Normotensive male Wistar–Kyoto rats (WKY) were allocated to three groups. Three analogous groups of spontaneously hypertensive rats (SHR) were also formed. The WKY and SHR rats received copper in a standard daily dose—6.5 mg/kg CuCO3 or CuNPs (100% replacement) or 3.25 mg/kg CuCO3 plus 3.25 mg/kg CuNPs (50% replacement)—for 8 weeks. Next, blood, heart, small intestine, liver, and kidney samples were collected. The activity of alanine aminotransferase, aspartate aminotransferase, creatine kinase, and gamma-glutamyl transferase and the content of creatinine and urea acid were measured in the plasma. The collected tissues were subjected to a histological evaluation, and redox status parameters  were determined. The replacement of CuCO3 with CuNPs in the diet may exacerbate the negative changes induced by hypertension in the heart, liver, and intestines. However, it seems that it is only in the case of the liver where the observed changes may be due to an increase in oxidative reactions resulting from the inclusion of CuNPs.The aim of this experiment was to test the effect of the partial or complete replacement of traditional CuCO Hypertension is one of the most common chronic diseases of the modern world and significantly increases the risk of premature death . HyperteIt is believed that the effects of increased oxidative stress accompanying hypertension can be mitigated by strengthening the body’s antioxidant defences . One of 3. This compound is relatively poorly absorbed by the body. Only 15% of Cu from inorganic forms is absorbed by the body, while the remaining 85% is excreted. For this reason, alternative forms of copper are being sought that would be better utilized and thus positively influence Cu-dependent metabolic processes, including antioxidant defences. One of the most interesting options seems to be Cu nanoparticles (CuNPs) [3 with CuNPs in the diet of rats reduced the oxidation and nitration of proteins and DNA in the blood [The bodies of humans and animals cannot accumulate copper reserves, so a steady source of it must be provided in the diet. The standard form of Cu in diet supplements for both people and animals is the inorganic form, CuCOhe blood , as wellhe blood . Therefohe blood ,19,20,21he blood . Thus, ihe blood , which c3) in their diet with Cu nanoparticles (CuNPs) may reduce oxidation reactions and their adverse effects on these organs. The aim of the experiment was to test the effect of the partial (50%) or complete (100%) replacement of traditional CuCO3 in the diet of rats with copper in the form of CuNPs on selected blood biochemical parameters, redox status, and the histomorphometry of the liver, kidneys, intestine, and heart.Therefore, we postulated that in hypertensive rats, there is an increased induction of oxidative processes that negatively affect the functionality of the liver, kidneys, intestine, and heart, and the partial or complete replacement of the standard form of Cu (CuCO2/g specific surface area (SSA), spherical morphology, 0.19 g/cm3 bulk density, 8.9 g/cm3 true density) were purchased from SkySpring Nanomaterials Inc., . A stock solution (5 g/L) was prepared in rapeseed oil, and about 9% of the NPs were dissolved as Cu (II) ions; thus, the final suspension contained both NPs and released copper species. The zeta potential of the copper NP suspension was determined to be −30.3 mV ) and −38.3 mV (pH 5), and the size was 104 nm (in rapeseed oil), determined by dynamic light scattering with a Zetasizer Nano ZS  [Metal-based copper nanoparticles  .n = 10 each) and individually fed experimental diets for 8 weeks under standard laboratory conditions. Three analogous groups of spontaneously hypertensive rats (SHR/NCrl) were formed . Only male rats were studied to enable comparison with the previous experiments. The experiment was carried out in compliance with the European Guidelines for the Care and Use of Laboratory Animals and was approved by the Local Ethics Committee for Animal Experiments in Olsztyn Local Animal Care and Use Committee . The study was carried out in compliance with the ARRIVE guidelines. Every effort was made to minimize the suffering of the animals used in the experiment. The rats were fed additional copper in a standard daily dose of 6.5 mg/kg, either as carbonate salt (CuCO3 6.5) or metal NPs (NP6.5). The third group (CuCO3 3.25 + NP3.25) additionally received 3.25 mg of copper NPs plus 3.25 mg of copper carbonate. CuCO3 was added as a dietary ingredient in the mineral mixture, while the copper nanoparticles were supplied together with dietary rapeseed oil (for the safety of the person preparing the diets) [Twenty-four-week-old normotensive Wistar–Kyoto (WKY/NCrl) rats from Charles River  were allocated randomly to 3 groups (e diets) . Semi-pue diets) .n = 8 for each group). At the end of the experiment, the rats were fasted for 24 h and anaesthetized intraperitoneally with ketamine and xylazine ; X, 10 mg/kg BW) according to the recommendations for the anaesthesia and euthanasia of experimental animals. Then, blood was collected from the vena cava and centrifuged at 3000× g for 10 min to separate the plasma, which was further stored at −80 °C until analysis. Finally, the rats were euthanized by cervical dislocation, and the heart, small intestine, liver, and kidney were removed. Samples of the heart, small intestine, liver, and kidney were preserved for histopathological examination, and homogenates from these organs were prepared and stored at −80 °C until analysis.All physiological measurements were made separately for each animal , aspartate aminotransferase (AST), creatine kinase (CK), and gamma-glutamyl transferase (GGT), as well as the content of creatinine (CREAT) and urea acid (UA), in the plasma were measured using an automatic biochemical analyser .Indicators of antioxidant status, i.e., the activity of superoxide dismutase (SOD) and catalase (CAT) and the concentrations of malondialdehyde (MDA) and total glutathione (GSH + GSSG), were determined in the homogenates of the heart, small intestine, liver, and kidney according to the procedure described in .Samples of the heart, small intestine, liver, and kidney were cut in half lengthwise and fixed for 24 h in 5% formalin, pH = 7.2. Within 24 h, the fixed tissue fragments were passed through increasing concentrations of alcohol solutions, acetone, and xylene into paraffin blocks in a tissue processor . Paraffin-embedded microscope sections 5 μm thick were stained with haematoxylin and eosin (HE staining). A morphometric evaluation of the organs was carried out using a computer-assisted microscopic image analysis system. The system included a light microscope  with a digital camera  and a PC with image-analysis software .n = 10) were considered as experimental units. The data were subjected to two-way ANOVA to examine the following effects: (a) C, the main effect of the Cu source (CuCO3 6.5 or CuNP6.5 or CuCO3 3.25 +CuNP3.25); (b) R, the main effect of the rat strain (normotensive WKY or spontaneously hypertensive SHR); and (c) the interaction between C and R (C×R). When a significant C×R interaction effect was noted (F test), the treatment means were separated using the post-hoc Tukey test. All calculations were performed using the generalized linear model (GLM) procedures of the STATISTICA software ver. 13.1 . Data variability was expressed as means with a pooled standard error of the mean (SEM), and p < 0.05 was considered statistically significant.For the analysis of parameters, individual rats , while the CREAT level was lower  than that of WKY rats (The activity of ALT and CK was higher in the plasma of the SHR rats in comparison to the WKY rats (WKY rats .3) with CuNPs in the diet of rats increased the activity of AST and CK in the plasma . In the case of 100% replacement of CuCO3 with CuNPs, a reduction in ALT activity (p = 0.023) was noted in the plasma of rats relative to those for which the CuCO3 additive was only partially (50%) replaced with CuNPs  or completely replaced with CuNPs resulted in an increase in SOD activity (p < 0.001) in the heart. In addition, 50% replacement of CuCO3 with CuNPs was found to reduce CAT activity in the heart tissue, while 100% replacement increased it (p = 0.007) (The administration of a diet in which the standard addition of CuCO= 0.007) .3 was fully replaced with CuNPs showed higher levels of MDA and GSH + GSSG . SOD activity (p < 0.001) was also increased in the liver of rats receiving a diet in which CuCO3 was 50% replaced with CuNPs; however, it was reduced when CuCO3 was 100% replaced with CuNPs. Both 50% and 100% replacement of CuCO3 with CuNPs reduced CAT activity (p < 0.001) in the liver of rats  in the kidney. In the case of GSH + GSSG, this effect was more pronounced when CuCO3 was completely replaced with CuNPs, while the effect on SOD activity was more pronounced in the case of 50% replacement. Only 100% replacement of the standard form of copper (CuCO3) with CuNPs caused an increase in CAT activity (p = 0.008) in the kidney (3 was completely replaced with CuNPs, (p < 0.001; The replacement of 50% or 100% of the CuCOe kidney . In addiThe histological images in 3 in their diet revealed low-grade generalized congestion and fatty degeneration (3 was 50% replaced with CuNPs; however, local foci of fatty degeneration were much more numerous (3 in their diet, as in the case of the WKY rats, the histological image of the heart muscle revealed low-grade congestion and generalized fatty degeneration (3 with CuNPs resulted in severe local congestion and numerous small foci of fatty degeneration in the heart muscle of SHR rats (The histological image of the heart muscle from WKY rats receiving 6.5 mg CuCOneration A. The genumerous B. In thenumerous C. In theneration D. The reSHR rats E. On theSHR rats F.3 (3 was replaced with CuNPs showed no significant pathological changes in the tissue (3 was fully replaced with CuNPs (3 showed no pathological changes (3 additive with CuNPs in the diet of SHR rats revealed isolated lesions in the mucous membrane and the base of the villi, as well as single gaps in the tips of the villi (3 was fully replaced with CuNPs resulted in numerous lesions in the mucous membrane and the base of the villi (The histopathological examination of the intestine revealed generally isolated extensive lesions in the base of the villi in WKY rats receiving the recommended copper additive in the standard form of CuCO3 A. The hie tissue B, as in th CuNPs C. The hi changes D. The pahe villi E. A diethe villi F.3 (3 was completely replaced with CuNPs showed numerous large foci of fatty degeneration (3 exhibited characteristics of fatty degeneration in combination with isolated foci of congestion (3 with CuNPs in the diet of SHR rats resulted in the appearance of scattered small foci of fatty degeneration in combination with isolated large foci, as well as low-grade congestion in the tissue (3 with CuNPs caused numerous extensive foci of fatty degeneration in the liver (The histopathological examination of the liver of rats revealed generalized congestion in the tissue and isolated foci of fatty degeneration in the WKY group receiving the recommended copper additive in the standard form of CuCO3 A. The hi3 B. The lineration C. The lingestion D. The pae tissue E. The cohe liver F.3 or a diet in which 50% or 100 % of the CuCO3 was replaced with CuNPs (3 additive showed numerous local foci of congestion in combination with local foci of fatty degeneration (3 with CuNPs in the diet of SHR rats caused local foci of fatty degeneration in combination with generalized congestion of the kidney (The histopathological evaluation of the kidney revealed generalized congestion of the tissue in combination with local foci of fatty degeneration, both in the WKY rats receiving a diet that contained the recommended copper additive in the standard form of CuCOth CuNPs A–C.The hneration D. Both 5e kidney E,F, whice kidney F.It seems important to emphasize that the pharmacological preparations used to euthanize the rats may have had some effect on the results of the biochemical analysis of the blood and the occurrence and severity of pathological changes in the organs. While this hypothesis cannot be verified at this stage of research, a thorough analysis of the results requires that this potential influence be mentioned.The available literature indicates that hypertension can lead to damage to numerous internal organs. The most susceptible, however, seem to be the heart, central and peripheral arteries, kidneys, central nervous system, and eyes ,27. Ther3 with CuNPs increase fatty degeneration, and 50% replacement of CuCO3 with CuNPs additionally increases congestion in the hearts of both normotensive and hypertensive rats. The discrepancies observed between the results of our own research and that of Jing et al. [The results of our study in rats showed that both 50% and 100% replacement of the standard Cu source in the diet with CuNPs resulted in damage to the heart muscle, as indicated by increased CK activity in the tissue. Similarly, Jing et al.  noted a g et al.  may be dg et al.  used a sg et al.  indicate3 with CuNPs in the diet increased ALT activity in the plasma, which indicates that as the level of CuNPs in the diet is increased, the severity of tissue damage increases. This is fully confirmed by the histological image of the liver from the experimental rats. It should be noted, however, that in hypertensive rats, both 50% and 100% replacement of CuCO3 with CuNPs caused visible morphological changes in the liver, and their severity increased with the dose of nanoparticles. In normotensive rats, pathological changes only appeared when CuCO3 was entirely replaced with CuNPs. The results of the histopathological examination of the small intestine were also interesting; they showed that 50% and 100% replacement of CuCO3 with CuNPs in the diet of normotensive rats did not worsen, and even improved, the morphology of the intestines, whereas in hypertensive rats, it increased the severity of the pathological changes in the form of damage to the basement membrane and villi. In light of these findings, it seems likely that the body’s response to CuNPs may depend on the blood pressure level. Hypertension seems to make the liver and intestines more sensitive to the toxic effects of CuNPs, most likely due to an overall increase in metabolism, and thus in the metabolism of CuNPs.According to the available literature, Cu in the form of nanoparticles can also have toxic effects on the liver . Eid et 3 with CuNPs in the diet had no effect on this organ in both normotensive and hypertensive rats.The results of studies by Mohammadyari et al.  and LiaoInternal organ damage observed in patients with hypertension is believed to be an effect of haemodynamic overload caused by increased blood pressure . However3 [3 with CuNPs in the diet of rats increased SOD activity in the heart. The complete replacement of CuCO3 with CuNPs additionally increased CAT activity in this tissue. This suggests that CuNPs beneficially stimulate antioxidant defences, which is additionally evidenced by the lack of negative changes in the glutathione and MDA levels. Similarly, Jing et al. [3 with CuNPs, due to the lack of a negative effect on the activity of antioxidant enzymes or an increase in the lipid peroxidation processes, this could not be linked to the disturbances in the antioxidant response. This suggests that the heart and intestinal damage observed in our study in the groups receiving CuNPs was not induced by free radicals.Copper, as a cofactor of Cu/Zn SOD and by binding to ceruloplasmin, enhances the body’s antioxidant defences. On the other hand, as a transition metal, it undergoes Fenton and Haber–Weiss reactions, thereby increasing the synthesis of free radicals. According to the available literature, CuNPs, owing to their better bioavailability and greater reactivity, can have a much stronger effect on redox status than their standard counterparts, such as CuCO3 ,17. Our g et al.  found no3 with CuNPs reduced CAT activity while increasing SOD activity in the liver. The uncoordinated activity of the antioxidant enzymes SOD and CAT observed in the liver of rats receiving the lower level of CuNPs may thus increase oxidative stress, as the increased dismutation of O2− by SOD significantly increases the concentration of H2O2, which can lead to tissue damage if there is no compensation by the reduced CAT activity [3 with CuNPs, there was a reduction in CAT activity accompanied by an increase in MDA and GSH levels in the tissue. While the increase in the level of glutathione, an endogenous antioxidant, is somewhat surprising and difficult to explain, the increase in lipid peroxidation and the accompanying reduction in CAT activity indicates a disturbance in the antioxidant defences of the liver. This suggests that the liver damage observed in our study, confirmed by the biochemical analysis and histopathological examination, may be directly linked to the pro-oxidative effects of CuNPs. Moreover, it is highly likely that the CuNPs used in hypertensive individuals, instead of mitigating the adverse changes in the morphology and function of the liver caused by hypertension, may only exacerbate them. Nanoparticles are very small in size; owing to this, they have the ability to penetrate biological membranes, block ion channels, inhibit enzymatic proteins, and interact with genetic material, which may result in the development of oxidative stress [According to the available literature, the liver is particularly susceptible to the toxic effects of CuNPs ,34. Reseactivity . Moreovee stress . Researce stress , which ue stress  reportede stress  found th3 with CuNPs in the diet of rats adversely affected antioxidant defences and increased lipid peroxidation in the kidney [3 with CuNPs, irrespective of blood pressure. The complete replacement of CuCO3 with CuNPs additionally increased CAT activity in the kidney. This suggests that the experimental factor had a beneficial effect on antioxidant status, which increased with the level of CuNPs. The simultaneous improvement in the biochemical parameters of the kidneys due to the inclusion of CuNPs in the diet suggests that they can be used to mitigate the negative effects of hypertension in this tissue.Lei et al.  showed te kidney . The res3 with CuNPs in the diet of rats may exacerbate the negative changes induced by hypertension in the heart, liver, and intestines. Nevertheless, it seems that it is only in the case of the liver where the changes observed may be due to an increase in oxidative reactions resulting from the inclusion of CuNPs in the diet of rats.The results of our study indicate that the replacement of CuCO


Twenty sows were allocated to one of two groups: (1) 1 day after weaning  and (2) 8 days after weaning . The basis of the sow diet was SK-1 compound feed, balanced in terms of nutrients and energy in accordance with modern standards and the recommended feeding regimen. Sows blood samples were taken and analyzed for the metabolites of nitrogenous, carbohydrate-lipid, and mineral metabolism and indicators of antioxidant status. The results showed that, in group 2, the total protein content was 89.07 g/l, which is 10.2% higher than that in group 1 (p < 0.05); it was mainly achieved due to the globulin fraction. The urea increased by 19.1% (p < 0.05), but the concentrations of magnesium and chlorides decreased by 20.2% (p < 0.01) and 5.43% (p < 0.05), as well as the alkaline phosphatase and ALT activities decreased by 42.5% (p < 0.05). Strong positive correlations of the ceruloplasmin with total protein (0.672) and very strong with globulins (0.780) were observed. There was a strong negative correlation between the AST activity and the TBA-AP content, as well as the values of phospholipids and TAWSA. There are moderate negative correlations of the TBA-AP with magnesium, TAWSA and ALT activity, and moderate positive correlations of the TBA-AP with total protein, albumin, triglycerides, and cholesterol. The revealed tendencies and dependencies will serve as the theoretical basis for the development of practical methods for regulating the level of free-radical reactions.The aim of our research was to determine the content of protein, carbohydrate, lipid, and mineral metabolites, as well as an antioxidant status of the sow's blood after weaning and to calculate the correlation between these parameters. The experiment was carried out on twenty clinically healthy crossbred sows (Yorkshire  For the timely prevention and elimination of metabolic disorders, as well as to ensure the optimal metabolic status and productive health of farm animals, it is necessary to constantly monitor the state of metabolism and conduct preventive or therapeutic  measures. In this regard, a study of the physiological and biochemical status (PhBS) of the organism of productive animals is relevant from the animal health point of view. Special attention in this matter should be paid to females in the “special” periods of physiological stress (such as pregnancy and lactation).It is important to highlight that sow health plays a fundamental role in producing healthy offspring ; herds wγ-glutamyltransferase , alkaline phosphatase , alanine aminotransferase , and lactate dehydrogenase (LDH). These effects indicated the activation of protein and carbohydrate metabolism, including glycolysis and other major biochemical processes. The authors [It is well-known , 4 that Along with the study of all physiological and biochemical indicators that characterize the state of metabolism in the body, it is relevant to assess the pro- and antioxidant status of the body, whose indicators are known to be interrelated with animal health, including the state of the immune and reproductive systems .The researchers pay attention to the study of the indicators of lipid peroxidation (LPO) and antioxidant status in the body of pigs of different sex and age groups , 6. ThusIn the case of negative environmental influences on productive animals, various dysfunctions occur, and then the coming pathology can cause various damages to pig breeding and animal husbandry , 8. It iShabunin S. V. and coauthors  presenteGalochkin V. A. and coauthors  showed tAccording to Tregubova N.V. and coauthors , animalsThe researchers also concern the study of the state of the antioxidant system in the animal's body at various diseases .The authors are comparing the LPO processes that are occurring in the body of different breed animals. The blood samples of clinically healthy animals of different breeds contain a certain background of free radicals  and products of oxidative modification of major proteins. The highest level of products of lipid peroxidation and oxidative modification of proteins, as well as a high iron content, was recorded for pigs .According to the authors , the detOther authors compared the indicators of antioxidant protection in the body of “repair pigs” and sows of 2 and 4-5 farrowing. The activity of some antioxidant defense (AOD) enzymes was lower in repair pigs, which indicates that the enzymatic link of AOS is less effective in these animals. This was also consistent with the indicators of oxidative damage. The combined results of studies, including the analysis of systemic markers of oxidative stress, showed that sows of 4-5 farrows more effectively counteract the harmful effects of oxidative stress .Vencova I. Yu. studied the changes of the antioxidant status depending on the physiological state of animals . Usenko Thus, the study of biochemical processes, including the state of the antioxidant system, is relevant for assessing the general physiological and biochemical status of the body and monitoring the health status of animals of various age and sex and physiological groups. Particular attention here should be paid to the parent herd, on the health of which the quality of the offspring depends.Given the fact that the metabolic status and indicators of the antioxidant defense of the body affect the health of animals, the state of the immune system, and reproductive function, it is of interest to study these markers at various stages of the reproductive cycle. The obtained data can serve for the accumulation of a database for the development of reference intervals in pigs of different physiological conditions. In addition, there are few data in the literature that characterize the correlation between metabolic parameters and indicators of antioxidant protection. This is important for determining new markers of the state of the antioxidant defense of the body.Despite the great biological significance, the processes of free radical lipid oxidation and the state of the body's antioxidant defense system during the development of adaptive and pathological processes in pigs, as well as their relationship with blood biochemical parameters, have not yet been sufficiently studied. One of the central issues of the participation of free radical reactions in physiological processes is to clarify the possibility and mechanisms of their regulation. In this regard, it is interesting to study the correlation between blood biochemical parameters and indicators of the state of the body's antioxidant system.In the studies of modern authors, attention is paid to biochemical indicators in the body of sows, but there is not enough data on the state of the antioxidant system at various stages of the reproductive cycle. We believe that the study of indicators of antioxidant protection in the body of sows, both separately and in conjunction with biochemical markers, will be useful for monitoring the health of animals and developing reference intervals for monogastric animals of different sex, age, and technological groups.The aim of our research was to determine the content of protein, carbohydrate, lipid, and mineral metabolites, as well as an antioxidant status of the sow's blood (after weaning) and to calculate the correlation between these parameters. × Landrace). Twenty sows were allocated to one of two groups: (1) one day after weaning (group 1) and (2) 8 days after weaning (group 2).Sows were maintained at a breeder facility. The experiment was carried out on twenty clinically healthy crossbred sows . The research was approved by the bioethical commission of the L.K. Ernst Federal Research Center for Animal Husbandry .The basis of the diet was SK-1 compound feed, balanced in terms of nutrients and energy in accordance with modern standards and the recommended feeding regimen . The comBlood collections were carried out mid-morning. Blood samples were collected from the jugular vein from each sow on the same day. An 18 g needle was used to collect blood samples into 2 × 8 ml VACUETTE® serum tubes with blood clotting activator  that were centrifuged within 4 h of collection at 5000 g for 5 min. Samples were sent to the laboratory  and analyzed on an automatic biochemical analyzer ChemWell  using reagents from Analyticon Biotechnologies AG (Germany), Spinreact (Spain) and Deacon (Russia).Methods used: total protein—by the biuret method (9104), albumin—by the colorimetric method (9136), globulins—by calculation, A/G—by calculation, urea—according to Berthelot (1001329), creatinine—by the Jaffe kinetic method (448), alanine aminotransferase (ALT)—by the UV kinetic (1187), aspartate aminotransferase (AST)—by the UV kinetic (1177), alkaline phosphatase (ALP)—by the UV kinetic (1625), glucose—by the enzymatic-glucose oxidase (4341), Triglycerides—by the enzymatic-colorimetric method (41031), phospholipids—by the enzymatic-colorimetric method (1001140), total bilirubin—by the Walters and Gerarde method (804), cholesterol—by the enzymatic-colorimetric method (41021), calcium—by the O-cresolphthalein complexone method (10100), phosphorus—by the colorimetric method (1914), and magnesium—by the colorimetric method (1001280).μmol⁄ml. The activity of ceruloplasmin (CP) was measured by the method of Revin [Lipid peroxidation (LPO) level in serum samples was measured by the standard method (reaction with the thiobarbituric acid) using kits “Agat-Med” (Russia). The values of the thiobarbituric acid active products (TBA-AP) were expressed in of Revin .3. An amount of 2.2 mmol/dm3 phosphoric acid solution was used as an “eluent.” The results of measuring the total antioxidant activity of the samples were statistically processed using the MS Excel program.The total amount of water-soluble antioxidants (TAWSA) was measured by the amperometric method using the device “TsvetYauza-01-AA” . The TAWSA values were determined by measuring the strength of the electric current arising during the oxidation of molecules on the surface of the working electrode at a potential of ∼500 mV. TAWSA was measured equivalent to gallic acid as in . For thiThe ratio of TBA-AP to CP was calculated by the authors.M) and standard error (±SEM). Pearson correlation test was used to determine relationship between the obtained biochemical parameters. All the data were analyzed by using the software packages “Microsoft Office Excel 2003” and “R Studio” (version 1.1.453) (https://rstudio.com). The results of the statistical analysis were considered significant at p < 0.05.The quantity data are presented as mean value . This was mainly achieved due to the globulin fraction (45.21 g/l in group 2 versus 39.11 g/l in group 1) at p < 0.05. With an increase in the number of days after weaning, the body of sows also increases the urea content by 19.1% at p < 0.05. The content of metabolites of carbohydrate-lipid metabolism—cholesterol and triglycerides—was also higher in the group of sows 8 days after weaning, compared with the animals of the other group.As the first results of the conducted biochemical studies, some differences in the studied parameters between two groups of animals were established . Thus, ip < 0.01), chlorides by 5.43% (p < 0.05), and the activity of alkaline phosphatase by 42.5% (p < 0.05) in sow's group 2 as compared to the first one.As for the metabolites of mineral metabolism, in our studies, we noted a decrease in the concentration of magnesium by 20.2% (p < 0.01), as well as the amount of TBA-active products, by the lengthening of the period after weaning. At the same time, the content of water-soluble antioxidants in animal's blood increased only slightly (by 6.4%) (The parameters of LPO and AOD in the body of sows are presented in by 6.4%) . The conby 6.4%) , probablAfter conducting a correlation analysis between the studied parameters, the moderate and strong positive correlations were observed between the serum concentrations of ceruloplasmin and total protein (0.672) or globulin (0.780) . A moderWe found a weak negative correlation between the serum content of ceruloplasmin and magnesium and a positive correlation of the same degree between the content of ceruloplasmin and urea, ceruloplasmin, and cholesterol.Thus, during lactation and after weaning of piglets, the biochemical processes of sow's body are changing significantly. It is well known , 33 thatAuthors of  studied These data are proved by authors of  who stud2+ and Cu2+ ions, removing them from the fenton reaction, which is one of the key processes in the initiation of LPO [p < 0.01). Thus, the concentration of ceruloplasmin, which plays an important role in the antioxidant defense of the body, significantly increases in sow's blood after weaning. It is known that healthy sows (after weaning of piglets) are characterized by optimal lipid composition of red blood cells and low activity of lipid peroxidation in them [Ceruloplasmin is currently considered as one of the main antioxidants of blood plasma . In addin of LPO . In our  in them . Our dat in them , who obs in them . ProbablAnalyzing the nature of changes of such an indicator as TBA-AP, it is necessary to take into account its priority value in comparison with other LPO indicators since the composition of TBA-AP includes a number of highly reactive compounds that act on all cell structures. According to the modern data, these compounds provide a multifactorial phenomenon, which is defined as endogenous intoxication . In our Thus, after the termination of lactation in sows, the state of the LPO and AOP systems also changes. This is expressed in an increase of a ceruloplasmin content in the blood after weaning of piglets, which indicates an increase in the level of AOP after the termination of lactation. A small decrease in the ratio of TBA-AP to CP in sow's group 2 due to the high activity of ceruloplasmin  shows thBerchieri-Ronchi C. B. and coauthors  showed tWe observed moderate positive correlations between the content of ceruloplasmin and total protein and strong positive between ceruloplasmin and globulins . This isInteresting in our opinion are the correlations between the content of magnesium in the blood and the concentration of TBA-AP. It is known that magnesium participates in the stabilization of cell membranes, acting as a membrane- and “cytoprotective” factor. It is known that the composition of TBA-AP includes a number of highly reactive compounds that act on all cell components, including DNA, and lead to disorganization of the cell membrane structure . It is oThe revealed tendencies and dependences will serve as the theoretical basis for the development of practical methods for regulating the level of free-radical reactions , which is of great practical importance.The authors are conducting further studies of genetic and phenotypic factors that affect the blood parameters of sows and the state of the antioxidant defense system in different periods of the reproductive cycle. In this context, it would be important to estimate the correlation between the content of some other biochemical parameters in the sow's blood , for the perspective of managing the processes of adaptation and increasing the resistance of animals, stimulating growth and development, and increasing their productivity.


PLOS ONE journal was notified about the omission of the minimal dataset. The missing information can be viewed below. The data has been assessed by senior members of the journal staff and a member of the PLOS ONE Editorial Board. With this correction, the relevant data are now provided.After the publication of this article , the PLOS1 FileThis file includes the minimal dataset underlying Tables 1, 2, 4, and 5.(XLSX)Click here for additional data file.


Supercritical fluid extraction is a powerful analytical tool and it is willingly used by researchers for the isolation of various components from different matrices. In our study, the carbon dioxide in the supercritical state was used for the extraction of oils from blackcurrant and black cumin seeds. To determine the optimal conditions for the process , the method of statistical experiment planning and the Box–Behnken design was applied and the yield of the oils and the content of fatty acids (FAs) were taken into consideration. It has been found that an increase in pressure causes an increase in extraction yield (W), and an increase in temperature, both at constant pressure and time, does not significantly change the yield value. Optimal yield values were obtained for both materials under almost similar extraction parameters: 306 bar/ 43 min/ 50 °C (blackcurrant) and 282 bar/ 40 min/ 50 °C (black cumin). The influence of the above parameters  on the content of FAs in the extracts has a slightly different trend. The use of supercritical carbon dioxide for the extraction of blackcurrant and black cumin seeds allowed for high process yield and high-quality, rich in polyunsaturated fatty acids oils which can be used as a substrate or final product for industry.  Currently, products that are described as “natural” are experiencing a renaissance. Their main sources are primarily plants containing different various bioactive ingredients. They include fiber, fatty acids , phytosterols, tocopherols, carotenoids, vitamins and polyphenolic compounds with antioxidant properties. Individually or in groups, these components have a significant impact on the work of the heart and the blood pressure in the cardiovascular system, the digestive system, the skeletal system (including bones and teeth), the body’s lipid metabolism, and its natural resistance (immune system), showing gastro-protective, antithrombotic, anti-inflammatory, antidiabetic, antiviral, antibacterial and antifungal effects ,7,8,9,10An important element is therefore the stage of developing effective and selective methods for the isolation of bio-ingredients from natural products. There are several methods to isolate, from plant raw materials, chemical compounds, or mixtures of biologically active compounds showing the desired effects. Different techniques are used depending on what type of compound or group of compounds will be isolated. In addition to conventional methods, such as maceration ,12, distSupercritical fluid extraction is a powerful analytical tool, and it is willingly used by researchers for the isolation of various components from different matrices ,19. It aOne of the advantages of the fluid in the supercritical state is its selectivity, which depends primarily on the extraction parameters, as well as on the composition and structure of the plant material. All known processes occurring during SFE extraction were presented by Ramsey et al. 1998) in his book at the end of the 1990s , and are98 in hisThe purpose of our study was to investigate the supercritical carbon dioxide extraction of oils under different conditions of temperature, pressure and extraction time. Two crop species seeds with different oil content were used as a model. One of them was blackcurrant seeds, a by-product of berry processing, which are unusual for obtaining an oil extract. The second was black cumin seeds, which are a typically oily plant from the buttercup family (Ranunculaceae or crowfoot family). An experimental plan was created for both materials. Over the last years, a growing application of the statistical design of experiments is observed as an extremely useful method for the optimization of technological processes ,23. To dTaking into account the variable extraction parameters such as temperature, pressure, time –, their mutual dependencies, and the resulting changes in the solubility of the matrix components, it was decided to conduct several supercritical extraction experiments on a laboratory scale using the selected seed material. The unique fatty acid composition (high level of polyunsaturated fatty acids in both blackcurrant and black cumin seeds and desirable ω3/ω6 FAs ratio with a high concentration of unique gamma-linolenic acid in black currant), in combination with the high content of lipid-soluble antioxidants, makes these seeds valuable materials. The yield (efficiency) of the process and the content of fatty acids (FAs) in the obtained oil extracts were selected for evaluation. 2, and only a small amount of data on the extraction of blackcurrant seeds. In addition, only a few of them indicate the effect of the applied changes in the parameters both on the efficiency of the process and on the content of fatty acids. Moreover, and as far as we know, there are no reports regarding the optimization of FAs content in extracts obtained from blackcurrant and black cumin seeds with scCO2 using response surface methodology (RSM). In the literature, there are some data on the optimization of the black cumin seed extraction process using scCOIt also seems interesting to compare, in this form, the results obtained for the seeds of a typical oily plant with the results for the berry seeds with a different biochemical composition and different climatic requirements. The obtained results and the mathematical description of the parameters correlation presented in the work provide valuable information for other researchers and entities interested in using selected seeds for the purposes of the food, cosmetics, medicine and pharmacy industries.The obtained results can also be used to transfer the experiment to a larger extraction scale. However, here one should remember the interactions and complicated processes between liquid, gas and supercritical phases, as well the limitations of the laboratory scale equipment, where there is no possibility to isolate water from the obtained extract. The use of statistical/mathematical methods allows for a lower total cost of the optimization procedure by the lower consumption of plant material and selection of the optimal working time. Moreover, in relation to the cited literature, selected other values of the conducted experiments parameters cover a fairly wide range of changes, especially in the area of pressure. However, these are not the values of the high pressure area, for which appropriate and often expensive equipment is necessary. Therefore, they can be performed on the equipment available in the laboratory. The proposed time range is also reasonable and allows the research to be carried out during a routine working day.CP—extraction yield (%) and CFA—fatty acid content (% w/w).The complete matrix of the experimental design and the results of extraction of blackcurrant seeds with supercritical carbon dioxide obtained during the Box–Behnken design (BBD) implementation are shown in CP) and for the total content of fatty acids in the extracts (CFA) together with basic statistical data describing the correctness of fitting general square models to experimental data are presented in The regression coefficients of equations in the form of second-order polynomials for the yield of extraction of blackcurrant seeds with supercritical carbon dioxide  was statistically insignificant (p > 0.05). The coefficients of determination R2 and R2 adjusted have values above 0.9, which proves a high correlation of input and response data. The 2 predicted had a low value. Therefore, a reduction of statistically insignificant parameters was made, allowing for the simplification of the model while maintaining its statistical significance. The results of the analysis for the reduced models are presented in However, not all factors of the obtained mathematical models were statistically significant, and the coefficient of the R2 predicted coefficient for extraction yield from 0.4985 to 0.8429, and for the content of fatty acids in the extract from 0.6123 to 0.8325. In addition, in the case of reduced models, a very low value of the “p” parameter was obtained, confirming that the developed models are statistically significant. The lack of fit is statistically insignificant in both cases. It can therefore be concluded that the developed mathematical models are suitable for describing the extraction yield and the content of fatty acids in the extract, within the established range of input parameters variability.The reduction of statistically insignificant components resulted in an improvement of the RX2) and extraction time (X3), and the quadratic terms of pressure (X22) and time (X32). In contrast, significant model expressions for the fatty acid content of the extract are the linear term of pressure (X2), interactions between temperature and pressure (X1X2), interactions between pressure and extraction time (X2X3), and quadratic terms of pressure (X22) and time (X32).A statistical analysis of the regression coefficients showed that the significant expressions of the model for the extraction yield of blackcurrant seeds are the linear term of pressure (The figures below show the response surface plots. X1), pressure (X2) and time (X3) on the yield of blackcurrant seed extraction (WCP) with supercritical carbon dioxide carried out in the ASFE MV-10 Waters system.FA) is shown in The influence of the same extraction parameters on the content of fatty acids in the extract (CCP and CFA as a function of coded (1) (3) and uncoded (2) (4) variables, respectively.Below is the final form of the reduced mathematical model for the WThe yield of blackcurrant seed extraction:Total content of fatty acids in blackcurrant seed extract:Both the codes of the selected parameters, the equation describing the polynomial, and the statistical analysis of the results have the same wording as in the above description of blackcurrant seeds extraction. BC–extraction yield (%) and CFA–total fatty acid content (% w/w).BC) and for the total content of fatty acids in the extracts (CFA) were determined, and a statistical analysis of these models was performed. Statistical analysis results for full quadratic models are presented in On the basis of the experimental data contained in p-value in the case of a mathematical model in the form of a second-order polynomial for the extraction yield of black cumin seeds and the content of fatty acids is 0.0042 and 0.0025, respectively. This means that the obtained models are statistically significant. The coefficient of determination R2 and R2 adjusted have a value of ≥0.90, which indicates a high correlation of input and response data. The However, not all factors of the obtained mathematical models for the extraction yield of black cumin seeds and the content of fatty acids were statistically significant, as in the case of blackcurrant. Therefore, a reduction of statistically insignificant parameters was also made here, allowing for the simplification of the model, while maintaining its statistical significance. The results of the analysis for the reduced models are presented in 2 predicted coefficient for extraction yield from 0.4259 to 0.8271, and for the content of fatty acids from 0.6265 to 0.8686. In the case of the reduced model, a small p-value was obtained, confirming that the developed models are statistically significant. In addition, the lack of fit was statistically insignificant. Similar results for black cumin extraction yield  were presented by Gawron et al. [The reduction of statistically insignificant components resulted in an improvement of the Rn et al. .X2) and extraction time (X3), in addition to the quadratic terms of pressure (X22) and time (X32). In contrast, the significant model expressions for the fatty acid content of the extract are the linear terms of pressure (X2) and extraction time (X3), temperature and time interactions (X1X3), pressure and time interactions (X2X3), and the quadratic term of pressure (X22).Statistical analysis of the regression coefficients showed that the significant expressions of the model for the extraction yield of black cumin seeds are the linear terms of pressure  and the content of fatty acids in the extract (b) and the experimental data are shown in BC) as a function of variables X1, X2 and X3. The influence of temperature, pressure and extraction time on the content of fatty acids in the extract (CFA) is shown in BC and CFA in coded form (5) (7) and uncoded form (6) (8) are presented below.The final reduced mathematical models for response variables WBlack cumin seeds extraction yield:Total content of fatty acids in black cumin seeds extract:CP) as a function of temperature (T) and pressure (p), increasing the temperature value in one area of constant pressure (p) does not practically change the yield value. The increase in the yield is evident with the increase in pressure, which can be explained by the simultaneous increase of the solvent density. According to the literature [2 density, the higher the solubility of compounds and chemical substances contained in the test material. On the other hand, the lower the pressure and the higher the temperature, the CO2 will have a lower density, but also a higher diffusion coefficient and permeability. Too high a diffusion coefficient may then hinder or exclude fractionation, because compounds with different solubility  mix easily.As is shown in terature ,26,27, tIn the case of the dependence of extraction yield on temperature (T) and process time (t) b, increaFrom CP value is 11.05%.The optimal process parameters for the extraction yield, as determined on the basis of the developed model, are a pressure of 306 bar, a time of 43 min, and a temperature of 50 °C. The obtained WFA) as a function of temperature (T) and pressure (p), increasing the pressure value to approx. 230 bar, both at low and high temperatures, causes an overall increase in FAs content in the obtained oil extracts. The maximum is obtained in the range of pressure changes of 230 ± 25 bar, as well as temperature changes of 55 to 60 °C. Keeping the pressure value constant above 230 bar, but lowering the temperature from 55 to 40 °C, the FAs content decreases slightly, maintaining a constant level above 93.7% over a large area. By increasing the pressure to 230 bar, the fatty acid content changes over a wider percentage range (from 75.9 to 96.7–99.6) at higher temperature (60 °C) than at a lower one of 40 °C (from 81.9–84.8 to 93.7–96.7). An increase in pressure causes a faster increase in the total FAs content at lower temperatures.As can be seen in In the case of the dependence of the FAs content on the temperature (T) and process time (t) b, increaFrom FA value obtained then is 98.61% w/w.The optimal process parameters for the content of fatty acids in the blackcurrant seed extract are a pressure of 301 bar, a time of 18 min, and a temperature of 60 °C. The CBC) as a function of temperature (T) and pressure (p) and increasing the value (T) in one area of constant pressure does not significantly change the extraction yield, generating a slightly lower value at a higher temperature. However, with the increase in pressure, an increase in the extraction yield is observed, which can be explained, as in the case of blackcurrant, by the increase in the density of the solvent (CO2). The maximum yield is recorded for the temperature range of 45–55 °C and the pressure of 240–320 bar.As is shown in In the case of the dependence of extraction yield on temperature (T) and process time (t) b, increaBC value on the mutual correlations between changes in pressure (p) and changes in the extraction time (t). The lower the values of both parameters, the lower the yield evidently is. Both the pressure and the time changes, in the indicated ranges of the BBD experiments (130–330 bar and 5–45 min), generate a comparable change in yield value of 200% (from 0.0–7.5% to 15.0–22.5%). The yield values above 30%, for both 330 bar and 230 bar, can only be observed from 22 and 25 min, respectively.In BC value obtained then is 38.82%. The optimal process parameters for the extraction yield are the pressure of 282 bar, a time of 40 min and a temperature of 50 °C. The WIn general, the results of the implementation of the BBD plan show that the extraction yield of black cumin seeds ranges from 6.34 to 37.58%, depending on the parameters of the experiment. The average value for the eight higher values is 35.11%. The mean value is 13.96% for seven samples taken either at low pressure (130 bar) or at a short time (5 min). Similar extraction yield values (6.13–31.15%) were presented by Gawron et al. (2021) ,28.FA) as a function of temperature (T) and pressure (p) for black cumin extract samples, increasing the pressure value to 330 bar, both at low and high temperature, causes a general symmetrical decrease in the content of FAs in the obtained oil extracts to approx. 56.7–63.3% w/w. The maximum CFA (90.0–96.7% w/w) is obtained in a fairly narrow range of pressure changes, from 163 ± 11 bar, but at any temperature in the range of 40–60 °C. By keeping the pressure value at any level between 240 and 130 bar, a high content of FAs (above 83.3%) is maintained, regardless of the temperature in the range of 40–60 °C.As can be seen from The effect of temperature (T) and time (t) on the content of fatty acids in the black cumin seeds extracts is shown in FA.The dependence of the FAs content on the mutual correlation between changes in pressure (p) and changes in extraction time (t) is shown at FA value obtained is 99.04%.The optimal process parameters for the content of fatty acids in the black cumin seed extract are the pressure of 135 bar, time of 5 min and temperature of 60 °C. The CThe obtained results can be used to transfer the experiment to a larger scale . However, when comparing the obtained values, one should remember the homogeneity and amount (mass) of the extraction sample, as well as all processes and interactions between the supercritical phase and the liquid and gas phases, which are difficult to define.2 it is Tc = 31.1 °C and pc = 73.8 bar), scientists notice various properties of the supercritical fluid, which may affect the kinetics and dynamics of the extraction process and thus change the solubility of the substances contained in the sample matrix [Although the plant material may come from one purchase lot, a complete transfer in the results will not be accurately reflected from one scale to another. Even at a certain short distance of the operating point (40 °C) from the critical point . Due to this different solubility of individual compounds contained in the extracts, it is possible to partially separate them. As a result of this process, plant oil fractions enriched or depleted in certain bio-components can already be obtained at the production stage . 2 extraction process, it is possible to modify the composition of the obtained oil, striving to obtain the maximum amount of product (emphasizing extraction yield–By changing the parameters of the scCO2), and this results in higher solvation capacity in supercritical carbon dioxide. As a consequence, the greater dissolution of oil components occurs at higher pressure [The observed increase in the total FAs content with increasing pressure, at constant temperature and constant extraction time, can be explained by the fact that higher pressure increases the density of the solvent . This is because, in addition to the general process conditions, the solute’s solubility in the supercritical solvent is also determined by its vapor pressure, molecular weight, and polarity. The higher vapor pressure of the solute facilitates its removal from the matrix . In turn2 extraction at 35 °C and increasing pressure, from two tested FAs, the oleic acid (282 g/mol) was more soluble than stearic acid (284 g/mol). This increase in solubility was due to the presence of a double bond in the structure of oleic acid, which significantly lowers its melting point. However, further studies also showed that the effect of the presence of a double bond on solubility is mainly due to the difference in the physical state of the solute . When both solutes were in the liquid state, their solubilities were similar.As reported by Güçlü-Üstündag O. and Temelli, F. (2000) , in a hoAn increase in solubility with a decrease in molecular weight, however, cannot be observed in a series of unsaturated FAs or their esters, where the introduction of a double bond only slightly changes the solubility. According to Gustinelli G. et al. (2018),  α-linole2 than triglycerides [2 extraction resulted in oils with a higher content of ω-3 and ω-6 acids than the lower pressure at a constant temperature –a berry, perennial plant from the gooseberry family, while the other was the black cumin (Nigella sativa L.), an annual oil plant from the buttercup family. Both species have been cultivated for utility purposes since the 15th–16th centuries, and their medicinal properties, as plants in the wild, were used even earlier.As a model raw material for research, the seeds of two different plants were selected. One was the blackcurrant  and Zielony Klub, Kielce (blackcurrant seeds). The seeds were ground before extraction in an IKA laboratory mill. The appearance of the seeds is shown in The extraction process was carried out in the Chromatographic Laboratory of the Research Laboratory of Plant Raw Materials and Products of the Łukasiewicz Research Network–New Chemical Syntheses Institute. A Waters SFE MV-10 laboratory supercritical extractor  presented in 2 flow, the valves are automatically opened and the extraction is started. After the process is completed, the system automatically goes to the balancing stage, during which the ABPR valve (back pressure regulator) reduces the system pressure to atmospheric pressure. The obtained extract flows directly into the fraction collector.The process begins with filling the prepared sample (ground or crushed) into the extraction vessel. The vessel is then placed in the extraction oven and the selected parameters are set. After reaching the set values of T, p and COThe scale of extraction is smaller, which allows the use of a small amount of sample  and less media consumption. A small amount of the obtained extract, usually a few milliliters, can sometimes be as a disadvantage of this technique, but this amount depends on the type of matrix and extraction parameters.The extraction process of the selected seeds was carried out using the Box–Behnken design (BBD) for three independent variables: temperature of 40, 50 and 60 °C; pressure values of 130, 230 and 330 bar, and extraction time of 5, 25 and 45 min, respectively. The weight of a single charge into the 5 mL extraction vessel was 7.0 g and 3.6 g for black currant and black cumin seeds, respectively. The carbon dioxide flow for both matrices was set at 7 mL/min to allow the obtained results comparison.Carrying out the process under the coded parameter given in X1, X2, X3–independent variables, respectively: temperature (T), pressure (p) and time (t), β–coefficients in the equation defining: equation constant (β0), main effects of parameters , their interaction  and quadratic terms .The influence of the extraction parameters of both seeds on the response variables was approximated by a second-order polynomial regression model described by the following equation:The analysis of variance (ANOVA) was used for the statistical evaluation of the obtained results. The F test was performed to assess the significance of the estimated coefficients in the model. The test of statistical significance was based on error criteria with a confidence level of 95%. A regression analysis was performed using Design Expert 11.0.6.0 .The FA analysis method consists in the indirect determination of the content of individual fatty acids (FA) in the form of methyl esters (FAMEs) obtained by esterification with methanolic solution of trimethylsulfonic hydroxide (TMSH). Samples of both oil extracts were prepared according to ISO 12966, part 1–4. A 10 (±2) mg sample was dissolved in 500 µL of tert-butyl methyl ether and mixed for 5 min. Next, 250 µL of TMSH solution (0.25 M in methanol) was added, and the sample was shaken for 5 min to obtain methyl derivatives. The components were analyzed on GC/MSD. The quantitation analysis was based on analytical standards  and external calibration curves for each compound separately. The obtained FAMEs value were converted to FAs by molecular weight and dilution factor. The total yield of the extraction process was determined by gravimetric measurement of the mass of the obtained extract and expressed as a percentage of the initial mass of seeds constituting the extraction vessel charge.A gas chromatography analysis was performed on Agilent equipment  with a single quadrupole mass spectrometer detector (MSD 5975), split/splitless injector and an Agilent J&W GC capillary column type HP-88 for FAMEs separation. Both GC and MSD systems were controlled by MSD ChemStation, version E.02.02.1431 . The examples of fatty acid chromatograms obtained during chromatography analysis are presented in The use of carbon dioxide in the supercritical state for the extraction of blackcurrant and black cumin seeds allowed for high process yield and high-quality, rich in polyunsaturated fatty acids oils which can be used as a substrate or final product in several industries.X2) and extraction time (X3), and the quadratic terms of pressure (X22) and time (X32). An increase in pressure causes an increase in extraction yield (W), and an increase in temperature, both at constant pressure and time, does not significantly change the yield value. For both materials, the optimal yield values were obtained under almost similar extraction parameters: 306 bar/ 43 min/ 50 °C (blackcurrant) and 282 bar/ 40 min/ 50 °C (black cumin).All listed parameters, temperature, pressure and extraction time have a similar effect on the yield of blackcurrant and black cumin seeds extraction obtained in a laboratory supercritical extractor (ASFE MV-10 Waters). In both examples, the significant expressions of the model are the linear terms of pressure (X2), the quadratic term of pressure (X22), and the pressure–time interactions (X2X3). However, in the case of black cumin, the interactions of temperature and time (X1X3) were also significant, and in the case of blackcurrant, the quadratic term of time (X32) was also significant, which is reflected in the obtained response surface plots. The content of FAs in black cumin extracts decreases with increasing pressure and increasing extraction time. When the process temperature increases, the FAs content in black cumin extracts increases, but only when the extraction time is relatively short (up to about 30 min) and decreases when the time is longer. The influence of the above parameters  on the content of FAs in the extracts has a slightly different trend. In both examples, the significant expressions of the model are the linear term of pressure (


The progress of space science and technology has ushered in a new era for humanity’s exploration of outer space. Recent studies have indicated that the aerospace special environment including microgravity and space radiation poses a significant risk to the health of astronauts, which involves multiple pathophysiological effects on the human body as well on tissues and organs. It has been an important research topic to study the molecular mechanism of body damage and further explore countermeasures against the physiological and pathological changes caused by the space environment. In this study, we used the rat model to study the biological effects of the tissue damage and related molecular pathway under either simulated microgravity or heavy ion radiation or combined stimulation. Our study disclosed that ureaplasma-sensitive amino oxidase (SSAO) upregulation is closely related to the systematic inflammatory response  in rats under a simulated aerospace environment. In particular, the space environment leads to significant changes in the level of inflammatory genes in heart tissues, thus altering the expression and activity of SSAO and causing inflammatory responses. The detailed molecular mechanisms have been further validated in the genetic engineering cell line model. Overall, this work clearly shows the biological implication of SSAO upregulation in microgravity and radiation-mediated inflammatory response, providing a scientific basis or potential target for further in-depth investigation of the pathological damage and protection strategy under a space environment. During human evolution, disease is often accompanied by the existence of pain and death. In today’s world, heart disease, diabetes, neurodegenerative disease, tumor, and other major diseases are serious threats to human health ,2,3,4. IWith the continuous advancement in the major scientific and technological projects of manned spaceflight, mankind has turned the space program to planetary exploration, aiming to send astronauts to Mars and the Moon ,27,28. HIn this work, we investigated the biological effects of the tissue damage and related molecular pathway in a simulated space environment exposed rat model and genetic engineered cell lines. By using histomorphology, biochemical analysis, and Western blot verification, the correlation of SSAO on the inflammatory response in damaged tissues under simulated microgravity and heavy ion radiation was studied. At the same time, the molecular mechanisms behind the pathological phenotype changes were profiled in detail. Our study disclosed that SSAO upregulation is closely related to the systematic inflammatory response  in rats under a simulated aerospace environment. In particular, the simulated space environment leads to significant changes in the level of inflammatory genes in the heart tissues and systemic circulation, thus causing different inflammatory responses among different tissues, which might be related to altering the expression and activity of SSAO. In summary, this study provides a scientific basis for further in-depth investigations of space environment-caused pathological damage, benefiting the development of an effective biomedical protecting strategy for manned space exploration.We first evaluated the physiological and pathological indications of the spatial environment rat model. The results of the rat body weight can be seen in In order to further evaluate the effect of systemic inflammatory response on different tissues caused by the simulated space environment, the immunohistochemistry staining method was used to evaluate the pathological characteristics of tissues from the rats in our experimental model. The results of the hematoxylin and eosin (H&E) staining are shown in Based on the correlation between SSAO and tissue inflammatory damage, we evaluated the expression level and enzyme activity of SSAO in different tissues. The expression of all SSAO proteins in the heart tissue was also significantly increased in the treatment group compared with the CON group, but there was no change in the renal tissue a. ComparIn order to further detect the correlation between SSAO and tissue cell damage, we constructed the system of SSAO overexpression and detected intracellular and extracellular SSAO from different tissue sources after the upregulation of SSAO. We made the recombinant AAV vector overexpress the SSAO in different target cells including human cardiomyocyte AC16 and human embryonic kidney HEK293 cells . The RT-PCR results showed that the levels of inflammatory cytokines IL-6 and TNF-α were significantly upregulated a. The exIn the present study, we determined the damaging effects of the simulated space environment on the body, and confirmed the damaging changes of the simulated space environment on the myocardial inflammatory response. Therefore, we used a combined model of total body irradiation plus tail crane for 21 days to study the changes of the long-term space flight on the body, providing a new angle for exploring the protection and prevention of the body in the space environment. The spatial composite model in this study corresponds to the actual situation of the blood’s redistribution due to the microgravity conditions. As the dynamic center of blood transport in the body, the heart is bound to undergo corresponding changes as the mechanical signals change, and radiation in the environment of long-term space flight will also cause changes in the body ,46.Furthermore, the compound condition model had an impact on the growth of the rat body and the spleen, and the serum results showed that the simulated spatial compound condition can cause changes in the serum levels of SSAO and inflammatory factors in rats. Compared with the control group, the content of IL-6 in the serum in the compound condition group significantly increased, while the content of TNF-α did not, and the content and activity of SSAO also obviously increased. There was no obvious correlation between the change trend of SSAO in the serum and TNF-α, while the change in SSAO level was highly consistent with IL-6. This indicates that SSAO in serum also has a great correlation with inflammation and has a synergistic effect with IL-6, which may jointly mediate the occurrence of inflammation. The pathological sections of various organs showed that the sensitivity of different organs to the combined conditions of radiation and microgravity was different, which may be related to the structure, composition, volume, location, and other characteristics of different tissues. Changes in cardiovascular function induced by microgravity in space can further lead to physiological problems during later flight. The damaged cardiovascular system may be more susceptible to the effects of space radiation, and the synergistic effect of both may make cardiovascular damage more pronounced. The pathological section results of this study showed that after the combined action of radiation and microgravity, the inflammatory reaction was more serious than the control group, and the pathological injury of the heart was the most serious, which resulted in myocardial fibrosis. In this study, no significant pathological changes were observed in the kidney tissues of rats in the control group or the combined group of radiation and microgravity, indicating that the kidney was less sensitive to radiation and microgravity under the simulated space conditions. Our preliminary results show that SSAO varies in the heart and kidney, and is associated with the expression of inflammatory cytokines . HoweverBased on the experimental results of the in vivo model, we also explored the effects of SSAO changes on the AC16 cardiomyocytes and HEK293 kidney cells in the in vitro model. In this study, after the infection of the HEK293 cells and AC16 cells under the optimal experimental conditions, the extracellular secretion of the SSAO protein in the HEK293 cells obviously increased while decreased in the AC16 cells, but the intracellular expression level of the SSAO protein in the AC16 cells and HEK293 cells both increased significantly. Compared with the AAV negative control group, the SSAO overexpression cell model was successfully constructed. However, the intracellular and extracellular expression of SSAO were obviously different. The results showed that after SSAO overexpression, the number of HEK293 cells and AC16 cells decreased, indicating that SSAO virus infection had an inhibitory effect on cell proliferation. In HEK293 cells, there were significant differences in the activity of the apoptotic factor, and the level of the apoptotic factor was downregulated while no significant difference was shown. The expression levels of the apoptosis promoter Caspase 8, and apoptotic factor Caspase 3/7 were both significantly increased in the AC16 cells, which indicated that the overexpression of SSAO would cause damage to the AC16 cells. Further study results showed that from the transcription level of inflammatory factors IL-6 and TNF-α greatly increased after the overexpression of SSAO in the AC16 cells, which showed that SSAO significantly upregulated the RNA level of inflammatory factor IL-6 and upregulated the RNA level of inflammatory factor TNF-α. However, after the overexpression of SSAO in the HEK293 cells, the level of inflammatory cytokines IL-6 and TNF-α decreased or had no significant changes. Therefore, it can be considered that the excessive intracellular expression of SSAO will regulate the transcription of inflammatory factors at the transcriptional level, thus causing inflammatory effects and inducing tissue damage. SSAO is a potential target for the treatment of inflammation, and further research is of great significance for the improvement in biological protection strategies for astronauts under long-term flight conditions.n = 12): control, radiation, and tail crane combined. Animals in the radiation and tail crane combined group had total-body radiation at the radiation dose of 0.25 Gy/min. The radiation source was 12C6+ heavy ion rays from Wuwei Heavy Ion Therapy Center in Lanzhou, with the energy of 260 MeV, the energy transfer line density (LET) of 13.9, and the total radiation dose of 0.5 G). Animals in the radiation and tail crane combined group were housed individually in cages at about 30–35 degrees head-down tilt with their tails in such a position that only the forelimbs were free for locomotion and could touch the bottom of the cage /min/g protein. SSAO activity was determined by the difference between the production rates of hydrogen peroxide with and without semicarbazide.SSAO activity was determined as a result of the turnover of hydrogen peroxide, which was measured by the Amplex Red Monoamine Oxidase Assay Kit . Briefly, 100 g of protein was incubated at room temperature with clorgyline 1 M (monoamine oxidase-A inhibitor), pargyline 3 M (monoamine oxidase-B inhibitor), benzylamine 2 mM (substrate of SSAO), Amplex Red reagent 100 M, and horseradish peroxidase 1 U/mL, with or without semicarbazide 1 mM. Absorbance at 570 nm was measured every 5 min for 30 min. A standard curve was plotted using different solutions with known hydrogen peroxide concentrations. The production rate of hydrogen peroxide was calculated and expressed as [H2.The human cardiac myocytes AC16 and the human embryonic kidney cell line HEK293 were obtained from the Cell Center of Peking Union Medical College . DMEM or 1640 supplemented with 10% heat-inactivated fetal bovine serum , 100 units/mL penicillin, and 100 μg/mL streptomycin  was used for cell culture. The culture was maintained at 37 °C in a humidified incubator containing 5% CO4 cells (treated with or without irradiation) were collected at different time points  and lysed in the provided reagent, which contained the profluorescent substrate with an optimized bifunctional cell lysis/activity buffer for Caspase 3/7 or Caspase 8, and then incubated at room temperature for 2 h before being read in a fluorometer at 484/530 nm. The relative caspase activity was given to evaluate the apoptosis level.Caspase 3/7 and Caspase 8 activation were performed using the ApoOne Homogenous Caspase-3/7 Assay and Caspase-8 Assay , respectively, according to the manufacturer’s instructions. Briefly, 1 × 10t-test and ANOVA assay. Differences were considered statistically significant at p < 0.05.All experiments were performed in triplicate each time. The results were shown as the mean values ± standard deviation (SD). The error bars represent the SD of at least three replicates in three independent experiments. Statistical significance was evaluated by the Student’s


Severe acute respiratory syndrome-related coronavirus (SARS-CoV-2), which still infects hundreds of thousands of people globally each day despite various countermeasures, has been mutating rapidly. Mutations in the spike (S) protein seem to play a vital role in viral stability, transmission, and adaptability. Therefore, to control the spread of the virus, it is important to gain insight into the evolution and transmission of the S protein. This study deals with the temporal and geographical distribution of mutant S proteins from sequences gathered across the US over a period of 19 months in 2020 and 2021. The S protein sequences are studied using two approaches: (i) multiple sequence alignment is used to identify prominent mutations and highly mutable regions and (ii) sequence similarity networks are subsequently employed to gain further insight and study mutation profiles of concerning variants across the defined time periods and states. Additionally, we tracked the variants using visualizations on geographical maps. The visualizations produced using the Directed Weighted All Nearest Neighbors (DiWANN) networks and maps provided insights into the transmission of the virus that reflect well the statistics reported for the time periods studied. We found that the networks created using DiWANN are superior to commonly used approximate distance networks created using BLAST bitscores. The study offers a richer computational approach to analyze the transmission profile of the prominent S protein mutations in SARS-CoV-2 and can be extended to other proteins and viruses. The COVID-19 pandemic has caused over 230 million infections globally with a fatality rate of approximately 2% as of September 2021 ,5. SubunVarious studies confirm the major role of the S protein in SARS-CoV-2 pathogenesis ,8. HenceThe role of S protein as an antigen and the rising number of mutations in the S protein have resulted in vaccines that focus on targeting this protein ,13. SomeVariants are commonly studied using phylogenetic tree analysis. The Pango network is a highly regarded tool to identify and name lineages of SARS-CoV-2 and uses a hierarchical system to study the virus evolution . AnotherTo understand the distribution of mutations in the S protein, we carried out an extensive analysis of S protein sequences reported in the US in 2020 and 2021. In this work, using the large amount of data available in the NCBI Virus repository , we perfThe DiWANN model was applied on short sequence repeats in a previous study and has proven to be more effective than threshold based networks for short sequences. This study deals with much longer sequences (about 20–25 times longer) comprising of amino acids. Additionally, we apply various network visualization techniques to elucidate transmission of the virus. The results were supplemented by maps created using Tableau to show the distribution of variants across the states. Taken together, the analysis demonstrates a new approach to study the transmission of variants across a geographical area over time.The SARS-CoV-2 S protein is 1273 amino acids (aa) in length. It consists of a signal peptide (aa 1-13), the S1 subunit aa 14-685), and the S2 subunit (aa 686-1273) , and the,34. The Mutations in the S protein can be observed by aligning sequences or calculating distance (dissimilarity) between the reported sequences. There are various distance metrics and algorithms to determine similarity, including Levenshtein distance (edit distance) ,38, HammDistance measures are used by SSN-based approaches to compute the dissimilarity between sequences. The most similar sequences are then connected by edges. Sequence similarity can, for instance, be used to identify homologous proteins or genes  that shaThe DiWANN network is much sparser than a basic SSN, while still capturing the core structural elements of the sequence dataset . The undDiWANN has several modeling and algorithmic strengths that make it a preferable approach for constructing SSNs compared to k-nearest neighbors (kNN) and all nearest neighbors (ANN) approaches. The kNN approach relies on selection of a threshold which can be difficult to determine. A threshold too large creates a dense network, which can be computationally expensive to work with, while a threshold too low can lose important structure. The ANN approach avoids these threshold issues, however, computing distances for large sequences can still be prohibitively costly, leading to similarity networks to be built with approximate distance metrics, which can be unreliable. The DiWANN network is both inherently sparse (for most datasets) and does not rely on approximate distance metrics. In this study, we create DiWANN networks to visualize the distribution of prominent mutations across the US in a manner that facilitates analysis of the spread of the virus over time.In this study, we extracted over 100,000 complete nucleotide sequences for the S protein from the NCBI Virus repository . The SARTo better visualize and understand the variations in the protein and the transmission pattern, we divided the dataset into eleven time periods. The divisions were made to correspond to the spread of the virus and measures taken to control the spread in the country. The first time period ranged from 1 January 2020 to 20 March 2020 (T1) and represented the initial stages of the pandemic. The second time period ranged from 21 March 2020 to 30 April 2020 (T2) and represents the stay-at-home order which lasted for more than a month. Many states started reopening in the first week of May. Therefore, we considered the third time period from 1 May 2020 to 20 September 2020 (T3). The fourth time period extended from 21 September 2020 to 31 December 2020 (T4). The fifth time period lasted from 1 January 2021 to 31 January 2021 (T5). There were many new mutations arising every month after T5 and hence, every month has been considered as a time period after T5 for better analysis of distribution of the mutations. Therefore, the month of February 2021 was considered as T6, March 2021 as T7, April 2021 as T8, May 2021 as T9, June 2021 as T10, and July 2021 as T11.This temporal division allowed for more useful analysis of the transmission profile of the virus in the US compared to an analysis that does not use temporal divisions. Relevant statistics of the sequences in our dataset are provided in We performed multiple sequence alignment (MSA) to observe mutations in the S protein sequences of SARS-CoV2. The alignment tool Clustal Omega  was usedSequence alignment tools are ill-suited for visualizing distance (dissimilarity) among all the sequences in the dataset while also taking the temporal and geographical information into consideration. In contrast, sequence similarity networks can provide meaningful insights into the relationship between the sequences and can also be used to depict temporal and geographical distribution. SSNs are networks in which the nodes are sequences and edges depict a closely related pair of sequences. This class of networks can aid biologists in finding useful starting points for analyzing sequences belonging to different groups or families . In thisWe created a DiWANN network for the sequences in each time period. The nodes are colored to indicate the geographical source of the sequences. In particular, we color the sequences reported from the same geographical area (state) the same color. Since some states had only a few samples reported, we colored only the states that had sufficient data. Consequently, the colored states are Florida, Washington, California, Wisconsin, Massachusetts, Michigan, and New York. The other states are simply divided into East and West. We consider the states to the west of Minnesota as the West region and the rest as East. There were a few sequences without a state label; we included these in our analysis as they still contain pertinent information about the sequences in the country. Those sequences have been labeled as ‘USA’ for the purpose of coloring the nodes.An important note is that the location assigned to a node (sequence) is the state where the sequence first occurred. There could be more than one state where a sequence was reported, but for this network analysis, we considered only the first occurrence. However, the count of the occurrence of each sequence is taken into consideration, utilizing the whole dataset. The entire dataset is also considered while visualizing the distribution of variants across the US.Both the temporal and geographical distribution of the prominent mutations obtained from the sequence alignment were studied using the DiWANN networks. A network was created for each time period. The individual time period networks were visualized to show the location and the variant using different node colors and node shapes, respectively. We supplement the SSN models with geographic maps for each time period, showing where particular spike protein mutations appeared across the country over time. The maps were created using Tableau .Finally, we create SSNs using a threshold-based approach and compare them to the DiWANN networks. The thresholds for creating the SSNs are the bitscore values obtained by using Basic Local Alignment Search Tool (BLAST) . The BLAWe encountered more than 400 mutations while performing MSA of the S protein sequences. The mutations in T1 were mostly found in single isolates. T2, T3, T4 and T5 had a significant number of mutations that occurred in more than one sequence, and periods T6 and later had many occurrences of several mutations. The earliest most notable variant in the S protein was D614G (aspartic acid at residue 614 replaced by glycine). Residue 614 of the spike protein is part of the carboxy(C)-terminal region of the S1 subunit and belongs to the region that directly associates with S2. This variant occupied a large portion of detected mutants in the US since March 2020.Other potentially significant mutations observed in the first three time periods include L5F, V6F, S221L, A570V, and P1263L as shown in We observed several new mutations in T4 and T5 sequences including S13I, W152C, V382L, L452R, Q677H and P681H as shown in We found over 30 mutations in the receptor binding domain (RBD). Among these, A344S, V483A and A522V were dominant in the first 3 time periods T1-T3, but were soon dominated by the mutations V382L, L452R and A520S in T4 and T5. Mutation L452R became more common after T5 and got characterized as an important mutation in the Delta variant. Additionally, new mutations became dominant after T5, including K417T, S477N and S494P in T6, T7 and T8, and R346K, T478K, E484K and N501Y in T9, T10 and T11. Period T11 had a significant increase in several mutations, including K77T, G142D, A222V, T478K, T859N and D950N, most of which are characteristic mutations of the Delta variant. Overall, the number of mutations, especially in subunit S1 increased after T5.The results from MSA show that the D614G variant became well established by the period T3 and the Delta variant spread rapidly across the US. The DiWANN networks we constructed for the D614G and Delta variants provide insight into the transmission trend of the variants over time and across states. The networks also provide relative occurrences of sequences depicted via node sizes. Therefore, the central nodes in the large components (cores) of the networks are larger in size compared to the surrounding nodes due to high occurrences. These nodes usually represent the D614G or Delta variant sequences. The D614G variant spread very quickly, and glycine (G) has become the most prevalent aa at residue 614 over time as shown in the networks in At the start of the pandemic, in T1, there was a similar distribution of the two variants, but G started increasing in representation in T2, and became the dominant variant by T3. The networks corresponding to T1 and T2 had two cores, which represented the two variants (D and G at residue 614), however, the networks for T3 and T4 have just one large component representing aa G at residue 614. The emergence of the variant can be observed well in these 4 time periods, after which there is almost no occurrences of the D variant. Since we colored the nodes depending on the state in which the sequence first occurred, the networks depict the transmission pattern in the country over time.While performing MSA, we observed that the number of mutations and the frequency of these mutations started to rise in 2021. The four variants: Alpha, Beta, Delta and Gamma occurred in significant numbers in period T6 and later. The bar plots in The Centers for Disease Control and prevention (CDC) had classified variants Alpha, Beta, Delta and Gamma as variants of concern (VOC). However, CDC downgraded Alpha, Beta and Gamma to variants being monitored (VBM) in September 2021; Delta was downgraded to a VBM in April 2022. The distribution of the Delta variant in the US during periods T6–T8 can be seen in the DiWANN networks in The DiWANN networks consider only the unique sequences, that is the first occurrence of the sequences. This provides us with information about the sequences and the mutations in the spike protein. However, to analyze the distribution of the Delta variant in the US, we have created geographical maps for the time periods T6, T8, T9 and T11 that utilize the entire dataset and not just the unique sequences. As motivated in the Introduction, we use the network model DiWANN because it is better suited for our dataset and analysis compared to alternative SSN models. To verify this, we compare it against the use of SSN based on threshold values and scores using BLAST . We creaIn this study, we analyzed SARS-CoV-2 S protein mutations and studied the temporal and geographical distribution of some highly transmissible variants in the US. The logical division of the dataset into time periods depicted a story of how the S protein in the virus evolved over time, despite the counter measures taken around the world. We initially divided the time periods by month, which was a finer division for the year 2020, however, we found that dividing the year according to the stages in the pandemic gave us better insights into viral transmission across the country. Additionally, during the early months of the pandemic, there were not many variants and so the chosen time periods depicted a better story.The MSA results indicate that the virus mutated faster in the later stages of the pandemic than in the earlier stages, making it of paramount importance that we keep track of the changes that are potentially increasing the viral infectivity and transmissibility. Further, analysis using MSA showed that there have been more mutations since T6 (February 2021), which corresponds to the deployment of vaccines, indicating that the virus is adapting to selective pressure. Additionally, more heavily mutated SARS-CoV-2 variants have been emerging over time . OverallVariant D614G had been established by the end of 2020 as can be seen from The networks for T3 and T4 have a single large component primarily represented by the variant D614G across the states. Studies suggest an increase in viral infection as a result of the mutation at residue 614 due to the reduction in shedding of S1 and an increase in the incorporation of the total S protein into the virus . This coOther important variants were studied in a similar manner. From The temporal distribution of the Delta variant shows how the occurrence of this variant increased over time and has been high since T9 (May 2021). The T9, T10, and T11 networks in Another interesting observation about the distribution of Delta variant is that the variant was initially found more in the west  and became dominant across the US by T11. The T6 network in The geographical maps in Our study involved a large number of S protein sequences collected over a period of 19 months. The data provided significant insights into the transmission and mutations of the virus. The approach used is useful in deriving information to be considered while targeting regions of the S protein for treatments and analyzing pathogenesis of the virus. However, a limitation of this analysis is that the completeness of the data obtained from NCBI Virus depends on the states reporting the sequences. There were a good number of sequences from most highly populated states, but some states reported only a few sequences or did not report consistently with time. Therefore, our results rely on the available data, but we believe they provide a picture of the temporal and geographical distribution of mutations in the spike protein across the US.The study demonstrates how an approach using the DiWANN model and analysis can be effective in providing insights about the transmission of a virus. We applied this approach to identify trends over time periods (January 2020–July 2021) that had some established facts regarding the virus, enabling us to verify that our approach provided valid results. Additionally, studying the pandemic right from the start till July 2021 provided us with a broad picture of the spread of different variants, especially the Delta variant, which was the most severe variant among people who were not vaccinated. This effective approach combines temporal and geographical data and can potentially be used to study other variants of SARS-CoV-2 or other infectious diseases in future works.We chose a DiWANN SSN as our model for this analysis over a threshold-based SSN using either an exact or approximate similarity metrics because it offered better results while keeping computational cost low; in particular, exact threshold-based SSN is overly costly to construct and approximate threshold-based SSN can provide inferior results . AdditioWe found DiWANN to be better suited for this dataset which consists of quite similar sequences as all are spike proteins and fairly conserved. However, although the DiWANN network is much faster to construct than a threshold-based SSN using an exact similarity measure, the approach may still be computationally intensive for large datasets. In such cases, some approximations may become necessary.With the continuously growing outbreak of SARS-CoV-2, understanding the biology of the infection is of paramount importance. Study of the spike (S) protein and its mutations is key to understanding viral transmission and pathogenicity. In this study, we combine two approaches to study mutations in the S protein, namely, multiple sequence alignment and network analysis. Prominent mutations were identified by performing MSA on the collected sequences from the NCBI Virus repository. The temporal and geographical distribution of two important variants, namely the D614G variant and the Delta variant were studied using a variant of sequence similarity networks called DiWANN. Each stage of the pandemic has its own story which we observed in the DiWANN networks, verifying the soundness of the implemented approach. Our computational approach provided richer insights into the behavior of the virus over time and can be used to gain insights about other variants, proteins or viruses.