AU760814B2 - Fungal sulphur source and method of using the same - Google Patents

Description

WO 98/42326 PCT/AU98/00075 -1- Fungal Sulphur Source and Method of Using the Same Background The present invention relates to a sulphur source for fungi and a method for using the same to promote the growth of fungi in the rumen of a ruminant animal. More particularly, the present invention relates to a method for promoting the growth of anaerobic fungi in the rumen of animals fed on low sulphur content diets.

The rumen or forestomach is an organ found in the digestive tract of certain herbivorous mammals. The rumen is located prior to the gastric stomach and is the site where digestion and fermentation of plant material occurs through the activity of microbial populations.

Anaerobic fungi, bacteria and protozoa represent the three major groups of microorganisms in the rumen. The anaerobic fungi make a vital contribution to the digestion of plant fibre and in particular, plant fibre in poor or low quality feed. Poor quality feed often lacks one or more dietary components which may affect the microbial populations in the rumen. In particular, when ruminant animals consume a low sulphur content diet the population of anaerobic fungi in the rumen may be significantly reduced. A reduction in the fungi population can hamper the digestion of feed which in turn may reduce feed intake and cause the animal to have a reduced productivity.

Fungi in the rumen require sulphur in reduced form to meet their growth requirements. When sulphur is administered to the rumen as dietary sulphur it is mostly degraded to sulphide by bacteria and used by fungi and other microorganisms in the rumen. Any remaining sulphide is transported across the rumen wall and out of the rumen. Thus, sulphide does not persist in the rumen.

WO 98/42326 PCT/AU98/00075 -2- The effect of low sulphur diets on the population of anaerobic fungi in the rumen was observed in 1983. Since that time a number of sulphur sources have been used to supplement the diet of ruminant animals fed on low sulphur diets. For example, sulphur in the form of sodium sulphate has been added directly to feed sources such as pastures with low sulphur contents, and the sulphur containing amino acid, methionine, has been administered to animals as a feed supplement.

Whilst the prior art sulphur sources outlined above have been found to promote the growth of ruminant fungi and increase the intake of feed by ruminant animals, they are degraded to sulphide by bacteria in the rumen at a rate that is greater than the rate at which the fungi in the rumen use the resulting sulphide.

Thus, a large amount of the sulphide produced is not used by the fungi.

The rate of degradation of the prior art sulphur sources, and to a certain extent their mode of administration, means that relatively large amounts need to be administered to deliver an effective dose of sulphur to fungi in the rumen of a ruminant animal. The large amounts of the sulphur sources used cause the methods to be relatively expensive and inefficient.

The present invention seeks to overcome or at least partially alleviate the problems identified above.

Summary of the Invention The present invention provides a method for promoting the growth of at least one anaerobic fungus in the rumen of a ruminant animal, the method comprising the step of administering to the rumen an effective amount of a degradation resistant sulphur source.

Throughout this specification, unless the context requires otherwise, the word "comprise", or variations such as "comprises" or "comprising" will be understood WO 98/42326 PCT/AU98/00075 -3to imply the inclusion of a stated integer or group of integers, but not the exclusion of any other integer or group of integers including method steps.

Further, throughout the following description reference will be made to "ruminant animals" which will be understood to include all ruminant and ruminant-like animals, being members of the Order Artiodactyl and having a pre-gastric fermentation in a rumen (for ruminant animals), or a similar part of the digestive tract such as the paunch (for ruminant-like animals) For the purposes of the present invention the term "degradation resistant" is to be interpreted in terms of the microbial population in the rumen. Thus, a "degradation resistant sulphur source" is a sulphur source that is degraded by microorganisms in the rumen, such as bacteria, to sulphide at a slower rate than the prior art sulphur sources so that it remains available to fungi for a longer period of time.

The degradation resistant sulphur source may be varied. Preferably, the degradation resistant sulphur source is 3-mercaptopropanoic acid (MPA) or a functional equivalent thereof. Functional equivalents for the purposes of the present invention include; the salts of MPA with monovalent and divalent cations such as sodium, potassium, calcium, copper, zinc and magnesium salts; the esters of MPA incorporating alkyl groups such as methyl, ethyl and butyl groups and; the dimer form of MPA, in which two MPA monomers are linked by a disulphide bond.

The sulphur source may be administered in any of a number of ways apparent to one skilled in the art. Preferably, the sulphur source is administered intraruminally or orally as a feed supplement. When the sulphur source is administered intra-ruminally, it is preferably administered using a controlled release device such as the device described in Australian patent 555998.

WO 98/42326 PCT/AU98/00075 -4- The degradation resistant nature of the sulphur source of the present invention allows it to be administered in smaller doses and less often relative to other sulphur sources. In this respect, the sulphur source of the present invention is degraded by microorganisms such as bacteria in the rumen at a slower rate than the prior art sulphur sources and thus continues to be available in the rumen for longer periods of time.

The effective dose may be varied and may be at least partially dependent on the type of ruminant animal receiving the dose and the amount of sulphur the animal is extracting from its regular intake of feed. In this respect, it is expected that the larger the animal receiving the dose, the larger the dose of the sulphur source required to constitute an effective amount will be. Similarly, the higher the sulphur content of a given feed, the smaller the dose of the sulphur source required to constitute an effective amount will be. It will be appreciated that the optimum dose for a particular animal could be readily determined by one skilled in the relevant art.

Preferably, the effective dose of MPA is such that the concentration of MPA in the rumen is approximately 0.2mM to 8mM. Even more preferably, the effective dose of MPA is such that the concentration of MPA in the rumen is approximately 0.2 to 4mM. In one particularly preferred form, the effective dose of MPA is such that the concentration of MPA in the rumen is approximately 0.75 to The effective dose required to produce the MPA concentrations outlined above varies. Preferably, and when the ruminant animal is a sheep, the effective dose is approximately 25-200 mg Sulphur/day or more preferably, approximately 190mg.Sulphur/day. For example, when the sulphur source is MPA and the ruminant animal is a sheep fed on a low sulphur diet (approximately 0.06% sulphur), the effective dose of MPA is preferably approximately 190mg Sulphur/day.

WO 98/42326 PCT/AU98/00075 The method of the invention makes it possible for animals to have an improved productivity. In this respect, and as will be illustrated in the examples below, animals to which the method is applied show improved voluntary feed intake, dry matter digestibility and dry matter digested. The improvement in these characteristics correlates to improved productivity compared to those same animals when not subjected to the method of the invention.

The present invention also provides a veterinary preparation comprising a degradation resistant sulphur source and a suitable carrier.

The present invention will now be described with reference to the following examples. The description of the examples is in no way to limit the scope of the preceding description.

Examples Example 1: In vitro growth of a representative anaerobic fungus (Neocallimastix sp.LM1) using different compounds as the sole source of sulphur.

Fungi were tested by subculture in media that had only trace levels of sulphur.

The medium was a modification of standard media for the growth of anaerobic fungi with the following changes: the media contained no rumen fluid nor peptone (an amino acid mix without sulphur amino acids was added); yeast extract is added at 17mg I 100ml (rather than 52mg I 100ml for standard media); e a trace minerals mix and vitamins mix were added; WO 98/42326 PCT/AU98/00075 -6no sulphur reductant was included but a sterile solution of titanium nitrilotriacetate was added to sterilised media (at final concentration of 1mM) as the reductant; subculture media had cellobiose added at 0.2% concentration and added for the test media; and media were prepared with a CO 2 /carbonate buffering system and dispensed in Hungate test tubes and autoclaved.

The test compounds were added at a concentration of 0.5mM and the test tubes injected with a 5% inoculum of fungi. The fungi were subcultured twice in the media with the final culture containing 0.5% carbohydrate. After five days of incubation at 39 0 C the pH of the media were determined. As the fungi are fermentative, with the major end-products organic acids, the pH of the media decreases with increasing growth: the more the growth the greater the change of pH (ApH). To make comparisons easier the ApH has been multiplied by a 100.

Any ApH (x100) below 15 represents negligible growth.

The majority of test compounds were also screened in a previous experiment.

Where compounds were tested previously the results have been adjusted relative to a positive control (sodium sulphide).

The ability of a range of sulphur containing compounds to act as the sole sulphur source is given in Table 1. MPA gave growth comparable to sodium sulphide and somewhat better than cysteine. Dimethyl disulphide gave better growth and this may be due to this compound containing two sulphur atoms.

WO 98/42326 WO 9842326PCT/AU98/00075 -7- TABLE 1 Test Compound Relative Growth (p x100) Dimethyl disulfide 96 3-Mercaptopropafloic acid 79 Sodium Sulphiide Cystine HCI)56 Methyl 3-mercaptopropafloic acid 47 Mercaptopropalesulfoflic acid Homocysteine thiolactone 41 Homocysteine Methionine 39 2-Mercaptopropaloic acid 31 Methionine sulfoxide Thio I collate (Sodium) Methyl cysteine 1 Dimethyl sulfoxide Ethanethiol Mercaptoethane sulfinic acid 11 Cystathionine9 Hypotaurine Glutathione Thioacetamide Sodium Sodium thiosulfate Cysteic acid Methionine sulfone Sodium sulfite4 Dimethyl sulfide Taurine Methyl thiogI collate Thiourea2 Potassium Thiocyanate 2 Bis-Imethylthiol methanle 0 Ethionine Propanedithiol0 Thioacetic acid Dithiothreitol Mercaptoethylanime 0 WO 98/42326 PCT/AU98/00075 -8- Example 2: Digestion of MPA in the rumen.

The degradation of MPA and the sulphur amino acid cysteine in rumen fluid was determined by in-vitro incubations. The compound was added to low sulphur media (described in Example 1) but with the carbohydrate source being filter paper strips (1%wt/vol). A heavy inoculum of rumen digesta was added and the tubes incubated. At different time periods samples were taken and frozen until analyses. Degradation was determined by measuring the accumulation of sulphide in the medium. Sulphide was determined by the methylene blue reaction where the reaction product was separated and measured by HPLC.

The results of incubating 10mM solutions of cysteine or MPA are shown in Figure 1. The rate and extent of degradation of MPA is significantly less than that of cysteine. Similar results were obtained when digesta was taken from sheep that had been infused with MPA for 10 days.

Example 3: In vitro growth of selected fungi to different doses of MPA This example determines if non-indigenous fungi that are being used for inoculation experiments have a different response in growth to the sulphur supplement MPA.

Five isolates were recovered in standard media from cryopreservation and one isolate was obtained from the culture collection. Table 2 below indicates nature of isolates.

Table 2 Isolate Genus Animal Location LM1 Neocallimastix Sheep Prospect NSW SM1 Piromyces Sheep Prospect NSW Piromyces Cattle Prospect NSW TZB2 Piromyces Zebra Tipperary Nth Territory TGB1 Neocallimastix Gemsbok Tipperary Nth Territory WO 98/42326 PCT/AU98/00075 -9- Each isolate was transferred from standard media to a modification of the low sulphur media described in Example 1. Carbohydrates (glucose cellobiose) were added to give either 0.2% (in subculture phase or 0.5% (in test phase) For each isolate replicate tubes of low sulphur media containing the following MPA concentrations 0.2, 0.8mM) were prepared by inoculating with dilutions of a 40mM stock MPA solution made up in boiled/Nitrogen bubble water and filter sterilised]. Triplicate tubes of the three concentrations were inoculated with each isolate and after 3-4 days subcultured using the tubes with the best apparent growth. This set-up was repeated again and then isolates were inoculated into media containing 0.5% carbohydrate and the following MPA concentrations (mM; the subculture used in square brackets), 0 0.1, 0.2 0.4, 0.8, 1.2, 1.6 This was to be the final assessment however as some growth was obtained in the 0 controls so all tubes were subcultured again into the same type of media. After a weeks incubation growth was determined by the decrease in media pH as described in Example 1.

The result from the final series of media with 0.5% CHO is shown in Figures 2a and 2b. Figure 2a shows the growth responses of the Neocallimastix isolates LM1 from sheep(*) and TGB1 from gemsbok Figure 2b shows the Piromyces isolates SM1 from sheep CS15 from cattle and TZB2 from zebra Although there was better growth of the Neocallimastix sp there was no clear difference between the sheep and non-sheep isolates in these results.

Example 4: The effect of different doses of MPA on fungal numbers in the rumen.

Sheep were fed alkali treated straw supplemented with minerals and urea but having a low sulphur content (0.052% sulphur) and infused with increasing levels of MPA directly into the rumen. After seven days on the same concentration of MPA the numbers of fungal zoospores in the rumen were determined by culture.

WO 98/42326 PCT/AU98/00075 Six cross-bred sheep [wethers] having rumen cannula were moved metabolism cages equipped with automatic feeders. They were given eight equal buckets of feed a day (three hourly) and offered water ad-lib. The sheep were offered 1.1kg of a low sulphur feed (see below) After a period of three weeks on the diet rumen digesta was collected and rumen fluid (150ml)was taken for zoospore counts. (An extra 100ml of rumen digest was taken from each animal combined and mixed and 100ml returned to each rumen to minimise differences between animals.) Zoospores were assessed by standard methods for handling anaerobic fungi. Serial dilutions of rumen fluid were prepared using an anaerobic diluent. Agar roll tubes were inoculated using a syringe with 0.2ml of either a dilution of rumen fluid or undiluted rumen fluid. The media used was one mimicking the rumen environment but without added rumen fluid and with antibiotics (penicillin and streptomycin) added to stop overgrowth by rumen bacteria. After incubation the number of thallus forming units were counted and the number of zoospores in the rumen fluid calculated.

After sampling the infusion was begun by giving one days dose of MPA into the rumen and then starting the infusion ie infusion was at the rate of 48.5ml/day using Palmer syringe pumps. The sheep were initially infused at the rate of 0.4mMoles sulphur per day per 4 litres (ie 0.1mM in rumen) This was equivalent to 12.67mg Sulphur/day.

The same protocols were used with successively higher doses. The following doses were tested (given as rumen concentration and mg/d of sulphur): 0 (first sample); 0.1mM [12.67];0.25mM 0.35mM 0.5mM 0.75mM 1.0mM [126.7]; 1.5mM [190]; 5.0mM [633.3]. Each infusion was for one weeks before sampling rumen fluid and infusing the higher dose. A control period of one week with no infusion was done after infusion with 95mg Sulphur/d MPA and 4 of six sheep had no cultivable fungi with two having 1.7 thallus forming units/ml.

WO 98/42326 PCT/AU98/00075 -11 Wheat straw was milled and approximately 40kg quantities were transferred from the Gell feed mill to a mixer where 4 I of 40% NaOH was added while mixing. The feed was bagged (approximately two bags per 40kg) and left for several days before a minerals mix was added. Two bags were placed in the mixer and the compounds in Table 3 were added.

Table 3 Compound I 9Kg Urea 22.0 Trace Mineral Solution mK ZnS04H20p.0.

nS 4.H20 MnS04.... H2 CuSO 4 .5H 2 0 20 N a 2M o 4 8 c §u H..2 N a 2 M o 4 KI 0.067 Trace elements were made up in 140 ml in water and were added to the urea which was previously dissolved in 25 Litres of water. This solution was then added to two bags of alkali treated feed while mixing. The remaining dry chemicals were mixed and the appropriate amount added (1.638kg) onto the mixing feed. The feed was re-bagged into the original bags and left open for several weeks before the bags were tied shut.

The overall response can be seen in Figure 3. There is a clear dose response between the concentration of MPA in the rumen and the numbers of zoospores found in rumen fluid. Four of the six animals had levels of fungi (=1000/ml) commonly seen in normal sheep at a dose of 0.75mM (95mg S/day). In subsequent experiments with diets having higher S content fungal numbers did WO 98/42326 PCT/AU98/00075 -12not change as quickly. The optimum level of MPA of 1.5mM (190mg S)lday is likely to be affected by the very low S content of this diet.

Example 5: The effect of MPA on digestion and rumen microbiology Feed was prepared as previously described (see example Initially 22 sheep were fed 1 kg of alkali straw/day in the animal house for 4 days to select out sheep not eating the diet. 19 sheep were weighted and transferred to metabolism cages and feed the alkali straw diet ad-lib. Sheep were fed and on the following day the feed refusal weighted and if less then 100 g remained a further 100g was fed; if 100-200g of feed remained the sheep was given the same amount of feed; if over 200g of feed was left the sheep was given 100g less feed.

Over the following week another 6 sheep were moved out of the experiment (usually due to very low or erratic intakes). Infusion of MPA was done as previously described (see example 4) with a constant dose rate of 6mMoles Isheepday. This dose [192mg Sulphur/day/sheep] was expected to give a intraruminal concentration of 1.5mM MPA and was given to seven of fourteen sheep for 21 days. After 14 days infusion the collection period of 7 days commenced.

Feed refusals were weighted, recorded, and a sample of up to 200g /day pooled for subsequent analyses. Spilt feed was collected daily, weighted, recorded, and discarded. Faeces were collected with separators that excluded urine. The faeces were weighed and sub-sampled and pooled for subsequent analyses.

Pooled faeces and feed were stored at -20 0 C and then dried in a hot air oven (faeces were initially dried at 700C at 100C cooled and weighted to determine dry weight (DM).

At the end of the collection period samples of rumen digesta were taken for microbiology and other analyses. Rumen fluid was collected and fungal zoospores and total bacteria assessed as described in example 4 (the total bacteria were assessed on the same medium but without antibiotics).

Cellulolytic bacteria were assessed by a most probable number count. Rumen fluid was serially diluted and subsamples of each dilution inoculated into WO 98/42326 PCT/AU98/00075 -13culture tubes containing an anaerobic medium containing rumen fluid and strips of filter paper. After incubation the presence of cellulolytic bacteria was detected by degradation of the paper and the numbers of positive tubes compared to tables to give a number of these bacteria in the original sample.

Following the last sampling the infusion was stopped in the first group of sheep and on the following day the infusion treatment was begun in the other group of seven sheep. The second period (the crossover) was conducted in the same way as the first period with the same samples being taken at the same times.

With the exception of one animal (which went off feed in the week preceding the non-infused collection period and rebounded during collection) there was a strong, positive, response in digestive performance see Table 4) and to the numbers of rumen fungi to infusion by MPA. (see Table Table 4: Effect of MPA on Feed intake and digestibility (MPA infused at 192mg Sulphur/sheep/day) Sheep Intake DM (gld) Digestibility DM) Digested DM (gld) Ta MPA Control MPA Control MPA Control 3009 1485 1356 9.6 48.119 48.117 0.002 714.69 652.25 9.67 2621 635 531 19.6 54.648 47.189 7.459 347.27 250.79 38.47 2509 805 581 38.6 51.522 43.723 7.799 414.97 254.10 63.31 2611 703 565 24.5 50.799 45.780 5.019 357.26 258.65 38.13 3017 1163 903 28.8 51.891 44.097 7.795 603.65 398.16 61.61 2520 1221 977 24.9 45.708 43.098 2.610 558.00 421.10 32.61 3012 983 736 33.5 52.812 51.359 1.453 518.94 378.00 37.28 2976 1389 1080 28.6 58.076 52.964 5.112 806.76 572.01 41.04 2492 1126 955 17.9 53.589 46.127 7.462 603.24 440.51 36.94 2616 1347 1142 18.0 46.808 44.264 2.544 630.64 505.49 24.76 3018 1288 1108 16.2 51.433 50.989 0.444 662.31 564.96 17.23 2997 1250 902 38.6 53.840 50.985 2.855 673.01 459.88 46.34 3010 1265 992 27.5 51.933 47.156 4.777 657.21 467.79 40.49 Aver. 1128 910 26.1 51.6 47.37 4.26 581 433 36.76 n=13 +SD 257 238 8.4 3.2 3.2 2.71 133 122 13.41 1 increase of intake in individual sheep after infusion of MPA Table 5: Effect of MPA on Feed intake and microbiota (MPA infused at 192mg Suiphur/sheeplday) Total Bacteria Sheep Tag 3009 2521 2509 25111 3017 Intake DM (gid) MPA Control 1485 1356MR 635 531 805 581 703 565 1163 903 9.6 19.6 38.6 24.5 28.8 MPA. Control l og 5383 533 1.0 4Z900- 401 1.1 2567 5 2.7 7083 25 2.5 3050 32 2.

-MPA I(Control I lgJL 6.33 113.83 -0.34_ 150 1 8.37 1 0.10 Cellulotytic Bacteria (x iO) -MPA [Control l og I.) 18.37 1 4.42 10.62 10.50 8.07 6.83 3.93 15.00 41 r fl 0.31 -0.34 0.556 4.426 14 1.837 1.837 3.219

I

-0.52 0.38 -0.24 2520 1221 97 1 249 1 A. 7283_ n M 1 17 31 1 8 1R 1 0 0 7 3012 2976 2492 2515 3018 2997 3010 Aver.

(n=13) 983 1389 1126 1347 1288 736 33.5- 1080 28.6 955 17.9 1 142 181.0 1108 16.2 92 38.6 92 ]27.5 90 25.1 7250 2200 6167 j 1350 4417 1 683 0.05 0.5 7.17 6.33 0.7 0.8I A 1, 5.87 j 3.3 12.00 6.2 0.25 0.29 0.29 1.08 18 j-.4 092 0.696 12 3.219 I0.0556 1 1.76 1 UU I f 1 6467 1 11167 1 5887 I,~15 81 u 6.-25- 47I1717 0.6 10.83 5.07 j0.33 1 3.219 0.556 0.76 17I1183 1.0 10.67 .517 J0.31 1 3.219 .0.322 1.00 1150 1.20 929 7.81 0.10 j4.05 1.59 j0.40 5887 0.24 4.28 1 1.11 0.00 257 1 zm 41it. 1 245 35 increase of intake in individual sheep after infusion of MPA loglO increase in individual sheep after infusion of MPA WO 98/42326 PCT/AU98/00075 General Materials and Methods used in Examples Media for growth of anaerobic fungi (and rumen bacteria when antibiotics not included).

Mix the solutions and ingredients of basal medium in the order given in Table 6 and dissolve the solids. Add the four carbon sources listed in Table 7 to make medium Boil the mixture for 5 min in a suitable glass vessel with a narrow neck a 150-ml heat-resistant conical flask for a 100 ml quantity of medium), add 1 ml fresh reducing solution (see footnote to Table 6 for composition) for each 100 ml medium.

Bubble a gentle stream of CO 2 through the medium with the vessel in an ice bath until the liquid is cold, i.e. 0 4°C. About 15 min is adequate for 100 ml but a longer time is required to cool larger volumes of media. The colour of the medium will change from pink (oxidized resazurin) to colourless (reduced). At this stage, the pH of the medium is measured and should be in the range 6.4 6.8 before dispensing the medium. A medium with a pH of greater than 6.8 will probably respond to continued bubbling with CO 2 Medium with a pH value that is much below 6.4 is likely to be faulty and should be remade. Reduced medium is dispensed into Hungate tubes or other appropriate vessels [Hungate tubes are screw-capped test tubes (16 mm diameter) and are supplied by Bellco Glass Incorporated (Vineland, New Jersey, catalogue number 2047), they are used in conjunction with butyl rubber septa and screw caps obtained from the same source]. Sterilize the medium by autoclave at 120 0 C for 15 min.

To make a broth medium for maintenance of cultures, dispense 5 ml under CO 2 into each Hungate tube which already contains 2.5 mg agar (added as a small aliquot of a molten agar solution). Cap and seal as before and autoclave. Disperse the agar through the medium while it is still warm by gently inverting the tubes several times.

WO 98/42326 PCT/AU98/00075 -16- Antibiotic solution Benzyl penicillin (12 mg/ml) and streptomycin sulphate (2 mg/ml) are dissolved in distilled water which has been previously boiled for 5 min and bubbled with a gentle stream of N 2 until cooled. The antibiotic (pen-strep) solution is filter sterilized (a membrane with pores of 0.2 pm) whilst being gassed with N 2 Store the solution in a closely capped bottle, under N 2 at 4*C for up to 5 d. A mixture of lincomycin HCI (1mg/ml) and chloramphenicol succinate (0.42mg/ml) is made and sterilized in the same manner.

Anaerobic Dilution Solution (ADS) Combine all components listed in Table 8 and boil the solution for 5 min. Add 1 ml freshly prepared reducing solution per 100 ml (see footnote to Table Bubble the medium with CO 2 on ice until the liquid is cold. Dispense exactly 9.0 ml under CO 2 into Hungate tubes (see section seal with butyl rubber septa and screw caps.

Autoclave at 120C for 15 min. Prepared ADS sometimes has a white precipitate at the bottom of the tubes when hot. The precipitate redissolves upon gentle mixing of the cooled ADS. The final pH of ADS is about 6.2.

Aqar roll tubes Roll tubes are prepared by dispensing 2.8 ml medium 10X into Hungate tubes which already contain 60 mg agar powder (Oxoid Limited, Basingstoke, Hampshire, England) and are being flushed with a gentle stream of CO 2 Alternatively, agar roll tubes can be prepared in rimless, thick-walled test tubes fitted with black butyl rubber stoppers by adjusting the volume of medium to match the capacity of the tubes. The roll tubes are sterilized in an autoclave for 15 min at 120 0 C. Hold these tubes of molten agar medium in a water bath at 52 55 0 C and add 0.3 ml fresh penstrep antibiotic solution to each by means of a sterile disposable 2-ml syringe (and gauge needle) about 30 min before the tubes will be used. Do not hold these WO 98/42326 PCT/AU98/00075 -17tubes around 55°C for longer than overnight as the agar will become partly hydrolysed.

Zoospore counts by aqar roll tubes The number of zoospores in samples of strained ruminal fluid can be used to follow zoosporogenesis in the rumen and it provides a relative estimate of the size of the anaerobic fungal population.

Strain a sample of ruminal digesta through an open weave cloth, and collect the filtered fluid in a C0 2 -filled flask.

Serially dilute the fluid ten-fold in anaerobic dilution solution (ADS) to a final dilution of 10 3 To do this, transfer 1 ml volumes using 1 ml disposable sterile syringes fitted with sterile 23 gauge (0.65mm) needles to tubes containing 9 ml ADS, starting with fluid to form the 10 1 dilution. Use a clean syringe at each dilution step.

Using a 1 ml syringe (and 23 ga. needle, as before) inoculate three roll tubes each with 0.2 ml of 10- 3 dilution. Immediately roll these tubes horizontally on ice until the agar sets. With the same syringe, inoculate three more roll tubes with 0.2 ml of 10- 2 dilution and roll them. Repeat with 10 1 dilution and finally with undiluted fluid, if appropriate.

Incubate the roll tubes vertically at 39°C for 5 d. Count the total number of fungal colonies growing in each tube and use the inoculum volume (0.2 ml) and the dilution value 0.1, 00.1 or 0.001) to calculate the number of fungal zoospores per ml of original fluid.

WO 98/42326 PTA9/07 PCT/AU98/00075 18- Table 6. Composition of liquid basal medium for anaerobic fungi Solution or Component(s) Concentration Quantity per ingredient or source or proportion 100 ml medium in solution Mineral solution I K 2

HPO

4 3.0 g/l 15 ml Mineral solution 11 KH 2

PO

4 1.0 g NaCI

(NH

4 2 S0 4 6.0 g CaCI 2 .2H- 2 0 0.8 g MgC1 2 .6H 2 0 1.0 g/l 15 ml Redox indicator resazurin 1.0 g/l 0.1 ml solution Haemin solution haemin 0.5 g NaOH 2.0 g/l 0.2 ml Volatile fatty acids acetic 17 ml propionic 6 ml n-butyric 4 ml i-butyric 1 ml n-valeric 1 ml i-valeric 1 ml DL-2-methyl-butyric 1 ml 0.31 ml Total 31m1 Carbonate solution Na 2

CO

3 80 g/l 7 ml Yeast extract Oxoid a 0.05 g Peptone "Tryptone", Oxoida 0.2 g WO 98/42326 PCT/AU98/00075 -19aOxoid, Basingstoke, UK Reducing solution: Dissolve 0.625 g cysteine-HCI in 24 ml distilled water, adjust pH to 11 with 10 M NaOH. Add 0.625 g Na 2 S.9H 2 0, dissolve, and boil immediately before use. Make fresh reducing solutions for each batch of basal medium.

Table 7. Composition of medium Table 8. Composition of Anaerobic Dilution Solution (ADS) Component Volume (ml) Mineral solution 1a 15.0 Mineral solution II 15.0 Resazurin aqueous) 0.1 Na 2

CO

3 aqueous) 3.8 Distilled water 66.1 aThe composition of Mineral solutions I and II are given in Table 6.

The present invention includes within its scope adaptations and modifications apparent to one skilled in the art.

Claims (11)

1. A method for promoting the growth of at least one anaerobic fungus in the rumen of a ruminant animal, the method comprising the step of administering to the rumen an effective amount of 3-mercaptopropanoic acid (MPA) or a functional equivalent thereof.

2. A method according to claim 1, wherein the functional equivalent is selected from the group comprising; a salt of MPA with monovalent and divalent cations, an ester of MPA incorporating an alkyl group, and the dimer form of MPA.

3. A method according to claim 2 wherein the salt of MPA is a sodium, potassium, calcium, copper, zinc or magnesium salt.

4. A method according to claim 2 wherein the ester of MPA incorporates an alkyl group selected from the group comprising; methyl, ethyl and butyl groups. A method according to any one of the preceding claims wherein the MPA or functional equivalent thereof is administered intra-ruminally.

6. A method according to claim 5 wherein the MPA or functional equivalent thereof is administered using a controlled release device.

7. A method according to any one of claims 1 to 4 wherein the MPA or functional equivalent thereof is administered orally.

8. A method according to any one of the preceding claims wherein the effective dose of MPA or functional equivalent thereof is such that the concentration of MPA or functional equivalent thereof in the rumen is approximately 0.2mM to 8mM. AMENDED SHEET (Article 34) (IPEA/AU) -21

9. A method according to any one of claims 1 to 7 wherein the effective dose of MPA or functional equivalent thereof is such that the concentration of MPA or functional equivalent thereof in the rumen is approximately 0.2 to 4mM. A method according to any one of claims 1 to 7 wherein the effective dose of MPA or functional equivalent thereof is such that the concentration of MPA or functional equivalent thereof in the rumen is approximately 0.75 to

11. A method according to any one of claims 1 to 7 wherein the effective dose of MPA or functional equivalent thereof is approximately 25-200 mg Sulphur/day

12. A method according to any one of claims 1 to 7 wherein the effective dose of MPA or functional equivalent thereof is approximately 95-190mg Sulphur/day.

13. A method according to any one of claims 1 to 7 wherein the effective dose of MPA or functional equivalent thereof is approximately 190mg Sulphur/day. 15 14. A method according to claim 1 substantially as herein described with reference to the examples. Dated this THIRTEENTH day of MARCH 2003. Commonwealth Scientific and Industrial Research Organisation and Australian Wool Innovation Applicants Wray Associates Perth, Western Australia S• Patent Attorneys for the Applicants