Supplementation with MitoTEMPO before cryopreservation improves sperm quality and fertility potential of Piedmontese beef bull semen.

The purpose of this study was to improve the quality of frozen-thawed Piedmontese bull semen by incorporating MitoTEMPO (MT) in extended semen before cryopreservation. Semen was collected from 4 fertile bulls, using an artificial vagina, once weekly for 6 consecutive weeks. Semen samples were pooled, diluted with Bullxcell® extender, and supplemented with different concentrations of MT (0 as control, 5, 10, 20, 40, and 80 µM) before cooling, equilibration, and freezing procedures. The frozen-thawed semen was assessed for motility, vitality, acrosome intactness, plasma membrane integrity, DNA integrity, apoptosis, mitochondrial membrane potential, intracellular ROS level and in vitro fertilizing capability. The results showed that MT at concentrations of 10, 20, and 40 µM improved the total, progressive, and rapid motility directly after thawing while, at the highest tested concentration (80 µM), it decreased the progressive and rapid motility after 1, 2, and 3 h of incubation. The sperm kinetics including STR and LIN were noticeably increased at concentrations of 10, 20, and 40 µM directly after thawing (0 h), whereas the MT effect was variable on the other sperm kinetics during the different incubation periods. MitoTEMPO improved the sperm vitality at all tested concentrations, while the acrosomal and DNA integrity were improved at 20 µM and the mitochondrial membrane potentials was increased at 80 µM. The cleavage and blastocyst formation rates were significantly increased by using semen treated with 20 µM MT compared with controls. These findings suggest a potential use of MT mainly at a concentration of 20 µM as an additive in the cryopreservation media of bull semen to improve sperm quality.

In vitro effects of different ozone preparations on microorganisms responsible for endometritis in the mare.

Infectious endometritis is considered one of the major causes of infertility and it can affect up to 60% of barren mares. It is characterized by the presence of one or more microorganisms in the reproductive tract and it is treated with the administration of antibiotics, ecbolic agents and uterine lavages. Ozone, thanks to its antimicrobial properties that are based on its high oxidative potential, could represent an effective alternative treatment for endometritis. The aim of this study was to test in vitro the bactericidal and fungicidal properties of different ozone formulations, either as gas (experiment 1) or dissolved in two liquid matrices (experiment 2), specifically distilled water or oil (Neozone 4000, Cosmoproject, Parma, Italy), onto 6 different species of microorganisms isolated from mares with clinical endometritis, namely Escherichia coli, Staphylococcus aureus, Streptococcus equi subsp. Zooepidemicus, Pseudomonas aeruginosa, Klebsiella pneumoniae and Candida albicans. In the first experiment, 3 clinical antibiotic-resistant strains per each species were exposed to different conditions: to O2O3 gas mixtures (15 and 40 µg/ml for 1, 3 and 5 min), to 100 % O2 or left untreated. The results showed a reduction of the microbial count of over 99,9% for every pathogen, time and concentration of O2O3 gas mixtures tested. Furthermore, gaseous ozone showed both a time-dependant effect (5 vs 3 vs 1 min of exposure) and a concentration-dependant effect (40 vs 15 µg/ml) at 1 and 3 min, while after 5 min no differences were observed. In the second experiment, minimum inhibitory concentration (MIC), and minimum bactericidal/fungicidal concentration (MBC, MFC) of ozonated distilled water and ozonated oil were evaluated. Ozonated oil showed a bactericidal/fungicidal activity against all the strains tested (MIC range 12.5-25 % v/v, MBC/MFC range 12.5-50 % v/v) while ozonated distilled water didn't show an observable antimicrobial effect, discouraging its use as an antimicrobial agent for the treatment of endometritis. The results of this in vitro study indicate that both gaseous ozone and ozonated oil exerted remarkable antimicrobial activities and are promising alternative treatments for infectious endometritis, even when caused by antibiotic-resistant bacteria, and encourage further experiments in an effort to scale down or even prevent the use of antibiotics in equine reproduction.

Safety and Effects of a Commercial Ozone Foam Preparation on Endometrial Environment and Fertility of Mares.

Mares' subfertility represents a complex diagnostic and therapeutic challenge and both clinical and subclinical endometritis are considered major causes of impaired fertility. Thanks to its properties, ozone has a big potential as a treatment for equine endometritis. Therefore, the aim of this study is to describe the safety and the effects on endometrium and reproductive parameters of mares of a commercial ozone foam preparation (Riger Spray®). Twenty-four mares were treated during estrus: ozone group with an intrauterine instillation of ozone foam preparation (OG, n=16) and control group with 20 ml of lactated Ringer's solution (CG, n=8). Samples for endometrial cytology were collected before the ozone treatment (T0), after 24 h (T1), after one week (T2), two weeks (T3), and when the subsequent estrous phase was detected (T4). Furthermore, samples for histological examination and uterine swab for bacteriological examination were collected at T0 and T4. At T1, a statistically significant increase of endometrial inflammation in the OG mares compared to T0 (P<.05) and to CG at same time point (P<.05) was observed, but it was already resolved at T2. No differences in endometrial inflammation in CG, biopsy grade before and after the treatment in the two groups, number of mares pregnant at the end of the season and number of mares pregnant at the first cycle were observed. However, the number of inseminations required for pregnancy tended to be lower (P=.0711) in the OG (1.69±0.06) than in CG mares (2.60±0.89).

Effect of Epidermal Growth Factor (EGF) on Cryopreserved Piedmontese Bull Semen Characteristics.

The purpose of this study was to determine the effect on fresh and post-thaw beef bull semen quality of the supplementation of epidermal growth factor (EGF) to the semen extender at various concentrations (0-control, 50, 100, 200, and 400 ng/mL). For 8 weeks, sperm was collected from four fertile bulls, yielding a total of 32 ejaculates. Semen samples were pooled, diluted with Bullxcell® extender, and then cooled, equilibrated, and frozen. After thawing, semen was tested for motility and velocity parameters. Furthermore, semen was evaluated for vitality, integrity, mitochondrial and antioxidant (SOD) activities, mucus penetration distance, and in vitro fertilizing capability. The supplementation with EGF prior to cryopreservation improved the total sperm motility at various concentrations over long incubation periods (from 1 to 4 h). Interestingly, EGF addition improved both progressive and rapid motility, particularly at 50, 200, and 400 ng/mL. In addition, EGF, primarily at 200 and 400 ng/mL, significantly increased several velocity parameters after different incubation periods. We can conclude that adding EGF to bull sperm extender before cryopreservation has a positive stimulatory effect on sperm motility without affecting vitality, integrity, or in vitro fertilizing capability.