Use of enzymes as feed additives

A number of enzymes are already used commercially as feed additives, particularly in non-ruminant (pig and poultry) feeds, to increase the digestion and subsequent absorption of nutrients⁽ Reference Bedford and Schulze ^{8 –} Reference Humer, Schwarz and Schedle ^{10 )}. They are mainly used to improve the digestion of feed components that the animals cannot normally digest or are only able to digest fairly poorly, such as complex carbohydrates and phytate. By increasing the digestibility of the feed, more nutrients enter the body and less pass through in the faeces, resulting in increased growth for the same level of feed intake, hence improving FE.

A number of enzyme feed additives are commercially available to improve the digestibility of cereal carbohydrates, particularly targeting xylans and arabinoxylans present in the cell walls⁽ Reference Masey O'Neill, Smith and Bedford ^{9 )}. By digesting these important structural carbohydrates in the cell wall, that then allows the animals’ own carbohydrate-digesting enzymes (e.g. α-amylase) better access to the main starch stores within the plant cells. Secondly, the digestion reduces the viscosity problems associated with arabinoxylans and β-glucans⁽ Reference Masey O'Neill, Smith and Bedford ^{9 )}. A number of studies have shown improved FE and/or FCR of pigs and chickens when these enzymes are added to the feed. For example, xylanase supplementation of feed was shown to improve FCR (1·41 v. 1·56 in controls) in broiler chickens by increasing weight gain, but not affecting feed intake⁽ Reference Amerah, Mathis and Hofacre ^{11 )}. As well as increasing the digestibility of the carbohydrate component of the feed and reducing the viscosity, there are suggestions that these carbohydrate-degrading enzymes might have prebiotic actions on the gut microflora via the oligosaccharides they produce⁽ Reference Masey O'Neill, Smith and Bedford ^{9 )}. This could be another potential mechanism for their effects on FE. The absorption of nutrients across the gut is also known to affect production of gut peptides, which can subsequently alter gut motility and feed intake. Indeed xylanase supplementation of feed has been shown to increase plasma  peptide YY levels in broiler chickens⁽ Reference Singh, Masey O'Neill and Ghosh ^{12 )} and we have recent data showing effects of xylanase supplementation on plasma peptide YY, gastric inhibitory polypeptide and glucagon-like peptide-1 concentrations in young pigs⁽ Reference May, O'Sullivan and Brameld ^{13 )}. Hence, the regulation of gut peptides and their subsequent effects on gut motility, feed intake and/or nutrient utilisation might be additional, alternative mechanisms for the effects of these carbohydrate-degrading enzymes on FE.

Phytase is another enzyme used commercially in non-ruminant (pig and poultry) feeds⁽ Reference Humer, Schwarz and Schedle ^{10 )}. Phytase digests phytate (also called phytic acid or inositol hexakisphosphate), the main storage form for phosphorus in plants. Phytate (hexakisphosphate) is inositol with six phosphate groups attached and phytase is able to cleave individual phosphate groups, thereby releasing them for absorption and use by the animal. Phytase supplementation results in greater absorption of phosphorus and calcium from the feed in broiler chickens and pigs⁽ Reference Simons, Versteegh and Jongbloed ^{14 )}, resulting in increased growth and reduced FCR. However, the increased growth may not simply be due to increased absorption of these important micronutrients. Chicken studies⁽ Reference Liu, Ru and Li ^{15 )} have shown that high levels of phytate in the diet inhibit pepsin and trypsin activities and therefore inhibit protein digestion and amino acid absorption, resulting in increased FCR. Inclusion of phytase as well as high phytate in the diet reduced the inhibitory effect on proteolysis, resulting in improved (reduced) FCR⁽ Reference Liu, Ru and Li ^{15 )}.

Both of these feed additive enzymes have positive effects on FE in pigs and chickens fed cereal-based diets. They do so by different mechanisms, meaning their benefits are likely to be additive, but importantly they may allow the use of poorer quality (i.e. human inedible) feed ingredients, an important consideration for future sustainability and food security. These and other enzymes are also being investigated for use in ruminants⁽ Reference Masey O'Neill, Bedford, Walker, Garnsworthy and Wiseman ^{16 )}.

Use of growth promoters/metabolic modifiers/anabolic agents

There are three main classes of growth promoters⁽ Reference Sillence ^{17 )} : β-adrenergic agonists (BA), anabolic steroids and growth hormone (GH, also called somatotropin). They all improve FE in livestock to some extent and this is associated with increased lean mass (particularly skeletal muscle) and reduced fat mass⁽ Reference Sillence ^{17 )}. Indeed they have all been in the news at different times in relation to their illegal use as performance enhancing drugs in sportsmen and women. Their effects on muscle and fat mass were first discovered in the 1950s (anabolic steroids) or 1980s (BA and GH) and a number of commercial products are currently licenced for livestock production around the world⁽ Reference Sillence ^{17 )}, although they are all banned in the European Union (EU). For example, ractopamine and zilpaterol (both BA) are licenced for use in pigs and/or cattle in North and South America, South Africa, India and Australia, but not China. Similarly, the anabolic steroid mix of trenbolone acetate and oestradiol is licenced for use in beef cattle in North and South America, South Africa, India, Australia and China and GH (either bovine or porcine somatotropin) is licenced for use in dairy cattle or pigs in the same areas. We were unable to find information for other parts of the world (e.g. Northern Africa and other parts of Asia); so to our knowledge only the EU has a total ban on the use of these agents in livestock production. This is despite much of the early research work being carried out in the EU, especially the UK, and the original scientific reports suggesting their use was safe⁽ Reference Lamming, Ballarini and Baulieu ^{18 )}, as long as appropriate guidelines were followed (e.g. a withdrawal period prior to slaughter).

At the University of Nottingham, we have been comparing the molecular modes of action of BA and GH in both sheep⁽ Reference Al-Doski, Parr and Hemmings ^{19 –} Reference Parr, Al-Doski and Hemmings ^{21 )} and pigs⁽ Reference Brameld, Atkinson and Saunders ^{22 –} Reference Sensky, Jewell and Ryan ^{24 )} combining transcriptomic and metabolomics technologies in a systems biology approach to identify novel mechanisms to achieve the same effects. Ultimately the aim is to identify novel target genes/proteins to develop more acceptable drugs or for targeted breeding or nutritional manipulations. We have made good progress and have identified up-regulation of the serine biosynthesis pathway⁽ Reference Al-Doski, Parr and Hemmings ^{19 ,} Reference Parr, Al-Doski and Hemmings ^{21 ,} Reference Brameld, Ryan and Williams ^{23 )} and a number of other novel changes in response to BA and/or GH treatments. We are currently performing proof-of-principle studies to determine whether the novel genes we have identified really do regulate growth, body composition and/or FE. If successful, the next stage will be to use this information to develop breeding strategies, new dietary regimens or drugs that result in improved FE in livestock.

For proof-of-principle studies we often utilise transgenic animals (mainly mice) where the gene of interest is either overexpressed or knocked out/down (i.e. genetic manipulation (GM)), often in a tissue-specific manner. This is done to investigate whether manipulation of the specific gene results in the predicted changes in tissue growth and/or metabolism, as well as changes in FE or whole-body energy expenditure. Such studies cannot be performed in cultured cells, so must be done in animals. Although technically challenging, GM can now be achieved in livestock⁽ Reference Niemann, Kuhla and Flachowsky ^{25 )}, so that it will theoretically be possible to produce herds of transgenic livestock. Indeed the Chinese government is funding work using GM aimed at developing new breeds of livestock for agricultural use in the future, including research into their safety^{( 26 )}. One of the main advantages of GM over conventional animal breeding is that GM speeds up the process and is more gene-specific; whereas conventional breeding, while very successful over the past 50 years, can result in unwanted side effects, both on animal welfare and also product quality. The halothane pig⁽ Reference Rosenvold and Andersen ^{27 )} and Callipyge sheep⁽ Reference Tellam, Cockett and Vuocolo ^{28 )} are prime examples of this. Both have increased growth rates, particularly muscle, but one (halothane) results in highly stressed pigs and both result in poorer meat quality.

Molecular studies of low residual feed intake animals

The concept of low and high RFI has progressed rapidly over recent years⁽ Reference Herd, Oddy and Richardson ^{29 ,} Reference Sartin ^{30 )}. Studies are being carried out around the world aimed at identifying specific genes (or markers) for improved FE in virtually all livestock species (cattle, pigs, sheep and poultry). The genetic approach has been to identify markers (quantitative trait loci or SNP) of low RFI for subsequent use in selective breeding programmes. For example, a Chinese group⁽ Reference Luo, Sun and Ma ^{31 )} recently identified a SNP in a microRNA (miR-1596) gene in chickens that resulted in reduced expression of miR-1596 in livers and was associated with low RFI. Interestingly, they suggested that there were more than seventy target genes for miR-1596⁽ Reference Luo, Sun and Ma ^{31 )}, which were mainly involved in energy metabolism, apoptosis and immune responses, with some being important proteins for assembling mitochondria.

We collaborated with another Chinese group⁽ Reference Jing, Hou and Wu ^{32 )}, to investigate differential gene expression in skeletal muscle from pigs with low v. high RFI using a deep sequencing (RNAseq and miRNAseq) approach. A number of mRNA (IGF2, FABP3 and PGC1a) and miRNA (miR1, miR30, miR10b and miR145) were found to be differentially expressed, but importantly the majority of mitochondrial genes were down-regulated. The data suggested that low RFI was linked with changes in expression of mRNA and miRNA associated with increased muscle growth and reduced mitochondrial activity in skeletal muscle⁽ Reference Jing, Hou and Wu ^{32 )}.

Effects on mRNA or miRNA associated with mitochondria appear to be a recurring theme in the low RFI studies⁽ Reference Bottje and Kong ^{33 ,} Reference Grubbs, Huff-Lonergan and Gabler ^{34 )} and this agrees with some of our growth promoter studies, where we also see down-regulation of a number of genes associated with mitochondria, including both tricarboxylic acid cycle and oxidative phosphorylation genes (JM Brameld, T Parr et al., unpublished results).

Once again the genes being identified in these various RFI studies could be potential targets for novel drugs, dietary regimens or GM in animals, as well as being used for conventional breeding strategies to improve FE in livestock.