Holding effect in a commercial OPU-ICSI program: a retrospective study

Abstract

In vitro embryo production has been rapidly evolved in horses, since the immature oocytes could be viably held overnight and shipped for intracytoplasmic sperm injection (ICSI). However, in facilities where ovum pick-up (OPU) and ICSI are performed in situ, direct in vitro maturation (IVM) can be performed. In a recent study, holding of oocytes followed by extended IVM (36h) resulted in increased embryo development compared to direct IVM for 26 to 28h. Nevertheless, the effect of holding was not evaluated separately in a clinical ovum pick-up (OPU)-ICSI program. Therefore, the current study aimed to determine whether holding oocytes prior IVM could affect their developmental competence. Data from 253 OPU-ICSI sessions at Ghent University (136 mares and 54 stallions) were analysed retrospectively. Collected oocytes were submitted to direct IVM (DM:1152) (TCM-199 Earl's salts (Gibco) containing 10% (v/v) FBS (Gibco), 9.4 µg/mL follicle-stimulating hormone, and 1.88 µg/mL luteinizing hormone (Stimufol, Reprobiol, Ouffet, Belgium)) or kept in commercial holding medium (HM:1371) (Emcare, Agtech, Zulte, Belgium) at room temperature for 17–24 h in a 5 mL Falcon® Test Tube (Corning Europe, Lasne, Belgium) prior to IVM. After an average of 28.4 h of IVM (DM: 28.2; HM: 28.6), oocytes with a polar body werefertilized by piezo-drill ICSI. The zygotes were cultured in DMEM/F-12 (Gibco) with 10% (v/v) FBS under oil (38.2°C, 5% O 2 , 5% CO 2 , and 90% N 2 ). Cleavage was evaluated 2 or 3 days after ICSI, and blastocyst development was monitored daily from day six up to day 13. For statistical analysis, generalized linear model were used to test the effect of holding on maturation, cleavage, and blastocyst rate. Always, the oocyte/ zygote was considered as the unit of interest, and results are expressed as least square means ± standard error. No significant differences were observed between treatments for maturation (DM: 64.9±1.4%; HM: 67.0±1.3; p=0.3), cleavage (DM: 62.7±1.9%; HM: 66.8±1.6; p=0.1), and blastocyst rates (DM: 19.0±1.4%; HM: 17.0±1.2; p=0.3). Likewise, comparable kinetics of embryo development were obtained with 9.08 days in average for DM and 9.33 for HM (p = 0.08). In conclusion, oocyte holding prior to IVM did not affect developmental competence when an average IVM duration of 28h was maintained. Therefore, the observed increased embryo development rates in HM vs DM reported previously primarily resulted from an extended maturation time rather than from the inclusion of an oocyte holding step. Other variables, such as the type of sperm, the effect of the mare, the time of the year could be included in further analyses.