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.

The aim of this work was to evaluate the efficiency of different FSH doses and FSH coasting times before ovum pick-up (OPU) on follicular growth and oocyte competence in buffalo. Experiment 1 involved two different FSH treatments: 40 mg FSH given three (FSH3) or six (FSH6) times, 2 days after dominant follicle removal were tested, with OPU carried out after 40–44 h of coasting. In experiment 2, OPU was carried out after FSH6 protocol followed by 28–32 h (C1), 40–44 h (C2), or 64–68 h (C3) of coasting time. Cumulus oocyte complexes (COCs) were classified, in vitro matured, fertilized, and cultured. The results demonstrated that FSH6 increased the total number of follicles, the number and percentages of medium and large follicles, the number and the proportion of good quality oocytes, and the number of grade 1,2 and fast-developing blastocysts compared to the control. C3 decreased the percentage of good quality oocyte and blastocyst rates compared to C1 and C2. A higher percentage of fast blastocysts and average number of grade 1,2 blastocysts was observed in C1 compared to C3, with intermediate values found in C2. The improved efficiency in terms of blastocyst yields suggests the use of FSH6 + C1 protocol for ovarian superstimulation in buffalo.

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.

Over the years, regenerative therapies have emerged as promising alternatives for persistent breeding-induced endometritis. In vitro studies testing the effects of these therapies on equine endometrial cells are still scarce. This study aimed to evaluate in vitro the effect of Wharton's jelly (WJ) mesenchymal stromal/stem cell (MSCs)-derived conditioned medium (WJ-CM) and platelet-rich plasma (PRP) on equine endometrial cells, with or without lipopolysaccharide (LPS)-induced inflammation. The WJ-CM was obtained after 24 h of starvation in Ringer's lactate of WJ-MSCs and PRP was prepared using the double centrifugation. Endometrial epithelial cells obtained from 3 diestrus mare uteri at slaughterhouse were treated for 24 h according to six experimental groups: DMEM standard complete medium (CTRL); 10 ng/mL LPS (LPS); 10 % WJ-CM (CM); 5 % PRP (PRP); 10 ng/mL LPS and 10 % WJ-CM (LPS + CM); 10 ng/mL LPS and 5 % PRP (LPS + PRP). After 6, 12, and 24 h, endometrial cells were evaluated for viability (apoptosis and necrosis), mitochondrial activity and reactive oxygen species (ROS) generation. PGE-2 and IL-10 concentrations in spent medium were measured. The WJ-CM alone did not affect endometrial cell viability and prevented the detrimental effect of LPS on endometrial cells; it suppressed the production of PGE-2. PRP had a deleterious effect on endometrial cell viability, induced the secretion of PGE-2, as well as increased mitochondrial activity and ROS production. Endometrial benefits of the WJ-CM treatment are evident even after an LPS challenge, while unexpectedly PRP showed a deleterious effect.