Introduction
In human nutrition, diet plays a significant role in maintaining health status and daily performance. Despite the successes in prevention of atherosclerosis, coronary artery disease is still responsible for one of every three cases of death. It is now well known that cholesterol plaques which are derived from plasma oxidized low-density lipoprotein cholesterol tend to
increase the incidence of coronary heart disease [13]. The high concentration of plasma low-density lipoproteins is a prerequisite for most forms of atherosclerosis [1]. Numerous studies have shown that dietary cholesterol can increase cir-culating LDL-cholesterol [8]. In recent years, humans have been paying more attention to health, thus lowering consumption of cholesterol enriched food. Egg is considered as one of the major sources of human dietary cholesterol.
SUMMARY
The present study was conducted to evaluate whether dietary pumpkin seed oil would affect laying performance, egg quality, egg cholesterol content and the egg yolk fatty acid composition in Brown layer hens. For that, 72 Brown layers, 28- week-old, were divided into 4 equal groups (n = 18) of similar mean weight according to the added doses (0, 10, 20 or 40 g/kg) of pumpkin seed oil into the standard ration for 8 weeks. Laying performance was assessed by recording initial and final body weights, weight gains, food intake and food conversion ratio, egg weight, egg production daily, egg cha-racteristics, as well as the plasma glucose, cholesterol and triglyceride concentrations and the egg cholesterol content and the fatty acid composition. The dietary pumpkin seed oil inclusion has not significantly affected the growth performances, the laying performance or the egg quality even if the overall number and weight of eggs and also the thickness and the weight of shell tended to slightly decrease. Whereas the plasma concentrations of tri-glycerides remained similar in the 4 groups, the cholesterolemia tended to decline according to the dose added to the ration and the glycaemia was significantly depressed in supplemented hens (P < 0.05). In parallel, the egg yolk cholesterol content was significantly lowered in birds receiving the pumpkin seed oil in a dose related manner and the yolk fatty acid composition was significantly altered, showing increase of behenic acid with the highest dose of pumpkin seed oil, and marked increase of unsaturated acids, parti-cularly of oleic and linolenic acids whatever the dose. These results show that pumpkin seed oil can be added to the ration without significantly altering the growth and laying performance in order to produce eggs enriched in unsaturated acids and made thin in cholesterol.
Keywords: Brown layers, pumpkin seed oil, dietary inclusion, egg production, egg quality, glycaemia, cholesterolemia, egg cholesterol, fatty acid composition.
RÉSUMÉ
Intérêt de l’addition d’huile de pépins de citrouille dans la ration des poules pondeuses dans la production d’œufs pauvres en cholestérol et riches en acides oléique et linolénique
Cette étude a été menée afin d’évaluer les effets potentiels de l’huile de pépins de citrouille sur les performances de ponte, les qualités des œufs et leur teneurs dans le jaune en cholestérol et en acides gras chez des poules pondeuses Brown. Pour cela, 72 poules de 28 semaines ont été répartis en 4 groupes égaux (n = 18), homogènes en poids, en fonction de la dose d’huile de pépins de citrouille (0, 10, 20 ou 40 g/kg) ajoutée à une ration standard pendant 8 semaines. Les performances des poules ont été déterminées en mesurant les poids initiaux et finaux, les gains de poids, l’ingéré alimentaire et l’efficacité alimentaire, le poids des œufs, la production journalière en œufs, leurs caractéristiques, ainsi que les concentrations plasmatiques de glucose, de cholestérol et de triglycérides, et les teneurs du jaune en cholestérol et en acides gras. L’addition d’huile de pépins de citrouille dans la ration n’a affecté ni les performances de croissance ni les performances de ponte même si le nombre total d’œufs, leur poids moyen ainsi que l’épaisseur et le poids de la coquille ont tendu à diminuer. Alors que les concentrations plasmatiques de triglycérides sont restées similaires dans les 4 groupes, la cholestérolémie a eu tendance à diminuer en fonction de la dose reçue et la glycémie a signi-ficativement diminué chez les poules supplémentées (P < 0.05). En parallè-le, la teneur en cholestérol du jaune était significativement plus faible chez les oiseaux supplémentés de façon dose-dépendante et la composition en acides gras a aussi été significativement modifiée, présentant une augmen-tation de la proportion en acide béhénique lors de l’administration de la plus forte dose et une nette accumulation des acides gras insaturés, en particulier des acides oléique et linolénique, quelque soit la dose d’huile de pépins de citrouille. Ces résultats montrent que l’huile de pépins de citrouille peut être ajoutée à la ration sans altérer les performances des poules afin de produire des œufs enrichis en acides gras insaturés et appauvris en cholestérol.
Mots clés : Poules pondeuses Brown, huile de pépins de citrouille, inclusion alimentaire, production en œufs, qualités des œufs, glycémie, cholestérolémie, teneur en cholestérol dans l’œuf, composition en acides gras.
cholesterol low and oleic and linolenic acid
rich egg production in layer hens
L. ÇELIK1*, H.R. KUTLU1, Z. ŞAHAN1, A. BOZKURT KIRAZ1, U. SERBESTER2, A. HESENOV3, A. TEKELI4
1Department of Animal Science, University of Çukurova, 01 330 Adana, TURKEY. 2Bor Vocational Training School, University of Niğde, 51 700 Niğde, TURKEY. 3Department of Chemistry Science, University of Çukurova, 01 330 Adana, TURKEY. 4Department of Animal Science, University of Yüzüncüyıl, 65 080 Van, TURKEY.
Since cholesterol in eggs accounts for more than 50% of daily take-up, people are advised to reduce their egg consumption to control blood cholesterol concentration. Those people having cholesterol problem but enjoying with egg consumption seem to have a demand for eggs low in cholesterol. Designing such egg could satisfy people having cholesterol problem or potentially susceptible for coronary hard disease and also globally beneficial to the public's health. For this reason, many experiments have been conducted to decrease the egg cholesterol content using natural organic or inorganic sources like, fibre [19], oils [30], copper [17] and garlic [10]. However, very limited success accompanied these experiments.
Pumpkin seed oil is a natural source rich in antioxidants and polyunsaturated fatty acids. It can supply for essential fatty acids but also for β-carotenes, lutein γ and β-tocopherols, phytosterols and selenium [2] that act synergistically in animal tissues to constitute an important antioxidant defence system against reactive oxygen species mediated lipid peroxidation of lipoproteins [1, 11, 22]. It has also been claimed that the pumpkin seed oil exhibits anti-hypercholesterolemic [1], anti-oxidant [2] and anti-arthritis [11] effects in several species of growing monogastric animals. Pumpkin seed oil has also been reported to increase glucagon sensitivity and alter responsiveness to insulin in laying hens [27].
On the other hand, it is well known that the fatty acid com-position of egg yolk is influenced by the diet of the laying hens [21]. Most of the fatty acids in pumpkin seeds are monounsaturated and polyunsaturated fatty acids (PUFA), which belongs to omega-6 groups [4] (n-6 PUFA). Besides its putative positive effects on laying performance and egg quality, the dietary inclusion of pumpkin seed oil would strengthen the egg yolk contents of n-6 PUFA and of anti-oxidants. However, very limited information have been reported on dietary use of pumpkin oil in layers and its pos-sible effects on laying performance and also egg quality in the present literature.
The aim of the present study was, therefore, to investigate whether inclusion of dietary pumpkin seed oil may improve the laying performance and egg qualities, contribute to reduce the cholesterolemia and the cholesterol content of the egg yolk and may increase the unsaturated fatty acid content in egg yolk.
Materials and Methods
EXPERIMENTAL ANIMALS AND PROTOCOL DESIGN Prior to the trial, seventy-two, 28-week-old Brown-Nick layers were fed ad libitum with a standard layer diet for two weeks period, during which daily egg production and egg weight were recorded. At the beginning of the trial, when the birds were 30 weeks old, all the birds were divided into 4 experimental groups of similar mean body weight and egg production level, comprising 18 birds each. The birds were fed with standard layer diets containing 0, 10, 20 or 40 g/kg pumpkin seed oil for an 8 weeks long period. The standard layer diet was formulated using 40 g/kg vegetable oil. The oil
inclusion to the treatment diets was balanced with soya (0 to 40 g/kg) and pumpkin seed oil (40 to 0 g/kg) as given in Table I.
The birds were housed in individual layer cages of three-tier battery blocks in a complete randomized design at a conven-tional ambient temperature (20-22°C) with a relative humi-dity of 60-70%. Light was provided 16 hours (from 05.00 to 21.00) each day. The experimental procedure was approved by the Animal Care and Ethics Committee of Çukurova University, Adana, Turkey.
PERFORMANCE AND EGG QUALITY ANALYSIS
The laying performance was determined daily by measu-ring food intake, egg mass, food conversion ratio (egg mass / food intake) and egg production (in house, number and weight). Egg quality was estimated by measuring the egg-shape index (width / length), the shell weight, the breaking strength, the shell thickness, the egg weight, the yolk weight, the albumen weight, the albumen height, the yolk index (weight / height) and the yolk colour score (Roche, Yolk Colour Fan) of each egg obtained on the third day of every week. Shell samples from top, middle and bottom of the egg were measured for thickness using a micrometer and the mean was calculated prior to the statistical analysis.
CHEMICAL ANALYSIS
Five birds from each group were randomly chosen and blood samples were collected by puncture of the vena brachialis biweekly throughout the experiment. After centrifugation (1 500g, 5 minutes, 4°C), plasmas were carefully harvested and stored at -20°C until analysis. The plasma concentrations of the glucose, the cholesterol and the triglycerides were measured with commercial kits (glucose GOD-PAP; Roche Diagnostics, GmbH, Germany, cholesterol CHOD-PAD; Roche Diagnostics, GmbH, Germany, triglyceride GPO-PAP; Roche Diagnostics, GmbH, Germany) on an automated KEYLAB LiquiVet Analyzer.
The egg yolk cholesterol content [5] was weekly determined in eggs from hens used for the blood biochemistry analysis using a specific kit (Boehringer Manheim Gmbh Biochemica). Briefly, the egg yolks were hard-boiled for 10 minutes, homogenised and allotted in 0.1g yolk fractions. After lipid extraction with isopropanol (4 mL, incubation at 37°C for 10 minutes and centrifugation at 1 500 g for 5 minutes at 37°C) [5], the yolk cholesterol concentration was determined in the filtered samples by UV spectrophotometer using a commercial specific kit (Boehringer Manheim Gmbh Biochemica).
The egg yolk fatty acid composition was evaluated by per-forming fatty acid analysis at the beginning and the end of the experiment. Firstly, the lipid extraction from the egg yolk was carried out according to the method of FOLCH et al. [12] and then, the fatty acid profiles was determined by gas chromatography, using a Shimadzu Model 14A, under the following operating conditions: fused silica capillary column
(60 m x 0.25 mm x 0,2 μm; SPTM-2380); Helium as the carrier
gas with a flow rate of 1 ml/minute; oven temperature, initially 80°C for 5 minutes, increased up to 230°C at 2.8°C/min and maintained at 230°C for 5 minutes; injection port and detector temperatures: 225°C and 250°C, respecti-vely. The injection volume was 1 μL of sample solution. The retention time of methyl ester standards (1000 ppm, 500 ppm, 250 ppm, 125 ppm and 62.5 ppm) were used to identify peaks.
STATISTICAL ANALYSIS
The data obtained in the experiment were analysed using the General Linear Models (GLM) and orthogonal
polyno-mial of SAS [29]. Linear, quadratic and cubic effects were determined by orthogonal polynomial contrasts. Differences were considered as significant when P value was less than 0.05.
Results
The laying performance and the egg qualities observed in laying hens according to the dietary pumpkin seed oil inclusion were summarized in the Table II. The addition of the pumpkin seed oil in the aliment has not significantly affected the growth and the laying performances of the layers although the weight gain for 8 weeks, the egg number and the egg mass recorded during the experimental period have appeared
Dietary treatment groups (pumpkin seed oil (g/kg)
0 10 20 40
Ingredients (g/kg)
Corn 487.35 487.35 487.35 487.35
Soybean meal (440 g/kg CP) 248.20 248.20 248.20 248.20
Corn gluten meal (550 g/kg CP) 30.00 30.00 30.00 30.00
Wheat bran (140 g/kg CP) 53.41 53.41 53.41 53.41
Meat bone meal (320 g/kg CP) 50.00 50.00 50.00 50.00
Limestone 80.00 80.00 80.00 80.00 Dicalcium phosphate 2.12 2.12 2.12 2.12 Soda 3.00 3.00 3.00 3.00 Salt 1.66 1.66 1.66 1.66 Methionine 0.76 0.76 0.76 0.76 Vitamin premix1 2.00 2.00 2.00 2.00 Mineral premix2 1.50 1.50 1.50 1.50 Soya oil 40.00 30.00 20.00
-Pumpkin seed oil - 10.00 20.00 40.00
Chemical composition(g/kg) Dry matter 880.90 Crude protein 180.00 Crude fibre 37.70 Ether extract 69.40 Crude ash 137.00 Lysine 8.80 Methionine 3.60 Methionine + Cystein 6.70 Arginin 12.00 Tryptophan 1.90 Calcium 38.40 Available P 4.30 Sodium 2.00 Metabolisable energy (MJ/kg) 11.97
TABLEI: Composition and nutrients contents of the experimental diets given to 30 week old Brown-Nick layers (n = 72) for 8 weeks.
CP: crude protein; 1
: Each 2 kg of vitamin premix contains vitamin A (6 000 000 IU), vitamin D3 (800 000 IU), vitamin E (14 000 mg), vitamin K3 (1600 mg), vitamin B1 (1250 mg), vitamin B2 (2800 mg), niacin (8000 mg), Ca-D-pantothenate (4000 mg), vitamin B6 (2000 mg), vitamin B12 (6 mg), folic acid (400 mg), d-biotin (18 mg), vitamin C (20 000 mg) and choline chloride (50 000 mg); 2: Each kg of mineral premix contains manganese (80
slightly lowered in supplemented birds. The egg qualities (egg weight, weights of the shell, yolk and albumen, egg yolk score, egg dimensions, yolk and albumen indexes and egg resistance) have not been significantly modified in layer hens fed with diets containing pumpkin seed oil but, never-theless, the egg weight, the weight and the thickness of the shell have tended to weakly decrease and the yolk index was the highest in birds fed with diets containing 1% pumpkin seed oil and the lowest in birds fed with diets containing 4% pumpkin seed oil (P < 0.05, quadratic effect).
As shown in Table III, whereas the plasma concentrations of triglycerides were not significantly changed with the dietary pumpkin seed oil inclusion, the glycaemia was significantly decreased in birds fed with standard diets containing 10 and 20 g/kg of pumpkin seed oil compared to the controls which received no pumpkin seed oil (P < 0.05, quadratic effect) and the cholesterolemia tended to gradually decrease (but not significantly) in a dose related manner. In the same way, the yolk cholesterol content (expressed as mg/g of egg) was signi-ficantly and markedly diminished in eggs from pumpkin seed oil supplemented hens according to the added dose (P < 0.05). As far as the fatty acid composition of the egg yolk was concerned (Table IV), it was observed that in eggs from the supplemented birds whatever the dose, the total proportions of saturated fatty acids were slightly decreased compared to
controls (P > 0.05): while the percentage of the major satu-rated fatty acid, the palmitic acid, was unchanged, the stearic acid percentage tended to decrease, but not significantly and the behenic acid (C22:0) percentage was significantly increased in eggs from birds fed with diets containing 4% pumpkin seed oil (P < 0.05, quadratic effect). In parallel, the global pro-portions of unsaturated acids were increased (P > 0.05): the preponderant oleic acid was highly significantly accumulated (P < 0.01) and the proportions of minority polyunsaturated acids were also increased (linolenic acid: P < 0.05; lignoseric acid: not significant) while, in contrast, the percentage of the linoleic acid tended to decrease according to the dose of pumpkin seed oil in the ration.
Discussion
In the present study, the dietary inclusion of pumpkin seed oil in the standard diet has not affected the growth parameters and the egg production but has slightly modified some egg qualities and the composition of the egg yolk. Very few data were found in the literature on the effects of the dietary inclusion of pumpkin seed oil on egg quality in hens [25, 26]. PAL [26] has observed significant increments of the yolk weight in laying hens feeding with diet containing 40 g/kg
Dietary pumpkin seed oil (g/kg) SED P value Significance level of factor effects
0 10 20 40 Linear Quadratic Cubic
Laying performances
Initial body weight (g/bird) 1828.00 1796.22 1794.17 1806.94 18.36 NS NS NS NS Final body weight (g/bird) 1948.88 1923.17 1905.89 1924.67 19.48 NS NS NS NS
Weight gain (g) 120.88 126.95 111.72 117.73 1.13 NS NS NS NS
Food intake (FI) (g/bird/8 weeks) 6468.76 6732.94 6547.00 6524.76 61.30 NS NS NS NS Egg mass (EM) (g/bird/8 weeks) 3338.56 3260.17 3260.15 3257.27 44.01 NS NS NS NS Food conversion ration (EM / FI) 0.52 0.48 0.50 0.50 0.00 NS NS NS NS
Mean egg weight (g/day) 62.20 59.91 59.55 59.69 0.64 NS NS NS NS
Number of eggs (/bird/8 weeks) 53.35 52.83 52.89 51.39 0.49 NS NS NS NS
Egg qualities
Egg weight (g/egg) 63.08 62.40 62.62 62.58 0.50 NS NS NS NS
Shell weight (g/egg) 7.13 7.14 7.02 6.97 0.06 NS NS NS NS
Yolk weight (g/egg) 15.39 15.06 15.25 15.41 0.14 NS NS NS NS
Albumen weight (g/egg) 40.56 40.19 40.35 40.20 0.37 NS NS NS NS
Egg yolk score 11.38 11.25 11.21 11.18 0.06 NS NS NS NS
Egg width (mm) 44.23 44.13 44.24 44.05 0.12 NS NS NS NS
Egg height (mm) 56.60 56.39 56.67 56.73 0.21 NS NS NS NS
Shape index (%) 78.18 78.29 78.11 77.70 0.25 NS NS NS NS
Egg yolk index 44.88 45.57 45.03 44.04 0.20 <0.05 NS <0.05 NS
Albumen index 8.04 8.66 7.99 7.76 0.20 NS NS NS NS
Haugh unit 79.03 81.56 78.88 78.10 0.78 NS NS NS NS
Breaking strength (kg/cm2) 2.31 2.20 2.01 2.23 0.05 NS NS NS NS
Shell thickness (μm) 390.74 393.95 389.55 387.08 2.01 NS NS NS NS
TABLEII: Laying performance and egg qualities according to the dietary inclusion of pumpkin seed oil in laying hens for 8 weeks. Results are expressed as means SED (standard error of difference between means).
pumpkin seed oil and he has also reported that the yolk weight and the yolk percentage were increased when birds were dietary co-supplemented with the vitamin E (60 IU/kg) and the pumpkin seed oil. However, in the present study, the yolk weight was not significantly affected by inclusion of pumpkin seed oil in the ration. MAHMOUD et al. [18] demonstrated that the egg size was reduced when the egg yolk cholesterol content was decreased by 20-30% in layer hens, and that sometimes, the egg production was completely stopped. In the current study, although it tended to progres-sively declined according to the pumpkin seed oil dose added into the ration, the cholesterolemia was not significantly modified but the yolk cholesterol content was significantly decreased by about 10.4%. The egg dimensions were not significantly influenced by the dietary treatments, nevertheless, the egg production evaluated through the number of eggs during the 8 weeks long period, the egg mass and the daily mean egg weight tended to be lowered in supplemented birds whatever the dose used compared to the controls. No com-parable data could be found in the literature on the effects of dietary inclusion of pumpkin seed oil on egg cholesterol
contents in hens. Our study should be considered as a first pilot study showing the influence of different doses of dietary pumpkin seed oil on the egg cholesterol amount in the laying hens. The data evidenced a significant linear decrease of the biochemical parameter according to the dose of pumpkin seed oil added to the ration. As 95% of yolk total lipids, including cholesterol, derived from triacylglycerol-rich lipo-proteins synthesized in the liver and rapidly transferred into developing yolks from the plasma over a period of several days before ovulation [14], the reduction in egg cholesterol content could be attributed to inhibition of enzyme activities and hepatic cholesterol biosynthesis or to over-expression of lipoprotein receptors in liver by pumpkin seed oil or by its active derived compounds. On the other hand, it was pre-viously demonstrated that pumpkin seed kernels were greatly enriched in phytosterols (265 mg / 100g) [28]. Phytosterols are vegetal compounds which exhibit a similar-cholesterol chemical structure and PHILLIPS et al. [28] have shown that these compounds were able to reduce cholesterolemia as well as the risk for some cancer types and to enhance the immune function. Several theories have been proposed for
Dietary pumpkin seed oil (g/kg) SED P value Significance level of factor effects
0 10 20 40 Linear Quadratic Cubic
Plasma glucose (mmol/L) 15.65 14.50 14.43 15.07 0.19 < 0.05 NS < 0.05 NS
Plasma cholesterol (mmol/L) 3.37 3.17 3.26 2.98 0.18 NS NS NS NS
Plasma triglycerides (g/L) 10.29 10.63 10.44 10.13 0.47 NS NS NS NS
Egg yolk cholesterol content
mg/L 1912.5 1842.7 1789.6 1781.9 31.3 NS NS NS NS
mg / g egg 12.01 11.69 11.32 10.88 0.20 < 0.05 < 0.05 NS NS
TABLEIII: Blood biochemistry and egg yolk cholesterol content according to the dietary inclusion of pumpkin seed oil in laying hens for 8 weeks. Results are expressed as means SED (standard error of difference between means).
NS: not significant.
Dietary pumpkin seed oil (g/kg) SED P value Significance level of factor effects
0 10 20 40 Linear Quadratic Cubic
Fatty acids (%)
Myristic acid 0.27 0.29 0.28 0.31 0.001 NS NS NS NS
Palmitic acid 25.91 25.49 25.75 26.04 0.230 NS NS NS NS
Stearic acid 9.27 8.89 8.56 8.13 0.100 NS NS NS NS
Behenic acid 1.75 1.75 1.77 1.92 0.031 < 0.05 NS < 0.05 NS
Total saturated acids 37.20 36.41 36.36 36.40 0.311 NS NS NS NS
Oleic acid 39.47 43.62 43.53 44.65 0.320 < 0.01 < 0.01 NS NS
Linoleic acid 22.74 19.36 19.50 18.25 0.267 NS NS NS NS
Linolenic acid 0.23 0.24 0.24 0.26 0.001 < 0.05 < 0.05 NS NS
Hexaenoic acid 0.21 0.19 0.22 0.18 0.001 NS NS NS NS
Lignoseric acid 0.14 0.18 0.15 0.27 0.022 NS NS NS NS
Total unsaturated acids 62.80 63.60 63.64 63.61 0.608 NS NS NS NS
TABLEIV: Fatty acid composition of the egg yolk according to the dietary inclusion of pumpkin seed oil in laying hens for 8 weeks. Results are expressed as means SED (standard error of difference between means).
explaining the hypocholesterolemic effects of the phytosterols. They would decrease the intestinal cholesterol absorption by co-crystallisation with the cholesterol in the gastrointestinal tract forming poorly absorbable mixed crystals [33]. In a second proposed mechanism, as phytosterols are less hydro-soluble than the cholesterol and present a stronger affinity for micelles, they would displace the cholesterol from the intestinal micelles and would be preferentially taken up by the enterocytes. This competition in the micelles prevents the cholesterol to reach the absorption site [6, 24, 33]. Moreover, phytosterols would also directly decrease the hepatic choles-terol biosynthesis by down-regulating the expression of enzymes such as those involved in the terpene synthesis and the 3-hydroxy-3-methyl glutaryl coenzyme A (HMGCoA) reductase, the rate limiting enzyme in the cholesterol synthesis [3]. It is well known that phytosterols are structurally analogous to cholesterol and have been shown to substantially reduce intestinal cholesterol absorption by 30-40%. Phytosterol-enriched foods have been reported to reduce plasma total cholesterol [31] without affecting plasma concentrations of other lipids, i.e. triglycerides or HDL-cholesterol [20]. It could be speculated that pumpkin seed oil, through its phy-tosterol content (265 mg/100g [28]), reduces cholesterol absorption from gut which in turn lowers body pools and enhances synthesis rate throughout the desuppression of cel-lular hydroxy-methylglutaryl-CoA (HMG-CoA) reductase activity [16]. AL-ZUHAIRU et al. [1] demonstrated that the simultaneous administration of pumpkin seed oil and of inhi-bitor drugs of the HMGCoA reductase has markedly reduced the cholesterolemia in hypercholesterolemic rabbits. Pumpkin seed oil has also been reported to contain vitamin B15, also called pangamic acid [15]. It was previously shown that vita-min B15 caused a decrease of the cholesterol contents in the stomach wall, the intestines and the liver in rabbits [23]. In accordance with that, it was observed in the present study that the cholesterolemia gradually decreased in a dose-dependent manner in hens fed with standard layer diets containing pumpkin seed oil, although the variations were not statistically significant. The present study indicates that some compounds from the pumpkin seed oil may act as moderate hypocholesterolemic agents for blood and also egg yolk.
A possible mechanism for pumpkin seed oil’s anti-hyper-cholesterolemic effect was related to the high enrichment with unsaturated fatty acids [1]. Because unsaturated fatty acids occupy a greater area than saturated acids, they alter the spatial configuration of the lipids into which they are incorporated; as a result, fewer lipid molecules can be accommodated by the apolipoproteins of the low-density lipoproteins (LDL), and consequently the lipid content of the lipoprotein is lowered [32]. It could therefore be speculated that the improvements with egg cholesterol may be attributed to pumpkin seed oil’s unsaturated fatty acid composition on cholesterol metabolism and to its high phytosterol content [28] which is known to reduce cholesterol concentrations in blood and egg yolk [31].
The results of the present study showed that dietary inclusion of pumpkin seed oil has also affected the composition in fatty acids of the egg yolk. Firstly, the proportions of saturated acids were poorly affected but the percentage of the saturated
long chain docosanoic acid, the behenic acid (C22:0) in egg yolk was markedly increased when hens received the diets containing 4% pumpkin seed oil. The behenic acid is known as a cholesterol-raising fatty acid in humans [7]. However, the behenic acid is poorly absorbed [9] and because of its chain length and its low bioavailability compared with other fatty acids (for example, palmitic acid), it has been speculated that behenic acid does not significantly affect the egg yolk cholesterol concentrations in laying hens. Secondly, it was observed that egg yolks were enriched in unsaturated acids, particularly in oleic and linolenic (C18:3, Δ9, 12, 15) acids when pumpkin seed oil was included to the ration instead of the soya oil. By contrast, the linoleic acid (C18:2, Δ9, 12) percentage has tended to decrease according to the added doses. In agreement with that, Pal [25] reported that the die-tary inclusion of pumpkin seed oil increased the ratio of n-6 to n-3 PUFA. The dietary inclusion of pumpkin seed oil in layer hens induced a decrease in the total monounsaturated fatty acids and an increase of the total polyunsaturated fatty acids with a strengthening of the n-3 fatty acids [26]. The effect of the pumpkin seed oil on the fatty acid composition of the egg yolk would be directly devoted to its great amount in unsaturated acids (more than 70% of fatty acid composition) [1] and the main fatty acids of pumpkin seed oil belongs to the n-6 type polyunsaturated linoleic acid (18:2n-6) like in corn and sunflower oils [26]. As PUFA are largely susceptible to oxidative damage due to high number of double bounds in the molecules [34], the high amount of unsaturated acids probably reflect the occurrence of some protection mecha-nisms of the fatty acids from oxidation and an increased oxi-dative stability of the egg yolk from pumpkin seed oil sup-plemented hens.
Increasing the level of the dietary pumpkin seed oil inclusion has caused a quadratic change (P < 0.05) in glycaemia whereas the plasma triglyceride concentration was unchanged. PAL [25] has previously reported that, 60 minutes after the injection of glucagon, the glycaemia was higher in hens supplemented with pumpkin seed oil than in hens not supplemented or sup-plemented with cod liver oil whereas the plasma triglyceride and lipoprotein concentrations were not modified. Hens fed with diets containing 4% pumpkin seed oil, with a high level of linoleic acid (C18:2, n-6) [27] showed prolonged plasma glucose response to the glucagon administration compared to hens fed with low-fat control diet or cod liver oil diets [27]. Diets containing n-6 PUFA-rich oils seem to improve the glucagon sensitivity and to alter the insulin responsiveness.
As a conclusion, the pumpkin seed oil can replace soya oil in the diet of laying hens at 1%, 2% and 4% without decreasing the performance parameters and the egg quality parameters are not changed by inclusion of pumpkin seed oil into the ration, except for the yolk index which is increased. The yolk composition is modified: the cholesterol content (mg/g egg) linearly decreases after 8 weeks long supplementation and the proportions of unsaturated fatty acids (particularly of n-3 and n-9 unsaturated acids) increase. Furthermore, the dietary pumpkin seed oil inclusion directly and indirectly affects the studies are required for identifying the active compounds and the involved mechanisms, particularly on the cholesterol bio-synthesis and on the hormone (glucagon, insulin) secretion.
Acknowledgements
The authors are grateful to the Scientific and Technical Research Council of Turkey (TUBITAK, 106O066) and Çukurova University Research Fund for their financial support.
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