Heterogeneity determination of bovine leukemia virus genome in Santa Catarina state, Brazil / Determinação da heterogeneidade do genoma do vírus da leucose enzoótica bovina no estado de Santa Catarina, Brasil

Bovine leukemia virus (BLV) is a member of Retroviridae family, genus Deltaretrovirus, and the main viral agent responsible for economic loses in dairy herds. Some studies have been carried out about BLV genotypes, and at least seven genotypes were found out in samples of different regions of the world. The objective of this study was to identify BLV samples from seropositive dairy cattle in Santa Catarina state, Brazil, using molecular techniques. Blood samples were collected (454) from dairy cattle from 31 different farms, and serology using agar gel immunodiffusion test (AGID) was performed. After that, 191 seropositive samples were submitted to DNA extraction, and in 77 samples the polymerase chain reaction (PCR) for amplification of a 440 bp fragment of the env gene was performed. Nineteen DNA samples were subjected to restriction fragment length polymorphism (RFLP) analysis by digestion of the PCR fragment by five restriction endonucleases - BamHI, HaeIII, Tru9I, TaqI, and MwoI. It was found 42% seropositive animals (191/454) and 68% positives of the farms (21/31). The PCR showed 80.5% (62/77) of animals positive. The RFLP analysis identified five different genotypes dispersed by Santa Catarina state, with the highest prevalence for genotype X (47.4%). Overall, our results identified the viral genotypes present in dairy cattle and the prevalence of new variants in representative farms from Santa Catarina state.(AU)

O bovine leukemia virus (BLV) é um membro da família Retroviridae, gênero Deltaretrovirus, e o principal agente viral causador de perdas econômicas em rebanhos leiteiros. Diversos estudos têm sido feitos sobre os genótipos de BLV, e foram encontrados pelo menos sete em amostras de diferentes partes do mundo. O objetivo deste estudo foi realizar a caracterização molecular de amostras de BLV de bovinos leiteiros soropositivos no estado de Santa Catarina. Foram coletadas 454 amostras de sangue de bovinos de 31 propriedades, e fez-se inicialmente a sorologia por meio do teste de imunodifusão em gel de ágar. Após a sorologia, 191 amostras soropositivas foram então submetidas à extração de DNA, e em 77 amostras se realizou a reação da polimerase em cadeia (PCR), para a amplificação de um fragmento de 440 pb do gene env. Dezenove amostras foram submetidas à análise do polimorfismo dos fragmentos de restrição por digestão do fragmento da PCR por cinco enzimas de restrição: BamHI, HaeIII, Tru9I, TaqI e MwoI. Os resultados obtidos na sorologia apontaram 42% de animais soropositivos (191/454) e 68% de propriedades positivas (21/31). Na PCR, 80,52% (62/77) dos animais apresentaram-se positivos. A análise do polimorfismo dos fragmentos de restrição identificou cinco genótipos circulantes no estado, e a maior prevalência foi observada no genótipo X (47,4%). Este estudo permite-nos conhecer alguns dos genótipos virais presentes em bovinos leiteiros do estado de Santa Catarina, bem como identificar a existência de novas variantes e sua prevalência atual, e os resultados são úteis para futuros estudos epidemiológicos.(AU)

Heterogeneity determination of bovine leukemia virus genome in Santa Catarina state, Brazil / Determinação da heterogeneidade do genoma do vírus da leucose enzoótica bovina no estado de Santa Catarina, Brasil

Bovine leukemia virus (BLV) is a member of Retroviridae family, genus Deltaretrovirus, and the main viral agent responsible for economic loses in dairy herds. Some studies have been carried out about BLV genotypes, and at least seven genotypes were found out in samples of different regions of the world. The objective of this study was to identify BLV samples from seropositive dairy cattle in Santa Catarina state, Brazil, using molecular techniques. Blood samples were collected (454) from dairy cattle from 31 different farms, and serology using agar gel immunodiffusion test (AGID) was performed. After that, 191 seropositive samples were submitted to DNA extraction, and in 77 samples the polymerase chain reaction (PCR) for amplification of a 440 bp fragment of the env gene was performed. Nineteen DNA samples were subjected to restriction fragment length polymorphism (RFLP) analysis by digestion of the PCR fragment by five restriction endonucleases - BamHI, HaeIII, Tru9I, TaqI, and MwoI. It was found 42% seropositive animals (191/454) and 68% positives of the farms (21/31). The PCR showed 80.5% (62/77) of animals positive. The RFLP analysis identified five different genotypes dispersed by Santa Catarina state, with the highest prevalence for genotype X (47.4%). Overall, our results identified the viral genotypes present in dairy cattle and the prevalence of new variants in representative farms from Santa Catarina state.(AU)

O bovine leukemia virus (BLV) é um membro da família Retroviridae, gênero Deltaretrovirus, e o principal agente viral causador de perdas econômicas em rebanhos leiteiros. Diversos estudos têm sido feitos sobre os genótipos de BLV, e foram encontrados pelo menos sete em amostras de diferentes partes do mundo. O objetivo deste estudo foi realizar a caracterização molecular de amostras de BLV de bovinos leiteiros soropositivos no estado de Santa Catarina. Foram coletadas 454 amostras de sangue de bovinos de 31 propriedades, e fez-se inicialmente a sorologia por meio do teste de imunodifusão em gel de ágar. Após a sorologia, 191 amostras soropositivas foram então submetidas à extração de DNA, e em 77 amostras se realizou a reação da polimerase em cadeia (PCR), para a amplificação de um fragmento de 440 pb do gene env. Dezenove amostras foram submetidas à análise do polimorfismo dos fragmentos de restrição por digestão do fragmento da PCR por cinco enzimas de restrição: BamHI, HaeIII, Tru9I, TaqI e MwoI. Os resultados obtidos na sorologia apontaram 42% de animais soropositivos (191/454) e 68% de propriedades positivas (21/31). Na PCR, 80,52% (62/77) dos animais apresentaram-se positivos. A análise do polimorfismo dos fragmentos de restrição identificou cinco genótipos circulantes no estado, e a maior prevalência foi observada no genótipo X (47,4%). Este estudo permite-nos conhecer alguns dos genótipos virais presentes em bovinos leiteiros do estado de Santa Catarina, bem como identificar a existência de novas variantes e sua prevalência atual, e os resultados são úteis para futuros estudos epidemiológicos.(AU)

In vitro development and cell allocation after aggregation of syngeneic wild type and fluorescence-expressing bovine cloned embryos

Background: The in vitro production (IVP) of embryos by in vitro fertilization or cloning procedures has been known to cause epigenetic changes in the conceptus that in turn are associated with abnormalities in pre-and postnatal development. Handmade cloning (HMC) procedures and the culture of zona-free embryos in individual microwells provide excellent tools for studies in developmental biology, since embryo development and cell allocation patterns can be evaluated under a wide range of embryo reconstruction arrangements and in in vitro embryo culture conditions. As disturbances in embryonic cell allocation after in vitro embryo manipulations and unusual in vivo conditions during the first third of pregnancy appear to be associated with large offspring, embryo aggregation procedures may allow a compensation for epigenetic defects between aggregated embryos or even may influence more favorable cell allocation in embryonic lineages, favoring subsequent development. Thus, the aim of this study was to evaluate in vitro embryo developmental potential and the pattern of cell allocation in blastocysts developed after the aggregation of handmade cloned embryos produced using syngeneic wild type and/or transgenic somatic cells. Materials, Methods & Results: In vitro-matured bovine cumulus-oocyte complexes (COC) were manually bisected after cumulus and zona pellucida removal; then, two enucleated hemi-oocytes were paired and fused with either a wild type (WT) or a GFP-expressing (GFP) fetal skin cell at the 11th and 19th passages, respectively. Following chemical activation, reconstructed cloned embryos and zona-free parthenote embryos were in vitro-cultured in microwells, for 7 days, either individually (1 x 100%) or after the aggregation of two structures (2 x 100%) per microwell, as follows: (G1) one WT cloned embryo; (G2) two aggregated WT embryos; (G3) one GFP cloned embryo; (G4) two aggregated GFP embryos; (G5) aggregation of a WT embryo and a GFP embryo; (G6) one parthenote embryo; or (G7) two aggregated parthenote embryos. Fusion (clones), cleavage (Day 2), and blastocyst (Day 7) rates, and embryonic cell allocation were compared by the x² or Fisher tests. Total cell number (TCN) in blastocysts was analyzed by the Student's test (P < 0.05). Fusion and cleavage rates, and cell allocation were similar between groups. On a per WOW basis, development to the blastocyst stage was similar between groups, except for lower rates of development seen in G3. However, when based on number of embryos per group (one or two), blastocyst development was higher in G1 than all other groups, which were similar between one another. Cloned GFP embryos had lower in vitro development to the blastocyst stage than WT embryos, which had more TCN than parthenote or aggregated chimeric WT/GFP embryos. Aggregated GFP embryos had fewer cells than the other embryo groups. Discussion: The in vitro development of GFP cloned embryos was lower than WT embryos, with no effects on cell allocation in resulting blastocysts. Differences in blastocyst rate between groups were likely due to lower GFP-expressing cell viability, as GFP donor cells were at high population cell doublings when used for cloning. On a per embryo basis, embryo aggregation on Day 1 resulted in blastocyst development similar to non-aggregated embryos on Day 7, with no differences in cell proportion between groups. The use of GFP-expressing cells was proven a promising strategy for the study of cell allocation during embryo development, which may assist in the elucidation of mechanisms of abnormalities after in vitro embryo manipulations, leading to the development of improved protocols for the in vitro production (IVP) of bovine embryos.

In vitro development and cell allocation after aggregation of syngeneic wild type and fluorescence-expressing bovine cloned embryos / In vitro development and cell allocation after aggregation of syngeneic wild type and fluorescence-expressing bovine cloned embryos

Materials, Methods & Results: In vitro-matured bovine cumulus-oocyte complexes (COC) were manually bisected after cumulus and zona pellucida removal; then, two enucleated hemi-oocytes were paired and fused with either a wild type (WT) or a GFP-expressing (GFP) fetal skin cell at the 11th and 19th passages, respectively. Following chemical activation, reconstructed cloned embryos and zona-free parthenote embryos were in vitro-cultured in microwells, for 7 days, either individually (1 x 100%) or after the aggregation of two structures (2 x 100%) per microwell, as follows: (G1) one WT cloned embryo; (G2) two aggregated WT embryos; (G3) one GFP cloned embryo; (G4) two aggregated GFP embryos; (G5) aggregation of a WT embryo and a GFP embryo; (G6) one parthenote embryo; or (G7) two aggregated parthenote embryos. Fusion (clones), cleavage (Day 2), and blastocyst (Day 7) rates, and embryonic cell allocation were compared by the 2 or Fisher tests. Total cell number (TCN) in blastocysts was analyzed by the Student´s test (P 0.05). Fusion and cleavage rates, and cell allocation were similar between groups. On a per WOW basis, development to the blastocyst stage was similar between groups, except for lower rates of development seen in G3. However, when based on number of embryos per group (one or two), blastocyst development was higher in G1 than all other groups, which were simi

Background: The in vitro production (IVP) of embryos by in vitro fertilization or cloning procedures has been known to cause epigenetic changes in the conceptus that in turn are associated with abnormalities in pre- and postnatal development. Handmade cloning (HMC) procedures and the culture of zona-free embryos in individual microwells provide excellent tools for studies in developmental biology, since embryo development and cell allocation patterns can be evaluated under a wide range of embryo reconstruction arrangements and in in vitro embryo culture conditions. As disturbances in embryonic cell allocation after in vitro embryo manipulations and unusual in vivo conditions during the fi rst third of pregnancy appear to be associated with large offspring, embryo aggregation procedures may allow a compensation for epigenetic defects between aggregated embryos or even may infl uence more favorable cell allocation in embryonic lineages, favoring subsequent development. Thus, the aim of this study was to evaluate in vitro embryo developmental potential and the pattern of cell allocation in blastocysts developed after the aggregation of handmade cloned embryos produced using syngeneic wild type and/or transgenic somatic cells.Materials, Methods & Results: In vitro-matured bovine cumulus-oocyte complexes (COC) were manually bisected after cumulus and zona pellucida removal; then

Mice Embryos Cryopreservation: Vitrification or Ultra-Rapid Freezing?

The importance of mice as animal model for research has promoted the surge of many strains with important characteristics which need to be preserved. Embryo cryopreservation appears as the most suitable technique. However, until now there is not an effective methodology for this specie. This study aimed to evaluate three methods for mice embryos cryopreservation.

Mice Embryos Cryopreservation: Vitrification or Ultra-Rapid Freezing?

The importance of mice as animal model for research has promoted the surge of many strains with important characteristics which need to be preserved. Embryo cryopreservation appears as the most suitable technique. However, until now there is not an effective methodology for this specie. This study aimed to evaluate three methods for mice embryos cryopreservation.(AU)

Viabilidade de fibroblastos bovinos submetidos a diferentes estratégias de criopreservação / Bovine fibroblast viability after cryopreservation using distinct protocols

A avaliação da viabilidade de células animais submetidas à criopreservação é importante para estudos em cultivo celular, aplicação em clonagem animal e para o estabelecimento e manutenção de bancos genéticos. O objetivo do trabalho é otimizar uma metodologia de criopreservação de células somáticas bovinas utilizando diferentes recipientes e crioprotetores. Para tanto, três experimentos foram conduzidos utilizando células fibroblásticas de origem cutânea foram obtidas por meio de biópsia auricular de uma fêmea da raça Flamenga. No primeiro experimento, as células foram criopreservadas em criotubos na concentração fixa de 1x106 células/mL em três soluções de congelamento (1) meio de cultivo +10% de DMSO; (2) meio de cultivo + 10% de Propileno glicol; (3) meio de cultivo + 10% de Etileno glicol. No segundo experimento, o congelamento foi realizado com as mesmas soluções crioprotetoras utilizadas no experimento um, porém as células foram criopreservadas em palhetas de 0,25 mL e 0,5 mL. No terceiro experimento, foram testadas diferentes concentrações celulares, (0,33x106 cel/mL, 1x106 cel/mL, 3x106 cel/mL e 5x106 cel/mL) que foram criopreservadas em palheta de 0,5 mL utilizando-se meio de cultivo + 10% propileno glicol. Em todos os experimentos, as células foram descongeladas em banho maria a 37 oC para avaliação da sobrevivência celular pelo método de azul de Tripan e avaliação da curva de crescimento celular após 72 h de cultivo, além disso, no primeiro experimento foi realizado o teste de population doubling time (PDT) e no segundo e terceiro experimento o grupo controle foi realizado com células criopreservadas em criotubo utilizando a solução de congelamento composta meio de cultivo +10% de DMSO. Os dados foram analisados através da análise de variância com comparações pareadas entre grupos pelo teste de Tukey, com nível de significância de 5%. No primeiro experimento, não houve diferença significativa entre os grupos utilizando diferentes crioprotetores e o tempo de divisão celular estimado pelo PDT variou de 15,9 a 16,5 horas. No segundo experimento, a taxa de sobrevivência pós descongelamento, considerando as palhetas de 0,25 e 0,5 mL, não apresentou diferença significativa entre os grupos, independente do crioprotetor utilizado. Já na curva de crescimento, a sobrevivência das células criopreservadas em palhetas de 0,25 mL apresentou um desempenho estatisticamente inferior às congeladas em palhetas de 0,5 mL e criotubos. Neste experimento todos os grupos apresentaram resultados de sobrevivência significativamente inferiores ao grupo controle. No terceiro experimento as concentrações celulares que apresentaram melhores resultados de sobrevivência após o descongelamento foram de 0,33x106 cel/mL e 1x106 cel/mL. Com base nos resultados obtidos conclui-se que é possível utilizar palhetas para o congelamento de células e que as palhetas de 0,5 mL apresentaram resultados compatíveis aos obtidos com criotubos. Da mesma forma o uso de PG pode ser uma alternativa viável para o congelamento de células somáticas

The viability evaluation of animal cells subjected to different cryopreservation strategies is important for studies in cell culture, application in animal cloning and the establishment and maintenance of gene banks. The aim of this study is optimize a method for cryopreservation of bovine somatic cells in different containers using different cryoprotectants. For this purpose, three experiments were conducted using bovine skin fibroblast cells obtained by ear biopsy explantation by ear biopsy of a female Flemish breed. In the first experiment the cells were cryopreserved in cryotubes at fixed concentration of 1x106 cells/mL and in three freezing culture medium (1) culture medium +10% DMSO, (2) culture medium + 10% propylene glycol, (3) culture medium + 10% ethylene glycol. In the second experiment the freezing was performed with the same solutions used in experiment 1, however, the cells were cryopreserved in 0.25 mL and 0.5 mL straws. In the third experiment cells in different concentrations, (0.33x106 cells/mL, 1x106 cells/mL, 3x106 cells/mL and 5x106 cells/mL) were cryopreserved in 0.5 mL straws with culture medium + 10% propylene glycol. In all experiments, the cells were thawed in waterbath at 37 oC for evaluation of cell survival by trypan blue method and by the cell growth curve after 72 h in culture, moreover, in the first experiment was carried out the population doubling time test (PDT). The data were analyzed by analysis of variance with pairwise comparisons between groups by the Tukey test, with significance level of 5%. In first experiment there was no significant difference between groups using different cryoprotectants and the time of cell division, estimated by PDT, ranged from 15.9 to 16.5 hours.In the second experiment no significant difference in the survival rate between groups was observed, regardless of the cryoprotectant used, and considering the 0.25 and 0.5 mL straws. In the growth curve, the survival performance of cells cryopreserved in 0.25 ml straws was statistically inferior to that frozen in 0.5 mL straws and cryovials. In this experiment all groups had significantly lower survival results campared to the control group. In the third experiment, the cell concentrations that presented a better survival rate after thawing were 0.33x106cells/ mL and 1x106 cells /mL. Based on the results obtained it follows that straws can be used for freezing cells and the straws of 0.5 mL showed results consistent with those obtained by cryotubes. Likewise the use of PG can be a viable alternative for the freezing somatic cells

In vitro development and cell allocation after aggregation of syngeneic wild type and fluorescence-expressing bovine cloned embryos / In vitro development and cell allocation after aggregation of syngeneic wild type and fluorescence-expressing bovine cloned embryos

Materials, Methods & Results: In vitro-matured bovine cumulus-oocyte complexes (COC) were manually bisected after cumulus and zona pellucida removal; then, two enucleated hemi-oocytes were paired and fused with either a wild type (WT) or a GFP-expressing (GFP) fetal skin cell at the 11th and 19th passages, respectively. Following chemical activation, reconstructed cloned embryos and zona-free parthenote embryos were in vitro-cultured in microwells, for 7 days, either individually (1 x 100%) or after the aggregation of two structures (2 x 100%) per microwell, as follows: (G1) one WT cloned embryo; (G2) two aggregated WT embryos; (G3) one GFP cloned embryo; (G4) two aggregated GFP embryos; (G5) aggregation of a WT embryo and a GFP embryo; (G6) one parthenote embryo; or (G7) two aggregated parthenote embryos. Fusion (clones), cleavage (Day 2), and blastocyst (Day 7) rates, and embryonic cell allocation were compared by the 2 or Fisher tests. Total cell number (TCN) in blastocysts was analyzed by the Student´s test (P 0.05). Fusion and cleavage rates, and cell allocation were similar between groups. On a per WOW basis, development to the blastocyst stage was similar between groups, except for lower rates of development seen in G3. However, when based on number of embryos per group (one or two), blastocyst development was higher in G1 than all other groups, which were simi

Background: The in vitro production (IVP) of embryos by in vitro fertilization or cloning procedures has been known to cause epigenetic changes in the conceptus that in turn are associated with abnormalities in pre- and postnatal development. Handmade cloning (HMC) procedures and the culture of zona-free embryos in individual microwells provide excellent tools for studies in developmental biology, since embryo development and cell allocation patterns can be evaluated under a wide range of embryo reconstruction arrangements and in in vitro embryo culture conditions. As disturbances in embryonic cell allocation after in vitro embryo manipulations and unusual in vivo conditions during the fi rst third of pregnancy appear to be associated with large offspring, embryo aggregation procedures may allow a compensation for epigenetic defects between aggregated embryos or even may infl uence more favorable cell allocation in embryonic lineages, favoring subsequent development. Thus, the aim of this study was to evaluate in vitro embryo developmental potential and the pattern of cell allocation in blastocysts developed after the aggregation of handmade cloned embryos produced using syngeneic wild type and/or transgenic somatic cells.Materials, Methods & Results: In vitro-matured bovine cumulus-oocyte complexes (COC) were manually bisected after cumulus and zona pellucida removal; then