Elevated blood urea nitrogen alters the transcriptome of equine embryos.

High blood urea nitrogen (BUN) in cows and ewes has a negative effect on embryo development; however, no comparable studies have been published in mares. The aims of the present study were to evaluate the effects of high BUN on blastocoele fluid, systemic progesterone and Day 14 equine embryos. When a follicle with a mean (±s.e.m.) diameter of 25±3mm was detected, mares were administered urea (0.4g kg-1) with sweet feed and molasses (n=9) or sweet feed and molasses alone (control; n=10). Blood samples were collected every other day. Mares were subjected to AI and the day ovulation was detected was designated as Day 0. Embryos were collected on Day 14 (urea-treated, n=5 embryos; control, n=7 embryos). There was an increase in systemic BUN in the urea-treated group compared with control (P<0.05), with no difference in progesterone concentrations. There were no differences between the two groups in embryo recovery or embryo size. Urea concentrations in the blastocoele fluid tended to be higher in the urea-treated mares, with a strong correlation with plasma BUN. However, there was no difference in the osmolality or pH of the blastocoele fluid between the two groups. Differentially expressed genes in Day 14 embryos from urea-treated mares analysed by RNA sequencing were involved in neurological development, urea transport, vascular remodelling and adhesion. In conclusion, oral urea treatment in mares increased BUN and induced transcriptome changes in Day 14 equine embryos of genes important in normal embryo development.

Are mule pregnancies really longer than equine pregnancies? Comparison between mule and equine pregnancies.

In equine management, it is important to predict the approximate foaling date of mares to monitor parturition and allow early identification and intervention of problems during the perinatal period. There are no studies comparing accurate gestational length (GL) when mares are carrying mule foals and no controlled comparisons between GL of mares pregnant with equine or mule foals. Therefore, the objectives of this study were to compare GL of mares pregnant with equine or mule foals and establish normal reference values for GL of mares pregnant with male and female mules. Gestational length of Mangalarga Paulista breed mares pregnant with equine (n = 54) or with mule (n = 54) foals during the breeding seasons of 2007 to 2016 was evaluated. The mean GL was 347.2 ± 1.4 days (range of 326-368 days) and 341.1 ± 1.6 days (range of 307-360 days) for equine and mule pregnancies, respectively. The normal GL reference for mule pregnancies was 316.9-365.3 days. Therefore, GL of equine pregnancies was longer than of mule pregnancies. Gestational length was not different when pregnancies resulted in females or males within each group. This study established an important reference value for normal GL of mule pregnancies, which can be used by practitioners to estimate and predict foaling dates more accurately.

Artificial activation of bovine and equine oocytes with cycloheximide, roscovitine, strontium, or 6-dimethylaminopurine in low or high calcium concentrations.

Knowledge on parthenogenetic activation of oocytes is important to improve the efficiency of nuclear transfer (NT) and intracytoplasmic sperm injection (ICSI) because artificial activation of oocyte (AOA) is an essential step to achieve embryo production. Although different procedures for AOA have been established, the efficiency of in vitro production of embryos remains low, especially in equines and Bos taurus bovines. In an attempt to improve the techniques of NT and ICSI in bovine and equine species, we tested different combinations of drugs that had different mechanisms of action for the parthenogenetic activation of oocytes in these animals. The oocytes were collected, in vitro matured for 24 to 30 h and activated artificially, in the presence of low or high concentrations of calcium, with combinations of calcium ionophore (ionomycin) with cycloheximide, roscovitine, strontium, or 6-dimethylaminopurine (6-DMAP). For assessment of activation rates, oocytes were stained with Hoechst 33342 and observed under an inverted microscope. We showed that all combinations of drugs were equally efficient in activating bovine oocytes, with the best results obtained when high concentrations of calcium were adopted. For equine oocytes, high concentrations of calcium were not beneficial for the parthenogenetic activation and the combination of ionomycin with either 6-DMAP or roscovitine was effective in inducing artificial activation of oocytes. We believe that our preliminary findings provide some clues for the development of a better AOA protocol to be used with these species.

Successful equine in vitro embryo production by ICSI - effect of season, mares' age, breed, and phase of the estrous cycle on embryo production.

This retrospective study aimed at identifying factors that contribute to the success of equine in vitro embryo production by intracytoplasmic sperm injection (ICSI). A total of 7993 ovum pick-up (OPU) sessions were performed, totaling 2540 donor mares and semen from 396 stallions. Oocytes were aspirated at multiple sites in Brazil and were sent to the laboratory, within 6 h from OPU, in pre-maturation medium where they were in vitro matured (IVM) followed by ICSI and in vitro embryo culture for 7-8 days. The number of recovered oocytes, matured oocytes, cleaved embryos and blastocysts were used to explore the effect of age and breed of the donor mare, time of year in which the mare was aspirated and phase of the estrous cycle on the day of follicular aspiration. Mares between 6 and 15 years old were superior to other age groups in most parameters evaluated, including the average number of blastocysts per OPU. The impact of age was similar when evaluated within two breeds, American Quarter Horse (AQHA) and Warmblood mares. We observed that breed (AQHA, Warmblood, Crioulo, Lusitano and Mangalarga) had an important effect on most of the parameter evaluated, including number of oocytes recovered, blastocysts produced per OPU, and blastocyst rates. The overall impact of season was less pronounced than age and breed, with the only statistically significant difference being a higher rate of oocyte maturation during the summer season. Finally, most of the parameters evaluated were superior in follicular phase mares, with or without dominant follicle than luteal phase mares. In conclusion, this retrospective study revealed that breed, age, season and stage of estrous at the time of OPU are all important parameters for the success of equine embryo production by ICSI. This technology enables producing embryos all-year-round from mares of different breeds and ages from OPU-derived oocytes collected at multiple sites.

Comparative stereological evaluation of the term allantochorion membrane in the mare pregnant with mule foals and equine foals.

Mules are derived from crossing horse mares with a donkey, in which the interest is due to gentleness and ability to work and equestrian sports. As the placenta is responsible for fetal development and maturation, knowing its typical microstructure allows us to understand how fetomaternal interactions occur in this interspecific pregnancy. Thus, the study performed a comparative stereological evaluation of volumetric composition and fetomaternal contact surface in the uterine body (UB), gravid uterine horn (GUH), and nongravid uterine horn (NGUH) of Mangalarga Paulista mare's term allantochorion membrane in mule and equine pregnancies. In equine gestation, the UB microcotyledon surface density was negatively correlated with the NGUH absolute area and the total volume of microvilli. In mule gestation, the base width and the number of microcotyledon were negatively correlated with the height and number of microcotyledons in the NGUH. Mule also showed a negative correlation between (1) the UB microcotyledon surface density and the GUH microcotyledons number per unit of membrane length, (2) the GUH total volume and the NGUH microcotyledon number. Such differences demonstrate a compensatory mechanism in conversion capacity among macrocompartments. A trend toward a greater total volume of allantoid vessels and total volume of allantoid mesoderm in UB microvilli was found in the equine and mule groups, respectively. There was a significant increase in the base width of microcotyledons in the NGUH of mules versus horses. These finds possibly influence the exchange capacity of each placental microregion and suggest a difference between mule versus horse term allantochorion membrane.

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

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

Postnatal Dynamics of Circulating Steroid Hormones in Mule and Equine Neonates.

It is necessary to study hormonal patterns from mules to recognize alterations and neonatal maladaptation. Our objective was to evaluate concentrations of hormones in mule (n = 6) and equine foals (n = 6). Blood was collected at T0, 1, 6 and 12 h after birth. Hormone concentrations were evaluated using liquid chromatography tandem mass spectrometry. Effects of time, group and interactions and regression analysis were evaluated (p < 0.05). There was a cubic and quadratic decline in mule and equine foals, respectively, for 3ß,20α-dihydroxy-DHP. Mule foals were born with lower circulating 3ß,20α-dihydroxy-DHP concentrations, which might be related to progestogen concentrations in mares with a hybrid placenta. Corticosterone and cortisol concentrations remained unchanged for the first hour post-foaling then declined in mule and equine foals (p < 0.0001). Dehydroepiandrosterone was the main androgen present. There was a decrease in dihydrotestosterone at 12 h (p = 0.002). Differences in the temporal patterns of secretion within each steroid class, pregnanes, corticoids, and androgens, suggest they were derived from different tissue sources, presumptively the placenta, adrenals and gonads of the fetus/neonate, respectively. Mule and horse foals were born without evidence of testosterone secretion. For the first time, steroid hormone levels were measured in neonatal mules, and this will provide insight into neonatal physiology that differs from equine and allow us to gain an understanding of mules that have rarely been studied. Further studies are needed to elucidate the effects of hybrid pregnancies in the steroid endocrinology of neonates.

Prenatal or postnatal corticosteroids favor clinical, respiratory, metabolic outcomes and oxidative balance of preterm lambs corticotherapy for premature neonatal lambs.

Challenges of the extrauterine environment can be life threatening for a premature fetus with inadequate fetal maturity. Maternal corticosteroids therapy is widely employed to induce fetal pulmonary maturation. Nevertheless, whenever therapeutic pregnancy interruption has to be performed in a time manner insufficient to treat the dam, postnatal corticotherapy can be considered an alternative. However, it is not known if antenatal and postnatal corticotherapy can improve similarly neonatal outcomes and pulmonary function. This research aimed to analyze antenatal and postnatal corticotherapy on premature lambs vitality, pulmonary functioning, metabolic and oxidative status. Lambs were evaluated according to the mode of treatment: Prenatal Corticosteroid Group (8 lambs born after maternal betamethasone treatment 48 h prior to birth), Postnatal Corticosteroid Group (9 lambs subjected to betamethasone treatment 10 min after birth) and Control Group (5 lambs remained untreated). Lambs were medically followed-up from birth to 72 h thereafter through a complete physical examination, as well as lactatemia, glycaemia, blood acid-base balance and antioxidant status. Treated lambs had higher vitality score than the Control Group. Heart rate was higher in postnatal therapy compared to prenatal treatment. Respiratory rate and rectal temperature were higher in treated groups. Treated lambs had hyperglycemia, while the Postnatal Group had higher lactatemia than the Control Group. The Prenatal Group had lower and normal pCO2 from 60 min onwards. The Postnatal Group had higher superoxide dismutase activity than untreated lambs. In conclusion, prenatal and postnatal betamethasone treatments favor neonatal clinical outcome, respiratory function, glucose homeostasis and oxidative balance.

Evaluation of Blood Glucose and Lactate Concentrations in Mule and Equine Foals.

Information about mule physiology is scarce. Glucose and lactate serve as prognostic tools in neonates; thus, real-time evaluations would be beneficial. Our main objective was to measure glucose and lactate concentrations from healthy mule and equine foals from birth to 720 hours. Glucose and lactate concentrations were evaluated with a benchtop Randox Daytona analyzer (LAB) using plasma and with an Accutrend Plus system (ACP) using whole blood. Data were analyzed using PROC MIXED (P < .05), intraclass coefficient correlation and Bland-Altman analysis. Glucose and lactate concentrations from mule and equine foals were different when evaluated with LAB, but there was no difference when evaluated with ACP. Glucose pattern of variation, evaluated with both analyzers for mule and equine foals, had an increase, with subsequent decrease, whereas lactate pattern of variation had initial higher values with subsequent decrease. Intraclass coefficient correlation for glucose was low and moderate for lactate. Results of glucose and lactate with ACP from our experimental neonates were not highly correlated with LAB. However, the ACP had the same pattern of variation for glucose and lactate, thus can still be used clinically for immediate evaluations if the technique is standardized with the specific samples that will be used.

Transplacental transmission of Theileria equi in mules: Should we worry?

Equine piroplasmosis, disease globally responsible for significant economic losses, is caused by the transmission of the hemoprotozoa Theileria equi and Babesia caballi by ticks. Transplacental transmission has been reported previously in horses, which is not the case for mules´ foals. The present study investigated, by Real Time-PCR (RT-PCR), the presence of both Theileria equi and Babesia caballi in newborn mules before the ingestion of colostrum. Blood samples of 18 mares and their respective mules´ foals were collected and analyzed by RT-PCR. Only one mare was positive for B. caballi. In contrast, 13 mares were positive for T. equi, with two foals (11.1%) positive for the same protozoon, both of affected mares. We may conclude that transplacental transmission of Theileria equi also occurs in mules which may be an important concern while breeding such animals.

Redox, acid-base and clinical analysis of preterm and term neonatal lambs.

During pregnancy, fetal lambs are exposed to low oxygen tension. Thus, an effective antioxidant mechanism is partially developed which sensitizes fetus to oxidative stress. Consequently, term and preterm neonates are susceptible to molecular and cellular injury caused by oxygen species (ROS). This study aimed to evaluate the development of antioxidant enzymes and oxidative profile of preterm (135 days of pregnancy) and term (145 days of pregnancy) neonatal lambs, correlating with clinical analysis. Preterm lambs had significantly (P ≤ 0.05) lower score of vitality (4.00 ± 1.10), bradycardia (99 ± 34 bpm) and bradypnea (13 ± 10 mpm). However, both groups were normothermic and euglycemic. Preterm group had low blood pH (7.07 ± 0.10) and both groups had hypercapnia, more severe in preterm group (85.52 ± 18.65 mmHg). In addition, premature newborns had lower pO2 (10.67 ± 5.65 mmHg) and SO2 (6.17 ± 5.85%) values. No significant difference (P ≥ 0.05) on antioxidant enzymes and oxidative stress were verified among experimental groups, although glutathione peroxidase negatively correlated with Apgar score, heart rate, SO2 and pO2. Our data show that preterm neonates are less adapted to the odds of labor and to overcome the immediate changes of extra-uterine life. Furthermore, we verified an influence of glutathione peroxidase in controlling oxidative stress, which highlights mature enzymatic mechanisms of cell redox, even in premature lambs.

Use of bayesian inference to correlate in vitro embryo production and in vivo fertility in zebu bulls.

The objective of this experiment was to test in vitro embryo production (IVP) as a tool to estimate fertility performance in zebu bulls using Bayesian inference statistics. Oocytes were matured and fertilized in vitro using sperm cells from three different Zebu bulls (V, T, and G). The three bulls presented similar results with regard to pronuclear formation and blastocyst formation rates. However, the cleavage rates were different between bulls. The estimated conception rates based on combined data of cleavage and blastocyst formation were very similar to the true conception rates observed for the same bulls after a fixed-time artificial insemination program. Moreover, even when we used cleavage rate data only or blastocyst formation data only, the estimated conception rates were still close to the true conception rates. We conclude that Bayesian inference is an effective statistical procedure to estimate in vivo bull fertility using data from IVP.

Placental efficiency in the equine species: which factors may be related? / Eficiência placentária na espécie equina: quais fatores podem estar relacionados?

This review intends to show the main factors that may affect intrauterine growth and the placentals nutrients supply to the fetus, morphofunctional aspects placental efficiency, correlating them with performance indexes, such as breed, age and pluriparious. This study aims to discuss the interrelationships that determine greater placental efficiency, proposing a comparative parallel between various species in order to understand more about the placental efficiency in the equine species.

Esta revisão de literatura propõe levantar os principais fatores que podem influenciar o crescimento intrauterino e o suprimento de nutrientes placentários para o feto, abordando aspectos morfofuncionais relacionados à eficiência placentária, correlacionando-os com aspectos zootécnicos, como raça, pluriparidade e idade das fêmeas gestantes. O objetivo foi estabelecer as interrelações que determinem maior eficiência placentária, propondo um paralelo comparativo entre espécies a fim de ser entendida a eficiência placentária na espécie equina.

Correlations between testicular hemodynamic and sperm characteristics in rams / Correlações entre a hemodinâmica testicular e as características espermáticas em carneiros

The testicular artery is responsible for the blood supply that reaches the testis and has great importance in heat radiation. Vascular changes in the testis may lead to damage in sperm production, reflected in sperm motility and morphology. The aim of the present study was to evaluate correlations between testicular vascularity and sperm characteristic. Eight adult Santa Ines rams showing different reproductive status were used. The testicular vascularity and sperm characteristics were evaluated fortnightly during 90 days. Color Doppler ultrasonography was used to evaluate the testicular hemodynamic. Resistance index (RI) and pulsatility index (PI) of the testicular artery were evaluated by spectral-Doppler mode. The color-Doppler mode was used to evaluate the blood flow of the pampiniform plexus and testicular parenchyma. The semen analyses assessed were volume, concentration, motility, and morphology. The data were submitted to Pearson´s linear correlations test (p 0.05 was considered significant). No correlations were found between motility and testicular hemodynamic. The percentage of total sperm defects was positively correlated to left and right parenchymal score and to left RI and PI. On the other hand, the pampiniform plexus score was positively correlated to the number of colored pixels and negatively correlated to the RI and PI, for both sides. This study showed that the increase of sperm defect can be related to increase of testicular blood flow; however, more studies are need.

A artéria testicular é responsável pelo fluxo de sangue que chega aos testículos e tem grande importância na termorregulação. Mudanças vasculares nos testículos podem levar à queda da produção espermática, refletindo na motilidade e morfologia. O objetivo deste trabalho foi avaliar as correlações entre a vascularização testicular e as características espermáticas. Foram utilizados oito carneiros adultos Santa Inês com diferentes status reprodutivos. A vascularização testicular e as características seminais foram avaliadas por um período de 90 dias. A ultrassonografia Doppler colorida foi utilizada para avaliar a hemodinâmica testicular. Foram calculados os índices de resistência (RI) e os índices de pulsatilidade (PI) com o modo espectral do Doppler. O modo colorido do Doppler foi utilizado para analisar o fluxo sanguíneo do plexo pampiniforme e do parênquima testicular. As características seminais avaliadas foram o volume, concentração, motilidade e morfologia. Os dados foram submetidos ao teste de correlação linear de Person (P 0.05 foi considerado significativo). Não foram encontradas relações entre motilidade e a hemodinâmica testicular. A porcentagem de defeitos totais correlacionou-se positivamente com o escore de vascularização dos parênquimas direito e esquerdo, e com o RI e PI esquerdos. Também o escore de vascularização dos plexos se correlacionou positivamente com a média de pixels e negativamente com o RI e PI, de ambos os lados. Este trabalho mostrou que o aumento de defeitos espermáticos pode estar correlacionado com o aumento do fluxo sanguíneo nos testículos; contudo, mais estudos são necessários.