Avaliação de diferentes crioprotetores intra e extracelulares na criopreservação de sêmen de touros / Evaluation of different intra and extracellular cryoprotectants on bull semen cryopreservation

Em um delineamento experimental usando o fatorial 3x2, três crioprotetores internos, glicerol (GLI), etilenoglicol (EG) e dimetilformamida (DMF), e dois externos, gema de ovo (GEMA) e lipoproteína de baixa densidade (LDL), avaliaram-se a motilidade ao descongelamento de GLI-GEMA 53,9±1,96, sendo superior aos demais tratamentos (P<0,05). Na avaliação de morfologia ao descongelamento, não houve diferença (P>0,05) entre os tratamentos EG-GEMA 68,3±1,58, EG-LDL 72,2±2,39 e DMF-GEMA 68,7±1,67 que foram mais altos que os demais (P<0,05). A avaliação de integridade de membrana por fluorescência ao descongelamento GLI-GEMA 34,2±2,28 e EG-GEMA 30,9±1,32 não diferiram entre si (P>0,05), mas foram mais elevados que os demais (P<0,05), enquanto que a HOST dos tratamentos DMF-GEMA 13,6±1,30 e DMF-LDL 9,8±0,78 diferirem entre si (P<0,05) e foram mais baixas que as demais (P<0,05). O uso de etilenoglicol associado à gema de ovo pode ser uma alternativa ao uso de glicerol nos protocolos de congelamento de sêmen de touros.

The experiment was designed as 3 x 2 factorial design, with three internal cryoprotectants, glycerol (GLY), etileneglycol (EG) and dymethilformamide (DMF) and two external, egg yolk (YOLK) and density low lipoproteina (LDL). The motility at thawing for GLY-YOLK (53.9±1.96) was higher than other treatments (P<0.05). The percentage of cells with normal morphology at thawing was not different between EG-YOLK (68.3±1.58), EG-LDL (72.2±2.39) and DMF-YOLK (68.7±1.67), but they were higher than the others (P<0.05). The evaluation of membrane integrity through fluorescent probes at thawing indicate that GLY-YOLK (34.2±2.28) and EG-YOLK (30.9±1.32) were not different (P>0.05), but were higher than the others (P<0.05). The evaluation of membrane integrity through hypoosmotic swelling test (HOST) indicate that DMF-YOLK (13.6±1.30) and DMF-LDL (9.8±0.78) were different (P<0.05) and lower than the others (P<0.05). The use of ethylene glycol associated to egg yolk can be a viable alternative to the use of glycerol in bull semen freezing protocols.

Effect of recombinant bovine somatotropin on plasma concentrations of insulin-like growth factor I, insulin and membrane integrity of bull spermatozoa.

This study aimed to evaluate the effect of the exogenous recombinant bovine somatotropin (rbST) on plasma concentrations of insulin-like growth factor I (IGF-I), insulin and semen quality of bulls. Twenty bulls (Aberdeen Angus and Brangus) were divided by breed into two groups. Placebo group was injected with NaCl 0.9% (s.c.) and treatment group with rbST (s.c., 500 mg) at days 0 and 14 of the experiment. Immediately after semen collection, blood samples were taken on days 0, 14, 28, 42 and 56 of the experiment. Semen was also collected on day 70 of the experiment. Evaluation of sperm motility was performed at pre-freezing and post-thawing stage, whereas assessment of sperm membrane integrity was performed after freezing and thawing. Analysis of data revealed that the effect of treatment and treatment-by-collection day on plasma concentrations of IGF-I and insulin was not significant. However, mean plasma concentrations of IGF-I and insulin were affected (p < 0.0001) by days of blood sampling. Effect of treatment and treatment-by-collection day on motility of spermatozoa was similar (p > 0.05) at pre-freezing and post-thawing stage. Intactness of plasmalemma and tail membrane of spermatozoa at post-thawing stage was higher (p < 0.05) in rbST-treated group than in control. In conclusion, rbST did not affect plasma concentrations of IGF-I and insulin, however, it did improve post-thaw sperm membrane integrity.

Oocistos deCryptosporidium parvum em fezes de bezerro infectado experimentalmente / Cryptosporidium parvum oocysts in the feces of experimentally infected calf

A dose of 5.0x106 Cryptosporidium parvum oocysts was inoculated in a newborn calf. After the inoculation, the feces were daily collected and the presence of oocysts was examined on slides using 0.17% green malachite dye. The total yield reached 1.5x1010 oocysts, with a peak production on the 7th day, confirming the infectious process and the role of calf infection in the potential risk for environmental contamination.

Transient high-level expression of beta-galactosidase after transfection of fibroblasts from GM1 gangliosidosis patients with plasmid DNA.

GM1 gangliosidosis is an autosomal recessive disorder caused by the deficiency of lysosomal acid hydrolase beta-galactosidase (beta-Gal). It is one of the most frequent lysosomal storage disorders in Brazil, with an estimated frequency of 1:17,000. The enzyme is secreted and can be captured by deficient cells and targeted to the lysosomes. There is no effective treatment for GM1 gangliosidosis. To determine the efficiency of an expression vector for correcting the genetic defect of GM1 gangliosidosis, we tested transfer of the beta-Gal gene (Glb1) to fibroblasts in culture using liposomes. Beta-Gal cDNA was cloned into the expression vectors pSCTOP and pREP9. Transfection was performed using 4 microL lipofectamine 2000 and 1.5-2.0 microg DNA. Cells (2 x 10(5)/well) were harvested 24 h, 48 h, and 7 days after transfection. Enzyme specific activity was measured in cell lysate and supernatant by fluorometric assay. Twenty-four hours after transfection, treated cells showed a higher enzyme specific activity (pREP9-beta-Gal: 621.5 +/- 323.0, pSCTOP-beta-Gal: 714.5 +/- 349.5, pREP9-beta-Gal + pSCTOP-beta-Gal: 1859.0 +/- 182.4, and pREP9-ss-Gal + pTRACER: 979.5 +/- 254.9 nmol x h-1 x mg-1 protein) compared to untreated cells (18.0 +/- 3.1 for cell and 32.2 +/- 22.2 nmol x h-1 x mg-1 protein for supernatant). However, cells maintained in culture for 7 days showed values similar to those of untreated patients. In the present study, we were able to transfect primary patients' skin fibroblasts in culture using a non-viral vector which overexpresses the beta-Gal gene for 24 h. This is the first attempt to correct fibroblasts from patients with GM1 gangliosidosis by gene therapy using a non-viral vector.

Transient high-level expression of ß-galactosidase after transfection of fibroblasts from GM1 gangliosidosis patients with plasmid DNA

GM1 gangliosidosis is an autosomal recessive disorder caused by the deficiency of lysosomal acid hydrolase ß-galactosidase (ß-Gal). It is one of the most frequent lysosomal storage disorders in Brazil, with an estimated frequency of 1:17,000. The enzyme is secreted and can be captured by deficient cells and targeted to the lysosomes. There is no effective treatment for GM1 gangliosidosis. To determine the efficiency of an expression vector for correcting the genetic defect of GM1 gangliosidosis, we tested transfer of the ß-Gal gene (Glb1) to fibroblasts in culture using liposomes. ß-Gal cDNA was cloned into the expression vectors pSCTOP and pREP9. Transfection was performed using 4 µL lipofectamine 2000 and 1.5-2.0 µg DNA. Cells (2 x 10(5)/well) were harvested 24 h, 48 h, and 7 days after transfection. Enzyme specific activity was measured in cell lysate and supernatant by fluorometric assay. Twenty-four hours after transfection, treated cells showed a higher enzyme specific activity (pREP9-ß-Gal: 621.5 ± 323.0, pSCTOP-ß-Gal: 714.5 ± 349.5, pREP9-ß-Gal + pSCTOP-ß-Gal: 1859.0 ± 182.4, and pREP9-ß-Gal + pTRACER: 979.5 ± 254.9 nmol·h-1·mg-1 protein) compared to untreated cells (18.0 ± 3.1 for cell and 32.2 ± 22.2 nmol·h-1·mg-1 protein for supernatant). However, cells maintained in culture for 7 days showed values similar to those of untreated patients. In the present study, we were able to transfect primary patients' skin fibroblasts in culture using a non-viral vector which overexpresses the ß-Gal gene for 24 h. This is the first attempt to correct fibroblasts from patients with GM1 gangliosidosis by gene therapy using a non-viral vector.

Speech rehabilitation after laryngectomy with the amatsu tracheoesophageal shunt.

The Amatsu vocal rehabilitation technique is a tracheoesophageal shunt using a posterior tracheal flap associated with a sphincter made of esophageal muscular wall. Since march 1991 the procedure was done in 33 men and 2 women. ages ranging from 30 to 78 years. Previous radiotherapy, hypopharynx lesions, need for postoperative radiotherapy or use of myocutaneous flap were not considered contraindications. Vocalization was achieved in 76% of our patients and the quality was considered superior than that obtained by the esophageal voice. In only one case the shunt had to be closed surgically because of aspiration. In conclusion, the Amatsu tracheoesophageal shunt is an inexpensive technique that obtain a good quality of vocal rehabilitation in a high percentage of patients, it has few complications and it should be considered for all candidates to a laryngectomy, mainly for those with a good prognosis and a desire to return to their social environment.

[Bacterial contamination of breast milk collected through manual expression and stored at room temperature] / Contaminação bacteriana do leite humano coletado por expressão manual e estocado à temperatura ambiente.

OBJECTIVES: To the determine the bacterial contamination profile of unheated expressed breast milk, collected without rigid hygienic precautions and stored at room temperature for nine hours. The purpose was to give poor lactating mothers the alternative of storing their own milk out of refrigerator. A research on cultural, social and economical aspects as well as on donators knowledge about breastfeeding was considered necessary. METHODS: 35 donators were interviewed and an experimental investigation was performed with 33 samples of breast milk stored at room temperature (17 masculine C to 30.5 masculine C) and bacteriologically analyzed at zero, three, six and nine hours after collection. The same breast milk was stored at refrigerator (2 masculine C to 6 masculine C) as a control procedure. Total count of bacterial contents and identification of Staphylococcus aureus and Escherichia coli were evaluated.RESULTS: The enterviews revealed the low socio-economical and cultural level of lactating mothers and their little experience in expressing, collecting and using their own milk. Bacteriological data analysis showed mesophyllous average of 7.1x10(3)UFC/mL, acceptable outline of bacterial contamination, despite the use of a simplified hygiene technique. After nine hours, samples stored at room temperature showed final average of bacterial contents similar to the first ones (7.3x10(3)UFC/mL) and without relevant statistic differences from the ones kept under refrigeration (p=0.05) for studied bacterias.CONCLUSION: This study shows that it is possible to use unprocessed breast milk for babýs consumption if it is stored at room temperatures until nine hours after it has been collected. However, mothers have to be told about the possibility of storing breast milk for babies later consumption.