Porcine semen microbiota analysis in relation to sperm quality in semen extender coupled with gentamicin

The present study was carried out to (i) investigate the association between boar semen microbiota and semen qualities and (ii) determine the relationship between boar bacteria contamination and longevity of boar semen preserved for 3 days in BTS with and without gentamicin. A total of 20 Duroc ejaculates were collected from standard quality boars (n=10) and high-quality ones (n= 10). Besides 3 ml of fresh semen of each ejaculate was subjected to 16S rRNA gene sequencing and bioinformatics including alpha diversity, beta diversity, and taxonomic mapping, each ejaculate was diluted in two conditions of Beltsvile Thawing Solution (BTS) extender: CONTROL: semen was diluted in BTS without gentamicin and TREATMENT: the semen was diluted in BTS supplemented with 0.25 g/l of gentamicin to produce doses of semen containing 3 billion of spermatozoa in 100 ml. During 3 storage days, the sperm motility, sperm viability, acrosome integrity, sperm membrane permeability, mitochondrial activity, and total aerobic bacteria count was evaluated. Semen microbiota analysis indicated that Lactobacillales (25.2 %), Enterobacterales (10.3 %) and Bacteroidales (9.3 %) were the most dominant bacteria. While 88.3 % of standard quality semen clustered into the top 25% of lowest Lactobacilales, 100% of high-quality samples clustered in the third quartile. Although no differences in the diversity in microbiota composition were detected across semen quality samples (P > 0.05), the negative linear relationship between Lactobacillales and Enterobacterales: y = 507.3 - 0.5x, R2 = 0.24 (P < 0.001), the differences in the abundance of lesser dominant bacteria and the correlation among them were detected (P < 0.05). Bacterial culture and semen evaluation revealed that along with a higher sperm viability detected in high quality samples compared to standard quality ones (83.8 ± 1.5 % versus 69.4 ± 1.5 %, respectively, P < 0.001) was the lower total bacteria count (CFU/ ml, log10) (3.7 ± 0.2 log versus 4.4 ± 0.2 log, respectively, P = 0.019) and a negative linear relationship established: y = 102. 8 – 6.4x, R2 = 0.25 (P = 0.026). All ejaculates were contaminated with three to ten bacterial types belonging to Enterobacteriaceae Pseudomonadaceae, Pasteurellaceae family. While Staphylococcus spp. (20.5 %), Micrococcus spp. (12.6 %), and Escherichia coli (11.7 %) accounted for the highest percentage, the lower percentage ones such as Klebsiella oxytoca (3.1 %), Acinetobacter spp. (0.9 %) and Bachybacterium conlomeratum (0.2 %) were also isolated. There were no differences in Staphylococcus spp. count and Proteus spp. count compared between standard and high-quality ejaculates (P < 0.05) although these were most frequently isolated. Under the bacteriostatic activity of the antibiotic, the total CFU/ml in the diluted semen with antibiotic was lower than in the diluted one without antibiotic and fresh semen (2.0 versus 2.9 and 4.0 log, respectively, P < 0.001). The total bacterial count in the TREATMENT group was lower than in the CONTROL group (1.9 ± 0.1 versus 3.9 ± 0.1 respectively, P < 0.001) on each day of preservation (P< 0.001). In CONTROL group, the total CFU/ ml, log10 on day 2 (4.3 log) and day 3 (5.3 log) was higher than on day 0 (2.9 log) and day 1 (3.2 log) (P < 0.001), but no significant differences was detected in TREATMENT group (P > 0. 05). Generally, although no significant difference regarding sperm plasma membrane permeability and mitochondrial activity was detected (P > 0.05), the sperm motility, viability and acrosome integrity in the TREATMENT group were higher than those in the CONTROL group (P < 0.05) during preservation time across boar ejaculates. While all measured sperm parameters in the TREATMENT group were higher than those in the CONTROL group (P < 0.001) examined on high-quality samples, only sperm mitochondrial activity in the CONTROL group was higher than TREATMENT group (60.2 ± 3.1% versus 58.7 ± 3.1%, respectively, P < 0.05) examined on standard quality samples. On days 1, 2 and 3 of storage, no differences between the CONTROL and TREATMENT groups were detected among standard quality samples (P > 0.05). However, the significant differences were registered on Day 2 and Day 3 between the CONTROL and TREATMENT groups across high-quality samples (P > 0.05). In conclusion, porcine semen microbiota analysis can be used to access the semen quality and adding the current antibiotic dose significantly controlled the bacteria contamination in stored semen as well as can improve the semen qualities collected from high-quality boars but not standard quality ones during preservation time.