Seasonality does not influence cortisol or testosterone production, or seminal quality of stallions located at low latitudes.

The effects of seasonality on the reproduction of stallions vary based on the latitude. Although previous studies have shown the influence of seasonality in raw semen quality in south-eastern Brazil, data regarding the influence of seasonality in cooled and frozen stored semen in Brazil is limited. Therefore, in this study, we have analysed if seasonality influences the hormone production (i.e., cortisol and testosterone), spermatogenesis, and quality of fresh, cooled, and frozen semen of stallions in central Brazil, and established the season most suitable for semen cryopreservation in a latitude of 15°S. Ten stallions were followed-up for one year, which was divided into two seasons, namely, drought, and rainy. Fresh, cooled, and frozen-thawed semen samples were assessed using CASA and flow cytometry. Additionally, the temperature and humidity index (THI) was calculated to determine the thermal stress. Although the THI varied between the two seasons, no thermal stress was observed throughout the year, nor were there differences in the physiological parameters of the stallions or plasma cortisol or testosterone levels. Furthermore, differences were not detected in total and progressive motility, sperm capacitation, and sperm membrane integrity, as well as in the number of live sperm with intact acrosomes and high mitochondrial membrane potential, between the two seasons in the fresh and frozen-thawed semen. Our data suggest that semen can be effectively collected and cryopreserved throughout the year within central regions of Brazil.

Effectiveness of Ozone Therapy in The Treatment of Endometritis in Mares.

Endometritis is defined as inflammation of the endometrium that may be acute or chronic, infectious, or non-infectious. Endometritis is an important cause of subfertility in mares. Considering the antimicrobial characteristics, immune-stimulating ability, and low cost of ozone (O3) therapy, we aimed to evaluate the effectiveness of intrauterine O3 therapy as an alternative treatment for endometritis in mares. Twenty-five mares with a known reproductive history of uterine infection and inflammation were allocated into three groups: Group 1 (control; n = 7), uterine lavage with Ringer's lactate solution; Group 2 (O3-gas; n = 9), uterine lavage with Ringer's lactate solution followed by uterine insufflation with O2-O3 gas mixture containing 40 µg O3 mL-1 for 10 minute; and Group 3 (O3-oil; n = 9), uterine infusion of ozonized sunflower oil. Uterine inflammation was evaluated through a uterine cytological examination (cytobrush) and uterine culture (swab) for microbiological content before and after all treatments. In assessments of uterine cytology, the average number of neutrophils/field changed from 9.14 ± 3.02 to 7.71 ± 3.59 in the control group, from 10.67 ± 3.84 to 2.89 ± 3.59 in the O3-gas group, and from 6.44 ± 2.79 to 6.55 ± 7.18 in the O3-oil group post-treatment. The pre- and post-treatment findings in the mares treated with ozonized gas were significantly different (P < .05), unlike the findings for the mares in the control and O3-oil groups. All mares (25/25) showed a positive uterine culture before treatment. After treatment, the percentage of mares showing positive culture results was 57%, 11%, and 22% in the control, O3-gas, and O3-oil groups, respectively. Our results showed the effectiveness of two groups (O2-O3 gas mixture and ozonized sunflower oil) for the treatment of uterine infections in mares. Thus, direct intrauterine O3 gas infusion has been shown to be effective in treating endometritis in mares, reducing both inflammation and uterine infection.

Aspects of Breeding Stallion Management with Specific Focus on Animal Welfare.

Risk prevention is often counterproductive to stallions' living conditions when assessed under welfare aspects. In the wild, stallions live in social groups, but under domestic conditions, the majority of breeding stallions live in individual boxes with limited social contact. This stimulates aggressiveness and the incidence of stereotypic behavior. While racehorse stallions start their breeding career after having finished their performance career, riding horse stallions are often simultaneously used for breeding and performance. Training, performance, and the associated stress are unlikely to impair semen characteristics, but performing stallions may benefit from more opportunities for social contact. With regard to accommodation of stallions in single boxes, changes in the construction of the partition between them may help to improve social contact from merely visual to much more physical. This will help to reduce social isolation but requires careful observation if neighbors tend to either sympathize or fight with each other. Careful organization of the barn is thus of great importance. Under certain conditions, even keeping adult stallions in groups on spacious pastures is possible. Interestingly, the frequency of agonistic interactions usually decreases quickly and remains low after successful group integration. General changes in the husbandry of young stallions and at their transfer into the future career may help to produce stallions with better social skills and facilitate accommodation under improved welfare conditions.

Fetal development and blood hematological-biochemical parameters in Campeiro and Pantaneiro foals.

For a long time, Pantaneiro and Campeiro breeds were raised only within their places of origin. Consequently, there are few of these horses; therefore, establishing reproductive and clinical standards for these animals is necessary to implant new biotechnologies for reproduction to preserve their genetics. This study aimed to perform a descriptive evaluation of fetal age determination by fetus ocular orbit measurement in mares of the Campeiro and Pantaneiro breeds. We also evaluated sequential changes in hematology and biochemistry for foals from birth to six months of life by counting red blood cells, hemoglobin concentration, mean corpuscular volume, mean corpuscular hemoglobin concentration, leukocytes, neutrophils, lymphocytes, eosinophils, monocytes, basophils, platelet count, fibrinogen, albumin, plasma protein, creatinine, aspartate aminotransferase, alkaline phosphatase, gamma-glutamyl transferase, and globulins. There was no significant difference in relation to fetal gestational age and ocular orbit between the two breeds (P = 0.578). There was no significant difference in the hematological parameters between the Campeiro and Pantaneiro foals, but there were differences in the means and changes in the blood variables when compared to the literature. These hematological and biochemical variations provide useful information for clinical evaluations from Campeiro and Pantaneiro foals up to six months of age.

Effects of environmental temperature and season on hair coat characteristics, physiologic and reproductive parameters in Shetland pony stallions.

We hypothesized that housing of stallions in a thermoneutral temperature zone during autumn and winter does not only influence metabolism and hair shedding but also improves the characteristics of raw and processed semen. Fertile Shetland pony stallions were followed from October to June. This time coincided with the seasons autumn, winter and spring. Ponies were kept in outside paddocks (group CON, n = 8) or in indoor stables (group ST, n = 8) from October to March when ST stallions returned to outdoor paddocks, but ponies remained in the same groups. The rectal temperature was measured once weekly. Heart rate, heart rate variability, testosterone and cortisol concentration in blood as well as quality and length of the coat were determined. Semen was collected once weekly and raw semen characteristics were analyzed. The characteristics of cooled-stored and cryopreserved semen were determined once monthly. During the stabling period, environmental temperature for group ST averaged 13.6 ± 2.3 and for group CON 5.6 ± 4.2 °C. The mean rectal temperature was higher (p < 0.05) in ST than in CON stallions. All hair coat parameters underwent seasonal changes (p < 0.001) and differed between groups (p < 0.05) with shorter guard hair, slower hair regrowth and earlier hair change in ST stallions. Season influenced heart rate which was highest in autumn, lowest in winter and intermediate in spring but did not differ between groups. Testosterone and cortisol concentrations in blood as well as sexual behavior underwent seasonal changes but did not differ between CON and ST stallions. Gel-free semen volume and total sperm count were influenced by season (p < 0.01) and showed a more pronounced increase from winter to spring in CON than in ST stallions (p < 0.05) while no differences with regard to sperm concentration in raw semen were detected. Progressive motility of spermatozoa in raw semen was highest in spring (p < 0.05) but not affected by group. In cooled-stored and cryopreserved semen, neither season nor group affected total motility, progressive motility or membrane integrity. In conclusion, environmental temperature during autumn and winter had clear results on body temperature as well as hair coat characteristics in Shetland stallions. Simultaneously determined effects on semen characteristics were minimal indicating that reproductive function in the horse is more dependent on day length i.e. the geophysical year than on other environmental factors.

Cooled-storage of equine semen does not induce major changes in sperm DNA methylation.

A decrease in fertility of equine semen during cooled-storage so far has mainly been attributed to changes in sperm membrane function. In the present study we hypothesized that cooled-storage also changes the sperm DNA methylation level. For this purpose, semen was collected from 10 fertile stallions and processed for cooled-storage at 5 °C. Two final concentrations, 50 × 106 and 100 × 106 cells/mL, were used. Semen was analyzed for total motility, progressive motility, membrane integrity, phosphatidylserine translocation (PST), mitochondrial membrane potential and chromatin condensation, immediately after processing and at 24 h-intervals until 72 h of storage. DNA cytosine methylation was assessed by ELISA after DNA extraction and denaturation. DNA methylation did neither change over time nor was affected by semen concentration. Total motility, progressive motility, membrane integrity, PST, mitochondrial membrane potential and chromatin condensation changed over storage time, but no differences between semen concentrations could be demonstrated. The results demonstrate that cooled-storage of equine semen does not induce changes in sperm DNA cytosine methylation. In cooled-semen of good quality, a concentration of 100 × 106 sperm/mL does not affect semen longevity. It can be concluded that a better fertility of cooled-stored than cryopreserved stallion semen is at least in part a result of only minor influences of cooled-storage on DNA integrity and methylation.

Efficacy of 2,6-dichlorophenol lure to control Dermacentor nitens.

This study was carried out with the objective of evaluating the efficacy of a 2,6-dichlorophenol (2,6-DCP) lure to control Dermacentor nitens (Acari: Ixodidae). Slow-release formulations of the pheromone formulated with and without cypermethrin were prepared. Olfactometer bioassays were used to define the best dose of the pheromone and to evaluate the effect of cypermethrin with 2,6-DCP attractiveness. Sexually active males were released 15 cm from 2 cmx1 cm pieces of polypropylene treated with different odors: 2,6-DCP in a liposphere system (1.5, 30 and 300 microg--without cypermethrin and 30 microg--with cypermethrin) and 2,6-DCP in hexane (30 microg). The tests were conducted 7 and 15 days after the preparation of the odor sources. The percentages of males that oriented, or showed directional movement toward the stimulus, and their tracks were recorded. Lures (10 cmx2 cm pieces of polypropylene) treated with the best dose of the liposphere formulation (30 microg) were prepared. The lures were evaluated in horses that had been artificially infested with D. nitens. The horses' ears were infested with 3000 D. nitens larvae per ear, once weekly for 4 weeks. The animals were divided into three groups: control, 2,6-DCP and 2,6-DCP+cypermethrin. On day 0, the lures of their respective treatments were attached to the horses' napes. From days 6 to 20 after attachment, female ticks of 4 mm or over in length were counted on the ears, every 2 or 3 days. Olfactometer analysis showed higher orientation to 30 microg dose and more prolonged release of the pheromone in the liposphere formulation than in hexane; cypermethrin did not interfere with the attractive effect of the pheromone. The lures were efficient in the first 10 days after attachment, when the mean number of females was higher in the control group (24.9) than in 2,6-DCP and cypermethrin (5.4), and 2,6-DCP (9.2) groups. After that period the number of females was similar in the control and treated groups. These results indicate that 2,6-DCP lures used in a liposphere formulation can control D. nitens for at least 10 days by preventing its copulation. However, further evaluation of longer-term pheromone release under natural conditions is needed in order to validate this kind of control. In addition, the use of extra lures on the horse's tail may help to control populations on the hindquarters.