Risk of contamination of germplasm during cryopreservation and cryobanking in IVF units.

Cryopreservation of sperm, embryos and, more recently, oocytes plays an important and increasing role in assisted reproduction, due to improvements of old, and introduction of new technologies. In parallel, concerns are increasing about the technical and biological safety of these procedures. However, published data regarding the confirmed or theoretical hazards of these procedures are sparse and sometimes contradictory.

Disinfection procedures for controlling microorganisms in the semen and embryos of humans and farm animals.

Semen and embryos generated by assisted reproductive techniques (ARTs) may be contaminated with numerous microorganisms. Contamination may arise from systemic or local reproductive tract infections in donors or the inadvertent introduction of microorganisms during ARTs, and may lead to disease transmission. This review describes sanitary procedures which have been investigated to ascertain whether they are effective in rendering semen and embryos free of pathogenic microorganisms, including internationally adopted washing procedures, which can be supplemented by antibiotics and enzymatic treatments.

Risk of transmission of Mycobacterium avium ssp. paratuberculosis by embryo transfer of in vivo and in vitro fertilized bovine embryos.

Over a 5-year interval, experiments were conducted to determine if Mycobacterium avium ssp. paratuberculosis (Map) is associated with in vivo and in vitro fertilized (IVF) embryos and whether it can be transmitted by embryo transfer. The present studies included: collection of embryos from five asymptomatic, naturally infected donors and transfer to uninfected recipients; collection of oocytes from two naturally infected donors with overt clinical signs; exposure of in vivo and IVF embryos to Map and transfer to uninfected recipients; and the inoculation (transfer) of "clean" IVF embryos to the uterine lumen of infected cows.

Non-transmission of bacterial and viral microbes to embryos and semen stored in the vapour phase of liquid nitrogen in dry shippers.

Cryopreservation and storage of germplasm is an important factor in the prevention of disease transmission by embryo transfer and artificial insemination. Here we report the results of an investigation on transmission of selected bacterial and viral pathogens by the vapour phase of liquid nitrogen (VPLN) to embryos and semen in dewars designed for short-term storage and transportation of biological specimens. In this study transmission of Pseudomonas aeruginosa, Escherichia coli, Staphylococcus aureus, BVDV, and BHV-1 was examined from: (1) contaminated dry shippers to germplasm; (2) between contaminated and non-contaminated cryopreserved germplasm; and (3) between stock culture of pathogenic agents and germplasm.

Experimental microbial contamination and disinfection of dry (vapour) shipper dewars designed for short-term storage and transportation of cryopreserved germplasm and other biological specimens.

Cryopreservation, storage and transport of cryopreserved germplasm without the risk of disease transmission is of great concern to animal and human health authorities. Here we report on the efficacy of microbial decontamination of the liquid nitrogen (LN) dry (vapour) shippers used for short-term storage and transportation of germplasm and other biological specimens. Dry shippers containing either a hydrophobic or a non-hydrophobic LN absorbent were experimentally contaminated with high titers of cultures of Pseudomonas aeruginosa, Escherichia coli, Staphylococus aureus, bovine viral diarrhea virus (BVDV) and bovine herpesvirus-1 (BHV-1).

An attempt to render oocytes and embryos free from the porcine circovirus type 2 after experimental in vitro exposure.

The purpose of this study was to investigate the possibility of rendering oocytes and embryos free of porcine circovirus type 2 (PCV2). Groups of cumulus oocytes complexes, cumulus free oocytes, and embryos 3 to 5 d post breeding were exposed to PCV2 (10(5) TCID50/mL) prior to disinfection by washing and different combinations of enzymatic treatments. The study suggests that under the in vitro conditions used, standard washing procedures with, or without, trypsin or incorporating pronase or hyaluronidase and DNase/RNase in the treatment was not effective in rendering oocytes and embryos free from PCV2 nucleic acid.

Observations on the fertilization and development of preimplantation bovine embryos in vitro in the presence of Tritrichomonas foetus.

Tritrichomonas foetus, a world-wide distributed parasitic protozoan is a cause of infertility and abortion. There is no documented information on the susceptibility of bovine embryos to the parasite. To determine the effect of T.

Microbial contamination of embryos and semen during long term banking in liquid nitrogen.

We report on microbial contamination of embryos and semen cryopreserved in sealed plastic straws and stored for 6-35 years in liquid nitrogen. There were 32 bacterial and 1 fungal species identified from randomly drawn liquid nitrogen, frozen semen, and embryos samples stored in 8 commercial and 8 research facility liquid nitrogen (LN) tanks. The identified bacteria represented commensal or environmental microorganisms and some, such as Escherichia coli, were potential or opportunistic pathogens for humans and animals.

Adherence of bovine viral diarrhea virus to bovine oocytes and embryos with a hardened zona pellucida cultured in vitro.

The purpose of this study was to investigate the adherence of bovine viral diarrhea virus (BVDV) to bovine mature, or immature, cumulus-free oocytes and to in vitro fertilized embryos, maintained in vitro in a ligated bovine oviduct to allow for the hardening of the zona pellucida. Incubation of the oocytes and embryos in the oviduct for 5 h caused hardening of the zona pellucida as measured by resistance to pronase digestion (which increased from approximately 3 min to 7 h; P > 0.001).

Bovine immunodeficiency virus in relation to embryos fertilized in vitro.

The association of bovine immunodeficiency virus (BIV) with embryos derived by in vitro fertilization from oocytes of experimentally infected heifers or oocytes/embryos exposed to the virus in vitro was investigated. Using a nested-PCR assay, proviral DNA of BIV was not detected in follicular fluid or in embryos derived from BIV-infected donors. In vitro exposure of oocytes to BIV during maturation or insemination with BIV-infected semen resulted in zona pellucida-intact embryos testing negative for BIV provirus.

Experimental collection and transfer of embryos from bovine immunodeficiency virus (BIV) infected cattle.

Three experiments were conducted to determine whether the lentivirus, bovine immunodeficiency virus (BIV) is likely to be transmitted via embryo transfer. In the first experiment, embryos collected from BIV-negative heifers were exposed in vitro to BIV for 24 h, washed and then tested for the presence of the provirus. In the second experiment, embryos obtained from BIV-negative heifers were transferred to the uterine horns of BIV-infected heifers; 24 h later these embryos were recovered and tested for the presence of BIV.

Effect of Mycoplasma bovis and Mycoplasma bovigenitalium in semen on fertilization and association with in vitro produced morula and blastocyst stage embryos.

Frozen-thawed bovine semen contaminated with Mycoplasma bovis (M. bovis) or Mycoplasma bovigenitalium (M. bovigenitalium) at either a high (10(6) CFU/mL) or low (10(4) CFU/mL) concentration was used for bovine oocyte insemination.

The effect of high concentrations of cryoprotectants on the passage of bovine viral diarrhea virus through the zona pellucida of in vitro fertilized embryos.

The effect of high concentrations of cryoprotectants on the passage of bovine viral diarrhea virus (BVDV) through the zona pellucida (ZP) of intact bovine embryos during the pre-freezing step of cryopreservation was investigated in a series of experiments. In vitro fertilized (IVF) embryos at the blastocyst stage were exposed to 10(6) TCID50 BVDV (non-cytopathic NY-1 strain) in a 30% suspension of either ethylene glycol, glycerol, DMSO, or 2 M sucrose in physiological saline for 10 min at 20 degrees C. Subsequently, the embryos were washed free of residual unbound viral particles, and the ZP of some embryos were removed by micromanipulation.

Sanitary status of oocytes and embryos collected from heifers experimentally exposed to Leptospira borgpetersenii serovar hardjobovis.

In a preliminary trial and three experiments, a total of 30 Holstein heifers were experimentally infected with a culture of Leptospira borgpetersenii serovar hardjobovis via one or more routes (uterine, cervical supraconjunctival, intranasal) and oviductal and uterine fluids recovered post-mortem or in vivo following superovulation with FSH. All routes of administration were effective in establishing Leptospira infection in the reproductive tract and Leptospira were identified in the oviductal and uterine fluids of all 30 heifers by microscopy. The incidence of infection was confirmed by positive identification of serum antibodies by the microscopic agglutination test (MAT).

Leptospira borgpetersenii serovar hardjo type hardjobovis in bovine embryos fertilized in vitro.

The association of Leptospira borgpetersenii serovar hardjo type hardjobovis with bovine embryos produced by in vitro fertilization was examined by scanning electron microscopy (SEM) and transmission electron microscopy (TEM). Morula stage embryos with an intact zona pellucida (ZP) were exposed to this spirochete for 24 h in culture medium, washed by the standard washing procedure as recommended by the International Embryo Transfer Society, and then examined. SEM showed typical helicoid leptospires on the surface and in the pores of the ZP.