# Taxonomic Profile of Cultivable Microbiota from Adult Sheep Follicular Fluid and Its Effects on In Vitro Development of Prepubertal Lamb Oocytes

## TL;DR

This study explores the presence of bacteria in adult sheep follicular fluid and their effects on the development of prepubertal lamb oocytes in vitro.

## Contribution

The study is the first to detect bacteria in adult sheep follicular fluid and report specific species like Streptococcus infantarius subsp. infantarius and Burkholderia cepacia.

## Key findings

- Bacteria were detected in adult sheep follicular fluid for the first time.
- Bacterial metabolites showed variable short- and long-term effects on oocyte maturation and embryonic development.
- One supernatant reduced bioenergetic parameters and cleavage rates in prepubertal lamb oocytes.

## Abstract

Most of the studies conducted to date on the ovarian follicular fluid (FF) microbiome have been performed in humans with few studies in farm animals. Prepubertal lamb oocytes (PLOs) allow for applications in the juvenile in vitro embryo transfer (JIVET) technology and have translational relevance as models for human pediatric onco-fertility. However, despite extensive research effort, they still display lower developmental competence compared to their adult counterparts, due to the incomplete/perturbed nuclear/cytoplasmic maturation. Following the hypothesis that the FF of adult subjects could contain the microbiota metabolites essential for PLO acquisition of developmental competence, the aims of this study were to perform the taxonomic profiling of adult sheep FF cultivable bacteria, by combining 16s rRNA gene sequencing and (targeted) culturomics and to evaluate the functional effects of bacterial cell-free supernatants on PLO in vitro maturation and developmental potential. For the first time, we detected bacteria presence in adult sheep FF and demonstrated the variable short- and long-term effects of bacterial metabolites on PLO maturation and embryonic development.

The aims of the present study were to analyze the taxonomic profile and to evaluate the functional effects of sheep FF cultivable microbiota on prepubertal lamb oocytes PLOs developmental potential. Ovarian FFs were recovered from slaughtered adult sheep via the aspiration of developing follicles and used for microbiota propagation. Bacterial pellets underwent 16S rRNA gene sequencing and targeted culturomics, whereas cell-free supernatants were used as supplements for the in vitro maturation (IVM) of slaughtered PLOs. For the first time, bacteria presence in adult sheep FF was detected, with the first report of Streptococcus infantarius subsp. infantarius (as a species) and Burkholderia cepacia (as a genus and species) in either animal or human FF. The short- and long-term effects of bacterial metabolites on PLO maturation and embryonic development were demonstrated. As short-term effects, the addition of FF microbiota metabolites did not affect the oocyte nuclear maturation and mitochondria distribution pattern, except in one of the examined supernatants, which reduced all quantitative bioenergetic/oxidative parameters. As long-term effects, one of them reduced the total cleavage rate after in vitro embryo culture (IVC). In conclusion, microbiota/bacteria are present in adult sheep FF and may influence reproductive outcomes in vitro. Future studies may reveal the beneficial in vitro effects using the microbiome from preovulatory follicles.