Effect of Monensin and Fish Oil Supplementation on Biohydrogenation and CLA Production by Rumen Bacteria In vitro When Incubated with Safflower Oil *

An in vitro study was conducted to examine the effect of monensin or fish oil addition on bio-hydrogenation of C18unsaturated fatty acids and CLA production by mixed ruminal bacteria when incubated with safflower oil. Commercially manufactured concentrate (1%, w/v) with safflower oil (0.2%, w/v) were added to mixed solution (600 ml) of strained rumen fluid and McDougalls artificial saliva (control). Monensin (Rumensin, 10 ppm, w/v, MO), mixed fish oil (0.02%, w/v, absorbed to 0.2 g alfalfa hay, FO) or similar amounts of monensin and fish oil (MO+FO) to MO and FO was also added into the control solution. All the culture solutions prepared were incubated in the culture jar anaerobically at 39°C up to 12 h. Higher pH (p<0.047) and ammonia concentration (p<0.042) were observed from the culture solution containing MO at 12 h incubation than those from the culture solutions of control or FO. The MO supplementation increased (p<0.0001-0.007) propionate proportion of culture solution but reduced butyrate proportion at 6 h (p<0.018) and 12 h (p<0.001) of incubations. Supplementation of MO or MO+FO increased (p<0.001) the proportions of C18:2. The MO alone reduced (p<0.022-0.025) the proportion of c9,t11-CLA compared to FO in all incubation times. The FO supplementation increased the proportion of c9,t11-CLA. An additive effect of MO to FO in the production of c9,t11-CLA was observed at 6 h incubation. In vitro supplementation of monensin reduced hydrogenation of C18-UFAs while fish oil supplementation increased the production of CLA. (Asian-Aust. J. Anim. Sci. 2005. Vol 18, No. 2 : 221-225)


INTRODUCTION
Attempts to increase the CLA levels in ruminant products have been made on a dietary manipulation by forage intake level or forage to concentrate ratio (Chouinard et al., 1998b;Wang et al., 2003) due to the beneficial health effects from CLA in cancer (Ha et al., 1987), atherosclerosis (Lee et al., 1994) and immunity (Michal et al., 1992).Selection of oil source (Wang et al., 2002a), supplementation type of oil (Wang et al., 2002b) and pH effect (Wang and Song, 2003) also have been important in the CLA production.
Meanwhile, supplementation of monensin which is the ionophoric antibiotics has increased the ratio of propionate to acetate (Newbold et al., 1993;Yang and Russell, 1993) while reduced CH 4 production, thus improved energetic efficiency in beef cattle (Spears and Harvey, 1984).Dietary ionophores has also been known to alter lipid metabolism in the rumen.Fellner et al. (1997) found an increased CLA proportion from the infusion of monensin (2 µg/ml) in the in vitro study compared to that without it.Dhiman et al. (1999) also reported that monensin supplementation (250 mg/cow/day) to the diets enhanced CLA contents in milk.Thus, the use of monensin in association with lipid supplementation may be one of the ways to increase the CLA proportion.
In addition, fish oil which is generally high in ω-3 fatty acids may also contribute to the ruminal production of CLA.The CLA concentration in milk fat increased when lactating dairy cows were fed diets supplemented with fish oil (Chouinard et al., 1998a;Dhiman et al., 1999).The effect was even greater when the diet was supplemented with fish meal and monensin together (Dhiman et al., 1999).
However, since much of the CLA found in milk is actually synthesized within the mammary gland from C 18:1 trans-11 through the action of stearoyl-CoA desaturase (Griinari and Bauman, 1999), effects of monensin and fish oil in the CLA production by the rumen microbes are still required to determine.The present study, therefore, was conducted to examine the effect of monensin and/or fish oil on the hydrogenating characteristics of C 18 -unsaturated fatty acids (UFAs) and the formation of CLA by rumen bacteria.

Preparation of rumen fluid
Rumen contents were collected at 3 h after morning feeding (0700) from two ruminally cannulated Holstein cows fed 5 kg of corn silage (60%) and concentrate (40%) for the middle lactating period on a DM basis twice daily in an equal portion and were mixed in equal portions.Analyzed contents of crude protein, ether extracts and neutral detergent fiber in concentrate were 15.8, 3.57 and 38.2%, respectively.The rumen contents were brought to the laboratory a thermos in 20 min after it was taken and were blended in a Waring blender (Fisher 14-509-1) for 20 seconds at high speed to detach the bacteria from the feed particles, and were strained through 12 layers of cheesecloth to remove the feed particles and protozoa.The strained rumen fluid was flushed with CO 2 .

Preparation of culture solution and its incubation
Strained rumen fluid was mixed with McDougalls artificial saliva (1948) at the ratio of 1:1 under flushing of CO 2 .Six grams of commercially manufactured concentrate (1% of culture solution, w/v, as-fed basis) for the growing dairy cattle with 1.2 g of safflower oil (0.2%, w/v) absorbed to 2 g ground (1 mm) alfalfa hay (2.45% EE, DM basis) were added to the 600 ml mixed solution of rumen fluid and McDougalls artificial saliva in the glass culture jar (control), and CO 2 was flushed into the culture solution for 1 minutes.Monensin (Rumensin  , 10 ppm, w/v, MO), mixed fish oil (0.02%, w/v, absorbed to 0.2 g alfalfa hay, FO), and similar amounts of monensin and fish oil (MO+FO) to MO and FO were added into the control solution, respectively.The culture jar was covered with a glass lid equipped with stirrer and was placed into a water-bath (39°C).Culture solution was again flushed with CO 2 through glass tube connected to the jars for 1 min, and was incubated up to 12 h.Stirring speed during incubation was adjusted to 120 times/min.The incubation of culture solution was done three times under the similar condition.

Sampling and analysis
pH of culture solution was measured at the incubation times of 3, 6 and 12 h by inserting the prove of pH meter into the culture solution in the jar, and 5 ml culture solution was collected for ammonia and VFA analysis.All samples collected were kept frozen at -20°C until analyzed.Ammonia concentration was determined by the method of Fawcett and Scott (1960) using the spectrophotometer (DU-650).Four mls culture solution were mixed with 1 ml 25% phosphoric acid and 0.5ml pivalic acid solution (2%, w/v) as an internal standard.The mixed solution was centrifuged at 15,000×g for 15 min, and the supernatant was used to determine the concentration and composition of VFA using gas chromatograph (GC, HP 5,890 II, Hewlett Packard Co.).In addition, fifty ml incubation solution was also collected at the incubation times of 3, 6 and 12 h, freeze dried and lipids were extracted using Folch's solution (Folch et al., 1957).Methylation of the lipids extracted followed the method of Lepage and Roy (1986) prior to injecting into the GC using a fused silica capillary column (100 m×0.25 mm, i.d.×0.20 µm thickness, Supelco, SPTM-2,560, USA).Fatty acid composition of safflower oil and fish oil was also analyzed by the same method as for the culture solution and shown in Table 1.

Statistical analysis
The results obtained were subjected to least squares analysis of variance according to the general linear models procedure of SAS (1985) and the data among treatments were compared using S-N-K Test (Steel and Torrie, 1980).

RESULTS
Higher pH (p<0.047, Figure 1) and ammonia concentration (p<0.042, Figure 2) were observed from the culture solution containing MO at 12 h incubation than those from control or FO.Supplementation of MO did not affect the VFA concentration, but increased (p<0.0001-0.007)propionate (C 3 ) proportion over the all collection times of culture solution while reduced butyrate (C 4 ) proportion at 6 h (p<0.018) and 12 h (p<0.001) of incubations (Table 2).Concentration and proportion of VFA were not greatly affected by FO supplementation.Supplementation of MO or MO-FO lowered (p<0.001-0.002) the proportion of C 18:0 throughout the incubation, while enhanced the proportions of C 18:1 at 12 h (p<0.035), and C 18:2 at 3 h (p<0.001) and 6 h (p<0.0009) of culture solution (Table 3).Supplementation of MO alone reduced (p<0.022-0.025) the proportion of c9,t11-CLA compared to FO in all incubation times but increased (p<0.025) the proportion of c9,t11-CLA compared to that of control at 12 h incubation.Reversed trends to the profiles of most fatty acids in culture solution containing MO, however, were obtained from the supplementation of FO (Table 3).An additive effect of MO to FO in the production of c9,t11-CLA was only observed at 12 h incubation.Production of t10,c12-CLA was relatively small compared to c9,t11-CLA and its proportional trend for all incubation times as influenced by supplementation sources followed the cases of c9,t11-CLA.

DISCUSSION
The patterns of the changes with incubation time in pH, ammonia concentration and VFA indicate the normal fermentation and they responded to the supplemented sources.The data of pH (Figure 1) and proportions of C 3 and C 4 (Table 1) in culture solution in the present study may indicate that MO (10 ppm, w/v) affected the fermentation as it probably reduce the Gram-positive bacterial growth which are sensitive to MO (Russell, 1987).
In general, fish oil contains relatively long-chain polyunsaturated fatty acids (PUFAs, Table 1).But effect of fish oil on the ruminal bio-hydrogenation of the PUFAs has not been well defined.Further study, therefore, is required to examine how the fish oil act on lipolysis, hydrogenation of UFAs and CLA production by rumen bacteria.

Table 1 .
Fatty acid composition (% of total) of oils used in the Figure 2. Ammonia concentration in culture solution.

Table 2 .
Concentration and molar proportion of VFA in culture solution when incubated with safflower oil

Table 3 .
Composition (%) of C 18 -fatty acids in culture solution when incubated with safflower oil