A candidate gene approach to identify DNA markers associated with meat quality and production traits: investigation of several cathepsin genes in Italian heavy pigs.

The cathepsin enzymes represent an important family of lysosomal proteinases with a broad spectrum of functions in many, if not in all, tissues and cell types. In addition to their primary role during the normal protein turnover, they possess highly specific proteolytic activities, including antigen processing in the immune response and a direct role in the development of obesity and tumours. In pigs, the involvement of cathepsin enzymes in proteolytic processes have important effects during the conversion of muscle to meat, due to their influence on meat texture and sensory characteristics, mainly in seasoned products. Their contribution is fundamental in flavour development of dry-curing hams. However, several authors have demonstrated that high cathepsin activity, in particular of cathepsin B, is correlated to defects of these products, such as an excessive meat softness together with abnormal free tyrosine content, astringent or metallic aftertastes and formation of a white film on the cut surface. Thus, investigation of their genetic variability could be useful to identify DNA markers associated with these dry cured hams parameters, but also with meat quality, production and carcass traits in Italian heavy pigs. Unfortunately, no association has been found between cathepsin markers and meat quality traits so far, in particular with cathepsin B activity, suggesting that other genes, besides these, affect meat quality parameters. Nevertheless, significant associations were observed with several carcass and production traits in pigs. A recent study has demonstrated that different single nucleotide polymorphisms (SNPs) localized in cathepsin D (CTSD), F (CTSF), H and Z genes were highly associated with growth, fat deposition and production traits in an Italian Large White pig population. The aim of this thesis was to confirm some of these results in other pig populations and identify new cathepsin markers in order to evaluate their effects on cathepsin activity and other production traits. Furthermore, starting from the data obtained in previous studies on CTSD gene, we also analyzed the known polymorphism located in the insulin-like growth factor 2 gene (IGF2 intron3-g.3072G>A). This marker is considered the causative mutation for the quantitative trait loci (QTL) affecting muscle mass and fat deposition in pigs. Since IGF2 maps very close to CTSD on porcine chromosome (SSC) 2, we wanted to clarify if the effects of the CTSD marker were due to linkage disequilibrium with the IGF2 intron3-g.3072G>A mutation or not. In the first chapter, we reported the results from these two SSC2 gene markers. First of all, we evaluated the effects of the IGF2 intron3-g.3072G>A polymorphism in the Italian Large White breed, for which no previous studies have analysed this marker. Highly significant associations were identified with all estimated breeding values for production and carcass traits (P<0.00001), while no effects were observed for meat quality traits. Instead, the IGF2 intron3-g.3072G>A mutation did not show any associations with the analyzed traits in the Italian Duroc pigs, probably due to the low level of variability at this polymorphic site for this breed. In the same Duroc pig population, significant associations were obtained for the CTSD marker for all production and carcass traits (P < 0.001), after excluding possible confounding effects of the IGF2 mutation. The effects of the CTSD g.70G>A polymorphism were also confirmed in a group of Italian Large White pigs homozygous for the IGF2 intron3-g.3072G allele G (IGF2 intron3-g.3072GG) and by haplotype analysis between the markers of the two considered genes. Taken together, all these data indicated that the IGF2 intron3-g.3072G>A mutation is not the only polymorphism affecting fatness and muscle deposition in pigs. In the second chapter, we reported the analysis of two new SNPs identified in cathepsin L (CTSL) and cathepsin S (CTSS) genes and the association results with meat quality parameters (including cathepsin B activity) and several production traits in an Italian Large White pig population. Allele frequencies of these two markers were evaluated in 7 different pig breeds. Furthermore, we mapped using a radiation hybrid panel the CTSS gene on SSC4. Association studies with several production traits, carried out in 268 Italian Large White pigs, indicated positive effects of the CTSL polymorphism on average daily gain, weight of lean cuts and backfat thickness (P<0.05). The results for these latter traits were also confirmed using a selective genotype approach in other Italian Large White pigs (P<0.01). In the 268 pig group, the CTSS polymorphism was associated with feed:gain ratio and average daily gain (P<0.05). Instead, no association was observed between the analysed markers and meat quality parameters. Finally, we wanted to verify if the positive results obtained for the cathepsin L and S markers and for other previous identified SNPs (cathepsin F, cathepsin Z and their inhibitor cystatin B) were confirmed in the Italian Duroc pig breed (third chapter). We analysed them in two groups of Duroc pigs: the first group was made of 218 performance-tested pigs not selected by any phenotypic criteria, the second group was made of 100 Italian Duroc pigs extreme and divergent for visible intermuscular fat trait. In the first group, the CTSL polymorphism was associated with weight of lean cuts (P<0.05), while suggestive associations were obtained for average daily gain and backfat thickness (P<0.10). Allele frequencies of the CTSL gene marker also differed positively among the visible intermuscular extreme tails. Instead, no positive effects were observed for the other DNA markers on the analysed traits. In conclusion, in agreement with the present data and for the biological role of these enzymes, the porcine CTSD and CTSL markers: a) may have a direct effect in the biological mechanisms involved in determining fat and lean meat content in pigs, or b) these markers could be very close to the putative functional mutation(s) present in other genes. These findings have important practical applications, in particular the CTSD and CTSL mutations could be applied in a marker assisted selection (MAS) both in the Italian Large White and Italian Duroc breeds. Marker assisted selection could also increase in efficiency by adding information from the cathepsin S genotype, but only in the Italian Large White breed.

Alternative genotyping method for the single nucleotide polymorphism A2959G (AF159246) of the bovine CAST gene

The objective of this work was to genotype the single nucleotide polymorphism (SNP) A2959G (AF159246) of bovine CAST gene by PCR-RFLP technique, and to report its use for the first time. For this, 147 Bos indicus and Bos taurus x Bos indicus animals were genotyped. The accuracy of the method was confirmed through the direct sequencing of PCR products of nine individuals. The lowest frequency of the meat tenderness favorable allele (A) in Bos indicus was confirmed. The use of PCR-RFLP for the genotyping of the bovine CAST gene SNP was shown to be robust and inexpensive, which will greatly facilitate its analysis by laboratories with basic structure.

Alternative genotyping method for the single nucleotide polymorphism A2959G (AF159246) of the bovine CAST gene

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

Encurtamento pelo frio de fibras musculares oxidativas de bovinos pela técnica de NADH-TR

Carcaças bovinas resfriadas rapidamente podem apresentar uma contração muscular conhecida como encurtamento pelo frio. Esse fenômeno, prejudicial à textura da carne, ocorre principalmente nas fibras musculares oxidativas. O objetivo deste trabalho foi disponibilizar uma ferramenta analítica para distinguir essas fibras e determinar com maior precisão a contração do tecido muscular pela mensuração do comprimento dos sarcômeros. Amostras do músculo Longissimus dorsi de 12 novilhas nelore foram coletadas. As amostras obtidas de uma das meias-carcaças foram submetidas a uma refrigeração rápida, e as amostras provenientes da outra meia-carcaça à refrigeração lenta. Foi testado um método analítico, baseado na técnica de coloração por Nicotinamida Adenina Dinucleotídeo - Tetrazolium Redutase (NADH-TR), para mensurar, por microscopia, o comprimento dos sarcômeros das fibras vermelhas. Foram determinadas as velocidades de queda de pH e temperatura, a área do olho de lombo (AOL) e a força de cisalhamento. Os resultados demonstraram que a temperatura é o principal fator responsável pelo comprimento do sarcômero quando a velocidade de resfriamento é rápida, sendo essa influência menor quando a queda de temperatura é mais lenta. Desta forma, demonstrou-se que a técnica de coloração com NADH-TR é capaz de detectar a ocorrência do encurtamento pelo frio nos músculos esqueléticos de bovinos.

Fatores que influenciam a textura da carne de novilhos Nelore e cruzados Limousin-Nelore

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

MÉTODO SIMPLES PARA ESTIMAR ENCURTAMENTO PELO FRIO em CARNE BOVINA

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

Espessura da lâmina de cisalhamento na avaliação instrumental da textura da carne

A padronização da textura é um dos principais problemas relacionados à qualidade da carne bovina. Por isso, a avaliação objetiva da maciez, por métodos mecânicos, vem sendo estudada há muitos anos. Para que a avaliação instrumental da textura seja uma ferramenta efetiva nos estudos envolvendo a maciez da carne, é necessário minimizar as causas de variação envolvidas na análise. Atualmente, a força de cisalhamento Warner-Bratzler é o método mais amplamente empregado para essa finalidade. O protocolo padrão dessa técnica analítica preconiza a utilização de uma lâmina de cisalhamento de 1,016mm de espessura. Neste trabalho, foram comparados dados de textura de carne bovina obtidos com a lâmina de cisalhamento padrão Warner-Bratzler, de 1,016mm de espessura, e uma lâmina mais grossa, de 3,05mm de espessura. Após o abate, amostras de músculo Longissimus dorsi, semelhantes em relação aos teores de umidade, lipídeos e proteínas, foram submetidas a duas diferentes temperaturas de resfriamento, atingindo diferentes velocidades de queda de temperatura e, consequentemente, diferentes graus de contração do tecido muscular, o que foi confirmado pela determinação do comprimento dos sarcômeros. Os resultados demonstraram uma maior sensibilidade da lâmina padrão Warner-Bratzler na detecção de variações da textura da carne, em função da velocidade de resfriamento.

Óleo de linhaça como principal fonte lipídica na dieta de frangos de corte

Pós-graduação em Ciência Animal - FMVA

Efeito do óleo de linhaça no desempenho, características de carcaça e qualidade de carne de frangos de corte

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

Pesos de abate e uréia protegida na dieta de terminação em confinamento de bovinos Nelore não castrados

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Cozinha a baixa temperatura:

A dissertação “Cozinha a Baixa Temperatura: Aplicação ao caso da Confeção de Carne de Novilho” inicia-se pelo enquadramento histórico e respetiva conceptualização. São enumerados e explicados os intervenientes e procedimentos na Cozinha a Baixa Temperatura-Cozinha em Vácuo. Enquanto estudo, a cozinha a baixa temperatura foi alvo de críticas positivas no que confere aos benefícios nutricionais, à maximização das potencialidades do produto (conservação, textura, sabor, entre outros…), ao incremento das vantagens organizacionais e económicas (gestão de cozinhas/restaurantes); alertando para o fato de que um incorreto embalamento comprometerá as vantagens da técnica em estudo, ou seja são enumerados ainda todos os requisitos necessários a uma correta e eficaz cozinha em vácuo. Os diferentes tipos de cozinha a vácuo são igualmente abordados (indireta e dupla cozedura), bem como a influência da relação existente entre o tempo e a temperatura e as consequentes modificações químicas e fisiológicas. No intuito de introduzir uma vertente prática e enquanto projeto pessoal e profissional foi desenvolvida uma máquina para cozinhar a baixa temperatura (roner). Neste âmbito foram enumerados os materiais utilizados e explicado o seu funcionamento. Porque conceber um roner implica testá-lo, foi então decidido utilizar carne de vitela (capitação 100gr) durante 1 hora com temperaturas entre os 54 ºC e os 70 ºC. Foram realizados testes de textura em todas as amostras de carne recorrendo ao texturómetro TA XT Plus e respetivo software para o tratamento dos resultados. Em suma, analisando os resultados obtidos, a relação tempo e temperatura origina modificações químicas e fisiológicas na carne, ou seja uma carne a 54 ºC aporta uma menor gelatinização do colagénio originando uma carne menos macia, de tom rosa avermelhado, onde se dá uma menor contração das fibras musculares e que consequentemente origina uma menor quantidade de sucos expelidos da carne; já o contrário acontece numa confeção a 60 ºC, em que se verifica um tom rosa uniforme onde acontece uma maior contração das fibras musculares originando uma maior quantidade de sucos expelidos pela carne. Os resultados dos ensaios instrumentais de textura mostraram que a carne confecionada a 60 ºC se apresentou menos firme e menos dura, e portanto mais macia.

Qualidade da carne de ovinos puros e cruzados criados no Semiárido Nordestino brasileiro.

Resumo: A procura por carne de qualidade tem proporcionado ao setor de ovinocultura a adoção por técnicas de produção que possam contribuir para disponibilizar ao mercado produtos que atendam as necessidades dos consumidores, que se tornam cada vez mais exigentes quanto à aquisição de produtos que possam trazer benefícios a saúde. Assim, objetivou-se determinar a qualidade da carne de ovinos puros e cruzados criados no semiárido Nordestino. Utilizaram-se 30 cordeiros machos e fêmeas, oriundos dos cruzamentos Morada Nova x Morada Nova, Rabo Largo x Morada Nova e Santa Inês x Morada Nova, alojados em baias e alimentados com dieta a base de capim Canarana, milho farelo de soja e calcário. Os animais foram abatidos com peso médio de 24 kg de peso vivo e as carcaças mantidas em câmara frigorífica a 4?C, por 24 horas. Após esse período, foi seccionado o músculo Longissimus dorsi que foi embalado, identificado e armazenado a 20?C. Foram determinados os atributos físicos de capacidade de retenção de água, perda de peso por cocção e força de cisalhamento; os atributos químicos como umidade, proteína, cinza, lipídios e colesterol; avaliações subjetivas da carcaça como cor, marmoreio, textura da carne, distribuição da gordura, espessura da gordura e área de olho de lombo; os sensoriais de dureza, suculência, sabor, aroma e aceitação global e o perfil de ácidos graxos. O delineamento utilizado foi em esquema fatorial 3x2 (três grupos genéticos e dois sexos) e as médias comparadas pelo teste de Duncan a 5%. Não houve diferença (p>0,05) entre os genótipos para a composição química da carne, exceto para a variável lipídio (p<0,05). Para as características subjetivas da carcaça o genótipo influenciou (p<0,05) nas variáveis cor, marmoreio e textura da carne. O genótipo promoveu efeito (p<0,05) para as características físicas de capacidade de retenção de água e perda de peso por cocção. Os atributos sensoriais não sofreram efeitos (p>0,05) dos genótipos avaliados. Os genótipos influenciaram (p<0,05) as concentrações dos ácidos graxos saturados, monoinsaturados e poli-insaturados assim como, nas relações ?3:?6 e AGM:AGS. O fator sexo influenciou (p<0,05) nas variáveis umidade, força de cisalhamento, dureza, ácidos graxos desejáveis e relação ?3:?6. O cruzamento entre raças nativas mostrou-se com potencial para a produção de carne de qualidade, a raça Morada Nova melhorou os atributos físico-químicos da carne e em seu perfil lipídico e, o fator sexo melhorou o atributo maciez dando destaque a carne das fêmeas. Abstract: The demand for quality meat products has given the sheep industry sector productions techniques that can contribute providing the market with products that supply the needs of consumers require, who are becoming more demanding as the acquisition of differentiated and bring health benefits. The objective was to evaluate the quality of the pure sheep meat and cross bred in the semiarid Northeast of Brazil. It was used 30 male lambs and female, that came from crosses Morada Nova x Morada Nova, Rabo Largo x Morada Nova and Santa Ines x Morada Nova, housed in pens and fed a diet of grass Canarana, corn soybean meal and limestone. The animals were slaughtered at average weight of 24 kg, live weight, and carcasses kept in refrigeration chamber at 4 ° C for 24 hours. After this period the Longissimus dorsi muscle was selected, packaged, labeled and stored at 20˚C. The physical attributes were determined such as water holding capacity, cooking weight loss and shear force; the chemical attributes such as moisture, protein, ash, lipids and cholesterol; Subjective evaluations such as color, marbling, meat texture, fat distribution, fat thickness and loin eye area; the hardness sensory, juiciness, flavor, aroma and global acceptance and fatty acid profile. The design was a 3x2 factorial arrangement (three genetic groups and two genders) and the measures compared by Duncan test at 5%. There was no difference (p> 0.05) between genotypes for the chemical composition of meat, except for the lipid variable (p <0.05). To the subjective characteristics of the carcass, the genotype influenced (p <0.05) in the variables color, marbling and meat texture. The genotype promoted significant effect (p <0.05) for the physical characteristics, water retention capacity and weight loss on cooking. The sensory attributes did not suffer significant effects (p> 0.05) of the evaluated genotypes. The genotypes influenced (p <0.05) in the concentrations of saturated fatty acids, monounsaturated and polyunsaturated as well as in the relations ω3: ω6 and AGM:AGS. The gender factor influenced (P <0.05) in the variables moisture, shear strength, hardness, desirable fatty acids and relation ω3: ω6. The cross between native breeds showed up with potential for quality meat production, the Morada Nova improved the physical and chemical attributes of the flesh and in their lipid profiles and sex factor improved softness attribute highlighting the meat of females.

Meat quality of ten cattle breeds of the Southwest of Europe

A sample of about 70 young bulls of each of ten beef cattle breeds reared in their typical production systems has been characterised with respect to meat quality traits. Breeds included were Asturiana de los Valles, Asturiana de la Montaña, Avileña-Negra Ibérica, Bruna dels Pirineus, Morucha, Pirenaica and Retinta from Spain, and Aubrac, Gasconne and Salers from France. As was previously showed regarding carcass traits, there exist large differences both between and within breed – systems. In general, rustic breeds tended to present darker and redder meats with higher haematin contents, whereas less precocious and more specialised breeds showed brighter meats with bigger water losses. Protein content was similar, whereas intramuscular fat presented the larger variations both between and within breeds. ICDH content was higher in the more rustic breeds, showing the predominantly oxidative character of their fibres. Texture measurements showed in general large within breed-system variations, the differences between breeds being less evident. Within breed-system, daily gain weight was positively associated with brighter and tender meats. In the range studied, increasing slaughter weight within breed did not have influence on meat quality. Conformation was related to lower water holding capacity and less dry matter and intramuscular fat, as well as to a lower haematin content giving brighter meats. The increase in fatness scores was related to an augment of toughness in meat specialised breeds, although in Avileña-Negra Ibérica breed fatness carcasses were related to more tender meats. Redness parameter a* was positively related to fatter animals and opposed to conformation, the opposite being true for the L* (lightness) parameter. There was a small trend of fatter carcasses to be related to oxidative fibres, whereas the muscular fibres of the more conformed carcasses were more glycolytic. Texture measurements maximum load, maximum stress and toughness were very closely related. Shear force was opposed to tenderness. Losses at cooking were opposed to juiciness, but only in the Spanish breeds. No consistent trends regarding relationships between carcass characteristics and tenderness could be observed. Overall acceptability was primarily related with tenderness and flavour, and later on juiciness.

Effect of magnesium sulphate and L-tryptophan and genotype on the feed intake, behaviour and meat quality of pigs

Sixty-nine entire male pigs with different halothane genotype (homozygous halothane positive – nn –, n=36; and homozygous halothane negative – NN-, n=33) were fed with a supplementation of magnesium sulphate (Mg) and/or L-tryptophan (Trp) in the diet for 5 days before slaughter. Animals were housed individually and were submitted to stressful ante mortem conditions (mixed in the lorry according to treatments and transported 1 hour on rough roads). Individual feed intake was recorded during the 5-d treatment. At the abattoir, pig behaviour was assessed in the raceway to the stunning system and during the stunning period by exposure to CO2. Muscle pH, colour, water holding capacity, texture and cathepsin activities were determined to assess meat quality. The number of pigs with an individual feed intake lower than 2 kg/d was significantly different among diets (P&0.05; Control: 8.7 %; Mg&Trp: 43.5 %; Trp: 17.4 %) and they were considered to have inadequate supplement intake. During the ante mortem period, 15.2 % of pigs included in the experiment died, and this percentage decreased to 8.7 % in those pigs with a feed intake & 2kg/day, all of them from the stress-sensitive pigs (nn). In general, no differences were observed in the behaviour of pigs along the corridor leading to the stunning system and inside the CO2 stunning system. During the stunning procedure, Trp diet showed shorter periods of muscular excitation than control and Mg&Trp diets. The combination of a stressful ante mortem treatment and Mg&Trp supplementation led to carcasses with high incidence of severe skin lesions. Different meat quality results were found when considering all pigs or considering only those with adequate supplement intake. In this later case, Trp increased pH45 (6.15) vs Control diet (5.96) in the Longissimus thoracis (LT) muscle (P&0.05) and pH at 24h (Trp: 5.59 vs C: 5.47) led to a higher incidence of dark, firm and exudative (DFD) traits in SM muscle (P&0.05). Genotype affected negatively all the meat quality traits. Seventy-five percent of LT and 60.0 % of the SM muscles from nn pigs were classified as pale, soft and exudative (PSE), while none of the NN pigs showed these traits (P&0.0001). No significant differences were found between genotypes on the incidence of DFD meat. Due to the negative effects observed in the Mg&Trp group in feed intake and carcass quality, the utilization of a mixture of magnesium sulphate and tryptophan is not recommended.

Effect of magnesium sulphate and L-tryptophan and genotype on the feed intake, behaviour and meat quality of pigs

Sixty-nine entire male pigs with different halothane genotype (homozygous halothanepositive – nn-, n=36; and homozygous halothane negative – NN-, n=33) were fed with a supplementation of magnesium sulphate (Mg) and/or L-tryptophan (Trp) in the diet for 5days before slaughter. Animals were housed individually and were submitted to stressful ante mortem conditions (mixed in the lorry according to treatments and transported 1h on rough roads). Individual feed intake was recorded during the 5-day treatment. At the abattoir, pig behaviour was assessed in the raceway to the stunning system and during the stunning period by exposure to CO2. Muscle pH, colour, water holding capacity, texture and cathepsin activities were determined to assess meat quality. The number of pigs with an individual feed intake lower than 2kg/day was significantly different among diets (P<0.05; Control: 8.7%; Mg&Trp: 43.5%; Trp:17.4%) and they were considered to have inadequate supplement intake. During the antemortem period, 15.2% of pigs included in the experiment died, and this percentagedecreased to 8.7% in those pigs with a feed intake > 2kg/day, all of them from thestress-sensitive pigs (nn). In general, no differences were observed in the behaviour ofpigs along the corridor leading to the stunning system and inside the CO2 stunningsystem. During the stunning procedure, Trp diet showed shorter periods of muscularexcitation than control and Mg&Trp diets. The combination of a stressful ante mortemtreatment and Mg&Trp supplementation led to carcasses with high incidence of severeskin lesions. Different meat quality results were found when considering all pigs orconsidering only those with adequate supplement intake. In this later case, Trp increased pH45 (6.15) vs Control diet (5.96) in the Longissimus thoracis (LT) muscle (P<0.05) and pH at 24h (Trp: 5.59 vs C: 5.47) led to a higher incidence of dark, firm and dry (DFD) traits in SM muscle (P<0.05). Genotype affected negatively all the meat quality traits. Seventy-five percent of LT and 60.0% of the SM muscles from nn pigs wereclassified as pale, soft and exudative (PSE), while none of the NN pigs showed these traits (P<0.0001). No significant differences were found between genotypes on the incidence of DFD meat. Due to the negative effects observed in the Mg&Trp group in feed intake and carcass quality, the utilization of a mixture of magnesium sulphate and tryptophan is not recommended