Background: 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 non-invasive sex detection of in vitro produced buffalo embryos by analyzing metabolites in spent CM. 
 
Methods: 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 &micro;l of CM. On day 7, embryos and spent medium were collected and stored at -20oC. 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. 
 
Result: 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.

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

Background: The utilization of in vitro fertilization (IVF) and in vitro embryo production (IVEP) has increased significantly as an effective tool of assisted reproductive technology. Sperm capacitation is a process of membrane maturation allowing the acrosome reaction to take place. Ionomycin is a calcium ionophore that induces the acrosome reaction by increasing calcium cycling across phospholipid membranes. The aim of this study was to enhance the cleavage rate and development of bovine embryos.&nbsp; 
 
Methods: Bovine ovaries were collected from the central slaughter house. Oocytes were obtained from follicles and were let to mature for 24 h. Frozen bull semen thawed in 37&deg;C water and transferred to a discontinuous percoll gradient (Sigma GE17-0891-01). The spermatozoon pellet was separated into four groups after resuspension in bovine in vitro fertilization media (BO-IVF): control without additives; with DMSO and with ionomycin (25 and 50 nM) and kept in an incubator for three hours. The oocytes matured under commonly used media (TCM-199) for 24 h. The mature oocytes were then fertilized for 18-24h. The embryos were cultured in SOF medium until the seventh day to study cleavage rate and blastocyst rate.&nbsp; 
 
Result:&nbsp;The results indicated that 50 nM ionomycin supplementation considerably reduced cleavage while DMSO and 25 nM ionomycin supplementation had no effect on cleavage percentage. Moreover, when compared to the other groups, ionomycin at 25 nM significantly increased blastocyst development. In conclusion, the highest rate of blastocyst development was observed in the capacitation medium supplemented with 25 nM ionomycin, apparently due to the importance of calcium and metabolism in spermatozoa for fertilization and embryo development.

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

The Role of Ionomycin in the Improvement of in vitro Fertilization and Increasing the Rate of Bovine Embryo Development