Animal selection for the main sampling experiment

The objective of the animal selection was to obtain rumen samples from animals that were representative of the national dairy herd. The ruminally fistulated Friesian dairy cows used in this experiment were kept at Dairy NZ’s Lye and Scott Farms (Vaile Road, Hamilton, New Zealand). From the nine available animals, six were selected

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(four for sampling and two in reserve). Unfortunately, cow A (2225) died after the first sampling, and the reserve cow E (3122) was used as its replacement. The second reserve cow F (4109) was not used for any analyses. The age of the cows and their milk production were taken into account when selecting the cows to be sampled. At least one animal born in each of the years 2002, 2003 and 2004 were selected, and the individuals with the highest and lowest milk yields were also selected (Table 4-1). DNA for DGGE was extracted from freeze dried rumen contents from all nine animals and DGGE of the V3 region of the 16S rRNA gene showed remarkable similarity between all of the cows. The six animals selected spanned the diversity observed among the nine animals considered (Figure 4-1). Whole rumen contents from the cows selected were pooled, and the solid digesta separated and washed four times using the same method as the trial cultivation to obtain the washed digesta fraction. Cell counts done on this fraction were comparable to those obtained in wash experiment two and the trial cultivation, at 1.9u 1011/g of digesta or 2.7 u 1011_{/g of rumen content.}

Table 4-1. Milk production values of the nine animals under consideration for sampling.

Cow ID Date of birth Milk yield

L/daya Milk solids kgb Selected animal code 2225 6/08/2002 16.1 303.31 A 2628 13/07/2002 15.4 382.66 3122 29/06/2003 17.3 341.93 E 3517 16/07/2003 17.1 360.94 B 3950 20/07/2003 13.1 364.62 4109 8/07/2004 13.6 329.17 F 4110 10/07/2004 11.6 320.4 C 4512 15/07/2004 13.2 341.68 4519 22/07/2004 13.1 345.14 D

a _{Average daily milk yield over seven days taken from 13-02-2009.}

b _{Total milk solids (fat + protein) collected for the lactation up until 20-02-2009.}

Data obtained from personal communications with Dr Garry Waghorn, DairyNZ, Hamilton, NZ.

129 Figure 4-1. Comparison of DGGE profiles of bacterial 16S rRNA genes (V3 region) generated from DNA extracted from rumen contents of nine animals under consideration for sampling. Differences between the patterns were analysed using the Jaccard correlation and pattern relatedness is shown by branch length using the UPGMA method. DGGE gel courtesy of Dong Li (AgResearch, Grasslands, NZ)

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4.3.1 Sampling and media

All culture tubes containing the RM02 medium were coded according to the month of the sampling:

M – May 2009 A – August 2009 N – November 2009 F – February 2010 L – May 2010 (last)

Media other than RM02 are collectively referred to as the ‘other’ media and RM02 as the ‘main’ medium. All media were prepared using the same anaerobic techniques and (with the exception of 98-5) supplemented with a sugar and vitamin substrate mix containing; D-glucose, D-cellobiose, D-xylose, L-arabinose, Na L-lactate,

casamino acids, bacto peptone, yeast extract and vitamin mix. For RM02 and RM02- based media, this mix was dissolved in clarified rumen fluid and designated GenRFV. For the media already containing rumen fluid (BY and clarified rumen fluid), the mix was dissolved in water. The sample code for the type of media and sampling month is summarised in Table 4-2.

At the August 2009 sampling, three other media were compared with RM02. These media were designed to test whether adding a solid surface for bacteria to attach to would increase their culturability. The nomenclature for these ‘other’ media was as follows:

YA – RM02 medium with the addition of 0.5% w/v bacteriological agar YG – RM02 medium with the addition of 0.5% w/v ground dried ryegrass YS – RM02 medium with the addition of 2-5 cm of sisal string

YR – RM02 medium (control)

A control of RM02 medium was included, using the same dilution range and number of tubes inoculated as the ‘other’ media. With YA medium, the agar set at bottom of the tube while the top remained liquid. Growth of bacteria was often observed at the solid- liquid interface.

At the November sampling, two new media were used. These media were selected to explore whether culturability and isolate diversity could be increased using a more

131 complex medium and one with different substrates. The nomenclature for these ‘other’ media was as follows:

XB – BY medium

XP – RM02 medium with the addition of 0.08% w/v pectin (from apple)

At the February sampling, two new media were used. The nomenclature for these ‘other’ media was as follows:

WC – CRF medium, clarified rumen fluid (100%)

WT – RM02 medium with the addition of titanium (III) NTA solution

Many of the WT media tubes changed colour from clear to pale yellow over 2-6 weeks of incubation and a loose white precipitate formed. In some batches, but not others, WC medium formed a dark brown precipitate. These batches were not used for the isolation experiments in case poor growth was obscured.

At the May 2010 sampling two new media were used. The nomenclature for these ‘other’ media was as follows:

V9 – Medium 98-5

VC – CRF+sisal, Clarified rumen fluid (100%) with the addition of sisal string

Each combination of sampling month, animal and medium was treated as a separate cultivation experiment.

4.3.2 Cultivation dilution range

The blended plant-adherent fraction was diluted before being inoculated into culture media, to obtain an inoculum with a theoretical 0.2 viable cells per tube (which should give rise to growth in 20% of the inoculated tubes). A series of 4-fold dilutions were used to inoculate the growth media. As it was established which dilutions consistently produced the expected number of growth-positive tubes, fewer dilutions were needed to obtain the same number of cultures in RM02 medium (Table 4-2). During the first May sampling, 60 tubes were inoculated for each of five dilutions of the inoculum from each animal. For the August sampling, 60 or 90 tubes were inoculated for each of four dilutions of the inoculum from each animal. For the November, February, and final May samplings, 60 tubes were inoculated for each of three dilutions of the inoculum from each animal. The previously untested ‘other’ media had the

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potential to increase or decrease the culturability, so the larger range of five dilutions (20 tubes each) of the inoculum from each per animal was used. For each sampling, a dilution that covered the targeted percentage (20%) of growth-positive tubes was obtained (Table 4-3). The most probable number (MPN) was calculated from the numbers of growth positive tubes in each dilution. A summary showing the average of the MPN values and their associated confidence intervals for each medium type are shown in Figure 4-2. Tubes of a dilution set that received close to the theoretical 0.2 viable cells per tube were selected for subculture.