ניסוי מס' 2


בחינת ההשפעה של תוספת תערובת שמנים עתירת קשרים כפולים למנה כולית קונוונציונלית על ריכוז החל"צ בחלב

A milk fat CLA peak response to oil supplementation of a TMR fed to lactating cows


D. Ben-Ghedalia,a∗ E. Yosef,a J. Miron,a R. Solomon,b M. Nikbahata


aAgricultural Research Organization, Bet Dagan 50250, Israel
bMinistry of Agriculture, Extension Service, Bet Dagan 50250, Israel


Abstract
An experiment was conducted to study the effects of adding a highly unsaturated oil mixture at 5% dry matter (DM) of a total mixed ration (TMR) fed to lactating cows on the lactational performance and milk fat conjugated linoleic acid (CLA). Ten lactating multiparous Holstein cows were divided into two groups of five cows each, similar in average stage of lactation, daily milk yield and body weight at the onset of the experiment. All the cows were held in one shaded corral yard as a single group and had free access to water. Along the entire experimental period, the two groups of cows received a commercial TMR. The control group was on that ration only, and the treatment group received the TMR plus a mixture of: sunflower oil + fish oil + linseed oil at the ratios of, 6:3:1, respectively, at 5% of the ration DM. During the first week of the experiment, the treatment cows were adapted to the oil mixture by increasing gradually the oil content of the TMR up to 5% DM basis. This feeding pattern lasted for six weeks during which milk was sampled at weekly intervals. The addition of the mixture of highly unsaturated oils to the TMR at 5% DM, resulted in a decrease in the digestibility of the ration and particularly of the fibrous fraction, and a decrease in feed intake, milk production and milk fat. Only minor changes in CLA content occurred in the control milk fat during the experiment. But there was a CLA fast response in the oil treatment which developed during the 1st week to a peak of 27.5g CLA per kg milk fat, followed by a steep decrease during the 2nd week to 12.3 g/kg, and reaching the nadir of 5.5 g/kg at the end of the experiment. It appears that at an early stage of the experiment, the flow of vaccenic acid from the rumen was disturbed in the oil treatment.

Abbreviations: CLA, conjugated linoleic acid; FA, fatty acids.
Key words: Dietary oil; Dairy cow; Performance; CLA

Introduction
The lowest CLA concentration in natural milk able to exert a pharmacological impact on human health, and the way to produce that milk continuously under intensive conditions by high lactating cows, are among the major un-resolved issues in the CLA research area. Pasture as the only or major source of feed, proved to be almost the only nutritional management for producing CLA- enriched milk at a steady level for an extended period of time (Kelly et al., 1998; White et al., 2001; Schroeder et al., 2003). However, milk production by pasture fed cows is in the range of 15 – 25 kg/d with a CLA content of 10 – 20 mg/g FA (Dhiman et al., 1999; Ward, et al., 2003). This pattern of performance does not coincide with the aspiration for intensive production of high CLA milk.
Usually, high level of milk production is attained by indoors TMR feeding, but the milk CLA under these conditions is in the range of 3 – 5 mg/g FA (Ben-Ghedalia, et al., 2002; Loor, et al., 2003; AbuGhazaleh et al., 2004). Green forage is low in lipids, usually in the range of 30g/kg DM and low in linoleic acid (Elgresma et al., 2003; Loor et al., 2003; Whiting et al., 2004). Linoleic acid is required for the production of vaccenic acid (VA) in the rumen which is turned into CLA in the mammary gland (Corl et al., 2000). Thus, the positive CLA- green- pasture effect is yet unexplained. The reason why the same forage while fed virtually as conserved or as a part of a TMR, does not elicit a CLA positive effect, is not thoroughly understood (Elgersma et al., 2004).
Oils and extruded oilseeds, mostly extruded soybeans were used for fortifying TMRs with linoleic acid, aiming at an increase in milk CLA beyond the normal level of 0.3% of milk FA (Kelly et al., 1998a; Solomon, et al., 2000; AbuGhazaleh et al., 2003). These studies, as many other Latin square designed, short term experiments, showed a remarkable effect of the oil supplemented TMRs on milk CLA. Most of the information in the literature, originates from short term, largely Latin square-designed experiments presenting average figures for entire experimental periods. However, there is little data based on experiments conducted over extended periods of time with oil supplemented TMRs. The objective of this study was to follow up during an extended period of time at weekly intervals, the changes in milk CLA of cows fed oil supplemented TMR.


Materials and methods
Cows, diets, and sampling procedures
Ten lactating multiparous Holstein cows (average of 2.7 lactations) were divided into two groups of five cows each, similar in average (means ± SE) stage of lactation (137 ± 5.3 DIM), daily milk yield (38.2 ± 0.75 kg) and body weight (630 ± 8.2 kg), at the onset of the experiment. The cows were housed in the ARO experimental dairy barn (Bet-Dagan, Israel) which is equipped with cow recognition system and individual feeders on top of weighting balances that enable computerized monitoring of voluntary feed intake for each individual cow. All the cows were held in one shaded corral yard as a single group and had free access to water. Along the entire experimental period, the two groups of cows received a commercial TMR whose composition is shown in Table 1. The control group was on that ration only, and the treatment (oil) group received the TMR plus a mixture of: sunflower oil + fish oil + linseed oil at the ratios of, 6:3:1, respectively, at 5% of the ration DM. The fatty acid composition of the oils is shown in Table 2. During the first week of the experiment, the treatment cows were adapted to the oil mixture by increasing gradually the oil content of the TMR up to 5% DM basis. This feeding pattern lasted for six weeks during which milk was sampled at weekly intervals. Rations were fed once daily at 10:00 for ad libitum intake, allowing for 5 to 10% orts. Individual feed intakes were recorded and TMR were sampled daily. The TMR samples were pooled on a weekly basis to produce seven composites for each dietary treatment. Dry matter intake (DMI) was determined by oven drying of a portion of the TMR weekly samples at 105oC for 24h. The weekly TMR samples were oven dried at 60oC for 48h, ground through a 1-mm screen and used for the determination of in vitro digestibility and chemical analyses.
Cows were milked three times a day at 7:00, 14:00 and 21:00. Milk yield was recorded daily by automatic meter (Afimilk, Israel). Milk samples were collected during three sequential milkings on a weekly basis every Wednesday of the experimental period. Every set of fresh milk samples for each cow was stored at 4oC until analyzed for fat, protein, and lactose by infrared analysis (AOAC, 2000; using Milkoscan 4000, Foss Electric, Hillerod, Denmark). Another set of individual milk samples was used for analyzing fatty acids composition.


Chemical analyses
Samples of dried TMR (seven samples for each TMR) collected on a weekly basis were assayed in triplicates for DM and organic matter (OM), (AOAC, 2000); crude protein according to the Kjeldahl method (AOAC, 2000) and NDF according to Van Soest et al. (1991), employing the -amylase procedure and Ancom apparatus (Ancom, USA) for extraction and filtering.
In vitro digestibility of the DM and NDF of the TMR samples was determined in triplicates according to the two-stage fermentation technique of Tilley and Terry (1963) by using the two sources of rumen inocula from both the control and the oil fed cows.
Fatty acids composition of the weekly milk samples of each individual cow was determined in duplicates. Fat was extracted from milk samples by boiling in a detergent solution as described by Hurley et al. (1987). The extracted milk fat samples and the individual oils fed, were weighed, capped under argon gas and stored at -20oC until further analysis. The fat samples were derivatized to methyl esters by mixing 30 mg of fat with 5 ml of 4% HCl-methanol. Heptadecanoic acid was used as an internal standard. The methyl esters were extracted with hexane and the extract was washed twice with distilled water and dried over anhydrous sodium sulfate. Fat samples were analyzed by gas chromatography (Model 5890 Series II, Hewlett-Packard Co., Wilmington, DE) fitted with a flame ionization detector. Samples containing methyl esters in hexane (1 to 3 µl) were directly injected through the split-less injection port onto Chrompack, CP-Sil 88 capillary column (100m X 0.25 mm i.d, Supelco Inc., Bellefonte, PA). Oven temperature was programmed from 50 to 2000C in three steps at the rate of 100C per minute: to 500C for 1'; to 1600C for 45'; and the 3rd increment up to 2000C, lasting for 22'.


Calculations and statistical analysis
Daily fat corrected milk production (3.5% FCM) was calculated according to the equation used by the Dairy Milk Association, Israel:
FCM (kg) = 0.342 x milk (kg)+16.216 x milk fat (kg).
Differences in the in vitro digestibility values between the two TMRs as well as data of DMI, milk yield and composition, and milk fatty acid composition of the individual cows were statistically analyzed by ANOVA using the GLM procedure of SAS (SAS, 1996). Data are presented as means + standard error of the means (SEM). Significance was declared at P< 0.05.


Results and discussion
The mixture of oils used in this experiment consisted of sunflower oil + fish oil + linseed oil at the ratios of: 6+3+1. Table 2 shows that sunflower oil is rich in linoleic acid, linseed oil in linolenic acid and fish oil in poly-unsaturated fatty acids. This mixture was designed to form a FA profile which will both contribute the precursor for the production of vaccenic acid in the rumen on one hand, and protect that intermediate from being further hydrogenated to stearic acid in the rumen, on the other hand (Chilliard etal., 2001; Harfoot et al., 1973; Polan et al., 1964). However, although the poly-unsaturated FA of fish oil, C20:5 (EPA) and C22:6 (DHA) are known as promoters of milk CLA, they are also recognized as milk fat depressors (Chilliard et al., 2001). The performance data shown in Table 3 coincide with the above-mentioned. Generally, milk fat depression following the inclusion of oils at levels higher than 2%, are not uncommon in the literature (Whitlock et al., 2002). However, the effect of supplementing the TMR with 5% DM basis of that mixture, was expressed also in a remarkable decrease in feed intake (Table 3). A reduction in feed intake was found also by Loor et al.(2002) on a basal ration supplemented with 3.3% canola oil (DM basis). It should be mentioned however, that our study was conducted on the Mediterranean coast during the hot summer months of July-August, characterized by high temperature (26-38OC) and humidity (60-80%). The climatic factor was probably responsible for the overall performance of the cows and played some role also in facilitating the "oil effect". The significant reduction in feed intake by the oil-treatment cows was expressed in a lower milk production, which while combined with the lower milk fat, resulted in lower daily FCM yield. The patterns of these effects along the 7 weeks of the experimental period are shown in Figures 1-3. Figure 1 shows that the reduction in feed intake reached a plateau from the 2nd week on. A steep and similar decrease in milk yield in all the cows during the 1st week of the experiment as shown in Figure 2 was probably associated with the harsh summer conditions during that period of time. From that week on, there was a consistent gap in average daily milk yield between the treatments, in favor of the control. Figure 3 shows that in the oil treatment, the milk fat depression effect was immediate and from the 1st week on, milk fat fluctuated between 2.3 to 2.9%. Figure 4, shows the changes in the content of CLA in milk fat of the control and treatment cows along the 7 weeks of the experimental period. As expected, only minor changes in CLA content occurred in the control milk fat. But in the milk fat of the oil treatment, there was a fast response which developed during the 1st week to a peak of 27.5 mg CLA per kg milk fat, followed by a steep decrease during the 2nd week to 12.3 mg/kg, and reaching the nadir of 5.5 mg/kg at the end of the experiment. A vast amount of CLA data in the literature originate from Latin square designed studies and are expressed as average figures obtained during short experimental periods (AbuGhazaleh et al., 2003; Kelly et al., 1998; Loor et al., 2002; Whitlock et al., 2002; Zheng et al., 2005;). Milk fat CLA, time-dependent behavior as affected by a dietary treatment, can not be derived from such studies. This parameter is important in order to find out whether the effect of a given dietary treatment on the content on milk fat CLA, is transitory or consistent along time. It might be argued whether the peak effect found in this study is the result of the particular conditions, including the high level and highly unsaturated dietary oil, or not. However, some of our unpublished data obtained with Israeli commercial diets, show that such peak effects are not uncommon even at lower levels and less unsaturated dietary oils. The above-mentioned is supported by data from AbuGhazaleh et al. (2004) which conducted an experiment over an extended period of 10 weeks with cows fed a 50% forage diet supplemented with 2% oil from extruded soybeans plus 0.5% fish oil. In their study, CLA peaked at the 3rd week to 1.4%, decreased to 1% of milk fat at the 5th week and remained steady at this level thereafter. An experiment by Ryhanen et al. (2005) conducted over 14 weeks with rations containing ~50% grass silage DM plus ~2% rapeseed oil resulted in milk fat CLA fluctuating around 1%. Changes in milk FA composition of the oil treatment cows are shown in Table 4. FA profiles of the control milk were fairly stable along the experiment and very similar to that of the 0 week of the oil treatment. The effects of the oil treatment were expressed in a decrease in the short chain FA synthesized in the mammary gland, a decrease in stearic acid and an increase in oleic acid. These changes were predictable as shown also and discussed by Chilliard et al. (2001) and Loor et al. (2002). Table 5 shows the in vitro digestibility of the experimental rations. The oil mixture added to the TMR resulted in a considerable decrease in the digestibility of the ration with particular effect on the fibrous fraction. This was most likely the result of the inhibitive effect of the oil, exerted on the fibrolytic rumen bacteria including the Butyrivibrio species which are involved both in CW degradation and in the biohydrogenation of linoleic acid to vaccenic acid (Harfoot and Hazlewood, 1997; Kim et al., 2000). Although the final step of CLA production occurs in the mammary gland, a continuous undisturbed flow of vaccenic acid from the rumen is apparently the key issue shaping the final concentration of CLA in milk.


Conclusions
The addition of a mixture of highly unsaturated oils to a TMR at 5% DM, resulted in a decrease in the digestibility of the ration and particularly of the fibrous fraction, and a decrease in feed intake, milk production and milk fat. There was a peak effect of the oil ration on the milk fat CLA, reaching 27.7mg/g after 7 days of adaptation, followed by a fast decrease after 14 days, down to 5.5mg/g at the end of the 7 weeks of the experiment. It appears that at an early stage of the experiment, the flow of vaccenic acid from the rumen was disturbed in the oil treatment. Factors such as ration composition, the level oil added to the TMR, and the FA profile, are of major importance and should be considered in order to ensure a steady and consistently increased level of CLA in milk.


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