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J. Anim Sci. 2008. 86:187-196. doi:10.2527/jas.2007-0085
© 2008 American Society of Animal Science
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ANIMAL NUTRITION

Supplemental algal meal alters the ruminal trans-18:1 fatty acid and conjugated linoleic acid composition in cattle1

M. M. Or-Rashid*,2,3, J. K. G. Kramer*, M. A. Wood{dagger} and B. W. McBride{dagger}

* Food Research Program, Agriculture and Agri-Food Canada, Guelph, Ontario, Canada N1G 5C9; and {dagger} Department of Animal and Poultry Science, University of Guelph, Guelph, Ontario, Canada N1G 2W1


   Abstract
Top
 Abstract
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS AND DISCUSSION
 LITERATURE CITED
 
The effects of dietary algal supplementation, a source of docosahexaenoic acid, on the fatty acid profile of rumen lipids in cattle were evaluated, with special emphasis on CLA and trans fatty acids produced by rumen microbes. A diet based on corn silage was fed with supplements containing the following: 1) no algal meal and fed at 2.1 kg of DM/d (control), 2) algal meal and fed at 1.1 kg of DM/d (low algal meal), 3) algal meal and fed at 2.1 kg of DM/d (medium algal meal), and 4) algal meal and fed at 4.2 kg of DM/d (high algal meal). A modified lipid extraction procedure was developed to analyze the lipid changes in rumen fluid. The percentage of stearic acid (18:0) in rumen fluid was decreased by algal meal supplementation (P < 0.001) compared with control and was linearly dependent on the level of algal meal supplementation (P = 0.005). Total trans-18:1 in rumen fluid of cattle fed the control diet was 19% of total fatty acids. Addition of algal meal increased (P < 0.001) total trans-18:1 up to 43%, mostly due to 18:1 trans-10 that increased (P = 0.002) to 29.5% of total rumen fatty acids. This increase in 18:1 trans-10 seems to suggest a change in the rumen microbial population. Vaccenic acid (18:1 trans-11) increased quadratically (P = 0.005) with increasing level of algal meal supplementation in the diets. The total CLA content was low in the control (<0.9%) and increased with dietary algal meal addition, although not significantly; the greatest level was 1.5% with the medium algal meal diet. The increase of rumenic acid (cis-9, trans-11 CLA) was quadratic (P = 0.05) with algal meal supplementation, whereas trans-10, cis-12 CLA increased linearly with increased level of algal meal from 0.08 to 0.13% (P = 0.03). The ratio of trans-11 (cis-9, trans-11 CLA + 18:1 trans-11) to trans-10 (trans-10, cis-12 CLA + 18:1 trans-10) decreased from 2.45 to 0.77, 0.87, and 0.21 for the control, low algal meal, medium algal meal, and high algal meal diets, respectively. The content of docosahexaenoic acid in rumen fluid increased (P = 0.002) from 0.3 to 1.4% of total fatty acids with increasing level of algal meal supplementation in the diets. Our results suggest that algal meal inhibits the reduction of trans-18:1 to 18:0, giving rise to the high trans-18:1 content. In conclusion, algal meal could be used to increase the concentration in rumen contents of trans-18:1 isomers that serve as precursors for CLA biosynthesis in the tissues of ruminants.

Key Words: algal meal • conjugated linoleic acid • docosahexaenoic acid • rumen fluid • vaccenic acid


   INTRODUCTION
Top
 Abstract
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS AND DISCUSSION
 LITERATURE CITED
 
Milk and meat fats from ruminant animals play an important role in human nutrition (Parodi, 2004Go). In recent years, there have been attempts to increase the amount of rumenic acid, the cis-9, trans-11 isomer of CLA, and vaccenic acid (VA, 18:1 trans-11) in ruminant fat because of their reported health benefits (Lock and Bauman, 2004Go). Lipids in cattle rations are extensively hydrolyzed in the rumen, and the unsaturated fatty acids, specifically linoleic (18:2n-6) and linolenic acid (18:3n-3), are isomerized and then reduced via trans-octadecenoic acids (18:1) to stearic acid (18:0) by rumen bacteria (Kepler et al., 1966Go; Harfoot and Hazlewood, 1997Go). The major CLA isomer in ruminant products such as milk, meat, and butter is cis-9, trans-11 CLA, produced mainly by {Delta}9 desaturation from VA in tissues of ruminants and the mammary gland (Bauman and Griinari, 2003Go).

Many recommendations have been made by health regulatory agencies to increase the intake of n-3 PUFA, such as eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) in the human diet (National Academy of Sciences, 2002Go). The potential benefits associated with increased n-3 PUFA intake include protection against cardiovascular disease, atherosclerosis, improved neural development and visual acuity, reduced inflammation, arrhythmia, and circulating triglyceride levels (Knapp et al., 2003Go). A number of sources have been used to enrich milk and muscle fat with DHA and EPA, such as fish oil (Enser et al., 1999Go; Wachira et al., 2002Go; AbuGhazaleh et al., 2003Go), fish meal (Wright et al., 2003Go; AbuGhazaleh et al., 2004Go), and algae (Franklin et al., 1999Go; Papadopoulos et al., 2002Go).

The inclusion of fish products in the rations of ruminants was reported to increase the concentration of CLA and VA in duodenal or omasal contents compared with control (Shingfield et al., 2003Go; Lee et al., 2005Go; Loor et al., 2005aGo). The increased flow of VA from the rumen is desirable, because it would result in an increase of cis-9, trans-11 CLA in the meat and milk of ruminants; VA is converted to cis-9, trans-11 CLA by the action of {Delta}9-desaturase (Bauman and Griinari, 2003Go). It was also reported that animals fed high-concentrate diets (Piperova et al., 2002Go) showed 5 times more accumulation of 18:1 trans-10 in rumen than animals consuming low-concentrate diets. However, the effect of algae on ruminal fatty acid composition has not been reported. Therefore, the objective of the current study was to evaluate the ruminal fatty acid changes, especially CLA, VA, and 18:1 trans-10, as a result of supplementing algal meal.


   MATERIALS AND METHODS
Top
 Abstract
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS AND DISCUSSION
 LITERATURE CITED
 
Management of Animals and Rumen Fluid Collection

Animals were cared for and handled in accordance with the Canadian Council on Animal Care regulations, and the University of Guelph Animal Care Committee reviewed and approved the experiment and all procedures carried out in the study. Four Holstein cows (620 ± 34 kg of BW) housed in a tie-stall facility at the Elora Dairy Research Center (University of Guelph) were used in a 4 x 4 Latin square design with 3-wk periods. Weeks 1 and 2 were for adaptation to the new feed levels, and the third week was the collection period. The animals were fed a diet based on corn silage, for ad libitum intake (allowing for a 5% refusal; Table 1Go), twice daily at 0800 and 1500 and had unlimited access to fresh water. Orts were collected before the morning feeding. The algal meal and the control formulations were pelleted and fed as a top-dress at each feeding (Table 2Go). The algal pellet was fed at 3 levels. The low level of algal meal (L-Alg) was fed at 1.1 kg/d (DM basis), the medium level of algal meal (M-Alg) at 2.1 kg/d (DM basis), and the high level of algal meal (H-Alg) at 4.2 kg/d (DM basis). The control pellet was offered at the same inclusion level as the M-Alg treatment (2.1 kg/d on DM basis). The diets provided 0.0 (control), 2.52 (L-Alg), 4.82 (M-Alg), or 9.64 (H-Alg) g of DHA/d. The algal meal was a partially delipidated residue of Crypthecodinium cohnii obtained from Martek Biosciences Corporation (Columbia, MD). Rumen fluid was taken during each collection week (on the last day), 3 to 4 h after the morning feeding, via stomach tube. Approximately 500 mL of fluid was collected and then filtered using 4 layers of cheesecloth. Aliquots of samples were frozen at –20°C for subsequent analyses.


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  		Table 1. Basal diet ingredients and chemical analysis
 

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  		Table 2. Ingredients and chemical analysis (DM basis) for algal and control pellets
 
Preparation and Derivatization of the Sample for Fatty Acid Analysis

Rumen fluid was thawed and placed in 2.0-mL Eppendorf tubes. One gram of zirconium beads (0.5 mm and 0.1 mm in a 1:1 ratio by weight) was added into the Eppendorf tube containing rumen fluid and shaken for 2.5 min twice, with a 5-min interval, at room temperature on a reciprocating shaker (Mini-bead-beater-8, Biospec Products Inc., Bartlesville, OK) at the maximum speed for mechanical disruption of the ruminal contents. Contents of the tubes were then transferred into a 15-mL culture tube equipped with a screw cap and Teflon liner. Two portions of 1.9 mL of methanol were added to the Eppendorf tube to ensure complete transfer. The content of the culture tube was well mixed by vortexing and allowed to stand at room temperature for 1 h. After 1 h, 1.9 mL of chloroform, 1.8 mL of water, and 0.1 mL of 3 M aqueous HCl were added, then mixed again by vortexing and centrifuged (at 150 x g for 3 min). The acid was added to ensure the pH of the extract was slightly acidic (pH 5.0 to 6.0). The chloroform layer (lower phase) containing the esterified lipid and FFA was removed. The methanol-water phase was extracted again using chloroform (1.9 mL). The chloroform phases were combined, dried over anhydrous Na2SO4, and passed through a Pasture pipette containing a glass wool plug into a vial. Chloroform was removed from the vial under a stream of N2. Three drops of benzene were added to dissolve the lipids, and then 1 mL of 1 M NaOH (dissolved in 95% ethanol) was added into the vial, vortex-mixed, and kept at room temperature overnight in a dark place.

The following day, 1.0 mL of 3 M aqueous HCl was added and extracted 2 times with hexane (1.0 mL each time). After evaporation of hexane under N2, 0.8 mL of benzene and 0.2 mL of methanol were added. After mixing, 7 to 8 drops of TMS-diazomethane (TCI America, Portland, OR) were added into the vial and shaken lightly and occasionally over a 60-min period at room temperature (caution: TMS-diazomethane is explosive). After 60 min, a few drops of glacial acetic acid were added to inactivate the derivatizing agent (i.e., until the yellow color disappeared). Water (0.5 mL) and 1.0 mL of hexane were added and vortexed. The hexane layer was removed, and the extraction was repeated with the same amount of hexane. The combined hexane extracts (about 2.0 mL) were condensed to 70 to 80 µL under a stream of N2 and then applied to a Silica Gel-G thin-layer chromatography (TLC) plate (Fisher Scientific, Ottawa, Ontario, Canada) that was developed using the solvent mixture of hexane/diethyl ether/acetic acid (90:10:1 by volume). The fatty acid methyl esters (FAME) band was removed from the TLC plate after visualization with 2',7'-dichlorofluorescein. The FAME were recovered from the silica using hexane (about 7.0 mL). The hexane eluate containing the FAME was reduced using a stream of N2 to 100 µL and analyzed by GLC. For mechanical disruption of the microbial cells, we did not use a sonicator, as commonly used for disrupting the cell, because an early study suggested that it destroyed 50 to 60% of total unsaturated fatty acids (our unpublished data).

Analysis of Fatty Acid by GLC

The FAME analysis was performed using a Hewlett-Packard Model 5890 Series II GLC (Palo Alto, CA) equipped with a split-splitless injector at 250°C, a flame ionization detector at 250°C, and a CP Sil 88 column (100 m, 0.25 mm, 0.2-µm film thickness, Varian Inc., Mississauga, Ontario, Canada). Hydrogen was used as the carrier gas at a constant flow rate of 1 mL/min. The temperature of the GLC oven was set to 45°C for 4 min, increased at 13°C/min to 175°C and held for 27 min, and again increased at the rate of 4°C/min to a final temperature of 215°C and held for 35 min (Kramer et al., 2001Go; Cruz-Hernandez et al., 2004Go). Agilent Technologies ChemStation software (Version A.10, Mississauga, Ontario, Canada) was used for data analysis. A 1-µL sample was injected using the splitless mode set at 0.3 min. Peaks were routinely identified by comparison of retention times with FAME standards (GC 463, UC-59M, 21:0, 23:0, and 26:0, Nu-Check-Prep Inc., Elysian, MN). The individual isomers of 18:1 were determined as follows: the temperature of the GLC oven was maintained at 45°C for 4 min, increased to 163°C at a rate of 13°C/min and held for 40 min, and again increased at the rate of 4°C/min to a final temperature of 215°C and held for 23 min. Peaks of 18:1 isomers were identified by comparison to published data (Kramer et al., 2002Go; Shingfield et al., 2003Go; Loor et al., 2004Go). Fatty acid composition was expressed as a percentage of total fatty acids.

Chemical Analyses

Feed samples were pooled weekly and analyzed for DM content by drying in an oven at 60°C for 48 h (AOAC, 1990Go). A subsample was ground using a Wiley mill with a 1-mm screen (Thomas-Wiley, Philadelphia, PA) and stored at –20°C until analyzed. The feed samples were analyzed for CP using the Kjeldahl procedure (AOAC, 1990Go) and for ADF (AOAC, 1990Go), NDF (Goering and Van Soest, 1970Go), and crude fat (AOAC, 1990Go). Representative portions of each pellet mixture (about 10 g) were extracted with chloroform/methanol (1:1), and the total lipids were transesterified using HCl/methanol (Cruz-Hernandez et al., 2004Go). All FAME were purified by TLC before analysis by GLC in duplicate (see above).

Statistical Analysis

Statistical analyses were conducted using the GLM procedure (SAS Inst. Inc., Cary, NC). The model used was: Yijk = µ + Ci + Pj + Tk + eijk, where Y = the dependent variable; µ = the true mean; C = the effect of animal (i = 1 to 4); P = the effect of period (j = 1 to 4); T = treatment (k = control, low, medium, or high); and eijk = the random residual error. A term was originally included in the model for carryover, but it was not statistically significant and was removed from the model. Results were analyzed using orthogonal contrasts to determine linear and quadratic effects of increasing level of algal pellet and to detect any difference between the control diet and the average effect of the 3 algal treatments. Significant data were presented as P < 0.05 unless otherwise stated. Means of individual fatty acids were the average of 4 measurements.


   RESULTS AND DISCUSSION
Top
 Abstract
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS AND DISCUSSION
LITERATURE CITED
 
The ingredient and chemical compositions of the basal diet and of the control and algal pellets are shown in Tables 1Go and 2Go. Control and algal pellets contained similar DM, CP, and crude fat but differed in their ADF and NDF contents. The basal diet consisted of about 90% corn silage and high-moisture corn. The major fatty acid was palmitic acid (16:0) and was similar in both the control and algal pellets (30.2% average; Table 3Go). The second most abundant dietary fatty acid was oleic acid (18:1 cis-9), with concentrations of 26.0 and 25.4% of total fatty acids for control and algal pellets, respectively. The control pellet contained greater concentrations of linoleic (18:2n-6) and linolenic (18:3n-3) acids compared with the algal pellet. Docosahexaenoic acid (22:6n-3) and very small amounts of docosapentaenoic acid (22:5n-3) were present in the algal pellet (Table 3Go). There was no EPA (20:5n-3) in either the control or algal pellet; however, all treatments contained small amounts of trans-16:1 and trans-18:1 fatty acids but no odd-chain or branched-chain (iso or anteiso) fatty acids.


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  		Table 3. Fatty acid composition of control and algal pellets (% of total fatty acids)
 
Rumen fluid was sampled from each animal during the last day of the collection week via stomach tube. Sampling was undertaken at the same time during the third week (on the last day) and at the same time (3 to 4 h) after feeding for each animal on each diet. Fatty acid profiles of rumen samples could change before feeding and during the postprandial period (Devillard et al., 2004Go; Or-Rashid et al., 2007Go). Therefore, the relevance of our observations (i.e., fatty acid composition of rumen fluid) might be limited to feeding time.

SFA

Inclusion of algal meal in the diet did not alter the content of palmitic acid (16:0) in rumen fluid, and there was no difference between control and the average effect of algal meal treatments (Table 4Go). The percentage of stearic acid (18:0) was decreased by algal meal supplementation (P < 0.001) compared with control and linearly decreased with algal meal supplementation (P = 0.005). The greatest level of algal meal addition (H-Alg) decreased the 18:0 content by 5.5 times relative to control. The inclusion of algal meal concomitantly increased the total trans-18:1 from 19 to 43% of total fatty acids. The large change in the relative content of total trans-18:1 and 18:0 in rumen contents has been attributed to an inhibition of the reduction step of trans-18:1 to 18:0 by long-chain PUFA present in fish oil (Shingfield et al., 2003Go; AbuGhazaleh and Jenkins, 2004Go; Lee et al., 2005Go; Loor et al., 2005aGo). Control rumen fluid had a greater (P < 0.001) percentage of total SFA compared with the algae treatments, and total SFA linearly decreased with level of algal meal supplementation (P < 0.001, Table 4Go).


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  		Table 4. Least squares means for fatty acids of rumen fluid (% of total fatty acids) from animals fed control pellets or 3 levels of algal meal
 
Odd-Chain and Branched-Chain Fatty Acids

The percentage of iso-14:0 was less for diets supplemented with algae than for control (P = 0.004, Table 4Go). On the other hand, the percentages of iso-13:0, anteiso-13:0, 13:0, iso-15:0, anteiso-15:0, 15:0, iso-16:0, iso-17:0, and anteiso-17:0 were not significantly altered (P > 0.05) with dietary algal meal supplementations (Table 4Go). Both odd-chain and branched-chain fatty acids are synthesized by rumen microbes and form important constituents of microbial lipids (O’Kelly and Spiers, 1991Go). According to Sauvant and Bas (2001)Go, dietary fiber content was positively related to the percentage of odd-chain and branched-chain fatty acids within ruminal bacteria. Others have shown an increase in the percentage of odd-chain and branched-chain fatty acids in duodenal lipids with dietary supplementation of unsaturated oils (Sauvant and Bas, 2001Go; Loor et al., 2004Go). In the current study, long-chain PUFA, such as DHA present in algae, might alter the percentage of some branched-chain fatty acids but none of the odd-chain fatty acids in the rumen fluid were altered significantly.

cis-18:1 Isomers

The percentage of total cis-18:1 (Table 4Go) was increased by algal meal supplementation compared with control (P = 0.04). The predominant cis-18:1 isomer that responded to algal meal supplementation was 18:1 cis-9 (P = 0.02; Table 5Go). The percentage of 18:1 cis-9 in rumen fluid was 46.4% greater (P = 0.02) for the average of algal meal diets compared with the control diet (Table 5Go). The greater proportion of 18:1 cis-9 in the ruminal content of animals fed the algal rations may indicate that algae partially protected 18:1 cis-9 from microbial biohydrogenation. Loor et al. (2005b)Go also reported less biohydrogenation of 18:1 cis-9 when fish oil was fed (64% biohydrogenation) compared with feeding linseed oil (84% biohydrogenation). However, no similar information is available in the literature for algae feeding. In contrast, Lee et al. (2005)Go and Shingfield et al. (2003)Go found no differences in the 18:1 cis-9 content in the duodenal or omasal flow, respectively, with addition of fish oil to the diet. The percentage of 18:1 cis-12 was less for diets supplemented with algae than for control (P = 0.05, Table 5Go).


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  		Table 5. Effect of algae supplementation on the positional isomers of 18:1 monoenes in rumen fluid of animals fed control pellets and 3 levels of algal meal (% of total fatty acids)
 
18:3 Isomers and Nonconjugated 18:2 Fatty Acids

Addition of algal meal in diets resulted in a proportional increase (P = 0.03) in 18:2 trans-9, trans-12 and 18:2 trans-11, cis-15 (Table 4Go). Rumen microbes responsible for the reduction of trans-18:1 are also capable of hydrogenating nonconjugated cis/trans-18:2 isomers (Shingfield et al., 2003Go), and algal meal supplementation inhibits the metabolism of these fatty acids. Linoleic acid accounted for 67.7 to 78.1% of the total nonconjugated 18:2 fatty acids in the rumen, whereas the trans-11, cis-15 isomer accounted for 5.4 to 8.4%. Kemp et al. (1975)Go reported the production of 18:2 trans-11, cis-15 during biohydrogenation of 18:3n-3 in vitro. The percentage of 18:3n-3 was greater in rumen fluid for animals fed the control diet compared with that in animals fed the algal meal diets (P = 0.02), which is in keeping with the lower level of 18:3n-3 in the algal meal than in the control supplement (Table 3Go).

trans-18:1 Isomers

The percentage of total trans-18:1 in the rumen fluid of animals fed the control diet was relatively high (19% of total fatty acids, Table 4Go), and this appears to be due to the highly fermentable carbohydrate in the diet. Similar levels of total trans-18:1 were found in duodenal lipids of ruminants fed high levels of fermentable carbohydrate as 74% corn (18% total trans-18:1; Sackmann et al., 2003Go) and 32.4% ground wheat (15.5% total trans-18:1; Loor et al., 2004Go). Greater levels of total trans-18:1 in the duodenal flow were also obtained by addition of fish oil (250 g of fish oil/d, 38% total trans-18:1; Shingfield et al. 2003Go) or linseed oil to low-fiber diets (a 65% concentrate diet plus 3% linseed oil, 28.8% total trans-18:1; Loor et al., 2004Go). In the current study, supplementation with algal meal resulted in an increase (P < 0.001) of total trans-18:1 (from 19 to 43% of total fatty acids, Table 4Go). There were also linear increases (P = 0.01) in total trans-18:1 in rumen fluid from animals fed more algae. Some of the individual trans-18:1 isomers increased linearly (e.g., 18:1 trans-10) or quadratically (e.g., 18:1 trans-11, and 18:1 trans-13 and -14), and 18:1 trans-10 became the major trans-18:1 isomer in the rumen fluid of all algal meal-fed groups (Table 5Go). The percentage of 18:1 trans-10 was increased by algal meal supplementation compared with control (P = 0.002) and was linearly dependent on the level of algal meal supplementation (P = 0.006). Vaccenic acid (18:1 trans-11) was the major trans-18:1 isomer in animals fed the control diet, but it showed a quadratic response to increasing algal supplementation (P = 0.005). In animals fed the control diet, 18:1 trans-10 was 2.3 times lower than VA but started to increase upon algal meal supplementation, which may indicate a shift in microbial population within the rumen induced by algal meal supplementation. The proportion of 18:1 trans-10 in the duodenal (forage to concentrate 65:35; Loor et al., 2004Go) or the omasal flow (forage to concentrate 60:40; Shingfield et al., 2003Go) when greater-forage diets were fed was much smaller in comparison to our results. The ratio of 18:1 trans-10 to 18:1 trans-11 was 0.21 and 0.12 in the latter 2 studies, respectively, which compares to a ratio of 5.3 for our H-Alg diet. Under normal rumen conditions, dietary 18:2n-6 is mainly converted to VA via cis-9, trans-11 CLA (Harfoot and Hazlewood, 1997Go). On the other hand, the trans-10, cis-12 CLA from 18:2n-6 has been considered to be the main intermediate in the formation of 18:1 trans-10 in the rumen by bacteria such as Megasphaera elsdenii (Kim et al., 2002Go) or propionic bacteria (Wallace et al., 2006Go). However, under certain conditions, isomerization of oleic acid (18:1 cis-9) was also reported to contribute to 18:1 trans-10 production (Mosley et al., 2002Go). Of the other trans-18:1 isomers, only 18:1 trans-9 showed an increase with addition of algal meal, whereas 18:1 trans-12, 18:1 trans-13 + 18:1 trans-14, 18:1 trans-15, and 18:1 trans-16 appeared unaffected with supplementation of algal meal (Table 5Go).

CLA Isomers

The total CLA percentage in rumen fluid showed a quadratic effect (P = 0.07), although not significant, in animals supplemented with algal meal (low level to high level, Table 4Go). A quadratic effect (P = 0.05) of increasing dietary algae supplement was also detected for cis-9, trans-11 CLA (Table 6Go). Although the trans-10, cis-12 CLA isomer remained a minor CLA component (0.05 to 0.13%), it did increase in the rumen fluid with algal meal supplementation (P = 0.01) and linearly increased with level of algal meal supplementation (P = 0.03, Table 6Go). A number of trans, trans-CLA isomers were found in the rumen fluid regardless of diet. The trans-9, trans-11 + trans-10, trans-12 CLA isomers were decreased linearly (P = 0.02) with increased level of algal supplementation. The content of cis-9, trans-11 CLA, the major CLA isomer, ranged from 64.4 to 74.8% of total CLA, whereas the trans-10, cis-12 isomer accounted for 5.7 to 7.3% of total CLA. Kucuk et al. (2001)Go reported a decrease of trans-10, cis-12 CLA and an increase of cis-9, trans-11 CLA in the duodenum with increased levels of dietary forage. On the other hand, AbuGhazaleh et al. (2002)Go found a 2.9- to 4.0-times increase in both cis-9, trans-11 and trans-10, cis-12 CLA in ruminal digesta when fish oil was added to the diet of cows, and Lee et al. (2005)Go found a doubling of these CLA isomers in the duodenal flow by addition of fish oil to steers. These reports, in addition to the current study, suggest that the CLA profile in ruminal digesta was affected by the forage-to-concentrate ratio of the diet, the type and level of oil supplementation, and the interaction between these factors. To our knowledge, no in vivo experiment has been conducted to evaluate the content of cis-9, trans-11 and trans-10, cis-12 CLA in response to graded levels of dietary algal meal supplementation. The low levels of CLA isomers observed in rumen fluid should not be confused with the much greater levels of cis-9, trans-11 CLA and trans-7, cis-9 CLA generally observed in the milk and meat fats of these animals, because the major site of CLA synthesis is the tissues of ruminants (Bauman and Griinari, 2003Go).


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  		Table 6. Effect of algae supplementation on the isomers of CLA in rumen fluid of animals fed control pellets and 3 levels of algal meal (% of total fatty acids)
 
Feeding high-concentrate diets appears to have a direct impact on the rumen biohydrogenation and the inherent rumen microbial populations (Latham et al., 1972Go; Tajima et al., 2001Go; Loor et al., 2004Go). The ratio of cellulolytic (primarily Butyrivibrio fibrisolvens) to propionogenic, lactogenic, and amylolytic bacteria in the rumen of lactating cows was effectively reduced when animals were fed high-concentrate supplements (Latham et al., 1972Go). Butyrivibrio fibrisolvens has been shown to synthesize mainly cis-9, trans-11 CLA from 18:2n-6 (Kepler et al., 1966Go; Kim et al., 2000Go). However, high grain supplementation to ruminants results in an increase in the population of other bacteria (i.e., M. elsdenii) with a resultant increase in the production of trans-10, cis-12 CLA (Kim et al., 2002Go). Megasphaera elsdenii, a gram-negative cocci, is more resistant to unsaturated fatty acids such as 18:2n-6 than B. fibrisolvens (Henderson, 1973Go) that have a gram-positive-type cell wall (Cheng and Costerton, 1977Go). The growth of gram-positive bacteria is inhibited by long-chain fatty acids to a greater extent than gram-negative bacteria (Galbraith and Miller, 1973Go; Henderson, 1973Go). This phenomenon suggests that inclusion of dietary long-chain PUFA, such as DHA from algal meal, might have changed the ruminal environment sufficiently to decrease the growth of B. fibrisolvens, and that shift caused the large production of 18:1 trans-10 via trans-10, cis-12 CLA within the rumen. Another factor that may have changed the ruminal environment to produce more 18:1 trans-10 could have been the presence of essential oil in the algal meal. Liersch (1976)Go reported that the presence of essential oil in algae and gram-positive bacteria have generally been shown to be more sensitive to essential oil than gram-negative bacteria (Smith-Palmer et al., 1998Go).

DHA and DHA Metabolites

Rumen digesta contained DHA in proportion to the level of algal meal supplementation (P = 0.002, Table 4Go). Eicosapentaenoic acid was not detected in the rumen fluid consistent with the absence of this fatty acid in the algal product from C. cohnii (Table 3Go) as reported by Barclay et al. (1998)Go. The percentage of 22:5n-3 in the rumen fluid was low, but it increased linearly (P = 0.03) with increased levels of algal meal in the diets (Table 4Go). Trace amounts of this fatty acid were present in the algae product (Table 3Go), and some have suggested that 22:5n-3 could arise from retro conversion of DHA (Barclay et al., 1998Go).

According to AbuGhazaleh and Jenkins (2004)Go, DHA inhibits the reductase enzyme within certain rumen microorganisms causing the accumulation of trans-18:1 fatty acids in the rumen. Such an effect could explain the observed increases in the concentration of trans-18:1 and CLA in rumen digesta when animals were supplemented with the algal meal, a source of DHA (Tables 4Go, 5Go, and 6Go).

Other researchers have found an increment of trans-18:1 and CLA in rumen or duodenal contents in response to the inclusion of fish oil in dairy rations (Shingfield et al., 2003Go; Lee et al., 2005Go; Loor et al., 2005aGo). However, depending on the diet composition and the ratio of dietary components, the increase in the content of trans-18:1 and CLA in milk and meat may not be the desired VA (18:1 trans-11) and rumenic acid (cis-9, trans-11 CLA) if trans-18:1 isomers other than VA are largely produced by rumen microbes. For example, feeding diets with a high concentrate-to-forage ratio, and including oils high in unsaturated fatty acids in the diet, have been shown to increase 18:1 trans-10 rather than 18:1 trans-11 (Griinari et al., 1998Go; Sackmann et al., 2003Go; Shingfield et al., 2005Go). To our knowledge, no studies have compared the relative rate of DHA inhibition on the different trans-18:1 isomers or which rumen microbes are involved. Such a study could explain the relative changes of different trans-18:1 isomers in rumen digesta more clearly.

Ratios of VA/18:0 and -trans-11/trans-10s Indicators of Changed Rumen Microbial Populations

An increase (P < 0.001) in the VA/18:0 ratio was observed with dietary inclusion of the algal meal in the diets (Figure 1Go), and the VA/18:0 ratio was linearly dependent on the level of algal meal supplementation (P = 0.01). This is an indication of incomplete biohydrogenation of unsaturated fatty acids with algae supplementation. AbuGhazaleh et al. (2002)Go also found that inclusion of fish oil into dairy rations resulted in greater VA/18:0 ratios in the rumen. However, their ratio would be slightly greater, because the 18:1 trans-10 and 18:1 trans-11 isomers were not well separated.


Figure 1
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  		Figure 1. Ratio of selected trans-containing fatty acids in rumen fluid. One ratio consisted of 11 trans-containing fatty acids (18:1 trans-11 and cis-9, trans-11 CLA) and 10 trans-containing fatty acids (18:1 trans-10 and trans-10, cis-12 CLA), the other of vaccenic acid (18:1 trans-11) and stearic acid (18:0) in the lipids of rumen fluid. The SEM were 0.59 and 0.08, respectively. Control = no algal meal (2.1 kg/d); L-Alg = low level of algal meal (1.1 kg/d); M-Alg = medium level of algal meal (2.1 kg/d); H-Alg = high level of algal meal (4.2 kg/d).

 
The major trans-11 fatty acids in milk fat produced during normal rumen metabolism are 18:1 trans-11 and cis-9, trans-11 CLA, whereas a shift in rumen metabolism generates trans-10 fatty acids, specifically 18:1 trans-10 and trans-10, cis-12 CLA (Cruz-Hernandez et al., 2004Go, 2006Go; Kramer et al., 2004Go). We have used the trans-11/trans-10 ratio in rumen fluid of cattle fed any supplement to determine a change in the rumen bacterial population (Cruz-Hernandez et al., 2006Go). In the current study, the trans-11/trans-10 ratio decreased from 2.45 to 0.21 with increasing dietary algae supplementation (Figure 1Go) as the percentage of 18:1 trans-10 increased from 3.5 to 29.5% (Table 5Go). Loor et al. (2004)Go showed that cattle fed a concentrate diet increased the content of 18:1 trans-10 by 13.8 times in duodenal digesta compared with a high-roughage-based control diet. The trans-11/trans-10 ratio decreased from 14.1 (high-roughage diet) to 1.3 (low-roughage diet; Loor et al., 2004Go). In contrast, Shingfield et al. (2003)Go did not observe an increase of 18:1 trans-10 relative to 18:1 trans-11 in omasal digesta when cattle were fed a fish oil supplement using a basal diet consisting of 60% grass silage and 40% concentrate. However, when Shingfield et al. (2005)Go included a basal diet rich in corn products in a repeat study, they were able to observe a similar increase in the levels of 18:1 trans-10 compared with 18:1 trans-11. The differences in response appeared to be related to differences in the basal diet. A diet with low fiber and high in concentrate and PUFA alters the rumen microbial population resulting in the production of more 18:1 trans-10 and trans-10, cis-12 CLA, whereas a diet high in fiber increases the production of 18:1 trans-11 and cis-9, trans-11 CLA within the rumen.

In conclusion, we observed high contents of total trans-18:1 (19% of total fatty acids) and 18:1 trans-10 isomer (3.5% of total fatty acids) in rumen fluid, which likely reflect the impact of our corn-based diet on the rumen microbial population. Addition of algal meal as a source of DHA inhibited the reduction of trans-18:1 to 18:0 and increased the levels of total trans-18:1 to 43% of total fatty acids and 18:1 trans-10 to 29.5% of total fatty acids, whereas VA increased up to the M-Alg diet and then decreased with H-Alg. Even though the relative rate of DHA inhibition of the reduction of different trans-18:1 isomers is not known, the large increase of trans-18:1 isomers suggests that reduction of most of these isomers (including 18:1 trans-10) was effectively inhibited in rumen microbes. The cis-9, trans-11 CLA was increased by 2 times with M-Alg compared with the control. The total CLA content in the rumen fluid of animals fed the control ration averaged less than 1% of total fatty acids, and the feeding of M-Alg nearly doubled this amount. The DHA content in the rumen increased in response to algal meal supplementation. Our results showed that the residual algal meal contained sufficient DHA to increase the trans-18:1 isomers by inhibiting the biohydrogenation of trans-18:1 isomers to 18:0.


   Footnotes
 
1 We would like to thank the staff at the Elora Dairy Cattle Research Centre for their help, Martek Biosciences Corporation for supplying the algal meal, Dairy Farmers of Ontario (BWM, MMO-R, and JKGK), the Ontario Ministry of Agriculture and Food, and the Natural Sciences and Engineering Research Council of Canada for funding (BWM) and Ousama AlZahal and Nicholas Odongo (both from Department of Animal and Poultry Science, University of Guelph, Guelph, Ontario, Canada) for statistical consulting. Back

3 Current address: Department of Animal and Poultry Science, University of Guelph, Guelph, ON, Canada N1G 2W1. Back

2 Corresponding author: morrashi{at}uoguelph.ca

Received for publication February 7, 2007. Accepted for publication September 27, 2007.


   LITERATURE CITED
Top
 Abstract
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS AND DISCUSSION
 LITERATURE CITED
 


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