Changes in Bull Semen Metabolome in Relation to Cryopreservation and Fertility

SIMPLE SUMMARY: Although semen cryopreservation has facilitated the diffusion of artificial insemination and in vitro fertilization in cattle, it still represents a major factor affecting sperm fertility. It is known that cryopreservation induces the loss of fertility-associated proteins, while the effect on metabolites has not been evaluated, although several compounds affect sperm physiology and fertility. The aim of the present work was to study the metabolome in bovine sperm and seminal plasma after cryopreservation and to correlate the metabolic profile of high- and low-fertility bulls in order to identify fertility markers. The analysis, carried out by liquid chromatography–mass spectrometry methods, revealed differences in metabolite contents between fresh and cryopreserved semen, both at cellular and plasmatic levels. Interestingly, metabolites showing variation have important functions related to fertility. In addition, the study highlighted the differences in lipid profile between high- and low-fertility bulls. The identification of new potential fertility markers is of high economic impact. In addition, it opens the way for the development of corrective strategies to improve the fertility of low-fertility bulls. ABSTRACT: Semen cryopreservation determines several sperm damages, including the loss of fertility-associated proteins. The purpose of the study was to compare the metabolite contents in bovine sperm and seminal plasma before and after cryopreservation, and between high- and low-fertility bulls in vitro. Forty-eight ejaculates, collected from eight bulls (six per bull), were analyzed by liquid chromatography–mass spectrometry. Cryopreservation resulted in an over-expression of lysophosphatidylcholine (0:0/18:2(9Z,12Z)) in seminal plasma. In addition, higher levels of glycine betaine and pyro-l-glutaminyl-l-glutamine were observed in cryopreserved compared to fresh spermatozoa. The fresh seminal plasma of high-fertility bulls showed an over-expression of l-acetylcarnitine, glycerol tripropanoate, 2,3-diacetoxypropyl stearate and glycerophosphocholine, and an under-expression of lysophosphatidylcholine and butyrylcarnitine, compared to low-fertility bulls. Higher levels of glycerophosphocholine and lysophosphatidylcholine (16:0/0:0) were recorded in fresh spermatozoa from high-fertility bulls. In high-fertility bulls, a greater content of glycerophosphocholine and lower levels of butyrylcarnitine, glycine betaine and l-carnitine were found in cryopreserved seminal plasma, and lower levels of glycine betaine were detected in cryopreserved spermatozoa. In conclusion, cryopreservation affects bovine semen metabolome at both plasmatic and cellular compartments, and metabolic profile differs between high- and low-fertility bulls.