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.

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Season clearly influences oocyte competence in buffalo (Bubalus bubalis); however, changes in the oocyte molecular status in relation to season are poorly understood. This study characterizes the microRNA (miRNA) and transcriptomic profiles of oocytes (OOs) and corresponding follicular cells (FCs) from buffalo ovaries collected in the breeding (BS) and non-breeding (NBS) seasons. In the BS, cleavage and blastocyst rates are significantly higher compared to NBS. Thirteen miRNAs and two mRNAs showed differential expression (DE) in FCs between BS and NBS. DE-miRNAs target gene analysis uncovered pathways associated with transforming growth factor β (TGFβ) and circadian clock photoperiod. Oocytes cluster in function of season for their miRNA content, showing 13 DE-miRNAs between BS and NBS. Between the two seasons, 22 differentially expressed genes were also observed. Gene Ontology (GO) analysis of miRNA target genes and differentially expressed genes (DEGs) in OOs highlights pathways related to triglyceride and sterol biosynthesis and storage. Co-expression analysis of miRNAs and mRNAs revealed a positive correlation between miR-296-3p and genes related to metabolism and hormone regulation. In conclusion, season significantly affects female fertility in buffalo and impacts on oocyte transcriptomic of genes related to folliculogenesis and acquisition of oocyte competence. Water buffalo (Bubalus bubalis) is an important livestock resource for both developing and developed countries. The major factor affecting buffalo farming profitability is reproductive seasonality, resulting in cycles of calving and milk production. Buffalo is a short-day breeder, with increased fertility in response to decreasing day length 1,2. This photoperiod-dependent seasonality pattern is more pronounced as distance from the equator, together with variations in the light/dark ratio, increases. In Italy, in order to satisfy market demand, out of breeding mating strategy (OBMS), consisting in interrupting sexual promiscuity or the use of artificial insemination (AI) during the breeding season (BS), is commonly utilized 2. The OBMS improves the distribution of calving throughout the year, but it reduces fertility 3. Longer post-partum anoestrus periods as well as higher incidence of embryonic mortality are observed in months with increasing daylight length and particularly in midwinter , which coincides with the transition to seasonal anoestrus at Italian latitudes 1,4. The embryonic mortality is due to inadequate luteal growth and function, resulting in reduced progesterone secretion 5. This has a negative impact on embryo growth, associated with alterations in transcriptomic and proteomic profiles of the embryos and chorioamnios/caruncles 6,7 , which ultimately impair embryo attachment to the uterine endometrium.

The reduced oocyte competence recorded during the non-breading season (NBS) is one of the key factors affecting the profitability of buffalo farming and limits the IVEP efficiency. The purpose of this experiment was to evaluate whether season influences the lipid content within the ovarian follicle in the Italian Mediterranean buffalo. Abattoir-derived ovaries were collected during the breeding season (BS) and the NBS, and different matrices (follicular fluid, oocytes, cumulus and follicular cells) were recovered. After the extraction of the apolar fraction, all samples were analyzed by H1 nuclear magnetic resonance and FF samples by gas chromatography–mass spectrometry. Seasonal differences in lipid composition were observed in all matrices. In particular, during the NBS, the triglyceride content was higher in the follicular fluid and in the oocytes but reduced in the follicular cells. Both cholesterol and phospholipids were reduced in the follicular fluid and follicular cells during the NBS. Furthermore, the total amount of non-esterified fatty acids was significantly increased in the follicular fluid. The seasonal variation in lipid profile of the follicle may, in part, account for the reduced buffalo oocyte competence during the NBS, due to the critical role played by lipids in regulating ovarian functions.

Sperm cryopreservation is essential for assisted reproduction; however, freezing induces biochemical and physical damage to the sperm membrane structures, negatively affecting sperm fertilising ability (Castro et al. 2006 J. Anim. Sci. Biotechnol. 5, 1-9). Metabolomics is the study of small molecules, commonly known as metabolites, which play essential roles in biological systems. Emerging research in the field of metabolomics showed a potential role in identifying male fertility biomarkers (Bieniek et al. 2016 Asian J. Androl. 18, 426-433). Metabolites present in the seminal plasma play several roles related to sperm motility, sperm membrane protection from oxidative stress, and regulation of metabolic activity (Therien et al. 1995 Biol. Reprod. 52, 1372-1379). However, the extent of metabolite changes in seminal plasma during cryopreservation is still not well known. Therefore, the aim of this study was to evaluate the differences of the seminal plasma metabolites between fresh and frozen bovine sperm. Four ejaculates from eight Holstein (Bos taurus) bulls (4-6 years age) maintained at an authorised national semen collection center (Centro Tori Chiacchierini, Civitella D’Arna, Italy) under uniform management conditions were collected weekly using an artificial vagina (IMV Technologies). Each ejaculate was split into two aliquots (one fresh and one frozen) at −20°C for at least 2h. Seminal plasma from fresh (immediately) and frozen-thawed semen was separated from sperm by centrifugation (1600×g for 10min), and supernatants were then transferred to a 2-mL tube and stored at −80°C until use. After methanol extraction of metabolites, samples were centrifuged at 6400×g for 30min at 4°C, and supernatants were analysed using a liquid chromatography-mass spectrometry (LC-MS; Agilent 1260 HPLC; Agilent Technologies) system with an autosampler and a binary pump coupled to an Agilent Q-TOF 6540 (Agilent Technologies). Univariate analyses of the data were performed using bioinformatics approaches. The differences between fresh and frozen seminal plasma samples were analysed using a paired Student's t-test. Twenty-three metabolites were found in both fresh and frozen seminal plasma samples, out of which eight were significantly different (P<0.05) between the two groups; among these, hypotaurine, ceramide, and isoindoline were identified. In particular, hypotaurine and ceramide were downregulated, whereas isoindoline was upregulated in fresh samples compared with frozen samples. To the best of our knowledge, this is the first study to identify seminal plasma metabolites in fresh and frozen samples by using LC-MS. In order to ensure the reliability and accuracy of results, the validation of the method used in the analysis of biosamples is still in progress, as is the evaluation of the metabolic profile in sperm cells. In conclusion, the results of this study suggest that LC-MS can be a promising screening tool to detect metabolites potentially associated with bull fertility.