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Article

Experimental Evidence Reveals Both Cross-Infection and Cross-Contamination Risk of Embryo Storage in Liquid Nitrogen Biobanks

1
Instituto de Ciencias Biomédicas, Facultad de Veterinaria, Universidad Cardenal Herrera-CEU, CEU Universities, Avenida Seminario s/n, 46113 Moncada, Spain
2
Instituto de Ciencia y Tecnología Animal, Universitat Politècnica de València, 46022 Valencia, Spain
*
Author to whom correspondence should be addressed.
Animals 2020, 10(4), 598; https://doi.org/10.3390/ani10040598
Received: 8 March 2020 / Revised: 30 March 2020 / Accepted: 31 March 2020 / Published: 1 April 2020
(This article belongs to the Section Animal Reproduction)
This study was conducted to demonstrate the potential hazards of cross-infection and cross-contamination of embryos during storage in liquid nitrogen biobanks. For the harmless and successful cryopreservation of embryos, the vitrification method must be chosen meticulously to guarantee not only a high post-thaw survival of embryos, but also to reduce the risk of disease transmission when those embryos are in storage for long periods.
In recent decades, gamete and embryo cryopreservation have become routine procedures in livestock and human assisted reproduction. However, the safe storage of germplasm and the prevention of disease transmission continue to be potential hazards of disease transmission through embryo transfer. This study aimed to demonstrate the potential risk of cross-infection of embryos from contaminated liquid nitrogen, and cross-contamination of sterile liquid nitrogen from infected embryos in naked and closed devices. Additionally, we examined the effects of antibiotic-free media on culture development of infected embryos. The study was a laboratory-based analysis using rabbit as a model. Two experiments were performed to evaluate both cross-infection (liquid nitrogen to embryos) and cross-contamination (embryos to liquid nitrogen) of artificially inoculated Salmonella Typhimurium, Staphylococcus aureus, Enterobacter aerogenes, and Aspergillus brasiliensis. Rapid cooling through vitrification was conducted on rabbit embryos, stored for a year, thawed, and cultured. In vivo produced late morulae–early blastocyst stages (72 h) embryos were used (n = 480). Embryos were cultured for 1 h in solutions with and without pathogens. Then, the embryos were vitrified and stored in naked and closed devices for one year in two liquid nitrogen biobanks (one pathogen-free and the other artificially contaminated). Embryos were warmed and cultured for a further 48 h, assessing the development and the presence of microorganism (chromogenic media, scanning electron microscopy). Embryos stored in naked devices in artificially contaminated liquid nitrogen became infected (12.5%), while none of the embryos stored in closed devices were infected. Meanwhile, storage of artificially infected embryos incurred liquid nitrogen biobank contamination (100%). Observations by scanning electron microscopy revealed that all the microorganisms were caught in the surface of embryos after the vitrification-thawed procedure. Nevertheless, embryos cultured in antibiotics and antimycotic medium developed to the hatched blastocyst stage, while artificially infected embryos cultured in antibiotic-free medium failed to develop. In conclusion, our findings support that both cross-contamination and cross-infection during embryo storage in liquid nitrogen biobanks are plausible. So, to ensure biosafety for the cryogenic storage, closed systems that avoid direct contact with liquid nitrogen must be used. Moreover, it seems essential to provide best practice guidelines for the cryogenic preservation and storage of gametes and embryos, to define appropriate quality and risk management procedures. View Full-Text
Keywords: microorganisms; pathogen transmission; vitrification; bacteria; fungi; embryo; naked device; closed device microorganisms; pathogen transmission; vitrification; bacteria; fungi; embryo; naked device; closed device
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MDPI and ACS Style

Marin, C.; Garcia-Dominguez, X.; Montoro-Dasi, L.; Lorenzo-Rebenaque, L.; Vicente, J.S.; Marco-Jimenez, F. Experimental Evidence Reveals Both Cross-Infection and Cross-Contamination Risk of Embryo Storage in Liquid Nitrogen Biobanks. Animals 2020, 10, 598. https://doi.org/10.3390/ani10040598

AMA Style

Marin C, Garcia-Dominguez X, Montoro-Dasi L, Lorenzo-Rebenaque L, Vicente JS, Marco-Jimenez F. Experimental Evidence Reveals Both Cross-Infection and Cross-Contamination Risk of Embryo Storage in Liquid Nitrogen Biobanks. Animals. 2020; 10(4):598. https://doi.org/10.3390/ani10040598

Chicago/Turabian Style

Marin, Clara, Ximo Garcia-Dominguez, Laura Montoro-Dasi, Laura Lorenzo-Rebenaque, José S. Vicente, and Francisco Marco-Jimenez. 2020. "Experimental Evidence Reveals Both Cross-Infection and Cross-Contamination Risk of Embryo Storage in Liquid Nitrogen Biobanks" Animals 10, no. 4: 598. https://doi.org/10.3390/ani10040598

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