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Threonine:lysine ratio for Japanese quail hen diets
©2013 Poultry Science association, inc.
Threonine:lysine ratio for Japanese quail hen diets Matheus Ramalho de Lima,*1 Fernando Guilherme Perazzo Costa,† Ricardo Romão Guerra,† José HumbertoVilar da Silva,† Carlos Bôa-Viagem Rabello,‡ Maria Angélica Miglino,§ Gledyson Bruno Vieira Lobato,† Severino Bernardino Sena Netto,† and Leonilson da Silva Dantas†
Primary Audience: Poultry Farmers, Poultry Nutritionists SUMMARY the objective of this study was to evaluate threonine requirements for laying Japanese quail fed with different threonine:lysine ratios through measurement of zootechnical and histological performance. a total of 288 Japanese quail at 125 d of age were distributed in a totally randomized design, with 6 treatments and 6 replications of 8 each. the treatments consisted of a basal diet made of corn, soybean meal, and corn gluten meal, supplemented with l-lysine, dlmethionine, l-tryptophan, l-isoleucine, and l-valine to meet the laying Japanese quail essential amino acid needs, except for threonine. the basal diet was supplemented with 0.00, 0.04, 0.08, 0.12, 0.16, and 0.20% of l-threonine in substitution for l-glutamate acid, with the objective of reaching levels of 0.66, 0.70, 0.74, 0.78, 0.82, and 0.86% of threonine in the feed, resulting in threonine:lysine ratios of 66, 70, 74, 78, 82, and 86%. Zootechnical and histological indexes of the digestive and reproductive system were evaluated. the amino acid ratio alteration assessed in this study influenced performance and histology of the quail through improving productive performance at a 78% threonine:lysine ratio. Key words: egg production, threonine, histological response 2013 J. appl. Poult. Res. 22:260–268 http://dx.doi.org/10.3382/japr.2012-00670
DESCRIPTION OF PROBLEM the excellent productive performance of Japanese quail, which begin laying between 35 and 40 d of age and have an average production of 300 eggs/hen per year, need a small area for production (400 bird/m2), and have a low 1
Corresponding author: [email protected]
investment with rapid return of capital invested [1], has stimulated the development of a quail industry in Brazil. despite such great economic importance, few studies have been performed in Brazil on the nutritional requirements of quail with feed formulation for quail performed based on suggested requirements [2, 3] or extrapola-
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*Federal University of West of Para, Biodiversity and Forest Institute, 68040-410, Santarém, Pará, Brazil; †Federal University of Paraiba, Department of Animal Science, PB 079, Km 1/i92, 58397000, Campus II, Areia, Paraiba, Brazil; ‡Federal Rural University of Pernambuco, Department of Animal Science, Recife, Pernambuco, Brazil 52171-900; and §Faculty of Veterinary Medicine and Animal Science, Sao Paulo University, Sao Paulo, Brazil 05508-270
Ramalho de Lima et al.: THREONINE:LYSINE RATIO
MATERIALS AND METHODS The study was performed at the Poultry Research Center of the Federal University of Paraiba (Research Farm of Poultry CCA/UFPB) at Areia, Paraiba. A total of 288 Japanese quail at 125 d of age were distributed in a complete randomized design among 6 treatments with 6 replications of 8 quail each. The treatments consisted of a basal diet formulated based on corn, soybean meal, and corn gluten meal, supplemented with l-lysine, dl-methionine, ltryptophan, l-isoleucine, and l-valine to meet the essential amino acid needs of the Japanese quail hen except for threonine [2]. The project had ethical approval from the Animal Use and Care Committee of the Federal University of Paraiba, Brazil. The quail were allocated among
40 × 40 × 20 cm cages in clay-tile sheds with 2 water troughs. The birds received 17 h of light per day. The average temperature and RH were 27°C and 88%, respectively. The basal diet was supplemented with 0.00, 0.04, 0.08, 0.12, 0.16, and 0.20% l-threonine to substitute l-glutamic acid with the objective of reaching levels of 0.66, 0.70, 0.74, 0.78, 0.82, and 0.86% threonine in the diet, resulting in the threonine:lysine ratios of 0.66, 0.70, 0.74, 0.78, 0.82, and 0.86 or 66, 70, 74, 78, 82, and 86% (Table 1). l-Glutamic acid was used to make the diets isonitrogenous. The dietary level of soybean oil and of the inert material was added in substitution to l-glutamic acid that was reduced with the increase in the levels of threonine in the experimental diets, making all diets isonitrogenous and isoenergetic. Zootechnical and histological evaluations were performed. In the zootechnical evaluations, the feed intake (g/bird), egg production (%), egg weight (g/egg), egg mass (g/egg production), feed conversion per egg mass (g/g), feed conversion per dozen eggs (g/g), relative albumen weight (%), percent yolk weight (%), percent shell weight (%), yolk pigmentation score, egg specific gravity (g/cm3), and eggshell thickness (mm) were assessed. The period of evaluation of egg production was divided into 5 periods of 21 d each. At the end of each period, the feed left in each feed trough was collected for the calculation of feed intake. Egg collection was performed twice a day at 1000 and 1600 h, and mortality was recorded. Percent egg production was calculated by dividing the total quantity of eggs per pen by the number of quail, correcting for mortality. The eggs of the last 3 d of each period were weighed individually to obtain the average weight of the eggs. Egg mass was the product of the production of eggs and average weight of the eggs per pen. The feed conversion per egg mass was the ratio of feed intake to egg mass, and feed conversion per dozen eggs was the ratio of feed intake to egg production multiplied by 12. For the histological analyses, at termination of the experiment when the quail were 230 d of age, the collection of the material was performed from fragments of the digestive (duodenum and liver) and reproductive (magnum and uterus) systems of 10 animals per treatment. Those sections were immersed in methacarn fix-
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tion from laying quail or broilers. One of the few exceptions of data obtained from experiments performed in Brazil are the Tables of Nutritional Recommendations for Japanese and European quail [4, 5]. In addition to contemplating nutritional recommendations for laying, broiler, and breeder quail in their poultry recommendations, Rostagno et al. [5] also included Japanese quail in their latest edition. However, Silva and Costa [4] included more information with higher reliability to quail producers. Using inadequate levels of amino acids can induce a lower performance by quail because amino acid deficiency results in protein synthesis limitation when unused excess amino acids result in more energy diverted to support nitrogen excretion. Beside the reduction in energy used for production, there is potential environmental pollution due to an increase in nitrogen excretion. However, it can be noticed that progressive reduction in CP in the diet can induce a situation in which other amino acids such as threonine become limiting to support better performance. Thus, to reach an increase in quail performance with low-protein diets, it is important to establish requirements of all essential amino acids with greater precision. The objective of the current study was to determine the requirement of threonine for laying Japanese quail fed diets with different threonine:lysine ratios through zootechnical and histological performance measurements.
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262 Table 1. Feed and chemical composition of the experimental diets
Threonine:lysine ratio Item
0.70
0.74
0.78
0.82
0.86
63.62 18.32 5.87 5.41 1.38 0.29 0.35 0.08 0.02 0.18 0.11 0.30 0.10 0.10 0.10 3.50 — 0.26 —
63.62 18.32 5.87 5.41 1.38 0.29 0.35 0.08 0.02 0.18 0.11 0.30 0.10 0.10 0.10 3.50 0.01 0.21 0.04
63.62 18.32 5.87 5.41 1.38 0.29 0.35 0.08 0.02 0.18 0.11 0.30 0.10 0.10 0.10 3.50 0.02 0.16 0.08
63.62 18.32 5.87 5.41 1.38 0.29 0.35 0.08 0.02 0.18 0.11 0.30 0.10 0.10 0.10 3.50 0.03 0.10 0.12
63.62 18.32 5.87 5.41 1.38 0.29 0.35 0.08 0.02 0.18 0.11 0.30 0.10 0.10 0.10 3.50 0.04 0.05 0.16
63.62 18.32 5.87 5.41 1.38 0.29 0.35 0.08 0.02 0.18 0.11 0.30 0.10 0.10 0.10 3.50 0.04 — 0.20
2.95 18.6 2.50 0.35 0.15 0.21 0.53 1.00 0.70 0.19 0.92 0.90 1.26 0.66
2.95 18.6 2.50 0.35 0.15 0.21 0.53 1.00 0.70 0.19 0.92 0.90 1.26 0.70
2.95 18.6 2.50 0.35 0.15 0.21 0.53 1.00 0.70 0.19 0.92 0.90 1.26 0.74
2.95 18.6 2.50 0.35 0.15 0.21 0.53 1.00 0.70 0.19 0.92 0.90 1.26 0.78
2.95 18.6 2.50 0.35 0.15 0.21 0.53 1.00 0.70 0.19 0.92 0.90 1.26 0.82
2.95 18.6 2.50 0.35 0.15 0.21 0.53 1.00 0.70 0.19 0.92 0.90 1.26 0.86
1
Vitamin premix per kilogram of feed: vitamin A, 15,000,000 IU; vitamin D3, 1,500,000 IU; vitamin E, 15,000 IU; vitamin B1, 2.0 g; vitamin B2, 4.0 g; vitamin B6, 3.0 g; vitamin B12, 0.015 g; nicotinic acid, 25 g; pantothenic acid, 10 g; vitamin K3, 3.0 g; and folic acid, 1.0 g. 2 Mineral premix per kilogram of feed: Mn, 60 g; Fe, 80 g; Zn, 50 g; Cu, 10 g; Co, 2 g; I, 1 g, Se, 250 mg; and vehicle quantity sufficient to 500 g.
ation (60% methanol, 30% chloroform, and 10% acetic acid) for 12 h, then transferred to 70% alcohol. These organs were selected because their activities are very important in absorption and metabolism of nutrients and egg production. For optical microscopy, inclusions of the fragments in paraplast were made. Serial sections of the fragments with 5 µm thickness were performed. The following histological staining was performed: hematoxylin and eosin, periodic acid Schiff (PAS), and Masson’s trichrome. The pho-
tomicrographs were captured with the aid of a micro camera attached to a microscope (Olympus BX-51) and the images were digitalized on software KS 400.3 (Zeiss 4.3). The results were statistically analyzed using the SAS program [6]. The regression analysis was performed using linear and quadratic effects to estimate the digestible threonine requirements while considering the significance level, determination coefficient value, and the biological response of the Japanese quail.
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Ingredient, % Corn Soybean meal Corn gluten meal Limestone Dicalcium phosphate Salt l-Lysine dl-Methionine l-Tryptophan l-Isoleucine l-Valine l-Arginine Choline Vitamin mix1 Mineral mix2 Soybean oil Pure river sand l-Glutamate acid l-Threonine Calculated composition, % ME, kcal/kg CP Calcium Available phosphorus Sodium Chloride Potassium Lysine Methionine + cystine Tryptophan Valine Isoleucine Arginine Threonine
0.66
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Table 2. Feed intake (FI), egg production (EP), egg weight (EW), egg mass (EM), feed conversion per egg mass (CEM), and feed conversion per dozen eggs (CDE) of Japanese quail hen fed graded threonine:lysine ratio Item Threonine:lysine, % 66 70 74 78 82 86 SEM P-value Linear Quadratic REQ6
FI, g/d
EP,1 %
20.85 20.70 20.44 21.01 20.58 20.76 0.1300
78.45 79.86 81.55 82.38 81.93 79.07 0.4308
0.133 0.087 —
0.092 0.009 77.13
EW,2 g/egg
EM,3 g
9.71 9.74 9.76 9.77 9.75 9.74 0.0252
7.62 7.77 7.95 8.05 7.99 7.7 0.0417
0.102 0.007 75.12
0.073 0.005 78.22
CEM,4 g/g 2.73 2.66 2.57 2.61 2.57 2.69 0.0194 0.053 0.002 79.61
CDE,5 g/g 0.319 0.311 0.305 0.304 0.302 0.309 0.0022 0.032 0.002 81.00
Ŷ = −0.0334x2 + 5.1524x – 116.45, R2 = 0.91. Ŷ = −0.0004x2 + 0.0601x + 7.4249, R2 = 0.95. 3 Ŷ = −0.0035x2 + 0.5476x – 13.108, R2 = 0.93. 4 Ŷ = 0.0013x2 – 0.207x + 10.565, R2 = 0.88. 5 Ŷ = 0.0001x2 – 0.0162x + 0.9412, R2 = 0.96. 6 Requirement of threonine:lysine ratio according to derivation of polynomial equation. 2
RESULTS AND DISCUSSION Feed intake was not influenced (P > 0.05) by dietary level of threonine. However, there was a significant (P < 0.05) quadratic effect of treatment on variables presented in Table 2 with an estimated optimum threonine:lysine ratio of 77, 75, 78, 80, and 81% for egg production, egg weight, egg mass, feed conversion per egg mass, and feed conversion per dozen eggs, respectively. The threonine:lysine ratios used in this study did not significantly (P > 0.05) modify internal or external egg quality (Table 3).
The intestinal villus of the quail fed diets with higher threonine:lysine ratio were wider with more ramifications (Figure 1A, 1B) and had a greater number of more active goblet cells (Figure 1C, 1D). The folds of the magnum of the quail hens subjected to the treatment with higher threonine:lysine ratio were more active as observed related to the gray staining (Figure 1E, 1F) and the epithelium of these folds have more mucus-producing cells (Figure 1G, 1H). There were more secondary folds in the uterus of the Japanese quail hen fed submitted to the higher threonine:lysine ratio (Figure 1I, 1J).
Table 3. Relative weight of albumen (PALB), yolk (PYK), and eggshell (PSH); yolk pigmentation score (PIG); specific gravity (SG); and eggshell thickness (EST) of Japanese quail hens fed graded levels of threonine:lysine ratio Item Threonine:lysine, % 66 70 74 78 82 86 SEM P-value Linear Quadratic
PALB, %
PYK, %
PSH, %
PIG
SG, g/cm3
EST, mm
57.45 57.46 57.11 56.76 56.87 57.86 0.2074
30.39 29.26 28.78 29.66 29.57 28.38 0.2162
8.24 8.58 8.45 8.55 8.73 8.51 0.0475
5.96 6.11 6.07 6.11 6.06 6.18 0.0249
1.073 1.071 1.073 1.073 1.075 1.073 0.0003
0.233 0.241 0.238 0.243 0.242 0.241 0.0013
0.178 0.242
0.187 0.225
0.141 0.214
0.133 0.142
0.094 0.073
0.088 0.101
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In the treatments with higher threonine:lysine ratios, the magnum tubular glands appeared in greater numbers, in a more active functional stage, and of superior quantity of albumen than the other treatments (Figure 1E, 1F). The increase in the threonine:lysine ratio, mainly 82%, also increased mucus production by the magnum epithelium, protecting its mucosa (Figure 1G, 1H). That diet also increased the quantity of uterus secondary folds in these quail (Figure 1I, 1J).
Despite the noticeable results due to increased threonine:lysine ratio in laying quail, however, those diets worsen the hepatic steatosis commonly found in the liver of laying birds, because such pathogenesis were more intense in the ratio of 0.86 (Figures 2A, 2B, 2C). Along with hepatic steatosis, it was observed that there were small deposits of parenchyma collagen (Figure 2E, 2F) in the liver of birds of the group that received feed with higher proportions of threonine (82 and 86%), whereas in the other groups the collagen was found only around normal blood vessels (Figure 2D). The liver of the animals consuming the 66% threonine:lysine ratio had a higher affinity to PAS (Figure 2G), indicating a greater accumulation of glycogen in comparison with the liver of quail fed higher than 66% threonine:lysine ratio (Figures 2H, 2I). Samadi and Liebert [7] found that genotype, sex, age, and efficiency of dietary threonine utilization affect avian threonine requirement without affecting protein deposition, suggesting the constant need to reevaluate dietary threonine recommendations for quail hens. The NRC [2] recommends 0.74% threonine, whereas INRA [3] recommends 0.58% for this amino acid. Furthermore, Silva and Costa [4] recommended a feed with at least 0.79 and 0.67% of total and digestible threonine, respectively. Meanwhile, Rostagno et al. [5] recommended a feed for 177g quail hens to contain 0.66% digestible threonine. Using diets based on soybean meal and casein [8], others [9–11] estimated levels of 0.11, 0.64, and 0.63% threonine, respectively. When evaluating the influence of supplementing l-threonine in the feed of meat-producing Japanese quail, Baylan et al. [12] used 6 levels of total threonine in their feed that varied from 0.81 to 1.06%, with 0.05% of threonine increments. The base feed contained 23% protein and 1.10% total lysine so that the supplementation of synthetic amino acid was used to obtain the levels of threonine determined by the treatment. Dietary threonine supplementation influenced live BW and feed conversion of quail, but did not influence weight of carcass parts. Their conclusion was that threonine supplementation does not affect growth and is related to a possible bird adaptation to threonine level in the feed. A similar study to the one of Baylan et al. [12] was performed by Umigi et al. [13], but in
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Figure 1. Photomicrograph of digestive and reproductive organs of Japanese quail hens fed a diet with a 66 and 82% threonine:lysine ratio. For A, C, E, G, and I photomicrographs, the Japanese quail hens were fed a diet with a 66% threonine:lysine ratio. The photomicrographs C, D, F, H, and J show Japanese quail hens fed a diet with an 82% threonine:lysine ratio. For A, B, I, and J, hematoxylin-eosin stain was used. For photomicrographs C, D, G, and H, periodic acid Schiff stain was used. For E and F photomicrographs, Masson’s trichrome stain was used. The bar for photomicrographs A, B, E, and F is equal to 500 µm. For C, D, I, and J photomicrographs, the bar is equal to 200 µm. For G and H photomicrographs, the bar is equal to 100 µm. Color version available in the online PDF.
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the laying stage. They [13] worked with 5 levels of digestible threonine varying from 0.65 to 0.85% with 0.05% digestible threonine increments. The diets were nutritionally balanced except for digestible threonine where protein and energy present in supplemented l-threonine was corrected by l-glutamic acid and vegetable oil replaced starch. The authors did not verify an effect on daily feed intake, corroborating data from this study, but the intake of threonine increased linearly. Thus, based on results obtained, Umigi et al. [13] concluded that digestible diet with 0.65% digestible threonine is sufficient to provide good performance and quality of eggs. In the current study, we evaluated the supplementation of threonine at levels from 0.66 to 0.86% in increments of 0.04% in the diet of Japanese quail hens providing threonine level 0.66, 0.70, 0.74, 0.78, 0.82, and 0.86%
and threonine:lysine ratio level of 66, 70, 74, 78, 82, and 86%. Although no feed intake effect was observed as by Umigi et al. [13], we observed a quadratic effect on production, egg weight, egg mass, feed conversion per egg mass, feed conversion per dozen egg at optimal threonine:lysine ratio of 77, 75, 78, 79, and 81%, respectively. Sá et al. [14], working with levels of threonine in feed of lightweight laying quail, did not find significant differences in egg weight, but there was an increase in egg mass when the threonine level was elevated. Thus, the results presented herein showed that the increase in threonine:lysine ratio in the feed of laying quail improved egg weight, egg mass, feed conversion per egg mass, and feed conversion per dozen egg as shown for lightweight quail hens [14] and, when evaluated, digestible threonine requirement for the same birds [15, 16]. As to
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Figure 2. Photomicrographs of the liver of Japanese quail hens in the group of diets having a 66% (A, D, and G), 82% (B, E, and H), and 86% (G, H, and I) threonine:lysine ratio. Hematoxylin-eosin stain (A, B, and C). Masson’s trichrome stain (D, E, and F). Periodic acid Schiff stain (G, H, and I). Bar = 200 µm. Color version available in the online PDF.
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The better performance of quail in relation to a higher supplementation of threonine can be explained by histological alterations found in the intestinal villi of the quail that received greater threonine:lysine ratios (78, 82, and 86%) in their feed. This rearranging of the mucosa demonstrated the physiological need to enlarge the absorptive surface of the small intestine in relation to a determined nutrient [18], in this case, threonine. These results are similar to those of Gomide-Junior [19], who reported that the intestinal mucosa responds to exogenous agents by means of morphological modifications in the height and number of the intestinal villus, depth of intestinal crypts, cellular proliferation, and number of dead cells by epithelial loss. The greater positively to placement of PAS in the intestinal epithelium with the increase of dietary threonine demonstrates that the goblet cells of the intestinal epithelium are greater in number and produce more mucus. This characteristic permits the feed bolus to pass through the intestine more easily, avoiding constipations and protecting the intestinal mucosa from damages caused by fasting or pathogenic agents [19]. Therefore, the increased threonine:lysine in a diet may also be beneficial to the health of the bird. The increase in the production of eggs due to the increase in threonine:lysine ratio in feed can also be explained by the occurrence of alterations in the physiology of the liver to deposit energy. Current results indicated that there was no large-scale energy deposition in birds that received a higher level of threonine and most of the energy was destined primarily to production. Horn et al. [20] suggested that threonine may influence the protection functions of the intestine and possibly affect the absorption of nutrients in general, so it is important to have an appropriate level of that amino acid to avoid problems of protection as well as reduced performance. Based on the changes in the tubular glands of the magnum, which were greater in number and in a more active functional stage, it can be concluded that these characteristic changes permit the eggs to be produced in a shorter period of time, increasing egg production, egg weight, egg mass, feed conversion per egg mass and dozen eggs. Characteristics such as the increase in the production of mucus in the epithelium of
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the quality of quail eggs, Umigi et al. [13] found no alterations in this parameter due to the levels of threonine in the diet, corroborating results obtained by Lima et al. [16]. Similarly, others [15] working with lightweight laying quail found no significant effect for the variables on the internal and external egg quality when the level of threonine was increased. However, Teixeira et al. [17] reported that increasing digestible threonine level in feed only influenced specific gravity. The same authors recommended 0.53% digestible threonine for white quail hens. Hence, threonine seems to influence quail performance with or without affecting the quality of the egg. However, histological differences in the quail reproductive organs were noticeable when feeding higher threonine:lysine ratio to Japanese quail hens. In this case, the tubular glands of the magnum are greater in number with a more active functional stage. Thus, producing a superior quantity of albumen that permits the production of eggs in a shorter period of time to increase egg production, egg weight, egg mass, feed conversion per egg mass and dozen eggs as presented in the current study. Moreover, it was noticed that an increase in threonine:lysine ratio also elevated the quantity of secondary folds in the uterus of laying Japanese quail. That characteristic response permitted the formation of the shell in a shorter time, increasing the production of eggs, because in its formation the egg needs to stay in the uterus for a variable amount of time for calcium carbonate to deposit. If that time is accelerated by increased threonine in the organ internal surface, then discoloration of the egg can be reduced, because in the majority of these eggs discoloration is caused by stress, forcing the bird to lay too soon, and with a more rapid formation of the shell that management problem can be alleviated. The authors found in the literature reports only about the effects of threonine supplementation on performance; however, we include information concerning the effects on the digestive and reproductive organs and histological changes in quail. Thus, we provide information that can affect the quail, giving greater harmony to the data and enabling a better understanding of the response of threonine in the feed of laying Japanese quail.
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Ramalho de Lima et al.: THREONINE:LYSINE RATIO
CONCLUSIONS AND APPLICATIONS
1. The increase in threonine:lysine ratio does not affect egg quality, but can cause significant effects on the digestive and reproductive organs of laying Japanese quail. 2. In general, there was an increase in zootechnical or performance indices, with threonine:lysine ratio of 78 or 0.78% threonine in the feed of Japanese quail hens having the best production index.
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3. Institut National de la Recherche Agronomique (INRA). 1999. Alimentação dos animais monogástricos: Suínos, Coelhos e Aves. 2nd ed. Roca, São Paulo, Brazil. 4. Silva, J. H. V., and F. G. P. Costa. 2009. Tabelas para codornas Japonesas e Européias. 2nd ed. Viçosa, Minas Gerais, Brazil. 5. Rostagno, H. S., L. F. T. Albino, and J. L. Donzele. 2011. Tabelas brasileiras para aves e suínos: Composição de alimentos e exigências nutricionais. Universidade Federal de Viçosa Viçosa, Minas Gerais, Brazil. 6. SAS Institute Inc. 1997. SAS User’s Guide: Statistics. SAS Inst. Inc., Cary, NC. 7. Samadi and F. Liebert. 2006. Modeling of threonine requirements in fast-growing chickens, depending on age, sex, protein deposition, and dietary threonine efficiency. Poult. Sci. 85:1961–1968. 8. Allen, N. K., and R. J. Young. 1980. Studies on the amino acid protein requirements of laying Japanese quail (Coturnix coturnix japonica). Poult. Sci. 59:2029–2037. 9. Shim, K. F. P., and A. Vohra. 1984. Review of the nutrition of Japanese quail. World’s Poult. Sci. J. 40:261–271. 10. Shrivastav, A. K., and B. Panda. 1990. A review of quail nutrition research in India. World’s Poult. Sci. J. 55:73–81. 11. Shim, K. F., and T. K. Lee. 1993. Effect of dietary essential amino acids on egg production of laying Japanese quail. Sing. J. Prim. Ind. 21:72–75. 12. Baylan, M., S. Canogullari, and T. Ayasan. 2006. Dietary threonine supplementation for improving growth performance and edible carcass parts in Japanese quail. Int. J. Poult. Sci. 5:635–638. 13. Umigi, R. T., S. L. T. Barreto, and J. L. Donzele. 2007. Níveis de treonina digestível em dietas para codornas japonesas em postura. Rev. Bras. de Zootec. 36:1868–1874. 14. Sá, L. M., P. C. Gomes, and P. R. Cecon. 2007. Exigência nutricional de treonina digestível para galinhas poedeiras no período de 34 a 50 semanas de idade. Rev. Bras. de Zootec. 36:1846–1853. 15. Lima, M. R., and F. G. P. Costa. 2009. Qualidade de ovos de poedeiras brancas alimentadas com diferentes relações treonina digestível: lisina digestível. CD-ROM in Proc. Congresso sobre Manejo e Nutrição de Aves e Suínos, CBNA, Campinas, São Paulo, Brazil. 16. Lima, M. R., F. G. P. Costa, and G. B. V. Lobato. 2010. Exigência de treonina para codornas japonesas, utilizandose dietas com diferentes relações treonina: lisina. Page 244 in Proc. Fourth International Symposium and Third Brazilian Congress on Quail Production. UFLA, Lavras, Minas Gerais, Brazil. 17. Teixeira, E. N. M., J. H. V. Silva, and M. R. Lima. 2005. Exigência de treonina digestível para poedeiras leves e semipesadas. Rev. Bras. de Ciên. Avíc. 7:131. 18. Aptekmann, K. P., S. M. Baraldi-Artoni, and M. A. Stefanini. 2001. Morphometric analysis of the intestine of domestic quails (Coturnix coturnix japonica) treated with different levels of dietary calcium. Anat. Hist. Embryol. 30:277–280. 19. Gomide-Junior, M. L., E. V. Sterzo, M. Macari, and I. C. Boleli. 2004. Use of scanning electron microscopy for the evaluation of intestinal epithelium integrity. Rev. Bras. de Zootec. 33:1500–1505. 20. Horn, N. L., S. S. Donkin, T. J. Applegate, and O. Adeola. 2009. Intestinal mucin dynamics: Response of
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the magnum and a greater quantity of secondary folds in the uterus permit formation of the shell in a shorter time, increasing the production of eggs because the egg, while forming, stays in the uterus for a variable amount of time for the deposition of calcium carbonate [21]. That deposition is accelerated by the increase in the internal surface of the organ. In relation to steatosis with the increase of threonine in feed, studies demonstrated that diet supplementation with methionine also induces the increase in liver fat [22]. This increase of hepatic steatosis could have been caused by the greater estrogen synthesis in the ovary to give support to the greater production of eggs [22], which contradicts the current results. In relation to the small deposits of parenchymal collagen observed in the liver of the quail that received feed with threonine:lysine ratios of 82 and 86%, it should be pointed out that although it is a small quantity, this effect denotes a fibrotic process resulting from the continuous hepatocyte regeneration process [23]. However, the average productive life of a quail is approximately 360 d, so these metabolic alterations do not generate productive, qualitative, and consequently economic losses. Furthermore, the group that more evidently presented these hepatic alterations was the one with 86% threonine:lysine ratio with suboptimal bird performance.
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268 broiler chicks and white Pekin ducklings to dietary threonine. Poult. Sci. 88:1906–1914. 21. King, A. S., and J. Mclelland. 1979. Form and Function in Quails. Academic Press, London, UK. 22. Bunchasak, C., and T. Silapasorn. 2005. Effects of adding methionine in low-protein diet on production performance, reproductive organs and chemical liver composition of laying quails under tropical conditions. Int. J. Poult. Sci. 4:301–308.
23. Fausto, N. 1986. New perspectives on liver regeneration. Hepatology 6:326–327.
Acknowledgments
Special thanks to Conselho Nacional de Desenvolvimento Científico e Tecnológico, Granja Fujikura (Suzano, São Paulo, Brazil), Guaraves Alimentos Ltda. (Guarabira, Paraiba, Brazil), and Ajinomoto Animal Nutrition (São Paulo, Brazil).
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Threonine:lysine ratio for Japanese quail hen diets Matheus Ramalho de Lima,*1 Fernando Guilherme Perazzo Costa,† Ricardo Romão Guerra,† José HumbertoVilar da Silva,† Carlos Bôa-Viagem Rabello,‡ Maria Angélica Miglino,§ Gledyson Bruno Vieira Lobato,† Severino Bernardino Sena Netto,† and Leonilson da Silva Dantas†
Primary Audience: Poultry Farmers, Poultry Nutritionists SUMMARY the objective of this study was to evaluate threonine requirements for laying Japanese quail fed with different threonine:lysine ratios through measurement of zootechnical and histological performance. a total of 288 Japanese quail at 125 d of age were distributed in a totally randomized design, with 6 treatments and 6 replications of 8 each. the treatments consisted of a basal diet made of corn, soybean meal, and corn gluten meal, supplemented with l-lysine, dlmethionine, l-tryptophan, l-isoleucine, and l-valine to meet the laying Japanese quail essential amino acid needs, except for threonine. the basal diet was supplemented with 0.00, 0.04, 0.08, 0.12, 0.16, and 0.20% of l-threonine in substitution for l-glutamate acid, with the objective of reaching levels of 0.66, 0.70, 0.74, 0.78, 0.82, and 0.86% of threonine in the feed, resulting in threonine:lysine ratios of 66, 70, 74, 78, 82, and 86%. Zootechnical and histological indexes of the digestive and reproductive system were evaluated. the amino acid ratio alteration assessed in this study influenced performance and histology of the quail through improving productive performance at a 78% threonine:lysine ratio. Key words: egg production, threonine, histological response 2013 J. appl. Poult. Res. 22:260–268 http://dx.doi.org/10.3382/japr.2012-00670
DESCRIPTION OF PROBLEM the excellent productive performance of Japanese quail, which begin laying between 35 and 40 d of age and have an average production of 300 eggs/hen per year, need a small area for production (400 bird/m2), and have a low 1
Corresponding author: [email protected]
investment with rapid return of capital invested [1], has stimulated the development of a quail industry in Brazil. despite such great economic importance, few studies have been performed in Brazil on the nutritional requirements of quail with feed formulation for quail performed based on suggested requirements [2, 3] or extrapola-
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*Federal University of West of Para, Biodiversity and Forest Institute, 68040-410, Santarém, Pará, Brazil; †Federal University of Paraiba, Department of Animal Science, PB 079, Km 1/i92, 58397000, Campus II, Areia, Paraiba, Brazil; ‡Federal Rural University of Pernambuco, Department of Animal Science, Recife, Pernambuco, Brazil 52171-900; and §Faculty of Veterinary Medicine and Animal Science, Sao Paulo University, Sao Paulo, Brazil 05508-270
Ramalho de Lima et al.: THREONINE:LYSINE RATIO
MATERIALS AND METHODS The study was performed at the Poultry Research Center of the Federal University of Paraiba (Research Farm of Poultry CCA/UFPB) at Areia, Paraiba. A total of 288 Japanese quail at 125 d of age were distributed in a complete randomized design among 6 treatments with 6 replications of 8 quail each. The treatments consisted of a basal diet formulated based on corn, soybean meal, and corn gluten meal, supplemented with l-lysine, dl-methionine, ltryptophan, l-isoleucine, and l-valine to meet the essential amino acid needs of the Japanese quail hen except for threonine [2]. The project had ethical approval from the Animal Use and Care Committee of the Federal University of Paraiba, Brazil. The quail were allocated among
40 × 40 × 20 cm cages in clay-tile sheds with 2 water troughs. The birds received 17 h of light per day. The average temperature and RH were 27°C and 88%, respectively. The basal diet was supplemented with 0.00, 0.04, 0.08, 0.12, 0.16, and 0.20% l-threonine to substitute l-glutamic acid with the objective of reaching levels of 0.66, 0.70, 0.74, 0.78, 0.82, and 0.86% threonine in the diet, resulting in the threonine:lysine ratios of 0.66, 0.70, 0.74, 0.78, 0.82, and 0.86 or 66, 70, 74, 78, 82, and 86% (Table 1). l-Glutamic acid was used to make the diets isonitrogenous. The dietary level of soybean oil and of the inert material was added in substitution to l-glutamic acid that was reduced with the increase in the levels of threonine in the experimental diets, making all diets isonitrogenous and isoenergetic. Zootechnical and histological evaluations were performed. In the zootechnical evaluations, the feed intake (g/bird), egg production (%), egg weight (g/egg), egg mass (g/egg production), feed conversion per egg mass (g/g), feed conversion per dozen eggs (g/g), relative albumen weight (%), percent yolk weight (%), percent shell weight (%), yolk pigmentation score, egg specific gravity (g/cm3), and eggshell thickness (mm) were assessed. The period of evaluation of egg production was divided into 5 periods of 21 d each. At the end of each period, the feed left in each feed trough was collected for the calculation of feed intake. Egg collection was performed twice a day at 1000 and 1600 h, and mortality was recorded. Percent egg production was calculated by dividing the total quantity of eggs per pen by the number of quail, correcting for mortality. The eggs of the last 3 d of each period were weighed individually to obtain the average weight of the eggs. Egg mass was the product of the production of eggs and average weight of the eggs per pen. The feed conversion per egg mass was the ratio of feed intake to egg mass, and feed conversion per dozen eggs was the ratio of feed intake to egg production multiplied by 12. For the histological analyses, at termination of the experiment when the quail were 230 d of age, the collection of the material was performed from fragments of the digestive (duodenum and liver) and reproductive (magnum and uterus) systems of 10 animals per treatment. Those sections were immersed in methacarn fix-
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tion from laying quail or broilers. One of the few exceptions of data obtained from experiments performed in Brazil are the Tables of Nutritional Recommendations for Japanese and European quail [4, 5]. In addition to contemplating nutritional recommendations for laying, broiler, and breeder quail in their poultry recommendations, Rostagno et al. [5] also included Japanese quail in their latest edition. However, Silva and Costa [4] included more information with higher reliability to quail producers. Using inadequate levels of amino acids can induce a lower performance by quail because amino acid deficiency results in protein synthesis limitation when unused excess amino acids result in more energy diverted to support nitrogen excretion. Beside the reduction in energy used for production, there is potential environmental pollution due to an increase in nitrogen excretion. However, it can be noticed that progressive reduction in CP in the diet can induce a situation in which other amino acids such as threonine become limiting to support better performance. Thus, to reach an increase in quail performance with low-protein diets, it is important to establish requirements of all essential amino acids with greater precision. The objective of the current study was to determine the requirement of threonine for laying Japanese quail fed diets with different threonine:lysine ratios through zootechnical and histological performance measurements.
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262 Table 1. Feed and chemical composition of the experimental diets
Threonine:lysine ratio Item
0.70
0.74
0.78
0.82
0.86
63.62 18.32 5.87 5.41 1.38 0.29 0.35 0.08 0.02 0.18 0.11 0.30 0.10 0.10 0.10 3.50 — 0.26 —
63.62 18.32 5.87 5.41 1.38 0.29 0.35 0.08 0.02 0.18 0.11 0.30 0.10 0.10 0.10 3.50 0.01 0.21 0.04
63.62 18.32 5.87 5.41 1.38 0.29 0.35 0.08 0.02 0.18 0.11 0.30 0.10 0.10 0.10 3.50 0.02 0.16 0.08
63.62 18.32 5.87 5.41 1.38 0.29 0.35 0.08 0.02 0.18 0.11 0.30 0.10 0.10 0.10 3.50 0.03 0.10 0.12
63.62 18.32 5.87 5.41 1.38 0.29 0.35 0.08 0.02 0.18 0.11 0.30 0.10 0.10 0.10 3.50 0.04 0.05 0.16
63.62 18.32 5.87 5.41 1.38 0.29 0.35 0.08 0.02 0.18 0.11 0.30 0.10 0.10 0.10 3.50 0.04 — 0.20
2.95 18.6 2.50 0.35 0.15 0.21 0.53 1.00 0.70 0.19 0.92 0.90 1.26 0.66
2.95 18.6 2.50 0.35 0.15 0.21 0.53 1.00 0.70 0.19 0.92 0.90 1.26 0.70
2.95 18.6 2.50 0.35 0.15 0.21 0.53 1.00 0.70 0.19 0.92 0.90 1.26 0.74
2.95 18.6 2.50 0.35 0.15 0.21 0.53 1.00 0.70 0.19 0.92 0.90 1.26 0.78
2.95 18.6 2.50 0.35 0.15 0.21 0.53 1.00 0.70 0.19 0.92 0.90 1.26 0.82
2.95 18.6 2.50 0.35 0.15 0.21 0.53 1.00 0.70 0.19 0.92 0.90 1.26 0.86
1
Vitamin premix per kilogram of feed: vitamin A, 15,000,000 IU; vitamin D3, 1,500,000 IU; vitamin E, 15,000 IU; vitamin B1, 2.0 g; vitamin B2, 4.0 g; vitamin B6, 3.0 g; vitamin B12, 0.015 g; nicotinic acid, 25 g; pantothenic acid, 10 g; vitamin K3, 3.0 g; and folic acid, 1.0 g. 2 Mineral premix per kilogram of feed: Mn, 60 g; Fe, 80 g; Zn, 50 g; Cu, 10 g; Co, 2 g; I, 1 g, Se, 250 mg; and vehicle quantity sufficient to 500 g.
ation (60% methanol, 30% chloroform, and 10% acetic acid) for 12 h, then transferred to 70% alcohol. These organs were selected because their activities are very important in absorption and metabolism of nutrients and egg production. For optical microscopy, inclusions of the fragments in paraplast were made. Serial sections of the fragments with 5 µm thickness were performed. The following histological staining was performed: hematoxylin and eosin, periodic acid Schiff (PAS), and Masson’s trichrome. The pho-
tomicrographs were captured with the aid of a micro camera attached to a microscope (Olympus BX-51) and the images were digitalized on software KS 400.3 (Zeiss 4.3). The results were statistically analyzed using the SAS program [6]. The regression analysis was performed using linear and quadratic effects to estimate the digestible threonine requirements while considering the significance level, determination coefficient value, and the biological response of the Japanese quail.
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Ingredient, % Corn Soybean meal Corn gluten meal Limestone Dicalcium phosphate Salt l-Lysine dl-Methionine l-Tryptophan l-Isoleucine l-Valine l-Arginine Choline Vitamin mix1 Mineral mix2 Soybean oil Pure river sand l-Glutamate acid l-Threonine Calculated composition, % ME, kcal/kg CP Calcium Available phosphorus Sodium Chloride Potassium Lysine Methionine + cystine Tryptophan Valine Isoleucine Arginine Threonine
0.66
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Table 2. Feed intake (FI), egg production (EP), egg weight (EW), egg mass (EM), feed conversion per egg mass (CEM), and feed conversion per dozen eggs (CDE) of Japanese quail hen fed graded threonine:lysine ratio Item Threonine:lysine, % 66 70 74 78 82 86 SEM P-value Linear Quadratic REQ6
FI, g/d
EP,1 %
20.85 20.70 20.44 21.01 20.58 20.76 0.1300
78.45 79.86 81.55 82.38 81.93 79.07 0.4308
0.133 0.087 —
0.092 0.009 77.13
EW,2 g/egg
EM,3 g
9.71 9.74 9.76 9.77 9.75 9.74 0.0252
7.62 7.77 7.95 8.05 7.99 7.7 0.0417
0.102 0.007 75.12
0.073 0.005 78.22
CEM,4 g/g 2.73 2.66 2.57 2.61 2.57 2.69 0.0194 0.053 0.002 79.61
CDE,5 g/g 0.319 0.311 0.305 0.304 0.302 0.309 0.0022 0.032 0.002 81.00
Ŷ = −0.0334x2 + 5.1524x – 116.45, R2 = 0.91. Ŷ = −0.0004x2 + 0.0601x + 7.4249, R2 = 0.95. 3 Ŷ = −0.0035x2 + 0.5476x – 13.108, R2 = 0.93. 4 Ŷ = 0.0013x2 – 0.207x + 10.565, R2 = 0.88. 5 Ŷ = 0.0001x2 – 0.0162x + 0.9412, R2 = 0.96. 6 Requirement of threonine:lysine ratio according to derivation of polynomial equation. 2
RESULTS AND DISCUSSION Feed intake was not influenced (P > 0.05) by dietary level of threonine. However, there was a significant (P < 0.05) quadratic effect of treatment on variables presented in Table 2 with an estimated optimum threonine:lysine ratio of 77, 75, 78, 80, and 81% for egg production, egg weight, egg mass, feed conversion per egg mass, and feed conversion per dozen eggs, respectively. The threonine:lysine ratios used in this study did not significantly (P > 0.05) modify internal or external egg quality (Table 3).
The intestinal villus of the quail fed diets with higher threonine:lysine ratio were wider with more ramifications (Figure 1A, 1B) and had a greater number of more active goblet cells (Figure 1C, 1D). The folds of the magnum of the quail hens subjected to the treatment with higher threonine:lysine ratio were more active as observed related to the gray staining (Figure 1E, 1F) and the epithelium of these folds have more mucus-producing cells (Figure 1G, 1H). There were more secondary folds in the uterus of the Japanese quail hen fed submitted to the higher threonine:lysine ratio (Figure 1I, 1J).
Table 3. Relative weight of albumen (PALB), yolk (PYK), and eggshell (PSH); yolk pigmentation score (PIG); specific gravity (SG); and eggshell thickness (EST) of Japanese quail hens fed graded levels of threonine:lysine ratio Item Threonine:lysine, % 66 70 74 78 82 86 SEM P-value Linear Quadratic
PALB, %
PYK, %
PSH, %
PIG
SG, g/cm3
EST, mm
57.45 57.46 57.11 56.76 56.87 57.86 0.2074
30.39 29.26 28.78 29.66 29.57 28.38 0.2162
8.24 8.58 8.45 8.55 8.73 8.51 0.0475
5.96 6.11 6.07 6.11 6.06 6.18 0.0249
1.073 1.071 1.073 1.073 1.075 1.073 0.0003
0.233 0.241 0.238 0.243 0.242 0.241 0.0013
0.178 0.242
0.187 0.225
0.141 0.214
0.133 0.142
0.094 0.073
0.088 0.101
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In the treatments with higher threonine:lysine ratios, the magnum tubular glands appeared in greater numbers, in a more active functional stage, and of superior quantity of albumen than the other treatments (Figure 1E, 1F). The increase in the threonine:lysine ratio, mainly 82%, also increased mucus production by the magnum epithelium, protecting its mucosa (Figure 1G, 1H). That diet also increased the quantity of uterus secondary folds in these quail (Figure 1I, 1J).
Despite the noticeable results due to increased threonine:lysine ratio in laying quail, however, those diets worsen the hepatic steatosis commonly found in the liver of laying birds, because such pathogenesis were more intense in the ratio of 0.86 (Figures 2A, 2B, 2C). Along with hepatic steatosis, it was observed that there were small deposits of parenchyma collagen (Figure 2E, 2F) in the liver of birds of the group that received feed with higher proportions of threonine (82 and 86%), whereas in the other groups the collagen was found only around normal blood vessels (Figure 2D). The liver of the animals consuming the 66% threonine:lysine ratio had a higher affinity to PAS (Figure 2G), indicating a greater accumulation of glycogen in comparison with the liver of quail fed higher than 66% threonine:lysine ratio (Figures 2H, 2I). Samadi and Liebert [7] found that genotype, sex, age, and efficiency of dietary threonine utilization affect avian threonine requirement without affecting protein deposition, suggesting the constant need to reevaluate dietary threonine recommendations for quail hens. The NRC [2] recommends 0.74% threonine, whereas INRA [3] recommends 0.58% for this amino acid. Furthermore, Silva and Costa [4] recommended a feed with at least 0.79 and 0.67% of total and digestible threonine, respectively. Meanwhile, Rostagno et al. [5] recommended a feed for 177g quail hens to contain 0.66% digestible threonine. Using diets based on soybean meal and casein [8], others [9–11] estimated levels of 0.11, 0.64, and 0.63% threonine, respectively. When evaluating the influence of supplementing l-threonine in the feed of meat-producing Japanese quail, Baylan et al. [12] used 6 levels of total threonine in their feed that varied from 0.81 to 1.06%, with 0.05% of threonine increments. The base feed contained 23% protein and 1.10% total lysine so that the supplementation of synthetic amino acid was used to obtain the levels of threonine determined by the treatment. Dietary threonine supplementation influenced live BW and feed conversion of quail, but did not influence weight of carcass parts. Their conclusion was that threonine supplementation does not affect growth and is related to a possible bird adaptation to threonine level in the feed. A similar study to the one of Baylan et al. [12] was performed by Umigi et al. [13], but in
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Figure 1. Photomicrograph of digestive and reproductive organs of Japanese quail hens fed a diet with a 66 and 82% threonine:lysine ratio. For A, C, E, G, and I photomicrographs, the Japanese quail hens were fed a diet with a 66% threonine:lysine ratio. The photomicrographs C, D, F, H, and J show Japanese quail hens fed a diet with an 82% threonine:lysine ratio. For A, B, I, and J, hematoxylin-eosin stain was used. For photomicrographs C, D, G, and H, periodic acid Schiff stain was used. For E and F photomicrographs, Masson’s trichrome stain was used. The bar for photomicrographs A, B, E, and F is equal to 500 µm. For C, D, I, and J photomicrographs, the bar is equal to 200 µm. For G and H photomicrographs, the bar is equal to 100 µm. Color version available in the online PDF.
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the laying stage. They [13] worked with 5 levels of digestible threonine varying from 0.65 to 0.85% with 0.05% digestible threonine increments. The diets were nutritionally balanced except for digestible threonine where protein and energy present in supplemented l-threonine was corrected by l-glutamic acid and vegetable oil replaced starch. The authors did not verify an effect on daily feed intake, corroborating data from this study, but the intake of threonine increased linearly. Thus, based on results obtained, Umigi et al. [13] concluded that digestible diet with 0.65% digestible threonine is sufficient to provide good performance and quality of eggs. In the current study, we evaluated the supplementation of threonine at levels from 0.66 to 0.86% in increments of 0.04% in the diet of Japanese quail hens providing threonine level 0.66, 0.70, 0.74, 0.78, 0.82, and 0.86%
and threonine:lysine ratio level of 66, 70, 74, 78, 82, and 86%. Although no feed intake effect was observed as by Umigi et al. [13], we observed a quadratic effect on production, egg weight, egg mass, feed conversion per egg mass, feed conversion per dozen egg at optimal threonine:lysine ratio of 77, 75, 78, 79, and 81%, respectively. Sá et al. [14], working with levels of threonine in feed of lightweight laying quail, did not find significant differences in egg weight, but there was an increase in egg mass when the threonine level was elevated. Thus, the results presented herein showed that the increase in threonine:lysine ratio in the feed of laying quail improved egg weight, egg mass, feed conversion per egg mass, and feed conversion per dozen egg as shown for lightweight quail hens [14] and, when evaluated, digestible threonine requirement for the same birds [15, 16]. As to
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Figure 2. Photomicrographs of the liver of Japanese quail hens in the group of diets having a 66% (A, D, and G), 82% (B, E, and H), and 86% (G, H, and I) threonine:lysine ratio. Hematoxylin-eosin stain (A, B, and C). Masson’s trichrome stain (D, E, and F). Periodic acid Schiff stain (G, H, and I). Bar = 200 µm. Color version available in the online PDF.
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The better performance of quail in relation to a higher supplementation of threonine can be explained by histological alterations found in the intestinal villi of the quail that received greater threonine:lysine ratios (78, 82, and 86%) in their feed. This rearranging of the mucosa demonstrated the physiological need to enlarge the absorptive surface of the small intestine in relation to a determined nutrient [18], in this case, threonine. These results are similar to those of Gomide-Junior [19], who reported that the intestinal mucosa responds to exogenous agents by means of morphological modifications in the height and number of the intestinal villus, depth of intestinal crypts, cellular proliferation, and number of dead cells by epithelial loss. The greater positively to placement of PAS in the intestinal epithelium with the increase of dietary threonine demonstrates that the goblet cells of the intestinal epithelium are greater in number and produce more mucus. This characteristic permits the feed bolus to pass through the intestine more easily, avoiding constipations and protecting the intestinal mucosa from damages caused by fasting or pathogenic agents [19]. Therefore, the increased threonine:lysine in a diet may also be beneficial to the health of the bird. The increase in the production of eggs due to the increase in threonine:lysine ratio in feed can also be explained by the occurrence of alterations in the physiology of the liver to deposit energy. Current results indicated that there was no large-scale energy deposition in birds that received a higher level of threonine and most of the energy was destined primarily to production. Horn et al. [20] suggested that threonine may influence the protection functions of the intestine and possibly affect the absorption of nutrients in general, so it is important to have an appropriate level of that amino acid to avoid problems of protection as well as reduced performance. Based on the changes in the tubular glands of the magnum, which were greater in number and in a more active functional stage, it can be concluded that these characteristic changes permit the eggs to be produced in a shorter period of time, increasing egg production, egg weight, egg mass, feed conversion per egg mass and dozen eggs. Characteristics such as the increase in the production of mucus in the epithelium of
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the quality of quail eggs, Umigi et al. [13] found no alterations in this parameter due to the levels of threonine in the diet, corroborating results obtained by Lima et al. [16]. Similarly, others [15] working with lightweight laying quail found no significant effect for the variables on the internal and external egg quality when the level of threonine was increased. However, Teixeira et al. [17] reported that increasing digestible threonine level in feed only influenced specific gravity. The same authors recommended 0.53% digestible threonine for white quail hens. Hence, threonine seems to influence quail performance with or without affecting the quality of the egg. However, histological differences in the quail reproductive organs were noticeable when feeding higher threonine:lysine ratio to Japanese quail hens. In this case, the tubular glands of the magnum are greater in number with a more active functional stage. Thus, producing a superior quantity of albumen that permits the production of eggs in a shorter period of time to increase egg production, egg weight, egg mass, feed conversion per egg mass and dozen eggs as presented in the current study. Moreover, it was noticed that an increase in threonine:lysine ratio also elevated the quantity of secondary folds in the uterus of laying Japanese quail. That characteristic response permitted the formation of the shell in a shorter time, increasing the production of eggs, because in its formation the egg needs to stay in the uterus for a variable amount of time for calcium carbonate to deposit. If that time is accelerated by increased threonine in the organ internal surface, then discoloration of the egg can be reduced, because in the majority of these eggs discoloration is caused by stress, forcing the bird to lay too soon, and with a more rapid formation of the shell that management problem can be alleviated. The authors found in the literature reports only about the effects of threonine supplementation on performance; however, we include information concerning the effects on the digestive and reproductive organs and histological changes in quail. Thus, we provide information that can affect the quail, giving greater harmony to the data and enabling a better understanding of the response of threonine in the feed of laying Japanese quail.
JAPR: Research Report
Ramalho de Lima et al.: THREONINE:LYSINE RATIO
CONCLUSIONS AND APPLICATIONS
1. The increase in threonine:lysine ratio does not affect egg quality, but can cause significant effects on the digestive and reproductive organs of laying Japanese quail. 2. In general, there was an increase in zootechnical or performance indices, with threonine:lysine ratio of 78 or 0.78% threonine in the feed of Japanese quail hens having the best production index.
REFERENCES AND NOTES 1. Albino, L. F. T., and S. L. T. Barreto. 2003. Codornas: Criação de codornas para produção de ovos e carne. Viçosa, Minas Gerais, Brazil. Editora Aprenda Fácil. 2. NRC. 1994. Nutrient Requirements of Poultry. 9th ed. Natl. Acad. Press, Washington, DC.
3. Institut National de la Recherche Agronomique (INRA). 1999. Alimentação dos animais monogástricos: Suínos, Coelhos e Aves. 2nd ed. Roca, São Paulo, Brazil. 4. Silva, J. H. V., and F. G. P. Costa. 2009. Tabelas para codornas Japonesas e Européias. 2nd ed. Viçosa, Minas Gerais, Brazil. 5. Rostagno, H. S., L. F. T. Albino, and J. L. Donzele. 2011. Tabelas brasileiras para aves e suínos: Composição de alimentos e exigências nutricionais. Universidade Federal de Viçosa Viçosa, Minas Gerais, Brazil. 6. SAS Institute Inc. 1997. SAS User’s Guide: Statistics. SAS Inst. Inc., Cary, NC. 7. Samadi and F. Liebert. 2006. Modeling of threonine requirements in fast-growing chickens, depending on age, sex, protein deposition, and dietary threonine efficiency. Poult. Sci. 85:1961–1968. 8. Allen, N. K., and R. J. Young. 1980. Studies on the amino acid protein requirements of laying Japanese quail (Coturnix coturnix japonica). Poult. Sci. 59:2029–2037. 9. Shim, K. F. P., and A. Vohra. 1984. Review of the nutrition of Japanese quail. World’s Poult. Sci. J. 40:261–271. 10. Shrivastav, A. K., and B. Panda. 1990. A review of quail nutrition research in India. World’s Poult. Sci. J. 55:73–81. 11. Shim, K. F., and T. K. Lee. 1993. Effect of dietary essential amino acids on egg production of laying Japanese quail. Sing. J. Prim. Ind. 21:72–75. 12. Baylan, M., S. Canogullari, and T. Ayasan. 2006. Dietary threonine supplementation for improving growth performance and edible carcass parts in Japanese quail. Int. J. Poult. Sci. 5:635–638. 13. Umigi, R. T., S. L. T. Barreto, and J. L. Donzele. 2007. Níveis de treonina digestível em dietas para codornas japonesas em postura. Rev. Bras. de Zootec. 36:1868–1874. 14. Sá, L. M., P. C. Gomes, and P. R. Cecon. 2007. Exigência nutricional de treonina digestível para galinhas poedeiras no período de 34 a 50 semanas de idade. Rev. Bras. de Zootec. 36:1846–1853. 15. Lima, M. R., and F. G. P. Costa. 2009. Qualidade de ovos de poedeiras brancas alimentadas com diferentes relações treonina digestível: lisina digestível. CD-ROM in Proc. Congresso sobre Manejo e Nutrição de Aves e Suínos, CBNA, Campinas, São Paulo, Brazil. 16. Lima, M. R., F. G. P. Costa, and G. B. V. Lobato. 2010. Exigência de treonina para codornas japonesas, utilizandose dietas com diferentes relações treonina: lisina. Page 244 in Proc. Fourth International Symposium and Third Brazilian Congress on Quail Production. UFLA, Lavras, Minas Gerais, Brazil. 17. Teixeira, E. N. M., J. H. V. Silva, and M. R. Lima. 2005. Exigência de treonina digestível para poedeiras leves e semipesadas. Rev. Bras. de Ciên. Avíc. 7:131. 18. Aptekmann, K. P., S. M. Baraldi-Artoni, and M. A. Stefanini. 2001. Morphometric analysis of the intestine of domestic quails (Coturnix coturnix japonica) treated with different levels of dietary calcium. Anat. Hist. Embryol. 30:277–280. 19. Gomide-Junior, M. L., E. V. Sterzo, M. Macari, and I. C. Boleli. 2004. Use of scanning electron microscopy for the evaluation of intestinal epithelium integrity. Rev. Bras. de Zootec. 33:1500–1505. 20. Horn, N. L., S. S. Donkin, T. J. Applegate, and O. Adeola. 2009. Intestinal mucin dynamics: Response of
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the magnum and a greater quantity of secondary folds in the uterus permit formation of the shell in a shorter time, increasing the production of eggs because the egg, while forming, stays in the uterus for a variable amount of time for the deposition of calcium carbonate [21]. That deposition is accelerated by the increase in the internal surface of the organ. In relation to steatosis with the increase of threonine in feed, studies demonstrated that diet supplementation with methionine also induces the increase in liver fat [22]. This increase of hepatic steatosis could have been caused by the greater estrogen synthesis in the ovary to give support to the greater production of eggs [22], which contradicts the current results. In relation to the small deposits of parenchymal collagen observed in the liver of the quail that received feed with threonine:lysine ratios of 82 and 86%, it should be pointed out that although it is a small quantity, this effect denotes a fibrotic process resulting from the continuous hepatocyte regeneration process [23]. However, the average productive life of a quail is approximately 360 d, so these metabolic alterations do not generate productive, qualitative, and consequently economic losses. Furthermore, the group that more evidently presented these hepatic alterations was the one with 86% threonine:lysine ratio with suboptimal bird performance.
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268 broiler chicks and white Pekin ducklings to dietary threonine. Poult. Sci. 88:1906–1914. 21. King, A. S., and J. Mclelland. 1979. Form and Function in Quails. Academic Press, London, UK. 22. Bunchasak, C., and T. Silapasorn. 2005. Effects of adding methionine in low-protein diet on production performance, reproductive organs and chemical liver composition of laying quails under tropical conditions. Int. J. Poult. Sci. 4:301–308.
23. Fausto, N. 1986. New perspectives on liver regeneration. Hepatology 6:326–327.
Acknowledgments
Special thanks to Conselho Nacional de Desenvolvimento Científico e Tecnológico, Granja Fujikura (Suzano, São Paulo, Brazil), Guaraves Alimentos Ltda. (Guarabira, Paraiba, Brazil), and Ajinomoto Animal Nutrition (São Paulo, Brazil).
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