THE LEVEL OF SELECTED BLOOD INDICATORS OF LAYING HENS FED WITH ADDITION OF AMARANTH GRAIN

Bożena Króliczewska¹, Wojciech Zawadzki², Anna Bartkowiak³, Teresa Skiba^4
1 Department of Animal Physiology, Wrocław University of Environmental and Life Sciences, Poland
² Department of Animal Physiology and Biostructure, Faculty of Veterinary Medicine, Wrocław University of Environmental and Life Sciences, Poland
³ Department of Animal Hygiene and Ichthyology, Wrocław University of Environmental and Life Sciences, Poland
⁴ Department of Animal Products Technology and Quality Management, Wroclaw University of Environmental and Life Sciences, Poland

ABSTRACT

32 weeks old laying hens were used in this study. The birds were divided into 4 groups: the control group (group I) fed with basal diet and experimental groups II, III and IV fed with basal diet supplemented by appropriately 2, 5 and 10% addition of amaranth grain. No statistically relevant changes in the haemoglobin level and the haematocrit volume in the birds’ blood were noted during the study. The erythrocyte count was diversified and ranged between 2.7 (group III) and 3.1 x 10¹² l^-1(group IV). As compared to the control group, a statistically significant difference in the total cholesterol level was noted for hens in group II. Falling tendency was observed for groups fed with diet supplemented by a larger share of amaranth grain, i.e. 5 and 10 % share. The largest fall of the LDL fraction of cholesterol, by appropriately 45.6 and 52.3 %, was shown for groups fed with 5 and 10% addition of amaranth grain (group III and IV). The level of HDL fraction of cholesterol was higher in the experimental groups, fluctuating around the level of 0.24-0.36 mmol l^-1, as compared to the control group with the level of 0.22 mmol l^-1The triglycerides content in respective groups of birds fed with addition of amaranth grain was significantly different (p≤0.05) as compared to the control group. The largest fall of triglycerides content was noted for group IV. The ALT activity in groups fed with 2 and 10% addition of amaranthus grain was higher in comparison to the control group, reaching respectively 44.8 and 36.9 IU l^-1, (p≤0.05) for experimental groups and 27.5 IU l^-1 for the control group. The ALT activity in the group fed with 5% addition of grain was comparable to the one noted for the control group, i.e. 20.5 IU l^-1. The highest level of aminotransferase (AST) activity, i.e. 466.14IU l^-1 , was observed for the group fed with the largest (10%) addition of amaranth grain and constituted difference that was statistically significant in comparison to the control group level, i.e. 373.71 IU l^-1, p ≤ 0.05. Elevated levels of glucose and protein were observed in experimental groups as compared to the control group.

Key words: amaranth, blood, lipids, laying hens.

INTRODUCTION

For many years scientists have conducted research looking for alternative plants, able to replace the species cultivated so far and make a valuable addition to the animal feed, enhancing at the same time animal health and productivity as well as the nutritional value of animal products. Amaranthus, known as purple amaranth (Amaranthus cruentus), a native plant of South America, seems to be such a plant.

This interest arises from its potential nutritional value. Compared to common cereals such as maize, wheat and rice, amaranth is reported to have high contents of protein and is relatively rich in lysine, tryptophan and methionine. Moreover, amaranth has also a higher concentration of soluble fiber than many cereals, such as weat, corn, or oat, high concentration of calcium, phosphorus, iron, potassium, zinc, vitamins E and B complex, squalene and a low level of antinutritional factors [1,2,13,14]. Chemical composition of amaranth grain was presented in Table 1.

Table 1. Chemical composition of amaranth grain [2]

Ingredient	Range
Crude proteina	12.5 – 17.0
Lysine	0.73 – 0.84
Tryptophan	0.18 – 0.28
Crude fibera	3.4 – 5.3
Total lipidsa	7.7 – 12.8
C16 fatty acids	16.83 – 23.83
C18:0 fatty acids	1.86 – 4.11
C18:1 fatty acids	20.29 – 35.46
C18:2 fatty acids	38.25 – 57.86
Crude asha	2.8 – 3.6
Tannin	0.043 – 0.13
Kb	290
Cab	175
Mgb	244
Feb	17.4
Nab	31
Cub	1.2
Znb	3.7
Mnb	4.6

a % of dry matter, b mg 100 g-1 dry matter

Applying amaranth grain to feeding animals has already been tested with rats [4,8,9,], pigs [21], rabbits [13] and broiler chickens [15]. Tillman and Waldroup obtained body mass increase of statistical significance (p≤0.05) in broilers fed with fodder supplemented by amaranth grain [18]. Faruga et al. showed that supplementation of diets for broiler chicken Hybro N and ISA Brown laying hens with flaked amaranth seeds had no negative effect on the digestability of nutrients, metabolic blood indicators and bird growth rate [6].

The health properties of diet supplements are proved by, among other things, the values of haematological and biochemical blood indices. Their level is influenced precisely by the applied diet, certainly apart from such factors as: age, sex, physiological status, raising technology and stress. Therefore, the above-presented data and relationships persuaded the authors of this work to start research into the influence on the value of selected haematological and biochemical parameters of blood in laying hens by supplementing their diet with amaranth grain.

MATERIALS AND METHODS

The investigations were conducted on 80 at the 32-week of age laying hens of ISA SHAVER Line. The hens were fed with basal diet complete feeds J-297 PROVIMI-ROLIMPEX S.A., Poland, containing 16.16% of crude protein and 22.61MJ of energy/kg. After two weeks of adaptation to the vivarian condition , the birds were divided into 4 groups (each consisting of 20 hens) in the following manner: control group (group I) fed with the basal diet and experimental groups II, III and IV fed with basal diet supplemented by appropriately 2, 5 and 10% addition of whole amaranth grain (Szarłat S.C., Łomża, Poland). Amaranth grains were mixed with the fodder until the mixture was homogenous. Hens were fed in accordance with the nutrition standards [16]. Diets and fresh water were provided ad libitum. Birds were maintained in electrically heated batteries.

After 8 weeks of the experiment, blood samples were taken V. jugularis into disposable heparinised test tubes. Blood samples were centrifugated at 3000xg for 10 min and plasma was collected. In the plasma total cholesterol (TC) was analyzed according to the method of Young [20], high-density lipoprotein (HDL), low-density lipoprotein (LDL) and triglycerides (TG) were analysed according to method of Fossati and Prencipe [7].

Ion concentration (Fe) and glucose levels were analysed according to method of Tietz [19], using analytical sets (BioSystems S.A., Spain). The activities of alanine aminotransferase (ALT), aspartate aminotransferase (AST) were analysed according method of Reitman-Frankel using kits from AQUA MEDICA.

Erythrocytes and leukocytes were counting in whole blood by routine method. Sample of blood was diluted using mixing pipette up o the 0.5 mark and then Hayem’s reagent (for red blood cells) or Türk’s solution (for white blood cells) up to the 101 mark. Blood cells were counted in a Thom-Zeiss chamber. The required cell type in a defined volume was counted and the number of cells per litre of blood was then calculated. Also, the haemoglobin content was determined with photocolorimetric method, using the Epoll-20 photometer and haematocrit with the microhematocrit method, using the haematocrit centrifuge.

All data were analyzed by one-way ANOVA [17] to test the effects of the dietary treatments at the significant level of P < 0.05. All data were expressed as means ± SD.

RESULTS AND DISCUSSION

The results of haematological and biochemical tests are shown in Tables 2 and 3. Feeding laying hens with fodder supplemented by amaranth grain did not significantly influence the haemoglobin content, determined at the level ranging from 8.7 (group IV) to 9.7 mmol l^-1 (group I), not distant from the standards presented by other researchers [12]. However, a tendency of haemoglobin content decreasing along with the increase of the amount of amaranth grain in the fodder was observed (Table 2). No statistically relevant changes in the haematocrit volume in the birds’ blood were noted. Similar results were obtained by Grajeta in her study on rats fed with fodder supplemented by amaranth grain [8]. The red blood cell count was diversified and ranged between 2.7 (group III) and 3.1 10¹² l^-1 (group IV). Higher levels of white blood cells were noted for groups fed on diet supplemented with the amaranth grain as compared to the control group. The highest leukocyte count was observed for groups (III and IV), on average appropriately 28.5 and 27.7 10⁹ l^-1, however these were not statistically significant differences (Table 2).

Table 2. Blood haematological indices of hens fed with addition of amaranth grain (average ± SD)

Parameter	Group I control group	Group II 2%	Group III 5%	Group IV 10%
Haemoglobin [g·l-1]	9.7 ± 0.3	9.6 ± 0.4	9.4 ± 0.2	8.7 ± 0.7
Haematocrit [l l-1]	0.3 ± 0.006	0.3 ± 0.02	0.3 ± 0.001	0.3 ± 0.008
Erythrocytes [1012 l-1]	2.8 ± 0.2	2.7 ± 0.3	2.7 ± 0.3	3.1 ± 0.3
Leukocytes [109 l-1]	18.4 ± 46.8	22.9 ± 7.7	26.9 ± 7.4	26.1 ± 7.2

The results of biochemical tests are shown in Table 3. Feeding laying hens with fodder supplemented by amaranth grains had generally beneficial influence on their blood lipid metabolism. A statistically significant difference in the total cholesterol level was noted for hens in group II as compared to the control group. Falling tendency was observed for groups fed with diet supplemented with larger share of amaranth grain, i.e. 5 and 10 %, (Table 3). Grajeta had observed a positive effect of reducing (by 14%) the cholesterol content in blood of rats fed on diet supplemented with amaranth grain, however, she obtained this result while applying 30% share of the grain [8].

Table 3. Blood haematological indices of hens fed with addition of amaranth grain (average ± SD)

Parameter	Group I control group	Group II 2%	Group III 5%	Group IV 10%
Total cholesterol [mmol l-1]	4.5 ± 1.2a	4.7 ± 1.0b	4.3 ± 1.2a	4.2 ± 1.5a
HDL [mmol l-1]	0.22 ± 0.06a	0.36 ± 0.07b	0.26 ± 0.04a	0.24 ± 0.08a
LDL [mmol l-1]	3.4 ± 1.044a	2.1 ± 0.306b	1.8 ± 1.1b	1.6 ± 0.4b
Triglycerides [mmol l-1]	19.4 ± 5.2 a	10.8 ± 5.0b	11.4 ± 6.3b	6.2 ± 2.8b
ALT [IU l-1]	27.5 ± 1.2a	44.8 ± 7.4 b	20.6 ± 7.9a	36.9 ± 3.9c
AST [IU l-1]	373.7 ± 0.9a	377.7 ± 5.1a	235.2 ± 7.3b	466.1 ± 12.8c
Glucose [mmol l-1]	13.8 ± 0.4a	14.6 ± 0.7 b	21.9 ± 1.4c	15.6 ± 0.4d
Protein [g l-1]	43.6 ± 2.2a	55.4 ± 3.2 b	54.0 ± 2.2b	45.0 ± 2.8a
Iron [µmol l-1]	110.8 ± 17.0	113.6 ± 22.7	129.1 ± 25.1	105.0 ± 22.8

abc significant differences at (p ≤ 0.05)

The results obtained by Grajeta also indicate positive changes in the LDL lipoprotein fraction of cholesterol for groups of laying hens fed on diet supplemented with amaranth grain [8]. The largest fall of this so-called ‘bad’ cholesterol fraction by appropriately 45.6 and 52.3 %, was shown for groups fed with 5 and 10% addition of amaranth grain (group III and IV). In their experiments on pigs, Zralý et al. confirm the above-drawn conclusions [21]. Also in their studies on rabbits, Plate and Arěas showed that groups of animals fed with amaranth grain cake had lower level of total cholesterol as well as its LDL fraction [13]. Research explicitly confirms the existence of a positive relationship between high level of cholesterol and its LDL fraction in blood and the circulatory system diseases [11]. Lowering these parameters in blood prevents their deposition on arterial walls and thus impedes atherosclerosis [10]. The authors’ own research also provided similar results confirming positive influence of amaranthus on metabolism of LDL fraction cholesterol and its level in the blood plasma.

In their study on rats, Chaturvedi et al. obtained results suggesting that addition of amaranth grain to the diet lowers the total level of cholesterol and LDL fraction cholesterol as well as increases the total level of HDL lipoprotein [3]. In the authors’ own research the level of this cholesterol fraction was higher in the experimental groups, fluctuating around the level of 0.24-0.36 mmol l^-1 as compared to the control groups with the level of 0.22 mmol l^-1. Also in case of this fraction of cholesterol, positive physiological tendency was observed.

Triglycerides present in serum are of exogenous and endogenous origin. Available literature provides little data on the content of triglycerides in the serum of poultry. However, interrelation between the decrease of their level and the age of birds can be observed [12]. In the authors’ own research, the content of triglycerides in respective groups of birds fed with addition of amaranth grain was significantly different (p≤0.05) as compared to the control group. The largest fall of triglycerides content was noted for group IV. Chaturvedi et al., indicated significant decrease of triglycerides level in blood serum of rats fed with fodder supplemented by amaranth seeds [3]. The same result was obtained by Czerwińska and Zralý in their research on pigs [4,21]. Whereas, Grajeta did not show any significant differences in the triglyceride level in blood of rats [9]. Chaturvedi et al., suggest that the fatty acids are factors of hypolipemic effect in the amaranth seeds [3]. Whereas, according to Danz and Lupton, it is dietary fibre because it is present in this plant in large amounts and its larger part is a water-soluble fraction [5].

In the authors’ own research the activity of two liver enzymes was marked, connected with fat metabolism and cholesterol synthesis. The first one is aspartate aminotransferase (AST). This is an intracellular enzyme, present in all tissues of particularly high concentration in myocardium, skeletal muscles, liver and brain. In poultry, its activity in blood serum is dependent upon the bird’s age, for it increases with age until reaching sexual maturity, to decrease in adult birds. The second marked enzyme was alanine aminotransferase (ALT), present mainly in liver. Its activity in hens’ blood serum also shows the growing tendency with age [12].

While analysing the content and normal level of liver enzymes, the change in activity of both ALT and AST was observed. The ALT activity in groups fed with 2 and 10% addition of amaranthus was higher in comparison to the control group, reaching respectively 44.8 and 36.9 IU l^-1, (p≤0.05) for experimental groups and 27.5 IU l^-1 for the control group. The ALT activity in the group fed with 5% addition of grain was comparable to the one noted for the control group, i.e. 20.5 IU l^-1. The highest level of aminotransferase (AST) activity, i.e. 466.14 IU l^-1, was observed for the group fed with the largest (10%) addition of amaranth grain and constituted difference that was statistically significant in comparison to the control group level, i.e. 373.71 IU l^-1, p ≤ 0.05. In their research on pigs, Zralý et al. obtained elevated level of ALT, whereas AST level remained unchanged [21]. The obtained results suggest that the polyunsaturated acids contained in the amaranth seed are the components of strong hypolipemic effect.

This research showed significant differences in the blood glucose level of the birds involved in this study. Elevated levels of this parameter were observed in experimental groups as compared to the control group. Different results were obtained by Zralý et al. [21] They showed falling level of blood glucose in pigs fed on diet supplemented by amaranth grain. Similar results were achieved by Roučkova et al. in their study on broiler chickens [15].

The content of total protein in the poultry’s blood serum increases with age. The content of protein in the experimental groups was higher in comparison to the control group. The obtained results fit the reference range for this species, amounting to from 32 to 62 g l^-1 [12]. Similar results were achieved by other researchers in their studies on pigs [21]. Whereas, Roučkova et al. did not show any influence of the amaranthus on the total content of protein in blood of broiler chickens [15].

No differences were discovered in the iron level contained in the birds’ blood, its content fluctuated around 105.0 to 129.1 µmol l^-1 which correlates with the unchanged content of haemoglobin.

CONCLUSIONS

Feeding hens on diet supplemented with amaranth grain did not negatively influence the selected haematological parameters of blood. Moreover, the generally beneficial influence of applying the amaranth seed on the lipid parameters of birds’ blood was observed. Also, changes in the activity of aminotransferases ALT and AST were noted, as well as the elevated levels of protein and glucose content in the groups fed with addition of amaranth grain.

REFERENCES

1. Berger A., Monnard I., Dionisi F., Gumy D., Hayes K.C., Lambelet P., 2003. Cholesterol-lowering properties of amaranth flakes, crude and refined oils in hamsters. Food Chemistry 81, 119-124.

2. Bressani R., Gonzales J.M., Zuniga J., Breuner M., Elias L.G., 1987. Yield, selected chemical composition and nutritive value of 14 selections of amaranth grain representing four species. J. Sci. Food Agric. 38, 347-356.

3. Chaturvedi A., Sarojini G., Devi N.L., 1993. Hypocholesterolemic effect of amaranth seeds (A. esculantus). Plant. Food. Hum. Nutr., 44, 63-70.

4. Czerwińska J., Bartnikowska E., Leontowicz H., Lange E.,Leontowicz M., Katrich E., Trakhtenberg S., Gorinstein S., 2004. Oat (Avena sativa L.) and amaranth (Amaranthus hypochondricus) meals positively affect plasma lipid profile In rats fed cholesterol-containing diets. J. Nutr. Biochem., 15, 622-629.

5. Danz R.A., Lupton J.R., 1992. Physiological effects of dietary amaranth (Amaranthus cruentus ) on rats. Cereal Foods World., 37(7), 489-494.

6. Faruga A., Mikulski D., Bobrzecka D., Mikulska M., Pudyszak K., 1998. Wykorzystanie nasion szarłatu (Amaranthus cruentus) w żywieniu kurcząt brojlerów i kur niosek [Utilization of Amaranth seeds (Amaranthus cruentus L. in the diets for broiler chickens and egg-laying hens] Zesz. Nauk. PTZ. Prz. Hod., 36, 287-295 [in Polish].

7. Fossati P, Prencipe L., 1982. Serum triglycerides determined calorimetrically with an enzyme that procedures hydrogen peroxide. Clin. Chem. 28, 2077.

8. Grajeta H., 1999. Effect of amaranth and oat on blood serum and liver lipids in rats depending on the kind of dietary fats. Nahrung., 43, 2, 114-117.

9. Grajeta H., 2000. Hipolipemiczne działanie ekspandowanych nasion szarłatu u szczurów doświadczalnych [A hypolypemic effect of amaranth (Amaranthus cruentus) expanded seeds in experimental rats]. Brom. Chem. Toks., 33, 1, 7-13 [in Polish].

10. Harborne J.B., Williams Ch.A., 2000. Advances in flavonoid research since 1992. Phytochem., 55, 481-504.

11. Marcone M.F., Kakuda Y., Yada R.Y., 2004. Amaranth as a rich dietary source of β-sitosterol and other phytosterols. Plant. Food. Hum. Nutr., 58, 207-211.

12. Mazurkiewicz M., 2005. Choroby drobiu [Poultry deseases]. AWA. Wrocław [in Polish].

13. Plate Andrea Y.A., Arėas Jose A.G., 2002. Cholesterol-lowering effect of extruded amaranth (Amaranthus caudatus L.) in hypercholesterolemic rabbits. Food Chem, 76, 1-6.

14. Ravindran V., Hood R.L., Gill R.J., Kneale C.R., Bryden W.L., 1996. Nutritional evaluation of grain amaranth (Amaranthus hypochondriacus) in broiler diets. Anim. Feed Sci. Technology 63, 323-331.

15. Roučkova J., Trčkova M., Herzig I., 2004. The use of amaranth grain in diets for broilers chickens and its effect on performance and selected biochemical indicators. Czech J. Anim. Sci., 49, 532-541.

16. Smulikowska S., 1994. Dodatki paszowe w żywieniu drobiu [Feed additives in poultry feeding]. IFŻZ im Jana Kielanowskiego PAN. Jabłonna [in Polish].

17. StatSoft, Inc.: Statistica for Windows, StatSoft, Inc., Tulsa OK., 1997. http://www.statsoft.com.

18. Tietz N. W., 1999. Clinical guide to laboratory tests, 3 ed. Saunders Co.

19. Tillman P.B., Waldroup P.W., 1987. Performance and yields of broilers fed extruded grain amaranth and grown to market weight. Poult. Sci., 67, 743-749.

20. Young D.S., 1995. Effects of drugs on clinical laboratory tests, 4 ed. AACC Press.

21. Zralý Z., Pisaŕĭková B., Trčková M., Herzig I., Juzl M., Simeonovová J., 2006. Effect of lupine and amaranth on growth efficiency, health, and carcass characteristics and meat quality of market pigs. Act.Vet. Brno., 75, 363-372.

 

Accepted for print: 18.04.2008

---

Bożena Króliczewska
Department of Animal Physiology,
Wrocław University of Environmental and Life Sciences, Poland
C. K. Norwida 31, 50-375 Wrocław, Poland
Phone: (+4871) 32 05 437
email: bozena.kroliczewska@up.wroc.pl

Wojciech Zawadzki
Department of Animal Physiology and Biostructure, Faculty of Veterinary Medicine, Wrocław University of Environmental and Life Sciences, Poland
C.K. Norwida 31
50-375 Wrocław
Poland
Phone: +48 71 320 5401
email: wojciech.zawadzki@up.wroc.pl

Anna Bartkowiak
Department of Animal Hygiene and Ichthyology,
Wrocław University of Environmental and Life Sciences, Poland
Chełmońskiego 38c, 50-630 Wrocław, Poland
Phone: (+4871) 32 05 861
email: anna.bartkowiak@up.wroc.pl

Teresa Skiba
Department of Animal Products Technology and Quality Management,
Wroclaw University of Environmental and Life Sciences, Poland
C.K. Norwida 25/27 , 50-375 Wrocław, Poland
Phone: (+4871) 32 05 217
email: teresa.skiba@up.wroc.pl

---

Responses to this article, comments are invited and should be submitted within three months of the publication of the article. If accepted for publication, they will be published in the chapter headed 'Discussions' and hyperlinked to the article.

---

Main  -  Issues  -  How to Submit  -  From the Publisher  -  Search  -  Subscription