The relationships of dairy ruminal odd- and branched- chain fatty acids to the duodenal bacterial nitrogen flow and volatile fatty acids

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The relationships of dairy ruminal odd- and branched- chain fatty acids to the duodenal bacterial nitrogen flow and volatile fatty acids Keyuan Liu , Yang Li , Guobin Luo , Hangshu Xin , Yonggen Zhang , Guangyu Li PII: DOI: Reference:

S1871-1413(19)30691-2 https://doi.org/10.1016/j.livsci.2020.103971 LIVSCI 103971

To appear in:

Livestock Science

Received date: Revised date: Accepted date:

15 May 2019 31 December 2019 10 February 2020

Please cite this article as: Keyuan Liu , Yang Li , Guobin Luo , Hangshu Xin , Yonggen Zhang , Guangyu Li , The relationships of dairy ruminal odd- and branched- chain fatty acids to the duodenal bacterial nitrogen flow and volatile fatty acids, Livestock Science (2020), doi: https://doi.org/10.1016/j.livsci.2020.103971

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Highlights 

The relationship of ruminal OBCFA (odd- and branched- chain fatty acids) with duodenal bacterial nitrogen and was significantly influenced by the forage ratio of diets.



Rumianl and duodenal volatile fatty acids were changed with feeding time.



Ruminal OBCFAs should be used to predict bacterial nigtrogen flow in the duodenum.

The relationships of dairy ruminal odd- and branchedchain fatty acids to the duodenal bacterial nitrogen flow and volatile fatty acids Keyuan Liua,b, Yang Lib, Guobin Luoc,b, Hangshu Xinb, Yonggen Zhangb*, Guangyu Lia*

a

Institute of Special Economic Animal and Plant Science, Chinese Academy of Agricultural

Sciences, Changchun 130112, China b

Department of Animal Science and Technology, Northeast Agricultural University, Harbin

150030, China c

Zhejiang NHU Company Ltd. Shaoxing 312500, China

*

Corresponding author: Department of Animal Science and Technology, Northeast Agricultural

University, Harbin 150030, China. Email: [email protected] *

Institute of Special Economic Animal and Plant Science, the Chinese Academy of Agricultural

Sciences, 4899 Juye Avenue, Changchun 130112, China. Email: [email protected] Keyuan Liu and Yang Li contributed equally to this work and they are both co-first authors.

Abstract The objectives of the present research were to investigate the changes in duodenal bacterial nitrogen flow and volatile fatty acids (VFAs) with the different dietary ratios of forage and concentrate (F:C) and to determine the relationship of duodenal bacterial nitrogen flow and volatile fatty acids with ruminal odd- and branched-chain fatty acids (OBCFAs). The experimental design was a 3×3 Latin square. Three rumen- and duodenal-fistulated dry Holstein cows were fed

three rations with different dietary ratios of forage and concentrate (F:C; 30:70, 50:50 and 70:30). The rumen and duodenal samples were collected every two hours over 3 consecutive days of each sampling period. The determined parameters included OBCFA profiles in the rumen, duodenal bacterial nitrogen flow, VFAs, ammonia nitrogen (NH 3-N) content and pH. The results showed that ruminal OBCFA contents, duodenal bacterial nitrogen flow, VFAs, ammonia nitrogen (NH3-N) and pH were significantly influenced by the different dietary F:C ratios and sampling times (P < 0.01). The ruminal contents of C15:0 and C15:0+C17:0 were positively related to the molar proportions of duodenal acetate but negatively correlated with the propionate, butyrate and NH3-N contents (P < 0.05). The ruminal C15:0/C17:0 ratio was positively correlated with the proportions of acetate and pH (P < 0.05) but negatively correlated with VFA contents in the duodenum (P < 0.05). The duodenal bacterial nitrogen flow was positively correlated with ruminal C11:0 and iso-C17:0

contents

(P

<

0.05)

but

negatively

correlated

with

C15:0/C17:0

and

iso-C15:0/iso-C17:0 (P < 0.05). These findings suggested that some ruminal OBCFAs are closely related to the microbial nitrogen and volatile fatty acids in duodenal flow and significantly changed with the proportion of forage in diets. Ruminal OBCFA have the potential to predict microbial nitrogen flow, and the predicted equations for duodenal microbial nitrogen flow were established with OBCFAs in the rumen. Keywords: Rumen odd- and branched-chain fatty acids, duodenal microbial nitrogen flow, volatile fatty acids

1. Introduction Microbial protein synthesis in the rumen is an excellent source of digestible protein for

ruminants (NRC, 2001; Firkins et al., 2007; Kand et al., 2018). The accurate estimation of microbial protein synthesis and out flow from the rumen is essential for proper formulation of diets for ruminants (Stern et al., 1994; Oldick et al., 1999; NRC, 2001). Due to the complexity of the rumen ecosystem, the techniques for assessing microbial protein synthesis in rumen have been improved by different methods (Dewhurst et al., 2000). Mcallan and Smith (1973) suggested that nucleic acids or their constituent purine or pyrimidine bases act as internal microbial markers of microbial protein synthesis. However, this method needed to clarify the composition of undetached microbes associated with rumen particles and confirm endogenous purine losses. Vlaeminck et al. (2006a) summarized that odd- and branched-chain fatty acids (OBCFAs) in ruminant milk have been increasingly considered as potential microbial nitrogen outflow markers in rumen. Ruminal OBCFAs are mainly present in bacterial membrane lipids (Kaneda, 1991) and are the major source of OBCFAs in ruminant milk. Therefore, ruminal and milk OBCFAs has potential as markers to quantify bacterial matter leaving the rumen (Vlaeminck et al., 2005). Vlaeminck et al. (2005) determined that C17:0 and total OBCFA contents were highly related to the duodenal flow of purine bases and uracil. The marker ratios for solely bacteria that were isolated from rumen contents were generally used to estimate of microbial protein content (Dewhurst et al., 2000). Vlaeminck et al. (2007) estimated N flows from the OBCFA pattern and some microbial bases and suggested that changes in proportions of solid- and liquid-associated rumen bacteria could impact on estimated duodenal flow of bacterial nitrogen. The ruminal nitrogen supply significantly influences microbial growth, diet fermentation, and digesta outflow (Maeng et al., 1976). In the rumen, organic matters is fermented by rumen microbes and produces volatile fatty acids (VFAs) (Dijkstra, 1994), which are mostly absorbed by

the rumen wall (Greenwood et al., 1997). A large amount of concentrates results in an increased production of VFAs in the rumen, and an increase in the osmolality of the rumen contents results in the extension of the abomasal wall (Van Winden et al., 2004). Many researchers have determined that short-chain fatty acids inhibit the motion of abomasal epithelial cells, resulting in digesta stasis (Van Winden et al., 2004). Additionally, unabsorbed VFAs into the duodenum affect the duodenal environment and nutrient digesion and absorption in the intestine. However, there are no reported studies on the metabolism of VFAs in the postruminal and duodenal regions. Hence, the first aim of the present study was to investigate the changing rules of microbial nitrogen flow and VFA contents in the duodenum over time. The second was to determine the relationships between ruminal OBCFAs and duodenal bases that are markers of the bacterial nitrogen flow and VFA contents using data from different dietary ratios of the F:C feeding ratios. If relationships were identified, prediction equations of the latter were developed for ruminal OBCFAs, which were used as an independent data set.

2. Material and methods 2.1. Experimental design, diets and sampling Three dry Holstein cows (600±24 kg BW) fitted with permanent fistulas in the rumen and proximal duodenum were used in a 3×3 Latin square (n=3). The cows were fed three total mixed rations (TMRs), which were formulated according to the dairy nutrient requirements of National Research Council (NRC, 2001), with different dietary F:C ratios (F:C; 30:70, 50:50, and 70:30). Each experimental period lasted for 3 weeks with the last week for sampling. The basal diets were offered in equal amounts twice daily at 0600 and 1800 h. The composition and nutrition levels of

the basal diets are shown in Table 1. Rumen and duodenal samples of the liquid phase were taken during the final week of each experimental period. The rumen and duodenal samples were collected every two hours over 3 consecutive days of each period (Bas et al., 2003). The ruminal contents were taken from the cranial ventral, caudal ventral, central and cranial dorsal and mixed thoroughly.

2.2. Samples analysis method The TMRs samples were air-dried at 60±5°C and then analysed for DM, and crude protein (CP), calcium (Ca) and total phosphorus (TP) following to the procedures of AOAC (1990). The acid detergent fibre (ADF) and neutral detergent fibre (NDF) concentrations were analysed according to Van Soest et al. (1991) using the Ankom system (Ankom 220 Fiber Analyzer; Ankom) with a heat-stable a-amylase and expressed exclusive of residual ash. Net energy for lactation (NEL) at a production level was calculated using a NRC summative approach from the dairy nutrient requirement (NRC, 2001). The rumen contents were passed through four layers of cheesecloth to remove particulate matter and then freeze-dried before analysis by GLC, as described by Vlaeminck et al. (2004) and the detailed description were showed on Zhang et al. (2016). In detail, 0.2 g of freezedried rumen contents were accurately weighed into 25-ml test tubes with screw caps, and 4.0 ml of 10% methanolic HCl was added. Additionally, 0.5 ml of nonadecanoic acid (concentration 1 mg/ml, Sigma, Bornem, Belgium) was used as the internal standard, and this mixture was then incubated at 55 °C for 2 h. After cooling, 5 ml of 6% potassium carbonate and 2 ml of hexane were added to the same tube to hydrolyse the samples, and then, the samples were centrifuged at 500 g for 2 min.

After transferring the upper layer to a new tube, the extracts were evaporated under N2. The residue was dissolved in 1 ml of hexane and analysed by gas chromatography (GC 2010, Tokyo, Japan) with an SP-2560TM column for fatty acid methyl esters (100 m × 0.25 mm×0.2 μm). For total fatty acid analysis, the injector temperature was maintained at 240 °C and the detector temperature was maintained at 240 °C. The initial oven temperature was held at 170 °C for 30 min, increased by 1.5 °C/min to 200 °C (held for 20 min) and then increased at 5 °C/min to 230 °C (held for 5 min). Highly pure nitrogen was the carrier gas. The injector pressure was held constant at 266.9 KPa. The duodenal samples of VFAs (acetate, propionate, and butyrate) were treated according to the description of Li and Meng (2006) and then analysed by a gas chromatography (GC 2010, Tokyo, Japan) with an FFAP capillary column (HP-INNOWAX, 30 m×0. 25 mm×0.2 μm). The pH value of duodenal sample was immediately determined in the obtained samples by a pH meter (Sartorius Basic pH Meter, Germany). The concentration of ammonia nitrogen (NH3-N) was determined by an ammonia-sensing electrode (Expandable Ion Analyzer EA 940, Orion, USA). The duodenal contents were freeze-dried and then analyzed the total N following to the method of AOAC (1990). The duodenal microbial bases were extracted from freeze-dried samples using perchloric acid, as described by Zinn and Owens (1986). In detail, the lyophilized rumen fluid samples (50 mg) were placed in screw-cap tubes and added 2.5 mL 0.6 M HClO4, and then incubated for 1 h. After cooling, the pH was adjusted to between 6.6 and 6.9 with KOH (8 M), and combined with 10 mM NH4H2PO4 to 10 mL. After centrifugation of samples at 500 g for 10 min, the supernatant was filtered through a 0.45 μm filter, and then analyzed by HPLC using a C18 column (5 μ, 250×4.6 mm, Diamonsil). The buffer solution (20 mM NH 4H2PO4) was run

isocratically at 1 mL/min, and the effluent was monitored at 254 nm. The concentrations of individual bases were determined from standard curve equations. The standards of individual bases (≥99.5%, Aladdin, China) were formulated to a concentration of 50 mg/L. The mixed solution which mixed by the standards bases solutions with same volume was serially diluted into 5 gradients and subsequently analyzed by HPLC which was same as the analysis method of duodenal samples. The standard curve equations calculated from data of analyzed by HPLC and their correspondent concentrations.

2.3. Statistical analysis All data statistical analyses were performed using SAS 9.2 (SAS Institute Inc., SAS Campus Drive, Cary, North Carolina, USA.). Univariate analysis Analysis of variance was done through the MIXED procedure of SAS 9.2. The mixed model procedure included the random effect of study as described by St-Pierre (2001). The effect of different dietary F:C ratio on OBCFAs, duodenal microbial bases, VFA, NH 3-N and pH value were estimated according to: Yijkl = μ + Ti + Sj + TS𝑖𝑗 + Ck + SC𝑗𝑘 + P𝑙 + ϵi𝑗𝑘𝑙 where Yijkl is the individual observation, μ the overall mean, Ti is the effect of dietary treatment (i = 3; F:C = 30:70, 50:50, and 70:30), Sj is the effect of sampling time, TS𝑖𝑗 ie the interaction between treatment and sampling time, Ck is the effect of cow, SC𝑗𝑘 is the interaction between sampling time and cow, P𝑙 is the effect of effect of experimental period, Ck is the effect of cow, and ϵi𝑗𝑘𝑙 the residual error. Effect of cow was treated as a random effect. For all

statistical analyses, significance was declared at P < 0.05. Correlation analysis The relationships between OBCFA profile as obtained from the total mixed duodenal contents and VFAs, NH3-N, pH value as well as microbial bases were analyzed by CORR PROC of SAS 9.2 using the Pearson correlation method. The correlations were declared significant at P ≤ 0.05. Linear regression analyses The data of OBCFA were considered as independent data set and the data of VFAs, NH3-N, pH value or duodenal microbial bases as dependent dataset. A multiple regression was applied using the STEPWISE method of REG procedure of SAS. The SLENTRY and START value were all 0.05. The equations were determined by least squares estimation (P < 0.05). The regression equations were evaluated based on the root mean square error (RMSE) and coefficient of multiple determinations (R2) of regression model. n

1 RMSE = √ × ∑(yi − ŷi )2 n i=1

where n is the number of observations, and yi and ŷi are the observed and predicted values , respectively.

3. Results 3.1. Changes of rumen OBCFA with different dietary F:C ratio The concentrations of OBCFA were significantly influenced by the ratios of forage in diets and sampling time (P < 0.01) (Table 2). The concentrations of C11:0 and C13:0 were frequently

more abundant at 0-4 h before feeding time, and C15:0 and C17:0 contents were greater during 2 h before and after feeding. The contents of iso-C15:0 and iso-C17:0 were often greater at 2-4 h before feeding time. However, the iso-C17:0 contents were lower at 4-6 h after feeding time. The concentrations of iso-C16:0 were often lower 0 h before feeding time and most abundant at 2 h before feeding time with diet ratios of 30:70 and 50:50, however, they were greatest at before evening feeding and least at 2 h after morning feeding with the diet ratio of 70:30. The contents of anteiso-C15:0 and anteiso-C17:0 were commonly greater 2-4 h after feeding. The total OBCFA contents were lowest at 2 h after morning feeding time with the diet ratio of 30:70, and they were greatest before evening feeding time with the diet ratio of 50:50. Furthermore, the total OBCFA contents were greatest at 4 h before evening feeding and lowest at morning feeding time with the diet ratio of 70:30.

3.2. Changes of duodenal microbial nitrogen flow with different dietary F:C ratio The concentrations of duodenal microbial bases and total N were changed with sampling time (Table 3) and significantly affected by the interaction of dietary F:C ratio and sampling time (P < 0.01). The contents of duodenal bases and total N were greater at 0-2 h before evening feeding time but lower at 6 h after morning feeding time in the cows fed a dietary F:C ratio of 30:70. However, adenine contents were lower at 2 h after morning feeding in the cows fed a dietary F:C ratio of 50:50. For the cows fed a dietary F:C ratio of 70:30, they had a greater level of duodenal bases before evening feeding time. The total N concentrations were much greater after morning feeding time with the diets ratio of 30:70 and 50:50, but they were greatest before evening feeding time with the diet ratio of 70:30.

3.3. Changes of duodenal VFA, NH3-N and pH with different dietary F:C ratio The duodenal VFA contents were influenced by the different dietary ratios of F:C (Table 4) (P < 0.05). The contents of acetate and TVFA were greater at 4-6 h before feeding time and often lower 4 h before evening feeding. However, the concentrations of propionate and butyrate were more abundant at 4 h and usually lower at 6 h after morning feeding. The pH values often were greater at 4-6 h after morning feeding and lower at 0-4 h before evening feeding. Meanwhile, the concentrations of duodenal NH3-N were increased at 2-4 hours after feeding and then decreased and increased at 6-8 h, and they were lowest before feeding with the exception of cows fed a dietary F:C ratio of 50:50.

3.4. Correlation between rumen OBCFA profiles and duodenal microbial nitrogen flow The relations of ruminal OBCFA contents to duodenal microbial bases concentrations and the ratios of duodenal microbial bases contents to total N which are showed in Table 5. The duodenal cytosine contents were positively and significantly related to the concentrations of C11:0, iso-C16:0, iso-C17:0, anteiso-C17:0, total branched-chain fatty acids (r = 0.20~0.44, P < 0.05), however, negatively correlated with C15:0, C15:0+C17:0, total odd-chain fatty acids, C15:0/C17:0 and iso-C15:0/iso-C17:0 (r = -0.21~-0.27, P = 0.01~0.03). The contents of uracil in duodenum were positively associated with the concentrations of C11:0, iso-C15:0, iso-C16:0, iso-C17:0, anteiso-C17:0, iso-C15:0+iso-C17:0, total iso-fatty acids and total branched-chain fatty acids in rumen (r = 0.21~0.51, P < 0.05). The ruminal C11:0 and iso-C17:0 contents were positively correlated with guanine contents in duodenum (r = 0.30~0.33, P = 0.001). However, the

C13:0, iso-C15:0, C15:0, C15:0+C17:0, total odd-chain fatty acids, C15:0/C17:0 and iso-C15:0/iso-C17:0 in rumen were negatively related to duodenal guanine contents (r = -0.20~-0.49, P < 0.05). The duodenal adenine concentrations were positively related to C11:0, iso-C16:0, iso-C17:0, anteiso-C15:0+anteiso-C17:0, total anteiso-fatty acids and total branched-chain fatty acids contents (r = 0.19~0.41, P < 0.05), and negatively linked with C15:0, C15:0+C17:0, total odd-chain fatty acids, C15:0/C17:0 and iso-C15:0/iso-C17:0 in rumen (r = -0.20~-0.33, P = 0.001~0.04). As regards the ratio of cytosine to total N in duodenum was positively related to C11:0, C13:0, iso-C16:0, iso-C17:0, anteiso-C17:0, total iso-fatty acids and total branched-chain fatty acids (r = 0.19~0.40, P < 0.05), but negatively associated with the ratio of C15:0/C17:0 and iso-C15:0/iso-C17:0 (r = -0.23~-0.31, P = 0.001~0.02). The ratio of uracil to total N in duodenum was positively correlated with C11:0, C13:0, iso-C15:0, iso-C16:0, iso-C17:0, anteiso-C17:0, iso-C15:0+iso-C17:0, total iso-fatty acids and total branched-chain fatty acids contents in rumen (r = 0.20~0.48, P < 0.05). The ruminal C11:0 and iso-C17:0 concentrations were positively related to the ratio of guanine to total N of duodenum (r = 0.26~0.35, P = 0.0002~0.01). However, the contents of iso-C15:0 and the ratios C15:0 to C17:0 and iso-C15:0 to iso-C17:0 in duodenum were negatively linked with the ratio of guanine to total N (r = -0.24~-0.53, P < 0.05). The ratio of adenine in duodenum was positively correlated with the ruminal C11:0, iso-C16:0 and iso-C17:0 contents (r = 0.22~0.37, P < 0.05), but negatively related to C15:0, C15:0+C17:0, total odd- chain fatty acids, C15:0/C17:0 and iso-C15:0/iso-C17:0 in rumen (r = -0.21~-0.35, P = 0.0003~0.03). The prediction equations of duodenal microbial bases established by the contents of ruminal OBCFA, and tested P < 0.0001 by the least square methods (Table 6). The R2 values of

predictions were about 0.30, and that of uracil prediction were more than 0.05.

3.5. Correlation between rumen OBCFA profiles and duodenal VFA, NH3-N and pH The relationships of rumen OBCFA and duodenal VFA contents are showed in Table 7. The concentrations of C15:0+C17:0, total odd-chain fatty acids and the ratio of C15:0 and C17:0 were positively relative to acetate content in duodenum (r = 0.22~0.30, P = 0.002~0.02). The duodenal propionate contents were negatively correlated with C15:0, C15:0+C17:0 and total odd-chain fatty acids concentrations and the ratio of C15:0 to C17:0 (r = -0.22~-0.29, P = 0.002~0.02). The duodenal butyrate contents were negatively linked with concentrations of C15:0, TOBCFA, C15:0+C17:0 and total odd-chain fatty acids, and the ratio of C15:0 to C17:0 (r = -0.19~-0.32, P = 0.001~0.048). As well as, there was a positively relationship between duodenal butyrate contents (r = 0.19, P = 0.04). The total VFA contents were positively associated with anteiso-C17:0 concentrations (r = 0.19, P = 0.04). The equations of duodenal VFA were established with ruminal OBCFA, whose accuracy were tested by the least square method (Table 8). The predicted equations were composed by C13:0 and C15:0.

4. Discussion The significant effect of the F:C ratio on the OBCFA composition of rumen bacteria has been observed by Vlaeminck et al. (2006b) and Zhang et al. (2016). The same result was obtained from this study, which probably reflected shifts in the bacterial populations (Vlaeminck et al., 2006b). The OBCFA profile of many cultivable rumen bacteria has been reviewed by Vlaeminck et al.

(2006a). Cellulolytic bacteria contain high amounts of iso-fatty acids, but amylolytic bacteria show low levels of branched-chain fatty acids and linear odd-chain fatty acids. However, there are only a number of limited bacterial species that have been studied, and it is not possible to describe the species composition of rumen samples (Vlaeminck et al., 2006b). The concentrations of individual OBCFAs were changed with rumen fermentation in this study. The feed amount, eating rate and feeding duration lead to circadian rhythms of nutrient intake (Nikkhah, 2014). Koike et al. (2003) indicated that variation in ruminal bacteria is related to the dietary compositions and transport in rumen, especially when the concentrate content is changed in the diets. Bryant and Robinson (1968) discussed that the bacterial populations are lowest at 1 h but are increased at 2.5-5.5 h after feeding. In this study, the contents of C11:0 and C13:0 decreased after feeding and then increased with a diet ratio of 30:70, which may be explained by previous research. Forage and water entering the rumen causes the dilution of rumen contents and temperature decreases (Leedle et al., 1982). Some bacterial populations increase to adapt to this change and produce the corresponding metabolite (Dawes and Ribbons, 1962). Hence, increased contents of C15:0 and C17:0 at 2 h after feeding were found in this study. Nikkhah et al. (2008) and Nikkhah et al. (2011) reported that 30:70 diets are consumed in the rumen within 3 h after dietary intake. Due to rumen content out flow and nutrition assimilated by the rumen wall, the concentrations of bacteria are greater before feeding than after feeding. Therefore, some OBCFAs were more abundant before feeding. Ruminal microbial ecology is complicated by the fact that there are many interrelationships among the various ruminal microbes (Russell and Baldwin, 1981). In the current study, the OBCFA contents were complex with rumen fermentation and after being fed different F:C diets.

Previous studies have investigated the effects of a range of supplements and forage types (Johnson et al., 1998), as well as feed restrictions (González-Ronquillo et al., 2004) and different F:C ratios, on duodenal microbial protein flow (Moorby et al., 2006). Smith and Mcallan (1974) indicated that ruminant species and time of sampling after feeding alters the RNA-N:total-N ratio. Zinn and Owens (1986) utilized the base contents and the ratio of base contents to total N to estimate the duodenal N flow. This result was highly related to that obtained using the tungstate method in cows fed high concentrate amounts. Castillo-Lopez et al. (2014) found that the duodenal N flow is elevated when evaluating bases and DNA. In addition, nonprotein purine also affects the estimated results (Belanche et al., 2011). Moorby et al. (2006) indicated that the total and microbial N flow to the duodenum increased linearly with decreasing F:C ratio diets. The total N and microbial bases were also significantly influenced by the different F:C ratio diets in this study. Nitrogen absorbed directly from the rumen and other stomach tissues into the body is balanced by the recycling of N from the body back into the rumen (Huntington, 1989). Hence, the N flow into the duodenum is similar to the N consumption. Microbial base concentrations varied in response to feeding time, with peaks occurring before morning feeding time, but they were lowest at 4 to 6 h after morning feeding. According to Bergman (1990), 75% of VFAs produced in the rumen are absorbed in the rumen, and the remaining VFAs are absorbed in subsequent parts of the digestive tract. Previous research has indicated that the omasum has a strong butyrate absorption ability (Tesseraud et al., 2003). In our study, the contents of VFAs in the duodenum were much lower than those in the rumen, and they were influenced by the ratio of forage in diets. The total contents of acetate, propionate and butyrate in the duodenum of dairy cows fed the greatest level of concentrates

diets were greater than in other dairy cows, which were similar to the contents in the rumen (Zhang et al., 2016). The dynamic changes of VFA contents in the duodenum were similar to those in the rumen (Sutton et al., 2003), suggesting that the substance metabolism in the duodenum was closely related to rumen fermentation. Most ammonia is absorbed in the N recycling system, and it plays important roles in the entire process of N digestion and metabolism for ruminants (Wang and Tan, 2013). Accordingly, the contents of NH3-N in the duodenum were much lower than those in the rumen in this research. Supplementation with nitrogenous compounds in the abomasum of animals fed low-quality forage could stimulate intake via nitrogen recycling (Rufino et al., 2016), which suggests that the postruminal nitrogen contents could affect the absorption of nitrogen in the rumen. Diurnal variation in OBCFAs clearly reflects the microbial colonization of newly ingested pasture (Sun and Gibbs, 2012). Ipharraguerre et al. (2007) documented that the flow of

bacterial N is moderately greater for omasal than duodenal sampling using purines as a marker (approximately 15%), but is much greater when

15

N is used as a marker

(approximately 45%). Vlaeminck et al. (2005) estimated the bacteria N flow using milk OBCFA contents, and their result was more accurate than that obtained by using DMI and different diet compositions. Vlaeminck et al. (2005) also found that the OBCFA contents in milk were significantly correlated with duodenal microbial base concentrations and diaminopimelic acid content. Vlaeminck et al. (2007) found that the estimations of duodenal N flow achieved by adenine, cytosine and OBCFAs were close to each other, in both in liquid phase bacteria and solid phase bacteria in the rumen. However, Dewhurst et al. (2000) indicated that the result of duodenal microbial protein evaluation is significantly influenced by the different rumen phases of microbial

origin, which reflects that the growth of different rumen phases of microbial origin and the availability of nutrition are due to different kinds of microbes (Volden et al., 1999; Carro and Miller, 2002). In addition, the effect of different rumen phases of microbial origin is greater than that of different diets (Rodríguez-Prado et al., 2004). In this research, the predicted equations for duodenal microbial bases from ruminal OBCFAs were composed of odd- and iso-chain fatty acids, which further explained the function of ruminal OBCFAs as a duodenal protein marker. Hristov (2007) obtained estimated results of microbial N flow in ruminal samplings similar to those of duodenal and reticular samplings. Predictions of duodenal purines based on milk secretion of C17:0 were more accurate than predictions based on milk secretion of OBCFA (Vlaeminck et al., 2005). Additionally, the equations in this research for purines contained iso-C17:0 in this research. These results suggested that metabolism of dairy fatty acids with 17 carbons is closely related to microbial quantity. Ruminal bacteria synthesize odd-chain fatty acids with short-chain fatty acids, and this process occurs through the action of fatty acid synthetase (Kaneda, 1991; Vlaeminck et al., 2006a). The balance of which primers are used may be a function of the relative availability of the substrates rather than a reflection of an altered specificity of the fatty acid synthetase (Fulco, 1983). In this study, the rumen OBCFA contents were negatively associated with propionate and butyrate in the duodenum in this study. This result may explain the use of propionate and butyrate in the synthesis of OBCFAs and may indicate that fatty acid metabolism in the duodenum is related to the rumen contents. The relations of ruminal OBCFAs with duodenal acetate were different than the relations of duodenal propionate and butyrate in our research. The results suggested that the acetate metabolism process was different from that of others in the duodenum.

Different VFAs arise from variations in substrate intake and bacterial populations, and the individual VFAs have distinct metabolic fates (Dijkstra et al., 2012). Nozière et al. (2010) observed increased propionate and decreased acetate formation upon an increase in DM intake. An increase in pH value causes the conversion of an NH4+ ion to NH3, which is then rapidly absorbed in the rumen (Tillman and Sidhu, 1969). The correlations of ruminal OBCFAs with NH3-N and pH were found to be opposite in the duodenum. The results suggested that the changes in NH3-N content and pH in the duodenum were related to those in the rumen. The prediction equations for VFAs in the duodenum from ruminal OBCFA were composed by C13:0 and C15:0, and the equations of VFAs of rumen from ruminal OBCFA also contained odd-fatty acids (Zhang et al., 2016). These results suggested that the odd-chain fatty acids in the rumen are obviously associated with short-chain fatty acids in the rumen and duodenum. 5. Conclusion The rumen OBCFA profiles were significantly influenced by the forage ratio of diets and changed with rumen fermentation. Moreover, different dietary F:C ratios significantly affected duodenal microbial nitrogen flow and VFA contents. In addition, several relationships existed between rumen OBCFAs and duodenal microbial nitrogen. Ruminal OBCFAs should be used to predict variables of duodenal microbial variables, and the prediction models foe microbial nitrogen flow were established with rumen OBCFAs. To increase the accuracy of these equations, however, larger numbers of animals should be tested in the experimental setting and under field conditions.

6. Acknowledgements

The research is supported by the National Key R&D Program of China (2018YFC1706600), the China Agriculture Research System (CARS-36), the Postdoctoral Foundation in Heilongjiang Province (LBH-Z17035), Young Talents "Project of Northeast Agricultural University" (18QC35) and Technology Research Project from Education Department of Heilongjiang Province (No. 12511036).

Conflict of interest The authors declare no conflicts of interest

Author Contribution Statement Conceptualization, Keyuan Liu; Data curation, Keyuan Liu, Yang Li and Guobin Luo; Funding acquisition, Yang Li, Hangshu Xin, Yonggen Zhang, and Guangyu Li; Writing-original draft, Keyuan Liu; Writing—review and editing, Keyuan Liu, Yonggen Zhang.

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Table 1. Feed ingredients and chemical composition (g/kg DM) of the rations F:C Ingredients Alfalfa hay Chinese wildrye Corn silage Distillers dried grains with soluble (DDGS) Wheat bran Corn grain Soybean meal CaHPO4 Limestone NaCl Premix

1)

30:70

50:50

70:30

12 156 132

24 272 204

161 255 284

164

112

68

60

50

28

405

229

105

51

92

89

1.2 14

2.2 10

5.2 0.0

1.6

1.6

1.6

3.2

3.2

3.2

7.27

7.19

7.10

Chemical composition NEL2) (MJ/kg) CP

152

153

147

NDF

436

530

564

ADF

181

266

327

Ca TP 1)

3)

6.3

6.2

6.2

3.7

3.7

3.6

Provided per kilogram of premix: Cu 4 560 mg, Mn 4 590 mg, Zn 12 100 mg, I 270 mg, Co 60

mg, VA 2000 000 IU, VD3 450 000 IU, VE 10 000 IU, VE 3 000 mg. 2)

NEL is calculated value and the other nutrient levels are measured values (NRC, 2001).

3)

TP means the contentration of total phosphorus in the rations.

Table 2. Changes of ruminal OBCFA profiles in rumen with different F:C ratios of diets (g/kg DM) Time

P

F:C

SEM 6h

8h

10 h

12 h

14 h

16 h

18 h

30:70

0.016 0.015 0.023 0.017 0.021 0.026 0.031

50:50

0.016 0.008 0.021 0.013 0.021 0.020 0.012

70:30

0.018 0.014 0.017 0.019 0.014 0.027 0.032

30:70

0.016 0.013 0.039 0.027 0.020 0.025 0.019

50:50

0.027 0.028 0.015 0.030 0.028 0.037 0.031

70:30

0.035 0.043 0.038 0.027 0.036 0.033 0.035

30:70

0.846 0.753 0.706 1.174 0.929 0.800 1.562

50:50

0.904 1.052 0.868 0.663 0.954 0.881 0.730

70:30

0.387 1.627 1.209 1.424 2.366 0.539 1.503

30:70

0.040 0.133 0.131 0.160 0.101 0.115 0.096

50:50

0.070 0.064 0.122 0.143 0.117 0.112 0.098

70:30

0.139 0.143 0.296 0.151 0.150 0.102 0.086

30:70

0.152 0.166 0.162 0.101 0.131 0.138 0.116

50:50

0.143 0.139 0.137 0.144 0.141 0.146 0.167

70:30

0.215 0.197 0.230 0.056 0.271 0.207 0.213

30:70

0.141 0.163 0.233 0.160 0.184 0.176 0.203

50:50

0.108 0.106 0.139 0.128 0.130 0.137 0.125

70:30

0.178 0.196 0.206 0.162 0.214 0.184 0.155

30:70

0.106 0.109 0.171 0.135 0.133 0.224 0.174

50:50

0.095 0.115 0.119 0.104 0.106 0.225 0.100

70:30

0.170 0.152 0.186 0.193 0.183 0.231 0.364

30:70

0.325 0.097 0.352 0.382 0.309 0.327 0.454

Anteiso-C15:0 50:50

0.341 0.421 0.324 0.177 0.243 0.383 0.198

70:30

0.187 0.559 0.146 0.170 0.157 0.428 0.360

30:70

0.027 0.082 0.077 0.034 0.045 0.050 0.055

Anteiso-C17:0 50:50

0.051 0.046 0.011 0.034 0.033 0.050 0.033

70:30

0.025 0.027 0.077 0.041 0.052 0.017 0.024

30:70

1.67

1.53

1.89

2.19

1.87

1.88

2.71

50:50

1.76

1.98

1.76

1.44

1.77

1.99

1.49

70:30

1.36

2.96

2.41

2.24

3.44

1.77

2.77

C11:0

C13:0

C15:0

C17:0

Iso-C15:0

Iso-C17:0

Iso-C16:0

TOBCFA

Diet

Time

D*T

0.001 <0.0001 <0.0001 <0.0001

0.002 <0.0001 <0.0001 <0.0001

0.054 <0.0001 <0.0001 <0.0001

0.007 <0.0001 <0.0001 <0.0001

0.012 <0.0001 <0.0001 <0.0001

0.009 <0.0001 <0.0001 <0.0001

0.006 <0.0001 <0.0001 <0.0001

0.020

0.003

<0.0001 <0.0001

0.003 <0.0001 <0.0001 <0.0001

0.060 <0.0001 <0.0001 <0.0001

D*T, the interaction of dietary F:C ratio and time of sampling; SEM, standard error of mean; TOBCFA, the total

contents of odd- and branched-chain fatty acids.

Table 3. Effect of dietary F:C ratio on duodenal microbial bases and N concentrations (g/kg DM) Time

P

F:C

SEM 6h

8h

10 h

12 h

14 h

16 h

18 h

30:70

0.43

0.67

0.95

1.27

0.73

0.84

0.79

Cytosine 50:50

0.48

0.78

0.47

0.63

0.69

1.04

0.95

70:30

0.77

0.67

0.63

0.71

0.54

0.80

1.06

30:70

0.16

0.35

0.64

1.25

0.27

0.42

0.70

50:50

0.21

0.40

0.26

0.26

0.35

0.44

0.56

70:30

0.35

0.65

0.37

0.36

0.27

0.61

1.15

30:70

0.88

1.33

1.79

1.80

1.54

1.77

1.33

50:50

0.84

1.46

0.72

1.32

1.49

1.59

1.21

70:30

0.71

1.37

1.09

1.35

1.23

1.23

1.60

30:70

0.31

0.73

0.35

1.47

0.85

0.81

0.57

50:50

0.31

1.19

0.25

0.63

0.63

0.71

1.04

70:30

0.44

0.73

0.53

0.51

0.63

0.72

1.44

30:70

39.79 46.88 52.23 46.46 50.93 47.08 47.99

50:50

42.78 39.27 39.86 42.98 43.93 44.52 45.93

70:30

34.29 44.74 45.16 39.26 37.33 44.14 44.37

Uracil

Guanine

Adenine

Total N

Diet

Time

D*T

0.07

0.0004

<0.0001

<0.0001

0.08

<0.0001

<0.0001

<0.0001

0.10

<0.0001

<0.0001

<0.0001

0.10

0.003

<0.0001

<0.0001

0.61

<0.0001

<0.0001

<0.0001

D*T, the interaction of dietary F:C ratio and time of sampling；SEM, standard error of mean.

Table 4. Changes of duodenal VFA and NH 3-N concentrations with different F:C ratios of diets Time

P

F:C

SEM 6h

8h

10 h

12 h

14 h

16 h

18 h

Diet

Time

D*T

30:70

725.6 622.0 459.0 721.1 741.8 712.2 691.6

50:50

692.1 617.2 588.2 647.8 684.1 517.3 511.6 12.90 <0.0001 <0.0001 <0.0001

70:30

639.6 672.8 524.3 716.3 701.4 631.2 693.4

30:70

219.7 291.0 431.6 212.4 199.7 226.9 239.8

50:50

238.0 302.3 324.9 287.0 248.7 361.1 385.9 10.93 <0.0001 <0.0001 <0.0001

70:30

279.1 253.7 373.3 224.3 235.7 287.4 242.3

Acetate (mmol/mol)

Propionate (mmol/mol) 30:70

54.6

86.9 109.3

66.5

58.5

60.9

68.6

50:50

70.0

80.5

87.0

65.2

67.1 121.6 102.6 2.36 <0.0001 <0.0001 <0.0001

70:30

81.2

73.5 102.4

59.3

62.9

Butyrate (mmol/mol) 30:70 TVFA (mol/L) 50:50 70:30

pH

NH3-N (mg/dL)

81.4

64.4

21.92 12.49 10.07 24.53 25.00 23.65 17.93 18.50 12.06 11.12 17.24 17.25 10.46

9.47 0.90 <0.0001 <0.0001 <0.0001

13.21 16.18 10.85 22.29 18.91 13.96 20.10

30:70

2.83

2.24

2.45

2.20

2.45

2.43

2.56

50:50

2.51

2.06

2.17

2.71

2.31

2.30

2.13 0.07

70:30

2.67

2.47

2.34

2.54

2.77

2.40

2.44

30:70

7.82

9.82

9.38

7.16 11.68 10.20

7.84

50:50

8.96

8.13

7.94

8.54 11.72 10.28

9.43 0.42 <0.0001 <0.0001 <0.0001

70:30

7.81

9.05

8.08

7.15 11.80 10.70

7.67

0.01

<0.0001 <0.0001

D*T, the interaction of dietary F:C ratio and time of sampling; SEM, standard error of mean; TVFA, the total

contents of acetate, propionate and butyrate in duodenum.

Table 5. Correlation between rumen OBCFA contents and duodenal microbial nitrogen flow Cytosine

Uracil

Guanine

Adenine

Cytosine/N

Uracil/N

Guanine/N

Adenine/N

r

P

r

P

r

P

r

P

r

P

r

P

r

P

r

P

0.44

<0.0001

0.51

<0.0001

0.31

0.001

0.41

<0.0001

0.40

<0.0001

0.48

<0.0001

0.26

0.01

0.37

<0.0001

C13:0

0.09

0.33

0.15

0.11

-0.24

0.01

0.08

0.43

0.20

0.03

0.20

0.04

-0.12

0.21

0.12

0.21

Iso-C15:0

-0.01

0.88

0.21

0.03

-0.25

0.01

-0.15

0.11

-0.01

0.89

0.22

0.02

-0.24

0.01

-0.16

0.11

Anteiso-C15:0

0.07

0.47

0.10

0.30

-0.03

0.74

0.18

0.07

0.07

0.50

0.09

0.34

-0.03

0.78

0.16

0.10

C15:0

-0.22

0.02

-0.17

0.08

-0.21

0.03

-0.26

0.01

-0.19

0.052

-0.16

0.11

-0.17

0.08

-0.25

0.01

Iso-C16:0

0.24

0.01

0.42 <0.0001

0.13

0.20

0.32

0.001

0.20

0.04

0.40

<0.0001

0.09

0.37

0.27

0.005

Iso-C17:0

0.29

0.003

0.43 <0.0001

0.32

0.001

0.21

0.03

0.32

0.001

0.44

<0.0001

0.35

0.0002

0.22

0.02

Anteiso-C17:0

0.27

0.004

0.35

0.0002

0.18

0.06

0.07

0.50

0.27

0.01

0.35

0.0002

0.17

0.08

0.07

0.48

C17:0

-0.02

0.82

-0.05

0.62

0.12

0.23

-0.11

0.24

-0.003

0.97

-0.04

0.71

0.14

0.15

-0.11

0.27

TOBCFA

-0.13

0.17

-0.04

0.66

-0.17

0.08

-0.17

0.07

-0.10

0.28

-0.03

0.74

-0.13

0.19

-0.17

0.08

C15:0+C17:0

-0.22

0.02

-0.17

0.08

-0.20

0.04

-0.27

0.005

-0.19

0.06

-0.16

0.11

-0.15

0.11

-0.26

0.01

Iso-C15:0+iso-C17:0

0.12

0.20

0.35

0.0002

-0.03

0.73

-0.01

0.91

0.14

0.15

0.36

0.0001

-0.01

0.89

-0.01

0.91

Anteiso-C15:0+anteiso-C17:0

0.12

0.23

0.16

0.10

-0.003

0.98

0.19

0.048

0.11

0.25

0.15

0.11

0.0001

1.00

0.17

0.07

TIFA

0.21

0.03

0.46

<0.0001

0.04

0.71

0.14

0.13

0.20

0.04

0.46

<0.0001

0.03

0.74

0.12

0.21

TAFA

0.12

0.23

0.16

0.096

-0.003

0.98

0.19

0.048

0.11

0.25

0.15

0.11

0.00

1.00

0.17

0.07

TOCFA

-0.21

0.03

-0.16

0.09

-0.20

0.04

-0.26

0.01

-0.18

0.07

-0.15

0.13

-0.15

0.11

-0.25

0.01

TBCFA

0.20

0.04

0.39

<0.0001

0.02

0.84

0.21

0.03

0.19

0.047

0.38

<0.0001

0.02

0.84

0.19

0.054

C15:0/C17:0

-0.21

0.03

-0.15

0.12

-0.28

0.003

-0.20

0.04

-0.23

0.02

-0.16

0.09

-0.29

0.003

-0.21

Iso-C15:0/iso-C17:0

-0.27

0.01

-0.13

0.17

-0.49

<0.0001

-0.33

0.001

-0.31

0.001

-0.14

0.14

-0.53

Anteiso-C15:0/anteiso-C17:0

-0.02

0.87

-0.11

0.27

-0.05

0.60

0.09

0.38

-0.02

0.83

-0.12

0.23

-0.06

C11:0

<0.0001 -0.35 0.56

0.07

0.03 0.0003 0.45

r, correlation coefficient; TIFA, the total contents of iso-fatty acids; TAFA, the total contents of anteiso-fatty acids; TOCFA, the total contents of odd-chain fatty acids; TBCFA,

the total contents of branched-chain fatty acids.

Table 6. Equations to prediction duodenal microbial nitrogen flow from rumen OBCFA Dependent variable

Prediction equations

RMSE

R2

Adj R2

P

Duodenal microbial bases (g/kg DM) Cytosine

Y= C1

+ 1 11 × C11 ×

Y=− Uracil

− 1 ×

‐ C1 +1

1 × C11

‐ C1

−

× C1

‐ C1

+

×

+1

0.37

0.35

<0.0001

0.19

0.61

0.58

<0.0001

0.26

0.49

0.47

<0.0001

0.31

0.34

0.33

<0.0001

0.39

0.34

0.32

<0.0001

0.52

0.35

0.33

<0.0001

0.67

0.29

0.28

<0.0001

0.69

0.31

0.29

<0.0001

×

+11 × ‐ C1

0.18

+

×

‐ C1 Y=1 ‐ C1

Guanine

−1

× C1

−1

×

−

× C1

+ 11 ×

× C11

−

×

−

×

‐ C1 Adenine

Y= C1

+ +

×

‐ C1

Duodenal microbial bases/N (g/100 g N) Cytosine/N

Uracil/N

Guanine/N

Adenine/N

Y=

+ 1

× C11

C1

+ 1 ×

‐ C1

Y=−

+

‐ C1 Y=

× C11

+1 −

+1

×

×

‐ C1

× C1

+1 1 ×

‐ C1 Y= C1

+ +

× C11 ×

−

‐ C1

R2, coefficient of determination; Adj R2, adjusted R2.

×

Table 7. Correlation between rumen OBCFA contents and duodenal VFA, NH3-N contents and pH Acetate r C11:0

P

Propionate r

P

Butyrate r

P

r

r

P

pH P

0.18 -0.08 0.42

-0.06

0.56

0.07 -0.07 0.47 -0.13 0.18

0.11

0.27

0.13

P

NH3-N r

0.0004 1.00 0.001 0.99 -0.01 0.95 0.18

TVFA

C13:0

-0.19

0.055 0.19

0.06

Iso-C15:0

0.06

0.56

-0.05

0.63 -0.09 0.36

0.08

0.42 -0.05 0.61

0.18

0.07

Anteiso-C15:0

0.06

0.53

-0.06

0.52 -0.06 0.56

0.09

0.36 -0.19 0.052

-0.12

0.20

C15:0

0.24

0.01

-0.23

0.01 -0.24 0.01

0.19 0.054 -0.20 0.03

0.12

0.21

Iso-C16:0

-0.10

0.28

0.10

0.29

0.11

0.28 -0.04 0.70 -0.13 0.18

0.01

0.88

Iso-C17:0

-0.08

0.42

0.09

0.36

0.04

0.72

0.02

0.82 -0.14 0.14

0.08

0.41

Anteiso-C17:0

-0.18

0.06

0.17

0.07

0.19

0.04

0.04

0.67 -0.04 0.65

-0.03

0.78

C17:0

-0.13

0.19

0.13

0.19

0.12

0.22 -0.05 0.64 -0.07 0.47

-0.13

0.18

TOBCFA

0.19

0.051 -0.18

0.06 -0.19 0.048 0.17

0.07 -0.23 0.02

0.09

0.33

C15:0+C17:0

0.22

0.02

-0.22

0.02 -0.22 0.02

0.18

0.06 -0.21 0.03

0.11

0.26

Iso-C15:0+iso-C17:0

0.004

0.97

0.01

0.93 -0.05 0.63

0.07

0.50 -0.10 0.29

0.17

0.09

Anteiso-C15:0+anteiso-C17:0

0.03

0.73

-0.03

0.72 -0.03 0.79

0.10

0.32 -0.20 0.04

-0.13

0.18

TIFA

-0.05

0.63

0.06

0.57

0.87

0.03

0.76 -0.14 0.15

0.13

0.19

TAFA

0.03

0.73

-0.03

0.72 -0.03 0.79

0.10

0.32 -0.20 0.04

-0.13

0.18

TOCFA

0.22

0.02

-0.22

0.02 -0.22 0.02

0.18

0.06 -0.21 0.03

0.11

0.26

TBCFA

-0.01

0.93

0.01

0.90 -0.01 0.94

0.08

0.41 -0.21 0.03

-0.004

0.97

C15:0/C17:0

0.30

0.002 -0.29 0.002 -0.32 0.001 0.21

0.03 -0.23 0.02

0.19

0.04

Iso-C15:0/iso-C17:0

0.07

0.46

-0.07

0.47 -0.07 0.45 -0.02 0.83

0.14

0.08

0.39

Anteiso-C15:0/anteiso-C17:0

0.09

0.35

-0.08

0.38 -0.10 0.28 -0.01 0.90 -0.06 0.54

-0.11

0.25

0.02

0.14

r, correlation coefficient; TVFA, the total contents of acetate, propionate and butyrate in duodenum; TIFA, the total

contents of iso-fatty acids; TAFA, the total contents of anteiso-fatty acids; TOCFA, the total contents of odd-chain fatty acids; TBCFA, the total contents of branched-chain fatty acids.

Table 8. Equations to predict duodenal metabolism parameters from rumen odd and branched-chain fatty acids Dependent variable

Prediction equations

Acetate (mmol/mol)

Y=

Propionate (mmol/mol)

Y=

Butyrate (mmol/mol)

Y=

+

NH3-N (mg/dl)

Y=

−

− +

1 × C1

+

1 × C1 × C1 × C1

R2, coefficient of determination; Adj R2, adjusted R2.

− −

RMSE

R2

Adj R2

P

× C1

6.85

0.14

0.12

0.0003

× C1

5.43

0.14

0.12

0.0004

1.52

0.13

0.12

0.001

1.68

0.04

0.03

0.03

× C1