Cold-induced damage in boar sperm cooled to 5°C in Lactose Egg Yolk (LEY) medium with different Glycerol concentrations.

Boar sperm is highly susceptible to cold damage. When temperature drops to 5°C, the plasmatic membrane is destabilized. The freezing process causes a reduction of the fertility window because frozen/thawed boar sperm has less survivability. The aim of this work was to analyze the effect on sperm characteristics and response to capacitation stimuli of cooling to 5°C using a controlled protocol. Also, we evaluated if the addition of Glycerol 2% or 3% at 5°C was able to modify these parameters. For this purpose, we assessed motility, plasmatic membrane integrity and acrosomal membrane status. Capacitation was induced using Tyrode´s capacitating medium (TCM) and assessed by chlortetracycline stain and induction of acrosomal reaction with Progesterone. Motility patterns were analyzed using a CASA system. These tests were performed at three different points of the freezing curve: 37°C; 17°C and 5°C. Response to TCM vs TBM was only significant at 37°C. While at 37°C and 17°C capacitated sperm was below 20%, at 5°C reached 50% both in the TBM and TCM. CASA analysis showed that spermatozoa exposed to TCM had higher LIN and WOB than those in TBM. All parameters were similar in the Glycerol concentrations studied. These results suggest that the chilling process may be causing an effect similar to cryocapacitation along the cooling curve, starting subtle at 17°C and reaching 50% of the sperm population at 5°C, being independent of Glycerol concentration.

Effect of Mineral Supplementation on the Macromineral Concentration in Blood in Pre- and Postpartum Blackbelly Sheep.

The objective of this study was to determine the effect of mineral supplementation on the serum concentration of calcium, phosphorus, and magnesium in pre- and postpartum Blackbelly sheep throughout three successive lambing periods under free grazing conditions in the Ecuadorian Amazon Region. The field work was carried out between January 2015 and February 2018 using 20 Blackbelly sheep belonging to the Centre for Research, Postgraduate Studies and Conservation of Amazon Biodiversity, Ecuador. The flock was randomly divided into two groups: Group 1 (G1) was fed with forage plus a supplementation (Pecutrin® Mineral supplement plus vitamins A, D3, and E. Bayer HealthCare) and Group 2 (G2) was fed only with forage without mineral supplementation. Three blood samples from the coccygeal vein were taken from each sheep 30 days before lambing, 30 days after, and 60 days after lambing. Concerning the average of calcium, significant differences were found at different times inside each group and also between them (p < 0.0001 in both cases). As for the phosphorus, significant differences were found between the means of the groups for all times from 30 days after the second lambing season (p < 0.05). It was observed that the groups differed significantly in terms on the average of magnesium (considering a significance level of 0.05) 30 days before the first lambing and at all times measured from the 30 days after the second lambing (p < 0.005). In this study, we showed that Blackbelly sheep raised under free grazing conditions in the Ecuadorian Amazon Region had very low serum calcium values, and supplementation was unable to improve them. Meanwhile, phosphorus and magnesium levels were below the required values, but after supplementation, they exceeded the minimum threshold. Mineral supplementation in the rearing of sheep in grazing systems is necessary during the entire production cycle, but it must be done taking into account the soil-plant-animal relationship specifically for the Amazonian Region systems.

Effect of different glycerol concentrations on phosphatidylserine translocation and mitochondrial membrane potential in chilled boar spermatozoa.

Boar spermatozoa are extremely sensitive to low temperatures and the cryopreservation causes dramatic changes in sperm survivability, but it is not clear which part of the cryopreservation process affects the most. The aim of this work was to assess early events of apoptotic changes as damage indicators in boar sperm cooled to 5 °C and exposed to different glycerol (GLY) concentrations. For this purpose, progressive sperm motility (CASA), plasmatic and acrosome membranes integrity (CFDA/PI; phase contrast), plasma membrane functionality (HOS), phosphatidylserine translocation (Annexin-V/FITC) and reduction of mitochondrial membrane potential (Ψm) (JC-10) were carried out at 37 °C, 17 °C and 5 °C in eight boar sperm pools. Afterwards, three aliquots were diluted in different freezing extenders (control: 0% GLY; A: 2% GLY and B: 3% GLY); sperm quality and early apoptotic changes were assessed. Motility was negatively affected during cooling to 5 °C. Furthermore, plasma membrane functionality was the most affected by cooling. The number of necrotic cells was higher at 5 °C. However, no differences were observed in phosphatidylserine translocation. The extender with 3% GLY at 5 °C presented better Ψm than 0 and 2% GLY. Based on this analysis, boar sperm cooling to 5 °C does not modify the rate of early apoptotic changes, although alterations in the Ñ°m were evident.

Effects of different sucrose concentrations on vitrified porcine preantral follicles: Qualitative and quantitative analysis.

The aim of the present study was to perform a qualitative and quantitative analysis of the effect of different sucrose concentrations combined with ethylene glycol in the preservation of vitrified porcine preantral follicles. Fragments of ovarian cortex were vitrified in cryotubes containing 200 µl of the vitrification solution (30% Ethylene Glycol; 20% Fetal Bovine Serum; 0 M-0.25 M - 0.75 M or 1 M sucrose) and stored in liquid nitrogen for a week. Histological analysis showed that after vitrification the number of normal follicles decreased compared to the fresh tissue (control). The percentage of normal primordial follicles was sucrose dose dependent. The percentage of normal primary follicles was similar in 0 M or 0.25 M sucrose, while higher concentrations (0.75 M and 1 M) increased significantly the percentage of abnormal follicles (p < 0.05). Morphometric analysis showed a statistically significant reduction in the total area of primordial follicles with 0.75 M sucrose and a significant increase in the cytoplasmic area of primordial follicles with 0 M sucrose (p < 0.05). The qualitative and the quantitative analysis appear to be a complementary tool when choosing a vitrification protocol. For our cryopreservation system - vitrification of ovarian cortex slices in cryotubes-the best vitrification medium was TCM 199-Hepes with 30% de ethylene glycol, 20% of Fetal Bovine Serum and 0 or 0.25 M sucrose. The present study shows that the use of high sucrose concentrations in the vitrification solution has a deleterious effect on the preservation of porcine preantral follicles contained in ovarian tissue. Consequently, its use at 0.75 M or 1 M wouldn't be recommended.

Funcion de distintas formas de fibronectina en el desarrollo de foliculos bovinos in vitro / Role of different forms of fibronectin in in vitro bovine follicular development

En el presente estudio, se analizó la posibilidad de que las distintas formas de fibronectina (FN), producidas como resultado de la maduración alternativa (alternative splicing) del mensajero, ejerzan funciones diferenciales en el desarrollo folicular. En particular se determinó la presencia de la región ED-I, ausente en la FN plasmática, tanto a nivel de mensajero como de proteína, durante este proceso. El análisis de los niveles de FN en fluidos foliculares correspondientes a distintas etapas de desarrollo mostró marcadas variaciones en la concentración de FN ED-I+ y los de permanecieron relativamente constantes. En folículos corespondientes a la fase de selección se observó una correlación inversa entre los niveles de FN ED-I+ y los de estradio (p<0.001). El tratamiento con estradiol no tuvo efecto sobre el splicing alternativo de FN en cultivos de células de la granulosa bovinas, mientras que el AMPc tuvo un efecto inhibitorio sobre la incorporación de ED-I. Por otra parte, el factor de crecimiento transformante tipo beta (TGF-beta) estimuló tanto la produción de FN total como la inclusión de la región ED-I. Este efecto fue verificado tanto a nivel de la proteína (Western blots) como del ARN mensajero (Northern blots). Un péptido correspondiente a la región ED-I tuvo un efecto estimulatorio sobre el crecimento de una línea de células de la granulosa bovinas (BGC-1) mientras que el péptido correspondiente e las regiones flanqueantes no tuvo efecto. Los datos presentados en este estudio plantean una nueva forma de regulación mediante la cual cambios cualitativos en la estructura primaria de la FN podrían mediar algunas de las acciones de gonadotrofinas y factores intraováricos durante el desarrollo folicular.

Funcion de distintas formas de fibronectina en el desarrollo de foliculos bovinos in vitro / Role of different forms of fibronectin in in vitro bovine follicular development

En el presente estudio, se analizó la posibilidad de que las distintas formas de fibronectina (FN), producidas como resultado de la maduración alternativa (alternative splicing) del mensajero, ejerzan funciones diferenciales en el desarrollo folicular. En particular se determinó la presencia de la región ED-I, ausente en la FN plasmática, tanto a nivel de mensajero como de proteína, durante este proceso. El análisis de los niveles de FN en fluidos foliculares correspondientes a distintas etapas de desarrollo mostró marcadas variaciones en la concentración de FN ED-I+ y los de permanecieron relativamente constantes. En folículos corespondientes a la fase de selección se observó una correlación inversa entre los niveles de FN ED-I+ y los de estradio (p<0.001). El tratamiento con estradiol no tuvo efecto sobre el splicing alternativo de FN en cultivos de células de la granulosa bovinas, mientras que el AMPc tuvo un efecto inhibitorio sobre la incorporación de ED-I. Por otra parte, el factor de crecimiento transformante tipo beta (TGF-beta) estimuló tanto la produción de FN total como la inclusión de la región ED-I. Este efecto fue verificado tanto a nivel de la proteína (Western blots) como del ARN mensajero (Northern blots). Un péptido correspondiente a la región ED-I tuvo un efecto estimulatorio sobre el crecimento de una línea de células de la granulosa bovinas (BGC-1) mientras que el péptido correspondiente e las regiones flanqueantes no tuvo efecto. Los datos presentados en este estudio plantean una nueva forma de regulación mediante la cual cambios cualitativos en la estructura primaria de la FN podrían mediar algunas de las acciones de gonadotrofinas y factores intraováricos durante el desarrollo folicular. (AU)