Uterine nitric oxide levels and isofluopredone treatment effect in mares susceptible to persistent post-breeding endometritis

Transient endometritis is a normal consequence of breeding and results from uterine contamination with both semen and bacteria. The modulation of the inflammatory response with the use of isoflupredone has been proposed as efficient for the treatment of endometritis by increasing pregnancy rates. The aim of the current study was to determine the effects of isoflupredone on nitric oxide (NO) levels in uterine samples from mares susceptible to persistent postbreeding endometritis, presenting or not the infectious process. Seven consecutive estrous cycles were induced in 11 mares, being the first one used as control (no treatment). All mares were submitted to the following four treatments: treatment 1: control, treatment 2: glucocorticoid (GC) treatment (20 mg isoflupredone acetate) every 12 h, for three consecutive days, treatment 3: infected treatment (intrauterine infusion of 1x109 CFU/ml Streptococcus equi subsp. zooepidemicus), treatment 4: combination of GC + infected treatment (infusion of bacteria 24 h after the first GC treatment). At 12 h after the end of each treatment, uterine samples were collected by flushing and NO was determined. After nitrate reduction, total nitrite was determined by spectrophotometer. No significant differences on nitric oxide concentration were verified by analysis of variance in the different experimental groups. It is concluded that the use of isoflupredone did not alter the nitric oxide concentration in uterine flushing’s from susceptible mares 12 h after treatment.

Uterine nitric oxide levels and isofluopredone treatment effect in mares susceptible to persistent post-breeding endometritis

Transient endometritis is a normal consequence of breeding and results from uterine contamination with both semen and bacteria. The modulation of the inflammatory response with the use of isoflupredone has been proposed as efficient for the treatment of endometritis by increasing pregnancy rates. The aim of the current study was to determine the effects of isoflupredone on nitric oxide (NO) levels in uterine samples from mares susceptible to persistent postbreeding endometritis, presenting or not the infectious process. Seven consecutive estrous cycles were induced in 11 mares, being the first one used as control (no treatment). All mares were submitted to the following four treatments: treatment 1: control, treatment 2: glucocorticoid (GC) treatment (20 mg isoflupredone acetate) every 12 h, for three consecutive days, treatment 3: infected treatment (intrauterine infusion of 1x109 CFU/ml Streptococcus equi subsp. zooepidemicus), treatment 4: combination of GC + infected treatment (infusion of bacteria 24 h after the first GC treatment). At 12 h after the end of each treatment, uterine samples were collected by flushing and NO was determined. After nitrate reduction, total nitrite was determined by spectrophotometer. No significant differences on nitric oxide concentration were verified by analysis of variance in the different experimental groups. It is concluded that the use of isoflupredone did not alter the nitric oxide concentration in uterine flushings from susceptible mares 12 h after treatment.(AU)

Skim milk-egg yolk based semen extender compensates for non-enzymatic antioxidant activity loss during equine semen cryopreservation EN

Cryopreservation exposes spermatozoa to stressful conditions, leading to reduced cell viability. Several studies propose that overproduction of reactive oxygen species and decreased antioxidant capacity of semen may increase the damaging effects of the technique. The objective of this work was to evaluate the influence of a skim milk-egg yolk based semen extender on enzymatic and non-enzymatic antioxidant activity in equine semen cryopreservation. Fifteen ejaculates from six fertile Criollo stallions were cryopreserved using a commercial citrate-Hepes, egg yolk, skim milk and glycerol extender. Activities of catalase, glutathione peroxidase and superoxide dismutase and total radical-trapping antioxidant potential were assessed in raw semen, semen diluted in extender and thawed semen. All three enzymes showed higher activities in raw semen than in diluted or in thawed semen (P < 0.01), but enzyme activities did not differ significantly between diluted and thawed semen samples (P > 0.05). Non-enzymatic antioxidant defenses did not differ among any of the stages in the cryopreservation process (P > 0.05). In conclusion, the present study shows that dilution of semen with skim milk-egg yolk based extender after centrifugation compensates for the non-enzymatic antioxidant protection (but not enzymatic antioxidant defense) lost with seminal plasma removal. The absence of correlation between seminal and antioxidant parameters suggests that the compensation was enough for semen protection against oxidative stress, or antioxidant protection plays a minor role on semen from fertile stallions.(AU)

Skim milk-egg yolk based semen extender compensates for non-enzymatic antioxidant activity loss during equine semen cryopreservation

Cryopreservation exposes spermatozoa to stressful conditions, leading to reduced cell viability. Several studies propose that overproduction of reactive oxygen species and decreased antioxidant capacity of semen may increase the damaging effects of the technique. The objective of this work was to evaluate the influence of a skim milk-egg yolk based semen extender on enzymatic and non-enzymatic antioxidant activity in equine semen cryopreservation. Fifteen ejaculates from six fertile Criollo stallions were cryopreserved using a commercial citrate-Hepes, egg yolk, skim milk and glycerol extender. Activities of catalase, glutathione peroxidase and superoxide dismutase and total radical-trapping antioxidant potential were assessed in raw semen, semen diluted in extender and thawed semen. All three enzymes showed higher activities in raw semen than in diluted or in thawed semen (P 0.05). Non-enzymatic antioxidant defenses did not differ among any of the stages in the cryopreservation process (P > 0.05). In conclusion, the present study shows that dilution of semen with skim milk-egg yolk based extender after centrifugation compensates for the non-enzymatic antioxidant protection (but not enzymatic antioxidant defense) lost with seminal plasma removal. The absence of correlation between seminal and antioxidant parameters suggests that the compensation was enough for semen protection against oxidative stress, or antioxidant protection plays a minor role on semen from fertile stallions.

Recombinant expression and purification of the bovine acidic Seminal Fluid Protein

The acidic Seminal Fluid Protein (aSFP), a 12.9 kDa protein is a maker for bovine semen freezability possibly due to its antioxidant activity and effect on sperm mitochondrial function. However, its precise function on sperm preservation during freezing thaw is poorly understood. The use of recombinant DNA technology allows new approaches on the study of function and structure of proteins, and its production in procaryote systems offers several advantages. The present work describes the recombinant expression of the bovine aSFP and its binding properties. A cDNA library from the bovine seminal vesicle was used as template for amplification of the aSFP coding region. The amplicon was cloned into a pET23a (+) vector and transformed into E.coli BL21 pLysS strain. The recombinant expression was obtained in E coli. One step ion immobilized affinity chromatography was performed, resulting in high yield of purified protein. To determine the bioactivity of the r aSFP, the protein was incubated in different concentrations with 10 7 spermtozoa at 37°C for 5 h. Western blotting and fluorescence microscopy analyses showed the ability of the recombinant aSFP to attach to the spermatozoa. Based on our results, the described method can be used to obtain mg levels of recombinant aSFP.