Genome transfer technique for bovine embryo production using the metaphase plate and polar body.

Despite many studies in humans and mice using genome transfer (GT), there are few reports using this technique in oocytes of wild or domestic animals. Therefore, we aimed to establish a GT technique in bovine oocytes using the metaphase plate (MP) and polar body (PB) as the sources of genetic material. In the first experiment, GT was established using MP (GT-MP), and a sperm concentration of 1 × 106 or 0.5 × 106 spermatozoa/ml gave similar fertilization rates. The cleavage rate (50%) and blastocyst rate (13.6%) in the GT-MP group was lower than that of the in vitro production control group (80.2% and 32.6%, respectively). The second experiment evaluated the same parameters using PB instead of MP; the GT-PB group had lower fertilization (82.3% vs. 96.2%) and blastocyst (7.7% vs. 36.8%) rates than the control group. No differences in the amount of mitochondrial DNA (mtDNA) were observed between groups. Finally, GT-MP was performed using vitrified oocytes (GT-MPV) as a source of genetic material. The cleavage rate of the GT-MPV group (68.4%) was similar to that of the vitrified oocytes (VIT) control group (70.0%) and to that of the control IVP group (81.25%, P < 0.05). The blastocyst rate of GT-MPV (15.7) did not differ neither from the VIT control group (5.0%) nor from the IVP control group (35.7%). The results suggested that the structures reconstructed by the GT-MPV and GT-PB technique develop in embryos even if vitrified oocytes are used.

Maturation system affects lipid accumulation in bovine oocytes.

This study evaluated the effects of three maturation systems, namely invitro (MatV) and invivo (MatS) systems, as well as intrafollicular transfer of immature oocytes (IFIOT; MatT), on the accumulation of lipid droplets in bovine oocytes. Lipids were evaluated using confocal microscopy and transmission electron microscopy. The expression of genes related to lipid metabolism, namely acyl-CoA synthetase short chain family member 2 (ACSS2), ELOVL fatty acid elongase 1 (ELOVL1) and fatty acid binding protein 3 (FABP3), was quantified by quantitative polymerase chain reaction. The mean (±s.d.) area occupied by lipids in immature oocytes (13±2%) was similar to those matured invivo (MatS, 16±2%; MatT, 12±2%). However, there was a significant increase in lipids in oocytes in the MatV group (24±2%) compared with all other groups (P<0.001). In the ultrastructural evaluations, MatV oocytes also showed the highest lipid content. The expression of ELOVL1 and FABP3 was similar in the MatS and IFIOT groups. However, transcript levels of ACSS2 were lower in IFIOT than MatV oocytes. These results indicate, for the first time, that oocytes matured by IFIOT are similar to those matured invivo with regard to lipid accumulation, which indicates better quality than those matured invitro.

Adição da proteína específica do oviduto de porcas (pOSP) e da melatonina em meios de maturação e o efeito na clivagem in vitro de embriões suínos / Addition of the oviduct specific protein of porcine (pOSP) and melatonin in maturation media and their effect on the in vitro production of pig embryos

No presente estudo, utilizou-se a melatonina e a proteína específica do oviduto (pOSP) nos meios de maturação in vitro. Foram avaliadas a expansão do complexo cumulus-ovócito (CCOs), as concentrações intracelulares de espécies reativas de oxigênio (ROS) e o desenvolvimento embrionário nos diferentes grupos (C = controle; T1 = somente com melatonina; T2 = com melatonina e pOSP e T3 somente com pOSP). No tocante à expansão do CCOs, houve diferença (P<0,05) dos valores obtidos no grupo C em relação aos valores médios dos grupos T1, T2 e T3, porém não houve diferença entre os valores obtidos nos tratamentos (P>0,05). Na dosagem de ROS, não houve diferença entre os valores médios obtidos no grupo C (26,4±10,9) e o valor verificado no grupo T1 (23,4±7,8), porém no grupo T2 (21,3±9,7) o valor médio mostrou-se satisfatório em relação ao valor do grupo C. No entanto, o valor médio do grupo T3 (16,6±10,5) foi o que demonstrou resultado mais satisfatório quando comparado aos demais grupos (P<0,05). A produção de embriões foi avaliada por meio da taxa de clivagem. Não houve diferença (P >0,05) entre os valores obtidos entre o grupo C (48,9 %) e os valores verificados nos grupos T1 (51,5 %), T2 (50 %), T3 (57,7 %), nem destes entre si. Este estudo permitiu concluir que a proteína específica do oviduto recombinante e a melatonina foram eficientes em melhorar a expansão dos CCOs. Além disso, as células tratadas com pOSP mostraram-se com menor quantidade de ROS, podendo a pOSP ser considerada um antioxidante proteico.(AU)

The present study used melatonin and recombinant oviduct specific protein (pOSP) in in vitro maturation medium (IVM). The expansion of the cumulus-oocyte complexes (COCs), the intracellular concentrations of reactive oxygen species (ROS) and embryo development of the different groups were evaluated (C = control; T1 = melatonin; T2 = melatonin and pOSP and T3 = pOSP). Regarding the COCs expansion, the groups T1, T2 and T3 showed satisfactory results compared with group C (P<0.05), but there was no difference between treatments (P>0.05). In the ROS dosage, there was no difference between the mean values obtained in group C (26.4 ± 10.9) and group 1 (23.4 ± 7.8). However, in group 2 (21.3 ± 9.7), the average value was found to be satisfactory in relation group C. Despite that, the average value of treatment 3 (16.6 ± 10.5) was the most satisfactory result found compared to the other groups (P<0.05). The production of embryos was evaluated by cleavage rate, there was no difference between the values obtained in group C and the values recorded in groups T1 (51.5 %), T2 (50 %), T3 (57.7 %), and among them. This study showed that the pOSP and the melatonin were effective in the improvement of the expansion of COCs cells. In addition, the cells that were treated with pOSP presented a lower amount of ROS, allowing the pOSP to be considered a proteic antioxidant.(AU)

Influência do Hormônio do Crescimento na concentração de testosterona plasmática e nas características seminais de touros jovens e adultos da raça Nelore / Influence of Growth Hormone on plasma testosterone concentration and seminal characteristics of young and adult Nellore bulls

Estudou-se o efeito do Hormônio do Crescimento bovino (bGH) sobre os parâmetros seminais e a concentração sérica de testosterona de touros da raça Nelore. Dezesseis touros foram distribuídos em um delineamento fatorial 2 x 2 (duas idades e dois níveis de r-bST), com quatro animais em cada tratamento. As aplicações de r-bST foram realizadas a cada 14 dias, totalizando nove aplicações por animal, em um período experimental de 120 dias. As coletas de sêmen foram realizadas a cada 15 dias e, para determinar as concentrações de testosterona, foram realizadas coletas de sangue a cada quatro horas, num total de 24 horas, e a cada 30 dias a partir da primeira aplicação de r-bST. O sêmen dos touros adultos tratados com r-bST apresentaram maior motilidade e vigor (P<0,05) em relação aos animais controles (79,70±11,56% vs 64,06±23,65%) e (3,80±0,88 vs 2,92±1,25), respectivamente; entretanto, as demais características seminais não foram influenciadas (P>0,05) pelo tratamento com r-bST. Os aspectos físicos do sêmen dos touros jovens não foram afetados (P>0,05) pelo tratamento com r-bST. Entretanto o tratamento com r-bST diminuiu os defeitos morfológicos menores (delgado, gigante normal, pequeno normal, globoso, cabeça isolada normal, abaxial, cauda dobrada e enrolada levemente e gota distal) em relação aos jovens não tratados (P<0,05). As concentrações séricas de testosterona foram influenciadas (P<0,05) pelo tratamento, aos 120 dias após a primeira aplicação do r-bST, e apresentaram tendência de três picos no período de 24 horas.

The aim of this trial was to study the effect of Growth Hormone (GH) on the seminal parameters and testosterone profile from Nellore bulls. Sixteen bulls were allocated in a 2 x 2 factorial arrangement (two ages and two r-bST levels), with four animals per treatment. The r-bST was injected every 14 days, with a total of nine injections per animal, during the 120 days of the experimental period. The semen collections were done every 15 days and blood samples were collected in four hour intervals for 24 hours, and in 30 day intervals from the first r-bST injection for testosterone profile. The semen of adult bulls treated with r-bST showed greater MOT and VIG (P<0.05) in relation to the control (79.70±11.56% vs 64.06±23.65%) and (3.80±0.88 vs 2.92±1.25) respectively; however, the other evaluated seminal parameters were not affected (P>0.05) by the r-bST treatment. The physical measurements of the semen of young animals was also unaffected (P>0.05) by r-bST injection. However, the treatment with r-bST decreased the number of minor morphologic defects (narrow head, giant head, small normal head, round head, free normal head, abaxial implantation, simple bent tail and distal droplet) compared to untreated young bulls (P<0.05). The serum testosterone concentration was influenced (P<0.05) by treatment, at 120 days after the first injection of r-bST and they showed a tendency of three picks in a 24 hour period of sampling.