Comparative transcriptome profile of embryos at different developmental stages derived from somatic cell nuclear transfer (SCNT) and in-vitro fertilization (IVF) in riverine buffalo (Bubalus bubalis).

Somatic cell nuclear transfer (SCNT) is a very important reproductive technology with many diverse applications, such as fast multiplication of elite animals, the production of transgenic animals and embryonic stem (ES) cells. However, low cloning efficiency, a low live birth rate and the abnormally high incidence of abnormalities in the offspring born are attributed to incomplete or aberrant nuclear reprogramming. In SCNT embryos, the aberrant expression pattern of the genes throughout embryonic development is responsible for the incomplete nuclear reprogramming. The present study was carried out to identify the differential gene expression (DEGs) profile and molecular pathways of the SCNT and IVF embryos at different developmental stages (2 cell, 8 cell and blastocyst stages). In the present study, 1164 (2 cell), 1004 (8 cell) and 530 (blastocyst stage) DEGs were identified in the SCNT embryos as compared to IVF embryos. In addition, several genes such as ZEB1, GDF1, HSF5, PDE3B, VIM, TNNC, HSD3B1, TAGLN, ITGA4 and AGMAT were affecting the development of SCNT embryos as compared to IVF embryos. Further, Gene Ontology (GO) and molecular pathways analysis suggested, SCNT embryos exhibit variations compared to their IVF counterparts and affected the development of embryos throughout the different developmental stages. Apart from this, q-PCR analysis of the GDF1, TMEM114, and IGSF22 genes were utilized to validate the RNA-seq data. These findings contribute valuable insights about the different genes and molecular pathways underlying SCNT embryo development and offer crucial information for improving SCNT efficiency.

Effects of MEK1/2 inhibitor U0126 on FGF10-enhanced buffalo oocyte maturation in vitro.

Fibroblast growth factor 10 (FGF10) plays critical roles in oocyte maturation and embryonic development; however, the specific pathway by which FGF10 promotes in vitro maturation of buffalo oocytes remains elusive. The present study was aimed at investigating the mechanism underlying effects of the FGF10-mediated extracellular regulated protein kinases (ERK) pathway on oocyte maturation and embryonic development in vitro. MEK1/2 (mitogen-activated protein kinase kinase) inhibitor U0126, alone or in combination with FGF10, was added to the maturation culture medium during maturation of the cumulus oocyte complex. Morphological observations, orcein staining, apoptosis detection, and quantitative real-time PCR were performed to evaluate oocyte maturation, embryonic development, and gene expression. U0126 affected oocyte maturation and embryonic development in vitro by substantially reducing the nuclear maturation of oocytes and expansion of the cumulus while increasing the apoptosis of cumulus cells. However, it did not have a considerable effect on glucose metabolism. These findings suggest that blocking the MEK/ERK pathway is detrimental to the maturation and embryonic development potential of buffalo oocytes. Overall, FGF10 may regulate the nuclear maturation of oocytes and cumulus cell expansion and apoptosis but not glucose metabolism through the MEK/ERK pathway. Our findings indicate that FGF10 regulates resumption of meiosis and expansion and survival of cumulus cells via MEK/ERK signaling during in vitro maturation of buffalo cumulus oocyte complexes. Elucidation of the mechanism of action of FGF10 and insights into oocyte maturation should advance buffalo breeding. Further studies should examine whether enhancement of MEK/ERK signaling improves embryonic development in buffalo.

Enhancing the quality of inferior oocytes of buffalo for in vitro embryo production: The impact of melatonin on maturation, SCNT, and epigenetic modifications.

Success of animal cloning is limited by oocyte quality, which is closely linked to reprogramming ability. The number of layers of cumulus cells is typically used to assess the quality of oocyte; a minimum of one-third of collected cumulus-oocyte complexes (COCs) are discarded as inferior oocytes because they have less cumulus cells. Melatonin, which has been recognised for its ability to sequester free radicals and perform multiple functions, has emerged as a potentially effective candidate for enhancing inferior oocytes quality and, consequently, embryo development competency. The current study investigates to improve the quality of inferior oocytes by supplementation of melatonin (10-9 M) during in vitro maturation (IVM) and subsequent cloned embryo production and its mechanism. The results indicate that melatonin supplementation significantly (p<0.05) enhances inferior oocytes maturation, reduces oxidative stress by reducing ROS levels, and improves mitochondrial function by boosting GSH levels. The melatonin treatment (10-9 M) enhances the expression of SOD, GPx1, GDF 9, BMP 15, ATPase 6, and ATPase 8 in inferior oocytes. Furthermore, melatonin treatment increases the total cell number in the treated groups, promoting cloned blastocyst formation rates derived from inferior oocytes. Furthermore, compared to the control, 10-9 M melatonin supplementation enhances H3K9ac acetylation and lowers H3K27me3 methylation in cloned blastocysts derived from inferior oocytes. In conclusion, 10-9 M melatonin supplementation during IVM increased inferior oocyte maturation and promoted cloned buffalo embryo development by lowering oxidative stress and promoting epigenetic alterations. These studies show that melatonin may improve the quality of poor oocytes and buffalo cloning.

Serum biochemical profile of neonatal buffalo calves / Perfil bioquímico sérico de bezerros bubalinos no período neonatal

Serum blood samples from 50 Murrah buffalo calves were examined in this study. The animals were allocated into three groups according to the number of parturitions of their mothers: G1 (n= 15) calves from primiparous buffaloes, G2 (n= 19) calves from buffaloes with two to four parturitions, and G3 (n= 16) calves from buffaloes with five or more parturitions. Blood samples were taken at birth, before colostrum ingestion, at 24h, 48h, and 72h after birth, and at 7, 14, 21, and 30 days after birth for determination of levels of gammaglutamyl transferase (GGT), alkaline phosphatase (ALP), aspartate aminotransferase (AST), creatine kinase, total protein, albumin, globulins (including immunoglobulin G), iron, total calcium, ionized calcium, phosphorus, sodium, and potassium. The age of the calves was found to influence all of the biochemical parameters, with the exception of ionized calcium and potassium in the calves in groups G1 and G3. The calving order was found to influence AST, GGT, total protein, albumin, and globulins, including IgG. The high serum ALP activity in the first two days after birth indicates that measurement of the levels of this enzyme may be used as an indirect method of assessing passive immunity transfer.(AU)

Amostras de sangue de 50 bezerros de búfalo Murrah foram examinados nesse estudo. Os animais foram distribuídos em três grupos de acordo com a paridade de suas genitoras: G1 (n=15) bezerros de búfalas primíparas, G2 (n=19) bezerros de búfalas com 2 a 4 gestações, e G3 (n=16) bezerros de búfalas com cinco ou mais gestações. Amostras de sangue foram colhidas ao nascimento, antes da ingestão de colostro e 24h, 48h, e 72h após o nascimento e 7, 14, 21 e 30 dias após nascimento para determinar níveis de gammaglutamil transferase (GGT), fosfatase alcalina (ALP), aspartato aminotrasferase (AST), creatina quinase, proteínas totais, albumina, globulina (inclusive imunoglobulina G), ferro, cálcio total, cálcio ionizado, fósforo, sódio e potássio. A idade dos bezerros influenciou todos os parâmetros bioquímicos, exceto cálcio ionizado e potássio nos bezerros dos grupos G1 e G3. A ordem de nascimento influenciou AST, GGT, proteínas totais, albumina e globulinas, inclusive IgG. Intensa atividade ALP no soro nos primeiros dois dias após nascimento indica que medidas dos níveis dessa enzima podem ser utilizados como método indireto de avaliar transferência passiva de imunidade.(AU)

Morphological aspects of buffaloes (Bubalus bubalis) umbilical cord / Aspectos Morfológicos do cordão umbilical de búfalos (Bubalus bubalis)

Buffalo is an important livestock resource, with a great participation in agricultural systems, providing milk, meat, and work power. Umbilical cord is responsible for maternal-fetal nutrients exchange during pregnancy, and its alterations can compromise the fetal development. We investigated ten pregnant uteruses collected from cross-bread buffaloes in different stages of gestation. Pregnancy and fetal age was determined by measuring the apex sacral length and development period was calculated by previously published formula. Umbilical cords were measured for length determination. Umbilical cord vascular net and anastomosis were observed by injection of Neoprene latex. Histological sections of the umbilical cord were studied after stain with HE, picrossirius, toluidine blue, orceine, and PAS reaction. Buffaloes' umbilical cord was formed by two central arteries, an allantois duct and two peripheral veins. The artery wall was composed by large quantity of collagen, elastic fibers, fibroblasts and large number of vasa vasorum. The allantois duct was located between the arteries and presented a great number of small nourishing vessels. Small nourishing vessels should be carefully considered to avoid to be mistaken to the arterials and veins vasa vasorum. Medium length of umbilical cord from buffalos was 11.8cm (minimum of 6.8cm and maximum of 17.4cm).

Búfalo é uma importante fonte de recurso nos rebanhos animais, apresentando uma grande participação na agropecuária, provendo leite, carne e força de trabalho. O Cordão umbilical é responsável pela troca de nutrientes materno-fetais durante a gestação, e suas alterações podem comprometer o desenvolvimento fetal. Nós investigamos dez úteros gravídicos de búfalos de raças cruzadas em fases diferentes de gestação. O período de gestação e a idade fetal foram determinados pelo comprimento ápice sacral, aplicando fórmulas previamente estabelecidas. Posteriormente mediu-se o comprimento do cordão umbilical. A rede vascular do cordão umbilical e anastomoses foram observadas por injeção ou látex de neoprene. O cordão umbilical foi estudado a partir de cortes histológicos, corados por HE, picrossirius, azul de Toluidina, orceína e reação histoquímica de PAS. O cordão umbilical de búfalos é formado por duas artérias centrais, ducto alantóide e duas veias periféricas e apresentam forma de ampulheta. A parede da artéria umbilical é composta por grande quantidade de fibras colágenas e elásticas, fibroblastos e um grande número de vasa vasorum. O ducto alantóide fica alocado entre as artérias e apresenta um grande número de pequenos vasos nutritivos. Os vasos nutritivos devem ser cuidadosamente identificados para evitar-se confundi-los com vasa vasorum. O comprimento médio do cabo de cordão umbilical dos búfalos era 11.8cm (mínimo de 6.8cm e máximo de 17.4cm).

Fator de crescimento fibroblástico básico e seus receptores em relação à atividade proliferativa na placenta bubalina em diferentes fases da gestação / Basic fibloblast growth factor and its receptors in relation to proliferative activity in the buffalo placenta during gestation

Estudou-se a distribuição espaço-temporal do fator de crescimento fibroblástico básico (bFGF), do receptor 1 do fator de crescimento fibroblástico (FGFR1) e do receptor 2 do fator de crescimento fibroblástico (FGFR2) na placenta bubalina, correlacionando-a à proliferação celular. Para a detecção do bFGF, FGFR1, FGFR2 e antígeno Ki-67, colheram-se 12 placentas de búfalas nos terços inicial, médio e final da gestação, em abatedouros, e realizaram-se testes de imunoistoquímica. Detectou-se e avaliou-se a expressão do bFGF, do FGFR1, do FGFR2 e do antígeno Ki-67 ao longo da gestação. No compartimento fetal da placenta, observaram-se correlações positivas entre a expressão do bFGF e Ki-67, entre FGFR1 e Ki-67 e entre FGFR2 com Ki-67 (r=0,313, 0,358 e 0,384, respectivamente). No epitélio e estroma maternos observaram-se altas correlações entre FGFR1 e Ki-67 (r=0,739 e r=0,511, respectivamente). Os resultados sugerem envolvimento do bFGF, FGFR1 e FGFR2 na proliferação do trofoblasto enquanto no compartimento materno da placenta bubalina apenas o FGFR1 atuaria como modulador dessa atividade.

The space-temporal expression of basic fibroblast growth factor (bFGF), fibroblast growth factor receptor 1 (FGFR1) and fibroblast growth factor receptor 2 (FGFR2) in buffalo placenta and correlation to proliferative activity was studied. For the localization of bFGF, FGFR1, FGFR2 and Ki-67, 12 buffalo placentas from initial, middle and final gestational thirds were collected and immunohistochemistry tests were performed. Expression of bFGF and its receptors was detected and analyzed from the initial third until the end of gestation. In the fetal compartment, positive correlations were observed between the expression of bFGF and Ki-67, FGFR1 and Ki-67, besides FGFR2 and Ki-67 (r=0.313, 0.358 and 0.384, respectively). High correlations were found between FGFR1 and Ki-67 in maternal epithelium and stroma (r=0.789 and r=0.511, respectively). The results suggest that bFGF, FGFR1 and FGFR2 may be involved in the modulation of trophoblast proliferation, whereas maternal compartment proliferation in the buffalo placenta would only be modulated by FGFR1.

Polimorfismo cromossômico em búfalos (Bufalus bubalis) / Chromosomic polymorphism in buffaloes (Bubalus bubalis)

Para estudar o polimorfismo cromossômico em Bubalus bubalis, 152 animais com características híbridas provenientes da Ilha de Marajó, Regiäo Amazônica, foram citogeneticamente analisados. Nesta amostra, nenhum animal com 48 cromossomos foi encontrado e indivíduos com 2N=50 predominaram (88,81 por cento). Estes resultados, somados às características híbridas, advogam a favor de seleçäo contra a fusäo tandem, responsável pela reduçäo do número cromossômico