Metabolomics signatures on the influence of holding time on increasing boar spermatozoa cryotolerance

Author(s)

Mariana Andrade Torres

• Document type: Doctoral Thesis
• Press: São Paulo.
• Institution: Universidade de São Paulo (USP). Faculdade de Medicina Veterinária e Zootecnia (FMVZ/SBD)
• Defense date: 2020-01-10

Examining board members

Marcilio Nichi; Maíra Bianchi Rodrigues Alves; Ivan Cunha Bustamante Filho; Eneiva Carla Carvalho Celeghini; Edson Guimarães Lo Turco

• Advisor: André Furugen Cesar de Andrade; Heidge Fukumasu

Abstract

Cryopreservation of boar semen is still a major challenge due to differences in the cryotolerance potential of ejaculates influencing post-thawed semen quality. Holding time (HT) is an alternative used in cryopreservation protocols to improve the quality of thawed semen, it is characterized by semen refrigeration at 17ºC before cryopreservation. However, the ideal period of holding is unknown. During this period, unknown interactions occur between spermatozoa and seminal plasma (and extender) that can alter the cryotolerance potential differently between high and low freezing ejaculates. To understand these interactions, the use of metabolomics as a molecular tool is essential, since metabolomics is considered the biochemical phenotype of cells/fluids at a given time. The present study aimed to 1) verify which is the ideal HT period for cryopreservation; 2) check if ejaculate freezability is affected by the use of HT; and 3) investigate the effects of HT on the ejaculate freezability on the metabolome of spermatozoa and seminal plasma (SP). For the first study, the semen was cryopreserved using seven different HT periods (0, 4, 8, 12, 24, 28 e 32 hours). The results of thawed semen showed that for total and progressive motility and the integrity of the plasma and acrosomal membranes, the best results were obtained after 24 hours of HT. For the second study, one sperm-rich fraction was collected from each of the 27 boars. These ejaculates were cryopreserved with (24 hours) and without (0 hours) the use of HT. After each HT period (0 and 24 hours) the SP and the spermatozoa were separated for further analysis of the metabolome. Of the total ejaculates, five were classified as high (EAC) and five as low (EBC) freezability based on the loss of total motility and integrity of the plasma membrane. An interaction was observed between the use of HT and the freezing of ejaculates, that is, EACs are only able to demonstrate their freezability potential if cryopreserved after 24 hours at 17ºC. Also, metabolomic analyzes of PS and spermatozoa were performed before and after HT of EAC and EBC. It was possible to describe that the sperm\'s metabolomic signature is due to the differences in freezability inherent to the ejaculate, characterized by changes in the abundance of some metabolites between the experimental groups. The SP metabolomic signature is characterized by the intrinsic differences to HT, characterized by changes in the abundance of metabolites between ejaculates cryopreserved with or without HT. Thus, the interaction between the differences in the metabolites of sperm and PS may be responsible for explaining the results observed in the physiology of thawed sperm. (AU)

• FAPESP's process: 16/09441-8 - Metabolomics sgnatures on the influence of holding time on increasing boar spermatozoa cryotolerance
• Grantee: Mariana Andrade Torres
• Support Opportunities: Scholarships in Brazil - Doctorate