text:
Methane Emissions, Performance and Carcass Characteristics of Different Lines of Beef Steers Reared on Pasture and Finished in Feedlot

Simple Summary The livestock sector is a significant contributor to global greenhouse gas emissions, with cattle representing 62% of CO2 emissions. Genetic selection is a tool that has the potential to reduce emissions from rumen fermentation and can be adopted in grazing and feedlot systems. Therefore, the purpose of this study was to evaluate steers from a new line of the Canchim breed (5/8 Charolais, 3/8 Zebu) derived by genetic selection improvement, and formed and maintained as a closed herd since 1998, in regards to performance, carcass dressing, and enteric methane emissions, when compared with the old line formed and maintained as a closed herd since 1960. Animals from the new line of the Canchim breed should be used for rearing on pasture to obtain greater liveweight gain. However, genetic improvement in Canchim breed does not guarantee animals with lower methane emissions under grazing conditions. For the finishing feedlot phase, increased liveweight gain, improved feed conversion, and a better carcass dressing percentage are obtained using animals from the new line of Canchim breed. The new line also leads to higher daily feed intake and higher daily liveweight gain, but with the same methane emissions per kg of animal liveweight than the other lines. Abstract The present study aimed to investigate whether different lines of a composite breed (5/8 Charolais × 3/8 Zebu), formed at different times, and genetically improved, would result in differences in animal performance, enteric methane emissions, and carcass traits. Forty-six Canchim steers (15 months, 280 ± 33 kg liveweight) from three different lines were used: old, new, and their cross. These three breed lines were considered the treatments (arranged in four randomized blocks based on initial liveweight) and were evaluated under grazing and feedlot conditions in relation to the performance and emission of enteric methane. During the grazing period, the new line was found to be superior to the old only in relation to the average daily liveweight gain (0.692 vs. 0.547 kg/day), and with no differences in relation to the cross line (0.692 vs. 0.620). In the feedlot finishing phase, only the average daily liveweight gain was significantly higher in the new line compared to the cross and old line (1.44 vs. 1.32 and 1.23 kg/day). The new and cross lines demonstrated higher dry matter intake when compared to the old line (10.25 and 10.42 vs. 9.11 kg/day), with the crossline animals demonstrating the best feed conversion. The new line showed higher enteric methane emissions compared to the old line (178 vs. 156 g/day). The line had an effect on the carcass dressing of the animals, with greater fat thickness in carcasses from the new and cross lines than the old line (4.4 and 3.8 vs. 3.2 mm). Canchim cattle selected for improved productive performance characteristics does not guarantee animals with lower methane emissions under grazing conditions; while in feedlots, can lead to increased daily feed consumption, and hence, to higher emissions of methane.

Introduction
Livestock farming is a fundamental activity for maintaining the current society. By 2050, the world's population is expected to reach 9.1 billion people. As a result, agricultural activities must grow accordingly in order to sustainably meet this growing food demand [1]. According to Oliveira Silva et al. [2], ruminant animals are the largest cause of agricultural externalities in terms of land use and emissions of greenhouse gas (GHG), such as methane (CH 4 ). In 2010, agriculture accounted for 5.5 gigatonnes of carbon dioxide equivalent (GtCO 2 eq) of global emissions, with 2.1 GtCO 2 eq of this total resulting from of enteric fermentation by these animals [3].
The approach adopted by the Intergovernmental Panel on Climate Change (IPCC) for GHG inventories, while adequate, does not take into account the mitigating options that exist within each activity. Cattle reared on pasture, for example, have significantly lower emissions when we take into account atmospheric carbon dioxide (CO 2 ) that is absorbed by grasses and added to the stock of carbon (C) in the soil [4]. Considering that approximately 3.7 billion hectares of the earth's surface are occupied by pasture, the potential for sequestration varies between 0.01 and 0.3 GtC/year [5]. Almeida and Medeiros [6] suggest the use of strategic feedlots for a short period during the finishing of beef cattle as an alternative for mitigation of GHG emissions. If the animal reaches slaughter weight earlier, its life cycle will be shorter, and consequently, it will emit less GHG. A similar effect occurs when more efficient animals are used [7].
The hypothesis of the present study was: Canchim breed cattle genetically improved for performance traits, even within lines of the same breed, will result in differences in animal performance, enteric methane emissions, and carcass traits. Therefore, the purpose of this study was to evaluate steers from different lines of the Canchim breed (5/8 Charolais, 3/8 Zebu) resulting from genetic selection programs, in regards to performance, carcass dressing, and enteric CH 4 emissions.

Experimental Design and Treatments
The experiment took place between December 2014 and September 2015 at the Brazilian Agricultural Research Corporation (Embrapa Southeast Livestock, São Carlos, São Paulo). The trial was approved by the local committee for Ethics in Animal Experimentation (CEUA) (protocol number 03/2014).
Forty-Six Canchim steers (composite breed of 5/8 Charolais and 3/8 Zebu) from three distinct lines were used: old (n = 13), new (n = 20) and cross (n = 13, a result from the crossing of animals from the old and new lines). These three breed lines were considered the treatments. According to recommendations by Morris [8], within the breed lines, animals were arranged in four randomized blocks based on initial liveweight. Four pasture areas and four pens in feedlot were used to receive one block of each treatment (breed lines). Each area or pen comprised one block with the three treatments. The final arrangement is presented in Table 1.

Description of the Canchim Breed Lines
The Canchim breed was developed by the Brazilian Ministry of Agriculture at the then São Carlos Experimental Station (now Embrapa Southeast Livestock), from the 1940s, through the alternate crossing between Charolais and Zebu (Guzera, Indubrasil, and Nelore breeds). The overall objective was to take advantage of the favorable effects of heterosis (hybrid vigor), to complement the desirable characteristics of Charolais (high growth rate and good meat quality), and Zebu (adaptation to the tropical conditions), and to facilitate the introduction of Charolais genes into Brazilian beef production systems through the use of the Canchim breed [9]. The old line has been kept at Embrapa Southeast Livestock as a closed herd since 1953. It was originally formed from 53 Charolais bulls imported from France between 1922 and 1950 that were mated with Zebu females [10,11]. The breed's genetic basis was limited to these resources until the early 1990s, when, after approval by the Ministry of Agriculture, Livestock and Supply, the process of formation of new lines was approved at the request of the Brazilian Association of Canchim Breeders. The development of the new line began at that time, when the genetic base of the breed began to enlarge with the use of Charolais bulls imported from different countries, and from breeding programs with proven genetic merit, and at that time, exclusively mated with Nellore females. After the establishment of the new line, it has been kept closed since 1998 [12]. Animals from the cross line began to be bred from 1998 to 2010 [13], when the group was closed. The aim was to study the effect of hybrid vigor obtained from the crossing of the old and new line.
From their formations, breeding animals of the respective lines were mated based on Expected Differences in Progeny (EPDs), provided by the Geneplus-Embrapa breeding program, where the following selection traits were used: birth weight, scrotal circumference, coat scores, belly button, cold shape, weaning liveweight, yearling liveweight, and age at first calving [10]. Regarding the relative selection pressure, the "Geneplus" Genetic Qualification Index (GQI) for the Canchim breed is: 0.15 BW + 0.20 MA+ 0.30 YL + 0.20 YCC + 0.15 YSC (where BW is birth weight; MA is maternal ability; YL is yearling liveweight; YCC is yearling carcass conformation; YSC is yearling scrotal circumference). In the experimental herd of Embrapa Southeast Livestock, since 2013, EPDs for BW, belly button and pelage are being controlled (values close to zero) and EPDs for YL, YCC, and YSC with greater emphasis on the use of animals above the average EPD value. Since 2013, through new experiments, unconventional criteria were studied to provide future production systems with heat tolerant animals, with good resistance to parasites, early maturing, and with better carcass finishing. Animals from different lines have been studied at Embrapa Southeast Livestock since 2011 [13], with the objective of comparing the productive characteristics of the three lines, as closed experimental herds.

Pasture Characteristics and Measurements
The animals within their respective lines were ranked according to their initial liveweight (to avoid competition), so that the heaviest animals of each line were placed with the heaviest of the other two lines, the intermediate with the intermediate and the lightest with the lightest, respectively, being then allocated to one of the four pasture areas (considered as a block) by drawn. Each area of pasture was composed of four modules with 12 strips each, following a variable rotational grazing method. Drinking water and a trough for mineral supplementation was always available. The pasture system was composed of Panicum maximum L. (cv. Tanzania), which received nitrogen fertilization after the animals were moved on from each strip, totaling 400 kg of N/ha per year.
The grazing period adopted was three days per strip with a rest period of 33 days, totaling 36 days per cycle. The pasture rearing period (165 days) permitted the observation of 4 full grazing cycles (4 × 36 d) plus 21 grazing days on the fifth grazing cycle (the last five strips were not grazed in this last grazing cycle). The stocking rate was adjusted following the put-and-take technique [14], to ensure a similar forage offer between the modules. To monitor liveweight (LW) gain, the animals were weighed after a fasting period of 16 h, at the beginning and at the end of the grazing phase, and without fasting every 36 days within this interval.
Forage mass production was determined directly by the quadrat method, where the frame had a size of 1.0 m 2 , and a random choice of collection points (9 points per paddock). The material collected above the residue (40 cm) recommended height, according Barbosa et al. [15], was weighed, and a sub-sample taken to determine the dry matter was dried in an oven at 65 • C for 72 h. The mean forage yield during the five cycles evaluated was 2550 ± 648 kg DM/ha.

Animal Characteristics and Measurements
The animals from the experimental Canchim herd were reared at Embrapa Southeast Livestock, born between August and November 2013. At the start of the experiment, their LW was 280 ± 33 kg and were 15 ± 3 months of age, with 13 animals from the old line, 20 from the new line, and 13 from the cross line.
During the pasture rearing period, dry matter intake (DMI) was estimated individually using the prediction model proposed by Azevêdo et al. [16], considering the determined average LW (ALW) and average daily gain (ADG), where ALW is the average of the initial and final LWs during the pasture phase (used for calculating the ADG): This prediction model was adopted considering that it has been proposed based on a meta-analysis of 561 results obtained in 27 studies (thesis and dissertations) published by the Federal University of Viçosa and the University of São Paulo and is known as: "BR-CORTE" [17]. Although prediction models by the National Research Council (NRC) [18,19] can be applied in Brazilian studies, those models have been developed using mostly Bos taurus cattle data and, besides that, the database included results obtained from animals that had received anabolic steroids prohibited in Brazil since 1961. Studies that evaluated NRC [19] prediction models concluded that the equations proposed to predict DMI were not totally adequate and the development of new and more precise equations was suggested [20]. Valadares Filho et al. [21] also reported a lack of fit in models proposed by the NRC [18,19], when predicting DMI by beef cattle in tropical conditions. Ribeiro et al. [22] evaluated the prediction of DMI by genetic groups of Zebu cattle using models by the NRC [19], the Cornell Net Carbohydrate and Protein System -CNCPS 5.0 [23] and BR-CORTE [17], concluding that the Brazilian model [17] (BR-CORTE, 2006) was more accurate for beef cattle in tropical conditions.

Methane Emissions Sampling at Grazing
Emissions of enteric methane were measured using the sulphur hexafluoride (SF 6 ) tracer gas technique [24][25][26], whereby each of the 24 animals from the new and old lines (the 12 heaviest animals from the new line and the old line) were assessed daily for 24 h for 5 consecutive days in April 2015. Animals from the cross line were not evaluated (it was expected to obtain an intermediary result and the SF 6 technique is both costly and laborious). As such, this study aimed to quantify only the emissions of the new and old lines. The steers were fitted with halters and sampling canisters for gas collection 15 days before sampling in order to allow the animals to adapt to the devices, and thereby facilitate handling during the sampling period. Permeation tubes filled with known quantities of SF 6 and release rates (1431 ± 59 ng/min) were administered orally to each of the 24 animals, 15 days prior to sampling, in order to allow the flow of tracer gas to stabilize in the rumen.
The animals were equipped with gas collection halters attached to pre-evacuated polyvinyl chloride (PVC) sampling canisters, made to permit 50% filling in 24 h. The collections started at 7:00 a.m., with the animals brought in from their pasture strip to the handling center in order to facilitate the exchange of equipment. The pressures of the sampling canisters were measured immediately after completing the 24-h collection period to evaluate their quality. If the final pressure was below that expected, this could mean that the sampling line was blocked or disconnected, while greater expected pressure could mean that leakage occurred at some point in the system. In the case of either of these situations, the halter and sampling canisters were replaced. After exchanging the collection containers, the animals were returned to their original pasture strip.
Daily samples were also collected to determine the concentration of CH 4 and SF 6 in the environment. The pressures, dates, and times of removal of the containers in both cases were noted for control.

Feedlot Phase: Diet and Animals Measurements
The feedlot finishing phase lasted for 105 days and occurred between June and September 2015, when the animals reached average weights of 354 ± 41 kg. The feedlot consisted of 4 collective pens of 396 m 2 , each equipped with drinking water, methane sampler (GreenFeed), and 2 automated troughs (GrowSafe Systems Ltd., Airdrie, Alberta, Canada), where they were offered water and feed ad libitum. The animals were allocated, respecting the same order as the 4 pasture areas ( Table 1). The duration of the adaptation period was 20 days, with the animals receiving only corn silage in the first 10 days, 50% of concentrate with corn silage during the next 10 days, and 100% concentrate at the end of this period. The dietary composition is described in Table 2. The experimental diet was offered twice daily at 8:00 a.m. and 2:00 p.m. Samples of the complete diet were collected on a weekly basis to determine the DM of the diet, using an oven at 65 • C for a period of 72 h. To measure the intake, the steers were fitted with a half-duplex electronic identification (EID) ear tag (Allflex Brazil, Joinville, SC, Brazil), and each automated trough was equipped with an antenna to detect animal presence, load cells to measure the feed amount, and a grid with adjustable vertical bars to allow only one animal at a time. The acquisition software continuously recorded the feed disappearance data and the presence of an animal at the trough when the EID transversed the vertical plane of the grid. The sum of the amounts of each visit over a 24-h period defined the daily feed intake [27].
Animals were weighed at the beginning and end of the feedlot period after a fasting (16 h) period, and at 28-day intervals during this period without fasting for monitoring of liveweight gain. The level of carcass fat of the animals was monitored in vivo using the ultrasound technique, with the images provided by an Aquila model Pie Medical appliance, equipped with a 3.5 MHz, 18 cm linear transducer, and using vegetable oil as the coupling agent. All measurements were taken between the 12th and 13th ribs on the Longissimus thoracis muscle, following the methodology of Herring et al. [28]. ADG, feed conversion (FC), and feed efficiency (FE) were then determined based on the measurements of liveweight gain and dry matter intake in feedlot.

Methane Emissions Sampling in the Feedlot
To determine enteric methane emissions during the finishing stage, four units of GreenFeed (GF) (C-lock Inc., Rapid City, SD, USA) automated system were used in the experimental feedlot area, with the equipment installed on the sides of the barns occupied by animals of the old and new lines. During the first seven days, access to the device remained unrestricted in order to encourage the animals to visit it. Once each animal, belonging to each plot, had been identified by the system, the access to the device was limited to restrict access to one animal at a time. The equipment was programmed using software provided by C-Lock Inc. to provide a maximum of six rotations of the feed dosing cup, with approximately 55 g per rotation, and intervals of 45 s between rotations, permitting a maximum provision of 330 g of pellet per visit. A maximum of five visits per day were permitted, with a minimum interval of 4 h and 45 min required between visits. Thus, if an animal returned to the device in a period shorter than this interval, the pellets were not dispensed.
The pellets used were commercial feed pellets (Presence do Campo, InVivo Nutrição e Saúde Animal, Paulínia, São Paulo, Brazil), chosen for their high fiber content and guaranteed content levels of 13% moisture, 14% crude protein, 3% ether extract, 14% mineral matter, 20% crude fiber, 1.2% calcium, and 0.6% phosphorus. Operation of the device was triggered by animals inserting their heads into the trough, and a radio frequency identification sensor (RFID) identified the chip fixed to the ear of the animal. Gas sampling was only initiated when the animal's head was in the correct position, as identified by an infrared sensor, resulting in the distribution of pellets to attract and encourage the animal to keep its head in the correct position. This factor, alongside wind speed and direction, determined the success of the reading since the position of the animal's head and the wind may have varied, but only data with uninterrupted measurements were considered valid for statistical analysis. Periodical cleaning and calibration of the equipment was carried out to guarantee the quality of gas measurements.
The CH 4 emissions were calculated using the CH 4 :SF 6 ratio in the sampling canister, with each of the gases corrected for background concentrations, together with the predetermined permeation rate of the SF 6 capsules. In the following equation, M indicates the sample measured from the animal and BG indicates the background concentration: Furthermore, RCH 4 (g/d) is the ruminal CH 4 emission rate calculated in g/d; RSF 6 depicts the release rate measured from the SF 6 capsule (mg/d); MWCH 4 is the molecular weight of CH 4 (16) and MWSF 6 is the molecular weight of SF 6 (146). The CH 4 concentrations are expressed in ppm and SF 6 concentrations in ppt. The factor 1000 converts the units so that RCH 4 is expressed in g/d.
The flow of enteric methane emitted by the animal during the finishing stage was calculated, considering the methane concentration of the sample and the volume of air displaced by the extractor fan. Real-time communication between the GF units and the software provided by C-Lock Inc. on the internet was possible via a Wi-Fi network. Methane emission was expressed in the following different units: grams per day (g/day), kilograms of methane per kilogram of dry matter intake (kg/kg DMI), kilograms per kg of average daily gain (kg/kg ADG), grams per kg of liveweight (g/kg LW), and methane yield (YM%) as the percentage of gross energy lost in the form of CH 4 (as proposed by Intergovernmental Panel on Climate Change-IPCC [31]). To calculate YM (%), the daily emission (CH 4 g/d) transformed into unit kg (CH 4 (g/d)/1000), was divided by the amount of energy of 1 kg of CH 4 (55.6 MJ/kg). The value obtained was divided by the gross energy ingested (DMI/GE of 1 kg of the diet = 18.4 MJ/kg DM) and multiplied by 100; thus, obtaining the value of YM in percentage. For this calculation, the gross energy content of the diet (MJ/kg MS) was determined using a calorimeter pump (IKA C 200, Staufen, Germany); sub-samples were collected from the samples used to determine dry matter, and ground to 2 mm, before being shaped into pellets and burned inside the pump, according to AOAC recommendations [32].

Carcass Data Collection
The animals were slaughtered at the end of the feedlot phase, when they happened to have an average of 488 ± 48 kg of body weight and 4 ± 1.5 mm subcutaneous fat thickness, as measured by ultrasonography. The slaughter was carried out in accordance with Brazilian government inspection procedures.
After 16 h of fasting (water and feed), the animals were weighed to obtain the shrunk body weight (SWB). At slaughter, the animals were stunned with a captive bolt gun and then exsanguinated through jugular section. Carcasses were weighed before being stored in a cold chamber for 24 h, to obtain the hot carcass weight (HCW). After this interval, a sample of the Longissimus thoracis muscle was taken between the 12th and 13th ribs of the left half carcass for real rib eye area (REA) and fat thickness (FT) measurements, using tracing paper and reticulated grid (cm 2 ), and a millimeter ruler, respectively. The weights of the main cuts hind quarter (HQ), front quarter (FQ), and spare ribs (SR) were also taken. The carcass dressing percentage (CD) were calculated as the ratio between the HCW and the SBW.

Statistical Analyses
Data were analyzed using the Statistical Analysis System 9.3 package (SAS Inst. Inc., Cary, NC, USA). The experimental unit was the individual animal for the analyzed variables. The presence of discrepant information (outliers) and normality of residuals (Shapiro-Wilk) were tested using the PROC UNIVARIATE procedure. Performance data (initial LW, final LW, ADG, DMI, FC, FE) and data relating to CH 4 emissions (g/day; kg/year; kg/kg DMI; kg/kg ADG; g/kg LW; YM (%)) were analyzed using the PROC MIXED procedure for mixed models. Fifteen different covariance structures were tested, and the best-fitting statistical model was selected [33] based on the lowest value of Corrected Akaike Information Criterion (AICC). The model included the effect of treatment (line), period (pasture and feedlot) as a fixed effect, and random of block (area repetition).
The slaughter (HQ; FQ; SR; FT; REA) were analyzed using the same procedure for mixed models, but when the normality of the residuals was not met, the Poisson distribution-a method available in Proc Glimmix, a component of SAS-was also tested. The Tukey's test was used to compare the treatments for all variables, considering a level of significance of 5%.

Results
The results obtained for the performance variables during the pasture rearing period are described in Table 3. The lines did not differ in initial LW, final LW, or DMI (p > 0.05), averaging 280.0 ± 32.6, 374.9 ± 42.9, and 6.3 ± 1.5 kg, respectively. The line of the animals had a significant effect on ADG (p < 0.001); the animals from the new line presented higher averages than the old line, while the animals belonging to the cross line did not differ from those of the new lines. The results for enteric methane emission variables corresponding to the pasture rearing period are described in Table 4. The line of the animals had no effect on methane emissions (p < 0.05) for the different units, averaging 203.4 ± 51.9 g/d, 0.331 ± 0.2 kg/kg ADG, and 0.560 ± 0.1 g/kg LW. In the 105-day feedlot finishing period, the line used had an effect on the variables of ADG, daily DMI (p < 0.05), and FC (p < 0.01) ( Table 5). The new line was superior (p < 0.05) in terms of ADG compared to the old line, while the cross line did not differ from the other two lines in relation to these variables. The highest daily DMI were observed in animals belonging to the new and cross lines in relation to the old line. The cross line presented a higher (p < 0.05) average FC compared to new and old lines. Methane emissions from the feedlot finishing phase are described in Table 6. The line of the animals had an effect (p < 0.05) on CH 4 emissions expressed in grams per day (g/day). Animals belonging to the new line emitted more methane (p < 0.05) than the old line when this variable was expressed in kilograms per year and grams per day. There were no differences in the methane emissions per DMI, ADG, LW, and YM, averaging, 0.017 ± 0.004 kg/kg DMI, 0.137 ± 0.1 kg/kg ADG, 0.375 ± 0.1 g/kg LW, and 5.13 ± 1.3%, respectively.  Table 7 shows the averages for CY, HQ, FQ, SR, FT, and REA. The line had a significant effect (p < 0.05) on the dressing of the animals given that FT was greater in the carcasses from the new line than the old line, while the FT measured from the cross line carcasses did not differ (p > 0.05) from the carcasses of the new and old lines. The genetic line did not affect any of the other variables measured on the carcass, although SR tended (p = 0.058) to be lower in the old line compared with the other two lines.

Discussion
The superior daily LW gain displayed by the new line during the rearing period was a result of the use of improved bulls to form this line, since feed intake did not differ between the animals of the two lines, as was also the case in relation to the cross line. According to Alencar [34], the Canchim breed's performance characteristics generally show medium to high values for heritability, indicating that selection results in good genetic progress. This was backed-up by the superior performance of the new line in this study. Animal performance is an important aspect when the purpose of cattle rearing is meat production. Usually, the evolution of the animals is evaluated based on liveweight gain, which can be measured at different stages of the animal's life (breeding, post-weaning, and finishing).
In a study of Canchim cattle in an exclusive grazing regime, Vieira [35] observed an ADG of 0.375 kg/day, lower than the figure obtained in this study for the new line (0.692 kg/day), old line (0.620 kg/day), and their cross (0.547 kg/day), grazing a pasture with high nutritional value and high availability. Vieira's [35] study was one of the pioneering studies in relation to the performance of the Canchim breed, where genetic progress in the breed had previously been based solely on observations of the breeding of the different combinations of breeds, and not on the result of an already established selection process, without any perspectives for formation of a new line. Alencar et al. [36], comparing Canchim and Nellore cattle breeds of both sexes under a grazing regime, found differences in ADG and identified higher gains for Canchim males in relation to the other categories. Goulart et al. [37], working with Nellore and crossbreeds (Nellore, Canchim × Nellore, Angus × Nellore, and Simental × Nellore) reared on pasture, also observed different ADG (0.210, 0.290, 0.300, and 0.330 g/day, respectively), where the Nellore group presented lower ADG in relation to the three crossed groups, whilst for the variables FE and FC, significant differences were not found.
In the feedlot finishing phase, the highest ADG was presented by the new line, which can be explained by their higher feed intake and lower value for FC. The old line, in spite of its FC being equivalent to that of the new line, did not obtain similar ADG due to lower DMI. The cross line animals, despite having higher DMI than the old line, did not gain more liveweight because their FC was worse than the old line. Fernandes et al. [38], working in feedlots with Canchim animals of different sexual condition (castrated/not castrated males and females), observed significantly different ADG, DMI, and FC. Differences in these same characteristics were also observed by Rubiano et al. [39], who studied Nellore, Canchim, and the crossbred between these breeds in feedlots. Increased ADG is expected when there is higher DMI, since these characteristics are positively correlated [40]. The effect of the Charolais breed on performance was also confirmed by Crews et al. [41], who studied genetic parameters between growth and carcass traits of Charolais animals and stated that the heritability of these traits is moderate to high in this breed, indicating that selection can be used to improve both growth patterns and the conformation of the carcass.
The Canchim breed displays additive genetic variation in relation to LW gain, with improvements in this characteristic as time progresses, as described by Chaves et al. [42], and this was also confirmed by the results of the animals of the new line in this study. Barichello [43] also stated that the genetic selection process applied to the Canchim breed has been providing progress in LW and slaughter conformation characteristics. In the classic study reported by Koch et al. [44], they describe how selection for LW gain is effective and leads to improved FE, in addition to an increase in feed intake. These effects were observed in this study in the new line (formed from improved 1990s Charolais bulls with proven genetic merit) when compared to the old line (closed line since 1960).
Regarding enteric methane emissions in the pasture phase, different studies have compared the emissions obtained using the SF 6 tracer gas technique with results obtained from the same animals in respiration chambers, and observed average variations in emissions of 7.5% either way [45]. As such, the technique provides greater variability in results causing the need to increase the number of animals necessary to identify treatment differences [46]. Twelve animals per treatment measured using the SF 6 technique in this experiment is not a small number, but may not have constituted enough animals to detect differences in methane production between the new and old line. According to Mercadante et al. [47], there is no evidence that more efficient animals produce less enteric methane, even if they display lower feed consumption and performance equivalent to animals considered inefficient. Sakamoto et al. [48], evaluated emissions from Canchim cattle reared in different pasture systems (intensive, extensive, integrated crop-livestock, integrated crop-livestock-forest and silvopastoral) and did not find significant differences in CH 4 emissions in grams per day.
The higher emissions of enteric methane from the new line animals in the feedlot can be related to the higher DMI, since there is a directly proportional relationship between increased consumption and increased methane production [49,50]. This finding was also reported by Moss et al. [51], who highlighted the fact that methane production is linked to DMI, but this variable should only be used with single DM sources or diets, as in this study. It is also important to note that other authors attest to a strong correlation (r > 0.60) between methane production and DMI [31,40]. Hegarty et al. [52], working in feedlots with Angus lines selected for low or high residual fed intake (RFI), observed significant differences in relation to DMI and daily methane production; the animals that presented higher DMI emitted greater quantities of enteric methane. However, the authors did not observe differences in ADG and methane production expressed in relation to ADG or DMI, as was also the case in this study. Genetic selection for lower methane production can produce undesirable effects on animal productivity, as this characteristic has a strong correlation (0.79 to 0.86) with growth characteristics [53]. In this study, despite the old line emitting less methane, it also demonstrated lower ADG and higher FC.
Although a higher emission is expected as the animals grow [54], in this experiment we observed higher emissions in the pasture period. The difference between pasture and feedlot periods can be explained by the different methods used for measuring the CH 4 . According to Hammond et al. [55], the emissions can be higher using SF 6 technique rather than GF automated system, but there is a significant correlation (0.60) between the two methods. Another factor is the different diets in the two periods; feeding grain-based diets can lower enteric CH 4 emission when compared with forage-based diets [29], which is in agreement with the present experiment, since the emissions decreased when the animals went from the pasture to feedlot. The gross energy content in the diet also supports this statement, as the animals had more than twice the energy available in the feedlot (8.7 ± 0.1 vs. 18.4 ± 0.1 MJ/kg DM). In general, the average methane emissions determined in this experiment (67 kg/year) are consistent with estimates made using the IPCC's Tier 2 methodology (64 kg/year) [56], slightly above the Brazilian national average, estimated at 60 kg/year [57,58].
The greater FT in steers of the new line in this study may be linked to the higher ADG of the animals during the rearing period on pasture and feedlot finishing. According to Di Marco et al. [59], breed, dietary history, and increase of liveweight gain are characteristics that determine the amount of subcutaneous fat deposited. A few studies show results related to fat deposition: Fernandes et al. [38], evaluating the performance and carcass characteristics of castrated or non-castrated males, plus females of the Canchim breed, detected similar deposition of subcutaneous fat between the different categories evaluated. The Canchim breed displays delayed fat deposition, with this feature becoming more significant as the animal nears adult liveweight and, according to Alencar et al. [60], the use of the Charolais breed provides a higher growth rate and larger size, in addition to other features, with subsequent deposition of fat at a later stage.
Despite the differences in FT observed between the new and old lines, all met the minimum of 3 mm required by slaughterhouses. The FT has an important role in the process of transforming muscle into meat and is a determining factor in its quality since it acts as a thermal insulator to ensure that the temperature decrease occurs more slowly, protecting the carcass [61,62], and preventing the shortening of muscle fibers by the cooling action [63]. This change began in the early 1990s, where practically all beef came from lean male animals of 3 to 5 years of age [39]. Since then, the reduction in the age at slaughter, associated with the genetic potential of the animals, has been an important step towards producing meat with greater efficiency and of higher quality, satisfying the final consumer.

Conclusions
Reared on pasture, the steers of the new genetic line of the Canchim breed grow faster and emit the same amount of enteric methane as those of the old line. Additionally, when finished in feedlot, the steers of the new line grow faster than the steers from the old line, but emit a higher amount of methane per day. In the feedlot phase, the animals from the new and the crossed lines have a higher dry matter consumption than those of the old line, whilst the new and the old lines are more efficient in converting the feed consumed into liveweight, compared with the cross line steers. Moreover, better carcass dressing can be obtained by using the new lines of the Canchim breed, when reared on pasture and finished in feedlot. These results are evidence of the impact that the genetic improvement exerted on the Canchim composite breed have on animal performance and methane emissions.


s2fieldsofstudy:
Agricultural And Food Sciences
Biology

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

text:
Physical Performance of Broiler Chickens Affected by Dietary Biological Additives. J. World Poult. Res., 10 (3): 443-450. DOI: https://dx.doi.org/10.36380/jwpr.2020.51;

The current study aimed to evaluate the effects of Saccharomyces cerevisiae yeast as a probiotic, compared to sodium butyrate as an organic acid on the productive performance of broiler chickens, with special attention to their economic efficiency. Therefore, 270 of one-day-old Hubbard broiler chickens were divided into 5 groups. The first group included chickens receiving basal ration without any treatment (and considered as a control group). The second group was composed of chickens treated with 0.2 g SB/kg, the third group embraced chickens treated with 0.3 g SB/kg, the fourth group included chickens treated with 0.2 g SC/kg, and the fifth group consisted of chickens treated with 0.3 g SC/kg. The obtained results showed that administration of sodium butyrate or yeast showed a significant improvement of final body weight (BW), body weight change, feed conversion ratio and performance index from third to fifth weeks of age. Nevertheless, all treated groups showed an insignificant effect in feed intake, compared to control group. Furthermore, the dietary addition of Saccharomyces cerevisiae was detected more profitable than sodium butyrate addition. Accordingly, it can be concluded that sodium butyrate and yeast can be successively used as a natural substitute for antibiotic growth-promoting agents in the broiler chickens. Yeast can be considered as the most important alternative followed by sodium butyrate.

INTRODUCTION
The poultry industry is now considered as one of the most important industries related to food security for peoples worldwide. One of the most important pillars of this industry is nutrition, which accounts for 75 to 80% of the industry's costs (El-Kholy et al., 2018). So, the concern was to devise strategies that support this pillar, and contribute to its development (Eltazi et al., 2014). Nowadays, one of the most important strategies is feed additives, because it is of the utmost importance in the poultry production (El-Kholy et al., 2019;Omar, 2020). Feed additives are aimed primarily for improving the physical performance (PR) of birds such as increasing body weight (BW), BW change and improving feed conversion ratios. In addition, they play a vital role in improving the productive and economic efficiency of poultry farms (Omar, 2020). In recent times, use of probiotics and organic acids were considered as natural feed additives used for broiler production. One of the probiotics is Saccharomyces cerevisiae (SC) which is produced from malted grains fermentation, also known as ''baker's yeast'', and it is most widely used as dietary growth promoters for animals (Gao et al., 2008;Omar, 2020). Moreover, SC is a rich source of protein, vitamin B complex, trace minerals and many other useful factors (Sun et al., 2020). Sodium butyrate (SB) is a recently used organic acid in broiler chickens' diet for realizing optimum performance (Awaad et al., 2019;Lan et al., 2020). It is rapidly absorbed to provide energy for the epithelial cells (Lan et al., 2020), and promote sodium and water absorption (Friedel and Levine, 1992). It increased the epithelial cell growth and the proliferation index in the intestinal crypts (Lan et al., 2020), also it had a trophic effect on the gut mucosa. Addition of these acidifiers broiler's diet enhanced nutrient utilization, growth and feed efficiency (Lan et al., 2020). Also, SB improved the balance of the intestinal microflora which led to a positive DOI: https://dx.doi.org/10.36380/jwpr.2020.51 impact on the host's health (Candela et al., 2010). In poultry production, dietary acidifier addition did not gain as much attention as in swine production (Dehghani-Tafti and Jahanian, 2016). The previous studies on SC and SB in broiler chickens mainly focused on their phenotypic effects individually. So the aim of the present work was to study the effects of dietary administration of SC in comparison to SB on the growth performance in broiler chickens by estimation of both phenotypic changes in BW, BW change, feed intake, feed conversion ratio, relative growth rate and performance index, and economic efficiency.

Ethical approval
The current study protocol used in this study was endorsed by the Animal Care and Use Committee of Damietta University, Damietta, Egypt.

Materials
The current experiment was conducted at a private commercial poultry farm under supervision of Poultry Production Department, Faculty of Agriculture, Damietta University, Damietta Governorate, Egypt. The period of study extended from twenty sixth of February, 2019 till second of March, 2019 to demonstrate the effects of dietary addition of probiotic (Saccharomycis cerevisiae; "SC") and organic acids (Sodium Butyrate, "SB") on some productive efficiencies of broiler chickens.

Birds and experimental design
Total of 270 one day-old Hubbard broiler chickens with an initial body weight (IBW, g) of 47.43 ± 0.16 gram supplied by commercial hatchery (El-Aml Hatching Company, Damietta) were used in this study. Chickens were individually weighed and assigned randomly to 5 equal experimental groups of 54 birds in each. Chickens of each group were subdivided into 3 replicates of 18 birds in each, and housed in floor pens. All birds were kept under the same managerial conditions. Feed and water were offered ad libitum throughout the experimental period (1 to 5 weeks of age). The first experimental group was fed with control diet while, the other four groups were fed with the basal diet with 0.2 and 0.3 g for each of SC and SB per kilogram body weight. For each treatment group, both levels of SC and SB were added to the basal diet, and subsequently mixed and stirred with a mixer. The birds were fed on starter and finisher ration according to NRC (1994). The basal diets composition are tabulated in Table  1.

Management
The birds were housed in a clean and well ventilated farm that was previously disinfected and prepared for receiving birds for the experiment. Birds were randomly housed in trial pens (2 m×2.10 m×3 m) with stocking density of 15/m2. The ambient temperature during brooding was 35 °C ± 1 at one-day-old of age, and gradually decreased to 25 °C ± 1 on day 21, and then kept constant. The birds were subjected to light schedule similar to commercial condition; 23 hours light from oneday-old birds until seventh day to give them enough time to find out feed and water, followed by 20 hours light from eighth day to the end of the experiment (35 days of age). Broiler chickens were vaccinated with mix (Infectious Bronchitis "IB" Ma5 + Newcastle clone 30) on day 7, and Gumboro D78 vaccine at 14 days of age, and they were replicated at 21 days of age. Finally, the birds were vaccinated against Newcastle (live clone 30) at 28 days of age.

Performance parameters
The averages of body weight (BW, g), body weight change (BWC, g), feed intake (FI, g) and feed conversion ratio (FCR) which were evaluated according to the method described as follow: Average body weight (BW, g): The chickens were weighted individually at the beginning of the experiment, afterward chickens were weekly weighted and the live body weight change was taken; Body weight change (BWC, g): it was calculated as differences between two successive weights; Body weight change: W2 -W1; Where: W1 is the weight at any week, and W2 is the weight at the next week; Feed intake (FI, g): it was calculated by difference between the weight of the offered feed/week and the remained part, and then divided by the birds number in each group to measure the weekly FI per bird; FCR: it was calculated by dividing the amount of feed consumed (g) during the week by the gain in weight (g) during the same week; Performance index (PI): it was calculated by adopting the below formula proposed by Bird (1955); PI: Body weight gain (g) × FCR.

Partial budget analysis
The economics of feeding diet inclusion with SC and SB were calculated on the basis of overall cost of inputs, i.e. the cost of chickens, feeds, labor, medicines and other miscellaneous cost. Final live weight of the bird was considered for calculating the gross return per bird and net profit per bird.

Statistical analysis
All data were expressed as mean ± standard error (SE) by one-way ANOVA with dietary treated addition as the main factor using statistical software of SPSS Version 25 (IBM SPSS, 2017) which used the general linear model (GLM) procedure based on the following model: Y ij : μ + T i + e ij ; where, Yij: Observation of the j th chickens in the treatment i; μ: Overall mean; T i : Effect of the treatments (i,: 1, 2, 3, 4 and 5); e ij : Random error component. A probability of P ≤ 0.05 was required for statements of significance.

Effects of dietary biological addition on average body weight of broiler chickens:
Average body weight (BW) of broiler chickens as affected by SC and SB addition are presented in indicating that birds were randomly distributed into the experimental treatments. During the starter period (0 to 2 weeks), dietary treatments did not affect (P ≥ 0.05) chickens' body weight. At the end of 3 weeks of ages, BW for chickens only treated with SC groups (T4 and T5) significantly (P ≤ 0.01) increased in comparison with the other groups. However, during the finisher (4 to 5 weeks) periods, dietary addition of SC and SB, significantly (P ≤ 0.05) increased the live BW. At the end of 5 weeks of age (marketing age), BW for groups fed with diets of T2, T3, T4 and T5 was higher (0.1, 8.4, 14.7 and 23.5 percent, respectively) as compared to those fed with control basal diet. Also, results reported in this study clearly indicated that, addition of SC had the significantly (P ≤ 0.01) highest FBW as compared to SB in all experimental period except of 1 to 2 weeks which had no significant (P ≥ 0.05) differences.

Effect of dietary biological addition on average body weight change of broiler chicks
Chickens treated with SC or SB (0.2 or 0.3 g/kg) showed a significant increase (P ≤ 0.05) in the BWC in comparison with the control chickens for all experimental periods, except for the first and the second period (Table  3). Within the treated chickens, chickens treated with SC (0.3 g/kg) showed the highest BWC followed by chickens treated with SC (0.2 g/kg) then chickens treated with SB from third to fifth weeks.

Effects of dietary biological addition on feed intake of broiler chicks
The effect of dietary addition of SC and SB on the FI of broiler chickens, as seen in table 4, showed an insignificant (P ≥ 0.05) effect through the different weeks of age.

Effects of dietary biological addition on feed conversion ratio of broiler
Feed conversion ratio (FCR) was significantly (P≤0.01) improved from the third week until the end of experiment, and overall FCR at fifth week of age, and the value was being greater for T1 and T2 than T3, T4 and T5 (Table 5). A significant improvement in FCR was recorded in the treated groups with SC or SB as compared to the untreated group. Within the treated chickens, chickens treated with SC (0.3 g/kg) showed the lowest FCR followed by chickens treated with SC (0.2 g/kg), then chickens treated with SB at the third, fifth and 1 to 5 weeks of age. In general, high level addition of SC showed the best FCR compared to other treated groups.

Effects of dietary biological addition on performance index of broiler
Chickens treated with SC or SB (0.2 or 0.3 g/kg) showed a significant (P ≤ 0.01) increase in the PI in comparison with the control chickens (T1) for all experimental period except for the first and second period (Table 7). Also, PI showed an insignificant difference (P ≥ 0.05) among treatments in the first and second period. Within the treated chickens, chickens treated with SC (0.3 g/kg) showed the highest PI followed by chickens treated with SC (0.2 g/kg), then chickens treated with SB at third, fourth, fifth and 1 to 5 weeks of age. In general, high level addition of SC (T5) showed the best PI in compared to other treated groups.

Partial budget analysis
Data concerning economical evaluation are summarized in table 8. The highest net revenue, economic efficiency and relative economic efficiency were obtained for T5 group followed by T4, T3, T2 compared to the lowest values which were detected in T1 (control group).

DISCUSSION
In order to determine the productive effect of dietary addition of either SC or SB in broiler chickens was performed as natural and environmentally friendly alternatives. It was found that the birds treated with either SB or SC had a significant improvement effect on the broiler chickens' growth during all experimental period except for the first and second period. The insignificant differences in the results obtained during the first and second periods could be explained as the functionality of intestines of day-old chickens, and the activity of the digestive enzymes were not sufficiently developed (Ravindran, 2003). All these results were in agreement with findings of Sun et al. (2020) and Omar (2020) in broiler chickens. In contrary to the present results, no negative effect on BW, BWC and FCR were detected when broiler chickens had either supplements of SC (Eltazi et al., 2014;Devi et al., 2019) or organic acid (Denli et al., 2003;Lan et al., 2020). This might be due to kind and concentration of Saccharomyces. The result for FCR was in agreement with Mulatu et al. (2019) who reported a significant improvement in feed per gain ratio. In addition, many authors reported significant and better FCR on either SC (Mulatu et al., 2019;Sun et al., 2020) or SB addition in the diet of commercial broiler chickens (Awaad et al, 2019;Lan et al., 2020). In contrary to this result, Devi et al. (2019) and Al-Khalaifa et al. (2019) illustrated that dietary addition of SC had no impact on FCR.
In addition, Aghazadeh et al. (2012) recorded that dietary SB supplementation during the study period of 35 days had no effect on average FCR. These different results may be due to the available contents of SC or SB, animal age, health status and environment hygiene. The relative growth rate (RGR) result could be speculated that this was due to growth-stimulating effect of SC in broiler chickens as mentioned in the effects of SC in final BW and BWC (Tables 2 and 3). Higher PI was observed in group T5 which might be due to the effect of SC in final BW, and better FCR as compared to the other treatment groups in the current study. Comparable findings were discovered by Devi et al. (2019) Hernandez et al. (2013) who found that dietary addition of SC or SB, respectively, increased significantly the FI of broiler chickens.
In addition, the current study became in confrontation with those of Mulatu et al. (2019) who showed the lowest fed intake in all chickens received probiotic, and highest in the untreated group. So, the dietary addition of either SB or SC did not affect the FI of broiler chickens, but they increased the body weight gain significantly indicating an improved feed efficiency. This improvement in PR of broiler chickens in treated groups compared to control may be related to SC constituent with many metabolites like peptides, organic acids oligosaccharides, organic acids and flavor possibly some unidentified growth factors which were proposed to deliver useful responses in poultry production (Gao et al., 2008;Sun et al., 2020). These results also agreed with Markovic et al. (2009) demonstrating that dietary addition of SC improved broiler PR as SC improved the intestinal mucosal aspects, and produced new epithelial cells in the intestinal crypts, and migrated along the villi to the top. For instance, the significant (P ≤ 0.01) increase of PR in SB groups as compared to the control one, may be discussed from the point of view that SB associated with direct or indirect modulation of insulin-like growth factor (GF) production by insulin which can be related to impulse of muscle development (Mátis et al., 2019). The present results indicated an improvement in the net profit for chickens fed with diets containing of either SC or SB compared to those fed with diets with no additive (control). The present study was in agreement with those of Mulatu et al. (2019) and Devi et al. (2019) who indicated that the ration containing SC addition was potentially profitable than untreated one (control). Therefore, T5 appeared to be cost-effective in economic parameters used in the study. In the identical pattern, Omar (2020) verified that the supplementation of dried brewer's yeast as 0.4 % for broiler breeds changed into more economically profitable than 0.2% and untreated. Also, Mátis et al. (2019) illustrated that dietary addition of sodium butyrate as organic acid gave the best economic profits compared to the control group (untreated) on broiler production.

CONCLUSION
From this trial, it was concluded that sodium butyrate can be used as a growth promoter in chickens as it improves the final body weight, body weight change, feed conversion ratio and relative growth rate by increasing intestinal absorption surface. But when sodium butyrate compared to yeast, yeast achieve the highest improvements. Also, the dietary addition of yeast and sodium butyrate for broiler breed was more economic than the control group.


text:
The Effect of Chromium and Herbicide on the Growth of Aspergillus terreus Isolated from Soil Environment

The release of various chemical substances by human activities in soil leads to change its soil properties and affect the life of microbes, plants and animals including human. Microorganisms, especially fungi play an important role in soil fertility and various biochemical features, which is affected by the contamination of heavy metals and Organo-chlorine compounds including herbicides. Therefore, the present study was focused on the bio-diversity of fungi from soil samples collected from leather industry (6 samples) and paddy field (6 samples) environment. Further, the frequently occurring species named Aspergillus terreus from the above soil was studied in vitro for the growth tolerance of chromium and herbicide (Pretilachlor) at various concentrations amended in PDA medium. The results indicated that the fungi such as A. niger, A. glaucus, A. terreus, A. nidulans, A. flavus, curvularia sp, Penicillium sp and sterile mycelia fungi were mainly present in the above soil samples. For quantitative pattern, leather industry soil showed 4,100 cfu/g to 30,600 cfu/g with an average of 16,896 cfu/g whereas the paddy field soil had 14,700 cfu/g to 33,720 cfu/g with an average of 22,215cfu/g. However, A. terreus and A. niger were present remarkably in the above soil samples. Hence, for the study of in vitro tolerance of chromium by A. terreus, the increased concentrations such as 100ppm to 500ppm, the growth observed as in the decreasing level, whereas above 500ppm, growth is completely suppressed. But, for the herbicide amendment in Potato dextrose broth, the fungi able to grow up to 1000ppm with decreasing level. The present study concludes that the species A. terreus could be an effective microorganism to degrade herbicide (pretilachlor) and moderately for chromium in the soil environment.

INTRODUCTION
Soil is the major component of earth's ecosystem which contains organic matter, minerals, gases and large numbers of macro and microorganisms. The diversity of microorganisms such as bacteria and fungi is essential when monitoring environmental influences on soil quality and its atmosphere is supported by several interactions among its physical, chemical and biological components (Chandrashekar et al., 2014). There are many reports on biodiversity and influence of different species of fungi on various soil locations were available at global level which indicates the soil fungi are ubiquitous and inhabit different types of soils including polluted soil environment (Frac et  There are many factors causing land pollution and the most common among them are the release of effluents from various type of industries and usage of chemical in the agricultural practices. The discharge of industrial effluents especially without treatment may have profound influence on physio-chemical and biological properties of soil related to soil fertility (Akpomie et al., 2016). The effluent from leather processing Industries is a major cause that produces huge volume of wastewater contain poisonous heavy metals especially chromium that normally discharged to irrigated agricultural lands. This tannery wastewater contains some proteins from animal skins that are released during enzymatic processing of leather but also contaminants such as salts and chromium (Cr), that might affect soil processes and crop production. According to various reports, the leather industry globally generates up to 5.4 million tons of solid and liquid waste each year which is one of the major pollution in soil environment. Noorjahan (2014) has studied the physicochemical characteristic identification of fungi and Biodegradation of Industrial effluent. According to Rasool and Irum, (2014) studied the isolaltion microorganism from industrial polluted soil reference to tannery effluent and reported that the filamentous fungi including varius species of Aspergillus and Pencillium grown and found resist to the tannery effluent.
The use of agricultural chemicals such as chemical fertilizer, pesticide and herbicide becomes an important issue in agricultural soil environment. The intensive use of pesticides causes concern both to public and scientists all over the world. Herbicides also used nowadays worldwide and the residues found variable toxicity. These herbicides could then accumulate to toxic levels in the soil and become harmful to microorganisms, plant, wild life and proved remain the most effective, efficient and economical way to control weeds. For this reason, several biological techniques involving biodegradation of organic compounds by microorganisms have been developed. The use of microorganisms such as different species of bacteria and fungi either naturally occurring or introduced, to degrade pollutants is called bioremediation. Therefore, certain fungi represent a powerful prospective tool in soil bioremediation and some species have already been patented. In vitro studies of degradation of the pesticide Chlorpyrifos conducted by Jayaraman et al., (2012) reported Trichoderma virede was more effective followed by T. harzianum and consortium of both the organisms. In contrast to the fungal suppression, Sclerotium, Trichoderma, Rhizoctonia, Penicillium and Aspergillus sp. showed enhancement at higher concentration of herbicide as well as other chemicals . According to Chukwuma (2015), the microbial biomass of an agricultural soil plays an essential role in degradation and mineralization of nutrient element and supply to plants through recycling. Whereas,certain herbicide exhibited the inhibitory effect on the mycelia growth of fungi. (Paula et al., 2007), studied the degradation of herbicide by fungi was increased from 14.42% to 35.01% after 10 days period . Two isolates of Aspergillus fumigatus and A. niger (87, 79%) were found to be highly degrading the herbicide (butachlor) and form high level of biomass respectively (Sangeetha et al., 2018). Treatment of soil with glyphosphate degrading fungus would be useful in some areas where this herbicide is extensively used (Arfarita et al., 2014). Paraquat, Glyphosate and Atrazine showed higher inhibition potency on certain fungal mycelial growth than Primextra and Linuron (Chukwuma., 2015). Bacterial degradation of s-triazines and parachlorobenzene (PCB) is described and novel strategies to improve bioremediation are discussed (Michael et al., 2010). bioremediation of pesticides by the isolate microorganisms from soil could be used for remediation of pesticides contaminated soil and water (Nilesh et al., 2016).
Based on the above scientific background and the significance of chemical pollution such as chromium and herbicide released in the soil environment, the present study is focused with the aim of analyzing the soil samples for the occurrence of fungal species in the industry environment and paddy field environment soil. Further, the frequently dominating species of fungi such as Aspergillus species to be studied for in vitro growth tolerance against the chromium and herbicide (pretilachlor) amended nutrient medium. The results of the study will provide an idea of fungal tolerance of the above environment and lead to biodegradation aspects.

MATERIALS AND METHODS Collection of soil samples
The soil samples (12 Nos.) were collected from Leather industry environment located in Vellore district and the paddy filed environment located in Thiruvallur and Kancheepuram districts of Tamil Nadu during January to March 2019. The soil samples about 200 g were randomly collected by digging the soil about 5 inch depth by using cleaned spatula and metal spoons and packed in pre-cleaned polythene bags (LDPE bags) and tightly tied with rubber bands. Different soil samples collected in polythene bags were labeled properly with sample details such as source and date of collection and brought to the laboratory immediately in the laboratory for further analysis within a week time.

Determination of moisture content
The moisture content of the soil samples were determined by standard oven-drying method (Mishra, 1968). Exactly 5g of freshly collected soil sample was weighed in a pre-weighed empty glass Petri-dish and the initial weight was noted. The petri-dish with soil sample was placed in a hot air oven for 2 hours at 105°C. After drying, the soil sample of the petri-dish was cooled to room temperature and weighed for loss of weight i.e final weight. The weight of the Petri-dish with soil sample and the percentage of moisture content was determined. The following standard formula was used for the calculation of moisture content percentage.
% of soil moisture = X100 Initial weight (Petridish with soil) -Final weight Weight of soil sample taken (5) Analysis of fungi from the soil samples Different soil samples collected in the present study were analysed for the presence of fungi by serial dilution plating method using Pototo Dextrose Agar (PDA) is a nutrient medium (Walksmann,1950;Warcup,1950). Sodium chloride solution (0.9% w/v) was used as a saline for preparation of soil dilution. For the serial dilution method, 1gm of the soil sample was dispersed thoroughly in 10ml of sterile saline. From this sample solution, 1ml was transferred to 9 ml sterile saline solution by using micropipette. The resulting solution was mixed well and from this 1ml was pipetted out into a test tube containing 9 ml sterile saline. Likewise the sample dilutions were made up to the 10 -5 and the dilutions of 10 -2 , 10 -3 and 10 -4 were used for plating on PDA medium. About 1ml of soil suspension of each concentration was added and spreaded on sterile petri-dish containing antibiotic amended PDA medium by spread plate method. Replicates were maintained for each dilution of the samples. The inoculated PDA agar plates were incubated at 30+/-1 o C in an Incubator for 3 to 7 days till the growth and sporulation of individual species of fungi were occurred. Then the culture plates were taken for observation of both qualitative and quantitative pattern of fungi. The observation was done and the data was recorded for interpretation of the results.

Qualitative and Quantitative study of fungi from soil samples
The occurrence of individual species of fungi present in various soil samples were observed and identified for the qualitative pattern. The colonies present in the sample inoculated culture plates were isolated by pure culture method and observed for the colony morphology and microscopical characters. The characteristic features such as colony colour, morphology, growth and appearance of the colony in PDA and CDA medium; microscopic characters include hyphae structure, conidiophore structure, structure and shape of conidiosporangium, number of sterigmata, conidia shape and structure of the individual fungal species were recorded for further identification. Then the individual species of fungi were identified by using standard identification manuals for Aspergillus, Pencillium and Hyphomycetes fungi (Raper and Fennel, 1965;Raper, Fennel and Thom, 1949; Barnett & Hunter, 1971;Ellis, 1971).
The characteristic features of individual species of fungi are given below:

Aspergillus niger
Initially white and the colony appear with black heads after 3 rd day, rapid in growth, cottony in texture, reverse off-white to light yellow in colour, mycelium septate, conidiophore arise from fertile branch and with terminal globose shaped sporangium, sporangium bearing with phialides or sterigmata in biseriate and release globoid conidiospores, rich in spore production.

A. flavus
Colony white mycelium initially and with sparse green to normal green coloured heads with conidophores, rapid growth, radial pattern, cottony texture, reverse off white to light yellow colour, exudate produced in some conditions, septate hyphae, globose sporangium, sterigmata biseriate, round shapeds condiospores.

A. glaucus
Fast growing spreading rapidly, colony gray green to yellow colour after sporulation, reverse yellow to orange colour, exudate appear sometimes, spread fast on the surface of the culture medium, globose heads or sporangium, sterigmata uniseriate, globose conidiospores.

A. nidulans
White mycelium with dark green coloured conidiophores, restricted growth or slow growing, often white batches of mycelium appear in morphology, exudate appeared, smaller heads, globose to subglobose heads, biseriate sterigmata, globose to elliptical spores appear.

A. terreus
Colony appear initially in white mycelium and light brown to chocolate brown, velvette appearance, restricted growth to rapid growth, centre of the colony with dense condiophores with mycelium in the margin, conidiophores small, sporangium clavate to subclavaate shaped, sterigmata biseriate, dense and appear as flame from the vesicle, conidia small in size, ovate to globose.

Penicillium sp.
Colony in grey green to bluish green with white mycelium in the margin, yellow to off white in colour, mycelium dense and septate, short branches of condiophore, small sporangial heads, Pialide long with 2 to 3 seriate, conidia elliptical to subglobose.

Pyricularia sp.
Colony grey to black colour, loosly grown mycelium, reverse of the colony appear in black, septate hyphae, Conidia are solitary, pyriform to obclavate, narrowed toward tip, rounded at the base, 2-septate, hyaline to pale brown, with a distinct basal hilum, sometimes with marginal frill.

Trichoderma viride
The colony appears to be a bit granular on PDA, with green conidia distributed throughout with white mycelium. An irregular yellow zone without conidia was present around the inoculum. Some white pustules were also found growing on the geen mat of conidia, The conidia of T. viride were appear in clusters, globose, light green in colour, phialides were arranged in divergent groups of 2-4,

White non-sporulating fungus
White bushy mycelial colony, sometime dense and spread throught the culture medium, septate to non septate mycelium without any fertile branches. Hence, it is sterial colony.
After the complete growth of fungi from individual sample plated on agar plates, the culture plates were observed for the presence of various fungi grown. The individual colonies of fungi present in each culture plate plated with 1 ml of soil sample were counted and the total number of colonies of fungi were calculated with reference to the dilution made. The population or total number of fungi present in soil sample is expressed as colony forming units (Cfu/g) of soil sample. The total number of fungi and number of individual species of fungi present in 1 g of soil sample were recorded and calculated for the percentage occurrence.

Maintenance of fungal cultures
The frequently occurred individual species of fungi such as A. terreus, A. niger, A. flavus and Penicillium sp. occurred from soil samples were isolated and cultured in the agar slants containing PDA by subculture method. The sub cultured agar slants were incubated for the complete growth of fungi. Then the pure culture of fungi were labeled and stored in refrigerator for further reference in the laboratory and an in vitro growth experiments.

In vitro evaluation of A. terreus for Chromium (Chromium III sulphate) tolerance
Based on the frequency occurrence and the population of individual species of fungi from the soil samples of leather industry environment, the species Aspergillus terreus found to be a potential fungus. Therefore, the ability of growth of A. terreus isolated from the above soil samples was cultured in the Chromium amended (Chromium III sulphate -Cr 2 (SO 4 ) 3 Potato dextrose broth (PDB) at the concentration level of 100ppm, 200ppm, 300ppm, 400ppm and 500ppm. The inoculated culture medium flasks were kept in a shaker as well as at stable condition at room temperature for 5 to 7 days period. After 7 days period, the cultures were killed and the mycelium were filtered through whatman No.1 filter paper and dried at 70°C in hot air oven for 60 mins. Then the mycelial dry weight was recorded. Control PDB medium without Chromium amendment was inoculated with the above fungus and run the experiment parallel to the above.

In vitro evaluation of A.terreus for Herbicide (pretilachlor) tolerance
As mentioned above the frequently occurred species A. terreus was isolated from the soil samples of Paddy field environment and inoculated in the Herbicide (pretilachlor) amended PDB medium at the concentration level of 100ppm, 200ppm, 300ppm, 400ppm, 500ppm and 1000ppm. The inoculated culture medium flasks were kept in shaker as well as at stable condition at room temperature for 5 days period. After 5 days, the cultures were killed and filtered through what man No.1 filter paper and dried at 70°C in hot air oven for 60 min. Then the mycelial dry weight was recorded and the results were noted. Control medium without herbicide amendment was inoculated with the above fungus and run the experiment parallel to the above experiments.

Statistical analysis
Sufficient duplicates were made in each experiment and the mean values were calculated and +/-SD values were expressed.

RESULTS AND DISCUSSION Sample details
Totally 12 soil samples including 6 from leather Industry environment of vellore district and 6 soil samples from paddy field environment of Thiruvallur District of Tamil Nadu were collected in the present study. The details of samples collected from different sources and their moisture content percentage were presented in the Table 1.

Moisture content of soil samples
The moisture content of soil samples from both the sources was determined and found * -IS-1 to IS-6 -Industry environment soil samples * -PS -71 to PS -12 -Paddy field soil soil samples in the range of 6.80% to 55.5% with an average of 19.64% for 12 soil samples. The highest moisture was observed in sample No.7 collected from paddy field soil as 55.4% and the lowest moisture as 6.8% in the sample No. 10 which also from paddy field soil. The frequency is as 4 samples less than 10%, 3 samples 10 to 20%, 3 samples 20 to 40% and 1 sample showed above 50% of moisture. The moisture content of different soil samples collected from the agricultural fields were found from 11.0% to 26.3% in which the groundnut and turmeric field show higher moisture as 26.0% and 26.3% and the lowest level was observed in sugarcane and tapioca soil with 11.0% and 17.0% respectively (Jayaraman et. al., 2018). However, in the present study, the higher moisture found in rice field soil is quite natural because of irrigation. The pattern of the moisture content of the individual soil samples were presented in Fig. 1.

Analysis of fungi in soil samples
The individual soil samples collected from different environment of Leather industry and paddy field soil were analysed for the presence of qualitative and quantitative pattern of fungi. Out of 12 soil samples analysed, 8 samples were contaminated with higher population (15000 cfu/g to 35,000 cfu/g) with various individual species of fungi and remaining 4 samples occurred with moderate number (4,000 -15,000 cfu/g) of population of fungi. For the quantitative    (23%), Sugarcane field (17%), Tapioca and waste land soil each with 14% and the turmeric field soil has the lowest occurrence as 4% of fungal population. The details of occurrence of total number of fungi were presented in Table 2, and the percentage occurrence of individual species of fungi from the soil samples were presented in Fig. 2.
The overall percentage occurrence of individual species of fungi from the soil samples collected from different sites of leather industry environment and paddy field soil environment were calculated and compared. In Leather Indsutry environment, A. terreus was the dominant fungus which occupies 46.6% followed by A. flavus (16.4%) and Penicillium species (10.2%), whereas other species of fungi were present less than 10%. The paddy field soil shows the dominant species of fungi with A. niger (27%) followed by A. terreus (16.6%) and A. flavus (16.1%). Other species of fungi were in lower level of occurrence (< 10%). The details were represented in the Fig. 3 and Fig. 4.

In vitro studies for Chromium (Chromium III sulphate) tolerance of Aspergillus terreus
The results of the in vitro study on the growth tolerance of A. terreus to Chromium showed that the growth was decreased from lower concentration (100ppm) to higher concentration (500ppm). Whereas, there was no growth of fungi observed at 500ppm concentration of PDB medium. The results were same in both shaking culture condition as well as at stable incubation condition except the amount of biomass of fungi. The mycelial dry weight of the fungi observed in the range from 310 mg/100ml at 100ppm to 20 mg/100ml at 400ppm. But no growth was observed at 500 ppm in both the incubation condition. This indicates that the presence of chromium in the nutrient medium disturb the growth metabolism of fungi and control the growth. The results were detailed in the table 3 and fig. 5. The requirement of Chromium as an essential micronutrient for the growth of many microorganisms for the maintenance of normal glucose, cholesterol and fatty acid metabolism up to certain level was reported in previous studies (Thacker et al., 2005). The results of the present study is in accordance with the findings of (Loreta Levinskaite, 2015) where certain soil fungi able to accumulate the Cr(IV) in their biomass especially on their cell wall up to certain concentration.

In vitro studies for Herbicide (pretilachlor) tolerance of Aspergillus terreus
For the in vitro tolerance study of A.terreus in herbicide (pretilachlor) amended potato Dextrose Broth (PDB) Medium, the results showed the gradual decrease of growth from 100ppm to 500ppm and 50% reduction in growth at 1000ppm of shaking and stable condition of culture when compared with control. The reduction of growth of fungi is quite rapid in shaking incubation condition than the inoculum kept in stable incubation condition. Degradation of herbicide (butachlor) by two isolates of fungi namely Aspergillus fumigatus and A. niger (87, 79%) was studied by Sangeetha et al., (2018) and found to be highly degrading and form high level of biomass respectively. Paraquat, Glyphosate and Atrazine showed higher inhibition potency on certain fungal mycelial growth than Primextra and Linuron also were reported (Chukwuma., 2015). The details of growth of fungi in herbicide amended PDB were shown in table 4 and Fig. 6.

CONCLUSION
From the present study it is concluded that the soil samples of leather industry environment as well as paddy field environment contain versatile group of fungi which mainly comprised of Aspergillus and Penicillium species. Among the species of fungi encountered in both the soil environment, Aspergillus terreus was the dominant species in Leather industry environment followed by Aspergillus flavus , Penicillium sp. and Aspergillus niger. In the case of paddy field soil, even though A.niger was the dominant species followed by A. flavus, A. terreus also occurred with significant level of population. However, it is also concluded that from the observations of in vitro experimental study as the species A. terreus found to be a effective growth organism for tolerance of chromium (Chromium III sulphate) amended Further, for the herbicide supplemented growth medium, A. terreus able to tolerate and grown upto 1000 ppm of herbicide concentration. Therefore, it is concluded from the present study, as the species of A.terreus can be used as a biological degrading agent for the polluted soil with chromium and herbicide contamination soil environment after conducting sufficient field trials.