Abstract
The study investigated 1) the effect of in vitro pre-treatment of sunflower meal (SM) and wheat middlings (WM) using fiber degrading enzymes (FDE) without or with protease on mono-sugars release, organic acid concentration (SCFA), organic matter solubilisation, protein solubilization and apparent disappearance (AD) of neutral detergent fiber (NDF). Each feedstuff was mixed with 1% of FDE without or with protease in a ratio of 1:4 wt/wt for feedstuff:water, incubated for 24h and 48h at 40˚C, and freeze-dried. 2) Impact of the inclusion of pre-treated sunflower (TSM) and wheat middling (TWM) in a maize-soybean meal (MSBM) ration on growth performance, tibia attributes, apparent retention (AR) of components and caecal sugars and short-chain fatty acids production. A total of 288 Ross-708 d old male broiler chicks were placed in cages (6 birds/cage) based on body weight (BW) and allocated to 6 diets in a completely randomized design giving 8 replicates per diet. The diets were: I) PC, a MSBM, positive control (PC), II) NC, PC plus untreated sunflower meal (USM) and wheat middling (UWM), and III) 4 test diets in which USM and UWM were replaced with TSM and TWM at 25% (N25), 50% (N50), 75% (N75) and 100% (N100). For pre-treatment, each feedstuff was mixed with 1% of FDE in a ratio of 1:2 wt/wt for feedstuff:water, incubated for 24h at 40˚C, and oven-dried. All diets had titanium dioxide (0.3%) for AR determination. The body weight (BW), feed intake (FI), and mortality were recorded. Excreta was collected and frozen (-20℃). The data were subjected to PROC GLIMMIX of SAS 9.4, and LS means were separated using the Tukey test (P<0.05). The analyses of freeze-dried samples showed improved in-vitro solubilisation of organic matter, increased mono-sugars, organic acids concentration, protein solubilization, and AD of NDF in both SM and WM. Analyses of oven-dried samples showed that pre-treatment increased soluble protein (SP) and decreased neutral detergent protein (NDF-P) and NDF in TWM. Compared to USM, TSM had less SP while NDF-P and NDF were greater. The overall BW and BW gain (BWG) of NC were lesser than PC throughout the study (P ≤0.001). The N75 and N100 BW and BW gain were less than PC (P ≤0.001) throughout the trial. Overall feed intake was not different during the starter, finisher, and overall period (P ≥0.217) relative to PC and NC. Also, feed conversion ratios were not different throughout the study (P ≥0.151). The NC tibia attributes were not different from PC throughout the trial; test diets tibia length was lesser than PC (P = 0.005) and had a linear decrease response with higher inclusion levels (P = 0.004) on d42. The test diets did not affect the AR of CP (P = 0 <0.001); there was decreased apparent NDF retention on d 21 and d 42 (P ≤0.001) and apparent gross energy retention on d 21 and d 42 (P ≤ 0.001) compared to PC. The NC and TD did not affect caecal sugars and SCFA production throughout the feeding period. In conclusion, pre-treating increased in vitro digestibility of sunflower meal and wheat middling, but their pre-treatment did not improve broiler chicken performance and AR of components.
Key Words: Fiber Degrading Enzymes; Broiler; Performance; Component Retention
Table of Contents
Acknowledgments iii
Dedication iv
List of Tables ix
List of Figures xi
Chapter One: Introduction
1.1 Background 3
1.2 Problem statement 5
1.2 Objectives 6
1.2.1 Broad objective 6
1.2.2 Specific objectives 7
1.3 Hypothesis (HO) 7
1.4 Justification 8
Chapter Two: Literature Review
2.1 The poultry industry in Kenya 9
2.2 Use of feed additives in poultry 9
2.2.1 Enzymes 9
2.3 Use of wheat and its by-products in poultry diets 11
2.3.1 Non-starch polysaccharides in wheat milling byproducts 12
2.5 Use of sunflower seed cake in poultry diets 14
2.6 Enzymatic pre-treatment/pre-treating of feeds 15
2.7 Fiber and Chicken gut microflora 18
2.8 Short-chain fatty acids in poultry 19
Chapter Three. The effects of pre-treating wheat middlings and sunflower meal with enzymes on crude protein, apparent disappearance of crude fiber, and concentration of mono-sugars, and organic acids
Abstract 21
3.0 Introduction 23
3.1 Materials and Methods 25
3.1.1 Laboratory analyses 29
3.1.2 Data analysis 30
3.3 Results and discussion 30
3.4.0 Conclusions 47
3.4.1 Recommendations 47
Chapter Four: The effect of inclusion of sunflower meal and wheat middlings pretreated with fiber degrading enzymes in maize-soybean meal diets on growth performance, organ weights, and tibia attributes in broiler chicken
Abstract 48
4.0 Introduction 50
4.1 Materials and Methods 51
4.1.1 Ingredients and pre-treatment 51
4.1.2. Wheat middlings and sunflower meal 51
4.1.3 Diets formulation 52
4.1.4 Birds, housing, and experimental design 52
4.1.5 Measurements of growth performance and sampling 53
4.1.6 Sample processing and laboratory analyses 53
4.2.0 Calculations and statistical analyses 54
4.3.0 Results and Discussion 56
4.3.1 Ingredients and diets 56
3.2 Growth performance 59
4.3.3 Organ weights 66
4.3.4 Tibia attributes 69
4.4.0 Conclusions 71
4.4.1 Recommendations 71
Chapter Five: Effects of inclusion of sunflower meal and wheat middlings pre-treated with fiber degrading enzymes on apparent retention of components and concentration of mono- sugars and short chain fatty acids in ceca digesta of broiler chickens
5.0 Abstract 72
5.1 Introduction 74
5.2.0 Materials and Methods 75
5.2.1 Ingredients and Pre-Treatment 75
5.2.2 Diets formulation 75
5.2.3 Samples Collection 75
5.2.4 Samples Preparation and Chemical Analysis 75
5.3.0 Calculations and Statistical Analysis 77
5.4.0 Results and Discussion 78
5.4.0 Conclusion and recommendation 88
Chapter Six. General discussion, conclusions and recommendations
6.1.0 General conclusions 91
6.2.0 General recommendations 91
References 93
List of Tables
Table 3. 1 Analysed chemical composition of wheat middlings pre-treated with or without fiber degrading enzymes on a dry matter basis 322
Table 3. 2 The pH of and apparent disappearance of NDF, CP, and DM of wheat middlings pre- treated with or without fiber degrading enzymes 34
Table 3. 3 Effects of fiber degrading enzymes on mono sugars and organic acids production in supernatant of pre-treated wheat middlings over time 38
Table 3. 4 Analyzed composition of sunflower meal treated with or without exogenous enzymes on a dry matter basis 40
Table 3. 5 The pH and apparent disappearance (%) of NDF, CP, and DM of sunflower meal pre-treated with fiber degrading enzymes without or with protease 43
Table 3. 6 Effects of fiber degrading enzymes with or without protease on mono sugars and organic acids concentration in the supernatant of pre-treated sunflower meal over time 46
Table 4. 1Composition of experimental diets1, as fed basis 55
Table 4. 2 Analysed composition of enzyme-treated and untreated ingredients, as fed basis 57
Table 4. 3. Analyzed composition of experimental diets1, as fed basis 58
Table 4. 4. Growth performance of broiler chickens fed maize-soybean meal-based diets with pre-treated sunflower meal and wheat middlings 61
Table 4. 5. The economic analysis of the cost of feed intake expenditure cost of body weight gain per kilogram of FI 66
Table 4. 6. Organ weights (% body weight) in broiler chickens fed maize-soybean meal-based diets with pre-treated sunflower meal and wheat middlings 68
Table 4. 7. Tibia characteristics in broiler chickens fed maize-soybean meal-based diets with pre- treated sunflower meal and wheat middlings 70
Table 5. 1. Coefficients of apparent retention of components, metabolizable energy (AME) and excreta moisture content in broiler chickens fed maize-soybean meal-based diets with pre-treated sunflower meal and wheat middlings 83
Table 5. 2. Differences in the analyzed chemical composition of dietary treatments against positive control in starter and finisher phases on a DM basis. 84
Table 5. 3. Concentration (µmol/g) of sugars and short-chain fatty acids (SCFA) in ceca digesta of broiler chickens fed maize-soybean meal-based diets with pre-treated sunflower meal and wheat middlings 87
List of Figures
Figure 3. 1 wheat middlings pre-treatments flowchart. 27
Figure 3. 2 sunflower meal pre-treatments flowchart 28
Chapter One
Introduction
1.1 Background
The poultry industry is an essential component of the food sector. Their products form an essential source of protein in the diets, especially for people in developing countries, and as such, their feed conversion efficiency is paramount (Mottet & Tempio, 2017; Sell-Kubiak et al., 2017). The production of broilers has seen a tremendous increase since the 1980s due to the awareness of the nutritional attributes of poultry meat compared to other meat protein sources (Horn & Süt, 2014).
In developing countries, Kenya included, the poultry industry is being confronted by various challenges, including the high cost of maize and soybean used as conventional feed ingredients (Alagawany et al., 2015). Maize and soybean are also consumed as human food in these countries (Mottet & Tempio, 2017). Due to the lack of quality feed ingredients at affordable prices, poultry production in these countries is low compared with developed countries ( Ravindran, 2013). This has prompted an urgent search for affordable and nutritious feeds. Since feed accounts for approximately 70% of total poultry production cost, researchers have given great attention to using agro-industrial by-products as alternative feed resources (Alagawany et al., 2018; Alagawany & Attia, 2015). The use of by-products has been intensified by the shortage of conventional raw materials such as maize, which is now also used to produce biofuels (Zhang & Lis, 2020), and soybean price keeps on fluctuating due to fuel market volatility, thus becoming unreliable (Mutisya et al., 2021).
In Kenya, the common poultry feed ingredients are maize and maize milling by-products (maize germ), soybean meal, and fish meal (‘omena’) (Abro et al., 2020). Most feed ingredients are selected based on the availability, nutrients they can provide, the presence or absence of anti- nutritional factors, palatability or their effect on voluntary feed intake and cost (Raza et al., 2019).
Apart from whole cereals, their milling by-products such as wheat middling’s (also known as pollard), wheat bran, and rice bran are used in developing countries to lower the cost of production by acting as a source of energy (Bhattacharjee & Dey,2012; Supriyati et al., 2015; Osunbami, 2021). By-products such as sunflower seed cake, rapeseed meal, canola meal have also attracted attention as potential targets for replacing soybean meal in broiler diets (Biesek et al., 2020; Mbukwane et al., 2022).
The main limitation of using the agricultural by-products is the presence of anti-nutritional factors: cellulose, hemicellulose, and non-starch-polysaccharides (NSP), for which poultry lacks endogenous enzymes that can hydrolyze them (Raza et al., 2019). This has led to the development of exogenous enzymes that can hydrolyze these NSP, thus allowing nutrients to the bird (Fafiolu et al., 2015). The use of enzymes has enabled the poultry industry to save billions of shillings and, at the same time, reduce environmental pollution from unutilized nutrients in poultry faecal wastes (Dosković et al., 2013). Numerous studies have shown that poultry performance and feed conversion ratio improve when diets containing high NSP such as sorghum, barley, and wheat or triticale are supplemented with exogenous enzymes. Saleh et al. (2020) showed that the performance of birds feeding on olive seed cake was improved when an exogenous enzyme was added to it.
Enzymes have also been used in poultry diets to improve broiler performance and gut health (Kiarie et al., 2013). Exogenous enzymes help fight coccidiosis through short-chain fatty acids production in the intestines and overcome coccidia’s adverse effects on bones (Kiarie & Mills, 2019). Although the use of enzymes in poultry feeds has become a norm, there are physiological limitations such as pH and feed gut transit time (less than 90 minutes in proventriculus/gizzard (Ravindran, 2013) that affects their response (Rodrigues & Choct, 2018). These limits affect even highly digestible ingredients containing starch as only about 90% is digested; it would still be unrealistic to expect 100% nutrient digestion after directly supplementation enzymes (Ravindran, 2013). Researchers are now attempting to use feeds pretreated with enzymes to overcome these limitations (Rahimi et al., 2020). Due to the reduced retention time and the increased use of byproducts high in crude fiber, there is a limited time for enzyme-substrate interaction when exogenous enzymes are directly mixed (premixing) with poultry feeds (Ravindran, 2013). Pre- treatment with FDE of these ingredients high in crude fiber outside the bird GI system would provide a controlled environment which is optimum for fiber degrading enzymes to hydrolyse the crude fiber (Rahimi et al., 2020).
1.2 Problem statement
For quality broiler meat that is affordable to be produced there is a need for a nutritionally balanced diet that is cost-effectively formulated as feeds account for greater than 60% of total variables of broiler production (Sonkar et al., 2020). However, the poultry industry faces a feed shortage due to overreliance on maize and soybean meal which are only produced by a few countries (Wahyono & Utami, 2018). In Kenya, maize, fish meal, and soybean meal are the primary energy and protein sources used in poultry feeds and are mostly imported (Abro et al., 2020; Gakuya et al., 2014). Byproducts such as sunflower meal and wheat middlings, which are relatively cheap, are increasingly being used to partially replace soybean and maize, respectively, thus reducing the cost of broiler production (Gerzilov & Petrov, 2022; Abudabos, 2016; Mbukwane et al., 2022). Sunflower meal has particularly been pointed out as the soybean alternative in regions that don’t produce enough (Ditta & King, 2017a).
With the evolution of intensive feeding, the role of the gizzard and crop has been ignored as they are now more of transit organs, which affects feed retention time in the bird and enzyme activity (Kiarie & Mills, 2019). Due to the reduced retention time and the increased use of byproducts high in crude fiber, there is a limited time for enzyme-substrate interaction when exogenous enzymes are directly mixed (premixing) with poultry feeds (Ravindran, 2013). Most enzymes have an optimum pH of between 4.0-6.0, and in the chicken gut, the pH varies a lot (gizzard 2.0-4.0 and small intestine pH between 6.5-7.5) (Svihus, 2011; Ndou et al., 2015). Some exogenous enzymes, such as phytase, are only considered effective in the crop (where the pH is optimum) (Sommerfeld et al., 2018). Hence more research is needed on a combination of feeding strategies, exogenous enzymes, and the structure of feeds to overcome the gut limitations (Kiarie & Mills, 2019; Rodrigues & Choct, 2018). Pretreatment of meals with heat, 0.5% HCl and enzymes is an effective method of ensuring nutrients are available to chickens more than conventional methods of enzyme application (pre-mixing) alone (Rahimi et al., 2020). Still, more studies on pretreatments are required to provide practical ways of decreasing antinutrients in feeds (Rahimi et al., 2020).
The above shows that there is a need to enhance the use of non-conventional animal feed ingredients to improve their nutritional quality, ensure stability in the price and supply of mixed feeds. This would further alleviate hunger, malnutrition, and poverty as there would be affordable, high-quality protein from broilers. Agricultural byproducts such as wheat pollard, rice bran, and sunflower seed cake readily available in Kenya would be suitable byproducts whose nutritional value can be improved through enzymatic pretreatment.
1.2 Objectives
1.2.1 Broad objective
To evaluate the effect of enzymatic pre-treatment of high crude fiber feedstuffs on nutrient digestibility and performance of broiler chickens.
1.2.2 Specific objectives
1. To evaluate the effect of in vitro pre-treatment of sunflower meal (SM) and wheat middlings (WM) on mono-sugars, organic acids and short-chain fatty acid concentration, protein solubilisation and apparent disappearance of neutral detergent fiber.
2. To evaluate the effect of inclusion of enzymes pre-treated SM and WM in a maize-soybean meal diet on growth performance, breast muscle weight, and tibia attributes of broiler chicken.
3. To evaluate the effect of inclusion of enzymes pre-treated SM and WM in a maize-soybean meal diet on apparent retention of components, cecal sugars, and short-chain fatty acids production in broiler chicken.
1.3 Hypothesis (HO)
1. Pre-treating sunflower meal (SM) and wheat middlings (WM) has no effect on mono sugars, organic acids, and short-chain fatty acids concentration in the supernatant, protein solubilization apparent disappearance of neutral detergent fiber.
2. Inclusion of enzymes pre-treated SM and WM in a maize-soybean meal diet has no effect on growth performance, breast muscle weight, and tibia attributes of broiler chicken.
3. Inclusion of enzymes pre-treated SM and WM in a maize-soybean meal diet has no effect on apparent retention of components, cecal sugars, and short-chain fatty acids production in broiler chicken.
1.4 Justification
Maize and soybean meal are the commonly used conventional poultry feed ingredients due to their superior nutritional quality. These traditional ingredients are expensive due to competition between humans and animals for the same ingredients (te Pas et al., 2021; Woyengo et al., 2014), and maize is currently being used to produce biofuels (Woyengo et al., 2014). Due to the unavailability of conventional ingredients at a relatively low price, there is a need to seek other alternatives (Laudadio et al., 2014). Most developing countries have started focusing on using agricultural by-products, which are abundant, as alternatives to conventional feedstuffs (Alshelmani et al., 2021). However, these by-products are rich in non-starch polysaccharides (NSP) such as mannans, arabinose, and xylans which suppresses the bird’s performance (Aftab & Bedford, 2018), because of increased gut viscosity (Singh & Kim, 2021a). The poultry industry has recognized the value of exogenous enzymes in improving the growth performance and efficiency of feed utilization in the non-conventional feedstuffs ( Adeola & Cowieson, 2011; Alagawany et al., 2018; Cozannet et al., 2017 ).
There is a short retention time of feeds in the poultry digestive system, and therefore the results of enzymes directly pre-mixed with feeds can vary (Sacranie et al., 2017). The use of exogenous enzymes in pre-treatment has the potential to solve these problems (Denstadli et al., 2007; Matshogo et al., 2021). Boroojeni et al. (2017) conducted an experiment showing that enzymatic pretreatment of peas drastically improved standardized ileal digestibility of nutrients in turkey.
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