Livestock Science 164 (2014) 39–45

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Livestock Science

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Using archaeol to investigate the location of in the ruminant digestive tract

C.A. McCartney a,b,n, I.D. Bull b, R.J. Dewhurst a,1 a Teagasc, Animal and Grassland Research and Innovation Centre, Grange, Dunsany, Co. Meath, Ireland b School of Chemistry, University of Bristol, Cantock's Close, Bristol, BS8 1TS, UK article info abstract

Article history: The quantification of archaeol, a , may provide an alternative Received 28 August 2013 method to estimate methanogen abundance. The focus of this study was to determine the Received in revised form location of methanogens in the ruminant digestive tract using this biomarker. Archaeol 13 February 2014 was quantified in samples obtained from four lactating cows with rumen cannulae that Accepted 24 February 2014 grazed on either white clover (WC) or perennial ryegrass (PRG) in a changeover design study with three 3-week periods. Faeces were collected over the final 5 d of each period Keywords: and total rumen contents (TRC) were obtained on the final 2 days (day 1: 9 am; day Archaeol 2: 3 pm). Solid-associated microbes (SAM) and liquid-associated microbes (LAM) were Faeces also isolated from the TRC. Concentrations of archaeol in the TRC showed a significant diet Methanogen by time interaction, which may be related to diurnal grazing patterns and different rumen Rumen conditions associated with PRG or WC diets. There was significantly more archaeol associated with SAM than LAM, which may reflect difficulties of methanogen proliferation in the liquid phase. Faeces had higher concentrations of archaeol than SAM and LAM which was unexpected, although, losses of methanogens may have occurred during isolation (i.e., attachment to protozoa and very small particles), or the methanogens associated with SAM may have been underestimated. There was no significant relation- ship between faecal and TRC archaeol concentrations. Finally, there was a significant positive relationship between rumen pH and concentrations of archaeol in SAM and LAM, which may be caused by pH and/or WC diet effects. In conclusion, archaeol is potentially a useful alternative marker for determining the abundance of methanogens in the ruminant digestive tract. This work has also highlighted the difficulties associated with methanogen quantification from microbial isolates, and the need for more representative rumen sampling in future studies. & 2014 Elsevier B.V. All rights reserved.

1. Introduction

This study investigated an alternative approach to study methanogen abundance involving the use of the lipid biomarker archaeol (2,3-diphytanyl-O-sn-). This is n Corresponding author. Present address: Rowett Institute of Nutrition a core membrane lipid that is ubiquitous in methanogens, and and Health, University of Aberdeen, Greenburn Road, Aberdeen, AB21 is readily quantified by gas mass spectro- þ 9SB, UK. Tel.: 44 1224 438713. metry. Previous studies have already assessed archaeol as a E-mail address: [email protected] (C.A. McCartney). 1 Present address: SRUC, King's Buildings, West Mains Road, potential faecal biomarker for methanogens and methane Edinburgh, EH9 3JG, UK. production (Gill et al., 2011; McCartney et al., 2013a), where http://dx.doi.org/10.1016/j.livsci.2014.02.020 1871-1413 & 2014 Elsevier B.V. All rights reserved. 40 C.A. McCartney et al. / Livestock Science 164 (2014) 39–45 a significant positive relationship was found between total samples were sent to NMR laboratories (National Milk methanogens and methane production when looking at Records plc, Chippenham, UK) for infrared milk analysis of treatment means. Archaeol has also recently been assessed milk fat, protein and lactose. in parallel with quantitative real-time PCR (qPCR) techniques Herbage samples (20 random snips to 6 cm) were and it was found that archaeol was a useful complementary collected daily and bulked weekly for feed composition approach for estimating methanogen abundance (McCartney analysis (described below). Cows were dosed via the et al., 2013c). rumen cannula with plastic pellets of different colours to The purpose of this study was to use archaeol as a total identify their faeces in the field (pellets were removed methanogen proxy to provide insights into the location prior to analysis). Faecal samples were collected daily from and abundance of methanogens in the digestive tract of the field over 5 days at the end of each experimental cows grazing either perennial ryegrass (PRG) or white period (bulked proportional sample of all faeces deposited clover (WC). Archaeol was quantified in samples from over the previous 5 days) and then lyophilized and finely the rumen, including total rumen contents, liquid- ground for analysis. The dried faecal samples were stored associated microbes (LAM) and solid-associated microbes at room temperature. Rumen contents were obtained by (SAM), and in faeces. total rumen evacuation on the final 2 days of each period (day 1: 9 am; day 2: 3 pm). A 5% sub-sample was taken throughout the evacuation procedure (every twentieth 2. Materials and methods bail) and composited for subsequent analysis and the remaining rumen contents were returned immediately 2.1. Animal study on completion of the evacuation procedure. The weight, DM and NDF content of the total rumen contents was This study was conducted under a license issued under determined. Rumen fluid samples were taken using auto- the UK Animal (Scientific Procedures) Act (1986). Four matic samplers every 2 h for the 2 days prior to rumen lactating Holstein–Friesian cows that had previously been evacuation for ammonia and VFA analysis. The pH was prepared with rumen cannulae (Bar Diamond, Parma, ID) recorded (Oakton PC510 pH meter; Eutech Instruments BV, were used. At the start of the experiment the animals had Nijkerk, The Netherlands) twice a day at morning and an average BW of 694 kg (s.d.¼46.4), a BCS of 2.7 evening milking on the final 2 days of the experimental (s.d.¼0.51) and were 104 days in milk (s.d.¼23.6). The period. All chemical analysis of feed and rumen contents BCS was determined according to Mulvany (1977). Animals was performed using methods outlined by Dewhurst et al. were strip-grazed on pure stands of either perennial (2000). Briefly, the DM was determined by toluene dis- ryegrass (Lolium perenne cv. Fennema) or white clover tillation, VFA concentrations were determiend using gas (Trifolium repens cv. AberHerald) that had been re-growing chromatography, crude protein was determined using a for 21–27 days in a changeover design with three 3-week Kjeldahl procedure with Cu catalyst on ‘Kjeltec’ equipment periods. In period 1, two animals grazed WC and 2 animals (Perstorp Analytical Ltd., Berkshire, UK). Detergent fibre grazed PRG. In period 2, two animals switched diet, and in analyses, NDF (with amylase) and ADF were determined period 3 all animals switched diet. As a result two animals using ‘Fibretec’ equipment (Perstorp Analytical Ltd., Berk- received the same diet in periods 1 and 2. Measurements shire, UK). Water-soluble carbohydrate were concentration were taken from all four animals in each period, resulting was determined using an automated anthrone method in six measurement periods per dietary treatment. All (Method 9a; Technicon Industrial Systems, Tarrytown, NY), cows were supplemented with 4 kg/d of pelleted concen- and starch by incubating with buffered amyloglucosidase trates (Table 1) and received 25 g/d of poloxalene (Bloat prior to the automated anthrone method. Rumen ammonia Guard, Agrimin Ltd., Brigg, Lincolnshire, UK). Cows were concentration was determined using a test kit (No. 66-55; milked twice-daily (approx. 08:00 and 16:00 h) with milk Sigma-Aldrich Co. Ltd., Poole, UK) on a discrete analyser. yields recorded electronically (Tru-Test NZ Ltd., Auckland, In vitro DOMD based on incubation with pepsin–cellulase NZ) and samples for analysis of milk components taken on was performed according to Jones and Hayward (1975). four consecutive milkings at the end of each period. Milk At the end of the experiment the animals had an average Table 1 bodyweight of 665 kg (SD¼29.7) and an average BCS of 2.8 Chemical composition of the feeds used in this study. (SD¼0.34).

Component (g/kg DM, Perennial White Concentrates 2.2. Isolating SAM and LAM unless stated otherwise) ryegrass clover

DM (g/kg) 113 94.0 870 SAM and LAM fractions were obtained from the whole OM 899 886 914 rumen contents collected at the 9 am rumen evacuation NDF 486 231 279 following the protocol of Merry and McAllan (1983). ADF 269 211 142 Ether extract 30.6 19.9 54.1 To obtain the LAM fraction, total rumen contents were Crude protein (N 6.25) 216 309 200 hand squeezed through 4 layers of cheesecloth to obtain Starch ––283 1000 mL of liquid before centrifugation for 10 min at low Neutral cellulase-gammanase ––823 speed (500g). The supernatant was then centrifuged at digestibility high speed (25,000g) for 25 min. After discarding the Digestible organic matter 663 749 – Water-soluble carbohydrates 96.1 51.0 89.2 supernatant, the remaining pellet washed in saline (9 g NaCl/L) before another high-speed centrifugation step for C.A. McCartney et al. / Livestock Science 164 (2014) 39–45 41

25 min. The supernatant was discarded and the pellet (REML) analysis of linear mixed models with ‘diet’ ‘time’ washed in distilled water. After one final high-speed as fixed effects and ‘period’þ‘cow’ as random effects. centrifugation step for 15 min, the supernatant was dis- ‘Time’ was defined as either morning (approx. 9 am) or carded and the remaining pellet freeze-dried before sto- afternoon (approx. 3 pm). ‘Diet’, ‘time’ and their interaction rage at 20 1C. To obtain the SAM fraction, 500 g of rumen each used one d.f., there were 2 d.f. for period and 3 d.f. contents that had previously been retained within the for cow, leaving 15 residual d.f. REML was also used to cheese cloth was gently washed with saline (9 g NaCl/L). analyse milk yield and composition data with ‘diet’ as a This was then hand squeezed twice before processing with fixed effect and ‘period’þ‘cow’ as random effects. In this 320 mL of saline in a stomacher (Seward Ltd., West Sussex, case there were 1 d.f. for diet, 2 d.f. for period, 3 d.f. for cow UK) for 5 min. After the contents were centrifuged at low and 5 residual d.f. Comparisons of archaeol concentration in speed, with the resultant supernatant then centrifuged at the different fractions used REML, with sample type as a high speed. After discarding the supernatant, the pellet fixed effect (1 d.f. for sample type and 22 residual d.f.). was washed with saline then centrifuged at high speed for A repeated measured analysis of variance (procedure ARE- 25 min. The pellet was then re-washed with distilled PMEASURES) was applied to rumen ammonia and VFA data water, and centrifuged at high speed for 15 min. After with 12 time points, with ‘diet’ ‘time’ as fixed effects and discarding the supernatant, the remaining pellet was ‘period’þ‘cow’ as random effects. Simple linear regression freeze-dried prior to storage at 20 1C. analysis was also used to explore relationships between archaeol concentrations with rumen pH. All statistical 2.3. Archaeol analysis analyses were carried out using Genstat (14th Edition, VSN International Ltd). A P-value of o0.05 was considered o Quantification of archaeol was achieved according to the significant, and a P-value of 0.1 was considered a trend. protocol of McCartney et al. (2013a).Insummary,around 300 mg of dried, ground faeces and total rumen contents were weighed in triplicate, and around 200 mg of the dried 3. Results LAM or SAM fraction was weighed singly. An internal standard m 1,2-di-O-hexadecyl-rac-glycerol (43.4 g) was added to the The chemical composition of feeds is provided in weighed samples prior to extraction. The total lipid extract Table 1. The WC had a high CP content, low NDF content (TLE) was extracted using a modified monophasic extraction and was highly digestible, whereas the PRG diet also had a procedure, followed by removal of sugar- and phosphate-head relatively high CP content, but had moderate levels of NDF groups by acid methanolysis. The TLE was chromatographi- and was moderately digestible. The average milk yield was cally separated over an activated silica column to isolate a 26.4 kg/d (SD¼4.17), and the average milk fat, protein and fraction containing hydroxylated compounds, which were lactose content of the milk was 3.38% (SD¼0.50), 3.06% analysed as the corresponding trimethylsilyl ethers by (SD¼0.23) and 4.47% (SD¼0.13), respectively. There were GC/MS. Archaeol was identified on the basis of its character- no significant effects of dietary treatments on milk yield istic mass spectrum and retention time, and quantification and composition was achieved by using the internal standard and an external All of the rumen measurements were significantly calibration curve of 1,2-di-O-phytanyl-sn-glycerol normalized affected by the diet consumed, though in the case of faecal against the internal standard. archaeol concentration, this was most evident as a sig- nificant ‘diet’ ‘time’ effect (Table 2). Animals grazing WC 2.4. Statistical analysis had a lower pH, lower total rumen volume and lower NDF content in their rumen contents than animals grazing PRG. One sample of rumen contents was lost prior to Total volume of rumen contents was also affected by ‘time’, analysis, so all data from this cow-period were excluded being higher for the 3 pm measurements compared to the from subsequent analysis, hence there were 6 replicates 9 am measurements. for PRG and 5 replicates for WC. Analysis of variance for There were significant effects of diet on rumen ammonia-N effects on archaeol, rumen contents (kg), DM of rumen and VFA concentrations (Table 3). The higher VFA concen- contents, rumen DM (kg), NDF concentrations and rumen trations reflect the higher rate of fermentation of WC in pH were conducted using residual maximum likelihood comparison with PRG (Dewhurst et al., 2009), whilst the

Table 2 Effects of diet and sampling time on the quantity of rumen digesta, rumen pH and the concentrations of NDF and archaeol in rumen digesta.

Perennial ryegrass White clover SED (Diet Time) Significance (P)

AM PM AM PM Diet Time Diet Time

NDF (g/kg DM) 489 498 402 404 14.8 o0.001 0.550 0.735 Archaeol (mg/kg DM) 4.73 5.17 4.75 4.04 0.3686 0.074 0.738 0.037 pH 6.28 6.07 5.98 5.87 0.152 0.036 0.143 0.643 Rumen contents (kg) 73.6 83.9 59.3 68.3 6.461 0.008 0.044 0.882 DM of rumen contents (g/kg) 120 129 115 112 5.80 0.016 0.427 0.162 Rumen contents (kg DM) 8.81 10.8 6.54 7.38 0.533 o0.001 0.002 0.153 42 C.A. McCartney et al. / Livestock Science 164 (2014) 39–45

Table 3 Effects of diet and sampling time on concentrations of ammonia-N (mg/L) and VFA (mmol/L), as well as molar proportions of VFA (mmol/mol) in rumen fluid.

Perennial ryegrass White Clover SED (Diet Time) Significance (P)

Diet Time Diet Time

Ammonia-N (mg/L) 227 312 44.1 o0.001 0.004 0.558 Total VFA (mmol/L) 107 119 12.3 0.021 0.067 0.630 Acetic acid (mmol/mol) 661 646 14.8 0.020 0.225 0.532 Propionic acid (mmol/mol) 181 190 7.30 0.020 0.034 0.284 iso-Butyric acid (mmol/mol) 11.6 13.5 1.27 0.003 0.190 0.335 n-Butyric acid (mmol/mol) 122 119 6.0 0.079 0.084 0.624 iso-Valeric acid (mmol/mol) 13.7 17.6 1.47 o0.001 0.046 0.366 n-Valeric acid (mmol/mol) 12.0 15.0 1.13 o0.001 0.119 0.623

Table 4 Effects of diet on archaeol concentration (mg/kg DM) of the different sample types analyzed.

Perennial ryegrass White clover SED Significance (P)

Feces 11.6 10.5 1.05 0.351 Total rumen contents 4.97 4.35 0.342 0.126 Liquid-associated microbial fraction 3.93 3.35 0.440 0.236 Solid-associated microbial fraction 7.99 5.15 1.181 0.016

Fig. 1. Relationship between rumen pH and the concentration of archaeol (mg/kg DM) in the liquid- and solid-associated microbial fraction. higher rumen ammonia-N also reflects the higher N content of There was no significant relationship between faecal WC. Sampling time only affected concentrations of ammonia, archaeol and total rumen archaeol concentrations (P¼0.521). propionic acid and iso-valeric acid, and there were no sig- Regression analysis showed a significant relationship nificant ‘diet’ ‘time’ interactions. between average rumen pH and archaeol concentration in The average coefficient of variation for the archaeol the SAM and LAM fractions (Fig. 1). There were no significant triplicate measurements for total rumen contents and faeces relationships between average rumen pH and total archaeol was 7.79% (SD¼0.55). The concentrations of archaeol (mg/kg concentration in the whole-rumen contents (WRC) (P¼0.130) DM) found in total rumen contents, faeces, and SAM and or faecal archaeol concentration (P¼0.687). LAM fractions are presented in Table 4. Faeces contained the highest concentration of archaeol, followed by the SAM 4. Discussion fraction, total rumen contents, and then the LAM fraction. Archaeol concentration in the SAM fraction was significantly 4.1. Rumen measurements higher than in the LAM fraction (LAM¼3.64 mg/kg DM; SAM¼6.67 mg/kg DM; SED¼0.587; Po0.001). Archaeol Conditions in the rumen were altered when animals concentration in the faeces was significantly higher than in grazed predominantly on WC instead of PRG. The lower the total rumen contents (total rumen contents¼4.60 mg/kg NDF content of the white clover herbage was paralleled by DM; faeces¼11.08 mg/kg DM; SED ¼0.7224; Po0.001). lower concentrations in the total rumen contents. WC also C.A. McCartney et al. / Livestock Science 164 (2014) 39–45 43 led to significantly lower average rumen pH at both important for and consequent growth of sampling times. The lower pH with WC is probably due methanogens (Van Nevel and Demeyer, 1996). However, to the effect of high fermentation rates and subsequent the liquid-phase lacks particulate (or matrix) material on higher VFA production (Van Kessell and Russell, 1996; Lana which these associations could occur. Furthermore, the et al., 1998). VFA concentrations were also higher for WC in rate of passage of the liquid fraction from the rumen is this study. Much of the removal of VFAs by passive higher than for the solid fraction (typical values being 7.5 diffusion through the rumen epithelium is a consequence vs. 3.5% h1; Evans, 1981a,b). This may affect methanogen of rumen movements, which is triggered by the presence abundance in the liquid fraction as methanogens are slow of particulate matter in the rumen (Lana et al., 1998). Less growing (Pei et al., 2010) and therefore they may not have efficient removal of VFAs on a WC diet could be explained time to proliferate before they are effectively ‘washed out’ by the reduced amounts of ruminal digesta and therefore from the rumen. VFAs can accumulate in the rumen and reduce pH further. Another explanation for lower pH could be the reduced 4.4. Comparison of archaeol concentration in faeces levels of rumination and saliva production with WC and isolated microbial fractions (Rutter et al., 2002). Faeces had a higher concentration of archaeol (mg/kg 4.2. Archaeol in rumen digesta DM) than the SAM and LAM fractions. This was unex- pected as pure microbial fractions should contain much Concentrations of archaeol in rumen digesta showed a higher levels of methanogens (and archaeol) than faeces. significant ‘diet’ by ‘time’ interaction. This effect is probably It is unlikely that hindgut fermentation would have con- related to the diurnal grazing patterns and the different tributed to the archaeol content in faeces, as a previous rumen conditions associated with PRG or WC diets. Cows study by Gill et al. (2010) found no detectable levels of tend not to graze at night and have a major grazing bout after archaeol in the faeces of animals that carry out extensive morning milking (Rutter et al., 2002), so morning samples hindgut fermentation (i.e., horses). In addition, the con- include a lot of material that has been in the rumen over- tribution of archaeol by non-methanogenic is also night, whilst the afternoon samples include relatively much possibly negligible, as studies have found o0.05% non- more recently-ingested herbage. The low concentrations of methanogenic Archaea in the rumen (Shin et al., 2004; Lee archaeol in the afternoon for cows grazing WC suggest that et al., 2012). One explanation for the discrepancy is that methanogens proliferated more slowly with this diet. This there must be other methanogen fractions in the rumen may be the consequence of a number of factors, including the that are not represented by LAM or SAM. For instance, lower pH, lower NDF or higher rumen passage rates. Whilst protozoa losses could occur when the rumen contents we may expect higher DMI for legumes (Dewhurst et al., were strained through the cheesecloth, as the pore size of 2009), intake was not recorded in this study and the absence cheesecloth is typically around 50 mm, whereas protozoa of milk production responses suggests that any increase can range 15–250 mm(Mould et al., 2005). Loss of proto- would have been modest. In this study, the pH of the rumen zoal associated methanogens could be considerable, as it is contents for animals grazing WC was at the lower end of the estimated that methane production from protozoa- normal range, which would inhibit methanogens (Janssen, associated methanogens is as much as 37% (Finlay et al., 2010) with a smaller and less active population as a con- 1994). The main source of the discrepancy, however, is sequence (Van Kessell and Russell, 1996). Furthermore, the likely to be the inefficient detachment of solid-adherent high passage rate of legumes from the rumen (Dewhurst micro-organisms from rumen solids. Legay-Carmier and et al., 2009) restricts the opportunity for methanogens to Bauchart (1989) used diaminopimelic acid (DAPA) as a colonize the feed substrate. Higher growth rates are required bacterial marker and found that 50–60% of rumen for methanogens to maintain themselves in the rumen at could not be detached from rumen solids. Furthermore, higher passage rates (Janssen, 2010). very fine particles (o0.1 mm) were associated with 70–80% of total rumen bacteria, of which only 12–15% of 4.3. Comparison of archaeol concentration in SAM and LAM bacteria could be extracted. Similarly, Martín-Orúe et al. (1998) found that only 17–21% of bacteria could be There was a higher concentration of archaeol, and by extracted from the solid fraction of rumen digesta. The implication methanogens, in the SAM fraction in compar- colonisation of methanogens on feed is mostly in associa- ison with the LAM fraction. This is in agreement with the tion with bacteria, so failure to isolate the bacterial qPCR studies of Gu et al. (2011); Pei et al. (2010) and population could also mean that methanogens were not Morgavi et al. (2012) that reported a higher abundance of isolated. This problem is exacerbated because bacteria and methanogens associated with the solid rather than the archaea associated with small particles that have been liquid fraction. present in the rumen for a longer period of time are most The lower abundance of methanogens in the LAM likely to leave the rumen via the reticulo-omasal orifice fraction in this study probably reflects the difficulties of (Poppi et al., 1981). methanogens establishing and proliferating in the liquid Since methanogens are slow growing, it seems possible phase. This is because methanogens are most efficient that there would be higher levels on small particles that when located closely to symbiotic fermentative microbes have been present in the rumen for longer – further in a matrix (Conrad et al., 1985) as it is required for them exacerbating the under-sampling of archaea. Sampling of to carry out ‘interspecies hydrogen transfer’ which is SAM and LAM at a single time of day (9 am) is a limitation 44 C.A. McCartney et al. / Livestock Science 164 (2014) 39–45 of this study, which could explain some of the discrepancy relationship found for the concentration of archaeol in in archaeol concentration between microbial fractions and LAM. These results are in agreement with a previous study faeces; future work should consider diurnal variation in by Hook et al. (2011) who showed a positive correlation the composition of SAM and LAM. Furthermore, the between rumen pH and methanogens per grams of wet archaeol concentrations in the faeces are more likely to weight (pgww) for rumen solids (P¼0.018; R2¼0.97), but be associated with methanogen concentrations in digesta no relationship between pH and methanogens pgww in in the reticulum (i.e., that is about to leave the reticulo- rumen fluids. It was clear there was an inhibition of rumen) as opposed to concentrations in the WRC. This is methanogens by lower pH in these fractions. This may because digesta from the reticulum is very similar to that have been caused by the reduced dehydrogenase activity found in the duodenum (Hristov, 2007) and no further of the fermentative bacteria via the increased partial selective retention of particles occurs after this site pressure of hydrogen in the rumen. As a consequence, less

(Ahvenjärvi et al., 2001). products of fermentation (i.e., CO2) would have been available to the methanogens for growth (Janssen, 2010). 4.5. Comparison of archaeol concentration in whole rumen Variation in relationships between archaeol concentra- contents and faeces tion and rumen pH for the different fractions may be related to differences in the composition of the cell There was no significant relationship between archaeol membrane. For example, some methanogens may contain concentration in whole rumen contents and faeces, even at a higher proportion of tetraethers in their cell membrane the individual animal level. Furthermore, there were sig- (Schouten et al., 2013). Furthermore, we have shown nificant differences between observed faecal archaeol significant effects of diet forage/concentrate ratio on the concentrations and ‘expected’ faecal archaeol concentra- archaeol/tetraether ratio in faeces (McCartney et al., tions (obtained by adjusting the total rumen archaeol for 2013d). In theory, the methanogens could adapt their the estimated digestibility of the feed (Table 1) and membranes to contain more tetraethers in more ‘challen- disappearance of feed post-rumen (Agricultural Research ging’ rumen environments in order to minimise the loss of Council, 1980)). The expected and actual faecal archaeol protons and thus conserve energy. In this case, the liquid concentrations were 9.12 and 11.4 mg/kg DM respectively fraction may contain a higher proportion of tetraethers in for the PRG diet, and 8.60 and 10.7 mg/kg DM respectively order for the slow-growing methanogens to survive with a for the WC diet. Faecal archaeol was approximately 25% higher flow rate through the rumen. higher than expected. Both of these observations point to The poorer relationship between pH and archaeol the lack of a strong relationship between the population of concentration in LAM may be due to the considerably methanogens present in the rumen and the numbers of lower abundance of methanogens associated with the fluid methanogens leaving the rumen. Possible explanations for fraction. Alternatively, it may be that SAM are more this effect could include differences in the selective reten- sensitive to the effects of the WC diet and low pH. tion of digesta (and thus methanogens) in the rumen. Interestingly, no significant effect of pH was seen for A number of factors could influence this selectivity, includ- archaeol concentration in the WRC. This may be because ing diet, dry matter intake, protozoal population size and methanogens in the loosely-adherent SAM and LAM frac- physiological effects (e.g., stage of lactation; McCartney tions are more sensitive to changes in the rumen than et al., 2013b). Another potential reason for higher faecal methanogens found elsewhere such as protozoa and small, archaeol concentrations is the heterogeneous nature of the highly digested particles. For example, protozoa have a rumen contents. Ahvenjärvi et al. (2001) argued that stabilizing effect on pH (Veira et al., 1983) so effects will a heterogeneous distribution of rumen microbial markers not be as large. In addition, the high density of microbes in in different particle size fractions would lead to incorrect the small, highly digested particles may exhibit a protec- total rumen estimates. In particular, inconsistent distribu- tive mechanism against pH. For example, Li et al. (2001) tion of the very fine particles (o0.1 mm) which tend to found that a higher density of microbes and the produc- settle in the reticulum (Hristov, 2007) could skew micro- tion of biofilms improved cell survival when exposed to bial abundance estimates as they make up 70–80% of total low pH. rumen bacteria (Legay-Carmier and Bauchart, 1989). Variation in the abundance of microbes within different 5. Conclusions sites in the rumen may explain why there was no clear relationship between TRC and faecal archaeol concentra- The quantification of archaeol from the ruminant tions. It is likely that archaeol concentrations from reticu- digestive tract has provided an insight into the abundance lum digesta would have a better relationship with faecal and kinetics of methanogens when animals were consum- archaeol concentrations because the reticulum contains a ing a WC based diet in comparison to a PRG diet. The higher proportion of material that is ready to be expelled combination of the assumed higher passage rates from the rumen. (Dewhurst et al., 2009) and low rumen pH for the WC diet reduced archaeol concentrations in all samples stu- 4.6. Relationships between archaeol concentration died, and particularly affected archaeol concentration in in microbial fractions and rumen pH SAM. Archaeol concentrations in the isolated microbial fractions were lower than expected, which highlights the There was a strong relationship between rumen pH and importance of representative rumen sampling and the the concentration of archaeol in SAM, with a weaker efficient extraction of methanogens from the feed. C.A. McCartney et al. / Livestock Science 164 (2014) 39–45 45

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