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Vitamin C prevents the effects of high rearing temperatures on the quality of broiler thigh meat1
Vitamin C prevents the effects of high rearing temperatures on the quality of broiler thigh meat1 I. B. Ferreira,∗ J. B. Matos Junior,∗ S. Sgavioli,∗ T. I. Vicentini,∗ V. S. Morita,∗ and I. C. Boleli∗,2 ∗
Department of Animal Morphology and Physiology, School of Agricultural and Veterinary Sciences, S˜ ao Paulo State University, Jaboticabal, 14884-900 S˜ ao Paulo, Brazil hot rearing temperatures reduced the muscle fiber area (cold = 5.413 μm2 , control = 5.612 μm2 , hot = 4.448 μm2 ) (P < 0.05) without altering meat quality (P > 0.05). Hot rearing temperatures increased the cooking loss (cold = 30.10%, control = 33.66%, hot = 37.01%), shear force (cold = 3.05 kgf cm−2 , control = 3.43 kgf cm−2 , hot = 4.29 kgf cm−2 ) and redness (a∗ : cold = 4.63, control = 3.55, hot = 3.20) in the over-thigh meat of broilers from eggs incubated at 37.5o C, increasing the area of muscle fibers, while cold rearing temperatures diminished cooking loss and shear force, reducing the muscle fiber area (P < 0.05). Incubation at 39o C and 39o C+vitamin C prevented the effects of hot and cold rearing temperatures, by diminishing and increasing the muscle fiber area, respectively.
Key words: heat stress, meat, muscle growth, poultry, vitamin C 2015 Poultry Science 94:841–851 http://dx.doi.org/10.3382/ps/pev058
INTRODUCTION
characteristic red, reddish, or white coloring, respectively (Peter et al., 1972; Banks, 1992). Glycolytic metabolism following the bird’s death decreases the meat’s muscular pH, and causes the muscular toughness that affects its sensory and functional properties, and the products resulting from its processing (Froning et al., 1978; Richardson, 1995). Thus, muscles with a greater glycolytic capacity may have a greater and faster postmortem glycolysis, accelerating and accentuating the drop in pH as well as the meat toughness. Posthatch muscle growth in chickens involves marked fiber hypertrophy, since muscle hyperplasia occurs only during the fetal period of incubation (Moss, 1968; Kang et al., 1985; Rehfeldt et al., 2000; Alves et al., 2012). Differences in the rate of post-hatching hypertrophy between types of fibers may make the muscle more or less glycolytic and alter meat quality. Environmental factors such as temperature can alter the characteristics and hypertrophic growth of the skeletal muscle fibers, thus altering meat quality (Dransfield and Sosnicki, 1999; Lefaucheur, 2010). The strains of broilers selected for greater and faster growth have a high rate of metabolic heat production, which makes them more sensitive to high rearing temperatures, as they have difficulty maintaining normothermia
Color, tenderness, succulence, and flavor are features that determine the consumer’s choice for a particular meat. These features are developed postmortem and are influenced by the preslaughter fibrillar and connective tissue composition of the skeletal muscles (Chambers et al., 1989; Sams, 1999). Intramuscular connective tissue determines meat toughness, while fiber contractile and metabolic characteristics determine color, pH, tenderness, and succulence (Cuvelier et al., 2006; Vestergaard et al., 2000). Mixed muscles have slow-twitch fibers (with oxidative metabolism) and fast-twitch fibers (with oxidative–glycolytic or glycolytic metabolism), while white muscles only have fast-twitch fibers (with oxidative–glycolytic and/or glycolytic metabolism); these fibers give the meats their C 2015 Poultry Science Association Inc. Received September 12, 2014. Accepted December 23, 2014. 1 The authors would like to thank the Funda¸c˜ao de Amparo a Pesquisa do Estado de S˜ ao Paulo (FAPESP, process no. 2010/15280-0) for their financial support of this study. The authors declare that they have no conflict of interest. 2 Corresponding author: [email protected]
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ABSTRACT We investigated the effects of incubation temperatures and vitamin C injections into eggs (treatments: 37.5o C, 39o C, 39o C+vitamin C) on resultant chick pectoralis major and sartorius muscle fiber hypertrophy, as well as their effects on the quality of breast and over-thigh meat of broilers reared under cold, control, or hot temperatures. Incubation at 39o C increased the shear force and reduced meat redness in breast meat (P < 0.05). Vitamin C prevented these high temperature incubation effects [shear force (kgf cm−2 ): 37.5o C = 2.34, 39o C = 2.79, 39o C+vitamin C = 2.44; redness: 37.5o C = 2.64, 39o C = 1.90, 39o C+vitamin C = 2.30], but reduced water content (37.5o C = 74.81%, 39o C = 74.53%, 39o C+vitamin C = 69.39%) (P < 0.05). Cold rearing temperatures increased breast meat redness (a∗ : cold = 2.78, control = 2.12, hot = 1.98), while
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Nutritional programming has also been associated with phenotypic changes in the offspring, including changes in meat quality (e.g., Chen et al., 2009; Wei et al., 2011; Maiorano et al., 2012), but to the best of our knowledge, associations between egg nutrition and incubation temperature have not been investigated. Supplementing broiler diet with vitamin C was shown to be effective in increasing heat resistance during rearing (Sahin et al., 2003; Vaz, 2006) and in reducing the adverse effects of heat stress on weight gain and meat quality (Imik et al., 2012). However, it is not known whether continuous high temperature exposure during incubation together with increased vitamin C influences the effects of rearing temperature on the quality of broiler meat. While incubation temperature manipulation has recently been studied as a tool to induce thermotolerance post-hatch (Tzschentke and Plagemann, 2006; Tzschentke et al., 2004; Tzschentke, 2007), and considering that continuous high temperatures increase embryonic mortality and decrease hatchability (Decuypere et al., 1979; Suarez et al., 1996), in the present study, we examined the effects of continuous high incubation temperature with vitamin C supplement (injected into eggs) on hatchability, chick weight, pectoralis major and sartorius muscle fiber hypertrophy, and the quality of breast and thigh meat in broilers raised in cold, control, or hot conditions.
MATERIALS AND METHODS Study Conditions The study protocol was determined following the Ethical Principles in Animal Experimentation ´ (Princ´ıpios Eticos na Experimenta¸c˜ao Animal) drafted by the Brazilian College of Experimentation (Col´egio Brasileiro de Experimenta¸c˜ao, COBEA) and was approved by the Ethics Committee on Animal Use ´ (Comiss˜ao de Etica no Uso de Animais, protocol no. 7377/10) of the College of Agricultural and Veterinary Sciences (Faculdade de Ciˆencias Agr´arias e Veterin´ arias) of the Universidade Estadual Paulista (Jaboticabal Campus), Jaboticabal, S˜ ao Paulo, Brazil. Fertile broiler eggs (n = 3,000; Cobb 500) obtained from a commercial hatchery (Globoaves, Itirapina, SP, Brazil) from 47-week-old breeders were weighed and divided into 3 incubation treatments with 1,000 eggs each: 1) eggs without vitamin C injection and incubated at 37.5o C (control group), 2) eggs without vitamin C injection and incubated at 39o C, and 3) eggs with 6 μg/100 μL vitamin C injection and incubated at 39o C. For each incubation treatment, the eggs were distributed into 5 incubators (5 replicates with 200 eggs each) with automatic temperature control, relative humidity, and egg turning (every 2 h) (Premium Ecol´ ogica IP200, Belo Horizonte, MG, Brazil). The average weight of eggs per replicate and treatment was 67 ± 2 g. All eggs were kept at 60% relative humidity until day 18,
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and homeostasis (Macari et al., 2002). Compared to birds raised under low temperatures, birds raised under high temperatures have reduced performance and lower hypertrophy of the fast-twitch flexor hallucis muscle fibers with glycolytic metabolism (Sartori et al., 2001). Relative to birds raised under normal conditions, on the other hand, birds raised under high temperatures are subject to increased luminosity, cooking loss, and shear force, as well as decreased pH and redness (indicated by the a∗ index) in pectoral and thigh muscles (Zhang et al., 2012). In ducks subjected to cold, an increase in oxidative activity and in the proportion of oxidative fibers occurs in skeletal muscles (Duchamp et al., 1992), resulting in redder meat. Several studies have analyzed whether short or long and continuous or intermittent thermal programming during distinct periods of in ovo development can induce posthatch thermotolerance. For example, intermittent heat exposure (3h/day at 39.5◦ C) during the embryonic (1 to 8 days of incubation), fetal (16 to 18 days of incubation) phase, or both did not induce thermotolerance in chickens; however, intermittent heat exposure during the fetal phase increased breast muscle growth without altering pH or reducing meat moisture (Collin et al., 2007). High incubation temperatures (38.5o C) from the seventh to the 10th day, however, reduced the area of breast fibers in broilers (Werner et al., 2010). Continuous high temperature exposure (24h at 39.5o C) from days 7 to 16 of incubation impaired broiler chick performance but improved thermoregulation in response to heat conditions by reducing heat production (Piestun et al., 2008). In broilers exposed to hot conditions from day 21 age, on the other hand, relative breast muscle weight and the percentage of large-diameter fibers were increased until day 35 age (Piestun et al., 2011). Finally, eggs exposed to 38.1◦ C through day 5 incubation showed improved hatchability, body weight gain, and relative increased breast muscle weight (Piestun et al., 2013). Although previous studies have suggested that prenatal thermal programming can induce thermotolerance in bird species (Yahav et al., 2004b,a; Piestun et al., 2008, 2011), which can be explained by an increase in thermolysis capacity or a reduction in the thermogenic potential (Yahav et al., 2005). These data indicate that phenotypic changes in broilers can be induced by continuous or intermittent thermal challenges, applied during the embryonic and/or fetal phase of their ontogenetic development. Although in this study we manipulated incubation temperature to analyze its role in increasing heat tolerance, we are interested in whether exposure to high temperatures during incubation could make the broiler chickens more sensitive to cold-temperature conditions, negatively impacting their survival, performance, and meat quality when exposed to low rearing temperatures. This is an important question because temperature variations from one climatic zone to another (and across and within seasons, mainly in winter and spring) may impair posthatching growth.
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Table 1. Formulation and analysis of the basal diet. Ingredient (%)
Grower diet (d 22 to 42)
60.81 35.15 1.63 0.84 0.42 0.25 0.29 0.08 0.01 0.50
63.74 29.79 3.12 1.16 0.76 0.44 0.21 0.23 0.04 0.01 0.50
Calculated composition2 Crude protein (%) Metabolizable energy (kcal/kg) Calcium (%) Sodium (%) Available phosphorus (%) Methionine + cystine (%) Methionine (%) Lysine (%) Threonine (%) Tryptophan (%) Arginine (%)
21.27 2,883 0.85 0.19 0.42 0.88 0.56 1.22 0.79 0.24 1.32
18.86 3,121 0.69 0.20 0.32 0.77 0.49 1.05 0.68 0.21 1.16
1 Provided (per kilogram of diet): starter diet: vitamin A 7,000 IU, vitamin D3 3,000 IU, vitamin E 25 IU, vitamin K 0.98 mg, vitamin B1 1.78 mg, vitamin B2 9.6 mg, vitamin B6 3.5 mg, vitamin B12 10 μ g, folic acid 0.57 mg, biotin 0.16 mg, niacin 34.5 mg, calcium pantothenate 9.8 mg, copper 0.12 g, cobalt 0.02 mg, iodine 1.3 mg, iron 0.05 g, manganese 0.07 g, zinc 0.09 mg, organic zinc 6.75 mg, selenium 0.27 mg, choline 0.4 g, growth factor 30 mg, coccidicide 0.1 g, methionine 1.68 g; grower diet: vitamin A 7,000 U.I., vitamin D3 3,000 U.I., vitamin E 25 IU, vitamin K 0.98 mg, vitamin B1 1.78 mg, vitamin B2 9.6 mg, vitamin B6 3.5 mg, vitamin B12 10 μ g, folic acid 0.57 mg, biotin 0.16 mg, niacin 34.5 mg, calcium pantothenate 9.8 mg, copper 0.12 g, colbalt 0.02 mg, iodine 1.3 mg, iron 0.05 g, manganese 0.07 g, zinc 0.09 mg, organic zinc 6.75 mg, selenium 0.27 mg, choline 0.6 g, growth factor 7.5 mg, coccidicide 0.1g, methionine 1.4g. 2 Calculated values according to Rostagno et al. (2011).
after which 70% relative humidity was applied and egg rotation was stopped. The eggs were injected with an aqueous vitamin C solution (6 μg/100 μL) prior to the start of incubation, based on our previous study showing that 6 mg/egg vitamin C injections pre-incubation do not alter broiler chick hatchability (Sgavioli et al., 2013). Eggs were positioned horizontally, the injection site was cleaned with 100% ethanol, and the eggshells were punctured with a sterile needle [Injex, 13 × 0.38 (27.5 G 1/2 in.)]. Next, a vitamin C solution (Synth, 99% purity, Diadema, Brazil) was injected into the albumen near the end of the egg opposite to the air chamber, about 6 mm beneath the shell. After injection (100 μL), the hole was sealed with a label identifying the treatment and trial. The fresh vitamin C solution was prepared with autoclaved Mili-Q water and kept in a dark bottle, and in a dark environment. The lights were kept off during injections. At hatching, a total of 540 1-day-old chicks (Cobb) (180 chicks from each incubation treatment, with 36 birds per incubation treatment replicate) were distributed into 3 rearing temperature groups: cold, control (the temperature recommended for this strain), and hot. Temperatures from the first to sixth week for each rearing temperature were 1) cold: 32, 30, 26, 22, 18, and 14o C; 2) control: 32, 31, 29, 27, 25, and 23o C, and 3) hot: 32o C during the entire period. Chicks were housed
in one of 3 identical climatic chambers (6.5 × 9 × 2.8 m) with automatic control of temperature and light regime (12L:12D). There was one chamber per rearing temperature and 15 plots/chamber (1 × 2.5 m), and chicks were equally distributed in 5 replicates of 12 birds per incubation treatment. The average body weight of chicks per replicate and incubation treatment was 50 ± 2.5 g. The mean relative humidity inside the chambers during the study period was approximately 57%. Chicks were raised from the first to the 42nd day of life and received water and food ad libitum until age 42 day. The diet was based on corn and soybean meal (Table 1), was altered for the initial (1 to 21 days) and final (22 to 42 days) rearing stages, and met the birds’ nutritional requirements, as described in Rostagno et al. (2011). Chicks were vaccinated against Marek’s disease and Avian Bouba, as well as against Gumboro disease and Newcastle disease during rearing, following the vaccination program recommended for this strain.
Meat Quality Birds were killed on day 42 of rearing and quality analyses of the breast and thigh meat were conducted 24 h postmortem using a total of 90 animals, comprising 10 birds/treatment (2 per trial). The birds were killed
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Starter diet (d 1 to 21)
Corn Soybean meal 45% Soybean oil Dicalcium phosphate Limestone Sodium chloride L -Lysine–HCl (78%) DL -Methionine (99%) L -Threonine Antioxidant (i.e., BHT) Vitamin–mineral supplement1
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Muscle Fiber Morphometry Muscle samples At 42 days age, the pectoralis major (white) and sartorius (mixed) muscles were quickly removed from 5 birds/treatment, which had previously been weighed and killed by electrosensitization followed by bleeding. Samples were taken from the pars sternobranchialis of the pectoralis major muscle and the middle region of the sartorius muscle, and immediately frozen in n-hexane cooled in liquid nitrogen; they were then kept in a freezer at −70◦ C until histochemical and immunohistochemical processing. Histochemical and immunohistochemical processing Fiber size was evaluated from the crosssectional (transverse) area by freeze-cutting 8 to 12μm thick transverse and semiserial sections with a microtome–cryostat at −20o C and submitting them to the NADH tetrazolium reductase (TR) technique and indirect immunohistochemical marking of slow-twitch fibers. These techniques identified three types of fibers: slow-twitch oxidative (SO), fast-twitch oxidative–glycolytic (FOG), and fast-twitch glycolytic (FG) fibers. For histochemical processing with the NADH–TR technique, sections were kept at room temperature for 30 min for drying and fixation on the histological slide. Then, following the method described by Dubowitz and Brooke (1984), sections were incubated with a solution containing NADH (reduced form: 0.08%) and nitro blue tetrazolium (0.1%) (Sigma, St. Louis, MO) in Tris–HCl 0.2 M, pH 7.4, for 40 minutes at 37o C.
Next, the sections were washed with distilled water (3 × 5 min), fixed in 5% formaldehyde buffered at pH 7.0 for 5 minutes, and rinsed again with distilled water (3 times). They were then dehydrated in a series of increasing ethanol concentrations (60, 70, 80, 90, and 100%), diaphanized in xylene (100%), and mounted with Entellan. Oxidative, oxidative–glycolytic, and glycolytic fibers were identified according to their dark, intermediate, and light coloring, respectively. For immunohistochemical processing, the general procedure ´ of DAngelis et al. (2005) was followed. Briefly, 8-μm thick transverse serial sections were obtained from the same sample used for histochemical analyses. The sections were subjected to indirect immunohistochemistry (peroxidase–antiperoxidase) for the marking of fibers containing slow-twitch myosin. Initially, the sections were kept for 30 minutes at room temperature for drying and adherence to histology slides. Next, they were fixed in 3.5% formalin for 30 minutes, washed in 0.1 M phosphate buffer (pH 7.2; 2 × 2 min each), and incubated with 3% hydrogen peroxide in methanol for 5 minutes to block endogenous peroxidase. The sections were washed once again with phosphate buffer and incubated with nonspecific rabbit serum (1:50 in phosphate buffer) for 30 minutes in a humid chamber at 4◦ C to control the background. The serum was removed and the sections were incubated with slow-twitch antimyosin monoclonal primary antibody produced in mice (1:400 in phosphate buffer) (NOQ7.5.4D, Sigma, St. Louis, MO) for 150 min in a humid chamber at 4◦ C. Once again, the sections were washed with phosphate buffer (2 × 4 min each, under gentle agitation) and then incubated with peroxidase-conjugated goat antimouse IgG secondary antibody (1:1,000 in phosphate buffer) (Sigma, St. Louis, MO) for 90 min in a humid chamber at 4◦ C. A new wash with phosphate buffer was then conducted (2 × 4 min each). The antigen– antibody complexes were revealed by incubation of the sections with diaminobenzidine (0.2 mg/1.5 mL 3,3 diaminobenzidine per 0.1 M phosphate buffer, pH 7.2) (Sigma, St. Louis, MO) at room temperature, containing 2 to 3 drops of hydrogen peroxide. The staining intensity was visually inspected and development was blocked by washing the sections with phosphate buffer. Subsequently, the sections were dehydrated in a series of increasing ethanol concentrations (60, 70, 80, 90, and 100%), diaphanized in xylene (100%), and mounted with Entellan. Slow-twitch fibers had a golden color with slow-twitch antimyosin antibody, while fast-twitch fibers were not marked. The negative control of the antigen–antibody reaction was conducted by omitting incubation with primary antibody.
Hatchability, Body Weight, Feed Intake, and Feed Conversion Rates Hatchability was determined as a function of the total number of fertile eggs. The chicks were weighed
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by electrosensitization followed by bleeding. Prior to slaughter, the broilers were fasted for 10 h. The pH was determined by directly inserting an electrode into the muscles using a digital pH meter (Testo, model Testo106, Lenzkirch, Germany). Meat color was measured from the inner portion of the breast and over-thigh using Chrome Meter CR-300 equipment (Konica Minolta Sensing, Osaka, Japan) and the CIELAB trichromatic system, which determines lightness (L∗ ), redness (a∗ ), and yellowness (b∗ ) values. The water-holding capacity was measured by press loss: 1 g from each meat sample was pressed with a 10-kg weight for 5 minutes at 25o C; each sample was subsequently reweighed to calculate water-holding capacity, and this value was expressed as a percentage of the initial weight. To determine cooking losses, samples were placed in plastic bags and cooked in a water bath at 85◦ C for 30 minutes at a final internal temperature of 75 to 80◦ C. After releasing the exuded water and cooling to reach room temperature, they were again weighed and compared to the initial weight (Cason et al., 1997). Warner–Bratzler shear force was measured on the cooked samples previously used in the cooking loss analysis using a Texture Analyzer TAXT2i (Godalming, Surrey, United Kingdom) and expressed as kilograms per square centimeter (Lyon et al., 1998).
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immediately after their plumage was dry. Broiler weight was measured on day 42 on a replicate basis. Amounts of feed offered to each replicate were recorded weekly, and uneaten feed in each replicate was weighed (from 1 to 42 d). Individual bird feed intake for each replicate was determined by dividing the cumulative feed intake by the total number of living birds in the replicate. Feed conversion ratio was calculated by dividing the amount of feed intake by the body weight gain (grams per gram).
Statistical Analyses
RESULTS Hatchability, Body Weight, Feed Intake, and Feed Conversion Hatchability was lower for the high-temperature groups (with and without vitamin C) than for the control group group (control: 84.50a ± 1.12%, 39o C: 74.70b ± 1.20%, 39o C+vitamin C: 74.40b ± 9.24%, P < 0.0189). However, absolute chick weight at hatch was not altered by these incubation treatments relative to the control groups (control: 51.04 ± 2.24 g, 39o C: 50.22 ± 1.28 g, 39o C+vitamin C: 50.39 ± 2.12 g, P = 0.6718). At the end of the study period, the body weight, feed intake, and feed conversion rates were not affected by either incubation treatment (P = 0.99, P = 0.48 and P = 0.42, respectively) or by the interaction between incubation treatments and rearing temperatures (P = 0.81, P = 0.18, P = 0.37, respectively). However, the rearing temperature affected the 3 variables, which were lower under hot rearing temperatures than under cold or control temperatures (these last 2 variables did not differ from each other; P < 0.05, Figure 1).
Breast Characteristics Table 2 shows the effects of the incubation treatments and the rearing temperatures on meat quality and fiber area of the breast muscle (pectoralis major). The cooking loss, pH, L∗ , and b∗ values were not influenced by the incubation treatments or the rearing temperatures
Figure 1. Effects of rearing temperatures (cold, control, and hot) on broiler body weight at 42-days-old (A), feed intake (g per bird) from day 1 to 42 age (B), and feed conversion (C). Bars with distinct letters indicate a difference between means (P < 0.05).
(P > 0.05). Shear force and water-holding capacity were affected only by the incubation treatments (P < 0.05), while a∗ was affected by both the incubation treatments and the rearing temperatures (P < 0.05). Shear force was greater under incubation at 39o C than at 37.5o C or 39o C+vitamin C. The water-retention capacity was similar for incubation at 37.5o C and at 39o C, and both of these were greater than at 39o C+vitamin C. Redness (a∗ ) was similar for incubation at 37.5o C and at 39o C+vitamin C, and was greater in both of these treatments than at 39o C. Redness was also greater under cold rearing temperatures than under hot or control temperatures, neither of which affected a∗ . The FG fiber area of the pectoralis major muscle was not affected by incubation temperature (P > 0.05),
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All data were assessed with one-way ANOVA using the general linear model procedure of the SAS software package (SAS Institute, 2002), and statistical significance was set at P ≤ 0.05. For hatchability and chick weight at hatch, we verified the effects of each of the incubation treatments (37.5o C, 39o C, 39o C+vitamin C). For body weight, feed intake, feed conversion, fiber area, and meat quality characteristics, we assessed the effects of incubation treatments (I), rearing temperatures (R: cold, control, and hot) and the respective interactions (IR) using the following model: Yijk = μ + Ii + Rj + (IR)ij + eijk. For differences among groups, mean comparisons were conducted with Tukey’s tests.
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FERREIRA ET AL. Table 2. Effects of incubation treatments and rearing temperatures on pectoralis major meat quality and cross-sectional area of muscle fibers in 42-day-old broilers. Variables1
CL (%)
SF (kgf cm−2 )
pH
WHC (%)
CSAF (μ m2 )
Color L∗
a∗
b∗
Incubation treatments (IT) 37.5o C 27.12 26.38 39o C 39o C+vitamin C2 26.19 Rearing temperatures (RT) Cold 25.77 Control3 27.55 Hot 26.48 P-values IT 0.7560 RT 0.4170 IT x RT 0.5355 11.50 CV (%)4
2.34b 2.79a 2.44b
6.01 6.03 6.04
74.81a 74.53a 69.39b
52.34 51.95 52.34
2.64a 1.90b 2.30ab
4.16 4.48 4.59
5,033 5,325 5,115
2.63 2.49 2.43
6.04 6.03 6.00
72.29 72.91 73.68
51.09 51.84 53.56
2.78a 2.12b 1.98b
4.23 4.80 4.20
5,413a 5,612a 4,448b
0.0013 0.2799 0.0784 20.43
0.6639 0.5240 0.4964 1.82
< 0.0001 0.1392 0.6914 4.98
0.9456 0.1269 0.1008 5.63
0.0211 0.0075 0.1478 24.54
0.8022 0.5463 0.2083 31.08
0.8032 0.0329 0.2675 24.11
Table 3. Effects of incubation treatments and rearing temperatures on over-thigh meat quality and cross-sectional area of sartorius muscle fibers in 42-day-old broilers. Variables1
CL (%)
Incubation treatments 37.5o C 33.30 33.33 39o C 31.39 39o C-vitamin C2 Rearing temperatures Cold 31.99 33.73 Control3 Hot 32.52 P-values IT 0.0909 RT 0.2666 IT x RT 0.0039 CV (%)4 7.68
SF (kgf cm−2 )
WHC (%)
pH
Color L∗
a∗
b∗
3.59 3.53 3.67
70.75 72.18 71.42
6.19 6.16 6.13
53.74 53.76 55.65
3.70 3.81 3.90
5.43 5.80 6.01
3.47 3.70 3.60
71.67 72.14 70.51
6.16 6.17 6.16
53.23 54.58 55.04
4.63a 3.55a,b 3.20b
5.24 6.39 5.57
0.8592 0.4812 < 0.0001 18.02
0.1183 0.0768 0.0017 4.36
0.6828 0.9808 0.3938 2.38
0.3079 0.4310 0.0957 5.96
0.8554 0.0100 0.2745 28.40
0.7164 0.2464 0.0914 29.79
1 CL: cooking losses. SF: shear force. WHC: water holding capacity. L∗ : lightness. a∗ : redness. b∗ : yellowness. 2 39o C+vitamin C: incubation at 39o C plus intra eggs injection of vitamin C. 3 Control: standard rearing temperature for lineage. 4 CV: coefficient of variation. a,b Means with different superscripts differ significantly (P < 0.05).
but was modified by rearing temperature (P < 0.05): it was smaller in chickens raised under hot temperatures than under control or cold temperatures, with no difference in fiber area observed between broilers raised under these last 2 temperatures.
Over-Thigh Characteristics Table 3 shows the effects of the treatments during incubation and rearing temperatures on over-thigh meat quality. Neither the pH nor the L∗ or b∗ indices were altered by incubation treatment or rearing temperature, and there were also no interactions between these vari-
ables (P > 0.05). Redness (a∗ ) was only affected by rearing temperature (greater under cold than hot temperatures), while there were no differences in a∗ under thermoneutral conditions relative to cold and hot temperatures. There were significant interactions between incubation treatments and rearing temperature for cooking loss, shear force, and water-retention capacities (P < 0.05). Specifically, rearing temperature only affected the cooking loss of the over-thighs of broilers from eggs incubated at 37.5o C, which was greater for broilers raised under hot (relative to cold) temperatures, and did not differ from broilers raised under control temperatures (Table 4). Incubation
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1 CL: cooking losses. SF: shear force. WHC: water holding capacity. L∗ : lightness. a∗ : redness. b∗ : yellowness. CSAF: cross-sectional area of the fibers. 2 39o C+vitamin C: incubation at 39o C plus intra eggs injection of vitamin C. 3 Control: standard rearing temperature for lineage. 4 CV: coefficient of variation. a,b Means with different superscripts differ significantly (P < 0.05).
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TEMPERATURE AND VITAMIN C IN BROILER MUSCLES Table 4. Interaction between incubation treatments and rearing temperatures for over-thigh meat quality of 42-day-old broilers. Variables1
Incubation treatments
Rearing temperatures (o C) 2
Cold o
B
Control
A,B
P-values
Hot
CL (%)
37.5 C 39o C 39o C+vitamin C P-values
30.10 33.36 32.50 0.1868
33.66 33.94 33.55 0.9770
37.01A,a 32.69a,b 27.75b 0.0006
0.0050 0.7812 0.0598
SF (kgf cm−2 )
37.5o C 39o C 39o C+vitamin C P-values
3.05B 3.82A 3.53 0.0635
3.43A,B 4.00A 3.68 0.2204
4.29A,a 2.78B,b 3.71a,b < 0.0001
0.0011 0.0007 0.8408
WHC (%)
37.5o C 39o C 39o C+vitamin C P-values
70.65A,B,b 74.03a 70.32b 0.0066
72.78A 72.41 71.24 0.0802
68.43B 70.11 72.83 0.1564
0.0071 0.4525 0.0962
treatments also affected the cooking loss of the overthighs of broilers raised under hot temperatures. Cooking loss was greater for broilers from eggs incubated at 37.5o C than at 39o C and injected with vitamin C, but it did not differ from that of broilers from eggs incubated at 39o C and not injected with vitamin C. Shear force was affected by rearing temperature in broilers from eggs incubated at 37.5 and 39o C. In the first group, the shear force did not differ between broilers raised under control temperatures and those raised under the other 2 temperatures, but was greater in broilers raised under hot relative to cold temperatures. In broilers from eggs incubated at 39o C, on the other hand, the shear force was lower for broilers raised under hot temperatures than for those raised under cold or control temperatures. Only in broilers raised under hot temperatures did incubation temperature affect shear force, which was greater in broilers from eggs incubated at 37.5o C than at 39o C, but not in broilers from eggs incubated at 39o C and injected with vitamin C. The interaction also revealed effects of rearing temperature and incubation treatments on the water-retention capacity of the over-thigh meat only in broilers from eggs incubated at 37.5o C, which was greater under hot than control rearing temperatures. Chickens raised under cold temperatures did not have altered water-retention capacity, which was lower when raised under hot temperatures than under control temperatures. Incubation treatments affected water-retention capacity only in broilers raised under cold temperatures. Retention capacity was greater in broilers from eggs incubated at 39o C than those incubated at 37.5o C and at 39o C that were also injected with vitamin C; these last 2 conditions did not differ from each other in terms of water retention. Tables 5 and 6 present the results obtained for the mean cross-sectional area of sartorius muscle fibers in
Table 5. Effects of incubation treatments and rearing temperatures on mean cross-sectional area of sartorius muscle fibers in 42-day-old broilers. Fiber types1
SO (μ m2 )
Incubation treatments 2,346 37.5o C 39o C 3,048 39o C+vitamin C2 2,917 Rearing temperatures Cold 2,547 Control3 2,859 Hot 2,905 P-values IT 0.0515 RT 0.4240 IT x RT 0.0242 CV (%)4 29.03
FOG (μ m2 )
FG (μ m2 )
2,656 3,360 2,750
3,299 3,753 3,036
2,964 2,842 2,961
3,570 3,371 3,138
0.0401 0.8905 0.0004 26.98
0.0419 0.4179 0.0064 22.18
1 SO: slow oxydative fiber. FOG: fast oxydative and glycolytic fiber. FG: fast glycolytic fiber. 2 39o C+vitamin C: incubation at 39o C plus intra eggs injection of vitamin C. 3 Control: standard rearing temperature for Cobb lineage. 4 CV: coefficient of variation (P < 0.05).
42-day-old broilers. There was a significant interaction between incubation treatments and rearing temperatures for the area of SO, FOG, and FG fibers (P < 0.05) (Table 5). This interaction (Table 6) revealed that rearing temperature did not alter the area of FG fibers, but affected the area of SO and FOG fibers, and only in broilers from eggs incubated at 37.5o C. Incubation treatments altered the area of SO, FOG, and FG fibers of broilers raised under cold temperatures, and the area of FOG and FG fibers of broilers raised under hot temperatures. Among broilers raised under cold temperatures, the area of SO fibers was larger in birds from eggs incubated at 39o C than at 37.5o C, whether or not they received a vitamin C injection. The area of FOG and
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1 CL: cooking losses. SF: shear force. WHC: water holding capacity. 39o C+vitamin C: incubation at 39o C plus intra eggs injection of vitamin C. 2 Control: standard rearing temperature for Cobb lineage. a,b Means (columns) with different superscripts differ significantly (P < 0.05). A,B Means (rows) with different superscripts differ significantly (P < 0.05).
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FERREIRA ET AL. Table 6. Interaction between incubation treatments and rearing temperatures for cross-sectional area of the sartorius muscle fibers in 42-day-old broilers. Fiber types1
Incubation treatments
Rearing temperatures (o C) Cold
Control3
Hot
P-values
SO (μ m2 )
37.5o C 39o C 39o C+vitamin C2 P-values
1,545B,b 3,019a 3,077a 0.0064
2,268A,B 3,018 3,291 0.1293
3,227A 3,107 2,382 0.2133
0.0082 0.9800 0.1894
FOG (μ m2 )
37.5o C 39o C 39o C+vitamin C P-values
1,791B,b 3,944a 3,156a,b 0.0005
2,336A,B 3,347 2,843 0.1431
3,843A,a 2,789a,b 2,250b 0.0098
0.0006 0.0822 0.1963
FG (μ m2 )
37.5o C 39o C 39o C+vitamin C P-values
2,831b 4,229a 3,649a,b 0.0193
3,165 4,020 2,926 0.0644
4,052a 3,010a,b 2,534b 0.0154
0.0577 0.0517 0.0699
FG fibers was larger in broilers from eggs incubated at 39o C than at 37.5o C, and there was no difference in the values recorded for broilers from these 2 treatments and those incubated at 39o C and injected with vitamin C. Among broilers raised under hot temperature, the areas of FOG and FG fibers were influenced by incubation at 39o C only when the eggs also received a vitamin C injection, and these areas were smaller in these broilers than in those incubated at 37.5o C.
DISCUSSION In this study, we studied 1) whether injection of vitamin C in eggs reduces the effects of incubation at high temperatures on hatchability and chick weight; 2) whether continuous high incubation temperatures with or without preincubation injections of vitamin C into the eggs changes performance, muscle fiber hypertrophy, and meat quality of the breast and over-thigh of broilers reared at high temperatures; and 3) the effects of these incubation treatments when broilers are reared under cold-temperature conditions. Our results showed that high incubation temperatures with or without vitamin C injection into the eggs reduced or eliminated the effects of the high rearing temperature on the meat quality of the over-thigh. This involved changes in muscle fiber hypertrophy but did not alter zootechnical variables (except for hatchability, which was reduced).
Hatchability, Body Weight, Feed Intake, and Feed Conversion Although vitamin C has been attributed an antistressor role (Pardue and Thaxton, 1986; Mahmoud et al., 2004), its injection into the egg did not prevent
the decrease in hatching rate caused by high temperature incubation. In the current study, cold rearing temperatures did not influence broiler performance, while hot rearing temperatures reduced broiler feed intake, feed conversion, and weight gain, as previously reported (Aksit et al., 2006; Imik et al., 2012). High incubation temperatures (with or without vitamin C injections into the egg) did not reduce or prevent the negative effects of hot rearing temperatures on broiler performance, nor did they influence body weight, feed intake, and conversion of broilers reared under cold temperatures. These results showed that high-temperature exposure during the whole incubation period did not grant the broilers a pre-adaptation to hot rearing temperatures, nor did it make them more sensitive to cold-rearing conditions.
Breast Characteristics Although no effect of high incubation temperatures plus vitamin C on performance was shown, we observed smaller effects of incubation treatments and/or rearing temperatures on a few meat quality characteristics, and on the size of FG fibers in broiler breasts. To the best of our knowledge, this is the first study indicating that an increase in the amount of vitamin C in the egg affects the quality of broiler breast meat as well as the effects caused by thermal stress. High incubation temperatures increased the shear force and reduced a∗ , making the breast meat tougher and less red without altering its cooking loss, pH, or luminosity. Injecting vitamin C into the eggs prevented the effects of high-temperature incubation. Nonetheless, the capacity for water retention decreased by approximately 5% in breast meat, making it less succulent. Vitamin C is required for amino acid and mineral metabolism, as well as for hormonal
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1 SO: slow oxydative fiber. FOG: fast oxydative and glycolytic fiber. FG: fast glycolytic fiber. 2 39o C+vitamin C: incubation at 39o C plus intra eggs injection of vitamin C. 3 Control: standard rearing temperature for Cobb lineage. a,b Means (columns) with different superscripts differ significantly (P < 0.05). A,B Means (rows) with different superscripts differ significantly (P < 0.05).
TEMPERATURE AND VITAMIN C IN BROILER MUSCLES
2012) and present high metabolic plasticity, perhaps the redder coloring we observed in the present study could have been caused by the increased vascularity of the fibers associated to metabolic changes. Further, the lower hypertrophy of the breast muscle fibers of broilers raised under hot temperatures may have been caused by the reduced feed intake and body weight reported in this study, as well as by the reduced amino acid and mineral metabolism resulting from insufficient endogenous vitamin C availability (caused in turn by the higher heat-induced demand) (Thornton, 1961; Pardue and Thaxton, 1986; Kutlu and Forbes, 1993). Our data differ from those observed by Aksit et al. (2006), who reported increased redness in the breast meat of broilers reared under hot temperatures.
Over-Thigh Characteristics In the present study, we also observed an interaction of the effects of incubation and rearing temperatures on meat quality parameters, and muscle fiber size in broiler over-thigh meat. High incubation temperatures reduced over-thigh meat shear force in broilers raised under hot temperatures and increased the water-retention capacity in broilers raised under low temperatures, making the meat more tender and succulent, respectively. These high incubation temperature effects on meat quality were eliminated when vitamin C was injected into the egg, confirming the important role of vitamin C in heat stress. As mentioned above, several authors reported that an increase in the muscle fiber area in a transverse cut negatively affects meat texture and succulence, reducing cooking loss and water-retention capacity, and increasing the shear force (Seideman and Crouse, 1986; Rehfeldt et al., 2000). However, this was not observed in the current study in broilers raised under hot temperatures and incubated at 37.5o C or at 39o C and injected with vitamin C. In contrast, however, we observed an increase in the cross-sectional area of the fibers (slowtwitch oxidative, fast-twitch oxidative–glycolytic, and fast-twitch glycolytic) together with an increase in the water-retention capacity of the over-thigh meat of broilers from eggs that were incubated at high temperatures and raised under cold temperatures. In the present study, hot rearing temperatures increased the redness (a∗ ) of the thigh meat, in agreement with previous work by Zhang et al. (2012) in which redness was associated with an increase in the amount of oxidative and oxidative–glycolytic fibers. In addition, the hot rearing temperature reduced thigh meat tenderness and succulence of broilers from eggs incubated at the usual temperature (37.5o C) due to the increased cooking loss and shear force, and the decreased water retention caused by the greater hypertrophic growth of the SO and FOG fibers (as well as FG fibers, if we consider 6% significance). On the other hand, cold-rearing temperatures caused the opposite effect and improved meat quality while reducing the area of fibers in the
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production linked to stress resistance (Abidin and Khatoon, 2013). Heat conditions increase the utilization and demand for vitamin C, making its endogenous production by chickens insufficient for homeostasis maintenance (Thornton, 1961; Pardue and Thaxton, 1986; Kutlu and Forbes, 1993). Under these conditions, exogenous vitamin C can play an important role in the maintenance of physiological processes. In line with this view, the pre-incubation administration of vitamin C into the egg in this study appears to have supplied, at least in part, the demand for vitamin C caused by exposure to heat. Thus, in line with previous studies (see, Mckee et al., 1997; Lin et al., 2006; Attia et al., 2009), our results highlight the potential role of vitamin C in reducing or eliminating heat stress effects, as well as the potential use of intra-egg injections of vitamin C before incubation. Nonetheless, more detailed studies should be conducted to better understand the biological processes involved in the antiheat effects of vitamin C on pectoral muscles. Several authors have reported that a larger area of myofibers in a transverse cut implies a lower waterretention capacity and greater shear force (Larzul et al., 1997; Maltin et al., 1997), which promote meat toughness (Seideman and Crouse, 1986; Rehfeldt et al., 2000); furthermore, the higher the muscle’s glycolytic content (caused by an increase in fiber number or size), the greater the cooking losses, lightness (L∗ ), paleness (b ), and shear force, and the lower the pH and the water-retention capacity, which affects texture and succulence. According to Berri et al. (2007), an increase in the size of broiler breast muscle fibers increased the pH and improved water-retention capacity and meat quality. In the current study, the area of FG fibers in the pectoralis major muscle was not affected by high incubation temperatures with or without vitamin C injection into the egg; this is in contrast to data reported by Werner et al. (2010), who showed an increase in fiber size promoted by high temperature from the seventh to the 10th day of incubation. Therefore, our data show that the effects of high incubation temperature together with vitamin C on broiler breast meat did not result in the alteration of the hypertrophic growth of the muscle fibers, and that this lack of fiber alteration may explain the lack of alteration in pH, cooking losses, and luminosity. In the present study, broiler breast meat was redder (a∗ ) under cold-rearing temperatures, while the transverse area of the glycolytic fibers was smaller under hot-rearing temperatures. Acquisition of red color by white muscles was also observed by Hirabayashi et al. (2005) in the breast muscles of chicks exposed to coldtemperature conditions during incubation. Duchamp et al. (1992) reported that birds submitted to cold temperatures show an increase in oxidative activity and in the proportion of oxidative fibers in skeletal muscles. Considering that the pectoralis major muscle in broilers contains glycolytic fast-twitch fibers that grant the meat a white color (Rosser et al., 1996; Alves et al.,
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transverse cut. These opposing effects were likely due to the greater feed intake in broilers raised under cold temperatures. The inverse relation between muscle fiber size and meat quality in over-thighs of broilers from eggs incubated at the control temperature (37.5o C) and raised under either cold or hot temperatures is in agreement with results reported for breast meat by Berri et al. (2007). Even just the effects of rearing temperature on fiber hypertrophy and meat quality on the over-thighs of broilers from eggs incubated at the control temperature are an important contribution of the current study. To the best of our knowledge, this is the first study showing that continuous high incubation temperatures prevent the effects of thermal stress during rearing on the quality of thigh meat in broilers.
High incubation temperature affects the shear force, water-retention capacity, and cooking loss in chicken meat. In addition, it prevents toughness and the reduction in succulence caused by high rearing temperatures in the thighs of chickens from eggs incubated at the control temperature. Further, it improves the succulence and tenderness of thigh meat, in chickens raised under cold and hot temperatures, respectively. Vitamin C prevents the effects of high incubation temperature on meat quality and on the area of FOG and FG fibers of chickens raised under cold temperatures, as well as the shear force in chickens raised under hot temperatures. High incubation temperature during the fetal phase plus vitamin C injection into the egg prevents the thermal stress effects of high rearing temperatures.
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CONCLUSIONS
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Department of Animal Morphology and Physiology, School of Agricultural and Veterinary Sciences, S˜ ao Paulo State University, Jaboticabal, 14884-900 S˜ ao Paulo, Brazil hot rearing temperatures reduced the muscle fiber area (cold = 5.413 μm2 , control = 5.612 μm2 , hot = 4.448 μm2 ) (P < 0.05) without altering meat quality (P > 0.05). Hot rearing temperatures increased the cooking loss (cold = 30.10%, control = 33.66%, hot = 37.01%), shear force (cold = 3.05 kgf cm−2 , control = 3.43 kgf cm−2 , hot = 4.29 kgf cm−2 ) and redness (a∗ : cold = 4.63, control = 3.55, hot = 3.20) in the over-thigh meat of broilers from eggs incubated at 37.5o C, increasing the area of muscle fibers, while cold rearing temperatures diminished cooking loss and shear force, reducing the muscle fiber area (P < 0.05). Incubation at 39o C and 39o C+vitamin C prevented the effects of hot and cold rearing temperatures, by diminishing and increasing the muscle fiber area, respectively.
Key words: heat stress, meat, muscle growth, poultry, vitamin C 2015 Poultry Science 94:841–851 http://dx.doi.org/10.3382/ps/pev058
INTRODUCTION
characteristic red, reddish, or white coloring, respectively (Peter et al., 1972; Banks, 1992). Glycolytic metabolism following the bird’s death decreases the meat’s muscular pH, and causes the muscular toughness that affects its sensory and functional properties, and the products resulting from its processing (Froning et al., 1978; Richardson, 1995). Thus, muscles with a greater glycolytic capacity may have a greater and faster postmortem glycolysis, accelerating and accentuating the drop in pH as well as the meat toughness. Posthatch muscle growth in chickens involves marked fiber hypertrophy, since muscle hyperplasia occurs only during the fetal period of incubation (Moss, 1968; Kang et al., 1985; Rehfeldt et al., 2000; Alves et al., 2012). Differences in the rate of post-hatching hypertrophy between types of fibers may make the muscle more or less glycolytic and alter meat quality. Environmental factors such as temperature can alter the characteristics and hypertrophic growth of the skeletal muscle fibers, thus altering meat quality (Dransfield and Sosnicki, 1999; Lefaucheur, 2010). The strains of broilers selected for greater and faster growth have a high rate of metabolic heat production, which makes them more sensitive to high rearing temperatures, as they have difficulty maintaining normothermia
Color, tenderness, succulence, and flavor are features that determine the consumer’s choice for a particular meat. These features are developed postmortem and are influenced by the preslaughter fibrillar and connective tissue composition of the skeletal muscles (Chambers et al., 1989; Sams, 1999). Intramuscular connective tissue determines meat toughness, while fiber contractile and metabolic characteristics determine color, pH, tenderness, and succulence (Cuvelier et al., 2006; Vestergaard et al., 2000). Mixed muscles have slow-twitch fibers (with oxidative metabolism) and fast-twitch fibers (with oxidative–glycolytic or glycolytic metabolism), while white muscles only have fast-twitch fibers (with oxidative–glycolytic and/or glycolytic metabolism); these fibers give the meats their C 2015 Poultry Science Association Inc. Received September 12, 2014. Accepted December 23, 2014. 1 The authors would like to thank the Funda¸c˜ao de Amparo a Pesquisa do Estado de S˜ ao Paulo (FAPESP, process no. 2010/15280-0) for their financial support of this study. The authors declare that they have no conflict of interest. 2 Corresponding author: [email protected]
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ABSTRACT We investigated the effects of incubation temperatures and vitamin C injections into eggs (treatments: 37.5o C, 39o C, 39o C+vitamin C) on resultant chick pectoralis major and sartorius muscle fiber hypertrophy, as well as their effects on the quality of breast and over-thigh meat of broilers reared under cold, control, or hot temperatures. Incubation at 39o C increased the shear force and reduced meat redness in breast meat (P < 0.05). Vitamin C prevented these high temperature incubation effects [shear force (kgf cm−2 ): 37.5o C = 2.34, 39o C = 2.79, 39o C+vitamin C = 2.44; redness: 37.5o C = 2.64, 39o C = 1.90, 39o C+vitamin C = 2.30], but reduced water content (37.5o C = 74.81%, 39o C = 74.53%, 39o C+vitamin C = 69.39%) (P < 0.05). Cold rearing temperatures increased breast meat redness (a∗ : cold = 2.78, control = 2.12, hot = 1.98), while
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Nutritional programming has also been associated with phenotypic changes in the offspring, including changes in meat quality (e.g., Chen et al., 2009; Wei et al., 2011; Maiorano et al., 2012), but to the best of our knowledge, associations between egg nutrition and incubation temperature have not been investigated. Supplementing broiler diet with vitamin C was shown to be effective in increasing heat resistance during rearing (Sahin et al., 2003; Vaz, 2006) and in reducing the adverse effects of heat stress on weight gain and meat quality (Imik et al., 2012). However, it is not known whether continuous high temperature exposure during incubation together with increased vitamin C influences the effects of rearing temperature on the quality of broiler meat. While incubation temperature manipulation has recently been studied as a tool to induce thermotolerance post-hatch (Tzschentke and Plagemann, 2006; Tzschentke et al., 2004; Tzschentke, 2007), and considering that continuous high temperatures increase embryonic mortality and decrease hatchability (Decuypere et al., 1979; Suarez et al., 1996), in the present study, we examined the effects of continuous high incubation temperature with vitamin C supplement (injected into eggs) on hatchability, chick weight, pectoralis major and sartorius muscle fiber hypertrophy, and the quality of breast and thigh meat in broilers raised in cold, control, or hot conditions.
MATERIALS AND METHODS Study Conditions The study protocol was determined following the Ethical Principles in Animal Experimentation ´ (Princ´ıpios Eticos na Experimenta¸c˜ao Animal) drafted by the Brazilian College of Experimentation (Col´egio Brasileiro de Experimenta¸c˜ao, COBEA) and was approved by the Ethics Committee on Animal Use ´ (Comiss˜ao de Etica no Uso de Animais, protocol no. 7377/10) of the College of Agricultural and Veterinary Sciences (Faculdade de Ciˆencias Agr´arias e Veterin´ arias) of the Universidade Estadual Paulista (Jaboticabal Campus), Jaboticabal, S˜ ao Paulo, Brazil. Fertile broiler eggs (n = 3,000; Cobb 500) obtained from a commercial hatchery (Globoaves, Itirapina, SP, Brazil) from 47-week-old breeders were weighed and divided into 3 incubation treatments with 1,000 eggs each: 1) eggs without vitamin C injection and incubated at 37.5o C (control group), 2) eggs without vitamin C injection and incubated at 39o C, and 3) eggs with 6 μg/100 μL vitamin C injection and incubated at 39o C. For each incubation treatment, the eggs were distributed into 5 incubators (5 replicates with 200 eggs each) with automatic temperature control, relative humidity, and egg turning (every 2 h) (Premium Ecol´ ogica IP200, Belo Horizonte, MG, Brazil). The average weight of eggs per replicate and treatment was 67 ± 2 g. All eggs were kept at 60% relative humidity until day 18,
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and homeostasis (Macari et al., 2002). Compared to birds raised under low temperatures, birds raised under high temperatures have reduced performance and lower hypertrophy of the fast-twitch flexor hallucis muscle fibers with glycolytic metabolism (Sartori et al., 2001). Relative to birds raised under normal conditions, on the other hand, birds raised under high temperatures are subject to increased luminosity, cooking loss, and shear force, as well as decreased pH and redness (indicated by the a∗ index) in pectoral and thigh muscles (Zhang et al., 2012). In ducks subjected to cold, an increase in oxidative activity and in the proportion of oxidative fibers occurs in skeletal muscles (Duchamp et al., 1992), resulting in redder meat. Several studies have analyzed whether short or long and continuous or intermittent thermal programming during distinct periods of in ovo development can induce posthatch thermotolerance. For example, intermittent heat exposure (3h/day at 39.5◦ C) during the embryonic (1 to 8 days of incubation), fetal (16 to 18 days of incubation) phase, or both did not induce thermotolerance in chickens; however, intermittent heat exposure during the fetal phase increased breast muscle growth without altering pH or reducing meat moisture (Collin et al., 2007). High incubation temperatures (38.5o C) from the seventh to the 10th day, however, reduced the area of breast fibers in broilers (Werner et al., 2010). Continuous high temperature exposure (24h at 39.5o C) from days 7 to 16 of incubation impaired broiler chick performance but improved thermoregulation in response to heat conditions by reducing heat production (Piestun et al., 2008). In broilers exposed to hot conditions from day 21 age, on the other hand, relative breast muscle weight and the percentage of large-diameter fibers were increased until day 35 age (Piestun et al., 2011). Finally, eggs exposed to 38.1◦ C through day 5 incubation showed improved hatchability, body weight gain, and relative increased breast muscle weight (Piestun et al., 2013). Although previous studies have suggested that prenatal thermal programming can induce thermotolerance in bird species (Yahav et al., 2004b,a; Piestun et al., 2008, 2011), which can be explained by an increase in thermolysis capacity or a reduction in the thermogenic potential (Yahav et al., 2005). These data indicate that phenotypic changes in broilers can be induced by continuous or intermittent thermal challenges, applied during the embryonic and/or fetal phase of their ontogenetic development. Although in this study we manipulated incubation temperature to analyze its role in increasing heat tolerance, we are interested in whether exposure to high temperatures during incubation could make the broiler chickens more sensitive to cold-temperature conditions, negatively impacting their survival, performance, and meat quality when exposed to low rearing temperatures. This is an important question because temperature variations from one climatic zone to another (and across and within seasons, mainly in winter and spring) may impair posthatching growth.
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Table 1. Formulation and analysis of the basal diet. Ingredient (%)
Grower diet (d 22 to 42)
60.81 35.15 1.63 0.84 0.42 0.25 0.29 0.08 0.01 0.50
63.74 29.79 3.12 1.16 0.76 0.44 0.21 0.23 0.04 0.01 0.50
Calculated composition2 Crude protein (%) Metabolizable energy (kcal/kg) Calcium (%) Sodium (%) Available phosphorus (%) Methionine + cystine (%) Methionine (%) Lysine (%) Threonine (%) Tryptophan (%) Arginine (%)
21.27 2,883 0.85 0.19 0.42 0.88 0.56 1.22 0.79 0.24 1.32
18.86 3,121 0.69 0.20 0.32 0.77 0.49 1.05 0.68 0.21 1.16
1 Provided (per kilogram of diet): starter diet: vitamin A 7,000 IU, vitamin D3 3,000 IU, vitamin E 25 IU, vitamin K 0.98 mg, vitamin B1 1.78 mg, vitamin B2 9.6 mg, vitamin B6 3.5 mg, vitamin B12 10 μ g, folic acid 0.57 mg, biotin 0.16 mg, niacin 34.5 mg, calcium pantothenate 9.8 mg, copper 0.12 g, cobalt 0.02 mg, iodine 1.3 mg, iron 0.05 g, manganese 0.07 g, zinc 0.09 mg, organic zinc 6.75 mg, selenium 0.27 mg, choline 0.4 g, growth factor 30 mg, coccidicide 0.1 g, methionine 1.68 g; grower diet: vitamin A 7,000 U.I., vitamin D3 3,000 U.I., vitamin E 25 IU, vitamin K 0.98 mg, vitamin B1 1.78 mg, vitamin B2 9.6 mg, vitamin B6 3.5 mg, vitamin B12 10 μ g, folic acid 0.57 mg, biotin 0.16 mg, niacin 34.5 mg, calcium pantothenate 9.8 mg, copper 0.12 g, colbalt 0.02 mg, iodine 1.3 mg, iron 0.05 g, manganese 0.07 g, zinc 0.09 mg, organic zinc 6.75 mg, selenium 0.27 mg, choline 0.6 g, growth factor 7.5 mg, coccidicide 0.1g, methionine 1.4g. 2 Calculated values according to Rostagno et al. (2011).
after which 70% relative humidity was applied and egg rotation was stopped. The eggs were injected with an aqueous vitamin C solution (6 μg/100 μL) prior to the start of incubation, based on our previous study showing that 6 mg/egg vitamin C injections pre-incubation do not alter broiler chick hatchability (Sgavioli et al., 2013). Eggs were positioned horizontally, the injection site was cleaned with 100% ethanol, and the eggshells were punctured with a sterile needle [Injex, 13 × 0.38 (27.5 G 1/2 in.)]. Next, a vitamin C solution (Synth, 99% purity, Diadema, Brazil) was injected into the albumen near the end of the egg opposite to the air chamber, about 6 mm beneath the shell. After injection (100 μL), the hole was sealed with a label identifying the treatment and trial. The fresh vitamin C solution was prepared with autoclaved Mili-Q water and kept in a dark bottle, and in a dark environment. The lights were kept off during injections. At hatching, a total of 540 1-day-old chicks (Cobb) (180 chicks from each incubation treatment, with 36 birds per incubation treatment replicate) were distributed into 3 rearing temperature groups: cold, control (the temperature recommended for this strain), and hot. Temperatures from the first to sixth week for each rearing temperature were 1) cold: 32, 30, 26, 22, 18, and 14o C; 2) control: 32, 31, 29, 27, 25, and 23o C, and 3) hot: 32o C during the entire period. Chicks were housed
in one of 3 identical climatic chambers (6.5 × 9 × 2.8 m) with automatic control of temperature and light regime (12L:12D). There was one chamber per rearing temperature and 15 plots/chamber (1 × 2.5 m), and chicks were equally distributed in 5 replicates of 12 birds per incubation treatment. The average body weight of chicks per replicate and incubation treatment was 50 ± 2.5 g. The mean relative humidity inside the chambers during the study period was approximately 57%. Chicks were raised from the first to the 42nd day of life and received water and food ad libitum until age 42 day. The diet was based on corn and soybean meal (Table 1), was altered for the initial (1 to 21 days) and final (22 to 42 days) rearing stages, and met the birds’ nutritional requirements, as described in Rostagno et al. (2011). Chicks were vaccinated against Marek’s disease and Avian Bouba, as well as against Gumboro disease and Newcastle disease during rearing, following the vaccination program recommended for this strain.
Meat Quality Birds were killed on day 42 of rearing and quality analyses of the breast and thigh meat were conducted 24 h postmortem using a total of 90 animals, comprising 10 birds/treatment (2 per trial). The birds were killed
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Starter diet (d 1 to 21)
Corn Soybean meal 45% Soybean oil Dicalcium phosphate Limestone Sodium chloride L -Lysine–HCl (78%) DL -Methionine (99%) L -Threonine Antioxidant (i.e., BHT) Vitamin–mineral supplement1
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Muscle Fiber Morphometry Muscle samples At 42 days age, the pectoralis major (white) and sartorius (mixed) muscles were quickly removed from 5 birds/treatment, which had previously been weighed and killed by electrosensitization followed by bleeding. Samples were taken from the pars sternobranchialis of the pectoralis major muscle and the middle region of the sartorius muscle, and immediately frozen in n-hexane cooled in liquid nitrogen; they were then kept in a freezer at −70◦ C until histochemical and immunohistochemical processing. Histochemical and immunohistochemical processing Fiber size was evaluated from the crosssectional (transverse) area by freeze-cutting 8 to 12μm thick transverse and semiserial sections with a microtome–cryostat at −20o C and submitting them to the NADH tetrazolium reductase (TR) technique and indirect immunohistochemical marking of slow-twitch fibers. These techniques identified three types of fibers: slow-twitch oxidative (SO), fast-twitch oxidative–glycolytic (FOG), and fast-twitch glycolytic (FG) fibers. For histochemical processing with the NADH–TR technique, sections were kept at room temperature for 30 min for drying and fixation on the histological slide. Then, following the method described by Dubowitz and Brooke (1984), sections were incubated with a solution containing NADH (reduced form: 0.08%) and nitro blue tetrazolium (0.1%) (Sigma, St. Louis, MO) in Tris–HCl 0.2 M, pH 7.4, for 40 minutes at 37o C.
Next, the sections were washed with distilled water (3 × 5 min), fixed in 5% formaldehyde buffered at pH 7.0 for 5 minutes, and rinsed again with distilled water (3 times). They were then dehydrated in a series of increasing ethanol concentrations (60, 70, 80, 90, and 100%), diaphanized in xylene (100%), and mounted with Entellan. Oxidative, oxidative–glycolytic, and glycolytic fibers were identified according to their dark, intermediate, and light coloring, respectively. For immunohistochemical processing, the general procedure ´ of DAngelis et al. (2005) was followed. Briefly, 8-μm thick transverse serial sections were obtained from the same sample used for histochemical analyses. The sections were subjected to indirect immunohistochemistry (peroxidase–antiperoxidase) for the marking of fibers containing slow-twitch myosin. Initially, the sections were kept for 30 minutes at room temperature for drying and adherence to histology slides. Next, they were fixed in 3.5% formalin for 30 minutes, washed in 0.1 M phosphate buffer (pH 7.2; 2 × 2 min each), and incubated with 3% hydrogen peroxide in methanol for 5 minutes to block endogenous peroxidase. The sections were washed once again with phosphate buffer and incubated with nonspecific rabbit serum (1:50 in phosphate buffer) for 30 minutes in a humid chamber at 4◦ C to control the background. The serum was removed and the sections were incubated with slow-twitch antimyosin monoclonal primary antibody produced in mice (1:400 in phosphate buffer) (NOQ7.5.4D, Sigma, St. Louis, MO) for 150 min in a humid chamber at 4◦ C. Once again, the sections were washed with phosphate buffer (2 × 4 min each, under gentle agitation) and then incubated with peroxidase-conjugated goat antimouse IgG secondary antibody (1:1,000 in phosphate buffer) (Sigma, St. Louis, MO) for 90 min in a humid chamber at 4◦ C. A new wash with phosphate buffer was then conducted (2 × 4 min each). The antigen– antibody complexes were revealed by incubation of the sections with diaminobenzidine (0.2 mg/1.5 mL 3,3 diaminobenzidine per 0.1 M phosphate buffer, pH 7.2) (Sigma, St. Louis, MO) at room temperature, containing 2 to 3 drops of hydrogen peroxide. The staining intensity was visually inspected and development was blocked by washing the sections with phosphate buffer. Subsequently, the sections were dehydrated in a series of increasing ethanol concentrations (60, 70, 80, 90, and 100%), diaphanized in xylene (100%), and mounted with Entellan. Slow-twitch fibers had a golden color with slow-twitch antimyosin antibody, while fast-twitch fibers were not marked. The negative control of the antigen–antibody reaction was conducted by omitting incubation with primary antibody.
Hatchability, Body Weight, Feed Intake, and Feed Conversion Rates Hatchability was determined as a function of the total number of fertile eggs. The chicks were weighed
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by electrosensitization followed by bleeding. Prior to slaughter, the broilers were fasted for 10 h. The pH was determined by directly inserting an electrode into the muscles using a digital pH meter (Testo, model Testo106, Lenzkirch, Germany). Meat color was measured from the inner portion of the breast and over-thigh using Chrome Meter CR-300 equipment (Konica Minolta Sensing, Osaka, Japan) and the CIELAB trichromatic system, which determines lightness (L∗ ), redness (a∗ ), and yellowness (b∗ ) values. The water-holding capacity was measured by press loss: 1 g from each meat sample was pressed with a 10-kg weight for 5 minutes at 25o C; each sample was subsequently reweighed to calculate water-holding capacity, and this value was expressed as a percentage of the initial weight. To determine cooking losses, samples were placed in plastic bags and cooked in a water bath at 85◦ C for 30 minutes at a final internal temperature of 75 to 80◦ C. After releasing the exuded water and cooling to reach room temperature, they were again weighed and compared to the initial weight (Cason et al., 1997). Warner–Bratzler shear force was measured on the cooked samples previously used in the cooking loss analysis using a Texture Analyzer TAXT2i (Godalming, Surrey, United Kingdom) and expressed as kilograms per square centimeter (Lyon et al., 1998).
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immediately after their plumage was dry. Broiler weight was measured on day 42 on a replicate basis. Amounts of feed offered to each replicate were recorded weekly, and uneaten feed in each replicate was weighed (from 1 to 42 d). Individual bird feed intake for each replicate was determined by dividing the cumulative feed intake by the total number of living birds in the replicate. Feed conversion ratio was calculated by dividing the amount of feed intake by the body weight gain (grams per gram).
Statistical Analyses
RESULTS Hatchability, Body Weight, Feed Intake, and Feed Conversion Hatchability was lower for the high-temperature groups (with and without vitamin C) than for the control group group (control: 84.50a ± 1.12%, 39o C: 74.70b ± 1.20%, 39o C+vitamin C: 74.40b ± 9.24%, P < 0.0189). However, absolute chick weight at hatch was not altered by these incubation treatments relative to the control groups (control: 51.04 ± 2.24 g, 39o C: 50.22 ± 1.28 g, 39o C+vitamin C: 50.39 ± 2.12 g, P = 0.6718). At the end of the study period, the body weight, feed intake, and feed conversion rates were not affected by either incubation treatment (P = 0.99, P = 0.48 and P = 0.42, respectively) or by the interaction between incubation treatments and rearing temperatures (P = 0.81, P = 0.18, P = 0.37, respectively). However, the rearing temperature affected the 3 variables, which were lower under hot rearing temperatures than under cold or control temperatures (these last 2 variables did not differ from each other; P < 0.05, Figure 1).
Breast Characteristics Table 2 shows the effects of the incubation treatments and the rearing temperatures on meat quality and fiber area of the breast muscle (pectoralis major). The cooking loss, pH, L∗ , and b∗ values were not influenced by the incubation treatments or the rearing temperatures
Figure 1. Effects of rearing temperatures (cold, control, and hot) on broiler body weight at 42-days-old (A), feed intake (g per bird) from day 1 to 42 age (B), and feed conversion (C). Bars with distinct letters indicate a difference between means (P < 0.05).
(P > 0.05). Shear force and water-holding capacity were affected only by the incubation treatments (P < 0.05), while a∗ was affected by both the incubation treatments and the rearing temperatures (P < 0.05). Shear force was greater under incubation at 39o C than at 37.5o C or 39o C+vitamin C. The water-retention capacity was similar for incubation at 37.5o C and at 39o C, and both of these were greater than at 39o C+vitamin C. Redness (a∗ ) was similar for incubation at 37.5o C and at 39o C+vitamin C, and was greater in both of these treatments than at 39o C. Redness was also greater under cold rearing temperatures than under hot or control temperatures, neither of which affected a∗ . The FG fiber area of the pectoralis major muscle was not affected by incubation temperature (P > 0.05),
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All data were assessed with one-way ANOVA using the general linear model procedure of the SAS software package (SAS Institute, 2002), and statistical significance was set at P ≤ 0.05. For hatchability and chick weight at hatch, we verified the effects of each of the incubation treatments (37.5o C, 39o C, 39o C+vitamin C). For body weight, feed intake, feed conversion, fiber area, and meat quality characteristics, we assessed the effects of incubation treatments (I), rearing temperatures (R: cold, control, and hot) and the respective interactions (IR) using the following model: Yijk = μ + Ii + Rj + (IR)ij + eijk. For differences among groups, mean comparisons were conducted with Tukey’s tests.
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FERREIRA ET AL. Table 2. Effects of incubation treatments and rearing temperatures on pectoralis major meat quality and cross-sectional area of muscle fibers in 42-day-old broilers. Variables1
CL (%)
SF (kgf cm−2 )
pH
WHC (%)
CSAF (μ m2 )
Color L∗
a∗
b∗
Incubation treatments (IT) 37.5o C 27.12 26.38 39o C 39o C+vitamin C2 26.19 Rearing temperatures (RT) Cold 25.77 Control3 27.55 Hot 26.48 P-values IT 0.7560 RT 0.4170 IT x RT 0.5355 11.50 CV (%)4
2.34b 2.79a 2.44b
6.01 6.03 6.04
74.81a 74.53a 69.39b
52.34 51.95 52.34
2.64a 1.90b 2.30ab
4.16 4.48 4.59
5,033 5,325 5,115
2.63 2.49 2.43
6.04 6.03 6.00
72.29 72.91 73.68
51.09 51.84 53.56
2.78a 2.12b 1.98b
4.23 4.80 4.20
5,413a 5,612a 4,448b
0.0013 0.2799 0.0784 20.43
0.6639 0.5240 0.4964 1.82
< 0.0001 0.1392 0.6914 4.98
0.9456 0.1269 0.1008 5.63
0.0211 0.0075 0.1478 24.54
0.8022 0.5463 0.2083 31.08
0.8032 0.0329 0.2675 24.11
Table 3. Effects of incubation treatments and rearing temperatures on over-thigh meat quality and cross-sectional area of sartorius muscle fibers in 42-day-old broilers. Variables1
CL (%)
Incubation treatments 37.5o C 33.30 33.33 39o C 31.39 39o C-vitamin C2 Rearing temperatures Cold 31.99 33.73 Control3 Hot 32.52 P-values IT 0.0909 RT 0.2666 IT x RT 0.0039 CV (%)4 7.68
SF (kgf cm−2 )
WHC (%)
pH
Color L∗
a∗
b∗
3.59 3.53 3.67
70.75 72.18 71.42
6.19 6.16 6.13
53.74 53.76 55.65
3.70 3.81 3.90
5.43 5.80 6.01
3.47 3.70 3.60
71.67 72.14 70.51
6.16 6.17 6.16
53.23 54.58 55.04
4.63a 3.55a,b 3.20b
5.24 6.39 5.57
0.8592 0.4812 < 0.0001 18.02
0.1183 0.0768 0.0017 4.36
0.6828 0.9808 0.3938 2.38
0.3079 0.4310 0.0957 5.96
0.8554 0.0100 0.2745 28.40
0.7164 0.2464 0.0914 29.79
1 CL: cooking losses. SF: shear force. WHC: water holding capacity. L∗ : lightness. a∗ : redness. b∗ : yellowness. 2 39o C+vitamin C: incubation at 39o C plus intra eggs injection of vitamin C. 3 Control: standard rearing temperature for lineage. 4 CV: coefficient of variation. a,b Means with different superscripts differ significantly (P < 0.05).
but was modified by rearing temperature (P < 0.05): it was smaller in chickens raised under hot temperatures than under control or cold temperatures, with no difference in fiber area observed between broilers raised under these last 2 temperatures.
Over-Thigh Characteristics Table 3 shows the effects of the treatments during incubation and rearing temperatures on over-thigh meat quality. Neither the pH nor the L∗ or b∗ indices were altered by incubation treatment or rearing temperature, and there were also no interactions between these vari-
ables (P > 0.05). Redness (a∗ ) was only affected by rearing temperature (greater under cold than hot temperatures), while there were no differences in a∗ under thermoneutral conditions relative to cold and hot temperatures. There were significant interactions between incubation treatments and rearing temperature for cooking loss, shear force, and water-retention capacities (P < 0.05). Specifically, rearing temperature only affected the cooking loss of the over-thighs of broilers from eggs incubated at 37.5o C, which was greater for broilers raised under hot (relative to cold) temperatures, and did not differ from broilers raised under control temperatures (Table 4). Incubation
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1 CL: cooking losses. SF: shear force. WHC: water holding capacity. L∗ : lightness. a∗ : redness. b∗ : yellowness. CSAF: cross-sectional area of the fibers. 2 39o C+vitamin C: incubation at 39o C plus intra eggs injection of vitamin C. 3 Control: standard rearing temperature for lineage. 4 CV: coefficient of variation. a,b Means with different superscripts differ significantly (P < 0.05).
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TEMPERATURE AND VITAMIN C IN BROILER MUSCLES Table 4. Interaction between incubation treatments and rearing temperatures for over-thigh meat quality of 42-day-old broilers. Variables1
Incubation treatments
Rearing temperatures (o C) 2
Cold o
B
Control
A,B
P-values
Hot
CL (%)
37.5 C 39o C 39o C+vitamin C P-values
30.10 33.36 32.50 0.1868
33.66 33.94 33.55 0.9770
37.01A,a 32.69a,b 27.75b 0.0006
0.0050 0.7812 0.0598
SF (kgf cm−2 )
37.5o C 39o C 39o C+vitamin C P-values
3.05B 3.82A 3.53 0.0635
3.43A,B 4.00A 3.68 0.2204
4.29A,a 2.78B,b 3.71a,b < 0.0001
0.0011 0.0007 0.8408
WHC (%)
37.5o C 39o C 39o C+vitamin C P-values
70.65A,B,b 74.03a 70.32b 0.0066
72.78A 72.41 71.24 0.0802
68.43B 70.11 72.83 0.1564
0.0071 0.4525 0.0962
treatments also affected the cooking loss of the overthighs of broilers raised under hot temperatures. Cooking loss was greater for broilers from eggs incubated at 37.5o C than at 39o C and injected with vitamin C, but it did not differ from that of broilers from eggs incubated at 39o C and not injected with vitamin C. Shear force was affected by rearing temperature in broilers from eggs incubated at 37.5 and 39o C. In the first group, the shear force did not differ between broilers raised under control temperatures and those raised under the other 2 temperatures, but was greater in broilers raised under hot relative to cold temperatures. In broilers from eggs incubated at 39o C, on the other hand, the shear force was lower for broilers raised under hot temperatures than for those raised under cold or control temperatures. Only in broilers raised under hot temperatures did incubation temperature affect shear force, which was greater in broilers from eggs incubated at 37.5o C than at 39o C, but not in broilers from eggs incubated at 39o C and injected with vitamin C. The interaction also revealed effects of rearing temperature and incubation treatments on the water-retention capacity of the over-thigh meat only in broilers from eggs incubated at 37.5o C, which was greater under hot than control rearing temperatures. Chickens raised under cold temperatures did not have altered water-retention capacity, which was lower when raised under hot temperatures than under control temperatures. Incubation treatments affected water-retention capacity only in broilers raised under cold temperatures. Retention capacity was greater in broilers from eggs incubated at 39o C than those incubated at 37.5o C and at 39o C that were also injected with vitamin C; these last 2 conditions did not differ from each other in terms of water retention. Tables 5 and 6 present the results obtained for the mean cross-sectional area of sartorius muscle fibers in
Table 5. Effects of incubation treatments and rearing temperatures on mean cross-sectional area of sartorius muscle fibers in 42-day-old broilers. Fiber types1
SO (μ m2 )
Incubation treatments 2,346 37.5o C 39o C 3,048 39o C+vitamin C2 2,917 Rearing temperatures Cold 2,547 Control3 2,859 Hot 2,905 P-values IT 0.0515 RT 0.4240 IT x RT 0.0242 CV (%)4 29.03
FOG (μ m2 )
FG (μ m2 )
2,656 3,360 2,750
3,299 3,753 3,036
2,964 2,842 2,961
3,570 3,371 3,138
0.0401 0.8905 0.0004 26.98
0.0419 0.4179 0.0064 22.18
1 SO: slow oxydative fiber. FOG: fast oxydative and glycolytic fiber. FG: fast glycolytic fiber. 2 39o C+vitamin C: incubation at 39o C plus intra eggs injection of vitamin C. 3 Control: standard rearing temperature for Cobb lineage. 4 CV: coefficient of variation (P < 0.05).
42-day-old broilers. There was a significant interaction between incubation treatments and rearing temperatures for the area of SO, FOG, and FG fibers (P < 0.05) (Table 5). This interaction (Table 6) revealed that rearing temperature did not alter the area of FG fibers, but affected the area of SO and FOG fibers, and only in broilers from eggs incubated at 37.5o C. Incubation treatments altered the area of SO, FOG, and FG fibers of broilers raised under cold temperatures, and the area of FOG and FG fibers of broilers raised under hot temperatures. Among broilers raised under cold temperatures, the area of SO fibers was larger in birds from eggs incubated at 39o C than at 37.5o C, whether or not they received a vitamin C injection. The area of FOG and
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1 CL: cooking losses. SF: shear force. WHC: water holding capacity. 39o C+vitamin C: incubation at 39o C plus intra eggs injection of vitamin C. 2 Control: standard rearing temperature for Cobb lineage. a,b Means (columns) with different superscripts differ significantly (P < 0.05). A,B Means (rows) with different superscripts differ significantly (P < 0.05).
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FERREIRA ET AL. Table 6. Interaction between incubation treatments and rearing temperatures for cross-sectional area of the sartorius muscle fibers in 42-day-old broilers. Fiber types1
Incubation treatments
Rearing temperatures (o C) Cold
Control3
Hot
P-values
SO (μ m2 )
37.5o C 39o C 39o C+vitamin C2 P-values
1,545B,b 3,019a 3,077a 0.0064
2,268A,B 3,018 3,291 0.1293
3,227A 3,107 2,382 0.2133
0.0082 0.9800 0.1894
FOG (μ m2 )
37.5o C 39o C 39o C+vitamin C P-values
1,791B,b 3,944a 3,156a,b 0.0005
2,336A,B 3,347 2,843 0.1431
3,843A,a 2,789a,b 2,250b 0.0098
0.0006 0.0822 0.1963
FG (μ m2 )
37.5o C 39o C 39o C+vitamin C P-values
2,831b 4,229a 3,649a,b 0.0193
3,165 4,020 2,926 0.0644
4,052a 3,010a,b 2,534b 0.0154
0.0577 0.0517 0.0699
FG fibers was larger in broilers from eggs incubated at 39o C than at 37.5o C, and there was no difference in the values recorded for broilers from these 2 treatments and those incubated at 39o C and injected with vitamin C. Among broilers raised under hot temperature, the areas of FOG and FG fibers were influenced by incubation at 39o C only when the eggs also received a vitamin C injection, and these areas were smaller in these broilers than in those incubated at 37.5o C.
DISCUSSION In this study, we studied 1) whether injection of vitamin C in eggs reduces the effects of incubation at high temperatures on hatchability and chick weight; 2) whether continuous high incubation temperatures with or without preincubation injections of vitamin C into the eggs changes performance, muscle fiber hypertrophy, and meat quality of the breast and over-thigh of broilers reared at high temperatures; and 3) the effects of these incubation treatments when broilers are reared under cold-temperature conditions. Our results showed that high incubation temperatures with or without vitamin C injection into the eggs reduced or eliminated the effects of the high rearing temperature on the meat quality of the over-thigh. This involved changes in muscle fiber hypertrophy but did not alter zootechnical variables (except for hatchability, which was reduced).
Hatchability, Body Weight, Feed Intake, and Feed Conversion Although vitamin C has been attributed an antistressor role (Pardue and Thaxton, 1986; Mahmoud et al., 2004), its injection into the egg did not prevent
the decrease in hatching rate caused by high temperature incubation. In the current study, cold rearing temperatures did not influence broiler performance, while hot rearing temperatures reduced broiler feed intake, feed conversion, and weight gain, as previously reported (Aksit et al., 2006; Imik et al., 2012). High incubation temperatures (with or without vitamin C injections into the egg) did not reduce or prevent the negative effects of hot rearing temperatures on broiler performance, nor did they influence body weight, feed intake, and conversion of broilers reared under cold temperatures. These results showed that high-temperature exposure during the whole incubation period did not grant the broilers a pre-adaptation to hot rearing temperatures, nor did it make them more sensitive to cold-rearing conditions.
Breast Characteristics Although no effect of high incubation temperatures plus vitamin C on performance was shown, we observed smaller effects of incubation treatments and/or rearing temperatures on a few meat quality characteristics, and on the size of FG fibers in broiler breasts. To the best of our knowledge, this is the first study indicating that an increase in the amount of vitamin C in the egg affects the quality of broiler breast meat as well as the effects caused by thermal stress. High incubation temperatures increased the shear force and reduced a∗ , making the breast meat tougher and less red without altering its cooking loss, pH, or luminosity. Injecting vitamin C into the eggs prevented the effects of high-temperature incubation. Nonetheless, the capacity for water retention decreased by approximately 5% in breast meat, making it less succulent. Vitamin C is required for amino acid and mineral metabolism, as well as for hormonal
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1 SO: slow oxydative fiber. FOG: fast oxydative and glycolytic fiber. FG: fast glycolytic fiber. 2 39o C+vitamin C: incubation at 39o C plus intra eggs injection of vitamin C. 3 Control: standard rearing temperature for Cobb lineage. a,b Means (columns) with different superscripts differ significantly (P < 0.05). A,B Means (rows) with different superscripts differ significantly (P < 0.05).
TEMPERATURE AND VITAMIN C IN BROILER MUSCLES
2012) and present high metabolic plasticity, perhaps the redder coloring we observed in the present study could have been caused by the increased vascularity of the fibers associated to metabolic changes. Further, the lower hypertrophy of the breast muscle fibers of broilers raised under hot temperatures may have been caused by the reduced feed intake and body weight reported in this study, as well as by the reduced amino acid and mineral metabolism resulting from insufficient endogenous vitamin C availability (caused in turn by the higher heat-induced demand) (Thornton, 1961; Pardue and Thaxton, 1986; Kutlu and Forbes, 1993). Our data differ from those observed by Aksit et al. (2006), who reported increased redness in the breast meat of broilers reared under hot temperatures.
Over-Thigh Characteristics In the present study, we also observed an interaction of the effects of incubation and rearing temperatures on meat quality parameters, and muscle fiber size in broiler over-thigh meat. High incubation temperatures reduced over-thigh meat shear force in broilers raised under hot temperatures and increased the water-retention capacity in broilers raised under low temperatures, making the meat more tender and succulent, respectively. These high incubation temperature effects on meat quality were eliminated when vitamin C was injected into the egg, confirming the important role of vitamin C in heat stress. As mentioned above, several authors reported that an increase in the muscle fiber area in a transverse cut negatively affects meat texture and succulence, reducing cooking loss and water-retention capacity, and increasing the shear force (Seideman and Crouse, 1986; Rehfeldt et al., 2000). However, this was not observed in the current study in broilers raised under hot temperatures and incubated at 37.5o C or at 39o C and injected with vitamin C. In contrast, however, we observed an increase in the cross-sectional area of the fibers (slowtwitch oxidative, fast-twitch oxidative–glycolytic, and fast-twitch glycolytic) together with an increase in the water-retention capacity of the over-thigh meat of broilers from eggs that were incubated at high temperatures and raised under cold temperatures. In the present study, hot rearing temperatures increased the redness (a∗ ) of the thigh meat, in agreement with previous work by Zhang et al. (2012) in which redness was associated with an increase in the amount of oxidative and oxidative–glycolytic fibers. In addition, the hot rearing temperature reduced thigh meat tenderness and succulence of broilers from eggs incubated at the usual temperature (37.5o C) due to the increased cooking loss and shear force, and the decreased water retention caused by the greater hypertrophic growth of the SO and FOG fibers (as well as FG fibers, if we consider 6% significance). On the other hand, cold-rearing temperatures caused the opposite effect and improved meat quality while reducing the area of fibers in the
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production linked to stress resistance (Abidin and Khatoon, 2013). Heat conditions increase the utilization and demand for vitamin C, making its endogenous production by chickens insufficient for homeostasis maintenance (Thornton, 1961; Pardue and Thaxton, 1986; Kutlu and Forbes, 1993). Under these conditions, exogenous vitamin C can play an important role in the maintenance of physiological processes. In line with this view, the pre-incubation administration of vitamin C into the egg in this study appears to have supplied, at least in part, the demand for vitamin C caused by exposure to heat. Thus, in line with previous studies (see, Mckee et al., 1997; Lin et al., 2006; Attia et al., 2009), our results highlight the potential role of vitamin C in reducing or eliminating heat stress effects, as well as the potential use of intra-egg injections of vitamin C before incubation. Nonetheless, more detailed studies should be conducted to better understand the biological processes involved in the antiheat effects of vitamin C on pectoral muscles. Several authors have reported that a larger area of myofibers in a transverse cut implies a lower waterretention capacity and greater shear force (Larzul et al., 1997; Maltin et al., 1997), which promote meat toughness (Seideman and Crouse, 1986; Rehfeldt et al., 2000); furthermore, the higher the muscle’s glycolytic content (caused by an increase in fiber number or size), the greater the cooking losses, lightness (L∗ ), paleness (b ), and shear force, and the lower the pH and the water-retention capacity, which affects texture and succulence. According to Berri et al. (2007), an increase in the size of broiler breast muscle fibers increased the pH and improved water-retention capacity and meat quality. In the current study, the area of FG fibers in the pectoralis major muscle was not affected by high incubation temperatures with or without vitamin C injection into the egg; this is in contrast to data reported by Werner et al. (2010), who showed an increase in fiber size promoted by high temperature from the seventh to the 10th day of incubation. Therefore, our data show that the effects of high incubation temperature together with vitamin C on broiler breast meat did not result in the alteration of the hypertrophic growth of the muscle fibers, and that this lack of fiber alteration may explain the lack of alteration in pH, cooking losses, and luminosity. In the present study, broiler breast meat was redder (a∗ ) under cold-rearing temperatures, while the transverse area of the glycolytic fibers was smaller under hot-rearing temperatures. Acquisition of red color by white muscles was also observed by Hirabayashi et al. (2005) in the breast muscles of chicks exposed to coldtemperature conditions during incubation. Duchamp et al. (1992) reported that birds submitted to cold temperatures show an increase in oxidative activity and in the proportion of oxidative fibers in skeletal muscles. Considering that the pectoralis major muscle in broilers contains glycolytic fast-twitch fibers that grant the meat a white color (Rosser et al., 1996; Alves et al.,
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transverse cut. These opposing effects were likely due to the greater feed intake in broilers raised under cold temperatures. The inverse relation between muscle fiber size and meat quality in over-thighs of broilers from eggs incubated at the control temperature (37.5o C) and raised under either cold or hot temperatures is in agreement with results reported for breast meat by Berri et al. (2007). Even just the effects of rearing temperature on fiber hypertrophy and meat quality on the over-thighs of broilers from eggs incubated at the control temperature are an important contribution of the current study. To the best of our knowledge, this is the first study showing that continuous high incubation temperatures prevent the effects of thermal stress during rearing on the quality of thigh meat in broilers.
High incubation temperature affects the shear force, water-retention capacity, and cooking loss in chicken meat. In addition, it prevents toughness and the reduction in succulence caused by high rearing temperatures in the thighs of chickens from eggs incubated at the control temperature. Further, it improves the succulence and tenderness of thigh meat, in chickens raised under cold and hot temperatures, respectively. Vitamin C prevents the effects of high incubation temperature on meat quality and on the area of FOG and FG fibers of chickens raised under cold temperatures, as well as the shear force in chickens raised under hot temperatures. High incubation temperature during the fetal phase plus vitamin C injection into the egg prevents the thermal stress effects of high rearing temperatures.
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CONCLUSIONS
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