Cooling rate modifies the location of aquaporin 3 in spermatozoa of sheep and goat.

The freeze-thawing process induces osmotic changes that may affect the membrane domain location of aquaporins' (AQP) in spermatozoa. Recent studies suggest that changes in AQP3 localization allows better sperm osmo-adaptation, improving the cryoresistance. Ultra-rapid freezing is an alternative cryopreservation technique that requires less equipment than conventional freezing, and it is faster, simpler and can be used in the field. This study aimed to determine the influence of freezing-thawing rates (slow (control) vs. ultra-rapid) on AQP3 expression and location in the spermatozoa from small ruminants (sheep and goats) and its relationship with sperm cryo-damage. Spermatozoa were collected from 10 Merino rams and 10 Murciano-Granadina bucks. The presence and distribution of AQP3 were assessed by Western blotting and immunocytochemistry (ICC), employing a commercial rabbit polyclonal antibody. Sperm motility was CASA system-analyzed, and membrane and acrosome integrity assessed by fluorescence (PI/PNA-FITC). Western blotting did not detect a significant effect of freezing-thawing rate on the amount of AQP3 while ICC found freezing-thawing rate affecting AQP3 location (P < 0.05). In both species, the percentages of spermatozoa showing AQP3 in the post-acrosome region, mid-piece, and principal piece of the tail were greater in samples cryopreserved by slow freezing-thawing (control) than ultra-rapid freezing-thawing rates (P < 0.05). Spermatozoa cryopreserved using ultra-rapid freezing-thawing showed decrease motility, plasma membrane, and acrosome integrity (P < 0.05), which might be related, at least in part, to a lower expression of AQP3. In conclusion, the cooling rate modifies the location of AQP3 in spermatozoa of sheep and goat, which might be associated with sperm cryosurvival.

Colostrum Quality Assessment in Dairy Goats: Use of an On-Farm Optical Refractometer.

Failure of passive immunity transfer is one of the main causes of increased susceptibility to infectious agents in newborn kids. To ensure successful transfer of passive immunity, kids need to be fed high-quality colostrum, containing an adequate concentration of IgG. This work evaluated the quality of colostrum obtained in the first 3 days postpartum from Malagueña dairy goats. The IgG concentration in colostrum was measured using an ELISA as a reference method, and it was estimated by optical refractometer. Colostrum composition in terms of fat and protein was also determined. The mean concentration of IgG was 36.6 ± 2.3 mg/mL, 22.4 ± 1.5 mg/mL and 8.4 ± 1.0 mg/mL on days 1, 2 and 3 after parturition, respectively. Brix values obtained using the optical refractometer were 23.2%, 18.6% and 14.1% for days 1, 2 and 3, respectively. In this population, 89% of goats produced high-quality colostrum with IgG concentrations of >20 mg/mL on the day of parturition, but this percentage declined dramatically over the following 2 days. The quality of the fresh colostrum estimated with the optical refractometer was positively correlated with those obtained using ELISA (r = 0.607, p = 0.001). This study highlights the importance of feeding first-day colostrum to newborn kids and demonstrates that the optical Brix refractometer is suitable for the on-farm estimation of IgG content in colostrum.

Periovulatory Hormonal Profiles after Estrus Induction and Conception Rate by Fixed-Time AI in Payoya Goats during the Anestrous Season.

Sexual activity in domestic goats is positively influenced by reducing the photoperiod. Various protocols have therefore been developed in goats for the induction and synchronization of estrus during those months in which their sexual activity is reduced. The present observational study evaluates the periovulatory hormonal profile in Payoya goats (n = 24), during a non-favorable photoperiod (i.e., spring), being treated for estrus induction. The treatment comprised the vaginal insertion of sponges impregnated with progestogen (fluorogestone acetate, FGA), together with cloprostenol and equine chorionic gonadotrophin (eCG), 48 h before the end of the treatment. When the treatment ended, the plasma concentrations of the LH, FSH, progesterone and estradiol were determined. The goats were inseminated 46 h after the sponge withdrawal, and a pregnancy diagnosis was carried out 40-45 days after the insemination. Various parameters were monitored, such as the peaks of luteinizing hormone (LH), follicle-stimulating hormone (FSH) and estradiol, and their respective intervals, in reference to the time of the sponge withdrawal. The conception rate was 62.5%, and the kidding rate was 50%. The results record the hormonal release pattern after the estrus synchronization treatment based on the FGA, and the differences between the pregnant and non-pregnant goats. The findings suggest that the LH peak produced after the estrus synchronization treatment, both in terms of the amplitude and the time of increment, is involved in the reproductive failure detected.

Frozen dog spermatozoa are negatively affected during storage at -80, -21 and -8 ºC.

Cryopreservation in liquid nitrogen (LN2) allows for semen to be stored for long periods of time while there is sustaining of sperm viability. In this study, there was assessment of effects induced by different storage temperatures on cryopreserved dog spermatozoa. After cryopreservation at -196 °C, sperm samples were transferred to storage conditions of -80, 21 or -8 °C. Sperm motility, morphology, viability, acrosome integrity, mitochondrial membrane potential and DNA fragmentation were determined in samples stored at -196 °C (evaluation time =0 h), and then after 12 h and 1, 4, 7 and 15 d of storage at 80, -21 and -8 °C. In samples stored at -80 °C, sperm morphology, viability, acrosome integrity, mitochondrial membrane potential and DNA fragmentation did not differ at successive evaluation times. Progressive motility was less (P < 0.05) after 12 h and total motility after 4 d of storage at -80 ºC as compared with that of the 0 h sample. With storage at the other temperatures (-21 and -8 ºC), there was a reduction of mean values for sperm total and progressive motility, viability and mitochondrial membrane potential after 12 h of storage at these temperatures. Results, therefore, indicate the use of ultra-freezers at -80 ºC to store frozen dog semen allows for maintenance of sperm characteristics for at least 15 d but motility is sustained for only 1 d. Neither of the -21 or -8 ºC storage temperatures were effective for storing of frozen dog sperm and retaining viability.

Vitrification induces critical subcellular damages in ram spermatozoa.

The aim of the present study was to analyse morphological variations in ovine spermatozoa subjected to different cryopreservation protocols using high resolution imaging techniques. Ejaculates were pooled and diluted in Tris-based extender. Aliquots containing 300 × 106 spz/ml were prepared and evaluated a) after the semen collection and pooling, b) after conventional freezing, c) after vitrification of samples maintained at room temperature (22 °C) prior to vitrification, and d) after vitrification of samples maintained at 5 °C prior to vitrification. Sperm motility, acrosome integrity, DNA fragmentation and morphology were assessed. Subcellular sperm changes were assessed and described by light microscopy, scanning electron microscopy (SEM) and transmission electron microscopy (TEM). The maintenance of spermatozoa at 5 °C prior to vitrification and the use of 0.4 M sucrose pointed out lower dimensions of area, length and width than fresh, frozen and sperm maintained at 22 °C prior to vitrification. It was observed that the head width and length are significantly higher (P < 0.0001) in fresh spermatozoa than in the vitrified sperm samples. It could be hypothesized that greater intracellular fluid loss during vitrification could prevent damages in the spermatozoon throughout the reduced ice crystals formation, but mainly by the reduction of extracellular ice crystals due to the physical properties modification obtained when high concentrations of sugars are added. This is the first ultramicroscopic study carried out in ovine vitrified spermatozoa, which confirms the functional sperm alterations previously detected.