Comparative Analysis of Culture Media and ATR-FTIR Metabolomics for Non-invasive Sex Determination in In vitro Produced Buffalo Embryos

Production of female offspring is vital for maximizing milk yield in the dairy industry, making embryo sexing essential. Metabolic differences between male and female embryos may be reflected in the culture medium (CM). This study explored noninvasive sex detection of in vitro produced buffalo embryos by analyzing metabolites in spent CM.

Buffalo ovaries were collected from a slaughterhouse and oocytes were matured and fertilized in vitro. Presumptive zygotes were cultured in either a sequential medium, Synthetic oviduct fluid (SOF) or a single-step medium, Potassium Simplex Optimized Medium (KSOM). After 5 days, morulae were placed in 25 μl of CM. On day 7, embryos and spent medium were collected and stored at -20°C. Embryo sex was determined using PCR. Spent media were analyzed using Attenuated Total Reflection - Fourier Transform Infrared Spectroscopy (ATR-FTIR) spectroscopy to detect sex-specific metabolites.

This approach enabled non-invasive identification of embryo sex and revealed the impact of different culture media on sex ratio. The findings highlight the potential of spent CM analysis for predicting embryo sex, offering significant benefits for the dairy industry.