Adriana Pires Neves

Enzymatic scavengers in the epididymal fluid: Comparison between pony and miniature breed stallions

The use of stallion semen collected from cauda epididymis for AI has increased due to the new protocols available for cryopreservation. Preserving the genetic material from valuable males that suffer sudden death or other events that prematurely end the stallions reproductive life is an important strategy for Stud breeding management. While protecting spermatozoa from oxidative stress and infectious agents, the epididymis promotes the enhancement of sperm cell morphology and changes in membrane protein profile, increasing its fertility potential. The epididymal fluid must be a balanced redox environment to allow sperm preservation and protein-protein and protein-lipids interactions to quantify. The aim of this study was quantify the enzymatic ROS scavengers in epididymal fluid of pony and miniature breed stallions. Epididymides from 8 pony stallions and 12 miniature breed stallions were dissected and fluid from caput, corpus and cauda epididymis collected. Spermatozoa were separated of epididymal fluid by 2-step centrifugation. The activities of catalase, superoxide dismutase (SOD) and glutathione peroxidase (GPx) were measured and compared between stallion groups and epididymal regions. The three enzymes were present in all epididymal regions tested, with higher activities of catalase and SOD in cauda epididymis in miniature breed stallions (P<0.05). GPx activity was higher in caput epididymis in pony stallions (P<0.05), however with no difference to fluid from cauda epididymis of both breeds. These results show a difference in antioxidant enzymatic scavengers between pony and miniature breed stallions. Also, our data confirm the protective role of cauda epididymis, preserving spermatozoa integrity from oxidative damage. As glutathione peroxidase is involved in several signaling pathways, its constant activity during epididymal transit corroborates the importance of this enzyme for spermatozoa maturation.

Seminal Plasma: Effect on Motility, Membrane Functionality, and Spermatic Chromatin Dispersion of Equine Sperm Treated with N-acetyl-L-cysteine at 5°C

Background: N-acetyl-L-cysteine (NAC) is a low molecular weight thiol studied as an antioxidant for stallion semen preservation without changes on sperm viability. Equine seminal plasma is rich in sulfur proteins (cysteine residues) named CRISPS, which, when combined with sulfur-containing antioxidants, can enhance the appearance of DNA lesions. The aim of this study was to assess and compare the effect of different concentrations of NAC by evaluating motility, membrane function and sperm chromatin integrity of equine semen cooled at 5°C in 50% of seminal plasma. Materials, Methods & Results: Nine ejaculates from 9 stallions were divided into 4 aliquots, diluted and divided in nonsupplemented skim milk group (0.0 mM), or supplemented with 5.0, 2.5 and 0.5 mM NAC. Evaluations were made at 0 h, 24 h and 48 h of cooling, except for motility which was evaluated only up to 24 h. The 0.5 (59.7 μM2) and 5.0 mM NAC (55.5 μM2) groups showed similar areas of sperm chromatin dispersion among all groups. However, the area of chromatin dispersion between the non-supplemented group was higher = 65.3 μM2 than the group supplemented with 2.5 mM. The percentage of cells with a functional plasma membrane was similar between supplemented and non-supplemented (0.0 mM) groups, but higher (P < 0.05) in the 0.5 mM NAC (39.7 and 39.8%, respectively) than that of 2.5 mM (34.5%) and 5.0 mM (34.2%) concentrations. Progressive motility was similar among all groups supplemented with NAC. The 0.5 mM NAC group showed 35.2% motile cells while the non-supplemented group exhibited 36.2%. Although 50% seminal plasma was used, NAC did not affect sperm chromatin integrity. Discussion: Seminal plasma interfered more in the results of different concentrations of NAC. This statement is proven by the motility analysis where all NAC concentrations showed similar results. Plasma percentage higher than 20% in diluted semen causes deleterious effects on sperm, such as decreased motility and fertilizing capacity. The membrane analysis in our study was compromised because NAC (2.5 to 5.0 mM) showed high osmolarity. As this was not adjusted, it affected the result. The 2.5 mM NAC group showed a lower area of sperm chromatin dispersion than none-treated sperm, although showing similar results to the other treatments. In a study with semen of Mangalarga Marchador stallions, the 2.5 mM of NAC was able to protect sperm membrane integrity. However, in another study, where semen was kept cooled between 5 and 15°C, no change was observed on sperm quality over different concentrations of NAC. This reinforces that 2.5 mM of NAC provides adequate protection to semen exposed to harmful conditions.The high percentage of plasma associated with this sulfur antioxidant did not compromise DNA integrity, as NAC concentration used was 100 times less than the concentration needed to induce DNA lesions.

Beyond Fifty Shades: The Genetics of Horse Colors

Since the dawn of horse domestication, coat colors have always fascinated humankind. In the last century, knowledge of genetics and development of scientific tools have become powerful enough so that the effects of many DNA mutations could be critically studied. Coat color nomenclature varies according to countries and breed associations; in addi‐ tion, many factors can modify the color of the coat, such as sun exposure, age, sex, and nutritional status of the animal. Nevertheless, horses are capable of producing only two pigments. Several genes have been indicated as putative to coat color modification, alter‐ ing the basic color by dilution, redistribution, or lacking of pigments.