Centrifugation through a low density colloid as an alternative to antibiotics in semen extenders: a controlled insemination trial

Abstract

Antibiotics are added to semen extenders to inhibit seminal bacterial growth, but this non-therapeutic use of antibiotics goes against recommendations for prudent antibiotic stewardship. Colloid centrifugation could provide an alternative method for reducing the bacterial load in stallion semen. Previous studies have shown that centrifugation through a low density colloid reduces the bacterial count of stallion semen considerably and sperm quality was not adversely affected by the procedure (Malaluang et al. Theriogenology 2024;216:111-117. The objective of the current study was to assess the fertilising capacity of stallion sperm samples prepared by Single Layer Centrifugation through Equicoll in an artificial insemination (AI) trial. Semen samples were collected from 4 stallions and pooled. The pool was divided into two portions. One portion was prepared as standard AI doses in EquiPlus with antibiotics, with 200 million sperm per dose, while the other half was extended in EquiPlus without antibiotics for preparation by centrifugation through a low density colloid. For the latter, 20 mL extended semen at a sperm concentration of 100 million/mL was layered over 15 mL 20% Equicoll in a 50 mL tube. After centrifuging for 20 min at 300 RCF, the supernatant was removed and the sperm pellet resuspended in EquiPlus without antibiotics to provide 200 million spermatozoa per insemination dose. The ovaries of pony mares were examined per rectum. When a follicle of more than 35 mm diameter was seen on ultrasound, 1000 iu hCG (Chorulon) was administered to induce ovulation and the mares were inseminated 24 h later. The mares were randomly assigned to two groups, one group (23 mares) being inseminated with the control doses (with antibiotics) and the other group (23 mares) receiving doses without antibiotics. Mares were scanned after a further 14 days and the presence of an embryonic vesicle in the uterus was noted. The results were as follows: an embryonic vesicle was detected in 19 of 23 control mares and 17 of the 23 mares receiving the colloid centrifuged samples. No mares developed a uterine infection. Since the outcome of the inseminations was similar in both groups, we can conclude that centrifugation through a low density colloid could replace antibiotics in semen extenders without having a detrimental effect on inseminated mares.

Thanks to the Linnea and Axel Ericsson Foundation, Stockholm, (DL) and Fonds Spéciaux de la Recherche 2024, Veterinary Faculty of Liege (SD) for financial support.