Non Technical Summary
In 2013, zilpaterol hydrochlorides (Zilmax) was banned from a major meatpacker due to the possibility of adverse impacts such as losing hooves during the hot climate. This may also reflect that more consumers are reluctant to eat meat produced using growth-promoting technologies like a hormonal steroid hormone and beta-agonist, as they are worried about its long-term effects and animal welfare issues. For these reasons, phytochemicals currently attract animal scientists' attention because of its potential alternatives to existing growth promotants and antibiotics in animal agriculture. This is a relatively new area of research, and there are a lot of opportunities to be industrialized in animal agriculture. More than 70% of animal feed companies are willing to use some kinds of alternative feed additives (Lillehoj et al., 2018).Growth promoting techniques, including hormonal implants and beta-adrenergic agonists, have been used in the US livestock industry for several decades. These technologies allow for maximum productivity via improving skeletal muscle fiber hypertrophy, feed efficiency, and carcass characteristics in the livestock species, including cattle, sheep, poultry, and swine. Amongst various synthetic beta-adrenergic agonists (β-AA), ractopamine hydrochloride (OptaFlexx, Elanco; RAC) and zilpaterol hydrochlorides (Zilmax, Merck; ZH) are the most widely used/investigated agents in feedlot beef cattle. In the US, Over 70% of the cattle population is finished on a beta-agonist. These products are orally administrated during the finishing stage of fattening (less than 30 days). These agents reported regulating the cells' signaling pathways in the target tissues, such as skeletal muscle and adipose tissue, by binding specific beta-adrenergic receptors. This leads to an increase in lean yield (skeletal muscle mass) and feed efficiency by promoting protein accretion, satellite cell activity, and altering energy manipulation (Johnson et al., 2014). Ferulic acid (4-hydroxy-3-methoxy cinnamic acid), a natural phenolic compound found in the plant's cell wall, seems to possess similar effects to this commercial β-AAs in the skeletal muscle. FA is a potential activator of beta-2 adrenergic receptor (β2-AR) like ZH does (Wu BN, 1998). Freeform of FA have high availability for absorption and subsequently interact with target tissues (Adam et al., 2002) and is a potential inducer of skeletal muscle growth with various health benefits (Zhao and Moghadasian, 2008).Sustainable animal agriculture has been emphasized in the last decades. Especially for managing the uncertainty of meat production in the pandemic, the importance of producing animal protein using the local resources is necessary. Our current research plan is targeting to utilize locally available ferulic acid sources, barley and brewers spent grain (BSG), as a source of phytochemicals. In addition to this, the utilization of low-quality (e.g., damaged or vomitoxin-infected) barley can be used for animal feed. It will offset losses by damaged gains for grain producers while reducing the feed cost for livestock producers. According to this USDA report (2019), barley that is feed-grade (not suitable for human consumption, but acceptable for use in animal feed) can be heavily discounted so that producers receive half the price of barley that is acceptable for feeding (United States Department of Agriculture, 2019). Feed-grade barley can be valued as low as $2-3 per bushel in some instances, depending on current market value and discounts for low test weight, disease, sprouting, etc. Brewers' spent grain (BSG) is also available year-round in the Michigan area at a relatively low price. Utilizing beer byproducts also help to make an extra profit to local business. A combination of high-ferulic acid-containing barley and BSG expect to maximize starch utilization and skeletal muscle growth, thus improving feed efficiency and ultimately reducing production cost.
Animal Health Component
Research Effort Categories
Goals / Objectives
The objectives of the study are to; Determine the regulatory effects of ferulic acids on the formation of muscle hypertrophy, and adipose cell accumulation in bovine satellite cells and adipose cells via a beta-adrenergic receptors signaling pathwayDetermine how feeding free ferulic acids and ferulic acid-rich grains (barley and its byproduct; BSG) influence steer performance, carcass characteristics, and sera metabolite responses, along with gene expression and protein level of the Longissimus muscle (LM) in a long-term manner (120 days)
Experiment 1: primary cell cultures Bovine satellite cells will be isolated from longissimus dorsi (LD) six-month-old crossbred calves (n=4) using a 6 mm Bergstrom biopsy needle following the method described (Kim et al., 2018; Kim et al., 2020a). Isolated cells will be incubated at 38? with a humidified atmosphere of 5% CO2.Phase I: satellite cells activity Does dependent ferulic acids: various doses (0, 0.1, 1, 10, and 100 µM) of free ferulic acids will be added in the basal medium (10% FBS/ DMEM). Cells will be incubated for 48h and harvested for mRNA gene expression (Myf5, Pax7, Pax3, myogenin, and beta-adrenergic receptors (β1 and β2)) and protein level. Satellite cells will also be plated on the three-well microscopy glass slides for histology.Phase II: skeletal muscle fiberOnce mononucleated satellite cells reach 70-80% of confluency, myogenic differentiation will be induced by 5% horse serum/ DMEM. Various doses (0, 0.1, 1, 10, and 100 µM) of free ferulic acids will be added along with differentiation media. After 72 h of incubation, the cell will be harvested and used for mRNA gene expression (myogenin, myosin heavy chain I, II, and IIX, beta-adrenergic receptors (β1 and β2), cAMP, Akt, PI3K, mTOR) and protein levels. Histology of beta-adrenergic receptors (β1 and β2) will also be conducted to confirm mRNA and protein data.Phase III: adipose cells: bovine subcutaneous and intramuscular adipose cells Bovine preadipocytes will be isolated from the subcutaneous adipose tissue of eighteen-month-old crossbred steers (n=3) following the method previously described (Kim et al., 2020b). Preadipocytes will be incubated in 10% FBS/DMEM for 4 days. Once cells reach 80-90% of confluency, media will be changed with differentiation media containing 3% FBS, insulin, pioglitazone, hydrocortisone, and oleic acid. Free ferulic acids will be mixed with differentiation media in a dose-dependent manner (0, 0.1, 1, 10, and 100 µM). After 72h of incubation, cells will be harvested and used for mRNA gene expressions (C/EBPα, β, PPARγ, δ, SCD) and protein levels.Experiment 2: Animal feeding/ LM biopsy studyThirty-six Angus × Simmental steers (estimated initial average BW = 950-1000 lb) will be assigned to 1 of 3 treatments (n =36, 9/ treatment, expected daily gain: 2.5-2.7 lb).The three experimental treatments include:A corn-based diet with distillers' grain (Cont)Cont diet with FA 2000mg (FA2000)Cont diet with FA 4000mg (FA4000)Barley based diet with brewers' spent grain (BS)- expected to contain over 6000mg of FA/daySteers will be allocated into three pens per treatment. Previous research suggests that we need to sample 10 steers/treatments in order to identify statistically meaningful differences in sera metabolites and LM mRNA values. The current protocol calls for these measures to be collected on 12 steers per treatment. Data will be analyzed using a model appropriate for a completely randomized design with repeated measures over time, and steer will serve as the experimental unit.Upon arrival at the feedlot, steers will be offered a new diet gradually over 28-d, allowing 3-5 d of adaptation to 30, 60, 75, 80, 85% concentrates on a diet DM basis. Steers will be fed ad libitum, using clean bunk management. All steers will be fed 1× daily at 0800h. Feed samples will be collected from the bunk, and chemical values will be measured by proximate analysis of bunk samples on a dry matter basis except for Diet DM. Limestone and ionophore (Monensin) will be fed to protect unwanted pH changes in the rumen.Individual rumen fluid samples using a stomach tube (JørgenKruuse A/S, Langeskov, Denmark) will be obtained on d 0, 56, and 108 for analysis of free ferulic acid. Approximately 50 mL of rumen contents will be taken by inserting a stomach tube.Biopsies of longissimus muscle (LM) and whole blood will be collected from each steer on d 0, 28, 56, 84, and 108 relatives to the timing of study initiation. The LM biopsy will be collected by aseptic procedures using a 6 mm Bergstrom biopsy needle, and then samples will be immediately snap-frozen in liquid nitrogen (LN2) and stored at -80 C until subsequent analyses. The stored sample will later be used for mRNA gene expression (β1-AR, β2-AR, IGF-1, Akt, mTOR, PPARα, β) and protein levels. Sera will be stored in duplicate 2 mL aliquots at -20°C until subsequent analysis. Sera will be used to quantify circulating concentrations of circulating IGF-1, Non-esterified fatty acids, and free ferulic acid. All data repeated over time will be analyzed with the appropriate design for repeated measures over time.Carcass data will be collected at the end of the trial (d 120). After animals are harvested, carcasses will chill for at least 24 h. Traits will include hot carcass weight (HCW), dressing percentage (DP), 12th rib fat thickness, longissimus muscle area, percent kidney/pelvic/heart fat (KPH), USDA calculated yield grade, marbling score, lean maturity (meat color), and skeletal maturity. After grading, strip loin samples will be collected and used for pH, Warner-Bratzler shear force (i.e., instrumental tenderness, 14-day of age), instrumental color evaluation, and meat composition (moisture, protein, fat, fatty acid profile).Longissimus muscle will also be collected for muscle fiber typing. Samples will be cross-sectioned and mounted on positively charged glass slides. Satellite cell population and density (DAPI staining) and myosin heavy chains (MHC type I, IIA, and IIX) will be determined by the immunostaining technique. The image will be taken under the fluorescence microscope and will be counted and enumerated as a percentage of the total number of muscle fibers. The cross-sectional area of each fiber will also be measured.