Semen parameters and fertility of cooled stallion semen extended with sodium caseinate and phosphocaseinate based extenders

Aim was to compare 2 commercially available equine semen extenders based on phosphocaseinate (INRA 96 IMV, US) and sodium caseinate associated with cholesterol-cyclodextrin (Botugold, Botupharma, US), on sperm parameters and fertility of stallion semen after cooling. We hypothesized that use of a sodium caseinate cholesterol-enriched extender and seminal plasma removal would result in superior parameters in cooled stallion semen. In Experiment 1, 45 ejaculates collected from 9 mature stallions were extended to 50 x 106 /ml (SP group) with INRA96 or Botugold and processed through cushion centrifugation (CC group) (1000 g x 20 minutes) before resuspension at 100 x 106 /ml with respective extender. Noncentrifuged and CC samples were placed in 3 (Equitainer, Botuflex, and Equine-Express II) passive cooling devices for 24 or 48 hours. Seminal parameters assessed at 0, 24, and 48 hours, included total motility (TM), progressive motility (PM) with CASA (Spermvision, MOFA, Verona, WI), and membrane integrity and mitochondrial membrane potential with spectral flow cytometry (Zombie Green and Mitotracker Deep Red). In experiment 2, mares (n = 12 x 2 estrous cycles) were bred with 1 billion total sperm from 1 fertile stallion, 40 hours postinduction of ovulation with gonadotrophin releasing hormone agonist, histrelin. Semen was collected, extended in INRA96 and Botugold, and stored for 48 hours in Equitainer, and mares were randomly bred with 1 extender in an alternate order. Mares had transcervical embryo flushing performed on day 8 postovulation. Data were analyzed using mixed models (R project) and Tukey’s as posthoc. Addition of cholesterol to sodium caseinate extender, resulted in superior (p < 0.05) semen parameters. There were no differences between container type. Cushion centrifugation increased (p < 0.05) sperm kinetics parameters and mitochondrial membrane potential and not (p > 0.05) plasma membrane integrity (Table). In Experiment 2, embryo recovery rates were identical (p > 0.05) between extenders (50%). In conclusion, we inferred that novel extender (Botugold) is a suitable commercially available product that can be used for stallion semen cooling with different containers, with superior sperm parameters than traditional INRA96 and at least with comparable fertility.

Keywords: Stallion, semen, extender; cryopreservation, andrology

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.