Heparin sodium

Blood samples were treated with 10 IU/mL heparin sodium (Nacalai Tesque, Kyoto, Japan). Plasma was obtained by centrifuging the blood at 1000 x g for 15 min at 4°C. Activities of plasma alanine aminotransferase (ALT) and aspartate aminotransferase (AST), as well as plasma triacylglycerol (TG), were measured using kits (L-Type Wako ALT, L-Type Wako AST, and L-Type Wako TG M, respectively), each obtained from FUJIFILM Wako Pure Chemical.

Sperm suspensions derived from one bull alone or two different bulls were used in this experiment. The suspensions were incubated for 1, 2, 3, 4, 5, 24, or 48 h in non-capacitation or capacitation medium at 38.5 °C with 5% CO₂. A small amount of each suspension was added to a glass slides, covered with glass coverslips, then observed under an inverted fluorescence microscope (BZ-X710, KEYENCE, Osaka, Japan). We obtained images from random fields (size: 1085 × 1500 μm), and counted the number of unagglutinated sperm, head-to-head agglutination of two sperm, and head-to-head agglutination of three or more sperm. Note that we counted the number of agglutinations but not the sperm number constituting an agglutination. At least 100 single sperm cells or sperm agglutinations were counted, and the percentage of each of the above three groups at each timepoint were calculated.
To examine the effect of heparin on bovine sperm agglutination, we added a final concentration of 100 μg/mL heparin sodium (Nacalai Tesque, Kyoto, Japan) to sperm suspensions before incubation or after two hours of incubation. Sperm samples were then incubated in non-capacitation medium supplemented with or without heparin for 0, 2, or 4 h. After incubation, the sperm agglutination rate was determined as above-mentioned.