Studies on in vitro production of equine embryos by Intracytoplasmic Sperm Injection (ICSI) using non-sorted, or sex-sorted, frozen/thawed stallion sperm: Effects on post-thaw sperm quality, cleavage and blastocyst rates, and characterization of cellular events during the first 24 hours post-fertilization via confocal microscopy.

Overview

• This study examines the impact of using non-sorted (NS) versus sex-sorted (SS) frozen/thawed stallion sperm on sperm quality, embryo development rates after Intracytoplasmic Sperm Injection (ICSI), and cellular events during the first 24 hours after fertilization in horses.

Objective

• To compare the post-thaw sperm quality between NS and SS frozen/thawed stallion sperm.
• To evaluate the effects of sperm sorting on fertilization success, cleavage, and blastocyst formation rates after ICSI.
• To characterize the early cellular events post-fertilization using confocal microscopy.

Background

• ICSI is a common assisted reproductive technique used in equine breeding utilizing frozen/thawed stallion sperm.
• Sex-sorted sperm traditionally yield lower embryo cleavage and blastocyst formation rates compared to non-sorted sperm.
• A new sex-sorting technology (Genesis III) has been recently commercialized and requires evaluation for performance.

Experiment 1: Post-Thaw Sperm Quality Comparison

• Comparison between NS-F/T and SS-F/T stallion sperm using Genesis III sorting:
• Sperm motility was significantly higher in NS-F/T (41%) than SS-F/T (12%).
• Sperm viability rates were similar between NS-F/T (54%) and SS-F/T (57%).
• DNA integrity, assessed by Sperm Chromatin Structure Assay (SCSA), showed a higher percentage of sperm with DNA susceptible to denaturation in SS-F/T (80%) compared to NS-F/T (5%).

Experiment 2: Embryo Development Assessment After ICSI

• Using the same sperm groups, cleavage and blastocyst formation rates were evaluated post-ICSI:
• Cleavage rates were higher for NS-F/T sperm (71%) compared to SS-F/T sperm (48%).
• Blastocyst rates per injected oocyte were greater in NS-F/T (26%) than SS-F/T (15%).
• Blastocyst rates per cleaved embryos were similar between groups (NS-F/T 37% vs. SS-F/T 32%).
• Blastocyst development timing (days 7 to 10 post-ICSI) showed no significant differences between NS and SS groups.
• The average number of blastocysts produced per ICSI cycle was comparable (NS-F/T 1.4 vs. SS-F/T 0.7).

Experiment 3: Early Cellular Events Post-ICSI

• The initial 24 hours after fertilization were examined through laser confocal microscopy to study:
• Sperm DNA decondensation.
• Sperm aster formation (microtubule organization important for pronuclei development).
• Pronuclei formation and apposition, metaphase plate formation, and mitotic division timing.
• Results showed similar progression for oocytes fertilized with either NS-F/T or SS-F/T sperm:
• 50-80% had pronuclear formation within 8 hours post-ICSI.
• Pronuclear apposition or metaphase plate formation occurred by 12-18 hours.
• First mitotic division observed by 24 hours.

Conclusions and Implications

• NS-F/T sperm exhibit better post-thaw motility and DNA integrity than SS-F/T sperm using the new Genesis III technology.
• The higher DNA denaturation susceptibility correlates with reduced cleavage and blastocyst rates when using sex-sorted sperm, indicating possible compromised sperm quality despite similar viability.
• Embryos from both sperm types that did cleave have similar developmental competence to blastocysts and time to reach key developmental milestones.
• The study provides important insights into the limitations of sex-sorted sperm in equine ICSI and identification of critical early fertilization events, guiding improvements in assisted reproduction techniques.