The use of reduced glutathione (GSH) as antioxidant for cryopreserved sperm in dogs / O uso da glutationa reduzida (GSH) como antioxidante na criopreservação seminal em cães

The aim of this study was to evaluate the effect of supplementation with different concentrations of reduced glutathione GSH (0; 5; 7.5; 10mM) in the extender for cryopreservation in dogs with evaluations performed after glycerolization (chilled) and thawing (thawed). For this purpose, we used 8 dogs and two semen collections were performed in a weekly interval, totaling 16 semen samples. The sperm were analyzed by automatic sperm motility (CASA) and flow cytometry analysis of mitochondrial potential (JC1 dye) and membrane/acrosome integrity (FITC-PI dyes). We evaluated subjectively the membrane and acrosome integrity, mitochondrial activity and DNA integrity. Seminal plasma was evaluated for lipid peroxidation (TBARS concentration). Chilled and thawed samples supplemented with 7.5 and 10mM of GSH had lower percentage of sperm with high (DAB - Class I) and medium (DAB - Class II) mitochondrial activity. And 10mM of GSH had higher percentage of low mitochondrial activity (DAB - Class III). Moreover, thawed samples of 10mM of GSH had high DNA fragmentation rates. Probably by a reductive stress effect on mitochondria which lead to an increase in reactive oxygen species, and a mitochondrial malfunction.(AU)

O objetivo deste estudo foi avaliar o efeito da suplementação com diferentes concentrações de glutationa reduzida (GSH - 0; 5; 7,5; 10mM) para criopreservação em cães com avaliações realizadas após glicerolização (refrigeração) e descongelação. Para tal, foram utilizados oito cães e foram realizadas duas coletas de sêmen em intervalo semanal, totalizando 16 amostras de sêmen. Foram avaliadas a motilidade espermática computadorizada (CASA) e a análise de citometria de fluxo do potencial mitocondrial (sonda JC-1) e integridade da membrana/acrossomal (sonda FITC-PI). Subjetivamente foi avaliada a integridade da membrana plasmática e do acrossomal, atividade mitocondrial e integridade do DNA. O plasma seminal foi avaliado quanto à peroxidação lipídica (concentração de TBARS). As amostras refrigeradas e descongeladas suplementadas com 7,5 e 10mM de GSH apresentaram menor porcentagem de espermatozoides com alta atividade mitocondrial (DAB - Classe I) e média (DAB - Classe II). Na concentração de 10mM de GSH, apresentaram maior porcentagem de baixa atividade mitocondrial (DAB - Classe III). Além disso, amostras descongeladas de 10mM de GSH apresentaram taxas de fragmentação de DNA elevadas, provavelmente por efeito de estresse redutivo sobre as mitocôndrias que elevam as espécies reativas de oxigênio e disfunção mitocondrial.(AU)

Hematological and immunological development from birth to six months of age in Holstein calves / Desenvolvimento hematológico e imunológico em bezerros Holandeses do nascimento aos seis meses de idade

The hematological and immunological development of calves from birth to 6 months of age was performed by hemogram and cellular phenotype. Ten male Holstein calves were assessed in 13 moments: before colostrum intake (D0), every 2 days until the 10th day of life (D2 to D10), at the 15th day after birth (D15), and then monthly up to 6 months (D180). Calves presented hemoconcentration on day (D) 0 and showed a gradual decrease in hematimetric rates until D180. The inversion of the neutrophilic for lymphocytic profile was observed on D4. The percentage of CD3+ cells on D10 was higher than D30 up to D180. The number of CD4+ and CD8+ cells did not change between time points. The number of CD21+ lymphocytes was significantly higher at early time points of D0 up to D15, compared on D30 until D150. In conclusion, the neonatal period was marked by stress leukogram in the first 4 days, and low number of B lymphocytes. These might be risk factors for bacterial infections responsible for navel inflammation and diarrhea. The increase in the number of B cells from 30 days of age demonstrated that the calves were functional and able to generate an immune response.(AU)

O desenvolvimento hematológico e o imunológico de bezerros do nascimento aos seis meses de idade foram avaliados por meio hemograma e imunofenotipagem dos linfócitos. Para tanto, 10 bezerros machos Holandeses foram avaliados em 13 momentos: antes da ingestão do colostro (D0), a cada dois dias até o 10º dia (D2 a D10), 15º dia (D15) e, em seguida, mensalmente até seis meses de idade (D180). Os bezerros apresentaram hemoconcentração em D0 e decréscimo gradual dos índices hematimétricos até D180. A inversão do perfil neutrofílico para linfocítico foi observada em D4. Os valores de CD3+ (%) em D10 foram maiores que aqueles observados em D30 até D180. CD4+ e CD8+ não apresentaram alterações entre os momentos. Os linfócitos B CD21+ aumentaram significativamente a partir dos primeiros momentos D0 até D15, em comparação com os momentos subsequentes D30 até D150. Em conclusão, o período neonatal foi caracterizado pelo leucograma de estresse nos primeiros quatro dias e o baixo número de linfócitos B. Esses fatos podem ser considerados como fatores de risco para infecções bacterianas responsáveis pelas inflamações umbilicais e pela diarreia. Por outro lado, o aumento de células B após os 30 dias de vida demonstrou que os bezerros foram capazes de gerar uma resposta imune.(AU)

Passive adaptation to stress in adulthood after short-term social instability stress during adolescence in mice.

This study reports that short-term social instability stress (SIS) in adolescence increases passive-coping in adulthood in male mice. Short-term SIS decreased the latency of immobility and increased the frequency and time of immobility in tail suspension test. These findings support the hypothesis that adolescent stress can induce a passive adaptation to stress in adulthood, even if it is a short period of stress.

Long-lasting monoaminergic and behavioral dysfunctions in a mice model of socio-environmental stress during adolescence.

Adolescence is one of the critical periods of development and has great importance to health for an individual as an adult. Stressors or traumatic events during this period are associated with several psychiatric disorders as related to anxiety or depression and cognitive impairments, but whether negative experiences continue to hinder individuals as they age is not as well understood. We determined how stress during adolescence affects behavior and neurochemistry in adulthood. Using an unpredictable paradigm (2 stressors per day for 10days) in Balb/c mice, behavioral, hormonal, and neurochemical changes were identified 20days after the cessation of treatment. Adolescent stress increased motor activity, emotional arousal and vigilance, together with a reduction in anxiety, and also affected recognition memory. Furthermore, decreased serotonergic activity on hippocampus, hypothalamus and cortex, decreased noradrenergic activity on hippocampus and hypothalamus, and increased the turnover of dopamine in cortex. These data suggest behavioral phenotypes associated with emotional arousal, but not depression, emerge after cessation of stress and remain in adulthood. Social-environmental stress can induce marked and long-lasting changes in HPA resulting from monoaminergic neurotransmission, mainly 5-HT activity.

Lung hyperinflation stimulates the release of inflammatory mediators in spontaneously breathing subjects

Lung hyperinflation up to vital capacity is used to re-expand collapsed lung areas and to improve gas exchange during general anesthesia. However, it may induce inflammation in normal lungs. The objective of this study was to evaluate the effects of a lung hyperinflation maneuver (LHM) on plasma cytokine release in 10 healthy subjects (age: 26.1 ± 1.2 years, BMI: 23.8 ± 3.6 kg/m²). LHM was performed applying continuous positive airway pressure (CPAP) with a face mask, increased by 3-cmH2O steps up to 20 cmH2O every 5 breaths. At CPAP 20 cmH2O, an inspiratory pressure of 20 cmH2O above CPAP was applied, reaching an airway pressure of 40 cmH2O for 10 breaths. CPAP was then decreased stepwise. Blood samples were collected before and 2 and 12 h after LHM. TNF-á, IL-1â, IL-6, IL-8, IL-10, and IL-12 were measured by flow cytometry. Lung hyperinflation significantly increased (P < 0.05) all measured cytokines (TNF-á: 1.2 ± 3.8 vs 6.4 ± 8.6 pg/mL; IL-1â: 4.9 ± 15.6 vs 22.4 ± 28.4 pg/mL; IL-6: 1.4 ± 3.3 vs 6.5 ± 5.6 pg/mL; IL-8: 13.2 ± 8.8 vs 33.4 ± 26.4 pg/mL; IL-10: 3.3 ± 3.3 vs 7.7 ± 6.5 pg/mL, and IL-12: 3.1 ± 7.9 vs 9 ± 11.4 pg/mL), which returned to basal levels 12 h later. A significant correlation was found between changes in pro- (IL-6) and anti-inflammatory (IL-10) cytokines (r = 0.89, P = 0.004). LHM-induced lung stretching was associated with an early inflammatory response in healthy spontaneously breathing subjects.

Lung hyperinflation stimulates the release of inflammatory mediators in spontaneously breathing subjects.

Lung hyperinflation up to vital capacity is used to re-expand collapsed lung areas and to improve gas exchange during general anesthesia. However, it may induce inflammation in normal lungs. The objective of this study was to evaluate the effects of a lung hyperinflation maneuver (LHM) on plasma cytokine release in 10 healthy subjects (age: 26.1 +/- 1.2 years, BMI: 23.8 +/- 3.6 kg/m(2)). LHM was performed applying continuous positive airway pressure (CPAP) with a face mask, increased by 3-cmH(2)O steps up to 20 cmH(2)O every 5 breaths. At CPAP 20 cmH(2)O, an inspiratory pressure of 20 cmH(2)O above CPAP was applied, reaching an airway pressure of 40 cmH(2)O for 10 breaths. CPAP was then decreased stepwise. Blood samples were collected before and 2 and 12 h after LHM. TNF-alpha, IL-1beta, IL-6, IL-8, IL-10, and IL-12 were measured by flow cytometry. Lung hyperinflation significantly increased (P < 0.05) all measured cytokines (TNF-alpha: 1.2 +/- 3.8 vs 6.4 +/- 8.6 pg/mL; IL-1beta: 4.9 +/- 15.6 vs 22.4 +/- 28.4 pg/mL; IL-6: 1.4 +/- 3.3 vs 6.5 +/- 5.6 pg/mL; IL-8: 13.2 +/- 8.8 vs 33.4 +/- 26.4 pg/mL; IL-10: 3.3 +/- 3.3 vs 7.7 +/- 6.5 pg/mL, and IL-12: 3.1 +/- 7.9 vs 9 +/- 11.4 pg/mL), which returned to basal levels 12 h later. A significant correlation was found between changes in pro- (IL-6) and anti-inflammatory (IL-10) cytokines (r = 0.89, P = 0.004). LHM-induced lung stretching was associated with an early inflammatory response in healthy spontaneously breathing subjects.

Cohabitation with a B16F10 melanoma-bearer cage mate influences behavior and dendritic cell phenotype in mice.

This study evaluated the effects of cohabitation with a B16F10 melanoma-bearer cage mate on behavior and immune functions in mice. Five different experiments were conducted. In each of them, the female mice were divided into two groups: control and experimental. One mouse of each control pair was kept undisturbed and called "companion of health partner" (CHP). One mouse of each experimental pair was inoculated with B16F10 cells and the other, the subject of this study, was called "companion sick partner" (CSP). On Day 20 of cohabitation, behavior and immune parameters from CHP and CSP mice were analyzed. In comparison to the CHP, the CSP mice: (1) presented an increased general locomotion in the open field and a decreased exploration time and number of entries in the plus-maze open arms; (2) had an enhanced expression of the CD80 costimulatory molecule on Iab(+)CD11c(+) spleen cells, but no differences were found on lymph nodes cells; (3) presented an altered differentiation of bone marrow cells in the presence of GM-CSF, IL-4, and LPS in vitro, resulting in a lower percentage of Iab(+)CD80(+) cells; (4) had a deficit in the establishment of a Delayed Type of Hypersensitivity to ovalbumin, which was associated to an in vitro proliferation of an IL-10-producing lymphocyte subpopulation after ovalbumin stimulation. Corticosterone levels detected on Day 20 of cohabitation were similar in CHP and CSP mice. It is shown here that DCs phenotype in mice is affected by conditions associated with behavioral alterations indicative of an anxiety-like state induced by the cohabitation with a tumor-bearer conspecific. This phenomenon occurred probably through a nondependent corticosterone mechanism.

Técnica de citometria de fluxo para avaliação da produção de espécies reativas de oxigênio pelas células do líquido sinovial de eqüinos / Cytometry flow techniques for evaluation of reactive oxygen species produced by equine synovial cells fluid

Using the cytometry flow analysis and adapting the method proposed by Hasui et al. (1985), it was possible to quantify the reactive oxygen species (ROE) produced by distinct cell populations of the synovial fluid from rigid and inflamed joints in horses, and to identify an increase in the polymorphonuclear cells population densities in the inflamed joints. These data strengthen the premise that cytometry flow techniques can contribute for future researches on the articular cartilage degeneration in horses.

Effects of prenatal stress on stress-induced changes in behavior and macrophage activity of mice.

The present study analyzed the effects of maternal stress on behavior and macrophage activity of mice. Pregnant mice received a daily footshock (0.2 mA) from gestational days 15 (GD15) to 19. Experiments were performed on male offspring, challenged or not with another footshock (0.2 mA) on postnatal day 30 (PND30) or 60. The following results were obtained for maternal stress: (1) increment in locomotor activity of juvenile but not of adult mice observed in both open-field and plus-maze; (2) increment in rearing frequency of juvenile but not of adult mice observed in the open-field; (3) decrement in macrophage spreading of adult but not of juvenile mice; (4) abolishment of postnatal footshock effects in both macrophage spreading on PND30 and macrophage nitric oxide (NO) production on PND60; (5) reversion of postnatal footshock effects on H(2)O(2) spontaneous and PMA-induced release by macrophage on PND30; (6) modification of postnatal stress effects on macrophage phagocytosis on PND60. These changes were unrelated to differences in gestational parameters and did not reflect altered maternal-pup interactions or nutritional factors. The observed data provide experimental evidence that maternal stress alters behavior, and macrophage activity at the same time and in the same litter. These data were discussed in the light of possible neuroimmune interactions that involve catecholaminergic pathways.

Effects of maternal stress on anxiety levels, macrophage activity, and Ehrlich tumor growth.

The present study analyzed the effects of maternal stress on behavior and immune function of mice. Pregnant mice received a daily footshock (0.4 mA) from gestational day 15 (GD 15) to GD 19. Experiments were performed on male offspring aged 2 months. The following results were obtained for offspring from dams stressed during pregnancy: (1) decreased locomotor activity observed in the open-field central zone; (2) decreased number of entries into the open arms of the plus-maze and decreased time spent in the exploration of these arms; (3) decreased macrophage spreading and phagocytosis, but no changes in macrophage NO(2)(-) production; (4) increased growth of both the ascitic and solid forms of Ehrlich tumor. These changes were unrelated to differences in gestational parameters and did not reflect altered maternal-pup interactions or nutritional factors. The observed data provide experimental evidence that maternal stress alters stress/anxiety levels, macrophage activity and Ehrlich tumor growth at the same time and in the same litter. The data were discussed in the light of possible neuroendocrine-immune system interactions.

Diazepam effects of peritoneal macrophage activity and corticosterone serum levels in Balb/C mice.

In the present experiment we investigate the effects of diazepam on macrophage activity and serum corticosterone levels in mice. Adult mice were treated with diazepam (1.5 mg/kg/day - group E) or with control solution (group C1) for 7 days; some animals were only handled, receiving no treatment (group C2). Oral onco-BCG was used for peritoneal macrophage activation. Diazepam treatment: 1-decreased macrophage spreading and phagocytosis; 2-decreased the concentrations of H2O2 spontaneously but not phorbol myristate-acetate-induced release. In relation to mice of group C1, diazepam treatment increased the serum levels of corticosterone. No differences were detected between data of groups C1 and C2 both for macrophage activity and serum corticosterone levels. The present data were explained on the basis of a synergistically action for diazepam through peripheral type binding sites (PBR) present in both adrenals and macrophages, stimulating adrenal glucocorticoid production and altering the macrophage cytokine network.

Copper toxicosis in sheep: a case report.

Two ewes died after being fed rabbit ration and mineral salts designated for cows. This food is known to contain copper levels above that indicated for sheep. Blood counts revealed anemia, and serological tests showed high blood urea nitrogen, glutamic pyruvic transaminase and glutamic oxalacetic transaminase values. At necropsy, the animals had hepatopathy and nephropathy. Hepatic copper levels were 414 ppm. Histological examination revealed chronic active hepatitis with bile casts and tubular nephrosis. The liver, spleen and kidneys had hemosiderosis. This was attributed to the massive hemolysis caused by the copper. The liver failure and hemolytic anemia caused death in these sheep.

Perinatal astemizole exposure in the rat throughout gestation: long-term behavioral and anatomic effects associated with reproduction.

Astemizole (ATZ), a non-sedative antihistamine, which antagonize histamine at the level of H1 receptor, was administered daily to female Wistar rats as a 10-mg/kg dose throughout pregnancy. ATZ exposure reduced offspring body weight and delayed the pinna detachment and startle reflex without any modification of dams body weight during gestation. Long-term disruption of male reproductive behavior was seen in experimental animals, whereas female sexual behavior was not modified. In addition, no motor alterations were observed in female or males in adulthood. Testis wet weight was reduced, but no modifications were detected in vasa deferentia or seminal vesicle. We proposed that ATZ administration during pregnancy causes several effects mainly of a sexual nature by interfering either with the hormonal mechanism involved in the central nervous system masculinization or by a direct action of the drug on pups during the development.

Behavioral effects of acute and long-term administration of catnip (Nepeta cataria) in mice.

Catnip or catmint (Nepeta cataria) is a plant used extensively to treat human diseases and in toys for pets. We investigated the effects of acute and long-term administration of the plant on some behaviors of mice. The plant was fed as 10% of the normal diet for 2 h/d for 1 or 7 d. Acute and long-term dosing increased both rearing and locomotion frequencies observed in an open field. Acute exposure to catnip increased stereotyped behavior and susceptibility to seizures, did not interfere with haloperidol-induced catalepsy, and decreased sleeping time after sodium pentobarbital administration. Long-term exposure induced tolerance to stereotypic behavior, catalepsy and sleeping time, and increased the susceptibility to seizures induced by picrotoxin and strychnine. An amphetamine-like effect of catnip was suggested to explain the acute effects, while dispositional and functional adaptative changes were considered involved with the long-term effects.