Lipidomic, metabolomic and functional status analysis of sperm from bulls with different fertility rates and susceptibility to cryopreservation profiles

Author(s)

Roberta Ferreira Leite

• Document type: Doctoral Thesis
• Press: São Paulo.
• Institution: Universidade de São Paulo (USP). Faculdade de Medicina Veterinária e Zootecnia (FMVZ/SBD)
• Defense date: 2022-03-23

Examining board members

Marcilio Nichi; Pietro Sampaio Baruselli; Daniel Rettori; Mateus Jose Sudano; Edson Guimarães Lo Turco

• Advisor: Marcilio Nichi

Abstract

Fertility and sperm quality and viability post-cryopreservation are fundamental to the animal production industry and are related to the integrity and function of all sperm structures. Besides, the technologies used on livestock to increase animal reproductive rates depend on sperm cryopreservation. However, the use of sperm from bulls with subfertility issues or from fertile bulls with a high loss of sperm viability during the cryopreservation process implies great losses for the livestock industry. The aim of this study was the association of functional and molecular analysis for the evaluation of sperm from bulls with different fertility rates and cryopreservation resistance profiles to identify functional and metabolic profiles from bulls with infertility and cryopreservation issues. The study was divided into two chapters, the first related to the fertility rate of bulls used in FTAI programs and the second to bull sperm with higher and lower susceptibility to cryopreservation. In both, sperm samples were analyzed to access their functional status (morphology, kinetics, plasma and acrosomal membrane integrity, mitochondrial activity, DNA integrity and oxidative status analysis) and their metabolic profiles (by LC-MS/MS analysis). The statistical analysis included: differences between groups by T-test, correlation, principal component analysis (PCA) and partial least squares discriminant analysis (PLS-DA). In the first chapter, an interaction analysis was performed before comparing the variables between the groups and, considering the PCA results, a ROC curve analysis was also made with the metabolites detected. In this chapter, cryopreserved samples from Angus (Bos taurus) and Nellore (Bos indicus) bulls with known fertility history in FTAI programs were analyzed in a 2x2 factorial design, considering the breed (Angus x Nellore), the fertility factor (high vs low) as well as the interaction between those factors. High fertility groups were constituted of 40 Angus sperm samples and 44 Nellore sperm samples, and the low fertility groups of 40 Angus sperm samples and 28 Nellore sperm samples (n=152). Results showed a clear difference between the Angus and Nellore breeds, mainly in the lipid and metabolic profiles. Regarding the differences between the high and low fertility groups, the functional and metabolic results indicated the presence of mitochondrial disorders, oxidative stress, and damage to sperm structures, especially in low fertility Angus. The metabolic profile difference between high and low fertility groups allowed the identification of potential fertility biomarkers with high accuracy, sensitivity and specificity values. In chapter 2, sperm samples collected on the same day from bulls under the same management and fertility profile were used. Samples were divided into two groups, with higher and lower susceptibility to cryopreservation, considering a reduction of more or less than 20% in post-cryopreservation sperm motility. Samples of eight bulls with a homogeneous distribution of breeds were selected for each group. Functional analysis was performed only on cryopreserved sperm samples, while mass spectrometry analysis was performed on seminal plasma and pre and post-cryopreservation sperm cells. Functional results showed a great difference between the groups regarding energy metabolism status, ROS presence and integrity of cellular structures. Regarding the lipid and metabolic profile analysis, there were also differences between the groups in all biological samples analyzed, which corroborated their functional status. It is important to note, however, the metabolic profile difference between the groups observed in pre-cryopreservation sperm. This difference may indicate that the preservation of sperm viability during cryopreservation depends much more on its ability to absorb and use these metabolites before the dilution, cooling and freezing process. Considering that fertility and sperm susceptibility to cryopreservation are multifactorial, the results obtained in this study with the correlation between functional and metabolic profiles, provide a better understanding of the pathways involved in the sperm energy metabolism and oxidative status, as well indicate potential biomarkers for early identification of bulls with subfertility or high susceptibility to cryopreservation problems. (AU)

• FAPESP's process: 19/20114-7 - Lipidomic, metabolomic and sperm functional characteristics analysis of bulls with different fertility rates and cryotolerance profiles
• Grantee: Roberta Ferreira Leite
• Support Opportunities: Scholarships in Brazil - Doctorate