Chemically assisted enucleation results in higher G6PD expression in early bovine female embryos obtained by somatic cell nuclear transfer.

Despite extensive efforts, low efficiency is still an issue in bovine somatic cell nuclear transfer (SCNT). The hypothesis of our study was that the use of cytoplasts produced by chemically assisted enucleation (EN) would improve nuclear reprogramming in nuclear transfer (NT)-derived embryos because it results in lower damage and higher cytoplasm content than conventional EN. For that purpose, we investigated the expression of two X-linked genes: X inactive-specific transcript (XIST) and glucose 6-phosphate dehydrogenase (G6PD). In the first experiment, gene expression was assessed in day-7 female blastocysts from embryonic cell NT (ECNT) groups [conventional, ECNT conv; chemically assisted, ECNT deme (demecolcine)]. Whereas in the ECNT conv group, only one embryo (25%; n=4) expressed XIST transcripts, most embryos showed XIST expression (75%; n=4) in the ECNT deme group. However, no significant differences in transcript abundance of XIST and G6PD were found when comparing the embryos from all groups. In a second experiment using somatic cells as nuclear donors, we evaluated gene expression profiles in female SCNT-derived embryos. No significant differences in relative abundance (RA) of XIST transcripts were observed among the groups. Nonetheless, higher (p<0.05) levels of G6PD were observed in SCNT deme and in vitro-derived groups in comparison to SCNT conv. To know whether higher G6PD expression in embryos derived from SCNT chemically assisted EN indicates higher metabolism in embryos considered of superior quality or if the presence of higher reactive oxygen species (ROS) levels generated by the increased oxygen consumption triggers G6PD activation, the expression of genes related to stress response should be investigated in embryos produced by that technique.

Karyoplast exchange between strontium- and 6-DMAP-parthenogenetically activated zygotes of cattle.

Ooplasmic factors drive nuclear organization after fertilization and are also important for re-programming in nuclear transfer procedures, in which artificial activation is essential for reconstructed embryos to progress in development. The present research evaluated the effect of pronuclear transfer (PT) between zygotes parthenogenetically activated with ionomycin followed by strontium (S) or 6-DMAP (D) on early embryonic development. PT was performed in the same zygote to obtain embryos in control groups (S-PT and D-PT) and between zygotes activated with S and D to achieve embryos with differentially activated cytoplasm (C) and nucleus (N) (SCDN and DCSN). PT procedure did not affect cleavage and blastocyst rates, respectively, in PT control groups compared to non-manipulated control (S-PT: 73.6% and 7.3% compared with S-Control: 77.9% and 7.8%; and D-PT: 73.3% and 31.7% compared with D-Control: 83.1% and 41.5%). Cleavage, eight-cell, and blastocyst rates, respectively, were similar between SCDN (76.5%, 36.4%, and 6.8%) and DCSN (69.5%, 25.0%, and 4.9%) embryos. Developmental rates in SCDN were similar to S-PT, but inferior to D-PT. Developmental arrest up to eight-cell stage was greater in SCDN and DCSN than in S-PT and D-PT. In conclusion, karyoplast exchange between parthenogenetic zygotes activated with strontium and 6-DMAP can lead to nuclear-cytoplasmic incompatibilities and affect embryonic development to the eight-cell and blastocyst stages.

Demecolcine effects on microtubule kinetics and on chemically assisted enucleation of bovine oocytes.

This study aimed to evaluate the effect of demecolcine, a microtubule-depolymerizing agent, on microtubule kinetics; to determine the best concentration of demecolcine as a chemically assisted enucleation agent in metaphase I (MI) and metaphase II (MII) bovine oocytes, and to evaluate the embryonic development after nuclear transfer (NT) using chemically assisted enucleation of recipient oocytes. Oocytes in vitro matured for 12 h (MI) and 21 h (MII) were exposed to several concentrations of demecolcine and evaluated for enucleation or membrane protrusion formation. Demecolcine concentration of 0.05 microg/mL produced the highest rates of enucleation in group MI (15.2%) and protrusion formation in group MII (55.1%), and was employed in the following experiments. Demecolcine effect was seen as early as 0.5 h after treatment, with a significant increase in the frequency of oocytes with complete microtubule depletion in MI (58.9%) and MII (21.8%) compared to initial averages at 0 h (27.4% and 1.9%, respectively). Microtubule repolymerization was observed when MII-treated oocytes were cultured in demecolcine-free medium for 6 h (42.4% oocytes with two evident sets of microtubules). Chemically assisted enucleated oocytes were used as recipient cytoplasts in NT procedures to assess embryonic development. For NT, 219 of 515 oocytes (42.5%) formed protrusions and were enucleated, and reconstructed, resulting in 58 nuclear-transferred one-cell embryos. Cleavage (84.5%) and blastocyst development (27.6%) rates were assessed. In conclusion, demecolcine can be used at lower concentrations than routinely employed, and the chemically assisted enucleation technique was proven to be highly efficient allowing embryonic development in bovine.

Contagem de células somáticas em amostras de leite cru na região de Catalão, GO / Counting of somatic cells in raw milk samples in the region of Catalão, GO

O presente trabalho teve como objetivo, identificar a contagem celular somática (CCS) nas amostras obtidas de propriedades rurais com diferentes tipos de ordenha do município de Catalão-GO. A pesquisa desenvolveu-se em 15 fazendas, sendo cinco com ordenha manual, cinco com ordenha mecânica em circuito fechado e cinco com ordenha mecânica com balde ao pé. As vacas estavam no meio da lactação e todas foram escolhidas aleatoriamente. O número de amostras em cada fazenda foi em torno de 30, sendo uma amostra por vaca, totalizando 150 amostras por tipo de ordenha. O leite foi colhido com o auxílio de um copo coletor e posteriormente identificado e enviado para análise. O diagnóstico da qualidade do leite foi realizado através da contagem eletrônica de células somáticas (CCS), pelo aparelho Fossomatic 5000 Basic e os teores dos componentes foram analisados utilizando-se o Milkscan 4000. Entre as variáveis tipo de ordenha e mastite subclínica, houve relação signfiicativa (p=0,07). A ordenha manual obteve 21 por cento (32) das amostras com CCS acima de 300.000 células/mL, enquanto a ordenha mecância circuito fechado a presentou 23 por cento (34) da amostras e a ordenha mecânica balde ao pé obteve 32 por cento (48) das amostras com CCS >300.000 células/mL. Entre as variáveis tipo de ordenha e CCS preconizada pelo PNMQL, não houve relação significativa (p>0,05). De acordo com a análise estatística, não existiu correlação entre os componentes do leite (gordura, proteína, lactose e EST)e CCS.

Prevalência de resíduos de antibióticos em amostras de leite cru na região do Triângulo Mineiro, MG / Prevalence of residues of antibiotics in samples of raw milk in the region of the Triângulo Mineiro, MG

A cadeia produtiva do leite no Brasil vem enfrentando mudanças rápidas e significativas nos últimos anos. Assim, a qualidade do leite tem recebido crescente atenção por parte dos vários segmentos do setor lácteo. O objetivo da presente pesquisa foi avaliar a qualidade do leite produzido na região do Triângulo Mineiro, observada pela Contagem de Células Somáticas (CCS) e pela presença de resíduos de antibióticos. A pesquisa foi desenvolvida em 21 fazendas, com diferentes tipos de ordenha: manual, mecânica circuito fechado e “balde ao pé”, com sete propriedades cada. As colheitas de leite foram realizadas em cada fazenda, durante a ordenha da tarde, aleatoriamente de 30 animais, na quantidade de 50ml e acondicionadas em frascos, com conservante e identificados com código de barras para cada animal, para análise de CCS. Cerca de 2 ml de leite de cada animal foi colhido para formar uma amostra composta, acondicionada em frasco, sem conservante, para análise de resíduo de antibiótico. O diagnóstico da qualidade do leite foi feito pelo aparelho SOMACOUNT 300*¹ (CCS) e pelo DELVOTEST (“kit” para detecção de resíduos de antibióticos (beta-lactâmicos) foi da ordem de 33,3 por cento, sendo todas essas amostras de leite com CCS acima de 750.000 células/ml. Observou-se que, das amostras que apresentaram resíduos de antibióticos, 23,8 por cento foram da ordenha circuito fechado; as amostras de leite da ordenha manual foram negativas.