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Developing scoring functions to assess soil quality at a regional scale in rangelands of SW Spain

The drawing of maps of soil quality at a large scale is increasingly being more useful to land planners and stakeholders. Nevertheless, it involves different methodological steps from the description of soil profiles in the field until the regional mapping of integrative soil quality index (IQI) values. The development of proper scoring functions is a paramount task for the calculation of these IQI values since every parameter needs to be standardized accordingly and weighting factors are usually estimated by multivariate techniques. The main goal of this study was to map soil quality in the Spanish region of Extremadura (commonly known by its rangelands called dehesas). To do that, i) we gathered information from 194 soil profiles described throughout the region, ii) we calculated the weighting factors of ten meaningful parameters used as indicators by using multivariate techniques (Principal Component Analysis, PCA; and Analytic Hierarchy Process, AHP), and iii) we developed standard scoring functions (SSFs) that represent the singularity of every variable (less is better, more is better). We established upper and lower limits for standardizing the values of each indicator properly. Regarding weighting factors, soil texture was highlighted by the PCA and nutrients by the AHP. Once IQI values were calculated, two regional maps of soil quality were drawn by using interpolation methods (ordinary kriging). The IQI maps showed remarkable spatial differences in soil quality presumably induced by land management. We conclude this methodology could be useful and we encourage other colleagues to test its effectiveness in places where soil data are available.

INTRODUCTION
Soil quality can be defined as the capacity that a soil has to function properly within its ecosystem limits, i.e., providing food for people and animals, storing and cleaning water, keeping good habitat conditions for several species, etc. (Bünemann et al., 2018). The generalized interest of scientists for measuring soil quality emerged in the early 1990s after many decades of research focused on the estimation of erosion rates as the way of evaluation of the land management practices effects (Andrews et al., 2004). Nevertheless, the assessment of soil quality has been a controversial topic that has always required a wide knowledge of the system object of study (Liu et al., 2017).
The use of specific indicators is the most consistent methodology known so far for assessing soil quality (Laudicina et al., 2017). In addition, Zornoza et al. (2015) remarked the importance of adopting soil quality indicators of different natures (physical, chemical, and biological origins) to achieve a holistic point of view. These indicators must be selected and undoubtedly interpreted, and after that, the most important task is the integration in a comprehensive index (Andrews et al., 2002). This indexing process requires indicators that have been previously scored and combined (Shiri et al., 2017). Therefore, a soil quality index should return a single value from a sum or multiplication (using weights or not) from different indicators scored (Mukherjee and Lal, 2014). One of the most known indexes is the integrative quality index (IQI) proposed by Qi et al. (2009).
The weighting method is a permanent challenging question since the variety of criteria can be used (Lima et al., 2013). The weights of each variable are usually obtained from statistical-based procedures (e.g., multivariate analysis). Principal Component Analysis (PCA), for instance, has been frequently used in the process of obtaining a minimum data set (MDS), i.e., to reduce the total number of indicators (Stefanoski et al., 2016). Meanwhile, the Analytic Hierarchy Process (AHP) has been already utilized for many authors (Ying et al., 2007) to determine the weight in the evaluation of environmental quality at a regional scale.
The standardization of each value is also a controversial topic since the statistical heterogeneity of the parameters that are usually considered as indicators. The most common procedure is to convert their real values into values within a range from 0 to 1 (0 = lowest quality and 1 = highest quality) by using commonly known methods such as min-max scaling and/or z-score standardization. Pulido et al. (2017), for instance, classified real values in five classes following a rationale based on a Likert scale.
The development of standard scoring functions (SSFs) allows, therefore, obtain more accurate standardized values since they are based on the previous knowledge of every variable (e.g., bulk density is better when its values decrease meanwhile soil organic carbon is better when its values increase). They have been widely used for assessing soil quality in agricultural soils of many parts of the world (Li et al., 2018). Although their calculation is based on consistent methods largely validated at a regional scale, at least in agricultural soils, there are no published studies (up to our knowledge) on rangelands, where millions of people depend directly on keeping soils in good conditions that can guarantee enough pasture production for feeding their livestock. Pulido et al. (2017) assessed soil quality at a farm-scale in rangelands of Extremadura Region (Spain). Nevertheless, they did not use both weighting factors and scoring functions for calculations. In addition, there is not a regional map that allows the assessment of soil quality at a large scale. For these reasons, the main goal of this study was to draw maps of soil quality for Extremadura. To do that, we calculated weighting factors by using multivariate techniques (PCA and AHP) and to develop SSFs for every indicator.
As indicators, we selected ten soil properties (sampling at the layer 0.00-0.30 m): sand, silt, clay, bulk density (BD), total nitrogen, available phosphorous (P), potassium (K), pH(H 2 O), soil organic carbon (SOC), and cation exchange capacity (CEC). We hypothesize the AHP generates more realistic maps than PCA since we can select the most influencing factors from our knowledge on the spatial variability of the soils of the study area.

Study area
This study was carried out in the Autonomous Region of Extremadura (SW Spain) (Figure 1). Extremadura covers 41,633 km 2 (8.2 % of Spain) where the 50-60 % out of its territory is occupied by rangelands and grasslands, the remaining land is agricultural (Guadiana River) and mountainous areas (Spanish Central System at the North). The rangelands are located at an elevation ranging from 300 to 700 m a.s.l. dominated by undulated terrains (2-8 % of inclination) over old erosion surfaces of shales, schists, greywackes, and granites. The climate is Mediterranean with dry and warm summers from June to September. Soils are sandy loam-textured, shallow, and poor in nutrients the main soil types present in the area are: Leptosols and (Epileptic) Cambisols (IUSS Working Group WRB, 2015).
Extremadura is inhabited by more than one million people (2 % out of whole Spain), recording a population density of ≈25 people km -2 , spread out by two provinces (Cáceres and Badajoz) and 388 municipalities. Extremadura's population has still a noticeably rural character since more than 25 % of people are living in places with a population lower than 10,000 inhabitants. Thirteen percent of active people work in the primary sector (mainly agriculture and livestock husbandry) providing 6 % of the regional gross domestic product (GDP). So, although rangelands do not suppose a significant contribution in terms of GDP, they are particularly interesting in terms of livestock production (735 tons of meat per year), biodiversity conservation (habitat of special protection by the European Union), and rural idiosyncrasy.

Soil profile description
The soil profile dataset is composed of 194 soil profile descriptions: 47 out of them were conducted during the fieldwork of the Ph.D. thesis of the first author (Pulido Fernández, 2014), 116 during former research projects between 2003 and 2006, and 31 descriptions were taken from the Extremenian Catalog of Soils.

Work scheme and rationale
Figure 2 shows a conceptual scheme in which the rationale of this study can be understood. The core of this study is the Qi et al. (2009) equation for calculating the integrative quality index (IQI) and the final target is the drawing of maps of soil quality at a regional scale. Since we selected ten indicators from our previous knowledge, and we gathered information from about 194 soil profiles described throughout Extremadura, the first step was the calculation of weighting factors for each indicator. For doing that, we used multivariate Rev Bras Cienc Solo 2020;44:e0200090 techniques such as Principal Component Analysis (PCA) and Analytic Hierarchy Process (AHP). Secondly, we developed standard scoring functions (SSFs) to standardize every indicator. We distinguished two types of functions: less is better (L) and more is better (M). Finally, two IQIs were calculated for the 194 sampling points and these values were interpolated (ordinary kriging) to obtain two regional maps of soil quality.

Weighting factors
We utilized two sets of weighting factors, one obtained from the PCA and the other one from the AHP. Both analyses were made by using IBM SPSS 22.0 and MO Excel 2013 software packages, respectively.
The PCA weighting factors were assigned from the commonality of each indicator calculated according to equation 1.

Weighting factor Ix = (Commonality Ix)/(Sum of every communalities)
Eq. 1 The weighting factors of the AHP set were obtained following these methodological steps (Saaty, 1990

Standard scoring functions
We applied standard scoring functions (SSFs) (Mukherjee and Lal, 2014) in which the calculation of the upper and lower limits are needed. Since there is no information about the upper and lower thresholds in the specific rangeland areas of Extremadura, the minimum and maximum observational values of the variables in the region were considered as lower and upper thresholds, respectively. Regarding the fact that the range of soil reaction can affect soil quality, the values of the range had a score equal to 1 and increasing the distance from this range (more or less), the score decreased. The final results were classified as "less is better" and "more is better".

Soil quality mapping
Once every indicator was both standardized (range: 0-1) by the scoring functions and its two weighting factors (PCA and AHP) were assigned, the two IQI (Qi et al., 2009) values (PCA and AHP) of each one of the 194 sampling points were calculated. These values were interpolated by using ordinary kriging, embedded in the Geostatistical Wizard Tool by ArcGIS 10.5 (ESRI), and the resulting raster was clipped to the boundaries of the region of Extremadura. Finally, the two maps generated were interpreted to decide which one is more reliable.

Descriptive statistics of the whole dataset
The total dataset is comprised of ten soil quality indicators reporting information about soil texture, acidity, cations and nutrients contents, and bulk density. Table 1 summarizes the descriptive statistics of the 194 soil profiles at a sampling layer from 0.00-0.30 m. Soil texture is characterized by 14.1, 38, and 47.9 % of clay, silt, and sand contents, respectively. It is important to highlight that extreme maximum values can also be found (45.0 % clay, 62.7 % silt, and 82.1 % sand), being clay and silt contents the most variable.
The mean pH(H 2 O) values were close to 5.6, with maximum values of 8.2 and a minimum of 4.1, giving us information about the acidity of the rangeland's soils. The average value of CEC was 10.65 cmol c kg -1 , reaching maximum values of 31.54 cmol c kg -1 and minimum ones of 3.33 cmol c kg -1 . Total N reaches mean contents of 0.14 %, increasing in some places till 0.49 %. The mean Olsen-P value was 17.08 mg kg -1 and available K 0.49 mg kg -1 . These values are much related to the low SOC (1.9 %) found in the dehesas.

Correlations among variables
The Pearson linear correlation coefficients of the soil quality indicators are shown in figure 3. Regarding soil texture variables (clay, silt, and sand contents) silt and sand showed a high negative correlation (r = -0.85). This fact is due to the dominance of loam soils with slight variations in texture, depending on a higher content of sand (sandy loam) or silt (silty loam). Clay, by its part, was positively correlated with the cation exchange capacity (r = 0.70). Soil compaction (expressed as bulk density) was negatively correlated with almost all variables. Chemical and biological properties were logically correlated in a positive way among them and negatively with bulk density and the sand content.
The CEC seemed to be the variable that explains the best the existing relationships between every indicator. It was positively correlated to the pH (r = 0.43) and with the parameters usually utilized as fertilizers: N (r = 0.36), P (r = 0.41), and K (r = 0.40). Contrariwise, it was negatively correlated to bulk density (r = -0.29) that was also negatively correlated to N (r = -0.47). Remarkable are also the role played by P and SOC as indicators of fertility. They were positively correlated between them (r = 0.37) and kept positive correlations with K (K-P: 0.53; K-SOC: 0.34) and N (N-P: 0.29; N-SOC: 0.42).

Principal component analysis
The PCA sorted the ten variables studied according their importance. The CEC and clay content showed the highest predictive power (0.779 and 0.684, respectively) while the lowest values were recorded with SOC and bulk density (0.365 and 0.317, respectively). Nutrients content (N, P, and K) showed importance lower than soil texture, pH, and CEC. However, their variables showed similar power values of prediction (Figure 4).    (Burt, 2004); pH (1:2.5 soil/water ratio); cation exchange capacity (CEC) (MAPA, 1982); total nitrogen according to Kjeldahl (1883); phosphorous according to Olsen et al. (1954); available potassium according to MAPA (1982); soil organic carbon according to Walkley and Black (1934); and bulk density according to Coile (1936). SD: standard deviation; CV: coefficient of variation.

Standard scoring functions, weight assignment and integrative quality index
Each indicator was transformed and normalized in a range from 0 to 1 and their functions varied according to their logical behavior, i.e., properties that improve if their values  increase ("more is better" pattern, M) and those that improve when their values decrease ("low is better" pattern, L). The detailed equations are shown in the table 3, including the upper and lower limits within the system to function properly.
The weight of each indicator is calculated by its communality obtained by two ways: PCA and AHP analysis (table 4). In the PCA, soil texture, pH, CEC, and available K obtain a weight higher than 0.100, meanwhile, in the AHP, the highest weights are obtained by the available K and Olsen-P.
Then, in table 5, we show the descriptive statistics of the integrated quality index in the 194 sites according to its weights returned by PCA and AHP analysis, respectively. The PCA returned an IQI with mean values higher than AHP but this last shows a wider range of qualities (0.154-0.705). Figures 5 and 6 show the spatial distribution of IQI throughout Extremadura using PCA and AHP, respectively. In the first map (PCA), the IQI values ranged from 0.203 to 0.682, meanwhile in the second one (AHP), the IQI values ranged from 0.154 to 0.705. This higher data range of AHP could be influenced by the higher weights obtained for the contents of phosphorus (0.220) and potassium (0.201).    In both maps, the province of Badajoz (southern half of the territory) evidences a higher quality in its soils, particularly using AHP procedure. The soils with lower values (green colors) correspond to the northern part of the region (mountainous areas in which data of soil properties are scarce) and eastern part of the province of Badajoz dominated by treeless grasslands. Remarkable differences have been observed in the province of Cáceres (northern half of the region) in which rangeland soils are mostly considered as of medium quality (predominance of yellow instead of red color).

DISCUSSION
This study can be interpreted as an up-scaling (from farm to the regional scale) of previous studies carried out by Pulido et al. (2017). The role played by potassium and clay (in sandy/silty loam soils) looks keys in the understanding on soil quality in rangelands of SW Spain. From a methodological point of view, we propose an index for assessing soil quality more sensitive (weight vs. average values), integrative (more indicators), and easy to use (there are much information free on Internet). The pros and constraints of using different analysis (PCA vs. AHP) in the scoring process have been also addressed.
The analysis performed in this study evidenced a great importance of some couples of variables such as CEC, clay (PCA), phosphorous, and potassium (AHP) on soil quality. These findings were in consonance with two indicators (CEC and K) also proposed by Pulido et al. (2017) in their research at a farm scale. Nevertheless, it keeps alive the never ended discussion about the origin of a good soil. That is, it shows a high quality because belong to areas with deep soils rich in clay and nutrients (e.g., on alluvial deposits) or its quality has been improved by land management practices such as a long-term fertilization (animal excreta, improved pastures, etc.) or avoiding soil erosion.
Phosphorous is considered by a majority of regional farmers and extensionists (personal communications in several meetings) as the key nutrient for fertilizing natural pastures  due to their rapid effects on pasture production (Barbosa et al., 2009;Recena et al., 2017). Potassium has been hardly ever considered in any soil indicators-based system in rangeland areas. Pulido et al. (2017) selected it due to its strong correlations with pasture production as a good explaining factor of its spatial variability regardless rainfall amounts. Potassium is commonly known for increasing the tolerance of plants against droughts and frosts (Behera and Shukla, 2015;Blanchet et al., 2017), which are recurrent phenomena in inner Mediterranean regions such as Extremadura (Schnabel, 1997).
The role of clay on soil quality in this land system can be controversial. It is obvious that clay contributes to a higher retention of cations (Sulieman et al., 2018) and some nutrients (Hosseini et al., 2017) due to its surface negative charge, although a high content of clay and silt can increase soil bulk density and consequently induce soil compaction (Bienes et al., 2016). Anyway, the affecting power of clay has been neglected by Pulido et al. (2017) due to the low range of soil texture studied at a farm scale (mostly sandy-loam texture).
Soils with high percentages of sand usually show high bulk density values because their soil density is naturally higher, and it does not mean soil compaction (Dexter, 2004). In addition, sandy soils in Extremadura are logically heavier because quartz is abundant in their mineralogy.
The positive correlation between clay and CEC has been also confirmed by other authors in arid and semi-arid environments (Koganti et al., 2017). It can be an indication of a positive effect of clay on fertility retaining cations and nutrients (although not for soil organic carbon and nitrogen) and reducing the acidity of soils (Silva et al., 2018). Nevertheless, the range of clay content has not been so large in this study.
The PCA also confirmed the logical negative effect of bulk density, considered as land degradation indicator by Pulido et al. (2017). These latter only found evidences of the negative effect of bulk density on pasture production (Pulido et al., 2018) although it still remains unclear if it is a consequence of a reduction in the fertility or in the capacity of storing water. Authors such as Montagu et al. (2001), Anderson et al. (2006), Kraaijvanger et al. (2015), and Bogunovic et al. (2017), among others, have already found evidences of a reduction in fertility induced by soil compaction.
In rangelands, soil compaction generally reduces fertility by two interconnected processes: animal trampling and the removal of tree and pasture litter accumulated on the soil surface (Pulido-Fernández et al., 2013). Under circumstances of overgrazing animal trampling exerts a pressure on soil comparable to heavy machinery that makes decrease its porosity and increases its penetration resistance limiting grasses root activity (Greenwood and McKenzie, 2001;Bilotta et al., 2007). It also increases runoff and soil erosion as well as the washing of nutrients (Bogunovic et al., 2020).
Although the lithology dominant in Extremenian rangelands are siliceous rocks (shales and granites) and the dominant land management is the livestock husbandry, we consider the necessity of combining in further research data taken at a regional scale with studies based on local knowledge from a farm scale. In addition, the values returned in this IQI should be compared with topographic factors (Manandhar and Odeh, 2014), soil maps (Stahr and Clemens, 2017;Nabiollahi et al., 2018), and information provided by the Spanish National Inventory of Soil Erosion, among other sources, to detect regional patterns of soil quality or particular influences. This IQI should be also validated in other areas with different dominant land uses such as vineyards, olive groves, or chestnut tree orchards.

CONCLUSIONS
We drew two regional maps (Extremadura, SW Spain) of soil quality by interpolating 194 punctual values of an integrated quality index (IQI) calculated both from two sets of weighting factors (PCA and AHP) and two types (less is better, much is better) of standard scoring functions (SSFs). Methodologically speaking, it supposed the transformation of 194 soil profile descriptions into valuable maps of soil quality. The most reliable map seemed to be that obtained from the AHP method since the weighting of indicators was more focused on the content of nutrients, which it is one of the limiting factors that explains the geographical distribution of rangelands. This study represents a methodological advance regarding previous studies aimed at a farm scale but further research is still needed in terms of spatialization of soil properties considering also topographical and managerial factors.


extfieldsofstudy: Physics Chemistry

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

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The effect of iron powder as oxygen absorber active packaging on fish oil total oxidation value

The high content of unsaturated fatty acids causes fish oil can be easily damaged by oxidation. Direct oxygen contact with food is the initial stage of food damage by oxygen. The use of active packaging aims to extend the shelf life, maintain the sensory and quality of the packaged material. Oxygen absorber commonly used in the market is iron powder. This study aims to determine the potential use of iron powder as an active oxygen absorbent material in the packaging in reducing the total value of oxidation of fish oil during the storage period. This study used a completely randomized design with 6 treatments. Fish oil stored in a Polyethylene Terephthalate packaging bottle and placed in a refrigerator at 23°C for 21 days. The addition of iron powder on fish oil packs is 2.7 grams, 5.4 grams, 8.1 grams, and 10.7 grams with negative controls in the form of pure fish oil and positive control in the form of packaged fish oil. The results showed that the addition of 10.7 grams of iron powder as an oxygen absorber to the active packaging of fish oil had a greater ability to reduce the total value of fish oil oxidation during storage (P <0.05).

Introduction
The use of packaging in food products initially only aims to wrap and protect food from external contamination [1]. Food packaging continues to grow as a response to consumer needs and the trend of the food industry to food products that use less preservative, fresh, long shelf life and maintained quality [2].
Active packaging aims to extend the shelf life and maintain or even improve the quality of packaged food products [3]. Active packaging is added to help improve packaging capabilities in maintaining quality, safety and sensory aspects of foodstuffs [4]. Active ingredients added to the packaging material, on the packaging surface, in multilayer structures or into special elements which will later be included in food packaging such as sachets, labels and bottle caps [2]. According to Mills [5], oxygen is one of the main factors causing damage to food. The high content of unsaturated fatty acids (Polyunsaturated Fatty Acid) causes fish oil to be easily damaged by oxidation, resulting in the decreased shelf life of fish oil products [6]. Damage to fish oil products due to direct contact with oxygen is expected to be reduced using oxygen absorber based packaging on fish oil packaging. The active ingredient that is commonly applied as an oxygen absorber on active packaging is iron powder [7]. The application of 0.3 grams of iron powder reacted with metal halide and alkaline earth metal can absorb the entire remaining oxygen contained in a 500 cc capacity with oxygen content up to 0.0041 moles [8]. Based on this theory, it is necessary to measure the total oxidation value in fish oil, which in the packaging is added to the substance of iron powder as an oxygen-absorbing active ingredient.

Research materials
This research was conducted at the Chemistry and Analysis Laboratory and Microbiology Laboratory of the Faculty of Fisheries and Marine Airlangga University in July to August 2018. The materials used were samples of Scott's Emulsion packaged fish oil produced by PT. Combiphar, packaged iron powder (Fresh One®) produced by PT. Sigmaco Saksama Image, pure fish oil obtained from PT. Blambangan Foodpackers Indonesia. The tools used include plastic Polyethylene Terephtalate (PET) bottles, analytic balance (pioneer ohauss) with an accuracy of 0,0001 grams, burette (Duran Schott), fume hood (FH-120G type), refrigerators (GEA Pharmaceutical), and spectrophotometer (Human x-ma 1200).

Experimental design
This study used a completely randomized design (CRD), 6 treatments with each treatment having 7 replications. Treatment A is packaged fish oil without the addition of iron powder on the packaging, Treatment B is pure fish oil without the addition of iron powder on the packaging, Treatment C is pure fish oil with the addition of 2.7 grams of iron powder on the packaging, Treatment D is pure fish oil with addition 5.4 grams of iron powder on the packaging, Treatment E is pure fish oil with the addition of 8.1 grams of iron powder on the packaging, and Treatment F is pure fish oil with the addition of 10.8 grams of iron powder on the packaging. The treatment unit for fish oil is each contained in a 200 ml dark Polyethylene Terephthalate (PET) bottle and placed in the refrigerator at 23 o C for 21 days.

Acid value analysis
Acidity values were analyzed based on the AOCS Ca 5a-40 method [9]. Determination of the degree of acidity is carried out by titrating hydroxide (KOH) against the sample using the principle amount of potassium hydroxide (KOH) needed (mg) to neutralize 1 gram of fat.

Free fatty acid value analysis
Free fatty acids percentage was analyzed by the AOCS Ca 5a-4 method [9] by weighing 10 grams of oil then adding 25 ml of 96% alcohol, and heated in a water bath for 10 minutes at 40 o C. Phenolphtalein is used as the indicator and titrated with 0.1 N KOH until a pink color appears [9].

Total plate count analysis
Total Plate Count analysis is a quantitative method used to determine the number of microbes in a sample. Total Plate Count is determined by calculating the number of microbes in a product that grows on the agar medium with the specified temperature and incubation time [10]. Calculation of Total Plate Count using the common media Plate Count Agar. The number of colonies that grow is calculated by the number of microbial Colony Forming Units.

Result and discussion
The quality of samples fish oil was measured before being given the treatment of adding the iron powder to the packaging are shown in Table 1. Analysis of the Peroxide Value, p-Anisidine Value, and Total Oxidation Value are shown in Table 2. Homogeneity test on the analysis of Peroxide Value (0.089>0.05). p-Anisidine Value (0.855>0.05). And Total Oxidation Value (0.146>0.05) showed that the groups of data tested were homogeneous and the population variance was homogenous. The results of the analysis using variance showed that the addition of iron powder had a significant effect on the Peroxide Value. p-Anisidine Value. and the Total Oxidation Value in fish oil. Data analysis of Acid Value and Free Fatty Acid Percentage are shown in Table 3. The homogeneity test on the analysis of Acid Value (0.253>0.05) and Free Fatty Acid Value (0.505>0.05) showed that the groups of data tested were homogeneous, and the population variance was homogenous. The results of the analysis using variance showed that the addition of iron powder had a significant effect on Acid Value and Free Fatty Acid Value. Total Plate Count in pure fish oil and packaged fish oil after storage treatment has a mean value that is not significant and tends to be the same.  Based on the research conducted after the storage process and the treatment of iron powder addition on fish oil packaging. it is known that peroxide value has fluctuating values. The initial peroxide value of pure fish oil (13.32±0.11 meq/kg) is higher than the packaged fish oil peroxide value (2.10±0.20meq/kg). Treatments E and F have lower peroxide value compared to treatments C. D. and B (controls). Table 2 showed that the amount of iron powder added to fish oil packaging is not linear with fish oil peroxide value decrease. Lower peroxide value numbers do not always indicate that the oxidation process is running at an early stage. but it can also be caused by products are oxidized by fat which has decomposed into other compounds at an advanced level [11]. According to Samples [12]. in the early stages of the oxidation process, there will be a continuous increase in peroxide value numbers until it reaches the maximum condition. After achieving maximum conditions, this will trigger an increase in the reaction speed of the secondary production of the oxidation process. so that the peroxide level will decrease.
Numbers of pure fish oil p-Anisidine value and packaged fish oil p-anisidine value increased after the storage period. Table 2 showed p-Anisidine value decreases inversely with the amount of iron powder added to each treatment. The p-Anisidine number in treatments C. D. E. and F has a lower number of p-Anisidine than treatment B as control of pure fish oil stored without the addition of fish oil during the storage period. From the results obtained. It is known that a bigger amount of iron powder added causes the value of the p-Anisidine number to decrease. The number of p-Anisidine is measured by compound decomposes peroxide number. The decomposition of hydroperoxide components forms malonaldehyde compounds as a result of secondary oxidation [13]. Based on the research carried out the total oxidation value of fish oil treated with the addition of iron powder with different concentrations on the packaging has a fluctuating value because the dominant total oxidation value is fluence by peroxide numbers whose it is values are also volatile. However, packaged fish oil total oxidation value. Which has antioxidant content continues to experience an increase in value compared to its initial quality. This shows that even though antioxidants have been added. Fish oil still can oxidation during the storage period.
Based on the research conducted, acid numbers and free fatty acid levels decreased inversely with the amount of iron powder added to each treatment. The acid numbers and free fatty acid levels in treatments C. D. E also F have a lower value than treatment B as a negative control of pure fish oil stored without the addition of iron powder on the packaging. Table 3 showed that the greater the amount of iron powder added causes the value of the free fatty acid level to decrease. According to Christie et al. [14], the level of free fatty acid is an indicator of the oxidation process in oils that produce other organic acid components. The decrease in free fatty acid levels is due to the free fatty acid component undergoing an autooxidation process at the termination stage. The termination phase describes free fatty acids into aldehydes and ketones.
The free fatty acid level is a tertiary product of the oxidation process, which shows the occurrence of oxidative and hydrolytic rancidity. The level of free fatty acids is a fatty acid that is not bound as triglycerides. Free fatty acids are produced from oxidation or hydrolysis. The results of the reaction from the hydrolysis process are glycerol and free fatty acids. Factors that influence the hydrolysis process include water acidity and catalyst (enzyme). Increased levels of free fatty acids cause a decline in taste and odor in fish oil [15].
Based on the test of the total plate count in Table 4 it is known that there is no significant difference in the number of bacteria in fish oil. According to Rorong et al. [16], the form of damage to oil is mainly caused by the interaction of oxygen in the air against fat. Fat decomposition by microbes can only occur when there are water and nitrogen compounds while oil oxidation by oxygen in the air (autooxidation) occurs spontaneously with the speed of the oxidation process based on the type of fat [16].

Conclusion
The addition of iron powder to fish oil packaging has a significant effect on fish oil total oxidation value during the storage period as evidenced by the secondary oxidation product indicator in the form of panisidine, which decreases with an increasing amount of iron powder added to fish oil packaging. The addition of 10.8 grams of iron powder on fish oil packaging can inhibit the addition of p-anisidine numbers to point 17.74 mEq/kg for 21 days of storage. However. Further research is needed to periodically measure peroxide numbers because of its fluctuating value. Periodic measurement of peroxide numbers aims to determine the cycle of up and down peroxide numbers through the maximum and minimum values of peroxide value during the storage period.


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CHANGES IN YIELD AND CHEMICAL COMPOSITION OF THREE-YEAR-OLD SHORT-ROTATION PLANTATIONS OF Dipteryx panamensis IN COSTA RICA

Research and implementation of energy crops using short-rotation woody species (SRWC) are increasing in Latin America, especially for biomass production and use in bioenergy. For this purpose, one of the main factors to consider is species assessment. Therefore, the objective of the present study was to evaluate the growth characteristics, the production and distribution of biomass, and the chemical, physical, and energetic properties of the biomass of three-year-old Dipteryx panamensis plantations in SRWC in three diff erent spacings (1.0x2.0 m, 1.0x0.5 m and 1.0x1.0 m) and establish the best spacing condition for this tropical species. The results showed that the production of biomass varies between 1.1 and 42.36 t.ha, and that 33-44% of the production are concentrated in the leaves. At three years, the SRWC presented 50% mortality, with a diameter of 4.8 cm at 30 cm-height from the ground, with total tree heights ranging from 5.17 to 6.98 meters. The evaluation of the biomass showed a calorifi c value between 18.9 and 19.4 MJ/kg, less than 1.81% of ash content and 86% of volatile content. As for the eff ect of the spacing, the green density of the wood and the moisture content increased with increasing spacing, while spacings of 1.0x1.0 m and 1.0x0.5 m showed the best behaviour regarding annual biomass production.

1.INTRODUCTION
High and variable cost of fossil fuels due to geopolitical factors, together with global warming (Hauk et al., 2014), have forced the creation of new environmental policies amongst which the promotion of renewable alternative energy sources stands out (Inglesi-Lotz, 2016). This type of energy source is one of the most abundant in the world (Pereira and Costa, 2017).
Forest residues resulting from harvest and sawmill processing wastes area important for energy production, followed by agroforestry residues or short rotation woody crops (SRWC) (Moya et al., 2019). The latter have become increasingly important, since they are highly productive plantations whose purpose is to use the stem and branches for the production of energy (Zamora et al., 2015).
In general, SRWC are associated with planting densities higher than 5000 trees/ha (Dickmann, 2006). The fi rst experiences in SRWC establishment used spacings oriented to wood production, that is, 3x3 m, 4x3 m and 4x4 m (Silva et al., 2015). Another advantage of using SRWC is that in addition to biomass production, SRWC provide other ecological benefi ts, such as carbon capture and habitat for various wild species (Volk et al., 2011). From the point of view of reproduction, these systems can use genetically improved and easy to propagate species, with short cutting cycles between 3 to 10 years (Vance et al., 2014).
Species selection, planting density, annual increments and rotation are important at the time of establishing a SRWC (Volk et al., 2011). Regarding the planting density, densities above 5000 trees/ha are necessary depending on the dimensions needed (Dickman, 2006). With respect to annual increments and rotations, some authors point out that, at the age of 3, production should be around 5 to 50 t.ha -1 (Dickman, 2006;Acuña et al., 2017;Tenorio et al., 2016aTenorio et al., , 2018. Dipteryx panamensis, a native species that had not been studied in SWRC, was tested in the present study. D. panamensis is a dense wood that shows initial fast growth, with potential for reforestation for sawlog production in Costa Rica (Tenorio et al., 2016a). Harvesting this species is currently forbidden. D. panamensis is one of the endemic species of the region (Redondo-Brenes, 2007), with basic wood density above 0.7 (Tenorio et al., 2016a). Recent studies have shown that D. panamensis presents adequate properties for energy production, and that torrefaction processes increase its energy performance (Gaitán-Alvarez et al., 2017).
Although D. panamensis is used in reforestation for timber production (Redondo-Brenes, 2007), there is lack of information on its performance in SRWC systems for biomass production (Moya et al., 2019. Therefore, the objective of this study was to evaluate the growth characteristics of the tree, the production (tons) and distribution of biomass, and the chemical, physical and energy properties of the biomass of a three yearold Dipteryx panamensis plantations in SRWC at three diff erent spacings (1.0x2.0 m, 1.0x0.5 m and 1.0x1.0 m) in Costa Rica and establish the best spacing condition for this tropical species

Location and conditions of the plantation
The study was carried out within a trial of 3-yearold Dipteryx panamensis (Figure 1a-b), located in the northern zone of Costa Rica. The trial consisted of 3 diff erent spacings: (i) 1.0 x 2.0 m (low density, 5000 trees/hectare), (ii) 1.0 x 1.0 m (medium density, 10000 trees/hectare) and (iii) 1.0 x 0.5 m (high density, 20,000 trees/hectare), thus obtaining 3 diff erent treatments. Each of the planting spacings was represented in 3 blocks, resulting in 3 random repetitions with experimental units of 10 x 10 m of each spacing. Each repetition consisted of 100 m 2 plot ( Figure 1a).

Tree sampling in the plantation and sampling in the tree
In each spacing, 15 trees in total were sampled (5 trees x 3 repetitions), making a total of 45 sampled trees for the whole study. Each tree was felled at ground level and its total height and diameter at 30 cm above the ground were measured (Figure 1c). This height was defi ned because the 1.3 m of tree height were not presented in all sampled trees. Following, the branches and the leaves were separated from the tree stem and each part was weighed separately. This was denominated as green weight. Then, 2 samples 10 cm long each were extracted from the stem free of leaves and branches, in three sections at diff erent tree heights (at the base of the tree, at 50% height and at the total height) ( Figure 1c). The sampling tree was applied according to previous researches published by Tenorio et al. (2016a;2018)

Determination of moisture content and biomass production
The biomass moisture content (MC) was determined for each part of the tree (leaves, branches and stem). A sample approximately 150 g of leaves and branches was collected and dried at 60 °C for 72 hours and then weighed to obtain the dry weight. As for the stem MC, one of the 3 samples cut from three diff erent heights of the stem was used. Each sample was removed the bark, each of these parts (bark and stem) was weighed in green condition and then dried for 72 hours at 60 °C avoiding the material degradation, to obtain the dry weight of the stem and the bark. With green and dry weights of stem, bark, leaves and branches we proceeded to calculate the MC.
Once the MC for each part of the tree was obtained, dry biomass production was calculated using as a reference the MC and the weight in green condition of each part of the tree at the time of sampling (Equation 1). Then with these data the percentage of each of the parts (stem, leaves and branches) was calculated as the ratio of the weight of each part and the dry biomass of the whole tree, expressed as a percentage. These data were extrapolated to hectares in the diff erent planting spacings.

Determination of specifi c gravity and green density
The specifi c gravity (SG) represents the biomass relative to the green weight of the sample. It was calculated only for the stem, using the other section of the sample extracted from 3 diff erent tree heights. The bark was removed, weighed and the volume by water displacement was determined. The pieces were then placed in an oven for 24 h at 103 °C, and the weight was recorded before and after drying. SG was determined according to ASTM D-4442 (ASTM, 2007), while the density was determined in freshly cut condition (green mass and volume).

Determination of the chemical and energy characteristics of the biomass
The percentage of carbon (C), hydrogen (H) and nitrogen (N), lignin, cellulose were determined only to stem part of trees. The stem from 3 diff erent tree heights from the fi ve trees per each sampled block and spacing was used to get milled material, these milled material were air dry until it reached 12% moisture content. The material was then sieved through 40-60 meshes (425 and 250 μm). Regarding the chemical characteristics, the percentage of C, H and N were determined utilizing the Elemental Analyser , model Vario Micro Cube. Also the cellulose and lignin percentage of the material were obtained; the lignin were calculated using three samples of 2 mg of the milled material according to norm TAPPI T222 om-02 (TAPPI, 2002), and the cellulose were calculated using three samples of 2 mg of the milled material according to the procedure describe by Seifert (1960). Regarding the energy characteristics, the gross calorifi c value (GCV) and the percentages of ash and volatile material were determined. As for the GCV, a portion of 10 g of the milled material was brought to a humidity condition of 0%. Then, a sample of each repetition of 2 grams was selected and the GVC was determined by means of the Parr's Calorifi c Test, in accordance with ASTM D-5865 (ASTM, 2004). These tests were performed on a 6725 Micro-Parr model calorimeter pump at a temperature of 20 °C. The percentage of ashes was determined by ASTM D-1102 (ASTM, 2013a). For volatiles percentage 3 random samples of 2 g each were selected again and determinations were performed following the procedure described in ASTM D-1762 (ASTM, 2013b). For chemical and energy characteristics, three samples of approximately 2 g of the previously sieved material were used for each of the blocks studied (3 samples x 3 blocks x 3 spacings = 27 samples).

Statistical analysis
The statistical analysis consisted of, fi rstly, the determination of the normality and homoscedasticity of the variance analysis model for all the measured variables. Next, the descriptive statistics were determined for each of the variables, and then the characterization of the variables. An analysis of variance was conducted to determine the eff ect of the plantation spacing, followed by Tukey's multiple comparison analysis to verify signifi cant diff erences between the spacings, using the averages of the measured variables. The SAS program (SAS Institute Inc., Cary, NC) with α=0.05 confi dence level was utilized to perform these analyses.  Table 1 shows the initial density, the density at the time of sampling and the mortality rate of the plantations at the diff erent spacings. Table 1 shows that the highest mortality at 3 years of age is approximately 50%, being higher in spacing 1.0 x 1.0 m. In relation to the morphological dimensions of the tree, the diameter at 30 cm-height from the ground at 3 years of age, in each spacing, registered values between 47 and 48 mm, with no signifi cant diff erences between the three spacings ( Table 1). As for the total height, D. panamensis trees in spacing 1.0 x 0.5 m were statistically higher close to 7 m (Table 1).

Biomass production
The lowest biomass production, with and without leaves, was observed in spacing 1.0 x 2.0 m, followed by spacing 1.0 x 1.0 m, with the highest biomass production in spacing 1.0 x 0.5 m ( Table 2). Regarding the stem biomass production, spacing 1.0 x 2.0 m presented the statistically lower biomass production with respect to the other two spacings (Table 2). In relation to the biomass from the leaves and branches, the highest yield was obtained with the density 1.0 x 0.5 m, this being statistically diff erent from the other spacings, which did not show statistical diff erences in the production of leaves and branches ( Table 2).
As regards to biomass distribution in the diff erent parts of the tree, in spacing 1.0 x 2.0 the highest percentage of biomass production was in the leaves, followed by the stem, branches and bark (Figure 2). In spacings 1.0 x 1.0 m and 1.0 x 0.5 m, most biomass production came from the stem, followed by the leaves, then the bark and lastly, the branches (Figure 2).

Chemical characteristics
Regarding the content of cellulose and lignin, no signifi cant diff erences were found between the three spacings (Table 2).. Statistical diff erences in the percentages of N, C and the C/N ratio were observed. There were no statistical diff erences between the 1.0 x 1.0 and 1.0 x 0.5 m spacing, only in the 1.0 x 2.0 m ( Table 2). The highest value of N percentage was obtained in spacing 1.0 x 2.0 m and 1.0 x 1.0 m, whereas in spacing 1.0 x 2.0 m the N percentage was statistically diff erent from spacing 1.0 x 0.5 m ( Table 2). The lowest percentage of C was obtained by trees planted at a distance of 1.0 x 2.0 m, being statistically diff erent from the other two densities, which did not present statistical diff erences (Table 2). In relation to the C/N ratio, the highest value was shown by the spacing 1.0 x 0.5 m, being statistically diff erent from the other spacings, which did not show diff erences between them (Table 2).

Physical and energy characteristics
With regard to the physical properties of the trees, the density of recently felled wood was higher in spacings 1.0 x 2.0 m and 1.0 x 1.0 m, with no statistical diff erences between those spacings, while spacing 1.0 x 1.0 m showed no diff erences with 1.0 x 0.5 m (Figure 3a). The trees in the three spacings were on average statistically similar regarding the specifi c gravity (Figure 3b).
Regarding the energy properties of the biomass, spacings 1.0 x 0.5 m and 1.0 x 1.0 m did not show statistical diff erences in MC, while spacing 1.0 x 0.5 m presented statistically lower MC than the previous spacings ( Figure  3c). The highest value of GCV was obtained in the spacings 1.0x1.0 m and 1.0 x 0.5 m, without diff erences between them, while the spacing 1.0 x 1.0 m presented GCV value statistically diff erent to the spacing 1.0 x 2.0 m ( Figure 3d). As for the ash content, the highest value was obtained with biomass from the spacing 1.0 x 2.0 m, which was statistically diff erent from the other planting densities, which shows the lower ash content value (1.0 x 1.0 and 1.0 x 1.5 m) (Figure 3e). The percentage of volatile materials was higher in the biomass planted at 1.0 x 1.0 m, while the remaining spacings were statistically lower, showing no statistical diff erence (Figure 3f).

Plantation characteristics and biomass production
Firstly, D. panamensis is catalogued as a slow growing species in plantations (Piotto et al., 2003), according to the results in diameter growth if compared with other woody trees growing under plantation conditions. For example, Tenorio et al. (2018) report a diameter of 72.43 mm for Gmelina arborea in 3-year-old SRWC plantations, for the same densities, which surpasses the diameter of 47.08 mm found in D. panamensis.
It is expected that the higher the planting density (narrow spacings), the smaller the diameter of the trees, due to the competition between neighbouring trees (Gspaltl et al., 2013). However, this did not occur in the densities of the plantations studied, where the diameters at the base of the trees in the diff erent spacings showed no signifi cant diff erences (Table 1). This lack of diff erence is probably because in D. panamensis, with high density and slow growth rates), the levels of competition among 3-year-old trees is still low as to produce diff erences in the diameter of the trees (Redondo-Brenes, 2007). At early ages, as in D. panamensis in this study, crown intertwining among trees (the visible evidence that competition begins) barely started, therefore competition was low (Li et al., 2017).
However, although no diff erences in the diameters were observed (Table 1), tree height showed diff erences in the spacing 1.0x0.5 m. Physiologically, the tree will focus its development mainly on stability and subsistence in the fi rst years of age, in order to survive (Li et al., 2013), so that at early ages growth will be greater in height (Table 1) in those spacings that produce greater competition, such as 1.0x0.5 m.
Another signifi cant aspect in the fi rst 3 years of development of the plantation of D. panamensis under the SRWC system, is the high mortality occurring in the 3 spacings studied, from 49.00 to 52.67% (Table 1), which is common in D. panamensis plantations for wood production (Piotto et al., 2003). Therefore, the high mortality obtained in D. panamensis SRC was infl uenced by lack of weed management during the fi rst year of establishment, which in these biomass production systems is highly important (Dickmann, 2006).
The variation of the biomass values (1.11 to 42.36 t.ha -1 ) in the 3 spacings studied (Table 2) is lower than found by Tenorio et al. (2016aTenorio et al. ( , 2018 in SRWC of 3-year-old Gmelina arborea in Costa Rica, Populus sp SRWC in Chile (Carmona et al., 2015). These diff erences appear mostly because D. panamensis is a slow growing species (Piotto et al., 2003). However, despite its low biomass production, D. panamensis exhibits high SG in planted trees (Tenorio et al., 2016b), thus concentrating the biomass in less volume, which is important during processing, densifi cation and biomass transportation (Dickmann, 2006). Added to this, the potential of this species for energy production or biomass thermochemical processes has been demonstrated (Gaitán-Alvarez et al., 2017).
Despite the high leaf percentage (Table 2, Figure 2) and its physiological importance (Pleguezuelo et al., 2015), the stem sections (bark and branches) concentrate a high percentage of the total biomass, with diff erences occurring between spacings (Table 2, Figure 2). In general, in the SRWC systems, a high percentage of the biomass is expected to be concentrated in the stem and branches (Dickmann, 2006). An increase in biomass production in the stem reduces maintenance costs, since the canopy closes faster, preventing weed growth in the plantation (Hauk et al., 2014). Therefore, the spacings with the highest percentage of biomass in the tree stem and branches are the most appropriate. The spacing 1.0x2.0 m, with the highest percentage of leaf production (Figure 2), is inconvenient for biomass production, while the spacings 1.0x1.0 m and 1.0x0.5 m are more appropriate since higher proportion of the biomass is concentrated in the stem and lower percentage in the leaves (Figure 2).

Chemical, physical and energy characteristics of the biomass
The little variation in the chemical properties in the diff erent spacings (Table 2) is explained by the fact that these characteristics are infl uenced by variations between sites, topography and climatic conditions (Stolarski et al., 2017). As the present trial locates in one site, with the same topography and soil conditions, few signifi cant diff erences are to be expected between the spacings ( Table 2). The contents of cellulose and lignin (Table 2) were lower than those reported by Gaitan-Alvarez et al., (2017) for biomass from trees growing in natural forests or older plantations, which is explained by the age of the trees, since it is expected that the chemical composition increased with the age (Gaitán-Alvarez et al., 2017). The genetic variation was not possible because it was planted same genetic material.
Lower density in green condition occurs with the greater spacing (Figure 3a and 3b), as could be observed in the present study. This can be explained by the fact that trees in wider spacings have less competition, therefore develop anatomical structures with greater cell wall or greater pore frequency or pore diameter (Aloni, 2001). This results in a lower moisture percentage ( Figure  3a, 3c). In contrast, high density in green condition is related to the water present in the wood, which has larger spaces to contain water (Aloni, 2001), resulting in higher MC, as occurs in spacings 1.0x2 .0 m and 1.0x1.0 m (Figure 3c).
On the other hand, the GCV agrees with values reported in literature on fast growing plantations of around 5 years of age of Eucalyptus (Gominho et al., 2012) and G. arborea (Tenorio and Moya, 2013). In relation to the high GCV obtained in spacing at 1.0x1.0 m (Figure 3d, Table 2), it appears to be infl uenced by a low ash percentage ( Figure 3e) and higher volatile content (Figure 2f).
The ash content is a parameter of high interest in biomass. High ash content refl ects more residual material that cannot be burned during burning (Nielsen et al., 2009). Thus, the high value of ashes obtained in spacing 1.0x2.0 m is a negative factor compared to the other spacings studied (1.0x1.0 and 1.0x0.5 m), where less ash content was found (Figure 3e). This means that this spacing (1.0x2.0 m) would produce high ash content. Added to this negative factor, spacing 1.0x2.0 m presented higher MC (Figure 3c Finally, according to the above mentioned biomass characteristics of Dipterx panamensis in a SRWC system for biomass production, it is a species that shows optimal physical, chemical and energy properties to be used for the production of biomass for energy purposes, since they are comparable to other tropical species used as biomass (Gaitán-Alvarez et al., 2017;Moya et al., 2019). However, the properties evaluated present some diff erences by spacing. There is higher production of stems as well as biomass production per hectare in spacings 1.0x1.0 and 1.0x0.5 m (Figure 2.3). Still, these production levels must be evaluated in other aspects, such as the economic aspect, since these values of biomass production per hectare are below the average found in other tropical species in SRWC systems (Eloy et al., 2016). Nevertheless, unlike the previous species, the biomass of D. panamensis has lower MC.

5.CONCLUSION
The characteristics presented previously of the biomass of Dipterx panamensis in a SRWC system for biomass production, determine that it is a species that has optimal physical, chemical and energy properties to be used for biomass production for energy purposes compared to other species used for the same purpose. The biomass extracted from trees growing in the short rotation crop system shows high value of specifi c gravity, low MC, accessible percentages of ash and volatile contents, as well as optimal values of GCV, comparable with other forest species used for biomass production. We observed higher biomass production in the stem and branches, in the spacings 1.0x1.0 and 1.0x0.5 m, at the age of 3 years. Although the biomass production per year is lower than that reported in other tropical timber species, the spacings at 1.0x1.0 m and 1.0x0.5 m show annual biomass production from 12.09 to 28.40 t.ha -1 , which is a good result if compared with species in other regions of the world. Concluding that Dipteryx panamensis plantations at spacing of 1.0 x 1.0 and 1.0 x 0.5 m works well for biomass production.


s2fieldsofstudy:
Agricultural And Food Sciences
Biology

text:
Isolation and genotyping of Mycobacterium bovis in suggestive lesions of tuberculosis in cattle slaughtered in the state of Ceará, Brazil1

RESUMO.[Isolamento e tipificação genética de Mycobacterium bovis em lesões sugestivas de tuberculose em bovinos abatidos no estado do Ceará.] A tuberculose bovina (TB) é uma zoonose causada pelo Mycobacterium bovis, o qual induz ao desenvolvimento de lesões nodulares e granulomatosas em vários tecidos do animal. O reconhecimento dessas lesões macroscópicas sugestivas durante a inspeção sanitária post mortem em matadouros fornece um diagnóstico presuntivo, sendo necessário a utilização de testes complementares para confirmação da doença. O objetivo deste trabalho foi verificar ABSTRACT.Ferreira F.F., Oliveira M.L.M., Alzamora Filho F., Santos B.R., Araújo L.C.S.S.C., Santos E.S.V., Gomes H.M. & Costa J.N. 2020. Isolation and genotyping of Mycobacterium bovis in suggestive lesions of tuberculosis in cattle slaughtered in the state of Ceará, Brazil. Pesquisa Veterinária Brasileira 40(11):863-870. Agência de Defesa Agropecuária do Estado do Ceará, Avenida Dr. José Martins Rodrigues 150, Edson Queiroz, Fortaleza, CE 60811-520, Brazil. E-mail: felipefrancelinovet@gmail.com Bovine tuberculosis (BTB) is a zoonosis caused by the bacterium Mycobacterium bovis, which induces the development of nodular and granulomatous lesions in various animal tissues. The recognition of these suggestive gross lesions during postmortem sanitary inspection in slaughterhouses provides a presumptive diagnosis, which requires the use of complementary tests to confirm the disease. This study aimed to verify the occurrence of BTB in cattle slaughtered in slaughterhouses in the state of Ceará, Brazil, using bacteriological and molecular methods. To this end, suggestive lesions were analyzed on carcasses condemned by the “Serviço de Inspeção Estadual” (SIE). The samples were submitted to microbiological analysis using culture media and specific staining followed by spoligotyping molecular technique for identification and genotyping of the mycobacteria. Occurrence of lesions suggestive of BTB was verified in bovine carcasses (0.071%) from different municipalities of the state. These lesions were located mainly in the lung (95.12%), lymph nodes (58.53%), and liver (36.58%). Microbiological culture showed bacterial isolation (17.94%), with the growth of colonies showing morphological and tannic characteristics belonging to genus Mycobacterium spp. Genetic polymorphism analysis identified M. bovis in all isolates, which were discriminated into six spoligotypes (SB0121, SB0295, SB1064, SB0120, SB0870, and SB0852). These profiles have been described in Brazil and several areas of the world, except for profiles SB1064 and SB0852, which were described in the country for the first time. The results show that the association of the diagnostic methods used was the basis for the first study on identification of mycobacteria found in the state, which may provide a database for the epidemiological study of BTB in the state of Ceará.

INTRODUCTION
Bovine tuberculosis (BTB) is an infectious and contagious disease of progressive character and chronic evolution caused mainly by the bacterium Mycobacterium bovis. It is characterized by development of nodular and granulomatous lesions in different organs, mainly in the lung, since the main form of transmission occurs through inhalation of aerosols (Paes & Franco 2016). BT is of great importance in public and animal health due to its zoonotic potential, determining a situation of high morbidity and mortality in humans (Pérez-Lago et al. 2014). In addition, it causes marked economic losses in cattle herds as a result of animal death, reduced milk production and weight gain, reproductive changes, animal disposal, and carcass condemnation (Pacheco et al. 2009, Paes & Franco 2016. Brazil has the largest commercial cattle herd globally, with approximately 214.89 million head (IBGE 2017). Consequently, animal products destined for the domestic and foreign markets must present high quality and low health risk.
onducting epidemiological surveys in slaughterhouses is an indicator of BT prevalence in cattle destined for slaughter and, therefore, of the probability of human exposure through infected meat consumption (Biffa et al. 2010). In the state of Ceará, surveillance for BT in slaughterhouses has not been reported. Only one epidemiological survey on BT has been identified in the state of Ceará. This survey was carried out in rural settlements in the municipality of Quixerambim, in which 248 cattle were submitted to tuberculization and no positive animals were identified. However, it cannot be said that there is no tuberculosis in cattle in the surveyed region, as other factors may have contributed to test negativity. These factors include the voluntary adherence of the participating owners and the drought that spread in the region, which may have contributed to the death of debilitated and sick animals, limiting the spread of the disease (Nascimento 2014).
It is known that results obtained from surveillance in slaughterhouses can indicate BT health status in the herd. However, due to the similarity between the disease's lesions and other nodular and granulomatous inflammatory processes, it is often impossible to identify all sick animals (Roxo 1997, Mendes et al. 2013. In general, recognition of gross lesions associated with BT during routine health inspection in slaughterhouses has been the only measure used to diagnose the disease, which may incur errors in its detection, especially in paucibacillary lesions. It is recommended that complementary diagnostic tests be used in these lesions to minimize these misunderstandings (Furlanetto et al. 2012).
Microbiological culture of the etiologic agent is the certified method for direct diagnosis. However, it presents low sensitivity and prolonged time between primary isolation and final identification of the species (Corner 1994). In contrast, an alternative that enables rapid identification of mycobacteria is the application molecular techniques, which can distinguish between different strains, allowing increased sensitivity and specificity and a decrease in the number of diagnoses of this infection (Ramos et al. 2015). Some studies have demonstrated the possibility of using polymerase chain reaction (PCR) and spoligotyping techniques to detect and type M. bovis in fragments of lesions collected in slaughterhouses (Rodriguez et al. 2004, Costa et al. 2012, Parreiras et al. 2012, Alzamora Filho et al. 2014b.
This study aimed to analyze the occurrence of BTB in the state of Ceará through the isolation, identification, and genetic characterization of M. bovis from gross lesions suggestive of tuberculosis in cattle slaughtered in slaughterhouses under the "Serviço de Inspeção Estadual".

MATERIALS AND METHODS
Between August 2017 and September 2018, slaughter and postmortem inspection of cattle were monitored by the "Serviço de Inspeção Estadual" (SIE) of the state of Ceará in three slaughterhouses located in the municipalities of Iguatu and Juazeiro do Norte and of Maracanaú in the metropolitan region of Fortaleza, south and center-south of the state. Data on the animals' origin were obtained from the "Guia de Trânsito Animal" (GTA). An additional assessment of cattle traceability was carried out by analyzing the health records of producers and rural properties obtained from the "Agência de Defesa Agropecuária do Estado do Ceará", Ceará state (ADAGRI 2019).
According to methods recommended by Ministério da Agricultura, Pecuária e Abastecimento (MAPA) through the "Regulamento da Inspeção Industrial e Sanitária de Produtos de Origem Animal" -RIISPOA (Brasil 2017), samples of lesions suggestive of BTB were collected by veterinary medical inspectors during routine health inspection of beef. Data on the characteristics and location of these lesions were recorded on slaughter follow-up forms.
For bacteriological exams, the collected samples were placed in sterile universal type collectors, identified, and stored at -20°C or in saturated sodium borate decahydrate solution (Na2B4O7 • 10H2O, 140g/L), which preserves mycobacteria for up to 60 days at room temperature (Richards & Wright 1983). Subsequently, the samples were sent to the "Laboratório de Micobacterioses" of the "Universidade Estadual de Santa Cruz" (LAMVET-UESC), Ihéus, state of Bahia, for processing and bacteriological analysis. Initially, the tissue fragments were decontaminated using 1.5% 1-hexadecylpyridinium chloride (HPC) for 30 min. Then they were inoculated in Stonebrink-Leslie and Lowenstein-Jensen culture media and incubated at 37 °C for up to 90 days. After the cultivation period, the isolated colonies were stained using the Ziehl-Neelsen (ZN) method for staining characterization of alcohol-acid resistant bacilli (AARB) (Rodriguez 2005, Brasil 2008. From the isolates characterized as AARB, a bacterial suspension was performed in 200μL of Tris-EDTA (TE) buffer solution (10mM Tris-HCl; 1mM EDTA; pH 8.0) to inactivate the bacterium, and later DNA extraction was obtained by thermal lysis through incubation at 90 °C for 30 min, followed by centrifugation (Shi et al. 2018). The resulting DNA was taken to the "Laboratório de Biologia Molecular" applied to mycobacteria of the "Fundação Oswaldo Cruz" (FIOCRUZ -Rio de Janeiro), where molecular analyses were carried out.
Identification of Mycobacterium bovis was confirmed through molecular typing, and analysis of genetic polymorphism was performed using the Spoligotyping technique, as described by Kamerbeek et al. (1997). Direct region amplification was performed in a final volume of 50 μL with 0.2 mM containing dNTP (10mM Tris-HCl, pH 8.0; 50mM KCl), 1.5mM MgCl2, 20 pmol of primers for each primer (biotinylated DRa and DRb), and 1U of Taq DNA polymerase and 10ng of DNA. The polymerase chain reaction (PCR) consisted of an initial denaturation at 96 °C for 3 min., 20 cycles of amplification at 96 °C for 1 min., 55 °C for 1 min, and 72 °C for 30 s, followed by a final extension at 72 °C for 5 min. M. bovis BCG DNA was used as a positive control, whereas ultrapure water was the negative control. The PCR products were membrane hybridized containing 43 oligonucleotides of known spacer sequences. After incubation with streptavidinperoxidase, the spacers were detected by chemiluminescence. The polymorphic patterns obtained in hybridization were compared with an international database for M. bovis available in the literature (Kremer et al. 1999, Van Embden et al. 2000, Haddad et al. 2001). The nomenclature adopted for the spoligotypes obtained was the same as that available in the Sitvit Web database (Machado et al. 2014) and on the website 8 .

RESULTS
In the study period, 57,347 cattle were evaluated during the inspection routine for health on ante-mortem examination, but did not undergo tuberculosis screening tests. Of these, 41 animals (0.071%) presented lesions suggestive of tuberculosis at postmortem inspection and had their carcasses condemned in whole or in part by the SIE inspectors. Considering the gross findings of the official service, the percentage of condemnation (slaughtered cattle condemned because of lesions suggestive of BTB) was 0.32% (24/7374), 0.06% (15/25694), and 0.01% (2/24279) in the three slaughterhouses under the SIE of the state of Ceará (Table 1). The analysis of the mandatory animal health documents (GTA) showed that the sampled cattle belonged to herds from 13 municipalities located in different regions of the state of Ceará: Fortaleza, Caucaia, Cascavel, Ocara, Tauá, Saboeiro, Quixelô, Jucás, Iguatu, Icó, Cedro, Cariús, and Crato, with a positive diagnosis of BTB 8 Available at <www.mbovis.org> confirmed in three of these municipalities (Jucás, Iguatu, and Quixelô), as shown in Figure 1.
The condemned animals presented firm, nodular lesions, focal to multifocal, with a purulent or caseous aspect, presence of fibrous capsule, in some cases showing areas of calcification in the center of the lesion, evidenced by knife creaking at cut. These lesions, present in one or more tissues of the same animal, were located mainly in the lungs (95.12%) and lymph nodes (58.53%), as well as on the head, neck, thoracic lymph nodes, and liver (36.58%). In addition, two cattle were identified with suggestive nodules in the spleen and kidney, each associated with liver and lung injuries (Table 2). At the gross examination  2.43 *N = 41 bovines; in total, 80 fragments of lesions located in different organs were collected, and one or more lesions suggestive of BTB were collected from the same animal (sample). of the lung lesions, the initially noticeable nodules on the lobes surface were deepened by cutting the parenchyma. Bovine carcasses that showed lesions not suggestive of BTB, considered only as common or nonspecific lymphadenitis by the SIE evaluation, did not have their samples collected.
Of the samples of presumptive BTB lesions collected by the official service, two were discarded due to tissue autolysis, and 39 were processed and cultured. The bacteriological analysis revealed bacterial isolation in 17.94% (7/39) of the samples, with growth of small, rounded, cream-yellowish colonies with irregular edges and granular surface in the Stonebrink-Leslie culture medium, with average observed time for the appearance of colonies of 28 days-minimum of 16 days and maximum of 32 days. Of the seven positive samples in the Stonebrink-Leslie medium, growth of colonies was observed in three samples in the Lowenstein-Jensen culture medium, with an average time observed for the appearance of colonies of 45 days (minimum 32 days and maximum 64 days). All smears from the 10 isolated colonies presented alcohol-acid resistant bacilli (AARB + ) when subjected to Ziehl-Neelsen (ZN) staining.
Considering the stratification by sex and age group of cattle with positive bacteriological diagnosis for Mycobacterium spp., it was found that 85.71% (6/7) of the animals were female, and only 14.29% (1/7) were male. The age group most affected was over 36 months, with 83.33% (5/6) of positive females having this age. Of the positive animals, only one young female (16.67%), aged 13-24 months, was affected by the disease. The municipality of Iguatu stood out as the most affected by BTB, representing the origin of 71.42% (5/7) of the positive cattle diagnosed in the study, and the positive animals were all from different herds and properties (Table 3).
In the genetic material of the isolates submitted to the spoligotyping technique, six spoligotypes were identified as Mycobacterium bovis. The studied profiles showed deletions in spacers 3, 9, 16, and 39-43, characterizing the pathogen. Of the seven samples, three showed two different spoligotypes each, which justifies seven positive animals and 10 isolates. Six spoligotypes previously identified in the databases were observed in the 10 AARB isolates. Three of them formed clusters of genotypes and three had a single profile. Three AARB isolates had spoligotype profile SB0121, two presented SB0295, two showed SB1064, and the single profiles SB0120, SB0852, and SB0870 were found in one isolate (Table 4).

DISCUSSION
This study found that, according to the SIE, based on the occurrence of gross lesions suggestive of BTB (0.071%), this disease is present in the cattle herd of the state of Ceará. Condemnation of cattle carcasses with suspicion of BTB were previously reported by Baptista et al. (2004), who found a prevalence of 0.07% of suggestive lesions in animals slaughtered in Minas Gerais. There is a wide variation in BTB prevalence from postmortem inspection of cattle in Brazil (0.17 to 5.16%). This difference may be related to the degree of regional development, especially concerning sanitary measures in the herds (Araújo 2004).
In this study, the carcass condemnation rate (CCR) per establishment ranged from 0.01 to 0.32%. These data are probably related to the slaughtering particularities in each establishment, such as type of exploitation, density, and health practices. In the slaughterhouse with the lowest CCR (SIE -A), in addition to having a higher slaughtering volume, most of the animals were originated from extensively reared beef herds, a category in which BTB presents sporadic occurrence  ■■□■■■■■□■■■■■■□■■□■■■■■■■■■■■■■■■■■■■□□□□□ 1 Iguatu * International name given by Mbovis.org. and SitVitWeb, ** Black and white boxes indicate the presence and absence, respectively, of the specific spacer in position 1-43 in the direct repeat (DR) locus. (Rodriguez 2005). In contrast, in the establishment with the highest CCR (SIE -B), this value is possibly related to the increased slaughter of animals discarded from dairy or mixed herds concentrated in the region, cases in which the probability of BTB occurrence increases (Costa et al. 2012). Moreover, detection of lesions suggestive of BTB depends on the time available to scrutinize the carcass. A short time may be insufficient to identify discreet lesions, and this identification may also be hindered by lack of inspection experience. In this way, the slaughter inspection accuracy probably varies between regions and slaughterhouses (Perrez et al. 2002, Alzamora Filho et al. 2014a).
In the present study, it was observed that the organ most affected by lesions suggestive of BTB was the lung, followed by the lymph node chain, and the liver, which is in agreement with results obtained in other studies (Alzamora Filho et al. 2014a. The more significant occurrence of lung lesions may be directly related to inhalation of contaminated aerosols, which are eliminated through the cough or nasal discharge of infected animals, occurring mainly in dairy herds as a result of high population density and confinement (Paes & Franco 2016). The predominant lesions in this study are in line with the description of focal, multifocal, or diffuse nodular lesions in affected organs (Costa et al. 2013), with a high occurrence of caseous and calcified lung injuries ). In the generalized form of BTB, dissemination of the bacillus reaches several organs such as the spleen, udders, and kidneys , corroborating the gross findings of detected renal and splenic lesions.
Regarding the bacteriological analysis, considering the characteristics of the colonies described by Corner (1994), colonies compatible with Mycobacterium bovis were isolated in only seven samples, which reflected a sensitivity of 17.94% for this method. Due to the characteristics of the lesions obtained in slaughterhouses, many isolations of the pathogen were expected. As verified by Fráguas et al. (2008), in the present study, the low sensitivity of the bacteriological culture may be associated with the decontamination techniques used in the processing of samples. These techniques may have contributed to death of the bacilli and the consequent low index of mycobacterial isolation, especially in paucibacillary samples. Low rates of mycobacterial isolation have been reported in previous studies addressing lesions suggestive of BTB (Pinto et al. 2002, Araújo et al. 2005. It can also be explained by the occurrence of nodular and granulomatous inflammatory processes determined by other etiologies, with specific characteristics, and undifferentiated gross features of BTB, such as lymphosarcoma, actinomycosis, actinobacillosis, and nocardiosis (Roxo 1997, Mendes et al. 2013, Paes & Franco 2016. The absence or deficiency of viable pathogens in suggestive gross lesions may also be associated with the difficulty of the pathogen to multiply in culture, because the lesions present a bacillary concentration below 10,000 CFU/mL (Zanini et al. 2001), and the death of mycobacteria after promoting the lesion by defending the organism from the chronic nature of the disease (Pinto et al. 2002, Fráguas et al. 2008. A reduced number of M. bovis isolates have also been observed in studies that bacteriologically evaluated both lesions suggestive of BTB and nonspecific lymphadenitis during postmortem examination (Furlanetto et al. 2012, Alzamora Filho et al. 2014a. Considered the gold standard for BTB diagnosis, higher isolation rates are expected in bacteriological culture, with mycobacterial growth rates that can vary from 60.0 (Nassar et al. 2007) to 68.75% (Silva et al. 2018) of the suggestive lesions. In contrast, despite the low frequency of positive samples verified in the present study, ZN staining revealed AARB + in 100% of the isolates, confirming the high identification specificity of the mycobacterial method (Corner 1994).
In Brazil, BTB is endemic and affects many dairy properties, since animals are subjected to confinement and high metabolic demand (Brasil 2006). The occurrence of lesions suggestive of BTB had a confirmatory diagnosis for M. bovis in municipalities in the center-south region of Ceará (Fig.1B), a critical dairy basin in the state, with emphasis on the municipality of Iguatu, which is among the 10 municipalities with highest production (IPECE 2018). Among the positive cattle, females aged >36 months were the most affected, suggesting that they were culling cows with advanced age for reproduction, probably from dairy or mixed herds located in the affected region. Similar to our results, Grisi Filho et al. (2011), when evaluating animals condemned for BTB according to the isolation of M. bovis in the state of São Paulo, reported greater positivity in females (58.89%). Other studies conducted with female bovines slaughtered at old age, which demonstrated a high occurrence of lesions suggestive of BTB followed by significant positivity rates in bacterial isolation, have also been found in the literature (Damina et al. 2011, Okeke et al. 2014. Those authors suggested that the findings may be related to the chronic character of the disease and the increased exposure of the animals to mycobacteria over time, considering that females have a longer productive life.
In association with classic bacteriological isolation, the use of complementary molecular methods to identify mycobacteria is more sensitive and efficient for a definitive diagnosis of BTB. Spoligotyping is a molecular technique that allows simultaneous detection and typification of the M. tuberculosis complex (Kamerbeek et al. 1997). It is referred as the technique of choice for comparing the strain with few copies of IS 6110. Another advantage is the differentiation between M. bovis and M. tuberculosis due to the absence and presence, respectively, of spacers 39 to 43 (Rodriguez et al. 2004). Research on spoligotypes was a vital tool to identify and typify the genotypes of M. bovis present in cattle herds in the state of Ceará. In the present study, six spoligotypes were identified: SB0121, SB0295, SB1064, SB0120, SB0852, and SB0870 with deletions in spacers 3, 9, 16, and 39-43, which is characteristic of M. bovis strains (Kamerbeek et al. 1997, Haddad et al. 2004. Spoligotype SB0121 was the most frequent in the present study (30%), with a cluster in three isolates registered in the municipality of Iguatu. This predominant genetic profile is in line with the results obtained in other studies in Brazil (Parreira et al. 2012, Alzamora Filho et al. 2014b, Ramos et al. 2014, Cazola et al. 2015, Carvalho et al. 2016). In addition to being described as the most frequent spoligotype in Brazil, Mexico (Zumárraga et al. 2013), and Portugal (Ghavidel et al. 2018), it has also been reported in countries such as Argentina, Belgium, Germany, Spain, France, United Kingdom, Italy, Sweden, Tunisia, Uruguay, the United States, and South Africa, as verified in the Sitvit Web database (Machado et al. 2014).
The profiles SB0120, SB0870, and SB0852 were isolated, each in different samples from the municipality of Iguatu. Spoligotype SB0120 was the most common circulating type on several continents (Ghavidel et al. 2018). In Brazil, it was identified in the states of Paraíba and the Federal District (Parreiras et al. 2012), Bahia (Costa et al. 2012), and São Paulo , having importance in other regions of the world. This spoligotype is widespread in several European countries, being mostly described in France with a prevalence of 26% of the isolates (Haddad et al. 2001). However, it has also been found in Africa and Asia (Sitvit Web). In addition, cases of human tuberculosis caused by M. bovis (spoligotype SB0120) have been reported in the United Kingdom (Gibson et al. 2004), indicating the zoonotic potential of this profile. Spoligotype SB0870, in turn, was reported in a single isolate identified in a sample obtained in the state of São Paulo (Rocha 2013), as well as in some European countries such as Germany, Denmark, and France (Sitvit Web). Spoligotype SB0852 has been reported only in France and the United States (Sitvit Web).
With the molecular discrimination of spoligotypes SB1064 and SB0852, the present study seems to have generated unprecedented results in identifying the polymorphism of genetically typified M. bovis isolates in Brazil. This study seems to be the first report of these spoligotype profiles in the country.
The identification of M. bovis through bacteriological and molecular methods confirmed the existence of BTB focus properties in the state of Ceará. The tracking of the bovines' origin with confirmatory diagnosis, associated with the evaluation of the transport history of the focus properties, allowed us to identify that the animals of these herds were entered or discharged from properties from 17 different municipalities in the state of Ceará. Bovines with lesions suggestive of BTB originating from six of these municipalities were identified (Fig.1A).
The focus properties showed several cattle, between 52 and 457 animals, with characteristics of mixed or dairy herds. In general, these properties acquired cattle from other rural properties through intra and/or inter-municipal transport, and were characterized by intense movement of animals due to the accentuated inter-municipal cattle trade. In this internal traffic, it is not mandatory to carry out screening exams for GTA issuance. In addition, there was interstate transport of cattle, including the egress of animals to the states of Paraíba and Bahia, and admission from the state of São Paulo -this official transport with exams and GTA issuance. However, there may be unrecorded irregular transport of animals without compliance with sanitary requirements. Previous studies pointed out BTB prevalence or occurrence with a confirmatory diagnosis of M. bovis in these states , Alzamora Filho et al. 2014a, Ramos et al. 2018. This suggests a possible route for the movement of infected animals between the state of Ceará and these geographic regions, especially with the states of Bahia (Costa et al. 2012, Alzamora Filho et al. 2014a) and São Paulo , where the presence of M. bovis strains similar to those identified in this work were previously identified (SB0120, SB 0121, and SB0295).
The transit of cattle with BTB between properties, regions, or countries represents a potentially critical factor for the transmission of this disease (Skuce et al. 2012), which can be confirmed by the genetic diversity of M. bovis isolates from bovine carcasses in the state of Ceará. The risk factors frequently identified and described for the disease include free movement of possibly infected animals, occurrence of the disease on the property and in neighboring areas, number of animals, type of exploration, and use of the intensive system, in which herds are kept together for long periods, and entry of animals from herds with a history of BTB (Skuce et al. 2012, Bahiense et al. 2016, Barbieri et al. 2016. Additionally, the history of cattle transported to local livestock events, such as livestock shows and events (vaquejadas), was also verified in focus properties of the present study, which are characterized as important areas of risk for BTB due to the agglomeration of animals of different origins (Brasil 2006).

CONCLUSIONS
The association of post mortem BTB diagnosis techniques with molecular genotyping techniques were the basis of the first study to identify mycobacteria in the state of Ceará, and this work was the starting point for the creation of a database of great relevance to the epidemiological study of this disease in the state.
In addition, among the genetically typified Mycobacterium bovis isolates, two spoligotypes that had not yet been reported in Brazil were identified (SB1064 and SB0852). Therefore, this study is the first report on these profiles in the country.