Rumen microbial population dynamics in ... - Wiley Online Library

Letters in Applied Microbiology 2005, 41, 97–101

doi:10.1111/j.1472-765X.2005.01707.x

Rumen microbial population dynamics in response to photoperiod N.R. McEwan1, L. Abecia1,2, M. Regensbogenova1,3, C.L. Adam4, P.A. Findlay4 and C.J. Newbold1,5 1

Gut Microbiology and Immunology Division, Rowett Research Institute, Aberdeen, UK, 2Departamento Produccioń Animal y Ciencia de los Alimentos, University of Zaragoza, Zaragoza, Spain, 3Institute of Animal Physiology, Kosice, Slovakia, 4Energy Balance and Obesity Division, Rowett Research Institute, Aberdeen, UK, and 5Institute of Rural Sciences, University of Wales, Aberystwyth, Wales, UK

2004/0701: received 18 June 2004, revised 14 October 2004 and accepted 19 October 2004

ABSTRACT N . R . M C E W A N , L . A B E C I A , M . R E G E N S B O G E N O V A , C . L . A D A M , P . A . F I N D L A Y A N D C . J . N E W B O L D . 2005.

Aims: This work was carried out to determine if there was a difference in the microbial population of the rumen associated with daylength at which sheep are housed. Methods and Results: Denaturing gradient gel electrophoresis (DGGE) was used to study the ciliate and bacterial diversity in the rumen of Soay rams kept in long day (16 h light) or short day (8 h light) photoperiods. Bacterial diversity varied according to the daylength conditions where the host animal was housed, as did total volatile fatty acids (VFA) concentrations. No differences associated with daylength were detected in ciliate diversity, branched VFA concentrations or the ruminal ammonia concentrations. Conclusions: As diets had identical composition, yet voluntary intakes levels were higher during long days, it is proposed that the differences in bacterial populations arise because of the differences in amount of food consumed. Significance and Impact of the Study: The outcome of this study demonstrated that factors beyond dietary composition must be taken into account when trying to study microbial populations, even in what can be considered a fairly constant environment. Keywords: daylength, DGGE, rumen microbes, soay sheep, VFA.

INTRODUCTION Soay sheep are a primitive domestic breed introduced to Soay Island in the St Kilda archipelago of western Scotland about 4000 years ago (O’Brien 2000). They are renowned for being hardy, and so able to be maintained with minimal husbandry, consequently in many cases these animals can exist as a semi-feral population. This minimal husbandry resulted in many of the traits found in the progenitor animals of modern domestic sheep being retained. For example, although all temperate breeds of sheep are seasonal and respond to changes in photoperiod to a greater or lesser extent, Soays remain particularly photosensitive, as they have been under no human-induced productivity-driven selection against this trait. Correspondence to: Dr Neil R. McEwan, Rowett Research Institute, Aberdeen AB21 9SB, UK (e-mail: [email protected]).

ª 2005 The Society for Applied Microbiology

The persistence of a high photosensitivity in a domestic animal is of particular interest in nutritional studies as daylength changes have been shown to have an influence on appetite levels. Notably, voluntary food intake of seasonal ruminants (sheep, red deer) increases in long days (spring– summer) and decreases in short days (autumn–winter) even when the food supply is constant and unlimited (Kay 1979). The animals feed preferentially during daylight hours so that they ingest food faster and over a longer period in long days than in short days (Rhind et al. 2002). Various studies have suggested that rumen capacity and metabolic rate are greater in summer long days than in winter, but changes in retention time are equivocal and rumen motility apparently does not alter with season (reviewed in Rhind et al. 2002). Changes to the diet have previously been shown to influence the microbial composition of the rumen by the use of molecular ecological techniques such as single strand

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conformation polymorphisms (SSCP) (McEwan et al. 2002) or denaturing gradient gel electrophoresis (DGGE) (Kocherginskaya et al. 2001; Regensbogenova et al. 2004). These changes were detected following: dietary supplementation with essential oils; use of a growth-promoting antibiotic; and variation in the protein content of the diet. In all cases it was previously anticipated that these dietary variations might lead to a change in the microbial population. However, it is unknown if altering the amount of food an animal is consuming, assuming the animal is maintained on at least a maintenance level diet, is likely to affect the composition of the major microbial population in the rumen. All the above criteria meant that an inbred, highly photosensitive line of sheep such as the Soay breed provides a unique opportunity to assess the effects of daylength associated levels of food intake on the microbial population of the rumen in a way that is free of factors such as dietary composition, seasonal temperature, restrictions on dietary intake and to a lesser than normal extent, genetic variation in the host animal. M A T E R I A LS A N D M E T H O D S A flock of Soay sheep were maintained as a semi-feral population. Rams aged c. 18 months were removed from the flock and housed in individual pens for 12 weeks in either long day photoperiod (16 h constant light and 8 h darkness), or short day photoperiod (8 h constant light and 16 h darkness). Animals in both groups were given unrestricted access to a complete diet (500 kg hay, 299Æ5 kg barley, 100 kg molasses, 91 kg fish meal and 9Æ5 kg vitamin and mineral supplement – all values are per tonne of dry weight matter). All procedures received prior approval from the Institute’s Animal Ethical Review Committee. Sheep were killed by an overdose of sodium pentobarbitone administered intravenously. Rumen fluid and digesta were collected immediately after slaughter and stored at )20C until ready for analysis. Rumen fluid was filtered through a double layer of muslin to remove plant material prior to nucleic acid isolation (Eschenlauer et al. 1998). DNA was isolated from the microbial fraction using a QIAamp DNA Stool Mini Kit (Qiagen Ltd, West Sussex, UK) following the manufacturer’s instructions. Approximately 200 bp of the bacterial 16S rDNA gene was amplified using the primers: forward primer: 5¢-TAC GGG AGG CAG CAG-3¢; reverse primer: 5¢-ATT ACC GCG GCT GCT GG-3¢ (Kay 1979), with a GC-clamp at the 5¢ terminus of the forward primer: 5¢-CGC CCG CCG CGC GCG GCG GGC GGG GCG GGG GCA CGG GGG G-3¢. Approximately 200 bp of rumen ciliate 18S rDNA gene was amplified using the primers: forward primer: 5¢-GGT GGT GCA TGG CCG-3¢ and reverse

primer: 5¢-AAT TGC AAA GAT CTA TCC C-3¢ (Rhind et al. 2002), with a GC-clamp at the 5¢ terminus of the reverse primer: 5¢-CGC CCG CCG CGC CCC GCG CCC GGC CCG CCG CCC CCG CCC GGG GCC-3¢ (Muyzer et al. 1998). For the ciliate studies, PCR was performed using the following steps: 1 cycle (94C for 4 min, 60C for 30 s, 72C for 1 min); 35 cycles (94C for 1 min, 60C for 30 s, 72C for 1 min); 1 cycle (94C for 1 min, 60C for 30 s, 72C for 10 min). For the bacterial studies, PCR was performed using the following steps: 1 cycle (94C for 4 min, 60C for 1 min, 72C for 1 min); 30 cycles (94C for 1 min, 60C for 1 min, 72C for 1 min); 1 cycle (94C for 1 min, 60C for 1 min, 72C for 5 min). Amplicons were analysed by electrophoresis on a 2Æ5% agarose gel and visualized after staining with ethidium bromide. DGGE was performed on the DCodeTM Universal Mutation Detection system (16 cm system; Bio-Rad, Hemel Hempstead, UK). Samples from seven sheep from each of the conditions were used for DGGE analysis. DGGE parallel gradient gels ranged from 20 to 35% (6% acrylamide), run at 130 mV, 200 mA, for 6 h at 60C. DNA was visualized by staining with SYBR Gold nucleic acid gel stain (Molecular Probes, Eugene, OR, USA). DGGE gels were scored for the presence of absence of bands at different migration distances within the gels. Minkowski metrics (e.g. Euclidean distances) between profiles is a popular method of analysis (Dıéz et al. 2001) and this method was applied using binary scoring within an Excel spreadsheet (McEwan 2004). As the data used here were scored as binary scores (i.e. a band which is present scored 1, and absence of a band at this mobility position in another lane scored 0), the distances measured were the Hamming Distances between different profiles. Rumen digesta samples were analysed for VFA content by gas chromatography as previously described (Newbold et al. 1995). Ruminal concentrations of ammonia were determined using an automated form of the phenol-hypochlorite method (Whitehead et al. 1967). Differences between the two treatments were assessed using F-test supported two-tailed T-tests. RESULTS Sheep exposed to 16 h light per day consumed an average of 1277 g dry matter (DM) per day (SEM ¼ 71Æ8 g), as opposed to 729 g DM (SEM ¼ 36Æ3 g) per day for the sheep exposed to only 8 h light per day, during the final week before being killed. Their average body weights at slaughter were 43Æ0 kg (SEM ¼ 1Æ14 kg) and 38Æ8 kg (SEM ¼ 1Æ37 kg), respectively, and their weights when first brought in from the field before the experiment were 32Æ0 kg (SEM ¼ 0Æ86 kg) and 32Æ2 kg (SEM ¼ 1Æ08 kg) respectively.

ª 2005 The Society for Applied Microbiology, Letters in Applied Microbiology, 41, 97–101, doi:10.1111/j.1472-765X.2005.01707.x

PHOTOPERIOD AND RUMEN MICROBES

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Long day 05 Long day 07 Long day 03 Long day 01 Long day 02 Long day 04 Short day 03 Short day 01 Short day 07 Short day 02 Short day 05 Short day 04 1·0 Short day 06

Fig. 1 DGGE profiles of the major bacterial population present in the rumen of sheep fed on the same diet but having different lengths of daylength. (S, short day – 8 h light per day; L, long day – 16 h light per day)

Amplification of bacterial samples gave the profile seen in Fig. 1. It is clear that there was considerable variation between lanes. By performing a pair-wise Hamming distance analysis on the lanes present it was shown that there was a split between the animals on days of 8 h light and animals on 16 h light, with the exception of a single animal which was on the short light period clustering with the samples from animals on long daylengths (Fig. 2). Amplification of ciliate samples gave a profile where there appeared to be minimal diversity (Fig. 3). By performing a pair-wise Hamming distance analysis on the lanes present on the ciliate gel, no difference between animals on days of 8 h light and animals on 16 h light was observed (Fig. 4). From Fig. 2 it can be seen that there was a change in the bacterial population of the rumen that was associated with the daylength of the host animal, but that this was not observed in the ciliate population. The data in Table 1 demonstrate that effects on the rumen composition were not restricted to bacterial diversity, with the non-branched and total VFA concentrations being different in the two populations, although no differences were detected in the

Fig. 2 Relationship between the DGGE banding patterns detected in samples from sheep on different lengths of daylength using bacterial primers. ‘Short day 01’ refers to sheep 1 from the short day group, etc.

branched VFA concentrations or the abundance of ammonia. DISCUSSION Previous molecular studies on shifts in rumen populations tended to focus on changes in the composition of the diet (Kocherginskaya et al. 2001; Tajima et al. 2001; Regensbogenova et al. 2004). However, this work used a diet which was kept constant in terms of its composition, and the variation was restricted to the level of voluntary intake of the host animal. It was already known that Soay rams housed under opposite photoperiods differ profoundly in physiological and endocrine status (Lincoln and Richardson 1998). For example in short days they are reproductively active, with high circulating concentrations of testosterone, and concentrations of prolactin are low, whereas in long days they are reproductively inactive, with low testosterone, and prolactin concentrations are high. In addition, as demonstrated by the present rams, voluntary food intake increased in long days and decreased in short days. It has also been suggested that


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S Long day 01 Short day 02 Long day 03 Long day 02 Long day 07 Long day 06 Short day 07 Short day 06 Short day 05 Short day 03 1·0 Short day 04 Long day 04 Long day 05

Fig. 3 DGGE profiles of the major ciliate population present in the rumen of sheep fed on the same diet but having different lengths of daylength. (S, short day – 8 h light per day; L, long day – 16 h light per day)

Fig. 4 Relationship between the DGGE banding patterns detected in samples from sheep on different lengths of daylength using ciliate primers

Table 1 Chemical composition of rumen samples

rumen capacity and metabolic rate are greater in summer long days than in winter (reviewed Rhind et al. 2002). Here we describe a clear change in ruminal properties associated with the differences in daylength where the host animal was housed. There has been a change in the distribution of major bacterial species present, the concentration of the unbranched VFAs, and the total VFA concentrations. However, there was no change in either the ammonia concentration, or the concentration of branched VFAs (both of which are only present in the rumen as a result of protein breakdown), and the ciliate population. It seems likely that the changes in VFAs were a consequence of the change in the bacterial population. The cause of the dichotomy between the bacterial populations was less clear. A number of hypotheses may be proposed which could explain these results, although they can generally be classified into one of three criteria. i As there are hormonal changes associated with photoperiodism (Lincoln and Richardson 1998), it is possible that at least one of these altered components in the blood is able to transverse the rumen wall, influence the rumen environment and have an effect on the bacterial composition.

Mean values Mean values for sheep on for sheep on 8 h daylength 16 h daylength SED Caprioic acid (mmol l)1) 0Æ333 1Æ072 Valeric acid (mmol l)1) iso-Butyric acid (mmol l)1) 2Æ004 2Æ250 iso-Valeric acid (mmol l)1) Propionic acid (mmol l)1) 15Æ7 11Æ5 Butyric acid (mmol l)1) 55Æ9 Acetic acid (mmol l)1) All VFA (mmol l)1) 88Æ6 151 Ammonia (mg N l)1)

0Æ635 1Æ534 2Æ035 1Æ843 27Æ2 21Æ4 94Æ8 149Æ4 146

0Æ230*** 0Æ399*** 0Æ513 0Æ693 7Æ7**** 7Æ0**** 25Æ6**** 40Æ2**** 27Æ7

*P < 0Æ05, **P < 0Æ02, ***P < 0Æ01, ****P < 0Æ001.

ii It is possible that there is a rate-limiting dietary component for a particular population of bacteria; this might be exhausted at the lower intake level (in short days) resulting in suppression of numbers of this population, but not in the sheep with the higher intake in long days. iii Alternatively, it is possible that there is a breakdown product from part of the diet, with a relatively slow


PHOTOPERIOD AND RUMEN MICROBES

rumen outflow rate, that is toxic towards a particular population of bacteria. Only at an elevated intake level, such as that in the long daylength animals, does this toxicity exist. Of course the above hypotheses are not mutually exclusive and a combination of causes may explain the observed differences. In conclusion, we report for the first time an effect of photoperiod on the bacterial population composition of the rumen ecosystem, which is independent of dietary composition, and it is proposed that the differences are most likely because of the photoperiod-driven differences in food intake. ACKNOWLEDGEMENTS The Rowett Research Institute receives funding from the Scottish Executive Environmental and Rural Affairs Department. This investigation has been supported by a European Union funded thematic research programme – ERCULE (European rumen ciliate collection QLRI-CT 2000 01455). Martina Regensbogenova and Leticia Abecia were recipients of Training Studentships under the Rowett Research Institute’s Marie Curie Training award (HPMTCT-2001-00409). REFERENCES Dıéz, B., Pedro´s-Alio´, C., Marsh, T.L. and Massana, R. (2001) Application of denaturing gradient gel electrophoresis (DGGE) to study the diversity of marine picoeukaryotic assemblages and comparison of DGGE with other molecular techniques. Appl Environ Microbiol 67, 2942–2951. Eschenlauer, S.C.P., McEwan, N.R., Calza, R.E., Wallace, R.J., Onodera, R. and Newbold, C.J. (1998) Phylogenetic position and codon usage of two centrin genes from the rumen ciliate protozoan, Entodinium caudatum. FEMS Microbiol Lett 166, 147– 154.

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Kay, R.N.B. (1979) Seasonal changes of appetite in deer and sheep. Agric Res Counc Res Rev 5, 13–15. Kocherginskaya, S.A., Aminov, R.I. and White, B.A. (2001) Analysis of the rumen bacterial diversity under two different diet conditions using denaturing gradient gel electrophoresis, random sequencing, and statistical ecology approaches. Anaerobe 7, 119–134. Lincoln, G.A. and Richardson, M. (1998) Photo-neuroendocrine control of seasonal cycles in body weight, pelage growth and reproduction: lessons from the HPD sheep model. Comp Biochem Physiol 119C, 283–294. McEwan, N.R. (2004) Minkowski metrics – an easy approach for the molecular ecologist. Reprod Nutr Dev 44, S9. McEwan, N.R., Graham, R.C., Wallace, R.J., Losa, R., Williams, P. and Newbold, C.J. (2002) Effect of essential oils on ammonia production by rumen microbes. Reprod Nutr Dev 42, S65. Muyzer, G., Brinkhoff, T., Nu¨bel, U., Santegoeds, C., Scha¨fer, H. and Wawer, C. (1998) Denaturing gradient gel electrophoresis (DGGE) in microbial ecology. In Molecular Microbial Ecology Manual, Vol. 3.4.4. ed. Akkermans, A.D.L., van Elsas, J.D. and de Bruijn, F.J. pp. 1–27. Dordrecht, the Netherlands: Kluwer Academic Publishers. Newbold, C.J., Wallace, R.J., Chen, X.B. and McIntosh, F. (1995) Different strains of Saccharomyces cerevisiae differ in their effects on ruminal bacterial numbers in vitro, in sheep. J Anim Sci 73, 1811– 1818. O’Brien, S.J. (2000) Adaptive cycles: parasites selectively reduce inbreeding in Soay sheep. Trends Genet 15, 7–9. Regensbogenova, M., Pristas, P., Javorsky, P., Moon-van der Staay, S.Y., van der Staay, G.W.M., Hackstein, J.H.P., Newbold, C.J. and McEwan, N.R. (2004) Assessment of ciliates in the sheep rumen by DGGE. Lett Appl Microbiol 49, 144–147. Rhind, S.M., Archer, Z.A. and Adam, C.L. (2002) Seasonality of food intake in ruminants: recent developments in understanding. Nutr Res Rev 15, 43–65. Tajima, K., Aminov, R.I., Nagamine, T., Matsui, H., Nakamura, M. and Benno, Y. (2001) Diet-dependent shifts in the bacterial population of the rumen revealed with real-time PCR. Appl Environ Microbiol 67, 2766–2774. Whitehead, R., Cooke, G.H. and Chapman, B.T. (1967) Problems associated with the continuous monitoring of ammoniacal nitrogen in river water. Anal Chem 11, 377–380.
