Blastocyst production by conventional in vitro fertilization (cIVF) in horses: Effects of sperm storage method, incubation timing of cool-stored semen before gamete co-incubation, and comparisons between cIVF and intracytoplasmic sperm injection (ICSI).

Abstract: Currently, Intracytoplasmic Sperm Injection (ICSI) on in vitro-matured equine oocytes obtained by transvaginal oocyte aspiration (TVA) is the method of choice for in vitro production of equine blastocysts in a clinical setting. A protocol for conventional in vitro fertilization (cIVF) using either fresh or frozen/thawed stallion sperm incubated under capacitating conditions (Tyrode's Albumin Lactate Pyruvate medium + penicillamine, hypotaurine, epinephrine; FERT-PHE) has been recently reported. Several factors that may impact the clinical applicability of this cIVF protocol have yet to be studied. In Experiment 1, a comparison between fresh, cool-stored, or frozen/thawed sperm from a fertile stallion (OP) for cIVF was performed. Differences on cleavage rate (embryos with ≥8 blastomeres at day 5 of culture), blastocyst rate (blastocysts/cleaved embryos), and the number of blastocysts per mare TVA/cIVF cycle were not detected among fresh (68 % [38/56]; 18 % [7/38]; 1), cool-stored (61 % [34/56]; 21 % [7/34]; 1), and frozen/thawed sperm (54 % [29/54]; 21 % [6/29]; 1), respectively (P > 0.05). In Experiment 2, the effect of incubation time (22 vs. 10 h) that cool-stored sperm were exposed to the FERT-PHE medium before gamete co-incubation was studied. Differences on cleavage rate (45 % [29/64] vs. 56 % [47/84]), blastocyst rate/cleaved embryos (28 % [8/29] vs. 19 % [9/47]), and number of blastocysts per mare TVA/cIVF cycle (1.1 vs. 1.3) were not detected between treatment groups (P > 0.05). In Experiment 3, frozen/thawed sperm from 2 fertile stallions (OP vs. HW) was processed to compare cIVF and ICSI cycles. Differences on cleavage rates were not detected between stallions in cIVF (OP: 41 % [16/39] vs. HW: 52 % [34/65]; P > 0.05) or ICSI cycles (OP: 34 % [22/64] vs. HW: 53 % [19/36]; P > 0.05); nor detected when the stallion values were combined: cIVF (48 % [50/104]) vs. ICSI (41 % [41/100]; P > 0.05). Differences on blastocyst rate/cleaved embryos were not detected between stallions in cIVF (OP: 13 % [2/16] vs. HW: 26 % [9/34]) or ICSI cycles (OP: 45 % [10/22] vs. HW 42 % [8/19]; P > 0.05); but when stallion values were combined, blastocyst rate/cleaved embryos was lower for cIVF (22 % [11/50]) than for ICSI (44 % [18/41]) cycles (P < 0.05). The number of blastocysts per mare TVA cycle was lower for stallion OP in cIVF (0.4) than for stallion HW in ICSI (2; P < 0.05), while similar for stallions OP and HW in cIVF cycles (1.5), and stallion OP in ICSI cycles (1.4; P > 0.05). Six blastocysts from cIVF (n = 3) or ICSI (n = 3) cycles produced with frozen/thawed sperm from stallion HW were vitrified, warmed, and transferred into recipient mares. All mares were confirmed pregnant at days 14, 28, and 45 of gestation. In conclusion, in this study, the production of cIVF-derived blastocysts was similar for fresh, cool-stored, or frozen/thawed sperm from a fertile stallion. Reducing the sperm incubation timing in FERT-PHE from 22 to 10 h did not affect blastocyst production by cIVF using cool-stored sperm. Finally, in this study, an overall lower blastocyst rate per cleaved embryo was observed in two fertile stallions when utilizing frozen/thawed sperm for cIVF compared to ICSI. Further studies are warranted to understand additional factors that may affect the production of equine blastocysts by cIVF in a clinical setting.