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.

Antioxidantes associados à pressão hidrostática sobre a viabilidade embrionária pós-desvitrificação / Association between antioxidant and hidrostatic pressure on the embryonic viability after

Avaliou-se o efeito de dois antioxidantes no cultivo de embriões pós-desvitrificação, associados ounão à pressão hidrostática (PH) em três experimentos. O primeiro avaliou a interação entre PH e antioxidante(β-mercaptoetanol - BME, cisteamina - CYST e BME + CYST). O segundo, similar ao primeiro, vitrificou osembriões uma hora após passarem ou não pela PH. Os parâmetros foram taxas de eclosão e degeneração com24 e 48 h após passar pela PH (experimento 1) e 12, 24, 48 e 72 h pós-desvitrificação (experimento 2). Oexperimento 3 avaliou a taxa de prenhez de embriões cultivados por 12 h com/sem BME. O primeiroexperimento não demonstrou interação entre os tratamentos. No segundo, os resultados foram similares para BX.O tratamento BME + CYST obteve melhor taxa de eclosão dos BL com 48 e 72 h (76,04%). O mesmo fatoocorreu para a taxa de degeneração às 24 h (BME + CYST = 7,29%; 32,29% controle). Ao transferir os embriões(n = 55), percebeu-se uma similaridade em todos os grupos (38,9% CONT; 16,7% VITRIF e 31,6% BME). Opresente trabalho mostrou que o uso da pressão hidrostática e de BME e CYST não influencia as taxas deeclosão, degeneração e de prenhez de embriões vitrificados.

This study aimed to evaluate different antioxidants in embryo culture after vitrification, with or withoutthe previous use of hydrostatic pressure (PH). Three experiments were designed to evaluate the interactionbetween PH and antioxidants (β-mercaptoethanol - BME, cysteamine - CYST and BME + CYST) in fresh andvitrified in vitro produced embryos. In experiment 1 hatching and degeneration rates were evaluated at 24 and48 h after passing through the PH and in experiment 2 the same parameters were evaluated at 12, 24, 48 and72 h after heating. The last step of the study evaluated the pregnancy rate of vitrified embryos, cultured for 12 hwith / without BME. The first experiment showed no difference between treatments. The second found similarresults for all parameters evaluated in BX embryos. Note that the BME + CYST treatment obtained a betterhatching rate in BL with 48 and 72 h (76.04%) than the control group (45.83%). The same behavior wasobserved in degeneration 24 h, where the BME + CYST group was 7.29% against 32.29% in the control.However, the pregnancy rates (55 embryo transfers) were not different between control fresh, control vitrifiedand BME groups (38.9%, 16.7% and 31.6%, respectively). This study showed that the use of hydrostaticpressure and antioxidant had no effect on the evaluated parameters.

Antioxidantes associados à pressão hidrostática sobre a viabilidade embrionária pós-desvitrificação / Association between antioxidant and hidrostatic pressure on the embryonic viability after

Avaliou-se o efeito de dois antioxidantes no cultivo de embriões pós-desvitrificação, associados ounão à pressão hidrostática (PH) em três experimentos. O primeiro avaliou a interação entre PH e antioxidante(β-mercaptoetanol - BME, cisteamina - CYST e BME + CYST). O segundo, similar ao primeiro, vitrificou osembriões uma hora após passarem ou não pela PH. Os parâmetros foram taxas de eclosão e degeneração com24 e 48 h após passar pela PH (experimento 1) e 12, 24, 48 e 72 h pós-desvitrificação (experimento 2). Oexperimento 3 avaliou a taxa de prenhez de embriões cultivados por 12 h com/sem BME. O primeiroexperimento não demonstrou interação entre os tratamentos. No segundo, os resultados foram similares para BX.O tratamento BME + CYST obteve melhor taxa de eclosão dos BL com 48 e 72 h (76,04%). O mesmo fatoocorreu para a taxa de degeneração às 24 h (BME + CYST = 7,29%; 32,29% controle). Ao transferir os embriões(n = 55), percebeu-se uma similaridade em todos os grupos (38,9% CONT; 16,7% VITRIF e 31,6% BME). Opresente trabalho mostrou que o uso da pressão hidrostática e de BME e CYST não influencia as taxas deeclosão, degeneração e de prenhez de embriões vitrificados.(AU)

This study aimed to evaluate different antioxidants in embryo culture after vitrification, with or withoutthe previous use of hydrostatic pressure (PH). Three experiments were designed to evaluate the interactionbetween PH and antioxidants (β-mercaptoethanol - BME, cysteamine - CYST and BME + CYST) in fresh andvitrified in vitro produced embryos. In experiment 1 hatching and degeneration rates were evaluated at 24 and48 h after passing through the PH and in experiment 2 the same parameters were evaluated at 12, 24, 48 and72 h after heating. The last step of the study evaluated the pregnancy rate of vitrified embryos, cultured for 12 hwith / without BME. The first experiment showed no difference between treatments. The second found similarresults for all parameters evaluated in BX embryos. Note that the BME + CYST treatment obtained a betterhatching rate in BL with 48 and 72 h (76.04%) than the control group (45.83%). The same behavior wasobserved in degeneration 24 h, where the BME + CYST group was 7.29% against 32.29% in the control.However, the pregnancy rates (55 embryo transfers) were not different between control fresh, control vitrifiedand BME groups (38.9%, 16.7% and 31.6%, respectively). This study showed that the use of hydrostaticpressure and antioxidant had no effect on the evaluated parameters.(AU)

Buffalo (Bubalus bubalis) pre-antral follicle population and ultrastructural characterization of antral follicle oocyte.

The main objectives of the present study were to determine the ultrastructural modifications occurring in the oocyte during late folliculogenesis and to estimate pre-antral follicle population in buffalo. Half the collected ovaries were fixed and prepared for optic microscopy; the antral follicles from the other ovaries were measured and individually punctured. The cumulus-oocyte complexes (COCs) were processed for transmission electron microscopy. The number of pre-antral follicles in buffalo ovaries was estimated at 19 819 structures. Cumulus-oocyte complexes derived from 1-mm antral follicle had an eccentrical nucleus and compact corona radiata, ooplasm vilosities were fully embedded in zona pellucida (ZP) and a well-defined junction could be observed. Mitochondria were predominantly round and well distributed in ooplasm, as were small lipid vacuoles. In COCs derived from 2-mm antral follicles, the initial formation of perivitelline space was observed. The nucleus was peripherally located and the number of pleomorphic mitochondria increased. Cortical granules were clustered at oocyte periphery and lipid vacuoles increased in number and size. In COCs derived from 6-mm antral follicles, the organelles were located mainly in the perinuclear region. Golgi complexes and smooth endoplasmic reticulum (SER) were more developed. Mitochondria migrated to the cortical region and lipid vacuoles migrated to the medullar region. In COCs derived from 10-mm antral follicles, the lipid vacuoles coalesced and occupied the medullar region of the oocyte, together with a well-developed SER. Mitochondria were pleomorphic and located at the oocyte periphery. In conclusion, the morphological differences described in this paper could be responsible for some functional differences observed in in vitro embryo production and follicular dynamics for buffalo, when compared with cattle.