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.

Cryopreservation of dog epididymal spermatozoa by conventional freezing or ultra-rapid freezing with nonpermeable cryoprotectant.

This study was aimed to assess the effectiveness of two methods for cryopreservation of dog epididymal spermatozoa, one by conventional freezing (CF) with shortening both equilibration and cooling times, and the other by ultra-rapid freezing (URF) with nonpermeable cryoprotectant. Sixty epididymides were recovered from thirty orchiectomized adult dogs and the sperm samples were retrieved by retrograde flushing using TCG-EY (tris, citric acid, glucose + 20% egg yolk) extender and then 20 pools were conformed. Each pool was divided into 2 aliquots and then cryopreserved by CF and URF methods respectively. The CF method maintained the cooled-pool samples for 2h (1h without and 1h with 5% glycerol) and then were frozen by liquid nitrogen (LN2) vapors for 2 min. The URF method cryopreserved the cooled-pool samples using TCG-EY+250 mM sucrose, equilibrating during 30 min (5 °C) and submerging 30-µL drops directly in LN2. The results showed that the URF method produced a lower percentage of total and progressive motilities and acrosome integrity (P < 0.05) than the CF method. However, the kinetic variables (curvilinear and straight-line velocities, straightness, linearity, wobble, amplitude of lateral head displacement, and beat-cross frequency) and plasma membrane integrity did not differ (P > 0.05) between both cryopreservation methods. Unlike the URF method, the width, area and perimeter of sperm head were reduced after the CF method (P < 0.05). In conclusion, despite the low motility achieved after the ultra-rapid freezing method, the similar values of kinetic, viability and head morphometric dimensions to those obtained after conventional freezing, suggest that ultra-rapid freezing with sucrose may be a useful alternative for the cryopreservation of canine epididymal sperm.

Methods of collection, extender type, and freezability of semen collected from creole bulls raised in the tropical highlands of Ecuador.

This study was conducted to determine the best combination between two collection method and two extenders in the cryopreservation of semen from creole bulls adapted to highlands of the Ecuadorian Andes. Sixty ejaculates from three adult Creole bulls were evaluated after collection by artificial vagina (AV) and electroejaculation (EE). Semen samples were split into two aliquots and diluted with a soy lecithin extender (Andromed®; A) or an egg yolk-containing extender (Triladyl®; T) and packed in straws of 0.25 ml with 20 × 106 sperms. Optical microscopy and computer-assisted semen analysis system (CASA) were used to evaluate semen quality characteristics. The effects of collection methods and extender type as well as its interaction were evaluated by a factorial ANOVA and Bonferroni's test. Semen samples collected with EE and frozen with T (EE-T) and A (EE-A) had greater proportion of spermatozoa with optical assessed individual progressive motility (IPM), plasmatic membrane intact (HosT), and lower tail abnormalities than those obtained with AV and frozen with the same extenders (AV-T and AV-A); however, differences were significant only between EE-A and AV-T. CASA assessment indicated that the total mobility (TM) was greater (P < 0.05) in semen samples diluted with T, although these samples had a greater proportion (P < 0.05) of sperms with local motility (LM) and fewer immobile sperms (IS), than those extended with A. Generally, semen samples obtained with EE or AV and diluted with T seems to be the best option to ciopreserve gametes of Creole bulls raised in highlands of Ecuadorian Andes.