Cows as Bioreactors for the Production of Nutritionally and Biomedically Significant Proteins.

Dairy and beef cattle make a vital contribution to global nutrition, and since their domestication, they have been continuously exposed to natural and artificial selection to improve production characteristics. The technologies of transgenesis and gene editing used in cattle are responsible for generating news characteristics in bovine breeding, such as alteration of nutritional components of milk and meat enhancing human health benefits, disease resistance decreasing production costs and offering safe products for human food, as well as the recombinant protein production of biomedical significance. Different methodologies have been used to generate transgenic cattle as bioreactors. These methods include the microinjection of vectors in pronuclear, oocyte or zygote, sperm-mediate transgenesis, and somatic cell nuclear transfer. Gene editing has been applied to eliminate unwanted genes related to human and animal health, such as allergy, infection, or disease, and to insert transgenes into specific sites in the host genome. Methodologies for the generation of genetically modified cattle are laborious and not very efficient. However, in the last 30 years, transgenic animals were produced using many biotechnological tools. The result of these modifications includes (1) the change of nutritional components, including proteins, amino acids and lipids for human nutrition; (2) the removal allergic proteins milk; (3) the production of cows resistant to disease; or (4) the production of essential proteins used in biomedicine (biomedical proteins) in milk and blood plasma. The genetic modification of cattle is a powerful tool for biotechnology. It allows for the generation of new or modified products and functionality that are not currently available in this species.

Domestication strategies for the endangered catfish species Pseudopimelodus mangurus Valenciennes, 1835 (Siluriformes: Pseudopimelodidae).

Wild fish domestication can be considered a strategic approach to endangered species conservation, supporting studies and reducing economic and environmental costs. Three of the most important strategies in the domestication processes of fish are the adaptation of wild fish to captivity, the reproduction of the adapted fish and the production and maintenance of the young individuals. That being said, the present study is divided in three experiments: the 1st aimed to adapt wild Pseudopimelodus mangurus to captivity environment using different feeding approaches and a prophylactic strategie; the 2nd aimed to reproduce the adapted individuals from the 1st experiment; and the 3rd aimed to train the P. mangurus juveniles to accept commercial diets. The 1st and 2nd experiments were successful at the maintenance and artificial reproduction of P. mangurus kept in tanks between the reproductive seasons. The results suggest that the reproductive performance of animals kept in captivity (initial relative fertility-IRF = 609.25 ± 36.6 eggs/g) was similar (p > 0,05) to the performance found in wild individuals (IRF = 679.21 ± 45.66 eggs/g). Feed training of P. mangurus juveniles (3rd experiment) was also conducted, evaluating three feeding treatments with different concentrations of bovine heart and ration. At the end of the experiment, the treatment containing half bovine heart and half commercial feeding resulted in the highest values of weight gain (0.10 ± 0.16 g), specific growth rate (0.37 ± 0.11 mm), length (47.78 ± 2.35 mm) and growth (2.15 ± 2.27 mm), suggesting reasonable acceptability to artificial diets in the cultivation of this species. As conclusion, the present study contributes with the development of techniques for the domestication of fresh water fish species with commercial value or andangered of extinction, showing the domestication and reproduction of wild P. mangurus in captivity. However, more studies have to be conducted in order to improve the acceptance of artificial feeding by juveniles and to increase their survival rate.

Domestication strategies for the endangered catfish species Pseudopimelodus mangurus Valenciennes, 1835 (Siluriformes: Pseudopimelodidae) / Estratégias de domesticação das espécies ameaçadas de peixe-gato Pseudopimelodus mangurus Valenciennes, 1835 (Siluriformes: Pseudopimelodidae)

Wild fish domestication can be considered a strategic approach to endangered species conservation, supporting studies and reducing economic and environmental costs. Three of the most important strategies in the domestication processes of fish are the adaptation of wild fish to captivity, the reproduction of the adapted fish and the production and maintenance of the young individuals. That being said, the present study is divided in three experiments: the 1st aimed to adapt wild Pseudopimelodus mangurus to captivity environment using different feeding approaches and a prophylactic strategie; the 2nd aimed to reproduce the adapted individuals from the 1st experiment; and the 3rd aimed to train the P. mangurus juveniles to accept commercial diets. The 1st and 2nd experiments were successful at the maintenance and artificial reproduction of P. mangurus kept in tanks between the reproductive seasons. The results suggest that the reproductive performance of animals kept in captivity (initial relative fertility-IRF = 609.25 ± 36.6 eggs/g) was similar (p > 0,05) to the performance found in wild individuals (IRF = 679.21 ± 45.66 eggs/g). Feed training of P. mangurus juveniles (3rd experiment) was also conducted, evaluating three feeding treatments with different concentrations of bovine heart and ration. At the end of the experiment, the treatment containing half bovine heart and half commercial feeding resulted in the highest values of weight gain (0.10 ± 0.16 g), specific growth rate (0.37 ± 0.11 mm), length (47.78 ± 2.35 mm) and growth (2.15 ± 2.27 mm), suggesting reasonable acceptability to artificial diets in the cultivation of this species. As conclusion, the present study contributes with the development of techniques for the domestication of fresh water fish species with commercial value or andangered of extinction, showing the domestication and reproduction of wild P. mangurus in captivity. However, more studies have to be conducted in order to improve the acceptance of artificial feeding by juveniles and to increase their survival rate.

A domesticação de peixes selvagens pode ser considerada uma abordagem estratégica para a conservação de espécies ameaçadas, apoiando estudos e reduzindo custos econômicos e ambientais. Três das estratégias mais importantes para o processo de domesticação de peixes são a adaptação dos peixes ao cativeiro, a reprodução dos peixes adaptados e a produção e manutenção dos indivíduos jovens. O presente estudo está dividido em três partes: a 1ª objetivou adaptar Pseudopimelodus mangurus selvagens ao ambiente de cativeiro usando diferentes abordagens alimentares e uma estratégia profilática; o 2º objetivou reproduzir os indivíduos adaptados do 1º experimento; e o 3º teve como objetivo treinar os juvenis de P. mangurus para aceitar dietas comerciais. O 1o e 2o experimento obteveram sucesso na manutenção e reprodução artificial de P. mangurus mantidos em tanques entre as estações reprodutivas. Os resultados sugerem que o desempenho reprodutivo dos animais mantidos em cativeiro (fertilidade inicial relativa-FIR = 609,25 ± 36,6 ovos / g) foi similar (p> 0,05) ao dos indivíduos selvagens (FIR = 679,21 ± 45,66 ovos / g). O treinamento alimentar de P. mangurus juvenis (3º experimento) também foi realizado avaliando-se 3 tratamentos alimentares com diferentes concentrações de coração bovino e ração. Ao final do experimento, o tratamento contendo metade coração bovino e metade ração gerou os maiores valores de ganho de peso (0,10 ± 0,16 g), taxa de crescimento específico (0,37 ± 0,11 mm), comprimento (47,78 ± 2,35 mm) e crescimento (2,15 ± 2,27 mm), sugerindo razoável aceitabilidade para dietas artificiais no cultivo desta espécie. Como conclusão, o presente estudo contribui com o desenvolvimento de técnicas para a domesticação espécies de peixes de água doce, de interesse comercial ou ameaçados de extinção, mostrando a domesticação e reproduçao de em cativeiro P. Mangurus selvagens. No entanto, mais estudos devem ser conduzidos no intuito de aumentar a aceitação de dietas comerciais pelos juvenis e melhorar sua taxa sobrevivência.

In vivo phagocytosis and hematology in Astyanax altiparanae, a potential model for surrogate technology / Fagocitose in vivo e hematologia em Astyanax altiparanae, um potencial modelo para tecnologia de propagação mediada

Abstract Although the potential of surrogate propagation technology for aquaculture and conservation of Neotropical fish, the poor understanding of the host immune system may results in rejection and destruction of the donor material. Thus, it is necessary to study and to develop methods to evaluate the effects of immunosuppressive drugs employment and to evaluate the immunocompatibility between donor and receptor. Thus, the present study aimed to optimize a methodology to assess in vivo phagocytosis in Astyanax altiparanae using Saccharomyces cerevisiae and to evaluate their hematological response resultant from the inflammatory induction. To this, S. cerevisiae were labeled with Congo red and injected in the coelomic cavity of A. altiparanae at the concentration of 2.5 x 106 cells mL-1. A PBS solution and a non-injected group were kept as control. Fish blood was sampled and the phagocytic capacity and index were determined at 1, 2, 3 and 6 h post-injection (hpi). The yeast injection successfully stimulated phagocytosis, with the best result for phagocytosis assessment after 2 hpi. Moreover, it was achieved a high traceability of phagocytized and non-phagocytized yeast under optic microscopy analysis due to the Congo red labeling. The hematological profile was similar to usually observed in early infections, indicating lymphocyte migration to inflammatory site and increase in number of circulating phagocytes due to natural response to inflammatory stimulus. In conclusion, our method was efficient to assess in vivo phagocytosis in A. altiparanae and will be an important tool to evaluate the efficacy of immunosuppressive drugs in this species. Additionally, these results may serve as support for further studies in fish immunocompetence, both in laboratory and in field conditions.

Resumo Apesar do potencial apresentado pela tecnologia de propagação mediada para a aquicultura e conservação de peixes Neotropicais, o pobre entendimento do sistema imune do hospedeiro pode resultar na rejeição e destruição do material do doador. Com isso, se fazem necessários o estudo e o desenvolvimento de métodos para análise tanto dos efeitos de drogas imunossupressoras quanto para a avaliação da imunocompatibilidade entre doadores e receptores. Logo, o presente estudo teve como objetivo aperfeiçoar um método para analisar a fagocitose in vivo em Astyanax altiparanae usando Saccharomyces cerevisiae marcado e avaliar seu perfil hematológico resultante da indução inflamatória. Para isso, S. cerevisiae foram marcados com vermelho Congo e injetados na cavidade celomática dos A. altiparanae na concentração de 2,5 x 106 células.mL-1. Peixes injetados com PBS e peixes não injetados foram mantidos como controle. Sangue foi colhido e a capacidade fagocítica e o índice fagocítico foram determinados após 1, 2, 3 e 6 horas após à injeção (hpi). A injeção de levedura estimulou a fagocitose com sucesso, com o melhor resultado atingido após 2 hpi. Ainda, foi observada uma alta rastreabilidade das leveduras fagocitadas e não fagocitadas sob microscopia óptica devido à marcação com vermelho Congo. O perfil hematológico foi similar ao observado usualmente em infecções recém-induzidas, indicando migração de linfócitos ao sítio inflamatório e aumento no número de fagócitos circulantes devido à resposta natural ao estímulo inflamatório. Como conclusão, nosso método foi eficiente para analisar a fagocitose in vivo em A. altiparanae e será uma ferramenta importante para a avaliação de eficácia de drogas imunossopressoras para esta espécie. Em adição, estes resultados podem contribuir para futuros estudos em imunocompetência em peixes, tanto em âmbito laboratorial quanto a campo.

In vivo phagocytosis and hematology in Astyanax altiparanae, a potential model for surrogate technology.

Although the potential of surrogate propagation technology for aquaculture and conservation of Neotropical fish, the poor understanding of the host immune system may results in rejection and destruction of the donor material. Thus, it is necessary to study and to develop methods to evaluate the effects of immunosuppressive drugs employment and to evaluate the immunocompatibility between donor and receptor. Thus, the present study aimed to optimize a methodology to assess in vivo phagocytosis in Astyanax altiparanae using Saccharomyces cerevisiae and to evaluate their hematological response resultant from the inflammatory induction. To this, S. cerevisiae were labeled with Congo red and injected in the coelomic cavity of A. altiparanae at the concentration of 2.5 x 106 cells mL-1. A PBS solution and a non-injected group were kept as control. Fish blood was sampled and the phagocytic capacity and index were determined at 1, 2, 3 and 6 h post-injection (hpi). The yeast injection successfully stimulated phagocytosis, with the best result for phagocytosis assessment after 2 hpi. Moreover, it was achieved a high traceability of phagocytized and non-phagocytized yeast under optic microscopy analysis due to the Congo red labeling. The hematological profile was similar to usually observed in early infections, indicating lymphocyte migration to inflammatory site and increase in number of circulating phagocytes due to natural response to inflammatory stimulus. In conclusion, our method was efficient to assess in vivo phagocytosis in A. altiparanae and will be an important tool to evaluate the efficacy of immunosuppressive drugs in this species. Additionally, these results may serve as support for further studies in fish immunocompetence, both in laboratory and in field conditions.

Effects of supplementation of medium with different antioxidants during in vitro maturation of bovine oocytes on subsequent embryo production.

The aim of this study was to assess the effects of different antioxidants on the levels of reactive oxygen species (ROS) and glutathione (GSH) in oocytes during in vitro maturation (IVM), as well as on the production of embryos. Oocyte of slaughterhouse-derived cattle ovaries were placed in IVM with different antioxidants: quercetin (2 µM), cysteamine (100 µM), carnitine (0.5 mg/ml), vitamin C (50 µg/ml) or resveratrol (2 µM). Oocytes matured without any antioxidant supplementation were used as control. The oocytes were assessed for maturation rates and for ROS and GSH levels by fluorescence staining in 2',7'-dichlorodihydrofluorescein diacetate and Cell Tracker Blue, respectively. Embryo production was assessed in terms of cleavage, blastocysts and hatching rates and embryo cell numbers. The results expressed in arbitrary fluorescence units showed ROS reduction (p < .05) in the groups with quercetin (27.5 ± 3.4), vitamin C (27.1 ± 3.0) or resveratrol (28.1 ± 4.7), in comparison with those with cysteamine (34.9 ± 4.5), carnitine (34.6 ± 3.8) or to the control group (36.5 ± 5.2). GSH levels increased (p < .05) in cysteamine (63.5 ± 5.5) or carnitine (60.8 ± 4.4) groups in comparison with quercetin (52.7 ± 5.1), vitamin C (53.0 ± 3.8), resveratrol (53.1 ± 4.4) or to the control (49.6 ± 4.5). Nuclear maturation cleavage and hatched blastocysts rates did not differ (p > .05) between groups. However, blastocyst rates after in vitro fertilization in quercetin (53.5 ± 3.9%), vitamin C (52.1 ± 3.1%) resveratrol (54.2 ± 4.0%), cysteamine (52.4 ± 2.7%) or carnitine (54.2 ± 3.1%) groups were higher (p < .05) than in the control (47.2 ± 2.7%). Total cell numbers in embryos from the vitamin C, resveratrol, cysteamine or carnitine groups were higher than in quercetin and control groups, which were similar to each other. The results suggest that using antioxidants during IVM may reduce oxidative stress either by decreasing ROS levels directly or by increasing GSH levels in oocytes, depending on the type of antioxidant used. Overall, oxidative stress control during IVM with the antioxidants examined here improved blastocyst development with similar efficacy.

In vivo phagocytosis and hematology in Astyanax altiparanae, a potential model for surrogate technology

Abstract Although the potential of surrogate propagation technology for aquaculture and conservation of Neotropical fish, the poor understanding of the host immune system may results in rejection and destruction of the donor material. Thus, it is necessary to study and to develop methods to evaluate the effects of immunosuppressive drugs employment and to evaluate the immunocompatibility between donor and receptor. Thus, the present study aimed to optimize a methodology to assess in vivo phagocytosis in Astyanax altiparanae using Saccharomyces cerevisiae and to evaluate their hematological response resultant from the inflammatory induction. To this, S. cerevisiae were labeled with Congo red and injected in the coelomic cavity of A. altiparanae at the concentration of 2.5 x 106 cells mL-1. A PBS solution and a non-injected group were kept as control. Fish blood was sampled and the phagocytic capacity and index were determined at 1, 2, 3 and 6 h post-injection (hpi). The yeast injection successfully stimulated phagocytosis, with the best result for phagocytosis assessment after 2 hpi. Moreover, it was achieved a high traceability of phagocytized and non-phagocytized yeast under optic microscopy analysis due to the Congo red labeling. The hematological profile was similar to usually observed in early infections, indicating lymphocyte migration to inflammatory site and increase in number of circulating phagocytes due to natural response to inflammatory stimulus. In conclusion, our method was efficient to assess in vivo phagocytosis in A. altiparanae and will be an important tool to evaluate the efficacy of immunosuppressive drugs in this species. Additionally, these results may serve as support for further studies in fish immunocompetence, both in laboratory and in field conditions.

Resumo Apesar do potencial apresentado pela tecnologia de propagação mediada para a aquicultura e conservação de peixes Neotropicais, o pobre entendimento do sistema imune do hospedeiro pode resultar na rejeição e destruição do material do doador. Com isso, se fazem necessários o estudo e o desenvolvimento de métodos para análise tanto dos efeitos de drogas imunossupressoras quanto para a avaliação da imunocompatibilidade entre doadores e receptores. Logo, o presente estudo teve como objetivo aperfeiçoar um método para analisar a fagocitose in vivo em Astyanax altiparanae usando Saccharomyces cerevisiae marcado e avaliar seu perfil hematológico resultante da indução inflamatória. Para isso, S. cerevisiae foram marcados com vermelho Congo e injetados na cavidade celomática dos A. altiparanae na concentração de 2,5 x 106 células.mL-1. Peixes injetados com PBS e peixes não injetados foram mantidos como controle. Sangue foi colhido e a capacidade fagocítica e o índice fagocítico foram determinados após 1, 2, 3 e 6 horas após à injeção (hpi). A injeção de levedura estimulou a fagocitose com sucesso, com o melhor resultado atingido após 2 hpi. Ainda, foi observada uma alta rastreabilidade das leveduras fagocitadas e não fagocitadas sob microscopia óptica devido à marcação com vermelho Congo. O perfil hematológico foi similar ao observado usualmente em infecções recém-induzidas, indicando migração de linfócitos ao sítio inflamatório e aumento no número de fagócitos circulantes devido à resposta natural ao estímulo inflamatório. Como conclusão, nosso método foi eficiente para analisar a fagocitose in vivo em A. altiparanae e será uma ferramenta importante para a avaliação de eficácia de drogas imunossopressoras para esta espécie. Em adição, estes resultados podem contribuir para futuros estudos em imunocompetência em peixes, tanto em âmbito laboratorial quanto a campo.

Domestication strategies for the endangered catfish species Pseudopimelodus mangurus Valenciennes, 1835 (Siluriformes: Pseudopimelodidae)

Abstract Wild fish domestication can be considered a strategic approach to endangered species conservation, supporting studies and reducing economic and environmental costs. Three of the most important strategies in the domestication processes of fish are the adaptation of wild fish to captivity, the reproduction of the adapted fish and the production and maintenance of the young individuals. That being said, the present study is divided in three experiments: the 1st aimed to adapt wild Pseudopimelodus mangurus to captivity environment using different feeding approaches and a prophylactic strategie; the 2nd aimed to reproduce the adapted individuals from the 1st experiment; and the 3rd aimed to train the P. mangurus juveniles to accept commercial diets. The 1st and 2nd experiments were successful at the maintenance and artificial reproduction of P. mangurus kept in tanks between the reproductive seasons. The results suggest that the reproductive performance of animals kept in captivity (initial relative fertility-IRF = 609.25 ± 36.6 eggs/g) was similar (p > 0,05) to the performance found in wild individuals (IRF = 679.21 ± 45.66 eggs/g). Feed training of P. mangurus juveniles (3rd experiment) was also conducted, evaluating three feeding treatments with different concentrations of bovine heart and ration. At the end of the experiment, the treatment containing half bovine heart and half commercial feeding resulted in the highest values of weight gain (0.10 ± 0.16 g), specific growth rate (0.37 ± 0.11 mm), length (47.78 ± 2.35 mm) and growth (2.15 ± 2.27 mm), suggesting reasonable acceptability to artificial diets in the cultivation of this species. As conclusion, the present study contributes with the development of techniques for the domestication of fresh water fish species with commercial value or andangered of extinction, showing the domestication and reproduction of wild P. mangurus in captivity. However, more studies have to be conducted in order to improve the acceptance of artificial feeding by juveniles and to increase their survival rate.

Resumo A domesticação de peixes selvagens pode ser considerada uma abordagem estratégica para a conservação de espécies ameaçadas, apoiando estudos e reduzindo custos econômicos e ambientais. Três das estratégias mais importantes para o processo de domesticação de peixes são a adaptação dos peixes ao cativeiro, a reprodução dos peixes adaptados e a produção e manutenção dos indivíduos jovens. O presente estudo está dividido em três partes: a 1ª objetivou adaptar Pseudopimelodus mangurus selvagens ao ambiente de cativeiro usando diferentes abordagens alimentares e uma estratégia profilática; o 2º objetivou reproduzir os indivíduos adaptados do 1º experimento; e o 3º teve como objetivo treinar os juvenis de P. mangurus para aceitar dietas comerciais. O 1o e 2o experimento obteveram sucesso na manutenção e reprodução artificial de P. mangurus mantidos em tanques entre as estações reprodutivas. Os resultados sugerem que o desempenho reprodutivo dos animais mantidos em cativeiro (fertilidade inicial relativa-FIR = 609,25 ± 36,6 ovos / g) foi similar (p> 0,05) ao dos indivíduos selvagens (FIR = 679,21 ± 45,66 ovos / g). O treinamento alimentar de P. mangurus juvenis (3º experimento) também foi realizado avaliando-se 3 tratamentos alimentares com diferentes concentrações de coração bovino e ração. Ao final do experimento, o tratamento contendo metade coração bovino e metade ração gerou os maiores valores de ganho de peso (0,10 ± 0,16 g), taxa de crescimento específico (0,37 ± 0,11 mm), comprimento (47,78 ± 2,35 mm) e crescimento (2,15 ± 2,27 mm), sugerindo razoável aceitabilidade para dietas artificiais no cultivo desta espécie. Como conclusão, o presente estudo contribui com o desenvolvimento de técnicas para a domesticação espécies de peixes de água doce, de interesse comercial ou ameaçados de extinção, mostrando a domesticação e reproduçao de em cativeiro P. Mangurus selvagens. No entanto, mais estudos devem ser conduzidos no intuito de aumentar a aceitação de dietas comerciais pelos juvenis e melhorar sua taxa sobrevivência.

Effect of low-level laser therapy on the gene expression of collagen and vascular endothelial growth factor in a culture of fibroblast cells in mice.

Low-level laser therapy treatment (LLLT) is widely used in rehabilitation clinics with the aim of accelerating the process of tissue repair; however, the molecular bases of the effect of LLLT have not been fully established. The aim of the present study was to evaluate the influence of the exposure of different doses of LLLT on the expression of collagen genes type I alpha 1 (COL1α1) and vascular endothelial growth factor (VEGF) in the fibroblast cells of mice (L929) cultivated in vitro. Fibroblast cells were irradiated with a Gallium-Arsenide laser (904 nm) every 24 h for 2 consecutive days, stored in an oven at 37 °C, with 5% CO2 and divided into 3 groups: G1-control group, G2-irradiated at 2 J/cm(2), and G3-irradiated at 3 J/cm(2). After irradiation, the total RNA was extracted and used in the complementary DNA (cDNA) synthesis. The gene expression was analyzed by real-time polymerase chain reaction. The cells irradiated in G2 exhibited a statistically significant growth of 1.78 in the expression of the messenger RNA (mRNA) of the COL1α1 gene (p = 0.036) in comparison with G1 and G3. As for the VEGF gene, an increase in expression was observed in the two irradiated groups in comparison with the control group. There was an increase in expression in G2 of 2.054 and G3 of 2.562 (p = 0.037) for this gene. LLLT (904 nm) had an influence on the expression of the genes COL1α1 (2 J/cm(2)) and VEGF (2 e 3 J/cm(2)) in a culture of the fibroblast cells of mice.

Maturação in vitro de oócitos bovinos em meios suplementados com quercetina e seu efeito sobre o desenvolvimento embrionário / In vitro maturation of bovine oocytes in medium supplemented with quercetin, and its effect on embryonic development

A quercetina é um flavonoide, amplamente encontrada em frutas, vegetais, grãos, flores, com elevada concentração no vinho tinto, e tem sido caracterizada funcionalmente pela atividade antioxidante. Para avaliação da maturação nuclear e do desenvolvimento embrionário bovino, os oócitos foram maturados por 22h na presença de quercetina (0,4, 2, 10 e 50µM), cisteamina (100µM) e na ausência dos antioxidantes. Os oócitos maturados foram corados com Hoechst para avaliação da maturação in vitro. Para avaliação do desenvolvimento embrionário, os oócitos foram fertilizados e cultivados in vitro, as taxas de desenvolvimento embrionário foram determinadas no sétimo dia de cultivo e o percentual de eclosão e o número de células dos embriões no oitavo dia. Os níveis de glutationa (GSH) dos oócitos foram mensurados por emissão de fluorescência com CMF2HC. A porcentagem de maturação nuclear (±89%) não diferiu entre os grupos. O desenvolvimento embrionário variou entre os tratamentos, o percentual de blastocisto foi superior (P<0,05) nos grupos tratados com 0,4, 2, 10 e 50∝M de quercetina (56,9, 59,5, 53,6 e 49,6%, respectivamente) e com 100∝M de cisteamina (50,4%) em relação ao grupo controle (42,3%). Na comparação entre os dois antioxidantes, a quercetina (0,4 e 2µM) foi superior na produção de embriões (56,9 e 59,5%, respectivamente) em comparação com cisteamina (50,4%). As taxas de embriões eclodidos foram similares (P>0,05) entre os grupos (±63,0%). O número médio de células dos embriões também foi similar entre os grupos (±233). Os níveis intracelulares de GSH foram superiores nos oócitos maturados com cisteamina, mas similares entre os oócitos tratados com quercetina e o controle. A suplementação da maturação in vitro com antioxidantes melhora as taxas de blastocistos. A quercetina foi superior à cisteamina, que, por sua vez, foi superior ao controle. Mas os níveis de GSH foram superiores somente nos oócitos tratados com cisteamina.

Quercetin is a flavonoid widely found in fruit, vegetables, grains and flowers, with a high concentration in red wine, and has been functionally characterized by its antioxidant activity. For assessment of nuclear maturation and bovine embryo, oocytes were matured for 22h in the presence of quercetin (0.4, 2, 10 and 50µM), cysteamine (100µM) and in the absence of antioxidants. The matured oocytes were stained with Hoechst to evaluate the in vitro maturation. To assess embryonic development, oocytes were fertilized and cultured in vitro and rates of embryo development were obtained in the seventh day of culture and the percentage of hatching and the number of cells on eighth day embryos. The levels of glutathione (GSH) of the oocytes were measured by fluorescence emission with CMF2HC. The percentage of nuclear maturation (±89%) did not differ between groups. Embryonic development varied between treatments, the percentage of blastocyst was higher (P<0.05) in the groups treated with 0.4, 2, 10 and 50∝M of quercetin (56.9, 59.5, 53.6 and 49.6%, respectively) and 100 ∝M cysteamine (50.4%) compared to the control group (42.3%). Comparing the two antioxidants, quercetin (0.4 to 2µM) was superior in embryo production (56.9 and 59.5% respectively) compared with cysteamine (50.4%). The rates of hatched embryos were similar (P>0.05) between groups (±63.0%). The average number of embryo cells was also similar in both groups (±233). The intracellular GSH levels were higher in oocytes matured with cysteamine, but similar between the oocytes treated with quercetin and control. The supplementation of matured in vitro with antioxidants improves blastocyst rates. Quercetin was greater than cysteamine, which in turn was superior to the control. However, GSH levels were higher in oocytes treated only with cysteamine.

Breeding of transgenic cattle for human coagulation factor IX by a combination of lentiviral system and cloning.

Recombinant coagulation factor IX must be produced in mammalian cells because FIX synthesis involves translational modifications. Human cell culture-based expression of human coagulation factor IX (hFIX) is expensive, and large-scale production capacity is limited. Transgenic animals may greatly increase the yield of therapeutic proteins and reduce costs. In this study, we used a lentiviral system to obtain transgenic cells and somatic cell nuclear transfer (SCNT) to produce transgenic animals. Lentiviral vectors carrying hFIX driven by 3 bovine ß-casein promoters were constructed. Bovine epithelial mammary cells were transduced by lentivirus, selected with blasticidin, plated on extracellular matrix, and induced by lactogenic hormones; promoter activity was evaluated by quantitative PCR. Transcriptional activity of the 5.335-kb promoter was 6-fold higher than the 3.392- and 4.279-kb promoters, which did not significantly differ. Transgenic bovine fibroblasts were transduced with lentivirus carrying the 5.335-kb promoter and used as donor cells for SCNT. Cloned transgenic embryo production yielded development rates of 28.4%, similar to previous reports on cloned non-transgenic embryos. The embryos were transferred to recipient cows (N = 21) and 2 births of cloned transgenic cattle were obtained. These results suggest combination of the lentiviral system and cloning may be a good strategy for production of transgenic cattle.

ß-casein gene expression by in vitro cultured bovine mammary epithelial cells derived from developing mammary glands.

Epithelial cells from mammary gland tissue that are cultured in vitro are able to maintain specific functions of this gland, such as cellular differentiation and milk protein synthesis. These characteristics make these cells a useful model to study mammary gland physiology, development and differentiation; they can also be used for production of exogenous proteins of pharmaceutical interest. Bovine mammary epithelial cells were cultured in vitro after isolation from mammary gland tissue of animals at different stages of development. The cells were plated on Petri dishes and isolated from fibroblasts using saline/EDTA treatment, followed by trypsinization. Cells isolated on plastic were capable of differentiating into alveolus-like structures; however, only cells derived from non-pregnant and non-lactating animals expressed ß-casein. Real-time qPCR and epifluorescence microscopy analyses revealed that alveolus-like structures were competent at expressing Emerald green fluorescent protein (EmGFP) driven by the ß-casein promoter, independent of ß-casein expression.