Maataloustieteellinen Aikakauskirja Vol. 62: 423—433, 1990 Distillers feeds and feed fractions of barley in the diets of laying hens MATTI NÄSI University of Helsinki, Department of Animal Husbandry 00710 Helsinki, Finland Abstract. Two experiments using 551 and 537 LSK-61 WL laying hens in the tests were conducted toevaluate hen performance and egg quality when graded levels of barley or wheat distillers feeds (BDDGS, WDDGS) from conventional process and barley protein and fibre (BP, BF) from integrated starch-ethanol production were incorporated into the diets. In the first exp. hens were fed diets containing 200 g/kg diet of WDDGS or 100 or 200 g BDDGS either with or without cellulase addition, respectively. In the second trial hens were fed diets with 50 or 100 g BP as protein supplement or 100 or 200g/kg diet BF with or without multien- zyme (Avizyme) addition. Each diet with similar contents of ME, CP, lysine and S-amino acids was fed to hens from 34 to 58 wk of age following 4-wk pretreatment and 2-wk transition periods. There were only small dietary effects and no significant differences in performance due to treatment in either trial. The production level was rather high; on average the laying rate was 82.3 and 84.5 %, feed intake 119 g and 118 g/d and FCR 2.42 and 2.37 kg feed/kg eggs in exp. 1 and 2, respectively, indicating no adverse effects of the supplements used. A linear decrease (P < 0.01) in egg weight and yolk colour intensity (P <0.01) was found in hens fed on diets with BP, while shell-% was linear improved PcO.Ol) in both BP and BF diets. Dis- tillery feeds and barley feed fractions could be used in laying hens up to 200 g/kg without any reduction in production and up to two thirds of soybean protein could be replaced in diets fortfied with pure amino acids. The treatment of DDGS with cellulase or supplementation of multienzyme in BF diet had no effect on performance except that yolk colour was lighter in group on multienzyme treated BF diet. Index words: distillery feeds, barley protein, fibre, laying hen Introduction Conventional distillers dried grains with solubles (DDGS) has long been known to be a useful feed ingredient for inclusion in poul- try diets. No undesirable effects have been reported from feeding diets containing from 100 to 200 g/kg DDG or DDGS mainly from maize based ethanol production. In most of the reports, a significant improvement in 423 JOURNAL OF AGRICULTURAL SCIENCE IN FINLAND https://www.c-info.fi/en/info/?token=D5bUQQTz4KjaJn6S.GR8zf86YkcaNjJZTbk76DA.MKCHmMgE_mnrtxAiItpew4z9ad6zO9E4fFDykdJo70xBUX_8Ng-ubFRxE-gZj5J8xfKw55yWmFkCBqkkeo9zzMFRNrsLltNO6wV3L-ifOrqPLMdhdCLq-Ks0gCL_r9SqE2644ROgxrqKodDXDFFr_2SBQjPWpszyeruYN48 growth, egg production, feed utilization and hatchability has been observed in favor of the diets containing distillers feeds over the con- trol diets (review of Jensen 1985). DDGS ap- pears to contain some unidentified nutritional factors which affect interior egg quality, liver lipid accumulation, hormone balance and cal- cium metabolism in laying hens (Jensen 1985). Barley distillers feeds from the tradi- tional ethanol process have been found to have a relatively low nutritive value due to their denatured protein and high fibre content (Näsi 1985). In the integrated production of starch and ethanol, barley by-products suit- able for use in the diets of both ruminants and monogastrics have been obtained (Nasi 1988 a, 1989, Huhtanen et ai. 1988, 1989). Barley protein showed high digestibility and, when fortified with pure lysine, gave a nitro- gen balance in growing pigs similar to that of the isonitrogenous soybean-barley diet. Up to two-thirds of soybean protein could be re- placed with barley protein without differences in the performance of growing pigs (NAsi 1989). The objectives of this study were to com- pare various diets composing by-products from distillery and starch process using bar- ley as a raw material with respect to laying hen performance and egg quality and to study the effect of enzyme addition. Materials and methods Feeds The distillery feeds in the first experiment were obtained from the conventional distillery process (Alko Ltd., Koskenkorva) using de- hulled barley or wheat as raw material (BDDGS, WDDGS). BDDGSC treated with cellulase prior to cooking was compared with the untreated material. Previously assayed apparent metabolizable energy values were 12.1, 10.9 and 10.5 MJ/kg DM for BDDGS, BDDGSC and WDDGS, respectively (Kiiski- nen 1987). In the second trial barley protein and fibre fractions from the integrated etha- nol-starch process (Alko Ltd., Rajamäki) were used. The chemical composition of the feed ingredients is shown in Table 1. Kiiskinen (1988) found the assayed nutrient digestibili- ties of barley protein (BP) and fibre (BF) to be 0.874, 0.539 for crude protein, 0.849, 0.549 for ether extract and 0.833, 0.480 for carbo- hydrates, respectively. AMEn values were 14.9 and 9.1 MJ/kg DM for BP and BF, respec- tively, which were employed in formulation of the feed mixtures. In the both trials there were six diets: the control diet and five diets formulated with the intentionof achieving a similar concentrations of energy, crude protein, amino acids and minerals to meet the requirements (Salo et Table 1. Chemical composition by-products from distillery and starch production. Composition Barley Wheat Barley e/ke DM* K DDGS DDGSC DDGS protein fibre Dry matter 900 942 928 939 958 Ash 55 77 38 42 38 Crude protein 344 332 415 355 159 Ether extract 84 94 63 56 72 Crude fibre 78 71 99 14 114 NFE 439 427 385 533 617 NDF 380 333 409 5 507 ADF 220 214 182 136 ADL 96 113 87 24 Lysine 9.4 5.7 6.4 12.0 4.9 Cystine 5.4 5.8 7.3 8.3 3.1 Methionine 3.3 2.2 3.0 6.6 2.3 Threonine 13.3 12.5 12.9 12.8 4.7 Arginine 14.4 12.0 14.2 15.1 7.7 424 425 Table 2. Composition experimental diets containing various distillery by-products fed to layers in experiment 1. Diet no 12 3 4 5 6 DDGS supplement CONT BDDGS BDDGS BDDGSC BDDGSC WDDGS Level in diet, g/kg 100 200 100 200 200 Ingredients Barley 361 309 266 317 284 292 Oats 170 170 170 170 170 170 Wheat 120 120 120 120 120 120 Soybean meal 141 98 53 94 45 19 Fish meal 25 25 25 25 25 25 Meat meal 25 25 25 25 25 25 Barley DDGS 100 200 Barley DDGS, cellul. 100 200 Wheat DDGS 200 Grass meal 20 20 20 20 20 20 Fat mixture 40 36 30 35 27 31 Calcium carbonate 82 82 78 79 69 82 Hostaphos 6 5 4 5 4 4 Trace element mix 5 5 5 5 5 5 Vitamin mix 5 5 5 5 5 5 Methionine 0.5 0.6 0.7 0.7 0.9 0.9 L-lysine 1.9 Calculated composition, g/kg Crude protein 170 176 182 176 183 187 Digestible protein 145 145 145 145 145 145 ME MJ/kg DM 11.2 11.2 11.2 11.2 11.2 11.2 Lysine 9.1 8.6 8.0 8.2 8.0 8.0 S-amino acids 6.0 6.1 6.2 6.1 6.3 6.4 Calcium 35 35 33 34 33 35 Phosphorus 6.3 6.4 6.5 6.4 6.5 6.4 Analysed composition, g/kg DM Dry matter 901 894 896 898 904 898 Ash 136 125 123 126 124 127 Crude protein 190 196 206 193 200 206 Ether extract 68 72 72 71 69 68 Crude fibre 65 67 70 69 72 69 NFE 541 540 529 541 535 530 Amino acids Arginine 13.5 12.8 11.5 11.8 10.6 10.1 Cystine 3.7 3.9 3.7 3.7 4.2 4.0 Histidine 4.7 4.6 4.1 4.6 4.2 4.1 Isoleucine 7.4 7.9 7.6 7.2 7.1 7.1 Leucine 14.4 15.9 15.5 14.5 14.4 14.8 Lysine 10.5 10.1 8.8 8.7 7.9 7.2 Methionine 4.0 4.4 4.4 4.1 4.1 4.2 Phenylalanine 9.3 9.8 9.4 8.8 9.4 9.4 Serine 9.1 9.6 9.3 9.4 9.5 10.0 Threonine 7.5 7.7 7.5 7.5 7.5 7.3 Tyrosine 6.2 5.9 6.5 5.7 5.4 5.5 Valine 9.0 9.9 9.9 9.1 9.4 9.6 al. 1982). The control diets were normal com- mercial layer mixtures having 170 g crude pro- tein and 11.2 MJ ME/kg DM. In the first ex- periment 100 and 200 g/kg diet BDDGS un- treated or treated with cellulase enzyme and 200 g WDDGS replaced soybean meal and barley to give mixtures with similar nutrient contents. Coefficients to correct for rather low protein digestibility of distillery feeds found in pigs (Näsi 1985) were used in the formu- 426 Table 3. Composition experimental diets containing barley feed fractions from integrated starch-ethanol process fed to layers in experiment 2. Diet no 12 3 4 5 6 Barley fraction CONT Barley protein Barley fibre Enzym. Level in diet, g/kg 50 100 100 200 200 Ingredients Barley 437 427 420 286 136 136 Oats 150 150 150 150 150 150 Dehulled oats 100 100 100 150 200 200 Soybean meal 121 87 51 107 94 94 Fish meal 25 25 25 25 25 25 Meat and bone meal 20 20 20 20 20 20 Barley protein 50 100 Barley fibre 100 200 200 Grass meal 40 40 40 40 40 40 Fat mixture 27 20 13 40 53 53 Calcium carbonate 36 38 38 37 37 37 Ouster shell 30 30 30 30 30 30 Dicalcium phosphate 5 4 3 5 6 6 Sodium chloride 111111 Trace element mix 6 6 6 6 6 6 Vitamin mix 1.7 1.7 1.7 1.7 1.7 1.7 Methionine 0.6 0.5 0.6 0.5 0.6 L-lysine 0.4 Calculated composition, g/kg Dry matter 889 892 894 900 810 910 Crude protein 160 160 161 162 164 164 ME MJ/kg DM 10.8 10.8 10.9 10.9 11.1 11.1 Lysine 9.0 7.6 7.6 7.9 7.8 7.8 S-amino acids 6.5 6.6 6.8 6.7 7.0 7.0 Calcium 32 33 33 33 33 33 Phosphorus 6.3 6.3 6.4 6.3 6.3 6.3 Analysed composition, g/kg DM Dry matter 901 899 900 908 913 916 Ash 114 114 109 116 118 112 Crude protein 186 183 183 185 185 178 Ether extract 71 66 61 89 109 108 Crude fibre 61 59 57 72 71 68 NFE 568 578 590 538 517 534 NDF 165 180 159 218 217 209 ADF 53 57 54 74 72 69 Amino acids Arginine 10.6 10.3 9.7 10.8 11.1 10.2 Cystine 3.5 3.7 3.9 3.7 3.8 3.7 Histidine 4.0 3.9 3.9 4.0 4.1 3.9 Isoleucine 6.9 6.5 6.6 6.6 6.8 6.5 Leucine 13.0 12.5 12.6 12.7 12.8 13.4 Lysine 8.9 8.0 8.0 8.7 8.8 8.2 Methionine 5.3 3.8 4.0 3.9 4.2 3.5 Phenylalanine 8.2 8.3 8.2 8.0 8.1 8.1 Serine 8.3 7.9 7.7 8.1 8.1 7.9 Threonine 6.7 6.3 6.4 6.5 6.7 6.4 Tyrosine 6.0 6.0 5.8 5.9 6.1 5.8 Valine 8.5 8.3 8.4 8.4 8.6 8.4 lation of the diets (0.69, 0.56 and 0.73 for pro- tein in BDDGS, BDDGSC and WDDGS, respectively). In the second experiment, two diets replaced either one-third and two-thirds of soybean protein with barley protein (50 and 100 g/kg). In two other diets barley fibre was substituted for barley at the level of 100 or 200 g/kg. Ad- dition of multienzyme preparate (Avizyme, Cultor, Ltd. Helsinki) was compared in diet containing 200 g/kg BF. Feed fat was used to equalize the ME-content and pure lysine and methionine in fortifying the amino acid com- position in mixtures. The feed mixtures were prepared by Suomen Rehu Oy and they were in granular form. The composition of the feeds and their nutrient contents are given in the Tables 2 and 3. Animals and management The separate performance trials were car- ried out with 551 and 537 Finnish hybrid LSK 61 laying hens in each experiment. The hens were housed in stair model cages with three birds in each. The hens were fed pretreatment diet for four weeks followed by distribution according to the pretrial laying rate in ex- perimental groups consisting of six replicates of 14—16 hens per treatment. The tests lasted six 28-day periods from 34 to 58 weeks of age of the hens. The diets were fed ad libitumand feed con- sumption was recorded for each subgroup for a 28-day period. Feed was added to feeders quantitatively as required, and weighed back at 4-wk intervals. Intake was calculated (g hen-1 d-1 ) on a 28-d basis. Egg production was recorded daily by weighing and counting the eggs. The feed ingredients and experimen- tal mixtures were analysed according to stan- dard methods. Egg shell weight, albumen height, Haugh units and yolk pigmentation were also deter- mined. Egg quality measurements in exp. 2 were made once in pretreatment periods and three times in comparison periods on two con- secutive days consisting assays of 144 and 432 eggs, respectively. Eggs were weighed in- dividually after collection. The eggs were then broken out onto a glass sheet and the albu- men height was measured by a micrometer in order to calculate Haugh units. Yolk colour was scored by comparing the yolks using Roche scale. The dryed egg shells were weighed following day. The presence of blood and meat spots in the eggs was also recorded. Analysis of variance was performed on the 28-day period data as a split-plot design, with diet as the treatment and period as the split plot, using the statistical package of WSYS (Vilva 1988). Results and discussion Experiment 1 The chemical composition of the mixtures were close to the values calculated. Lysine content of mixture with WDDG was lower than expected although pure lysine was added and analysed values of amino acids were used in formulation. The explanation for this er- ror remains uncertain. Crude protein varied between mixtures because different coeffi- cients for crude protein digestibility were ap- plied according to previous trials made in pigs (Näsi 1985) to equalize the available protein supply. The crude fibre content between mix- tures was similar although distillery feeds con- tained NDF 333—409 g/kg. Acid detergent lignin contents were high in distillery feeds (87 —113 g/kg), but the anal- ysis may reflect a high concentration Maillard products. The production results in experiment 1. are presented in Table 4. for distillery feed com- parisons. There were no statistically signifi- cant differences in performance of hens be- tween treatments. Mean production of hens fed BDDGS was numerically higher than the control and other distillery feed groups, but the difference was non significant (P>0.05). Hens on diets supplemented with cellulase treated BDDGS or WDDGS tended to have slightly lower egg production and feed con- version probably indicating deficiencies in amino acids and protein supply. The high egg production level, on average 82.3 % in hens fed diets with distillery feeds, was equal to control. It is notable that up to two thirds soy- 427 Table 4. Performance of layers fed diets containing various distillery by-products, experiment 1. Diet no 12 3 4 5 6 SEM DDGS supplement CONT BDDGS BDDGS BDDGSC BDDGSC WDDGS Level in diet, g/kg 100 200 100 200 200 Number of hens Beginning of expt. 91 93 93 92 91 91 End of expt. 89 89 90 92 88 88 Mortality, % 2.2 4.3 3.2 0 3.3 3.3 Egg production Laying rate, % Standard, period 92.0 92.1 92.0 92.0 91.9 92.0 Transition period 93.4 93.1 93.3 91.0 92.5 91.0 Test period (168 d) 82.7 84.2 83.0 80.5 82.6 80.5 0.56 Eggs, g/hen/d 49.2 50.5 49.7 48.4 49.4 48.1 0.35 Mean egg weight, g 59.6 59.8 60.0 60.3 60.0 60.0 0.13 Feed intake Feed, g/hen/d Standard, period 107.6 105.5 109.6 108.1 106.9 107.2 Test period 116.5 119.3 120.3 118.6 119.6 118.6 0.65 DM, g/hen/d 105.0 106.6 107.7 106.4 108.1 106.5 0.58 Protein, g/hen/d 20.0d 20.9b <: 22.2" 20.5 cd 21.6ab 21.9 s 0.12 ME MJ/hen/d 1.22 1.24 1.25 1.23 1.25 1.23 0.007 Feed conversion Feed, kg/kg eggs 2.38 2.38 2.43 2.46 2.43 2.48 0.021 Feed DM, kg/kg eggs 2.14 2.13 2.17 2.21 2.19 2.23 0.019 Feed CP, g/kg eggs 408 d 418cd 449 ab 425 bcd 438aIK 458" 3.7 Feed ME MJ/kg eggs 24.9 24.7 25.2 25.6 25.3 25.8 0.022 Means with different letters were significantly different: a ~d (PcO.Ol) bean meal protein could be replaced with dis- tillery feeds and only methionine supplemen- tation was necessary to maintain protein qual- ity. The layers consumed diets including DDGS a littlebit more than that of control mixture. Feed conversion efficiency was not reduced by distillers feeds. The energy values measured in a previous digestibility trial (Kiiskinen 1987) corre- sponded well with the present production results. ME-values for DDGS from dehulled barley and that of wheat without bran have been reported as 11.5 and 12.2 MJ/kg DM, respectively (Askbrant and Thomke 1986). These are higher than values for present dis- tillery feeds, but Pettersson et ai. (1987) found a much higher value of 13.7 for de- hulled barley distillers spent grains. The raw material and the process differences cause variability in energy value. The protein digestibilities of the batches of DDGS in this study were 0.662, 0.622 and 0.713 for barley, cellulase treated barley and wheat distillery feeds (Kiiskinen 1987), the values being quite close those assayed with pigs (Näsi 1985), which were applied in the feed formulation. The reported protein digest- ibility of distillers feeds has varied considera- ble due to differences in cooking, distillation and dehydration processes. Higher digestibil- ities than the present study were reported by Askbrant and Thomke (1986) and Petters- son et ai. (1987), the values being 0.7—0.8. Protein digestibility has a great effect on pro- tein utilization of DDGS. Low digestibility value indicate also the deterioration of ami- no acids and their availability. Present performance results from experi- ments with laying hens are in agreement with the other reports which have shown that rela- tively high levels of DDGS can be incorpo- rated into properly balanced diets. Jensen (1985) reported that laying hens could be fed up to 200 g/kg DDGS in isocaloric diets with 428 no adverse effect on egg production or other performance characteristics. Matterson et al. (1966) showed that the diets containing 100—200 g/kg DDGS supplied adequate es- sential amino acids to layers and that sup- plemental pure lysine did not produce an ad- ditional response. However, lysine is a criti- cal amino acid when using DDGS, and when 100 g/kg DDGS has been added to wheat- based diets, lysine supplementation has been necessary to obtain optimum performance (Jensen 1985). In the present study only methionine was added in barley DDGS diets and only lysine in the wheat DDGS diet which had lower lysine content in oder to obtain con- tents of the layers requirements. Bossard et al. (1981) compared the use of 0, 100, 200 and 300 g/kg DDGS in cornsoybean meal diets for laying hens. The diets were isonitrogenous and isocaloric and were balanced for lysine. No difference in rate of egg production was ob- served, but birds fed the higher levels had lower egg weight, suggesting that some of other amino acids may have been out of bal- ance. In the present study hens fed diets with DDGS tended to lay heavier eggs than the con- trol group. Diets containing DDGS or other fermenta- tion by-products have been reported to signifi- cantly improve interior egg quality (Jensen et al. 1978, Sauveur 1981). Identification of the activity in DDGS affecting interior egg quali- ty has not yet been achieved. The possibility that the trace mineral content of DDGS might be involved was suggested by Jensen (1985). Inclusion of DDGS has resulted in a signifi- cant reduction in liver lipid accumulation in caged laying hens compared to birds fed sim- ple maize-soybean meal diet (Jensen 1985). Also DDGS supplements have been shown to affect calcium metabolism of hens and im- provements in egg breaking-strength have been observed (Jensen 1985). Mortality of the hens was low during this 6-month experi- ment. Cannibalism was rare which is usual in this poulty house. Table 5. Performance of layers fed with diets containing barley feed fractions from integrated starch-ethanol proc- ess, experiment 2. Diet no 12 3 4 5 6 SEM Barley fraction CONT Barley protein Barley fibre Enzym. Level in diet, g/kg 50 100 100 200 200 Number of hens Beginning of expt. 88 88 87 90 90 88 End of expt. 86 82 82 88 87 85 Mortality, % 2.3 6.8 5.7 2.2 3.3 3.4 Egg production Laying rate, % Standard, period 93.7 93.7 93.7 93.7 93.7 93.7 Transition period 92.1 91.3 91.9 92.9 93.1 92.7 Test period (168 d) 84.5 83.3 85.2 84.3 85.2 84.6 0.98 Eggs, g/hen/d 50.5 49.2 49.8 49.8 50.3 50.2 0.60 Mean egg weight, g 59.8 59.1 58.6 59.2 59.6 59.3 0.30 Feed intake Feed, g/hen/d Standard, period 118.0 115.2 115.6 117.8 117.9 116.8 Test period 119.6 119.7 116.8 116.2 118.5 118.3 2.38 DM, g/hen/d 107.7 107.6 105.1 105.5 108.1 108.4 2.15 Protein, g/hen/d 20.0 19.7 19.2 19.5 20.0 19.3 0.39 ME MJ/hen/d 1.29 1.30 1.27 1.27 1.31 1.31 0.02 Feed conversion Feed, kg/kg eggs 2.37 2.44 2.35 2.33 2.36 2.36 0.05 Feed DM, kg/kg eggs 2.14 2.19 2.11 2.12 2.15 2.16 0.05 Feed CP, g/kg eggs 397 402 386 392 399 385 8.50 Feed ME MJ/kg eggs 25.6 26.4 25.5 25.5 26.1 26.1 0.56 429 Experiment 2 Performance results from the experiment 2 are shown in Table 5. Laying rate changed be- tween groups from 83 to 85 % and feed con- version from 2.33 to 2.44 kg/kg eggs, al- though there were no statistically significant dietary effects. Barley protein had high pro- tein digestibility (0.87) and an AME-value of 14.9 MJ/kg DM (Kiiskinen 1988). It appears from these production data that barley pro- tein fortified with lysine and methionine could be a substitute for a major proportion of the protein of SBM without an adverse effect on performance. This is in accordance with the observation of Kiiskinen (1988) who sug- gested that BP is suited to broiler finisher diets fortified with lysine and methionine and can replace SBM up to a level of s*—lo °7o. Simi- larly barley protein showed high digestibility and if fortified with pure lysine gave a nitro- gen balance in growing pigs similar to that of isonitrogenous soybean-barley diet. Up to two-thirds of soybean protein could be replaced with barley protein without a differ- ence in the performance of growing pigs (Näsi 1989). Barley fibre had rather low protein digesti- bility (0.54) and an AME-value of 9.1 MJ/kg DM (Kiiskinen 1987). Those assayed values were in accordance with the production data; BF could replace barley in isocaloric layer diets without adverse effects. NDF contents of these BF-diets were over 200 g/kgand when dietary fibre increases nutrient availability decreases (Janssen and Carre 1985, Krog- dahl 1986), but the characteristics of the fibre are of great importance. A feedstuff similar to barley fibre is corn gluten feed (CGF) a coproduct of the wet milling indus- try of maize starch and sugar (Anon. 1982). Sibbald (1986) reported that TME values for 12 samples of CGF ranged from 8.01 to 11.6 MJ/kg DM and digestibility of lysine and methionine from 0.66 to 0.81 and from 0.79 to 0.89, respectively. Lysine and tryptophan were equally limiting in CGF and the bioavail- ability of lysine by chick assay varied consider- Table 6. Quality parameters of eggs layed by hens fed diets containing barley feed fractions from integrated starch-ethanol process, experiment 2. Statistical significance of effect Diet no 12 3 4 5 6 Protein supplement Fibre supplement Enzyme Barley fraction CONT Barley protein Barley fibre Enzym. supplement Level in diet, g/kg 50 100 100 200 200 SEM Lin Quad SEM Lin Quad. SEM Sign. Egg weight, g 60.4 59.6 58.7 60.0 59.6 60.1 0.44 ** NS 0.42 NS NS 0.43 NS Shell weight, g 5.50 5.48 5.49 5.61 5.60 5.56 0.05 NS NS 0.05 NS NS 0.05 NS Shell- % 9.11 9.20 9.35 9.36 9.41 9.26 0.07 ** NS 0.07 ** NS 0.07 NS Albumen height, mm 6.8 6.7 7.0 7.0 6.8 7.0 0.12 NS NS 0.12 NS NS 0.12 NS Haugh number 81.6 81.1 83.4 83.2 81.8 83.2 0.81 NS NS 0.79 NS NS 0.80 NS Yolk colour 9.0 8.7 8.6 8.9 8.9 8.5 0.09 ** NS 0.08 NS NS 0.08 ** * P<0.05, * P