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SummaryInformation on donkey sperm bioenergetics, kinetics and oxidative status is scarce even though crucial for development of reproductive technologies and germplasm conservation. For these reasons, it is interesting to monitor sperm kinetics, bioenergetics, and oxidative status during sperm storage at +4°C and with several sperm extenders and concentrations. Donkey semen was collected from three jackasses, three times each. It was diluted with four extenders (Kenney, Equiplus, INRA96 or Hippex), set at three sperm concentrations (30, 50 or 70 × 106 spermatozoa/ml) and evaluated for its functionality after 0, 3, 24, 48 and 72 h storage at +4°C. Sperm kinetics was analyzed by Sperm Computer Analysis; sperm bioenergetics was assessed by mitochondrial membrane potential (MMP); sperm oxidative status was evaluated by lipid peroxidation (LPO), anti-LPO potential and nitroblue tetrazolium (NBT) assays. Incubation produced a progressive (P < 0.01) decline in sperm kinetics and MMP, whereas parameters related to oxidative status either increased (LPO, NBT) or decreased (anti-LPO). The anti-LPO potential was the index better related to sperm motility and kinetics. Extenders proved to be differently (P < 0.01) effective in preserving sperm kinetics, MMP, and oxidative status. The concentration of 30 × 106 spermatozoa/ml provided an optimum preservation of sperm functions. Significant correlations emerged between most parameters examined. This study identified reference criteria for storing donkey spermatozoa at +4°C. A low sperm concentration together with a proper extender are crucial requirements for optimum sperm cryopreservation efficiency. Field trials are, however, required to validate these findings, making them operational in practice.

SummaryDuring follicular development, granulosa cells undergo functional and structural changes affecting their steroidogenic activity. Oestrogen synthesis mainly occurs in the endoplasmic reticulum and relies on aromatase activity to convert androgens that arise from theca cells. In the present study, indicators of mitochondria-related steroidogenic capacity, as steroidogenic acute regulatory (StAR) protein expression and mitochondrial membrane potential (MMP), have been evaluated in bovine granulosa cells (GCs) and related to follicle growth and atresia. Atresia was estimated by morphological examination of follicle walls and cumulus–oocyte complexes (COC) and assessed by terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick end labeling (TUNEL) assay for apoptosis detection. Bovine ovarian follicles were macroscopically classified according to their atresia grade and grouped into small, medium or large follicles. After follicle opening, the COCs were morphologically classified for follicle atresia and the GCs were collected. Granulosa cells were fixed for immunofluorescence (IF) and TUNEL assay, frozen for western blotting (WB) or freshly maintained for MMP analyses. StAR protein expression was assessed using both IF and WB analyses. The follicle atresia grade could be efficiently discriminated based on either follicle wall or COC morphological evaluations. Granulosa cells collected from small non-atretic follicles showed a higher (P P

SummarySeveral genetic and physiological factors increase the risk of DNA damage in mammalian oocytes. Two critical events are: (i) meiosis progression, from maturation to fertilization, due to extensive chromatin remodelling during genome decondensation; and (ii) aging, which is associated with a progressive oxidative stress. In this work, we studied the transcriptional patterns of three genes,RAD51,APEX-1andMLH1, involved in DNA repair mechanisms. The analyses were performed by real-time quantitative PCR (RT-qPCR) in immature andin vitromatured oocytes collected from 17 ± 3-month-old heifers and 94 ± 20-month-old cows. Batches of 30–50 oocytes for each group (three replicates) were collected from ovarian follicles of slaughtered animals. The oocytes were freed from cumulus cells at the time of follicle removal, or afterin vitromaturation (IVM) carried out in M199 supplemented with 10% fetal calf serum, 10 IU luteinising hormone (LH)/ml, 0.1 IU follicle-stimulating hormone (FSH)/ml and 1 μg 17β-oestradiol/ml. Total RNA was extracted by Trizol method. The expression of bovineGAPDHgene was used as the internal standard, while primers for bovineRAD51,APEX-1andMLH1genes were designed from DNA sequences retrieved from GenBank. Results obtained indicate a clear up-regulation ofRAD51,APEX-1andMLH1genes after IVM, ranging between two- and four-fold compared with germinal vesicle (GV) oocytes. However, onlyRAD51showed a significant transcript increase between the immature oocytes collected from young or old individuals. This finding highlightsRAD51as a candidate gene marker for discriminating bovine immature oocytes in relation to the donor age.