DOI: 10.1111/jpn.12158
ORIGINAL ARTICLE
The metabolic profile of growing lambs fed diets rich in unsaturated fatty acids jtowski1, M. Stanisz1, M. Szumacher-Strabel3, G. Czyz_ ak-Runowska2, R. Steppa1, K. Szkudelska2, J. Wo 3 A. Cieslak , M. Markiewicz-Kez szycka4 and M. Pietrzak2 University of Life 1 Department of Small Mammal Breeding and Raw Materials of Animal Origin, Faculty of Animal Breeding and Biology, Poznan Sciences, Złotniki, Poland University of Life Sciences, Poznan , Poland 2 Department of Animal Physiology and Biochemistry, Faculty of Animal Breeding and Biology, Poznan University of Life Sciences, Poznan , Poland, 3 Department of Animal Nutrition and Feed Management, Faculty of Animal Breeding and Biology, Poznan and 4 Department of Animal Sciences, Institute of Genetics and Animal Breeding of the Polish Academy of Sciences, Jastrzez biec, Poland
Summary The effect of two diets enriched with unsaturated fatty acids – one containing the addition of dried distillers grains with solubles (DGS) and the other the addition of false flax – Camelina sativa cake (CS) – on some metabolic parameters and hormone concentration in growing lambs was determined in this experiment. A total of 21 ram lambs of the Polish Whiteheaded mutton sheep were divided into three groups (the control, receiving DGS and CS). The diets were administered to animals for 6 weeks. During the experiment, peripheral blood was collected. Glucose (GL), total cholesterol (CH), triglycerides (TG), free fatty acids (FFA), insulin (IN), leptin (LE), triiodothyronine (T3) and thyroxine (T4) were assayed in serum. The age-dependent reduction in CH and TG limited by both experimental diets were observed. A significant increase in FFA concentration was observed in samples collected in the last, that is, third, time period. This was most probably caused by a 12-h pre-slaughter fasting period. A significantly lower dynamic of FFA increase in that experimental period was found in animals receiving the experimental feed. Insulin concentration in DGS-receiving lambs was increased, in contrast to the CS-receiving lambs, in which it was lower when compared to the control. LE concentration was decreased by both experimental diets, more markedly in the DGS-receiving animals. No differences between the experimental groups and the control were observed in T3 and T4 concentrations. The effect of 12-h pre-slaughter fasting was statistically highly significant for the levels of examined blood markers and hormones, except for TG and IN in the group of lambs receiving the experimental diet with CS. Keywords lambs, hormone, fatty acids, metabolic profile, feeding jtowski, Department of Small Mammal Breeding and Raw Materials of Animal Origin, Faculty of Animal Breeding and Biology, Correspondence J. Wo University of Life Sciences, Złotniki, 62-002 Suchy Las, Poland. Tel/Fax: +48 61 812 55 20; E-mail: [email protected] Poznan Received: 26 October 2013; accepted: 4 December 2013
Increasing interest may be observed worldwide in the so-called functional food, containing components having a health-promoting (probiotic) effect on the human organism (Pikul et al., 2008; van Arendonk, 2011; Markiewicz-Kez szycka et al., 2013). A diet with excessive fat contents contributes to the development of many civilization-related diseases, such as obesity, diabetes and cardiovascular diseases. Both Camelina sativa cake (CS) and dried distillers grains with solubles (DGS) contain unsaturated fatty acids, being potential
good sources of energy in the feed ration, and may influence metabolic changes in the organism, at the same time improving the composition of fatty acids in animal products (Cais-Sokoli nska et al., 2011; Szumacher-Strabel et al., 2011). Although some studies have been published indicating an advantageous effect of administration of the above-mentioned feed additives on the fat composition of products of animal origin (Szumacher-Strabel et al., 2011), there are no available papers discussing their effect on the metabolic profiles of animals. The aim of this study was to investigate the effect of the two different experimental
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Introduction
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diets, rich in unsaturated fatty acids, on selected parameters of the metabolic profile in growing slaughter lambs. Material and methods
intestine per day. Throughout the fattening period, lambs had free access to water. Fattening started when lambs were 66 1 days old at body weight of 24.6 0.84 kg. After a 6-week fattening period, lambs were slaughtered at the age of 110 1 days and at average body weight of 38.2 0.73 kg.
Animals and study design
Fattening was performed on a total of 21 ram lambs of the Polish Whiteheaded mutton sheep (Barczak et al., 2009). Within a week from weaning, lambs were divided into three groups, two experimental and one control, comprising seven animals each, placed into 21 individual box feeders and administered varied feeding regimes. The components differentiating concentrates included contents of rapeseed meal as well as the addition of dried distillers grains with solubles (DGS) or the addition of Camelina sativa cake (CS), Table 1. Every day of the fattening, period lambs were also fed ad libitum alfalfa/grass silage of the following basic composition (in%): dry matter 51.26, crude ash 7.42, crude protein 8.14, crude fibre 11.31 and crude fat 1.46. The diets were formulated using the INRA (1993) system to meet the animals’ nutrient requirements, average for 35 kg of body weight and 400 g/ day of growth: 1.18 unit for meat production per day and 139 g protein truly digestible in the small
Experimental protocol
*UFM – unit for meat production (1820 kcal EN) per kg of d.m. †PDIN – protein truly digestible in the small intestine depending on NH3 N amount per kg of d.m. ‡PDIE – protein truly digestible in the small intestine depending on energy amount per kg of d.m.
Samples of concentrates and alfalfa/grass silage were collected every week and analysed according to the AOAC (2007) for dry matter (DM; method no. 934.01), ash (AS; method no. 942.05) and crude fibre (method no. 978.10). Crude protein (CP) was determined with the use of a Kjel-Foss Automatic 16210 analyzer (A/SN. Foss Electric, Hillerød, Denmark; method no. 976.05), while crude fat (CF) was tested with a Soxtec System HT analyzer (Tecator AB, Horanas, Sweden; method no. 973.18). NDF was determined by the method of van Soest et al. (1991). Organic matter was calculated from the difference between dry matter and ash. The fatty acid composition of CS and DGS after extraction was determined in a gas chromatograph (GS; Varian Star CP 3800; Varian Analytical Instruments, Walnut Creek, CA, USA) equipped with a flame ionization detector and a silica capillary column CP-Sil 88 (100 m 9 0.25 mm id, 0.20 lm film thickness; Chrompack, Varian, Middleburg, the Netherlands), according to the protocol described by Cieslak et al. (2009). Hydrogen was used as a carrier gas at a constant flow of 30.0 ml/min and a split ratio of 1:10. The oven temperature was programmed as follows: initially 175 °C for 25 min, then increasing at 5 °C/min to 235 °C. The fatty acid peaks were identified by comparison with the retention times of known standards (FAME Mix; Supelco, Belfonte, OK, USA) and according to Collomb and B€ uhler (2000). Fatty acid standards (Supelco) were used to create calibration curves for the quantification of C18:0, C18:1 trans 9, C18:1 cis 9 and C18:2. Peripheral blood was collected from lambs three times, first on the day preceding the beginning of the administration of the experimental feed, the second time at day 21 of fattening and the third time at the day of slaughter, with successive collections denoted with numbers 1, 2 and 3. The first and second blood collections were performed at 3–4 h after the morning feeding time. Blood from the third collection (day of slaughter) was obtained after 12-h pre-slaughter fasting. Blood collected to sterile test tubes was centrifuged at 4000 g for 10 min, and thus, obtained serum was frozen at a temperature of approximately 20 °C.
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Table 1 Ingredients, basic composition and nutritive value of three concentrates Group Ingredient (g/kg of d.m.)
Control
CS 12%
DGS 12%
Wheat Wheat bran Rapeseed extracted meal Camelina sativa cake (CS) Dried distillers grains (DGS) Minerals and vitamins Basic composition (%) Dry matter Organic matter Crude ash Crude protein Crude fibre Crude fat Neutral-detergent fibre Nutritive value UFM* PDIN† [g] PDIE‡ [g]
700 80 200 0 0 20
700 80 80 120 0 20
700 80 80 0 120 20
88.49 83.25 5.24 17.20 5.13 3.46 15.39
91.63 87.50 4.13 17.95 4.83 4.45 15.71
88.60 83.60 5.00 16.20 5.10 2.60 15.84
1.12 115.4 106.8
1.12 116.1 107.4
1.11 103 102
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Concentrations of the following components were determined in blood serum: glucose (GL), cholesterol (CH), triglycerides (TG), free fatty acids (FFA) and hormones: insulin (IN), leptin (LE), triiodothyronineT3 and thyroxine-T4. Concentrations of the T3 and T4 hormones, in contrast to the other components, were determined twice, that is, on the day preceding the administration of the experimental feed and on the day of slaughter. Insulin was determined radioimmunologically (RIA) using a kit specific for the rat hormone (Rat Insulin; Millipore, St. Charles, MO, USA). The specificity of this test to sheep insulin reaches 100%. The intra-assay coefficient was 4.6%. Leptin was also assayed by the RIA method using the Multi-Species Leptin kit (Millipore), which is suitable for sheep plasma. The intra-assay coefficient was 3.6%. Triiodothyronine-T3 and thyroxine-T4 were determined with the use of RIA kits (DIAsourceImmunoAssays S.A., Nivelles, Belgium). The intra-assay coefficients were 5.6% and 3.2% respectively. Glucose was detected photocolorimetrically by means of the enzymatic method with glucose oxidase, peroxidase and o-dianisidine. The free fatty acid concentration in serum was determined according to Duncombe (1964), while triglyceride concentration by the colorimetric Hatzsch condensation method (Foster and Dunn, 1973). Total cholesterol was assayed by the enzymatic method developed by Richmond (1973) with cholesterol esterase and oxidase.
The metabolic profile of growing lambs
Table 2 Concentration of some FA in CS and DGS FA (g 100 g
1
of identified fatty acids)
C16: 0 (palmitic) C18: 1 cis-9 (oleic) C18: 2 cis-9 cis-12 (linoleic; n-6) C18: 3 cis-9 cis-12 cis-15 (linolenic; n-3)
CS
DGS
7.3 16.1 26.2 38.8
13.4 25.8 55.17 1.8
In our experiment, both experimental diets were rich in unsaturated fatty acids. CS and DGS used in this experiment contained, among others, fatty acids shown in the Table 2. Considering the effects of fodder composition on growing lambs, it is very important to take into consideration that some biochemical parameters assayed in the serum may be significantly modified depending
on the time of blood collection. An example of such a parameter may be provided by, for example, glucose concentration. The reducing effect of blood collection time on glucose concentration (Table 3), especially between day 21 and the last day of the study, was observed in the control group and both experimental groups, while no differences were observed between the groups receiving CS and DGS. In ruminants, such a result may be a consequence of the change in the nutrition regime after weaning. The resulting amounts of lipids in blood are observed due to the high (approximately 6%) lipid concentration in ewes’ milk (Ashton et al., 1964). An increased content of lipoproteins was also found in these animals (Kubasek et al., 1974). After this period until approximately day 100 of life, a reduced blood lipid concentration is detected (Leat and Gillman, 1964) and it may be a result of weaning. In our study, this specific agedependent decrease in lipid concentration was also observed – total cholesterol was reduced, especially during the first 3 weeks of the experiment, and triglycerides were being reduced throughout the entire period (6 weeks) of the experiment. The parameters related to lipid metabolism were significantly influenced by the diet composition. In the groups of animals consuming the experimental diets, no increase was found in CS and DGS, or total cholesterol concentration during the second 3 weeks. It was detected in the last day of the study, in comparison with the control group, and the value of this parameter was the highest in the group consuming CS. The level of triglycerides in blood of examined animals varied depending on sampling time. The downward trend for TG concentration with successive sample collection date and thus with the age of animals was observed in animals from the control group. In lambs from both experimental groups following its significant reduction at day 21 of the experiment (p < 0.01), its level in successive days remained on a similar, stable level (p > 0.05). Although the content of polyunsaturated fatty acids (PUFA) in both CS and DGS diets was different, as CS is rich in n-3 PUFA and DGS in n-6 PUFA, this difference was not important concerning the effect on blood cholesterol and triglycerides. However, cholesterol concentration remained slightly
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Statistical analysis
The effect of the experimental feed on concentrations of blood markers and hormones was estimated by the GLM procedure of SAS ver. 9.1 (2000), based on the unitrait linear model, with the sampling time 9 feeding treatment interaction. The results were expressed as the mean standard error of means (SEM). As calculated interactions between blood sample collection and the nutrition factor proved to be non-significant (p > 0.05), they were not included in the table. Results and discussion
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Table 3 Concentration of blood markers and hormones in successive blood samplings (mean SEM) Blood marker/ blood samplings† GL (mM) 1 2 3 CH (mM) 1 2 3 TG (mM) 1 2 3 FFA (mM) 1 2 3 IN (ng/ml) 1 2 3 LE (ng/ml) 1 2 3 T3 (nM) 1 3 T4 (nM) 1 3
Group Control (C)
CS
DGS
Significance of differences between groups
4.56 0.18 a 4.40 0.18 4.08 0.18 b
4.54 0.18 A 4.64 0.18 A 3.90 0.18 B
4.34 0.18 4.71 0.18 A 4.09 0.18 B
2.28 0.16 B 1.47 0.16 A 1.46 0.16 A
2.43 0.16 Aa 1.59 0.16 B 1.91 0.16 b
2.54 0.16 A 1.09 0.16 B 1.72 0.16 C
CS-DGS* C-CS*
3.38 0.13 A 2.76 0.13 B 2.22 0.13 C
3.17 0.13 B 2.53 0.13 A 2.65 0.13 A
3.13 0.13 A 2.65 0.13 B 2.62 0.13 B
C-CS*, C-DGS*
0.17 0.03 A 0.14 0.03 A 0.90 0.03 B
0.16 0.03 A 0.17 0.03 A 0.56 0.03 B
0.18 0.03 A 0.17 0.03 A 0.67 0.03 B
C-CS**, C-DGS**, CS-DGS*
0.72 0.09 a 0.96 0.09 Ab 0.66 0.09 B
0.67 0.09 0.62 0.09 0.53 0.09
0.65 0.09 Aa 1.19 0.09 Ba 0.91 0.09 b
C-CS**, CS-DGS**’ C-DGS* CS-DGS**, C-DGS*
1.35 0.09 1.40 0.09 1.18 0.09
1.29 0.09 A 1.22 0.09 A 0.82 0.09 B
1.44 0.09 A 1.42 0.09 A 0.42 0.09 B
C-CS**, C-DGS**, CS-DGS**
2.49 0.13 A 1.67 0.13 B
2.78 0.13 A 1.64 0.13 B
2.81 0.13 A 1.64 0.13 B
82.26 6.13 94.03 6.13
88.68 6.13 105.10 6.13
97.18 6.13 100.52 6.13
GL, glucose; CH, cholesterol; TG, triglycerides; FFA, free fatty acids; IN, insulin; LE, leptin; T3, triiodothyronine; and T4, thyroxine. In columns, AB (ab) values designated with different capital (small) letters differ significantly at p ≤ 0.01 (p ≤ 0.05). In rows, **p ≤ 0.01, *p ≤ 0.05. †1 – day of the beginning of administration of experimental feed; 2–21st day of fattening; 3–42nd day of fattening (day of slaughter).
increased after consumption of the n-3 PUFA-rich CS diet in comparison with DGS. Other experiments revealed that depending on the source of unsaturated fatty acids, their effect on lipid parameters may vary. In their review, Roche and Gibney (2000) described (although not in lambs) that n-3 polyunsaturated fatty acids (PUFAs) from fish oil reduced plasma triglyceride concentrations. In the experiments performed by Ponnampalam et al. (2001), total cholesterol and HDL-cholesterol levels were greater when compared to the control in lambs receiving protected sunflower meal, but they were lower in lambs consuming n-3 PUFA-rich fish oil or fish oil with protected sunflower meal. In the same study, no alterations in plasma triglycerides were detected. Unlike cholesterol and triglycerides, an opposite effect of blood collection time on free fatty acid (FFA)
concentrations was observed in the performed experiment. This parameter was not altered during the first 3 weeks, but a marked increase was observed in the second period of the study in all groups of animals, which could have been the effect of 12-h preslaughter fasting of animals. It is difficult to explain this effect as there are limited literature data concerning this issue. Lindsay and Leat (1975) reported that FFA are not altered depending on age in sheep, while Nelson (1973) gathered that the reason of this effect in sheep could be connected with the absorption of lipids as fatty acids. On the other hand, the increase in fatty acid concentrations may be due to the change of the manner of digestion from non-ruminant to ruminant. Holcombe et al. (1992) studied the level of nonesterified fatty acids (NEFA) in serum of lambs weaned at 42 days of age and observed a twofold
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increase in this parameter 1 day after weaning. Moreover, NEFA remained significantly increased for the first week after weaning (Holcombe et al., 1992), which what was probably connected with the response of animals to decreased energy intake. However, in our experiment in the groups of animals receiving the CS and DGS diets, the increase in FFA was substantially limited when compared to the control. This effect was probably related to the presence of unsaturated fatty acids in the experimental diets. Moreover, the CS diet, rich in n-3 PUFA, exerted a stronger diminishing effect on plasma FFA concentration in lambs in comparison with that of DGS. It is obvious that n-3 PUFA may increase the activity of adiponectin, which can stimulate fatty acid oxidation by muscle cells (Tishinsky et al., 2012). Moreover, it was established by Priore et al. (2012) that n-3 PUFA from fish oil increases the expression of carnitine/acylcarnitine translocase – a pivotal protein responsible for fatty acid transport to mitochondria, where they are oxidized. Recapitulating, the results obtained in our experiment suggest that in weaned ruminants, thus also lambs, we can observe age-dependent changes in blood lipids, that is, total cholesterol and triglycerides, and their increased utilization accompanied by reduced concentrations in blood. Reduced glycemia also appears in growing animals due to the change in their nutrition and metabolism. The applied experimental diets, that is, CS, rich in PUFA n-3 and DGS, rich in PUFA n-6, alleviate the decrease in blood lipid levels, but have no effect on glycemia. In agreement with our results, Cieslak et al. (2003) revealed that supplementation of lambs’ diets with components rich in unsaturated fatty acids may increase the energy value of the diet without causing significant changes in blood parameters. It is concluded that an additional source of energy containing polyunsaturated fatty acids does not cause metabolic disorders. As Camelina sativa cake is the source of primarily n-3 linolenic acid, whereas dried distillers grains with solubles of n-6 linolenic, and n-6 linoleic acid is converted to arachidonic acid, a precursor for the 2 series of prostaglandins, whereas n-3 linolenic acid is converted to eicosapentaenoic acid, a precursor of the 3-antagonistic series of prostaglandins, the mode of action on the evaluated blood parameters does not change. Apart from metabolic parameters, blood concentrations of some hormones regulating metabolism, that is, insulin, leptin, T3 and T4, were also assayed in the performed experiment (Table 3). The first 21 days of the experiment had a significant effect on an increase in the concentration of insulin in blood serum of
lambs from the C and DGS groups. Its concentration in animals from the above-mentioned groups after 12h pre-slaughter fasting (blond samplings 3) was next reduced by 0.30 and 0.28 ng/mol (p < 0.01 and p < 0.05) respectively. In contrast, no effect of feeding was found on IN level in lambs from group CS (p > 0.05). However, insulin concentration in the group receiving DGS, increased after the first 3 weeks of the study, decreased until the end of the experiment, but it was still substantially increased in comparison with the residual groups. This effect seemed to be partial due to glucose concentration in the DGS group, which was increased after the first 3 weeks of the experiment and was decreasing until the last day, but in the case of glucose, no differences were observed between the experimental groups and the control. In relation to the content of both diets consumed by lambs, it may be suggested that probably n6 PUFA could be responsible for the observed effect on the hormone concentration in blood. The literature data describing differences in blood hormone concentration after consumption of a diet rich in n-6 PUFA in ruminants are rather scanty, but there are more data concerning the effects of an n-3 PUFA-rich diet. In the experiment performed on growing lambs by Kim et al. (2007), the dietary n-6:n-3 fatty acid ratio had no effect on concentration of either IGF-1 or insulin in plasma in these animals. As it was shown by Ponnampalam et al. (2001), feeding lambs with a diet supplemented with fish oil as a source of n-3 PUFA resulted in a significant reduction in the diet-induced increase in insulin concentration when compared to the control. Baur et al. (1998) established that in in vivo and in vitro experiments on humans and rats, unsaturated fatty acids supplemented in the diet were incorporated to the muscle membranes, they improved insulin action and the binding capacity of the hormone. In our experiment, this hypothesis could be viable for lambs receiving the n-3 PUFA-rich CS diet, as insulin concentration in this group was lower when compared to the control throughout the experiment. Still no explanation was provided why insulin concentration was greater in the DGS-receiving animals, but the greater content of n-6 PUFA and the diminished n-3 PUFA content in the DGS diet could probably be due to this effect. The age of lambs had no effect on leptin concentration, as it was observed in the control group during the 6 weeks of the experiment. However, the composition of the diet had an important effect on leptin concentration in the last period of the study, as both experimental diets, that is, CS and DGS, significantly decreased hormone concentration. Blood leptin
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concentration can be dependent on both adiposity and nutrition, although Ehrhardt et al. (2003) established that in the early post-natal life of lambs the effects of nutrition are predominant. There is another explanation for the reduced leptin concentration – this effect may result from a decreased insulin concentration, as insulin is an important physiological stimulator of leptin secretion in ruminants (Chilliard et al., 2001). However, in our experiment, this supposition should be excluded, as in the DGS group, the highest concentration of insulin was assayed after slaughter when compared to the control or the CS group. The data describing the effects of PUFA content on leptin plasma concentration in other animals are also insufficient, as it was reported that in rats, n-3 with PUFA supplementation of the high-fat diet the plasma leptin concentration was lower, similarly as the leptin mRNA in adipose tissue (Ukropec et al., 2003). In our experiment, the n-3 PUFA-rich CS diet induced a decrease in plasma leptin concentration. The concentration of T3 decreased with age from the first to the last day of the study, but no significant alterations were induced depending on diet composition. There were no age-related changes and no effect of diet composition on T4 concentration in blood. The age-related changes in T3 concentration observed in our study are in accordance with literature data, where plasma T3 levels in lambs after feeding were higher in lambs in the first days of life and they decreased with age (Symonds et al., 1989). The lack of differences between the experimental groups and the control makes it possible for us to conclude that the alleviating effect on metabolic parameters, observed in blood after the consumption of both experimental diets, that is, CS and DGS, is not a consequence of changes in the thyroid hormone concentration and probably they are not related to changes of metabolic rates in the experimental animals. References van Arendonk, J. A. M., 2011: Opportunities for animal breeding to meet the challenges of the future. Nauka Przyroda Technologie 5, 3 #30. Ashton, W. M.; Owen, J. B.; Ingleton, J. W., 1964: A study of the composition of Clun Forest ewe’s milk. Journal of the Science of Food and Agriculture 63, 85–89. Association of Official Analytical Chemists, 2007: Official Methods of Analysis. In: W. Horwitz, W. Latimer (eds), 18th edn. AOAC, Gaithersburg, MD, pp. 27–31.
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Conclusion Summing up the presented study, it may be stated that the administration of feed mixtures containing CS and DGS in fattening of lambs had an effect on the levels of blood metabolic indexes and hormones. The applied experimental diets alleviate the decrease in blood lipid levels, but have no effect on glycemia. Preslaughter fasting had an important effect on leptin concentration in the experimental groups, as both experimental diets, that is, CS and DGS, in contrast to the diet of the control (C), significantly decreased hormone concentration. The age-dependent reduction in CH and TG limited by both experimental diets was observed. The IN concentration in DGS-receiving lambs was increased, in contrast to CS-receiving lambs, which was lower in this group compared to the control. The LE concentration was decreased by both experimental diets, more markedly in DGS-receiving animals. The effect of 12-h pre-slaughter fasting was statistically highly significant for the level of analysed blood markers and hormones, except for TG and IN in the group of lambs receiving experimental diets with CS. Acknowledgements This research was conducted under the Project ‘Biofood – innovative, functional animal products’, financed by the Polish Ministry of Economy within the European Regional Development Fund 2007– 2013, Operating Program Innovative Economy no. POIG 01.01.02-014-090/09. Conflict of interest The authors have no conflict of interest to declare whatsoever.
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Kubasek, F. O. T.; Leat, W. M. F.; Buttress, N., 1974: Plasma lipoproteins of the suckling lamb compared with adult sheep. Proceedings of the Nutrition Society 33, 47A–48A. Leat, W. M. F.; Gillman, T.; 1964: Intestinal absorption and clearance of lipids in ruminants and non-ruminants. In: R. M. C. Dawson, D. N. Rhodes (eds.), Metabolism and Physiological Significance of Lipids. John Wiley and Sons Ltd, London, pp. 257. Lindsay, D. B., Leat, W. M. F., 1975: Carbohydrate and lipid metabolism. In: M. H. Blunt (ed.), The Blood of Sheep. Composition and Function. Springer-Verlag, Berlin-Heidelberg-New York, pp. 45–62. Markiewicz-Kez szycka, M.; W ojtowski, J.; Kuczy nska, B.; Puppel, K.; Czy_zak-Runowska, G.; Bagnicka, E.; Strzałkowska, N.; J ozwik, A.; Krzy_zewski, J., 2013: Chemical composition and whey protein fraction of late lactation mare milk. International Dairy Journal 31, 62–64. Nelson, G. J., 1973: – The lipid composition of plasma lipoprotein density classes of sheep, Ovis aries. Comparative Biochemistry and Physiology 46B, 81–91. Pikul, J.; W ojtowski, J.; Dank ow, R.; Kuczy nska, B.; Łojek, J., 2008: Fat content and fatty acids profile of colostrum and milk of primitive Konik horses (Equus caballus gmelini Ant.) during six months of lactation. Journal of Dairy Research 75, 302–309. Ponnampalam, E. N.; Sinclair, A. J.; Egan, A. R.; Blakeley, S. J.; Li, D.; Leury, B. J., 2001: Effect of dietary modification of muscle long-chain n-3 fatty acid on plasma insulin and lipid metabolites, carcass traits, and fat deposition in lambs. Journal of Animal Science 79, 895–903. Priore, P.; Stanca, E.; Gnoni, G. V.; Siculella, L., 2012: Dietary fat types differently modulate the activity and expression of mitochondrial carnitine/acylcarnitine translocase in rat liver. Biochimica et Biophysica Acta 1821, 1341– 1349.
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Richmond, W., 1973: Preparation and properties of cholesterol oxidase from Nocardia sp. and application to the enzymatic assay of total cholesterol in serum. Clinical Chemistry 19, 1350–1356. Roche, H. M.; Gibney, M. J., 2000: Effect of long-chain n-3 polyunsaturated fatty acids on fasting and postprandial triacylglycerol metabolism. American Journal of Clinical Nutrition 71(1 Suppl), 232S– 237S. SAS. 2000: SASâ Users Guide, Version 9.1, SAS Institute Inc., Cary, NC. van Soest, P. J.; Robertson, J. B.; Leis, B. A., 1991: – Methods for dietary fiber, neutral detergent fiber, and non-starch polysaccarides in relation to animal nutrition. Journal of Dairy Science 74, 3583–3597. Symonds, M. E.; Andrews, D. C.; Johnson, P., 1989: The endocrine and metabolic response to feeding in the developing lamb. Journal of Endocrinology 123, 295– 302. Szumacher-Strabel, M.; Cieslak, A.; Zmora, P.; Pers-Kamczyc, E.; Bieli nska, S.; Stanisz, M.; W ojtowski, J., 2011: Camelina sativa cake improved unsaturated fatty acids in ewe’s milk. Journal of the Science of Food and Agriculture 91, 2031–2037. Tishinsky, J. M.; Gulli, R. A.; Mullen, K. L.; Dyck, D. L.; Robinson, L. E., 2012: Fish oil prevents high-saturated fat dietinduced impairments in adiponectin and insulin response in rodent soleus muscle. American Journal of PhysiologyRegulatory Integrative and Comparative Physiology 302, R598–R605. Ukropec, J.; Reseland, J. E.; Gasperikova, D.; Demcakova, E.; Madsen, L.; Berge, R. K.; Rustan, A. C.; Klimes, I.; Drevon, C. A.; Seb€ okova, E., 2003: The hypotriglyceridemic effect of dietary n-3 FA is associated with increased beta-oxidation and reduced leptin expression. Lipids 38, 1023–1029.
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ORIGINAL ARTICLE
The metabolic profile of growing lambs fed diets rich in unsaturated fatty acids jtowski1, M. Stanisz1, M. Szumacher-Strabel3, G. Czyz_ ak-Runowska2, R. Steppa1, K. Szkudelska2, J. Wo 3 A. Cieslak , M. Markiewicz-Kez szycka4 and M. Pietrzak2 University of Life 1 Department of Small Mammal Breeding and Raw Materials of Animal Origin, Faculty of Animal Breeding and Biology, Poznan Sciences, Złotniki, Poland University of Life Sciences, Poznan , Poland 2 Department of Animal Physiology and Biochemistry, Faculty of Animal Breeding and Biology, Poznan University of Life Sciences, Poznan , Poland, 3 Department of Animal Nutrition and Feed Management, Faculty of Animal Breeding and Biology, Poznan and 4 Department of Animal Sciences, Institute of Genetics and Animal Breeding of the Polish Academy of Sciences, Jastrzez biec, Poland
Summary The effect of two diets enriched with unsaturated fatty acids – one containing the addition of dried distillers grains with solubles (DGS) and the other the addition of false flax – Camelina sativa cake (CS) – on some metabolic parameters and hormone concentration in growing lambs was determined in this experiment. A total of 21 ram lambs of the Polish Whiteheaded mutton sheep were divided into three groups (the control, receiving DGS and CS). The diets were administered to animals for 6 weeks. During the experiment, peripheral blood was collected. Glucose (GL), total cholesterol (CH), triglycerides (TG), free fatty acids (FFA), insulin (IN), leptin (LE), triiodothyronine (T3) and thyroxine (T4) were assayed in serum. The age-dependent reduction in CH and TG limited by both experimental diets were observed. A significant increase in FFA concentration was observed in samples collected in the last, that is, third, time period. This was most probably caused by a 12-h pre-slaughter fasting period. A significantly lower dynamic of FFA increase in that experimental period was found in animals receiving the experimental feed. Insulin concentration in DGS-receiving lambs was increased, in contrast to the CS-receiving lambs, in which it was lower when compared to the control. LE concentration was decreased by both experimental diets, more markedly in the DGS-receiving animals. No differences between the experimental groups and the control were observed in T3 and T4 concentrations. The effect of 12-h pre-slaughter fasting was statistically highly significant for the levels of examined blood markers and hormones, except for TG and IN in the group of lambs receiving the experimental diet with CS. Keywords lambs, hormone, fatty acids, metabolic profile, feeding jtowski, Department of Small Mammal Breeding and Raw Materials of Animal Origin, Faculty of Animal Breeding and Biology, Correspondence J. Wo University of Life Sciences, Złotniki, 62-002 Suchy Las, Poland. Tel/Fax: +48 61 812 55 20; E-mail: [email protected] Poznan Received: 26 October 2013; accepted: 4 December 2013
Increasing interest may be observed worldwide in the so-called functional food, containing components having a health-promoting (probiotic) effect on the human organism (Pikul et al., 2008; van Arendonk, 2011; Markiewicz-Kez szycka et al., 2013). A diet with excessive fat contents contributes to the development of many civilization-related diseases, such as obesity, diabetes and cardiovascular diseases. Both Camelina sativa cake (CS) and dried distillers grains with solubles (DGS) contain unsaturated fatty acids, being potential
good sources of energy in the feed ration, and may influence metabolic changes in the organism, at the same time improving the composition of fatty acids in animal products (Cais-Sokoli nska et al., 2011; Szumacher-Strabel et al., 2011). Although some studies have been published indicating an advantageous effect of administration of the above-mentioned feed additives on the fat composition of products of animal origin (Szumacher-Strabel et al., 2011), there are no available papers discussing their effect on the metabolic profiles of animals. The aim of this study was to investigate the effect of the two different experimental
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Introduction
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diets, rich in unsaturated fatty acids, on selected parameters of the metabolic profile in growing slaughter lambs. Material and methods
intestine per day. Throughout the fattening period, lambs had free access to water. Fattening started when lambs were 66 1 days old at body weight of 24.6 0.84 kg. After a 6-week fattening period, lambs were slaughtered at the age of 110 1 days and at average body weight of 38.2 0.73 kg.
Animals and study design
Fattening was performed on a total of 21 ram lambs of the Polish Whiteheaded mutton sheep (Barczak et al., 2009). Within a week from weaning, lambs were divided into three groups, two experimental and one control, comprising seven animals each, placed into 21 individual box feeders and administered varied feeding regimes. The components differentiating concentrates included contents of rapeseed meal as well as the addition of dried distillers grains with solubles (DGS) or the addition of Camelina sativa cake (CS), Table 1. Every day of the fattening, period lambs were also fed ad libitum alfalfa/grass silage of the following basic composition (in%): dry matter 51.26, crude ash 7.42, crude protein 8.14, crude fibre 11.31 and crude fat 1.46. The diets were formulated using the INRA (1993) system to meet the animals’ nutrient requirements, average for 35 kg of body weight and 400 g/ day of growth: 1.18 unit for meat production per day and 139 g protein truly digestible in the small
Experimental protocol
*UFM – unit for meat production (1820 kcal EN) per kg of d.m. †PDIN – protein truly digestible in the small intestine depending on NH3 N amount per kg of d.m. ‡PDIE – protein truly digestible in the small intestine depending on energy amount per kg of d.m.
Samples of concentrates and alfalfa/grass silage were collected every week and analysed according to the AOAC (2007) for dry matter (DM; method no. 934.01), ash (AS; method no. 942.05) and crude fibre (method no. 978.10). Crude protein (CP) was determined with the use of a Kjel-Foss Automatic 16210 analyzer (A/SN. Foss Electric, Hillerød, Denmark; method no. 976.05), while crude fat (CF) was tested with a Soxtec System HT analyzer (Tecator AB, Horanas, Sweden; method no. 973.18). NDF was determined by the method of van Soest et al. (1991). Organic matter was calculated from the difference between dry matter and ash. The fatty acid composition of CS and DGS after extraction was determined in a gas chromatograph (GS; Varian Star CP 3800; Varian Analytical Instruments, Walnut Creek, CA, USA) equipped with a flame ionization detector and a silica capillary column CP-Sil 88 (100 m 9 0.25 mm id, 0.20 lm film thickness; Chrompack, Varian, Middleburg, the Netherlands), according to the protocol described by Cieslak et al. (2009). Hydrogen was used as a carrier gas at a constant flow of 30.0 ml/min and a split ratio of 1:10. The oven temperature was programmed as follows: initially 175 °C for 25 min, then increasing at 5 °C/min to 235 °C. The fatty acid peaks were identified by comparison with the retention times of known standards (FAME Mix; Supelco, Belfonte, OK, USA) and according to Collomb and B€ uhler (2000). Fatty acid standards (Supelco) were used to create calibration curves for the quantification of C18:0, C18:1 trans 9, C18:1 cis 9 and C18:2. Peripheral blood was collected from lambs three times, first on the day preceding the beginning of the administration of the experimental feed, the second time at day 21 of fattening and the third time at the day of slaughter, with successive collections denoted with numbers 1, 2 and 3. The first and second blood collections were performed at 3–4 h after the morning feeding time. Blood from the third collection (day of slaughter) was obtained after 12-h pre-slaughter fasting. Blood collected to sterile test tubes was centrifuged at 4000 g for 10 min, and thus, obtained serum was frozen at a temperature of approximately 20 °C.
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Table 1 Ingredients, basic composition and nutritive value of three concentrates Group Ingredient (g/kg of d.m.)
Control
CS 12%
DGS 12%
Wheat Wheat bran Rapeseed extracted meal Camelina sativa cake (CS) Dried distillers grains (DGS) Minerals and vitamins Basic composition (%) Dry matter Organic matter Crude ash Crude protein Crude fibre Crude fat Neutral-detergent fibre Nutritive value UFM* PDIN† [g] PDIE‡ [g]
700 80 200 0 0 20
700 80 80 120 0 20
700 80 80 0 120 20
88.49 83.25 5.24 17.20 5.13 3.46 15.39
91.63 87.50 4.13 17.95 4.83 4.45 15.71
88.60 83.60 5.00 16.20 5.10 2.60 15.84
1.12 115.4 106.8
1.12 116.1 107.4
1.11 103 102
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Concentrations of the following components were determined in blood serum: glucose (GL), cholesterol (CH), triglycerides (TG), free fatty acids (FFA) and hormones: insulin (IN), leptin (LE), triiodothyronineT3 and thyroxine-T4. Concentrations of the T3 and T4 hormones, in contrast to the other components, were determined twice, that is, on the day preceding the administration of the experimental feed and on the day of slaughter. Insulin was determined radioimmunologically (RIA) using a kit specific for the rat hormone (Rat Insulin; Millipore, St. Charles, MO, USA). The specificity of this test to sheep insulin reaches 100%. The intra-assay coefficient was 4.6%. Leptin was also assayed by the RIA method using the Multi-Species Leptin kit (Millipore), which is suitable for sheep plasma. The intra-assay coefficient was 3.6%. Triiodothyronine-T3 and thyroxine-T4 were determined with the use of RIA kits (DIAsourceImmunoAssays S.A., Nivelles, Belgium). The intra-assay coefficients were 5.6% and 3.2% respectively. Glucose was detected photocolorimetrically by means of the enzymatic method with glucose oxidase, peroxidase and o-dianisidine. The free fatty acid concentration in serum was determined according to Duncombe (1964), while triglyceride concentration by the colorimetric Hatzsch condensation method (Foster and Dunn, 1973). Total cholesterol was assayed by the enzymatic method developed by Richmond (1973) with cholesterol esterase and oxidase.
The metabolic profile of growing lambs
Table 2 Concentration of some FA in CS and DGS FA (g 100 g
1
of identified fatty acids)
C16: 0 (palmitic) C18: 1 cis-9 (oleic) C18: 2 cis-9 cis-12 (linoleic; n-6) C18: 3 cis-9 cis-12 cis-15 (linolenic; n-3)
CS
DGS
7.3 16.1 26.2 38.8
13.4 25.8 55.17 1.8
In our experiment, both experimental diets were rich in unsaturated fatty acids. CS and DGS used in this experiment contained, among others, fatty acids shown in the Table 2. Considering the effects of fodder composition on growing lambs, it is very important to take into consideration that some biochemical parameters assayed in the serum may be significantly modified depending
on the time of blood collection. An example of such a parameter may be provided by, for example, glucose concentration. The reducing effect of blood collection time on glucose concentration (Table 3), especially between day 21 and the last day of the study, was observed in the control group and both experimental groups, while no differences were observed between the groups receiving CS and DGS. In ruminants, such a result may be a consequence of the change in the nutrition regime after weaning. The resulting amounts of lipids in blood are observed due to the high (approximately 6%) lipid concentration in ewes’ milk (Ashton et al., 1964). An increased content of lipoproteins was also found in these animals (Kubasek et al., 1974). After this period until approximately day 100 of life, a reduced blood lipid concentration is detected (Leat and Gillman, 1964) and it may be a result of weaning. In our study, this specific agedependent decrease in lipid concentration was also observed – total cholesterol was reduced, especially during the first 3 weeks of the experiment, and triglycerides were being reduced throughout the entire period (6 weeks) of the experiment. The parameters related to lipid metabolism were significantly influenced by the diet composition. In the groups of animals consuming the experimental diets, no increase was found in CS and DGS, or total cholesterol concentration during the second 3 weeks. It was detected in the last day of the study, in comparison with the control group, and the value of this parameter was the highest in the group consuming CS. The level of triglycerides in blood of examined animals varied depending on sampling time. The downward trend for TG concentration with successive sample collection date and thus with the age of animals was observed in animals from the control group. In lambs from both experimental groups following its significant reduction at day 21 of the experiment (p < 0.01), its level in successive days remained on a similar, stable level (p > 0.05). Although the content of polyunsaturated fatty acids (PUFA) in both CS and DGS diets was different, as CS is rich in n-3 PUFA and DGS in n-6 PUFA, this difference was not important concerning the effect on blood cholesterol and triglycerides. However, cholesterol concentration remained slightly
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Statistical analysis
The effect of the experimental feed on concentrations of blood markers and hormones was estimated by the GLM procedure of SAS ver. 9.1 (2000), based on the unitrait linear model, with the sampling time 9 feeding treatment interaction. The results were expressed as the mean standard error of means (SEM). As calculated interactions between blood sample collection and the nutrition factor proved to be non-significant (p > 0.05), they were not included in the table. Results and discussion
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Table 3 Concentration of blood markers and hormones in successive blood samplings (mean SEM) Blood marker/ blood samplings† GL (mM) 1 2 3 CH (mM) 1 2 3 TG (mM) 1 2 3 FFA (mM) 1 2 3 IN (ng/ml) 1 2 3 LE (ng/ml) 1 2 3 T3 (nM) 1 3 T4 (nM) 1 3
Group Control (C)
CS
DGS
Significance of differences between groups
4.56 0.18 a 4.40 0.18 4.08 0.18 b
4.54 0.18 A 4.64 0.18 A 3.90 0.18 B
4.34 0.18 4.71 0.18 A 4.09 0.18 B
2.28 0.16 B 1.47 0.16 A 1.46 0.16 A
2.43 0.16 Aa 1.59 0.16 B 1.91 0.16 b
2.54 0.16 A 1.09 0.16 B 1.72 0.16 C
CS-DGS* C-CS*
3.38 0.13 A 2.76 0.13 B 2.22 0.13 C
3.17 0.13 B 2.53 0.13 A 2.65 0.13 A
3.13 0.13 A 2.65 0.13 B 2.62 0.13 B
C-CS*, C-DGS*
0.17 0.03 A 0.14 0.03 A 0.90 0.03 B
0.16 0.03 A 0.17 0.03 A 0.56 0.03 B
0.18 0.03 A 0.17 0.03 A 0.67 0.03 B
C-CS**, C-DGS**, CS-DGS*
0.72 0.09 a 0.96 0.09 Ab 0.66 0.09 B
0.67 0.09 0.62 0.09 0.53 0.09
0.65 0.09 Aa 1.19 0.09 Ba 0.91 0.09 b
C-CS**, CS-DGS**’ C-DGS* CS-DGS**, C-DGS*
1.35 0.09 1.40 0.09 1.18 0.09
1.29 0.09 A 1.22 0.09 A 0.82 0.09 B
1.44 0.09 A 1.42 0.09 A 0.42 0.09 B
C-CS**, C-DGS**, CS-DGS**
2.49 0.13 A 1.67 0.13 B
2.78 0.13 A 1.64 0.13 B
2.81 0.13 A 1.64 0.13 B
82.26 6.13 94.03 6.13
88.68 6.13 105.10 6.13
97.18 6.13 100.52 6.13
GL, glucose; CH, cholesterol; TG, triglycerides; FFA, free fatty acids; IN, insulin; LE, leptin; T3, triiodothyronine; and T4, thyroxine. In columns, AB (ab) values designated with different capital (small) letters differ significantly at p ≤ 0.01 (p ≤ 0.05). In rows, **p ≤ 0.01, *p ≤ 0.05. †1 – day of the beginning of administration of experimental feed; 2–21st day of fattening; 3–42nd day of fattening (day of slaughter).
increased after consumption of the n-3 PUFA-rich CS diet in comparison with DGS. Other experiments revealed that depending on the source of unsaturated fatty acids, their effect on lipid parameters may vary. In their review, Roche and Gibney (2000) described (although not in lambs) that n-3 polyunsaturated fatty acids (PUFAs) from fish oil reduced plasma triglyceride concentrations. In the experiments performed by Ponnampalam et al. (2001), total cholesterol and HDL-cholesterol levels were greater when compared to the control in lambs receiving protected sunflower meal, but they were lower in lambs consuming n-3 PUFA-rich fish oil or fish oil with protected sunflower meal. In the same study, no alterations in plasma triglycerides were detected. Unlike cholesterol and triglycerides, an opposite effect of blood collection time on free fatty acid (FFA)
concentrations was observed in the performed experiment. This parameter was not altered during the first 3 weeks, but a marked increase was observed in the second period of the study in all groups of animals, which could have been the effect of 12-h preslaughter fasting of animals. It is difficult to explain this effect as there are limited literature data concerning this issue. Lindsay and Leat (1975) reported that FFA are not altered depending on age in sheep, while Nelson (1973) gathered that the reason of this effect in sheep could be connected with the absorption of lipids as fatty acids. On the other hand, the increase in fatty acid concentrations may be due to the change of the manner of digestion from non-ruminant to ruminant. Holcombe et al. (1992) studied the level of nonesterified fatty acids (NEFA) in serum of lambs weaned at 42 days of age and observed a twofold
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The metabolic profile of growing lambs
increase in this parameter 1 day after weaning. Moreover, NEFA remained significantly increased for the first week after weaning (Holcombe et al., 1992), which what was probably connected with the response of animals to decreased energy intake. However, in our experiment in the groups of animals receiving the CS and DGS diets, the increase in FFA was substantially limited when compared to the control. This effect was probably related to the presence of unsaturated fatty acids in the experimental diets. Moreover, the CS diet, rich in n-3 PUFA, exerted a stronger diminishing effect on plasma FFA concentration in lambs in comparison with that of DGS. It is obvious that n-3 PUFA may increase the activity of adiponectin, which can stimulate fatty acid oxidation by muscle cells (Tishinsky et al., 2012). Moreover, it was established by Priore et al. (2012) that n-3 PUFA from fish oil increases the expression of carnitine/acylcarnitine translocase – a pivotal protein responsible for fatty acid transport to mitochondria, where they are oxidized. Recapitulating, the results obtained in our experiment suggest that in weaned ruminants, thus also lambs, we can observe age-dependent changes in blood lipids, that is, total cholesterol and triglycerides, and their increased utilization accompanied by reduced concentrations in blood. Reduced glycemia also appears in growing animals due to the change in their nutrition and metabolism. The applied experimental diets, that is, CS, rich in PUFA n-3 and DGS, rich in PUFA n-6, alleviate the decrease in blood lipid levels, but have no effect on glycemia. In agreement with our results, Cieslak et al. (2003) revealed that supplementation of lambs’ diets with components rich in unsaturated fatty acids may increase the energy value of the diet without causing significant changes in blood parameters. It is concluded that an additional source of energy containing polyunsaturated fatty acids does not cause metabolic disorders. As Camelina sativa cake is the source of primarily n-3 linolenic acid, whereas dried distillers grains with solubles of n-6 linolenic, and n-6 linoleic acid is converted to arachidonic acid, a precursor for the 2 series of prostaglandins, whereas n-3 linolenic acid is converted to eicosapentaenoic acid, a precursor of the 3-antagonistic series of prostaglandins, the mode of action on the evaluated blood parameters does not change. Apart from metabolic parameters, blood concentrations of some hormones regulating metabolism, that is, insulin, leptin, T3 and T4, were also assayed in the performed experiment (Table 3). The first 21 days of the experiment had a significant effect on an increase in the concentration of insulin in blood serum of
lambs from the C and DGS groups. Its concentration in animals from the above-mentioned groups after 12h pre-slaughter fasting (blond samplings 3) was next reduced by 0.30 and 0.28 ng/mol (p < 0.01 and p < 0.05) respectively. In contrast, no effect of feeding was found on IN level in lambs from group CS (p > 0.05). However, insulin concentration in the group receiving DGS, increased after the first 3 weeks of the study, decreased until the end of the experiment, but it was still substantially increased in comparison with the residual groups. This effect seemed to be partial due to glucose concentration in the DGS group, which was increased after the first 3 weeks of the experiment and was decreasing until the last day, but in the case of glucose, no differences were observed between the experimental groups and the control. In relation to the content of both diets consumed by lambs, it may be suggested that probably n6 PUFA could be responsible for the observed effect on the hormone concentration in blood. The literature data describing differences in blood hormone concentration after consumption of a diet rich in n-6 PUFA in ruminants are rather scanty, but there are more data concerning the effects of an n-3 PUFA-rich diet. In the experiment performed on growing lambs by Kim et al. (2007), the dietary n-6:n-3 fatty acid ratio had no effect on concentration of either IGF-1 or insulin in plasma in these animals. As it was shown by Ponnampalam et al. (2001), feeding lambs with a diet supplemented with fish oil as a source of n-3 PUFA resulted in a significant reduction in the diet-induced increase in insulin concentration when compared to the control. Baur et al. (1998) established that in in vivo and in vitro experiments on humans and rats, unsaturated fatty acids supplemented in the diet were incorporated to the muscle membranes, they improved insulin action and the binding capacity of the hormone. In our experiment, this hypothesis could be viable for lambs receiving the n-3 PUFA-rich CS diet, as insulin concentration in this group was lower when compared to the control throughout the experiment. Still no explanation was provided why insulin concentration was greater in the DGS-receiving animals, but the greater content of n-6 PUFA and the diminished n-3 PUFA content in the DGS diet could probably be due to this effect. The age of lambs had no effect on leptin concentration, as it was observed in the control group during the 6 weeks of the experiment. However, the composition of the diet had an important effect on leptin concentration in the last period of the study, as both experimental diets, that is, CS and DGS, significantly decreased hormone concentration. Blood leptin
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concentration can be dependent on both adiposity and nutrition, although Ehrhardt et al. (2003) established that in the early post-natal life of lambs the effects of nutrition are predominant. There is another explanation for the reduced leptin concentration – this effect may result from a decreased insulin concentration, as insulin is an important physiological stimulator of leptin secretion in ruminants (Chilliard et al., 2001). However, in our experiment, this supposition should be excluded, as in the DGS group, the highest concentration of insulin was assayed after slaughter when compared to the control or the CS group. The data describing the effects of PUFA content on leptin plasma concentration in other animals are also insufficient, as it was reported that in rats, n-3 with PUFA supplementation of the high-fat diet the plasma leptin concentration was lower, similarly as the leptin mRNA in adipose tissue (Ukropec et al., 2003). In our experiment, the n-3 PUFA-rich CS diet induced a decrease in plasma leptin concentration. The concentration of T3 decreased with age from the first to the last day of the study, but no significant alterations were induced depending on diet composition. There were no age-related changes and no effect of diet composition on T4 concentration in blood. The age-related changes in T3 concentration observed in our study are in accordance with literature data, where plasma T3 levels in lambs after feeding were higher in lambs in the first days of life and they decreased with age (Symonds et al., 1989). The lack of differences between the experimental groups and the control makes it possible for us to conclude that the alleviating effect on metabolic parameters, observed in blood after the consumption of both experimental diets, that is, CS and DGS, is not a consequence of changes in the thyroid hormone concentration and probably they are not related to changes of metabolic rates in the experimental animals. References van Arendonk, J. A. M., 2011: Opportunities for animal breeding to meet the challenges of the future. Nauka Przyroda Technologie 5, 3 #30. Ashton, W. M.; Owen, J. B.; Ingleton, J. W., 1964: A study of the composition of Clun Forest ewe’s milk. Journal of the Science of Food and Agriculture 63, 85–89. Association of Official Analytical Chemists, 2007: Official Methods of Analysis. In: W. Horwitz, W. Latimer (eds), 18th edn. AOAC, Gaithersburg, MD, pp. 27–31.
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Conclusion Summing up the presented study, it may be stated that the administration of feed mixtures containing CS and DGS in fattening of lambs had an effect on the levels of blood metabolic indexes and hormones. The applied experimental diets alleviate the decrease in blood lipid levels, but have no effect on glycemia. Preslaughter fasting had an important effect on leptin concentration in the experimental groups, as both experimental diets, that is, CS and DGS, in contrast to the diet of the control (C), significantly decreased hormone concentration. The age-dependent reduction in CH and TG limited by both experimental diets was observed. The IN concentration in DGS-receiving lambs was increased, in contrast to CS-receiving lambs, which was lower in this group compared to the control. The LE concentration was decreased by both experimental diets, more markedly in DGS-receiving animals. The effect of 12-h pre-slaughter fasting was statistically highly significant for the level of analysed blood markers and hormones, except for TG and IN in the group of lambs receiving experimental diets with CS. Acknowledgements This research was conducted under the Project ‘Biofood – innovative, functional animal products’, financed by the Polish Ministry of Economy within the European Regional Development Fund 2007– 2013, Operating Program Innovative Economy no. POIG 01.01.02-014-090/09. Conflict of interest The authors have no conflict of interest to declare whatsoever.
Barczak, E.; Wolc, A.; W ojtowski, J.; osarz, P.; Szwaczkowski, T., 2009: Sl Inbreeding and inbreeding depression on body weight in sheep. Journal of Animal and Feed Sciences 18, 42–50. Baur, L. A.; O’Connor, J.; Pan, D. A.; Kriketos, A. D.; Storlien, L. H., 1998: The fatty acid composition of skeletal muscle membrane phospholipids: Its relationship with type of feeding and plasma glucose levels in young children. Metabolism 47, 106–112. Cais-Sokoli nska, D.; Majcher, M.; Pikul, J.; Bieli nska, S.; Czauderna, S.; W ojtowski, J., 2011: The effect of Camelina sativa
cake diet supplementation on sensory and volatile profiles of ewe’s milk. African Journal of Biotechnology 10, 7245–7252. Chilliard, Y.; Bonnet, M.; Delavaud, C.; Faulconnier, Y.; Leroux, C.; Djiane, J.; Bocquier, F., 2001: Leptin in ruminants. Gene expression in adipose tissue and mammary gland, and regulation of plasma concentration. Domestic Animal Endocrinology 21, 271–295. Cieslak, A.; Potkanski, A.; SzumacherStrabel, M., 2003: The influence of fat supplementation to sheep diets on the glucose concentration and other
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