extfieldsofstudy: Biology Medicine

text:
Suitability of layer-type male chicks for capon production

ABSTRACT The aim of this study was to determine the effect of age and caponization on the growth performance and carcass quality characteristics of Leghorn cockerels. The experiment was conducted on 224 Leghorn cockerels. At 8 wk of age, 112 birds were surgically castrated by a qualified veterinarian in accordance with Commission Regulation (EC) No. 543/2008. The birds were divided into 2 sex categories (with 8 replications per group and 14 birds per replication). The birds were raised to 28 wk of age, and were fed commercial diets ad libitum. From 12 wk of age, at 4-wk intervals, 8 intact cockerels and 8 capons (1 bird per replication) were selected randomly and slaughtered. Caponization had a beneficial influence on the feed conversion ratio (FCR). FCR (kg/kg) based on body weight (BW) gain, carcass weight gain, and edible weight gain was lower in capons from 24 wk of age (P < 0.05), and FCR based on lean weight gain was lower in capons from 21 wk of age (P < 0.05). The content of edible components expressed as a percentage of the total BW of cockerels and capons was similar in the corresponding age groups. Caponization had no effect on the total lean meat content of the carcass (P = 0.744), but differences were found between the weights of breast muscles and leg muscles. In week 24 and 28, the weight of breast muscles was higher in capons than in cockerels (P < 0.05). Cockerels had higher leg muscle weight than capons, and significant differences were noted in week 16 and in 28 (P < 0.05).

INTRODUCTION
The elimination of day-old cockerels of layer breeds poses a problem for the poultry industry (Leenstra et al., 2011;Soisontes, 2015;Brümmer et al., 2017). Several solutions have been proposed, including the development of reliable and rapid methods for sexing chick embryos (Krautwald-Junghanns et al., 2018), and effective and humanitarian euthanasia of layer-type male chicks (Gurung et al., 2018). However, social concerns and expectations should also be taken into account. Raising unwanted cockerels for meat is the approach that has met with the highest degree of social acceptance (Bruijnis et al., 2015;Brümmer et al., 2017;Mueller et al., 2018). Due to genetically determined C The Author(s) 2019. Published by Oxford University Press on behalf of Poultry Science Association. This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/ 4.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited. For commercial re-use, please contact journals.permissions@oup.com. lower body weight (BW) gain and higher feed intake per unit of gain, the process of raising layer-type cockerels for meat takes longer and is less profitable compared with broiler chickens (Koenig et al., 2012;Choo et al., 2014). Leenstra et al. (2011) andBrümmer et al. (2017) investigated potential alternatives to the killing of day-old male chicks of layer breeds and found that some consumers declared their willingness to pay a premium for eggs and chicken meat to prevent the above practice. In Asia and Africa, the carcasses of male chicks of layer breeds and traditional native breeds are popular among consumers (Jatarusitha et al., 2008, Khobondo et al., 2015. According to Leenstra et al. (2011), layer cockerels need around 18 wk to attain 2.0 kg BW. Soisontes (2015) reported that in Thailand, layer-type male chicks used for meat production were raised for approximately 60 D to achieve slaughter weight of 0.8 to 1.2 kg. According to other authors, however, extensively raised native chickens achieve BW of 1.0 to 1.5 kg at 4 to 5 mo of age (Leotharakul et al., 2002). An increase in blood testosterone levels, observed in growing cockerels, is an important consideration since it may be associated with antagonistic and/or aggressive behaviors (Queiroz and Cromberg, 2006;Essien et al., 2012). Undesirable behavioral changes 3345 can be eliminated by surgical or chemical castration (Quaresma et al., 2017). Caponization is carried out to produce meat with exceptional flavor (Díaz et al., 2010;Calik et al., 2015;Adamski et al., 2016;Amorim et al., 2016). Previous research revealed increased deposition of fat in caponized birds, in particular abdominal fat and mesenteric fat, which can decrease carcass dressing percentage . On the other hand, the accumulation of lipids in the muscle tissue of capons improves the palatability of meat (Amorim et al., 2016;Gesek et al., 2017). Lin and Hsu (2002) and Gesek et al. (2017Gesek et al. ( , 2019a demonstrated that low androgen levels affected the diameter of muscle fibers, which was smaller in the breast muscles of capons compared with intact males. In Europe, the meat of capons of selected traditional breeds is appreciated by some consumer groups for its tenderness and flavor, but it is more expensive than meat from broiler chickens and organic chickens (Muriel Duran, 2004;Franco et al., 2016).
A few studies have investigated the suitability of layer-type male chicks for capon production. Chen et al. (2010) found that caponization increased final BW and decreased the feed conversion ratio (FCR) in Single Comb White Leghorn chickens. Symeon et al. (2012, Lohman Silver, Lohman Tierzucht) reported that caponization had no significant effect on the growth performance of male chicks of a layer line and suggested that they could be used for the production of capon meat, appreciated by consumers as a niche product. However, consumers' interest in niche food products and their prices are difficult to estimate. Consumers' preferences change over time in response to both increasing awareness of animal welfare and sociocultural factors affecting their food choices (Soisontes, 2015;Long and Blok, 2017;Bosona and Gebresenbet, 2018). In view of the above, the suitability of layer-type male chicks for meat production should be analyzed, and their optimal slaughter age should be established.
The aim of this study was to determine the effect of age and surgical caponization on the growth performance and carcass quality characteristics of Leghorn cockerels.

MATERIALS AND METHODS
The experiment was conducted on 224 Leghorn cockerels (supplied by the National Research Institute of Animal Production in Chorzelów, Poland). Oneday-old birds were weighed and marked with wing tags. The birds were raised in the experimental center of the Department of Commodity Science and Animal Improvement of the University of Warmia and Mazury in Olsztyn (Poland), to 28 wk of age, and were fed commercial diets ad libitum (Table 1). At 8 wk of age, 112 birds were surgically castrated by a qualified veterinarian in accordance with Commission Regulation (EC) No. 543/2008. The procedure was approved by the Local Ethics Committee in Olsztyn, Poland. The birds were divided into 2 sex categories (with 8  replications per group and 14 birds per replication). From 12 wk of age, at 4-wk intervals, 8 intact cockerels and 8 capons (1 bird per replication) were selected randomly and slaughtered (electrical stunning followed by cutting the jugular vein). Carcasses were eviscerated after the removal of heads (between the occipital condyle and the atlas) and feet (at the carpal joint).
The digestive tract, liver, heart, and abdominal fat were removed. Live BW was determined before slaughter, and carcass weight was determined after bleeding and plucking. The weights of the head, feet, hot carcass, heart, liver, gizzard, gastrointestinal tract (including the contents and peri-intestinal fat, excluding the gizzard), kidneys, trachea, lungs, and abdominal fat were also determined. Carcasses were chilled at 4 • C for around 18 h and were divided into primal cuts (neck, wings, legs, back, and breast) which were subjected to detailed dissection (Zio lecki and Doruchowski, 1989).
To determine the percentage of edible components and non-edible components in total BW, the weights of all components were added up. The total weight of edible components and non-edible components is not equal to the BW of birds before slaughter. The difference results from loss during post-slaughter Figure 1. Blood testosterone levels in cockerels and capons (ng/mL; mean ± SD). Values followed by different letters (age) or * ("sex") differ significantly: α = 0.05 processing and dissection: drip loss, evaporation, and drying. The total amount of loss was comparable across the age groups of cockerels and capons.
At 4 and 8 wk of age, blood samples were collected from 8 randomly selected birds to determine testosterone levels. In successive weeks (from 12 to 28 wk), at 4-wk intervals, blood samples were collected from 8 cockerels and 8 capons (one bird per pen) into test tubes containing heparin. Freshly collected blood was centrifuged twice (MPW-350R centrifuge, MPW MED INSTRUMENTS; 5 min, 10 000 rpm), and each time the supernatant was transferred to 1.5 mL Eppendorf Safe-Lock micro test tubes with an Eppendorf automatic electronic pipette. Plasma samples were frozen at -72 • C in 1.5 mL Eppendorf Safe-Lock micro test tubes in the Kaltis 390 ultralow temperature laboratory freezer. Testosterone levels were analyzed by radioimmunoassay (RIA) with the use of commercial kits supplied by DIAsource TESTO-RIA-CT (DIAsource ImmunoAssays S.A., Belgium).
The statistical analysis involved the determination of arithmetic means (X) and standard deviations (SD). The data were analyzed by 1-way ANOVA (age or caponization) and 2-way ANOVA (age x caponization). The significance of differences in mean values between age groups was determined by Duncan's test. All calculations were performed using Statistica 2010 software. Significance was set at P ≤ 0.05.

RESULTS
Average plasma testosterone levels in cockerels reached 0.14 ng/mL at 4 wk of age and 0.18 ng/mL at 8 wk of age (castration). Testosterone concentration increased to 1.55 ng/mL in control group (intact) cockerels at 28 wk of age. The highest increase in testosterone levels (by 1.05 ng/mL, Figure 1, P < 0.05) was observed between week 12 and 16. In caponized birds, average testosterone concentration between week 12 and 28 was 0.13 ng/mL, and it was significantly lower than in cockerels (Figure 1, P < 0.05).
Daily feed intake (DFI, g/bird) increased with age (P = 0.038, Table 2). In cockerels, DFI values continued to increase until the end of the experiment (from 61 g in week 12 to 127 g in week 28, P < 0.01). In capons, DFI values continued to increase until week 24 (from 57 g in week 12 to 100 g in week 24). In general, caponization had no effect on the DFI (P = 0.184), but in week 28, DFI was significantly higher in cockerels than in capons (age x caponization interaction, P = 0.021).
FCR (kg/kg) based on BW gain, carcass weight gain, edible weight gain, and lean weight gain increased with age in both cockerels and capons (P < 0.01, Table 2). Caponization had no effect on the FCR, but at 28 wk of age, FCR based on edible weight gain was higher in cockerels than in capons (age x caponization interaction, P = 0.036) by 0.34 kg (9.11 kg/kg in cockerels vs. 8.77 kg/kg in capons, P < 0.05). FCR based on lean weight gain was higher in cockerels than in capons (age x caponization interaction, P = 0.032), by 0.35 kg in week 24 (12.08 kg/kg in cockerels vs. 11.72 kg/kg in capons, P < 0.05) and by 0.66 kg in week 28 (12.23 kg/kg in cockerels vs. 11.57 kg/kg in capons, P < 0.05).
At 8 wk of age (before castration), the average BW of birds was 595 g. Caponization had no significant effect on BW, carcass weight, or the weights of tissue components (lean meat, skin with subcutaneous fat, bones), which were affected by age (except in week 16, Table 3, P < 0.05).
Caponization had no effect on the total lean meat content of the carcass (P = 0.744, Table 3), but differences were found between the weights of breast muscles ( Figure 2a) and leg muscles (Figure 2b). In week 24 and 28, the weight of breast muscles was higher in capons than in cockerels by 35.5 g (300.6 and 265.1 g, Figure 2a, P < 0.05) and 24.2 g (316.9 and 292.7 g, Figure 2a, P < 0.05), respectively. Cockerels had higher leg muscle weight than capons, and significant differences were noted in week 16 (257.1 g in cockerels vs. 215.6 g in capons, Figure 2b, P < 0.05) and in week 28 (406.6 g in cockerels vs. 356.3 g in capons, Figure 2b, P < 0.05).
Caponization had no influence on the total weight of giblets (heart + gizzard + liver, P = 0.132), which was affected by age (P < 0.000), but had a significant effect on heart weight and gizzard weight (Table 3). Heart weight was lower in capons than in cockerels (P = 0.000), whereas gizzard weight was higher in capons than in cockerels (P = 0.006). Between week 12 and 28, heart weight increased from 5.8 to 12.5 g in cockerels (P < 0.05), and from 5.3 to 9.2 g in capons (P < 0.05). From 12 wk of age, gizzard weight remained stable in cockerels (age x caponization interaction, P = 0.002), and it increased from 25.6 to 43.7 g in capons (P < 0.05). Liver weight increased from 23.3 g in week 12 to 30.9 g in cockerels (P < 0.05), and from 21.2 to 31.8 g in capons (P < 0.05). Caponization had no significant effect on liver weight (P = 0.342). The weights of edible and non-edible components increased with age (Table 3, P < 0.000), and were comparable in cockerels and capons at corresponding ages. Between week 12 and 28, the weight of edible components increased from 608.5 to 1330.2 g in cockerels (P <0.05), and from 589.3 to 1331.3 g in capons (P < 0.05). The weight of non-edible components increased from 523.6 to 846.2 g in cockerels (P < 0.05), and from 501.2 to 868.8 g in capons (P < 0.05). Table 4 presents carcass dressing percentage and the percentage content of edible and non-edible components in the total BW of Leghorn cockerels and capons. Dressing percentage was similar in cockerels and capons at corresponding ages, and it increased until week 28 in cockerels (from 62.2% in week 12 to 70.9% in week 28, P < 0.05), and until week 20 in capons (from 62.8% in week 12 to 69.70% in week 20, P < 0.05).
The percentage of edible and non-edible components in the total BW of cockerels and capons was affected by age (P < 0.000), but not by caponization (Table 4).

DISCUSSION
The results of this study are difficult to compare with the findings of other authors due to considerable differences in capon production systems, age at caponization, length of the rearing period, breed, and diet. Chen et al. (2010) analyzed Single Comb White Leghorn chickens that were caponized at 12 wk of age and reported higher feed intake and lower FCR in capons between week 16 and 26. Calik et al. (2017) compared Rhode Island Red (R-11) cockerels and capons and reported lower FCR in the latter. In the present study, a tendency toward lower DFI was observed in capons, particularly at the end of rearing (week 24 to 28), compared with intact males. Unfortunately, there are no published data regarding FCR values calculated based on carcass weight gain, edible weight gain, or lean weight gain in caponized birds. In our study, FCR based on BW gain, carcass weight gain, and edible weight gain was lower in capons between week 24 and 28, and FCR based on lean weight gain was lower between week 21 and 24 and 24 to 28 (Table 2). Higher feed intake in intact males may be associated with their higher maintenance energy requirements due to increased activity and behavioral changes (ruffling feathers, chasing and pushing other birds around, antagonistic behaviors; Queiroz and Cromberg, 2006) induced by an increase in testosterone levels from 12 wk of age (Figure 1). Removal of the gonads in males also entails other consequences that are reflected in the bird's habits (Gryzińska et al., 2011). FCR values are lower in capons than in cockerels but considerably higher than in broiler chickens (Murawska and Bochno, 2007;Koenig et al., 2012). The temperament of individual birds also changes-they become more sleepy and quiet. In a study by Soma (2006), capons did not crow, did not try to mate with females, and did not fight aggressively with other males. Caponized birds are more gentle and docile, which was also observed in our study, and this could contribute to improving the efficiency of feed utilization. Table 3. Body weight, carcass weight, weights of tissue components and organs in Leghorn cockerels and capons (g; mean ± SD). Caponization did not cause an increase in the BW of birds or the weights of major tissue components except abdominal fat (Table 3). No differences in final BW between cockerels and capons were observed by Symeon et al. (2012, males of a layer line) and Miguel et al. (2008, Castellana Negra -native Spanish chickens).
The percentage share of edible components was comparable in Leghorn capons and capons of a native dual-purpose breed known as the Green-legged Partridge. Zawacka et al. (2018) demonstrated that in Green-legged Partridge capons (caponization at 8 wk of age), edible components accounted for approximately 50% of total BW in week 12 and 55.5% in week 28. In the current study, edible components accounted for 52.4% of the total BW of Leghorn capons in week 12 and 57.4% in week 28. In Leghorn capons, the above values resulted from a relatively low percentage of abdominal fat in total BW. Previous research shows that abdominal fat usually has a high share of total BW in capons, ranging from 3.7 to 4.5% (Terčič et al., 2007) to nearly 5% (Calik et al., 2015) depending on breed and slaughter age.
In layer-type male chicks, the content of leg muscles increases and the content of breast muscles decreases with age (Murawska et al., 2005). Such a trend was Table 4. Carcass dressing percentage, and the percentage content of edible and non-edible components in the total body weight of Leghorn cockerels and capons (%; mean ± SD). a-d Mean values in a row (age) followed by different letters are significantly different at α = 0.05. * Mean values in the age groups of cockerels and capons are significantly different at α = 0.05.

Figure 2.
Arithmetic means for the weight of (a) breast muscles and (b) leg muscles (g) in cockerels and capons, subject to the age of birds (g, mean ± SD). Values followed by different letters (age) or * ("sex") differ significantly: α = 0.05 also observed in this study in Leghorn cockerels, but not in capons which were characterized by an increase in the content of lean meat located in the breast. This could indicate that testosterone exerts varied effects on different muscle groups in birds (Chen et al., 2007).
According to Mahmud et al. (2013), low levels of sex steroids in capons can increase fat deposition and affect the weight and function of bodily organs. In the current study and in experiments conducted by other authors (Miguel et al., 2008;Symeon et al., 2012), heart weight was lower in capons than in cockerels, and no differences were found in gizzard weight or liver weight. Chen et al. (2006) observed higher gizzard weight and liver weight in capons. A histopathological analysis of selected organs in Leghorn cockerels and capons revealed that surgical castration did not induce any pathological changes except increased lipid accumulation (Gesek et al., 2019b).
In conclusion, caponization had a beneficial influence on feed intake and FCR in layer-type (Leghorn) cockerels, but it did not contribute to an increase in BW or the weights of major tissue components. The content of edible components expressed as a percentage of the total BW of cockerels and capons was similar in the corresponding age groups. From 20 wk of age, the percentage of breast muscles in total BW was higher, and the percentage of leg muscles in total BW was lower in capons than in cockerels. The carcasses of Leghorn capons may arouse interest among consumers, but future demand is difficult to estimate.


extfieldsofstudy: Medicine Biology

provenance: AgriculturalAndFoodSciences,2019-2020-Agricultural And Food Sciences-2019.jsonl.gz.gz

text:
Survival of entomopathogenic nematodes in oil emulsions and control effectiveness on adult engorged ticks (Acari: Ixodida)

Abstract Although their control is based on chemical products, the infestations by ticks (Ixodes scapularis Say) are causing great losses and damages in the livestock production worldwide. In this study, the survival of the entomopathogenic nematodes Heterorhabditis bacteriophora, Steinernema carpocapsae, and Steinernema websteri in vegetal oil suspension at concentrations of 13% and 33% and their effectiveness to control ticks at concentrations of 50 ± 5 and 100 ± 10 nematodes in oil suspensions of Cymbopogon citratus, Pelargonium sp, Juniperus virginiana, Rosa sp, and Mentha piperita were evaluated in lab conditions. In field conditions, the Lethal Concentration (LC90) of S. websteri in oil suspensions of J. virginiana and C. citratus in dogs infested with ticks was evaluated. In the laboratory, it was found that an oil emulsion of C. citratus and J. virginiana at 13% maintained the survival of S. carpocapsae, H. bacteriophora, and S. websteri from 55% to 60% for a period of 96 hr. The combination of the S. websteri nematode with 50 or 100 nematodes in oil emulsions of J. virginiana at 33% presented a control effectiveness of 80–100% in adult ticks 24 hr post-application. In field, the LC90 of 119 juveniles of S. websteri in oil emulsions of J. virginiana at 33% on domestic dogs presented an accumulated and a control effectiveness of 89% after 96 hr post-application. The combined application of J. virginiana and S. websteri could be a good alternative for the control of ticks. It was observed that the time of contact and the type of vegetable oil were crucial factors to increase the effectiveness of control.

The ticks (Acari: Ixodidae) are widely distributed in the tropical, subtropical, and warm areas and are able to parasite vertebrates. In animals, the ticks climb from their feet to the ears, neck and the perianal zone. In these places, the skin is thinner which makes their feeding easier. As many other parasites, they can spread easily throughout their hosts because they hold onto them during long periods and when they finish eating they then drop off the host and go to a new place. This is very important for the dynamic of the illnesses they trans-mit and the risk of incursion of new pathogens in areas where they were absent (Díaz et al., 2012). It is estimated that around one billion of bovine livestock are found in tropical and subtropical zones exposed to the infestations by ticks and/or illnesses transmitted by them, causing great losses in livestock production (Cruz et al., 2016).
In Mexico, the conventional method to control the ticks is using ixodicides, chemical products that are available in the national market. However, the risk of generating populations of ticks resistant to the ixod-icides is higher if its use is not handled as indicated by the standards (Rodríguez-Vivas et al., 2014). Nowadays, there are very few studies on the biological control of ticks using entomopathogenic nematodes (Kocan et al., 1998).
Entomopathogenic nematodes (EPNs) are efficient agents of biological control, and are compatible with some chemical pesticides (Kaya and Gaugler, 1993). The effectiveness of the EPNs in tick's control depends on the time of permanency of the formulation on the pest's body because the infective juveniles (IJs) require enough time to find a host and need protection against adverse abiotic factors such as solar radiation and low moisture. Therefore, when the EPNs are applied in aqueous suspension, the efficiency generally decreases. In this work, we aimed to identify the most beneficial combination of a vegetable oil emulsion to extend the shelf-life of EPNs at room temperature and to increase their infectivity on ticks in laboratory and field evaluations.
The compatibility of some vegetable oils with the nematode Heterorhabditis bacteriophora has been reported (Krishnayya and Grewal, 2002;Alves et al., 2017). Furthermore, owing to its viscosity level, the oil sticks for some time and its evaporation is slower compared with water. These physicochemical characteristics can be exploited in the application of EPN by delaying its desiccation and improving their interaction with the insect-pest.
Hence, in an effort to increase the survival time and infectivity of EPNs on ticks, this study aimed to evaluate: (i) the effect of five vegetable oil emulsions on the survival and infectivity of S. carpocapsae, S. websteri, and H. bacteriophora on ticks in laboratory; and (ii) the control effectiveness (CE) of application of EPNs in oil emulsion on ticks present in infested dogs (Canis lupus familiaris) in field conditions.

Description of the area of study
This work was carried out under lab and field conditions. The lab experiments were done in the Laboratorio de Nematodos Entomopatógenos at CIIDIR-Oaxaca and the field experiments were done in Santa Cruz Xoxocotlán, Oaxaca, Mexico (17° 01¢ 35²N, 96° 44¢ 00²O, 1,523 m altitude.

Reproduction of entomopathogenic nematodes
The EPNs Heterorhabditis bacteriophora 18S and 28S HB1 Strain (Poinar, 1975), Steinernema car-pocapsae 18S Access Gen Bank AF121049.1 (Weiser, 1955) were provided by the Department of Entomology of the University of California, Davis, USA, and Steinernema websteri 28SR and 28SF Access AY84172.1 (Cutler and Stock, 2003) is an native nematode that was isolated from soil in plantations of Agave angustifolia Haw by Delgado-Gamboa et al. (2015). The juveniles were reproduced on last instars of the wax moth Galleria mellonella, following the method proposed by Kaya and Stock (1997). The temperature and relative humidity (RH) in laboratory were 19 to 25 °C and 37 to 64%, respectively. Three days after the inoculation, the infected larvae were set in White traps with filter paper discs (90 mm of diameter, Whatman No.1). The emerging IJs were collected and maintained in distilled water in culture bottles of 250 mL and stored at 12 ± 1 °C for 24 hr before use.

Collection of ticks
It was done by visual and tactile inspection of the presence of ticks in the infested dogs' bodies. Adult ticks were collected by following the method proposed by Bonilla-Cárdenas et al. (2014), which consisted of holding the parasite with the index finger and thumb to separate it from the body of the animal pulling in a contrary direction of hair growth to avoid damages on it. In total, 60 adult ticks were collected and were kept in plastic containers of 100 mL which contained a piece of wet cotton to avoid dehydration of the organisms and to provide a suitable micro-environment to maintain their nonparasitic cycle. Furthermore, some 2 mm-holes were made in the lid for air exchange.

Survival assays of entomopathogenic nematodes in emulsions
Two emulsions at two different vegetable oil concentrations (13 and 33%) were made. The amount of EPNs in each emulsion was 400 ± 20 IJs, but different amounts of oil and water were used (Table 1). Steinernema websteri -AD (control) 0 119 ± 10 2,500 These were placed in plastic petri dishes (55 mm of diameter), which were stored at room temperature (22 ± 3 °C and 37-64% RH). The survival of IJs in oil emulsions was assessed every 24 hr during 6 d. The observation and counting of living and dead IJs was done using a stereoscopic microscope. The IJs were considered alive if they had mobility in the head and tail by themselves (Peters, 2016).

Control effectiveness on ticks under laboratory conditions
The CE effectiveness of S. websteri, S. carpocapsae, and H. bacteriophora nematodes in oil emulsions of C. citratus and J. virginiana, both at 33% of concentration, was assessed. The concentrations of EPNs were 50 ± 5 and 100 ± 10 IJs/tick. The ectoparasites were set individually in petri dishes with filter paper. The inoculation was performed after 12 hr with the oil emulsion of EPNs. The conditions of the ticks were registered every 24 hr until accumulating 100% of mortality; 120 adult ticks were used for this study. The cadavers were collocated in White traps to determine the cause of the death throughout necropsy tests, verifying if there were any EPNs, and the Koch's postulates were performed (Lacey and Solter, 2012).

Control effectiveness on ticks in C. lupus
The EPNs were applied in oil emulsions of J. virginiana and C. citratus at 33% concentration in selected areas of infested domestic dogs, following the Mexican Standard NOM-006-ZOO (1993). The dogs were examined to identify and count the ticks. In total, 10 dogs with an individual weight of about 55 kg that were infested by ticks were selected, and four to six ticks per zone (feet, chest, cross, rump, muscular back, and ears) were located for the bioassay. A total of 10 infested dogs with ticks with application of an aqueous suspension containing S. websteri were established as a control. The application of the oil emulsions with EPNs on the infested areas was done in one single occasion. A disposable plastic syringe (BD Plastipak ® of 5 mL) was used to apply the treatments on the infested areas so that the emulsion could penetrate the dogs' fur and skin. The control was treated with the same concentration of EPNs, but without vegetable oil. Evaluations were performed to all the fallen ticks or to the ones that presented signs of infection by EPNs, and the mortality of the ectoparasites was registered every 24 hr for 4 d. To verify infection by EPNs, each cadaver was washed twice with distilled water, transferred to White traps and incubated at 22 ± 3 °C until the IJs emerged.

Experimental design
The experiments of survival of EPNs, the CE on adult ticks and the final composition of the evaluated treatments in the lab were done by duplicate with five repetitions per treatment. The field experiment on infested dogs with engorged adult ticks was done in a single occasion (Table 1).

Statistical analysis
To determine the mortality of ticks, percentage data were transformed with the arcsine function. Then, variance analysis was carried out to establish the differences between the means by the Tukey test (P < 0.05). The LC 90 was determined by Probit analysis of the data from the lab bioassay, which was used for the application of the EPNs in the effectiveness trials in the field. All the analyses were done in the statistic software SAS ® (Version 9.4; SAS Institute Inc., Cary, NC, USA).

Survival of entomopathogenic nematodes in vegetable oil emulsions
The survival of S. websteri, S. carpocapsae, and H. bacteriophora was heterogeneous among the different oil emulsions because there was a wide variation from 6.14 to 60%. The lowest survival was obtained with the M. piperita oil emulsion and Rose oil emulsion. The most favorable emulsions for high survival percentages of IJs were the ones prepared with J. virginiana and C. citratus, the first one with a 60% and the second one with 55% survivorship, but without statistical differences between them (P > 0.05). However, they were statistically different (α < 0.005) from the emulsions of Pelargonium, Rose, and M. piperita (Fig. 1).
It was observed that, because of desiccation, the emulsions began to lose mass and they hardened 72 hr after elaborated. Based on the survival of IJs, the data suggest that the vegetable oils of C. citratus and J. virginiana were the ones with the highest compatibility with the EPNs. The survival percentages of the nematodes S. websteri, H. bacteriophora, and S. carpocapsae in the oil emulsions of C. citratus and J. virginiana were the highest ones (Fig. 2). S. carpocapsae 400 had the highest survival percentage in J. virginiana oil emulsion and C. citratus, with 95 and 88.7%, respectively. The second best treatment was the combination nematode S. websteri 400 in J. virginiana oil emulsions or C. citratus oil, with a variation of 46.66 to 58.80%. The combination with the lowest IJs survival percentage was H. bacteriophora in J. virginiana or C. citratus oil emulsions. The treatment with H. bacteriophora was statistically different (P = 0.05) from the treatments with the nematodes S. websteri and S. carpocapsae.

Mortality of engorged adult ticks treated with EPN in laboratory
It can be observed that the control effectiveness (CE) of the EPNs on ticks in a period of 24 hr varied from 50 to 100%. As can be seen in Figure 3, applying the concentration of 50 IJs/tick, the treatment with the nematode S. websteri in J. virginiana oil emulsions was the one with the highest CE (80%), followed by S. carpocapsae also with the J. virginiana oil emulsion, S. crpocapasae in C. citratus emulsion, and H. bacteriophora in J. virgijiana oil emulsion. All of them had a CE higher than 70%. There were statistical differences (P = 0.05) in CE among the treatments mentioned above, as well as for the combinations of S. websteri in C. citratus (60%) oil emulsion, and H. bacteriophora in oil emulsion of C. citratus (50%). After 48 hr of the application, the CE increased in all the treatments. The nematode S. websteri in J. virginiana oil emulsion reached a 100% of CE; that is why it was statistically different (P < 0.05) from the treatments S. websteri-C. citratus, S. carpocapsae-J. virginiana and H. bacteriophora-J. virginiana and H. bacteriophora-C. citratus, which reached 80% of CE (Fig. 3).
As can be seen in Figure 4, the concentration of 100 IJs/tick gave a CE of 100% in treatments with S. websteri-J. virginiana and S. carpocapse-J. virginiana, while three treatments showed a CE of 90% (S. websteri-C. citratus, H. bacteriophora-J. virginiana, and H. bacteriophora-C. citratus). In this assay, it was found a statistical difference (P < 0.05) among the treatments mentioned above and the treatment with S. carpocapsae-C. citratus, which only controlled 50% of the ticks. After 48 hr, it was found that the treatment S. websteri-C. citratus reached a CE of 100% on ticks, and the treatments H. bacteriophora-J. virginiana and H. bacteriophora-C. citratus accumulated a 90% of CE. A statistical difference (P < 0.05) was found between the treatments with S. carpocapsae-C. citratus and the rest of the treatments. All the combinations with a concentration of 100 IJs/tick are considered a suitable alternative for controlling these parasites. In the infectivity of ticks by EPNs verification tests, it was observed a limited emergence of IJs, which could be due to the chitinous characteristics of ticks.

Control effectiveness on ticks in infested dogs
The treatment that produced the highest CE was S. websteri at 119 IJs/tick in the J. virginiana oil emulsion, which after 24 hr had 68% CE; after 72 hr it increased to 80%, and after 96 hr it reached 89%. The treatment with the nematode S. websteri at the same concentration in the C. citratus oil emulsion caused 57.50% mortality in a period of 24 hr and after 72 hr the solution reached 85%, being the most effective.
The control S. websteri-distilled water reached 42.5% of mortality on the ectoparasites in 24 hr, and increased to 60% in 48 hr. At the end of the evaluation  period, the maximum CE with this treatment was 72%. This result could be due to the effect of the applied nematode (Fig. 5).

Discussion
The survival of EPNs improved according to the essential oil used for the preparation of the emulsions. There exist just a few reported studies on the behavior of EPN survival in vegetable oil emulsions. Alves et al. (2017) prepared an Aureo ® vegetable oil emulsion containing 2,000 IJs of Heterorhabditis sp. (strain CB40) which increased its viability in 87.4% with regard to the control in water.
Considering that the storage temperature of the oil emulsions was stable (23 ± 3 °C) and below 35 °C, the results suggest that the IJs of H. bacteriophora and S. carpocapsae survived because they have their own biological fitness for desiccation tolerance, especially in emulsions with C. citratus and J. virginiana, both at 13% concentration.
It is known that heat and desiccation cause negative effects in the survival of the EPNs, mainly in field applications (Ruiz-Vega et al., 2011). Dito et al. (2016)   established that the survival of the nematodes is closely linked to their metabolism, where temperature is a determinant variable. Temperature increases the mobility of the IJs and their lipids expenditure, which causes starvation and physical damage and the increment of mortality (Cagnolo and Campos, 2008).
Tolerance to heat and desiccation in the nematodes S. carpocapsae and H. bateriophora is often reported, but there is variability among strains of the same species; however, for S. websteri, there are no published studies about their tolerance to heat and desiccation. Ruiz-Vega et al. (2011) reported a 70% survival of H. bacteriophora at 35 °C, while in a range of water activity (A w ) of 0.86 to 0.97 its survival went from 23.6 to 59.3%. This affirmation was confirmed by Ulu and Susurluk (2014), as they found the same tendency in relation to heat and desiccation in 10 strains of H. bacteriophora isolated from different climatic regions in Turkey.
In this study it was observed that from the lowest concentration of IJs used, which was of 50 IJs/ tick and that caused 66.66% of mortality, the CE increased when the concentration of IJs was higher. There are no registers of studies of S. websteri against these ectoparasites, but it was the most effective nematode against the engorged adult ticks. The LC 50 and the LC 90 estimated were 32.2 IJs/tick and 119 IJs/tick, respectively. These are the lowest when compared with Freitas-Ribeiro et al. (2005) who estimated the LD 50 and the LD 90 of S. carpocapsae (all strain) for a period of CE of 3 d in 156 IJs and 5,001 IJs, respectively.
The EPNs are effective agents for biological control of engorged adult ticks. Some CE reports in laboratory with H. bacteriophora and S. carpocapsae have been published, but there are not any reported results on the CE of the S. websteri nematode on adult or nymphs ticks. Regarding S. carpocapsae, it was considered a moderately effective nematode for control of adult ticks and similar results have been found by other authors. Zhioua et al. (1995) found that S. carpocapsae was a pathogen for the full female ticks (Ixodes scapularis Say), although nothing was said about the concentrations of the nematode used. In laboratory trials at 25 ± 1 °C, Butt et al. (2016) found that S. carpocapsae (Turkish strain) at a concentration of 4,000 IJs was slightly more infectious against engorged adults (30-40%) than against nymphs (36%), but it required an exposure period of 14 d to the IJs; however, there was not any emergence of EPNs from the infected cadavers. The exposition of Boophilus microplus during 72 hr to the concentration of 6,000 IJs was more effective against male and female engorded adults (>90%) after 7 to 6 d under lab conditions at 27 ± 1 °C and 80% relative humidity (Freitas-Ribeiro et al., 2005).
The percentage of control of adult female engorged ticks of Rhipicephalus microplus exposed to the nematode H. bacteriophora (HP88) in a concentration of 300 IJs/tick was of 95.6% (Monteiro et al., 2013). The authors applied the nematodes H. bacteriophora (HP88) and H. indica (LPP1) in concentration of 300 IJs combined with Lippia sidoides essential oil on engorged adult female ticks of Rhipicephalus microplus at 27 ± 1 °C and a relative humidity of 80 ± 10%, which controlled effectively the ectoparasites (>99%).
In the laboratory, Monteiro et al. (2013) found that a concentration of 300 IJs/female of H. bacteriophora (HP88) was enough to cancel 100% of the oviposition of the non-parasitic phase of R. microplus with a time of exposure of 48 hr, which suggests that the longer times of exposure permit the nematode to localize and penetrate the host.
The application of EPNs in insect cadaver formulations in laboratory conditions (27 ± 1 °C and RH of 80 ± 10%) on engorged R. microplus females was tested by Monteiro et al. (2014) and found a higher cumulative mortality using H. bacteriophora HP88 and S. carpocapsae all, with 100 and 80.6% CE, respectively. Using H. bacteriophora Poinar (VS strain) and S. carpocapsae Weiser (All strain) against R. microplus (Deutch strain), the maximum mortality reported by Singh et al. (2018a) was 15 and 20%, respectively. In bioassays in petri dishes containing a 1.0-ml distilled water suspension at concentrations of 1,250, 2,500, and 5,000 IJs per dish, the maximum mortality of R. microplus ticks was 85% at the largest concentration of S. carpocapsae Weiser (All strain) and 45% with the largest concentrations of H. bacteriophora (Singh et al., 2018b).
In our study, the application of EPNs in oil and water emulsions on the domestic dogs' fur increased the CE on the adult ticks. Even though vegetable oils and EPNs have been used for the control of adult ticks in laboratory (Samish and Glazer, 2001;Ellse and Wall, 2014), the studies about their combined application in the field on domestic dogs are limited.
The action of some vegetable oils against ticks is through repellency (Benelli and Pavela, 2017), while the action of the EPNs is by pathogenicity and virulence (Samish and Glazer, 2001). The concentration of citronela oil (Corymbia citriodora (Hook.) at 1% applied against Dermacentor reticulatus Fab. was able to repel 76.6 to 86.9% of ticks. Against Amblyoma americanum L., the concentration of Juniperus communis L. and Juniperus chinensis L. oils were able to repel 50% of the ectoparasites in 15 min.
As it was showed in the results obtained in this study, the contact time with the insect-pest is a determinant factor to increase the CE of the EPNs, which agrees with Monteiro et al. (2012) and Hill (1998). The last author explains that the exposure time of the ticks to the EPNs for a short period reduces the number of IJs that successfully penetrate in the engorged female ticks reducing the mortality rate of the host.

Conclusions
The combined application of vegetable oil and EPNs could be a good alternative for the control of the ticks, as it was observed that the time of contact and the kind of oil are the determinant factors to increase CE.
The oil emulsion of C. citratus and J. virginiana at 13% favored the survival of the nematodes (S. carpocapsae, H. bacteriophora, and S. websteri) in 55 and 60% for a period of 96 hr, respectively.
In lab applications, the combination of the nematode S. websteri (in doses of 50 and 100 IJs) in oil emulsions of J. virginiana at 33% concentration was highly lethal on engorged adult ticks in the first 24 hr after the application, with a CE of 80 to 100%.
In field applications on domestic dogs, it was found that the DL 90 of 119 IJs of S. websteri in oil emulsions of J. virginiana (33%) had the best performance with an accumulated CE of 89%, 96 hr after being applied.
The results suggest that the treatment with the nematode S. websteri in oil emulsions of J. virginiana or C. citratus at a concentration of 100 IJs per tick has the right potential to obtain a good percentage of control in dogs with presence of ticks, because it only required from 24 to 48 hr to be effective.


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Effect of Supplemental Cyanocobalamin on the Growth Performance and Hematological Indicators of the White Pekin Ducks from Hatch to Day 21

Simple Summary Vitamin B12 plays a key role in the normal functioning of the brain and nervous system as well as creation and regulation of nucleic acids (DNA and RNA). Furthermore, vitamin B12 plays a significant role in fatty acid metabolism and energy generation. Deficiency of vitamin B12 in animals may lead to weakness and anemia because it is involved in the formation of hemoglobin, which transports oxygen to body cells and red blood cells. Similarly, deficiency of vitamin B12 may also lead to hyperhomocysteinemia (increased level of homocysteine in the blood) which may depress immunity and cause cardiovascular diseases. There is no literature available regarding cyanocobalamin requirement for Pekin ducks. Therefore, the aim of our study is to determine its requirement. However, we find that cyanocobalamin has no influence on growth performance (weight gain), but it has more effect on hematological indicators (blood). On the basis of growth performance and hematological indicators we suggest that 0.02 mg cyanocobalamin/kg of feed is the dietary requirement of male Pekin ducks from hatch to day 21. Abstract The experiment was conducted to evaluate the requirement of cyanocobalamin of male Pekin ducks from hatch to 21 days of age. A total of three-hundred-eighty-four, one-day-old meat-type male Pekin ducks were randomly allocated to six treatments, i.e., dietary cyanocobalamin (vitamin B12) concentrations of 0.00, 0.02, 0.04, 0.06, 0.08 and 1.00 mg/kg, respectively in their feed. Each treatment had eight replicated pens with eight ducks for each pen. Feed and water were provided ad libitum. The experiment was conducted for 21 days. Different growth parameters including average daily weight gain (ADG), average daily feed intake (ADFI), feed conversion ratio (FCR), and hematological indicators were evaluated because, on the basis of hematological indicators, the health and nutritional status of an animal can be accessed. It is observed that supplemental cyanocobalamin has no significant effect on ADG, ADFI, and FCR but it improves hematological parameters such as white blood cells, red blood cells, and its indices and platelet counts compared to the control group (p < 0.05). On the basis of growth performance and hematological indicators it is concluded that 0.02 mg cyanocobalamin/kg of feed is the dietary requirement of male Pekin ducks from hatch to day 21 of age.

Introduction
Growth of ducks is faster than all other poultry species [1]. To bias the increased growth rate, these have been genetically selected like all the other poultry species [2]. Numerous studies have suggested that vitamin B12 is necessary for chick growth and egg hatchability [3][4][5][6][7]. Deficiency of vitamin B12 has a critical impact on growth and existence of rats. It is also reported that the addition of high levels of protein in a diet with a deficiency of vitamin B12 causes high mortality [8]. In another study, Dryden and Hartman (1971) observed a steady increase in growth rate with every increased unit of protein content that might not be associated with the breakdown of surplus nitrogen but with complications in the disposal of the carbon skeleton of certain or total amino acids of added protein contents in vitamin B12 deficient diets [9].
The natural vitamin contents of diets depend upon ingredients and compositions that can vary considerably between particular feedstuffs based on the season, region, and processing conditions which can induce natural variations in vitamin contents. Natural dietary contents rarely provide the intakes thought to fulfill the needs of birds to meet their normal requirements and to provide a margin of safety to meet extra metabolic demands imposed by stress and other factors [10]. Whitehead (2002) [11] demonstrated that diets supplemented with vitamins play a vital role for disease prevention and treatment. He also illustrated that biological functions are carried out by the involvement of vitamins that allow an animal to use energy and proteins for growth, health, maintenance, reproduction, and feed conversion [11]. Poultry intake B12 either by feed supplementation or ingesting feces. This vitamin plays a vital role in the nervous system and proper brain functioning, homocysteine metabolism, energy metabolism, normal blood functions, cell division, and in the immune system [12]. Similarly it is also anti-anemic in function [13]. Vitamin B12 acts as a co-factor for L-methylmalonyl-CoA mutase and methionine synthase. Methionine synthase increases the rate of homocysteine conversion to methionine which is further required for DNA and RNA synthesis while L-methylmalonyl-CoA mutase converts L-methylmalonyl-CoA to Succinyl-CoA which is a crucial biochemical reaction in fat and protein metabolism. Succinyl-CoA is also required for hemoglobin synthesis [14].
Ceca of the birds synthesize vitamin B12 by using cobalt, but its production is lower than daily requirements, and suggestions were given to supplement diets with vitamin B12 [15]. In another study, researchers reported that for an increased growth rate and maximum feed utilization, a growing ration fortified with vitamin B12 had appreciable effects but it may not be optimum for achieving maximum feed efficiency [16]. Folic acid, vitamin B12, and dietary total non-structural carbohydrates have no effect on milk production and milk total solid yield. There was also a non-significant difference observed on average daily gains among the calves supplemented with these vitamins [17]. Vitamin B12 deficiency also caused health problems in many ways, from hematological manifestations to neurological disorders, varied as leucoenia, numbness in fingers, unsteadiness of gait, fasciculation, and macrocytic anemia [18]. Erythrocytes depend on vitamin B12 for their maturation and proliferation. Therefore vitamin B12 deficient erythrocytes cannot mature, resulting in hemolysis and hyperbilirubinemia [19].
Blood parameters are indices of the internal environment of the living body and they also indicate the health status of ducks [20]. To improve animals' productivity, it is important to understand their physiology and hematological characteristics. For the establishment of a diagnostic baseline for blood characteristics, hematological studies are usually undertaken on farm animals as routine management practices [21,22]. Hematological constituents serve as authentic tools for monitoring animal health because they usually reflect the physiological reactions of an animal to its external and internal environment [23,24]. Hematological indicators are very important as they designate an animal's health and nutritional status [25]. These parameters in poultry are also sensitive to stress reactions [26]. Hematological indicators are considered to be biomarkers for the immune system [27]. There is a limited literature available for hemogram parameters in vitamin B12 deficiency [28]. That is why we investigate these hematological indicators on different levels of vitamin B12 in Pekin ducks. The National Research Council (NRC) (1994) also has no data regarding the vitamin B12 requirement of meat-type Pekin ducks for growth and hematological indicators. Therefore, one of the main goals of this study is to estimate the requirement of cyanocobalamin as a supplement for the growth performance and hematology of male Pekin ducks from hatch to 21 days of age.

Study Design
All procedures of this study were permitted by the Animal Care and Welfare Committee of the Institute of Animal Science, Chinese Academy of Agricultural Sciences (No. IASCAAS-AE-03, No. 20180416). A dose-response experiment with six supplemental vitamin B12 levels (0, 0.02, 0.04, 0.06, 0.08 and 1.00 mg/kg) was designed. Vitamin B12 deficient basal diet was prepared (Table 1) and it was comprised of 0.00 mg/kg total vitamin B12, according to the data of feed ingredients of the NRC (1994).

Chicks and Diets
The basal diet was formulated first and then six experimental diets were added with various supplemental levels of crystalline vitamin B12. A total of 384 one-day-old male White Pekin ducks with average body weights around 54 g were divided into 6 experimental groups and each group was replicated 8 times with 8 birds per pen. These ducklings were reared in steel cages with plastic floors (200 × 100 × 40 cm), from hatch to 21 days of age. They were raised and allocated randomly to an environmental control shed. The birds were offered feed and water ad libitum. Provision of water was done by using a drip-nipple, and feed was offered in pellet form. Birds were provided 24-h lighting, and temperature was maintained at 33 • C with relative humidity at 20% from 1 to 3 days of age, then temperature was decreased steadily to room temperature while relative humidity gradually increased to 65% until birds were 21 days of age.

Growth Performance and Hematological Parameters
On the 21st day, the average daily weight gain, average daily feed intake, and feed conversion ratio (FCR) of ducks from every pen were calculated using the following formula.
Feed conversion ratio (FCR) = Feed intake Weight gain Feed intake and FCR were all corrected for mortality.

Sampling and Analysis
After fasting 12 h, two ducks from each pen were selected randomly for blood collection on the basis of average body weight gain of the corresponding pen. Three to four milliliters of blood were collected in an Ethylenediaminetetraacetic Acid (EDTA)-containing tube from the jugular vein of each selected bird. These blood samples were analyzed by using an automated hematological analyzer (ABX PENTRA DX 120, Munich, Germany).

Statistical Analysis
Data were analyzed using SPSS Statistics 22.0 (Statistical Packages for the Social Sciences, released December 2013, Armonk, New York, USA). The mean comparison test was applied by using Duncan's new multiple-range test to find out the significant differences among the applied treatments at 5% the level of significance (p < 0.05).

Growth Performance
The effect of dietary cobalamin on average daily weight gain (ADG), average daily feed intake (ADFI), and feed conversion ratio (FCR) of the experiment is described in Table 2. ADG, ADFI, and FCR showed non-significant results among the vitamin B12 supplemented groups and the control group (p > 0.05), as presented in Table 2.

White Blood Cells
On day 21, ducks from the control group had significantly lower (p < 0.05) values for the white blood cell count (WBC) and had significantly higher (p < 0.05) values for the percentage of intermediate cells (MID %) than the supplemented groups. However, there were no further significant differences among the vitamin B12 supplemented groups. The absolute value of granulocytes (GRA) and percentage of granulocytes (GRA %) were lower for the control (C) group as compared to the vitamin B12 supplemented groups, although these values were not statistically significant (p > 0.05). There were non-significant results observed for the absolute value of lymphocytes (LYM), percentage of lymphocytes (LYM %), and absolute value of intermediate cells (MID), as presented in Table 3.

Red Blood Cells
At the end of the experiment, ducks with different levels of vitamin B12 had higher significant (p < 0.05) values for red blood cells (RBC), hemoglobin (Hb), hematocrit (HCT), mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), and standard deviation of RBC distribution width (RDW-SD) compared to the control group (Table 4). By the end of the experiment, mean corpuscular hemoglobin concentration (MCHC) and coefficient of variation in RBC width (RDW-CV) values of the control ducks had increased more significantly (p < 0.05) than the treated ducks. The values of RBC, Hb, HCT, MCV, MCH, and RDW-SD of the control ducks were significantly lower (1.4 to 1.8 10 12 /L, 82.4 to 116.9 g/L, 0.17 to 0.26 L/L, 107.5 to 148.9 fL, 54.3 to 66.6 pg, and 43.2 to 63.9%) than supplemented ducks respectively. Results also concluded that vitamin B12 plays a vital role in the synthesis of blood necessary for the different proper and optimal types of functions for the ducks and their well-being (Table 4).

Platelet Count
The values of the platelets indices by day 21 are shown in Table 5. The values of the platelet count, thrombocytocrit, and mean platelet volume of the control group had significantly lower values than the supplemented groups, (41.3 to 84.6 10 9 /L, 0.05 to 0.11 L/L and 11.4 to 13.7 fL) respectively, whereas the values of platelet distribution width of the control group were significantly higher (51.9 to 22.9%) than treated groups (Table 5).

Discussion
The influence of different levels of vitamin B12 on growth performance and hematological analysis was studied in Pekin ducks during the starter phase (0-21 days). There is no data available on the requirement of vitamin B12 in the Pekin duck, also the literature regarding vitamin B12 and Pekin ducks is limited.
In our study there were no significant differences found for the average daily feed intake and average daily weight gain in the group without vitamin B12 supplementation and groups supplemented with vitamin B12 in the Pekin ducks, respectively. The mean ADG, ADFI, and FCR in the present study is similar to that described in ducks earlier with vitamin B12 [12]. Our results also match the findings of other researchers who found that increasing levels of vitamin E in the diet had no significant effect on live body weight gain, body weight gain, feed intake, and FCR [29].
Without supplementation of vitamin B12 in the feed, the ducks did not show any clinical signs related to vitamin deficiency because the liver can preserve vitamin B12 for long periods of time, even feeding a vitamin B12 deficient diet. They further illustrated that about 2-5 months may be required for the elimination of B12 preserved by hens to such an extent that progeny will hatch with a low reserve of vitamin B12 [30].
Vitamin B12 had no effect (Table 2) on growth performance (p > 0.05). This might be due to the short period of the experiment (21 days) and its very low requirement for proper growth. In the present study, ducklings hatched from parent flocks which were feeding according to the nutrient requirements of breeder ducks. Therefore, it can be assumed that the storage of vitamin B12 in the egg yolk was stabilized, and the freshly hatched ducklings had an optimum B12 depot in the liver and in the last part of the yolk sac, and that the liver maintained the level of vitamin B12 for 21 days in the ducklings even without its supplementation in the diet [12].
Supplementation of vitamin B12 had no significant effect on the production and fertility of eggs. It was also revealed that vitamin B12 deficiency in the breeder diet had adverse effect on growth of chicks and when the chicks were collected from breeders during hatching they were supplemented with vitamin B12 but their deficiency was fulfilled up to some extent. It is therefore concluded that the supplementation of vitamin B12 in the diet of hens was more effective than the chick diet for the growth in their early stage [7].
However, limited research work has been done on hematological profiles and limited information is available on these parameters in ducks. Vitamin B12 is essential for proper red blood cell production, neurological function, and DNA formation [31][32][33][34]. The effects of vitamin B12 on the hematological indicators are shown in (Tables 3-5). The hematological indicators weremeasured in WBCs, RBCs, and platelets after both the addition and subtraction of vitamin B12. Vitamin B12 supplemented groups showed significantly higher values than the control group (p < 0.05).
Vitamin B12 works as a co-factor for methionine synthase and L-methylmalonyl-CoA mutase. Methionine synthase speeds up the transformation of homocysteine to methionine. Methionine is mandatory for the creation of S-adenosylmethionine, which is a universal methyl donor for nearly 100 different substrates, including DNA, RNA, hormones, proteins, and lipids. L-methylmalonyl-CoA mutase converts L-methylmalonyl-CoA to Succinyl-CoA in the degradation of propionate, which is a critical biochemical reaction in fat and protein uptake. Succinyl-CoA is necessary for synthesis of hemoglobin [35].
Dietary supplementation with a combination of probiotics and prebiotics markedly increased the PCV, RBC, and Hb of guinea fowls [36]. Molasses also had significant effect on MCV and platelets in broiler chickens during hot dry seasons. Presence of high MCV may indicate an active erythropoiesis, as MCV is thought to be the average size of an individual erythrocyte [30]. Younger erythrocytes are larger than older ones [37,38]. Betaine had highly significant effects on hematological parameters in the meat-type ducks under stress conditions; specially values of RBC, HCT, HGB, MCV, MCHC, RDW, platelets PLT, PCT, and MPV were significantly higher (p < 0.05) in betaine added groups as compared to the control group but no further significant differences were found among the betaine supplemented groups [39]. Similarly the control group had significantly lower (p < 0.05) values for RBC, Hb, PCV, MCV, and MCH in different breeds of Omani goats from treated groups which were receiving dietary cobalt and cutaneous injections of hydroxycobalamin [40]. Hemorrhagic anemia and hemolytic anemia are caused by the decreased value of RBCs, whereas the decrease of hemoglobin causes microcythemia. It is also reported that hematocrit and packed red cells volume have an immense correlation with each other. Consequently, when the mean hematocrit decreases, the levels of hematoglobin and hemoglobin becomes lower [39]. In a study, MPV was found to be lower in patients with vitamin B12 deficiency than in the patients without vitamin B12 deficiency as a result of production of smaller platelets [18,41].

Conclusions
To the best of our knowledge, this is the first time that someone determined the dietary requirement of cyanocobalamin for male Pekin ducks during the starter phase on the basis of growth performance and hematological indicators. However, we found that cyanocobalamin had a non-significant effect on growth performance, but it had more significant improvements on hematological indicators. On the basis of our findings we suggest that 0.02 mg cyanocobalamin/kg of the feed may be supplemented to improve hematological variables of Pekin ducks from hatch to day 21.


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Temporal Behavior of Tree Communities in Different Soil Classes

The objective of this work was to evaluate if the soil heterogeneity expressed by the different soil classes would condition tree communities with different dynamic behaviors. The data were collected from 25 plots sized 20 × 20 (400 m2), 10 of them with Inceptisols, 9 with Ultisols, 5 with Entisols B and 1 with Entisols A. All individuals with DBH ≥ 5.0 cm in the plots were sampled in 2000, 2005, 2009 and 2015. Rates of mortality, recruitment, gain, loss and turnover dynamics in number of individuals and basal area were estimated for each soil class. The tree communities present in the soil classes showed no differences in the dynamic behavior to the point of expressing edaphic fragment heterogeneity. The differences are associated with stochastic fluctuations in rates caused by small disturbances, suggesting little influence of the soils on the dynamics of small-scale communities.

INTRODUCTION AND OBJECTIVES
Tropical forests are dynamic systems that exhibit variations throughout space and time. In semi-deciduous forests, this variation has been associated with a history of fragment disturbance and environmental heterogeneity caused by water availability, topography, successional stages and soil types (Botrel et al., 2002;Meyer et al., 2015;Morel et al. 2016;Oliveira-Filho et al., 2007;Terra et al., 2015).
Among the factors that promote environmental heterogeneity, soil stands out as one of the main determinants of tropical forests on local scales. On this scale, the specific characteristics of each habitat assume a special role in conditioning vegetation. The abundance and distribution of tree individuals, the richness and diversity of species and the tropical forest's structure are strongly influenced by soil resource availability (nutrients and water) on small scales (Bolhman et al., 2008;Martins et al., 2015;Peña-Claros et al., 2012).
Variations in soil characteristics may also cause differences in the tree community's dynamics. Growth, mortality and recruitment rates are commonly higher in environments with higher resource availability, such as fertility and humidity, resulting in faster turnover rates (Martins et al., 2015;Missio et al., 2016;Pontara et al., 2016;Russo et al., 2008), while slow growth, low mortality and recruitment of individuals are observed in poor-resourced soils (Martins et al., 2015;Missio et al., 2016;Pontara et al., 2016;Russo et al., 2008).
In this sense, our study aims to analyze the dynamic behavior of a semi-deciduous seasonal forest with different soil types after 15 years of monitoring. Therefore, we hypothesized that soil heterogeneity, expressed by different soil classes, conditions tree communities with different dynamic behaviors.

Study area
The study area comprises a fragment of Seasonal Semi-deciduous Forest located on the riverbanks of Ingaí River, municipality of Ingaí, in the Alto Rio Grande region of southern Minas Gerais, and at the geographical coordinates 21° 24' S and 44° 55' W. The fragment has a total area of 16.85 ha, with an altitude varying between 870 and 890 m (Botrel et al., 2002).
The study area has no cutting history, only the removal of some trees at the edge of the forest in the 1950s (Botrel et al., 2002), and presence of domestic animals, such as cattle and horses, which were very common until 2009. Currently, only some trails, which may exist due to the sporadic presence of these animals and visiting fishermen, have been observed in the fragment. The matrix in the fragment's surroundings is formed by other small fragments and by anthropized areas (pastures and eucalyptus, among others).
According to the Köppen classification, the climate of the region is Cwa; a subtropical climate with dry winter and rainy summer (Dantas et al., 2007). The average annual temperature is 20°C, with maximum average of 28.1°C and minimum of 9.3°C; the average annual rainfall corresponds to 1476 mm (Oliveira-Filho, 2010).

Edaphic fragment characterization
The forest fragment features four soil types: Gleico Eutrophic Tb Fluvent Entisols (Entisols A); Typic psamitic Fluvent Entisols (Entisols B); Typic Tb Dystrophic Haplic Cambisols (Inceptisols); and Typic haplortox soil (Ultisols) (Botrel et al., 2002). Entisol A was sampled from only 1 plot, located on the lower alluvial terraces, in which the soil remains saturated or flooded for a longer period of the year (poorly drained) and with medium texture. Entisol B was sampled from 5 plots, being present in higher alluvial terraces with occasional floods and with sandy and poorly-drained texture. Inceptisols occurred in 10 plots above the alluvial terraces or adjacent to the river, where the margin is steeper. It has medium to clayey texture and drainage ranging from imperfect to moderately drained. Ultisol was sampled from 9 plots in higher areas further from the river. This soil has medium texture and drainage ranging from well to markedly drained. The variations in soil type, fertility, humidity and relief suggest the presence of a gradient: Ultisols < Inceptisols < Entisols B < Entisols A; which corresponds to an increase in water and nutrient availability (Botrel et al., 2002).

Monitoring the tree community
The first study of the tree community was conducted between August 1999 and August 2000, when 25 permanent 20 × 20 m plots were allocated, totaling 1 ha of sampled area. The layout of the plots is detailed in Botrel et al. (2002). All tree individuals in the plots with DBH (diameter at breast height) ≥ 5.0 cm were sampled, received numbered aluminum plates and were identified (Botrel et al., 2002). Individuals with multiple stems were recorded when the quadratic root of the DBH was ≥ 5.0 cm (Scolforo & Mello, 2006). The plots were measured again in the years 2005, 2009 and 2015, with the objective of re-measuring the surviving individuals, estimating dead individuals and including those that met the minimum inclusion criterion.

Tree community dynamics by soil class
The tree community dynamics were analyzed for each soil class according to the rates of: Equation 1 (mortality of individuals) and Equation 2 (recruitment of individuals); Equation 3 (basal area loss); and Equation 4 (basal area gain). These formulas were obtained according to Sheil et al. (1995) and Sheil & May (1996): The turnover rates in number of trees (Equation 5) and in terms of basal area (Equation 6) were determined from the formulas described by Phillips (1996) and Phillips & Gentry (1994): In the equations, N 0 , N t , N m and N r represent the numbers of initial, final, dead and recruited tree individuals in time interval (t), respectively, while BA 0 , BA t , BA m , BA d , BA r and BA g represent the initial alive, final alive and dead basal areas of the trees, the basal area lost with the decrease in survivors, the basal area of the recruits and the basal area gained with the increase in survivors, respectively.
The tree individuals' mortality and recruitment rates were corrected according to Lewis et al. (2004) (Equation 7) due to time irregularities between the sampling periods: λ': corrected dynamic rates; λ: verified dynamic rates.

Statistical analyses
The data had their normality verified by the Shapiro-Wilk test (Zar, 2010). Structural and dynamic parameters were analyzed to verify differences within the soil classes between the studied time periods (2000-2005; 2005-2009 and 2009-2015), and the space between the soil classes at each monitoring interval. In the temporal comparisons, the parameters of basal area and abundance of individuals were compared using the paired T-test at a significance level of 5% (Zar, 2010), while for the spatial comparisons, the basal area and abundance of individuals were compared by the analysis of variance (ANOVA) followed by Tukey's test at a significance level of 5%. The changes over time and between soil classes in relation to the number of dead, recruits, losses (baseline area of the deceased individuals plus the decrease in survivors) and gains (baseline area of the recruits plus the increase in survivors) were verified using the non-parametric Kruskal-Wallis test with the Mann-Whitney test, considering a significance level of 5% (Zar, 2010). The turnover in numbers of individuals and in basal area were compared over time and between the soil classes also by the analysis of variance (ANOVA) followed by Tukey's test at a significance level of 5%. To meet the statistical assumptions, the turnover rates were transformed by the equation "arcsen = root (×/100)" (Zar, 2010). The Entisols A class was removed from the temporal comparisons and from the comparisons between environments since it did not feature repetitions, and thus was analyzed and discussed separately. The statistical analyses were performed using the PAST v.3.14 software (Hammer et al., 2001). In the comparisons between them, the abundance of individuals was significantly higher in Ultisols in the four studies, Entisols B having featured an abundance of individuals that was similar to that shown by Ultisols in 2009 (Table 1).
The number of deaths in Ultisols and Entisols B was significantly higher in the third interval, resulting in rates of 2.93 and 6.65 %.year -1 , respectively. The mortality in Ultisols was also significant in the second interval (2.97 %.year -1 ). Nonetheless, the comparisons between soil classes did not show significant differences in the number of deaths for any of the monitoring intervals (Table 1).
The recruitment of individuals showed no temporal differences in any soil class, and they also did not differ from one another ( Table 1). The turnover in number of individuals showed temporal differences in Ultisols, where the second interval had a significantly lower turnover rate; and in Entisols B, with the turnover rate of 2009-2015 having been higher than that of 2005-2009. In the comparisons between classes, the environments differed in the third interval, the turnover in number of individuals having been faster in Inceptisols and Entisols B (Table 1).
As for the biomass descriptors, basal area showed a significant increase between 2000 and 2005 and from 2005 to 2009 in the soil classes of Ultisols, Inceptisols and Entisols B. However, no significant differences in basal area in the period between 2009 and 2015 were found for any soil class. The basal area was significantly higher in Entisols B in 2005, and no other significant differences were found between soils for the other years of monitoring (Table 1).
The loss in basal area was significantly higher for the third interval in Ultisols, resulting in a rate of 1.97 %.year 1 , and in Entisols B, with a rate of 3.62 %.year -1 . In the comparisons between classes, differences were observed between 2000 and 2005, when the loss was lower in Ultisols, and between 2009 and 2015, when it was higher in Entisols B (Table 1).
The basal area gains showed no differences over the monitoring periods for any soil class (Table 1). Among the environments, differences were verified only in the first interval, in which Entisols B had a superior gain in basal area. The basal area turnover was significantly higher in the second interval of Ultisols. Differences in classes were observed between 2005 and 2009, the fastest turnover in basal area having been found in Ultisols; and between 2009 and 2015, with Entisols B having shown a faster turnover than Inceptisols (Table 1). Table 1. Structural parameters and dynamic rates of the arboreal communities present in the classes of Ultisols, Inceptisols and Entisols B at the Seasonal Semi-deciduous Forest fragment in the municipality of Ingaí, MG. Capital letters represent the significant statistical difference (p≤0.05) between the environments in each monitoring interval, and lowercase letters, the differences over time for each soil class.   The only sample of Entisols A was characterized by high dynamic rates. In the first interval, the high rates were associated with the mortality of individuals and losses in basal area (20.09 and 20.60 %.year -1 , respectively). In the second interval, the processes were reversed, with a high recruitment of individuals and gains in basal area (19.73 and 21.35 %.year -1 , respectively), while in the third period, the demographic rates were low, but the basal area gain remained high (7.91 %.year -1 ) ( Table 2).  The tree communities in the soil classes had a similar temporal behavior characterized by the self-thinning of trees. In the communities submitted to self-thinning, mortality rates are higher than recruitment rates, which reduces the density of individuals and causes a shift in balance in favor of basal area gains, which provides an increase in or stability of the tree community's basal area (Higuchi et al., 2008;Meyer et al., 2015).

Rates
The similar temporal behavior among tree communities shows that, despite the existence of a fertility and texture gradient between the soil classes, the dynamic processes may not be conditioned by soil heterogeneity, since the observed differences do not describe any pattern associated with soil conditions. Other studies carried out on local scales have also verified similar dynamic behaviors among tree communities in different soil classes (Higuchi et al., 2008;Meyer et al., 2015;Toledo et al., 2011). The soils' influence on the forest dynamics has been mainly associated with large fertility variations on regional scales, with higher rates being verified in soils with greater resource availability Quesadas et al., 2009). However, on local scales, Toledo et al. (2011) state that soil fertility variations may have a modest effect on forest dynamics. On this scale, the reduced area favors lower heterogeneity due to the more similar physical conditions (light, water, soil, etc.) (Costa et al., 2012). On the other hand, on larger area size entails greater environmental heterogeneity (Cavender-Bares et al., 2009).
The low influence of soils on the tree community's dynamics verified on local scales (Higuchi et al., 2008;Meyer et al., 2015;Toledo et al., 2011;in the present study) indicates that the soils play a more important role in structuring these communities than in the dynamic processes that modify them. This edaphic 6/8 Floresta e Ambiente 2019; 26(4): e20170754 Santos ABM, Carvalho WAC, Morel JD, Santos R, Figueiredo MAP, Cirne-Silva TM, Souza CR heterogeneity would mainly influence the abundance and spatial distribution of the species in the plant communities (Bohlman et al., 2008;Pan et al., 2013;Peña-Claros et al., 2012), selecting the individuals that are more adapted to the environmental conditions based on the niche need of each species (Begon et al., 2007;Poulos & Camp, 2010). The abundance and distribution of the species in the study area mainly reflect variations in water and nutrient availability (Botrel et al., 2002), demonstrating the importance of the soil in structuring this community.
On the other hand, the temporal behavior would be conditioned by agents that alter the successional advance of the forests, such as disturbances. Disturbances are events that drive changes in the forests due to resource changes (Davis & Moritz, 2001;Denslow et al., 1998), in substrate availability or in the physical environment (Davis & Moritz, 2001), thus reducing density and resulting in considerable losses of basal area, which opens spaces for the recruitment of individuals and recovery of biomass (Felfili, 1995;Chazdon et al., 2007;Sheil et al., 2000;Swanson et al., 2011). However, the absence of major disturbances that would promote the resumption of forest succession attenuates the role of soils in the studied fragment, since they would act to select the species with colonization potential in each environment. In tropical forests, the onset of succession is not stochastic, but rather conditioned by processes, such as plant-plant facilitation interactions, and local environmental conditions, such as soil characteristics, which determine the successional process in these forests (Lebrija-Trejos et al., 2010;Martins et al. 2015).
The absence of major disturbances associated with the the fragment's protection favors the successional advance of the tree communities standing over the soil classes. Seasonal tropical forests usually exhibit structural parameters of advanced successional stages around 40-45 years after the area's abandonment, when the late colonizing species, which grow at a slower pace, become dominant or reach significant development rates (Aide et al., 1996;Lebrija-Trejos et al., 2010;Morel et al., 2016;Toniato & Oliveira-Filho, 2004). The advanced successional stage of the tree communities in the fragment shows that they are already structured with the species that compose them and already adapted to the local environmental conditions, being dominated by species with long life cycles that have remained in the community for a long time in the absence of great disturbances, thereby attenuating the role of the soils in the selection of species. Thus, the differences among soil classes may be associated with stochastic fluctuations in dynamic rates (Carvalho & Felfili, 2011), which are caused by small localized disturbances originated from natural disturbances, such as seasonal flooding in plots near the river bank (Silva et al., 2011).
River banks are environments characterized as dynamic due to the influence of a number of factors that alter their configuration and the patterns of plant communities (Kyle & Leishman, 2009). One of these factors is the river water's action on the slope's concave margin, which promotes the continuous removal of sediments (Christofoletti, 1980). In Entisols B, this action resulted in the collapse of part of a plot between 2009 and 2015, and in the death of several tree individuals. Another factor is the occurrence of seasonal floods caused by the overflow of the riverbed and/or elevation of the water levels during periods of intense precipitation (Almeida Júnior et al., 2009;. Seasonal flooding represents an important disturbance that changes the dynamics of Entisols A. Floods increase the mortality of individuals and the loss of basal area (Niu et al., 2014;Silva et al., 2011), thus making room for the recruitment of individuals (Felfili, 1995) and causing instability in the dynamics of forests bordering water courses.

CONCLUSIONS
The tree communities did not show differences in dynamic behavior to the point of expressing edaphic fragment heterogeneity. The differences found are probably more associated with stochastic fluctuations caused by minor disturbances, suggesting lesser influence of the soils on the dynamics of communities on small scales. On this scale, soils may play a more important role in structuring the community, influencing parameters such as the abundance and distribution of species. However, in this fragment, this influence may be attenuated by the advanced stage of succession and the absence of major disturbances.


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Antimicrobial Effects of the Nanoemulsion of Rosemary Essential Oil Against Important Foodborne Pathogens

Background: The purpose of this study was to determine the effect of rosemary essential oil (REO) nanoemulsion against some important food borne pathogens.

Methods: Antibacterial effects of REO and REO nanoemulsion were determined using Agar disc diffusion, Broth microdilution and Steam phase diffusion methods against Staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa, Shewanella SP, Listeria monocytogenes and Salmonella enteritidis.

Results: Antibacterial effect of REO and REO nanoemulsion was increased with concentration enhancing of REO. There was no significant antibacterial activity in the effectiveness of nanoemulsion on the studied bacteria in comparison with REO in both disk diffusion and steam phase diffusion methods. MIC and MBC analysis of REO and prepared REO nanoemulsion showed that REO and its nanoemulsion have inhibited all studied bacteria. REO showed better inhibitory effects. REO and nanoemulsion of rosemary essential oil have the greatest effect on Shewanella SP., L. monocytogenes, S. aureus, S.enteritidis, E. coli and P. aeruginosa, respectively.

Conclusion: In total, it can be said that REO and its nanoemulsion are desirable to inhibit the growth of food borne pathogens and can be a good choice as antimicrobial agents in food industry to enhance safety and extend foods’ shelf life.

Introduction
Recently, there has been growing demand for safe foods across the world due to the increased prevalence of severe foodborne diseases, which are caused by the consumption of food products contaminated with food pathogens. The Center for Disease Control and Prevention (CDC) has reported that approximately 48 million individuals are affected by foodborne diseases each year in the United States. Several methods have been developed to increase food safety, such as thermal processing and use of chemical preservatives [1,2].
Each of these methods has specific advantageous and limitations; for instance, heating may corrupt foodborne pathogens and spoilage bacteria, while it could also decrease the nutritional value of food products. There have been growing concerns about consumers due to the long-term use of chemical preservatives in large quantities as it may cause various disorders (e.g., cancer) [2]. Therefore, trends to use natural preservatives have extended as an alternative by manufacturers and consumers since they increase the shelf life and safety of food products with no side-effects [2]. Essential oils (EOs) and herbal extracts are considered to be excellent natural preservatives for food products. These compounds are aromatic, oily liquids, which are extracted from plants and commonly used as flavoring agents in food [3].Numerous studies have confirmed the antimicrobial effects of EOs against foodborne pathogens and spoilage microorganisms [4,5]. In this regards, the scientific community and food industry have attempted to use potential preservatives of natural origins in various food products.
Rosemary essential oil (REO) is a bioactive compound with remarkable antifungal, antibacterial, and hepatoprotein properties. REO is extracted from rosemary (Rosmarinus officinalis), which is a perennial herb of the Lamiaceae family and widely used for the preservation and flavoring of food products [6,7]. However, direct addition of aromatic plants or their derivatives to food as preservatives is mainly limited due to their flavor, which influences the organoleptic attributes of food at bioactive effective doses. On the other hand, the dispersion of EOs in water-based products is rather difficult [7]. Special methods have been developed in order to overcome these limitations, such as encapsulation and nanotechnology.
Nano-scale manipulation of materials has created novel opportunities for new research and interdisciplinary scientific cooperation [8]. The particle size of bioactive compounds changes to less than 100 nanometers by nanotechnology, which could also be applied to carry EOs, compromise their adverse effects, and optimize the interactions of EOs with food components [9].
In recent years, nanotechnology has expanded rapidly in food industry [9]. This technology is highly capable of extending the shelf life of food products, production of new foods, and optimizing the taste, texture, and sensory attributes of food [10]. Furthermore, nanoemulsions, which are in the form of oil in water emulsions with the droplet size of less than 100 nanometers, have been identified as a transmission system for the bioactive compounds that are produced by nanotechnology with unique features such as transparency, stability, and high performance [8].
Given the importance of EOs in the prevention of foodborne diseases and food spoilage, and since no reports have been published regarding the antimicrobial effects of REO nanoemulsion against foodborne pathogens, the present study aimed to determine the effects of REO nanoemulsion against some important foodborne pathogens.

Essential Oil Extraction
Rosemary plant (Rosmarinus officinalis) was purchased a local market in Zanjan, Iran. The plants were washed and dried in shade at room temperature for seven days. REO was extracted from the grinded, dried plants using the hydrodistillation method and Clevenger's apparatus (KOL, Behr, Germany) at the temperature of 100 ˚C for three hours. Afterwards, the EO was collected and dried using anhydrous sodium sulfate, filtered through 0.22-μm filters, and stored in a sterile bottle made of dark glass at the temperature of 4 ºC for further analysis [11].

GC-MS Analysis
An equipped gas chromatograph (AGILENT 7890 B, Santa Clara, CA) with an HP-5MS column (length: 60 m, film thickness: 0.25 mm, I.D.: 0.25 mm), which was coupled to a mass spectrometer (AGILENT 5977A), was applied to determine the chemical composition of REO using the method described by Raeisi et al. (2016) with some modifications.
The flow rate of helium (He2) as the carrier gas was 1.5 ml/min. The initial temperature of the column was maintained at 50 °C for five minutes and programmed to rise gradually to 150 °C at the 5°C/min gradient (holding time: 5 min), while it increased to 300°C with the gradient of 20 °C/min (holding time: 5 min). The temperature of the injector and detector was set at 270 °C and 250 °C, respectively. The injected volume and split ratio were determined to be one microliters and 1:50, respectively. The obtained mass spectra were compared to those available in the database (Wiley-VCH 2001 data /NIST Ver 11, 2017) in order to identify the existing compounds [12]. A loopful of the stock culture was transferred to the brain heart infusion (BHI) broth (Merck, Darmstadt, Germany) and incubated at the temperature of 35 ºC for 18-24 hours so as to prepare fresh suspensions of the tested bacteria. Following that, the freshly prepared suspensions were set to the absorbance of 0.08-0.1 at 600 nanometers in order to obtain an enumeration equal to 108 CFU/ml of the bacteria using a spectrophotometer (Milton Roy Company, Warminster, USA). In addition, bacterial count was confirmed by the tenfold serial dilution and surface spreading of the bacteria on the plate count agar media and incubation at the temperature of 35 ºC for 24 hours [13].

Preparation of the REO Nanoemulsion
At this stage, the high-amplitude ultrasonic method was used for the preparation of the REO nanoemulsion. To this end, various concentrations of REO and Tween 80 with Span 80 as the surfactant were mixed ( Table 1). The combination of REO and surfactant was mixed on a hot plate using a magnetic stirrer for 10 minutes. Afterwards, the solution was initially blended and homogenized using a high-speed homogenizer (model: Silent Crusher M, Heidolph, Germany) at 9,000 rpm for 20 minutes. Following that, sonication was performed for three minutes at the maximum rate. Table 1 shows the REO nanoemulsion with various formulations. As can be seen, the prepared nanoemulsions had a transparent structure with no turbidity. All the prepared formulations were preserved for up to five months at room temperature (25 °C) and refrigerated temperature (4 °C). No changes were observed in the particle size and polydispersity index (PDI), confirming the long-term sustainability of the nanoemulsions and specified maintenance period at room and refrigerated temperatures.

Determining the Size of the Nanoemulsion Particles
The particle size of the nanoemulsion was determined using the dynamic light dispersion device (ZEN 3600, Malvern, Investor Company, GB) at the temperature of 25 °C. To this end, one milliliter of each sample (diluted with deionized water 20 times) was poured into the cell of the target device [14].

Antibacterial Effects of the REO Nanoemulsion
In the current research, three methods were used to assess the antibacterial effects of REO and REO nanoemulsion.

Agar Diffusion Assay (Disk-Diffusion Method)
In the present study, the disk-diffusion method was applied to determine the antibacterial effects of REO and REO nanoemulsion. Briefly, 100 microliters of each of the mentioned bacterial suspensions containing 10 7 CFU/ml were spread on the Müller-Hinton agar culture media (Merck, Darmstadt, Germany), and sterile disks (diameters: 6-10 mm) were transferred onto the culture media containing the bacteria. Afterwards, 10 microliters of various concentrations of filtered REO and REO nanoemulsion (1%, 2%, 4%, and 8%) were transferred onto the disks, and the agar medium plates were incubated at the temperature of 37 °C for 24 hours. Finally, the antibacterial activity of REO and REO nanoemulsion was determined by measuring the growth inhibition zone (GIZ) of each bacterium.
In this process, disks containing chloramphenicol (30 u/g) were used as the positive control [15].

Minimum Inhibitory Concentration (MIC) and Minimum Bactericidal Concentration (MBC)
To determine the minimum inhibitory concentration (MIC), the broth microdilution method was used as recommended by the CLSI with some modifications. Briefly, 160 microliters of the BHI broth was added to each well of a 96-well microplate. Following that, 20 microliters of the each fresh overnight bacterial inoculum (5 ×10 6 CFU/ml set by 0.5 McFarland standard tubes) and 20 microliters of each concentration of the REO and REO nanoemulsion solutions (0.5%, 1%, 2%, 4%, and 8%) were added. A well containing 20 microliters of the inoculum of each bacterium and 180 microliters of the BHI broth was considered as the positive control, and another well containing 180 microliters of the BHI broth and 20 microliters of filtered REO was considered as the negative control.
The microplates were incubated at the temperature of 37 °C for 24 hours. The minimum concentration of the EOs visibly showing bacterial inhibition growth was considered as the MIC. The wells without bacterial growth (predetermined 99% reduction of bacterial population) were defined as the minimum bactericidal concentration (MBC) of REO and REO nanoemulsion [16].

Steam-phase Diffusion Method
The steam-phase diffusion method was applied in accordance with the method described by Masoomi et al. (2016). Initially, 100 microliters of the bacterial suspension with the inoculation dose of 107 CFU/ml was spread on the BHI agar culture medium. Following that, 0.5 milliliter of a semi-melted agar medium was placed at the center of the plate door. In addition, a sterile blank disk was placed on this section, and 20 microliters of the mentioned pure bioactive materials (REO and REO nanoemulsion) was added to the blank disk. The culture plates were incubated at the temperature of 37 °C for 24 hours, and the GIZ (mm) indicated the steam diffusion rate of the REO and REO nanoemulsion [17].

Statistical Analysis
All the experiments were conducted in triplicate. Data analysis was performed using the mean and standard deviation (SD) of the variable in Microsoft Excel software (2010).
According to the information in Table 2, monoterpene and monoterpenoid were the main components of REO, which is inconsistent with the results of the previous studies in this regard ( Table 3). The discrepancy in the chemical constituents of EOs could be attributed to several factors, including the differences in climate, geographical conditions, plant parts, cultivation conditions, cultivar type, planting season, methodology and duration of extraction, and duration and condition of EO storage [12]. Table 1 shows various formulations of the REO nanoemulsion. Based on the mean size and PDI, the optimal prepared nanoemulsion was formulation No. 11, which contained 5% (w/w), 3.75% (w/w), and 2.5% (w/w) of REO, Tween 80 and Span 80, respectively, with the mean particle size of 61.73 ± 0.94 nanometers and mean PDI of 0.230 ± 0.009.

Nanoemulsion and Particle Size Characteristics
The most important stage in the present study was the accurate formulation and proper production of oil in water nanoemulsion. If the formulation was prepared improperly, the emulsion system would collapse [22]. The foremost parameters that influenced the chemical characteristics and stability of the nanoemulsion included the homogenization pressure, rotation rate, and concentration of the surfactants [23]. The mean particle size largely depended on the surfactant-to-oil ratio (SOR), which decreased with increased SOR. In a study in this regard, Martin Piñero et al.
(2018) reported that obtaining smaller particle sizes than 200 nanometers is possible with the 30% increase of the surfactant and reduction of the EO concentration [24].
Similarly, Restrepo et al. (2018) reported the mean particles size of rosemary nanoemulsion to range from 164 ± 9 to 676 ± 26 nanometers [25].  3.3. Antimicrobial Properties of the REO and REO Nanoemulsion Table 4 shows the growth inhibitory effects of the REO and REO nanoemulsion against E. coli, S. enteritidis, Shewanella sp., L. monocytogenes, S. aureus, and P. aeruginosa based on the agar disk-diffusion and steamphase diffusion methods. Table 5 shows the MIC and MBC of the REO and REO nanoemulsion against these pathogens based on the broth microdilution method.
The findings of the current research indicated no significant antibacterial effects of the nanoemulsion on the studied bacteria compared to the REO using the diskdiffusion and steam-phase diffusion methods (Tables 4 & 5). In the nanoemulsion particles, the EO droplets were covered by a liquid phase containing the surfactant. Therefore, the antibacterial effects of the EO were not observed in the nanoemulsion escape phase as was found in pure EO (Table 5) [17].
Several reports have denoted the antibacterial effects of REO. In a study conducted by Raeisi et al. (2016) regarding the antimicrobial effects of REO against foodborne pathogens, the mean diameters of the GIZ formed by pure REO on E. coli, L. monocytogenes, S. typhimurium, and S. aureus were estimated at 23.2 ± 0.4, 31.3 ± 0.3, 21.4 ± 0.2, and 30.4 ± 0.3 millimeters, respectively [12]. In another study in this regard, Fu et al. (2007) reported that the mean GIZ and antibacterial effects of REO against S. aureus, E. coli, P. aeruginosa were 18.5 ± 1.3, 10.0 ± 0.8, and 6.0 ± 0 millimeters, respectively [26]. The results of the aforementioned studies are inconsistent with the current research.
In reported to be 9.72 ± 0.3 millimeters and zero based on the disk-diffusion and steam-phase diffusion methods, respectively [17]. On the other hand, the EO surrounded by the surfactant layer in the nanoemulsion particles could not penetrate into the vapor phase, and the antibacterial effects of the REO became subsequently weaker compared to the pure EO [17].
In the present study, the MIC and MBC analysis of REO and the prepared REO nanoemulsion indicated that REO and its nanoemulsion inhibited all the studied bacteria, while REO exhibited more significant inhibitory effects (Table 4). This is inconsistent with the results obtained by , which demonstrated the four-fold antibacterial activity of EO nanoemulsion compared to the bulk oil against E. coli and S. typhi based on the MIC and MBC measurements [28]. The difference in this regard could be due to the active binding sites of the bioactive ingredients of EO with the applied surfactant, as well as the inhibition of bringing the EO into the proximity of the bacterial cell membrane [29]. On the other hand, the results of the present study demonstrated that P. aeruginosa had lower sensitivity to REO, which is in line with the findings of Jawad et al. (2018) and Thanh et al. (2017) [18,30].
According to the findings of the current research, the REO and REO nanoemulsion most significantly affected Shewanella sp., L. monocytogenes, S. aureus, S. enteritidis, E. coli, and P. aeruginosa, respectively. In addition, the results of all the antimicrobial experiments indicated that the REO and REO nanoemulsion had more significant antibacterial effects against gram-positive bacteria (Shewanella sp., L. monocytogenes, and S. aureus) compared to the gram-negative bacteria (S. enteritidis, E. coli, and P. aeruginosa). In gram-positive bacteria, ion permeability enhanced the cell membrane due to the direct contact of the lipophilic components of the EO with the phospholipid layer of the cell membrane, and the absence of an outer phospholipid membrane facilitated intracellular excretion [15].

Conclusion
According to the results of the microdilution and diskdiffusion assays, both the REO and REO nanoemulsion exerted more significant antibacterial effects against grampositive bacteria compared to the gram-negative bacteria. However, no significant differences were observed in the antibacterial effects of REO nanoemulsion and REO based on the disk-diffusion and steam-phase diffusion methods. In conclusion, it could be stated that REO and REO nanoemulsion are proper candidates for the growth inhibition of foodborne pathogens and could be used as effective antimicrobial agents in food industries for the improvement of food safety and to extend the shelf life of food products.