Sperm-bound antisperm antibodies are associated with poor cryosurvival of stallion sperm

Semen freezing is a common practice in equine reproduction. However, ~ 30 - 40% stallions’ semen does not survive cryopreservation. Reason for poor cryosurvival is not clearly understood. Antisperm antibodies (ASAs) are associated with poor cooling ability of stallion sperm. We hypothesized that presence of sperm-bound ASAs is associated with poor cryosurvival. Objective of this study was to evaluate ASA binding in stallions with semen with good versus poor cryosurvival. Ejaculates from stallions (n = 21) were extended in INRA96 to 40 x 10 6 sperm/ml and shipped overnight to laboratory in a passive cooling device (Equitainer® ). After arrival, each ejaculate was divided into 3 aliquots, centrifuged, resuspended in Botucrio ® (BC; Botupharma, Scottsdale, AZ), EZ Mixin Cryomax Modified French™ (MFR; ARS, Chino, CA) or EZ Mixin Cryomax Lactose-EDTA™ (LE; ARS) and frozen following manufacturer’s instructions. Semen was stored in liquid nitrogen until evaluation. One straw from each aliquot was thawed at 38C for 30 seconds and was assessed for total and progressive sperm motility (CASA). In addition, acrosomal integrity (FITC-PNA/PI), percentage of apoptotic and necrotic sperm (Annexin V/PI), and percentage of sperm with IgG and IgA binding (antiequine IgG and IgA) were evaluated with flow cytometry. Postthaw motility was considered acceptable if PM  30%. Semen considered as good cryosurvival if they had acceptable postthaw motility with at least 2 extenders.

There was no difference in ASA binding among semen extenders and data were pooled. Semen with good cryosurvival (n = 13) had a lower percentage of IgG (4.1 ± 0.5%) and IgA bound sperm (2.9 ± 0.3%) compared to semen with poor cryosurvival (n = 8; IgG 13.5 ± 2.3%, IgA 11.2 ± 1.7%) (p < 0.0001; Student’s t-test). None of the semen with good cryosurvival were ASA positive. However, 43.5% of semen with poor cryosurvival were ASA positive (p < 0.0001, Chi Square). There was a negative correlation between percentage of IgG and IgA bound sperm, and total motility (IgG: p = 0.0045,

R 2 = -0.36; IgA: p = 0.0003, R 2 = -0.45) and progressive motility (IgG: p = 0.0005, R 2 = -0.44;

IgA: p < 0.0001, R 2 = -0.49), and a positive correlation between percentage of IgG bound sperm and percentage of sperm with damaged acrosomes (IgG: p = 0.049, R 2 = -0.36). In summary, presence of ASAs was associated with poor cryosurvival of stallion sperm.

Keywords: Stallion, semen, antisperm antibodies, cryopreservation, freezing

This manuscript was originally published in the journal Clinical Theriogenology Vol 12(3) Sept 2020.  Clinical Theriogenology is the official journal of the Society for Theriogenology (SFT) and the American College of Theriogenologists (ACT).  This content has been reproduced on the IVIS website with the explicit permission of the SFT/ACT.