The effect of diet of the donor cows on in vitro measurements of methane production from wheat and corn incubated in various forage-to-grain ratios.

BACKGROUND: Supplementation of ruminant diets with wheat and corn grains influences ruminal fermentation. In vitro fermentation is a methodology that can be used to screen feeds for their potential to produce enteric methane. However, there is evidence that the diet of the donor cows could impact the results of in vitro analysis. This research investigated the in vitro fermentation of wheat and corn grain when incubated in ruminal fluid from cows fed different grain types and different forage-to-grain ratios. RESULTS: The type of grain fed to the donor cows, as well as forage-to-grain ratio, affected the outcome of fermentation of wheat and corn grain. Differences in methane production (MP) between grains were only observed when incubated with ruminal fluid adapted to each specific grain type. Increasing proportions of wheat but not of corn decreased in vitro MP in a linear manner compared with MP produced from forage only. CONCLUSIONS: Wheat grain has a greater in vitro antimethanogenic effect than corn. However, to detect the different fermentations between wheat and corn, grains should be incubated in ruminal fluid from cows adapted to that specific grain type. © 2019 Society of Chemical Industry.

Phyto-oestrogens affect fertilisation and embryo development in vitro in sheep.

Phyto-oestrogens such as isoflavones are natural compounds that can profoundly affect reproductive function. In the present study, we tested whether including isoflavone compounds (genistein, biochanin A, formononetin) in the maturation medium would affect the outcomes for ovine oocytes in vitro. Each isoflavone compound was evaluated at five concentrations (0, 2.5, 5, 10, 25µgmL-1) and the entire protocol was repeated four times. Cumulus-oocyte complexes were randomly allocated to the treatments, then fertilised and cultured in vitro. Compared with control (0µgmL-1), the lower concentrations of isoflavone (2.5, 5 and 10µgmL-1) had no detectable effect on the rates of cleavage or embryo development, or on embryo total cell counts (TCC). However, the highest concentration (25µgmL-1) of all three isoflavones exerted a variety of effects (P<0.05): genistein decreased cleavage rate, blastocyst rate and blastocyst efficiency (blastocysts produced per 100 oocytes); biochanin A decreased cleavage rate and blastocyst efficiency; and formononetin decreased blastocyst rate and blastocyst efficiency. Biochanin A (25µgmL-1) reduced embryo TCC specifically at the hatched blastocyst stage (P<0.05). We conclude that the presence of isoflavones at 25µgmL-1 during IVM decreases the cleavage rate and inhibits blastocyst hatching.

Exploiting Compositionally Similar Grape Marc Samples to Achieve Gradients of Condensed Tannin and Fatty Acids for Modulating In Vitro Methanogenesis.

Ruminants produce large amounts of the greenhouse gas, methane, which can be reduced by supplementing feed with products that contain anti-methanogenic compounds, such as the solid winemaking by-product, grape marc. The aim of this study was to exploit compositional differences in grape marc to better understand the roles of condensed tannin and fatty acids in altering methanogenesis in a ruminant system. Grape marc samples varying in tannin extractability, tannin size and subunit composition, and fatty acid or tannin concentrations were selected and incubated in rumen fluid using an in vitro batch fermentation approach with a concentrate-based control. Four distinct experiments were designed to investigate the effects on overall fermentation and methane production. Generally, fatty acid concentration in grape marc was associated with decreased total gas volumes and volatile fatty acid concentration, whereas increased condensed tannin concentration tended to decrease methane percentage. Smaller, extractable tannin was more effective at reducing methane production, without decreasing overall gas production. In conclusion, fatty acids and tannin concentration, and tannin structure in grape marc play a significant role in the anti-methanogenic effect of this by-product when studied in vitro. These results should be considered when developing strategies to reduce methane in ruminants by feeding grape marc.

Dose-response effects of dietary pequi oil on fermentation characteristics and microbial population using a rumen simulation technique (Rusitec).

The effect of increasing the concentration of commercial pequi (Caryocar brasiliense) oil on fermentation characteristics and abundance of methanogens and fibrolityc bacteria was evaluated using the rumen simulation technique (Rusitec). In vitro incubation was performed over 15 days using a basal diet consisting of ryegrass, maize silage and concentrate in equal proportions. Treatments consisted of control diet (no pequi oil inclusion, 0 g/kg DM), pequi dose 1 (45 g/kg DM), and pequi dose 2 (91 g/kg DM). After a 7 day adaptation period, samples for fermentation parameters (total gas, methane, and VFA production) were taken on a daily basis. Quantitative real time PCR (q-PCR) was used to evaluate the abundance of the main rumen cellulolytic bacteria, as well as abundance of methanogens. Supplementation with pequi oil did not reduce overall methane production (P = 0.97), however a tendency (P = 0.06) to decrease proportion of methane in overall microbial gas was observed. Increasing addition of pequi oil was associated with a linear decrease (P < 0.01) in dry matter disappearance of maize silage. The abundance of total methanogens was unchanged by the addition of pequi oil, but numbers of those belonging to Methanomassiliicoccaceae decreased in liquid-associated microbes (LAM) samples (P < 0.01) and solid-associated microbes (SAM) samples (P = 0.09) respectively, while Methanobrevibacter spp. increased (P < 0.01) only in SAM samples. Fibrobacter succinogenes decreased (P < 0.01) in both LAM and SAM samples when substrates were supplemented with pequi oil. In conclusion, pequi oil was ineffective in mitigating methane emissions and had some adverse effects on digestibility and selected fibrolytic bacteria.

Products derived from olive leaves and fruits can alter in vitro ruminal fermentation and methane production.

BACKGROUND: The industrial processing of olive generates a high quantity of by-products. The objective of this study was to examine the effects of products derived from olive trees, i.e. leaves, fruits or kernels as a sole substrate (part A), and crude extract from leaves combined with a substrate (part B) on rumen microbial fermentation in an in vitro batch fermentation system. In this study, total gas production, methane production, and concentrations of volatile fatty acids (VFA) and ammonia in ruminal fluid were measured. RESULTS: In part A, in vitro fermentation of leaves or fruits yielded a gas and total VFA production that were comparable with control substrate, while most of them produced significantly less methane (up to 55.6%) when compared to control substrate. In part B, amongst leaf extracts, only addition of chloroform extract reduced methane production, which was also associated with a decrease (P < 0.01) in gas production. This effect was associated with a significant reduction (P < 0.01) in acetate to propionate ratio and ammonia production, but not in reduction in VFA concentrations. CONCLUSION: Olive leaf and olive leaf chloroform extract reduced ammonia production and increased the molar proportion of propionate in the rumen and can assist in developing novel feed additives for methane mitigation from the rumen. © 2016 Society of Chemical Industry.

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 nation's 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. © 2017 Society of Chemical Industry.

Bioactive fractions from the pasture legume Biserrula pelecinus L. have an anti-methanogenic effect against key rumen methanogens.

Methanogenic archaea (methanogens) are common inhabitants of the mammalian intestinal tract. In ruminants, they are responsible for producing abundant amounts of methane during digestion of food, but selected bioactive plants and compounds may inhibit this activity. Recently, we have identified that, Biserrula pelecinus L. (biserrula) is one such plant and the current study investigated the specific anti-methanogenic activity of the plant. Bioassay-guided extraction and fractionation, coupled with in vitro fermentation batch culture were used to select the most bioactive fractions of biserrula. The four fractions were then tested against five species of methanogens grown in pure culture. Fraction bioactivity was assessed by measuring methane production and amplification of the methanogen mcrA gene. Treatments that showed bioactivity were subcultured in fresh broth without the bioactive fraction to distinguish between static and cidal effects. All four fractions were active against pure cultures, but the F2 fraction was the most consistent inhibitor of both methane production and cell growth, affecting four species of methanogens and also producing equivocal-cidal effects on the methanogens. Other fractions had selective activity affecting only some methanogens, or reducing either methane production or methanogenic cell growth. In conclusion, the anti-methanogenic activity of biserrula can be linked to compounds contained in selected bioactive fractions, with the F2 fraction strongly affecting key rumen methanogens. Further study is required to identify the specific plant compounds in biserrula that are responsible for the anti-methanogenic activity. These findings will help devise novel strategies to control methanogen populations and activity in the rumen, and consequently contribute in reducing greenhouse gas emissions from ruminants.

Eremophila glabra reduces methane production and methanogen populations when fermented in a Rusitec.

Eremophila glabra Juss. (Scrophulariaceae), a native Australian shrub, has been demonstrated to have low methanogenic potential in a batch in vitro fermentation system. The present study aimed to test longer-term effects of E. glabra on rumen fermentation characteristics, particularly methane production and the methanogen population, when included as a component of a fermentation substrate in an in vitro continuous culture system (Rusitec). E. glabra was included at 150, 250, 400 g/kg DM (EG15, EG25, and EG40) with an oaten chaff and lupin-based substrate (control). Overall, the experiment lasted 33 days, with 12 days of acclimatization, followed by two periods during which fermentation characteristics (total gas, methane and VFA productions, dry matter disappearance, pH) were measured. The number of copies of genes specifically associated with total bacteria and cellulolytic bacteria (16S rRNA gene) and total ruminal methanogenic archaeal organisms (the methyl coenzyme M reductase A gene (mcrA)) was also measured during this time using quantitative real-time PCR. Total gas production, methane and volatile fatty acid concentrations were significantly reduced with addition of E. glabra. At the end of the experiment, the overall methane reduction was 32% and 45% for EG15 and EG25 respectively, compared to the control, and the reduction was in a dose-dependent manner. Total bacterial numbers did not change, but the total methanogen population decreased by up to 42.1% (EG40) when compared to the control substrate. The Fibrobacter succinogenes population was reduced at all levels of E. glabra, while Ruminococcus albus was reduced only by EG40. Our results indicate that replacing a portion of a fibrous substrate with E. glabra maintained a significant reduction in methane production and methanogen populations over three weeks in vitro, with some minor inhibition on overall fermentation at the lower inclusion levels.

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.

Antibacterial compounds from the Australian native plant Eremophila glabra.

Recent reports of Eremophila glabra (R.Br.) Ostenf. (Scrophulariaceae) displaying antibacterial activity has led us to investigate the bioactive secondary metabolites responsible for this activity. Bioassay-directed fractionation of solvent extracts prepared from the leaves of E. glabra led to the isolation of seven serrulatane diterpenes, three flavonoids and the caffeoyl ester disaccharide verbascoside. Among these, four serrulatanes, namely 18-acetoxy-8, 20-dihydroxyserrulat-14-en-19-oic acid (14), 18,20-diacetoxy-8-hydroxyserrulat-14-en-19-oic acid (16), 8,18,20-triacetoxyserrulat-14-en-19-oic acid (17) and 18-acetoxy-8-hydroxyserrulat-14-en-19-oic acid (18) are described for the first time, while 8,20-diacetoxyserrulat-14-en-19-oic acid (3), 8,18,20-trihydroxyserrulat-14-en-19-oic acid (5) and 20-acetoxy-8-hydroxyserrulat-14-en-19-oic acid (19) were previously reported. All three flavonoids hispidulin (12), jaceosidin (13) and cirsimaritin (15) are known but reported for the first time in E. glabra. All compounds were tested in an agar diffusion antimicrobial assay against Staphylococcus aureus (NCTC 10442) and Staphylococcus epidermidis (ATCC 14990). Compounds 12, 13, 17, 18 and 19 exhibited moderate activity, with minimum inhibitory concentrations (MICs) ranging from 32 to 512µg/mL. Compound 19 demonstrated the highest activity against S. epidermidis ATCC 14990 with MIC of 32µg/mL, while 13 demonstrated the highest activity against S. aureus NCTC 10442 with MIC of 128µg/mL.

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.

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.

Climate Clever Clovers: New Paradigm to Reduce the Environmental Footprint of Ruminants by Breeding Low Methanogenic Forages Utilizing Haplotype Variation.

Mitigating methane production by ruminants is a significant challenge to global livestock production. This research offers a new paradigm to reduce methane emissions from ruminants by breeding climate-clever clovers. We demonstrate wide genetic diversity for the trait methanogenic potential in Australia's key pasture legume, subterranean clover (Trifolium subterraneum L.). In a bi-parental population the broadsense heritability in methanogenic potential was moderate (H2 = 0.4) and allelic variation in a region of Chr 8 accounted for 7.8% of phenotypic variation. In a genome-wide association study we identified four loci controlling methanogenic potential assessed by an in vitro fermentation system. Significantly, the discovery of a single nucleotide polymorphism (SNP) on Chr 5 in a defined haplotype block with an upstream putative candidate gene from a plant peroxidase-like superfamily (TSub_g18548) and a downstream lectin receptor protein kinase (TSub_g18549) provides valuable candidates for an assay for this complex trait. In this way haplotype variation can be tracked to breed pastures with reduced methanogenic potential. Of the quantitative trait loci candidates, the DNA-damage-repair/toleration DRT100-like protein (TSub_g26967), linked to avoid the severity of DNA damage induced by secondary metabolites, is considered central to enteric methane production, as are disease resistance (TSub_g26971, TSub_g26972, and TSub_g18549) and ribonuclease proteins (TSub_g26974, TSub_g26975). These proteins are good pointers to elucidate the genetic basis of in vitro microbial fermentability and enteric methanogenic potential in subterranean clover. The genes identified allow the design of a suite of markers for marker-assisted selection to reduce rumen methane emission in selected pasture legumes. We demonstrate the feasibility of a plant breeding approach without compromising animal productivity to mitigate enteric methane emissions, which is one of the most significant challenges to global livestock production.