Produção in vitro de embriões de mini vacas / In vitro embryo production of miniature cattle

With a focus on productivity, Brazil has advanced research and application of reproductive biotechnologies. However, much remains to be explored, such as in vitro embryo production and transfer in miniature breeds. Thus, the objective of the present work is to report the in vitro production of miniature bovine embryos and transfer to conventional sized cows located in Palmas/To. Nine miniature Jersey-Holstein cross breed donors were aspirated to obtain approximately 13 viable oocytes per female. The oocytes were matured, fertilized and cultured the in vitro, where the production of viable embryos was 23.2%. On day 7 of development (D7) the embryos were transferred to 17 crossbred dairy recipients and after 30 days of innovulation by ultrasound examination a conception rate of 76.4% was found. Thus, the technique of in vitro production and embryo transfer is viable for the reproduction of mini dairy cows, despite the lack of data regarding the work performed on small animals that serve as comparative parameters for the results achieved in the study.(AU)

Produção in vitro de embriões de mini vacas / In vitro embryo production of miniature cattle

With a focus on productivity, Brazil has advanced research and application of reproductive biotechnologies. However, much remains to be explored, such as in vitro embryo production and transfer in miniature breeds. Thus, the objective of the present work is to report the in vitro production of miniature bovine embryos and transfer to conventional sized cows located in Palmas/To. Nine miniature Jersey-Holstein cross breed donors were aspirated to obtain approximately 13 viable oocytes per female. The oocytes were matured, fertilized and cultured the in vitro, where the production of viable embryos was 23.2%. On day 7 of development (D7) the embryos were transferred to 17 crossbred dairy recipients and after 30 days of innovulation by ultrasound examination a conception rate of 76.4% was found. Thus, the technique of in vitro production and embryo transfer is viable for the reproduction of mini dairy cows, despite the lack of data regarding the work performed on small animals that serve as comparative parameters for the results achieved in the study.

Genetic engineering of Theileria parva lactate dehydrogenase gene: a new anti-theilerial target

Theileria parva is the causative agent of East Coast Fever (ECF), a tick borne disease, which results in major economic losses in cattle. Major problems in dealing with this illness are the high cost of drugs, development of resistance, and absence of effective vaccines. Thus, exploiting new targets for cost effective and higher therapeutic value drugs are imperative. Glycolysis is the main pathway for generation of ATP in T. parva, given its development inside erythrocytes. Thus, the enzymes of this pathway may prove potential targets for designing new-generation anti-theilerials. Lactate dehydrogenase of T. parva (TpLDH) has the highest activity of all glycolytic enzymes and thus we selected this enzyme as the potential therapeutic target. Our study is the first to report the isolation, removal of introns through directed mutagenesis, and cloning of TpLDH and showing that amino acid insertions or deletions most notably corresponded to a 5-amino acid sequence (Asn-91A, Glu-91B, Glu-91C, Trp-91D, Asn-91E) between Ser-91 ve Arg-92 of the enzyme. This region is also present in other apicomplexan such as Babesia bovis, a pathogen of cattle and Plasmodium falciparum, a human pathogen. Providing as the attachment site for the enzyme inhibitors and not being present in LDH of respective hosts, we propose this site as an attractive drug target. The work here is expected to lead new studies on detailed structural and kinetic aspects of apicomplexan LDHs and development of new inhibitors.(AU)

Genetic engineering of Theileria parva lactate dehydrogenase gene: a new anti-theilerial target

Theileria parva is the causative agent of East Coast Fever (ECF), a tick borne disease, which results in major economic losses in cattle. Major problems in dealing with this illness are the high cost of drugs, development of resistance, and absence of effective vaccines. Thus, exploiting new targets for cost effective and higher therapeutic value drugs are imperative. Glycolysis is the main pathway for generation of ATP in T. parva, given its development inside erythrocytes. Thus, the enzymes of this pathway may prove potential targets for designing new-generation anti-theilerials. Lactate dehydrogenase of T. parva (TpLDH) has the highest activity of all glycolytic enzymes and thus we selected this enzyme as the potential therapeutic target. Our study is the first to report the isolation, removal of introns through directed mutagenesis, and cloning of TpLDH and showing that amino acid insertions or deletions most notably corresponded to a 5-amino acid sequence (Asn-91A, Glu-91B, Glu-91C, Trp-91D, Asn-91E) between Ser-91 ve Arg-92 of the enzyme. This region is also present in other apicomplexan such as Babesia bovis, a pathogen of cattle and Plasmodium falciparum, a human pathogen. Providing as the attachment site for the enzyme inhibitors and not being present in LDH of respective hosts, we propose this site as an attractive drug target. The work here is expected to lead new studies on detailed structural and kinetic aspects of apicomplexan LDHs and development of new inhibitors.(AU)

Efficient edition of the bovine PRNP prion gene in somatic cells and IVF embryos using the CRISPR/Cas9 system.

The recently developed engineered nucleases, such as zinc-finger nucleases, transcription activator-like effector nucleases, and clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated nuclease (Cas) 9, provide new opportunities for gene editing in a straightforward manner. However, few reports are available regarding CRISPR application and efficiency in cattle. Here, the CRISPR/Cas9 system was used with the aim of inducing knockout and knock-in alleles of the bovine PRNP gene, responsible for mad cow disease, both in bovine fetal fibroblasts and in IVF embryos. Five single-guide RNAs were designed to target 875 bp of PRNP exon 3, and all five were codelivered with Cas9. The feasibility of inducing homologous recombination (HR) was evaluated with a reporter vector carrying EGFP flanked by 1 kbp PRNP regions (pHRegfp). For somatic cells, plasmids coding for Cas9 and for each of the five single-guide RNAs (pCMVCas9 and pSPgRNAs) were transfected under two different conditions (1X and 2X). For IVF zygotes, cytoplasmic injection was conducted with either plasmids or mRNA. For plasmid injection groups, 1 pg pCMVCas9 + 0.1 pg of each pSPgRNA (DNA2X) was used per zygote. In the case of RNA, two amounts (RNA1X and RNA2X) were compared. To assess the occurrence of HR, a group additionally cotransfected or coinjected with pHRegfp plasmid was included. Somatic cell lysates were analyzed by polymerase chain reaction and surveyor assay. In the case of embryos, the in vitro development and the genotype of blastocysts were evaluated by polymerase chain reaction and sequencing. In somatic cells, 2X transfection resulted in indels and large deletions of the targeted PRNP region. Regarding embryo injection, higher blastocyst rates were obtained for RNA injected groups (46/103 [44.6%] and 55/116 [47.4%] for RNA1X and RNA2X) than for the DNA2X group (26/140 [18.6%], P < 0.05). In 46% (26/56) of the total sequenced blastocysts, specific gene editing was detected. The total number of genetic modifications (29) was higher than the total number of gene-edited embryos, as three blastocysts from the group RNA2X reported more than one type of modification. The modifications included indels (10/56; 17.9%) and large deletions (19/56; 33.9%). Moreover, it was possible to detect HR in 1/8 (12.5%) embryos treated with RNA2X. These results report that the CRISPR/Cas9 system can be applied for site-specific edition of the bovine genome, which could have a great impact on the development of large animals resistant to important zoonotic diseases.

Genética molecular na seleção caprina / Caprina molecular genetics in goat selection

O desenvolvimento científico e os avanços da genética molecular, tem permitido progressos significativos nos estudos de seleção com a espécie caprina, ainda que recente. Objetivou-se descrever o processo evolutivo das pesquisas com as tecnologias genéticas, esclarecer as utilidades aplicacionais dos marcadores moleculares e como as diferentes técnicas estão sendo utilizadas na identificação de genes de interesse para a caprinocultura, como também, as perspectivas do seu uso na seleção. As técnicas utilizadas através dos marcadores moleculares têm sido, satisfatoriamente aplicadas, pois possibilita realizar a seleção de animais cada vez mais cedo dentro do rebanho. A importância da investigação neste campo, é indiscutível, pois graças à tecnologia molecular tem abastecido um elevado número de marcadores de DNA, que vem gerando informações úteis, de forma gradativa e acumulativa, para a seleção genômica como uma nova ferramenta no melhoramento animal.(AU)

Genética molecular na seleção caprina / Caprina molecular genetics in goat selection

O desenvolvimento científico e os avanços da genética molecular, tem permitido progressos significativos nos estudos de seleção com a espécie caprina, ainda que recente. Objetivou-se descrever o processo evolutivo das pesquisas com as tecnologias genéticas, esclarecer as utilidades aplicacionais dos marcadores moleculares e como as diferentes técnicas estão sendo utilizadas na identificação de genes de interesse para a caprinocultura, como também, as perspectivas do seu uso na seleção. As técnicas utilizadas através dos marcadores moleculares têm sido, satisfatoriamente aplicadas, pois possibilita realizar a seleção de animais cada vez mais cedo dentro do rebanho. A importância da investigação neste campo, é indiscutível, pois graças à tecnologia molecular tem abastecido um elevado número de marcadores de DNA, que vem gerando informações úteis, de forma gradativa e acumulativa, para a seleção genômica como uma nova ferramenta no melhoramento animal.

Transgênese em caprinos / Transgenesis in goats

Entre todos os mamíferos transgênicos produzidos até hoje, a espécie caprina tem representado um excelente modelo em transgênese quando são considerados os fatores como a demanda do mercado para a proteína, o volume de leite produzido por lactação e as taxas reprodutivas. Várias proteínas recombinantes foram obtidas a partir de cabras transgênicas, e entre estas, a antitrombina humana, foi a primeira proteína recombinante de origem animal a ser aprovada como um medicamento para a utilização clínica em seres humanos. Esta revisão tem por objetivo apresentar o estado-da-arte em caprinos transgênicos dando especial ênfase aos resultados obtidos por nosso grupo.

Among all the transgenic mammalians produced so far, goats have represented an excellent model of transgenesis when considering the factors such as the market demand for protein, volume of milk produced per lactation and reproductive rate. Various recombinant proteins have been obtained from the transgenic goats, and among these, human antithrombin was the first recombinant protein of animal origin to be released as a drug for the clinical use in humans. This review aims to present the state-of-the-art in transgenic goats with special emphasis on results obtained by our group.

Genetic engineering of livestock to improve human health: The human lysozyme transgenic goat model

Background: Transgenic animals have been generated for a variety of purposes including research tools, medical models, bioreactors (dairy animals producing human pharmaceuticals in their milk or in the egg whites of chickens) and for production agriculture (animals with increased growth, decreased environmental pollution, disease resistance), which includes the generation of animals designed to benefit human health. For example, transgenic goats expressing human lysozyme in their milk are being used as a model method to supply milk with antibacterial properties to help fight diarrheal illnesses in children. Lysozyme is a naturally occurring antimicrobial found in human milk at much higher levels than in the milk of dairy goats and cows. Lysozyme serves as part of the natural defense system against infection and also helps establish a healthy gut microbiota in the infant. We hypothesized that the presence of increased levels of lysozyme in the milk of dairy goats could offer several benefits that affect human health, including the promotion of a healthy gut microbiota and associated benefits such as improved growth and resistance to intestinal infections. Review: Research with this line of transgenic goats over the last 12 years has demonstrated that the presence and expression of the human lysozyme transgene is not detrimental to the animals themselves and that the milk can indeed act in an antimicrobial fashion when consumed by pigs, a model animal for human health, and impact the state of the intestine in a positive manner. Pigs consuming milk from human lysozyme transgenic goats had significantly lower levels of coliforms and E. coli in their intestine than did pigs consuming milk from non-transgenic control animals. In addition to bacterial changes, intestinal tissue of pigs consuming milk from lysozyme transgenic goats had a significantly larger surface area with significantly fewer intraepithelial lymphocytes and an elevated level of expression of the anti-inflammatory cytokine TGF-â1 compared to control-fed animals, all indicators of a healthier intestinal tract. Metabolite profile analysis demonstrated significant differences in the levels of 18 metabolites in the serum of pigs fed lysozyme milk with the direction of changes beneficial to the health of the animal. Finally, pigs consuming milk from lysozyme transgenic goats were more resistant to infection when challenged with an enteropathogenic E. coli, indicating a protective effect of lysozyme milk. In the semi-arid northeast of Brazil, 89 of 1000 children die before they reach the age of 2 years and 17% of these deaths can be attributed to diarrhea. The use of genetically engineered animals containing increased levels of lysozyme in their milk is a novel and simple approach to fight this problem. Work will be presented outlining the characterization of these animals and the impact of consuming the milk with the goal of one day having the milk available as a preventative or treatment agent for diarrheal illnesses. Conclusions: Genetic engineering is a viable approach to produce animal food products that can be used to improve human health. Brazil's acceptance of this technology has positioned itself at the forefront to demonstrate to the world a new tool to help fight common diarrhea and its impact on the growth and development of children.