2. Universidad Cardenal Herrera-CEU

Leishmaniasis and toxoplasmosis are two of the most common parasitic zoonoses. Leishmaniasis is endemic to 98 countries around the world, whereas toxoplasmosis is widely distributed throughout the world, causing significant health expenditure. Horses can play a relevant role in the transmission of the disease, being a silent reservoir, as clinical signs are not common. Serum samples from 166 horses living in eastern Spain (Mediterranean basin) were analysed to determine the presence of antibodies against Leishmania spp. and T. gondii by ELISA (Enzyme-linked Immunosorbent Assay.) The risk factors evaluated were the geographical area and the relative humidity and average temperature, and epidemiological factors such as sex, reproductive status, age, breed, morphotype, living with other domestic animals, use and access to the outdoors. Seroprevalence of Leishmania spp. and T. gondii infection was found 28.92%, and 16.27% respectively, whereas co-infection of the two parasites was found only in two males. Leishmania seroprevalence was high in castrated males and several mesodolichomorphic equine breeds used for teaching, as well as in outdoor animals. The most elevated seroprevalence was found in winter with higher levels of rainfall, whereas high seroprevalence of T. gondii was found in crossbreeding animals and those used for breeding. High seroprevalence of Leishmania spp. and T. gondii was found in horses of the Mediterranean basin. These data suggest that horses can act as a silent reservoir and that this species has high potential for transmission to humans, outdoor animals and in geographical areas with high average rainfall.

Bronchoalveolar lavage fluid (BALF) cytology is used for the diagnosis of non-infectious lower airway inflammation in equids. Discrepancies have been reported in the differential cell count when different staining methods were used both in humans and horses. The objective of this study was to compare the results of BALF cytology in donkeys using four different staining methods: modified May-Grunwald Giemsa (mMGG), Diff-Quick (DQ), Toluidine blue (TB) and Perls Prussian blue (PPB). Nine healthy Amiata female donkeys were enrolled. The BAL procedure was performed as previously described and pairs of cytocentrifuged BALF slides were stained with each method. No differences between mMGG and DQ were found for macrophages, neutrophils, and eosinophils, while differences were found in mast cell count between DQ vs.TB, but not between mMGG vs. DQ or mMGG vs. TB. Finally, no differences were obtained in the differential count for hemosiderophages comparing mMGG, DQ and PPB. The mMGG appears to be an excellent stain for the identification of all possible cell types, including mast cells in the BALF of donkeys. DQ, if used alone, may lead to inappropriate identification of mast cells. These results are consistent with the literature on BALF staining methods in horses.

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.