Marin Clara, Garcia-Dominguez Ximo, Montoro-Dasi Laura, Lorenzo-Rebenaque Laura, Vicente José S, Marco-Jimenez Francisco
Instituto de Ciencias Biomédicas, Facultad de Veterinaria, Universidad Cardenal Herrera-CEU, CEU Universities, Avenida Seminario s/n, 46113 Moncada, Spain.
Instituto de Ciencia y Tecnología Animal, Universitat Politècnica de València, 46022 Valencia, Spain.
Animals (Basel). 2020 Apr 1;10(4):598. doi: 10.3390/ani10040598.
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 Typhimurium, , , and . 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.
近几十年来，配子和胚胎冷冻保存已成为家畜和人类辅助生殖中的常规程序。然而，种质的安全储存以及疾病传播的预防仍然是通过胚胎移植传播疾病的潜在风险。本研究旨在证明受污染液氮中胚胎交叉感染的潜在风险，以及在裸露和封闭装置中受感染胚胎对无菌液氮的交叉污染。此外，我们研究了无抗生素培养基对受感染胚胎培养发育的影响。该研究是以兔子为模型进行的基于实验室的分析。进行了两项实验，以评估人工接种鼠伤寒沙门氏菌后胚胎的交叉感染（液氮到胚胎）和交叉污染（胚胎到液氮）情况。对兔胚胎进行玻璃化快速冷却，储存一年，解冻并培养。使用体内产生的晚期桑葚胚 - 早期囊胚阶段（72小时）的胚胎（n = 480）。将胚胎在有和没有病原体的溶液中培养1小时。然后，将胚胎玻璃化并在两个液氮生物样本库（一个无病原体，另一个人工污染）中的裸露和封闭装置中储存一年。胚胎解冻后再培养48小时，评估发育情况和微生物的存在（显色培养基、扫描电子显微镜）。储存在人工污染液氮中裸露装置中的胚胎被感染（12.5%），而储存在封闭装置中的胚胎均未被感染。同时，人工感染胚胎的储存导致液氮生物样本库污染（100%）。扫描电子显微镜观察显示，在玻璃化 - 解冻程序后，所有微生物都附着在胚胎表面。然而，在含有抗生素和抗真菌剂的培养基中培养的胚胎发育到孵化囊胚阶段，而在无抗生素培养基中培养的人工感染胚胎未能发育。总之，我们的研究结果支持在液氮生物样本库中胚胎储存期间交叉污染和交叉感染都是可能的。因此，为确保低温储存的生物安全性，必须使用避免与液氮直接接触的封闭系统。此外，似乎有必要为配子和胚胎的低温保存和储存提供最佳实践指南，以确定适当的质量和风险管理程序。
