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(Journal of Nutrition. 1999;129:1579-1584.)
© 1999 The American Society for Nutritional Sciences


Nutrient Metabolism

Conjugated Linoleic Acids Alter Milk Fatty Acid Composition and Inhibit Milk Fat Secretion in Dairy Cows1 ,2

P. Yvan Chouinard*,3, Louise Corneau*,3, David M. Barbano{dagger}, Lloyd E. Metzger{dagger} and Dale E. Bauman*,4

Departments of * Animal Science and {dagger} Food Science, Cornell University, Ithaca, NY 14853

4To whom correspondance should be addressed.


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Conjugated linoleic acids (CLA) have positive health effects in experimental models. Our objective was to determine the effect of CLA supplementation on milk of dairy cows. A commercial source of CLA was infused abomasally to by-pass rumen fermentation. The supplement contained 61.2% CLA; the major CLA isomers were cis/trans 8,10, cis/trans 9,11, cis/trans 10,12 and cis/trans 11,13. Four Holstein cows were used in a 4 x 4 Latin square design. Treatments were 5-d infusions of 0, 50, 100 and 150 g/d of CLA supplement. Infusion increased milk fat content of CLA from 6.8 mg/g fat (zero dose) to 63.6 mg/g fat (highest dose). All of the major CLA isomers in the supplement were transferred to milk fat in a dose-dependent manner. Apparent efficiency of transfer to milk fat was 22.5, 22.5, 10.2 and 26.3% for cis/trans 8,10, cis/trans 9,11, cis/trans 10,12 and cis/trans 11,13, respectively. CLA infusion had no effect on milk protein and little effect on milk yield (21.5, 20.4, 20.9 and 18.3 kg/d for 0, 50, 100 and 150 g/d CLA supplement, respectively). However, CLA infusion dramatically reduced milk fat. On average, the content and yield of milk fat were reduced by 52 and 55%, respectively. The role of specific CLA isomers and mechanism(s) for the reduction in milk fat have not been established, although the pattern of milk fatty acids demonstrated effects were most pronounced on de novo fatty acid synthesis and the desaturation process. Overall, dietary supplemention of CLA increased milk fat content of CLA, altered milk fatty acid composition and markedly reduced the content and yield of milk fat.


KEY WORDS: • cows • conjugated linoleic acid • lactation • fatty acids • milk fat


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Conjugated linoleic acids (CLA)5 represent a mixture of positional and geometric isomers of octadecadienoic acid with conjugated double bonds. CLA have been demonstrated to have a range of positive health effects in experimental models. These include suppression of carcinogenesis (Belury 1995Citation , Ip et al. 1994Citation ), antiobesity effect (Park et al. 1997Citation ), modulation of the immune system (Cook et al. 1993Citation ) and reductions in atherogenesis (Nicolosi et al. 1997Citation ) and diabetes (Houseknecht et al. 1998Citation ). The specific CLA isomer(s) responsible for these various biological effects has not been clearly established, although cis-9, trans-11 CLA is thought to be key in the anticarcinogenic effects (Ha et al. 1990Citation , Ip et al. 1991Citation ).

Food products from ruminants, particularly dairy products, are the major dietary source of CLA for humans. CLA are intermediates in the biohydrogenation of linoleic acid, and it is generally accepted that CLA in ruminants originate from the incomplete biohydrogenation of unsaturated fat by rumen bacteria (Kelly et al. 1998Citation ). However, recent work has demonstrated that cows can also synthesize CLA from trans-11 octadecenoic acid, another intermediate in the rumen biohydrogenation process (Griinari et al. 1998bCitation ). The major CLA isomer in food products from ruminants is cis-9, trans-11, although other CLA isomers are present and these may vary under different rumen conditions (Griinari et al. 1997aCitation , Ha et al. 1989Citation ).

CLA levels in food products derived from ruminants can be altered by affecting ruminal production of CLA or trans-11 18:1, or by dietary supplement with these fatty acids. Our objective was to examine the effect of dietary supplementation of CLA on performance and milk composition of dairy cows, and the efficiency with which CLA were transferred to milk fat. To do this, we utilized a commercial source of CLA that represented a mixture of CLA isomers; the CLA supplement was infused abomasally to by-pass rumen fermentation processes.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
All procedures involving animals were approved by the Cornell University Institutional Animal Care and Use Committee.

Four multiparous Holstein cows (258 ± 43 d postpartum; mean ± SD) fitted with rumen fistulas were used. Cows were fed a total mixed diet formulated according to the Cornell Net Carbohydrate and Protein System (Fox et al. 1992Citation , Russell et al. 1992Citation , Sniffen et al. 1992Citation ). The major forage component was chopped alfalfa hay and the major concentrate component was cracked shelled corn (Table 1Citation ). The diet contained 16.3% crude protein and net energy for lactation of 6.54 MJ/kg (dry matter basis), and was balanced for protein, energy, minerals and vitamins to meet the predicted requirements (NRC 1989Citation ). Cows were fed ad libitum with equal portions of feed were offered every other hour to minimize postprandial effects on nutrient supply. In addition, water was available at all times.


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Table 1. Ingredient and chemical composition of diet

 
Cows were randomly assigned to treatments in a 4 x 4 Latin square design. Treatments were abomasal infusion of 0, 50, 100 and 150 g/d of a commercial source of CLA (CLA-60; Natural Lipids, Hovdebygda, Norway). The infusion periods lasted for 5 d with a 4-d interval between infusion periods. To provide a slow and continuous release of CLA into the abomasum, the CLA supplement was infused in an emulsion with skim milk. Three separate skim milk plus CLA emulsions (38 kg/emulsion) were prepared containing 1, 2 and 3% CLA supplement. Initially, the materials for each batch of emulsion were prepared by mixing the appropriate amount of the CLA supplement and skim milk (at 74°C) with a high shear mixer (model AXR, Silverson Machines, Wateside, Chesham, Bucks, United Kingdom). Each mixture was passed through a two-stage homogenizer (model 75E, Manton Gaulin Manufacturing, Everett, MA) with a first stage pressure of 13.8 MPa (2000 psi) and a second stage pressure of 3.45 MPa (500 psi). The emulsions were cooled to 10°C in an ice bath. This process produced a liquid for infusion in which the lipid remained uniformly dispersed. The CLA supplement concentration in each emulsion was determined by the difference in total solids content [AOAC method #990.20] between the emulsion and the original skim milk. The actual concentrations of CLA supplement were 1.02, 1.93 and 2.93% for the 1, 2 and 3% emulsions, respectively.

Abomasal infusions involved a 0.5-mm (i.d.) Nalgen tubing that passed through the rumen fistula and sulcus omasi into the abomasum as previously described (Griinari et al. 1997bCitation ). Emulsions were continuously infused by a peristaltic pump (Harvard Apparatus, South Natick, MA) calibrated to deliver 5 L/d. A sanitized carboy served as the reservoir for the emulsion; the carboy and emulsions were replaced daily.

Cows were milked at 600 and 1800 h daily. At each milking, yield was determined and milk was sampled. One aliquot was stored at 4°C with a preservative (bronopol tablet; D&F Control System, San Ramon, CA) until analyzed for fat and protein content by infrared analysis (New York DHI, Ithaca, NY). Because of the low milk fat content observed for some milk samples, we verified the accuracy of the infrared analysis of milk fat according to Association of Official Analytical Chemists [method #989.05; (AOAC 1995Citation )] and results were in agreement (±3%). A second set of milk samples without preservative was combined proportionately to milk yield on an individual cow basis for each day and stored at -20°C until analyzed for fatty acid composition.

For fatty acid analysis, an aliquot of thawed milk (~30 mL) was centrifuged at 17,800 x g for 30 min at 8°C, and then 300–350 mg of fat cake were removed. Lipid extraction of milk fat was performed according to Hara and Radin (1978)Citation using a hexane/isopropanol (3:2, v/v) solution (18 mL/g of fat cake) followed by a 67 g/L sodium sulfate solution (12 mL/g of fat cake). Milk fatty acids were transesterified with sodium methoxide prepared in our laboratory according to the method of Christie (1982)Citation with modifications. The sodium methylate was obtained from Fluka Chemie Ag (Buchs, Switzerland). Hexane (2 mL) was added to 40 mg of butter oil followed by 40 µL of methyl acetate. After the mixture was vortexed, 40 µL of methylation reagent (1.75 mL methanol:0.4 mL of 5.4 mol/L sodium methylate) was added. The mixture was vortexed and allowed to react for 10 min; then 60 µL of termination reagent (1 g oxalic acid/30 mL diethyl ether) was added. The sample was then centrifuged for 5 min at 2400 x g at 5°C leaving a clear layer of hexane; an aliquot of the hexane was taken and used directly for chromatographic determination.

Fatty acid methyl esters (FAME) in hexane were then injected (split inlet 87.5:1) into a gas chromatograph (Hewlett Packard GCD system HP G1800 A; Avondale, PA) equipped with an electron ionization detector. Separation of FAME was performed with a Supelcowax-10 fused silica capillary column [60 m x 0.32 mm (i.d.), with 0.25-µm film thickness] (Bellefonte, PA). Helium was used as the carrier gas. The oven temperature was programmed from 50 to 190°C at 10°C/min and held for 41.5 min. Injector and detector were at 250°C. Peak area was measured using a HP G107A GCD integrator. Each peak was indentified and quantified using pure methyl ester standards (Nu-Chek-Prep, Elysian, MN).

For the fatty acid analysis of CLA supplement, free fatty acids were methylated using 1% sulfuric acid in methanol as described by Christie (1989)Citation . Fatty acid profiles were determined following the gas chromatographic procedure described for milk fat. With our particular column and conditions, we were able to obtain partial separation of distinct peaks of the major CLA isomers cis/trans (c/t) 8,10, c/t 9,11, c/t 10,12, and c/t 11,13. We identified these peaks using pure standards (Nu-Chek-Prep, Matreya, Pleasant Gap, PA) and various enrichments of CLA isomers (Natural Lipids). Further, we validated our ability to determine specific amounts of each CLA isomer from comparisons with the various CLA isomer mixtures (Natural Lipids).

Data were analyzed as a 4 x 4 Latin square design using the general linear model models procedure of SAS (1989)Citation according to the following model:

where Yijkl is the observation, µ is the overall mean, Ti is the treatment (i = 1, 2, 3 and 4), Pj is the period (j = 1, 2, 3 and 4), Ck is the cow (k = 1, 2, 3 and 4) and Eijkl is the residual error. Contrasts were used to test the linear, quadratic or cubic effect of the dose of CLA-60 infusion. Values in the text are presented as means ± SEM.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The CLA supplement contained 61.2% CLA and was comprised of a mixture of four major CLA isomers (Table 2Citation ). The actual rates of infusion of CLA supplement were 0, 51.2 ± 3.7, 94.3 ± 5.1 and 147.1 ± 6.6 g/d during the four periods of the experiment for the 0, 50, 100 and 150 g/d treatments, respectively. Thus, actual amounts of CLA isomers infused for the treatments were 0, 31.3, 57.7 and 90.0 g/d. When expressed on the basis of intake, the three levels of CLA isomers represented ~0.14–0.42% of total dry matter.


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Table 2. Fatty acid profile composition of the conjugated linoleic acids (CLA) supplement1

 
Infusion of the CLA supplement had no effect on dry matter intake, which averaged 21.5 kg/d (Table 3Citation ). Moreover, the CLA supplement had minimal effects on milk yield except at the highest dose (150 g/d). CLA supplement infusion also had no effect on milk protein content and yield. In contrast, abomasal infusion of CLA supplement resulted in a dramatic reduction of milk fat synthesis. The reduction in milk fat was essentially maximized at the lowest level of CLA supplement infusion (31.3 g/d of total CLA isomers); this represented a reduction of >50% for both milk fat content and milk fat yield across the three levels of CLA infusion.


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Table 3. Dry matter intake, milk yield, and concentration and yield of milk constituents of four lactating cows during abomasal infusion of a conjugated linoleic acid (CLA) supplements12

 
Figure 1Citation shows the temporal pattern of milk fat content at each of the twice daily milkings during the 5-d infusion and the 4-d postinfusion intervals. The reduction in milk fat was already maximized after 5–6 milkings (d 3 of infusion). During the postinfusion interval, milk fat progressively increased, but it had not fully returned to normal by 4 d postinfusion. This residual effect resulted in a milk fat content that averaged only 2.8% on d 0 of the infusion periods (Fig. 1)Citation . This is lower than typical for the cows used in this study; milk fat content averaged 3.3% for the four cows during the week preceeding the initial infusion period. Thus, estimates of the magnitude of milk fat reduction by CLA infusion represent an underestimate.



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Figure 1. Temporal pattern of milk fat content in dairy cows during abomasal infusion of 0, 50, 100 and 150 g/d of a supplement of conjugated linoleic acids. Infusions lasted for 5 d, followed by a 4-d postinfusion interval. Values represent means for four cows; SEM for the percentage of milk fat ranged from 0.08 to 0.20. Treatments differed (P < 0.05) from the 3rd through the 18th milking.

 
The infusion of the CLA supplement decreased the milk fat content of short- and medium-chain length fatty acids (4:0–14:0), as well as 16:0 (Table 4Citation ). The proportions of most of these fatty acids (6:0–14:0) were altered in a quadratic pattern with the decrease already maximized at the lowest level of CLA supplementation. Moreover, the proportions of 18:0, 18:2 and 18:3 were increased by the infusion of CLA supplement, whereas 18:1 remained unchanged. The ratios of 14:0 to 14:1, 16:0 to 16:1 and 18:0 to 18:1 were increased linearly with increasing amounts of CLA supplement infused.


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Table 4. Milk fatty acid profile of four lactating cows during abomasal infusion of conjugated linoleic acid (CLA) supplements12

 
Infusion of the CLA supplement markedly increased the CLA content of milk fat (Fig. 2Citation ). All of the major CLA isomers present in the CLA supplement were transferred to milk fat in a dose-dependent manner. The proportion of each individual isomer increased gradually over the 5-d infusion interval and had generally reached a plateau by d 4. The apparent transfer efficiency of the abomasally infused CLA isomers (amount of isomer excreted in milk fat as a percentage of the amount infused) was ~22–26% for most of the isomers present in the CLA supplement (Fig. 3Citation ). The exception was the cis/trans 10,12 CLA isomer, which was transferred to milk fat with a much lower efficiency (10.2%).



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Figure 2. Temporal pattern of milk fat content of cis/trans (c/t) 8,10 conjugated linoleic acid (CLA) (panel A), c/t 9,11 CLA (panel B), c/t 10,12 CLA (panel C), and c/t 11,13 CLA (panel D) during abomasal infusion of 0, 50, 100 and 150 g/d of a CLA supplement in four dairy cows. Panel A: SEM ranged from 0.14 to 0.76 mg/g fat, and treatment effect was significant (P < 0.05) for d 1–9. Panel B: SEM ranged from 0.56 to 0.97 mg/g fat, and treatment effect was significant (P < 0.05) for d 2–9. Panel C: SEM ranged from 0.10 to 0.56 mg/g fat, and treatment effect was significant (P < 0.05) for d 1–9. Panel D: SEM ranged from 0.11 to 1.26 mg/g fat, and treatment effect was significant (P < 0.05) for d 1–9.

 


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Figure 3. Apparent transfer efficiency of abomasally infused conjugated linoleic acid isomers into milk fat of dairy cows. Each column represents the mean (± SD) for transfer, calculated on d 4 and 5 of infusion for four cows at the three levels of conjugated linoleic acid (CLA) supplement infusion.

 

    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The CLA content of milk and milk products can be altered by affecting ruminal production of CLA or trans-11 18:1, or by dietary supplementation with these fatty acids. In this study, we examined the efficiency with which a supplement of CLA was transferred to milk fat. Although the technology exists to protect dietary compounds such as CLA from rumen alterations (Doreau et al. 1997Citation ), we infused the CLA supplement abomasally to by-pass rumen fermentation processes. The addition of the CLA supplement increased the milk fat content of CLA in a dose-dependent manner from ~7 mg/g fat at the zero dose to 64 mg/g fat at the high dose of 150 g/d of CLA supplement (90 g/d of actual CLA isomers). The milk content of CLA had typically reached a constant value by 4–5 d after initiation of the daily CLA supplement infusion. Milk fat normally contains cis-9, trans-11 as the major CLA isomer, whereas the CLA supplement contained a mixture of isomers with cis/trans 8,10, cis/trans 9,11, cis/trans 10,12 and cis/trans 11,13 as the major isomers (Table 2)Citation . We observed that all of the major isomers in the CLA supplement were transferred to milk fat in a dose-dependent manner (Fig. 2)Citation . However, there were differences among the isomers in the efficiency of transfer. Only ~10% of the dietary supplement of cis/trans 10,12 CLA isomer was transferred to milk fat, whereas the cis/trans 8,10, cis/trans 9,11 and cis/trans 11,13 CLA isomers were transferred to milk fat with over twice the efficiency (22–26%) (Fig. 3)Citation .

The abomasal infusion of the CLA supplement had a dramatic effect on milk fat yield. Across all CLA doses, the content and yield of milk fat were reduced by 52 and 55%, respectively (Table 3)Citation . This was surprising because earlier studies in which lactating rats were fed a CLA supplement at 0.25 and 0.50% of diet dry matter showed no indication of reduction in milk fat secretion as indicated by growth rates of the nursing pups (Chin et al. 1994Citation ). Loor and Herbein (1998)Citation also observed a reduction in milk fat in dairy cows that received a 24-h abomasal infusion of CLA (100 g). In our study, the reduction in milk fat was of a similar magnitude at all levels of CLA supplement infusion; the low dose of 50 g/d of CLA supplement (31.3 g/d of CLA isomers) represented a dietary level of CLA isomers of 0.14% on a dry matter basis.

Certain dietary situations reduce milk fat in lactating cows; this is referred to as milk fat depression or low milk fat syndrome (Davis and Brown, 1970Citation , Sutton, 1989Citation ). High concentrate diets, addition of rumen-active fats and diets in which the forage is of small particle size are examples of conditions that lead to decreased milk fat yield. Typically, these situations cause reciprocal changes in fat synthesis in which mammary synthesis of milk fat is reduced and fat accretion in adipose tissue is increased (Davis and Brown 1970Citation , Griinari et al. 1997a and 1997bCitation Citation , Sutton 1989Citation ). It is not known whether a reciprocal increase in fat synthesis occurred in adipose tissue from cows exhibiting the CLA-induced reduction in milk fat. However, reciprocal changes may not have occurred in lactating cows because dietary CLA supplements (mixture of isomers) have been shown to decrease body fat accretion in several species of growing animals, including mice (DeLany et al. 1998Citation , Park et al. 1997Citation ) and pigs (Cook et al. 1998Citation , Dugan et al. 1997Citation , Dunshea et al. 1998Citation ). If fat accretion was decreased or not altered in the CLA supplement-infused cows, we would expect voluntary intake to decrease by an amount energetically equal to the reduction in milk fat. We observed minimal changes in voluntary intake over the 5-d infusion period (Table 3)Citation , but longer-term studies would be required to assess this adequately.

The effect of the CLA supplement on the performance of lactating cows was specific for milk fat; the yield of milk protein was not affected, and effects on milk yield were minimal except at the highest CLA dose (Table 3)Citation . The milk fat of dairy cows contains a distinct pattern of fatty acids that originates from de novo fatty acid synthesis, uptake of preformed fatty acids and desaturation of a portion of the long-chain fatty acids. The reduction in milk fat due to the CLA supplement was relatively greater for the short- and medium-chain fatty acids (Table 4)Citation . These represent the portion synthesized de novo by the mammary epithelial cells. In addition, {Delta}-9 desaturase catalyzes the desaturation of longer-chain fatty acids, and the ratios of 14:0 to 14:1, 16:0 to 16:1 and 18:0 to 18:1 were all increased with CLA supplement infusion (Table 4)Citation . This raises the possibility that the mechanism of the CLA inhibition involves both de novo fatty acid synthesis and the desaturation process. However, it is important to recognize that the full complement of fatty acids is required to obtain a pattern of triglycerides with appropriate fluidity characteristics. Thus, the specific site(s) at which CLA inhibits milk fat synthesis could involve any of these pathways as well as the esterification, packaging and secretion of milk fat triglycerides.

It is also not known whether the inhibition of fat synthesis is specific for one or more of the CLA isomers. We do not think that the cis-9, trans-11 CLA isomer is involved because we have observed that milk fat content of cis-9, trans-11 CLA is not correlated with fat content of milk (Griinari et al. 1997aCitation ). Furthermore, Lee et al. (1998)Citation observed that a dietary supplement containing a mixture of CLA isomers decreased rat liver expression of {Delta}-9 desaturase mRNA, but the effect involved isomers other than cis-9, trans-11 CLA. Recently, we have shown that the classical diet-induced milk fat depression is associated with an increase in the milk fat content of trans-10 18:1 (Griinari et al. 1998aCitation ). Thus, it may be that the specific CLA isomers affecting fat synthesis in the mammary gland (lactation) and adipose tissue (growth) are those with a trans-10 double bond (cis-8, trans-10 CLA and trans-10, cis-12 CLA).

Overall, this study demonstrated that the milk fat content of CLA can be increased via CLA supplement infusion. However, the most pronounced effect of the CLA supplement was to cause a reduction in milk fat yield of >50% and a shift in the fat composition to more long-chain fatty acids. The possibility of specific roles for the different CLA isomers and the specific site(s) of inhibition of mammary synthesis of milk fat remain to be elucidated.


    ACKNOWLEDGMENTS
 
The assistance of Kathryn Jarrett and Yvonne Feldpausch, Joanna Lynch and Dottie Ceurter is greatly appreciated. The authors also gratefully acknowledge the donation of CLA supplement by Natural Lipids LTD and protein supplement by Taylor By-Products.


    FOOTNOTES
 
1 Presented in part at the Cornell Nutrition Conference, October 1997, Rochester, NY [Griinari, J. M., Chouinard, P. Y. & Bauman, D. E. (1997) Trans fatty acid hypothesis of milk fat depression, revised. Proceedings, pp. 208–216]. Also presented in part in abstract form at 93rd Annual Meeting of the American Dairy Science Association, July 1998, Denver, CO [Chouinard P. Y., Corneau L., Bauman D. E., Barbano D. M. & Metzger L. E. (1998) Milk yield and composition during abomasal infusion of conjugated linoleic acid in dairy cows, J. Dairy Sci. 81 (suppl. 1): 353 (abs.)]. Back

2 Supported in part by Dairy Management, (Rosemont, IL), Northeast Dairy Foods Research Center and the Cornell Agricultural Experiment Station. Back

3 Current address: Département des sciences animales, Pavillon Paul-Comtois, Université Laval, Québec, Canada G1K 7P4. Back

5 Abbreviation used: c, cis; CLA, conjugated linoleic acids, FAME, fatty acid methyl esters; t, trans. Back

Manuscript received October 16, 1998. Initial review completed January 13, 1999. Revision accepted April 27, 1999.


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 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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