The effect of heparin, caffeine and calcium ionophore A23187 on in vitro induction of the acrosome reaction in frozen-thawed bovine and caprine spermatozoa.

The effect of heparin (5 IU), caffeine (5 mM) and calcium-ionophore A23187 (0.1 mM) on motility and in vitro induction of the acrosome reaction in glass wool filtered frozen-thawed bull and goat semen was studied. The motile spermatozoa fraction was obtained after glass wool filtration of frozen-thawed semen. The seminal plasma was removed from filtered semen by centrifugation, and the sperm pellet was resuspended in Sperm-TALP medium. Samples of treated and untreated control semen of both species were incubated at 37 degrees C. At 1, 15 and 30 min of incubation the proportions of progressively motile and acrosome-reacted spermatozoa were assessed. Trypan blue and Giemsa stain was used to differentiate live and dead spermatozoa having undergone acrosome reaction. Glass wool filtration enhanced the proportion of motile spermatozoa from 43% to 62% in the bovine and from 41% to 60% in the caprine. Whereas the effect of incubation with caffeine, heparin and calcium-ionophore on spermatozoan motility was negligible, the treatment of semen with calcium-ionophore resulted in a significantly improved percentage of live spermatozoa with true acrosome reaction at all stages of incubation, both in the bovine and the caprine.

Comparison of quality and freezability of water buffalo semen after washing or Sephadex filtration.

Split aliquots of pooled buffalo semen samples were processed before freezing 1) by washing twice with Tris-citric acid buffer by centrifugation and re-suspension to the original volume in the same buffer, or 2) or by passage through a G-15 Sephadex column. The effect of these procedures on progressive motility, percentages of live spermatozoa, sperm abnormalities and intact acrosomes and release of glutamate oxatoacetate transaminase (GOT) into the medium were assessed after extension, after equilibration and after 18 to 24 h or 15 d of frozen storage. Prior to extension, gel filtration reduced sperm concentration and enhanced progressive motility, whereas washing produced little effect on these attributes. Except in the case of GOT release, which was significantly (P < 0.05) lower after the washing of semen (34.3 +/- 16.40) than the filtering of semen (45.7 +/- 12.35), the 2 procedures did not cause significant effects (P > 0.05). Damage to spermatozoa due to freeze-processing was also similar in the 2 treatments, and the extent of beneficial effect in improved motility and live spermatozoan numbers after thawing was also similar.

Effect of glycerolization procedure and removal of seminal plasma on post-thaw survival and got-release from Boer goat spermatozoa.

Forty ejaculates (20 for each of 2 experiments) were collected from 4 Boer goat bucks at weekly intervals to study the effect of glycerolization procedure and removal of seminal plasma on progressive motility, percent live spermatozoa and release of glutamic oxaloacetic transaminase (GOT) before and after the freezing of semen. Stepwise glycerolization at 37 degrees C gave higher progressive motility and percentage of live spermatozoa both before freezing and after thawing than one-step glycerolization at 37 degrees C or stepwise extension with glycerol being added after cooling to 5 degrees C. The GOT-release was reduced before freezing and after thawing of semen with stepwise glycerolization (P<0.05). Progressive motility and the percentage of live spermatozoa were higher (P<0.05) after the freezing of whole semen than in washed spermatozoa. The concentration of GOT in the extra-cellular fluid was lower in washed spermatozoa prior to freezing (P<0,05); but after thawing, the washed spermatozoa released more GOT than spermatozoa in whole semen. Removal of seminal plasma prior to freezing spermatozoa in an extender containing egg yolk had an unfavorable effect on their post-thaw motility and integrity.

Computer-assisted motility assessment of spermatozoa from fresh and frozen-thawed semen of the bull, boar and goat.

Generally, both subjective and computer-assisted (HTM-2000 motility analyzer) assessment of sperm motility in fresh and in frozen-thawed semen of bulls, boars and bucks yields comparable results. However, the use of a motility analyzer renders consistently more accurate estimates, especially when that motility is vigorous as in fresh bull semen.

The effect of zwitterion buffers on the freezability of Boer goat semen.

Twenty semen samples with mass activity greater than +3 were collected from six healthy, mature Boer goat bucks. Each ejaculate was divided into four equal parts and extended at 37 degrees C in Tris, Test, Tes and Bes buffers containing egg yolk and glycerol. Semen was placed into medium size French strawsand after 2 hours of equilibration at 5 degrees C, frozen in the vapour phase and stored in liquid nitrogen for 7 days at -196 degrees C. Progressive motility, the number of live spermatozoa and glutamic oxaloacetic transaminase (GOT) release were studied after the initial extension, after equilibration and after 15 minutes and 7 days of freezing of semen. Semen samples when extended with Tris yolk glycerol showed significantly (P<0.01) higher progressive motility and live spermatozoa than when extended with the other zwitterion buffer-based extenders. The change of extenders did not influence the release of GOT at various stages of freezing of semen (P>0.05).

Seasonal variation in freezability of buffalo semen.

Fifty-six buffalo semen ejaculates of mass activity greater than plus three on a five-point scale (28 each in summer and winter seasons) were frozen in Tris yolk glycerol (TY-G) extender with four hours equilibration time. Motility of semen was checked after first extension (initial motility), after equilibration time, 15 minutes after freezing, and 30 days after freezing. Motility increased during winter season. Significant differences (P<0.01) in motility after freezing were observed between summer and winter seasons.

Effect of different extenders on glutamic oxalacetic transaminase (GOT) and glutamic pyruvic transaminase (GPT) release from frozen buffalo semen.

Sixty buffalo semen samples (motility greater than 60%) were frozen in 3 extenders, viz., Tris yolk glycerol (TY-G), Citric acid whey glycerol (CAW-G) and Egg yolk glucose sodium bicarbonate glycerol (EYGSB-G) for studying the release of GOT and GPT enzymes in the extracellular fluid during pre-freezing (after first extension) and post-freezing (15 minutes and 30 days after freezing). Release of GOT and GPT enzymes was less in TY-G than CAW-G and EYGSB-G extenders. Significant differences (P<0.01) in GOT and GPT release were observed between extenders and bulls at various stages of freezing of semen.

Effect of different equilibration times and extenders on deep freezing of buffalo semen.

Thirty semen collections from 3 Murrah buffalo bulls were frozen in Tris yolk glycerol (TY-G) and Citric acid whey glycerol (CAW-G) extenders using 2, 4 and 6 hours equilibration times and 7 percent glycerol level. Sperm motility after freezing was studied at an interval of 15 minutes 7 days and 30 days storage in liquid nitrogen. Sperm survivability was found to be better at all the stages of deep-freezing using 4 hours equilibration time. Significant differences (P<0.01) were observed between extenders and equilibration times.