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The main goals of this study were to document and compare the normal ranges of testicular haemodynamic parameters in pre- (aged 8-12 months; n = 4) and postpubertal (aged 24-259 months; n = 16) Miranda donkeys in the breeding season, and to correlate animal biometric data and testicular Doppler indices with basic semen quality parameters of sexually mature jacks. Colour and pulsed-Doppler ultrasonography were employed to assess blood flow in the left and right distal supratesticular artery (DsTA) and their marginal branches (marginal arteries-MA). Peak systolic velocity (PSV), end diastolic velocity (EDV), pulsatility index (PI) and resistive index (RI) were evaluated in both blood vessels, and TAMV (time-averaged mean velocity), TABF (total arterial blood flow) and TABF rate (TABF-R) were calculated for MA. The mean diameter of MA was greater (p < 0.05; 0.24 ± 0.05 vs. 0.19 ± 0.05 cm; mean ± SD) but TABF-R was less (p < 0.05; 0.004 ± 0.004 vs. 0.02 ± 0.01 mL/s/cm 3 ) in sexually mature donkeys compared with prepubertal animals. Apart from RI values for the right testicle of prepubertal donkeys, PI and RI were consistently greater (p < 0.05) in DsTA compared with MA. Significant correlations were found among select biometric and haemodynamic attributes of the testes (height, width and length, TV, TTV and PSV-ST) and ejaculate characteristics (volume, sperm defects-total, head and midpiece) in sexually mature donkeys (n = 8). The present results highlight the importance of scrotal ultrasonography for the reproductive assessment of jacks and provide reference values, based on the available subpopulation of Miranda donkeys that can be used in their clinical and reproductive management and research, or conservation programmes., (© 2024 Wiley‐VCH GmbH. Published by John Wiley & Sons Ltd.)

The donkey has always been a notable working animal and its importance as a companion animal has been growing over the last few years. However, there are only a few studies about cardiology in this species. Therefore, this study aimed to evaluate the influence of age, sex, training, and bodyweight on cardiac dimension in mixed-breed donkeys. Forty-five clinically and para-clinically healthy mixed-breed donkeys were included, and B-mode and M-mode echocardiographic measurements were recorded. Multivariate linear regression analysis was performed to identify the effect of age, sex, bodyweight, and training on cardiac measurements. Most of the echocardiographic measurements showed a strong statistically significant correlation with bodyweight. Linear regression analysis between echocardiographic measurements and bodyweight was performed to obtain the values of the intercept and slope of the linear equation to calculate the echocardiographic measurements as a function of bodyweight. This is the first study reporting a strong linear correlation between echocardiographic measurements and bodyweight and reporting a correlation between training and echocardiographic parameters in donkeys, suggesting that this variable should be considered when assessing cardiac dimensions in these animals., (© 2024. The Author(s).)

The welfare of donkeys remains a compelling subject for researchers, with limited literature available on the response of the donkey cardiovascular system during strenuous exercise. The study aimed to address two primary objectives. Firstly, to assess the reliability of wearable devices in detecting heart rate (HR) and ECG readings. Secondly, to determine HR, locomotor and cardiac troponin 1 (cTnI) levels in donkeys during exercise. A total of seven donkeys were outfitted with two systems for ECG measurements, namely Equimetre and the Standard base apex, to enable a comparison between the two. Additionally, fifteen apparently healthy donkeys equipped with Equimetre were divided into two groups: the race group (R), consisting of donkeys trained for racing, and the non-race group (NR), comprising donkeys used for regular riding. The results indicated a level of agreement between the two devices in intervals R-R (P =  < 0.0001), S-T (P = 0.0002), Q-T(P = 0.0003), P-R (P = 0.0037), segment P-R (P = 0.0023) and HR (P =  < 0.0001) at rest. This suggested that Equimetre can provide a level of accepted ECG reading in donkey. No significant difference in heart response and locomotor parameters between donkey groups, although this finding needs further studies to verify it and to understand the dynamics of donkey. This study demonstrates the feasibility of Equimetre in detection HR and present initial data of heart response and locomotor in donkeys during exercise., (© 2024. The Author(s).)

Two Poitou donkey jennies were presented for clinical oocyte recovery and embryo production via intracytoplasmic sperm injection (ICSI). Both jennies underwent transvaginal ultrasound-guided follicle aspiration on two occasions. Recovered oocytes were held overnight then placed into maturation culture, using standard methods for mare oocytes. On the first replicate for both jennies, the oocytes were divided into two groups; one group was denuded and examined at 30 h culture (standard culture duration for mare oocytes) and the second was denuded and examined at 36 h culture. No oocytes with polar bodies were observed at either time. The oocytes were maintained in maturation culture until 46 h, at which time oocytes with polar bodies were observed. Semen was then prepared; oocytes underwent ICSI approximately 48 h after being placed into maturation culture. On the second replicate for both jennies, oocytes were cultured for maturation for 42 h, then denuded and subjected to ICSI at 46 h. Sperm preparation, injection and embryo culture were performed as for mare oocytes. Blastocyst rates per injected oocyte were 8/19 (42 %) overall, being 4/12 and 4/7 for the first and second TVAs, respectively. Blastocysts were vitrified. Three blastocysts were warmed and transferred to Poitou donkey jenny recipients. One embryonic vesicle was visualized on ultrasonography on embryo Day 12, which increased in size on Day 13 but was not present when examined on Day 14. These results demonstrate that oocyte recovery and ICSI are efficient for production of Poitou donkey blastocysts. To the best of our knowledge, this is the first report of production of blastocysts via ICSI in the Poitou donkey, and the first report of transfer of ICSI-produced embryos in the donkey. Further work is needed on factors affecting pregnancy after embryo transfer in the donkey., Competing Interests: Declaration of competing interest The authors declare that they have no conflicting interests., (Copyright © 2024. Published by Elsevier Inc.)

In East Africa, community-based conservation models (CBCMs) have been established to support the conservation of wildlife in fragmented landscapes like the Tarangire Ecosystem, Tanzania. To assess how different management approaches maintained large herbivore populations, we conducted line distance surveys and estimated seasonal densities of elephant, giraffe, zebra, and wildebeest in six management units, including three CBCMs, two national parks (positive controls), and one area with little conservation interventions (negative control). Using a Monte-Carlo approach to propagate uncertainties from the density estimates and trend analysis, we analyzed the resulting time series (2011-2019). Densities of the target species were consistently low in the site with little conservation interventions. In contrast, densities of zebra and wildebeest in CBCMs were similar to national parks, providing evidence that CBCMs contributed to the stabilization of these migratory populations in the central part of the ecosystem. CBCMs also supported giraffe and elephant densities similar to those found in national parks. In contrast, the functional connectivity of Lake Manyara National Park has not been augmented by CBCMs. Our analysis suggests that CBCMs can effectively conserve large herbivores, and that maintaining connectivity through CBCMs should be prioritized., (© 2024. The Author(s).)

Eastern Africa is home to the largest terrestrial migrations on Earth. Though these migratory systems have been well studied for decades, little is known of their antiquity and evolutionary history. Serially sampled strontium stable isotopes ( 87 Sr/ 86 Sr) from tooth enamel can be used to track migration in mammals. Here we analyse 87 Sr/ 86 Sr for 79 bovid and equid individuals representing 18 species from four localities in Kenya to characterize prehistoric migratory systems during the Last Glacial Period (115-11.7 ka). Of the species analysed, 16 lack definitive evidence for migration, including blue wildebeest (Connochaetes taurinus), Thomson's gazelle (Eudorcas thomsonii) and plains zebra (Equus quagga), which are long-distance migrants today in the Greater Serengeti Ecosystem and historically in the Athi-Kapiti Plains. Only two species, the extinct wildebeests Rusingoryx atopocranion and Megalotragus sp., were migratory. These findings suggest a possible alternative narrative about ecosystem dynamics during the Last Glacial Period and shed light on the behaviour of both extant and extinct species at this time. In particular, these results indicate that migratory behaviour in extant species either emerged during the Holocene or was more spatiotemporally constrained in the past. Our results contribute to a growing body of evidence suggesting that the structure and function of geologically recent large mammal communities in eastern Africa differed considerably from those observed in the present day., (© 2024. The Author(s), under exclusive licence to Springer Nature Limited.)

There are two species of free-roaming feral equids in North America: horses (Equus caballus) and donkeys or "burros" (E. asinus). Both species were introduced as domestic animals to North America in the early 1500s and currently inhabit rangelands across the western United States, Canada, and all continents except Antarctica. Despite their global distribution, little is known about their fine scale spatial ecology. Contemporary research tools to assess space use include global positioning system (GPS) tracking collars, but older models were problematic due to stiff collar belting causing poor fit. We tested modern designs of GPS collars on n = 105 horses and n = 60 burros for 4 years in five populations (3 horse, 2 burro) across the western United States, to assess whether collars posed welfare risks to horses or burros. We found no difference in survival of collared versus uncollared mares and jennies, and no difference in survival of their foals. In 4036 of 4307 observations for horses (93.7%) and 2115 of 2258 observations for burros (93.6%), collars were observed symmetrical, maintaining proper fit on the neck. Fur effects from collars (sweaty neck, indented fur, broken fur) were seen in 3% of horse observations and 25% of burro observations. Superficial effects (chafes and marks on skin surface) were seen in 2% of horse observations and 11% of burro observations; no severe effects from collars were seen. Body condition was not affected by collars; mean body condition of collared horses was 4.70 ± 0.54 (mean ± s.d) and 4.71 ± 0.65 for collared burros. Behavior results indicated minimal effects; collared horses stood slightly more than uncollared, and collared burros stood and foraged more in one population, but not in the other. For 6.3% of observations of horses and 6.4% of observations of burros, we found an effect of time wearing a collar on the cumulative sum of fur effects which increased over time (burros: rs = 0.87, P = <0.0001; horses: rs = 0.31, P = 0.002). Burros also showed an increase over time in the number of superficial effects, but horses did not. Collars occasionally moved into the wrong position, shifting forward over the ears; we observed this on 19 horses and 1 burro. Of those, most collars went over the ears in summer (n = 12). All collars were equipped with a remote release mechanism as well as a timed-release mechanism for redundancy, thus removed when observed in wrong position to avoid rubbing or discomfort. Our finding of no consequential physical effects in 98% of horse observations, and 89% of burro observations suggests the consequences of collars on free-roaming equid welfare and survival is biologically insignificant, although collars should be monitored regularly and continue to be equipped with a remote release mechanism to remove a collar if needed. With frequent welfare-driven, visual monitoring, collaring of free-roaming equids can be a safe and useful tool to increase our understanding of their spatial ecology, demography, habitat use, behavior, and interactions with other wildlife., Competing Interests: The authors have declared that no competing interests exist., (Copyright: This is an open access article, free of all copyright, and may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone for any lawful purpose. The work is made available under the Creative Commons CC0 public domain dedication.)

The aim of this study was to evaluate two cryoprotectants, dimethylformamide (DMF) and methylformamide (MF) in two concentrations (5 and 7 %) in vitro in donkey semen using a rapid freezing technique and the effect on pregnancy rates in mares. Twenty-four ejaculates from 8 jacks (n = 8; r = 3) were divided into 4 extenders: BotuSemen Gold with 5 % or 7 % MF and 5 % or 7 % DMF, all containing 11 % lactose, 20 % egg-yolk and 0.5 % Equex. Post-thaw evaluations included: sperm motility, membrane function and acrosome status. A linear mixed effect model was used to test the effect of different freezing media on semen parameters. No differences were observed between the 4 freezing media used, for any of the seminal parameters (P > 0.05). However, samples with 5 % DMF showed the highest percentages of sperm with acrosomes and functional membranes (DMF: 5 %: 53.67 ± 22.01; 7 %: 33.92 ± 23.4; MF: 5 %: 44.5 ± 20.46; 7 %: 38.75 ± 27.4) (Data: mean ± SD; P > 0.05). Hence, thirty mares were inseminated: 15 with 5 % DMF and 15 with 7 % DMF. The pregnancy rate was 46 % (7/15) and 0 % (0/15) using the extender with 5 % or 7 % DMF, respectively (P = 0.003). To conclude, the use of 5 % or 7 % of MF or DMF did not affect the in vitro parameters. Despite the lack of differences in vitro with the two DMF concentrations, in vivo results only showed pregnancies when using 5 % DMF. Thus, the results of this study demonstrate the importance of accompanying in vitro semen evaluations with studies that evaluate post-insemination pregnancy rates., Competing Interests: Declaration of competing interest None of the authors has any conflict of interest to declare., (Copyright © 2024. Published by Elsevier Inc.)

Optimal foraging theory predicts that animals maximise energy intake by consuming the most valuable foods available. When resources are limited, they may include lower-quality fallback foods in their diets. As seasonal herbivore diet switching is understudied, we evaluate its extent and effects across three Kenyan reserves each for Critically Endangered eastern black rhino (Diceros bicornis michaeli) and Grevy's zebra (Equus grevyi), and its associations with habitat quality, microbiome variation, and reproductive performance. Black rhino diet breadth increases with vegetation productivity (NDVI), whereas zebra diet breadth peaks at intermediate NDVI. Black rhino diets associated with higher vegetation productivity have less acacia (Fabaceae: Vachellia and Senegalia spp.) and more grass suggesting that acacia are fallback foods, upending conventional assumptions. Larger dietary shifts are associated with longer calving intervals. Grevy's zebra diets in high rainfall areas are consistently grass-dominated, whereas in arid areas they primarily consume legumes during low vegetation productivity periods. Whilst microbiome composition between individuals is affected by the environment, and diet composition in black rhino, seasonal dietary shifts do not drive commensurate microbiome shifts. Documenting diet shifts across ecological gradients can increase the effectiveness of conservation by informing habitat suitability models and improving understanding of responses to resource limitation., (© 2024. The Author(s).)

Competition, facilitation, and predation offer alternative explanations for successional patterns of migratory herbivores. However, these interactions are difficult to measure, leaving uncertainty about the mechanisms underlying body-size-dependent grazing-and even whether succession occurs at all. We used data from an 8-year camera-trap survey, GPS-collared herbivores, and fecal DNA metabarcoding to analyze the timing, arrival order, and interactions among migratory grazers in Serengeti National Park. Temporal grazing succession is characterized by a "push-pull" dynamic: Competitive grazing nudges zebra ahead of co-migrating wildebeest, whereas grass consumption by these large-bodied migrants attracts trailing, small-bodied gazelle that benefit from facilitation. "Natural experiments" involving intense wildfires and rainfall respectively disrupted and strengthened these effects. Our results highlight a balance between facilitative and competitive forces in co-regulating large-scale ungulate migrations.

Lions have long been perceived as Africa's, if not the world's, most fearsome terrestrial predator, 1 , 2 , 3 , 4 , 5 , 6 , 7 , 8 , 9 the "king of beasts". Wildlife's fear of humans may, however, be far more powerful and all-prevailing 1 , 10 as recent global surveys show that humans kill prey at much higher rates than other predators, 10 , 11 , 12 due partly to technologies such as hunting with dogs or guns. 11 , 13 , 14 , 15 We comprehensively experimentally tested whether wildlife's fear of humans exceeds even that of lions, by quantifying fear responses 1 in the majority of carnivore and ungulate species (n = 19) inhabiting South Africa`s Greater Kruger National Park (GKNP), 9 , 15 , 16 , 17 using automated camera-speaker systems 9 , 18 at waterholes during the dry season that broadcast playbacks of humans, lions, hunting sounds (dogs, gunshots) or non-predator controls (birds). 9 , 19 , 20 , 21 , 22 Fear of humans significantly exceeded that of lions throughout the savanna mammal community. As a whole (n = 4,238 independent trials), wildlife were twice as likely to run (p < 0.001) and abandoned waterholes in 40% faster time (p < 0.001) in response to humans than to lions (or hunting sounds). Fully 95% of species ran more from humans than lions (significantly in giraffes, leopards, hyenas, zebras, kudu, warthog, and impala) or abandoned waterholes faster (significantly in rhinoceroses and elephants). Our results greatly strengthen the growing experimental evidence that wildlife worldwide fear the human "super predator" far more than other predators, 1 , 19 , 20 , 21 , 22 , 23 , 24 , 25 , 26 , 27 , 28 and the very substantial fear of humans demonstrated can be expected to cause considerable ecological impacts, 1 , 6 , 22 , 23 , 24 , 29 , 30 , 31 , 32 , 33 , 34 , 35 presenting challenges for tourism-dependent conservation, 1 , 36 , 37 particularly in Africa, 38 , 39 while providing new opportunities to protect some species. 1 , 22 , 40 ., Competing Interests: Declaration of interests The authors declare no competing interest., (Copyright © 2023 Elsevier Inc. All rights reserved.)

Background: Epidural analgesia using the alpha-2 agonist detomidine (DE), alone or in combination with lidocaine (LD), is frequently employed for standing surgical procedures in horses, but its use has not been evaluated in donkeys., Methods: In a randomised controlled prospective trial, 24 healthy adult donkeys were assigned to four groups (n = 6), each receiving 40 μg/kg of DE, 0.22 mg/kg of LD, combined DE and LD (DELD) or 0.9% sterile normal saline epidurally. After epidural injection of each treatment, the onset, degree and duration of sedation and anatomical extension of anti-nociception were observed., Results: DE and DELD treatments resulted in complete bilateral analgesia with loss of sensation in the tail, perineum, inguinal area, chest and the caudal aspect of the upper pelvic limb, and extended distally to the dorsal metatarsal area. DE and DELD resulted in a significantly (p < 0.05) longer duration of anti-nociception (110 ± 15.4 min and 141.6 ± 14.7 min, respectively) than LD (75.8 ± 4.9 min). The DELD duration of sedation was significantly (p < 0.05) longer than the DE duration (118.3 ± 19.4 min and 108.3 ± 7.5 min, respectively)., Limitation: The current study's main limitation is using only one dose of DE., Conclusion: DE and DELD produced a very effective, safe and acceptable sedative and analgesic effect in the perineal and inguinal regions of donkeys., (© 2022 British Veterinary Association.)

Cryopreservation of semen is an important technique to preserve genetic material. Yet, pregnancy rates in jennies after artificial insemination with frozen-thawed donkey semen are poor. This condition has been attributed to the impact of permeable cryoprotectants, that could cause high post-breeding endometritis. Removal of seminal plasma (SP) prior to semen freezing process is another contributing factor. SP is involved in a multitude of sperm functions and events preceding fertilization and has a mediating effect of sperm capacitation and postcoital uterine inflammatory response. The aim of this study was to evaluate different alternatives in donkey semen cryopreservation with permeable, non-permeable cryoprotectants, BSA and SP. Thirty ejaculates from 10 donkeys were cryopreserved with different combinations of dimethylformamide (DMF, 5%), sucrose (SUC, 200 mM) and homologous SP (10%): DMF (T1), DMF/SP (T2), SUC/BSA (T3), SUC/BSA/SP (T4), DMF/SUC/BSA (T5), DMF/SUC/BSA/SP (T6), DMF/BSA (T7) and DMF/BSA/SP (T8). After thawing, sperm motility and kinetics were assessed by computerized semen analysis. Sperm vitality (SV) was evaluated by fluorescence microscopy, functional membrane integrity (FMI) by the HOST test, abnormal morphology by eosin-nigrosin staining and sperm membrane stability by flow cytometry. For statistical analysis, sperm quality indexes (SQi) were obtained, general linear models were carried out and mean comparisons were made by the Tukey test. T1, T2, T5, T6, and T7 had higher and equivalent results for motility, most kinetic parameters and function membrane integrity. Cryopreservation of donkey semen without permeable cryoprotectant (T3 and T4) showed a reduction in motility, kinetics, SV, FMI and SQi. T5 showed a reduction in progressive motility, sperm velocities, IMF and SQi compared to other DMF treatments. T6 and T8 achieved higher SQi values compared to T1, but they were not different compared to T2 and T7. T1 had a smaller sperm population with low-M540 compared to T3. It is concluded that the use of permeable cryoprotectant is essential to achieve higher post-thaw quality of donkey semen. In addition, the combined use of BSA, SUC and/or PS may provide additional sperm protection compared to the individual use of DMF., (© 2022 Wiley-VCH GmbH.)

The study aimed to evaluate the effects of packing (load carrying) on rectal and body surface temperatures and their circadian rhythmicity in donkeys during the hot-dry season. Twenty pack donkeys of both sexes (15 males and 5 non-pregnant females), aged 2-3 years, with average weight of 93 ± 2.7 kg and divided into two groups randomly, served as experimental subjects. Group 1 donkeys (packing + trekking) were subjected to packing in addition to trekking, while group 2 (trekking only) did not carry any load. All the donkeys were trekked, covering a distance of 20 km. The procedure was repeated three times within the week, one day apart. During the experiment, dry-bulb temperature (DBT), relative humidity (RH) and temperature-humidity index (THI), wind speed and topsoil temperature were recorded; and rectal temperature (RT) and body surface temperature (BST) were measured before and immediately after packing. Starting from 16 h after the last packing, circadian rhythms of RT and BST were recorded at 3-h intervals for 27-h period. The RT and BST were measured by digital thermometer and non-contact infrared thermometer, respectively. The DBT and RH, especially after packing (35.83 ± 0.2 °C and 20.00 ± 0.0%, respectively), were outside the thermoneutral zone for donkeys. The RT value (38.63 ± 0.1 °C) in packing + trekking donkeys recorded immediately (15 min) after packing was higher (P < 0.05) than the value obtained in trekking only donkeys (37.27 ± 0.1 °C). The overall mean RT recorded during the continuous 27-h period of measurement, starting from 16 h after the last packing procedure was higher (P < 0.05) in packing + trekking donkeys (36.93 ± 0.2 °C) compared with trekking only donkeys (36.29 ± 0.3 °C). The BSTs recorded in both groups were higher (P < 0.05) immediately after packing when compared with pre-packing values, but insignificant 16-h post-packing. The RT and BST values were generally higher in the photophase and lower during the scotophase in both groups of donkeys during the continuous recordings. The eye temperature was the closest to the RT, followed by scapular temperature, while the coronary band temperature was the farthest. The mesor of RT in packing + trekking donkeys (37.06 ± 0.2 °C) was significantly higher than in donkeys that were trekked only (36.46 ± 0.1 °C). The amplitude of RT in trekking only donkeys (1.20 ± 0.1 °C) was wider (P < 0.05) than that obtained in packing + trekking donkeys (0.80 ± 0.1 °C). The acrophase and bathyphase occurred later in packing + trekking donkeys (18:10 ± 0.3 h and 6:10 ± 0.3 h, respectively), compared to the trekking only donkeys (16:50 ± 0.2 h and 4:50 ± 0.2 h, respectively). In conclusion, exposure to thermally stressful prevailing environmental conditions during packing increased the body temperature responses, especially in packing + trekking donkeys. Packing significantly influenced the circadian rhythms of body temperatures in working donkeys, as evidenced by differences in circadian rhythm parameters in packing + trekking group, compared with trekking only donkeys during the hot-dry season., Competing Interests: Declaration of competing interest The authors have declared that no competing interest exists., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

In most animals, pain can compromise physiological functions and delay healing so, rapid detection of pain through behavior and inflammatory reaction with biomarkers are necessary. This study aimed to evaluate pain, physiological variations and Acute Phase Proteins (APP) in donkeys undergoing orchiectomy technique by inguinal access. For this research, 15 male northeastern donkeys kept in extensive management were selected, with a mean age of 4.5±3.1 years. All animals had the same anesthetic protocol, using dissociative anesthesia and local block with lidocaine, followed by orchiectomy by inguinal access. Due to their predisposition to complications, the inguinal technique is the most indicated to minimize complications and excessive inflammation in donkeys' orchiectomy, the donkeys were evaluated regarding behavioral assessment of pain, hematological parameters, APP and the surgical wound, during 0 hour, 24 hours, 48 hours and 72 hours. As for the physiological parameters and APP, no significant differences were observed between times, due to the use of nonsteroidal antiinflammatory drugs. In the macroscopic evaluation of the surgical wound, it was observed that there were no significant differences between the times, with animals presenting mean scores of 1.8±0.414, in 48 hours 1.6 ± 0.507, and in 72 hours 1.6 ± 0.507. Most animals had mild to moderate edema in the scrotum and foreskin regions. As for pain assessment, the average scores were between 2 and 3, representing mild and moderate pain, not requiring intervention. However, further research is needed to elucidate the behavior of PFAs in the face of variables and the creation of new pain scales for animals raised in an extensive system., (Copyright © 2023 Elsevier Inc. All rights reserved.)

Endometritis is a common disease in donkeys that causes economic losses to donkey farms and the common cause is bacterial infection. Uterine flush fluid proteomics has been used to study protein biomarkers associated with endometritis in mares. As a convenient diagnostic tool, serum proteomics has not been studied yet in equine species with endometritis. This study is aiming to evaluate the serum proteomics in jennies with and without endometritis and identify potential proteins as biomarker for endometritis diagnosis. Nine donkeys recruited into this study were diagnosed of bacterial (Escherichia coli) endometritis and nine healthy jennies were selected as control. Blood samples of each donkey was collected, and serum was separated from each sample. Peptides samples extracted from the serum were analyzed using nano-ultrahigh-performance liquid chromatography-tandem mass spectrometry in data-independent acquisition mode. Protein identification and quantification were performed followed by differential and functional analysis. Of 579 proteins identified in all jennies, 12 proteins were exclusively identified in jennies with endometritis (group E) including myeloperoxidase and Ras-related protein Rab-1B, which might be associated with bacterial infection. There were 11 differentially expressed proteins detected between the two groups of jennies with 4 downregulated proteins and 7 upregulated proteins in jennies with endometritis. Some upregulated proteins along with the GO and KEGG annotation indicated inflammatory response against uterine infection. Characteristic serum proteins identified in jennies with endometritis were associated with inflammation or bacterial infection. These proteins might be potential biomarkers for endometritis diagnosis in jennies., (Copyright © 2023. Published by Elsevier Inc.)

The study aimed to evaluate the effects of melatonin administration on rectal and body surface temperature (RT and BST, respectively) responses and their circadian rhythmicity in donkeys subjected to packing (load carrying) during the hot-dry season. Twenty donkeys were divided into two equal groups randomly: Groups 1 (packing + melatonin) and 2 (packing - melatonin), subjected to packing and both covered 20 km. The RT, BST, and thermal environmental parameters were measured before and after packing. The procedure was carried out three times within the week, one day apart. This was followed 16-h after the last (third) packing procedure by 27-h recording period of all the parameters at 3-h intervals. The RT (37.77 ± 0.1℃) recorded in packing + melatonin donkeys was lower (P < 0.05) than in (packing - melatonin) (38.29 ± 0.1℃) post-packing, while the BSTs in packing + melatonin donkeys were lower than in (packing - melatonin) donkeys, especially the neck (33.07 ± 0.6℃ vs 35.4 ± 0.7℃, respectively) and coronary band (30.58 ± 0.5℃ vs 33.38 ± 0.7℃, respectively) temperatures. In both groups 16-h post-packing, overall mean RT and BST values were not different, except the coronary band temperature (26.61 ± 0.8℃) that was lower (P < 0.05) in packing + melatonin donkeys than (packing - melatonin) donkeys (28.78 ± 1.4℃). Melatonin-exerted biphasic effects on circadian rhythms of RT and BSTs by reducing body temperatures during the photophase and increasing the values during the scotophase in pack donkeys. In conclusion, melatonin may enhance packing output in donkeys during the hot-dry season., (© 2022. The Author(s) under exclusive licence to International Society of Biometeorology.)

In impoverished nations, donkeys help people make a living. The welfare of donkeys in Sudan is neglected compared with developed countries. However, there is no information available about donkey welfare in Nyala. This study aimed to assess the welfare of donkeys in Nyala. Donkeys were divided into two groups (n = 50), working and nonworking. The donkeys' physical, emotional, and clinical parameters and an owner's interview were assessed. There were significant differences in body condition scores: 37% and 47% of working and nonworking donkeys, respectively, had ideal body condition scores, while 13% of working donkeys were emaciated. Of the working and nonworking 33% and 19% had hoof problems, respectively. Fifteen percent of working donkeys had ocular discharge, and 25% had wounds. In working donkeys, 7% and 5% depressive and aggressive behavioral responses, respectively, were observed. Furthermore, there were significant differences in tools used for hitting donkeys, with 33% and 17% of owners using a stick and whip, respectively, for hitting working donkeys. Eighty-two percent of owners feed their donkeys one to three times daily (50% working and 32% nonworking donkeys), and free access feeding was only observed in 18% of nonworking donkeys. There were no significant differences (P > .06) in emotional parameters. We conclude that working donkeys suffer from multiple welfare problems more than nonworking donkeys in Nyala. More awareness, veterinary services, and research are needed to improve donkey care in Nyala., (Copyright © 2022. Published by Elsevier Inc.)

To study the effects of exercise on donkey semen parameters, Dezhou donkey (Equus asinus) jackasses (n = 18) were assigned to three groups: control (CN, n = 6), 1-hour exercise (1 h EX, n = 6) and 2-hour exercise (2 h EX, n = 6) groups. They were exercised with a walking machine for 3 days/week for 8 weeks. Semen parameters and reproductive hormones were evaluated weekly. The heart rate, rectal temperature, blood haematology indexes, and the body condition score (BCS) were also measured. Results showed that the ejaculation volume increased while the sperm concentration decreased after exercise. However, other sperm parameters were unaffected. Reproductive hormones at resting period were rarely affected by exercise. The red blood cell number increased (P = .023), while the mean erythrocyte hemoglobin concentration decreased (P = .045) after the 8-week exercise in the 2 h EX group compared with the CN. After the 8-week exercise, the resting heart rate of the two exercise groups decreased significantly compared with the CN. The BCS in the CN group increased after the experiment (P = .024). In conclusion, we found that exercise had positive effects on some physiological indices but did not affect semen parameters expect for volume and concentration of the jackass., (Copyright © 2022 Elsevier Inc. All rights reserved.)

Sperm cryoinjuries caused by cryopreservation restrict the application of donkey frozen semen in artificial insemination (AI). Identification of differentially represented proteins in fresh and frozen-thawed spermatozoa is of great significance to optimize the cryopreservation process and modify the component of cryopreservation extender. In this study, protein samples prepared from fresh (F) and frozen-thawed (FT) donkey spermatozoa were compared. 2682 proteins were quantitatively identified by tandem mass spectrometry (TMT) polypeptide labeling technique and LC-MS/MS method, of which 28 were more abundant in thawed samples and 147 in fresh spermatozoa. The differential abundant proteins (DAPs) were analyzed by bioinformatics. Most of the DAPs in intensive bioinformatic analysis were involved in the process of regulation of biological process and metabolism. Functional protein analysis showed that DAPs process mainly protein hydrolase activity and oxidoreductase activity. Cellular Component analysis showed that DAPs were related to vesicle transport and membrane system. This is the first analysis and study on differential proteomics of donkey sperm proteins before and after cryopreservation, which has a certain guiding significance for studying the mechanism of sperm damage caused by cryopreservation and improving the freezing and thawing procedure. SIGNIFICANCE: In recent years, the commercial value of donkey products has been discovered. Improving the breeding efficiency of donkeys can save the stock of donkeys which is decreasing rapidly, and allow people to continuously benefit from the nutritional value brought by donkey milk. Sperm cryopreservation technology has laid the foundation for encouraging the spread of artificial insemination in donkey reproduction, but the freezing and thawing process causes damage to sperm, which dramatically reducing the viability of frozen sperm and leading to low fertility. At present, the mechanism of damage to donkey sperm caused by cryopreservation is still unclear, and studying this mechanism can provide a direction for improving the quality of frozen semen. Protein is a potential key factor affecting sperm cryopreservation activity. Studying changes in the sperm proteome during cryopreservation can provide promising evidence for revealing sperm cryopreservation damage, which is of great significance for optimizing the cryopreservation process, improving the composition of cryopreservation extender, and seeking directions for improving the quality of frozen semen., (Copyright © 2022 Elsevier B.V. All rights reserved.)

Kisspeptins (KPs) are the most potent stimulating neurotransmitters of GnRH release, and consequently KP administration triggers LH and/or FSH release. In small ruminants, KP or its analogs induced an LH surge followed by ovulation in both cyclic and acyclic animals, while in the mare KP only increased LH plasma levels but failed to induce ovulation. This study in jennies compares the endocrinological effects, ovulatory and pregnancy rates of the KP analog C6 and the GnRH analog buserelin acetate. The ovarian activity of nine Amiata jennies was monitored daily by transrectal ultrasound for three complete estrous cycles. Jennies in estrus were assigned, to one of three treatment groups: 50 nmol of the KP analog C6 (injected twice, 24 h apart, C6 group); 0.4 mg buserelin acetate (injected once, Bu group); and 2 mL of saline (injected once, CTRL group). Blood samples were collected at Day-1 (-24 h) Day0 (h0, before treatment), h2, h4, h6, h8, h10, h24 (before second treatment with C6), h26, h28, h30, h32, h34, h48 and every 24 h until ovulation. Jennies were inseminated once at h24 with fresh extended semen from a donkey stallion. Pregnancy diagnoses were performed 14 days after ovulation. On days 5, 10, and 14 after ovulation, for every CL the cross-sectional area (CSA) and the vascularized area (VA) were recorded by color doppler ultrasound and measured. Significantly higher plasma LH levels were found after induction between the Bu and CTRL groups at h6 and h8 (P < 0.05), while tendentially higher differences were found between the Bu/C6 groups and CTRL at h10. Five/9, 4/9, and 2/9 jennies ovulated between 24 and 48 h after induction from the Bu, C6, and CTRL groups respectively, (P > 0.05). Correlations between corpora lutea CSA and VA with serum progesterone concentration were r = 0.31, P = 0.01, r = 0.38, P = 0.01, respectively. Pregnancy rates after artificial insemination did not differ among groups (CTRL: 6/9, 66.7%; C6: 7/9, 77.8%; Bu: 6/9, 66.7%; P > 0.05). Ovulation rates after C6 treatment were comparable to that of Bu, although not different from the CTRL. Pregnancy rates were comparable to the literature in terms of fresh extended donkey semen in every group. This study suggests that stimulation of the Kp system in jennies, in contrast to findings observed in mares, induces ovulation. Further studies using higher doses and/or more animals are needed to better characterize the efficacy of C6 in jennies., (Copyright © 2022 Elsevier Inc. All rights reserved.)

Multiple hypotheses have been proposed for possible functions of zebra stripes. The most thoroughly experimentally supported advantage of zebra stripes is their visual unattractiveness to horseflies (tabanids) and tsetse flies. We propose here a plausible hypothesis why biting horseflies avoid host animals with striped pelages: in sunshine the temperature gradients of the skin above the slightly warmer blood vessels are difficult to distinguish from the temperature gradients induced by the hairs at the borderlines of warmer black and cooler white stripes. To test this hypothesis, we performed a field experiment with tabanids walking on a host-imitating grey test target with vessel-mimicking thin black stripes which were slightly warmer than their grey surroundings in sunshine, while under shady conditions both areas had practically the same temperature as demonstrated by thermography. We found that horseflies spend more time walking on thin black stripes than surrounding grey areas as expected by chance, but only when the substrate is sunlit. This is because the black stripes are warmer than the surrounding grey areas in the sun, but not in the shade. This is consistent with the flies' well-documented attraction to warmer temperatures and provides indirect support for the proposed hypothesis. The frequent false vessel locations at the numerous black-white borderlines, the subsequent painful bitings with unsuccessful blood-sucking attempts and the host's fly-repellent reactions enhance considerably the chance that horseflies cannot evade host responses and are swatted by them. To eliminate this risk, a good evolutionary strategy was the avoidance of striped (and spotted) host animals., (© 2022. The Author(s).)

The aim of the study was to evaluate oxidative stress and behavioural responses of donkeys during the cold-dry and hot-dry seasons. Thirty donkeys divided into three groups of ten donkeys each according to their age served as experimental subjects. During each season, behavioural and blood parameters of the donkeys were evaluated three times with an interval of one week at 06:00 h, 12:00 h and 18:00 h (GMT+1). During the hot-dry season, the peak dry-bulb temperature (DBT) value of 36.00 ± 1.00°C was recorded at 12:00 h, indicating that the season induced heat stress. The time spent feeding demonstrated diurnal fluctuations in adult, yearling and foal donkeys, with the highest time occurring at 12:00 h in both seasons. The malondialdehyde (MDA) concentrations in adults (2.34 ± 0.14 nMol/L), yearlings (2.28 ± 0.19 nMol/L) and foals (3.48 ± 0.17 nMol/L) during the hot-dry season were significantly ( P < 0.05) higher, when compared to the corresponding values (0.96 ± 0.09, 1.18 ± 0.10 and 1.82 ± 0.10 nMol/L, respectively) recorded during the cold-dry season. At 0.3% NaCl-concentration, the percentage haemolysis obtained during the hot-dry season in adult (90.33 ± 0.49%), yearling (91.24 ± 0.88%) and foal (97.44 ± 0.24%) donkeys were significantly ( P < 0.05) higher than the corresponding values (78.53 ± 0.98, 78.67 ± 0.58 and 88.23 ± 0.47%, respectively), recorded during the cold-dry season. In conclusion, the hot-dry season induced heat stress in the donkeys, resulting in a decrease in time spent on feeding and oxidative stress, as evidenced by high erythrocyte MDA concentration and percentage haemolysis.

Background: Discrimination and perception of emotion expression regulate interactions between conspecifics and can lead to emotional contagion (state matching between producer and receiver) or to more complex forms of empathy (e.g., sympathetic concern). Empathy processes are enhanced by familiarity and physical similarity between partners. Since heterospecifics can also be familiar with each other to some extent, discrimination/perception of emotions and, as a result, emotional contagion could also occur between species., Results: Here, we investigated if four species belonging to two ungulate Families, Equidae (domestic and Przewalski's horses) and Suidae (pigs and wild boars), can discriminate between vocalizations of opposite emotional valence (positive or negative), produced not only by conspecifics, but also closely related heterospecifics and humans. To this aim, we played back to individuals of these four species, which were all habituated to humans, vocalizations from a unique set of recordings for which the valence associated with vocal production was known. We found that domestic and Przewalski's horses, as well as pigs, but not wild boars, reacted more strongly when the first vocalization played was negative compared to positive, regardless of the species broadcasted., Conclusions: Domestic horses, Przewalski's horses and pigs thus seem to discriminate between positive and negative vocalizations produced not only by conspecifics, but also by heterospecifics, including humans. In addition, we found an absence of difference between the strength of reaction of the four species to the calls of conspecifics and closely related heterospecifics, which could be related to similarities in the general structure of their vocalization. Overall, our results suggest that phylogeny and domestication have played a role in cross-species discrimination/perception of emotions., (© 2022. The Author(s).)

Once believed uniquely human, the capacity to reason is now investigated in a wide range of species. One component of this ability, inference by exclusion, has been traditionally explored through the cups task, where 2 containers are presented but only 1 covers a food reward (if Cup A is empty, then choose Cup B). Often based on low-level cognitive mechanisms (learning), performance on this task can also reflect reasoning processes in some individuals. Limited taxonomic investigation, however, has hindered our understanding of the factors that contribute to the evolution of reasoning in animals. Using the cups task, we first investigated the ability of 14 semiwild donkeys to locate a food reward by using visual or acoustic cues alone (pretest phase). Although all subjects failed the task when provided with visual cues, 7 donkeys succeeded in the acoustic modality. We thus tested the capacity of donkeys to make choices by exclusion in the acoustic modality (test phase). Three conditions were presented, where subjects received information about either both containers (full information condition), solely the empty container (exclusion condition), or no information (control condition). Most subjects chose the correct container in the full information condition, whereas they chose randomly in the control condition. Except for 1 individual, donkeys improved their performance across trials in the exclusion condition. Our study contributes to the very small body of literature on exclusion based on acoustic cues in animals and paves the way to further experiments on the cognitive processes underlying exclusion performance in donkeys. (PsycInfo Database Record (c) 2022 APA, all rights reserved).

Rancho La Brea (California, USA) is the most emblematic Quaternary fossiliferous locality in the world, since both the high number and diversity of the specimens recovered and their excellent preservational quality. In the last decades, paleobiological and paleoecological knowledge of the different groups of mammals from this site has increased notably; however, some aspects have not yet been inquired or there is little information. In this work we provide information on one of the most abundant mammals of this site, the equid Equus occidentalis, based on the study, from osteohistological and histotaphonomic perspectives, of thin sections of different limb bones. On the one hand, from an osteohistological viewpoint, we observe that the distribution and characterization of bone tissues in the different skeletal elements are, in general lines, similar to that mentioned for other extant and extinct equids. Cyclical growth marks allowed us to propose preliminary skeletochronological interpretations. On the other hand, from a taphonomic viewpoint, we note that all the samples reflect an excellent preservation of the bone microstructure, slightly altered by different pre- and post-burial processes. The variations recorded evidence different taphonomic history and preservation conditions among pits. This is the first study including fossil material from Rancho La Brea exclusively based on the analysis of the bone microstructure features., Competing Interests: The authors have declared that no competing interests exist.

Effective conservation and management of threatened wildlife populations require an accurate assessment of age structure to estimate demographic trends and population viability. Epigenetic aging models are promising developments because they estimate individual age with high accuracy, accurately predict age in related species, and do not require invasive sampling or intensive long-term studies. Using blood and biopsy samples from known age plains zebras (Equus quagga), we model epigenetic aging using two approaches: the epigenetic clock (EC) and the epigenetic pacemaker (EPM). The plains zebra EC has the potential for broad application within the genus Equus given that five of the seven extant wild species of the genus are threatened. We test the EC's ability to predict age in sister taxa, including two endangered species and the more distantly related domestic horse, demonstrating high accuracy in all cases. By comparing chronological and estimated age in plains zebras, we investigate age acceleration as a proxy of health status. An interaction between chronological age and inbreeding is associated with age acceleration estimated by the EPM, suggesting a cumulative effect of inbreeding on biological aging throughout life., (© 2021. The Author(s).)

Pregnancy rates after embryo transfer (ET) are disappointing in donkey species. This study aims to report two successful ET of mini-donkey embryos using Brazilian Northeastern jennies as recipients. Eighteen embryo flushes were performed 9 days post-ovulation in two non-pregnant mini-donkeys jennies (11 and 7 cycles per jenny). Eleven embryos (61%, 11/18) were collected and transferred to Brazilian Northeastern jennies 4-6 days post-ovulation by conventional (n = 6) or an alternative (n = 5) technique. The alternative method consisted of inserting a Polansky equine vaginal speculum smeared with lubricant in the vagina of the recipient jenny. The arms of the speculum were extended to allow the visualization of the cervix. Then, using an adapted crafted, elongated, toothed tissue grasping forceps, the external cervical os was held, and the cervix was gently pulled backward, aiming to straight the cervical canal. The ET gun was inserted through the vagina and cervix by visual inspection, and the embryo was released into the uterine lumen. All embryos collected were Grade 1 and classified as Expanded Blastocysts. No jennies become pregnant after conventional ET (0/6), whereas two recipient jennies (40%, 2/5) become pregnant and delivered offspring in the following year after ET using the alternative technique. In conclusion, Brazilian Northeastern jennies can be used as embryo recipients using the alternative method proposed in the present study. However, further investigations are needed to improve the knowledge and results of ET in donkey species., (© 2021 Wiley-VCH GmbH.)

Artificial insemination (AI) with cryopreserved semen is an important tool to preserve endangered species, including European donkey breeds. Sperm vitrification is an alternative method to conventional freezing using high cooling rates and non-permeable cryoprotectant agents (CPAs). In donkeys, sperm vitrification was firstly developed in spheres by directly dropping the sperm (30 µl) into the liquid nitrogen. The vitrification media contained a combination of sucrose and bovine serum albumin as non-permeable CPAs and resulted in better sperm parameters after warming than extenders containing glycerol. Thereafter, sperm vitrification was optimized using an aseptic protocol, which consists of volumes up to 160 µl vitrified at 300 million sperm/ml using 0.25-ml straws with outer covers, obtaining similar sperm parameters as conventional freezing for total motility (52.7 ± 15.6% versus. 58.2 ± 16.1%), progressive motility (44.3 ± 15.0% versus. 44.7 ± 18.2%) and plasma membrane integrity (49.2 ± 11.2% versus. 55.4 ± 9.0%), respectively. In order to vitrify larger volumes of sperm, a procedure using 0.5-ml straws was evaluated; however, this methodology failed when compared to conventional freezing or other vitrification protocols, obtaining poor sperm quality after warming. Recently, a new methodology was developed for warming 0.25-ml straws in a water bath and after AI using the vitrified sperm, the uterine inflammatory response solved faster, and pregnancy rates were greater (22%) than frozen semen (10%) but not statistically different. In conclusion, all these findings confirm that sperm vitrification can be performed in donkeys as an alternative to conventional freezing for AI in jennies., (© 2021 Wiley-VCH GmbH.)

There is a long-lasting debate about the possible functions of zebra stripes. According to one hypothesis, periodical convective air eddies form over sunlit zebra stripes which cool the body. However, the formation of such eddies has not been experimentally studied. Using schlieren imaging in the laboratory, we found: downwelling air streams do not form above the white stripes of light-heated smooth or hairy striped surfaces. The influence of stripes on the air stream formation (facilitating upwelling streams and hindering horizontal stream drift) is negligible higher than 1-2 cm above the surface. In calm weather, upwelling air streams might form above sunlit zebra stripes, however they are blown off by the weakest wind, or even by the slowest movement of the zebra. These results forcefully contradict the thermoregulation hypothesis involving air eddies., (© 2021. The Author(s).)

Variation in donkey sperm freezability (capacity to withstand freeze-thawing) between ejaculates is a limitation for sperm cryopreservation. Seminal plasma proteins are essential for sperm function and also related to individual differences in sperm freezability. A Tandem Mass Tag (TMT) peptide labeling combine with a LC-MS/MS approach was conducted to quantitatively identify the seminal plasma proteins differentially abundant in ejaculates with optimal freezability characteristics (GFE) compared with those with suboptimal freezability characteristics (PFE). A total of 866 proteins were identified, and 99 ejaculates were in larger abundance in GFE samples. Differentially abundant proteins (DAPs) were subjected to intensive bioinformatic analysis. The majority of DAPs were involved in metabolic processes, oxidation-reduction processes and biological regulation. Results from functional protein analysis suggested that proteins functioned in oxidoreductase activity and acid phosphatase activity. This is the first report where there were analyses of the proteome of seminal plasma from donkey ejaculates with different freezability and to identify candidate proteins that could be used to explore the molecular mechanism related to donkey sperm cryotolerance. In this study, there also was elucidation of biomarkers for the early identification and selection of donkeys with optimal semen freezability., (Copyright © 2021 Elsevier B.V. All rights reserved.)

This article provides an overview of initial assessment and management of common emergency presentations in donkeys and mules. The principles are similar to those in horses (and ponies), but clinicians must be aware of differences in recognition of signs of pain/disease, approach to handling, pharmacology of some drugs, and subtle differences in the physiology and local anatomy in donkeys and mules. The epidemiology of common disease presentations will vary between pet/companion or working/farmed donkeys and mules. Regular dental checks, deworming, vaccination, and monitoring of behavior and quality of life are important aspects of preventive care., Competing Interests: Disclosure The authors have nothing to disclose., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Over the last several hundred years, donkeys have adapted to high-altitude conditions on the Tibetan Plateau. Interestingly, the kiang, a closely related equid species, also inhabits this region. Previous reports have demonstrated the importance of specific genes and adaptive introgression in divergent lineages for adaptation to hypoxic conditions on the Tibetan Plateau. Here, we assessed whether donkeys and kiangs adapted to the Tibetan Plateau via the same or different biological pathways and whether adaptive introgression has occurred. We assembled a de novo genome from a kiang individual and analyzed the genomes of five kiangs and 93 donkeys (including 24 from the Tibetan Plateau). Our analyses suggested the existence of a strong hard selective sweep at the EPAS1 locus in kiangs. In Tibetan donkeys, however, another gene, i.e., EGLN1 , was likely involved in their adaptation to high altitude. In addition, admixture analysis found no evidence for interspecific gene flow between kiangs and Tibetan donkeys. Our findings indicate that despite the short evolutionary time scale since the arrival of donkeys on the Tibetan Plateau, as well as the existence of a closely related species already adapted to hypoxia, Tibetan donkeys did not acquire adaptation via admixture but instead evolved adaptations via a different biological pathway.

Background: Whole milk is a good source of all the nutrients, and it also contains a sufficient number of vitamins to permit regular the growth of the neonate. Dairy cow milk can create allergy in infants less than 12 months old because of the high caseins and β-lactoglobulin content. In these circumstances, donkey milk can represent a good replacement for dairy cows' milk in children affected by Cow Milk Protein Allergy (CMPA) because of its close chemical composition with human milk, mainly due to its low protein and low mineral content. Milk vitamin content is highly variable among mammalian species and it is strictly correlated with the vitamin status and the diet administered to the mother. Fat-soluble vitamins content in donkey milk is, on average, lower compared to ruminants' milk, while vitamin C content determined in donkey milk is higher compared to dairy cows' milk, showing a great similarity with human milk. In donkey milk, the content of vitamins of the B-complex such as thiamine, riboflavin, niacin, pyridoxine, and folic acid is higher compared to human milk. The use of donkey milk as a new functional food must be further evaluated in interdisciplinary clinical trials in which pediatricians, dietitians, and food scientists must be involved to deepen the knowledge about the positive health impact of donkey milk in different sensitive people, especially children and the elderly.

With increasing livestock numbers, competition and avoidance are increasingly shaping resource availability for wild ungulates. Shifts in the dietary niche of wild ungulates are likely and can be expected to negatively affect their fitness. The Mongolian Gobi constitutes the largest remaining refuge for several threatened ungulates, but unprecedentedly high livestock numbers are sparking growing concerns over rangeland health and impacts on threatened ungulates like the Asiatic wild ass (khulan). Previous stable isotope analysis of khulan tail hair from the Dzungarian Gobi suggested that they graze in summer but switch to a poorer mixed C3 grass / C4 shrub diet in winter, most likely in reaction to local herders and their livestock. Here we attempt to validate these findings with a different methodology, DNA metabarcoding. Further, we extend the scope of the original study to the South Gobi Region, where we expect higher proportions of low-quality browse in the khulan winter diet due to a higher human and livestock presence. Barcoding confirmed the assumptions behind the seasonal diet change observed in the Dzungarian Gobi isotope data, and new isotope analysis revealed a strong seasonal pattern and higher C4 plant intake in the South Gobi Region, in line with our expectations. However, DNA barcoding revealed C4 domination of winter diet was due to C4 grasses (rather than shrubs) for the South Gobi Region. Slight climatic differences result in regional shifts in the occurrence of C3 and C4 grasses and shrubs, which do not allow for an isotopic separation along the grazer-browser continuum over the entire Gobi. Our findings do not allow us to confirm human impacts upon dietary preferences in khulan as we lack seasonal samples from the South Gobi Region. However, these data provide novel insight into khulan diet, raise new questions about plant availability versus preference, and provide a cautionary tale about indirect analysis methods if used in isolation or extrapolated to the landscape level. Good concordance between relative read abundance of C4 genera from barcoding and proportion of C4 plants from isotope analysis adds to a growing body of evidence that barcoding is a promising quantitative tool to understand resource partitioning in ungulates., Competing Interests: Sampling of khulan tail hair in the SGR happened within the framework of the Oyu Tolgoi’s Core Biodiversity Monitoring Program, implemented by the WCS through a cooperative agreement with Sustainability East Asia LLC. There are no patents, products in development or marketed products to declare. This does not alter our adherence to PLOS ONE policies on sharing data and materials.

Although donkeys have been domesticated for over 6,000 years, limited information is available concerning their reproductive physiology, especially under intensive rearing conditions. The aims of this experiment were to study follicular dynamics and reproductive hormone variation in jennies during the inter-ovulatory interval in different seasons. A total of 12 continuous cycles of six Dezhou Black (DB) donkey jennies were examined in four different seasons. The diameters of the six largest follicles of each jenny were measured daily by ultrasonography, and blood samples were collected at fixed times for reproductive hormone assays. The results demonstrated that most jennies displayed regular oestrous cycles in all seasons. The follicular dynamics were similar in Spring, Summer and Winter, while the jennies had longer oestrous cycles with delayed follicular deviation and dominant selection in Autumn. At least two follicular waves were observed in each oestrous cycle, throughout the study, but two jennies presented oestrous cycles with three follicular waves in the Autumn. The numbers of follicular waves were consistent with the numbers of FSH surges. Oestrous characteristics of the jennies in a large herd were also analysed. The results showed that the rates of regular oestrous cycles were 83.1% (265/319), 89.6% (215/240), 80.2% (235/293) and 77.1% (178/231), with 26.4% (70/265), 19.5% (42/215), 22.1% (52/235) and 23.0% (41/178) double ovulation rates in Spring, Summer, Autumn and Winter, respectively. The results presented may be useful for donkey farms in the design of breeding strategies., (© 2020 Wiley-VCH GmbH.)

Equid breeds originating from the Iberian Peninsula and North Africa are believed to have genetically contributed to the formation of breeds and ecotypes from Brazil. The country has numerous breeds and ecotypes of horses and donkeys but there are no extensive studies on maternal genetic diversity and their origins. This study reports the results of the first genetic analysis of all horse and donkey breeds/ecotypes from Brazil based on sequences of the mitochondrial DNA control region (D-loop) whose main objective was to characterize the genetic variation in these animals. These analyses will contribute to the understanding of the current population structure and diversity of breeds/ecotypes of horses and donkeys raised in the Brazil. We analyzed 310 D-loop sequences representing 41 breeds/ecotypes of Equus caballus and Equus asinus, including 14 native horse breeds/ecotypes, 3 native donkey breeds/ecotypes and 24 cosmopolite horse breeds. The results revealed that the breeds are well structured genetically and that they comprise different groups. A total of 80 and 14 haplotypes were identified for horses and donkeys, respectively. Most of the horse mtDNA haplotypes were shared by many breeds, whereas donkey mtDNA haplotypes seemed to be more group-especif. Some groups presented a low intrabreed distance and/or a low haplotype/nucleotide diversity such as Lavradeiro, Crioulo, Piquira and Percheron horses and Brazilian donkey. Thus, specific actions must be designed for each population. The different levels of genetic diversity provided important information for conservation resource management of adapted groups as well as for mating orientation of breed associations. Some autochthonous ecotypes require attention because of their low genetic variability., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Wild ungulates are an important part of terrestrial ecosystems and play a critical role in maintaining ecosystem health and integrity. In many grassland ecosystems that are habituated by wild ungulates, the coexistence of domestic ungulates has created a conflict over grazing resources. Solving this conflict requires a balanced and sustainable policy that satisfies both the needs of wildlife protection and food production. Here, we assess the optimal grassland livestock carrying capacity of an alpine grassland on the Qinghai-Tibet Plateau, given the coexistence of wild populations of kiangs (Equus kiang) and Tibetan gazelles (Procapra picticaudata), two key species grazing in this region. We use kriging and the MaxEnt method to estimate the population sizes of kiangs and Tibetan gazelles in Maduo County, Qinghai Province. We then convert the estimated population size of the two species into sheep units and calculate the residual carrying capacity for livestock grazing. We show that after accounting for the grazing need for kiangs and Tibetan gazelles, grassland in Maduo is capable of supporting 420,641 sheep units, which is slightly more than the current livestock population. However, the residual carrying capacity is highly uneven across the region, and overgrazing is found in many areas of Maduo, especially in northern Maduo. This research provides a useful framework for planning sustainable livestock farming for the Qinghai-Tibet Plateau and other regions facing wildlife-livestock conflict.

The purpose of this study was to analyse the effects of season, age, gonad and accessory sex glands on semen characteristics of jackass and to construct multivariate regression models to predict semen quality. In autumn, spring and summer, semen characteristics of 30 sexually mature donkeys (1,014 ejaculations) were analysed to investigate the effect of seasons on semen quality, and gonad and accessory sex gland parameters of 12 jackasses were measured immediately after ejaculation by ultrasonography to investigate the effect of seasons on reproductive organ size. Semen (598 ejaculates), gonad and accessory sex gland parameters of 40 jackasses aged between 3 and 7 years were analysed in autumn to investigate the effects of age and reproductive organ size on semen quality and to construct multivariate models. To verify the accuracy of the models, semen (476 ejaculates), gonad and accessory sex gland parameters of 20 jackasses were measured from March to June. Results revealed that semen, gonad and accessory sex gland parameters were not affected by season and age. Progressive motility (PM) was positively correlated with long axis of the spermatic cord (LASC) and negatively correlated with percentages of sperm abnormality (PSA). Total sperm count (TSC) was positively correlated with testis circumferences (TC) and cross-sectional area of cauda epididymis (CSACE). TC, CSACE, LASC and PSA were included into multivariate models to predict PM, TSC and functional sperm count (FSC) in 20 jackasses (PM = 72.332 + 0.428 LASC - 0.441 PSA; TSC = -169.929 + 8.728 TC + 0.253 CSACE; FSC = -206.645 + 8.788 TC + 0.258 CSACE). The predicted and observed values corresponded well. In conclusion, the tested models can be used for predicting semen quality of donkey., (© 2020 Wiley-VCH GmbH.)

Background: This study aims to determine the effects of transportation on the nasal microbiota of healthy donkeys using 16S rRNA sequencing., Results: Deep nasal swabs and blood were sampled from 14 donkeys before and after 21 hours' long-distance transportation. The values of the plasma hormone (cortisol (Cor), adrenocorticotrophic hormone (ACTH)), biochemical indicators (total protein (TP), albumin (ALB), creatinine (CREA), lactic dehydrogenase (LDH), aspartate transaminase (AST), creatine kinase (CK), blood urea (UREA), plasma glucose (GLU)) and blood routine indices (white blood cell (WBC), lymphocyte (LYM), neutrophil (NEU), red blood cell (RBC), hemoglobin (HGB)) were measured. 16S rRNA sequencing was used to assess the nasal microbiota, including alpha diversity, beta diversity, and phylogenetic structures. Results showed that levels of Cor, ACTH, and heat-shock protein 90 (HSP90) were significantly increased (p < 0.05) after long-distance transportation. Several biochemical indicators (AST, CK) and blood routine indices (Neu, RBC, and HGB) increased markedly (p < 0.05), but the LYM decreased significantly (p < 0.05). Nine families and eight genera had a mean relative abundance over 1%. The predominant phyla in nasal microbiota after and before transportation were Proteobacteria, Firmicutes, Actinobacteria, and Bacteroidetes. Transportation stress induced significant changes in terms of nasal microbiota structure compared with those before transportation based on principal coordinate analysis (PCoA) coupled with analysis of similarities (ANOSIM) (p < 0.05). Among these changes, a notably gain in Proteobacteria and loss in Firmicutes at the phylum level was observed., Conclusions: These results suggest transportation can cause stress to donkeys and change the richness and diversity of nasal microbiota. Further studies are required to understand the potential effect of these microbiota changes on the development of donkey respiratory diseases.

Tim Caro introduces why zebras are striped., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Donkey milk is an ideal substitute for human milk owing to its similar composition. Nevertheless, changes in the composition and related metabolic pathways of free fatty acids (FFA) in donkey milk between colostrum and mature milk have not been studied well. In this study, metabolomic methods based on gas chromatography tandem time-of-flight mass spectrometry (GC-TOF-MS) were used to explore and compare FFA in donkey colostrum (DC) and mature milk (DMM). A total of 24 FFA were characterized and quantified in DC and in DMM. Of these, 11 FFA differed significantly between DC and DMM, and there were 6 key differential metabolic pathways. These results demonstrated that the composition of FFA in donkey milk changed with lactation stage. The interactions and metabolic pathways were further analyzed to explore the mechanisms that altered the milk composition during lactation. Our results provide insights into the changes in milk of the nonruminant mammals during lactation. The results provide practical information for the development of donkey milk products and a foundation for future research on specific milk nutrients., (Copyright © 2020 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.)

Sertoli cells produce anti-Müllerian hormone (AMH) and number of these cells is associated with numbers of sperm produced. The study aim was to quantify AMH concentrations in serum and seminal plasma of donkeys during puberty, and to correlate the values with those for testicular width and semen quality of sexually mature males. Blood was collected from five donkeys every second month from 4 to 24 months of age, and then once at 40 months of age. Semen was collected once monthly, from 13 to 19 and 23-25 months of age. There was quantification of AMH concentrations in serum and seminal plasma. During puberty, there was a redirection of AMH secretion from the blood stream into seminal plasma. In serum, AMH concentrations increased during puberty with a maximal concentration at 16 months and the changes were similar for seminal plasma with a maximal concentration at 18 months of age. Serum AMH concentrations from 14-20 were greater than at 12 or 22 months of age. Maximal serum AMH concentrations were associated with testicular width at 24 months (r = 0.97, P = 0.005), but not with sperm count, sperm motility or percentage of sperm with normal morphology at 42 months of age. There were no significant correlations among values for AMH concentrations in seminal plasma during puberty and values for any of the seminal variables., Competing Interests: Declaration of Competing Interest The authors declare that they have no competing interests., (Copyright © 2020 Elsevier B.V. All rights reserved.)

This study sought to determine whether sperm irradiation using a light emission diode (LED) at 620-630 nm affects the motility, membrane integrity (viability), mitochondrial activity and intracellular levels of reactive oxygen species (ROS) in fresh diluted and liquid-stored donkey semen. With this purpose, sixteen ejaculates (eight fresh diluted and eight cooled-stored) were collected from eight adult jackasses. Fresh semen samples were diluted in Kenney extender and stimulated with red-light after collection, whereas cooled semen was stored at 4 °C for 24 h after dilution and then irradiated. In all cases, semen samples were packed into 0.5-mL transparent straws, which were then randomly divided into control and 19 treatments: six consisted of single red-light exposure, and the other 13 involved irradiation at light-dark-light intervals. Upon irradiation, sperm motility, membrane integrity mitochondrial membrane potential (MMP) and intracellular levels of superoxide anion (·O 2 - ) and hydrogen peroxide (H 2 O 2 ) were evaluated. While specific light-patterns increased both sperm motility and mitochondrial activity, they did not affect sperm membrane integrity and had no clear impact on intracellular ROS levels. The effects of irradiation patterns differed between fresh and cooled semen since, whereas 1 and 4 min patterns induced the greatest increments in the total and progressive motility of fresh semen, 4 min, 4-1-4 and 4-4-4 were the most suitable for cooled-stored samples. In both fresh diluted and cooled-stored semen, the motility increase observed after light-stimulation for 4 min was concomitant with changes in the percentages of spermatozoa with high mitochondrial membrane potential. In summary, this study shows, for the first time, that specific irradiation patterns increase sperm motility and mitochondrial activity in the donkey. Furthermore, the precise effect of red-light appears to depend on the specific functional status of cells, with separate effects on fresh and cooled samples., Competing Interests: Declaration of competing interest The authors declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of the research reported herein., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Assisted reproduction of endangered equids, such as Persian onagers (Equus hemionus onager), is vital for species conservation. Little is known about Persian onager reproductive functions, including functions of the uterine endometrium. Recently, successful cryopreservation of the domestic mare endometrium was reported, but there is no information on cryo-sensitivity or in vitro culture of endometrial tissues of any non-domestic equid. In the present study, endometrial explants from Persian onagers were cryopreserved and cultured in vitro for 5 days. There was no difference between endometrial explants when 10% and 20% dimethyl sulfoxide (DMSO) was used for cryopreservation. Cell viability and structural integrity were comparable to fresh tissue. Abundance of estrogen receptor-α (ESR1) and progesterone receptor (PGR) mRNA transcript in endometrial explants was less in most treatment groups compared to the fresh tissue control. There was variation in E-cadherin mRNA abundance in endometrial explants among treatment groups with some treatment groups having a lesser abundance compared to the control group. The abundance of Ki67 mRNA transcript of endometrial explants was not different among treatment groups compared to the control group. Results indicate that DMSO is a suitable cryoprotectant for the Persian onager endometrium, and in vitro culture in a liquid-gas interface can maintain Persian onager endometrial explants for as long as 5 days. Findings allow for a greater understanding of reproductive mechanisms in vitro for this endangered species and other domestic equids including 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., (Published by Elsevier B.V.)

Aim: To evaluate the sedative and clinical effects of I/V xylazine, detomidine, medetomidine and dexmedetomidine in miniature donkeys. Methods: Seven clinically healthy, male adult miniature donkeys with a mean age of 6 years and weight of 105 kg, were assigned to five I/V treatments in a randomised, cross-over design. They received either 1.1 mg/kg xylazine, 20 μg/kg detomidine, 10 μg/kg medetomidine, 5 μg/kg dexmedetomidine or saline, with a washout period of ≥7 days. The degree of sedation was scored using a 4-point scale by three observers, and heart rate (HR), respiration rate (RR), rectal temperature and capillary refill time (CRT) were recorded immediately before and 5, 10, 15, 30, 60, 90 and 120 minutes after drug administration. Results: All saline-treated donkeys showed no sedation at any time, whereas the donkeys treated with xylazine, detomidine, medetomidine and dexmedetomidine had mild or moderate sedation between 5 and 60 minutes after treatment, and no sedation after 90 minutes. All animals recovered from sedation without complication within 2 hours. The mean HR and RR of saline-treated donkeys did not change between 0 and 120 minutes after administration, but the mean HR and RR of donkeys treated with xylazine, detomidine, medetomidine and dexmedetomidine declined between 5 and 60 minutes after drug administration. The mean rectal temperature of all treated donkeys did not change between 0 and 120 minutes after administration. The CRT for all donkeys was ≤2 seconds at all times following each treatment. Conclusions and clinical relevance: Administration of xylazine at 1.1 mg/kg, detomidine at 20 μg/kg, medetomidine at 10 μg/kg and dexmedetomidine at 5 μg/kg resulted in similar sedation in miniature donkeys. Therefore any of the studied drugs could be used for sedation in healthy miniature donkeys.

The aim of this study was to compare the embryonic and early fetal development of horse embryos between recipient mules and mares from day 10-60 of pregnancy, in addition to hormonal (eCG and progesterone), ovarian, and uterine characteristics for approximately 4 months. Embryo donor mares (n = 5) and two groups of recipients (acyclic mules, n = 7; cyclic mares, n = 7) were used. Donor mares were monitored daily by transrectal ultrasonography and inseminated using fresh semen. Cyclic recipient mares were synchronized with the donor's ovulation using PGF2α and deslorelin acetate. Mules were prepared for the embryo transfers with estrogen and progestagen. Embryo collection and transfer were performed 8 days after ovulation of the donor mares. Pregnancy diagnosis with ultrasonography began 1 day after embryo transfer. After pregnancy confirmation, the recipient mules received long-acting progesterone once weekly for at least 120 days. The first day of detection (day 10) of an embryonic vesicle (EV) was similar between mules and mares. A period of extensive intrauterine mobility of the embryonic vesicle was observed similarly in mules and mares from days 10-17. The day of fixation of the EV in mules tended to be 1-day earlier than in mares; however, the diameter and growth rate of the EV did not differ between the two species. The embryo proper was first detected at day 20, and the crown-rump, width, and diameter were similar between the two recipient types. The heartbeat and allantoic sac tended to be detected 1 day later in mules than in mares, while the umbilical cord was first observed around day 40 in both species. Besides the expected differences found in ovarian aspects and eCG production, similar endometrial diameter, uterine tone and echotexture, and progesterone levels were seen between the two types of recipients. In conclusion, striking ultrasound similarities in equine embryo and fetal development, and uterine characteristics were seen between mules and mares used as recipients of horse embryos., Competing Interests: Declaration of competing interest The authors declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of this work., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Osteoarthritis (OA) is one of the most degenerative joint diseases in both human and veterinary medicine. The objective of the present study was the early diagnosis of OA in donkeys using a reliable grading of the disease based on clinical, chemical, and molecular alterations. OA was induced by intra-articular injection of 25 mg monoiodoacetate (MIA) as a single dose into the left radiocarpal joint of nine donkeys. Animals were clinically evaluated through the assessment of lameness score, radiographic, and ultrasonographic findings for seven months. Synovial fluid and cartilage samples were collected from both normal and diseased joints for the assessment of matrix metalloproteinases (MMPs) activity, COL2A1 protein expression level, and histopathological and immunohistochemical analysis of Caspase-3. Animals showed the highest lameness score post-induction after one week then decreased gradually with the progression of radiographical and ultrasonographic changes. MMP activity and COL2A1 and Caspase-3 expression increased, accompanied by articular cartilage degeneration and loss of proteoglycan. OA was successfully graded in Egyptian donkeys, with the promising use of COL2A1and Caspase-3 for prognosis. However, MMPs failed to discriminate between early and late grades of OA.