Ultrastructural characteristics and immune profile of equine MSCs from fetal adnexa.

Both in human and equine species, mesenchymal stem cells (MSCs) from amniotic membrane (AM) and Wharton's jelly (WJ), may be particularly useful for immediate use or in later stages of life, after cryopreservation in cell bank. The aim of this study was to compare equine AM- and WJ-MSCs in vitro features that may be relevant for their clinical employment. MSCs were more easily isolated from WJ, even if MSCs derived from AM exhibited more rapid proliferation (P < 0.05). Osteogenic and chondrogenic differentiation were more prominent in MSCs derived from WJ. This is also suggested by the lower adhesion of AM cells, demonstrated by the greater volume of spheroids after hanging drop culture (P < 0.05). Data obtained by PCR confirmed the immunosuppressive function of AM and WJ-MSCs and the presence of active genes specific for anti-inflammatory and angiogenic factors (IL-6, IL 8, IL-ß1). For the first time, by means of transmission electron microscopy (TEM), we ascertained that equine WJ-MSCs constitutively contain a very impressive number of large vesicular structures, scattered throughout the cytoplasm. Moreover, an abundant extracellular fibrillar matrix was located in the intercellular spaces among WJ-MSCs. Data recorded in this study reveal that MSCs from different fetal tissues have different characteristics that may drive their therapeutic use. These finding could be noteworthy for horses as well as for other mammalian species, including humans.

Therapeutic Application of Extracellular Vesicles Derived from Mesenchymal Stem Cells in Domestic Animals.

Recently, the therapeutic potential of extracellular vesicles (EVs) derived from mesenchymal stem cells (MSCs) has been extensively studied in both human and veterinary medicine. EVs are nano-sized particles containing biological components commonly found in other biological materials. For that reason, EV isolation and characterization are critical to draw precise conclusions during their investigation. Research on EVs within veterinary medicine is still considered in its early phases, yet numerous papers were published in recent years. The conventional adult tissues for deriving MSCs include adipose tissue and bone marrow. Nonetheless, alternative sources such as synovial fluid, endometrium, gingiva, and milk have also been intermittently used. Fetal adnexa are amniotic membrane/fluid, umbilical cord and Wharton's jelly. Cells derived from fetal adnexa exhibit an intermediate state between embryonic and adult cells, demonstrating higher proliferative and differentiative potential and longer telomeres compared to cells from adult tissues. Summarized here are the principal and recent preclinical and clinical studies performed in domestic animals such as horse, cattle, dog and cat. To minimize the use of antibiotics and address the serious issue of antibiotic resistance as a public health concern, they will undoubtedly also be utilized in the future to treat infections in domestic animals. A number of concerns, including large-scale production with standardization of EV separation and characterization techniques, must be resolved for clinical application.

Could assisted reproductive techniques affect equine fetal membranes and neonatal outcome?

Embryo transfer (ET) and intracytoplasmic sperm injection (ICSI) are widely used in equine species, but their effects on fetal adnexa and neonates have not been investigated yet. The aim of this study was to retrospectively evaluate whether pregnancies obtained by ET or ICSI could be associated with the presence of macroscopic alterations of fetal membranes (FM) and umbilical cord (UC) and if the use of these techniques could influence neonatal outcome. Sixty-six light breed mares hospitalized at the Veterinary Teaching Hospital, University of Bologna, for attending delivery were included in the study. Mares were divided into Artificial Insemination (AI; 32/66 mares, 48 %), Embryo Transfer (ET; 12/66 mares, 18.2 %) and Intracytoplasmic Sperm Injection (ICSI; 22/66 mares, 33 %) groups. All the medical reports of mares and their foals were reviewed and data about mare, pregnancy, foaling, fetal membranes, umbilical cord and foal were recorded. The occurrence of dystocia resulted statistically different between AI group and ICSI group (p = 0.0066), and between AI group and ET group (p = 0.044). Macroscopic examination of FM revealed alterations in 30/66 mares (46 %): 8/32 in AI (25 %), 7/12 in ET (58 %) and 15/22 in ICSI (68 %) with significant lower incidence in AI compared to ET (p = 0.04) and ICSI (p = 0.002) groups. Alterations reported were chorionic villi hypoplasia, chorioallantois edema, allantois cysts, necrotic areas and greenish-grey concretions. Total length of UC resulted significantly shorter in ICSI group (49 ± 9 cm; p < 0.03) compared to AI (60 ± 17 cm) and ET (59 ± 15 cm). However, there were no differences in the incidence of foals' diseases at birth and in foals' survival among groups (p > 0.05). The results demonstrate that transfer of in vivo or in vitro produced embryos may lead to alterations of placental development, as observed in other species, without being associated with a higher incidence of neonatal morbidity and mortality. Further studies about trophoblast development, FM histological evaluation, and placental gene expression should be carried out to clarify the mechanisms underlying the placental alterations.

On-field Gross Morphology Evaluation of Dromedary Camel (Camelus dromedarius) Fetal Membranes.

The dromedary camel (Camelus dromedarius) fetal membranes, commonly referred to as "the placenta", are epitheliochorial, diffuse, and microcotyledonary, similarly to the mare's placenta. The evaluation of the placenta is an essential component of the neonatal evaluation in the equine species. However, post-partum or post-abortion placental assessment in dromedary camels is unfortunately too frequently neglected and, to the best of the authors' knowledge, the dromedary camel species lacks a comprehensive description of the normal placenta's gross morphology. In order to facilitate its on-field evaluation, the current study describes the macroscopic features of the placenta of the dromedary camel after full-term pregnancy and spontaneous parturition.

Gross and histological examination of Wharton's Jelly in the equine umbilical cord.

Wharton's jelly (WJ) is fundamental for the well-being of the fetus, binding to the umbilical vessels and protecting them from twisting and compression. Gross and microscopic studies have been undertaken on the umbilical cord (UC) of human placentae in both normal and high-risk pregnancies, however there is limited research on equine UC. The aim of this study was to describe microscopically and immunohistochemically the equine UC in normal pregnancies, with particular attention to WJ. Forty-seven healthy mares, with no complications during pregnancy, admitted to the hospital for attending delivery were enrolled. Clinical data was collected at foaling on foal health and placental characteristics. UC samples were collected from three sites (amniotic, allantoic and in the region of vein anastomosis) for histology. The thickness of different layers of arteries and veins and WJ in different UC portions were measured (µm). Wharton's Jelly was weighted (g) and its sections were stained with Masson's trichrome, orcein technique and silver impregnation. Immunohistochemistry was undertaken using antibodies raised-against collagen type I, V, VI and fibrillin. Forty-seven UCs, from 19 colt and 28 filly foals, were analyzed for WJ weight and 8/47 UCs were examined histologically. Warton's jelly was only found in the amniotic portion of the UC closest to the foal's abdomen. The weight of WJ (4.0 ± 3.3 g) did not vary between colts and fillies and it was not correlated with any of the clinical or UC parameters measured. The tunica media of arteries and veins was thicker in the amniotic portion of the UC, as described in human UCs in late pregnancy. This finding could be an adaptation to aid in resisting compression because of fetal movements and UC twisting. The umbilical vein was thicker than the umbilical arteries in the tunica media and tunica adventitia in the sections examined throughout the length of the cord. This preliminary study describes gross and histological WJ's structure in the equine species. However, further studies are required to better characterize UC's changes throughout pregnancy and in the presence of mare's or fetal disease.

Lidocaine constant rate infusion in isoflurane anesthetized neonatal foals.

Introduction: In horses, lidocaine infusion is administered intraoperatively for analgesia and for a reduction of inhalant anaesthetic requirement. The objective of the study was to describe the anaesthetic effects of lidocaine infusion in isoflurane anaesthetised foals. Methods: Twelve foals (<3 weeks old) undergoing surgery were included in the study (LIDO group). Foals were premedicated with midazolam and butorphanol IV, anaesthesia was induced with ketamine and propofol IV and maintained with isoflurane. Lidocaine was administered intraoperatively at 0.05 mg/kg/min. Also, the anaesthetic records of 11 foals in which lidocaine was not administered intraoperatively were retrospectively evaluated and they were considered as a historical control group (HC). Heart rate (HR), mean arterial pressure (MAP) and fraction of expired isoflurane were monitored continuously. Time of extubation, time to reach sternal recumbency and standing were recorded. The quality of recovery was assessed. Results: HR decreased in both groups compared with baseline values and intraoperatively the differences were statistically significant (p = 0.01 and p = 0.03 respectively in the LIDO and HC groups). Intraoperatively the HR was significantly lower in the LIDO group (71.2 ± 13.4 bpm) compared with the HC group (87.1 ± 17.7 bpm) (p = 0.0236). The number of foals requiring inotropic support (LIDO n = 7 and HC n = 9) was not statistically associated with the treatment group (p = 0.371). The extubation time, the time to reach the sternal recumbency and the quality of recovery did not differ significantly between the two groups (p = 0.7 and p = 0.6 respectively). Discussion: In conclusion, in anaesthetised foals the addition of lidocaine does not provide a sparing effect on isoflurane requirement, and it does not interfere with the quality of recovery, however it decreases significantly the HR, which is pivotal in foals for the maintenance of cardiac output and peripheral perfusion. Therefore, a continuous patient monitoring is essential.

Assessment of the microvascular perfusion using sidestream dark-field imaging in healthy newborn foals.

BACKGROUND: Different methods to measure tissue perfusion are available in equine neonatology, but they are not representative of microvascular derangements. OBJECTIVE: To evaluate the feasibility of the sidestream dark-field (SDF) capillaroscopy to visualize the capillary microvasculature in conscious newborn foals, the differences between two imaging sites and times of measurements, and the measurements' reproducibility. METHODS: Seventeen healthy newborn foals were enrolled. Three sites at the upper and lower lip mucosa were assessed by SDF, using a hand-held capiscope, at 24 h and at 4-5 days after birth. Videos were assessed independently by two observers for quality and for semiquantitative calculation of microvascular parameters, including vascular density (VD), microvascular flow index (MFI), proportion of perfused vessels (PPV), and functional capillary density (FCD). Data were analyzed using unpaired and paired Student's T-tests to assess differences between sites and time-points. Bland-Altman plots and intraclass correlation coefficient (ICC) were used to assess measurement reproducibility. RESULTS: Differences were found between the upper and the lower lip for VD at both 24 h and 4-5 days, and for FCD at 24 h, and between the two time-points for PPV at the lower lip. ICC for measurement reproducibility was good for all parameters (0.64-0.79) for the lower lip, and was good for VD and FCD (0,76-0,79) and fair to moderate for MFI and PPV (0.31-0.41) for the upper lip. CONCLUSIONS: Measurement of the capillary microvasculature is feasible in the conscious newborn foal. The lower lip has the best measurement reproducibility. Further investigations are warranted in cardiovascularly compromised cases, particularly in septic foals.

Management of uroperitoneum through combination of conservative and surgical treatments in two colts.

Background: Ruptures of the urinary bladder and urachus are the most frequent cause of uroperitoneum in foals. Surgical correction is often the first treatment choice, however, nonsurgical methods, such as urine removal via urinary catheters and abdominal drains, have been successfully performed in foals. Case Description: Two foals were referred to the Equine Perinatology Unit for suspicion of uroperitoneum. The diagnosis was confirmed by hematobiochemical and ultrasound examinations, thus cystorrhaphy and cystoplasty were attempted. Surgeons found a lesion in the dorsocranial margin of the bladder (Case 1) and a tear in the pelvic urethra (Case 2); in the first case, the defect was routinely repaired, while the last lesion was impossible to repair due to its localization. A urinary catheter was left in place in both cases. Uroperitoneum recurred 72 hours after the surgery in both foals: a second surgical correction was not recommended due to the localization of the tears and conservative treatment, with the placement of a 32F chest tube in the most ventral part of the abdomen, was preferred. Abdominal drains were removed 5-7 days after surgery, while urinary catheters were left in place for up to 7-8 days. Colts' conditions improved during hospitalizations. Two months after bladder surgery, Case 1 was euthanized due to multiple adhesions between the small intestine and the abdominal wall. Case 2 was still alive one year postoperatively. Conclusion: Although it cannot be considered the first choice for the treatment of uroperitoneum in the foal, nonsurgical treatment was successful in both cases in the short-term follow-up. However, the prognosis should be cautious due to the risk of long-term complications. Conservative management may be used to manage bladder/urethral tears that cannot be solved by surgery.

Shipping Temperature, Time and Media Effects on Equine Wharton's Jelly and Adipose Tissue Derived Mesenchymal Stromal Cells Characteristics.

To use Mesenchymal Stromal Cells (MSCs) in equine patients, isolation and expansion are performed in a laboratory. Cells are then sent back to the veterinary clinic. The main goal of storage conditions during cell transport is to preserve their biological properties and viability. The aim of this study was to evaluate the effects of storage solutions, temperature and time on the characteristics of equine adipose tissue and Wharton's jelly-derived MSCs. We compared two different storage solutions (plasma and 0.9% NaCl), two different temperatures (4 °C and room temperature) and three time frames (6, 24, 48 h). Cell viability, colony-forming units, trilineage differentiation, the expression of CD45 and CD90 antigens and adhesion potentials were evaluated. Despite the molecular characterization and differentiation potential were not influenced by storage conditions, viability, colony-forming units and adhesion potential are influenced in different way, depending on MSCs sources. Overall, this study found that, despite equine adipose tissue MSCs being usable after 24 h of storage, cells derived from Wharton's jelly need to be used within 6 h. Moreover, while for adipose cells the best conservation solutions seems to be plasma, the cell viability of Wharton's jelly MSCs declined in both saline and plasma solution, confirming their reduced resistance to conservation.

Dystocia in the Standardbred Mare: A Retrospective Study from 2004 to 2020.

Dystocia as a prolonged stage II parturition (>30 min) was associated with a higher risk of complications. The hypothesis of the study was that any type of dystocia could affect the foal's health, even when the stage II was <30 min. Clinical reports on 222 Standardbred mares and their foals hospitalized at the Veterinary Teaching Hospital of the University of Bologna from 2004 to 2020 were reviewed. Mares were divided into the Eutocia Group (165, eutocic delivery) and the Dystocia Group (57, dystocic delivery). The incidence of dystocia was 4.9%. Stage II was longer in the Dystocia Group (median 20 min) than in the Eutocia Group (median 12 min). All occurrences of dystocia were retrospectively classified into three categories of severity: mild, moderate and severe dystocia. The occurrence of postpartum complications in mares and neonatal diseases and failure of passive transfer of immunity in foals was higher in the Dystocia Group. Foal venous lactatemia and serum creatine kinase were significantly higher in the Dystocia Group (median 3.9 mmol/L; 262 UI/L respectively) than in the Eutocia Group (median 3.1 mmol/L; 187 UI/L respectively). The APGAR score was lower in the Dystocia Group (median 8) than in the Eutocia Group (median 10) and significantly lower in severe dystocia (median 3). The duration of stage II should not be considered the only parameter of dystocia in mares: even a rapid resolution of dystocia could pose health risks to the foal and the mare.

High-Risk Pregnancy Is Associated With Increased Alpha-Fetoprotein Concentrations in the Amniotic Fluid and Foal Plasma.

This study aimed to determine alpha-fetoprotein (AFP) concentrations in amniotic fluid, plasma of mares and respective foals: carrying normal pregnancies and delivering healthy foals (n = 20; Group 1); carrying apparently normal pregnancies and delivering sick foals (n = 15; Group 2); carrying high-risk pregnancies and delivering sick foals (n = 14; Group 3). High-risk pregnancy was defined by a history of premature udder development/lactation or increased of the combined thickness of the uterus and placenta, or vulvar discharge and/or mares' systemic illness. Sick foals were affected by neonatal encephalopathy, sepsis, prematurity/dysmaturity, or hypoxic-ischemic encephalopathy. Based on histological examination of the chorioallantois, AFP trend was analyzed in pregnancies with pathologic (PFM) and normal fetal membranes (NFM). Concentrations of AFP were measured using a commercially available immunoassay previously validated for horses. Mares' plasma AFP did not change during the last 15-20 days of pregnancy in the three groups, and there was no difference among them. Amniotic fluid AFP was higher in Group 3 (P = .014). Foals' plasma AFP concentration was higher from birth to 72hours in foals of Group 2 and 3 than in healthy ones, and foals of Group 3 had the highest value. The strong association (r = 0.84; P < .0001) between AFP in amniotic fluid and foals' plasma at birth is likely due to the presence of AFP in fetal urine. AFP was higher in pregnancy with PFM than with NFM in mare's plasma at admission (P = .031), amniotic fluid (P = .004), foal's plasma at birth (P = .002), at 24 (P = .005) and at 72 hours of life (P = .004). AFP is higher in pregnancy with histopathological lesions of the chorioallantois providing the evidence of the differences between pregnancy with a normal placental barrier and the more compromised ones. The increased AFP concentration in the amniotic fluid and plasma of high-risk foals suggests upregulation.

Hair Cortisol and DHEA-S in Foals and Mares as a Retrospective Picture of Feto-Maternal Relationship under Physiological and Pathological Conditions.

Equine fetal hair starts to grow at around 270 days of pregnancy, and hair collected at birth reflects hormones of the last third of pregnancy. The study aimed to evaluate cortisol (CORT) and dehydroepiandrosterone-sulfate (DHEA-S) concentrations and their ratio in the trichological matrix of foals and mares in relation to their clinical parameters; the clinical condition of the neonate (study 1); the housing place at parturition (study 2). In study 1, 107 mare-foal pairs were divided into healthy (group H; n = 56) and sick (group S; n = 51) foals, whereas in study 2, group H was divided into hospital (n = 30) and breeding farm (n = 26) parturition. Steroids from hair were measured using a solid-phase microtiter radioimmunoassay. In study 1, hair CORT concentrations measured in foals did not differ between groups and did not appear to be influenced by clinical parameters. A correlation between foal and mare hair CORT concentrations (p = 0.019; r = 0.312, group H; p = 0.006; r = 0.349, group S) and between CORT and DHEA-S concentrations in foals (p = 0.018; r = 0.282, group H; p < 0.001; r = 0.44, group S) and mares (p = 0.006; r = 0.361, group H; p = 0.027; r = 0.271, group S) exists in both groups. Increased hair DHEA-S concentrations (p = 0.033) and decreased CORT/DHEA-S ratio (p < 0.001) appear to be potential biomarkers of chronic stress in the final third of pregnancy, as well as a potential sign of resilience and allostatic load in sick foals, and deserve further attention in the evaluation of prenatal hypothalamus-pituitary-adrenal (HPA) axis activity in the equine species. In study 2, hormone concentrations in the hair of mares hospitalized for attended parturition did not differ from those that were foaled at the breeding farm. This result could be related to a too brief period of hospitalization to cause significant changes in steroid deposition in the mare's hair.

Morphological study of equine amniotic compartment.

Exfoliative cytology of human amniotic fluid (AF) has been extensively studied since 1940s, but no data exist in equine species. The AF compartment represents the environment in which the foetus grows and matures, and its composition changes, reflecting foetal well-being and development. The aim of this study was to describe for the first time the morphology of equine AF cells and amniotic membrane (AM) with light microscopy (LM) and transmission electron microscopy (TEM). AF was collected at parturition within 5 min after the appearance of the AM with a 60 mL syringe from 34 mares and samples of AM were collected from a subset of 7 mares with normal pregnancy hospitalized for attended parturition. For LM observation, a sample of cytocentrifuged fresh AF was stained with May-Grünwald Giemsa and AM sections were stained with H-E. For TEM observation, AF and AM were fixed, embedded in epoxy resins, then sectioned and stained with uranyl acetate and lead citrate solutions. Nucleated and anucleated squamous cells with basophilic cytoplasm, intensely basophilic cornified cells, polymorphonuclear cells, and clusters of eosinophilic amorphous substance were observed. Cells presumably derived from tracheal epithelium and small round nucleated cells with eosinophilic cytoplasm presumably derived from amniotic or urinary epithelium were occasionally found. Lamellar body-like structures (LBs) were present in some epithelial cells. In AM, epithelial, basal and mesenchymal layers were clearly visible with both techniques as previously described. Epithelial cells had several cytoplasmic vacuolization and microvilli were present on apical surface. The connective tissue presented fibroblasts, mesenchymal and rare polymorphonuclear cells, surrounded by abundant extracellular matrix, with distribution of collagen fibres. This is the first report about equine amniotic compartment description by LM and TEM. As recently reported in human medicine, the AM could be a second potential source of pulmonary surfactant, given the finding of LBs inside the cells which could have the same function as in humans. Further studies in samples collected at different gestational ages could increase the knowledge of AF cells and their modification during pregnancy, as well as a better comprehension of the role of AM as a secondary source of pulmonary surfactant in the horse. The diagnostic evaluation of AF cellular composition in high-risk pregnancies may also be investigated.

Study on NGF and VEGF during the Equine Perinatal Period-Part 1: Healthy Foals Born from Normal Pregnancy and Parturition.

The importance of trophic factors, such as nerve growth factor (NGF), vascular endothelial growth factor (VEGF), and brain-derived neurotrophic factor (BDNF) during the perinatal period, is now emerging. Through their functional activities of neurogenesis and angiogenesis, they play a key role in the final maturation of the nervous and vascular systems. The present study aims to: (i) evaluate the NGF and VEGF levels obtained at parturition from the mare, foal and umbilical cord vein plasma, as well as in amniotic fluid; (ii) evaluate NGF and VEGF content in the plasma of healthy foals during the first 72 h of life (T0, T24 and T72); (iii) evaluate NGF and VEGF levels at parturition in relation to the selected mares' and foals' clinical parameters; (iv) evaluate the relationship between the two trophic factors and the thyroid hormone levels (TT3 and TT4) in the first 72 h of life; (v) assess mRNA expression of NGF, VEGF and BDNF and their cell surface receptors in the placenta. Fourteen Standardbred healthy foals born from mares with normal pregnancies and parturitions were included in the study. The dosage of NGF and VEGF levels was performed using commercial ELISA kits, whereas NGF, VEGF and BDNF placental gene expression was performed using semi-quantitative real-time PCR. In foal plasma, both NGF and VEGF levels decreased significantly over time, from T0 to T24 (p = 0.0066 for NGF; p < 0.0001 for VEGF) and from T0 to T72 (p = 0.0179 for NGF; p = 0.0016 for VEGF). In foal serum, TT3 levels increased significantly over time from T0 to T24 (p = 0.0058) and from T0 to T72 (p = 0.0013), whereas TT4 levels decreased significantly over time from T0 to T24 (p = 0.0201) and from T0 to T72 (p < 0.0001). A positive correlation was found in the levels of NGF and VEGF in foal plasma at each time point (p = 0.0115; r = 0.2862). A positive correlation was found between NGF levels in the foal plasma at T0 and lactate (p = 0.0359; r = 0.5634) as well as between VEGF levels in the foal plasma at T0 and creatine kinase (p = 0.0459; r = 0.5407). VEGF was expressed in all fetal membranes, whereas NGF and its receptors were not expressed in the amnion. The close relationship between the two trophic factors in foal plasma over time and their fine expression in placental tissues appear to be key regulators of fetal development and adaptation to extra-uterine life.

Study on NGF and VEGF during the Equine Perinatal Period-Part 2: Foals Affected by Neonatal Encephalopathy.

Neonatal Encephalopathy (NE) may be caused by hypoxic ischemic insults or inflammatory insults and modified by innate protective or excitatory mechanisms. Understanding the underlying pathophysiology is important in formulating a rational approach to diagnosis. The preliminary aim was to clinically characterize a population of foals spontaneously affected by NE. The study aimed to: (i) evaluate nerve growth factor (NGF) and vascular endothelial growth factor (VEGF) levels in plasma samples obtained in the affected population at parturition from the mare's jugular vein, umbilical cord vein and foal's jugular vein, as well as in amniotic fluid; (ii) evaluate the NGF and VEGF content in the plasma of foals affected by NE during the first 72 h of life/hospitalization; (iii) evaluate NGF and VEGF levels at birth/admission in relation to selected mare's and foal's clinical parameters; (iv) evaluate the relationship between the two trophic factors and thyroid hormone levels (TT3 and TT4) in the first 72 h of life/hospitalization; and (v) assess the mRNA expression of NGF, VEGF and brain-derived neurotrophic factor (BDNF), and their cell surface receptors, in the placenta of mares that delivered foals affected by NE. Thirteen affected foals born from mares hospitalized for peripartum monitoring (group NE) and twenty affected foals hospitalized after birth (group exNE) were included in the study. Dosage of NGF and VEGF levels was performed using commercial ELISA kits, whereas NGF, VEGF, and BDNF placental gene expression was performed using a semi-quantitative real-time PCR. In group NE, NGF levels decreased significantly from T0 to T24 (p = 0.0447) and VEGF levels decreased significantly from T0 to T72 (p = 0.0234), whereas in group exNE, only NGF levels decreased significantly from T0 to T24 (p = 0.0304). Compared to healthy foals, a significant reduction of TT3 levels was observed in both NE (T24, p = 0.0066; T72 p = 0.0003) and exNE (T0, p = 0.0082; T24, p < 0.0001; T72, p < 0.0001) groups, whereas a significant reduction of TT4 levels was observed only in exNE group (T0, p = 0.0003; T24, p = 0.0010; T72, p = 0.0110). In group NE, NGF levels were positively correlated with both TT3 (p = 0.0475; r = 0.3424) and TT4 levels (p = 0.0063; r = 0.4589). In the placenta, a reduced expression of NGF in the allantois (p = 0.0033) and a reduced expression of BDNF in the amnion (p = 0.0498) were observed. The less pronounced decrease of the two trophic factors compared to healthy foals, their relationship with thyroid hormones over time, and the reduced expression of NGF and BDNF in placental tissues of mares that delivered affected foals, could be key regulators in the mechanisms of equine NE.

Development of a Composite Pain Scale in Foals: A Pilot Study.

Prompt pain management is crucial in horses; however, tools to assess pain are limited. This study aimed to develop and pilot a composite scale for pain estimation in foals. The "Foal Composite Pain Scale" (FCPS) was developed based on literature and authors' expertise. The FCPS consisted of 11 facial expressions, 4 behavioural items, and 5 physical items. Thirty-five pain-free foals (Control Group) and 15 foals experiencing pain (Pain Group) were used. Foals were video-recorded at different time points: the Control Group only at inclusion (C), while the Pain Group at inclusion (T1), after an analgesic treatment (T2), and at recovery (T3). Physical items were also recorded at the same time points. Videos were scored twice by five trained observers, blinded to group and time points, to calculate inter- and intra-observer reliability of each scale item. Fleiss' kappa values ranged from moderate to almost perfect for the majority of the items, while the intraclass correlation coefficient was excellent (ICC = 0.923). The consistency of FCPS was also excellent (Cronbach's alpha = 0.842). A cut-off ≥ 7 indicated the presence of pain. The Pain Group scores were significantly higher (p < 0.001) than the Control Group and decreased over time (T1, T2 > T3; p = 0.001). Overall, FCPS seems clinically applicable to quantify pain and improve the judgment of the quality of life in foals, but it needs modifications based on these preliminary findings. Consequently, further studies on a larger sample size are needed to test the feasibility and validity of the refined FCPS.

Activities of matrix metalloproteinase-2 and -9 in amniotic fluid at parturition in mares with normal and high-risk pregnancy.

The matrix metalloproteinases (MMPs) are a family of enzymes involved in extracellular matrix remodeling. MMPs are secreted in a latent form and activated by local and infiltrating cells. MMP-2 and -9 are the most studied in reproduction and have been detected in bovine, ovine, equine and human placenta. There is only one study on MMPs in the equine amniotic fluid (AF) reporting a decrease in the activity of MMP-2 in case of premature delivery. The aim of this study was focused on MMP-2 and -9 activity in AF collected at parturition from mares with normal or high-risk pregnancy. High-risk pregnancy was defined as a history of premature udder development/lactation, increase of combined thickness of the uterus and placenta, vulvar discharge and/or mare's systemic illness. The diagnosis of placental insufficiency was confirmed retrospectively after macroscopic and histopatologic examination of the placenta. AF was collected by needle puncture of the amnion within 5 min after its appearance through the vulva. The activity of MMP-2 and -9 was analyzed by in-gel zymography allowing the evaluation of both latent and active forms. Twenty mares with normal pregnancy (group 1) and 8 mares with high-risk pregnancy (group 2) were included. All mares in group 2 had a high-risk pregnancy with a diagnosis of placental insufficiency associated with placental villous hypoplasia, placentitis or placental edema. The bands relative to latent and active forms of MMP-2 were clearly visible in both groups and the activity of latent (P = 0.010) and active (P = 0.004) forms was lower in the AF samples of group 2. The band of the latent form of MMP-9 was visible in 17/20 samples of group 1, while it was completely absent in all samples of group 2. In contrast, the band of the active form was clearly visible and with a greater activity in AF samples of group 2 (P = 0.002). Placental dysfunction seems to induce a lower MMP-2 activity and a higher MMP-9 activity through the release of pro-inflammatory cytokines. Because fetal pulmonary secretions are a likely source of gelatinases in AF during late gestation, the increased MMP-9 activity could be related to fetal distress. These data provide a starting point to better understand the role of MMPs in equine pregnancy, although it should be confirmed in a larger and more homogeneous population of mares with high-risk pregnancy.

Changes in the Fecal Microbiota Associated with a Broad-Spectrum Antimicrobial Administration in Hospitalized Neonatal Foals with Probiotics Supplementation.

There is a wide array of evidence across species that exposure to antibiotics is associated with dysbiosis, and due to their widespread use, this also raises concerns also in medicine. The study aimed to determine the changes on the fecal microbiota in hospitalized neonatal foals administered with broad-spectrum antimicrobials and supplemented probiotics. Fecal samples were collected at hospital admission (Ta), at the end of the antimicrobial treatment (Te) and at discharge (Td). Feces were analysed by next-generation sequencing of the 16S rRNA gene on Illumina MiSeq. Seven foals treated with IV ampicillin and amikacin/gentamicin were included. The mean age at Ta was 19 h, the mean treatment length was 7 days and the mean time between Te and Td was 4.3 days. Seven phyla were identified: Actinobacteria, Bacteroidetes, Firmicutes, Fusobacteria, Proteobacteria, TM7 and Verrucomicrobia. At Ta, Firmicutes (48.19%) and Proteobacteria (31.56%) were dominant. The alpha diversity decreased from Ta to Te, but it was the highest at Td. The beta diversity was higher at Ta than at Te and higher at Td than at Te. An increase in Akkermansia over time was detected. The results suggest that the intestinal microbiota of neonatal foals rapidly returns to a high diversity after treatment. It is possible that in foals, the effect of antimicrobials is strongly influenced or overshadowed by the time-dependent changes in the developing gut microbiota.

Fetal Congenital Diaphragmatic Hernia and Hydramnios in a Quarter Horse Mare.

Hydramnios is an excessive accumulation of fluid within the amniotic compartment. It is a rare condition in mares, often associated with fetal anomalies. Hydrops of fetal membranes predisposes to the rupture of the prepubic tendon, and many authors suggest the induction of parturition to preserve mare's reproductive career. This report presents the case of a 15-year-old multiparous Quarter Horse mare, referred at 268 days of gestation for suspected hydrops. Repeated ultrasonographic exams confirmed an increase in the depth of the amniotic fluid and reduced fetal viability. During the hospitalization, the mare developed a partial rupture of the prepubic tendon. In this case, a conservative approach was elected, and the mare was treated with nonsteroidal anti-inflammatory drugs (NSAIDs) and an abdominal support bandage. At 327 days of gestation, the mare gave birth to a foal with APGAR score 1. The resuscitation attempt was unsuccessful, and the foal died immediately. A post-mortem examination diagnosed a congenital diaphragmatic hernia (CDH) with pleuroperitoneal diaphragmatic eventration.

Transabdominal ultrasonographic measurement of caudal vena cava to aorta derived ratios in clinically healthy neonatal foals.

BACKGROUND: Ultrasonographic measurement of the vena cava and aorta (Ao) diameters and their ratios have been suggested to be a reliable way of quantifying hypovolemia. OBJECTIVE: To evaluate the feasibility and reliability of an ultrasonographic technique for measurement of Ao and caudal vena cava (CVC) and derived ratios using three different acoustic windows in a population of healthy neonatal foals. Correlation between Ao and CVC measurements and ratios and foals' age or bodyweight were also investigated. METHODS: In 14 healthy foals aged less than 7 days, the diameters of the Ao and of the CVC in long and short axis were measured by two observers from images obtained using three different ultrasonographic imaging planes (left dorsal, left ventral and right views). The Ao and CVC cross-sectional area and the CVC/Ao diameter and area ratios were calculated. Image quality was subjectively assessed. Intraobserver and interobserver reliabilities for image quality scores and measurements were evaluated between the two observers. Simple linear regression models were used to identify correlations between the CVC/Ao measurements and ratios and the age and bodyweight of the foals. RESULTS: The left ventral view showed the highest reliability. A correlation between bodyweight and the short axis measurement of the CVC was found (R2  = 0.385; p = 0.018). Age was positively correlated with the long axis of measurement of the CVC (R2  = 0.426; p = 0.011) and CVC/Ao diameter ratio (R2  = 0.625; p = 0.001). CONCLUSIONS: The left ventral view allows the Ao and CVC cross sections to be easily visualized and measured in neonatal foals in right lateral recumbency.