Experimental groups were fed a diet supplemented with CuNPs (6.5 or 13 mg/kg) and combined with different types of fibre . After the feeding period, blood and small intestine samples were collected for further analysis. Replacing CuCO3 by CuNPs in the diet positively reduced the level of lactic acid and apoptosis markers in the small intestine; however, it also resulted in the intensification of DNA oxidation. The most beneficial effect on DNA repair mechanisms is related to inulin, while pectin has the greatest ability to inhibit inflammatory processes that induce the apoptotic death of cells in the small intestine. Our results suggest that dietary fibre supplementation protects the small intestine against potentially harmful, oxidative effects of CuNPs by intensifying the intestinal barrier.The aim of the study was to verify the hypothesis regarding the effect of recommended (6.5 mg/kg) or enhanced (13 mg/kg) level of CuNPs in the diet in combination with different types of dietary fibre—cellulose (control), inulin, pectin or psyllium—on selected biological parameters of intestinal integrity in rats. Rats were randomly divided into 10 groups. The first two groups were fed a control diet that contained cellulose, and a mineral mixture with standard or enhanced content of CuCO Importantly, Cu is a cofactor of numerous metabolic enzymes, engaged in the energy production in the respiratory chain, free radical deactivation, the maintenance of connective tissue and the proper function or modulation of nerve conduction as a result of the conversion of dopamine to norepinephrine ,4,5,6,7.3 ,9,10. Ho3 ,9,10. We3 [3 , but mos3 [3  and redu3 [3 . Neverth3 [3 ,15,16,173 [3 . Due to Dietary fibre is one of the most important components of the diet, apart from nutrients and microelements. It is defined as parts of plants or analogous carbohydrates that are resistant to digestion and absorption in the small intestine of humans and animals, with the participation of endogenous enzymes, and are completely or partially fermented in the large intestine . DietaryTherefore, we assumed that the dietary combination of CuNPs with different types of fibre—neutral cellulose (control), prebiotic inulin, sticky pectin or swelling psyllium—would affect the physiological intestinal response, thereby regulating the metabolic effect of CuNPs in the body. The aim of the study was to verify the hypothesis regarding the effect of recommended Cu/kg concentration (6.5 mg) or the two times higher (13.0 mg) level of CuNPs in the diet, in combination with different types of dietary fibre  on selected biological parameters of intestinal integrity and histology of the small intestine in rats.2/g specific surface area (SSA), spherical morphology, 0.19 g/cm3 bulk density, 8.9 g/cm3 true density) were obtained from Sky Spring Nanomaterials Inc. . CuNPs with the same properties were also used in our previous studies [Copper nanoparticles . In the diets with Cu-NP the mineral mixture was deprived of CuCO3 and the copper nanoparticles were added to the diet, along with rapeseed oil (as an emulsion) for operator safety. The control dietary fibre, α-cellulose, was added at 8% of the diet, while the experimental fibre preparations  were added at 6% of the diet at the expense of cellulose preparation  were fed a standard semi-purified rat diet supplemented with two CuNPs doses  and combined with different types of dietary fibre. The control diet contained a mineral mixture with standard and enhanced content of CuCOparation . The expg, 10 min, 4 °C). Plasma samples were kept frozen at −80 °C until assay.Body weight (BW) was monitored at the start and end of the study for each animal, while the diet consumption was checked daily. Before the termination of the study, the rats were starved for 8 h but had free access to water. On the last day, the animals were subjected to body fat and lean tissue content analysis with the aid of the time-domain nuclear magnetic resonance (NMR) protocol . The principle of the analysis is that the tissue contrast is high between fat and muscle based on relative relaxation times. Next, the rats were anaesthetised i.p. with ketamine and xylazine in 0.9% NaCl  according to the anaesthesia and euthanasia guidelines for laboratory rodents. Following laparotomy, blood samples were drawn from the caudal vena cava into heparinised tubes, and finally the rats were euthanised by cervical dislocation. After that, the small intestine, heart, kidneys and spleen were dissected and weighed. Samples of the small intestine were preserved for histopathological examination, and intestinal tissue homogenates were prepared and stored at −80 °C until analysis. Blood plasma was prepared by solidification and low-speed centrifugation , while the dry matter (DM) concentration in the digesta was analysed by sample drying at 105 °C to a constant weight. The apparent digesta viscosity was measured in the supernatant fraction using the cone/plate viscometer .TM, Tecan Group Ltd., Männedorf, Switzerland).In the blood plasma, the level of selected indicators of intestinal integrity—lactic acid (LA) and diamine oxidase (DAO)—was determined using a commercial measurement enzyme-linked immunosorbent assay (ELISA) kit, following the protocol provided by the manufacturer . Absorbances were measured at 450 nm via ELISA reader .In homogenates prepared from the small intestine, the level of 8-hydroxydeoxyguanosine (8-OHdG), apurinic/apyrimidinic endonuclease 1 (APE-1), 8-oxoguanine DNA glycosylase (OGG1), lactic acid (LA), diamine oxidase (DAO) and the level of caspase 3 and caspase 8 were determined. These parameters were determined using a commercial measurement enzyme-linked immunosorbent assay (ELISA) kit, following the protocol provided by the manufacturer . Absorbances were measured at 450 nm via ELISA reader (SunriseZO-1), occludin (OCLN), trefoil factor 2 (TFF2) and 8-oxoguanine glycosylase (OGG1)—were designed using Primer 3 software  and synthesised by Genomed . The sequences are shown in RNA from the small intestine was extracted using Trizol Reagent , according to the manufacturer’s protocol. The isolated RNA yield was estimated spectrophotometrically , with integrity assessed electrophoretically by separation on 0.8% agarose gel. For complementary cDNA synthesis, 1 µg of RNA was reverse transcribed using the NG dART RT kit , according to the manufacturer’s instructions. Specific primers for evaluation of genes expression—zonula occludens-1  and β-actin (ACTB) genes were selected as endogenous controls to normalise gene expression.Real-time PCR was performed on Quantabio thermocycler  and using SG qPCR Master Mix (2×)  according to the following protocol: one cycle at 95 °C for 10 min , followed by a PCR including 35 cycles at 95 °C for 20 s (denaturation), 59–62 °C for 20 s  and 72 °C for 30 s (elongation). A melting curve analysis was performed over 50–72 °C at 0.3 °C/s intervals. Negative controls without the cDNA template were provided. Real-time PCR was performed in duplicate. Normalised gene expression was calculated using the 2Histopathological examinations of small intestine samples from rats were performed according to the procedure described by Cholewińska et al. .3 and containing cellulose as the main dietary fibre source) with the aid of a t-test. Similarly, the t-test was used to compare the experimental groups fed diets with CuNPs H dose with the control CH group fed diet with 13 mg/kg Cu from CuCO3 and containing cellulose as the main dietary fibre source. Differences with p ≤ 0.05 are considered to be significant.The STATISTICA software, version 12.0 , was used to determine the differences among treatment groups. Two-way ANOVA was applied to assess the effects of main factors CuNPs dose  and dietary fibre type , followed by Duncan’s multiple range test. Additionally, each experimental group fed CuNPs L dose was compared with the control C group  from CuCO3 with 8% of cellulose as dietary fibre source; the CN group was fed a diet with a supplementation of 6.5 mg Cu/kg from CuNPs with 8% of cellulose dietary fibre source; the PN group was fed a diet with a supplementation of 6.5 mg Cu/kg from CuNPs with 2% of cellulose and 6% of pectin dietary fibre source; the JN group was fed a diet with a supplementation of 6.5 mg Cu/kg from CuNPs with 2% of cellulose and 6% of inulin dietary fibre source; and the SN group was fed a diet with a supplementation of 6.5 mg Cu/kg from CuNPs with 2% of cellulose and 6% of psyllium dietary fibre source).OGG1 gene in the CN, JN and SN experimental groups; the TFF2 gene in the CN, PN and SN experimental groups; and the ZO-1 gene in the CN, PN and JN experimental groups compared to control group was found  with 8% of cellulose as a dietary fibre source; the CNH group was fed a diet with a supplementation of 13 mg Cu/kg from CuNPs, with 8% of cellulose as a dietary fibre source; the PNH group was fed a diet with a supplementation of 13 mg Cu/kg from CuNPs with 2% of cellulose and 6% of pectin as a dietary fibre source; the JNH group was fed a diet with a supplementation of 13 mg Cu/kg from CuNPs with 2% of cellulose and 6% of inulin as a dietary fibre source; the SNH group was fed a diet with a supplementation of 13 mg Cu/kg from CuNPs with 2% of cellulose and 6% of psyllium as a dietary fibre source). in the blood plasma of rats is related to the fact that the use of a higher dose of CuNPs resulted in an increase in the content of this indicator in rats from the CN group, which was not observed when using a higher dose of CuNPs in the PN, JN and SN groups (p = 0.020) in the tissue of the small intestine of rats is due to the fact that the use of a higher dose of CuNPs resulted in an increase in the level of this enzyme in rats from the PN, JN and SN groups, which was not observed with the use of higher dose CuNPs in the CN group. In turn, the interaction observed for the content of lactic acid (p = 0.019) in the small intestine of rats is related to the fact that the use of a higher dose of CuNPs resulted in a decrease in the content of this indicator in the PN and SN groups, which was not observed when using a higher dose of CuNPs in the CN and JN groups (OGG1 expression level (p = 0.037) is related to the fact that the use of a higher dose of CuNPs resulted in a decrease in these parameters in rats from the PN group, which was not observed when using a higher dose of CuNPs in the CN, JN and SN groups . Increasing the level of CuNPs from 6.5 to 13 mg/kg diet in the rat diet resulted in an increase DAO levels in the blood  compared to the control receiving cellulose as a standard dietary fibre source  compared to cellulose only. The addition of pectin or inulin lowered the pH of the ileal contents of rats (p < 0.001) also. Supplementation of pectin or psyllium also resulted in an increase in ileal viscosity , while the inclusion of pectin to the diet contributed to a decrease in the level of caspase-3 (p = 0.043) in the small intestine compared to being control fed a diet containing cellulose only (ZO-1 (p = 0.013) gene expression than in the control rats was also observed  on maintaining a proper intestinal barrier.In the assessment of the intestinal barrier function, diamine oxidase (DAO) is often considered. This enzyme catalyses the decomposition reaction of histamine in the digestive tract, which is responsible for the development of an allergic reaction. Under physiological conditions, a relatively high level of DAO is found in the small intestine, while in blood plasma it is very low. When the intestinal mucosa is damaged, e.g., as a result of ischemia, hypoxia, contact with a harmful factor present in food or tissue nutrition disorders, an inflammatory reaction is triggered with the release of histamine. Due to the necessity of its deactivation, a decrease in the level of DAO in the intestinal mucosa is observed, resulting in a simultaneous decrease in the level of this indicator in the blood . Our res3 with CuNPs resulted in a reduction in the level of lactic acid in the small intestine and blood serum as well. In light of the above-mentioned reports, this effect may be considered positive. In addition, the increased lactic acid level in the intestine may result in acidification of the environment. In the case of the large intestine, this effect may be desirable, because it protects it, among others, from colonisation by pathogenic bacteria [Lactic acid is the end product of glucose oxidation in anaerobic glycolysis . It may bacteria , while, bacteria . Acidifibacteria .3 by CuNPs resulted in an increase in the level of 8-OHdG in the small intestine, which suggests the intensification of oxidative processes in the examined tissue. Interestingly, the inclusion of the recommended level of CuNPs in the diet of rats contributed to a downregulation of OGG1 gene expression in the small intestine, which, however, did not translate into a decrease in the amount of functional OGG1 protein. It was also not found that increasing the level of CuNPs in the diet of rats from 6.5 to 13 mg Cu/kg of diet resulted in a deterioration of the oxidoreductive status and weakening of DNA repair mechanisms in the small intestine. However, an increase in the OGG1 protein level was noted, with no changes in OGG1 gene expression and 8-OHdG content in rats supplemented with inulin. This suggests that excessive oxidation of nitrogenous bases does not occur, and thus, the OGG1 enzyme is not significantly used for their repair. This allows us to assume that among all the tested forms of fibre, inulin best protects DNA against the harmful effects of free radicals, which may potentially be formed as a result of including CuNPs in the diet. The beneficial effect of the combined use of pectin with a higher level of CuNPs is evidenced by the observed DxF interaction for the level of APE-1 and for the expression of the OGG1 gene in the small intestine, resulting from the fact that only in the case of combining pectin as a source of fibre with the addition of a higher level of CuNPs, an increase in the level of APE-1 and downregulation of OGG1 were noted. Although an increase in the APE-1 level  may indicate an increased oxidation of this acid [Among all organs, the tissues of the gastrointestinal tract, especially the small intestine, is most exposed to the potentially harmful effects of xenobiotics entering the body through diet. It is assumed that this harmful effect is very often associated with the increased synthesis of free oxygen radicals. These, in turn, contribute to the occurrence of oxidative stress, resulting in damage to cellular macromolecules, including DNA modifications such as the oxidation of nitrogenous bases . In ordehis acid . However3 by CuNPs resulted in a decrease in caspase 3 and caspase 8 levels in the small intestine of rats, which seems to be a very beneficial phenomenon, and proves that this supplement does not induce significant negative changes in the cells of the small intestine. It also allows us to assume that the previously mentioned induction of oxidative DNA damage as a result of including CuNPs in the diet of rats was not serious enough to lead to significant changes in the genome of intestinal cells, which would result in their being directed to the apoptotic pathway. Moreover, it was not found that increasing the level of CuNPs in the diet contributed to the occurrence of negative changes in the level of the tested caspases. Regardless of the level of CuNPs introduced into the rats’ diet, no increase in the synthesis of caspases was found as a result of inulin and psyllium inclusion in the diet, and the addition of pectin reduced the level of caspase 3 in the wall of the small intestine. This suggests that, among the studied forms of fibre, pectin best protects the cells of the small intestine against damage that could lead to their apoptosis.In the event of damage to important cellular macromolecules, the programmed cell death may be activated. Caspases play an important role in this multi-stage process . The resZO-1 gene, which is expressed especially in the intestine, kidneys, liver, lungs and brain [TFF2 may be expressed in the wall of the small intestine. Interestingly, the TFF2 protein is involved in intestinal defence and repair mechanisms, while TFF2 overexpression is often observed in the case of significant tissue damage, infection or neoplastic changes in the within the digestive tract [3 by CuNPs in the diet of rats resulted in TFF2 and ZO-1 downregulation, with no changes in the expression level of OCLN. However, there was no effect of doubling the level of CuNPs in the diet—in relation to the nutritional recommendations—on the level of expression of the analysed genes of the intestinal barrier in rats. The obtained results can be interpreted in two ways, because the downregulation of TFF2 gene expression by CuNPs seems to be beneficial, but a decrease in the level of ZO-1 may indicate that CuNPs increase intestinal permeability and, thus, deteriorate the intestinal barrier. The explanation of the obtained results may be related to the formation of a functional protein, which reduces the expression of the TFF2 and ZO-1 mRNA pools. The results of our study also showed that the inclusion of dietary fibre in the form of psyllium in the diet of rats increased the ZO-1 gene expression in the small intestine, which, in light of reports by other authors, is an effect conducive to maintaining the proper integrity of the intestinal barrier [The intestinal barrier is crucial for maintaining intestinal homeostasis, which determines the proper supply of the body with nutrients and the prevention of intestinal diseases. It consists of the apical cell membrane and intercellular tight junctions (TJ) of enterocytes. Tight junctions are protein complexes resulting from the interaction between members of the claudin family, zonula occludens and MARVEL tight junction proteins (TAMP) . ZO-1 isnd brain . There and brain ,62. It ind brain . The resve tract . The res barrier ,62.3 and cellulose as a source of fibre. We observed no adverse changes in the morphology of the small intestine in rats receiving a diet containing the 6.5 mg/kg of CuNPs and the addition of cellulose (control group) or pectin. For rats fed a diet containing the same level of CuNPs but supplemented with inulin or psyllium, single tissue defects were observed at the top of the intestinal villi. Similar changes were intensified by the inclusion of a higher level of CuNPs (13 mg/kg) in the diet, regardless of the form of fibre used, with the most significant and numerous losses of the top part of the intestinal villi observed in the group receiving cellulose in the diet. The obtained results indicate that the replacement of CuCO3 by CuNPs had a beneficial effect on the morphology of the intestines. In turn, changes in the top parts of the villi observed in groups of rats fed a diet containing an increased level of CuNPs, regardless of the fibre source used, seem to be related to the direct effect of Cu in the form of nanoparticles. However, the lack of any pathological changes at the base of the villi and in the mucosa of the small intestine, regardless of the type of dietary fibre, as well as the lack of deterioration of indicators proving the integrity of the intestinal barrier, do not seem to confirm this assumption. Moreover, our previous studies did not show any pathological changes in the small intestine tissue of healthy, normotensive rats, in which both 50% and 100% of the traditional CuCO3 was replaced by CuNPs. In the histological image of the small intestine of rats receiving the recommended level of Cu in the diet only in the form of CuCO3, isolated extensive changes in the base of intestinal villi were found [Finally, the results of histopathological examination indicate the normal structure of the small intestine with the presence of small tissue defects in the apical parts of the villi in rats receiving a standard diet containing the recommended level of Cu in the form of CuCOre found . In ligh2+ ions, and this effect is stronger the higher their dose and the longer the exposure time. The authors observed that increased production of free oxygen radicals resulted in damage to the mitochondrial membrane and reduced viability of small intestine cells exposed to CuO NPs [2+ ions. However, the authors did not record a similar phenomenon in intestinal juice [2+ ions from CuO NPs in the intestinal juice [3 [2+) with free carboxyl groups, which reduces their bioavailability [Numerous reports in the literature indicate that the use of Cu nanoparticles in animal nutrition may be associated with many risks, such as their increased accumulation in internal organs ,17. Hens CuO NPs . Lee et al juice . Accordial juice . The resjuice [3 , which, juice [3 , should juice [3 ,23,24,25juice [3 , mineraljuice [3  observedlability ,65. Furtlability ,26,66. Ilability . Taken t3 by CuNPs in the diet of rats positively reduced the level of lactic acid and markers of apoptotic cell death in the small intestine; however, it resulted in the intensification of DNA oxidation. Increasing the level of CuNPs from 6.5 to 13 mg Cu/kg of diet had no negative effect on the physiological intestinal response. Our results indicate that the most visible and beneficial effect on DNA repair mechanisms is related to inulin, while pectin has the greatest ability to inhibit inflammatory processes that induce apoptotic death of cells in the small intestine. The obtained results suggest that dietary fibre supplementation in rats’ diet effectively protects the small intestine against potentially harmful, oxidative effects of CuNPs by intensifying the intestinal barrier, and this may finally translate into a beneficial regulation of their metabolic effect.To conclude, replacing CuCO


Fatty acids from the omega-3 family play an important role in both human and animal organisms, but they are not synthesized in the body and must be provided with diet. This study examined an effect of ethyl esters of linseed oil rich in alpha-linolenic acid supplemented to healthy beagle dogs on the fatty acid profile of their blood serum, erythrocyte membranes and hair sebum. The treatment resulted in a decrease in the content of saturated fatty acids exhibiting adverse effects and an increase in the level of beneficial unsaturated acids, especially these from the omega-3 family.The aim of this study was to determine the effect of supplementation with ethyl esters of linseed oil on the fatty acid profile in hair sebum, blood serum and erythrocyte membranes in healthy dogs. The material for the study included hair and blood samples of adult beagle dogs. The experiment was performed in two periods: summer and winter. Each time it lasted 16 weeks. During the first 8 weeks, the dogs received a supplement, the amount of which was determined individually so that the ratio of α-linolenic acid (ALA) to linoleic acid (LA) together in the feed and supplement was 1:1. Hair coat and blood samples were collected on days 0, 56 and 112; i.e., before the start of supplementation, after 8 weeks of supplementation and 8 weeks after supplementation was completed. The study included a determination of the fatty acid profile with a particular emphasis on polyunsaturated fatty acids (PUFAs) and the ratio of omega-6 to omega-3 in hair sebum, blood serum and erythrocyte membranes. As a result of supplementation, a significant decrease in saturated acids and an increase in unsaturated acids was observed in hair sebum both in summer and winter and especially in omega-3 fatty acids; i.e., α-linolenic (ALA) and its derivatives eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA). The same relationships were observed in blood serum and in erythrocyte cell membranes in all the studied periods. Additionally, 8 weeks after the end of supplementation, the level of polyunsaturated fatty acids was still higher compared to the period before supplementation. Moreover, the supplementation resulted in a favorable decrease in the ratio of omega-6 to omega-3 acids in the tested samples, which persisted even after the withdrawal period. Polyunsaturated fatty acids (PUFAs) of the omega-6 and omega-3 families, known as essential fatty acids (EFA), play a significant role in the proper development and functioning of the body ,2,3,4. DFatty acids from the exogenous omega-6 and omega-3 families together with the endogenous omega-9 family compete for the same enzymes in the conversion process ,10,14,15An increase in alpha-linolenic acid (α-linolenic (ALA)) content in the diet increases the level of EPA and DHA in cell membranes and concurrently reduces AA content, but this process in dogs is considered inefficient ,18,19,20Numerous studies conducted on mammals have shown that a diet rich in linseed or linseed oil may cause an ALA, EPA, DPA (docosapentaenoic acid) and LA (linoleic acid) increase and an AA decrease in both plasma and erythrocyte membranes, but it does not affect the changes in DHA levels ,23,24,25Apart from compounds showing beneficial effects on the organism, linseed oil also contains anti-nutritive substances that may show negative effects ,29,30. AEthyl esters of higher fatty acids obtained as a result of a transesterification reaction are characterized by better bioavailability to the organism in comparison to triglycerides. Ethyl esters of linseed oil are an excellent source of PUFAs, especially in about 50–60% of α-linolenic acid. Unlike linseed oil, they are free of harmful substances. Due to lower oxygen solubility in esters, they are also more durable and less susceptible to oxidation, epoxidation and peroxidation processes ,32.The aim of the study was to determine the effect of linseed oil ethyl esters on the changes in the fatty acid profile in the hair sebum, blood serum and erythrocyte membranes of healthy dogs.The experiment was conducted on 8 healthy adult beagle dogs  weighing 9–13 kg and kept in the Department of Reproduction and Clinic of Farm Animals, Faculty of Veterinary Medicine, Wrocław University of Life Sciences. Throughout the whole period of the experiment, the dogs stayed in partially roofed outdoor enclosures with access to insulated shelters. The dogs had all the current vaccinations and were dewormed. The health status of the dogs was monitored daily, and the body weight was measured weekly. The food ration was set individually for each dog to ensure proper body weight and fitness. The dogs were fed twice a day (in the morning and in the afternoon). They had constant access to fresh water.According to the opinion of the 2nd Local Ethical Committee for Experiments on Animals, Wroclaw University of Environmental and Life Sciences, Poland, the study did not require the approval of the Committee under the Act of 15 January 2015 on the protection of animals used for scientific and educational purposes .The experiment was conducted in two periods: summer (June to September 2015) and winter (November 2015 to February 2016). In both periods, the length of the experiment was 16 weeks, and the preparation period was an additional 8 weeks before the trial began. During the preparation period, the pet dry food that was used throughout the experiment was introduced. The average nutritional value of the food is shown in After the preparation period, all dogs received an additional supplement in the form of ethyl esters of linseed oil administered individually and orally using a syringe once a day after morning feeding for 8 weeks. In order to determine the correct amount of the supplement, the content of fatty acids in the food and supplement was determined by means of gas chromatography . SynthesThe fatty acid profile was determined in the Laboratory of Chromatography and Meat Evaluation, Institute of Animal Breeding, Wrocław University of Environmental and Life Sciences, Poland. The amount of the supplement was determined so that the ratio of ALA to LA acids in total in the food and supplement was as close as possible to 1:1. The average amount of the supplement was 35 mL/day/dog. Hair coat and blood samples for the fatty acid profile, blood count and biochemical indices analysis were collected three times in both periods from all dogs participating in the experiment: week 0 , week 8  and week 16 .The hair coat was collected using a surgical clipper (3M) on the left side at the level of the last rib by cutting square patches measuring 5 × 5 cm. The samples were packed in tightly closed containers and stored at −20 °C until the marking was carried out. Blood was collected from the cephalic vein of the front leg. Each time, 5 mL of blood was collected from one dog into the test tubes with potassium EDTA. After collection, the blood was centrifuged and divided into serum and morphotic elements and then was stored at −20 °C until the time of analysis.Sebum fat was extracted with ether using the Soxhlet method. Fatty acid methyl esters were obtained using 2M KOH solution in methanol. The profile of the fatty acids in the obtained fat samples was determined using an Agilent 7890A gas chromatograph  with a flame ionization detector (FID) .Fat present in serum and erythrocytes was extracted using the Folch method. Fatty acid methyl esters were obtained using 2M KOH solution in methanol. The fatty acid profile in the obtained fat samples was determined using an Agilent 7890A gas chromatograph  with an FID detector . The determinations were made in the following conditions: HP-88 capillary column (Agilent)—100 m long and 0.25 mm diameter at an initial temperature of 50 °C and with temperature increments of 3 °C/min to 220 °C; temperature of the dispenser—270 °C.The identification of the obtained fatty acid peaks was performed via comparison with the retention times of the Sulpeco 37 fatty acid methyl ester standards from Sigma.Blood count and biochemical indices were determined by the commercial veterinary laboratory VetLab sp. z o.o .The obtained results were analyzed statistically by using the Statistica ver. 13.1 package. The normality of the data distribution was evaluated via the Shapiro–Wilk test. Results concerning the fatty acid profile in hair coat sebum, serum and erythrocyte membranes were evaluated by using a two-factor analysis of variance.The health status of dogs was constantly monitored during each experiment. Also, the body weight remained stable during the whole study. All dogs willingly consumed the food and supplement. They did not have any undesirable gastrointestinal clinical signs . They maintained proper physical condition throughout the whole period of the experiment.The results of the blood count and biochemical indices are presented in The results for changes in fatty acid composition in the sebum are presented in Both in summer and winter, a significant increase in the contents of LA AA, ALA, EPA and DHA was observed at week 8 compared to the beginning of supplementation.At week 16 of both periods, a significant increase in the contents of ALA and DHA was observed in relation to week 0. The contents of LA and AA in summer as well as AA and EPA in winter also increased.The total sum of omega-6 and omega-3 series acids increased significantly as a result of supplementation (week 8) in both periods. In relation to the beginning of the experiment (week 0), their significantly higher level was observed at week 16 of the experiment, except for omega-6 in winter.The ratio of omega-6 to omega-3 acids both in summer and in winter significantly decreased at week 8. At week 16, it was still at a significantly lower level compared to week 0.Considering the differences in particular weeks between the seasons, significantly higher levels of PUFAs were noted in summer in weeks 0 and 8 as well as in the case of MUFAs in week 16. The contents of LA (week 8), AA (week 0), EPA and DHA were significantly higher in particular weeks in winter, while the opposite was true for ALA (weeks 0 and 16) and AA (weeks 8 and 16). The level of PUFAs in week 8 was significantly higher and in week 16 lower in winter compared to summer. The level of Omega-6 acids was significantly higher in winter in weeks 0 and 6, while the content of omega-3 acids in week 16 was higher in summer. Finally, the ratio of omega-6 to omega-3 acids was significantly higher in winter at the beginning of supplementation .The results for changes in the fatty acid composition in the blood serum are presented in At week 8 of the summer period of the experiment, a significant increase in the contents of LA, AA, ALA, EPA and DHA was observed. However, at week 16 of the same experiment, only the levels of EPA and DHA were still significantly higher compared to week 0.At week 8 of the winter period of the experiment, the levels of ALA, EPA and DHA increased significantly, but at week 16 of the experiment only the level of DHA was significantly higher compared to week 0.As a result of supplementation with ethyl esters of linseed oil (week 8), the level of omega-3 series acids increased significantly both in the experiments carried out in summer and winter, and at week 16 of the experiment, they were still at a significantly higher level in relation to week 0. The level of omega-6 series acids increased significantly only at week 8 of the summer period.At week 8 of both experimental periods, the ratio of omega-6 to omega-3 acids decreased significantly. At week 16 of the experiment, its value increased compared to day 56, but it was still lower compared to week 0.There were also significant differences between the seasons examined. The level of MUFAs at week 16 was significantly higher in summer. A similar trend was found for LA (week 8) as well as EPA. In turn, the levels of ALA (week 16), AA and DHA (weeks 0 and 8) were significantly higher in winter. The content of Omega-3 acids was significantly higher in winter at weeks 0 and 8, while the ratio of omega-6 to omega-3 was statistically higher in summer at week 0 .The results for changes in the fatty acid composition in the erythrocyte membranes are presented in Both in the summer and winter periods of the experiment, a significant increase in the contents of AA, ALA, EPA and DHA was observed at week 8. At week 16 of both experimental periods, the levels of AA, ALA, EPA and DHA were significantly higher compared to week 0 of the experiment.The sum of omega-6 acids increased significantly.As a result of supplementation with ethyl esters (week 8), the sum of omega-3 series acids increased significantly in both periods of the experiment, and at week 16 it was still at a higher level compared to week 0.In both experimental periods, the ratio of omega-6 and omega-3 acids at week 8 decreased significantly, and at week 16 it was still lower than at week 0.Considering differences between summer and winter, in the case of erythrocyte membranes, only the level of MUFAs at week 16 was significantly higher in winter compared to summer .Polyunsaturated fatty acids of the omega-6 and omega-3 series are essential for the proper development of the young organism and good health maintenance ,2,3,4. DThe authors of numerous studies have shown that 8-week supplementation with various oils (those of both vegetable and fish) brings significant changes in the fatty acid profile in erythrocyte membranes ,27 or inStoeckel et al.  showed tMany authors have suggested that increasing ALA in the diet may cause an increase in EPA and DHA in the body membranes of dogs, but this process is considered inefficient ,18,19,20The preference for ALA as a substrate by Δ6-desaturase may be associated with an increase in EPA and DHA and a decrease in the level of AA in the fatty acid profile of erythrocyte membranes. Additionally, the fact that LA is not converted makes it beneficial for the skin and hair coat . In a stA study conducted by Stoeckel et al.  showed tThe fatty acid profile in erythrocyte membranes is considered to be a model for the membranes of other cells of an organism ,46. ThisStudies on dogs indicate that they are also a model for human fatty acid metabolism studies due to their similarity in PUFA conversion ,24,49.The small number of dogs participating in the experiment and the lack of a control group were limitations of this study. However, the participation of dogs of one breed that were fed in the same way and kept in one environment makes the results more convincing. Further research including a larger number of healthy dogs from one breed and more experimental groups is needed in the future.In conclusion, this study examined the effect of linseed oil ethyl esters on the changes in fatty acid profile in the hair sebum, blood serum and erythrocyte membranes in healthy dogs. As a result, a significant decrease in saturated fatty acids and an increase in unsaturated fatty acids in the fatty acid profiles of the hair coat sebum, blood serum and erythrocyte membranes in dogs participating in the experiment were observed as a result of ethyl ester of linseed oil supplementation. In particular, the contents of ALA and its derivatives; i.e., EPA and DHA, increased significantly, which contributed to the decrease in the ratio of omega-6 to omega-3 family acids. The conducted study may confirm that a diet in which the ratio of ALA to LA is 1:1 may facilitate the conversion of ALA to EPA and DHA, which is extremely important for the proper functioning of the body.


In the last decade, research centered around the fault diagnosis of rotating machinery using non-contact techniques has been significantly on the rise. For the first time worldwide, innovative techniques for the diagnosis of rotating machinery, based on electrical motors, including generic, nonlinear, higher-order cross-correlations of spectral moduli of the third and fourth order , have been comprehensively validated by modeling and experiments. The existing higher-order cross-correlations of complex spectra are not sufficiently effective for the fault diagnosis of rotating machinery. The novel technology CCSM3 was comprehensively experimentally validated for induction motor bearing diagnosis via motor current signals. Experimental results, provided by the validated technology, confirmed high overall probabilities of correct diagnosis for bearings at early stages of damage development. The novel diagnosis technologies were compared with existing diagnosis technologies, based on triple and fourth cross-correlations of the complex spectra. The comprehensive validation and comparison of the novel cross-correlation technologies confirmed an important non-traditional novel outcome: the technologies based on cross-correlations of spectral moduli were more effective for damage diagnosis than the technologies based on cross-correlations of the complex spectra. Experimental and simulation validations confirmed a high probability of correct diagnosis via the CCSM at the early stage of fault development. The average total probability of incorrect diagnosis for the CCSM3 for all experimental results of 8 tested bearings, estimated via 6528 diagnostic features, was 1.475%. The effectiveness gains in the total probability of incorrect diagnosis for the CCSM3 in comparison with the CCCS3 were 26.8 for the experimental validation and 18.9 for the simulation validation. The effectiveness gains in the Fisher criterion for the CCSM3 in comparison with the CCCS3 were 50.7 for the simulation validation and 104.7 for the experimental validation. Pumps, gear motors, and cranes are examples of IM applications. Most IMs are required to operate for a long time between maintenance actions. That is why effective condition monitoring is needed in industrial applications of motors ,6,7,8,9,For bearing diagnosis in an industrial environment, vibration-based methods are still the most used. However, they require the installation of expensive vibration sensors on the engine, making these methods invasive, inconvenient, and costly. vibration methods are well known and have been used for a long time to detect bearing damage ,24,25,26Motor current signature analysis (MCSA) is the subject of much up-to-date research, as it has become a non-invasive substitute to vibration-based methods for IM bearing diagnosis. This method significantly reduces the cost of diagnosis, as it does not require the installation of expensive piezoelectric accelerometers on a motor. MCSA can be performed remotely, as the IM current can be measured with only access to the power supply line of a motor. IM current signals are frequently used for detecting electrical faults in motors and faults in coupled gearboxes . Early dSpectral component frequencies used in IM bearing diagnosis are estimated using models that have been improved over time. Studies ,36 have The other approach to monitoring IM bearing condition is presented in ref. ,31, in wThe authors of ref.  proposedTo improve the reliability of MCSA-based IM bearing damage diagnosis, new and robust signal processing techniques are sought. Recent research in this area considered using the wavelet transform ,36,37,38In ref. , the conMCSA-based higher-order spectral techniques were employed in ref. ,4,33,34 An MCSA-based method that involved instantaneous frequency analysis was proposed in ref. . In ref.The Park vector approach used in MCSA-based IM bearing diagnosis allows the enhancement of components related to bearing damage, which slightly increases the poor SNR. After Park vector estimation, various sophisticated methods of signal processing can be used for the detection of fault-related components ,35. ThisThe authors of ref. ,2 proposThus, the main novelties here include novel validation by comprehensive modeling and experiments of novel, nonlinear, higher-order cross-correlations for damage diagnosis of rotating machinery based on electrical motors.novel comparison of the CCSM3 and CCCS3 technologies via comprehensive modeling trialsnovel comparison of the CCSM4 and CCCS4 technologies via comprehensive modeling trialsnovel comparison of the CCSM3 technologies and CCCS3 technologies via comprehensive experimental trialsThe objectives of the research are as follows:perform a novel comprehensive validation of the CCSM3 and CCSM4 via modeling trials for pristine and faulty mechanical componentsperform a novel comparison of the CCSM3 with the CCCS3 and the CCSM4 with the CCCS4 via modeling trials for pristine and faulty mechanical componentsperform novel comprehensive validation of the CCSM3 via experimental trials for pristine and faulty motor bearings, using motor current signal processingperform a novel comparison of the CCSM3 with the CCCS3 via experimental trials for pristine and faulty motor bearings, using motor current signal processingOther novelties of the research are as follows:n is described by Equation (1) [The first higher-order spectral signal processing technologies examined in this paper are the cross-correlations of spectral moduli (CCSM) proposed in ref. . The CCSEquation  [1].(1)The CCSM of orders three (CCSM3) and four (CCSM4) are expressed by Equations (2) and (3), respectively :(2)CCSM3n is described by Equation (4):The second higher-order spectral signal processing technologies examined in this paper are the cross-correlation of complex spectra (CCCS). An unnormalized version of these technologies is proposed in ref. . NormallThe CCCS of orders three (CCCS3) and four (CCCS4) are expressed by Equations (5) and (6), respectively.In order to estimate diagnostic features, the CCSM3/CCSM4 and CCCS3/CCCS4, the discrete time domain signal has to be divided into time segments of a predefined duration. The following step is to perform the selected frequency transform or time–frequency transform of each time segment. In this paper, the Fourier transform is used. The final step is to estimate the CCSM3/CCSM4 and CCCS3/CCCS4 via Equations (2), (3), (5) and (6), respectively.To evaluate the diagnostic effectiveness of the technologies, histograms of the considered diagnostic features under various settings are evaluated. Then, each histogram is approximated by the normal  probability density function. The normal probability density function of random variable X with mean (μ) and standard deviation (σ) is stated as follows:rN and prN are the total numbers of correct diagnosis for the defective bearing and new bearing, respectively; rNt and prNt are the total numbers of diagnostic features for the defective bearing and new bearing, respectively.To compare the CCSM with the CCCS, two estimates related to a diagnosis accuracy are used. The first estimate is the total probability of correct diagnosis (TPCD) described by Equation (8) and the second estimate is the Fisher criterion (FC) described by Equation (9). A threshold-based decision-making rule (an aqua line is used to indicate the diagnostic threshold) is employed via the Bayesian criterion  for the To compare the proposed CCSM and CCCS techniques, healthy and damaged mechanical components were modeled. A generic bilinear oscillator was used to model these components. A generic dynamic bi-linear oscillator used for modeling is widely employed for modeling various electromechanical components of industrial electromechanical systems and complex structures, e.g., sub-harmonic resonances of offshore structures , loadingThe equation for the bilinear oscillator is shown below :(10)d2ydThe random initial phase is uniformly distributed in the range . Equations (10)–(12) are used to describe a mechanical component with and without damage. The stiffness of positive displacement 3) below :(13)ωo=ωfor healthy componentsfor damaged components with relative damage severity of 5%for damaged components with relative damage severity of 10%A total of 300 time domain realizations were simulated via Equations (10)–(13) to investigate the cross-correlation techniques, including:These signals were simulated using resonance frequencies The following parameters were used to evaluate the cross-correlation techniques: the internal window was 2 s, the frequency resolution was 0.5 Hz, the time window was the Hanning window, and the overlapping of internal windows was 0%. Gaussian white noise was added to each simulated signal to simulate an early stage of damage.The modeling flow chart for the CCSM3/CCCS3 and CCSM4/CCCS4 is given below in The CCSM3 and CCCS3 are functions of three independent frequencies. To estimate the diagnostic effectiveness of the CCSM3 and CCCS3, four sets of harmonics were investigated using the simulated signals from components with resonance frequencies To estimate the CCSM3 and CCCS3, SNR of 22 dB was used for 5% relative damage severity and SNR of 18 dB was used for 10% relative damage severity. The main parameter that affects an accurate histogram evaluation is the bin number. The bin number is selected to accurately extract the shape of the histograms, using the following methodology. For each pair of the obtained modeling histograms in The superiority of the CCSM3 over the CCCS3 can be seen in The Fisher criteria also validated the higher performance of the CCSM3 over the CCCS3 for all investigated cases. The CCSM3′s Fisher criterion result range was 3–8, as shown in The simulation results generated from processing the modeled bilinear signals with resonance frequencies of 14 and 30 Hz, with and without damage at relative severities of 5% and 10%, and with the addition of white Gaussian noise demonstrated that the CCSM3 technique was highly effective in detecting nonlinearities resulting from component faults. The performance of the CCSM3 technology was compared to the performance of the CCCS3 technology using the TPCDs and Fisher criteria, which confirmed the superiority of the CCSM3 in diagnosing early-stage faults. Therefore, the CCSM3 technology was an efficient technique for identifying incipient faults.The CCSM4 and CCCS4 are functions of four independent frequencies. The CCSM4 and CCCS4 were estimated using the following harmonic sets: , ,  and . The fourth cross-correlations for harmonic set  were estimated for the four resonance harmonics: the fundamental resonance harmonic employed twice, the second resonance harmonic, and the third resonance harmonic. The triple cross-correlations for harmonic set  were estimated for the four resonance harmonics: the fundamental resonance harmonic, the second resonance harmonic, the fourth resonance harmonic, and the fifth resonance harmonic. The fourth cross-correlations for harmonic set  were estimated for the four resonance harmonics: the fundamental resonance harmonic, the second resonance harmonic, the third resonance harmonic, and the fourth resonance harmonic. Finally, the fourth cross-correlations for harmonic set  were estimated for the four resonance harmonics: the fundamental resonance harmonic, the third resonance harmonic, the fourth resonance harmonic, and the fifth resonance harmonic. These sets of resonance harmonics were investigated using resonance frequencies To estimate the CCSM4 and CCCS4, SNR of 24 dB was used for 5% relative damage severity and SNR of 19 dB was used for 10% relative damage severity for resonance frequencies of 14 and 30 Hz The superiority of the CCSM4 over the CCCS4 is shown in The simulation results acquired by processing the modeled bilinear components with resonance frequencies of 14 and 30 Hz, at fault severity ratios of 5% and 10%, and in the presence of white Gaussian noise suggested that the CCSM4 technology was proficient in detecting nonlinearities resulting from component faults. The TPCDs and Fisher criteria were utilized to compare the performance of the CCSM4 to the CCCS4, revealing the superior diagnostic capability of the CCSM4 in identifying early-stage faults. Therefore, the CCSM4 technology was an efficient technique for diagnosing incipient faults.The laboratory test rig used in this research is described in detail in ref. ,2. A phoAll measurements used in this experiment were made under the conditions of a full motor load. All experimental tests were performed with 6204C3 Koyo bearings. One of the new tested bearings is shown in The acquired data for each diagnostic case included three digital standard current signals of three phases of the induction motor. These signals were not normalized. The length of each signal was 65 s acquired with a sampling frequency of 65 kHz. The current signals were captured for stationary motor operation under full load conditions. The main component of each signal was the 50 Hz supply current component. Graphical illustrations of fragments of the recorded signals for the damaged and pristine bearings are presented in RSH) frequency and the parameters of the used IM constructions [RSH frequency, Equation (14) was used for each RSH, The captured signals were split into 13 non-overlapping time segments of 5 s. The Blackman time window was used for each segment of the signal. The frequency of the IM shaft rotation ructions ,76. Aftetimation ,76:(14)fThe values of these frequencies are needed to estimate the characteristic frequencies related to bearing damage. Next, the complex spectral amplitude for the CCCS3 and the modulus of the complex spectral amplitude for the CCSM3 from the current spectra were obtained for three bearing defect-related characteristic frequencies for each segment. The following step is to use Equation (2) for the CCSM3 estimation and Equation (5) for the CCCS3 estimation. The final step is to calculate the mean values of the CCSM3 and CCCS3 for three phases of the captured IM current. The values of the CCSM3 and CCCS3, obtained via the described algorithm are used as diagnostic features for IM bearing diagnostics and for the comparison of the CCSM3 and CCCS3.The bearing defect frequencies are described by the following equations ,2,19: of bearings were split into two cases: two bearings with outer race defects and two bearings with inner race defects. Each pair of bearings was examined separately and the obtained results were averaged. The recorded signals were processed via the examined CCSM3 and CCCS3 technologies in order to make a comparison.In the current stage, the acquired data were processed on a separate computer via Matlab R2021a. The computational/measurement complexity are discussed below. All data in the experimental part were processed on PC with Intel Core i7-2600 CPU and 8 GB of RAM . The computation time on this hardware acquired with the Matlab cpu time command was 1.9812 s for 0% overlapping and 3.3696 s for 50% overlapping. It took 123.5 s to calculate 51 diagnostic features. If the CCSM3 algorithm software was organized, using the calculation of the CCSM3 during the sample intervals, then the CCSM3 feature could be estimated in “real time.” The signals were the three phases of the motor current, the length was 65 s, the sampling frequency was 65,536 Hz, and 24 bits were used for the sampling.The total number of operations acquired with Matlab socFunctionAnalyzer  was 310,778,788 for the original 65,536 Hz sampling frequency, 65 s of signal duration, and 5 s duration of time segments with no overlapping. The number of operations could be significantly decreased by decreasing the sampling frequency used in this experiment. After downsampling the signal by 2  the total number of operations was decreased to 155,458,468. The total number of operations for further downsampling is presented in the Based on the theoretical basis, the number of operations can be estimated as follows.segment current data into K non-overlapped segments of N samples eachestimate DFT coefficients for each segmentcalculate CCSM3 estimates for all segments via multiplications of moduli of the selected DFT coefficients and average these estimates over K segmentsConsidering a conventional method of CCSM3 estimation, the main method for estimation of the CCSM3 is the direct method. Extension of this method to the CCSM4 or to any higher CCSM order can be made easily. Considering the direct method of estimation of the numerator of the CCSM3 for one current phase  for one diagnostic feature, the following steps should be employed:The number of multiplication operations to estimate the DFT for each segment is Finally, ignoring a small number of addition operations for the averaging of the CCSM3 over K segments and a small number of multiplication operations for obtaining the CCSM3 for each segment, the total number of multiplication operations for K segments is K × Using the above methodology to estimate the total number of operations for calculation of the numerator of the CCSM3, the numbers of multiplication and addition operations were 78,040,269 and 156,080,538, respectively, for the processing parameters employed in the experimental trials, i.e., the segment size was 5 s, the number of segments was 13, and the sampling frequency was 65.536 kHz with 0% segment overlapping. Therefore, the total number of operations  for calculation of the numerator of the CCSM3 was 234,120,807.Components with the frequency related to bearing inner race defects, described by Equation (16), did not directly appear in the IM current spectrum. The frequencies of the components of the IM current related to bearing inner race defects can be described by Equation (17) ,36:(17)fThe bearings used in this part of experiment were the pairs of bearings in1, p1 and in2, p2. The results obtained in this part were estimated with the use of 84 components, which were used to calculate the CCSM3 and CCCS3 for 28 different combinations of three components for each bearing. The 84 components were obtained with the For each Components with the frequency related to bearing outer race defects described by Equation (14) did not directly appear in the IM current spectrum. The frequencies of the components of the IM current related to bearing outer race defects can be described by Equation (17) ,28,36: and 24-bit DAQ are minimal requirements for current data capturethe computational complexity of the estimations of the proposed technologies. This limitation is easy to overcome via powerful IT tools.The presented higher-order spectral technologies are very powerful technologies for performing damage diagnosis in various electromechanical systems using motor current signature analysis (MCSA). The proposed technologies effectively work even for signals with low signal-to-noise ratio. For the presented research, the main limitations are as follows:The proposed technologies are novel, as these technologies do not use a phase spectrum. This non-traditional outcome will open the door for wide industrial implementation of the novel cross-correlations of spectral moduli for predictive maintenance of various rotating machinery. Therefore, the future scope of the research is to prove its effectiveness for other MCSA and vibration applications.The results of this study are significant for damage diagnosis. The proposed cross-correlations of spectral moduli present a novel conceptualization and will make a considerable impact on damage diagnosis in electrical and mechanical engineering via motor current signature analysis, vibration analysis, ultrasound analysis, etc.


The hardness of heat  treated CoCrMo ingots, recycled by electron beam melting and refining (EBMR) of a technogenic CoCrMo material  under different process conditions (temperature and residence time) is examined. The heat treatment consists of two-step heating up to temperatures of 423 K and 1343 K and retention times of 40 and 60 min, respectively. The influence of various loads  on the hardness of the CoCrMo alloy, recycled by EBMR, before and after heat treatment is studied. It has been found that regardless of the EBMR process conditions, the obtained samples after heat treatment have similar hardness values (between 494.2 HV and 505.9 HV) and they are significantly lower than the hardness of the specimens before the heat treatment. The highest hardness (600 HV) is measured in the alloy recycled at 1845 K refining temperature for 20 min. This is due to the smaller crystal structure of the resulting alloy and the higher cobalt content. The results obtained show that the heat treatment leads to considerable changes in the microstructure of the CoCrMo ingots recycled by EBMR. With the increase of the e-beam refining temperature, after the heat treatment, the grains’ size increases and the grains’ shape indicates an incomplete phase transition from γ-fcc to ε-hcp phase. This leads to a slight increase in the hardness of the alloy. Over the last decades, CoCrMo superalloys have found wide application in medicine for the manufacture of dental and medical  implants. This is due to their high resistance to wear and corrosion, good mechanical properties and biocompatibility with the human body .The global market for CoCrMo alloys is constantly growing . It is eCoCrMo alloys can be produced using a traditional melting and casting method and other alternative methods such as vacuum casting, laser-based powder bed fusion of metals (PBF-LB/M), computer-aided design/computer-aided manufacturing, laser sintering, etc. ,6,7,8,9.The microstructure and mechanical properties of the CoCrMo alloys are strongly related to their chemical and phase composition, which can vary significantly depending on the method of their production, the cooling rate of the melt, the content of other alloying elements, etc. .Usually, CoCr alloys contain two types of crystal structures that determine their physical, chemical and mechanical properties: a cobalt-rich face-centered cubic γ-fcc phase stable at high-temperature and a low-temperature cobalt-rich hexagonal close-packed ε-hcp phase ,14,15.The temperature of the γ-fcc to ε-hcp phase transformation can be changed due to the presence of other alloying elements in the alloy . Element2O3) layer [Cobalt is the main metal that determines the mechanical properties  of the CoCrMo alloy, while chromium provides biocompatibility and corrosion resistance by forming a protective oxide (Cr3) layer .The impact of the cobalt content on the hardness of different commercial brands of CoCrMo alloy is investigated in . It is fThe influence of other alloying elements  on the hardness of CoCrMo alloy is investigated in . CoCrNi CoCrNi, CoCrW, and CoCrMo alloys are characterized by different ratios of the γ-fcc and ε-hcp phases, which determine their mechanical properties . Nickel The conducted research ,19 showsA comparative analysis of the mechanical properties  before and after heat treatment at a temperature of 1493 K for 1, 2, and 4 h of a standard CoCrMo alloy (ASTM F75) produced by the selective laser melting (SLM) method is conducted in . The autThe impact of loads of 0.98 N, 1.96 N, 2.94 N, 4.9 N, and 9.8 N on the hardness of a CoCrMo alloy obtained by traditional casting and using the SLM method is examined in . The harThe influence of production parameters such as laser power, feed rate and scan speed on the hardness of the resulting alloy is examined in . FurtherThe mechanical properties of several CoCrMo alloys produced under the electron beam melting process before and after heat treatment are tested in . The heaIn our previous work , based o−3 Pa and the refined melt is formed in a water-cooled copper crucible with a movable bottom connected to a draw mechanism [The microstructure and the hardness of the initial technogenic CoCrMo alloy and ingots obtained after recycling by EBMR, before and after heat treatment, are studied. echanism .The melting parameters in the furnace (refining temperature T and residence time τ) and the chemical composition of the initial alloy and the resulting ingots are given in The heat treatment of the initial alloy and the ingots obtained after recycling by EBMR includes the following stages. First, the samples are placed in a muffle furnace at a temperature of 294 K and heated to T = 423 K. At this temperature, the ingots are held for 40 min then they are heated to a temperature of 1343 K and retained for 60 min. The heating rate of the samples in the interval from 423 K to 1343 K is 9.2 grad/min. The heat treatment is carried out in an argon atmosphere.3 is used for etching [The preparation of the specimens for the metallographic examination involves the standard procedure of grinding, polishing and etching. A Glyceregia reagent prepared from 15 mL HCl, 10 mL glycerol, and 5 mL HNO etching . The timA Leica DM2500  light microscope with a digital camera Leica EC3  was used to study the macrostructure of the obtained specimens. The Leica LAS software  was used for image processing.The hardness of the studied samples is determined by the Vickers method using a Micro-Hardness Tester HV-1000 apparatus. The following loads are applied during the measurements: 0.98 N, 1.96 N, 2.94 N, 4.9 N, and 9.8 N.Microscopic examinations have been performed to determine the influence of the heat treatment of CoCrMo alloy on the macro and microstructure. A light microscope which allows the examination of alloy morphology, macro and microstructure characteristics, determination of grains’ shape, inclusions’ distribution, presence of mechanical defects, etc. was used.The phase transitions in CoCrMo alloys during cooling can be estimated using the binary phase diagram Co-Cr, calculated using Thermo-Calc and PBIN database ,15,21.The microstructure of technogenic  CoCrMo alloy after heat treatment—two-step heating to a temperature of 423 K and 1343 K and retention of 40 and 60 min, respectively, is shown in It can be seen that after the heat treatment, the initial alloy has a metastable dendritic matrix, and chemical and intermetallic compounds are precipitated along its boundaries. The formation of a dendritic structure proceeds parallel to the solid/liquid border surface in the direction of heat removal. Large crystals (150–200 μm) are observed, surrounded by crystallized intermetallic melts and chemical compounds formed by the Fe, Mn, Si, Nb, and W elements present in the alloy.In Since the samples obtained under different EBMR conditions were heat-treated under the same conditions , it can A comparative analysis of the structure of the CoCrMo alloys obtained at a minimum (T = 1830 K for 20 min) and at a maximum EBMR operating temperature (T = 1860 K for 20 min) after the heat treatment e,h indic1.8Cr phase, which can be obtained after the eutectoid decomposition of an ε-phase → Co3Cr+Co2Cr at T < 973 K in the presence of Mo and Si [After the alloy’s heat treatment (Co-02-ann), the dendritic structure is broken. The formation of a finer-grained structure in the Co-02-ann sample is most likely due to the higher Si content in the sample and formation of Coo and Si ,23,26,27One of the main parameters determining the practical applicability of CoCrMo alloys in the dental industry is their hardness. From a practical point of view, it is important to determine the influence of the melting parameters (refining temperature and retention time) on the hardness of the ingots obtained after recycling by EBMR. The hardness of the initial CoCrMo alloy and of the ingots obtained in the electron beam furnace was measured during the research. The melting conditions and chemical compositions of the ingots are given in The influence of load 0.98 N, 1.96 N, 2.94 N, 4.9 N, and 9.8 N on the hardness of the resulting ingots is determined. The hardness is measured using the Vickers method, with nine measurements made at each load. The calculated mean hardness values and standard deviations are given in The influence of the technological parameters of EBMR of the technogenic CoCrMo alloy and the cobalt content on the hardness of the resulting CoCrMo ingots under different loads is shown in 3Cr + Co2Cr) and the formation of finer grains [It can be seen that at refining temperatures of 1830 K and 1860 K for the same retention time (τ = 20 min), the hardness of the resulting ingots (measured at the highest load 9.8 N) increases from 520.3 ± 15.3 HV to 550.8 ± 21.9 HV. The highest hardness 600.0 ± 14.0 HV) is measured for the Co-02 ingot obtained at a refining temperature of 1845 K and a residence time of 20 min. This can be explained by the eutectoid decomposition of the ε-phase  and the ingots obtained after recycling by EBMR  is also investigated. The mean hardness values measured at different loads and the calculated standard deviations of the heat-treated CoCrMo ingots are given in The analysis of the resulting dependences (the hardness as a function of the load) shows that after the heat treatment of the alloy, its hardness decreases slightly compared to the initial material. The average hardness values for loads higher than 1.96 N remain almost constant and are 504.9 HV and 493.3 HV before and after the heat treatment, respectively.The influence of load on the hardness of CoCrMo alloys recycled by EBMR and after their heat treatment is shown in The analysis of the resulting dependences  shows thThe microstructure and, respectively, the mechanical properties of CoCrMo alloys, are determined by the speed of two main processes—cooling and solidification . Figure The same figure  comparesTherefore, regardless of the different conditions for obtaining the CoCrMo alloy, the necessary hardness can be achieved through appropriate heat treatment and cooling rate.As the e-beam refining temperature increases, at the same time of retention, the hardness of the recycled CoCrMo alloys, measured at the highest load of 9.8 N, increases from 520.3 HV to 550.8 HV. Extension of the residence time leads to an increase in the hardness of the alloy. The highest hardness (600 HV) is measured in the alloy recycled at 1845 K and a retention time of 20 min. This is due to the smaller crystal structure of the resulting alloy and the higher cobalt content.It has been found that regardless of the EBMR processing conditions, after the heat treatment, the hardness of the ingots varies within very narrow limits (from 494.2 HV to 505.9 HV) and is significantly lower than the hardness of the samples before their heat treatment.It has been found that the heat treatment process leads to considerable changes in the microstructure of the CoCrMo alloys recycled by EBMR. With the increase of the e-beam refining temperature, after the heat treatment, the grain size increases from ~50 to ~200 μm. The grains’ shape indicates an incomplete phase transition from γ-fcc to ε-hcp phase and this leads to a slight increase in the hardness of the alloy.It has been confirmed that the base metal that determines the hardness of CoCrMo alloys is cobalt. As the content of this metal in the alloy increases, its hardness also increases.Based on the conducted research regarding the impact of different electron beam melting parameters (temperature and residence time) and the heat treatment (under the same conditions) of ingots recycled by EBMR on the hardness of the CoCrMo alloys, the following conclusions can be made:Through appropriate heat treatment, desired hardness of CoCrMo alloys recycled from a technogenic material  by EBMR can be achieved.


The liver has a huge impact on the functioning of our body and the preservation of homeostasis. It is exposed to many serious diseases, which may lead to the chronic failure of this organ, which is becoming a global health problem today. Currently, the final form of treatment in patients with end-stage (acute and chronic) organ failure is transplantation. The proper function of transplanted organs depends on many cellular processes and immune and individual factors. An enormous role in the process of acceptance or rejection of a transplanted organ is attributed to, among others, the activation of the complement system. The aim of this study was the evaluation of the concentration of selected biomarkers’ complement system activation ) in the serum of patients before and after liver transplantation . The study was conducted on a group of 100 patients undergoing liver transplantation. There were no complications during surgery and no transplant rejection in any of the patients. All patients were discharged home 2–3 weeks after the surgery. The levels of all analyzed components of the complement system were measured using the ELISA method. Additionally, the correlations of the basic laboratory parameters—C-reactive protein (CRP), hemoglobin (Hb), total bilirubin, alkaline phosphatase (ALP), alanine aminotransferase (ALT), aspartate aminotransferase (AST), gamma-glutamyl transpeptidase (GGTP), and albumin—with the parameters of the complement system  were determined. In our study, changes in the concentrations of all examined complement system components before and after liver transplantation were observed, with the lowest values before liver transplantation and the highest concentration two weeks after. The direct increase in components of the complement system  24 h after transplantation likely affects liver damage after ischemia-reperfusion injury (IRI), while their increase two weeks after transplantation may contribute to transplant tolerance. Increasingly, attention is being paid to the role of C3a and CRP as biomarkers of damage and failure of various organs. From the point of view of liver transplantation, the most interesting correlation in our own research was found exactly between CRP and C3a, 24 h after the transplantation. This study shows that changes in complement activation biomarkers and the correlation with CRP in blood could be a prognostic signature of liver allograft survival or rejection. Approximately 8000 liver transplants are performed each year in the world according to the Organ Procurement and Transplantation Network, and the three-year patient survival outcome is approximately 80%. The increasing number of patients undergoing liver transplantation from year to year implies a global challenge and the need to search for new, more effective, and safer methods of achieving immune tolerance in this group of patients after the transplantation of this organ. It also gives hope to many patients waiting for effective therapy to regenerate the damaged liver earlier and avoid last-resort transplantation. The proper function of the transplanted organ depends on many cellular and immunological processes and individual features. It is believed that factors contributing to the migration, adhesion, differentiation, and survival of cells significantly impact the liver transplant process. Therefore, it is postulated that the presence of an immunologically incompatible organ and its damage in the mechanism of ischemia-reperfusion injury (IRI) may be the result of the impaired activation of chemokines ,2,3 and Anaphylatoxins of the complement system contribute to inflammation and adaptive immune responses ,24. TheySalerno et al.  observedShah et al.  also resC5b-9 (membrane attack complex—MAC—C5b-9) is a final product of the complement system and plays an essential role in the immune system . Recent Many parameters are necessary for the clinic to monitor the liver and the patient’s condition after transplantation. In the face of the above facts and public health, the aim of this study was to assess the changes in the concentration of selected components of the complement system, molecules directly related to the activity of the nonspecific immune response, in the serum of 100 patients before and after liver transplantation . At this stage of the research, we decided to determine the levels of three biomarkers of complement system activation , which are involved in initiating inflammatory processes that may lead to rejection of the transplanted organ. Additionally, the correlations of the basic laboratory parameters with the parameters of the complement system  were determined. The study was conducted on a group of 105 patients (45 women and 60 men) aged 20 to 68 years undergoing liver transplantation at the Department and Clinic of General, Transplant, and Liver Surgery of the Medical University of Warsaw. There were no complications during surgery and no transplant rejection in 100 patients . During the planned collection period, 4 out of 5 patients did not live to the second time point, i.e., 24 h after transplantation, while the last patient died and did not live to the third time point (2 weeks after transplantation). All patients without complications were discharged home 2–3 weeks after the surgery. Demographic and laboratory data on analyzed patients are summarized in To determine the examined factors, approximately 8 mL of venous blood was collected from patients in tubes with a clotting activator to obtain a serum fraction. The material was collected immediately before liver transplant surgery, on the first day after surgery, and two weeks after transplantation. The peripheral blood was then centrifuged and transferred to new tubes and kept at −80 °C until the samples were transported on dry ice to the Medical Analytics Department of the Pomeranian Medical University, where all analyses were performed.The concentration of selected components of the complement system was measured in all samples, both before and after liver transplantation. For C3a and C5a, it was necessary to evaluate the levels of their breakdown products, C3a-desArg and C5a-desArg, because both C3a and C5a are unstable in vivo and are very quickly converted by endogenous carboxypeptidase N to these less active but much more stable forms . TherefoCRP was determined by an immunoturbidimetric assay in the serum on the Cobas 6000 module c501 biochemistry analyzer from Roche . Other parameters, such as total bilirubin, ALP, ALT, AST, GGTP, and albumin, were determined in the serum using the spectrophotometric method and the Cobas 6000 module c501 biochemistry analyzer from Roche . Similarly, optical detection in EDTA whole blood determined Hb using the MEK-7300 hematology analyzer . All determinations were performed in the Central Laboratory of the University Clinical Center of the Medical University of Warsaw according to the manufacturer’s instructions at the same time points (before transplantation (0 h), 24 h after transplantation, and 2 weeks after transplantation).The results were evaluated using the RStudio program and the Statistica PL 13 statistical package . The Anderson–Darling test was performed to assess the distribution of variables. Due to the fact that the compared factors were time-related variables, the analysis of their differences was based on paired tests. The Friedman ANOVA test and the Kendall compliance test were used to determine possible changes in all tested factors’ concentrations. The Wilcoxon signed-rank test was performed to assess the differences in parameter concentrations. Additionally, the correlations of basic laboratory parameters  with the tested components of the complement system were performed using Spearman’s correlation test.Our studies showed that the highest C3a-desArg concentrations occur two weeks after liver transplantation (5869 ng/mL), while the lowest occur directly before transplantation (4639 ng/mL) .p < 0.0001)  .p < 0.0001) (Our studies showed that the highest C5a-desArg concentrations occur two weeks after liver transplantation (100 ng/mL), while the lowest occur before transplantation (42 ng/mL) . The ana 0.0001) .p < 0.0001) (Our studies showed that the highest sC5b-9 concentrations occur two weeks after liver transplantation (1901 ng/mL), while the lowest occur before transplantation (574 ng/mL) . The ana 0.0001) .During the planned collection period, four out of five patients did not live to the second time point, i.e., 24 h after transplantation, while the last patient died and did not live to the third time point (2 weeks after transplantation). Demographic and laboratory data on analyzed patients are summarized in Our studies on the five patients showed that the concentrations of C3a, C5a, and sC5b-9 before transplantation were 6408 ng/mL, 66 ng/mL, and 985 ng/mL, respectively . Only onAs part of the statistical analysis, Spearman’s correlation test was performed for laboratory parameters  with the tested complement system components .No correlation was observed with any of the components of the complement system tested in the case of ALP, total bilirubin, GGTP, and albumin.p = 0.029)  A.p = 0.006 for ALT, r = −0.3070, p = 0.002 for AST)  B,C. p = 0.010) and sC5b-9  in the same period  or autoimmune diseases such as primary biliary cirrhosis (PBC) ,24,30. Ronholm et al.  studied The research shows that all three pathways of the complement system may play an essential role in the IRI mechanism in the newly transplanted liver. Activation of C3 and C5, and consequently the anaphylatoxins C3a and C5a, triggers the inflammatory response and formation of sC5b-9 . IncreasThe balance between immunity and tolerance usually determines the acceptance or rejection of a transplanted organ . StudiesWe would also like to pay attention to studies on a small group of patients who died in the peri-transplant period that show a tendency in which the concentrations, regardless of the tested component of the complement system, are initially higher than in the group of patients without complications. It is quite interesting, but we did not decide to perform statistical analyses due to the very small research group, and in the future, we will plan to perform these analyses on a larger group of deceased patients. On the other hand, based on our previous studies in a group of healthy people , we alsoIn our research, we also noticed a few interesting correlations of laboratory indicators with the components of the complement system we studied. In the cases of ALT and AST, we observed negative correlations between the levels of these liver enzymes and the concentration of C3a before transplantation. The liver enzymes AST and ALT are important biomarkers of liver damage or cirrhosis. Due to their intracellular nature during disease, they are released into the serum due to necrosis and apoptosis of hepatocytes . The C3 p <0.0001) more than 3-fold increase in CRP levels 24 h after transplantation, compared to the baseline level (From the point of view of liver transplantation, the CRP correlation with C3a, occurring 24 h after the liver transplantation, seems to be the most interesting. CRP is an acute-phase protein, synthesized by the liver, the synthesis of which begins approximately 6 h after the inflammatory injury .CRP is ane level , which pIn addition, increasing attention is being paid to the role of C3a and CRP as biomarkers of damage and failure of various organs. Gombos et al.  show thaThe lowest concentrations of the studied factors—C3a-desArg, C5a-desArg, and sC5b-9—occur right before transplantation and then grow to reach the highest values two weeks after. The lowest pre-transplantation values are a consequence of the primary disease causing the reduction in the synthesis of components of the complement system. We suspect that transplantation itself activates the complement system, which causes a direct increase in the concentration of its components and may lead to liver damage caused by the IRI mechanism. In turn, we assume the highest concentrations of the factors found two weeks after transplantation may contribute to the emergence of transplant tolerance, but this aspect requires further research. Therefore, we wonder whether the observation of changes in the concentration of complement system components could help monitor the function of the transplanted liver after surgery. In addition, the correlation of CRP with C3a 24 h after transplantation may suggest that the latter protein may behave as an acute phase protein in a transplant and may be an additional biomarker of the stratification risk of transplant acceptance/rejection, and in our particular case, the prognostic signature of liver allograft survival. There is no doubt that a better understanding of the role of the various components of the complement system and its interaction with other parameters could help develop new therapies that suppress complement components and improve graft survival.


The treatment of inoculation of white cast iron with carbide precipitations that consist of increasing the number of primary austenite grains is not as well-known as the treatment of inoculation of gray cast iron in which the number of eutectic grains increases. In the studies included in the publication, experiments were carried out using the addition of ferrotitanium as an inoculant for chromium cast iron. The Cellular Automaton Finite Elements (CAFE) module of ProCAST software was used in order to analyze the formation of the primary structure of hypoeutectic chromium cast iron in a casting of various thicknesses. The modeling results were verified using Electron Back-Scattered Diffraction (EBSD) imaging. The obtained results confirmed obtaining a variable number of primary austenite grains in the cross-section of the tested casting, which significantly affects the strength properties of the obtained chrome cast iron casting. Therefore, the microstructure of the casting walls of various dimensions will differ not only in the size of a given phase but also in the phase composition. In general, the structure of white chromium cast iron can be classified according to the range of alloy chemistry, phase growth rate and temperature gradient. These parameters allow hypoeutectic, eutectic and hypereutectic structures to be obtained in cast iron.As a pre-eutectic phase and as a phase included in the eutectic grain, there is Mhttps://www.en-standard.eu/din-en-12513-founding-abrasion-resistant-cast-irons/ accessed on 5 March 2023 or https://sklep.pkn.pl/pn-en-12513-2011e.html accessed on 5 March 2023). Based on the data presented in 7C3 type carbides, thereby increasing its share in the metal matrix. Molybdenum is introduced into high-chromium cast iron to prevent the separation of pearlite in the metal matrix of high-chromium cast iron. If pearlite appears in the metal matrix of this cast iron, it will mean a significant decrease in the abrasion resistance of the cast iron.To obtain the required hardness given in 2C and 7C3 types and is partially dissolved in a solid solution. Type 15-3 cast iron can be used in the unfinished state when the wall thickness of the casting is small, with the possible use of stress relief annealing at a temperature of 200–250 °C. To obtain a high hardness value, this type of cast iron is hardened in air and then possibly tempered. In general, it must be said that the PN-EN 12513 standard for high-chromium cast iron castings allows the following types of heat treatment: tempering, hardening, hardening and tempering, soft annealing, soft annealing and hardening, soft annealing, hardening and tempering. However, in the case of the need for mechanical treatment of castings made of high chromium cast iron, soft annealing is recommended. If the order concerns castings after soft annealing, it is the client who is responsible for the correct course of the subsequent treatments: hardening and tempering.In the group of high-chromium cast iron resistant to abrasive wear, there is a widespread type 15-3 chromium-molybdenum cast iron (known as Climax Alloy) containing 14 ÷ 16 wt% chromium and 2.4 ÷ 3 wt% molybdenum. Grades of this type of cast iron are included in the US standard ASTM A532 as Class II Types B and D and designated as 15% Cr-Mo-LC (low carbon) and 15% Cr-Mo-HC (high carbon). In fact, it is a grade of white martensitic cast iron and its equivalent, according to the PN-EN 12513 standard, is a grade classified in the group of high-chromium white cast iron as EN-GJN-HV600(XCr14) with some modifications in the composition of alloying elements such as wt% Ni + wt% Mo + wt% Cu. It must be noted that this cast iron shows very good resistance to abrasive wear while maintaining good plastic properties and good corrosion resistance. The molybdenum in this alloy is bound in the form of carbides of the MoChromium cast iron is used in the production of castings that must be characterized by very good wear resistance and good corrosion resistance.The casts produced may be several millimeters thick, for example, the cast wall of a defibrating machine used for the production of wood pulp for the production of MDF plates, which can be up to several dozen millimeters, or the cast of slurry pump bodies used in mines. In articles ,4, the aThe phase composition of chromium cast iron described above results from the theoretical analysis of cross-sections of the polythermal triple phase equilibrium system of Fe-C-Cr alloys determined for the designed chemical composition.In the literature, another important approach to the inoculation procedure can be found, which consists of the production of other types of carbides in the structure of chrome cast iron, for example, TiC, NbC and others ,16,17,18In the Laboratory of Computer Modelling of Crystallization Processes, modeling of the primary structure was carried out for the thickness of 20 mm, 30 mm and 40 mm. The model was carried out for a fragment of an infinite plate with the thickness mentioned above, located between the two layers of bentonite mass, 50 mm thick each, as in The length of the sample in the direction of heat dissipation (L) is 20, 30 and 40 mm, respectively. The cross-section of the area for which the results of the microstructure simulation with the CAFE method were presented has dimensions of 10 × 10 mm b. In theFew examples are known of the application of the CAFE method to simulate the microstructure of hypoeutectic alloys ,29,30. TThe model does not take into account the possibility of nucleation and grain growth processes in the area beyond the outer boundaries of the buffer layers. For this reason, the microstructure of the outer zone of the buffer zone obtained in the simulation is not a good representation of the microstructure of the primary grains. The purpose of using such a buffer area was to eliminate the impact on the microstructure of the analyzed area of the inability to account for the influence of nucleation and grain growth in the area located beyond the outer lateral surface of these buffer areas. When analyzing the simulation results, the microstructure of the buffer zone was not taken into account. All simulation results shown below were obtained in the analysis area with a 10 × 10 mm cross-section.The sample is located between the two layers of molding sand of the same cross-section and 50 mm thickness in the direction of heat dissipation on each side. The adiabatic condition was assumed on the side surfaces of the entire temperature field modeling area. On the front walls of the molding sand area (15 × 15 mm), the condition of air cooling was adopted.For the prediction of the primary grains structure, the Cellular Automata technique is used in the ProCAST software. The processes of the nucleation and growth of the solid phase grains from the liquid are analyzed based on a simulated temperature field. Detailed information on the simulation method using the cellular automaton technique can be found in the publications ,33,34,353; c—heat capacity, J/(kg × K);ρ—density, kg/mT—temperature, K; τ—time, s;λ—thermal conductivity coefficient, W/(m × K);q—rate of heat of crystallization release (source function), W/m3;div and grad—mathematical operators of divergence and gradient.For the prediction of the temperature field, the Fourier equation is solved numerically by using the Finite Elements Method as follows:T. The model assumes that the dependence of the fraction of substrates for the heterogeneous nucleation of primary austenite grains on the undercooling at which their activation and nucleation occur follows the Gaussian statistical law. The parameters of this normal distribution are the mean value of undercooling (ΔmT), at which the rate of increase in the number of growing grains with increasing undercooling increases more rapidly, and the standard deviation (ΔTσ).In the model, 3D approach is used for the estimation of the primary austenite grain number and shape with an account of the local undercooling at the solid-liquid interface Δn, on which the primary austenite grains nucleate and grow, increases until maximum undercooling is reached before recalescence. The rate of nucleation with increasing undercooling is calculated based on the probability density function of the normal distribution as follows:nmax is the maximum number of heterogeneous nucleation substrates generated in the alloy as a result of the inoculation treatment.With undercooling increasing, the number of activated nucleation substrates The number of grains is calculated by integrating Equation (2) during the period of increasing undercooling to a maximum value. Increasing the temperature during recalescence, which causes a decrease in undercooling, terminates the nucleation.a2 and a3 are growth kinetics coefficients.The velocity of the interface migration at the dendrite grain tip  and a Cellular Automata (CA) technique for primary microstructure prediction is coupled by the source function term of Equation (1).3 to 6840 kg/m3. The corresponding changes in the thermal conductivity coefficient in the same range of temperature changes were 23,000 to 25,100 W/(m·K), and the changes in specific heat were 905 J/(kg·K) to 810 J/(kg·K).The initial alloy temperature was assumed to be 1400 °C, while the initial molding sand temperature was 20 °C. The density of the analyzed alloy used to simulate the temperature field by means of Equation (1) in the temperature range of 1200 to 1400 °C varies from 7180 kg/mTo verify the results of the simulation, chromium cast iron was melted. Melts were carried out at Odlewnie Polskie S.A. in Starachowice. A medium-frequency induction furnace with a capacity of 120 kg located in the Research and Development Centre of Foundry Components “OBRKO” was used for the tests. Plates with dimensions of 100 × 100 mm and thicknesses of 10, 20, 30 and 40 mm were cast. The appearance of the research casting is shown in The EBSD microstructure analysis was performed for three thicknesses: 20, 30 and 40 mm. The chemical composition of the inoculated cast iron is presented in -from a 40 mm plate, the center of the sample is 0.5 of the thickness of the plate;-from a 30 mm plate, the center of the sample is 0.18 of the thickness of the plate;-from a 20 mm plate, the center of the sample is 0.12 of the thickness of the plate.From the cast plates, samples were cut for analysis on a scanning microscope with an EBSD detector as follows:Primary microstructure analysis was performed using a high-resolution scanning electron microscope FEI Quanta 3D FEGSEM with the EDAX Trident system  located in the Institute of Metallurgy and Materials Engineering, Polish Academy of Sciences in Kraków.9 to 1011 are shown in The results of the simulation with a given number of nucleation sites from 109 m−3), there is no zone of equiaxial fine crystals near the casting surface. In this zone, columnar grain structure prevails. The depth of the zone of columnar grains is similar for each analyzed variant. Increasing the number of nucleation substrates results in a marked increase in the number of fine equiaxial grains near the casting surface  and the volumetric grain density NV (6) were calculated according to the formula. The calculation results are presented in W—number of whole grains in the area; NP—number of grains cut by sides of the area; NR—number of grains in corners of the area; NA—surface grain density 1/mm2; P—area mm2; NV—volumetric grain density 1/mm3.N—number of grains in the area; NThe FEI Quanta 3D FEGSEM high-resolution scanning electron microscope was used for the analysis. On the basis of the results, pictures of EBSD microstructures and simulation imaging were matched. The results are shown in Research has shown that the use of cellular automaton simulation allows analysis of the number of primary austenite grains produced in the structure of a chrome cast iron casting. In addition, the images from the simulation clearly show a large influence of the casting wall thickness on its structure. The number of primary austenite grains at the edge of the wall increases significantly in relation to the central part of the casting. Therefore, the grain measurements made on the central surface of the casting are inadequate for the number of grains located on the edge of the casting.Simulation with a cellular automaton in the ProCAST program allows for predicting the number of primary austenite grains in chrome cast iron by controlling the number of nucleation sites on the cross-section of the casting.The number of grains determined by the ProCast simulation, after verifying it at selected points of the cross-section of the casting using the EBSD method, allows for faster determination of the number of grains than in the case of using only the EBSD method over the entire cross-sectional area of the casting.