2. Universidad Cardenal Herrera-CEU

Ovum pick-up (OPU) by transvaginal ultrasound guided follicle aspiration in mares is a common assisted reproductive technique used for oocyte recovery and in vitro production of horse embryos. There has been relatively little research into the factors influencing oocyte recovery in OPU from live mares. The objective of this study was to compare oocyte recovery and morphology of ultrasound-guided follicle puncture and aspiration in live mares and in postmortem excised ovaries, in order to validate an experimental model for research purposes of the efficiency of OPU in mares. Data from OPU performed in 12 mares from a commercial program (follicle numbers, oocyte recovery and oocyte morphology) were compared to that obtained from ultrasound-guided follicle puncture of 13 postmortem excised ovaries from slaughtered mares processed within 2 h of slaughter. In both groups, the OPU was performed by the same operator using the same equipment and OPU technique. The recovered oocytes per aspirated follicle was higher (P < 0.05) in the postmortem group (105/166, 63.2 %) than in live mares (138/261, 52.9 %). There was more (P < 0.05) expanded cumulus oocyte complexes in the postmortem than in the live mares (18 % vs. 2.9 %). Several oocytes (5 oocytes from 81 aspirated follicles) were found in the leaked fluid which overflowed during follicle flushing of postmortem ovaries. In conclusion, the higher recovery rate obtained in the excised ovaries and the finding of oocytes in the leaked fluid during OPU, suggests that there is still room for improvement in the in vivo OPU technique. Utilizing postmortem excised ovaries could offer an alternative for further research into factors affecting oocyte recovery and oocyte leakage during OPU procedures.

Toxoplasmosis is one of the most common parasitic zoonoses and represents a significant health risk for humans, especially for immunodeficient patients. The main transmission route is by oral uptake of oocysts and consumption of undercooked meat of infected animals. Different species have been evaluated as possible reservoirs of the parasite, but few studies have been carried out to examine the role of horses in transmission of the disease. Given the proximity of these animals to humans and the widespread consumption of their meat in many countries, including the Mediterranean basin, it is important to determine the prevalence of T. gondii infection in this species. In this study, blood samples from 105 horses were collected and the presence of T. gondii was evaluated by serological and molecular methods. Antibodies against T. gondii of 12 horses (11.43%) were detected by enzyme-linked immunosorbent assay (ELISA), whereas 29 horses (27.62%) showed positive for PCR. Seroprevalence was related to use of the animals, being higher in horses used for dressage than in others. Purebreds had higher seroprevalence than crossbred animals. No differences between breed, sex or age were found. The results of this study confirm the presence of T. gondii infection in horses, highlighting the need to analyse the meat of this species before human consumption and to control of this infection in horses, as they could be an important reservoir of this zoonotic parasite.

Leishmaniosis is a zoonosis caused by Leishmania spp., an intracellular protozoan parasite. This parasite is transmitted by sandflies, and the disease is endemic in the Mediterranean basin. In recent years, the number of species which could be a reservoir of the parasite is increased. One of the most relevant species is the horse, due to their contact with humans and ability to control the disease, thus being a possible silent reservoir. In this study, we have analyzed the prevalence and factors related to L. infantum infection in healthy horses in the Mediterranean region. Epidemiological data and serum samples were obtained from 167 apparently healthy horses, and the presence of L. infantum was evaluated via the ELISA method and real-time PCR. The results show 27.5% of prevalence and that the main factors related to infection are equine breed, morphotype, outdoor living, use, and season. In conclusion, the prevalence of L. infantum infection in apparently healthy horses from eastern Spain (Mediterranean basin) is elevated. To control this zoonosis, it would be advisable to carry out more studies on this and other species that could be silent reservoirs of the parasite, as well as carry out measures such as the use of repellents on a regular basis.

Background: In modern times, horses are utilized not only for labour and transportation purposes but also for recreational activities such as competition and pleasure riding. In these various pursuits, the role of vision plays a crucial role. Electroretinography is the most used test to diagnose diseases of the retinal outer segment. There is a wide variety of devices to perform the electroretinography differing one from each other in the corneal electrode and the light stimulation. The Koijman electrode has been tested in dogs but not in horses. The main purpose of this study was to compare electroretinography parameters from horses sedated with detomidine alone or in combination with butorphanol, during a standardized protocol using the Koijman electrode and RETI-port® system. Seven mares were allocated to the detomidine and detomidine plus butorphanol group in a randomised, controlled, crossover study. Friedman and Willcoxon-signed ranked tests were used to compare the electroretinogram parameters. A Student's t-test was used to compare differences in the number of artefacts to valid values ratio obtained under both sedation protocols. Results: Dark adaptation peaked after 16 min under scotopic conditions in both groups. No significant differences in electroretinogram parameters between groups were observed. During the mixed rod and cone response evaluation under scotopic conditions, all mares made a movement of the head resulting in a high number of artefacts. The detomidine plus butorphanol group showed a non-significant tendency to have fewer artefacts and a longer duration of sedation compared to the detomidine group. Conclusions: Detomidine alone or combined with butorphanol may be suitable to use Koijman electrode and the RETI-port® to perform a standardized long protocol in horses with some adaptations.

Two experiments were conducted in the Northern (UK) and Southern (Brazil) hemispheres to determine the effect of season (month of conception) on the development of supplementary CL (SCL) and the relationship with pregnancy loss. In experiment 1, 199 pregnancies were followed between Day 14 and term, to determine the number of SCL and pregnancy viability (Northern Hemisphere). From the 199 pregnancies, 178 were obtained from inseminations during the breeding season (March–September), while the rest, 21 pregnancies resulted from conceptions in the non-breeding season (October to February). Pregnancies conceived in the breeding season were more likely (P < 0.01) to have at least 1 SCL (75.8 %, 135/178) than pregnancies from the non-breeding season (33 %, 7/21). However, the pregnancy loss between Days 35 and 120 of pregnancy in mares with no SCL was similar (3.5 %, 2/57; P >0.1) than from mares with SCL (1.4 %, 2/142). In Experiment 2 (Southern Hemisphere), three groups of recipients were used based on their ovarian activity at the time of embryo transfer: Anestrus (n = 8), transitional (n = 7) and cyclic (n = 7) recipient mares. While all transitional and cyclic mares developed at least 1 SCL, only 50 % of anestrous recipients (4/8) developed SCL by 120 of gestation. In conclusion, the development of SCL in pregnant mares is influenced by the time of season of conception, therefore it appears to be regulated by the photoperiod and the endogenous seasonal variation in gonadotropin concentrations. Mares with no SCL were not at increased risk of pregnancy loss.

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.

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-17β (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.

Equine ventricular repolarisation wave can be influenced by many physiological and pathological factors. T wave abnormalities have been related to a shorter time for ventricular filling, reduced stroke volume, cardiac output and exercise performance in racehorses. The present research performed electrocardiographic recordings in 14 four-year-old male Andalusian horses, when they were untrained and after three months of an aerobic training programme. Leads I, aVF, V10, V1R, V3R, V1L and V3L were used. It was aimed to assess the incidence of abnormal T waves in this breed, according to the criteria of abnormalities established for other equine breeds, to evaluate if the changes induced by training in the T wave are the same that those reported in the athletic heart syndrome in canine and human athletes and, to analyse if the abnormal T waves could have been related to changes in the plasma concentrations of Na, K, and Cl, since these electrolytes are involved in the cardiac electrical processes. It was found out that the incidence of abnormal T waves was quite high in the Andalusian breed, especially in the precordial leads. Moreover, the incidence of abnormal T waves increased in precordial leads and decreased in lead I and aVF after training. The abnormal T waves after training were shorter and had the same voltage, changes which were different to those presented for the athlete’ heart syndrome. The horses with abnormal T waves had higher plasma K concentrations, both before and after training. Plasma Na and Cl concentration at rest decreased after training. Plasma Na concentrations were positively related to T wave duration and negatively to T wave voltage.

Plasma K accumulation during exercise results from the balance between exchange through biological membranes (mainly muscle fibres and erythrocytes), distribution to other tissues and the haemoconcentration. In the present study, the effect of exercise and training on plasma K concentrations and its relationships with other physiological variables have been analysed in two equine breeds. Twenty male Andalusian (AN) and ten Angloarabian (AA) horses, 7 females and 3 males, were subjected to two standardised exercise tests, composed of four workloads, before and after training. Heart rate (HR) was monitored and venous blood was withdrawn at rest, before each exercise level and during recovery. The following parameters were analysed: packed cell volume (PCV), plasma K, lactate (LA) and total protein (TPP). Furthermore, the horses were filmed and three kinematic parameters were studied: stride duration (SD), frequency (SF) and length (SL). Exercise induced an increase in K from 6 and 8 m/sec in AA and AN horses respectively, a steady-state until the end of the exercise and a decrease after 2 min of recuperation. Some interbreed differences existed, with higher K levels in the AN horses, due to the higher relative exercise intensity, stride frequency and haemoconcentration. K was correlated with HR, PCV, TPP, SL, SD and SF. Training caused a decrease in K in AN, but not in AA horses. Plasma K seems to be a good indicator of the physical effort intensity, fitness and training degrees, but it was not related to the magnitude of the glycolytic response to exercise.

El objetivo de este trabajo ha sido describir un sistema de pun-tuación objetivo, a partir de índices de funcionalidad cardiovas-cular y metabólica, que permita la discriminación entre caba-llos según su potencial atlético. Se han estudiado 45 caballosadultos, Pura Raza Española (PRE), machos. Tras un calenta-miento al trote (4 m/s), los animales realizaron un test de ejer-cicio, a velocidades de 5, 6,7y8m/s, cubriendo 1.000 m encada carga de esfuerzo. Se monitoreó la frecuencia cardiaca(FC) y se extrajeron muestras de sangre venosa en reposo,tras el calentamiento, después de cada fase del test y a los 2,4, 6, 8, 10, 15 y 30 minutos de una recuperación activa. Ensangre entera, se determinó la concentración de hemoglobina(HB) y el valor hematócrito (HTO). En plasma, se midieron losniveles de lactato (LA). Los índices de funcionalidad oxidativoshan sido: HBo’ y HTOo’ (HB y HTO a 8 m/s), V150 y VLA2 (ve-locidad a 150 lpm y 2 mmol/L de LA respectivamente), FCLA2(FC a 2 mmol/L de LA) y LA150 (LA a 150 lpm). Como índicesglicolíticos-mixtos, se han considerado: FCmáx (FC máxima),FCo’ (FC a 8 m/s), LAmáx (LA máximo), V200 y VLA4 (veloci-dades a 200 lpm y 4 mmol/L de LA), FCLA4 (FC a 4 mmol/Lde LA) y LA200 (LA a 200 lpm). Los índices que mejor han dis-criminado a los caballos PRE según su nivel de forma físicahan sido VLA2, LA150 y HTOo’ (oxidativos) y FCo’, VLA4 yV200 (glicolíticos-mixtos). VLA2, VLA4 y V200 estuvieron co-rrelacionados positivamente con el potencial físico y FCo’,HTOo’ y LA150, de modo negativo.