7 research outputs found
Efeito da selecção genética na expressão e actividade das enzimas reguladoras do metabolismo intermediário, da estrutura muscular e da função testicular: do Oryctolagus cuniculus algirus ao Oryctolagus cuniculus cuniculus
Tese de Doutoramento em Ciência e Tecnologia AnimalO objectivo deste trabalho é a identificação da actividade e da expressão genética de enzimas reguladoras do metabolismo intermediário. O efeito da restrição alimentar na expressão proteica do músculo e na actividade angiogénica testicular também é abordada. São utilizadas duas raças de coelhos: oryctolagus cuniculus algirus, o coelho bravo (WR) e uma raça de carne de alta produtividade, oryctolagus cuniculus cuniculus (coelho NeoZelandês-NZ). Os grupos experimentais foram sujeitos a restrição alimentar (30% da alimentação ad libitum) durante 30 dias, resultando numa perda de peso de 17.7% nos NZ (n=11) e 21.7% nos WR (n=10). Os grupos controlo foram alimentados ad libitum (NZ, n=10; WR, n=9). Um grupo com restrição mais acentuada (NZHR-34.7%perda de peso) foi utilizado no estudo da angiogénese testicular. Os nossos resultados das actividades das enzimas sugerem que os NZ apresentam um melhor controlo da glicemia e uma maior disponibilidade energética dos substratos através das acções das enzimas HK, PFK e PK. Os resultados do metabolismo proteico mostram que o grupo WR apresenta um nível de actividade inferior na desaminação do glutamato. Apesar dos WR apresentarem um nível superior do mRNA da HL, foram encontrados níveis semelhantes dos NEFA em ambos os grupos. Este perfil enzimático do metabolismo proteico e lipídico pode explicar os melhores desempenhos da raça NZ. Os nossos resultados mostram que o nível do mRNA pode não ser indicador fiável da actividade das respectivas enzimas. Das hormonas estudadas verifica-se que a insulina desempenha um papel na homeostase da glicemia, o T3 e o IGF-1 parecem desempenhar funções importantes na produtividade aumentada da raça NZ, com o T3 a intervir nos mecanismos de poupanças energéticas nos animais em restrição e o IGF-1 com a conhecida função anabólica no metabolismo proteico. Os nossos resultados relativos à expressão proteica do músculo permitiram-nos identificar níveis diferentes de proteínas com funções metabólicos/fisiológicos (enolase e PQ) e estruturais (MHC, MLC, actina) relevantes, tal como também a expressão de proteínas menos conhecidas (LIM e α-crystallin chain B). Este estudo também indicou que diversas proteínas musculares podem ser usadas como marcadores válidos para a perda de peso. O estudo da angiogénese testicular mostra um aumento na área microvascular do testículo e na mitogénese de BAEC nos coelhos NZHR. Apesar de não haver diferenças na concentração de DNA testicular entre os grupos, a restrição alimentar diminuiu os níveis de RNA e de proteína face aos controlo. Nenhuma diferença foi observada entre os grupos na % de túbulos seminíferos com células espermáticas e nem na área ocupada pelos túbulos seminíferos. No entanto, a testosterona sérica foi marcadamente inferior no grupo NZHR comparado com NZC e NZR. Estes resultados sugerem que a angiogénese pode desempenhar um papel na disfunção testicular causada pela subnutrição e na preservação das funções reprodutivas em coelhos sujeitos a restrição alimentar.ABSTRACT : The aim of this work is the identification of intermediate metabolism enzyme gene expression and activity enhancing the improvement of meat production due to genetic selection. The effect of food restriction in muscle protein expression and on testicular angiogenic activity will also be studied. Two different breeds of rabbits were used: oryctolagus cuniculus algirus, a wild rabbit breed and a highly meat selected breed, oryctolagus cuniculus cuniculus (New Zealand rabbit). In order to achieve these objectives both groups were subjected to a restricted diet (30% of ad libitum feeding) during 30 days, resulting in a 17.7% weight loss in the NZ rabbit (n=11) and 21.7% in the WR (n=10). Control groups were fed ad libitum (NZ, n=10; WR, n=9). A more restricted group was used (NZHR-34.7% weight loss) in the testicular angiogenic study. Our results regarding the enzymes activities, suggest that NZ rabbits have a better glycemia control and greater energetic substrate availability through HK, PFK and PK actions. The nitrogen metabolism results show that WR group has a lower activity level in the deamination of glutamate. Although the WR group showed a higher mRNA HL, similar NEFA levels were found in both experimental groups. This enzyme profile concerning nitrogen and lipid metabolism can explain better performances shown by the NZ breed. Our results show that mRNA transcriptions may not be reliable indicators of respective enzyme activity. The hormone results show that apart from insulin with a role in glycemia homeostasis, T3 and IGF-1 seem to play a relevant role in the enhanced productivity in the NZ breed, with T3 intervening in the energy saving mechanisms of the restricted diet animals and IGF-1 with the known protein anabolic function. Our protein expression results of muscle showed that we were able to identify and detect differential expression levels of several proteins with relevant metabolic/physiological (enolase, PK) and structural (MHC, MLC, Actin) roles, as well as others less familiar such as LIM protein and α-crystallin chain B. This study also indicates that several muscle proteins can be used as valid markers for weight loss. The work on testicular angiogenesis showed an increase in testicular microvascular area and mitogenesis of BAEC in NZHR rabbits. Despite no change in testicular DNA concentration among groups, feed restriction decreased both RNA and protein over control. No treatment differences in the % of seminiferous tubules filled with sperm cells, as well as the area occupied by seminiferous tubules were observed. Nevertheless, serum testosterone was markedly less in group NZHR compared to NZC and NZR. These results suggest that angiogenesis may play a role in overcoming testicular nutritional impairment and preservation of reproductive function in rabbits subjected to food restriction.This work was funded by Fundação para a Ciência e Tecnologia, grant SFRH/BD/4943/2001
and by CIISA-FMV
The Effect of Weight Loss on the Muscle Proteome in the Damara, Dorper and Australian Merino Ovine Breeds.
Seasonal Weight Loss (SWL) is an important constraint, limiting animal production in the Tropics and the Mediterranean. As a result, the study of physiological and biochemical mechanisms by which domestic animal breeds respond to SWL is important to those interested in animal breeding and the improvement thereof. To that end, the study of the proteome has been instrumental in gathering important information on physiological mechanisms, including those underlying SWL. In spite of that, little information is available concerning physiological mechanisms of SWL in production animals. The objective of this study was to determine differential protein expression in the muscle of three different breeds of sheep, the Australian Merino, the Dorper and the Damara, each showing different levels of tolerance to weight loss (low, medium and high, respectively). Per breed, two experimental groups were established, one labeled "Growth" and the other labeled "Restricted." After forty-two days of dietary treatment, all animals were euthanized. Muscle samples were then taken. Total protein was extracted from the muscle, then quantified and two-dimensional gel electrophoresis were conducted using 24 cm pH 3-10 immobiline dry strips and colloidal coomassie staining. Gels were analyzed using Samespots® software and spots of interest were in-gel digested with trypsin. The isolated proteins were identified using MALDI-TOF/TOF. Results indicated relevant differences between breeds; several proteins are suggested as putative biomarkers of tolerance to weight loss: Desmin, Troponin T, Phosphoglucomutase and the Histidine Triad nucleotide-binding protein 1. This information is of relevance to and of possible use in selection programs aiming towards ruminant animal production in regions prone to droughts and weight loss
Two-dimensional gel electrophoresis.
<p>Example of a 2D gel for a merino ram gastrocnemius muscle, showing the spots that were selected for identification. Molecular mass markers (MM, vertical scale), as well as the isoelectric point (pI, horizontal scale are also presented. Two-dimensional electrophoresis was carried out in linear 24 cm Immobiline DryStrips (pH 3–10). Gels were stained with colloidal Coomassie Blue G-250.</p
Plasma concentrations of leptin and insulin (mean (s.e.)) in three breeds of sheep fed a diet maintaining or restricting growth.
<p>Plasma concentrations of leptin and insulin (mean (s.e.)) in three breeds of sheep fed a diet maintaining or restricting growth.</p
Live weight changes (kg ± standard deviation) for Damara, Dorper and Merino breeds assigned to either a restricted or growth diet.
<p>Live weight changes (kg ± standard deviation) for Damara, Dorper and Merino breeds assigned to either a restricted or growth diet.</p
Two-dimensional gel electrophoresis.
<p>Examples of 2D gels obtained for the muscle tissue of Merino, Damara and Dorper rams. Molecular mass markers (MM, vertical scale), as well as isoelectric point (horizontal scale) are also presented. Two-Dimensional electrophoresis was carried out in linear 24cm Immobiline dry strips (pH 3–10). Six hundred micrograms of protein were loaded per gel. Staining was performed with Colloidal Coomassie Blue G-250.</p