354 POOR EMBRYO DEVELOPMENT AFTER ICSI WITH DOMESTIC CAT TESTICULAR SPERM IS OVERCOME BY CENTROSOME AND MIDPIECE REPLACEMENT

Abstract

Testicular sperm from the cat can be used via intracytoplasmic sperm injections (ICSI) to produce embryos in vitro, but the proportion of morulae and blastocysts is less than that obtained using ejaculated sperm. Compromised embryo development has been linked to the inability of the cat testicular sperm centrosome to form a normal sperm aster during the first cell cycle post-ICSI. The aim of the present study was to improve embryo development after ICSI with a testicular spermatozoon by centrosome/midpiece replacement from an ejaculated spermatozoon. Sperm suspensions used for ICSI (fresh testicular sperm from one adult testis vs. frozen-thawed sperm from one ejaculate in each replicate; four replicates) were sonicated for 3 s at setting 3 using a 60 Sonic Dismembrator (Fisher Scientific, Pittsburgh, PA, USA) to separate heads from centrosome/midpieces. Control immunostaining with monoclonal centrin antibodies revealed that centrosomes were attached to midpieces and were separated from sperm heads after sonication. A single sperm head (testicular, T_h; ejaculated, E_h) was carefully positioned proximal to a single centrosome/midpiece (testicular, T_c; ejaculated, E_c) before injection into an oocyte with a visible polar body (in each replicate, n = 18 oocytes injected with each T_hE_c, E_hE_c, E_hT_c, or T_hT_c combination; four replicates). Injected oocytes then were activated with 7% ethanol and cultured in vitro in Ham's F10 (38.5°C, 5% CO₂ in air). Percentages of first cleavages were recorded from 20 to 32 h post-activation (hpa). Nuclear status of uncleaved oocytes was evaluated at 48 hpa, and embryo development was assessed after 7 days of in vitro culture. Values were expressed as mean ± standard deviation and analyzed by ANOVA. None of the uncleaved oocytes were activated. Percentages of cleaved oocytes (relative to the total number of injected oocytes) were not different (range, 61-65%; P > 0.05) among combinations of sperm heads and centrosome/midpieces. The mean times of the first cleavage, however, were earlier (P < 0.05) after combinations with ejaculated centrosome/midpieces (T_hE_c, 25.1 ± 1.1 h; E_hE_c, 25.0 ± 0.9 h) than with testicular centrosome/midpieces (E_hT_c, 29.2 ± 0.8 h; T_hT_c, 29.3 ± 1.2 h). Percentages of morulae produced were higher (P < 0.05) after combinations with ejaculated centrosome/midpieces (T_hE_c, 15.3 ± 2.7%; E_hE_c, 16.7 ± 2.1%) than with testicular centrosome/midpieces (E_hT_c, 8.3 ± 3.1%; T_hT_c, 6.9 ± 2.2%). Likewise, percentages of blastocysts were higher (P < 0.05) after combinations with ejaculated centrosome/midpieces (T_hE_c, 12.5 ± 3.0%; E_hE_c, 13.9 ± 2.8%) than with testicular centrosome/midpieces (E_hT_c, 5.6 ± 3.3%; T_hT_c, 6.9 ± 2.9%). These results demonstrated that: (1) kinetics of the first cell cycle and the success of embryonic development were determined by centrosome/midpiece source; and (2) poor developmental potential of domestic cat testicular sperm could be circumvented by centrosome/midpiece replacement from an ejaculated spermatozoon.