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Time course of in vitro capacitation of frozen and unfrozen bovine spermatozoa
THERIOGENOLOGY
TIHE COURSEOF IB VITRO CAPACITATIOM OF FROZBBBAD UIWROZ~ BOVIIWSPERMATOZOA M. B. Wheeler and G. E. Seidel, Jr. Animal Reproduction Laboratory Colorado State University Fort Collins, CO 80523 USA In vitro fertilization is an important tool for studying gamete Methods for effective capacitation of bull interactions in cattle. sperm in vitro are essential for use of this technology on a wide scale. Applications include understanding. molecular mechanisms of fertilization, evaluation of fertility of semen, circumventing and production of embryos for further genetic infertility, manipulation. An in vitro zona-penetration assay was used to determine the time required to capacite bull sperm in vitro. Oocytes were harvested from l-20 mm follicles of bovine slaughterhouse ovaries Ejaculates were collected from three bulls; one stored at -20C. half of each ejaculate was frozen in egg yolk-citrate extender at a concentration of lo8 motile sperm/ml, and the other half was washed three times by centrifugation at 500 g for 5 min and capacitated in modified Tyrode’s medium (TALP) for up to 30 hours at After thawing, the 38C at a concentration of 20 x IO6 sperm/ml. frozen semen was washed and incubated in the same manner. After sperm were added to droplets containing various incubation intervals, The sperm oocytes to achieve a final concentration of lo6 sperm/ml. Oocytes then were and oocytes were incubated together for 3 hours. examined microscopically at 320 X for sperm penetration of the zona Data pellucida with the aid of a micromanipulator to rotate the ova. The percentages of were analyzed by factorial analysis of variance. ova that had at least one sperm penetrate the zona pellucida are presented in the table. Percentage
Treatment Fresh semen Frozen semen bci
0
of
Penetrated
6
Ovaa
Time (hr) 12
24
lb 17b
n = 150 ova per group Values within rows without (P<.Ol ,Tukey’s test).
30 25d Od
a common superscript
differ
was significant The fresh-frozen treatment by time interaction the penetration profiles of the two (P<.Ol) indicating that There were no significant bull treatments were different over time. effects. We conclude that unfrozen semen requires a longer Fur thermore, the frozen-thawed capacitation time than frozen semen. semen deteriorates more rapidly than unfrozen semen.
21.6
JANUARY
1986VOL. 25 NO.1
TIHE COURSEOF IB VITRO CAPACITATIOM OF FROZBBBAD UIWROZ~ BOVIIWSPERMATOZOA M. B. Wheeler and G. E. Seidel, Jr. Animal Reproduction Laboratory Colorado State University Fort Collins, CO 80523 USA In vitro fertilization is an important tool for studying gamete Methods for effective capacitation of bull interactions in cattle. sperm in vitro are essential for use of this technology on a wide scale. Applications include understanding. molecular mechanisms of fertilization, evaluation of fertility of semen, circumventing and production of embryos for further genetic infertility, manipulation. An in vitro zona-penetration assay was used to determine the time required to capacite bull sperm in vitro. Oocytes were harvested from l-20 mm follicles of bovine slaughterhouse ovaries Ejaculates were collected from three bulls; one stored at -20C. half of each ejaculate was frozen in egg yolk-citrate extender at a concentration of lo8 motile sperm/ml, and the other half was washed three times by centrifugation at 500 g for 5 min and capacitated in modified Tyrode’s medium (TALP) for up to 30 hours at After thawing, the 38C at a concentration of 20 x IO6 sperm/ml. frozen semen was washed and incubated in the same manner. After sperm were added to droplets containing various incubation intervals, The sperm oocytes to achieve a final concentration of lo6 sperm/ml. Oocytes then were and oocytes were incubated together for 3 hours. examined microscopically at 320 X for sperm penetration of the zona Data pellucida with the aid of a micromanipulator to rotate the ova. The percentages of were analyzed by factorial analysis of variance. ova that had at least one sperm penetrate the zona pellucida are presented in the table. Percentage
Treatment Fresh semen Frozen semen bci
0
of
Penetrated
6
Ovaa
Time (hr) 12
24
lb 17b
n = 150 ova per group Values within rows without (P<.Ol ,Tukey’s test).
30 25d Od
a common superscript
differ
was significant The fresh-frozen treatment by time interaction the penetration profiles of the two (P<.Ol) indicating that There were no significant bull treatments were different over time. effects. We conclude that unfrozen semen requires a longer Fur thermore, the frozen-thawed capacitation time than frozen semen. semen deteriorates more rapidly than unfrozen semen.
21.6
JANUARY
1986VOL. 25 NO.1