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Introduction
The use of low-protein diets (LPD) in fattening pigs is a viable option for reducing nitrogen emission into the environment, which reduces the amount of greenhouse gases and soil contamination (Osada et al 2011). In addi tion, LPD enable to obtain a similar growth performance compared to standard diets when supplemented with crystalline AA (He et al 2016, Jiao et al 2016, Peng et al 2016). However, the reduction of more than 4-6% of crude protein (CP) in the diet affects growth performance and digestive enzymatic production (He et al 2016). Therefore, it is necessary to find alternative additives that compensate for the reduction of protein ingredients and the amount of CP in pig diets, increasing AA digestibility.
Addition of protease might improve availability of protein in pig diets through an increment on protein digestibility and amino acid (AA) availability in the gas trointestinal tract of pigs (Wang et al 2011, Guggenbuhl et al 2012). Nevertheless, in some cases, using protease in LPD for weaning, growing and finishing pigs did not improve growth performance or carcass characteristics variables (Reyna et al 2006, Zamora et al 2011). The variability in protease effectiveness was attributed to the age of pigs, the use of different types of ingredients, the source of protease (Adeola and Cowieson 2011), and the high degradation and inactivation of these enzymes in the gastrointestinal tract (Pan et al 2016). To surpass this variability of results using protease for pig diets, recent technological advances have developed coated (protected) protease (Xu et al 2017), which works under different conditions in the gastrointestinal tract (Pan et al 2016). Thus, using protected protease in pig diets may improve digestibility of AA which, in turn, is reflected in a better growth performance (Zuo et al 2015).
This improved protein and AA digestibility and avail ability in the gastrointestinal tract of pigs (Wang et al 2011, Guggenbuhl et al 2012) means that the supplementation of protected protease could compensate for the reduction of AA in diets for pigs when fed LPD. For these reasons, the objective of this research was to evaluate the addition of protected protease to low AA concentration (low-crude protein) diets in 25-100 kg barrows in terms of growth performance, carcass characteristics, and plasma urea nitrogen concentration.
Material and methods
The experimental procedures were performed accord ingly to the recommendations of the International Guiding Principles for Biomedical Research Involving Animals1, and observing the standards for ethics, biosafety, and animal well-being of the Colegio de Postgraduados, Mexico, under the Official Mexican Regulation (Norma Oficial Mexicana, 1999) for the use of animals in experimentation.
The experiment was conducted in the Swine Unit of the Experimental Farm at the Colegio de Postgraduados, located in Montecillo, Estado de Mexico (98° 48’ 27’’ W and 19° 48’ 23” N). The climate is temperate, semi-arid, with an average annual temperature of 15.9 °C, infrequent frosts, average annual rainfall of 686 mm and an altitude of 2241 m (García 1988).
Forty eight hybrid (Landrace x Yorkshire x Duroc; 27.42 ± 3.48 kg initial body weight and 60 days-old) bar rows were used, housed in individual pens, each equipped with a single feeder and nipple drinker. Water and feed were provided ad libitum.
The experiment consisted in the evaluation of six treat ments (T). Three decreasing levels of protein (standardized ileal digestible (SID) basis AA levels; control, medium, and low), with or without the addition of protease were evaluated (0.03% of protease was added, according to the amount indicated by the JEFO Company; Poultry Grow 250®, Streptomyces griseus, Type XIV, Puebla, Mexico) in pigs diet. Three stages were evaluated (growing, 25-50 kg body weight; finishing I, 50-75 kg; finishing II, 75-100 kg; Table 1, Table 2, Table 3 respectively), changing the feed for each stage accordingly to the average body weight of the pigs, trying to follow the recommendations of the NRC (2012) for each stage. Forty-eight barrows were distributed (each pig was considered a replicate) in a completely randomised design with a factorial arrangement of treatments; there were eight replicates (barrows) per treatment. The AA or CP (low-protein diets; LPD) reduction was achieved by decreasing the lysine content by 0.05% and 0.10% relative to control diet, with a proportionate reduction in concentrations of the remaining AA in the diet, trying to maintain the ratio relative to Lys (ensuring that all AA were supplied, at least the Lys proportion).
Table 1 Composition of diets for growing barrows (25-50 kg BW) fed low-protein diets supplemented with protease.
Table 2 Composition of diets for finishing I barrows (50-75 kg BW) fed low-protein diets supplemented with protease.
Table 3 Composition of diets for finishing II barrows (75-100 kg BW) fed low-protein diets supplemented with protease.
The nutritional values of the control treatment were established as recommended by the NRC (2012) to cover or exceed the requirements for each stage of growth. The diets for each stage were formulated with the Solver command2 using the least-cost feed formulation method.
Chemical analysis
Crude protein content of diets for each stage was determined by the Macrokjeldahl method (AOAC 2005), calcium and phosphorus content by atomic absorption spectrophotometry (Karl et al 1979) using a Perkin Elmer 4000 Model (Series Lambda 2, Perkin Elmer Inc., Norwalk, CT, USA). On the last day of the experiment, blood samples (5 mL; pre-prandial 08:00 h) were collected by vena cava puncture in live pigs, using Vacutainer tubes without anticoagulant (BD Vacutainer®), and stored at 4 °C. The blood samples were centrifuged (SIGMA 2-16 k, Germany) at 3500 g for 20 min to obtain blood serum. Serum samples were stored in Eppendorf tubes at -20 °C in a freezer (SANYO MDF-436, USA) until determination of plasma urea (Chaney and Marbach 1962).
Variables
Growth performance variables were: average daily feed intake (ADFI), average daily gain (ADG), feed:gain ratio (FGR), fat-free lean gain (FFLG) and final body weight (BW). Carcass characteristics determined were: backfat thickness (BT), longissimus muscle area (LMA), lean meat percentage (LMP); and plasma urea nitrogen (PUN) concentration. The BT and LMA were measured using real-time ultrasound (SonoVet 600, MEDISON: Medison, Inc., Cypress, CA, USA) at the 10th rib on the first and last day of the experiment. The BT, LMA, initial and final BW data were used to determine FFLG and LMP, following the procedure indicated by Burson and Berg (2001): Lb. lean=5.7769+(0.401 x warm carcass wt., lbs)-(18.838 x 10th rib fat depth, in.)+(4.357 x 10th rib loin muscle area, sq. in.)+(1.006 x sex of pig) (barrow=1, gilt=2)).
Statistical analysis
The experimental design was completely randomised with a factorial arrangement of treatments, where the factors were three CP levels and two protected protease levels (with and without). Shapiro-Wilk and Levene’s test were used to check normal distribution and homogeneity of variance for all variables. Data were analyzed with the GLM procedure, and Tukey’s test (P≤0.05) was used to compare treatment means (Statistical Analysis System 2010. Inc. Cary, NC, USA). The initial body weight was used as a covariate (P≤0.05).
Results
Low-protein diets
No differences (P>0.05) were detected between treat ments for FGR, LMP, BT and LMA due to the protein level in the diet for growing (Table 4) and finishing II barrows (Table 6). An improved response was observed in ADG, final BW, ADFI and FFLG in growing barrows fed 16.17 % and 15.49 % CP compared to 16.86 % CP (P≤0.05) (Table 4). In finishing II barrows, values for ADG and final BW improved (P≤0.05) when using 12.08 % CP in the diet compared to 12.86 % and 13.63 % CP. The FFLG was greater (P≤0.05) when using 12.86 % and 12.08 % CP compared to control diet (13.63 % CP) (table 6). There were no effects (P>0.05) of protein level on any growth performance variables in finishing I (50-75 kg) barrows (Table 5).
Table 4 Productive performance, carcass characteristics and plasma urea nitrogen concentration of growing barrows (25-50 kg) fed three levels of crude protein and two levels of protease.
Table 5 Productive performance, carcass characteristics and plasma urea nitrogen concentration of finishing I barrows (50-75 kg) fed three levels of protein and two levels of protease.
Protease
The addition of protease to diets during three growth phases (growing, finishing I and finishing II) did not change (P>0.05) the growth performance and carcass characteristics of barrows (Table 4, Table 5 and Table 6, respectively).
In the growing stage, the control diet (16.86 % CP) with added protease reduced BT (P≤0.05); the protease interaction with the CP level had no effect (P>0.05) on other variables (Table 4). There were no effects (P>0.05) of the interaction between protein level and protease ad dition on growth performance and carcass characteristics in finishing I and finishing II barrows (Tables 5 and Table 6).
Plasma urea nitrogen
The addition of dietary protease in the three phases of growth increased PUN concentration (P≤0.05) of barrows; while protein reduction reduced PUN concentration in all three phases (P≤0.05). In growing barrows, the lowest concentration of this metabolite occurred with the lowest CP level (15.49 %) and the addition of protease (P≤0.05). In finishing I barrows, the lowest PUN concentration was also detected with the lowest CP concentration (13.63 %) regardless the addition or not of protease (P≤0.05). Finishing II barrows showed lower PUN (P≤0.05) with the lowest PC concentration (12.08 %) as in the previous growth stages.
Discussion
The NRC (2012) does not establish a CP value for diets formulation, so, its concentration in the diet is the result of the AA level. The concentrations of lysine, methionine, threonine, tryptophan and other AA were reduced in the diets used in this study, trying to maintain the proportion of AA with respect to lysine (Tables 1-3), which reduced the CP level of the diet. However, under the concept of minimum cost formulation of diets for of some AA (NRC 2012) than trying to accomplish the lower (goal) values (Dubeau et al 2011). In our study, the reduction of phenylalanine, arginine, isoleucine, leucine and valine was limited because the basal ingredients (sorghum grain-soybean meal) have a high concentration of these AA.
Low-protein diets
The results indicate that it is feasible to improve or maintain growth performance variables when the amount of AA in barrow diets is reduced, as long as the lysine:AA ratio is maintained. Results of other studies showed that finishing pigs fed LPD, with an unchanged AA ratio per CP unit, had similar and even improved growth variables (Gallo et al 2014, Tous et al 2014).
In contrast, Qin et al (2015), Jiao et al (2016) and Peng et al (2016) reported that these growth variables were unaffected when CP content was reduced, but the requirement for the more limiting AA (Lys, Met, Trp, Thr) must be reached in the diet. The use of lower levels of AA in our experiment with a performance similar to that of the control protein diet, leads us to determine that the nutritional requirement values coincide with the rec ommendations for pigs with low genetic potential for lean growth, when comparing CP and lysine concentrations evaluated in our study with the NRC (2012) and Brazilian tables (Rostagno et al 2017).
In the present research, the decrease in CP as a result of AA reduction showed beneficial or null effects on the growth performance. Gloaguen et al (2014) and González et al (2016) reported that a drastic reduction in dietary CP should not be made, because lowering CP by more than three percentage units affected ADG, ADFI, FGR, final BW and FFLG. The negative response to LPD may be attributed to AA deficiency, because CP reduction limits the concentration of AA and the amount of nitrogen nec essary for the synthesis of non-essential AA (Gloaguen et al 2014).
Consistent with results of our study, Zamora et al (2011) and Qin et al (2015) confirmed that decreasing CP by less than two percentage units in diets for fattening pigs did not affect carcass characteristics. This may be a result of better balance of AA for protein synthesis and, therefore, AA are not required as an energy source, reflected by similar LMP values with different CP concentrations (Orlando et al 2007). However, González et al (2016) and Figueroa et al (2012) observed that in nursery and finishing pigs, decreasing dietary CP by more than three percentage units had a negative effect on BT, LMP and LMA. Reduction in FFLG and increase in BT in pigs fed LPD were attributed to the greater availability of energy for adipose tissue accretion, due to an imbalance between a greater amount of available energy and a deficiency of AA (Gómez et al 2002).
Protease
Various reports (Zuo et al 2015, Pan et al 2016, Yu et al 2016) have demonstrated the efficacy of adding protected protease to pig diets, increasing CP digestibility and AA availability, improvements in the plasma concentration of total protein, increased pepsin enzyme activity in the stomach, as well as increased pancreatic amylase and trypsin. The combination of an increase in CP digestibility and lower consumption could reflect improvements in the growth performance of pigs fed protease (Mc Alpine et al 2012, O’Shea et al 2014). These changes resulted in a better productive performance in both weaning and nursery pigs, which does not have adequate protease production (Zuo et al 2015, Greiner et al 2016).
Previous research showed that supplementation of LPD with protease did not improve growth performance in fattening pigs (Morales et al 2002, Reyna et al 2006, Zamora et al 2011). Besides, Mc Alpine et al (2012) and O’Shea et al (2014) observed that the use of protected protease in pig diets could reduce ADG in response to a reduction of ADFI.
This phenomenon could be explained because the pro tease hydrolyses the protein in the small intestine, which releases components that can be absorbed, and increases the ileal digestibility of protein, increasing the amount of available N from the diet (Mc Alpine et al 2012, O’Shea et al 2014). Additional nutrients released could trigger a feedback mechanism to reduce feed intake because of a glucostatic and/or aminostatic response that could create nutrient imbalance in the gastrointestinal tract of pig (Nortey et al 2007).
Plasma urea nitrogen
Decreasing dietary protein in pigs reduced the con centration of PUN (Qin et al 2015, Peng et al 2016). This lower PUN concentration is associated with a decrease in metabolic heat production associated with pig metabolism derived from lower amount of total nitrogen and hence, lower synthesis and excretion of urea originated by the AA excess when fed a standard CP diet, indicating better utilization of nitrogen by pigs fed LPD (Qin et al 2015). The increase in PUN concentration, which was observed in the present research when protease was added to the diet, may be due to greater digestibility of the CP diet and increased AA availability (Reyna et al 2006). The effect of the protease cannot be determined because reduction of protein never decreased growth performance of pigs. Thus, the extra amino acid available from the inclusion of the protease will be catabolized without further effect.
Based on the results of this study, we conclude that the reduction of the concentration of AA in the diet (low CP) improves or maintains growth performance and reduce the plasma urea nitrogen in 25-100 kg hybrid barrows compared to standard CP diets, however, the lysine:AA ratio in the diet must be maintained. The addition of protected protease to pig diets does not show any effect on productive performance, because reduction of protein never decreased growth performance of pigs.
