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Journal of Animal Science - Article

 

 

This article in

  1. Vol. 86 No. 12, p. 3568-3574
     
    Received: Mar 20, 2008
    Published: December 5, 2014


    2 Corresponding author(s): sealy@ufl.edu
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doi:10.2527/jas.2008-1049

Differential response of cull cow muscles to the hypertrophic actions of ractopamine-hydrogen chloride1

  1. J. M. Gonzalez,
  2. R. D. Dijkhuis,
  3. D. D. Johnson,
  4. J. N. Carter and
  5. S. E. Johnson2
  1. Department of Animal Sciences, University of Florida, Gainesville 32611

Abstract

Ractopamine-HCl (RAC) is a β-adrenergic agonist with variable effects on cattle performance and carcass variables. Cull cows fed RAC (200 mg · head−1 · d−1) demonstrate an increased size of type I and II muscle fibers that does not translate into a larger ribeye area. The objective of this study was to examine the dose-dependent effects of RAC on cull cow muscle morphometrics. Eighty-eight cull beef cows representing 2 breed types (n = 44 each) were fed 0, 100, 200, and 300 mg · head−1 · d−1 of RAC for the last 28 d of a 54-d feeding period. On d 54, cows were slaughtered, and samples of the LM and semimembranosus muscle (SM) from 16 randomly selected carcasses (n = 4 per treatment) were taken for measurement of β 2-adrenergic receptors and type I, IIA, and IIX myosin heavy chain (MyHC) gene expression. Twenty-four hours postmortem, LM, SM, infraspinatus (INF), and vastus lateralis samples from 40 randomly selected carcasses (n = 10 per treatment) were obtained and frozen for immunohistochemical analysis. Muscle fiber cross-sectional area and diameter, MyHC isoform expression, and fiber-associated nuclei numbers were measured. Ractopamine dosage exhibited differential effects on muscle morphometrics and MyHC gene expression. Muscle fiber cross-sectional area and diameter were increased (P < 0.05) by RAC in INF type I and IIA fibers and SM type IIA fibers. Ractopamine increased (P < 0.05) MyHC type IIX mRNA and tended to increase (P < 0.10) β2-adrenergic receptors in the SM; a change in mRNA abundance was not detected for either gene in the LM. Treatment with RAC decreased (P < 0.05) fiber-associated nuclei numbers in the INF, vastus lateralis, and LM but did not affect (P > 0.05) MyHC or β-adrenergic receptor expression. These results indicate that cull cow feeding programs may consider supplementing RAC as a means of adding value to cuts within the chuck, such as the INF.


INTRODUCTION

Ractopamine-HCl (RAC) is a β-adrenergic agonist (β AR) approved for use in beef cattle in the United States. β-Adrenergic agonists improve feedlot performance in growing beef steers and heifers as evidenced by increased ADG and feed efficiency (Avendano-Reyes et al., 2006; Walker et al., 2006). However, cattle fed RAC demonstrate only modest improvements in carcass traits (Gruber et al., 2007). Heavier HCW with no changes in loin eye area (LEA), yield grade, or measures of fat deposition were observed in steers fed RAC (Winterholler et al., 2007). Others report an increase in both HCW and LEA in RAC-fed steers (Gruber et al., 2007). A similar tempered response is evident in heifers. Ractopamine augmented heifer feedlot performance measures, HCW, LEA, and yield grade (Sissom et al., 2007). By contrast, Walker et al., (2006) found no improvement in HCW, LEA, or yield grade in heifers receiving RAC. The disparity in RAC effects in fed cattle remains unresolved.

Individual tissue responses to RAC are a consequence of β AR isoform expression and numbers. Swine adipocytes express an equal percentage of β 1- and β 2-adrenergic receptors, whereas muscle fibers express pre-dominately the β 2-adrenergic receptor (Spurlock et al., 1994; Liang and Mills, 2002; Sillence et al., 2005). Interestingly, β-adrenergic receptor density is decreased in backfat depots in pigs fed RAC but not in skeletal muscle (Spurlock et al., 1994). Downregulation of β-adrenergic receptor may account for the loss of a lipolytic effect over time of RAC feeding. In cattle, transcripts for all 3 β-adrenergic receptor isoforms are present in skeletal muscle (Walker et al., 2007). Ractopamine-HCl supplementation to steers and heifers causes a reduction in β 2-adrenergic receptor mRNA with no effect on either β1 or β3-adrenergic receptor expression (Sissom et al., 2007; Winterholler et al., 2007). The ability of RAC supplementation to downregulate muscle β2-adrenergic receptors in cull cows is unknown. Due to the subtle improvements in lean deposition in cattle, a more thorough investigation of RAC effects on muscles of varying fiber type composition is required. The objective of this study was to examine the effects of varying concentrations of RAC on fiber hypertrophy and β-adrenergic receptor gene expression from muscles located in the fore- and hindquarter of cull beef cows.

MATERIALS AND METHODS

This experiment was approved by the University of Florida Institutional Animal Care and Use Committee.

Animals and Diets

Eighty-eight Beefmaster and Angus-type cull cows were stratified by breed and BW to one of 4 RAC supplementation treatments. Cows consumed a concentrate diet (Table 1) ad libitum for 54 d before slaughter. Ractopamine-HCl (0, 100, 200, and 300 mg · head−1 · d−1) was supplemented during the final 28 d on feed as a pelleted type B premix that consisted of wheat middlings (97.6%) and RAC (2.4%; Optaflexx 45, Elanco Animal Health, Greenfield, IN). The appropriate concentration of RAC of each treatment group premix was formulated based on a projected DMI of approximately 13.6 kg · head−1 · d−1. The initial BW of the 4 treatment groups were 426.3, 436.9, 418.8, and 439.0 kg for the 0, 100, 200, and 300 groups, respectively. At the end of the feeding portion, cows weighed 490.0, 483.1, 466.7, and 497.8 kg for the 0, 100, 200, and 300 groups, respectively.

Slaughtering and Sample Collection

Cows were slaughtered at a commercial USDA-inspected facility. Within 60 min of exsanguination, portions of the LM and semimembranosus muscle (SM) from 4 randomly selected animals per group (n = 16) were collected and frozen in liquid nitrogen for RNA extraction. Twenty-four hours postmortem, whole muscles of the LM, SM, infraspinatus (INF), and vastus lateralis (VL) were transported to the University of Florida Meats Laboratory. Two 1-cm3 portions of each muscle from 10 randomly selected cows per group (n = 40) were suspended in OCT tissue freezing medium (Fisher Scientific, Hampton, NH), frozen by submersion in super-cooled isopentane, and stored at −80°C.

Immunohistochemistry

The methods used by Gonzalez et al. (2007) were followed for immunohistochemical staining. Briefly, two 12-μ m serial cryosections were collected on frost-resistant slides (Fisher Scientific). Nonspecific antigen sites were blocked with 5% horse serum in PBS. Cryosections were incubated in primary antibodies for 60 min at room temperature. Primary antibodies and dilutions were α-dystrophin (Abcam, Cambridge, MA) 1:50, myosin heavy chain (MyHC) type I (BAD.5, Developmental Studies Hybridoma Bank, University of Iowa, Iowa City) hybridoma supernatant, and MyHC type IIA (SC.71, Developmental Studies Hybridoma Bank, University of Iowa) hybridoma supernatant. After washing with PBS, tissues were incubated in secondary antibodies for 45 min at room temperature. Labeled secondary antibodies included rabbit anti-mouse AlexaFluor 568 (Invitrogen, San Diego, CA) for α-dystrophin detection and goat anti-rat biotin (Vector Laboratories, Burlingame, CA) followed by streptavidin AlexaFluor 488 (Invitrogen) for MyHC isoform detection. Hoechst 33245 was used to detect nuclei. After a final PBS wash, slides were coverslipped and fluorescence was visualized using an Eclipse TE 2000-U microscope (Nikon, Lewisville, TX) equipped with an X-Cite 120 epifluorescence illumination system (EXFO, Mississauga, Ontario, Canada). Images were captured using a DXM 1200F digital camera (Nikon) and analyzed for individual muscle fiber area and diameter using the NIS-Elements software (Nikon). For each animal, a minimum of 1,000 fibers were measured and analyzed. The region constrained by α-dystrophin immunostaining defined individual fibers for cross-sectional area (CSA) and diameter measurement. Fiber-associated nuclei (FAN) were identified as Hoechst 33245-labeled nuclei lying adjacent to the α-dystrophin border. The number of fibers located within each micrograph was counted to determine the number of nuclei per fiber CSA.

RNA Extraction and Real-Time PCR Analysis

Five hundred milligrams of muscle was homogenized in 10 mL of STAT-60 (Tel-Test Inc., Friendswoods, TX) with a mechanical tissue disruptor. Two milliliters of chloroform was added, and the upper aqueous layer containing nucleic acids was collected by centrifugation. Ribonucleic acid was precipitated by isopropanol and centrifugation (10,000 × g, 10 min). The nucleic acid pellet was washed with 70% ethanol and air-dried. Pellets were resuspended in sterile-filtered, double-distilled water and further purified using the PureLink Micro-to-Midi Total RNA Purification System (Invitrogen). Purity of the RNA was evaluated by spectroscopy with all samples exhibiting an optical density 260:280 greater than 1.9. Integrity of RNA was verified by the presence of intact ribosomal RNA bands after electrophoresis through ethidium bromide-impregnated agarose gels. Aliquots of RNA were stored at −80°C.

One microgram of total RNA was treated with RNase-free DNase (Promega, Madison, WI) to remove trace genomic DNA contamination. Subsequently, the RNA was reverse-transcribed with MMLV-Reverse Transcriptase (Ambion, Austin, TX) and random hexamers at 42°C for 60 min. Complementary DNA from 50 ng of RNA was amplified with SYBR Green PCR Master Mix (Applied Biosystems, Foster City, CA) and the appropriate forward and reverse primers (20 pM; Table 2) in an ABI 7300 Real-Time PCR System (Applied Biosystems). Thermal cycling parameters included a denature step of 95°C for 10 min and 40 cycles of 15 s at 95.0°C and 1 min at 55.0°C. A final dissociation step included 95°C for 15 s, 60°C for 30 s, and 95°C for 15 s. Serial dilutions of pooled samples were used to generate standard curves to ensure generation of cycle threshold values that were within the linear range of amplification (Castellani et al., 2004).

Statistics

Use of carcass as the experimental unit and data were statistically analyzed similar to Wheeler et al. (1990) and Gonzalez et al. (2007). Data for gene expression and FAN numbers were analyzed as a split-plot design using the PROC MIXED procedure (SAS Inst. Inc., Cary, NC). The whole plot consisted of breed type, RAC treatment, and whole plot error. The whole plot error consisted of breed type × RAC treatment. The subplot was muscle, breed type × muscle, RAC treatment × muscle interaction, and the subplot error. The subplot error was comprised of the 3-way interaction between breed type, RAC treatment, and muscle. Muscle fiber CSA and diameter data were analyzed using a split-split-plot design. The whole plots and subplots were the same as the split-plot analysis. The sub-sub-plot consisted of muscle fiber type and the remaining interactions. The 4-factor interaction between breed type, RAC treatment, muscle, and fiber type was used as the sub-subplot error. Pairwise comparisons between the least squares means of the factor levels were computed by using the PDIFF option of the LSMEANS statement.

RESULTS

Cryosections from each of the 4 muscles were immunostained for MyHC type I and IIA isoforms. Morphometrics for type I and II fibers from the LM, VL, SM, and INF were measured (Table 3). Ractopamine fed at a rate of 100 or 300 mg · head−1 · d−1 (RAC-100 and RAC-300, respectively) altered (P < 0.05) the fiber type composition in all muscles examined. Ractopamine fed at 200 mg · head−1 · d−1 (RAC-200) caused an increase (P < 0.05) in the percentage of type IIA fibers in the LM, SM, and VL. By comparison to the LM, the SM and VL had the largest fiber type distribution shifts. By contrast, a shift toward more (P < 0.05) type I fibers in the INF was measured in cull cows fed RAC-200 and RAC-300. RAC-200 tended to increase (P = 0.14) the diameter and CSA of the type I fibers within the VL and significantly increase (P = 0.05) type IIA fibers within the SM. An increase (P < 0.05) in the dimensions of both type I and IIA fibers was observed within the INF of cull cows fed RAC-100; no changes (P > 0.05) in fiber CSA or diameter were observed within the LM, SM, or VL.

As reported previously, RAC-200 does not increase the calculated LM myonuclear domain (Gonzalez et al., 2007). Fewer (P < 0.05) myonuclei per fiber were observed in the LM and VL of cull cows fed RAC-200; no changes (P > 0.05) were found in the INF or SM (Table 4). Cows fed RAC-100 contained fewer (P < 0.05) FAN in the INF, LM, and VL than controls. The RAC-300 cull cows contained fibers with fewer FAN within the LM and VL.

Semiquantitative real-time PCR indicated that RAC supplementation at any concentration did not change (P > 0.05) the amount of detectable β2-adrenergic receptors and MyHC type I, IIA, or IIX mRNA in the LM (Table 5). Compared with controls, RAC decreased (P < 0.05) β2-adrenergic receptors and MyHC type I and IIX mRNA content in the SM when fed at 100 mg · head−1 · d−1 (Table 5). Ractopamine-HCl supplementation at 200 mg · head−1 · d−1 tended to increase both β2-adrenergic receptors and MyHC type IIX mRNA abundance in the SM.

DISCUSSION

Ractopamine-HCl supplementation to growing cattle offers an advantageous improvement in performance variables including increased ADG and decreased G:F (Walker et al., 2007). However, these performance measures translate into only modest improvements in carcass traits. The tempered responses to RAC are further confounded by sex. Heifers fed RAC (200 mg · head−1 · d−1) require less feed per unit of BW gain but do not differ from control heifers with regard to ADG, DMI, or carcass variables (Walker et al., 2006; Sissom et al., 2007). In a similar manner, nonimplanted heifers fed both 200 and 300 mg · head−1 · d−1 of RAC during the final 28 d of a 42-d feeding trial demonstrated no improvements in live performance variables or carcass weight (Quinn et al., 2008). The beef cull cows fed RAC in the current study demonstrated no improvements in either feedlot performance (Carter and Johnson, 2007) or carcass traits (Dijkhuis et al., 2008). Thus, the lack of an improved performance response is not due to the age of our animals but may be related to animal sex.

Ractopamine concentration was sufficient to cause a biological response in cull cows as evidenced by an increase in muscle fiber CSA (Gonzalez et al., 2007). However, larger muscle fiber CSA stimulated by RAC fed at the recommended dose of the manufacturer (200 mg · head−1 · d−1) does not translate into larger ribeye area (Carter et al., 2006). The present study addressed the possibility that a greater concentration of RAC may be required to elicit a global improvement on muscle cell size in aged cows. Ractopamine supplementation at a rate of 300 mg · head−1 · d−1, a concentration within the recommended feeding guideline, provoked a response no different than the conventional 200 mg · head−1 · d−1 feeding rate. The measured CSA of LM type I and II fibers did not differ between controls, RAC-100, or RAC-200. The shift in fiber type in RAC-treated animals indicates that the feed additive is bioactive. Assuming an equivalent rate of absorption and cellular delivery, no change in fiber morphometrics would suggest that the limitation to further increases in muscle growth are not a consequence of insufficient RAC concentration.

The lack of a robust increase in fiber size is not likely attributable to low numbers of β2-adrenergic receptors. β2-Adrenergic receptor is the preferred receptor for RAC in swine tissues (Mills et al., 2003), and the receptor is present in skeletal muscles of cattle (Bridge et al., 1998). Our results indicate that the LM and SM transcribe the β2-adrenergic receptor gene and transcript abundance is not diminished in response to RAC. Indeed, SM β2-adrenergic receptor mRNA concentrations tend to be increased by RAC-200, as reported by others (Sissom et al., 2007; Winterholler et al., 2007). Because receptor protein expression was not determined in any of these studies, it remains possible that female cattle are refractile to the positive effects of RAC at the cellular level due to low abundance β AR numbers or deficits in the signal transduction system, or both.

As reported in swine (Depreux et al., 2002; Gunawan et al., 2007), RAC initiated a shift in fiber types from slow to fast. A reduction in the percentage of type I slow fibers is evident in the LM, SM, and VL with the relative decline between muscles variable. In the LM, RAC-100 is sufficient to elicit a reduction in the percentage of type I fibers with no further decline with increased RAC consumption. By contrast, the SM and VL are highly variable in their response to RAC. In both muscles, RAC-200 elicited the greatest effect with approximately 30% of the type I fibers transitioning to a fast isoform. In the SM, the increased number of type II fibers is associated with greater CSA and a tendency toward greater amounts of MyHC type IIX mRNA. Further increases in RAC did not exacerbate the shift but caused a dampened response. The slight increase in type I fibers in the SM and VL of cows receiving RAC-300 versus RAC-200 may indicate a downregulation of key mediator(s) of RAC effects. The identity of the intracellular mediators of RAC signals in bovine muscle fibers is unknown at this time.

The divergence in responses to RAC among muscles is further exemplified by fiber-type shifts in the INF. The INF, a forelimb muscle, is nearly a 50:50 mix of slow and fast fibers. It was found that RAC-100 caused a reduction in the numbers of oxidative type I fibers, as expected. However, RAC-200 behaved in the opposite manner, with a significant increase in the numbers of slow fibers. Additionally, RAC-300 did not differ from RAC-200 in these measured variables. Feeding RAC-100 also caused a 65 to 70% increase in the size of the INF muscle fiber independent of metabolic enzyme classification. The unexpected shift from fast to slow fiber types also was noted in the SM of cows fed RAC-100 without a change in CSA. Interestingly, the larger SM fiber CSA in RAC-100 cows is associated with a reduction in MyHC gene expression. The dichotomy of larger fibers with less MyHC mRNA suggests that the increased size may reflect a reduction in protein degradation or prolonged half-life of the contractile protein. At the very least, these results underscore the complexity of RAC effects in cattle.

The results presented in this study support our previous work demonstrating that fiber size increases without an increase in myonuclei numbers (Gonzalez et al., 2007). Each fiber contains hundreds of myonuclei, and the ratio of nuclei:cytoplasm, or myonuclear domain, must remain constant (Aberle et al., 2001). O’Connor and Pavlath (2007) described muscle fiber growth as having an initial phase characterized by enhanced transcription and translation, leading to increased protein accretion and a small expansion of the myonuclear domain. After this initial growth, fusion of satellite cells must occur to combat the threshold or ceiling established by myonuclear domain and facilitates additional increases in fiber CSA. However, O’Connor et al. (2007) concluded that increases in nuclear content are not needed to induce skeletal muscle growth, and strong evidence is provided by the ability of β-AR to promote protein synthesis without the addition of myonuclei. The mechanism underlying the increase in muscle fiber size in response to RAC has been linked to altered protein turnover. In pigs, poultry, lambs, and cattle, numerous β-AR including RAC, cimaterol, and clenbuterol increased skeletal muscle hypertrophy in the absence of increases in either DNA content or myonuclear number (Beermann et al., 1987; Smith et al., 1987; Gwartney et al., 1992; Dunshea et al., 1993). Pigs fed RAC demonstrate an increase in fractional protein synthesis rates (Dunshea et al., 1993, 1998; Williams et al., 1994). To date, direct measurement of protein turnover rates in cattle receiving RAC has not been reported. Thus, in cattle, RAC likely stimulates muscle fiber growth by a change in protein synthesis or degradation rates, or both.

In conclusion, ractopamine supplementation to cattle causes a biological effect on muscle fiber isoform distribution and size at concentrations ranging from 100 to 300 mg · head−1 · d−1. Cull beef cows fed RAC-100 responded in a manner similar to conventional RAC-200 as measured by a shift in muscle fiber isotypes. Interestingly, the lesser concentration of RAC improved fiber size only in the INF, a muscle characterized by a greater proportion of red fibers. Cull cow feeding programs may consider supplementing RAC-100 as a means of adding value to cuts within the chuck, such as the INF.


View Full Table | Close Full ViewTable 1.

Composition of basal diet (DM basis)1

 
Soybean hulls 21.1
Citrus pulp 19.7
Cracked corn 14.4
Wheat middlings 14.2
Cottonseed hulls 12.7
Cottonseed meal 7.0
Liquid molasses 7.0
Vitamins and minerals 2.1
Tallow 1.3
Urea 0.4

View Full Table | Close Full ViewTable 2.

Sequence of bovine-specific PCR primers used for determination of the expression of mRNA for β1- and β2-adrenergic receptors and myosin heavy chain isoforms

 
18S
    Forward 5′-GTAACCCGTTGAACCCCATT-3′
    Reverse 5′-CCATCCAATCGGTAGTAGCG-3′
β2
    Forward 5′-TCATGTCGCTTATTGTCCTGG-3′
    Reverse 5′-TCATGTCGCTTATTGTCCTGG-3′
Myosin heavy chain
    MYO21 5′-ATCCAGGCTGCGTAACGCTCTTTGAGGTTGTA-3′
    MYO1112 5′-CACTTGCTAACAAGGACCTCTGAGTTC-3′
    MYO2093 5′-CTTTCCTCATAAAGCTTCAAGTTCTGACC-3′
    MYO4054 5′-TGCTGCTCTCAGGCCCCTGCCACCTT-3′

View Full Table | Close Full ViewTable 3.

LSMEANS of muscle fiber myosin heavy chain isoform distribution, cross-sectional area, and diameter from 4 muscles of cull cows fed 3 concentrations of ractopamine-HCl

 
INF
    CON 51.1a 4,545 ± 384a 74 ± 4a 48.9a 2,554 ± 384a 56 ± 4a
    100 45.7c 6,432 ± 385b 88 ± 4b 54.3c 3,871 ± 385b 68 ± 4b
    200 54.3b 3,955 ± 384a 69 ± 4a 45.7b 2,617 ± 384a 55 ± 4a
    300 54.8b 4,927 ± 384a 77 ± 4a 45.2b 3,100 ± 384ab 61 ± 4a
LM
    CON 34.4a 2,109 ± 385 49 ± 4 65.6a 3,124 ± 384 60 ± 4
    100 32.6b 2,753 ± 385 57 ± 4 67.4b 3,116 ± 384 61 ± 4
    200 32.2b 2,833 ± 385 58 ± 4 67.8b 3,473 ± 384 64 ± 4
    300 32.6b 2,090 ± 385 50 ± 4 67.4b 3,591 ± 384 65 ± 4
SM
    CON 34.0a 1,980 ± 385 49 ± 4 66.0a 3,127 ± 354a 61 ± 4a
    100 39.5b 2,067 ± 386 50 ± 4 60.5b 3,820 ± 384ab 67 ± 4ab
    200 23.6c 2,368 ± 385 54 ± 4 76.4c 4,175 ± 384b 71 ± 4b
    300 27.5d 1,937 ± 385 47 ± 4 72.5d 3,674 ± 384ab 64 ± 4ab
VL
    CON 35.3a 2,284 ± 384x 52 ± 4x 64.7a 3,046 ± 384 60 ± 4
    100 31.0b 2,722 ± 385xy 56 ± 4xy 69.0b 3,554 ± 384 65 ± 4
    200 24.6c 3,044 ± 385y 61 ± 4y 75.4c 3,658 ± 384 66 ± 4
    300 29.1d 2,222 ± 385x 51 ± 4x 70.9d 3,322 ± 384 62 ± 4

View Full Table | Close Full ViewTable 4.

Myonuclei per fiber cross-section from cull cows fed 4 different concentrations of ractopamine-HCl muscle1

 
INF
    0 1.87 ± 0.05a
    100 1.70 ± 0.06b
    200 1.78 ± 0.06ab
    300 1.81 ± 0.06ab
LM
    0 1.35 ± 0.06a
    100 1.07 ± 0.06b
    200 1.15 ± 0.06b
    300 1.13 ± 0.05b
SM
    0 1.55 ± 0.06
    100 1.64 ± 0.07
    200 1.68 ± 0.06
    300 1.52 ± 0.06
VL
    0 2.06 ± 0.06a
    100 1.50 ± 0.07b
    200 1.82 ± 0.06c
    300 1.86 ± 0.06c

View Full Table | Close Full ViewTable 5.

Real-time PCR ΔCT values for β2-adrenergic receptor and myosin heavy chain isoform expression from the LM and semimembranosus of cull-cows fed 4 concentrations of ractopamine-HCl1

 
Muscle2
    LM
        0 10.24 11.71 14.55 20.95
        100 10.11 11.50 13.10 22.10
        200 9.84 9.94 12.27 20.19
        300 10.62 10.34 12.74 21.55
    SM
        0 10.35a,x 9.79a 11.12x 13.67a,x
        100 13.11b 13.17b,x 14.59y 21.01b
        200 8.80a,y 9.31a 11.22x 10.86a,y
        300 8.63a,y 10.39a,y 11.96x 12.57a
        SEM 0.88 1.27 1.35 1.11
Slope3 −3.10 −3.45 −3.57 −3.22

 

References

Footnotes