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ANIMAL NUTRITION |
* Department of Animal and Poultry Science, and
Department of Food Science, University of Guelph, Guelph, Ontario, Canada N1G 2W1
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Abstract |
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 Top Abstract INTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONLITERATURE CITED |
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Key Words: fish oil • drinking water • milk fatty acid • dairy cow
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INTRODUCTION |
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TopAbstractINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONLITERATURE CITED |
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; Murphy, 1992). Yet, intake of water by lactating dairy cows has been largely overlooked. Ingested water is assumed to equilibrate with ruminal fluid (Cafe and Poppi, 1994) although 18 to 80% of ingested water bypassed the rumen (Woodford et al., 1984; Zorrilla-Rios et al., 1990).
The transition period can be divided into 2 phases: 5 to 7 d prepartum, characterized by a 30% reduction in DMI (Grummer, 1995), and 0 to 21 d postpartum, when intake increases rapidly. Typically, fats are fed to increase dietary energy density (NRC, 2001), but fat supplementation has other potential benefits [e.g., increasing the unsaturated fatty acid (FA) content of milk fat (Middaugh et al., 1988; Stegeman et al., 1992)]. Considering the prepartum reduction in DMI, the rumen bypass potential of water, and advantages of using water as a delivery vehicle for nutrients (Osborne et al., 2002), use of water as a delivery vehicle for enrichment of n-3 PUFA such as eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) in milk may be warranted. However, there is no report on whether fish oil (FO) supplemented in the drinking water would bypass the rumen and increase the rate of transfer of dietary n-3 PUFA into milk.
Conventionally, FO is top-dressed on the total mixed ration (TMR) resulting in its FA being extensively bio-hydrogenated in the rumen (Griinari and Bauman, 1999; Bauman and Griinari, 2003). We hypothesized that FO in drinking water of cows would bypass the rumen and increase n-3 PUFA in milk. Our objective was to determine effects of supplementing FO in drinking water of dairy cows on production performance and milk FA composition compared with top-dressing FO on the TMR.
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MATERIALS AND METHODS |
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TopAbstractINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONLITERATURE CITED |
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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.
Sixteen lactating multiparous Holstein dairy cows (741 ± 84 kg BW; 60 ± 2.3 d in milk, mean ± SD) housed in a tie-stall facility at the Elora Dairy Research Center, University of Guelph (Guelph, Ontario, Canada) were used in the study. The study was conducted in a completely randomized design with repeated measurements. The cows were offered a TMR (Table 1) for ad libitum intake, allowing for 5 to 10% refusal. The TMR was offered twice daily at 0830 and 1300, and DMI was monitored daily throughout the experiment. The cows were blocked by days in milk and allocated to 1 of 2 treatments: 10 g of menhaden FO [specific gravity (H2O = 1): 0.93; Omega Protein Inc., Reedville, VA] per kg of DM top-dressed on the TMR (FOT), or 2 g of menhaden FO/L delivered in the drinking water (FOW). The trial lasted for 5 wk: a 1-wk pretreatment adjustment period and 4 wk of treatment.
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The cows were milked in their stalls twice daily at 0500 and 1500. Milk samples were collected from the morning milking and preserved with 2-bromo-2-nitro-propane-1-2-diol, and the samples were immediately submitted to the Central Milk Testing Laboratory (Laboratory Services Division, University of Guelph, Ontario, Canada) for compositional analysis. A second set of milk samples without preservative was also collected daily from the morning milking and stored at –70°C for milk FA analysis. Blood samples were obtained weekly via puncture of a coccygeal artery or vein before the morning feeding into 10-mL evacuated tubes (Becton Dickinson Vacutainer Systems, Franklin Lakes, NJ). Samples were allowed to stand at room temperature for 30 min and then centrifuged at 2,500 x g for 15 min in a refrigerated centrifuge (Beckman, Model TJ-6, Palo Alto, CA) at 4°C. Serum was separated and transferred to 7-mL, plastic scintillation vials and stored at –70°C until analysis.
Chemical Analysis
Representative samples of the TMR were collected from the data ranger (American Calan, Northwood, NH) 3 times/wk before top-dressing the FO, pooled weekly and stored at –20°C until analyzed. Orts from individual cows were weighed each morning before feeding, and representative samples were collected and stored at –20°C until analyzed. The TMR and orts were analyzed for DM by oven-drying at 60°C for 48 h (method 930.15; AOAC, 1990). The dried TMR samples were then ground to pass through a 1-mm screen (Wiley Mill, Arthur H. Thomas, Philadelphia, PA), and the chemical composition was determined in duplicate at a commercial laboratory (Agri-Food Laboratories, Guelph, Ontario, Canada). Analytical DM content was determined by oven-drying at 135°C for 2 h (method 3.002; AOAC, 1990), OM by ashing at 500°C for 16 h (method 942.05; AOAC, 1990), and CP by use of Leco FP 428 nitrogen analyzer (Leco Corporation, St. Joseph, MI; method 4.2.08; AOAC, 1990). The samples were also analyzed for ether extract (method 920.39; AOAC, 1990), ADF (method 973.18c; AOAC, 1990), and NDF (Van Soest et al., 1991) using 
-amylase (Sigma No. A3306, Sigma Chemical Co., St. Louis, MO), sodium sulfite, and correction for ash concentration adapted for an Ankom 200 fiber analyzer (Ankom Technology, Fairport, NY). The Ca, P, K, Mg, and Na were determined by inductively coupled plasma spectroscopy (method 945.46; AOAC 1990). Milk samples were analyzed for true protein and fat using a near infrared analyzer (MilkoScan 4000, Foss NIR Systems Inc., Hillerod, Denmark).
Analysis of FA Composition
Frozen serum and milk samples were thawed in a water bath at 38°C, and the milk samples were pooled weekly according to method 925.21 (AOAC, 1990). Lipids were extracted with chloroform:methanol:water in the ratio of 1:1:0.9 using a modification of the method described by Bligh and Dyer (1959). The total lipids were methylated using NaOCH3 as the catalyst (Cruz-Hernandez et al., 2004) and analyzed directly by GLC. Fatty acid methyl ester 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 x 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., 2001; Cruz-Hernandez et al., 2004). Agilent Technologies ChemStation software (Version A.10, Palo Alto, CA) was used for data analysis. A 1-µL sample was injected using the splitless mode set at 0.3 min. Peaks were identified by comparison of retention times with FA methyl ester 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., 2002; Shingfield et al., 2003; Loor et al., 2004). Fatty acids are reported as g/100 g of total FA.
Calculations and Statistical Analysis
The transfer coefficient of DHA, docosapentanoic acid (DPA), or EPA was calculated as
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Milk FA yield was estimated as described by Glasser et al. (2007), whereas FA intake was calculated as DMI x concentration of FA in the diet (g/kg).
The ANOVA for averages of DMI, milk yield, and serum and milk FA and FA transfer coefficients were conducted using the MIXED procedure (SAS Inst. Inc., Cary, NC) using the model Yij = µ + i + βj + βij + 
ij, where Yij = the dependent variable, µ = the overall mean, i = the effect of treatment (i = 1, 2), βj = the effect of time (j = 1, 2, 3, 4), βij = the effect of the treatment x time interaction (ij = 1, 2, ....., 8), and ij = the random residual error. The effects of treatment were considered fixed, and cow within treatment was included as a random effect. To determine time-dependent changes, and interactions between time and treatment, the effects of treatments over time were evaluated using orthogonal contrasts. Day in milk and pre-treatment DMI, milk yield, and milk fat percentage were used as covariates. Effects were considered significant at a probability of P < 0.05. Differences among treatment means were tested for significance using Tukey’s multiple range test. Data are expressed as mean ± SEM, which represents the pooled SEM for the model.
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RESULTS AND DISCUSSION |
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TopAbstractINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONLITERATURE CITED |
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The FA composition of the menhaden FO is presented in Table 2. Dry matter intake, water intake, milk yield, and milk components are presented in Table 3. No interaction of treatment x sampling time was observed; therefore, treatment means across all sampling times are reported. Supplementing FO in the drinking water of dairy cows had no effect (P 
0.19) on DMI, water intake, or milk yield compared with top-dressing it on the TMR. Dry matter and water intakes remained relatively uniform throughout the trial. Similar water intake patterns have been reported for lactating dairy cows fed saturated and unsaturated FA supplements (Harvatine and Allen, 2006). Other studies have shown reduced DMI when FO was fed at 3% of ration DM (Donovan et al., 2000; Whitlock et al., 2002).
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). In general, oils with a high degree of unsaturation disturb rumen fermentation and fiber digestibility, leading to lower acetate production and milk fat synthesis (Coppock and Wilks, 1991). Pretreatment milk fat percentages were 3.45 vs. 3.59% ± 0.28, FOT vs. FOW, respectively, whereas the pretreatment milk fat yields were 1,272 vs. 1,534 g ± 276, FOT vs. FOW, respectively. This milk fat depression is consistent with other studies where FO has been fed (Cant et al., 1997; Jones et al., 1998; Whitlock et al., 2002). High concentrate diets, addition of oil seeds (Middaugh et al., 1988), and other rumen-active fats cause milk fat depression in lactating cows (Sutton, 1989). The protein content and yield in milk were not affected (P 0.15) by either treatment or over time, consistent with previous studies (Jones et al., 2000; Donovan et al., 2000; Whitlock et al., 2002), but contrary to Cant et al. (1997) who observed a reduction in milk protein content when FO, monensin, or both were added to the diet of dairy cows. Milk and Serum FA Composition
Milk and serum FA, 18:1 isomers, and CLA contents (g/100 g of total FA) are summarized in Tables 4 to 10. The major characteristic of ruminant milk fat is the high proportion of saturated FA (Palmquist et al., 1993; see Table 4). The contents of the short-chain saturated FA 7:0, 8:0, 9:0, 10:0, and 12:0 in the milk of FOT cows were lower (P < 0.05) than for FOW cows. Short-chain FA (4:0 to 12:0) are considered products of de novo synthesis within the mammary gland using acetate as the precursor. As mentioned, milk fat content was reduced linearly over time (Table 3). This was associated with altered rumen biohydrogenation characterized by a shift in major biohydrogenation pathways: decreased formation of 18:1 trans-11 (vaccenic acid) and increased formation of 18:1 trans-10 in the rumen (Griinari and Bauman, 1999) via trans-10, cis-12 CLA as a direct inhibitor of milk fat synthesis in the mammary gland (Bauman and Griinari, 2003).
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; Wattiaux and Grummer, 2004). Irrespective of the treatments, the concentration of 18:0 in serum was 66% higher than in milk (Tables 4 and 5). Under normal conditions 18:0 is one of the major FA that are transferred without alteration into milk lipid (Duncan and Garton, 1963; Mansbridge and Blake, 1997). The contents of 20:0 and 20:1 in the milk of FOW cows were lower (P < 0.05) than for FOT cows. There was no treatment difference (P 0.60) in the content of 20:0 or 20:1 in serum (Table 5). The contents of nervonic acid (24:1n-9) in the milk of FOW cows were higher (P < 0.05) than for FOT cows although the concentrations were very low in both groups. The concentration of 24:1 in serum was 10 times higher than in milk, but there was no difference (P = 0.33) between treatments.
The concentrations of the highly unsaturated FA arachidonic acid (20:4n-6), EPA (20:5n-3), DPA (22:5n-3), and DHA (22:6n-3) in milk were not (P 0.16) affected by treatment (Table 4). Normally, DHA occurs only in trace amounts (
0.01%) in milk fat of cows fed conventional diets, whereas EPA and DPA are present at about 0.05 and 0.08%, respectively (Wright et al., 2007). By supplementing 2 g of FO/L in the drinking water of dairy cows in the current study, the yields of EPA (P = 0.16) and DPA (P = 0.17) in milk were 13 and 12% higher, respectively, for FOW compared with FOT (Table 6).
Offer et al. (1999) reported transfer coefficients of less than 0.03 for PUFA of chain length C20. Cant et al. (1997) reported transfer efficiencies of 0.09 and 0.16 for EPA and DHA, respectively, whereas Wright et al. (1999) reported that the transfer of EPA and DHA declined from 0.27 to 0.07, and 0.34 to 0.11, respectively, as intakes increased from 1.6 to 16 g/d. Palmquist and Griinari (2006) reported that only 10 mg of DHA/g of intake, 57 mg of EPA/g of intake, and 110 mg of DPA/g of intake appeared in milk compared with the 17, 34, and 214 mg/g of DHA, EPA, and DPA intake, respectively, in the drinking water of the cows in the current study. We had hypothesized that FO supplemented in the drinking water of cows would bypass the rumen and increase the milk FA contents of n-3 PUFA such as EPA and DHA. The transfer coefficients for EPA, DPA, and DHA from diet to milk fat were not different (P 0.16) between treatments (Table 6). It appears the amount of FO added in the current study did not bypass the rumen as hypothesized, and based on milk fat data, it appears both modes of delivery of FO affected rumen function in a similar manner resulting in similar transfer coefficients between treatments for EPA, DPA, and DHA. We however concur with Palmquist and Griinari (2006) that a more precise approach for determining transfer efficiencies is to use different levels of FO in the diet and determine the slope of the regression of the amount of FO FA in milk vs. intake instead of using single concentrations of the individual FA in diet and milk.
The contents of 18:1 trans-12, 18:1 trans-13 and -14, and 18:1 trans-16 in the milk of FOW cows were lower (P < 0.05) than for FOT cows (Table 7). Fish oils in lactation diets have been shown to modify ruminal biohydrogenation and increase 18:1 trans isomers in milk fat (Pennington and Davis, 1975; Kalscheur et al., 1997). It has been suggested that the very long-chain FA of FO inhibit the final biohydrogenation step to 18:0, thereby maximizing yield of 18:1 trans intermediates (Wonsil et al., 1994; Shingfield et al., 2003; Lee et al., 2005). The concentration of 18:1 cis-9 in milk was highest among the MUFA in both groups. In milk, vaccenic acid was the main 18:1 trans isomer in both treatment groups (Table 7). The concentration of 18:1 trans-11 was higher in milk than in serum (Tables 7 and 8), which was surprising because 70 to 80% of 18:1 trans-11 in blood is converted to cis-9, trans-11 CLA in the mammary tissue, which should lead to a depletion of 18:1 trans-11 in milk (Griinari et al., 1998). The higher concentration of 18:1 trans-11 in milk may reflect differential esterification or availability of other FA for esterification. Similarly, the concentration of 18:1 trans-10 (Tables 7 and 8) and total 18:1 trans (Tables 4 and 5) in milk were higher than in serum. Differences in saturated and unsaturated FA concentrations between serum and milk (Tables 4 and 5) indicate the existence of desaturation activity in the mammary gland (Chilliard et al., 2000; Voigt and Hagemeister, 2001). The concentration of 18:1 trans-10, 18:1 trans-11, and total 18:1 trans increased quadratically (P < 0.001) with time (data not shown).
Several CLA isomers and their precursors were also identified. Generally, the concentration of cis-9, trans-11 CLA; trans-7, cis-9 CLA; and total CLA was higher in milk than in serum (Tables 9 and 10). The contents in milk of trans-9, trans-11 plus trans-10, trans-12 CLA in FOW cows were lower (P < 0.05) than for FOT cows (Table 9). All the other CLA identified were not (P 0.08) affected by treatments. The trans-10, cis-12 CLA was present in milk in low concentrations in both treatments (Table 9). This CLA isomer has been reported to depress milk fat (Peterson et al., 2002). In the current study, milk fat percentage and yield were numerically lower for FOT cows, although not significantly different from FOW cows (Table 3). The concentration of trans-10, cis-12 CLA was also lower during diet-induced milk fat depression than when comparable depression of milk fat was induced by postruminal trans-10, cis-12 CLA infusions (Bauman and Griinari, 2003), suggesting that other biohydrogenation intermediates may also have contributed to the reduction in milk fat secretion. Shingfield et al. (2003) reported that FO may reduce the flow of trans-10, cis-12 CLA leaving the rumen and suggested that the decrease in milk fat content in response to FO was associated with increased milk fat 18:1 trans-10 concentrations that arose from increased ruminal formation of this biohydrogenation intermediate. However, in an experiment to examine the effect of 18:1 trans-10 on milk fat synthesis, Lock et al. (2007) infused pure 18:1 trans-10 in the abomasum of dairy cows and reported that although 18:1 trans-10 was taken up by the mammary gland and transferred to milk fat, it had no effect on milk fat synthesis even when provided at a dose 10 times greater that the effective dose of trans-10, cis-12 CLA. Perfield et al. (2007) suggested that an increase in milk fat content of trans-9, cis-11 CLA was associated with diet-induced milk fat depression. These authors provided evidence of a role for this isomer in milk fat depression based on the 15% reduction in milk fat yield with abomasal infusion of a CLA enrichment that supplied 5 g/d of trans-9, cis-11 CLA.
The concentration of vaccenic acid in milk was not different between FOT and FOW (P = 0.81; Table 7). It has been suggested that FO reduces the final biohydrogenation step where vaccenic acid is converted to stearic acid (Griinari and Bauman, 1999). Similarly, DHA has been shown to promote vaccenic acid accumulation in mixed ruminal cultures when incubated with linoleic acid (AbuGhazaleh and Jenkins, 2004).
The effect of FO on the concentration of cis-9, trans-11 CLA in milk for both treatments increased quadratically (P = 0.003) with time, consistent with previous studies. Whitlock et al. (2002) reported that the level of cis-9, trans-11 CLA in milk decreased after 14 d when FO and extruded soybeans were fed, whereas AbuGhazaleh et al. (2004) reported that the concentration of cis-9, trans-11 CLA in milk increased until d 21 and then declined thereafter. In the rumen, cis-9, trans-11 CLA is formed primarily from isomerization of dietary linoleic acid (18:2n-6) during the first step of biohydrogenation (Harfoot and Hazlewood, 1988).
In conclusion, there were no differences between the 2 modes of administration of FO (10 g FO per kg of DM top-dressed on the TMR vs. 2 g of FO/L metered in the drinking water) on DMI, water intake, or milk yields. Although it appears that the amount of FO added in the study did not bypass the rumen as hypothesized, these results suggest that drinking water can be an effective alternative for supplementing FO in the diet of dairy cows. Further research is warranted to explore the use of drinking water as a vehicle for supplying nutrients to dairy cows, especially during the transition period when DMI is normally reduced.
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Footnotes |
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3 Current address: Agriculture and Agri-Food Canada, Lethbridge Research Centre, Lethbridge, Alberta, Canada T1J 4B1.
2 Corresponding author: vosborne{at}uoguelph.ca
Received for publication June 11, 2007. Accepted for publication November 19, 2007.
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LITERATURE CITED |
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TopAbstractINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONLITERATURE CITED |
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