318 EFFECT OF SPERM PRETREATMENT WITH ISOBUTYLMETHYLXANTHINE AND METHYL-β-CYCLODEXTRIN ON THE EFFICIENCY OF BOVINE INTRACYTOPLASMIC SPERM INJECTION

Abstract

The efficiency of intracytoplasmic sperm injection (ICSI) in bovines is lower than in other species. We propose that in vitro sperm capacitation could optimize the ICSI in cattle. The aim was to evaluate the effects of isobutylmethylxanthine (IBMX) and methyl-β-cyclodextrin (MβCD) on the sperm capacitation and in vitro development of embryos generated by ICSI. Frozen-thawed spermatozoa (3–5 × 10⁶ cells mL^–1) were pre-incubated for 2 h at 38.5°C, 5% CO₂ in defined medium (Sp-TLP/PVA) supplemented with MβCD (1 mM) or IBMX (0.4 mM) (capacitating conditions). The untreated control group (UTG; not supplemented) and vehicle group (VG) were incubated for 2 h. The non-capacitating control group (NCG) was not supplemented (neither vehicle nor IBMX or MβCD) and not incubated. The sperm viability and capacitation {intracellular calcium [Ca²⁺]_i, plasma membrane fluidity (PMF), and acrosomal reaction} were evaluated by flow cytometry (n = 3 biological replicates). For the ICSI procedure, only motile spermatozoa were selected. After ICSI, oocytes were activated with ionomycin + cycloheximide. Culture was performed at 38.5°C, 5% CO₂, 5% O₂, 90% N₂, saturation humidity in KSOM base medium. Data were analysed by ANOVA and Scheffe's test. Pronuclear formation was evaluated by a chi-square test with Bonferroni's correction. Significance was set at P < 0.05. Pretreated spermatozoa showed lower (P < 0.05) viability (49 and 67% for IBMX and MβCD, respectively) compared with the NCG (89%), UTG (80%), and VG (78%). The [Ca²⁺]_I analysed by median fluorescence intensity (MFI) was lower (P < 0.05) in NCG (117 MFI) with respect to UTG (127 MFI), VG (124 MFI), IBMX (126 MFI), and MβCD (131 MFI). The PMF increased (P < 0.05) with IBMX (115 MFI) and MβCD (106 MFI) compared with NCG (70 MFI), UTG (89 MFI), and VG (65 MFI). Acrosome reaction improved with capacitating treatments with respect to both control groups (16, 23, 8, 4, and 3% for IBMX, MβCD, UTG, VG, and NCG, respectively). Analysis of capacitating v. non-capacitating conditions on ICSI efficiency revealed that the fertilization rate, assessed by pronuclear formation, was higher (P < 0.05) in ICSI-MβCD (76%; n = 46) compared with ICSI-IBMX (55%; n = 53) and ICSI-NCG (50%; n = 44). Nevertheless, there were no differences among groups in cleavage (Day 3): 85, 86, and 84% and blastocyst rates (Day 8): 19, 25, and 18% for ICSI-IBMX (n = 8), ICSI-MβCD (n = 7), and ICSI-NCG (n = 7), respectively. The parthenogenetic and sham injection groups yielded a lower rate of cleavage (73 and 53%, respectively) and blastocyst (13% and 10%, respectively). The results demonstrated an improvement of the fertilization rate of bovine embryos generated by ICSI using sperm capacitated by MβCD pretreatment. However, more studies are necessary to improve in vitro developmental potential of these embryos to the blastocyst stage.

Frigorífico Temuco and funding support from FONDECYT 1120241 CONICYT-Chile are gratefully acknowledged.