Cattle breed type and anabolic implants impact calpastatin expression and abundance of mRNA associated with protein turnover in the longissimus thoracis of feedlot steers

Abstract

Two methods that the beef cattle industry can use to improve efficiency, sustainability, and economic viability are growth promotants and crossbreeding cattle of different breed types. In the United States, over 90% of cattle receive an anabolic implant at some point during production resulting in an overall increase in skeletal muscle growth. Recent research suggests that the two main cattle breed types, Bos indicus and Bos taurus, respond differently to anabolic implants. The objective of this study was to characterize changes that occur in skeletal muscle following implanting in Bos indicus influenced steers or Bos taurus steers. Twenty steers were stratified by initial weight in a 2 × 2 factorial design examining two different breeds: Angus (AN; n = 10) or Santa Gertrudis influenced (SG; n = 10), and two implant strategies: no implant (CON; n = 10) or a combined implant containing 120 mg TBA and 24 mg E2 (IMP; n = 10; Revalor-S, Merck Animal Health). Skeletal muscle biopsies were taken from the longissimus thoracis (LT) 2 and 10 d post-implantation. The mRNA abundance of 24 genes associated with skeletal muscle growth were examined, as well as the protein expression of µ-calpain and calpastatin. Succinate dehydrogenase mRNA abundance was impacted (P = 0.05) by a breed × treatment interaction 2 d post-implanting, with SG-CON having a greater increased abundance than all other steers. A tendency for a breed × treatment interaction was observed for calpain-6 mRNA (P = 0.07), with SG-CON having greater abundance than AN-CON and SG-IMP. Additionally, calpastatin protein expression was altered (P = 0.01) by a breed × treatment interaction, with SG-CON and SG-IMP steers having increased expression (P = 0.01) compared with AN-CON steers. At 2 d post-implanting, a breed × treatment interaction was observed with SG-CON steers having greater (P = 0.05) mRNA abundance of mitogen-activated protein kinase compared with AN-CON steers. Furthermore, breed affected (P = 0.05) calpastatin abundance with AN steers having increased (P = 0.05) abundance 2 d post-implanting compared with SG steers. Meanwhile, implants tended to affect (P = 0.09) muscle RING finger protein-1 mRNA abundance, with CON steers having increased (P = 0.09) abundance compared with that of IMP steers. These findings suggest that cattle breed type and anabolic implants impact calpastatin expression and mRNA abundance associated with protein turnover in the LT of feedlot steers 2 and 10 d post-implantation.

Anabolic implants and cattle breed type interact with each other to cause changes in mRNA abundance in the longissimus thoracis that are related to protein turnover.

Calpastatin expression of the longissimus thoracis is altered by an interaction between cattle breed type and anabolic implants.

Introduction

The cattle industry can take advantage of both heterosis gained through crossbreeding and the use of growth promotants to help further improve environmental and economic sustainability of the beef industry (Capper and Hayes, 2012). The two main cattle breed types, Bos indicus and Bos taurus are physiologically different from one another (Frisch, 1987; Marshall, 1994). Bos indicus cattle tend to be better adapted to higher temperatures, nutritional stress (Forbes et al., 1998), and are more disease resistant than Bos taurus (Glass et al., 2005), while consuming less water (Winchester and Morris, 1956; Forbes et al., 1998). However, in the United States, taurine breeds, including the Angus (AN) and Hereford breeds, are typically favored by producers as they are known to have improved carcass characteristics and temperaments (Cooke, 2014). When utilizing Bos indicus genetics, producers have concerns related to growth, carcass characteristics, and animal temperament (Cooke, 2014; Wright et al., 2018). However, these negative traits can be minimized by crossbreeding Bos taurus with Bos indicus cattle (Elzo et al., 2016). The Santa Gertrudis (SG) breed is one example of these crosses, being a composite breed composed of 5/8 Shorthorn (a Bos taurus breed) and 3/8 Brahman (a Bos indicus breed) (Ferraz et al., 2000).

In the United States, over 90% of cattle on feed receive an anabolic implant at some point during the production cycle (APHIS, 2013). Anabolic implants are used to increase growth and efficiency of beef cattle, where on average, anabolic implants improve average daily gain by 18%, feed efficiency by 6%, and feed intake by 6% (Duckett and Pratt, 2014). Anabolic implants are typically composed of the steroid hormone estradiol (E2), and the synthetic testosterone analogue trenbolone acetate (TBA) (Smith and Johnson, 2020). However, the physiological and molecular mechanisms that anabolic implants operate through to improve skeletal muscle growth have yet to be determined (Reichhardt et al., 2021b).

Recent research has demonstrated that anabolic implants and cattle breed type interact, suggesting that there is a need to identify optimal anabolic implant protocols for different cattle breed types (Reichhardt et al., 2021a; Rivero et al., 2021). However, the extent of these interactions have yet to be fully characterized. In this study, we hypothesized that due to the innate physiological differences between the two cattle breed types, anabolic implants would elicit changes in skeletal muscle of feedlot steers in a breed dependent manner resulting in increased growth. The objective of this trial was to identify changes that occur in mRNA abundance and protein expression of the longissimus thoracis (LT) of feedlot steers sired by two different beef breeds 2 and 10 d post-implantation to better understand the interactions that occur between cattle breed type and anabolic implants.

Materials and Methods

All live animal procedures and protocols for this experiment were approved by the Utah State University Institutional Animal Care and Use Committee (IACUC Protocol #2817).

Animal management

The animals used in this research were part of a previously published trial (Reichhardt et al., 2021a). Treatments and feedlot performance parameters of this trial have been previously published (Reichhardt et al., 2021a). Briefly, this trial was conducted at the Utah State University feedlot and used a 2 × 2 factorial design. Twenty steers, 10 AN sired (362 ± 5.7 kg) and 10 SG sired (365 ± 8.5 kg), that had not previously received any growth promotants, were initially stratified by weight. All steers used in the trial were out of commercial Angus dams, and three unrelated sires of each breed type. Before beginning the trial, each steer received an electronic and visual ear tag. Steers were assigned to one of two implant treatments: 1) no implant (CON; n = 10) or 2) a combined implant containing 120 mg TBA and 24 mg E2 (IMP; n = 10; Revalor S, Merck Animal Health). Steers were randomly placed into one of four covered pens each equipped with two GrowSafe bunks. Each pen housed a total of 15 steers. Steers utilized in this trial were housed with other steers of similar size from the Utah State University beef herd. Steers always had free choice access to water. All steers were fed the same typical feedlot ration. Rations were stepped up between 10% and 12% (DM basis) concentrate every 10 d from a backgrounding ration consisting of 40% (DM basis) concentrate to a finishing ration consisting of 86% (DM basis) concentrate (Supplementary Table 1) over a 41-d period.

Sample collection and processing

Skeletal muscle samples were collected from the LT on day 2 and 10 post-implanting by clinical veterinarians at Utah State University using previously published methods (Thornton et al., 2012). In brief, a 30 × 30 cm square area was shaved and prepped following standard procedures. The area was injected with 20 mL of 2% lidocaine hydrochloride in an inverted L pattern. A 10 cm oblique skin incision was made in the longissimus thoracis muscle. Samples were taken on the same side of the animal. The day 2 samples were taken above the 11th rib, and the day 10 samples were taken from above the 13th rib. A Wheatlander retractor was used to open the incision and a 2 × 2 × 2 cm muscle biopsy was obtained using a Metzenbaum scissor. The incision in the muscle was closed using 0 chromic gut suture in an interrupted cruciate pattern. The incision was closed using 0 braunamid suture in a continuous ford interlocking suture pattern. The wound was then sprayed with an antiseptic spray. Skeletal muscle biopsy samples were then flash frozen in liquid nitrogen and stored at −80 °C for subsequent analyses.

Isolation of RNA, quantification, and cDNA synthesis

Flash frozen skeletal muscle samples were ground under liquid nitrogen using mortar and pestle. Isolation of RNA was performed using TriZol (Invitrogen, Carlsbad, CA, USA) as per the manufacturer’s protocol and as previously described (Gardner et al., 2021). Isolated RNA was quantified using a Take3 plate on a BioTek all-in-one microplate reader with Gen5 2.0 software (BioTek Instruments, Winooski, VT, USA). All RNA samples were treated with deoxyribonuclease (Ambion, Foster City, CA, USA) before beginning cDNA synthesis using a high-capacity cDNA reverse transcription kit (Applied Biosystems, Foster City, CA, USA) following the manufacturer’s protocol.

Fluidigm reverse transcription qPCR

The 48.48 Dynamic Array Integrated Fluidic Circuit (Fluidigm, San Francisco, CA, USA) was utilized for quantitative gene expression as previously described (Suasnavas et al., 2015). A total of 24 genes were targeted for analysis and examined on a single chip (Supplementary Table 2). Primer sets were designed and validated by Fluidigm. In brief, following the Fluidigm protocol, a specific target amplification (STA) was performed to enrich each sample for target-specific cDNA prior to quantitative PCR. For STA thermal cycling, each reaction consisted of 1.25 µL of primer mix, 2.5 µL of the Taqman PreAmp Master Mix (Applied Biosystems), and 1.25 µL of cDNA. Enzyme activation took place at 95 °C for 10 min and then the amplification was done for 10 cycles (95 °C for 15 s then 60 °C for 4 min). The Fluidigm IFC chip was then run on the Biomark thermocycler/detection module. Genes were normalized to the housekeeping gene eukaryotic translation elongation factor 1 alpha 2 (EEF1A2) using the per sample ΔC_t method. Eukaryotic translation elongation factor 1 alpha 2 mRNA abundance was checked, and neither breed, treatment, nor their interaction impacted (P > 0.10) mRNA abundance of EEF1A2. Furthermore, EEF1A2 has been previously published and used as a housekeeping gene when evaluating beef cattle skeletal muscle (Wellmann et al., 2021). Abundance of mRNA was determined by analyzing the relative expression of each sample calculated as 2^{-relative threshold cycle (ΔCt)}.

Protein extraction

For the analysis of calpastatin and calpain-1 abundance, frozen biopsy samples collected at 2- and 10-day post-treatment were homogenized at 100 mg/mL with a solubilization buffer composed of 2 M thiourea, 50 mM Tris-HCl, 3% (w/v) SDS, 8 M urea, and 75 mM dithiothreitol (pH 6.8) (Warren et al., 2003). Samples were subjected to a bead-beating homogenizer and heated at 60 °C for 10 min. Then, samples were centrifuged at 10,000 ×g for 10 min at room temperature. Resulting supernatants were transferred to new tubes and utilized for protein concentration determination using the RC DC protein assay kit (BioRad Laboratories, Hercules, CA, USA). Following protein determination, samples were diluted to the same protein concentration by the solubilization buffer with 0.05% (w/v) bromophenol blue added. All samples were stored at −80 °C until loaded in gels.

SDS-PAGE and immunoblotting

Calpastatin and calpain-1 abundance were determined with SDS-PAGE followed by immunoblotting as previously described (Dang et al., 2020). Samples were thawed at room temperature and heated at 60 °C for 10 min in a dry heating block. Self-casted 8% polyacrylamide gels (26.7% [v/v] 30% acrylamide/0.8% bisacrylamide, 0.37 M Tris, pH 8.8, 0.1% [w/v] SDS, 0.13% [w/v] ammonium persulfate, and 0.07% [v/v] TEMED) were used for the separation of calpastatin and calpain-1. Four samples from each breed at the same time point were included on one gel along with a reference sample and a protein standard. Separated proteins were transferred to nitrocellulose membranes and reversibly stained with Ponceau S. UVP Chemstudio Imaging System and software (Analytik Jena, Upland, CA, USA) was used to image and quantify total protein within each lane. Membranes were destained and subsequently blocked with 1.5% (w/v) casein in PBS-T (phosphate-buffered saline and 0.1% [v/v] tween-20) for 1 h at 25 °C. All membranes were immunoblotted with primary antibodies at 4 °C overnight. Primary antibodies were diluted with PBS-T at specified ratios. Calpastatin primary antibody was diluted at 1:1,000 (MA3-944, Thermo Scientific, Rockford, IL, USA), while calpain-1 was diluted at 1:2,000 (MA3–940, Thermo Scientific). Membranes were washed three times (each for 5 min) with PBS-T and incubated with fluorescent secondary antibodies (CF680, Biotium Inc., Fremont, CA, USA) for 1 h at room temperature. Final imaging took place using the same imaging system and software mentioned above. Band intensities were quantified and normalized to the intensity of the total protein within each lane.

Statistical analysis

Statistical analysis for all data was performed using the MIXED procedure of SAS (version 9.4; SAS Inst. Inc., Cary, NC, USA) with implant, breed, and their interaction as fixed effects, and animal and pen as a random effect in the model. All data are presented as the least square mean ± SEM. When treatment differences were found to be significant (P ≤ 0.05), least square means (LS means) were separated using Tukey–Kramer adjustments. Significance was determined at P ≤ 0.05 and tendencies were declared at 0.05 < P ≤ 0.10.

Results

The mRNA abundance of 24 genes analyzed in this study were split into four groups. The groups include abundance of mRNA associated with skeletal muscle differentiation, protein turnover, skeletal muscle metabolism, and receptors that are associated with growth. Protein expression was examined for two proteins, calpain-1 and calpastatin.

Abundance of mRNA associated with skeletal muscle differentiation

Abundance of four different genes relative to skeletal muscle differentiation were investigated 2 and 10 d post-implanting (Table 1). No breed × treatment interactions (P > 0.10) were observed for any of the genes investigated at either time point (Table 1). Two days post-implanting, there was no difference (P > 0.10) in mRNA abundance of any of the genes relative to breed or implant (Table 2). However, 2 d post-implanting SG steers had a greater (P = 0.05) Paired box transcription factor 7: Myoblast differentiation factor 1 (PAX7:MYOD) ratio compared with AN steers. Furthermore, 10 d post-implanting, it was found that CON steers had greater (P = 0.05) abundance of myogenic regulatory factor 5 (MYF5) than IMP steers. None of the other genes investigated on day 10 were affected (P > 0.10) by breed or treatment (Table 1).

Table 1.

Abundance of mRNA associated with skeletal muscle differentiation from longissimus thoracis skeletal muscle of feedlot steers of different cattle breed types following implanting

Steers (n)	Implant and breed treatments1				SEM	P-values2
Gene3	AN-IMP	SG-IMP	AN-CON	SG-CON		Breed	Trt	B × T
	5	5	5	5
PAX7
Day 2	53.98	56.76	45.14	21.89	33.47	0.77	0.54	0.71
Day 10	10.73	15.52	18.41	30.54	7.15	0.26	0.13	0.58
MYF5
Day 2	14.11	3.27	10.07	13.03	5.06	0.46	0.59	0.21
Day 10	10.38	9.98	20.84	38.47	10.02	0.41	0.05	0.32
MYOD
Day 2	11.80	7.74	7.15	6.89	2.57	0.39	0.27	0.44
Day 10	21.02	30.82	32.66	14.92	12.29	0.75	0.87	0.29
MYOG
Day 2	8.75	6.57	10.01	4.45	3.38	0.23	0.88	0.54
Day 10	6.26	6.39	4.32	139.6	65.52	0.32	0.33	0.32
PAX7:MYOD
Day 2	0.08	0.33	0.14	0.53	0.15	0.05	0.39	0.63
Day 10	0.15	0.06	0.22	0.14	0.08	0.27	0.29	0.99

Implant treatments administered on day 0 include the following: no implant + Angus (AN-CON), no implant + Santa Gertrudis (SG-CON), Revalor-S + Angus (AN-IMP; 120 mg trenbolone acetate + 24 mg estradiol) and Revalor-S + Santa Gertrudis (SG-IMP; 120 mg trenbolone acetate + 24 mg estradiol).

P-values indicate the effect of Breed, Treatment (TRT), or B × T (Breed × Treatment). Skeletal muscle biopsies were collected on day 2 and 10 post-implanting, and RNA isolation and quantitative PCR were performed as described in the Materials and Methods. All data are presented as the least square means ± SEM.

PAX7, paired box transcription factor 7; MYF5, myogenic regulatory factor 5, MYOD, myoblast differentiation factor 1; MYOG, myogenin.

Italics indicate P-values for the different effects (breed, trt and trtxbreed).

Table 2.

Abundance of mRNA associated with protein turnover from longissimus thoracis skeletal muscle of feedlot steers of different cattle breed types following implanting

Steers (n)	Implant and Breed Treatments1				SEM	P-values2
Gene3	AN-IMP	SG-IMP	AN-CON	SG-CON		Breed	Trt	B × T
	5	5	5	5
CAPN6
Day 2	53.46	15.35	21.77	151.92	42.26	0.29	0.24	0.07
Day 10	56.13	38.21	45.54	57.86	13.15	0.84	0.74	0.29
CAST
Day 2	49.79	30.25	42.17	28.00	7.83	0.05	0.52	0.72
Day 10	26.85	47.46	26.03	32.99	10.63	0.22	0.48	0.53
mTOR
Day 2	90.09	60.18	55.27	56.57	17.63	0.45	0.31	0.41
Day 10	56.77	60.02	38.94	74.59	17.31	0.31	0.93	0.39
MAPK
Day 2	22.84xy	21.71xy	16.50x	44.10y	7.87	0.10	0.28	0.05
Day 10	19.95	29.22	29.12	31.08	5.55	0.34	0.35	0.53
ATROG-1
Day 2	60.39	61.41	84.11	78.48	13.47	0.87	0.15	0.81
Day 10	64.75	89.25	81.69	96.44	18.82	0.32	0.53	0.79
MURF-1
Day 2	53.85	53.11	52.24	75.21	8.48	0.23	0.26	0.20
Day 10	73.79	70.19	98.02	105.61	16.05	0.91	0.09	0.74
FOXO3
Day 2	76.48	64.28	123.20	83.28	28.97	0.35	0.23	0.59
Day 10	55.37	71.68	65.61	86.27	17.21	0.26	0.41	0.88

Implant treatments administered on d 0 include: no implant +Angus (AN-CON), no implant + Santa Gertrudis (SG-CON), Revalor-S + Angus (AN-IMP; 120 mg trenbolone acetate + 24 mg estradiol) and Revalor-S + Santa Gertrudis (SG-IMP; 120 mg trenbolone acetate + 24 mg estradiol).

P-values indicate the effect of Breed, Treatment (TRT), or B × T (Breed × Treatment). Skeletal muscle biopsies were collected on day 2 and 10 post-implanting, and RNA isolation and quantitative PCR were performed as described in the Materials and Methods. All data are presented as the least square means ± SEM.

CAPN6, calpain 6; CAST, calpastatin; mTOR, mammalian target of rapamycin; MAPK, mitogen-activated kinase; ATROG1, atrogin 1; MURF1, muscle ring finger protein 1; FOXO3, forkhead box O3.

Different letters indicate a tendency between time points (0.05 < P ≤ 0.10).

Italics indicate P-values for the different effects (breed, trt and trtxbreed).

Abundance of mRNA associated with protein turnover

Abundance of mRNA associated with skeletal muscle protein turnover were examined in the LT of AN and SG steers 2 and 10 d post-implanting (Table 2). Two days post-implanting, there was a breed × treatment interaction, with SG-CON steers having a greater (P = 0.05) mRNA abundance of mitogen-activated protein kinase (MAPK) than AN-CON steers. Additionally, abundance of Calpain-6 (CAPN6) tended to be altered (P = 0.07) by a breed × treatment interaction 2 d post-implanting. However, when LS Means were examined, the means were not different (P > 0.10) from one another despite SG-CON having numerically increased abundance of CAPN6 compared with SG-IMP steers (Table 2). Furthermore, breed affected (P = 0.05) Calpastatin (CAST) abundance. Two days post-implanting, CAST abundance was greater (P = 0.05) in AN steers than in SG steers (Table 2). Anabolic implants did not alter (P > 0.10) mRNA abundance of CAST on either day 2 or day 10. Ten days post-implanting, there was a tendency for CON steers to have greater (P = 0.09) mRNA abundance of muscle RING finger protein 1 (MURF-1). Anabolic implants, breed, or their interaction did not alter (P > 0.10) mRNA abundance of any other genes investigated 10 d post-implanting (Table 2).

Abundance of mRNA associated with skeletal muscle metabolism

Abundance of mRNA of nine genes associated with muscle metabolism was investigated in the LT of AN and SG steers 2 and 10 d post-implanting (Table 3). There was a breed × treatment interaction (P = 0.05) in respect to succinate dehydrogenase (SDHA) mRNA abundance (Table 3). When LS Means were examined, none of the means were different (P > 0.10) from one another, however, SG-CON had numerically increased abundance of SDHA compared with AN-CON steers. There were no other interactions (P > 0.10) regarding mRNA associated with skeletal muscle metabolism observed on either day (Table 3). Additionally, neither breed nor treatment affected (P > 0.10) abundance of any of the genes investigated on either day (Table 3).

Table 3.

Abundance of mRNA associated with skeletal muscle metabolism from longissimus thoracis skeletal muscle of feedlot steers of different cattle breed types following implanting

Steers (n)	Implant and Breed Treatments1				SEM	P-values2
Gene3	AN-IMP	SG-IMP	AN-CON	SG-CON		Breed	Trt	B × T
	5	5	5	5
MHC I
Day 2	266.72	230.33	199.06	164.17	50.74	0.66	0.26	0.72
Day 10	308.59	273.19	249.17	316.28	82.60	0.84	0.91	0.49
MHC IIa
Day 2	99.29	75.01	65.00	58.87	16.56	0.33	0.11	0.53
Day 10	114.14	132.94	127.21	102.70	29.99	0.93	0.79	0.51
MHC IIx
Day 2	445.61	361.86	309.57	257.19	114.26	0.56	0.31	0.89
Day 10	591.44	385.96	551.07	688.26	133.90	0.80	0.34	0.21
AKT
Day 2	26.44	26.24	21.47	26.11	4.51	0.64	0.59	0.61
Day 10	25.59	33.34	31.08	10.15	7.06	0.26	0.39	0.93
LDHA
Day 2	87.24	100.85	94.62	80.21	23.85	0.98	0.77	0.53
Day 10	148.73	107.89	113.62	104.80	32.74	0.48	0.58	0.64
GAPDH
Day 2	275.32	256.61	268.35	228.0	69.95	0.67	0.79	0.87
Day 10	381.15	353.67	356.92	356.45	109.60	0.90	0.92	0.91
CS
Day 2	137.18	76.62	118.95	117.29	20.61	0.16	0.59	0.18
Day 10	74.97	98.60	105.38	126.76	19.17	0.26	0.14	0.95
SDHA
Day 2	45.37	35.95	18.74	60.79	13.36	0.94	0.23	0.05
Day 10	44.37	36.06	43.57	66.17	14.93	0.65	0.36	0.34

Implant treatments administered on d 0 include: no implant +Angus (AN-CON), no implant + Santa Gertrudis (SG-CON), Revalor-S + Angus (AN-IMP; 120 mg trenbolone acetate + 24 mg estradiol) and Revalor-S + Santa Gertrudis (SG-IMP; 120 mg trenbolone acetate + 24 mg estradiol).

P-values indicate the effect of Breed, Treatment (TRT), or B × T (Breed × Treatment). Skeletal muscle biopsies were collected on day 2 and 10 post-implanting, and RNA isolation and quantitative PCR were performed as described in the Materials and Methods. All data are presented as the least square means ± SEM.

MHCI, myosin heavy chain I; MHCII X, myosin heavy chain IIX; AKT, protein kinase B; LDHA, lactate dehydrogenase A; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; CS, citrate synthase; SDHA, succinate dehydrogenase A.

Different letters indicate a difference (P < 0.05) between time points.

Different letters indicate a tendency between time points (0.05 < P ≤ 0.10).

Italics indicate P-values for the different effects (breed, trt and trtxbreed).

Abundance of mRNA of receptors that are associated with growth

Abundance of the oxytocin receptor (OXTR), insulin-like growth factor-1 receptor (IGF-1R), and the vitamin D receptor (VDR) were examined 2 and 10 d post-implanting in AN and SG steers (Table 4). Implant, breed, or their interaction had no effect (P > 0.10) on the IGF-1R at either timepoint (Table 4). There was no interaction or implant effect (P > 0.10) regarding OXTR abundance. However, 10 d post-implanting the OXTR was affected by breed, with SG steers having increased (P = 0.03) mRNA abundance compared with AN steers. Ten days post-implanting, there was a breed × treatment interaction, with the VDR having greater (P = 0.03) mRNA abundance in the SG-CON steers than all other steers (Table 4).

Table 4.

Abundance of receptor mRNA associated with skeletal muscle growth from longissimus thoracis skeletal muscle of feedlot steers of different cattle breed types following implanting

Steers (n)	Implant and breed treatments1				SEM	P-values2
Gene3	AN-IMP	SG-IMP	AN-CON	SG-CON		Breed	Trt	B × T
	5	5	5	5
OXTR
Day 2	56.58	19.56	69.90	99.98	50.91	0.94	0.24	0.37
Day 10	0x	23.21xy	0x	88.58y	32.01	0.03	0.15	0.31
IGF-1R
Day 2	59.65	23.46	25.83	18.00	14.00	0.15	0.19	0.35
Day 10	21.75	23.01	31.87	37.65	10.08	0.72	0.19	0.80
VDR
Day 2	77.05	25.26	62.18	104.46	48.13	0.92	0.47	0.29
Day 10	6.93a	22.91a	4.66a	114.93b	22.54	0.01	0.04	0.03

Implant treatments administered on day 0 include the following: no implant +Angus (AN-CON), no implant + Santa Gertrudis (SG-CON), Revalor-S + Angus (AN-IMP; 120 mg trenbolone acetate + 24 mg estradiol) and Revalor-S + Santa Gertrudis (SG-IMP; 120 mg trenbolone acetate + 24 mg estradiol).

P-values indicate the effect of Breed, Treatment (TRT), or B × T (Breed × Treatment). Skeletal muscle biopsies were collected on day 2 and 10 post-implanting, and RNA isolation and quantitative PCR were performed as described in the Materials and Methods.

OXTR, oxytocin receptor; IGF-1R, insulin-ike growth factor 1 receptor; VDR, vitamin D receptor.

Different letters indicate a difference (P < 0.05) between time points.

Different letters indicate a tendency between time points (0.05 < P ≤ 0.10). All data are presented as the least square means ± SEM.

Italics indicate P-values for the different effects (breed, trt and trtxbreed).

Protein expression of Calpain-1 and Calpastatin

Relative abundance of CAST (Figure 1) and calpain-1 (CAPN1; Figure 2), were evaluated within and between SG and AN group at 2 and 10 d post-treatment. Two days post-implanting, CAST expression was affected by a breed × treatment interaction (P = 0.01; Figure 1A) with SG-CON and SG-IMP having a greater expression of CAST than AN-CON. Furthermore, 10 d post-implanting, CAST expression was affected by breed with SG steers having increased (P < 0.0001) expression of CAST compared with AN steers (Figure 1B). However, CAPN1 evaluation showed no differences (P ≥ 0.11) in relative abundance when accounting for breed, treatment, or their interactions on 2 (Figure 2A) or 10 d (Figure 2B) post-implanting.

Figure 1.

Calpastatin (CAST) protein expression was determined on (A) day 2 and (B) 10 post-implanting by SDS-PAGE and Immunoblotting as described in the Materials and Methods. Steers were stratified by weight and by breed; Angus (AN) or Santa Gertrudis (SG) and assigned to one of two treatments: (1) Control, no implant (CON), or (2) implanted with 120 mg trenbolone acetate and 24 mg estradiol (IMP). Data represent CAST abundance and are presented as LS mean ± SEM.

Figure 2.

Calpain-1 (CAPN1) protein expression was determined on (A) day 2 and (B) 10 post-implanting by SDS-PAGE and Immunoblotting as described in the Materials and Methods. Steers were stratified by weight and by breed; Angus (AN) or Santa Gertrudis (SG) and assigned to one of two treatments: (1) Control, no implant (CON), or (2) implanted with 120 mg trenbolone acetate and 24 mg estradiol (IMP). Data represent CAPN1 abundance and are presented as LS mean ± SEM.

Discussion

Improving our understanding of skeletal muscle growth is imperative to continue to produce environmentally and economically sustainable products, as skeletal muscle eventually becomes the marketable end product; meat (England et al., 2013). Anabolic implants are currently one of the best tools that beef producers can utilize to improve skeletal muscle growth in an environmentally and economically sustainable manner (Crawford et al., 2022), as anabolic implant improve important animal production traits as previously discussed (Duckett and Pratt, 2014). Furthermore, Bos taurus and Bos indicus cattle breeds are known to be physiologically different from one another (Frisch, 1987), with the Santa Gertrudis is a composite breed composed of both Bos indicus cattle (3/8 Brahman) and Bos taurus cattle (5/8 Shorthorn) (Ferraz et al., 2000). When Santa Gertrudis bulls are crossed with Angus cows, the resulting calves are approximately 3/16ths Bos indicus, and the remaining percentage is Bos taurus. This use of crossbreeding Bos indicus with Bos taurus helps to mitigate some of the negative effects that are commonly associated when using Bos indicus genetics within the herd, (Cooke, 2014; Elzo et al., 2016; Wright et al., 2018), while allowing for the preservation of the advantageous traits of the Bos indicus (Forbes et al., 1998). Additionally, research has emerged demonstrating that skeletal muscle between Bos indicus influenced animals and Bos taurus animals is intrinsically different (Wright et al., 2018), with research from our group and others finding that anabolic implant protocol and cattle breed types interact, leading to questions regarding the optimization of implant protocols for specific breed types (Reichhardt et al., 2021a; Rivero et al., 2021). However, it has been found that anabolic implants do not alter performance in a breed dependent manner between Continental and British feedlot cattle (Boles et al., 2009). Therefore, the interactions this trial found are most likely due to the inclusion of Bos indicus genetics, and not a result of crossbreeding different taurine breeds. It is important to note that interpretation of the results of this study are somewhat limited due to the small sample size utilized, however, to the authors’ knowledge, this is one of the first studies examining the changes that occur in skeletal muscle in response to anabolic implants, and the resulting relationship with breed type. Better understanding this relationship between cattle breed types and anabolic implant protocols will allow for management strategies to be developed so that cattle producers may further improve environmental and economic sustainability of their operations.

The steers used in this trial were part of a previously published performance trial (Reichhardt et al., 2021a). In brief, it was found that average daily over the course of the trial was greater in AN steers when compared with SG steers. Furthermore, numerically anabolic implants improved average daily gain by 7% in AN steers and 13% in SG steers. The SG-CON steers numerically gained the least amount of weight throughout the course of the trial. This translated into economic return increasing the most in AN-IMP steers, then AN-CON and SG-IMP steers, with the least amount of economic return for the SG-CON steers. Additionally, serum haptoglobin concentrations were altered by anabolic implants in a breed dependent manner with 28 d post-implanting AN-CON steers having the lowest circulating serum haptoglobin concentration (Reichhardt et al., 2021b).

Mammalian muscle fiber number is largely fixed at birth; therefore, hypertrophy of existing muscle fibers is the primary mechanism for postnatal growth (Hawke and Garry, 2001; Li et al., 2011; Yablonka-Reuveni, 2011). Hypertrophy does eventually require additional nuclei from satellite cells for muscle growth to occur (Hawke and Garry, 2001; Li et al., 2011; Yablonka-Reuveni, 2011; Dayton and White, 2014). Satellite cells are muscle precursor cells that proliferate, then differentiate and fuse with existing muscle fibers to support post-natal hypertrophy (Li et al., 2011). Differentiation and phenotypic maturation are necessary for satellite cells to fuse (Gonzalez et al., 2020). Markers of differentiation in skeletal muscle include increased expression of MYOD, MYF5, and myogenin, and decreased expression of PAX7 (Halevy et al., 2004; Yablonka-Reuveni et al., 2008; Yablonka-Reuveni, 2011). Furthermore, research has found that ¾ Bos taurus and ¼ Bos indicus crossbred animals possess more type IIa fibers and less type IIx fibers than purebred animals of either breed type (Wright et al., 2018), with type IIa fibers being known to have more satellite cells (Holterman and Rudnicki, 2005).

To better understand the relationship between cattle breed type and anabolic implants, the first group of genes investigated in this study was related to skeletal muscle differentiation. This study found that 10 d post-implanting, CON steers had a greater mRNA abundance of MYF5 compared with IMP steers. When primary bovine satellite cells are induced to differentiate and are treated with TBA, MYF5 mRNA abundance is increased by 4 h post-treatment when compared with control-treated cultures (Reichhardt et al., 2021b). However, by 12 h post-treatment, MYF5 mRNA abundance is drastically decreased when compared with that of control cultures (Reichhardt et al., 2021b). This suggests that TBA accelerates satellite cells through the myogenic lineage. In this study, the opposite was observed. This could have been due to differences in the time points assessed and also the inherent differences between in vitro and in vivo studies. Additionally, examining the PAX7:MYOD ratio can help determine the current activity within the satellite cell pool. In this study, it was found that SG steers had a greater PAX7:MYOD ratio than AN steers. This indicates that SG steers had a greater proportion of satellite cells remaining in the proliferation phase. Furthermore, CAPN6 mRNA abundance tended to be increased in SG-CON steers 2 d post-implanting. In mouse satellite cells, loss of function of CAPN6 results in accelerated differentiation into myotubes, suggesting that CAPN6 is a suppressor of satellite cell differentiation (Tonami et al., 2013). Taken together, these findings suggest that Bos indiucs influenced steers may have decreased satellite cell differentiation when compared with Bos taurus steers, however future work is warranted in this area to assess the relationship between cattle breeds and their satellite cell pools.

Protein turnover is intimately related to skeletal muscle growth as growth is defined as the difference between protein synthesis and protein breakdown (Owens et al., 1993). Mitogen activated protein kinase is a marker of cellular proliferation and an upstream regulator of the mTOR pathway via the MAPK/ERK pathway (Cargnello and Roux, 2011). Two days post-implantation, SG-CON steers had a greater mRNA abundance of MAPK than AN-CON steers. It is important to note that MAPK is primarily regulated by phosphorylation and dephosphorylations of the protein, which was not addressed in this study (Cargnello and Roux, 2011). Additionally, a tendency was observed in the current trial for CON steers to have increased abundance of MURF-1 when compared with IMP steers regardless of breed. Muscle RING finger protein-1 triggers skeletal muscle protein degradation by ubiquitination (Koyama et al., 2008). In cattle, it has been found that E2 promotes protein synthesis and decreases protein degradation (Kamanga-Sollo et al., 2010), with literature consistently demonstrating that E2 increases protein synthesis rates of fused bovine satellite cells in vitro (Kamanga-Sollo et al., 2010; Kamanga-Sollo et al., 2017). Overall, these findings suggest that the hormones present in anabolic implants may decrease protein breakdown regardless of cattle breed type. Additionally, CAST and CAPN1 can be used as identifiers of meat tenderness due to their relationship with protein degradation (Casas et al., 2006). Abundance of CAST was impacted by breed in the current trial, with AN steers having increased abundance compared with SG steers 2 d post-implanting, however by 10 d post-implanting the SG steers had numerically greater mRNA abundance of CAST compared with AN steers. Furthermore, it has been suggested that increasing Bos indicus percentage leads to an increase in CAST activity resulting in lower tenderness values (Ferguson et al., 2000). This may be explained by the fact that calpastatin has been found to be differentially controlled at different levels, including transcription and translation (Parr et al., 2004). Additionally, mRNA abundance can be influenced by the stability of the transcript and rate of rate of transcription (Greenbaum et al., 2002). Calpastatin has been found to be a long-lived protein, but the mRNA is rather short lived (Barnoy et al., 2000), suggesting that mRNA abundance and protein expression of calpastatin will not always be correlated from samples taken at the same time point.

Calpastatin is an inhibitor of CAPN1 (Koohmaraie, 1996), with research finding that as the percentage of Bos indicus influence increases, CAPN1 autolysis decreases (Ramos et al., 2018; Wright et al., 2018). The current trial found that CAST protein expression was affected by a breed × treatment interaction with SG-CON and SG-IMP steers having greater expression than AN-CON steers 2 d post-implanting, while 10 d post-implanting SG steers had greater expression of CAST than AN steers. This corroborates the finding from other research groups that increasing Bos indicus percentage leads to an increase in CAST activity (Ferguson et al., 2000). Collectively, these data suggest that both anabolic implants and cattle breed type may alter protein turnover early on in the feedlot stage of production, which may eventually lead to alterations in meat quality, specifically tenderness. Therefore, future work is necessary in this area to identify the relationship between cattle breed type, anabolic implants, and protein turnover.

Succinate dehydrogenase has been identified as a predictor of meat tenderness in beef (Morzel et al., 2008). Increasing the usage of anabolic implants and increasing the percentage of Bos indicus influenced genetics both independently raise concerns as they relate to meat quality (Montgomery et al., 2001; Elzo et al., 2012), especially tenderness, however, in the current trial skeletal muscle biopsies were taken early in the feedlot phase of production. Succinate dehydrogenase was affected by a breed × treatment interaction within the current trial, however, the LS Means were not different from one another, which is most likely explained due to the small sample size used in the trial. Nevertheless, AN-IMP had numerically greater abundance of SDHA compared with AN-CON steers, while SG-CON steers had a greater abundance of SDHA compared with SG-IMP steers. This suggests that anabolic implants may alter SDHA in a breed dependent manner. Succinate dehydrogenase is one of the mitochondrial enzymes of the TCA cycle in skeletal muscle, with SDHA being greater in oxidative fibers and lowest in glycolytic fibers (Peter et al., 1972). As previously discussed, ¾ Bos taurus and ¼ Bos indicus crossbred animals possess more type IIa fibers and less type IIx fibers (Wright et al., 2018). Type IIa fibers have been known to have greater SDHA activity than type IIx fibers in cattle (Picard and Gagaoua, 2020). These preliminary findings warrant further research to determine whether mitochondria and muscle fiber type are involved in the interaction between cattle breed type and anabolic implants.

The oxytocin receptor and VDR have been linked to both skeletal muscle growth and the physiological response of anabolic implants (Jager et al., 2011; Zhang et al., 2018; Reichhardt et al., 2021a). In this study, it was found that SG steers had increased abundance of OXTR in the LT. To the authors’ knowledge, the effect of cattle breed type on circulating oxytocin has yet to be investigated. However, oxytocin has been found to directly inhibit the proteolytic activities of the lysosomal and proteasomal systems in rat oxidative skeletal muscle by suppressing atrogene expression through stimulation of Akt/FoxO signaling (Costa et al., 2021). Furthermore, oxytocin has been found to increase fusion indexes in bovine satellite cells induced to differentiate (Zhang et al., 2018). The role that oxytocin and the OXTR are appearing to play in skeletal muscle growth suggests that oxytocin and the OXTR may partially explain the differences observed in skeletal muscle growth between the two cattle breed types. Additionally, it has been found that vitamin D and its receptor are required for adult mammalian skeletal muscle growth and maintenance (Ceglia, 2008). Previous work completed by our group has found that circulating serum 25-HydroxyVitamin D was affected by cattle breed type and anabolic implants, with non-implanted SG steers having lower concentrations than non-implanted AN steers (Reichhardt et al., 2021a). In this study, SG-CON steers had an increased mRNA abundance of VDR 10 d post-implanting than all other steers in the current trial. Most likely, this is explained due to the fact the SG-CON steers had a lower concentration of 25-HydroxyVitamin D, resulting in an upregulated abundance of the VDR. Additionally, in rats, it has been found that overexpression of the VDR stimulates skeletal muscle hypertrophy (Bass et al., 2020). This suggests a critical role for VDR in skeletal muscle growth. Additional studies are needed to clarify the role of VDR in bovine skeletal muscle growth.

Summary

In summary, the findings of this research suggest that anabolic implants and cattle breed type impact calpastatin expression and mRNA abundance associated with protein in the LT of feedlot steers. More specifically, anabolic implants and cattle breed type interact with each other to alter mRNA abundance of SDHA and CAST, while protein expression of CAST is impacted by this interaction with SG-CON and SG-IMP having greater expression than AN-CON steers. Future work is warranted to improve our understanding of interactions between cattle breed type and anabolic implants so that proper implant protocols can be developed to better improve environmental and economical sustainability without sacrificing meat quality.

Glossary

Abbreviations

CAPN1

µ-calpain

CAPN6

Calpain-6

CAST

Calpastatin

EEF1A2

eukaryotic elongation factor 1 alpha 2

E2

Estradiol

IGF-1R

insulin-like growth factor-1 receptor

LT

longissimus thoracis

MAPK

mitogen-activated protein kinase

MYOD

myoblast differentiation factor 1

MYF5

myogenic regulatory factor 5

MURF-1

muscle RING finger protein-1

OXTR

oxytocin receptor

PAX7

paired box transcription factor 7

SDHA

succinate dehydrogenase

SEM

standard error of the mean

STA

specific target amplification

TBA

trenbolone acetate

VDR

vitamin D receptor

Conflict of Interest Statement

The authors declare no real or perceived conflicts of interest.