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Journal of Animal Science - Animal Growth, Physiology, and Reproduction

Grain feeding coordinately alters expression patterns of transcription factor and metabolic genes in subcutaneous adipose tissue of crossbred heifers12

 

This article in JAS

  1. Vol. 91 No. 6, p. 2616-2627
     
    Received: Sept 10, 2012
    Accepted: Feb 25, 2013
    Published: November 25, 2014


    3 Corresponding author(s): tdb0006@auburn.edu
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doi:10.2527/jas.2012-5846
  1. C. N. Key*,
  2. S. D. Perkins*,
  3. C. L. Bratcher*,
  4. L. A. Kriese-Anderson* and
  5. T. D. Brandebourg 3
  1. Department of Animal Sciences, Auburn University, Auburn, AL 36849

Abstract

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.



INTRODUCTION

Heifers comprise one third of the annual United States beef harvest (USDA, 2009). Despite producing greater quality grades than male counterparts, heifer carcasses are less valuable because heifers are less efficient, produce lighter HCW, and generally are less tender than steers at similar ages (Choat et al., 2006; Tatum et al., 2007). Given the tendency for greater marbling in heifers, the potential exists to eliminate this valuation gap if strategies could be devised to improve efficiency and tenderness.

Marbling or intramuscular fat (IMF) is positively associated with palatability and achieving adequate IMF is essential to attaining acceptable quality grades (Savell et al., 1987). Cattle are currently finished on high energy, grain-based diets to achieve this goal, but increasing competition for inputs threatens the viability of this strategy (Martin, 2010). Complicating efforts to improve production efficiency, IMF accretion occurs predominantly during the finishing stage in cattle at a time when significant quantities of visceral and subcutaneous fat have already accumulated on the carcass (Allen, 1976; Hood, 1982).

Transcriptional networks coordinate adipose tissue hyperplasia and hypertrophy in rodent models (Siersbaek et al., 2012). Unfortunately, knowledge of factors controlling these processes and how production practices influence such factors is limited in cattle, especially concerning mechanisms that control the timing and extent of depot development (Smith et al., 2012). Consequently, it has proven difficult to devise strategies that enhance marbling and simultaneously limit adipose tissue accretion in undesirable depots that negatively impact feed efficiency and yield grade in cattle. Therefore, our objectives were to determine the association of days on feed (DOF) and grass versus grain finishing on growth performance, meat quality, and the expression of candidate regulatory genes in subcutaneous adipose tissue of heifers.


MATERIALS AND METHODS

All procedures involving animals were approved by the Auburn University Animal Care and Use Committee.

Animals and Design

Crossbred beef heifers (n = 24) were selected from the resident herd at the Alabama Agricultural Experiment Station E.V. Smith Research Center Beef Unit at 397 ± 24 d of age and were stratified across 4 groups (n = 6) based on BW, height, age, breed type, frame score, ultrasound 12th rib backfat (BF), ultrasound ribeye area (REA), and percentage intramuscular fat so that no initial differences existed across groups (Table 1). There were 4 Gelbvieh-sired heifers in the initial cohort. Each experimental group contained a Gelbvieh-sired heifer with the remaining 5 heifers being Angus-sired. To test the effects of age and diet on indices of meat quality and the expression of regulatory genes in subcutaneous adipose tissue, cattle weaned at uniform BW and backgrounded on a summer perennial pasture mix [bermudagrass, Cynodon dactylon (L.) Pers.; and bahiagrass, Paspalum notatum Flugge] and then reared and harvested as follows: Group 1: heifers were harvested on initiation of the study to serve as a baseline for comparison; Group 2: heifers were harvested after 99 d on ryegrass pasture; Group 3: forage-finished heifers were maintained on ryegrass pasture for the duration of the trial (218 d); Group 4: grain-finished heifers were maintained on ryegrass pasture for 99 d, then transitioned to a dry lot for an additional 119 d, where they were given ad libitum access to a grain-based ration consisting of a combination of 90% whole corn and 10% corn gluten feed (CP = 11.2%; TDN = 85%) fed in troughs under and open barn. Forage quality was assessed by proximate analysis at regular intervals during the study (Table 2). Heifer BW and hip height measurements were performed monthly. Real-time ultrasound measurement (of heifers for rump fat, 12th rib fat thickness, LM area, and percentage intramuscular fat in the LM was performed initially to allow stratification. All real-time ultrasound measurements were recorded by the same Ultrasound Guidelines Council certified technician using an Aloka SSD-500V with a 17.3-cm transducer (Hitachi Aloka Medical, Ltd., Wallingford, CT). At the end of the feeding period, cattle were transported to Auburn University Lambert Powell Meats Laboratory, where they were humanely harvested under USDA-FSIS inspection.


View Full Table | Close Full ViewTable 1.

Means ± SEM for initial age and carcass characteristics of heifers by group1,2

 
Variable Group 1 Group 2 Group 3 Group 4 SEM P-value
Ryegrass, d 0 99 218 99
Grain, d 0 0 0 119
Age, d 407 400 393 386 9.89 0.51
BW, kg 288 268 275 284 8.36 0.31
Height, cm 122 121 122 121 1.58 0.92
Frame score 5.2 5.0 5.3 5.1 0.33 0.97
Ultrasound backfat, mm 0.085 0.115 0.098 0.117 0.14 0.23
Ultrasound REA,3 cm2 43.8 44.5 42.5 45.4 2.43 0.83
Intramuscular fat, % 3.6 3.7 3.6 3.6 0.25 0.97
1Analysis of initial group data was not significant between groups for any trait, n = 6.
2Group 1 = control (0 d, initial heifers), Group 2 = 99 d on ryegrass, Group 3 = 218 d on ryegrass, Group 4 = 99 d on ryegrass followed by 119 d on grain.
3Ultrasound REA = ultrasound ribeye area adjusted for rump fat thickness.

View Full Table | Close Full ViewTable 2.

Nutritional characteristics of ryegrass1,2

 
Variable February March April May June July
DM 370.8 746.7 604.4 1540.0 2286.6 1660.0
DM, % 22.1 45.8 17.1 22.2 24.5 47.4
CP 21.4 10.7 13.3 8.4 8.2 7.0
NDF 33.9 28.9 43.0 51.1 63.4 65.9
ADF 18.9 16.5 25.2 27.6 35.1 37.5
ADL 0.54 N/A 0.39 1.36 1.70 3.63
RFV 203.6 245.1 149.9 122.7 89.7 84.3
Low, °C –0.6 4.6 10.2 17.7 20.2 22.3
High, °C 11.6 17.6 23.7 30.2 30.0 35.1
1RFV = relative feed value (100 = nutritive quality of late bloom alfalfa).
2Average daily low (Low) and high (High) temperatures recorded in Celsius at the Milstead Weather Station, Shorter, AL.

Chemical Analysis of Forage

Forage samples were collected throughout the trial in conjunction with monthly cattle weigh dates. Heifers were stocked at a density of 1 animal/0.40 ha and randomly distributed across 4 paddocks with heifers from each treatment and breed equally represented in each paddock. Forage quantity was uniform across paddocks. Forage samples were randomly selected across each pen with 4 quadrats collected per sample and placed in brown paper bags. Samples were then dried for 48 h at 55°C in a convection oven (Model 420, NAPCO, Winchester, PA) and weighed before and after drying (equilibrated to room temperature for 24 h) to determine DM. Dry samples were ground in a Thomas-Willey mill (Model 4, Thomas Scientific, Swedesboro, NJ) to pass through a 1-mm mesh screen, labeled, and placed in sealed plastic containers. Chemical analyses were conducted on ground samples to determine DM, ash, NDF, ADF, and CP. Methods described by AOAC (1998) were used to determine DM, ash, and CP. Nitrogen was determined using a Leco TruSpec (Leco Corporation, St. Joseph, MI) and multiplied by 6.25 to estimate CP. Forage NDF and ADF were determined on samples according to Van Soest et al. (1991) using an Ankom200 fiber analyzer and Ankom F57 filter bags (Ankom Technology Corp., Fairport, NY)

Carcass Fabrication

Animals were harvested after the treatment DOF at the Auburn University Lambert-Powell Meats Lab, under USDA-FSIS inspection. Hot carcass weight was recorded after harvest, and carcasses were chilled at 2 ± 1°C for 24 h. At 24 h postmortem, carcass pH was recorded in the left side round using a pH Spear probe (Oakton Instruments, Vernon Hills, IL), and each carcass was split between the 12th and 13th ribs for evaluation of BF, REA, KPH, skeletal maturity, lean maturity, average maturity, and marbling. Longissimus muscles were evaluated for objective color measurements at the 12th and 13th rib interface using a Hunter Miniscan XE Plus (Hunter Lab, Reston, VA) for Hunter L*, a*, and b* values. The Miniscan was calibrated according to manufacturer’s recommendations and used a D65 light source, a 10° viewing angle, and a 35-mm viewing area. Two LM steaks from each carcass were removed after aging for 7 d at 2 ± 1°C for Warner-Bratzler shear force (WBS) evaluation and frozen at 20°C until further analysis. Steaks were packaged in vacuum-sealed (Ultravac UV21C; Kansas City, MO) bags individually.

Shear Evaluation

Steaks were thawed under refrigeration at 4 ± 2°C for 24 h before cooking. All samples were cooked on a clam-shell-style grill (Calphalon Removable Plate Grill, Caphalon, Perrysburg, OH), preheated to ∼177°C. Steaks were cooked for 7 min, resulting in an internal temperature of 71°C. Temperature was monitored with copper constantan thermocouple wire inserted into the geometric center of the steak and attached to a hand-held Omega data logger HH309A temperature recorder (Omega, Stamford, CT). Steaks were allowed to cool for 15 min and then covered in aluminum foil and chilled at 4°C for 24 h. Six cores (1.27 cm in diam.) were removed from each steak with a brass cork borer (Model 1601A Series Brass Cork Borer, Boekel Scientific, Feasterville, PA), parallel to the muscle fiber orientation. Each core was sheared once at its center, perpendicular to the muscle fiber orientation, using a TA-XT2i Texture Analyzer shear machine (Texture Technologies Corp., Scarsdale, NY). The peak force measurements were then averaged from the 6 cores from each steak to be used for statistical analysis. The probe was programmed to be lowered 30 mm after detection of resistance. The penetration speed was 3.3 mm/s with a posttest speed of 10 mm/s and a pretest speed of 2.0 mm/s.

Gene Expression Analysis

Upon exsanguination, subcutaneous adipose tissues were immediately collected, snap frozen in liquid nitrogen, and stored at –80°C until mRNA analysis. To facilitate this, a plug of SCF and LM was removed by cutting through the hide immediately on verifying that the animal had expired. Tissue samples were then processed immediately in a wet lab adjacent to the harvest floor. Processed samples were thoroughly snap-frozen by emersion in liquid N and buried in dry ice within 5 to 10 min of exsanguination. Total RNA was extracted from adipose tissue using a 2-step purification protocol with total RNA first being extracted from whole tissue using RNAzol RT (MRC Inc., Cincinnati, OH) followed by purification using RNAeasy spin columns (QIAGEN Inc., Valencia, CA) according to the manufacturers’ recommendations. The RNA was quantified using a BioTek Synergy 4 plate reader using the Take3 system (BioTek U.S., Winooski, VT), with all samples exhibiting an optical density (OD) 260/280 between 1.8 and 2.0 and an OD 260/230 value between 1.8 and 2.2. Spectral scans ranging from 200 to 400 nm further verified sample purity as all RNA samples produced smooth curves exhibiting 1 peak at 260 nm. Total RNA integrity was accessed both visually by resolving 2 μg of RNA on a denaturing formaldehyde gel containing ethidium bromide and by determining an RNA Integrity Number (RIN) using an Agilent 2100 bioanalyzer (Agilent Technologies, Inc., Clara, CA). All samples demonstrated sharp ribosomal bands with a 28S to 18S ratio > 1 and RIN values > 7.0 and were thus judged intact and nondegraded. Total RNA was then reverse transcribed using Superscript II reverse transcriptase (Promega Inc, Madison, WI) and oligo-dT primers. Real-time PCR was performed on the resultant cDNA using a Roche Lightcycler 480 Real-time PCR machine and LightCycler 480 SYBR Green I Master Mix (Roche Applied Science, Indianapolis, IN) according to manufacturer’s recommendations. All PCR reactions were performed using intron-spanning primers under optimized conditions with primer efficiencies ranging between 90 and 101% (Table 3) as verified with standard curves. Product purity was assessed by melting curve analysis and expected amplicon sizes were verified on a 2% agarose gel stained with ethidium bromide. Values were normalized to eukaryotic translation initiation factor 3 subunit K (EIF3K) mRNA expression. The EIF3K mRNA abundance represent an appropriate control as the efficiency of the EIF3K primers was 100% (Table 3) and EIF3K mRNA expression was not different between any groups tested (P < 0.93). Data are expressed as fold change relative to baseline and calculated according to Pfaffl, 2010.


View Full Table | Close Full ViewTable 3.

Oligonucleotide PCR primers

 
Gene1 Accession No. Primer sequence (5ʹ→3ʹ) Orientation Efficiency
%
PPARγ NM_181024.2 CGC ATG CCA CAG GCC GAG AA forward 97
CCG TCA AGA TCG CCC TCG CC reverse
BMP2 NM_001099141.1 AAG ATG AGC ACA GCT GGT CAC AGA forward 99
TCT TAC AGC TGG ACT TGA GGC GTT reverse
SMAD1 NM_001076223.2 ACC TCC GCA CAA AGA AGC TAA GGA forward 100
TTC ACC AAA GCA TCA ACG GCC TTC reverse
SREBP1c NM_001113302.1 CCA GCT GAC AGC TCC ATT GA forward 96
TGC GCG CCA CAA GGA reverse
SHH AF144100.1 TCA GAG GTG CAA GGA CAA GCT GAA forward 91
ACT GGT TCA TCA CGG AGA TGG CTA reverse
COUPTF1 NM_175804.2 AAC TTA CAC ATG CCG TGC CAA CAG forward 91
AGG CAC TTC TTA AGG CGG CAG TAT reverse
COUPTF2 NM_174402.3 CCC ATG TGG AAA GCT TGC AGG AAA forward 93
TCC AAA TCG TGT TGG TTG GTT GGG reverse
PREF1 AB050725.1 TTG CAA CCA GCA GGC GGT GT forward 101
GGG AGG GGA GGT TGG CGA CT reverse
WNT5a NM_001205971.1 ATT TCT CTC CTT CGC CCA GGT TGT forward 93
TGG TCC TGA TAC AAG TGG CAG AGT reverse
LPL NM_001075120.1 ACA CAG CTG AGG ACA CTT GCC forward 94
GCC ATG GAT CAC CAC AAA GG reverse
FAS AF285607.2 ACC TCG TGA AGG CTG TGA CTC A forward 90
TGA GTC GAG GCC AAG GTC TGA A reverse
SCD NM_173959.4 TCC TGT TGT TGT GCT TCA TCC forward 97
GGC ATA ACG GAA TAA GGT GGC reverse
EIF3K NM_001034489.2 TGA CAG ACA GCC AGC TAA AGG TGT forward 100
TCT TCT CCA CGA TGT TCT TGG GCT reverse
1Bone morphogenic protein 2 (BMP2), SMAD family member 1 (SMAD1), 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), preadipocyte secreted factor-1 (PREF-1), wingless-type MMTV integration site family, member 5a (WNT5a), lipoprotein lipase (LPL), fatty acid synthase (FAS), stearoyl-coenzyme A desaturase (SCD), eukaryotic translation initiation factor 3 subunit K (EIF3K).

Statistical Analysis

Growth and carcass traits were analyzed as a completely randomized design using a general linear model (SAS Inst. Inc., Cary, NC). Animal served as the experimental unit. Independent variables included group, breed, and the interaction of group and breed using this model: yijk = μ + groupi + breedj + group * breedij + eijk. Least squares means were used to separate means using a significance level of α = 0.05. Changes in gene expression were calculated from the cycle threshold, after correction using EIF3K expression and analyzed using the Pair Wise Fixed Reallocation Randomisation Test of REST-MCS v.2.0 (http://rest.gene-quantification.info/, verified 29 April 2013). Correlations between gene expression levels in subcutaneous adipose tissue samples were also determined using the Proc Corr procedures of SAS.


RESULTS

Growth Performance

When randomly assigning animals to experimental groups, heifers were stratified based on age, breed type, and carcass composition. As a result, there were no differences in initial age, BW, height, frame score, BF, REA, or intramuscular fat percentage between groups at the initiation of the growth trial (Table 1).

The effect of DOF on growth performance is indicated in Table 4. As expected, HCW increased with DOF across groups (P < 0.0001) though final frame score was not different (P < 0.95). Likewise REA increased 32% in heifers during the first 99 d (P < 0.0002) but REA did not increase substantially with further DOF. As expected, a temporal pattern of adipose tissue development emerged with increasing DOF as KPH and BF approached maximal levels in heifers by 99 d, but marbling continued to increase with DOF throughout the study. Overall, BF increased 777% (P < 0.0001), KPH increased 264% (P < 0.0001), and marbling score increased 27% (P < 0.0009). Consistent with these changes in composition of gain, ADG decreased with DOF (P < 0.0001).


View Full Table | Close Full ViewTable 4.

Least squares means ± SEM for performance, carcass traits, Warner-Bratzler shear (WBS), objective Hunter score color measures, and cook loss of heifers by group1,2

 
Variable3 Group 1 Group 2 Group 3 Group 4 SEM P-value
Ryegrass, d 0 99 218 99
Grain, d 0 0 0 119 d
Days on feed, d 0 99 218 218
ADG, kg·d–1 3.28a 2.06b 2.04b 0.11 0.0001
Final frame score 5.58 5.72 5.62 5.35 0.42 0.95
HCW, kg 308c 494b 548a 576a 19 0.0001
Backfat, mm 0.9c 6.4b 7.0b 12.3a 0.8 0.0001
Marbling score 386c 444b 494a 539a 23 0.0009
REA, cm2 47.0b 61.8a 69.4a 65.2a 3.0 0.0002
KPH, % 0.76c 1.92b 2.01b 2.26a 0.11 0.0001
Yield Grade 1.57c 2.32b 2.23b 3.11a 0.20 0.0001
24h pH 5.74 5.76 5.90 5.71 0.07 0.31
L*, lightness 34.4 36.7 35.8 38.7 1.4 0.19
a*, redness 15.8c 19.3b 20.1b 22.5a 0.8 0.0001
b*, yellowness 12.0c 15.0b 15.0b 17.0a 0.8 0.002
Cook loss, % 26.5a 11.0b 8.3b 8.2b 2.8 0.0001
WBS, kg 4.31a 4.75a 2.67b 2.54b 0.22 0.0001
1Group means within a row with different superscripts differ (P < 0.05), n = 6.
2Group 1 = control (0 d, initial heifers), Group 2 = 99 d on ryegrass, Group 3 = 218 d on ryegrass, Group 4 = 99 d on ryegrass followed by 119 d on grain.
3REA = ribeye area. Backfat is presented as adjusted backfat. Cook loss percentage is expressed on a wt/wt wet basis.
4Marbling Score: 300 = Traces; 400 = Slight; 500 = Small.

The effect of forage (Group 3) or grain (Group 4) on growth performance is also indicated in Table 4. There were no differences observed for ADG (P < 0.89), HCW (P < 0.26), frame score (P < 0.85), or REA (P < 0.38). However, grain finishing significantly increased BF 76% (P < 0.0004) and KPH 12% (P < 0.05), and numerically increased marbling score 5% (P < 0.14).

Meat Quality

As expected, yield grade increased (P < 0.0014) whereas cook loss percentage decreased (P < 0.001) with DOF. However, 24-h pH was unaffected (P < 0.31). Objective Hunter color score was affected by DOF as both a* (P < 0.0001) and b* (P < 0.0016) were increased, though L* values were unchanged (P < 0.19) with age. The WBS decreased 37% as DOF increased (P < 0.0089), indicating that after 218 d, heifers produced steaks that would be perceived as more tender. When considering marbling scores, cook loss percentage and WBS data across forage-fed-only groups, results indicate carcasses from heifers reared on pasture for 218 d displayed improved meat quality compared with their grass-fed counterparts slaughtered at fewer days on feed.

Grain finishing increased yield grade 39% (P < 0.001) and all Hunter color scores, L* (P < 0.03), a* (P < 0.03), and b* (P < 0.05), but did not affect 24-h pH (P < 0.88), cook loss percentage (P < 0.98), or WBS (P < 0.44) compared with forage-finished heifers, suggesting grain finishing resulted in fatter carcasses but did not necessarily effect sensory characteristics.

Gene Expression

Figure 1 illustrates the effect of DOF on gene expression in subcutaneous adipose tissue of heifers reared on grass. The expression of PPARγ mRNA increased 4.6-fold in heifers fed ryegrass for 99 d versus heifers harvested at the initiation of the feeding trial, whereas this effect was blunted in heifers reared on ryegrass pastures for 218 d. Likewise, SMAD family member 1 (SMAD1) expression increased 1.6-fold with 99 d on pasture, and 3.7-fold with 218 d on pasture, relative to the control group. Expression of bone morphogenic protein 2 (BMP) mRNA was unchanged in heifers fed ryegrass for 99 d, but surprisingly decreased 80% in heifers maintained on pasture for 218 d. Expression of sterol regulatory element binding protein 1c (SREBP1c) mRNA was increased only in heifers fed on pasture for 218 d (2.6-fold). Meanwhile, the mRNA abundance of sonic hedgehog (SHH), chicken ovalbumin protein transcription factor 1 (COUP-TF1), chicken ovalbumin protein transcription factor 2 (COUP-TF2), and preadipocyte secreted factor-1 (PREF-1) was significantly decreased 78, 52, 45, and 76% with 99 d on ryegrass, though the effect was again diminished in heifers reared on pasture for 218 d. The expression of wingless-type Mouse mammary tumor virus (MMTV) integration site family, member 5a (WNT5a) was increased 1.3- and 2.4-fold with 99 and 218 DOF, respectively. Meanwhile, the mRNA abundance of metabolic genes lipoprotein lipase (LPL), stearoyl-coenzyme A desaturase, (SCD) and fatty acid synthase (FAS) remained relatively constant across DOF, being no different than the control group in heifers that grazed ryegrass pastured for 99 and 218 d.

Figure 1.
Figure 1.

The effect of grazing heifers on ryegrass for 0, 99, or 219 d on subcutaneous adipose tissue gene expression for transcriptional modulators (A) PPARγ, (B) bone morphogenic protein 2 (BMP2), and (C) SMAD family member 1 (SMAD1), (D) sterol regulatory element binding protein 1c (SREBP-1c), (E) sonic hedgehog (SHH), (F) chicken ovalbumin protein transcription factor 1 (COUP-TF1), (G) chicken ovalbumin protein transcription factor 2 (COUP-TF2), (H) preadipocyte secreted factor-1 (PREF-1) and metabolic markers, (I) lipoprotein lipase (LPL), (J) stearoyl-coenzyme A desaturase (SCD), (K) fatty acid synthase (FAS), and (L) wingless-type MMTV integration site family, member 5a (WNT5a). Expression was determined by real-time PCR. Values were normalized to EIF3K expression. Data is expressed as fold change relative to baseline (d 0) and calculated according to Pfaffl (2010). Bars denoted by * and ** differ (P < 0.05), n = 6.

 

Figure 2 illustrates the effects of finishing diet on gene expression. The expression of PPARγ mRNA was 25-fold greater, BMP2 mRNA was 21.4-fold greater, and SMAD1 mRNA was 5.2-fold greater in the subcutaneous adipose tissue of grain-fed animals relative to heifers finished on grass. Meanwhile, the expression of SREBP1c mRNA was 72% less, SHH mRNA was 97% less, COUP-TF1 mRNA was 84% less, COUPTF2 mRNA was 68% less, and PREF1 mRNA was 90% less in the subcutaneous adipose tissue of grain-fed animals than heifers finished on grass, whereas there was no difference in WNT5a mRNA expression. These observed differences in mRNA expression for transcription factors were associated with a 16.2-fold increase in LPL mRNA abundance, 84.3-fold increase in SCD1 mRNA, and a 2.9-fold increase in FAS mRNA in the subcutaneous adipose of grain-fed animals relative to heifers finished on grass.

Figure 2.
Figure 2.

The effect of finishing heifers on either ryegrass or grain-based diets on subcutaneous adipose tissue mRNA expression for (A) PPARγ, (B) bone morphogenic protein 2 (BMP2), and (C) SMAD family member 1 (SMAD1), (D) sterol regulatory element binding protein 1c (SREBP-1c), (E) sonic hedgehog (SHH), (F) chicken ovalbumin protein transcription factor 1 (COUP-TF1), (G) chicken ovalbumin protein transcription factor 2 (COUP-TF2), (H) preadipocyte secreted factor-1 (PREF-1), (I) lipoprotein lipase (LPL), (J) stearoyl-coenzyme A desaturase (SCD), (K) fatty acid synthase (FAS), and (L) wingless-type MMTV integration site family, member 5a (WNT5a). Expression was determined by real-time PCR. Values were normalized to EIF3K expression; n = 6. Data is expressed as fold change relative to Group 3 and calculated according to Pfaffl, 2010. Bars denoted by * and ** differ (P < 0.05), n = 6.

 

Pearson correlation coefficients and associated P-values for adjusted BF and mRNA expression of target genes in subcutaneous adipose tissue are reported in Table 5. The mRNA abundance of SCD1 (P < 0.039) but neither the expression of LPL mRNA nor FAS mRNA significantly correlated with BF thickness. The expression of PPARγ and SMAD1 both positively correlated with BF thickness, though BMP2 and SREBP1c mRNA abundance did not significantly correlate with this carcass parameter. The expression of SHH was negatively correlated with BF (P < 0.05), whereas the expression of COUPTF1 (P < 0.11) and COUPTF2 (P < 0.07) mRNA abundances tended to be negatively correlated. The expression of WNT5a mRNA was not significantly correlated with BF. This was further confirmed by examining the correlation of PPARγ mRNA, the master regulator of adipogenesis, with expression of other genes examined in the study. Abundance of PPARγ mRNA was positively correlated with BMP2, SMAD1, LPL, FAS, and SCD1 mRNA expression, but were negatively correlated with mRNA expression of SHH, COUPTF1, COUPTF2, and PREF1.


View Full Table | Close Full ViewTable 5.

Correlation of subcutaneous adipose tissue gene expression and adjusted backfat thickness1

 
Gene ADJ_BF2 PPARγ BMP2 SMAD1 SREBP1c SHH COUPTF1 COUPTF2 PREF1 WNT5a LPL FAS SCD
PPARγ 0.518 0.776 0.680 –0.289 –0.900 –0.859 –0.890 –0.749 –0.066 0.836 0.567 0.926
P < 0.01 P < 0.0002 P < 0.003 P < 0.26 P < 0.0001 P < 0.0001 P < 0.0001 P < 0.0002 P < 0.788 P < 0.0001 P < 0.011 P < 0.001
BMP2 0.286 0.776 0.216 –0.266 –0.638 –0.516 –0.525 –0.241 –0.524 0.859 0.418 0.726
P < 0.24 P < 0.0002 P < 0.42 P < 0.29 P < 0.003 P < 0.024 P < 0.021 P < 0.32 P < 0.021 P < 0.0001 P < 0.07 P < 0.0004
SMAD1 0.539 0.680 0.216 –0.148 –0.798 –0.687 –0.779 –0.823 0.623 0.410 0.321 0.757
P < 0.035 P < 0.003 P < 0.42 P < 0.58 P < 0.0001 P < 0.002 P < 0.0002 P < 0.0001 P < 0.008 P < 0.10 P < 0.20 P < 0.0004
SREBP1c 0.231 –0.289 –0.266 –0.148 0.309 0.320 0.363 0.289 –0.013 –0.179 –0.024 –0.186
P < 0.36 P < 0.26 P < 0.29 P < 0.58 P < 0.212 P < 0.20 P < 0.14 P < 0.25 P < 0.96 P < 0.48 P < 0.92 P < 0.46
SHH –0.429 –0.900 –0.638 –0.789 0.309 0.922 0.932 0.846 0.027 –0.800 –0.631 –0.934
P < 0.05 P < 0.0001 P < 0.003 P < 0.0001 P < 0.21 P < 0.0001 P < 0.0001 P < 0.0001 P < 0.90 P < 0.0001 P < 0.003 P < 0.0001
COUPTF1 –0.373 –0.859 –0.516 –0.687 0.320 0.922 0.906 0.894 –0.165 –0.668 –0.548 –0.839
P < 0.11 P < 0.0001 P < 0.02 P < 0.002 P < 0.20 P < 0.0001 P < 0.0001 P < 0.0001 P < 0.49 P < 0.001 P < 0.01 P < 0.0001
COUPTF2 –0.410 –0.890 –0.525 –0.779 0.363 0.932 0.906 0.865 –0.108 –0.707 –0.536 –0.894
P < 0.07 P < 0.0001 P < 0.02 P < 0.0002 P < 0.14 P < 0.0001 P < 0.0001 P < 0.0001 P < 0.65 P < 0.0005 P < 0.01 P < 0.0001
PREF1 –0.416 –0.749 –0.241 –0.822 0.289 0.846 0.894 0.865 –0.414 –0.444 –0.458 –0.730
P < 0.05 P < 0.0002 P < 0.32 P < P < 0.25 P < 0.0001 P < 0.0001 P < 0.0001 P < 0.07 P < 0.05 P < 0.04 P < 0.0003
WNT5a 0.201 –0.066 0.524 0.623 –0.013 0.027 –0.165 0.108 –0.414 –0.450 –0.137 –0.092
P < 0.40 P < 0.79 P < 0.02 P < 0.008 P < 0.96 P < 0.91 P < 0.49 P < 0.65 P < 0.07 P < 0.05 P < 0.57 P < 0.70
LPL 0.261 0.836 0.859 0.410 –0.179 –0.800 –0.668 –0.707 –0.445 –0.450 0.711 0.889
P < 0.27 P < 0.0001 P < 0.0001 P < 0.10 P < 0.48 P < 0.0001 P < 0.001 P < 0.0005 P < 0.05 P < 0.05 P < 0.0004 P < 0.0001
FAS 0.214 0.567 0.418 0.321 –0.024 –0.630 –0.548 –0.536 –0.458 –0.137 0.711 0.715
P < 0.37 P < 0.01 P < 0.07 P < 0.21 P < 0.92 P < 0.003 P < 0.01 P < 0.01 P < 0.04 P < 0.57 P < 0.0004 P < 0.0004
SCD 0.462 0.923 0.723 0.757 –0.187 –0.924 –0.839 –0.894 –0.730 –0.092 0.889 0.715
P < 0.039 P < 0.0001 P < 0.0004 P < 0.0004 P < 0.46 P < 0.0001 P < 0.0001 P < 0.0001 P < 0.0003 P < 0.70 P < 0.0001 P < 0.0004
1Pearson correlation coefficients and associated P-values for adjusted backfat and subcutaneous adipose tissue mRNA expression of PPARγ, bone morphogenic protein 2 (BMP2), SMAD family member 1 (SMAD1), 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), preadipocyte secreted factor-1 (PREF-1), wingless-type MMTV integration site family, member 5a (WNT5a), lipoprotein lipase (LPL), fatty acid synthase (FAS), and stearoyl-coenzyme A desaturase (SCD); n = 24.
2ADJ_BF = ultrasound carcass 12th rib fat thickness adjusted for rump fat thickness.


DISCUSSION

There is significant economic pressure for producers to finish cattle to a marbling endpoint that merits quality grades of USDA Prime or Choice (Anderson, 2012). However, a lack of knowledge concerning the mechanisms underlying the timing and extent of adipose tissue depot development in cattle has hampered efforts to enhance IMF and simultaneously limit fat depots, such as trim and KPH that negatively impact feed efficiency and yield grade (Hausman et al., 2009; Du et al., 2010; Smith et al., 2012). The present study indicates carcass adiposity increases and meat quality improves with increasing DOF in heifers grazing for up to 218 d, and the greatest degree of SCF, KPH, and IMF accretion occurred in grain-finished heifers. These changes in carcass adiposity correlated with increases in mRNA for transcription factors PPARγ, BMP2, and SMAD1 in subcutaneous adipose tissue concomitant with decreases in mRNA for SHH, COUP-TF1, COUP-TF2, and PREF-1 in this fat depot. Expression levels for metabolic genes (LPL, SCD1, FAS) were largely unaffected by DOF, but were significantly increased by grain finishing relative to grass finishing. The coordinate expression of genes examined in this study suggests unifying signals exist which dictate the degree to which adipose tissue accumulation occurs in cattle by directing stimulatory and inhibitory gene networks in tandem.

Though heifers deposit more IMF, they are less efficient, produce lighter HCW, and generally are less tender than steers at similar ages and therefore produce carcasses less valuable than their steer counterparts (Choat et al., 2006; Tatum et al., 2007). However, given the tendency for greater marbling in heifers, the potential exists to eliminate this valuation gap if strategies could be devised to improve efficiency and tenderness. Therefore, our first objective was to determine the effect of DOF and grass versus grain finishing on growth performance and meat quality in heifers. Consistent with previous studies examining the effect of increasing time on forage in both steers and heifers, DOF was associated with increased HCW, REA, adiposity (as estimated by KPH and BF), marbling score, and USDA yield grade concomitant with a decreased ADG (May et al., 1992; Van Koevering et al., 1995; Winterholler et al., 2007; Roberts et al., 2009). Likewise responses to grain or grass finishing were comparable to previous results, as USDA yield grade, KPH, and BF were greater in grain-finished heifers relative to their grass-fed counterparts, though no difference in HCW or REA were evident between heifers due to finishing strategy (Schaake et al., 1993; Muir et al., 1998; French et al., 2000; Roberts et al., 2009). Yield grade was lower in forage-fed cattle as in previous studies largely because grain-finished heifers were significantly fatter (Bidner et al., 1986; McMillin et al., 1990; Kerth et al., 2003; Roberts et al., 2009). Several studies have observed greater marbling scores in grain versus grass fed animals when DOF are held constant (Reagan et al., 1981; Crouse et al., 1984; Bidner et al., 1986). In the present study, marbling score was numerically greatest in the grain-finished animals, though differences between grass- and grain-finished heifers were not significant. These results agree with 2 recent studies conducted at the EV Smith Experiment Station, in which no differences in marbling were observed between rye-grass and grain-finished Angus-sired steers (Kerth et al., 2007; Roberts et al., 2009). Nonetheless, KPH and BF were greater in grain- versus grass-fed cattle, consistent with previous studies that indicate grain-fed cattle are fatter than grass-fed counterparts even if the effect on marbling has been variable (Schaake et al., 1993; Muir et al., 1998; French et al., 2000; Kerth et al., 2007; Roberts et al., 2009). Forage quality declined as the trial progressed into the warm season and high daily temperatures may have depressed DMI, and these issues may have decreased performance in heifers and influenced marbling deposition. Nonetheless, significant and expected differences were observed across groups as heifers were significantly fatter with increasing DOF, and grain-finished heifers achieved a BF thickness of 12 mm. Overall, growth responses and carcass characteristics observed in the present study are comparable with similar studies in the literature.

Due to increased fattening and USDA yield grade and the decreased ADG associated with longer feeding durations, it may be detrimental to feed heifers for more than 100 d beyond the attainment of a typical stocker weight (May et al., 1992; Anderson and Gleghorn, 2007; Winterholler et al., 2007). However, although similar changes in these variables were observed in the current study, they were accompanied by a significant decrease in shear force and cook loss, and increased marbling score through 218 d, suggesting heifers produced steaks that would be perceived as more tender. Therefore, grazing heifers beyond 100 d past attainment of desired stocker weights may confer an advantage by promoting greater meat quality. Color measures indicated that DOF were associated with potential yellowing of IMF, but overall did not indicate quality differences between steaks harvested from grain- or grass-finished animals. These results agree with previous studies in which forage-finished steers had lower subcutaneous fat and marbling scores, but displayed no difference in tenderness or sensory scores relative to grain-finished steers (Bidner et al., 1986; Roberts et al., 2009). Taken together, these data indicate grain finishing did not improve tenderness measures or marbling score compared with finishing cattle on ryegrass pasture. Although profitability has been positively associated with increased DOF, largely due to increased USDA quality grades, extending the finishing period in feedlot systems confers substantial risk, given the volatility of feed costs (Anderson and Gleghorn, 2007). However, extending DOF in a forage-based production system would not carry this related risk, and could represent a viable strategy to increase sustainability of cow-calf operators because it may be possible to achieve significant improvement in meat quality and allow operators to finish culled heifers and receive a greater return on receipts.

Our second objective was to investigate the effect of DOF (forage) and grain finishing on expression of genes in subcutaneous adipose tissue that are known to regulate adipogenesis in mammals. Fat deposition in cattle results from the additive contributions of increases in adipocyte number and size (Allen, 1976; Hood, 1982). Studies using in vivo rodent models and clonal preadipocyte cell lines have yielded a molecular framework for the mechanisms controlling the recruitment, proliferation, and differentiation of preadipocytes. In the current model of adipogenesis, the sequential expression of C/EBPβ, PPARγ, and C/EBPα results in transactivation of adipocyte-specific genes such as the insulin receptor (IR), glucose transporter 4 (GLUT4), SCD1, LPL, and FAS, leading to terminal differentiation of preadipocytes, an ability to respond to homeorhetic hormones such as insulin, and the induction of metabolic pathways related to lipid metabolism (Lazar, 2002). Presently, PPARγ is considered the master regulator of adipocyte differentiation, whereas C/EBPα is thought to potentiate differentiation by upregulating genes that confer insulin sensitivity on the adipocyte (Hamm et al., 1999; Lazar, 2002; Rosen, 2002). Additionally, sterol regulatory element-binding protein (SREBP)-1c is believed to potentiate adipogenesis both by upregulating PPARγ expression and by increasing availability of ligands for PPARγ through upregulation of genes involved in lipid metabolism (Iijima et al., 1998; Fajas et al., 1998).

The regulation of adipogenesis can also be inhibitory. For instance, PREF1 inhibits adipogenesis in part by blocking transcription of C/EBPβ, thus preventing initiation of the stimulatory transcription factor cascade necessary to trigger the adipogenic gene program (Smas and Sul., 1993; Sul., 2009). Another antiadipogenic transcription factor, COUP-TF, has been implicated in mediating the inhibitory action of retinoids and conjugated linoleic acids in primary cultures of porcine preadipocytes, and this action appears to be mediated primarily through antagonizing PPARγ (Brandebourg and Hu, 2005a, 2005b; Xu et al., 2008; Okamura et al., 2009). Recruitment of preadipocytes from mesenchymal stem cells appears regulated by the expression of morphogenic proteins such as Wnts, SHH, and BMPs. Both Wnts and SHH likely prevent adipogenesis, in part through activation of COUP-TF proteins, though each also likely signal via other mechanisms as well (Shang et al., 2007; Okamura et al., 2009; Du et al., 2010). Both BMP2 and 4 antagonize this action by promoting C/EBP expression through a Smad1-mediated mechanism, ultimately leading to upregulation of PPARγ and stimulation of the adipogenic program (Tseng et al., 2008). Thus, both factors that decide the fate of mesenchymal stem cells and factors acting directly to alter the preadipocyte transcriptome regulate adipogenesis.

In the present study, morphoregulatory and transcription factor genes involved in preadipocyte recruitment and differentiation displayed expression patterns that suggest the presence of both stimulatory and inhibitory regulatory gene networks in bovine adipose tissue. The mRNA abundances of PPARγ, BMP2, and SMAD1 were significantly increased in response to grain feeding relative to grass-finished heifers whereas PPARγ, and SMAD1 mRNA expression was increased by DOF relative to baseline mirroring increases in BF observed in these groups. On the other hand, mRNA abundance for SHH, COUP-TF1, COUP-TF2, and PREF-1 were significantly depressed in grain-finished animals relative to their grass-finished counterparts, and were generally reduced by increasing DOF compared with baseline, though due to large variation in expression of these transcription factors within grass-finished animals, the expression of COUP-TF1 and COUP-TF2 was not significantly different than baseline in grass-finished animals on d 218. Correlation analysis further supports the presence of regulatory gene networks, as strikingly strong correlations between individual genes were observed across groups. For instance, PPARγ was positively correlated with BF and a subset of genes implicated in stimulating adipose tissue accretion, including BMP2, SMAD1, LPL, FAS, and SCD. Meanwhile, PPARγ was negatively correlated with a subset of genes implicated in negatively regulating adipogenesis in rodent models, including SHH, COUPTF1, COUPTF2, and PREF1.

A role for PPARγ in stimulating adipogenesis in cattle is supported by the detection of PPARγ mRNA in bovine adipose tissue and the ability of PPARγ agonists to stimulate the differentiation of primary bovine preadipocytes in culture (Sundvold et al., 1997; Chung et al., 2006; Grant et al., 2008; Ortiz-Colon et al., 2009). In the present study, PPARγ expression was positively correlated with BF across all heifers, and was greatest in the fattest heifers. Abundances of PPARγ mRNA increased with 99 d, but decreased as BF leveled with further DOF. Meanwhile, grain finishing dramatically increased PPARγ expression versus grass-finished heifers. Smith et al. (2012) observed a similar response to age in grain-fed steers, though the effect of forage versus grain was not compared.

In the current study, the expression of SCD, LPL, and FAS was relatively constant in SCF across DOF but mRNA abundances of all 3 genes were dramatically increased in grain versus grass-finished heifers. These responses were consistent with the effects of age on the expression of SCD mRNA in SCF across multiple studies of grain-fed steers (Martin et al., 1999; Chung et al., 2006; Brooks et al., 2011; Smith et al., 2012). The effect of grain versus grass finishing in the present study was also consistent with responses reported by Duckett et al. (2009), where grain finishing increased SCD and FAS mRNA 46- and 9-fold, respectively, versus pasture-fed steers. In the current study, SCD and FAS mRNA abundances were increased 84- and 2.9-fold by grain finishing. Thus, it appears that the expression of metabolic genes remain relatively constant in SCF of growing heifers; however, switching from forage to a grain-finishing diet results in the upregulation of these genes. Together, these studies suggest a consistent response for PPARγ and SCD in SCF of both steers and heifers across various forage paradigms, and in response to transitioning from pasture to dry lots. The results of the current study further support a role for these genes in the regulation of adipose tissue mass in cattle.

Expression data for BMP2, SREBP-1c, SHH, PREF1, COUPTF1, and COUPTF2 represent novel observations in bovine adipose tissue. Based on rodent models, we hypothesized that BMP2 and SREBP-1c expression would be increased in fatter heifers, whereas the expressions of WNT5a, SHH, PREF1, COUPTF1, and COUPTF2 mRNA would be decreased as BF thickness increased. Using real-time PCR, Minoshima et al. (2001) demonstrated an age-dependent suppression of PREF1 expression in bovine visceral adipose tissue when comparing expression in samples harvested from neonatal and 30-mo-old cattle. Our findings extend these results, as PREF1 mRNA was negatively correlated with adjusted BF and decreased in SCF across DOF and in response to grain finishing. Indeed, consistent with our hypothesis, the expressions of SHH, COUPTF1, and COUPTF2 were generally reduced by DOF, with an even more striking reduction manifested in response to grain finishing. Thus, these data implicate several novel genes as negative regulators of bovine SCF development, as their expressions decreased with increasing SCF mass.

Potential roles for WNT5a, BMP2, and SREBP-1c in the regulation of subcutaneous adipose tissue development in heifers are less clear, based on gene studies from the current experiment. For instance, BMP2 mRNA was clearly induced in the SCF of grain-finished heifers relative to their grass-finished counterparts, and BMP2 expression was strongly correlated with PPARγ across all heifers, suggesting BMP2 does play a role in stimulating bovine adipogenesis. However, puzzlingly, BMP2 expression was decreased in heifers allowed to graze for 218 d. Meanwhile, both WNT5a and SREBP-1c expression increased in heifers across DOF, but were unchanged and decreased respectively in the SCF of grain-fed heifers versus grass-finished ones. Furthermore SREBP-1c expression was correlated with neither adjusted BF thickness nor PPARγ expression. These observations suggest that SREBP-1c did not play a role in regulating adipogenesis under the conditions studied in the current trial, whereas BMP2 may have played a smaller role than rodent models predict. It is clear that SREBP-1c functions to sense intracellular lipid concentrations and to regulate lipid and cholesterol metabolism accordingly. For instance, SREBP-1c has been demonstrated to play a role in regulating adipose tissue metabolism in lactating dairy cows, and SREBP-1c expression was upregulated in redifferentiated cultures of dedifferentiated bovine adipocytes, though the effect in these cultures was highly variable (Taniguchi et al., 2008; Rincon et al., 2011). It is possible that conditions of the current study would not elicit a robust SREBP-1c response, and its role in adipogenesis may have been thus obscured. However, contrary to our hypothesis, the current study does not provide definitive evidence for roles of WNT5a and SREBP-1c in regulating SCF development in heifers.

Implications

Feeding heifers for >100 DOF conferred a meat quality advantage, and this may be a practical solution for increasing the sustainability and profitability of forage-based production systems such as those used by cow-calf operators. This is the first study that implicates a role for BMP2, SMAD1, SHH, COUPTF1, COUPTF2, and PREF1 in the regulation of bovine subcutaneous adipose tissue development. The patterns of expression of morphoregulatory and transcription factor genes measured in subcutaneous adipose tissue as cattle fattened illustrates the presence of stimulatory and inhibitory regulatory gene networks that appear coordinately regulated in opposing fashions. Better understanding the regulation of adipose tissue development in cattle will help us devise an effective methods to control adipose tissue development in a depot-specific fashion so that beef products can be provided that are more acceptable to consume and more cost-effective to produce.

 

References

Footnotes


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