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.

In vitro culture, PCR , and nested PCR for the detection of Theileria equi in horses submitted to exercise

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

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)

Seed and seedling surface-sterilization for in vitro culture of tillandsia gardneri (Bromeliaceae)

Tillandsia gardneri is a bromeliad with ornamental value and a wide geographical distribution over Brazil. However, due to habitat loss and illegal overcollection in the wild it is included as a vulnerable species in the official list of endangered plants of the State of Rio Grande do Sul, Brazil. The development of a protocol for T. gardneri seed propagation in vitro may be useful for reintroducing plants in their natural habitats, and for germplasm conservation. A difficult problem encountered during the establishment of an in vitro culture is explants disinfection, especially when working with endangered species, from which explant availability is restricted. Thus, the establishment of a sterilization protocol is crucial for the initiation and success of a micropropagation system for T. gardneri. The objective of this study was to evaluate the effect of sodium hypochlorite concentration and exposure time in seed and seedling surface disinfection, tissue sensitivity and development. Sodium hypochlorite solutions (10 or 20%/5, 10 or 15 min; 25%/5 or 10 min; and 50%/5 min) were effective in eliminating seed superficial contaminants. There was no significant difference among the effective sterilization treatments in relation to seed germination (%), and seedling length and number of leaves, after 120 days in vitro. Also, no damage to seed and seedling tissues were observed. Surface sterilization of seedlings, for initiation of an in vitro culture, required higher concentrations of sodium hypochlorite (25%/15 min; 20 or 50%/5, 10 or 15 min; and 40%/5 and 10 min) for controlling fungal and yeast contamination, compared to seed sterilization. No significant differences among these treatments were found in relation to seedling length and number of leaves, after 60 days in vitro.

Effects of ascorbic acid on in vitro culture of bovine preantral follicles

The objective of this study was to evaluate the effects of adding ascorbic acid to the media for in vitro culture of cattle ovarian fragments and to determine their effects on growth activation and viability of early-stage follicles. The ovarian cortex was divided into small fragments; one fragment was immediately fixed (control) and the other fragments were cultured in minimum essential medium (MEM) supplemented or not with various doses of ascorbic acid. Ovarian tissue was processed for histology, transmission electron microscopy (TEM) and immunohistochemical demonstration of proliferating cell nuclear antigen (PCNA). Compared with control fragments, the percentage of primordial follicles was reduced (p < 0.05) and the percentage of growing follicles had increased (p < 0.05) in cultured cortical fragments, independent of the tested medium or incubation time. Furthermore, compared with control tissue, culture of ovarian cortex for 8 days reduced the percentages of healthy, viable follicles (p < 0.05), but not when cultures were supplemented with 25, 50 or 100 μg/ml of ascorbic acid. Ultrastructural and immunohistochemical analysis of 8 day cultured ovarian cortical fragments, however, showed the integrity and viability of follicles only when fragments were cultured in presence of 50 μg/ml of ascorbic acid. In conclusion, this study demonstrated that addition of ascorbic acid to MEM at a concentration of 50 μg/ml not only stimulates the activation of 8 day in vitro cultured cattle primordial follicles and subsequent growth of activated follicles, but also safeguards the viability of these early-stage follicles. © 2012 Copyright Cambridge University Press.

Supplementation with the histone deacetylase inhibitor trichostatin A during in vitro culture of bovine embryos

Summary Trichostatin A (TSA) is a histone deacetylase inhibitor that induces histone hyperacetylation and increases gene expression levels. The aim of the present study was to establish a suitable condition for the use of TSA in in vitro cultures of bovine embryos, and to determine whether TSA would increase blastocyst rates by improvement of chromatin remodelling during embryonic genome activation and by increasing the expression of crucial genes during early development. To test this hypothesis, 8-cell embryos were exposed to four concentrations of TSA for different periods of time to establish adequate protocols. In a second experiment, three experimental groups were selected for the evaluation of embryo quality based on the following parameters: apoptosis, total cell number and blastocyst hatching. TSA promoted embryonic arrest and degeneration at concentrations of 15, 25 and 50 nM. All treated groups presented lower blastocyst rates. Exposure of embryos to 5 nM for 144 h and to 15 nM for 48 h decreased blastocyst hatching. However, the terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling assay (TUNEL) assay revealed similar apoptosis rates and total cell numbers in all groups studied. Although, in the present study, TSA treatment did not improve the parameters studied, the results provided background information on TSA supplementation during in vitro culture of bovine embryos and showed that embryo quality was apparently not affected, despite a decrease in blastocyst rate after exposure to TSA. © Cambridge University Press 2011.

Lethal effect of high concentrations of Parecoxib and Flunixin meglumine on the in vitro culture of bovine embryos

Since cyclooxygenase (COX) inhibitors have been pointed out as potential treatments to increase pregnancy rates after embryo transfer, the present experiment aimed to evaluate the effects of flunixin meglumine (FM) and parecoxib (P), a COX-1 and 2 or COX-2 specific inhibitor, respectively, on the development of bovine embryos until the hatched blastocyst stage. In vitro produced bovine embryos were cultured in media with different concentrations of FM (0.14; 1.4; 14; 140 or 1400 mu g/ml) or P (0.09; 0.9; 9; 90 or 900 mu g/ml) and the production rates were evaluated. Concentrations of FM <= 14 mu g/ml and P <= 90 mu g/ml did not impair embryo development, although compiled data from non-lethal FM concentrations (<= 14 mu g/ml) indicated a toxic effect enough to decrease the hatching rate of blastocysts. Concentrations of FM at 140 and 1400 mu g/ml and P at 900 mu g/ml were lethal as no cleavage was detected on presumptive zygotes.

Effects of gaseous atmosphere and antioxidants on the development and cryotolerance of bovine embryos at different periods of in vitro culture

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

Pulp fruit added to culture medium for in vitro orchid development

As an additive in in vitro culture media, fruits have a great potential for facilitating economical orchid production because of lower technology requirements and the ease of obtaining raw materials to formulate culture media. We studied the in vitro growth of Cattleya bicolor Lindl. grown in a simplified culture medium supplemented with different kinds of fruit pulp. The experimental design was completely randomised, with eight seedlings per replication and ten replications per treatment, for a total of 80 seedlings per treatment. The culture medium was made using 150 g L -1 of pulp (without peel or seed) from the following fruits: ripe Santa Cruz tomatoes (Solanum lycopersicum L.), dwarf bananas (Musa cavendishii L.) of intermediate ripeness, light green chayote (Sechium edule (Jacq.) Sw), ripe papaya (Carica papaya L.) or green coconut (Cocos nucifera L.).The treatment control was MS 50 %. The treatments and the control were kept in a growth chamber for seven months before evaluating seedling survival percentage, shoot height, number of leaves, rooting percentage, root number, root length and dry masses of shoot and roots. The highest percentages of seedling survival were obtained using MS 50 %, banana and coconut medium. The seedling survival and rooting percentages illustrate that it is possible to emphasise the culture medium MS 50% and the culture medium supplemented with coconut on the most traditional culture medium with banana or tomato pulp. For the in vitro development of Cattleya bicolor Lindl., a simplified culture medium supplemented with coconut pulp is the most suitable for use as an alternative to MS 50%. A simplified culture medium supplemented with papaya pulp is not recommended for the in vitro development of Cattleya bicolor Lindl.

In vitro Survival of Follicles Collected from Domestic Cats' Ovaries at Different Stages of Oestrous Cycle and Cultured with IGF-1

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)