Obesity and type 2 diabetes (T2DM) are major public health concerns associated with serious morbidity and increased mortality. Both obesity and T2DM are strongly associated with adiposopathy, a term that describes the pathophysiological changes of the adipose tissue. In this review, we have highlighted adipose tissue dysfunction as a major factor in the etiology of these conditions since it promotes chronic inflammation, dysregulated glucose homeostasis, and impaired adipogenesis, leading to the accumulation of ectopic fat and insulin resistance. This dysfunctional state can be effectively ameliorated by the loss of at least 15% of body weight, that is correlated with better glycemic control, decreased likelihood of cardiometabolic disease, and an improvement in overall quality of life. Weight loss can be achieved through lifestyle modifications  and pharmacotherapy. In this review, we summarized different effective management strategies to address weight loss, such as bariatric surgery and several classes of drugs, namely metformin, GLP-1 receptor agonists, amylin analogs, and SGLT2 inhibitors. These drugs act by targeting various mechanisms involved in the pathophysiology of obesity and T2DM, and they have been shown to induce significant weight loss and improve glycemic control in obese individuals with T2DM. Nowadays, the role of insulin and its influences are continuously gaining attention since metabolic disorders have reached global pandemic proportions. In fact, insulin resistance (IR) is clinically associated with several metabolic disorders, including glucose intolerance, dyslipidemia, hyperuricemia, and hypertension. Hence, there is an urgent need to establish the mechanisms of IR and possible pharmacological treatments for these metabolic conditions. Beside being correlated to different metabolic syndromes, IR is also one of the major underlying mechanisms responsible for type 2 diabetes mellitus (T2DM), which consists of an array of multiple and complex disorders characterized by hyperglycemia and resulting from the combination of different metabolic and homeostatic disturbances that are sustained over time. Despite significant investments in clinical care, research, and public health interventions, T2DM is on the rise, and there does not appear to be any sign of a reduction in the rate of increase. Nearly 90% of the reported 537 million cases of diabetes worldwide suffer from T2DM, and these numbers are expected to rise to 783 million by 2045 . Indeed,1c).As just mentioned, IR characterized the pathophysiology of T2DM, which results in early hyperinsulinemia and a steady loss in the ability of pancreatic cells to make insulin. Prolonged suboptimal glycemic control increases the risk of both microvascular  and macrovascular complications . However, despite being recognized as a complex disease, T2DM is diagnosed solely on persistently elevated levels of glycemia or glycated hemoglobin  and the availability of new therapies contributed to the reduction of rates of major lower limb amputations and mortality from vascular causes . Notwithstanding the encouraging outcomes, newer lines of evidence point to a shift in management trends and the rise of obesity as a novel treatment target.Obesity is a metabolic disease and seems to be correlated with the rising burden of T2DM ,9. As a Several studies support the benefit of a sustained loss (at least 15%) of body weight, which exerts major benefits on T2DM-related endpoints and cardiovascular events . ChanginIn more detail, white adipose tissue (WAT) is a remarkably complex organ that plays a significant role as a key energy reservoir. However, in obese and diabetic subjects, WAT often becomes highly dysfunctional, leading to adipocyte hypertrophy, visceral adiposity, and ectopic fat deposition, which result in the development of cardiometabolic syndromes . AdiposiInterestingly, one of the hallmarks of obesity-linked inflammation in WAT is the increased number of macrophages, which are also involved in the development of IR . In addiBesides the strong relationship between insulin resistance and obesity , weight Therefore, a weight-centric, or even better, adipose-centric intervention would slow the disease course and benefit other associated cardiovascular risk factors, preventing long-term microvascular and macrovascular complications of T2DM ,11. Determining the degree to which an individual is overweight or obese is essential to applying the most suitable guidelines for obesity management. However, the use of BMI-based criteria to determine eligibility for weight loss interventions is flawed because it excludes a significant percentage of the T2DM population from treatment, particularly those whose ethnic or racial backgrounds are more likely to develop metabolic comorbidities despite having a BMI below the obesity threshold . In ever1c value without glucose-lowering medications for at least three months [Another effective opportunity lies in bariatric surgery, also known as metabolic surgery. The growing consensus that bariatric surgery is an enduring treatment for obese patients is reflected in the rise in the number of procedures. Moreover, bariatric surgery is supported as an anti-diabetic intervention for people with T2DM and obesity by the International Diabetes Federation and the American Diabetes Association ,22. Indee months ,21. Thise months ,25. Addie months .The four most common bariatric operations performed worldwide are laparoscopic sleeve gastrectomy, laparoscopic Roux-en-Y gastric bypass, laparoscopic adjustable gastric banding, and duodenal switch. Among these surgeries, new research indicates that the bypass technique may result in better long-lasting weight reduction and glycemic management, while the sleeve procedure is linked to fewer reoperations . However®) Montgomery, AL, USA, liraglutide (Saxenda®) Bagsvaerd, Denmark, and semaglutide (Wegovy®) Bagsværd, Denmark. Orlistat is an inhibitor of gastrointestinal lipase, and it is able to reduce the absorption of ingested fat by approximately 30% [®) Campbell, CA, USA [®) San Diego, CA, USA [Until now, there are no available pharmacotherapies that are able to replicate such results; still, several agents have received regulatory authorities’ approval for chronic weight management . Currenttely 30% . As a retely 30% . These d CA, USA . The forBesides these obesity pharmacotherapies, several glucose-lowering agents that are FDA-approved for the treatment of T2DM have been recognized to promote weight loss . These iMetformin, represented in Metformin works by inhibiting mitochondrial glycerophosphate dehydrogenase, indirectly activating adenosine monophosphate-activated protein kinase (AMPK), and lowering cytosolic dihydroxyacetone phosphate while increasing the cytosolic NADH/NAD ratio . As a reStudies also show that metformin promotes weight loss and decreases food intake. This is justified by the AMPK-dependent effect in the brain, where metformin suppresses orexigenic peptides, neuropeptide Y, and agouti-related proteins, while the decrease in food intake is due to an increase in the expression of the leptin receptor gene in the arcuate nucleus to reduce central leptin sensitivity .Additionally, chronical metformin treatment increases growth differentiation factor 15 (GDF15) and GLP1 levels, two key mediators of metformin-induced weight loss ,28. MetfAccording to the Diabetes Prevention Study (DPP), which is the largest study to show the weight benefits of metformin, the average weight reduction after one year on the medicine is only 2.1 kg . In 2 yeGiven the safety and tolerability of metformin, as well as its mild weight-loss effect, the FDA has not approved metformin as a weight-loss agent. Nevertheless, it is currently and commonly used off-label in individuals who are at high risk for metabolic problems and who cannot tolerate alternative therapies. As a matter of fact, the 2016 American Association of Clinical Endocrinologists (AACE) guidelines on obesity management recommend the use of metformin (as well as acarbose and thiazolidinediones) in obese patients with evidence of prediabetes or insulin intolerance that does not respond to lifestyle medications or other anti-obesity medications  .Incretin-based therapies are a prominent approach to successfully managing obesity and metabolic disorders such as T2DM. Patients who suffer from T2DM fail to achieve glycemic control due to a desensitization of incretin receptors ,39,40. NIncretin hormones include GLP1 and the glucose-dependent insulinotropic polypeptide hormone (GIP). These enteroendocrine hormones are released from the gut in response to intraluminal carbohydrates and fats .They act mainly on the pancreas, where they exert their insulinotropic activity. Concomitantly, they play a direct role in several biological pathways involved in T2DM and obesity, such as satiety and lipolytic activity . The numGLP1 also exerts several cardiovascular effects, including improving endothelial function and reducing blood pressure. Moreover, GLP1 inhibits glucagon production from pancreatic α-cells, which helps lower hepatic glucose production and enhance peripheral glucose absorption . The netUnlike GLP1, GIP may stimulate glucagon secretion at lower glucose levels and promote lipid synthesis and storage in adipose tissue, which can increase fat accumulation and body weight gain . This haHowever, the administration of GIP receptor (GIPR) agonists  is reported to lead to a reduction in caloric intake and body weight ,49. The ®) San Diego, CA, USA, albiglutide (Tanzeum®) Middlesex, UL, USA, dulaglutide (Trulicity®) Indianapolis, IN, USA, and lixisenatide (Adlyxin®) Paris, France. Furthermore, as already mentioned, among the approved GLP1RAs, some drugs, namely liraglutide and semaglutide  at 26 weeks and 4.7% (5.0 kg) at 56 weeks ,55,56, wMoreover, obese people can lose up to 12.5 kg in total during the run-in phase of a low-calorie diet, of which half was observed during the liraglutide 3.0 mg treatment period .The indirect impact of improved glucose management on body weight homeostasis may be the cause of the difference in weight reduction between patients with T2DM and subjects with obesity.Regarding semaglutide, the peptide backbone was modified at position 8, where an aminoisobutyric acid (Aib) was introduced instead of an Ala and an acetylated Lys26. Structure-activity investigations proved that C18 diacid together with a γGlu and two oligo (ethylen glycol) linkers resulted in the highest albumin affinity combined with GLP1R potency. Clinical studies demonstrated that semaglutide has a similar safety profile to other GLP1RAs while offering better weight reduction than liraglutide ,60,61. ABesides these GLP1RAs, dual GIP/GLP1RAs have been investigated as well, such as tirzepatide (LY3298176) , which i1c value (2–2.3% reduction) than did 1.0 mg of semaglutide  over 40 weeks in addition to metformin [In the SURPASS phase 3 clinical trial program, 5, 10, and 15 mg of Tirzepatide have been investigated for patients suffering from T2DM. All three doses led to a larger reduction in body weight (from 8.2% up to 11.9% for the highest dose) and an improved HbAetformin .1c of 1.2% with a dose of 300 mg of cotadutide [The design of GLP1 and glucagon dual agonists has also been explored. This combination aims to take advantage of the satiety effect while balancing insulin secretion mediated by GLP1 and glucagon’s mobilization of hepatic glucose. The most promising compound is MEDI0832 (cotadutide), a synthetic peptide that results in side chain modification of the primary sequence of glucagon in order to confer GLP1R activity, in addition to an esterification with a palmitic fatty acid to facilitate albumin binding. This novel peptide is more potent toward the GLP1R, with a 3–4 times lower efficacy at the GLP1R compared with native GLP1, and an 8-fold lower potency at the GCGR (glucagon receptor) compared with natural glucagon. Cotadutide is under clinical development by AstraZeneca and is currently in Phase II of clinical studies . Obese ptadutide . Table 3Incretin hormones are only some of the released peptides from the gastrointestinal system in response to food intake. Among the plethora of satiety hormones, there is amylin , a 37-amino acid peptide, co-stored and co-secreted with insulin by pancreatic islet β-cells. IAPP relays signals to the hypothalamic nuclei and other areas of the subcortical areas of the brain, resulting in increased feelings of satiety and fullness. In addition, IAPP plays also a major role in glucose homeostasis by slowing gastric emptying, suppressing glucagon secretion, and exerting anorexigenic effects by working synergistically with leptin . FurtherIn a 26-week clinical study, 4.5 mg of cagrilintide (weekly dosed) achieved 7.8% mean weight loss versus placebo. In addition, the result obtained with 3.5 mg of cagrilintide was also greater than the 6.0% mean weight loss achieved with liraglutide at 3.0 mg . Contrary to a placebo, weight loss with cagrilintide persisted for the full 26 weeks of therapy without hitting a plateau. Similarly to the outcomes with liraglutide at 3.0 mg, weight reductions following cagrilintide treatment were followed by an overall improvement in TFEQ (Three-Factor Eating Questionnaire) scores for cognitive restraint, emotional eating, and uncontrolled eating in the same trial. A further step along the way to identifying a compound able to manage weight and glycemic targets in people with T2DM has been to evaluate the combination between 2.4 mg of semaglutide (GLP1RA) and different doses of cagrilintide . The concomitant treatment of cagrilintide and semaglutide, in comparison to semaglutide alone, provided effective weight loss (15.4% up to 17.1%). Furthermore, glycemic control was improved regardless of the cagrilintide dose .Other glucose-lowering agents with weight-loss propriety are the sodium-glucose cotransporter 2 inhibitors (SGLT-2i). At the moment, there are several SGLT-2i available on the market namely, canagliflozin, dapagliflozin, empagliflozin, and ertugliflozin , or in dThis class of small molecules is characterized by an insulin-independent mechanism and normalizes glycemia by avoiding the reuptake of filtered glucose in the kidney. In addition to the common primary outcome of glycemic control, this mechanism of action leads to further beneficial effects such as a reduction in blood pressure and weight ,83. SeveFurthermore, SGLT-2i reduces blood leptin, increases adiponectin levels, and shows cardiovascular protective properties, in particular in cases of heart failure, by reducing the adipose accumulation in the myocardium . The degThus, pharmacotherapy with SGLT-2i is recommended for T2DM subjects who have indicators of high risk of renal or heart failure, although treatment with SGLT-2i is correlated with an increased risk of urinary and genital infections .Obesity is a highly multifaceted chronic disease condition that poses a serious public health issue. Furthermore, it is associated with an increased risk of IR, T2DM, and several cardiovascular and chronic inflammatory diseases. As a matter of fact, obesity and T2DM are strictly interconnected since they share key pathophysiological mechanisms. Intensive dietary and lifestyle modifications can be effective for both obese and T2DM subjects by ameliorating glycemic control and producing weight loss. Such beneficial effects can also be achieved through existing pharmacological approaches that treat both obesity and T2DM and are already available on the market. When the weight loss, is at least 15% of the initial bodyweight, it can lead to significant improvement in the metabolic status and can also induce remission. However, better solutions are required for successful, significant, and long-lasting weight loss since maintaining a healthy weight is not so simple and there is a huge unmet need for effective pharmacotherapy with minimal side effects.Nonetheless, it is important to underline that using BMI-based criteria to establish eligibility for weight loss interventions has several flaws. BMI is an inadequate indicator of a person’s health risks since it does not directly evaluate body fat mass or its potential for harm. Addressing obesity should focus on a more adiposopathy-based approach. Therefore, considering adiposity health should be essential, even if efforts are still needed to easily detect adipose tissue pathology. The progress toward personalized medicine in order to better define obesity and provide individualized treatment choices is a challenge to prioritize. Furthermore, it is also important to recognize that more efforts are needed to understand the complex link between adiposopathy, obesity, and T2DM in order to identify new possible targets and develop new effective active compounds while avoiding the rise of both obesity and T2DM.


To understand abnormal thyroid function and its associated factors among medical staff in radiotherapy departments.2 test and binary logistic regression.Data related to medical staff in radiotherapy departments who met the inclusion and exclusion criteria were analyzed from September to December 2022 in Shenyang, Liaoning Province, including basic personal and lifestyle habits, work information, and scores on the Depression Self-Assessment Scale, Perception Stress Scale, and the Generalized Anxiety Scale. Data analysis was performed using the χAmong 484 medical staff in the radiotherapy department, 147 (30.4%) had abnormal thyroid function. Binary logistic regression analysis showed that age, years of work, smoking, occupational exposure, smoking, late-night snacking habits, depression, and stress perception were factors associated with abnormal thyroid function among medical staff in radiotherapy departments; on the other hand, physical exercise was a protective factor.The positive rate of thyroid dysfunction among medical personnel in the radiology department is relatively high. It is necessary to strengthen health education and awareness among relevant practitioners and improve the coverage of occupational disease prevention and control education. Abnormal thyroid function is closely associated with prolonged occupational exposure, which is more common among medical staff in radiation therapy units . It is aResearch studies have found that the incidence of thyroid function abnormalities among medical staff is between 4 and 30% , 7, showCurrently, abnormal thyroid function is becoming an important public health and occupational health problem threatening the health of radiologists. Although medical staff are aware of the hazards of low-dose radiation and thyroid disease, they need to analyze potential risk factors and put interventions and preventive measures in place. This study investigates the association between abnormal thyroid function and its associated factors among medical staff in radiotherapy departments, especially exposure to lifestyle, psychological regulation, and behavioral patterns, to provide a scientific basis for preventing and controlling occupational disease hazards.This study adopted a cross-sectional design, using the random number table method to randomly select 10 hospitals from 37 public tertiary A hospitals in Shenyang, Liaoning Province, from September to December 2022. Approximately 50 medical staff from the radiation therapy departments of the 10 hospitals were randomly sampled, and all individuals who met the standards were included in the target population.N = 400 × Q/P (Q = 1-P and P was the incidence rate). Due to the lack of a unified incidence rate of abnormal thyroid hormone levels, after consulting Chinese domestic literature  Aged ≥18 years; (2) Abnormal function as determined by two or more internists by thyroid color Doppler ultrasound and serum thyroid hormone tests ; (3) Voluntary participation in this study and no participation in similar studies within 6 months; and (4) No mental disorders caused by cerebrovascular diseases, physical infections, or other physical diseases. Exclusion criteria: (1) Family history of thyroid disease, history or other autoimmune diseases; (2) Have a history of pituitary or hypothalamic diseases; (3) Have a history of severe parathyroid disease; (4) Chronic consumptive diseases  and malignant tumors; (5) Recent history of acute and severe infections; (6) Take or use drugs that affect thyroid hormones, such as phenytoin sodium, Carbamazepine, Amiodarone, Lithium carbonate, and iodine-containing contrast agent, and (7) Pregnant women. The study was approved by the hospital's ethical review committee, and all participants gave informed consent to the study content.The sample size of the study was calculated using the formula: terature , the detThe questionnaire covered basic personal details, living habits, and work information. The basic personal information and living habits included gender, age, education, family history of thyroid disease, alcohol consumption, smoking, frequency of physical exercise, and whether there was a night snacking habit. The basic work information included years of work, title, type of occupation, whether the night shift was on rotation, and whether occupational exposure occurred.*g. Approximately 500 uI of serum was collected and placed in an EP tube. Simultaneously, a backup was created and immediately frozen in a freezer at −20°C for testing. In this study, five indicators of thyroid function in the serum of the participants were detected by Roche E411 automatic electrochemiluminescence immune analyzer and its detection reagent. All experimental reagents were calibrated using a standard calibration solution, and those that passed the calibration were used on the machine. The products were tested daily for quality control before the experiments to ensure that each experiment was within the quality control range. The reference ranges of the blood biochemical indexes were: TSH 0.27–4.2 mIU/L, FT4 12–22 pmol/L, FT3 3.1–6.8 pmol/L, TPOAb 3–34 mIU/L, and TGAb 0–115 mIU/L. Two internists jointly determined whether the thyroid function was abnormal based on the blood biochemical index results according to the Chinese Guidelines for the Diagnosis and Treatment of Thyroid disease.The five indicators of thyroid function included free triiodothyronine (CFT3), free thyroxine (CFT4), thyroid stimulating hormone (TSH), thyroglobulin antibody (TGAb), and thyroid peroxidase antibody (TPOAb). Approximately 3–5 ml of fasting peripheral blood was collected from the study participants and placed in a non-anticoagulant tube. It was let to stand at room temperature for 30 m and centrifuged at 3,500 rpm for 10 m, with a centrifugal force of 1,095The scale consists of seven symptoms and one symptom-related degree of difficulty item that assessed the severity and functional impact of the seven anxiety symptoms over the past 2 weeks . Each syThe scale was developed based on the nine symptoms of DSM-IV depressive disorder and was widely used in scientific research and clinical practice because of its brevity and ease of use . The totThe perceived stress scale (PSS) was developed by Cohen et al.  in 1983 2 test. The measured variables were expressed as mean ± standard deviation, and a t-test was used for comparison between groups. Univariate and multivariate logistic regression were used to determine the factors influencing abnormal thyroid function.All collected data were analyzed using SPSS 26.0 software. The count variables were expressed as percentages, and comparisons between groups were made using the χA total of 500 questionnaires were issued, and 484 valid questionnaires were returned, including 147 with abnormal thyroid function, with a detection rate of 30.4%. Hyperthyroidism, hypothyroidism, subclinical hyperthyroidism, and subclinical hypothyroidism were detected in 15, 28, 37, and 67 cases, with detection rates of 10.2%, 19.0%, 25.2%, and 45.6%, respectively. The age range of medical staff in the radiotherapy department was 21–37 years old, with an average of 29.0 ± 8.6 years. There were 283 (58.5%) female and 201 (41.5%) male staff. Approximately 289 participants mainly had irregular eating habits, accounting for 51.9%. In addition, 224 (46.3%) and 226 (46.7%) medical doctors had smoking and drinking habits, respectively. Approximately 216 (44.6%) participants never exercised, 112 (23.1%) occasionally exercised, and 156 (32.3%) always exercised. Their occupational types were predominantly physicians 168 (34.7%), in addition to 99 (20.5%) nurses, 122 (25.2%) physiatrists, 46 (9.5%) technicians, and 49 (10.1%) other staff. Approximately 287 (59.3%) of the staff had a history of occupational exposure. Most of the participants had worked for 5 years or less, and those with intermediate and below job titles were predominant. There were 319 (65.9%) medical staff involved in night shifts and 165 (34.1%) staff without night shifts. The details are shown in Reviewing the depression, anxiety, and stress assessments in both groups, the results showed that the PHQ-9, GAD-7, and PSS scores were significantly higher in the abnormal thyroid functioning group than in the non-abnormal group, and the differences were statistically significant. The details are shown in The results of univariate logistic regression analysis showed that age, years of work, type of occupation, job title, night shift, occupational exposure, late-night snacking habit, smoking, family history, physical exercise status, GAD-7, PHQ-9, and PSS were associated with abnormal thyroid function among medical staff in the radiotherapy department. The details are shown in p < 0.1 in the univariate logistic regression analysis as independent variables. The results showed that age, years of work, occupational exposure, smoking, late-night snacking habits, depression, and stress perception were associated factors for abnormal thyroid function among medical staff in the radiotherapy department: physical exercise, on the other hand, was a protective factor for abnormal thyroid function. For details, see A multivariate logistic regression analysis was performed with abnormal thyroid function  as the dependent variable and factors with The thyroid gland is the largest endocrine gland in adults, which mainly regulates the growth and development of the human body, and affects emotions and the metabolism of sugar, fat, and protein. The prevalence of thyroid disease is increasing year by year . This stThe results of the multivariate analysis showed that age and working hours were associated factors for abnormal thyroid function in medical staff in radiation therapy units—the older the medical staff in radiation therapy units and the longer the working years, the higher the positive rate of abnormal thyroid function among them, which is generally consistent with previous studies , 32. TheThe thyroid gland is sensitive to ionizing radiation, and long-term exposure can lead to dysfunction and teratogenic effects . RadiatiThe results of this study showed that the risk of thyroid function abnormalities was higher in radiotherapy medical staff who smoked than in nonsmokers and that their serum TSH levels were significantly higher than in nonsmokers . This isMedical staff who exercise daily are less likely to have abnormal thyroid function than those who exercise less than twice a week, which may be related to continuous and regular exercise reducing resting T4, T3, and thyroid-stimulating hormone levels. Further, it has been noted that exercise can alter the levels of zinc in the body, which is considered an important substance involved in synthesizing and processing thyroid hormones , 45.This study is an important addition to the epidemiology of thyroid disorders. With the rising incidence of cancer, the issue of abnormal thyroid function among medical staff in radiotherapy units deserves attention and prevention. Age, years of work, smoking, occupational exposure, late-night snacking habits, depression, and perceived stress were found to be risk factors for abnormal thyroid function among radiotherapy medical staff, with physical activity as a protective factor. The study still has the following limitations. First, the sample size was limited, and all were from the same study center, subject to selection bias. Second, cross-sectional studies, which do not provide good evidence of causality, are necessary to conduct cohort studies to assess the effect of each factor more clearly on abnormal thyroid function.In conclusion, the positive rate of thyroid dysfunction among medical personnel in the radiology department is relatively high. It is necessary to strengthen health education and awareness among relevant practitioners and improve the coverage of occupational disease prevention and control education. Hospitals should conduct regular training to enhance the radiation protection awareness of medical personnel, focusing on protecting the thyroid area and improving protective conditions. Medical staff should cultivate good living habits and maintain their mental health.The original contributions presented in the study are included in the article/The studies involving humans were approved by the Ethics Committee of The First Hospital of China Medical University. The studies were conducted in accordance with the local legislation and institutional requirements. The participants provided their written informed consent to participate in this study. Written informed consent was obtained from the individual(s) for the publication of any potentially identifiable images or data included in this article.ZR and PS: full access to all the data in the study, take data analysis, and supervision. YR: conceptualization, methodology, and writing—original draft. XB: software and validation. GL: writing—reviewing and editing. All authors approved the submitted version.


Although the role of vitamins in the human body is proven, guidelines for patients with chronic kidney disease (CKD) remain unclear. This narrative review summarizes the findings of 98 studies of CKD and the effects of vitamin D, B, C, A, E, and K supplementation on patients on dialysis for CKD, with the aim of summarizing the existing guidelines. The findings are promising, showing the potential effectiveness of vitamin supplementation with, for example, vitamins B, D, or C. However, recommendations are still ambiguous, especially in the case of vitamins A and K, due to the potential toxicity associated with higher doses for patients. Continued research is needed to rigorously evaluate the effectiveness and carefully consider the potential risks of some vitamin supplementation for patients with CKD. Due to increasing life expectancy and the significant development of society, there has been a notable rise in the prevalence of civilization diseases worldwide. Chronic kidney disease (CKD) is considered one of them. It is usually associated with obesity, hypertension, and diabetes ,2. Body This article aims to present a narrative review of the effect of vitamin supplementation with vitamins such as D, B, C, A, E, and K on patients on dialysis due to chronic kidney disease.Chronic kidney disease (CKD) is a significant public health problem worldwide ,16. Out Studies show that HHcy is a significant risk factor for the development of atherosclerotic processes that lead to cardiovascular disease (CVD)  and stroThe persistent elevation of parathyroid hormone (PTH) causes the development of osteitis fibrosa cystica and osteoarthritis . In addiKidneys are an important source of antioxidant enzymes, e.g., glutathione peroxidase, which is why pro-oxidant levels in CKD are elevated . For thiPatients with CKD on hemodialysis (HD) are characterized by an excessive increase in oxidative stress, which is associated with progressive deterioration in kidney function and, consequently, higher mortality rates . In addiSome disorders, such as nutritional deficiencies, recurrent vomiting, appetite disorders and progressive anorexia, muscle cramps, bone diseases, and insomnia, can be minimized with appropriate diet and supplementation ,26. CKD The guidelines of the European Society for Clinical Nutrition and Metabolism (ESPEN) recommend exact doses of vitamin supplements, which mostly correspond to the recommended daily dose for healthy adults. However, these guidelines on supplementation do not always coincide with the loss of vitamins during dialysis. Therefore, they will not always be appropriate for CKD patients. Due to the risk of toxicity with some vitamin levels being too high, care should be taken when supplementing ,27 Tabl. NeverthVitamin D is found in the body as 25-hydroxycholecalciferol or 1,25-dihydroxycholecalciferol  ,29,30, aIt is important in bone formation, immunity, vascular and nervous systems, and reproduction . VitaminVitamin D deficiency is associated with an increased risk of mortality and secondary hyperparathyroidism (SHPT) . Since tIn a meta-analysis of 10 prospective studies, Pilz et al. confirmed that the risk of death from any cause in patients with CKD increases by 14% for every 10 ng/mL reduction in vitamin D levels . TherefoThe KDIGO group recommends vitamin D supplementation but does not specify which measures to take or the optimal strategy for restoring vitamin D levels ,38. Somep = 0.001), and this difference persisted until the end of the study (12 weeks) [Many scientists have studied the effectiveness of cholecalciferol. Based on their results, it can be concluded that vitamin D supplementation in this form is effective and safe ,30,34,402 weeks) . Similar2 weeks)  and Okša2 weeks) . They sh2 weeks) . However2 weeks) . Matias 2 weeks) .Ziyad Al-Aly’s study on the effects of ergocalciferol on serum concentrations of 25-hydroxyvitamin D and intact plasma parathyroid hormone shows that an increase in 25-hydroxyvitamin D levels greater than 5 ng/mL (12 nmol/L) is associated with a significant probability of a more than 30% decrease in intact plasma PTH levels. This means that the administration of ergocalciferol has a beneficial effect on PTH levels . Blair eArmas et al. compared the effectiveness of vitamin D2 with vitamin D3. They showed that vitamin D2 is one-third less effective than vitamin D3. Therefore, doctors should consider abandoning vitamin D2 supplementation in favor of the stronger vitamin D3 . In contThere is growing evidence of the usefulness of vitamin D supplementation in dialysis patients, who are the most likely to be deficient in vitamin D. However, there is still no data indicating which dose would be most effective ,36,40,48The effects depend on the dose of vitamin D, the type of vitamin D compounds, the duration of the study, and the population studied ,34 Figu.Dogan et al. investigated the effect of monthly oral supplementation of 300,000 IU of vitamin D3  on markers of uremic bone disease (UBD), such as PTH and alkalinophosphatase. In conclusion, the authors suggest that treatment with oral cholecalciferol results in a statistically significant reduction in serum PTH levels, but does not result in a statistically significant change in Ca or P . SimilarGuillaume Jean sought to evaluate the efficacy and safety of a monthly dose of cholecalciferol  in patients with a vitamin D (HD) deficiency undergoing hemodialysis. At the end of the study, of the 107 patients, 91% had serum 25(OH)D levels higher than the target level (>75 nmol/L). Serum calcitriol 2D) levels also increased. However, no significant changes were observed regarding calcemia, phosphatemia, blood pressure, serum albumin, hemoglobin, and C-reactive protein. Based on this and other studies, it can be concluded that the long-term monthly administration of cholecalciferol orally is a safe and effective method for correcting vitamin D deficiency ,48,50. TB vitamins are very important for the proper functioning of the body. They are involved in metabolism and immune function and contribute to overall growth and development ,13. CurrThe suspicion of the inadequate intake of B vitamins in patients with CKD, especially those on dialysis, is due to the fact that they have a lower appetite and a low protein supply in their diet . At the It has been shown that the expression of thiamine and folic acid carriers is significantly reduced in an animal model of CKD . TherefoThiamine is a hydrophilic B vitamin involved in many metabolic functions  that serIn their cross-sectional study of 288 dialysis patients, Saka et al. assessed the relationship between thiamine levels in the blood and other clinical parameters. In total, 30 patients (12.4%) had lower blood thiamine levels than the lower normal limit. Thiamine blood levels correlated with age, body mass index, and Barthel score (BI). The proportion of patients with end-stage CKD with low thiamine blood levels is high. Low physical performance—a low BI score—is an independent risk factor for thiamine deficiency. The authors point out that physicians should be aware of the possible occurrence of thiamine deficiency in patients with end-stage CKD, especially those with low physical function .Although the data do not indicate that all patients are thiamine deficient, there are indications that there is a risk of insufficient concentrations in patients with CKD . It is aRiboflavin is a hydrophilic vitamin of group B. It is necessary for oxidation-reduction reactions . After pPorrini et al. studied patients with advanced CKD who were not on dialysis using the α-erythrocyte glutathione reductase (a-EGR) stimulation index to assess riboflavin counts. In this study, 8% of patients had elevated levels of a-EGR, indicating riboflavin deficiency. When the protein intake prescribed to these patients was intentionally reduced compared to their usual intake, the incidence of elevated a-EGR increased from 8% to 25% and 41% . TherefoNiacin is a hydrophilic B vitamin that is consumed as a nicotinic acid amide from animal sources or in the form of nicotinic acid from plant sources. Niacin becomes active in humans when it is converted to nicotinamide adenine dinucleotides or nicotinamide adenine dinucleotide phosphate . Niacin Kang et al. found that because a low dose of niacin improves dyslipidemia, lowers serum phosphorus levels, and increases the GFR with rare adverse effects, low-dose supplementation may be considered in patients with CKD . HoweverVitamin B6 occurs in vivo as six compounds: pyridoxal, pyridoxine, and pyridoxamine, and as the 5′-phosphates of these three compounds. Pyridoxal-5-phosphate (PLP) is a cofactor for many enzymes, especially those involved in amino acid metabolism. PLP is necessary for (d)-aminolevulinate synthase to initiate heme synthesis ,14.Vitamin B6 losses during dialysis are still a matter of controversy . A 35% dKopple et al. and Podda et al. showed that many patients with CKD have suboptimal serum vitamin B6 levels ,65. KoppMany drugs and other compounds  can interfere with the work or metabolism of pyridoxine and thus increase the likelihood of vitamin B6 deficiency. This is particularly likely in patients with CKD because they may often be prescribed these medications, and they show lower vitamin B6 intake with increased demand. These data suggest that patients with stage 3 or higher CKD are at an increased risk of vitamin B6 deficiency and should therefore take the appropriate supplementation to reduce CV risk ,38. BothCobalamin is a nutrient that plays a key role in human health. It is essential as a cofactor for enzyme methionine synthase and other biochemical reactions such as the beta-oxidation of fatty acids, DNA synthesis, and red blood cell production. Cobalamin is one of the most complex coenzymes in nature .In healthy adults, vitamin B12 deficiency is extremely rare. Even if a healthy person consumed insufficient amounts of vitamin B12 for a period of 3 years, he would not have inadequate levels of this vitamin. However, these data do not necessarily apply to patients with CKD because there are no data on the amount of vitamin B12 stored in the body in patients with this disease . TherefoIn contrast, some authors state that vitamin B12 deficiency is a common feature of patients with CKD due to elevated concentrations of holotranscobalamin (TC2). The kidney plays an important role in the metabolism of TC2 because it is filtered into the glomeruli, and from there, it is reabsorbed into the proximal tubule. Defects in protein resorption in the proximal tubule can therefore lead to the biologically active loss of TC2 in the urine. Functional vitamin B12 deficiency has been observed despite normal total B12 levels, which may explain the increased leakage of TC2 in the urine, lower absorption of TC2 in the proximal tubule, and the lower cellular absorption of TC2 .When deciding on supplementation, it is important to consider the fact that high levels of vitamin B12 can be harmful to people with CKD. This is related to the metabolism of cyanide, which is impaired in these individuals due to a reduced glomerular filtration rate. Cyanocobalamin, the most common form of vitamin B12 replacement, is metabolized to active methylcobalamin by releasing small amounts of cyanide. Under normal conditions, methylcobalamin binds to cyanide, converting it to harmless cyanocobalamin. However, in patients with CKD, this process is disturbed. In addition, excessive amounts of cyanocobalamin supplementation may release cyanide ions that are not excreted and contribute to complications such as uremic neuropathy ,67.Folic acid (FA) is pteroylmonoglutamic acid. FA deficiency causes megaloblastic anemia. Since the human body is unable to synthesize folic acid, it must be supplied through the diet . Folic aThe poor binding of FA to plasma proteins results in significant losses during each dialysis session. After a single procedure, a 37% decrease in plasma FA concentration was described. According to the available evidence, folate supplementation at a dose of 1 mg/day should prevent deficiencies in hemodialysis patients. Interestingly, increasing the dose to 2 mg/day results in a five-fold increase in plasma FA concentrations. High doses of folic acid have been used to reduce cardiovascular complications in patients with end-stage renal disease (ESRD) due to its effect on homocysteine methylation. However, the benefits of this practice have never been confirmed .It has been shown that patients with CKD demonstrate anionic inhibition of the membrane transport of 5-MTHF with a simultaneous decrease in the intracellular rate of folate incorporation. Therefore, it is suggested that the level of folate measured in the blood of people with uremia does not reflect its intracellular use since its uptake is altered due to anionic inhibition . In a raThe benefits of folate supplementation in people with impaired renal function do not appear to correlate fully with a decrease in serum homocysteine (Hcy) levels . The maiThe minimum dose of folic acid to achieve Hcy reduction has been debated. ESRD patients without diabetes may respond to a daily dose of 5 mg FA, but diabetic patients with ESRD may need up to 15 mg to lower Hcy levels by more than 20% and to see benefits related to CVD risk, regardless of FA enhancement [The DIVINE study examined the effects of high-dose therapy with FA (40 mg/day), vitamin B12 (1000 mg/day), and vitamin B6 (2 mg/day) in patients with diabetic nephropathy. It showed that this treatment regimen does not increase survival or slow the progression of ESRD. Instead, it leads to a higher incidence of cardiovascular events and a greater decrease in the eGFR. However, such results may be influenced by its suboptimal study design, such as the lack of division of patients with CKD and ESRD .Another study looked at the effects of B12 and FA supplementation on CVD after 24 months of treatment. The results indicated that FA reduced the risk of CVD by 15%. Greater benefits were seen in those studies where treatment was longer than 24 months .The role of folic acid and vitamin B12 supplementation in reducing mortality and preventing progression to ESRD has not yet been determined ,19. AccoVitamin C, or ascorbic acid (AA), is capable of inhibiting the oxidation of other compounds by donating a maximum of two electrons. After donating one electron, ascorbic acid becomes a free radical known as semidehydroascorbic acid. After receiving the second electron, semidehydroascorbic acid is converted into dehydroascorbic acid . ThroughAccording to some studies, including Morena et al., AA loss during a single dialysis session was about 28%, with some results reaching as high as 60%. Based on these studies, it can be concluded that the loss of vitamin C during each dialysis session is significant . Some auOver the years, indications for ascorbate supplementation have been formulated very carefully. This is due to oxalate, which is a metabolite of AA. A large amount in the body can cause hyperoxalemia, which can be considered a uremic toxin. Oxalate levels in HD patients are twice as high as in non-dialysis patients, and can be up to seven times higher when supplemented with vitamin C. Therefore, high doses of vitamin C are not recommended in patients with advanced CKD ,76. Canap = 0.001) compared with a negligible score in the other groups. In addition, they reported that both vitamin C alone and the rutin/vitamin C combination showed a protective role against lipid peroxidation, as evidenced by reduced levels of MDA. Finally, rutin had a beneficial synergistic effect with vitamin C in reducing TG and TNF-α levels and increasing HDL-C levels [One randomized, prospective trial looked at the effects of rutin and vitamin C compared to vitamin C alone on oxidative stress and inflammation in HD patients. It studied 105 HD patients that were divided into three groups. Group 1 received the rutin/vitamin C combination, group 2 received AA (1 g), and group 3 was the control group. At the beginning and end of the study, serum malondialdehyde (MDA), glutathione peroxidase (GPx), high-sensitivity C-reactive protein (hs-CRP), tumor necrosis factor α (TNF-α), lipid profile levels, and the erythrocyte sedimentation rate were evaluated. The authors showed that vitamin C significantly increased serum GPx in group 2  are increased in CKD, which may partly explain the elevated plasma concentrations in patients with CKD. National Health and Examination Survey III data showed a link between elevated serum creatinine and elevated vitamin A levels. This finding confirms previous studies that described elevated vitamin A levels in non-dialysis patients with CKD, ESRD patients, and kidney transplant recipients. Since serum vitamin A levels begin to increase with increasing creatinine, there appears to be no need to provide additional vitamin A to patients with CKD, except in some specific cases .Espe et al. found a strong association between low levels of retinol and the risk of sudden cardiac death and fatal infection. One possible interpretation is that lower levels of retinol are a marker of inflammation .In dialysis patients, plasma vitamin A levels are elevated, and vitamin A deficiency is rarely observed. In fact, even a small level of vitamin A supplementation can cause vitamin A toxicity. Therefore, the guidelines for vitamin A supplementation recommend not exceeding the daily dose in healthy people  ,86.Vitamin E is a lipophilic molecule that is typically found in cell membranes. It comes in four forms: a-tocopherol, b-tocopherol, g-tocopherol, and d-tocopherol ,87.The role of oxidative stress as a pathological factor in several disease states is becoming increasingly apparent, and vitamin E supplementation is also being considered as a potential treatment for this condition ,89,90 duAccording to some studies, in non-diabetic patients with mild to moderate CKD, a treatment strategy with pravastatin, vitamin E, and Hcy reduction therapy (vitamin B12 and folic acid) leads to a significant reduction in the progression of carotid stenosis and a significant improvement in endothelial function and urinary albumin excretion. However, no significant effect on the eGFR was observed .In HD patients and peritoneal dialysis (PD), serum a-tocopherol levels can range from low or normal to increased .Numerous studies have shown that vitamin E supplements reduce oxidative stress ,83,91.Vitamin K participates in the post-translational carboxylation of specific glutamic acid (Gla) residues in proteins , allowing it to bind to calcium and interact with other compounds. This is necessary for blood coagulation and bone mineralization . VitaminSince vitamin K binds to lipoproteins, no loss is expected during the dialysis procedure . InitialUntil recently, routine vitamin K supplementation was not recommended in dialysis patients. However, it is now possible to identify functional vitamin K deficiency by measuring non-carboxylated fractions of vitamin K-dependent proteins . More reSome studies suggest that a high menaquinone intake in the general population has a beneficial effect on CVD and bone density, leading to a lower risk of bone fracture. However, the results are not conclusive . A studyKnowledge about the need for vitamin supplementation among patients with CKD remains incomplete ,27. The The topic of vitamin A, E, and K supplementation remains controversial. Some papers suggest that supplementation should be undertaken and is beneficial. On the other hand, because of possible toxicity, some suggest the opposite. This leads to cautious recommendations for supplementation with these vitamins ,11,38,83Based on current evidence, the individualized approach towards vitamin supplementation is recommended. Folic acid should be supplemented cautiously because a high intake may mask signs of anemia and progressive neurological disorders. Further research is needed to precisely define the demand for these essential nutrients in patients with CKD ,27,95.


Currently, there is a lack of diagnostic methods for liver fibrosis in veterinary medicine. Two-dimensional shear wave elastography is a non-invasive diagnostic technique widely used in human medicine. However, there is a limited amount of relevant research in veterinary medicine, with only a few studies available. These studies suffered from small sample sizes, and the results of different experiments contradict each other. The aim of this study was to measure the range of liver stiffness in healthy dogs and to investigate the factors influencing it. We found that liver stiffness in healthy dogs was 3.96 ± 0.53 kPa, which was influenced by anesthesia and measurement site. These findings provided a theoretical basis and data support for its application in veterinary clinical practice. At the same time, the determination of health values contributed to the subsequent study of two-dimensional shear wave elastography in dogs.(1) Background: Two-dimensional shear wave elastography (2D-SWE) is a non-invasive method widely used in human medicine to assess the extent of liver fibrosis but only rarely applied to veterinary medicine. This study aimed to measure liver stiffness in healthy dogs and investigate the factors that impacted 2D-SWE measurement. (2) Methods: In total, 55 healthy dogs were enrolled and subjected to 2D-SWE measurements before and after anesthesia administration. Post-anesthesia 2D-SWE measurements and computerized tomography (CT) scans were obtained. (3) Results: The liver stiffness range in healthy dogs was 3.96 ± 0.53 kPa. In a stratified analysis based on confounding factors, liver stiffness was influenced by measurement site and anesthesia, but not by sex. No correlation was observed between liver stiffness and weight or liver CT attenuation. (4) Conclusions: 2D-SWE is feasible for liver stiffness measurement in dogs. Anesthesia and measurement site are sources of variability. Therefore, these factors should be considered while recording 2D-SWE measurements. Our data on liver stiffness in healthy dogs can serve as the basis for future studies on 2D-SWE to assess pathological conditions in dogs. Liver stiffness is one of the most important indicators of liver disease, as changes in liver stiffness often accompany the progression of liver disease . StudiesFor this reason, a non-invasive imaging diagnostic technique, elastography, was first introduced in human medicine. Elastography includes strain elastography (SE), acoustic radiation force impulse imaging (ARFI), transient elastography (TE), point shear wave elastography (p-SWE), and 2D-SWE. The type of force application is one of the crucial factors in the categorization of elastography, and it can be further divided into two categories, quasistatic and dynamic excitation methods, based on the method of force application. In the quasistatic excitation method, pressure is applied to the target tissue using a transducer or physiological motion to induce deformation, as in SE. On the other hand, the dynamic excitation method generates stimulation either mechanically on the body surface or using acoustic radiation force internally, as in TE and 2D-SWE ,5,6.2D-SWE is a promising technology to measure liver stiffness. As one of the earliest elastography techniques, SE can only provide qualitative or semi-quantitative measurements of tissue stiffness. It assesses the elasticity of the target tissue by comparing it to the surrounding healthy tissue. The application of acoustic radiation force has made it possible to quantitatively measure tissue stiffness values. TE, as a shear wave imaging technique, can only measure the average hardness along a single measurement line. In contrast, 2D-SWE not only allows quantitative measurement of liver stiffness but also allows the placement of multiple regions of interest (ROI) in the target area, thereby expanding the measurement range and providing more representative data. 2D-SWE integrates conventional ultrasound and color-coded tissue stiffness in real time to effectively avoid the inclusion of non-targets, thereby producing more reliable results. 2D-SWE allows real-time placement of the ROI under the guidance of conventional ultrasound, avoids blind inspection, and provides improved accuracy while reducing operational difficulties. Studies have shown that an operator can be considered a 2D-SWE expert after 300 cases of conventional ultrasound experience or more than 50 cases of elastography experience. Moreover, the use of 2D-SWE is not limited by the presence of ascites. 2D-SWE is an excellent diagnostic imaging technique and offers promising clinical applications in veterinary medicine ,8,9.2, where ρ is the tissue density, defined as 1.00 kg/m3 [As a form of shear wave imaging, 2D-SWE directly measures the physical quantity of the shear wave velocity itself. The process of performing 2D-SWE measurements typically begins through the application of an acoustic radiation force to multiple points within a larger ROI. The arrival time of shear waves generated at each focal point along a transverse line is then determined. This collected information is used to calculate the shear wave velocity , which is then automatically converted to Young’s modulus  in most cases using the equation E = 3ρC00 kg/m3 ,11,12.The measurement results are encoded by the ultrasound machine into color images that can be overlaid on B-mode grayscale images or displayed side-by-side with B-mode images. The transparency and color display scale can be adjusted. By placing the ROI at the desired location, quantitative statistical data of Young’s modulus of the target tissue, such as mean, standard deviation, minimum and maximum values, can be obtained ,13.Liver biopsy is still considered as the gold standard for diagnosis of liver diseases, but its invasive nature can lead to complications such as bleeding, pain, and other complications . ImagingThere is limited published literature on the use of 2D-SWE in veterinary medicine and only a few articles have investigated its feasibility and repeatability in measuring canine liver stiffness. Recently published 2D-SWE studies in veterinary medicine have failed to fully investigate confounding factors such as measurement site, state of anesthesia, and the effect of spontaneous breathing, and to measure liver stiffness in healthy dogs. Further, the results of these studies are contradictory, owing to small sample sizes ,23,24.Herein we aimed to measure liver stiffness in a sufficient number of healthy dogs to establish a range of healthy values and to aid in the diagnosis of liver fibrosis and other liver diseases in dogs. Identifying an appropriate range of liver stiffness values in healthy dogs will facilitate future research on the use of the 2D-SWE technique in dogs. To this end, we determined the feasibility of 2D-SWE to measure liver stiffness in 55 healthy dogs and investigated the effects of several factors on 2D-SWE measurements. Previous studies have shown that 2D-SWE had good applicability and accuracy in diagnosing liver disease in human medicine ,25. In aFifty-five healthy dogs  were included in this study. There were 19 mixed-breed dogs, 28 spaniels, 1 German shepherd, 1 labrador, 3 reindeers, and 2 beagles, with an average age of 3 years (range 1–4 years) and a median weight of 13 kg (range 4.5–32.5 kg). All dogs were deemed to be healthy, as per physical examination, routine blood tests, blood biochemistry , abdominThis study was approved by the Ethics Committee of Nanjing Agricultural University.Liver stiffness in dogs was measured using 2D-SWE in the awake state. Intravenous anesthesia was administered, and 2D-SWE measurements and abdominal CT scans were performed. Liver stiffness and CT attenuation were measured six times on each side of the liver lobes when 2D-SWE measurements and CT scans were completed. All CT scans and 2D-SWE measurements were performed in the same anesthesia event.2D-SWE measurements were obtained for each experimental dog in both the awake and anesthetized states.Prior to examination in the awake state, dogs were fasted for more than 12 h, and 2D-SWE measurements were performed using a Philips ultrasound scanner  and a c5-1 probe (1–5 mHz), as previously described ,27,28,29To maintain uniformity in measurement results, the ultrasound parameters for 2D-SWE were set to the system’s default configuration and no adjustments were made during the measurement process. The dual-screen mode of 2D-SWE was selected on the ultrasound machine, which displayed the confidence map on the left and the stiffness map on the right. The confidence map converted the confidence level of the measurements into different colors, which were overlaid on the ultrasound greyscale map. Green was regarded as highly reliable data, while red indicated unreliable data. The stiffness map represented tissue stiffness, with blue and red indicative of the lowest and highest stiffness, respectively . The absThe 2D-SWE measurements were conducted in the left lateral and right lateral positions to measure the liver stiffness of the left and right lobe, respectively. Measurements were repeated with different frames and ROIs until six valid observations were obtained. The mean values of SWEs were representative of the liver modulus values.2 were continuously monitored.All animals were anesthetized with an intravenous dose of xylazine hydrochloride (0.85 mg/kg), tiletamine (4 mg/kg), and midazolam (0.2 mg/kg). During the anesthesia, heart rate, blood pressure, oxygen saturation, and end-tidal COA Hitachi CT scanner was used with the following scan parameters: slice thickness of 1.25 mm to 2.50 mm, tube voltage of 120 kV, tube current of 200 mA, rotation time of 0.75 s, pitch of 1.0625, and collimation of 1.25 × 16.Animal positioning and CT scan results are shown in t-tests.Data were presented as mean ± standard deviation (SD) and analyzed using independent samples p = 0.05), 90% power). Four healthy dogs were used in the pre-experimentation stage. The mean difference in liver stiffness values for the experimental dogs before and after anesthesia was 2.71 kPa (4.05 kPa vs. 6.76 kPa), with standard deviations of 104% and 141% for liver stiffness before and after anesthesia, respectively. Based on this, PASS 15 calculated that a sample size of 15 animals would result in >90% power using an independent sample t-test with a 5% type 1 error. The sample size was determined by pre-experimentation, experimental design, and conventional numerical settings using PASS 15  Microsoft Corp., Redmond, WA, USA). All measurements were expressed as Young’s modulus, and the data were analyzed by the Shapiro–Wilk test. Correlation analysis was used to determine the relationship between weight and liver stiffness. A value of p > 0.05). However, the liver stiffness was significantly higher in the left lobe than in the right lobe in the anesthesia state (p < 0.01).The liver stiffness in healthy dogs was 3.96 ± 0.53 kPa. We compared the liver stiffness between the left and right lobes in the awake and anesthesia states. . No signp < 0.01 for the right liver lobe, p < 0.01 for the left liver lobe). The color changes in the stiffness map reflect the impact of anesthesia on liver stiffness in healthy dogs. After anesthesia, the color of the stiffness map shifted from a uniform light blue to a mixture of blue and green .Sex did not significantly affect liver stiffness in healthy dogs in either the awake or anesthesia states . No signp > 0.05 for the awake state, p > 0.05 for the anesthesia state).We conducted a correlation analysis to explore the effect of weight on liver stiffness . The resp = 0.637 for the left liver lobe, p > 0.05 for the right liver lobe, and p >0.05 for the entire liver).We determined the correlation of liver CT attenuation with liver stiffness . No signIn the present study, we investigated the applicability of 2D-SWE to assess liver stiffness in healthy dogs. The effects of anesthesia, measurement site, sex, and weight on the results were investigated, and the correlation between liver CT attenuation and liver stiffness was explored. The aim was to measure liver stiffness in a sufficient number of healthy dogs to establish a range of healthy values and to aid in the diagnosis of liver fibrosis and other liver diseases in dogs.Unlike humans, dogs were not cooperative during examinations. Anesthesia is occasionally necessary in veterinary medicine to perform liver 2D-SWE. Tamura et al. identified motion as an important limitation in 2D-SWE measurements in dogs . 2D-SWE In our study, the liver stiffness of anesthetized dogs was significantly higher than that of awake dogs. This result is similar to a previously published study but different from the results of another study ,23. ThisIn our study, the stiffness of the right liver lobe was slightly higher than that of the left lobe, but there was no significant difference. Interestingly, a significant difference was observed in stiffness between the right and left liver lobes in the anesthesia state. Consistent with the above speculation, we believe that this observation was related to changes in the liver blood perfusion. The anesthesia protocol used in this study might lead to differential changes in perfusion between the left and right liver lobes. The comparison of the changes in the liver stiffness before and after anesthesia made it clear that the right lobe experienced a greater increase in stiffness than the left lobe. Given the relatively poor distensibility of the liver capsule, we speculate that this phenomenon is attributed to the greater increase in the blood perfusion to the right lobe. Unfortunately, there are no reported studies on this topic. Given the time constraints and other factors, we were unable to thoroughly investigate this issue, and it warrants future study.The results of this study indicated that sex had no significant effect on liver stiffness in healthy dogs, and that body weight was not significantly correlated with liver stiffness. Thus, our results are applicable across different sexes and body weights of dogs. In clinical practice, there is no need for concern about the influence of these two factors on measurement results. However, it is important to note that the dogs included in this experiment had a BCS of 3, which eliminated the effect of obesity on the results. The applicability of these results to obese dogs, which are commonly encountered in clinical settings, needs to be confirmed. Unfortunately, there are no published reports investigating the effect of obesity on 2D-SWE measurements in canine livers, and further studies are warranted to investigate this.The lack of correlation between liver CT attenuation and liver stiffness indicated that 2D-SWE technology cannot be replaced by CT. In addition, 2D-SWE has several advantages over existing clinical imaging diagnostics in veterinary medicine. First, it offers high sensitivity and specificity and has been considered one of the preferred non-invasive imaging techniques for the clinical assessment of liver fibrosis in human medicine ,37,38. SThe present study has some limitations. First, the proportion of large dogs in this study was low, which may have influenced our findings. Therefore, the results of this study may not apply to large dogs. Second, all the data were measured by a single imaging physician which did not allow for the assessment of inter-observer variability. Third, the dogs in this study were not histologically examined to determine the health status of the liver, and this was presumed by physical examination, blood test, CT scan, and ultrasound. However, the dogs were relatively young, and no clinical signs or abnormalities of illness were found.Here we demonstrated the applicability of 2D-SWE to measure dogs’ liver stiffness with high reproducibility. Liver stiffness was measured in 55 healthy dogs, and the results provided statistical support for the use of 2D-SWE in veterinary medicine. The range of liver stiffness in healthy dogs established herein will be useful in the diagnosis of liver fibrosis and other liver diseases in dogs. In addition, our study lays the foundation for future studies using 2D-SWE to diagnose pathological conditions that increase liver stiffness in dogs.Anesthesia was deemed as a source of variability in liver stiffness. However, the effect of anesthesia on the liver stiffness of healthy dogs might be related to the anesthesia protocol used. The measurement site also affected 2D-SWE values and this effect was also, at least in part, related to the state of anesthesia. Therefore, these two factors should be interpreted and considered while recording 2D-SWE measurements in dogs.


Gracilaria lemaneiformis (GL), industrial synthetic astaxanthin , Phaffia rhodozyma  and Haematococcus pluvialis  at their optimal supplementation amounts. Furthermore, the SA80, PR80, and HP120 groups performed better in all aspects, including survival, growth, immune response, antioxidant activity, thermal resistance, and disease resistance, compared with the GL group. The PR80 and HP120 group also had a better growth performance than the SA80 group. In terms of heat stress and bacterial challenge, abalone in the PR80 group showed the strongest resistance. Overall, 80 mg/kg astaxanthin supplementation from Phaffia rhodozyma was recommended to obtain a more effective and comprehensive outcome. This study contributes to the discovery of the optimum dietary astaxanthin supplementation source for abalone, which is helpful to improve the production efficiency and economic benefits of abalone. Future research can further explore the action mechanism and the method of application of astaxanthin to better exploit its antioxidant role.Dietary antioxidant supplementation, especially astaxanthin, has shown great results on reproductive aspects, egg quality, growth, survival, immunity, stress tolerance, and disease resistance in aquatic animals. However, the effects of dietary astaxanthin supplementation from different sources are still unknown. A comprehensive comparison of survival, growth, immune response, antioxidant activity, thermal resistance, disease resistance, and intestinal microbial structure was conducted in dietary antioxidant supplementation from the sources of  Seriola dumerili, curcumin supplementation) [Litopenaeus vannamei, vitamin E supplementation) [Pagrus major, selenium nanoparticle supplementation) [Eriocheir sinensis, zinc supplementation) [The excessive production of reactive oxygen species (ROS) caused by a loss of cell redox balance events can cause damage to DNA, proteins, lipids, and carbohydrates of the organism, finally resulting in cell death and tissue damage. It is well-established that antioxidants can inhibit the production of free radicals and eliminate the impairment of oxidative stress . Except ntation) , whitelentation) , red seantation) , and Chintation) . The addCarotenoid is a kind of natural functional pigment that has been used as a dietary antioxidant supplement for aquaculture, playing other important biological functions besides colorants ,8,9. Amo, Haliotis discus hannai, is a common economic shellfish that possesses a high nutritional and medicinal value [Pacific abaloneal value ,18. It ial value ,20. It ial value ,22,23. Aal value ,25,26. TH. discus hannai; a significant growth advantage was not found in the results compared with other extracts within the group, with only an observable influence on shell color. The study of Ma et al. [Dietary antioxidant supplementation, especially astaxanthin, could be a gateway to solutions or mitigation, ensuring smooth production and output increase through use of its excellent biological functions. So far, a few researchers have attempted to investigate this. Lim and Lee  first ada et al.  made anoH. pluvialis and the red yeast P. rhodozyma are economical and efficient alternative sources of astaxanthin [The market is currently mainly dominated by industrial synthetic astaxanthin; however, with an increase in the rapidly growing demand, the green microalgae axanthin ,30. TherH. discus hannai) were all provided by Fuda Abalone Aquafarm Co., Ltd. . All abalones came from the same batch of artificial hatching and were later raised in the same culturing environment. Prior to the experiment, they were transferred to a cement pond (8 m × 2.2 m × 0.8 m) and acclimated for 15 days to adapt to the experimental diets. The pond was a flowing water system and was aerated all the time, and two-thirds of the seawater was replaced once a day to maintain good water quality. The water quality was controlled under the following conditions: temperature 22.5–23.5 °C, dissolved oxygen 7.24–7.36 mg/L, salinity 31–32‰, and pH 7.6–8.0, respectively.The experimental abalones  were randomly selected and divided into 4 diet groups . Experimental diets were fed by hand until apparent satiation once a day at 18:00, and the feeding trial lasted for 100 days. Meanwhile, mortality was recorded daily. Culture management and condition control were consistent with the acclimatization period protocol described in 2.1.Gracilaria lemaneiformis . The astaxanthin supplemented in the three experimental diets was from three different sources, respectively: industrial synthetic astaxanthin , Phaffia rhodozyma  and wall-broken Haematococcus pluvialis dry powder . The addition amounts of each were chosen according to their respective optimal amounts based on previous pre-experiments (unpublished): 800 mg/kg industrial synthetic astaxanthin supplementation , 20 g/kg Phaffia rhodozyma supplementation , 4 g/kg wall-broken Haematococcus pluvialis dry powder supplementation .In the present study, four diet groups were designed, including three isonitrogenous and isolipidic  experimental diets and a natural diet, All experimental diets were provided by Fuzhou Promarine Biotechnology Co., Ltd. . Fish meal, soybean meal, and wheat gluten were used as the main protein sources. Soy lecithin was used as the main lipid source. Kelp powder, high-gluten flour, dextrin, and α-starch were used as the main carbohydrate sources. All ingredients were ultra-micronized and screened using a 100 µm mesh, and then mixed. Experimental diets sources were finally pressed into flakes (2 cm × 2 cm) using a tablet press and dried at 60 °C for 24 h. Dried flakes were stored at −20 °C. Detailed information on the diets is shown in Proximate compositions of the diets were analyzed according to the methods described by the Association of Official Analytical Chemists AOAC . Crude pAfter the trial, all abalones went through a 3-day starvation period to make their intestines empty. Individual quantity, size, and weight were determined first, and then, the survival rate (SR), daily increment in shell length (DISL), weight gain rate (WGR), specific growth rate (SGR), and visceral–somatic index (VIS) were calculated. The calculation formulas are as follows:g, 10 min) to obtain serum. Following this, hepatopancreas and intestines from the abalones were collected from the and frozen in liquid nitrogen. All serum and tissue samples were stored at −80 °C for further analysis.Twelve individuals were randomly selected from each group, and approximately 1 mL of hemolymph was collected from the foot muscle using a sterilized syringe. The hemolymph samples were immediately centrifuged  of nine-fold of their weight. The homogenate was centrifuged at 3000× All the physiological and biochemical indexes were determined using commercial assay kits  according to the manufacturer’s instructions. In detail, the glucose (GLU) and cortisol (COR) contents and alkaline phosphatase (AKP) and lysozyme (LZM) activity of the serum were analyzed using the corresponding kits . Total antioxidant capacity (T-AOC), catalase (CAT), superoxide dismutase (SOD), and glutathione peroxidase (GSH-PX) activity, and the malondialdehyde (MDA) content of the hepatopancreas, were analyzed using the corresponding kits .At the end of the feeding trial, fourteen abalones were selected from each group for heat-resistance evaluation. The heat resistance of abalone was evaluated by the heat adhesion duration (HAD) proposed by Yu et al. ; a lesseVibrio harveyi AP37, at a lethal concentration of 50% , was adopted in the bacterial challenge experiment. There was no feed for 168 h after being injected with 50 µL activated bacteria, and mortality was recorded at 9:00 a.m. and 6:00 p.m., respectively. The choice of V. harveyi AP37, the concentration determination, and activation method were all based on research by Zou et al. [At the end of the feeding trial, 45 abalones  were selected from each group for the evaluation of disease resistance. u et al. .V. harveyi AP37 at a sublethal concentration (3.0 × 106 CFU/mL) was adopted here. Among the selected abalones, thirty individuals were injected with 50 µL of V. harveyi AP37 suspension, and the remaining ten individuals were used as the negative control by being injected with an equal volume of sterile seawater. Three abalones per group were sampled each time at 0 (negative control group), 12, 24, 48, 72, and 96 h after injection, and 2 mL hemolymph of the individual was collected and kept on ice. Immune responses in the hemolymph were characterized using flow cytometry  by measuring the total hemocyte counts (THC) and mortality rate, phagocytic activity, and the production of reactive oxygen species (ROS). The determination methods for all hemolymph parameters referred to the protocols described by Zou et al. [Another forty abalones per group were collected for further disease-resistance evaluation through hemolymph immune responses, and u et al. .® DNA PCR-Free Sample Preparation Kit  was used for the library construction. Finally, sequencing was performed using NovaSeq6000.Four abalones per group were used for total DNA extraction according to the CTAB method, and agarose gel electrophoresis was used to detect its concentration and purity. The genomic DNA was amplificated through PCR using the specific primers 515 F and 806 R. After mixing and purifying PCR products, a TruSeqThe raw data were quality-controlled using fastp to obtain clean data according to Bokulich et al. . All effp value < 0.05 was recognized as statistically significant.All data analyses were conducted using SPSS 20.0 . The results were presented as means ± standard error (SE). To investigate differences among groups, on the premise of passing the normality test and homogeneity-of-variance test, a one-way analysis of variance (ANOVA) was employed. When there were statistically significant differences, Duncan’s multiple-range test was used to compare the means of different groups. In addition, the heat adhesion duration and survival-rate curves were conducted with an overall comparison using the Log Rank Test. A Phaffia rhodozyma and Haematococcus pluvialis sources (PR80 and HP120 group) compared with the industrial synthetic source (SA80 group). Significantly, the WGR in PR80 and HP120 groups grew by approximately 110% while the SA80 group also had about an 80% increase compared to the GL group. Also, the SGR of abalone in the PR80 and HP120 groups were boosted by nearly 75% compared to the GL group. As for the shell growth (FSL and DISL), the DISL in all dietary supplementation groups, in particular, rose to more than twice as much as the GL group. In addition, only the increase in the PR80 and HP120 groups reached significance in VIS compared with the GL group.After a 100-day feeding period, the dietary supplement of astaxanthin from the three sources all had obvious positive effects on the shell and weight growth  compared with the natural diet (GL group), but not in the survival rate, which all remained at a high level (>90%) . In termp > 0.05). The content of the COR of abalone from all supplementation groups was significantly lower compared with the GL group (p < 0.05). The AKP and LZM activity of abalone had the same trend, with all astaxanthin supplementation groups having significantly higher activity than the GL group.Four serum immune indexes of abalone were given in p < 0.05). There was no significant difference between all the treatment groups (p > 0.05).As the results of the hepatopancreas antioxidant enzyme activity show in H. discus hannai fed diets with astaxanthin from different supplementation sources, under heat-resistance evaluation, during the experiment. The attachment rate of abalone in the GL group first reached half at about 4 h, and the maximum HAD was also the least among all the groups. The HADs of fifty percent attachment in the SA80 and HP120 groups were similar, and both were between 5 and 6 h. The attachment rate of abalone in PR80 dropped to 50% at approximately 6.5 h, and possessed the longest HAD at about 10 h.V. harveyi AP37 infection, the survival rates of abalone in all astaxanthin-supplementation groups were significantly higher than that in the GL group (below 50%). The survival rates in the PR80 and HP120 groups were both still above 75%, and were 82% in the PR80 group and 75% in HP120 group, respectively. The lowest survival rate was seen in the SA80 group among the three astaxanthin-supplementation groups. At the end of the trial, the correlation of the survival rate among all groups still stayed the same; PR80 maintained the top at about 66%, followed by the HP120 and SA80 groups.The survival rate of abalone under the bacterial challenge experiment was shown in p > 0.05). At 12 h of the insertion, THC in all groups showed remarkable growth, but all supplementation groups presented a significantly higher number than the GL group (p < 0.05), among which the PR80 group achieved the top level. Up until 24 h after the challenge, all groups almost obtained peak values, and then showed a decrease with increasing challenge time. The relationship between groups did not change much; the THC in PR80 was always the maximum, followed by that of HP120 and then SA80. As shown in p > 0.05).The results on hemocyte mortality were shown in p < 0.05). After the challenge for 24 h, a surge in ROS in all groups started to appear, and those of the PR80 and HP120 groups rose more and were significantly higher than the SA80 and GL groups (p < 0.05). The ROS of abalone in the PR80 and HP120 groups then reduced from 48 h after the challenge until the three treatment groups showed no significant differences at the time of 96 h (p > 0.05), while they were still significantly higher than the control (p < 0.05).As shown in p > 0.05). There was a significant gap in phagocytic activity of all the treatment groups from that of the GL group at the time of 12 h. Then, they all peaked at the time of 24 h, and then, a decline was witnessed at a later time. The phagocytic activity of the PR80 group always maintained the highest position since the increase began.The phagocytic activity of abalone under the bacterial challenge is shown in The sequences were clustered into 576-3167 OTUs  for samples based on a 97% similarity level . As showp < 0.05), but not significantly higher than the PR80 group (33.03%). The relative abundances of proteobacteria in the GL and HP120 groups (20.20% and 21.87%) were significantly lower than those of the SA80 and PR80 groups (34.56% and 33.86%) (p < 0.05).The relative abundances of the top 10 bacterial communities at the phylum level were illustrated in Mycoplasma, Psychrilyobacter, and Vibrio also had the dominant relative abundances in all groups. Furthermore, unidentified_Chloroplast, Ralstonia, and Burkholderia-Caballeronia-Paraburkholderia dominated in the GL group, Serratia dominated in the HP120 group, and unidentified_Chloroplast, Ralstonia dominated in the SA80 group, whereas unidentified_Chloroplast, Ralstonia and Serratia dominated in the PR80 group. In addition, the relative abundances of Mycoplasma in the GL and HP120 groups (48.61% and 34.66%) were significantly higher than those of SA80 (13.08%) (p < 0.05). As for Serratia and Vibrio, the GL group had the least relative abundance (p < 0.05), 0.2% and 2.05%, respectively. The most abundant Psychrilyobacter was seen in the HP120 group (2.54%), and there was no significant difference among other groups (p > 0.05).At the genus level, the relative abundances of the top 10 bacterial communities of different supplementation-source groups are shown in p < 0.05).As shown in Furthermore, these biomarkers in the groups were higher for genetic information processing, cell motility, signal transduction, neurodegenerative diseases, metabolism, etc., according to the function-prediction analysis . The cluSerratia, and Psedomonadaceae in Proteobacteria (p < 0.05).Significantly different species of the intestinal microbiota of abalone fed diets with astaxanthin from different supplementation sources at different levels were found and are shown in Pelteobagrus fulvidraco). Regarding sea bass Lateolabrax maculatus, dietary supplementation of H. pluvialis improved the growth performance and antioxidant capacity of the fish. An increase was also witnessed in their immune response and bacterial resistance. Kheirabadi et al. [Litopenaeus vannamei significantly improved its immune response; it could also increase its antioxidant capacity and resistance to white spot syndrome virus (WSSV). In our study, astaxanthin from industrial synthesis, P. rhodozyma, and H. pluvialis supplementation in the abalone diet performed better in all aspects, including survival, growth, immune response, antioxidant activity, thermal resistance, and disease resistance, compared with common algae feeding. This is quite similar to previous studies on other aquatic animals. Aquatic animals are unable to synthesize carotenoids, so they need to be added into their feed ,36. Astai et al.  added rei et al.  indicateH. pluvialis and synthetic astaxanthin on carotenoid composition, concentration, esterification degree, and astaxanthin isomers in the ovaries, hepatopancreas, carapace, and the epithelium of Chinese mitten crab (Eriocheir sinensis), and the results suggest that H. pluvialis is more effective in astaxanthin accumulation, and it is better at increasing the carotenoid and astaxanthin isomer composition compared with synthetic astaxanthin. Xie et al. [H. pluvialis supplementation in golden pompano (Trachinotus ovatus). Astaxanthin supplementation from two sources all provided the promotion of growth performance, antioxidant capacity, and anti-inflammation, but there were no significant differences between them. The effects of natural astaxanthin from H. pluvialis and synthetic astaxanthin supplementation were also evaluated on the ridgetail white prawn . The results illustrated that natural astaxanthin from H. pluvialis showed superiority in growth performance, pigmentation, and astaxanthin content than that from synthetic astaxanthin. In our study, as for survival and growth performance, astaxanthin from the two natural sources caused better growth performances in abalone than the industrial synthetic source. In terms of heat stress and bacterial challenge, abalone fed dietary astaxanthin from Phaffia rhodozyma showed the strongest resistance. In brief, the supplementation effects of different astaxanthin sources were different, even among the natural sources.Although there were some differences among them because of the supplementation level and the sources of supplementation, these results contribute to improving our understanding of the addition of astaxanthin in aquaculture. Certainly, some researchers have noticed the problem of the sources of supplementation, like our study. For instance, Su et al.  comparede et al.  comparedIt is clear that astaxanthin dietary supplementation significantly altered the gut microbiota structure of abalone according to the alpha diversity and beta diversity data in this study. However, when relying on clustering results and diversity data, the fact that the intestinal microbiota structure of the HP120 group was not affected too much and was similar to that of the GL group may look confusing. The intestinal microbiota structure of SA80 and PR80 changed a lot and influenced the level and manner to a similar extent, clustering into a group in clustering analysis. The variation in the intestinal microbiota structure cannot match the results of other production performances like growth, stress resistance, disease resistance, and immunity. The intestinal microbiota structure of the HP120 group was similar to the GL group, but the three supplementation groups all achieved better performances compared to the GL group. It is highly possible that HP120 achieved positive effects through other approaches, so we focused on the other two astaxanthin supplementation groups in which the intestinal microbiota may have made greater contributions. Hence, we discovered all the significantly different species of the intestinal microbiota of abalone among groups at different levels. The situation that the relative abundance of the PR80 group was significantly higher than that of the SA80 group was especially notable because the effects of the PR80 group were better than the SA80 group. Serratia, and Psedomonadaceae in Proteobacteria. Proteobacteria and Firmicutes were the dominant phylum in most aquatic animals’ intestines [P. rhodozyma supplementation (80 mg/kg astaxanthin) in the abalone diet obtained a better production performance because of the high percentage of intestinal Proteobacteria, which had a stronger ability to utilize carbohydrates and an increased FE than the added synthetic astaxanthin in the abalone diet. A significantly higher relative abundance of Firmicutes was also observed in the PR80 group. Some studies have evidenced that a higher relative abundance of Firmicutes means a higher energy-harvesting ability, specifically reflected in a higher fatty-acid-absorption and lipid-metabolism ability [P. rhodozyma supplementation caused a higher abundance of Firmicutes, and the digestion and metabolism of lipids in the host were strengthened, finally resulting in a better growth performance. According to our results, the relative abundance in the PR80 group being significantly higher than that in the SA80 group was found in Oscillospirales, Ruminococcaceae, and Staphylococcaceae in Firmicutes and Yersiniaceae, testines ,44,45. Ptestines ,47. A retestines  also rep ability ,50,51. ISerratia, Yersiniaceae, and Psedomonadaceae belonging to Proteobacteria showed a higher abundance in the PR80 group compared to the synthetic astaxanthin supplementation group. There has been limited research to understand the function and effect of Serratia in animals’ gut. Present acknowledgements about Serratia have been divergent: some studies have held the view that Serratia is a Gram-negative opportunistic pathogen, and can injure intestinal epithelial cells causing gastrointestinal problems, and has consequences for the host’s physiology and health [Serratia can activate innate immune responses of Drosophila through Toll and Imd immune-signaling pathways to achieve the goal of anti-Plasmodium and antibacterial immune defense. Also, the study of Nie et al. [Serratia can produce a bioactive substance Prodigiosin (PG), a red microbial pigment with a tetrapyrrole ring structure. It has multiple biological functions, including anticancer, antimalarial, antibacterial, and immunomodulatory functions. Therefore, combining the results of this experiment, the absolute predominance of Serratia in the PR80 group compared to the SA80 group should result in a positive influence on abalone, such as representing antibacterial, and immunomodulatory function, so that advantages can be seen in the heat-resistance evaluation experiment and the bacterial challenge experiment. The significantly different species Psedomonadaceae also belongs to Proteobacteria. In terms of insect biology, gut Pseudomonas in olive fly guts can help hydrolyze the proteins of olive flesh [In detail, d health ,53,54. Od health  illustrae et al.  proved tve flesh . This reve flesh ,59. As aP. rhodozyma astaxanthin. As for another significantly different species Firmicutes, Ruminococcaceae participates in the degradation and fermentation of polysaccharides and fibers . Its maiThe intestinal microbe Lactococcus also cannot be ignored; related results are shown in the LEfSe analysis. Lactococcus was the only biomarker in the PR80 group, which may help to explain the reason why comprehensive advantages were presented in the PR80 group. According to extensive reports, Lactococcus is a marker of the health of the intestines, and can promote the recovery of blood metabolites  and the H. pluvialis only possesses the  isomer, while yeast P. rhodozyma has the  isomer. However, all three types of optical isomers can be found in synthetic astaxanthin, at a proportion of 1:2:1 of   and  isomers [Astaxanthin supplementation from different sources has been proved to impose influence on abalone by different pathways and approaches; however, what caused the addition of the ‘same’ astaxanthin to cause different results? Astaxanthin has three configurational isomers, including stereoisomers  and ) and a geometric isomer ). There are different isomers contained in different astaxanthin sources.  isomers ,73,74.In addition, astaxanthin exists in free and esterified forms, because it can be esterified in hydroxyl groups with different fatty acids or without esterification or combined with proteins . InteresTherefore, the best form of astaxanthin should depend on the demand: it needs to be accurately, efficiently, and sustainably produced in the service industry. In brief, isomer differences and their associations with compounds may be the cause of production performance differences .Phaffia rhodozyma, and Haematococcus pluvialis into the abalone diet performed better in all aspects including survival, growth, immune response, antioxidant activity, thermal resistance, and disease resistance, compared with common algae feeding. As for survival and growth performance, astaxanthin sources from Phaffia rhodozyma and Haematococcus pluvialis had better growth performances than the industrial synthetic source. In terms of heat stress and bacterial challenge, abalone fed a diet of dietary astaxanthin from the Phaffia rhodozyma source showed the strongest resistance. Overall, 80 mg/kg astaxanthin supplementation from Phaffia rhodozyma is recommended to achieve a more effective and comprehensive outcome.The supplementation of astaxanthin from industrial synthesis,


Reticulitermes amamianus (Ra) and R. speratus (Rs)  and demonstrate that the soldier sex ratio and worker population size of hybrid colonies skew to colonies of king’s species, while the soldier proportion skew to queen’s species. The survival rate of offspring resulting from interspecies hybridization was significantly higher for mRa × fRs than for mRs × fRa. The results of this study demonstrate the asymmetric influence of kings and queens on caste determination and colony growth, which can contribute to our better understanding of parental influence on the colony dynamics of social insects.Altruistic caste, including worker and soldier (derived from worker), plays a critical role in the ecological success of social insects. The proportion of soldiers, soldier sex ratios, and the number of workers vary significantly between species, and also within species, depending on colony developmental stage and environmental factors. However, it is unknown whether there are sex-linked effects from parents on controlling the caste fate or not. Here, we compared soldier sex ratios, soldier proportions, and population size among a four mating types of  In the ecological and evolutionary progression of social insects, division of labor plays an integral role, as progeny differentiate into either reproductive (nymph) or functionally sterile castes –3. WorkeReticulitermes termites. Accumulating evidence suggests that hybrid termite colonies can inherit phenotypic traits from both parental species [Reticulitermes soldiers differentiate from fourth- or fifth-instar workers in mature colonies  and R. speratus (Rs). First, we collected colonies of these two species in the field and compared the sex ratio of soldiers in mature field colonies, finding that the soldier sex ratio of R. speratus colonies was skewed towards females, while that of R. amamianus colonies was skewed towards males. Thus, we compared soldier sex ratio, soldier proportion, and number of workers among four combinations of parent species: mRa × fRa, mRa × fRs, mRs × fRa, and mRs × fRs . We performed two rounds of observations and data collection: first in colonies aged two years, and second in the colonies aged two and a half years.In this study, we investigated sex-linked effects from parents on the colony characteristics by using hybrid colonies of R. amamianus or R. speratus colonies were collected in pine or Japanese cedar forests in Kagoshima, Hyogo, Kyoto, Shiga, and Hokkaido, Japan, from 2019–2021 . Twenty eggs were randomly extracted from each colony and transferred into a dish (34 mm diameter × 10 mm height) with BPC medium and 50 male workers. The male workers were selected from a different colony of the same cross.The hatching period in  35 days , so we r 35 days , 22, 23.R. amamianus and R. speratus against the null hypothesis assuming that the numbers of males and females were equal. The total number of male and female soldiers in each species were used for the analyses.Exact binomial tests were applied to compare the observed numerical soldier sex ratio in field-collected colonies in Comparison of the numerical soldier sex ratio, soldier ratio, offspring survival rate, or offspring sex ratio among four combinations of parent species  was performed with generalized linear models (GLMMs) with a binomial distribution. In the models, the objective variable was the numerical sex ratio of soldiers , soldier ratio (number of soldiers vs workers), offspring survival rate (number of surviving vs dead larvae), or offspring sex ratio , the explanatory variable was the combination of parent species, and colony ID was treated as a random factor. Comparison of the number of workers among the four combinations of parent species was performed with a GLMM with Poisson distribution. In the model, the objective variable was the number of workers, the explanatory variable was the combination of parent species, and colony ID was treated as a random factor. We also performed GLMMs which included a species of king × queen interaction term instead of the combination of parent species to account for sex and species-specific effect. Likelihood ratio tests (LRTs) were used to determine the statistical significance of each explanatory variable.Exact binomial tests were applied to compare the observed numerical offspring sex ratio in each combination of parent species against the null hypothesis assuming that the numbers of males and females were equal. The total number of male and female offspring in each combination of parent species were used for the analyses.p < 0.05 was considered to indicate statistical significance.All analyses were performed by the software R v4.2.3 , with thR. amamianus colonies, the mean numerical sex ratio of soldiers  was significantly skewed toward males .In the field collected p < 0.05, χ2 = 0.728, df = 1, p = 0.394; 2.5-year-old colonies: GLMM, LRT: χ2 = 0.435, df = 1, p = 0.510).The hybrid experiment showed that the species of king had a significant effect on the soldier sex ratio .The species of queen had a significant effect on the soldier proportions in 2-year-old colonies . In colonies where the queen is R. amamianus, the species of king had a significant effect , while in colonies where the queen is R. speratus, the effect was not significant.The pattern was similar in 2.5-year-old colonies, but there was a significant interaction effect between the species of king and queen .The species of king had a significant effect on the number of workers in 2-year-old colonies . In colonies where the king is R. amamianus, the species of queen had a significant effect on the proportion of soldiers , while in colonies where the king is R. speratus, the effect was not significant.In 2.5-year-old colonies, there was a significant interaction between the species of king and queen , while there was no statistically significant bias in the larval sex ratio in mRa × fRs , mRs × fRa , and mRs × fRs . All the offspring were differentiated into workers, and no nymphs were found.There was no significant difference in worker sex ratio among the colony types  as well as socioenvironmental factors . Parent-offspring communication, exemplified by trophallaxis, plays a pivotal role in determining soldier caste . HoweverReticulitermes termites, it is known that epigenetic factors inherited from the king and queen can influence the caste determination of offspring (whether they follow the worker pathway or nymph pathway) [Reticulitermes genus. In the case of R. speratus and R. virginicus, which are AQS species [Phenotypic traits in offspring of various organisms can be influenced by heritable factors beyond DNA sequences , 30. Sevpathway) , 34, and species , 36, the species . The ideS1 Text(PDF)Click here for additional data file.S1 Fig(TIF)Click here for additional data file.S1 Dataset(XLSX)Click here for additional data file.S2 Dataset(XLSX)Click here for additional data file.