Skeletal muscle growth characteristics and myogenic stem cell activity in broiler chickens affected by wooden breast.

The degenerative myopathy known as wooden breast (WB) has been increasingly observed in the breast muscle (PM) of commercial broilers during the last decade. Previous research has demonstrated that WB may be induced or ameliorated by modulating dietary digestible Lys (dLys) concentrations. Two concurrent experiments (Exp) were conducted to verify the effects of feeding 2 diets formulated to 75% and 100% of recommended dLys concentrations from 15 to 25 d of age on production responses and WB incidence (Exp 1), and the characterization of muscle stem cell activity in broilers affected by WB (Exp 2). At 25 and 43 d of age, birds were injected with 5΄-bromo-2΄-deoxyuridine (BrdU) prior to the collection of PM tissue to label mitotically active cells. Muscle samples were processed for cryosectioning and immunofluorescence staining and microscopy in order to determine myofiber cross-sectional area (CSA), to enumerate Myf-5+ and Pax7+ myogenic stem cell populations, and to determine the mitotic activity (BrdU+) of these populations. The reduced dLys diet produced broilers with differing (P < 0.001) incidences of WB within the same flock (Exp 1), with some detrimental effects on performance and processing characteristics. In Exp 2, broilers with severe WB had increased numbers (P = 0.016) and proportions (P = 0.022) of mitotically active, myogenic stem cells, as well as increased proportions (P < 0.05) of large CSA myofibers relative to broilers unaffected by WB at 25 d of age. At 43 d of age, broilers affected by severe WB had a greater (P = 0.011) total population of myogenic stem cell types (Myf-5+, Pax7+, or Myf-5+:Pax7+) and a concurrent increase (P = 0.007) in the mitotic activity (Myf-5+:BrdU+, and Pax7+:BrdU+, and Myf-5+:Pax7+:BrdU+) of these cells. Additionally, a greater (P < 0.05) proportion of small CSA myofibers was observed in broilers with severe WB. These results provide evidence that myofiber CSA, as well as the heterogeneity and mitotic activity of myogenic stem cell populations were altered in the presence of WB.

Mitochondrial DNA sequence and phylogenetic evaluation of geographically disparate Sus scrofa breeds.

Next generation sequencing of mitochondrial DNA (mtDNA) facilitates studies into the metabolic characteristics of production animals and their relation to production traits. Sequence analysis of mtDNA from pure-bred swine with highly disparate production characteristics (Mangalica Blonde, Mangalica Swallow-bellied, Meishan, Turopolje, and Yorkshire) was initiated to evaluate the influence of mtDNA polymorphisms on mitochondrial function. Herein, we report the complete mtDNA sequences of five Sus scrofa breeds and evaluate their position within the phylogeny of domestic swine. Phenotypic traits of Yorkshire, Mangalica Blonde, and Swallow-belly swine are presented to demonstrate their metabolic characteristics. Our data support the division of European and Asian breeds noted previously and confirm European ancestry of Mangalica and Turopolje breeds. Furthermore, mtDNA differences between breeds suggest function-altering changes in proteins involved in oxidative phosphorylation such as ATP synthase 6 (MT-ATP6), cytochrome oxidase I (MT-CO1), cytochrome oxidase III (MT-CO3), and cytochrome b (MT-CYB), supporting the hypothesis that mtDNA polymorphisms contribute to differences in metabolic traits between swine breeds. Our sequence data form the basis for future research into the roles of mtDNA in determining production traits in domestic animals. Additionally, such studies should provide insight into how mtDNA haplotype influences the extreme adiposity observed in Mangalica breeds.

Effect of residual feed intake on hypothalamic gene expression and meat quality in Angus-sired cattle grown during the hot season.

The relationship between heat stress, meat quality, and residual feed intake (RFI) is unknown in growing steers. To address this issue, high RFI (HRFI) and low RFI (LRFI) individuals were compared by assessing RFI in 48 Angus-sired steers during a 70-d feeding trial conducted during July through September to identify steers with calculated RFI at least 2 SD apart. The association of RFI with indices of meat quality and expression of genes within hypothalamic and adipose tissue was then determined in LRFI and HRFI steers. While on test, feed intake was recorded daily with BW and hip heights recorded every 14 d. Ultrasound measurements of rib eye area (REA) and backfat (BF) were recorded initially and before harvest. Carcass and growth data were analyzed using a mixed model with RFI level (LRFI and HRFI) as the independent variable. The least square means for RFI were -1.2 and 0.99 kg DMI/d, respectively, for the LRFI and HRFI cohorts (P < 0.0001). Dry matter intake was higher for the HRFI individuals versus the LRFI steers (P < 0.0001) while on-test gain was not different (P < 0.95). Marbling score was greater in LRFI than HRFI steers (P < 0.05). However, there were no differences in REA (P < 0.53), BF (P < 0.65), yield grade (P < 0.24), or objective Hunter color measures between LRFI and HRFI steers indicating there was no consistent relationship between RFI and indices of meat quality. Hypothalamic neuropeptide Y (NPY), agouti related protein (AGRP), relaxin-3 (RLN3), melanocortin 3 receptor, and relaxin/insulin-like family peptide receptor 1 (RXFP1) mRNA were expressed 280, 185, 202, 183, and 163% greater, respectively (P < 0.01), while proopiomelanocortin (POMC) mRNA was expressed 42% lower in LRFI than HRFI animals (P < 0.05). Hypothalamic GnRH mRNA expression was 67% lower while gonadotropin inhibiting hormone (GnIH) mRNA was 209% higher in LRFI than HRFI animals (P < 0.01). Pituitary expression of FSHß and LHß correlated to hypothalamic GnRH levels (P < 0.05) indicating changes in gene expression within the hypothalamus had functional consequences. Leptin mRNA expression levels were not different between adipose tissue of LRFI or HRFI steers (P < 0.84). These data indicate that animals with superior RFI evaluated during warm conditions have higher expression of orexigenic neuropeptide genes independent of the expression of adipose-derived leptin. Furthermore, the gonadotropin axis may also influence feed efficiency under these conditions.

Residual feed intake studies in Angus-sired cattle reveal a potential role for hypothalamic gene expression in regulating feed efficiency.

Mechanisms underlying variation in residual feed intake (RFI), a heritable feed efficiency measure, are poorly understood while the relationship between RFI and meat quality is uncertain. To address these issues, 2 divergent cohorts consisting of High (HRFI) and Low (LRFI) RFI individuals were created by assessing RFI in 48 Angus-sired steers during a 70 d feeding trial to identify steers with divergent RFI. The association of RFI with indices of meat quality and expression of genes within hypothalamic and adipose tissue was then determined in LRFI and HRFI steers. While on test, feed intake was recorded daily with BW and hip heights recorded at 14 d intervals. Ultrasound measurements of rib eye area (REA) and backfat (BF) were recorded initially and before harvest. Carcass and growth data were analyzed using a mixed model with RFI level (LRFI, HRFI) as the independent variable. The least-square means (lsmeans) for RFI were -1.25 and 1.51 for the LRFI and HRFI cohorts (P < .0001). Dry matter intake was higher for the HRFI individuals versus the LRFI steers (P < .0001) while on test BW gain was not different between the 2 groups (P < 0.73). There were no differences detected in marbling score (P < 0.93), BF (P < 0.61), REA (P < 0.15), yield grade (P < 0.85) or objective Hunter color measures between LRFI and HRFI steers indicating that there was no relationship between RFI and meat quality. Neuropeptide-Y (NPY), relaxin-3 (RLN3), melanocortin 4 receptor (MC4R), and GnRH mRNA expression was 64%, 59%, 58%, 86% lower (P < 0.05), respectively, while gonadotropin inhibiting hormone (GnIH) and pro-opiomelanocortin (POMC) mRNA expression was 198% and 350% higher (P < 0.01) in the arcuate nucleus of LRFI steers. Expression of agouti-related protein (AGRP), relaxin/insulin-like family peptide receptor 1 (RXFP1), and melanocortin 3 receptor mRNA was similar between LRFI and HRFI animals. Pituitary expression of FSHß (P < 0.03) and LHß (P < 0.01) was correlated to hypothalamic GnRH levels suggesting that changes in gene expression within the arcuate nucleus had functional consequences. Leptin mRNA expression was 245% higher in the adipose tissue of LRFI steers consistent with lower levels of NPY and higher expression of POMC in their hypothalami. These data support the hypothesis that differences in hypothalamic neuropeptide gene expression underlie variation in feed efficiency in steers while the gonadotropin axis may also influence feed efficiency.

Grain feeding coordinately alters expression patterns of transcription factor and metabolic genes in subcutaneous adipose tissue of crossbred heifers.

The ability to improve meat quality and production efficiency in cattle is limited by an inability to enhance marbling and simultaneously limit undesirable adipose tissue accretion. The objective of this study was to examine expression of regulatory genes in subcutaneous (SCF) adipose tissue of heifers in response to increasing days on feed (DOF) and finishing strategy. Crossbred heifers (n = 24) were allotted as follows: Group 1 = 0 d, Group 2 = 99 d on winter annual ryegrass (grass; Lolium multiflorum Lam.), Group 3 = 218 g on grass, Group 4 = 99 d on grass followed by 119 d on grain. Adipose tissue samples were collected at time of harvest and frozen. Carcass characteristics were measured 24 h postharvest. As expected, HCW (P < 0.0001), ribeye area (REA; P < 0.0002), backfat (BF; P < 0.0001), KPH (P < 0.0001), and marbling score (P < 0.0009) increased with DOF though frame score was not different (P < 0.95). Average daily gain decreased with DOF (P < 0.0001). Yield grade increased (P < 0.0014) but cook loss percentage decreased (P < 0.001) with DOF without changes in 24-h pH (P < 0.31). Interestingly, Warner-Bratzler shear force (WBS) was decreased with DOF (P < 0.0089). Meanwhile, BF (P < 0.01) and KPH (P < 0.05) were greater, whereas marbling values trended greater in grain versus grass-finished heifers. Neither ADG (P < 0.89), HCW (P < 0.26), frame score (P < 0.85), nor REA (P < 0.38) were different between these groups. Grain finishing increased yield grade (P < 0.001) but did not affect 24-h pH (P < 0.88), cook loss percentage (P < 0.98), or WBS (P < 0.44) compared with grass-finished heifers. The expression of PPARγ, bone morphogenic protein 2 (BMP2), and SMAD family member 1 (SMAD1) mRNA was upregulated in response to DOF and grain finishing, whereas sterol regulatory element binding protein 1c (SREBP-1c), sonic hedgehog (SHH), chicken ovalbumin protein transcription factor 1 (COUP-TF1), chicken ovalbumin protein transcription factor 2 (COUP-TF2), and preadipocyte factor-1 (PREF-1) mRNA was decreased in response to DOF and grain finishing. These changes were associated with increased expression of lipoprotein lipase (LPL), stearoyl-coenzyme A desaturase (SCD), and fatty acid synthase (FAS) mRNA. In summary, increasing DOF was associated with improved meat quality whereas gene expression studies suggest several novel genes are associated with subcutaneous adipose tissue development in growing and finishing cattle.

Isomer-specific regulation of differentiating pig preadipocytes by conjugated linoleic acids.

Conjugated linoleic acids are a group of geometric and positional isomers of linoleic acid that decrease body fat in growing animals by a poorly understood mechanism. The objective of this study was to investigate the isomer-specific effect of CLA on the proliferation and differentiation of pig preadipocytes in primary culture. The effect of CLA on preadipocyte proliferation was determined using cleavage of the tetrazolium salt, WST-1, as a marker for proliferation. Preadipocyte number was decreased in a dose-dependent fashion by trans-12,cis-10 CLA (P < 0.05). No other fatty acid affected preadipocyte number. Differentiation was monitored on d 10 after induction morphologically, enzymatically, and by measuring the mRNA abundance of key adipogenic transcription factors. Both a crude CLA preparation containing a mixture of CLA isomers (CLA-mix) and the pure trans-10,cis-12 CLA isomer inhibited glycerol-3-phosphate dehydrogenase (GPDH) activity in a dose-dependent fashion, with trans-10,cis-12 CLA being more potent (P < 0.01) than the CLA-mix. Cis-9,trans-11 CLA failed to decrease GPDH activity; however, increasing concentrations of cis-9,trans-11 CLA tended to blunt the inhibitory effect of trans-10,cis-12 CLA on GPDH activity (P < 0.09), suggesting that cis-9,trans-11 CLA may antagonize the action of trans-10,cis-12 CLA in porcine adipocytes. Finally, the isomer-specific effect of CLA on adipogenic transcription factor gene expression was investigated. Trans-10,cis-12 CLA decreased expression of peroxisome proliferator-activated receptor gamma (PPAR gamma; P < 0.01) and sterol regulatory element-binding protein-1c (SREBP-1c; P < 0.05) mRNA, while failing to alter the expression of CCAAT/enhancer binding protein alpha (C/EBPalpha) mRNA. Interestingly, both the CLA-mix and the trans-10,cis-12 CLA isomer increased the mRNA abundance of chicken ovalbumin upstream promoter transcription factor 1 (COUP-TF; P < 0.002). No other fatty acid affected COUP-TF mRNA levels. Collectively these data support the concept that CLA decreases fat accretion in pigs, in part by inhibiting preadipocyte proliferation and differentiation, with trans-10,cis-12 CLA being an active isomer eliciting these effects. Furthermore, trans-10,cis-12 CLA inhibits porcine preadipocyte differentiation by a mechanism that involves the down-regulation of PPARgamma and SREBP-1c mRNA. This mechanism is independent of changes in C/EBPalpha mRNA abundance and may involve COUP-TF.

Regulation of differentiating pig preadipocytes by retinoic acid.

All-trans retinoic acid (ATRA) potently inhibits the differentiation of porcine preadipocytes in primary culture; however, the mechanism by which ATRA exerts this effect in pigs is poorly understood. The objective of this study was to use retinoid receptor-specific ligands to investigate the mechanism underlying the antiadipogenic action of retinoids in cultured pig preadipocytes by identifying the retinoid receptor mediating this action and examining the effect of retinoids on the expression of key adipogenic transcription factors. Stromal-vascular cells were harvested from porcine adipose tissue and cultured in serum-free medium. Glycerol-3-phoshphate dehydrogenase (GPDH) activity, a late marker of preadipocyte differentiation, was decreased (P < 0.01) by the addition of 0 to 10 microM of either ATRA, a nonspecific agonist for both the retinoic acid receptor (RAR) and the retinoid X receptor (RXR) or the selective RAR agonist, 4-(E-2-[5,6,7,8-tet-rahydro-5,5,8,8-tetramethyl-2-naphthalenyl]-1-propenyl) benzoic acid (TTNPB). Addition of increasing amounts of Ro-61, a RAR-specific antagonist (0 to 10 microM) prevented ATRA and TTNBP from decreasing GPDH activity. Addition of methoprene acid, an RXR-specific agonist, increased (P < 0.01) GPDH activity. Preadipocytes were then continuously treated with 10 nM of TTNPB in the presence or absence of 1 microM Ro-61, and mRNA was isolated on d 2 and 8. Addition of TTNPB decreased (P < 0.001) the expression of peroxisome proliferator-activated receptor gamma (PPARgamma), sterol regulatory element-binding protein-1c (SREBP-1c), retinoid X receptor alpha (RXRalpha), and adipocyte fatty acid binding protein (aP2) mRNA transcripts, whereas these effects were prevented by the presence of Ro-61. Interestingly, TTNBP increased (P < 0.001) the mRNA abundance of the orphan nuclear receptor chicken ovalbumin upstream promoter-transcription factor 1 (COUP-TF1), whereas Ro-61 prevented this increase. These changes were independent of alterations in the mRNA abundances of the retinoic acid receptor alpha, and CCAAT/enhancer binding protein alpha and beta (C/EBPbeta; C/EBPalpha) genes. These results indicate that retinoic acid inhibits porcine preadipocyte differentiation by a mechanism that involves activation of the RAR and downregulation of PPARgamma, RXRalpha, and SREBP-1C mRNA. This mechanism is independent of changes in C/EBPbeta and C/EBPalpha mRNA abundance and may involve COUP-TF.