BAI Sarvvl, CAO Zhi-jun, , JIN Xin, WANG Ya-jing, , YANG Hong-jian, , LI Sheng-li,

1 State Key Laboratory of Animal Nutrition, College of Animal Science and Technology, China Agricultural University, Beijing 100193, P.R.China

2 Beijing Engineering Technology Research Center of Raw Milk Quality and Safety Control, College of Animal Science and Technology, China Agricultural University, Beijing 100193, P.R.China

Abstract Five lactating Holstein cows in a 5×5 Latin square experiment were fed five high-concentrate total mixed rations (TMRs)to investigate the effects of step-wise improvement of forage combination on ruminal and milk fatty acid profiles. The ratio of concentrate to forage was fixed as 61:39, and the step-wise improvement of forage combination was applied as: TMR1,a ration containing corn stover; TMR2, a ration containing corn stover and ensiled corn stover; TMR3, a ration containing ensiled corn stover and Chinese wild ryegrass hay (Leymus chinensis); TMR4, a ration containing the ryegrass hay and whole corn silage; TMR5, a ration containing the ryegrass hay, whole corn silage and alfalfa hay. The TMRs were offered to the cows twice daily at 0700 and 1900 h. The entire experiment was completed in five periods, and each period lasted for 18 days. Diurnal samples of rumen fluids were collected at 0100, 0700, 1300 and 1900 h (day 16); 0300, 0900, 1500 and 2100 h (day 17); and 0500, 1100, 1700 and 2300 h (day 18). The step-wise improvement of forage combination increased energy and crude protein contents and decreased fibre content. As a result, the step-wise improvement of forage combination increased dry matter intake and milk yield (P＜0.05). The step-wise improvement increased dietary content of linolenic acid (C18:3n-3), but did not alter dietary proportions of palmitic acid (C16:0), stearic acid (C18:0), oleic acid (C18:1cis-9),linoleic acid (C18:2n-6) and arachidic acid (C20:0). In response to the forage combination, ruminal concentration of C16:0,C18:2n-6 and C18:3n-3 linearly increased against their dietary intakes (P＜0.10). The step-wise improvement increased milk contents of C10:0, C12:0, C14:0, C16:0, C18:2n-6 and C18:3n-3 (P＜0.10) and decreased milk contents of C18:0 and C18:1cis-9 (P＜0.05). Milk yields of C16:0, C18:1cis-9, C18:2n-6 and C18:3n-3 were linearly increased by the increase of these fatty acids in the rumen (R2≥0.79, P＜0.05), and milk yields of C18:2n-6 and C18:3n-3 were also positively correlated with dietary intake of these fatty acids (R2≥0.85, P＜0.05). The step-wise improvement increased the transfer efficiencies from feed to milk for C18:2n-6 from 11.8 to 14.2% and for C18:3n-3 from 19.1 to 22.3%. In a brief, along with the step-wise improvement of forage combination, more dietary linoleic and linolenic acids might escape microbial hydrogenation in the rumen and consequently accumulated in milk fat though these fatty acids were present in low concentrations in ruminal fluids. The step-wise improvement of forage combinations could be recommended as a dietary strategy to increase the transfer efficiency of linoleic and linolenic acids from feed to milk.

Keywords: forage combination, lactating cow, ruminal fatty acid, milk fatty acid

1. lntroduction

In recent decades, milk has been increasingly recommended as a component of a healthy diet, not only in childhood but also adulthood in China. Linoleic (C18:2n-6) and linolenic (C18:3n-3) fatty acids are generally considered the most valuable nutrients in milk fat. The deficiency of C18:2n-6 is believed to be associated with various adverse consequences, including poor growth, reproductive failure and fatty liver disease, etc., and C18:3n-3 deficiency may cause impaired vision, polydipsia (Connoret al. 1992) and fatal ischemic heart disease (Huet al. 1999). Mammary uptake of nutrients, biosynthetic pathways and the impact of diet on milk composition have been discussed in detail in previous studies (Chilliardet al. 2007; Elgersma 2015).

Various strategies have been proposed to increase the unsaturated fatty acid (mainly C18:2n-6 and C18:3n-3)content of raw milk in dairy cows. These strategies include the improving ration formulation (e.g., adding plant oil and marine oil) and grazing strategies (Abughazalehet al. 2002;Looret al. 2005; Royet al. 2006; Sun and Gibbs 2012).Chilliardet al. (2001) in a review summarized that dietary fat supplementation would be an effective strategy to improve milk yield and milk fatty acid profiles in lactating cows.However, less knowledge is available to address the role of forage lipids in regulating C18 fatty acid content in the rumen and milk. In feeding practice of ruminant animals, forage should be considered not only a low-cost ration ingredient but also a source of dietary unsaturated fatty acids. For instance, corn silage is rich in C18:2n-6 (Benchaaret al.2007), and alfalfa hay is a good forage source of C18:3n-3 for ruminants (Boufaiedet al. 2003). Kala? and Samková(2010) in a review noted that the milk ratio of unsaturated to saturated fatty acids was high in dairy cows grazed in grasslands or fed legume forage than that in cows fed silage or hay-dominated diets.

Short and medium fatty acids (C4–C14) in milk and half of C16 fatty acids that werede novosynthesized in mammary glands from acetate and β-hydroxybutrate originated from dietary carbohydrate fermentation products in the rumen,and they were absorbed in blood through the ruminal wall(Wikinget al. 2010). The other half of C16 fatty acids and most C18 fatty acids in milk were originated from the diet and adipose tissue and uptaked from blood (M?nsson 2008;Wikinget al. 2010). As reported in previous studies, not only fatty acid metabolism (e.g., lipolysis and biohydrogenation)in the rumen but also dietary forage species and composition influenced the fatty acid profile of milk (Van Dorlandet al.2008; Jenkinset al. 2008; Wikinget al. 2010). As dairy production has developed rapidly, forage costs have increasingly become a major factor influencing raw milk profitability. In order to achieve a comparatively high milk yield, most small- and medium-sized dairy farms often adopt a high concentrate to forage ratio in crop residue-based rations. In many large-scale intensive dairy farms, alfalfa hay, which is increasingly imported from the USA in recent years, has been used as important forage source (Yanget al. 2013). In the present study, five high-concentrate total mixed rations (TMRs) were formulated with a step-wise improvement of forage combinations, and the objective was to determine the effects of the step-wise improvement on fatty acid profiles in the rumen and milk of dairy cows and then attempted to explore associations between dietary,ruminal and milk fatty acid profiles.

2. Materials and methods

This experiment was conducted at the State Key Laboratory of Animal Nutrition of China Agricultural University (Beijing,China). The use of dairy cows were approved by the Animal Care and Use Committee of China Agricultural University,and the experimental procedures applied in the present study were in accordance with the university’s guildlines for animal research.

2.1. Animals

Five Holstein cows in early lactation (days in milk: (60±13)days, mean±standard deviation), with an average body weight (543±25) kg, average daily milk yield (20±2) kg and fitted with a permanent ruminal cannula (Type 2C; Bar Diamond Inc., Parma, ID) were used as the experimental animals. The cows were housed in 10-m2individual tie stalls, with rubber mats and individual feeding facilities.The animals had free access to water and were fed TMRs twice daily at 0700 and 1900 h, as described in Section 2.2.

2.2. Experimental design

A 5×5 Latin square experiment was designed to study the effects of five TMRs using step-wise improvement in forage combinations on ruminal and milk fatty acid profiles(Table 1). The five TMRs were formulated in fixed ratios of concentrate to forage of 61:39. The combination treatments on dry matter basis were as follows: 390 g kg–1of corn stover dry matter (TMR1), which represented a typical ration commonly used on small-scale dairy farms (dairy herds less than 10 heads) in the Shuangcheng region of Heilongjiang Province of China; 195 g kg–1of corn stover and 195 g kg–1of ensiled corn stover (TMR2), which represented a typical ration commonly used on small- to medium-scale dairy farms(dairy herds less than 100 heads) located in Hohhot region of Inner Mongolia Autonumous Region of China; 195 g kg–1of ensiled corn stover and 195 g kg–1of Chinese wild ryegrass(TMR3), which represented a typical ration commonly used on medium-scale dairy farms (dairy herds less than 500 heads) in the Wulanchabu Plateau of Inner Mongolia of China; 195 g kg–1of Chinese wild ryegrass DM and 195 g kg–1of whole corn silage (TMR4), which represented a typical ration commonly used on medium- to large-scale dairy farms (dairy herds less than 1 000); and 134 g kg–1of Chinese wild ryegrass plus 128 g kg–1of whole corn silage plus 128 g kg–1of alfalfa hay (TMR5), which represented a typical ration commonly used on large-scale intensive dairy farms (dairy herds greater than 1 000) located in suburban regions close to Beijing.

The chemical composition and fatty acid profile of each TMR are shown in Table 2. The entire experiment was completed in five periods, with each period lasting 18 days(15 days of adaptation and 3 days of sample collection)according to previous study (Wikinget al. 2010). In each period, the TMRs contained 5–10% orts in the first week,and feeding amount was then restricted in the subsequent days. The ingredient mix was adjusted based on a weekly analysis of forage DM. The feed intake and milk yield of each cow were recorded daily during the last 3 days of each experimental period.

2.3. Sampling procedure

Representative samples of corn stover, ensiled corn stover,Chinese wild ryegrass hay, whole corn silage, alfalfa hay and concentrate were collected in each period, dried at 65°C for 48 h and then ground using a standard Aoll mill (model AK-400B Aoll Co., Ltd., Wenling, China) and passed through a 1-mm screen. All the samples were stored at –20°C for later chemical analysis.

During each experimental period, after adaptation for 15 days to the new TMR, sampling was continuously conducted for 3 days. To minimize sampling stress to the cows, the ruminal contents were sampled at 0100, 0700,1300 and 1900 h (day 16); 0300, 0900, 1500 and 2100 h(day 17); and 0500, 1100, 1700 and 2300 h (day 18). The samples were collected from different compartments of the rumenviaa ruminal cannula, mixed thoroughly and then filtered through two layers of muslin. The samples were then centrifuged at 1 000×g for 30 min at 4°C. The supernatant samples of the ruminal liquid fraction without digesta particles were immediately stored at –20°C for later analysis of fatty acid composition.

The daily milk yield of each cow in each period was recorded. Milk samples were collected in vials that contained a preservative (dichromate potassium, K2Cr2O7)at 0500 and 1700 h each day on the last 3 days of each 18-day period. One aliquot of milk sample was taken for analysis of milk fat content and stored at 4°C. The other aliquots of milk samples were used for later analysis of fattyacid composition and stored at –20°C.

Table 1 Ration formulation on dry matter basis (g kg–1) of five experimental total mixed rations (TMRs) with step-wise improved forage combination1)

Table 2 Chemical composition and fatty acid profile of five experimental total mixed rations (TMRs) with step-wise improved forage combination1)

2.4. Chemical analysis

Following the methods of the AOAC (1999), DM (method 930.5), crude protein (CP, method 984.13), ether extract(method 920.39) and ash (method 942.05) contents of the feed samples were analysed. The contents of neutral detergent fibre (NDF) and acid detergent fibre (ADF) were analysed following the method of Van Soestet al. (1991).Fatty acid contents of feed samples were analysed as described by Buet al. (2007).

The pH in rumen fluids was measured immediately after collection at each sampling time of each cow using a hand-held pH electrode (Model pH B-4; Shanghai Chemical,Shanghai, China). After thawing, the filtered ruminal fluid samples (2 mL) were added to 200 μL of nonadecanoic acid(C19:0) as an internal standard (Sigma Co., USA) and then extracted twice with hexane:isopropanol (3:2, v/v). The extracted solution was transferred to a 30-mL tube, and the samples were esterified using the method of Krameret al. (1997).

Milk fat content was determined using mid-infrared analysis (Foss Milk?Scan, Foss Food Technology Corp.,Eden Prairie, MN, USA). The analysis of fatty acids in milk samples was performed as described by Buet al. (2007).Briefly, thawed milk samples obtained from individual cows in each period were centrifuged at 5 000×g for 20 min at 8°C to separate fat from milk, and 20 mg of the milk fat was transferred to a 30-mL high-temperature resistant tube,followed by the addition of 200 μL of C19:0 as an internal standard and was esterified using the method of Krameret al. (1997).

Fatty acid methyl esters were detected using a gas chromatograph (Model GC1200; Wufeng Co., Shanghai,China), which was equipped with a 100-m wall-coated open tubular CP-Select capillary column (internal diameter,0.25 mm; film thickness, 0.20 μm) and a flame ionization detector. Then, 1.0 μL of fatty acid methyl ester sample was injected at a 50:1 split ratio. The column oven temperature program was as follows: initial temperature of 90°C for 8 min, then increased from 90 to 220°C at 2°C min–1, and finally held at 220°C for 24 min. Pure N2at a constant inlet pressure of 0.2 MPa was used as the carrier gas. Both the injector and detector temperatures were maintained at 250°C. Peaks for individual fatty acids were identified by the retention time and comparison to known commercially prepared fatty acid methyl ester mix standards (Supelco,Bellefonte, PA, USA).

2.5. Calculations and statistical analysis

The yield of 3.5% fat corrected milk (FCM kg d–1) was determined according to Maynardet al. (1979):

3.5%FCM=0.35×Milk yield (kg d–1)+15.0×Fat yield (kg d–1)

The transfer efficiency of C18:2n-6 or C18:3n-3 from diet to milk was calculated as follows:

Transfer efficiency (%)=Milk yield of target unsatuated fatty acid (g d–1)/Dietary intake of target unsaturated fatty acid (g d–1)×100

Data in the experiment were analysed as a 5×5 Latin square design using PROC GLMMIX of SAS (2002)according to the following equation:

Where,Yijkwas the response variable,μwas the overall mean,Tiwas the fixed effect of the treatment (i=TMR1 to 5),Pjwas the random effect of the period (j=1 to 5), Ckwas the random effect of the cow (k=1 to 5) andeijkwas the residual error. First-order autoregressive and compound symmetry (homogenous and heterogeneous) were tested as covariance structures, and the covariance structure with the lowest Akaike’s Information Criterion was retained in the final model. Least squares means were compared using Tukey’s procedure. The relationships of fatty acids among dietary intake, rumen and milk were analysed with the regression(REG) procedure of SAS (2002). Significance was declared atP＜0.05 unless otherwise noted.

3. Results and discussion

3.1. DMl and milk performance

In the present study, the step-wise improvement of forage combination increased energy and crude protein contents and decreased NDF contents of the TMRs. As a result, the step-wise improvement significantly increased DMI though the increase of milk yield was not significant in the present study (see Table 3). Dietary non-fibre carbohydrate (NFC)in comparison with NDF has low physical fill in the rumen and a high outflow rate (Allen 1996), and could explain why the step-wise improvement increased DMI. Hristovet al.(2004) noted that milk yield was positively correlated with DMI in dairy cows. Thus, the increase of dietary energy and crude protein intake should be the main reason why the step-wise improvement of forage combinations increased milk yield and 3.5% FCM in the present study, and this was consistent with a previous study that increasing dietary crude protein quadratically increased milk yield in lactating cows(Colmenero and Broderick 2006).

3.2. Ruminal and milk fatty acid profiles

Ruminal microorganisms consist of bacteria (1010–1011cells mL–1), archaea (107–109cells mL–1), protozoa (104–106cells mL–1) and fungi (103–106cells mL–1) (Wright and Klieve 2011). In the rumen, lipolysis of dietary glycolipids,phospholipids and triglycerides leads to free fatty acids, of which unsaturated fatty acid are hydrogenated to a large extent (Buccioniet al. 2012; Elgersma 2015). Levels of odd chain fatty acids are useful markers of the extent of microbial colonization on the digesta in the rumen (Kimet al.2005). In the present study, the step-wise improvement in forage combination increased odd chain fatty acids C11:0,C13:0 and C15:0 in the rumen, implicating that the extent of microbial lipolysis of dietary lipids in the rumen might be increased in response to the increase of microbial colonization as reflected by odd chain fatty acids.

Dschaaket al. (2011) noted that C16:0 and C18:0 were major long-chain fatty acids in ruminal fluid, accounting for 21–24% and 36–39% of total fatty acids, respectively. In the present study, C16:0 and C18:0 in the rumen accounted for approximately 33–35% and 31–36% of total fatty acids,respectively (see Table 4). The step-wise improvement of forage combination increased dietary C16:0 intake, and this could explain why C16:0 in rumen (Fig. 1-A) and milk(Fig. 2-A) was linearly increased against dietary C16:0 intake, and similar results were also observed for C18:2n-6(Fig. 1-B) and C18:3n-3 (Fig. 1-C). Previous studies noted that a comparatively large part of unsaturated C18 in diets was biohydrogenated to C18:0 by ruminal microbes(Destaillatset al. 2005; Or-Rashidet al. 2011), and these results could explain why C18:0 content was higher than that of the other fatty acids in the rumen (Table 4).

Table 3 Milk performance and fatty acid intake of dairy cows fed high-concentrate total mixed rations (TMRs) with step-wise improved forage combination1)

Medium-chain fatty acids (mainly C10:0, C12:0 and C14:0) and about half of C16:0 in milk that arede novosynthesized in the mammary gland from acetate and β-hydroxybutyrate are generated through fermentation of dietary feed in the rumen (M?nsson 2008). As shown in Table 5, the step-wise improvement of forage combination increased milk yields of C10:0, C12:0, C14:0 and C16:0,and this could be due to an increase inde novosynthesis of milk fat precursors of acetate and β-hydroxybutyrate in the mammary gland. ?rskovet al. (1969) reported that acetate infusions increased milk C12:0, C14:0 and C16:0 contentsand decreased milk C18:1 content, and the decrease of milk C18:1cis-9 percentage was also observed in the present study. Most C18:0 in milk are the ruminal end product of unsaturated C18 hydrogenation (Chilliardet al. 2007). The present decrease of milk C18:0 percentage in response to the step-wise improvement in forage combinations could be caused by the decrease of unsaturated C18 biohydrogenation. Among long-chain fatty acids, C18:2n-6 and C18:3n-3 are essential fatty acids that are beneficial to human health. These fatty acids in milk are derived from dietary sources and transported to the mammary gland(Kholifet al. 2014). In the present study, the step-wise improvement of forage combination increased dietary intake of C18:2n-6 and C18:3n-3 (Table 3) and then increased milk yield of these fatty acids (Fig. 3). The results obtained in the present study further confirmed that dietary fatty acid levels were closely associated with milk fatty acid levels.

Table 4 pH and fatty acid concentrations (mg mL–1) in rumen fluids of lactating cow fed high-concentrate total mixed rations (TMR)with step-wise improved forage combination1)

Fig. 1 The change of rumen fatty acid content against dietary fatty acid intake in lactating cows fed high-concentrate total mixed rations (TMRs) with step-wise improved forage combination (TMR1, the ration containing corn stover; TMR2, the ration containing corn stover and ensiled corn stover; TMR3, the ration containing ensiled corn stover and Chinese wild ryegrass hay; TMR4, the ration containing Chinese wild ryegrass hay and corn silage; TMR5, the ration containing corn silage, Chinese wild ryegrass and alfalfa hay).

Fig. 2 The change of milk fatty acid yield against rumen fatty acid concentration in lactating cows fed high-concentrate total mixed rations (TMRs) with step-wise improved forage combination (TMR1, the ration containing corn stover; TMR2, the ration containing corn stover and ensiled corn stover; TMR3, the ration containing ensiled corn stover and Chinese wild ryegrass hay; TMR4, the ration containing Chinese wild ryegrass hay and corn silage; TMR5, the ration containing corn silage, Chinese wild ryegrass and alfalfa hay).

3.3. Transfer efficiency of C18:2n-6 and C18:3n-3 from feed to milk

Milk from cows fed legume hays or silages generally contains higher levels of polyunsaturated fatty acids (C18:2n-6 and C18:3n-3). Benchaaret al. (2012) reported that increased transfer efficiency of C18:2n-6 and C18:3n-3 from feed to milk was in line with higher dietary intakes of these fatty acids. In the present study, the step-wise improvement of forage combination decreased fibre content in the TMRs and the transfer efficiency of both C18:2n-6 and C18:3n-3 from feed to milk (Table 5). Similarly, Alzahalet al. (2009) studied the effect of dietary fiber level on milk fat concentration and fatty acid profile of cows fed diets containing low levels of polyunsaturated fatty acids and found that decreasing dietary fibre content also increased the transfer efficiency of C18:2n-6 from 13.74 to 16.99% and C18:3n-3 from 11.96 to 15.03% as calculated. In the present study, the transfer efficiency of C18:2n-6 was slightly lower than that reported by Alzahalet al. (2009) and close to that of Halmemies-Beauchet-Filleauet al. (2014) who reported that the transfer efficiency of C18:2n-6 was increased from 11.4 to 15.7%.Khiaosa-Ardet al. (2010) noted that the transfer efficiency of C18:3n-3 from feed to milk was increased from 3.6 to 15% in cows fed isoenergetic diets differing in proportion and origin of concentrates and roughages. In the present study, the C18:3n-3 transfer efficiency from feed to milk was comparatively greater than the efficiencies reported inthe above-mentioned studies. Besides the contribution of dietary fatty acid intake, the results obtained in the present study provide evidence that step-wise improvement in forage combinations indeed enhanced the transfer efficiency of

linoleic and linolenic acids from feed to milk by decreasing dietary fibre content.

Table 5 Milk fatty acid profile, yield and transfer efficiency of dairy cows fed high-concentrate total mixed rations (TMRs) with step-wise improved forage combinations1)

Fig. 3 The change of milk fatty acid yield against dietary fatty acid intake in lactating cows fed high-concentrate total mixed rations(TMRs) with step-wise improved forage combination (TMR1, the ration containing corn stover; TMR2, the ration containing corn stover and ensiled corn stover; TMR3, the ration containing ensiled corn stover and Chinese wild ryegrass hay; TMR4, the ration containing Chinese wild ryegrass hay and corn silage; TMR5, the ration containing corn silage, Chinese wild ryegrass and alfalfa hay).

4. Conclusion

The step-wise improvement in forage combinations increased milk yield, as well as total fatty acids in milk. When the dietary fibre content was decreased, more dietary linoleic and linolenic acids escaped microbial hydrogenation in the rumen and consequently accumulated in milk fat, although concentrations of these fatty acids were low in ruminal fluids.The step-wise improvement in forage combinations applied in the present study can be recommended as a dietary strategy to increase the transfer efficiency of linoleic and linolenic acids from feed to milk.

Acknowledgements

This work was supported by the National Natural Science Foundation of China (31772628) and the National Key Basic Research Program of China (2011CB100801).