ear velocity, and lateral head displacement, it is concluded that post-thaw quality of buffalo semen can be improved using the Tris–TCA buffering system. q 2000 Elsevier Science B.V. All rights
reserved.
Keywords: Buffalo — spermatozoa; Buffers
1. Introduction
The first short-term storage extender for bull spermatozoa consisted of sodium sulfate Ž
. Ž
. Na SO , glucose and peptone Milovanov and Selivanova, 1933 . This was replaced
2 4
Ž .
by sodium citrate Salisbury et al., 1941 due to its properties of being a chelating agent and preventing peroxide formation in mammalian spermatozoa. Later, Tris-based exten-
der was shown to be superior to sodium citrate for the cryopreservation of bull Ž
. spermatozoa Davis et al., 1963 , presumably due to its better buffering capacity.
Ž .
Buffers containing zwitterions Bes, Hepes, Mes, Mops, Pipes, Tes and Tricine had p K
values closer to the optimal freezing pH of 7.0 and were more stable than Tris in
a
Ž .
the temperature shift Good et al., 1966 . The bull spermatozoa diluted in zwitterions when titrated to physiological pH with either Tris, NaOH or KOH were more motile but
Ž .
released more glutamic oxaloacetic transaminase GOT than sperm diluted in phosphate Ž
. Ž
or citrate Graham et al., 1972 . In the same study, Tes–Tris Tes buffer titrated to Tris; .
pH s 7.0 yielded the most satisfactory buffering system for diluting bull spermatozoa for freezing. Since then, little development of other buffers for preservation of bull
Ž .
spermatozoa has been reported Graham, 1978 . Development of a suitable buffering system for cryopreservation of buffalo spermato-
zoa has been in progress for sometime and the results are equivocal. Post-thaw motility was higher when buffalo spermatozoa were extended in Tris compared to citrate, citric
Ž acid whey or lactose Chinnaiya and Ganguli, 1980; Heuer, 1980; Matharoo and Singh,
. 1980; Tuli et al., 1981 . However, motility of frozen-thawed spermatozoa did not vary
Ž when they were extended in Tris compared to lactose and citrate Dhami and Kodagali,
. Ž
. Ž
. 1990 , milk Dhami et al., 1996 , or skim milk and citrate Kakar and Anand, 1981 .
Ž .
The release of enzymes, GOT and glutamic pyruvic transaminase GPT , was less due to Ž
Tris compared to citric acid whey or sodium bicarbonate after freezing Tuli et al., .
Ž .
1982 . In contrast, Chinnaiya et al. 1979 reported higher release of these enzymes in Tris compared to citric acid whey. The release of enzymes has been shown to be
Ž .
associated with sperm cell injury Pace and Graham, 1970 . Fertility of buffaloes inseminated with frozen-thawed semen was significantly higher when Tris was used
Ž .
compared to lactose or citrate Dhami and Kodagali, 1990 but did not differ when Ž
. compared with milk extender Dhami et al., 1996 . The introduction of computer-as-
Ž .
sisted semen analyzer CASA has provided a more precise quantification of sperm Ž
. characters than subjective measures in bulls Budworth et al., 1988; Anzar et al., 1991 .
The present study was designed to compare the post-thaw motion characteristics, plasma Ž
. membrane integrity and acrosome morphology between the conventional Na-citrate
w Ž
. Ž
. and zwitterion buffers
Tris–citric acid TCA , Tris–Tes
TEST and Tris–Hepes
Ž .x
HEPEST in buffalo spermatozoa to identify the suitable buffering system.
2. Materials and methods
