Luteal tissue blood flow and side effects of horse-recommended luteolytic doses of dinoprost and cloprostenol in donkeys.

This study assessed luteolysis and side effects in jennies receiving standard horse-recommended doses of cloprostenol and dinoprost. Sixteen cycles of eight jennies were randomly assigned in a sequential crossover design to receive dinoprost (5 mg, i.m.) and cloprostenol (0.25 mg, i.m.) at 5-d post-ovulation. B-mode and Doppler ultrasonography were employed to assess luteal tissue size and blood flow before (-15 min and 0h) and after (0.5, 1, 2, 3, 4, 5, 6, 7, 8, 12, 24, and 48h) administering PGF2α. Immunoreactive progesterone concentrations were assayed at similar timepoints via RIA. Side effects such as sweating, abdominal discomfort, and diarrhea were scored at 15-min-intervals for 1h after PGF2α. Data normality was assessed with the Shapiro-Wilk's test. Luteal tissue size and blood flow were analyzed using PROC-MIXED and post-hoc by Tukey. Non-parametric tests analyzed side effect variables. The luteal blood flow increased overtime by 27% at 45 min and peaked by 49% at 3 h for dinoprost, and conversely, it increased by 14% at 30 min and peaked at 39% at 5h for cloprostenol (P<0.05). Luteal blood flow was reduced by 50%, 25%, and 10% on both groups at 8, 12, and 24h (P<0.05). Immunoreactive progesterone concentrations decreased in 0.5h for dinoprost and 1h for cloprostenol and gradually decreased by 48h (P<0.05). Dinoprost induced greater sudoresis scores, while cloprostenol resulted in greater abdominal discomfort and diarrhea scores (P<0.05). In conclusion, dinoprost and cloprostenol effectively induced luteolysis with distinct side effects; this could guide practitioners' case selection to use one or another PGF2α.

Effect of cloprostenol on luteolysis and comparison of different estrus synchronization protocols in jennies.

Estrus synchronization is necessary for intensive donkey farming. Studies on estrus synchronization in jennies are, however, scarce. We aimed to investigate the susceptibility of the donkey corpus luteum to cloprostenol and design a successful estrus synchronization protocol. Firstly, the effects of different cloprostenol doses and the timing effect of cloprostenol treatment on estrous cycle was investigated. The time from treatment to luteolysis, the ovulation interval, pre-ovulatory diameter, and ovulation rates were compared between groups. Secondly, to identify the best protocol, eight estrus synchronization protocols from three categories were examined. In the first category, jennies in groups A (n = 55) and B (n = 30) received a progesterone releasing intra-vaginal device (JVID®) and cloprostenol treatment. In the second category (group C to F), jennies were pretreated with deslorelin, and then treated with JVID and cloprostenol, including groups C (n = 50), D (n = 50), E (n = 70), and F (n = 65). In the third category, jennies were treated with deslorelin and cloprostenol, including groups G (n = 40) and H (n = 40). Comparisons were made among groups regarding the degree of synchronization, ovulation, and pregnancy rates. Treatment with 0.4 mg cloprostenol on the third day following ovulation minimized the length of the luteal phase and estrous cycle. Synchronization rate varied from 60.0% to 88.6% among groups and was highest in group E. Pregnancy rates did not differ among the eight protocols. In conclusion, cloprostenol effectively induced luteolysis in jennies and a treatment protocol combining deslorelin, cloprostenol, and JVID is efficient for estrus synchronization in donkeys.

Evaluation of Ultrasound Measurement of Subcutaneous Fat Thickness in Dairy Jennies during the Periparturient Period.

The body condition score (BCS) represents a practical but subjective method for assessing body fat reserves. Real time ultrasonography (RTU) has been proposed as an accurate method to objectively measure subcutaneous fat (SF) thickness and predict body fat reserves in cows, horses and donkeys. The aim of the present study was to describe RTU measures of SF thickness during periparturient period in jennies. The present prospective cohort study evaluated six dairy jennies. SF RTU were performed at 15 and 7 days before the presumptive delivery, and 2, 15 and 30 days after delivery. A portable ultrasound machine and multifrequency linear transducer (5-7.5 MHz) was used. RTU images were obtained in six sites (S1-S6). Results at each time point were reported as mean ± standard deviation and compared through time. A total of 180 images were evaluated. RTU technique was easy to perform and well tolerated. No statistically significant differences were found of each site during time, except for S2 and S6a: S2 at T2 and S6a at T1 were significatively different to values obtained at T5. The RTU mean values were above those reported by others, suggesting major physio-logical challenges related to energy balance and fat mobilization in pregnant jennies bred for milking production. BCS and sites through observational time have shown a good and reliable association. Our study could give preliminary indications on fat reserves in different body locations evaluated thanks to RTU and it show no significative variation of SF thickness, in pregnant and lactating jennies.

The efficiency of intrauterine infusion of platelet-rich plasma in the treatment of acute endometritis as assessed by endoscopic, Doppler, oxidative, immunohistochemical, and gene expression alterations in jennies.

This study used autologous platelet-rich plasma (PRP) to treat acute endometritis in jennies with follow-up for alterations in uterine hemodynamics, endoscopic, immunohistochemistry, oxidant/antioxidant imbalance, pro-inflammatory regulatory molecules, and transmembrane mucin expressions. Ten jennies suffering from endometritis (acute type; n = 10) were included in the study. PRP was prepared from each animal and two intrauterine infusions one week apart were administrated. Examination and follow-up were done physically, ultrasonographically, endoscopically and samples were taken for histopathology, immunohistochemistry, and bacteriological examination. Blood and uterine fluid samples were taken to estimate biochemical and oxidative stress alterations. Expression of TRAF6 and MUC1 genes was investigated in uterine fluid, at days -1 (day of diagnosis establishment), 7, 14, and 21. Uterine bacteriological examination showed a decrease in bacterial isolates after PRP treatment. The uterine thickness and uterine vascular perfusion as illustrated by color Doppler ultrasonography were significantly decreased in jennies treated by PRP. Uterine spectral wave pattern showed a significant linear increase in pulsatility index only. Three weeks after first PRP treatment, white light endoscopic examination revealed normal uterine body mucosa and uterine horn folds. A high nuclear factor (NF-κB) expression was seen in the mononuclear cells. A significant reduction in oxidative stress biomarkers in both serum and uterine fluid was recorded after PRP treatment. The TRAF-1 gene expression significantly decreased gradually after intrauterine PRP infusion. The MUC-1 gene expression significantly decreased gradually after intrauterine PRP infusion. Both genes were within normal levels by week 3. Endometritis in jennies is associated with an oxidative process, alterations in serum biochemical parameters, Doppler indices, endoscopic appearance, high NF-κB expression, and upregulation of TRAF-1 and MUC-1 expressions. Two intrauterine infusions of autologous PRP restored normal endometrial appearance after acute endometritis.

Hormonal Management for the Induction of Luteolysis and Ovulation in Andalusian Jennies: Effect on Reproductive Performance, Embryo Quality and Recovery Rate.

Two prostanglandins (luprostiol, LUP, and dinoprost, DIN) and two ovulation-inducing agents (human Chorionic Gonadotropin, hCG, and deslorelin, DES) were evaluated for luteolysis and estrus induction, and for ovulation induction, respectively, in embryo donor jennies. Twenty-six fertile Andalusian jennies were used. In Experiment 1, jennies (n = 112 cycles) were randomly treated with either LUP or DIN after embryo flushing. In Experiment 2, donors (n = 84 cycles) were randomly treated with either hCG or DES to induce ovulation. No differences were found between prostaglandins for all variables studied (prostaglandin-ovulation interval (POI), interovulatory interval (IOI), embryo recovery rate (ERR), positive flushing rate (PFR) and embryo grade (EG)). The ovulation rate was similar for hCG and DES (60.9% vs. 78.7%). However, the interval to ovulation (ITO) was affected (62.61 ± 7.20 vs. 48.79 ± 2.69 h). None of the other variables studied (ERR, PFR and EG) were affected (p > 0.05), except for embryo quality (p = 0.009). In short, both prostaglandins evaluated are adequate to induce luteolysis and estrus. Both ovulation-inducing agents hastened ovulation, but DES seems to be more effective than hCG. Follicular diameter affected the interval from treatment to ovulation, and high uterine edema was related to low embryo quality.

Assessment of serum amyloid A concentrations and biochemical profiles in lactating jennies and newborn Ragusano donkey foals around parturition and one month after foaling in Sicily.

A proper knowledge of biochemical parameters and inflammatory markers like serum amyloid A (SAA) is crucial in the monitoring of the first post-partum period in equids. Since no information is available on SAA for donkeys at this stage, 50 animals including jennies (n.10) and newborn foals (n.10) within 48 hr from foaling, and jennies (n.10) and foals (n.20) after 30 days from parturition were enrolled in the study to assess routine biochemical profile including SAA. Jennies showed higher alkaline phosphatase and lower bilirubins and cholesterol at 30 days of lactation compared to post-partum. Neonatal donkey foals showed significant higher concentrations of sodium, alkaline phosphatase, lactic dehydrogenase, blood urea nitrogen, creatinine and albumin within 48 hr of age, whilst higher values of phosphate and triglycerides were observed in older foals of 30 days of age. Significant higher SAA concentrations were recorded during the peripartum period in both jennies (25.95 ± 14.98 µg/ml) and newborn donkey foals (37.44 ± 19.75 µg/ml) compared to SAA values recorded in lactating jennies (2.38 ± 1.78 µg/ml) and in donkey foals (16.04 ± 18.14 µg/ml) at 30 days after parturition. The assessment of SAA in jennies and donkey foals around parturition and one month after foaling represents a valuable tool for the monitoring of health status during this stage when animals have to face with new challenges like the peak of lactation and extrauterine life adaptation respectively.

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

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

Natural Occurrence of Ochratoxin A in Blood and Milk Samples from Jennies and Their Foals after Delivery.

An assessment of the natural ochratoxin A (OTA) exposure of seven Martina Franca jennies was carried out by analyzing blood and milk samples collected close to and after delivery. A total of 41 and 34 blood samples were collected from jennies and foals, respectively, and analyzed by ELISA. A total of 33 milk samples were collected from jennies and analyzed by the HPLC/FLD method based on IAC clean-up. Furthermore, 53 feed samples were collected from January to September and analyzed by a reference method (AOAC Official Method No. 2000.03) for OTA content. Feed samples showed OTA levels up to 2.7 ng/g with an incidence of 32%, while the OTA incidence rate in jennies' blood samples was 73%, with a median value of 97 ng/L and concentrations ranging from

Label-Free Mass Spectrometry-Based Quantitative Proteomics Analysis of Serum Proteins During Early Pregnancy in Jennies (Equus asinus).

Early pregnancy in jennies is routinely determined by palpation per rectum or ultrasonography and also by detecting steroid hormone and chorionic gonadotropin levels in the blood, plasma, and serum. Herein we applied label-free mass spectrometry-based quantitative proteomics to identify serum proteins that were differentially expressed between early pregnant (day 45 after ovulation) and non-pregnant jennies. Bioinformatics analysis allowed illustration of pathways potentially involved in early pregnancy. We identified 295 proteins from a total of 2,569 peptides. Twenty-five proteins (22 upregulated and three downregulated) were significantly differentially expressed between the early pregnant and non-pregnant groups. The majority of the differentially expressed proteins were involved in defense response, early embryonic development, and hormone signaling pathways. Furthermore, functional protein analyses suggested that proteins were involved in binding, enzyme inhibitor activity, and enzyme regulator activity. Five serum proteins-granulin precursor/acrogranin, transgelin-2, fibronectin, fibrinogen-like 1, and thrombospondin 1-can be considered as novel, reliable candidates to detect pregnancy in jennies. To the best of our knowledge, this is the first study to use label-free mass spectrometry-based quantitative proteomics to analyze serum proteins during early pregnancy in jennies. Our results should facilitate the identification of valuable pregnancy diagnostic markers in early pregnant jennies.

Endometrial Cytology During the Different Phases of the Estrous Cycle in Jennies: New Evidences.

Since in the mare and other animal species such as bitches and cats, the endometrial cell pattern varies depending on the phase of the estrous cycle, the aim of this study was to describe and quantify the endometrial cytological (EC) findings in cycling jennies. EC of eight nonpregnant jennies by cytobrush (CB) at diestrus (day 1 and day 14) and estrous (day 21) were evaluated. All slides were stained with Wright´s stain and microscopically examined at both 400× and 1000× magnification. Seven high-power fields (400×) were assessed in each smear and the endometrial epithelial cells and neutrophils (PMNs) were counted. Endometrial epithelial cells were classified as intact, distorted or fragmented and, on the basis of the presence of dense groups, in monolayer or single clusters. Cytoplasmic characteristics, such as vacuolation or streaming and size, form, position of nuclear characteristics, including karyorrhexis, were recorded. Background aspect, as clear, proteinaceous, or debris, was also considered. In general, sampling by CB provided a yield of cells and clumped endometrial epithelial cells in many smears, being more abundant in estrus than early and late diestrus. Individual endometrial epithelial cells, during estrous, presented a columnar morphology, ciliated or not ciliated and basal nuclei. During diestrus phase, endometrial epithelial cells presented a more cuboidal ciliated or not ciliated morphology. Moderate amount of proteinacious material and red blood cells (RBC) was also observed. Non variation in the percentage of PMNs during diestrus was obtained, but lower and segmented PMNs in CB smears were shown in estrous. This study provides new insights on the physiological changes of endometrial epithelial cells in cycling jennies during the estrus cycle. The CB technique represents a suitable and adequate method for endometrial evaluation, taking into account cytological and/or cytopathological purposes also in jennies.

Characterization of the ovarian preantral follicle populations and its correlation with age and nutritional status in Brazilian Northeastern donkeys (Equus assinus).

This study aimed to characterize the ovarian follicular population and to determine its correlations with the age and nutritional status of donkeys of the Northeastern Brazilian breed. A total of 10 females with a mean age of 5.1±3.2years were submitted to ovariectomy by videolaparoscopy to obtain the ovaries. In the laboratory, the ovaries were sectioned into 6-12 fragments of approximately 7mm in diameter, which were fixed in Carnoy, dehydrated in increasing concentrations of alcohol (85%, 95% and absolute), clarified using xylol and embedded in blocks of histological paraffin. The blocks were cut in sections of 7µm and each 120th section was mounted on a slide for observation using optical microscopy. The follicle counting and identification allowed the characterization of the population of the preantral follicles. A total of 21.135±10.821 preantral follicles was counted, of which, 91.3% were primordial, 8.3% were primary follicles and 0.4% were secondary follicles. There were no differences between the two ovaries of each animal regarding the follicular population (P>0.05). There was a rate of 9.77% degenerated follicles. Values of 0.99% follicles containing two oocytes were also identified and classified as multi-oocyte follicles, always of the primordial category. The thickness of the granulosa cell layer was 1.85µm±1.39, 3.56µm±2.08 and 21.85µm±17.27, for primordial, primary and secondary follicles, respectively. There was a significant inverse correlation (r=-0.66; P<0.001) between the age of the animals and the population of ovarian follicles. A negative influence of the weight and body score on the ovarian follicular population was also observed, when donkeys had very little or a great amount of body condition. This is the first study to describe the morphometric characteristics and estimation of the population of preantral ovarian follicles in Northeastern Brazilian donkey, showing that number of preantral follicles decreased with increasing age of the animals and this finding may be affected by nutritional status of the animals.

Strategies to improve the fertility of fresh and frozen donkey semen.

Fertility rates of donkey semen in jennies are lower compared to mares. The aims of this study were to evaluate different sperm cryopreservation methods and insemination strategies to improve the fertility of donkey semen in jennies. Three experiments were performed: (1) the comparison of two freezing methods of donkey semen (conventional method and automated method); (2) the determination of a suitable insemination dose of fresh donkey semen for jennies and mares; and (3) the influence of the semen deposition site on fertility of jennies inseminated with frozen donkey semen. For experiment 1, no differences were observed in total motility, angular velocity, curvilinear velocity, straight-line velocity, and plasma membrane integrity between samples frozen with the conventional (Styrofoam box) and the automated method (TK 4000C). However, the automated method provided higher values of progressive motility and rapid cells in frozen-thawed samples in comparison with the conventional method (P < 0.05). For experiment 2, mares were bred using 500 × 10(6) fresh sperm (M); and jennies using 1 × 10(9) (J1) or 500 × 10(6) fresh sperm (J5). Pregnancy rates in M, J1, and J5 were 93% (14/15), 73% (11/15), and 40% (6/15), respectively. When using different insemination doses, 500 × 10(6) or 1 × 10(9) sperm, no significant difference was observed in pregnancy rates of mares (M, 14/15) and jennies (J1, 11/15). Furthermore, there was no significant difference between the two insemination doses in jennies. However, with an insemination dose of 500 × 10(6) fresh sperm, the pregnancy rates were significantly higher in mares (M, 14/15) than in jennies (J5, 6/15; P < 0.05). For experiment 3, the inseminations were carried out in the uterine body (UB) or in the uterine horn of jennies with frozen-thawed donkey semen. No pregnancies were achieved with inseminations performed in the UB (0/12). The pregnancy rate for uterine horn group was 28.26% (13/46) and thus significantly higher than the UB group (0%; 0/12; P < 0.05). In conclusion, the automated method showed higher values on progressive motility and rapid cells parameters compared to the conventional method and can be used as an alternative for freezing donkey semen. The increase in the number of sperm cells per insemination dose using fresh donkey semen improved the fertility rates in jennies. The deep horn inseminations using frozen-thawed donkey semen increased the pregnancy rate in jennies, and the multiple inseminations may be an option to improve the fertility rates of donkey semen in jennies.