CC BY
39veterinary sciences
Yaremchuk V. Y.
PhD student, Slivinska L. G.
DVM, Professor, Lukashchuk B.O.
PhD, assistant Stepan Gzhytskyi National University of Veterinary Medicine and Biotechnologies,
Lviv, Ukraine
DOI: 10.24412/2520-2480-2020-2880-4-9 LIPID METABOLISM PARAMETERS IN LAYING HENS WITH HEPATOSIS
The article presents the results of lipid metabolism assessment in laying hens with determination of new hepatospecific tests for the diagnosis offunctional and morphological changes in the liver. To achieve this goal, two groups of clinically healthy laying hens (control, n = 10) and two groups of laying hens with hepatosis (experimental, n = 10) of the cross "Lohmann Brown" aged 224 and 300 days were formed. The evaluation criteria were the results of biochemical analysis of the blood serum. The level of lipoproteins (cholesterol, HDL, LDL, VLDL, triglycerides) and bile acids were determined.
According to the results, the most informative diagnostic criterion to determine the nature and extent of the pathological process is a significant reduction in total cholesterol, the content of which in the experimental groups from the control groups in the two age groups with a high degree of reliability (p <0.001).
An increased concentration of triglycerides in the serum in laying hens was established aged 224 days (27.4%; p <0.001) and 300 days (21.3%; p <0.001) comparing to the group of clinically healthy, which indicates an increase in their synthesis and development of fatty liver disease.
In addition, increased of other lipid profile parameters in laying hens with hepatosis was established. At 224 days of age, the content of HDL, LDL and VLDL by 4.9 %, 60.5 % and 52 % compared to the control group. In laying hens aged 300 days, these parameters in the experimental group were also increased. Namely, HDL - by 8.3%, LDL - 13.9% and VLDL - 25.6%, respectively, compared to the control group of this age. Such changes occur when the increase in lipogenesis exceeds the ability to synthesize and secrete lipoproteins.
Increase serum concentration of bile acids in laying hens aged 224 days in 2.3 times and 300 days - 2.1 times was established. It indicates a violation of the secretion of bile acids by hepatocytes and a decrease in the functional capacity of the liver.
Basing on the results of research, changes in the lipid spectrum of the blood are an important additional diagnostic evaluation criterion of the liver functional state disorders in laying hens with hepatosis.
Key words: laying hens, hepatosis, liver, blood serum, cholesterol, triglycerides, lipoproteins.
Introduction
Lipids play an important role in liver functional state disorders. This is due to their participation in the metabolic processes of the body and the peculiarities of their metabolism in determining the functional state of the organism, individual cells and subcellular elements, as well as in assessing the quality of products obtained from poultry. Therefore, the study of patterns of changes in lipid metabolism and its regulation in poultry in industrial production is important due to determination of their influence on the growth, development, functional state of the body, absorption of feed nutrients, productivity and quality of products [9, 12].
In poultry, as in other animals, the most involved organ in metabolic processes is the liver [6]. The main metabolic pathways, such as glycolysis, glucogenesis, tricarboxylic acid cycle, glycogenesis, glycogenolysis, peptose phosphate cycle are the most active in the liver. Lipid metabolism is also extensive in the liver, including the export of accumulated and de novo synthesized cholesterol, triacylglycerol and phospholipids in the form of low-density lipoproteins, very low-density lip-oproteins and high-density lipoproteins. In a liver lengthening and shortening of carboxylic chains of
fatty acids, their dehydrogenation, p-oxidation, synthesis and breakdown of lipids take place [15].
Lipids play an important role in egg formation, as most yolk precursors are synthesized in the liver and transferred to the follicle as very low-density lipopro-teins [17]. Therefore, the study of changes in lipid metabolism in hens during intensive egg-laying is of scientific and practical interest.
Lipoprotein metabolism involves two cycles - endogenous and exogenous, the integrating organ of which is the liver. During the endogenous cycle, lipids synthesized in the liver are transported to peripheral tissues for disposal. During this process, VLDL, LDL and HDL are formed.
Lipid transport in blood is carried out in the form of lipoproteins among which allocate: chylomicrons (CM), p-lipoproteins of very low density (VLDL) or pre-P-LP, low-density lipoproteins (LDL) or P-LP, high-density lipoprotein (HDL) or a-LP, and fatty acids in combination with albumins. Each class of blood lip-oproteins performs specific functions. Very low-density lipoproteins are structures synthesized in the liver from triacylglycerols, phospholipids, and cholesterol and perform transport of the latter to extrahepatic tissues. They are synthesized mainly in the liver and are
the main transport form of endogenous lipids from the liver to peripheral tissues. Low and high density lipo-proteins exchange cholesterol and its ethers between the liver and peripheral tissues. The exploitation of LDL is mainly carried out in the liver, adrenal glands and adipose tissue. In the cell, cholesterol is incorporated in the membrane in those areas where it is needed, and excess intracellular cholesterol is esterified [18].
The analysis of bile acids is one of the new tests for diagnosing liver function in poultry. The advantage of this analysis is that it specifically indicates functional changes in the liver. Bile acids are formed in the liver from cholesterol and excreted in the intestine, where they are involved in the breakdown of fats. They determine the amount of bile outflow, as well as control the excretion of bile from cholesterol, bilirubin, phospho-lipids and a number of other substance from the bile [3].
An important condition for the optimal course of physiological processes in the whole organism is the interaction of bile acids with the molecules of lipids and proteins - the main components of cell membranes. This interaction affects the transport of various compounds across the membrane and the activity of enzymes, which leads to changes in the intensity of metabolism in cells [16].
Lipid metabolism in poultry is disturbed during many diseases of various etiologies and often general obesity and hepatosis are established in laying hens [4]. According to the literature, excessive accumulation of lipids in the liver develops "fatty liver syndrome" (Couch, 1956), which leads to the development of hepatosis. This pathology is characterized by a sudden decrease in productivity, the liver becomes loose, yellow, high in lipids and clinically manifested by severe general obesity of the abdominal cavity. It should be noted that hepatosis often has a subclinical course. Because in the early stages of the disease dystrophic processes are still reversible, so timely diagnosis of the pathology is important for effective treatment and prevention of poultry hepatosis [5, 10].
The purpose of our research
The aim of our research was to evaluate the lipid metabolism in laying hens and to establish new hepato-specific tests for the diagnosis of functional and morphological changes in the liver.
Material and Methods
Experimental studies were performed on laying hens of the "Lohmann Brown" breed aged 224 and 300 days, which were kept at the poultry farm LLC (LIMITED LIABILITY COMPANY) Agrofirm "Zagai" in Kamiyanka-Buzkyi district of Lviv region. Poultry keeping - cage, 5-tier and 3-tier batteries are equipped with feeders and drinkers, planting density of 10 and 5 heads in the cage. Laying hens at the poultry
farm were kept on the main ration (MR) provided by the technological map for the use of this breed of poultry. Two groups of laying hens aged 224 and two groups aged 300 days were formed. The first group included clinical healthy laying hens (control, n = 10), the second - hens with hepatosis (experimental, n = 10).
Blood for the study was obtained in vivo from the subclavian vein, in compliance with the rules of aseptic and antiseptic. Serum biochemical analysis was performed in 10 hens from each group. Laboratory tests were made on the basis of the laboratory of clinical and biological research at State Scientific-Research Control Institute of Veterinary Medicinal Products and Feed Additives.
Serum content of total cholesterol, triglycerides, high-density lipoproteins, low-density lipoproteins, and very low-density lipoproteins were determined. The study was performed using a semi-automatic biochemical analyzer «HumaLyzer 3000» using Human Diagnostics Worldwide (Germany) reagents.
Experimental study was performed in accordance to bioethical standards in relation to animals that meet the requirements of the Law of Ukraine № 3447-4 "On protection of animals from cruel treatment", the provisions of European Convention for the Protection of Vertebrate Animals, used for Experimental and Scientific Purposes (Strasbourg, 1986).
The results of biochemical studies are presented in accordance with the International System of Units recommended for use in clinical laboratory practice. The analysis of research results was performed using a program package Statistica 6.0 software (Stat Soft, Tulsa, USA). Probability differences were assessed by Student's t-test.
Results
Among the informative parameters, which characterise the state of lipid metabolism is cholesterol level. We established, that its content in laying hens of the control and experimental group at the age of 224 days was 3.8 ± 0.09 and 2.7 ± 0.13 mmol/L, respectively, and at the age of 300 days - 3.7 ± 0.10 and 2.4 ± 0.06 mmol/L. The total cholesterol content of the experimental group was lower at the age of 224 and 300 days by 40.7 % and 54.2 % (p<0.001), respectively, compared with the control group. In the two study groups, the age dynamics of the decrease in this parameter is observed, which indicates changes in the functional state of the liver. Low parameters of serum cholesterol may be due to impaired esterification of its esters by hepatocytes.
Parameters of cholesterol content in laying hens with hepatosis has a tend to decrease both in age and in comparison with similar parameters of clinically healthy (Fig. 1).
3,8
3,7
224 days 300 days
■ Control ^ Experimental Fig. 1. The content of serum total cholesterol in laying hens (n = 10), mmol/L
Triglycerides are one of lipid fractions. An increase in their level is mainly of alimentary origin and is often observed in fatty hepatosis, which confirms the results of our study (Fig. 2). The level of triglycerides in the control group at the age of 224 days was 12.4±0.29, 300 days - 13.6±0.30 mmol/L and in the experimental group - 15.8 ± 0.27 and 16.5 ± 0.43 mmol/L, respectively. Evaluation of changes in the
20
concentration of serum triglycerides in sick laying hens, determined increased levels (p <0.001) in poultry aged 224 days - 27.4 % and 300 days - 21.3 %, compared to the values in clinically healthy. Increased synthesis of triglycerides leads to a decrease in the rate of their removal from the liver, resulting in the development of fatty infiltration and fatty degeneration of the liver.
15,8
16,5
15 10 5 0
224 days 300 days
■ Control j Experimental Fig. 2. The concentration of serum triglycerides in laying hens (n = 10), mmol/L
Between the content of total cholesterol and the concentration of triglycerides in the serum of laying hens with hepatosis (Fig. 3) a strong negative correlation (224 days - r = -0.979; 300 days - r = -0.949) was
established. It indicates an increase lipolysis activity, accumulation of triglyceride fat vacuoles in liver cells and can serve as an early diagnostic test for hepatosis.
17,5
224 days
17,0 16,5 16,0 15,5 15,0 14,5 14,0
2,0
2,2
2,4
2,6
2,8
3,0
R2 = 0,9586
3,2
3,4
19 18,5 18 17,5 17 16,5 16 15,5 15 14,5 14
300 days
2,1
2,2
2,3
2,4
2,5
R2 = 0,902
2,6
2,7
2,8
Fig. 3. Correlation between serum total cholesterol and triglycerides in laying hens with hepatosis aged 224 and 300 days
2
We also studied the lipid profile in serum of sick and healthy laying hens of different ages. The lipid spectrum is presented in table 1.
High-density lipoproteins (HDL) help to remove excess cholesterol from the bloodstream into the liver, where it is processed and broken down. The content of HDL in the serum of laying hens with hepatosis did not change significantly in any age group. At the age of 224 days, this parameter was 4.9 % higher in sick laying hens. At 300 days of age, the HDL content of the experimental group was 8.3% higher than in the control group, but 65.4 % lower than in sick laying hens of age 224 days.
Low-density lipoproteins (LDL) are the main transporters of cholesterol in the body. Assessing the nature of this fraction in the serum of laying hens of the experimental group, established increase in poultry
aged 224 days - by 60.5 % (p<0.001) and 300 days -by 13.9 % (p<0.01), compared to the values of this parameter in the control group. In laying hens aged 300 days, this parameter was 68.3 % lower (p<0.001) in the experimental group and 19.4 % (p<0.001) in the control group comparing to the parameter at the age of 224 days.
The significant increase of very low-density lipo-proteins (VLDL) was established in a sick hens (p<0.001). This parameter was 52 % higher in the experimental group in laying hens at the age of 224 days and 25.6 % at the age of 300 days. Such changes indicate a sharp increase in lipogenesis that contributes to the development of fatty degeneration of the liver. However, with age, a decrease in VLDL in the control and experimental groups was 5 % and 27.5 %, respectively.
Table 1
The content of serum lipoproteins in laying hens (n = 10)
Animal age, days Animal group Parameters
HDL, mmol/L LDL, mmol/L VLDL, mmol/L
224 Control 0,41±0,009 0,43±0,012 2,71±0,024
Experimental 0,43±0,008 0,69±0,015 *** 4,12±0,075 ***
300 Control 0,24±0,012 0,36±0,009 2,58±0,119
Experimental 0,26±0,016 0,41±0,011 ** 3,24±0,097***
Note: ** - p<0,01; *** - p<0,001- compared to the parameters of control group
Studies of the concentration of serum bile acids in sick laying hens established a significant increase of their levels (Fig. 4). This can be explained by impaired bile acids secretion by hepatocytes.
The concentration of bile acids in the control group at the age of 224 days was 35.7±1.25, 300 -47.1±3.17 ^mol/L and in the experimental group -82.1±2.45 and 94.9 1.05 ^mol/L, respectively. It should
be noted that at the age of 300 days, the average value of this parameter increased in both sick and clinically healthy poultry compared to laying hens aged 224 days. Namely, by 1.2 times (p<0.001) and by 1.3 times (p<0.01), respectively. We also diagnosed that a bile acids concentration in laying hens at age 224 days in-crised 2.3 times (p<0.001) and 300 days - 2.1 times (p<0.001) comparing to the group of clinically healthy.
100 80 60 40 20 0
94,9
82,1
224 days 300 days
■ Control f Experimental Fig. 4. The concentration of serum bile acids in laying hens (n = 10), fimol/L
Discussion
Modern rates of rearing laying hens have a significant impact on metabolic processes of the poultry body [5,7,19]. Therefore, the study of lipid metabolism and hepatic disorders of lipids are the key points in improving growth of poultry and increasing its productivity.
Studies in poultry have revealed a large number of pathologies associated with dysfunction and morphological changes in the liver. Among them, the leading place is occupied by hepatosis in laying hens [5,15]. As dystrophic changes in the liver increase, its functional state decreases [6]. The reaction to hypoglycemia is a mobilization of fat from the fat depot, its entry into the liver and increased fatty infiltration of the body. Hyper-cholesterolemia contributes to this process. The accumulation of cholesterol in the liver reduces the formation of phospholipids in it and thus inhibits the release of fat from the liver. Fatty hepatosis occurs when the intake of fatty acids exceeds the ability of hepato-cytes to metabolize them and secrete them into the blood as part of triglycerides [3].
Decreased fatty acid synthesis in the liver leads to increased formation of triglycerides. Along with this, the formation of lipoproteins in the liver - the main transport form of triglycerides - is inhibited. Intake of hepatotropic poisons inhibits the synthesis of apolipo-protein, which is the part of lipoproteins. That's why, the transport of triglycerides is inhibited and they accumulate in hepatocytes [15,1].
One of the informative parameters of the state of lipid metabolism is cholesterol, which is a component of cell membranes, a precursor of steroid hormones and bile acids. This parameter characterizes the lipid status and metabolic processes in the body. According to the authors [8, 11], a decrease in concentration of cholesterol in the blood serum is registered during hepatitis, hepatodystrophy due to a decrease in the synthetic function of hepatocytes and changes in bile acid metabolism.
Increased synthesis of triglycerides and decreased synthesis of phospholipids indicates a violation of fat metabolism, "delayed release of fat from the liver" [4].
Serum lipoproteins play an important role in the redistribution of cholesterol between the intestine, liver, blood cells, vascular wall, and other organs and tissue systems [16]. The main donors and acceptors of cholesterol in blood plasma are LDL and HDL. In ad-
dition to transport and antioxidant functions, LDLs perform a regulatory function. The uptake of oxidized LDL by macrophages reduces the chemotactic activity of cells and changes their secretory activity, which may be one of the mechanisms of inflammatory granuloma formation. Such action of oxidized LDL also causes the accumulation of macrophages (foam cells) in the lumen of blood vessels. Redistribution of lipoprotein fractions, increase in their content in the blood, and their oxidative damage, especially LDL, can be considered as an important link in the pathogenesis of atherosclerosis. In this case, LDL and VLDL are atherogenic fractions that contribute to the deposition of cholesterol in the vascular wall, and HDL - antiatherogenic. They are able to remove cholesterol from cells by diffusion from the cell surface.
According to the literature [1, 2,14], serum TG, LDL and HDL concentrations are important diagnostic parameters of the lipid profile. The synthesis of adipose tissue, fat deposition and the formation of yolk in poultry is depends on their content in the serum. Most fatty acids are produced in the liver and transported via LDL or chylomicrons for storage in adipose tissue as triglycerides [13].
With increase of the mass of adipose tissue, free fatty acids release increases it due to the activation of lipolysis. Intensive intake of free fatty acids (FFA) from adipose tissue to the liver leads to an increase synthesis of VLDL in hepatocytes [11,17].
Decreased liver function, which occurs during many diffuse liver diseases, leads to hepatocytes damage, which disrupts the absorption of bile acids from the blood. This explain the increase in the concentration of bile acids in the blood serum in laying hens with hepa-tosis [3].
Conclusion
Determination of the serum lipid profile in laying hens with hepatosis is a diagnostic criterion for establishing disorders of lipid metabolism and determination of the course of pathological processes in the liver. The results of studies in laying hens with hepatosis indicate a significant decrease in cholesterol and an increase in triglycerides, HDL, LDL, VLDL and bile acids comparing to clinically healthy. Probable changes between all studied parameters of lipid metabolism were established and the dynamics of gradual decrease of choles-
terol and liporoteins with the age of poultry was observed. These changes can be explained by a more intense metabolism in younger laying hens.
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