2. Universidad Cardenal Herrera-CEU

The present experiment aimed at determining whether the timing of the maternal recognition of pregnancy (MRP) was specific to individual mares by determining when luteostasis, a failure to return to oestrus, reliably occurred in individuals following embryo reduction. Singleton (n = 150) and synchronous twin pregnancies (n = 9) were reduced in 10 individuals (5–29 reductions/mare) at pre-determined time points within days 10 (n = 20), 11 (n = 65), 12 (n = 47), 13 (n = 12) or 14 (n = 15) of pregnancy. Prior to embryo reduction, the vesicle diameter was measured in 71% (106/150) of the singleton pregnancies. The interovulatory interval (IOI) was recorded on 78 occasions in seven of the mares in either non-pregnant cycles (n = 37) or those in which luteolysis followed embryo reduction (n = 41). The earliest time post-ovulation at which the embryo reduction resulted in luteostasis in an individual was 252 h (mid-Day 10). Consistency in luteostasis following embryo reduction showed individual variation between mares (272–344 h). Binary logistic regression analysis showed an individual mare effect (p < 0.001) and an effect of the interval post-ovulation at which embryo reduction was undertaken (p < 0.001). However, there was no significant effect of vesicle diameter at the time of embryo reduction (p = 0.099), nor a singleton or twin pregnancy (p = 0.993), on the dependent of luteolysis or luteostasis. The median IOI between individual mares varied significantly (p < 0.05) but was not correlated to the timing of MRP. The timing of MRP varied between the mares but was repeatable in each individual. The factors and mechanisms underlying the individuality in the timing of MRP were not determined and warrant further study.

It is believed that during the spring transition, the developing follicle tends to grow more slowly, persist longer and grow to a larger diameter prior to ovulation than at subsequent oestrus periods. A general suspicion, that the first ovulation of the year is less fertile than subsequent ovulations could be explained by a slower growth rate of the ovulatory follicle during transition with the consequent production of a subfertile oocyte. By detailed serial examination of the same group of Irish Draught mares over three winter/spring periods, no significant difference was found in either growth rate or pre-ovulatory diameter when compared with subsequent ovulations. Mean growth rates over the ten days prior to ovulation were 2.20 mm/day (range 1.18 to 3.64) and 2.19 mm/day (range 1.25 to 3.41) for first and subsequent ovulations respectively. Mean maximum pre-ovulatory diameters were 44.7 mm (range 35 to 59) and 43.5 mm (range 31 to 57.5) for first and subsequent ovulations respectively. The impression gained by practitioners that the first follicle develops more slowly during the transition to the first ovulation of the season may be due to less frequent examinations and consequently a failure to observe and record that follicles may grow and then regress during this period. The largest follicle observed a few days previously is not necessarily the same large follicle found at a later examination.

Las prostaglandinas (PGE2 y PGF2α) ejercen un rol esencial en el proceso de la ovulación en los mamíferos. El objetivo de este experimento fue determinar si la inyección intrafolicular con prostaglandina F2 alfa (PGF2α) administrada en el período preovulatorio inducía la ovulación normal y formación del cuerpo lúteo, confirmado más tarde con la presencia de una vesícula embrionaria. Se utilizaron 6 yeguas mestizas en dos ciclos estrales cada una, con un diseño crossover. El tracto reproductivo de las yeguas fue ecografiado cada 24 h desde el día 15 del ciclo (Día 0 = día de la ovulación) hasta el momento de la punción folicular (Hora 0). La punción ecoguiada del folículo preovulatorio (diámetro ≥ 35mm y presencia de moderado edema uterino) se realizó a través del flanco, inyectando 750 μg de PGF2α diluido en 0,5 ml de agua estéril (grupo tratado n=6) o placebo (0,5 ml de agua de inyección) (grupo control n=6). Previo a la punción, las yeguas fueron inseminadas con semen fresco. Se obtuvieron imágenes ultrasonográficas cada 12 h del folículo punzado y se tomaron muestras de sangre cada 48 h de la vena yugular para determinar los niveles plasmáticos de progesterona. Cinco de las seis yeguas (83%) del grupo tratado, tuvieron fallas en la ovulación y sólo una de las yeguas ovuló pero no se obtuvo preñez. De acuerdo con estos resultados, la inyección intrafolicular con PGF2α no permitiría la ovulación normal y, contrariamente a lo esperado, induciría la formación de folículos hemorrágicos anovulatorios. Además, se observó un retraso en la elevación de progesterona en la única yegua que ovuló. / Prostaglandins (PGE2 and PGF2α) play an essential role in the ovulation process in mammals. The objective of this experiment was to determine whether intrafolicular injection with prostaglandin F2 alpha (PGF2α) administered in the pre-ovulation period induced normal ovulation and corpus luteum formation, it was later confirmed with the presence of an embryonic vesicle. Six mixedbreed mares were used in two estrous cycles each, with a crossover design. The ultrasonographic examination of the reproductive tract was performed every 24 hours from day 15 of the cycle (Day 0 = day of ovulation) until the time of follicular puncture (Hour 0). The ultrasound guided puncture of the preovulatory follicle (diameter ≥ 35 mm and presence of moderate uterine edema) was performed through the flank, injecting 750 μg of PGF2α diluted in 0.5 ml of sterile water (treated group n = 6) or placebo (0.5 ml of injection water) (control group n = 6). Prior to puncture, the mares were inseminated with fresh semen. Ultrasound images were obtained every 12 h of the punctured follicle and blood samples were taken every 48 h from the jugular vein to determine plasma progesterone levels. Five of the six mares (83%) of the treated group had ovulation failures and only one of the mares ovulated but no pregnancy was obtained. According to these results, intrafolicular injection with PGF2α at the concentration used would not allow normal ovulation and contrary to expectations would induce the formation of anovulatory hemorrhagic follicles. In addition, a delay in progesterone rise was observed in the only mare that ovulated.

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.

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.

Dopamine (DA) is an important endogenous catecholamine that exerts generalized effects on both neuronal (as a neurotransmitter) and non-neuronal tissues (as an autocrine or paracrine agent). In the central nervous system (CNS), DA binds to specific membrane receptors present in neurons and plays a key role in the control of motor activity, learning, cognition, affectivity and attention. Horses can also present with hyper- and hypodopaminergic conditions, including stereotypic behaviors and pituitary pars intermedia dysfunction and Parkinsonian’s syndrome, respectively. DA biosynthesis also occurs in peripheral tissues, and receptors in various organs such as the kidney, pancreas, lungs and blood vessels outside the CNS have been detected. DA emulates the actions related to the sympathetic nervous system (SNS), promoting the increase in heart rate, blood pressure, electrolyte balance and gastrointestinal (GI) motility. In fact, GI alterations in dopaminergic transmission have been directly or indirectly related to hypomotility and/or postoperative ileus (POI). On the other hand, there are physiological factors, such as breed, age, exercise and reproductive status that modify DA concentrations. In reproduction, the administration of DA antagonists in the middle/end of the spring and anestrus transition period advances the first ovulation of the year in mares. This chapter offers a brief description of the importance of DA as a neurotransmitter and peripheral hormone. Special attention is paid to: (1) functional alterations that occur in the brain and GI tract in various diseases and (2) current therapy to correct alterations in DA systems.

Los folículos hemorrágicos anovulatorios (HAF) son la patología anovulatoria más común en yeguas. Dicha condición es comparable a la que padecen las mujeres (folículos luteinizados anovulatorios), siendo una causa importante de infertilidad en medicina humana. La causa exacta por la que se producen estos folículos es todavía una incógnita, pero son muchos los factores que influyen en la inhibición de la ruptura folicular. Estudios en yeguas y en otras especies han demostrado que la inhibición de la síntesis de prostaglandinas (PG) mediante administración de antiinflamatorios no esteroideos (AINEs) en el periodo periovulatorio ha producido un alto porcentaje de folículos luteinizados anovulatorios. De forma fisiológica es la hormona luteinizante (LH) la que inicia la cascada de eventos que tienen como fin la ovulación, entre los cuales está la síntesis de PG dentro del folículo preovulatorio. Otro elemento que parece estar implicado en la formación de HAF son los niveles altos de prolactina como se ha demostrado en otras especies como la rata y el conejo. Un estudio reciente obtuvo resultados similares de producción de folículos luteinizados anovulatorios en yeguas mediante la administración de sulpirida, un antagonista de la dopamina que eleva la prolactina. El objetivo de esta tesis doctoral fue valorar el efecto de la prolactina y las prostaglandinas E2 y F2α sobre la ovulación y fertilidad en la yegua. La administración de sulpirida durante el periodo periovulatorio produjo un aumento de las concentraciones plasmáticas de prolactina. Sin embargo, esta hiperprolactinemia no tuvo ningún efecto sobre la ovulación ni sobre la formación de folículos hemorrágicos anovulatorios. En yeguas tratadas con AINEs en el periodo periovulatorio, la administración intrafolicular de PGE2 y PGF2α restableció la ovulación dentro de las 12 h tras la inyección, evitando la formación de folículos luteinizados anovulatorios. Además, supuso la correcta liberación y fertilización del ovocito, ya que las yeguas quedaron gestantes. Este mismo protocolo se empleó en yeguas al inicio del estro. En la mayoría de las yeguas se produjo el colapso folicular dentro de las 12 h siguientes a la administración del tratamiento. Esta fase del estudio reafirma el papel de las prostaglandinas en el proceso de ruptura de la pared y colapso folicular y sugiere que este hecho puede suceder incluso antes de la última fase de la onda preovulatoria de LH.