Delta-9-tetrahydrocannabinol decreases masticatory muscle sensitization in female rats through peripheral cannabinoid receptor activation.

BACKGROUND: This study investigated whether intramuscular injection of delta-9-tetrahydrocannabinol (THC), by acting on peripheral cannabinoid (CB) receptors, could decrease nerve growth factor (NGF)-induced sensitization in female rat masseter muscle; a model which mimics the symptoms of myofascial temporomandibular disorders. METHODS: Immunohistochemistry was used to explore the peripheral expression of cannabinoid receptors in the masseter muscle while behavioural and electrophysiology experiments were employed to assess the functional effects of intramuscular injection of THC. RESULTS: It was found that CB1 and CB2 receptors are expressed by trigeminal ganglion neurons that innervate the masseter muscle and also on their peripheral endings. Their expression was greater in TRPV1-positive ganglion neurons. Three days after intramuscular injection of NGF, ganglion neuron expression of CB1 and CB2, but not TPRV1, was decreased. In behavioural experiments, intramuscular injection (10 µL) of THC (1 mg/mL) attenuated NGF-induced mechanical sensitization. No change in mechanical threshold was observed in the contralateral masseter muscles and no impairment of motor function was found after intramuscular injections of THC. In anaesthetized rats, the same concentration of THC increased the mechanical thresholds of masseter muscle mechanoreceptors. Co-administration of the CB1 antagonist AM251 blocked the effect of THC on masseter muscle mechanoreceptors while the CB2 antagonist AM630 had no effect. CONCLUSIONS: These results suggest that reduced inhibitory input from the peripheral cannabinoid system may contribute to NGF-induced local myofascial sensitization of mechanoreceptors. Peripheral application of THC may counter this effect by activating the CB1 receptors on masseter muscle mechanoreceptors to provide analgesic relief without central side effects. SIGNIFICANCE: Our results suggest THC could reduce masticatory muscle pain through activating peripheral CB1 receptors. Peripheral application of cannabinoids could be a novel approach to provide analgesic relief without central side effects.

Duplex real-time PCR assay using SYBR Green to detect and quantify Malayan box turtle (Cuora amboinensis) materials in meatballs, burgers, frankfurters and traditional Chinese herbal jelly powder.

The Malayan box turtle (Cuora amboinensis) (MBT) is a vulnerable and protected species widely used in exotic foods and traditional medicines. Currently available polymerase chain reaction (PCR) assays to identify MBT lack automation and involve long targets which break down in processed or denatured tissue. This SYBR Green duplex real-time PCR assay has addressed this research gap for the first time through the combination of 120- and 141-bp targets from MBT and eukaryotes for the quantitative detection of MBT DNA in food chain and herbal medicinal preparations. This authentication ensures better security through automation, internal control and short targets that were stable under the processing treatments of foods and medicines. A melting curve clearly demonstrated two peaks at 74.63 ± 0.22 and 78.40 ± 0.31°C for the MBT and eukaryotic products, respectively, under pure, admixed and commercial food matrices. Analysis of 125 reference samples reflected a target recovery of 93.25-153.00%, PCR efficiency of 99-100% and limit of detection of 0.001% under various matrices. The quantification limits were 0.00001, 0.00170 ± 0.00012, 0.00228 ± 0.00029, 0.00198 ± 0.00036 and 0.00191 ± 0.00043 ng DNA for the pure meat, binary mixtures, meatball, burger and frankfurter products, respectively. The assay was used to screen 100 commercial samples of traditional Chinese herbal jelly powder from eight different brands; 22% of them were found to be MBT-positive (5.37 ± 0.50-7.00 ± 0.34% w/w), which was reflected through the Ct values (26.37 ± 0.32-28.90 ± 0.42) and melting curves (74.63-78.65 ± 0.22°C) of the amplified MBT target (120 bp), confirming the speculation that MBT materials are widely used in Chinese herbal desserts, exotic dishes consumed with the hope of prolonging life and youth.

Severe acute malnutrition in Asia.

Severe acute malnutrition (SAM) is a common condition that kills children and intellectually maims those who survive. Close to 20 million children under the age of 5 years suffer from SAM globally, and about 1 million of them die each year. Much of this burden takes place in Asia. Six countries in Asia together have more than 12 million children suffering from SAM: 0.6 million in Afghanistan, 0.6 million in Bangladesh, 8.0 million in India, 1.2 million in Indonesia, 1.4 million in Pakistan, and 0.6 million in Yemen. This article is based on a review of SAM burden and intervention programs in Asian countries where, despite the huge numbers of children suffering from the condition, the coverage of interventions is either absent on a national scale or poor. Countries in Asia have to recognize SAM as a major problem and mobilize internal resources for its management. Screening of children in the community for SAM and appropriate referral and back referral require good health systems. Improving grassroots services will not only contribute to improving management of SAM, it will also improve infant and young child feeding and nutrition in general. Ready-to-use therapeutic food (RUTF), the key to home management of SAM without complications, is still not endorsed by many countries because of its unavailability in the countries and its cost. It should preferably be produced locally from locally available food ingredients. Countries in Asia that do not have the capacity to produce RUTF from locally available food ingredients can benefit from other countries in the region that can produce it. Health facilities in all high-burden countries should be staffed and equipped to treat children with SAM. A continuous cascade of training of health staff on management of SAM can offset the damage that results from staff attrition or transfers. The basic nutrition interventions, which include breastfeeding, appropriate complementary feeding, micronutrient supplementation, and management of acute malnutrition, should be scaled up in Asian countries that are plagued with the burden of malnutrition.

Troubleshooting the rabbit ferric chloride-induced arterial model of thrombosis to assess in vivo efficacy of antithrombotic drugs.

INTRODUCTION: The FeCl3-induced arterial model of thrombosis is one of the most widely used animal models to assess arterial efficacy of new antithrombotic drug candidates. This model is well-established in rodents but in a less extent in the rabbit. In this work, we present a methodology for a rabbit FeCl3-induced arterial model of thrombosis derived from our troubleshooting which allows the generation of reliable efficacy data for new antithrombotic drug candidates. METHODS: Rabbits were administered with heparin 4.5U/kg/min, argatroban 10µg/kg/min or saline by intravenous infusion. The blood flow was monitored using a Doppler flow probe. The time from the application of FeCl3 to the recorded zero blood flow was defined as the time to occlusion, with a maximum recording time of 60min post-FeCl3 application. After 30min of infusion, thrombosis was induced by wrapping a FeCl3-saturated filter paper around the carotid artery caudal to the flow probe. Animals were subject to exclusion criteria based on the visual aspect of the artery FeCl3-induced injury and based on changes in blood flow upon FeCl3 application. RESULTS: Following the application of FeCl3, a mean time to occlusion for saline, heparin and argatroban of 24.3±1.8, 52.5±4.8 and 53.5±4.5min was obtained, respectively. Mean time to occlusion for heparin and argatroban administered groups was significantly different when compared to the saline-treated group (p<0.05). These results for the test compounds represent approximately 80% of the maximum possible prolongation. DISCUSSION: The rabbit FeCl3-induced arterial model of thrombosis presented in this paper derived from our troubleshooting is sensitive and reproducible for the generation of accurate and reliable efficacy data in the assessment of new antithrombotic agents in preclinical drug development.

An optimized method to assess in vivo efficacy of antithrombotic drugs using optical coherence tomography and a modified Doppler flow system.

INTRODUCTION: Animal models of venous and arterial thrombosis are extremely useful to study the efficacy of antithrombotic agents. Variability in efficacy data is often observed in those preclinical studies. The goal of this study was to optimize the methodology for assessing antithrombotic drug efficacy by the use of optical coherence tomography (OCT) and a modified Doppler flow system in rat models of thrombosis. METHODS: Thrombus formation was assessed in both the rat venous and arterial ferric chloride (FeCl(3)) models of thrombosis. In the venous model, thrombus volume post-treatment was measured using OCT, and data were correlated against the thrombus weight. In the arterial model, the time to occlusion was measured using a Doppler flow probe connected to a perivascular flow module which allowed the reporting of dynamic blood flow data every 30s. Heparin (130 or 165U/kg), argatroban (4.5mg/kg), bivalirudin (1.3mg/kg) or saline were administered intravenously. RESULTS: In the venous model, for all treatment groups a strong linear correlation (R(2)=0.998) was observed between thrombus volume measured by OCT and thrombus weight. In the arterial model, using a high sampling rate of a dynamic blood flow using a modified Doppler flow system provided data accuracy and precision of the time to occlusion measurement. DISCUSSION: This study demonstrates that OCT is a powerful tool for the assessment of antithrombotic drug efficacy. Furthermore, it shows that a high Doppler sampling rates of dynamic blood flow leads to data accuracy and precision.

A comparative evaluation of multiple micronutrient and iron-folic acid supplementation during pregnancy in Pakistan: impact on pregnancy outcomes.

BACKGROUND: Maternal micronutrient deficiencies are widespread in Pakistan and are potentially associated with maternal undernutrition and intrauterine growth retardation. Intervention strategies largely consist of administration of iron-folic acid supplements during pregnancy. OBJECTIVE: We evaluated the acceptability of multiple micronutrient supplementation and its potential benefits on pregnancy outcomes and maternal micronutrient status in a cohort of pregnant women in rural and urban Sindh through a cluster-randomized design. METHODS: We randomly assigned 2378 pregnant women to receive either iron-folic acid or multiple micronutrient supplements. The supplements were administered fortnightly by community health workers who performed home visits to assess tolerance and observe the mothers. RESULTS: The women in both groups consumed about 75% of the supplements provided, and few reported adverse effects such as vomiting, abdominal pain, etc. There was a small (70 g) but significant increase in birthweight among infants of mothers receiving multiple micronutrients as compared with infants of mothers receiving iron-folic acid supplements (2.95 +/- 0.6 vs. 2.88 +/- 0.5 kg, p = .01). This translated into a 10% reduction (p < 0.17) in the proportion of low-birthweight infants among infants of mothers receiving multiple micronutrients. Although stillbirth rates were comparable in the two groups, the early neonatal mortality rate in the group receiving multiple micronutrients was higher, although not significantly, than that in the group receiving iron-folic acid (43.2 vs. 23.5 deaths per 1000 live births; RR = 1.64; 95% CI, 0.94 to 2.87). Comparable reductions in anemia (hemoglobin < 11 g/dL) were observed, although the proportion with low iron stores (assessed by serum ferritin) was lower in the iron-folic acid group in the postnatal period. Although the proportion of women with subclinical vitamin A deficiency after supplementation did not differ between the two groups, the iron-folic acid group had a higher proportion with lower serum zinc levels in the immediate postpartum period. CONCLUSIONS: These data suggest that multiple micronutrient supplements are well tolerated during pregnancy, but the effect on birthweight is modest. The observed effect on early neonatal mortality suggests the need for further studies and careful assessment of the intervention in health system settings.

Diphenylmethyl selenocyanate inhibits DMBA-croton oil induced two-stage mouse skin carcinogenesis by inducing apoptosis and inhibiting cutaneous cell proliferation.

Numerous epidemiological and experimental studies have showed the inverse relationship between dietary selenium intake and different types of cancer. Continuous efforts are going on to develop suitable organoselenium compounds, which can be used as cancer chemopreventive agents for human. In the present study, a synthetic organoselenium compound diphenylmethyl selenocyanate was evaluated for its ability to arrest cell proliferation and to induce apoptosis against 7,12-dimethylbenz[a]anthracene-croton oil induced two-stage mouse skin carcinogenesis model. Reduction in the incidence and number of papilloma, the preneoplastic lesion, was considered to be the mean of assessment. Significant decrease in the level of cell proliferation (p<0.01) and significant enhancement in the level of apoptosis (p<0.01) were found. Caspase-3, which contribute a part in the process of cellular apoptosis to prevent further cellular differentiation was also elevated significantly (P<0.01) during the treatment with the Se compound. These observations seem to be correlated with the significant reduction in the corresponding number of skin papilloma formation after 12 weeks of experiment. Thus the compound, diphenylmethyl selenocyanate may be considered for further research to establish it as an effective cancer chemopreventive agent.

Prevalence of home deliveries and antenatal care coverage in some selected villages.

A cross sectional study was conducted in 14 selected villages of Gazipur Thana with the aim to find the prevalence of home deliveries and ante natal care coverage. All those females who delivered in the recent past (within last three months) from the selected villages were the study population Data were collected by two trained interviewers with a semi-structured interview schedule. Study result shows that 83% of the respondents received ante natal check-up throughout their last pregnancy. Most of this group (88.5%) received this care from "Health Assistant or 'Family Welfare visitor's while only 3.1% received care from graduate doctors. Out of 505 respondents 91.3% of the respondents was found to have delivered at home while only 8.7% at institutions. Study findings also suggested that there was association of place of delivery with level of education and family income of the respondents. Also there was associations of utilisation of antenatal care with level of education and family income of the respondents.

Kinetic analyses of alcohol-induced potentiation of the response of GABA(A) receptors composed of alpha(1) and beta(1) subunits.

To investigate the kinetics of both the potentiation and desensitization of the response of ionotropic GABA receptors (GABA(A) receptors) in the presence of various compounds, we expressed receptors composed of alpha(1) and beta(1) subunits by injecting cells with the cRNAs synthesized from cloned bovine GABA(A) receptor cDNAs and measured the electrical responses of the cells electrophysiologically with or without the compounds. The potentiation of the GABA(A) receptor-mediated response was quantitatively analyzed using a simple model with the assumption that the receptors have two identical binding sites for GABA molecules with a dissociation constant of K(1), and one potentiation site for the compound with a dissociation constant of K(p), and that the binding of the compound to the potentiation site only increases the affinity of the GABA binding sites, changing K(1) to K(1p). The estimated K(p) and K(1p) were dependent on the functional groups and the chain length of the compounds. These results could be satisfactorily analyzed using this simple model. The potentiation of the GABA(A) receptor-mediated response by the components of essential oils used for aromatherapy was also examined. These compounds accelerated the decay of the response, possibly due to desensitization of the receptors, which was also analyzed on the basis of the model.

Correlates of perceived malarial episodes and treatment-seeking behavior in a malaria-endemic rural area in Bangladesh.

Malaria is one of the most serious diseases of developing countries. In Bangladesh the estimated population at risk of malaria was calculated to be 103.7 million. This study, carried out in 1995 in villages of the malaria-endemic south-eastern part of Bangladesh aimed to identify the correlates of perceived malarial episodes and healthcare-seeking behavior. Data were collected from villagers and healthcare providers by interviewing. Seventeen percent of the study population reported an episode of malaria during the two months prior to the survey. Males reported more malarial episodes than females; irregular visitors to the jungle and day laborers reported higher prevalence of illness than their regular counterparts. Ninety-nine percent of those who reported suffering from malaria consulted a village healthcare provider within 21 days of the onset of symptoms. Contact rate was higher for those living in highlands, the economically better-off and those aged 10-14 years. The education of the household head, location of the house, the age of the individual, the duration of treatment and the kind of medication suggested were significantly associated with treatment compliance. There is a need to raise awareness about prevention and appropriate management of malarial episodes.

Simultaneous vitamin A administration at routine immunization contact enhances antibody response to diphtheria vaccine in infants younger than six months.

A randomized, double-blind, placebo-controlled trial was conducted to evaluate the effect of simultaneous vitamin A supplementation and diphtheria, pertussis and tetanus (DPT) vaccination on the antibody levels. Infants aged 6-17 wk (n = 56) were randomly given 15 mg oral vitamin A or placebo at the time of their DPT immunization. Three such doses were given at monthly intervals. Immunoglobulin (Ig) G antibodies to diphtheria, pertussis and tetanus were assayed on enrollment and 1 mo after the third dose. Baseline antibody concentrations to diphtheria, pertussis and tetanus did not differ between the vitamin A-supplemented and placebo-treated groups. The postdose antibody to diphtheria level was significantly greater in the vitamin A than in the placebo-treated group. The geometric mean +/- SEM antibody levels (mg/L) were 22.9 +/- 1.2 and 11.0 +/- 1.3 in the vitamin A and placebo groups, respectively (P = 0.029). The postsupplementation concentrations of antibodies to pertussis and tetanus did not differ between the two groups. These results suggest that antibody response to diphtheria vaccination was potentiated by simultaneous vitamin A administration and DPT immunization.

A single amino acid change in the acetylcholinesterase-like domain of thyroglobulin causes congenital goiter with hypothyroidism in the cog/cog mouse: a model of human endoplasmic reticulum storage diseases.

Newly synthesized thyroglobulin (Tg), the major secretory glycoprotein of the thyroid gland, folds and homodimerizes in the endoplasmic reticulum (ER) before its export to the site of iodination, where it serves as the precursor for thyroid hormone synthesis. In families with defective Tg export, affected individuals suffer from a thyroidal ER storage disease characterized by a distended thyrocyte ER containing misfolded Tg, along with induced ER molecular chaperones. Inherited as an autosomal recessive trait, deficient Tg causes congenital hypothyroidism in newborns that, if untreated, results in goiter along with serious cognitive and growth defects. Recently, a similar phenotype has been observed in inbred cog/cog mice, although the precise molecular defect has remained undefined. Here, we have isolated and cloned a full-length 8.5-kb Tg cDNA from cog/cog mice and unaffected isogenic AKR/J mice. Comparison of the complete sequences reveals that cog/cog mice express a Leu-2263 --> Pro missense mutation in the acetylcholinesterase-homology domain of Tg. Heterologous expression studies in COS cells indicate that cog Tg exhibits a severe defect in exit from the ER. Site-directed mutagenesis of cog Tg to convert the single amino acid back to Leu-2263 restores normal Tg secretion. We conclude that the cog mutation in Tg is responsible for this ER storage disease that causes thyroid dyshormonogenesis.

Biodisponibilidade de fósforo de fosfatos de rocha e do termofosfato magnesiano para frangos de corte / Phosphorus bioavailability of rock phosphate with thermic treatment for broilers

Determinou-se a biodisponibilidade de fósforo (BDP) de diferentes fontes desse mineral obtidas em relaçäo ao fosfato bicálcico Mitsui (20,0 por cento de P), cuja BDP foi considerada, arbitrariamente, como sendo 100 por cento, com o emprego de método da abscissa para calcular as BDP, usando-se o teor de cinzas ósseas (TCO). Foram utilizados 360 pintos de um dia, machos da linhagem "Hubbard", distribuídos ao acaso, por sorteio dirigido, em nove tratamentos com quatro repetiçöes de dez aves por gaiola. Quatro tratamentos foram destinados à obtençäo da curva padräo por meio de regressäo linear, em níveis crescentes de P (0,0 por cento; 0,15 por cento; 0,25 por cento; 0,35 por cento) adicionando-se fosfato bicálcico Mitsui à dieta básica. Os demais tratamentos corresponderam à adiçäo de 0,15 por cento de P à dieta básica, proveniente dos fosfatos-teste: fosfatos de Araxá com concentraçöes diferentes de P (8,5 por cento; 10,0 por cento; 11,5 por cento; 13,0 por cento) e o termofosfato magnesiano (8 por cento de P). As dietas foram calculadas de forma a suprir as exigências nutricionais das aves, exceto em P. No 21§ dia, cinco aves de cada repetiçäo foram abatidas para a retirada do fêmur esquerdo, a partir do qual foram determinados os TCO. Pela regressäo linear "y=28,64 + 44,05x", R2= 86,59 por cento, foram obtidas as seguintes BDP (por cento) em relaçäo ao fosfato bicálcico Mitsui: fosfato de Araxá (8,5 por cento de P) - 58,79 por cento; fosfato de Araxá (10,5 por cento de P) - 68,74 por cento; fosfato de Araxá (11,5 por cento de P)-25,70 por cento; fosfato de Araxá (13,0 por cento de P) - 60,54 por cento e termofosfato magnesiano (8,0 por cento de P) - 20,39 por cento. Os fosfatos naturais de rocha näo säo aconselhados como fonte de fósforo para alimentaçäo de frangos de corte

Efeitos da concentraçäo de fósforo disponível durante o pico de postura sobre o desempenho e a qualidade do ovo em duas linhagens de poedeiras comerciais leves / Effects of different concentrations of available phosphorus on the performance and egg quality of two strains of layers during the peak of production

Este trabalho teve como objetivo estudar os efeitos de diferentes concentraçöes de fósforo disponível (Pd) sobre o desempenhio e qualidade dos ovos de duas linhagens de poedeiras leves durante o pico de postura. Foram utilizadas 864 poedeiras leves, sendo 432 da linhagem Shaver White e outras 432 da linhagem Isa Babcock B-300, da 22ª à 40ª semana de idade. As aves receberam dieta à base de milho e farelo de soja com níveis de 0,15; 0,25; 0,35 e 0,45 por cento de Pd, com 15,75 por cento de PB, 3,82 por cento de Ca e 2705 kcal EM/kg. O delineamento experimental foi o inteiramente ao acaso, num esquema fatorial 4 x 2 x 6 (nível de Pd x linhagem x período). Maior produçäo de ovos foi obtida com dieta com 0,39 por cento de Pd e maior consumo de raçäo com a dieta com 0,45 por cento de Pd, para ambas as linhagens. Concentraçöes de 0,45 e 0,26 por cento de Pd resultaram em melhor peso dos ovos e melhor conversäo alimentar, respectivamente, para a linhagem Shaver, näo havendo influência das concentraçöes de Pd sobre estas variáveis para Babcock. Maior unidade Haugh do ovo foi observada com os níveis de 0,35 e 0,15 por cento de Pd para a Shaver e Babcock, respectivamente. Melhores resultados para espessura da casca do ovo foram obtidos com o consumo da raçäo de 0,45 por cento até a 3ª semana e níveis decrescentes após esta fase

Biodisponibilidade de fósforo em seis fontes fosfatadas para frangos de corte / Phosphorus bioavailability of six phosphates for broilers

Determinou-se a biodisponibilidade de fósforo (BDP) de diferentes fontes deste mineral obtidas em relaçäo ao fosfato bicálcico Mitsui (20,0 por cento de P), cuja BDP foi considerada, arbitrariamente, como sendo 100 por cento. O método utilizado para calcular as BDP foi o da abscissa, utilizando-se o teor de cinzas ósseas (TCO). Foram utilizados 400 pintos de um dia de idade, machos, da linhagem "Hubbard", pesados e distribuídos ao acaso, em 10 tratamentos com quatro repetiçöes de 10 aves por gaiola. Quatro tratamentos foram destinados à obtençäo da curva padräo por meio de regressäo linear em níveis crescentes de P (0,0; 0,15; 0,25; 0,35 por cento) adicionando-se fosfato bicálcico Mitsui à dieta básica. Os demais tratamentos corresponderam a adiçäo de 0,15 por cento de P à dieta básica, proveniente dos fosfatos teste: fosfato de Araxá (10,4 por cento de P), termofosfato magnesiano (8,0 por cento de P), fosfato de Patos de Minas (11,0 de P), fosforindus (16,0 por cento de P), superfosfato simples (10,0 por cento de P) e superfosfato triplo (15,3 por cento de P). As dietas foram calculadas de forma a suprir as exigências nutricionais das aves, exceto em P. No 28§ dia, cinco aves de cada repetiçäo foram abatidas para a retirada do fêmur esquerdo, a partir do qual foram determinados o TCO. Pela regressäo linear y=27,34+57,25x,R ao quadrado=86,44 por cento foram obtidas as seguintes BDP (porcentagem) em relaçäo ao fosfato bicálcico Mitsui: fosfato de Araxá - 69,08 por cento; termofosfato magnesiano - 18,80 por cento; fosfato de Patos de Minas - 40,75 por cento; fosforindus - 20,26 por cento; superfosfato simples - 85,12 por cento; superfosfato triplo - 119,95 por cento. O superfosfato triplo foi o que obteve melhores resultados, sendo indicado para raçöes de frangos de corte

Efeito de vários níveis de manganês e fósforo disponível sobre a produçäo e qualidade de ovos em poedeiras / Effect of several levels of manganese and available phosphorous on egg production and egg shell quality

O efeito dos níveis de fósforo disponível na dieta (0,25 por cento, 0,35 por cento e 0,45 por cento) e a suplementaçäo de manganês (25, 50 e 75 mg/kg) para poedeiras comerciais foram avaliados por um período de oito semanas (42 a 50 semanas de idade). Todas as dietas experimentais possuíam 2678kcal EM/kg, 15,6 por cento PB e 4,12 por cento Ca. O manganês (Mn) foi suplementado com sulfato de manganês (MnSO4 5H2O). Aumentando o nível de fósforo e manganês na dieta näo se observou efeito significativo (P>0,05) no consumo de raçäo e conversäo alimentar por kg de massa de ovo. A produçäo de ovos e o peso do ovo foram (P<0,05) afetados pelo nível de manganês na dieta, obtendo-se melhor resultado com 50 e 75 mg/kg de Mn, respectivamente, mas näo com os níveis de fósforo. A espessura da casca do ovo näo foi influenciada (P>0,05) pelos níveis de Mn, mas o aumento dos níveis de fósforo disponível de 0,25 para 0,45 por cento reduziu a espessura da casca do ovo