Rocz. Nauk. Zoot., 45, 1 (2018) 25–46
EFFECT OF FEED ENZYMES ON PERFORMANCE, CARCASS QUALITY AND ILEAL AMINO ACID DIGESTIBILITY IN BROILER CHICKENS FED DIETS CONTAINING EXTRACTED
RAPESEED MEAL*
F r a n c i s z e k B r z ó s k a , B o g d a n Ś l i w i ń s k i
National Research Institute, Animal Production Institute, Faculty of Nutrition Physiology, 32-083 Balice by Krakow
e-mail: franciszek.brzoska@izoo.krakow.pl
The studies were carried to determine the effect of exogenous feed enzymes on body weight, mortality, feed conversion and carcass quality (exp. 1), biochemical parameters of blood plasma as well as ileal amino acid digestibility (exp. 2) in broiler chickens fed with mash mixes containing 11% (starter mix) and 18% (grower mixture) of solvent-extracted rapeseed meal. In a growth trial, a batch of 1 day-old sexed Ross 308 chickens were divided into 5 dietary treatments with 8 replicates per sex and 8 chickens per pen, based on the following design: positive control group with solvent-extracted soybean meal (SBM) – without enzymes; negative control group with rapeseed meal (RSM) – without enzymes; experimental groups with solvent-extracted rapeseed meal (RSM), supplemented with the enzymes endo-β-1,4-xylanase and endo-1,3(4)-β-glucanase (group RSM + e KG), serine protease (group RSM + e P), and all the enzymes (RSM + e KG + e P). Apparent ileal digestibility of amino acids (AA) in the four grower diets: SBM, RSM, RSM + e KG, RSM + e P (experiment 2) was determined with Ross 308 (n=320) roosters. The recipe composition and nutritional value of the feed were the same in both experiments. Feeding chickens with feed mixes containing rapeseed meal as compared to feed mixes containing soybean meal reduced the body weight of broilers from 2466 g to 2182 g (P˂0.05). The body weight of chickens receiving compound feed with enzymes did not differ significantly from the weight of broilers fed with rapeseed meal without enzymes. Feed with rapeseed meal without enzymes (RSM) significantly increased the mortality of chickens as compared to the group containing soybean meal in the fodder (SBM, P˂0.05). The aAddition of enzyme preparations of endo-β-1,4-xylanase and endo-1,3 (4) -β-glucanase and serine protease significantly reduced broiler mortality (P˂0.05). Feeding mixtures containing rapeseed meal without and with the addition of enzymes significantly reduced feed intake (P˂0.05). There was no significant influence of experimental factors on slaughter yield and content of elements in broiler carcasses. Feeding broilers with feed containing rapeseed without and with the addition of enzymes significantlydecreased the content of spare fat in carcasses (P˂0.05). There was also no influence of experimental factors on the chemical composition of broiler muscles, except for the significant reduction of ash in broiler muscles receiving mixes with rapeseed feed without and with the addition of enzymes as well as a significant effect on biochemical parameters of blood plasma. Feeding broilers with fodder with rapeseed meal compared to mixes with soybean meal
* The work was performed as a part of the statutory activity of the research task 05-009.1 in years
significantly reduced the digestibility of exogenous and endogenous amino acids, with the exception of methionine and cystine (P˂0.05). The addition of endo-β-1,4-xylanase and endo-1,3 (4) -β-glucanase to the mixture with rapeseed meal significantly decreased the digestibility of arganine and proline, and increased methionine digestibility (P˂0.05). The addition of serine protease increased the digestibility of isoleucine, lysine, methionine, valine, without significantly altering the digestibility ratios of the remaining amino acids.
Key words: rapeseed meal, enzymes, growth, slaughter efficiency, digestibility, amino acids
Extracted rapeseed meal is a by-product of oilseed rape seeding. About 1170 thousand tons of rapeseed meal are produced in Poland, including 600-700 thousand tons of meal for fodder purposes per year (Dzwonkowski et al., 2016). The anti-nutritive factor of rapeseed fodder, which decreases its nutritional value to the greatest extent along with glucosinolates, are non-starch polysaccharides (NSP). It has been shown that post-extracted rapeseed meal of Canadian Canola varieties contains from 20 to 40% of NSP (Slominski and Campbell, 1990, Bach Knudsen, 1997, Slominski et al., 1999). Non-cellulose NSP is 13-16% of rapeseed meal and contains arabinose (33%), xylose (13%), galactose (13%), glucose (5%), mannose (3%), ramnose ( 2%), fucose (2%) and uronic acids (30%) (Slominski and Campbell, 1990). The non-starch polysaccharides of rapeseed meal were also to be divided according to their chemical structure into pectins (including homogalacturon, ramnogalactouranon, arabinan and arabinogalactan), hemicellulose (including xylogglycol, glucouranoxylate, galactomannans) and cellulose (Pustjens et al., 2013; 2014). The rapeseed cover consists mainly of arabinoxylans, (1-3,1-4) -β-glucans and a small amount of cellulose, heteromannans and esterified phenolic acids (Rybka, 1994). Non-starch polysaccharides, mainly soluble in water, are hydrolysed to a small extent by the bacterial flora of the gastrointestinal tract, bind water and increase the viscosity of the digestive tract, reducing digestion of starch, proteins and fat (Annison and Choct, 1991; Smith and Annison, 1996; Smulikowska et al., 1998). Chickens do not have enzymes capable of hydrolysing non-starch polysaccharides in the absorptive part of the small intestine, and their partial decomposition takes place in the cecum under the influence of bacterial microflora.
The prohibition of the use of genetically modified feed in Poland forces searching for methods to increase the share of alternative feed materials for GM soybean meal for monogastric animals, including broiler chickens. The recommended level of rapeseed meal in feed mixes for starter broilers is 5-6%, and in mixers grower-finisher 10-12% (Pastuszewska et al., 1992; Smulikowska and Rutkowski, 2005), although these recommendations apply generally to broilers, and not individual genetic hybrids of broilers. The excess of the recommended level of rapeseed meal in broiler feeds reduces the use of energy
and amino acids, reduces the growth and worsens the quality of post-slaughter carcasses (Michalik-Rutkowska, 2016).
In order to increase the digestibility and use of rapeseed meal for fodder purposes, enzymes were used in broiler nutrition, aimed at decomposition of non-starch polysaccharides, peptides and proteins (Pustjens et al., 2014, de Vries et al., 2014). Attempts have also been made to increase the susceptibility of NSP to rapeseed meal against enzymes by crushing and hydrothermal treatment although excessive temperatures lead to the degradation of proteins and certain amino acids (McDugall et al., 1996) and heating increased the viscosity of the digestive content (de Vries et al., 2012) and negatively influenced the digestibility of nutrients and their absorption (Smith and Annison, 1996). The efficiency of feed enzymes in the impact on rapeseed meal assessed in broiler feeding experiments did not give satisfactory results (Kocher et al., 2000, de Vries et al., 2014).
The new generation enzymatic preparations combine the activity of several enzymes. The mechanism of their operation consists in the partial decomposition of non-starch polysaccharides and the decrease in viscosity of the digestive tract in the small intestine. As a result of hydrolysis of non-starch polysaccharides, endogenous enzymes of birds, especially pancreatic enzymes, can more easily penetrate into the cells and hydrolyse the nutrients contained in them, including proteins. Enzymes of endo-β-1,4-xylasease and endo-1,3 (4) -β-glucanase by decomposing NSP can improve the calorific value of feed mixtures, and serine protease enzymes can improve the use of amino acids in compound feeds, which can be a premise to attempts to increase the share of rapeseed meal in mixtures for broiler chickens.
The purpose of the study was to assess the effectiveness of feeding the broiler chickens with the addition of new generation enzyme preparations with endo-β-1,4-xylanase and endo-1,3 (4) -β-glucanase activity and serine protease introduced separately or jointly into feeding stuffs with an abnormal content of post-extraction rapeseed meal.
Materials and methods
The chicken living conditions and all the applied experimental procedures were approved by the 2nd Local Ethical Committee for Experiments on Animals in
Krakow.
Experiment 1 - growth
The growth experiment was carried out on sexed broiler chickens Ross 308, divided into two positive and negative control groups and three experimental groups. Each group consisted of chickens of both sexes, assigned at 8 replicas for each sex, and each repetition consisted of 8 birds. The positive control group received a standard feed mix with soybean meal (SBM) and the control group had
a negative mix with rapeseed meal (RSM). The experimental groups received a mixture with an identical composition of feed materials as in the case of the negative control group (RSM, tab. 1) with addition of (1) Novagro WXVP preparation (group RSM + e KG) containing endo-β-1,4-xylanase (EC 3.2.1.8) with an activity of 40,000 FXU/kg produced by Aspergillus Niger microorganism (CBS 109.713) and endo -1,3 (4) -β-glucanase (WE 3.2.1.6) with activity 2000 FBG/kg, produced by the Trichoderma longibrachiatum microorganism (ATCC 2106) (EURO-Lex-32005R1458 - PL), (2) Ronozyme® ProAct preparation (group RSM + e P) containing the serine protease enzyme (EC 3.4.21) with activity 75,000,000 PROT/kg of the preparation. It is a microbial enzyme, a pancreatic chymotrypsin analogue produced by a modified strain of Bacillus
licheniformis (DSM 19670). The last experimental group contained both
preparations together (RSM + e KG + e P). Novagro preparation was used according to the manufacturer's recommendation in the amount of 3.5 kg per ton of compound feed. Ronozyme® ProAct preparation was used in accordance with the manufacturer's recommendations in the amount of 200 g/t of feed.
Loose starter mixes (for the first breeding period, 1-21 days) and grower type mixes (for the second breeding period, 22-42 days) were prepared. The cereal component of the mixtures was maize or maize plus wheat, and the protein components in the negative control group and in the experimental groups were also dried distillers (DDGS) and fodder yeasts - materials necessary to balance the level of total protein in the broiler diets (Tab. 1). The feed was made at the Experimental Mixture Institute of the National Research Institute of Animal Production in Aleksandrowice according to recipes developed and optimised using the WinPasze PRO 3.6 software. The nutrients of protein components of compound feed (soybean meal, rapeseed meal, dried distillery, fodder yeast) were analytically determined according to AOAC (2009) and entered into the WIN-Pasze software for the optimisation of compound feed recipes.
The chickens were kept in boxes on litter of deciduous tree shrubs and fed with loose feed mixtures fed at will. During the first six days of rearing, the feed was served on flat trays, and from day 7 in vertical feeds filled once a day. Water was supplied from the central water supply system through a system of pipes and droplet drinkers. There were two drinkers for each of the boxes. The nesting density of the chicks was 15 items/m2 in the first breeding period, which corresponded to the load of 30-33 kg body weight chickens/m2 at the final rearing period. During the first three days of life, the birds received anti-diarrheal preparations (Scanoflox 10% in an amount of 1 ml/1 water). On day 7, an aqueous solution of the Gumboro vaccine, and on day 14 the vaccine against peptic fever (Bio-Vac ND-IB preparation) was administered. The room temperature was brought to 34°C within three days before settling and maintained at this level for the first 5 days and then gradually lowered to 24°C in the last week of chicken rearing. The room was provided with appropriate air exchange conditions and constant bulb lighting with the required intensity, taking into account the requirements of the broilers for light.
Table 1. Feed materials and nutrient content of diets for broiler chickens – experiments 1 and 2
Feed materials (%)
Feedingstuffs and groups
starter grower
SBM RSM SBM RSM
Maize 56,08 47,81 51,70 41,50
Wheat - - 10,00 10,0
Solvent soybean meal 36,00 20,00 30,00 11,00 Solvent-extracted rapeseed meal - 11,00 - 18,0
DDGS - 8,00 - 8,00 Fodder yeast - 4,50 - 2,00 Rapeseed oil 3,50 5,00 4,00 6,00 Dicalcium phosphate 2,00 1,50 1,60 1,20 Calcium carbonate 1,30 1,10 1,50 1,00 Fodder salt 0,30 0,30 0,30 0,30 L-Lysine HCl (78%) 0,10 0,17 0,18 0,30 DL-Methionine (99%) 0,22 0,22 0,22 0,20 Mineral-vitamin premix* 0,50 0,50 0,50 0,50 Enzymes - +/- - +/- Calculated nutrients (g/kg) Metabolizable energy (MJ/kg) 12,6 12,6 13,0 13,0 Dry matter 885 889 886 890 Crude protein 222 222 200 200 Crude fat 61,2 82,2 65,9 92,1 Crude fibre 26,7 38,2 26,3 39,0 Starch 370 33,0 390 34,0 Crude ash 26,9 33,5 26,6 33,5 Calcium 10,0 10,0 10,1 10,0 Available phosphorus 4,00 4,00 3,93 4,00 Lysine 12,10 12,10 12,00 12,00 Methionine 5,50 5,50 5,26 5,26 Tryptophan 2,30 2,00 2,30 2,00 Threonine 8,00 8,00 8,00 8,00
* 1 kg premix DKA Starter 0.5% contains: vit. A – 13 5000 IU; vit. D3 – 250 IU; vit. E – 40 mg; vit. B1 – 3.25 mg; vit. B2 – 7.5 mg; vit. B6 – 5 mg; B12 – 0.0323 mg; vit. K3 – 6 mg; biotin – 0.15 mg; nicotinic acid – 45 mg; calcium pantothenate –15 mg; folic acid – 1.5 mg; choline chloride – 100 mg; Mn – 100 mg; Cu – 1.75 mg; Fe – 76.5 mg; Se – 0.275 mg; I – 1 mg; Zn – 75 mg; Co – 0.4 mg; Endox (antioxidant) – 125 mg; Sincox (coccidiostat) – 1 g; Ca – 0.679 g.
1 kg premix DKA Grower 0.5% contains: vit. A – 12 000 IU; vit. D3 – 250 IU; vit. E – 40 mg; vit. B1 – 2 mg; vit. B2 – 7.25 mg; vit. B6 – 4.25 mg; B12 – 0.03 mg; vit. K3 – 2.25 mg; biotin – 0.1 mg; nicotinic acid – 40 mg; calcium pantothenate – 12 mg; folic acid – 1.0 mg; choline chloride – 450 mg; Mn – 100 mg; Cu – 1.75 mg; Fe – 76.5 mg; Se – 0.275 mg; I – 1 mg; Zn – 75 mg; Co – 0.4 mg; Endox (antioxidant) – 125 mg; Sincox (coccidiostat) – 1 g; Ca – 0.79 g.
During the experiment, the feed intake was determined, weighing the uneaten feed in individual boxes on a daily basis. To determine the rate of chicken growth, 40 randomly chosen 1-day-old chickens were weighed and all the chickens were weighed after 12 hours of starving at the age of 21 and 42 days. The body weight of 1-day-old chickens was on average 39.2 ± 4.6 g. Daily survival of birds in all groups was controlled. Taking into account the weight of dead chickens, a unitary feed conversion was calculated. On the 43rd day of the experiment, after the
completion of rearing, 10 chickens of both sexes (5/5) were randomly selected from each group. After being starved, the birds were weighed and then slaughtered by stupefaction with an electric impulse and bleeding. During bleeding into heparinised tubes, blood samples were collected to obtain plasma for chemical analysis. In fresh plasma, the glucose level was determined and the remaining part was frozen for further analysis. After scalding and mechanical removal of the feathers and the head, the carcasses were eviscerated. The mass of the warm carcass, the mass of the stomach, liver and storage fat were defined as the sum of perigastric fat and the epiphyseal fat. Based on the slaughter mass of chickens and the weight of the warm carcass with offals and joints, the slaughtering yield was calculated. The carcasses were cooled for 24 hours at 5°C, and then the right half of the carcass was dissected. The dissection consisted in the formulation and weighing of the carcass elements of culinary importance, including the breast muscles and leg muscles. The slaughter yield and the percentage share of carcass elements in the warm mass (liver, stomach) and in chilled mass (breast muscles, leg muscles) were calculated. The dissection was performed according to the procedure described by Zgłobica and Róża (1972). Representative muscle samples of both types were collected for chemical analyses. The samples were ground and frozen at -18°C. The analyses were carried out after 30 days of refrigerated storage of samples.
Experiment 2 - digestibility
Apparent ileal digestibility of amino acids (AA) in the four grower diets: SBM, RSM, RSM + e KG, RSM + e P type grower (tab. 1) were determined on 320 Ross 308 roosters divided into 4 groups, in 8 replicas of 10 pieces in each. The birds’ living conditions, feeding habits as well as the prescription composition and nutritional value of feeds used in the respective groups were the same as in the growth experiment. Chromium trioxide (Cr2O3) in the amount of 5 g/kg of the
diet, as a digestibility marker, was introduced into the batch of ready-made grower feeds fed in the last week of rearing. On the 35th day all the birds were slaughtered
by intraperitoneal injection of Morbital (sodium pentobarbital). After the blooding of the carcasses and dissection of the abdominal walls, the final part of the small intestine (ileum) was dissected from the Meckel diverticulus (remnants of the biliary-intestinal tract) to the point of 20 mm before the joining of the cecum with the large intestine, and then gently squeezed the nutrient into a plastic container. The procedure and the sequence of procedures for acquiring intestinal contents were in line with the description given by Kadim and Moughan (1997). The
content taken from particular individuals was combined (replicates, n = 10) and frozen at -18 ° C. After 20 days, the material was thawed, lyophilised and subjected to chemical analyses for dry matter, amino acids and added chromium
Chemical analyses and calculations
The content of basic chemical components in the feed materials as well as the muscles and tissues of the chickens was determined in accordance with the methods given in the AOAC analytical norms (2006). The level of starch in the compound feed was determined by the polarimetric method (PN-R-64785: 1994). The concentration of nutrients and the level of metabolic energy in diets, calculated on the basis of the formulas given in the European Tables of Energy Value of Feed for Poultry (ETEVPF, 1989) were calculated as the sum of these values for individual feed materials. The level of glucose in the blood plasma was determined using the enzymatic method using glucose oxidase. Analyses of the total protein, triglycerides, total cholesterol and high molecular weight (HDL) content were performed by enzymatic-colorimetric method using diagnostic kits from Cormay Diagnostyka Polska.
The content of chromium in the feed and the intestinal content was determined after wet mineralisation of the samples in a mixture of nitric acid and perchloric HNO3/HClO4 (1: 1.5) (Saha and Gilbreath, 1991). Before analysing
amino acids in feed and intestinal contents all the samples were hydrolysed in 6 N HCl at 110°C for 22 hours (AOAC, 2006). For the determination of methionine and cystine, pre-oxidation of samples in performic acid was performed. The amino acids were determined by high-performance liquid chromatography on a Beckman 126 AA System Gold analyser. The amino acid content was corrected for incomplete recovery from hydrolysis. The apparent intestinal amino acid digestibility (AID) coefficients contained in individual compound feeds were calculated according to the following formula (Kadim and Moughan, 1997):
AID (%) = 100 – [Crd x AAtj) / (Crtj x AAd)] x 100
where:
Crd and Crtj – the content of the indicator (Cr) in the dry mass of the diet
and intestinal content;
AAtj and AAd – the amino acid content in the intestinal dry matter,
respectively.
Statistical data analysis
Data on chickens mortality were transformed according to the equation x = log (x + 2) applied for the percent mortality rates. The transformed data was subjected to statistical analysis. The significance of differences between groups for the parameters of production efficiency was calculated using the Tukey test for 5% probability. For the digestibility of amino acids, the analysis of variance was performed, identifying differences between the groups by the Fisher test
(NIR) for 5% probability. Between the groups fed with rapeseed meal without and with the addition of feed enzymes, the smallest significant difference was calculated using the orthogonal contrast method. The calculations were performed using the SAS 9.3.TS Level 1 MO computer program.
Results
Basic production parameters and biochemical blood indicators
The introduction of post-extraction rapeseed meal without and with the addition of enzymes to the feed mixtures significantly reduced the weight of the broiler chickens in comparison with the soybean mixture (P˂0.05, tab. 2). There were no significant differences in the body weight of broilers between the control group (RSM) without enzymes and groups with β-1,4-xylanase and endo-1,3 (4) -β-glucanase (group RSM + e KG) and with protease serine (group RSM + e P). The mix with rapeseed meal (RSM) significantly increased the mortality of chickens (P˂0.05) as compared to the mixture with soybean meal (P˂0.05), whereas the compound feed with serine protease (RSM + e P) and a mixture with both preparations enzymes significantly reduced the mortality rate (P˂0.05). Feeding broilers with feed mixtures containing rapeseed meal without and with the addition of both enzyme preparations significantly reduced the intake of compound feeds in a relative value as compared to a feed mixture containing soybean meal on average by 8.7% (P˂0.05). The use of feed in the negative control group was 3.6% higher than in the positive control group, and the use of feed enzymes significantly reduced the feed conversion rates, on average by 9.7% among the experimental groups receiving enzymes as compared to the negative control group not receiving the enzyme addition (P˂0.05). The feeding of feed mixes with the addition of endo-β-1,4-xylanase and endo-1,3 (4) -β-glucanase significantly reduced the feed conversion rate as compared to the broilers receiving compound feed with soybean meal and rapeseed meal without enzymes. The feeding of fodder mixtures containing rapeseed meal without enzymes significantly reduced the European Efficacy Index (EIEP), due to the high mortality of chickens in this group while mixtures containing both enzyme preparations used separately and together significantly increased it as compared to the group of broilers receiving the mixture with rapeseed meal without enzymes (P˂0.05).
There were no significant differences in the slaughter weight, the weight of chilled carcasses and the slaughter yield of broiler chickens between the broiler groups (Tab. 3). There were also no significant differences between the broiler groups in the mass of breast muscles, leg muscles, as well as stomachs and liver in the percentage share of the carcass weight. The feeding of compound feed without enzymes to the broilers with endo1,4-xylanase and endo-1,3 (4) -β-glucanase and serine protease significantly reduced the content of spare fat in carcasses and the percentage of fat in the weight of the chilled carcass (P˂ 0.05).
The chemical composition of breast muscles and leg muscles in particular broiler groups was at the same level (Tab. 4). There were no significant differences in the content of dry matter, total protein and crude fat content among particular groups. Experimental factors, including rapeseed meal and the enzyme serine protease in broiler mixtures, decreased the content of crude ash in the breast muscles and the muscles of broiler legs. Significantly higher content of crude ash was found in broiler breast muscles of chicken receiving a mixture with the content of soybean meal and a mixture with the content of rapeseed meal with the addition of endo-β-1,4-xylanase and endo-1,3 (4) -β-glucanase (P˂0.05). The above values may have resulted from the different assimilation of mineral components of feed mixtures, although this remains a research hypothesis.
Table 2. Performance results of broiler chickens fed diets containing soybean meal and solvent-extracted rapeseed meal with and without enzymes
Item Body weight (g) Mortality (%) Feed consumption (g/bird/42 days) Feed conversion (kg/kg BWG) 21 day 42 day SBM 718 a 2466 a 4,8 b 4567 a 1,84 b RSM 651 b 2182 b 7,3 a 3728 b 1,91 a RSM + e KG 690 ab 2292 b 6,3 a 3950 b 1,63 d RSM + e P 688 ab 2288 b 1,6 c 4083 b 1,75 c RSM + e KG + e P 694 ab 2243 b 0,0 c 4080 b 1,82 b SEM 9 27 0,8 75 0,02 P-value 0,1740 0,0070 0,015 0,0030 0,0002
Orthogonal contrasts (P-value):
SBM vs RSM 0,0151 0,0004 0,0005 0,0440 0,2300
RSM vs RSM + e KG 0,1421 0,1352 0,0004 0,0360 0,0002
RSM vs RSM + e P 0,1650 0,1511 0,0019 0,2970 0,0020
RSM vs RSM + e KG + e P 0,1080 0,3981 0,0014 0,7060 0,1340
Abbreviations: SBM – soybean meal; RSM – solvent-extracted rapeseed meal; e KG – enzymes endo-β-1,4- xylanase and endo-1,3(4)-β-glucanase; e P – enzyme serine protease; SEM – standard error of the mean; P-value – probability value of an F-test ; BWG – body weight gain; a, b, c – P<0.05 (Tukey’s test).
The analysis of basic blood plasma indices did not show any significant differences in their level depending on the type of compound feed and enzyme preparation used in the mixtures (Tab. 5).
Apparent digestibility of amino acids
The apparent intestinal digestibility of the amino acids of blends containing post-extraction rapeseed meal without and with the addition of enzyme preparations was significantly lower than the digestibility of the amino acids of the feed mixture containing soybean meal, with the exception of cystine (P˂0.05; Tab. 6). The average values for the digestibility of exogenous amino acids in particular groups were, as follows: 87, 82, 81 and 84%, and for endogenous amino acids were, respectively, as follows: 86, 81, 79 and 82%. The addition of enzyme preparations of endo-β-1,4-xylanase and endo-1,3 (4) -β-glucanase significantly increased methionine digestibility and decreased proline digestibility (P˂0.05). It did not significantly affect the digestibility of other amino acids. The addition of the serine protease enzyme to the feed mix significantly increased the digestibility of isoleucine, lysine, methionine, valine and alanine (P˂0.05), without significantly affecting the digestibility of the remaining amino acids.
Discussion of the results Body weight, slaughter yield, mortality and feed use
Birds and mammals do not have endogenous enzymes in the digestive tract that break down non-starch polysaccharides contained in feed of plant origin, including 1,4-β-xylanase and 1,3-β-glucanase bonds. Hence, the technology for the production of feed enzymes from microorganisms, bacteria and fungi having enzymes that degrade this type of bonds (Headon and Walsh, 1994; Groves, 1997). The main purpose of using feed enzymes is to improve the digestion process of non-starch polysaccharides (NSP), to release the energy found in them as well as to lower the viscosity of the digestive content reducing digestion and absorption of nutrients. Academic literature on the use of feed enzymes in Poland, but also in Germany, concerns mainly the improvement of the nutritional value of cereals, including rye, triticale, barley and oats, to increase their share in the production of complete compound feed for poultry and pigs (Szymczyk et al., 2005; Amerah, 2015), as well as to improve the nutritional value of dried distillers (Świątkiewicz and Koleski, 2006, Świątkiewicz et al., 2013). A separate group is devoted to the use of phytic phosphorus from feed of plant origin by broilers using the phytase enzyme (Józefiak et al., 2010, Banaszkiewicz, 2012). Another group consists of the literature on the use of enzymes to increase the nutritional value of feeds containing post-extraction meal (Alloui et al., 1994; Szczurek and Koreleski, 1998; Szczurek, 2008).
This issue was examined as a part of work on post-extraction substitution of soybean meal with rapeseed feed in compound feed for broilers (Ramesh et al., 2006, de Vries et al., 2014; Pustjens et al., 2014). Rapeseed meal contains high levels of non-starch polysaccharides (NSP). The level of NSP in rapeseed feed is estimated by 20 to 40% by various authors (Slominski and Campbell, 1990, Bach Knudsen, 1997, Slominski et al., 1999). They form the cell wall structures of the
seed capsule remaining after the extrusion of rapeseed oil from seeds, including cellulose, hemicellulose and pectins associated with lignine and other NSP components (Siddiqui and Wood, 1977). Shahidi (1990) determined the composition of the carbohydrate fraction of rapeseeds as 4-5% cellulose, 4-5% pectin and 3% hemicellulose, and 45-50% fat fraction and 10% soluble sugars. It was assumed that the use of endo-β-1,4-xylanase and endo-1,3 (4) -β-glucanase potentially capable of hydrolysis of the cell wall fraction and NSP would release a part of the glucose contained therein. Inside the cells there is also a part of peptides and proteins for release of which the serine protease enzyme was used. Some of the proteins contained in the cell walls of plants close the way for enzymes of endogenous birds. It was assumed that the life of endo- β-1,4-xylanase and endo-1,3 (4) -β-glucanase together with the serine protease would increase the release of inaccessible proteins for broilers and perhaps increase the digestibility of the amino acids of the diet containing post-extraction meal rapeseed.
Less known are the reactions during the thermal extraction and toasting of rapeseed meal which may limit the absorption of glucose from cell wall polysaccharides and amino acids from peptides and proteins remaining in the blast after the oilseed rapeseed oil has been emptied. Szczurek and Koreleski (1998) used the proteolytic enzyme preparation and a multi-enzyme preparation containing xylanase and glucanase for testing superheated rapeseed meal in compound feed. They found that the proteolytic enzyme improved the production parameters of broilers, and the effectiveness of its use improved with the simultaneous use of non-starch polysaccharide degradation enzymes.
The research results obtained in our work indicate that the substitution of soybean meal with rapeseed meal at the level of 11% in the first phase of rearing and 18% of the feed mixture in the second phase of broiler breeding without the addition of enzymes significantly reduced the chicken body mass. The use of enzyme preparation containing endo-β-1,4-xylanase and endo-1,3 (4) -β-glucanase and a preparation containing the enzyme serine protease separately as well as both enzyme preparations together increased the broilers’ body weight by about 4.2 %, on average, although the differences found between groups of chickens fed without and with the addition of enzymes, in both periods of their rearing, were statistically insignificant. These results confirm, therefore, the earlier results obtained by Szczurek and Koreleski (1998) that enzymes of the genus endoβ1,4xylanase and endo1,3 (4) -β-glucanase, but also the serine protease can release a part of the dietary and amino acids energy contained in non-starch polysaccharides (NSP), which may translate into slightly increased growth of the body weight of broilers as well as reduced broiler mortality.
The issue of the influence of feed enzymes on the efficiency of broiler production has not been fully recognised. Simbaya et al. (1996) showed that the serine protease improved the growth of broilers fed diets containing rapeseed meal obtained from the Canola rape whereas the complex of commercial carbohydrase-type enzymes, although increasing the solubility of the cell wall polysaccharide
fraction in vitro, did not significantly affect the rate of growth and mass of broilers. In the work of Banaszkiewicz et al. (2013) it was shown that the addition of endo-1,4-β xylanase from Aspergillus oryzae to a compound feed for broilers containing 15% rapeseed cake of Kana variety did not significantly increase the body weight of 21-day-old chickens, with a tendency increasing in feed intake and digestibility of protein, fat and phosphorus. In other studies, Guenter et al. (1998), using the additive of enzymes described as carbohydrases in the soybean mixture with rapeseed meal, have obtained a significant increase in the weight of broilers.
The use of abnormal quantities of rapeseed meal in compound feed without enzymes and with the addition of endo1,4-xylanase and endo-1,3 (4) -β-glucanase and serine protease separately as well as both formulations together in our studies significantly increased the mortality of chickens as compared to broilers fed with mixtures containing soybean meal. In the Canadian authors' research, however, it was found that the rate of NSP fraction degradation with enzymes at broilers is low and ranges from 3 to 6% (Meng and Slominski, 2005).
In this work, serine protease obtained from the Bacillus lichenififormis microorganism added to the feed mixture significantly reduced the mortality of broilers while the simultaneous use of three enzymes, endo-β-1,4-xylanase and endo-1,3 (4) -β-glucanase, and serine protease, eliminated chicken mortality rate completely. The above results indicate that due to the need to limit the mortality of chickens, it would be advisable to combine in the multienzyme preparations the activity of xylanases and glucanases with proteolytic enzymes. The broiler survival rate is an element of the algorithm to assess the production yield of meat chicken. The smaller it is the higher the production yield of broilers, which is confirmed by the results of this work. Our results indicate that the addition of formulations containing endo-β-1,4-xylanases and endo-1,3 (4) -β-glucanase and the serine protease preparation significantly reduces the feed conversion rate of the broilers if they were used individually. The feed conversion index is also significantly reduced. These results are not confirmed by the results of de Vries et al. (2014). Investigating the effect of pectinolytic enzymes on NSP degradation of feed mixtures with rapeseed meal, they showed that this factor did not affect the feed intake, body weight or the feed conversion. The interaction between the enzyme addition and the degree of grinding of the feed mixture and the size of its particles has not been demonstrated. The reason for the divergent test results may be the different origin of individual enzymes as well as the different amount of activity units used in the studies. There are dozens of enzyme preparations authorised for use by feed law, differing in the species of the microorganism from which enzymes and enzymatic activity concentration have been obtained.
Quality of carcasses and meat composition
The quality assessment of broiler carcasses is closely related to slaughter body weight. It was demonstrated that the conversion of soybean meal in complete mixtures for broilers to rapeseed meal significantly reduced the weight of carcasses, but did not significantly differentiate the slaughter yield.
Michalik-Rutkowska (2016) showed that the conversion of soybean meal into rapeseed meal, at the level of 4% (starter) and 12.6% (grower) of compound feed significantly reduces body mass, increasing broiler mortality. The results of research on the production efficiency of feed enzymes in broiler nutrition are not clear. Kocher et al. (2001) found that rapeseed meal used as a substitute for soybean meal in mixtures containing sorghum improved slaughter efficiency and breast mass to the level of indicators obtained on mixtures containing soybean meal.
The results of our work indicate that the use of enzymes in compound feed did not significantly differentiate slaughter efficiency, muscle mass as well as stomach and liver mass. These parameters are closely related to the mass of the carcass and the genome of chickens, and environmental factors such as feed materials that form a complete mix have little effect on the differentiation of the proportion of individual carcass elements in broilers. The use of feed enzymes in this work did not significantly differentiate the quality of carcasses. The part of the carcass remaining under the influence of nutrition is the amount of the fat of the epiphyseal and perigastric. When broilers were fed with rapeseed meal, the content of spare fat was significantly reduced as compared to a mixture containing soybean meal, and feed enzymes reduced its content in comparison to control carcasses without enzymes. Similar results were found when enzymatic preparations of various configurations containing: β-glucanase were added to compound feed with the participation of barley and triticale with different fat content; β-glucanase, cellulase and xylanase and xylanase, α-amylase, pectinase, protease and β-glucanase (Kondzielska and Pisarski, 2000). They found that preparations with the content of β-glucanase significantly reduced the fat content in broiler muscles, to the greatest extent in chickens fed with fatty mixtures. Under the influence of enzyme preparations, the content of dry matter, total protein and crude ash in the carcasses did not change.
In our work, there were no significant differences in the chemical composition of broiler muscles due to the composition of mixtures and the use of feed enzymes. There were no significant differences in the biochemical parameters of chickens on the basis of blood plasma analyses.
Amino acids digestibility
The increase in the body weight of broiler chickens is mainly determined by the intestinal digestibility of amino acids, including exogenous amino acids. The obtained results showed that substitution of soybean meal in compound feed with rapeseed meal at the level of 11% (starter) and 18% (grower) significantly reduces the apparent digestibility of all amino acids, with the exception of methionine and cystine. These results are consistent with the results of the research carried out by Michalik-Rutkowska (2016), who investigated the digestibility of protein and amino acids at three levels of substitution of soybean meal in compound feed using rapeseed meal and expeller. It is believed that the factors that reduce intestinal digestibility of amino acids, including lysine, are seed oil extraction and
subsequent fermentation of meal (Newkirk and Classen, 1999; Newkirk et al., 2000).
The addition of enzyme preparations of endo-β-1,4-xylanase and endo-1,3 (4) -β-glucanase significantly increased methionine digestibility and decreased proline and arginine digestibility. It did not significantly affect the digestibility of other amino acids. The addition of serine protease increased the digestibility of isoleucine, lysine, methionine, valine, without significantly altering the digestibility ratios of the remaining amino acids. The substitution of soybean meal in compound feed significantly reduced digestibility of lysine. Considering that lysine is the most important amino acid determining muscle growth and protein synthesis in chicken muscles, the deficiency of digestible lysine in Mushtag et al. studies (2007) contributed to the reduction in the weight of the broiler negative control group and other groups receiving feed enzymes, which was confirmed in our work.
To sum up, it should be stated that the use of feed enzymes of the endo-β-1,4-xylanase and endo-1,3 (4) -β-glucanase as well as serine protease in compound feed for broilers containing 11% rapeseed meal ( starter) and 18% (grower) as well as DDGS and fodder yeast, in barothermically non-processed feed mixes, does not significantly increase chicken body weight and carcasses in comparison to diets without these enzymes. Simultaneous administration of these enzymes improved intestinal apparent amino acid digestibility as compared to diets without enzymes, although it was significantly lower, except for methionine and cystine as compared to the digestibility of amino acids of the diets containing extracted soybean meal.
Acknowledgement
The authors express their sincere thanks to Witold Szczurek, PhD, for the instructions and assistance with collecting the samples for the digestibility of amino acids as well as in statistical interpretation of the obtained data. We would also like to thank the technical staff of the Central Laboratory of the Animal Production Institute for carrying out chemical analyses of feed, muscle and blood plasma.
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FRANCISZEK BRZÓSKA, BOGDAN ŚLIWIŃSKI
Effect of feed enzymes on performance, carcass quality and ileal amino acid digestibility in broiler chickens fed diets containing solvent-extracted rapeseed meal
SUMMARY
The aim of the study was to determine the effect of exogenous feed enzymes on body weight, mortal- ity, feed conversion, carcass quality (exp. 1) as well as ileal amino acid digestibility (exp. 2) in broiler chickens fed mash diets containing 11% starter and 18% grower solvent-extracted rapeseed meal. In a growth trial, a batch of day-old sexed Ross 308 chickens were divided into 5 dietary treatments with 8 replicates per sex and 8 chickens per pen, based on the following design: positive control group with solvent-extracted soybean meal (SBM) – without enzymes; negative control group with rapeseed meal (RSM) – without enzymes; experimental groups with solvent-extracted rapeseed meal (RSM), supple- mented with the enzymes endo-β-1,4-xylanase and endo-1,3(4)-β-glucanase (group RSM + e KG), serine protease (group RSM + e P), and all the enzymes (RSM + e KG + e P). Apparent ileal digestibility of amino acids in the four grower diets: SBM, RSM, RSM + e KG, RSM + e P (exp. 2) was determined with 320 Ross 308 cockerels aged 2-4 weeks, which were fed the same diets as in exp. 1.
The rapeseed meal diets fed to the chickens caused a significant decrease in their body weight com- pared to the group fed the soybean meal diet (P˂0.05). The body weight of the chickens receiving the diets with enzymes did not differ significantly from that of the chickens receiving no enzymes. Feeding the rapeseed meal diet without enzymes (RSM) caused a significant increase in chicken mortality com- pared to the positive control group (SBM, P˂0.05). The diet with endo-β-1,4-xylanase and endo-1,3(4)-β- glucanase and the diet with serine protease significantly decreased it (P˂0.05). Feeding the rapeseed meal diet with and without enzymes caused a significant decrease in feed intake (P˂0.05). Feed conversion was significantly better for the rapeseed meal diet with enzymes than for the diet without enzymes (P˂0.05). Treatment factors, rapeseed meal and feed enzymes had no effect on the dry matter and crude protein con- tent of the breast and leg muscles, and on the level of biochemical blood plasma indicators while reducing the level of carcass abdominal fat (P<0.05).
Feeding broilers the rapeseed meal diets compared to the soybean meal diet caused a significant decrease in the digestibility of essential and non-essential amino acids except for methionine and cystine (P˂0.05). Endo-β-1,4-xylanase and endo-1,3(4)-β-glucanase supplemented to the rapeseed meal diet significantly decreased arginine and proline digestibility, and increased methionine digestibility (P˂0.05). The serine protease supplement significantly increased the digestibility of isoleucine, lysine, methionine and valine (P˂0.05), without causing significant differences in the digestibility coefficients of the other amino acids.
