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The objective of this study was to determine if the addition of glycerol to the diet of dairy cows would stimulate milk protein yield in the same manner as the addition of corn grain. Twelve multiparous lactating dairy cows at 81 ± 5 d in milk were subjected to 3 dietary treatments in a replicated 3 × 3 Latin square design for 28-d periods. The diets were a 70% forage diet considered the basal diet, the basal diet with 19% ground and high-moisture corn replacing forages, and the basal diet with 15% refined glycerol and 4% added protein supplements to be isocaloric and isonitrogenous with the corn diet. Cows were milked twice a day and samples were collected on the last 7 d of each period for compositional analysis. Within each period, blood samples were collected on d 26 and 27, and mammary tissue was collected by biopsy on d 28 for Western blot analysis. Dry matter intake increased from 23.7 kg/d on the basal diet to 25.8 kg/d on the corn diet and 27.2 kg/d on the glycerol diet. Dry matter intake tended to be higher with glycerol than corn. Milk production increased from 39.2 kg/d on the basal diet to 43.8 kg/d on the corn diet and 44.2 kg/d on the glycerol diet. However, milk yield did not differ between corn and glycerol diets. Milk lactose yields were higher on the corn and glycerol diets than the basal diet. Milk fat yield significantly decreased on the glycerol diet compared with the basal diet and tended to decrease in comparison with the corn diet. Mean milk fat globule size was reduced by glycerol feeding. Milk protein yield increased 197 g/d with addition of corn to the basal diet and 263 g/d with addition of glycerol, and the glycerol effect was larger than the corn effect. The dietary treatments had no effects on plasma glucose concentration, but plasma acetate levels decreased 27% on the glycerol diet. Amino acid concentrations were not affected by dietary treatments, except for branched-chain amino acids, which decreased 22% on the glycerol diet compared with the corn diet. The decreases in plasma acetate and branched-chain amino acid concentrations with glycerol and the larger effects of glycerol than corn on milk protein and fat yields suggest that glycerol is more glucogenic for cows than corn grain.
). The mechanism responsible for this stimulation of milk protein yield is unclear. The effect of grain feeding may be explained in part by an increase in microbial protein outflow from the rumen (
), presumably through the insulin signaling cascade inside mammary epithelial cells. Essential amino acids and insulin are both activators of the mechanistic target of rapamycin complex 1 (mTORC1) in mammary epithelial cells (
Essential amino acids regulate both initiation and elongation of mRNA translation independent of insulin in MAC-T cells and bovine mammary tissue slices.
) that may be responsible for nutritional stimulation of milk protein synthesis. Glucose, glucose precursors, and grains also depress milk fat production (
Studies of secretion of milk of low fat content by cows on diets low in hay and high in concentrates. V. The importance of the type of starch in the concentrates.
) that may become available as a feed ingredient for cattle as a co-product of biodiesel manufacture. As a glucogenic feedstuff, glycerol has several features in common with corn grain. Degradation of glycerol in the rumen increases the proportions of propionate and butyrate at the expense of acetate (
Supplemental cracked corn or wheat bran for steers grazing endophyte-free fescue pasture: Effects on live weight gain, nutrient quality, forage intake, particulate and fluid kinetics, ruminal fermentation, and digestion.
), a portion of which may enter the bloodstream. In a similar fashion, approximately half of the glycerol consumed is absorbed across the rumen wall into blood, where it becomes available for hepatic gluconeogenesis (
). Because of these similarities in fermentation pattern and glucogenicity, glycerol may be comparable to glucose or corn grain in its effect on milk component yields.
Whether addition of glycerol to the diet stimulates milk protein yield the way addition of corn grain does has not been evaluated to our knowledge. In several experiments, isocaloric inclusions of refined glycerol up to 15% of diet DM have not affected intake, rumen microbial protein yield, or milk protein yield (
The effects of added glycerol or unprotected free fatty acids or a combination of the two on silage intake, milk production, rumen fermentation and diet digestibility in cows given grass silage based diets.
. Hypercaloric additions of glycerol to the diet, instead of isocaloric substitutions, have been made at much smaller doses and in the context of preventing metabolic disorders associated with the transition into lactation (
). These additions have not resulted in changes in DM intakes or milk component yields. When glycerol was included at 20 g/L in the drinking water of transition cows, for a glycerol intake equal to 1.3 kg/d or 10% of DMI, ad libitum consumption of the diet decreased so that NEL intake was not affected and milk component yields remained unchanged (
Effects of supplementing glycerol and soybean oil in drinking water on feed and water intake, energy balance and production performance in periparturient dairy cows.
The effects of added glycerol or unprotected free fatty acids or a combination of the two on silage intake, milk production, rumen fermentation and diet digestibility in cows given grass silage based diets.
). Effects on milk yield and composition may be difficult to detect with statistical significance when glycerol replaces grain isocalorically, but they may be easier to detect when glycerol is added hypercalorically to a diet. For use as a major feed ingredient in milking cow rations throughout lactation, it would be useful to know if addition of glycerol can stimulate daily milk yield, and if it stimulates milk protein and depresses milk fat production in the manner of a corn grain addition. Our hypothesis was that the fermentability and glucogenicity of glycerol would lead to a milk protein stimulation and milk fat depression when added to increase the energy density of lactating cow diets. Our objective was to compare the hypercaloric effects of corn and glycerol on milk production and composition and mammary mTORC1 signaling.
MATERIALS AND METHODS
Animals, Housing, and Diets
The Animal Care Committee at the University of Guelph approved all experimental procedures in this study. Twelve multiparous, lactating Holstein cows (674 ± 66 kg BW; 81 ± 5 DIM) were housed in a tie-stall barn and given free access to feed and water throughout the study. Cows were assigned to 3 dietary treatments (Table 1) in a replicated 3 × 3 Latin square design of three 28-d periods. The 3 diets were a 70% forage basal TMR (BAS), the basal TMR with 19% of the forages substituted for corn grain (CG), and the basal TMR with 19% of the forages substituted for refined glycerol from a soybean oil source (99.7% glycerol; Palmera G997U, KLK Oleo, Selangor, Malaysia) plus protein supplements (GLYC). The CG and GLYC diets were formulated to be isocaloric and isonitrogenous and to contain 30% NDF, compared with 38% NDF for the BAS diet.
Table 1Ingredient and chemical composition (% of DM unless otherwise noted; mean ± SD; n = 3) of experimental diets
BAS = 70% forage basal TMR; CG = basal TMR with 19% of the forages substituted for corn grain; GLYC = basal TMR with 19% of the forages substituted for refined glycerol from a soybean oil source plus protein supplements.
1 BAS = 70% forage basal TMR; CG = basal TMR with 19% of the forages substituted for corn grain; GLYC = basal TMR with 19% of the forages substituted for refined glycerol from a soybean oil source plus protein supplements.
The amounts of feed offered and refused were recorded daily for each individual cow to estimate feed intakes. Orts were sampled from each cow and pooled on the last week of each period. Samples of each diet were collected daily, pooled by period, and submitted for nutrient composition analyses by wet chemistry at a commercial laboratory (Agri-Food Labs, Guelph, ON, Canada). Dry matter contents of feed and orts samples were determined using a forced-air oven at 60°C for 24 h.
Cows were milked at 0500 and 1530 h daily, and milk yields were recorded. Milk samples were collected from each milking during the last 7 d of each period and submitted for compositional analysis by infrared spectroscopy (Laboratory Services Division, University of Guelph, ON, Canada). Fresh milk samples from d 23, 25, and 27 of each period were also subjected to analysis of fat globule size distribution by integrated light scattering (Mastersizer S, Malvern Instruments, Southborough, MA) according to
. Average volume-weighted diameter (d4,3) was calculated as ΣNidi4/ΣNidi3, and average surface-weighted diameter (d3,2) was calculated as ΣNidi3/ΣNidi2, where Ni is the number of fat globules with diameter di. Specific area of fat globules was calculated as 6/(0.92 × d3,2), and fat globule membrane yields were calculated according to
as specific area (m2/g) × 10 nm membrane thickness × milk fat yield (g/d).
Energy balance of cows was calculated as NEL intake − NEL milk − NEL maintenance, where NEL intake was calculated from observed DMI and calculated NEL content; NEL milk was obtained from the regression equation of
Blood samples were collected by tail venipuncture on d 26 and 27 of each period and centrifuged at 2000 × g for 10 min. Plasma was transferred to polypropylene tubes for storage at −20°C. Samples were thawed and analyzed by enzyme-linked spectrophotometry for glucose, acetate, BHB, nonesterified fatty acids (NEFA) and triacylglycerol (TAG) as described by
On the last day of each period, following the morning milking, mammary tissue was collected by biopsy from one hindquarter of the 6 cows comprising 2 of the 3 × 3 Latin squares. Cows were sedated with 0.5 mL of xylazine i.v., ketoprofen (3 mg/kg of BW) was administered intramuscularly, and 5 mL of lidocaine was injected subcutaneously at the biopsy site. Tissue was collected aseptically using the device of
. Samples were immediately rinsed with saline, snap-frozen in liquid N2, and stored at −80°C for further processing and analysis.
Approximately 500 mg of mammary tissue were homogenized in 1 mL of RIPA lysis buffer (1% Triton X-100, 0.1% SDS, 50 mM Tris-HCl pH 8.0, 150 mM NaCl, and 0.5% sodium deoxycholate) containing protease and phosphatase inhibitor cocktail (Thermo Scientific, Nepean, ON), and supernatants were recovered after centrifuging at 13,000 × g for 15 min at 4°C. Supernatants were analyzed for DNA concentration with the Qubit dsDNA BR assay using a Qubit 2.0 Fluorometer (Life Technologies Inc., Burlington, ON, Canada), RNA concentration with the Qubit RNA BR assay (Life Technologies Inc.), and protein concentration with the BCA Protein Assay Kit (Pierce, Rockford, IL) using bovine serum albumin as a standard.
Supernatants of mammary samples were subjected to Western blotting as described by
with modifications. Briefly, samples containing 20 µg of protein, along with BLUeye Prestained Protein Ladder (FroggaBio, Toronto, ON, Canada), were separated by 10% SDS-PAGE and transferred onto polyvinylidene difluoride membranes (Millipore, Mississauga, ON, Canada) before incubation with rabbit monoclonal antibodies against ribosomal S6 kinase 1 (S6K1; no. ab9366, Abcam, Cambridge, MA), phosphorylated S6K1 (Thr389, no. ab2571, Abcam), eukaryotic initiation factor 2α (eIF2α; no. 9722; Cell Signaling Technology Inc., Danvers, MA), phosphorylated eIF2α (Ser51, no. 3597, Cell Signaling), eIF4E-binding protein 1 (4EBP1; no. 9452, Cell Signaling Technology Inc.), phosphorylated 4EBP1 (Ser65, no. 9451, Cell Signaling Technology Inc.), protein kinase R-like endoplasmic reticulum kinase (PERK; no. 3192, Cell Signaling Technology Inc.), phosphorylated PERK (Thr980, no. 3179, Cell Signaling Technology Inc.), eIF2Bε (no. ab32713, Abcam), phosphorylated eIF2Bε (Ser539, no. ab4775, Abcam), AMP-activated protein kinase α (AMPKα; no. 2603, Cell Signaling Technology Inc.), or phosphorylated AMPKα (Thr172, no. 2535, Cell Signaling Technology Inc.). Appropriate portions of each membrane were also probed for β-actin (no. ab6276, Abcam), as a loading control. Membranes were then washed, incubated at room temperature for 1 h with horseradish peroxidase–linked anti-rabbit IgG (no. 7074, Cell Signaling), and developed using enhanced chemiluminescence (Amersham, Arlington Heights, IL). Blot densities in scanned images were determined by ImageLab software (Bio-Rad Laboratories Inc., Mississauga, ON) and normalized to the corresponding β-actin blot density. Phosphorylation state of each protein was calculated as the ratio of phosphorylated to total protein blot densities.
Statistical Analysis
Mean performance and plasma metabolite observations during the last week of each period and Western blot results were subjected to ANOVA using PROC MIXED of SAS (SAS Institute Inc., Cary, NC) according to the following model:
where μ = overall mean, cowi = random effect of cow (i = 1 to 12), Perj = fixed effect of period (j = 1 to 3), Trtk = fixed effect of treatment (k = 1 to 3), and εijk = residual error. Least-squares treatment means were separated using the pdiff option of SAS when overall treatment P < 0.10. Differences were considered significant at P ≤ 0.05 and tendencies at 0.05 < P ≤ 0.10.
RESULTS
Intake and Lactational Performance
A difference in DMI was found between the BAS diet compared with CG and GLYC treatments (Table 2). On a DM basis, cows offered CG ate 2.1 kg/d more than cows fed the BAS diet (P < 0.01), and cows offered GLYC ate 3.5 kg/d more than those given the BAS diet (P < 0.01). The 1.4 kg/d higher DMI on GLYC compared with CG tended to be significant (P = 0.06).
Table 2Dry matter intake, milk yield and composition, and NEL balance of lactating dairy cattle (n = 12) during the last 7 d of being fed BAS, CG, and GLYC diets
BAS = 70% forage basal TMR; CG = basal TMR with 19% of the forages substituted for corn grain; GLYC = basal TMR with 19% of the forages substituted for refined glycerol from a soybean oil source plus protein supplements.
Means within a row with different superscripts are significantly different (P ≤ 0.05).
0.03
<0.01
a–c Means within a row with different superscripts are significantly different (P ≤ 0.05).
1 BAS = 70% forage basal TMR; CG = basal TMR with 19% of the forages substituted for corn grain; GLYC = basal TMR with 19% of the forages substituted for refined glycerol from a soybean oil source plus protein supplements.
Cows that were fed the CG diet produced 4.6 kg/d more milk than those on the BAS diet (P < 0.01), and cows that were fed GLYC produced 5.0 kg/d more (P < 0.01). Milk yield did not differ between GLYC and CG. Daily protein yield increased by 197 g/d (P < 0.01) and 263 g/d (P < 0.01) for the CG and GLYC diets, respectively, in comparison with the BAS diet. The protein yield on GLYC was 66 g/d higher than on CG (P = 0.05). Milk fat yields were not affected by addition of corn to the BAS diet, but GLYC depressed milk fat yield by 140 g/d (P = 0.02) compared with BAS and tended to depress milk fat yield by 101 g/d compared with CG (P = 0.08). Daily lactose yield increased by 283 g/d on the CG diet (P < 0.01) and 285 g/d on the GLYC diet (P < 0.01) compared with the BAS diet.
Protein percentage increased by 0.14 (P < 0.01) and 0.25 (P < 0.01) units in milk from cows fed CG and GLYC, respectively, compared with BAS (Table 2). Protein percentage was higher on GLYC than CG (P = 0.04). Milk fat percentage decreased by 0.43 (P < 0.001) or 0.69 (P < 0.001) units with addition of CG or GLYC to the BAS diet and was lower with GLYC compared with CG (P = 0.05). Lactose content of milk increased by 0.14 (P < 0.01) and 0.10 (P < 0.01) percentage units for CG and GLYC diets, respectively.
Energy-corrected milk yield increased 2.4 kg/d (P = 0.02) between CG and BAS diets and tended to increase 1.7 kg/d (P = 0.07) between GLYC and BAS diets. The ECM yield did not differ between CG and GLYC (P = 0.46).
The NEL balance of cows on the BAS diet was negative, increased 4.2 Mcal/d (P < 0.01) to a positive value when CG was fed, and increased 7.3 Mcal/d (P < 0.01) when GLYC was fed. The NEL balance was higher with GLYC than CG (P = 0.02). Efficiencies of dietary NEL output in milk were lower than BAS for both CG and GLYC diets (P < 0.03) and tended to be lower on GLYC versus CG (P = 0.06).
Mean milk fat globule diameter decreased by 6% (P < 0.01) between BAS and GLYC diets on a volume-weighted basis, and 7% (P = 0.01) on a surface area–weighted basis (Table 3). Between BAS and CG diets, volume-weighted mean fat globule size tended to decrease (P = 0.10) and surface area–weighted mean diameter decreased significantly (P = 0.03). Mean fat globule size did not differ between CG and GLYC diets (P > 0.22). The smaller fat globule sizes on GLYC compared with BAS resulted in a greater membrane content per gram of milk fat, expressed either as specific area (m2/g fat; P = 0.01) or specific weight (g/kg fat; P = 0.01). Likewise, membrane content of milk fat tended to be higher on CG versus BAS (P = 0.06). However, daily milk fat globule membrane yield was reduced by GLYC because of the depression in milk fat yield.
Table 3Milk fat globule sizes and membrane yields from cows (n = 12) fed BAS, CG, and GLYC diets
BAS = 70% forage basal TMR; CG = basal TMR with 19% of the forages substituted for corn grain; GLYC = basal TMR with 19% of the forages substituted for refined glycerol from a soybean oil source plus protein supplements.
Means within a row with different superscripts are significantly different (P ≤ 0.05).
0.7
0.03
a,b Means within a row with different superscripts are significantly different (P ≤ 0.05).
1 BAS = 70% forage basal TMR; CG = basal TMR with 19% of the forages substituted for corn grain; GLYC = basal TMR with 19% of the forages substituted for refined glycerol from a soybean oil source plus protein supplements.
2 d4,3 = ΣNidi4/ΣNidi3 and d3,2 = ΣNidi3/ΣNidi2, where Ni is the number of fat globules with diameter di.
Plasma glucose, BHB, NEFA, and TAG concentrations were not significantly affected by corn or glycerol addition to the BAS diet (Table 4). However, a trend was found for a decrease in plasma acetate concentrations with GLYC compared with either BAS or CG (P = 0.06).
Table 4Plasma concentrations of metabolites in lactating dairy cattle (n = 12) fed BAS, CG, and GLYC diets
BAS = 70% forage basal TMR; CG = basal TMR with 19% of the forages substituted for corn grain; GLYC = basal TMR with 19% of the forages substituted for refined glycerol from a soybean oil source plus protein supplements.
Means within a row with different superscripts are significantly different (P ≤ 0.05).
0.16
0.06
BHB, mM
1.16
0.91
0.90
0.10
0.15
Fatty acids, μM
83
87
56
13
0.17
Triacylglycerol, μM
93
99
76
9
0.20
a,b Means within a row with different superscripts are significantly different (P ≤ 0.05).
1 BAS = 70% forage basal TMR; CG = basal TMR with 19% of the forages substituted for corn grain; GLYC = basal TMR with 19% of the forages substituted for refined glycerol from a soybean oil source plus protein supplements.
Addition of corn to the BAS diet tended to increase His (P = 0.08) and Gly (P = 0.07) concentrations in plasma but had no effects on any other AA (P > 0.33). In contrast, the GLYC diet caused a decrease in each of the 3 branched-chain AA (BCAA: Ile, Leu, and Val) concentrations in plasma compared with either BAS or CG diets (P ≤ 0.03; Table 5). The remaining AA were not affected by GLYC (P > 0.10).
Table 5Plasma concentrations of free amino acids (μM) in lactating dairy cattle (n = 12) fed BAS, CG, and GLYC diets
BAS = 70% forage basal TMR; CG = basal TMR with 19% of the forages substituted for corn grain; GLYC = basal TMR with 19% of the forages substituted for refined glycerol from a soybean oil source plus protein supplements.
a,b Means within a row with different superscripts are significantly different (P ≤ 0.05).
1 BAS = 70% forage basal TMR; CG = basal TMR with 19% of the forages substituted for corn grain; GLYC = basal TMR with 19% of the forages substituted for refined glycerol from a soybean oil source plus protein supplements.
2 3-MeHis = 3-methylhistidine.
3 BCAA = branched-chain amino acids (Ile, Leu, and Val).
4 Non-BC-EAA = nonbranched-chain EAA (Arg, His, Lys, Met, Phe, Thr, and Trp).
Addition of corn and glycerol to the BAS diet did not stimulate changes in phosphorylation status or abundance of proteins associated with mammary translation signaling pathways (Table 6). Total AMPK tended to be elevated with CG compared with BAS and GLYC (P = 0.06).
Table 6Total cell signaling protein abundances (normalized to β-actin) and their phosphorylation states (phosphorylated protein normalized to total) in mammary tissue of lactating cows (n = 6) fed BAS, CG, and GLYC diets
BAS = 70% forage basal TMR; CG = basal TMR with 19% of the forages substituted for corn grain; GLYC = basal TMR with 19% of the forages substituted for refined glycerol from a soybean oil source plus protein supplements.
Means within a row with different superscripts are significantly different (P ≤ 0.05).
0.07
0.06
Phosphorylation state
0.67
0.35
0.48
0.19
0.39
a,b Means within a row with different superscripts are significantly different (P ≤ 0.05).
1 BAS = 70% forage basal TMR; CG = basal TMR with 19% of the forages substituted for corn grain; GLYC = basal TMR with 19% of the forages substituted for refined glycerol from a soybean oil source plus protein supplements.
Figure 1Representative Western blot images of phosphorylated and total forms of signaling proteins in mammary tissue of cows fed basal (B), corn grain (C), and glycerol (G) diets for 28 d (means from all cows presented in Table 6). 4EBP1 = eukaryotic initiation factor 4E binding protein 1; Akt = Ser/Thr protein kinase B; AMPK = AMP-activated protein kinase; eIF2α = eukaryotic initiation factor 2 subunit α; eIF2Bε = eukaryotic initiation factor 2B subunit ε; PERK = PKR-like endoplasmic reticulum kinase; S6K1 = ribosomal protein S6 kinase 1.
As the demand for biodiesel fuels increases, glycerol, as a co-product of biodiesel manufacture, will be increasingly available. Glycerol has been used as a feed additive in transition cow diets since the early 1950s to prevent the metabolic disorder of ketosis in dairy cattle (
). An increased interest exists for glycerol as a routine ingredient in dairy cattle rations, and information on its feeding value is needed. Several studies have been conducted in which refined glycerol replaced a portion of the concentrate up to 15% of DMI (
The effects of added glycerol or unprotected free fatty acids or a combination of the two on silage intake, milk production, rumen fermentation and diet digestibility in cows given grass silage based diets.
). Effects on milk yield and composition were not detected, indicating that glycerol is equivalent in value to the ingredients replaced. Statistical standards to declare a lack of effect are not as well established as those to declare a significant difference (e.g., P < 0.05), so we chose to design an experiment that offered a greater opportunity to detect lactational effects of glycerol by adding it to increase the energy density of the diet (i.e., hypercalorically). Addition of 15% glycerol increased DMI and yields of milk, protein, and lactose and decreased milk fat yield. Addition of corn at a caloric equivalent to glycerol also increased DMI and yields of milk, protein, and lactose, but protein yield did not go up as high and fat yield did not decrease.
Milk Protein Stimulation
Glycerol is a glucogenic substance similar to corn in that a portion is fermented in the rumen to propionate and a portion is converted to glucose postruminally (
). Glycerol appears to exert a stronger effect on rumen VFA profile than grains though, because isocaloric substitution for corn or barley increases the rumen propionate concentration and decreases acetate (
The effects of added glycerol or unprotected free fatty acids or a combination of the two on silage intake, milk production, rumen fermentation and diet digestibility in cows given grass silage based diets.
). The tendency for a lower concentration of plasma acetate in GLYC cows versus those on BAS or CG may be related to such a ruminal effect. However, the net splanchnic release of acetate into circulation in lactating cows is highly positively related to rumen-fermentable OM intake (
), which was higher on GLYC versus BAS, so acetate entry was not likely responsible for the 24% drop in plasma acetate concentration. Rather, the concentration drop may have been a consequence of diversion of plasma acetate into adipose lipids such as occurs during glucose infusion (
). The change in NEL balance from a negative value on the BAS diet to a positive value on the GLYC diet and the tendency for a more positive NEL balance on GLYC versus CG support this contention.
Total DMI was stimulated by addition of either glycerol or corn, and it tended to be higher with glycerol. Most studies have shown no effect on DMI when glycerol, refined or crude, is isocalorically and isonitrogenously substituted into diets (
The effects of added glycerol or unprotected free fatty acids or a combination of the two on silage intake, milk production, rumen fermentation and diet digestibility in cows given grass silage based diets.
). Supplementation of 1,300 g/d glycerol through the drinking water depressed intake of solid feed by transition cows but total NEL intake was not affected (
Effects of supplementing glycerol and soybean oil in drinking water on feed and water intake, energy balance and production performance in periparturient dairy cows.
). The stimulation of DMI by both CG and GLYC may be related to the decrease in NDF content of the diets compared with BAS because of a fill effect that is relieved under approximately 35% dietary NDF (
The effects of added glycerol or unprotected free fatty acids or a combination of the two on silage intake, milk production, rumen fermentation and diet digestibility in cows given grass silage based diets.
). Inconsistent reports of a milk protein effect may be related to an inability to detect small changes that occur with isocaloric replacement studies.
Increasing glucogenic energy supply through concentrate feeding or postruminal carbohydrate infusion stimulates milk protein production (
Nutritional stimulation of milk protein yield of cows is associated with changes in phosphorylation of mammary eukaryotic initiation factor 2 and ribosomal S6 kinase 1.
). Accordingly, the increase in milk protein yield caused by GLYC may be related to its glucogenicity, although it may also have been a consequence of the higher CP intake due to the stimulation of DMI and the unexpectedly higher CP percentage of the GLYC diet. However, none of the essential amino acid concentrations in plasma were increased by glycerol addition to the BAS diet and, in fact, concentrations of each of the BCAA were decreased in plasma. A drop in BCAA concentrations is typical of a postruminal glucose or euglycemic insulin effect. During prolonged insulin infusion into lactating cows or goats, plasma concentrations of essential amino acids decrease dramatically and the BCAA are reduced the most (
). The reduction of plasma BCAA concentrations in the cows that were fed GLYC, but not those fed CG, suggests that glycerol elicits more of a glucose–insulin response than corn grain. The tendency for a stimulation of DMI with GLYC versus CG, resulting in cows on the GLYC diet consuming more glucogenic energy than cows on the CG diet, may have contributed to the larger glucose–insulin response. The higher glucogenicity may be responsible for the greater stimulation of milk protein yield by GLYC compared with CG.
We hypothesized that the milk protein stimulation from glucose or glucogenic feedstuffs is mediated by mTORC1 or integrated stress response (ISR) pathways in the mammary glands. However, the only change in phosphorylation state of key mTORC1 and ISR participants was an increase in peIF2α, which is inhibitory to protein synthesis. Stimulation of protein synthesis in muscle and liver of growing animals in the postprandial period following consumption of a meal is due to activation of intracellular mTORC1 by insulin and amino acids (
). Initiation of translation is also regulated by kinases of the ISR that phosphorylate eIF2α and thereby inhibit ribosome recruitment in response to amino acid insufficiency, unfolded protein accumulation in the ER, and other stressors (
). In mammary cells in vitro, stimulators of protein synthesis such as insulin, IGF-1, glucose, and amino acids increase the phosphorylation state of mTORC1 targets and decrease phosphorylation of ISR targets within minutes (
Essential amino acids regulate both initiation and elongation of mRNA translation independent of insulin in MAC-T cells and bovine mammary tissue slices.
). Within 2 d of commencing postruminal infusions of carbohydrate or amino acids into cows, phosphorylation states of mammary mTORC1 and ISR targets also change in accord with the increases in milk protein yield that occur (
Nutritional stimulation of milk protein yield of cows is associated with changes in phosphorylation of mammary eukaryotic initiation factor 2 and ribosomal S6 kinase 1.
). However, abomasal infusions of amino acid mixtures lacking single essential amino acids had no effect on phosphorylation state of eIF2α in mammary tissue collected after 5 d of infusion, and S6K1 phosphorylation and eIF2Bε abundance both increased with no change in milk protein yield (
Essential amino acid infusions stimulate mammary expression of eukaryotic initiation factor 2Bε but milk protein yield is not increased during an imbalance.
). These responses suggest that factors other than mTORC1 and ISR signaling may be responsible for long-term control of milk protein synthesis. Likewise, the current finding of no change in phosphorylation state of key participants in mTORC1- and ISR-mediated regulation of mRNA translation after 28 d of feeding milk protein–stimulating diets indicates that alternative hypotheses for the yield increases must be sought.
Milk Fat Depression
The observed decrease in milk fat yield and percentage from cows fed the GLYC diet in comparison to both BAS and CG diets lies in accordance with a previous study that reported a decrease in milk fat percentage due to isocaloric glycerol at an intake of 2.7 kg/d (
The effects of added glycerol or unprotected free fatty acids or a combination of the two on silage intake, milk production, rumen fermentation and diet digestibility in cows given grass silage based diets.
), our study differs in that glycerol was added hypercalorically. Increasing the concentrate portion of a cow's diet so that NEL intake increases causes a decrease in milk fat yield, concentration, or both (
Studies of secretion of milk of low fat content by cows on diets low in hay and high in concentrates. V. The importance of the type of starch in the concentrates.
SREBP1 and thyroid hormone responsive Spot 14 (S14) are involved in the regulation of bovine mammary lipid synthesis during diet-induced milk fat depression and treatment with CLA.
). The decrease in plasma acetate concentration we observed could also contribute to a glycerol-induced milk fat depression because the effects of CLA, propionate, and acetate on milk fat production are additive (
Average milk fat globule size decreased significantly on GLYC compared with BAS. The membrane that surrounds lipid droplets in milk is a rich source of sphingolipids that possess anticancer properties, are anti-inflammatory, and help stimulate development of the central nervous system (
). Factors that regulate globule membrane content of milk as it is expressed from the cow's udder have been studied very little. Globule size is positively correlated with daily milk fat yield (
). For example, including unsaturated fat in the diet of cows or offering fresh pasture to cows decreases milk fat yield and also decreases average fat globule size (
Elevated concentrate-to-forage ratio in dairy cow rations is associated with a shift in the diameter of milk fat globules and remodeling of their membranes.
), even though milk fat yield was not depressed. The observation that cows in early lactation with high plasma insulin concentrations have higher ratios of phospholipid to TAG in their milk than low-insulin cows (
) suggests that insulin reduces milk fat globule size. Although we did not detect an effect of CG on milk fat globule size, GLYC caused a significant decrease, again supporting the assertion that glycerol is more glucogenic than corn grain.
CONCLUSIONS
Addition of glycerol to a high-forage diet for lactating dairy cows exerted similar effects on milk composition as addition of corn grain, but the stimulation of milk protein yield and the depression of milk fat yield were greater with glycerol. Reductions in plasma acetate and BCAA concentrations on the GLYC diet, but not the CG diet, suggest that glycerol is more glucogenic than corn grain, which may have been responsible for the larger effects on milk component yields.
ACKNOWLEDGMENTS
The authors thank Laura Wright and staff at the Ponsonby Dairy Research Station (Ponsonby, ON, Canada) for their technical help and animal care during the experiment. The authors also acknowledge the financial support of Dairy Farmers of Canada (Mississauga, ON, Canada), NSERC Canada (Ottawa, ON, Canada), and the Ontario Ministry of Agriculture, Food and Rural Affairs (OMAFRA; Guelph, ON, Canada). R.V. Curtis was the recipient of an OMAFRA HQP Scholarship.
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Nayananjalie W.A.D.
Escobar J.
Hanigan M.D.
Essential amino acids regulate both initiation and elongation of mRNA translation independent of insulin in MAC-T cells and bovine mammary tissue slices.
Elevated concentrate-to-forage ratio in dairy cow rations is associated with a shift in the diameter of milk fat globules and remodeling of their membranes.
Studies of secretion of milk of low fat content by cows on diets low in hay and high in concentrates. V. The importance of the type of starch in the concentrates.
Essential amino acid infusions stimulate mammary expression of eukaryotic initiation factor 2Bε but milk protein yield is not increased during an imbalance.
SREBP1 and thyroid hormone responsive Spot 14 (S14) are involved in the regulation of bovine mammary lipid synthesis during diet-induced milk fat depression and treatment with CLA.
Supplemental cracked corn or wheat bran for steers grazing endophyte-free fescue pasture: Effects on live weight gain, nutrient quality, forage intake, particulate and fluid kinetics, ruminal fermentation, and digestion.
The effects of added glycerol or unprotected free fatty acids or a combination of the two on silage intake, milk production, rumen fermentation and diet digestibility in cows given grass silage based diets.
Effects of supplementing glycerol and soybean oil in drinking water on feed and water intake, energy balance and production performance in periparturient dairy cows.
Nutritional stimulation of milk protein yield of cows is associated with changes in phosphorylation of mammary eukaryotic initiation factor 2 and ribosomal S6 kinase 1.
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