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Follicular fluid is formed from the transudation of theca and granulosa cells in the growing follicular antrum. Its main function is to provide an optimal intrafollicular microenvironment to modulate oocyte maturation. The aim of this study was to determine the metabolomic profile of preovulatory follicular fluid (PFF) in jennies. For this purpose, PFF was collected from 10 follicles of five jennies in heat. Then, PFF samples were analysed by nuclear magnetic resonance (NMR) and heteronuclear single quantum correlation (2D 1H/13C HSQC). Our study revealed the presence of at least 27 metabolites in the PFF of jennies (including common amino acids, carboxylic acids, amino acid derivatives, alcohols, saccharides, fatty acids, and lactams): 3-hydroxybutyrate, acetate, alanine, betaine, citrate, creatine, creatine phosphate, creatinine, ethanol, formate, glucose, glutamine, glycerol, glycine, hippurate, isoleucine, lactate, leucine, lysine, methanol, phenylalanine, proline, pyruvate, threonine, tyrosine, valine, and τ-methylhistidine. The metabolites found here have an important role in the oocyte development and maturation, since the PFF surrounds the follicle and provides it with the needed nutrients. Our results indicate a unique metabolic profile of the jennies PFF, as it differs from those previously observed in the PFF of the mare, a phylogenetically close species that is taken as a reference for establishing reproductive biotechnology techniques in donkeys. The metabolites found here also differ from those described in the TCM-199 medium enriched with fetal bovine serum (FBS), which is the most used medium for in vitro oocyte maturation in equids. These differences would suggest that the established conditions for in vitro maturation used so far may not be suitable for donkeys. By providing the metabolic composition of jenny PFF, this study could help understand the physiology of oocyte maturation as a first step to establish in vitro reproductive techniques in this species J.C. was funded by the project “Demetra” (Dipartamente di Eccellenza 2018–2022, CUP_C46C18000530001), Italian Ministry for Education, University and Research. I.M.-R. was awarded a scholarship from the Spanish Ministry of Science, Innovation and Universities (BES-2017- 081962). I⋅Y.-O. was funded by the Secretary of Higher Education, Science, Technology and Innovation (SENESCYT), Ecuador (Scheme: Programa de Becas Internacionales de Posgrado 2019; Grant: CZ02- 000507-2019). The authors also acknowledge the support from the Ministry of Science and Innovation, Spain (Grant: AGL2017-88329-R), the Catalan Agency for Management of University and Research Grants, Regional Government of Catalonia, Spain (Grant: 2017-SGR-1229); and the Catalan Institution for Research and Advanced Studies, Spain (ICREA)

Fatty acid based lipidomic analysis has been widely used to evaluate health status in human medicine as well as in the veterinary field. In equine species there has been a developing interest in fertility and sperm quality of the stallions. Fatty acids being the principal components of the membranes play an active role in the regulation of the metabolic activities and their role on the spermiogenesis seem to be of great importance for the resulting quality of the sperm and as a consequence of the fertility of the animal. With the application of widely used lipidomic techniques, this study was aimed to: a) evaluate for the first time the fatty acid content of spermatozoa’s membranes of 26 healthy male Martina Franca donkeys and its possible influence on the sperm parameters and b) the evaluation of the composition of the red blood cells’ membrane. For both the spermatozoa and the erythrocytes, the PUFA omega-6 are the principal components of the Total PUFA content (40.38% and 47.79% respectively), however on the spermatozoa’s membranes DPA is the predominant one (27.57%, ) while in the erythrocyte’s membranes DPA is not present and linoleic is the one found in higher concentrations (46.28%). For the spermatozoa, stearic acid levels as well as EPA have demonstrated a positive correlation with motility (%). Progressive motility (%) is positively associated with oleic acid. the data obtained offer information on the structure of the sperm cells as well as the erythrocytes of healthy male MF donkeys and are reflecting the metabolic transformations of the spermatozoa’s membrane during the maturation period. These results provide a deeper understanding of the distribution of the fatty acid content on the spermatozoa’s membrane and their role in sperm parameters that are highly associated with fertility. &nbsp

The genotoxic and nephrotoxic mycotoxin Ochratoxin A (OTA) has also been reported to have adverse effects on oocyte maturation and embryo development. Previous studies on the effects of OTA on female fertility have used micromolar concentrations, but no information is available to date on effects in a more relevant nanomolar range. This study used a juvenile sheep model to evaluate the effects of oocyte exposure to low levels of OTA on maturation, fertilization, and embryo development. Further, it was investigated whether different mechanisms of action of OTA could be responsible for varying toxic effects at different levels of exposure. Cumulus-oocyte-complexes (COCs) were exposed to 10 μmol/L–0.1 nmol/L OTA during in vitro maturation and evaluated for cumulus viability, oocyte maturation, and bioenergetic/oxidative status. COCs were subjected to in vitro fertilization, embryo culture, and embryo quality assessment via morphology, viability, bioenergetic/oxidative status, and time-lapse monitoring. At micromolar concentrations, OTA induced cytotoxic effects, by reducing cumulus expansion and oocyte maturation. OTA altered temporospatial dynamics of zygote pronuclear formation and embryo morphokinetics. Blastocysts, even morphologically normal, were found to undergo collapse events, which were probably related to boosted blastocyst mitochondrial activity. At nanomolar concentrations, OTA did not affect COC morpho-functional parameters, but impaired oocyte ability to prevent polyspermy and increased blastocyst apoptosis. In conclusion, in the female germ cell, cytotoxic nonspecific effects characterize OTA-induced toxicity at high exposure levels, whereas fine tuning-mode effects, not associated with altered cell viability and integrity, characterize OTA toxic action at low levels. Electronic supplementary material The online version of this article (10.1007/s12550-020-00410-y) contains supplementary material, which is available to authorized users.

The reagent urinary test strips (TS) marketed for urines represent the first-line diagnostic tool in many instances. Therefore, the aim of the study was to assess the usefulness of TS for the on-field evaluation of fetal fluids’ composition in newborn dogs at elective caesarean section. Of a total of 137 puppies born at term, 127 survived and 10 did not survive. One hundred and thirteen amniotic and 107 allantoic samples from surviving newborns were collected, and 8 amniotic and allantoic fluids were collected from the non-surviving newborns and assessed by strips. Significantly lower amounts of amniotic glucose and higher amounts of amniotic and allantoic nitrites, amniotic protein, allantoic urobilinogen, and amniotic bilirubin concentrations were found in non-surviving when compared to surviving newborns. In the surviving ones, higher specific gravity and bilirubin concentrations, and lower pH, were found in allantoic than in amniotic fluids. Higher amniotic and allantoic glucose concentrations, higher amniotic and allantoic pH, and lower amniotic and allantoic protein concentrations were found in medium/large- than in small-sized puppies. The TS allowed the quick evaluation of fetal fluids in puppies at birth. The differences between surviving and non-surviving puppies seem to suggest that the on-site analysis of fetal fluids’ composition by TS could represent a first-line diagnostic tool in the field of canine neonatology, allowing the quick recognition of puppies needing assistance as a complementary tool for clinical evaluation.

Seminal plasma (SP) of donkey stallions was evaluated using various oxidative stress parameters as well as protease and protease inhibitor activities. SP was obtained by nine donkey stallions. In addition, one donkey stallion with non-obstructive azoospermia was enrolled in this study. Free radical scavenging activity (FRSA), the ferric reducing ability of plasma (FRAP), total antioxidant capacity (TAC), and total thiol level (TTL) were highly correlated with each other and with the protease inhibitor activity. However, only FRAP, TAC, and the nitrate/nitrite concentration (NOx) were significantly correlated with sperm concentration, production, and kinetics. Protease inhibitor activity was highly correlated with sperm concentration and production; however, it did not correlate with sperm kinetics. The azoospermic stallion produced a lower amount of semen than the normospermic stallions and its SP showed a lower antioxidant activity when evaluated with FRAP, TAC, and TTL as well as a higher NOx and a lower protease inhibitor activity. In conclusion, the evaluation of SP oxidative profile by FRAP, TAC, and NOx may provide reliable information on donkey sperm quality whereas protease inhibitor activity may play a role as a marker of the sperm concentration in this species.

Simple Summary Mammary gland tumours are the most common canine neoplasms. They account for 25–50% of all tumors diagnosed in bitches. Metastases and recurrences develop in about 35–70% of bitches following excision. The presence of regional lymph node metastases is a relevant factor affecting prognosis and treatment in cases of mammary gland tumors. The sentinel lymph node (SLN) is the first lymph node (or nodes) in the regional lymphatic basin that receives lymphatic flow from the primary neoplasm. As mammary gland tumors mainly spread by lymphatic vessels invasion, conceptually, distant metastasis should not be present if the SLN does not have evidence of a tumor burden. In the present study, an indirect lymphography was used for sentinel lymph node mapping in a cohort of 14 adult female dogs with 24 mammary tumors involving the third to the fifth mammary gland. Contrast was injected around the mammary tumor, and lymph nodes that picked up the contrast were visible after 24 h. The results of this study indicate that the lymph drainage pattern of the neoplastic mammary gland may be different for each tumor. The knowledge of the SLN of the neoplastic mammary glands should be of high importance for the surgeon not only for performing the most adequate surgical excision but also for determining an accurate post-surgical prognosis. Abstract Mammary gland tumors are the most common canine neoplasms. They account for 25–50% of all tumors diagnosed in bitches. Metastases and recurrences develop in about 35–70% of bitches following excision. The presence of regional lymph node metastases is a relevant factor affecting prognosis and treatment in cases of mammary gland tumors. The sentinel lymph node (SLN) is the first lymph node (or nodes) in the regional lymphatic basin that receives lymphatic flow from the primary neoplasm. The aim of this study is to investigate the SLN with indirect lymphography for a mammary tumor in dogs. The knowledge of the precise drainage pattern and SLN of the neoplastic mammary glands would provide clinically relevant information to the surgeon and to the oncologist, and it would be of high importance for the surgeon not only for performing the most adequate surgical excision but also for determining an accurate post-surgical prognosis.

Juvenile in vitro embryo technology (JIVET) provides exciting opportunities in animal reproduction by reducing the generation intervals. Prepubertal oocytes are also relevant models for studies on oncofertility. However, current JIVET efficiency is still unpredictable, and further improvements are needed in order for it to be used on a large-scale level. This study applied bioengineering approaches to recreate: (1) the three-dimensional (3D) structure of the cumulus–oocyte complex (COC), by constructing—via bioprinting technologies—alginate-based microbeads (COC-microbeads) for 3D in vitro maturation (3D-IVM), (2) dynamic IVM conditions, by culturing the COC in a millifluidic bioreactor, and (3) an artificial follicular wall with basal membrane, by adding granulosa cells (GCs) and type I collagen (CI) during bioprinting. The results show that oocyte nuclear and cytoplasmic maturation, as well as blastocyst quality, were improved after 3D-IVM compared to 2D controls. The dynamic 3D-IVM did not enhance oocyte maturation, but it improved oocyte bioenergetics compared with static 3D-IVM. The computational model showed higher oxygen levels in the bioreactor with respect to the static well. Microbead enrichment with GCs and CI improved oocyte maturation and bioenergetics. In conclusion, this study demonstrated that bioengineering approaches that mimic the physiological follicle structure could be valuable tools to improve IVM and JIVET.

In a temperature-increasing scenario, due to global warming, the individual thermic resilience of the male assumes a crucial role in the reproductive efficiency of a male since the thermic stress, such as the inability of the male to reduce body or regional temperature on a physiological level, impairs testicular function. In this study, the effect of the environmental conditions on the fresh semen quality, in terms of volume, concentration, total sperm in the ejaculate, total motility, normal morphology, membrane integrity, and discarding rate, were compared longitudinally in Belgian Blue (BB) and Brown Swiss (BS) bulls. The environmental conditions, summarized in the mean temperature-humidity index (THI), were calculated on the day of collection, as well as 7 days (epididymal maturation), 35 days (late spermatogenesis), and 70 days (early spermatogenesis) before the collection, to reflect spermatogenesis time. Our findings showed that limited seasonal effects were present in the semen quality of BS bulls. On the other hand, in BB bulls lower semen quality was found between July and November, with a different timing depending on the seminal parameter. This effect of the season on BB semen parameters appears to be related to the THI. The data presented in this study shows that the temperature and humidity, summarized in THI, could affect the semen quality of the bull on breed basis, given that volume, concentration, total sperm in the ejaculate, total motility, membrane integrity, and sperm normal morphology were significantly reduced by an increasing THI in the Belgian Blue bulls, but not in Brown Swiss bulls.

During capacitation, sperm undergo a myriad of changes, including remodeling of plasma membrane, modification of sperm motility and kinematic parameters, membrane hyperpolarization, increase in intracellular calcium levels, and tyrosine phosphorylation of certain sperm proteins. While potassium channels have been reported to be crucial for capacitation of mouse and human sperm, their role in pigs has not been investigated. With this purpose, sperm samples from 15 boars were incubated in capacitation medium for 300 min with quinine, a general blocker of potassium channels (including voltage-gated potassium channels, calcium-activated potassium channels, and tandem pore domain potassium channels), and paxilline (PAX), a specific inhibitor of calcium-activated potassium channels. In all samples, acrosome exocytosis was induced after 240 min of incubation with progesterone. Plasma membrane and acrosome integrity, membrane lipid disorder, intracellular calcium levels, mitochondrial membrane potential, and total and progressive sperm motility were evaluated after 0, 120, and 240 min of incubation, and after 5, 30, and 60 min of progesterone addition. Although blocking potassium channels with quinine and PAX prevented sperm to elicit in vitro capacitation by impairing motility and mitochondrial function, as well as reducing intracellular calcium levels, the extent of that inhibition was larger with quinine than with PAX. Therefore, while our data support that calcium-activated potassium channels are essential for sperm capacitation in pigs, they also suggest that other potassium channels, such as the voltage-gated, tandem pore domain, and mitochondrial ATP-regulated ones, are involved in that process. Thus, further research is needed to elucidate the specific functions of these channels and the mechanisms underlying its regulation during sperm capacitation This research was supported by the Ministry of Science and Innovation, Spain (Grants: RYC‐2014‐15581 and AGL2017‐88329‐R), Regional Government of Catalonia, Spain (Grant: 2017‐SGR‐1229) and the University of Teramo, Italy (Assegno di Ricerca; Rector’s Decree 251 (2018),CUN07‐VET/10)

Simple Summary Diagnosis of prostatic neoplasia in dogs remains critical since none of the diagnostic methods routinely performed seem to have sufficient sensitivity or specificity. In recent years, contrast-enhanced ultrasound of the prostate gland has been successfully performed in intact dogs. However, data regarding the performance of the technique in castrated dogs have not been provided, despite the incidence of prostate neoplasia potentially being higher after castration. The purpose of the present study was to evaluate the application of contrast-enhanced ultrasound in castrated dogs and to describe the vascular pattern of the canine prostate. Prior to contrast-enhanced ultrasound evaluation, B-mode ultrasound was performed to assess prostate size and parenchymal features. In all cases, perfusion kinetics of the contrast agent within the prostate was analyzed, with the aim of detecting any areas with increased microvessel density. None of the dogs had any ultrasonographically detected pathology. Contrast ultrasound allowed an improved and enhanced ability to evaluate the prostate compared with B-mode ultrasound. This technique is not yet to become a first-choice diagnostic tool; however, since the results of this study are favorable, it seems likely that contrast ultrasound will have an important role in the future for the detection of prostate pathologies in neutered dogs. Abstract Prostatic neoplasia (PN) occurs in 5–7% of dogs with prostatic disease, with castrated dogs having the same or higher prevalence when compared to intact dogs. Considering the promising results achieved by performing contrast-enhanced ultrasound (CEUS) in intact dogs to detect PN, the present study aimed to acquire data on the prostatic perfusion pattern in neutered dogs. CEUS was performed in 64 neutered dogs, using a 5–7.5 MHz linear transducer with coded harmonic capability, dedicated analytical software, and a second-generation contrast agent, SonoVue. After B-mode evaluation was performed to assess mean prostate volume, the CEUS examination was undertaken. The flow of contrast agent was visible 10 s after injection. The subcapsular vessels were highlighted and produced rapid peripheral rim enhancement. Subsequently, the contrast agent reached the prostatic urethra via the parenchymal arterioles and gradually reached the entire prostate. Perfusion peak intensity (PPI) and time to peak (TTP) values were respectively 45.3% and 34.1 s. The measured parameters were compared with those obtained in previous studies on intact dogs with normal and with pathological patterns. In this study, CEUS showed features that may be promising for its use as a diagnostic tool for early detection of PN in neutered dogs.

Sperm cryopreservation represents a powerful tool for horse breeding. To improve the efficiency of artificial insemination in the horse using cryopreserved spermatozoa, an adequate understanding of the underlying biophysical properties that affect sperm cryosurvival needs to be reached yet. In this pilot study, we described isolation and analysis of the main fatty acids from sperms of stallions classified as good and poor freezers (7 GF and 5 PF, according to sperm motility and viability, before and after cryopreservation). Fatty acid profiles were only assessed in pre-thaw sperms. Eight main fatty acids were identified, using gas chromatography, and their contents were expressed as percentage of the total lipid content. We found that lauric, myristic and oleic acid (C12:0, C14:0 and C18:1n9c) turned out to be about 2-fold more abundant in the sperm cells of the GFs compared with PFs. Moreover, we described for the first time the presence of a very high amount of a trans geometrical isomer of linoleic acid, linolelaidic acid (C18:2n6t), in pre-thaw PF spermatozoa. Notably, we found in fresh sperms of PF stallions a ratio of unsaturated fatty acids to saturated fatty acids which was twice that of those of GF group, suggesting a positive effect of a high saturated-to-unsaturated fatty acid ratio for the “freezability” of equine spermatozoa. Finally, principal component analysis (PCA) confirmed the relationships between specific fatty acids and cryotolerance of equine spermatozoa, also providing a graphical classification and additional information about the dominant variables governing the classification process.

In donkeys, the use of frozen-thawed sperm for artificial insemination (AI) leads to low fertility rates. Furthermore, donkey sperm produce a large amount of reactive oxygen species (ROS), and post-AI inflammation induces the formation of neutrophil extracellular traps (NETosis), which further generates many more ROS. These high ROS levels may induce lipid peroxidation in the sperm plasma membrane, thus affecting its integrity. Enzymatic and non-enzymatic antioxidants, mainly found in the seminal plasma (SP), are responsible for maintaining the redox balance. However, this fluid is removed prior to cryopreservation, thereby exposing sperm cells to further oxidative stress. The exogenous addition of antioxidants to the freezing medium can reduce the detrimental effects caused by ROS generation. Therefore, the aim of this study was to evaluate how the addition of different reduced glutathione (GSH) concentrations (control, 2 mM, 4 mM, 6 mM, 8 mM, and 10 mM) to fresh sperm affect their cryotolerance. Total and progressive motility, kinematic parameters and motile sperm subpopulations were significantly (p < 0.05) different from the control in treatments containing 8 mM and 10 mM GSH, but not at lower concentrations. Plasma and acrosome membrane integrity, mitochondrial membrane potential (MMP) and intracellular superoxide levels (O2−) were not affected (p > 0.05) by any GSH concentration. Interestingly, however, the addition of 8 mM or 10 mM GSH reduced (p < 0.05) the percentages of viable sperm with high overall ROS levels compared to the control. In conclusion, frozen-thawed donkey sperm are able to tolerate high GSH concentrations, which differs from what has been observed in other species. This antioxidant capacity suggests that ROS could be important during post-AI and that the impact of using exogenous antioxidants like GSH to improve the sperm resilience to freeze-thawing is limited in this species I.Y.-O. was funded by the Secretary of Higher Education, Science, Technology and Innovation (SENESCYT), Ecuador (Scheme: Programa de Becas Internacionales de Posgrado 2019; Grant: CZ02-000507-2019). J.C. was funded by the “Demetra” project of the Department of Excellence Postdoctoral fellowship, University of Teramo, Italy (Scheme: Assegno di Recerca 2021). The authors also acknowledge the support from the Ministry of Science and Innovation, Spain (Grant: AGL2017-88329-R) and the Regional Government of Catalonia (2017-SGR-1229

Pain management is necessary for all surgical procedures. Little scientific evidence about drug efficacy in donkeys is available. The aim of this study was to evaluate the analgesic effect of butorphanol in donkeys undergoing orchiectomy under total intravenous anaesthesia with guaifenesin-ketamine-detomidine. A randomized blinded prospective clinical trial (Protocol n. 2021/0000338), was carried out on 18 clinically healthy donkeys undergoing bilateral orchiectomy. Patients were assigned to Group D (n = 8) or Group DB (n = 10) if receiving intravenous detomidine or detomidine-butorphanol respectively, before induction of general anaesthesia with ketamine-diazepam. Intraoperative muscle relaxation, nystagmus, palpebral reflex, heart and respiratory rate, and non-invasive blood pressure were evaluated every 2 min, time to prepare the patient, duration of surgery and anaesthesia and recovery score were recorded. Group D had significantly longer surgical time, higher heart rate, higher systolic and mean blood pressure (p &lt, 0.05, repeated measure ANOVA), increased muscle rigidity and expression of palpebral reflex (p &lt, Mann–Whitney U test) than group DB. Top-ups with thiopental were statistically higher in Group D. Butorphanol and detomidine together produced a more stable anaesthetic plan. The low dosage of opioid and alpha-2-agonists and reduced rescue anaesthesia are responsible for a safer and more superficial anaesthesia, which is mandatory under field conditions.

The mitochondrial DNA (mtDNA) D-loop of endangered and critically endangered breeds has been studied to identify maternal lineages, characterize genetic inheritance, reconstruct phylogenetic relations among breeds, and develop biodiversity conservation and breeding programs. The aim of the study was to determine the variability remaining and the phylogenetic relationship of Martina Franca (MF, with total population of 160 females and 36 males), Ragusano (RG, 344 females and 30 males), Pantesco (PT, 47 females and 15 males), and Catalonian (CT) donkeys by collecting genetic data from maternal lineages. Genetic material was collected from saliva, and a 350 bp fragment of D-loop mtDNA was amplified and sequenced. Sequences were aligned and evaluated using standard bioinformatics software. A total of 56 haplotypes including 33 polymorphic sites were found in 77 samples (27 MF, 22 RG, 8 PT, 19 CT, 1 crossbred). The breed nucleotide diversity value (π) for all the breeds was 0.128 (MF: 0.162, RG: 0.132, PT: 0.025, CT: 0.038). Principal components analysis grouped most of the haplogroups into two different clusters, I (including all haplotypes from PT and CT, together with haplotypes from MF and RG) and II (including haplotypes from MF and RG only). In conclusion, we found that the primeval haplotypes, haplogroup variability, and a large number of maternal lineages were preserved in MF and RG, thus, these breeds play putative pivotal roles in the phyletic relationships of donkey breeds. Maternal inheritance is indispensable genetic information required to evaluate inheritance, variability, and breeding programs.

The neonatal response to stress was reported to be related to the type of delivery, that is elective or emergency cesarean sections (ELCS and EMCS, respectively). Nitric oxide (NO) is also reported to be related to uterine inertia, and high levels of NO metabolites (NOs) are associated with physical and emotional stress. The study aimed to assess the concentrations of cortisol (C) and NOs in the amniotic fluid of puppies delivered by ELCS or EMCS. In total, 32 puppies were delivered by ELCS and 22 by EMCS. ANCOVA showed an effect of the ELCS vs. EMCS on both amniotic NOs (p &lt, 0.001) and C (p &lt, 0.001) concentrations. Lower amniotic C concentrations were found at increasing Apgar score (p &lt, 0.001). Higher amniotic NOs concentrations were associated to increasing mother’s parity (p &lt, 0.001), puppies’ birthweight (p &lt, 0.001), and time of labor within the EMCS group (p &lt, 0.05). A positive correlation between birthweight and amniotic NOs concentrations was also found (p &lt, 0.05) in the EMCS group. Due to the possible concurrence of several compartments (maternal, maybe placental, and fetal) to the final amniotic fluid composition, the definition of the role played by the three compartments in the higher C and NOs concentrations found in amniotic fluids collected from puppies delivered by EMCS than ELCS needs further clarification.

In humans and animals there is a popular belief that spontaneous parturition, and therefore pregnancy length, could be influenced by the lunar cycle, but results from humans are often impaired by multiple variables of the studied population. In domestic animals those problems can be limited by studying restricted population, such as a single breed, reared under uniform conditions. The present study aimed to assess the possible influence of the lunar cycle on pregnancy length in Martina Franca jennies belonging to a single breeding farm. The retrospective study was performed on data collected from 96 Martina Franca healthy jennies with a normal singleton pregnancy course. The present study showed that, whit lunar cycle divided in 4 lunar phases, neither lunar phase at ovulation, nor lunar phase at foaling significantly influenced pregnancy length (371.8±6.45 days). Pregnancy length was not significantly influenced also by the month of the year in which ovulation and spontaneous foaling occurred, as well as by jennies’ age and foal’s sex. In conclusion, pregnancy length in Martina Franca jennies is very variable (335-395 days) but does not seem to be influenced by lunar phases at ovulation or at foaling, month of ovulation and foaling, and by jennies’ age and foal’s sex. Further studies are needed to clarify the reasons for this pregnancy length variability in Martina Franca donkey breed.

This study aimed to evaluate donkey seminal plasma metabolites and relate this information to the main characteristics of sperm quality. Sperm kinetics from 10 donkey stallions were analyzed with a computerized system at the time of collection (T0) and after 24 h storage at 4 &deg, C (T24). Seminal plasma was frozen at &minus, 80 &deg, C for subsequent proton nuclear magnetic resonance (1H NMR) spectroscopy. On three stallions, semen collection was repeated monthly for three times and sperm analysis also included mitochondrial activity and oxidative status. One stallion was azoospermic and a second semen collection was performed after one month. In the seminal plasma, 17 metabolites were identified, their levels showed numerous significant variations between the azoospermic and the normospermic individuals and grouped in well-defined clusters in a multivariate analysis. Comparing individuals with high and low sperm motility, the only discriminating metabolite was phenylalanine, whose levels were lower in the latter, as in the azoospermic individual. Phenylalanine was also the only metabolite highly correlated with all sperm kinematic parameters at T24. In conclusion, the present study has provided relevant information on the chemical characteristics of donkey semen, identified relationships between seminal metabolites, semen parameters, and sperm kinetics, and offered insights for future technological applications.

The Martina Franca donkey (MFd) is one of the largest Italian donkey breeds, considered as endangered breed. To support the conservation strategies, knowledge about the physiologic hematological parameters of MFds is needed. The aims of the study were to determine reference value for hematological and major serum parameters in a population of healthy MFds and to estimate the influence of age on these parameters. Eighty-one clinically healthy MFds (17 males and 64 females) in different ages were enrolled: group A (foals, n° 16, animals < 1 year old) group B (young animals, n° 36, from 1 to 3 years old), and group C (adult animals, n° 29, over 3 years old). Red blood cell count (RBC); hematocrit value (HCT); hemoglobin concentration (HGB); mean corpuscular volume (MCV); mean corpuscular hemoglobin (MCH); hemoglobin concentration distribution width (HDW); RBC distribution width (RDW); total white blood cell (WBC); WBC differential count for neutrophils, lymphocytes, monocytes, eosinophils and basophils, and platelets (PLT); mean platelet volume (MPV); platelet volume distribution width; and plateletcrit (PCT) were analyzed. The biochemistry panel included aspartate aminotransferase (AST), alanine aminotransferase (ALT), alkaline phosphatase (ALP), gamma glutamyl transferase (GGT), total serum protein (TP), albumin (ALB), cholesterol (CHOL), triglyceride (TGL), blood urea nitrogen (BUN), creatinine (CREA), glucose (GLU), Ca2+, phosphorus, Na+, Cl−, K+, and Mg2+. The effect of age on hematological parameters was investigated using one-way ANOVA test. Age of donkeys does not influence total WBC, HGB, HCT, platelet count and MPV, and PCT (P> 0.05). Some leukocyte populations such as eosinophils, monocytes, and basophils showed age-linked variations (P< 0.05). RBC count, RDW, and HDW decrease with age whereas MCV and MCH increase. Na+, K+, Cl−, Ca2+, phosphorus, ALP, GGT, CREA, GLUC, and CHOL decrease with age (P< 0.05), while AST and TP showed an increase with aging (P< 0.05). ALB reaches the lowest values in young donkeys and returns to values of foals in older animals (P< 0.05). Finally, a difference among groups for BUN and TGL was not found (P< 0.05). The results suggest how even for the MFd breed, age is a variable that affects different hematological and biochemical parameters. Compared to other donkey and horses, the MFd breed showed some differences that clinicians involved during conservation strategies need to be consider.

Eight Martina Franca pregnant jennies were selected in order to evaluate the transfer of colostral antibodies against equine herpesvirus type 1 in their relative foals after immunization with a commercial inactivated vaccine, compared with an unvaccinated group. Samples of serum and colostrums/milk were collected from jennies and foals under study starting from 10 min before and up to 21 days after the foaling. Specific anti-EHV-1 antibody titers were evaluated by means of a serum neutralization test, and the results obtained from both groups were analyzed. The serological titers in the vaccinated jennies was significantly higher (p < 0.01). No significant differences were found in the specific time-point intervals in both groups examined (p > 0.05). The antibody titers in milk at the time of delivery and subsequent withdrawal (T0 and T1) were very high in both groups, but no significant differences were found between the two groups (p > 0.05). In the foal sera, a significant difference was found between foals in the vaccinated group compared with those in the unvaccinated group (p < 0.05). Finally, a significant correlation (p < 0.05) was observed between the antibody titers found in serum and colostrum of jennies and the foal titers in the first time-point sampling (up to 12 h after foaling). The results confirm a substantial homology in the antibody production compared with other most investigated equids, highlighting the efficacy of the vaccination against EHV-1 of the jennies to ensure the protective immunity to their foals during the first weeks after delivery.

Maternal inheritance is an indispensable aspect in donkey rare breed population biodiversity management and breeding programs. It is a challenge to characterize breeds genetic inheritance using morphology and historical records, we study mtDNA, to overcome those limitations. The mitochondrial DNA (mtDNA) sequencing is a highly informative system to investigate maternal lineages and breed linkage such as molecular evolution and phylogenetic relationships. Martina Franca, Ragusano, Pantesco and Catalonian donkey mtDNA sequencing analyses were used to study intraspecific genetic diversity and population structure, and to reconstruct phylogenetic relations among these geographically isolated breeds.A wide lost in variability among all breeds emerged. In this scenario, the primeval haplotypes, higher haplogroups variability and larger number of maternal lineages are preserved in Martina Franca and Ragusano. Accordingly, a putative pivotal role in the phyletic relationship is likely for such breeds.Given the level of endangerment undergone by these breeds, some actions are necessary to ensure their longtime survival and conservation. Improving the reproduction and management of existing populations, clarifying their historic interactions by studying the genetic status of their populations, extending and improving monitoring maternal lineages represent valid options.

Egg yolk (EY) is conventionally used to reduce sperm cryodamage, however, there has not be evaluation of whether there is a dose-dependent effect with inclusion of EY in semen extender. To enhance the knowledge about the protective effect of EY during cryopreservation of dog semen, a specific study was designed to evaluate the dose-dependent protection of the EY against osmotic and cryogenic damage of dog sperm. In the first experiment, sperm stored in an extender that contained graded EY concentrations (0 %, 5 %, 10 %, and 20 %) were diluted with hypo- or hyper-osmotic solutions (final osmolality of 75, 150, 300, 500, 1000 mOsm/kg). Results from sperm kinetic, membrane integrity (MI), mitochondrial activity, and normal morphology evaluations indicated osmotic stress has especially marked effects on the kinetic capacity of spermatozoa, however, there were no direct effects on mitochondrial activity. In both hypo- and hyper-osmotic conditions, EY had a protective effect regardless of concentration. In the second experiment, semen samples were diluted in extenders at increasing EY concentrations (0 %, 5 %, 10 %, and 20 %) and cryopreserved. Effects on sperm kinetics, membrane and acrosome integrity and mitochondrial membrane potential indicated there was improved sperm viability after thawing when the EY concentration was 5 % and 10 %, and lesser viability when it was 20 %. These results indicate, for the first time, that EY reduces osmotic and cryogenic damage when used at 5 % or 10 % concentrations, and that these concentrations can be used to protect dog spermatozoa more effectively than the conventionally used concentration (20 %).

Mules (Equus mulus) are interspecific hybrids derived from crossing horse (Equus caballus) mares with jackasses (Equus asinus). In Italy, the production of mules is very important for the special environmental areas, where they are used as working animals, instead of the forbidden agricultural machines. Although many horse mares carry mule pregnancy successfully at term, low fertility of mares used for producing mules was reported, but limited data about reproductive efficiency in mares carrying mule pregnancy, especially when age and reproductive status are concerned, are available. Therefore, the present study aimed to assess the effect of age and of reproductive status on some reproductive indices of horse mares carrying mule pregnancy, in the final purpose to better clarify factors affecting reproductive performances in the valuable production of mules as working animals under special environmental areas in Italy. The study, performed on 96 draft mares inseminated with Martina Franca donkey semen, showed a satisfactory 89.6% 14 days PO pregnancy rate, lower pregnancy losses (19.8%) than those previously reported for mule pregnancies, leading to satisfactory foaling rates (71.9%), similar to those reported for intraspecific horse pregnancies. A clear, significant detrimental effect of mare's age on pregnancy losses, foaling rate, and on number of estrous cycles needed to achieve pregnancy, was found. Reproductive status also affected the reproductive indices, with significantly higher pregnancy losses and lower foaling rate in barren mares, reopening the question about the possible lower reproduction efficiency in horse mares carrying mule pregnancy. The present study results, although mostly descriptive, add useful information about some factors affecting reproductive performances in the valuable production of mules as working animals under special environmental areas in Italy., Horse mare, Age, Mule pregnancy, Reproductive status, Reproductive indices, Animal science, Animal breeding, Animal physiology, Veterinary medicine