Caldeira Jéssica Lobo Albuquerque, Faria Ana Carolina Silva, Diaz-Miranda Edgar Andres, Zilch Tiago Jaquel, da Costa Caliman Sanely Lourenço, Okano Denise Silva, Guimarães José Domingues, Pena Junnia Luisa, Barbosa Wagner Faria, Junior Abelardo Silva, Chang Yung-Fu, Moreira Maria Aparecida Scatamburlo
Department of Veterinary, Division of Preventive Medicine and Public Health, Universidade Federal de Viçosa, University Campus, PH Rolfs Avenue, 36570-000, Vicosa, Minas Gerais, Brazil.
Department of Veterinary, Division of Animal Reproduction, Universidade Federal de Viçosa, University Campus, PH Rolfs Avenue, 36570-000, Vicosa, Minas Gerais, Brazil.
Theriogenology. 2021 Feb;161:228-236. doi: 10.1016/j.theriogenology.2020.12.007. Epub 2020 Dec 9.
Mycobacterium avium subsp. paratuberculosis (MAP) is responsible for Paratuberculosis mainly affecting domestic ruminants. The interaction between MAP and sperm and/or germ cells has not yet been established, however the adherence between MAP and the host cell surface is associated to the 85 complex proteins that bind to the host cell's fibronectin. Therefore, this study aimed to evaluate the binding of MAP to bovine sperm and to verify changes in these cells by the presence of MAP before and after sperm cryopreservation. Polyclonal antibodies to MAP 85 complex proteins were produced and utilized in the analyzes. Two Nelore bulls were used for semen collection and MAP dilutions (10-10 CFU/mL) were inoculated in the samples; sperm motility and vigor were evaluated using light microscopy at different times before and after cryopreservation and in the presence and absence of the antibodies 85A and 85B. Interaction of MAP and sperm, interaction of MAP and sperm in the presence of Ab 85A and in the presence of Ab 85B were analyzed by scanning electron microscopy. The viability of MAP after sperm cryopreservation were evaluated by plating the samples after thawing. It was observed that sperm in the presence of MAP shows a decrease in motility and vigor, and that the higher the MAP concentration, the lower the sperm performance. It was possible to determine the viability of MAP after cryopreservation in samples of higher concentrations, which demonstrates the potential of transmission of this pathogen through artificial insemination. The interaction of MAP with bovine sperm occurs mainly in the midpiece and may be linked to the proteins 85A and 85B present in the MAP membrane.
副结核分枝杆菌（MAP）是引起主要影响家养反刍动物的副结核病的病原体。MAP与精子和/或生殖细胞之间的相互作用尚未明确，但MAP与宿主细胞表面的黏附与结合宿主细胞纤连蛋白的85复合体蛋白有关。因此，本研究旨在评估MAP与牛精子的结合情况，并验证在精子冷冻保存前后MAP的存在对这些细胞的影响。制备了针对MAP 85复合体蛋白的多克隆抗体并用于分析。使用两头内洛尔公牛采集精液，并在样本中接种MAP稀释液（10-10 CFU/mL）；在冷冻保存前后的不同时间，以及存在和不存在抗体85A和85B的情况下，使用光学显微镜评估精子活力和活率。通过扫描电子显微镜分析MAP与精子的相互作用、在抗体85A存在下MAP与精子的相互作用以及在抗体85B存在下MAP与精子的相互作用。通过解冻后对样本进行平板接种来评估精子冷冻保存后MAP的活力。观察到在有MAP存在的情况下精子活力和活率下降，且MAP浓度越高，精子性能越低。在较高浓度的样本中能够确定冷冻保存后MAP的活力，这表明该病原体通过人工授精传播的可能性。MAP与牛精子的相互作用主要发生在中段，可能与MAP膜中存在的85A和85B蛋白有关。
