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Conjugated Linoleic Acid Increases in Milk from Cows Fed Condensed Corn Distillers Solubles and Fish Oil¹

M. Bharathan^*,2, D. J. Schingoethe^*,3, A. R. Hippen^*, K. F. Kalscheur^*, M. L. Gibson and K. Karges

^* Dairy Science Department, South Dakota State University, Brookings 57007-0647
Poet Nutrition, Sioux Falls, SD 57104

³ Corresponding author: david.schingoethe{at}sdstate.edu

	ABSTRACT

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS AND DISCUSSION CONCLUSIONS ACKNOWLEDGEMENTS REFERENCES

 
Twelve lactating Holstein cows were randomly assigned to 1 of 4 experimental diets in a replicated 4 x 4 Latin square design with 4-wk periods to ascertain the lactational response to feeding fish oil (FO), condensed corn distillers solubles (CDS) as a source of extra linoleic acid, or both. Diets contained either no FO or 0.5% FO and either no CDS or 10% CDS in a 2 x 2 factorial arrangement of treatments. Diets were fed as total mixed rations for ad libitum consumption. The forage to concentrate ratio was 55:45 on a dry matter basis for all diets and the diets contained 16.2% crude protein. The ether extract concentrations were 2.86, 3.22, 4.77, and 5.02% for control, FO, CDS, and FOCDS diets, respectively. Inclusion of FO or CDS or both had no effect on dry matter intake, feed efficiency, body weight, and body condition scores compared with diets without FO and CDS, respectively. Yields of milk (33.3 kg/d), energy-corrected milk, protein, lactose, and milk urea N were similar for all diets. Feeding FO and CDS decreased milk fat percentages (3.85, 3.39, 3.33, and 3.12%) and yields compared with diets without FO and CDS. Proportions of trans-11 C18:1 (vaccenic acid), cis-9 trans-11 conjugated linoleic acid (CLA; 0.52, 0.90, 1.11, and 1.52 g/100 g of fatty acids), and trans-10 cis-12 CLA (0.07, 0.14, 0.13, and 0.16 g/100 g of fatty acids) in milk fat were increased by FO and CDS. No interactions were observed between FO and CDS on cis-9 trans-11 CLA although vaccenic acid tended to be higher with the interaction. The addition of CDS to diets increased trans-10 C18:1. Greater ratios of vaccenic acid to cis-9 trans-11 CLA in plasma than in milk fat indicate tissue synthesis of cis-9 trans-11 CLA in the mammary gland from vaccenic acid in cows fed FO or CDS. Feeding fish oil at 0.5% of diet dry matter with a C18:2 n-6 rich source such as CDS increased the milk CLA content but decreased milk fat percentages.

Key Words: fish oil • condensed corn distillers solubles • conjugated linoleic acid • cow

	INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS AND DISCUSSION CONCLUSIONS ACKNOWLEDGEMENTS REFERENCES

 
Studies conducted at South Dakota State University showed that the conjugated linoleic acid (CLA) content of milk fat from cows fed sources of n-6 fatty acids such as linoleic and linolenic acids can be further increased by feeding fish oil (FO) in combination with the plant oils and the increase in CLA was more than the additive effect of the FO and plant oils high in linoleic acid (Whitlock et al., 2002; AbuGhazaleh et al., 2003a). Fish oil is more effective than plant oils to increase cis-9 trans-11 CLA in milk fat (Offer et al., 1999; Donovan et al., 2000; Chouinard et al., 2001).

Fish oil contains virtually no CLA but stimulates rumen bacteria to make increased amounts of CLA from fat sources that are high in linoleic acid such as soybeans, sunflower seeds, and possibly corn oil found in CDS (AbuGhazaleh et al., 2002; Whitlock et al., 2002) and only a small amount of FO is needed with the high linoleic acid source (Whitlock et al., 2006). Greater amounts of the n-6 fatty acids linoleic, linolenic, as well as the n-3 fatty acids eicosapentaenoic acid (EPA; C20:5n-3) and docosahexaenoic acid (C22:6n-3), which are not synthesized in ruminant tissues, need to be supplied in diets for them and CLA to appear in milk in increased concentrations.

Byproducts of ethanol production include condensed corn distillers solubles (CDS), which provide an excellent source of energy, especially fat, and distillers grains with solubles (DGS); both are relatively inexpensive sources of protein and energy. The major fatty acid in CDS is linoleic acid, which accounts for approximately 52% of the total fatty acids (DaCruz et al., 2005; Sasikala-Appukuttan et al., 2008). Feeding 5 or 10% CDS to lactating cows increased production in the study by DaCruz et al. (2005); greater amounts may also be fed (Sasikala-Appukuttan et al., 2008). The objective of this research was to assess and compare the response in the CLA content of milk fat when CDS was fed with or without fish oil to lactating dairy cows. We hypothesized that feeding a small amount of fish oil (as a modifier of ruminal biohydrogenation) with a source of linoleic acid such as CDS (as a substrate) would increase the CLA content of the milk and the content of its precursor vaccenic acid (VA) more than feeding the CDS alone.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS AND DISCUSSION CONCLUSIONS ACKNOWLEDGEMENTS REFERENCES

 
Experimental Plan
This experiment was performed at South Dakota State University Dairy Research and Training Facility with all procedures conducted under approval of the South Dakota Animal Care and Use Committee. The objective of this experiment was to assess and compare the effect of feeding FO or CDS or both on DMI, milk production, and milk and plasma fatty acid composition. Twelve mid-lactation Holstein cows (8 multiparous and 4 primiparous), averaging 124 (±10) DIM at the start of the experiment were assigned to 1 of the 4 experimental diets in a replicated 4 x 4 Latin square design with a 2 x 2 factorial arrangement of treatments. Cows were blocked by parity, production, and DIM, and then, within blocks (squares), assigned at random to experimental diets. Each experimental period consisted of 4 wk, with wk 1 and 2 for adaptation and wk 3 and 4 for data collection. Cows were housed in a free-stall barn and adapted to Calan doors (American Calan Inc., Northwood, NH) 1 wk before start of the experiment. Diets were fed as TMR for ad libitum consumption using the Calan Broadbent feeder door and box system.

The experimental diets consisted of 1) control (no FO and no CDS), 2) control diet with 0.5% fish oil (FO), 3) control diet with 10% CDS supplying 2% dietary fat (CDS), and 4) control diet with 10% CDS and 0.5% fish oil (FOCDS). All diets (Table 1) were balanced to provide recommended total daily nutrients for a dairy cow producing 45 kg of milk/d (NRC, 2001). Diets were formulated to contain 17% CP using ground shelled corn and soybean meal as the base ingredients of the concentrate mix; CDS and FO replaced portions of these ingredients in the treatment diets. The FO (Menhaden) was included in the concentrate mix. The CDS was stored in a tank of approximately 1,100 kg capacity at the farm and mixed thoroughly by circulation for 1 h before incorporating into the diet. The proportion of diet ingredients was a 55:45 blend of forage and concentrate on a DM basis. The forage portion of all diets, containing 27.5% alfalfa hay and 27.5% corn silage (DM basis), was premixed in a mixer wagon (1999 NDE 500, Westside Implement, Clark, SD). Concentrate mix and CDS were added to the Calan Data Ranger (American Calan Inc.) after addition of premixed forages at the time of feeding. Cows were milked 3 times a day at 0600, 1400, and 2100 h, with milk production recorded daily.

Milk samples were collected at all 3 milking times for 2 consecutive days during wk 3 (d 20 and 21) and 4 (d 27 and 28) of each period. Composites of milk samples were made by day and sent to Heart of American DHI Laboratory (Manhattan, KS) for analysis using AOAC (2002) approved methods. Analysis of milk fat, protein, and lactose were done by near infrared spectroscopy (Bentley 2000 Infrared Milk Analyzer, Bentley Instruments, Chaska, MN), while MUN concentration was determined using chemical methods based on a modified Berthelot reaction (Chaney and Marbach, 1962; ChemSpec 150 Analyzer, Bentley Instruments). Somatic cell counts were determined with a flow cytometer laser (Somacount 500, Bentley Instruments). Energy-corrected milk was determined using the equation [(0.327 x kg of milk) + (12.95 x kg of fat) + (7.2 x kg of protein)] (Orth, 1992).

Cows were weighed on 3 consecutive days before the beginning of the trial and on the last 3 d of each period. At the start of the trial and end of each period, BCS were recorded by 3 independent individuals. Body condition scores were based on a scale of 1 to 5, with 5 representing obese and 1 representing emaciated (Wildman et al., 1982).

Rumen fluid was collected on 2 d in wk 4 of each period, approximately 3 h postfeeding by applying vacuum pressure to an esophageal tube with a suction strainer on the rumen end. The first 200 to 300 mL of rumen fluid was discarded before sample collection to minimize contamination with saliva. Ten-milliliter aliquots of rumen fluid samples were mixed with 2 mL of 25% (wt/vol) meta-phosphoric acid and placed immediately into storage tubes and stored at –20°C until analysis. Rumen liquor was analyzed for ammonia nitrogen concentration (Chaney and Marbach, 1962) and VFA (Ottenstein and Bartley, 1971) using GC (model 6890, Hewlett-Packard, Palo Alto, CA) with a flame-ionization detector. The injector port was at a temperature of 250°C with a split ratio of 100:1 using a column 15 m in length and 0.25 mm in diameter (Nukal, Supelco Inc., Bellefonte, PA). Flow rate was 1.3 mL/min of helium, and the detector and column were maintained at 225 and 130°C, respectively.

Blood samples were collected from a coccygeal vessel approximately 3 h after feeding on the last day of wk 4 of each period. Blood was drawn into K₃ EDTA vacuum tubes (Becton Dickinson and Co., Franklin Lakes, NJ). The samples were immediately placed on ice and were taken to the laboratory where they were centrifuged (500 x g) for 20 min to separate the plasma. Plasma was stored at –20°C until further analysis.

Fatty Acid Analysis.
Feed fatty acids were extracted and prepared as butyl esters (Abu-Ghazaleh et al., 2002) for analysis using GC (model 6890, Hewlett-Packard, Palo Alto, CA). The samples were analyzed using a flame-ionization detector. The injector port was set at a temperature of 230°C with a split ratio of 100:1. The column was 100 m long and had an inside diameter of 0.25 mm (Supelco 2560, Supelco Inc.). The flow rate was 2.0 mL/min of helium; the detector was maintained at 250°C. Initial temperature was 50°C held for 1 min, and then increased to 145°C at a rate of 5°C/min, and held for 30 min. The temperature was then increased to 190°C at the rate of 10°C/min and held for 30 min, and finally, the temperature was increased 5°C/min to 210°C, and held for 35 min. The total run per sample was 129 min and fatty acids were identified based on elution patterns of known fatty acids. Standard mixtures of fatty acids (FIM-FAME-7, Matreya Inc., Pleasant Gap, PA; and GLC-68D, Nu-Chek Prep Inc., Elysian, MN) and standards for cis-9 trans-11 and trans-10 cis-12 CLA (Matreya Inc.) were analyzed for identification of the fatty acid elution positions.

Composites of milk samples for each of wk 3 and wk 4 were made for each cow and period and prepared for milk fatty acid composition analysis using an adapted method (Loor et al., 2005) originally described by Sukhija and Palmquist (1988), except that it was modified to form butyl esters (Abu-Ghazaleh et al., 2002). An internal standard C13:1 was used. The samples were then analyzed by GC (model 6890, Hewlett Packard) as described for feed fatty acids.

Plasma samples were analyzed for fatty acids using GC (model 6890, Hewlett-Packard). Fatty acids from 1 mL of whole plasma were butylated with n-butanol, followed by acetylation with acetyl chloride as described above without prior extraction. The fatty acid C13:1 was used as the internal standard. Samples were kept for incubation at 100°C for 90 min, followed by the addition of 6% potassium carbonate and 1 mL of hexane at room temperature. Samples were centrifuged and washed 3 times with double distilled water. Fatty acid butyl esters recovered in 1 mL of hexane were transferred to a GC vial. Five microliters of butyl esters in hexane were injected at a 20:1 split ratio. The injector port was at a temperature of 250°C with a split ratio of 20:1.

Statistical Analysis
Weekly means of DMI, milk yield, milk composition, and fatty acid profiles of milk for each period were used for statistical analysis. Rumen liquor and blood collected on d 28 of each period were used for statistical analysis. The design of this experiment was a 4 x 4 Latin square with a 2 x 2 arrangement of treatments. The data were analyzed using the MIXED procedures of SAS (SAS Institute, 1999) with the model:

where µ = overall mean, P_i = effect of period (i = 1 to 4), C_j(Rm) = effect of cow nested within parity (j = 1 to 12), FO_k = effect of FO (k = 1 to 2), CDS_l = effect of CDS (l = 1 to 2), (FO_k x CDS_l) = interaction of FO_k and CDS_l, R_m = effect of parity (m = 1 to 2), (FO_k x R_m) = interaction of FO_k and parity m, (CDS_l x R_m) = interaction of CDSl and parity m, (FO_k x CDS_l x R_m) = interaction of FO_k, CDS_l, and parity m, and e_ijklm = random residual error. Cow within parity was the random effect. Parity remained in the model for all variables, although interactions with parity that were not significant were dropped from the model. There were no interactions of main effects with period for any variable; therefore, they were not included in the model. Significance was declared at P < 0.05, and tendency was indicated at P < 0.10.

	RESULTS AND DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS AND DISCUSSION CONCLUSIONS ACKNOWLEDGEMENTS REFERENCES

 
Nutrient Content of Diets
Nutrient composition of diet components and TMR are shown in Table 2. Dry matter content of the diets varied from 55.8 to 45.5%. As expected, inclusion of CDS in diets decreased the DM content to 45.5% and remained similar among CDS diets. Diets were formulated to be isonitrogenous (17%); however, analyses were slightly lower at 16.2 to 16.3% CP, because of less protein in the alfalfa hay than anticipated. Diets were similar in NDF, ADF, and lignin. As intended, the ether extract contents were higher for diets containing FO and CDS. Fatty acid compositions of TMR, concentrate mixes, forages, FO, and CDS (Table 3) were typical of what was expected. The fat in CDS was approximately 52% C18:2, the major precursor for CLA. The n-3 fatty acids C22:5 and C22:6 were detected only in FO diets.

Milk fat percentage and milk fat yield decreased (P < 0.01) in cows fed FO compared with those not fed FO (3.26 vs. 3.59%). Decreased milk fat percentage and fat yield (P < 0.01 and 0.04, respectively) were observed for cows fed CDS compared with cows not fed CDS (3.23 vs. 3.62%). These results agreed with the findings of previous research with CDS (Da Cruz et al., 2005). When FO is fed to dairy cows, milk fat depression is usually observed (Cant et al., 1997; Donovan et al., 2000). Inclusion of FO or CDS or both to the diets resulted in lowered milk fat percentage, which may be due to the rapid availability of oil in the rumen and its potential effect on fiber digestibility (Jenkins and Jenny, 1989). Ruminal VFA data (reported later) did not indicate differences in fiber digestibility, although that was not measured. The oil in CDS is likely more available to interfere with ruminal fiber digestibility and to be more available for ruminal biohydrogenation than is the oil in DGS. This may be why milk fat depression may occur when feeding CDS but does not usually occur when feeding DGS in diets that contain sufficient amounts of forage fiber (Anderson et al., 2006).

Milk protein percentage was not affected by the addition of FO, although CDS tended (P = 0.10) to decrease milk protein percentage. Milk protein yield and MUN were not affected by the addition of FO or CDS to the diets.

The addition of FO did not affect milk lactose percentage; however, addition of CDS to the diets decreased milk lactose percentage (P < 0.05) only when also in the presence of FO, but the lactose yield was not affected by the addition of FO or CDS or both to the diets. Although some of the above values were statistically significant, the numerical values were very small and likely not biologically significant.

The addition of FO, CDS, or both did not affect feed efficiency. However, feed efficiency for multiparous cows fed CDS was greater (P < 0.05) than for primiparous cows fed CDS (1.65 vs. 1.37, respectively). This likely reflected that a portion of the diet fed to primiparous cows was used for growth rather than for milk, and may indicate that multiparous cows have been mobilizing more tissues (NRC, 2001).

Fatty Acid Composition of Milk
Inclusion of FO, CDS, or both in the diets resulted in marked changes in fatty acid composition of milk fat (Table 5). The total concentration of saturated fatty acids decreased (P = 0.01) from 64.7 to 62.1 g/100 g of fatty acids fed FO and decreased (P < 0.01) from 66.4 to 60.4 g/100 g of fatty acids when fed CDS. These results were expected because inclusion of C18:2-rich sources like CDS in dairy cow diets often increases the unsaturated fatty acid concentration in milk fat (Sasikala-Appukuttan et al., 2008). Other studies also documented the increase in unsaturated fatty acids in milk fat by the addition of FO or C18:2-rich sources such as extruded soybeans (Schingoethe et al., 1996; Abu-Ghazaleh et al., 2002; Whitlock et al., 2002). In this study, 2% of fat in the CDS diets supplied as CDS (rich in C18:2) contributed to the increase in total unsaturated fatty acids in these diets.

Concentrations of long-chain fatty acids (C18:0 to C22:6) in milk fat were not affected by the addition of FO to the diets but increased (P < 0.01) from 34.3 to 40.6 g/100 g of fatty acids with the supplementation of CDS to the diets. These results agreed with the findings of Whitlock et al. (2006), who used 0.33 and 0.67% fish oil and Sasikala-Appukuttan et al. (2008), who fed 10% CDS in dairy cow rations.

The concentrations of C18:1 and C18:2 isomers in milk fat are presented in Table 6. The major isomer of CLA in milk cis-9 trans-11 C18:2 increased (P < 0.01) from 0.82 to 1.21 g/100 g of fatty acids when FO was added to the diets. Similarly, an increase (P < 0.01) from 0.71 to 1.32 g/100 g of fatty acids was noted when CDS was added to the diets. Even though the inclusion of both FO and CDS increased the concentration of cis-9 trans-11 CLA, the increase was additive with either FO or CDS alone, with no interaction of FO and CDS. Based on the research of Whitlock et al. (2002), an interaction of FO and CDS might have been expected to occur. Cis-9 trans-11 C18:2 typically comprises 85 to 95% of the CLA in ruminant milk fat (Chin et al., 1992), which was also true in this experiment.

The concentrations of VA (trans-11 C18:1) in the milk fat were also increased (P < 0.01) when FO (1.26 vs. 1.94 g/100 g of fatty acids) or CDS (0.99 vs. 2.21 g/100 g of fatty acids) were added to the diets (Table 6). Supplementation of both FO and CDS to the diets also showed a tendency (P = 0.07) for VA to increase in milk. The increase over the control due to both FO and CDS (1.9 g/100 g of fatty acids) tended (P < 0.07) to be greater than the additive effect of FO and CDS (1.2 g/110 g of fatty acids), a response that was hypothesized to occur based on the response when cows were fed fish oil and extruded soybeans (Whitlock et al., 2002). Much of the increase in cis-9 trans-11 C18:2 CLA in the current study was likely due to the endogenous synthesis from trans-11 C18:1 in the mammary gland by ⁹-desaturase (Griinari et al., 2000). This close relationship between concentrations of VA as a precursor for synthesis of CLA in milk fat across the diets also explains the increased concentration of cis-9 trans-11 CLA in milk.

The second major CLA isomer, trans-10 cis-12 C18:2, also increased (P < 0.01) when FO or CDS was added to the diets. No FO by CDS interaction was noticed for trans-10 cis-12 CLA in this experiment. The current findings are similar to other studies involving diet-induced milk fat depression (Donovan et al., 2000; Da Cruz et al., 2005). Trans-10 cis-12 CLA is considered to be a potential inhibitor of milk fat synthesis as demonstrated with postruminal infusion studies (Baumgard et al., 2002). Previous studies (Whitlock et al., 2002) demonstrated that feeding FO to dairy cows could increase the concentration of trans-10 cis-12 CLA in milk. This correlated with the observed milk fat depression when FO or CDS was added to the diets in the current study. The isomer trans-10 C18:1 was also attributed to cause milk fat depression (Griinari et al., 1998); however, the infusion study by Lock et al. (2007) demonstrated that trans-10 C18:1 had no effect on milk fat depression. The concentration of trans-10 C18:1 in milk also increased (P < 0.01) when CDS was added to the diets in this study. It increased when CDS was supplemented, whereas little change occurred when FO was added to the diets. Although an increase in trans-10 C18:1 was observed with the inclusion of both FO and CDS in diets, no significant FO by CDS interaction was noted. Increased trans-10 cis-12 C18:2 together with a greater concentration of total C18:1 trans fatty acids were related to the milk fat depression with supplementation of the diets with FO or CDS.

The concentrations of EPA in milk fat increased (P < 0.01) when FO was added to the diets whereas it decreased (P < 0.01) with CDS diets (Table 5). Docosahexaenoic acid concentration tended to be greater (P = 0.07) in milk from cows fed FO; however, docosahexaenoic acid decreased (P < 0.05) with dietary supplementation of CDS. Total n-3 fatty acids in milk increased (P = 0.01) with the addition of FO to the diets. These results agreed with the findings of Whitlock et al. (2006), who observed an increase in total n-3 fatty acids when small amounts of FO was fed to dairy cows. Un-identified fatty acids, which are reported as others, also increased (P < 0.01) with the addition of FO or CDS or both to the dairy cow diets.

Plasma Fatty Acid Composition
Unlike milk, plasma fatty acid composition (Table 7) did not differ much between diets except for some key fatty acids, which agreed with the results of Loor et al. (2005). Greater concentrations of C16:1, trans-11 C18:1, cis-11 C18:1, C18:3n-3, C20:0, C20:1, C22:5n-3, C22:6n-3, and total n-3 were observed in plasma as a result of adding FO to the diets. Feeding FO also resulted in decreased concentrations of C18:0, C18:3n-6, and C20:3; however, the concentrations of EPA, CLA isomers, medium-chain, long-chain, unsaturated, and saturated fatty acids were not affected compared with no FO diets. Supplementation with CDS resulted in greater (P < 0.01) plasma concentrations of trans-10 C18:1 and trans-11 C18:1 compared with no CDS diets. Lower concentrations of trans-6 C18:1, C18:3n-6, and C22:4 were observed in cows fed CDS compared with cows fed no CDS. The proportions of C18:3n-3 tended to increase in CDS-fed cows. No FO by CDS interaction was observed for any of the key fatty acids.

Rumen Liquor Analysis
The results (Table 8) indicated that rumen ammonia concentrations were within a normal range and did not differ among treatments because of similar CP content of diets. Ruminal pH was measured and was similar for all diets; however, the data are not reported because pH values obtained via esophageal samples are not always accurate. Ruminal proportions of propionate, isobutyrate, isovalerate, total VFA, and acetate to propionate ratio were similar among diets (P > 0.05). Ruminal proportions of acetate were unaffected by the addition of FO; however, acetate tended to (P = 0.08) decrease when CDS was added to the diets. Inclusion of CDS to the diets increased (P < 0.01) the ruminal proportions of butyrate; however, no effect was found with the addition of FO. Supplementation of FO to the diets increased (P < 0.01) ruminal proportions of valerate. In general, the ruminal ammonia and VFA data did not appear to influence milk fatty acid data, which was the main thrust of this experiment.

	CONCLUSIONS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS AND DISCUSSION CONCLUSIONS ACKNOWLEDGEMENTS REFERENCES

	ACKNOWLEDGEMENTS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS AND DISCUSSION CONCLUSIONS ACKNOWLEDGEMENTS REFERENCES

 
The authors gratefully acknowledge the farm crew at the South Dakota State University Dairy Research Unit for care of the cows and assistance with data collection. The authors also wish to acknowledge the South Dakota Corn Utilization Council (Sioux Falls, SD) for partial financial support, Poet Nutrition (Sioux Falls, SD) for supplying CDS, and Omega Protein International (Hammond, LA) for menhaden fish oil.

	FOOTNOTES

 
¹ Published with the approval of the director of the South Dakota Agricultural Experiment Station as Publication Number 3618 of the Journal series.

² Current address: Dairy Science Department, Virginia Polytechnic Institute and State University, Blacksburg 24061.

Received for publication December 11, 2007. Accepted for publication February 26, 2008.

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TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS AND DISCUSSION CONCLUSIONS ACKNOWLEDGEMENTS REFERENCES

 


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