Bacterial development in semen stored at ambient temperature in INRA96®

Abstract

Sperm motility and mitochondrial activity can be maintained at room temperature for 24h in INRA96®. However bacterial development remains possible. Furthermore, very few studies evaluated the sensitivity of bacteria to the antibiotics included in INRA96. In experiment 1, semen from 8 stallions (28 ejaculates) was diluted to 20 × 106 sperm/mL in INRA96. The total amount of bacteria, dead or alive, was evaluated using Easykit6® (IMV-Technologies) immediately after dilution, then after 24h and 48h. Bacterial concentration remained stable during 48h cooled storage in Neopor® box (Minitube): mean±sem 9.8±2 × 105, 8.5±2 × 105, 8.4±12 × 105 bacteria/mL at 0h, 24h, 48h, respectively. Bacterial concentration significantly increased after 48h at ambient temperature between 13 and 26°C: 9.7±2 × 105a, 10±2 × 105ab, 28±6 × 105b bacteria/mL at 0h, 24h, 48h, and was significantly higher at ambient temperature than in cooled samples both after 24h and 48h. However, when data were analysed individually, the significant increase occured only for 3 of the 8 stallions. In experiment 2, semen from 7 stallions with 3 ejaculates each was evaluated in duplicate after 0h, 24h and 48h at ambient temperature. Easykit6® showed a significant increase in bacterial concentration for 2 stallions. Conventional bacteriology demonstrated an increase in CFU concentration for 3 stallions including the 2 mentioned above, and a decrease for another stallion. For example, stallion S showed 8±8a, 592±298ab, 8567±4493bCFU/mL at 0h, 24h, 48h, respectively, whereas stallion L showed 1259±504b, 92±53a,117±72aCFU/mL. Sixteen bacterialspecies (38 strains) and one yeast were identified. All the strains of Enterococcus fecalis, Listeria grayi, Streptococcus zooepidemicus, and some strains of Aerococcus viridans, Aeromonas salmonicida, Pasteurella multocida, Pseudomonas sepacia, Staphylococcus hominis, Staphylococcus xylosus, were resistant to both antibiotics of INRA96: 0.028g/L penicillinG and 0.077g/L gentamicin. Ten of the 20 strains identified at 0h and resistant to those antibiotics were not observed anymore at 24h. Eighteen of the 20 strains identified at 48h were resistant to those antibiotics. Eight of them have not been detected at 0h. Candida famata and the 17 bacterial strains identified at 0h and sensitive to those antibiotics were not observed anymore at 24h.The antibiotics and amphotericin B present in INRA96 seem enough to prevent microbial development at ambient temperature, provided that micro-organisms are sensitive to those molecules. Competition occurs between strains: those sensitive to the antibiotics of the extender cannot develop during storage, whereas others, even under the level of detection in raw semen, grow during storage because they are resistant. Bacterial concentration was not significantly correlated with sperm quality (motility, morphological abnormalities). Such bacterial level may not affect spermatozoa. Whether it could induce endometritis in inseminated mares needs investigation. Financial support: IFCE scientific council (Spermicrobes).