Intracytoplasmic sperm injection (ICSI) has been a valuable tool in many species because of its ability to overcome male factor infertility problems and eliminate risk of polyspermy; more recently, it been used to improve genome editing technologies. However, limited success with bovine ICSI has hindered these applications in cattle. Numerous treatments have been used to increase the success rate, with marginal improvement. Replicating events synonymous with fertilization, such as the acrosome reaction, may improve fertilization with bovine ICSI. Progesterone (P4) is naturally found in both the cumulus cells surrounding the oocyte and follicular fluid released at ovulation and activates a physiological pathway within sperm to induce an acrosome reaction, a crucial process in fertilization. Progesterone induction of the acrosome reaction as a sperm pretreatment for ICSI has not yet been evaluated in cattle. In this study, frozen–thawed bovine sperm was used. Sperm were first thawed, washed, and separated via gradient to obtain the motile population before capacitation with heparin over 4h before treatment with or without P4 (10 μM) for 15min. To measure the acrosome reacting population, sperm were stained with fluorescein isothiocyanate-conjugated peanut agglutinin (FITC-PNA) and observed over 2h using cytometric flow analysis and fluorescent microscopy to measure the population undergoing and completing an acrosome reaction. For ICSI, a total of 220 oocytes were used, and both treated and nontreated sperm were incubated for 1h before injection to allow for completion of an acrosome reaction. Fertilization rates were measured by observing pronuclear formation and an absence of sperm 18h after ICSI. Means of acrosome reacting population gathered by flow cytometry and microscopy were analysed by ANOVA. Differences in fertilization rates between groups post ICSI were analysed using a Yates’ corrected chi-squared test. A significantly higher proportion of P4 treated versus control sperm initiated an acrosome reaction at hour 1 (80.2%±4.2 vs. 19%±9.1), which increased to (89.3%±3.9 vs. 29%±8.3) after 2h. P4 also increased the percentage of sperm that completed an acrosome reaction, from 50% (±5.1) at hour 1 and 62.5% (±7.4) at hour 2. Only 14.2% (±3.6) completed acrosome reactions in sperm not treated with P4 by hour 2. Motile sperm in both groups did not decrease over the 2-h incubation time period (P<0.05). Progesterone treatment increased the percentage of fertilized embryos after ICSI, with 38.1% fertilized compared with 10.1% with heparin-treated control injections (P<0.001). These results show that P4 has effects on bovine sperm that allow for higher rates of fertilization after ICSI by utilising the sperm’s physiological response to progesterone. Further embryo development using ICSI with P4-treated sperm, or additional physiologically similar treatments, should continue to be assessed.
EXCESSIVE TRIMMING OF CUMULUS CELLS IS ASSOCIATED WITH LOWER FERTILIZATION RATES AFTER CONVENTIONAL INSEMINATION
