Fertility of sorted sperm has been low compared to unsorted control sperm, due partly to mechanical damage during sperm sorting by flow cytometry. Lowering system pressure improved both sperm quality and fertility in IVF. The present study evaluated the effect of system pressure during sperm sorting and extended maturation of oocytes on development of embryos after ICSI. Sperm from each of three bulls were stained with 125μM Hoechst 33342 for 45min at 34°C, sorted into X- and Y-chromosome bearing populations at 95% accuracy with the pressure of SX MoFlo® sorters at 40 or 50psi, and then cryopreserved. Fifty bovine oocytes obtained from slaughterhouse ovaries were placed per well with 1mL of CDM1 supplemented with 0.5% FAF-BSA, 2mM glucose, 50ngmL−1 EGV, 15ngmL−1 NIDDK-0FSH-20, 1μgmL−1 USDA-LH-B-5, 1μgmL−1 E2 and 0.1mM cysteamine, and then matured for 24 or 30h at 38.5°C, 5% CO2 in air. Cumulus cells of matured oocytes were removed by vortexing, and oocytes with a polar body were selected. Motile sperm from sorted frozen-thawed semen were recovered by centrifugation through 2mL each of 45 and 90% Percoll, and the concentration was adjusted to 4×106mL−1. Matured oocytes were divided into two injection groups, ICSI and sham injection, using a Piezo injection system. The outer diameter of the sperm injection pipette was 8–10μm. All manipulations were performed at room temperature (24–25°C). After injection, oocytes were activated with 5μM ionomycin for 4min, cultured in 50μL of CDM1 at 38.5°C under 5% CO2, 5% O2 and 90% N2, and assessed for degeneration/cleavage at 72h post-injection. Uncleaved oocytes from ICSI and sham-injected groups were stained with orcein and evaluated for fertilization status. Cleaved embryos were further cultured, and blastocyst development was evaluated on Day 7.5 after injection. Data were subjected to ANOVA;; the arcsin transformation was used for percentage data, and main effect means are presented. With 24h matured oocytes, there were no differences (P>0.1) between sperm sorted at 40v. 50psi for degeneration, cleavage or blastocyst rates, nor was there pressure×bull interaction. There were significant effects of bulls for all responses studied (P<0.05). When injected with sperm sorted at 40psi, oocytes matured for 30h resulted in higher cleavage and lower degeneration rates than 24h-matured oocytes (22.9 and 13.7% v. 12.2 and 22.0%, respectively, P<0.05), with no difference (P>0.1) in blastocyst rate. Overall blastocyst development was higher in ICSI than in sham injection (7.5 v. 1.3%, P<0.05). When uncleaved oocytes from 24h maturation were evaluated for fertilization status, ICSI showed a higher percentage with two polar bodies and/or decondensed sperm compared to sham injection (15.7 v. 1.7%, P<0.05). With 30h matured oocytes there was no difference in fertilization status between those groups. We conclude that there was no difference in cleavage or development to blastocysts after ICSI using motile sperm that had been sorted at 40 or 50psi.
334INTRACYTOPLASMIC SPERM INJECTION (ICSI) OF BOVINE OOCYTES WITH SEXED
SPERM SORTED AT DIFFERENT PRESSURES
