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Determination of sex ratio of bovine embryos produced in vitro
Increase of bovine embryos produced by in vitro fertilization (IVF) has been seen. The main reason for producing in vitro fertilized embryos in Korea has been to utilize the genetics of cows with higher carcass grade. Ovaries are collected from the cows in the slaughter house and the information on the carcass grade of the cow can be traced. Embryos produced from cows with higher carcass grade have been favored by the farmers. PCR has been one of the main techniques for sex determination of embryos targeting various genes. Bovine sex determining region Y (SRY) is specific to Y chromosome. However, it requires a control gene for PCR, if the embryo is female. In comparison to SRY, amelogenin can be amplified from male or female embryos with different fragment sizes due to differential splicing in all bovidae. The goal of this study was to determine whether there are any differences in the sex ratio of embryos produced in vitro and to compare the efficiency of sex determination using PCR. Ovaries of Hanwoo were collected and transported to the laboratory in thermal bottles. For in vitro maturation, oocytes were collected from the follicles with less than 8 mm of diameter and placed in either the Brackett & Oliphant media (BO), Tissue culture medium-199 (TCM-199), or IVMD101 media, containing 3% fetal bovine serum (FBS), 0.5 mg/ml FSH, 0.5 mg/ml LH, and 1 mg/ml estradiol-17β. For IVF, frozen sperm from Hanwoo bulls were used. After 22-24h IVF, embryos were transferred and cultured either in BO or TCM-199 with 10% FBS until the embryos were hatched. Hatched blastocysts were stored in PBS frozen, and later thawed and treated with embryo lysis buffer. After isolating genomic DNA, it was used for PCR using primers for casein beta (CSN2), as PCR control, or for male specific SRY primers. Alternatively, primers for amelogenin were used. Sex of embryos was determined and the sex ratio was analyzed. Out of 94 embryos, sex of 83 embryos (88.3%) was determined and there were 40 male embryos (48.2%) and 43 female embryos (51.8%). Sex of 31 embryos was determined using both SRY and amelogenin. Among those, 17 embryos were determined as having identical sex, while 1 embryo was determined as having different sex, and the sex of 11 and 2 embryos were determined only by amelogenin or SRY primers, respectively. In conclusion, the success of determining the sex of embryos by PCR was relatively high. Using amelogenin primer for PCR tends to be more efficient than SRY primer in determining the sex. Slightly higher ratio of female embryos was different from previous years and the cause for the difference may require further investigation.
