2. Universidad Cardenal Herrera-CEU

In some species, female steroid hormones modify the profile of acute phase proteins (APPs) during the estrous cycle and pregnancy, according to the ovulation, embryonic implantation and placental development; however, nowadays there’s no experimental evidence for equine species. Objectives of this study were: to compare the serum amyloid A (SAA), haptoglobin (Hp) and C-reactive protein (CRP) concentrations between cyclic and pregnant mares, and to analyze the influence of estradiol-17β (E2) during estrous cycle or estrone sulfate (E1) during pregnancy, and progesterone (P4) on these proteins to assess their potential role to identify the cyclicity or pregnancy in Spanish mares. Blood samples were taken from 20 Purebred Spanish mares on the day of ovulation (day 0), on days +5 and +16 post-ovulation, and then, monthly during the whole pregnancy. SAA, Hp and CRP did not change between day 0, +5 and +16 post-ovulation days. P4 concentrations were significantly higher on day +16 than on days +5 and 0; and E2 concentrations were significantly higher on day 0 than day +5. On the other hand, pregnancy was characterized by a progressive increase in the Hp, variable modifications of E1 and P4 concentrations, without changes in SAA and CRP. The absence of significant differences in the APPs between days 0, +5 and +16, suggested that these proteins cannot be used as biomarkers of diagnosis of heat or preg- nancy in Spanish mares, at least early, since the Hp later increases during the gestation. Nevertheless, it is possible to use them for comparative purposes with other equine breeds, as supervisor instrument of health status in breeding females as diagnostic tools to monitor pregnancy’s development and/or subclinical reproductive inflammations, that could lead to the early embryonic death.

In cycling females, the periovulatory period is characterized by stimulation of the hypothalamic pituitary adrenal (HPA) axis. The aim of present study was to analyze the pattern and interrelationships among adrenocorticotropic hormone (ACTH), cortisol (CORT), aldosterone (ALD) and electrolytes (sodium—Na+, potassium—K+ and chloride—Cl􀀀) during periovulatory period in cycling mares. Venous blood samples were obtained daily from a total of 23 Purebred Spanish broodmares, aged 7.09 2.5 years, from day 􀀀5 to day +5 of estrous cycle, considering day 0, the day of ovulation. Plasma ACTH was measured by a fluorescent immunoassay kit, serum CORT and ALD by means of a competitive ELISA immunoassay, and plasma Na+, K+ and Cl􀀀 were quantified by an analyzer with selective electrodes for the three ions. ACTH showed higher concentrations at day 0 compared to days 􀀀5 to 􀀀1 and +1 to +3 (p < 0.05). CORT showed higher concentrations at day 0 compared to days 􀀀5 to 􀀀2 and +1 to +5 (p < 0.05). ALD showed higher concentrations at day 0 compared to days 􀀀5 to 􀀀2 (p < 0.05) and +2 (p < 0.05). Na+ and Cl􀀀 showed higher concentrations at day 0, compared to day 􀀀5 and +5. K+ showed lower concentrations at day 0 compared to day +1 (p < 0.05). The significant correlations obtained between ACTH and CORT (r = 0.20) and between ACTH and ALD (r = 0.32) suggest that although ACTH may have an effect both on CORT and ALD, there are other very important determinants that could be considered. Hence, it is possible to presume that the pituitary adrenocortical response and ALD may be involved in the ovulatory mechanisms without a direct relation with electrolyte pattern.

The hypothesis of this study was to investigate if the presence of corpus luteum (CL) in one ovary could modify the hormonal content of follicular fluid (FF) in the follicles. Sixty ovaries were taken after the slaughter of 30 clinically healthy mares. In relation to the sizes, the follicles were classified into three di erent categories, as small (20–30 mm), medium (31–40 mm) and large ( 41 mm). Blood samples were collected from the jugular vein of mares before their slaughter, and then the FF samplings were extracted from each single follicle. The ovaries that were collected were classified into two groups, according to the presence (CL-bearing) or absence (non-CL-bearing) of CL. The serum and FF samples were analysed for progesterone (P4), oestradiol-17 (E2), testosterone (T), androstenedione (A4) and dehydroepiandrosterone (DHEA). Intrafollicular P4 concentrations in large follicles of CL-bearing groups were lower than for non-CL-bearing ones. Intrafollicular E2 concentrations increased with the increase of the follicle diameter in both groups, CL-bearing and non-CL-bearing. However, in the FF with a large and medium follicle size, E2 concentrations were significantly higher in non-CL-bearing groups than in CL-bearing groups. T and A4 significantly increased in the large and medium follicle sizes when compared to the small follicle sizes in both groups, but higher concentrations in the non-CL-bearing group were obtained. Intrafollicular DHEA significantly decreased with the increase of the follicular diameter in both groups. Steroid hormones in FF dynamically changed, according to the presence or not of CL in the ovary. This study brings new knowledge on the role of the CL in the follicular hormonal composition in mares.

Since in the mare and other animal species such as bitches and cats, the endometrial cell pattern varies depending on the phase of the estrous cycle, the aim of this study was to describe and quantify the endometrial cytological (EC) findings in cycling jennies. EC of eight nonpregnant jennies by cytobrush (CB) at diestrus (day 1 and day 14) and estrous (day 21) were evaluated. All slides were stained with Wright´s stain and microscopically examined at both 400 and 1000 magnification. Seven high-power fields (400 ) were assessed in each smear and the endometrial epithelial cells and neutrophils (PMNs) were counted. Endometrial epithelial cells were classified as intact, distorted or fragmented and, on the basis of the presence of dense groups, in monolayer or single clusters. Cytoplasmic characteristics, such as vacuolation or streaming and size, form, position of nuclear characteristics, including karyorrhexis, were recorded. Background aspect, as clear, proteinaceous, or debris, was also considered. In general, sampling by CB provided a yield of cells and clumped endometrial epithelial cells in many smears, being more abundant in estrus than early and late diestrus. Individual endometrial epithelial cells, during estrous, presented a columnar morphology, ciliated or not ciliated and basal nuclei. During diestrus phase, endometrial epithelial cells presented a more cuboidal ciliated or not ciliated morphology. Moderate amount of proteinacious material and red blood cells (RBC) was also observed. Non variation in the percentage of PMNs during diestrus was obtained, but lower and segmented PMNs in CB smears were shown in estrous. This study provides new insights on the physiological changes of endometrial epithelial cells in cycling jennies during the estrus cycle. The CB technique represents a suitable and adequate method for endometrial evaluation, taking into account cytological and/or cytopathological purposes also in jennies.

In some species, catecholamines in follicular fluid (FF) are related to local physiological events responsible for the regulation of ovarian functions and oocyte maturation. The aim of the present study was to determine and compare intrafollicular and systemic concentrations of dopamine (DA), noradrenaline (NA) and adrenaline (AD) in cycling mares. Sixty ovaries were collected during breeding season from 30 mares raised for slaughterhouse meat production, with clinically normal reproductive tracts, were evaluated. Blood samples were collected prior to slaughter. Follicles were classified into three categories in relation to size: small (20–30 mm; n = 20), medium ( 31–40 mm; n = 20) and large ( 41 mm; n = 20). Follicular fluid (FF) samples were extracted from each follicle. Intrafollicular DA, NA and AD concentrations were significantly higher than the systemic concentrations (p < 0.05). Intrafollicular DA concentrations were higher in medium than small and large follicles (p < 0.05). Intrafollicular NA concentrations were higher in small than medium and large follicles (p < 0.05). Intrafollicular AD concentrations were higher in large than small and medium follicles (p < 0.05). Follicle diameter was significantly and negatively correlated with NA and AD (p < 0.05). A significant correlation of the same hormone concentration in FF and in systemic fluid was observed (p < 0.05). In summary, the FF can serve as an intraovarian catecholamine-storing compartment, with the ability to release neurotransmitters in a regulated way. These results provide novel insights into the neuronal nature of the follicle, suggesting the involvement of catecholamines in normal ovarian functions in mares.