Comparison of two methods of seminal plasma removal on buffalo (Bubalus bubalis) sperm cryopreservation.

Cryopreservation causes damage to spermatozoa, and methods minimizing this damage are therefore needed. Although much discussed, seminal plasma removal has become an alternative to improve sperm quality and viability after freezing and has been applied to different species in attempt to obtain good results. The objective of this study was to evaluate semen quality in buffaloes submitted to two methods for seminal plasma removal (filtration and centrifugation). Semen samples were collected from seven Murrah buffalo bulls (Bubalus bubalis) once a week for 8 weeks. Each ejaculate was divided into three groups: control (presence of seminal plasma), centrifugation and filtration. Sperm kinetics was evaluated with the computer-assisted sperm analysis (CASA) system. Plasmalemma and acrosomal membrane integrity, mitochondrial membrane potential and reactive oxygen species (ROS) were measured by flow cytometry, and lipid peroxidation was evaluated by the thiobarbituric acid reactive substances (TBARS) assay. Seminal plasma removal did not improve sperm kinetics compared to the control group. Centrifugation increased the number of cells with damaged acrosomal membranes (0.77 ± 0.05) and filtration caused greater plasmalemma and acrosomal membrane damage (22.18 ± 1.07). No difference in the mitochondrial membrane potential was observed between groups. In contrast, ROS production was higher in the centrifugation group compared to the control and filtration groups, although no differences in TBARS formation were detected. In conclusion, seminal plasma removal did not improve the quality of thawed buffalo semen compared to control in terms of sperm kinetics, membrane integrity, mitochondrial membrane potential or lipid peroxidation.

Transporte de oócitos bovinos em meio de maturação por diferentes períodos de tempo sem controle da atmosfera gasosa / Transport of bovine oocytes in maturation medium for different periods of time whithout controlled gaseous atmosphere

Avaliou-se a viabilidade do transporte de oócitos em meio quimicamente definido, e analisou-se a necessidade da adição ou não de hormônios neste meio. Os oócitos do grupo-controle (0h) foram maturados por 24h em estufa de CO2, e os dos grupos experimentais foram transportados em incubadora portátil. No experimento I, as taxas de clivagem foram similares (P>0,05) para os grupos 0h (59,7 por cento), 3h (53,5 por cento) e 9h (48,8 por cento), e houve redução nos grupos 6h (46,1 por cento) e 12h (43,8 por cento). Essas taxas foram semelhantes entre os grupos 3h, 6h, 9h e 12h. A produção de blastocistos não foi diferente (P>0,05) para os grupos 0h (38,0 por cento), 3h (32,3 por cento), 6h (27,3 por cento) e 9h (24,8 por cento), e houve redução no grupo 12h (18,9 por cento). Essas taxas foram semelhantes entre os grupos 6h, 9h e 12h. No experimento II, não houve diferença (P>0,05) entre as taxas de clivagem para os grupos 0h (71,4 por cento), 3h (70,3 por cento), 6h (56,0 por cento) com hormônios, e os grupos 3h (64,8 por cento) e 6h (54,1 por cento) sem hormônios. A produção de blastocistos foi similar (P>0,05) para os grupos 0h (46,1 por cento), 3h com hormônios (45,8 por cento) e 3h sem hormônios (41,1 por cento), porém houve redução nos grupos 6h com hormônios (35,5 por cento) e 6h sem hormônios (33,5 por cento). Essas taxas foram semelhantes entre os grupos 3h sem hormônios e 6h com e sem hormônios. Estes resultados indicam que é possível otransporte de oócitos bovinos por um período de até nove horas, e que a adição de hormônios neste meio não influencia os índices de clivagem e de blastocistos.

The viability of the transport of the bovine oocytes was evaluated in chemically defined medium and the need for the addition or not of hormones in this medium was analyzed. The oocytes in the control group (0h) were matured for 24h in CO2 incubator, and in experimental groups they were transported in portable incubator. In experiment I, the cleavage rates were similar (P>0.05) to the groups 0h (59.7 percent), 3h (53.5 percent), and 9h (48.8 percent), but they decreased in groups 6h (46.1 percent) and 12h (43.8 percent), however, these rates were similar among the groups 3h, 6h, 9h, and 12h. The production of blastocysts was not different (P>0.05) for groups 0h (38.0 percent), 3h (32.3 percent), 6h (27.3 percent), and 9h (24.8 percent), but there was a reduction in the 12h group (18.9 percent). These rates were similar among the groups 6h, 9h and 12h. In experiment II, no significant difference (P>0.05) was observed among the rates of cleavage for the groups 0h (71.4 percent), 3h with (70.3 percent) and without hormones (64.8 percent), and 6h with (56.0 percent) and without hormones (54.1 percent). The production of blastocysts was similar (P>0.05) for groups 0h (46.1 percent) and 3h with (45.8 percent) and without hormones (41.1 percent), but decreased in groups 6h with (35.5 percent) and without hormones (33.5 percent). These rates were similar among the groups 3h without, 6h with and without hormones. These results indicate the possibility of the transport of bovine oocytes up to 9h, and the addition of hormones in this medium does not influence the rates of cleavage and blastocysts.

Efeito do estradiol e da progesterona no desenvolvimento e na qualidade de embriões bovinos produzidos in vitro / Effect of estradiol and progesterone on development and quality of bovine embryos produced in vitro

Avaliaram-se o desenvolvimento e a qualidade de embriões bovinos, cocultivados com células epiteliais do oviduto bovino (CEOBs) expostas ou não ao estradiol e à progesterona. Os ovócitos foram maturados in vitro por 24h e, então, fertilizados utilizando-se sêmen congelado, em estufa de CO2 a 5 por cento e 38,5ºC. As CEOBs foram cultivadas em TCM-199 com ou sem estradiol (E2) (24 horas), nas mesmas condições da maturação e fertilização in vitro (MIV e FIV), e, em seguida, adicionadas aos diferentes grupos em CR2 com ou sem progesterona (P4) (G1=P4+E2); (G2=E2); (G3=P4) e (G4=controle). Após 18h da FIV, as células foram cultivadas nos diferentes sistemas. Nenhuma diferença (P>0,05) foi observada nas taxas de clivagem entre G1, G2 e G4 (53,5 por cento; 56,3 por cento; 51,7 por cento) e nos padrões de blastocistos (BLs) (29,3 por cento; 31,2 por cento, 28,7 por cento). Índices menores (P<0,05) foram obtidos no G3 para ambas as variáveis (34,5 por cento; 16,4 por cento). G1 e G2 apresentaram taxas de eclosão maiores (P<0,05) que os outros grupos (23,3 por cento; 23,2 por cento), sendo G4 (19,3 por cento) diferente de G3 (16,1 por cento). Em G1, G2 e G3, o número de células nos BLs aumentou 125,9; 128,4 e 123,6, respectivamente (P<0,05), em relação ao G4 (112,5). Conclui-se que o tratamento das CEOBs com o E2, nas primeiras 24 horas de cultivo, pode ser usado isoladamente ou em combinação com a progesterona, a fim de melhorar a qualidade de embriões bovinos produzidos in vitro.

The development and quality of bovine embryos co-cultivated with bovine oviduct epithelial cells (BOEC) supplemented or not with estradiol and progesterone were evaluated. Oocytes were matured and fertilized in vitro (MIV/FIV) (5 percentCO2/38.5ºC). The BOEC were cultivated in TCM-199 with or without estradiol (E2) (24h), in the same conditions of MIV/FIV. Presumptive zygotes were transferred to BOEC in suspension after in vitro maturation and fertilization of bovine oocytes with thawed percoll-selected sperm. The zygotes were cultivated in CR2 medium containing or not progesterone (P4) (G1=P4+E2), (G2=E2), (G3=P4), and (G4=control). No significant differences (P>0.05) were found in the cleavage rates among G1, G2, and G4 (53.5 percent, 56.3 percent, and 51.7 percent) as well as in relation to the blastocystis (BL) rates (29.3 percent, 31.2 percent, and 28.7 percent). However, significant differences (P<0.05) were observed in the G3 for both variables (34.5 percent and 16.4 percent). G1 and G2 showed hatching rates higher (P<0.05) than the other groups (23.3 percent; 23.2 percent), being G4 (19.3 percent) different from G3 (16.1 percent) (P<0.01). In the groups G1, G2, and G3, the total cell number of the BL increased (125.9, 128.4, and 123.6) (P<0.05) compared to G4 (112.5). These results demonstrate that the treatment of the BOEC with estradiol in first the 24 hours of culture can be used separately or in combination with the progesterone to improve the quality of bovine embryos produced in vitro.