Functional response of systemic and intrafollicular placental growth factor in cycling mares.

The aim of the study was to assess the physiological reference values for systemic and intrafollicular placental growth factor (PlGF) concentrations in different categories of follicular sizes in cycling mares, according to progesterone (P4) and oestradiol (E2) patterns. Sixty ovaries were taken after slaughter from 30 clinically healthy mares. Regarding their size, the follicles were classified into three different categories, i.e. small (20-30 mm), medium-sized (31-40 mm) and large (≥41 mm), and follicular fluid (FF) was sampled from each single follicle. Intrafollicular PlGF concentrations were significantly increased in larger and medium-sized follicles compared to small follicles, and their values were 1.48 and 1.36 times higher than the systemic values, respectively. On the other hand, systemic PlGF concentrations were 1.3 times higher than those in the FF of follicles of small size. Intrafollicular P4 concentrations were significantly higher in larger follicles than in small ones, and their concentrations were 6.74 and 3.42 times higher than the systemic values, respectively. Intrafollicular E2 concentrations were significantly higher in large and medium-sized follicles than in follicles of small size, and their concentrations were 21.1, 15.4 and 8.35 times higher than the systemic values, respectively. Intrafollicular and systemic PlGF concentrations were strongly and positively correlated; nevertheless, no correlations between intrafollicular and systemic steroid hormones, PlGF and follicle diameters, PlGF and E2, or PlGF and P4 were observed. This represents the first study to characterise systemic and intrafollicular PlGF concentrations in cycling normal mares, providing evidence that the bioavailability of this factor in follicles of medium and large sizes was higher than in small follicles, independently of steroid hormone concentrations. Further studies are needed to assess the presumable implications of PlGF in follicular angiogenesis in mares, similar to those already observed in women and primates.

Intrafollicular and systemic serotonin, oestradiol and progesterone concentrations in cycling mares.

The hypothesis that a local serotonergic network might also exist in the follicle of mares remains poorly documented, with exception for humans and laboratory species. For this reason, the aim of the present study was to clarify this possibility, investigating intrafollicular serotonin concentrations of the cycling mare at ovulation time. Sixty ovaries collected from 30 clinically healthy mares of slaughterhouse meat production with clinically normal reproductive tracts after slaughtering were evaluated. Blood samples were taken prior to sacrifice. Follicles were classified in three categories in relation to size, as small (20-30 mm), medium (31-40 mm) and large (>41 mm), and the follicular fluid samples were extracted from each follicle. Intrafollicular and systemic serotonin (5-HT), oestradiol-17ß (E2 ) and progesterone (P4 ) were determined by means of enzyme-linked immunosorbent assay and RIA, respectively. Intrafollicular 5-HT, E2 and P4 concentrations were higher than systemic ones (p < .05). 5-HT concentrations increased in larger compared to medium follicles, without differences compared to small size follicles (p < .05). 5-HT and E2 (r = .79) and 5-HT and P4 (r = .79; p < .05) were positively correlated. 5-HT and P4 concentrations in follicular fluid increased progressively with the increase in follicular size (p < .05). Follicle diameter and E2 (r = .85) and P4 (r = .68) were correlated (p < .05). Since serotonin interacts with steroids, its role on steroidogenesis during growth of the dominant follicle may be suggested.

Effect of age on androgens pattern in cyclic mares.

Androgens are produced in both sexes. In females produced by the adrenal gland and the ovaries they play a crucial role in regulating ovarian function, estrogen synthesis and follicular growth. Age leads to a reduction in androgen concentrations, although, at present, these mechanisms are not elucidated in mares. The objective of this study was to evaluate the concentrations of testosterone (T), androstenedione (A4) and dehydroepiandrosterone (DHEA) in mares of different ages. Blood samples were drawn from seventy cyclic Spanish Purebred mares belonging to five age groups: 3-5 years, 6-9 years, 10-13 years, 14-16 years and > 16 years. The concentrations of T, A4 and DHEA were determined by EIA, validated specifically for horses. Mares aged 3-5, 6-9 and 10-13 years had higher T concentrations (P < 0.05) than mares aged >16 years, and mares aged 6-9 years had also higher concentrations than those 14-16 years old (P < 0.05). A4 concentrations were lower (P < 0.05) in mares >16 years old when compared with those of other age groups. DHEA concentrations were lower (P < 0.05) in mares 14-16 years and > 16 years old when compared with those of other age groups. DHEA was positively correlated with T (r = 0.61; P < 0.05) and A4 (r = 0.51; P < 0.05). Age induces reduction in androgens' synthesis in physiologically cyclic Spanish Purebred mares. These physiological variations must be duly considered for a correct and objective interpretation of the analytical data.

Correlation between erythrocyte parameters and iron status in cyclic Spanish Purebred mares.

The aim of this study was to evaluate the serum iron status and its relation to hematological indexes in cyclic mares. Blood samples were taken from 40 Spanish Purebred mares on days - 5, 0, + 5 and + 16 of their cycle. Concentration of transferrin (TRF) was significantly lower on day 0 than on days + 5 and + 16, transferrin saturation (TSAT) decreased significantly on days 0 and + 16 compared to day - 5, total iron-binding capacity (TIBC) on day + 16 was significantly higher than those on days - 5 and 0, and on day + 5 it was also significantly higher than that on day 0, unsaturated iron-binding capacity (UIBC) was reduced on day + 16 compared to days - 5 and 0, red blood cell (RBC) count on day + 16 was higher than that on days - 5 and 0 (p < 0.05), with no differences in the concentration of hemoglobin (HB) and packed cell volume (PCV). TRF and TIBC (r = 0.95), RBC and HB (r = 0.64), RBC and PCV (r = 0.78), and HB and PCV (r = 0.63) were positively and significantly correlated (P < 0.05). The estrous cycle in the Spanish Purebred mare is characterized by an increase in TRF and TIBC during the follicular phase and an increase in TSAT, UIBC and RBC in the luteal phase, without changes in other hematological parameters. The coordinated activity of these parameters guarantees an adequate iron (Fe) transfer and utilization during follicular development, ovulation, and the luteal period in the mare. Therefore, the estrous cycle must be considered in the evaluation of the mare's iron status, in light of significant changes observed both in early and at late luteal phases. The magnitude of these changes and the meaning to the physiology of the mares showed that in cyclic mares, hematological parameters and indicators of iron status evolve differently depending on the phase of the cycle, and their interpretation can help to veterinarians involved in equine practice.

Effect of age on amplitude of circulating catecholamine's change of healthy cyclic mares.

Catecholamines (CATs) are neurotransmitters and allostatic hormones whose plasma concentrations are physiologically modified in various species such as human, rats, mice and donkeys, with advancing age. However, currently these mechanisms are less well elucidated in horses and more specifically in mares. The hypothesis of this study was that, as in afore mentioned species, the CATs could experience physiological changes with advancing age. The objective of this study was to evaluate the concentrations of adrenaline (A), noradrenaline (NA), dopamine (DA), and serotonin (5-HT) in mares of different ages. Blood samples were drawn from 56 non-pregnant Spanish Purebred mares belonging to four different age groups: 6 to 9 years, 10 to 12 years, 13 to 16 years and > 16 years. The concentrations of A, NA, DA, and 5-HT were determined by competition EIA-Technical 3-CAt EIA, specifically validated for horses. Mares aged > 16 years showed lower A, DA, and 5-HT but higher NA concentrations than 6-9, 10-12, and 13-16 years (p < 0.05). Mares of 13-16 years showed lower A and higher NA than 6-9 and 10-12 years (p < 0.05). A and NA (r=-0.72; p < 0.05), and NA and 5-HT (r=-0.67; p < 0.05) were negatively correlated, and A and 5-HT (r = 0.74; p < 0.05) were positively correlated. Advanced age leads to a predominance of sympathetic nervous activity and lower serotonergic activity in non-pregnant mares.

Short Road Transport and Slaughter Stress Affects the Expression Profile of Serotonin Receptors, Adrenocortical, and Hematochemical Responses in Horses.

Horse transport is considered a cause of stress in animals and is known to affect the 5-HT concentrations in both the brain and other tissues. The aim of this research was to investigate the effect of horse transportation and slaughter stress on plasma serotonin's concentration and the expression levels of the related 5-HT1B and 5-HT2A receptors in PBMCs. Furthermore, the IL-12 levels and a variety of blood parameters, including triglycerides, total cholesterol, glucose, aspartate aminotransferase, creatine phosphokinase, lactate dehydrogenase, were also considered. This research was carried out on 32 horses submitted to short road transport of 40 km to slaughter. Blood samples were collected in baseline conditions (T0) and 24 h later, after they were slaughtered (T1). The results showed a significantly increased expression of 5-HT1B and 5-HT2A receptors and a significantly decreased expression of IL-12 in PBMCs at T1 vs. T0. Furthermore, a significant increase in cortisol and glucose concentrations, and LDH activity was observed at T1. In contrast, a significantly lower circulating 5-HT concentration was observed at T1 vs. T0. These results indicate that the stress induced by transport and slaughter stimuli led to the serotoninergic system's activation, suggesting that the expression of serotonin receptors could be used as a pivotal marker of stress, with potential applications for the improvement of elective protocols to observe the guidelines relating to transported horses.

Estrogen-iron axis in cyclic mares: Effect of age.

In woman and in animal models, estrogens are involved in iron (Fe) homeostasis supporting the hypothesis of the existence of an "estrogen-iron axis". Since advancing age leads to a decrease in estrogen levels, the mechanisms of Fe regulation could be compromised. In cyclic and pregnant mares, to date, there is evidence linking the iron state with estrogens pattern. Then, the objective of this study was to determine the relationship among Fe, ferritin (Ferr), hepcidin (Hepc) and estradiol-17ß (E2) in cyclic mares with advancing age. A total of 40 Spanish Purebred mares of different ranges of age was analyzed: 4-6 years (n = 10), 7-9 years (n = 10), 10-12 years (n = 10), and >12 years (n = 10). Blood samples were obtained on days -5, 0, +5 and + 16 of the cycle. Compared to mares of 4-6 years, serum Ferr was significantly higher (P < 0.01) and Fe significantly lower (P < 0.01) in mares >12 years of age. Hepc was significantly higher in mares >12 years (P < 0.01) than in those 7-9 years of age. E2 levels were higher in mares of 7-9 years (P < 0.01) than in 4-6 and >12 years of age. Fe and Ferr were negatively correlated with Hepc (r = -0.71 and r = -0.02, respectively). E2 was negatively correlated with Ferr and Hepc (r = -0.28 and r = -0.50, respectively), and positively with Fe (r = 0.31). There is a direct relationship between E2 and Fe metabolism, mediated by the inhibition of Hepc in Spanish Purebred mares. The reduction of E2 decreases the inhibitory effects on Hepc, increasing the levels of stored Fe and mobilizing less the free Fe in circulation. Based on the fact that ovarian estrogens participate in changes in the parameters indicative of iron status with age, the existence of an "estrogen-iron axis" in the mares'estrous cycle could be considered. Future studies are required to clarify these hormonal and metabolic interrelationships in the mare.

Hematological indexes and iron status in pregnant mares.

During pregnancy, iron requirements are increased to meet optimal placental and fetal growth and the expansion of the maternal red-cell mass and to prevent complications related to the mother's iron deficiency anemia. Red-cell parameters and iron status provide consistent additional information for diagnosis of iron deficiency conditions. The aim of this study was to evaluate the serum iron status and its relation to hematological indexes in pregnant mares. Blood samples were taken from 31 Spanish Purebred mares over 11 months of pregnancy. Concentrations of iron (Fe), ferritin (Ferr), transferrin (T), and total iron-binding capacity (TIBC) increased significantly and unsaturated iron-binding capacity (UIBC) decreased as the pregnancy progressed without changes in red blood cell (RBC) count, hemoglobin (HB) concentration, packed cell volume (PCV), and transferrin saturation (TSAT). Fe and Ferr were positively correlated (r=0.21). Fe and T (r=0.69) and Fe and TSAT (r=0.94) were positively correlated, and Fe and UIBC were negatively correlated (r=-0.69). T and TIBC were positively correlated (r=1.00). Pregnancy in the Spanish Purebred mare is characterized by a progressive increase in Fe, Ferr, T, and TIBC and a decrease in UIBC without modification in hematological indexes. Hematological parameters and iron status seem to indicate a sufficiency for Fe transport and its related mobilization and utilization during gestation in Spanish Purebred mares.

Changes of Hepcidin, Ferritin and Iron Levels in Cycling Purebred Spanish Mares.

Several studies have demonstrated that in woman the sex hormones such as estrogen (E2) and progesterone (P4) influence iron (Fe) regulation, contributing to variations in Fe parameters along the menstrual cycle. These mechanisms based on the regulation of hepcidin (Hepc) which limits Fe availability during the cycle, remain poorly characterized in healthy mares. The objective of this study was to establish the relationship between Hepc, Fe, ferritin (Ferr), and the primary ovarian hormones E2 and P4 in cycling Purebred Spanish mares. Blood samples were taken from 31 Purebred Spanish mares day -5, on day 0, day +5 and day +16 of the cycle. Fe and Ferr significantly increased and Hepc decreased during pre- and ovulatory periods. The secretion peak of estradiol-17ß (E2) was reached on day 0 and progesterone (P4) between days +5 and +16. Fe and Ferr were positively correlated (r = 0.57). Fe and Ferr were negatively correlated with Hepc (r = -0.72 and r = -0.02, respectively). E2 and P4 were negatively and positively correlated with Hepc (r = -0.753 and r = 0.54, respectively). In cycling Purebred Spanish mares there is a measurable relationship between steroid hormones and systemic Fe metabolism. Estrogenic dominance in the pre- and ovulatory period allows for a more effective iron status, mediated by hepcidin inhibition. However, P4 during the luteal phase substantially reduces serum Fe and iron stores, possibly related to Hepc stimulation. Future research is required to clarify the relationship between steroid hormones and iron metabolism at the molecular level in equids.

Contribution of Hemogram Plan in the Horse's Clinical Evaluation.

The interpretation of the blood count is essential to help the equine clinician in the diagnosis, prognosis, patient management, and control of equine diseases. Hematologic alterations often reflect the condition of the individual or an overall response to a pathological situation. A thorough clinical examination of the patient is essential to correctly interpret the hematological results. The most common abnormalities in the erythrogram are mainly anemia and polycythemia. The frequent causes of anemia in horses are acute and chronic blood loss, hemolytic anemia, and anemia caused by chronic disease. Evaluation of leukogram, including a total white cell count, a differential cell count, absolute numbers of specific leukocytes can help identify abnormalities that may suggest specific diseases such as a viral or bacterial infection, inflammatory disorders or even a neoplastic process. The platelet count is most often used to monitor or diagnose conditions that cause too much bleeding related with thrombocytopenia; it can be due to multiple mechanisms such as reduction of thrombopoiesis (myeloptisis, myelofibrosis, myeloproliferative disease, and idiopathic medullary aplasias or due to the effect of mielosuppresive drugs), increased peripheral destruction of platelets (immune mediated thrombocytopenia), consumption (dissemined intravascular coagulation) sequestration of the spleen and loss of platelets by idiopathic origin.

Hepcidin, ferritin and iron homeostasis in pregnant Spanish Purebred mares.

During pregnancy, maternal erythropoietic expansion and fetal development require greater mobilization of available iron (Fe) stores. These adjustments in Fe metabolism in humans and rodents are largely mediated by the hormone hepcidin (Hepc), which controls the expression of ferroportin (Fpn), a transporter responsible for exporting Fe from stores to extracellular fluid and plasma. These mechanisms based on the regulation of Hepc on the availability of Fe during gestation in healthy mares remain unknown. The objective of this study was to determine the existence of interrelationships among concentrations of Hepc, ferritin (Ferr), Fe, and estrone (E1) and progesterone (P4) in Spanish Purebred mares along the whole gestation. Blood samples were taken from 31 Spanish Purebred mares each month, during 11 months of pregnancy. Fe and Ferr significantly increased and Hepc decreased during pregnancy (P < 0.05). The secretion peak of estrone (E1) was reached in the 5th month and progesterone (P4) between the 2nd and 3rd months of gestation (P < 0.05). Fe and Ferr were weakly positively correlated (r = 0.57; P < 0.05). Fe and Ferr were negatively correlated with Hepc (r = -0.80 and r = -0.67, respectively) (P < 0.05). P4 was positively correlated with Hepc (r = 0.53; P < 0.05). Pregnancy in the Spanish Purebred mare was characterized by a progressive increase in Fe and Ferr and a reduction in Hepc concentrations. E1 was partially responsible for the suppression of Hepc; on the other hand, P4 induced its stimulation during pregnancy in the mare.

Adaptive Response of Estrogen-Iron Axis in Pregnant Purebred Spanish Mares of Different Age.

The maintenance of iron (Fe) homeostasis is vital for the physiological function along life. In sexually mature humans and experimental animals, estrogens downregulate hepcidin (Hpc) expression, in order to improve the intestinal absorption and to mobilize Fe stores for maternal erythropoietic expansion and placental development. However, changes of these mechanisms related to regulation of Hpc on the availability of Fe during gestation with advancing age in mares, remain unknown. The objective of this study was to evaluate the interrelationships between serum Fe, Ferritin (Ferr) and Hpc with estrone (E1) and estradiol-17ß (E2) concentrations in pregnant mares of different ages. Blood samples were taken from 40 pregnant Spanish Purebred mares belonging to four different age groups, 10 subjects for each group: 4 to 6 years, 7 to 9 years, 10 to 12 years, and >12 years were used in this study. Fe concentrations of 4 to 6 and 7 to 9 years groups were higher (P < .01) than 10 to 12 and >12 years groups. Ferr concentrations of 4 to 6 years group were higher (P < .01) than other groups. Hpc concentrations increased and E1 decreased (P < .01) in >12 years group compared to other age groups. E2 concentrations of 7 to 9, 10 to 12 and >12 years groups were higher (P < .01) than those of 4 to 6 years group; 7 to 9 years group had higher E2 concentrations (P < .01) than >12 years group. Fe and Ferr were negatively correlated with Hpc (r = -0.81 and r = -0.67, respectively). E1 and E2 were negatively correlated with Fe (r = -0.23 and r = -0.11, respectively). E2 was positively correlated with Hpc (r = 0.78). In pregnant Spanish Purebred mare, the increase of estrogens, according to the more efficient iron status in response to Hpc inhibition and consequent mobilization of circulating and iron reserve, shows the existence of "estrogen-iron axis" in young mares. Nevertheless, these mechanisms are reversed in old mares, suggesting a less efficient iron metabolism with advancing age. It is hoped that new investigations are needed to understand in depth and clarify further the complex metabolic and hormonal mechanisms involved also in equine species.

Biochemical and Hematological Indexes of Liver Dysfunction in Horses.

In the present review, the authors, based on the multiple functions performed by the liver, analyze the multiple biochemical and hematological changes as an expression of altered liver function in the horse. The liver performs important metabolic functions related to the synthesis, degradation, and excretion of various substances. Modification of these functions can be evaluated and diagnosed by determining serum concentrations of several serum analytes, including enzymes and other endogenous substances. Hepatocellular enzymes, such as sorbitol dehydrogenase-SDH and glutamate dehydrogenase-GLDH, are released following hepatocellular necrosis. Hepatobiliary enzymes, such as γ-glutamyl transferase-GGT, increase in response to necrosis, cholestasis, and other alterations in bile conducts. Serum concentrations of mainly endogenous and exogenous substances that the liver should synthesize or eliminate, such as proteins (albumin and globulins), bile acids, urea, glucose, total and direct bilirubin, and coagulation factors, and fibrinogen should be included in the liver function test profile. The interpretation of laboratory tests of liver function will allow the diagnosis of functional loss of the organ. Some of the analytes considered provide information on the prognosis of liver disease. This review will provide an accurate and objective interpretation of the common biochemical and hematological tests in use in the diagnosis of equine hepatic disease patients, aiding still further the veterinary activity on the applied equine clinical cases.

Successful Pregnancy Outcome in Mares: The Potential Role of Body Conditional Score, Age and Biochemical Parameter's Adjustments.

The objective of this research was to evaluate the changes in body condition score (BCS), age and the main biochemical parameters in pregnant Spanish Purebred mares. A total of 45 pregnant Spanish broodmares has been studied, classified in three different groups: group A (n = 15), aging 14-18 years old and BCS lower than 4 of 10, group B (n = 15), with ages of 4 to 8 years and a BCS of 5-6/10, and group C (n = 15), aging between 6 and 11 years and a BCS of 8 of 10. Three pregnancy periods were considered: the 1st period, from the 1st to the 3rd, the 2nd from the 4th to the 6th, and the last period, from the 7th month of pregnancy to delivery. Blood samples were taken every month along the pregnancy. The following plasma biochemical parameters were measured: triglycerides, total cholesterol, high density lipoprotein-cholesterol, low density lipoprotein-cholesterol, total, direct and indirect bilirubin, aspartate aminotransferase, lactate dehydrogenase, γ-glutamyl transferase, alkaline phosphatase, total plasma proteins, albumin, glucose, urea and creatinine. Pregnancy in the Spanish broodmare induced hyperlipidemia with hypertriglyceridemia and increased plasma concentrations of total, direct and indirect bilirubin (P < .05). Older pregnant mares showed biochemical changes probably consistent with decreased organ functionality (P < .05). Obesity did not influence lipidic metabolism during pregnancy (P > .05).

Changes of acute-phase proteins, glucose, and lipid metabolism during pregnancy in lactating dairy cows.

The study aimed to evaluate the effects of different stages of lactation (0 to >  300 d) and pregnancy (0 to >  180 d) on serum amyloid A (SAA), C-reactive protein (CRP), glucose, total cholesterol (TCho), and triglyceride (TG) concentrations in dairy cows of different breeds. Thus, 40 healthy multiparous cows (10 Holstein, 10 Simmental, 10 Brown, and 10 Modicana) were randomly selected, and blood samples were collected once every 60 d for 1 year. Overall, SAA and CRP serum concentrations progressively increased and became more variable along the lactation, peaking at >  240-300 d, and then decreased in the last period ( >  300 d). Along pregnancy, SAA and CRP initially increased, with the highest concentrations at >  60-120 d, and then decreased until the last phase of pregnancy ( >  180 d). However, lactation and gestation phases did not significantly affect SAA and CRP when all the cows were analyzed together. A significant and positive correlation was observed between SAA and CRP both along lactation ( r = 0.89 ; p < 0.0001 ) and pregnancy ( r = 0.91 ; p < 0.0001 ). Breeds only showed differences in CRP levels along gestation ( p = 0.0102 ), due to a peak registered at 0-60 d in Holstein cows. In pregnant cows, glucose was positively correlated with SAA ( r = 0.43 ; p = 0.0017 ) and CRP ( r = 0.42 ; p = 0.0019 ). Hence, these significant and positive relationships reflect the physiological adaptations of the dairy cows along both gestational and lactational dynamics, suggesting that these proteins may also be involved in non-pathological processes. In this perspective, this study established that the obtained response markedly varies among healthy individuals along lactation and gestation and thus that the physiological range of acute-phase proteins (APPs) is wide; this makes it difficult to use these proteins as a marker of different physiological reproductive and productive periods.

Changes in Hematological and Biochemical Profiles in Ovariohysterectomized Bitches Using an Alfaxalone-Midazolam-Morphine-Sevoflurane Protocol.

The aim of this study was to monitor hematochemical changes during and after OHE in bitches. Twenty-four females were anesthetized with alfaxalone, midazolam, morphine and sevoflurane. Blood samples were taken before anesthesia (T0), at 30 (T1), and 60 min (T2), at 3 (T3), 6 (T4), 12 (T5), and 24 h (T6), and at 3 (T7) and 7 days (T8) from the start of surgery. Red blood cells (RBC) and packed cell volume (PCV) decreased significantly from T1 to T5 and hemoglobin (HB) concentration from T4 to T6. Both the white blood cell (WBC) and neutrophil (NFS) count increased significantly from T3 to T6, monocyte (MON) from T2 to T5, and eosinophil (EOS) at T5. Platelet (PLT) and plateletcrit (PCT) significantly decreased at T5 and increased from T6 to T8; platelet distribution width (PDW) increased significantly from T3 to T6. Creatine kinase (CK) activity increased significantly from T5 to T7. Glucose (GLU) concentrations increased significantly at T2 and P from T2 to T3. TG levels decreased from T2 to T4 and blood urea nitrogen (BUN) levels from T1 to T7, subsequently increasing until T8. Changes possibly resulting from stress and surgical trauma, as well as hemodilution and splenic storage, are due to anesthesia and surgery. In healthy bitches, these changes tend to gradually stabilize after the ending of OHE. A post-operative follow-up is essential to detect possible post-operative complications.

Systemic and Intrafollicular Androgen Concentrations in Cycling Mares.

The evidence that androgens regulate the folliculogenesis supports the hypothesis that intrafollicular testosterone (T), androstenedione (A4) and dehydroepiandrosterone (DHEA) could be modified along follicular growth. The objective of this study was to evaluate the changes and related relationships between systemic and intrafollicular T, A4 and DHEA in post-deviation and impending ovulation follicles. Sixty ovaries were taken after the slaughter of 30 clinically healthy mares. In according to the sizes, the follicles were classified in 3 different categories, as small (20-30 mm), medium (31-40 mm) and large (≥ 41 mm), and the follicular fluid (FF) samplings were extracted from each single follicle. Intrafollicular concentrations of T, A4, and DHEA were significantly higher than systemic ones. Intrafollicular and systemic T and A4 concentrations were strongly and positively correlated, and DHEA negatively. A4 was the predominant androgen in FF. T and A4 were positively and DHEA negatively correlated with the follicular diameter. T and A4 significantly increased in large and medium than small follicle sizes. DHEA was significantly higher in small than medium and large follicle size. The increase of intrafollicular androgens suggests the presence of androgenic environment based in the biotransformation of DHEA in A4 and later in T in the follicles, and the progressive oestradiol-17ß (E2) production with the advance of follicular growth. The evidence of significant correlations between systemic and intrafollicular androgens considerably helps to understanding more deeply the role of these steroids in the physiology of follicular development in the mare, adding a new segment of scientific literature.

Endocrine and Electrolyte Balances during Periovulatory Period in Cycling Mares.

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.

Interrelationship between reproductive hormones and acute phase proteins during estrous cycle and pregnancy in Spanish purebred broodmares.

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-17ß (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 pregnancy 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.

Can the Presence of Ovarian Corpus Luteum Modify the Hormonal Composition of Follicular Fluid in Mares?

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 different 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-17ß (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.