This article was published in the March 2021 issue of Pet Food Processing. Read it and other articles from this issue in our March digital edition.

Protein is considered an essential nutrient; however, like humans, companion animals do not have a requirement for protein, but rather a requirement for indispensable amino acids (AA) and sufficient nitrogen. Indispensable AAs must be supplied in the diet because companion animals cannot synthesize them. Sufficient intake of indispensable AAs, as well as adequate provision of non-essential AAs, which can be synthesized by the animal if enough nitrogen and carbon sources are provided in the diet, allows for the maintenance of whole-body protein reserves (i.e. lean muscle) and the synthesis of secondary metabolites (i.e. the skin pigment melanin is synthesized from phenylalanine and tyrosine) (Biourge and Sergheraert 2002).

Current recommendations for indispensable AA requirements established by the National Research Council (NRC) for adult dogs are based off limited studies where minimum requirements of the majority of indispensable AAs —histidine, isoleucine, leucine, lysine, phenylalanine, tyrosine, threonine, tryptophan, and valine — are based only on the lowest concentrations reported in a doctoral dissertation and one empirical peer-reviewed report (NRC 2006). In these studies, adult dogs were fed low-crude protein diets for an extended period of time and displayed no observable clinical signs of AA deficiency leading to definition of the minimum requirements.

Both studies that contributed to the recommendations of these AA requirements in adult dogs used the beagle as a representation of all dog breeds (Ward 1976) (Sanderson, et al. 2001). This one breed serves as a basis for suggested requirements of a number of indispensable AAs for adult dogs of all breeds and breed sizes, so these reports do not account for potential differences among dog breeds in physical characteristics (e.g. conformation), genetics, or lifestyles (e.g. activity level). These differences may also cause variation in the way a particular breed utilizes dietary AAs, as well as the concentration of each indispensable AA that the breed needs to maintain an ideal bodyweight and/or sustain metabolic processes and may also affect the dogs’ metabolism through a myriad of different processes.

Currently, the most conventional and commonly utilized method for estimating AA requirements is measuring nitrogen balance and growth performance in growing animals fed diets containing various levels of the specific AA being estimated and with all other dietary components held constant (NRC 2006) (Milner 1979) (Burns and Milner 1982) (Czarnecki and Baker 1982). Nitrogen balance techniques consist of determining the difference between dietary nitrogen intake and nitrogen excretion (Tessari 2006). While these techniques may be practical for generating estimates of AA requirements for growing animals, results from these studies must be carefully considered when extrapolating these requirements to adult dogs (Just 1982). For example, while growth is typically correlated to nitrogen retention in growing animals (Baker 1986), changes in nitrogen balance, bodyweight, and whole-body protein stores occur at slower rates in adult animals since they are no longer growing (Moughan 1995). As well, this methodology neglects the secondary metabolic utilization of an AA, which may have a more profound effect on the requirements of adult animals than for growing animals (Moughan 1999). Therefore, alternative and more sensitive techniques for determining AA requirements in adult dogs should be considered in the future.


Beyond the recommendations

AAFCO in the United States and FEDIAF in Europe make recommendations for dietary AAs which are similar to those established by the NRC. However, they have been scaled up to account for ingredient-to-ingredient differences in AA digestibility (the amount of an AA that is transported out of the gut and into the body) and bioavailability (the amount of an AA that can be used for protein synthesis in the body).

“Determining amino acid digestibility and bioavailability of different ingredients is important when formulating diets,” wrote Crosbie, et al., University of Guelph.

Determining AA digestibility and bioavailability of different ingredients is important when formulating diets. This is, in part, because many AA minimum requirements presented by the NRC are also based upon data from studies utilizing purified diets with highly digestible ingredients, such as crystalline AAs. Crystalline AAs are assumed to be 100% bioavailable while commercial diets are typically formulated with natural intact-based protein sources that are generally assumed to have a protein digestibility of approximately 80% (NRC 2006). However, AA digestibility does not necessary correlate to nitrogen digestibility. The most accurate method for determining AA digestibility is measurement of ileal AA digestibility by collecting digesta from the end of the small intestine (NRC 2006). This method determines what proportion of the AAs supplied in the diet are absorbed by the animal, since protein is digested, and AAs are absorbed before the end of the small intestine. However, this method is invasive, requiring surgical insertion of a cannula at the end of the small intestine, and for this reason, is no longer done in dogs. Alternate methods for determining digestibility of AAs in dogs have been used with the most common in the pet food industry being the assessment of total-tract AA digestibility (NRC 2006). While certainly less invasive, as this method only requires collection of feces, it overestimates AA digestibility from the diet as the feces will also include AAs produced by the bacteria in the large intestine and endogenous losses (e.g. enzymes, sloughed cells, bacteria, and mucus) (NRC 2006). As digestibility is often considered an indication of bioavailability, this demonstrates that bioavailability may also be overestimated when extrapolated from total tract digestibility.

Processing and storage of ingredients can also affect the bioavailability of an AA even if it is digestible. For example, lysine can react with reducing sugars in carbohydrates in the presence of heat (referred to as Maillard reaction products) and during alkali treatments (formation of cross-linked AAs) (Fernandez and Parsons 1996) (González-Vega, et al. 2011). These products can be absorbed, but bound lysine cannot be used by protein synthesis. Therefore, digestibility values of an AA may remain the same, but bioavailability may be reduced.Amino acids. names and structures

Digestibility of indispensable AAs in intact protein ingredients can be further affected by other dietary components such as fermentable carbohydrates. Fermentable carbohydrates reduce ileal digestibilities, nitrogen retention, and average daily gain in pigs (Myrie, et al. 2008). The addition of dietary pectin has been reported to reduce total tract crude protein digestibility in dogs (Silvio, et al. 2000). In addition to reducing ileal digestibility of the AA, fermentable fibers such as oligosaccharides result in greater colonic weight of rats (Campbell, et al. 1997), which may affect the utilization of AAs, specifically those that are used in considerable amounts by the gut (e.g. methionine) (Shoveller, et al. 2003) (Riedijk, et al. 2007). Therefore, it is important to know not only what indispensable AAs are provided by specific protein ingredients but also the digestibility of these indispensable AAs on their own and in the presence of other ingredients to ensure the requirements for these indispensable AAs can be met accurately. This also provides a further case for additional supplementation of crystalline AAs when meeting indispensable AA requirements through only intact ingredients.

Regardless of what method is used to determine AA digestibility and bioavailability in intact ingredients, processing (especially heat treatment) can impact digestibility/bioavailability of AAs, as many ingredients require processing treatments before consumption to improve digestibility and eliminate bacteria. A trend in commercial dog diets, especially grain-free diets, is the use of legumes (such as peas, chickpeas, and lentils) to supply carbohydrates and protein. Legumes cannot be consumed in their native form and must be processed before consumption. Leguminous ingredients in their raw forms contain anti-nutritional factors (ANF). Of the ANFs found in legumes, trypsin and chymotrypsin inhibitors, tannins, and phytate can all decrease protein and AA availability (Gilani, et al. 2005). The inhibitors of serine proteases (trypsin and chymotrypsin) are the most important enzyme inhibitors in legumes with respect to protein digestion (Belitz and Weder 1990). These enzyme inhibitors inhibit the function of trypsin and chymotrypsin enzymes, decreasing the overall digestion of proteins and subsequently the availability of AAs for absorption (Singh and Basu 2012). Phytate, while primarily associated with minerals, can also bind and decrease the availability of proteins in the gastrointestinal tract (Singh and Basu 2012). Tannins can negatively affect protein digestibility through the precipitation of proteins in the gastrointestinal tract reducing availability for absorption (Singh and Basu 2012) (Gilani, et al. 2005). Therefore, importance is placed on processing pulses to enhance their digestibility and suitability in pet foods.

While processing of legumes can include methods such as dehulling, roasting, boiling, and pressure-cooking, the most common method utilized in the pet industry is extrusion for their inclusion into dry commercial dog diets. The high pressure and heat used in the extrusion process not only allows for the inclusion of legumes in kibble but is necessary to reduce ANFs and may result in improved protein and indispensable AA contents, digestibility, and protein quality (Malcolmson and Han 2019). Caution must be taken as these high-heat processing methods may also result in decreased protein and indispensable AA digestibility, which may be attributed to the formation of Maillard reaction products, as well as thermal cross linking of AAs (Khattab, et al. 2009). Despite this, the reduction or destruction of ANFs from these processing methods is generally greater compared to reduction in AA content and overall results in a net positive change in protein and AA availability.


Supplying adequate AAs

As mentioned, using nitrogen balance techniques to determine minimum requirements of indispensable AAs does not account for secondary metabolic utilization of an AA (Moughan 1999).   Supplying adequate indispensable AAs to adult dogs goes beyond purely supporting protein synthesis, as most AAs have secondary roles through synthesis of secondary metabolites. For example, tryptophan is an indispensable AA for dogs and while its primary role is in its contribution to protein synthesis, tryptophan is also vital for a variety of secondary metabolic processes, including the synthesis of both serotonin and melatonin (Triebwasser, et al. 1976) (Richard, et al. 2009) (Freeman, et al. 2013).

Tryptophan is the sole precursor of serotonin, a neurotransmitter involved in the modulation of numerous central nervous system functions, such as mood, aggression, anxiety, and motor behaviors, (Richard, et al. 2009) (Sandyk 1992), as well as the regulation of the gastrointestinal environment (Gainetdinov, et al. 1999) (Mohammad-Zadeh, et al. 2008) (O’Mahoney, et al 2015). Serotonin can also be used to synthesize melatonin in the pineal gland, a pathway that is influenced by a dog’s circadian rhythm, with the activity of the enzymes responsible being greater at night and reduced during daylight (Szeitz and Bandiera 2107). Another example is phenylalanine, another indispensable AA for dogs (NRC 2006). Phenylalanine is required for protein synthesis and for the synthesis of tyrosine, but it also plays a vital role in the synthesis of catecholamines (such as dopamine, epinephrine, and norepinephrine), which are important to regulate the stress-response (Fernstrom and Fernstrom 2007). Phenylalanine and tyrosine also play a role in the maintenance of coat color via production of a compound called eumelanin, a form of melanin (Biourge and Sergheraert 2002). When black-coated dogs have a deficiency in phenylalanine and tyrosine in the diet, it can be easily observed as these dogs will develop a reddish-brown haircoat (Biourge and Sergheraert 2002). While these are only a few examples of secondary metabolites synthesized from indispensable AAs, this illustrates that many play an important role in other vital metabolic processes and often these are noticeable to the dog owner.

“The importance of supplying sufficient quantities of amino acids in dog diets is clearly much more nuanced than purely supplying enough dietary protein,” wrote Crosbie, et al., University of Guelph.

The importance of supplying sufficient quantities of AAs in dog diets is clearly much more nuanced than purely supplying enough dietary protein. To ensure diets are formulated with the intention of promoting health and longevity in our pets, it is critical that more attention is paid to the indispensable AA profile of specific protein ingredients, the digestibility of these indispensable AAs, the processing necessary to ensure the protein source is suitable for diet formulation, and the metabolic role(s) played by each indispensable AA. In contrast to the information available on other species, such as agricultural animals, on what variables affect AA requirements, the information available in dogs and among dog breeds is sparse. Optimization of protein and AA delivery needs to be improved to support the long-term sustainability of the pet food industry.

References cited in this article are available upon request.

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