Breast muscle white striping and serum corticosterone reduced in broilers exposed to laser environmental enrichment

Abstract

Genetic selection for breast yields and fewer days to market has inadvertent effects on broiler meat quality. Woody breast (WB) and white striping (WS) are pectoralis major myopathies prevalent in commercial broilers. Effects of voluntary exercise on these disorders, specifically, are unknown. A second-generation laser enrichment device shown to induce activity in Ross 308 and 708 birds was implemented using 1,360 Ross 708 broilers randomly assigned to laser enrichment or control for 49 d. Laser-enriched birds were exposed to 6-min laser periods 4 times daily. Seventy focal birds were gait and contact dermatitis scored weekly. Blood was collected wk 5 to 7 from 56 broilers for serum corticosterone, myoglobin, and troponin. Seventy broilers were sampled for breast muscle width, fillet dimensions, and WB and WS at wk 6 and 7. One and 2-day postmortem, fillet compression force and water-holding capacity were measured. Serum corticosterone was reduced by up to 21% in laser-enriched birds wk 5 to 7 (P < 0.01). Serum myoglobin was increased in laser-enriched broilers by 5% on wk 5 (P < 0.01) but increased in control birds wk 6 to 7 by up to 13% (P < 0.01). Serum troponin was reduced in laser-enriched broilers by 9% at wk 5 (P < 0.01). Laser exposure increased breast width and fillet weight at d 42 by 1.08 cm (P < 0.05) and 30 g (P < 0.05). At d 49, fillet height was increased 0.42 cm in laser-enriched birds (P < 0.05). Laser enrichment reduced severe WS incidence at d 42 by 24% (P < 0.05) and on d 49 by 15% (P < 0.10). Severe WB score was numerically reduced by 11% in laser enrichment on d 42 and 18% on d 49 (P > 0.05). Water-holding capacity was improved in laser-enriched breasts (P < 0.01) and expression of myostatin and insulin-like growth factor 2 were increased on d 49 (P ≤ 0.01. Laser enrichment reduced markers of stress and muscle damage while improving breast muscle quality and is therefore a potential effective enrichment for commercial broilers.

INTRODUCTION

The broiler chicken pectoralis major myopathies known as woody breast (WB) and white striping (WS) have become dominant meat quality concerns in today's poultry industry (Baldi et al., 2020). A wide breadth of research has focused on these abnormalities, particularly WB, investigating potential causes, identifying biomarkers, determining meat quality effects, and studying interventions to prevent or reduce severity (Kuttappan et al., 2016). A single, irrefutable cause of either myopathy has not been established in the literature, but heavier broilers and thicker, heavier breast fillets are consistently linked with both disorders (Mudalal et al., 2014; Kuttappan et al., 2017a). Downgrades due to WB and WS have been estimated to cost the U.S. industry over $200 million annually and concerns for the welfare of WB-affected broilers are widely expressed, hence there is a sense of urgency and relevance in controlling or preventing these myopathies (Kuttappan et al., 2016).

WB is primarily a texture concern associated with hard, rigid fillets and connective tissue replacement of muscle fibers and is also frequently described as exudative and pale in color, while WS is a primarily visual issue hallmarked by infiltration of white striations of fat and connective tissue parallel to muscle fibers (Tijare et al., 2016). Breast fillets affected by WB show higher razor and shear force than normal, while both myopathies have been associated with reduced meat color and water-holding capacity, higher ultimate pH, and increased drip and cook loss (Trocino et al., 2015; Soglia et al., 2016; Tijare et al., 2016). A general loss of functional protein replaced by fat and connective tissue characterize both conditions (Kuttappan et al., 2017b; Soglia et al., 2017). This is an outcome of breast muscle fiber hypertrophy, enhanced through genetic selection for breast muscle yield in commercial broilers. Hypertrophy exceeds connective tissue and circulatory support needed for healthy muscle growth and repair mechanisms. Resulting muscle cell degradation and death lead to fibrosis and adiposis, or the replacement of muscle fibers with interstitial connective tissue and fat (Velleman, 2019).

Histologically, fast-growing broilers with concurrent WB and WS show increased muscle fiber size, fibrosis, and adiposity, but decreased capillary density compared to both slow-growing broilers and fast-growing birds without WB and WS (Pampouille et al., 2019). In WB specifically, this lack of sufficient capillary to muscle fiber ratio means reduced blood supply, therefore diminished delivery of oxygen and nutrients leading to the well-established hypoxic and oxidative stress state in WB-affected muscle (Lilburn et al., 2019; Maharjan et al., 2020). Multiple biomarkers of abnormal metabolism and tissue necrosis have been identified: Mutryn et al. (2015) characterized a variety of changes in gene expression of myopathy-affected muscle indicating hypoxia, attempted fiber-type switching, and oxidative stress. Papah and Abasht (2019) observed altered gene expression in WB-affected tissue showing dysregulated lipid metabolism. Soglia et al. (2016) observed higher Na and Ca in WB and WS-affected breasts, along with lower muscle pyruvate kinase and creatine kinase. Significantly decreased glycogen content and markers of oxidative stress, altered glucose metabolism, and tissue degradation were reported by Abasht et al. (2016).

However, identifying potential biomarkers in live, growing broilers is of interest from an early detection standpoint. Shifts in circulation of myopathy-affected broilers have been detected. Maharjan et al. (2020) observed elevated plasma metabolites indicative of vascularity problems, localized hypoxia, and muscle atrophy. Kong et al. (2021) identified multiple plasma proteins and metabolites that differed in WB-affected commercial broilers, possible WB predictors, as early as 4 wk of age. Santos et al. (2021) detected increased plasma creatine kinase, aspartate transaminase, and lactate dehydrogenase, believed to be indicative of muscle damage, in conventional broilers with the greatest occurrence of breast myopathies. Circulating corticosterone (CORT) has been reported to be significantly higher in birds affected by WB and concurrent WB and WS, identifying CORT as a biomarker and indicating a stress state of myopathy-affected birds. This outcome highlights a myopathy-related detraction from animal welfare (Kang et al., 2020).

These muscle disorder effects on broiler welfare and behavior are under-studied. Work by Gall et al. (2019) linked WB with pulmonary disease, dorsal recumbency, and mortality as birds neared market weight. The authors summarized that broilers with WB cannot relax the pectoralis major muscle, therefore the pectoralis minor cannot sufficiently contract to raise the wing and right the bird, leaving them vulnerable to dorsal recumbency and death. Ross et al. (2020) did not observe an effect of WB presence or severity on broiler activity measured through automated tracking software, but the authors concluded that a more detailed behavior analysis is warranted. A recent examination of environment and animal management effects on WB by Che et al. (2022) showed that transportation and processing variables including dead on arrival, loading time, length of time in lairage, and season (summer vs. winter) were associated with increased WB occurrence. This is a critical analysis showing that not only can WB negatively influence animal welfare, but management impacting animal welfare in turn influences WB.

An analysis of voluntary exercise effects on WB and WS is largely missing from the current literature. Yin et al. (2015) showed that inducing exercise in broiler chickens increased muscle regulatory factor expression in breast and thigh muscles as well as enhanced muscle fiber diameter. The authors concluded this was an outcome of the well-understood effects of exercise on hypertrophy, but also because exercise stimulates capillary development leading to increased nutrient and oxygen delivery to skeletal muscle (Fluck, 2006). Mattioli et al. (2017) reported that serum creatine kinase, indicative of muscle damage, as well as worsened oxidative status measured through serum reactive oxygen species were increased in fast and slow-growing broilers forced to exercise 1 h daily. The authors summarized that forcing exercise in fast-growing broilers induces oxidative stress and negatively impacts animal welfare, but meat quality outcomes were not collected. Previous work by the current authors utilizing a visual form of environmental enrichment for broilers, a laser device activated 4 times daily for 4-min periods, increased voluntary physical activity, significantly increased breast muscle width, and numerically reduced WB score at 6 and 7 wk of age compared to the control without negatively impacting animal welfare outcomes (Meyer et al., 2021a,b). However, these studies did not include lab-based measurements of meat quality, an analysis of WS prevalence, or serum biomarkers of muscle damage and stress.

Therefore, the aims of the current work were to introduce a second-generation laser enrichment device to Ross 708 broilers for lengthened, 6-min exposure periods, to measure WB and WS occurrence and severity as well as breast muscle gene expression, stress and muscle damage serum markers, and outcome-based animal welfare indicators.

MATERIALS AND METHODS

All live bird procedures were approved by the Iowa State University Institutional Animal Care and Use Committee, IACUC #19-322.

Animals

A total of 1,360 mixed sex Ross 708 broilers were obtained from Welp Hatchery (Bancroft, IA) and transported to the Iowa State University Poultry Research and Teaching Farm on day of hatch for a 49-day grow-out. Chicks were randomly assigned to floor pens and weighed as a pen upon arrival (average starting BW 44.07 ± 0.92 g). Seventy birds were randomly selected on d 0 as focal birds (n = 5 birds/pen; 7 pens/treatment), identified with wing-bands, and marked on the back with unique colors of livestock spray paint reapplied weekly.

Housing and Feeding

Birds were housed in 40 pens of 34 birds/pen (1.22 by 2.44 m/pen) across 2 rooms in barn. Approximately 10 cm of fresh wood shavings were provided as bedding over concrete flooring, and PVC pipe dividers with mesh walls (1.22-m height) separated pens. One room contained 20 laser-enriched pens (exposed to laser device), and the other contained 20 control pens (no laser exposure), with an anteroom separating so no laser exposure to the control was possible. Environmental conditions and management were kept consistent across rooms, with birds gradually adjusted from 24 h light on d 0 (30–40 lux) to 20 h light (20–30 lux) d 8 to 49. Chicks were brooded with 2-heat lamps/pen (22.9-cm reflectors with porcelain socket) using 125-W heat bulbs (Sylvania, Wilmington, MA) for the first week of life. Ambient temperatures were maintained at approximately 32°C to 34°C wk 1 to 2, 27°C to 29°C wk 3 to 4, and 21°C to 23°C wk 5 to 7; high and low temperatures and humidity were monitored daily using a digital hygrometer and recorded by research staff. Broilers were fed standard mash diets formulated according to Ross 708 guidelines for starter (d 0–14), grower (d 14–28), finisher 1 (d 28–42), and finisher 2 (d 42–49) performance phases (Table 1A–D). Birds were fed ad libitum out of 2 hanging feeders/pen (Brower Equipment, Houghton, IA) raised to accommodate bird height. Water was provided from a hanging nipple water line (approximately 4 nipples/pen; source: Story County rural water) raised as needed to accommodate bird height.

Table 1.

(A–D) Diet formulations and proximate analyses fed to Ross 708 broilers over (A) starter, (B) grower, (C) finisher 1, and (D) finisher 2 performance periods.

A
Ingredients (%)	Starter diet
Corn	55.30
Soybean meal 48	37.36
Soy oil	1.97
Salt	0.40
DL methionine	0.33
Lysine HCl	0.25
Threonine	0.15
Limestone	1.20
Dicalcium phosphate	2.01
Choline chloride 60	0.40
Vitamin premix1	0.63
Calculated values (%)
Fat	4.54
Crude protein	23.15
ME (kcal/kg)	3000
Digestible lysine	1.31
Digestible arginine	1.39
Digestible threonine	0.93
Analyzed values
Dry matter	87.55
Crude fat	4.86
Crude protein	22.83
Gross energy (kcal/kg)	3877
B
Ingredients (%)	Grower diet
Corn	58.69
Soybean meal 48	33.40
Soy oil	2.98
Salt	0.40
DL methionine	0.30
Lysine HCl	0.23
Threonine	0.15
Limestone	1.01
Dicalcium phosphate	1.81
Choline chloride 60	0.40
Vitamin premix1	0.63
Calculated values (%)
Fat	5.59
Crude protein	21.50
ME (kcal/kg)	3100
Digestible lysine	1.19
Digestible arginine	1.28
Digestible threonine	0.87
Analyzed values
Dry matter	87.65
Crude fat	5.84
Crude protein	20.69
Gross energy (kcal/kg)	3940
C
Ingredients (%)	Finisher 1 diet
Corn	62.69
Soybean meal 48	28.55
Soy oil	4.00
Salt	0.40
DL methionine	0.30
Lysine HCl	0.22
Threonine	0.15
Limestone	1.02
Dicalcium phosphate	1.65
Choline chloride 60	0.39
Vitamin premix1	0.63
Calculated values (%)
Fat	6.67
Crude protein	19.50
ME (kcal/kg)	3200
Digestible lysine	1.06
Digestible arginine	1.14
Digestible threonine	0.79
Analyzed values
Dry matter	87.58
Crude fat	7.15
Crude protein	19.06
Gross energy (kcal/kg)	3959
D
Ingredients (%)	Finisher 2 diet
Corn	66.28
Soybean meal 48	25.46
Soy oil	3.55
Salt	0.40
DL methionine	0.25
Lysine HCl	0.25
Threonine	0.15
Limestone	1.10
Dicalcium phosphate	1.54
Choline chloride 60	0.40
Vitamin premix1	0.63
Calculated values (%)
Fat	6.34
Crude protein	18.30
ME (kcal/kg)	3200
Digestible lysine	1.01
Digestible arginine	1.05
Digestible threonine	0.75
Analyzed values
Dry matter	87.54
Crude fat	6.71
Crude protein	17.63
Gross energy (kcal/kg)	3966

¹Vitamin and mineral premix provided per kg of diet: selenium 250 μg; vitamin A (retinyl acetate) 8,250 IU; cholecalciferol (vitamin D₃) 2,750 IU; α-tocopherol acetate (vitamin E) 17.9 IU; menadione 1.1 mg; vitamin B₁₂ 12 μg; biotin 41 μg; choline 447 mg; folic acid 1.4 mg; niacin 41.3 mg; pantothenic acid 11 mg; pyridoxine 1.1 mg; riboflavin 5.5 mg; thiamine 1.4 mg; iron 282 mg; magnesium 125 mg; manganese 275 mg; zinc 275 mg; copper 27.5 mg; iodine 844 μg.

Laser Device and Periods

Ten patented enrichment devices (updated from initial laser designs containing 2 lasers previously described by Meyer et al., 2019, 2021b) were designed to cover 2 neighboring pens. Each laser device consisted of 5 synchronized red lasers projected from a flat-faced mobile unit mounted on a 2.4-m tall wooden structure placed above 2 pens. The lasers projected in the direction of both pen floors and moved slowly in a random, circular pattern for 6-min “laser periods” at: 05:30, 11:30, 17:30, and 23:30 daily for the trail entirety. Researchers and staff did not enter laser or control rooms of the barn during the entire 4 h daily containing the 6-min laser periods.

Contact Dermatitis

Focal birds (n = 70) were scored weekly for breast blister and footpad dermatitis presence. Scores were conducted at the same day and time weekly by 1 researcher throughout the study. Breast blisters were scored present/absent based on methods by Greene et al. (1985), where blisters would be considered present when equal to or larger than 1.30 cm² or when there were scabs on breast skin. Footpad dermatitis was scored pass/fail using the American Association of Avian Pathologists Paw Scoring system (AAAP, 2015).

Gait Score

Focal birds (n = 70) were gait scored weekly using methods previously described by the current authors (Meyer at al., 2021a). The same researcher conducted all gait scores. Birds were removed from their pen in groups of 5 (5 focal birds/pen) and placed on a plywood runway in groups of 2 to 3. Briefly, broilers walked 1.50 m independently or were gently encouraged to walk by tapping on the bird's back side. Each bird was considered to have completed the gait score when both feet had crossed the 1.50 m mark. Gait score was adapted from National Chicken Council guidelines (National Chicken Council, 2020): 0 indicated the ability to walk 1.5 m with no signs of lameness, 1 indicated the ability to walk 1.50 m but showed unevenness in steps or sat down at least once, and 2 indicated a bird that did not walk 1.50 m.

Performance

All birds were weighed as a pen at the end of each performance phase to calculate weight gain and average body weight, and feed intake (FI) was recorded throughout. Feed conversion ratio (FCR; kg:kg) was calculated by pen and averaged per bird each performance phase: starter (wk 1–2), grower (wk 2–4), finisher 1 (wk 4–6), and finisher 2 (wk 6–7).

Serum Analysis

Blood samples were taken from 56 randomly selected broilers (28 birds/treatment) on wk 5, 6, and 7 at 12:00 pm on the same day each week by 2 researchers. Birds were calmly caught in their home pen, carried right-side-up, and held lying flat against a table by 1 researcher. Blood was drawn immediately form the brachial wing-vein by the other researcher. Blood was collected into serum separation tubes (BD Vacutainer, Franklin Lakes, NJ) and centrifuged at 1,000 × g for 15 min. Serum was collected and separated for CORT, myoglobin, and troponin type I 2a (troponin) ELISA. The CORT assay was conducted using serum diluted 1:10 in sterile PBS, and myoglobin and troponin assays were conducted using serum diluted 1:4 in sterile PBS. All ELISA were completed in duplicate following manufacturer protocols for the Chicken CORT Competitive ELISA, Chicken Myoglobin Competitive ELISA, and Chicken Troponin Competitive ELISA, respectively (MyBioSource, San Diego, CA).

Breast Muscle Yield and Meat Quality

At d 42 and 49, a subset of 70 birds (35/treatment; focal birds at d 49) was measured for width of the complete pectoralis major muscle using a seamstress tape measure. Then the same 70 birds each timepoint were harvested and the left breast fillet was dissected and weighed. One researcher assigned each fillet a WB score using a 3-point tactile scale based on the scoring system by Kuttappan et al. (2016). Briefly, normal breasts retained flexibility throughout, moderate WB were hardened through cranial region, and severe WB maintained no flexibility and were rigid throughout. WS score was assigned on the same fillets by the same researcher using a 3-point visual scale. Normal fillets showed no WS, moderate WS had visible, thin white lines covering less than half of the fillet, and severe WS had thin to thick white lines covering more than half of the fillet (modified from Kuttappan et al., 2016).

Scored fillets were placed in 4°C cold storage overnight and mechanical compression force was measured on raw samples 1-day postmortem using methods by Sun et al. (2018). Briefly, each fillet was compressed 3 times on different points across the cranial region using a 6.5 mm flat probe on a TA.XT Plus Texture Analyzer (Texture Technologies Corp., Surrey, UK). Trigger force of 5 g was used, probe height was set at 55 mm, pre- and postprobe speeds were 10 mm/s, and the test speed was 5 mm/s. Mean compression force of the 3 tests per fillet was used for data analysis. Following texture analysis, fillets were returned to cold storage overnight and analyzed for water-holding capacity 2-day postmortem. Ten grams of minced tissue in duplicate were centrifuged at 25,000 × g for 20 min, supernatant was removed, and weight of the remaining solid was measured. Percent centrifugation loss was calculated, a negative indicator of water-holding capacity of the meat, as follows:

$$\%\text{Centrifugation}\text{loss}=\left[\left(\text{initial}\text{weight}-\text{post}\text{-}\text{spin}\text{weight}\right)/\left(\text{initial}\text{weight}\right)\right]\times 100$$

Breast mRNA Isolation and qPCR

On d 42 and 49 following WB and WS scoring, the cranial region (approximately 52 cm² segment) from a subset of right breast fillets was collected (n = 30 samples/timepoint). Samples were placed in RNAlater Stabilization Solution (Invitrogen, Waltham, MA) overnight, then removed from the solution and placed in −80°C for future analysis. RNA was isolated from 60 breast muscle samples across both timepoints using the RNeasy Fibrous Tissue Kit (Qiagen, Germantown, MD). Fifteen breast muscle samples/treatment/timepoint were therefore analyzed. Isolated RNA was DNAse treated and diluted to 50 ng/uL for One-Step SYBR Green quantitative PCR (Qiagen, Germantown, MD), tested against 6 muscle-growth and regulation primers of interest and the ubiquitously expressed chicken housekeeping gene, 28s. Genes of interest included: myoblast determination protein 1 (MyoD); myogenin (MyoG); muscle regulatory factor 4 (MRF4); myostatin (MSTN), insulin-like growth factor 1 (IGF1); and insulin-like growth factor 2 (IGF2). Means are reported as adjusted Ct values, calculated as follows:

$$40-\left[\left(\text{MeanC}{\mathrm{t}}_{\text{test}\text{gene}}\right)\right]+\left[\left(\text{Media}{\mathrm{n}}_{28\mathrm{s}}\right)-\left(\text{MeanC}{\mathrm{t}}_{28\mathrm{S}}\right)\right]$$

Statistical Analysis

Because laser treatment was applied to an entire room, room within barn was confounded by treatment, hence was not included in the model, but research conditions were kept as identical as possible between rooms. All data were analyzed using SAS version 9.4 (SAS Institute Inc., 2016; SAS Institute Inc., Carey, NC). PROC UNIVARIATE was used to assess the distribution of data prior to analysis. Contact dermatitis occurrence and gait scores greater than 0 were too low to analyze statistically, hence data are presented descriptively as number of affected birds per treatment by week. WB and WS scores were analyzed as counts of scores by treatment using a likelihood ratio chi-square and are reported as percent distribution of scores by treatment and timepoint. The remaining data were analyzed using PROC MIXED with the main effect of treatment, and qPCR data included the effect of day of sampling and the treatment by day interaction. LSMeans and SEM are reported, a value of P ≤ 0.05 was considered significant and P ≤ 0.10 is reported as a trend.

RESULTS

Contact Dermatitis

No breast blisters were detected on any birds in the study. Foot pad dermatitis was absent wk 1 to 5. On wk 6 and 7, 4 out of 35 control focal birds (11.4%) and 2 out of 35 laser-enriched focal birds (5.7%) scored fail for footpad dermatitis.

Gait Score

All focal birds received a gait score of 0 wk 1 to 5. On wk 6, 4 out of 35 control birds (11.4%) and 2 out of 35 laser-enriched focal birds (5.7%) received a score of 1. On wk 7, one control bird (2.9%) received a score of 1 and 4 of 35 control focal birds (11.4%) received a gait score of 2, while 2 laser-enriched focal birds (5.7%) received a score of 1.

Performance

There were no significant treatment effects on FI, BW gain, BW, or FCR in any performance period (Table 2).

Table 2.

Ross 708 broiler performance outcomes averaged per bird by main effect of treatment.

Performance outcome	Control	Laser	SEM	P value
Feed intake, kg
Starter (g)	471	472	8.88	0.947
Grower	1.55	1.53	0.01	0.340
Finisher 1	2.37	2.35	0.02	0.457
Finisher 2	1.37	1.38	0.02	0.748
Body weight gain, kg
Starter (g)	394	395	3.5	0.849
Grower	1.07	1.06	0.01	0.475
Finisher 1	1.39	1.37	0.02	0.512
Finisher 2	0.96	0.94	0.02	0.425
Body weight (kg)
d 14	0.44	0.44	0.003	0.895
d 28	1.50	1.50	0.01	0.816
d 42	2.88	2.86	0.02	0.564
d 49	3.77	3.81	0.03	0.362
FCR
Starter	1.20	1.19	0.02	0.960
Grower	1.45	1.45	0.01	0.975
Finisher 1	1.71	1.71	0.02	0.961
Finisher 2	1.43	1.45	0.03	0.714

Serum

Laser treatment decreased serum CORT at all timepoints analyzed. Laser-enriched broiler serum CORT was decreased by 7.3% at wk 5 of age (P < 0.001), by 9.9% at wk 6 (P = 0.071), and by 21% at wk 7 (P < 0.001) compared to the control (Table 3). Serum myoglobin was increased in laser-enriched broilers at wk 5 by 5.4% compared to the control (P < 0.001) but decreased in the laser treatment wk 6 and 7, maintaining a 7.6% decrease at wk 6 (P < 0.001), and a 12.9% decrease at wk 7 of age (P < 0.001) compared to the control (Table 3). Serum troponin was affected by treatment at wk 5, where laser-enriched broilers showed a 6.8% decrease (P = 0.005). Serum troponin trended to be impacted by treatment at wk 7, where laser-enriched birds showed a 2.2% increase (P = 0.062) compared to control birds (Table 3).

Table 3.

Ross 708 broiler serum corticosterone, myoglobin, and troponin collected from 28 birds/treatment weekly, wk 5 to 7¹.

Serum measure	Control	Laser	SEM	P value
Corticosterone (ng/m)
Wk 5	28.93a	26.82b	0.10	<0.001
Wk 6	37.29	33.59	1.32	0.071
Wk 7	28.02a	22.14b	0.13	<0.001
Myoglobin (ng/mL)
Wk 5	184.31b	194.34a	1.27	<0.001
Wk 6	210.35a	194.27b	1.76	<0.001
Wk 7	167.46a	145.88b	3.87	<0.001
Troponin (pg/mL)
Wk 5	3697a	3447b	62.48	0.005
Wk 6	3131	3174	32.68	0.339
Wk 7	3172	3241	26.91	0.062

¹Different superscript letters (a,b) indicate treatment differences within the same timepoint P ≤ 0.05.

Breast Muscle Yield and Meat Quality

Pectoralis major width was increased in laser-enriched broilers by 1.08 cm, a 4.5% increase, compared to control birds at d 42 (P = 0.003) and was not affected by treatment at d 49 (Table 4). Breast fillet weight was increased by 30 g, an 8.4% increase, in laser-enriched broilers at d 42 (P = 0.054) and was not impacted at d 49 (Table 4). The height of the left breast fillet was not altered at d 42 but was increased by 0.42 cm or 9.7% at d 49 (P = 0.039; Table 4). Percent centrifugation loss, a measure with an inverse relationship with water-holding capacity of meat, was not affected by treatment at d 42 but was positively decreased in laser-enriched breast fillets at d 49 by 19.5% (P = 0.008). Mechanical compression force of fillets was not affected by treatment at either timepoint analyzed (Table 4).

Table 4.

Breast muscle yield and meat quality outcomes analyzed with the main effect of treatment¹: breast width, breast weight and height, compression force, and water-holding capacity².

Breast muscle outcome	Control	Laser	SEM	P value
Live bird measure
Breast width, cm
d 42	24.17b	25.25a	0.25	0.003
d 49	25.99	26.47	0.27	0.208
Breast fillet measure
Fillet weight, kg
d 42	0.36b	0.39a	0.01	0.054
d 49	0.48	0.48	0.01	0.896
Fillet height, cm
d 42	4.39	4.23	0.15	0.228
d 49	4.34b	4.76a	0.14	0.039
Compression force, g (1-day postmortem)
d 42	2662	2705	127.5	0.810
d 49	3470	3754	147.6	0.178
Centrifugation loss, % (2-day postmortem)
d 42	5.50	5.58	0.46	0.903
d 49	5.51a	4.43b	0.28	0.008

¹Different superscript letters (a,b) indicate treatment differences within the same timepoint P ≤ 0.05.

²Negative indicator of water-holding capacity.

WB scores were not affected by treatment at wk 6 or 7, but numerical shifts existed. There was a 14.2% increase in moderate scores and an 11.5% decrease in severe scores in laser-enriched broiler breasts at d 42 (P = 0.41; Figure 1A and B). At d 49, there were 13.6% more moderate scores and 18.4% fewer severe scores observed in laser-enriched broiler breasts compared to the control (P = 0.24; Figure 2A and B). WS scores were affected by treatment at d 42, where laser-enriched broiler breasts received 23% greater scores of moderate and 24% fewer scores of severe than the control (P = 0.04; Figure 3A and B). This shift in scores continued through d 49, where laser-enriched breasts received 22.4% more moderate scores and 15.4% less severe scores compared to the control (P = 0.08; Figure 4A and B).

Figure 1.

Ross 708 broiler woody breast palpation scores conducted on left fillets at d 42. Scores are presented as percent distribution by treatment (P = 0.41).

Figure 2.

Ross 708 broiler woody breast palpation scores conducted on left fillets at d 49. Scores are presented as percent distribution by treatment (P = 0.24).

Figure 3.

Ross 708 broiler visual white striping scores conducted on left fillets at d 49. Scores are presented as percent distribution by treatment (P = 0.04).

Figure 4.

Ross 708 broiler visual white striping scores conducted on left fillets at d 49. Scores are presented as percent distribution by treatment (P = 0.08).

Breast Muscle Myogenic Gene Expression

Treatment affected expression of MSTN (P = 0.01), with breast tissue sampled from laser-enriched broilers showing 2% upregulated expression on d 42 and 6.5% upregulated expression on d 49 compared to control birds (Figure 5A and B). Treatment also affected relative expression of IGF2 (P = 0.001), with 3% increased expression on d 42 and 10% increased expression on d 49 in laser-enriched broiler breast tissue vs. the control (Figure 5A and B). Day of sampling was significant for expression of MyoG (P < 0.001), where relative expression was increased by 11% on d 49 vs. d 42, regardless of treatment. Day of life also affected MSTN expression (P = 0.001), with 5% reduced expression on d 49 compared to d 42. There was a trend for an effect of day of breast sampling on MRF4 expression (P = 0.06), with 4% increased expression on d 49 compared to d 42. There was a trend for a day by treatment interaction for IGF2 expression alone (P = 0.08).

Figure 5.

Expression of 6 myogenic genes of interest reported as adjusted Ct values relative to the chicken housekeeping gene, 28s. qPCR was performed using RNA extracted from broiler breast tissue at (A) d 42 and (B) d 49, and values are reported as LSMeans by day with the main effect of treatment. Different letters indicate means that differ significantly within a gene (P ≤ 0.05).

DISCUSSION

Pectoralis major myopathies, while inarguably meat quality concerns, are widely believed to impact broiler welfare. We hypothesized that factors influencing broiler welfare such as environment and husbandry may also induce myopathy. Briefly, a general physiological response to acute environmental stress, such as bird handling, is increased hypothalamic-adrenal axis activity and elevated circulating glucocorticoids aimed to regulate metabolism (Fallahsharoudi et al., 2015). The dominant adrenal glucocorticoid released due to adrenocorticotropic hormone and corticotropin-releasing hormone in chickens is CORT, previously shown to increase in circulation as a result of stressors such as increased temperature, restraint, transport, greater stocking density, and high ammonia levels (Scanes, 2016). This is therefore one of the most common physiological parameters used to measure stress, and serum CORT has been validated to most accurately indicate circulating CORT levels in broilers (Weimer et al., 2018). Relative to muscle biology, glucocorticoids induce protein catabolism and degradation of skeletal muscle (Scanes, 2016).

Elegant work by Kang et al. (2020) showed higher circulating CORT levels in WB and WS-affected birds than unaffected birds, and downregulated gene expression of glucocorticoid-receptors in affected breast muscle, indicating a chronic stress state in myopathic birds. The authors suggested that the elevation of glucocorticoids, which function to break down muscle protein to provide substrates and prioritize glucose production in response to stress, induces muscle atrophy and may then trigger WB and WS. Kang et al. (2020) also identified CORT as a potential biomarker for these disorders. In the current work, broilers provided with laser enrichment had significantly decreased serum CORT consistently wk 5 to 7 of life compared to control broilers without laser exposure. Reduced serum CORT has been previously reported as a positive effect of auditory environmental enrichment in young chicks, but not adult broilers by Hafizah et al. (2015). Aviary-housed laying hen blood CORT was decreased when hens were provided with pumice stone or alfalfa hay physical enrichments (Son et al., 2022). Broiler plasma CORT was reduced in birds reared with UV light vs. those raised under LED light control, leading the authors to conclude that UV-enriched environments reduce bird stress state (House et al., 2020). Therefore, while sensory and physical forms of enrichment have previously been linked with reduced CORT, and WB and WS myopathies have been associated with increased circulating CORT, the current work is novel in simultaneously measuring reduced myopathy and lower serum CORT as a direct result of environmental enrichment.

Potentially, the visual enrichment provided daily improved animal welfare state through reducing stress, which in turn prevented severe myopathy and improved breast meat quality, measured through improved WB and WS scores. Contact dermatitis and gait scores were not altered, but number of birds affected with footpad dermatitis was numerically reduced in the laser treatment at wk 6 and 7, and no laser-enriched broilers ever received a gait score of 2, compared to 4 control birds receiving this score at wk 7. These outcomes lend support to the conclusion that laser enrichment positively impacted welfare when considered with the CORT data. As the enrichment device utilized here also increased broiler active behavior (M. M. Meyer et al., unpublished data), we must also consider potential exercise effects on breast muscle biology.

Skeletal muscle is understood to be a dynamic tissue that adapts to physical exercise in multiple ways, including increased substrate and oxygen delivery to the tissue along with activated glycolytic and oxidative metabolism. The muscle achieves increased nutrient demands through vasodilation, activated in part by nitric oxide which increases up to 200% as a result of exercise (Suhr et al., 2013). Nitric oxide's role as a potent vasodilator has been long-established in humans (Radegran and Saltin, 1999), and more recently, dietary supplements have been introduced to maximize this effect (Sylla et al., 2018). Recent work by the current authors showed that supplementing an inositol-stabilized arginine silicate ingredient known to boost circulating nitric oxide levels in humans to broiler chickens numerically reduced WB severity, concluded to be an outcome of vasodilation (M. M. Meyer and E. A. Bobeck, unpublished data) Therefore, we speculate that the reduced severity in myopathies observed in the current work may also be considered an outcome of exercise-induced vasodilation providing oxygen and nutrients to the growing breast muscle, preventing hypoxia and oxidative stress characteristic to these disorders.

In terms of molecular analysis, 2 genes showed upregulated expression in laser-enriched broiler breast tissue: MSTN and IGF2. Myostatin is well-known to be a negative regulator of skeletal muscle growth and its expression has been shown to be reduced in the breast muscles of commercial broiler chickens compared to a lower yielding, slower growing breed (Dou et al., 2018). Lassiter et al. (2019) reported that breast tissue from high feed efficiency male broilers showed downregulation of the myostatin pathway compared to a lower feed efficiency divergent line; the authors concluded that these changes in gene expression support muscle development and protein synthesis. Decreased expression of MSTN is believed to be a result of genetic selection for fast growth rate and large breast muscle size, and the upregulated MSTN observed in laser-enriched breast tissue in conjunction with reduced WS and WB may therefore be an unexpected, positive outcome indicating altered, potentially more healthy regulatory mechanisms in breast muscle growth. The concurrent upregulation of IGF2 expression may support this hypothesis, as IGF2 has been shown to be increased in fast-growing broiler breast muscle previously (Zhang et al., 2019) linking it with positive, posthatch muscle growth as well as embryonic muscle development (Vaccaro et al., 2022).

While exercise is linked to improved leg heath in broiler chickens, whether induced through forced locomotion or an enriched environment (Reiter and Bessei, 2009; Guz et al., 2021), physical activity has also been linked with reduced growth performance and indicators of muscle damage and oxidative stress (Mattioli et al., 2017; Guz et al., 2021). The worsened antioxidant status and increased serum creatine kinase activity observed by Mattioli et al. (2017) in fast-growing broilers forced to exercise was believed to be evidence of muscle protein turnover and metabolic stress in characteristically inactive birds. However, laser enrichment induced voluntary activity without impacting growth performance, and has previously been shown to stimulate locomotion while improving Ross 308 (Meyer et al., 2019) and Ross 708 (Meyer et al., 2021b) performance. We have therefore speculated that laser enrichment-driven exercise does not demand energy allocation to a level that detracts from protein accretion. Further, breast muscle yields were increased in laser-enriched broilers in the current study, measured through both width and weight, at 6-wk harvest.

Serum myoglobin and troponin were analyzed to determine the presence of muscle damage and to identify potential biomarkers to WB and WS. Myoglobin is a cardiac and skeletal muscle-specific protein released during muscle injury or damage (Brancaccio et al., 2010; Gondal et al., 2021), and troponin I type 2a is a fast-twitch skeletal muscle-specific protein involved the troponin complex, which regulates calcium-binding for muscle contraction (Brotto et al., 2005), also indicative of muscle damage when present in serum. Neither of these proteins have been previously studied in terms of broiler muscle damage, specifically. Laser-enriched broilers showed increased serum myoglobin at 5 wk of life but maintained reduced myoglobin wk 6 to 7 of life compared to the control, while laser-enriched broilers had reduced serum troponin at wk 5 of life compared to the control. It is unclear why myoglobin was increased while troponin was decreased at wk 5 alone in laser-enriched broiler serum. Severity of WB scores were numerically reduced at both wk 6 and 7 timepoints, but breast tissue was not sampled and scored for myopathy at wk 5. Further examination with breast scoring timepoints earlier in grow-out is warranted to determine if these proteins may serve as potential biomarkers. However, based on concurrent improved WS and WB scores with reduced circulating myoglobin at 6 and 7 wk of life, myoglobin measured in broiler serum may serve as an indicator of myopathy in birds nearing market weight.

In summary, these data provide a unique insight into the effects of environmental enrichment and exercise on both stress and breast muscle disorders in fast-growing broilers. Voluntary locomotion stimulated through visual enrichment lowered circulating CORT without impacting outcome-based measures of animal welfare or growth performance. Further, laser enrichment increased breast muscle yields at 6 wk of age and reduced WS severity in breast fillets at 6 and 7 wk harvest. WB scores were numerically improved at 6 and 7 wk while water-holding capacity of laser-enriched broiler fillets was improved at 7 wk harvest compared to the control. These data provide evidence that improved welfare state and voluntary activity may mitigate pectoralis major myopathies, supporting our hypothesis that animal welfare contributes to breast muscle quality. Additionally, our work provides further evidence that serum CORT is a potential biomarker for WB and WS in finishing broilers.