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Chemical contaminants migration from food contact materials into aqueous extracts

. Cardboard packaging is widely used in the food industry, especially as secondary or tertiary packaging, or as transport packaging. In most cases, these packs may contain certain chemical contaminants that can be accidentally transferred to packaged foods. These contaminants include Bisphenol A (BPA) and Formaldehyde, which can cause significant adverse effects, especially to vulnerable persons. For this reason, the purpose of this study was to analyze by a UV - VIS spectrophotometric method the content of BPA and Formaldehyde from 17 corrugated board samples from Romanian producers. BPA concentrations varied between 0.044 mg/dm 2 and 0.090 mg/dm 2 , while Formaldehyde concentrations were lower than 0.048 mg/dm 2 . The results were compared with the legislation in force to establish their compliance for contact with food.

Introduction
Nowadays, there has been an increase in the production of packaging materials for all industries. This is due to the fact that each product is sold in a package. Packaging has a vital importance especially in the food industry where they are used as food contact materials (FCM), whose role is to preserve, to protect, to promote the food products throughout the food chain [1].
According to EU Regulation no. 1935/2004 [2], FCM must not release chemicals that may endanger consumers health, affect product sensory or nutritional properties, or alter food composition [3].
These chemicals, called food contact chemicals (FCC), are chemical constituents from food contact materials that are added to these packaging, and which, under certain conditions, can migrate into the packaged food product [4].
The main types of FCM used in the food industry are represented by plastics, glass, paper and cardboard, metallic materials [5]. These products must be safe for food contact. Their compliance with regulations in force, depending on the type of material is necessary to be tested [1].
The widespread use of paper and cardboard packaging, as primary, secondary or tertiary packaging, is due to the low price, reduced weight, availability on the packaging market, ease of storage, but also good mechanical properties [5].
In connection with substances that can migrate from this type of packaging, their number is quite large. Formaldehyde and Bisphenol A (BPA) are two chemical compounds ( Figure 1) which can cause adverse effects on the consumers health. Most of the chemical contaminants that can migrate, including formaldehyde and BPA, come either from the printing inks, or from the adhesives used in the process of obtaining the packaging [1]. These compounds can migrate from the packaging into the product under certain conditions, being influenced by direct or indirect contact with the food, their chemical properties, their initial concentration, contact time and temperature [1].
The aim of this study was to assess the possible contamination with BPA and Formaldehyde of several types of cardboard from different Romanian producers, in order to evaluate the compliance with the legislation in force.

Sample description
A total of 17 samples of corrugated cardboard (CB) were used in this study. These samples, presented in Table 1 were collected from different Romanians producers.

Equipment
BPA and Formaldehyde analysis was performed by UV/VIS molecular spectroscopy, using a Jasco v550 UV/VIS Spectrophotometer, provided with a deuterium lamp and a wavelength range between 190 nm and 900 nm. A GFL type water bath (Burgwedel, Germany) for preparing the solutions and an OHAUS analytical balance for weighing the samples were also used.

Preparation of water extract
Sampling, preparation and extraction of samples were performed according to SR EN 645: 1997 standard [6]. For the preparation of the extract, 10 grams (± 0.1 g) of each sample were used. Samples were cut into small pieces, were immersed in 200 mL water, and after 24 hours at 23°C (± 2 °C), the extract is filtered, brought to a 250 mL volumetric flask and then analyzed.

Preparation of standard solutions
For BPA calibration curve, 0.1 g of BPA was weighted in a volumetric flask and then filled up to 100 ml with ethanol. The solution was diluted 100 times with ultrapure water for obtaining solution B. The standard solutions were prepared, from solution B, according to Table 2. Calibration curve for formaldehyde was performed according to SR EN 1541: 2003 [7]: 5 mL of formaldehyde solution are diluted up to 1000 mL in a volumetric flask (Solution I) with ultrapure water. Then, another 20 times dilution is performed for obtaining Solution II, which, finally is diluted 100 times to obtain Solution III. 1 mL of the final solution contains approximately 0.001 mg of formaldehyde. The calibration solutions were prepared, from solution III, as presented in Table 3.

Calibration
Calibration curve for each element was performed using reagents described above. The calibration curve for BPA and Formaldehyde reveal a good linearity over the whole range of concentrations. Also, for both chemical contaminants a recovery of over 95% and a regression coefficient higher than 0.995 was obtained. The limits of detection and quantification for the analyzed contaminants were 0.01 mg/L, respectively 0.03 mg/L for BPA and 0.004 mg/L and 0.012 mg/L for Formaldehyde.

Sample preparation
10 ml of extract were acidified with HNO3 65% and boiled for 25 minutes. After cooling, 1 ml of KOH 50% was added in order to complete reaction. If BPA is present the solution is turning yellow. The solution is then measured at 430 nm. The control solution was prepared in the same way, except for the 10 ml which were of ultrapure water. Formaldehyde reacts with 2,4-Pentandione, in the presence of ammonium acetate, forming 3,5-diacetyl-1,4-dihydrolutidine. The extract is then measured at 410 nm wavelength. Preparation of calibration solutions and samples was performed according to SR EN 1541:2003 [7]. (1).

C = (Cs-Cc) x 1000/V0
(1) C is BPA concentration (in mg/L), Cs is the BPA concentration of the sample (mg/10 ml extract), Cc is the BPA concentration of the control (mg/10 ml standard solution), V0 is the volume of extract used (10 mL).
Formaldehyde concentration calculation was performed using Equation (2).
Cf is Formaldehyde concentration (in mg/dm2), C is formaldehyde concentration from calibration curve (in mg), V0 is the total volume of the extract (250 mL), V1 is the volume taken for testing (25 mL), b is sample weight (in g/m2) and G is the mass of the sample (in grams).

Results and discussion
BPA analysis method is based on the reaction between BPA and HNO3 with the formation of aromatic compounds (ortho-phenols) of yellow color. Analyzing the results presented in Figure 2, the BPA concentrations varied between 0.044 mg/dm2 and 0.090 mg/dm2. According to CEPI Guide, 2012 [8], the maximum allowed limit for BPA in paper and cardboard intended for food contact is 0.1 mg/dm2. All values obtained are below the limit.
Regarding formaldehyde, according to the EC Resolution ResAP version 3/2007 [9], the maximum allowed limit is 2.5 mg/dm2. As can be observed analyzing the results in Table  4, all values are much lower than the limit.
The highest values for both BPA and Formaldehyde were found in sample 13, which is a 3-layer corrugated board. However, the values obtained are higher than in the case of samples 10 and 17 which are cardboard samples consisting of 5 layers. This may be due to the raw material (paper) from which the layers were obtained.

Conclusions
In this article a simple and rapid method was used for BPA and Formaldehyde analysis in corrugated cardboard used as FCM.
All the results obtained are below the maximum allowed limits, for both BPA and Formaldehyde. Regarding the BPA content, the concentrations found were higher than those in the literature [10,11]. The differences may occur due to the analytical methods used.
In the case of formaldehyde, the values obtained are comparable with some results from the literature [12]. One reason could be that the UV-VIS method is the standardized method for determining the formaldehyde in paper and board used for contact with food. The


extfieldsofstudy: Medicine Mathematics

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

text:
Nutrient requirements and evaluation of equations to predict chemical body composition of dairy crossbred steers

Objective Objectives were to estimate energy and protein requirements of dairy crossbred steers, as well as to evaluate equations previously described in the literature (HH46 and CS16) to predict the carcass and empty body chemical composition of crossbred dairy cattle. Methods Thirty-three Holstein×Zebu steers, aged 19±1 months old, with an initial shrunk body weight (BW) of 324±7.7 kg, were randomly divided into three groups: reference group (n = 5), maintenance level (1.17% BW; n = 4), and the remaining 24 steers were randomly allocated to 1 of 4 treatments. Treatments were: intake restricted to 85% of ad libitum feed intake for either 0, 28, 42, or 84 d of an 84-d finishing period. Results The net energy and the metabolizable protein requirements for maintenance were 0.083 Mcal/EBW0.75/d and 4.40 g/EBW0.75, respectively. The net energy (NEG) and protein (NPG) requirements for growth can be estimated with the following equations: NEG (Mcal/kg EBG) = 0.2973(±0.1212) ×EBW0.4336(±0.1002) and NPG (g/d) = 183.6(±22.5333)×EBG−2.0693(±4.7254)×RE, where EBW, empty BW; EBG, empty body gain; and RE, retained energy. Crude protein (CP) and ether extract (EE) chemical contents in carcass, and all the chemical components in the empty body were precisely and accurately estimated by CS16 equations. However, water content in carcass was better predicted by HH46 equation. Conclusion The equations proposed in this study can be used for estimating the energy and protein requirements of crossbred dairy steers. The CS16 equations were the best estimator for CP and EE chemical contents in carcass, and all chemical components in the empty body of crossbred dairy steers, whereas water in carcass was better estimated using the HH46 equations.

and Title of the manuscript: Nutrient requirements and evaluation of equ 24 composition of dairy crossbred steers. 25

ABSTRACT 27
Objective: Objectives were to estimate energy and protein requireme 28 well as to evaluate equations previously described in the literature (H 29 carcass and empty body chemical composition of crossbred dairy catt 30 be explained nature of equations shortly or spell out because these appe 31 Methods: Thirty-three Holstein×Zebu steers, aged 19±1 months old, w 32 (BW) of 324±7.7 kg, were randomly divided into three groups: refere 33 level (1.17% BW; n = 4), and the remaining 24 steers were randomly 34 Treatments were: intake restricted to 85% of ad libitum feed intake for 35 d finishing period.  G, empty body gain; and RE, retained energy. Crude protein (CP) and ether extract (EE) chemical tents in carcass, and all the chemical components in the empty body were precisely and accurately imated by CS16 equations. However, water content in carcass was better predicted by HH46 equation.

nclusion:
The equations proposed in this study can be used for estimating the energy and protein uirements of crossbred dairy steers. The CS16 equations were the best estimator for CP and EE mical contents in carcass, and all chemical components in the empty body of crossbred dairy steers, ereas water in carcass was better estimated using the HH46 equations.

INTRODUCTION
It is recognized that dairy (Holstein and Holstein crossbreeds) and beef cattle exhibit differences regarding their nutritional requirements [1,2]. However, despite the large participation of male dairy cattle in feedlots in US and Brazil [3][4][5], most of nutritionists in US (92.2%) and Brazil (71.7%) use some version beef cattle NRC as source of information on nutrient requirements to formulate diets [3,6]. Therefore, the establishment of proper nutritional requirements for male dairy cattle and its crossbreeds is necessary and consequently ensures their subsequent productive performance and farmers profitability.
In this regard, quantifying body composition is a major obstacle when estimating nutrient requirements. The direct determination of body composition, which is based on the separation and dissection of all body constituents, and analysis of its individual constituents, is the most accurate method available, to generate reliable data. However, this process is lengthy, laborious, and expensive. Due to these challenges, indirect methods have been developed for estimating body composition [7][8][9].
Among the indirect methods available, the method that predicts body composition from equations based on the 9th to 11th rib section composition, which was first proposed by Hankins and Howe (HH46) [7], is wide spread due the ease of use, reduced cost involved, and speed of the process. However, Paulino et al [10] and Marcondes et al [11] unanimously concluded that the equations developed by HH46 [7] were not fully applicable to predict body composition of Zebu cattle. Prediction equations to estimate the growth pattern and composition of gain of Holstein×Zebu crossbreds have not been reported. In this respect, Costa e Silva et al (CS16) [2] developed equations for estimating body composition of crossbred dairy cattle. Nonetheless, they still need to be evaluated with an independent database in order to be widely and reliably adopted. Therefore, testing those equations for crossbred dairy animals should provide useful information about their utility for such animals. Thus, the objectives were to estimate energy and protein requirements of dairy crossbred steers, as well as to evaluate the goodness of fit of prediction equations developed by HH46 [7] and CS16 [12] to predict the carcass and empty body chemical composition of crossbred dairy cattle.

MATERIALS AND METHODS
The animal care and use committee of the Universidade Federal de Viçosa approved all procedures involving animals (protocol number 12/2016).
The steers were housed in 6 m 2 individual pens with a concrete floor, which were equipped with individual feeders and continuous flow concrete drinkers. The experimental diet was the same for all treatments. The diet was calculated according to BR CORTE recommendations [2] to provide approximately 11% crude protein (CP) on a DM basis and to support an average daily gain (ADG) of 1.4 kg. The diet contained (DM basis) 40% corn silage and 60% of a concentrate mix ( Table 1).
The feed was provided once a day (0800 h) as total mixed ration, and the steers had free access to clean water. The diet for R0 steers was adjusted daily to provide orts of approximately 2% (as fed basis) of the total offered, and orts were mixed with the new feed that was offered in the next day. Briefly, bunks were evaluated at 0700 h daily to quantify refusals. According to the amount of refusals, the total mixed ration was reduced (more than 2% orts at morning evaluation) or increased (less than 2% orts at morning evaluation) to reach ad libitum intake. Silage was sampled daily and stored at -20°C until further analysis. Silage samples were combined weekly (percent as-fed basis), dried in a forced-air oven (55°C) for 72 h, and ground through a 1-mm screen (Fortinox, Piracicaba, SP, Brazil). The individual ingredients used to prepare the concentrate were sampled directly from the feed mill silos on the days the concentrate was mixed. These ingredients were analyzed individually and used to calculate the diet composition.

Digestibility trials, slaughter and sampling procedures
To evaluate apparent total-tract digestibility and consequently the total digestible nutrients (TDN), two digestibility trials were performed, from d 25 to 27 and d 73 to 75, by collect- ing the total feces excreted over three consecutive days [13]. In brief, the feces were collected using dustpans and then stored in plastic buckets at room temperature. At the end of each collection day, feces were quantified and mixed. Subsamples of approximately 300 g of feces (on an as-is basis) were saved each day. The fecal samples were dried in a forcedair oven (55°C) for 72 h, and ground through a 1-mm screen (Fortinox, Brazil). Furthermore, each period, an individual composite fecal sample from the 3 d of collection was prepared for each animal based on the DM content of the feces collected on the individual days. The final composite samples represented the 3-d collection of feces on a proportional DM basis. All animals were slaughtered after 84 d. Steers were fasted for 14 h before slaughter. The slaughter was performed by stunning using the captive bolt technique followed by jugular vein exsanguination. The digestive tract of each steer was emptied and washed, and each organ was weighed separately. The weight of the heart, lungs, liver, spleen, kidneys, internal fat (fat surrounding kidneys, pelvis, and heart), diaphragm, mesentery, tail, trachea, esophagus, and reproductive tract along the weight of the washed gastrointestinal tract were added to that of the other body parts (head, hide, limbs, and blood) to determine the non-carcass components weight. The non-carcass weight was added to that of the carcass to determine the empty body weight (EBW). Blood samples were collected immediately after slaughter. Samples of blood, organs and viscera, head and limbs, and hide were placed in aluminum trays and freeze-dried (Liotop, LP 510 model, São Carlos, SP, Brazil).
The carcass of each steer was split in to 2 halves along the spine, and then cooled at -4°C for 24 h. After 24 h, the carcass halves were weighed. The 9th to 11th rib section was obtained from the left half, as recommended by HH46 [7], and subsequently dissected into bone, fat and muscle tissues. The rest of the left carcass was completely dissected into bone, muscle, and fat tissues, and the 9th to 11th rib section was summed, to obtain the full carcass composition. Dissected samples were weighed, individually ground, packed in aluminum trays, and freeze-dried (Liotop, LP 510 model, Brazil).
All lyophilized samples from the steer's body were ground in a Wiley mill (Fortinox, Brazil) using liquid N. Four subsamples per steer, named "carcass", "non-carcass", "muscle plus fat-rib section", and "bones-rib section" were taken for further determination of the chemical composition. The carcass sample was comprised of the lyophilized samples of bone and muscle plus fat from the carcass. The non-carcass sample was comprised of blood, head and limbs, organs and viscera, and hide. The muscle plus fat-rib section was comprised of muscle and fat from the 9th to 11th rib section, and the bones-rib section contained the bone sample from the 9th to 11th rib section.

Laboratory analysis and calculations
Individual feed ingredients and feces were analyzed for DM, organic matter (OM), CP, and ether extract (EE) according to the AOAC [14,15]. The quantification of neutral detergent fiber (NDF) was performed without the addition of sodium sulfite, but with the addition of thermostable α-amylase to neutral detergent (Ankom Tech. Corp., Fairport, NY, USA). The NDF concentrations were corrected for ash [16] and residual N compounds [17]. The non-fiber carbohydrates were calculated as proposed by Detmann and Valadares Filho [18]. The TDN of the diet was estimated by the sum of digestible nutrients as proposed by NRC [19].
Digestible energy (DE) intake was obtained by multiplying digestible nutrients by their respective energy values [19]. The metabolizable energy intake (MEI) was estimated as 82% DE [20]. The metabolizable protein intake (MPI) was obtained by adding the true digestible microbial CP (TDM-CP) and the digestible RUP (DRUP) intake. The microbial CP (MCP) was calculated as proposed by Valadares Filho et al [2]. In addition, we assumed that the MCP is composed of 80% of AA and that these AA have an 80% digestibility [19]. The DRUP intake was estimated based on the difference between CP intake and MCP and multiplied by its intestinal digestibility (80%) [19].
Carcass, non-carcass, muscle plus fat-rib section, and bones-rib section samples were analyzed for DM [14], and CP and EE content [21]. The relationship between the shrunk BW (SBW) and EBW was calculated to convert SBW to EBW, while the relationship between the ADG and empty body gain (EBG) was calculated to convert ADG to EBG.
Whole-body energy content (EC) of each steer was obtained from the whole-body content for CP and fat by using their caloric equivalent [22]. The whole-body retained energy (RE) was calculated as the difference between the final and initial EC of each steer. Heat production (HP) was estimated as the difference between MEI and EC. Then, the net energy requirement for maintenance (NE M ) was estimated to be the intercept (β 0 ) of the exponential non-linear regression between HP and MEI [4]: where HP (Mcal/EBW 0.75 /d), MEI (Mcal/EBW 0.75 /d), β0 and β1 = regression parameters; and e = the Euler number (2.718281). Also, the ME requirement for maintenance (ME M , Mcal/EBW 0.75 /d) was estimated by the iterative method, with ME M considered to be the value where MEI equals HP [2]. The efficiency of utilization of ME M (k M ) was calculated as the ratio between the NE M and ME M [2].
The EC as a function of EBW of the animals, was estimated according to the exponential model [22]: The net energy requirement for gain (NE G ) was obtained by the derivative of the equation above [22]: where NE G (Mcal/kg of EBG), EBW (kg). The efficiency of utilization of ME G (k G ) was estimated to be the slope (β1) [2].
The metabolizable protein requirement for maintenance (MP M ) was estimated to be the intercept (β0) of the linear regression between MPI and EBG divided by average metabolic EBW (EBW 0.75 ) [2], as follows: where MPI = metabolizable protein intake (g/d), EBG (kg/d), and β0 and β1 = regression parameters.
To calculate the net protein requirement (NP G ), we adjusted the model: where NP G = net protein requirement for gain (g/d), EBG = empty body gain (kg/d), RE = retained energy (Mcal/d), and β1 and β2 = regression parameters. The efficiency of metabolizable protein utilization for gain (k PG ) was estimated to be the slope (β1) of the equation: where RP = whole-body retained protein (g/EBW 0.75 /d), MPI = metabolizable protein intake (g/EBW 0.75 /d), and β0 and β1 = regression parameters.

Evaluated equations
The descriptive statistics of the variables used to estimate the carcass and empty body chemical composition are presented in Table 2. The equations developed CS16 [12] were used to estimate carcass and empty body chemical composition using the 9th to 11th rib section ( Table 3). The equations developed   HH46 [7] were used to estimate carcass chemical composition using the 9th to 11th rib section only. It is important to highlight that the present data was independent of those used to develop the predictive equations from HH46 [7] and CS16 [12].

Statistical analysis
The relationship between the SBW and EBW and the ADG and EBG, and data on energy and protein requirements were analyzed as linear and nonlinear models built through the REG and NLIN procedures of SAS (version 9.4, SAS Inst. Inc.; Cary, NC, USA), respectively, in which the nonlinear model was adjusted with the Gauss-Newton method. The body components estimated by HH46 [7] and CS16 [12] equations were compared with the observed values using the following regression model: y = β 0 +β 1 ×x, where x = predicted values; y = observed values; β 0 and β 1 = intercept and slope, respectively. The regression was evaluated according to the following statistical hypothesis: H 0 : β 0 = 0 and H 0 : β 1 = 1, and H a : not H 0 . If the null hypotheses were not rejected, it could be concluded that the equations accurately and precisely estimate the body components of Holstein×Zebu steers. Slope and intercept were separately evaluated to observe possible errors in the equations.
Estimates were also evaluated using the mean square error of prediction (MSEP) and its components [23]: where x are predicted values; y are observed values; MSEP is the mean squared error of prediction; SB is the squared bias; MoF is the component relative to the model of random fluctuation; MaF is the component relative to the magnitude of random fluctuation; s x and s y are the standard deviations of the predicted and observed values, respectively, and r is the Pearson linear correlation coefficient between the predicted and observed values. The prediction of efficiency was determined by estimating the correlation and concordance coefficient or reproducibility index (CCC) as described by Tedeschi [24]. Values of CCC below 0.4, between 0.4 and 0.7, and above 0.7 were considered low, medium, and high, respectively. All calculations were obtained using the Model Evaluation System (MES) [24]. For comparisons, the level of 0.05 was established as the critical level of probability for type I error.

Energy and protein requirements
The average ratio between EBW and SBW and EBG and ADG observed in this experiment were 0.88 and 0.93, respectively (R 2 of 0.98 and 0.97, respectively). The relationship between HP and MEI was described by the equation:  The k G was 0.35, and this value was obtained based on the slope of the linear relationship between RE and MEI.

RESULTS AND DISCUSSION
The MP M was 4.40 g/EBW 0.75 , and this value was obtained based on a linear regression between MPI and EBG divided by the average metabolic EBW. The NP G were estimated from the model: The average ratio between EBW and SBW and EBG and ADG obse 253 and 0.93, respectively (R² of 0.98 and 0.97, respectively). The male dairy cattle (Holstein and Holstein crossbreeds) play im 273 . The k PG was 0.39, and this value was obtained based on the slope of the linear regression between RP and MPI.
The male dairy cattle (Holstein and Holstein crossbreeds) play important participation in feedlots in Brazil and the USA [3][4][5]. However, although it is acknowledged that dairy and beef cattle exhibit differences regarding their nutritional requirements [1,2], most of nutritionists use some version beef cattle NRC as source of information on nutrient requirements to formulate diets [3,6]. In this context, we compared energy and protein requirements estimated by equations proposed in this study with the values estimated by the last update of beef cattle NRC (NASEM) [1], which have maintained the same recommendations of the previous version [20] for energy and protein requirements.
Considering a steer with 416 kg of SBW, EBW of 365, shrunk body gain of 1.38 kg/d, EBG of 1.29 kg/d (the average of the steers in this study), and equivalent EBW (EQEBW) of 355 kg (EQEBW was calculated considering the BW at maturity of 532 kg and standard reference weight of 517 [2] [1] were approximately 17 and 59 g/d below the value calculated by equations developed in the current study. The most substantial differences between the requirements estimated by the equations proposed in this study and NASEM [1] are regarding NE G and NP G . The composition of EBG is the main determinant for estimating the nutrient requirements for BW gain, and it depends on the physiological maturity of the cattle, which is affected by sex and breed [25]. Differently from our database, the equations recommended by NASEM [1] are derived from studies based on body composition of Bos taurus beef purebreds and their crossbreds. Differences in maturity degree of the different genetic groups reflect different body composition in animals with the same BW [2]. Thus, for animals with the same BW and weight gain rate, which is the case of the example above, higher energetic and lower protein concentrations are expected in the gain, and consequently greater energy and lower protein requirements for gain in animals from genetic groups with lower weight at maturity (medium-frame steers; i.e. beef steers), compared with genetic groups with later maturity (large-frame steers; i.e. dairy steers) [2]. Therefore, the differences observed between the nutrient requirements obtained in the current study and those values estimated by NASEM [1] might be due cattle genetics. Nevertheless, it is important to highlight that the dietary management applied to animals in the current study may also have influenced the nutrient requirement estimations.

Carcass chemical composition
The HH46 [7] equation did not correctly estimate carcass chemical content of CP and EE (Table 4) because it rejected the null hypothesis of a respective intercept and slope equal to 0 and 1 (p<0.05). Comparing the maximum values estimated with the observed values, HH46 [7] equations overestimated CP and EE by 8.60% and 16.6%, respectively. Good precision and accuracy were observed when estimating water carcass content with the HH46 [7] equation, as CCC was close to 1; and it did not reject the null hypothesis of a respective intercept and slope equals to 0 and 1 (p>0.05). However, CS16 [12] equations had problems of adjustment for carcass water content according to the slope of regression analysis (p<0.05), indicating poor accuracy.
The CS16 [12] equations accurately and precisely estimated CP and EE in carcasses, as the null hypothesis of a respective intercept and slope equal to 0 and 1 (p>0.05) were not rejected; and CCC was close to 1 (CCC of 0.91 and 0.91 for CP and EE, respectively). The accuracy of prediction equations tends to decrease when they are used to estimate carcass composition of animals from genetic groups different from the ones used to generate the equation in the first place [26,27]. The HH46 [7] equations were developed based on carcass chemical composition of Bos taurus beef purebreds and their crossbreds. At the same BW, beef carcasses (Angus) have greater soft tissue to bone ratio, and proportionally more fat than dairy carcasses (Holstein) [28]. These differences are likely because genetic selection in dairy cattle has been focused on milk production rather than tissues deposition. Therefore, the presence of the Holstein breed in the genotype of the animals of the current study, may have resulted in a different growth and conformation pattern when compared to breeds selected for beef production and their crossbreds. Similar to our results, Nour and Thonney [29] pointed out that differences among breed types must be considered for greater accuracy and precision of prediction when using HH46 [7] equations to predict carcass composition of Angus and Holstein steers. Nevertheless, it is important to highlight that the dietary management applied to animals in the current study may also have influenced the body composition obtained. The CS16 [12] equations were developed using a database composed by animals Holstein×Zebu, which probably resulted in the best goodness of fit for CP and EE content in carcass among the equations evaluated. Therefore, CS16 [12] equations are more appropriate to predict CP and EE, whereas HH46 [7] equations better explained the variation in carcass water content of Holstein×Zebu steers.

Empty body chemical composition
The CS16 [12] equations did not reject the null hypothesis of a respective intercept and slope equal of 0 and 1 (p>0.05), and presented high R 2 (R 2 of 0.97, 0.96, and 0.99 for CP, EE, and water, respectively) and CCC (CCC of 0.85, 0.96, and 0.98 for CP, EE, and water, respectively) values for all chemical contents evaluated (Table 4).
Moreover, the prediction errors for EE and water empty body contents were mostly (more than 75% of MSEP) associated with random errors, rather than central tendency, and due to linear regression or systematic bias. However, most of prediction error for CP in the empty body (more than 80% of MSEP) was associated with the SB.
The CS16 [12] equations are part of the suggested equations in the 3rd edition of Nutrient Requirements of Zebu and Crossbred Cattle [2] for estimating body composition of Zebu and crossbred cattle. The CS16 [12] equations were developed after authors verified that equations generated based on a database composed by Zebu cattle (mainly Nellore) and their crosses with Bos taurus beef breeds (Angus or Simmental) [30] did not correctly estimate body composition of crossbred dairy cattle. However, until the present study, CS16 [12] equations had not been evaluated with an independent database.
In the current study, CS16 [12] equations accurately estimated all chemical components of empty body. However, reduced goodness of fit was observed when predicting CP content. The CS16 [12] developed equations using body composition data from crossbred dairy cattle, which were composed by 80 bulls, 56 steers, and 44 heifers. The greater proportion of bulls in the database of CS16 [12] could explain the reduced goodness of fit in predicting empty body CP in the current study, as bulls usually have greater CP content and accretion rates than steers [25]. However, when predicting empty body CP sex was not shown to be significant [12].
In Moreover, our results suggested that there may be differences between the NE G and NP G of beef and dairy crossbred steers. Thus, the equations described above may be used to calculate the nutrient requirements of Holstein×Zebu steers instead of using recommendations based on values from beef cattle.
The CP and EE chemical contents in carcass, and all chemical components in the empty body were precisely and accurately estimated by CS16 [12] equations. However, water content in carcass was better predicted by HH46 [7] equation. Therefore, the equations proposed by CS16 [12] were the best estimator for CP and EE in carcass of crossbred dairy steers, whereas water in carcass is better estimated using equations proposed by HH46 [7]. The equations proposed by CS16 [12] can be used to estimate all chemical components in the empty body.

CONFLICT OF INTEREST
We certify that there is no conflict of interest with any financial organization regarding the material discussed in the manuscript.


text:
Performance of immunohistochemistry versus real-time PCR method for detecting mycobacterial infections of cattle  screened by comparative tuberculin test

In addition to the fifty years since the test-and-slaughter program began in Iran and despite a significant reduction in the disease prevalence, positive tuberculosis cases are still being isolated from livestock farms across the country. Tests with 100% sensitivity and specificity are essential features for bovine tuberculosis diagnosis. The relationship between real-time PCR and immunohistochemistry (IHC) as two essential laboratory methods in the diagnosis of bacterial infections were aimed to evaluate single intradermal comparative tuberculin test (SICTT) results. One hundred thirty-eight cows in two groups were examined: Reactors (108 cows) and clean (as a control group; 30 cows). In the reactor group, 58(54.00%) cows were Mycobacterium bovis positive, 46(43.00%) were Mycobacterium avium subsp. paratuberculosis (MAP) positive, and 11(10.00%) were Mycobacterium tuberculosis positive. 32(55.00%) cows were co-infected with M. bovis and MAP and 5(4.55%) cows were co-infected with Mycobacterium tuberculosis and MAP in this group. Of 50 M. bovis negative cows of reactor group were 14(28.00%) MAP positive and 36(72.00%) negative, as well. Concurrent infection with all was observed in one reactor case. Comparing IHC and real-time PCR for the detection of bovine tuberculosis and Johne’s disease showed very good agreement (Kappa values 0.81 - 1.00). The results also provided further confirmation on IHC and real-time PCR as a sensitive and reliable diagnostic screening approach for detection of bovine tuberculosis. The use of one laboratory method to detect bovine tuberculosis is not sufficient alone.

Introduction
Mycobacterium tuberculosis complex (MTC) causes tuberculosis in humans and animals, one of the top 10 causes of death in the world, and millions of people continue to fall sick each year. According to the World health organization (WHO) in 2017, Globally, 10.00 million people spread the disease and it caused 1.30 million deaths (16.00%). 1 The acquired immunodeficiency syndrome (AIDS) co-infection, the failure of Another significant mycobacterial disease, namely paratuberculosis or Johne's disease (JD), is a chronic and progressive granulomatous enteritis of ruminants caused by Mycobacterium avium subsp. Paratuberculosis (MAP) and a disease affecting multiple species with socioeconomic and/or public health importance. It may be associated with the etiology of Crohn's disease in humans. 1 Although paratuberculosis available vaccines show a reduction in disease incidence, they are unable to prevent bacterial shedding or subsequent new cases in the herd. Unfortunately, these vaccines can interfere with the results of the tuberculin test. 3,4 A comparison between tuberculin test methods has shown that the use of comparative intradermal test method vs single intradermal test, decrease the percentage of cross-reaction from 6.55% to 0.15%. 5 Besides, in affected animals, the incubation period is 2 to 5 years (but up to 10 years), and the subclinical form of MAP infection usually appears several years before the clinical form (diarrhea, reduced milk production, cachexia and severe emaciation) leading to death since no cure is available. 6 In recent years, the tuberculin skin test was the only tool for detecting mycobacterial infections especially in latent cases of infection. The tuberculin skin test is an intradermal injection of purified protein derivative (PPD) that is a complex of several antigens including antigens shared with non-tuberculous mycobacteria. The PPD is injected below the epidermal layer that causes the inflammatory reaction (a delayed-type hypersensitivity reaction) and results in the characteristic indurated area at the injection site. The tuberculin skin test has limitations including technical problems in administration, boosting of the immune response in the test reputation, false positive and false negative results, complicated, subjective interpretation and requirement in the second visit. Other methods, such as acid-fast staining and isolation of Mycobacterium spp. in culture, have many limitations. The acid-fast staining detection limit is more than 10 4 bacilli per slide. Mycobacterial culture requires several weeks, and in a few bacilli, its sensitivity is low. 7 In Iran, the testand-slaughter program has been launched following the world plan since 1967. 8 Unfortunately, in addition to the fifty years since the program began, and despite a significant reduction in the disease prevalence, 8 still positive cases of tuberculosis are being isolated from livestock farms across the country. The success of the test-and-slaughter program for bovine tuberculosis control is directly dependent on the diagnostic ability of the laboratory tests. 9 Adopting tests with 100% sensitivity and specificity, high operating speed and low cost are essential features for a bovine tuberculosis diagnosis. 10 The use of real-time polymerase chain reaction (PCR) and immunohistochemistry (IHC) methods has been considered by researchers in the last decades. Direct realtime PCR has some advantages over the detection of mycobacterial species in animal tissue samples, which takes a few hours and highly analytical specificity. However, cross-reaction with unwanted microorganisms (contamination) is one of its main limitations. 11 The IHC is a powerful technique for detection and scoring of cellular macromolecules along with the microscopic observation of lesions in fresh or formalin-fixed tissue samples. Especially in the formalin-fixed form, it is a safe method to study many high-risk pathogens like mycobacteria in the laboratory. Human error, damage to some epitopes because of over-fixation, and difficulty in quantifying results are the most considerable limitations of IHC. 12 The relationship between real-time PCR and IHC as two essential laboratory methods in diagnosis of bacterial infections were aimed to evaluate single intradermal comparative tuberculin test (SICTT) results and to evaluate the SICTT sensitivity and specificity in Khorasan-Razavi province, Iran.

Materials and Methods
Samples. The minimum sample size (N) needed in this study was calculated using the following formula with a 95.00% confidence limit, 22.00% estimated prevalence (P), and 5.00% Tolerable error: 13 N= 1.96 2 P (1-P) / 0.05 2 Thirty healthy and one hundred eight positive reactor cattle screened by SICTT for tuberculosis were included in this study. After inspection of carcasses, samples of ileum and bronchial, mediastinal and mesenteric lymph nodes were taken. After dividing the samples into two parts, the first part of the specimen was sent to the molecular laboratory of the Khorasan-Razavi Veterinary Office, ice packed. The second part was placed in 10.00% neutral buffered formalin (NBF; Sigma-Aldrich, St. Louis, USA) and sent to the above histopathological laboratory.
Preparation of tissues for histopathological examinations. After fixation in NBF, the specimens were placed in suitably labeled cassettes to segregate them from other specimens. For dehydration, series of increasing alcohol (Sigma-Aldrich) concentrations were used to avoid excessive distortion of the tissue, and then cleared with xylene (Merck, Darmstadt, Germany), and infiltrated with paraffin wax (Sigma-Aldrich). The infiltrated tissues were then embedded into wax blocks. Cutting of paraffinembedded tissues was performed at 5.00 µm. Paraffin sections were properly mounted onto poly-L-lysine (Sigma-Aldrich) coated glass slides.
Immunohistochemistry. The tissue sections were deparaffinized and hydrated in water. One part trypsin concentrate (Abcam, Boston, USA) with one part buffer were mixed. Sections were incubated for 5 -10 min at 37.00 ˚C to retrieve mycobacterium antigens. Then, they were rinsed thoroughly in Tris buffered saline (TBS; Sigma-Aldrich) and proceeded with the immunostaining procedure. The slides were washed two times for 5 min in TBS plus 0.025% Triton X-100 (Sigma-Aldrich) and then blocked in 10.00% normal serum with 1.00% BSA (Sigma-Aldrich) in TBS for 2 hr at room temperature. The slides were drained for a few seconds and wiped around the sections with tissue paper. Then, the rabbit primary antibody (anti-M. bovis, Bioss Inc., Woburn, USA /anti-MT, Abcam /anti-MAP, Daco, Glostrup, Denmark) was diluted to the manufacturer's recommendations (1/200), and the sections were incubated overnight at 4.00 ˚C. After two times for 5 min TBS plus 0.025% Triton X-100, the slides were incubated in 0.30% H2O2 (Sigma-Aldrich) in TBS for 15 min. After two times washing in TBS buffer, for enzymatic detection, enzyme-conjugated secondary antibody goat anti-rabbit (HRP; Abcam) was diluted in TBS with 1.00% BSA (1/200), applied to slides and incubated for 1 hr at room temperature. The samples were rinsed with in buffer TBS plus 0.025% Triton X-100, three times for 10 min, and then 3, 3-diamino-benzidine (DAB; Sigma-Aldrich) chromogen was used for 10 min at room temperature. The DAB produced a brown precipitate (where the secondary HRP antibody binds to the primary) that was insoluble in alcohol, xylene and other organic solvents most commonly used in the laboratories. The slides were rinsed in running tap water for 10 min and stained with nuclear counterstained Mayer's hematoxylin (Sigma-Aldrich) for better visualization of the tissue morphology. Dehydration, clearance, and mounting using a compatible mounting medium were carried out. Positive and negative controls were used with all samples. The analysis was performed in a blinded manner.
DNA extraction. Fifty mg of each sample were placed into 2.00 mL Eppendorf microcentrifuge 5420 tubes (Eppendorf, Hamburg, Germany) and digested by adding 200 µL of tissue lysis buffer (4.00 M Urea, 200 mM Tris, 20.00 mM NaCl, 200 mM EDTA, pH 7.40 at 25.00 ˚C) containing 40.00 µL proteinase K (200 µg mL -1 ; Roche, Indianapolis, USA). Then each tube was incubated at 55.00 ˚C for 72 hr. Boiling (for 10 min) and immediately freezing (at -20.00 ˚C for 30 min) were performed three times. According to the manufacturer ' s instructions, the extraction process was carried out using a DNA extraction kit (High pure PCR template preparation kit; Roche). Eluted DNA was stored at -80.00 ˚C.
Real-time PCR. Real-time PCR was carried using a LightCycler ® 480 (Roche) and LightCycler ® 480 DNA SYBR Green I Master Mix (Roche). The oligonucleotides INS-1 and INS-2 were used to amplify a 245-bp fragment of the IS6110 sequence present in members of the MTC, Jb21 and Jb22 were used to amplify a 500-bp fragment from the RvD1Rv2031c sequence present in members of M. bovis. P90 and P91 were used to amplify a 400-bp fragment from the IS900 sequence present in members of MAP (Table  1). 10,[14][15][16] Each reaction mixture of 20.00 μL was containing 10.00 µL of SYBR Green I Master Mix (Roche), 5.00 mM MgCl2 (Roche), 2.00 µL of each primer, 5.00 µL of the template and molecular grade water up to 20.00 μL. The amplifications were carried out with the two following cycles: An initial step of 95.00 ˚C for 10 (Table 1). If samples' results were negative for a target, the simultaneous DNA process control needed to be positive to prove that there was material in the reaction and that the real-time PCR was not inhibited. Positive and negative controls were used to assess accuracy in all PCR run. Real-time PCR data were analyzed to determine whether a significant difference was existed between samples and controls and to calculate the double delta cycle threshold (Ct) analysis by Livak and Schmittgen. 17 Statistical analysis. The results of the real-time PCR and IHC assay were compared to those of SICTT using agreement and kappa tests. Analyses were done using SPSS Software (version 19.0; IBM, Armonk, USA).
Real-time PCR results. One hundred thirty-eight cows were examined in two groups: The reactor group (108 cows) and the clean group (30 cows; base of negative response to SICTT and the absence of clinical signs and no report of macroscopic lesions in the last two years as a control group). The results are shown in Table 2. In the reactor group, 58(54.00%) cows were M. bovis positive, 46(43.00%) cows MAP positive, and 11(10.00%) MTC positive (Fig. 1). 32(55.00%) cows were co-infected with M. bovis and MAP, and 5(4.55%) cows were co-infected with MTC and MAP in this group. Out of 50 M. bovis negative cows of reactor group, 14(28.00%) were MAP positive, and 36(72.00%) negative. Concurrent infection with all was observed in one reactor case. In the clean group, all samples were negative ( Table 2). The sensitivity, specificity, positive predictive value (PPV) and negative predictive value (NPV) of SICTT using real-time PCR as the gold standard for detection of M. bovis, MTC and MAP in tissue sections are shown in Table 3 Table 3. The agreement between the SICTT for M. bovis, MTC, MAP and IHC was fair (κ = 0.34, 95.00% CI, 0.242 to 0.498, p < 0.0005), very good (κ = 1.00, 95.00% CI, 1.158 to 0.842, p < 0.0005) and moderate (κ = 0.52, 95.00% CI, 0.872 to 1.088, p < 0.0005), respectively. No false-positive signals were founded (Fig. 2).

Discussion
Even though the test and slaughter programs based on SICTT and slaughter surveillance were adopted for eradication of M. bovis infection, the prevalence of infection has not substantially been diminished in several regions of Iran such as Khorasan-Razavi province. Slaughter surveillance for detection of mycobacterial infection requires infected cattle with visible macroscopic lesions, however, in many cases small lesions at early stages of infection are not visible at the abattoir among standard inspection in reactor animals. [19][20][21] In this study, 108 positive reactors were inspected and 26(24.00%) showed macroscopically visible lesions, 82(76.00%) no macroscopic lesion, and no lesion was seen in the clean group. These results were similar to other countries like UK and Ireland that 50.00 -80.00% of reactor animals had no visible lesions. [22][23][24][25][26] Pal et al. showed that the probability of missing bovine tuberculosis lesions during routine slaughterhouse inspection was 95.00%. 27 Earlier studies indicated these probabilities as 84.00%, 28 70.00%, 24 and 85.00%. 29 These results also revealed a much low sensitivity of meat inspection to detect the tuberculous lesion, and it was depended on the anatomical sites and method of examination. 27,[30][31][32] Factors including early infection, examination technique, location of the lesion, latent infection, and infection with other mycobacteria are the most important reasons. 33 In the present study, most lesions were in the lung (bronchial and mediastinal) lymph nodes, head and diaphragm, respectively. These results were similar to previous findings, which reported that many macroscopic visible lesions occurred in the respiratory system, 20,34-37 however, differed from that reported mesenteric lymph nodes by Ameni et al. 38 Generally, locations of tubercles depend on the route of infection, and transmission by ingestion is usually observed in calves and in the respiratory tract in cattle. 29 Out of 108 extracted DNA of reactor cows, 58(54.00%) were positive using the species-specific primers Jb21 and Jb22. Rodríguez et al. obtained 100% concordance between culture and PCR with primers JB21/JB22. 39 Meikle et al. reported that PCR was a more specific and sensitive method than culture. 40 Vitale et al., Tortoli et al. and Mustafa et al. proposed that PCR was the gold standard for identifying mycobacterium species. 7,41,42 In this study base on real-time PCR, 11(10.00%) reactor cows were MTC positive using primers INS1/INS2. Only 4 cases of 11 MTC positive cattle had a macroscopically visible lesion. It might be due to the transmission of MTC between farmers and cattle. In similar studies, Ameni et al. reported five out of the 16 isolates from farmers and cattle were members of the MTC. 38 Romero et al. reported MT infections in three cattle farms in Spain with humans as the source. 43 Ameni et al. at a preliminary study reported approximately 27.00% in grazing cattle in Ethiopia. 38 Thakur et al. in a study reported four cases of MT infections in India. 44 The results of this study and similar findings indicated the necessity of considering veterinary health measures in dairy farms to control bovine tuberculosis in humans.
The results of real-time PCR with primers P90/P91 showed that out of the total cows studied, 46(33.00%) were infected with MAP, which showed high contamination of Khorasan-Razavi province farms. Also, there were no macroscopic visible lesions in 28(60.00%) of these MAP positive cows that might be due to subclinical infection. Fathi et al. compared three methods in diagnosis of John's disease and concluded that molecular technique could be more valuable than culture. 45 In another study, 90 dairy cattle were tested by Johnin test and PCR. The results showed that the PCR technique was able to identify suspected cases. Out of 108 reactor cows, 32 cases of M. bovis and MAP co-infection, four cases of MT and MAP co-infection and one case of co-infection with all three mycobacteria were observed. Our results were similar to that of Kennedy et al. They reported that coinfection with M. bovis and MAP on a single animal induced cross-reactivity with PPD and then false responses to many types of diagnostic tests were observed. 46 Brahma et al. showed that M. bovis infection increased the animal susceptibility to MAP infection or vice versa, and coinfection was occurred with both agents. 2 Byrne et al. highlighted the co-infection of M. bovis and MAP infection as a potential factor in identifying and removing infected animals from endemic cattle farms. 26 Alvarez et al. described an increase in false-negative results in SICTT. 47 Amadori et al. showed when MAP was administered prior to M. bovis, animals did not react or poorly reacted to the skin test compared to M. bovis only. 48 The results of these researches showed the need for comprehensive and thorough research to identify the interactive effects of mycobacterial co-infection on routine diagnostic methods of tuberculosis.
In this study, the results of IHC confirmed real-time PCR findings in the reactor group and showed very good agreement (κ = 1.00, 95.00% CI, 1.135 to 0.865, p < 0.0005). In the clean group, 4(13.00%) MAP positive cows detected by IHC were negative in real-time PCR and SICTT. It might be due to the low concentration (under 8.00 pg µL -1 ) of DNA in samples that remained undetected by PCR or due to early infection with MAP. 2 In this group, real-time PCR and IHC showed very good agreement (κ = 0.94, 95.00% CI, 1.085 to 0.795, p < 0.0005). It showed the role of IHC in the diagnosis of MAP infection compared to the tuberculin test and real-time PCR. In several studies, the important role of IHC in the diagnosis of mycobacterial infections in formalin-fixed paraffin-embedded tissues was reported. 49 In another study, there was 100% agreement between the real-time PCR and IHC for detection of MTC and 94.00% for detection of MAP. 2 Our results were different from study of Mustafa et al. that reported an 87.00% agreement between IHC and PCR as the gold standard. 7 In Iran, in 1967, the test-and-slaughter program was instituted nationally based on the SICTT using both tuberculins provided by Razi Vaccine and Serum Research Institute (RVSRI), for intensive farms as well as their surrounding traditional farms. The operation of this program since 1981 has decreased the frequency of bovine tuberculosis in the cattle population of Iran from > 5.00% to < 0.14%. 8 In this study, the sensitivity and specificity of SICTT for M. bovis were calculated 53.70% and 100%, MTC 100% and 100%, and MAP 60.50% and 100%, respectively. These results were similar to Alvarez et al. study in Spain that the sensitivity and specificity of SICTT were highly variable (sensitivity v = 40.10 -92.20% and specificity > 99.00%). 47 Similarly, Schiller et al. in a review of many international studies, reported 55.10 -93.50% sensitivity and 88.80 -100% specificity for SICCT. 50 According to a study by O'Hagan et al., in the British Isles, the sensitivity of the SICCT test was 88.61%, and the median estimated for the specificity was 99.80% that was different from our results in the specificity. 25 In some circumstances, anergy and immunosuppression in the advanced stages of tuberculosis, could explain the decreased insensitivity. This low sensitivity is an essential issue for the success of the test-and-slaughter program that should be considered in the control program.
The success of the test-and-slaughter program for bovine tuberculosis control directly depends on the diagnostic ability of the laboratory tests. The SICTT is limited in its sensitivity and specificity especially in association with another Mycobacterium spp. In this study, one of the most important points was coinfection with two or more mycobacterium. This means that using one laboratory method to detect bovine tuberculosis alone is not sufficient, and it must use two or more laboratory methods. The results also provided further confirmation of IHC and real-time PCR as a sensitive and reliable diagnostic screening approach for the detection of bovine tuberculosis.


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Environmental Improvement of Saline and Alkali Soils in the Kamyshlovskaya Valley and Revival of Natural Grassland Efficiency

Agricultural use of saline soils is possible only through implementing reclamation measures on their desalinization. The removal of salt from soils is a cost-demanding and time-consuming process. Expenses related to the treatment of saline soils are connected with a desalinization method and include costs of water supply to a saline area and its drainage. A reasonable project cost, as well as efficient water consumption depends on the use of natural environment conditions. The reported paper presents the assessment of water recourses in the drainage area of the valley valley, it is focused on the restoration of water bodies in the Kamyshlovsky log, stabilization of their balance, and environmental improvement; the study suggests economic perspectives of saline and alkali soils. The latter is essential to revive the efficiency of natural grasslands in the valley and its use as a disposal of production and household waste water from surrounding rural settlements.

Introduction
Geographically, the Kamyshlovsky log stretches over the territory of two states (the Republic of Kazakhstan and Russia); it crosses the north-east of North Kazakhstan Region in Aiyrtau District, Esil District, Akkayin District, and Magzhan Zhumabaev District and passes into Omsk Region, extending over Isilkulsky District, Moskalensky District, Maryanovsky District, and Omsk District ( Figure 1). A territory represents dry lowland with numerous lakes in the valley of the Kanyshlovsky log. A total length of the valley from Bolshoy Tarangul Lake is around 380 km, its width ranges 1.5 to 20 km, and the valley cut into the present day topography is up to 15…17 m.
The surface of the valley is a wavy and poorly dissected lowland plain with numerous shallow holes filled with waterlakes [1]. The valley slopes gently to the north-east like the inclination of underlying rocks that used to be a bank of a fresh-water basin in the Tertiary.
Highest areas are located in the south-west of the valley along the boundary with Akmola Region, where the river Kamysakty has its source at elevations of 200 m and higher. Further north-east, absolute heights decrease to 112 m near Bulaevo, North-Kazakhstan and to 100 m near the urban-type settlement of Maryanovka, Omsk Region. To date, the economic use of lands in the Kamyshlovsky log is concentrated on its terraces and slopes and comprises individually used farmland for grain and feed crops or natural pastures grazed by domesticated livestock. The efficiency of agriculturally used lands on these areas is not similar. For instance, arable lands of high terraces differ from zones of the watershed and generally depend on moisture conditions of the areas. Along the Kamishlovsky log there is a strip of saline meadow soils, solod soils, and meadow alkaline soils. The productivity of agricultural fields on terraces creating a gentler slope is lower. It is related to the salinization of soils, which becomes more severe close to the river plain, at a height of 2…3 m from the water edge of lakes they are so poorly productiveeven in years with high rainfall amounts a maximum of 3-4 centner/ha (if recalculated to hay)that they are used for cattle grazing. As for the development of plant communities they have a low value (a content of uneatable species is up to 60%). The reason is salty soils; their percentage is as high as 0.6 to 1.3% in a 0…50 cm layer on sides. Besides it, vast areas on the valley bottom are confined to almost saline and alkaline soils. Typical alkali soils are found around salt lakes.
The efficient land use on this territory is possible provided that desalination actions of saline and alkaline soils are taken; in this case we may increase the productivity of natural grasslands on a total area of above 25 thousand hectares and produce hay of a high quality (15-20 centner/ha). The most optimal measure to reduce a mineralization level of soils is supposed to be the restoration of water bodies aimed at the stabilization of their water-salt balances. Undoubtedly, it will have a positive effect on the efficiency revival of natural meadows in the valley.

Hydrographic and hydrologic features of the Kamyshlovsky log
In hydrology, there are mostly bodies with standing water in the valley; a water system represents a chain of lakes with varying sizes and depths, as well as degrees of mineralization. Most lakes in the river basin are without outflow except for the river mouth region with the river Kamyshlovka, which enters the river Irtysh on the left bank at a distance of 1822 km from the river mouth. Water bodies of the rest part have mainly no outlets, only in high-water years some of them may be filled with melt water in spring and turn into running water habitats.
Areas of lake surfaces vary 0.1 to 40 km 2 (Lake Bolshoy Tarangul). A lot of lakes have low and gently sloping shores, and vast areas get overflooded under a high water level rise, some of them get connected with neighboring water bodies at a distance of several kilometers. As a spring flood is over, water levels of lakes fall because of evaporation, many of lakes dry out. Catchment areas of many lakes 3 are smallseveral tens of square kilometers, and the biggest part of their surfaces (50-60%) were ploughed up. Watersheds are not distinct, therefore, poorly recognizable on topographic maps.
Lakes in the valley are mainly salt lakes with a significant area varying 1300 m 2 to 4200 m 2 and a depth of 0.5…1.0 m. A value of total mineralization is in a range 93 to 15 g/l according to the outcomes of survey expeditions carried out by employees of Omsk State Agrarian University in different years. It is related to wide-range water level fluctuations in lakes; in particular, in shallow ones with a large surface area.
Besides salt lakes there are also small but deep (to 2…4 m) fresh-water lakes: Zheltoye, Pokrovka, Presnoye, Pitnoye, Zhelandy, Balykty, etc. A total mineralization level in these lakes varies 0.36 to 3.03 mg/l of chloride and sodium composition with a high concentration of Са and НСО3. It relates to the fact that these lakes are deeply cut into the topography and fed by surface streams and subsurface water.
A pattern of fresh-water and salt lakes on the territory is irregular; sometimes they are located close to each other. Fresh-water lakes, in contrast to salt-water lakes, are weeded by marginal aquatic plants, e.g. Phragmites.
For the water balance of a majority of lakes on the territory of interest a lacking outlet is typical, except for infrequent high-water years. The most significant water flowing out was recorded in 1865, when water reached the station of Polydino of the Omsk Railways. Thus, a principal input in the lake balance consists of snowmelt flowing from the catchment area and precipitation on the lake surface.
Rain water with a percentage of 80% in an annual precipitation sum for many years penetrates into soil and evaporates from it without creating a surface runoff because of hot summers and soil dryness. Only lakes with deep basins are fed with ground water.
A main output component of the lake water balance is evaporation on the water surface, which is constant during a year. Within a cold period evaporation on the snow surface is insignificant. Expeditions of State Hydrological Institute carried out in 1956 and 1957 [2] showed an evaporation discharge in the snowmelt time (April) is around 8-10 mm and 550-700 mm within a warm period, respectively.
Lakes are filled when the snowmelt period begins in the first half of April and lasts 10-12 days till the end of Aprilfirst decade of Mai. A water level rise in the period of seasonal flood is in a range of 0.3…0.4 m on average and 0.1…0.2 m in low-water years. After filling a high level of water remains in lakes during 2…4 and up to 15…20 days. In the middle of Mai a level of lake water begins to fall gradually and continuously; in summer and autumn it decreases by 0.3…0.5 m on average. The precipitation on the lake surface in a warm year period reduces considerably a water level falling-down rate. The lowest possible level of water in lakes is usually registered in late October. Every year at this time shallow lakes tend to dry out. A level of deeper lakes, which remain filled with water till the end of autumn, hardly changes during winter time. Water bodies with a depth below 1 m freeze solid to the bottom every year.

Restoration of Lakes
Lakes in the valley of Kamyshlovka form a complex hydrological system. In the 1920-s they used to be 1.5…2.0 m deep with a water mineralization value of 1.4…6.0 g/l, while nowadays most of them are shallow-water pounds with a depth of 0.4…0.7 m and in certain periods a water mineralization value ranges up to 50 g/l or even 70 g/l. Moreover, many of them dried out completely during drought years (1951…1953, 1974…1975, 1998…2000 and 2010) [3] leaving in the lakebed a 1.0…1.5 cm layer of salts being blown by the wind. Lakes Peschanoye and Severnoye, Maryanovsky District, Lake Kamyshlovoye, Moskalensky District (Elita), etc., tend to dry out every 4-6 years.
A hydrological analysis shows no changes in the total amount of moisture coming to the territory for many years. A water balance of lakes suffers mainly from man-made transformations in the water catchment structure provoked by economic use of lands and road construction of higher categories. Numerous shallow dams in ravines caused the shrinkage of natural water catchment areas, in several cases more than twice. In these conditions a poor natural runoff (7…10 mm) in the residual catchment areas is localized in depressions and fails in reaching lakes. In years with a less changed runoff a degree of water mineralization in lakes remains almost constant 5…15 g/l, in shallow and deep lakes (with a surface up to 20…40 ha) -1.2…2.0 g/l. Then, a changing hydrological regime led to a dramatically risen water mineralization value in lakes. For instance, a water mineralization level in Lake Solenoye was 11.46 g/l in August 1948 and 14.57 g/l in Lake Krivoye in the same period, whereas in 1953 these values increased to 19.82 g/l and 28.5 g/l, respectively.
The growth of water mineralization is hardly related to a substantial increase of salt resources. A principal factor responsible for mineralization increase in lakes is supposed to be the evaporation on water surfaces, which are too large in changed runoff conditions.
As known, in the Kamyshlovskaya valley there is no runoff into lakes during low-water years with a small amount of rainfall. Naturally, a water level in lakes falls, as well as their volume decreases, at the same time reducing water levels and volumes inevitably provoke the growth (approximately by two times) of water mineralization in lakes and water salinization in the floodplain for ground waters lying near lakes at a depth of 1.0…1.2 m make an important contribution to the evaporation and accumulation of salts in soil. As estimated, a share of salts in the surface of some areas ranges up to 6% and a level of mineralization is 60 g/l.
Therefore, a floodplain part of the valley may be used in economy provided that the desalinization of lands is carried out, and, more importantly, a hydrological regime of lakes is stabilized alongside with their demineralization [4].
Water demineralization in water bodies represents the most complex and challenging issue because it is necessary to drain salt water out of them and supply fresh water from other sources. In this case a reasonable use of fresh water resources from the local runoff makes a problem less difficult and furthers the efficient utilization of the surface runoff.
To assess a possibility to rely on a local runoff in the economic revival of the Kamyshlovka valley we estimated hydrological parameters of the runoff and water balance in sections formed when arranging the valley economy. For this purpose we selected 6 sections within the boundaries of Omsk Region, here, a zero point is coincident with the western boundary of Lake Plovinnoye near the settlement of Pervotarovka, Isilkulsky District. Other sections are determined according to inter-farm dams. The data obtained show insignificant amounts of an annual runoff (Table 1), even under assumption, a natural drainage area is taken into account when calculating a volume of inflow; in fact it is almost twice smaller than values presented (Figure 1).
To evaluate an alternative of stabilizing the water balance of lakes and demineralizing them, a prediction calculation for Lake Piketnoye is conducted (a factual drainage area FВ=56 km 2 and lake surface area fо=9.25 km 2 ). As an additional inflow element we considered purified production and household sewage, which also helps demineralize water in the lake. A volume of the lake with a depth of 0.6 m is determined to be WОЗ=3.7 mln.m 3 . For an average mineralization value of water 20 g/l resources of salts in the lake are SОЗ = 74•10 3 t.
As known, a seriously shrinking real drainage area because of a decreasing runoff has a consequence that significant lake surfaces dry out and a value of their mineralization rises. In such MPETAT 2020 IOP Conf. Series: Materials Science and Engineering 941 (2020) 012011 IOP Publishing doi:10.1088/1757-899X/941/1/012011 5 cases it makes sense to reduce a lakescape via deepening. Here, the deepening is planned to 3.0 m and in future the lake can be used for fish-breeding. A surface of the lake will get smaller to 1.23 km 2 .
We discussed all possibilities salts can be input into this water body: -an amount of salts from the drainage area for a layer Y = 7 mm and a value of mineralization qСТ = 0.25 kg/m 3 is determined SСТ = 95.85 t; -an annual amount of salts supplied with the precipitation for a layer КХ = 320 mm, qОС = 0.05 g/l -SОС = 19.63 t; -a salt input of Neogenic and Quaternary deposits for a module of subterranean runoff МНЧ = 0.08 l/s per km 2 or 2.52 mm of a runoff layer for a drainage area FНЧ = 54.77 km 2 , a value of mineralization qНЧ =1.5 g/l is as much as SНЧ = 207.03 t. -a salt input of Oligocene waters for a runoff module МОЛ = 0.01 l/s km 2 or 0.32 mm and a level of mineralization qОЛ =5 g/l -SОЛ = 87.63 t. In this case a total amount of salts is calculated: ∑ = ст + ос + НЧ + ол = 95.85 + 19.63 + 207.03 + 87.63 = 410.142 t.
Taking into account the evaporation on the lake surface 0.861•10 6 m 3 and a volume of water remained in the lake WОЗ =3.7•10 6 m 3 , a level of mineralization qОЗ = 20 g/l, an averaged value of mineralization will be q'ОЗ =19.37 g/l in autumn.
Furthermore, in the valley there is a flow of infrabed water in the alluvial apron with an average width 12 m and averaged filtration coefficient 1.5 m/d. In this case a volume of annual ground water input from this part is as high as ПР = 44.6 m 3 .
The cut of evaporation losses and involvement of the infrabed flow will support a positive balance of the lake, and a water mineralization value in the restored watercourse will be ОЗ ′′ = 18.644 / . In the practice of water use we know that amounts of water taken from water sources should be brought back after certain qualitative or quantitative transformations [5]. Unless water-draining systems are built, there is a problem of sewage recycling. In this case, sewage is stored in special underground tanksseptic system and discharged then. A problem of production and household sewage recycling is to be solved in settlements located along the Kamyshlovsky log, ground waters are frequently overfilled with drained water, what leads to overflooding. In these conditions a use of water bodies located in the valley as an intake structure of purified household sewage can make an additional contribution to the solution of a problem of overflooding. In its turn the discharge of biologically purified water [6] cuts a mineralization value of water bodies. On average, one lake can receive 800 m 3 per day or 292000 m 3 per annum sewage produced by one farm and one farm center; with this volume a value of lake water mineralization in the first year is 1г = 17.65 g/l.
Taking into consideration an average year with all its input and output components of water-salt balance, we can determine a mineralization value of water in the water body. As calculated, a further year reduces a mineralization value of lake water to q2Г = 15.36 g/l. Further estimations show that in five years a value of mineralization ranges to q5Г =12.23 g/l given that an optimal reorganization plan of water bodies is realized and additional water runoff is taken into account, in 27 years it stabilizes with a value of 4.5…5.5 g/l.

Conclusion
The biggest territory of the Kamyshlovsky log is covered with a chain of shallow lakes with no outlets and different values of mineralization; their water levels fall dramatically after seasonal floods and many of them even dry out. Water bodies with a high level of mineralization are the reason for the expansion of fruitless saline and alkaline soils, their salinity increases near the floodplain. The environmental improvement and revival of natural grasslands efficiency in the valley are possible if a soil mineralization parameter is reduced through the restoration of water bodies and stabilization of their water balance.
The desalinization of lakes via deepening and reducing their lakescapes, as well as supply of purified sewage from rural settlements is an effective step to improve the condition of soils, although it is a long-term process. A proposed model for Lake Piketnoye is similar to the drainage of 18 lakes located further upstream.


extfieldsofstudy: Medicine Biology

text:
Metabolic profile according to the parity and stage of lactation of high-performance Holstein-Friesian cows

Objective The aim of the study was to determine the effect of parity and the stage of lactation on the metabolic profile of cows based on the basic chemical milk components and the blood parameters. Methods The study material consisted of high-yielding Holstein-Friesian cows. In total, 473 cows were examined. According to the parity, cows were divided into four groups: primiparous (P), and multiparous in the second (M2), in the third (M3), and in subsequent lactations (M4). The feeding of cows was based on total mixed ration (TMR) ad libitum. Milk and blood samples were collected individually from each cow three times per standard lactation period. Results Greater exacerbation of changes in the dynamics of the blood plasma parameters examined was proved for multiparous cows. The highest value of β-hydroxybutyrate acid (0.946 mmol/L) was found for multiparous cows from group M3 at the beginning of lactation. However, it was still in the normal range. The results showed aspartate aminotransferase, and gamma-glutamyl transferase (GGT) activities in dairy cows during lactation had significant variations taking in to account stage of lactation. The highest activity of GGT was found in the group of the oldest cows and measured from 26.36 U/L at the beginning of lactation to 48.75 U/L at the end of the lactation period. Conclusion The time-related changes in the concentrations of the biochemical parameters described differ markedly among lactating cows, though the housing conditions on the research dairy farm are highly standardised. This indicates that the ability to cope with metabolic stress is mainly affected by the individual predispositions of cows and feed nutrient supply in different stage of lactation. Especially, the feed nutrient supply (in net energy for lactation), which was the best in TMR 1 in comparison TMR 3.

INTRODUCTION
The knowledge of a metabolic changes in dairy cows, on their diagnostics and detection, is extremely important. These metabolic changes are closely related to clinical and subclini cal diseases after calving, lactation and reproductive performance-factors that significantly shape the profitability of milk production. Varying metabolic blood and milk traits have been shown to be related to energy balance in dairy cows. They commonly experience an unbalanced energy status in the early lactation stage, and this condition can lead to the onset of several metabolic disorders [1,2]. Metabolic profile testing is a valid tool to monitor and detect the most common early lactation disorders. The term 'metabolic profile' refers to the analysis of milk and blood biochemical parameters that are useful to assess and prevent metabolic and nutritional disorders in dairy herds.
Unfortunately, blood sampling and analysis is timeconsuming and expensive, and the procedure itself is invasive and stressful for the cows. As em phasised by Puppel et al [3], routine identification of biomarkers accurately characterising the physiological status of an animal is crucial for decisive strategies. The milk fat/protein ratio (FPR) can be used as an indi cator of nutritional problems and energy deficit in lactating dairy cattle. Negative energy balance can be associated with increased milk fat content due to adipose tissue mobilisation, and decreased milk fat content due to a shortage of glucose for milk protein synthesis in the udder. A FPR greater than 1.5 is considered a risk factor for metabolic problems, such as ketosis. However, if the ratio drops to 1.0 or below, the herd is considered at risk of subacute ruminal acidosis. The frequent and regular milking process of dairy cows creates the opportunity to obtain samples at any stage of lactation. Routine identification of biomarkers accurately characteris ing the physiological status of an animal is crucial for decisive strategies [3,4]. As demonstrated by Jenkins et al [5], the FPR can be used to screen cows for subclinical ketosis, but not in the final diagnostic for administration of treatments. The best diagnostic tools are the blood parameters that may reflect the metabolic profile and the nutrient status of the cow, such as nonesterified fatty acid (NEFA), βhydroxybu tyrate acid (BHBA), as well as enzymes and proteins that reveal liver status. Maintenance of the metabolic balance in highyielding cows is very difficult. This applies mainly to cows after the peak lactation (120 to 150 days postpartum) [6]. However, currently there has also been a greater inten sity of clinical changes, even in younger cows. This leads to the elimination of dairy cows with high genetic potential, and thus results in large economic losses for dairy farmers. Biochemical blood indicators, like the activity of some liver enzymes such as aspartate aminotransferase (AST, EC 2.6.1.2) and gammaglutamyl transferase (GGT, EC 2.3.2.2) in blood serum, are used for the evaluation of metabolic balance [7]. The hepatic enzymes such as AST and GGT are the two main, sensitive enzymes that primarily reflect hepatocellular necrosis, and cholestasis, respectively, so are proposed to be two of the common parameters for the detection and diagnosis of liver failure [8,9]. The biochemical tests evalu ated showed larger specificity than sensitivity regarding the detection of hepatic injuries in healthy dairy cattle. This enzyme catalyses the transfer of an alphaamino group from an amino acid to an alphaketo acid and is widely distributed in animal tissues. The high specificity of the GGT serum ac tivity allows its use in the detection of chronic hepatic lesions that may be related to a negative impact on the performance of cattle herds [10,11]. Some earlier studies found relation ships between markers of oxidative stress of highyielding dairy cows in different stages of lactation, and their impact on the quality of milk [12,13]. However, association between biochemical parameters of blood in animals of different ages and at different stages of lactation was not examined in this experimental herd. Thus, the aim of the study was to deter mine the effect of parity number and stage of lactation on the metabolic profile indicators of highperformance Hol steinFriesian (HF) cows. This study attempted to provide a complete picture of the dynamics of selected biochemical blood parameters in cows of varying ages from the start of lactation to the dry period. This complete picture will illus trate the normal range of the most commonly used serum biochemical parameters during the different reproductive stages. The ranges of biochemical parameters indicated should be considered as guidelines for the management strategies of animals during their productive lives [14]. These guide lines should guarantee the fulfilment of metabolic needs of the animals and the high quality of their milk, and, as a con sequence, the reduction of economic losses.
The aims of the study were to give the normal range of the most commonly used serum biochemical parameters of HF cows during the different reproductive stage; and to examine the usefulness of metabolic status and physiological changes in lactating dairy cows as diagnostic tools in nutritional evalu ations of the dairy herd.

Animal care
All applicable international, national and/or institutional guildelines for the care and use of animals were followed. The authors carefully considered and sufficiently addressed potential ethical issues. All personnel engaged in the use of live animals received appropriate education and training. During the study, only routine zootechnical and veterinary medical diagnostic procedures were applied to the animals. These procedures included body condition scoring (BCS). Blood samples were collected from the tail vein by an autho rised veterinary technician. Milk samples were taken during routine milking. All animal procedures in this study were approved by the National Commission for Ethics of Animal Experimentation, III Local Ethics Committee for Animal Research (Warsaw, Poland); permission no. 10/2011.

Farm and management
This study was conducted on the Warsaw University of Life Sciences Agricultural Experimental Farm (in the Mazovia region, Poland; latitude: 52°07′ N; longitude: 21°15′ E) with an average stock of 620 HF cattle and a mean 305d milk yield per cow of 11,250 kg. The cows were milked twice a day in a herringbone milking parlour at 5 to 6 am and 5 to 6 pm. The cows were kept in a freestall dairy shed with free access to water and fed total mixed ration (TMR) diet. The TMR diet was provided ad libitum and formulated using the system of the French National Institute for Agricultural Re search (INRA) to provide adequate milk energy and milk production for a mean live weight of 638±34 kg. During the lactation period, TMR was adjusted based on the milk per formance and lactation stage. The ingredient composition of TMR diets, and nutrient supply with balance between ani mals observed in the experiment, is presented in Table 1.

Experimental design
The studies were conducted for five years. The experiment was conducted on 473 HF cows (starting the experiment at 15 days after calving) used a completely randomized design. Representative milk and blood samples were collected indi vidually from each cow, three times per standard lactation period (≤100 days of lactation, the initial stage of lactation; from 101 to 200 days of lactation, the middle stage of lactation; ≥201 days of lactation, the final stage of lactation). Accord ing to the parity, the cows were divided into four groups: P1, primiparous (n = 154); M2, multiparous in the second lacta tion (n = 114); M3, multiparous in the third (n = 125); and M4, cows in subsequent lactations (n = 80).
Milk was placed in sterile bottles, preserved with 2Bromo 2nitropropano1,3diol (Bentley, Warsaw, Poland) and immediately submitted to the Animal Breeding Department Milk Testing Laboratory.
Serum blood samples were obtained from the coccygeal vein by an authorised veterinary technician (empty test tubes for clotted blood and test tubes containing lithium heparin were used). Blood was collected before morning feeding. After collection, the samples were transported on ice, under chilled conditions (0°C to 6°C) to the Department of Pathol ogy and Veterinary Diagnostics, Laboratory of Faculty of Veterinary Medicine.
Levels of the following blood serum parameters were esti mated with the help of UVVIS spectrophotometer Ultrospec 2000 (Pharmacia Biotech, New York, NY, USA): the level of BHBA was examined with the enzymatic method test (Ran dox, Crumlin, Antrim, UK); the level of AST and NEFA was examined with the colorimetric method (Pointe Scientific, Warsaw, Poland) described by Sakowski et al [6]. Energy re serves were estimated based on the BCS, using a scale from 1 (thin) to 5 (obese), with 0.25 intervals [15].

Calculation
All the data were analysed by analysis of variance procedure of IBM SPSS, Statistics 25 (IBM, Armonk, NY, USA). The analysis of variance was performed to determine the influ ence of the parity and the stage of lactation on daily milk yield (kg), the cow' s body condition score (point), fat (%), pro tein (%) milk and selected blood parameters. It included the fixed effect the stage of lactation (S j ) and random effect of a cow (C k ) and was calculated with the model: where Y ij = dependent variable for kth cow at the ith parity (i = P, primiparous; M2, multiparous in second lactation; M3, multiparous in third lactation; and M4, subsequent lactation), in three lactation periods (j ≤100 days; 101 to 200 days; ≥201 days of lactation); μ = overall mean; P i = effect of parity; S j = effect of the stage of lactation; C k = random effect of the cow k; and e ijk = experimental error, P i ×S j = interaction.
Data are presented as least square means (LSM) and stan dard errors. Separation of LSM for significant effects was accomplished using the Tukey's test within the mixed proce dure of SAS. In all analyses, the significance level was set at

RESULTS
The animals used in this study did not display any obvious signs of illness in the prepartum period, therefore all the cows were classified as healthy before calving. Cytological quality of milk of examined cows expressed by means of SCC dur ing the test milkings did not go beyond the requirements of EU law and was below 400 (thousand cm 3 ). Table 2 shows the influence of the parity and the stage of lactation on the daily milk yield and BCS. The average daily milk yield and protein content differs significantly between production periods, and between the parities of cows. The cows from the M3 group produced more milk during the first 100 days compared to the younger cows. The peak lac tation of M3 cows was clearly observed before 100 days postpartum, and their daily milk production reached about 42.0 kg. Multiparous cows in the third and subsequent lacta tions were characterised by a higher milk yield (41.6 and 38.4 kg/d) compared to the P1 (32.7 kg/d) and M2 cows (35.5 kg/d). At the same time, a relationship between the parity and the cows' condition was observed. The lowest BCS values were characteristic for cows from the M3 group and measured below 3.0 points (2.78). The smallest devia tion for the BCS was found in primiparous cows (Table 2). In the critical period for highyielding multiparous cows (from 0 to 200 days of lactation), increased from 2.80 to 3.0 points in the BCS of M3 cows was recorded.
Concentration of protein was highest in the milk of M4 cows (from 3.34 to 3.94 g/100 g). An increase milk produc tion in early lactation (to 100 days postpartum) was observed, but this was limited to cows in third lactation and higher, when the FPR was optimal at 1.28 to 1.37 in the early stage of lactation (Table 3).
During the remainder of the study period differences in NEFA concentrations were observed between these four groups of cows; however, there was an effect of the stage of lactation for all cows ( Table 4). The NEFA concentration in blood constantly decreased from 101 to 200 days of lacta tion. The highest level of NEFA was observed in the early lactation, mainly for cows from the M2 group (0.532 mmol/ L). However, postpartum blood NEFA levels at the early lac tation were lower for the P and M3 group, compared to the M4 group (0.318 and 0.420 vs 0.495 mmol/L).
BHBA levels during the standard lactation periods showed significant differences. Postpartum blood BHBA levels at the early lactation were lower for M2 cows compared with M3 cows (0.886 mmol/L vs 0.946 mmol/L) ( Table 4). The mea sured concentration of BHBA showed a constant decrease of values in the blood plasma of multiparous cows (M2, M3, and M4) from the beginning of lactation until over 200 days postpartum. In case of primiparous cows, the BHBA value remained at a similar level throughout the lactation, reach ing the highest value (0.781 mmol/L) at the end of lactation.
Blood plasma GGT was significantly affected by the lacta tion stages succeeding the parity number (Table 5). Higher GGT activity was determined in the blood of M4 group cows, compared to the P1, M2, and M3 goups. The lowest GGT activity was found in the blood of primiparous cows at the early lactation and measured 22.36 U/L. The highest activity of GGT was characteristic for the oldest cows and measured from 26.36 U/L at the beginning of lactation to 48.75 U/L at the end of the lactation period. Multiparous cows were characterised by a greater exac erbation of changes in the dynamics of the blood plasma parameters examined, particularly in the AST and GGT, BHBA, NEFA levels.
The level of creatinine (CR) in the blood plasma differed significantly between production periods and between the parities of cows. The greatest range in the CR concentration was found in the blood of M4 group cows, and ranged from 0.48 to 1.00 mg/L. The results indicated that the ability to cope with metabolic stress is dependent on the parity and the stage of lactation.
Greater exacerbation of changes in the dynamics of exam ined blood plasma parameters has been proven, particularly in the AST, GGT, BHBA, and NEFA levels in the group of multiparous cows.

DISCUSSION
The increasing lactation performance of dairy cows over the last few decades is closely related to higher nutritional re quirements. A TMR is the best method of feeding cows that combines feeds formulated to a specific nutrient content into a single feed mix. The mix contains the following feeds: forages, grains, protein feeds, minerals, vitamins and feed  . The feed nutrient supply in net energy for lactation (NEL) was better in TMR 1 in comparison to other, espe cially TMR 3. In whole period of lactation the total feed intake was in groups M2, M3, M4 significantly better about 2 kg (dry matter) then in P group cows. Also the better feed nu trient supply in cows with influenced a body condition index during 100 days of lactation. At the beginning of lactation in group P or M2 was observed better body score condition in dex in comparison to group M3 (Table 2). There are very few reports that describe changes in TMR to levels of biochemi cal blood parameters in cows, because it is a highly invasive study. In one interesting study focused on the effects of a specially designed energyprotein supplement (EPS) to TMR on milk composition and preventing metabolic diseases in highyielding dairy cows [16]. Moreover, EPS had a positive impact on the milk yield of cows. Systematic and advanced quantitative monthly monitoring of metabolic profile of high performance cows kept in large herds fed a properly balanced mixed food ration is a useful tool for assessing animal wel fare and health, as well as for early diagnosis of many health problems [4].
According to the available literature about taking milk from ruminants, the age and stage of lactation has a signifi cant impact on both the milk chemical composition and somatic cell count [6,17,18]. A single milk sample can be sufficient to provide valuable information.
For example, the FPR is a valuable indicator of lipomo bilisation and negative energy balance of cows [19]. In the herd studied there were no deviations from the norm in terms of FPR. The optimum condition of cows at calving should be 3.5 points BCS, and this score is the most beneficial for primi parous cows, as well as for the release of energy from fat tissue after calving and subsequent replenishment of re serves [20]. This allows them to achieve high performance during lactation and minimises the risk of metabolic dis eases. Postpartum BCS levels at early lactation were lower for primiparous compared to those of multiparous cows in the third and subsequent lactations 3.47 vs 2.78 respectively ( Table 2).
In whole experimental period the interesting tendency of changes the biochemical parameters in blood were observed (Tables 4, 5). In group M3 in beginning of lactation the con centration of BHB in blood serum was higher, on the level 0.946 mmol/L and in parallel the NEFA concentration was lower in comparison to other groups multiparous cows ( Table  4). The concentration of both metabolite markers in blood correspondences to values presented in references for healthy cows (without ketosis) [2124]. Furthermore, the concentra tion of NEFA in blood serum in group primiparous cows was lower in comparison to other groups multiparous cows at beginning of lactation, which were fed TMR1. During next phases of lactation the content of NEFA in blood serum was not changed between groups except for cows from the M3 group. The results showed AST and GGT activities in dairy cows during lactation showed significant variations taking in to account stage of lactation. Jóźwik et al [7] proved that the AST activities in the blood serum were lower in dairy cows with production 7,000 kg per lactation compared dose with 10,000 kg milk. Additionally, Sakowski et al [6] found signifi cant negative correlation between average daily milk yield and AST concentration in blood. Determination of GGT and AST concentrations is helpful for assessing the liver function of cows with abomasal displacement, which confirms the re lationship of dislocations with hepatic lipidosis and hepatocyte damage. Liu et al [11] reported that detection of ALT, AST, and GGT activities in milk may be an alternative way to moni tor the liver function of cows.
Nogalski et al [21] reported that the levels of NEFA and BHBA were the highest in the blood of cows with a retained placenta in the first week of lactation (condition >4 points BCS), at the levels of 1.41 and 1.65 mmol/L and for ketosis 1.09 and 1.59 mmol/L respectively. Varying metabolic blood and milk traits have been shown to be related to the cow's age and lactation stage [2,6,22].
These studies have shown that the lowest concentrations of blood parameters were found in the first lactation and in creased with the increasing parity number.
In their study, Vanholder et al [22] observed that the risk of developing subclinical and clinical ketosis was higher in the second lactation and in older cows, compared with heifers. They found that in cases of clinical ketosis the risk was higher only after the third parity. In this study, a decrease of AST values could be observed in the blood plasma of M2 and M4 cows. The AST decreased constantly from the beginning until the end of lactation. It was observed that especially in the last 100 days of lactation, the AST activity in the blood plasma was statistically much higher compared to the middle of the lactation. Kurek and Stec [23] showed significantly higher levels of AST in the blood plasma after calving in younger cows (2.5 to 4 years) than in older cows (6 to 10 years). Doornenbal et al [24] reported in their study, that AST levels differed greatly between cows of different ages, but generally increased with age from 123.8 for heif ers to 132.7 U/L for six to 10yearold cows. The increasing AST concentration in the blood serum could indicate a grow ing intensity of metabolic changes, mainly of proteins, in the period from 120 to 150 days and more than 250 days postpartum [6].
In this study, the mean values of GGT activities measured at the beginning of lactation (up to 100 days) were much lower than activities obtained for other lactation stages for cows from P1 and M4 groups. Much higher GGT activities were determined in the blood of the M4 group compared to the values obtained for P1 and M2 cows. This corresponds with the suggestion reported by Jóźwik et al [7], who observed that serum GGT is higher in the later days of lactation. Liu et al [11] indicated much higher levels of GGT in milk than in plasma, and hence concluded that a cow's liver functions can be studied on the basis of the concentration of liver en zymes in milk. Serum NEFA concentration is the result triglycerides breakdown in the adipose tissue in response to a negative energy balance [22]. Circulating NEFAs are ab sorbed and metabolised for energy by the liver and other tissues. The results of this research showed that the level of NEFA increased significantly postpartum and at the end of the lactation, which was particularly visible in M3 cows. A similar conclusion has been reported by Kurek and Stec [23]. The authors observed a constant decrease of NEFA values in the blood plasma from 100 to over 200 days of lactation (Table  3). Similarly, BjerreHarpøth et al [25] and Nogalski et al [21] have shown that in dairy cows, the highest NEFA level in plasma is observed in the first weeks of lactation, to com pensate for the accompanying negative energy balance.
The concentration of BHBA, measured in this study, showed a constant decrease in the blood plasma of multiparous cows (M2, M3, and M4) from the beginning until the end of lac tation. Kapusta et al [13] and Kurek and Stec [23] showed in their study, that the BHBA reaches the highest concentration only between day 29 and day 70 of lactation, that is, at peak lactation. Sakowski et al [6] reported that BHBA level in blood was negatively correlated with the lactation stage.
The metabolic status, i.e. NEFA and BHBA concentration in blood plasma of P cows, characterized by lowest produc tion at early stage of lactation (≤100) were lower than in all other compared groups. It was probably the effect of the highest positive net balance supply of the NEL energy in their diet comparing to more productive cows form other studied groups. Similar relationships were observed in GGT and AST results These results also help to better follow and understand the metabolic pathways and metabolic profile of highyielding HF cows in relation to parity and stage of lactation. Moreover, the data obtained could be used as valuable, additional data to support herd health programs on dairy farms.
The timerelated changes in the concentrations of the biochemical parameters described differ markedly among lactating cows, though the housing conditions on the re search dairy farm are highly standardised. This indicates that the ability to cope with metabolic stress is affected by the individual predispositions of cows, and feed nutrient supply and is also dependent on the parity number, as evi denced by the reported significance. The analysis indicated that as many factors as possible should be considered, even if they are not very selective.