The relationship between post-thaw sperm quality parameters and blastocyst production following Intracytoplasmic Sperm Injection (ICSI) of in vitro-matured equine oocytes.

Abstract: In vitro production of equine embryos by Intracytoplasmic Sperm Injection (ICSI) is commonly utilized to maximize the availability of stallion sperm, particularly from stallions with limited semen supply. The relationship between standard measures of sperm quality in frozen/thawed semen (i.e., sperm motility, normal morphology, DNA quality) and embryo production after ICSI of in vitro-matured equine oocytes has not been thoroughly studied. In this study, frozen/thawed semen from 47 stallions utilized in a commercial ICSI program was analyzed for post-thaw sperm total and progressive motility (determined both subjectively and by Computer-Assisted Sperm Analysis [CASA]), morphology features (Differential Interference Contrast [DIC] microscopy), and DNA damage (Sperm Chromatin Structure Assay - SCSA) before and after sperm selection by swim-up. Morphologically normal-motile sperm were selected following swim-up and injected by Piezo-driven ICSI into 485 in vitro-matured equine oocytes from 58 mares (85 cycles). The relationship between sperm quality characteristics (before and after swim-up) and cleavage (>8 blastomeres at day 5 post-ICSI), or blastocyst development (from day 7-10 post-ICSI), was investigated using a backward stepwise logistic regression analysis. No sperm quality parameter was associated with cleavage (P > 0.05). Post-thaw sperm quality parameters related to blastocyst production included mean (±SD) total motility (TMOT) before swim-up, determined subjectively (34 ± 13 %; odds ratio [OR]: 1.20, 95 % confidence interval [CI]: 1.02-1.42), or by CASA (29 ± 13 %; OR: 1.08, CI: 1.01-1.28); and morphologically normal (NORMAL) sperm before swim-up (57 ± 11 %; OR: 1.31, CI: 1.10-1.57). In contrast, before swim-up, mean (±SD) proximal droplets (5 ± 4 %; OR: 0.63, CI: 0.41-0.94), coiled tails (2 ± 1 %; OR: 0.25, CI: 0.08-0.63), and COMPαt (16 ± 8 %; OR: 0.74, CI: 0.57-0.96) were negatively associated with blastocyst production (P < 0.05). Threshold values for post-thaw TMOT (determined subjectively or by CASA) and NORMAL, before swim-up, that were associated with a higher likelihood of blastocyst production were 44 % and 56 %, respectively. In addition to these sperm quality parameters, the number of injected oocytes, but no mare age nor number of recovered cumulus-oocyte-complexes (COCs), was associated with blastocyst production (OR: 2.76, CI: 1.43-5.31), while none of these parameters were associated with cleavage (P > 0.05). The current study provides clinically relevant data on post-thaw sperm quality measures that can be assessed to determine the fertility potential of frozen-thawed stallion sperm for ICSI.