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An indirect immunofluorescence assay to assess sperm cryodamage using anti-bull sperm monoclonal antibodies
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Theriogenology AN INDIRECT IMMUNOFLUORESCENCE ASSAY TO ASSESS SPERM CRYODAMAGE USING ANTI-BULL SPERM MONOCLONAL ANTIBODIES L.A. Johnson’, J.D. Ambrose’, R. Rajamahendran’, C.Y.G. Lee’ Departments of ‘Animal Science and Obstetrics & Gynecology University of British Columbia, Vancouver, B.C. Canada V6T 124
Simple yet reliable techniques for assessing cryodamage in frozen thawed bull spermatozoa are unavailable. Our lab has generated 11 anti-bull sperm monoclonal antibodies (mAbs) that recognize specific intra-acrosomal antigens (Ambrose etal., 1994; Biol. Reprod. 50 suppl. 1:2OO). Preliminary investigations revealed that these mAbs cannot access their cognate antigens in spermatozoa with intact membranes, but can in membrane damaged spermatozoa. Since it is known that sperm membrane integrity is affected during the freeze-thaw process associated with cryopreservation, we hypothesize that our mAbs may bind to spermatozoa with compromised membrane integrity. Therefore, this study was undertaken to evaluate the mAbs IBS-1, IBS-5, and IBS-10 (selected for their unambiguous binding pattern) as markers to assess such cryodamage. Fresh and frozen semen samples from the same ejaculate from each of five Holstein bulls were tested in duplicate for mAb binding. Washed spermatozoa were incubated in phosphate buffered saline + 5% BSA (5-106 sperm/ml) with each of the three mAbs for 30 min at 39 degrees C, 5% CO*, and processed for an indirect immunofluorescence assay. Fluorescent isothiocyanate conjugated goat anti-mouse IgG+IgM was used as the Samples were scored for fluorescence (mAb binding) by two second antibody. individuals, each counting approximately 300 spermatozoa in randomly selected fields. Ail three mAbs showed significant (PcO.05) increases in their binding to the frozenthawed as opposed to fresh spermatozoa in four of the five bulls tested. The results for one of the mAbs, IBS-1, are given. Table 1. Results of mAb IBS-1 binding to fresh Vs. frozen-thawed spermatozoa. Bull
% mAb binding to fresh spermatozoa
% mAb binding to frozenthawed spermatozoa
1
8.6 + 2.5
16.7 It 0.6
2
22.2 f 2.8
28.0 k 0.8
12.4 + 0.8
30.4 f 5.3
4
16.4 + 1.7
25.4 + 2.1
5
15.6 f 0.8
28.6 f 2.7
There is evidence of binding differences between bulls. The results of this preliminary study suggest that these mAbs could be useful markers to assess cryodamage in bull spermatozoa. Further studies are underway.
Theriogenology AN INDIRECT IMMUNOFLUORESCENCE ASSAY TO ASSESS SPERM CRYODAMAGE USING ANTI-BULL SPERM MONOCLONAL ANTIBODIES L.A. Johnson’, J.D. Ambrose’, R. Rajamahendran’, C.Y.G. Lee’ Departments of ‘Animal Science and Obstetrics & Gynecology University of British Columbia, Vancouver, B.C. Canada V6T 124
Simple yet reliable techniques for assessing cryodamage in frozen thawed bull spermatozoa are unavailable. Our lab has generated 11 anti-bull sperm monoclonal antibodies (mAbs) that recognize specific intra-acrosomal antigens (Ambrose etal., 1994; Biol. Reprod. 50 suppl. 1:2OO). Preliminary investigations revealed that these mAbs cannot access their cognate antigens in spermatozoa with intact membranes, but can in membrane damaged spermatozoa. Since it is known that sperm membrane integrity is affected during the freeze-thaw process associated with cryopreservation, we hypothesize that our mAbs may bind to spermatozoa with compromised membrane integrity. Therefore, this study was undertaken to evaluate the mAbs IBS-1, IBS-5, and IBS-10 (selected for their unambiguous binding pattern) as markers to assess such cryodamage. Fresh and frozen semen samples from the same ejaculate from each of five Holstein bulls were tested in duplicate for mAb binding. Washed spermatozoa were incubated in phosphate buffered saline + 5% BSA (5-106 sperm/ml) with each of the three mAbs for 30 min at 39 degrees C, 5% CO*, and processed for an indirect immunofluorescence assay. Fluorescent isothiocyanate conjugated goat anti-mouse IgG+IgM was used as the Samples were scored for fluorescence (mAb binding) by two second antibody. individuals, each counting approximately 300 spermatozoa in randomly selected fields. Ail three mAbs showed significant (PcO.05) increases in their binding to the frozenthawed as opposed to fresh spermatozoa in four of the five bulls tested. The results for one of the mAbs, IBS-1, are given. Table 1. Results of mAb IBS-1 binding to fresh Vs. frozen-thawed spermatozoa. Bull
% mAb binding to fresh spermatozoa
% mAb binding to frozenthawed spermatozoa
1
8.6 + 2.5
16.7 It 0.6
2
22.2 f 2.8
28.0 k 0.8
12.4 + 0.8
30.4 f 5.3
4
16.4 + 1.7
25.4 + 2.1
5
15.6 f 0.8
28.6 f 2.7
There is evidence of binding differences between bulls. The results of this preliminary study suggest that these mAbs could be useful markers to assess cryodamage in bull spermatozoa. Further studies are underway.