212 Equine IVF and ICSI can benefit from microfluidics-based sperm selection

In vitro embryo production (IVEP) is used in horses worldwide. Traditionally, IVF in horses has been restricted to intracytoplasmic sperm injection (ICSI), although recent advances in conventional IVF bring new possibilities for the equine industry. Sperm selection is key in the success of both techniques, especially in ICSI, in which a sperm is manually selected by a person, eliminating natural selection. In IVF, protocols using frozen-thawed sperm are still under development, and sperm selection is critical as only high-quality gametes can survive the prolonged capacitation culture. The objective of this study was to evaluate the use of frozen-thawed sperm selection with microfluidics for equine IVP. Frozen semen from three stallions was selected using a microfluidics-based device (Zymot Multi 850 μL, Zymot Fertility, Inc.). Briefly, a 1/7 cut of a 0.5-mL straw was thawed and diluted in G-MOPS-10% fetal bovine serum, loaded in the microfluidic chamber, and incubated for 30 min (37°C, room air). Quality parameters such as kinetics (CASA), membrane functionality by hypo-osmotic swelling test, acrosome integrity, and sperm morphology with Spermac Stain Kit (Minitube International), mitochondrial membrane potential with JC-1 staining, and viability using eosin-nigrosin stain were assessed before (PR) and after (PT) sperm selection. A minimum of 200 cells were counted per test. Lastly, frozen-thawed sperm selected with microfluidics was used for IVEP in two separate trials (ICSI and IVF). Statistical analysis was performed using JMP Pro18. Total motility was significantly higher PT (34 ± 9% PR, 53 ± 12% PT; mean ± SED; P = 0.01) but not progressive motility (PM, 22 ± 5% PR, 37 ± 17% PT; P = 0.1). There was no statistical difference in membrane functionality, mitochondrial membrane potential, and sperm morphology between PR and PT assessments (P > 0.01). However, a higher proportion of acrosome intact sperm was observed in PT samples (38 ± 6% PR, 54 ± 12% PT; P = 0.02). Oocytes obtained from transvaginal aspiration were used for IVF (n = 18), as described by Felix et al. (2022 Biol. Reprod. 107, 1551–1564) and modified for frozen-thawed semen, and ICSI (n = 28), as described by Martin-Pelaez et al. (2023 Theriogenology 205, 1–8). Cleavage rate was 80% for IVF and 60% for ICSI. Blastocyst rates were 33% and 28.2% for IVF/ICSI, respectively. In our study, parameters such as motility and acrosome integrity were superior on samples after microfluidic selection. The integrity of acrosomal enzymes is paramount for adequate IVF, a parameter that might be of benefit for this selection method, as well as a superior motility. There was no significant difference in any other parameters measured, but the trend observed was consistent with current literature. This might be due to a stallion-specific influence or a low sample number, as we observed numerical differences in PR and PT samples, with a higher numerical value in PT samples. Further studies to evaluate these parameters are needed. Nonetheless, we showed that production of embryos using conventional IVF and ICSI is possible with frozen-thawed sperm selected by a commercially available microfluidics device.