De Nardi et al. BMC Veterinary Research (2016) 12:29 DOI 10.1186/s12917-016-0653-4 RESEARCHARTICLE Open Access Metagenomic analysis of rumen microbial population in dairy heifers fed a high grain diet supplemented with dicarboxylic acids or polyphenols Roberta De Nardi1, Giorgio Marchesini1, Shucong Li2, Ehsan Khafipour2, Kees J. C. Plaizier2, Matteo Gianesella1, Rebecca Ricci1, Igino Andrighetto1 and Severino Segato1* Abstract Background: The aim of this study was to investigate the effects of two feed supplements on rumen bacterial communities of heifers fed a high grain diet. Six Holstein-Friesian heifers received one of the following dietary treatments according to a Latin square design: no supplement (control, C), 60 g/day of fumarate-malate (organic acid, O) and 100 g/day of polyphenol-essential oil (P). Rumen fluid was analyzed to assess the microbial population using Illumina sequencing and quantitative real time PCR. Results: The P treatment had the highest number of observed species (P < 0.10), Chao1 index (P < 0.05), abundance based coverage estimated (ACE) (P < 0.05), and Fisher’s alpha diversity (P < 0.10). The O treatment had intermediate values between C and P treatments with the exception of the Chao1 index. The PCoA with unweighted Unifrac distance showed a separation among dietary treatments (P = 0.09), above all between the C and P (P = 0.05). The O and P treatments showed a significant increase of the family Christenenellaceae and a decline of Prevotella brevis compared to C. Additionally, the P treatment enhanced the abundance of many taxa belonging to Bacteroidetes, Firmicutes and Tenericutes phyla due to a potential antimicrobial activity of flavonoids that increased competition among bacteria. Conclusions: Organic acid and polyphenols significantly modified rumen bacterial populations during high-grain feeding in dairy heifers. In particular the polyphenol treatment increased the richness and diversity of rumen microbiota, which are usually high in conditions of physiological rumen pH and rumen function. Keywords: Ruminal acidosis, Rumen microbiota, Fumarate-malate, Polyphenol, Heifers Background of excessive amounts of organic acids (volatile organic The rumen is an anaerobic fermentation chamber hous- acid (VFA) and lactate) [2–4]. High concentrations of ing diverse microorganisms such as bacteria, protozoa, VFA and lactate can exceed the buffering capacity of the fungi and viruses. Bacteria play a key role in rumen fer- rumen resulting in major changes in the rumen environ- mentation, which in turn greatly impact production and ment, such as a pH depression, and reduction in the health of dairy cows. [1]. High-grain diets used to im- populations of many beneficial bacteria [1, 5, 6]. Large prove performance of dairy cows alter microbial com- individual variability of the rumen bacterial community, munities in the rumen and the symbiosis between the even in animals feeding on the same ration have been host and these communities by causing the production reported. [7, 8]. Other studies [1, 6, 7] showed that diet- ary changes, like an increased inclusion of cereals in the ration, lead to shifts in the microbial community even * Correspondence: [email protected] 1Department of Animal Medicine, Production and Health, University of though the ruminal microbiota was able to maintain a Padova, Legnaro, PD 35020, Italy stable core of bacterial taxa. The populations of starch- Full list of author information is available at the end of the article © 2016 De Nardi et al. Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. De Nardi et al. BMC Veterinary Research (2016) 12:29 Page 2 of 9 fermenting and lactic acid-utilizing bacteria increase of the acetate-to-propionate ratio due to an increase of when high starch diets are fed [9]. An excessive depres- the numbers of lactate-consuming and propionate- sion in rumen pH causes a reduction in the number of producing bacteria [17]. cellulolytic bacteria [2], major shifts in bacterial popula- A previous study described the effect of organic acid tions, and increases the lysis of Gram-negative bacteria (O) and polyphenols (P) on reticular pH drop and acute resulting in a higher concentration of free lipopolysac- phase response in dairy heifers fed a high grain diet [23]. charide (LPS) endotoxins in rumen digesta [2, 10, 11]. In this manuscript we report the effects of these supple- When the translocation of free LPS from the rumen ments on rumen bacterial populations in dairy heifers and/or lower gut into the interior circulation occurs, an fed a high-grain diet. immune response in the host is induced, causing meta- bolic alterations. Moreover, the increase in proportion of Results bacteria such as Enterobacteriacae during high starch Illumina sequencing produced 317,369 sequences across feeding heightens the presence of virulence factors (fim- treatments. The average numbers of sequences were brial adhesins, heat-stable and heat-labile toxins and in- 16,413, 18,814 and 28,247 for the C, O and P treatment, flammatory peptides), which have the potential to cause respectively. The number of the generated sequences inflammation [2, 10, 12]. Nutritional strategies to pre- was not affected by treatment (P = 0.60). vent the onset of SARA and translocation of LPS in The P treatment led to the highest richness of the dairy cows are based on feeding sufficient amounts of rumen microbial population (Table 1), characterized by physically effective fiber (peNDF) and modulating the the highest number of observed species (P < 0.10) and amount of easily degradable starch in the diet [13]. In the highest value of ACE (P < 0.05). The O treatment addition the use of feed supplements to enhance the resuted in intermediate values between P and C for both rumen microbial community and subsequently ruminal the number of species observed and the index ACE. fermentation has also been suggested for this purpose. The Chao1 index was higher during the P treatment These supplements include the use of yeasts, like Sac- (P < 0.05) compared to the C and O treatments. More- charomyces cerevisiae, strain CNCM I-1077 [14], pro- over, Fisher’s alpha diversity tended (P <0.10) to be biotic bacteria, like Megasphaera elsdenii, strain H6F32 higher during the P treatment compared to the C treat- [15], and dicarboxylic acids [16], flavonoids [17] and es- ment, whereas the O resulted in intermediate values. sential oils [18]. It has been suggested that the dicarbox- The Shannon and Simpson indices were not affected by ylic acids malate and fumarate attenuate the ruminal pH treatments. drop during high grain feeding [16], and that these acids The PERMANOVA analysis showed a significant (P < increase the activity of the succinate-propionate meta- 0.10) effect of treatments on Bray-Curtis and unweighted bolic pathway in several rumen bacteria, resulting in in- Unifrac distances (Table 2). In addition, differences were creased lactic acid uptake and production of propionate observed in bacterial population among periods (P =0.02 [19, 20]. In in vitro rumen fermentative experiments, and P = 0.06, respectively). However the interaction of several essential oils (EO), or blends of EO have been treatment and period (D × P) on these measures was demonstrated to enhance rumen fermentation [18, 21]. not significant pointing out the lack of a carry-over However, few in vivo studies have investigated the effects effects of treatments. The multiple-comparisons based of EO on rumen fermentation and bacterial populations on Bray-Curtis distance showed only a tendency (P = [22]. Furthermore, the effects of addition of polyphenolic 0.11) towards a significant difference between dietary compounds like flavonoids to diets of dairy cows may in- treatments (C vs P), while the multivariate analysis clude prevention of the pH reduction and the decrease based on unweighted Unifrac distance indicated a Table 1 Number of observed species, richness (Chao1 and ACE) and diversity estimators (Shannon, Simpson and Fisher’alfa) in control (C), organic acid (O) and polyphenols (P) dietary treatments Observed species Richness Diversity Chao1 ACE Shannon Simpson1 Fisher’s alpha Control 2,116β 3,420b 3,731b 5.9 0.99 645β Organic acid 2,255α, β 3,834b 4,214a, b 6.1 0.99 691α, β Polyphenols 2,742α 7,164a 6,927a 6.1 0.98 890α SEM 188.7 653.1 711.8 0.22 (0.99–0.98) 78.2 P-value 0.08 0.03 0.06 0.90 0.93 0.07 1Statistical analysis was conducted on natural logarithm (ln) transformed data that are presented as ln back transformed and 95 %-confidence interval in brackets a, bWithin column indicate statistical differences (P < 0.05); α, βWithin column indicate statistical differences (P < 0.10) De Nardi et al. BMC Veterinary Research (2016) 12:29 Page 3 of 9 Table 2 PERMANOVA analysis of the effect of dietary treatments, Illumina sequencing detected 32 classes, 55 orders, periods and heifers on rumen bacteria dissimilarities based on 87 families and 88 genera. The predominant sequences Bray-Curtis and unweighted Unifrac (>1 %) within Bacteroidetes belong to order Bacteroi- Source Bray-curtis Unweighted unifrac dales (22.7 %), genus Prevotella (21.2 %), genus Paludi- pseudo-FP-value pseudo-FP-value bacter (2.24 %), family BS11 (2.04 %), family S24-7 Dietary treatment 1.208 0.10 1.155 0.09 (1.77 %), and genus CF231 (1.06 %).