Essential oils for dairy calves: effects on performance,scours, rumen fermentation and intestinal fauna

The first cause of death of dairy calves is often diarrhea which is mainly caused by pathogenic bacteria, which can result in excessive use of antibiotics. However, facing the increase concern by the industry and consumers, the use of antibiotics not only to control pathogens, but also to manipulate growth, has become a challenge. Alternative additives, such essential oils, have the potential to decrease antibiotic use, without reducing performance or increasing mortality of dairy calves. The objective of this study was to evaluate the use of a commercial blend of essential oils, incorporated into the calf starter and/or milk replacer to monitor the effect on overall calf performance, fecal scores and rumen fermentation parameters. A total of 30 Holstein calves received 6 l/day of a liquid diet, consisting of a commercial milk replacer containing 20% CP : 15% fat (EE). Calves had free choice access to water and calf starter. Weaning occurred at week 8, and calves were followed until the 10^th week of age. Calves were assigned to one of the three treatment groups in a randomized block design. Treatments: (1) control without essential oils supplementation (C); (2) essential oils blend in the milk replacer at 400 mg/kg (MR) and (3) essential oils blend in the milk replacer (200 mg/kg) and starter feed (200 mg/kg) (MRS). From the 2^nd week, calves were weighed and body measurements were taken, while concentrate intake and fecal scores were monitored daily. Blood samples were drawn weekly for determination of glucose and β-hydroxybutyrate. Fecal samples were collected weekly and analyzed for lactic acid bacteria and Enterobacteria; and ruminal fluid for determination of pH, short chain fatty acids, ammonia-N and counts of amylolytic and cellulolytic bacteria, and protozoa. Performance, fecal scores and intestines microorganisms were not affected by the essential oils supplementation. Ruminal and blood parameters were also not affected, with the exception the rumen ammonia-N concentration, with higher values when essential oils were supplemented in a combination of milk replacer and starter feed. Most of the evaluated parameters were affected by age of calves, mainly as a response to the increase in concentrate intake as animals’ aged. Essential oils are promising substitutes for antibiotics. However, the dose and routes of administration deserve further studies, allowing a better animal performance and health to be achieved.     

Quantification by real-time PCR of cellulolytic bacteria in the rumen of sheep after supplementation of a forage diet with readily fermentable carbohydrates: effect of a yeast additive

AIM: To examine the effect of concentrate and yeast additive on the number of cellulolytic bacteria in the rumen of sheep. METHODS AND RESULTS: Fibrobacter succinogenes, Ruminococcus albus and Ruminococcus flavefaciens were quantified using real-time PCR (targeting 16S rDNA) in parallel to cellulolytic flora enumeration with cultural techniques. Whatever the conditions tested, R. flavefaciens was slightly more abundant than F. succinogenes, with both species outnumbering R. albus. Before feeding, the shift from hay to hay plus concentrate diet had no effect on rumen pH and on the number of the three specie; while after feeding, the concentrate-supplemented diet induced a decrease (-1 log) of the number of the three species concomitant with the rumen acidification. Overall, the presence of the live yeast resulted in a significant increase (two- to fourfold) of the Ruminococci. CONCLUSION: The use of real-time PCR allowed us to show changes in the number of cellulolytic bacterial species in vivo in response to diet shift and additives that could not be as easily evidenced by classical microbial methods. SIGNIFICANCE AND IMPACT OF THE STUDY: This study contributes to the understanding of the negative impact of readily fermentable carbohydrates on rumen cellulolysis and the beneficial effect of yeast on rumen fermentation.     

Effects of essential oils on ruminal microorganisms and their protein metabolism

A commercial blend of essential oil (EO) compounds was added to a grass, maize silage, and concentrate diet fed to dairy cattle in order to determine their influence on protein metabolism by ruminal microorganisms. EO inhibited (P < 0.05) the rate of deamination of amino acids. Pure-culture studies indicated that the species most sensitive to EO were ammonia-hyperproducing bacteria and anaerobic fungi.     

Effects of Essential Oils on Ruminal Microorganisms and Their Protein Metabolism

A commercial blend of essential oil (EO) compounds was added to a grass, maize silage, and concentrate diet fed to dairy cattle in order to determine their influence on protein metabolism by ruminal microorganisms. EO inhibited (P < 0.05) the rate of deamination of amino acids. Pure-culture studies indicated that the species most sensitive to EO were ammonia-hyperproducing bacteria and anaerobic fungi.     

The effect of dietary concentrate and soya oil inclusion on microbial diversity in the rumen of cattle

Aims: Methane emissions from ruminants are a significant contributor to global greenhouse gas production. The aim of this study was to examine the effect of diet on microbial communities in the rumen of steers. Methods and Results: The effects of dietary alteration (50 : 50 vs 90 : 10 concentrate–forage ratio, and inclusion of soya oil) on methanogenic and bacterial communities in the rumen of steers were examined using molecular fingerprinting techniques (T‐RFLP and automated ribosomal intergenic spacer analysis) and real‐time PCR. Bacterial diversity was greatly affected by diet, whereas methanogen diversity was not. However, methanogen abundance was significantly reduced (P = 0·009) in high concentrate–forage diets and in the presence of soya oil (6%). In a parallel study, reduced methane emissions were observed with these diets. Conclusions: The greater effect of dietary alteration on bacterial community in the rumen compared with the methanogen community may reflect the impact of substrate availability on the rumen bacterial community. This resulted in altered rumen volatile fatty acid profiles and had a downstream effect on methanogen abundance, but not diversity. Significance and Impact of the Study: Understanding how rumen microbial communities contribute to methane production and how these microbes are influenced by diet is essential for the rational design of methane mitigation strategies from livestock.     

Toxicity of some terpenoids of essential oils of Xylopia aethiopica from Cameroon against Sitophilus zeamais Motschulsky

Abstract:  The acute toxicity of essential oils from the whole fruit (EF) or from the fibres of the fruit (FF) of the local aromatic plants Xylopia aethiopica Dunal (Annonaceae) collected in north Cameroon was evaluated on Sitophilus zeamais adults. A concentration of 1 ml of essential oil per 100 g of maize seeds was tested to determine weevil mortality after 24 h of exposure. Under these conditions, the essential oil derived from both EF and FF of X. aethiopica led to 100% mortality. In a second step, proportions of active compounds present in the oil of both EF and FF of X. aethiopica were quantified. The toxicity of the four main compounds was tested against S. zeamais : α -pinene, β -pinene, Δ-3-carene and terpinen-4-ol according to their proportion in the essential oil of the concerned plant part. β -pinene and terpinen-4-ol were responsible for 50% of the mortality at the proportion found in EF and FF essential oils respectively. When mixed, a synergic effect of the compounds was observed that restored the mortality percentage observed for the crude oil. It appears that X. aethiopica essential oil could be a potential source of natural and low-cost insecticide to control storage pests.     

Correlation between microbial enzyme activities in the rumen fluid of sheep under different treatments

Five total mixed rations prepared from finger millet (Eleusine Coracana) straw as a roughage (48%) and mixed concentrate (52%), supplemented with a 1% isoacid mixture (i-C4, i-C5, C5 and phenylacetic acid in equal proportions) or oil (groundnut oil, 5% more than the control) or urea (5% more nitrogen than the control), and protein (groundnut cake, 5% more nitrogen than the control) were given in a Latin square experiment to sheep. Enzymatic activities were estimated for urease, cellulase, protease, amylase, and lipase in various fractions of rumen fluid on the one hand and rumen microbial biomass on the other hand. Rumen samples were taken 3-4 hours after feeding and mixed rumen bacteria were separated as a strained rumen fluid without protozoa (SRFWP), cell free rumen fluid (CFRF) and enzymes associated with the bacteria cell (EABC). Samples of SRFWP and EABC contained higher enzyme activities than CFRF. Depending on the type of enzymes in each fraction, some significant coefficient of determination (r2) was seen. These values showed very close cooperative action between proteolytic and amylolytic enzymes under the experimental conditions, or perhaps the presence of some species of bacteria with both activities. Lipolytic bacteria are completely specialized for lipase production only (P < 0.05). The results showed oil, isoacid and crude protein enhanced microbial production (P < 0.05) and this can change the pattern of enzymes in the rumen of sheep.     

Antibacterial Action of Essential Oils of Artemisia as an Ecological Factor: II. Antibacterial Action of the Volatile Oils of Artemisia tridentata (Big Sagebrush) on Bacteria from the Rumen of Mule Deer

Rumen microorganisms of wild and captive deer were subjected to increasing amounts of volatile oils. The oils had a marked antibacterial effect on the rumen bacteria when the concentration reached approximately 16 muliters of oil per 10 ml of rumen fluid nutrient broth. The gross reactions of rumen bacteria obtained from wild, as well as captive, deer to the volatile oils seemed to be of the same magnitude; thus no adaptation by the bacteria to the oils was apparent.     

Validation of in sacco method: influence of sampling site,nylon bag or rumen contents,on fibrolytic activity of solid-associated microorganisms

Three ruminally cannulated cows, fed twice daily with a 70:30 forage:concentrate diet, were used to investigate the differences in fibrolytic activity of solid-associated microorganisms between nylon bags and rumen contents. Two different grass hays (regrowth and late harvested) were incubated in ruminal nylon bags. After 2 h or 23 h incubation time, pH was measured in bags and rumen contents, and enzymes of solid-associated microorganisms were extracted from bag residues and surrounding digesta by grinding, freezing, defrosting and sonication. Xylanase, avicelase, β-D-xylosidase and β-D-glucosidase activities were measured. Activities were lower in bag residues than in rumen digesta, and differences were greater after 2 h than after 23 h incubation time. Causes of these differences are discussed. For each incubation time and each enzyme, the differences in solid-associated microorganisms activities between rumen and bags contents were independent of the quality of hay in the bag. Thus the lower fibrolytic activity inside the bags may account for an underestimation of in vivo ruminal fiber degradation by the in sacco method, but this underestimation may be similar whatever the nature and content of forage cell walls.     

Evidence of Increased Diversity of Methanogenic Archaea with Plant Extract Supplementation

This study evaluated the effects of selected essential oils on archaeal communities using the ovine rumen model. Forty weaned Canadian Arcott ewes, fed with barley-based diet, were allotted to one of three essential oil supplementation treatments or a control (10 ewes per treatment) for 13 weeks. The treatments were cinnamaldehyde, garlic oil, juniper berry oil, and a control with no additive. Rumen content was sampled after slaughter and grouped by treatment by combining subsamples from each animal. DNA was extracted from the pooled samples and analyzed for methanogenic archaea using quantitative polymerase chain reaction, denaturing gradient gel electrophoresis, cloning, and sequencing. Our results suggest that the total copy number of archaeal 16S rRNA was not significantly affected by the treatments. The phylogenetic analysis indicated a trend toward an increased diversity of methanogenic archaea related to Methanosphaera stadtmanae, Methanobrevibacter smithii, and some uncultured groups with cinnamaldehyde, garlic, and juniper berry oil supplementation. The trends in the diversity of methanogenic archaea observed with the essential oil supplementation may have resulted from changes in associated protozoal species. Supplementation of ruminant diets with essential oils may alter the diversity of rumen methanogens without affecting the methanogenic capacity of the rumen.     

Comparison of Rumen Microbial Inhibition Resulting from Various Essential Oils Isolated from Relatively Unpalatable Plant Species

Essential oils were isolated from eight plant species which were relatively unpalatable to sheep and deer. The inhibitory potency of these essential oils upon sheep and deer rumen microorganisms was compared, in terms of total gas and volatile fatty acid (VFA) production, by use of an anaerobic manometric technique. Inhibitory effects of oils from the eight plant species may be placed in four groups: (i) essential oils from vinegar weed (Trichostema lanceoletum) and California bay (Umbellularia californica) inhibited rumen microbial activity most; (ii) lesser inhibition was exhibited by rosemary (Rosmarinus officinalis) and California mugwort (Artemisia douglasiana) oils, followed by (iii) blue-gum eucalyptus (Eucalyptus globulus) and sagebrush (Artemisia tridentata) oils; and (iv) oils from Douglas fir (Psuedotsuga menziesii) and Jerusalem oak (chenopodium botrys) resulted in the least inhibition, when 0.3 ml of each oil was used. A highly significant correlation coefficient (r = 0.98(**)) between total gas and VFA production indicated the validity of either method to measure the activity of rumen microorganisms. Our results are discussed in relation to the hypothesis that the selectivity and voluntary consumption of ruminants are related to the characteristic odor and antibacterial action of essential oils isolated from relatively unpalatable plant species.     

Antifungal activity of essential oils evaluated by two different application techniques against rye bread spoilage fungi

AIMS: To study how antifungal activity of natural essential oils depends on the assay method used. METHODS AND RESULTS: Oils of bay, cinnamon leaf, clove, lemongrass, mustard, orange, sage, thyme and two rosemary oils were tested by two methods: (1) a rye bread-based agar medium was supplemented with 100 and 250 microl l-1 essential oil and (2) real rye bread was exposed to 136 and 272 microl l-1 volatile oil in air. Rye bread spoilage fungi were used for testing. Method 1 proved thyme oil to be the overall best growth inhibitor, followed by clove and cinnamon. On the contrary, orange, sage and rosemary oils had very limited effects. Mustard and lemongrass were the most effective oils by the volatile method, and orange, sage and one rosemary showed some effects. Oil compositions were analysed by gas chromatography-mass spectrography. CONCLUSIONS: Antifungal effects of the essential oils depended on the application method. Larger phenolic compounds such as thymol and eugenol (thyme, cinnamon and clove) had best effect applied directly to medium, whereas smaller compounds such as allyl isothiocyanate and citral (mustard and lemongrass) were most efficient when added as volatiles. SIGNIFICANCE AND IMPACT OF THE STUDY: This study proves that the method used for screening essential oils as potential antimicrobials should correspond with the application sought.     

Antimicrobial activity of essential oils and structurally related synthetic food additives towards selected pathogenic and beneficial gut bacteria

AIMS: To assess the potential of essential oils and structurally related synthetic food additives in reducing bacterial pathogens in swine intestinal tract. METHODS AND RESULTS: The antimicrobial activity of essential oils/compounds was measured by determining the inhibition of bacterial growth. Among 66 essential oils/compounds that exhibited > or =80% inhibition towards Salmonellatyphimurium DT104 and Escherichia coli O157:H7, nine were further studied. Most of the oils/compounds demonstrated high efficacy against S. typhimurium DT104, E. coli O157:H7, and E. coli with K88 pili with little inhibition towards lactobacilli and bifidobacteria. They were also tolerant to the low pH. When mixed with pig cecal digesta, these oils/compounds retained their efficacy against E. coli O157:H7. In addition, they significantly inhibited E. coli and coliform bacteria in the digesta, but had little effect on the total number of lactobacilli and anaerobic bacteria. CONCLUSIONS: Some essential oils/compounds demonstrated good potential, including efficacy, tolerance to low pH, and selectivity towards bacterial pathogens, in reducing human and animal bacterial pathogens in swine intestinal tract. SIGNIFICANCE AND IMPACT OF THE STUDY: This study has identified candidates of essential oils/compounds for in vivo studies to develop antibiotic substitutes for the reduction of human and animal bacterial pathogens in swine intestinal tract.     

Rumen chemical and bacterial changes during stepwise adaptation to a high-concentrate diet in goats

The correlation between rumen chemical and bacterial changes was investigated during a four periodical stepwise adaptation to a high-concentrate diet (concentrate level at 0%, 30%, 50% and 70% for diet I to IV, respectively) in goats. The results showed that ruminal pH decreased from 6.7 to 5.5 after switching from diet I to II, and was maintained at about 5.5 on diet III. Denaturing gradient gel electrophoresis results showed that the rumen bacterial community was relatively stable during the initial three feeding periods, except for the appearance of three bands when diet changed from I to II, suggesting that an appropriate concentrate level can promote the proliferation of some bacteria. After 12 days of feeding diet III, total volatile fatty acid (VFA) concentration and butyrate proportion decreased. At days 2 and 3 of feeding diet IV, ruminal pH declined sharply to 5.3 and 4.7, respectively, and total VFA concentration decreased further while lactic acid concentration increased markedly, suggesting a relation between lactic acid accumulation and ruminal pH decline. At the same time, many bacteria disappeared, including most fibrolytic-related bacteria while Streptococcus bovis and Prevotella-like species dominated. Interestingly, Succinivibrio dextrinosolvens-like species maintained throughout the experiment, suggesting its tolerance to low pH. In conclusion, rumen bacterial community was relatively stable feeding 0% to 50% concentrate diets, and it was observed that appropriate concentrate levels in the diet could increase the diversity of rumen bacteria. However, concentrate-rich diets caused lactic acid accumulation and low ruminal pH that caused the disappearance of most fibrolytic-related bacteria sensitive to low pH while S. bovis and genus Prevotella persisted.     

不同精粗比底物下瘤胃真菌和纤维降解细菌共培养发酵特性及菌群变化

采用体外厌氧共培养技术,研究了瘤胃真菌和纤维降解细菌在不同精粗比(A组为全粗料,B组3∶7,C组5∶5,D组7∶3,E组为全精料)底物下菌群变化及其共培养发酵特性。结果表明:与0h相比,发酵至24h时B组和C组的厌氧真菌数量有较大幅度的上升,A组和D组则有所下降,E组未检测到真菌生长;纤维降解细菌随精粗比的增加呈上升趋势。发酵至48h时,各组均未检测到真菌生长;从A组到C组细菌数量呈上升趋势,此后急剧下降。DGGE结果表明,A、B和C组(精粗比低于5∶5)的DGGE图谱相似,有11条共有条带,但是当精粗比上升到7:3时,条带数目显著下降。随精料比例的增加,整个发酵期共培养系统中pH值显著下降(P<0.05)。整个发酵期间,共培养系统发酵产生的VFA主要为乙酸,丙酸和丁酸的量较少,乙酸与丙酸比值从A组到C组呈下降趋势,此后呈上升趋势。随精料比例的上升,发酵48h时总挥发性脂肪酸浓度从A组到C组呈上升趋势,此后呈下降趋势。发酵48h的羧甲基纤维素酶活和木聚糖酶活均以A组最高,而α-淀粉酶活从A组到D组逐渐增大,而E组最低,仅为B、C、D组的1/4~1/3。     

Evidence for existence of lectins on the ruminal bacteria from steers fed roughage and concentrate diets

Mixed rumen bacteria, isolated by centrifugation from the rumen of steers fed a roughage (R) or concentrate (C) diet, were used to determine if lectins are present on rumen bacteria, based on hemagglutination (HA) and HA inhibition assays in vitro. Rumen bacteria from steers fed either diet agglutinated erythrocytes from cattle, sheep, pigs, and rats, suggested that lectins exist on rumen bacteria. Bacterial HA titers from steers receiving the R diet were much higher (p<0.001) than those from steers fed the C diet, depending on the erythrocyte source used. Centrifugation at 20,000xg for 30 min fractionated the rumen bacteria into upper (U) and lower (L) layers. The HA titers of the U bacterial fractions were significantly higher (p<0.001) than those of the L fractions. A remarkable reduction or complete disappearance of HA titers following treatment of rumen bacteria with protease, trypsin, sodium dodecyl sulfate (SDS) or sodium periodate indicates that rumen bacterial lectins are probably glycoproteins. Lectin specificity for saccharides (galactose, lactose, N-acetyl-D-galactosamine, methyl-alpha-D-galactopyranoside and methyl-beta-D-galactopyranoside) and glycoproteins (mucin, fetuin, and thyroglobulin) was found in the RU, RL, and CU bacterial fractions; no specific binding was determined in the CL fractions. The potential role of lectins in mediating the attachment of rumen bacteria to feed particles, rumen epithelia and other microorganisms is discussed.     

Interaction of air ions and bactericidal vapours to control micro-organisms

AIMS: The aim of this study was to investigate the antibacterial activity of candles containing specific-antibacterial compounds, such as essential oils and their constituent compounds. The importance of the ionization products from the flame and the aerial concentration of the volatile compounds were investigated. METHODS AND RESULTS: Agar plates inoculated with Escherichia coli (DH5alpha) or Staphylococcus aureus (NCTC strain number 8532) were exposed in a large air-tight chamber to candle flames combined with the volatile bactericidal compounds beta-pinene and orange oil. A steady decline in E. coli numbers was observed over time because of the effect of a candle flame. This was significantly increased by the addition of volatile oils. The number of S. aureus colonies was not reduced by a plain candle, but significant reductions were caused following exposure to beta-pinene and orange oil candles. As aerial concentration of the volatiles was increased the viability of E. coli and S. aureus declined. Ionization products from the flame made a significant contribution to the observed effects, as intercepting the ions on a grounded grid over the agar plates allowed at least 20% more cells to survive. CONCLUSIONS: This study demonstrates the antibacterial properties of ionization products from a candle flame, and that this effect can be significantly increased by the addition of specific-antibacterial compounds, such as orange oil and beta-pinene. The role of both the ionization products from the candle flame and the concentration of volatile compounds released are important to the effect. SIGNIFICANCE AND IMPACT OF THE STUDY: The technique described here offers a new and novel technique for reducing the concentration of bacteria on surfaces.     

Influence of ciliate protozoa on biochemical changes and hydrolytic enzyme profile in the rumen ecosystem

AIMS: To assess the effect of presence or absence of rumen protozoa on fermentation characteristics and enzyme profile in growing lambs. METHODS AND RESULTS: Weaner lambs (G1, G2, G3, G4, G5 and G6 groups) were defaunated by oral administration of sodium laurel sulphate (at 8 g 100 kg(-1) body weight). The lambs of G4, G5 and G6 groups were refaunated. The roughage and concentrate ratio in the diet of G1 and G4, G2 and G5, and G3 and G6 were 50:50 (R1), 65:35 (R2) and 80:20 (R3), respectively. Daily dry matter intake was similar in defaunated and faunated lambs. However, digestibility of organic matter (OM), cellulose and gross energy were lower in defaunated lambs while crude protein (CP) digestibility was similar in both defaunated and faunated lambs. The rumen pH and NH3-N were lower (P < 0.01) while TVFA, total-N and TCA-ppt-N were higher (P < 0.01), in defaunated lambs. Ruminal activity of carboxymethyl cellulase was lower (P < 0.01) in defaunated lambs and amylase, xylanase, protease and urease were similar in faunated and defaunated lambs. Nutrient utilization, rumen metabolites and ciliate protozoal count were higher, whereas digestibility of fibre fractions was lower in high rather than low concentrate fed lambs. The rumen protozoa present before defaunation were B-type and the protozoa which re-established on refaunation were also B-type. CONCLUSIONS: Absence of ciliate protozoa decreased nutrient digestibility and increased ruminal TVFA and total-N with lower NH3-N concentration, indicating better energy and protein utilization in defaunated lambs. SIGNIFICANCE AND IMPACT OF THE STUDY: Defaunation improved energy and protein utilization in lambs.     

Antimicrobial activity of essential oils isolated from Portuguese endemic species of Thymus

AIMS: Thymus species are wild species mostly found in the arid lands of Portugal. Possible antimicrobial properties of Thymus essential oils have been investigated. The chemical composition of the essential oils and the antimicrobial activity of Thymus mastichina (L) L. subsp. mastichina, T. camphoratus and T. lotocephalus from different regions of Portugal were analysed. METHODS AND RESULTS: Hydrodistillation was used to isolate the essential oils and the chemical analyses were performed by gas chromatography (GC) and GC coupled to mass spectrometry. The antimicrobial activity was tested by the disc agar diffusion technique against Candida albicans, Escherichia coli, Listeria monocytogenes, Proteus mirabilis, Salmonella spp. and Staphylococcus aureus. Pure linalool, 1,8-cineole and a mixture (1 : 1) of these compounds were included. Linalool, 1,8-cineole or linalool/1,8-cineole and linalool/1,8-cineole/linalyl acetate were the major components of the essential oils, depending on the species or sampling place. The essential oils isolated from the Thymus species studied demonstrated antimicrobial activity but the micro-organisms tested had significantly different sensitivities. CONCLUSIONS: The antimicrobial activity of essential oils may be related to more than one component. SIGNIFICANCE AND IMPACT OF THE STUDY: Portuguese endemic species of Thymus can be used for essential oil production for food spoilage control, cosmetics and pharmaceutical use. Further studies will be required to elucidate the cell targets of the essential oil components.