Ovine placental explants: A new ex vivo model to study host‒pathogen interactions in reproductive pathogens

15 páginas, 9 figuras, 2 tablas.

Reproductive failure is one of the main performance constraints in ruminant livestock. Transmissible agents such as Toxoplasma gondii and Neospora caninum are commonly involved in the occurrence of abortion in ruminants, but little is known about the mechanisms involved. While in vivo models are optimal for the study of abortion pathogenesis, they have a high economic cost and come with ethical concerns. Unfortunately, alternative in vitro models fail to replicate the complex in vivo placental structure. To overcome the limitations of currently available models, we developed an ex vivo model based on the cultivation of fresh and cryopreserved sheep placental explants, enabling the biobanking of tissues. Reproducible and simple markers of tissue integrity (histology, RNA concentrations), viability (resazurin reduction), and functionality (synthesis of steroid hormones) were also investigated, allowing a clear quality assessment of the model. This work shows that, similar to fresh explants, tissues cryopreserved in ethylene glycol using slow freezing rates maintain not only their structure and function but also their receptivity to T. gondii and N. caninum infection. In addition, the findings demonstrate that explant lifespan is mainly limited by the culture method, with protocols requiring improvements to extend it beyond 2 days. These findings suggest that cryopreserved tissues can be exploited to study the initial host‒pathogen interactions taking place in the placenta, thus deepening the knowledge of the specific mechanisms that trigger reproductive failure in sheep. Importantly, this work paves the way for the development of similar models in related species and contributes to the reduction of experimental animal use in the future.

This work has been funded by the Madrid Government (Comunidad de Madrid, Spain) under the Programme to Stimulate Research for Young Doctors (5th Regional Programme of Research and Technological Innovation) (PR65/19–22457) and the PLATESA2-CM P2018/BAA4370 project. This project has also been funded by the Spanish Ministry of Science and Innovation (PID2019-104713RB-C21). RA was financially supported by a grant from the Spanish Ministry of Science, Innovation and Universities (PRE2020-092101). IPF was supported by a research programme funded by the Madrid Government (Comunidad de Madrid, Spain, Programa de Atraccion ´ de Talento, 2018T2/BIO10170). The funding bodies were not involved in the design of the study, the collection, analysis, and interpretation of data, or in writing the manuscript. The authors would like to thank Barbara ´ Pérez-Arroyo for technical assistance during the study

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