Estimation of the standardized ileal digestible lysine requirement and the ideal ratio of threonine to lysine for late finishing gilts fed low crude protein diets supplemented with crystalline amino acids

Animal Feed Science and Technology 201 (2015) 46–56

Contents lists available at ScienceDirect

Animal Feed Science and Technology journal homepage: www.elsevier.com/locate/anifeedsci

Estimation of the standardized ileal digestible lysine requirement and the ideal ratio of threonine to lysine for late finishing gilts fed low crude protein diets supplemented with crystalline amino acids W.F. Ma, X.F. Zeng, X.T. Liu, C.Y. Xie, G.J. Zhang, S.H. Zhang, S.Y. Qiao ∗ State Key Laboratory of Animal Nutrition, China Agricultural University, No.2, Yuanmingyuan West Road, Beijing, 100193, PR China

a r t i c l e

i n f o

Article history: Received 11 June 2014 Received in revised form 24 September 2014 Accepted 27 September 2014 Keywords: Gilts Low-protein Standardized ileal digestibility Lysine Threonine

a b s t r a c t Two experiments were conducted to investigate the effects of various standard ileal digestible (SID) lysine (Lys) levels and SID threonine (Thr) to Lys ratios on the performance and carcass characteristics of finishing gilts receiving low crude protein (CP) diets supplemented with crystalline amino acids (CAA). In Exp. 1, 108 gilts (87.8 ± 5.9 kg) were randomly allotted to one of six diets which consisted of a high CP (135 g/kg) diet with 6.1 g/kg SID Lys or five low CP (100 g/kg) diets providing SID Lys levels of 4.9, 5.5, 6.1, 6.7 and 7.3 g/kg, respectively. Gilts were housed in three pigs per pen with six pens per treatment. At the end of the 28 days experiment, 36 gilts (one pig per pen) with average body weight (BW) of 116 kg were killed to evaluate carcass traits. The SID Lys levels required to maximize average daily gain (ADG) and optimize feed conversion ratio (FCR) as well as to minimize serum urea nitrogen (SUN) levels were 5.7, 5.8 and 6.1 g/kg using a linear-break point model and 6.5, 6.5 and 6.6 g/kg using a quadratic model. The fat-free lean gain tended to increase linearly with the increase in dietary SID Lys levels from 4.9 to 7.3 g/kg when gilts receiving a low CP diet (linear effect, P=0.06). In Exp. 2, 90 gilts (90.6 ± 5.7 kg) were utilized in another dose–response study. Based on the Lys requirement estimated in Exp. 1, dietary treatments were formulated to contain 5.1 g/kg SID Lys to ensure the dietary Lys level was marginally deficient for late finishing gilts. Graded levels of crystalline Thr (0, 0.3, 0.6, 0.9 or 1.2 g/kg) were added to the basal diet providing SID Thr to Lys ratios of 0.54, 0.60, 0.66, 0.72 or 0.78, respectively. Each diet was fed to six pens with three gilts per pen. At the end of Exp. 2, 30 gilts (one pig per pen) were slaughtered to evaluate carcass traits (average BW = 118 kg). The optimum SID Thr to Lys ratios to maximize ADG and FCR as well as to minimize SUN levels were 0.61, 0.63 and 0.64 using a linear-break point model and 0.70, 0.75 and 0.74 using a quadratic model. With the exception of L* light (linear effect, P<0.05), no effect on carcass characteristics was observed with increasing dietary SID Thr to Lys ratio. © 2015 Published by Elsevier B.V.

Abbreviations: AA, amino acid; ADFI, average daily feed intake; ADG, average daily gain; BW, body weight; CAA, crystalline amino acids; CP, crude protein; FCR, feed conversion ratio; Lys, lysine; SID, standard ileal digestible; SUN, serum urea nitrogen; Thr, threonine. ∗ Corresponding author. Tel.: +86 10 6273 1456; fax: +86 10 6273 3688. E-mail address: qiaoshy@mafic.ac.cn (S.Y. Qiao). http://dx.doi.org/10.1016/j.anifeedsci.2014.09.025 0377-8401/© 2015 Published by Elsevier B.V.

W.F. Ma et al. / Animal Feed Science and Technology 201 (2015) 46–56

47

1. Introduction Considering the entire growth phase, late finishing pigs have the highest feed intake and poorest feed conversion. Suggested dietary manipulations for feed formulation include the use of phase feeding and low-protein diets supplemented with crystalline amino acids (CAA) to achieve economic and environmental benefits (Prandini et al., 2013). Lysine (Lys) and threonine (Thr) are often the first- or second-limiting amino acid (AA) for finishing pigs fed a maizesoybean meal based diet (Saldana et al., 1994; Shelton et al., 2011). The requirement for Lys and Thr can be significantly influenced by body weight (BW), which may be due to the increased proportion of protein deposition and maintenance for amino acid requirements for pigs moving towards the finishing period (Friesen et al., 1994; Hahn and Baker, 1995). A large deficiency of Lys and Thr may appear by reducing dietary soybean meal concentration, which may lead to poor performance in finishing pigs. This can be overcome by supplementing the diet with CAA but these tend to be expensive and it is important to ensure appropriate levels of CAA are used in the diet (Zhang et al., 2013). In late finishing period, gilts usually need higher amino acid levels than barrows because of the greater lean growth rate and reduced feed intake of gilts (Friesen et al., 1994; King et al., 2000). It is important to recognize that Lys and Thr requirements should be evaluated separately based on gender of pigs in order to feed the animal closer to its requirement. The NRC (2012) estimates the Lys requirement of 100–135 kg gilts to be 6.4 g/kg standard ileal digestible (SID) Lys while the National Swine Nutrition Guide (2010) estimates the SID Lys requirement of 100–120 kg gilts to be 6.9 g/kg for high lean gain lines and 5.9 g/kg for medium lean gain lines. The similar estimates of ratio of SID Thr to Lys ranged from 0.66 to 0.68 for late finishing gilts in NRC (2012) and National Swine Nutrition Guide (2010). However, the recommended estimates were based on predictable model and there were limited empirical studies in finishing pigs, especially on the basis of SID AA (NRC, 2012). The aims of this study were to determine the SID Lys requirement and optimum dietary Thr to Lys ratio for late finishing gilts fed low crude protein (CP) diets supplemented with CAA. 2. Materials and methods All experimental procedures and animal care were approved by the China Agricultural University Animal Care and Use Committee (Beijing, China). 2.1. Animals, housing and dietary treatments Two trials were conducted to determine the SID Lys requirement and the optimum dietary SID Thr to Lys ratio for late finishing gilts (Duroc × Yorkshire × Landrace) fed low CP diets supplemented with CAA. Both experiments were conducted at the Pig Research Facility at the Swine Nutrition Research Centre of the National Feed Engineering Technology Research Centre (Chengde, Hebei Province, China). Gilts were placed in partially steel-slatted concrete floored pens (2.4 m × 1.8 m) that provided 1.3 m2 per pig in a finishing facility. Each pen was equipped with a stainless steel self-feeder and a nipple drinker. Pens of pigs (three gilts per pen) were allotted to one of six (Exp. 1) or five (Exp. 2) dietary treatments in a randomized complete block design with six replicates per treatment and had free access to water and feed. At the beginning and end of the experiments, all gilts were weighed after an overnight fast (feeders were cleaned out) and feed disappearance was determined to determine average daily gain (ADG), average daily feed intake (ADFI) and feed conversion ratio (FCR). The experimental diets were formulated based on maize, wheat bran and soybean meal (Exp. 1) or maize and wheat bran (Exp. 2). With the exception of Thr (Exp. 2), the ratios of the remaining indispensable SID AAs to SID Lys in the experimental diets were formulated to meet 110% of the recommendations of NRC (2012). The SID AA content for all experimental diets were estimated by multiplying the analyzed total AA levels in maize, soybean meal and wheat bran by the SID coefficients of the corresponding AA in those feedstuffs obtained from NRC (2012) and summing the values. The efficiency of the utilization of CAA was assumed to be 100% (Tuitoek et al., 1997). 2.1.1. Experiment 1 This study was conducted to determine the optimum SID Lys requirement for finishing gilts (88–116 kg) fed low protein diets supplemented with CAA. One hundred and eight finishing gilts were allotted to one of six dietary treatments including a high CP (135 g/kg) diet with 6.1 g/kg SID Lys or five low CP (100 g/kg) diets (supplemented with Lys, Thr, methionine, tryptophan, isoleucine and valine) providing SID Lys levels of 4.9, 5.5, 6.1, 6.7, 7.3 g/kg, respectively (Tables 1 and 2). Alanine and corn starch were added to produce isonitrogenous for low CP diets. Gilts were housed three pigs per pen with six pens per treatment for a 28 days trial. 2.1.2. Experiment 2 Experiment 2 was another dose–response study lasting for 28 days conducted to determine the optimum dietary SID Thr to Lys ratio for 90–118 kg gilts fed low CP diets supplemented with CAA. Ninety finishing gilts were allotted to one of five dietary treatments. The experimental diets were formulated to provide SID Lys of 5.1 g/kg ensuring that Lys was marginally deficient for pigs based on the results in Exp. 1. Crystalline L-Thr was added to the basal diet to formulate dietary SID Thr to

48

W.F. Ma et al. / Animal Feed Science and Technology 201 (2015) 46–56

Table 1 Ingredient composition of the experimental diets used to determine the optimal standardized ileal digestible (SID) threonine (Thr) to lysine (Lys) ratio for finishing gilts (Exp. 1 and Exp. 2; as-fed basis)a . Ingredients, g/kg

Normal-CP

Maize Soybean meal (439 g/kg CP) Wheat bran Corn starch Limestone Dicalcium phosphate Salt Vitamin-mineral premixa l-Lysine HClc (788 g/kg Lys) d,l-Methionined (990 g/kg Met) l-Threonined (990 g/kg Thr) l-Tryptophand (990 g/kg Trp) l-Isoleucined (990 g/kg Iso) l-Valined (990 g/kg Val) l-Alanined (990 g/kg Ala)

Basal diet for Exp. 2b

Exp. 1

774.6 140 60.0 0 3.50 11.5 4.00 5.00 1.40 0 0 0 0 0 0

SID Lys, g/kg 4.9

5.5

6.1

6.7

7.3

836.2 0 123 5.20 4.00 11.5 4.00 5.00 3.60 0.50 1.30 0.30 0.60 0.30 4.50

836.2 0 123 4.00 4.00 11.5 4.00 5.00 4.30 0.80 1.70 0.40 1.00 0.70 3.40

836.2 0 123 2.00 4.00 11.5 4.00 5.00 5.10 1.20 2.20 0.50 1.30 1.20 2.80

836.2 0 123 1.10 4.00 11.5 4.00 5.00 5.90 1.60 2.50 0.60 1.60 1.50 1.50

836.2 0 123 0 4.00 11.5 4.00 5.00 6.60 2.00 3.00 0.80 2.00 1.90 0

839.3 0 130 0 4.00 11.5 4.00 5.00 3.80 0.60 0.40 0.40 0.70 0.30 0

a Premix provided the following per kg of complete diet for finishing pigs: vitamin A, 5512 IU; vitamin D3, 2200 IU; vitamin E, 30 IU; vitamin K3, 2.2 mg; vitamin B12, 27.6 ␮g; riboflavin, 4 mg; pantothenic acid, 13.8 mg; niacin, 30 mg; choline chloride, 400 mg; folacin, 0.7 mg; vitamin B1, 1.5 mg; vitamin B6, 3 mg; biotin, 44 ␮g; Mn, 40 mg; Fe, 75 mg; Zn, 75 mg; Cu, 100 mg; I, 0.3 mg; Se, 0.3 mg. b l-Thr was added at 0, 0.3, 0.6, 0.9 and 1.2 g/kg of the diet to provide SID Thr to Lys ratios of 0.54, 0.60, 0.66, 0.72, and 0.78, respectively. c Provided by DaCheng Group, ChangChun, China. d Provided by Health & Nutrition of Evonik Industries AG Germany.

Lys ratios of 0.54, 0.60, 0.66, 0.72 and 0.78 (Tables 1 and 3), respectively. Crystalline methionine, tryptophan, isoleucine and valine were also supplemented in the experimental diets. 2.1.3. Collection and sampling At the termination of the experiment, blood samples were collected from six gilts per treatment (one pig per pen) after an overnight fast. Gilts were bled via jugular vein puncture using uncoated vacutainer tubes (Becton Dickinson Vacutainer Systems, Franklin Lakes, NJ). After blood collection, blood samples were quickly centrifuged at 3000 × g (Heraeus Biofuge 22R Centrifuge, Hanau, Germany) for 10 min and the serum was stored at −80 ◦ C until analyzed for serum AA and serum urea nitrogen (SUN). Table 2 Chemical analysis and calculated nutritional content of the experimental diets used to determine the optimal standardized ileal digestible (SID) lysine (Lys) ratio for finishing (88–116 kg) gilts (Exp. 1; as-fed basis)a . Normal-CP

4.9

5.5

6.1

6.7

7.3

101.0 3.54 5.60 3.84 4.55 1.04 4.36

101.2 3.87 6.27 4.17 5.07 1.00 4.80

101.1 4.18 6.86 4.59 5.49 1.11 5.23

101.7 4.55 7.49 4.95 5.86 1.30 5.69

101.7 4.88 8.07 5.39 6.29 1.30 6.13

13.6 2.48

13.3 2.48

13.3 2.48

13.3 2.48

13.3 2.48

13.3 2.48

4.43 6.11 3.81 3.91 1.25 5.25

2.99 4.87 3.19 3.49 1.03 3.61

3.31 5.54 3.51 4.00 1.04 4.04

3.62 6.11 3.92 4.40 1.16 4.46

3.99 6.74 4.29 4.77 1.37 4.93

4.32 7.31 4.72 5.19 1.47 5.36

Chemically determined values, g/kg Crude protein 134.7 Isoleucine 5.22 Lysine 7.16 Methionine + cystine 4.59 Threonine 5.23 Tryptophan 1.36 Valine 6.35 Calculated values DE, MJ/kgb NE, Mcal/kgb SID amino acids, g/kgc Isoleucine Lysine Methionine + cystine Threonine Tryptophan Valine a

SID Lys, g/kg

Values are based on a composite sample obtained weekly. Digestible energy (DE) and Net energy (NE) content of the diets were calculated using energy values for the ingredients obtained from the NRC (2012). c Values for standardized ileal digestible (SID) concentrations of amino acids for the diets were estimated using standardized ileal digestible coefficients for the various ingredients provided by NRC (2012). b

W.F. Ma et al. / Animal Feed Science and Technology 201 (2015) 46–56

49

Table 3 Chemical analysis and calculated nutritional content of the experimental diets used to determine the optimal standardized ileal digestible (SID) threonine (Thr) to lysine (Lys) ratio for finishing (90–118 kg) gilts (Exp. 2; as-fed basis)a . SID Thr to Lys ratios 0.54

0.60

0.66

0.72

0.78

Chemically determined values, g/kg Crude protein Isoleucine Lysine Methionine + cystine Threonine Tryptophan Valine

95.4 3.69 5.94 4.08 3.78 1.23 4.68

95.9 3.64 5.94 4.04 4.10 1.27 4.68

96.2 3.67 5.97 4.01 4.41 1.27 4.63

95.6 3.63 5.97 4.06 4.71 1.24 4.63

95.6 3.63 5.96 4.03 5.12 1.28 4.72

Calculated values DE, MJ/kgb NE, Mcal/kgb

13.3 2.48

13.3 2.48

13.3 2.48

13.3 2.48

13.3 2.48

3.07 5.11 3.36 2.77 1.07 3.71

3.07 5.11 3.36 3.06 1.07 3.71

3.07 5.11 3.36 3.39 1.07 3.71

3.07 5.11 3.36 3.68 1.07 3.71

3.07 5.11 3.36 3.97 1.07 3.71

SID amino acids, g/kgc Isoleucine Lysine Methionine + cystine Threonine Tryptophan Valine a

Values are based on a composite sample obtained weekly. Digestible energy (DE) and Net energy (NE) content of the diets were calculated using energy values for the ingredients obtained from the NRC (2012). c Values for standardized ileal digestible (SID) concentrations of amino acids for the diets were estimated using standardized ileal digestible coefficients for the various ingredients provided by NRC (2012). b

One pig per pen (six gilts per treatment) was randomly selected for slaughter to measure carcass traits at a pen mean weight of approximately 116 (Exp. 1) or 118 kg (Exp. 2) after an overnight fast. Thirty-six (Exp. 1) or 30 gilts (Exp. 2) were weighed on day 28 and slaughtered on day 29. Gilts were killed under commercial conditions at the Beijing Langzhong Meat Processing Facility (Beijing, China) and hot carcass weight was immediately recorded following slaughter. Dressing percentage was determined as the ratio of hot carcass weight to live weight. Carcass length was measured from the anterior edge of the symphysis pubis to the cranial edge of the first rib adjacent to the thoracic vertebra. The right carcass was split and then cut between the 10th and 11th rib to allow measurement of longissimus muscle area, fat depth, 45-min and 24-h pH (hand-held pH meter, Model 2000, VWR Scientific Products Co., South Plainfield, NJ). Carcass fat-free lean gain was calculated using the equations of the National Pork Producers Council (NPPC, 1994). Longissimus muscle samples were also taken for analysis. Drip loss was calculated by hanging a loin section (100 g, Longissimus muscle sample) in an inflated and closed plastic bag for 24 h at 4 ◦ C (King et al., 2000). Muscle marbling was determined according to NPPC (1994) guidelines and the CIELAB L* (lightness), a* (redness), and b* (yellowness) color was determined by Colorimeter (Chromameter, CR410, Minolta, Japan). 2.2. Chemical analyses The CP content (AOAC method 984.13) in the experimental diets was analyzed according to AOAC (2003) procedure. The AA content in the feeds (except methionine, cystine, and tryptophan) were detected by ion-exchange chromatography using a Hitachi L-8800 AA Analyzer (Tokyo, Japan) following acid hydrolysis with 6N HCl. Dietary methionine and cysteine were measured by acid hydrolysis with HCl after an oxidation step for quantification of total sulphur, and tryptophan was determined using reverse-phase HPLC (Waters 2690, Milford, MA) after an alkaline hydrolysis at 120 ◦ C for 16 h. Serum AA concentrations were determined by ion-exchange chromatography (S-433D Amino Acid Analyzer, Sykam, Germany) as described by Sedgwick et al. (1991). SUN concentration was determined with a blood urea nitrogen color test kit (Nanjing Jiancheng Bioengineering Institute, Nanjing, China). 2.3. Statistical analyses Data were analyzed by the GLM procedures of SAS (Statistical Analysis System, Version 6.12, 1998) using a randomized complete block design. Means are expressed as least squares means with pen as the experimental unit. An alpha level of P<0.05 was the criterion for statistical significance. Polynomial contrasts were performed to determine linear and quadratic relationships. Estimates of AA requirements for optimum performance, SUN and carcass traits were determined by subjecting the data to least squares, broken-line methodology [y = L + U × (R − x), where (R − x) is = 0 when x > R, L = plateau, U = slope, R = breakpoint; Robbins et al., 2006] as well as determining the asymptote of the quadratically fitted line using the NLIN procedure of SAS.

50

W.F. Ma et al. / Animal Feed Science and Technology 201 (2015) 46–56

Table 4 Effect of standardized ileal digestible (SID) lysine (Lys) in the diet on the performance and SUN of finishing (88–116 kg) gilts (Exp. 1)a . Normal-CP

Average daily gain, kg Average daily feed intake, kg Feed conversion ratio Daily SID lysine intake, g Serum urea nitrogen, mmol/L a b c

1.01 3.17 3.14 19.4 4.17

SEMb

SID Lys, g/kg 4.9

5.5

6.1

6.7

7.3

0.932 3.42 3.68 16.8 2.18

0.984 3.37 3.42 18.5 1.78

1.01 3.21 3.18 19.6 1.19

0.998 3.38 3.38 22.6 1.41

0.994 3.27 3.29 23.8 1.39

0.018 0.090 0.096 0.586 0.171

P valuec Linear

Quadratic

0.032 0.291 0.018 <0.001 <0.001

0.010 0.483 0.007 <0.001 <0.001

Data are means of six replicates per treatment. Standard error of mean. Linear and quadratic contrasts for effect of SID Lys levels.

3. Results 3.1. Experiment 1 The same ADG (1.01 kg/d) and non-significant differences in FCR (3.14 vs. 3.18) were obtained in gilts receiving the control diet and the low protein diet containing 6.1 g/kg SID Lys. Daily Lys intake increased (linear and quadratic effect, P<0.05) from 16.8 to 23.8 g/d as the SID Lys increased from 4.9 to 7.3 g/kg. Increasing SID Lys increased ADG (linear and quadratic effect, P<0.05) and also improved FCR (linear and quadratic effect, P<0.05). SUN decreased as the SID Lys increased (linear and quadratic effect, P<0.05) (Table 4). Muscle pH, carcass length and hot carcass dressing percentage (yield) were not affected by dietary SID Lys content. Longissimus muscle area (linear effect, P<0.05), marbling (linear and quadratic effect, P<0.05) and L* lightness (linear effect, P<0.05) increased as the dietary SID Lys increased from 4.9 to 7.3 g/kg (Table 5). Serum concentrations of Lys, alanine, valine and isoleucine were increased with the corresponding AA supplementation in low CP diets (linear and quadratic effect, P<0.05) (Table 6). The linear broken-line estimated the optimum dietary SID Lys as 5.7, 5.8 and 6.1 g/kg to maximize ADG, optimize FCR and minimize SUN. The quadratic analysis model estimated the optimum SID Lys as 6.5, 6.5 and 6.6 g/kg to maximize ADG, optimize FCR and minimize SUN (Figs. 1–3). 3.2. Experiment 2 According to the conclusion of Exp. 1, the dietary SID Lys requirement was no less than 5.7 g/kg for late finishing gilts using ADG as the response criterion. Therefore, gilts in Exp. 2 with a similar weight range as Exp. 1 were provided marginally deficient SID Lys levels of 5.1 g/kg (approximately 10% less than Lys requirement estimate which was determined using the linear-break point analysis). Increased ratios of dietary SID Thr to Lys from 0.54 to 0.78 resulted in an increase in ADG (linear and quadratic effect, P<0.05) and an improvement in FCR (linear and quadratic effect, P<0.05) (Table 7). SUN (linear and quadratic effect, P<0.05) Table 5 Effect of standardized ileal digestible (SID) lysine (Lys) in the diet on the carcass traits of finishing gilts (Exp. 1)a . Normal-CP

Carcass traits Dressing percentage, % Carcass length, cm Tenth rib fat depth, cm Longissimus muscle area, cm2 Fat-free lean gain, g/d Fat-free lean percentage, % Muscle quality Marbling Drip loss % pH45 min pH24 h L* Light a* Redness b* Yellowness a b c

SEMb

SID Lys 0.49

0.55

0.61

0.67

0.73

74.0 83.4 2.85 43.0 331 47.5

73.5 84.6 2.97 41.9 255 46.8

73.4 82.4 3.09 42.3 259 46.4

74.5 82.6 2.82 42.9 263 47.6

74.5 82.6 2.75 43.5 296 48.0

74.0 83.6 2.59 44.9 345 48.8

2.68 3.83 6.11 5.66 41.0 8.21 3.86

3.44 4.17 6.19 5.52 41.3 9.25 4.61

2.90 3.81 6.2 5.69 40.0 8.00 4.17

2.82 4.20 6.11 5.43 42.4 8.76 4.60

2.72 3.98 6.06 5.53 44.1 9.63 4.31

2.74 4.07 6.05 5.47 43.1 9.08 4.55

Data are means of six replicates per treatment. Standard error of mean. Linear and quadratic contrasts for effect of SID Lys levels.

P valuec Linear

Quadratic

0.820 1.55 0.238 1.07 37.2 1.03

0.408 0.716 0.119 0.017 0.061 0.058

0.606 0.532 0.276 0.051 0.121 0.154

0.151 0.439 0.109 0.153 0.890 0.590 0.221

0.005 0.988 0.246 0.579 0.023 0.522 0.996

0.003 0.984 0.518 0.841 0.081 0.675 0.767

W.F. Ma et al. / Animal Feed Science and Technology 201 (2015) 46–56

51

Table 6 Effect of dietary standardized ileal digestible (SID) lysine (Lys) on the serum amino acid concentrations of finishing (88–116 kg) gilts (Exp. 1)a . Normal-CP

SEMb

SID Lys 0.49

Serum essential amino acids, nmol/mL Arginine 366 Histidine 326 Isoleucine 143 Leucine 335 Lysine 867 Methionine 87 Phenylalanine 172 Threonine 285 Tryptophan 78 Valine 146

0.55

0.61

0.67

0.73

P valuec Linear

Quadratic

253 249 119 218 744 85 132 272 72 111

264 245 130 249 798 90 126 314 70 155

229 234 161 205 873 108 109 307 75 183

276 292 157 283 849 106 116 327 80 166

226 282 170 236 858 101 129 350 78 176

32 41 15 38 42 8 13 41 6 18

0.687 0.385 0.009 0.524 0.011 0.072 0.706 0.208 0.231 0.026

0.875 0.643 0.032 0.794 0.012 0.086 0.441 0.458 0.494 0.025

Serum non-essential amino acids, nmol/mL Alanine 530 956 Asparate 18 17 Glutamine 274 344 Glycine 1448 1330 Cystine 89 62 Proline 2187 2460 Serine 304 298 Tyrosine 370 274

939 18 400 1810 78 2083 293 260

870 18 338 1671 78 2150 294 211

721 21 292 1649 78 2339 297 235

784 18 310 1803 73 2062 304 262

69 3 50 209 10 283 36 24

0.011 0.552 0.280 0.205 0.491 0.514 0.884 0.520

0.040 0.808 0.538 0.346 0.424 0.769 0.969 0.176

a b c

Data are means of six replicates per treatment. Standard error of mean. Linear and quadratic contrasts for effect of SID Lys levels.

Table 7 Effect of standardized ileal digestible (SID) threonine (Thr) to lysine (Lys) ratio in the diet on performance and SUN of finishing (90–118 kg) gilts (Exp. 2)a . SEMb

SID Thr to Lys ratio

Average daily gain, kg Average daily feed intake, kg Feed conversion ratio Daily SID threonine intake, g Serum urea nitrogen, mmol/L a b c

0.54

0.60

0.66

0.72

0.78

0.891 3.56 4.03 9.80 2.57

0.970 3.48 3.58 10.6 1.60

0.990 3.38 3.42 11.4 1.06

0.982 3.35 3.43 12.3 0.729

0.983 3.18 3.27 12.7 0.847

0.027 0.147 0.197 0.514 0.160

P valuec Linear

Quadratic

0.034 0.058 0.011 <0.001 <0.001

0.021 0.168 0.024 <0.001 <0.001

Data are means of six replicates per treatment. Standard error of mean. Linear and quadratic contrasts for SID Thr to Lys ratios.

Fig. 1. Fitted broken line analysis (—) and quadratic model (—) plot of average daily gain plotted against standardized ileal digestible lysine levels (Exp. 1).

52

W.F. Ma et al. / Animal Feed Science and Technology 201 (2015) 46–56

Fig. 2. Fitted broken line analysis (—) and quadratic model (—) plot of feed conversion ratio plotted against standardized ileal digestible lysine levels (Exp. 1).

decreased as dietary SID Thr to Lys ratio increased (Table 7). With the exception of L* light (linear effect, P<0.05), no effects on carcass characteristics were observed with the increase in dietary SID Thr to Lys ratio (Table 8). Supplementation with Thr increased the level of serum Thr (linear and quadratic effect, P<0.05) and alanine concentration (linear effect, P<0.05) (Table 9). Linear-break point analysis estimated the break points of dietary SID Thr to Lys ratio as 0.61, 0.63 and 0.64 to maximize ADG, optimize FCR and minimize SUN, respectively. Quadratic analysis estimated the optimum SID Thr to Lys ratios as 0.70, 0.75 and 0.74 to maximize ADG, optimize FCR and minimize SUN, respectively (Figs. 4–6). 4. Discussion Lowering the dietary CP level by using supplemented CAA is not only a common economic strategy to reduce nitrogen excretion of finishing pigs but also to improve carcass traits with no effect on pig performance (Kerr et al., 1995). Feed formulators often attempt to decrease dietary CP levels by decreasing the content of soybean meal (Galassi et al., 2010; Prandini et al., 2013) and this may result in reductions in the AA concentration in the diet. Thus, knowledge of the appropriate SID Lys requirement and AA to Lys ratio plays a very important role in reducing feed costs with maintaining animal performance (Zhang et al., 2013; Tous et al., 2014). During the late finishing period, Lys requirement was largely influenced by lean protein deposition (Gu et al., 1992; Friesen et al., 1994), which indicated that the lean growth rate could be a very important parameter for finishing pigs. In the present study, the gilts fed a low CP, AA-supplemented diet could achieve the similar performance compared with the gilts fed the control diet. However, the SID Lys level of 5.7 g/kg was sufficient to maximize the performance, while the fat-free lean gain tended to increase linearly by dietary SID Lys level increasing from 4.9 to 7.3 g/kg for gilts fed the low CP diets. There may be a consensus that gilts tend to get fatter when receiving a low CP diet (Kerr et al., 1995, 2003) and increasing the dietary Lys level was beneficial for lean protein deposition of pigs. In previous studies, Tous et al. (2014) also concluded that adequate

Fig. 3. Fitted broken line analysis (—) and quadratic model (—) of serum urea nitrogen plotted against standardized ileal digestible lysine levels (Exp. 1).

W.F. Ma et al. / Animal Feed Science and Technology 201 (2015) 46–56

53

Table 8 Effect of standardized ileal digestible (SID) threonine (Thr) to lysine (Lys) ratio lysine (Lys) in the diet on carcass traits of finishing gilts (Exp. 2)a SEMb

SID Thr to Lys ratios

Carcass traits Dressing percentage, % Carcass length, cm Tenth rib fat depth, cm Longissimus muscle area, cm2 Fat-free lean gain, g/d Fat-free lean percentage, % Muscle quality Marbling Drip loss, % pH45 min pH24 h L* Light a* Redness b* Yellowness a b c

0.54

0.60

0.66

0.72

0.78

74.0 84.5 2.73 41.6 201 46.5

73.4 82.7 2.85 42.3 228 47.3

73.6 83.3 2.87 42.6 231 47.4

74.1 82.2 2.98 41.9 212 46.7

73.9 83.8 2.83 42.6 240 47.6

3.15 4.56 6.16 5.49 37.8 8.42 2.36

3.12 4.33 6.36 5.56 38.8 8.52 2.68

3.30 4.30 6.25 5.62 39.9 8.45 2.86

3.13 4.19 6.27 5.63 38.7 8.74 2.39

3.17 4.53 6.38 5.54 41.5 8.86 3.17

P valuec Linear

Quadratic

0.58 1.22 0.12 1.44 35.86 0.46

0.805 0.631 0.392 0.715 0.561 0.333

0.828 0.462 0.405 0.924 0.838 0.606

0.15 0.39 0.11 0.08 0.85 0.69 0.30

0.912 0.871 0.297 0.576 0.013 0.605 0.163

0.916 0.755 0.585 0.399 0.045 0.866 0.373

Data are means of six replicates per treatment. Standard error of mean. Linear and quadratic contrasts for SID Thr to Lys ratios.

Fig. 4. Fitted broken line analysis (—) and quadratic model (—) of average daily gain plotted against standardized ileal digestible threonine to lysine ratios (Exp. 2).

Fig. 5. Fitted broken line analysis (—) and quadratic model (—) of feed conversion ratio plotted against standardized ileal digestible threonine to lysine ratios (Exp. 2).

54

W.F. Ma et al. / Animal Feed Science and Technology 201 (2015) 46–56

Fig. 6. Fitted broken line analysis (—) and quadratic model (—) of serum urea nitrogen plotted against standardized ileal digestible threonine to lysine ratios (Exp. 2).

levels of dietary Lys had positive effects on carcass traits for 62–120 kg barrows receiving a low CP, AA-supplemented diet. Cline et al. (2000) showed that estimated fat-free lean gain increased linearly as the dietary total Lys level increased from 8.0 to 1.4 g/kg for 54–116 kg gilts. However, no improvement was observed in carcass traits by increasing Lys to calorie ratio for 100–120 kg barrows and gilts in Main et al. (2008). In previous studies, limited data was validated on the basis of SID AA. Shelton et al. (2011, 2012) concluded that the optimum SID Lys level for 84–100 kg and 102–125 kg gilts appeared to be 8.9 and 7.8 g/kg, respectively. Compared with the current study, the different conclusion may be due to the variation of lean protein deposition capacity of gilts among the experiments. The SID Thr requirement of pigs was estimated using a prediction model and represented relative to Lys (de Lange et al., 2001). The Thr to Lys ratio could be strongly influenced by variation in stage of growth (Russell et al., 1986; Li et al., 1999; de Lange et al., 2001; Kerr et al., 2003; Ettle et al., 2004; Wecke and Liebert, 2010) and tend to increase moving towards the finishing period, which may be due to the increased proportion of Thr needed for maintenance for finishing pigs (Hahn and Baker, 1995). However, very few empirical studies have been conducted to determine the optimum SID Thr to Lys ratio for late finishing pigs. Pedersen et al. (2003) and Plitzner et al. (2007) reported the optimum SID Thr to Lys ratio for 60–110 kg Table 9 Effect of dietary standardized ileal digestible (SID) threonine (Thr) to lysine (Lys) ratio on the serum amino acid concentrations of finishing (90–118 kg) gilts (Exp. 2)a . SEMb

SID Thr to Lys ratio 0.54

0.6

0.66

0.72

0.78

201 338 295 180 410 90 123 87 51 63

209 360 320 183 469 85 122 139 54 69

207 249 332 183 452 84 115 160 57 83

211 242 302 161 481 88 112 201 53 62

Serum non-essential amino acids, nmol/mL Alanine 708 852 Asparate 26 25 Glutamine 392 403 Glycine 2095 1763 Cystine 221 235 Proline 2235 2305 Serine 301 256 Tyrosine 280 238

873 27 355 2043 218 2286 302 271

917 27 423 2089 228 2146 308 319

897 24 400 1869 229 2107 254 224

Serum essential amino acids, nmol/mL Arginine 253 Histidine 307 Isoleucine 286 Leucine 181 Lysine 448 Methionine 96 Phenylalanine 130 Threonine 72 Tryptophan 54 Valine 62

a b c

Data are means of six replicates per treatment. Standard error of mean. Linear and quadratic contrasts for SID Thr to Lys ratios.

P valuec Linear

Quadratic

25 55 24 19 24 14 14 22 6 70

0.343 0.224 0.367 0.527 0.185 0.582 0.309 <0.001 0.841 0.369

0.071 0.286 0.382 0.661 0.354 0.759 0.602 <0.001 0.964 0.264

67 3 36 176 27 167 43 42

0.039 0.864 0.740 0.769 0.886 0.430 0.715 0.797

0.059 0.860 0.885 0.956 0.990 0.634 0.866 0.804

W.F. Ma et al. / Animal Feed Science and Technology 201 (2015) 46–56

55

pigs was 0.62–0.64. Frantz et al. (2005) determined an optimum SID Thr to Lys ratio of 0.67 for 75–105 kg pigs. Thus the results in the present study were similar compared with previous studies. The appropriate Lys requirement and optimum Thr to Lys ratio had mostly been determined using ADG or FCR as the response criteria (Saldana et al., 1994; Shelton et al., 2011). However, recent studies indicated that SUN concentration could also be used to evaluate the AA requirement of pigs, which could reflect the efficiency of nitrogen utilization and can be significantly influenced by the quality of dietary protein (Coma et al., 1995; Kohn et al., 2005) and was decreased until the first-limiting amino acid reached requirements (Brown and Cline, 1974; Cameron et al., 2003). In the present study, the AA requirements were determined by setting Lys or Thr as the first limiting amino acid in the experimental diets, thus SUN could be a very strong response criteria. The effects on carcass traits by dietary level of Lys and Thr were also measured in the present study. The results indicated that carcass traits can be regulated by adjusting dietary Lys levels rather than Thr and various Thr to Lys ratios appeared to have little effect on carcass characteristics except meat color. In accordance with the present results, previous studies showed that the effect of Thr to Lys ratio on carcass characteristics was much lower than the effect on performance (Plitzner et al., 2006, 2007). Serum amino acid concentrations could produce reference patterns that may be used for protein quality evaluation (Keith et al., 1972). In the present study, gilts fed the low CP diet tended to obtain lower serum levels of arginine, histidine, phenylalanine and tyrosine compared with gilts fed the control diet, which was possibly because that the levels of these amino acids were relatively higher in soybean meal, compared with that in maize. As noted above, soybean meal was not included in the low CP diets. In addition, the changes of serum levels of Lys, valine, isoleucine, and alanine in Exp. 1 and Thr in Exp. 2 were expected to correspond to the change of the dietary AA level in experimental diets. However, the change in serum alanine level related to the different SID Thr to Lys ratios needs further investigation. The concentrations of serum AA in the present study did not reflect the optimum Thr to Lys ratio directly and this may be due to the difference in the length of the experimental period and diverse times for animal bleeding (Gloaguen et al., 2012). In dose–response study, the statistical methodology has also been shown to affect the determination of AA requirement. In broken-line regression model, the break point of two lines was selected as the minimum nutrient requirement for the theoretical average pig, which appeared to underestimates the nutritional requirement for pigs within the population (Baker, 1986). However, the quadratic model was used to estimate the AA requirement to reach 100% of the maximum response, however, the requirement was often overestimated (Baker, 1986). In accordance with the present results, previous study also demonstrated that broken-line models always resulted in lower estimates compared with quadratic models (Kendall et al., 2007). For the purpose of comparison, both models were chosen to determine the AA requirement in the present study. 5. Conclusions The optimum SID Lys to maximize ADG and optimize FCR as well as to minimize SUN levels were 5.7, 5.8 and 6.1 g/kg using a linear-break point model and 6.5, 6.5 and 6.6 g/kg using a quadratic model for 88–116 kg gilts. The fat-free lean gain tended to increase linearly with the increase in dietary SID Lys level from 4.9 to 7.3 g/kg for gilts fed a low CP diet. Linear-break point analysis estimated the optimum SID Thr to Lys ratio for 90–118 kg gilts as 0.61, 0.63 and 0.64 whereas the quadratic analysis estimated the optimum SID Thr to Lys ratio as 0.70, 0.74 and 0.72 to maximize ADG, minimize FCR and SUN, respectively, which is similar to the recommend ratios in NRC (2012) and National Swine Nutrition Guide (2010). Conflict of interest We confirm that there are no conflicts of interest associated with this publication and there has been no significant financial support for this work that could have influenced its outcome. Acknowledgments This study was supported by Modern Agro-Industry Technology Research System of Beijing and the National Basic Research Program of China (2012CB124704). The authors thank the DaCheng Group, (ChangChun), China and Health & Nutrition of Evonik Industries (AG Germany) for providing supplemental amino acids. References Baker, D.H., 1986. Problems and pitfalls in animal experiments designed to establish dietary requirements for essential nutrients. J. Nutr. 116, 2339–2349. Brown, J.A., Cline, T.R., 1974. Urea excretion in pig-indicator of protein quality and amino-acid requirements. J. Nutr. 104, 542–545. Cameron, N.D., McCullough, E., Troup, K., Penman, J.C., 2003. Serum urea concentration as a predictor of dietary lysine requirement in selected lines of pigs. J. Anim. Sci. 81, 91–100. Cline, T.R., Cromwell, G.L., Crenshaw, T.D., Ewan, R.C., Hamilton, C.R., Lewis, A.J., Mahan, D.C., Southern, L.L., 2000. Further assessment of the dietary lysine requirement of finishing gilts. J. Anim. Sci. 78, 987–992. Coma, J., Carrion, D., Zimmerman, D.R., 1995. Use of plasma urea nitrogen as a rapid response criterion to determine the lysine requirement of pigs. J. Anim. Sci. 73, 472–481. de Lange, C., Gillis, A.M., Simpson, G.J., 2001. Influence of threonine intake on whole-body protein deposition and threonine utilization in growing pigs fed purified diets. J. Anim. Sci. 79, 3087–3095.

56

W.F. Ma et al. / Animal Feed Science and Technology 201 (2015) 46–56

Ettle, T., Roth-Maier, D.A., Bartelt, J., Roth, F.X., 2004. Requirement of true ileal digestible threonine of growing and finishing pigs. J. Anim. Physiol. Anim. Nutr. 88, 211–222. Friesen, K.G., Nelssen, J.L., Unruh, J.A., Goodband, R.D., Tokach, M.D., 1994. Effects of the interrelationship between genotype, sex, and dietary lysine on growth performance and carcass composition in finishing pigs fed to either 104 or 127 kilograms. J. Anim. Sci. 72, 946–954. Frantz, N.Z., Tokach, M.D., Dritz, S.S., Usry, J.L., Goodband, R.D., DeRouchey, J.M., Nelssen, J.L., Jones, C.L., 2005. The optimal true ileal digestible (TID) lysine and threonine requirement for finishing pigs from 36 to 60 and 77 to 105 kg. J. Anim. Sci. 832, 68. Galassi, G., Colombini, S., Malagutti, L., Crovetto, G.M., Rapetti, L., 2010. Effects of high fibre and low protein diets on performance, digestibility, nitrogen excretion and ammonia emission in the heavy pig. Anim. Feed. Sci. Technol. 161, 140–148. Gloaguen, M., Le Floc’H., N., Corrent, E., Primot, Y., van Milgen, J., 2012. Providing a diet deficient in valine but with excess leucine results in a rapid decrease in feed intake and modifies the postprandial plasma amino acid and alpha-keto acid concentrations in pigs. J. Anim. Sci. 90, 3135–3142. Gu, Y., Schinckel, A.P., Martin, T.G., 1992. Growth, development, and carcass composition in five genotypes of swine. J. Anim. Sci. 70, 1719–1729. Hahn, J.D., Baker, D.H., 1995. Optimum ratio to lysine of threonine, tryptophan, and sulfur amino acids for finishing swine. J. Anim. Sci. 73, 482–489. Keith, M.O., Owen, B.D., Christen, D.A., 1972. Determination of methionine requirement of growing pigs using serum free amino-acids. Can. J. Anim. Sci. 52, 163–169. Kendall, D.C., Gaines, A.M., Kerr, B.J., Allee, G.L., 2007. True ileal digestible tryptophan to lysine ratios in ninety- to one hundred twenty-five-kilogram barrows. J. Anim. Sci. 85, 3004–3012. Kerr, B.J., Mckeith, F.K., Easter, R.A., 1995. Effect on performance and carcass characteristics of nursery to finisher pigs fed reduced crude protein, amino acid-supplemented diets. J. Anim. Sci. 73, 433–440. Kerr, B.J., Yen, J.T., Nienaber, J.A., Easter, R.A., 2003. Influences of dietary protein level, amino acid supplementation and environmental temperature on performance, body composition, organ weights and total heat production of growing pigs. J. Anim. Sci. 81, 1998–2007. King, R.H., Campbell, R.G., Smits, R.J., Morley, W.C., Ronnfeldt, K., Butler, K., Dunshea, F.R., 2000. Interrelationships between dietary lysine, sex, and porcine somatotropin administration on growth performance and protein deposition in pigs between 80 and 120 kg live weight. J. Anim. Sci. 78, 2639–2651. Kohn, R.A., Dinneen, M.M., Russek-Cohen, E., 2005. Using blood urea nitrogen to predict nitrogen excretion and efficiency of nitrogen utilization in cattle, sheep, goats, horses, pigs, and rats. J. Anim. Sci. 83, 879–889. Li, D.F., Xiao, C.T., Qiao, S.Y., Zhang, J.H., Johnson, E.W., Thacker, P.A., 1999. Effects of dietary threonine on performance, plasma parameters and immune function of growing pigs. Anim. Feed. Sci. Technol. 78, 179–188. Main, R.G., Dritz, S.S., Tokach, M.D., Goodband, R.D., Nelssen, J.L., 2008. Determining an optimum lysine: calorie ratio for barrows and gilts in a commercial finishing facility. J. Anim. Sci. 86, 2190–2207. NPPC, 1994. Pork Composition and Quality Assessment Procedures. Natl. Pork Producers Council, Des Moines, IA. NRC, 2012. Nutrient Requirements of Swine, 11th ed. National Academy Press, Washington, DC. National Swine Nutrition Guide, 2010. National, Swine Nutrition Guide Tables on Nutrient Recommendations, Ingredient Composition, and Use Rates. US Pork Center of Excellence, Ames, IA. Pedersen, C., Lindberg, J.E., Boisen, S., 2003. Determination of the optimal dietary threonine:lysine ratio for finishing pigs using three different methods. Livest. Prod. Sci. 82, 233–243. Plitzner, C., Windisch, W., Ettle, T., 2006. Experimental study on the requirement of threonine in finishing pigs. Die Bodenk 57, 161–168. Plitzner, C., Ettle, T., Handl, S., Schmidt, P., Windisch, W., 2007. Effects of different dietary threonine levels on growth and slaughter performance in finishing pigs. Czech. J. Anim. Sci. 52, 447–455. Prandini, A., Sigolo, S., Morlacchini, M., Grilli, E., Fiorentini, L., 2013. Microencapsulated lysine and low-protein diets: effects on performance, carcass characteristics and nitrogen excretion in heavy growing-finishing pigs. J. Anim. Sci. 91, 4226–4234. Robbins, K.R., Saxton, A.M., Southern, L.L., 2006. Estimation of nutrient requirements using broken-line regression analysis. J. Anim. Sci. 84, E155–E165. Russell, L.E., Easter, R.A., Gomezrojas, V., Cromwell, G.L., Stahly, T.S., 1986. A note on the supplementation of low-protein, maize-soya-bean meal diets with lysine, tryptophan, threonine and methionine for growing pigs. Anim. Prod. Sci. 42, 291–295. Saldana, C.I., Knabe, D.A., Owen, K.Q., Burgoon, K.G., Gregg, E.J., 1994. Digestible threonine requirements of starter and finisher pigs. J. Anim. Sci. 72, 144–150. Sedgwick, G.W., Fenton, T.W., Thompson, J.R., 1991. Effect of protein precipitating agents on the recovery of plasma free amino acids. Can. J. Anim. Sci. 71, 953–957. Shelton, N.W., Tokach, M.D., Dritz, S.S., Goodband, R.D., Nelssen, J.L., DeRouchey, J.M., 2011. Effects of increasing dietary standardized ileal digestible lysine for gilts grown in a commercial finishing environment. J. Anim. Sci. 89, 3587–3595. Shelton, N.W., Tokach, M.D., Dritz, S.S., Goodband, R.D., Nelssen, J.L., DeRouchey, J.M., Usry, J.L., 2012. Effects of porcine circovirus type 2 vaccine and increasing standardized ileal digestible lysine:metabolizable energy ratio on growth performance and carcass composition of growing and finishing pigs. J. Anim. Sci. 90, 361–372. Tous, N., Lizardo, R., Vil AF B., Gispert, M., Font-i-Furnols, M., Esteve-Garcia, E., 2014. Effect of reducing dietary protein and lysine on growth performance, carcass characteristics, intramuscular fat, and fatty acid profile of finishing barrows. J. Anim. Sci. 92, 129–140. Tuitoek, K., Young, L.G., DeLange, C., Kerr, B.J., 1997. The effect of reducing excess dietary amino acids on growing-finishing pig performance: an evaluation of the ideal protein concept. J. Anim. Sci. 75, 1575–1583. Wecke, C., Liebert, F., 2010. Optimal dietary lysine to threonine ratio in pigs (30–110 kg BW) derived from observed dietary amino acid efficiency. J. Anim. Physiol. An. Nutr. 94, 277–285. Zhang, G.J., Xie, C.Y., Thacker, P.A., Htoo, J.K., Qiao, S.Y., 2013. Estimation of the ideal ratio of standardized ileal digestible threonine to lysine for growing pigs (22-50 kg) fed low crude protein diets supplemented with crystalline amino acids. Anim. Feed. Sci. Technol. 180, 83–91.