l-carnitine enhances the kinematics and protects the sperm membranes of chilled and frozen-thawed Peruvian Paso horse spermatozoa.

l-carnitine (LC) transports fatty acids to the mitochondria for energy production, reducing lipid availability for peroxidation through ß-oxidation. This research examines the effect of LC supplementation to two skimmed milk-based extenders on the cryosurvival of chilled (5°C) and frozen-thawed Peruvian Paso horse spermatozoa .An initial experiment determined the optimal LC concentration (0, 1, 5, 10, 25, and 50 mM) when added to INRA-96® and UHT (skimmed milk + 6% egg yolk) extenders, using nine ejaculates from three stallions chilled for up to 96 h. Subsequently, the effect of 25 mM LC supplementation (the optimal concentration) on chilling (INRA-96) and freezing (INRA-Freeze®) extenders was evaluated using eight pooled samples from sixteen ejaculates (2 ejaculates/pool) from four stallions. Results indicated that all LC concentrations produced significantly higher values (P<0.05) for kinematic variables (total [TM] and progressive motilities, curvilinear [VCL] and straight-line [VSL] velocity, and beat-cross frequency [BCF]), and the integrity of plasma/acrosome membranes (IPIA) compared to non-supplemented chilled sperm samples for up to 96 h with both extenders. Moreover, the use of 25 mM LC was more efficient (P<0.05) in preserving the post-chilled values of velocity, BCF, and IPIA for the long term than lower LC concentrations (1-10 mM). Post-thaw values of total motility, the amplitude of lateral head displacement (ALH), and IPIA were significantly improved (P<0.05) when INRA-Freeze extender was supplemented with 25 mM LC. In conclusion, supplementation of l-carnitine to skimmed milk-based extenders enhanced kinematic variables and protected the membrane integrity in chilled and frozen-thawed Peruvian Paso horse spermatozoa.

Effect of Melatonin and Caffeine Supplementation to Freezing Medium on Cryosurvival of Peruvian Paso Horse Sperm Using a Two-Step Accelerating Cooling Rate.

This research examined the antioxidant and cryoprotective effects of melatonin (ME) and caffeine (CAF) supplementation in freezing medium on the cryosurvival of Peruvian Paso horse sperm using a two-step accelerating cooling rate. Twenty ejaculates from four adult and fertile stallions were recovered, initially diluted with INRA-96®, and finally frozen with INRA-Freeze® with either no supplementation (as control), 1 µM ME, or 2 mM CAF using a two-ramp freezing system content inside a cryogenic-box and liquid nitrogen vapors. The sperm kinematic parameters and integrity of the plasma and acrosomal membranes of fresh semen and cryopreserved samples were evaluated using the CASA system (SCA-Evolution® 2018) and PI/fluorescein isothiocyanate-conjugated peanut (Arachis hypogaea) agglutinin double fluorescent test, respectively. The oxidative stress of post-thaw sperm samples was also assessed using the CellRox Deep Red fluorescence test. The results showed that curvilinear velocity and average-path velocity were greater (p < 0.05) after freezing with CAF than the control group. In addition, there were significance differences (p < 0.01) between stallions (1-4) in post-thaw kinematic parameters regardless of ME or CAF addition. Both ME and CAF improved (p < 0.05) the proportion of sperm with intact plasma membranes and intact acrosomes. Nevertheless, neither CAF nor ME improved the oxidative stress after the cryopreservation process.

Intrauterine therapy with ozone reduces subclinical endometritis and improves reproductive performance in postpartum dairy cows managed in pasture-based systems.

New postpartum strategies have been developed in dairy cows to ameliorate uterine health and reproductive performance, especially the first service conception rates. This study aimed to assess the effect of intrauterine therapy with ozone (IUTO) in early postpartum on subclinical endometritis prevalence and reproductive parameters in dairy cows under commercial farm conditions. For this purpose, eighty clinically healthy cows with a body condition score between 3.0 and 3.5, from four dairy farms, were randomly allocated into two groups: ozone therapy group (OG, n = 40), which were subjected to IUTO, and control group (CG, n = 40). Content of uterine polymorphonuclear (PMN) leukocytes and subclinical endometritis (SE) percentage were assessed at 35 days after calving by uterine cytology. A second cytology was performed 72 h after IUTO. Reproductive parameters such as interval calving to first service (IFS), number of services per conception (nSC), interval calving to conception (ICC) and first service conception rate (FSCR) were analysed. The second endometrial cytology demonstrated that IUTO reduced (P < 0.01) both PMN (3.7 ± 1.4 vs. 7.6 ± 1.1%) and SE (5.0 vs. 50.0%) percentages compared with CG. Likewise, after ozone treatment, both nSC (2.1 ± 0.3 vs. 3.1 ± 0.2; P < 0.01) and ICC (126.2 ± 9.7 vs. 149.0 ± 9.0; P = 0.0672) decreased, and FSCR increased (50.0 vs. 16.2%; P < 0.01) compared with CG. In conclusion, intrauterine ozone therapy applied at 35 days after calving reduced subclinical endometritis prevalence and improved reproductive performance in postpartum dairy cows managed in a pasture-based system.

Supplementing a skimmed milk-egg yolk-based extender with L-carnitine helps maintain the motility, membrane integrity and fertilizing capacity of chilled ram sperm.

This study examines the effect of L-carnitine (LC) on chilled ram semen stored for up to 96 hr. Semen samples were collected, placed in a skimmed milk + 6% egg yolk extender, pooled, aliquoted and diluted with the same extender supplemented with different LC concentration: 0 (control), 1 mM (LC1), 2.5 mM (LC2.5), 5 mM (LC5), 7.5 mM (LC7.5) or10 mM (LC10). Sperm kinetics and membranes (plasma, acrosome and mitochondrial) were examined using the CASA system and triple fluorescence staining (PI/ PNA-FITC/Mitotracker). The progressive motility was greater (p < .05) with LC7.5 treatment than the control sperm at 96 hr. The curvilinear velocity (p < .01) and the percentage of sperm with intact membranes (plasma/acrosome/mitochondria) (p < .01) were greater with all LC treatments than the control group at all times. Straight line velocity was greater (p < .01) with LC5 and LC7.5 treatments than the control group after 48 hr. The LC5 group also returned lower ALH values (p < .05) than these seen for the control groups after 48 hr. The fertilizing capacity of LC5 samples stored at 15°C for 2 hr (LC5-15°C-2h) and at 5°C for 24 hr (LC5-5°C-24h) was tested in three ewe groups via cervical fixed-time artificial insemination. In two groups, the fertilizing capacity of the LC5-5°C-24h was reduced (p < .001). In the remaining group, however, no significant difference was seen between the LC5-15°C-2h and LC5-5°C-24h sperm in this respect (pregnancy rates 52.4% versus 42.8%; p > .05). Overall, the present results suggest that supplementing skimmed milk-egg yolk-based extenders with LC has a positive effect on chilled sperm variables and fertilizing capacity.