In the dairy and beef cattle industries, artificial insemination and embryo transfer are common. Many studies have demonstrated a skewed sex ratio in the calves derived from assisted reproductive technologies. During the period between insemination and calving, insemination condition and the environment in which embryo development occurs are believed to affect the sex of an embryo. This short review looks at studies of factors that have been reported to affect the primary sex ratio of bovine embryos, including the timing of insemination, sperm and oocyte status, and maternal conditions.

Although most sexual dimorphism appears after gonadal differentiation, some occur at earlier stages of development. One example of this phenomenon is the skewing of the sex ratio of embryos due to the glucose concentration of the culture medium. This skewing occurs because male and female embryos differ in their abilities to metabolize glucose. Another example is the sexually dimorphic expression of the bovine embryo-derived signal for maternal recognition of pregnancy, IFNT. The difference in expression of X-linked genes between male and female embryos, that results from incomplete X-chromosome inactivation, is considered to be involved, either directly or indirectly, in the sexual dimorphism that appears during early development.

Loop-mediated isothermal amplification (LAMP) is a novel DNA amplification method that amplifies a target sequence specifically under isothermal conditions. The LAMP product is detected by the turbidity of the reaction mixture without electrophoresis. We have developed a rapid sexing method for bovine preimplantation embryos using LAMP. Sexing is performed utilizing two LAMP reactions, male-specific and male-female common reactions, after DNA extraction. The time needed for sexing is <1 h. Sixty-one fresh sexed embryos, of which 23 and 38 had been judged as male and female, respectively, were transferred to recipient animals (one embryo per animal). The pregnancy rate was 57.4% (35/61) and all calves born were of the predicted sex. Therefore, LAMP-based embryo sexing accurately determined gender and was proven to be suitable for field application. In this review, we describe the procedure for detecting male-specific DNA sequence by LAMP for the rapid identification of freemartins. In addition, we also describe an efficient procedure for embryo sexing of water buffalo (Bubalus bubalis) with LAMP.

Sperm sexing using flow cytometry and cellsorting technology is now available for many mammalian species with approximately 90% accuracy. This technique has been applied only in the cattle livestock industry on a large-scale commercial basis. Frozen straw doses containing sexed bull sperm (usually marketed as ‘sexed semen’) have been sold in many countries for artificial insemination (Al) use. However, the cell-sorting process damages sperm physically/physiologically consequently compromising fertility results compared to conventionally processed sperm when used for Al or in vitro fertilization (IVF). Also, the production of sexed sperm is still limited and costly. Despite these biological and economic restrictions, sexed semen is widely accepted and the industry is awaiting further refinements to it because of growing demand.

The embryo transfer technology represents a powerful tool for the acceleration of various breeding programs in cattle. In cattle breeding, it is important to know the sex of embryos in order to improve the genetic potential of the herd. Normally, only five to six calves can be obtained during the reproductive life-time of a cow. However, calving rates can be increased by superovulation and embryo transfer program. If embryo sexing techniques are applied for the program, embryos with the preferred sex can be obtained from cows with high genetic performance. This is a potent way to improve the genetic background and production of animals. However, the reliability, cost, practicality and time required for this approach must be considered as well. The present paper discusses the current status and the future of bovine embryo transfer using sex determined embryos.

After screening 408 crude drugs, we found that Rizoma Arisaematis increased the cell numbers of mouse blastocysts developed from in vivo zygotes. We examined the effects of Rizoma Arisaematis on the in vitro development of mouse zygotes and embryos produced by ICSI and SCNT, as well as on fetal development. Mouse zygotes were cultured in media containing a water-soluble extract of Rizoma Arisaematis at various concentrations, and the potential of zygotes to develop into blastocysts and the cell numbers of blastocysts were examined. In addition, the effects of Rizoma Arisaematis on the in vitro and in vivo developmental potential of embryos produced by ICSI and nuclear transfer were examined. In vitro treatment of zygotes with Rizoma Arisaematis increased the cell numbers of blastocysts. The proportions of the blastocysts that implanted and developed into fetuses were slightly higher in the blastocysts which were developed from zygotes treated with Rizoma Arisaematis than those of the control. The Rizoma Arisaematis treatment of mouse fetal fibroblast cells or embryos produced by ICSI and SCNT did not affect the growth of the cells, or the in vitro development of the zygotes. The present study demonstrated that Rizoma Arisaematis improved the quality of embryos developed from in-vivo zygotes, but not that of embryos produced by ICSI and nuclear transfer.

Expression of GATA6 has been found during embryonic development in many mammalian species. In mouse embryonic development, GATA6 is a primitive endoderm (PrE) marker. However, the expression and effect of GATA6 in ruminant ungulates has not been well characterized. In this report, the expression of GATA6 mRNA was examined in the uteri of ruminants. GATA6 mRNA was detected in days 17, 20, and 22 (day 0=day of estrus) bovine conceptuses and in days 15, 17, and 21 ovine conceptuses. In both cases, GATA6 mRNA increased on day 22 or 21 after conceptus attachment to the uterine epithelium. GATA6 mRNA was also detected in bovine trophoblast CT-1 or F3 cells and ovine trophoblast oTr cells. In transient transfection analyses using the upstream region of the bovine IFNT gene (bIFNT, IFN-tau-c1), GATA6 overexpression was effective in the up-regulation of the bIFNT construct that had been transfected into human choriocarcinoma JEG3 or bovine ear-derived fibroblast EF cells. The observation in which GATA6 increased IFNT transcription suggests that in addition to lineage specification, GATA6 may have acquired ruminant-specific functions involving the control of trophoblast gene expression.