1. Investigación

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.

El óptimo crecimiento fetal depende de la eficiente función placentaria. El factor de crecimiento placentario (PlFG) favorece la angiogénesis y vascularización de la placenta, asegurando el efectivo flujo sanguíneo placentario esencial para mantener el transporte activo de moléculas y la síntesis de hormonas esteroideas. Por otro lado, los componentes del eje somatotropo (GH e IGF-1), la ACTH, el CORT y las catecolaminas influyen notablemente sobre el desarrollo placentario y el crecimiento fetal. Sin embargo, hasta la actualidad estos mecanismos permanecen desconocidos en la yegua. Por este motivo, los objetivos planteados en la presente investigación han sido los siguientes: 1) Establecer valores de referencia para las concentraciones de neurotransmisores (ADR, NORADR, 5-HT y DA), hormonas hipofisarias (ACTH y GH), placentarias (A4, DHEA, T, E1S y P4), adrenales (CORT) y PlFG e IGF-1 en yeguas gestantes sanas; 2) Examinar si la gestación en la yegua PRE induce modificaciones laboratoriales en las dinámicas de los componentes anteriormente descritos, y 3) Analizar si los cambios hormonales de GH, ACTH, andrógenos, E1S y P4 están implicados fisiológicamente en las modificaciones analíticas que experimentan los diversos neurotransmisores y ambos factores de crecimiento en la yegua gestante PRE. Se ha estudiado un total de 33 yeguas PRE con edades comprendidas entre 5 y 17 años, durante la gestación. Se obtuvieron muestras de sangre venosa por las mañanas con una frecuencia mensual. Las muestras fueron almacenadas en tubos de vidrio con activadores de la coagulación y gránulos PS para desuerado. Las concentraciones séricas de ADR, NORADR, DA y 5-HT fueron determinadas mediante técnicas inmunoenzimáticas (EIA) de competición validadas específicamente para la especie equina (3-CAt EIA; Demeditec, Diagnostics GmbH, Germany; Serotonin-EIA; Demeditec, Diagnostics GmbH, Germany). La cuantificación de ACTH y GH fue realizada utilizando EIA de competición estreptavidina-biotina (Phoenix peptide ACTH human Fluorescent Immunoassay Kit FEK-001-01). Los niveles de IGF-1 se analizaron mediante ELISA de competición (DRG® InternationalInc, IGF-1 equino, EIA-3982, USA). La detección del PlGF se realizó utilizando EIA de tipo sandwich (Demeditec DEE01). Las concentraciones de A4, T, DHEA, E1S, P4 y CORT fueron determinadas mediante técnicas EIA utilizando anticuerpos policlonales obtenidos y caracterizados en el Departamento de Fisiología Animal de la Facultad de Veterinaria de la Universidad Complutense de Madrid. El inicio de la gestación representa un incremento fisiológico de las concentraciones de ADR, NORADR, DA, P4 y ACTH. Esta elevación de las catecolaminas ADR y NORADR alcanza un pico de secreción máximo simultáneo al incremento de 5-HT, GH, IGF-1, A4, DHEA, E1S y CORT hacia la mitad de la gestación. La preñez tardía constituye un periodo caracterizado por el descenso de NORAD, DA, 5-HT, ACTH y GH e incremento de T. A diferencia de la mujer y otras especies animales en las que mayoritariamente la P4 y el E1S se consideran los principales reguladores de la secreción hipotalámica de NORADR e hipofisaria de GH, la falta de relación entre dichos parámetros sugiere que la síntesis de NORADR, GH e IGF-1 es independiente de la dinámica esteroidea durante la gestación. Aunque las concentraciones de A4, DHEA, T, E1S y P4 muestran un patrón similar al PlFG, la expresión y la síntesis de este factor parecen no estar sometidas a control hormonal de origen placentario. En conclusión, la gestación representa el incremento fisiológico de la actividad de los ejes simpático-adrenal, hipotalámico-hipofisario y somatotropo en la yegua PRE. Los factores y hormonas integrantes en estos ejes junto a las hormonas placentarias y el PlGF podrían ser considerados como marcadores predictivos de funcionalidad placentaria y del avance correcto de la gestación. / The optimal fetal growth depends on the efficient placental function. Placental growth factor (PlFG) favors angiogenesis and vascularization of the placenta, ensuring effective placental blood flow, essential to maintain active transport of molecules and synthesis of steroid hormones. The components of the somatotrope (GH and IGF-1), ACTH, CORT and catecholamines strongly influence on development of the placenta fetal growth. However, these mechanisms remain unknown in the mare. For this reason, the objectives of the present research were: 1) To establish reference values for neurotransmitter concentrations (ADR, NORADR, 5-HT and DA), pituitary hormones (ACTH and GH), placental (DHEA, T, E1S and P4), adrenals (CORT), PlFG and IGF-1 in healthy pregnant mares; 2) Examine whether gestation in the Spanish Purebred mare induces laboratorial modifications in the dynamics of the previously described components, and 3) Analyze whether the hormonal changes of GH, ACTH, androgens, E1S and P4 are physiologically involved in the analytical modifications experienced by the several neurotransmitters and both growth factors in the Spanish Purebred broodmares. A total of 33 Spanish Purebred broodmares with ages between 5 and 17 years, during gestation have been analyzed. Venous blood samples were collected in the mornings on a monthly frequency. Samples were stored in glass tubes with coagulation activators and PS granules to obtaining serum. Serum concentrations of ADR, NORADR, DA and 5-HT were determined using competition immunoenzymatic techniques (EIA) specifically validated for equine species (3-CAt EIA, Demeditec, Diagnostics GmbH, Germany, Serotonin-EIA, Demeditec, Diagnostics GmbH, Germany). Quantification of ACTH and GH was performed using streptavidin-biotin competition EIA (Phoenix peptide ACTH human Fluorescent Immunoassay Kit FEK-001-01). IGF-1 levels were analyzed by competition ELISA (DRG® InternationalInc, equine IGF-1, EIA-3982, USA). Detection of PlGF was performed using sandwich EIA (Demeditec DEE01). The concentrations of A4, DHEA, T, E1S, P4 and CORT were determined using EIA techniques using polyclonal antibodies obtained and characterized in the Department of Physiology of the Faculty of Veterinary of the Complutense University of Madrid. The onset of gestation represents a physiological increase on ADR, NORADR, DA, P4 and ACTH concentrations. This elevation on catecholamines ADR and NORADR reaches a peak of maximal secretion simultaneously with the increase of 5-HT, GH, IGF-1, A4, DHEA, E1S and CORT towards the middle of gestation. Late pregnancy is a period characterized by the decrease of NORAD, DA, 5-HT, ACTH and GH and increase of T. Unlike women and other animal species in which P4 and E1S are considered the main regulators of NORADR hypothalamic and GH pituitary secretion, the lack of relationship between these parameters suggests that the synthesis of NORADR, GH and IGF-1 is independent of the steroid dynamics during pregnancy. Although the concentrations of A4, DHEA, T, E1S and P4 show a pattern similar to PlFG, the expression and synthesis of this factor appear not to be subject to placental hormonal control. In conclusion, gestation represents the physiological increase of the activity of the sympathetic-adrenal, hypothalamic-pituitary and somatotrope axes in the PRE mare. Factors and hormones in these axes together placental hormones and PlGF could be considered as predictive markers of placental function and correct progression of gestation.