A case of a twin surviving to term following the abortion of its co-twin at 9 months in an Arabian mare.

Twin pregnancy in mares is one of the leading causes of abortions. Abortion invariably impacts both fetuses. This report describes an unusual case of a twin surviving to term following the abortion of its co-twin at 9 months in a 7-year-old Egyptian Arabian mare. At the time of abortion at 9 months of gestation, the size of the aborted fetus was equivalent to one of approximately 5 months of age while the age of the live co-twin was 9 months. Both fetuses were males. A skin sample was collected from the aborted fetus and hair samples were collected from the dam, sire and live foal for parentage analysis. The parentage analysis confirmed that both fetuses were by the same dam and sire stallion. The authors suggest several scenarios to explain this condition. This report describes a unique case of a twin surviving to term following the abortion of its co-twin at 9 months in a mare.

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.

Genome Sequence Comparisons between Small and Large Colony Phenotypes of Equine Clinical Isolates of Arcanobacterium hippocoleae.

Arcanobacterium hippocoleae is a Gram-positive fastidious bacterium and is occasionally isolated from the reproductive tract of apparently healthy mares (Equus caballus) or from mares with reproductive tract abnormalities. Apart from a few 16S rRNA gene-based GenBank sequences and one recent report on complete genome assembly, detailed genomic sequence and clinical experimental data are not available on the bacterium. Recently, we observed an unusual increase in the detection of the organism from samples associated with mare reproductive failures in Atlantic Canada. Two colony morphotypes (i.e., small, and large) were detected in culture media, which were identified as A. hippocoleae by MALDI-TOF mass spectrometry and 16S rRNA gene sequencing. Here, we report the whole genome sequencing and characterization of the morphotype variants. The genome length of the large phenotypes was between 2.42 and 2.43, and the small phenotype was 1.99 Mbs. The orthologous nucleotide identity between the large colony phenotypes was ~99%, and the large and small colony phenotypes was between 77.86 and 78.52%, which may warrant the classification of the two morphotypes into different species. Phylogenetic analysis based on 16S rRNA genes or concatenated housekeeping genes grouped the small and large colony variants into two different genotypic clusters. The UvrA protein, which is part of the nucleotide excision repair (NER) system, and 3-isopropoylmalate dehydratase small subunit protein expressed by the leuD gene were identified as potential virulence factors in the large and small colony morphotypes, respectively. However, detailed functional studies will be required to determine the exact roles of these and other identified hypothetical proteins in the cellular metabolism and potential pathogenicity of A. hippocoleae in mares.

Streptococcus equi subsp. zooepidemicus: High molecular diversity of Argentinian strains isolated from mares with endometritis.

Streptococcus equi subsp. zooepidemicus (S. zooepidemicus) is a mucosal commensal of the lower genital tract in horses and is the most isolated bacterium causing endometritis in mares. The aim of this study was to determine the molecular diversity of S. zooepidemicus obtained from endometritis in mares in Buenos Aires province, Argentina. Thirty isolates obtained from the uterus of mares in 2005 and 2017 were studied. The MLST scheme was applied to identify the Argentinian genotypes and the clonal relationships and patterns of evolutionary descent were identified using the eBURST algorithm - goeBURST. Twenty six different Sequence types (STs) were identified, being only 11 of them previously reported in horses and also, from several host species and tissues. The other 15 STs were reported in Argentinian reproductive strains of mares in our study for the first time. The genotypes obtained from uterus in Argentina were not evenly distributed when all the published S. zooepidemicus STs were analysed, thus, it was not possible to establish that the same lineage circulates in our equine population. The fact that the identified genotypes were also reported in other countries, diverse samples and host species suggest that there is not a host, and an anatomical niche adaptation. Finally, the isolation of the same genotype in the vagina/clitoris and the uterus of the same mare highlights the versatility of S. zooepidemicus and its role as an opportunistic pathogen.

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 breeding season and age on follicular dynamics and hemodynamics in embryo donor mares subjected to luteolysis after embryo flushing.

Background: Mares are the only companion animals simulating women in the large diameter of their follicles. Horses start reproduction at the age of three years, and some of them live for >30 years, so aging influences their reproductive capacity. Mares are sensitive to summer heat stress as they can sweat like humans. Aim: The current work aimed to study the effects of age (young versus senile), season (cold versus hot), and the hormonal treatments during embryo collection on the dominant and subordinate follicular dynamics and hemodynamics and circulating ovarian hormones in embryo donor mares ovulated twice spontaneously before inducing ovulation for flushing embryos. Methods: Spontaneous oestrous cycles were studied for young mares (<10 years; N = 6) or senile (>20 years; N = 5) during months of the cold season (November to April) and hot season (May to August). In young embryo donor mares, oestrous cycles after inducing ovulation and luteolysis were studied using Doppler ultrasound. Estradiol (E2), progesterone (P4), nitric oxide (NO), total cholesterol, and lactate dehydrogenase (LDH) were measured in blood serum. Results: A decrease in the dominant follicle antrum diameter (p > 0.05) and LDH (p = 0.016) was observed after inducing luteolysis in young embryo donor mares. Both human chorionic gonadotropin (hCG) and PGF2α treatments increased dominant follicle area (p = 0.0001), antrum area (p = 0.001), perimeter (p = 0.001), granulosa area (p = 0.0001), cholesterol (p = 0.0001), NO (p = 0.0001), and E2 (p = 0.0001). The dominant follicle area, antrum area, perimeter, color area, granulosa area, LDH, cholesterol, NO, and E2 increased (p = 0.0001) during the oestrous cycles of the hot season, but the circulatory % (p = 0.0001) declined. Senile mares had lower dominant follicle area (p = 0.002), antrum area (p = 0.0001), granulosa area (p > 0.05), LDH (p = 0.001), cholesterol (p = 0.0001), NO (p = 0.0001), and E2 (p = 0.0001) but higher circulatory % (p = 0.0001) and color area % (p = 0.023). The dominant follicle possesses the largest diameter, area, perimeter, granulosa area, and color area but the lowest circulatory % during spontaneous oestrous cycles, after inducing ovulation, or luteolysis with significant effects of the day of the spontaneous oestrous cycles on their dynamics and hemodynamics. Conclusion: During hot months, mares treated with hCG ovulated 24 hours later and prostaglandin-induced luteolysis was followed by new ovulation five days later. Follicles ovulated during the hot months were larger than those ovulated during the cold months and both had nearly the same color area %. Senile mares ovulated follicles with a lower area and antrum area but a higher color area %, so senile mares can be used as embryo or oocyte donors during the hot season.

Embryo transfer into anestrous recipient mares prepared using clomiphene citrate and short-acting progesterone.

Embryo transfer (ET) is an important technology for genetic improvement programs in the equine industry. Many protocols have been used for preparation of recipients. This study evaluates a new protocol for preparation of acyclic embryo recipient mares using clomiphene citrate (CC) and short-acting oral progesterone (Altrenogest). Seventy-two native breed recipient mares were divided into 2 groups. Group 1(G1) included 60 non-cyclic mares with follicular structures ≤ 15 mm in diameter that received CC and Altrenogest. Group 2 (G2) included 12 cyclic recipient mares that served as a control group. G1 mares were treated with oral CC at a dose of 250 mg for 4 days followed by oral administration of Altrenogest for 6 days. Ultrasonography was carried out for evaluation of uterine echotexture response to hormonal treatment, and pregnancy diagnosis post ET. Serum estradiol and progesterone concentrations were also assessed. All data were statistically analyzed. The results revealed that the serum progesterone concentrations in G1 were higher than G2 (P < 0.001). There was no difference in the estradiol concentrations between both groups during diestrus phase (P > 0.05). The pregnancy rate was higher in G1 (83.3 %) than G 2 (66.6 %). In conclusion, using oral CC and Altrenogest, as a new protocol, was effective for preparation of acyclic recipient mares in this study.

Multi-locus sequence typing and in vitro antimicrobial resistance of equine Streptococcus equi subspecies zooepidemicus strains.

Streptococcus equi subsp. zooepidemicus (S. zooepidemicus) is one of the most important pathogens frequently associated with the main causes of equine infertility. In this study, we surveyed 22 strains of S. zooepidemicus collected during 2021 from cervico-uterine swabs of mares with endometritis. The genetic variability of the isolated strains was studied by multi-locus sequence typing (MLST) from whole-genome sequencing (WGS) data. The average length of reconstructed genomes was 2,088,286 bp (95% CI: 2,061,569 bp-2,114,967 bp), which was expected for S. zooepidemicus genomes. The assembled genomes were assigned to sequence types (STs) using the S. zooepidemicus scheme targeting seven loci (arcC, nrdE, proS, spi, tdk, tpi, yqiL) available in PubMLST database. MLST revealed a wide variability of STs with two (9.1%) novel STs identified in this study, precisely ST521 with two isolates and ST522 with one isolate. Furthermore, 4/22 (18.2%) isolates were assigned to ST92, 3/22 (13.6%) to ST205, 2/22 (9.1%) to ST475, and one strain (4.5%) for each of the following STs: ST10, ST30, ST39, ST49, ST101, ST132, ST147, ST314, ST369, ST467. Isolates were also tested for antimicrobial resistance using Kirby-Bauer disk diffusion method. Resistance to amoxicillin-clavulanate, ampicillin, amikacin, gentamicin, streptomycin, enrofloxacin, sulfamethoxazole-trimethoprim, tetracycline, oxytetracycline represented the most common resistance profile (13/22, 59.1%). No correlation between specific ST and antimicrobial resistance profile was found. Our study provides a comprehensive insight into the epidemiology, ST diversity and antimicrobial resistance profile of S. zooepidemicus strains, isolated in Italy, causing subfertility problems in mares.

Recombinant Salmonella enterica OmpX protein expression and its potential for serologically diagnosing Salmonella abortion in mares.

Background and Aim: Salmonella abortion in mares is caused by Salmonella enterica subspecies enterica serovar abortus equi infection and is characterized by premature (abortion) or non-viable fetus birth. Although all horses are susceptible to infection, the condition is more often clinically manifested in pregnant mares, with most abortions recorded in young females. In addition, nonspecific clinical disease signs and poorly sensitive and effective bacteriological diagnostic methods hinder rapid and reliable infection diagnoses. Immunochemical methods such as enzyme-linked immunosorbent assay (ELISA) and immunochromatography assays can facilitate effective and rapid diagnoses. However, they require highly specific and active antigens and antibodies. This study aimed to generate a recombinant S. enterica outer membrane protein X (OmpX) and evaluate its suitability for serological diagnosis of Salmonella abortion in mares. Materials and Methods: Outer membrane protein X from the S. enterica antigen was synthesized de novo and expressed in Escherichia coli using the pET28 vector. Transformed E. coli cells were cultured under different conditions to detect recombinant OmpX (rOmpX) expression, and rOmpX purification and refolding were both conducted using metal affinity chromatography. Refolded and purified rOmpX was characterized by western blotting, liquid chromatography with tandem mass spectrometry, and ELISA. Results: After optimized rOmpX expression, a 23 kDa molecular weight protein was identified. Amino acid sequence analysis using Mascot program suggested that these peptides were the OmpX protein from S. enterica. High specificity and diagnostic efficiency were recorded when rOmpX was used in ELISA against 89 serum samples from aborted and contact mares. Conclusion: Recombinant outer membrane protein, in comparison to the O antigen, demonstrated better diagnostic characteristics against sera from mares who aborted and contact horses.

Progesterone Device Use Improves Ovum Pick-Up Efficiency in Acyclic Donors.

The aim of this study was to evaluate follicular dynamics and ovum pick-up (OPU) efficacy in untreated mares or mares treated with an intravaginal progesterone (P4) device during seasonal anestrus (acyclic) and during the breeding season (cyclic). Six mares (mean age = 5 years), were recruited into an ovum pick-up scheme that was performed every 14 days with and without the P4 device, during the acyclic and cyclic phases. Aspirations amounted to seven procedures with or without the P4 device during each phase. Five ultrasound assessments were performed at each interval between the OPUs. Data on follicular number and diameter as well as the numbers of recovered and the percentage of recovered oocytes were also collected. The number of follicles from mares in the acyclic phase was higher (P < .005) regardless of the treatment. However, the follicular diameter was smaller for the P4 group (P < .005) from the 2nd to the 5th evaluation post-OPU procedure. The percentage of oocytes recovered during the acyclic phase was higher for mares treated with the P4 device (P < .005). The P4 device resulted in follicles with smaller diameters and facilitated OPU efficacy.

Equine Placentitis in Mares Induces the Secretion of Pro-Inflammatory Cytokine eIL-1ß and the Active Extracellular Matrix Metalloproteinase (MMP)-9.

Equine placentitis is characterized by infection and inflammation of the placenta. Different biomarkers associated with this inflammatory response have been evaluated in experimentally induced equine placentitis, but not in pregnant mares with spontaneous placentitis. The aim of the current study was to determine the concentration of eIL-1ß and the activity of proMMP-2 and proMMP-9 in the serum of healthy mares and mares with placentitis on days 240 and 320 of gestation to explore whether these biomarkers are associated with equine maternal placentitis and/or with the birth of an infected or inviable foals. Serum samples were collected from sixteen pregnant English Thoroughbred mares, retrospectively classified as follows: (1) healthy mares with full-term gestation; and (2) mares with ultrasonographic signs of placentitis. The health of each foal was examined at birth, and it was decided to classify the cases into four groups: (1) healthy mares delivering a healthy foals (HM-HF, n = 6); (2) mares with USP delivering a healthy foal (USP-HF, n = 3); (3) mares with USP delivering a live septic foal (USP-LSeF, n = 4); and (4) mares with USP delivering a dead foal (USP-DF, n = 3). eIL-1ß was quantified by ELISA, and proMMP-2 and proMMP-9 activity by gelatin zymography electrophoresis. In healthy mares, the serum concentrations of eIL-1ß underwent a significant 16.5-fold increase from day 240 to day 320 of gestation. Although similar results were found in the mares with ultrasonographic signs of placentitis that delivered a healthy foal, those delivering a live septic or nonviable foal exhibited much higher concentrations of eIL-1ß. proMMP-2 and proMMP-9 activity was not associated with maternal placentitis, foal infection, or death. Hence, the presence of placentitis severe enough to affect the health of the foal can be confirmed or discarded by determining the eIL-1ß concentration in mares that have shown ultrasonographic signs of placentitis.

Antimicrobial Resistance and Extended-Spectrum Beta-Lactamase Genes in Enterobacterales, Pseudomonas and Acinetobacter Isolates from the Uterus of Healthy Mares.

Antibiotic-resistant bacteria are a growing concern for human and animal health. The objective of this study was to determine the antimicrobial resistance and extended-spectrum beta-lactamase genes in Enterobacterales, Pseudomonas spp. and Acinetobacter spp. isolates from the uterus of healthy mares. For this purpose, 21 mares were swabbed for samples, which were later seeded on blood agar and MacConkey agar. The isolates were identified using MALDI-TOF and the antimicrobial susceptibility test was performed using the Kirby-Bauer technique. To characterize the resistance genes, a polymerase chain reaction (PCR) scheme was performed. Of the isolates identified as Gram-negative, 68.8% were Enterobacterales, represented by E. coli, Enterobacter cloacae, Citrobacter spp., and Klebsiella pneumoniae; 28.1% belonged to the genus Acinetobacter spp.; and 3.1% to Pseudomonas aeruginosa. A 9.3% of the isolates were multidrug-resistant (MDR), presenting resistance to antibiotics from three different classes, while 18.8% presented resistance to two or more classes of different antibiotics. The diversity of three genes that code for ESBL (blaTEM, blaCTX-M and blaSHV) was detected in 12.5% of the strains. The most frequent was blaSHV, while blaTEM and blaCTX-M were present in Citrobacter spp. and Klebsiella pneumoniae. These results are an alarm call for veterinarians and their environment and suggest taking measures to prevent the spread of these microorganisms.

Use of Doppler ultrasonography for selection of recipients in embryo transfer programs in horses.

We aimed to evaluate the impact of corpus luteum (CL) and uterine characteristics accessed by B-mode and Color-Doppler ultrasonography in recipient mares at the time of embryo transfer (ET) on pregnancy outcomes. Recipient mares (n = 110), between days 3-9 after spontaneous ovulation, received a fresh embryo. Immediately before ET, the reproductive system was assessed by transrectal palpation for the following parameters: uterine tone (0-3), CL echogenicity (0-6), CL type (homogeneous, trabecular or anechoic center), luteal area (cm2), uterine echogenicity (0-3), uterine edema (0-3), luteal blood perfusion (0-100%) and uterine blood perfusion (1-4). Additionally, a blood sample was collected by puncture of the jugular vein for plasma P4 dosage. Retrospectively, recipients were classified according to the luteal area (small [≤ 6 cm2] or large [> 6 cm2]), luteal blood perfusion (low [≤ 55%] or high [> 55%]), and plasma concentration of P4 (low ≤ 9.98 ng/mL or high > 9.98 ng/mL). Pregnancy diagnosis was performed at 12 and 30 days of gestation. Luteal blood perfusion was significantly higher (P = 0.04) in pregnant recipients (n = 83) than in non-pregnant recipients (n = 27). Overall P/ET was higher (P ≤ 0.02) in mares with high luteal blood perfusion and high P4. Luteal blood perfusion was the most adequate significant (P = 0.01) predictor of pregnancy compared with the luteal area and plasma P4 concentration. Only luteal blood perfusion showed a linear (P = 0.03) and cubic (P = 0.004) effect on P/ET. In conclusion, CL blood perfusion determined by color-Doppler can be used in real-time to select recipients with the greatest chance of maintaining pregnancy in equine ET programs.

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.

Plasma Estradiol Profile After Administration of Different Types of Estradiol Esters in Acyclic Mares.

Although the use of different estradiol esters has been extensively studied in hormonal protocols in cows, such information is lacking in mares. The present study aimed to assess the effects of treatment with the same doses and administration frequency of estradiol cypionate, estradiol benzoate and 17ß estradiol on plasma estradiol (E2) concentrations of acyclic mares and correlate the E2 profile to the endometrial edema score. Sixteen treatments were performed in 14 mares randomly divided into three groups: EB (n = 5), EC (n = 5), and 17ß (n = 6), receiving 10 mg on day 0 (D0), 6 mg on D1, and 4 mg on D2 of estradiol benzoate, estradiol cypionate, and estradiol 17ß, respectively. Blood samples, rectal palpations, and ultrasound evaluations were performed once daily, starting before the first estradiol treatment (D0) until edema disappearance or D8. Moderate to high edema was observed in all groups 24 hours after the first estradiol administration. Edema persisted above score 2 until D7, D5, and D4 in groups EC, EB, and 17ß, respectively. Higher edema was found on D2 in EB group compared to 17ß, on D6 and D7 in EC compared to 17ß, and on D8 in EC compared to EB (p ≤ .05). Maximum E2 concentrations were detected on D1 in groups: EB, showing a sharp decrease from D2 to D3 (p < .0001); and 17ß, where no differences were observed between treatment days (p ≥ .05). In the EC group, maximum concentrations were observed on D2, which remained high from D2 to D4 compared to the other days (p < .0001). Plasma concentration of E2 was higher in EB than the other groups on D0 and D1, and EB>EC>17ß on D2. Plasma E2 concentrations and edema score were positively correlated, being moderate in groups EB and EC and weak in the 17ß group. In conclusion, the most pronounced plasma E2 concentration was observed in EB group 24 hours after the administration of the first dose. Estradiol concentrations peaked 48 hours after EC administration, while a distinct peak was not observed in a 24h interval evaluation in the 17ß group. Moreover, high edema does not necessarily reflect on high E2 concentrations in acyclic mares.

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.

Genotypic and Phenotypic Characterization of Escherichia coli Isolates Recovered from the Uterus of Mares with Fertility Problems.

Escherichia coli is the bacterial pathogen most frequently associated with mare infertility. Here, we characterized 24 E. coli strains isolated from mares which presented signs of endometritis and infertility from a genotypic and phenotypic point of view. The majority of the isolates belonged to phylogenetic group B1 (9/24, 37.5%). Regarding antibiotic resistance profiles, 10 out of 24 (41.7%) were multidrug-resistant (MDR). Moreover, 17 out of 24 (70.8%) were strong or moderate biofilm producers, and of these eight were MDR strains. Interestingly, 21 out of 24 (87.5%) E. coli strains were phenotypically resistant to ampicillin and 10 of them were also resistant to amoxicillin with clavulanic acid. Regarding the presence of selected virulence factors, 50% of the examined strains carried at least three of them, with fimH detected in all strains, and followed by kpsMTII (11/24, 45.9%). No strain was able to invade HeLa cell monolayers. No relevant differences for all the investigated characteristics were shown by strains that grew directly on plates versus strains requiring the broth-enrichment step before growing on solid media. In conclusion, this work provides new insight into E. coli strains associated with mares' infertility. These results broaden the knowledge of E. coli and, consequently, add useful information to improve prevention strategies and therapeutic treatments contributing to a significant increase in the pregnancy rate in 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.

The relationship between Th1/Th2 cytokine polarization and milk composition in the postnatal period in Kyrgyz mares and foals.

Immune compatibility between mare and foal is one of the important topics of reproductive immunology. At this point, although there are many studies on antibodies, there are not many publications on the relationship between the cytokine levels of mare, foal and milk and the effects of milk composition on this relationship. Here we investigate the relationship between Th1/Th2 cytokine balance and milk composition in Kyrgyz mares and foals. Samples were taken soon after the foal was born and on days 5, 10 and 20 after birth. Th1 (IFN-γ, IL-2, TNF-α) and Th2 (IL-4, IL-5, IL-10) cytokine levels in blood samples were measured using a commercial ELISA kit. Dry matter, fat, protein and non-fat dry matter ratios were determined in the mare's milk and colostrum. We detected no Th1/Th2 polarization in the mare's milk on the day of the foal's birth (day 0) and day 10, but there was Th1 polarization on day 5 and Th2 cytokine polarization on day 20. There was no polarization in the blood sera of the mares on days 0, 5, and 10, and Th1 cytokine polarization was also detected on day 20. We detected no Th1/Th2 cytokine polarization in the blood sera of the foals on any of the days. Dry matter (19.66 ± 0.39%), protein (16.56 ± 0.18%), fat (2.13 ± 0.17), and non-fat dry matter (17.59 ± 0.44%) were higher in colostrum than the other days. When the correlations between cytokine levels in milk samples and milk composition were examined, there was a positive correlation between IL-5 level and protein ratio on day 10. We detected a positive correlation between IL-2 level and the fat rate on day 20. There was a positive correlation between the IL-2 level and the non-fat dry matter ratio on day 20. Further studies are now needed to determine the relationship between the changes in the composition of mare's milk in the postnatal period and Th1/Th2 cytokine polarization in mares and foals at the time of birth and in the postnatal period. Our finding that protein and fat ratios in mare's milk in the postnatal period are positively correlated with IL-2 levels should be considered in terms of foal and human nutrition.

Seasonal Variation of Melatonin Concentration and mRNA Expression of Melatonin-Related Genes in Developing Ovarian Follicles of Mares Kept under Natural Photoperiods in the Southern Hemisphere.

This study investigated the seasonal variations in mRNA expression of FSH (Fshr), LH (Lhr) receptors, melatonin (Mt1 and Mt2) receptors, melatonin-synthetizing enzymes (Asmt and Aanat) and melatonin concentration in developing follicles from mares raised in natural photoperiods. For one year, ultrasonographic follicular aspiration procedures were performed monthly, and small (<20 mm), medium (20 to 35 mm) and large (>35 mm) follicles were recovered from five mares. One day before monthly sample collections, an exploratory ultrasonography conducted to record the number and the size of all follicles larger than 15 mm. The total number of large follicles were higher during the spring/summer (8.2 ± 1.9) than during autumn/winter (3.0 ± 0.5). Compared to autumn/winter seasons, there was an increase of Fshr and Aanat mRNA expressions in small, medium and large follicles, an increase of Lhr and Asmt mRNA expressions in medium and large follicles and an increase of Mt1 and Mt2 mRNA expressions in small and large follicles during spring/summer. The melatonin levels in follicular fluid were also higher during the spring/summer seasons. The present data show that melatonin locally upregulates the mRNA expression of Mt1 and Mt2 receptors and melatonin-forming enzymes in mare developing follicles during reproductive seasons.