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Dietary supplementation of Rosmarinus officinalis L. leaves in sheep affects the abundance of rumen methanogens and other microbial populations

Dietary supplementation of Rosmarinus officinalis L. leaves in sheep affects the abundance of... Background: Rumen microbiome has a great influence on ruminant health and productivity. Different plant extracts have been tested for their ability to modulate the rumen microbiome to improve feed digestion and fermentation. Among the evaluated plant extracts, essential oils, tannins, and saponins appeared to have positive effects on rumen protein metabolism, volatile fatty acids production, and methane and ammonia production. Methods: The objective of this study was to evaluate the effect of rosemary (Rosmarinus officinalis L.) leaves and essential oils on rumen microbial populations. Four ruminally cannulated sheep were used in a 4×4 Latin square design fed (21 d/period): 1) a control diet composed of alfalfa hay and concentrate pellet (CTR), 2) CTR supplemented with 7 g/d/sheep of rosemary essential oil adsorbed on an inert support (EO), 3) CTR with 10 g/d/sheep of dried and ground rosemary leaves (RL), and 4) CTR with 10 g/d of dried and ground rosemary leaves pelleted into concentrate (RL pellet). Abundance of total bacteria, archaea, protozoa, and some select bacterial species or groups was quantified using qPCR, while the community of bacteria and archaea was profiled using denaturing gradient gel electrophoresis. Results: No difference in abundance was noted for total bacteria, protozoa, or Ruminococcus flavefaciens between the control and the treatments, but the rosemary leaves, either in loose form or in pellet, decreased the abundance of archaea and the genus Prevotella (P < 0.001). The rosemary leaves in loose form also decreased (P <0.001) the abundance of Ruminococcus albus and Clostridium aminophilum, while the EO increased (P < 0.001) the abundance of Fibrobacter succinogenes. The community of bacteria and archaea was not affected by any of the supplements. Conclusions: Being able to affect the abundance of several groups of rumen microbes that are known to be involved in degradation of protein and fiber and production of methane and ammonia, rosemary leaves may be used to modulate rumen microbiome and its function. Keywords: Archaea, Essential oil, Plant extracts, Rosemary, Rumen microbiome Background outputs also represent a loss of dietary energy and nitro- The ruminant livestock sector contributes significantly gen, which are otherwise redirected to animal produc- to global emission of greenhouse gas (GHG) as methane tion. Both methane emission and nitrogen excretion and nitrous oxide, the latter of which is produced from result from feed fermentation by rumen microbiome. the nitrogen (as urea and ammonia) excreted by rumin- Several compounds or substances have been tested as ant animals [1, 2]. Both the methane and nitrogen dietary supplements for their ability to modulate the composition and metabolic activities of rumen micro- biome and to mitigate methane emission and nitrogen * Correspondence: cobellis.gabriella@gmail.com excretion from ruminant animals [1, 2]. Among them, Department of Veterinary Medicine, University of Perugia, via S. Costanzo 4, plant extracts, such as essential oils, tannins, and sapo- 06126 Perugia, Italy Department of Animal Sciences, The Ohio State University, 2029 Fyffe Road, nins, seem to have positive effects on rumen protein Columbus, OH 43210, USA © 2016 Cobellis 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. Cobellis et al. Journal of Animal Science and Biotechnology (2016) 7:27 Page 2 of 8 metabolism, volatile fatty acids (VFA) production, me- hay and 400 g/d of concentrate pellet (CTR), 2) CTR thane and ammonia production [3–5]. However, the di- supplemented with 7 g/d of rosemary essential oil (EO) lemma is that they often exert adverse effects on feed adsorbed onto inert material consisting of calcium car- intake, digestion, and rumen fermentation when added bonate and calcium and potassium aluminosilicate, 3) at concentrations high enough to achieve substantial or CTR with 10 g/d of dried and ground rosemary leaves desirable reduction in methane production, while they (RL), and 4) CTR with 10 g/d of dried and ground rose- have little effect when added at permissive concentra- mary leaves pelleted into concentrate (RL pellet). The tions that do not reduce animal productivity [2]. New diet composition was reported in Table 1. Feed was of- dietary intervention strategies are being sought after, in- fered twice daily in equal meals (8:00 and 16:00 h), and cluding using combinations of different inhibitors that each treatment lasted for 21 d. Different forms of rose- have additive inhibition to methane production by mary supplementations (dry ground leaves, pelleted rumen microbiome [6, 7]. leaves, and essential oil extract) were evaluated because Rosemary (Rosmarinus officinalis L.) is an evergreen they differed in composition of secondary metabolites. perennial shrub belonging to the Lamiaceae family and Based on the rosemary EO content determined in a pre- rosemary leaves are commonly used as a food seasoning. vious study [8], the amount of each form of rosemary The secondary metabolites in rosemary are well known, supplementation fed to each sheep was calculated to give with major compounds including monoterpenoids, such a dose of EO (0.05 g/kg of dry matter). To facilitate the as α-pinene, β-pinene, camphene, 1–8 cineole, camphor, introduction and mixing of the supplement in both the borneol, bornyl acetate and verbenone, and phenolic di- EO and RL diets, all feed ingredients were subjected to a terpenes, such as carnosol, carnosic acid, rosmanol, epir- rough grinding. Rumen content was sampled from each osmanol, isorosmanol, methyl carnosate, and rosmarinic sheep before morning feeding from 3 different sites of acid [8]. Some of these compounds have antimicrobial the rumen after 21 days of adaptation on each diet and and antioxidant activities. Several studies have evaluated frozen immediately at −80 °C until analyses. rosemary essential oil as feed additive using in vitro rumen fermentation [9–13], but these studies focused Metagenomic DNA extraction and quantitative real-time on the effect on feed digestion, not methane production PCR analyses or ammonia production. In a previous study using sheep The rumen samples were processed as described by [8], the authors found that rosemary leaves pelleted into Mosoni et al. [14]. Briefly, the frozen rumen content concentrate (RL pellet) contained less phenols, but more samples were thawed at 4 °C overnight. For each rumen flavonoids, rosmarinic acid, and total antioxidant activity sample, 5 g of solid phase and 5 g of liquid phases were than the same rosemary leaves that were not pelleted combined with 10 mL of sterile distilled water and ho- (RL) and rosemary essential oil. Carnosic acid was de- mogenized for 10 min using a Stomacher (PBI Inter- tected in the RL pellet and the RL diets, but not rose- national, Milan, Italy). The homogenate was centrifuged mary essential oil. Rumen pH, VFA, and lactic acid at 6,500 × g at 4 °C for 30 min and the supernatant was concentrations of the sheep were not affected by RL, RL discarded. Metagenomic DNA extraction was performed pellet, or rosemary essential oil, but CP and DM degrad- using 0.25 g of the pellet obtained after centrifugation ability and ammonia concentration (a tendency) were according the method of Yu and Morrison [15]. DNA decreased by RL pellet [8]. The objective of this study quality was evaluated using agarose gel (1 %) electro- was to evaluate the two forms of rosemary leaves (RL phoresis and DNA yield was quantified using NanoDrop and RL pellet) and rosemary essential oil for their effect 2000 (Thermo Scientific, Wilmington, USA). The DNA on select rumen bacterial populations and methanogens. samples were stored at −20 °C until analysis. All quantitative PCR (qPCR) analyses were performed Methods on a Stratagene Mx3000p system (Stratagene Corpor- Animals, diets, experimental design, and sampling ation, La Jolla, USA). Total bacterial population was The animals, diet, and feeding have been described quantified using a TaqMan assay, while the abundance previously [8]. Briefly, four Bergamasca x Appenninica of archaea, protozoa, and select bacterial species were ruminally cannulated sheep (6 years old, with a mean quantified using SYBR Green-based specific qPCR body weight of 60.5 ± 3.4 kg) were used in a replicated assays. The primers and some of the PCR conditions 4×4 Latin square design. The experimental procedures used are shown in Table 2. One sample-derived qPCR and animal care conditions were approved by the standard was prepared for each target bacterial group Bioethics Committee of the University of Perugia and using the respective specific PCR primer pair and a com- authorized by the Italian Ministry of Health. The ani- posite metagenomics DNA sample that was prepared by mals were kept in four individual pens, with each pen pooling an equal amount of all DNA samples as fed one of the four treatment diets: 1) 1.5 kg/d of alfalfa described previously [16]. After purification using a PCR Cobellis et al. Journal of Animal Science and Biotechnology (2016) 7:27 Page 3 of 8 Table 1 Ingredients (% as fed basis) and chemical composition eliminate the effect from potential primer dimers [16]. (g/100 g) of the concentrates used in the experimental diets (by Each qPCR assay was performed in triplicate for all sam- Cobellis et al. [8]) ples and the respective qPCR standards using the same Item Diet master mix and the same qPCR plate. The absolute Ingredients CTR RL pellet RL EO abundance of each bacterial group was expressed as log of rrs gene copies/g of rumen content. Wheat bran 40.00 30.00 39.00 39.30 10 Wheat flour middlings 17.80 24.30 17.35 17.49 Corn grain 10.00 10.00 9.75 9.82 PCR-DGGE analysis Sunflower meal 14.90 14.90 14.53 14.64 Denaturing gradient gel electrophoresis (DGGE) was used to evaluate the overall response of bacterial and ar- Soybean meal 5.00 6.00 4.87 4.91 chaeal communities to rosemary supplements as de- Calcium carbonate 4.20 4.20 4.09 4.13 scribed previously [17, 18]. Briefly, the V3 hypervariable Dehydrated alfalfa meal 3.50 3.50 3.41 3.44 region of the 16S rRNA gene of bacteria and archaea Beet protein concentrate 2.00 2.00 1.95 1.96 was amplified using bacterium- and archaeon-specific Sugar cane molasses 2.00 2.00 1.95 1.96 primers, with a 40-bp GC clamp added to the 5′ end of Vitamin-mineral supplement 0.60 0.60 0.60 0.60 the forward primer (Table 2). The PCR and DGGE con- ditions and the gel image analysis were essentially the Rosemary leaves - 2.50 2.50 - same as described previously [19]. Rosemary essential oil - - - 1.75 Chemical composition Analysed Statistical analysis All data were analysed as a 4 × 4 Latin square using the Dry matter 92.88 92.78 92.84 92.96 ANOVA procedure of SAS [20]. The statistical model in- Crude protein 18.40 18.44 18.09 18.08 cluded sheep, period, dietary treatment, and residual Crude fat 3.08 3.21 3.12 3.05 error. Fixed effects included period and dietary treat- Ash 9.84 9.98 9.79 11.24 ment. Sheep was the random effect. The abundances of NDF 29.65 29.52 29.79 29.13 rumen microbial populations (rrs gene copy number/g ADF 10.65 11.17 10.99 10.47 of rumen content) were first log-transformed prior to statistical analysis to improve normality. Overall differ- Lignin sa 3.14 2.99 3.38 3.08 ences between the means were evaluated using a Tukey Ca 0.80 0.70 0.79 0.79 test. Data were reported as least squares means ± stand- P 0.75 0.68 0.75 0.74 ard error. Differences were considered to be significant Na 0.28 0.27 0.27 0.28 when P ≤ 0.05. Based on the intensity and migration of Calculated the DGGE bands, a principal component analysis (PCA) Lys 0.70 0.71 0.68 0.69 was performed using the PC-ORD program as described by Patra and Yu [21] to analyze DGGE results. Met 0.29 0.29 0.28 0.28 Met + Cys 0.58 0.58 0.56 0.57 Choline 0.14 0.14 0.14 0.14 Results Supplied per kilogram of diet: Vitamin A, 18,000 I.U. (retinol); Vitamin D , 3 Abundance of rumen microbial populations 2,100 I.U.; Vitamin E, 21 mg (α-tocopheryl acetate); Fe, 29 mg; Co, 0.75 mg; Mn, The results of the qPCR are shown in Table 3. Overall, 39 mg; Zn, 150 mg; Se, 0.06 mg. CTR: control; RL pellet: CTR plus 10 g/d of the abundance of total bacteria, protozoa, and Rumino- dried and ground rosemary leaves pelleted into concentrate; RL: CTR plus 10 g/d of dried and ground rosemary leaves; EO: CTR plus 7 g/d of rosemary coccus flavefaciens was not affected by any of the rose- essential oil adsorbed on an inert support mary supplements. The abundance of archaea and Prevotella spp. was, however, significantly decreased (P Purification kit (Qiagen, Valencia, USA) and quantifica- < 0.001) by the two diets containing rosemary leaves (RL tion using a Quant-iT dsDNA broad-range assay kit or RL pellet). The RL diet also decreased (P < 0.001) the (Invitrogen, Carlsbad, USA), rrs gene copy number con- abundance of Ruminococcus albus and Clostridium ami- centration of each qPCR standard was calculated from nophilum. The rosemary EO did not affect any of the its length and the mass concentration. Tenfold serial di- quantified rumen microbial populations except for lutions of each purified standard were prepared in Tris- Fibrobacter succinogenes, which was increased (P < EDTA buffer prior to qPCR assays. In the SYBR-based 0.001) compared to the control. The abundance of total qPCR assays, one 86 °C for 30 s step was added to each archaea was decreased by both RL and RL pellet but not cycle and fluorescence signal was acquired at 86 °C to by the rosemary essential oil. Cobellis et al. Journal of Animal Science and Biotechnology (2016) 7:27 Page 4 of 8 Table 2 Primers used to quantify ruminal microbes (qPCR) and to profile bacterial and archaeal communities (DGGE) Organisms Primers Sequences (5′→ 3′) Annealing temperature, Amplicon length, References °C bp Real-time PCR Total bacteria 27f AGA GTT TGA TCM TGG CTC AG 55 1535 [41] 1525r AAG GAG GTG WTC CAR CC Total bacteria Eub358f TCC TAC GGG AGG CAG CAG T 60 448 [42] Eub806r GGA CTA CCA GGG TAT CTA ATC CTG TT TaqMan 6-FAM-5′-CGT ATT ACC GCG GCT GCT GGC 70 probe AC-3′-TAMRA Archaea ARC787f ATT AGA TAC CCS BGT AGT CC 60 272 [16] ARC1059r GCC ATG CAC CWC CTC T Protozoa 316f GCT TTC GWT GGT AGT GTA TT 54 223 [43] 539r CTT GCC CTC YAA TCG TWC T Fibrobacter succinogenes Fs219f GGT ATG GGA TGA GCT TGC 63 446 [44] Fs654r GCC TGC CCC TGA ACT ATC Ruminococcus flavefaciens Rf154f TCT GGA AAC GGA TGG TA 55 295 [44] Rf425r CCT TTA AGA CAG GAG TTT ACA A Ruminococcus albus Ra1281f CCC TAA AAG CAG TCT TAG TTC G 55 175 [44] Ra1439r CCT CCT TGC GGT TAG AAC A Prevotella spp. BAC303f GAA GGT CCC CCA CAT TG 56 418 [45] BAC708r CAA TCG GAG TTC TTC GTG Clostridium aminophilum C.amin-57 F ACG GAA ATT ACA GAA GGA AG 57 560 [46] C.amin-616R GTT TCC AAA GCA ATT CCA C PCR-DGGE Total bacteria GC-A357f CCC TAC GGG GCG CAG CAG 61→ 56 °C 194 [17] 519r GWA TTA CCG CGG CKG CTG Archaea GC-RC344f ACG GGG YGC AGC AGG CGC GA 61→ 56 °C 191 [18] 519r GWA TTA CCG CGG CKG CTG FAM: 6-carboxyfluorescein; TAMRA: 6-carboxytetramethylrhodamine Table 3 Effects of different rosemary supplements on select rumen microbial groups (log rrs copies/g) Diet SEM P-value CTR RL pellet RL EO Total Bacteria 11.03 11.01 10.89 11.01 0.06 0.1994 a b b a Archaea 8.86 8.69 8.64 8.80 0.05 <0.001 ab b b a Protozoa 8.26 7.71 7.97 8.75 0.15 <0.001 a b b a Prevotella spp. 9.92 9.68 9.72 9.90 0.12 <0.01 b ab b a Fibrobacter succinogenes 6.86 6.90 6.88 7.00 0.11 <0.001 a a b a Ruminococcus albus 7.62 7.67 7.27 7.64 0.16 <0.001 ab a a ab Ruminococcus flavefaciens 7.40 7.59 7.59 7.43 0.16 <0.05 a a b a Clostridium aminophilum 7.05 7.16 6.62 7.11 0.37 <0.001 a,b Means with different letters within a row differ significantly (P ≤ 0.05) CTR control; RL pellet: CTR plus 10 g/d of dried and ground rosemary leaves pelleted into concentrate; RL: CTR plus 10 g/d of dried and ground rosemary leaves; EO: CTR plus 7 g/d of rosemary essential oil adsorbed on an inert support; NS not significantly Cobellis et al. Journal of Animal Science and Biotechnology (2016) 7:27 Page 5 of 8 Community profiles of bacteria and archaea Discussion The DGGE profile of bacteria showed a large number In recent years, extracts from a variety of plants have of bands and a complex pattern (Fig. 1a). Differences been evaluated for their ability to modulate rumen in intensity of some bands were noted, but little dif- microbiome, feed digestion, and rumen fermentation. ference in banding patterns was visible between the Some plant compounds have been revealed to affect the control and the treatments, suggesting minimal effects abundance and/or the activity of rumen archaea, of the rosemary supplements on the ruminal bacterial protozoa, and specific bacteria populations [5, 21–24]. community of the sheep. The first three principal Rosemary contains a number of antimicrobial monoter- components (PC’s) together explained 71.61 % of the penoids and phenolic diterpenes and rosemary supple- total variation (Fig. 1b and c). No clear separation of ments can potentially modify rumen functions. Their bacterial community profiles between the control and biological activity can be variable but several studies any of the treatments along the first principal compo- documented their specific activity on growth and energy nent (PC1) axis that explained more than 50 % of the metabolism of microbial cells [5]. total variation. No separation of bacterial community A large number of in vitro and in vivo studies have profiles was seen along the second principal compo- been performed to test the ability of plant extracts to nent (PC2) axis or the third principal component modulate rumen microbiome, to our knowledge; (PC3) axis, which explained 12.46 and 6.75 % of the however, this is the first in vivo study to evaluate the total variation, respectively. effects of rosemary supplementations in different forms The DGGE profiles of archaea showed a small on rumen bacteria and archaea using cultivation- number of bands, and no difference in number or in- independent molecular biology techniques. In a previous tensity of bands was noticeable between the control study, we showed that rosemary leaves and essential oil and any of the treatments (Fig. 2a). The PC1, PC2, decreased CP and DM digestibility and tended to lower and PC3 together explained 78.26 % of the total vari- rumen ammonia concentration in sheep [8]. In the ation, and no separation of the archaeal community present study, the abundance of Prevotella and protozoa profiles was evident among the four different diet (though only numerically) was lowered by rosemary groups (Fig. 2b and c). leaves, ether in pellet (RL pellet) or in loose form (RL), Fig. 1 DGGE profiles (a) and PCA plots (b and c) of bacteria. S1, S2, S3, and S4: sheep 1, 2, 3, and 4, respectively. CTR (●): control; RL pellet (■): CTR plus 10 g/d of dried and ground rosemary leaves pelleted into the concentrate; RL (♦): CTR plus 10 g/d of dried and ground rosemary leaves; EO (▲): CTR plus 7 g/d of rosemary essential oil adsorbed on an inert support Cobellis et al. Journal of Animal Science and Biotechnology (2016) 7:27 Page 6 of 8 Fig. 2 DGGE profiles (a) and PCA plots of archaea (b and c). S1, S2, S3, and S4: sheep 1, 2, 3, and 4, respectively. CTR (●): control; RL pellet (■): CTR plus 10 g/d of dried and ground rosemary leaves pelleted into the concentrate; RL (♦): CTR plus 10 g/d of dried and ground rosemary leaves; EO (▲): CTR plus 7 g/d of rosemary essential oil adsorbed on an inert support and RL also decreased the population of C. aminophi- acid and carnosol, while rosmarinic acid has no anti- lum (Table 3). Because members of Prevotella and microbial activity against selected bacteria. However, a protozoa are the mainly proteolytic microbes and C. few studies showed that both rosmarinic and carnosic aminophilum is one the three well documented hyper- acids have antioxidant and antimicrobial activities [31, 32], ammonia-producing bacterial species [25], the decreased and interestingly, rosemary extracts with similar rosmari- CP digestibility observed in the previous study [8] might nic acid content but different ratios of two phenolic diter- be attributed to the decrease of these microbial groups. penes, carnosic acid and carnosol, differed in antibacterial By the same token, the lower DM degradation could be activities. These observations suggest chemical interac- related to the reduction of the abundance of Prevotella tions among different secondary metabolites and such and Ruminococcus albus (Table 3). interaction may affect the antimicrobial potency of rose- The abundance of the analyzed microbial groups was mary extracts. In addition, some of the monoterpenoids in affected by any of the rosemary leaves, but essential oil rosemary essential oil have a fairly broad range of anti- at the tested dose increased the abundance of F. succino- microbial activity [31, 33–35]. Some of these compounds genes (Table 3). The differential effects between rose- are chemically instable and/or high volatile [36]. The lack mary leaves and essential oil could be related to of effect of the rosemary essential oil observed in the differences in the chemical composition of their anti- present study might result from loss of some instable or microbial compounds, with phenolic diterpenes, such as volatile antimicrobial compounds. carnosic acid and rosmarinic acid, being rich in rose- The rosemary leave supplementation, either in loose mary leaves, while rosemary essential oil is rich in form or in pellet, decreased the abundance of archaea. monoterpenoids [8]. According to a number of studies However, the magnitude of the decrease in archaeal [26–28], rumen microbes can adapt to essential oils, abundance is relatively small. Ohene-Adjei et al. [37] especially at low levels. One mechanism is reduction of suggested that a reduction in the abundance of meth- active components of essential oils to inert alcohols by anogen could be observed after prolonged inhibition of some microbes [29]. Indeed, Bernardes et al. [30] methane synthesis. Indeed, lack of decrease in archaeal showed that the antimicrobial activity of extracts from abundance by anti-methane inhibitors has been observed rosemary leaves could be ascribed mainly to carnosic in short-term in vitro incubation [21, 22, 38]. In Cobellis et al. Journal of Animal Science and Biotechnology (2016) 7:27 Page 7 of 8 addition, although not reaching statistical significance, Authors’ contributions GC, GA and CF performed the animal study and collected the rumen the rosemary leaves also only lowered the abundance of samples. MTM, GA and ZY were responsible for experimental design. GC rumen protozoa, potentially decreasing protozoa- performed all laboratory analysis and ZY supervised all laboratory work. associated methanogens and their contribution to me- MTM, CF and GC performed statistical analysis. GC and ZY drafted and revised the manuscript. All authors read and approved the final thane production. Furthermore, the ability of rosemary manuscript for publication. leaves to decrease the abundance of R. albus, a hydrogen-producing bacterial species, points toward a Acknowledgements potential to lower production of hydrogen, the electron This research was sponsored by the IZSUM 004/09 RC project funded by the donor of methanogenesis. Therefore, rosemary leaves, Italian Ministry of Health (MinSal). G. Cobellis’s tenure at The Ohio State University was supported by a grant from the University of Perugia (PhD as suggested by some authors for other plant extracts research project in Animal Health, Livestock Production and Food Safety, [3, 37–39], may directly inhibit methanogenic archaea XXVIII cycle). This work was partially supported by the National Institute of and inhibit some microbial metabolic processes con- Food and Agriculture, U.S. Department of Agriculture, under award number 2012-67015-19437. The authors would like to thank G. Alunni for help in tributing to methane production. laboratory analyses and G. Ponti, A. Mazzoccanti and G. Covarelli for As revealed by DGGE analyses, no significant effect on assistance and care of the animals. Conagit S.p.A. and APA-CT srl are the overall bacterial or archaeal communities was noted gratefully acknowledged for providing technical support and advice in formulating experimental feeds. from any of the rosemary supplements at the tested doses (Figs. 1 and 2). The lack of apparent broad effect Received: 23 October 2015 Accepted: 17 April 2016 on bacterial or archaeal communities is consistent with the similar total bacterial abundance in the control and the rosemary treatments. DGGE can only detect some References predominant members of microbial communities, and 1. Hristov AN, Oh J, Firkins JL, Dijkstra J, Kebreab E, Waghorn G, et al. 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Free Radical Res. 2006;40:223–31. and we will help you at every step: 33. Smith-Palmer A, Stewart J, Fyfe L. Antimicrobial properties of plant essential • We accept pre-submission inquiries oils and essences against five important food-borne pathogens. Lett Appl Microbiol. 1998;26:118–22. � Our selector tool helps you to find the most relevant journal 34. Elgayyar M, Draughon FA, Golden DA, Mount JR. Antimicrobial activity of � We provide round the clock customer support essential oils from plants against selected pathogenic and saprophytic � Convenient online submission microorganisms. J Food Prot. 2001;64:1019–24. 35. Santoyo S, Cavero S, Jaime L, Ibanez E, Senorans FJ, Reglero G. Chemical � Thorough peer review composition and antimicrobial activity of Rosmarinus officinalis L. essential � Inclusion in PubMed and all major indexing services oil obtained via supercritical fluid extraction. J Food Prot. 2005;68:790–5. � Maximum visibility for your research 36. Turek C, Stintzing FC. Stability of essential oils: a review. Compr Rev Food Sci Food Saf. 2013;12:40–53. Submit your manuscript at www.biomedcentral.com/submit http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Journal of Animal Science and Biotechnology Springer Journals

Dietary supplementation of Rosmarinus officinalis L. leaves in sheep affects the abundance of rumen methanogens and other microbial populations

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Springer Journals
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Copyright © 2016 by Cobellis et al.
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Life Sciences; Agriculture; Biotechnology; Food Science; Animal Genetics and Genomics; Animal Physiology
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2049-1891
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10.1186/s40104-016-0086-8
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27123239
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Abstract

Background: Rumen microbiome has a great influence on ruminant health and productivity. Different plant extracts have been tested for their ability to modulate the rumen microbiome to improve feed digestion and fermentation. Among the evaluated plant extracts, essential oils, tannins, and saponins appeared to have positive effects on rumen protein metabolism, volatile fatty acids production, and methane and ammonia production. Methods: The objective of this study was to evaluate the effect of rosemary (Rosmarinus officinalis L.) leaves and essential oils on rumen microbial populations. Four ruminally cannulated sheep were used in a 4×4 Latin square design fed (21 d/period): 1) a control diet composed of alfalfa hay and concentrate pellet (CTR), 2) CTR supplemented with 7 g/d/sheep of rosemary essential oil adsorbed on an inert support (EO), 3) CTR with 10 g/d/sheep of dried and ground rosemary leaves (RL), and 4) CTR with 10 g/d of dried and ground rosemary leaves pelleted into concentrate (RL pellet). Abundance of total bacteria, archaea, protozoa, and some select bacterial species or groups was quantified using qPCR, while the community of bacteria and archaea was profiled using denaturing gradient gel electrophoresis. Results: No difference in abundance was noted for total bacteria, protozoa, or Ruminococcus flavefaciens between the control and the treatments, but the rosemary leaves, either in loose form or in pellet, decreased the abundance of archaea and the genus Prevotella (P < 0.001). The rosemary leaves in loose form also decreased (P <0.001) the abundance of Ruminococcus albus and Clostridium aminophilum, while the EO increased (P < 0.001) the abundance of Fibrobacter succinogenes. The community of bacteria and archaea was not affected by any of the supplements. Conclusions: Being able to affect the abundance of several groups of rumen microbes that are known to be involved in degradation of protein and fiber and production of methane and ammonia, rosemary leaves may be used to modulate rumen microbiome and its function. Keywords: Archaea, Essential oil, Plant extracts, Rosemary, Rumen microbiome Background outputs also represent a loss of dietary energy and nitro- The ruminant livestock sector contributes significantly gen, which are otherwise redirected to animal produc- to global emission of greenhouse gas (GHG) as methane tion. Both methane emission and nitrogen excretion and nitrous oxide, the latter of which is produced from result from feed fermentation by rumen microbiome. the nitrogen (as urea and ammonia) excreted by rumin- Several compounds or substances have been tested as ant animals [1, 2]. Both the methane and nitrogen dietary supplements for their ability to modulate the composition and metabolic activities of rumen micro- biome and to mitigate methane emission and nitrogen * Correspondence: cobellis.gabriella@gmail.com excretion from ruminant animals [1, 2]. Among them, Department of Veterinary Medicine, University of Perugia, via S. Costanzo 4, plant extracts, such as essential oils, tannins, and sapo- 06126 Perugia, Italy Department of Animal Sciences, The Ohio State University, 2029 Fyffe Road, nins, seem to have positive effects on rumen protein Columbus, OH 43210, USA © 2016 Cobellis 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. Cobellis et al. Journal of Animal Science and Biotechnology (2016) 7:27 Page 2 of 8 metabolism, volatile fatty acids (VFA) production, me- hay and 400 g/d of concentrate pellet (CTR), 2) CTR thane and ammonia production [3–5]. However, the di- supplemented with 7 g/d of rosemary essential oil (EO) lemma is that they often exert adverse effects on feed adsorbed onto inert material consisting of calcium car- intake, digestion, and rumen fermentation when added bonate and calcium and potassium aluminosilicate, 3) at concentrations high enough to achieve substantial or CTR with 10 g/d of dried and ground rosemary leaves desirable reduction in methane production, while they (RL), and 4) CTR with 10 g/d of dried and ground rose- have little effect when added at permissive concentra- mary leaves pelleted into concentrate (RL pellet). The tions that do not reduce animal productivity [2]. New diet composition was reported in Table 1. Feed was of- dietary intervention strategies are being sought after, in- fered twice daily in equal meals (8:00 and 16:00 h), and cluding using combinations of different inhibitors that each treatment lasted for 21 d. Different forms of rose- have additive inhibition to methane production by mary supplementations (dry ground leaves, pelleted rumen microbiome [6, 7]. leaves, and essential oil extract) were evaluated because Rosemary (Rosmarinus officinalis L.) is an evergreen they differed in composition of secondary metabolites. perennial shrub belonging to the Lamiaceae family and Based on the rosemary EO content determined in a pre- rosemary leaves are commonly used as a food seasoning. vious study [8], the amount of each form of rosemary The secondary metabolites in rosemary are well known, supplementation fed to each sheep was calculated to give with major compounds including monoterpenoids, such a dose of EO (0.05 g/kg of dry matter). To facilitate the as α-pinene, β-pinene, camphene, 1–8 cineole, camphor, introduction and mixing of the supplement in both the borneol, bornyl acetate and verbenone, and phenolic di- EO and RL diets, all feed ingredients were subjected to a terpenes, such as carnosol, carnosic acid, rosmanol, epir- rough grinding. Rumen content was sampled from each osmanol, isorosmanol, methyl carnosate, and rosmarinic sheep before morning feeding from 3 different sites of acid [8]. Some of these compounds have antimicrobial the rumen after 21 days of adaptation on each diet and and antioxidant activities. Several studies have evaluated frozen immediately at −80 °C until analyses. rosemary essential oil as feed additive using in vitro rumen fermentation [9–13], but these studies focused Metagenomic DNA extraction and quantitative real-time on the effect on feed digestion, not methane production PCR analyses or ammonia production. In a previous study using sheep The rumen samples were processed as described by [8], the authors found that rosemary leaves pelleted into Mosoni et al. [14]. Briefly, the frozen rumen content concentrate (RL pellet) contained less phenols, but more samples were thawed at 4 °C overnight. For each rumen flavonoids, rosmarinic acid, and total antioxidant activity sample, 5 g of solid phase and 5 g of liquid phases were than the same rosemary leaves that were not pelleted combined with 10 mL of sterile distilled water and ho- (RL) and rosemary essential oil. Carnosic acid was de- mogenized for 10 min using a Stomacher (PBI Inter- tected in the RL pellet and the RL diets, but not rose- national, Milan, Italy). The homogenate was centrifuged mary essential oil. Rumen pH, VFA, and lactic acid at 6,500 × g at 4 °C for 30 min and the supernatant was concentrations of the sheep were not affected by RL, RL discarded. Metagenomic DNA extraction was performed pellet, or rosemary essential oil, but CP and DM degrad- using 0.25 g of the pellet obtained after centrifugation ability and ammonia concentration (a tendency) were according the method of Yu and Morrison [15]. DNA decreased by RL pellet [8]. The objective of this study quality was evaluated using agarose gel (1 %) electro- was to evaluate the two forms of rosemary leaves (RL phoresis and DNA yield was quantified using NanoDrop and RL pellet) and rosemary essential oil for their effect 2000 (Thermo Scientific, Wilmington, USA). The DNA on select rumen bacterial populations and methanogens. samples were stored at −20 °C until analysis. All quantitative PCR (qPCR) analyses were performed Methods on a Stratagene Mx3000p system (Stratagene Corpor- Animals, diets, experimental design, and sampling ation, La Jolla, USA). Total bacterial population was The animals, diet, and feeding have been described quantified using a TaqMan assay, while the abundance previously [8]. Briefly, four Bergamasca x Appenninica of archaea, protozoa, and select bacterial species were ruminally cannulated sheep (6 years old, with a mean quantified using SYBR Green-based specific qPCR body weight of 60.5 ± 3.4 kg) were used in a replicated assays. The primers and some of the PCR conditions 4×4 Latin square design. The experimental procedures used are shown in Table 2. One sample-derived qPCR and animal care conditions were approved by the standard was prepared for each target bacterial group Bioethics Committee of the University of Perugia and using the respective specific PCR primer pair and a com- authorized by the Italian Ministry of Health. The ani- posite metagenomics DNA sample that was prepared by mals were kept in four individual pens, with each pen pooling an equal amount of all DNA samples as fed one of the four treatment diets: 1) 1.5 kg/d of alfalfa described previously [16]. After purification using a PCR Cobellis et al. Journal of Animal Science and Biotechnology (2016) 7:27 Page 3 of 8 Table 1 Ingredients (% as fed basis) and chemical composition eliminate the effect from potential primer dimers [16]. (g/100 g) of the concentrates used in the experimental diets (by Each qPCR assay was performed in triplicate for all sam- Cobellis et al. [8]) ples and the respective qPCR standards using the same Item Diet master mix and the same qPCR plate. The absolute Ingredients CTR RL pellet RL EO abundance of each bacterial group was expressed as log of rrs gene copies/g of rumen content. Wheat bran 40.00 30.00 39.00 39.30 10 Wheat flour middlings 17.80 24.30 17.35 17.49 Corn grain 10.00 10.00 9.75 9.82 PCR-DGGE analysis Sunflower meal 14.90 14.90 14.53 14.64 Denaturing gradient gel electrophoresis (DGGE) was used to evaluate the overall response of bacterial and ar- Soybean meal 5.00 6.00 4.87 4.91 chaeal communities to rosemary supplements as de- Calcium carbonate 4.20 4.20 4.09 4.13 scribed previously [17, 18]. Briefly, the V3 hypervariable Dehydrated alfalfa meal 3.50 3.50 3.41 3.44 region of the 16S rRNA gene of bacteria and archaea Beet protein concentrate 2.00 2.00 1.95 1.96 was amplified using bacterium- and archaeon-specific Sugar cane molasses 2.00 2.00 1.95 1.96 primers, with a 40-bp GC clamp added to the 5′ end of Vitamin-mineral supplement 0.60 0.60 0.60 0.60 the forward primer (Table 2). The PCR and DGGE con- ditions and the gel image analysis were essentially the Rosemary leaves - 2.50 2.50 - same as described previously [19]. Rosemary essential oil - - - 1.75 Chemical composition Analysed Statistical analysis All data were analysed as a 4 × 4 Latin square using the Dry matter 92.88 92.78 92.84 92.96 ANOVA procedure of SAS [20]. The statistical model in- Crude protein 18.40 18.44 18.09 18.08 cluded sheep, period, dietary treatment, and residual Crude fat 3.08 3.21 3.12 3.05 error. Fixed effects included period and dietary treat- Ash 9.84 9.98 9.79 11.24 ment. Sheep was the random effect. The abundances of NDF 29.65 29.52 29.79 29.13 rumen microbial populations (rrs gene copy number/g ADF 10.65 11.17 10.99 10.47 of rumen content) were first log-transformed prior to statistical analysis to improve normality. Overall differ- Lignin sa 3.14 2.99 3.38 3.08 ences between the means were evaluated using a Tukey Ca 0.80 0.70 0.79 0.79 test. Data were reported as least squares means ± stand- P 0.75 0.68 0.75 0.74 ard error. Differences were considered to be significant Na 0.28 0.27 0.27 0.28 when P ≤ 0.05. Based on the intensity and migration of Calculated the DGGE bands, a principal component analysis (PCA) Lys 0.70 0.71 0.68 0.69 was performed using the PC-ORD program as described by Patra and Yu [21] to analyze DGGE results. Met 0.29 0.29 0.28 0.28 Met + Cys 0.58 0.58 0.56 0.57 Choline 0.14 0.14 0.14 0.14 Results Supplied per kilogram of diet: Vitamin A, 18,000 I.U. (retinol); Vitamin D , 3 Abundance of rumen microbial populations 2,100 I.U.; Vitamin E, 21 mg (α-tocopheryl acetate); Fe, 29 mg; Co, 0.75 mg; Mn, The results of the qPCR are shown in Table 3. Overall, 39 mg; Zn, 150 mg; Se, 0.06 mg. CTR: control; RL pellet: CTR plus 10 g/d of the abundance of total bacteria, protozoa, and Rumino- dried and ground rosemary leaves pelleted into concentrate; RL: CTR plus 10 g/d of dried and ground rosemary leaves; EO: CTR plus 7 g/d of rosemary coccus flavefaciens was not affected by any of the rose- essential oil adsorbed on an inert support mary supplements. The abundance of archaea and Prevotella spp. was, however, significantly decreased (P Purification kit (Qiagen, Valencia, USA) and quantifica- < 0.001) by the two diets containing rosemary leaves (RL tion using a Quant-iT dsDNA broad-range assay kit or RL pellet). The RL diet also decreased (P < 0.001) the (Invitrogen, Carlsbad, USA), rrs gene copy number con- abundance of Ruminococcus albus and Clostridium ami- centration of each qPCR standard was calculated from nophilum. The rosemary EO did not affect any of the its length and the mass concentration. Tenfold serial di- quantified rumen microbial populations except for lutions of each purified standard were prepared in Tris- Fibrobacter succinogenes, which was increased (P < EDTA buffer prior to qPCR assays. In the SYBR-based 0.001) compared to the control. The abundance of total qPCR assays, one 86 °C for 30 s step was added to each archaea was decreased by both RL and RL pellet but not cycle and fluorescence signal was acquired at 86 °C to by the rosemary essential oil. Cobellis et al. Journal of Animal Science and Biotechnology (2016) 7:27 Page 4 of 8 Table 2 Primers used to quantify ruminal microbes (qPCR) and to profile bacterial and archaeal communities (DGGE) Organisms Primers Sequences (5′→ 3′) Annealing temperature, Amplicon length, References °C bp Real-time PCR Total bacteria 27f AGA GTT TGA TCM TGG CTC AG 55 1535 [41] 1525r AAG GAG GTG WTC CAR CC Total bacteria Eub358f TCC TAC GGG AGG CAG CAG T 60 448 [42] Eub806r GGA CTA CCA GGG TAT CTA ATC CTG TT TaqMan 6-FAM-5′-CGT ATT ACC GCG GCT GCT GGC 70 probe AC-3′-TAMRA Archaea ARC787f ATT AGA TAC CCS BGT AGT CC 60 272 [16] ARC1059r GCC ATG CAC CWC CTC T Protozoa 316f GCT TTC GWT GGT AGT GTA TT 54 223 [43] 539r CTT GCC CTC YAA TCG TWC T Fibrobacter succinogenes Fs219f GGT ATG GGA TGA GCT TGC 63 446 [44] Fs654r GCC TGC CCC TGA ACT ATC Ruminococcus flavefaciens Rf154f TCT GGA AAC GGA TGG TA 55 295 [44] Rf425r CCT TTA AGA CAG GAG TTT ACA A Ruminococcus albus Ra1281f CCC TAA AAG CAG TCT TAG TTC G 55 175 [44] Ra1439r CCT CCT TGC GGT TAG AAC A Prevotella spp. BAC303f GAA GGT CCC CCA CAT TG 56 418 [45] BAC708r CAA TCG GAG TTC TTC GTG Clostridium aminophilum C.amin-57 F ACG GAA ATT ACA GAA GGA AG 57 560 [46] C.amin-616R GTT TCC AAA GCA ATT CCA C PCR-DGGE Total bacteria GC-A357f CCC TAC GGG GCG CAG CAG 61→ 56 °C 194 [17] 519r GWA TTA CCG CGG CKG CTG Archaea GC-RC344f ACG GGG YGC AGC AGG CGC GA 61→ 56 °C 191 [18] 519r GWA TTA CCG CGG CKG CTG FAM: 6-carboxyfluorescein; TAMRA: 6-carboxytetramethylrhodamine Table 3 Effects of different rosemary supplements on select rumen microbial groups (log rrs copies/g) Diet SEM P-value CTR RL pellet RL EO Total Bacteria 11.03 11.01 10.89 11.01 0.06 0.1994 a b b a Archaea 8.86 8.69 8.64 8.80 0.05 <0.001 ab b b a Protozoa 8.26 7.71 7.97 8.75 0.15 <0.001 a b b a Prevotella spp. 9.92 9.68 9.72 9.90 0.12 <0.01 b ab b a Fibrobacter succinogenes 6.86 6.90 6.88 7.00 0.11 <0.001 a a b a Ruminococcus albus 7.62 7.67 7.27 7.64 0.16 <0.001 ab a a ab Ruminococcus flavefaciens 7.40 7.59 7.59 7.43 0.16 <0.05 a a b a Clostridium aminophilum 7.05 7.16 6.62 7.11 0.37 <0.001 a,b Means with different letters within a row differ significantly (P ≤ 0.05) CTR control; RL pellet: CTR plus 10 g/d of dried and ground rosemary leaves pelleted into concentrate; RL: CTR plus 10 g/d of dried and ground rosemary leaves; EO: CTR plus 7 g/d of rosemary essential oil adsorbed on an inert support; NS not significantly Cobellis et al. Journal of Animal Science and Biotechnology (2016) 7:27 Page 5 of 8 Community profiles of bacteria and archaea Discussion The DGGE profile of bacteria showed a large number In recent years, extracts from a variety of plants have of bands and a complex pattern (Fig. 1a). Differences been evaluated for their ability to modulate rumen in intensity of some bands were noted, but little dif- microbiome, feed digestion, and rumen fermentation. ference in banding patterns was visible between the Some plant compounds have been revealed to affect the control and the treatments, suggesting minimal effects abundance and/or the activity of rumen archaea, of the rosemary supplements on the ruminal bacterial protozoa, and specific bacteria populations [5, 21–24]. community of the sheep. The first three principal Rosemary contains a number of antimicrobial monoter- components (PC’s) together explained 71.61 % of the penoids and phenolic diterpenes and rosemary supple- total variation (Fig. 1b and c). No clear separation of ments can potentially modify rumen functions. Their bacterial community profiles between the control and biological activity can be variable but several studies any of the treatments along the first principal compo- documented their specific activity on growth and energy nent (PC1) axis that explained more than 50 % of the metabolism of microbial cells [5]. total variation. No separation of bacterial community A large number of in vitro and in vivo studies have profiles was seen along the second principal compo- been performed to test the ability of plant extracts to nent (PC2) axis or the third principal component modulate rumen microbiome, to our knowledge; (PC3) axis, which explained 12.46 and 6.75 % of the however, this is the first in vivo study to evaluate the total variation, respectively. effects of rosemary supplementations in different forms The DGGE profiles of archaea showed a small on rumen bacteria and archaea using cultivation- number of bands, and no difference in number or in- independent molecular biology techniques. In a previous tensity of bands was noticeable between the control study, we showed that rosemary leaves and essential oil and any of the treatments (Fig. 2a). The PC1, PC2, decreased CP and DM digestibility and tended to lower and PC3 together explained 78.26 % of the total vari- rumen ammonia concentration in sheep [8]. In the ation, and no separation of the archaeal community present study, the abundance of Prevotella and protozoa profiles was evident among the four different diet (though only numerically) was lowered by rosemary groups (Fig. 2b and c). leaves, ether in pellet (RL pellet) or in loose form (RL), Fig. 1 DGGE profiles (a) and PCA plots (b and c) of bacteria. S1, S2, S3, and S4: sheep 1, 2, 3, and 4, respectively. CTR (●): control; RL pellet (■): CTR plus 10 g/d of dried and ground rosemary leaves pelleted into the concentrate; RL (♦): CTR plus 10 g/d of dried and ground rosemary leaves; EO (▲): CTR plus 7 g/d of rosemary essential oil adsorbed on an inert support Cobellis et al. Journal of Animal Science and Biotechnology (2016) 7:27 Page 6 of 8 Fig. 2 DGGE profiles (a) and PCA plots of archaea (b and c). S1, S2, S3, and S4: sheep 1, 2, 3, and 4, respectively. CTR (●): control; RL pellet (■): CTR plus 10 g/d of dried and ground rosemary leaves pelleted into the concentrate; RL (♦): CTR plus 10 g/d of dried and ground rosemary leaves; EO (▲): CTR plus 7 g/d of rosemary essential oil adsorbed on an inert support and RL also decreased the population of C. aminophi- acid and carnosol, while rosmarinic acid has no anti- lum (Table 3). Because members of Prevotella and microbial activity against selected bacteria. However, a protozoa are the mainly proteolytic microbes and C. few studies showed that both rosmarinic and carnosic aminophilum is one the three well documented hyper- acids have antioxidant and antimicrobial activities [31, 32], ammonia-producing bacterial species [25], the decreased and interestingly, rosemary extracts with similar rosmari- CP digestibility observed in the previous study [8] might nic acid content but different ratios of two phenolic diter- be attributed to the decrease of these microbial groups. penes, carnosic acid and carnosol, differed in antibacterial By the same token, the lower DM degradation could be activities. These observations suggest chemical interac- related to the reduction of the abundance of Prevotella tions among different secondary metabolites and such and Ruminococcus albus (Table 3). interaction may affect the antimicrobial potency of rose- The abundance of the analyzed microbial groups was mary extracts. In addition, some of the monoterpenoids in affected by any of the rosemary leaves, but essential oil rosemary essential oil have a fairly broad range of anti- at the tested dose increased the abundance of F. succino- microbial activity [31, 33–35]. Some of these compounds genes (Table 3). The differential effects between rose- are chemically instable and/or high volatile [36]. The lack mary leaves and essential oil could be related to of effect of the rosemary essential oil observed in the differences in the chemical composition of their anti- present study might result from loss of some instable or microbial compounds, with phenolic diterpenes, such as volatile antimicrobial compounds. carnosic acid and rosmarinic acid, being rich in rose- The rosemary leave supplementation, either in loose mary leaves, while rosemary essential oil is rich in form or in pellet, decreased the abundance of archaea. monoterpenoids [8]. According to a number of studies However, the magnitude of the decrease in archaeal [26–28], rumen microbes can adapt to essential oils, abundance is relatively small. Ohene-Adjei et al. [37] especially at low levels. One mechanism is reduction of suggested that a reduction in the abundance of meth- active components of essential oils to inert alcohols by anogen could be observed after prolonged inhibition of some microbes [29]. Indeed, Bernardes et al. [30] methane synthesis. Indeed, lack of decrease in archaeal showed that the antimicrobial activity of extracts from abundance by anti-methane inhibitors has been observed rosemary leaves could be ascribed mainly to carnosic in short-term in vitro incubation [21, 22, 38]. In Cobellis et al. Journal of Animal Science and Biotechnology (2016) 7:27 Page 7 of 8 addition, although not reaching statistical significance, Authors’ contributions GC, GA and CF performed the animal study and collected the rumen the rosemary leaves also only lowered the abundance of samples. MTM, GA and ZY were responsible for experimental design. GC rumen protozoa, potentially decreasing protozoa- performed all laboratory analysis and ZY supervised all laboratory work. associated methanogens and their contribution to me- MTM, CF and GC performed statistical analysis. GC and ZY drafted and revised the manuscript. All authors read and approved the final thane production. Furthermore, the ability of rosemary manuscript for publication. leaves to decrease the abundance of R. albus, a hydrogen-producing bacterial species, points toward a Acknowledgements potential to lower production of hydrogen, the electron This research was sponsored by the IZSUM 004/09 RC project funded by the donor of methanogenesis. Therefore, rosemary leaves, Italian Ministry of Health (MinSal). G. Cobellis’s tenure at The Ohio State University was supported by a grant from the University of Perugia (PhD as suggested by some authors for other plant extracts research project in Animal Health, Livestock Production and Food Safety, [3, 37–39], may directly inhibit methanogenic archaea XXVIII cycle). This work was partially supported by the National Institute of and inhibit some microbial metabolic processes con- Food and Agriculture, U.S. Department of Agriculture, under award number 2012-67015-19437. The authors would like to thank G. Alunni for help in tributing to methane production. laboratory analyses and G. Ponti, A. Mazzoccanti and G. Covarelli for As revealed by DGGE analyses, no significant effect on assistance and care of the animals. Conagit S.p.A. and APA-CT srl are the overall bacterial or archaeal communities was noted gratefully acknowledged for providing technical support and advice in formulating experimental feeds. from any of the rosemary supplements at the tested doses (Figs. 1 and 2). The lack of apparent broad effect Received: 23 October 2015 Accepted: 17 April 2016 on bacterial or archaeal communities is consistent with the similar total bacterial abundance in the control and the rosemary treatments. DGGE can only detect some References predominant members of microbial communities, and 1. Hristov AN, Oh J, Firkins JL, Dijkstra J, Kebreab E, Waghorn G, et al. 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Published: Apr 27, 2016

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