Egg Yolk-Supplemented Tris-Citric Acid Extender Improves the Prefreezing and Post-Thaw Sperm Quality Indices of Guinea Pig (Cavia porcellus) Epididymal Spermatozoa.

This study aimed to assess the suitability of egg yolk (EY) supplementation to a tris-citric acid-based extender on cryosurvival of guinea pig (Cavia porcellus) epididymal spermatozoa. Two synthetic-based extenders, tris-citric acid-glucose plus 20% EY (TCG-EY) and tris-citric acid-fructose (TCF) both with 5% glycerol, were compared. Thirty-two epididymides were recovered from 16 adult guinea pig males by gonadectomy, and then the sperm samples were retrieved by retrograde flushing using TCG-EY and TCF extenders for left or right epididymis, respectively. TCG-EY and TCF sperm samples were frozen in static liquid nitrogen vapors through a two-step cooling procedure. Before freezing, the percentage of progressive sperm motility and sperm with intact plasma and acrosome membranes from TCG-EY sperm samples were higher (p < 0.05) than those diluted with TCF. Post-thaw sperm kinematic variables and membrane integrity were drastically reduced (p < 0.001) compared with prefreezing samples, regardless of extender type. The post-thaw plasmatic and acrosome membrane integrity from TCG-EY sperm samples was higher (p < 0.05) than those from TCF samples. Except for the length, the morphometric head dimensions of sperm diluted with TCG-EY or TCF did not vary (p > 0.05) after the freezing-thawing process compared with the prefreezing samples. In conclusion, despite greater cell cryoinjury with both extenders, the EY supplementation exerted greater cell membrane protection before and after the freezing-thawing process. This research shows an in-depth analysis of guinea pig sperm cryopreservation; however, more studies are recommended.

Accuracy of ultrasound and visual inspection in antral follicular count in crossbred Holstein cows raised under grazing systems at high altitude.

The objective of this research was to investigate the diagnostic accuracy of post-mortem ultrasound in antral follicle count (AFC) determination and compare it with visual AFC in grazing crossbred Holstein cows, at high altitude in Ecuador. Pre-mortem blood from 80 cows was collected, and AFC and ovarian characteristics were analysed post-mortem by ultrasound and visual techniques. AFC counts were stratified as high, medium or low by terciles. Mean AMH concentration in pre-mortem blood was 280.1 ± 15.53 pg/mL. The AFC obtained by visual inspection (26.9 ± 9.49 follicles) was 23.8% higher than by ultrasound (20.5 ± 7.53 follicles) in all ovaries. Body condition score, age and weight of the cattle did not interact with the count technique. In the low AFC group, visual inspection and ultrasound provided similar AFC results. However, in the Medium- and High-AFC groups, AFC by ultrasound was 14.9% lower than AFC by visual inspection. We confirm that ultrasound can be used with great accuracy for AFC >3 mm (close to the resolution limit) in grazing crossbred Holstein cows at high altitude.

Relationship between the concentration of anti-Müllerian hormone and antral follicle count in pasture-managed Holstein cows in the high tropics.

We evaluated the relationship between plasma levels of anti-Müllerian hormone (AMH) and the number of antral follicles at the restart of the follicular wave in crossbred Holstein cows reared under extensive grazing systems over 2500 m above sea level. The study included 140 cows from 15 farms that were in average at the 75.3 ± 2.10 d post partum. Animals were synchronized according to the following regime: day 0 = intravaginal progesterone releasing device (IPD) + estradiol benzoate (EB); day 7: withdrawal of IPD + prostaglandin; and day 8: EB, for restart of the follicular wave on day 11. On this day 11, antral follicle counts (AFCs) were made by transrectal ultrasound, and a plasma sample was taken for the determination of AMH. The mean AMH plasma level was 0.06 ± 0.03 ng/ml and the mean AFC was 17.26 ± 0.38 follicles. A strong positive linear correlation was found between these two variables (r = 0.783, r = 0.613, P < 0.0001). Cows were categorized according to AMH concentration as high (>0.09 ng/ml), intermediate (0.09-0.05 ng/ml) or low (<0.05 ng/ml). Cows with high AMH presented a higher AFC (25.0 ± 2.21 follicles) than those with low AMH (14.08 ± 2.68 follicles; P < 0.001. Our results suggest that the cut-off value of AMH = 0.09 ng/ml may be useful for selecting donors in multiple ovulation embryo transfer programs involving cows with these characteristics. Our data further suggest that AMH plasma concentration correlates with AFC and can be used as an endocrine biomarker of the number of antral follicles present at a given moment of the estrous cycle in crossbred Holstein cows raised at altitudes above 2500 m.

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.