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.

The effects of macromolecular and serum supplements and oxygen tension during bovine in vitro procedures on kinetics of oocyte maturation and embryo development

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

The effects of ovalbumin as a protein source during the in vitro production of bovine embryos

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

The effect of interaction between macromolecule supplement and oxygen tension on bovine oocytes and embryos cultured in vitro

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

Comparison of microbial numbers in soils by using various culture media and temperatures

The influence of different media and incubation temperatures on the quantification of microbial populations in sorghum, eucalyptus and forest soils was evaluated. Microbial growth was compared by using complex (tryptone soybean agar, TSA, casein-starch, CS, and Martin) and saline (Thorton, M3, Czapeck) media and incubation temperatures of 25 and 30° C. Higher numbers of total bacterial. and fungal colony-forming units (CFU) were observed in sorghum soils, and of spore-forming and Gram-negative bacteria in forest soils than other soils. Actinomycetes counts were highest in forest soil when using CS medium at 30° C and in sorghum soil at 25° C in M3 medium. Microorganism counts were dependent on the media and incubation temperatures. The counts at temperatures of 30° C were significantly higher than at 25° C. Microbial quantification was best when using TSA medium for total. and spore-forming bacteria, Thorton for Gram-negative bacteria, M3 for actinomycetes, and Martin for fungi. © 2005 Elsevier GmbH. All rights reserved.

Estimação de fatores de correção do perímetro escrotal para idade e peso corporal em touros jovens da raça Nelore

Neste trabalho foram estudados os efeitos de idade e peso sobre o perímetro escrotal (PE) de touros jovens da raça Nelore e estimados os fatores de correção (FC) do PE para esses efeitos. As informações utilizadas pertencem aos Registros de Produção da Associação Paraguaia dos Criadores de Nelore, obtidos entre 1986 e 1997, de 110 criatórios, totalizando 7458 observações. Os coeficientes de regressão que permitiram o cálculo dos FCs para o PE foram estimados pelo método dos quadrados mínimos, empregando-se um modelo que considerou o grupo contemporâneo (animais nascidos no mesmo ano, estação, rebanho, avaliados na mesma data e do mesmo grupo de manejo alimentar na desmama e no sobreano), o efeito linear da idade em dias e os efeitos linear e quadrático do peso aos 570 dias de idade (P570). O efeito da idade sobre o PE foi igual a 0,0383(±0,0024) cm/dia e do P570 sobre o PE corrigido para essa idade, 0,0708(±0,0049) cm/kg, efeito linear, e -0,0000413(±0,0000074) cm/kg², efeito quadrático. A idade e o peso do animal foram importantes fontes de variação a serem removidas do PE, como indicativo da precocidade sexual de touros de sobreano.

Dinâmica do peso e da condição corporal e eficiência reprodutiva de ovelhas da raça Santa Inês e mestiças Santa Inês-Suffolk submetidas a dois sistemas de alimentação em intervalos entre partos de 8 meses

O trabalho teve como objetivo comparar a eficiência produtiva e reprodutiva de ovelhas em dois sistemas de alimentação. Noventa e oito matrizes da raça Santa Inês e trinta e nove mestiças Suffolk (3/4 Suffolk + 1/4 Santa Inês) foram divididas em dois tratamentos: tratamento 1 (T1) - mantidas em pastagem, com suplementação de silagem de capim elefante na seca invernal, e tratamento 2 (T2) -mantidas em pastagem, com suplementação de silagem de capim elefante na seca invernal e de concentrado três semanas antes e durante a estação de monta, três semanas antes do parto e durante a lactação. Verificou-se diferença (P<0,05) entre peso inicial (PI) (52,5kg e 54,33kg), e peso final (PF) (53,38kg e 55,76kg) para T1 e T2, respectivamente. Houve efeito de genótipo (P<0,05), sendo PI 51,46 kg para a raça Santa Inês (SI) e 55,38kg para as mestiças Santa Inês-Suffolk (SF). Para PF, observou-se 52,36kg para a Santa Inês e 56,78kg na mestiça Santa Inês-Suffolk. O peso pré-parto (PPP) diferiu (P<0,05) apenas entre as estações, tendo sido de 65,23kg na estação reprodutiva I (2 a 4/2002), 58,15kg na estação reprodutiva II (10 a 12/2002) e 59,73kg na estação reprodutiva III (6 a 8/2003). No peso pós-parto (PPART), também ocorreram diferenças (P<0,05) entre a raça Santa Inês (53,59kg) e a mestiça Santa Inês-Suffolk (57,05kg); no peso aos 30 dias de lactação (P30d) a Santa Inês registrou 52,94kg e a mestiça Santa Inês-Suffolk 55,45kg. O peso aos 70 dias (P70d) de lactação foi para a Santa Inês de 50,83kg e de 53,22 kg para a mestiça Santa Inês-Suffolk; e o peso aos 100dias (P100d) de lactação foi de 51,55kg e de 53,61kg para a Santa Inês e para a mestiça Santa Inês-Suffolk, respectivamente. A condição corporal inicial (CCI) foi maior (P<0,05) para o T2 2,47 do que para o T1 2,16. Na condição corporal final (CCF), 2,19 e 2,6, respectivamente para T1 e T2, mas os tratamentos não diferiram na condição corporal pré-parto (CCPP). A CCI 2,4 para a mestiça Santa Inês-Suffolk foi (P<0,05) em relação a Santa Inês 2,22. A CCF da mestiça Santa Inês-Suffolk de 2,49 também foi maior (P<0,05) que da Santa Inês que obteve 2,3, mas não diferiram na CCPP. em relação as três estações reprodutivas, apenas na estação I a CCI 2,55, CCF 2,8 e a CCPP 3,03 foram maiores (P<0,05). Já as estações reprodutivas II= 2,47 e III= 2,1 diferiram somente na CCPP que foi menor na estação III. Na estação I e T1 ambos os genótipos obtiveram 72,5% de fertilidade e o T2 apresentou para as mestiças Santa Inês-Suffolk 77% e para a Santa Inês 88%. Na estação II o T1 obteve para as mestiças Santa Inês-Suffolk 42% e para as Santa Inês 38% e o T2 resultou em 56 e 50% para as mestiças Santa Inês-Suffolk e Santa Inês respectivamente. Na estação III a fertilidade do T1 foi para as mestiças 60% e para as Santa Inês 54% e o T2 87% e 76% para as mestiças e Santa Inês respectivamente. Encontrou-se diferença na prolificidade, entre os tratamentos, sendo na estação I a prolificidade foi de 1,20 e 1,55, na estação II foi 0,90 e 1,03 e na estação III obteve-se 1,11 e 1,14, respectivamente para o T1 e T2. Concluiu-se que a suplementação melhorou o desempenho reprodutivo das ovelhas.

Effects of fetuin on zona pellucida hardening, fertilization and embryo development in cattle

Mammalian oocytes can undergo spontaneous meiotic maturation when they are liberated from their follicles and cultured in vitro; however, the zona pellucida (ZP) becomes resistant to chymotrypsin digestion, or hardens, when spontaneous maturation occurs in serum-free medium. Schroeder et al. [Biol. Reprod. 43 (1990) 891] described that fetuin, a component of fetal calf serum (FCS), inhibits ZP hardening during oocyte maturation. The aim of this experiment was to study the effect of the presence of cumulus cells and addition of hormones to maturation media on bovine zona hardening and embryo development in medium with and without fetuin. In Experiment 1, different concentrations of fetuin were added to the maturation medium. The time necessary for digestion of 50% of the ZP (d50) was not different when oocytes were matured in presence of 10% FCS, 1 mg/ml polyvinyl alcohol (PVA), or 4, 1 and 0.25 mg/ml of fetuin; cleavage rates were also similar. However, significantly more blastocysts (P < 0.05) were formed when FCS was used compared to PVA and 0.25 mg/ml of fetuin. In Experiment 11, we examined the influence of the presence of cumulus cells and hormones during the maturation of oocytes in media with PVA, BSA, FCS and fetuin. The d50 was significantly higher (P < 0.05) when oocytes were matured in presence of cumulus cells. The cleavage rate of cumulus-intact oocytes was similar for all groups. However, when oocytes were partially stripped before maturation, the cleavage rate was significantly higher (P < 0.05) when FCS or fetuin was used. In both stripped and non-stripped groups, significantly more blastocysts (P < 0.05) were formed when oocytes were matured with FCS compared to BSA and PVA. These results indicate that zona hardening, as described for mouse and human oocytes, does not have a large effect on bovine cumulus-intact oocytes. Apparently fetuin can be used as a substitute for FCS during bovine oocyte maturation, since it leads to similar developmental rates as FCS in intact and partially stripped oocytes. (C) 2002 Published by Elsevier B.V. B.V.

Viability and cell cycle analysis of equine fibroblasts cultured in vitro

This experiment aimed to study equine fibroblasts in culture analyzing and the cell cycle and viability of cells pre- and post-freezing. Skin fragments were obtained from 6 horses and cultured in DMEM high glucose + 10% FCS in 5% CO(2) until the beginning of confluence. Two passages were performed before freezing. Cells subjected to serum starvation (0.5% FCS) were analyzed for viability and cell cycle at 24, 48, 72, 96, 120, 144 and 168 h of culture. For the confluent groups, cells were analyzed at the moment they achieved confluence. Cellular viability was assisted with Hoescht 33342 and propidium iodide. The analysis of apoptosis/necrosis and cell cycle was performed using a flow cytometer (FACS Calibur BD(A (R))) after staining the cells with annexin V and propidium iodide. Both optical microscopy and flow cytometry confirmed that cellular viability was similar for serum starvation and confluent groups (average 84%). Similarly, both methods were efficient to synchronize the cell cycle before freezing. However, after thawing, serum starvation, for more than 24 h, was superior to culture for synchronizing cells in G0/G1 (69% x 90%). The results of this experiment indicate that equine fibroblasts can be efficiently cultured after thawing.

Lipid content and apoptosis of in vitro-produced bovine embryos as determinants of susceptibility to vitrification

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

Post-thaw viability of in vivo-produced canine blastocysts cryopreserved by slow freezing

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Intracranial variables in propofol or sevoflurane-anesthestized dogs subjected to subarachnoid administration of iohexol

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

Melatonin in maturation media fails to improve oocyte maturation, embryo development rates and DNA damage of bovine embryos

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

THE EFFECT OF SERUM ON IN-VITRO MATURATION, IN-VITRO FERTILIZATION AND STEROIDOGENESIS OF BOVINE OOCYTES COCULTURED WITH GRANULOSA-CELLS

The influence of fetal calf serum alone (FCS) or associated with proestrous (FCS+PCS), estrous (FCS+ECS) or metaestrous (FCS+MCS) cow serum added to the culture medium and of the steroids produced by co-cultured granulosa cells were evaluated in terms of the in vitro maturation (TVM) and fertilization (IVF) of bovine oocytes. Supplementation of the medium with FCS+ECS and FCS+MCS resulted in higher proportions of oocytes that reached metaphase II (96.0% and 93.3%, respectively) and in higher proportions of embryos that reached the four- and eight-cell/morula stages (51.9% and 65.6%, respectively), whereas the supplementation with FCS and FCS+PCS resulted in only 79.2% and 67.5%, respectively, of matured oocytes and 26.7% and 34.3%, respectively, of cleaved embryos. These findings show that the best IVM and IVF were obtained at lower concentrations of estradiol produced by co-cultured granulosa cells (supplementation with FCS+ECS: 10.3 ng/ml and FCS+MCS: 2.1 ng/ml), whereas the worst-results in IVM and IVF occurred at higher concentrations of estradiol that were obtained with FCS (33.1 ng/ml) and FCS+PCS (19.9 ng/ml) supplementation. These data suggest an inhibitory effect of estradiol on resumption of oocyte meiosis in vitro.

In vitro Culture of Bovine Embryos in Murine ES Cell Conditioned Media Negatively Affects Expression of Pluripotency-Related Markers OCT4, SOX2 and SSEA1

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