Acute phase proteins and biomarkers for health in chickens

Acute phase proteins (APPs) are proteins synthesised predominantly in the liver, whose plasma concentrations increase (positive APP) or decrease (negative APP) as a result of infection, inflammation, trauma and tissue injury. They also change as a result of the introduction of immunogens such as bacterial lipopolysaccharide (LPS), turpentine and vaccination. While publications on APPs in chickens are numerous, the limited availability of anti-sera and commercial ELISAs has resulted in a lot of information on only a few APPs. Disease is a threat to the poultry industry, as pathogens have the potential to evolve, spread and cause rapid onset of disease that is detrimental to the welfare of birds. Low level, sub-acute disease with non-specific, often undiagnosed causes can greatly affect bird health and growth and impact greatly on productivity and profitability. Developing and validating methods to measure and characterise APPs in chickens will allow these proteins to be used diagnostically for monitoring flock health. Using immune parameters such as APPs that correlate with disease resistance or improvements in production and welfare will allow the use of APPs as selection parameters for breeding to be evaluated. For APPs to be useful parameters on which to evaluate chicken health, information on normal APP concentrations is required. Ceruloplasmin (Cp) and PIT54 concentrations were found to be much lower in healthy birds form commercial production farms than the reported normal values obtained from the literature. These APPs were found to be significantly higher in culled birds from a commercial farm and Cp, PIT54 and ovotransferrin (Ovt) were significantly higher in birds classified as having obvious gait defects. Using quantitative shotgun proteomics to identify the differentially abundant proteins between three pools: highly acute phase (HAP), acute phase (AP) and non-acute phase (NAP), generated data from which a selection of proteins, based on the fold difference between the three pools was made. These proteins were targeted on a individual samples alongside proteins known to be APPs in chickens or other species: serum amyloid A (SAA), C-reactive protein (CRP), Ovt, apolipoprotein A-I (apo-AI), transthyretin (Ttn), haemopexin (Hpx) and PIT54. Together with immunoassay data for SAA, Ovt, alpha-1-acid glycoprotein (AGP) and Cp the results of this research reveal that SAA is the only major APP in chickens. Ovotransferrin and AGP behave as moderate APPs while PIT54 and Cp are minor APPs. Haemopexin was not significantly different between the three acute phase groups. Apolipoprotein AI and Ttn were significantly lower in the HAP and AP groups and as such can be classed as negative APPs. In an effort to identify CRP, multiple anti-sera cross reacting with CRP from other species were used and a phosphorylcholine column known to affinity purify CRP were used. Enriched fractions containing low molecular weight proteins, elutions from the affinity column together with HAP, AP and NAP pooled samples were applied to a Q-Exactive Hybrid Quadrupole–Orbitrap mass spectrometer (Thermo Scientific) for Shotgun analysis and CRP was not identified. It would appear that CRP is not present as a plasma protein constitutively or during an APR in chickens and as such is not an APP in this species. Of the proteins targeted as possible novel biomarkers of the APR in chickens mannan binding lectin associated serine protease-2, α-2-HS-glycoprotein (fetuin) and major facilitator superfamily domain-containing protein 10 were reduced in abundance in the HAP group, behaving as negative biomarkers. Myeloid protein and putative ISG(12)2 were positively associated with the acute phase being significantly higher in the HAP and AP groups. The protein cathepsin D was significantly higher in both HAP and AP compared to the NAP indicating that of all the proteins targeted, this appears to have the most potential as a biomarker of the acute phase, as it was significantly increased in the AP as well as the HAP group. To evaluate APPs and investigate biomarkers of intestinal health, a study using re-used poultry litter was undertaken. The introduction of litter at 12 days of age did not significantly increase any APPs measured using immunoassays and quantitative proteomics at 3, 6 and 10 days post introduction. While no APP was found to be significantly different between the challenged and control groups at anytime point, the APPs AGP, SAA and Hpx did increase over time in all birds. The protein apolipoprotein AIV (apo-AIV) was targeted as a possible APP and because of its reported role in controlling satiety. An ELISA was developed, successfully validated and used to measure apo-AIV in this study. While no significant differences in apo-AIV plasma concentrations between challenged and control groups were identified apo-AIV plasma concentrations did change significantly between certain time points in challenged and control groups. Apoliporotein AIV does not appear to behave as an APP in chickens, as it was not significantly different between acute phase groups. The actin associated proteins villin and gelsolin were investigated as possible biomarkers of intestinal health. Villin was found not to be present in the plasma of chickens and as such not a biomarker target. Gelsolin was found not to be differentially expressed during the acute phase or as a result of intestinal challenge. Finally a proteomic approach was undertaken to investigate gastrocnemius tendon (GT) rupture in broiler chickens with a view of elucidating to and identify proteins associated with risk of rupture. A number of proteins were found to be differentially expressed between tendon pools and further work would enable further detailing of these findings. In conclusion this work has made a number of novel findings and addressed a number of data poor areas. The area of chicken APPs research has stagnated over the last 15 years with publications becoming repetitive and reliant on a small number of immunoassays. This work has sought to characterise the classic APPs in chickens, and use a quantitative proteomic approach to measure and categorise them. This method was also used to take a fresh approach to biomarker identification for both the APR and intestinal health. The development and validation of assays for Ovt and apo-AIV and the shotgun data mean that these proteins can be further characterised in chickens with a view of applying their measurement to diagnostics and selective breeding programs.

Feeding low protein diets to meat chickens: Effects on emissions of toxic and odorous metabolites

Low protein diet and odour emissions in meat chickens

Odour flux from litter of broiler chickens fed diets differing in protein levels and additives

The effect of dietary crude protein (CP) and additives on odour flux from broiler litter was investigated using 180 day-old Ross 308 male chicks randomly allocated to five dietary treatments with three replications of 12 birds each. A 5×3 factorial arrangement of treatments was employed. Factors were: diet (low CP, high CP, high CP+antibiotic, high CP+probiotic, high CP+saponin) and age (15, 29, 35 days). Low CP (LCP) and high CP (HCP) diets differed in CP levels by 4.5-5%. The low CP diets were supplemented with L-valine, L-isoleucine, L-arginine, L-lysine, D,L-methionine and L-threonine. The antibiotic used was Zn Bacitracin, the probiotic was a blend of three Bacillus subtilis strains and the saponin came from a blend of Yucca and Quillaja. Odorants were measured from litter headspace using a flux hood and selective ion flow tube mass spectrometry (SIFT-MS). Results were log tranformed and analysed by two-way ANOVA with repeated measures using JMP statistical software v.8, and means were separated by Tukey's HSD test at P<0.05.The results showed that LCP group produced lower flux of dimethyl amine, trimethyl amine, H2S, NH3 and phenol in litter compared to HCP group (P<0.05). Similarly, HCP+probiotic group produced lower flux of H2S (P<0.05) and HCP+saponin group produced lower flux of trimethylamine and phenol in litter compared to HCP group (P<0.05). The dietary treatments tended (P=0.065) to have higher flux of methanethiol in HCP group compared to others. There was a diet x age interaction for litter flux of diacetyl, acetoin, 3-methyl-1-butanol, 3-methylbutanal, ethanethiol, propionic acid and hexane (P<0.05). Concentrations of diacetyl, acetoin, propionic acid and hexane in litter were higher from LCP group compared to all other treatments on d 35 (P<0.05) but not on days 15 and 29. Thus, the low CP diet, Bacillus subtilis based probiotic and Yucca/Quillaja based saponin were effective in reducing the emissions of some key odorants from broiler litter.

Characterisation and quantification of changes in odorants from litter headspace of meat chickens fed diets varying in protein levels and additives

The effect of dietary crude protein (CP) and additives on odor flux from meat chicken litter was investigated using 180 day-old Ross 308 male chicks randomly allocated to five dietary treatments with three replicates of 12 birds each. A 5 × 3 factorial arrangement of treatments was employed. Factors were: diet (low CP, high CP, high CP+antibiotic, high CP+probiotic, high CP+saponin) and age (15, 29, 35 days). The antibiotic used was Zn bacitracin, the probiotic was a blend of three Bacillus subtilis strains and the saponin came from a blend of Yucca and Quillaja. Odorants were collected from litter headspace with a flux hood and measured using selective ion flow tube mass spectrometry (SIFT-MS). Litter moisture, water activity (Aw), and litter headspace odorant concentrations were correlated. The results showed that low CP group produced lower flux of dimethyl amine, trimethyl amine, H2S, NH3, and phenol in litter compared to high CP group (P < 0.05). Similarly, high CP+probiotic group produced lower flux of H2S (P < 0.05) and high CP+saponin group produced lower flux of trimethylamine and phenol in litter compared to high CP group (P < 0.05). The dietary treatments tended (P = 0.065) to have higher flux of methanethiol in high CP group compared to others. There was a diet × age interaction for litter flux of diacetyl, 3-hydroxy-2-butanone (acetoin), 3-methyl-1-butanol, 3-methylbutanal, ethanethiol, propionic acid, and hexane (P < 0.05). Concentrations of diacetyl, acetoin, propionic acid, and hexane in litter were higher from low CP group compared to all other treatments on d 35 (P < 0.05) but not on d 15 and 29. A high litter moisture increased water activity (P < 0.01) and favored the emissions of methyl mercaptan, hydrogen sulfide, dimethyl sulfide, ammonia, trimethyl amine, phenol, indole, and 3-methylindole over others. Thus, the low CP diet, Bacillus subtilis based probiotic and the blend of Yucca/Quillaja saponin were effective in reducing the emissions of some key odorants from meat chicken litter.

High-resolution electrophoretic procedures for the identification of five Eimeria species from chickens, and detection of population variation

To overcome limitations of conventional approaches for the identification of Eimeria species of chickens, we have established high resolution electrophoretic procedures using genetic markers in ribosomal DNA. The first and second internal transcribed spacer (ITS-1 and ITS-2) regions of ribosomal DNA were amplified by polymerase chain reaction (PCR) from genomic DNA samples representing five species of Eimeria (E. acervulina, E. brunetti, E. maxima, E. necatrix and E. tenella), denatured and then subjected to denaturing polyacrylamide gel electrophoresis (D-PAGE) or single-strand conformation polymorphism (SSCP) analysis. Differences in D-PAGE profiles for both the ITS-1 and ITS-2 fragments (combined with an apparent lack of variation within individual species) enabled the unequivocal identification of the five species, and SSCP allowed the detection of population variation between some isolates representing E. acervulina, which remained undetected by D-PAGE. The establishment of these approaches has important implications for controlling the purity of laboratory lines of Eimeria, for diagnosis and for studying the epidemiology of coccidiosis.

Effect of poultry viscera meal inclusion in broiler diets in different rearing periods on performance, carcass and parts yields

The objective of the present study was to evaluate the effect of the dietary inclusion of poultry viscera meal (VM) on broiler performance and carcass, parts, and abdominal fat yields in broilers by replacing a diet containing VM with a strictly vegetable diet and vice-versa. A number of 720 one-day-old broiler chicks were randomly distributed in 6 groups: G1-basal diet (BD) - corn and soybean based meal, with no VM from 1 to 42 days of age, G2- 8% VM diet from 1 to 42 days, G3- BD from 1 to 21 and 8% VM diet from 22 to 42 days, G4- BD from 1 to 35 and 8% VM diet from 36 to 42 days, G5- 8% VM diet from 1 to 21 days and BD from 22 to 42 days, G6- 8% VM diet from 1 to 35 and BD from 36 to 42 days. Average body weight, weight gain, feed intake, feed conversion ratio (FCR), production efficiency index, and mortality were determined from 1 to 42 days. There was no effect of treatments on performance or mortality, except for FCR, which was significantly better in the group fed VM from 1 to 35 days and withdrawn at the end of rearing (36-42 days). Also, there were no differences in carcass, parts, and abdominal fat yields, showing that VM in broiler diets does not influence yield parameters.

Characterization of heat waves affecting mortality rates of broilers between 29 days and market age

Climate may affect broiler production, especially where there are heat waves, which may cause high mortality rates due to the heat stress. Heat wave prediction and characterization may allow early mitigation actions to be taken. Data Mining is one of the tools used for such a characterization, particularly when a large number of variables is involved. The objective of this study was to classify heat waves that promote broiler chicken mortality in poultry houses equipped with minimal environmental control. A single day of heat, a heat-shock day, is capable of producing high broiler mortality. In poultry houses equipped with fans and evaporative cooling, the characterization of heat waves affecting broiler mortality between 29 days of age and market age presented 89.34% Model Accuracy and 0.73 Class Precision for high mortality. There was no influence on high mortality (HM) of birds between 29 and 31 days of age. Maximum temperature humidity index (THI) above 30.6 ºC was the main characteristic of days when there was a heat wave, causing high mortality in broilers older than 31 days. The high mortality of broilers between 31 and 40 days of age occurred when maximum THI was above 30.6 ºC and maximum temperature of the day was above 34.4 ºC. There were two main causes of high mortality of broilers older than 40 days: 1) maximum THI above 30.6 ºC and minimum THI equal or lower than 15.5 ºC; 2) maximum THI above 30.6 ºC, minimum THI lower than 15.5 ºC, and the time of maximum temperature later than 15:00h. The heat wave influence on broiler mortality lasted an average of 2.7 days.

Apparent metabolizable energy of microalgae (Spirulina sp.) for broilers under different methodologies.

2016

Estimativa de bem-estar de frango de corte em função da concentração de amônia e grau de luminosidade no galpão de produção

Brazil is an important poultry meat export country, and large parts of its destination are countries with specific rearing restrictions related to broiler s welfare. One of the aerial pollutants mostly found in high concentrations in closed poultry housing environment is ammonia. There are evidences that broilers welfare may be compromised by the continuous exposition to this pollutant in rearing housing. This research aimed to estimate broilers welfare reared under specific thermal environmental attributes and bird s density, as function of the ammonia concentration and light intensity inside the housing environment using the Fuzzy Theory. Results showed that the best welfare value (0.89 in the scale: 0-1) approximately 90% of the ideal was found in the conditions that associated the ideal thermal environment, with bird s density between 13-15 birds m-2, with values of the ammonia concentration in the environment below 5 ppm, and light intensity near 1 lx. Using the predictive method it was possible to estimate broilers welfare with relation to the ammonia concentration and light intensity in the housing.

Influência de diferentes materiais de cobertura no conforto térmico de instalações para frangos de corte no oeste paulista

Este trabalho apresenta um estudo da influência de diferentes materiais de cobertura no conforto térmico de instalações destinadas à criação de frangos de corte. A pesquisa foi desenvolvida no Câmpus Experimental da UNESP de Dracena - SP. Quatro protótipos em escala real foram construídos, com área de 28 m² cada, cobertos com telha reciclada à base de embalagens longa vida, telha cerâmica, telha cerâmica pintada de branco e telha de fibrocimento. Os dados foram coletados durante o período de inverno de 2007, totalizando 90 dias. Com esses dados, foram calculados os índices de conforto térmico Carga Térmica Radiante (CTR) e a variável ambiental (Ta). Uma análise estatística por inferência e descritiva foi realizada com os valores do índice de conforto térmico e da variável ambiental. Com os resultados obtidos, é possível afirmar que a telha reciclada apresentou índices de conforto térmico semelhantes àqueles encontrados para as telhas cerâmicas. O protótipo coberto com telha de fibrocimento apresentou os maiores índices, e o coberto com telha cerâmica branca, os menores índices de conforto térmico. No entanto para o período de inverno e para os horários avaliados, todas as instalações apresentaram índices de conforto térmico fora da zona de termoneutralidade do frango de corte.

Evaluation of recycled tiles and OSB ceiling materials in closed broiler house prototypes

This study aimed at evaluating the thermal performance of a modular ceiling system for poultry houses. The reduced- and distorted-scale prototypes used ceiling modules made of reforested wood and were covered with recycled long-life package tiles. The following parameters were measured for 21 days: tile internal surface temperature (ST), globe temperature and humidity index (WBGT), and radiant heat load (RHL). Measurements were made at times of highest heat load (11:00 am, 13:00 pm, and 03:00 pm). Collected data were analyzed by "R" statistics software. Means were compared by multiple comparison test (Tukey) and linear regression was performed, both at 5% significance level. The results showed that the prototype with the ceiling was more efficient to reduce internal tile surface temperature; however, this was not sufficient to provide a comfortable environment for broilers during the growout. Therefore, other techniques to provide proper cooling are required in addition to the ceiling.

Desempenho e rendimento de carcaça de frangos de corte alimentados com dietas contendo subprodutos do arroz formuladas com base nos conceitos de proteína bruta e ideal

Foi conduzido um experimento para avaliar a utilização de subprodutos do arroz em dietas formuladas com base nos conceitos de proteína bruta e ideal para frangos de corte de 1 a 42 dias de idade. Foram utilizados 720 pintos machos de 1 dia de idade da linhagem "Hybro", em delineamento inteiramente ao acaso, em esquema fatorial 3 × 2, composto de três dietas (sem subprodutos, farelo de arroz integral e quirera de arroz) e dois conceitos de formulação de rações (proteína bruta e ideal), totalizando seis tratamentos e quatro repetições de 30 aves. O ganho de peso, o consumo de ração e a conversão alimentar foram avaliados aos 21 e 42 dias e as características de carcaça aos 42 dias de idade. As aves alimentadas com dietas formuladas pelo conceito tradicional (baseado na proteína bruta) apresentaram melhor conversão alimentar e menor taxa de deposição de gordura abdominal.

Composição química, valores de energia metabolizável e aminoácidos digestíveis de subprodutos do arroz para frangos de corte

Dois experimentos foram conduzidos para determinar a composição química, os valores de energia e os coeficientes de digestibilidade dos aminoácidos, do farelo de arroz integral (FAI) e da quirera de arroz (QA). No primeiro estudo, foram utilizadas 144 aves, com 21 dias de idade, machos, linhagem Cobb, que tiveram suas excretas totalmente coletadas para determinação da energia metabolizável aparente (EMA) e energia metabolizável aparente corrigida (EMAn). O delineamento experimental foi inteiramente casualizado, com três tratamentos e seis repetições, com oito aves cada. No segundo experimento, foi utilizado o método de alimentação forçada para a determinação dos coeficientes de digestibilidade dos aminoácidos. O delineamento foi inteiramente casualizado, com dois alimentos e um jejum e seis repetições com um galo cada. Os valores de MS, PB, EE, FB, EMA e EMAn foram, respectivamente, para FAI: 88,6%; 11,8%; 15,3%; 10,2%; 2968kcal kg-1 e 2804kcal kg-1 e para QA: 93,5%; 9,1%; 0,73%; 0,45%; 3338kcal kg-1 e 3239kcal kg-1. Os valores médios encontrados dos coeficientes de digestibilidade de aminoácidos essenciais e não essenciais foram, respectivamente, de 75,9% e 73,9%, para FAI, e 77,9% e 76,5%, para QA. Embora tenham apresentado níveis inferiores de energia, FAI e a QA podem ser utilizados nas rações de aves em substituição ao milho, uma vez que tiveram níveis maiores de proteína bruta e aminoácidos digestíveis.

Níveis de lisina digestível para frangos de corte machos no período de 37 a 49 dias de idade

Foram utilizados 1.015 frangos de corte machos, linhagem Ross, dos 37 aos 49 dias de idade, para avaliar os efeitos de diferentes níveis de lisina digestível nas dietas experimentais. O delineamento experimental foi inteiramente casualizado, com 5 níveis de lisina digestível (0,90; 0,95; 1,00; 1,05; 1,10%), cada um com 7 repetições e 29 aves por unidade experimental. Utilizaram-se dietas isoenergéticas com 3.250 de kcal de EM/kg e isoprotéicas, com 18% de PB, à base de milho e farelo de soja. Foram avaliados o ganho de peso, o consumo de ração, a conversão alimentar, as características de carcaça, o rendimento de cortes, a composição e a deposição de nutrientes corporais. Os níveis de lisina digestível influenciaram, entre as características de desempenho, apenas a conversão alimentar, que melhorou de forma linear de acordo com níveis de lisina da ração. Das características de carcaça e rendimento de cortes, apenas gordura abdominal aumentou de forma quadrática conforme os níveis de lisina. Os níveis de lisina digestível tiveram efeito quadrático no teor da matéria mineral da carcaça, mas não influenciaram a composição química das vísceras e do sangue. Observou-se, contudo, tendência a aumento linear na deposição de proteína da carcaça e do corpo vazio com o aumento no nível de lisina digestível. Os resultados de desempenho indicam que o nível de lisina digestível da ração de frangos de corte machos no período de 37 a 49 dias de idade deve ser igual ou superior a 1,10%.

Lisina digestível e zinco quelado para frangos de corte machos: desempenho e retenção de nitrogênio na fase pré-inicial

Em dois ensaios experimentais, avaliaram-se níveis de lisina digestível (0,90; 1,00; 1,10; 1,20 e 1,40%) combinados a zinco quelado (43 e 253 ppm) para frangos de corte machos. Os períodos considerados foram: 1 a 11 dias de idade (desempenho) e 1 a 7 dias de idade (balanço de nitrogênio). O delineamento experimental utilizado foi de blocos ao acaso, em esquema fatorial 5 × 2. No ensaio de desempenho, utilizaram-se 7 repetições e 15 aves por unidade experimental e, no ensaio de digestibilidade, 6 repetições e 6 aves por unidade experimental. As dietas continham 2.960 de EM/kg e 21% de proteína bruta. Na fase de 1 a 11 dias de idade, não houve efeito de interação lisina digestível × zinco quelado no desempenho, mas verificou-se efeito quadrático dos níveis de lisina digestível sobre o peso final, o ganho de peso e o ganho de peso relativo e efeito linear crescente no consumo de ração. A conversão alimentar não diferiu com as variações dos níveis de lisina digestível e zinco na dieta. Nas condições de avaliação do desempenho, o nível ótimo de lisina digestível para frangos de corte machos mantidos em piso de concreto é de 1,19%. Na fase de 1 a 7 dias de idade, não houve influência da relação entre níveis de lisina digestível e zinco quelado no balanço de nitrogênio. A retenção de nitrogênio aumentou de forma linear crescente com o aumento da concentração de lisina digestível na dieta, o que está de acordo com o aumento linear do ganho de peso e da eficiência alimentar. A exigência de lisina digestível para frangos de corte machos de 1 a 7 dias de idade é igual ou superior a 1,40%.