Sperm factors associated with the production of equine blastocysts by intracytoplasmic sperm injection (ICSI) using frozen/thawed semen.

Abstract: Intracytoplasmic Sperm Injection (ICSI) using frozen/thawed sperm is a common procedure to obtain embryos from fertile or subfertile mares and stallions. Stallion-associated factors that impact the efficiency of ICSI have been studied less than those associated with the mare. Three experiments were conducted: Experiment 1: the effect of freezing extender composition and cryoprotectant; Experiment 2: the effect of sperm exposure to seminal plasma prior to freezing (ejaculated vs. epididymal sperm; two-freeze/thaw cycles each); and Experiment 3: the effect of sperm morphologic feature used for fertilization (normal vs. cytoplasmic droplet vs. bent tail); on the blastocyst rate after ICSI. In Experiment 1, stallion sperm was cryopreserved using commercially available extenders containing: a) 2% egg-yolk + milk + 4% glycerol (MFR5); b) 2% egg-yolk + milk + 2% glycerol + 3% methyl formamide (CMMFR5); c) 20% egg-yolk + 4.75% glycerol (LE); or d) 20% egg-yolk + 2% glycerol + 3% methyl formamide (CMLE). Sperm from each of the treatment groups were used for Piezo-driven ICSI on in vitro-matured equine oocytes (n = 321). Extender CMLE resulted in a lower cleavage rate (35%) than the other treatment groups (MFR5: 74%, CMMFR5: 62%, LE: 68%; P < 0.05). Extender MFR5 yielded a higher blastocyst rate per injected oocyte (21/82 [26%]) than the Groups LE (8/77 [10%]), CMLE (4/80 [5%]) or CMMFR5 (4/82 [5%]; P < 0.05). Extender MFR5 also yielded a higher blastocyst rate per cleaved oocyte (34%) than Groups LE, CMLE or CMMFR5 (15%, 14%, 8%; respectively P < 0.05). In Experiment 2, ejaculated (EJ) and epididymal (EPD) sperm from a fertile stallion which was initially cryopreserved in the CMLE extender, was thawed and re-cryopreserved in MFR5 extender for use in ICSI. Sperm from both groups (EJ vs. EPD) were used for ICSI on in vitro matured oocytes (n = 127). Differences were not detected for cleavage rate (EJ: 36/63 [57%] vs. EPD: 49/64 [77%]), blastocyst rate per injected oocyte (EJ: 11/63 [17%] vs. EPD: 11/64 [17%]), or blastocyst rate per cleaved oocyte (EJ: 31% vs. EPD: 22%) between treatment groups (P > 0.05). In Experiment 3, morphologically normal sperm (N), or sperm with proximal droplets (PD) or bent tails (BT), were obtained from a single fertile stallion and were used for ICSI on in vitro matured oocytes (n = 75). No differences were detected among treatment groups for cleavage rate (N: 19/25 [77%] vs. PD: 20/25 [88%] vs. BT: 18/25 [72%]), blastocyst rate per injected oocyte (N: 6/25 [24%] vs. PD: 5/25 [20%] vs. BT: 2/25 [8%]), and blastocyst rate per cleaved oocyte (N: 32% vs. PD: 23% vs. BT: 11%; P > 0.05). In conclusion, the current study indicates that freezing extender composition used for stallion sperm cryopreservation has an impact on the developmental competence of in vitro-matured equine oocytes after ICSI and in vitro culture. Furthermore, we were unable to detect differences on cleavage and blastocyst rates when performing ICSI when using: 1) ejaculated or epididymal sperm; or 2) sperm with different morphologic features. The results from the current study provide additional insight regarding stallion-related factors that should be considered when performing ICSI in horses.