The effect of reduced dietary glycine and serine and supplemental threonine on growth performance, protein deposition in carcass and viscera, and skin collagen abundance of nursery pigs fed low crude protein diets

Abstract

Thirty five barrows (initial body weight [BW]: 15.1 ± 1.0 kg) were used to determine the effect of partially replacing Gly + Ser with Thr in reduced crude protein (CP) diets on growth performance, protein deposition in carcass and viscera, and skin collagen abundance during the late nursery phase to 25 kg BW. Pigs were individually fed one of five iso-nitrogenous diets (n = 7) for 21 d. The basal diet met estimated essential amino acids (AA) requirements by using all essential AA plus Gly and Ser in free form (CON; 12.1% CP; as-fed, analyzed contents). The remaining four diets were formulated by reducing total Gly and Ser concentrations to 60% or 20% of the CON diet. The N removed with Gly and Ser was replaced with either crystalline Thr or Glu. Total analyzed Thr made up either 1.59% (T1; 12.5% CP) or 2.34% (T2; 12.2% CP) of the Thr-supplemented diets, and total analyzed Glu made up either 3.47% (G1; 12.7% CP) or 4.64% (G2; 12.9% CP) of the Glu-supplemented diets. Pigs were slaughtered on day 21 to determine body composition and skin collagen abundance via bright field microscopy. Overall, average daily gain (ADG) and G:F and final carcass weights were greater for pigs fed diets supplemented with Glu (G1 + G2) vs. those fed diets supplemented with Thr (T1 + T2; P < 0.05, P = 0.060, and P = 0.050 for ADG, G:F, and final carcass weight, respectively); intermediate values were observed for CON. Nitrogen retention in carcass plus viscera and the AA profile of deposited protein in the carcass were not influenced by dietary treatment. Pigs fed the T2 and G2 diets had greater retention of Thr (vs. CON and G2) and Glu (vs. CON and T2) in the viscera protein, respectively (P < 0.05). The apparent utilization efficiency of standardized ileal digestible Thr for protein deposition in carcass plus viscera was less for pigs fed T2 (15.1%) vs. those fed CON (56.7%) or G2 (58.6% ± 2.9%) diets (P < 0.001). Only pigs fed T1 had skin collagen abundance not different from CON; pigs fed G1, G2, and T2 had reduced skin collagen abundance compared with CON and T1 (P < 0.01). Using Glu as an N source when Gly and Ser were reduced to 60% and 20% of CON in reduced CP diets maintained ADG for pigs between 15 and 25 kg BW, whereas supplying Thr as a N source reduced ADG and carcass weight. When dietary Gly and Ser were supplied at 60% of CON, only Thr supplementation rescued skin collagen abundance. Therefore, supplemental Thr at excess levels is not sufficient to replace N from Gly and Ser in reduced CP diets fed to late nursery pigs, despite supporting skin collagen abundance as a secondary indicator of Gly status.

Introduction

Crystalline amino acids (CAA) can be used to reduce dietary crude protein (CP) concentrations to improve AA and N utilization efficiencies for protein gain and reduce the amount of N excreted into the environment (NRC, 2012; Millet et al., 2018). Using CAA supplementation, dietary CP can be reduced by up to 4 percentage units, without negatively affecting pig growth performance (Kerr et al., 1995; Toledo et al., 2014), though the total supply of nonessential AA (NEAA) from whole-protein sources is also reduced. In this situation, the endogenous synthesis of NEAA may be inadequate to support the optimal growth and secondary indicators of AA status. For example, Gly is necessary for collagen synthesis as well as for other metabolic fates (e.g., Ser, glutathione, creatine, and DNA synthesis; as reviewed by Wu et al., 2014). As Gly accounts for approximately 33% of AA residues in collagen (Gregg and Rogers, 1986), skin collagen abundance could be a sensitive indicator of Gly adequacy for other metabolic fates. Indeed, in nursery pigs, supplementing Gly but not Glu to low CP diets resulted in skin collagen abundance not different from pigs fed corn- and soybean meal-based diets (Silva et al., in press). Therefore, whether Gly is supplied via the diet or by the metabolism of the animal, sufficient quantities of Gly must be available to maximize muscle protein deposition and to maintain other important metabolic pathways.

Since crystalline Gly is not mass produced for inclusion in commercial swine diets, supplementation with crystalline Thr could be a relatively inexpensive and practical approach to supply Gly (indirectly) via the Thr dehydrogenase pathway (Le Floc’h et al., 1995) when feeding low CP diets. In this scenario, however, Thr must be supplemented above estimated requirements for whole-body protein deposition in order to satisfy the needs for both protein synthesis and conversion into Gly. Therefore, the objective of this study was to determine the effect of partially replacing Gly and Ser with Thr in reduced CP diets on growth performance, protein deposition in carcass and viscera, and skin collagen abundance of pigs during the late nursery period.

Materials and Methods

Animals and dietary treatments

The experimental protocol was approved by the University of Guelph Animal Care Committee and followed the Canadian Council of Animal Care guidelines for the care and use of farm animals (CCAC, 2009; AUP: e3786). Thirty-five Yorkshire × Landrace × Duroc barrows (initial body weight [BW] 15.1 ± 1.0 kg) from the Arkell Swine Research Facility, University of Guelph (Guelph, ON, Canada), were used for the study. Prior to the study, pigs were weaned at approximately 21 d of age (7.0 kg BW) and fed commercial nursery diets. Upon study initiation, pigs were housed individually but continued to receive ad libitum access to a standard nursery diet for 4 d. After the adaptation period, pigs were assigned to one of five dietary treatments; initial BW was balanced across dietary treatments and littermates were delegated to separate treatments. Pigs were fed in three equal meals per day at 0800, 1200, and 1700 hours to achieve intakes of 2.8 × estimated metabolizable energy requirements for maintenance (NRC, 2012). Pigs were weighed weekly to adjust feed allowance. Water was supplied with every meal in a ratio of 3:1 and was freely available from a nipple drinker in each pen for the duration of the study.

The five experimental diets were each formulated to meet estimated essential AA requirements for late nursery pigs (15 to 25 kg BW; NRC, 2012). Crystalline NEAA were included to generate a dietary AA profile similar to that in whole-body protein of pigs and to maintain an average essential AA N:total N ratio of 0.46 (Heger et al., 1998). The basal diet was semi-purified and met estimated essential AA requirements (CON; 12.1% CP as-fed; analyzed contents; Tables 1 and 2). The remaining four diets were formulated by reducing total Gly and Ser concentrations to 60% or 20%, respectively, of the CON diet. The N that was removed with Gly and Ser was replaced with either crystalline Thr or Glu, to maintain similar CP concentration as CON. Dietary Glu was selected as an N balancer to achieve isonitrogenous diets because it is the most abundant AA in the body and is easily synthesized from metabolic intermediates (e.g., α-ketogluterate) and, therefore, would likely have minimal effects on AA metabolism. Total analyzed Thr made up either 1.59% (T1; 12.5% CP; 2.8 × estimated Thr requirements) or 2.34% of the diet (T2; 12.2% CP; 4.1 × estimated Thr requirements), and total analyzed Glu made up either 3.47% (G1; 12.7% CP) or 4.64% of the diet (G2; 12.9% CP). Experimental diets were mixed individually, and the major components were added to the mixer independently, while additives (i.e., crystalline AA and ingredients with inclusion levels < 0.5%) were pre-mixed then added as a single ingredient to promote proper mixing. Representative subsamples of each diet were collected at diet manufacturing for chemical composition analyses.

Table 1.

Ingredient composition and nutrient concentrations of low CP diets with reduced concentrations of Gly and Ser and supplemental Thr or Glu (as-fed)

	Dietary treatment1
Item	CON	G1	G2	T1	T2
Ingredient composition,%
Cornstarch	63.16	62.06	62.04	62.85	62.54
Casein	3.00	3.00	3.00	3.00	3.00
Monocalcium phosphate	1.65	1.65	1.65	1.65	1.65
Limestone	1.23	1.23	1.23	1.23	1.23
Salt	0.44	0.44	0.44	0.44	0.44
Magnesium sulfate	0.10	0.10	0.10	0.10	0.10
Potassium sulfate	0.60	0.60	0.60	0.60	0.60
Swine premix2	0.60	0.60	0.60	0.60	0.60
Cellulose	3.00	3.00	3.00	3.00	3.00
Pectin	3.00	3.00	3.00	3.00	3.00
Sucrose	8.00	8.00	8.00	8.00	8.00
Soybean oil	4.00	4.00	4.00	4.00	4.00
l-Lys·HCl	1.15	1.15	1.15	1.15	1.15
Dl-Met	0.40	0.40	0.40	0.40	0.40
l-Cys·HCl	0.19	0.19	0.19	0.19	0.19
l-Trp	0.14	0.14	0.14	0.14	0.14
l-Ile	0.44	0.44	0.44	0.44	0.44
l-Val	0.54	0.54	0.54	0.54	0.54
l-Leu	0.88	0.88	0.88	0.88	0.88
l-His	0.31	0.31	0.31	0.31	0.31
l-Arg	0.42	0.42	0.42	0.42	0.42
l-Phe	0.46	0.46	0.46	0.46	0.46
l-Tyr	0.30	0.30	0.30	0.30	0.30
l-Pro	0.55	0.55	0.55	0.55	0.55
l-Asp	1.35	1.35	1.35	1.35	1.35
l-Thr3	0.56	0.56	0.56	1.57	2.58
l-Glu3	1.77	3.03	4.29	1.77	1.77
Gly3	1.21	0.73	0.24	0.73	0.24
l-Ser3	0.55	0.33	0.11	0.33	0.11
Total	100.00	100.00	100.00	100.00	100.00
Calculated nutrient contents4
CP,%	13.57	13.57	13.57	13.57	13.57
NE, kcal/kg	2,650	2,650	2,651	2,650	2,651
SID AA,%
Lys	1.11	1.11	1.11	1.11	1.11
Arg	0.50	0.50	0.50	0.50	0.50
His	0.38	0.38	0.38	0.38	0.38
Ile	0.57	0.57	0.57	0.57	0.57
Leu	1.11	1.11	1.11	1.11	1.11
Met	0.47	0.47	0.47	0.47	0.47
Met + Cys	0.61	0.61	0.61	0.61	0.61
Cys	0.14	0.14	0.14	0.14	0.14
Phe	0.59	0.59	0.59	0.59	0.59
Phe + Tyr	1.03	1.03	1.03	1.03	1.03
Thr	0.66	0.66	0.66	1.66	2.66
Trp	0.18	0.18	0.18	0.18	0.18
Val	0.70	0.70	0.70	0.70	0.70
Glu	2.27	2.89	3.52	2.27	2.27
Gly	1.24	0.76	0.28	0.76	0.28
Pro	0.97	0.97	0.97	0.97	0.97
Ser	0.67	0.45	0.23	0.45	0.23
Ala	1.15	1.15	1.15	1.15	1.15
Asp	1.50	1.50	1.50	1.50	1.50
E:TN5	0.43	0.43	0.43	0.49	0.54

¹CON, control; G1, Gly supplied at 60% of CON and replaced with Glu; G2, Gly supplied at 20% of CON and replaced with Glu; T1, Gly supplied at 60% of CON and replaced with Thr; T2 Gly supplied at 20% of CON and replaced with Thr.

²Supplied per kg of complete diet: vitamin A, 10,000 IU as retinyl acetate (2.5 mg) and retinylpalmitate (1.7 mg); vitamin D₃, 1,000 IU as cholecalciferol; vitamin E, 56 IU as dl-α- tocopherol acetate (44 mg); vitamin K, 2.5 mg as menadione; choline, 500 mg; pantothenic acid, 15 mg; riboflavin, 5 mg; folic acid, 2 mg; niacin, 25 mg; thiamine, 1.5 mg; vitamin B₆, 1.5 mg; biotin, 0.2 mg; vitamin B₁₂, 0.025 mg; Se, 0.3 mg from Na₂SeO₃; Cu, 15 mg from CuSO₄.5H₂O; Zn, 104 mg from ZnO; Fe, 100 mg from FeSO₄; Mn, 19 mg from MnO₂; and I, 0.3 mg from KI (DSM Nutritional Products Canada Inc., Ayr, ON).

³These AA were adjusted to generate the experimental diets.

⁴Calculated using ingredient values according to NRC (2012).

⁵Essential AA N: Total N ratio.

Table 2.

Analyzed crude protein, minerals, and total AA contents of the experimental diets (%, as-fed)

	Dietary treatment1
Item	CON	G1	G2	T1	T2
CP	12.11	12.73	12.88	12.46	12.24
Ca	0.74	0.77	0.69	0.69	0.67
P	0.32	0.26	0.25	0.24	0.25
Lys	1.06 (1.11)2	1.13 (1.11)	1.15 (1.11)	1.13 (1.11)	1.06 (1.11)
Arg	0.47 (0.50)	0.48 (0.50)	0.49 (0.50)	0.47 (0.50)	0.47 (0.50)
His	0.35 (0.38)	0.36 (0.38)	0.38 (0.38)	0.36 (0.38)	0.37 (0.38)
Ile	0.53 (0.57)	0.55 (0.57)	0.57 (0.57)	0.54 (0.57)	0.54 (0.57)
Leu	1.07 (1.11)	1.09 (1.11)	1.14 (1.11)	1.09 (1.11)	1.07 (1.11)
Met	0.41 (0.47)	0.41 (0.47)	0.41 (0.47)	0.40 (0.47)	0.39 (0.47)
Phe	0.56 (0.59)	0.59 (0.59)	0.61 (0.59)	0.59 (0.59)	0.56 (0.59)
Thr	0.58 (0.66)	0.62 (0.66)	0.75 (0.66)	1.59 (1.66)	2.34 (2.66)
Trp	0.15 (0.18)	0.15 (0.18)	0.16 (0.18)	0.15 (0.18)	0.15 (0.18)
Val	0.65 (0.70)	0.68 (0.70)	0.71 (0.70)	0.66 (0.70)	0.69 (0.70)
Glu	2.30 (2.29)	3.47 (3.54)	4.64 (4.79)	2.32 (2.29)	2.35 (2.29)
Gly	1.27 (1.25)	0.69 (0.77)	0.32 (0.29)	0.74 (0.77)	0.29 (0.29)
Pro	0.78 (0.97)	0.78 (0.97)	0.79 (0.97)	0.77 (0.97)	0.76 (0.97)
Ser	0.62 (0.68)	0.44 (0.46)	0.25 (0.25)	0.44 (0.46)	0.26 (0.25)
Ala	1.13 (1.15)	1.15 (1.15)	1.15 (1.15)	1.14 (1.15)	1.11 (1.15)
Asp	1.51 (1.50)	1.51 (1.50)	1.56 (1.50)	1.48 (1.50)	1.49 (1.50)
Cys	0.11 (0.14)	0.14 (0.14)	0.14 (0.14)	0.12 (0.14)	0.13 (0.14)

¹CON, control; G1, Gly supplied at 60% of CON and replaced with Glu; G2, Gly supplied at 20% of CON and replaced with Glu; T1, Gly supplied at 60% of CON and replaced with Thr; T2 Gly supplied at 20% of CON and replaced with Thr.

²Calculated nutrient contents are shown in parentheses.

Serial slaughter

Prior to implementing dietary treatments, an additional seven barrows with similar BW were slaughtered to determine initial body composition. At the end of the 21-d experimental period and during two consecutive days, pigs were slaughtered for the determination of physical body composition and carcass and viscera N and AA concentrations. Pigs were fasted for 15 h before they were weighed and a blood sample (10 mL) was collected via the orbital sinus into heparinized vials prior to electrical stunning, after which pigs were exsanguinated via severing major blood vessels in the neck. The heart, lungs, spleen, pancreas, kidneys, liver (drained gall bladder), and empty bladder were removed and weighed together, while the kidneys and liver were also weighed individually. The gastrointestinal tract was weighed separately after the contents were removed. The viscera fraction (including all aforementioned organs) and the carcass were placed in separate plastic bags per pig and stored at −20 °C for 2 wk before grinding. Whole carcasses (including head, skin, hair, feet, and hooves) and viscera were ground separately, as described previously (Totafurno et al., 2019); subsamples of ground carcass and viscera were collected for freeze drying and subsequent N and AA analyses. Blood samples were centrifuged for 20 min at 1,500 × g and 4 °C, plasma was aspirated, placed in microcentrifuge tubes, and frozen at −20 °C until further analysis.

From each pig, one skin sample (4 × 4 cm) was collected from the ham, superficial to the coxal joint, processed, and embedded as described previously (Kolarsick et al., 2011; Silva et al., in press). The Animal Health Histology Lab (University of Guelph, Guelph, ON, Canada) embedded each sample in paraffin, sliced and mounted the sections on microscope slides, and stained the sections using Picrosirius red for the visualization of collagen I and III fibers (Junqueira et al., 1979). Bright field microscopy (Leica Microsystems Model: DMR, Concord, ON, Canada) was used to estimate skin collagen abundance using 10× objective lens. Five pictures were taken per sample and analyzed using ImageJ software (Version 1.50 i; Rich and Whittaker, 2005; Schneider et al., 2012; Silva et al., in press).

Chemical analyses

Approximately, 100 g of subsampled feed from each diet was shipped to Evonik Nutrition & Care GmbH Analytical Services (Frankfurt, Germany) and analyzed for total AA contents by ion-exchange chromatography coupled with post-column derivatization with ninhydrin. AA were oxidized with performic acid, which was neutralized with sodium metabisulfite (Llames and Fontanie, 1994; AOAC, 2006; method 982.30). Diet samples were also sent to SGS Agrifood Laboratories (Guelph, ON, Canada) for dry matter (DM) (AOAC, 1997; method 930.15), P and Ca (AOAC, 1997; method 985.01), and N (via total combustion; LECO-FP 428; LECO Instruments Ltd., Mississauga, ON, Canada; AOAC, 1997; method 990.03) analyses. Samples of freeze-dried carcass and viscera were also sent to SGS Agrifood Laboratories (Guelph, ON, Canada) for DM and N analyses and to Evonik Nutrition & Care GmbH Analytical Services (Frankfurt, Germany) for total AA analyses (only for CON, G2, and T2), as described for the diets. Plasma free AA concentrations were analyzed according to Boogers et al. (2008) using Ultra Performance Liquid Chromatography and Empower Chromatography Data Software (Waters Corporation, Milford, CT).

Calculations and statistical analysis

Nitrogen retention in carcass and viscera were calculated as the difference in N contents in carcass and viscera for pigs slaughtered at the end and beginning of the experimental period, divided by the period length. The apparent efficiency of using standardized ileal digestible (SID) essential AA for protein deposition above maintenance (EAA_efficiency;%) was calculated as:

$${\displaystyle \begin{array}{l}{\displaystyle EAAefficiency=\frac{EAAretained}{SIDEAAint-\frac{EAAmaint}{EAAmainteff}}\times 100}\end{array}}$$

where EAA_retained is the increment of essential AA retained above the initial group, SID EAA_int is the estimated intake of dietary SID essential AA (g/d), EAA_maint are the losses of essential AA from the gastrointestinal tract and skin and hair relative to DM intake and metabolic BW (BW^0.75), respectively (NRC, 2012), and EAA_{maint eff} is the efficiency of using dietary SID essential AA for maintenance in growing pigs (NRC, 2012; Mansilla et al., 2017).

All statistical analyses were conducted using Proc GLIMMIX of SAS 9 (SAS Inst. Inc., Cary, NC) with dietary treatment as a fixed effect and pig as a random effect; initial BW was used as a covariate for final BW. When a significant treatment effect was detected, differences among individual means were assessed using the Tukey–Kramer post hoc test. For growth performance, physical body composition, and whole body N utilization, the main effect of N source was also tested (T1 + T2 vs. G1 + G2). Effects were considered significant at P < 0.05 and a trend when 0.05 ≤ P ≤ 0.10.

Results

The analyzed CP and AA of the experimental diets were generally comparable to calculated values (Tables 1 and 2). The exception was for Pro, where the analyzed values were approximately 27% less than calculated values for all diets; this was likely due to systematic error in Pro analysis. All pigs used in this experiment remained healthy throughout the study and readily consumed the experimental diets.

There were no differences in final BW or average daily feed intake (ADFI) over the 21-d experimental period among the treatment groups (Table 3). Pigs fed diets supplemented with Glu (G1 and G2) had greater average daily gain (ADG) (P < 0.05), G:F (P = 0.060), and final carcass weight (P = 0.050) compared with those fed diets supplemented with Thr (T1 and T2). No other treatment effects were observed for physical body composition, including for viscera, gastrointestinal tract, liver, and kidney weights at the end of the experimental period (day 21). SID N intake was greater for pigs fed G2 vs. those fed T1 (P < 0.05); intermediate N intakes were observed for CON, G1, and T2. Pigs fed diets supplemented with Glu had greater digestible N intakes than those fed diets supplemented with Thr (P < 0.05). The N retention in carcass and viscera and the apparent N utilization efficiency for N retention were not influenced by dietary treatment.

Table 3.

Growth performance, physical body composition, and N utilization of nursery pigs fed low CP diets with reduced concentrations of Gly and Ser and supplemental Thr or Glu

	Dietary treatment1						P-value
	CON	G1	G2	T1	T2	SEM2	Dietary treatment3	N-source4
Initial BW, kg	15.1	15.3	15.1	15.1	14.9
Final BW, kg	25.5	25.6	25.7	24.9	25.3	0.3	0.800	0.142
ADG; wk 1 to 3	497	500	508	469	488	14	0.800	0.041
ADFI; wk 1 to 3	874	879	878	873	875	13	0.488	0.353
G:F; wk 1 to 3	0.57	0.57	0.58	0.54	0.56	0.01	0.240	0.060
Physical body composition5
Carcass, kg	20.9	21.2	21.1	20.2	20.4	0.3	0.616	0.050
Viscera, g	3,073	2,938	3,148	3,030	3,235	80	0.757	0.470
Gastrointestinal tract, g	1,549	1,524	1,666	1,672	1,675	61	0.237	0.263
Liver, g	435	437	453	460	441	17	0.694	0.896
Kidneys, g	115	126	125	132	121	6	0.729	0.898
N utilization6
N intake, g/d7	14.3ab	14.9ab	15.0a	14.1b	14.2ab	0.2	0.031	0.002
Carcass N Retention, g/d	10.0	10.7	10.3	8.7	9.6	0.6	0.240	0.364
Viscera N Retention, g/d8	0.7	0.6	0.8	0.6	0.8	0.1	0.246	0.668
Carcass + viscera N retention, g/d	10.7	11.3	11.1	9.3	10.4	0.6	0.267	0.406
Apparent N utilization efficiency,%	74.3	75.7	73.5	66.3	73.2	3.5	0.419	0.789

¹CON, control; G1, Gly supplied at 60% of CON and replaced with Glu; G2, Gly supplied at 20% of CON and replaced with Glu; T1, Gly supplied at 60% of CON and replaced with Thr; T2 Gly supplied at 20% of CON and replaced with Thr.

²Maximum value of the standard error of the means.

³ P-value for one-way ANOVA.

⁴ P-value for the effect of N source (G1+ G2 vs. T1 + T2).

⁵Physical composition for pigs at the end of the experimental period (i.e., day 21).

⁶N utilization calculated over the entire experimental period (i.e., 21 d); DM basis.

⁷SID N intake (NRC, 2012).

⁸Visceral fraction included heart, lungs, spleen, pancreas, kidneys, liver (drained gall bladder), and empty bladder and gastrointestinal tract.

^a,bMeans followed by different superscripts in the same row are different according to a Tukey’s multiple range test (P < 0.05).

Pigs fed CON, G1, and G2 had lower plasma concentrations of Thr than those fed T1 and T2 (P < 0.05) and pigs fed T1 had lower plasma concentrations of Thr than those fed T2 (P < 0.05; Table 4). Pigs fed diets with Gly and Ser reduced to 20% of the CON diet (i.e., G2 and T2) had lower plasma concentrations of Gly than those fed CON or diets with Gly and Ser reduced to 60% of the CON diet (i.e., G1 and T1; P < 0.05). Dietary N source did not affect plasma AA concentration, except pigs fed Thr-supplemented diets had greater plasma Thr concentration than those fed Glu-supplemented diets (P < 0.001; data not shown). The AA profile of deposited protein in the carcass was not influenced by dietary treatment (Table 5). Pigs fed the T2 diet had greater retention of Thr in viscera protein vs. pigs fed CON or G2 diets (P < 0.05) and pigs fed the G2 diet had greater retention of Glu in viscera protein than pigs fed CON or T2 diets (P < 0.05; Table 6). Pigs fed G2 had greater Arg retention in visceral protein than pigs fed CON or T2 (P < 0.05). Pigs fed G2 had greater retention of Met in visceral protein than pigs fed CON (P < 0.05), while intermediate values were observed for T2. Pigs fed G2 or T2 diets had greater Phe retention in visceral protein than pigs fed CON and pigs fed G2 had lower Phe retention in visceral protein than pigs fed T2 (P < 0.05).

Table 4.

Plasma AA concentrations (μM) on day 21 for nursery pigs fed low CP diets with reduced concentrations of Gly and Ser and supplemental Thr or Glu

	Dietary Treatment1						P-value
	CON	G1	G2	T1	T2	SEM2	Dietary treatment3
Essential AA
Arg	182	176	184	211	274	83	0.914
His	70	58	67	81	61	7	0.138
Ile	86	76	78	77	73	6	0.633
Leu	123	123	123	121	116	7	0.954
Lys	227	198	149	188	163	19	0.061
Met	46	56	50	62	49	5	0.149
Phe	55	53	49	50	52	3	0.604
Thr	457c	416c	334c	2,927b	4,978a	247	<0.001
Trp	48	45	50	54	58	5	0.273
Val	261	252	246	260	254	11	0.869
NEAA
Ala	527	572	549	592	568	39	0.806
Asn	27	41	37	44	43	12	0.850
Asp	4	9	10	8	6	3	0.386
Cys	4	4	4	4	5	1	0.601
Gln	352	396	424	436	379	75	0.934
Glu	168	216	210	231	213	18	0.158
Gly	2,593a	2,062a	1,224b	2,036a	1,478b	158	<0.001
Pro	197	229	213	217	216	27	0.949
Ser	150	117	92	141	117	34	0.775
Tyr	66	57	60	66	53	4	0.186

¹CON, control; G1, Gly supplied at 60% of CON and replaced with Glu; G2, Gly supplied at 20% of CON and replaced with Glu; T1, Gly supplied at 60% of CON and replaced with Thr; T2 Gly supplied at 20% of CON and replaced with Thr.

²Maximum value of the standard error of the means.

³ P-value for one-way ANOVA.

^a-cMeans followed by different superscripts in the same row are different according to a Tukey’s multiple range test (P < 0.05).

Table 5.

The AA profile (g/100 g CP) of deposited protein in the carcass of nursery pigs fed low CP diets with reduced concentrations of Gly and Ser and supplemental Thr or Glu

	Dietary treatment1				P-value
	CON	G2	T2	SEM2	Diet3
Essential AA
Arg	6.47	6.45	6.50	0.13	0.966
His	2.80	2.53	2.48	0.13	0.298
Ile	3.54	3.20	2.99	0.16	0.157
Leu	6.40	5.91	5.68	0.24	0.210
Lys	6.94	6.23	6.05	0.28	0.170
Met	2.01	1.77	1.74	0.09	0.171
Phe	3.33	3.16	3.03	0.10	0.201
Thr	3.50	3.18	3.67	0.13	0.136
Trp	0.85	0.75	0.73	0.05	0.341
Val	4.23	3.99	3.81	0.12	0.164
NEAA
Ala	6.26	6.41	6.45	0.13	0.602
Asp	7.76	7.30	7.21	0.21	0.253
Cys	0.93	0.85	0.86	0.05	0.502
Glu	12.80	12.20	11.99	0.33	0.297
Gly	9.27	10.32	11.26	0.69	0.243
Pro	6.17	6.81	7.34	0.41	0.243
Ser	3.59	3.42	3.41	0.08	0.304

¹CON, control; G2, Gly supplied at 20% of CON and replaced with Glu; T2 Gly supplied at 20% of CON and replaced with Thr.

²Maximum value of the standard error of the means.

³ P-value for one-way ANOVA.

Table 6.

The AA profile (g/100 g CP) of deposited protein in the viscera of nursery pigs fed low CP diets with reduced concentrations of Gly and Ser and supplemental Thr or Glu¹

	Dietary treatment2				P-value
	CON	G2	T2	SEM3	Diet4
Essential AA
Arg	5.91b	6.48a	5.97b	0.01	0.021
His	2.27	2.15	2.41	0.13	0.434
Ile	2.98	3.53	3.51	0.13	0.056
Leu	6.97	7.11	7.42	0.19	0.338
Lys	6.05	6.31	5.85	0.24	0.454
Met	1.65b	1.96a	1.82ab	0.06	0.042
Phe	3.59c	3.94b	4.28a	0.10	0.002
Thr	3.60b	3.70b	4.75a	0.12	0.004
Trp	0.87	0.96	1.02	0.03	0.081
Val	4.67	4.90	5.16	0.13	0.081
NEAA
Ala	6.15	6.12	6.05	0.18	0.921
Asp	7.77	8.07	8.06	0.16	0.421
Cys	1.31ab	1.39a	1.17b	0.05	0.040
Glu	11.32c	12.51a	11.95b	0.20	0.026
Gly	8.16	8.14	7.18	0.44	0.227
Pro	5.56	5.43	5.72	0.32	0.823
Ser	3.81	3.84	3.87	0.08	0.878

¹Visceral fraction included heart, lungs, spleen, pancreas, kidneys, liver (drained gall bladder), and empty bladder and gastrointestinal tract.

²CON, control; G2, Gly supplied at 20% of CON and replaced with Glu; T2 Gly supplied at 20% of CON and replaced with Thr.

³Maximum value of the standard error of the means.

⁴ P-value for one-way ANOVA.

^a-cMeans followed by different superscripts in the same row are different according to a Tukey’s multiple range test (P < 0.05).

The apparent utilization efficiency of SID Thr for protein deposition in carcass plus viscera was greater for pigs fed the CON and G2 diets vs. those fed T2 (P < 0.001); no other treatment effects were observed for apparent AA utilization efficiencies for protein deposition in carcass plus viscera (Table 7). Only pigs fed T1 had skin collagen abundance not different from CON; pigs fed G1, G2, and T2 had reduced skin collagen abundance compared with CON and T1 (P < 0.01; Figure 1).

Table 7.

The apparent utilization efficiency of SID AA for whole body (carcass + viscera) protein deposition for nursery pigs fed low CP diets with reduced concentrations of Gly and Ser and supplemental Thr or Glu

		Dietary treatment1				P-value
		CON	G2	T2	SEM2	Diet3
	NRC, 2012 4
Arg	127.0	129.0	146.2	143.8	7.9	0.277
His	86.4	73.7	75.4	73.9	5.6	0.973
Ile	65.7	63.1	65.5	61.0	4.2	0.750
Leu	64.9	57.5	60.4	57.9	3.7	0.833
Lys	64.8	60.6	62.1	59.4	3.8	0.891
Met	63.1	40.5	41.0	39.6	2.4	0.927
Met + Cys	52.1	49.4	50.4	48.8	3.0	0.929
Phe	58.0	58.0	62.5	60.0	3.6	0.678
Thr	67.1	56.7a	58.6a	15.1b	2.9	<0.001
Val	69.1	63.1	67.6	64.4	3.9	0.714

¹CON, control; G2, Gly supplied at 20% of CON and replaced with Glu; T2 Gly supplied at 20% of CON and replaced with Thr.

²Maximum value of the standard error of the means.

³ P-value for one-way ANOVA.

⁴Biological maximum efficiency of AA deposition above maintenance for growing pigs according to the NRC (2012).

^a,bMeans followed by different superscripts in the same row are different according to a Tukey’s multiple range test (P < 0.05).

Figure 1.

Skin collagen abundance of nursery pigs fed low CP diets with reduced concentrations of Gly and Ser and supplemental Thr or Glu. CON, control; G1, Gly supplied at 60% of CON and replaced with Glu; G2, Gly supplied at 20% of CON and replaced with Glu; T1, Gly supplied at 60% of CON and replaced with Thr; T2 Gly supplied at 20% of CON and replaced with Thr.

Discussion

The objective of the present study was to explore the effect of reduced dietary Gly and Ser in low CP diets with either supplemental Thr or Glu on growth performance, protein retention, and skin collagen abundance of pigs between 15 and 25 kg BW. Providing supplemental dietary Thr is a potential means to supply Gly indirectly via the Thr dehydrogenase pathway (Le Floc’h et al., 1995) as crystalline Gly is not available for commercial use (Silva et al., in press). Indeed, when Gly and Ser were reduced to 60% of the CON diet, Thr but not Glu supplementation restored skin collagen abundance not different from pigs fed a diet with Gly and Ser supplemented to the same concentration as found in whole-body protein (i.e., CON). When dietary Gly and Ser were reduced to an extremely low level of 20% of the CON diet, Thr supplementation was not sufficient to rescue skin collagen abundance, despite increasing plasma Thr concentrations. Liver mitochondrial Thr dehydrogenase contributes 80% of Thr oxidation in pigs fed diets with low (0.68%) or high (0.81%) concentrations of Thr (Ballevre et al., 1990; Le Floc’h et al., 1997). In the current study, however, the Thr-supplemented diets contained 1.59% and 2.34% Thr, which could alter the partitioning of Thr between the Thr dehydrogenase and Thr dehydratase pathways, the latter of which does not produce Gly. In addition, the endogenous synthesis of Gly may have been hindered by substrate inhibition of Thr dehydrogenase (Cleland, 1979), particularly at the highest level of dietary Thr supplementation (2.34% analyzed Thr), which was in excess. Furthermore, the extent of Gly reduction in the diets with Gly and Ser reduced to 20% of CON may simply have been too extreme for Thr dehydrogenase to replenish Gly supply for skin collagen synthesis. Finally, the 3-wk experimental period could be insufficient to allow Thr dehydrogenase to adapt to high concentrations of Thr, as enzymes involved in AA catabolism have relatively long half-lives (Millward, 2003). One or a combination of these possibilities may explain why skin collagen abundance was rescued for pigs fed reduced CP diets with Thr supplementation at 2.8 × but not 4.1 × estimated requirements to replace N from Gly and Ser.

Glycine is an important element in animal metabolism including for the synthesis of purines, glutathione, polyphyrins, choline, creatine, and collagen protein (Wang et al., 2013, Li and Wu, 2018). Collagen enables the skin to resist mechanical pressures and also aids in rapid wound healing (Meyer et al., 1982). Thirty-three percent of AA in collagen protein are Gly (Gregg and Rogers, 1986), and skin has the highest collagen content by weight (74%; Schultz, 2011). Therefore, skin collagen abundance could be used as a sensitive measure of Gly adequacy and the availability of Gly for metabolic fates beyond muscle protein synthesis in growing pigs. Despite evidence indicating that the de novo synthesis of Gly is highly conserved in pigs and humans fed low CP diets (Gibson et al., 2002; Mansilla et al., 2017), it appears that the de novo synthesis of Gly is insufficient for nursery pigs fed low CP diets as skin collagen abundance was reduced (Silva et al., in press). In the aforementioned study, however, direct dietary supplementation of Gly restored skin collagen abundance (Silva et al., in press). In suckling piglets, oral dosing with Gly, above that present in sow’s milk, improved jejunal villus height and piglet ADG (Fan et al., 2019). Together with the current study, these outcomes imply that if Gly supply is insufficient for growth and secondary indicators of Gly adequacy, other metabolic pathways may also be perturbed, depending on how each pathway is prioritized. Determining Gly kinetics among these other metabolic fates, however, requires the use of isotopes and was beyond the scope of the current study.

Reducing dietary Gly and Ser to 60% and 20% of CON and replacing the N with Glu did not affect pig growth performance, but replacing the N with Thr reduced ADG, G:F, and final BW. Wang et al. (2010) reported that excess dietary Thr (1.24%) had detrimental effects on intestinal mucosal barrier function and growth performance of nursery pigs. Previous research suggests that the feed efficiency of pigs fed reduced-CP diets could be restored with Gly, but not Glu supplementation (in addition to supplementation with select essential AA; Powell et al., 2011; Hou et al., 2015). Others have also demonstrated that essential AA are not efficient in meeting the pig’s needs for nonspecific N, with growth performance also being negatively impacted (Heger et al., 1998). Conversely, in the current study, N retention in carcass plus viscera and N utilization efficiency were not affected when Gly and Ser N were replaced with either Glu or Thr, indicating that the differences in body weight gains were likely due to deposition of other chemical components in the carcass (e.g., lipid). With protein deposition in carcass plus viscera not different among treatment groups and presumably limited equally by N intake (i.e., indicated by apparent N utilization efficiency), it is feasible that excess Glu would contribute more energy for lipid deposition vs. Thr. Indeed, Glu oxidation yields 22.5 Adenosine triphosphate (ATP) per mol (after accounting for thermogenesis of urea production), while Thr oxidation yields 19 ATP per mol (Bender, 2012). Formulating diets on a net energy basis does not account for the fates of energy-providing nutrients during postabsorptive metabolism within the pig and would require complex models to determine the partitioning of specific nutrients among energy-yielding reactions and protein synthesis.

Despite no differences in the AA profile of carcass protein, the Thr deposited in visceral protein increased with Thr supplementation. In the small intestine, 16% of Thr is utilized for mucin protein synthesis, which is important for gut integrity (Bengmark and Jeppsson, 1995; Schaart et al., 2005). Indeed, using tracer kinetics, it was determined that approximately 30% of dietary essential AA were metabolized during first pass by intestinal mucosa, but for Thr, first-pass metabolism reached 60% of dietary supply (Stoll et al., 1998). In the current study, however, there was no improvement in visceral N retention when Thr was supplemented to replace 80% of dietary Gly and Ser, indicating that the surplus Thr present in the gastrointestinal tract did not result in greater Thr incorporation in visceral protein over the duration of the study. Alternatively, when cleaning the gastrointestinal tract, the mucin layer may have been dislodged and rinsed away, though mucin concentration was not measured in the current study. Furthermore, visceral Thr concentration increased with dietary supplementation of Thr and visceral Glu concentration increased with dietary supplementation of Glu, which could be explained by incomplete rinsing of digesta and blood from the gastrointestinal tract on the slaughter day or short-term storage of these AA in the mucosa of the small intestine.

In summary, using Glu as an N source when Gly and Ser were reduced to 60% and 20% of CON in low CP diets maintained ADG for pigs between 15 and 25 kg BW, whereas using Thr as an N source reduced ADG. When dietary Gly and Ser were supplied at 60% of CON, only Thr supplementation rescued skin collagen abundance, while there was no effect on protein retention in carcass and viscera. Therefore, supplemental Thr at 2.8 × estimated requirements is not adequate to replace N from Gly and Ser in low CP diets fed to late nursery pigs, despite supporting skin collagen abundance as a secondary indicator of Gly status, while the long-term effects of feeding such diets on carcass and meat quality should also be considered.

Glossary

Abbreviations

AA

amino acids

ADG

average daily gain

ADFI

average daily feed intake

ATP

adenosine triphosphate

BW

body weight

CAA

crystalline amino acids

CP

crude protein

DM

dry matter

NEAA

nonessential amino acids

SID

standardized ileal digestible

Conflict of interest statement

J.K.H. is an employee of Evonik Nutrition & Care GmbH. The remaining authors declare that they have no conflicts of interest.