Structural and functional characterization of BnSP-7, a Lys49 myotoxic phospholipase A(2) homologue from Bothrops neuwiedi pauloensis Venom

BnSP-7, a Lys49 myotoxic phospholipase A, homologue from Bothrops neuwiedi pauloensis venom, was structurally and functionally characterized. Several biological activities were assayed and compared with those of the chemically modified toxin involving specific amino acid residues, the cDNA produced from the total RNA by RT-PCR contained approximately 400 bp which codified its 121 amino acid residues with a calculated pi and molecular weight of 8.9 and 13,727, respectively. Its amino acid sequence showed strong similarities with several Lys49 phospholipase A, homologues from other Bothrops sp, venoms. By affinity chromatography and gel diffusion, it was demonstrated that heparin formed a complex with BnSP-7, held at least in part by electrostatic interactions. BnSP-7 displayed bactericidal activity and promoted the blockage of the neuromuscular contraction of the chick, biventer cervicis muscle. In addition to its in vivo myotoxic and edema-inducing activity, it disrupted artificial membranes, Both BnSP-7 and the crude venom released creatine kinase from the mouse gastrocnemius muscle and induced the development of a dose-dependent edema. His, Tyr, and Lys residues of the toxin were chemically modified by 4-bromophhenacyl bromide (BPB), 2-nitrobenzenesulfonyl fluoride (NBSF), and acetic anhydride (AA), respectively. Cleavage of its N-terminal octapeptide was achieved with cyanogen bromide (CNBr), the bactericidal action of BnSP-7 on Escherichia coli was almost completely abolished by acetylation or cleavage of the N-terminal octapeptide, the neuromuscular effect induced by BnSP-7 was completely inhibited by heparin, BPB, acetylation, and CNBr treatment. The creatine kinase releasing and edema-inducing effects were partially inhibited by heparin or modification by BPB and almost completely abolished by acetylation or cleavage of the N-terminal octapeptide, the rupture of liposomes by BnSP-7 and crude venom was dose and temperature dependent. Incubation of BnSP-7 with EDTA did not change this effect, suggesting a Ca2+-independent membrane lytic activity. BnSP-7 cross-reacted with antibodies raised against B. moojeni (MjTX-II), B. jararacussu (BthTX-I), and B. asper (Basp-II) myotoxins as well as against the C-terminal peptide (residues 115-129) from Basp-II. (C) 2000 Academic Press.

Biological and structural characterization of a new PLA(2) from the Crotalus durissus collilineatus venom

In the present article we report on the biological characterization and amino acid sequence of a new basic Phospholipases A(2) (PLA(2)) isolated from the Crotalus durissus collilineatus venom (Cdcolli F6), which showed the presence of 122 amino acid residues with a pI value of 8.3, molecular mass of 14 kDa and revealed an amino acid sequence identity of 80% with crotalic PLA(2)s such as Mojave B, Cdt F15, and CROATOX. This homology, however, dropped to 50% if compared to other sources of PLA(2)s such as from the Bothrops snake venom. Also, this PLA(2) induced myonecrosis, although this effect was lower than that of BthTx-I or whole crotoxin and it was able to induce a strong blockage effect on the chick biventer neuromuscular preparation, independently of the presence of the acid subunid (crotapotin). The neurotoxic effect was strongly reduced by pre-incubation with heparin or with anhydrous acetic acid and rho-BPB showed a similar reduction. The rho-BPB did not reduce significantly the myotoxic activity induced by the PLA(2), but the anhydrous acetic acid treatment and the pre-incu-bation of PLA(2) with heparin reduced significantly its effects. This protein showed a strong antimicrobial activity against Xanthomonas axonopodis passiflorae (Gram-negative), which was drastically reduced by incubation of this PLA(2) with rho-BPB, but this effect was marginally reduced after treatment with anhydrous acetic acid. Our findings here allow to speculate that basic amino acid residues on the C-terminal and molecular regions near catalytic site regions such as Calcium binding loop or rho-wing region may be involved in the binding of this PLA(2) to the molecular receptor to induce the neurotoxic effect. The bactericidal effect, however, was completely dependent on the enzymatic activity of this protein.

Effect of Environmental Temperature During the First Week of Brooding Period on Broiler Chick Body Weight, Viscera and Bone Development

O presente trabalho teve como objetivo estudar o efeito de diferentes temperaturas-ambiente durante a primeira semana de vida de pintos de corte sob parâmetros zootécnicos, desenvolvimento visceral e crescimento ósseo. Foram utilizados 240 pintos de um dia, alojados em 3 câmaras climáticas, com temperaturas constantes de 20, 25 e 35°C do 1° ao 7° dia de vida. Diariamente, o consumo de água e ração, bem como o peso vivo, o peso relativo do fígado, moela, coração, saco vitelino e bursa de Fabricius foram avaliados. A tíbia e o fêmur também foram pesados e o comprimento e espessura (diâmetro médio) mensurados. As aves criadas a 20°C ganharam menos peso e consumiram menos ração do que aves mantidas a 25°C e menos água do que aves mantidas a 35°C. O peso relativo do fígado, coração e moela foram afetados pela temperatura ambiente, entretanto, não foi observado efeito da temperatura de criação sobre o peso do saco vitelino e bursa de Fabricius. Os dados mostraram que todos os parâmetros ósseos pesquisados aumentaram com a idade das aves. A temperatura ambiente não afetou a espessura da tíbia e do fêmur, mas foi observado um aumento significativo no peso e comprimento dos ossos com o aumento da temperatura ambiente. Os resultados desse experimento mostraram que o estresse por frio (20°C) reduziu o crescimento ósseo bem como o peso vivo das aves, durante os primeiros sete dias após a eclosão.

Effect of temperature on incubation period, embryonic mortality, hatch rate, egg water loss and partridge chick weight (Rhynchotus rufescens)

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Effect of breeder age on eggshell thickness, surface temperature, hatchability and chick weigh

Two experiments were carried out to study the effect of breeder age on incubation parameters (hatchability, eggshell thickness, egg surface temperature and chick weight). In Exp. 1, fertile eggs (30- and 60-wk-old breeders) were incubated at three different temperatures (36.8, 37.8 and 38.8 ºC). Eggshell surface temperature was measured by attaching a thermocouple to the shell and data were collected in a datalogger every ten minutes. This study was conducted according to a 3 x 2 factorial design (three temperatures and two breeder ages). Data revealed that eggshell surface temperature changed according to incubation temperature, with the main increase occurring between 10 and 13 days of incubation, and that the maximum increase in eggshell surface temperature was not higher than +0.6 ºC, irrespective of incubator temperature. The incubator temperature affected total incubation period and hatchability (%) at 38.8 ºC, independent of breeder age. Heavier eggs resulted in heavier chicks, irrespective of incubator temperature. In Exp 2, the eggs (30- and 60-wk-old breeders) were incubated at 37.8 ºC and eggs characteristics (weight, specific gravity, total hatchability and chicks weight) were evaluated according to a randomized experimental design. The data showed that breeder age affected eggshell thickness and chick weight (heavier eggs resulted in heavier chicks), but not specific gravity, eggshell surface temperature or hatchability. The findings of this study revealed that hatchability can be influenced by incubation temperature, but not by the breeder age. Breeder age can affect eggshell thickness, egg weight and eggshell surface temperature, but not specific gravity.

Egg traits and physiological neonatal chick parameters from broiler breeder at different ages

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

beta(2)-Agonists and cAMP inhibit protein degradation in isolated chick (Gallus domesticus) skeletal muscle

1. The role of beta(2)-agonist and of cAMP in chick skeletal muscle proteolytic pathways and protein synthesis was investigated using an in vitro preparation that maintains tissue glycogen stores and metabolic activity for several hours.2. In extensor digitorum longus (EDL) muscle total proteolysis decreased by 15 to 20% in the presence of equimolar concentrations of epinephrine, clenbuterol, a selective beta(2)-agonist, or dibutyryl-cAMP. Rates of protein synthesis were not altered by clenbuterol or dibutyryl-cAMP.3. The decrease in the rate of total protein degradation induced by 10(-5) M clenbuterol was paralleled by a 44% reduction in Ca2+-dependent proteolysis, which was prevented by 10(-5) M ICI 118.551, a selective beta(2)-antagonist.4. No change was observed in the activity of the lysosomal, ATP-dependent, and ATP-independent proteolytic systems. Ca2+-dependent proteolytic activity was also reduced by 58% in the presence of 10(-4) M dibutyryl-cAMP or isobutylmethylxanthine.5. The data suggest that catecholamines exert an inhibitory control of Ca2+-dependent proteolysis in chick skeletal muscle, probably mediated by beta(2)-adrenoceptors, with the participation of a cAMP-dependent pathway.

Effects of egg storage time on spread of hatch, chick quality, and chick juvenile growth

A total of 1,800 incubating eggs produced by a commercial flock of Cobb broiler breeders was used to determine the effects of storage duration (3 or 18 d) on spread of hatch and chick quality. Chick relative growth (RG) at the end of 7 d of rearing was also determined as a measure of the chick performance. Chick quality was defined to encompass several qualitative characteristics and scored according to their importance. Eggs stored for 3 d hatched earlier than those stored for 18 d (P < 0.05). Hatching was normally distributed in both categories of eggs, and the spread of hatch was not affected by storage time (P = 0.69). Storage duration of 18 d reduced the percentage of day-old chick with high quality as well as average chick quality score (P < 0.05). RG varied with length of egg storage, quality of day-old chick, and the incubation duration (P < 0.05). Eighteen-day storage of eggs not only resulted in longer incubation duration and lower quality score but also depressed RG. Chick quality as defined in this study was correlated to RG and storage time. It was concluded that day-old chick quality may be a relatively good indicator of broiler performance. The results suggest however that in order to improve performance prediction power of chick quality, it would be better to define it as a combination of several qualitative aspects of the day-old chick and the juvenile growth to 7 d.

Immunohistochemical localization of nicotinic acetylcholine-receptor subunits in the mesencephalon and diencephalon of the chick (Gallus-gallus)

Monoclonal antibodies against two alpha-bungarotoxin-binding subunits (alpha-7 and alpha-8) of the nicotinic acetylcholine receptors (nAChRs) were used as immunohistochemical probes to map their distribution in the chick diencephalon and mesencephalon. The distribution of the alpha-7 and alpha-8 nAChR subunits was compared to the distribution of immunoreactivity produced by a monoclonal antibody against the beta-2 structural subunit of the nAChRs.Structures that contained high numbers of alpha-7-like immunoreactive (LI) somata included the intergeniculate leaflet, nucleus intercalatus thalami, nucleus ovoidalis, organum paraventricularis, nucleus rotundus, isthmic nuclei, nucleus trochlearis, oculomotor complex, nucleus interstitio-pretecto-subpretectalis, stratum griseum centrale of the optic tectum, and nucleus semilunaris. Neuropil staining for alpha-7-LI was intense in the nucleus dorsomedialis hypothalami, nucleus geniculatus lateralis ventralis, griseum tecti, isthmic nuclei, nucleus lentiformis mesencephali, nucleus of the basal optic root, and stratum griseum et fibrosum superficiale of the tectum. High numbers of alpha-8-LI somata were found in the stratum griseum et fibrosum superficiale of the tectum and the nucleus interstitio-pretecto-subpretectalis, and intense neuropil staining for alpha-8-LI was found in the dorsal thalamus, nucleus geniculatus lateralis ventralis, lateral hypothalamus, griseum tecti, nucleus lentiformis mesencephali, nucleus interpeduncularis, and stratum griseum et fibrosum superficiale of the tectum. High numbers of beta-2-LI somata were found only in the nucleus spiriformis lateralis, whereas neuropil staining for beta-2-LI was intense in the nucleus geniculatus lateralis ventralis, nucleus suprachiasmaticus, nucleus lateralis anterior, nucleus habenularis lateralis, area pretectalis, griseum tecti, nucleus lentiformis mesencephali, nucleus externus, and nucleus interpeduncularis, and in the stratum griseum centrale, stratum griseum et fibrosum superficiale, and stratum opticum of the tectum.These results indicate that there are major disparities in the localization of the alpha-bungarotoxin-binding alpha-7 and alpha-8 nAChR subunits and the beta-2 structural nAChR subunit in the chick diencephalon and mesencephalon. These nAChR subunits appear, however, to coexist in several regions of the chick brain.

Efeito do uso de probiótico sobre o desempenho e atividade de enzimas digestivas de frangos de corte

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Yeast extract and prebiotic in pre-initial phase diet for broiler chickens raised under different temperatures

Objetivou-se nesta pesquisa avaliar o desempenho, o rendimento de carcaça e a morfometria intestinal de frangos de corte criados em diferentes temperaturas e que receberam na fase pré-inicial ração contendo ou não extrato de leveduras e prebiótico. Foram utilizados 1.440 pintos machos de 1 dia de idade, criados em diferentes câmaras climáticas. As rações, acrescidas ou não de extrato de leveduras e prebiótico, foram oferecidas somente na fase pré-inicial (1 a 7 dias). A partir do oitavo dia, todas as aves receberam a mesma ração, reajustada de acordo com as recomendações usuais. Adotou-se o delineamento experimental inteiramente casualizado em arranjo fatorial 3 × 2 × 2, composto de três temperaturas de criação (calor, conforto e frio) e dois níveis de extrato de leveduras (com ou sem) e prebiótico (com ou sem). O desempenho das aves foi avaliado considerando o ganho de peso, o consumo de ração, a conversão alimentar e a viabilidade aos 42 dias de idade. Também foram avaliados o rendimento de carcaça e a morfometria intestinal. O calor ambiente prejudicou o desempenho e o rendimento de carcaça. A inclusão de prebiótico na ração pré-inicial aumentou o ganho de peso e melhorou a conversão alimentar das aves criadas no calor. A inclusão dos produtos na ração de frangos de corte criados em ambiente de calor e no frio tem efeito benéfico sobre as vilosidades das aves.

Salmonella spp investigation in transport boxes of day-old birds

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Efeito do processamento da soja integral sobre a energia metabolizável e a digestibilidade dos aminoácidos para aves

Essa pesquisa foi desenvolvida para se avaliar o valor nutricional da soja integral submetida a diferentes processamentos. Quatro ensaios de metabolismo foram conduzidos para determinar os coeficientes de digestibilidade da matéria seca (CDMS), do extrato etéreo (CDEE) e dos aminoácidos e os valores de energia metabolizável aparente e verdadeira da soja integral desativada (SID) e extrusada (SIE) e das misturas de farelo de soja com óleo degomado de soja (FSO) ou com óleo ácido de soja (FSOA). Nos ensaios um e dois, utilizou-se a metodologia tradicional de coleta total de excretas com pintos e galos, respectivamente. A metodologia da alimentação forçada com galos adultos intactos foi utilizada no ensaio três, e com galos cecotomizados, no ensaio quatro. Os CDMS e CDEE e a energia metabolizável determinados com galos foram superiores aos determinados com pintos. Os maiores valores de CDMS, CDEE e de energia metabolizável foram obtidos para FSO, seguidos pelos da SIE e FSOA, e os menores, para a SID. O processo de extrusão proporcionou melhores resultados no aproveitamento da gordura do grão de soja e, conseqüentemente, da energia. Entretanto, a digestibilidade dos aminoácidos não foi influenciada pelos processamentos. Os diferentes processamentos conferiram à soja integral características nutricionais que se distinguiram, principalmente quanto ao valor de energia metabolizável, que também variou com a idade das aves.

Desempenho, eficiência de utilização dos nutrientes e estrutura do trato digestório de pintos de corte alimentados na fase pré-inicial com rações de diferentes formas físicas

Este estudo foi realizado com o objetivo de avaliar o efeito da forma física da ração sobre o desempenho, a utilização dos nutrientes das rações e o desenvolvimento do trato disgestório de pintos de corte na primeira semana de vida. Foram utilizados 150 pintos machos Cobb distribuídos ao acaso em três tratamentos com cinco repetições de dez aves. Os tratamentos consistiram das formas físicas farelada, triturada e peletizada de uma ração pré-inicial comercial. Com exceção da moela, os órgãos do trato digestório e a estrutura morfométrica do intestino não foram influenciados pela forma física da ração. Maior digestibilidade do N foi obtida com a ração peletizada, enquanto a ração triturada possibilitou maior digestibilidade do EE. A energia metabolizável determinada para a ração farelada foi menor que a obtida nas demais formas físicas da ração. As aves alimentadas com ração farelada tiveram menor consumo de ração e ganho de peso e pior conversão alimentar. Ingeriram ainda menos energia, apresentaram menores retenções de proteína, gordura e energia corporal e utilizaram maior proporção da energia ingerida para produção de calor. As rações pré-iniciais na forma peletizada ou triturada possibilitam maior aproveitamento dos nutrientes da ração pelos pintos e melhor desempenho.