Small molecules and miRNAs as cryotolerance and fertility biomarkers in boar semen

• Grant number: 24/09891-0
• Support Opportunities: Regular Research Grants
• Start date: May 01, 2025
• End date: April 30, 2028
• Field of knowledge: Agronomical Sciences - Veterinary Medicine - Animal Reproduction
• Principal Investigator: André Furugen Cesar de Andrade
• Grantee: André Furugen Cesar de Andrade
• Host Institution: Faculdade de Medicina Veterinária e Zootecnia (FMVZ). Universidade de São Paulo (USP). São Paulo , SP, Brazil
• Associated researchers: Fabiana Fernandes Bressan ; Heidge Fukumasu ; Maíra Bianchi Rodrigues Alves ; Robert Knox ; Simone Maria Massami Kitamura Martins ; Theresa Casey

Abstract

The use of metabolomics and the micro-RNA profile evaluation to study andrology and sperm function in boars is still very scarce. In recent projects completed by our group, we were pleasantly surprised to find that the type and sometimes the number of small molecules and micro-RNAs vary between ejaculates with good freezing capacity and ejaculates with low capacity to withstand the freezing process (different cryotolerances). These changes can be monitored in the metabolites and micro-RNAs found in the sperm cell. In the present proposal, we intend to evaluate in a targeted way, qualitatively and quantitatively, the eight (8) metabolites and micro-RNAs that differed in the spermatozoa belonging to the ejaculates with higher and lower cryotolerance in the article by Torres et al., 2022 and by Pedrosa et al., 2021 (Proc. FAPESP 2016/24690-4), respectively. Additionally, it will be verified whether the presence of these small molecules and micro-RNA are maintained constant when several ejaculates are obtained from the same boar. To carry out this experiment, as many boars as necessary will be used to obtain ejaculates with cryotolerance differences. These will have their collected ejaculates processed, and a portion sent (1/3) refrigerated (17º C) to the laboratory where the cryopreservation will take place, and a portion sent (2/3) to the farms where they will be used in homospermic artificial inseminations. Aliquots of spermatozoa from these ejaculates will be collected, processed, and stored for future analysis of metabolomics and micro-RNAs. After thawing, computerized motility analyses and simultaneous plasma and acrosomal membrane integrity assessment by flow cytometry will be performed. In this way, two groups of ejaculates will be formed: 1) those with good cryotolerance: total motility > 50% and sperm with plasma and acrosomal membrane integrity > 40%; 2) those with bad cryotolerance with total motility <35% and sperm with plasma and acrosomal membrane integrity <30%. Ten representative animals will be selected to compose the groups. The samples of these twenty selected animals (previously stored) will then be subjected to metabolomics and micro-RNA analysis. The fertility history and the number of piglets born from the evaluated ejaculates will then be requested for the correlation analysis between these variables and the metabolites and micro-RNAs. At the end of the project, we intend to prove the following hypotheses: 1) It is possible to identify a pattern of small molecules that are identified in ejaculates with good and bad cryotolerance; 2) The quantification of the eight (8) selected metabolites allows the classification of good and bad cryotolerance ejaculates; 3) The amount of the eight (8) evaluated metabolites is kept constant between different ejaculates of the same boar. 4) Cryotolerance positively correlates with fertility and the number of piglets born (total and live). (AU)