Probable insect cocoon or pupation chamber in a channel‐fill sandstone bed of the Lower Cretaceous Jinju Formation,South Korea

An ellipsoidal chamber which is composed of a very thin organic layer and emplaced in an enlargement continuing upwards into a vertical burrow is found in thin sections of a fluvial channel‐fill sandstone bed of the Lower Cretaceous Jinju Formation in South Korea. Micromorphological analysis demonstrates that the ellipsoidal chamber is attributable to Fictovichnus. It can be interpreted as a free‐standing wall of a wasp cocoon attributable to Fictovichnus sciuttoi or Fictovichnus aragon, or an organic lining of chafer beetle or weevil pupation chamber attributable to Fictovichnus gobiensis. The analysis further shows that the vertical burrow and enlargement appear to reflect chafer and weevil larval behaviour, but the arrangement of the chamber within the enlargement appears to be the same as a wasp cocoon inside a cell. Such uncertainties of ichnotaxonomic assignment and putative producer suggest that the Jinju specimen may reflect an aberrant pupation behaviour of coleopterans or hymenopterans. This Early Cretaceous specimen may be the earliest record of an insect pupation structure probably associated with a burrow. Its atypical morphologies and very rare occurrence in a channel sandstone bed without other evidence of pedogenesis suggest that the Jinju specimen was constructed in incipiently and briefly exposed sediment, unlike typical insect nesting structures constructed in soil.     

Effects of isovalerate supplements on morphology and functional gene expression of rumen mucosa in pre- and post-weaning dairy calves

Isovalerate supplements could stimulate rumen development by improving morphology and function of rumen mucosa, and then promote the growth of calves. This study was done to evaluate the effects of isovalerate supplements on morphology and functional gene expression of rumen mucosa in dairy calves. In total, 48 Chinese Holstein male calves with 15 days of age and 45.1±0.36 kg of BW were randomly assigned to four groups. The treatments were: control, low-isovalerate, moderate-isovalerate and high-isovalerate with 0, 3, 6 and 9 g isovalerate per calf per day, respectively. Supplementary isovalerate was hand-mixed into milk in pre-weaning calves and into concentrate portion in post-weaning calves. The study consisted of a 15-day-adaptation period and a 60-day-sampling period. Calves were weaned at 60 days of age. Three calves were slaughtered from each of the four treatments at 30, 60 and 90 days of age. The weight of body and stomach were measured, samples of ruminal tissues and blood were analyzed. Total stomach weight, total stomach to BW ratio, rumen wall and keratinized layer thickness, serum growth hormone and IGF-1 for both pre- and post-weaning calves increased linearly with increasing isovalerate supplements. Rumen to total stomach weight ratio, the length and width of rumen papillae, and serum β-hydroxybutyrate increased linearly for post-weaning calves. However, abomasum weight to total stomach weight ratio decreased linearly for both pre- and post-weaning calves. The relative messenger RNA expression for growth hormone receptor, IGF-1 receptor and 3-hydroxy-3-methylglutaryl-CoA synthase 1 in rumen mucosa increased linearly for post-weaning calves. Our results suggested that isovalerate supplements promoted rumen development in a dose-dependent manner. The optimum dose was 6.0 g isovalerate per calf per day.     

Evaluation of the effects of different diets on microbiome diversity and fatty acid composition of rumen liquor in dairy goat

Fat supplementation plays an important role in defining milk fatty acids (FA) composition of ruminant products. The use of sources rich in linoleic and α-linolenic acid favors the accumulation of conjugated linoleic acids isomers, increasing the healthy properties of milk. Ruminal microbiota plays a pivotal role in defining milk FA composition, and its profile is affected by diet composition. The aim of this study was to investigate the responses of rumen FA production and microbial structure to hemp or linseed supplementation in diets of dairy goats. Ruminal microbiota composition was determined by 16S amplicon sequencing, whereas FA composition was obtained by gas-chromatography technique. In all, 18 pluriparous Alpine goats fed the same pre-treatment diet for 40±7 days were, then, arranged to three dietary treatments consisting of control, linseed and hemp seeds supplemented diets. Independently from sampling time and diets, bacterial community of ruminal fluid was dominated by Bacteroidetes (about 61.2%) and Firmicutes (24.2%) with a high abundance of Prevotellaceae (41.0%) and Veillonellaceae (9.4%) and a low presence of Ruminococcaceae (5.0%) and Lachnospiraceae (4.3%). Linseed supplementation affected ruminal bacteria population, with a significant reduction of biodiversity; in particular, relative abundance of Prevotella was reduced (−12.0%), whereas that of Succinivibrio and Fibrobacter was increased (+50.0% and +75.0%, respectively). No statistically significant differences were found among the average relative abundance of archaeal genera between each dietary group. Moreover, the addition of linseed and hemp seed induced significant changes in FA concentration in the rumen, as a consequence of shift from C18 : 2n-6 to C18 : 3n-3 biohydrogenation pathway. Furthermore, dimethylacetal composition was affected by fat supplementation, as consequence of ruminal bacteria population modification. Finally, the association study between the rumen FA profile and the bacterial microbiome revealed that Fibrobacteriaceae is the bacterial family showing the highest and significant correlation with FA involved in the biohydrogenation pathway of C18 : 3n-3.     

Effect of starter diet supplementation on rumen epithelial morphology and expression of genes involved in cell proliferation and metabolism in pre-weaned lambs

Starter feeding is usually used in lamb production to improve rumen development and to facilitate the weaning process, but molecular mechanism of which is not well understood. Therefore, the objective of this study is to investigate the effect of starter feeding on the expression of ruminal epithelial genes involved in cell proliferation, apoptosis and metabolism in pre-weaned lambs. We selected eight pairs of 10-day-old lamb twins. One twin was fed ewe milk (M, n=8), while the other was fed ewe milk plus starter (M+S, n=8). The lambs were sacrificed at 56 days age. Results showed that the lambs fed M+S had lower pH in the rumen and a higher concentration of acetate, propionate, butyrate and total volatile fatty acid (VFA). Compared with the M group, the concentration of β-hydroxybutyric acid in plasma had an increased trend, and the concentration of IGF-1 in plasma had an decreased trend in the M+S group. The length, width and surface of rumen papillae increased in the M+S group compared with the M group; this was associated with increased cell layers in the stratum corneum, stratum granulosum and total epithelia. Messenger RNA (mRNA) expression of proliferative genes of cyclin A, cyclin D1 and cyclin-dependent kinase 2 in the ruminal epithelia of M+S lambs was increased compared with M only lambs. The mRNA expression of apoptosis genes of caspase-3, caspase-8, B-cell lymphoma-2 (Bcl-2) and Bcl-2-associated X protein (Bax) in the M+S group was decreased compared with M group, but the ratio of Bcl-2 to Bax were not changed between the two groups. Expression of IGF-1 mRNA was decreased, but the mRNA expression of IGF-1 receptor was higher in ruminal epithelia in the M+S group. Furthermore, the mRNA expression of VFA absorption and metabolism genes of β-hydroxybutyrate dehydrogenase isoforms 1 and 3-hydroxy-3-methylglutaryl-CoA lyase had an increased trend in the M+S group than in the M group, but the mRNA expression of 3-hydroxy-3-methylglutaryl-CoA synthase isoform 1, monocarboxylate transporter isoform 1 and putative anion transporter isoform 1 had a decreased trend in the M+S group than in the M group. These results suggest that starter feeding increased proliferation and inhibited apoptosis of ruminal epithelial cells, and may promote the VFA metabolism in ruminal epithelium in pre-weaned lambs. These findings provide new insights into improving rumen development by nutritional intervention strategies in pre-weaned lambs.     

Cleavage of di- and tripeptides by Prevotella ruminicola

The final step in the conversion of protein to amino acids by the common Gram-negative rumen bacterium, Prevotella (formerly Bacteroides) ruminicola , is the cleavage of di- and tripeptides. Dipeptidase and tripeptidase activities were predominantly cytoplasmic, and toluene treatment increased the rate of Ala₂ and Ala₃ hydrolysis by whole cells, suggesting that transport limited the rate of hydrolysis of extracellular di- and tripeptides. The hydrolysis of Ala₂ and Ala₃ by whole cells was not affected by protonophores, ionophores or dicyclohexylcarbodiimide, but Ala₂ hydrolysis by EDTA-treated cells was inhibited by the Ca²⁺/H⁺ ionophore, tetronasin. Ala₃ hydrolysis was not affected by protonophores or ionophores in EDTA-treated cells. The dipeptidase of strain M384 was inhibited > 99% by 1,10-phenanthroline and 39% by EDTA but not other protease inhibitors, consistent with the enzyme being a metalloprotease. Tripeptidase was insensitive to protease inhibitors, except for a 33% inhibition by EDTA. Cleavage of tripeptides occurred at the bond adjacent to the N-terminal amino acid. Distinct di-, tri- and oligopeptidase peaks were obtained by anion-exchange liquid chromatography of disrupted cells. Banding patterns on native PAGE using activity staining also indicated that P. ruminicola M384 had separate single dipeptidase and tripeptidase enzymes which hydrolysed a range of peptides. The dipeptidase of strain M384 was different from other strains of P. ruminicola: strains GA33 and B₁4 had activities which ran at the same R_f; strain GA33 had another band of lower activity; strain 23 had two bands different from those of the other strains. The tripeptidases ran at the same R_f for the different strains. Dipeptidase activity of all strains was inhibited by 1,10-phenanthroline on gels. Gel permeation chromatography indicated that the M_r of the dipeptidases from strains M384 and B₁4 were 115 000 and 114 500 respectively, and 112 500 and 121 500 for the corresponding tripeptidases. Thus the metabolism of small peptides by P. ruminicola involves separate permeases and intracellular peptidases for di- and tripeptides.     

Degradation and utilization of xylans by the rumen anaerobe Prevotella bryantii (formerly P. ruminicola subsp. brevis) B(1)4

Freshly harvested whole cells from cultures of P. bryantii B(1)4 grown with oat spelt xylan (OSX) as an energy source showed less than 25% of the enzyme activity against OSX, and less than 15% of the activity against birchwood xylan (BWX) and carboxymethylcellulose, that was detectable in sonicated cell preparations. This indicates that much of this hydrolytic activity is either periplasmic, membrane-associated or intracellular and may be concerned with the processing of transported oligosaccharides.P. bryantii B(1)4 cultures were able to utilise up to 45% and 51% of the total pentose present in OSX and BWX, respectively, after 24 h, but could utilize 84% of a water-soluble fraction of BWX. Analysis of the xylan left undegraded after incubation with P. bryantii showed that while xylose and arabinose were removed to a similar extent, uronic acids were utilized to a greater extent than xylose. Predigestion of xylans with two cloned xylanases from the cellulolytic rumen anaerobe Ruminococcus flavefaciens gave little increase in overall pentose utilization suggesting that external P. bryantii xylanases are as effective as the cloned R. flavefaciens enzymes in releasing products that can be utilised by P. bryantii cells. The xylanase system of P. bryantiiis able to efficiently utilise not only xylo-oligosaccharides but also larger water-soluble xylan fragments.     

Anaerobic fermentation: Microbes from ruminants

Fed-batch fermentation of biomass could provide a route for direct conversion of renewable resources to commercially significant chemicals. The ecosystem in the forestomach (rumen) of ruminants provides a highly reduced environment (oxidation-reduction potential of ?250 to ?450 mV) in which anaerobic bacteria directly utilize cellulose, hemicellulose, and other fermentable biomass constituents to produce acetate, butyrate, propionate, methane and carbon dioxide at pH 5.7 to 7.3. The cellulose fermentation in the rumen is impacted by the physically and chemically heterogeneous character of the insoluble substrate, as well as the properties of the mixed culture responsible for fibre hydrolysis and carbohydrate utilization. The rumen system provides an interesting case study in the context of possible process concepts for direct fermentation of biomass to commercially important chemicals such as acetate, propionate, succinate, lactate and ethanol. The role of the chemical and physical characteristics of the substrate, the microbes in the rumen system and the metabolic pathways of soluble carbohydrates are discussed in the context of cellulose and hemicellulose fermentation.     

Parameters of fermentation and rumen microbiota of Nellore steers fed with different proportions of concentrate in fresh sugarcane containing diets

The objective of this study was to evaluate the effect of a fresh sugarcane-based diet and different roughage-to-concentrate ratios (70:30, 60:40, 40:60 and 20:80) on the rumen microbiota associated with rumen fermentation parameters and the intake and apparent digestibility of nutrients in Nellore steers. Eight rumen-cannulated Nellore steers (331 ± 8 kg BW) were distributed in a double 4 × 4 Latin square design balanced for the control of the residual effect. The ruminal pH decreased (p < 0.01) and the concentrations of N–NH₃, isovaleric and valeric acids increased linearly (p < 0.05) with an increase dietary concentrate level. Furthermore, an increased concentrate proportion reduced the population of Fibrobacter succinogenes and Ruminococus flavefaciens (p < 0.01) and increased the population of Selenomonas ruminantium and Megasphaera elsdenii (p < 0.01). The protozoa count revealed a predominance of the genus Entodinium. The synthesis of microbial N [g/d] and the efficiency of microbial synthesis [g of microbial N/kg of organic matter apparently digested in the rumen] increased as the proportion of concentrate was increased (p < 0.05). Therefore, it can be concluded that an increasing proportion of concentrate in sugarcane-containing diets enhances the synthesis of microbial protein and does not alter the fibre digestibility, although the population of fibre fermenting bacteria was reduced.     

The effect of rumen ciliates on chitinolytic activity,chitin content and the number of fungal zoospores in the rumen fluid of sheep

The objective of this study was to investigate the effect of selected protozoa on the degradation and concentration of chitin and the numbers of fungal zoospores in the rumen fluid of sheep. Three adult ewes were fed a hay-concentrate diet, defaunated, then monofaunated with Entodinium caudatum or Diploplastron affine alone and refaunated with natural rumen fauna. The average density of the protozoa population varied from 6.1 · 10⁴ (D. affine) to 42.2 · 10⁴ cells/ml rumen fluid (natural rumen fauna). The inoculation of protozoa in the rumen of defaunated sheep increased the total activity of chitinolytic enzymes from 2.9 to 3.6 μmol N-acetylglucosamine/g dry matter (DM) of rumen fluid per min, the chitin concentration from 6.3 to 7.2 mg/g DM of rumen fluid and the number of fungal zoospores from 8.1 to 10.9 · 10⁵ cells/ml rumen fluid. All examined indices showed diurnal variations. Ciliate population density was highest immediately prior to feeding and lowest at 4 h thereafter. The opposite effects were observed for the numbers of fungal zoospores, the chitin concentration and chitinolytic activity. Furthermore, it was found that chitin from zoospores may account for up to 95% of total microbial chitin in the rumen fluid of sheep. In summary, the examined ciliate species showed the ability of chitin degradation as well as a positive influence on the development of the ruminal fungal population.     

Short-chain fatty acids and CO2 as regulators of Na+ and Cl− absorption in isolated sheep rumen mucosa

Summary Unidirectional ²²Na⁺ and ³⁶Cl^– fluxes were determined in short-circuited, stripped rumen mucosa from sheep by using the Ussing chamber technique. In both CO₂/HCO _– ³ -containing and CO₂/HCO _– ³ -free solutions, replacement of gluconate by short-chain fatty acids (SCFA, 39 mM) significantly enhanced mucosal-toserosal Na⁺ absorption without affecting the Cl^– transport in the same direction. Short-chain fatty acid stimulation of Na⁺ transport was at least partly independent of Cl^– and could almost completely be abolished by 1 mM mucosal amiloride, while stimulation of Na⁺ transport was enhanced by lowering the mucosal pH from 7.3 to 6.5. Similar to the SCFA action, raising the PCO₂ in the mucosal bathing solution led to an increase in the amiloride-sensitive mucosal-to-serosal Na⁺ flux. Along with its effect on sodium transport, raising the PCO₂ also stimulated chloride transport. The results are best explained by a model in which undissociated SCFA and/or CO₂ permeate the cell membrane and produce a raise in intracellular H⁺ concentration. This stimulates an apical Na⁺/H⁺ exchange, leading to increased Na⁺ transport. The stimulatory effect of CO₂ on Cl^– transport is probably mediated by a Cl^–/HCO _– ³ exchange mechanism in the apical membrane. Binding of SCFA anions to that exchange as described for the rat distal colon (Binder and Mehta 1989) probably does not play a major role in the rumen.Abbreviations DIDS 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid - G _t transepithelial conductance (mS·cm^-2) - HSCFA undissociated short-chain fatty acids - J _ms mucosal-to-serosal flux (Eq · cm^-2 · h^-1) - J _net net flux (Eq · cm^-2 · h^-1) - J _sm serosal-to-mucosal flux (Eq · cm^-2 · h^-1) - PD transepithelial potential difference (mV) - SCFA dissociated short-chain fatty acids - SCFA short-chain fatty acids