BÁO CÁO KHOA HỌC VỀ NGHIÊN CỨU VÀ GIẢNG DẠY SINH HỌC Ở VIỆT NAM HỘI NGHỊ KHOA HỌC QUỐC GIA LẦN THỨ 5 DOI 10 15625/vap 2022 0069 EVALUATING CORPUS LUTEUM SIZE OF LOCAL VIETNAMESE CATTLE AND IDEALLY SYN[.]
BÁO CÁO KHOA HỌC VỀ NGHIÊN CỨU VÀ GIẢNG DẠY SINH HỌC Ở VIỆT NAM - HỘI NGHỊ KHOA HỌC QUỐC GIA LẦN THỨ DOI: 10.15625/vap.2022.0069 EVALUATING CORPUS LUTEUM SIZE OF LOCAL VIETNAMESE CATTLE AND IDEALLY SYNCHRONIZED TIMING FOR CLONED BOVINE EMBRYOS TRANSFER Nguyen Mai Phuong1,#, Do Minh Tan1,#, Nguyen Huu Hoang Minh1, Nguyen Tuan Anh1, Nguyen Gia Bao1, Pham Quoc Dinh1, Pham Minh Chien1, Pham Truong Duy1, Nguyen Kien Cuong2, Nguyen Van Thuan1,* Abstract In this study, we evaluated the effects of corpus luteum (CL) size of local Vietnamese cattle and determined the ideally synchronized timing for cloned bovine embryos transfer The results showed that there was a correlation between the size of corpus luteum of recipient cows and pregnancy rates after embryos transfer, in which the qualified recipient cow should consists of at least 1.7 cm in CL size at the time of transferring In addition, cloned bovine blastocyst at day was the ideal stage for embryos transfer that resulted in about 31.3 % of transferred cows have had pregnancy In conclusion, this was the first report demonstrated the essential requirements including CL size detection and ideally synchronized timing for improving the success of cloned bovine embryos transfer to local Vietnamese cattle Keywords: Cloned bovine embryos, corpus luteum, embryos transfer, local Vietnamese cattle INTRODUCTION The birth of Dolly the sheep marked a big breakthrough in mammals cloning technology (Wilmut et al., 1997) Since then, the technique has been successfully performed to produce various cloned species such as mice (Wakayama et al., 1998), cows (Kato et al., 1998), pigs (Onishi et al., 2000) and monkeys (Liu et al., 2018) Although there were many attemps to improve the success rates to term development, current cloning efficiency is still low The reasons are mainly caused by abnormal epigenetic modification during pre-implantation development of cloned bovine embryos Furthermore, the success of animal cloning techniques mammals is judged by the birth of healthy clones, with life cycles such as naturally born animals In addition to the embryo quality factors associated with an increase in the rate of success of cloning, embryo transfer technique is also one of the essential factors determines the success of cloning techniques in mammals In 1951, the first healthy cow was born by transferring embryo at day (Willett et al., 1951) However, the technique was still in impractical mean until 2003 that scientists International University, Vietnam National University, Ho Chi Minh City Nong Lam University, Ho Chi Minh City # Equally contributed to this work * Email: nvthuan@hcmiu.edu.vn 632 BÁO CÁO KHOA HỌC VỀ NGHIÊN CỨU VÀ GIẢNG DẠY SINH HỌC Ở VIỆT NAM successfully improved the efficiency and commercially applied into the market (Betteridge et al., 2003) Subsequently, bovine embryo transfer techniques were invested by researchers and experts to promote the implantation rate of the embryo for many applications, including cattle cloning It is well known that suitability of recipient cows (host mothers) is depended on the timing of estrous and the presence of a functional corpus luteum (CL) (Spell A et al., 2000) Estrous is defined as the cycle represents the cyclical pattern of ovarian activity that allow the establishment of pregnancy after mating While the function of CL is size-dependence The CL develops from ovarian follicle during estrous cycle; therefore, based on the size of the CL we are able to define whether the left or right bovine ovary is in the stage of estrous According to (Nogueira et al., 2012), the size of the CL is one of the factors affecting the pregnancy rate of cows During estrous, the size of the CL should be more than 1.5 cm and the position of the CL is also an extremely important factor for the conception rate of cloned bovine embryos because the bovine embryo will develop and form a fetus on the same side of the ovary with an ovulated egg, that is, the side where the corpus luteum is present Currently in Vietnam, the implementation of embryo transfer on cattle are still not popular and have not been widely applied in research Most of professional veterinarians were trained in the field of insemination that differs in the timing during estrous period and uterus location for injecting to recipients In addition, embryos transfer is very important in bovine cloning because it directly affects to the implantation ability of cloned bovine embryos and the total success to full-term development (Malin K et al., 2022) Therefore, optimization of bovine embryo transfer procedure from the preparation of estrous cows to determination the CL size of local Vietnamese cattle for transferring cloned embryos at ideally synchronized timing in uterus is extreme necessary In summary, this study compared the implantation ability and pregnancy rates after cloned embryo transfer to local Vietnamese cattle based on the relationship between CL size and embryo stage in order to establish the optimal embryo transfer procedure for improving post implantation development of cloned bovine embryos MATERIALS AND METHOD 2.1 Somatic cell nuclear transfer and production of cloned bovine embryos Sample preparation were performed as previous describe (Thuan et al., 2019) Briefly, cumulus-oocyte complexes (COCs) were cultured in in-vitro maturation medium covered with mineral oil in a humidified 5% CO2 incubator at 38.5 oC After 18-20 h, matured oocytes with first polar body (1st PB) from expanded COCs were collected by treatment with 0.1 % hyaluronidase and mouth pipetting Bovine lung tissues after washing were dissected into small pieces and were seeded in a cell culture disk of DMEM (Sigma-Aldrich, St Louis, MO, USA) supplemented with 10 % FBS, 100 IU/mL Penicillin in a humidified % CO2 incubator at 38.5 oC After reaching 80 % confluence, cells were passed and were freezed The cell culture at the passage 3-7 were thawed and treated with trypsin before nuclear transfer PHẦN NGHIÊN CỨU ỨNG DỤNG SINH HỌC PHỤC VỤ ĐỜI SỐNG VÀ PHÁT TRIỂN XÃ HỘI 633 SCNT was performed based on the established protocol from Cellular Reprogramming Laboratory (Thuan et al., 2019) In brief, matured oocytes were enucleated by aspirating the metaphase II chromosome in HEPES medium supplemented with 7.5 μg/mL cytochalasin B by the help of XYClone laser (Hamilton Thorne) to open the zona pellucida After that, donor cell nuclei were transferred to the enucleated oocytes by microinjection using the piezo pulse The reconstructed oocytes were activated by μM Ionomycin for mins at room temperature (RT), following treatment of mM 6-DMAP in mSOF medium for hours at 38.5 oC, % CO2 and 95 % humidified air Embryos then were in vitro developed (IVD) within the two-steps treatment of HDACi in droplets of mSOF supplemented 0.3 % BSA Cloned embryos were cultured up to days before embryo transfer 2.2 Preparation of estrous cattles for embryo transfer All recipient cows were raised at Yuri Farm, Ba Ria – Vung Tau province The cows were daily observed to determine the exact date of estrous base on a number of natural behaviors During this period, estrous cows often standing below to be ridden by other cows and these cows were labelled Another indicator was assessed by the appearance of vulva mucus With these signals, the cows were recorded in estrous on day zero Cloned bovine embryos at day or were transferred to the uterus of estrous cows on the timing around day or day 2.3 Detection of ovarian position and evaluation of corpus luteum size by ultrasound before embryo transfer Veterinarians used ultrasound machine KX5200 to check the condition of recipient cows before embryo transfer, by evaluating the characteristics of the CL/ovaries at that time and the condition state of the uterus The sizes of the CL were also used to determine the exact timing of estrous With the help of ultrasound machine, the CL size could be calculated by (length + width)/2 Furthermore, by day or of the estrous cycle, the cervix was completely closed Therefore, the introduction of the embryo transfer gun into the uterus of recipient cows requires highly specialized skills from a veterinary specialist 2.4 Cloned bovine embryos transfer Cloned bovine embryos at the blastocyst stage (day or day 7) were kept in mSOF medium in 0.5 mL tubes placed in a portable incubator This equipment aims to maintain a constant temperature (38.5 oC) during the transportation from the laboratory to the cow farm in Ba Ria Vung Tau province within up to hours At the farm, each bovine embryo was then transferred to the straw (IVM Technology 006430) The straw was placed inside the tube of the embryo transfer gun (IVM Technology 007240) After uterine examination to determine whether the recipient cow is eligible for embryo transfer by ultrasound method The veterinarian placed the gun directly into the vulva and moved it to the third stage of the surrogacy cow's uterine horn Simultaneously, the gun was moved to the side (left or right) of the horn where the ovary contains the CL Embryos were transferred to the uterine horns of recipient cows and pregnancy rates were checked at days 20th and 40th from the time of embryo transfer by ultrasound machine 634 BÁO CÁO KHOA HỌC VỀ NGHIÊN CỨU VÀ GIẢNG DẠY SINH HỌC Ở VIỆT NAM 2.5 Evaluation of implantation ability and pregnancy rates In the recent study, each recipient cow at day or day of estrous cycle was received cloned blastocyst derived either from our established optimal culture system We have performed around seven times embryos transfer (transfer to recipient cows) in each experimental group Veterinarians were able to use ultrasound machine to determine the location and appearance of embryos attached to the uterine horn Thereby, it was possible to determine the pregnancy rate after the day 20th from the time of embryo transfer Continuing to the day 40th, the uteruses of recipient cows were ultrasounded again to check the vitality of the cloned bovine embryo in which they could continue to implant or not Gradually, the external manifestations became more and more specific as follows: the left or right abdomen (the side of the horn of the uterus that is being transferred) got bigger, and veins appeared from the chest to the breast 2.6 Data analysis Data were analyzed by one-way ANOVA with Least-Significant Difference (LSD) test using IBM SPSS Statistics 20 software The probability P < 0.05 was considered a significant difference All the experiments were replicated at least times Data for analysis in Table 1, 2, were collected independently from different experiments In the Result 3.2 (Table 2), cloned bovine embryos were randomly selected (at day th or day 7th of embryo development) for embryo transfer to recipient cows of different groups of CL size Then the pregnancy rates were collected and the average rates among experimental groups were compared RESULT AND DISCUSSION 3.1 Effect of ultrasound examination of the position and size of the corpus luteum before embryo transfer on the pregnancy rate of cloned bovine embryos Figure The sizes of the corpus luteum (CL) were determined by ultrasound machine The CL sizes were 1.6 cm (A), 1.7 cm (B), 2.0 cm (C) Before embryo transfer, recipient cows were examined for a number of characteristic points of the ovaries and uterus to ensure that they meet the necessary requirements and conditions for receiving cloned bovine embryos In this experiment, results were obtained from groups: the first group without ultrasound test and the second group which involved in the use of ultrasound to examine before embryo transfer After the day 20th, we assessed the pregnancy rates by rectal examination Based on Table 1, the conception rate of the first group without the ultrasound machine detecting was very low PHẦN NGHIÊN CỨU ỨNG DỤNG SINH HỌC PHỤC VỤ ĐỜI SỐNG VÀ PHÁT TRIỂN XÃ HỘI 635 with only 5% In contrast, by the advantage of ultrasound machine to detect ovulation and corpus luteum formation, the pregnancy rate in day 20th was significantly increased (27.3%) At month 3rd, the latter rate reduced by nearly half with about 13.6% of recipient cows maintained the pregnancy, while a completely conception lost was observed in case of the former group Table Effects of using ultrasound method before embryo transfer on pregnancy rates of cloned bovine embryos Group Without Ultrasound With Ultrasound Total recipients 19 22 Day 20th 1(5.2)a 6(27.3)b Pregnancy rates (%) at Day 40th months a 0a 4(18.2)b 3(13.6)b Values with different superscripts within a column are significantly different (P < 0.05) 3.2 Effect of corpus luteum sizes derived from local vietnamese cattle on pregnancy rates of cloned bovine embryos According to the result in Table 2, there was a significant difference (P < 0.05) in the pregnancy rates at day 20th and day 40th between the group of recipient cows with 1.5 – 1.6 cm in CL size compared to the other groups In details, we could not obtain any pregnant cow after 20 days embryo transfer in the smallest CL group However, when CL reached to at least 1.7 cm, the ratios were observed at nearly similar rates (about 29 % at day 20th and 21 % at day 40th) among groups Spell A et al concluded that suitability of a potential embryo transfer recipient is determined by observed estrous and a palpable corpus luteum, regardless of size or quality (Spell A et al., 2000) In inconsistence with the report, our study proved that the ideal sizes (after 7-8 days from the beginning of estrous) of CL for embryos transfer were 1.7 to cm in diameter (Figure 1B, 1C), while the smaller sizes (1.5 to 1.6 cm in diameter) were at stage near to the estrous day (Figure 1A) An explanation for this may due to the difference of recipient cow breed, in which the physical properties and functional CL of local Vietnamese cattle were closely associated with the ability of receiving donor cloned embryos Thus, by applying the ultrasound machine to evaluate the size and location of CL in combination of the day of estrous and the condition of recipient cows right before embryos transfer, we successfully improved the implantation and pregnancy rate of cloned bovine embryos to nearly 30 % at day 20th Table Effects of corpus luteum sizes on pregnancy rate of cloned bovine embryos CL Size (cm) 1.5 – 1.6 1.7 – 1.8 1.9 – 2.0 Total recipients 10 14 Pregnancy rates (%) at Day 20 Day 40th a 0a 3(30)b 2(20)b b 4(28.6) 3(21.4)b th Values with different superscripts within a column are significantly different (P < 0.05) 636 BÁO CÁO KHOA HỌC VỀ NGHIÊN CỨU VÀ GIẢNG DẠY SINH HỌC Ở VIỆT NAM 3.3 Effect of embryo age for embryo transfer on pregnancy rate of cloned bovine embryos Table Effects of embryo age for embryo transfer on pregnancy rate of cloned bovine embryos Group Embryo at day 6th Embryo at day 7th Total recipient 16 18 th Day 20 5(31.3)a 3(16.7)b Pregnancy rates (%) at Day 40th months 4(25.0)a 3(18.8)a 3(16.7)b 0b Values with different superscripts within a column are significantly different (P < 0.05) In this experiment, we compared the effect of embryo age (or the ideal timing) for embryo transfer on the postimplantation development of cloned bovine embryos between the group of embryo transfer on day 5.5 – (early blastocyst stage) and embryo on day – 7.5 (hatching blastocyst) Based on the data showed in Table 3, the pregnancy rate at day 20th after embryo transfer in the group of early blastocyst stage was more than double that of group of embryos at day (31.3 % > 16.7 %, respectively, P < 0.05) Based on the results, there was a possibility that at early cloned blastocysts were more beneficial to hatch and implant in the uterus after embryos transfer comparing to hatching blastocyst According to the natural process of implantation, fertilized blastocyst will expand and hatch in the uterus On the other hand, cloned bovine blastocyst experience a different pattern for development, in which the early blastocyst will directly go to hatching with a slight expansion Therefore, transferring hatching blastocyst (late blastocyst) into the uterus might results in the reduction of implantation ability and subsequently decreased the pregnancy rates of cloned bovine embryos CONCLUSION This was the first report evaluated corpus luteum size of local Vietnamese cattle and ideally synchronized timing for cloned bovine embryos transfer In conclusion, it is much beneficial to apply ultrasound machine for detection of CL size and location during estrous cycle of recipient cows Transferring cloned bovine blastocyst at day of embryo development into the uterus of recipient cows that contained at least 1.7 cm in CL size can improve the implantation ability and the pregnancy rate of cloned bovine embryos, thus increase the total success of bovine cloning to full term development Acknowledgement: This study is funded by Ministry of Science and Technology of Vietnam under grant number ĐTĐL.CN-49/16 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