110 Absence of transmission of Mycoplasma bovis via naturally contaminated semen during in vitro fertilization

Abstract

Mycoplasma bovis (Mbo) has been isolated from genital tracts of bulls, and it can survive in processed semen. Experimental studies have shown that Mbo inoculation into the uterus or insemination with Mbo-infected semen can cause bursitis, salpingitis, abortion, and infertility. The control of Mbo is very difficult because of latent carrier animals, increasing resistance to antibiotics, and unavailability of effective vaccines. The aim of this study was to follow the passage of Mbo infection from naturally contaminated semen to transferable embryos during bovine in vitro embryo production (IVP). (Unless otherwise stated, all chemicals used were purchased from Sigma-Aldrich.) Two batches of slaughterhouse-derived oocytes were matured in tissue culture medium 199 (TCM-199) with glutamax-I (Gibco™; Invitrogen Corporation) supplemented with 0.25 mM sodium pyruvate, 100 IU mL⁻¹ penicillin, 100 µg mL⁻¹ streptomycin, 2 ng mL⁻¹ FSH (Puregon, Organon), 1 µg mL⁻¹ β-oestradiol (E-2257), and 10% heat-inactivated fetal bovine serum (FBS; Gibco™) for 24 h at 38.5°C in maximal humidity in 5% CO₂ in air. Matured oocytes were fertilized for 20 h in IVF-TL medium supplemented with 10 µg mL⁻¹ of heparin and 2 mM of PHE at 38.5°C in maximal humidity in 5% CO₂ in air, using spermatozoa per mL as a final concentration. The batches of oocytes were divided between uninfected IVP bull (N = 205) and naturally Mbo-infected AI bull (N = 690). Zygotes were cultured in G1/G2 media (Vitrolife) supplemented with bovine serum albumin, fatty acid free (4 mg mL⁻¹), at 38.5°C in maximal humidity in 5% O₂, 5% CO₂, and 90% N₂. Blastocysts were collected for Mbo cultures on Days 7 and 8 (IVF = Day 0). Samples of washed semen, fertilization medium, cumulus cells, culture medium, all wash media, and transferable embryos (with and without zona pellucidae) were collected for Mbo cultures. Half of the embryos were treated with trypsin according to IETS standards after the collections. The Mbo cultures were performed in accordance with procedures previously described by Bölske (1988 Zentralbl. Bakteriol. Mikrobiol. Hyg. A 69, 331-340), followed by detection with real-time PCR. Infection with Mbo does not seem to have negative effects on fertilization (cleavage rates: 77.1% and 89.0% for IVP and Mbo AI bulls, respectively) or embryo development rates (blastocyst rate: 26.3% and 32.5% for IVP and Mbo AI bulls, respectively). Following Mbo cultures, only washed semen was found to be Mbo positive via real-time PCR. We conclude that M. bovis is not likely transmitted in bovine IVP when using naturally infected semen.

We acknowledge Tiina Kortelainen for technical assistance and the Ministry of Agriculture and Forestry for funding.