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Percoll gradient selection of equine spermatozoa enhances ability to bind and penetrate the zona pellucida
Theriogenology41:158,
1994
PERCOLL GRADIENT SELECTION OF EQUINE SPERMATOZOA ENHANCES ABILITY TO BIND AND PENETRATE THE ZONA PELLUCIDA M. J. Arns and R. E. Shepherd Department of Animal Sciences and Industry Kansas State University Manhattan, KS 66506 USA We have previously reported (Buzby et al., 1993, J. Eq. Vet. SCi. 13:15-18) that sperm separation procedures influence spermatozoal motility during culture, and hypothesized that sperm separation through a Percoll discontinuous gradient eapaeitates equine spermatozoa. The present study was conducted to determine if Percoll gradient selection enhances ability of spermatozoa to bind and penetrate zonae pellucidae of nonviable equine oocytes. Ejaculates (n=4) were split into four treatments: TRT 1 sperm were diluted in Hams F-10 containing 3% BSA (SpermPrep, ZBL Laboratories, Lexington, K'Y), TRT 2 - sperm were diluted in a skim-milk glucose extender (E-Z Mixin, Animal Reproductive Systems, Chino, CA), TRT 3 - sperm were separated from their seminal plasma through a PercoU discontinuous gradient (Per-Ception, Fertility Technologies, Natick, MA) and resuspended in Hams F-10, and TRT 4 - sperm were resuspended in skim-milk glucose following Percoll selection. All treatments were diluted to a concentration of 1 X 106 sperm/ml prior to insemination of denuded equine oocytes. Ooeytes were harvested from frozen ovaries, washed 3X in PBS and IX with either Hams F-10 or skim-milk. Ooeytes (n = 10) were then placed into 100 IH of fresh diluent and inseminated with an equal volume from one of the four treatments. Oocytes were coincubated with spermatozoa for 4 hr at 37.5°C in a humidified atmosphere containing 5% CO 2. Ooeytes were then washed 5X through a small bore pipette to remove attached sperm. Oocytes were stained with Hoechst 33258 (Sigma Chemical Co., St. Louis, MO) to label spermatozoa, and then evaluated for sperm penetration to measure sperm capacitation. Analysis of variance procedures (SAS) were used to analyze transformed (log (x + 1)) count data, while Chi-square analysis was used to compare penetration rates. The results are shown below: Table 1. Mean (+--S.E.M.) number of sperm penetrating or bound to equine zonae. TRT
N
Total sperm bound/ooeyte
Total sperm penetrated/oocyte
% of oocytes penetrated
1
33
1.6 __+.3
.3 + .1
24 (8/33)
2
34
.1 + .1
0
0 (0/34)
3
33
7.1 + 1.1a
2.5 __+.4b
82¢ (27/33)
4
28
.6 __+.4
.1 __+.1
7 (2/28)
TRT 3 differs from other means within column ap<.03, bp<.06, cp<.001. A higher rate of sperm-oocyte interaction and sperm penetration existed for TRT 3 spermatozoa as compared to all other treatments. In addition, the percentage of oocytes penetrated was greater for oocytes inseminated with TRT 3 spermatozoa. Resuspension of spermatozoa in skim-milk glucose following Percoll separation (TRT 4) inhibited sperm-oocyte interaction. These results indicate that sperm separation by Percoll discontinuous gradient with subsequent resuspension of spermatozoa in Hams F-10 supports in vitro capacitation of equine spermatozoa.
158
Copyright © 1994 Butterworth-Heineman n
1994
PERCOLL GRADIENT SELECTION OF EQUINE SPERMATOZOA ENHANCES ABILITY TO BIND AND PENETRATE THE ZONA PELLUCIDA M. J. Arns and R. E. Shepherd Department of Animal Sciences and Industry Kansas State University Manhattan, KS 66506 USA We have previously reported (Buzby et al., 1993, J. Eq. Vet. SCi. 13:15-18) that sperm separation procedures influence spermatozoal motility during culture, and hypothesized that sperm separation through a Percoll discontinuous gradient eapaeitates equine spermatozoa. The present study was conducted to determine if Percoll gradient selection enhances ability of spermatozoa to bind and penetrate zonae pellucidae of nonviable equine oocytes. Ejaculates (n=4) were split into four treatments: TRT 1 sperm were diluted in Hams F-10 containing 3% BSA (SpermPrep, ZBL Laboratories, Lexington, K'Y), TRT 2 - sperm were diluted in a skim-milk glucose extender (E-Z Mixin, Animal Reproductive Systems, Chino, CA), TRT 3 - sperm were separated from their seminal plasma through a PercoU discontinuous gradient (Per-Ception, Fertility Technologies, Natick, MA) and resuspended in Hams F-10, and TRT 4 - sperm were resuspended in skim-milk glucose following Percoll selection. All treatments were diluted to a concentration of 1 X 106 sperm/ml prior to insemination of denuded equine oocytes. Ooeytes were harvested from frozen ovaries, washed 3X in PBS and IX with either Hams F-10 or skim-milk. Ooeytes (n = 10) were then placed into 100 IH of fresh diluent and inseminated with an equal volume from one of the four treatments. Oocytes were coincubated with spermatozoa for 4 hr at 37.5°C in a humidified atmosphere containing 5% CO 2. Ooeytes were then washed 5X through a small bore pipette to remove attached sperm. Oocytes were stained with Hoechst 33258 (Sigma Chemical Co., St. Louis, MO) to label spermatozoa, and then evaluated for sperm penetration to measure sperm capacitation. Analysis of variance procedures (SAS) were used to analyze transformed (log (x + 1)) count data, while Chi-square analysis was used to compare penetration rates. The results are shown below: Table 1. Mean (+--S.E.M.) number of sperm penetrating or bound to equine zonae. TRT
N
Total sperm bound/ooeyte
Total sperm penetrated/oocyte
% of oocytes penetrated
1
33
1.6 __+.3
.3 + .1
24 (8/33)
2
34
.1 + .1
0
0 (0/34)
3
33
7.1 + 1.1a
2.5 __+.4b
82¢ (27/33)
4
28
.6 __+.4
.1 __+.1
7 (2/28)
TRT 3 differs from other means within column ap<.03, bp<.06, cp<.001. A higher rate of sperm-oocyte interaction and sperm penetration existed for TRT 3 spermatozoa as compared to all other treatments. In addition, the percentage of oocytes penetrated was greater for oocytes inseminated with TRT 3 spermatozoa. Resuspension of spermatozoa in skim-milk glucose following Percoll separation (TRT 4) inhibited sperm-oocyte interaction. These results indicate that sperm separation by Percoll discontinuous gradient with subsequent resuspension of spermatozoa in Hams F-10 supports in vitro capacitation of equine spermatozoa.
158
Copyright © 1994 Butterworth-Heineman n