Use of a defined diluent increases the sex-sorting efficiency of stallion sperm.

The study proposed a novel, clear, diluent formula that improves the sex-sorting efficiency of stallion sperm compared to traditional milk-based diluents.

Overview of Research

This research aimed at formulating a clear diluent that improves the sex-sorting efficiency of stallion sperm. Their efforts were concentrated on increasing the flow cytometric sorting efficiency, which had previously been reduced due to the use of an opaque, skim milk-based diluent. The study was divided into three different experiments.

Experiment 1: Consideration of Different Diluents

• The researchers compared sperm incubated in five different diluents — no protein, skim milk, 0.25% Cohn’s Fraction V BSA, 0.5% BSA, or 1% BSA.
• Sperm were stored for 18 hours at 15 degrees Celsius, or examined shortly after being collected, before exposure to the diluents.
• The study found that sperm incubated in skim milk and 1% BSA had similar total and progressive sperm motilities and comparable acrosome integrity after 45 minutes of incubation.

Experiment 2: Optimization of Protein Source

• The second experiment involved the use of five different diluents — skim milk, BSA, fatty acid and endotoxin-free BSA (I-BSA), KnockOut™ Serum Replacement, and β-lactoglobulin.
• It was realized that I-BSA diluent was superior for maintaining sperm motility during incubation compared to skim milk.

Experiment 3: Effect of Diluents on Sorting

• In Experiment 3, the aim was to investigate the effect of the diluent on sorting, using different concentrations and incubation periods of H33342.
• The results revealed that the clear solution (1% BSA) improved the split ratio — the division of sperm into differently gendered divisions — when compared with the opaque solution (skim milk).
• Optimum staining time was noted as 45 minutes using 0.09 mM H33342.

Conclusion

The study concluded that the newly formulated diluent containing 1% fatty acid free, low endotoxin BSA was superior to traditional skim milk diluents as it improved sorting efficiency and motility characteristics of stallion sperm even after storage for 18 hours. This development opens up avenues for more effective sex selection in horse breeding.