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Influence of dietary fiber sources on the physiological and behavioral stress responses of Greylag geese (Anser anser)
C 2016 Poultry Science Association Inc.
Influence of dietary fiber sources on the physiological and behavioral stress responses of Greylag geese (Anser anser) L. W. He,1 Q. X. Meng, D. Y. Li, Y. W. Zhang, and L. P. Ren State Key Laboratory of Animal Nutrition, College of Animal Science and Technology, China Agricultural University, Beijing 100193, P. R. China
SUMMARY This experiment investigated the physiological and behavioral responses of Greylag geese (Anser anser) fed different fiber sources when exposed to the common stressors at harvest, primarily involving the procedures of fasting, transport, catching, etc. A total of 240 4-weekold Greylag geese were allocated to 4 diets with different fiber sources (corn straw silage [CSS], steam-exploded corn straw [SECS], steam-exploded wheat straw [SEWS], and steamexploded rice straw [SERS]). The birds were fed for 12 wk and harvested at 16 wk for slaughter. Blood samples designated to determine stress status were collected before (pre-stress) and after (post-stress) the harvest procedure was carried out. Irrespective of the dietary differences, the harvest procedure caused the concentration of superoxide dismutase (SOD) to drop (P < 0.01) 13.57% and increased (P < 0.01) that of creatine kinase (CK) by 186.21% (post-stress vs. pre-stress), inferring that the birds had experienced a severe stress. As to the dietary effects, the birds fed alternative fiber sources did not develop dissimilar (P > 0.05) pre-stress blood parameters and behavioral sensitivities, while the birds fed CSS had a higher (P < 0.05) poststress concentration of methane dicarboxylic aldehyde (MDA) than those offered SECS by 22.01%, suggesting that feeding Greylag geese alternative fiber sources influenced their stress response. Key words: Greylag goose, fiber source, harvest stress, blood parameter 2016 J. Appl. Poult. Res. 00:1–7 http://dx.doi.org/10.3382/japr/pfw023
DESCRIPTION OF PROBLEM Stress arises when individuals perceive that they cannot adequately cope with the demands being made on them or with threats to their well-being [1]. In response to such situations, a physiological stress response is initiated, consisting of activation of the hypothalamic– pituitary–adrenal (HPA) axis with release of catecholamines and corticosteroids [2] and 1
Corresponding author: [email protected]
becoming more alert in order to increase their survival [3]. In general, stress exerts to some extent adverse effects on the animal performance, food safety, and quality concerns [4], limiting the efficiency of production and causing economic loss to the farm [5]. Any improvement in production that can reduce stress is economically worthwhile. Generally, there are several blood parameters such as cortisone (CORT), superoxide dismutase (SOD), methane dicarboxylic aldehyde (MDA), and creatine kinase (CK) selected to detect the metabolic changes and
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Primary Audience: Flock Supervisors, Researchers, Nutritionists
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2
MATERIALS AND METHODS Experimental Management
Design
and
Animal
A total of 240 healthy 4-week-old Greylag geese (Anser anser) obtained from a commercial
hatchery [12] were randomly allocated to 4 diets differing in fiber source (corn straw silage [CSS], steam-exploded corn straw [SECS], steam-exploded wheat straw [SEWS], or steam-exploded rice straw [SERS]) [13]. The diets were formulated to meet the nutrient requirements of growing geese (Table 2) [14]. During the study (12 wk), the birds had access to pelletized feed and clean water ad libitum without artificial lightning [15]. This study was performed at the Animal Experimental Station of China Agricultural University, where all procedures were approved by the Animal Care and Use Committee [16].
Chemical Analysis of the Diets The chemical composition of the fiber sources (Table 1) and the diets (Table 2) were analyzed according to AOAC methodology [17]. Crude protein (CP) was analyzed using the combustion nitrogen analysis (FP-528, Leco, St. Joseph, Missouri) [18]. Crude fiber (CF) was measured by a fiber analyzer (A220, ANKOM Technology, Macedon NY) as well as neutral detergent fiber (NDF), acid detergent fiber (ADF), and lignin (ADL) [19]. Ether extract (EE) was analyzed by an extraction system (ANKOMXT10 EXTRACTOR, ANKOM Technology, Macedon NY). Calcium was determined by atomic absorption spectrophotometry (WFX320, BRAIC, Beijing, China), and phosphorus was determined by UV spectrophotometry (UVVIS 8500, Tianmei Scientific Instrument Co., Ltd., Shanghai, China) [20].
Evaluation of the Behavioral Sensitivity to Stress In order to evaluate the behavioral sensitivity to stress, the birds were placed separately into a plastic barrel (0.6 m in diameter, 0.8 m in height) at the end of the rearing period. Behavioral response was scored based on a 5-point scale (Table 3). A higher score means that the birds were more fearful of the unfamiliar condition and would be prone to suffer stress with high behavioral sensitivity.
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diagnose the stress status. Measuring HPA function is a standard approach to study stress and welfare in farm animals [6]. Throughout an animal’s life, it is inevitably exposed to various types of normal stress during transportation, weaning, vaccination, diet transition, etc. Transport is an integral part of today’s livestock industry [7] with animals being transported at least once during their production life for reasons such as assembly for harvest purposes [8], during which there would be a potential for the animals to experience stress, injury, even mortality due to limited access to feed and water, exposure to stimulus like noise and vibration, as well as poor handling and mixing with unfamiliar animals [9]. Moreover, it is reported that an animal’s fearfulness and its physiological stress response are positively related. Factors influencing an animal’s fear level can be its genetic makeup, unpredictable and uncontrollable early life experiences, and associative learning experiences [10]. Some species with a highly sustained fear level may have an increased vulnerability to stress in later life [11]. There are many potential factors contributing to the stress of poultry, such as nutrition, environment, management, and treatment. The diet could be a stressor when it is formulated with low digestibility and poor nutrients, which results in a lack of feed intake or biased nutrient ingestion. Alterations in physiology and behavior due to domestication could have influenced the adaptation to stress with modifications to endocrine and behavioral profiles [5]. Our hypothesis was that various dietary conditions (dietary components) in early life might result in different behavioral sensitivities along with different physiological stress responses in later life. Understanding how birds cope with common stress is fundamental for the improvement of their welfare. This experiment investigated the physiological and behavioral responses of Greylag geese (Anser anser) fed different fiber sources when exposed to common stressors at harvest.
HE ET AL.: STRESS RESPONSE IN GREYLAGS
3
Table 1. Chemical compositions of the 4 fiber sources (g/kg, as DM basis). Fiber source1 Item2
CSS
SECS
SEWS
SERS
DM CP CF EE NDF ADF ADL Ash
928.38 70.35 379.50 15.13 726.18 482.42 70.58 103.60
944.50 60.06 347.40 26.45 546.23 460.08 83.91 89.53
941.44 46.62 399.60 22.36 563.61 513.41 103.42 107.62
946.62 51.91 374.90 22.50 597.45 510.09 86.15 122.64
1
Table 2. Ingredients and nutrient compositions of experimental diets. Treatments1 Item Ingredient (g/kg) Corn Wheat bran Soybean meal DDGS2 Alfalfa particles Corn straw silage Steam-exploded corn straw Steam-exploded wheat straw Steam-exploded straw Lysine hydrochloride Methionine Sodium chloride Limestone Calcium hydrophosphate Active ferment3 Premix4 Chlortetracycline5 Calculated nutrient concentration (g/kg) Metabolizable energy (Kcal/kg) Lys Met Analyzed concentration (g/kg) Crude protein Crude fiber Calcium Total phosphorus 1
CSS
SECS
SEWS
SERS
350.0 50.0 210.0 30.0 30.0 300.0 3.0 2.0 3.0 4.0 6.0 1.0 10.0 1.0
350.0 50.0 210.0 30.0 30.0 300.0 3.0 2.0 3.0 4.0 6.0 1.0 10.0 1.0
350.0 50.0 210.0 30.0 30.0 300.0 3.0 2.0 3.0 4.0 6.0 1.0 10.0 1.0
350.0 50.0 210.0 30.0 30.0 300.0 3.0 2.0 3.0 4.0 6.0 1.0 10.0 1.0
2150 0.56 0.42
2150 0.56 0.42
2150 0.56 0.42
2150 0.56 0.42
16.25 14.88 0.50 0.38
16.39 12.33 0.42 0.40
16.14 14.10 0.44 0.38
16.76 12.83 0.43 0.40
CSS: Corn straw silage; SECS: Steam-exploded corn straw; SEWS: Steam-exploded wheat straw; SERS: Steam-exploded rice straw. 2 DDGS: Distillers dried grains with soluble. 3 Angel (golden), live yeast number not less than 2 billion per gram. 4 Provided per kg of diet: Cu,7.6 mg; Fe, 75.0 mg; Zn, 69.8 mg; I, 1.5 mg; Mn, 75 mg; Se, 0.2 mg; vitamin A, 8,328 IU; vitamin D3 , 3,987 IU; vitamin E, 59 IU; vitamin K 6.0 mg; vitamin B1 , 2.6 mg; vitamin B2 , 12 mg; vitamin B6 , 5.3 mg; vitamin B12 , 0.4 mg; nicotinic acid, 52.0 mg; folic acid, 1.3 mg; biotin, 0.6 mg. 5 Effective component, 15%.
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CSS: Corn straw silage; SECS: Steam-exploded corn straw; SEWS: Steam-exploded wheat straw; SERS: Steam-exploded rice straw. 2 DM: Dry matter; CP: Crude protein; CF: Crude fiber; EE: Ether extract; NDF: Neutral detergent fiber; ADF: Acid detergent fiber; ADL: Acid detergent lignin; Ash: Ash.
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4 Table 3. Ethogram used for the behavioral recordings of Greylag geese (Anser anser). Scoring 1 2 3 4
5
were purchased from Bio-Sino Bio-Technology and Science Inc., China [22].
Descriptions of behavioral responses Sits down quickly and keeps quiet without any scrabble after put into the barrel Moves slowly and stands looking around with slight scrabble after put into the barrel Wags head rapidly and pushes feet out with medium scrabble after put into the barrel Wags head rapidly and powerfully pushes feet out with severe scrabble and strong escaping intention after put into the barrel Constant and severe escape behavior
Statistical Analysis
Harvest Procedure and Blood Sampling To determine the physiological response to stress (fasting, capture, transport, crowding, fear, etc.), at 16 wk of age, one bird was randomly selected from each pen (60 pens in total) and was bled by venipuncture with a blood needle before a 12-h fasting period (i.e., pre-stress); after the fast, the birds were placed individually in bags with their heads provided an opening to the outside when transported half an h to the slaughter plant by platform trailer without shelter and then blood samples were collected again on the spot before slaughter (i.e., post-stress). Analysis of Blood Samples All the blood samples were prepared for blood serum and used to determine the parameters, including mainly CORT, SOD, MDA and CK, which were generally used to indicate stress status. The serum parameters were assayed by a testing agency (Beijing Sino-UK of Biological Technology, Beijing, China) [21] and the reagent kits
RESULTS AND DISCUSSION Irrespective of the dietary differences, the harvest stress resulting from the common potential stressors in practice decreased (P < 0.001) the concentration of SOD and increased (P < 0.001) that of CK in the blood (post-stress vs. pre-stress) along with no significant influence (P > 0.05) on the concentrations of CORT and MDA (Table 4), showing that the birds had suffered significant stress influence. As to dietary effects, no difference (P > 0.05) was found on the pre-stress blood parameters of growing Greylag geese fed alternative fiber sources, and they developed similar (P > 0.05) behavioral sensitivities (Table 5). The birds fed CSS had a higher (P < 0.05) post-stress concentration of MDA than those offered SECS, along with a similarity (P > 0.05) to those fed the other 2 fiber sources (SEWS and SERS), inferring that the birds fed different diets developed a dissimilar stress response; there was no difference
Table 4. Influence of harvest stress on the blood parameters of Greylag geese (Anser anser). Treatment Item1 CORT (ng/mL) SOD (U/mL) MDA (nmol/mL) CK (U/L) 1
Pre-stress
Post-stress
SEM2
61.35 84.46a 10.36 137.32b
61.96 73.00b 11.14 393.02a
0.76 1.46 0.33 21.86
CORT: Cortisone, SOD: Superoxide dismutase, MDA: Methane dicarboxylic aldehyde, CK: Creatine kinase. SEM: Standard Error of Mean. a,b Means in a row with uncommon superscripts differ significantly at P < 0.05. ∗∗∗ P < 0.001, NS = P ≥ 0.05. 2
P-value NS ∗∗∗
NS ∗∗∗
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A completely randomized design was applied to determine the dietary effects. Data were analyzed by the GLM procedure of SAS [23]. All values were calculated with the consideration of the average value of each pen as an experimental unit. Differences were considered significant when P < 0.05 and treatment means were compared using Duncan’s multiple range test.
HE ET AL.: STRESS RESPONSE IN GREYLAGS
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Table 5. Influence of dietary fiber source on the behavioral sensitivity and blood parameters (pre- and poststress) of growing Greylag geese (Anser anser). Treatments1 Item2
SECS
SEWS
SERS
SEM3
1.55
1.50
1.60
1.47
0.10
NS
60.44 85.18 10.07 133.97
60.50 85.33 9.64 128.61
61.35 85.25 10.87 147.09
63.10 81.88 10.98 140.98
1.58 3.69 0.81 7.37
NS NS NS NS
61.08 73.30 12.03a 444.31
61.36 73.22 9.86b 331.62
61.84 76.66 11.11a,b 414.98
63.56 70.44 11.31a,b 398.46
1.49 2.60 0.60 64.18
NS NS
P-value
∗
NS
1
CSS: Corn straw silage; SECS: Steam-exploded corn straw; SEWS: Steam-exploded wheat straw; SERS: Steam-exploded rice straw. 2 CORT: Cortisone, SOD: Superoxide dismutase, MDA: Methane dicarboxylic aldehyde, CK: Creatine kinase. 3 SEM: Standard Error of Mean. a,b Means in a row with uncommon superscripts differ significantly at P < 0.05. ∗ P < 0.05, NS = P ≥ 0.05.
(P > 0.05) among the other blood parameters (Table 5). At harvest, animals generally experience stress, injury, even mortality due to limited access to feed and water, exposure to stimulus like noise and vibration, as well as poor handling and mixing with unfamiliar animals [9]. The results of the present study showed that the common harvest procedure of fasting, catching, transport, etc., decreased (P < 0.01) the concentration of SOD and increased (P < 0.01) that of CK in the blood, inferring that the birds suffered significant harvest stress. Consistently, Toth et al. [24] reported that handling or catching the geese resulted in severe stress. It could be explained that stress would stimulate a significant increase in reactive oxygen to cause tissue damage, ultimately resulting in the skeletal muscle and cardiac muscle organ-specific enzyme CK being released into the general blood circulation [25]. Meanwhile, SOD, an important antioxidase that plays an important role in scavenging free radicals and recovering impaired tissue, consequently resulted in decreased concentration. However, the other 2 commonly used stress indicators, MDA and CORT, did not show a significant change after the stress procedure, inferring that MDA and CORT were not as sensitive to the stressors as SOD and CK. Mohammed et al. [26] reported
that force-feeding did not induce any significant increase in plasma CORT levels in ducks kept in individual cages — the same response as feather gathering [24]. In most cases, catching and handling of the animal can be stressful in itself [27]. The stress response system of birds modifies in domesticates [28]. A long-term adaptation to different diets generates various specific body status and intestinal microbial flora, consequently resulting in different physiological and behavioral responses to environmental changes and all kinds of stimuli. No difference was found in the behavioral sensitivities and prestress blood parameters among the birds fed different fiber sources in our study, implying that the birds behaved similarly to outside stimuli. Dietary fiber sources exerted no direct effect on the blood parameters when the complete diet was offered ad libitum, or the birds had rebuilt homeostasis during the long-time adaptation. In the present results, the birds fed the CSS had a higher post-stress MDA concentration than those offered the SECS. It might be explained as the birds fed CSS suffered more severe lipid oxidation, as MDA has been identified as the ultimate product of lipid peroxidation, inferring that the birds fed CSS experienced a heavier stress fluctuation. What makes the difference has not been delineated as yet. Possibly the difference of
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Behavioral sensitivity Pre-stress COR (ng/mL) SOD (U/mL) MDA (nmol/mL) CK (U/L) Post-stress COR (ng/mL) SOD (U/mL) MDA (nmol/mL) CK (U/L)
CSS
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6 feed intake accounts partially, as feed limitation would decrease the amount of mitochondria free radicals [29].
CONCLUSIONS AND APPLICATIONS
REFERENCES AND NOTES 1. Lazarus, R. S. 1966. Psychological Stress and the Coping Process. McGraw-Hill, NY, US. 2. Cockrem, J. F. 2007. Stress, corticosterone responses and avian personalities. J. Ornithol. 148:169–178. 3. Koolhaas, J. M., S. M. Korte, S. F. De Boer, B. J. Van Der Vegt, C. G. Van Reenen, H. Hopster, I. C. De Jong, M. Ruis, and H. J. Blokhuis. 1999. Coping styles in animals: Current status in behavior and stress-physiology. Neurosci. Biobehav. Rev. 23:925–935. 4. Speer, N. C., G. Slack, and E. Troyer. 2001. Economic factors associated with livestock transportation. J. Anim. Sci. 79:E166–E170. 5. Ericsson, M., A. Fallahsharoudi, J. Bergquist, M. M. Kushnir, and P. Jensen. 2014. Domestication effects on behavioural and hormonal responses to acute stress in chickens. Physiol. Behav. 133:161–169. 6. Morm`ede, P., S. Andanson, B. Aup´erin, B. Beerda, D. Gu´emen´e, J. Malmkvist, X. Manteca, G. Manteuffel, P. Prunet, and C. G. van Reenen. 2007. Exploration of the hypothalamic–pituitary–adrenal function as a tool to evaluate animal welfare. Physiol. Behav. 92:317–339. 7. Harris, T. 2001. The history and development of European and North American transport regulations and international trade issues. J. Anim. Sci. 79:E73–E85. 8. Smith, G. C., T. Grandin, T. H. Friend, D. Lay, Jr., and J. C. Swanson. 2004. Effect of transport on meat quality and animal welfare of cattle, pigs, sheep, horses, deer, and poultry. Review paper http://www.grandin.com/behaviour/effect of transport. 9. Schwartzkopf-Genswein, K. S., L. Faucitano, S. Dadgar, P. Shand, L. A. Gonz´alez, and T. G. Crowe. 2012. Road transport of cattle, swine and poultry in North America and its impact on animal welfare, carcass and meat quality: A review. Meat Sci. 92:227–243.
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1. The harvest procedure caused the concentration of SOD to drop (P < 0.01) 13.57% and increased (P < 0.01) that of CK by 186.21% (post-stress vs. pre-stress), inferring that the birds had experienced a severe stress influence. 2. Greylag geese (Anser anser) fed corn straw silage (CSS) had a higher (P < 0.05) poststress concentration of MDA than those offered steam-exploded corn straw (SECS) by 22.01%, suggesting that birds’ diets and dietary fiber sources may influence their stress response.
10. Barlow, D. H. 2000. Unraveling the mysteries of anxiety and its disorders from the perspective of emotion theory. Am. Psychologist. 55:1247. 11. Calandreau, L., A. Favreau-Peign´e, A. Bertin, P. Constantin, C. Arnould, A. Laurence, S. Lumineau, C. Houdelier, M. A. Richard-Yris, and A. Boissy. 2011. Higher inherent fearfulness potentiates the effects of chronic stress in the Japanese quail. Behav. Brain Res. 225:505–510. 12. Beijing Sing-An Poultry Breeding Base (Beijing, China). 13. four dietary treatments with 60 birds per group (15 replicates per group), which differed in dietary fiber source (corn straw silage [CSS], steam-exploded corn straw [SECS], steam-exploded wheat straw [SEWS], or steam-exploded rice straw [SERS]), accounting for 30% of the dietary components on an as-fed basis. 14. NRC. 1994. Nutrient Requirements of Poultry. National Academy Press, Washington, USA. 15. Greylags lived in an open-style building with a brickcovered floor, which was subdivided into 60 pens (1 × 9 m) with wire netting. Each pen was equipped with individual drinkers and feeders, and was half-shielded against rain and sunshine. 16. State Scientific and Technological Commission. 2013. Guide to the Care and Use of Experimental Animals. 3rd ed., Vol. 1, China Agricultural University, Beijing, China. 17. AOAC International. 2000. Official Methods of Analysis of AOAC International, 17th ed. AOAC, Int., Arlington, VA, USA. 18. Samples were analyzed for total N using the combustion nitrogen analysis (FP-528, Leco, USA); crude protein (CP) was calculated using the 6.25 nitrogen conversion factor (N × 6.25). 19. Crude fiber (CF) was measured by sequential extractions with dilute acid and alkali in a fiber analyzer (A220, ANKOM Technology, USA) followed by combustion at 550◦ C for 6 h as well as NDF, ADF, and ADL. NDF was assayed with use of an alpha amylase. Both NDF and ADF are expressed without residual ash. 20. Phosphorus was determined by UV spectrophotometry (UV-VIS 8500, Shanghai Tianmei Scientific Instrument Co., Ltd., Shanghai, China) using the phosphorus-vanadiummolybdate yellow colorimetric method. 21. The serum parameters were assayed by Beijing SinoUK of Biological technology, Beijing (China) using an AutoBiochemical Analyzer (Hitachi-7160, Hitachi Ltd., Japan) except for CORT, which was measured by an Auto-Radioimmuno Counter (r-911, Industrial Company of Science and Technology University of China, China). 22. The reagent kits were purchased from Bio-Sino BioTechnology and Science Inc., Beijing (China) and used as specification in detail. 23. SAS Institute Inc. 2002. The SAS System for Windows, Release 9.0. SAS Institute Inc., Cary, NC. 24. T´oth, P., L. B´odi, K. Maros, E. Sz˝ucs, and J. Janan. 2012. Blood corticosterone levels in growing geese around feather gathering. Acta Vet Hung. 60:477–487. 25. Apple, F. S., and M. K. McGue. 1983. Serum enzyme changes during marathon training. Am. J. Clin. Pathol. 79:716–719. 26. Mohammed, A. A., M. Abdel-Rahman, and M. H. Darwish. 2014. Force feeding as a Stress Factor on Muscovy Ducks. J. Advanced Veter. Res. 4:166–173.
HE ET AL.: STRESS RESPONSE IN GREYLAGS 27. Cook, C. J., and D. Mellor. 2000. Hands-on and hands-off measurement of stress. In:Moberg, G. P., and J. A. Mench (eds.) The Biology of Animal Stress. CABI Publishing, Wallingford. 28. K¨unzl, C., and N. Sachser. 1999. The behavioral endocrinology of domestication: A comparison between the domestic guinea pig (Cavia apereaf. porcellus) and its wild ancestor, the cavy (Cavia aperea). Horm. Behav. 35: 28–37.
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29. Chai, N. N. 1991. Nutrition, lipid peroxidation and atherosclerosis. Foreign Medi. Hyg. 2:91–96.
Acknowledgments The authors wish to express gratitude to Beijing Municipal Government ( 201202910411136) and Feed Utilization Technology Research & Demonstration of Northern Crop Straw (201503134) for funding this work.
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Influence of dietary fiber sources on the physiological and behavioral stress responses of Greylag geese (Anser anser) L. W. He,1 Q. X. Meng, D. Y. Li, Y. W. Zhang, and L. P. Ren State Key Laboratory of Animal Nutrition, College of Animal Science and Technology, China Agricultural University, Beijing 100193, P. R. China
SUMMARY This experiment investigated the physiological and behavioral responses of Greylag geese (Anser anser) fed different fiber sources when exposed to the common stressors at harvest, primarily involving the procedures of fasting, transport, catching, etc. A total of 240 4-weekold Greylag geese were allocated to 4 diets with different fiber sources (corn straw silage [CSS], steam-exploded corn straw [SECS], steam-exploded wheat straw [SEWS], and steamexploded rice straw [SERS]). The birds were fed for 12 wk and harvested at 16 wk for slaughter. Blood samples designated to determine stress status were collected before (pre-stress) and after (post-stress) the harvest procedure was carried out. Irrespective of the dietary differences, the harvest procedure caused the concentration of superoxide dismutase (SOD) to drop (P < 0.01) 13.57% and increased (P < 0.01) that of creatine kinase (CK) by 186.21% (post-stress vs. pre-stress), inferring that the birds had experienced a severe stress. As to the dietary effects, the birds fed alternative fiber sources did not develop dissimilar (P > 0.05) pre-stress blood parameters and behavioral sensitivities, while the birds fed CSS had a higher (P < 0.05) poststress concentration of methane dicarboxylic aldehyde (MDA) than those offered SECS by 22.01%, suggesting that feeding Greylag geese alternative fiber sources influenced their stress response. Key words: Greylag goose, fiber source, harvest stress, blood parameter 2016 J. Appl. Poult. Res. 00:1–7 http://dx.doi.org/10.3382/japr/pfw023
DESCRIPTION OF PROBLEM Stress arises when individuals perceive that they cannot adequately cope with the demands being made on them or with threats to their well-being [1]. In response to such situations, a physiological stress response is initiated, consisting of activation of the hypothalamic– pituitary–adrenal (HPA) axis with release of catecholamines and corticosteroids [2] and 1
Corresponding author: [email protected]
becoming more alert in order to increase their survival [3]. In general, stress exerts to some extent adverse effects on the animal performance, food safety, and quality concerns [4], limiting the efficiency of production and causing economic loss to the farm [5]. Any improvement in production that can reduce stress is economically worthwhile. Generally, there are several blood parameters such as cortisone (CORT), superoxide dismutase (SOD), methane dicarboxylic aldehyde (MDA), and creatine kinase (CK) selected to detect the metabolic changes and
Downloaded from http://japr.oxfordjournals.org/ at University of Florida on June 8, 2016
Primary Audience: Flock Supervisors, Researchers, Nutritionists
JAPR: Research Report
2
MATERIALS AND METHODS Experimental Management
Design
and
Animal
A total of 240 healthy 4-week-old Greylag geese (Anser anser) obtained from a commercial
hatchery [12] were randomly allocated to 4 diets differing in fiber source (corn straw silage [CSS], steam-exploded corn straw [SECS], steam-exploded wheat straw [SEWS], or steam-exploded rice straw [SERS]) [13]. The diets were formulated to meet the nutrient requirements of growing geese (Table 2) [14]. During the study (12 wk), the birds had access to pelletized feed and clean water ad libitum without artificial lightning [15]. This study was performed at the Animal Experimental Station of China Agricultural University, where all procedures were approved by the Animal Care and Use Committee [16].
Chemical Analysis of the Diets The chemical composition of the fiber sources (Table 1) and the diets (Table 2) were analyzed according to AOAC methodology [17]. Crude protein (CP) was analyzed using the combustion nitrogen analysis (FP-528, Leco, St. Joseph, Missouri) [18]. Crude fiber (CF) was measured by a fiber analyzer (A220, ANKOM Technology, Macedon NY) as well as neutral detergent fiber (NDF), acid detergent fiber (ADF), and lignin (ADL) [19]. Ether extract (EE) was analyzed by an extraction system (ANKOMXT10 EXTRACTOR, ANKOM Technology, Macedon NY). Calcium was determined by atomic absorption spectrophotometry (WFX320, BRAIC, Beijing, China), and phosphorus was determined by UV spectrophotometry (UVVIS 8500, Tianmei Scientific Instrument Co., Ltd., Shanghai, China) [20].
Evaluation of the Behavioral Sensitivity to Stress In order to evaluate the behavioral sensitivity to stress, the birds were placed separately into a plastic barrel (0.6 m in diameter, 0.8 m in height) at the end of the rearing period. Behavioral response was scored based on a 5-point scale (Table 3). A higher score means that the birds were more fearful of the unfamiliar condition and would be prone to suffer stress with high behavioral sensitivity.
Downloaded from http://japr.oxfordjournals.org/ at University of Florida on June 8, 2016
diagnose the stress status. Measuring HPA function is a standard approach to study stress and welfare in farm animals [6]. Throughout an animal’s life, it is inevitably exposed to various types of normal stress during transportation, weaning, vaccination, diet transition, etc. Transport is an integral part of today’s livestock industry [7] with animals being transported at least once during their production life for reasons such as assembly for harvest purposes [8], during which there would be a potential for the animals to experience stress, injury, even mortality due to limited access to feed and water, exposure to stimulus like noise and vibration, as well as poor handling and mixing with unfamiliar animals [9]. Moreover, it is reported that an animal’s fearfulness and its physiological stress response are positively related. Factors influencing an animal’s fear level can be its genetic makeup, unpredictable and uncontrollable early life experiences, and associative learning experiences [10]. Some species with a highly sustained fear level may have an increased vulnerability to stress in later life [11]. There are many potential factors contributing to the stress of poultry, such as nutrition, environment, management, and treatment. The diet could be a stressor when it is formulated with low digestibility and poor nutrients, which results in a lack of feed intake or biased nutrient ingestion. Alterations in physiology and behavior due to domestication could have influenced the adaptation to stress with modifications to endocrine and behavioral profiles [5]. Our hypothesis was that various dietary conditions (dietary components) in early life might result in different behavioral sensitivities along with different physiological stress responses in later life. Understanding how birds cope with common stress is fundamental for the improvement of their welfare. This experiment investigated the physiological and behavioral responses of Greylag geese (Anser anser) fed different fiber sources when exposed to common stressors at harvest.
HE ET AL.: STRESS RESPONSE IN GREYLAGS
3
Table 1. Chemical compositions of the 4 fiber sources (g/kg, as DM basis). Fiber source1 Item2
CSS
SECS
SEWS
SERS
DM CP CF EE NDF ADF ADL Ash
928.38 70.35 379.50 15.13 726.18 482.42 70.58 103.60
944.50 60.06 347.40 26.45 546.23 460.08 83.91 89.53
941.44 46.62 399.60 22.36 563.61 513.41 103.42 107.62
946.62 51.91 374.90 22.50 597.45 510.09 86.15 122.64
1
Table 2. Ingredients and nutrient compositions of experimental diets. Treatments1 Item Ingredient (g/kg) Corn Wheat bran Soybean meal DDGS2 Alfalfa particles Corn straw silage Steam-exploded corn straw Steam-exploded wheat straw Steam-exploded straw Lysine hydrochloride Methionine Sodium chloride Limestone Calcium hydrophosphate Active ferment3 Premix4 Chlortetracycline5 Calculated nutrient concentration (g/kg) Metabolizable energy (Kcal/kg) Lys Met Analyzed concentration (g/kg) Crude protein Crude fiber Calcium Total phosphorus 1
CSS
SECS
SEWS
SERS
350.0 50.0 210.0 30.0 30.0 300.0 3.0 2.0 3.0 4.0 6.0 1.0 10.0 1.0
350.0 50.0 210.0 30.0 30.0 300.0 3.0 2.0 3.0 4.0 6.0 1.0 10.0 1.0
350.0 50.0 210.0 30.0 30.0 300.0 3.0 2.0 3.0 4.0 6.0 1.0 10.0 1.0
350.0 50.0 210.0 30.0 30.0 300.0 3.0 2.0 3.0 4.0 6.0 1.0 10.0 1.0
2150 0.56 0.42
2150 0.56 0.42
2150 0.56 0.42
2150 0.56 0.42
16.25 14.88 0.50 0.38
16.39 12.33 0.42 0.40
16.14 14.10 0.44 0.38
16.76 12.83 0.43 0.40
CSS: Corn straw silage; SECS: Steam-exploded corn straw; SEWS: Steam-exploded wheat straw; SERS: Steam-exploded rice straw. 2 DDGS: Distillers dried grains with soluble. 3 Angel (golden), live yeast number not less than 2 billion per gram. 4 Provided per kg of diet: Cu,7.6 mg; Fe, 75.0 mg; Zn, 69.8 mg; I, 1.5 mg; Mn, 75 mg; Se, 0.2 mg; vitamin A, 8,328 IU; vitamin D3 , 3,987 IU; vitamin E, 59 IU; vitamin K 6.0 mg; vitamin B1 , 2.6 mg; vitamin B2 , 12 mg; vitamin B6 , 5.3 mg; vitamin B12 , 0.4 mg; nicotinic acid, 52.0 mg; folic acid, 1.3 mg; biotin, 0.6 mg. 5 Effective component, 15%.
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CSS: Corn straw silage; SECS: Steam-exploded corn straw; SEWS: Steam-exploded wheat straw; SERS: Steam-exploded rice straw. 2 DM: Dry matter; CP: Crude protein; CF: Crude fiber; EE: Ether extract; NDF: Neutral detergent fiber; ADF: Acid detergent fiber; ADL: Acid detergent lignin; Ash: Ash.
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4 Table 3. Ethogram used for the behavioral recordings of Greylag geese (Anser anser). Scoring 1 2 3 4
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were purchased from Bio-Sino Bio-Technology and Science Inc., China [22].
Descriptions of behavioral responses Sits down quickly and keeps quiet without any scrabble after put into the barrel Moves slowly and stands looking around with slight scrabble after put into the barrel Wags head rapidly and pushes feet out with medium scrabble after put into the barrel Wags head rapidly and powerfully pushes feet out with severe scrabble and strong escaping intention after put into the barrel Constant and severe escape behavior
Statistical Analysis
Harvest Procedure and Blood Sampling To determine the physiological response to stress (fasting, capture, transport, crowding, fear, etc.), at 16 wk of age, one bird was randomly selected from each pen (60 pens in total) and was bled by venipuncture with a blood needle before a 12-h fasting period (i.e., pre-stress); after the fast, the birds were placed individually in bags with their heads provided an opening to the outside when transported half an h to the slaughter plant by platform trailer without shelter and then blood samples were collected again on the spot before slaughter (i.e., post-stress). Analysis of Blood Samples All the blood samples were prepared for blood serum and used to determine the parameters, including mainly CORT, SOD, MDA and CK, which were generally used to indicate stress status. The serum parameters were assayed by a testing agency (Beijing Sino-UK of Biological Technology, Beijing, China) [21] and the reagent kits
RESULTS AND DISCUSSION Irrespective of the dietary differences, the harvest stress resulting from the common potential stressors in practice decreased (P < 0.001) the concentration of SOD and increased (P < 0.001) that of CK in the blood (post-stress vs. pre-stress) along with no significant influence (P > 0.05) on the concentrations of CORT and MDA (Table 4), showing that the birds had suffered significant stress influence. As to dietary effects, no difference (P > 0.05) was found on the pre-stress blood parameters of growing Greylag geese fed alternative fiber sources, and they developed similar (P > 0.05) behavioral sensitivities (Table 5). The birds fed CSS had a higher (P < 0.05) post-stress concentration of MDA than those offered SECS, along with a similarity (P > 0.05) to those fed the other 2 fiber sources (SEWS and SERS), inferring that the birds fed different diets developed a dissimilar stress response; there was no difference
Table 4. Influence of harvest stress on the blood parameters of Greylag geese (Anser anser). Treatment Item1 CORT (ng/mL) SOD (U/mL) MDA (nmol/mL) CK (U/L) 1
Pre-stress
Post-stress
SEM2
61.35 84.46a 10.36 137.32b
61.96 73.00b 11.14 393.02a
0.76 1.46 0.33 21.86
CORT: Cortisone, SOD: Superoxide dismutase, MDA: Methane dicarboxylic aldehyde, CK: Creatine kinase. SEM: Standard Error of Mean. a,b Means in a row with uncommon superscripts differ significantly at P < 0.05. ∗∗∗ P < 0.001, NS = P ≥ 0.05. 2
P-value NS ∗∗∗
NS ∗∗∗
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A completely randomized design was applied to determine the dietary effects. Data were analyzed by the GLM procedure of SAS [23]. All values were calculated with the consideration of the average value of each pen as an experimental unit. Differences were considered significant when P < 0.05 and treatment means were compared using Duncan’s multiple range test.
HE ET AL.: STRESS RESPONSE IN GREYLAGS
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Table 5. Influence of dietary fiber source on the behavioral sensitivity and blood parameters (pre- and poststress) of growing Greylag geese (Anser anser). Treatments1 Item2
SECS
SEWS
SERS
SEM3
1.55
1.50
1.60
1.47
0.10
NS
60.44 85.18 10.07 133.97
60.50 85.33 9.64 128.61
61.35 85.25 10.87 147.09
63.10 81.88 10.98 140.98
1.58 3.69 0.81 7.37
NS NS NS NS
61.08 73.30 12.03a 444.31
61.36 73.22 9.86b 331.62
61.84 76.66 11.11a,b 414.98
63.56 70.44 11.31a,b 398.46
1.49 2.60 0.60 64.18
NS NS
P-value
∗
NS
1
CSS: Corn straw silage; SECS: Steam-exploded corn straw; SEWS: Steam-exploded wheat straw; SERS: Steam-exploded rice straw. 2 CORT: Cortisone, SOD: Superoxide dismutase, MDA: Methane dicarboxylic aldehyde, CK: Creatine kinase. 3 SEM: Standard Error of Mean. a,b Means in a row with uncommon superscripts differ significantly at P < 0.05. ∗ P < 0.05, NS = P ≥ 0.05.
(P > 0.05) among the other blood parameters (Table 5). At harvest, animals generally experience stress, injury, even mortality due to limited access to feed and water, exposure to stimulus like noise and vibration, as well as poor handling and mixing with unfamiliar animals [9]. The results of the present study showed that the common harvest procedure of fasting, catching, transport, etc., decreased (P < 0.01) the concentration of SOD and increased (P < 0.01) that of CK in the blood, inferring that the birds suffered significant harvest stress. Consistently, Toth et al. [24] reported that handling or catching the geese resulted in severe stress. It could be explained that stress would stimulate a significant increase in reactive oxygen to cause tissue damage, ultimately resulting in the skeletal muscle and cardiac muscle organ-specific enzyme CK being released into the general blood circulation [25]. Meanwhile, SOD, an important antioxidase that plays an important role in scavenging free radicals and recovering impaired tissue, consequently resulted in decreased concentration. However, the other 2 commonly used stress indicators, MDA and CORT, did not show a significant change after the stress procedure, inferring that MDA and CORT were not as sensitive to the stressors as SOD and CK. Mohammed et al. [26] reported
that force-feeding did not induce any significant increase in plasma CORT levels in ducks kept in individual cages — the same response as feather gathering [24]. In most cases, catching and handling of the animal can be stressful in itself [27]. The stress response system of birds modifies in domesticates [28]. A long-term adaptation to different diets generates various specific body status and intestinal microbial flora, consequently resulting in different physiological and behavioral responses to environmental changes and all kinds of stimuli. No difference was found in the behavioral sensitivities and prestress blood parameters among the birds fed different fiber sources in our study, implying that the birds behaved similarly to outside stimuli. Dietary fiber sources exerted no direct effect on the blood parameters when the complete diet was offered ad libitum, or the birds had rebuilt homeostasis during the long-time adaptation. In the present results, the birds fed the CSS had a higher post-stress MDA concentration than those offered the SECS. It might be explained as the birds fed CSS suffered more severe lipid oxidation, as MDA has been identified as the ultimate product of lipid peroxidation, inferring that the birds fed CSS experienced a heavier stress fluctuation. What makes the difference has not been delineated as yet. Possibly the difference of
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Behavioral sensitivity Pre-stress COR (ng/mL) SOD (U/mL) MDA (nmol/mL) CK (U/L) Post-stress COR (ng/mL) SOD (U/mL) MDA (nmol/mL) CK (U/L)
CSS
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6 feed intake accounts partially, as feed limitation would decrease the amount of mitochondria free radicals [29].
CONCLUSIONS AND APPLICATIONS
REFERENCES AND NOTES 1. Lazarus, R. S. 1966. Psychological Stress and the Coping Process. McGraw-Hill, NY, US. 2. Cockrem, J. F. 2007. Stress, corticosterone responses and avian personalities. J. Ornithol. 148:169–178. 3. Koolhaas, J. M., S. M. Korte, S. F. De Boer, B. J. Van Der Vegt, C. G. Van Reenen, H. Hopster, I. C. De Jong, M. Ruis, and H. J. Blokhuis. 1999. Coping styles in animals: Current status in behavior and stress-physiology. Neurosci. Biobehav. Rev. 23:925–935. 4. Speer, N. C., G. Slack, and E. Troyer. 2001. Economic factors associated with livestock transportation. J. Anim. Sci. 79:E166–E170. 5. Ericsson, M., A. Fallahsharoudi, J. Bergquist, M. M. Kushnir, and P. Jensen. 2014. Domestication effects on behavioural and hormonal responses to acute stress in chickens. Physiol. Behav. 133:161–169. 6. Morm`ede, P., S. Andanson, B. Aup´erin, B. Beerda, D. Gu´emen´e, J. Malmkvist, X. Manteca, G. Manteuffel, P. Prunet, and C. G. van Reenen. 2007. Exploration of the hypothalamic–pituitary–adrenal function as a tool to evaluate animal welfare. Physiol. Behav. 92:317–339. 7. Harris, T. 2001. The history and development of European and North American transport regulations and international trade issues. J. Anim. Sci. 79:E73–E85. 8. Smith, G. C., T. Grandin, T. H. Friend, D. Lay, Jr., and J. C. Swanson. 2004. Effect of transport on meat quality and animal welfare of cattle, pigs, sheep, horses, deer, and poultry. Review paper http://www.grandin.com/behaviour/effect of transport. 9. Schwartzkopf-Genswein, K. S., L. Faucitano, S. Dadgar, P. Shand, L. A. Gonz´alez, and T. G. Crowe. 2012. Road transport of cattle, swine and poultry in North America and its impact on animal welfare, carcass and meat quality: A review. Meat Sci. 92:227–243.
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1. The harvest procedure caused the concentration of SOD to drop (P < 0.01) 13.57% and increased (P < 0.01) that of CK by 186.21% (post-stress vs. pre-stress), inferring that the birds had experienced a severe stress influence. 2. Greylag geese (Anser anser) fed corn straw silage (CSS) had a higher (P < 0.05) poststress concentration of MDA than those offered steam-exploded corn straw (SECS) by 22.01%, suggesting that birds’ diets and dietary fiber sources may influence their stress response.
10. Barlow, D. H. 2000. Unraveling the mysteries of anxiety and its disorders from the perspective of emotion theory. Am. Psychologist. 55:1247. 11. Calandreau, L., A. Favreau-Peign´e, A. Bertin, P. Constantin, C. Arnould, A. Laurence, S. Lumineau, C. Houdelier, M. A. Richard-Yris, and A. Boissy. 2011. Higher inherent fearfulness potentiates the effects of chronic stress in the Japanese quail. Behav. Brain Res. 225:505–510. 12. Beijing Sing-An Poultry Breeding Base (Beijing, China). 13. four dietary treatments with 60 birds per group (15 replicates per group), which differed in dietary fiber source (corn straw silage [CSS], steam-exploded corn straw [SECS], steam-exploded wheat straw [SEWS], or steam-exploded rice straw [SERS]), accounting for 30% of the dietary components on an as-fed basis. 14. NRC. 1994. Nutrient Requirements of Poultry. National Academy Press, Washington, USA. 15. Greylags lived in an open-style building with a brickcovered floor, which was subdivided into 60 pens (1 × 9 m) with wire netting. Each pen was equipped with individual drinkers and feeders, and was half-shielded against rain and sunshine. 16. State Scientific and Technological Commission. 2013. Guide to the Care and Use of Experimental Animals. 3rd ed., Vol. 1, China Agricultural University, Beijing, China. 17. AOAC International. 2000. Official Methods of Analysis of AOAC International, 17th ed. AOAC, Int., Arlington, VA, USA. 18. Samples were analyzed for total N using the combustion nitrogen analysis (FP-528, Leco, USA); crude protein (CP) was calculated using the 6.25 nitrogen conversion factor (N × 6.25). 19. Crude fiber (CF) was measured by sequential extractions with dilute acid and alkali in a fiber analyzer (A220, ANKOM Technology, USA) followed by combustion at 550◦ C for 6 h as well as NDF, ADF, and ADL. NDF was assayed with use of an alpha amylase. Both NDF and ADF are expressed without residual ash. 20. Phosphorus was determined by UV spectrophotometry (UV-VIS 8500, Shanghai Tianmei Scientific Instrument Co., Ltd., Shanghai, China) using the phosphorus-vanadiummolybdate yellow colorimetric method. 21. The serum parameters were assayed by Beijing SinoUK of Biological technology, Beijing (China) using an AutoBiochemical Analyzer (Hitachi-7160, Hitachi Ltd., Japan) except for CORT, which was measured by an Auto-Radioimmuno Counter (r-911, Industrial Company of Science and Technology University of China, China). 22. The reagent kits were purchased from Bio-Sino BioTechnology and Science Inc., Beijing (China) and used as specification in detail. 23. SAS Institute Inc. 2002. The SAS System for Windows, Release 9.0. SAS Institute Inc., Cary, NC. 24. T´oth, P., L. B´odi, K. Maros, E. Sz˝ucs, and J. Janan. 2012. Blood corticosterone levels in growing geese around feather gathering. Acta Vet Hung. 60:477–487. 25. Apple, F. S., and M. K. McGue. 1983. Serum enzyme changes during marathon training. Am. J. Clin. Pathol. 79:716–719. 26. Mohammed, A. A., M. Abdel-Rahman, and M. H. Darwish. 2014. Force feeding as a Stress Factor on Muscovy Ducks. J. Advanced Veter. Res. 4:166–173.
HE ET AL.: STRESS RESPONSE IN GREYLAGS 27. Cook, C. J., and D. Mellor. 2000. Hands-on and hands-off measurement of stress. In:Moberg, G. P., and J. A. Mench (eds.) The Biology of Animal Stress. CABI Publishing, Wallingford. 28. K¨unzl, C., and N. Sachser. 1999. The behavioral endocrinology of domestication: A comparison between the domestic guinea pig (Cavia apereaf. porcellus) and its wild ancestor, the cavy (Cavia aperea). Horm. Behav. 35: 28–37.
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29. Chai, N. N. 1991. Nutrition, lipid peroxidation and atherosclerosis. Foreign Medi. Hyg. 2:91–96.
Acknowledgments The authors wish to express gratitude to Beijing Municipal Government ( 201202910411136) and Feed Utilization Technology Research & Demonstration of Northern Crop Straw (201503134) for funding this work.
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