Difference in Developmental Kinetics of Y-Specific Monoclonal Antibody Sorted Male and Female In Vitro Produced Bovine Embryos

Sidrat, Tabinda,Kong, Rami,Khan, Abdul,Idrees, Muhammad,Xu, Lianguang,El Sheikh, Marwa,Joo, Myeong-Don,Lee, Kyeong-Lim,Kong, Il-Keun MDPI AG 2020 INTERNATIONAL JOURNAL OF MOLECULAR SCIENCES Vol.21 No.1

<P>Sex-related growth differences between male and female embryos remain an attractive subject for reproductive biologists. This study aimed to investigate the endogenous factors that play a crucial role in the pace of early development between male and female bovine embryos. Using sex pre-selected semen by Y-specific monoclonal antibodies for the production of bovine embryos, we characterized the critical endogenous factors that are responsible for creating the development differences, especially during the pre-implantation period between male and female embryos. Our results showed that at day seven, (57.8%) Y-sperm sorted in vitro cultured embryos reached the expanded blastocyst (BL) stage, whereas the X-sperm sorted group were only 25%. Y-BLs showed higher mRNA abundance of pluripotency and developmental competency regulators, such as Oct4 and IGF1-R. Interestingly, Y-sperm sorted BLs had a homogeneous mitochondrial distribution pattern, higher mitochondrial membrane potential (∆Ѱm), efficient OXPHOS (oxidative phosphorylation) system and well-encountered production of ROS (reactive oxygen species) level. Moreover, Y-blastocysts (BLs) showed less utilization of glucose metabolism relative to the X-BLs group. Importantly, both sexes showed differences in the timing of epigenetic events. All these factors directly or indirectly orchestrate the whole embryonic progression and may help in the faster and better quality yield of BL in the Y-sperm sorted group compared to the X counterpart group.</P>

Effect of charcoal:dextran stripped fetal bovine serum on <i>in vitro</i> development of bovine embryos

Mesalam, Ayman,Kong, Rami,Khan, Imran,Chowdhury, MMR,Choi, Byung-Hyun,Kim, Sung Woo,Cho, Kyu-Woan,Jin, Jong-In,Kong, Il-Keun Elsevier 2017 Reproductive biology Vol.17 No.4

<P><B>Abstract</B></P> <P>This study investigated the ability of charcoal:dextran stripped fetal bovine serum (CDS FBS) and heat-inactivated fetal bovine serum (HI FBS) to support <I>in vitro</I> development of bovine embryos. The developmental ability and quality of bovine embryos were determined by assessing their cell number, lipid content, mitochondrial activity, gene expression, and cryo-tolerance. The percentage of embryos that formed a blastocyst was significantly (<I>P<</I> 0.05) higher in medium containing CDS FBS than in medium containing HI FBS (42.84±0.78% vs. 36.85±0.89%, respectively). Furthermore, the beneficial effects of CDS FBS on embryos were associated with significantly (<I>P<</I> 0.05) increased mitochondrial activity, as identified by MitoTracker Green, as well as a reduced intracellular lipid content, as identified by Nile red staining, which increased their cryo-tolerance. Quantitative reverse transcription PCR showed that the mRNA levels of acyl-CoA synthetase long-chain family member 3, acyl-coenzyme A dehydrogenase long-chain, hydroxymethylglutaryl-CoA reductase, and insulin-like growth factor 2 receptor were significantly (<I>P<</I> 0.05) increased upon culture with CDS FBS. Moreover, the mRNA levels of sirtuin 1, superoxide dismutase 2, and the anti-apoptotic gene B-cell lymphoma 2 in frozen-thawed blastocysts were significantly (<I>P<</I> 0.05) higher in the CDS FBS-supplemented group than in the HI FBS-supplemented group, whereas that of the pro-apoptotic gene BCL2-associated X protein was significantly lower. Taken together, these data suggest that supplementation of medium with CDS FBS improves the <I>in vitro</I> developmental competence and cryo-tolerance of bovine embryos.</P>

OPU 유래 수정란 생산의 고능력 공란우의 반복 사용에 관한 연구

Byung-Hyun Choi,Bun-Young Park,Rami Kong,Chan-sang Park,Mi-Ju Son,Jong-In Jin,Il-Keun Kong 한국수정란이식학회 2018 한국수정란이식학회 학술대회 Vol.2018 No.11

현재까지 한우개량을 위해 가장 보편적으로 활용되고 있는 기술은 웅성 유전자원의 동결용 정액을 활용하는 인공수정기술로서 개량에 최소 6 세대 이상 즉, 암송아지만을 생산한다는 가정에서 15~20 년 이상의 시간이 소요될 뿐만 아니라 생산되는 산자 수 또한 한정적이다. 가축개량 효율을 높이기 위해 수정란이식 기술이 개발되었으며, 호르몬 투여에 의한 과배란 유래 체내 수정란 및 체외 수정란 생산 방법이 개발되어 활용되어 왔다. 체내 수정란 생산방법은 혈통관리는 정확하나 수정란 생산을 위해 호르몬 과다 투여에 의한 휴식기간 등이 필요하며 수정란의 생산량의 한계로 산업화 적용에 비효율적이고, 근래에 체외수정란을 활용되면서 도축 유래 수정란을 활용하는 경향이 있으나 친자 검정의 한계로 인한 친자 불일치 및 고도근친 위험성 등이 상존하고 있다. 이러한 문제를 극복하고 효율적인 이식 가능한 수정란의 생산이 가능한 OPU 유래 수정란 생산기술은 살아있는 공란우에서 난자를 채취하여 체외수정란을 생산함으로써 혈통관리가 정확하고 우수유전 자원을 선발 활용함으로써 계획 교배에 의한 근친도와 개량의 폭을 예측할 수 있는 장점이 있다. 또한 수정란의 생산 가능 량은 3~4 개월에 약 60 여 개의 이식 가능한 수정란을 대량 생산으로 산업화에 적합한 수정란 생산기술이다. 본 연구에서는 선발된 공란우의 반복 사용으로 우수 유전자원의 수정란을 대량생산에 활용하기 위해서 2013-2016 년까지 매년 3-4 개월 동안 주 2 회 채란 및 수정란을 생산하였고 채란 완료 후 일정 기간의 휴식한 다음 다시 채란에 활용으로 2 회 이상 수정란 생산으로 수정란의 반복 생산 가능성 및 생산 효율에 대하여 조사하였다. 2 반복의 공란우는 총 24 두와 3 반복은 7 두를 3-4 개월 동안/년 매주 2 회 총 1,626 회 채란으로 평균 3.6±2.9 개의 수정란이 생산되었다. 특히 채란 시 생산된 수정란은 채란 횟수당 1 회 반복에서 3.9±2.8 개, 2 회 반복은 3.3±2.7 개 및 3 회 반복은 3.5±3.3 개로 확인되었고, 3 회 이상의 반복 채란된 개체 7 두를 분석한 결과 1 회 반복 채란에서 평균 3.63±2.79 개, 2 회 반복은 평균 3.70±2.84 개 및 3 회 반복은 년 3.51±3.32 개로 반복 채란에 수정란 생산에 활용하였으며 반복 채란에 의한 수정란의 생산효율에는 유의적인 차이를 보이지 않았다. 따라서 가축개량을 위해 산업화에 가장 적합한 수정란 생산방법으로 유전능력이 우수한 한우에서 대량의 수정란을 생산하여 우량한우 집단구축을 위하여 약 3-4 개월 동안 매주 2 회 수정란을 생산하고 일정기간을 휴식한 다음 공란우로써 재활용 가능성을 확인하였고 또한 OPU 유래 수정란 생산방법은 우수한 유전자원을 보유한 개체를 연속적이며 반복적으로 활용할 수 있는 가능성을 확인하였다

Possibility of the preselected sex embryo production of elite cattle by using WholeMom

M.M.R. Chowdhury,Lianguang Xu,Rami Kong,Bun-Young Park,Byeong-Hyun Choi,Jong-In Jin,Il-Keun Kong 한국수정란이식학회 2018 한국수정란이식학회 학술대회 Vol.2018 No.11

Sex preselection has always generated great interest among livestock producers due to an increase in the profitability of the cattle industry through the production of offspring with desired sex, such as females for dairy or males for meat production. Among the prevalent sorting methods, the embryo developmental potential is still very low as expected, and there is distinguished evidence that sex sorting has a negative effect on sperm quality with an altered pattern of sperm motility, ultimately reducing lifespan. The consequence is a very low embryo development rate using sex-sorted semen, and its negative impact influences the progress of the dairy industry. Here, we established a new approach with reduced stress by using WholeMom® and observed no significant differences (P < 0.05) in early cleaving embryos between sorted X sperm and the control group, although there was a remarkable significant difference in embryos of the Y sperm group, 81.82 ± 2.71% vs. 87.44 ± 3.02% vs. 54.21 ± 2.21%, respectively. The percentage of embryos that developed into blastocysts (Day 7) was also significantly (P < 0.05) higher in the control and X Sperm group compared to the Y sperm group, 35.53 ± 1.92% and 29.76 ± 2.38% vs. 21.90 ± 1.54%. Moreover, B-SRY F2 and B-SRY R2 gene expression data exhibited 81.03% accuracy for the female embryos and 72.54% for the male embryos produced in vitro. And also the field trials for the heifer production using WholeMom by Artificial Insemination technique demonstrated 76% female and 24% male in vivo. In conclusion, the combination of pre-selected sex semen and OPU derived elite cattle embryo production is highly recommended to apply to the mass production in the dairy industry with rapid genetic up-gradation.

Ovum Pick-up 유래 수정란의 생산효율과 수정란 이식 성적 및 동향

Byung-Hyun Choi,Bun-Young Park,Rami Kong,Chan-sang Park,Mi-Ju Son,Jong-In Jin,Il-Keun Kong 한국수정란이식학회 2018 한국수정란이식학회 학술대회 Vol.2018 No.11

OPU 유래 수정란 생산 및 이식은 기존의 체내 및 체외 수정란 생산과 이식의 단점을 해결하고 한우 개량을 촉진 시킬 수 있는 기술이다. 특히 살아있는 공란우를 활용하므로 모계와 부계에 대한 혈통관리가 정확하고 또한 선발된 공란우와 계획 교배를 통하여 가장 적합한 동결정액을 사용하여 개량의 효과를 극대화가 가능하고 또한 현재까지 생산효율이 가장 높은 생산기술이다. 따라서 기존의 수정란 생산방법보다 공란우의 선발강도를 더 높일 수 있어 개량의 효과를 극대화 할 수 있는 장점이 있다. 본 연구실에서는 2009 년도부터 OPU 유래 수정란을 생산하여 이식하여 수정란이식이 산업화로 전개될 수 있도록 진행하고 있다. 특히 한우에 대한 우수성 확보, 개량 및 그 다음 세대 후대의 근친적인 문제가 발생되지 않도록 수정란이식에 의해 탄생된 송아지 혈통관리를 위한 친자검정으로 수정란에 대한 신뢰성을 높여 수정란 이식이 산업화가 될 수 있도록 진행하였다. 본 연구에 대한 조사는 2013 년도 7 개의 시·군을 시작으로 ‘14 년도 11 개, ‘15 년도 15 개, ‘16년도 17 개의 시·군으로 점진적으로 확대됨에 따라 공란우의 두수도 ‘13 년도 35 두를 시작으로 ‘16 년도는 71 두로 증가되었다. 조사된 4 년동안 각 지역에서 선발된 유전능력이 우수한 공란우는 총 211 두에서 수정란을 총 18,839 개, 두당 89.3 개를 생산하였다. 두당 생산 효율도 ‘13 도에는 회당 3.0±4.5 개에서 ‘16 년도에는 4.8±2.2 로 회당 이식 가능한 수정란의 생산량이 년도에 따라 꾸준히 향상되었다. 생산된 수정란은 자연발정 또는 발정동기화를 통하여 매주 2 회 이상 이식으로 ‘13 년 49.7%, ‘14 년 51.9%, ‘15 년 52.9%와 ‘16 년 47.9%의 수태율을 확인하였다. 이는 년도가 증가되면서 수정란의 생산성의 향상과 적정 수준의 수태율의 성과는 시술자와 농가의 적극적인 참여에 의한 결과로 판단되고 또한 유전능력이 우수한 수정란의 대량생산 및 공급으로 개량의 효과가 증가되고 우수한 한우 집단 구축으로 한우산업이 지속적으로 발전 할 것으로 판단된다.

Provision of the genetic improvement and reproductive performance of the Korean brindle cattle, Chikso

M.M.R. Chowdhury,Lianguang Xu,Rami Kong,Yeoung-Gyu Ko,Sung Soo Lee,Il-Keun Kong,Sung Woo Kim 한국실험동물학회 2019 한국실험동물학회 학술발표대회 논문집 Vol.2019 No.1

Effect of Serum and Serum Free Media on the Developmental Competence of OPU Derived Bovine IVP Embryo

Byung-Hyun Choi,Bun-Young Park,Rami Kong,Mi-Ju Son,Chan-Sang Park,Nyeon-Hak Shin,Hye-Young Cheon,Yeong-Rok Yang,Jin-Woo Lee,Jong-In Jin,Il-Keun Kong 한국동물생명공학회(구 한국동물번식학회) 2019 Journal of Animal Reproduction and Biotechnology Vol.34 No.4

Embryos produced with serum show the alterations in their ultrastructure, impaired compaction, abnormal blastulation, aberrant mRNA expression profiles and large calf syndrome with greater incidences of stillbirths and deaths after birth. The aim of the present study was to describe in vitro embryo production by analyzing embryo production, fetal production and pregnancy rate in free-serum medium. The OPU-IVP data used in this study from 2016. Approximately, sixteen cows (Hanwoo), which belonged to the Institute of Gyeongsang National University, were used. Two experimental group is used in this study. Serum groups were conducted in March to July and free-serum group was conducted in September to December. The recovered cumulus-oocyte complexes were morphologically classified to four grades based on the compaction of cumulus cells layers and homogeneity of the cytoplasm. The number of oocyte was significantly greater in serum groups than that in free-serum groups (29.61 ± 0.63 vs. 15.6 ± 0.62; p < 0.05). Between serum and free-serum groups indicate that average of 1st and 2nd grade oocytes were no difference (2.38 ± 1.67 vs. 2.38 ± 1.48; p > 0.05), but number of 3rd and 4th grade oocytes were greater in serum groups than that in free-serum groups (7.31 ± 7.64 vs. 5.60 ± 6.29; p < 0.05). Embryo cleaved competence was higher in rate in free-serum groups than that in serum groups (62.1% vs. 58.3; p < 0.05). However, blastocyst developmental rate was no difference between serum and free-serum groups (33.1% vs. 43.5%; p < 0.05). 986 recipients were used for embryo transfer. Pregnancy rate was indicated that between serum and free-serum group was no difference (54.6% vs. 56.3%; p < 0.05). In conclusion, we developed the free-serum system for production of in vitro bovine embryos in order to meet the developmental and qualitative requirements for large scale commercial use.

Improvement of in vitro produced bovine embryo quality using Charcoal:Dextran Stripped Fetal Bovine Serum on culture media

Mesalam Ayman,Lianguang Xu,Byung-Hyun Choi,Beon-Young Park,Rami Kong,Mi-Ju Son,Jong-In Jin,Sung Woo Kim,Il-Keun Kong 한국수정란이식학회 2017 한국수정란이식학회 학술대회 Vol.2017 No.05

This study investigated the effect of Charcoal:Dextran Stripped fetal bovine serum (CDS FBS) and heat-inactivated FBS (HI FBS) in embryo culture medium on their ability to support in vitro development of bovine embryos. The developmental ability and quality of bovine embryos were determined by assessing their cell number, lipid content, mitochondrial activity, gene expression, and cryo-tolerance. The percentages of embryos that underwent cleavage and formed a blastocyst were significantly (P<0.05) higher in medium containing CDS FBS than in medium containing HI FBS (42.84 ± 0.78% vs. 36.85 ± 0.89%, respectively). Furthermore, the beneficial effects of CDS FBS on embryos were associated with a significantly reduced intracellular lipid content, as identified by Nile red staining, which increased their cryo-tolerance. The post-thaw survival rate of blastocysts was significantly (P<0.05) higher in the CDS FBS than in the HI FBS group (85.33 ± 4.84% vs. 68.67 ± 1.20%). Quantitative real-time PCR showed that the mRNA levels of acyl-CoA synthetase long-chain family member 3, acyl-coenzyme A dehydrogenase long-chain, hydroxymethylglutaryl-CoA reductase, and insulin-like growth factor 2 receptor were significantly increased upon culture with CDS FBS. Moreover, the mRNA levels of sirtuin 1, superoxide dismutase 2, and anti-apoptotic associated gene B-cell lymphoma 2 in frozen-thawed blastocysts were significantly (P<0.05) higher in the CDS FBS group than in the HI FBS group, however, the mRNA level of the pro-apoptotic gene BCL2-associated X protein was significantly reduced. Taken together, these data suggest that supplementation of medium with CDS FBS improves in vitro bovine embryo developmental competence and cryo-tolerance.

Effect of OPU Session Periods on the Efficiency of In Vitro Embryo Production in Elite Korean Native Cow

Byung-Hyun Choi,Seok-Hwan Song,Bun-Young Park,Rami Kong,Mi-Ju Son,Chan-Sang park,Nyeon-Hak Shin,Hye-Young Cheon,Sung-Hoon Lee,Jong-In Jin,Jung-Gyu Lee,Il-Keun Kong 한국수정란이식학회 2018 한국동물생명공학회지 Vol.33 No.4

Up-to-date artificial insemination (AI) using frozen sperm consider as the most widely using technology for improvement of Korean Native Cow (Hanwoo) embryo production. However, it is time consuming, required at least 15~20 years to make more than 6 generations, and their offspring number is limited. To overcome such limitations, superovulation and in vitro fertilization have been developed. For superovulation, the number of produced embryos are not enough for commercialization and donor cows need rest period. This led to use of slaughterhouse ovary for in vitro fertilization, but it is impossible to repeat the collection from the same individual and it only can improve the genetic merits of offspring for one generation. Production of embryos using Ovum Pick-Up (OPU) technique, where oocytes can be repeatedly collected from living elite donor, might overcome these limitations. In this study, we investigated the possibility of using OPU technique from donors at different age and different session periods for mass-embryo-production. Oocytes were collected from 26 donor cows twice per week, 3 - 4 months per year, between 2013 and 2016. Results showed that, the average number of embryo produced in first year used donor was significantly higher than that in second year used donor (3.89 ± 2.85 vs 3.29 ± 2.70), however, there was no significant difference between third year used donor (3.51 ± 3.32) and other groups. Taken together, our data showed that repeated using of donor up to three years is possible for in vitro embryo mass-production. Moreover, OPU can be used as suitable embryo producing technique for livestock breed improvement.

Effect of OPU Session Periods on the Efficiency of In Vitro Embryo Production in Elite Korean Native Cow

Choi, Byung-Hyun,Song, Seok-Hwan,Park, Bun-Young,Kong, Rami,Son, Mi-Ju,park, Chan-Sang,Shin, Nyeon-Hak,Cheon, Hye-Young,Lee, Sung-Hoon,Jin, Jong-In,Lee, Jung-Gyu,Kong, Il-Keun The Korean Society of Embryo Transfer 2018 한국동물생명공학회지 Vol.33 No.4

Up-to-date artificial insemination (AI) using frozen sperm consider as the most widely using technology for improvement of Korean Native Cow (Hanwoo) embryo production. However, it is time consuming, required at least 15~20 years to make more than 6 generations, and their offspring number is limited. To overcome such limitations, superovulation and in vitro fertilization have been developed. For superovulation, the number of produced embryos are not enough for commercialization and donor cows need rest period. This led to use of slaughterhouse ovary for in vitro fertilization, but it is impossible to repeat the collection from the same individual and it only can improve the genetic merits of offspring for one generation. Production of embryos using Ovum Pick-Up (OPU) technique, where oocytes can be repeatedly collected from living elite donor, might overcome these limitations. In this study, we investigated the possibility of using OPU technique from donors at different age and different session periods for mass-embryo-production. Oocytes were collected from 26 donor cows twice per week, 3 - 4 months per year, between 2013 and 2016. Results showed that, the average number of embryo produced in first year used donor was significantly higher than that in second year used donor ($3.89{\pm}2.85$ vs $3.29{\pm}2.70$), however, there was no significant difference between third year used donor ($3.51{\pm}3.32$) and other groups. Taken together, our data showed that repeated using of donor up to three years is possible for in vitro embryo mass-production. Moreover, OPU can be used as suitable embryo producing technique for livestock breed improvement.