Milk fatty acid composition in response to reciprocal combinations of sunflower and fish oils in the diet

Animal Feed Science and Technology 131 (2006) 358–369

Milk fatty acid composition in response to reciprocal combinations of sunflower and fish oils in the diet D.L. Palmquist a,∗ , J.M. Griinari b a

Department of Animal Sciences, Ohio Agricultural Research and Development Center, The Ohio State University, Wooster, OH 44691, USA b Department of Animal Science, University of Helsinki, 00014 Helsinki, Finland Accepted 29 May 2006

Abstract This study investigated the hypothesis that a high linoleic acid vegetable oil fed in combination with a fish oil ruminal biohydrogenation modifier as a diet supplement would result in higher concentrations of vaccenic and rumenic acids in milk fat versus either fed alone. Four lactating cows were fed legume silage (600 g/kg diet DM) and a lactation concentrate (400 g/kg DM) supplemented with 30 g/kg diet DM of oil in a Latin square design with 3-week experimental periods. Fish oil was proportionately 0, 0.33, 0.67 or 1.0 of the oil supplement, with the balance as sunflower oil. The DM intake decreased linearly (P<0.04) with increasing concentrations of fish oil in the diet. Milk yield and its proportion of protein were unaffected by fish oil intake, whereas the proportion of milk fat increased linearly (P<0.02) with increasing fish oil. Proportions of milk fatty acids from C4 to 16:1 increased linearly (P<0.01) for all fatty acids except 14:1 (NS) as the proportion of fish oil increased. Conversely, 18:0 (0.001), cis-9 18:1 and 18:2 (P<0.01) decreased linearly with increasing fish oil, while trans-11 18:1 and cis-9, trans-11 18:2 were highest at intermediate levels of fish oil. Other identified trans isomers declined as fish oil increased. Arachidonic acid (AA; P<0.001), eicosapentaenoic acid (EPA) and docosapentaenoic acid (DPA; P<0.01) increased linearly in milk fat with increased fish oil feeding, whereas predictions were less robust for docosahexaenoic acid (DHA; P<0.05). Efficiency of transfer Abbreviations: AA, arachidonic acid; BH, biohydrogenation; CLA, conjugated linoleic acid; CP, crude protein; DHA, docosahexaenoic acid; DM, dry matter; DPA, docosapentaenoic acid; EPA, eicosapentaenoic acid; GLC, gas–liquid chromatography; VFA, volatile fatty acids ∗ Corresponding author. Tel.: +1 330 263 3795; fax: +1 330 263 3949. E-mail address: [email protected] (D.L. Palmquist). 0377-8401/$ – see front matter © 2006 Published by Elsevier B.V. doi:10.1016/j.anifeedsci.2006.05.024

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of fish oil fatty acids to milk fat, estimated by regressing their milk output on intake, ranged from <0.01 for DHA (P<0.10) to 0.79 for AA (P<0.01). Whereas only 10 mg of DHA/g intake appeared in milk, transfer of EPA was 57 mg/g intake (P<0.04) and for DPA it was 110 mg/g (P<0.01). Supplementing high linoleic vegetable oils with fish oil maximized concentrations of vaccenic and rumenic acids in milk fat, and transfer of eicosanoic fatty acids from diet to milk increased linearly as their intake increased. © 2006 Published by Elsevier B.V. Keywords: Milk fat; CLA; Vaccenic acid; Fish oil; Sunflower oil

1. Introduction Low rumen pH and fish oils in lactation diets modify ruminal biohydrogenation (BH) and can increase trans-18:1 isomers in milk fat (Pennington and Davis, 1975; Kalscheur et al., 1997). Lower rumen pH also influences the distribution of trans-18:1 isomers in milk fat (Piperova et al., 2002). Whereas oils high in 18:2 are excellent precursors for BH, the very long chain fatty acids of fish oil inhibit the final BH step to 18:0, thereby maximizing yield of trans-18:1 intermediates (Wonsil et al., 1994; Offer et al., 1999; Shingfield et al., 2003; Lee et al., 2005). As a precursor of rumenic acid (CLA; cis-9, trans-11 18:2) in milk fat, higher ruminal trans-11 18:1 leads to higher concentrations of rumenic acid (Griinari et al., 2000). At lower ruminal pH’s, the concentration of trans-10 18:1 increases, a condition associated with appearance of the trans-10, cis-12 18:2 isomer of CLA in milk fat, and milk fat depression (Bauman and Griinari, 2003), as it is a powerful inhibitor of milk fat synthesis (Baumgard et al., 2000). When milk fat depression is induced by fish oil supplements, increased trans-10 18:1 in milk fat occurs without an appreciable increase in the trans-10, cis-12 CLA isomer (Chilliard et al., 2001; Offer et al., 2001). In classical milk fat depression induced by high concentrate diets, de novo synthesis of short and medium chain fatty acids is decreased, and their yield is decreased proportionately more than the yield of long chain fatty acids of diet, or ruminal, origin (Bauman and Griinari, 2003). Diet-induced changes in BH are likely to reflect altered rumen microbial populations, but detailed information is limited. Wallace et al. (2005) characterized 69 different Butyrivibrio isolates, a major group of biohydrogenating bacteria, and showed that they could be divided into two distinct phylogenetic groups, which they called Groups ‘A’ and ‘B’. Group ‘A’ strains grew in higher concentrations (i.e., 200 ␮g/ml versus 5 ␮g/ml) of free linoleic acid, but linoleic acid isomerase activity was independent of phylogenetic position. A sub-group of Group ‘B’, containing Clostridium proteoclasticum, formed abundant stearate under specific growth conditions, whereas Group ‘A’ strains did not. Wallace et al. (2005) proposed that C. proteoclasticum is the long lost ‘Fusocillus’, shown to hydrogenate linoleic acid to stearate (Kemp et al., 1975). Group ‘A’ had low butyrate kinase and lipase activities, whereas these were high in Group ‘B’. Group ‘A’ bacteria produce butyrate by an acetyl-CoA/butyrylCoA transferase, forming free butyrate and acetyl-CoA from butyryl CoA (Diez-Gonzalez et al., 1999); in the presence of acetate the transferase activity is stimulated. In the present study, we postulated that feeding a combination of sunflower (substrate) and fish (modifier of rumen biohydrogenation) oils would provide optimum conditions to

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maximize ruminal trans-11 18:1 synthesis and resultant yields of rumenic acid in milk fat. Furthermore, we anticipated that fish oil-induced changes in biohydrogenation would alter volatile fatty acid (VFA) ratios in the rumen with the implication that shifts in microbial populations occurred.

2. Materials and methods Four ruminally cannulated cows in mid-lactation were fed four diets for ad libitum intake once daily in tie stalls. Diets were alfalfa silage:concentrate (60:40 dry matter (DM) basis) in a 4×4 Latin square experimental design with 3-week feeding periods. The composition of the concentrate mix (g/kg DM) was ground corn, 693; soya meal (440 g/kg crude protein (CP)) 184; oil, 75; dicalcium phosphate, 8.0; calcium carbonate, 10.0, magnesium oxide, 5.0, sodium bicarbonate 15.0; salt, trace minerals, vitamins, 10.0. Diets were fed as a total mixed ration with 30 g/kg DM of oil, consisting of 0, 10, 20 or 30 g/kg fish oil with the balance sunflower oil (Table 1). The DM and milk yield were measured daily and milk was sampled (weighted composite of the am and pm milkings) once weekly for content of protein and fat, as well as its fatty acid composition. Rumen contents were sampled at 2 and 6 h after feed was offered on the last day of each period to determine pH and VFA concentrations. Legume silage was sampled weekly and composited for analysis. Its DM was 590 g/kg, which contained 181 g CP/kg and 564 g NDF/kg. The DM of the ration was 710 g/kg. Milk Table 1 Composition (mg/g fatty acids) of the fatty acids in sunflower and fish oilsa Fatty acid

Sunflower oil

Fish oil

14:0 14:1 16:0 16:1 17:0 18:0 18:1 trans 18:1 cis-9 18:1 cis-11 18:2n − 6 18:3n − 3 20:0 20:1 20:2 20:4n − 6 20:3n − 6 20:5n − 3 22:5n − 3 22:6n − 3 Other

0.06b 0 68.5 0.7 0.7 44.2 0 192.4 8.0 639.6 15.1 3.3 2.1 – 0 – – – – 24.9

82.6 7.3 195.2 116.6 16.9 38.2 20.1 80.3 31.7 13.0 14.5 – 27.2 2.1 1.8 1.9 120.4 23.9 101.1 93.5

a b

Sunflower oil and Menhaden fish oil were both from Cereal By-Products, Mt. Prospect, IL, USA. Values represent duplicate analyses of two individual samples.

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fat and protein contents were determined by automated infrared analysis and VFA by Ohajuruka et al. (1991). Milk fat was collected from 10 ml of milk by centrifugation at 10,000×g for 10 min at 4 ◦ C. The fat cake was removed and stored in vials at −20 ◦ C. An aliquot of cake was transmethylated (Palmquist and Jenkins, 2003), using methyl-tert-butyl ether as the fat solvent. Methyl esters were analyzed by temperature-programmed GLC (Palmquist et al., 2004), with samples run isothermally a second time to optimize separation of trans-18:1 isomers, and proportions of individual trans isomers were then applied to the proportion of total trans-18:1 from the first run. Isomers were identified according to Precht and Molkentin (1996). Fatty acids are reported as mg/g of measured fatty acids.

3. Statistical methods The experimental design was a 4×4 Latin square with four levels of sunflower and fish oils in reciprocal amounts, with 30 g of total added oil/kg diet DM. Data from the last 2 weeks of each 3-week period were averaged for analysis by the PROC MIXED procedure of SAS (1999). Effects of level of fish oil were tested by linear and quadratic contrasts within SAS (1999).

4. Results and discussion The DM intake decreased linearly (P<0.04) with increasing concentrations of fish oil in the diet (Table 2), which is a typical response to fish oil supplementation (Cant et al., 1997; Shingfield et al., 2003). Milk yield and its protein concentration were unaffected by fish oil intake, as were yields of milk protein and milk fat. The diet supplying fish oil alone (i.e., 30 g of added oil/kg diet DM) resulted in a low milk fat proportion comparable to levels observed in other studies where fish oil has been fed to cows for at least 3 weeks (Abu-Ghazaleh et al., 2002; Chilliard et al., 2001; Offer et al., 1999, 2001; Shingfield et al., 2005). Increasing proportions of sunflower oil in the diet linearly decreased (P<0.02) milk fat to even lower proportions. Table 2 Dry matter intake and production of cows fed combinations of sunflower/fish oila Parameter

Dry matter intake (kg/day) Milk yield (kg/day) Milk fat (g/kg milk) Milk protein (g/kg milk) Milk fat (g/day) Milk protein (g/day) a b

Proportion of fish oil in oil supplement 0

0.33

0.67

1.00

17.2 22.1 20.6 29.8 430 650

17.2 21.6 22.8 29.2 495 620

15.5 20.3 26.2 28.8 532 585

14.5 19.8 26.4 29.5 501 546

All cows were fed 30 g oil/kg of diet dry matter. L = linear effect, Q = quadratic effect, and NS = P>0.10.

S.E.

Pb

1.34 2.83 3.03 1.35 73.8 73.8

<0.03L NS <0.02L <0.08Q NS NS

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Fig. 1. Fatty acid profile (g/100 g of total milk fatty acids) of milk fat from cows fed reciprocal amounts of fish oil and sunflower oil.

All reported fatty acids from 4:0 to 16:1 increased linearly (P<0.01), except 14:1 (NS), as the proportion of fish oil in the oil mixture increased (Fig. 1 and Table 3), suggesting that sunflower oil fatty acids, or their metabolites, more strongly inhibit de novo fatty acid synthesis in the mammary gland than fatty acids of fish oil, and/or fish oil fatty acids modified metabolites of sunflower oil fatty acids to less inhibitory products. Conversely, 18:0 (P<0.001), cis-9 18:1 and 18:2 (P<0.01) decreased linearly with increasing proportion of fish oil in the mixture and cis-11 18:1 increased linearly (P<0.001). The quadratic responses of trans-11 18:1 (P<0.01) and cis-9, trans-11 18:2 (P<0.05; Fig. 2a and b) are consistent with our hypothesis, also stated by Whitlock et al. (2003), that a combination of linoleic acid as substrate, and fish oil fatty acids as BH modifiers, would maximize formation of vaccenic acid in the rumen and rumenic acid in milk fat. Other identified trans-18:1 isomers declined as the proportion of fish oil increased. Decreasing proportions of total C18 fatty acids reflect their relatively low amounts in fish oil, and the very low amounts of 18:0 reflect strong inhibition of the final BH step in the rumen, also leading to less substrate in the mammary glands for -9 desaturase activity to synthesize cis-9 18:1. Whereas the increasing proportion of cis-11 18:1, synthesized by chain elongation of 16:1, closely follows the increasing amounts of 16:1 in the milk fat, cis-11 18:1 also occurred in measurable amounts in fish oil. Because both fatty acids have diet and tissue synthesis sources, no discussion of relative sources is possible from these data. High concentrations of linoleic acid, or other unsaturated fatty acids, inhibit further BH of trans-11 18:1 to stearic acid (Kepler et al., 1970). Evidence suggests that fish oil fatty acids may be most inhibitory in this respect, based on higher ruminal accumulations of trans-18:1 intermediates when fish oil, versus plant oil, is fed (Chilliard and Ferlay, 2004). Whereas there were large increases in trans-11 18:1 with fish oil supplementation, no increases in other trans monoenes occurred. As a result, the proportion of trans-11 18:1 of total trans-18:1 fatty acids reported (i.e., trans-6 to trans-12 18:1) increased from 0.51 in

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Table 3 Fatty acid concentrations (mg/g fatty acids) in milk fat with increasing fish oil in the diet Fatty acid

4:0 6:0 8:0 10:0 12:0 14:0 14:1 15:0 16:0 16:1 18:0 18:1 t6–8 18:1 t9 18:1 t10 18:1 t11 18:1 t12 18:1 c9 18:1 c11 18:2 18:3 CLA c9, t11 CLA t10, c12 20:4n − 6 20:5n − 3 22:5n − 3 22:6n − 3

Proportion of fish oil in oil supplement 0

0.33

0.67

1.00

33.4 13.7 6.4 13.3 17.8 77.8 8.8 10.8 224 19.8 108 8.4 8.1 43.1 72.4 10.2 224 8.1 34.8 8.4 40.2 1.1 0.2 1.2 0.6 ND

35.8 14.6 6.9 14.3 20.1 87.1 11.2 11.9 261 28.0 61.8 8.6 9.4 35.9 114 12.8 148 10.3 27.2 9.3 60.9 1.0 NDb 1.4 0.7 ND

49.6 21.5 10.8 21.6 25.0 100 10.0 13.2 283 36.1 36.5 4.3 6.9 27.6 136 10.9 85.8 12.6 20.4 8.3 58.4 1.0 1.3 5.8 1.7 0.6

52.7 25.0 13.6 27.7 31.4 110 9.4 15.6 309 44.5 36.2 2.3 4.3 31.3 79.8 9.5 76.8 20.6 22.9 8.8 34.3 1.2 1.5 6.8 3.0 0.9

S.E.

P

3.67 1.96 1.26 2.60 2.67 4.55 1.65 1.00 15.0 2.62 11.8 1.47 1.12 15.4 21.3 0.94 32.6 2.46 2.38 1.13 8.02 0.23 0.21 1.50 0.58 0.29

<0.001L <0.001L <0.001L <0.001L <0.01L <0.001L NSa <0.001L <0.01L <0.001L <0.001L <0.01L <0.02L NS <0.01Q NS <0.01L <0.001L <0.01L NS <0.01Q NS <0.001L <0.01L <0.01L <0.05L

L = linear effect and Q = quadratic effect. a Not significant. b Not detected.

cows fed sunflower oil only to 0.68, 0.78 and 0.72 when 0.33, 0.66 and 1.0 fish oil was fed, respectively, in the oil mixture. In addition, the proportion of trans-11 18:1 of total trans18:1 fatty acids appears to correspond closely with the degree of inhibition of trans-18:1 reduction estimated from the ratio of trans-18:1/(18:0 + cis-18:1 + trans-18:1) in milk fat (i.e., 0.33, 0.50, 0.64 and 0.58), respectively. The close agreement of the proportion of trans11 18:1 of total trans-18:1, and the estimated degree of trans-18:1 inhibition, suggest that as inhibition of trans-18:1 reduction approaches 1.0 (i.e., milk fat 18:0 + cis-18:1 approaches 0), the proportion of trans-11 18:1 of total trans-18:1 also approaches 1.0. The relationship could also suggest that trans-11 18:1 is a predominant trans-18:1 intermediate of rumen biohydrogenation and the majority of other trans-18:1 isomers in milk fat are likely to be produced as side reactions during trans-11 18:1 reduction. The possibility of trans-double bond migration as the likely mechanism for this conversion has been proposed (Griinari and Bauman, 1999). Because linoleic acid is the primary substrate for the cis-12, trans-11 isomerase (Kepler et al., 1970), and fish oil is a potent inhibitor of the conversion of trans-11 18:1 to stearic acid but is low in content of linoleic acid, we postulated that a combination

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Fig. 2. (a) Response of vaccenic acid (mg/g of milk fatty acids) in milk fat to increasing proportion of fish oil in the supplement (n = 16). (b) Response of CLA (mg/g of milk fatty acids) in milk fat to increasing proportions of fish oil in the supplement (n = 16).

of the oils would complementarily maximize vaccenic acid production, the primary source of CLA in milk fat (Palmquist et al., 2005). Data in Fig. 2 strongly support this conclusion. Arachidonic acid (AA, 20:4n − 6, P<0.001), eicosapentaenoic acid (EPA, 20:5n − 3) and docosapentaenoic acid (DPA, 22:5n − 3, P<0.01) increased linearly in milk fat with increasing proportion of fish oil in the oil mixture, whereas the increase was less robust for docosahexaenoic acid (DHA, 22:6n − 3; P<0.05). Transfer of dietary fish oil fatty acids from diet to milk fat, specifically EPA and DHA, is generally low, and reports of efficiency, usually estimated from single concentrations of the individual fatty acids in diet and milk, tend to be variable. Offer et al. (1999) reported transfers (Table 4) of less than 0.03 for Table 4 Comparison of transfers of EPA and DHA from diet to milk reported in some published studies and in this study Intake (g/day)

Cant et al. (1997) Wright et al. (1999) Offer et al. (1999) Shingfield et al. (2005) Present study a

Transfer coefficient

EPA

DHA

EPA

DHA

70 1.6, 5.5, 10.9 5–40 33 0–60

85 2.4, 8.2, 16.0 22 24 0–50

0.09 0.27, 0.13, 0.07 <0.03 0.007 0.057a

0.16 0.34, 0.20, 0.11 <0.03 0.007 0.0098

Calculated by regression analysis.

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Table 5 Regression coefficients for yields of individual fish oil fatty acids in milk vs. their intake Fatty acid

Slope

S.E.

Pa

20:1 20:4n − 6 20:5n − 3 22:5n − 3 22:6n − 3

0.041 0.79 0.057 0.11 0.0098

0.020 0.21 0.023 0.025 0.0053

<0.07 <0.01 <0.04 <0.01 <0.10

a

Probability that the slope is equal to zero.

polyunsaturated fatty acids of chain length ≥C20 , although it was 0.3 for DPA, suggested to be due to chain lengthening of 20:5n − 3. Conversely, Cant et al. (1997) reported 0.09 and 0.16 efficiencies for EPA and DHA, respectively. Shingfield et al. (2005) reported transfers of 0.008 and 0.009 for EPA and DHA when maize silage was fed, and values were even lower when grass silage was fed. A more precise approach is to determine the slope of the regression of the amount of fish oil fatty acids in milk versus intake. Wright et al. (1999) reported that 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/day, whereas recalculation of the data by regressing yield on intake suggests transfer efficiencies of 0.035 and 0.078, respectively, for EPA and DHA. Transfer increased linearly for most of the fish oil fatty acids measured (Table 5), and it differed considerably among fatty acids. The slopes of the regressions ranged from <0.01 for DHA (P<0.10) to 0.79 for AA (P<0.01). Whereas only 10 mg of DHA/g intake appeared in milk, transfer of EPA was 57 mg/g intake (P<0.04) and for DPA it was 110 mg/g (P<0.01). Although limited by the precision of measuring fish oil fatty acids in both the diet and milk, these transfers have a higher confidence than the single point estimates reported previously, and resulted in relatively high concentrations of n − 3 fatty acids in milk fat at the highest fish oil intake (Table 3). In our study, intakes of EPA and DHA ranged from 0 to 60 g/day, whereas intake of AA did not exceed 1 g/day, making the high transfer coefficient for AA suspect. Both lipolysis and BH rate decrease with increasing dietary concentrations of fish oil (Dohme et al., 2003), which contribute to their increased availability for incorporation into milk fat. Although it can be postulated that increased linoleic acid intake would increase de novo synthesis of AA, this does not occur (Palmquist and Schanbacher, 1991) because increased 18:2 and 18:3 intakes inhibit the -5 and -6 desaturases. In addition to the complementary effects of the two oils on CLA production, fish oil fatty acids were much less inhibitory to de novo synthesis of milk fatty acids when used in combination with sunflower oil (Table 3). The reason for this lower inhibitory effect of fish oil is not certain, but may relate to an effect on synthesis of trans-18:1 monoenes, either measured or not measured. The effect of fish oil on trans-18:1 is curvilinear, whereas the effect on de novo synthesis of fatty acids is linear. Fish oil replaced unsaturated C18 fatty acids of sunflower oil linearly and reduced the C18 fatty acid inhibition on de novo synthesis. The trans-10, cis-12 18:2 isomer, which inhibited milk fat synthesis when infused (Baumgard et al., 2000), remained unchanged at 1.0–1.2 mg/g of the milk fatty acids. The proportion of trans-10 18:1 was relatively high (28–43 mg/g milk fatty acids), with modest

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inverse relationship to milk fat proportion. A role for other biohydrogenation intermediates in regulation of milk fat synthesis cannot be excluded. It is somewhat surprising that supplementing a diet with sunflower oil at 30 g/kg diet DM produced the most severe depression in milk fat. When a diet based on grass silage was supplemented with increasing levels of sunflower oil up to 50 g/kg diet DM there was no appreciable effect on milk fat proportion (Shingfield and Griinari, unpublished). As well, concentrations of vaccenic and rumenic acids in milk fat when sunflower oil was fed without fish oil were higher than reported previously for unsaturated vegetable oil supplements (Kelly et al., 1998; Dhiman et al., 2000). This is most likely due to carryover effects in our Latin square design, as the 0 level of fish oil always followed the 1.0 level, except for the first experimental period. However, the main objective of the study was attained in that combinations of a high linoleic acid oil as substrate, and fish oil as BH modifier, resulted in higher concentrations of vaccenic and rumenic acids in milk fat than did either fed alone. At 2 h post-feeding, proportions of butyric acid in ruminal VFA were unusually high (Table 6) with 0.33 fish oil in the oil supplement, and differed (P<0.04) from 0 and 0.67 fish oil. All fish oil supplemented diets tended to have high butyrate proportions in ruminal VFA, compared to values of 100–120 mmol/mol reported for typical lactation rations (Ohajuruka et al., 1991), whereas effects were less definitive 6 h post-feeding. Higher rumi-

Table 6 Concentrations and proportions of ruminal VFA at 2 and 6 h post-feeding with increasing proportions of fish oil in the oil supplement S.E.

Pa

6.50 108

0.08 19.6

NS <0.02Q

Proportion of fish oil in oil supplement 0 2 h post-feeding pH Total VFA (mmoles/l) Proportions (mmol/mol) Acetic Propionic Butyric iso-Butyric Valeric iso-Valeric 6 h post-feeding pH Total VFA (mmoles/l) Proportions (mmol/mol) Acetic Propionic Butyric iso-Butyric Valeric iso-Valeric a

6.44 133

0.33 6.50 74.7

0.67 6.44 73.8

1.00

652 192 115 8.6 12.8 19.8

441 173 304 13.6 15.8 28.6

622 174 142 14.2 17.9 30.2

534 227 188 12.0 14.6 25.0

43 19 43 1.6 2.5 4.5

<0.02 <0.08Q <0.04 <0.04Q NS <0.11Q

6.16 121

6.04 106

6.20 123

6.25 128

0.15 10.8

NS NS

579 209 178 7.5 12.9 14.2

530 210 217 11.7 12.4 18.7

637 210 114 9.3 10.9 18.6

632 212 113 10.3 14.7 17.4

47 17 54 1.3 1.6 3.3

NS NS NS <0.08 <0.06Q NS

L = linear effect, Q = quadratic effect, and NS = P>0.10.

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nal butyrate proportions may reflect a selective increase in bacterial species tolerant to high concentrations of unsaturated fatty acids that are associated also with butyrate production (Wallace et al., 2005). In addition, decreased concentrations of acetate suggest that fish oil supplementation favored, although transiently, Butyrivibrio strains producing butyrate by the acetyl-CoA/butyryl-CoA transferase pathway. The biohydrogenation process requires conjugation of dienes only when they occur in an all-cis -2,5 or -9,12 pentadiene system (Palmquist et al., 2005), such as in linoleic and linolenic acids. Although linoleic acid is toxic, even to those organisms shown to be capable of BH (Kim et al., 2000), certain strains of Butyrivibrio spp. have increased tolerance to high concentrations of fatty acids; these use an alternate pathway for butyrate formation (Wallace et al., 2005), although it is not clear if butyrate synthesis is higher by the alternate pathway. Higher butyrate concentrations occurred immediately after feeding when the oil supplement contained 0.33 fish oil, which may be consistent with increased activity of Butyrivibrio spp. that tolerate high concentrations of unsaturated fatty acids. Whether these, or other mechanisms were responsible for increased butyrate concentrations remains to be determined.

5. Conclusions Feeding 30 g/kg DM oil in a lactation ration as a combination of sunflower oil and fish oils increased vaccenic and rumenic acids in milk fat to concentrations higher than either oil fed alone. Sunflower oil alone caused the greatest depression in milk fat proportion and yield. Increasing proportions of fish oil in the oil mixture supplement alleviated the depression, when expressed either as proportions and yields of de novo fatty acids or as milk fat proportion and yield. Transfer of dietary fish oil fatty acids to milk fat increased linearly as fish oil intake increased. Ruminal butyrate concentrations were higher than usually observed with lactation rations, and this may have been a consequence of increased populations of Butyrivibrio spp. that tolerate high concentrations of unsaturated fatty acids.

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