EFFECT OF REDUCING THE NUMBER OF SPERM CELLS (PER INSEMINATION), INCREASING ENERGY AND CRYOPROTECTING CONCENTRATIONS ON MOTION CHARACTERISTICS AND MEMBRANE INTEGRITY IN FROZEN THAWED BUFFALO SPERMATOZOA

The objective of this study was to find out the minimum number of sperm cells per dose of insemination that will not affect the conception rate, if levels of egg yolk and glycerol are increased. For this purpose, sperm motion characteristics and plasma membrane integrity in three concentrations of sperm cells per dose, each with two levels of egg yolk and glycerol were compared. Semen was collected from buffalo bulls using an artificial vagina. Split pool ejaculates possessing more than 60% visual sperm motility were diluted in Tris-citric acid (TCA) extender at 37°C, either in (1) 30-6-20 (Number of sperm cells in millions/0.5 ml insemi1:1ation dose-Glycerol%-Egg yolk %),(2) 15-6-20, (3) 7.5-6-20, (4) 30-10- 20, (5) 15-10-20, (6) 7.5-10-20, (7) 30-6-30, (8) 15-6-30, (9) 7.5-6-30, (10) 30-10-30, (11) 15-10-30 and (12) 7.5-10-30. Semen was cooled to 4°C in 2 hours, equilibrated at 4aC for 4 hours, filled in 0.5 ml straws and frozen in a programmable cell freezer ( + 4 to -15°C @ -3°C/minute and then to -80°C@ -10°C/minute) before plunging them into liquid nitrogen (-196°C). Thawing of frozen semen was performed after 24 hours at 37°C for 15 seconds. Sperm motion characteristics, including motilities (computer-assisted, linear and circular), velocities (straight-line, average path and curvilinear), and lateral head displacement (LHD) were assessed using computer assisted semen analyzer (CASA). Plasma membrane integrity was determined by using Hypo-Osmotic Swelling assay (HOS). Analysis of variance revealed that visual motility, computer-assisted motility, linear motility, circular motility, and plasma membrane integrity did not vary significantly when cell number was reduced from 30x106/dose to 15x106/dose. However, visual motility, computer-assisted motility and plasma membrane integrity were reduced significantly (P<0.05) when cell number was decreased to 7.5x106/dose. The velocities (straight- line, average path, curvilinear) and LHD did not vary significantly due to the reduction in number of sperm cells/dose, levels of glycerol and egg yolk. In conclusion, reduction of sperm number to 15x106 cells/dose did not affect sperm motion characteristics and plasma membrane integrity at both levels of glycerol and egg yolk, but when reduced to 7.5x106/dose, although linear and circular motilities, velocities and LHD were not affected, but visual and computer-assisted motilities, and plasma membrane integrity were reduced (P<0.05)