Effect of amino acid level and ratio on whole-body protein turnover in piglets

. The addition of lysine, methionine and threonine to the "ideal protein" diet in the low-protein diet contributed to increased protein deposition in the body of piglets due to protein-synthesizing activity. The optimal level and ratio of essential amino acids in low-protein diets provides an increase in the efficiency of the use of amino acids for the synthesis and deposition of proteins in the body of piglets. Based on the results of assessing the metabolism of proteins in the body and their turnover in pi glets (♂ Landrace × ♀ Large white) during the period of intensive rearing from 20 to 50 kg of live weight at an average daily gain of 500 g, the following norms for the concentration of nutrients in 1 kg of compound feed are proposed: 12.42 MJ exchange energy; 120.6 g crude protein; 9.10 g lysine; 5.90 g methionine + cystine; 6.09 g threonine at a lysine level of 0.73 g per MJ of metabolizable energy.


Introduction
In pig production, the dietary crude protein (CP) content can be reduced when the requirements for essential amino acids (EAA) and total nitrogen are met, because for pigs the need for dietary protein is essentially a need for amino acids (AA) [1,2].Characteristically, the level of digestibility of amino acids should correspond to the needs of animals with a minimum protein content in the diet.In this case, the possibility of increasing the efficiency of using cheaper feed in feeding pigs, in particular, cereal grains with a low protein level, is being solved [3].
The concept we are developing in assessing the need for "ideal protein" was formulated in the early 60s of the last century [4,5].At first, the protein of a chicken egg was considered to be the standard, and later, the total protein of the body of a growing and fattening animal began to be considered as ideal in terms of composition and ratio of amino acids.Undoubtedly, the development of this concept is of the greatest importance for data on determining the need for protein in rapidly growing new generation animals [6,7,8,9,10].However, it should be noted that the results obtained by researchers are rather contradictory.So in the studies of He et al. (2016) [11] reported a reduction in muscle mass and body protein deposition in pigs fed diets according to "ideal protein".On the other hand, Lee et al. (2022) [12] found no significant changes in muscle mass and body protein deposition in pigs fed low protein diets supplemented with amino acids to levels of "ideal protein" composition.In studies by Hewitt D. et al. (2020) [13], Wang Y. et al. (2018) [14] showed that crossbreeds of pigs that received diets at the level of "ideal protein" were characterized by increased nitrogen retention, against the background of increased protein biosynthesis and meat yield.The quantitative formation of a pool of proteins in the body of growing pigs depends on the intensity of their synthesis, decay, or a combination of these values.To study these issues, it is advisable to use the situation when there is an increase in the processes of synthesis or a decrease in the breakdown of protein in tissues.It has been proven that in intensively growing piglets, one of the factors limiting the intensity of protein synthesis processes is the amount and ratio of amino acids supplied from the gastrointestinal tract to the body's metabolic pool [15,16].It can be assumed that in the case of an adequate supply of the body of piglets with essential amino acids in the optimal ratio with an appropriate level of energy supply, it is possible to change the intensity and direction of metabolic processes in the direction of increasing protein synthesis, and, accordingly, allowing to fully realize the productive potential animals.

Purpose of the study
The aim of the work was to determine the optimal level and ratio of essential amino acids in low-protein diets and study their effect on body protein metabolism and measure their turnover in growing pigs.

Materials and methods
The experiment was carried out on cross-border piglets (♂ Landrace × ♀ Large white).According to the principle of paired analogues taking into account live weight, at the age of 65 days, 3 groups of piglets were formed 7 animals in each, fed 2 times a day (9.00 and 16.00) throughout the entire experiment.Group content in the cells, drinking from the car drinkers.The experiment lasted until the live weight of piglets 42-52 kg.
Animals of the 1st (negative control, A1) group received the main diet with a crude protein content of 11.91% without the addition of synthetic amino acids.Pigs of the 2nd (experimental, A2) group were supplemented with synthetic amino acids (threonine, lysine, methionine) to the above basic diet.For example, the level of lysine was adjusted to a level of 9.1 g per kg of feed (i.e.amino acid supplementation to the ideal protein level) (Table 1).In piglets of the 3rd group (A3), the content of crude protein in the diet was brought up to 15.37% by additional introduction of soybean meal into the compound feed.Compound feed of pigs of the 2nd and 3rd experimental groups contained the same amounts of limiting amino acids -lysine, methionine and threonine.The level of metabolic energy was the same in all study groups.At the same time, the relative levels of essential amino acids in relation to lysine and the ratio of lysine to metabolic energy in the diets of piglets during the growing period were determined (Table 1).
In order to characterize the intensity of growth and development of experimental animals, piglets were weighed at the beginning of the experiment and at the end of each age period.To characterize the assimilation of feed nitrogen and the efficiency of its use by pigs, a balance experiment was carried out on 3 animals from each group at the age of 120-125 days.
Most studies use the nitrogen metabolism model to measure protein turnover in the whole body [17], which is based on measuring the kinetics of metabolism of the introduced marker.To measure the rate of protein synthesis using this model, the total precursor flux, the rate of formation of the final products of nitrogen metabolism, and the rate of precursor secretion from the total fund into the gastrointestinal tract must be known.Methodological aspects of measuring the rates of protein synthesis and decomposition in the whole body using 15 N amino acids are considered in detail in [17].The results obtained by the researchers indicate that the use of different amino acids gives comparable data, despite the fact that the specificity of these amino acids as precursors for protein synthesis is not the same.
During the balance experiment, the rate of synthesis, decomposition, and deposition of total body proteins was determined in piglets at the end of the growing period according to the method [17] using labeled amino acid for nitrogen 15 N-glycine. 15N-glycine with an enrichment of 98 % atomic excess was administered per os in the amount of 3 mg 15 N per 1 kg of animal body weight for 7 days.Stool and urine samples for isotope analysis were taken in a separate trial with a daily interval in compliance with all requirements for working with stable isotopes.Feces collected during the N balance period were pooled, freeze-dried and stored at 4 °C for N determination.Urine collected was stored at −20 °C until analysis for N. Samples of diet, urine and feces were analyzed for N content by Kjeldahl method.The N-retention was calculated by minus N excretion (via feces and urine) from N intake.For isotopic studies from feces, urine taken on the 6th day of the balance experiment, nitrogen fractions were preparatively isolated using a Kjeltek instrument using the Kjeldahl method with washing the system with ethyl alcohol to isolate each sample.The 15 N content (in atomic percent) was measured on a DELTA V Plus isotope mass spectrometer.
The rates of synthesis, decomposition and deposition of proteins in the whole organism were calculated using the following formulas (1), ( 2 where V1 -protein synthesis, g / day; WF -taken nitrogen with feed, g / day; NF -taken 15 N with food; NO -natural enrichment (background, atomic percentage of excess); NM -( 15 N) atomic percentage of excess in urine; NK -( 15 N) atomic percentage of excess in feces; WM -nitrogen excreted in urine g / day; WK -nitrogen excreted with feces, g / day; V2protein breakdown, g / day; V3 -protein deposition, g / day.
The data for all parameters determined were analyzed statistically by one-way ANOVA of SPSS 11.0 software.A value of P≤0.05 was considered statistically significant.Data are presented as means ± standard error of the mean (SEM).

Results of study
Nitrogen retention and biological value were significantly increased by adding lysine, threonine, and methionine to the deficient diet to "adequate" levels, but were not further increased by higher protein levels.Rates of growth paralleled these changes.The poorer biological value of the unsupplemented diet A1 was shown also in a significantly higher excretion of urea N compared with diets A2 (Table 2).Nitrogen digestibility was not significantly affected by crude protein levels and the addition of lysine, threonine, and methionine.Both synthesis and breakdown of protein in the body increased markedly with "adequate" addition of lysine, threonine and methionine to the diet, but did not increase with excess crude protein (Table 3).
Based on the results of assessing the metabolism of proteins in the body and their turnover in piglets (♂ Landrace × ♀ Large white) during the period of intensive rearing from 20 to 50 kg of live weight at an average daily gain of 500 g, the following norms for the concentration of nutrients in 1 kg of compound feed are proposed: 12.42 MJ exchange energy; 120.6 g crude protein; 9.10 g lysine; 5.90 g methionine + cystine; 6.09 g threonine at a lysine level of 0.73 g per MJ of metabolizable energy.The ratio of threonine, methionine + cystine, valine, isoleucine, leucine, histidine, tryptophan, arginine and phenylalanine + tyrosine to lysine should be 67:100, 65:100, 69:100, 57:100, 100:100, 32:100, 18:100, 71:100 and 100:100 respectively.
It was concluded that the increase in the rate of protein growth observed when adding lysine, methionine and threonine to the diet to the "ideal protein" was due to a greater increase in the rate of protein synthesis than its degradation, and not a decrease in the rate of degradation.

Conclusion
The addition of lysine, methionine and threonine to the "ideal protein" diet in the lowprotein diet contributed to increased protein deposition in the body of piglets due to proteinsynthesizing activity.Thus, the optimal level and ratio of essential amino acids in lowprotein diets provides an increase in the efficiency of the use of amino acids for the synthesis and deposition of proteins in the body of piglets.

Table 2 .
Effect of amino acid level and ratio on nitrogen balance in piglets.

Table 3 .
Effect of amino acid level and ratio on whole-body protein turnover in piglets.synthesis rate; B -protein breakdown rate; NPG -net protein gain expressed in nitrogen; NF -nitrogen flux; EUN -endogenous urinary nitrogen.W0.75 -0.75 metabolic body weight.n = 4 in the group.* P≤0.05; ** P≤0.02 by the U-criterion when compared with A1 (negative control).