Development of a duplex qPCR assay with locked nucleic acid probes for A, B and E kappa-casein variants detection.

Milk proteins determine important milk technological characteristics. Among caseins, Ƙ-casein has been correlated with fat and protein content and cheese yield. Fourteen Ƙ-caseins variants have been described but the alleles A, B and E are the most important ones due to their frequency and/or influence on the technological aptitudes of milk. Therefore, in the present study two different duplex qPCR assays with locked nucleic acid probes (for positions 13104 and 13124 of the Ƙ-casein gene) were developed for the detection of A, B and E variants. Firstly, DNA isolation method from milk somatic cells and hair was optimised. The developed 13124-qPCR assay showed an increased sensitivity reaching up to 6.7 copies DNA copies/reaction at a 95% confidence level with A, B and E alleles reference samples. The 13104-qPCR assay reached up to 6.7 DNA copies/reaction for A allele reference sample and 67 DNA copies/reaction for B and E samples. Intra-assay variation results were below 6%. Applicability was determined using DNA samples from animals with known genotype for Ƙ-casein (AA, AB, BB, BE, AE, EE) and both assays were able to discriminate among the six genotypes with 100% accuracy. Thus, this qPCR method represents a sensitive and rapid option for the detection of Ƙ-casein alleles in both hair and milk samples.

Expression of key myogenic, fibrogenic and adipogenic genes in Longissimus thoracis and Masseter muscles in cattle.

Adipogenesis, myogenesis and fibrogenesis are related processes that can contribute to meat quality. Therefore, extending the knowledge of these processes would facilitate the identification of molecular markers that predict intramuscular fat accretion. The main purpose of this work, based on previous results, was to further study the expression of key genes related to adipogenic, myogenic, fibrogenic processes and some cytokines in Longissimus thoracis (LT) and Masseter (MS) muscles of Pirenaica and Holstein young bulls. Longissimus thoracis and MS muscles from Pirenaica (n = 4) and Spanish Holstein (n = 4) were sampled for proximate analysis, determination of adipocyte size distribution and expression of key candidate genes. Fat percentage was lower in LT than in MS muscle in Pirenaica young bulls (P = 0.023) and was higher in LT muscle in Holstein than in Pirenaica young bulls (P = 0.007). Gene expression analysis revealed that the mRNA level of myogenic differentiation 1 (MYOD) was higher in LT than in MS muscles in both groups of animals (P < 0.001) and that myostatin (MSTN) expression was also higher in LT than in MS muscle in Holstein bulls (P = 0.001). On the other hand, MSTN and PPARG showed higher expression in LT and MS in Pirenaica young bulls (P = 0.026), while the expression of fatty acid-binding protein 4 (FABP4) was higher in Holstein young bulls, also in both muscles (P < 0.001). The results suggested that the development of intramuscular adipose depot was directly related to the expression of adipogenic genes, such as FABP4, but inversely related to the expression of the cytokine MSTN and the myogenic gene MYOD, genes which showed a muscle-specific expression.

Expression of genes involved in adipogenesis and lipid metabolism in subcutaneous adipose tissue and longissimus muscle in low-marbled Pirenaica beef cattle.

The ability to accumulate intramuscular fat (IMF) is a highly variable characteristic in beef cattle. In breeds with a low tendency to accumulate IMF, this can lead to compromised meat quality because of the contribution of fat to such organoleptic attributes as juiciness and taste. This study considered adiposity and gene expression of some of the main markers involved in adipogenesis and lipid metabolism in the subcutaneous (SC) adipose tissue (AT) and the longissimus thoracis muscle (LM) and investigated differences in adipogenic regulation between the tissues during growth and fattening under different conditions. Pirenaica beef cattle were chosen for the study due to the breed's low tendency to accumulate IMF and the breed's regional importance. The young Pirenaica bulls used (n=16) were allocated to four groups and slaughtered at 6, 12 and 18 months. From 12 months onwards the bulls slaughtered at 18 months were fed diets having different energy densities. Backfat thickness increased from 6 to 12 months (P<0.05) but then was unchanged, while other fattening parameters such as percentage chemical fat and marbling did not vary. The adipose cell size distribution displayed a bimodal distribution for SC adipocytes and a unimodal distribution for IMF cells, suggestive of tissue-specific hyperplasia. Gene expression of peroxisome proliferator-activated receptor γ (PPARG), CCAAT/enhancer-binding protein α (CEBPA), sterol regulatory element-binding transcription factor 1 (SREBF1), wingless-type MMTV integration site family 10B (WNT10B), fatty acid-binding protein 4 (FABP4), acetyl Co-A carboxylase α, lipoprotein lipase and fatty acid synthase (FASN) were determined by real-time quantitative PCR. Expression did not differ between the experimental groups within the tissues but did differ between the tissues: PPARG, FABP4 and FASN were upregulated in the SC AT, while CEBPA, WNT10B and SREBF1 were upregulated in the LM. Although age and diet energy density did not have a significant effect on increasing the amount of IMF, these factors could have influenced adipocyte development in this tissue differently than in the SC AT. This was evidenced by the different size distributions of the cells in the two tissues, and the differing expression patterns of certain markers in the SC AT and the LM, which may indicate a differential role of PPARG and WNT10B in triggering adipocyte proliferation and fat accumulation capacity.

Effect of including linseed in a concentrate fed to young bulls on intramuscular fatty acids and beef color.

The effect of varying concentrate composition to include 5% linseed and 200 IU of vitamin E on the growth performance, fatty acid composition, and muscle color during shelf life was assessed in 46 young Pirenaica bulls finished to two fatness levels. Adding 5% linseed lowered the dressing rate without altering daily gain or carcass classification. It likewise did not alter the total saturated, monounsaturated, or polyunsaturated fatty acids in the intramuscular fat, though the percentage of α-linolenic acid and n-3 fatty acids increased significantly while the n-6 fatty acid to n-3 fatty acid ratio decreased. Higher subcutaneous fat cover depth at slaughter increased the total percentage of oleic acid and monounsaturated fatty acids without affecting the percentage of saturated or polyunsaturated fatty acids. Adding 200 IU of vitamin E in addition to linseed did not alter the color of film-wrapped fresh meat during storage in darkness.

Effect of whole linseed and rumen-protected conjugated linoleic acid enriched diets on feedlot performance, carcass characteristics, and adipose tissue development in young Holstein bulls.

Forty-eight young Holstein bulls (slaughtered at 458.6±9.79 kg body weight) were used to evaluate the effect of whole linseed and conjugated linoleic acid (CLA) supplementation on animal performance, adipose tissue development, and carcass characteristics. The animals were fed with one of four isoenergetic and isoproteic diets: control (0% linseed, 0% CLA), linseed (10% linseed, 0% CLA), CLA (0% linseed, 2% CLA), and linseed plus CLA (10% linseed, 2% CLA). Animal performance and carcass characteristics were unaffected by diet composition. Adding linseed or CLA to the concentrate diet did not result in significant differences in adipocyte size and number or lipogenic enzyme activity. However, while the frequency distribution of subcutaneous adipocyte diameters followed a normal distribution, the frequency distribution of intramuscular adipocyte diameters was not normal in any dietary group (skewness coefficients: 0.8, 1.2, 0.9, 0.8 for control, linseed, CLA, and linseed plus CLA, respectively; P<0.05), indicative of adipocyte proliferation in the intramuscular adipose tissue.

Predicting meat yields and commercial meat cuts from carcasses of young bulls of Spanish breeds by the SEUROP method and an image analysis system.

The SEUROP system is currently in use for carcass classification in Europe. Image analysis and other new technologies are being developed to enhance and supplement this classification system. After slaughtering, 91 carcasses of local Spanish beef breeds were weighed and classified according to the SEUROP system. Two digital photographs (a side and a dorsal view) were taken of the left carcass sides, and a total of 33 morphometric measurements (lengths, perimeters, areas) were made. Commercial butchering of these carcasses took place 24 h postmortem, and the different cuts were grouped according to four commercial meat cut quality categories: extra, first, second, and third. Multiple regression analysis of carcass weight and the SEUROP conformation score (x variables) on meat yield and the four commercial cut quality category yields (y variables) was performed as a measure of the accuracy of the SEUROP system. Stepwise regression analysis of carcass weight and the 33 morphometric image analysis measurements (x variables) and meat yield and yields of the four commercial cut quality categories (y variables) was carried out. Higher accuracy was achieved using image analysis than using only the current SEUROP conformation score. The regression coefficient values were between R(2)=0.66 and R(2)=0.93 (P<0.001) for the SEUROP system and between R(2)=0.81 and R(2)=0.94 (P<0.001) for the image analysis method. These results suggest that the image analysis method should be helpful as a means of supplementing and enhancing the SEUROP system for grading beef carcasses.

The effect of vitamin A supplementation on postnatal adipose tissue development of lambs.

Vitamin A (retinoic acid) is known to be an adipogenic factor influencing both in vitro and in vivo cell development. This study aimed to determine its effect on lamb adipose tissue development during the early phase of postnatal development until 100 d of age. Male lambs (n = 24) of the Rasa Aragonesa breed were used. At birth, lambs were assigned to 1 of 2 experimental groups: 1) the control (C) group, which received feed without vitamin A supplementation, and 2) the vitamin A (V) group, which received a supplement of 500,000 IU/animal twice per week from birth to slaughter. The effect of vitamin A supplementation was studied at 16.8 +/- 0.35 kg of BW (58 +/- 0.7 d of age) and at 27.8 +/- 0.78 kg of BW (101 +/- 6.5 d of age). The variables of lamb growth, carcass, LM area, and lipid content were analyzed. To study adipose tissue development, the amount of adipose tissue accumulated, the size and number of adipocytes, and lipogenic enzyme activities (glycerol 3-phosphate dehydrogenase, fatty acid synthase, and glucose 6-phosphate dehydrogenase) of the omental, perirenal, and s.c. depots were quantified. Results showed that vitamin A supplementation had no influence on growth, carcass variables, LM area, and lipid content during lamb growth but that the number of adipocytes in the perirenal depot was 30% greater in lambs of the V group (P < 0.05) and that these lambs had smaller adipocytes in the omental and perirenal depots (P = 0.06) at 28 kg of BW (101 d of age). These results suggest that the intake of this level of vitamin A during the whole period of growth of the lambs influenced the processes of hyperplasia and hypertrophy in the different adipose depots, depending on their degree of maturity.

Adipocyte cellularity in different adipose depots in bulls of seven Spanish breeds slaughtered at two body weights.

The influence of body weight (BW) at slaughter and genotype on adipocyte size and number in the omental (OM), perirenal (PR), subcutaneous (SC) and intermuscular (IM) adipose tissues was studied in 168 bulls of Spain's local Asturiana, Avileña, Morucha, Parda Alpina, Pirenaica, Retinta, and Rubia Gallega cattle breeds. The young bulls were slaughtered at two BWs, 320 and 540 kg. The results obtained showed the higher amounts of lipids that accumulated between 320 and 540 kg BW (P < 0.001) to be ascribable primarily to adipose cell hypertrophy, i.e. larger adipocyte size, in the OM and PR depots (P < 0.001). In addition to hypertrophy, there was also an increase (P < 0.001) in the number of adipose cells, i.e. hyperplasia, in the SC and IM adipose depots. Significant differences were observed when comparing the different genotypes, with the Morucha, Retinta and Avileña breeds having the highest amount of adipose tissue and the largest adipocytes. The Asturiana and Rubia Gallega breeds had the lowest amount of adipose tissue and the smallest adipocytes. The Pirenaica and Parda Alpina breeds had intermediate values in between the two groups identified above. In short, the results were indicative of different lipid deposition patterns in the different breeds depending on the individual growth and maturation rates in each. Similar findings were made when comparing the different adipose tissue depots, with adipocyte hypertrophy being the main factor responsible for lipid accumulation in the OM and PR depots, as opposed to adipocyte hyperplasia in the SC and IM depots.

Lipogenic enzyme activities in different adipose depots of Pirenaican and Holstein bulls and heifers taking into account adipocyte size.

The effects of sex, genotype, and adipose depot on lipogenic enzyme activity have been investigated in Holstein and Pirenaican bulls and heifers, taking into account differences in adipocyte size. Fifteen Pirenaican bulls and 15 heifers and 15 Holstein bulls and 13 heifers were fattened until slaughter (12 to 13 mo old and 450 to 500 kg of body weight). During the fattening period, animals had ad libitum access to commercial concentrates and straw. The 10th rib was dissected to determine the fat content. Adipocyte size and activities of the following lipogenic enzymes were determined: glycerol 3-phosphate dehydrogenase, fatty acid synthase, nicotinamide adenine dinucleotide phosphate (NADP)-malate dehydrogenase, glucose 6-phosphate dehydrogenase, and NADP-isocitrate dehydrogenase, in the omental, perirenal, subcutaneous, and intermuscular adipose depots, respectively. Because adipocyte mean cell volume varied with sex, breed, and depot, regression analyses of log(e) activity per cell and log(e) cell volume were used to compare activities per unit volume. Sex, breed and depot had no effect (P > 0.05) on the gradients of regressions, which did not differ significantly from 1. Thus, activity per unit volume did not vary with cell size. Consequently, sex, breed, and depot effects on the regression analyses were equivalent to effects on activity per unit volume. Females had greater amounts of fat in the 10th rib (P < 0.001), larger adipocytes (P < 0.001) and, in general, greater (P < 0.05) lipogenic activity per cell, even when adjusted for cell size, than males. These findings suggest that differences in adiposity between sexes are mainly due to females having a greater capacity for lipid synthesis, and hence, hypertrophy, than males. When adjusted for differences in carcass weight, Holsteins had larger adipocytes than Pirenaicans. The abdominal depots, omental and perirenal, had a greater adipocyte size (P < 0.001) and, in general, greater lipogenic enzyme activities per cell (P < 0.05) than the subcutaneous and intermuscular carcass depots. However, when activity per cell was adjusted for cell size, subcutaneous depots had greater fatty acid synthae, glucose 6-phosphate dehydrogenase, and NADP-malate dehydrogenase activities than omental and perirenal, indicating that other factors such as nutrient supply may restrict hypertrophy of carcass adipocytes.

Characteristics of Lacha and Rasa Aragonesa lambs slaughtered at three live weights.

A study was made of differences in the quality of meat from Lacha (L) and Rasa Aragonesa (RA) lambs slaughtered at 12, 24, or 36 kg live weight. Lambs from both breeds were weaned at 25 to 57 d, approximately 11.5 to 18.5 kg live weight, and fed concentrate and barley straw until slaughter at 24 and 36 kg live weight. Hot carcass weight, cold carcass weight, conformation, color, firmness, and thickness of backfat and color of rectus abdominis muscle were recorded on the carcass. Final pH (pHu), instrumental color (L*, a*, b*), myoglobin concentration, chemical composition, and water-holding capacity (WHC) of the longissimus muscle, shear force of the biceps femoris muscle, and iodine values and fatty acid composition of the i.m. and s.c. fat depots were determined. The percentage of fat in the longissimus muscle increased with live weight, and values for RA lambs were higher than those for L lambs. The WHC of meat from RA lambs was lower at 24 kg than at 12 or 36 kg slaughter weight. Live weight and breed had no effect on the shear force of the biceps femoris muscle. There was an increase in myoglobin concentration in the longissimus muscle with increased live weight in both breeds. The fatty acid content of s.c. and i.m. fat, which was not affected by breed, declined with the increase in slaughter weight. The polyunsaturated fatty acid content of the s.c. fat depot increased, whereas that of the i.m. fat depot decreased, with the increase in slaughter weight in both breeds. Subcutaneous fat had a higher content of heptadecanoic acid (17:0) than i.m. fat, and this increased with the increase in slaughter weight. In both depots, there was an increase in oleic acid (18:1) at 12 kg in RA lambs and at 24 kg in L lambs. In the s.c. fat depot, there was a progressive increase in linoleic acid (18:2) content with the increase in live weight in both breeds. There was a higher degree of unsaturation in the s.c. fat of RA lambs than in that of L lambs, which was reflected in the iodine value.