What are the phases of lipid peroxidation and their impact on broiler growth performance?

The complete oxidation of lipids, described as peroxidation, takes place over three phases; initialisation, propagation and termination with each step degrading and producing many different lipid peroxide compounds. Primary oxidation results in lipid hydroperoxides, reducing the quality of the feed lipid and forming both secondary and tertiary peroxidation products that have a detrimental impact on broiler growth performance.

A Review of Lipid Digestion, Metabolism and Nutritive Value in Broiler Diets

I. INTRODUCTION

In Australia, the cost of tallow and vegetable oil has more than doubled over the past two years (www.finvis.com), prompting broiler nutritionists to re-evaluate dietary energy content for meat-type chickens. Globally, a wide range of lipids with diverse chemical composition, are routinely included in poultry feed including soapstocks or acid oil, crude vegetable oil, hydrogenated material, recycled vegetable oil, restaurant grease, animal tallows and even products from the refining of oils such as bleaching earths, which are sometimes available as “dry fat” products (Wiseman, 1984, 1990; Krogdahl, 1985).

Fatty acids (FA) have four major physiological roles (Berg et al., 2019). Primarily, these are fuel molecules for the bird and are stored as triacylglycerols (also called neutral fats or triglycerides) which are uncharged esters of FA with glycerol. FA mobilised from stored triacylglycerols are oxidised to meet the energy needs of the bird. This is particularly important in migratory birds that cover vast distances across terrain where food is unavailable. Triacylglycerols are highly concentrated stores of metabolic energy since they are reduced and anhydrous. The energy yield from the complete oxidation of triacylglycerols is approximately 38 kJ/g in contrast with about 17 kJ/g for carbohydrates and protein (Berg et al., 2019). Carbohydrates are polar whilst lipids are non-polar and one gram of glycogen, for example, binds about two grams of water. Consequently, a gram of anhydrous lipid stores 6.75 times as much energy as a gram of hydrated glycogen. Secondly, FA are the building blocks of amphipathic molecules, namely phospholipids and glycolipids that are important components of cell bilayer membranes. Thirdly, the covalent attachment of FA to many proteins modifies and targets these to various membrane locations. Lastly, fatty acid derivatives serve as hormones and intracellular messengers.

II. DIGESTION, ABSORPTION AND LIPID ENERGY

Physiologically, the mechanism of fat digestion and absorption in non-ruminants has been well documented (Freeman, 1984). Lipid digestion occurs primarily in the duodenum and jejunum and consists of emulsification of dietary lipid by conjugated bile acids, followed by hydrolysis of triglycerides by endogenous pancreatic lipase. The resultant mixture consists essentially of 2-monoglycerides and free FA (FFA) whilst the subsequent absorbability of these is dependent upon their solubility in bile salt micelles. Lipids pose a special problem compared with carbohydrates and protein since they are not soluble in water. Bile acids coat ingested lipids and the ester bond of the lipids are orientated towards the surface of the bile salt-coated particle, rendering the bond more accessible to digestion by lipases in aqueous solution. Pancreatic colipase is required to bind lipase to the bile salt-coated particle to permit lipid degradation and the resulting product is transported across the plasma membrane in micelles (Berg et al., 2019; Rodriguez-Sanchez et al., 2021). Triacylglycerols are resynthesised from FA and monoacylglycerols in the intestinal mucosal cells and packaged into stable lipoprotein transport systems, known as chylomicrons, and transported to adipose tissue for storage. Tissues gain access to stored lipid through three stages of processing. Firstly, lipids are mobilised by degrading triacylglycerols into FA and glycerol and transported to the required site. Secondly, the FA are activated and transported into the cell mitochondria and thirdly, these are broken down in a step-by-step process into acetyl CoA which is then processed within the citric acid cycle (Berg et al., 2019).

Polar solutes are more readily incorporated into micelles which explains the superior absorbability and higher digestibility of unsaturated compared with saturated fats in broilers (Renner and Hill, 1961). Accordingly, oils have a higher dietary energy than hydrogenated or even partially hydrogenated fats (Wiseman, 1990). Instructively, a quantitative measurement of the degree of saturation described by Stahly (1984) and Wiseman (1990) is important and increasing the ratio of unsaturated to saturated dietary lipid was shown to be associated with a non-linear improvement in dietary energy. Additionally, an intact triglyceride is superior to hydrolysed lipid in terms of dietary energy (Young, 1961; Sklan, 1979) and increasing the proportion of FFA is associated with a linear reduction in lipid digestibility (Freeman, 1968). Based on the degree of saturation, proportion of FFA and, to a lesser extent, fatty acid chain length, in seminal work, Wiseman et al. (1998) were able to calculate the energy content of dietary lipids for both pigs and poultry. However, the Wiseman equation had a matrix assumption that lipid moisture content, impurities and unsaponifiable matter (MIU) totalled 20 g/kg. More recently, a correction factor was applied to the Wiseman equation, by extending the equation using a MIU (%) dilution constant (Wealleans et al., 2021). These authors calculated the constant as one minus MIU/100 and concluded that lipid apparent metabolizable energy (AME) variation was exaggerated by including MIU in the Wiseman equation. Interestingly, of the 724 commercially available fats and oils samples analysed in Wealleans et al., (2021), linseed oil had the largest range followed by poultry fat and beef tallow (Table 1). Unsaponifiable matter forms the largest component of MIU and is positively correlated to the levels of FFA that are known to have a pro-oxidant effect. Higher FFA increase sensitivity and rate of lipid oxidation, accompanied by a decline in the levels of poly-unsaturated fatty acids (Frega et al., 1999; Chen et al., 2011; Wealleans et al., 2021). In contrast to impurities, non-elutable material (NEM) represents the portion that cannot be used as an energy source by the animal. This is of particular importance for heat-damaged oils that are estimated to contain three to four times the levels of NEM than refined vegetable oil (Wiseman, 2017). Additionally, this has implications for recycled vegetable oils and oils that are recovered from further processing, for use in poultry feed, which is common practice in Australia. The NEM fraction is determined by exclusion gel chromatography and is considered by the European Parliament to be the best indicator of cooking oil degradation, particularly with respect to unsaturated fatty acids. In turn, the percentage of degraded fatty matter is calculated as the percentage of NEMminus the percentage of unsaponifiable matter (Boatella Riera and Codony, 2000). Other commonly used lipid quality measurements include colour, fatty acid profile, degree of unsaturation, or saturation, measured using iodine value (IV; titre) and measures of oxidative rancidity (Shurson et al., 2015).

III. OXIDATION AND RANCIDITY

Oxidative rancidity may occur prior to, or post-feed production and leads to the destruction of other fat-soluble nutrients such as vitamins A, D, E and K. Also, the greater the degree of unsaturation of lipid, the more prone to oxidation whilst some body lipid is required for fatsoluble vitamin storage (Kleyn and Chrystal, 2020; Wealleans et al., 2021). The complete oxidation of lipids, described as peroxidation, takes place over three phases; initialisation, propagation and termination with each step degrading and producing many different lipid peroxide compounds (Belitz et al., 2009). Primary oxidation results in lipid hydroperoxides, reducing the quality of the feed lipid and forming both secondary and tertiary peroxidation products (alcohols, ketones, aldehydes, hydrocarbons, epoxy compounds and volatile organic acids) that have a detrimental impact on broiler growth performance (Hung et al, 2017; Lindblom et al., 2019). At least 19 volatile compounds have been identified during linoleic acid peroxidation and the initial peroxides and aldehydes formed during primary oxidation are ultimately degraded as peroxidation continues (Belitz et al., 2009; Shurson et al., 2015). Dual evaluation of lipid peroxidation via firstly, indicative analyses that measure specific chemical compounds at time of sampling or secondly, predictive stability methods that test the ability of the lipid to withstand peroxidation, when exposed to standardised accelerated conditions, to induce peroxidation (Shurson et al., 2015).

Common indicators of peroxidation in feed lipids have been peroxide value (PV), panisidine value (AV) (deRouchey et al. 2004; Danowska‐Oziewicz et al., 2005) and thiobarbituric acid reactive substances (TBARS) (Liu et al., 2014). However, other measures such as the total oxidation value (TOTOX = AV + 2 × PV), conjugated dienes, triacylglycerol dimers and polymers, total carbonyls, hexanal value and oxirane value, have been occasionally used to assess lipid peroxidation (Seppanen, 2005) as well as assays that measure specific peroxidation compounds such as 2,4-decadienal (DDE) and 4-hydroxynonenal (HNE). In Wealleans et al., (2021) a PV for non-fish oils below five and TBARS below 0.5 would suggest no oxidation whilst 5 to 10 and 0.5 to 1, respectively, suggest first signs of oxidation. Oxidation has occurred at a PV of 10 to 20 (TBARS one to two) and strong oxidation above 20 and two TBARS respectively. Peroxidation compounds measured by PV, AV, TBARS, conjugated dienes, total carbonyls and hexanal are produced and subsequently degraded at various stages of the peroxidation process, making interpretation of results difficult and misleading (Shurson et al., 2015). Instructively, Gray (1978) concluded that there is no single chemical method that can entirely predict or explain changes in organoleptic properties of oxidised lipids, highlighting the importance of using multiple measures to assess the oxidative status of a lipid sample.

IV. FEED FORMULATION

Feed formulation assumes the apparent metabolizable energy (AME) assigned to individual feed ingredients is both linear and additive and it is unlikely that this is true. For example, in Kleyn and Chrystal (2020) composite data was used to calculate an “extra-calorific” effect of low levels of added dietary lipid (below 20 g/kg) and a non-linear decline in AME with increasing dietary lipid above 20 g/kg (Figure 1.). This is further complicated by the change in fat digestibility as broilers age and in Ravindran and Abdollahi (2021) the average of three lipid sources during the first seven days post-hatch was only 52.0% increasing to 79.9% by day 14 and reaching a maximum of 87.6% at day 21. Also, FFA may react with other nutrients to form soaps and compounds with minerals that may or may not be soluble. If insoluble salts are formed, both the mineral and the FA become unavailable to the bird and this is associated with reduced bone ash and calcium content (Kleyn and Chrystal, 2020). In high-yielding, modern broiler genotypes, the primary breeder recommendations for dietary AME have declined whilst digestible amino acid profiles have increased (Aviagen, 2022; Cobb-Vantress, 2022). This is due largely to the increased growth rate whereby the tangible amount of proportional dietary energy required for maintenance has declined. Also, selection for improved feed conversion has reduced the amount of total body lipid at maturity, and lipid has a worse feed conversion compared with lean gain. Furthermore, the heat increment of digestion of dietary lipid is lower than that of protein or carbohydrate and this has implications for formulating feed to net energy (NE), where NE is equivalent to the AME minus the heat increment of digestion (Wu et al., 2019). In Wu et al., (2019) an adjustment was made to AME, whereby dietary crude protein and crude fibre reduced NE whilst ether extract, used to measure dietary lipid, increased NE utilising metabolic closed-circuit calorimetry chambers, housing male Ross 308 broilers.

Interestingly, both primary breeders stipulate a minimum dietary linoleic acid for high yielding broilers; namely 12 g/kg from zero to 28 days post-hatch then 10 g/kg (Cobb-Vantress, 2022) and 12.5 g/kg from zero to 10 days post-hatch, then 12.0 g/kg from 11 to 24 days followed by 10 g/kg above 25 days post-hatch (Aviagen 2022). It would appear that the requirement is based on liver triene:tetraene ratio that changes at about 10 g/kg of dietary linoleic acid rather than broiler growth performance (Zornig et al., 2001). Instructively, in a series of 3 experiments with male Ross 208 chicks to 21 days post-hatch over two decades ago, Zornig et al., (2001) were able to demonstrate that broiler growth performance was very good at levels of 2.0 g/kg dietary linoleic acid.

In feed formulation, nutritional emulsifiers and biosurfactants such as lysophospholipids mimic the effect of natural bile salts and have their main effect on saturated fatty acids (C16:0 and C18:0). Lysophospholipids are more hydrophilic than phospholipids because they have a single FA residue per molecule and form spherical micelles in aqueous solution, leading to enhanced emulsification in the gastrointestinal tract. They may also play an important role in young broilers where bile production and recirculation are low (Ravindran, 2014). Studies have shown that lysophospholipids (lysolecithin) is more effective than bile and lecithin (Zhang et al., 2011; Zaefarian et al., 2015; Wealleans et al., 2020). In New Zealand studies, the addition of lysolecithin at 250 g/t to tallow and soyabean oil improved AME by 3.62% on average (from 13.54 to 14.03 MJ/kg, P < 0.05) in Ross 308 male broilers from one to 35 days post-hatch suggesting a raw material matrix value of 1960 MJ/kg for lysolecithin (Ravindran, 2014; Zaefarian et al., 2015). Lysolecithin has also been shown to enhance collagen expression and increase villus length in the jejunum of broiler chickens (Brautigan et al., 2017). Other commercially available emulsifiers include non-ionic liquids and powders and a novel glycolipid identified as sophorolipid (Kwak et al., 2022).

V. CONCLUSIONS

Dietary lipids cover a wide range of compounds and, a brief review is only able to cover some aspects of lipid nutrition without delving into detail. In least-cost linear feed formulation, energy remains the costliest component and, whilst it is treated as a “nutrient”, it comprises the energy released through the chemical oxidation of the feed. Both bird factors and feed factors play a role in the amount of energy available for broiler growth and maintenance as illustrated by Wu et al., (2019). Lipid digestion, absorption and metabolism are affected by numerous factors inherent in the quality of the dietary lipid used, level of saturation and the age of the broiler. The degree of oxidation, lipids damaged through heat, MIU and other NEM compounds are difficult to quantify but, they are vitally important in assessing the quality of the lipid used. The quality of dietary lipids therefore needs to be assessed using several measures. Furthermore, in feed formulation, response to added dietary lipid is quadratic, whilst bird age has an influence on lipid digestibility and resulting AME. Dietary lipid also has an influence on the NE, due to reduced HI of digestion, increasing the energy available for growth and maintenance in broilers, thus affecting its relative worth to other dietary energy components, particularly in hot climates. However, selected nutritional emulsifiers and biosurfactants may enhance the digestibility of dietary lipids, particularly with respect to young broilers.

Due to the primary breeder recommendations for dietary AME declining, added lipid has also declined in many broiler feeds raising implications for the EFA, linoleic acid. For maize/soyabean meal-based diets, it is unlikely the linoleic acid would be below 10 g/kg, but low-energy wheat/soyabean meal-based broiler diets regularly formulate to below this level and, may result in increased feed costs. For broiler feeds, removing the minimum constraint for linoleic acid in feed formulation should be considered. Also, some dietary lipid and adipose tissue in the bird are required for metabolism of the fat-soluble vitamins, A, D, E and K. In conclusion, adjusting accepted Wiseman calculations for dietary energy by including adjustments made for the portion of dietary lipid that does not contain an inherent AME value is prudent. However, further research is required to determine the levels of these diluents in commercially available feed lipids used in Australia, and establish the true energy values of these lipids more accurately.

Table 1 - Calculated minimum (Min.) and maximum (Max.) apparent metabolizable energy (AME) values based on Wiseman (1997) equation and analysed moisture, impurities and unsaponifiable matter (MIU) in broilers, at 10 and 53 days post-hatch (adapted from Wealleans et al., 2021).