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The Effect of Solids Dilution Rate and Oil Source on Trans C18:1 and Conjugated Linoleic Acid Production by Ruminal Microbes in Continuous Culture

Department of Animal Science, Food, and Nutrition, Southern Illinois University, Carbondale 62901

¹ Corresponding author: aabugha{at}siu.edu

	ABSTRACT

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION CONCLUSIONS REFERENCES

 
The objective of this study was to evaluate the effect of solids dilution rate (SDR) and oil source [soybean oil (SBO) or linseed oil (LSO)] on the ruminal production of trans C18:1 and conjugated linoleic acid (CLA). A dual-flow continuous culture system consisting of 4 fermenters was used in a 4 x 4 Latin square design with a factorial arrangement of treatments over 4 consecutive periods of 10 d each. Treatment diets (50:50 forage to concentrate) were fed at 120 g/d of dry matter (DM) in 3 equal portions. The concentrate mix contained 1% fish oil and either 2% SBO or 2% LSO on a DM basis. Treatments were as follows: 1) SBO at 6%/h SDR, 2) SBO at 3%/h SDR, 3) LSO at 6%/h SDR, and 4) LSO at 3%/h SDR. The oil source by SDR interaction was not significant for trans C18:1 and CLA fatty acids. The concentrations of trans C18:1 and vaccenic acid were greater in effluents when diets were supplemented with SBO vs. LSO (37.11 vs. 34.09 and 32.71 vs. 29.70 mg/g of DM, respectively) and at high SDR than low SDR (37.60 vs. 33.61 and 32.72 vs. 29.61 mg/g of DM, respectively). The concentration of cis-9, trans-11 CLA in effluents was also greater with SBO than LSO (0.81 vs. 0.40 mg/g of DM) supplementation and at high SDR than low SDR (0.68 vs. 0.54 mg/g of DM). Biohydrogenation of linoleic acid and linolenic acid increased at higher SDR within each oil treatment. Based on these results, SBO supplementation at high SDR enhances ruminal production of vaccenic acid, and therefore could potentially enhance cis-9, trans-11 CLA in milk fat through synthesis by ⁹-desaturase.

Key Words: oil source • solids dilution rate • conjugated linoleic acid • trans fatty acid

	INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION CONCLUSIONS REFERENCES

 
Conjugated linoleic acids (CLA) are a mixture of geometric and positional isomers of linoleic acid with conjugated unsaturated double bonds. Isomers of CLA have a wide range of beneficial effects, such as anticarcinogenic, antiatherogenic, antidiabetic, and antiobesity (Bauman et al., 2001). The major CLA isomer, cis-9, trans-11 (c9t11), is synthesized either in the rumen as an intermediate in the biohydrogenation (BH) of linoleic acid or in the tissues by ⁹-desaturase from vaccenic acid (VA; trans-11 C18:1), an intermediate in ruminal BH of both linoleic and linolenic acids (Harfoot and Hazlewood, 1988). Piperova et al. (2002) reported that approximately 90% of c9t11 CLA appearing in milk fat is synthesized in the mammary gland; thus, many efforts have addressed ways to increase VA flow from the rumen. The factors affecting the flow of CLA and trans C18:1, VA in particular, to the duodenum as a result of ruminal BH need to be elucidated to increase CLA concentration in milk and meat of ruminants used for human consumption.

Several researchers have reported an increase in milk VA and c9t11 CLA when soybean oil (SBO), sun-flower oil, linseed oil (LSO), and fish oil (FO) were added to dairy cow diets (Kelly et al., 1998; Chouinard et al., 2001; AbuGhazaleh et al., 2003b; Loor et al., 2005). High concentrations of VA in the duodenal digesta have been reported for ruminants consuming diets with either linoleic or linolenic acids as the major unsaturated fatty acids (FA; Sackmann et al., 2003). However, the production of VA and c9t11 CLA in response to dietary oil supplements high in linoleic and linolenic acids has not been always consistent. Kelly et al. (1998) showed that dietary supplementation with vegetable oils high in linoleic acid, rather than linolenic acid, resulted in the greatest increase in concentration of c9t11 CLA in milk fat. AbuGhazaleh et al. (2003b) and Ward et al. (2002) also reported highest milk VA and c9t11 CLA concentrations with diets supplemented with linoleic acid compared with linolenic acid oil source. However, Loor et al. (2004b, 2005) and Chow et al. (2004) reported no difference in VA and c9t11 CLA concentrations when sunflower oil and LSO were compared in vivo and in vitro. AbuGhazaleh et al. (2002) demonstrated that the increases in milk c9t11 CLA and VA concentrations were higher when diets rich in linoleic acid are supplemented with FO.

Solids retention time is known to modulate ruminal fermentation and microbial growth, which could lead to an alteration in the BH of unsaturated FA. There are limited data addressing the effect of solids retention time on trans C18:1 and CLA formation. Solids dilution rate (SDR), characterized by the outflow of materials from the rumen, is estimated to be from 2 to 12%/hr (Isaacson et al., 1975). The SDR can be highly variable in vivo because of differences in environment, physiological condition of the animal, dietary characteristics, level of feed intake, and many other factors. Therefore, continuous culture systems have been used to determine what effects changes in SDR, per se, have on rumen fermentation (Crawford et al., 1980a,b; Hoover et al., 1984; Martin and Jenkins, 2002; Qiu et al., 2004). Qiu et al. (2004) compared the effects of SDR of 8 and 4%/h on flows of VA and CLA. The researchers noted that at high SDR, CLA flow tended to increase compared with the low SDR. Martin and Jenkins (2002) reported a higher increase in the concentrations of trans C18:1 and c9t11 CLA in continuous cultures maintained at 0.10%/h liquid dilution rate (LDR) compared with 0.05%/h LDR. Recently, AbuGhazaleh et al. (2005) also reported less trans C18:1 formation from oleic acid when culture LDR decreased from 0.10 to 0.05%/h.

The objective of this study was to assess the effects of SDR, oil source, and their interaction on trans C18:1, VA in particular, and c9t11 CLA production in the rumen using a dual-flow continuous culture system.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION CONCLUSIONS REFERENCES

 
Experimental Design
Four dual-flow continuous culture apparatus, as described by Stern and Hoover (1990), were used in a 4 x 4 Latin square with a 2 x 2 factorial arrangement over 4 periods of 10 d each. Each experimental period consisted of 7 d for adaptation, with the last 3 d for sampling. Treatments used in this study were as follows: 1) SBO at 6%/h SDR, 2) SBO at 3%/h SDR, 3) LSO at 6%/h SDR, and 4) LSO at 3%/h SDR. The 6 and 3% SDR resulted in 16.7 and 33.3 h of solids retention time, respectively. Treatment diets (50% alfalfa pellets, 50% concentrate) were fed at 120 g/d of DM in 3 equal portions at 0800, 1500, and 2100 h. The concentrate mix contained 1% FO and either 2% SBO or 2% LSO (DM basis; Table 1).

Sample Collection and Analysis
Effluent from each fermenter was collected into 5-L plastic jugs submerged approximately three-fourths into a 4°C water bath. The solid and liquid effluent volumes were cataloged daily at 0800 h and discarded until the final 3 d of each period. On the last 3 d, the solid and liquid portions were combined and homogenized on a stir plate, and a 10% volume subsample was collected and stored at –5°C. Subsamples from each fermenter were composited for d 8, 9, and 10. Samples were thawed in a 50°C water bath, transferred into 250-mL plastic bottles, and centrifuged (Beckman J2-21, GMI Inc., Minneapolis, MN) at 36,000 x g for 15 min, after which the supernatant was removed. This process was repeated until all liquid in the thawed sample was removed. The bottle and fiber pellet were stored at –80 C° for 48 h, then freeze dried and ground to 1 mm using a Wiley mill (Arthur H. Thomas, Philadelphia, PA).

Samples of alfalfa pellets and concentrate were collected twice each period (d 5 and 10) and stored at –20°C until analyses. Samples were freeze dried for at least 48 h, then ground through a 2-mm screen of a standard Wiley mill (model 3; Arthur H. Thomas) and composited by period. Composites were analyzed for CP, NDF, and ADF (AOAC, 1997).

Feed and effluent samples were methylated according to Kramer et al. (1997) and analyzed for FA on a Shimadzu GC-2010 gas chromatograph (Shimadzu Scientific Instruments, Inc., Columbia, MD) equipped with a flame-ionization detector and 100-m SP-2560 fused silica capillary column (0.25 mm i.d. x 0.2 µm film thickness; Supelco Inc., Bellefonte, PA). The helium carrier gas was maintained at a linear velocity of 23 cm/s. The oven temperature was programmed to 165°C for 80 min, increased at 1.5°C/min to 180°C, and then increased at 5°C/min to a final temperature of 245°C, which was held for 9 min. The injector and detector temperatures were set at 255°C. Heptadecanoic acid (C17:0) was added to all samples as an internal standard after correcting for native C17:0. Peaks were identified by comparing the retention times with those of the corresponding standards (Nu-Chek Prep, Elysian, MN; Supelco; and Larodan Fine Chemicals, Malmo, Sweden).

Statistical Analysis
Data were analyzed with the PROC MIXED procedure of SAS 9.1 (SAS Institute, Cary, NC). Oil source, SDR, and their interaction were the fixed effect, whereas fermenter was the random effect in the statistical model. Least squares means and SEM are reported for all data. Significance was declared at P < 0.05.

	RESULTS

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The ingredient and chemical composition of diets are shown in Table 1. Dietary treatments had similar values for CP, ADF, NDF, and total FA. The dietary oil sources were chosen because of the differences in their FA compositions (Table 2). Linoleic acid was the major FA in SBO (52.9% of total FA), whereas linolenic acid was the major FA in LSO (55.7%; Table 2). Daily input of major unsaturated FA varied according to diet (Table 3). Total daily input of oleic, linoleic, and linolenic acids represented 17, 38, and 6% of total FA input, respectively, for diets supplemented with SBO. For the LSO diets, oleic, linoleic, and linolenic acids input represented 14, 19, and 33% of total FA input, respectively. The pH in fermenters was similar for all treatments (6.74, 6.60, 6.73, and 6.54 for treatments 1 through 4, respectively).

Dietary oil source and SDR also affected the concentration of other FA in effluent (Table 4). The concentration of trans-11 cis-15 C18:2 was greater (P < 0.05) with LSO (9.33 mg/g of DM) than with SBO (1.61 mg/g of DM) supplementation. Solid dilution rate had no effect on trans-11 cis-15 C18:2 concentrations. The concentrations of keto stearic acid and hydroxy stearic acid were affected (P < 0.05) by oil source and SDR. Greater concentrations for keto stearic acid and hydroxy stearic acid were seen with SBO (3.46 and 3.26 mg/g of DM, respectively) than with LSO (2.63 and 2.45 mg/g of DM, respectively) supplementation. The concentration of keto stearic acid was greater at 6% SDR (3.29 mg/g of DM) than at 3% SDR (2.81 mg/g of DM), whereas hydroxy stearic acid concentration was higher at 3% SDR (3.11 mg/g of DM) than at 6% SDR (2.61 mg/g of DM).

The effect of treatments on unsaturated C18 FA BH is presented in Table 5. The BH of oleic, linoleic, and linolenic acids was greater (P < 0.05) at 6% SDR than at 3% SDR. Addition of SBO to diets did not affect linoleic acid BH, whereas LSO addition increased linolenic acid BH.

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION CONCLUSIONS REFERENCES

The primary objective of this study was to investigate the effect of oil source and SDR on VA production in the rumen. Vaccenic acid, an intermediate in the BH of linoleic and linolenic acids, is the substrate for c9t11 CLA synthesis in the mammary gland by ⁹-desaturase (Bauman et al., 2001). Therefore, factors regulating VA synthesis and flow out of the rumen must be defined to maximize c9t11 CLA in ruminant foods. The greater concentration of VA with the SBO compared with the LSO diets suggests that either the pathway of BH for linoleic acid is more efficient than that of linolenic acid in producing VA, or the FO in LSO cultures may have inhibited the reduction of trans-11 cis-15 C18:2, the major nonconjugated trans C18:2 produced during the BH of linolenic acid into VA (Harfoot and Hazlewood, 1988), or both. Recently, AbuGhazaleh and Jenkins (2004a) suggested that docosahexaenoic acid (DHA) in FO promotes trans C18:1 and VA accumulation in rumen cultures, possibly by inhibiting the reductase enzyme in ruminal microorganisms responsible for the terminal hydrogenation of trans C18:1 to stearic acid. The possibility that the lower VA concentration in LSO effluent may have resulted from the effect of DHA on reductase enzyme activity is supported by the accumulation of trans-11 cis-15 C18:2 in the LSO effluent (Table 4). Under normal conditions, trans-11 cis-15 C18:2 is reduced to VA or cis-15 C18:1 by the reductase enzyme of rumen bacteria (Harfoot and Hazlewood, 1988). A similar accumulation in trans-11 cis-15 C18:2 was reported in vitro (AbuGhazaleh and Jacobson, 2007) and in vivo when dairy cows were fed a diet containing FO and LSO (Loor et al., 2004b). The inhibitory effect of DHA on the reduction of trans C18:1 into stearic acid may also explain the higher trans C18:1 concentration seen in this study compared with other in vitro (Troegeler-Meynadier et al., 2003; Qiu et al., 2004) and in vivo (Piperova et al., 2002; Loor et al., 2004a, 2005) studies.

Addition of linoleic and linolenic acids has been shown to increase VA concentration in rumen batch cultures (Troegeler-Meynadier et al., 2003; AbuGhazaleh and Jacobson, 2007), rumen digesta (AbuGhazaleh et al., 2003a), duodenal lipids (Loor et al., 2005), and milk fat (Chouinard et al., 2001). However, the level of increase in VA concentration with dietary linoleic and linolenic acids has not been always consistent. Although higher VA concentrations in milk fat (Chouinard et al., 2001; Ward et al., 2002) and rumen digesta (AbuGhazaleh et al., 2003a) were reported with dairy cows fed oils high in linoleic acid, Loor et al. (2004b, 2005) reported no difference in the duodenal flow of VA in dairy cows fed LSO and sunflower oil at 5% of dietary DM. Additionally, Chow et al. (2004) reported a similar increase in VA concentration in batch cultures when sunflower oil and LSO were added at 8% of feed DM. Discrepancies between these studies may have resulted from the presence of other unsaturated FA (primarily linoleic acid) in the linolenic acid diets, differences in diet composition, and (or) from the difference in the form of oil supplements (free oil vs. seeds).

Vaccenic acid concentration in effluent also increased as SDR increased. The effect of SDR on VA concentration may have resulted from the reduction in digesta retention time. Higher SDR decrease the opportunity for complete BH and therefore may increase the flow of BH intermediates. Approximately 80% of FA in the rumen are associated with rumen digesta (Harfoot and Hazlewood, 1988). The increase in VA concentration with high SDR may also be due to a shift in the bacterial population or bacterial enzyme activity. Low dilution rate was shown to have a negative effect on microbial growth (Martin et al., 2002), particularly on growth of cellulolytic bacteria (Latham et al., 1972). Crawford et al. (1980a) reported a significant drop in protozoa number in continuous fermenters as solids retention time decreased. Additionally, microbial protein synthesis also decreased as solids retention time increased (Crawford et al., 1980b). Using continuous cultures, Hoover et al. (1984) showed that, at a 0.04%/h dilution rate, lactic acid accounted for 53% of all organic acids, but as dilution rate increased to 0.08%/h, lactic acid was reduced to 28%. A further increase in dilution rate decreased lactic acid to 8%. The low dilution rate may modify the distribution (number, species) of cellulolytic bacteria colonizing the plant material. Cellulolytic bacteria, such as Butyrivibrio fibrisolvens, are indeed the main ruminal bacteria responsible for BH (Harfoot and Hazlewood, 1988). Additionally, at low dilution rate, the "balance" of microorganisms capable of carrying out various steps of BH may be altered, leading to changes in production of BH intermediates.

The concentration of c9t11 CLA was very low (<1%) in effluent relative to trans C18:1 (31%). Piperova et al. (2002) reported a flow of 0.24 to 0.53 g/d of c9t11 CLA compared with 57 to 120g/d for trans C18:1. Similar c9t11 CLA flow results were reported in steers (0.63 to 1.2 g/d; Sackmann et al., 2003) and in sheep (0.12 to 0.20 g/d; Kucuk et al., 2001). The studies of Harfoot and Hazlewood (1988) have shown that c9t11 CLA is rapidly metabolized in the rumen, and trans C18:1 accumulates during unsaturated FA BH. The higher concentration of c9t11 CLA with SBO treatments seen in this study is consistent with the findings of others (Loor et al., 2005; AbuGhazaleh and Jacobson, 2007). AbuGhazaleh et al. (2003a, b) showed that the increase in milk and rumen c9t11 CLA concentration is higher when diets with a high concentration of linoleic acid rather than linolenic acid are supplemented with FO. When LSO and sunflower oil were included in the diet of dairy cows at 5% of diet DM; the duodenal flow of c9t11 CLA with the sunflower oil diet was several fold higher compared with the LSO diet (Loor et al., 2005). The c9t11 CLA is an intermediate during the BH of linoleic acid, but not linolenic acid (Harfoot and Hazlewood, 1988). As with VA, increasing SDR increased the concentration of c9t11 CLA in effluent. Similarly, Martin and Jenkins (2002) observed a greater increase in c9t11 CLA concentration in fermenters maintained at 0.10%/h LDR compared with fermenters maintained at 0.05%/h. The c9t11 CLA is a transient intermediate during BH, and any increase in digesta rate of passage would potentially increase outflow.

The BH of linolenic acid increased with LSO supplementation. Other studies have also reported an increase in the BH of linolenic acid when linolenic acid source is used (Troegeler-Meynadier et al., 2003; Loor et al., 2004a). Linolenic is thought to be more toxic to rumen microbes and may be preferentially biohydrogenated more rapidly. Biohydrogenation of unsaturated C18 FA increased as SDR decreased. With a prolonged retention time, substrate would be available for a greater period for BH.

The greater concentration of hydroxy and keto stearic acids seen in the effluent of SBO diets resulted from the higher oleic acid content in SBO diets (Table 2). Recently, Jenkins et al. (2006) showed that the accumulation of hydroxy and keto stearic acids in the rumen is related to oleic acid input. AbuGhazaleh et al. (2005) showed that rumen microbes not only can biohydrogenate the double bond in oleic acid, but also can hydrate it, forming hydroxy and keto stearic acids. The biological and physiological consequences of consumption of hydroxy and keto stearic acids by humans are currently being examined.

	CONCLUSIONS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION CONCLUSIONS REFERENCES

 
Results from this experiment indicate that dietary oil source and SDR influence the production of VA and c9t11 CLA in the rumen. Higher concentrations of VA and c9t11 CLA are seen when a linoleic, rather than a linolenic acid, oil source is used in a diet containing FO. These results also show that reducing retention time of ruminal digesta increases VA and c9t11 CLA outflow. This study demonstrated that the best approach to increase ruminal VA and c9t11 CLA production, and potentially c9t11 CLA levels in ruminant food products, is to combine a high ruminal SDR with a linoleic acid oil.

Received for publication June 19, 2006. Accepted for publication October 16, 2006.

	REFERENCES

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION CONCLUSIONS REFERENCES

 


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