Zoey Durmic

In vitro screening of selected feed additives, plant essential oils and plant extracts for rumen methane mitigation

BACKGROUND Ruminants produce large quantities of methane in their rumen as a by-product of microbial digestion of feed. Antibiotics are added to ruminant feed to reduce wasteful production of methane; however, this practice has some downsides. A search for safer and natural feed additives with anti-methanogenic properties is under way. The objective of this research was to examine selected feed additives, plant essential oils and plant extracts for their anti-methanogenic potential in the rumen using an in vitro batch fermentation system. RESULTS A significant reduction (P < 0.05) in methane production was observed with nine feed additives (up to 40% reduction), all eight essential oils (up to 75% reduction) and two plant extracts (14% reduction) when compared to their respective controls. Amongst these, only an algal meal high in docosahexaenoic acid, preparations of Nannochloropsis oculata, calcareous marine algae, yeast metabolites and two tannins did not inhibit microbial gas and volatile acid production. CONCLUSIONS The current study identified some potent dietary ingredients or plant compounds that can assist in developing novel feed additives for methane mitigation from the rumen.

Screening of Australian plants for antimicrobial activity against Campylobacter jejuni.

Campylobacter jejuni is the most common cause of acute enteritis in humans, with symptoms such as diarrhoea, fever and abdominal cramps. In this study, 115 extracts from 109 Australian plant species were investigated for their antimicrobial activities against two C. jejuni strains using an in vitro broth microdilution assay. Among the plants tested, 107 (93%) extracts showed activity at a concentration between 32 and 1024 µg/mL against at least one C. jejuni strain. Seventeen plant extracts were selected for further testing against another six C. jejuni strains, as well as Campylobacter coli, Escherichia coli, Salmonella typhimurium, Bacillus cereus, Proteus mirabilis and Enterococcus faecalis. The extract from Eucalyptus occidentalis demonstrated the highest antimicrobial activity, with an inhibitory concentration of 32 µg/mL against C. jejuni and B. cereus. This study has shown that extracts of selected Australian plants possess antimicrobial activity against C. jejuni and thus may have application in the control of this organism in live poultry and retail poultry products. Copyright

Australian plants show anthelmintic activity toward equine cyathostomins in vitro.

Anthelmintic resistance in gastrointestinal parasites of horses is an increasing problem, particularly in cyathostomins, and there is a need to find alternative means for the control of these parasites. We screened crude extracts from 37 species of Australian native plants for their anthelmintic activity in vitro against cyathostomin larvae (development from egg to third larval stage), with the aim of identifying those species that may be suitable for incorporation into sustainable parasite management programs. Water extracts from seven species, namely Acacia baileyana, Acacia melanoxylon, Acacia podalyriifolia, Alectryon oleifolius, Duboisia hopwoodii, Eucalyptus gomphocephala and Santalum spicatum completely inhibited larval development (100% inhibition compared to the control), while another 10 species caused 90% inhibition at the initial screening concentration of 1400 μg of extractable solids/mL. The seven most potent extracts produced IC50 values (concentration of extract which resulted in a 50% inhibition of development) in the range 30.9-196 μg/mL. Fourteen extracts were incubated with polyvinylpolypyrrolidone (PVPP) before the assays, which removed the anthelmintic activity from 12 of these extracts, indicating that tannins were likely to be the bioactive compound responsible for the effect, while in two species, i.e. A. melanoxylon and D. hopwoodii, compounds other than tannins were likely to be responsible for their anthelmintic action. Our results suggest that a number of Australian native plants have significant anthelmintic activity against cyathostomin larval development in vitro. There is potential for these plants to be used as part of sustainable parasite control programs in horses, although more research is needed to identify the compounds responsible for the anthelmintic effects and confirm their activity in vivo.

Cryptosporidium homai n. sp. (Apicomplexa: Cryptosporidiiae) from the guinea pig ( Cavia porcellus )

The morphological, biological, and molecular characterisation of a new Cryptosporidium species from the guinea pig (Cavia porcellus) are described, and the species name Cryptosporidium homai n. sp. is proposed. Histological analysis conducted on a post-mortem sample from a guinea pig euthanised due to respiratory distress, identified developmental stages of C. homai n. sp. (trophozoites and meronts) along the intestinal epithelium. Molecular analysis at 18S rRNA (18S), actin and hsp70 loci was then conducted on faeces from an additional 7 guinea pigs positive for C. homai n. sp. At the 18S, actin and hsp70 loci, C. homai n. sp. exhibited genetic distances ranging from 3.1% to 14.3%, 14.4% to 24.5%, and 6.6% to 20.9% from other Cryptosporidium spp., respectively. At the 18S locus, C. homai n. sp. shared 99.1% similarity with a previously described Cryptosporidium genotype in guinea pigs from Brazil and it is likely that they are the same species, however this cannot be confirmed as actin and hsp70 sequences from the Brazilian guinea pig genotype are not available. Phylogenetic analysis of concatenated 18S, actin and hsp70 sequences showed that C. homai n. sp. exhibited 9.1% to 17.3% genetic distance from all other Cryptosporidium spp. This clearly supports the validity of C. homai n. sp. as a separate species.

Eremophila glabra is an Australian plant that reduces lactic acid accumulation in an in vitro glucose challenge designed to simulate lactic acidosis in ruminants

Lactic acidosis is a major welfare issue affecting animal health and production systems such as dairy and feedlot beef. We used two bioassays to identify bioactive plants of Australia with the potential to prevent acidosis in ruminants. In the first bioassay, a potentially acidotic environment was induced by adding glucose to rumen fluid and pH and gas production were used to estimate the effect on acid production and microbial fermentation after 5-h incubation. Australian plants (n = 104) were screened for their ability to prevent a decline in the pH without inhibiting normal gas production, and five plants namely Eremophila glabra, Kennedia eximia, Acacia saligna, Acacia decurrens and Kennedia prorepens with such properties were identified. We investigated further the two top ranking plants, E. glabra and K. prorepens, in the second bioassay to determine the extent of their effect in vitro, by extending the incubation to 24 h and measuring d-lactate, and volatile fatty acids (VFA) in addition to pH and gas production. These were measured at 0, 5, 10, 16 and 24 h after inoculation. Eremophila glabra maintained pH values that were higher and d-lactate concentrations that were lower than the control (P < 0.001), and comparable to the antibiotic-protected environment (AB; 12 μg of virginiamycin/ml). Eremophila glabra and AB treatments did not restrict fermentation, as judged by gas production and VFA. Kennedia prorepens slowed the decline in pH and reduced the accumulation of lactate but inhibited gas production. We concluded that, in vitro, E. glabra was effective at controlling events that can lead to acidosis and the effect was comparable to that of virginiamycin, while K. prorepens was less effective than E. glabra and also inhibited fermentation.

Investigating Eremophila glabra as a bioactive agent for preventing lactic acidosis in sheep.

The Australian native plant Eremophila glabra was tested as a potential agent for preventing lactic acidosis in sheep after it was observed to be effective against acidosis in vitro. Ruminally fistulated wethers were infused via rumen cannula with single doses of kibbled wheat (14 g/kg bodyweight) and either virginiamycin (Eskalin500; AB, 80 mg/kg of wheat plus 100 g milled oaten hay/kg of wheat, n = 6), E. glabra (EG, 100 g freeze-dried and milled leaf material per kg of wheat, n = 10) or milled oaten hay (Control, 100 g milled oaten hay/kg of wheat, n = 16). Rumen samples were collected immediately before infusion and then 2, 4, 6, 8, 12, 16 and 24 h after the infusion. The samples were analysed for pH, D-lactate, volatile fatty acids (VFA) and osmolality. Rumen pH and D-lactate values indicative of acidosis were detected in the Control and EG groups. The pH nadir of the rumen was 12 h after the wheat infusion, at which time the values in the EG (pH = 4.87) and Control (pH = 5.09) groups were lower (P < 0.05) than in the AB group (pH = 5.63) and the D-lactate concentrations were higher (P < 0.05) in the EG and Control groups (24 mmol/L and 15 mmol/L, respectively) than in the AB group (0.9 mmol/L). At the same time, total VFA concentration was higher (P < 0.05) in the AB group (102 mmol/L) than in the Control (65 mmol/L) and the EG (14 mmol/L) groups. Rumen osmolality did not differ between groups. Virginiamycin was effective at preventing lactic acidosis. However, the inclusion of dried leaves from E. glabra at a similar level that was effective in vitro did not prevent lactic acidosis in vivo, and the reasons behind this remain unclear. The study demonstrates the difficulty in converting in vitro results to in vivo and highlights the need to test the plant at higher doses in vivo.

Variability of in vitro ruminal fermentation and methanogenic potential in the pasture legume biserrula (Biserrula pelecinus L.)

Abstract. Biserrula (Biserrula pelecinus L.) is an important annual pasture legume for the wheatbelt of southern Australia and has been found to have lower levels of methane output than other pasture legumes when fermented by rumen microbes. Thirty accessions of the biserrula core germplasm collection were grown in the glasshouse to examine intra-specific variability in in vitro rumen fermentation, including methane output. One biserrula cultivar (Casbah) was also grown at two field locations to confirm that low methanogenic potential was present in field-grown samples. All of the biserrula accessions had significantly reduced methane [range 0.5–8.4 mL/g dry matter (DM)] output compared with subterranean clover (28.4 mL/g DM) and red clover (36.1 mL/g DM). There was also significant variation in fermentability profiles (except for volatile fatty acids) among accessions of the core collection. Methanogenic potential exhibited 86% broad-sense heritability within the biserrula core collection. The anti-methanogenic and gas-suppressing effect of biserrula was also confirmed in samples grown in the field. In conclusion, biserrula showed variability in in vitro fermentation traits including reduced methane production compared with controls. This bioactivity of biserrula also persists in the field, indicating scope for further selection of biserrula as an elite methane-mitigating pasture.

In vitro ruminal fermentation characteristics and methane production differ in selected key pasture species in Australia

Abstract. Thirteen current and potential pasture species in southern Australia were examined for differences in their nutritive values and in vitro rumen fermentation profiles, including methane production by rumen microbes, to assist in selection of pasture species for mitigation of methane emission from ruminant livestock. Plants were grown in a glasshouse and harvested at 7 and 11 weeks after sowing for in vitro batch fermentation, with nutritive values assessed at 11 weeks of growth. The pasture species tested differed significantly (P < 0.001) in methane production during in vitro rumen fermentation, with the lowest methane-producing species, Biserrula pelecinus L., producing 90% less methane (4 mL CH4 g–1 dry matter incubated) than the highest methane-producing species, Trifolium spumosum L. (51 mL CH4 g–1 dry matter incubated). Proxy nutritive values of species were found not to be useful predictors of plant fermentation characteristics or methane production. In conclusion, there were significant differences in fermentative traits, including methane production, among selected pasture species in Australia, indicating that the choice of fodder species may offer a way to reduce the impact on the environment from enteric fermentation.

Differences in the nutrient concentrations, in vitro methanogenic potential and other fermentative traits of tropical grasses and legumes for beef production systems in northern Australia

BACKGROUND In northern Australia, beef cattle grazed extensively on tropical rangelands are responsible for 5% of the nations total greenhouse gas emissions. Methane (CH4 ) is a potent greenhouse gas and in grazing ruminants might be mitigated by selecting forages that, when consumed, produce less CH4 when fermented by rumen microbes. This study examined variability in the in vitro fermentation patterns, including CH4 production of selected tropical grasses and legumes, to identify candidates for CH4 mitigation in grazing livestock in northern Australia. RESULTS Nutritive values and fermentation parameters varied between plant species and across seasons. Grasses with a relatively low methanogenic potential were Urochloa mosambicensis (wet summer), Bothriochloa decipiens (autumn), Sorghum plumosum (winter) and Andropogon gayanus (spring), while the legumes were Calliandra calothyrsus (wet summer and autumn), Stylosanthes scabra (winter) and Desmanthus leptophyllus (spring). There was some correlation between CH4 production and overall fermentation (volatile fatty acid concentrations) in grasses (R2 = 0.67), but not in legumes (R2 = 0.01) and there were multiple plants that had lower CH4 not associated with reduction in microbial activity. CONCLUSION Differences in nutrient concentrations of tropical grasses and legumes may provide opportunities for productive grazing on these pastures, while offering some CH4 mitigation options in the context of northern Australian extensive beef farming systems.

The type of forage substrate preparation included as substrate in a RUSITEC system affects the ruminal microbiota and fermentation characteristics

In vitro fermentation systems such as the rumen simulation technique (RUSITEC) are frequently used to assess dietary manipulations in livestock, thereby limiting the use of live animals. Despite being in use for nearly 40 years, improvements are continually sought in these systems to better reflect and mimic natural processes in ruminants. The aim of this study was to evaluate the effect of forage preparation, i.e., frozen minced (FM) and freeze-dried and ground (FDG), on the ruminal microbiota and on fermentation characteristics when included as a substrate in a RUSITEC system. A completely randomized design experiment was performed over a 15-day period, with 7 days of adaptation and an 8-day experimental period. Fermentation parameters (total gas, CH4, and volatile fatty acid production) were analyzed on a daily basis over the experimental period and the archaeal and bacterial microbiota (liquid-associated microbes [LAM] and solid-associated microbes [SAM] was assessed at 0, 5, 10, and 15 days using high-throughput sequencing of the 16S rRNA gene. Results from this study suggested a tendency (P = 0.09) of FM treatment to increase daily CH4 (mg/d) production by 16.7% when compared with FDG treatment. Of the major volatile fatty acids (acetate, propionate, and butyrate), only butyrate production was greater (P = 0.01) with FM treatment compared with FDG substrate. The archaeal and bacterial diversity and richness did not differ between the forage preparations, although feed particle size of the forage had a significant effect on microbial community structure in the SAM and LAM samples. The Bacteroidetes phylum was more relatively abundant in the FM substrate treatment, while Proteobacteria was enriched in the FDG treatment. At the genus-level, Butyrivibrio, Prevotella, and Roseburia were enriched in the FM substrate treatment and Campylobacter and Lactobacillus in the FDG substrate treatment. Evidence from this study suggests that forage preparation affects CH4 production, butyrate production, and the structure of the rumen microbiota during in vitro fermentation.