On the interaction of bovine serum albumin with ionic surfactants: Temperature induced EPR changes of a maleimide nitroxide reflect local protein dynamics and probe solvent accessibility

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Influence of the season of year, stage of lactation and milking time on the chloride content in bovine milk

From January to October 1995, chloride content determinations were accomplished in 2,218 samples of milk collected from 67 quarters of 17 lactating cows, in initial, middle and final stages of lactation, in the morning and afternoon milking. The highest means of chloride contents were observed among the milk samples collected in the final stage of lactation (0,203 gC1/100ml), in the summer (0,212 gC1/100ml) and in the morning milking (0,189 g C1/100ml). The differences observed between the chloride contents in the samples obtained in the different stages of lactation and the seasons of the year were statistically significant (P<0.0001). The gratest occornce of chloride at rates superior to 0.22 gC1100ml was observed in the final stge of lactation (31.4%), summer (44.2%) and after the morning milking (27.8%). These results evidentiate the influence of physiological and external factors (stage of lactation and season of the year) on the concentration of chloride of milk.

Bovine Biofertilizer and Water Regime Effects on Growth and Bulb Quality of Garlic

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

The kinetics of donor cell mtDNA in embryonic and somatic donor cell-derived bovine embryos

The mechanisms controlling the outcome of donor cell-derived mitochondrial DNA (mtDNA) in cloned animals remain largely unknown. This research was designed to investigate the kinetics of somatic and embryonic mtDNA in reconstructed bovine embryos during preimplantation development, as well as in cloned animals. The experiment involved two different procedures of embryo reconstruction and their evaluation at five distinct phases of embryo development to measure the proportion of donor cell mtDNA (Bos indicus), as well as the segregation of this mtDNA during cleavage. The ratio of donor cell (B. indicus) to host oocyte (B. taurus) mtDNA (heteroplasmy) from blastomere- (NT-B) and fibroblast- (NT-F) reconstructed embryos was estimated using an allele-specific PCR with fluorochrome-stained specific primers in each sampled blastomere, in whole blastocysts, and in the tissues of a fibroblast-derived newborn clone. NT-B zygotes and blastocysts show similar levels of heteroplasmy (11.0% and 14.0%, respectively), despite a significant decrease at the 9-16 cell stage (5.8%; p < 0.05). Heteroplasmy levels in NT-F reconstructed zygotes, however, increased from an initial low level (4.7%), to 12.9% (p < 0.05) at the 9-16 cell stage. The NT-F blastocysts contained low levels of heteroplasmy (2.2%) and no somatic-derived mtDNA was detected in the gametes or the tissues of the newborn calf cloned. These results suggest that, in contrast to the mtDNA of blastomeres, that of somatic cells either undergoes replication or escapes degradation during cleavage, although it is degraded later after the blastocyst stage or lost during somatic development, as revealed by the lack of donor cell mtDNA at birth.

Culture of mature trophoblastic giant cells from bovine placentomes

The mostly binucleate trophoblast giant cells (TGC) found in bovine placentomes, in addition to synthesizing and releasing hormones play an important role in fetal development and maternal adaptation to pregnancy. Placentomes from early gestation were collected, and for isolation of mature TGC, three cellular disaggregation methods, mechanical (MECH), enzymatic by trypsin (TRYP) or collagenase (COLL) were compared to each other. Further on, the cell survival in culture medium (DMEM) supplemented with either 10% fetal calf serum (FCS) or 10% serum replacement (SR) on culture plates free of any substrate was evaluated over a period of 90 days by trypan blue exclusion. The cells were further characterized by HOECHST 33342 nuclear staining, and immunocytochemical staining with monoclonal antibodies against vimentim and cytokeratin. A mean total rate of TGC survival of 82.56% was recorded. Statistical analysis showed significantly higher survival rates after enzymatic disaggregation with COLL (86.23%) than following MECH (80.38%) or TRYP (80.91%) treatment. Supplementation of DMEM with FCS resulted in significantly higher cellular survival rates (87.13%) when compared to the addition of SR (77.73%). Analysis of the influence of both, disaggregation method and medium supplementation on TGC survival revealed statistically significant differences between the following groups: MECH-SR (71.09%) was significantly lower than all other groups; TRYP-SR (78.03%) was significantly different from all other groups; TRYP-FCS (83.43%) and COLL-SR (84.08%) were significantly lower than MECH-FCS (89.98%) which together with COLL-FCS (88.25%) showed the highest cellular survival rate. In summary, our results show that TGC isolated from early gestation placentomes may be viable for more than 90 days of culture. However, whether these TGC produce placental lactogen throughout this period has yet to be determined. (c) 2006 Elsevier B.V. All rights reserved.

Steroidogenesis in cumulus cells of bovine cumulus-oocyte-complexes matured in vitro with BSA and different concentrations of steroids

The present in vitro experiments were designed to evaluate the ability of bovine cumulus-oocyte-complexes (COCs)to produce steroids and also to evaluate the modulatory effects of added estradiol, progesterone and testosterone on the steroidogenic activity of COCs. Considerable estradiol accumulation was observed in the control maturation medium for in vitro maturation of bovine COCs during the 24h of maturation (P < 0.05). When testosterone was added to the medium at various concentrations, a slight estradiol accumulation occurred, which, however, was lower (P < 0.05) than that observed in the control medium. Slight estradiol accumulation was observed in maturation medium containing progesterone at concentrations of 2.5, 5.0 and 10.0 mug/ml, but these increases were less (P < 0.05) than those observed in the control medium. However, in the presence of 1.0 mug/ml progesterone, estradiol accumulation was equal to that of the control medium (P > 0.05). Progesterone accumulation (P < 0.05) was observed in the control medium for in vitro maturation of bovine COCs. When estradiol was added to the maturation medium, progesterone accumulation was observed, but was significant (P < 0.05) only when the medium was supplemented with the lesser concentrations of estradiol utilized in the experiment (1.0 mug/ml). The results demonstrated that (1) cumulus cells of bovine COCs are able to secrete estradiol and progesterone in culture systems for in vitro maturation, and this steroidogenesis is modulated by the steroids progesterone, testosterone and estradiol, and (2) the addition of estradiol to the in vitro maturation medium of bovine oocytes should be reviewed, since cumulus cells of COCs have been demonstrated to secrete estradiol in the maturation medium. (C) 2002 Elsevier B.V. B.V. All rights reserved.

Activation and early parthenogenesis of bovine oocytes treated with ethanol and strontium

Efficient artificial activation is indispensable for the success of cloning programs. Strontium has been shown to effectively activate mouse oocytes for nuclear transfer procedures, however, there is limited information on its use for bovine oocytes. The present study had as objectives: (1) to assess the ability of strontium to induce activation and parthenogenetic development in bovine oocytes of different maturational ages in comparison with ethanol; and (2) to verify whether the combination of both treatments improves activation and parthenogenetic development rates. Bovine oocytes were in vitro matured for 24, 26, 28, and 30 h, and treated with ethanol (E, 7% for 5 min) or strontium chloride (S, 10 mM SrCl2 for 5 h) alone or in combination: ethanol + strontium (ES) and strontium + ethanol (SE). Activated oocytes were cultured in vitro in synthetic oviductal fluid (SOF) medium and assessed for pronuclear formation (15-16 h), cleavage (46-48 h) and development to the blastocyst stage (M). Treatment with ethanol and strontium promoted similar results regarding pronuclear formation (E, 20-66.7%; S, 26.7-53.3%; P > 0.05) and cleavage (E, 12.8-40.6%; S, 16.1-41.9%; P > 0.05), regardless of oocyte age. The actions of both strontium and ethanol were influenced by oocyte age: ethanol induced greater activation rates after 28 and 30 h of maturation (48.4 and 66.7% versus 20.0 and 23.3% for 24 and 26 It, respectively; P < 0.05) and strontium after 30 It (53.3%) was superior to 24 and 26h (26.7% for both). Blastocyst development rates were minimal in all treatments (0.0-6.3%; P > 0.05), however, when the mean (+/-S.D.) cell number in blastocysts at the same maturational period was compared, strontium treatment was superior to ethanol for activation rates (82 +/- 5.7 and 89.5 +/- 7.8 versus 54 and 61, at 28 and 30 h, respectively). Improved results were obtained by combined treatments. The combination of ethanol and strontium resulted in similar pronuclear formation (ES, 36.7-83.9%; SE, 53.1-90.3%) and cleavage rates (ES, 31.3-81.3%; SE, 65.6-80.7%). Regarding embryo development, there was no difference (P > 0.05) between treatments, and blastocysts were only obtained in treatment SE at 24 and 26 h (6.5% for both). It is concluded that, SrCl2 induces activation and parthenogenetic development in bovine oocytes. (C) 2003 Elsevier B.V. All rights reserved.

Sexing of murine and bovine embryos by developmental high-titer arrest induced by H-Y antisera

Murine and bovine embryos at the late morula stage were cultured in medium containing high-titer rat H-Y antisera. After 12 h of incubation, embryos blocked at the late morulae stage were classified as males and those at the blastocyst stage were classified as females. Sexing of murine embryos by PCR and cytogenetics revealed that 83% of the embryos classified as males and 82% of those classified as females had their sex correctly predicted (P < 0.05). Bovine embryos were transferred to recipient females. Pregnancy rates were 71.4% (10/14) for embryos classified as males and 68.8% (11/16) for embryos classified as females. The sex was correctly predicted for 80% (8/10) of the embryos classified as males and for 81.8% (9/11) of those classified as females (overall accuracy, 80.9%, P < 0.05). Therefore, the induction of developmental arrest by high-titer male-specific antisera was an efficient strategy for non-invasive embryo sexing. The procedure was straightforward and has considerable commercial potential for sexing bovine embryos. (c) 2004 Elsevier B.V. All rights reserved.

Parthenogenetic activation of bovine oocytes using single and combined strontium, ionomycin and 6-dimethylaminopurine treatments

In vitro-matured (IVM) bovine oocytes were activated with single and combined treatments of strontium (S), ionomycin (1) and 6-DMAP (D). Using oocytes IVM for 26 h, we observed that activation altered cell cycle kinetics (faster progression, MIII arrest, or direct transition from MII to pronuclear stage) when compared to in vitro fertilization. The effect of oocyte age on early parthenogenesis was assessed in oocytes IVM for 22, 26 and 30 h. Better results in pronuclear development were obtained in treatments ISD (81.7%) at 22 h; D (66.7%), IS (63.3%), ID (73.3%) and ISD (76.7%) at 26 h; and D (86.7%), IS (85.0%) and ID (78.3%) at 30 h. Higher cleavage occurred on ISD (80.0%) at 22 h; ID (83.3%) and ISD (91.7%) at 26 h; and 1 (86.7%), IS (90.0%), ID (85.0%) and ISD (95.0%) at 30 h. More blastocysts were achieved in ID (25.0%) and ISD (18.3%) at 22h; and in ID at 26h (45.0%) and 30h (50.0%). We also observed that IS allowed higher haploid (77.4%) embryonic development, whilst ID was better for diploid (89.1%) development. It was concluded that association of S and D without I was not effective for blastocyst development; treatments using S were less influenced by oocyte age, but when S was associated with D there was a detrimental effect on aged oocytes; treatment ISD promoted higher activation and cleavage rates in young oocytes and ID protocol was the best for producing blastocysts.

Oocyte activation and preimplantation development of bovine embryos obtained by specific inhibition of cyclin-dependent kinases

Realizaram-se dois experimentos para avaliar a eficiência da bohemina e roscovitina associadas à ionomicina para ativação partenogenética e desenvolvimento embrionário inicial de bovinos. No primeiro, foram testadas diferentes concentrações (0, 50, 75 ou 100µM) e diferentes tempos de exposição (2, 4 ou 6 horas) à bohemina ou à roscovitina na ativação de oócitos bovinos maturados in vitro (MIV) pré-expostos à ionomicina. Os melhores tratamentos, bohemina 75µM e roscovitina 50µM, ambos por seis horas, foram utilizados no segundo experimento, no qual oócitos bovinos MIV foram expostos à ionomicina seguido ou não pelo tratamento com inibidores específicos das quinases dependentes de ciclina (CDKI), e avaliados quanto à configuração nuclear, taxa de ativação e desenvolvimento até blastocisto. Os tratamentos combinados (ionomicina+CDKI) apresentaram melhor taxa de ativação (77,3%) e desenvolvimento embrionário inicial (35,2%) do que a ionomicina sozinha (69,4% e 21,9%, respectivamente), e também promoveram ativação mais uniforme (aproximadamente 90% de formação de um pronúcleo). Estes resultados demonstram que os CDKIs potencializam o efeito da ionomicina na ativação e desenvolvimento embrionário inicial e podem auxiliar na obtenção de protocolos de ativação mais eficientes, aumentando a capacidade de desenvolvimento de embriões produzidos por meio de biotécnicas reprodutivas.

Effect of insulin-like growth factor-1 during in vitro oocyte maturation and in vitro culture of bovine embryos

Avaliaram-se o efeito do IGF-I na maturação in vitro (MIV) (experimento I) e no desenvolvimento embrionário (DE) (experimento II) de oócitos bovinos fecundados in vitro, quanto às taxas de clivagem (TC), de blastocistos (TB) e de eclosão (TE). Para MIV, complexos cumulus-oócitos imaturos foram cultivados em meio TCM-199 suplementado com HEPES, bicarbonato e piruvato de sódio, aditivos, soro fetal bovino (meio B-199) e gonadotrofinas 14U/ml de PMSG e 7U/ml de hCG). Para o desenvolvimento embrionário, os oócitos/zigotos foram cultivados em meio B-199 com células epiteliais do oviduto bovino em suspensão sob óleo de silicone. As condições de cultivo in vitro para ambos os experimentos seguiram os tratamentos: 1- meio B-199 + 200 ng/ml IGF-I; 2- B-199 + 100 ng/ml IGF-I; 3- B-199 + 50 ng/ml IGF-I; 4- B-199 + 10 ng/ml IGF-I; 5- B-199 + 0 ng/ml IGF-I. Todas as culturas foram realizadas a 38,5° C em atmosfera com 5% de CO2 e os dados foram analisados pelo teste do qui-quadrado. No experimento I, não houve diferença (P>0,05) entre os tratamentos quanto às TC, TB e TE, quando o meio de MIV foi suplementado com IGF-I. No experimento II, a adição de IGF-I ao meio de DE resultou em aumento na TC (P<0,05) mas não influenciou a TB e a TE. A adição de 200 ng/ml de IGF-I ao meio DE melhorou a TC (71,1%) quando comparada com a TC dos grupos de 100 ng/ml de IGF-I (57,6%) ou controle (56,7%), entretanto não houve diferença quando comparada com a dos grupos de 50 ng/ml (69,4%) ou 10 ng/ml (73,1%) de IGF-I. Não houve efeito benéfico na adição de 10 a 200 ng/ml de IGF-I nos meios de MIV e de DE com relação ao desenvolvimento de embriões produzidos a partir de oócitos maturados e fecundados in vitro.

Comparative validation using quantitative real-time PCR (qPCR) and conventional PCR of bovine semen centrifuged in continuous density gradient

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)