STUDY OF THE SPERM MICROBIOME OF BULLS USING METAGENOMIC ANALYSIS

The study aimed to obtain information about the microbial communities in bull sperm, which is used for artificial insemination. The research included ten samples of cryopreserved bull sperm obtained from domestic breeding farms and 24 imported sperm samples. Next-generation sequencing technology on the Illumina Miseq platform was used for the analysis. Bioinformatics analysis was performed using the QIIME2 software package. The obtained whole-genome sequencing data showed a low level of biodiversity in the analysed samples. Diversity index values were determined for two groups of samples: sperm from domestic and foreign bulls. The Shannon index values were 1.40±0.12 and 1.30±0.08 for each group, respectively. Student's t-test calculations indicated no statistically significant differences in the α-biodiversity parameters between these groups. Up to 19 known bacterial types were detected in the samples, with only Proteobacteria, Firmicutes, Fusobacteria, Bacteroidetes, Actinobacteria, and Tenericutes present at frequencies exceeding 5%. Fusobacteria and Firmicutes dominated Russian bull sperm samples, while the predominant type in foreign bull sperm was Proteobacteria. Of the 18 detected bacterial classes, genetic material from 11 classes was found in at least one of the samples at a frequency exceeding 5%. Russian bull samples contained up to 13 genera of bacteria with genetic material above 1%, while foreign bull samples contained up to 15 genera. The predominant genus for domestic bull sperm samples was Fusobacterium, while for alien bull sperm, it was an unidentified genus of the Clostridiales order. The presence of representatives from the Campylobacter and Pseudomonas genera was also noted in the samples. Possible associations between the presence of specific bacterial taxa and morphofunctional indicators of bull sperm quality are discussed.

1. Braem G., Vliegher S.D., Verbist B., Heyndrickx M., Leroy F., Vuyst L.D., Culture-independent exploration of the teat apex microbiota of dairy cows reveals a wide bacterial species diversity, Vet Microbiol, 2012, Jun 15, Vol. 157 (3-4), pp. 383–90, DOI: 10.1016/j.vetmic.2011.12.031.

2. Hoque M.N., Istiaq A., Clement R.A., Sultana M., Crandall K.A., Siddiki A.Z., Hossain M.A., Metagenomic deep sequencing reveals association of microbiome signature with functional biases in bovine mastitis, Sci Rep, 2019, Vol. 9 (1), P. 13536, DOI: 10.1038/s41598-019-49468-4.

3. https://doi.org/10.1038/s41598-018-36673-w.

4. Santos T.M, Bicalho R.C., Diversity and succession of bacterial communities in the uterine fluid of postpartum metritic, endometritic and healthy dairy cows, PLoS One, 2012, Vol. 7, P. 53048.

5. Ong C.T., Turni C., Blackall P.J., Boe-Hansen G., Hayes B.J., Tabor A.E., Interrogating the bovine reproductive tract metagenomes using culture-independent approaches: a systematic review, Anim Microbiome, 2021, Jun 9, Vol. 3 (1), P. 41, DOI: 10.1186/s42523-021-00106-3.

6. Machado V.S., Oikonomou G., Bicalho M.L., Knauer W.A., Gilbert R., Bicalho R.C., Investigation of postpartum dairy cows' uterine microbial diversity using metagenomic pyrosequencing of the 16S rRNA gene, Vet Microbiol, 2012, Vol. 159, pp. 460–469.

7. Gonzalez Moreno C., Fontana C., Cocconcelli P.S., Callegari M.L., Otero M.C., Vaginal microbial communities from synchronized heifers and cows with reproductive disorders, J Appl Microbiol, 2016, Vol. 121, pp. 232–241.

8. Moretti E., Capitani S., Figura N., Pammolli A., Federico M.G., Giannerini V., Collodel G., The presence of bacteria species in semen and sperm quality, J Assist Reprod Gen, 2009, Vol. 26, pp. 47–56.

9. Smole I., Thomann A., Frey J., Perreten V., Repression of common bull sperm flora and in vitro impairment of sperm motility with Pseudomonas aeruginosa introduced by contaminated lubricant, Reprod Domest Anim, 2010, Vol. 45, pp. 737–742.

10. Baud D., Pattaroni C., Vulliemoz N., Castella V., Marsland B.J., Stojanov M., Sperm Microbiota and Its Impact on Semen Parameters, Front Microbiol, 2019, Feb 12, Vol. 10, P. 234.

11. Koziol J.H., Sheets T., Wickware C.L., Johnson T.A., Composition and diversity of the seminal microbiota in bulls and its association with semen parameters, Theriogenology, 2022, Apr 1, Vol.182, pp. 17–25.

12. Medo J., Žiarovská J., Ďuračka M., Tvrdá E., Baňas Š., Gábor M., Kyseľ M., Kačániová M., Core Microbiome of Slovak Holstein Friesian Breeding Bulls' Semen, Animals (Basel), 2021, Nov 22, Vol. 11 (11), P. 3331.

13. Cojkic A., Niazi A., Guo Y., Hallap T., Padrik P., Morrell J.M., Identification of Bull Semen Microbiome by 16S Sequencing and Possible Relationships with Fertility, Microorganisms, 2021, Nov 25, Vol. 9 (12), P. 2431.

14. The Tanner M. Young Biodiversity Calculator, available at: https://www.alyoung.com/labs/biodiversity_calculator.html (March 25, 2022).

15. Weng S.L., Chiu C.M., Lin F.M., Huang W.C., Liang C., Yang T., Yang T.L., Liu C.Y., Wu W.Y., Chang Y.A., Chang T.H., Huang H.D. Bacterial communities in semen from men of infertile couples: metagenomic sequencing reveals relationships of seminal microbiota to semen quality, PLoS One, 2014, Oct 23, Vol. 9 (10), P. 110152.

16. Gani M., Rao S., Miller M., Scoular S., Pseudomonas mendocina Bacteremia: A Case Study and Review of Literature, Am J Case Rep, 2019, Apr 5, Vol. 20, pp. 453–458.

17. Fedotova T.A., Shestakov A.G., Vasil'ev D.A., Vestnik Ul'yanovskoj GSKHA, 2019, No. 3 (47) pp. 116–123. (In Russ.)

18. Borunova S.M. Kompleksnaya ocenka morfofunkcional'nyh i mikrobiologicheskih pokazatelej polovyh kletok bykov-proizvoditelej (Comprehensive assessment of morphofunctional and microbiological parameters of germ cells of breeding bulls), Doctor's thesis of Biological Sciences, Moscow, 2019, 379 p. (In Russ.)