M. E. Doumit

MicroRNA transcriptome profiles during swine skeletal muscle development

BackgroundMicroRNA (miR) are a class of small RNAs that regulate gene expression by inhibiting translation of protein encoding transcripts. To evaluate the role of miR in skeletal muscle of swine, global microRNA abundance was measured at specific developmental stages including proliferating satellite cells, three stages of fetal growth, day-old neonate, and the adult.ResultsTwelve potential novel miR were detected that did not match previously reported sequences. In addition, a number of miR previously reported to be expressed in mammalian muscle were detected, having a variety of abundance patterns through muscle development. Muscle-specific miR-206 was nearly absent in proliferating satellite cells in culture, but was the highest abundant miR at other time points evaluated. In addition, miR-1 was moderately abundant throughout developmental stages with highest abundance in the adult. In contrast, miR-133 was moderately abundant in adult muscle and either not detectable or lowly abundant throughout fetal and neonate development. Changes in abundance of ubiquitously expressed miR were also observed. MiR-432 abundance was highest at the earliest stage of fetal development tested (60 day-old fetus) and decreased throughout development to the adult. Conversely, miR-24 and miR-27 exhibited greatest abundance in proliferating satellite cells and the adult, while abundance of miR-368, miR-376, and miR-423-5p was greatest in the neonate.ConclusionThese data present a complete set of transcriptome profiles to evaluate miR abundance at specific stages of skeletal muscle growth in swine. Identification of these miR provides an initial group of miR that may play a vital role in muscle development and growth.

Conditions for isolation and culture of porcine myogenic satellite cells

Myogenic satellite cells were isolated from semimembranosus muscles of 4-8 week-old pigs. Muscles were ground and incubated in 0.8 mg/ml Pronase solution for 40 min at 37 degrees C. Following enzymatic digestion, cells were separated from muscle debris by differential centrifugation and sequential filtering through 500 and 53 microns nylon mesh. Primary cultures grown in 16 mm diameter cell culture wells were used to evaluate five sera, media, and substrata for their ability to promote satellite cell proliferation and differentiation. Porcine satellite cell proliferation and myotube formation were optimized in cultures grown on gelatin-coated substratum in the presence of Minimum Essential Medium-alpha supplemented with 10% fetal bovine serum (FBS) (P less than 0.01). Maximum fusion was induced by 48 hr exposure to 2% FBS, horse serum, or lamb serum. These data 1) document the first evidence that myogenic satellite cells can be isolated from porcine skeletal muscle, and 2) identify culture conditions which optimize proliferation and myotube formation of porcine satellite cells.

Pork quality variation is not explained by glycolytic enzyme capacity

Our objective was to determine if increased glycolytic enzyme capacity accommodates rapid glycolysis, which leads to inferior pork color and water-holding capacity. Progeny from HAL-1843 free Duroc (n=16) or Pietrain (n=16) sires were harvested over a 2-week period. Coupled enzyme assays were used to quantify total capacity of pyruvate kinase (PK) and phosphofructokinase (PFK) in the sarcoplasmic fractions and crude homogenates of longissimus muscle (LM), respectively. Capacity of PK was not correlated with LM pH (20, 45, 180 min or 24 h), purge, drip loss, or CIE L* (P > 0.2). However, PFK capacity was inversely related to fluid loss (P<0.05). This finding was unexpected, but may result from PFK becoming partially denatured and inactivated by 20 min postmortem in samples that undergo a rapid pH decline. These data indicate that lighter pork color and reduced water-holding capacity are not associated with an increase in the capacity of enzymes that catalyze regulated steps of glycolysis.

Regulation of Adipogenesis and Key Adipogenic Gene Expression by 1, 25-Dihydroxyvitamin D in 3T3-L1 Cells

The functions of 1, 25-dihydroxyvitamin D (1, 25-(OH)2D3) in regulating adipogenesis, adipocyte differentiation and key adipogenic gene expression were studied in 3T3-L1 preadipocytes. Five concentrations (0.01, 0.1, 1, 10, 100nM) of 1, 25-(OH)2D3 were studied and lipid accumulation measured by Oil Red O staining and expression of adipogenic genes quantified using quantitative real-time PCR. Adipogenic responses to 1, 25-(OH)2D3 were determined on 6, and 12 h, and days 1-10 after induction of adipogenesis by a hormonal cocktail with or without 1, 25-(OH)2D3. In response to 1, 25-(OH)2D3 (1, 10, and 100 nM), lipid accumulation and the expression of PPARγ, C/EBPα, FABP4 and SCD-1 were inhibited through day 10, and vitamin D receptor expression was inhibited in the early time points. The greatest inhibitory effect was upon expression of FABP4. Expression of SREBP-1c was only affected on day 2. The lowest concentrations of 1, 25-(OH)2D3 tested did not affect adipocyte differentiation or adipogenic gene expression. The C/EBPα promoter activity response to 1, 25-(OH)2D3 was also tested, with no effect detected. These results indicate that 1, 25-(OH)2D3 inhibited adipogenesis via suppressing adipogenic-specific genes, and is invoked either during PPARγ activation or immediately up-stream thereof. Gene expression down-stream of PPARγ especially FABP4 was strongly inhibited, and we suggest that the role of 1, 25-(OH)2D3 in regulating adipogenesis will be informed by further studies of adipogenic-specific gene promoter activity.

Differentiation of bovine intramuscular and subcutaneous stromal-vascular cells exposed to dexamethasone and troglitazone

The objectives of these experiments were to compare differentiation of bovine stromal-vascular (S-V) cells isolated from i.m. and s.c. adipose tissues in response to a glucocorticoid and a peroxisome proliferator-activated receptor gamma agonist. Stromal-vascular cells were isolated from i.m. and s.c. fat depots of 3 Angus steers and propagated in culture. Cells were exposed to differentiation media containing 0.25 microM dexamethasone (DEX), a glucocorticoid analog, and 40 microM troglitazone (TRO), a peroxisome proliferator-activated receptor gamma agonist, or both. Cells treated with DEX and TRO had greater (P < 0.02) glycerol-3-phosphate dehydrogenase activity than control cells. No interactions between DEX, TRO, and depot (P > 0.59) or depot differences (P = 0.41) in glycerol-3-phosphate dehydrogenase activity were found. Morphological assessment of adipogenic colonies showed that DEX induced a 1.8-fold increase in the percentage of adipogenic colonies (P = 0.03), whereas TRO increased the proportion of adipogenic colonies by 1.9-fold (P = 0.02) compared with those not treated with DEX or TRO, respectively. Depots had a similar percentage of adipogenic colonies (P = 0.18); however, the percentage of differentiated cells within adipogenic colonies was found to be 6.4-fold greater in s.c. isolates compared with i.m. (P < 0.001). Addition of TRO increased the proportion of differentiated cells within colonies by 10-fold compared with those of nontreated colonies (P < 0.001), whereas the percentage of differentiated cells within adipogenic colonies only tended to be increased by DEX (P = 0.10). These data indicate that bovine i.m. and s.c. S-V cells are capable of enhanced differentiation in response to DEX and TRO, and these effects were additive. Most importantly, inherent differences in the capacity to differentiate exist between adipogenic bovine i.m. and s.c. S-V cells.

Influence of extended aging on beef quality characteristics and sensory perception of steaks from the gluteus medius and longissimus lumborum.

The objective was to determine the influence of post-fabrication aging (2, 14, 21, 42, and 63 days) on beef quality characteristics and consumer sensory perception of gluteus medius (GM) and longissimus lumborum (LL) steaks. Lipid oxidation and aerobic plate counts increased (P<0.05) with longer aging periods and retail display times. An aging period by day of retail display interaction (P<0.05) was observed for a* and b* values for both muscles and L* values for the LL. Warner-Bratzler shear force values decreased (P<0.05) with longer aging for the LL, while no difference was observed for the GM. Consumer panel results demonstrated that longer aging periods increased (P<0.05) tenderness of both muscles. Our results indicate that extended aging reduces retail color stability yet has positive effects on consumer perception of tenderness of beef loin muscles.

Adipocyte differentiation-specific gene transcriptional response to C18 unsaturated fatty acids plus insulin.

Adipocyte differentiation (AD) and AD-specific gene expression was studied in 3T3-L1 cells in response to oleic acid (OA) or linoleic acid (LA) alone and in combination with insulin. This system facilitated the study of key regulators of adipogenesis PPARγ and C/EBPα and other AD-specific genes, in the absence of dexamethasone (DEX) and isobutyl-1-methyl xanthine (IBMX) (components of the traditional AD medium, DMI). Lipid accumulation and expression levels of AD-specific genes were enhanced by both OA and LA in the presence of insulin but not by OA or LA alone. Gene expression levels of PPARγ, C/EBPα, FABP4, and SREBP1c induced by OA plus insulin, were comparable to DMI medium, by study day 10. The response to long-chain fatty acids (LCFA) plus insulin in the presence or absence of LY294002 demonstrated that the insulin-induced PI 3-kinase pathway regulates AD and AD-specific gene expression levels. Insulin treatment in the presence or absence of genistein suggested that genistein invoked inhibition of AD and AD-specific gene expression. In contrast when LCFA were also included with insulin, the presence of genistein invoked a pronounced and opposite effect on AD to that in the absence of LCFA. This effect may be modulated via C/EBPα as C/EBPα but not PPARγ expression patterns closely reflected the changes in AD. DMI invoked a rapid expression of all genes studied, and LCFA plus insulin invoke more gradual increases in gene expression, to similar levels to those invoked by DMI. The model system is valuable for study of transactivators and response elements of PPARγ and C/EBPα genes.

Bovine intramuscular, subcutaneous, and perirenal stromal-vascular cells express similar glucocorticoid receptor isoforms, but exhibit different adipogenic capacity.

Understanding preadipocyte differentiation in economically important adipose depots will facilitate efforts to selectively increase intramuscular (i.m.) lipid accretion in cattle. The objectives of this study were to determine if glucocorticoid receptor (GR) expression differs among bovine stromal-vascular (S-V) cells derived from i.m., subcutaneous (s.c.), and peri-renal (p.r.) adipose tissue, and to evaluate the effects of dexamethasone (DEX) on adipogenesis of these cell populations. Stromal-vascular cells isolated from i.m., s.c., and p.r. adipose tissues of 2 steers were propagated in culture and exposed to 0 or 250 nM DEX for 48 h. Cell lysates were subjected to GR immunoblot analysis, and immunoreactive protein bands of approximately 97, approximately 62, and approximately 48 kDa were detected and expressed relative to beta-actin immunoreactivity. The abundance of each GR immunoreactive protein was similar among S-V cell populations (P > 0.50). Dexamethasone exposure decreased the abundance of the approximately 97 and approximately 62 kDa GR immunoreactive bands in S-V cells from the 3 depots (P < 0.001), but did not affect the expression of the approximately 48 kDa band (P = 0.96). Stromal-vascular cells isolated from 3 steers were grown in culture, and upon confluence, were exposed to 0, 25, or 2,500 nM DEX for 48 h. After an additional 10 d in differentiation media, differentiation was determined by glycerol-3-phosphate dehydrogenase (GPDH) specific activity and oil red O staining. The extent of differentiation differed by depot (p.r. > s.c. > i.m.; P < 0.05). Compared with control, 2,500 nM DEX increased GPDH activity in S-V cells from all depots (P < 0.05), and no interaction between depot and DEX concentration was observed (P = 0.99). We observed an adipose tissue depot by DEX concentration interaction (P = 0.03) for S-V cells with large (> or = 10 microm-diameter) lipid droplets. The percentage of p.r. S-V cells with large lipid droplets increased in response to DEX in a linear manner (P < 0.02), but only increased greater than control in s.c. cells exposed to 2,500 nM DEX (P = 0.002). Dexamethasone did not significantly increase the percentage of i.m. S-V cells with large lipid droplets (P > 0.27). Collectively, these data demonstrate differences in adipogenic activity among bovine i.m., s.c., and p.r. S-V cells, but indicate no relationship between adipogenic activity and glucocorticoid receptor abundance or function.

Relationships among performance, residual feed intake, and product quality of progeny from Red Angus sires divergent for maintenance energy EPD.

Energy expenditure is a physiological process that may be closely associated with residual feed intake (RFI). The maintenance energy (ME(M)) EPD was developed by the Red Angus Association of America (RAAA) and is used as an indicator of energy expenditure. The objectives of this study were to evaluate and quantify the following relationships using progeny of Red Angus (RA) sires divergent for ME(M) EPD: 1) postweaning RFI and finishing phase feed efficiency (FE), 2) postweaning RFI and end-product quality, and 3) postweaning RFI and sire ME(M) EPD. A total of 12 RA sires divergent for ME(M) EPD were chosen using the RAAA-generated ME(M) EPD values and were partitioned into 2 groups: high ME(M) EPD (≥4 Mcal/mo) and low ME(M) EPD (<4 Mcal/mo), based on the breed average of 4 Mcal/mo. Commercial crossbred cows were inseminated to produce 3 cohorts of progeny, which were tested for postweaning RFI (cohorts 1, 2, and 3) and finishing phase FE (cohorts 1 and 3). Results indicate that postweaning RFI and finishing phase FE of steer progeny tended to be positively correlated (r = 0.38; P = 0.06) in cohort 1 and were positively correlated (r = 0.50; P = 0.001) in cohort 3. In addition, postweaning RFI was not phenotypically correlated (P > 0.05) with any carcass traits or end-product quality measurements. Sire ME(M) EPD was phenotypically correlated (P < 0.05) with carcass traits in cohort 1 (HCW, LM area, KPH, fat thickness, and yield grade) and cohort 2 (KPH and fat thickness). Since variation in measured LM area was not explained by the genetic potential of rib eye area EPD, and therefore, the observed correlation between sire ME(M) EPD and measured LM area may suggest an association between ME(M) EPD and LM area. A correlation (r = 0.24; P = 0.02) was observed between postweaning RFI and ultrasound intramuscular fat percentage in cohort 2 but was not detected in cohorts 1 or 3. In addition, no phenotypic relationship was observed (P > 0.05) between progeny postweaning RFI and sire ME(M) EPD. Therefore, results suggest 1) RFI measured during the postweaning growth phase is indicative of FE status in the finishing phase, 2) neither RFI nor sire ME(M) EPD negatively affected carcass or end-product quality, and 3) RFI and sire ME(M) EPD are not phenotypically associated.

Influence of extended aging on beef quality characteristics and sensory perception of steaks from the biceps femoris and semimembranosus.

The objective was to determine the influence of post-fabrication aging (2, 14, 21, 42, and 63days) on beef quality characteristics and consumer sensory perception of biceps femoris (BF) and semimembranosus (SM) steaks. Lipid oxidation and aerobic plate counts increased (P<0.05) with longer aging periods and retail display times. An aging period by day of retail display interaction (P<0.05) was observed for a* and b* values of the BF and SM. Warner-Bratzler shear force values decreased (P<0.05) with longer aging for the SM, while no difference was observed for the BF. Consumer panel results revealed that longer aging periods increased (P<0.05) acceptability of the SM, tenderness of both muscles, and tended to increase (P=0.07) juiciness of the SM. Our results show that extended aging reduces retail color stability yet has positive effects on consumer perception of tenderness of both muscles and overall acceptability of the SM.