334 INTRACYTOPLASMIC SPERM INJECTION (ICSI) OF BOVINE OOCYTES WITH SEXED SPERM SORTED AT DIFFERENT PRESSURES

Abstract

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 125 μM Hoechst 33342 for 45 min at 34°C, sorted into X- and Y-chromosome bearing populations at 95% accuracy with the pressure of SX MoFlo^® sorters at 40 or 50 psi, and then cryopreserved. Fifty bovine oocytes obtained from slaughterhouse ovaries were placed per well with 1 mL of CDM1 supplemented with 0.5% FAF-BSA, 2 mM glucose, 50 ng mL⁻¹ EGV, 15 ng mL⁻¹ NIDDK-0FSH-20, 1 μg mL⁻¹ USDA-LH-B-5, 1 μg mL⁻¹ E2 and 0.1 mM cysteamine, and then matured for 24 or 30 h 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 2 mL each of 45 and 90% Percoll, and the concentration was adjusted to 4 × 10⁶ mL⁻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 8–10 μm. All manipulations were performed at room temperature (24–25°C). After injection, oocytes were activated with 5 μM ionomycin for 4 min, cultured in 50 μL of CDM1 at 38.5°C under 5% CO₂, 5% O₂ and 90% N₂, and assessed for degeneration/cleavage at 72 h 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 24 h matured oocytes, there were no differences (P > 0.1) between sperm sorted at 40 v. 50 psi 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 40 psi, oocytes matured for 30 h resulted in higher cleavage and lower degeneration rates than 24 h-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 24 h 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 30 h 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 50 psi.