during IVF on the development of unstimulated prepubertal ewe oocytes

2004 ◽  
Vol 16 (9) ◽  
pp. 205 ◽  
Author(s):  
K. M. Morton ◽  
W. M. C. Maxwell ◽  
G. Evans
Keyword(s):  
Blastocyst Stage ◽  
Developmental Competence ◽  
Blastocyst Formation ◽  
In Vitro Development ◽  
Oocyte Collection ◽  
Chi Squared ◽  
Development In Vitro ◽  
Stage 1 ◽  
Vitro Development

The developmental competence of prepubertal oocytes can be increased by the administration of gonadotrophins prior to oocyte collection (1); but this is not possible with abattoir-sourced oocytes, and modifications to the IVP system may increase in vitro development. Experiments were conducted to determine the effects of FSH concentration (10, 20 or 60 μg mL-1) during IVM (5 replicates) and gamete co-incubation length (short: 2-3 h, long: 18-20 h) during IVF (6 replicates) on subsequent embryonic development. For both experiments ovaries were collected from prepubertal lambs (16-24 weeks) slaughtered at an abattoir and embryos produced in vitro (1). Data were analysed by chi-squared test. Oocyte cleavage at 48 hours post-insemination (hpi) was higher for oocytes matured in medium containing 20 (60/77; 77.9%) and 60 (56/73; 76.7%) than 10 μg mL-1 (40/67; 59.7%) FSH. Blastocyst formation (% cultured oocytes) on Day 7 (Day 0 = IVF) was higher for oocytes matured with 20 (31/77; 40.3%) than 10 (16/67; 23.9%) or 60 μg mL-1 (20/73; 27.4%). Oocyte cleavage at 48 hpi was reduced for short (36/57; 63.2%) compared with long (49/55; 89.1%) co-incubation, although blastocyst formation (% cultured oocytes; Day 7) did not differ between groups (22/57; 38.6% and 23/55; 41.8%, respectively). These results demonstrate that increasing the FSH concentration above normal levels during IVM of prepubertal lamb oocytes improves development in vitro. Gamete co-incubation length did not influence the proportion of oocytes progressing to the blastocyst stage. (1) Morton et al. (2003) Proc. Soc. Reprod. Fert. P18.

302 EFFECT OF GROWTH DIFFERENTIATION FACTOR 8 ON PORCINE OOCYTE IN VITRO MATURATION AND SUBSEQUENT EMBRYONIC DEVELOPMENT AFTER PARTHENOGENETIC ACTIVATION AND IN VITRO FERTILIZATION

2015 ◽  
Vol 27 (1) ◽  
pp. 240
Author(s):  
J. D. Yoon ◽  
E. Lee ◽  
S.-H. Hyun
Keyword(s):  
Embryonic Development ◽  
In Vitro Maturation ◽  
Developmental Competence ◽  
Blastocyst Formation ◽  
Vitro Fertilization ◽  
Treatment Groups ◽  
Nuclear Maturation ◽  
Intracellular Ros ◽  
Sperm Penetration

Growth differentiation factor 8 (GDF8) is a member of the transforming growth factor-β that has been identified as a strong physiological regulator. The purpose of this study was to investigate the effects of GDF8 on in vitro porcine oocytes maturation and subsequent embryonic development after pathenogenetic activation (PA) and in vitro fertilization (IVF). We investigated nuclear maturation, intracellular glutathione (GSH), reactive oxygen species (ROS) levels, sperm penetration (SP) analysis, and subsequent embryonic development after PA and IVF. Each concentration (0, 1, 10, and 100 ng mL–1) of GDF8 was added in maturation medium during process of in vitro maturation. Data were analysed by ANOVA followed by Duncan using SPSS (Statistical Package for Social Science) mean ± s.e.m. After 44 h of IVM, no significant difference was observed on nuclear maturation from the different concentration (0, 1, 10, and 100 ng mL–1) of GDF8 treatment groups (85.5, 85.9, 89.4, and 87.6%, respectively) compared with the control (P > 0.05). The 10- and 100-ng mL–1 GDF8-treated groups showed a significant (P < 0.05) decrease in intracellular ROS levels compared with other groups. The embryonic developmental competence after PA was affected with GDF8 treatment during IVM. The 10- and 100-ng mL–1 treatment groups showed significantly (P < 0.05) higher cleavage rates (67.5 and 69.1%, respectively) compared with control group (53.7%). The 10- and 100-ng mL–1 treatment groups also showed significantly (P < 0.05) higher blastocyst formation rates (50.5 and 52.7%, respectively) compared with other groups (34.5 and 35.8%). The IVF embryonic developmental competence also was affected with GDF8 treatment during IVM. The 10-ng mL–1 treatment group showed a significantly (P < 0.05) higher blastocyst formation rates and total cell number compared with control (21.5 and 131.3 v. 15.0 and 92.6%, respectively). Also, in the sperm penetration assessment, the 10- and 100-ng mL–1 treatment groups showed higher mono spermy ratio and fertilization efficiency (32.7 and 27.1, 32.0 and 26.5 v. 22.6 and 19.7%, respectively) than control, which was significant (P < 0.05). In conclusion, the treatment with 10 ng mL–1 of GDF8 during IVM improved the PA and IVF porcine embryo developmental competence by decreasing the intracellular ROS levels.This work was supported, in part, by a grant from the Next-Generation BioGreen 21 Program (No. PJ00956901), Rural Development Administration, and the National Research Foundation of Korea Grant funded by the Korean Government (NRF-2013R1A2A2A04008751), Republic of Korea.

176 Effect of epigallocatechin-3-gallate on bovine oocyte in vitro maturation, fertilization, and development

2019 ◽  
Vol 31 (1) ◽  
pp. 212
Author(s):  
Y. Honkawa ◽  
Y. Gen ◽  
S.-H. Hyon ◽  
C. Kubota
Keyword(s):  
Oocyte Maturation ◽  
In Vitro Maturation ◽  
Polar Body ◽  
Sperm Concentration ◽  
P Value ◽  
Bovine Oocyte ◽  
Vitro Fertilization ◽  
Significant Difference ◽  
Maturation Rate

Epigallocatechin-3-gallate (EGCG) is a major ingredient of catechin polyphenols, and a strong antioxidant compound. Huang et al. (2018 Asian-australas. J. Anim. Sci.) reported that adding 50μM EGCG can improve the bovine oocyte maturation rate. In this research, we investigated the effect of EGCG supplementation on different periods in bovine IVF. Cumulus-oocyte complex (COC) collected from ovaries of slaughtered cows were cultured in maturation medium (20 to 30 oocytes per 100-µL droplet), which consisted of TCM-199 with Earle’s salts and 25mM HEPES supplemented with 10% (vol/vol) fetal bovine serum (FBS), 1µg mL−1 oestradiol, 0.02mg mL−1 FSH, and antibiotics at 38.5°C in a humidified atmosphere of 5% CO2 in air for 24h (in vitro maturation, IVM). After IVM, COC were fertilized in the fertilization medium (modified Brackett-Oliphant media supplemented with 10 µgmL−1 heparin, 10mM caffeine, and 3mg mL−1 BSA) for 6h using semen of one bull at final sperm concentration of 1×107 mL−1 (IVF). After IVF, COC were denuded and cultured in culture medium [CR1aa supplemented with 10% (vol/vol) FBS and antibiotics] at 38.5°C in a humidified atmosphere of 5% O2, 5% CO2, and 90%N2 for 8 days (in vitro culture, IVC). The EGCG was supplemented at 10, 25, 50, and 100M in IVM medium; 25 and 50 µM in IVF medium; and 50 and 100 µM in IVC medium. After 24h in IVM medium, COC were denuded by pipetting, fixed in 3:1 ethanol:acetic acid for 24h and then checked for nuclear and polar body by using aceto-orcein stain. After 18h in IVF, the pronucleus in zygote was fixed in 3:1 ethanol:acetic acid for 24h and checked by aceto-orcein staining. Embryo development was evaluated by counting the total number of embryos that had reached compacted morula by 6 to 8 days after IVF. Significant differences were analysed by chi-squared test and residual analysis. A P-value&lt;0.05 was considered statistically significant. When EGCG was added to IVM, there was no significant difference of oocyte maturation rate between all concentrations (0v. 10v. 25v. 50v. 100 μM: 73.9% v. 56.7% v. 76.7% v. 72.7% v. 63.5%). When EGCG was added to IVF, there was no significant difference of fertilized rate (0v. 25v. 50 μM: 59.4% v. 73.7% v. 64.9%). When EGCG was added to IVC, there was no significant difference in development rate (0v. 50v. 100 μM: 26.2% v. 15.7% v. 22.0%). In this research, EGCG addition did not affect bovine in vitro fertilization.

ТРАНСВАГИНАЛЬНАЯ АСПИРАЦИЯ ФОЛЛИКУЛОВ И СПОСОБНОСТЬ OPU-ООЦИТОВ КОРОВ К ЭМБРИОНАЛЬНОМУ РАЗВИТИЮ В УСЛОВИЯХ IN VITRO

2019 ◽  
pp. 17-19
Author(s):  
G.N. SINGINA ◽  
V. HAVLICEK ◽  
N.P. TARADAYNIK ◽  
R.Y. CHINAROV ◽  
T.E. TARADAYNIK ◽  
...  
Keyword(s):  
In Vitro Fertilization ◽  
Embryo Development ◽  
In Vitro Maturation ◽  
Blastocyst Stage ◽  
Vitro Fertilization ◽  
Maturation In Vitro ◽  
Transvaginal Aspiration ◽  
Cattle Reproduction ◽  
Ovarian Follicular Growth

Представлены результаты трансвагинальной аспирации ооцитов коров, а также оценен их потенциал к эмбриональному развитию после оплодотворения в условиях in vitro. Донорами яйцеклеток являлись половозрелые телки симментальской породы в возрасте 1619 мес. Животныедоноры (n7) перед проведением процедуры Ovum Pickup (OPU) были гормонально обработаны с целью стимуляции роста фолликулов. Количество выделенных ооцитов от индивидуальных доноров составило в среднем 7,7 ооциткумулюсных комплексов (ОКК), что соответствовало степени извлечения 54,57,7. Доля ОКК хорошего качества, рассчитанная от общего числа извлеченных ОКК, между отдельными животными существенно не различалась (значения варьировали от 60,0 до 75,0) и в среднем составила 67,21,9. ОКК с признаками нормальной морфологии подвергали in vitro процедурам созревания, оплодотворения и последующего культивирования до стадии бластоцисты. Доля раздробившихся ооцитов и выход бластоцист после in vitro осеменения яйцеклеток коров равнялась 75,7 и 24,3, соответственно. В целом от одного донора за сессию OPU было получено 1,3 эмбриона на стадии бластоцисты, содержащих в среднем 89,8 ядра. Оцененный способ экстракорпорального оплодотворения OPUооцитов коров позволяет получать эмбрионы, пригодные для замораживания и трансплантации реципиентам и может быть использован в программах по воспроизводству желаемых генотипов у крупного рогатого скота.In the present work, we report the data on transvaginal aspiration of bovine ovarian follicles and estimation of in vitro embryo development competence of collected oocytes. The oocytes were collected by ovum pickup OPU from seven 1619 monthold Simmental heifers, previously hormonallytreated in order to stimulate ovarian follicular growth. In average, 7.7 oocytecumulus complexes (OCCs) per heifer per OPU session were collected that corresponded to 54.57.7 of recovery rate. Morphologically, 60.075.0 of OCCs were the good quality and this rate did not significantly differ between the animals. Good quality OCCs (total n37) were then subjected to in vitro maturation, in vitro fertilization and in vitro embryo development up to blastocyst stage. Cleavage and blastocyst rates were 75.7 и 24.3 , respectively. In total, 1.3 blastocysts were obtained per cow per OPU session in average these blastocysts contained 89.9 cells. In conclusion, we developed the methodology of in vitro fertilization of bovine OPUcollected oocytes that allowed obtaining the blastocysts potentially suitable for freezing and transplantation to recipients. This approach can be used to multiply desired genotypes in cattle reproduction.

scholarly journals Response of Bovine Cumulus–Oocytes Complexes to Energy Pathway Inhibition during In Vitro Maturation

Genes ◽  
2021 ◽  
Vol 12 (6) ◽  
pp. 838
Author(s):  
Paulina Lipinska ◽  
Ewa Sell-Kubiak ◽  
Piotr Pawlak ◽  
Zofia Eliza Madeja ◽  
Ewelina Warzych
Keyword(s):  
In Vitro Maturation ◽  
Ovarian Follicle ◽  
Cumulus Cells ◽  
Selective Inhibition ◽  
Blastocyst Stage ◽  
Vitro Fertilization ◽  
Glucose Inhibition ◽  
Maturation Medium ◽  
Pathway Inhibition

Glucose or fatty acids (FAs) metabolisms may alter the ovarian follicle environment and thus determine oocyte and the nascent embryo quality. The aim of the experiment was to investigate the effect of selective inhibition of glucose (iodoacetate + DHEA) or FA (etomoxir) metabolism on in vitro maturation (IVM) of bovine COCs (cumulus–oocyte complexes) to investigate oocyte’s development, quality, and energy metabolism. After in vitro fertilization, embryos were cultured to the blastocyst stage. Lipid droplets, metabolome, and lipidome were analyzed in oocytes and cumulus cells. mRNA expression of the selected genes was measured in the cumulus cells. ATP and glutathione relative levels were measured in oocytes. Changes in FA content in the maturation medium were evaluated by mass spectrometry. Our results indicate that only glucose metabolism is substantial to the oocyte during IVM since only glucose inhibition decreased embryo culture efficiency. The most noteworthy differences in the reaction to the applied inhibition systems were observed in cumulus cells. The upregulation of ketone body metabolism in the cumulus cells of the glucose inhibition group suggest possibly failed attempts of cells to switch into lipid consumption. On the contrary, etomoxir treatment of the oocytes did not affect embryo development, probably due to undisturbed metabolism in cumulus cells. Therefore, we suggest that the energy pathways analyzed in this experiment are not interchangeable alternatives in bovine COCs.

scholarly journals In vitro production of Sudanese camel (Camelus dromedarius) embryos from epididymal spermatozoa and follicular oocytes of slaughtered animals

2017 ◽  
Vol 20 (1) ◽  
pp. 95-101 ◽  
Author(s):  
A.E. Abdelkhalek ◽  
Sh.A. Gabr ◽  
W.A. Khalil ◽  
Sh.M. Shamiah ◽  
L. Pan ◽  
...  
Keyword(s):  
Culture Medium ◽  
Fertilization Rate ◽  
Blastocyst Stage ◽  
Dromedary Camel ◽  
In Vitro Production ◽  
Vitro Fertilization ◽  
Maturation Rate ◽  
Epididymal Spermatozoa ◽  
Vitro Production

Abstract Application of assisted reproductive technology in camelidea, such as artificial insemination (AI) and embryo transfer, has been slow in comparison to that for other livestock species. In Egypt, there are few attempts to establish in vitro maturation (IVM) and fertilization (IVF) techniques in dromedary camel. The present study was carried out to produce Sudanese camel embryos using in vitro matured oocytes and epididymal spermatozoa. Dromedary camel ovaries were collected from abattoirs and then, the oocytes were aspirated from all the visible follicles on the ovarian surface (~2-8 mm in a diameter). Meanwhile, Fetal Dromedary Camel Serum (FDCS) was obtained from camel fetuses after slaughtering. Thereafter, only Cumulus Oocyte Complexes (COCs) were matured in vitro in the Tissue Culture Medium (TCM-199) complemented with 10% FDCS. Spermatozoa required for in vitro fertilization were collected from testes (epididymal cauda) of the slaughtered camel bulls. The results clearly showed that the maturation rate of oocytes at metaphase II was about 59.5% while the fertilization rate was around 70.4%. Intriguingly, the embryo rates determined were 13.1%, in 2-cell; 0.0%, in 4-cell; 34.7%, in 8-16% cell; 39.1%, in morula and 13.1% in a blastocyst stage. This study represented a successful in vitro production of Sudanese dromedary camel embryos from epididymal sperm cells and in vitro matured oocytes recovered from slaughtered camels.

scholarly journals Development to Blastocyst Stage of Pig Oocytes Matured, Fertilized and Electroactivated In Vitro

2002 ◽  
Vol 45 (6) ◽  
pp. 547-556
Author(s):  
N. R. Mtango ◽  
M. D. Varisanga ◽  
D. Y. Juan ◽  
P. Wongrisekeao ◽  
T. Suzuki
Keyword(s):  
In Vitro Maturation ◽  
Blastocyst Stage ◽  
Developmental Competence ◽  
Cleavage Rate ◽  
Vitro Fertilization ◽  
Nuclear Maturation ◽  
Significant Difference ◽  
Activation Rates ◽  
Oviduct Fluid

Abstract. This study was designed 1) to determine the effectiveness of two in vitro maturation (IVM) media (tissue culture medium [TCM] and modified synthetic oviduct fluid supplemented with amino acids [mSOFaa]), 2) to compare the effects of two in vitro fertilization (IVF) media (modified Tris-buffered medium [mTBM] and mSOFaa) on the developmental competence of pig oocytes, and 3) to test the activation ability of IVM pig oocytes matured in TCM or mSOFaa, electroactivated and cultured in mSOFaa. The nuclear maturation rates were similar between IVM media (91.0 % vs. 89.0 %). A similar result was obtained when the activation rates were 54.2 % in TCM and 56.0 % in mSOFaa, and the blastocyst rates were 7.9 % and 6.1 %, respectively. There was no significant difference between mSOFaa and mTBM in the percentage of embryos with two pronuclei 33.2 % vs. 13.8 % or polypronuclei 5.3 % vs. 13.4 %. The cleavage rate was the same in both media. The medium mSOFaa gave a significantly higher (P< 0.05) blastocyst rate than mTBM (12.7 % vs. 3.9 %). We concluded that mSOFaa can enhance in vitro maturation, fertilization and culture of pig oocytes.

241 DIBUTYRYL CYCLIC AMP AND EGF-LIKE FACTORS SUPPORT IN VITRO MATURATION OF PORCINE OOCYTES IN A CHEMICALLY DEFINED MEDIUM WITHOUT GONADOTROPINS

2009 ◽  
Vol 21 (1) ◽  
pp. 218
Author(s):  
Y. Akaki ◽  
K. Yoshioka ◽  
H. Funahashi
Keyword(s):  
Cyclic Amp ◽  
In Vitro Maturation ◽  
Defined Medium ◽  
Blastocyst Stage ◽  
Developmental Competence ◽  
Chemically Defined Medium ◽  
Dibutyryl Cyclic Amp ◽  
Vitro Fertilization ◽  
Porcine Oocyte

Exposure of porcine oocyte–cumulus complexes (OCC) to gonadotropins induces meiotic resumption, but the details of this mechanism are still unknown. The present study was undertaken to examine combinational effects of EGF-like factors and dibutyryl cyclic AMP (dbcAMP) in a chemically defined medium on in vitro maturation (IVM) of porcine oocytes. The OCC were aspirated from 3- to 6-mm-diameter follicles of prepuberal ovaries and used in the current study. The basic culture medium was a chemically defined medium, Porcine Oocyte Medium (POM; Research Institute for the Functional Peptides, Yamagata, Japan). In the first experiment, various concentrations (0, 10, and 1000 ng mL–1) of EGF-like factors (EGF, amphiregulin, and betacellulin) were added to POM during an entire IVM period (44 h). In the second experiment, to determine the additive effect of EGF-like factors, each EGF-like factor with an effective concentration was combined with the others. In the last experiment, to examine the combined effect with dbcAMP, OCC were exposed to EGF (10 ng mL–1), amphiregulin (1000 ng mL–1), and dbcAMP (1 mm) during the first 20 h of IVM and then the culture was continued in the absence of EGF-like factors and dbcAMP. After culture, in all experiments, meiotic resumption and the progress of oocytes were examined after denuding, fixing, and staining. Statistical analyses was performed by ANOVA with a Bonferroni-Dunn post hoc test (significance, P < 0.05). In the first experiment, all treatments without supplementation with 10 ng mL–1 amphiregulin increased the incidence of oocytes maturing to the MII phase, as compared with controls (29.1 to 39.3% v. 11.1%, P < 0.05). In the second experiment, combinations with 2 kinds of EGF-like factor slightly (but not significantly) improved the percentage of oocytes at the MII stage (37.7 to 47.4%). In the last experiment, supplementation with 1 mm dbcAMP during the first 20 h of IVM, regardless of the presence of EGF-like factors, significantly increased the incidence of MII oocytes as compared with controls, whereas the incidence was the highest when 1 mm dbcAMP, 10 ng mL–1 EGF, and 1000 ng mL–1 amphiregulin were supplemented (75.5%). When those oocytes were cultured in a chemically defined medium after in vitro fertilization, the developmental competence of oocytes to the blastocyst stage (25.0%) was not different from oocytes matured in the presence of gonadotropins and dbcAMP during the first 20 h of IVM (17.3%). These observations indicate that supplementation of a chemically defined maturation medium with EGF-like factors and dbcAMP during the first 20 h of IVM can support the meiotic progress and developmental competence of porcine oocytes well. Currently, we are examining the developmental competence of those oocytes after embryo transfer. The results will be presented at the meeting. This study was supported by MAFF AgriBio1605.

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