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Reintroduced animals face disease risks, potentially impacting both the reintroduced and the local wildlife/domestic populations. This study focuses on the Asiatic wild asses (Equus hemionus) reintroduced to the Negev desert in southern Israel. Despite potential threats of disease spill-over to and from domesticated donkeys and horses in the area, there are no records of the gastrointestinal nematodes (GIN) of the wild ass population. We used DNA metabarcoding on fecal samples of wild asses collected across seasons and habitats, near water sources that they frequently use. Ten GIN species were detected in the feces, nine belonging to the family Strongylidae, which commonly infects and causes disease in equids worldwide, such as horses, zebras, and donkeys. Some of these Strongylidae species are also found in domesticated equids in Israel, thus raising concerns regarding potential parasite transmission between wild and domestic animals. The high prevalence of certain GIN species suggests frequent transmission, likely due to the congregation of the wild asses around water sources. While we observed statistically significant variations in some GIN species across seasons and habitats, we did not find clear overall differences between GIN communities. DNA metabarcoding proves to be a valuable tool for identifying GIN species in wild animals, with potential applications in monitoring their health and preventing disease transmission to and from domestic animals.

While the principle definitive host of the zoonotic cestode Echinococcus multilocularis in Europe is the red fox, several rodent species act as main intermediate hosts. Among others, e.g., humans, dogs, and pigs, also horses have been described to act as aberrant hosts in highly endemic regions. Here, a case of an E. multilocularis infection in a kulan (Equus hemionus kulan) is described. The five years old kulan from a zoo in Slovakia was transported to an animal park in Germany. The animal had to be euthanized within a few weeks after the import due to its poor general state of health. The pathological examination revealed a nodular mass in the liver as an incidental finding. By histological examination of the mass, a pyogranulomatous and necrotizing inflammation and intralesional fragments of amorphous eosinophil layers were detected. The suspected diagnosis of E. multilocularis infection was confirmed by PCR addressing parts of the genes 12S rRNA and the NADH dehydrogenase subunit 2, showing very high identities with isolates from France, Slovakia and the USA.

Equine Gastric Ulcer Syndrome (EGUS) occurs with variable prevalence in horses, donkeys, and mules. Due to the particularities of the mucous membranes, the syndrome is made up of Squamous Gastric Disease (ESGD) and Glandular Gastric Disease (EGGD). Given the multifactorial nature and multiple classification systems of the syndrome, significant differences have been reported between prevalence studies performed ante mortem, which are even more remarkable when compared with postmortem evaluations. This study aimed to determine the presence and grade of squamous gastric disease in horses, donkeys and mules immediately after slaughter. The postmortem examination considered the inspection of the squamous region (cardia, dorsal fundus, and margo plicatus) and the classification of the observed lesions. The general prevalence of ESGD in the entire population of study was 83.3 % (78 %, 89 %, and 83 % for horses, donkeys, and mules, respectively), compromising the margo plicatus in all cases. 75 % had more than 5 lesions and 50 % had deep lesions, lesions of varying severity and/or evidence of recent/active bleeding. The prevalence of ESGD was similar in horses, donkeys, and mules subjected to similar handling conditions prior to slaughter, including long-distance traveling, fasting, and stress factors., Competing Interests: Declaration of competing interest The authors declare that they have no know competing financial interest or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

Objective: To clinically evaluate previously developed pain scales [Donkey Chronic Pain Composite Pain Scale (DCP-CPS), Donkey Chronic Pain Facial Assessment of Pain (DCP-FAP) and combined Donkey Chronic Pain Scale (DCPS)], including behavioural and facial expression-based variables, for the assessment of chronic pain in donkeys., Study Design: Prospective, blinded clinical study., Animal: A group of 77 donkeys (34 patients and 43 healthy control animals)., Methods: Animals were assessed by two observers that were blinded to the condition of the animals., Results: Both DCP-CPS and DCP-FAP, and resulting combined DCPS scores, showed good interobserver reliability [intraclass correlation coefficient (ICC) = 0.91, 95% confidence interval (CI) = 0.86-0.95, p < 0.001; ICC = 0.71, CI = 0.50-0.83, p < 0.001 and ICC = 0.84, CI = 0.72-0.91, p < 0.001, respectively]. All scores (DCP-CPS, DCP-FAP and the resulting combined DCPS) were significantly higher for patients than for controls at all time points (p < 0.001 for all three scales). Sensitivity and specificity for identification of pain (cut-off value >3) was 73.0% and 65.1% for DCP-CPS, and 60.9% and 83.3% for DCP-FAP, respectively. For the combined DCPS, sensitivity was 87.0% and specificity 90.9% (cut-off value >6)., Conclusions and Clinical Relevance: Based on behavioural and facial expression-based variables, DCPS proved a promising and reproducible tool to assess different types of chronic pain in donkeys. The combination of behavioural and facial expression-based variables showed the best discriminatory characteristics in the current study. Further studies are needed for refinement of these tools., (Copyright © 2024 The Authors. Published by Elsevier Ltd.. All rights reserved.)

The analysis of stable isotopes in bone collagen allows us to infer the diet of the animals studied. This dataset consists of isotopic signatures (δ13C and δ15N) obtained by isotope ratio mass spectrometry from the skeletal remains of 42 equines (horse, ass and their hybrids) from the Can Roqueta site (Sabadell, Northeast Iberian Peninsula). Their chronology spans from Late Bronze Age to Late Roman Period, with particular emphasis on the Early Iron Age. These animals were found in storage silos and graves and were probably sacrificed as ritual offerings. The isotopic values are accompanied by data to assess the quality of the collagen analyzed. This fills a gap in equine isotopic values for this region and chronology, which may be of use to archaeologists interested in the study of livestock management or palaeodiet.

The digital stethoscope (DS) is a cost-effective single-lead digital stethoscope that allows simultaneous electrocardiographic (ECG) and phonocardiographic recordings on a smartphone. Despite its application in small animals and horses, there are currently no studies on its use in donkeys. The aim of this study was to evaluate the use of a new smartphone-based DS device in recording ECG tracings in donkeys. Standard base-apex lead ECG (sECG) and single-lead DS ECG (dECG) were simultaneously recorded for at least 30 s. Both sECG and dECG tracings were analysed by the same operator, recording heart rate, ECG waves and intervals, and the presence and duration of artefacts. Thirty-seven donkeys were included. The dECG tracings were interpretable in all the animals (100 %). The results showed perfect agreement between the sECG and dECG data for the classification of heart rhythm and P-wave polarity. Strong agreement was found in the evaluation of heart rate calculated manually and automatically by the smartphone app, QRS complex polarity, T wave polarity, and duration of the PR interval. However, no agreement was found in the evaluation of P wave duration, QRS complex duration and amplitude, and T wave duration and amplitude. In conclusion, although this is only a preliminary study, the DS was a valid, practical, and easy to use electrocardiographic tool for recording good-quality ECG tracings to assess the ECGs of donkeys in the field., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

Members of the Equus genus exhibit a fascinating capacity for hybridization, giving rise to healthy offspring. Mules, resulting from the mating of a mare with a jack, represent the most prevalent equid hybrid, serving diverse roles in our society. While in vitro embryo production, particularly through Intracytoplasmic Sperm Injection (ICSI), has rapidly gained significance in domestic horses, the in vitro production in other equids remains largely unexplored. Utilizing donkey sperm for fertilizing horse oocytes not only addresses this gap but also provides an opportunity to investigate donkey sperm's fertilization capability in vitro to further improve donkey ICSI. In this work, we initially studied the localization of donkey sperm Phospholipase C zeta (PLCζ) and assessed the sperm's capacity to induce pronuclear formation and maternal SMARCA4 recruitment upon injection into pig oocytes through ICSI. Subsequently, we investigated the injection of donkey sperm into horse oocytes, evaluating in vitro production up to the blastocyst stage using sperm from different jacks, including frozen and refrigerated samples. Distinct patterns of PLCζ localization were observed for donkey sperm cells compared to their horse counterparts. Additionally, donkey sperm exhibits a reduced ability to induce porcine oocyte activation. However, when injected into horse oocytes, donkey sperm demonstrated sufficient capability to induce oocyte activation as no discernible differences in cleavage or blastocyst rates are observed between in vitro produced mules and horse ICSI embryos. Our study not only delineates PLCζ localization in donkey sperm but also suggests potential differences in the ability to induce oocyte activation in pigs compared to horses while observing no distinctions in pronuclear recruitment of SMARCA4. Interestingly, donkey sperm remains sufficiently capable of inducing horse oocyte activation for in vitro mule blastocyst production., Competing Interests: Declaration of competing interest The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

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., Competing Interests: Declaration of competing interest None of the authors has any financial or personal relationships that could inappropriately influence or bias the content of the paper., (Copyright © 2024. Published by Elsevier Inc.)

This study aimed to compare the morphometry of horse and mule embryos. The study's hypothesis was that the micronuclei and nuclear fragmentation indexes are higher in mule embryos than in horse embryos. Twenty-two mares were randomly assigned in a crossover design to receive semen from a horse and a donkey; thirteen horse and thirteen mule embryos were obtained. Embryos were recovered eight days post-ovulation and classified according to the stage of development and quality with a score from 1 (excellent) to 4 (degenerate). Embryos were stained with Hoechst33342, and images were acquired with a fluorescence microscope. Nuclei were categorized as compact, mitotic, or fragmented; the fragmented and mitotic indexes were calculated based on their proportion over the total amount of nuclei counted. Embryo size and nuclear morphometry were assessed through ImageJ. Data analyses were carried out with GraphPad using ANOVA and T-test; significance was set at P < 0.05. The number of positive flushes in cycles bred with donkey or stallion semen did not differ when compared per cycle or per ovulation (13 vs. 12) (P > 0.05). One set of twins was recovered from a mare bred to the stallion that had a double ovulation; a mule and horse embryos were both recovered from eight mares. There was no difference in size between mule and horse embryos (915.5 ± 288 μm vs. 575.8 ± 69.6 μm) (P > 0.05) size of the study. The mule embryos scored between grade 1 (n = 9) and grade 2 (n = 4); similarly, the horse embryos scored between grade 1 (n = 6) and grade 2 (n = 7). The evaluation of the nuclear morphometry revealed that horse and mule embryos have a similar number of compact nuclei per sector (148.7 ± 6.8 nuclei/sector in mule embryos vs. 156.5 ± 8.5 nuclei/sector in horse embryos) (P > 0.05); however, the number of mitotic nuclei tended to be higher in mule embryos (5.2 ± 0.82) than in horse embryos (3.3 ± 0.3) (P = 0.08). The fragmented nuclei index was similar between mule (0.25 ± 0.1%) and horse (0.22 ± 0.1%) embryos (P = 0.4); the mitotic nuclei index was higher in mule embryos (3.2 ± 0.4%) than in horse embryos (2.2 ± 0.2%) (P = 0.02). In conclusion, embryo morphology of mares bred to a donkey and a horse shares similar nuclear ultrastructure features, except that mule embryos have a higher mitotic index., Competing Interests: Declaration of competing interest None., (Copyright © 2023 Elsevier Inc. All rights reserved.)

Ticks are important ectoparasites in equids, causing economic losses in animal husbandry in Iran and worldwide. This study was aimed to determine frequency and species diversity of hard ticks in equids in Ardabil province, during the four seasons in 2021. A total of 240 equids (187 horses, 53 donkeys) were randomly selected and examined. Ixodid ticks were collected from body surface of examined animals and identified. Of all examined equids, 32.5% horses, and 4.58% donkeys were infested with a total number of 412 ixodid ticks. Tick indices (tick number per animal) were 4.62. There was significant difference between prevalence and different age groups of infested animals. The highest prevalence of hard ticks was found in ≥3 years-old equids in the region. Of all examined ticks (412), three genera including Hyalomma spp., Rhipicephalus spp. .and Dermacentor spp. with four species. i.e. Hyalomma anatolicum (73.05%), H. marginatum (17.23%), Rhipicephalus bursa (7.76%) and Dermacentor marginatus (1.94%) were identified. Ixodid ticks' infestations occurred throughout the year with the highest prevalence in spring. The findings indicated that the equids had a high species diversity and ixodid tick infestation, necessitating additional research to identify any possible tick vectors for the hemoparasite transmission in equids of this region., Competing Interests: Declaration of competing interest The authors declare no conflict of interest., (Copyright © 2024 Elsevier B.V. All rights reserved.)

This study aimed to compare the efficacy of two pharmacological protocols for inducing ex copula ejaculation in donkeys. Seven healthy jacks (male donkeys) aged 4 to 20 years (median 8 years) and weighing 136.2±4.17 kg (mean±SE) were enrolled. Using a crossover design, each jack was subjected in a random order to two treatment protocols (IX and IDO) with an interval of 7 days between the two protocols. Each jack was orally administered 3 mg/kg imipramine hydrochloride followed 2 hours later by intravenous (IV) administration of 1.1 mg/kg xylazine hydrochloride (IX protocol) or 0.02 mg/kg detomidine hydrochloride and 20 IU total dose oxytocin (IDO protocol). The jacks were monitored for behavioral changes and ejaculation up to 3 hours from the beginning of each protocol. A total of 22 ex copula ejaculation replicates were attempted. Both protocols resulted in deep sedation and partial to complete penile protrusion in all jacks. There was no difference in the efficacy with the IX protocol inducing ejaculation in 1 of the 11 replicates and the IDO protocol inducing ejaculation in none of the 11 replicates. The results suggest that neither of the two tested pharmacological protocols were effective in inducing ex copula ejaculation in donkeys., Competing Interests: Declaration of competing interest The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Elsevier Inc. All rights reserved.)

In the last decades, western Countries increased their interest in innovative products like donkey milk and other activities carried out with donkeys (onotherapy, onotourism). Donkey milk is considered a high-added-value food and is very similar to human breast milk. It is also used as an ingredient in cosmetics. The growing public interest suggests the need for a pilot study on the sustainability of donkey milk production, according to Life Cycle Assessment (LCA) criteria. Milk was used as the Declared Functional Unit (DFU) and two different models were described, a Real Scenario Model (RSM, i.e. a farm with its declared milk yield), and an Increased Milk Production Model (IMPM, i.e., the same farm with theoretically increased milk yield). Allocation was applied both in RSM and IMPM; thus, different values of impact categories, i.e., Global Warming Potential (GWP, kg CO 2 equivalents), Acidification Potential (ACP, g SO 2 equivalents) and Eutrophication Potential (EUP, g PO 4 3- ) were observed. GWP improved after mass allocation and showed the lowest equivalents in IMPM, compared to economic and reference allocation criterion (P < 0.05). In RSM, allocations affected GWP in a different way: the smaller size of the DFU resulted in the largest estimation of CO 2 equivalents (P < 0.05) for reference allocation, whereas the mass allocation estimates were lower than with economic allocation (P < 0.05). ACP and EUP followed the same trends. No differences were found in IMPM results across the three allocation methods used. Moreover, mass allocation values recorded in RSM did not significantly differ from IMPM. ACP and EUP of RSM improved after economic allocation, although they were less sustainable (P < 0.05) than all IMPM values and RSM equivalents after mass allocation (P < 0.05). As expected, the theoretical model with increased milk yield improved the sustainability of the system. Both scenarios were affected by allocation criteria. In RSM, the economic and mass allocations described a representative scenario where donkey meat contributed to subtracting equivalents from milk (the main product). The present paper is a pilot study estimating for the first time the environmental impact of donkey milk production, with the aim to stimulate further research., (Copyright © 2023 The Author(s). Published by Elsevier B.V. All rights reserved.)

The application of embryo recovery and transfer technology in the donkey industry is far lower than that of horses and cattle. Sometimes the recovered embryos could not be transferred in time, which required embryo cryopreservation. The embryo cryopreservation technology is more conducive to the preservation and transportation of recovered embryos with excellent genetic traits. However, this technique for donkey embryos is not efficient and needs further optimization. The objective of this study was to evaluate the effect of different manual puncture methods on the viability and pregnancy rates of vitrified-thawed donkey embryos. A total of 117 donkey embryos were recovered on day 7-8 post-ovulation and were divided into four groups by random assortment. There were 28 embryos without puncture or cryopreservation (Control). 58 embryos were manually punctured using a 29G needle (VG, n = 29) or microneedle with a sharp tip of <10 μm (VM, n = 29), then vitrified in 15% EG + 15% DMSO + 0.5 M sucrose. Another 31 embryos were punctured with a microneedle and vitrified with 10% EG + 10% DMSO +0.5 M sucrose +2 mol/L proline (VMP). Both fresh embryos and vitrified-thawed embryos were incubated for 3 h (38.5 °C, 5% CO 2 in air) before embryo transfer. The results showed that the embryo recovery rates on day 7.5 and 8 were higher than day 7 (P < 0.05). After incubation, dead cells were assessed and the percentages of dead cells in VM and VMP were lower than that in VG (P < 0.05), although both were higher than those in Control (P < 0.05). The pregnancy rates after 23 days post transfer were assessed and the results showed that the pregnancy rate in VG (8.0%) was lower than that in Control (41.7%), VM (24.0%) and VMP (29.6%) (P < 0.05). No pregnancies resulted from the 10 embryos with diameters ≤650 μm in VG, which lower than either VM (33.3%) or VMP (38.9%) (P < 0.05). While, there was no difference in pregnancy rates among all vitrification groups when embryos were >650 μm in diameter (P > 0.05). In conclusion, the embryo recovery rate on day 7 after ovulation was relatively low, and it was more appropriate to extend it to day 8. Microneedle puncture could reduce embryo damage and achieve a higher pregnancy rate compared with 29G needles. Proline has the potential to improve donkey embryo cryopreservation., Competing Interests: Declaration of competing interest The authors declare no conflict of interest., (Copyright © 2023. Published by Elsevier Inc.)

Background: Perineural spread (PNS) of breast cancer to the brachial plexus is rare, with reports limited to cases only in female patients. This study aimed to determine the incidence of PNS in male compared with female patients., Methods: Adult breast cancer patients referred to a single institution between 1994 and 2022 were retrospectively reviewed for imaging or biopsy-confirmed cases of PNS to the brachial plexus. Two independent reviewers of articles published in any language between 1990 and 2022 in PubMed, Scopus, Embase, and Google Scholar performed a systematic literature review., Results: Of the 10,466 patients with breast cancer (10,355 female, 111 male) referred to a single institution, there were 42 female patients with PNS to the brachial plexus for an estimated incidence of 0.4%. In the same time period, there were 111 male patients treated for breast cancer, including 88 patients with Klinefelter's syndrome (KS); however, there were no cases of male patients with PNS. A systematic review of the world's literature did not identify male patients with PNS to the brachial plexus., Conclusions: There is a lack of any reported cases of PNS of breast cancer to the brachial plexus in men or patients with KS, who have a much higher incidence of breast cancer. This may reflect either the compounding rarity of these two events or point toward a distinct hormonally-driven link between breast cancer and PNS, highlighted by the disparity in PNS incidence in men and women., (Copyright © 2023 British Association of Plastic, Reconstructive and Aesthetic Surgeons. Published by Elsevier Ltd. All rights reserved.)

Asinina de Miranda is a protected donkey sub-species from the Mirandês plateau in northeastern of Portugal. Donkeys are animals that have substantially lost their place as working animals in modern society, this had led to a decrease in their population numbers. A need to preserve native species has led to the foundation of organizations like Associação para o Estudo e Proteção do Gado Asinino (AEPGA) and the development of studies regarding breed welfare, such as hematology. The IDEXX ProCyte Dx is a veterinary hematology analyzer validated for several species, but not for donkeys. The aim of this study was to validate the ProCyte Dx for Asinina de Miranda donkeys. The validation requires a controlled study of precision, carryover, linearity and comparison between the equipment and the manually obtained values for the leukocyte differential count and hematocrit. Results indicated coefficient of variation was good (below 5 %) for both the intra-assay and the inter-assay precision, except for basophils. Carryover was 0 % for all the parameters except platelets (5.88 %). Linearity showed a very high Pearson correlation coefficient, above 0.99, for erythrocytes, hematocrit, hemoglobin, leucocytes, neutrophils, lymphocytes, monocytes, eosinophils, platelets and plateletcrit. Comparison demonstrated excellent agreement for hematocrit (r s =0.96) and good Spearman rank correlation for neutrophils (r s =0.84) and lymphocytes (r s =0.90). Accuracy for total leukocyte count and platelets could not be determined. In conclusion, the ProCyte Dx seems appropriate to be used in Asinina de Miranda hematology., Competing Interests: Declaration of Competing Interest None of the authors has any financial or personal relationships that could inappropriately influence or bias the content of the paper., (Copyright © 2023 Elsevier Inc. All rights reserved.)

In horses and donkeys, age-related changes in hematological and biochemical parameters preclude the use of normal values of adults in the evaluation of foals. This study aimed to obtain data on hematological and biochemical parameters of mule foals from birth up to the second month of life and to assess age-related changes in order to determine if dedicated reference ranges are required in younger animals. Blood samples from seven healthy mule foals were obtained at birth before colostrum consumption, 24 h, 48 h of life, and then weekly until the second month of life. Results were expressed as mean and standard deviation or median, minimum, and maximum values if showing non-gaussian distribution. Kruskal-Walls and Dunn tests were used to verify the differences among sampling times. Significance was set at P < 0.05. Red blood cell count, packed cell volume and hemoglobin decreased from 24 h to one week of age. Mean corpuscular volume and mean corpuscular hemoglobin decreased over the first month. White blood cells increased from birth to seven days of life. Aspartate amino transferase increased while alkaline phosphatase decreased in the first week of life. Urea, creatinine, and lactate decreased, while glucose concentrations increased at 24 h. Ionized calcium and magnesium and total sodium and potassium showed no changes. In mule foals, several laboratory parameters may be the same or intermediate, lower or higher than in equine or donkey foals, but also compared to all other adult species. The preliminary results suggest that for mule foals, age influences hematological and biochemical parameters., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023. Published by Elsevier Inc.)

This study assessed luteolysis and side effects in jennies receiving standard horse-recommended doses of cloprostenol and dinoprost. Sixteen cycles of eight jennies were randomly assigned in a sequential crossover design to receive dinoprost (5 mg, i.m.) and cloprostenol (0.25 mg, i.m.) at 5-d post-ovulation. B-mode and Doppler ultrasonography were employed to assess luteal tissue size and blood flow before (-15 min and 0h) and after (0.5, 1, 2, 3, 4, 5, 6, 7, 8, 12, 24, and 48h) administering PGF 2α . Immunoreactive progesterone concentrations were assayed at similar timepoints via RIA. Side effects such as sweating, abdominal discomfort, and diarrhea were scored at 15-min-intervals for 1h after PGF 2α . Data normality was assessed with the Shapiro-Wilk's test. Luteal tissue size and blood flow were analyzed using PROC-MIXED and post-hoc by Tukey. Non-parametric tests analyzed side effect variables. The luteal blood flow increased overtime by 27% at 45 min and peaked by 49% at 3 h for dinoprost, and conversely, it increased by 14% at 30 min and peaked at 39% at 5h for cloprostenol (P<0.05). Luteal blood flow was reduced by 50%, 25%, and 10% on both groups at 8, 12, and 24h (P<0.05). Immunoreactive progesterone concentrations decreased in 0.5h for dinoprost and 1h for cloprostenol and gradually decreased by 48h (P<0.05). Dinoprost induced greater sudoresis scores, while cloprostenol resulted in greater abdominal discomfort and diarrhea scores (P<0.05). In conclusion, dinoprost and cloprostenol effectively induced luteolysis with distinct side effects; this could guide practitioners' case selection to use one or another PGF 2α ., Competing Interests: Declaration of Competing Interest None., (Copyright © 2023 Elsevier Inc. All rights reserved.)

Liver disease is common in equine practice, and treatment and prognosis are dependent on histopathologic examination of biopsies. Liver biopsy is invasive and expensive which restricts its use. Serum markers are used to predict hepatic fibrosis in humans. This study aimed to investigate the enhanced liver fibrosis (ELF) test, based on serum Hyaluronic Acid (HA), procollagen III N-terminal peptide (PIIINP), and tissue inhibitor of metalloproteinase 1 (TIMP-1) to detect hepatic fibrosis in equids. Four groups were included; two with increased serum concentrations of liver-derived enzymes and a liver biopsy (group H; 10 horses and ponies and group D; 10 donkeys) and two without any evidence of liver disease (group HC; 10 horses and ponies and group DC; 10 donkeys). All samples were analyzed for concentrations of HA, PIINP, and TIMP-1. Given the failure to detect TIMP-1 in most subjects, a novel eELF (equid ELF) score was calculated, based on HA and PIIINP. HA and PIIINP concentrations and the eELF score, were compared with determined hepatic fibrosis. HA, PIIINP, and eELF were significantly greater in horses and ponies with a histopathologic fibrosis score ≥ 2 compared with those < 2. A similar observation was found with donkeys for HA and eELF. A significant correlation was found between fibrosis score and HA, PIIINP, and eELF for horses and ponies, and between fibrosis score and HA and eELF in donkeys. Serum HA and the eELF score might be useful serum markers to predict and monitor hepatic fibrosis in horses, ponies, and donkeys., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier Inc. All rights reserved.)

Objective: To evaluate pathological changes in fossils from the Brazilian Intertropical Region (BIR), expanding the records of previously reported diseases for representatives of the Quaternary South American megafauna, including taxa not studied in previous works., Materials and Methods: We carried out a thorough macroscopic analysis of fifteen unpublished specimens belonging to representatives of the Quaternary megafauna of BIR to identify evidence of pathological alterations., Results: Alterations included: osteophytes in Toxodontidae, Megatheridae and E. laurillardi; rough subchondral bone, bone overgrowth and bone erosion in E. laurillardi; slit-shaped subchondral depressions in Equidae and E. laurillardi; and a triangular-shaped porous lesion in Mylodontidae., Conclusions: The alterations found allowed the recognition of the first cases of osteoarthritis for Toxodontidae and articular depressions for Equidae, and new cases of both diseases in Eremotherium laurillardi; a new case of osteochondritis dissecans for Mylodontidae; potential new cases of calcium pyrophosphate deposition and spondyloarthropathy for E. laurillardi SIGNIFICANCE: Our results provide additional evidence that calcium pyrophosphate deposition disease was widely spread among species of the South American megafauna and suggest that osteochondritis dissecans may have been relatively common among ground sloths., Limitations: The identification of calcium pyrophosphate deposition and spondyloarthropathy in E. laurillardi are quite tentative because the evidence found is ambiguous and the number of examined specimens is limited., (Copyright © 2023 Elsevier Inc. All rights reserved.)

Sarcocystis spp. and Toxoplasma gondii are two apicomplexan protozoa that infect a broad range of vertebrates, however, little is known about the infection of equids with these parasites. A total of 184 slaughtered equids from slaughterhouses of Bizerte and Tunis located in Northern Tunisia, were examined for meat infections with Sarcocystis spp. and T. gondii by PCR. The prevalence of infections with Sarcocystis spp. and T. gondii were 38% (95% CI: 31-45) and 39.7% (95% CI: 32.6-46.7), respectively. The highest prevalence of infection with Sarcocystis spp. was observed in donkeys (48.6%; 95% CI: 37.3-60) followed by mules (32.8%; 95%CI: 21.3-44.3), and horses (28.3%; 95% CI: 15.2-41.2) (P = .04). Similarly, the highest prevalence of infection with T. gondii was also observed in donkeys (66.2%; 95% CI: 55.4-77), followed by mules (18.7%; 95%CI: 9.2-28.3), and horses (26.1%; 95%CI: 13.4-38.8) (P < .001). The coinfection prevalence was estimated to be 17.4% (95%CI: 11.9-22.9). Taking into consideration that humans can be infected following consumption of infected equid meat with T. gondii and/or some Sarcocystis species, it is important to assess the risk of human infection. Thus, further studies are needed to better understand the epidemiology of these zoonoses., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier Inc. All rights reserved.)

Brucellosis in equines, including horses, donkeys, and mules, is characterized by abscesses in tendons, bursae, and joints. Reproductive disorders, which are common in other animals, are rare in both males and females. Joint breeding of horses, cattle, and pigs was found as the main risk factor for equine brucellosis, with the transmission from equines to cattle or among equines possible, although unlikely. Hence, evaluation of the disease in equines can be considered an indirect indicator of the effectiveness of brucellosis control measures employed for other domestic species. Generally, the disease in equines reflects disease status in the sympatric domestic species, mainly cattle. It is important to note that in equines, the disease has no validated diagnostic test, which limits the interpretation of available data. Finally, it is important to mention that equines also represent significant Brucella spp. infection sources for humans. Considering the zoonotic aspect of brucellosis, the significant losses due to infection, and the representativeness of horses, mules, and donkeys in the society, as well as the continuous efforts to control and eradicate the disease in livestock, in this review, we covered the various aspects of brucellosis in equines and compile the sparse and diffuse information on the subject., (Copyright © 2023. Published by Elsevier Inc.)

The purpose of the current study was to investigate the nasal colonization and drug resistance profile of Staphylococcus aureus, methicillin-resistant S. aureus (MRSA), and vancomycin-resistant S. aureus (VRSA) in donkeys (n =63), mules (n = 42), and horses (n = 98). MRSA and VRSA were confirmed based on phenotypic and molecular methods, followed by phylogenetic analysis. Furthermore, the association of various animal and management-based risk factors with S. aureus colonization was also evaluated. The presence of nuc gene on polymerase chain reaction showed an overall prevalence of 42.86% of S. aureus in equines. Based on Kirby-Bauer disc diffusion test, 26.44% of isolates showed resistance to vancomycin. Further, mecA and vanB genes were targeted which showed an overall 14.94% and 13.79% prevalence of methicillin and vancomycin-resistant isolates, respectively. The phylogenetic analysis revealed a significant variation of the study isolates with each other and with already reported sequences of mecA and vanB genes. Risk factor analysis revealed that raising purpose (P = .011), work intensity (P < .001), stocking density (P = .006), presence of other livestock animals in surroundings (P = .043), and common drinking water source (P = .023) as significant. Antimicrobial susceptibility testing of MRSA and VRSA isolates showed high resistance to various commonly used antibiotics. Furthermore, all the tested isolates showing resistance to three or more than three antibiotics were considered multiple drug-resistant. The current study manifests the molecular evidence of MRSA and VRSA isolated from equines in Pakistan which will help to address the emerging issue of multidrug resistance in S. aureus in equines and emphasizes the need for possible measures to tackle this issue., Competing Interests: Declaration of Competing Interest All authors have declared no conflict of interest in the submission/publication of this data., (Copyright © 2023 Elsevier Inc. All rights reserved.)

Major proteins of the seminal plasma in a variety of mammals such as bovine PDC-109, equine HSP-1/2, and donkey DSP-1 contain fibronectin type-II (FnII) domains and are referred to as FnII family proteins. To further our understanding on these proteins, we carried out detailed studies on DSP-3, another FnII protein of donkey seminal plasma. High-resolution mass-spectrometric studies revealed that DSP-3 contains 106 amino acid residues and is heterogeneously glycosylated with multiple acetylations on the glycans. Interestingly, high homology was observed between DSP-1 and HSP-1 (118 identical residues) than between DSP-1 and DSP-3 (72 identical residues). Circular dichroism (CD) spectroscopic and differential scanning calorimetric (DSC) studies showed that DSP-3 unfolds at ~45 °C and binding of phosphorylcholine (PrC) - the head group moiety of choline phospholipids - increases the thermal stability. Analysis of DSC data suggested that unlike PDC-109 and DSP-1, which exist as mixtures of polydisperse oligomers, DSP-3 most likely exists as a monomer. Ligand binding studies monitoring changes in protein intrinsic fluorescence indicated that DSP-3 binds lyso-phosphatidylcholine (K a  = 1.08 × 10 5  M -1 ) with ~80-fold higher affinity than PrC (K a  = 1.39 × 10 3  M -1 ). Binding of DSP-3 to erythrocytes leads to membrane perturbation, suggesting that its binding to sperm plasma membrane could be physiologically significant., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Musti J. Swamy reports financial support was provided by India Ministry of Science & Technology, Department of Science and Technology. Musti J. Swamy reports financial support was provided by University of Hyderabad., (Copyright © 2023 Elsevier B.V. All rights reserved.)

Brucellosis, a bacterial infection caused by Brucella spp., is a widespread zoonosis concerning human and animal health. In equines, brucellosis may occur asymptomatically or with clinical signs such as arthritis, bursitis, and tenosynovitis. This study aims to ascertain the overall seroprevalence of equine brucellosis and its related factors, including geographic region, serological detection method, equine's species, gender, age, and body condition. This is because equine brucellosis is a zoonotic infection with significant epidemiological implications. The systematic literature search was conducted from January 1, 1990, to April 1, 2022, in the following electronic databases: Google Scholar, Scopus, Pub Med, Science Direct, Web of Science, Embase, Springer, and ProQuest. Based on the preferred reporting items for systematic reviews and meta-analyses (PRISMA) and inclusion criteria, 49 qualified studies were acquired from several countries. Because Egger's test result and the asymmetry in the funnel plot indicated significant publication bias, the meta-analysis model was corrected using the trim-and-fill method. After correction, the pooled seroprevalence of equine brucellosis was 1.92% (95% CI, 0.67-3.79; estimated by the random effect model). Based on meta-regression analysis, the probable heterogeneity factors may be geographic areas (continents and countries). The brucellosis seroprevalence in equines was the lowest in Europe (0%, 95% CI, 0-0.1) and the highest in Asia (10.03%, 95% CI, 8-12.9). The highest overall estimate of seroprevalence of Brucella infection in equines based on a subset of serological tests was made using the serum agglutination test (10.2%, 95% CI, 7.7-13.4). The combined seroprevalence of brucellosis in donkeys (7.3%, 95% CI, 4.9-9.7) was higher than in other equine species. The overall seroprevalence of brucellosis in female equines was significantly higher than in males (OR = 1.437; 95% CI, 1.025-2.014); despite this, the overall seroprevalence had no significant difference based on other risk factors such as age and body condition. Understanding the epidemiology of this zoonotic infection in equines is critical for one-health integration. In addition, this study's results may be appropriate for controlling and managing equine brucellosis., (Copyright © 2023 Elsevier Inc. All rights reserved.)

The study aimed to evaluate effects of packing (load-carrying) and trekking on hematological and biochemical parameters in donkeys during the hot-dry season. Ten pack donkeys, aged 2-3 years, weighing 93 ± 2.7 kg were enrolled in the study and randomly divided into two groups of five animals each. Group 1 donkeys were subjected to packing + trekking while group 2 trekked only. All the donkeys trekked 20 km thrice, 1-day apart. Blood was collected before and after packing or trekking for hematological evaluation; and serum analyzed for electrolyte concentrations, and aspartate aminotransferase, creatine kinase and lactate dehydrogenase activities. Temperature-humidity index pre and postpacking was 70.00 ± 0.1 and 79.67 ± 0.3, respectively. The packed cell volume (30.93 ± 1.1%) was higher (P < .05) before packing than postpacking value (27.83 ± 0.7%) in group 1 donkeys. Hemoglobin concentration (9.01 ± 0.3 g/dL) decreased (P < .05) after trekking (7.81 ± 0.1 g/dL) in group 2 donkeys. Neutrophil count increased (P < .05) in group 1 donkeys after packing (from 8.63 ± 0.9 × 10 9 /L to 10.59 ± 1.2 × 10 9 /L). Lymphocyte count (8.58 ± 0.8 × 10 9 /L) decreased (P < .05), compared to postpacking (5.23 ± 0.5 × 10 9 /L) in group 1 donkeys. Postpacking neutrophil/lymphocyte ratio in group 1 donkeys (2.33 ± 0.4) was higher (P < .05) than in group 2 donkeys (1.31 ± 0.3). Electrolyte concentrations and enzyme activities did not change significantly. In conclusion, packing + trekking was more stressful to the donkeys than trekking only, evidenced by postpacking increased neutrophil count, neutrophil/lymphocyte ratio and phosphate ion concentration, but decreased lymphocyte count. If this stress is not mitigated, the performance of pack donkeys during hot-dry season may not be optimum., (Copyright © 2022 Elsevier Inc. All rights reserved.)

This study aimed to determine the associations between B-mode and Power-doppler ultrasonography and ovarian steroids of the periovulatory follicle and respective corpus luteum (CL) during luteogenesis and luteolysis in jennies. Twenty-four periovulatory follicles/estrus of correspondent one inter-ovulatory interval (n = 12 jennies) were assessed in the study. B-mode ultrasonography and teasing were carried out once day until the detection of a periovulatory follicle (≥28 mm, uterine edema, and signs of estrus). Thereafter, jennies were monitored at 4-hour-intervals by B-mode and Power-doppler ultrasonography. Closer to ovulation, jennies were hourly checked. Each CL was checked daily from luteogenesis to luteolysis. Plasma concentrations of estradiol and progesterone were assessed daily with chemiluminescence immunoassay. Granulosa echogenicity and thickness increased from -36 hour to -1 hour before ovulation in 70% of follicles (P < .05) and were strongly associated with impending ovulation (r = 0.80 and r = 0.70, respectively). The follicular-wall blood flow increased from -72 to -24 hour pre-ovulation, while the estradiol concentration declined from 42 pg/mL by -72 hour to 31.6 pg/mL by 24 hour before ovulation (P < .05). The vascularization of the periovulatory follicle decreased from 62% (-36 hour) to 37% (-1 hour) before ovulation (P < .05). The CL vascularization and progesterone concentration gradually increased, reaching the peak at 11- and 10-day after the ovulation, respectively (P < .05). The CL vascularization started to decline 3 day before luteolysis, while progesterone concentrations started to drop 4 day before luteolysis (P < .05). In conclusion, the structural changes of the periovulatory follicle detected on B-mode and Power-doppler can be used to detect impending ovulation in donkeys; however, Power-doppler, but not B-mode ultrasonography, can be used to assess CL function in jennies., (Copyright © 2023 Elsevier Inc. All rights reserved.)

The freezability difference between donkey ejaculates is a limiting factor of sperm cryopreservation. Our recent study shows that the freezability of donkey semen is related to the seminal plasma proteome. In this study, we aimed to identify the different abundance sperm proteins in good freezability ejaculates (GFEs) and poor freezability ejaculates (PFEs) using a Tandem Mass Tag (TMT) peptide labeling coupled with LC-MS/MS approach. A total of 2682 proteins were identified, among which 58 were significantly up-regulated in GFEs and 16 were down-regulated compared with PFEs. Bioinformatic analysis results revealed that the majority of different abundance proteins (DAPs) participated in copper and calcium binding, regulation of RNA biosynthetic process, positive regulation of innate immune response, and negative regulation of programmed cell death. KEGG pathway enrichment analysis showed the up-regulated proteins in GF group were mainly involved in N-Glycan biosynthesis and protein processing in endoplasmic reticulum. Our study was the first to analyze the proteome of sperm from donkey ejaculates with different freezabilities. The identified candidate proteins might be used to explore the molecular mechanism related to donkey sperm cryotolerance and might improve the screening of jacks with good sperm freezability. SIGNIFICANCE: Cryopreserved semen has been widely used in assisted reproductive technology. However, semen cryopreservation is a damaging process, which can cause oxidative stress, reduce sperm motility and motility. There are differences in sperm freezability reported to exist between or within breeds, and even between fractions coming from the same ejaculate. The freezability difference between donkey ejaculates is a limiting factor of sperm cryopreservation. The mechanisms that affect the freezing difference in sperm quality remain to be investigated, and freezability differences was found to be related to protein composition of spermatozoa. Some protein markers that can indicate good freezability or poor freezability semen have been identified in mammals. Until now, there is no information about the relationship between donkey spermatozoa proteome and freezability. Additional novel biomarkers of semen freezability in donkey spermatozoa are also needed. The identified candidate proteins might be used to explore the molecular mechanism related to donkey sperm cryotolerance and might improve the screening of jacks with good sperm freezability., Competing Interests: Declaration of Competing Interest The authors declare no competing financial interest., (Copyright © 2022 Elsevier B.V. All rights reserved.)

Multisystemic eosinophilic epitheliotropic disease (MEED) is a rare condition of equids characterized by eosinophilic infiltration of multiple organs. Clinical signs are variable depending on the affected organs. The most common clinical signs include chronic weight loss, diarrhoea and exfoliative dermatitis. Respiratory distress and raised liver enzymes are less frequently seen. The cause is unknown and the pathogenesis is poorly understood. There are less than 50 reported cases of horses with MEED. We now document the lesions in three donkeys with fluctuating or chronic loss of weight, lethargy, exfoliative dermatitis and peripheral eosinophilia. All three animals were euthanized due to poor prognosis and welfare concerns. Post-mortem examination revealed multiple white to tan, irregular masses composed of eosinophilic infiltrates, including eosinophilic granulomas in several organs, confirming the presence of MEED. To the best of our knowledge, MEED has not previously been reported in donkeys., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

The donkey milk has a remarkable similarity to human milk, in addition to its valuable nutritional composition and content of numerous immune factors. The donkey milk is the subject of research worldwide, and data from the literature suggest significant differences with respect to the contents of individual components. However, some basic characteristics of donkey milk have been established: low contents of fat and cholesterol, total proteins and casein and high contents of lactose, whey proteins, calcium, selenium, and Vitamin D3. The donkey milk is rich in various protective proteins (α-lactalbumin, lysozyme, lactoferrin, lactoperoxidase, and immunoglobulins), and shows strong antioxidant, antibacterial, antiviral, antifungal, hypoglycemic, antiparasitic, and antitumor activity. Donkey milk can be considered functional food, having in mind that its fatty-acid profile and content of essential fatty acids are beneficial for cardiac health. The total fat content in donkey milk is low compared to human milk, and for this reason it is necessary to enrich donkey milk with other fat in order to provide enough calories in a diet for children. Commercialization of donkey milk and dairy products is still limited due to low production levels, that is lack of products on the market, and lack of product information. Considering the research data from the literature, there is a need for human clinical trials in order to obtain a stronger evidence of the therapeutic properties of donkey milk., (Copyright © 2023 Elsevier Inc. All rights reserved.)

Equus members exhibit very divergent karyotype, genetic plasticity, and significant differences in their reproductive physiology. Despite the fact that somatic cell nuclear transfer and intracytoplasmic sperm injection (ICSI) has gained relevance in the last few years in horses, few reports have been published exploring ovum pick up (OPU) and in vitro maturation (IVM) of cumulus-oocyte complexes (COCs) in donkeys. Yet, some donkey species and breeds are considered endangered, and these assisted-reproductive technologies could help to preserve the genetic of valuable individuals. In this study, we tested the hypothesis that supplementation with jenny preovulatory follicular fluid (PFF) during IVM could improve oocyte developmental competence in the donkey. For this, in vitro nuclear maturation rates, cumulus cell expansion, and embryo development after ICSI of donkey COCs matured in culture media supplemented with fetal bovine serum (FBS) or donkey PFF, with a known metabolomic profile, were assessed. Time-lapse imagining was performed after ICSI of horse and donkey oocytes. Eight OPU sessions were done in five jennies with an average recovery rate of 69.2% (n = 45 COCs). Although lower cumulus cells expansion was observed in oocytes of PFF group (P = 0.0010), no significant differences were described in nuclear maturation rates and preimplantation embryo development between groups. Donkey ICSI embryos showed similar morphokinetics to horse ICSI embryos. Our study shows that supplementing IVM media with FBS or donkey PFF supports nuclear maturation and early preimplantation embryo development after ICSI in donkeys. To our knowledge, the present study is the first report of ICSI, time-lapse imaging and in vitro blastocyst production in donkey., Competing Interests: Declaration of competing interest The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Elsevier Inc. All rights reserved.)

Among infection diseases transmitted by arthropods, the equine vector-borne diseases transmitted by ticks represent an emerging problem worldwide due to their morbidity and mortality and, in some cases, to their zoonotic relevance. Understanding the host immune/inflammatory response to the pathogens is crucial to develop effective methods of diagnosis, control and treatment. This study aimed to evaluate red blood cells (RBC), hematocrit (Hct), hemoglobin concentration (Hb), mean cell volume (MCV), mean cell hemoglobin content (MCH), mean cell hemoglobin (MCHC), platelets (PLT), white blood cells (WBC), leukocytes populations, and serum protein pattern of Sardinian donkeys scored positive to tick-borne pathogens. Thirty donkeys were dived in five groups, six subjects each: Group 1, tested negative to pathogens (control group); Group 2, donkeys infected by Theileria equi; Group 3, donkeys infected by Ehrlichia equi; Group 4, donkeys infected by Rickettsia Rickettsii; Group 5, donkeys co-infected by T. equi, E. equi and R. Rickettsii. From donkeys blood samples were collected and haematological and serum protein profiles were assessed. One-way ANOVA showed lower red blood cells, haemoglobin, platelets, lymphocytes and neutrophils values, and higher eosinophils, basophils, serum total proteins, α2-, β- and γ- globulins values in donkeys infected by one or more pathogens compared to control group (P < .05). The study highlighted an immune and inflammatory response of donkeys positive to E. equi, T. equi, and R. Rickettsii and represents a starting point for further investigations in the field to fill the gaps currently existing on the complex interwoven relationship between the pathogen and host immune and inflammatory response in equids., (Copyright © 2022 Elsevier Inc. All rights reserved.)

Infundibular caries (IC) affects donkeys of all ages and causes pain, difficulty in mastication and weight loss. The study aimed to determine the epidemiology of IC in donkeys and examine the potential risk factors associated with the disease development. A total of 2118 donkeys in Inner Mongolia were sampled. The first section of the survey was performed by veterinarians and included the oral health of the donkeys. A survey on the donkey's signalment, diet, and husbandry was also conducted. The risk factors associated with IC were assessed using multivariable logistic regression analysis. Univariate analysis of 11 variables assessed revealed that only four factors, including age, type of forage, pasture time and water source, increased the risk of IC. The findings of this study enhance our knowledge of the management and prevention of IC in donkeys., (Copyright © 2022 Elsevier Inc. All rights reserved.)

This study aimed to evaluate the effect of different aging times (1, 6 and 14 days) on the tenderization rate and protein changes in ten Martina Franca donkey striploins using a proteomic approach. During aging, a progressive decrease in share force, hardness, gumminess, and chewiness together with an increase in myofibril fragmentation index were observed. Proteolysis monitored by immunoblotting revealed a progressive degradation of desmin and fast troponin-T over time and an increase of their degradation products up to 14 days aging. Proteomics revealed by means of two-dimensional electrophoresis 37 protein spots corresponding to 15 proteins to change significantly by increasing aging time. These proteins belong to three pathways, these being "muscle contraction, structure, and associated proteins" (9 proteins); ii) "energy metabolism" (5 proteins); and iii) "chaperone" (1 protein). This study is the first to highlight the possible role of interconnected pathways in driving the final quality of donkey meat and predicting its texture., Competing Interests: Declaration of Competing Interest The authors declare no conflict of interest., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

Serodiagnosis of surra, caused by Trypanosoma evansi, is still based on native antigens purified from bloodstream form of T. evansi grown in rodents. In order to investigate prospective diagnostic possibilities as an alternative for native antigens, we cloned, expressed 26 kDa calflagin protein containing 218 amino acids from T. evansi (Indian Strain) in Escherichia coli. The potential of recombinant calflagin (rCLF) protein as diagnostic antigen was evaluated in immunoblot and indirect ELISA using experimentally infected equine serum samples from 0 to 84 days post infection. The antibodies against T. evansi were detected with rCLF antigen in serum samples of experimentally infected equines as early as 10 days and 14 days post infection, using immunoblot and ELISA respectively. No cross-reactivity was observed with rCLF antigen in ELISA with different serum samples of equines positive for Equine herpesvirus 1, Burkholderia mallei, and Theileria equi infections. Several immunoreactive regions ranging from 10 to 28 kDa were detected using distinct T. evansi isolates (pony, cattle, donkey and camel origin) indicating presence of multiple calflagin family members in a single trypanosome. Indirect immunofluorescence antibody test with anti-CLF rabbit hyperimmune serum showed localisation of native immunogenic protein near attachment of flagellum. The rCLF protein was found to be a potential diagnostic candidate for distinguishing T. evansi positive and negative equine serum sample, suggesting that it could be used for serological surveys in animals for surra. In addition, it could be used with other potential diagnostic candidates to improve the diagnostic efficiency., Competing Interests: Conflict of interest statement The corresponding author on behalf of all authors of the manuscript declare that they have no competing interests., (Copyright © 2022 Elsevier B.V. All rights reserved.)

Animal sanctuaries are important organisations that promote animal welfare and health as well as social awareness. Following the appearance of several donkeys with severe and distinctive skin lesions in a sanctuary from western Spain in 2021, a multicomponent study was performed to rule out the possible causes. The lesions were mainly concentrated on the extremities and, to a lesser extent, on the face and/or chest. The use of cotton leggings on their extremities as an external barrier to prevent them from bites and treatments with antiparasitic, antiseptic washes and dermatitis lotion showed to be effective measures to improve the donkey lesions. Skin scraping was negative for any relevant causative agents. Histopathological examination of the lesions showed an inflammatory infiltrate at the superficial dermal level, compatible with diffuse chronic dermatitis. A field entomological study was conducted from May to October 2021 to ascertain if any Diptera was responsible for these lesions. Considerable numbers of the stable fly Stomoxys calcitrans were recorded in both sticky traps and decomposing straw bedding. Several species of hematophagous Diptera were also recorded in lower numbers in other traps placed on the farm facilities. According to the entomological data, the location of the bites and clinical signs, it was concluded that S. calcitrans was the main cause of the skin lesions in the donkeys. To our best knowledge, this is the first clinical case of donkeys affected by the stable fly in Spain. For differential diagnosis, a brief discussion of the evidence caused by other biting Diptera groups is provided., (Copyright © 2022. Published by Elsevier Inc.)

This study aimed to compare ultrasonographic features and steroid concentrations of jennies undergoing late-term pregnancy loss (n = 5) with gestationally age-matched health controls (n = 5). Transrectal ultrasonography of the combined thickness of uterus and placenta (CTUP) and fetal eyeball diameter was carried out at 15-day-intervals. Fetal heartbeat, aortic, and thorax diameters were determined by transabdominal ultrasonography at 30-day-intervals. Blood samples were collected simultaneous with each transrectal ultrasonography to determine progestogen and estradiol concentrations. Data were assessed for normality with Shapiro-Wilks. Data were log-transformed and analyzed with a mix model. Non-normally distributed data were analyzed with Kruskal-Wallis. Post-hoc analyzes were performed with Sidák's or Dunn's tests based on distribution. Gestational length between groups was compared with a t-test. Statistical significance was set at P < .05. The gestational length was shorter in jennies experiencing pregnancy loss (345 ± 32.3 vs. 365.4 ± 10.4 d P = .0009). Increasing gestational age (P < .0001) and pregnancy loss group (P = .004) had greater CTUP measurements with an interaction between them (P = .01). Fetal eyeball diameter increased with gestational age (P < .0001) but did not vary with group (P = .26), and there was no interaction between gestational age and group (P = .71). Fetal aortic and thorax diameters increased with gestational age (P < .0001), but an interaction between gestational age and group was only present with thorax diameter (P = .01). No effect of group was found for aortic (P = .78) or thorax (P = .86) diameters. Group (P = .06) and gestational age (P = .07) tended to be associated with an increased fetal heartbeat, but there was no interaction between them (P = .98). There was no effect of gestational age (P = .31), group (P = .19), or interaction between them for progestogens concentrations (P = .21). Estradiol concentration was not affected by gestational age (P = .76) or group (P = .51). In conclusion, late-term pregnancy loss was associated with increased CTUP measurements in donkeys., (Copyright © 2022 Elsevier Inc. All rights reserved.)

Practitioners are frequently requested to diagnose and stage pregnancy in donkeys with unknown breeding dates; however, scant work has been done to stage pregnancy in the species. Therefore, this study aimed to determine the association between measurements of fetal aortic, thoracic, and heartbeat with gestational age in donkeys carrying and delivering healthy foals. Multiparous Dezhou donkeys (n = 50) ranging from 4 to 16 years were enrolled in the study by 150 days of gestation. Transabdominal ultrasonography coupled with a 3.5 MHz sectorial convex transducer was performed at 30 day-intervals until delivery to obtain fetal aortic, thoracic, and fetal heartbeat measurements. Data were tested for normality with Shapiro- Wilk's test and then ANOVA and Tukey's. Statistical significance was set at P < .05. The mean duration of pregnancy was 356.6 ± 10.6 days (339-368). There were significant associations between gestational age and fetal aortic (r = 0.89) and thoracic (r = 0.88) measurements. Fetal heartbeat (r = -0.76) was negatively correlated with gestation length. The fetal aortic and thoracic measurements increased from the seventh month of gestation to the term (P < .001). The fetal heartbeat remained steady from 150 days to 270 days of gestation, then continuously decreased from 270 days until parturition (P < .001). In conclusion, fetal aortic and thoracic measurements are strongly associated with gestational age in donkeys. Fetal heartbeat decreases with advanced pregnancy., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022. Published by Elsevier Inc.)

In addition to providing individualized, specific, and ample nutritional compounds, donkey milk (DM) offers immunological modulation during health and disease. Recently, DM has attracted major interest in preparing infant formulas due to its similarity to human milk in terms of high protein and lactose content and low-fat concentration. The antimicrobial, anti-inflammation, antioxidant, and hypo-allergenicity properties of DM in human infants are well-documented. The purpose of this review is to summarize the knowledge of studies done in characterizing the composition of DM, including bioactive macronutrient levels influenced by the lactation status. The manufacture of DM-based food products and promising therapeutic applications in humans will also be discussed. The beneficial health effects of DM have been extensively studied as a valuable alternative source to breast milk. DM has proven to be a suitable nutrient to relieve milk-related allergies in human infants as opposed to cow's milk. Factors that influence the levels of macronutrients in DM include lactation status, processing, and manufacturing techniques. A wide variety of dairy products have been prepared using DM, such as cheese, ice cream, milk powder, novel functional fermented beverages, and milk powder for infant formulas. The bioactive macromolecules of DM exhibit antibacterial, antiviral, and antifungal effects as well as hypo-allergenicity, anti-inflammation, and antioxidant properties., (Copyright © 2022. Published by Elsevier Inc.)

Pythiosis is an endemic disease in northeastern Brazil and we now report the epidemiological, clinical and pathological findings in a retrospective study of naturally occurring cases in domestic animals. From January 1985 to December 2020, the Laboratory of Animal Pathology of the Federal University of Campina Grande examined 13,542 tissue samples from necropsies and biopsies. Among these samples, 306 were diagnosed as pythiosis: 195 cases in horses, 75 in sheep, 19 in dogs, six in mules, four in cattle, three in cats, two in goats, one in a donkey and one in an ostrich. Affected equids had lesions in the skin, mammary glands and nasal cavities. Affected sheep had cutaneous, nasal and digestive lesions while cattle and goats had cutaneous lesions. Carnivores developed lesions, mainly in the alimentary tract, of sufficient severity to cause death or result in euthanasia. The single affected bird had lesions in the alimentary tract and surgical excision resulted in remission. The disease had a long and life-threatening clinical course in most affected species but resolved spontaneously in cattle. Clinical signs were directly related to the location of the lesions, which were invariably characterized by chronic inflammation associated with intralesional hyphae. Veterinary clinicians and pathologists should be familiar with the clinicopathological features of pythiosis and the wide range of susceptible animal species., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

This study aimed to establish normal ranges and determine variables affecting the duration of gestation, placental and fetal eyeball measurements in donkeys. Jennies (n = 121) were submitted to transrectal ultrasonography at 15-day-intervals from 120 days of gestation until parturition. During each examination, variables assessed included: the combined thickness of uterus and placenta (CTUP), fetal eyeball diameter, amniotic membrane thickness, allantoic and amniotic fluids echogenicity, and chorioallantois edema. Parametric data were analyzed with t-test, linear regression, mixed models, ANOVA, Tukey's as posthoc, and non-parametric data with Kruskal-Wallis and Dunn's. Statistical significance was set at P < .05. The mean duration of gestation was 365.4 ± 10.4 days, and there were effects of the season (P < .001), sex of the foal (P = .001), but there was no interaction (P = .89). Spring-bred jennies had 16d longer gestation than summer- and fall-bred (P < .0001), and with no difference between the latter (P = .81). Colts had four days longer gestation than fillies (P = .0001). The proportion of colts (51%) and fillies (49%) were similar (P = .8). The CTUP measurements varied with gestation age (P < .0001), but there were no effects of the season (P = .68), sex of the foal, or interactions (P = .80). The incidence and scores of chorioallantois edema increased from 240 to 300 days (P < .001). Amniotic membrane thickness increased minimally between mid- and late-gestation (P = .02). The fetal eyeball was strongly associated with gestational age (r=0.78) but not with any other variable (P > .05). Allantoic fluid echogenicity did not vary throughout gestation (P = .21); however, the amniotic fluid echogenicity increased from mid-to late-gestation (P = .0001). In conclusion, the season and sex of the foal affected the duration of gestation, but no other variables. The CTUP displayed a sigmoid increase throughout pregnancy, and gestational age was the only variable affecting it. The incidence of chorioallantois edema and scores increased from 240 to 300 days of gestation. Fetal eyeball diameter was strongly associated with gestational age. Amniotic fluid echogenicity increased late in pregnancy, but not the allantoic fluid., (Copyright © 2022. Published by Elsevier Inc.)

The present study aimed to compare the sedative and analgesic effects of caudal epidural administration of lidocaine alone or in combination with four different α 2 -adrenergic agonists in Mediterranean miniature donkeys. A total of ten clinically healthy (five males and five females) Mediterranean miniature donkeys with an age of 5 ± 1 years, a weight of 100 ± 2 kg and a height at the withers of 0.8 ± 0.06 m (mean ± standard deviation) were used in experimental, crossover (Latin square), randomized, and blinded study. Animals were assigned to five treatment groups including lidocaine alone (0.22 mg kg -1 ), or associated with one among xylazine (0.17 mg kg -1 ), detomidine (30 μg kg -1 ), medetomidine (15 μg kg -1 ), dexmedetomidine (5 μg kg -1 ) with a minimum washout period of 8 days between treatments. The degree of sedation was investigated using a simple descriptive scale of 0-3. Sedation scores were compared at each time using nonparametric (Kruskal-Wallis and Mann-Whitney U) tests. Analgesia was assessed by pinprick test. Sedation was greater in lidocaine and/or α 2 -adrenergic agonist groups than in lidocaine group at 45-75 minutes after drug administration (P < .05). There were no significant differences among groups in time to onset of analgesia and ataxia and also in number of animals with complete perineal analgesia and ataxia. Duration of analgesia and ataxia were longer in lidocaine and/or α 2 -adrenergic agonist groups than in lidocaine (P < .05). There were no significant differences among lidocaine and/or α 2 -adrenergic agonist groups in sedation score and duration of analgesia and ataxia. No significant differences were observed in heart and respiratory rate and also rectal temperature at any time points between groups and within groups. Caudal epidural administration of α 2 - adrenergic agonists associated with lidocaine resulted in sedative effects on Mediterranean miniature donkeys, while lidocaine alone did not induce sedation. These drugs associations should be considered when superior analgesia is advocated., (Copyright © 2022. Published by Elsevier Inc.)

In the literature, the very low pregnancy rates after artificial insemination (AI) with frozen semen in donkeys were improved in one study after re-extension of the frozen-thawed semen in autologous seminal plasma. The aims of our study were (1) to describe in vitro post-thaw parameters of donkey jackass semen after re-extension in seminal plasma (SP) or in INRA96 and (2) to compare pregnancy rates in jennies bred with frozen-thawed semen using two different AI protocols. Semen collected from two Amiata donkey stallions, known to be fertile, was frozen in INRA96 supplemented with 2% egg yolk, and 2.2% glycerol. From the same jacks, seminal plasma was filtered, and stored at -20°C. According to the in vitro analysis, the post-thaw re-extension in SP some kinematic parameters decreased more over time. Two different AI protocols were tested: inseminating deeply into the uterine horn (DHAI) with or without thawed semen re-extension in SP. Higher pregnancy rates were obtained with re-extension in SP than when AIs were performed without SP. Further studies are needed to confirm the advantage of re-extension in autologous SP of donkey frozen-thawed semen before AI in jennies., (Copyright © 2022. Published by Elsevier Inc.)