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Journal of Animal Science - Animal Nutrition

An insufficient glucose supply causes reduced lactose synthesis in lactating dairy cows fed rice straw instead of alfalfa hay1

 

This article in JAS

  1. Vol. 94 No. 11, p. 4771-4780
     
    Received: May 04, 2016
    Accepted: Aug 04, 2016
    Published: October 13, 2016


    2 Corresponding author(s): liujx@zju.edu.cn
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doi:10.2527/jas.2016-0603
  1. B. Wang*†,
  2. F.-Q. Zhao*‡,
  3. B.-X. Zhang* and
  4. J.-X. Liu 2*
  1. * Institute of Dairy Science, MoE Key Laboratory of Molecular Animal Nutrition, College of Animal Sciences, Zhejiang University, Hangzhou 310058, P.R. China
     Beijing Key Laboratory for Dairy Cow Nutrition, College of Animal Science and Technology, Beijing University of Agriculture, Beijing 102206, P.R. China
     Laboratory of Lactation and Metabolic Physiology, Department of Animal and Veterinary Sciences, University of Vermont, 211 Terrill Building, 570 Main Street, Burlington, VT 05505

Abstract

The objective of the present study was to investigate the nutrient availability for milk production in the mammary gland of lactating cows fed different forage-based diets. The 3 diets contained 30% corn stover (CS), 30% rice straw (RS), or 23% alfalfa hay plus 7% Chinese wild rye hay (AH) as a forage source. All diets contained 15% of DM as corn silage and 55% of DM as concentrate. The percentage of milk lactose was always lower in the RS-fed cows than in the cows fed AH or CS during the 12-wk feeding trial (P < 0.01). Ruminal propionate concentrations were lower in the RS group than in the AH group (P = 0.03). The ratio of insulin to glucagon in the mammary venous plasma was greater in the AH group than in the CS or RS group (P = 0.04). The abundance of the pyruvate carboxylase mRNA in the liver was lower in the RS group than in the AH or CS group (P = 0.04), and the abundance of mitochondrial phosphoenolpyruvate carboxykinase, IGF-1 receptor, and phosphofructokinase-liver, phosphofructokinase-muscle, and phosphofructokinase-platelet mRNA in the liver were lower in the RS group than in the AH group (P < 0.05). The mammary glucose uptake was greater in the AH-fed cows than in the CS- or RS-fed cows (P = 0.02). The mRNA abundance of the glucose transporters in the mammary gland was similar among the 3 treatments. The mRNA abundance of α-lactalbumin in the mammary gland of the cows fed RS tended to be greater compared with that of the cows fed AH or CS. The milk potassium concentration was greater in the cows fed RS than those fed AH or CS (P < 0.01). In summary, the insufficient ruminal propionate concentrations in the cows fed RS were associated with lower gluconeogenesis in the liver, resulting in the shortage of glucose supply for mammary utilization.



INTRODUCTION

Alfalfa hay (AH), a widely used forage source, is produced in limited amounts in China, where its supply is largely dependent on its increased import from overseas nations (nearly 900 thousand metric tons in 2014). Cereal straws, such as corn stover (CS) and rice straw (RS), are produced abundantly all over the world (Pang et al., 2008) and can be used as alternative forage sources. However, we previously demonstrated (Wang et al., 2014) that the yield and concentration of lactose in milk were reduced in the cows fed RS instead of AH. The reason for this decrease was unclear.

Glucose is the major precursor of lactose synthesis in the mammary gland (Zhao, 2014). Because the mammary gland lacks glucose-6-phosphatase (G6Pase), it cannot synthesize glucose from other precursors (Scott et al., 1976). Therefore, the mammary gland is dependent on the arterial supply for glucose. In ruminants, the main source of glucose is hepatic gluconeogenesis from propionate, which is produced by fermentation in the rumen (Armstrong, 1965). The liver adjusts the rate of gluconeogenesis by changing the expression of key gluconeogenic enzymes, including G6Pase, pyruvate carboxylase (PC), mitochondrial phosphoenolpyruvate carboxykinase (PEPCK-m), and cytosolic phosphoenolpyruvate carboxykinase (PEPCK-c; Graber et al., 2010; Zachut et al., 2013). In the mammary gland, glucose is taken up by facilitative glucose transporters (GLUT) in the mammary epithelial cells (Zhao, 2014).

In mammary epithelial cells, glucose is primarily used in 3 processes: 1) lactose synthesis, 2) glycolysis, and 3) the pentose phosphate pathway (Zhao, 2014). The lactose synthesis pathway occurs in the Golgi vesicles during lactation (Mohammad et al., 2012) through the function of lactose synthase, which consists of the membrane-bound enzyme β-1,4-galactosyl transferase (B4GALT) and the milk protein α-lactalbumin (α-LA; Farrell et al., 2004). Lactose is the major osmotic substance in milk and accounts for approximately 50% of the total osmotic pressure of milk; the other 50% is contributed by milk ions, proteins, citrate, and other substances (Gáspárdy et al., 2004).

The objective of this study was to investigate the limiting factors that resulted in the lower milk lactose synthesis in dairy cows fed RS and CS compared with cows fed AH. We hypothesized that cows fed RS and CS have lower glucose availability to the mammary gland for lactose synthesis compared with the cows fed AH.


MATERIALS AND METHODS

Animals and Experimental Design

All experimental procedures were approved by the Animal Care Committee at Zhejiang University (Hangzhou, China) in accordance with the university’s guidelines for animal research. A detailed description of the experimental design and animal treatments has been previously reported (Wang et al., 2014). Briefly, 3 diets (Table 1) were formulated with 55% of DM as concentrate and 15% of DM as corn silage, but the 3 diets contained different forage sources (DM basis): 1) 23% AH and 7% Chinese wild rye hay, 2) 30% CS, and 3) 30% RS. Thirty Holstein cows (10 cows per group; means of 30.0 kg [SD 3.53] milk yield and 160 d in milk [SD 27.8]; mean parity = 3.4 [SD 1.57]) were randomly allocated to 1 of the 3 dietary treatments and fed for 14 wk, with the first 2 wk used as dietary adaption. The cows were fed and milked 3 times daily, at 0630, 1400, and 2000 h. At the end of experiment, 6 cows with medium milk yield were selected from each group and slaughtered for tissue collections.


View Full Table | Close Full ViewTable 1.

Ingredient and nutrient compositions of the experimental diets based on alfalfa hay (AH), corn stover (CS), or rice straw (RS)

 
Treatment
Item AH CS RS
Dietary ingredient, % of DM
    Ground corn grain 27.0 27.0 27.0
    Wheat bran 5.1 5.1 5.1
    Soybean meal 12.7 12.7 12.7
    Cottonseed meal 4.3 4.3 4.3
    Beet pulp 1.0 0.0 0.0
    Corn silage 15.0 15.0 15.0
    AH 23.0 0.0 0.0
    Chinese wild grass hay 7.0 0.0 0.0
    CS 0.0 30.0 0.0
    RS 0.0 0.0 30.0
    Urea 0.0 1.0 1.0
    Premix1 4.9 4.9 4.9
Nutrient composition, % of DM
    OM 92.0 ± 2.26 91.7 ± 1.69 90.6 ± 1.64
    CP 16.7 ± 0.22 16.2 ± 0.29 16.0 ± 0.46
    NDF 31.1 ± 1.73 36.3 ± 1.11 36.9 ± 1.26
    ADF 18.9 ± 1.07 19.5 ± 2.21 21.9 ± 2.53
    Nonfiber carbohydrate2 40.6 ± 3.29 36.0 ± 2.29 34.6 ± 3.66
    Na+ 0.28 0.25 0.26
    K+ 1.05 1.05 1.14
    Cl 0.49 0.61 0.54
    NEl, Mcal/kg 1.57 1.45 1.43
1Formulated to contain (per kilogram of DM) 174 g of zeolite powder, 1.25 g of yeast, 25 g of mold adsorbent (Solis Mos; Novus International Inc., St. Charles, MO), 21.44 g of KCl, 41.25 g of MgO, 150 g of NaCl, 187.5 g of NaHCO3, 84 g of Ca, 15 g of P, 125,000 IU of vitamin A, 750,000 IU of vitamin D3, 937.5 IU of vitamin E, 1,750 mg of Zn, 17.5 mg of Se, 28.75 mg of I, 375 mg of Fe, 15 mg of Co, 556.5 mg of Mn, and 343.75 mg of Cu.
2Nonfiber carbohydrate = 100 − % NDF − % CP − % ether extract − % ash.

Sampling, Measurements, and Analyses

The milk samples were collected on the third day of each week using milk-sampling devices (Waikato Milking Systems NZ Ltd., Waikato, Hamilton, New Zealand). Bronopol tablets were added to 1 milk sample (milk preservative; D & F Control Systems, San Ramon, CA), and then the samples were stored at 4°C for the subsequent lactose analysis by infrared analysis (Laporte and Paquin, 1999) with a spectrophotometer (Foss-4000; Foss Electric A/S, Hillerød, Denmark). Another fresh milk sample was collected on the third day of the final week, centrifuged at 3,000 × g at 4°C for 15 min, and then stored at −20°C for the subsequent analysis of the 6 major milk proteins [αs1-casein (CN), β-CN, κ-CN, αs2-CN, α-LA, and β-lactoglobulin (LG)] by reverse-phase HPLC (Agilent 1100; Agilent Technologies, Inc., Santa Clara, CA) using the method described by Bobe et al. (1998).

During the final week, the feed and milk samples were collected; the concentrations of Na+, K+, and Cl were measured by using atomic absorption spectrophotometry (DSI-903B; Shanghai Xunda Medical Instrument Co. Ltd., Shanghai, China) following the methods of Roche et al. (2005). The milk cation–anion difference (CAD; mM) was calculated using the equation by Chan et al. (2005):

The samples of rumen fluid were collected 3 h after the morning feeding using an oral stomach tube on d 6 of the final sampling week following the method described by Shen et al. (2012). The pH of the rumen fluid was immediately measured using a portable pH meter (Starter 300; Ohaus Instruments Co. Ltd., Shanghai, China). Then, the samples were placed in liquid N2, transferred to the laboratory, and stored at -20°C for subsequent analysis. Before analysis, the sample was thawed at 4°C and filtered through 2 layers of cheesecloth. The filtrate was then used to analyze the ammonia N and VFA concentrations. The ammonia N concentrations were determined by steam distillation into boric acid and titration with dilute hydrochloric acid. The VFA concentrations were determined using the method described by Hu et al. (2005) using a gas chromatograph (GC-8A; Shimadzu Corp., Kyoto, Japan).

The blood samples from the coccygeal artery and mammary vein were collected from the cows over 2 consecutive days on d 4 and 5 of the final week. The blood samples were collected every 6 h at 0600, 1200, 1800, and 2400 h on d 4 and at 0900, 1500, 2100, and 0300 h on d 5. The blood samples from the jugular vein were collected from the cows at 0900 h (3 h after morning feeding) on d 5. The cows were standing for at least 10 min prior to blood sampling to minimize the fluctuations in blood flow. The blood samples were collected into lithium heparin–containing vacuum tubes (Becton, Dickinson and Company, Franklin Lakes, NJ) and centrifuged at 3,000 × g for 15 min at 4°C. The plasma was pipetted into a new tube and stored at -80°C until further analysis. The plasma glucose concentrations were analyzed at each of the 8 sampling times and from a pooled plasma sample using an Auto Analyzer 7020 instrument (Hitachi High-Technologies Corporation, Tokyo, Japan) with a commercial colorimetric kit, as described by Delamaire and Guinard-Flament (2006). The clearance rate and mammary uptake of plasma glucose as well as the ratio of glucose uptake to the lactose secreted in milk were calculated from the pooled plasma samples based on the mammary plasma flow (MPF, Wang et al., 2016).

The concentrations of insulin (HY-0069), glucagon (HY-060), and IGF-I (HY-082) in the jugular vein, coccygeal artery, and mammary vein were analyzed using commercial kits (Sino-UK. Bio. Technol., Beijing, China). The insulin and glucagon kits used the standard double antibody RIA procedures described by Yalow and Berson (1996), whereas the IGF-1 kit used RIA procedures with ethanol–acetone–acetic acid extraction of the IGF-1 binding proteins, as described by Osgerby et al. (2002)

Analysis of the mRNA Abundance

The samples of the liver and mammary tissues were collected immediately after the animals (n = 6 for each group) were slaughtered (Krueger et al., 2010). Sterile surgical scissors were used to clip approximately 800 mg of samples that were quickly washed 20 times in ice-cold PBS (pH 7.2 to 7.4: NaCl 137 mM, KCl 2.7 mM, Na2HPO4 10 mM, and KH2PO4 2 mM). A portion of the tissue was immediately frozen in liquid N2 and stored at -80°C.

The tissue samples were pulverized in liquid N2 for RNA extraction. The tissue powder (approximately 100 mg) was then extracted with TRIZOL reagent (Invitrogen Corp., Carlsbad, CA) to isolate the total RNA. The cDNA were synthesized from the total RNA by reverse transcription using the PrimeScript first Strand cDNA Synthesis Kit (code number 6110A; Takara, Otsu, Japan). Quantitative real-time PCR was performed using the Applied Biosystems 7500 (Thermo Fisher Scientific Inc.,, Foster City, CA) and the 2x SYBR Premix Ex Taq (Tli RNaseH Plus) kit (code number RR420A; Takara). The PCR conditions were as follows: 1 cycle at 95°C for 10 min, 40 cycles of 95°C for 15 s, and 60°C for 34 s followed by a melting curve program (60 to 95°C). The primers for PC, G6Pase, PEPCK-m, PEPCK-c, IGF-1, the IGF-1 receptor (IGF-1R), B4GALT, α-LA, GLUT1, GLUT3, GLUT8, ubiquitously expressed transcript isoform (UX-T), ribosomal protein-encoding genes (RPS9), and β-actin were adapted from previous studies and are listed in Supplemental Table S1 (see the online version of the article at http://journalofanimalscience.org). The primers for phosphofructokinase-liver (PFK-L), phosphofructokinase-muscle (PFK-M), phosphofructokinase-platelet (PFK-P), and glucose-6-phosphate dehydrogenase (G6PD) were designed and are listed in Supplemental Table S2 (see the online version of the article at http://journalofanimalscience.org).

The relative changes in the mRNA levels for the individual genes were analyzed using the 2−ΔΔCT (in which CT stands for cycle threshold) method (Livak and Schmittgen, 2001), with UX-T, RPS9, and β-actin as internal controls. Beta-actin was excluded as an internal control for the liver samples (Bionaz and Loor, 2007).

Calculations and Statistical Analysis

Glucose utilization by the mammary gland was calculated as follows (Hanigan et al., 1998):in which mammary uptake is expressed in moles per day, arterial–venous difference is expressed in molar concentration, and MPF is expressed in liters per day, andin which mammary clearance rate is expressed in moles per day, arterial–venous difference and venous concentration are expressed in millimolar concentration, and MPF is expressed in liters per hour.

The variance of the data was analyzed using PROC MIXED from SAS (SAS Inst. Inc., Cary, NC). A randomized complete block design with repeated measures was used to analyze the changes in the lactose concentrations over the 14-wk feeding period and the changes in the arterial and venous glucose concentrations throughout an entire day, with the diet, time, and the interaction between the diet and time as the fixed effects and the cows within each diet as the random effect. The statistical model was as follows:in which Yijk is the dependent variable, μ is the overall mean, Bi is the random effect of block i, Tj is the diet effect, TMk is the time effect, TTMjk is the interaction between the diet and time, and Eijk is the error. The covariance structure with the least Akaike information criterion was used for the repeated-measures analysis. The results were reported as the least squares means. For the other data analyses, the time effect and interaction between diet and time were omitted. The main effects were defined as statistically significant at P ≤ 0.05. Trends were accepted at 0.05 < P ≤ 0.10.


RESULTS

Milk Lactose Content and Rumen Fermentation Characteristics

The changes in the milk lactose content over the 14-wk feeding trial are shown in Fig. 1. There were no differences between the 3 groups before the trial. However, after a 2-wk diet adaption, the lactose concentrations over the 12 wk of sampling were always lower in the cows fed the RS diet than those in the cows fed the AH or CS diets, with the exception of wk 5 (P = 0.10), 10 (P = 0.09), and 11 (P = 0.12), when the lactose concentrations in the RS-fed and CS-fed cows were not significantly different.

Figure 1.
Figure 1.

Changes in the milk lactose concentrations (%) of lactating cows fed diets with alfalfa hay (AH), corn stover (CS), or rice straw (RS) as the main forage source. The x-axis represents the weeks during the feeding experiment. The first 2 wk (−2 to −1) was considered the dietary adaptation feeding period. *During the indicated week, the lactose concentrations in the cows fed the RS diet were similar to those in the CS-fed cows (P > 0.05). The other weeks without symbols were different in the comparisons for CS vs. RS (P < 0.05). Across the sampling weeks, the lactose concentrations were similar in AH vs. CS (P > 0.05) but were different for AH vs. RS (P < 0.05).

 

The ruminal propionate concentrations were lower in cows fed RS than in cows fed AH (P = 0.03), with no difference between the AH and CS diets or the CS and RS diets (Table 2). The acetate-to-propionate ratio was higher in the cows fed RS than in the cows fed CS (P < 0.01). The total VFA (P = 0.05) and acetate concentrations (P = 0.04) were greater in the cows fed AH than in the cows fed CS. There were no differences in the rumen pH and butyrate concentrations among the 3 treatments.


View Full Table | Close Full ViewTable 2.

Rumen fermentation characteristics (3 h after the morning feeding) in dairy cows1 fed 3 experimental diets based on alfalfa hay (AH), corn stover (CS), or rice straw (RS) for 14 wk

 
Treatment
Item AH CS RS SEM P-value
pH 6.62 6.34 6.42 0.096 0.15
Ammonia nitrogen, mM 10.4b 13.1a 13.3a 0.69 0.02
Total VFA, mM 81.8a 66.5b 72.3ab 4.91 0.12
Acetate, mM 59.6a 45.6b 54.7ab 4.22 0.10
Propionate, mM 16.4a 15.0ab 13.0b 1.03 0.09
Butyrate, mM 5.82 5.85 4.62 0.60 0.28
Molar proportion, %
    Acetate 73.0ab 67.3b 75.9a 2.02 0.03
    Propionate 19.9ab 23.2a 18.0b 1.39 0.06
    Butyrate 7.07ab 9.52a 6.18b 1.02 0.10
    Acetate:propionate ratio 3.65ab 3.14b 4.22a 0.24 0.02
    Propionate:butyrate ratio 2.87 2.94 2.99 0.26 0.94
a,bMeans in the same row not bearing a common superscript letter are significantly different (P < 0.05).
1The number of cows for each group is 10.

Glucose Utilization in the Mammary Gland

The glucose concentrations in both the artery and the vein throughout an entire day were lower in the RS-fed cows than in the cows fed AH or CS, whereas there was no difference between the AH- and CS-fed cows (Fig. 2). The arterial–venous difference in the glucose concentrations, measured from the pooled blood samples over a 24-h period, was greater in the cows fed RS than in those fed CS (P = 0.03) but was not different between the cows fed AH and those fed CS or RS (Table 3). The mammary glucose uptake was much greater in the cows fed AH than in those fed CS (P < 0.01) or those fed RS (P < 0.01). The clearance rate of glucose was greater in cows fed AH than in those fed CS (P < 0.01) or RS (P = 0.02).

Figure 2.
Figure 2.

Changes in the arterial and venous glucose concentrations of lactating cows fed diets with alfalfa hay (AH), corn stover (CS), or rice straw (RS) as the main forage source throughout an entire day (starting at 0 h).

 

View Full Table | Close Full ViewTable 3.

Average plasma glucose concentrations measured from 8 sampling times over a 24-h period (every 3 h) across mammary gland of dairy cows1 fed 3 experimental diets based on alfalfa hay (AH), corn stover (CS), or rice straw (RS)

 
Treatment
Glucose AH CS RS SEM P-value
Arterial, mM 3.04 2.97 2.89 0.053 0.18
Venous, mM 2.48a 2.46a 2.25b 0.069 0.05
AV difference,2 mM 0.55ab 0.51b 0.64a 0.044 0.13
Uptake,3 mol/d 10.2a 5.58b 5.96b 0.870 <0.01
Clearance rate,4 L/h 171.3a 95.9b 112.8b 15.71 0.01
a,bMeans in the same row not bearing a common superscript letter are significantly different (P < 0.05).
1The number of cows for each group is 10.
2AV = arterial–venous.
3Uptake = AV difference (M) × mammary plasma flow (L/d).
4Clearance rate = AV difference (mM) × mammary plasma flow (L/h)/venous concentration (mM) × 100.

Blood Insulin, Glucagon, and IGF-1 Levels

The concentrations of insulin, glucagon, and IGF-1 in the jugular vein, coccygeal artery, and mammary vein are shown in Table 4. There were no differences in the glucagon concentrations in any of the blood samples among treatments. The insulin concentrations (P < 0.01) in the mammary vein were significantly greater in the cows fed AH than in those fed CS or RS, and in the mammary vein, the ratio of insulin to glucagon (P = 0.05) was also greater in the cows fed AH than in those fed CS or RS. The IGF-1 concentrations (P = 0.05) were greater in the coccygeal artery of the cows fed AH or CS than in those fed RS and tended to be greater in the jugular veins of the cows fed AH (P = 0.08) or CS (P = 0.09) than in those fed RS.


View Full Table | Close Full ViewTable 4.

The concentrations of insulin, glucagon, and IGF-1 in the jugular vein, coccygeal artery, and mammary vein of dairy cows1 fed 3 diets based on alfalfa hay (AH), corn stover (CS), or rice straw (RS)

 
Treatment
Item AH CS RS SEM P-value
Jugular vein
    Insulin, μIU/mL 15.0 16.0 12.5 1.30 0.18
    Glucagon, pg/mL 82.1 82.6 81.5 3.39 0.97
    IGF-1, ng/mL 200.2 198.2 173.5 9.91 0.13
    Molar insulin:glucagon ratio 4.45 4.71 3.81 0.42 0.33
Coccygeal artery
    Insulin, μIU/mL 16.6 16.7 15.6 0.81 0.59
    Glucagon, pg/mL 87.5 92.6 91.8 3.52 0.32
    IGF-1, ng/mL 211.7a 207.8a 181.1b 7.03 0.03
    Molar insulin:glucagon ratio 4.63 4.34 4.04 0.21 0.17
Mammary vein
    Insulin, μIU/mL 17.9a 14.5b 14.2b 0.81 <0.01
    Glucagon, pg/mL 88.5 88.1 85.7 3.49 0.83
    IGF-1, ng/mL 194.6 174.1 196.8 10.54 0.28
    Molar insulin:glucagon ratio 4.95a 4.03b 4.04b 0.28 0.05
a,bMeans in the same row not bearing a common superscript letter are significantly different (P < 0.05).
1The number of cows for each group is 6.

Gene Expression in the Liver and Mammary Gland

The gene expression in the liver is listed in Table 5. The abundance of the PC mRNA was greater in the cows fed AH (P = 0.017) or CS (P = 0.038) than in the cows fed RS, with no difference between AH- and CS-fed cows. The abundance of the PEPCK-m (P = 0.05), PFK-L (P = 0.04), PFK-M (P = 0.03), PFK-P (P = 0.04), and IGF-1R (P = 0.04) mRNA was greater in the cows fed AH compared with those fed RS, with no difference between those fed CS and those fed AH or RS. The abundance of the IGF-1 mRNA tended to be greater in the CS-fed cows (P = 0.07) or RS-fed cows (P = 0.08) compared with the AH-fed cows. There were no differences in the expression of the G6Pase and PEPCK-c mRNA among the 3 groups of cows.


View Full Table | Close Full ViewTable 5.

The mRNA abundance of genes related to gluconeogenesis and glycolysis in the liver of dairy cows1 fed 3 diets based on alfalfa hay (AH), corn stover (CS), or rice straw (RS)

 
Treatment
Genes2 AH CS RS SEM P-value
PC 17.2a 15.2a 6.73b 2.31 0.04
PEPCK-m 2.81a 2.38ab 1.61b 0.35 0.11
PEPCK-c 2.43 2.54 1.63 0.47 0.37
G6Pase 2.73 3.18 3.19 0.83 0.90
PFK-L 0.57a 0.34ab 0.17b 0.11 0.10
PFK-M 0.023a 0.014ab 0.010b 0.0031 0.06
PFK-P 0.081a 0.045ab 0.038b 0.0127 0.08
IGF-1 12.9 13.1 8.1 1.78 0.12
IGF-1R 31.9a 15.6ab 10.3b 6.39 0.09
a,bMeans in the same row not bearing a common superscript letter are significantly different (P < 0.05).
1The number of cows for each group is 6.
2PC = pyruvate carboxylase; PEPCK-m = mitochondrial phosphoenolpyruvate carboxykinase; PEPCK-c = cytosolic phosphoenolpyruvate carboxykinase; G6Pase = glucose-6-phosphatase; PFK-L = phosphofructokinase-liver; PFK-M = phosphofructokinase-muscle; PFK-P = phosphofructokinase-platelet; IGF-1R = IGF-1 receptor.

The abundance of glucose transporters (GLUT1, GLUT3, and GLUT8) and the B4GALT mRNA was similar in the mammary gland from each of the groups (Table 6). However, the abundance of the α-LA mRNA tended to be greater in the mammary gland of the cows fed CS than in those fed AH (P = 0.08).


View Full Table | Close Full ViewTable 6.

The mRNA abundance of genes related to glucose transport and glucose utilization in the mammary gland of dairy cows1 fed 3 diets based on alfalfa hay (AH), corn stover (CS), or rice straw (RS)

 
Treatment
Genes2 AH CS RS SEM P-value
GLUT1 0.099 0.080 0.091 0.0119 0.58
GLUT3 0.20 0.18 0.12 0.036 0.32
GLUT8 0.66 0.67 0.59 0.056 0.56
B4GALT 49.3 43.8 49.0 6.33 0.79
α-LA 58.1a 37.3b 51.9ab 6.73 0.19
a,bMeans in the same row not bearing a common superscript letter tend to be different (0.05 < P < 0. 10).
1The number of cows for each group is 6.
2GLUT = glucose transporter; B4GALT = β-1,4-galactosyl transferase; α-LA = α-lactalbumin.

Milk Protein Profiles and Milk Ions

The milk protein profiles and milk ion concentrations are presented in Table 7. There were no differences in the percentages of κ-CN, αs1-CN, αs2-CN, and α-LA among the 3 dietary treatments; however, the β-CN percentage was lower in the cows fed CS compared with the cows fed AH (P < 0.01) or RS (P < 0.01), and the β-LG percentage was greater in the cows fed CS compared with the cows fed AH (P < 0.01) or RS (P < 0.01). The milk Na+ concentration was higher in the cows fed RS than those fed CS (P = 0.01), and the milk K+ concentration was higher in the cows fed RS compared with the cows fed AH (P = 0.02) or CS (P = 0.05). Therefore, the sum of milk Na+ and K+ concentrations (P < 0.01) and CAD (P = 0.01) were higher in the cows fed RS compared with the cows fed AH or CS, with no difference between the cows fed AH and CS. There was no significant difference in Cl concentration among the 3 groups.


View Full Table | Close Full ViewTable 7.

The milk protein profile and ion concentrations in dairy cows1 fed 3 diets based on alfalfa hay (AH), corn stover (CS), or rice straw (RS)

 
Treatment
Item2 AH CS RS SEM P-value
Milk protein profiles, %
    κ-CN 8.74 7.97 7.52 0.602 0.37
    αs2-CN 11.63 11.18 10.51 0.589 0.42
    αs1-CN 30.28 31.92 32.48 0.835 0.18
    β-CN 34.3a 26.7b 35.4a 0.976 <0.01
    α-LA 5.23x 4.35y 5.00xy 0.360 0.23
    β-LG 9.82b 17.8a 9.07b 1.101 <0.01
Milk ions, mM
    Na+ 18.5ab 17.5b 19.7a 0.58 0.04
    K+ 44.3b 44.8b 47.2a 0.80 0.04
    Cl 27.6 27.4 27.8 0.23 0.44
    Na+ + K+ 62.7b 63.4ab 66.9a 0.84 <0.01
    CAD 35.2b 36.0b 39.1a 0.86 0.01
a,bMeans in the same row not bearing a common superscript letter are significantly different (P < 0.05).
x,yMeans in the same row not bearing a common superscript letter tend to be different (0.05 < P < 0. 10).
1The number of cows for each group is 10.
2CN = casein; α-LA = α-lactalbumin; LG = lactoglobulin; CAD = cation–anion difference ([Na+ + K+ − Cl] mM).


DISCUSSION

The milk lactose content is relatively constant during consecutive milkings from individual cows (Gibson, 1984) and is seldom changed by the diet, with the exception of severe underfeeding (Sutton, 1989). In the current study, when the cows were fed 30% (DM basis) RS as the forage source, the milk yield and lactose yield that has been published in Wang et al. (2014) presented a much lower value, and the lactose content also decreased over the 12-wk feeding period (Fig. 1). This study characterized the whole glucose metabolic flow from the glucose precursor generation, gluconeogenesis, hormone regulation, glucose transport, and metabolism in the mammary gland as well as the relation with output of other milk components in milk.

Propionate in the Rumen

Propionate is produced by microorganism-mediated fermentation in the rumen and is the major substrate of gluconeogenesis in the liver in ruminants (Armstrong, 1965). An increased propionate supply could increase gluconeogenesis (Oba and Allen, 2003). In this study, the RS-fed cows might have a reduced propionate supply in the liver for gluconeogenesis due to the lower ruminal propionate concentration.

Gluconeogenesis in the Liver

Glucose is primarily produced by gluconeogenesis in ruminant animals. Consistent with the reduced propionate substrate supply in the cows fed RS, both the arterial and venous glucose concentrations were significantly reduced in the RS-fed cows compared with the AH- and CS-fed cows (Table 3). The liver is the site of gluconeogenesis, which converts propionate to glucose (Armentano, 1992). The major enzymes in gluconeogenesis include G6Pase, PEPCK-m, PEPCK-c, and PC. It has been shown that the expression of the mRNA for the gluconeogenic enzymes in the liver reflects their activities (Greenfield et al., 2000). In the liver, the levels of the PEPCK-m mRNA increase during the transition period when the gluconeogenesis rate increases (Greenfield et al., 2000); the PC mRNA increases during severe feed restriction in midlactation dairy cattle (Velez and Donkin, 2005). In our study, expression of both the PEPCK-m and PC mRNA was dramatically reduced in the liver of the cows fed RS compared with the cows fed AH, suggesting that the RS-fed cows had a lower gluconeogenesis rate. It is worth noting that the glycolytic activity in the liver might also be lower in the cows fed RS compared with the cows fed AH, as shown by the mRNA levels of the phosphofructokinase (PFK), a key enzyme in glycolysis pathway (Table 5).

Hormonal Regulation

Several factors might be involved in the decreased liver gluconeogenesis enzyme expression in the cows fed RS. Nutritional factors, such as low propionate supply or negative energy balance, might play a role (Drackley et al., 2001; Wang et al., 2014). Other important factors might include endocrine factors. Insulin and glucagon are well-known hormones that regulate hepatic gluconeogenesis. Insulin directly inhibits the transcription and activity of gluconeogenesis enzymes, including phosphoenolpyruvate carboxykinase (PEPCK), whereas glucagon activates gluconeogenesis (O’Brien and Granner, 1991). The IGF-1 is also a potent inhibitor of PC and PEPCK expression in bovine hepatocytes (Wang et al., 2012).

In this study, the observed decreased arterial IGF-1 concentration and the downregulation of the IGF-1 mRNA levels in the livers of the cows fed RS may be attributed to the negative energy balance in these cows (Vandehaar et al., 1995; Wang et al., 2014). The changes in the levels of the IGF-1 and IGF-1R mRNA paralleled the changes in the coccygeal arterial IGF-1 concentrations, which supports the broadly held view that the liver is the primary source of the circulating IGF-1 protein (Radcliff et al., 2003). In addition, the glucose concentration in the mammary vein as well as the lactose content of milk were reflected by the mammary venous insulin:glucagon ratio but not by the insulin:glucagon ratio in the jugular vein or artery, which might indicate a better accuracy of mammary vein blood than arterial or jugular venous blood in determining the blood glucose status and glucose anabolism. On the other hand, observations from Kronfeld et al. (1963) suggested that hypoglycemia is a more direct cause of decreasing milk yield than is insulin. Therefore, the decreased milk lactose might be mainly attributed the short supply of glucose, and the regulation of insulin and glucagon may be reflective of the blood glucose concentration.

Glucose Utilization in the Mammary Gland

Consistent with the lower arterial and venous glucose concentrations as well as the lower MPF (Wang et al., 2016), the mammary glucose uptake was 30% lower in cows fed RS compared with cows fed AH. Our mammary glucose uptake data were consistent with a previous study (Cant et al., 2002) in which a mammary glucose uptake of approximately 7.2 mol/d is required to support a milk production of 14.4 kg/d, an amount that is similar to the milk yield of the cows in this study that were fed CS or RS. The lower concentrations of arterial and venous glucose and greater glucose arterial–venous difference and uptake in the mammary gland of the RS-fed cows did not appear to be due to the increased expression of the glucose transporters in the mammary gland because the expression of the GLUT1, GLUT3, and GLUT8 mRNA was not significantly different in the cows fed RS and AH, indicating that other factors, such as blood flow, must play a role (Zhao, 2014).

Mammary glucose uptake is achieved by the GLUT. Strong expression of GLUT1 and GLUT8 has been observed in the bovine mammary gland (Zhao, 2014). GLUT3 expression was observed in the sheep mammary gland (Tsiplakou et al., 2015). The similar levels of the GLUT mRNA for all treatments may indicate that the expression levels of the GLUT were not affected by the different arterial glucose supply and uptake in this study. The mRNA abundance level of GLUT1 was found to be greater than the other GLUT in mammary gland of dairy cows during lactation (Zhao, 2014). However, the lower relative abundance of GLUT1 mRNA than GLUT8 or GLUT3 observed in this study might be attributed to the late lactation stage.

In mammary alveolar epithelial cells, glucose is used for lactose synthesis, glycolysis, and the pentose phosphate pathway (Zhao, 2014). In lactating ruminants, up to 75% of the glucose taken up by the mammary gland is used for lactose synthesis (Chaiyabutr et al., 1980). Isotopic and balance data confirm that glucose is the main precursor of lactose and that the oxidation and transfer of glucose into lactose accounted for 69 to 98% of the glucose entry rate (Bickerstaffe et al., 1974). Therefore, the arterial glucose supply can determine lactose synthesis in the mammary gland. Many studies have also shown positive correlations between glucose uptake and lactose synthesis in the mammary gland (Cant et al., 2002; Mirzaei-Aghsaghali and Fathi, 2012). Therefore, it was very likely that the reduced lactose concentrations in the milk of cows fed RS compared with the AH-fed cows resulted from the reduced glucose uptake by the mammary gland. The lactose synthase activity might not play a role because mammary expression of 2 lactose synthase components, B4GALT and α-LA (Farrell et al., 2004), and milk α-LA were not different in the cows fed RS and AH (Tables 6 and 7).

Milk Osmotic Pressure

Lactose plays a major role in maintaining milk osmolarity and contributes to approximately 50% of the milk osmotic pressure, which is relatively consistent during the lactation period (Gáspárdy et al., 2004). Because of this role, the lactose yield is considered a major factor in determining the milk volume. Indeed, the milk yield was well correlated with the lactose yield in our study (Wang et al., 2014). However, the cows fed the RS diet had a lower milk lactose concentration compared with cows fed AH. These cows appeared to be in good health as demonstrated by the normal and relatively consistent DMI and low milk somatic cell count (Wang et al., 2014). Therefore, the lower milk lactose concentration does not appear to be a result of health issues in the cows but rather of the lower energy supply that restricted the lactose synthesis.

On the other hand, lactose is not the only factor that maintains milk osmolarity; milk ions are also involved. In the study of Roche et al. (2005), there was no significant change of milk Na+, K+, and Cl even under different dietary cation–anion treatments that yielded similar lactose contents; this may indicate the relatively constant biological functions of Na+, K+, and Cl and lactose in milk osmotic pressure. The lower milk osmolarity in response to the reduced milk lactose concentrations in the RS-fed cows might be compensated by the higher milk K+ concentrations (Table 7). The increased K+ concentrations in the RS diet might have resulted in the increased milk K+ concentration. Our observation was consistent with previous studies. There existed a significant negative correlation between the lactose concentrations and the milk Na+ + K+ + Cl concentrations (Oshima et al., 1980). It was reported that the changes in the milk Na+ and K+ concentrations were coordinated and opposite to the changes in the lactose concentrations, but not Cl concentration, in lactating dairy cows (Peaker, 2013).

Differences between the Rice Straw– and Corn Stover–Fed Cows

Although milk production in the CS-fed cows was as low as that in the RS-fed cows (Wang et al., 2014), the factors that caused the reduced milk production in the 2 groups appeared to be different. First, in contrast to the cows fed RS, the plasma glucose and milk lactose concentrations were not different between the CS- and AH-fed cows. There were also no differences in the rumen propionate concentrations between the CS- and AH-fed cows. These observations suggest that a limitation in the lactose precursor was not the major factor that caused the low milk production in the CS-fed cows. Second, although the rumen propionate concentrations in the CS- and AH-fed cows were similar, the rumen acetate and total VFA concentrations were significantly less in the cows fed CS compared with the cows fed AH. This observation may imply a more seriously limited dietary energy supply to the CS-fed cows. Third, the mammary glucose uptake and clearance rates were significantly less in the cows fed CS compared with the cows fed AH. These differences primarily resulted from the lower blood flow in the CS-fed cows, but this was not the case in the RS-fed cows. Finally, the milk protein profile was different in the cows fed CS compared with the cows fed AH and RS. These differences suggest that the milk protein synthesis is regulated by the nutrient supply, such as availability of glucose and individual AA.

Conclusions

Our study showed that the lower rumen propionate production and liver gluconeogenesis resulted in reduced lactose synthesis in the RS-fed cows compared with the AH-fed cows, which might result in reduced milk production in the RS-fed cows. However, a lower dietary energy supply and mammary blood flow might be the major factors for low milk production in the CS-fed cows. Therefore, it is important to provide compensatory nutrients when using RS and CS to replace AH as a forage source for lactating dairy cows. For RS-based diets, more glucogenic nutrients should be provided, whereas feedstuffs that yield more acetate might be helpful for cows fed CS-based diets.

 

References

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