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Development of a new extender for cryopreservation of boar semen: I) evaluation of the effects on spermatozoa, II) evaluation of inflammatory response in the reproductive tract, III) effects on pig fertility

Grant number: 22/09573-2
Support Opportunities:Regular Research Grants
Duration: May 01, 2023 - April 30, 2025
Field of knowledge:Agronomical Sciences - Veterinary Medicine - Animal Reproduction
Convênio/Acordo: Universidad de la Frontera
Principal Investigator:André Furugen Cesar de Andrade
Grantee:André Furugen Cesar de Andrade
Principal researcher abroad: Raul Sanchez Gutierrez
Institution abroad: Universidad de La Frontera (UFRO), Chile
Host Institution: Faculdade de Medicina Veterinária e Zootecnia (FMVZ). Universidade de São Paulo (USP). São Paulo , SP, Brazil
Associated researchers:Fabiana Fernandes Bressan ; Fabiola Alejandra Zambrano Quezada ; Felipe Andrés Pezo Fonseca ; Heidge Fukumasu ; Mabel Schulz ; Pamela Uribe Catalán

Abstract

The ideal method for artificial insemination (AI) would be the use of cryopreserved semen; however, it is not applied by the pig industry because similar results compared to refrigerated semen at 17°C have not been achieved in the pregnancy rate and the number of piglets per parturition. Nevertheless, cryopreservation is the most efficient method for long-term preservation of mammalian spermatozoa; however, freeze/thawing procedures strongly impair the sperm function in boar spermatozoa. Our research group has optimized the porcine semen cryopreservation, improving the critical factors in sperm function alteration, stabilizing the membrane with the saccharide trehalose, and decreasing the production of reactive and nitrogen species (ROS/RNS) with melatonin that antagonizes both ways. Along with improving freezing process conditions, it is necessary deeper knowledge of how intense the innate immunity response generated by the spermatozoa into the female's genital tract is. After AI, the number of spermatozoa in the sow's genital tract falls to 5-10% in a few hours, and up to 50% is eliminated due to the reflux of the extender medium. Another important cause of this rapid sperm decrease is the interaction with polymorphonuclear neutrophils (PMN) that migrate directly to the endometrium after AI, and within 2 h, their number reaches the same number of spermatozoa. This physiological response to clean the genital tract in preparation for the reception of the embryos can be exacerbated by AI with cryopreserved semen in many situations, such as; sperm with structural damage, apoptotic-like signals, dead sperm, or product associated with a cryopreservation medium. The PMN in contact with boar spermatozoa strongly induced the release of neutrophil extracellular traps (NETs), generated by the extrusion of DNA fibers in the extracellular medium, resulting in aggNETs, which efficiently entrapped numerous spermatozoa simultaneously, thereby neutralizing their independent progression. Also, the NETs released are glycolysis and ROS-dependent processes that can be reduced by using antioxidants. Hence, our hypothesis is that the optimized medium to cryopreserve sperm will attenuate the overactivation of innate immunity response, which will translate into the maintenance of a greater number of spermatozoa with preserved function post-AI process, which will increase the number of piglets using frozen/thawed semen. Therefore, the objective is to characterize the activation of the NETs process induced by cryopreserved spermatozoa and its impact on sperm function; and the use of NETs inhibitors or antioxidants as a strategy to improve reproductive outcomes during AI. This research, through an in vitro, ex vivo, and in vivo model, will evaluate the use of optimized cryopreserved sperm pretreated with antioxidant substances, membrane-stabilizing saccharides can reduce the extrusion of NETs and modify the response of innate immunity at the endometrial level. In addition, the modulation of the NETosis process with the addition of NETs inhibitors or antioxidants in the intrauterine insemination medium can be crucial to increasing the number of spermatozoa with intact functionality that can improve the rate of offspring born alive with AI with cryopreserved semen. This would allow modifying media used for intrauterine insemination in the different species of domestic animals, reducing the number of spermatozoa used during this procedure, and reducing costs, especially when using sexed semen. (AU)

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