Fats in poultry nutrition: Digestive physiology and factors influencing their utilisation

Accepted Manuscript Title: Fats in poultry nutrition: Digestive physiology and factors influencing their utilisation Author: V. Ravindran P. Tancharoenrat F. Zaefarian G. Ravindran PII: DOI: Reference:

S0377-8401(16)30029-3 http://dx.doi.org/doi:10.1016/j.anifeedsci.2016.01.012 ANIFEE 13457

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Animal

Received date: Revised date: Accepted date:

8-10-2015 13-1-2016 14-1-2016

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Please cite this article as: Ravindran, V., Tancharoenrat, P., Zaefarian, F., Ravindran, G., Fats in poultry nutrition: Digestive physiology and factors influencing their utilisation.Animal Feed Science and Technology http://dx.doi.org/10.1016/j.anifeedsci.2016.01.012 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Fats in poultry nutrition: Digestive physiology and factors influencing their utilisation V. Ravindrana* [email protected], P. Tancharoenrata, F. Zaefariana, G. Ravindranb a

Institute of Veterinary, Animal and Biomedical Science, Massey University,

Palmerston North 4442, New Zealand b

Institute of Food, Nutrition and Human Health, Massey University, Palmerston North

4442, New Zealand *

Corresponding author: Tel: +64 6 350 5528; fax: +64 6 350 5684

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Highlights 

Digestion and absorption of fat is a complex process.

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Emulsification is central to efficient fat digestion.

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Digestion is strongly related to the chemical structure of fats.

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The metabolisable energy of fat sources is variable.

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Nutritional emulsifiers can improve fat utilisation

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Abstract Fats and oils possess the highest caloric density of all known nutrients. In recent years, because of the ever-increasing energy costs, there is greater interest in maximising the use supplemental fats as nutritionists strive to increase the dietary energy density to meet the requirements of high-performing contemporary birds. To maximise their energy yielding potential, there is a need to better understand the physiological basis and factors affecting fat digestion. Compared to other macronutrients, the digestion and absorption of fats is a complex process and involve sequence of physicochemical events requiring breakdown to fat droplets, emulsification, lipolysis and micelle formation. Current knowledge of the principles of fat digestion and absorption in poultry is reviewed, along with factors influencing available energy content of supplemental fats. The supplemental fats are one of the most difficult ingredients to evaluate in terms of available energy. Important variables influencing the energy content of fats include age of the birds, degree of fat saturation, chain length, free fatty acids and fat inclusion level. Potential strategies to improve fat utilisation in poultry diets are also examined.

Abbreviations AME: apparent metabolisable energy FA: fatty acid FABP: fatty acid-binding protein FFA: free fatty acids HLB: hydrophilic-lipophilic balance MIU: moisture: insoluble impurities and unsaponifiables NSP: non-starch polysaccharides TME: true metabolisable energy 3

US: unsaturated: saturated.

Keywords: absorption; apparent metabolisable energy; digestion; emulsification; supplemental fats, poultry

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1. Introduction The term ‘fat’ is generally used as a synonym for lipid. Both terms describe a diverse variety of compounds that are insoluble in water, but dissolve in organic solvents such as chloroform, acetone, alcohol and diethylether. Lipids play an important role in the nutrition, biochemistry and physiology of animals (Brindley, 1984). From the nutritional point of view, lipids of importance are triglycerides, phospholipids, sterols and fat-soluble vitamins. Because of the rising cost, there is an increased interest in recent years in maximising the use of fat supplements in the diet as nutritionists strive to increase the dietary energy density to meet the requirements of fast growing birds. Fats are the preferred ingredients for this purpose as their energy value is at least twice as high as those of carbohydrates and protein (NRC, 1994). The dietary addition of fats also confers other advantages, including reduced dustiness, lower particle separation in mash diets, improved palatability, carriers for fat soluble vitamins, supply of the essential fatty acids (FA) and lubrication of feed milling equipments. Additionally, supplemental fat slows down the rate of feed passage through the digestive tract (Mateos and Sell, 1981b), allowing more time for better digestion and absorption of nutrients. A diverse array of fats and oils are available for use in feed manufacturing and these include restaurant greases (e.g. recovered frying oils; also known as yellow grease), rendering by-products (e.g. lard, tallow, mutton fat and poultry fat), vegetable oils (e.g. soybean oil, maize oil and palm oil), acidulated soapstocks (by-products of vegetable oil refining, mainly containing free FA), hydrogenated fats (fats or oils which are converted to saturated FA by the addition of hydrogen atom to double bonds of unsaturated FA), and acidulated soapstocks (free FA removed from the refining process by alkali and settled as alkali soaps). These fats and oils vary widely in terms of

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composition (Table 1). The choice of fat to be used, under a given commercial condition, is largely driven by its cost. Some FA are termed as essential for poultry because the birds are unable to synthesise or convert one FA to another FA within the same series (Enser, 1984). The essential FA include linoleic acid (C18:2), linolenic acid (C18:3) and arachidonic acid (C20:4) and need to be supplied in the diet. The deficiency of these essential FA may result in impairments in growth and immune system function. Symptoms of linoleic acid deficiency in poultry include retarded growth, increased water consumption and reduced resistance to diseases (Balnave, 1970). In male birds, deficiency symptoms also include lower testes weight and delayed development of secondary sexual characteristics. Decreased egg size is the major outcome of deficiency in laying hens (Watkins, 1991). To ensure adequate supply of these essential FA, a minimum inclusion level of 10 g/kg fat in poultry diets has been suggested by Leeson and Summers (2005). 20 to 50 g/kg fat is usually added in commercial poultry diets depending on the relative prices of fat and cereal grains. The addition of fat above 40 g/kg is generally avoided in pelleted diets because of the negative effects on pellet quality (Abdollahi et al., 2013a). With new technologies, however, it may be possible to add more than 40 g/kg fat in these diets. The influence of supplemental fats and oils on the carcass characteristics, particularly on fat deposition and carcass FA composition, of broiler chickens is well studied, but it is out of the scope of the current review. For example, Crespo and EsteveGarcia (2001) reported that broilers fed diets containing tallow had higher contents of saturated FA in the abdominal fat pad, thigh muscle and breast muscle than those fed diets supplemented with olive oil, sunflower oil and linseed oil. The observed changes in FA composition are due the direct incorporation of dietary FA into adipose tissues.

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Some FA are recognised as modulators of immune responses. Fritsche et al. (1991) reported that antibody titers to sheep red blood cells in pullets fed fish oil were higher than those fed lard, maize oil, canola oil and linseed oil. Dietary lipid source may influence immunocompetence via two primary mechanisms: (i) by affecting membrane FA composition and therefore the fluidity and function, and/or (ii) by affecting the inflammatory process and other cell-signaling pathways (Trushenski and Lochman, 2009). There is also current interest in the activity of medium-chain FA on gut microflora. Fatty acids, such as 1-monoglyceride of capric acid (monocaprin), have been found to be particularly effective in controlling Campylobacter jejuni (Thormar et al., 2006). Supplementation of 7 g/kg caprylic acid in feed reduced caecal counts of Campylobacter in broilers compared to the unsupplemented control (de los Santos et al., 2008). Despite their broad acceptance, feed grade fats remain the least understood of common feed ingredients. This lack of appreciation is entrenched in the diverse nature of fat sources, lack of uniformity and the complex nature of published data on the factors influencing their available energy content. The following is an overview of the current understanding of digestion and absorption of fats in poultry. Fat digestion and absorption occur in several steps and involve breakdown into droplets, emulsification, hydrolysis by pancreatic lipase and mixed micelle formation and, the movement of micelles towards gut epithelium and removal of end-products. These aspects have been considered in earlier reports (Freeman, 1984; Krogdahl, 1985; Drackley, 2000), which provide the background for the present paper. The intention of the present review is to link together these recent findings and reappraise various aspects of fat digestion in poultry diets and to reflect their implications. A detailed examination of the major

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factors limiting the digestion and available energy content of supplemental fats, and of potential strategies to improve lipid utilisation in poultry diets is considered. 2. Digestion and absorption of fats 2.1. Digestion of fats The major component in fats is triacylglycerol, which is a molecule of glycerol plus three FA. The term ‘fat’ refers to triacylglycerols that are solid at room temperature, whereas the term ‘oil’ refers to triacylglycerols that are liquid at room temperature (Enser, 1984). During digestion, two of the FA molecules from the triglyceride are removed, leaving a monoglyceride (a glycerol molecule with one FA attached). Thus, the hydrolysis of triglycerides produces a monoglyceride and two FA, which are the absorbable units of fat. 2.1.1 Role of gizzard Lipid digestion in poultry is initiated with the grinding action of in the gizzard. The feed entering the gizzard is reduced in size mechanically by grinding and vigorous mixing. A unique feature of birds is gut refluxes (known as reverse peristalsis) as an adaptation to fly to minimise the gut size and the action of gizzard controls these fluxes. Through these refluxes, the limitation of shorter digestive tract in birds is compensated and feed retention time is increased to provide more time for digestion. Reverse peristalsis in poultry occurs in three distinct regions of the gut, namely the gastric flux, the small intestinal flux and the cloaca-caecal flux (Duke, 1986; 1994). The activity of the gizzard controls the first two fluxes, with the first reflux moving the digesta between the gizzard and proventriculus once for each gastro-duodenal contraction cycle. Gizzard movements follow contractions of the proventriculus (Lentle et al., 2013). The second flux moves the digesta from the duodenum and jejunum back into the gizzard. The characteristic yellow staining of gizzard lining is an evidence of this reflux. In chickens, the reflux process is continuous, enabling penetration of the gizzard by duodenal 8

contents during the contractile period of the gizzard (Sklan et al., 1978). The presence of bile salts and monoglycerides from digesta refluxed from the duodenum initiates fat emulsification in the gizzard. This initiation is further facilitated by the proteolytic activity of pepsin in the proventriculus and gizzard which releases lipids from cell wall matrices. The acid conditions, peptic digests of protein and the mechanical activity of the gizzard serve further to disperse the lipids into a coarse emulsion. 2.1.2 Bile secretion Bile, excretory fluid of the liver, is formed in hepatocytes and then transported for storage in the gallbladder (Koeppen and Stanton, 2008) and delivered into the intestine at the duodenum. It contains bile pigments, bile salts, phospholipids, cholesterol, electrolytes and some proteins (Krogdahl, 1985). The primary components of the bile needed for lipid digestion are bile salts and phospholipids (Horace and Davenport, 1980). In poultry, bile salts are conjugated with taurine in the liver, which increases their solubility in water and also decreases the cellular toxicity of bile salts. The digestion and absorption of fats present unique problems because of their insolubility in water. Water and the fat do not mix, and the major function of bile is to reduce the tension at the oil-water interface to enable this mixing process. This step assists in the emulsification and activates pancreatic lipase as well as prevents denaturation of lipase when it leaves the surface of emulsified fat droplets (Chen et al., 1975). Bile salts are flat amphiphilic molecules, with one side being non-polar and hydrophobic surface (that interacts with water) and other side being polar and hydrophilic surface (that interacts with the oil phase of the emulsion). Because of this unique characteristic, bile salts lie at the water-lipid interface and do not penetrate deeply into either surface.

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Feed fat enters the intestine as rather big coagulated particles. The presence of bile imparts detergent-like effects on dietary lipids causing this coagulated mass to be broken into very fine droplets that are stable (i.e. prevent coalescence). The overall aim of this event is to increase the total surface area for the action of lipase. The stimulus for bile secretion is the presence of fat in the intestinal chyme. Bile secretion is reported to be influenced by the type and level of dietary fat (Lindsay et al., 1969). Secretion of bile is thought to be limited in young birds, especially during the first week after hatching (Noy and Sklan, 1995). Following fat digestion, released bile acids remain within the lumen and are recaptured through a process known as enterohepatic recycling (Horace and Davenport, 1980). This phenomenon refers to the circulation of bile from the liver, where it is produced, to the small intestine, where it aids in lipid digestion, and back to the liver via an active as well as a passive transport mechanism. Uptake of bile salts has been observed to occur from the jejunum and ileum in chickens, with rates of bile absorption being similar in these two segments. Passive diffusion accounts for most of the absorption of bile salts (Sklan et al., 1974). It is thought that around 95% of bile salt is re-circulated and that this recycling is critical for the efficient digestion and absorption of fats. Any impairment in bile re-circulation will have adverse effects on fat utilisation.

2.1.3. Pancreatic lipase Lipase is one of the digestive enzymes (trypsin, chymotrypsin, amylase and, phospholipases A1 and A2) secreted by pancreas. Specificity for the FA in the sn1- and sn3-positions of glycerol backbone is an important property of pancreatic lipase. The enzyme acts as a catalyst only when it appears on the surface of emulsified fat droplets along with bile salts and co-lipase, a co-factor present in pancreatic juice (Erlanson et al., 1973). Co-lipase itself has no enzyme activity but it is necessary to initiate the 10

activity of pancreatic lipase (Borgström and Erlanson, 1971). Co-lipase is rich both in hydrophobic and hydrophilic amino acids, and interacts with lipase to form a more hydrophobic, less-charged complex; this make it possible to maintain the lipase in an active configuration at the lipid-water interface and enable the lipase to reach its substrate. It is thought that the charge characteristics of co-lipase enable it to bind to the surface of fat droplets and act as an anchor for lipase allowing the enzyme to act on the triglycerides. Co-lipase and bile salts are competitive inhibitors for binding sites on the substrate. Pancreatic lipase activity is inhibited by the high concentrations of bile salts (Bosc-Bierne et al., 1984), but this is restored by co-lipase. The activity of lipase is reported to be influenced by the saturation of free FA generated by lipolysis. The presence of double bonds (unsaturation) causes changes in the three-dimensional structure, with each double bond resulting in a bend. van Kuiken and Behnke (1994) suggested that the FA binding site in lipase require the FA to bend at a 141° angle, but saturated FA have an angle of 180° which make them difficult to bind with lipase. As a result, unsaturated FA, which have an angle of approximately 141° at the site of double bond, have a greater ability to increase lipase activity compared to long chain saturated FA. The result being oleic (C18:1) and linoleic (C18:2) acids greatly increase lipase activity, whereas long chain saturated FA such as stearic acid (C18:0) have inhibitory effects (Larsson and Erlanson, 1981, 1986). The digestion of fat is greatly accelerated by the entry of digesta into the duodenum. The presence of fat in this segment of the gastrointestinal tract stimulates the secretion of cholecystokinin which in turn regulates the secretion of pancreatic juice and bile (Krogdahl, 1985). Cholecystokinin also stimulates the release of bile from the gall bladder (Wang and Cui, 2007).

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2.2. Absorption of fats The jejunum is the major site of digestion and absorption of fat in poultry (Hurwitz et al., 1973; Tancharoenrat et al., 2014), with the digestion continuing in the upper ileum (Figure 1). The absorption of fat is reported to be negligible in the hindgut (Renner, 1965). Tancharoenrat et al. (2014) observed differences between FA in terms of site of digestion and absorption. Linoleic acid was absorbed throughout the intestinal tract starting from the duodenum, whereas the absorption of palmitic, stearic and oleic acids started only in the jejunum. The exact reasons for these differences are not clear, but can be explained, in part, by the insufficiency of bile, as bile ducts in chickens enter only at the distal end of duodenal loop (Duke, 1986). In addition, passage time in the duodenum of chickens is very short (Ravindran, 2013), which may not give sufficient time to emulsify the saturated FA. The key to the absorption of lipolysis end-products (FA from the sn-1 and -3 positions, and the sn-2-monoacylglycerol) is the formation of mixed lipid-bile salt micelles, which is based on a complex series of chemical interactions. The FA and monoglycerides are spontaneously removed from the water-oil interface by incorporation into mixed micelles, which are water soluble aggregates of lipid molecules containing both polar and non-polar groups. Molecules are grouped in the micelles in such a way that the polar groups are on the outside in contact with the aqueous phase, while non-polar parts form the inner core. The FA liberated during digestion differ in their ability to form mixed micelles (Freeman, 1969), with unsaturated FA forming mixed micelles more readily than long chain saturated FA. The presence of just one double bond, due to its bending effect on the three-dimensional structure, is thought to be sufficient to enhance the ease with

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which micelles are formed. Another noteworthy feature is the ability of unsaturated FA to function as natural emulsifiers and form mixed micelles with saturated FA (Freeman, 1969). Micelles facilitate absorption by providing a high concentration of lipids in the unstirred water layer adjacent to the mucosal cells; when they come into contact with microvilli, they are disrupted and can be passively absorbed into the cells. Monoglycerides from the digestion of triglycerides also play an important role in fat absorption. In the absence of monoglycerides, nonruminants are not able to absorb many FA. Both bile salts and monoglycerides have portions of their molecular structure that can interact with aqueous systems (such as the fluid in the intestinal lumen) as well as lipids and form an interface between lipids and water. It must be noted, however, that the chemical considerations relating to micelle formation are quite complex and not well understood. Once hydrolysed, short-chain FA and monoglycerides need no emulsification and are absorbed passively from the intestinal lumen via the enterocytes (Pond et al., 2005). On the other hand, medium- and long-chain saturated FA, diglycerides, fat soluble vitamins and cholesteryl esters require solubilisation in the hydrophobic cores of mixed micelles (Davenport, 1980). These lipolytic molecules conglomerate into micelles with the hydrophobic components inwards and the hydrophilic components turned to the aqueous digesta fluid. The micelles make the fatty constituents soluble and enable them to move through the aqueous intestinal environment. Fatty acids incorporated into micelles are able to create a much higher diffusion gradient locally at the intestinal wall and then transported to the intestinal cells. The movement of FA through the cytosol of the absorptive cell seems to be influenced by a family of soluble intracellular proteins called FA-binding proteins

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(FABP; Ockner et al., 1972). In chickens, Katongole and March (1979) found that the concentration of FABP was highest in the proximal portion of the intestine. These proteins have greater affinity for unsaturated than for saturated FA and has almost no affinity for medium or short chain FA (Ockner and Manning, 1974). Fatty acid-binding proteins also function as a protective mechanism for the absorptive cell because free FA are potentially cytotoxic (Shiau, 1981). Within the enterocytes, monoglycerides and long chain FA are re-esterified, combined with free and esterified cholesterol, lipoprotein and phospholipids to form chylomicrons and secreted into lymphatic vessels. Since the lymphatic system of poultry is poorly developed, the chylomicrons are secreted directly to the portal circulation and are termed as portomicrons (Hermier, 1997). Portomicrons are transported to various tissues, particularly the liver, where lipids are used in the synthesis of various compounds required by the body such as lipoprotein and phospholipids, metabolised as source of energy or stored in tissues as fat depots (Scott et al., 1982). Overall, the digestion and absorption of fats is a complex process requiring adequate amounts of bile salts, pancreatic lipase and co-lipase. Lack of any one of these essentials will impair the digestion and absorption processes.

3. Endogenous fatty acid losses There is a continuous secretion of endogenous lipids into the lumen of the intestinal tract. The primary sources of this endogenous fat are bile and desquamated intestinal epithelial cells (Clement, 1980). These endogenous lipids mix with dietary lipids and, are partially digested and absorbed. The unabsorbed fraction passing beyond

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the ileum is a loss to the animal and the measurement of these inevitable losses is necessary to calculate the true digestibility of FA. Tancharoenrat rat et al. (2014) recently quantified the ileal endogenous losses of fat and FA in broilers to be 1,714 and 825 mg/kg DM intake, respectively (Table 2). The major saturated FA in the ileal endogenous fat were palmitic and stearic acids, whereas the main unsaturated FA were oleic, linoleic and arachidonic acids. Interestingly, the FA profile of ileal endogenous fat measured in their study corresponded closely to that of the bile, possibly suggestive of incomplete re-absorption of fat and FA in the bile. Only 48% of the endogenous fat was accounted by FA. The balance (52%) comes from non-FA sources (Tancharoenrat et al., 2014). The non-FA fraction may originate from bile acids, cholesterol, bile pigments, lipid-soluble intermediates and end products, and phospholipids in the bile (Tuchweber et al., 1999; McKee and McKee, 2009).

4. Apparent metabolisable energy of fats Because of its practical relevance, the apparent metabolisable energy (AME) of different supplemental fat sources has been determined in a number of studies and selected data are summarised in Table 3. The reported values are highly variable and difficult to deal with in practice, presenting a major problem for feed formulators. There is a large divergence in the efficiency with which various fats are digested by poultry and it is evident that the different fats are not chemically similar or biologically equivalent. The logical approach to better understand the differences involves an examination of factors influencing the AME and these include inter alia assay methodology, age of birds and fat characteristics.

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4.1. Methodology Evaluation of supplemental fats in terms of their true energy contribution to practical diets is a challenging exercise. Broadly two methodologies are used to determine the AME of fats in poultry diets and a discussion on the relative merits and drawbacks of these methods has been provided by Mateos and Sell (1981a) and Irandoust et al. (2012). In the first method, the digestibility of the fat is determined by the total collection of excreta and relating the dietary lipid intake to the lipid output in the excreta (the amount of lipid consumed minus the amount of lipid excreted divided by the amount of lipid consumed). Indigestible markers have also been used in some studies to determine the fat digestibility. The AME of the supplemental fat is then calculated by multiplying the gross energy content of the fat by the digestibility. In the second method, the fat is substituted into a basal diet at low levels. A test diet is then developed by replacing (weight/weight) the basal diet by the test fat and, the AME is calculated as the difference between the value determined for the basal diet and that for the test diet (basal diet plus test fat). Results obtained by these two methods, however, do not always agree (Mateos and Sell, 1981a) and, consequently, estimates of the AME of fats diets have been variable. Both these methods assume that lipid digestion is independent of the composition of the diet and that the supplemental fat does not alter the utilisation of other dietary constituents. These assumptions, however, are not always correct. The best known example is the interaction between wheat non-starch polysaccharides (NSP) and fat digestibility (Ward and Marquardt, 1983). Sibbald and Kramer (1978) observed that the true metabolizable energy (TME) of tallow was greater in maize- than in wheatbased diets. A synergism between saturated and unsaturated fatty acids, due to the

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natural emulsifying effects of the latter, is also recognised (Lall and Slinger, 1973; Sibbald, 1978) The determination of AME of supplemental fats also suffers from number of highly specific imperfections. First, because of their physical nature, they cannot be tested in isolation of other ingredients, needs to be assayed along with a basal diet and hence an assumption be made that there is no interaction between dietary components and the supplemental fat. However, as noted above, several cases of non-additivity are recognised. Second, fats can be included in the test diet only at low levels, typically at 30 to 50 g/kg, which represent the normal rate of fat inclusion in practical diets. Thus large errors of extrapolation, inherent in substitution methods, are unavoidable (Slinger and Sibbald, 1963a). Finally, the level of fat inclusion itself affects fat digestibility and impact the determined AME contents. Fats are more efficiently utilised at low rates of intake and, the consequence is that the determined AME would be higher at low inclusion levels and vice versa (Wiseman, 1984). Some researchers have used graded levels of fat inclusion and regression analysis to overcome the influence of inclusion levels (Veira et al., 2015). Wiseman et al. (1986) was of the opinion that, since lipids do not always respond in a linear fashion to increasing inclusion levels, that the AME must be determined at several inclusion levels to account for these effects. Overall, practical solutions to address the above limitations of fat AME determination are arduous and methodological differences will remain an important variable contributing to published AME content of supplemental fats.

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4.2. Bird-related factors 4.2.1. Age In the newly hatched chick, the ability to digest and absorb dietary fat is poorly developed. The secretion of bile appears to be the first limiting and lipase secretion, FABP synthesis or other physiological factors may be the next-limiting (Krogdahl, 1985). The biliary secretion is low in early life of chicks and increases with age. Smallwood et al. (1972) attributed the poor fat digestibility to inefficient recirculation of bile salts and the resultant small bile salt pool size in the young chick. It appears that the chicks are unable to replenish bile salts lost by excretion as readily as older birds (Serafin and Nesheim, 1967), because they have limited ability to synthesise bile acids. Although duodenal bile secretion is reported to increase more than 2-fold between d 4 and 7 and between d 7 and 10 posthatch (Noy and Sklan, 1995), it appears insufficient to support the emulsification needs. Nitsan et al. (1991) reported that the activity of all pancreatic enzymes (when expressed as units of activity per kilogram of bodyweight) increased with age, reaching a maximum on day 8 for the lipase. Noy and Sklan (1995) reported that the secretion of lipase, trypsin and amylase into the duodenum increased 20 to 100-folds between days 4 and 21 post hatch, but the increase in lipase activity was slower than those of other enzymes. Furthermore, although lipase secretion increases as the bird ages (Noy and Sklan, 1995), the secretion per unit of feed ingested may not be adequate for maximum fat digestion (Sklan, 2001). Krogdahl and Sell (1989) reported that the development of intestinal lipase activity is dependent on dietary fat level, with low activities being observed in birds fed diets containing low levels of fat. The synthesis of FABP has also been reported to be insufficient in very young birds, but increased after four weeks (Katongole and March, 1980).

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In general, the available data indicate that the ability to digest and absorb fat in chickens is poor at hatch, develops rapidly after week 1 and increase with advancing age (Carew et al., 1972; Renner and Hill, 1960). Renner and Hill (1960) investigated the utilisation of maize oil, lard and tallow by chickens at different ages and found that the ability to utilise tallow improved with age. The absorbability of tallow improved from 70% at 2 weeks of age to 82% at 8 weeks of age. However, the utilisation of maize oil and lard peaked at 6 weeks of age and declined two weeks later. The absorbability of maize oil was 94% at 2 weeks, increased to 98% at 4 and 6 weeks, and then decreased to 95% at 8 weeks of age. The absorbability of lard improved from 90% at 2 weeks to 95% at 6 weeks and then decreased to 92% at 8 weeks of age. Carew et al. (1972) determined the absorption of maize oil and beef tallow during the first 2 weeks of life. The ability to absorb maize oil and tallow was found to be low during the first week and increased during week 2. The absorbability of maize oil increased from 84 to 95%, while that of tallow increased from 40 to 79% between weeks 1 and 2. Wiseman and Salvador (1989) determined the AME of fats in broilers fed diets containing vegetable oil and tallow at 25, 50, 75, 100 and 125 g/kg inclusions at 2, 4, 6 and 8 weeks of age. The AME of both fat sources increased between 2 to 4 weeks of age, with no further increase thereafter. The increments were greater in tallow than in vegetable oil. In a subsequent study, Wiseman (1990) determined the AME of two dryemulsified fats (fat blended with bone solids, homogenised and then emulsified by spray drying). A maize-wheat-soy basal diet was supplemented with either emulsified fat A (blend of soybean oil and tallow) or emulsified fat B (tallow) at 25, 50, 75 100 and 125 g/kg. Diets containing fat A were fed to broilers aged 2, 4 and 6 weeks, while those containing fat B was fed to broilers of 3, 5 and 7 weeks of age. The results showed that

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the AME of both fats was higher in older birds and that the AME were highest at the lowest rate of inclusion (25 g/kg). The limited capacity of newly hatched chicks to digest fats has been confirmed in a recent study from our laboratory (Tancharoenrat et al., 2013; Table 4). In this study, the influence of age of birds on the AME and total tract fat digestibility of five fat sources (tallow, soybean oil, 50:50 blend of tallow and soybean oil, poultry fat and palm oil) in contemporary broiler genotypes was investigated. The AME of all fat sources was markedly lower during week 1, but increased during week 2. There were no further increases after week 2. The patterns observed for total tract digestibility were somewhat similar to those of AME. The major implication of these findings was that the use of a single AME value for all growth phases of broilers is fraught with serious flaws and the values determined with older birds are not applicable to young broilers, especially during week 1. These observations, considered together with previous data based on practical poultry diets (Thomas et al., 2008), emphasise the need to use age-dependant AME values for ingredients in feed formulations.

4.2.2. Gender, breed and species Differences in dietary nutrient requirements between different breeds, gender and poultry species are recognised. It follows that similar differences may exist in the efficiency of digestion of nutrients. Gender effects on the AME of oats, tallow and fish meal for chickens have been noted by Guirguis (1975; 1976), with the values being higher in females. In contrast, Yaghobfar (2001) determined the AME of maize for males and females of a layer (Rhode Island Red) and a broiler (Cornish) line, and found that gender had no effect. Zelenka (1997) investigated the effect of gender on the AME of two diets with different

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energy: protein ratios in broilers from 12 to 56 days of age. No differences were observed between the males and females. The effect of breed on energy utilisation was examined by Sibbald and Slinger (1963b). It was found that White Leghorns utilised more energy per unit of feed compared with White Rocks and attributed this to genetic differences in the ability to digest and absorb nutrients. Katongole and March (1980) determined the utilisation of tallow and maize oil in different genetic strains between weeks 3 and 11 of age and found that the absorbability of tallow and maize oil was higher in New Hampshires than in broiler-type or White Leghorns from 3 to 5 weeks of age. After 6 weeks, there were no differences between the three genotypes. In contrast, Young et al. (1963) fed White Plymouth Rock and Rhode Island Red x Barred Plymouth Rock crossbreds with diets containing 150 g/kg of either lard FA or tallow FA and reported no differences between the genotypes in terms of fat digestibility. Limited evidence suggests that chickens utilise lipids less efficiently than turkeys and ducks. Halloran and Sibbald (1979) reported that the AME content of lipid sources varied with age in broiler chickens, but was unaffected in turkey poults. Massab et al. (2010) observed that 1-week-old poults had markedly greater capacity to utilise lipids compared to 1-week-old broilers, but the differences disappeared by three weeks of age. A study by Martin and Farrell (1998) found that ducklings digested the lipids in rice bran better than broilers of equivalent age. It is possible that turkeys and ducks have greater capacity to produce sufficient quantities of bile and lipase than chickens, especially at early stages. 4.2.3 Intestinal infections Various intestinal disease conditions damage intestinal epithelium and consequently cause poor absorption of nutrients. Such diseases include necrotic

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enteritis, malabsorption syndromes and coccidiosis. It is often observed that fat digestion is impaired the most by epithelial damage. For example, Amerah and Ravindran (2015) observed that, compared to other nutrients, the digestibility of fat is more severely affected by coccidiosis. In their study, coccidiosis resulted in 30, 25, 19 and 96% reductions in the apparent ileal digestibility of dry matter, protein, starch and fat, respectively. The profound effect of coccidia challenge on fat digestibility suggests mechanisms in addition to the negative effects on intestinal structure and inflammation. Adams et al. (1996) reported a reduction in bile salt secretion during coccidia challenge. The mechanism by which coccidial challenge reduces bile salt secretion may be due to damage of sensors located in the crypts of the intestinal mucosa, which mediate the release of cholecystokinin. Cholecystokinin is responsible for the stimulation of gallbladder contraction and pancreatic enzyme secretion (Wang and Cui, 2007). 4.3. Diet-related factors 4.3.1. Degree of saturation of fatty acids Energy-yielding potential of a fat is markedly influenced by its chemical structure (Freeman, 1984; Krogdahl, 1985). Fatty acid composition and the length and saturation degree of the carbon chain all impact the digestion and absorption of fats. The term ‘saturated’ fats means the absence of double bonds, whereas ‘unsaturated’ indicates the presence of one or more double bonds. Knowledge of the FA composition of each fat source is equally important as knowledge of its carbon chain length, saturation degree and the position of the double bond. The degree of saturation of FA, in particular, has a major influence on the AME of fats (Wiseman et al., 1991). Animal fats such as tallow containing high amount of long-chain saturated FA (palmitic and stearic acids) are poorly digested and absorbed by poultry (Renner and Hill, 1961; Scott et al., 1982). Saturated FA require bile salts to emulsify them and to form micelles prior to digestion. Garrett and Young (1975) 22

reported that the solubilisation and absorption of saturated FA are more negatively affected in the absence of bile salts than those of unsaturated FA. Both palmitic and stearic acids are non-polar and cannot spontaneously form mixed micelles. They require the presence of conjugated bile salts and unsaturated FA to form the mixed micelles. On the other hand, vegetable oils contain high concentrations of unsaturated FA that are easily emulsified and better digested than tallow (Sklan, 1979). Ward and Marquardt (1983) determined the effects of chain length and degree of saturation on fat absorption in two separate experiments. In Experiment 1, chicks were fed either a wheat- or rye-based diet containing 50 g/kg of different pure glycerides [tristearin (C18:0), triolein (C18:1) or trilinolein (C18:2)]. It was found that the absorption of fat in diets with saturated fat (tristearin) was lower compared to those fed with unsaturated fats (triolein and trilinolein). In Experiment 2, chicks were fed wheat- or rye-based diets containing 50 g/kg pure saturated glycerides [tricaprylin (C8:0), trilaurin (C12:0), tripalmitin (C16:0) or tristearin (C18:0)]. The absorption of fat in birds fed diets with the short chain FA (tricaprylin) was higher than those of the other FA, demonstrating that longer the chain length, lower will be the absorption. There is evidence suggesting that blending of saturated and unsaturated fats may improve fat digestion and that there is a synergistic response with such blends (Lall and Slinger, 1973; Sibbald et al., 1962; Wiseman and Lessire, 1987). This phenomenon is particularly important for the absorption of long chain saturated FA. Sibbald (1978) studied the effect of blending soybean oil and tallow on the TME content. Tallow was blended with soybean oil at ratios of 100:0, 99:1, 98:2, 96:4, 92:8, 84:16, 68:32, 36:64 and 0:100. The TME at ratios of 100:0 and 99:1 were similar (33.1 MJ/kg). On the other hand, ratios of 98:2, 96:4, 92:8, 84:16, 68:32, 36:64 and 0:100 resulted in TME values of 33.6, 33.9, 34.4 , 35.0 , 35.3 and 37.4 MJ/kg, respectively.

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Wiseman and Lessire (1987) studied the effect of blends of tallow and rapeseed oil at five ratios (100:0, 95:5, 90:10, 80:20 and 0:100), supplemented at 40, 80 and 120 g/kg of a basal diet in 14-day broilers and adult roosters. The ratio of unsaturated to saturated (U:S) fats influenced the AME, with increasing proportions of unsaturated fat (rapeseed oil) being associated with higher values. It was also found that increasing ratios of rapeseed oil improved the digestibility of palmitic and stearic acids. Ketels and De Groote (1989) examined the relationship between the dietary ratio of U:S FA and observed that blending the vegetable oil with animal fat improved the utilisation of the latter. 4.3.2. Inclusion level of fat A comprehensive discussion on the effect of rate of fat inclusion on its utilisation is provided by Wiseman (1984). Fats may be more effectively utilised at lower levels of inclusion and, as noted previously, their inclusion rate is an important experimental variable in their AME determination. The negative effects of high inclusion rate have been demonstrated by Wiseman et al. (1986), who fed broilers a commercial fat blend substituted into a semi-synthetic fat–free basal diet and a practical basal diet at concentrations of 10 to 100 g/kg in 10 g/kg increments. The AME response to incremental inclusions of fat was negative and non-linear, and the negative effects were particularly evident when saturated lipids were evaluated with younger birds (Wiseman, 1984). The above results, however, are not without contradiction. There are some reports that conflict with the general thesis that AME is negatively influenced at higher fat inclusion levels. For example, Gomez and Polin (1974) reported no change, or slight improvement, in lipid digestibility with increasing levels of supplemental fats. Mateos and Sell (1981a) observed improvements in the AME of yellow grease when assayed at levels varying from 50 to 300 g/kg in laying hen diets. 24

In general, available data demonstrate that the higher degree of saturation, longer the FA chain length and higher the lipid inclusion levels, lower will be the digestibility of supplemental fats. It is noteworthy, however, the influence of these factors on the AME of lipids is specific to each fat source or blend (Wiseman et al., 1986), making an accurate prediction challenging. 4.3.3. The position of fatty acid Due to the specificity of pancreatic lipase, the positional distribution of FA within the glyceride molecule is recognised as an important factor contributing to differences in digestibility between fat sources. During digestion, triglycerides are hydrolysed by the action of pancreatic lipase at sn-1, 3 positions. End products of this process are two free FA and sn-2-monoacylglycerol. The fatty acid in sn-2 position is conserved during absorption and subsequently in its reassembly to triglycerides. The reported differences in fat digestibility between animal fats and vegetable oils are attributed, in part, to the fact that animal fats contain high proportions of saturated FA in 1 and 3 positions (Meng et al., 2004). Sibbald and Kramer (1977) reported that 73 to 81% of palmitic and stearic acids in beef tallow are located at these positions. These long-chain saturated FA will be better absorbed if located at the 2position instead of sn-1, 3 positions (Decker, 1996; Lin and Chiang, 2010), because sn2-monoacylglycerols are amphiphilic and natural emulsifiers and enhance FA incorporation into mixed micelles. This thesis is supported by Smink et al. (2008) who showed that the randomisation of palmitic acid to the sn-2 position of palm oil had a positive effect on its digestibility for broilers. 4.3.4. Quality of fats The quality of fat is a wider subject. In the feed industry, commonly used fat quality measurements are based on colour, degree of saturation (iodine value), saponification value, level of impurities and free FA (FFA). These indices are generally 25

used to ensure that the fat products meet trade specifications and provide no information on the relative feed value or energy content (Shurson et al., 2015). Impurities in the fats are usually measured as the total amount of moisture, insoluble impurities and unsaponifiables (collectively referred to as MIU; Leeson, 1993). These components, except for glycerol, contribute little or no energy to the diet and, more importantly, dilute the energy content of fats. The maximum acceptable value of moisture in common fats is 10 g/kg fat. Impurities are determined as the percentage insoluble fraction of the fat in petroleum ether and should be lower than 10 g/kg fat. Unsaponifiables, such as sterol, pigments and hydrocarbons, are substrates that are not saponified after treatment with caustic soda. The maximum accepted level of unsaponifiables is 10 g/kg fat. It must be noted, however, these acceptable limits do not apply to all fat sources; in particular, these are not applicable to restaurant greases, frying oils and acidulated soapstocks. Acid oil soapstocks often have a moisture content of more than 10 g/kg fat. Non-elutable material, which are defined as those not eluted from the column during determination by gas liquid chromatography (Edmunds, 1990), is another quality measure of fat (Wiseman, 1999). This fraction represents the total amount of non-nutritional materials in fats and includes the moisture, impurities, total oxidised and polymerised FA, unsaponifiables and glycerol, Fatty acids in fats are normally bound to triglycerides. When not attached to any molecules, they are referred to as FFA. Free FA content has been used frequently to assess damage to lipids used in human foods. The level of FFA is similarly considered as a sign of rancidity and also as an indicator of non-elutable materials by the animal feed industry. It is widely recognised that the digestibility and AME of fats are depressed with increasing concentrations of FFA (Freeman, 1976; Sklan 1979). This negative effect

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may be attributed to (i) a lack of glycerides in the intestinal lumen, causing incomplete micellar solubilisation of FFA (ii) their relatively higher hydrophobicity requiring more bile salts for emulsification, and/or (iii) their ability to more easily form insoluble soaps with divalent cations (Garrett and Young, 1975; Small, 1991). Sklan (1979) found that the absorption efficiency of fat was inversely related to its FFA content and that the negative effect was more pronounced in saturated fats. Wiseman and Salvador (1991) investigated the effect of FFA on the AME of diets containing tallow (TO) and tallow acid oil (TAO) blends (TO:TAO; 0.75:0.25, 0.50:0.50, 0.25:0.75), palm oil (PO) and palm acid oil (PAO) blends (PO:PAO; 0.75:0.25, 0.50:0.50, 0.25:0.75) and, soybean oil (SO) and soybean acid oil (SAO) blends (SO:SAO; 0.75:0.25, 0.50:0.50, 0.25:0.75) for 2- and 8-week old broilers. It was observed that increasing FFA concentrations decreased the AME in both age groups. Wiseman and Blanch (1994) fed broilers aged 12 and 52 days with a blend of coconut oil and palm kernel oil (CP; FFA content, 13.8 g/kg) and coconut oil and palm kernel acid oil (CPAO; FFA content, 839 g/kg). The two blends (CP:CPAO) were mixed in the following proportions: 75:25, 50:50 and 25:75. Five oils (coconut oil, palm kernel oil and the three mixtures) were included in a basal diet at 40, 80 and 120 g/kg. The fat with the lowest FFA had the highest AME (33.1 and 34.6 MJ/kg DM, respectively), while the fat containing the highest FFA had the lowest AME (25.8 and 33.0 MJ/kg DM, respectively) in both young and older birds. Several cheaper fat by-products (such as acidulated soapstocks) available to the feed industry contain high concentrations (> 500 g/kg) of FFA; however, unless well processed, their feed value may be questionable. Acidulated soapstocks (pH ≤ 5.0; low content of MIU) are acceptable ingredients, especially in broiler finisher and layer diets (Mateos et al., 2012). Irandoust et al. (2012) determined the AME of acidulated soybean

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oil soapstocks, containing 674 g/kg FFA, to be high (33.3 MJ/kg), in laying hens. In summary, when fat sources containing FFA are used in poultry diets, (i) they may be fed in fat blends that contain sufficient amounts of triglycerides, (ii) the proportion of FFA must be controlled and (iii) the U:S ratio of the final blend must be given due consideration. The absorption of FA, especially of saturated ones, is considerably lower when FA are in the free form compared to those from triglycerides. This was demonstrated by Vila and Esteve-Garcia (1996), who determined the effect of degree of saturation of FFA substituted for either tallow or sunflower oil on the fat digestibility in broilers. It was found that the substitution of saturated FFA for tallow or sunflower oil markedly depressed fat digestibility, whereas substitution of unsaturated FFA had no effect. Overall, the available information indicates that the reduction in digestibility with FFA is markedly greater in young birds and for more saturated fat sources. The feed value of fats is not only based on their energy potential, but also on their safety. Oxidative rancidity is not directly related to caloric values, but a major cause of loss of quality of the fat. Oxidative rancidity is a degradation process that occurs in unsaturated FA due to the oxidation of the double bond of triglycerides. This process affects the odour, colour and flavour, and consequently decreases the value of the fat. Rancidity in fat and oils is usually determined using the peroxide value and active oxygen method. Peroxide value is the widely used indicator of fat oxidation and expressed as meq of peroxide per kg fat. The acceptable range in animal diets is 10 to 20 meq/kg and an upper limit of 20 meq/kg is generally established. The rancidity of fats can change rapidly and must be stabilised with anti-oxidants as early in the distribution chain as possible (FAO, 1999).

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Vegetable oils which contain high amount of unsaturated FA tend to be more prone to oxidation, particularly when used for frying in restaurants. Such oxidised and polymerised fats can greatly compromise bird health, performance and product quality (Jensen et al., 1997) and caution must be exercised in using recovered or recycled oils in poultry diets. Cabel et al. (1988) fed broilers with diets containing oxidised poultry fat at levels of 0, 50, 100 and 175 meq/ kg fat. It was found that the body weights were lowest in birds fed diets containing 175 meq/kg. Similarly, Tavarez et al. (2011) reported that broilers fed diets supplemented with oxidised soybean oil at 180 meq/ kg fat had lower body weights and consumed less feed compared to those fed diets with 0 meq/kg fat. It was suggested that the oxidised oil contained substrates such as aldehydes, ketones and esters, which may have lead to the development of rancid flavours and odours, and reduced feed palatability. Iodine value is another method used to measure fat stability. Iodine value measures the level of unsaturation of fats and oils, and is expressed as grams of iodine absorbed per 100 g of sample. Unsaturated FA have higher iodine values compared to saturated FA (O’Brien, 2009). In brief, it is evident that issues relating to fat quality are complex and, little is known of the exact effects of each quality index on the AME of supplemental fat and subsequent bird performance. 4.3.5. Cereal base Available data indicate the existence of a significant interaction between cereal type and fat source in terms of fat digestion. Viscous cereals (wheat, barley and rye) contain high concentrations of soluble NSP such as arabinoxylans and β-glucans, which exhibit anti-nutritive activity in poultry diets (Annison, 1993; Choct, 1997). Antoniou et al. (1980) reported that the performance and fat digestion were markedly depressed in broilers fed rye-based diets containing tallow, but these effects were less significant in diets containing soybean oil. Dänicke et al. (1997) found that the broilers fed a rye29

based diet supplemented with soybean oil were heavier compared to those fed the diet supplemented with tallow. Ward and Marquardt (1983) reported that the combination of tallow and rye depressed the digestibility of fat more than the combination of tallow and wheat. The digestion of fat is affected more than that of other nutrients by viscous NSP and the digestion of saturated FA is affected more than that of unsaturated FA (Dänicke, 2001). Several possibilities may be proposed for these effects. (i) Higher intestinal viscosity, which slow down the gut motility and impair the diffusion and convective transport of droplets of emulsified fat, FA, mixed micelles, bile salts and lipase (Smulikowska, 1998), and (ii) Stimulation of microbial growth in the small intestine (Annison and Choct, 1991). Increased bacterial activity may increase the deconjugation of bile acids. Deconjugated bile cannot be reabsorbed and will be excreted. Poor digestion of fat, therefore, may occur due to the reduced recycling and the resultant low concentration of bile salts in birds fed diets containing high levels of NSP (Smits and Annison, 1996). As the saturated FA require conjugated bile salts to form the mixed micelles, this exacerbates the indirect effect of NSP on the digestion and absorption of FA. 4.3.6. Feed processing Limited experiments have been conducted to evaluate whether fat may become more digestible as a consequence of feed processing. Grinding of cereal grains is the first step in feed processing and it is believed that the lipid contained within the cereals is less utilised than supplemental lipids. Veira et al. (1997) observed that the lipid digestibility was lower for high-oil maize vs. yellow dent maize plus supplemental maize oil and attributed this finding to accessibility of lipids within the cell wall matrix. Jimenez-Moreno et al. (2009) found that steam-cooking of maize improved fat digestibility and, speculated that steam-cooking, by disrupting the cell wall matrix and 30

releasing encapsulated lipids, may enhance fat digestibility. Abdollahi et al. (2013b) reported that the effect of pelleting on the ileal digestibility of fat varied depending on the cereal base used. In maize-based diets, ileal digestibility of fat was improved by pelleting, whilst in wheat-based diets, pelleting reduced the digestibility. Similar findings in maize-based diets have been reported by Naderinejad et al. (2015), who speculated that most of the fat present in maize-based diets originated from intact fat contained within the cells and, that pelleting

disrupted the cell wall matrix, thus

increasing the accessibility of cellular contents to digestive enzymes. However, this was not the case in wheat- and sorghum-based diets, as most of dietary fat was provided by added soybean oil. Abdollahi et al. (2014) similarly observed that broilers fed sorghum-based pelleted diets exhibited lower ileal fat digestibility than those fed mash diets. 4.3.7 Dietary Ca levels Free FA, released during fat digestion, have the potential of reacting with divalent minerals, forming soluble or insoluble soaps. If insoluble soaps are formed, there is the possibility that both the FA and the mineral become unavailable to the bird (Leeson and Summers, 2005). It has been suggested that phytate, as Ca-phytate, may be involved in the formation of insoluble metallic soaps in the gut (Ravindran et al., 2000) and Cosgrove (1966) has described these ‘lipophytins’ as a complex of Ca/Mg-phytate, lipids and peptides. It therefore follows that diets with high dietary levels of Ca may increase the formation of lipophytins and lower the energy derived, especially from saturated animal fats. The positive effect of microbial phytase in improving the ileal fat digestibility in broilers (Camden et al., 2001; Zaefarian et al., 2013) lends some support to this thesis. Evidence suggests that the type of FA and dietary level of Ca impact soap formation and, the retention of fat and Ca. Atteh and Leeson (1983) fed broilers a basal 31

diet without supplemental FA and diets supplemented with 80 g/kg mixture of linoleic and oleic acids (2.5:1), oleic acid, palmitic acid or stearic acid at two levels of Ca (8 and 12 g/kg). Increasing Ca concentrations reduced fat retention only in birds fed the diet supplemented with palmitic acid. Birds fed diets with palmitic and stearic acids had higher concentrations of excreta soap than those fed diets supplemented with oleic acid and the mixture of linoleic and oleic acids. Atteh and Leeson (1984) examined the effect of FA saturation (80 g/kg mixture of oleic and palmitic acids, oleic acid or palmitic acid) and Ca level (8, 12 and 16 g/kg) on fat retention and excreta soap formation in broilers. Birds fed diets containing 16 g/kg Ca showed a significant decrease in fat retention. Significant interactions were observed between the FA saturation and Ca concentration for fat retention and excreta soap formation. Increasing the level of Ca above 8 g/kg resulted in higher excreta soap contents in diets supplemented with palmitic acid, whereas soap formation in birds fed the mixture of oleic and palmitic acids was increased only in diets containing 16 g/kg Ca. It was also found that the birds fed the diet with palmitic acid excreted more soap than those fed the mixture of oleic and palmitic acids and oleic acid at all three Ca concentrations. Similarly, Lin and Chiang (2010) found that increasing dietary Ca concentrations lowered fat digestibility and that the magnitude of reduction was markedly higher with saturated FA. Overall, these data indicate that the potential for insoluble soap formation is greater with saturated FA compared to unsaturated ones. Tancharoenrat and Ravindran (2014) evaluated three inclusion levels of tallow (0, 40 and 80 g/kg) and three dietary concentrations of Ca (7, 10 and 13 g/kg) in broiler starters fed maize-soy diets. The results showed that the total tract retention and ileal digestibility of fat were higher with supplementation of 40 g/kg of tallow compared to those of 0 and 80 g/kg tallow, and that high dietary Ca concentrations adversely affected

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the utilisation of fat and energy. Overall, these data indicate that the potential for insoluble soap formation is greater with saturated FA compared to unsaturated ones.

4.3.8 Anti-nutritional factors Numerous anti-nutritional factors, when present above their tolerance levels, can interfere with fat utilisation and these include tannins, trypsin inhibitors and various mycotoxins. For example, aflatoxicosis causes a lipid malabsorption syndrome coupled with decreased lipase secretion, lowered lipolysis, increased bile salt excretion, and impaired transport. A key effect of mycotoxins is lipid peroxidation in enterocytes leading to damage that substantially contribute to the malabsorption (Hamilton, 1977).

Strategies to improve fat utilisation 4.4. Introduction The digestion of fats is a relatively complex process that requires sufficient quantities of bile salts, which are essential for emulsification, and the lipase enzyme. It follows that any strategy to improve fat digestion must consider the supplementation of emulsifiers, enzyme or both. Another commonly employed strategy to improve fat digestion is to consider the use of fats containing high proportions of unsaturated FA or blends of fats. These strategies may be especially useful in young birds in which the ability to digest and absorb fat is not well developed (Carew et al., 1972; Wiseman and Salvador, 1991).

4.5. Supplemental enzymes 4.5.1. Lipases Lipases (acylglycerol acylhydrolases, EC 3.1.1.3) are ubiquitous enzymes widely distributed in the microbial, plant and animal kingdoms. Isolation and

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purification of lipases from these sources have been reported (Taipa et al., 1994), but research on the use of lipase supplementation in poultry diets is limited. Lipases tested in poultry evaluations have originated from two sources, namely crude porcine lipase (Polin et al., 1980) and microorganisms such as Rhizopus arrhizus, Aspergillus niger and Pseudomonas spp. (Kermanshahi et al., 1998). Mammalian lipases hydrolyse sn1and 3-positions of the triglyceride, whereas microbial lipases have a broad range of selectivity including the ability to hydrolyse sn1- and 3- positions (Carlier et al., 1991). Crude lipase preparations have been evaluated in poultry diets by several researchers. Polin et al. (1980) fed chicks with diets containing 40 g/kg tallow supplemented three levels of crude porcine lipase (0, 0.1, and 1 g/kg) and two levels of cholic acid (0 and 0.4 g/kg). Fat absorption from 2 to 9 days post-hatch was higher in birds fed diet supplemented with 1 g/kg of lipase compared to those fed 0 and 0.1 g/kg. However, the addition of cholic acid alone to the diet resulted in better fat absorption (82.6%) than the combination of cholic acid and 1 g/kg lipase (82.3%). DiMagno et al. (1977), however, observed that both porcine lipase and co-lipase are denatured by the acidic pH of upper digestive tract and will not have the desired activity when they reach the sites of fat digestion. Al-Marzooqi and Leeson (1999) evaluated the addition of pancreatin and crude porcine pancreatic preparation on the fat utilisation in young broilers in three experiments. In Experiment 1, a maize-based diet was supplemented with two levels of an animal-vegetable fat blend (40 and 80 g/kg) and three enzyme treatments (none, 7.14 g/kg crude pancreatic enzyme or 7.14 g/kg pancreatin) to study the effects of enzyme on the performance, fat digestibility and soap formation. Both enzymes improved fat digestibility and the AME compared to the unsupplemented control. Excreta soap formation in birds fed unsupplemented diets was higher than those fed diets with

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enzymes. However, enzyme supplementation reduced feed intake and weight gain of birds resulting in higher feed per gain. In Experiment 2, diets containing 40 g/kg animal-vegetable fat blend were supplemented with crude pancreatic enzyme at 0, 2.14, 4.29, 6.43, 8.57 and 10.07 g/kg and it was found that increasing enzyme additions increased fat digestibility and AME, and lowered soap formation. However, increasing levels of enzyme reduced feed intake and weight gain, and increased feed per gain. In Experiment 3, broilers were fed diets containing 40 g/kg animal-vegetable fat blend supplemented with ground dried crude porcine pancreas at 0, 3.21, 5.35, 7.50, 9.64, 11.78 and 13.92 g/kg. No significant effect was observed on the performance of broilers. It was concluded that fat digestion in broilers can be improved by lipase supplementation. It was speculated that the poor feed intake associated with lipase treatments may be due to contamination with cholecystokinin. In a follow-up series of studies, Al-Marzooqi and Leeson (2000) investigated the effect of crude porcine pancreatic enzyme supplementation in broilers. In Experiment 1, enzyme was used at graded levels of 0, 2.14, 4.29, 6.43, 8.57, and 10.71 g/kg to examine the effect on gut structure. Experiment 2 was designed to investigate the effect of lipase at 0, 2.68, 5.36, 8.04, 10.71, and 13.39 g/kg on gastric motility. No effect of the enzyme was found on gut morphology and gastric motility. In Experiment 3, the enzyme was supplemented at four levels (0, 3.75, 7.50, or 11.25 g/kg) in the broiler starter diet. Starter diets were fed from day 1 to 21 and then replaced with grower diets containing no enzyme. During the starter period, there was a linear decrease in feed intake and weight gain with increasing enzyme additions, but no differences in feed intake and weight gain were observed among treatment groups from 21 to 42 days of age.

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Studies evaluating pure forms of supplemental lipases are scant and, in general, have not to date yielded successful outcomes. Meng et al. (2004), for example, found no effect of lipase addition on the fat digestibility or AME in young broilers. These researchers suggested that the insufficiency of pancreatic lipase production may not be a factor contributing to incomplete fat digestion in young birds. 4.5.2. Glycanases As noted earlier, fat digestion suffers the most pronounced impairment in diets based on viscous cereals (Choct and Annison, 1992; Ward and Marquardt, 1983). Diets based on these cereals are routinely supplemented with exogenous glycanases (βglucanases and xylanases) to overcome the problem of digesta viscosity and, to improve nutrient metabolisability and bird performance. Dänicke et al. (1997) studied the interaction between fat source (100 g/kg soybean oil or tallow) and supplemental xylanase in rye-based diets and reported that the feed intake and live weight of broilers fed diets with soybean oil were higher than those fed diets with tallow. It was also shown that xylanase supplementation to tallow diets improved feed per gain to the same level as of birds fed soybean oil without enzyme. Enzyme addition decreased the viscosity of ileal digesta and improved fat digestibility compared to birds fed the unsupplemented diet (0.71 vs. 0.53). Langhout et al. (1997) investigated the effect of xylanase supplementation and fat source (65 g/kg of soybean oil or blend of 60 g/kg tallow and 5 g/kg soybean oil) on fat digestion in broilers fed wheat/ rye-based diets. It was reported that the xylanase increased fat digestibility and that the effect of enzyme was more pronounced in birds fed diets containing the blend compared to those fed diets containing soybean oil. Xylanase improved fat digestibility in the tallow blend (0.61 vs. 0.70), whereas the effect on fat digestibility in the soybean oil diet was small (0.78 vs. 0.80).

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Overall, these results demonstrated that the addition of xylanase to broiler diets based on viscous cereals and containing tallow resulted in greater improvements in fat digestibility.

5.3 Emulsifiers Fats are insoluble in water, do not solubilise in the aqueous phase of the gastrointestinal tract and needs to be emulsified before they can be hydrolysed by lipase. The ease of emulsification depends on the characteristics of the fat such as chain length, position of FA on the triglyceride and degree of fat saturation. As a polar amphipatic molecule, consisting of both hydrophilic and hydrophobic properties, an emulsifier (more accurately termed surfactant) is able to form a bridge between water- and fatsoluble materials, and improve fat utilisation, especially of animal fats. Emulsifiers may also play a particular role in overcoming the inadequacies of naturally low bile production and recirculation in young birds. Emulsifiers which are normally used in feed industry can be categorised into two groups, namely natural (such as bile and bile salts) and nutritional (such as lecithin and lysolecithin) emulsifiers.

5.3.1 Bile acid and salts Bile salts act as emulsifiers by reducing the tension of the oil-water interface and also activate pancreatic lipase as well as prevent denaturation of this enzyme when it leaves the surface of emulsified fat droplets. Bile salts are flat amphiphilic molecules with a hydrophobic surface on one side that interacts with the oil phase of the emulsion and a hydrophilic surface on the other that interacts with water (Chen et al., 1975). As noted previously, secretion of bile is thought to be limiting in young birds, especially during the first week of life, resulting in very low fat digestion (Krogdahl, 1985;Tancharoenrat et al., 2013). Young chicks are unable to replenish bile salts like the older birds and the decreased pool size of bile salts may also contribute to the 37

malabsorption of fat (Serafin and Nesheim, 1967, 1970). For this reason, bile acid derivatives such as bile salts and cholic acid have been evaluated in diets for young birds to improve fat digestion. Noy and Sklan (1995) reported that supplemental bile salts improved fat digestion in 7-d-old chicks. Similar results were also reported by Gomez and Polin (1976) who studied the absorption of tallow in chicks by adding bile acids (cholic and chenodeoxycholic acids) and bile salts (taurocholate) at three levels (0, 0.25 and 0.5 g/kg) to a maize-based diet containing 82 g/kg tallow. Addition of both supplements increased fat absorption at 7 and 19 days of age compared to birds fed the unsupplemented diet. The addition of cholic acid improved the absorption of tallow better than chenodeoxycholic acid and taurocholate. Polin et al. (1980) investigated the effect of bile acids on fat absorption in chicks by feeding diets containing 40 g/kg tallow supplemented with one of the bile acids (cholic acid, chenodeoxycholic acid, dehydrocholic acid or deoxycholic acid) or bile salt (sodium taurocholate) at 0.4 g/kg. The chenodeoxycholic acid supplemented group showed higher fat absorption compared to the unsupplemented control group during week 1 (0.90 vs. 0.84). At 3 weeks of age, fat absorption was higher in the cholic acid group compared to the control and chenodeoxycholic acid groups (0.87 vs. 0.85 and 0.81, respectively). Kussaibati et al. (1982) investigated the influence of synthetic bile salt addition on the digestibility of FA in an animal fat blend (150 g/kg inclusion). It was found that the digestibility of medium and long chain saturated FA (16:0 and 18:0) was improved by bile salt addition. On the other hand, the digestibility of short chain saturated FA (14:0) and long chain unsaturated FA (18:1 and 18:2) were already relatively high and was unresponsive to addition.

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Dietary supplementation of ox bile to improve fat utilisation was studied by Fedde et al. (1960) in two experiments. In Experiment 1, chicks were fed diets containing beef tallow (200 g/kg) supplemented without or with bile (5 g/kg). It was found that fat absorption in birds fed diets supplemented with bile was higher than those fed diets without bile. In Experiment 2, chicks were fed diets containing 200 g/kg beef tallow supplemented with increasing inclusions of bile (0, 0.5, 1, 5, 10, 20, 40 and 80 g/kg). The results showed that the body weight of birds fed diets with 40 and 80 g/kg bile were lower than the other groups. The weight of the gall bladder in birds fed diets supplemented with bile was higher than that of control birds. Fat absorption in birds fed diets supplemented with 5 g/kg bile was higher than those fed diets containing 0, 0.5, 1 and 20 g/kg bile, but there were no differences between birds fed 5, 10, 40 and 80 g/kg bile. Based on these results, it was speculated that the addition of exogenous bile may either aid directly in fat absorption or it may stimulate the liver cells to secrete more bile. Alzawqari et al. (2011) studied the effect of dried ox bile on the performance and fat digestibility in broilers by feeding diets containing tallow (50 g/kg) supplemented with three levels of bile (0, 2.5 and 5 g/kg). Supplementation of bile at 5 g/kg resulted in higher weight gain and lower feed per gain than at 0 and 2.5 g/kg inclusion. Fat digestibility coefficients at 0, 2.5 and 5 g/kg bile treatments were 0.59, 0.79 and 0.84, respectively. Although bile derivatives appear to have positive effects of fat digestion, it must be noted that the extent of improvement would be different for different bile salts. They are also expensive and, at the present time, their use in poultry diets is not cost effective and, thus, it is not of practical interest.

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5.3.2 Nutritional emulsifiers A long list of emulsifiers is approved for use in food applications to improve shelf stability of processed foods. Mono- and diglycerides (and their esters or derivatives) and refined lecithins are the most extensively used as natural emulsifiers or stabilisers in the food industry (Hasenhuettl, 2008). These food-grade emulsifiers are expensive and their use to improve fat utilisation in animal feeding is not economically viable. For use in animal feeds, a number of cheaper nutritional emulsifiers is commercially available. These fall into three main categories, namely, crude lecithins, lysolecithins and synthetic products such as glycerol polyethylene glycol ricinolate. When selecting a nutritional emulsifier, its degree of solubility in water or fat is important (Hasenhuettl, 2008) and this is measured as Hydrophilic-Lipophilic Balance (HLB). It is defined as the relative efficiency of the hydrophilic portion of the surfactant molecule to its lipophilic portion of the same molecule and assigned on an arbitrary scale of 0 to 20 (0 = very lipophilic and 20 = very hydrophilic). An emulsifier with high HLB, which is indicative of high water solubility, is desired in poultry diets, as birds drink twice the amount of water than feed and the feed contains only a small amount of fat. Crude lecithins, a co-product from the degumming of vegetable oils during the refining process, are a complex mixture of various species of surface-active phospholipids, consisting hydrophobic and hydrophilic portions. Thus lecithins are not uniform, standard materials, but natural mixtures of several components. A comprehensive overview of aspects of commercial lecithins is provided by Bueschelberger et al. (2015). Depending on the source of lecithin, the phospholipid profiles differ both in their phosphatidyl moieties (choline, inositol, ethanolamine and phosphatidic acid) and their FA composition (Jensen et al., 2015). 40

The potential of crude lecithins has been evaluated as an additive to improve fat digestion in young pigs (Overland et al., 1993; Soares and Lopez-Bote, 2002) and broilers (Polin, 1980). Polin (1980) supplemented a diet containing 40 g/kg tallow with 0.2, 2 and 20 g/kg lecithin, and reported that the absorption of tallow was increased in broilers fed 20 g/kg lecithin compared to those fed 0.2 and 2 g/kg. Lecithins can be used in poultry diets in substitution of other fat sources. In studies with layers, Mandalawi et al. (2015) observed that the replacement of pork fat by 40 g/kg crude lecithin (containing 923 g/kg fat), a by-product of the biodiesel industry from soybean oil, improved egg weight, egg yolk colour and the retention of dry matter, fat and gross energy. It was suggested that crude lecithin can be used as a lipid source for laying hens with beneficial effects on egg production. The benefits of feed grade lecithins include inter alia cost reduction, better handling, potential synergisms and improved fat digestibility because of its emulsifying effects, especially in young birds (Mateos et al., 2012). They are also good sources of energy (70% of the original soybean oil) and rich in essential FA, phosphorus, choline, vitamin E and inositol, but the high viscosity of the product is an issue in feed mixing (Mateos et al., 2012). The products commercially marketed as lysolecithins are mixtures of lysophospholipids and phospholipids. An important lysophospholipid component is lysophosphatidylcholine, which is the mono-acyl derivative of phosphatidylcholine produced by the action of enzyme phospholipase A2. Lysophospholipids are powerful biosurfactants and have a much higher HLB value than phospholipids. They have the ability to form small-sized micelles much more effectively than bile (Melegy et al., 2010). Othman et al. (2008), in a 35 day feeding trial, examined the addition of a commercial lysophosphatidylcholine (Lysoforte™; Kemin Animal Nutrition and Health,

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Singapore) to broiler diets containing 40 g/kg tallow or soybean oil. The results showed that the emulsifier addition was beneficial in terms of feed efficiency and AME in both tallow and soybean oil diets. Zhang et al. (2011) evaluated the effects of the same commercial emulsifier on the AME and performance of broilers fed diets with three fat sources (soybean oil, tallow and poultry fat) at 30 g/kg in starter diets and at 40 g/kg in grower diets. Supplementation of the emulsifier increased the weight gain in birds fed all three fat sources during the starter phase, but no differences were observed during the grower phase. Emulsifier tended to increase the AME during both the starter and grower phases, with the highest improvement determined in birds fed diets with poultry fat. In a recent study, Jensen et al. (2015) reported that soybean and rapeseed lysolecithins improved the AME and nitrogen retention of broiler diets and that the effects were greater in diets containing lard compared to soybean oil. Overall, the current evidence indicates that the use of nutritional emulsifiers represents an useful economic strategy to improve the energy value of lipids for poultry. 4.4.

Type of added fat

The animal fats widely used by the feed industry are poultry fat, lard and tallow, while the common vegetable oil products include are soybean oil, rapeseed oil and palm oil, and acidulated soapstocks. Numerous studies have examined the effects of using different types of fats and oils on fat digestion, AME and growth performance in broiler chickens (Zumbado et al., 1999; Pesti et al., 2002; Dei et al., 2006; Firman et al., 2008; also see Table 3). Overall, available data overwhelmingly highlight the poor fat utilisation in tallow by young, growing birds. From the point of cost reduction and practicality, blends of animal fats and vegetable oils represent an economic option for the poultry feed industry. Animal fats contain a high proportion of long chain saturated FA, while vegetable oils have a high

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proportion of unsaturated FA. As discussed previously, saturated FA are poorly digested compared with unsaturated FA. It is recognised that the utilisation of saturated fats can be improved by ensuring adequate amounts of unsaturated FA relative to saturated FA in the diet. The feed value of blends of animal fats and vegetable oils, with different ratios of U:S FA, has been evaluated by several researchers (Dänicke et al., 2000; Scaife et al., 1994; Wiseman et al., 1998; Wiseman and Lessire, 1987). As noted previously, the benefit of blending partly originates from the natural emulsifying effects of unsaturated FA. Ketels and De Groote (1989) reported that increasing U: S ratios increased the utilisation of saturated FA, but there was no effect on that of unsaturated FA. The most pronounced improvements were observed when the ratio was increased from 1.0 (tallow) to around 2.0. Beyond 2.0 ratio, a surplus of unsaturated FA resulted only in limited improvements. Leeson and Summers (2005) suggested that a ratio of 3:1 (U: S) is a good compromise for optimum fat digestibility in birds of all ages. It has been shown that the blending of animal fats with vegetable oils can produce a synergistic effect which can improve the utilisation of saturated fats (Lall and Slinger, 1973). Sibbald (1978) reported that the AME of blends of soybean oil and tallow was higher than the sum of the means of its components. Similarly, Muztar et al. (1981) found that the AME of blends of tallow and rapeseed soap stocks was 4% higher than that calculated from the AME of its components. Dänicke et al. (2000) fed broilers with rye-based diets containing 100 g/kg fat based on blends of beef tallow and soybean oil (0:100, 20:80, 40:60, 60:40, 80:20 and 100:0) which gave U:S ratios of 5.47, 3.23, 2.11, 1.45, 1.00 and 0.69, respectively. It was observed that increasing U: S ratios increased the AME and weight gain, and lowered feed per gain. In contrast, Preston et al. (2001) fed broilers wheat-based diets containing 60 g/kg tallow, soybean oil or a 2:1 blend of tallow and soybean oil and found that fat type influenced the digestion of fat,

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but no synergism was observed in the blend. Fat digestibility coefficients determined for soybean oil, blend and tallow were 0.85, 0.76 and 0.69, respectively. The basis for the synergism phenomenon appears to be due to the ability of polar solutes, such as unsaturated FA, to increase the micellar solubility of non-polar solutes, such as saturated FA, whereby the former assists in the absorption of otherwise poorly utilised latter (Wiseman et al., 1986). The extra-caloric value of fats, referred to in Table 3, has been attributed partly to these synergistic effects. However, the magnitude of synergism will depend on the inclusion level of fat and may be progressively smaller at higher inclusions (Mateos and Sell, 1981b). 4.5.

Dietary Ca concentrations

There is evidence suggesting that lower dietary Ca concentrations may be advantageous for the digestion of lipids, especially in diets containing high proportions of saturated FA (Tancharoenrat and Ravindran, 2014). Thus it may be argued that lipid utilisation may be maximised by maintaining the Ca levels as low as is realistically possible in broiler diets. Well-planned studies clearly are warranted to determine the dietary Ca concentrations to achieve optimum bone health as well as maximum utilisation of lipids. In this context, existing recommended requirements of both Ca and P for bone health and bird performance may also have to be re-defined. Limestone is usually the predominant source of Ca in poultry diets. It is likely that some of the effects of high dietary Ca on lipid digestion may be attributed to the reactivity of limestone, which has an extremely high acid binding capacity, and will tend to increase the pH of digesta along the gut. Conceivably the increase in digesta pH could increase Ca-phytate complex formation (Selle et al., 2009) and also potentially lower lipase activity. Future studies comparing interaction between fats and Ca sources (limestone vs. meat and bone meal) may provide insight on this hypothesis.

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6

Prediction of AME content of supplemental fats A critical discussion of the attempts to develop prediction equations to estimate

the AME content of supplemental fats is relevant, but is beyond the scope of this review. Although the presence of high MIU or oxidative stability is generally thought to be indicative of lower available energy, research shows that the ability of fat quality parameters to predict the AME appears to be poor (Huyghebaert et al., 1988; Pesti et al., 2002). These parameters may be useful, however, when considered along with chemical measures such FA profile (Huyghebaert et al., 1988). The FA composition data, and especially the U:S ratio, provide useful, though somewhat crude, estimates of AME content; however, as discussed above, this is only one of the many factors that determine the available energy. Complex multiple regression models based on FFA and contents of major FA (Huyghebaert et al., 1988) and more simpler equations based on the U:S ratio (Ketels and De Groote, 1989) and, U:S ratio and level of FFA (Wiseman et al., 1991) have also been reported. Although some of these predictive models are used in the feed industry to estimate fat AME, it must be recognised that they are over-simplistic and overlook a number of important parameters that contribute to the variations in AME content. As cautioned by Huyghebaert et al. (1988), such equations apply only to the fat types used in the development of prediction models and extrapolation to other structures may produce misleading results. 7.

Conclusions Compared to starch and protein, the digestion and absorption of fats is a

complex field of discipline because of the need to emulsification and micelle formation and, the large number of variables and interactions involved. This review presents an overview of the major steps involved in the digestion and absorption of fat. The wide

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variability in AME content of different supplemental fat sources is highlighted. Methodological differences are identified as an important factor contributing to the wide variability in the published AME contents. The efficiency of fat utilisation by poultry is dependent on number of factors, especially age of birds and, composition, inclusion level and quality of the fat. Studies to better understand these factors and to resolve their complexity are warranted to achieve the full benefits of supplemental fats. Of the various strategies available, the use of blended fats and nutritional emulsifiers are potentially useful to improve fat utilisation. CONFLICT OF INTEREST STATEMENT There is no conflict of interest.

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Figure Captions Figure 1. Digestion of fat, as proportion of total digestion determined at the lower ileum, along the small intestine of broilers fed diets supplemented with soybean oil (

)

or tallow ( ). Mean ± standard error. (UJ, upper jejunum; LJ, lower jejunum; UI, upper ileum; LI, lower ileum). Source: Tancharoenrat et al. (2014).

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Tables Table 1 Fatty acid composition (g/100g) of commonly used fats and oils in poultry dietsa Animal fats

Fatty acid (Carbon atom: Double bonds)

10:0 12:0 14:0 14:1 15:0 16:0 16:1 17:0 17:1 18:0 18:1 18:2 n-6 18:3 n-3 20:0 20:1 20:4 n-6 20:5 n-3 22:0 22:1 22:6 n-3 24:0 U:S ratiob a

Vegetable oils

Tallow (mutton)

Tallow (beef)

Lard

Poultry fat

Herring oil

Palm oil

Soybean oil

Maize oil

Sunflower oil

Rapeseed oil

Canola oil

0.2 0.3 5.2 0.3 0.8 23.6 2.5 2.0 0.5 24.5 33.3 4.0 1.3 0.74

0.1 3.2 0.9 0.5 24.3 3.7 1.5 0.8 18.6 42.6 2.6 0.7 0.2 0.3 1.1

0.1 0.1 1.5 0.1 26.0 3.3 0.4 0.2 13.5 43.9 9.5 0.4 0.2 0.7 1.4

0.1 0.8 0.2 0.1 25.3 7.2 0.1 0.1 6.5 37.7 20.6 0.8 0.2 0.3 2.0

6.2 12.7 7.5 1.1 12.9 1.1 0.7 15.1 0.3 6.8 22.0 5.8 3.1

0.1 1.0 44.4 0.2 0.1 4.1 39.3 10.0 0.4 0.3 0.1 0.99

0.1 10.6 0.1 0.1 4.0 23.2 53.7 7.6 0.3 0.3 5.5

0.1 10.9 0.2 0.1 2.0 25.4 59.6 1.2 0.4 0.1 6.4

0.1 7.0 0.1 0.1 4.5 18.7 67.5 0.8 0.4 0.1 0.7 6.8

0.1 3.8 0.3 1.8 18.5 14.5 11.0 0.7 6.6 0.5 41.1 1.0 11.6

3.5 0.2 1.5 60.1 20.1 9.6 0.6 1.4

Adapted from Tisch (2006) and Sauvant et al. (2004). Unsaturated to saturated fatty acid ratio.

2

67

0.3 0.2

15.3

Table 2 Ileal endogenous flow of fat and fatty acids (mean ± standard error), and the fatty acid profile of endogenous fat of broiler chickensa,b Ileal flow Profile of endogenous (mg/kg DM intake) fat (g/kg fat) Fat 1,714 ± 412.7 Saturated fatty acids C16:0 Palmitic C17:0 Margaric C18:0 Stearic

128 ± 30.1 16 ± 3.2 225 ± 65.5

74.6 9.5 131.4

Unsaturated fatty acids C18:1 Oleic C18:2 Linoleic C20:4 Arachidonic

125 ± 36.4 227 ± 76.8 103 ± 31.9

73.2 132.5 60.2

Total fatty acids

825 ± 243.9

481.0

a

From Tancharoenrat et al. (2014).

b

None of the other fatty acids were detected.

68

Table 3 Apparent metabolisable energy (AME) value of fat sources commonly used in poultry diets – select examples Beef tallow Beef tallow Beef tallow Beef tallow Beef tallow Beef tallow Tallow Tallow Lard (White grease) Lard Lard Lard Lard Palm oil Palm oil Palm oil Palm oil Palm oil Poultry fat Poultry fat Poultry fat Soybean oil Soybean oil Soybean oil Soybean oil Soybean oil Soybean oil Soybean oil Animal-vegetable blends Beef tallow-crude soybean oil Tallow-refined soybean oil Animal/vegetable blends Animal/vegetable blends By-product fats Yellow grease Yellow grease Yellow grease Acidulated soybean soapstock Acidulated soybean soapstock a

MJ/kg

Level of fat, g/kg a

Age of bird (days)

Reference

29.1-35.1 29.4 30.6 27.8-39.1 26.0 31.3 42.4b

30-100 70 70 20-60 90 40 40

24 17 44 19 24 18 Adult rooster

Guirguis (1976) Lessire et al.(1982) Lessire et al.(1982) Wiseman et al. (1986) Huyghebaert et al.(1988) Blanch et al. (1995) Blanch et al. (1996)

34.3 30.4 35.1 36.7

60 60 16.6 20-80

10 40 Adult Rooster 35

Pesti et al. (2002) Pesti et al. (2002) Firman et al. (2008) Veira et al. (2015)

24.6 43.6 27.1 21.9

90 40 60 60

24 Adult rooster 10 40

Huyghebaert et al. (1988)

38.0 37.4

70 70

17 44

40.5-42.7 35.7 44.1 46.5 40.0 38.2

20-60 90 40 60 60 35

19 24 Adult rooster 10 40 Adult rooster

Wiseman et al. (1986) Huyghebaert et al.(1988) Blanch et al. (1996) Pesti et al. (2002) Pesti et al. (2002) Irandoust et al. (2012)

32.8 40.0 43.3 41.3

90 60 60

24 10 40

Huyghebaert et al.(1988) NRC (1994) Pesti et al. (2002) Pesti et al. (2002)

44.8 40.4 34.5 32.9-33.3 35.7

60 60 16.6 35 20-80

10 40 Adult rooster Laying hen 35

Blanch et al. (1996) Pesti et al. (2002) Pesti et al. (2002) Lessire et al. (1982) Lessire et al. (1982)

Pesti et al. (2002) Pesti et al. (2002) Firman et al. (2008) Irandoust et al. (2012) Veira et al. (2015)

Inclusion level used in the test diet

b

Gross energy content of fats ranges between 38.1 and 40.6 MJ/kg; AME values above the gross

energy content are suggestive of ‘extra-caloric’ effects (Touchburn and Naber, 1966) due largely to the slowing effect of fats on passage rate (Mateos and Sell, 1981b), giving more time for the digestion and absorption of other energy-yielding nutrients.

69

Table 4. Influence of fat type and age of broilers on the AME (MJ/kg) of fatsa,b Fat type

Age (weeks) 1

2

3

5

Tallow

12.2

22.2

30.2

29.6

Soybean oil

18.8

34.2

38.4

37.1

Tallow: soybean oil

15.6

29.2

31.0

34.1

Poultry fat

17.0

32.7

35.0

35.1

Palm oilc

20.1

37.0

39.8

38.6

(50:50 blend)

a

Tancharoenrat et al. (2013).

b c

Fat type (P<0.0001); age of broilers (P<0.0001); Fat type x age (P>0.05).

Refined palm oil.

70