Vasectomy and Photoperiodic Regimen Modify the Protein Profile, Hormonal Content and Antioxidant Enzymes Activity of Ram Seminal Plasma.

This work aimed to determine the contribution of the testis and epididymis and the effect of the photoperiodic regimen on ram seminal plasma (SP). Semen was collected from 15 mature rams located in an equatorial (Colombian Creole and Romney Marsh, eight intact and two vasectomized) or a temperate climate (Rasa Aragonesa, three intact and two vasectomized). SP proteins were analyzed by Bradford, SDS-PAGE and difference gel electrophoresis (DIGE). Melatonin and testosterone concentrations were quantified by ELISA, and activity of glutathione peroxidase (GPx), glutathione reductase (GRD), and catalase by enzymatic assays. Vasectomy increased protein concentration and the intensity of high molecular weight bands (p < 0.001), with no differences between breeds. DIGE revealed the absence of six proteins in vasectomized rams: angiotensin-converting enzyme, lactotransferrin, phosphoglycerate kinase, sorbitol dehydrogenase, epididymal secretory glutathione peroxidase and epididymal secretory protein E1. Vasectomy also decreased melatonin concentrations in seasonal rams, and testosterone in all of them (p < 0.001), but did not affect antioxidant enzyme activity. Equatorial rams showed lower melatonin and testosterone concentration (p < 0.01) and catalase, but higher GPx activity (p < 0.05). In conclusion, vasectomy modifies the protein profile and hormonal content of ram seminal plasma, whereas the exposure to a constant photoperiod affects hormonal concentration and antioxidant enzymes activity.

Changes in melatonin concentrations in seminal plasma are not correlated with testosterone or antioxidant enzyme activity when rams are located in areas with an equatorial photoperiod.

In temperate climates, photoperiod and melatonin regulate ram reproduction, modulating hormonal secretions, sperm quality, and seminal plasma composition. Information on the effect of an equatorial photoperiod (12L:12D) on ram reproduction, however, is scarce, and no data on hormonal concentrations and antioxidant enzyme activity in seminal plasma have been reported. Thus, the variation was investigated of melatonin and its relationship with testosterone and antioxidant enzyme activity in the seminal plasma of three sheep breeds in Colombia, when there was a consistent photoperiod during two dry and two rainy seasons per year. Semen was collected once a week from 12 mature rams (four of each breed: Colombian Creole, Hampshire, and Romney Marsh). Seminal plasma was obtained by centrifugation. The concentration of melatonin and testosterone were quantified along with the enzymatic activity of glutathione peroxidase (GPx), glutathione reductase (GRD), and catalase (CAT). Correlation analyses between melatonin and testosterone concentrations or enzymatic activity were also performed. Melatonin concentration was affected by season (P < 0.05) but not breed, with lesser concentrations in the first rainy season. Testosterone concentration, however, was affected by breed and season, with greater concentrations (P < 0.01) in the Hampshire and Romney Marsh rams during the second dry season. Regarding antioxidant enzyme activity, there was only seasonal variation in GPx activity (P < 0.05). When correlation analyses were used for data assessments, there was a negative correlation between melatonin and testosterone concentrations in Hampshire rams. In conclusion, melatonin concentrations in seminal plasma of rams that were located in an area with an equatorial photoperiod was affected by the climatological season but there was no positive correlation with testosterone concentration or antioxidant enzyme activity.

Efecto de la adición de antioxidantes en los diluyentes para la preservación de semen ovino / Effect of adding antioxidants in diluents for the preservation of sheep semen / Efeito da adição de antioxidantes nos diluentes para preservação de sêmen ovino

Resumen El objetivo del presente estudio fue evaluar el efecto sobre la calidad espermática de tres substancias antioxidantes (50 µM de trolox/108 espermatozoides, 50 µg de catalasa/ml de eyaculado y cisteamina 5mM) en una base de diluyente comercial Triladyl™, bajo condiciones de refrigeración y congelación/descongelación en semen ovino. Se congeló semen de seis machos adultos en una base del diluyente comercial Triladyl™. El eyaculado de cada macho fue puesto en cuatro diferentes alícuotas: una para control, y a las demás se les adicionó con la base del diluyente 50 µM de trolox/108 espermatozoides, 50 µg de catalasa/ml de eyaculado y cisteamina 5 mM. La calidad espermática precongelación (5 °C) y poscongelación se evaluó con la ayuda de un sistema de análisis computarizado (IVOS II®), con lo cual se eliminó la subjetividad a la prueba. Se observó que 50 µM de trolox/108 SPZ y 50 µg de catalasa/ml tienen la capacidad de mantener valores (p < 0,05) superiores de motilidad total (64,26 ± 0,82 y 55,54 ± 0,85) y motilidad progresiva (47,26 ± 0,75 y 44,32 ± 1,13) en condiciones de precongelación, y para motilidad total (67,60 ± 1,91 y 63,47 ± 3,40), motilidad progresiva (50,87 ± 2,58 y 38,88 ± 2,31) y viabilidad (66,97 ± 2,25 y 61,16 ± 4,31) en poscongelación, comparados con los tratamientos control o la adición de 5 mM de cisteamina. La adición de 50 µM de trolox/108 espermatozoides y de 50 µg de catalasa/ml de semen mejora la motilidad total y progresiva del semen ovino refrigerado a 5 °C y la viabilidad en semen congelado/descongelado.

Abstract The present study aimed to evaluate the effect on sperm quality of three antioxidant substances (50 μM of trolox/108 spermatozoa, 50 μg of catalase/ml of ejaculate, and 5mM cysteamine) in a Triladyl™ commercial diluent base, under conditions of refrigeration and freezing/thawing in sheep semen. Semen from six adult males was frozen in a commercial Triladyl™ diluent base. The ejaculate of each male was placed in four different aliquots: one for control, and the other three with additional 50 μM of trolox/108 spermatozoa, 50 μg of catalase/ml of ejaculate, and 5 mM cysteamine, respectively, in the diluent base. Pre-freezing (5 °C) and post-freezing sperm quality was evaluated using a computerized analysis system (IVOS II®), which eliminated subjectivity during the test. It was observed that 50 μM of trolox/108 SPZ and 50 μg of catalase/ml are able to maintain higher values (p < 0.05) of total motility (64.26 ± 0.82 and 55.54 ± 0.85 ) and progressive motility (47.26 ± 0.75 and 44.32 ± 1.13) under pre-freezing conditions, and of total motility (67.60 ± 1.91 and 63.47 ± 3.40), progressive motility (50.87 ± 2.58 and 38.88 ± 2.31), and viability (66.97 ± 2.25 and 61.16 ± 4.31) in the post-freezing period, compared to control treatments or the addition of 5 mM of cysteamine. The addition of 50 μM of trolox/108 spermatozoa and 50 μg of catalase/ml of semen improves the total and progressive motility in ovine semen refrigerated at 5 °C, as well as viability in frozen/thawed semen.

Resumo O objetivo do presente estudo foi avaliar o efeito sobre a qualidade espermática de três substancias antioxidantes (50 µM de trolox/108 espermatozoides, 50 µg de catalasa/ml de ejaculado e cisteamina 5mM) numa base de diluente comercial Triladyl™, sob condições de refrigeração e congelamento/descongelamento em sêmen ovino. Sêmen de seis machos adultos foi congelado numa base do diluente comercial Triladyl™. O ejaculado de cada macho foi posto em quatro diferentes alíquotas: uma para controlo e nas outras foi adicionada a base do diluente 50 µM de trolox/108 espermatozoides, 50µg de catalasa/ml de ejaculado e cisteamina 5mM. A qualidade espermática precongelação (5 °C) e pós-congelação avaliou-se com ajuda de um sistema de análise computadorizado (IVOS II®), eliminando assim a subjetividade ao teste. Observou-se que 50 µM de trolox/108 SPZ e 50 µg de catalasa/ml têm a capacidade de manter valores (p < 0,05) superiores de motilidade total (64,26 ± 0,82 e 55,54 ± 0,85) e motilidade progressiva (47,26 ± 0,75 e 44,32 ± 1,13) em condições de pre-congelação, e para motilidade total (67,60 ± 1,91 y 63,47 ± 3,40), motilidade progressiva (50,87 ± 2,58 e 38,88 ± 2,31) e viabilidade (66,97 ± 2,25 e 61,16 ± 4,31) em pós-congelação, comparados com os tratamentos controle ou a adição< de 5 mM de cisteamina. A adição de 50 µM de trolox/108 espermatozoides e 50 µg de catalasa/ml de sêmen melhora a motilidade total e progressiva do sêmen ovino refrigerado a 5 °C e a viabilidade em sêmen congelado/descongelado.