Modeling Climate Change Effects on Genetic Diversity of an Endangered Horse Breed Using Canonical Correlations.

The historical increase in the occurrence of extreme weather events in Spain during the last thirty years makes it a perfect location for the evaluation of climate change. Modeling the effects of climate change on domestic animals' genetic diversity may help to anticipate challenging situations. However, animal populations' short life cycle and patent lack of historical information during extended periods of time drastically compromise the evaluation of climate change effects. Locally adapted breeds' gene pool is the base for their improved resilience and plasticity in response to climate change's extreme climatic conditions. The preservation of these domestic resources offers selection alternatives to breeders who seek such improved adaptability. The Spanish endangered autochthonous Hispano-Arabian horse breed is perfectly adapted to the conditions of the territory where it was created, developed, and widespread worldwide. The possibility to trace genetic diversity in the Hispano-Arabian breed back around seven decades and its global ubiquity make this breed an idoneous reference subject to act as a model for other international populations. Climate change's shaping effects on the genetic diversity of the Hispano-Arabian horse breed's historical population were monitored from 1950 to 2019 and evaluated. Wind speed, gust speed, or barometric pressure have greater repercussions than extreme temperatures on genetic diversity. Extreme climate conditions, rather than average modifications of climate, may push breeders/owners to implement effective strategies in the short to medium term, but the effect will be plausible in the long term due to breed sustainability and enhanced capacity of response to extreme climate events. When extreme climatic conditions occur, breeders opt for mating highly diverse unrelated individuals, avoiding the production of a large number of offspring. People in charge of domestic population conservation act as catalyzers of the regulatory changes occurring during breeds' climate change adaptive process and may identify genes conferring their animals with greater adaptability but still maintaining enhanced performance. This model assists in determining how owners of endangered domestic populations should plan their breeding strategies, seeking the obtention of animals more resilient and adapted to climate-extreme conditions. This efficient alternative is focused on the obtention of increased profitability from this population and in turn ensuring their sustainability.

Comparative Assessment of Oxidative and Antioxidant Parameters in Mule and Horse Neonates during Their First Month of Extrauterine Adaptation.

After parturition, a rapid transition occurs from the intrauterine to the extrauterine milieu, exposing neonates to physiological circumstances characterized by oxidative conditions that instigate the generation of reactive oxygen species. These free radicals play pivotal roles in physiological processes; however, an imbalance between their production and the removal of antioxidants can result in severe cellular damage. The main objective of this study was to compare the oxidative and antioxidant profiles in mule and horse neonates immediately post-parturition, as well as at subsequent time points (1, 6, 12, and 24 h, 7 and 30 days) during their extrauterine existence. The parameters assessed included the systemic concentrations of Thiobarbituric Acid Reactive Substances (TBARS) and carbonyl groups; the activities of the antioxidants superoxide dismutase (SOD) and glutathione peroxidase (GPx); and the levels of the total, indirect, and direct bilirubin. Our results showed no interaction effect between the neonatal groups and the assessed time points for the variables under investigation. Notably, the concentrations of TBARS, as a marker of lipid peroxidation, and bilirubin were consistently lower in the mules, whereas the glutathione peroxidase (GPx) activity exhibited higher levels in this group. The bilirubin levels were notably reduced in the mule neonates. The TBARS demonstrated a progressive decrease over the observation period in both groups, while the GPx activity remained relatively stable from birth to 7 days, with a substantial increase evident at the 30-day mark. Protein oxidation was not affected by the group and time, while for the SOD values, all times were statistically similar, except for the lower activity at T1h. Consequently, our findings lead us to the conclusion that neonatal mules and horses manifest distinct patterns of oxidative activity and antioxidant capacity during the initial month of their extrauterine existence, potentially indicative of different adaptation mechanisms to the extrauterine environment.

Transdermal Flunixin Meglumine as a Pain Relief in Donkeys: A Pharmacokinetics Pilot Study.

Recent approval of transdermal flunixin meglumine (FM) (Banamine®) in cattle has opened the door for the drug's potential application in other species. Transdermal FM could provide a safe and effective form of pain relief in donkeys. In order to evaluate the pharmacokinetics and effects of FM on anti-inflammatory biomarkers in donkeys, a three-way crossover study design was employed. In total, 6 healthy donkeys were administered transdermal (TD) FM at a dosage of 3.3 mg/kg, and oral (PO) and intravenous (IV) doses of 1.1 mg/kg body weight. Blood samples were collected over 96 h to determine the concentration of flunixin, 5OH flunixin, and eicosanoids (TXB2 and PGF2 alpha) using LC-MS/MS. The results indicated that both flunixin and 5OH flunixin were detectable in blood samples collected during TD. The elimination of the drug was slower following the TD route compared to PO and IV. TD administration significantly decreased TXB2 levels in non-stimulated serum from 1 to 96 h post-administration, while IV and PO resulted in TXB2 reduction for 1 to 8 h. A significant reduction in PGF2 alpha was observed in PO and IV 1 h after administration, while TD resulted in a gradual decline from 4 to 72 h. The study concluded that the off-label use of transdermal FM at 3.3 mg/kg could be effective in controlling inflammation in donkeys.

Expression of genes with biomarker potential identified in skin from DSLD-affected horses increases with age.

Degenerative Suspensory Ligament Desmitis (DSLD) negatively impacts connective tissues in horses, which often leads to progressive chronic pain and lameness. DSLD has been shown to be a systemic disorder that affects multiple body systems, including tendons, sclerae, and the aorta. Currently, the diagnosis is confirmed by post mortem histological examination of a tendon or suspensory ligament. Histology reveals inappropriate accumulations of proteoglycans in the tendons and other tissues in DSLD-affected horses. Unfortunately, there is no reliable method to diagnose DSLD in living horses. Recently, bone morphogenetic protein 2 (BMP2) was identified in active DSLD lesions. In addition, recent data from RNA sequencing (RNA-seq) showed overexpression of numerous genes, among them BMP2, FOS and genes for keratins in DSLD skin biopsies-derived RNA. We hypothesized that some of these genes can be used as biomarkers for diagnosis of DSLD in a panel. Overexpression of some of them was verified in quantitative real time PCR. Immunohistochemistry and RNAscope in-situ hybridization (ISH) assays were used to determine the level of overexpression of specific genes in skin biopsies from control and DSLD-affected horses. The RNAscope ISH assay has shown to be more reliable and more specific that immunohistochemistry. ISH confirmed a significant increase in KRT83 and BMP-2 in hair follicles in DSLD cases, as well as abnormally high expression of FOS in the epidermis, especially in aging horses. Because statistically relevant specificity and sensitivity was documented only for FOS and BMP2, but not KRT83 we recommend the use of FOS and BMP2 panel to diagnose DSLD. We conclude that a panel of two markers from the studied group (BMP2 and FOS) can serve as an additional diagnostic tool for DSLD in living horses, especially in older animals. Further studies are necessary to confirm if this biomarker panel could be used as a prospective tool to identify DSLD in horses as they age.

Measuring Biochemical Variables and Serum Amyloid A (SAA) in Working Mules in Central Italy.

According to FAO reports, the global mule population counts about 9 million mules. This hybrid cross of a male donkey and a female horse is mainly used for draft purposes because they are thought to be strong and hardy animals. Most consider mules to be less susceptible to disease and fatigue compared to horses. Therefore, the aim of this study was to investigate the effects of fieldwork on biochemical variables and serum amyloid A in working mules. Blood samples were collected from 10 healthy, female, working mules before and after 8 h of fieldwork. According to statistical analysis, a significant influence (p < 0.05) of fieldwork was found on mules' electrolyte profile with increased levels of sodium, chloride, and calcium, as well as on blood urea nitrogen and creatinine. After a day of fieldwork, serum sodium, chloride, calcium, urea, and creatinine concentrations were increased, supporting decreases in body water and renal blood flow. However, without comparison to a group of mules that were not exercised yet maintained under similar ambient conditions, it is uncertain whether these changes can be attributed to exercise. Further, no change in SAA concentration was found after exercise, indicating that the work performed did not result in systemic inflammation.

Serum Amyloid A as a Promising Biomarker in Domestic Animals' Reproduction: Current Knowledge and Future Perspective.

The investigation of acute phase proteins in veterinary medicine has opened the doors towards the identification and use of new markers for a timely assessment of health status in both companion and food-producing animals. The aim of this paper is to review the literature available on the use of serum amyloid A (SAA), an acute phase protein, for the diagnosis and monitoring of reproductive disorders in animals. This review critically appraises the usefulness of such marker in clinical practice and summarizes the current state of knowledge. Recent advances in the diagnosis and monitoring of reproductive diseases are presented, highlighting where SAA evaluation may enhance early diagnostic tools for dogs, cats, cattle, and equines.

One Hundred Years of Coat Colour Influences on Genetic Diversity in the Process of Development of a Composite Horse Breed.

Genetic diversity and demographic parameters were computed to evaluate the historic effects of coat colour segregation in the process of configuration of the Hispano-Arabian horse (Há). Pedigree records from 207,100 individuals born between 1884 and 2019 were used. Although coat colour is not a determinant for the admission of Hispano-Arabian individuals as apt for breeding, it may provide a representative visual insight into the gene contribution of Spanish Purebred horses (PRE), given many of the dilution genes described in Há are not present in the Arabian Purebred breed (PRá). The lack of consideration of coat colour inheritance patterns by the entities in charge of individual registration and the dodging behaviour of breeders towards the historic banning policies, may have acted as a buffer for diversity loss (lower than 8%). Inbreeding levels ranged from 1.81% in smokey cream horses to 8.80 for white horses. Contextually, crossbred breeding may increase the likelihood for double dilute combinations to occur as denoted by the increased number of Há horses displaying Pearl coats (53 Há against 3 PRE and 0 PRá). Bans against certain coat colours and patterns may have prevented an appropriate registration of genealogical information from the 4th generation onwards for decades. This may have brought about the elongation of generation intervals. Breeder tastes may have returned to the formerly officially-recognised coat colours (Grey and Bay) and Chestnut/Sorrel. However, coat colour conditioning effects must be evaluated timely for relatively short specific periods, as these may describe cyclic patterns already described in owners' and breeders' tastes over the centuries.

Initiative to Reduce Antibiotic Exposure of Asymptomatic Infants Born to Mothers with Intraamniotic Infection.

Infants born to mothers with intraamniotic infection (IAI) received antibiotic treatment per the Centers for Disease Control and Prevention and American Academy of Pediatrics guidelines in our neonatal intensive care unit (NICU) for early-onset bacterial sepsis evaluation. We conducted a quality improvement project to decrease antibiotic use and NICU admission in infants born to mothers with IAI. METHODS: We aimed to decrease the antibiotic exposure for asymptomatic infants born to mothers with IAI from 100% to 20% in 6 months. We obtained baseline data on these infants from January 2018 to January 2019, with the intervention starting in February 2019. A new standardized guideline to clinically monitor and follow laboratories on asymptomatic infants in couplet care was created with a multidisciplinary team's help and implemented after provider education. The team reviewed data monthly and used PDSA cycles to make necessary changes, including updating order sets, more educational handouts, and real-time coaching to both nurses and physicians. RESULTS: There was a dramatic decline (93%-0%) in antibiotic exposure and NICU admission after implementing this guideline. There was also a decrease in IAI diagnosis. There were no readmissions of infants for infection within 30 days of discharge, and there were no positive blood cultures. CONCLUSIONS: Implementing best antibiotic stewardship practices through a standardized guideline, testing, implementation of processes, and education by a multidisciplinary team limited the antibiotic exposure and NICU admissions for infants born to mothers with IAI with no known increase in readmissions.

Judgement Bias in Miniature Donkeys: Conditioning Factors and Personality Links.

Expectation-related bias may configure individuals' perception of their surrounding environment and of the elements present in it. This study aimed to determine the repercussions of environmental (weather elements) or subject-inherent factors (sex, age, or personality features) on judgment bias. A cognitive bias test was performed in eight Miniature jennies and four jacks. Test comprised habituation, training and testing phases during which subjects were trained on how to complete the test and scored based on their latency to approach an ambiguous stimulus. A questionnaire evaluating eleven personality features was parallelly completed by three caretakers, five operators and two care assistants to determine the links between personality features and judgment bias. Adjusted latencies did not significantly differ between sexes (Mann-Whitney test, p > 0.05). Although Miniature donkeys can discriminate positive/negative stimuli, inter-individual variability evidences were found. Such discrimination is evidenced by significant latency differences to approach positive/negative stimuli (33.7 ± 43.1 vs. 145.5 ± 53.1 s) (Mann-Whitney test, p < 0.05). Latencies significantly increased with patience, indicative of an expression of pessimism. Better understanding judgement bias mechanisms and implications may help optimize routine handling practices in the framework of animal welfare.

Selenium and Vitamin E Concentrations in Miranda Jennies and Foals (Equus asinus) in Northeast Portugal.

The increase in donkeys treated by practitioners in recent years has led to an increased interest in finding more information on basic biochemical preliminary reference values. The aims of this study were to measure Se and Vit E levels in plasma from Miranda jennies peripartum and postpartum and in their foals to compare blood profiles of the jenny and foal related to the overall foal's health. Twenty-two healthy peripartum and postpartum Miranda donkeys were sampled (12 jennies and 10 foals) in the northeast of Portugal (Atenor and Paradela) from May to November, 2018. Amounts of selenium in soil were significantly correlated (0.97) to concentrations of selenium in jennies (42.412 µg/L in Atenor and 9.612 µg/L in Paradela) and foals (19.378 µg/L in Atenor and 6.430 µg/L in Paradela). Selenium levels were lower in foals than adults and in males than females. Vitamin E was associated with overall foal health. Foals with a mean vitamin E of 3.585-5.307 mg/L showed signs of weakness, but carpal flexural deformities were observed when the average vitamin E was 11.520 mg/L. Low vitamin E levels (5.307 mg/L) in jennies were related to foal mortality. Diets, location, parity, and age affect blood profiles of jennies and, ultimately, foal health.

Discriminant Canonical Analysis of the Contribution of Spanish and Arabian Purebred Horses to the Genetic Diversity and Population Structure of Hispano-Arabian Horses.

Genetic diversity and population structure were analyzed using the historical and current pedigree information of the Arabian (PRá), Spanish Purebred (PRE), and Hispano-Arabian (Há) horse breeds. Genetic diversity parameters were computed and a canonical discriminant analysis was used to determine the contributions of ancestor breeds to the genetic diversity of the Há horse. Pedigree records were available for 207,100 animals born between 1884 and 2019. Nei's distances and the equivalent subpopulations number indicated the existence of a highly structured, integrated population for the Há breed, which is more closely genetically related to PRá than PRE horses. An increase in the length of the generation interval might be an effective solution to reduce the increase in inbreeding found in the studied breeds (8.44%, 8.50%, and 2.89%, for PRá, PRE, and Há, respectively). Wright's fixation statistics indicated slight interherd inbreeding. Pedigree completeness suggested genetic parameters were highly reliable. High GCI levels found for number of founders and non-founders and their relationship to the evolution of inbreeding permit controlling potential deleterious negative effects from excessively frequent mating between interrelated individuals. For instance, the use of individuals presenting high GCI may balance founders' gene contributions and consequently preserve genetic diversity levels (current genetic diversity loss in PRá, PRE, and Há is 6%, 7%, and 4%, respectively).

Donkey Epididymal Transport for Semen Cooling and Freezing.

The objectives of this study were to assess the cooling and freezing of donkey epididymal semen harvested immediately after castration (Experiment 1, n = 4) or after the shipment (24 or 48 h) of epididymides attached to testicles (Experiment 2, n = 14) or dissected apart (Experiment 3, n = 36). In each experiment, semen was frozen immediately (Non-Centrif) in an egg yolk-based semen extender (EY) or after processing through cushion-centrifugation (Centrif) while extended in a skim milk-based extender (SC). In all three experiments, cooled, pre-freeze, and post-thaw epididymal semen was assessed for total motility (TM), progressive motility (PM), plasma membrane integrity (PMI), and high mitochondrial membrane potential (HMMP). Data were analyzed with R using mixed models and Tukey's test as posthoc. Results showed that the cooling of epididymal semen up to 24 h after harvesting did not affect motility parameters or plasma membrane integrity; furthermore, in Experiment 3, the post-thaw evaluation of both Centrif and Non-Centrif achieved similar TM and PM. Collectively, the post-thaw results revealed low motility parameters across groups; while, the PMI and HMMP did not reflect this trend, and the values remained high, suggesting that there was a lack of epididymal sperm activation with either centrifugation or extenders. In summary, freshly harvested and cooled-shipped and cooled semen had satisfactory semen parameters. Future studies need to address donkey epididymal semen fertility in mares and jennies.

A Pilot Serosurvey for Selected Pathogens in Feral Donkeys (Equus asinus).

Recent removal and relocation of feral donkeys from vast public lands to more concentrated holding pens, training facilities, and offsite adoption locations raises several health and welfare concerns. Very little is known regarding the common equid pathogens that are circulating within the feral donkey population in and around Death Valley National Park, California, USA. The aim of this study was to utilize serologic assays to assess previous exposure of these donkeys to equine herpesvirus 1 (EHV-1), equine influenza (EIV), West Nile virus (WNV), and Borrelia burgdorferi (the causative agent of Lyme disease). The results of this study indicate that this feral equid population is mostly naïve and likely susceptible to these common equid pathogens upon removal from the wild.

Welfare Assessment and Identification of the Associated Risk Factors Compromising the Welfare of Working Donkeys (Equus asinus) in Egyptian Brick Kilns.

Donkeys are a cornerstone in human existence, having played an important role throughout history in different economic activities, such as working in brick kilns in Egypt. This study was conducted from January 2017 to the end of April 2017 in the El-Saf brick kilns, which are located to the south of the Giza Governorate and 57 Km away from Cairo. Physical clinical health and behavior data were collected from 179 donkeys spanning over a random sample of 20 brick kilns selected from the El-Saf brick kilns. Behavioral, physical health, harness, and environmental parameters were assessed and recorded. The study found that 80 ± 3% (n = 179) of kiln donkeys have some type of wound, and the most serious wound is a beating wound (49 ± 3.7%), which is caused by drivers hitting the donkeys. The drivers are mostly children, who have insufficient knowledge, skills, and attitudes to effectively communicate with their donkeys and no motivation to enhance the welfare of these equids. Other wounds are related to the harness, such as the breeching (10 ± 2.2%), saddle (43 ± 3.7%), neck collar (40 ± 3.6%), and shaft of the cart (12 ± 2.4%). A poor body condition was seen in 56 ± 3.7% of kiln donkeys. A correlation in terms of the prevalence of wounds was found between the body condition (p-value < 0.01) and/or cleanliness of the harness. There was a negative association between the body condition and wound prevalence in brick kilns (Pearson coefficient of correlation -0.71). The physical enviromental factors that affect the body condition of working donkeys are the working hours of donkeys/day, the number of donkeys in a kiln, the distance from loading to the oven, and the concentrated food/donkey (p-value < 0.01). These three variables can explain 78.85% of the variability in body conditions based on a 1-5 scale. In addition to health parameters, behavior parameters, such as the donkeys' general attitude, reaction to observers, and chin contact are associated with the body condition (p-value < 0.01). As a consequence, it is important for the owners of working donkeys to pay attention to their body condition in order to avoid compromising their body condition and welfare.

Development of a Donkey Grimace Scale to Recognize Pain in Donkeys (Equus asinus) Post Castration.

The objectives of this study were to establish a donkey ethogram, followed by a donkey grimace scale to be applied to donkeys pre- and post-castration and to test if there was a notable difference in scores based on observer knowledge, gender, and experience, which could reveal possible discomfort/pain. Nine healthy male adult donkeys were surgically castrated. Fifty-four photos were selected from frontal, lateral, and body views taken pre- and post-castration. Observers ranging from minimal to extensive knowledge and levels of experience based on education and hours/month spent with donkeys scored six photos/donkey on a scale of 0-2 (0 = not present, 1 = moderately present, 2 = obviously present). Scores were based on body language and facial parameters: Ears down, ears back, eye white showing, glazed look, orbital tightening, eyes round shape, nostril tension, eyes narrow shape, muzzle tension, and abnormal stance and overall perception of the animal being in pain. Level of experience and knowledge, as well as gender significantly (p < 0.001), affected observers' ability to accurately score images. The study suggests that the most significant indicators of pain in donkeys are overall appearance and abnormal body stance provided their sensitivity, specificity and accuracy values of 63.18%, 62.07%, and 62.60%, respectively.

Survey of Serum Amyloid A and Bacterial and Viral Frequency Using qPCR Levels in Recently Captured Feral Donkeys from Death Valley National Park (California).

Feral donkey removal from state land has raised concerns in terms of disease transmission between equine species. Disease outbreaks may occur as a result of the relocation of animals to new environments. Virus and bacteria DNA load and serum amyloid A derived from the pathogenic processes that they involve were measured in recently captured donkeys. Blood and nasal swabs were collected from 85 donkeys (Death Valley National Park, Shoshone, California); 24 were retested after 30/60 days in the Scenic (Arizona) long-term holding facility co-mingled with feral donkeys from Arizona and Utah. Quantitative Real-Time PCR (qPCR) was performed to detect viral and bacterial genomic material (equine influenza A [EIV], equine rhinitis A and B viruses, AHV-2, AHV-3, AHV-5 and EHV-1, EHV-4, Streptococcus equi subspecies equi and zooepidemicus,). Significant relations between behavior, body condition score, nasal discharge, and coughing were found in donkeys for which AHV-2 and Streptococcus zooepidemicus DNA was detected. Higher SAA concentrations were found in foals. AHV-2 and Streptococcus zooepidemicus DNA concentrations significantly differed between sampling moments (p < 0.05). In conclusion, donkeys do not appear to be a substantial risk for disease transmission to horses but could be if they carried strangles or other processes in which AHV-2 and Streptococcus zooepidemicus were involved.

Anthelmintic Efficacy and Pharmacokinetics of Ivermectin Paste after Oral Administration in Mules Infected by Cyathostomins.

Ivermectin (IVM) is an anthelmintic compound commonly used off-label in mules due to its broad-spectrum of activity. Despite the general use of IVM in mules with the same dose and route of administration licensed for horses, significant pharmacokinetic differences might exist between horses and mules, as already observed for donkeys. The aim of the present study was to evaluate the pharmacokinetic profile and anthelmintic efficacy of an oral paste of IVM in mules naturally infected with cyathostomins. Fifteen adult mules with fecal egg counts (FEC) ≥200 eggs per gram (EPG), with exclusive presence of cyathostomins, were included in the study. All mules were orally treated with IVM according to the manufacturer's recommended horse dosage (200 µg/kg body weight). FECs were performed before (day-10 and day-3) and after treatment at days 14 and 28 by using a modified McMaster method. The FEC reduction (FECR%) was also calculated. Blood samples were collected from five animals at various times between 0.5 h up to 30 days post treatment to determine pharmacokinetic parameters. The maximum IVM serum concentration (Cmax) was 42.31 ± 10.20 ng/mL and was achieved at 16.80 ± 9.96 hours post-treatment (Tmax), area under the curve (AUC) was 135.56 ± 43.71 ng × day/mL. FECR% remained high (>95%) until the 28th day.

Back in the saddle: student response to remote online equine science classes.

The COVID-19 pandemic has challenged professors and students of all disciplines to adjust quickly to remote online teaching and education platforms. In this new era of remote teaching, a greater challenge has been presented in the field of equine science; how to effectively share knowledge that is most often demonstrated by providing students access to live, in-person animal examples. Historically, students and teachers believed skill sets, which are vital for future careers in the industry (e.g., veterinarian) must be learned through hands-on experience. However, in-person methods were not available, so students were taught through the Zoom platform. Students enrolled in various levels of equine science classes were invited to complete a short voluntary questionnaire measuring their response and perception to equine courses taught in an entirely online remote setting by the same professor. One group was comprised of undergraduates majoring in the field (n = 44) in upper level equine science courses, Advanced Equine Reproduction Physiology and/or Equine Enterprise. These students, 41 females and 3 males, ranged in age from 20 to 25 yr, were provided a voluntary questionnaire seeking responses related to the perceived effectiveness and individual preferences of in-class lectures and in-person labs vs. remote online teaching practices. A similar questionnaire was offered on a volunteer basis to precollege students (n = 17). These students, female, high-school students from freshman to senior status (14-18 yr of age), were interested in equine science as a major at UC Davis in the future. This questionnaire evaluated their response to a 2-week remote synchronous online equine science course, which included multiple teaching methods, including lectures, mini labs, and full labs. Responses from both populations suggested that equine courses were perceived as effective when offered as online, remote courses. Live (synchronous) classes and labs offered on Zoom increased engagement and interaction, but students also appreciated the opportunity to access recorded materials. Students responded positively to online remote teaching and found courses to be effective for increasing their knowledge about equine science in an engaging manner, despite their continued preference for in-person instruction.

Donkey and Mule Behavior.

Donkeys and mules often are misunderstood because of their behavioral differences compared with horses. Working with these animals requires more patience and ability to notice the slightest changes in behavior to diagnose disease. Mules and donkeys form strong bonds and trust with familiar people and other equids. Training mules and hinnies from an early age is key to modifying behavior and acceptance of unfamiliar people. Reproductive behavior is different from that of horses and requires more patience when collecting jacks. Practitioners working with mules and donkeys should take a methodological approach and get to know the animal before performing examinations.