Development and pregnancy rates of Camelus dromedarius-cloned embryos derived from in vivo- and in vitro-matured oocytes

Son Young-Bum,Jeong Yeon Ik,Jeong Yeon Woo,Olsson Per Olof,Hossein Mohammad Shamim,Cai Lian,Kim Sun,Choi Eun Ji,Sakaguchi Kenichiro,Tinson Alex,Singh Kuhad Kuldip,Rajesh Singh,Noura Al Shamsi,Hwang Wo 아세아·태평양축산학회 2022 Animal Bioscience Vol.35 No.2

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 vitromatured 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. 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 vitromatured 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.

Effect of zeaxanthin on porcine embryonic development during <i>in vitro</i> maturation

Park, Seo-jin,So, Kyoung-Ha,Hyun, Sang-Hwan Elsevier 2017 JOURNAL OF BIOMEDICAL RESEARCH -ELSEVIER- ENGLISH Vol.31 No.2

<P>Zeaxanthin is a common carotenoid, which is a powerful antioxidant that protects against damage caused by reactive oxygen species. The aim of the present study was to investigate the effects of zeaxanthin supplementation on <I>in vitro </I>maturation of porcine embryo development. We investigated nuclear maturation, intracellular glutathione (GSH), and reactive oxygen species (ROS) levels during in vitro maturation, and subsequent embryonic development following parthenogenetic activation and <I>in vitro</I> fertilization (IVF). The oocytes were maturated and used at the metaphase II stage. After 42 hours of <I>in vitro</I> maturation, the zeaxanthin-treated group (0.5 mmol/L) showed significant increases in nuclear maturation (89.6%) than the control group (83.4%) (<I>P</I><0.05). The intracellular GSH levels increased significantly (<I>P</I><0.05) as zeaxanthin concentrations increased; ROS generation levels decreased with increased zeaxanthin concentrations, but there were no significant differences. There were no significant differences in subsequent embryonic development, cleavage rate, blastocyst stage rate, and total blastocyst cell numbers following parthenogenetic activation and IVF when in vitro maturation media was supplemented with zeaxanthin. These results suggest that treatment with zeaxanthin during in vitro maturation improved the nuclear maturation of porcine oocytes by increasing the intracellular GSH level, thereby slightly decreasing the intracellular ROS level.</P>

Epidermal Growth Factor(EGF)와 Transforming Growth Factor-$\alpha$(TGF-$\alpha$)가 돼지 난포란의 체외성숙에 미치는 영향

임정훈,박병권,이규승 한국동물번식학회 1997 Reproductive & developmental biology Vol.21 No.2

The present study examined the effects of epidermal growth factor(EGF) and transforming growth factor-$\alpha$(TGF-$\alpha$) on in vitro maturation of porcine follicular oocytes. Basic medium used TCM-HEPES, and oocytes cultured for 42 hours in vitro. The results obtained are as follows; 1. The nuclear maturation rates of EGF-treated groups(10ng/ml, 75.9% ; 30ng/ml, 69.2% ; 50ng/ml, 67.2% ; 100ng/ml, 71.0%) on the porcine oocytes cultured in medium without pFF in vitro were significantly(P<0.01) higher than those of non-treated group(57.1%). When the oocytes were cultured in media with 10%(v/v) pFF, the nuclear maturation rates of 30ng EGF/ml(77.1%) treated group were significantly(P<0.01) higher than those of non-(59.2%) and EGF-treated groups(10ng/ml, 65.4% ; 50ng/ml, 65.5% ; 100ng/ml, 70.4%). 2. The nuclear maturation rates of 30ng TGF-$\alpha$/ml treated group(71.9%) in media without pFF in vitro were significatnly(P<0.01) higher than those of non-(57.1%) and TGF-$\alpha$ treated groups(10ng/ml, 60.4% ; 50ng/ml, 65.4% ; 100ng/ml, 60.0%). When the oocytes were cultured in media with 10%(v/v) pFF, the nuclear maturation rates of 30 and 50ng TGF-$\alpha$/ml(77.4% and 79.6%) treated groups(10ng/ml, 64.2% ; 100ng/ml, 61.6%). 3. On the effect of EGF(30ng/ml) and/or TGF-$\alpha$(30ng/ml) treated groups in medium without pFF in vitro, the nuclear maturation rates indicated 57.3, 60.4, 75.9 and 79.7% in media with no EGF & TFG-$\alpha$, TGF-$\alpha$ only, EGF only nad EGF+TGF-$\alpha$ treated groups, respectively. The nuclear maturation rates in medium with EGF only or EGF+TGF-$\alpha$ were significantly(P<0.01) higher than those non- and TGF-$\alpha$ treated groups. When the oocytes were cultured in media with 10%(v/v) pFF, the nuclear maturation ratesof EGF+TGF-$\alpha$ treated group(75.9%) were significantly(P<0.01) higher than those of non-(59.2%), TGF-$\alpha$ only (64.2%) and EGF only(69.4%) treated groups.

The Suppression of Maturational Competence by Streptomycin during In vitro Maturation of Goat Follicular Oocytes

Kang, Jae Ku,Chang, Suk Min,Naruse, Kenji,Han, Jeung Whan,Park, Chang Sik,Jin, Dong Il Asian Australasian Association of Animal Productio 2004 Animal Bioscience Vol.17 No.8

Antibiotics are common additives in culture media during in vitro embryo development, but their effects on oocyte maturation in vitro have not been tested. The effects of penicillin, streptomycin and gentamicin on the maturational competence and subsequent development potential of goat follicular oocytes were examined after parthenogenetic activation in vitro. Maturation rates at 24 h after in vitro maturation, and parthenogenetic development at 48 h after activation, were evaluated by observing the protruding first polar body and the 4 cell stage cleavage, respectively. When streptomycin was present in the maturation medium, the percentages of matured oocytes 24 h after activation were significantly (p<0.01) lower than those from the other groups (42.5-45.7% vs. 69.1-73.8%). Penicillin and gentamicin treatment did not affect the maturation rates or the percentages reaching the 4 cell stage 48 h after activation. There was no significant difference in cleavage rates among the different antibiotic treatments 48 h after activation. Therefore, streptomycin suppresses the in vitro maturation of immature goat oocytes, but does not influence their subsequent development.

The Suppression of Maturational Competence by Streptomycin during In vitro Maturation of Goat Follicular Oocytes

Kang, Jae Ku,Chang, Suk Min,Naruse, Kenji,Han, Jeung Whan,Park, Chang Sik,Jin, Dong Il 충남대학교 형질전환복제돼지연구센터 2004 논문집 Vol. No.8

Antibiotics are common additives in culture media during in vitro embryo development, but their effects on oocyte maturation in vitro have not been tested. The effects of penicillin, streptomycin and gentamicin on the maturaltional competence and subsequent development potential of goat follicular oocytes were examined after parthenogenetic activation in vitro. Maturation rates at 24h after in vitro maturation, and parthenogenetic development at 48 h after activation, were evaluated by observing the protruding first polar body and the 4 cell stage cleavage, respectively. When streptomycin was present in the maturation medium, the percentages of matured oocytes 24h after activation were significantly(p<0.01) lower than those from the other groups(42.5-45.7% vs. 69.1-73.8%). Penicillin and gentamic in treatment did not affect the maturation rates or the percentages reaching the 4cell stage 48 h after activation. There was no significant difference in cleavage rates among the different antibiotic treatments 48 h after activation. Therefore, streptomycin suppresses the in vitro maturation of immature goat oocytes, but does not influence their subsequent development.

Effects of Hyaluronidase during In Vitro Maturation on Maturation and Developmental Competence in Porcine Oocytes

Ye-Eun Jeon,Yong Hwangbo,Hee-Tae Cheong,Choon-Keun Park 한국동물생명공학회(구 한국동물번식학회) 2019 Journal of Animal Reproduction and Biotechnology Vol.34 No.2

The aim of this study was to investigate effects of hyaluronidase during IVM on oocyte maturation, oxidative stress status, expression of cumulus expansion-related (PTX, pentraxin; GJA1, gap junction protein alpha 1; PTGS2, prostaglandin-endoperoxide synthase 2) and fatty acid metabolism-related (FADS1, delta-6 desaturase; FADS2, delta-5 desaturase; PPARα, peroxisome proliferator-activated receptor-alpha) mRNA, and embryonic development of porcine oocytes. The cumulus-oocyte complexes (COCs) were incubated with 0.1 mg/mL hyaluronidase for 44 h. Cumulus expansion was measured at 22 h after maturation. At 44 h after maturation, nuclear maturation, intracellular glutathione (GSH) and reactive oxygen species (ROS) levels were measured. Gene expression in cumulus cells was analyzed using real time PCR. The cleavage rate and blastocyst formation were evaluated at Day 2 and 7 after insemination. In results, expansion of cumulus cells was suppressed by treatment of hyaluronidase at 22 h after maturation. Intracellular GSH level was reduced by hyaluronidase treatment (p < 0.05). On the other hand, hyaluronidase increased ROS levels in oocytes (p < 0.05). Only PTGS2 mRNA was enhanced in COCs by hyaluronidase (p < 0.05). Population of oocytes reached at metaphase II stage was higher in control group than hyaluronidase treated group (p < 0.05). Both of cleavage rate and blastocyst formation were higher in control group than hyaluronidase group (p < 0.05). Our present results showed that developmental competence of porcine oocytes could be reduce by hyaluronidase via inducing oxidative stress during maturation process and it might be associated with prostaglandin synthesis. Therefore, we suggest that suppression of cumulus expansion of COCs could induce oxidative stress and decrease nuclear maturation via reduction of GSH synthesis and it caused to decrease developmental competence of mammalian oocytes.

Effects of BSA, PVA, Gonadotropins and Follicle Shell on In Vitro Maturation and In Vitro Fertilization of Porcine Oocytes

Pei-Qing Cong,Eun-Sook Song,Eui-Sook Kim,Zhao-Hua Li,Yong-Hua Zhang,Young-Joo Yi,Chang-Sik Park 한국동물생명공학회(구 한국동물번식학회) 2007 Reproductive & developmental biology Vol.31 No.2

This study was designed to evaluate effects of BSA, PVA, gonadotropins and follicle shell during IVM of porcine oocytes and subsequent development to the blastocyst stage after IVF. Cumulus oocyte complexes (COCs) were cultured in TCM-199 media containing 4 mg/ml BSA and 1 mg/ml PVA during IVM for 44 hr. To compare the effect of gonadotropins on oocyte maturation, COCs were cultured with FSH+LH, FSH, LH and FSH-LH-free media during IVM, respectively. Also, different number of follicle shells (0, 2, 4 and 6) was used to examine whether the presence of follicle shell in culture medium affects oocyte maturation. The percentages of fertilization and blastocyst formation, respectively, were higher in the medium containing the PVA (49.0 and 17.9%) than those containing the BSA (40.0 and 12.2%). Significantly higher rates of MII oocytes were in the presence of FSH+LH and FSH (88.6 and 85.1%) compared to other treatments (64.0 and 53.4% at LH and FSH-LH-free media). Co-culture with inverted follicle shells in 2 ml maturation medium enhanced the developmental competence of porcine oocytes. In conclusion, PVA could be used as a macromolecules instead of BSA, and FSH and follicle shell played important roles in maturation of porcine oocytes.

The role of sonic hedgehog signaling pathway in in vitro oocyte maturation

Sanghoon Lee,Jongki Cho 한국동물생명공학회(구 한국동물번식학회) 2021 Journal of Animal Reproduction and Biotechnology Vol.36 No.4

In vitro maturation (IVM) of oocytes is the procedure where the immature oocytes are cultivated in a laboratory until they are mature. Since IVM oocytes generally have low developmental competence as compared to those matured in vivo, development of an optimal IVM culture system by fine-tuning culture conditions is crucial to maintain high quality. In-depth knowledge and a deep understanding of the in vivo physiology of oocyte maturation are pre-requisites to accomplish this. Within ovarian follicles, various signaling pathways that drive oocyte development and maturation regulate interaction between oocytes and surrounding somatic cells. This review discusses the sonic hedgehog (SHH) signaling pathway, which has been demonstrated to be intimately involved in folliculogenesis and oocyte maturation. Advances in elucidating the role of the SHH signaling pathway in oocyte maturation will aid attempts to improve the current inferior in vitro oocyte maturation system.

The role of sonic hedgehog signaling pathway in in vitro oocyte maturation

이상훈,조종기 사단법인 한국동물생명공학회 2021 한국동물생명공학회지 Vol.36 No.4

In vitro maturation (IVM) of oocytes is the procedure where the immature oocytes are cultivated in a laboratory until they are mature. Since IVM oocytes generally have low developmental competence as compared to those matured in vivo, development of an optimal IVM culture system by fine-tuning culture conditions is crucial to maintain high quality. In-depth knowledge and a deep understanding of the in vivo physiology of oocyte maturation are pre-requisites to accomplish this. Within ovarian follicles, various signaling pathways that drive oocyte development and maturation regulate interaction between oocytes and surrounding somatic cells. This review discusses the sonic hedgehog (SHH) signaling pathway, which has been demonstrated to be intimately involved in folliculogenesis and oocyte maturation. Advances in elucidating the role of the SHH signaling pathway in oocyte maturation will aid attempts to improve the current inferior in vitro oocyte maturation system.

Culture Conditions for In Vitro Maturation of Abattoir Derived Oocytes of Native Zebu Cows of Bangladesh

Morshed, S.M. Niyaz,Bhuiyan, Mohammad Musharraf Uddin,Rahman, Mohammad Moshiur,Singha, Joydev Kumer,Juyena, Nasrin Sultana The Korean Society of Embryo Transfer 2014 한국동물생명공학회지 Vol.29 No.3

The objectives of the study were to determine an effective culture dish, culture duration and protein supplementation in medium for in vitro maturation (IVM) of oocytes of native zebu cows in Bangladesh. The ovaries of cows were collected from local slaughterhouse followed by aspiration of follicular fluid. The cumulus-oocyte-complexes (COCs) with more than 3 compact cumulus cell layers were cultured in tissue culture medium (TCM) 199 for maturation. The maturation of oocytes was determined by observing polar body under microscope. To determine an effective culture dish, 130 COCs derived from 48 ovaries in a well of 4-well dish and 102 COCs derived from 36 ovaries in drops covered with mineral oil within 35 mm petri dish were cultured for 24 hours. The rate of maturation of oocytes did not vary between 4-well dish ($51.3{\pm}15.0%$) and drops in petri dish ($52.4{\pm}11.6%$). To determine the effective culture duration, 185 COCs derived from 62 ovaries were cultured in drops for 18, 21, 24 and 27 hours. The rate of maturation of occytes ranged from $51.9{\pm}9.4%$ (18 hours) to $59.0{\pm}17.1%$ (27 hours) and the difference in maturation rate among different culture durations was not significant (P>0.05). To determine an effective protein supplementation, 63 oocytes from 19 ovaries were cultured separately in TCM 199 supplemented with either fetal bovine serum (FBS) or bovine serum albumin (BSA). The rate of maturation was significantly (P<0.01) higher in medium supplemented with FBS ($55.63{\pm}16.19%$) than that of BSA ($14.82{\pm}9.36%$). In conclusion, COCs of native zebu cows can be cultured for IVM either in 4-well culture dish or droplets in petri dish for 18 to 27 hours in medium supplemented with FBS.