Effect of Diludine on Laying Performance and Approach to Mechanism of the Effects in Hens
Abstract The experiment was conducted to study the effects of diludine on laying performance and egg quality in hens and approach to the mechanism of the effects by determining indexes of egg and serum parameters 1024 ROM hens were divided into four groups each of which included four replicates of 64 hens each, The treatment groups received the same basal diet supplemented with 0, 100, 150, 200 mg/kg diludine respectively for 80 d. The results were as follows. Addition of diludine to diet improved hens laying performance, of which 150 m g/kg treatment was best; its rate of lay was increased by 11.8% (p< 0.01), egg mass conversion was decreased by 10.36% (p< 0 01). Egg weights were increased with the increasing of diludine added. Diludine significantly decreased the serum concentration of uric acid (p< 0.01); adding diludine significantly decreased the serum Ca2+ and inorganic phosphate content, and increased activity of alkine phosphatase (ALP) of serum (p< 0.05), so it had significant effects on reducing egg breakage (p<0.05) and abnormality (p < 0.05); diludine significantly increased the albumen height. Haugh value (p <0.01), shell thickness and shell weight (p< 0.05), 150 and 200mg/kg diludine also decreased total choleasterol in egg yolk (p< 0 05), but increased egg yolk weight (p < 0.05). In addition, diludine could enhance the activity of lipase (p < 0.01), and decrease the content of triglyceride (TG3) (p<0.01) and cholesterol (CHL) (p< 0 01) in serum, it reduced percentage of abdominal fat (p< 0.01) and liver fat content (p< 0.01), had ability to prevent hens from fatty liver. Diludine significantly increased activity of SOD in serum (p< 0 01) when it was added to diet for more than 30d. Howeve, no significant difference was found in activities of GPT and GOT of serum between the control and treated group. It was inferred that diludine could prevent membrane of cells from oxidation
Key words Diludine; hen; SOD; cholesterol; triglyceride, lipase
The diludine is the novel non-nutritive anti-oxidation vitamin additive and has the effects [1-3] of restraining oxidation of the biological membrane and stabilizing the tissue of the biological cells, etc. In 1970s, the agricultural expert of Latvia in former Soviet Union found that diludine had the effects [4] of promoting growth of poultry and resisting freezing and ageing for some plants. It was reported that the diludine not only could promote growth of animal, but improve reproductive performance of animal obviously and improve pregnancy rate, output of milk, output of egg and hatching rate of the female animal [1, 2, 5-7]. The study of diludine in china was started from 1980s, and the majority of the studies about diludine in China are confined to using effect so far, and the few trials on laying fowl were reported. Chen Jufang (1993) reported that the diludine could improve the output of egg and the weight of the egg of the poultry, but did not deepen [5] the study of the mechanism of action thereof. Therefore, we implemented the systematic study of the effect and mechanism thereof by feeding the laying hens with the diet doped with the diludine, and one part of the result now is reported as follows:
Table 1 Composition and nutrient components of experiment diet
%
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Composition of diet Nutrient components
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Corn 62 ME③ 11.97
Bean pulp 20 CP 17.8
Fish meal 3 Ca 3.42
Rapeseed meal 5 P 0.75
Bone meal 2 M et 0.43
Stone meal 7.5 M et Cys 0.75
Methionine 0.1
Salt 0.3
Multivitamin① 10
Trace elements② 0.1
——————————————————————————————
① Multivitamin: 11mg of riboflavin, 26mg of folic acid, 44mg of oryzanin, 66mg of niacin, 0.22mg of biotin, 66mg of B6, 17.6ug of B12, 880mg of choline, 30mg of VK, 66IU of VE, 6600ICU of VD and 20000ICU of VA, are added to each kilogram of the diet; and 10g multivitamin is added to each 50kg of diet.
② Trace elements (mg/kg): 60 mg of Mn, 60mg of Zn, 80mg of Fe, 10mg of Cu, 0.35mg of I and 0.3mg of Se are added to each kilogram of the diet.
③ The unit of the metabolizable energy refers to M J/kg.
1. Materials and method
1.1 Test material
Beijing Sunpu Biochem. & Tech. Co., Ltd. should offer the diludine; and the test animal shall refer to the Roman commercial laying hens which are 300 days old.
Experiment diet: the test experiment diet should be prepared according to the actual condition during production on the basis of NRC standard, as shown in Table 1.
1.2 Test method
1.2.1 Feeding experiment: the feeding experiment should be implemented in the farm of Hongji Company in Jiande City; 1024 Roman laying hens should be selected and divided into four groups randomly and each for 256 pieces (each group should be repeated for four times, and each hen should be repeated for 64 times); the hens should be fed with the four diets with different contents of diludine, and 0, 100, 150, 200mg/kg of the feeds should be added for each group. The test was started on April 10, 1997; and the hens could find food and take water freely. The food taken by each group, the laying rate, the output of egg, the broken egg and the number of abnormal egg should be recorded. Moreover, the test was ended on June 30, 1997.
1.2.2 Measurement of egg quality: 20 eggs should be taken randomly when the test was implemented four 40 days in order to measure the indicators related to the egg quality, such as egg shape index, haugh unit, relative weight of the shell, the shell thickness, the yolk index, the relative weight of yolk, etc. Moreover, the content of cholesterol in the yolk should be measured by using the COD-PAP method in presence of Cicheng reagent produced by Ningbo Cixi Biochemical Test Plant.
1.2.3 Measurement of serum biochemical index: 16 test hens should be taken from each group each time when the test was implemented for 30 days and when the test is ended to prepare the serum after sampling the blood from the vein on the wing. The serum should be stored at the low temperature (-20℃) in order to measure relevant biochemical indexes. The abdominal fat percentage and the liver lipid content should be measured after slaughtering and taking out the abdominal fat and the liver upon completion of blood sampling.
The superoxide dismutase (SOD) should be measured by using saturation method in presence of the reagent kit produced by the Beijing Huaqing Biochem. & Tech. Research Institute. The uric acid (UN) in the serum should be measured by using the U ricase-PAP method in presence of the Cicheng reagent kit; the triglyceride (TG3) should be measured by using GPO-PAP one-step method in presence of the Cicheng reagent kit; the lipase should be measured by using the nephelometry in presence of the Cicheng reagent kit; the serum total cholesterol (CHL) should be measured by using the COD-PAP method in presence of the Cicheng reagent kit; the glutamic-pyruvic transaminase (GPT) should be measured by using the colorimetry in presence of the Cicheng reagent kit; the glutamic-oxalacetic transaminase (GOT) should be measured by using the colorimetry in presence of the Cicheng reagent kit; the alkaline phosphatase (ALP) should be measured by using the rate method in presence of the Cicheng reagent kit; the calcium ion (Ca2+) in serum should be measured by using methylthymol blue complexone method in presence of the Cicheng reagent kit; the inorganic phosphorus (P) should be measured by using the molybdate blue method in presence of the Cicheng reagent kit.
2 Test result
2.1 Effect to laying performance
The laying performances of different groups processed by using the diludine are shown in Table 2.
Table 2 Performance of hens fed with basal diet supplemented with four levels of diludine
| Amount of diludine to be added (mg/kg) | ||||
| 0 | 100 | 150 | 200 | |
| Feed intake (g) |  |
|||
| Laying rate (%) | ||||
| Average weight of egg (g) | ||||
| Ratio of material to egg | ||||
| Broken egg rate (%) | ||||
| Rate of abnormal egg (%) | ||||
From the Table 2, the laying rates of all groups processed by using diludine are improved obviously, wherein the effect when processed by using 150mg/kg is optimal (up to 83.36%), and the 11.03% (p<0.01) is improved compared with the reference group; therefore the diludine has the effect of improving the laying rate. Seen from the average weight of egg, the weight of the egg is increasing (p>0.05) along increasing diludine in the daily diet. Compared with the reference group, the difference among all processed parts of the groups processed by using 200mg/kg of diludine is not obvious when the 1.79g of feed intake is added averagely; however, the difference becomes more obvious gradually along with the increasing diludine, and the difference of the ratio of the material to egg among the parts processed is obvious (p<0.05), and the effect is optimal when 150mg/kg of diludine and is 1.25:1 which is reduced for 10.36% (p<0.01) compared with the reference group. Seen from the broken egg rate of all parts processed, the broken egg rate (p<0.05) can be reduced when the diludine is added to the daily diet; and the percentage of abnormal eggs is reduced (p<0.05) along increasing diludine.
2.2 Effect to egg quality
Seen from the Table 3, the egg shape index and the egg specific gravity are not affected (p>0.05) when the diludine is added to the daily diet, and the weight of the shell is increased along increasing diludine added to the daily diet, wherein the weights of the shells are increased for 10.58% and 10.85% (p<0.05) respectively compared with the reference groups when 150 and 200mg/kg of diludine are added; the thickness of the egg shell is increased along increasing diludine in the daily diet, wherein the thickness of the egg shell is increased for 13.89% (p<0.05) when 100mg/kg of diludine is added compared with the reference groups, and the thicknesses of the egg shells are increased for 19.44% (p<0.01) and 27.7% (p<0.01) respectively when 150 and 200mg/kg are added. The Haugh unit (p<0.01) is improved obviously when the diludine is added, which indicates that the diludine has the effect of promoting synthesis of thick albumen of egg white. The diludine has the function of improving the index of yolk, but the difference is not obviously (p<0.05). The contents of cholesterol of egg yolk of all groups are difference and can be obviously reduced (p<0.05) after adding 150 and 200mg/kg of diludine. The relative weights of the egg yolk are different from each other due to different amounts of diludine added, wherein the relative weights of the egg yolk are improved for 18.01% and 14.92% (p<0.05) when 150mg/kg and 200mg/kg compared with the reference group; therefore, the appropriate diludine has the effect of promoting synthesis of egg yolk.
Table 3 Effects of diludine on egg quality
| Amount of diludine to be added (mg/kg) | ||||
| Egg quality | 0 | 100 | 150 | 200 |
| Egg shape index (%) |  |
|||
| Egg specific gravity (g/cm3) | ||||
| Relative weight of egg shell (%) | ||||
| Thickness of egg shell (mm) | ||||
| Haugh unit (U) | ||||
| Egg yolk index (%) | ||||
| Cholesterol of egg yolk (%) | ||||
| Relative weight of egg yolk (%) | ||||
2.3 Effects to abdominal fat percentage and content of liver fat of the laying hens
See Figure 1 and Figure 2 for effects of diludine to abdominal fat percentage and content of liver fat of the laying hens
Figure 1 Effect of diludine on percentage of abodominal fat (PAF) of laying hens
| Percentage of abodominal fat | |
| Amount of diludine to be added |
Figure 2 Effect of diludine on liver fat content (LF) of laying hens
| Liver fat content | |
| Amount of diludine to be added |
Seen from Figure 1, the percentages of abodominal fat of the test group are reduced for 8.3% and 12.11% (p<0.05) respectively when 100 and 150mg/kg of diludine compared with the reference group, and the percentage of abodominal fat is reduced for 33.49% (p<0.01) when 200mg/kg of diludine is added. Seen from the Figure 2, the liver fat contents (absolutely dry) processed by 100, 150, 200mg/kg of diludine respectively are reduced for 15.00% (p<0.05), 15.62% (p<0.05) and 27.7% (p<0.01) respectively compared with the reference group; therefore, the diludine has the effect of reducing percentage of abodominal fat and the liver fat content of the laying content obviously, wherein the effect is optimal when 200mg/kg of diludine is added.
2.4 Effect to serum biochemical index
Seen from the Table 4, the difference among the parts processed during Phase I (30d) of SOD test is not obvious, and the serum biochemical indexes of all groups to which the diludine is added in Phase II (80d) of the test are higher than the reference group (p<0.05). The uric acid (p<0.05) in the serum can be reduced when 150mg/kg and 200mg/kg of diludine are added; while the effect (p<0.05) is available when 100mg/kg of diludine is added in Phase I. The diludine can reduce the triglyceride in the serum, wherein the effect is optimal (p<0.01) in the group when 150mg/kg of diludine is added in Phase I, and is optimal in the group when 200mg/kg of diludine is added in Phase II. The total cholesterol in the serum is reduced along increasing diludine added to the daily diet, more specifically the contents of total cholesterol in the serum are reduced for 36.36% (p<0.01) and 40.74% (p<0.01) respectively when 150mg/kg and 200mg/kg of diludine are added in Phase I compared with the reference group, and reduced for 26.60% (p<0.01), 37.40% (p<0.01) and 46.66% (p<0.01) respectively when 100mg/kg, 150mg/kg and 200mg/kg of diludine are added in Phase II compared with the reference group. Moreover, the ALP is increased along increasing diludine added to the daily diet, while the values of ALP in the group to which 150mg/kg and 200mg/kg of diludine are added are higher than the reference group (p<0.05) obviously.
Table 4 Effects of diludine on serum parameters
| Amount of diludine to be added (mg/kg) in Phase I (30d) of test | ||||
| Item | 0 | 100 | 150 | 200 |
| Superoxide dismutase (mg/mL) |  |
|||
| Uric acid | ||||
| Triglyceride (mmol/L) | ||||
| Lipase (U/L) | ||||
| Cholesterol (mg/dL) | ||||
| Glutamic-pyruvic transaminase (U/L) | ||||
| Glutamic-oxalacetic transaminase (U/L) | ||||
| Alkaline phosphatase (mmol/L) | ||||
| Calcium ion (mmol/L) | ||||
| Inorganic phosphorus (mg/dL) | ||||
| Amount of diludine to be added (mg/kg) in Phase II (80d) of test | ||||
| Item | 0 | 100 | 150 | 200 |
| Superoxide dismutase (mg/mL) |  |
|||
| Uric acid | ||||
| Triglyceride (mmol/L) | ||||
| Lipase (U/L) | ||||
| Cholesterol (mg/dL) | ||||
| Glutamic-pyruvic transaminase (U/L) | ||||
| Glutamic-oxalacetic transaminase (U/L) | ||||
| Alkaline phosphatase (mmol/L) | ||||
| Calcium ion (mmol/L) | ||||
| Inorganic phosphorus (mg/dL) | ||||
3 Analysis and discussion
3.1 The diludine in the test improved the laying rate, the weight of egg, the Haugh unit and the relative weight of the egg yolk, which indicated that the diludine had the effects of promoting assimilation of the protein and improving the amount of synthesis of thick albumen of egg white and protein of egg yolk. Further, the content of uric acid in the serum was reduced obviously; and it was generally acknowledged that the reduction of content of non-protein nitrogen in the serum meant that the catabolism speed of the protein was reduced, and the retention time of nitrogen was postponed. This result provided the basis of increasing protein retention, promoting laying of eggs and improving the weight of the egg of the laying hens. The result of the test pointed out that the laying effect is optimal when 150mg/kg of diludine was added, which was essentially consistent with the result [6,7] of Bao Erqing and Qin Shangzhi and acquired by adding diludine at the late period of laying hens . The effect was reduced when the amount of diludine exceeding 150mg/kg, which may be because the protein transformation [8] was affected due to excessive dosage and the excessive load of metabolism of the organ to the diludine.
3.2 The concentration of Ca2+ in the serum of the laying egg was reduced, the P in the serum was reduced in the beginning and the ALP activity was increased obviously in presence of diludine, which indicated that diludine affected metabolism of Ca and P obviously. Yue Wenbin reported that the diludine could promote absorption [9] of mineral elements Fe and Zn; ALP mainly existed in tissues, such as liver, the bone, the intestinal tract, the kidney, etc.; ALP in the serum was from the liver and bone mainly; ALP in the bone existed in the osteoblast mainly and could combine the phosphate ion with the Ca2 from the serum after transformation by promoting decomposition of phosphate and increasing the concentration of the phosphate ion, and was deposited on the bone in the form of hydroxyapatite, etc. in order to lead to reduction of Ca and P in the serum, which is consistent with increasing of egg shell thickness and relative weight of egg shell in the egg quality indicators. Moreover, the broken egg rate and the percentage of the abnormal egg were reduced obviously in terms of the laying performance, which also explained this point.
3.3 The abdominal fat deposition and the liver fat content of the laying hens were reduced obviously by adding diludine to the diet, which indicated that the diludine had the effect of restraining synthesis of fat in body. Further, the diludine could improve the activity of the lipase in the serum in the early stage; the activity of lipase was increased obviously in the group to which 100mg/kg of diludine was added, and the contents of the triglyceride and the cholesterol in the serum were reduced (p<0.01), which indicated that the diludine could promote decomposition of triglyceride and restrain synthesis of cholesterol. The fat deposition could be restrained because the enzyme of lipid metabolism in the liver [10,11], and reduction of cholesterol in the egg yolk also explained this point [13]. Chen Jufang reported that the diludine could restrain formation of fat in the animal and improve the lean meat percentage of the broilers and pig, and had the effect of treating fatty liver. The result of the test clarified this mechanism of action, and the dissection and observation results of the test hens also proved that the diludine could reduce occurrence rate of fatty liver of the laying hens obviously.
3.4 GPT and GOT are two important indicators reflecting the functions of the liver and the heart, and the liver and the heart may be damaged if the activities thereof are too high. The activities of GPT and GOT in the serum were not changed obviously when the diludine is added in the test, which indicated that the liver and the heart were not damaged; further, the measurement result of SOD shown that the activity of SOD in the serum could be improved obviously when the diludine was used for a certain time. SOD refers to the major scavenger of the superoxide free radical in body; it is of significance for maintaining integrity of the biological membrane, improving the organism immunity ability and maintaining health of the animal when the content of SOD in body is increased. Quh Hai, etc. reported that diludine could improve the activity of 6-glucose phosphate dehydrogenase in the biological membrane and stabilize the tissues [2] of the biological cell. Sniedze pointed out diludine restrained the activity [4] of NADPH cytochrome C reductase obviously after studying the relationship between the diludine and relevant enzyme in NADPH specific electron transfer chain in rat liver microsome. Odydents also pointed out diludine was related [4] to the composite oxidase system and the microsomal enzyme related to NADPH; and the mechanism of action of diludine after entering into animal is to play a role of resisting oxidation and protecting the biological membrane [8] by intercepting the activity of the electron transfer NADPH enzyme of the microsome and restraining the peroxidation process of the lipid compound. The test result proved that the protection function of diludine to the biological membrane from the changes of SOD activity to changes of activities of GPT and GOT and proved the study results of Sniedze and Odydents.
Reference
1 Zhou Kai, Zhou Mingjie, Qin Zhongzhi, etc. Study on diludine of improving reproductive performance of sheep J. Grass and Livestock 1994 (2): 16-17
2 Qu Hai, Lv Ye, Wang Baosheng, Effect of diludine added to daily diet to pregnancy rate and semen quality of meat rabbit. J. Chinese Journal of Rabbit Farming 1994(6): 6-7
3 Chen Jufang, Yin Yuejin, Liu Wanhan, etc. Test of expanded application of diludine as feed additive Feed Research 1993 (3): 2-4
4 Zheng Xiaozhong, Li Kelu, Yue Wenbin, etc. Discussion of application effect and mechanism of action of diludine as poultry growth promoter Feed Research 1995 (7): 12-13
5 Chen Jufang, Yin Yuejin, Liu Wanhan, etc. Test of expanded application of diludine as feed additive Feed Research 1993 (3): 2-5
6 Bao Erqing, Gao Baohua, Test of diludine for feeding breed Peking duck Feed Research 1992 (7): 7-8
7 Qin Shangzhi Test of improving productivity of breed meat hens in late period of laying by using diludine Guangxi Journal of Animal Husbandry & Veterinary Medicine 1993.9(2): 26-27
8 Dibner J Jl Lvey F J Hepatic protein and amino acid metabolian in poultry Poultry Sci 1990.69(7): 1188- 1194
9 Yue Wenbin, Zhang Jianhong, Zhao Peie, etc. Study of addition of diludine and Fe-Zn preparation to daily diet of laying hens Feed & Livestock 1997, 18(7): 29-30
10 Mildner A na M, Steven D Clarke Porcine fatty acid synthase cloning of a complementary DNA, tissue distribution of itsmRNA and suppression of expression by somatotropin and dietary protein J Nutri 1991, 121 900
11 W alzon R L Smon C, M orishita T, et a I Fatty liver hemorrhagic syndrome in hens overfed a purified diet Selected enzyme activities and liver histology in relation to liver honorrhage and resproductive performance Poultry Sci, 1993 72(8): 1479- 1491
12 Donaldson W E Lipid metabolism in liver of chicks response to feeding Poultry Sci. 1990, 69(7) : 1183- 1187
13 Ksiazk ieu icz J. K ontecka H, H ogcw sk i L A note on blood cholesterol as an indicator of body fatness in ducks Journal of Aninal and Feed Science, 1992, 1(3/4): 289- 294