Dietary fat lowers ileal endogenous amino acid losses in broiler chickens

ABSTRACT

1. An experiment was conducted to determine the effect of the source of fat (soybean oil or tallow) on the ileal endogenous amino acid (EAA) losses in broilers.

2. Three nitrogen (N)-free diets; a control diet with no added fat and test diets with 60 g/kg of either soybean oil or tallow were formulated. Titanium dioxide (5 g/kg) was added to all diets as an indigestible marker. Each diet was assigned to six replicate cages (eight birds per cage) from d 18 to 21 post-hatch. On d 21, the digesta were collected from the lower half of the ileum.

3. The endogenous losses of nitrogen and amino acids (AA) were lower (p = 0.08; p = 0.001) in broilers fed diets with soybean oil or tallow, respectively, compared to those fed the diet with no fat. Source of fat had no influence (p > 0.05) on EAA losses.

4. The most abundant AA in the ileal endogenous protein was glutamic acid, followed by aspartic acid, threonine, leucine, serine, valine and proline. In general, the concentrations of AA in the endogenous protein were lower (p < 0.05) with added fat. The exceptions were methionine, cysteine, proline and serine, which were unaffected. The effect of fat source on the AA contents of endogenous protein were inconsistent and differed depending on the AA.

5. The inclusion of fats decreased EAA losses which implied they have beneficial effects beyond direct energy contribution. It can be proposed that the reduction of EAA flow may be an additional mechanism contributing to the extra-caloric effect of dietary fats.

KEYWORDS:

• Amino acid
• broiler
• endogenous losses
• fat source
• soybean oil
• tallow

Introduction

The term endogenous amino acid (EAA) losses refers to those in the digesta leaving the terminal ileum, which are not of food origin. The measurement of EAA losses is needed to correct the apparent amino acid (AA) digestibility to true values (Lemme, Ravindran, and Bryden 2004; Ravindran and Bryden 1999). These losses represent a significant metabolic cost to the animal (Cowieson et al. 2009) and a better understanding of influencing factors is of fundamental interest to improve the nitrogen (N) utilisation efficiency of animals. A number of dietary factors have already been identified, including methodology, the amount and type of dietary protein and fibre, and presence of anti-nutritional factors (Adedokun et al. 2011; Ravindran 2021).

Animal fats and vegetable oils are energy dense, providing a unique and cost-effective opportunity to increase the energy supply in broiler diets. Typical fat inclusions in broiler diets range from 30 to 60 g/kg and even a small change in levels can have meaningful effects on energy supply. These two fat sources differ in their physical and chemical properties, and their digestion dynamics are inherently different. Unsaturated fatty acids dominate vegetable oils, whereas animal fats are more heavily composed of saturated fatty acids (Blanch et al. 1995). Saturated fatty acids are non-polar and so cannot form mixed micelles spontaneously and need bile salts for emulsification to form micelles prior to hydrolysis. However, unsaturated fatty acids in soybean oil are readily emulsified and, hence, better digested and absorbed (Ravindran et al. 2016).

A protein-sparing effect of fat in pig diets has been observed, which was presumed to be the result of slowing the rate of passage. Imbeah and Sauer (1991) and Cervantes-Pahm and Stein (2008) reported improvements (up to 4.9%) in the ileal digestibility of some AA when canola oil was added to diets for growing pigs. Li and Sauer (1994) found that AA ileal digestibility was linearly increased when canola oil inclusion was raised from 32 to 122 g/kg. Cowieson et al. (2010) similarly observed that addition of 20 g/kg soybean oil to a broiler starter diets increased ileal AA digestibility by an average of 3.5%. The greatest increase was seen in the digestibility of threonine, serine, isoleucine, aspartic acid, valine and glycine; the predominant AA in endogenous protein. These observations implied improvements in AA digestibility with added fat and it can be speculated that the mechanisms involved may, in part, reflect reductions in EAA losses. In addition, the composition and digestion kinetics of vegetable oils and animal fats differ (Tancharoenrat et al. 2014), and may be relevant on their influence on endogenous AA secretions and losses.

The present study tested the hypotheses that the addition of fat can lower ileal EAA losses in broilers and that, owing to differences in emulsification, digestion and absorption, the degree of reduction can be influenced by fat source.

Materials and methods

Ethical statement

The experimental procedures were approved by the Massey University ethics committee

Animal husbandry

Day-old male broilers (Ross 308) were raised in the floor until d 14 post-hatch and fed a commercial broiler starter diet (12.9 MJ/kg metabolisable energy, 226 g/kg crude protein, 11.0 g/kg Ca, 6.7 g/kg total P). On d 14, birds were transferred to grower cages and were maintained on the same diet until the introduction of test diets on d 18 post-hatch. Both the floor facility and cages were in an environmentally controlled house. Each cage was equipped with a linear feeder in the front and a nipple drinker with a cup in the back. The temperature was maintained at 31°C on d 1 and reduced to 20°C by d 21. A lighting schedule of 20 hours per day was applied by fluorescent tubes.

Three N-free diets, namely with no added fat source, 60 g/kg soybean oil or 60 g/kg tallow (Table 1), were developed. Titanium dioxide (5.0 g/kg) was included in the diets as an indigestible marker. On d 18, birds were individually weighed, and 144 birds were assigned to 18 cages containing eight birds each. Following 4 h of feed withdrawal, the assay diets were introduced and offered ad libitum. Water was available at all times.

Table 1. Ingredient composition (g/kg as fed) of the experimental diets.

	Control (No fat)	Soybean oil	Tallow
Dextrose	907	847	847
Soybean oil	0	60	0
Tallow	0	0	60
Cellulose	35	35	35
Sodium bicarbonate	3.0	3.0	3.0
Sodium chloride	3.0	3.0	3.0
Dicalcium phosphate	19.5	19.5	19.5
Limestone	13.2	13.2	13.2
Dipotassium phosphate	12.0	12.0	12.0
Vitamin premix^1	0.8	0.8	0.8
Trace mineral premix^1	1.5	1.5	1.5
Titanium dioxide	5.0	5.0	5.0
Calculated value
Apparent metabolisable energy, MJ/kg	14.0	15.4	15.0

^aSupplied per kg of diet: antioxidant, 100 mg; biotin, 0.2 mg; calcium pantothenate, 12.8 mg; cholecalciferol, 60 µg; cyanocobalamin, 0.017 mg; folic acid, 5.2 mg; menadione, 4 mg; niacin, 35 mg; pyridoxine, 10 mg; trans-retinol, 3.33 mg; riboflavin, 12 mg; thiamine, 3.0 mg; dl-α-tocopheryl acetate, 60 mg; choline chloride, 638 mg; Co, 0.3 mg; Cu, 3.0 mg; Fe, 25 mg; I, 1 mg; Mn, 125 mg; Mo, 0.5 mg; Se, 200 µg; Zn, 60 mg.

On d 21, all birds were euthanised by intravenous injection of sodium pentobarbitone and the contents of the lower ileum were collected. The ileum was divided into two halves and the contents were collected from the lower half towards the ileo-caecal junction (Ravindran and Bryden 1999). Digesta were flushed out with reverse-osmosis water, pooled within each cage, mixed, sub-sampled and lyophilised. Samples of diets and digesta were ground to pass through 0.5 mm sieve and stored in airtight plastic containers at 4°C until chemical analyses.

Chemical analyses

Fatty acid composition of the two fat sources was determined following the procedure of Sukhija and Palmquist (1988). The diet and digesta samples were analysed for dry matter, titanium dioxide, nitrogen (N) and AA, including sulphur-containing AA. Dry matter was determined by drying samples at 105°C for 16 h in a pre-weighed dried crucible in a convection oven (AOAC International 2016 method no: 930.15). Nitrogen was determined by using an N determinator (model FP-428, LECO® Corporation, St Joseph, MI). Titanium was determined as per the procedures of Short et al. (1996). For AA analysis, samples were prepared by hydrolysing the samples with HCl (containing phenol) for 24 h at 110 ± 2°C in glass tubes sealed under vacuum. Amino acids were then detected on a Waters ion exchange HPLC system, and the chromatograms were integrated using dedicated software (Maxima 820, Waters, Millipore, Milford, MA) with the AA identified and quantified using a standard AA solution (Pierce, Rockford, IL). Cysteine and methionine were analysed as cysteic acid and methionine sulphone by oxidation with performic acid for 16 h at 0°C and neutralisation with hydrobromic acid prior to hydrolysis.

The EAA flow at the terminal ileum was calculated as mg lost per kg dry matter intake (DMI) using the following formula (Moughan et al. 1990).

$\mathrm{Basal}\mathrm{endogenous}\mathrm{AA}\mathrm{flow}\left(\mathrm{mg}/\mathrm{g}\mathrm{DMI}\right)=\mathrm{Amino}\mathrm{acid}\mathrm{concentration}\mathrm{in}\mathrm{ileal}\mathrm{digesta}\left(\mathrm{mg}/\mathrm{kg}\right)\text{break}\times \left(\mathrm{Titaniu}{\mathrm{m}}_{\mathrm{Diet}}\left(\mathrm{mg}/\mathrm{kg}\right)/\mathrm{Titaniu}{\mathrm{m}}_{\mathrm{Digesta}}\left(\mathrm{mg}/\mathrm{kg}\right)\right)$

The AA concentrations were also calculated as percentage of crude protein (N x 6.25) and presented as g/100 g crude protein.

Statistical analysis

The data were analysed using the General Linear Model procedure (SAS 2015). Differences were considered significant at p < 0.05 and significant differences between means were separated by the Least Significant Difference test. Cage served as the experimental unit.

Results

The analysed fatty acid composition of soybean oil and tallow are presented in Table 2. The major fatty acids in both fat sources were palmitic (16:0), stearic (18:0), oleic (18:1) and linoleic (18:2) acids. Unsaturated fatty acids (18:1 and 18:2) were higher in soybean oil and saturated fatty acids (16:0 and 18:0) content were higher in tallow. The ratio of unsaturated to saturated fatty acids in soybean oil and tallow were 5.66 and 1.17, respectively.

Table 2. Fatty acid composition of soybean oil and tallow (g/kg fat, as received)¹.

	Soybean oil	Tallow
Moisture	ND^2	0.10
Saturated fatty acids
C8:0 Caprylic	ND	0.17
C10:0 Capric	ND	0.75
C12:0 Lauric	0.10	1.23
C13:0 Tridecanoic	ND	0.16
C14:0 Myristic	0.76	23.92
C16:0 Palmitic	103.4	197.81
C17:0 Margaric	1.28	9.68
C18:0 Stearic	40.81	156.8
C20:0 Arachidic	3.12	1.62
C21:0 Heneicosanoic	0.36	0.24
C22:0 Behenic	3.55	0.26
C24:0 Lignoceric	1.19	0.24
C23:0 Tricosanoic	0.46	0.11
Unsaturated fatty acids
C14:1 (n-5) Myristoleic	ND	2.83
C16:1 (n-9) Palmitelaidic	ND	0.50
C16:1 (n-7) Palmitoleic	0.82	26.84
C18:1 (n-9) Elaidic	0.14	2.34
C18:1 (n-7) Vaccenic	11.01	31.85
C18:1 (n-9) Oleic	205.31	308.9
C18:2 (n-6) Linoleic	574.04	46.84
C18:3 (n-6) Gamma linolenic	ND	0.50
C20:1 (n-9) Eicosenoic	4.19	4.70
C18:3 (n-3) Alpha linolenic	81.55	12.88
C20:2 (n-6) Eicosadienoic	0.47	1.76
C20:3 (n-6) Eicosatrienoic	ND	1.13
C20:3 (n-3) Eicosatrienoic	ND	0.59
C20:4 (n-6) Arachidonic	ND	2.52
C20:5 (n-3) Eicosapentaenoic	0.42	3.79
C22:5 (n-3) Docosapentaenoic	ND	2.52
C22:6 (n-3) Docohexaenoic	ND	6.42
∑ Saturated fatty acids	155.1	393.0
∑ Unsaturated fatty acids	877.4	457.9
Unsaturated/Saturated ratio	5.66:1	1.17:1

^aNot corrected for the negligible amount of moisture present in the tallow. ²Not detected.

The influence of dietary treatments on ileal EAA losses is summarised in Table 3. Addition of fat lowered the endogenous flows of N and all AA (p = 0.08 to p = 0.001, respectively). The reductions in ileal endogenous N losses due to the addition of soybean oil and tallow were 38.0 and 47.8%, respectively. The corresponding reductions in total EAA losses were 47.1 and 55.9%, respectively. The influence of the source of fat on EAA losses were similar (p > 0.05).

Table 3. Influence of fat type on the ileal endogenous nitrogen and amino acid flows (g/kg dry matter intake) in broilers¹.

	Control (No fat)	Soybean oil	Tallow	SEM^2	P<
Nitrogen	2.05^a	1.27^b	1.07^b	0.198	0.008
Indispensable amino acids
Arginine	0.65^a	0.34^b	0.26^b	0.080	0.009
Histidine	0.22^a	0.10^b	0.07^b	0.022	0.001
Isoleucine	0.63^a	0.30^b	0.24^b	0.081	0.009
Leucine	1.02^a	0.48^b	0.38^b	0.135	0.010
Lysine	0.64^a	0.27^b	0.21^b	0.076	0.003
Methionine	0.20^a	0.12^b	0.10^b	0.023	0.014
Phenylalanine	0.56^a	0.28^b	0.23^b	0.069	0.009
Threonine	0.84^a	0.49^b	0.45^b	0.084	0.009
Valine	0.78^a	0.40^b	0.33^b	0.092	0.007
Dispensable amino acids
Alanine	0.62^a	0.33^b	0.26^b	0.069	0.005
Aspartic acid	1.07^a	0.58^b	0.48^b	0.117	0.006
Cysteine^3	0.21^a	0.15^b	0.17^a^b	0.019	0.078
Glycine^3	0.62^a	0.33^b	0.29^b	0.060	0.003
Glutamic acid	1.57^a	0.84^b	0.66^b	0.195	0.011
Proline	0.69^a	0.39^b	0.37^b	0.074	0.014
Serine	0.74^a	0.43^b	0.41^b	0.074	0.010
Tyrosine	0.57^a	0.28^b	0.23^b	0.076	0.011
Total amino acids^4	11.56^a	6.12^b	5.10^b	1.309	0.006

^a,bMeans in a row with no common superscript differ (p<0.05).

¹Each value represents the mean of six replicates (eight birds per replicate). Measurements were made on d 21 post-hatch.

²Pooled standard error of mean.

³Semi-indispensable amino acids for poultry.

⁴Total endogenous flow of 17 amino acids.

The AA profile of endogenous protein (g/100 g crude protein), as influenced by fat addition, is shown in Table 4. In all three treatments, the most abundant AA in the ileal endogenous protein was glutamic acid, followed by aspartic acid, threonine, leucine, serine, proline and glycine. In general, the concentrations of AA in the endogenous protein were lowered (p < 0.05) by added fat. The exceptions were methionine, cysteine, proline and serine, which were unaffected. The effect of fat source on individual AA contents of the endogenous protein, however, were inconsistent and differed depending on each AA.

Table 4. Influence of fat type on the composition of endogenous protein (g/100 g crude protein)¹.

	Control (No fat)	Soybean oil	Tallow	SEM^2	P<
Indispensable amino acids
Arginine	4.46^a	4.22^ab	3.84^b	0.146	0.030
Histidine	1.59^a	1.29^b	1.58^a	0.126	0.001
Isoleucine	4.44^a	3.78^b	3.58^b	0.084	0.001
Leucine	6.89^a	6.05^b	5.63^c	0.154	0.001
Lysine	4.16^a	3.39^b	3.00^b	0.221	0.007
Methionine	1.45	1.45	1.39	0.112	0.915
Phenylalanine	3.94^a	3.51^b	3.47^b	0.064	0.001
Threonine	7.83^a	6.24^b	6.88^b	0.256	0.002
Valine	5.85^a	5.03^b	4.99^b	0.108	0.001
Dispensable amino acids
Alanine	4.35^a	4.15^a	3.78^b	0.083	0.001
Aspartic acid	7.91^a	7.41^b	7.17^b	0.133	0.004
Cysteine^3	1.82	1.94	2.02	0.260	0.093
Glycine^3	4.69^a	4.23^b	4.44^ab	0.118	0.047
Glutamic acid	10.74^a	10.50^a	9.71^b	0.249	0.027
Proline	5.45	4.98	5.63	0.286	0.253
Serine	5.99	5.49	6.26	0.308	0.229
Tyrosine	4.18^a	3.51^b	3.38^b	0.051	0.001

^a,b,cMeans in a row with no common superscript differ (P<0.05). ¹Each value represents the mean of six replicates (eight birds per replicate). ²Pooled standard error. ³Semi-dispensable amino acids.

Discussion

When N-containing diets are fed, the amount of nitrogenous materials leaving the ileum represents the net balance between N intake and secretions minus the absorption of dietary N and re-absorption of endogenous N (Moughan 2003). However, there is no N ingestion when a N-free diet is fed and that found in the digesta at terminal ileum is the arithmetic difference between the secretion and re-absorption of N of endogenous origin. Thus, the reduction in EAA losses with fat addition can only be explained by lower endogenous secretions, enhanced re-absorption, or both. Of these possibilities, lower secretion of endogenous N due to fat addition is counter-intuitive, whereas increased re-absorption provides a more likely explanation. Among the possible reasons for increased re-absorption, two are worth considering. First, the well-established deceleration in digesta transit time through the intestine with fat inclusion that results in digesta having a prolonged contact time with enzymes and absorptive sites. Mateos and Sell (1981) used this argument to account for the ‘extra-metabolic effect’ of dietary fats. It has been suggested that fats inhibit gastric emptying and slow the digesta passage rate (Mateos, Sell, and Eastwood 1982). It is possible that, through this mechanism, fats improve the utilisation of non-fat nutrients, including AA. Second, there may have been intestinal morphological changes due to added fat. Goda and Takase (1994) found that a high-fat diet increased the crypt depth and, length and proliferation of microvilli in the intestine of rats. A study by Li et al. (1990) similarly revealed that pigs fed a combination of soybean and coconut oils had longer villi and increased crypt depth when compared to those fed diets with no fat. An increase in the absorptive area could increase the efficiency of absorption, allowing better re-absorption of endogenous proteins in broilers fed diets with added fat.

Changes in the microbiome with added fat may be another possible cause. Microbial mass contributes substantially to the N content in the ileal digesta (Miner-Williams, Moughan, and Fuller 2009), but are not considered part of the endogenous N fraction. The caeca is the primary area of bacterial activity in poultry, but the small intestine has a significant presence of bacterial population (Bindari and Gerber 2022). The intestinal bacteria are particularly associated with mucosa (Tomas et al. 2016). Some bacterial species colonise the intestinal wall and use mucin a source of energy and AA (Dai et al. 2015; Pan and Yu 2014). They disrupt mucus production and the lower the release of digestive enzymes (Pan and Yu 2014; Tomas et al. 2016). It has been suggested that dietary fats may indirectly influence intestinal microbiome through its impact on intestinal transit time (Knarreborg et al. 2002). In the current study, however, changes in intestinal microbiome with fat addition were not measured.

The present results supported the test hypothesis in that the inclusion of dietary fat decreased the endogenous losses. However, no differences were observed between EAA losses in broilers fed diets with tallow and soybean oil, which suggested that, despite possible differences in digesta transit time, fat type had no effect on the EAA losses in broilers. These results further suggested that presence of fats can increase true AA digestibility. Studies in pigs have indicated that fat composition may be relevant (D. F. Li et al. 1990; Merriman et al. 2016). Li et al. (1990) found that improvements in AA digestibility with added fat sources were higher in those with unsaturated fatty acids.

The concentrations of EAA in the ileal digesta were within the range reported in the literature (Adedokun et al. 2011; Ravindran 2021). The higher proportions of glutamic acid, aspartic acid, threonine, leucine, serine, valine and proline determined in the endogenous protein were as expected. These seven AA accounted for 69–70% of the total in endogenous protein. The high proportions of glutamic acid, aspartic acid, threonine, serine and leucine and valine in the endogenous protein may have been due to their dominance in mucins and digestive enzymes, and the slower rate of re-absorption compared with other AA. The lower concentrations of Met and His can be attributed to the fact that these AA were absorbed in greatest proportions compared to the others (Ravindran 2021).

Endogenous proteins originate from various digestive secretions (bile, pancreatic enzymes and, gastric and intestinal secretions), mucoproteins and desquamated intestinal epithelial cells. It is known that these individual sources differ in their AA composition (Cowieson et al. 2009; Ravindran 2021; Tancharoenrat, Zaefarian, and Ravindran 2022). The current findings suggested that the profile of the EAA flow was altered by dietary fats, ostensibly due to changes in the relative contribution of its components. In general, the concentrations of AA in the endogenous protein were lowered by added fat. The exceptions were methionine, cysteine, proline and serine, which were unaffected. The effect of fat source on AA content in endogenous protein, however, were not consistent and differed depending on the AA. This inconsistency reflected the complexity of ascertaining the changes in the contribution of individual endogenous components.

A unique phenomenon associated with dietary fats is their ‘extra-caloric’ effect, wherein AME values are sometimes determined to be greater than the GE in the fat. This effect has been attributed to the decelerating effect of fats on digesta passage rate (Mateos and Sell 1981), giving more time for the digestion and absorption of other energy-yielding nutrients. The present work provided evidence for an additional mechanism, by which fat addition improved metabolisable energy through reductions in EAA losses. As discussed by Cowieson et al. (2009), the loss of EAA is nutritionally expensive because the synthesis and secretion of digestive enzymes and mucoproteins constitute a net energy cost to the animal.

In the current study, fat sources replaced (w/w) dextrose in the experimental diets resulting in increased AME contents (Table 1). This approach may be questioned because of the possibility that dietary energy content may influence endogenous AA flows. To the authors’ knowledge, however, no published reports are available on the effects of AME on endogenous secretions or protein losses in monogastric animals. The only alternative design would have been to balance the AME content by adjustments in starch or protein, but this would have confounded the stated aim.

Conclusions

To the authors’ knowledge, this is the first study examining the relationship between dietary fat and ileal EAA losses in birds. The hypotheses tested in the current research were that fat inclusion will lower the EAA losses and that the effects will be more pronounced with soybean oil. The former thesis was supported by the present results, but not the latter. Overall, the findings indicated that that the presence of fat in broiler diets lowers EAA flow, implying that dietary fats have a beneficial effects on N utilisation efficiency.

Disclosure statement

No potential conflict of interest was reported by the author(s).