Larvicidal evaluation of two novel cationic gemini surfactants against the potential vector of West Nile virus Culex pipiens Linnaeus (Diptera: Culicidae).

The development of insecticide resistance is a serious consequence of the widespread applications of synthetic insecticides. Recent studies have provided alternatives to currently available insecticides. Here, novel cationic gemini surfactants were synthesized to assess their insecticidal activities using laboratory and field strains larvae of Culex pipiens Linnaeus (Diptera: Culicidae). The efficacy of these surfactants was compared to that of clove oil and spinosad. The two surfactants G1 and G2 showed good insecticidal activities in laboratory strain with LC50 0.013 and 0.054 ppm, respectively, relative to spinosad with LC50 0.027 ppm, 48 h posttreatment. Although spinosad showed high efficiency against lab strain, it exhibited a high resistance ratio (RR) of 15.111 and 13.111 toward the field strain at 24 and 48 h posttreatment, respectively. The two gemini surfactants have a good safety profile and low RR (RR <5), which is close to clove oil; however, G1 and G2 presented high activities with 11,043.230 and 2658.648 folds, respectively, compared to clove oil. The treated Cx. pipiens larvae showed severe morphological malformations after treatment with gemini surfactants. The results of this study are promising in terms of developing novel, effective, affordable, and safe approaches for mosquito control strategies to reduce the risk of arbovirus transmission, which remains a global public health threat.

Bio-molluscicidal potential and biochemical mechanisms of clove oil and its main component eugenol against the land snail, Theba pisana.

The land snail, Theba pisana is a serious pest that adversely affects various crops in sustainable agriculture. Essential oils and their constituents represent an environmentally sound alternative to synthetic pesticides. Our study aimed to investigate the lethal and sub-lethal toxicity of clove oil and its main component eugenol to understand the mechanisms underlying its toxic action against T. pisana. The GC-MS profile of the clove oil composition was characterized. In the laboratory experiment, LD50 of clove oil and eugenol via the contact testing were determined after 48 and 72 h. Moreover, sub-lethal effects of clove oil or eugenol on the survivors following the exposure of snails to the 25 and 50% of the LD50/48 and 72 h were evaluated through using snail tissues for biochemical measurments. The GC-MS analysis showed that eugenol (64.87%) was the major constituent present in the oil. The results also showed that LD50 values at 48 and 72 h were 2006.5 and 1493.5 µg/g b.w for oil and 239.6 and 195.3 µg/g b.w for eugenol, respectively. Compared to control, the sub-lethal effects of clove oil or eugenol at 48 and 72 h showed a significant increase in reduced glutathione (GSH) levels. Catalase (CAT) and glutathione-S-transferase (GST) activities significantly elevated in oil- or eugenol-treated snails, except at low dose after 48 h. After two exposure times, snails exposed to oil or eugenol at both sub-lethal effects had considerably higher γ-glutamyltransferase (γ-GT) and aspartate aminotransferase (AST) activities. Moreover, markedly augmentation in alkaline phosphatase (ALP) and alanine aminotransferase (ALT) activities at all exposure times, with the exception of snails treated with low dose of eugenol after 48 h was observed. Both clove oil and eugenol at the tested doses caused a significant inhibition in acetylcholinesterase (AChE) activity at two exposure times. Our findings highlight the potential of clove oil and eugenol, as an efficient natural molluscicide alternative to its synthetic counterparts for snail control.

Extending shelf life and optimizing effective parameters by using clove oil (Syzygium aromaticum oleum)/orange oil (Citrus aurantium var dulcis oleum) in bread: thermal, morphological and sensory approach.

The aim of the present study was to extend the shelf life of bread by using organic additives and optimization techniques. Quadruple effects of temperature (-18, + 4, + 20 °C), clove oil (0, 1, 2%), orange oil (0, 1, 2%), residence time (7, 14, 21 days) were investigated to determine optimum bread as moisture response. Quadratic model was found to be suitable with an accuracy of 0.9854 for moisture response. Rheological properties remained intact for 7 days at 20 °C in bread with 1% clove oil and 1% orange oil added. The effect of 4 effective parameters from 3 levels was examined with Box-Behnken in Design Expert. Optimum and control samples at two different temperatures were determined with SEM, TG-DTA and polarized light microscope. Consumer survey was conducted as appearance, colour, smell for optimum, control samples for bread and cake. It was concluded that bread samples could be stored with clove and orange oil at + 20 °C with period of 7 days without losing their quality properties.

Antimicrobial Efficacy of Blended Essential Oil and Chlorhexidine against Periodontal Pathogen (P.gingivalis)-An In Vitro Study.

Background: Essential oils (EOs) have a considerable amount of therapeutic and preventive effect in treating dental diseases due to their wider potential as antibacterial and anti-inflammatory agents. EOs like virgin coconut oil, eucalyptus oil, peppermint oil thyme oil, and clove oil, when used in combination, may further have enhanced antimicrobial effects. However, limited information exists on the synergistic effect of these oils when used in combination, especially on the primary periodontal pathogen Porphyromonas gingivalis. Aim: The current study aims to compare the antimicrobial efficacy of commercially available EO on the periodontal pathogen, P. gingivalis, in comparison to chlorhexidine (CHX). Materials and Methods: Antimicrobial efficacy of EO and CHX was assessed at various concentrations against the periodontal pathogen P. gingivalis, by evaluating the minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC). Results: P. gingivalis was seen to be sensitive at a MIC of 100 µg/ml and 50 µg/ml concentration of the EO, which is regarded as the MIC of EO against P. gingivalis and CHX effectively inhibited microbial growth at 0.4 µg/ml. Conclusion: A combination of EOs possesses a potent antibacterial activity against P. gingivalis, and the antibacterial efficacy increases with increasing concentration of EOs.

Oral microbiota associated with gingiva of healthy, gingivitis and periodontitis cases.

Oral microbes coexist with each other in a symbiotic relationship or as commensals in healthy body. Teeth and oral cavity harbor diverse community of fungi and bacteria. This study focused on bacterial and fungal component of gingiva, where the last occupy little attention. In addition to study the antimicrobial activity of toothpastes, mouth washes and natural oils against microorganisms. Sixty swabs from outer surfaces of gingiva in healthy persons, as well as patients complaining of gingivitis and periodontitis were collected for fungal and bacterial analyses. Sensitivity of the isolated microorganisms to some pharmaceutical preparations and natural oils was also performed. Ten fungal and 9 bacterial species were identified. There is a highly significant variation in the frequency of Klebsiella pneumonia among healthy, gingivitis and periodontitis. Also, Candida tropicalis and cocci bacteria showed significant diversity among the three tested groups. Among pharmaceutical preparations (toothpastes and mouth washes) and natural oils, Paradontax, Hexitol and clove oil showed the best antimicrobial activity against tested fungal and bacterial strains. Although, minimum inhibition concentrations (MICs) of clove oil were high compared to Paradontax and Hexitol, nevertheless, it is highly recommended as both antifungal and antibacterial agent against oral pathogenic microorganisms, because it is a natural compound and nearly devoid of side effects.

Carcass characteristics and meat evaluation of cattle finished in temperate pasture and supplemented with natural additive containing clove, cashew oil, castor oils, and a microencapsulated blend of eugenol, thymol, and vanillin.

BACKGROUND: Forty crossbred steers were supplemented with different doses (from 0 control to 6000 mg/animal/day) of natural additive blend containing clove essential oil, cashew oil, castor oil, and a microencapsulated blend of eugenol, thymol, and vanillin for 80 days. Carcass characteristics, drip loss, and antioxidant activity were evaluated 24 h post mortem on longissimus thoracis, and the effects of aging (until 14 days) were evaluated for water losses (thawing/aging and cooking), texture, color, and lipid oxidation. RESULTS: The use of the natural additive blend did not modify (P > 0.05) carcass characteristics but did, however, modify body composition (P < 0.05). Drip losses were unaffected by the treatments tested (P > 0.05). There was an observed quadratic effect (P < 0.05) on losses from thawing/aging on the first day of storage. Regarding the effects of natural additives on cooking losses, there was a quadratic effect (P < 0.05) among the treatments on day 7 of aging. Differences between days of aging were only observed with control treatment. Shear force was similar among treatments on days 1 and 7 of aging. On day 14 a linear effect (P < 0.05) was observed. Also, a linear effect (P < 0.05) appeared on meat lightness, meat from the control group being clearer on day 1. No changes were observed in redness among treatments or days of storage (P > 0.05). Yellowness was not modified by the treatments (P > 0.05)but only by the days of storage in control and the lowest dosage used. CONCLUSION: The blend of natural additives has potential use in pasture feeding and could improve meat quality. However, doses should be adjusted. © 2021 Society of Chemical Industry.

In vitro inhibition of a multispecies oral cavity biofilm by Syzygium aromaticum essential oil.

OBJECTIVE: To evaluate the anti-fungal activity of Syzygium aromaticum essential oil and its inhibition of a multispecies biofilm in patients with oral candidiasis. BACKGROUND: Inhibiting biofilm formation on the denture surface is an important practice for preventing denture stomatitis. MATERIALS AND METHODS: The anti-fungal activity against Candida albicans and non-albicans Candida species was evaluated through the microdilution method to define Minimal Inhibitory (MIC) and Fungicidal (MFC) Concentrations. Time-kill assay assessed growth kinetics of C. albicans based on pre-determined time points (0, 1, 2, 4, 6 and 24 hours). A multi-species biofilm was formed using human saliva from patients with oral candidiasis and anti-biofilm activity determined by Colony Forming Units per milliliter (CFU/mL) count, fluorescence microscopy with calcofluor white to observe yeast presence and structure, and metabolic activity by XTT (2,3-Bis-(2Methoxy-4-Nitro-5-Sulfophenyl)-2H-Tetrazolium-5-carboxanilide) reduction assay. RESULTS: The essential oil showed an anti-fungal activity against all Candida species (MIC 500-1000 µg/mL, MFC 1000-2000 µg/mL), and the time-kill assay showed that 2000 µg/mL (from 2 hours onward) and 1000 µg/mL (from 4 hours onward) concentrations had substantially lower yeast growth than the negative control. In the biofilm analysis, the essential oil had a lower CFU/mL count and a biofilm metabolic activity (91.4%) than seen with its negative control, and in both analyses, the essential oil was not significantly different from the positive control (chlorhexidine). Morphological analysis showed amorphous and fragmented cellular structures after treatment with the essential oil. CONCLUSION: Syzygium aromaticum essential oil had anti-fungal activities, reduced the Candida growth kinetics substantially and inhibited the multi-species biofilm formation.

Antimicrobial behavior and mechanism of clove oil nanoemulsion.

Clove oil has many functions such as antibacterial, anti-inflammatory, anti-oxidation. In this experiment, a self-emulsification method was used to prepare clove oil nanoemulsion. And then filter paper diffusion method, minimum inhibitory concentration, and minimum bactericidal concentration were used to study the inhibitory behavior of clove oil nanoemulsion on Escherichia coli and Staphylococcus aureus. And explore the antibacterial mechanism by dynamically testing the content of nucleic acid and protein in the culture solution during the antibacterial process. The results show that when the surfactant content is 10 wt%, the hydrophile-lipophile balance (HLB) is 13.93, and the oil phase content is 2 wt%, a clove oil nanoemulsion with better dispersion and smaller average particle size can be prepared. The minimum inhibitory concentration (MIC) of clove oil nanoemulsion against Escherichia coli and Staphylococcus aureus is 0.5, 0.25 mg/mL, and the minimum bactericidal concentration (MBC) is 1, 2 mg/mL. The increase in protein content and the exponential growth of nucleic acid release also indicated that the clove oil nanoemulsion destroys the integrity of the cell membrane. The experimental results can provide a reference for the application of clove oil nanoemulsion in food, medicine and other fields.

Rhamnolipids and essential oils in the control of mosquito-borne tropical diseases.

The diseases transmitted by mosquito vectors are a great public health issue. Thus, effective vector control becomes the main strategy to reduce their prevalence. However, insecticide resistance has become a huge concern for the mitigation of mosquitoes; here, we propose the use of rhamnolipids in emulsion with clove oil against Aedes aegypti and Culex quinquefasciatus. The toxicity of rhamnolipids and clove oil to two species of mosquitoes transmitting tropical diseases was investigated. After 24 h, the LC50 was 140 mg/L when rhamnolipids were used and 154 mg/L when clove oil was used against Aedes aegypti larvae. In the case of Culex quinquefasciatus, the LC50 was 130 mg/L for rhamnolipids and 19 mg/L for clove oil. When the concentrations of the upper limits of one of the solutions (rhamnolipid or clove oil) were mixed, 100% mortality was obtained after 24 h. The bioassay of insecticidal action for solutions of rhamnolipids and clove oil in the lower limit, upper limit, and lethal concentration 50 to determine the effect on 50% of the population (KD50) achieved low results from KD50 to the upper limit compared to the other concentrations for both Aedes aegypti and Culex quinquefasciatus. The rhamnolipids and clove oil at the upper limit concentration had the greatest repellent activity against the two mosquito species. Bioassays using different concentrations of rhamnolipids revealed variations in the morphology of the intestinal epithelium (800 mg/L). A concentration of 900 mg/L led to the most severe morphological changes in the organization of the epithelium and the cells lining the intestines of these larvae. When larvae were exposed to a concentration of 1000 mg/L, the marginalization of chromatin in the nucleus of epithelial cells was very severe, indicating the onset of cell death.Key points• The toxicity of rhamnolipids and clove oil has a larvicidal, insecticidal, and repellent effect.• The combination of concentrations of these compounds enhances their action.• Different concentrations of rhamnolipids led to severe morphological changes in the organization of the epithelium and the cells and the intestines of larvae.

Synergistic bactericidal effect of clove oil and encapsulated atmospheric pressure plasma against Escherichia coli O157:H7 and Staphylococcus aureus and its mechanism of action.

We investigated the bactericidal effect of clove oil and encapsulated atmospheric pressure plasma (EAP), individually or in combination, against Escherichia coli O157:H7 and Staphylococcus aureus. The bactericidal effect of the combined treatment was also investigated in inoculated beef jerky. For both pathogens, clove oil and EAP single treatments resulted in less than 3.0-log reductions, whereas the combined treatment resulted in more than 7.5-log reductions. The disc-diffusion assay and gas chromatography-mass spectrometry showed no changes in both the clear zone diameter and chemical composition of clove oil before and after the EAP treatment. Significant changes in cell membrane permeability and cell morphology resulting from the combined treatment of clove oil and EAP were evidenced by increased in UV absorption of cell supernatants, increased cell staining with propidium iodide, and changes in cell structure revealed by transmission electron microscopy. The synergistic bactericidal effects of clove oil and EAP against both pathogens were also observed in inoculated beef jerky, but the treatments were less effective against S. aureus, presumably due to thicker peptidoglycan layer. Experiments also demonstrated that the synergistic bactericidal effects between clove oil and EAP are due to clove oil increasing the susceptibility of the bacteria to subsequent EAP treatment, and does not involve alteration of the antibacterial activity of clove oil by EAP.

Biological Properties and Prospects for the Application of Eugenol-A Review.

Eugenol is a phenolic aromatic compound obtained mainly from clove oil. Due to its known antibacterial, antiviral, antifungal, anticancer, anti-inflammatory and antioxidant properties, it has long been used in various areas, such as cosmetology, medicine, and pharmacology. However, high concentrations can be toxic. A dose of 2.5 mg/kg body weight is regarded as safe. This paper reviews the current state of knowledge regarding the activities and application of eugenol and its derivatives and recent research of these compounds. This review is based on information concerning eugenol characteristics and recent research from articles in PubMed. Eugenol remains of great interest to researchers, since its multidirectional action allows it to be a potential component of drugs and other products with therapeutic potential against a range of diseases.

Investigating Antiarthritic Potential of Nanostructured Clove Oil (Syzygium aromaticum) in FCA-Induced Arthritic Rats: Pharmaceutical Action and Delivery Strategies.

The combined application of clove oil in a lipid nanocarrier opens a promising avenue for bone and joints therapy. In this study, we successfully developed a tunable controlled-release lipid platform for the efficient delivery of clove oil (CO) for the treatment of rheumatoid arthritis (RA). The ultra-small nanostructured lipid carriers co-loaded with CO (CONCs) were developed through an aqueous titration method followed by microfluidization. The CONCs appeared to be spherical (particle size of 120 nm), stable (zeta potential of -27 mV), and entrapped efficiently (84.5%). In toluene:acetone:glacial acetic acid (90:9:1 percent v/v/v) solvent systems, high-performance thin layer chromatography (HPTLC) analysis revealed the primary components in CO as eugenol (RF = 0.58). The CONCs greatly increased the therapeutic impact of CO in both in vitro and in vivo biological tests, which was further supported by excellent antiarthritic action. The CONC had an antiarthritic activity that was slightly higher than neat CO and slightly lower than standard, according to our data. The improved formulation inhibited serum lysosomal enzymes and proinflammatory cytokines while also improving hind leg function. This study provides a proof of concept to treat RA with a new strategy utilizing essential oils via nanodelivery.

Anticancer Properties of Eugenol: A Review.

Conventional cancer treatments have shown several unfavourable adverse effects, as well as an increase in anticancer drug resistance, which worsens the impending cancer therapy. Thus, the emphasis is currently en route for natural products. There is currently great interest in the natural bioactive components from medicinal plants possessing anticancer characteristics. For example, clove (Syzygium aromaticum L.) (Family Myrtaceae) is a highly prized spice that has been historically utilized as a food preservative and for diverse medical uses. It is reckoned amongst the valued sources of phenolics. It is indigenous to Indonesia but currently is cultivated in various places of the world. Among diverse active components, eugenol, the principal active component of S. aromaticum, has optimistic properties comprising antioxidant, anti-inflammatory, and anticancer actions. Eugenol (4-allyl-2-methoxyphenol) is a musky oil that is mainly obtained from clove. It has long been utilized all over the world as a result of its broad properties like antioxidant, anticancer, anti-inflammatory, and antimicrobial activities. Eugenol continues to pique investigators' interest because of its multidirectional activities, which suggests it could be used in medications to treat different ailments. Anticancer effects of eugenol are accomplished by various mechanisms like inducing cell death, cell cycle arrest, inhibition of migration, metastasis, and angiogenesis on several cancer cell lines. Besides, eugenol might be utilized as an adjunct remedy for patients who are treated with conventional chemotherapy. This combination leads to a boosted effectiveness with decreased toxicity. The present review focuses on the anticancer properties of eugenol to treat several cancer types and their possible mechanisms.

Antibacterial and antioxidant activity of clove oil against Streptococcus iniae infection in Nile tilapia (Oreochromis niloticus) and its effect on hepatic hepcidin expression.

This study was designed to evaluate the modulating effect dietary clove essential oil (CL) has on the antioxidant and immunological status of Nile tilapia following Streptococcus iniae (Si) infection. Fish were placed on either control or (1.5 and 3%) CL-supplemented diets for 4 weeks. After sampling, the remaining fish in the control group were divided into 2 groups: an unchallenged (negative control) and an Si-challenged positive control. On the other hand, the remaining fish in CL-supplemented groups were challenged with Si, and mortality was checked for two weeks before the final sampling. Serum immunological parameters, tissue antioxidants, and oxidative stress markers were determined. Moreover, hepatic hepcidin expression was also measured in different groups. The obtained results showed improvements in blood phagocytic, bactericidal, lysozyme, and respiratory burst activities in CL-supplemented fish before and after the Si challenge. Si-challenge caused a remarkable increase in tissue malondialdehyde (MDA) levels that was inhibited by CL supplementation. The activities of glutathione peroxidase (GPx) and superoxide dismutase (SOD) in tissues were significantly elevated in a dose-dependent manner in CL-supplemented groups in both pre- and post-challenge experiments; renal SOD did not show any differences. Hepatic nitric oxide (NO) level was significantly decreased in CL-supplemented fish in a dose-dependent manner. In the post-challenge experiment, nitrosative stress was apparent in the liver and kidney; however, CL supplementation was sufficient to reverse it. Interestingly, a remarkable induction of the hepatic hepcidin expression was observed in all CL-supplemented groups in the pre-challenge experiment and Si-challenged fish, underscoring the role of CL as an antibacterial through inducing hepatic hepcidin expression to combat S. iniae infection. CL-supplementation was associated with lower mortality rates after Si-challenge, which was more pronounced in CL-3% supplemented fish. In conclusion, our results demonstrate that CL has a potent antioxidant role via increasing antioxidant enzymes' activities and antagonizing lipid peroxidation. Moreover, CL has an immune-stimulant effect by inducing the hepatic hepcidin expression and immunological markers in response to S. iniae infection.

Antifungal activity of the lemongrass and clove oil encapsulated in mesoporous silica nanoparticles against wheat's take-all disease.

Combined application of plant essential oils (EOs) with known antimicrobial effects and silica nanocapsules with high loading capacity and protection capability of the EOs make them proper candidates for creating environmentally friendly fungicides. In this study, EOs of the Lemongrass (LGO) and Clove (CO) were used against Gaeumannomyces graminis var. tritici (Ggt), a causal agent of take-all disease of wheat. To provide controlled delivery of the EOs, they were encapsulated into mesoporous silica nanoparticles (MSNPs) and then compared to the effects of pure EOs both in- vitro and in- vivo. MSNPs were synthesized via the sol-gel process. Various techniques such as Fourier transform infrared spectroscopy (FTIR), the Brunauer-Emmett-Teller (BET), thermogravimetric analysis (TGA), and UV-Vis spectroscopy were used to evaluate the successful loading of the EOs into the pore of MSNPs. The encapsulation efficiency (EE) was calculated as high as 84.24% for LGO and 80.69% for CO, while loading efficiency (LE) was determined 36% and 29% for LGO and CO, respectively. Scanning electron microscopy (SEM) and transmission electron microscopy (TEM) displayed spherical shapes and porous structures with average diameters of 50-70 nm. Recognition of the main components of the EOs via gas chromatographic-mass spectrometry (GC-MS) before and after the EO loading, detected eugenol and citral as the most frequent compounds in LGO and CO, respectively. For antifungal test in- vitro, selected concentrations of the pure EOs, EOs loaded in MSNPs (EOs- MSNPs) and Mancozeb ® fungicide based on pre-tests were mixed using potato dextrose agar (PDA). The inhibition percentage (IP) of fungal growth at each concentration, as well as minimum inhibition concentration (MIC) and minimum fungicidal concentrations (MFC) were obtained. The results indicated that antifungal effects in the encapsulated form increased by up to three times. In- vivo, the sterile wheat seeds were treated with pure EOs, EOs-MSNPs, and mancozeb at MFC concentration. Also, in order to keep on the EOs-MSNPs around the seeds, sodium alginate was used. The consequences of in- vivo experiments indicated that rate of disease control in presence of EOs-MSNPs and mancozeb was the same (~70%) and higher than pure EOs (LGO: 57.44%, CO: 49%). Also, improving the growth parameters in wheat plant, the covering of the EOs-MSNPs in alginate, had better control (84%) than that of EOs-MSNPs alone. Further, the release kinetics studies showed a gradual release of LGO and CO from MSNPs for four weeks in water and for five weeks in the soil-plant system. To the best of our knowledge, this is the first report of the control effect of LGO, CO, and their nanocapsule in MSNPs against the take-all disease of wheat. These results showed that the EOs-MSNPs can be a safe product for the efficient control of take-all disease in wheat crop.

Clove Oil (Syzygium aromaticum L.) Activity against Alicyclobacillus acidoterrestris Biofilm on Technical Surfaces.

Acidotermophilic bacteria Alicyclobacillus acidoterrestris is one of the main contaminants in the fruit industry forming biofilms which are difficult to remove from the production line by conventional methods. An alternative approach aims for the use of essential oils to prevent Alicyclobacillus biofilm development. The effect of clove essential oil on A. acidoterrestris biofilms on glass and polyvinyl chloride surfaces under static and agitated culture conditions was investigated by atomic force microscopy and the plate count method. The medium-flow and the type of technical surface significantly influenced A. acidoterrestris biofilm. The PVC was colonized in a greater extent comparing to glass. Clove essential oil in 0.05% (v/v) caused 25.1-65.0% reduction of biofilms on the technical surfaces along with substantial changes in their morphology by a decrease in the biofilm: height, surface roughness, and surface area difference. The oil also induced alteration in individual bacterial cells length and visible increase of their roughness. Clove essential oil seems to release EPS from biofilm and thus induce detachment of bacteria from the surface. Due to anti-A. acidoterrestris biofilm activity, the clove oil may be used in the juice industry to hinder a development of A. acidoterrestris biofilms on production surfaces.

Concentration effects of three common fish anesthetics on Pacific hagfish (Eptatretus stoutii).

The efficacy of three common fish anesthetics (clove oil, 2-phenoxyethanol, and tricaine methanesulfonate) was evaluated in the Pacific hagfish (Eptatretus stoutii). The overarching aim of our study was to identify the best anesthetic and concentration for the purposes of routine laboratory use of Pacific hagfish (i.e., short and consistent induction and recovery times and minimized stress and safety risk to hagfish). The objectives of our study were fourfold: (1) identify anesthetic stages of Pacific hagfish using clove oil anesthesia; (2) establish standardized anesthesia preparation procedures; (3) determine the optimal anesthetic and concentration for safely achieving stage V anesthesia; and (4) investigate the effects of repeatedly exposing Pacific hagfish to anesthesia. Experimental concentrations, ranging from 50 to 400 mg/L, of each anesthetic were tested on at least three Pacific hagfish individuals. We found the following: (1) Pacific hagfish exhibited similar stages of anesthesia to those described for bony fishes; (2) sufficient mixing of clove oil with seawater had a considerable effect on the consistency and timing of anesthetic induction; (3) concentration and anesthetic significantly impacted induction and recovery timing, whereas body mass had no impact on anesthetic trends; and (4) repeatedly exposing Pacific hagfish to optimal concentrations of clove oil or MS-222 had no effect on induction or recovery timing, whereas exposure number significantly impacted induction timing when using 2-PE. Due to consistent induction and recovery times, low risk of accidental overdose, and high safety margins for both handler and hagfish, we recommend 175 mg/L of clove oil as the ideal anesthetic and concentration for the routine laboratory use of Pacific hagfish.

Short communication: Evaluation of the efficacy of novel disbudding methods for dairy calves.

The objectives of this study were to (1) evaluate whether cryoablation or the administration of clove oil was as efficacious as cautery disbudding at preventing horn growth, and (2) evaluate whether the efficacy of cautery disbudding is affected by removing or leaving the horn bud tissue intact after disbudding of dairy calves. At approximately 4 d of age (4.0 ± 0.88 d of age, mean ± SD), 265 dairy heifer calves from 3 dairy farms (farm 1: n = 129 calves; farm 2: n = 109 calves; farm 3: n = 27 calves) were disbudded. Each calf had 1 of 4 treatments randomly assigned to each horn bud: (1) clove oil (0.5 mL) administered subcutaneously under the horn bud (CLOV, n = 135 buds); (2) a liquid nitrogen-filled probe applied to the horn bud area for 30 s (CRYO, n = 134 buds); (3) cautery disbudding using an electric hot-iron and the horn bud removed (BUDOFF, n = 130); or (4) cautery disbudding and the horn bud tissue left intact (BUDON, n = 131). Calves were assessed for signs of infection at the disbudding site frequently within the first month after disbudding. At approximately 6 mo of age (6 ± 2.2 mo, mean ± SD) calves were assessed for scur or horn growth. The disbudding procedures were considered successful if no scur or horn development was observed. Within the first month, 12% of disbudding wounds showed some indication of infection, such as pus, exudate, or swelling; of the infected buds, 52% were associated with the BUDON treatment, 27% with CLOV, 25% with BUDOFF, and 2% with CRYO treatments. At 6 mo of age, BUDOFF was the most effective method of preventing horn growth and CRYO was the least efficacious [mean percentage of success: BUDOFF: 100% (95% CI: 97.7-100.0); CRYO: 1% (95% CI: 0.2-5.3)]. Injecting clove oil under the horn bud was 87% (95% CI: 80.6-92.5) successful. Not removing the horn bud tissue after cautery disbudding reduced the efficacy of this method by 9% [91% success (95% CI: 83.8-95.7)]; moreover, this method was associated with more infection at the site of disbudding. It appears as though the clove oil treatment could be used as an alternative to cautery disbudding of dairy calves; however, further research is needed to evaluate the tissue damage and associated pain caused by clove oil and to refine this technique (i.e., administration methods to improve efficacy) before it could be considered an alternative to cautery.

The effects of commonly used anaesthetics on colour measurements across body regions in the poeciliid fish, Girardinus metallicus.

The effects of common anaesthetics on the hue, saturation and brightness measurements of the poeciliid fish Girardinus metallicus were investigated in two experiments. For both experiments the coloration of four body regions was measured from digital images of the same males obtained under three conditions: (1) control (in a water-filled chamber); (2) anaesthetised with MS-222; and (3) anaesthetised with eugenol (clove oil). In experiment 1 anaesthetised fish were photographed out of water. In experiment 2 all photographs were taken in a water-filled chamber. Anaesthetics altered coloration in both experiments. In the more methodologically consistent experiment 2 we found significantly different hue, increased saturation and decreased brightness in anaesthetic v. control conditions, consistent with darkening caused by the anaesthetics. The body regions differed in coloration consistent with countershading but did not differentially change in response to anaesthesia. These findings suggest that photographing fish in a water-filled chamber without anaesthetic is preferable for obtaining digital images for colour analysis and that multiple body regions of fish should be measured when assessing coloration patterns meaningful in behavioural contexts, to account for the gradients caused by countershading. We are encouraged that some researchers employ such methods already and caution against using anaesthetics except when absolutely necessary for immobilisation.

Essential Oils as Antimicrobial Agents-Myth or Real Alternative?

Herbs and the essential oils derived from them have been used from the beginning of human history for different purposes. Their beneficial properties have been applied to mask unpleasant odors, attract the attention of other people, add flavor and aroma properties to prepared dishes, perfumes, and cosmetics, etc. Herbs and essential oils (EOs) have also been used in medicine because of their biological properties, such as larvicidal action, analgesic and anti-inflammatory properties, antioxidant, fungicide, and antitumor activities, and many more. Many EOs exhibit antimicrobial properties, which is extremely important in fields of science and industry, such as medicine, agriculture, or cosmetology. Among the 250 EOs which are commercially available, about a dozen possess high antimicrobial potential. According to available papers and patents, EOs seem to be a potential alternative to synthetic compounds, especially because of the resistance that has been increasingly developed by pathogenic microorganisms. In this review we summarize the latest research studies about the most-active EOs that are known and used because of their antimicrobial properties. Finally, it is noteworthy that the antimicrobial activities of EOs are not preeminent for all strains. Further investigations should, thus, focus on targeting EOs and microorganisms.