Conductivity of mammary gland secretions is a sensitive and specific predictor of parturition in mares.

BACKGROUND: Accurate prediction of parturition is paramount to ensuring monitoring of delivery and preventing complications. Assessing the pH and electrolytes of the mammary gland secretions (MGS) helps detect impending parturition. As conductivity is related to electrolyte concentrations and pH, it could be a useful alternative for predicting impending parturition; however, this hypothesis warrants a critical assessment. OBJECTIVES: To assess the ability of conductivity, pH, and Brix in the MGS to predict parturition and to investigate their associations. STUDY DESIGN: Field study. METHODS: The MGS of periparturient mares (n = 241) was assessed daily for conductivity, pH, and Brix index from 320d until parturition. Receiving operating curve cut-off values for conductivity (≤4.8 mS/cm), pH (≤6.4), and Brix index (>23.6%) were used to calculate sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) for predicting parturition in ≤24 h. RESULTS: Impending parturition was associated with a reduction in conductivity and pH (p < 0.05), and conductivity was strongly correlated with pH (r = 0.88) and Brix (r = -0.80) (p < 0.05). Sensitivity, specificity, PPV, and NPV for parturition in ≤24 h for conductivity (82%, 91%, 77%, and 92%, respectively), pH (79%, 84%, 81%, and 71%, respectively), and Brix (72%, 79%, 66%, and 83%, respectively) were determined separated and pairwise. Of interest, the sensitivity, specificity, PPV, and NPV, of combining conductivity and pH, were 80%, 95%, 90%, and 88%, respectively. Conductivity (≤4.8 mS/cm) presented the greatest odds ratio for predicting parturition in ≤24 h, and coupling it with pH (≤6.4 pH units) doubled its odds ratio (i.e., 25.4-62.3). MAIN LIMITATIONS: Field study. CONCLUSION: The conductivity of MGS is a sensitive and specific method to predict parturition. This is the first large-scale study showing that a combination of conductivity and pH is useful for predicting parturition in mares. The methods employed can likely apply to other settings with similar results.

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.

Semen Quality of the First and Second Ejaculates Collected from Breeding Inactive Stallions after Cooling and Freezing.

This study aimed to assess the semen quality after the cooling and freezing of the first and second ejaculates of the season, which were collected 1 h apart. After collection (n = 40 ejaculates), the gel-free semen volume, concentration, total number of sperm, and sperm morphology were determined. An aliquot of each ejaculate was extended and cooled for 48 h; a second aliquot was cushion-centrifuged and cooled for 48 h; and a third aliquot was processed and then frozen. The total motility (TM) and progressive motility (PM), plasma membrane integrity (PMI), and high mitochondrial membrane potential (HMMP) were assessed pre-(0 h), 24 h, and 48 h post-cooling and before and after freezing. The second ejaculate had a lower gel-free semen volume (p = 0.026). The sperm concentration was greater in the first than in the second ejaculate (p < 0.001). The sperm morphology was similar between the ejaculates (p > 0.05). Cushion-centrifugation prevented a reduction in the TM, PM, and PMI over time (p < 0.05). The TM, PM, and PMI decreased after freezing but not between the ejaculates (p > 0.05). The first and second ejaculates of the season, which were collected 1 h apart, varied in quantity but not in quality after cooling and freezing.

The Temporal Associations of B-Mode and Power-Doppler Ultrasonography, and Ovarian Steroid Changes of the Periovulatory Follicle and Corpus Luteum During Luteogenesis and Luteolysis in Jennies.

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

Assessment of Fetal Aortic, Thoracic, and Heartbeat in Healthy Donkey Pregnancies.

Practitioners are frequently requested to diagnose and stage pregnancy in donkeys with unknown breeding dates; however, scant work has been done to stage pregnancy in the species. Therefore, this study aimed to determine the association between measurements of fetal aortic, thoracic, and heartbeat with gestational age in donkeys carrying and delivering healthy foals. Multiparous Dezhou donkeys (n = 50) ranging from 4 to 16 years were enrolled in the study by 150 days of gestation. Transabdominal ultrasonography coupled with a 3.5 MHz sectorial convex transducer was performed at 30 day-intervals until delivery to obtain fetal aortic, thoracic, and fetal heartbeat measurements. Data were tested for normality with Shapiro- Wilk's test and then ANOVA and Tukey's. Statistical significance was set at P < .05. The mean duration of pregnancy was 356.6 ± 10.6 days (339-368). There were significant associations between gestational age and fetal aortic (r = 0.89) and thoracic (r = 0.88) measurements. Fetal heartbeat (r = -0.76) was negatively correlated with gestation length. The fetal aortic and thoracic measurements increased from the seventh month of gestation to the term (P < .001). The fetal heartbeat remained steady from 150 days to 270 days of gestation, then continuously decreased from 270 days until parturition (P < .001). In conclusion, fetal aortic and thoracic measurements are strongly associated with gestational age in donkeys. Fetal heartbeat decreases with advanced pregnancy.

Fetoplacental Unit Ultrasonography and Immunoreactive Steroid Concentrations in Jennies Experiencing Late-Term Pregnancy Loss.

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

Transrectal ultrasonography of the caudal placental pole and fetal eyeball diameter and associations with the season, duration of gestation, placental weight, sex of the foal, and birthweight in donkeys☆.

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

Electrolytes and pH of Mammary Gland Secretions Assessments to Detect Impending Parturition and Associations With Placental and Neonate Features in Donkeys.

The objectives of this study were to determine (i) the usefulness of serial assessment of mammary gland electrolytes concentrations and correspondent pH to detect impending parturition in jennies; and (ii2) the associations between mammary gland secretions, and gestation length, foal sex, maternal, placental, and foal birth weights. Multiparous jennies (n = 37) were monitored daily from 350 to 355 days of gestation until parturition. The pH of mammary gland secretions was assessed daily with a hand-held device. Aliquots of mammary secretions were frozen daily and then retrospectively assessed for electrolyte concentrations (Ca2+, Mg2+, K+, and Na+) with an automated analyzer from five days until the last sampling pre-partum. Mammary gland electrolytes and pH were analyzed with mix-models. The gestational length, newborn, maternal, and fetal membranes weights were analyzed with t-test grouped by foal's sex. Associations across all variables were assessed with Pearson's coefficient of correlation. Sensitivity, specificity, and negative predictive value (NPV) and positive predictive values (PPV) were evaluated for pH (≤ 6.4), Ca2+ (>10 mmol/L), and a combination of both. Each jenny had pH profiles assessed visually and classified as fast pH drop (1), slow pH drop (2), and alkaline pH (3) as previously described for horses. The overall gestation length was 374 ± 8.7 days, ranging from 357 to 390 days. There were no differences for gestation lengths for jennies delivering colts (374 ± 2.1 range 357-385 days), versus the ones delivering fillies (373 ± 2.3 range 358-390 days) (P > .05). Of all the foals, there were 61.8% colts and 38.2% fillies. The ratio of foal birthweight with the dam's bodyweight was 9.7%, and the ratio with fetal membranes was 11%. The majority of parturitions happened during the night (91.9%). There was a significant reduction in Na+ and an increase in Ca2+, Mg2+, and K+ concentrations leading to foaling. The pH showed a 90% sensitivity for foaling within 24 hours, whereas the specificity was 70%, and the PPV and NPV values were 40% and 97%. Of interest, Ca2+ (>10 mmol/l) displayed a sensitivity and specificity of 71% and 85%, whereas the PPV and NPV were 72% and 84%. In the present study, jennies exhibited profiles 1 to 3 as previously described for mares. Herein, 65% of the jennies displayed profile 2 and foaled with a mean acidic pH of 6.4 ± 0.02. Conversely, 32% of the jennies showed a fast reduction in the pH of mammary secretion (profile 1) from day -1 (7.3 ± 0.2) to the day of foaling with an average pH of 6.6 ± 0.08. One jenny foaled with high and alkaline pH (i.e., 7.5). There were weak and negative correlations between pH and Ca2+, Mg2+, and K+ (P < .05). In addition, Ca2+ displayed a weak but significant correlation with Mg2+, Na+, and K+. In conclusion, daily pH measurements of the mammary gland secretions can predict foaling in jennies, whereas Ca2+ was not as useful. Contrary to horses that most mares present a fast pH profile, most jennies showed a slow pH profile. The sex of foal did not affect the gestational length and fetal/maternal and fetal membrane proportions in donkeys.

Daily Sperm Output, Spermatogenic Efficiency, and Sexual Behavior of Dezhou Donkey Jacks Mounting Jennies in Estrus.

This study aimed to assess the sexual behavior of jacks mounting jennies in estrus and determine the daily sperm output (DSO) and spermatogenic efficiency using two equations to calculate testicular volume (TV). Eight sexually rested mature jacks, aging 5 to 10 years old, had semen collected once a day for 10 consecutive days using jennies in good standing estrus for mounting. Sexual behavior and semen parameters were assessed during each collection. Testicular measurements of height, width, and length were taken immediately before the first semen collection, and these measurements were used to calculate TV. After that, the TV was used to predict the DSO. The average total sperm number (TSN) obtained on days 8 to 10 was deemed the actual DSO. Differences in the predicted vs. the actual DSO were used to calculate the spermatogenic efficiency. In addition, the actual DSO was also used to calculate the number of inseminating doses a jack could produce for both on- and off-site breeding. Jack's sexual behavior and sperm motility did not vary across collection days. Sperm concentration and TSN reduced over time (P < .05). The actual DSO was 9.1 ± 4.1 billion, and the predicted DSO varied from 4.7 to 18 billion. Spermatogenic efficiency varied from 50 to 150% based on jack and the equation used to calculate TV. The number of inseminating doses ranged from 15 to 47 at 300-500 million progressively motile sperm (pms)/dose for on-site breeding. In contrast, the number of breeding doses with cooled-shipped semen (1 billion pms/dose) varied from 4 to 14 doses across donkeys. In conclusion, sexual behavior was not affected by daily semen collections. Sexual rest did not affect sperm motility. The predicted DSO varied with the equation used to determine TV. Clinically normal donkeys have high spermatogenic efficiency, which confirms previous histology reports.

Effects of long-distance transportation on blood constituents and composition of the nasal microbiota in healthy donkeys.

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.