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.

Intraovarian influence of bovine corpus luteum on oocyte morphometry and developmental competence, embryo production and cryotolerance.

Three experiments were conducted to determine influence of the bovine corpus luteum (CL) on morphometric and functional characteristics of oocytes, and subsequent embryonic development. Cumulus-oocyte complexes were aspirated from two types of cows: 1) with a CL in one ovary (CL+) and without a CL in the contralateral ovary (CL-), 2) and from cows without CL in either ovary (C). Intracellular activity of the enzyme glucose-6-phosphate dehydrogenase (G6PDH), oocyte diameter and thickness of the zona pellucida were determined (Experiment 1). Then, the rate of in vitro oocyte maturation for each ovarian category was evaluated and oocyte diameter and zona pellucida thickness were measured after maturation (Experiment 2). In Experiment 3, in vitro embryo production and cryotolerance were assessed. The oocyte diameter was greater (P < 0.01) and the zona pellucida was thinner in CL+ than in CL- (P > 0.05) or C (P = 0.0131) ovaries. Activity of G6PDH was lower in oocytes from CL+ than CL- (P < 0.01) and C (P = 0.0148) ovaries. Rate of oocyte maturation, oocyte diameter and thickness of the zona pellucida after maturation did not differ among groups. Rate of cleavage was greater in zygotes from CL+ than from CL- or C (P < 0.01); and CL+ ovaries produced more total embryos on day 7 (P < 0.05) and more blastocysts (P < 0.01) than CL- and C ovaries. Rate of expansion and hatching of day-7 vitrified-warmed blastocysts at 24 and 48 h of culture did not differ among groups. In conclusion, oocytes collected from CL+ ovaries were larger and metabolically more prepared to continue maturation than those from ovaries lacking a CL. Also, rates of cleavage and yield of blastocysts were greater for oocytes from CL+ ovaries than from CL- and C ovaries. These findings indicate that a CL influenced oocyte developmental competence and embryonic development, presumably through intraovarian interactions.

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.