A Study on the Embryo Transfer of 2-Cell Stage Embryos in Mlac:WR

Abstract

Background and Objectives : The National Laboratory Animal Center, Mahidol University, aims to preserve the strains of Wistar rats (Mlac:WR) in the embryo bank. The benefits of embryo banking are genetic resources. To prevent the loss of laboratory animal strains due to genetic changes, failure of the reproductive system, failure of the animal husbandry system, infections in the laboratory animal colony, natural disasters, cessation of laboratory animal breeding, and reduction of laboratory animal breeding costs. Additionally, preserving laboratory animal strains in the embryo bank is beneficial in preventing a shortage of laboratory animals in Thailand and reducing the importation of expensive laboratory animals. Therefore, in this study, we attempt to study the superovulation protocol for the in vivo embryo production of 2-cell stage embryos in Mlac:WR rats to determine the percentage of 2-cell stage embryos (% 2-cell stage embryos). The 2-cell stage embryos were frozen using the vitrification method. After that, the vitrified 2-cell stage embryos were thawed to determine the percentage of survival rate (% survival), and then the surviving 2-cell stage embryos were used for embryo transfer to determine the percentage of newborns (% newborn).

Methodology : Mlac:WR rats were maintained in the animal room with strict hygienic conventional controlled temperature at 22±3ºC, 30-70% humidity, and 12 hours light conditions (lights on from 6:00 AM to 6:00 PM). Rats were supplied with a 5-7 ppm choline reverse osmosis water and standard diet (ad libitum). All animal procedures were conducted at the Association for Assessment and Accreditation of Laboratory Animal Care (AAALAC) International. This study was approved by the National Laboratory Animal Center-Animal Care and Use Committee (NLAC-ACUC, protocol no. RA2023-33). Eight female rats with 9-week-olds were induced to superovulate through an intraperitoneal (IP) injection of 175 IU/kg BW pregnant mare serum gonadotropin (PMSG) at 9:00-10:00 AM, followed by 300 IU/kg BW IP of human chorionic gonadotropin (hCG) 50 hours later (11:00-12:00 AM). These females were then mated with 12-week-old Mlac:WR male rats overnight. 46-47 hours after the hCG injection, female rats were euthanized by carbon dioxide. The oviducts were collected, and embryos were flushed out from the oviducts with M2 medium under a stereomicroscope. The 2-cell stage embryos with normal morphology were counted and washed in 50 µl of M2 medium 3 times. After that, 2-cell stage embryos were cultured in 150 µl of M16 medium covered with mineral oil in a CO₂ incubator (37ºC, 5% CO₂, 95% humidity). For the vitrification, the 2-cell stage embryos were pretreated with EFS20 solution (20% v/v ethylene glycol, 15% w/v Ficoll^® PM 70, 0.25 M sucrose) for 2 minutes and then frozen by the vitrification method using an EFS40 solution (40% v/v ethylene glycol, 30% w/v Ficoll^® PM 70, 0.5 M sucrose) in a 0.25 ml straw tube. After being stored in a liquid nitrogen tank for 7 days, the vitrified 2-cell stage embryos were thawed using 0.75 M and 0.25 M sucrose (thawing solution), respectively. The surviving 2-cell embryos were counted and cultured in M16 medium, waiting for embryo transfer. The Mlac:WR female rats with 8-week-olds were tested for the proestrus by vaginal smear technique after that set mating overnight with the 12-week-old vasectomized Mlac:WR male rats. Female rats with vaginal plugs or mated with vasectomized males were used as recipients. The surviving 2-cell stage embryos were used for transfer to the oviducts of recipients. The recipients were natural-born on days 19-20 after embryo transfer.

Main Results : The in vivo embryo production of 2-cell stage embryos in Mlac:WR female rats at 9-week-old by the superovulation technique. The results show that the total number of embryos harvested from the superovulation was 202 embryos, divided into 139 of 2-cell stage embryos (17.4±6.4), 18 of 4-cell stage embryos (2.3±0.9), and 45 of abnormal embryos (5.6±3.5). The percentage of 2-cell stage embryos was 68.8 (139/202). The 139 of 2-cell stage embryos were divided into 3 groups (47, 47, and 45 embryos) and frozen by vitrification for 3 tubes. After thawing the first tube, 45 of the 2-cell stage embryos were discovered under a stereomicroscope; after that, 13 of the 2-cell stage embryos were dead. The 32 of the 2-cell stage embryos were alive after thawing. The percentage of survival 2-cell stage embryos was 68.1 (32/47). The surviving 2-cell stage embryos were used for embryo transfer to 2 recipients (16 of 2-cell stage embryos per recipient). 19-20 days later, the number of newborns was 1 and 3 in recipients, respectively, with the percentage of newborns being 12.5 (4/32), 1 female, and 3 males.

Conclusions : The 2-cell stage embryos of Mlac:WR rats can be used to establish the embryo bank. Because of that, the percentage of 2-cell stage embryos obtained from in vivo embryo production and the percentage of survival 2-cell stage embryos after vitrification are relatively high, although the percentage of newborns after embryo transfer was low. However, this study was the first successful embryo transfer in Mlac:WR rats at the National Laboratory Animal Center. The knowledge and skills gained from this study will be used to improve the embryo transfer protocol. To successfully establish a rat embryo bank and promote the National Laboratory Animal Center as a source of learning on laboratory animal embryo bank techniques in Thailand.