Effects of dietary n-6/n-3 fatty acid ratio on nutrient digestibility and blood metabolites of Hanwoo heifers

In vivo trial

In vitro trial

Three diets with different ratios of n-6/n-3 FA (2.07, 5.18 and 7.37) as used in the in vivo trial were incubated with rumen fluid and Van Soest's medium (Van Soest et al. 1966) to study the rumen fermentation indices and FA profile of ruminal digesta in vitro. Rumen fluid was collected from two cannulated Hanwoo heifers fed rice straw and grain mixed at an 8:2 ratio. The heifers were fed at 08.00 and 17.00 hours daily. Rumen fluid was collected at 10.00, filtered through surgical gauze and mixed with Van Soest's medium in a 1:2 ratio. This mixture was flushed with carbon dioxide gas to maintain anaerobic condition as described by Adesogan et al. (2005). Substrates (0.5 g) and 40 mL of the inoculum were dispensed into culture bottles which were then sealed and incubated at 39°C for 48 h. Five replicates of each sample were evaluated as well as three blanks. Gas production was measured every 30 min for 48 h. The pH, ammonia-N and volatile fatty acids (VFA) were also analyzed. After incubation, the samples were filtered through Whatman filter paper (No 541) and residues were freeze-dried (LABCONCO, FreeZone 12 plus 7960047, Kansas City, MO, USA) and analyzed for DM concentration and FA profile. The pH of the filtrate was measured and the residue was centrifuged at 5645 × g for 15 min. Subsequently, ammonia-N and VFA were analyzed and DM digestibility (DMD) was calculated.

Chemical analysis

Feeds, orts and feces samples were dried at 60°C for 48 h, ground to pass the 1-mm screen of a grinder (Cutting Mill, SHINMYUNG ELECTRIC Co., Ltd, Gimpo, South Korea) and analyzed for DM, crude protein (CP), ether extract (EE), neutral detergent fiber (NDF) and acid detergent fiber (ADF). The CP was calculated as N × 6.25, after N was quantified on a N analyzer (B-324, 412, 435, 719 S Titrino, BUCHI, Flawil Switzerland). The EE was determined according to Association of Official Analytical Chemists (1995). The NDF and ADF were analyzed as described by Van Soest et al. (1991) using an Ankom Fiber Analyzer (A220, Ankom Technology, Macedon, NY, USA). Alpha-amylase and sodium sulfite were used for NDF determination and the results were expressed inclusive of ash.

The pH was measured with a pH meter (SevenEasy, Mettler Toledo, Greifensee, Switzerland) and ammonia-N was analyzed by colorimetry (Chaney & Marbach 1962). The concentrations of VFA were measured using high-performance liquid chromatography (HPLC) (L-2130; HITACHI, Tokyo, Japan) with an auto-sampler (L-2200; HITACHI, Tokyo, Japan), UV detector (L-2400; HITACHI, Tokyo, Japan) and a column (MetaCarb 87H, Varian, Middelburg, Netherlands) by the method of Muck and Dickerson (1988).

For FA analysis, diet (undried 20 g), plasma (2 mL) and in vitro rumen content (2 mL) were freeze dried (LABCONCO, FreeZone 12plus) and methylated using the direct methylation method described by Jenkins et al. (2001). The extracted FA methyl ester was analyzed with a gas chromatograph (Varian 450-GC, Varian) equipped with an auto-sampler (CP-8400; Varian), a flame ionization detector and a Varian capillary column (CP-Sil 88 for Fatty Acid Methyl Esters, 100 m × 0.25 mm × 0.2 μm). The carrier gas was nitrogen. The injector and detector were maintained at 230°C. The oven temperature was initially set at 120°C for 1 min, increased by 5°C/min up to 190°C, held at 190°C for 30 min, increased again by 2°C/min up to 220°C, and held at 220°C for 40 min. The peaks of samples were identified and concentrations calculated based on the retention time and peak area of known standards.

Plasma concentration of blood urea nitrogen (UREA/BUN kit; Roche, Mannheim, Germany) was determined. An electrochemiluminescence immunoassay (ECLIA) was used to determine the concentrations of plasma progesterone and insulin (Progesterone II and Insulin kit, respectively; Roche, Germany). Concentration of IGF-1 was determined using the immulite 2000 kit (Siemens, Malvern, PA, USA) following the chemiluminescence immumoassay (CLIA) technique. An enzymatic kinetic assay was used to determine plasma concentrations of glucose (GLU kit; Roche, Germany).

Statistical analysis

Data were analyzed using analysis of variance (ANOVA) with the GLM procedure of SAS (Statistical Analysis System Institute 2002). The IML procedure was used to generate coefficients for testing linear and quadratic effects of treatments with unequal spacing. Significance was declared at P < 0.05 and tendencies at P < 0.10.

Intake, digestibility and in vitro rumen fermentation

Dietary treatment had no effect (P > 0.10) on DM intake or in vivo apparent digestibility of nutrients (DM, CP, EE, NDF and ADF) (Table 3). However, as the dietary n-6/n-3 FA ratio increased, linear decreases were evident in in vitro digestibility of DM (P = 0.013), ruminal pH (P = 0.028) and concentrations of total VFA (P = 0.001) and propionate (P = 0.008). Quadratic changes were evident in concentrations of acetate (P = 0.004), iso-butyrate (P = 0.003), butyrate (P < 0.001), iso-valerate (P = 0.008) and acetate-to-propionate ratio (P = 0.027). Valerate and ammonia-N concentrations were unaffected by dietary treatment (P > 0.05).

Fatty acid profile

Increasing the dietary n-6/n-3 FA ratio linearly decreased plasma concentrations of C18:3n-3 (P = 0.008) and total n-3 FA (P = 0.029), but linearly increased the n-6/n-3 FA ratio (P = 0.007) (Table 4). However, dietary treatment did not affect concentrations of C18:2n-6, other individual FAs, total FA, saturated fatty acid (SFA), monounsaturated fatty acid (MUFA) and PUFA or PUFA-to-SFA ratio in plasma. Concentrations of C18:2n-6, total n-6 FA and n-6/n-3 FA ratio in rumen contents after 48 h of incubation increased linearly with increasing dietary n-6/n-3 FA ratio (Table 5). In contrast, C18:3n-3 and total n-3 FA decreased linearly and also quadratically as the dietary n-6/n-3 FA ratio increased. A quadratic change was also observed in residue C20:4n-6 and C14:0 concentrations.

Blood metabolites

Plasma concentrations of insulin (P = 0.022) and progesterone (P = 0.007) increased linearly with increasing ratio of dietary n-6/n-3 FA but concentrations of glucose, BUN and IGF-1 in plasma were unaffected (P > 0.05) (Table 6).

Fatty acid ratio (n-6/n-3)

The importance of dietary n-6/n-3 FA ratio in relation to human health was discussed earlier (Cook 1996; Simopoulos 2008). The typical n-6/n-3 FA ratios in modern Western diets are higher than those of humans and wild animals many centuries ago (Simopoulos 1991). Raes et al. (2004) noted that several animal experiments are being conducted to achieve n-6/n-3 FA ratio in animal products closer to <5:1 due to the potential health benefits. However, in ruminants, extensive biohydrogenation of unsaturated FA in the rumen often prevents animal products from reflecting the dietary FA. In the present study, the primary aim was to study how the variation in n-6/n-3 ratio affects rumen fermentation, digestibility and metabolic profile. The three different ratios (2.07, 5.18 and 7.37) tested in this study were achieved by strategic mixing of different oil sources. Earlier, Kim et al. (2007) used n-6/n-3 FA ratios of 2.3:1, 8.8:1 and 15.6:1, and Jalč et al. (2009) used ratios of 1:1, 3:1 and 5:1.

Intake, digestibility and in vitro fermentation indices

Feed intake and in vivo apparent total tract digestibility of nutrients were not affected by dietary n-6/n-3 FA ratio in this study. Similarly, Kim et al. (2007) reported that feeding diets with n-6/n-3 FA ratios of 2.3:1, 8.8:1, 12.8:1 and 15.6:1 had no effects on intake and digestibility of DM, EE and ADF by growing lambs. Jalč et al. (2009) also observed that in vitro degradation of DM, NDF and ADF were not affected by substrate n-6/n-3 FA ratios of 1:1, 3:1 and 5:1. In contrast, in the in vitro trial in this study, increasing the dietary n-6/n-3 FA ratio reduced in vitro dry matter digestibility (IVDMD) linearly. A similar numerical (P > 0.10) trend was also evident for in vivo DM and CP digestibility. The reasons for the decreases in digestibility are unclear since both n-6 and n-3 FAs exert antimicrobial effects on the rumen (Palmquist & Jenkins 1980; Jenkins 1993), which can result in reduced ruminal digestibility (Palmquist & Jenkins 1980). Nevertheless, the linear reduction in total VFA production in vitro was probably mediated by the same antimicrobial effects of the FA that reduced IVDMD. The specific and relative roles of either n-6 or n-3 FA on the reduction in digestibility and total VFA is unclear. Most of the PUFAs can exert toxic effects on rumen microbes, but C18:3n-3 FA is more lethal compared to C18:2n-6 (Zhang et al. 2008). Negative effects on nutrient digestibility by C18:3n-3-rich linseed or linseed oil have been reported (Martin et al. 2008), but such results contradict those in this study where the proportion of dietary C18:3n-3 FA decreased with increasing ratio of n-6/n-3 FA ratio in the diet (Table 2). As in this study, Whitney et al. (2000) reported a linear decrease in IVDMD with increasing level of dietary soybean oil, a rich source of C18:2n-6 FA in the diet of beef heifers. Hess et al. (2001) also observed decreased total tract organic matter and NDF degradability by increasing levels of soybean oil. It is likely that negative effects on digestibility are related to the ratio of n-6/n-3 FA rather than to the concentration of a particular n-6 or n-3 FA. However, more studies are needed to confirm this hypothesis.

Fatty acid profile

Transfer of dietary unsaturated FA to tissues in ruminants is challenging because of the extensive bio-hydrogenation of these FAs by the microbial community in rumen (Juárez et al. 2010). However, unsaturated FAs from the diet can escape, at least partially, from bio-hydrogenation in rumen and therefore, fatty acid composition of tissue lipids of ruminants can be influenced by the diet (Yeom et al. 2005). Many earlier reports suggested that feeding diets enriched with n-6 and/or n-3 FA increased the concentrations of these FAs in plasma, milk, muscles or other tissues of ruminants (Yeom et al. 2003; Herdmann et al. 2010; Zachut et al. 2010; Zhang et al. 2010; Juárez et al. 2011). In agreement, we also observed a corresponding linear increase in n-6/n-3 FA ratio in plasma as the ratio of n-6/n-3 FA in the diet increased in this study. However, only concentrations of C18:3n-3 and total n-3 FA in plasma were affected by the diet.

Blood metabolites

That plasma glucose concentration was unaffected by dietary FA treatments agrees with several previous studies (Oldick et al. 1997; Petit & Twangiramungu 2006; Andersen et al. 2008; Ghasemzadeh-Nava et al. 2011). Yet Mattos et al. (2004) reported decreased plasma glucose concentration in animals fed diets supplemented with fish oil (n-3 FA). This was mainly because the fish oil reduced DM intake through inhibitory effects of eicosapentaenoic acid (C20:5n-3) on phosphoenolpyruvate carboxykinase (Murata et al. 2001). In the present study, DM intake and C20:5n-3 concentrations were unaffected by diet.

As reported by Andersen et al. (2008) in pre- or postpartum cows fed a control diet instead of others high in unsaturated or saturated fat, BUN concentration, an indicator of protein status of animals, was not affected by dietary n-6/n-3 FA ratio. This may be because DM intake and in vivo DM and CP digestibility were unaffected by diet in that study. The concentration of IGF-1 was decreased by feeding a diet enriched with n-3 FA in the study of Bilby et al. (2006a) but increased by feeding one enriched with n-6 FA in a different study (Robinson et al. 2002). However, in the present study, the plasma IGF-1 concentration was unaffected by dietary n-6/n-3 FA ratio.

Plasma insulin concentration increased linearly with decreasing proportion of n-3 FA in this study. Usually, changes in plasma insulin concentration are associated with changes in glucose concentration in plasma (do Amaral 2008) but in this study, as in others (Selberg et al. 2004) glucose concentration was unaffected by the dietary FA composition but insulin concentration was. The response in this study may be due to the timing of the plasma sampling relative to exertion of the modulatory effects of plasma insulin on glucose or to the tight regulation of plasma glucose concentration (Kaneko 1989). The increase in plasma insulin concentration in this study may also be partly attributable to the decrease in the dietary n-3 FA concentration. Delarue et al. (2004) reported that n-3 FA can reduce plasma insulin concentrations in rodents by sustaining glucose transporter protein GLUT4 receptor in the muscle, by preventing decreased expression of GLUT4 in adipose tissue, and by inhibiting both activity and expression of liver glucose-6-phosphatase, which increases glucose uptake and metabolism. In agreement, Andersen et al. (2008) observed decreased insulin concentration in the plasma of prepartum dry cows supplemented with linseed oil, a rich source of n-3 FA. In addition, Mashek et al. (2005) reported that plasma insulin concentration decreased with intravenous infusion of linseed oil. Also, Xiao et al. (2006) stated that different FA profiles can affect glucose-induced insulin secretion by n-3 FA (linseed). However, Robinson et al. (2002) reported that plasma concentration of insulin was not affected by supplementation with n-3 or n-6 FA.

The linear increase in plasma progesterone concentration with increasing ratio of n-6/n-3 FA in this study is related more to the concentration of dietary n-3 FA than that of n-6 FA. In fact, it is evident that plasma progesterone concentration decreased linearly as dietary n-3 FA increased. Plasma progesterone concentrations were also reduced by feeding a diet enriched with n-3 FA instead of a control diet or one enriched with n-6 FA to cows in the mid luteal phase (Robinson et al. 2002). However, in the early luteal phase, no difference in progesterone concentrations was detected among cows fed diets enriched with n-3 and n-6 FA, although both groups had lower progesterone concentrations compared to those fed the control diet. Hinckley et al. (1996) also reported inhibitory effects of n-3 FA on bovine luteal cell progesterone secretion in vitro. The n-3 or n-6 FA either directly or indirectly (via prostaglandins) exert differential effects on ovarian steroid synthesis (Wathes et al. 2007). Although some researchers observed no change in luteal progesterone secretion in cows fed diets enriched with n-3 FA (Mattos et al. 2002; Ambrose et al. 2006; Bilby et al. 2006a), several mechanisms exist through which dietary PUFAs, especially n-3 FA, can decrease Prostaglandin F2alpha (PGF2α) secretion with a concomitant increase in luteal secretion of progesterone (Mattos et al. 2000). Therefore, the effects of n-6 and/or n-3 FA on plasma progesterone concentration are not consistent and may be related to other dietary or physiological factors. The ratio of n-6 and n-3 FA may have a unique role on ovarian steroid synthesis, which is different from the individual activities of these FA.