Microbial composition of goat buck's ejaculates is modified by the process of preparing and storing refrigerated semen doses

Abstract

Ejaculates present their own microbiota, and a link between ejaculates' microbiota and sperm quality and fertility exists. With the development of artificial insemination in animal breeding, ejaculates must be manipulated by diluting them with extenders and storing them at temperatures below body temperature. The effects that these processes have on the original semen microbiota have never been studied. This study explores the effects of the protocol for preparing refrigerated goat buck semen doses and storing on seminal microbiota. Semen from six adult goat bucks of the Murciano-Granadina breed (24 ejaculates) was used, cooled to 4 °C in a skimmed milk-based extender, and stored at this temperature for 24 h. Samples were taken in different steps: in the raw ejaculates (ejaculates), after dilution with the refrigeration extender (diluted), immediately after reaching 4 °C (chilled 0 h) and the samples refrigerated at 4 °C and stored at this temperature for 24 h (chilled 24 h). Sperm quality (motility and integrity of plasma and acrosomal membrane, and mitochondrial functionality) was also evaluated. Bacterial 16S rRNA sequencing was used to study the seminal microbiota. Our results indicated that both refrigeration and storage at 4 °C negatively affected sperm quality parameters. Preparing semen doses and their subsequent conservation caused a significant change in the bacterial community structure. Raw ejaculates showed a lower Pielou's evenness index than the other samples (diluted, chilled 0 h and chilled 24 h). Ejaculates also had a lower Shannon's diversity index (3.44) than the diluted semen (4.17) and the semen chilled for 24 h (4.43). Regarding beta diversity, significant differences were detected between ejaculates and the other treatments. Differences were also found in unweighted UniFrac distances between the semen chilled for 0 h and that chilled for 24 h. At the genus level, marked effects of preparing doses and their subsequent conservation were also evident: 199 genera that were absent in ejaculates were found in the semen chilled and stored for 24 h; 177 genera that were present in ejaculates disappeared after 24-h refrigeration. In conclusion, the extender and protocol for preparing refrigerated goat buck semen doses considerably modify microbial ejaculate composition.

Keywords: Caprine; Microbiota; Refrigerated semen; Semen doses; Sperm.