Threonine is an indispensable amino acid for pigs and is required for both maintenance and growth. It is the primary amino acid constituent of immunoglobulins and represents a significant portion of the proteins secreted by the small intestine (1). Threonine does not undergo transamination and there is no synthesis of threonine by the pigs. Consequently, all the threonine required by pig must be provide in the diet.

Many experiments were performed in order to determine the nutritional requirements of threonine, and most of them, through the empirical method. However, another method that might be employed to obtain the nutritional requirements of pigs is the factorial one. In that case, the animal’s nutritional requirement is defined according to well laid out physiological factors, such as the maintenance of protein structures and protein deposition (2). The estimated requirements for threonine by the factorial method suppose knowledge about maintenance requirement, rate of protein deposition and the post-absorptive utilization efficiency of threonine. Not all the threonine ingested by pig is recovered in body protein and the inefficiency is linked to the physiological processes, basal losses of amino acids of the integumentary and digestive system, nitrogen catabolism and protein turnover (3).

The knowledge of the amino acid efficiency for protein deposition is crucial to improve protein nutrition, which can result in economic improvements and reducing nitrogen losses in feces and urine. There are relatively few studies conducted to evaluate the marginal efficiency of threonine utilization by pigs. Thus, one study was conducted to determine the marginal efficiency of threonine utilization in pigs.

Animals and ambient condition. The experiment was performed with 12 castrated pigs of commercial line, in the finishing phase, and with an average live weight of 72±2 kg. The animals were housed in metabolic cages, with variable widths and heights in order to adjust the area to the weight of the animal, and kept in a controlled environment in order to achieve the average temperature of 22° Celsius.

Experimental design. The experiment was divided into two periods of 12 days (seven days of adaptation to the experimental conditions and five days of collection). The animals were distributed through a balanced changeover design (4) in four experimental diets, with a total of six replications per treatment and considering the animal as the experimental unit.

Diet characteristic. The diets were prepared using the concept of ideal protein in order to meet 30%, 45%, 60% and 70% of threonine nutritional conditions suggested by the NRC (3). The other amino acids were calculated to achieve a ratio of, at least, 15% of their requirements expressed in relation to the threonine (5). The calculated and centesimal composition of the experimental diet is presented in table 1.

Table 1 Table 1 Calculated and centesimal composition of pigs diets fed with increasing levels of standardized and digestible threonine.

Experimental procedure. The amount of feed was calculated to meet the consumption of 2.6 times the metabolizable energy for maintenance (250 kcal kg-1 BW0.60), considering an adjustment on daily feed intake according to the estimated weight gain of 0.8 kg per day. The food was distributed into four meals a day: at 08h:00, 11h:00, 13h:00, 18h:00 and water was provided ad libitum.

The feces were collected for five days according to the marker-to-marker approach using ferric oxide as an indigestible marker. The samples were packed into plastic bags and maintained in a freezer at -10° C. At the end of the experimental period, the feces were homogenized and samples of 0.5 kg were obtained, partially dried and grounded for further analysis.

The urine was drained into plastic containers containing 30 ml of sulfuric acid (H2SO4 3.5 M) to prevent bacterial contamination and nitrogen volatilization. The volume and urinary pH were measured every 12 hours, and a sample of 5% was collected and stored at 4° Celsius. The waste of feed and leftovers was determined to each animal. Subsequently, the feed leftovers were weighed, and the value was discounted from the animal’s feed intake.

The content of corn and soybeans amino acids used in the experimental diets were determined by liquid chromatography after acid hydrolysis. The dry matter of the ingredients, feces, feed and leftovers along with the nitrogen ingredients, feed, feces and urine, were determined by the AOAC methodology (6).

The retained nitrogen was obtained by the difference between the nitrogen intake and nitrogen eliminated in feces and urine (nitrogen balance). The protein deposition was calculated by multiplying the retained nitrogen by 0.0625 assuming that nitrogen constitutes 6.25% of the retained proteins (3).

The amount of threonine retained was calculated through the difference between the ingested standardized threonine and deposited threonine, assuming that it constitutes 3.79% of the body protein (7). The marginal efficiency of utilization of threonine was obtained by the angular coefficient of the line originated through the regression between the threonine retained and standardized digestible threonine intake (8).

Statistical analysis. The obtained data were submit to variance analysis by using a linear model containing animal, time and treatment as main effects. Subsequently, it was performed a procedure of linear regression with the adjusted means, and the use of the Minitab program (9).

Ethical aspects. The experimental protocol was reviewed and approved by the Ethics Committee on Animal Experimentation of the Federal University of Santa Maria (Opinion 005/2012).

The animals remained healthy during the experimental period. Feces and urine samples were obtained without any problem.

In our experiment the diet containing the highest ratio between standardized digestible threonine and metabolizable energy (STD: ME) presented a value of 1.4 g Mcal-1. The dry matter intake (DMI) differed between the treatments (p≤0.001).

There were differences (p≤0.001) in fecal nitrogen among treatments (Table 2) and was observed increased linearly (p≤0.001) in fecal nitrogen according to nitrogen intake with the slope of relationship being of 0.165 (SE=0.019). The nitrogen in urine (NU) was influenced by treatments (p≤0.001).

Table 2 Table 2 Dry matter intake, metabolizable energy intake and nitrogen balance of pigs consuming diets with increasing levels of standardized and digestible threonine (values expressed per day)

The N retention was linearly (p<0.001) associated with N intake. The slope of regression line relating NU and NI 0.254 (SE=0.026). The slope of relation between retained threonine (RT) and standardized digestible threonine (SDT) intake was 0.63 (SE=0.033).

One important assumption in empirical estimate of amino acids efficiency is that the amino acid test must be the first nutritional constraint in the experimental diets.

The ratio STD:ME is lower than the recommended for maximum protein deposition (2.0 g Mcal-1) of pigs with a live weight of 70 kg (3). In addition the amino acids others than threonine were supplies to exceed their requirements. This indicates that threonine was the first limiting nutritional factor for protein deposition.

The difference in DMI was not planned because the amount of DMI influences the content of endogenous losses of amino acids. However, since the difference between the lowest and highest consumption was only 1.3 % (Table 2), it is possible that the endogenous loss of threonine and other amino acids were barely affected.

The N found in feces indicating mean apparent N digestibility of experimental diets was 83.5%. The N in urine was according to the expectations since the urine is the main route of nitrogen excretion ingested in excess of requirements.

The results of N retention are in agreement with Libao-Mercado et al (8) and show that the threonine was first limiting to protein deposition in diets used in our experiment.

The slope of regression line relating NU and NI indicated that 25.4% of nitrogen consumed were eliminated through the urine. That value was bigger than 16% obtained by Heger et al (10) and smaller than value of 33% mentioned by Libao-Mercado (8). The differences may be associated with the diet composition once purified diets with protein of high biology value as used by Heger et al (10) has a trend to result a lesser proportion of nitrogen intake losses in urine.

When NU was associated with digestible nitrogen intake (DNI) we observed that the N eliminated in the urine due to non-use for protein deposition was 0.309 g kg-1 BW0.75 for each gram of DNI above of maintenance needs (Figure 1). The value was bigger than 16% obtained by Heger et al (11) who used purified diet containing a protein source of high biological value. The NU has origin from diet N that’s not used to protein synthesis, intestinal endogenous N secreted and no reabsorbed until the end of small intestine and N absorbed in large intestine. Therefore, these results emphasize again the importance of protein quality on improve N utilization in pig production (12).

The value of the relation between RT and STD intake indicates that marginal efficiency of SDT for protein deposition is 63% (Figure 2) which is lower than 73, 83 and 67 % found by de Lange et al (7), Heger et al (10) and Heger et al (13), respectively. These discrepancies can be explained at least partially because the differences in the methodological aspects found inter studies.

Figure 2 Figure 2 Relationship between standardized digestible threonine intake and threonine retention in pigs fed with increasing levels of threonine.

Assuming that 63% is the efficiency of standardized digestible threonine intake to protein retention in body protein implies that 37% of standardized digestible threonine is lost due to inevitable minimum catabolism that occurs even when the threonine intake limits protein deposition. The main physiologic determinants of inevitable minimum catabolism of amino acids in growing pigs are amino acid endogenous losses, physical amino acids losses with skin and hair and losses due to protein turnover (1). It is assumed that threonine is used with lower efficiency to protein deposition compared with other amino acids like lysine and valine, for example (5). Normally it is justified because the intestinal endogenous losses is the major contributing to inevitable catabolism and threonine is particularly important since its participation in intestinal protein mucin is high (14, 15).

The information about threonine deposition efficiency is essential to the development of requirement estimates by the factorial method, which in turn is the preferred method for estimating the nutritional requirements of pigs (16) and used in most of the simulation models of growth pigs (17).

In conclusion the marginal efficiency of utilization of 0.63 can be used to calculate the nutritional requirements of threonine by the factorial method.