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Journal of Animal Science - Animal Nutrition

Impact of improving dietary amino acid balance for lactating sows on efficiency of dietary amino acid utilization and transcript abundance of genes encoding lysine transporters in mammary tissue12

 

This article in JAS

  1. Vol. 94 No. 11, p. 4654-4665
     
    Received: June 03, 2016
    Accepted: Aug 20, 2016
    Published: October 13, 2016


    3 Corresponding author(s): trottier@msu.edu
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doi:10.2527/jas.2016-0697
  1. L. Huber*,
  2. C. F. M. de Lange*,
  3. C. W. Ernst,
  4. U. Krogh and
  5. N. L. Trottier 3
  1. * Department of Animal and Poultry Science, University of Guelph, Guelph, Ontario, N1G 2W1, Canada
     Department of Animal Science, Michigan State University, East Lansing 48824
     Department of Animal Science, Aarhus University, Foulum, Denmark

Abstract

Lactating multiparous Yorkshire sows (n = 64) were used in 2 experiments to test the hypothesis that reducing dietary CP intake and improving AA balance through crystalline AA (CAA) supplementation improves apparent dietary AA utilization efficiency for milk production and increases transcript abundance of genes encoding Lys transporter proteins in mammary tissue. In Exp. 1, 40 sows were assigned to 1 of 4 diets: 1) high CP (HCP; 16.0% CP, as-fed basis; analyzed concentration), 2) medium-high CP (MHCP; 15.7% CP), 3) medium-low CP (MLCP; 14.3% CP), and 4) low CP (LCP; 13.2% CP). The HCP diet was formulated using soybean meal and corn as the only Lys sources. The reduced-CP diets contained CAA to meet estimated requirements for essential AA that became progressively limiting with reduction in CP concentration, that is, Lys, Ile, Met + Cys, Thr, Trp, and Val. Dietary standardized ileal digestible (SID) Lys concentration was 80% of the estimated requirement. In Exp. 2, 24 sows were assigned to the HCP or LCP diets. In Exp. 1, blood samples were postprandially collected 15 h on d 3, 7, 14, and 18 of lactation and utilization efficiency of dietary AA for milk production was calculated during early (d 3 to 7) and peak (d 14 to 18) lactation. Efficiency values were estimated from daily SID AA intakes and milk AA yield, with corrections for maternal AA requirement for maintenance and AA contribution from body protein losses. In Exp. 2, mammary tissue was biopsied on d 4 and 14 of lactation to determine the mRNA abundance of genes encoding Lys transporter proteins. In peak lactation, Lys, Thr, Trp, and Val utilization efficiency increased with decreasing dietary CP (linear for Trp and Val, P < 0.05; in sows fed the MHCP diet vs. sows fed the HCP diet for Lys and Thr, P < 0.05). Total essential and nonessential 15-h postprandial serum AA concentrations increased with decreasing dietary CP (linear, P = 0.09 and P < 0.05, respectively), suggesting increased maternal body protein mobilization. Transcript abundance of several genes involved in Lys transport in mammary tissue did not differ between sows fed the LCP and HCP diets. Feeding lactating sows low-CP diets supplemented with CAA increases the efficiency of utilizing dietary Lys, Thr, Trp, and Val for milk protein production but is unrelated to abundance in mRNA of genes encoding Lys transport proteins in the mammary gland. Dietary Lys utilization for milk protein production in lactating sows appears to be optimized when crystalline Lys is included at a minimum of 0.10% in a diet containing 15.70% CP.



INTRODUCTION

The use of crystalline AA (CAA) allows for improvement of dietary AA balances in sow lactation diets and increases global N utilization efficiency for milk production (Huber et al., 2015), which is likely due to improvements in utilization efficiency of individual dietary AA. Except for Lys, the utilization efficiency of individual AA have not been empirically estimated (NRC, 2012), even though precise dietary formulations that are based on factorial approaches are dependent on valid AA efficiency estimates.

In addition to the need for generating such values, an understanding of mechanisms that may regulate AA utilization for milk protein production would allow the design of strategies to control AA efficiencies. The mammary gland is equipped with regulatory mechanisms that appear to modulate the extraction of circulating AA to meet the demands of milk protein synthesis (Bequette et al., 2000; Guan et al., 2004). Certain AA transporters act as nutrient sensors and can respond to chronic AA limitation by increasing gene expression and cellular AA extraction capacity, also known as adaptive regulation (as reviewed by Hyde et al., 2003). Circulating AA may also compete for transporters, which inhibits AA entry into the cell (Shennan et al., 1994) and may decrease AA (Lys) utilization efficiency for milk production when feeding diets with AA imbalances.

We hypothesized that lowering dietary CP concentration with incremental CAA supplementation meeting requirements of limiting AA would increase the apparent utilization efficiency of individual dietary AA for milk production and transcript abundance of genes encoding Lys transporter proteins in mammary tissue during early and peak lactation.

The objectives were to 1) estimate apparent dietary AA utilization efficiency for milk production in sows fed 4 diets containing reduced concentrations of CP and incremental levels of CAA and 2) determine the mRNA abundance of genes encoding Lys transporter proteins in mammary tissue during early and peak lactation.


MATERIALS AND METHODS

The experimental protocol was approved by the Michigan State University Institutional Animal Care and Use Committee (Animal use form number 09/12-176-00).

Animals and Feeding

Two experiments were conducted with 40 and 24 purebred multiparous (parity 2+) Yorkshire sows. Experiment 1 was conducted in 3 replicates with 12 or 16 sows per replicate and Exp. 2 was conducted in 2 replicates with 12 sows per replicate. Sows were selected at d 110 of gestation, blocked by parity, and randomly assigned to 1 of 4 dietary treatments in Exp. 1 or 1 of 2 dietary treatments in Exp. 2. Sows were housed in conventional farrowing crates, and litter sizes were standardized to 11 piglets within the first 24 h of birth via cross-fostering with the objective of weaning a minimum of 10 piglets in each litter. After farrowing, sows were progressively fed according to the NRC (2012) feed intake curve to reach a feed intake of 7.0 kg/d at d 12 of lactation and an average of 6.0 kg/d over the 21-d lactation period. Feed was provided in 3 equal meals per day, and intakes as well as refusals were measured daily. Water was freely available to both sows and piglets. Piglet and sow BW measurements in Exp. 1 are outlined in Huber et al. (2015). In Exp. 2, individual piglet BW were collected on d 1, 3, 7, 14, and 21 and sows were weighed on d 1 and 21. Injection of iron and surgical castration were conducted on d 1 and 7, respectively. Piglets were not supplied with creep feed.

Dietary Treatments

Ingredient and nutrient composition of diets for Exp. 1 are outlined in Huber et al. (2015) and presented in Table 1 for Exp. 2. Diets were formulated to satisfy estimated nutrient requirements for AA and other nutrients according to the NRC (2012) model based on the following parameters and previously reported piglet performance for this swine herd (Manjarín et al., 2012): mean sow BW at farrowing of 210 kg, litter size of 10 piglets, mean piglet gain of 282 g/d during a 21-d lactation period, mean feed intake of 6 kg/d, and minimum sow BW change with a protein:lipid energy ratio adjusted to near zero. In Exp. 1, sows were assigned 1 of 4 dietary treatments containing different CP concentrations (analyzed, as-fed basis), which are described in detail in Huber et al. (2015): 1) high CP (HCP; 16.0% CP), 2) medium-high CP (MHCP; 15.7% CP), 3) medium-low CP (MLCP; 14.3% CP), and 4) low CP (LCP; 13.2% CP). In Exp. 2, sows were assigned 1 of 2 dietary treatments containing 2 different CP concentrations (analyzed, as-fed basis): HCP (17.6% CP) or LCP (14.6% CP). The HCP diets were formulated using soybean meal and corn as the only sources of Lys. The other 3 diets had a decreasing inclusion of soybean meal, whereas CAA were added to meet the requirement of AA that became limiting (i.e., Lys, Ile, Met, Thr, Trp, and Val) as soybean meal was reduced; the LCP diet met the predicted N requirements (NRC, 2012). All diets were formulated to contain the same concentration of standardized ileal digestible (SID) Lys of 0.74%, a value corresponding to 20% below NRC (2012) predicted requirements, which was tested in Huber et al. (2015).


View Full Table | Close Full ViewTable 1.

Ingredient composition and nutrient content of experimental diets used in Exp. 2 (as-fed basis)

 
Item HCP1 LCP2
Ingredient composition, % (as-fed basis)
    Corn 64.47 67.39
    Soybean meal, dehulled, 48% CP 23.00 11.73
    Choice white grease 3.33 4.09
    Soy hulls 0 6.50
    Sugar food products3 5.00 5.00
    l-Lys HCl 0 0.33
    l-Ile 0 0.14
    dl-Met 0.03 0.14
    l-Thr 0.09 0.25
    l-Trp 0.01 0.07
    l-Val 0.16 0.36
    Vitamin premix4 0.25 0.25
    Mineral premix5 0.125 0.125
    Sow pack6 0.25 0.25
    Se 2007 0.068 0.068
    Sodium chloride 0.50 0.50
    Limestone 1.17 1.11
    Monocalcium phosphate 1.35 1.50
    Total 100.00 100.00
Calculated nutrient content8
    NE, kcal/kg 2,600 2,600
    CP, % 16.58 12.89
    SID9 Lys, % 0.74 0.74
    SID Ile, % 0.59 0.53
    SID Met, % 0.26 0.26
    SID Met + Cys, % 0.50 0.50
    SID Thr, % 0.59 0.59
    SID Trp, % 0.18 0.18
    SID Val, % 0.81 0.81
    SID Arg, % 0.95 0.63
    SID His, % 0.39 0.28
    SID Leu, % 1.27 0.97
    SID Phe, % 0.7 0.49
    SID Phe + Tyr, % 1.15 0.82
    STTD10 P, % 0.44 0.44
    Total Ca, % 0.88 0.89
    Fermentable fiber, % 10.25 10.00
    SID Lys/NE, g/Mcal 2.83 2.85
Analyzed nutrient content,11 %
    CP 17.62 14.63
    Lys 0.91 (0.86) 0.93 (0.84)
    Ile 0.73 (0.67) 0.68 (0.60)
    Met 0.29 (0.30) 0.28 (0.35)
    Met + Cys 0.55 (0.58) 0.50 (0.57)
    Thr 0.68 (0.70) 0.66 (0.68)
    Trp 0.22 (0.20) 0.22 (0.19)
    Val 0.93 (0.92) 0.98 (0.91)
    Arg 1.08 (1.03) 0.84 (0.69)
    His 0.45 (0.45) 0.36 (0.33)
    Leu 1.58 (1.45) 1.27 (1.12)
    Phe 0.86 (0.80) 0.68 (0.58)
    Phe + Tyr 1.44 (1.34) 1.17 (0.97)
1HCP = high CP (17.6% CP; as-fed basis; analyzed contents).
2LCP = low CP (14.6% CP).
3Sugar food product (International Ingredient Corporation, St. Louis, MO) to increase diet palatability; supplied, per kg, 2,842 kcal NE, 0.05% fermentable fiber, and 1.00% CP.
4Provided the following amounts of vitamins per kilogram of diet: 3,000 IU vitamin A, 300 IU vitamin D3, 20 IU vitamin E, 1 mg menadione (vitamin K), 20 μg vitamin B12, 4 mg riboflavin, 10 mg d-pantothenic acid, and 15 mg niacin.
5Provided the following amounts of trace minerals per kilogram of diet: 640 mg Fe as FeCO3, 260 mg Zn as ZnO, 36 mg Mn as MnO2, 20 mg Cu as CuCl2, and 0.58 mg I as ethylenediamine dihydriodide.
6Provided the following amounts of vitamins per kilogram of diet: 0.10 mg biotin, 250 mg choline as choline chloride, 0.75 mg folic acid, 2.3 mg vitamin B6 as pyridoxine HCl, 10 IU vitamin E as dl-tocopherol acetate, 90 μg Cr as chromium picolinate, and 23 mg carnitine as l-carnitine.
7Provided 0.5 mg of selenium per kg of diet as Na2Se.
8Based on nutrient content in feed ingredients according to the NRC (2012).
9SID = standardized ileal digestible (NRC, 2012).
10STTD = standardized total tract digestible (NRC, 2012).
11Calculated AA contents are shown in parentheses.

Blood Sampling, Serum Analysis, and Mammary Biopsy

In Exp. 1, blood was collected in the morning at 0700 h and 15 h after distributing the evening meal on d 3, 7, 14, and 18 of lactation by single jugular venipuncture. Blood samples (8.5 mL) were collected in vacutainers (RST Tube with Thrombin-Based Clot Activator and Polymer Gel; BD Medical Supplies, Franklin Lakes, NJ) and centrifuged for 20 min at 1,500 × g at 4°C. Serum was aliquoted in microcentrifuge tubes and stored at −20°C.

Frozen serum samples for AA analysis were selected from 6 sows per treatment whose feed intake was most similar to expected intake (i.e., according to the NRC [2012] predicted feed intake curve), to reduce possible variation in serum AA concentration due to feed intake. Samples were shipped to the Agricultural Experiment Station Chemical Laboratories (University of Missouri – Columbia, Columbia, MO) on dry ice for free AA analysis according to Spackman et al. (1958) and Deyl et al. (1986).

In Exp. 2, mammary biopsies were conducted as previously outlined (Kirkwood et al., 2007) on d 4 and 14 of lactation. On the biopsy day, sows were randomly assigned to 2 postfeeding treatment groups, with 6 sows per group: a 2-h postfeeding group fed a standardized meal size of 1.5 kg at 0600 h and a 15-h postfeeding group that received a meal on the evening before the biopsy day but were not fed immediately before the biopsy was collected. One piece of mammary tissue (between approximately 800 and 1,000 mg) was biopsied and rapidly dissected into 2 portions, which were immediately flash frozen in liquid N and subsequently stored at −80°C. All sows received 1 mL/100 kg BW Banamine (Merck Animal Health, Summit, NJ) immediately after mammary biopsy and at 24 and 48 h after biopsy, per institutional animal care and use protocol. Piglets were returned once sows were able to stand on their own, and sows were fed either the remainder of the meal (2-h postfeeding treatment group) or the entire meal (15-h postfeeding treatment group).

Gene Expression Analysis

Approximately 100 mg of flash-frozen tissue was homogenized and total RNA was purified according to manufacturer’s instructions (mirVana miRNA Isolation Kit; Ambion, Life Technologies Inc., Grand Island, NY). The RNA quality and quantity were determined (Agilent Bioanalyzer 2100 with the RNA 6000 Nano Labchip; Agilent Technologies, Palo Alto, CA), and integrity values averaged 9.5 with the minimum value being 8.3; 1 sample from the HCP group was removed because of a low integrity value of 4.7. Complementary DNA was synthesized using a High-Capacity cDNA Reverse Transcription Kit according to manufacturer’s instructions (Applied Biosystems, Life Technologies Inc., Grand Island, NY) and 2 μg of total RNA from each sample as the template. For each reaction, 0.5 μL ribonuclease inhibitor (Recombinant RNasin Ribonuclease Inhibitor; Promega Corporation, Madison, WI) was included. Final cDNA concentration was measured by spectrophotometry (NanoDrop 1000; Thermo Scientific, Waltham, MA) and then diluted to a working stock solution of 10 ng/μL and stored at −20°C.

The mRNA abundance was quantified using TaqMan Gene Expression Assays (Applied Biosystems, Life Technologies Inc.). Commercially available porcine gene-specific assays were used for SLC7A1 (CAT-1), SLC7A2 (CAT-2b), SLC7A7 (y+LAT1), SLC7A9 (b0,+AT), SLC6A14 (ATB0,+), SLC3A1 (rBAT), and SLC3A2 (4F2hc) and the reference genes VAPB and RSP23. Custom TaqMan assays were designed for SLC7A6 (y+LAT2) and the reference gene MRPL39 (see Table 2). Appropriate reference genes were selected based on the lowest average expression stability values reported by Manjarín et al. (2012). Assays for SLC7A1, SLC7A6, SLC7A7, SLC7A9, SLC6A14, SLC3A2, RPS23, and MRPL39 were not designed to span exon–exon junctions; however, the RNA extraction method contained a DNA digestion step to eliminate genomic DNA.


View Full Table | Close Full ViewTable 2.

Assay information (TaqMan Gene Expression Assays) for candidate and reference genes

 
Gene Protein Assay ID1 Custom assay
SLC6A14 ATB0,+ [Ss]03376400_u1
SLC7A9 b0,+AT [Ss]03386787_u1
SLC7A1 CAT-1 [Ss]03379090_u1
SLC7A2 CAT-2b [Ss]03389657_m1
SLC7A7 Y+LAT1 [Ss]03389674_g1
SLC7A6 Y+LAT2 CX064558.12
SLC3A1 rBAT [Ss]03818894_s1
SLC3A2 4F2hc [Ss]03377481_u1
VAPB Vesicle-associated membrane protein-associated protein B/C [Ss]03390804_m1
RSP23 Ribosomal protein 23 [Ss]03392259_g1
MRPL39 Mitochondrial ribosomal protein L39 Exon 23
1Assay identification number (ID) for TaqMan Gene Expression Assays (Applied Biosystems, Life Technologies Inc., Grand Island, NY).
2Accession number corresponding to the cDNA sequence tag deposited in the National Center for Biotechnology Information (Bethesda, MD) database used to construct a custom TaqMan assay.
3The human equivalent (NM_017446.3) was blasted against the pig genome, and a region of high alignment (exon 2) was used to construct a custom TaqMan assay.

Reverse transcription quantitative PCR reactions were performed using the ABI StepOne Plus Real-Time PCR System (Applied Biosystems, Life Technologies Inc.). The amplification program included 2 initial steps (50°C for 2 min and 95°C for 20 s) followed by 40 cycles (step 3; 95°C for 1 s and 60°C for 20 s). Reactions were conducted in duplicate with the 8 genes of interest and 3 reference genes analyzed per sample and 4 samples per plate.

Calculations and Statistical Analysis

Amino Acid Efficiencies.

The SID intakes for individual AA in Exp. 1 were calculated based on analyzed dietary AA concentration and estimated SID of AA-containing ingredients according to the NRC (2012). Individual apparent dietary AA utilization efficiencies for milk protein production (KAA; Eq. [1]) were calculated from estimated milk production using litter growth rate (LGR) and size between d 3 and 7 and between d 14 and 18 for early and peak lactation, respectively (NRC, 2012, Eq. [8–72]; Huber et al., 2015), and estimated AA content of milk protein (NRC, 2012, Table 2–11). Dietary AA intake was estimated using AA digestibility values according to the NRC (2012), accounting for maternal maintenance requirements (NRC, 2012, Eq. [8–40] and [8–41]), and the contribution of maternal body protein mobilization based on sow BW change (NRC, 2012; Huber et al., 2015). The efficiency of using dietary AA for maintenance (i.e., kmain; NRC, 2012, Table 2–12) and of using mobilized body AA for milk protein production (i.e., 0.87) were also included in the calculation. Because of the high variability in sow BW measurements (Huber et al., 2015), the average sow BW changes for the first (d 0 to 7) and last week (d 14 to 21), respectively, were used to calculate daily AA contribution from mobilized maternal body protein during early (d 3 to 7) and peak (d 14 to 18) lactation periods. The calculations were completed for each of the essential AA.

Transcript Abundance.

Candidate gene expression was normalized in Exp. 2 according to Manjarín et al. (2011, 2012; Eq. [2]):in which ∆Ctijk is the normalized target gene expression for the jth sow within the ith stage of lactation and the kth diet and CtCijk and CtRijk are the threshold cycle (Ct) values for candidate (C) and reference (R) genes, respectively.

Diet and stage of lactation effects were represented as fold changes by back transforming the estimated mean difference in Ct (ÄÄCt) between LCP- and HCP-fed sows and between d 4 and 14 of lactation, respectively, for each transporter of interest using the expression fold change = 2ÄÄCt (Livak and Schmittgen, 2001). When the interaction between dietary treatment and day of lactation was significant, data are presented for LCP- and HCP-fed sows on d 4 and 14 of lactation. The 95% confidence intervals of mean differences were also back transformed to provide confidence interval for the estimated fold change (Steibel et al., 2009). Fold changes with transformed confidence intervals spanning the value of 1.0 were considered not significant at α = 5%.

Statistical analyses for Exp. 1 were conducted as in Huber et al. (2015) using the mixed model procedure of SAS (SAS Inst. Inc., Cary, NC) with the repeated measure of day of lactation.

Linear and quadratic contrasts were constructed in Exp. 1 to compare dietary CP concentration in early and peak lactation. Multiple contrasts were also constructed to compare each diet with the subsequent reduced dietary CP concentration in early and peak lactation, respectively.

Statistics for sow and litter performance data in Exp. 2 were conducted using the mixed model procedure of SAS with fixed effects of dietary treatment and replicate and using the repeated measure of day of lactation. Regression variables included parity, sow initial BW for sow BW change over the 21-d lactation period, and piglet initial BW and litter size for piglet ADG and LGR. When appropriate, a reduced model was used. Statistics for gene expression were conducted in a similar manner using the mixed model procedure of SAS. Neither the effect of state (i.e., 15 versus 2 h after feeding) nor the interactive effect of state and dietary treatment was significant for any AA transporter gene expression; therefore, a reduced model with the fixed effects of dietary CP concentration, stage of lactation, and the interaction of the aforementioned was used.


RESULTS

Diet composition, nutrient analysis, and lactation performance data were previously published for Exp. 1 (Huber et al., 2015). Diet composition and nutrient analysis are presented in Table 1 for Exp. 2; calculated and analyzed nutrient values aligned well. In Exp. 1, 1 sow from each of the HCP and MLCP diets was removed due to illness, and therefore, any data collected from these sows were removed before analysis.

There was no effect of stage of lactation or interactive effect of dietary CP concentration and stage of lactation on individual apparent AA utilization efficiency for milk production in Exp. 1. Because sow N utilization was most sensitive to dietary treatment (Huber et al., 2015) and calculated AA utilization efficiency data were less variable in peak lactation, efficiency data are presented for both early and peak lactation. In early lactation, Leu utilization efficiency for milk production tended to increase (linear, P = 0.077; Table 3) with decreasing dietary CP concentration. In peak lactation, Arg, His, Ile, Leu, Phe, Phe + Tyr, and Trp utilization efficiency increased (linear, P < 0.05) with decreasing dietary CP concentration. Valine utilization efficiency tended to increase (linear, P = 0.061) and Met utilization efficiency tended to decrease (linear, P = 0.063, and quadratic, P < 0.05) with decreasing dietary CP. When applying specific comparisons between treatment diets in peak lactation, apparent dietary AA utilization efficiencies for milk production were as follows: sows fed the MHCP diet, compared with sows fed the HCP diet, had higher utilization efficiency for all essential AA (EAA; P < 0.05) except Trp, which remained unaffected; sows fed the MLCP diet, compared with sows fed the MHCP diet, had lower utilization efficiency for Met (P < 0.05); and sows fed the LCP diet, compared with sows fed the MLCP diet, had higher utilization efficiency for Arg, His, Leu, Phe, and Trp (P < 0.05).


View Full Table | Close Full ViewTable 3.

Apparent dietary AA utilization efficiency for milk protein production in sows fed high-CP (16.0%) or reduced-CP diets between d 3 and 7 of lactation (early lactation) and between d 14 and 18 of lactation (peak lactation) in Exp. 1

 
Diet1
P-value
Item HCP MHCP MLCP LCP NRC, 20122 SEM3 Linear Quadratic HCP vs. MHCP MHCP vs. MLCP MLCP vs. LCP
No. of sows 9 10 9 10
Early lactation (d 3–7)
    Arg 35.4 36.8 40.2 39.5 8.1 0.669 0.895 0.903 0.767 0.948
    His 58.4 65.7 68.6 73.6 8.6 0.212 0.896 0.552 0.807 0.682
    Ile 48.9 53.0 51.3 50.6 5.2 0.884 0.645 0.579 0.819 0.923
    Leu 46.4 53.9 53.7 59.6 4.8 0.077 0.877 0.283 0.981 0.389
    Lys 67.7 71.0 64.3 62.6 7.1 0.490 0.724 0.742 0.506 0.865
    Met 55.7 60.1 48.4 44.2 6.3 0.114 0.503 0.626 0.201 0.644
    Met + Cys 59.2 66.0 58.9 58.4 5.8 0.712 0.530 0.411 0.391 0.949
    Phe 42.0 48.2 49.6 53.1 4.9 0.126 0.787 0.379 0.848 0.614
    Phe + Tyr 55.3 60.4 61.2 60.4 7.4 0.630 0.695 0.629 0.943 0.944
    Thr 58.6 64.0 56.7 54.1 5.4 0.398 0.469 0.489 0.350 0.738
    Trp 49.9 54.3 48.7 51.8 3.5 0.995 0.850 0.377 0.264 0.536
    Val 44.5 46.6 42.6 39.4 4.6 0.352 0.995 0.748 0.541 0.623
    N 55.3 60.4 61.2 60.4 7.4 0.630 0.695 0.629 0.943 0.944
Peak lactation (d 14–18)
    Arg 31.7 41.0 43.8 53.6 81.6 2.8 <0.0001 0.919 0.021 0.470 0.015
    His 51.8 64.8 67.8 81.5 72.2 3.0 <0.0001 0.919 0.008 0.513 0.006
    Ile 43.1 50.6 51.0 54.3 69.8 2.3 0.002 0.356 0.022 0.909 0.281
    Leu 41.0 49.9 50.8 59.5 72.3 2.2 <0.0001 0.959 0.006 0.765 0.007
    Lys 59.9 68.4 65.4 67.6 67.0 3.0 0.123 0.274 0.042 0.453 0.580
    Met 50.0 58.9 48.8 47.0 67.5 2.3 0.063 0.028 0.010 0.003 0.622
    Met + Cys 52.6 61.6 57.1 57.9 66.2 2.4 0.284 0.091 0.011 0.185 0.810
    Phe 37.0 45.8 48.5 56.9 73.3 2.3 <0.0001 0.930 0.009 0.398 0.012
    Phe + Tyr 48.9 59.6 60.3 68.2 70.5 2.9 <0.0001 0.608 0.012 0.866 0.060
    Thr 52.6 60.0 57.4 59.3 76.4 2.4 0.103 0.248 0.032 0.428 0.559
    Trp4 44.2 48.8 46.6 52.3 67.4 2.0 0.017 0.758 0.108 0.426 0.045
    Val 39.3 45.0 44.5 45.0 58.3 2.0 0.061 0.566 0.041 0.855 0.875
    N 48.9 59.6 60.3 68.2 75.9 2.9 <0.0001 0.608 0.012 0.866 0.060
1HCP = high CP (16.0% CP; as-fed basis; analyzed contents); MHCP = medium-high CP (15.7% CP); MLCP = medium-low CP (14.3% CP); LCP = low CP (14.6% CP).
2Biological maximum AA utilization efficiency for milk protein production according to the NRC (2012).
3Standard error of the mean based on repeated measures analysis (largest value across treatments).
4Tryptophan utilization efficiency tended (P = 0.077) to be less in peak lactation than in early lactation.

Because there was no interactive effect of dietary CP concentration and day of lactation for serum AA concentration, only the main effects of dietary CP concentration and day of lactation are presented. Serum Lys and Thr concentrations decreased over the 21-d lactation period (linear, P < 0.0001, and quadratic, P < 0.0001; Fig. 1), as did serum His, Val, Phe and Tyr concentrations (linear, P < 0.01). Other EAA and nonessential AA (NEAA) showed similar declines in AA concentration across the 21-d lactation period (data not shown).

Figure 1.
Figure 1.

Postabsorptive serum concentration (μmol/L) of Lys, Thr, His, Val, Phe, and Tyr (panel A, B, C, D, E, and F, respectively) for sows fed high-CP (16.0%) or reduced-CP diets over the 21-d lactation period. Main effect of stage of lactation: P < 0.05. HCP = high CP (16.0% CP; as-fed basis; analyzed contents); MHCP = medium-high CP (15.7% CP); MLCP = medium-low CP (14.3% CP); LCP = low CP (14.6% CP); L = linear; Q = quadratic.

 

For EAA, serum Met, Thr, and Val concentrations increased (linear, P < 0.05) and total serum concentration of EAA tended to increase with decreasing dietary CP concentration (linear, P = 0.092; Table 4). Serum His concentration increased between 16.0 and 15.7% CP and decreased between 14.3 and 13.2% CP (quadratic, P < 0.05; Table 4).


View Full Table | Close Full ViewTable 4.

Serum concentration of essential, nonessential and selected AA metabolites for sows fed high-CP (16.0%) or reduced-CP diets in Exp. 11

 
Diet2
P-value
Item HCP MHCP MLCP LCP SEM3 Linear Quadratic
No. of sows 6 6 6 6
EAA,4 μmol/L
    Arg 212 198 213 196 14 0.542 0.892
    His 92 109 107 99 7 0.431 0.047
    Ile 96 94 92 94 5 0.682 0.719
    Leu 197 199 206 186 10 0.573 0.261
    Lys 137 119 123 153 17 0.455 0.107
    Met 46 53 56 57 4 0.026 0.407
    Phe 79 76 75 71 5 0.211 0.896
    Thr 175 203 215 250 10 <0.0001 0.722
    Trp 124 127 128 119 11 0.491 0.200
    Val 318 318 386 411 24 0.001 0.523
    Total EAA 1,489 1,494 1,605 1,642 88 0.092 0.818
NEAA,5 μmol/L
    Ala 575 556 617 679 32 0.017 0.221
    Asn 45 48 48 44 5 0.806 0.389
    Asp 31 30 32 33 5 0.390 0.499
    Cys 21 17 31 27 3 0.032 0.937
    Gln 565 550 605 562 38 0.786 0.706
    Glu 233 204 223 269 31 0.155 0.059
    Gly 894 1,006 1,091 1,053 58 0.042 0.211
    Pro 287 287 306 288 17 0.767 0.603
    Ser 134 129 132 123 10 0.329 0.809
    Tyr 96 105 94 69 7 0.006 0.021
    Total NEAA 2,847 2,919 3,176 3,145 116 0.038 0.665
Metabolite, μmol/L
    3-Methyl-histine 38 37 35 35 5 0.568 0.839
1Blood was sampled 15 h after the evening meal.
2HCP = high CP (16.0% CP; as-fed basis; analyzed contents); MHCP = medium-high CP (15.7% CP); MLCP = medium-low CP (14.3% CP); LCP = low CP (14.6% CP).
3Standard error of the mean based on repeated measures analysis (largest value across treatments).
4EAA = essential AA.
5NEAA = nonessential AA.

For NEAA, serum Ala, Cys, and Gly and total NEAA concentrations increased with decreasing dietary CP concentration (linear, P < 0.05; Table 4). Serum Glu concentration tended to increase (quadratic, P = 0.059) and serum Tyr decreased with decreasing dietary CP concentration (linear and quadratic, P < 0.05).

Lactation performance for Exp. 2 is presented in Table 5. Sow initial BW was greater in sows fed the LCP diet than in sows fed the HCP diet (P < 0.05) and litter size at weaning tended to be lower for sows fed the LCP diet than for sows fed the HCP diet (P = 0.052). Sow ADFI, litter performance (including piglet ADG and LGR), and sow BW change were not influenced by dietary treatment.


View Full Table | Close Full ViewTable 5.

Overall 21-d lactation performance for sows fed high- or low-CP diets in Exp. 2

 
Item HCP1 LCP2 SEM3 P-value4
No. of sows 12 11
Sow initial BW,5 kg 241 261 5 0.005
Sow ADFI, kg/d, as-fed basis 5.14 5.10 0.17 0.828
Litter size at weaning 10.1 9.4 0.3 0.052
Litter growth rate,6 kg/d 1.86 2.18 0.19 0.139
Piglet ADG,6 g/d 186 221 21 0.130
Sow BW change,6 kg −8.7 −9.1 3.4 0.935
1HCP = high CP (17.6% CP; as-fed basis; analyzed contents).
2LCP = low CP (14.6% CP).
3Standard error of the mean based on repeated measures analysis (largest value across treatments).
4Main effect of diet.
5Postfarrowing weight, d 1.
6Over the 21-d lactation period.

The mRNA abundance of candidate genes was not influenced by dietary state (i.e., 15 vs. 2 h after feeding; data not shown) or the main effect of dietary CP concentration (Fig. 2). The main effect of stage of lactation did not influence mRNA abundance of candidate genes, except for SLC6A14, which was 28% lower on d 14 compared with d 4 of lactation (P < 0.05; Fig. 3A). The interactive effect of dietary CP concentration and stage of lactation did not influence mammary mRNA abundance of candidate genes except for SLC7A6, whose expression was 32% lower in HCP- versus LCP-fed sows on d 4 of lactation, but no difference was observed between dietary treatments on d 14 of lactation (P < 0.05; Fig. 3B).

Figure 2.
Figure 2.

The mRNA abundance presented as fold changes (±SEM) for genes encoding Lys transporters in mammary tissue of lactating sows fed low-CP (17.6% CP) diets relative to sows fed high-CP (14.6% CP) diets.

 
Figure 3.
Figure 3.

The mRNA abundance presented as fold changes (±SEM) for SLC6A14 mRNA encoding the ATB+,0 transporter protein on d 4 relative to d 14 of lactation (P < 0.05; panel A) and SLC7A6 mRNA encoding the Y+LAT2 transporter protein for sows fed low-CP (17.6% CP) diets relative to sows fed high-CP (14.6% CP) diets on d 4 (P < 0.05) and 14 (P > 0.05) of lactation (panel B).

 

DISCUSSION

In a previous study (Huber et al., 2015), we reported that an improvement in dietary AA balance increased N utilization efficiency for milk protein production. The overall goal of the present study was to characterize the impact of dietary AA balance on utilization efficiency of dietary Lys and other AA for milk protein production. Except for Lys (for the MHCP, MLCP, and LCP diets) and His (for the LCP diet only) in peak lactation, our estimated efficiency values for EAA were lower than the biological maximum efficiency values published by the NRC (2012) for groups of sows. Nonetheless, except for Lys and Thr, there was a clear linear increase in utilization efficiency of all EAA for milk production in peak lactation, regardless if the SID EAA were supplied equally across dietary treatments (i.e., Met + Cys, Trp, and Val) or decreased concomitantly with reduced inclusion of soybean meal (i.e., Ile, Arg, His, Leu, and Phe), which is likely due to increased milk protein production (Huber et al., 2015) and increased milk protein production as well as reduced inclusion level, respectively. For Met, a quadratic decline in utilization efficiency was observed, which may be attributed to increasing dietary inclusion levels of crystalline Met and increased conversion of Met to Cys when reducing dietary CP concentration. A substantial improvement in Lys utilization efficiency was observed in sows fed the MHCP diet relative to those fed HCP diets in peak lactation. Therefore, a combination of both protein-bound and crystalline Lys at a minimum inclusion level of 0.10% in the diet appears optimal for lactating sows to maximize the efficiency of dietary Lys utilization, in the current study.

In a previous study (Huber et al., 2015), N utilization efficiency was most influenced by dietary AA balance in sows during peak lactation rather than early lactation. In the current study, the utilization efficiency of individual dietary AA was not influenced by stage of lactation. Variability associated with feed intake, BW change, and milk output was less in peak lactation, allowing for a more precise assessment of the effect of dietary AA balance on apparent AA utilization efficiencies.

Individual AA utilization efficiencies for milk production were calculated as outlined by the NRC (2012) and included corrections for the contribution of AA from maternal BW change and maternal maintenance requirements (NRC, 2012). Observed utilization efficiencies can be used to assess adequacy of dietary AA and N supply, where greater utilization efficiency values (i.e., values approaching the biological maximum) indicate a limiting supply of AA or N relative to demands for milk protein production. As N retention was reduced in sows fed the LCP diet during peak lactation (Huber et al., 2015), an AA or N may have been limiting maternal N retention as true milk protein yield increased. In sows fed the LCP diet during peak lactation, His, Lys, Phe + Tyr, and N were used at calculated efficiencies of 81.5, 67.6, 68.2, and 68.2%, respectively, which were approaching maximum biological efficiencies reported by the NRC (2012) of 72.2, 67.0, 70.5, and 75.9%, respectively, for populations of sows. When comparing observed with maximum biological efficiencies, His appears to be the first limiting AA in the LCP diet. It should be noted, however, that the NRC (2012) value for His is based on very limited empirical data, as is the case for several of the EAA (e.g., Phe, Phe + Tyr) and N. Additionally, His can be released from other body pools (i.e., mammary tissue [Trottier et al., 1997] and hemoglobin and carnosine [Heger et al., 2007]) making His utilization efficiency difficult to interpret. The observed efficiency value for Lys was similar for the MHCP, MLP, and LCP diets, suggesting that Lys remained limiting across all diets.

Serum AA concentrations did not mirror changes in AA efficiency. For both EAA and NEAA, serum concentrations somewhat increased as dietary CP was reduced. The increase in EAA corresponded mainly to those AA that were supplemented in their crystalline form. This was unexpected, given that blood samples were collected 15 h after feeding, but may indicate differential catabolism of AA based on time relative to feeding and AA supply (i.e., crystalline versus protein bound). The increase in NEAA was a result of Ala and Gly alone. Increased serum concentrations of Ala, Gly, Gln, and Ser seem to be key indicators of net AA mobilization from body protein pools (Reynolds et al., 1994). Therefore, feeding the LCP diet may have increased maternal body protein mobilization, which supports the reduced maternal N retention in LCP-fed sows (Huber et al., 2015). Only for Tyr, the concentration in serum linearly decreased with the reduction of dietary CP, which may simply reflect a reduction in intake of Tyr and Phe + Tyr. For several other EAA (i.e., Ile, Arg, Leu, and Phe), intake was also reduced with lowering dietary CP, whereas for these EAA, their serum concentrations were similar across dietary CP concentrations. It is possible that Phe was limiting for maternal body protein synthesis and conversion of Phe to Tyr was reduced.

Serum concentrations of all EAA and NEAA generally decreased as lactation advanced despite significant increase in feed and AA intake, hence likely reflecting a greater AA utilization as milk production increased. In addition, dietary AA may have been oversupplied relative to AA demand for milk protein production in early lactation. As milk production in early lactation was 30% less than in peak lactation and sows were fed according to the (energy) intake curve of the NRC (2012; Huber et al., 2015), dietary AA may have been supplied in excess relative to milk protein demand. There is also the possibility that the contribution of AA from uterine protein degradation after farrowing may be substantial (Theil, 2015). The uterus increases by approximately 250 g of protein between d 0 and 114 of gestation (NRC, 2012), with Lys composing 6.92% of uterine protein (NRC, 2012). It has been suggested that considerable amounts of Lys are released within the first 7 d after farrowing (Theil, 2015). At least for Lys and Thr in the current study, the d-3 serum concentrations (Fig. 1) were likely influential to the linear and quadratic decline across the 21-d lactation period. Without measurements of AA flux, dynamics of AA utilization and AA fate cannot be determined.

The relative differences in litter performance (i.e., piglet ADG and LGR) between sows fed the HCP and LCP diets were similar in Exp. 2 and Exp. 1. Therefore, the sows in Exp. 2 were reasonably representative of sows fed the HCP and LCP diets in Exp. 1 and can be used to explore the dietary effects on the expression of genes encoding cationic AA transporters in the mammary gland.

One of the key determinants of intracellular mammary AA availability for milk protein synthesis is the transport of AA across the blood facing basolateral membrane of mammary epithelial cells (Shennan et al., 1994; Calvert and Shennan, 1996). Several transporters have been characterized as playing a role in Lys transport, including Y+LAT1, Y+LAT2, CAT-1, CAT-2b, ATB0,+, and b0,+AT. Of those, the CAT-1 and CAT-2b proteins are involved in Arg transport across endothelial cells for nitric oxide production (Hatzoglou et al., 2004). Serum AA profiles may impact AA uptake by the mammary cells. For example, Guan et al. (2002) demonstrated that mammary net uptake of Lys is reduced in instances of excess dietary and circulating Val. Additionally, Manjarín et al. (2012) demonstrated increased Lys transport efficiency into the mammary gland in early lactation when crystalline Lys was included at 0.24% of the diet, and despite increased Lys extraction efficiency, there was no change in abundance of mRNA for genes encoding the transporters CAT-1, CAT-2b, ATB0,+, Y+LAT2, and b0,+AT. In that study, mammary tissue was sampled 12 to 14 h after feeding, voluntary feed intake was relatively low, and sow BW loss was high. In our studies, sow BW loss was notably less, milk production was higher, and mammary tissue was sampled at both 15 and 2 h after feeding. Neither the nutritional state (i.e., postprandial versus fed) nor dietary AA balance influenced mRNA abundance of mammary genes encoding for Lys transporter proteins in the present study. Therefore, expression of genes encoding AA transporters do not appear to be influenced by the availability of dietary AA in the short term (i.e., within 2 h of consuming a meal) or long term or by AA balance, at least within the dietary CP concentrations tested in this study. We and others did not quantify transporter protein abundance in response to diet in lactating sows; hence, the role of transporter activity (protein abundance) per se cannot be ruled out.

Because the expression of most genes involved in Lys transport were not significantly influenced by the balance of dietary AA, another mechanism must be responsible for the improved utilization efficiency with decreasing dietary CP for AA that were supplied equally across diets (i.e., Lys, Thr, Trp, and Val). An improvement in AA balance may reduce competitive inhibition among structurally similar AA that rely on the same transporters for transfer into mammary epithelial cells. Such AA antagonism has been demonstrated clearly for Lys and Arg in sow mammary tissue explants (Hurley et al., 2000) or for Lys and branched-chain AA (BCAA) in lactating rats (Shennan et al., 1994; Calvert and Shennan, 1996). In the HCP diet, Lys uptake may have been limited by the relatively high intake of Arg, Gln, Leu, Phe, Ala, and Asn, which have been shown in vitro to compete with Lys for access to AA transporters (Shennan et al., 1994). This competitive inhibition mechanism may explain the improvement in apparent dietary Lys utilization efficiency when comparing sows fed the HCP diet with sows fed the MHCP diet. Whether the parallel increase in utilization efficiency for other dietary EAA, namely Thr, Trp, and Val, may be related to improved transport for those EAA either via decreased competitive inhibition or AA transporter activity remains to be addressed in future studies. Dietary AA that are unable to enter tissues for protein synthesis due to AA imbalance will eventually be catabolized, which may shed some light regarding the lower dietary Lys utilization efficiency in the HCP-fed sows versus MHCP-fed sows. The dynamic nature of AA in blood relative to meal consumption and consequences on AA balance and AA delivery to the mammary epithelial cells deserves further consideration.

The single-limiting AA theory may be too simplistic for milk protein synthesis as previously suggested in lactating dairy cows (Appuhamy et al., 2011; Arriola Apelo et al., 2014) because the physiological effect of AA may influence protein synthesis (and degradation) in ways other than providing substrates for protein synthesis. In the current study, although BCAA were greater in the high-CP diets, circulating Val concentrations were greater in the reduced-CP diets. It has been previously demonstrated that excess BCAA in the mammary gland may be broken down and utilized for energy or for synthesis of NEAA, lactose, or lipids, which may also be a driver for milk production (Richert et al., 1998; Li et al., 2009). Also, Leu may stimulate protein synthesis in both muscle tissue and mammary epithelial cells (Garlick, 2005; Moshel et al., 2006). With these notions in mind, the competition and the control of dietary AA partitioning between maternal and milk protein pools warrant further elucidation. The partitioning of dietary AA has implications for first parity sows during lactation, as the maternal protein pool is still expanding and feed intake is generally reduced.

In conclusion, feeding lactating sows low-CP diets supplemented with CAA to optimize AA balance improves the efficiency of using dietary Lys, Thr, Trp, and Val for estimated milk protein production, despite being supplied in equal amounts across dietary protein levels. Dietary Lys utilization in lactating sows appears to be optimized for milk protein production when crystalline Lys is included at a minimum of 0.10% in a diet with 15.70% CP concentration. The expression of genes encoding Lys transporter proteins in the mammary gland does not appear to play a role the improvement in apparent dietary AA utilization efficiency associated with dietary AA balance. It remains unknown whether Lys transporter protein abundance is related to AA utilization efficiency or AA balance. Other mechanisms such as competitive inhibition may be modulating the utilization of dietary AA for milk protein production.

 

References

Footnotes


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