PREPARATION AND RECOVERY OF FROZEN-THAWED BOVINE SPERMATOZOA VIA VARIOUS SPERM SELECTION TECHNIQUES EMPLOYED IN ASSISTED REPRODUCTIVE TECHNOLOGIES

A number of semen manipulative techniques are currently available to r emove the undesirable spermatozoa, debris and other factors and to inc rease sperm quality. The use of motility stimulants such as caffeine o r others could optimize the recovery and quality of frozen-thawed sper matozoa processed by a variety of sperm selection techniques. Frozen-t hawed specimens from 5 bulls were slowly diluted and washed with Ham's F-10 medium containing 3% BSA (w/v) and 0 or 2mM caffeine. Aliquots c ontaining approximately 50 x10(6) total sperm cells were used for conv entional sperm wash, swim-up, Percoll density gradient centrifugation (80, 70, 55 and 40% Percoll gradients) and Sephadex (SpermPrep(TM)I) f iltration. Quantitative and qualitative characteristics of selected sp ermatozoa included: total sperm (x10(6)), percentage and grade (0 to 4 ) of motility, percentage of spermatozoa with coiled tails and respons e to the hypoosmotic swelling (HOS) rest (percentage of swollen sperma tozoa). When compared to washed specimens, fewer spermatozoa were reco vered via the swim-up, Percoll and SpermPrep(TM)I filtration methods. Quantitative and qualitative characteristics of these spermatozoa impr oved further after processing with Ham's F-10 containing 2 mM caffeine , followed by selection via the various techniques. Enhancement of spe rm motility, in conjunction with the most appropriate sperm selection technique, represents an efficient method for the recovery of spermato zoa with improved qualitative characteristics.