Insights on bovine genetic engineering and cloning

Transgenic technology has become an essential tool for the development of animal biotechnologies, and animal cloning through somatic cell nuclear transfer (SCNT) enabled the generation of genetically modified animals utilizing previously modified and selected cell lineages as nuclei donors, assuring therefore the generation of homogeneous herds expressing the desired modification. The present study aimed to discuss the use of SCNT as an important methodology for the production of transgenic herds, and also some recent insights on genetic modification of nuclei donors and possible effects of gene induction of pluripotency on SCNT.

Compounds used to produce cloned animals are genotoxic and mutagenic in mammalian assays in vitro and in vivo

The compounds 6-dimethylaminopurine and cycloheximide promote the successful production of cloned mammals and have been used in the development of embryos produced by somatic cell nuclear transfer. This study investigated the effects of 6-dimethylaminopurine and cycloheximide in vitro, using the thiazolyl blue tetrazolium bromide colorimetric assay to assess cytotoxicity, the trypan blue exclusion assay to assess cell viability, the comet assay to assess genotoxicity, and the micronucleus test with cytokinesis block to test mutagenicity. In addition, the comet assay and the micronucleus test were also performed on peripheral blood cells of 54 male Swiss mice, 35 g each, to assess the effects of the compounds in vivo. The results indicated that both 6-dimethylaminopurine and cycloheximide, at the concentrations and doses tested, were cytotoxic in vitro and genotoxic and mutagenic in vitro and in vivo, altered the nuclear division index in vitro, but did not diminish cell viability in vitro. Considering that alterations in DNA play important roles in mutagenesis, carcinogenesis, and morphofunctional teratogenesis and reduce embryonic viability, this study indicated that 6-dimethylaminopurine and cycloheximide utilized in the process of mammalian cloning may be responsible for the low embryo viability commonly seen in nuclear transfer after implantation in utero.

Casein fractions of ultra high temperature milk with different somatic cell counts

The objective of this work was to evaluate the effect of somatic cell counts (SCC) in casein fractions of ultra high temperature (UHT) milk. Raw milks were categorized in SCC groups of low (200,000-320,000 cells mL-1), intermediate (380,000-560,000 cells mL-1) and high cells (600,000-800,000 cells mL-1). Five replicates of UHT milks within each SCC category were analyzed for casein fractions after 8, 30, 60, 90 and 120 days of storage through high performance liquid chromatography. SCC showed effect only on beta-casein reduction. SCC in raw milk increases the proteolysis of UHT milk, as a consequence of beta-casein degradation.

alpha-Smooth muscle actin and proliferating cell nuclear antigen expression in focal segmental glomerulosclerosis: functional and structural parameters of renal disease progression

The aim of the present study was to investigate the expression of alpha-smooth muscle actin (alpha-SM-actin) and proliferating cell nuclear antigen (PCNA) in renal cortex from patients with focal segmental glomerulosclerosis (FSGS) and their correlations with parameters of renal disease progression. We analyzed renal biopsies from 41 patients with idiopathic FSGS and from 14 control individuals. The alpha-SM-actin immunoreaction was evaluated using a score that reflected the changes in the extent and intensity of staining in the glomerular or cortical area. The PCNA reaction was quantified by counting the labeled cells of the glomeruli or renal cortex. The results, reported as median ± percentile (25th; 75th), showed that the alpha-SM-actin scores in the glomeruli and tubulointerstitium from the renal cortex were 2.0 (2.0; 4.0) and 3.0 (3.0; 4.0), respectively, in patients with FSGS, and 0.5 (0.0; 1.0) and 0.0 (0.0; 0.5) in the controls. The number of PCNA-positive cells per glomerulus and graded field of tubulointerstitium from the renal cortex was 0.2 (0.0; 0.4) and 1.1 (0.3; 2.2), respectively, for patients with FSGS, and 0.0 (0.0; 0.5) and 0.0 (0.0; 0.0) for controls. The present data showed an increase of alpha-SM-actin and PCNA expression in glomeruli and renal cortex from FSGS patients. The extent of immunoreaction for alpha-SM-actin in the tubulointerstitial area was correlated with the intensity of proteinuria. However, there was no correlation between the kidney expression of these proteins and the reciprocal of plasma creatinine level or renal fibrosis. These findings suggest that the immunohistochemical alterations may be reversible.

Production of a cloned calf from a fetal fibroblast cell line

The present study examined the in vitro and in vivo development of bovine nuclear-transferred embryos. A bovine fetal fibroblast culture was established and used as nucleus donor. Slaughterhouse oocytes were matured in vitro for 18 h before enucleation. Enucleated oocytes were fused with fetal fibroblasts with an electric stimulus and treated with cytochalasin D and cycloheximide for 1 h followed by cycloheximide alone for 4 h. Reconstructed embryos were cultured for 7-9 days and those which developed to blastocysts were transferred to recipient cows. Of 191 enucleated oocytes, 83 (43.5%) were successfully fused and 24 (28.9%) developed to blastocysts. Eighteen freshly cloned blastocysts were transferred to 14 recipients, 5 (27.8%) of which were pregnant on day 35 and 3 (16.7%) on day 90. Of the three cows that reached the third trimester, one recipient died of hydrallantois 2 months before term, one aborted fetus was recovered at 8 months of gestation, and one delivered by cesarian section a healthy cloned calf. Today, the cloned calf is 15 months old and presents normal body development (378 kg) and sexual behavior (libido and semen characteristics).

Effect of sequential heat and cold shocks on nuclear phenotypes of the blood-sucking insect, Panstrongylus megistus (Burmeister) (Hemiptera, Reduviidae)

Thermal shocks induce changes in the nuclear phenotypes that correspond to survival (heterochromatin decondensation, nuclear fusion) or death (apoptosis, necrosis) responses in the Malpighian tubules of Panstrongylus megistus. Since thermal tolerance increased survival and molting rate in this species following sequential shocks, we investigated whether changes in nuclear phenotypes accompanied the insect survival response to sequential thermal shocks. Fifth instar nymphs were subjected to a single heat (35 or 40°C, 1 h) or cold (5 or 0°C, 1 h) shock and then subjected to a second shock for 12 h at 40 or 0°C, respectively, after 8, 18, 24 and 72 h at 28°C (control temperature). As with specimen survival, sequential heat and cold shocks induced changes in frequency of the mentioned nuclear phenotypes although their patterns differed. The heat shock tolerance involved decrease in apoptosis simultaneous to increase in cell survival responses. Sequential cold shocks did not involve cell/nuclear fusion and even elicited increase in necrosis with advancing time after shocks. The temperatures of 40 and 0ºC were more effective than the temperatures of 35 and 5ºC in eliciting the heat and cold shock tolerances, respectively, as shown by cytological analysis of the nuclear phenotypes. It is concluded that different sequential thermal shocks can trigger different mechanisms of cellular protection against stress in P. megistus, favoring the insect to adapt to various ecotopes.

Paullinia cupana Mart var. sorbilis, guaraná, reduces cell proliferation and increases apoptosis of B16/F10 melanoma lung metastases in mice

We showed that guaraná (Paullinia cupana Mart var. sorbilis) had a chemopreventive effect on mouse hepatocarcinogenesis and reduced diethylnitrosamine-induced DNA damage. In the present experiment, we evaluated the effects of guaraná in an experimental metastasis model. Cultured B16/F10 melanoma cells (5 x 10(5) cells/animal) were injected into the tail vein of mice on the 7th day of guaraná treatment (2.0 mg P. cupana/g body weight, per gavage) and the animals were treated with guaraná daily up to 14 days until euthanasia (total treatment time: 21 days). Lung sections were obtained for morphometric analysis, apoptotic bodies were counted to calculate the apoptotic index and proliferating cell nuclear antigen-positive cells were counted to determine the proliferation index. Guaraná-treated (GUA) animals presented a 68.6% reduction in tumor burden area compared to control (CO) animals which were not treated with guaraná (CO: 0.84 ± 0.26, N = 6; GUA: 0.27 ± 0.24, N = 6; P = 0.0043), a 57.9% reduction in tumor proliferation index (CO: 23.75 ± 20.54, N = 6; GUA: 9.99 ± 3.93, N = 6; P = 0.026) and a 4.85-fold increase in apoptotic index (CO: 66.95 ± 22.95, N = 6; GUA: 324.37 ± 266.74 AB/mm², N = 6; P = 0.0152). In this mouse model, guaraná treatment decreased proliferation and increased apoptosis of tumor cells, consequently reducing the tumor burden area. We are currently investigating the molecular pathways of the effects of guaraná in cultured melanoma cells, regarding principally the cell cycle inhibitors and cyclins.

REGγ is a strong candidate for the regulation of cell cycle, proliferation and the invasion by poorly differentiated thyroid carcinoma cells

REGγ is a proteasome activator that facilitates the degradation of small peptides. Abnormally high expression of REGγ has been observed in thyroid carcinomas. The purpose of the present study was to explore the role of REGγ in poorly differentiated thyroid carcinoma (PDTC). For this purpose, small interfering RNA (siRNA) was introduced to down-regulate the level of REGγ in the PDTC cell line SW579. Down-regulation of REGγ at the mRNA and protein levels was confirmed by RT-PCR and Western blot analyses. FACS analysis revealed cell cycle arrest at the G1/S transition, the MTT assay showed inhibition of cell proliferation, and the Transwell assay showed restricted cell invasion. Furthermore, the expression of the p21 protein was increased, the expression of proliferating cell nuclear antigen (PCNA) protein decreased, and the expression of the p27 protein was unchanged as shown by Western blot analyses. REGγ plays a critical role in the cell cycle, proliferation and invasion of SW579 cells. The alteration of p21 and PCNA proteins related to the down-regulation of REGγ suggests that p21 and PCNA participate in the process of REGγ regulation of cell cycle progression and cell proliferation. Thus, targeting REGγ has a therapeutic potential in the management of PDTC patients.

Qualidade de queijo minas frescal preparado com leite com diferentes quantidades de células somáticas

The objective of this work was to evaluate the effects of using bulk milk with different somatic cell counts (SCC) on the quality of minas frescal cheese. A randomized complete block design was used, with 3x5 factorial treatments, with three SCC levels (low, 125,000 cells mL-1; intermediate, 437,000 cells mL-1; and high, 1,053,000 cells mL-1) and five storage durations. Cheese was vacuum-packed in plastic bags and analyzed after 2, 9, 16, 23 and 30 days of storage at 4ºC. Somatic cell counts did not affect dry matter, fat, ash content, pH, free fatty acid concentrations and sensory parameters of minas frescal cheese. However, SCC in milk increased losses of protein in whey and decreased the cheese protein content. These changes did not affect the moisture-adjusted cheese yield and proteolysis during 30 days of storage. An interaction effect between SCC and time of storage was observed for firmness and sensory grades of cheeses. Results indicated that raw milk used to produce minas frescal cheese should not contain high SCC, in order to avoid lower acceptance of the product after 30 days of storage.

Avaliação da regeneração hepática com modulação pelo pré-condicionamento isquêmico após isquemia e reperfusão e hepatectomia parcial

OBJETIVO: Avaliar a regeneração hepática com modulação pelo pré-condicionamento isquêmico após isquemia, reperfusão e hepatectomia parcial. MÉTODOS: Foram usadas 24 ratas Wistar, de 12 semanas de idade, distribuídas randomicamente em quatro grupos: Grupo Controle (SHAM), Grupo Hepatectomia (HEP), Grupo Isquemia e Reperfusão (GIR) e Grupo Pré-condicionamento Isquêmico (PRE). Foi feita a análise das enzimas hepáticas ALT e AST, avaliação da regeneração através dos pesos inicial e final do fígado e da proliferação dos hepatócitos pela análise imunoistoquímica com o Proliferating Cell Nuclear Antigen (PCNA). RESULTADOS: Em todos os grupos ocorreu regeneração do fígado, não havendo significância estatística entre eles. Houve diferenças significativas em relação a ALT e AST entre os grupos HEP-SHAM, GIR-PRE, GIR-SHAM E PRE-SHAM (p< 0,05). Também houve diferença significativa em relação à marcação de PCNA do grupo SHAM quando comparado aos demais grupos (p< 0,05). CONCLUSÃO: O pré-condicionamento isquêmico diminuiu a lesão hepática, mas não influenciou na regeneração até 48 horas.

Sirolimus influence on hepatectomy-induced liver regeneration in rats

OBJECTIVE: To evaluate the influence of sirolimus on liver regeneration triggered by resection of 70% of the liver of adult rats.METHODS: we used 40 Wistar rats randomly divided into two groups (study and control), each group was divided into two equal subgroups according to the day of death (24 hours and seven days). Sirolimus was administered at a dose of 1mg/kg in the study group and the control group was given 1 ml of saline. The solutions were administered daily since three days before hepatectomy till the rats death to removal of the regenerated liver, conducted in 24 hours or 7 days after hepatectomy. Liver regeneration was measured by the KWON formula, by thenumber of mitotic figures (hematoxylin-eosin staining) and by the immunohistochemical markers PCNA and Ki-67.RESULTS: there was a statistically significant difference between the 24h and the 7d groups. When comparing the study and control groups in the same period, there was a statistically significant variation only for Ki-67, in which there were increased numbers of hepatocytes in cell multiplication in the 7d study group compared with the 7d control group (p = 0.04).CONCLUSION: there was no negative influence of sirolimus in liver regeneration and there was a positive partial effect at immunohistochemistry with Ki-67.

Aspectos histológicos do ovário de coelhas após criopreservação

OBJETIVO: avaliar a preservação folicular e as características celulares do tecido ovariano criopreservado, em coelhas. MÉTODOS: fez-se, sob anestesia, a ooforectomia direita de dez coelhas brancas, adultas. Dissecou-se o ovário mantendo-se o córtex com espessura de 1,5 milímetros. Fragmentou-se o tecido em pequenas secções, algumas para o estudo histológico de controle e outras destinadas à criopreservação pelo protocolo de congelamento lento. Passadas seis semanas efetuou-se o descongelamento e fez-se a avaliação histológica. As amostras do controle e do experimento, após processamento, foram coradas pela hematoxilina-eosina para identificação dos aspectos histológicos e submetidas à técnica imuno-histoquímica utilizando-se o PCNA (proliferating cell nuclear antigen) para a avaliação da viabilidade celular. Utilizaram-se os testes não paramétricos "comparação entre duas proporções" e Mann-Whitney e o teste paramétrico t de Student. RESULTADOS: observou-se que no tecido criopreservado só persistiram oócitos primordiais. Entre as alterações reversíveis identificaram-se: vacuolização citoplasmática em todas as amostras (p=0,039), lise estromal em 50% (p=0,648) e oócitos com contornos irregulares em 80% (p=0,007). Encontraram-se alterações irreversíveis como degeneração hialina e picnose em 30% das amostras (p=0,210). A análise imuno-histoquímica demonstrou os folículos, em diferentes estágios de desenvolvimento, no tecido não congelado e folículos primordiais no tecido criopreservado com positividade para o PCNA, indicando a presença de DNA ativo. CONCLUSÃO: no tecido ovariano criopreservado sobrevivem apenas os folículos primordiais; existem alterações histológicas reversíveis (vacuolização citoplasmática, lise estromal, fragmentação das células da granulosa e oócitos com contornos irregulares); alterações irreversíveis, em níveis não significantes (degeneração hialina e picnose) e presença de PCNA positivo em todos os folículos.

Records of performance and sanitary status from a dairy cattle herd in southern Brazil

Over the last decades, the emphasis on the health of dairy cows has changed from an individual to a herd level. In this scenario, the role played by the recording system and its interpretation by veterinarians has gained primordial importance. The records of productive and reproductive performance and of sanitary status from a southern Brazilian dairy cattle herd have been presented and discussed. The period of study was 2000-2009. Mean values per lactation period were 349D 8436M 290F 275P 201SCS (D: days in lactation, M: kg of milk yield, F: kg of fat, P: kg of protein and SCS: somatic cell score in 1000 cells/ml of milk). Major indexes of reproductive efficiency included age at first calving (31 months), services per conception (2.1), intercalving interval (428 days), calving to conception interval (146 days), mean annual rates of parturitions (76.2%), fetal losses (9.8-19.0%), and stillbirths (3.6%), apart of voluntary waiting period (94 days). Main information on sanitary status of the herd was associated with the mean prevalence of common disorders of dairy cattle such as anaplasmosis (29.8%), mastitis (27.8%), digital diseases (26.3%), ovarian cysts (21.3%), placental retention (19.7%), postpartum uterine infections (10.6%), and calf diarrhea (23.7%) and pneumonia (16.8%), among others. In addition, culling reasons (low reproductive performance [56.3%] and udder/mastitis problems [33.6%]), causes of cattle deaths (anaplasmosis [16.4%] and leukosis [11.4]), and the impact of cattle diseases such as tuberculosis, leukosis, and neosporosis on the herd have also been presented and succinctly discussed. Numbers between brackets represent rates accumulated in the 10-year period.

Bleaching of melanin in the epidermis of South American fur seal and its application on enzyme immunohistochemistry

The South American fur seal (Arctocephalus australis) is an amphibious marine mammal distributed along the Atlantic and Pacific coasts of South America. The species is well adjusted to different habitats due to the morphology of its fin-like members and due to some adaptations in their integumentary system. Immunohistochemical studies are very important to evaluate the mechanisms of skin adaptation due the differential expression of the antigens present in the tissue depending of the region of the body surface. However, its strongly pigmented (melanin) epidermis prevents the visualization of the immuno-histochemical chromogens markers. In this study a melanin bleaching method was developed aimed to allow the visualization of the chromogens without interfering in the antigen-antibody affinity for immunohistochemistry. The analysis of PCNA (proliferating cell nuclear antigen) index in the epidermis of A. australis by immunohistochemistry with diaminobenzidine (DAB) as chromogen was used to test the method. The bleaching of the melanin allowed to obtain the cell proliferation index in epidermis and to avoid false positive results without affecting the immunohistochemical results.

Breed and season influence on milk quality parameters and in mastitis occurrence

The aims of the present study were to evaluate the performance of Jersey and Holstein cows under different rainfall conditions (dry and rainy seasons) by monitoring aspects related to subclinical mastitis (somatic cell count, microbiological isolation, type of isolated pathogen), milk quality (lactose, protein, fat, total solids) and production (mean milk production) of both breeds. The study was carried out in a dairy farm located in the state of São Paulo, Brazil. Eight visitations were done to the farm, four in a period of high rainfall and four in a period of low rainfall. Milk samples were collected from 79 Holstein cows and 37 Jersey cows for electronic somatic cell count and determination of the main milk components (protein, fat, total solids, lactose). Milk fat, protein, total solids and production were influenced by breed and the season, with similar tendencies for both breeds in both seasons. Somatic cell count (SCC) showed similar results for both breeds. Holstein cows with intramammary infections (IMI) presented a higher increase in SCC when compared to Jersey cows (P<0.001). In the dry season, 53 animals had IMI in at least one month during the study, which 32 were Holstein and 21 were Jersey cows. In the rainy season, 65 animals had intramammary infection, being 43 Holstein and 22 Jersey cows. The frequency of IMI cases was larger in the rainy season than in the dry season. Jersey cows had a lower chance of showing IMI signs and symptoms than Holstein cows in the rainy season (odds ratio=0.52). The larger number of IMI cases in the rainy season may have led to a lower milk lactose rate for both breeds, thus milk lactose rate can be considered an indicator of IMI status. There was prevalence of contagious pathogens overall in the study. The applied model showed that environmental pathogens were more frequently isolated from the breed Jersey, regardless of the study season. There seems to be differences in the immune response of Jersey and Holstein breeds.