Endocrine-metabolic adaptations in Dorper ewes: comparison between single and twin pregnancies during gestation, parturition, and postpartum.

The experiment was conducted at Araí & Zumbi farm on sixty healthy Dorper ewes to compare blood glucose, hormonal profile, and insulin resistance evaluation in sheep from conception until 48 h postpartum in single and twin pregnancies. All experimental ewes raised under semi-intensive management system. Sixty animals were selected from 150 estrous synchronized and pregnant ewes. The animals were divided into two groups based on single (G1, n = 30) and twin pregnancies (G2, n = 30). Blood samples were collected at nine time points: immediately after fixed-time artificial insemination (D0); at 30 days (D30), 90 days (D90), 120 days (D120), 130 days (D130), and 140 days (D140) of pregnancy; on the delivery day (DD); and at 24 h (PD1) and 48 h (PD2) postpartum. The results of blood glucose, insulin, glucagon, cortisol, and thyroid hormones (T3 and T4) levels showed significant differences over the analyzed sample times; however, only cortisol showed differences within groups, with the G1 having higher values than the G2 group. The interaction of the groups × nine sample times showed a significant result (P = 0.001) only for glucagon. The number of fetuses directly interfered with the glucagon profile throughout gestation. The glucose, cortisol, glucagon, and the homeostatic model assessment for insulin resistance (HOMA IR) concentrations increased at DD and decreased at PD1 and PD2. T3 and T4 showed different behaviors among the sample times. T3 values presented a decrease from D0 to D90, followed by an increase from D90 to DD. Otherwise, for T4 values, a decrease from D90 to D130 was observed, followed by an increase from D130 to D140. Despite the changes found in the endocrine system and metabolism in Dorper ewes throughout pregnancy, the nutritional management ensured a healthy status during pregnancy, delivery, and postpartum in single and twin gestation, whose HOMA IR profiles remained identical.

Oxidative stress biomarkers in newborn calves: Comparison among artificial insemination, in vitro fertilization and cloning.

Oxidative stress occurs when there is greater than optimal production of reactive oxygen species (ROS) or an antioxidant system failure. Calves produced using in vitro fertilization (IVF) or cloning (CA) have greater mortality rates, with greater incidence of respiratory diseases, which could be explained by the deleterious outcomes from oxidative stress. Calves were studied that were produced using: artificial insemination (AI; n = 20), in vitro fertilization (IVF; n = 15) or cloning (CA; n = 15). Blood samples were collected at 6, 12, 24 and 48 h subsequent to the time of birth. The cloned calves had greater ROS production from lipid peroxidation, with greater thiobarbituric acid reactive substances. This factor was associated with a lesser amount of superoxide dismutase in the CA. Calves produced using IVF had a greater activity of catalase and glutathione peroxidase, either due to greater production of hydrogen peroxide or greater efficiency of enzymatic response of these neonates. Calves produced using AI had greater concentrations ​​of reduced thiol groups. These associated factors may indicate there is greater oxidative stress in calves produced by IVF and cloning than with use of AI, however in these calves there was an effective response to these oxidative stressors within 48 h subsequent to birth. Hence, calves produced using IVF and by cloning have greater ROS production when compared to calves produced using AI. The calves produced using IVF, however, had a greater enzymatic activity or were more efficient in adapting to ROS when compared to calves produced by cloning.

Evaluation of pulmonary maturity in bovine neonates: analysis of amniotic fluid / Avaliação maturidade pulmonar em neonatos bovinos: análise do líquido amniótico

Considering the representativeness of dairy cattle in our country, the concern about the mortality rates of the animals increases each time. Regarding to calf mortality, the Respiratory Distress Syndrome (RDS) has an important relevance during the neonatal period, and it is present in immature lungs. The amniotic fluid is in direct contact with the fetus, and it is able to offer evidence about his maturity. The aim of this study was to standardize the characteristics of the amniotic fluid, color, aspect, viscosity, quantification of lamellar body and surfactant evaluation by the Clements test and cytology, of term-born, mature and healthy calves. There were used 50 Black and White Holstein calves, which mothers were observed at calving in order to collect the amniotic fluid by puncture in the moment of exposure of the fetal membrane through the vaginal canal. Most amniotic fluid had a clear and hazy appearance due to varying degrees of viscosity and the presence or absence of clots. The Clements test could be adapted to the bovine species by the modification consisting in the addition of 3mL of amniotic fluid and 1mL of 95% ethanol. The methodology of the lamellar body count by the automated particle counter is not applicable for the bovine because of the small size of their lamellar body. The Nile Blue staining is unsatisfactory on predicting fetal maturity on the bovine species, different from cytology using Hematoxylin-Shorr stain. The presence of orange cells, increase in large amounts at the end of pregnancy. The cell stained orange counting, cells which are found in great amounts at the end of pregnancy. The present study stablished new parameters for evaluation of fetal and pulmonary maturity in the bovine species.(AU)

O objetivo desse estudo foi reunir novos dados práticos sobre a avaliação da maturidade pulmonar em neonatos bovinos, padronizando as características do líquido amniótico de bezerros maduros e hígidos, o que proporcionará a oportunidade de tratamento precoce dos animais prematuros, evitando prejuízos econômicos, principalmente quando consideramos os animais de alto valor genético. Amostras de líquido amniótico foram coletadas de 50 vacas da Raça Holandesa Preta e Branca. Corpos lamelares foram identificados por microscopia eletrônica de transmissão como estruturas de tamanho aproximado de 130nm, o que impede sua contagem em analisadores automáticos. O teste de Clements sofreu adaptações de técnica e se mostrou viável com a diluição de 3mL de líquido amniótico em 1mL de etanol a 95%. A citologia utilizando o método de Hematoxilina-Shorr, diferentemente do teste de Azul de Nilo, foi eficaz na identificação das células orangiofílicas, indicativas de maturidade fetal. Esses métodos mostraram-se originais e úteis ferramentas para a avaliação de maturidade pulmonar na espécie bovina, porém estudos com bezerros prematuros ainda são necessários.(AU)

Isolation, cultivation and immunofluorescence characterization of lamellar keratinocytes from equine hoof by using explants / Isolamento, cultivo e caracterização por imunofluorescência de queratinócitos lamelares de casco equino utilizando explantes

The importance of the hoof to the horse health is clear, and the current knowledge regarding the cellular aspects of hoof keratinocytes is poor. Studies on equine keratinocyte culture are scarce. Developing keratinocyte cultures in vitro is a condition for studies on molecular biology, cell growth and differentiation. Some methods have already been established, such as those for skin keratinocyte culture. However, few methodologies are found for lamellar keratinocytes. The objective of this study was to standardize the equine hoof keratinocyte isolation and cultivation, and then characterize the cell immunophenotype. For this, the primary culture method used was through explants obtained from three regions of the equine hoof (medial dorsal, dorsal, and lateral dorsal). After the cell isolation and cultivation, the cell culture and its explants were stained with anti-pan cytokeratin (pan-CK) (AE1/AE3), vimentin (V9), p63 (4A4), and Ki-67 (MIB-1) antibodies. Cells were grown to third passage, were positive for pan-CK, p63 and Ki-67, and few cells had vimentin positive expression. As for the explants, the epidermal laminae were not stained for vimentin or Ki-67. However, some cells presented positive pan-CK and p63 expression. This study demonstrated the viability of lamellar explants of equine hooves as a form of isolating keratinocytes in primary cultures, as well as characterized the proliferation ability of such keratinocytes in monolayers.(AU)

É notória a importância do casco na saúde dos equinos, mas o conhecimento em nível celular é pouco entendido. Estudos envolvendo o cultivo de queratinócitos equinos são escassos. Sabe-se que o desenvolvimento de cultivos de queratinócitos in vitro é uma condição para estudos sobre a biologia molecular, crescimento e diferenciação celular. Alguns métodos já estão estabelecidos, como para cultivo de queratinócitos de pele, mas poucas metodologias são encontradas para queratinócitos lamelares. O objetivo desse estudo foi padronizar o cultivo de queratinócitos provenientes de casco equino visando futuramente associar ao estudo da medicina regenerativa para assim estabelecer um modelo experimental in vitro e indicar o uso criterioso de terapias regenerativas para a laminite equina. Desta forma, o cultivo em monocamada e a caracterização de queratinócitos lamelares foram realizados. Para isso, o método de cultura primária utilizado foi através de explantes obtidos de três regiões do casco (dorso-medial, dorsal e dorso-lateral). As células foram caracterizadas para os marcadores anti pan-cytokeratin (AE1/AE3), vimentin (V9), p63 (4A4) e Ki-67 (MIB-1) nos cultivos e nos explantes. As células foram cultivadas até terceira passagem, tendo marcação positiva para pan-CK, p63 e Ki-67 e fraca marcação para vimentina. Já as lâminas epidermais não tiveram marcação de vimentin e Ki-67, porém marcaram acentuadamente para pan-CK e p63. Este estudo demonstrou a exiquibilidade do uso de explantes lamelares do casco de equinos, como forma de isolamento de queratinócitos em cultivos primários, bem como caracterizou a habilidade de proliferação desses queratinócitos em monocamada.(AU)