2. Universidad Cardenal Herrera-CEU

The effect of embryo reinsertion immediately after embryo flushing was studied. In Experiment 1, eight mares were used during 32 cycles (8 cycles in each group). For the first two groups, inseminated mares were flushed 8 days after ovulation and prostaglandin F2α was not administered: in group EF-ET (embryo flushing and embryo transfer) the embryo was reinserted in the same donor mare, while in the EF group, no further procedure was performed. In the third group (ET), non-inseminated mares (recipients) received a Day 8 embryo. Progesterone concentration was measured before EF/ET and 72 h after in the three groups. In Experiment 2, twelve mares were used during 17 cycles in two groups, EF-ET (n = 11) and ET (n = 6), as in Experiment 1, except that every mare was flushed 24 h after embryo transfer to retrieve the embryo. Fewer pregnancies resulted after transfer in EF-ET cycles (0/8, 0%) than in the ET group (6/8, 75%). Progesterone concentration decreased significantly (p = 0.05) 72 h after EF-ET but not in EF or ET cycles (p > 0.1). Three mares from the EF-ET showed full luteolysis and signs of endometritis. In Experiment 2, more (5/6; p = 0.08) grade 1 embryos were recovered in the ET compared to the EF-ET group (3/7); 4 embryos were graded 3–4 (were broken or had signs of degeneration) in the EF-ET group but none in the ET group. In both groups, capsule fragments were obtained as indicative of the presence of a recently destroyed embryo in the EF-ET (n = 3) and ET (n = 1) groups. Positive bacterial cultures were obtained in 2/11 and 1/6 embryo flushes from the EF-ET and ET groups, respectively.

The aims of this study were to determine the effect of the embryo flushing technique and the number of flushing attempts performed by operators of different experience on embryo recovery (ER). Ten non-lactating mares were inseminated with the same stallion in six cycles each (n = 60). Embryo flushing (EF) was performed 7–9 days after ovulation by three operators (OP; 20 EF cycles each): OP1 had performed >500 EF before the study, while OP2 and 3 had performed 0 EF. Each EF was performed with 2 flushing attempts (FA) using 1L of ringer's lactate “in-and-out” using two EF techniques: 1) uterine massage (UM): continuous ballottement and massage of the uterus per rectum during ringer lactate recovery, 2) gravity flow (GF): the ringer lactate was allowed to flow back without massaging the uterus. In both groups, 20 IU of oxytocin were administered at the second FA and the ringer lactate was allowed to remain in the uterus for 3 min before recovery. An extra FA was performed in each group using 0.5 L of ringer lactate and uterine massage. More embryos (P < 0.05) per ovulation were recovered in the UM (17/33, 0.51) than in the GF group (8/36, 0.22). For the UM group, 16/17 embryos (94.1 %) were recovered in the first FA, while only one embryo in the second FA (1/17, 5.9 %). In the GF group, 4 embryos were recovered in each FA. No embryo was found in the extra FA in the UM group, while seven additional embryos were found in the GF group (5/7 flushed by OP1; P < 0.05). The overall ER per cycle was 70, 40, and 45 % for OP1, 2 and 3, respectively. In conclusion, highest embryo recovery is achieved in EF performed with UM, with the majority of embryos being flushed in the first FA.

Background: PGF2α is commonly given at the end of embryo flushing (EF) to shorten the interval to the next oestrus and ovulation. Objectives: To determine the effect of repeated EF on plasma progesterone concentration, percentage of mares with endometritis, unwanted pregnancy and subsequent fertility in mares flushed without the use of PGF2α. Study design: Controlled experiments. Methods: Nine mares were inseminated in seven consecutive cycles (n = 63), to either perform an EF (n = 54) 7–9 days after ovulation or left pregnant (n = 9). PGF2α was not used to induce oestrus. Ultrasound examination and blood sampling were performed just before the EF and 72 h later to determine changes in progesterone concentration and signs of endometritis. Results: The overall percentage of positive EF/pregnancy was 55.5% (30/54) and 66.7% (6/9), respectively. The likelihood of pregnancy/positive EF in the first three cycles was 55.5% (15/29). This was not different (p > 0.1) from the fertility of the last four cycles (69.4%, 25/36). In five EF cycles (9.3%), mares had signs of endometritis and early luteolysis (progesterone <2 ng/mL) 72 h after EF. The reduction in progesterone concentration by 72 h after EF was greater (p < 0.05) for Day 9 (−2.3 ± 0.7 ng/mL) than Day 7 (−1.0 ± 0.8 ng/mL) or Day 8 (−1.3 ± 1.1 ng/mL) cycles. The progesterone concentration in non-flushed mares did not vary significantly during the sampled period (Day 7–12). There were 5 cycles in which the donor mare remained pregnant after the EF, although four were from a single mare. Main limitations: The mare population was limited to barren and maiden mares. The cycle order and operator allocation to each EF were not randomised. Conclusions: EF induces a subtle, but significant reduction in progesterone concentrations compared with non-EF cycles. However, the percentage of mares with EF-induced full luteolysis is low (9.3%). The fertility of mares after repeated EF without administration of PGF2α was unaffected; however, there is a considerable risk of unwanted pregnancy (5/27 = 18.5%) in donors from which an embryo was not recovered.

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.

Sheep estrous synchronization is mainly based on progestagen-impregnated sponges which could cause vaginitis. Several species of Lactobacillus used as probiotics are commonly used in the treatment or prevention of urogenital infections in humans. However, no studies have been performed to analyze the potential use of probiotics to prevent urogenital infections in sheep. A randomized controlled clinical trial was conducted with 21 one-year-old ewes to develop a model of probiotic infusion in vaginal sponges in order to study their influence in ewe’s vaginal microbiota, general health status, fertility and prolificity. Synchronization of estrus was based on intravaginal sponges for 14 days. Bacterial communities (Enterobacteriaceae and lactic acid bacteria) were highly fluctuating over time and between animals. The safety of probiotic infusion (mix of Lactobacillus spp. 60% L. crispatus, 20% L. brevis and 20% L. gasseri) in the vagina of healthy ewes was firstly confirmed. Neutrophils were observed in 80% (8/10) of the control ewes compared to 36% (4/11) of the ewes in the probiotic group 2 days after sponge removal (p = 0.056). Fertility in the control and probiotic groups was 60% (6/10) and 91% (10/11), respectively p = 0.097. These results suggest that Lactobacillus spp. infusion in the ewe’s vagina does not affect general health status or fertility.

Lactic acid bacteria (LAB) dominate human vaginal microbiota and inhibit pathogen proliferation. In other mammals, LAB do not dominate vaginal microbiota, however shifts of dominant microorganisms occur during ovarian cycle. The study objectives were to characterize equine vaginal microbiota in mares by culture-dependent and independent methods and to describe its variation in estrus and diestrus. Vaginal swabs from 8 healthy adult Arabian mares were obtained in estrus and diestrus. For culture-dependent processing, bacteria were isolated on Columbia blood agar (BA) and Man Rogosa Sharpe (MRS) agar. LAB comprised only 2% of total bacterial isolates and were not related to ovarian phases. For culture-independent processing, V3/V4 variable regions of the 16S ribosomal RNA gene were amplified and sequenced using Illumina Miseq. The diversity and composition of the vaginal microbiota did not change during the estrous cycle. Core equine vaginal microbiome consisted of Firmicutes, Bacteroidetes, Proteobacteria and Actinobacteria at the phylum level. At the genus level it was defined by Porphyromonas, Campylobacter, Arcanobacterium, Corynebacterium, Streptococcus, Fusobacterium, uncultured Kiritimatiaellae and Akkermansia. Lactobacillus comprised only 0.18% of the taxonomic composition in estrus and 0.37% in diestrus. No di erences in the relative abundance of the most abundant phylum or genera were observed between estrus and diestrus samples.

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.