Animals in the NEAD, NOAD, LEAD, and LOAD groups also received methimazole and cholecalciferol daily. Urinary, biochemical, hemodynamic, and electrocardiographic parameters and renal function were assessed. Samples of the liver, heart, kidney, and arteries were collected to investigate redox status and perform histopathological analyses. All of the groups developed dyslipidemia and hepatic steatosis. Only the NEAD group developed arterial lesions that were compatible with fatty streaks. Renal function was significantly impaired in the LEAD and NOAD groups. These results indicate a viable alternative to induce atherogenesis and hepato-renal impairment in female rats.Therapeutic approaches for the treatment of dyslipidemia and atherosclerosis have radically changed in recent decades. Part of this advance undeniably stems from basic biomedical research that has provided a better understanding and identification of new therapeutic targets. The aim of this work was to develop a model to induce atherogenesis and hepato-renal impairment in female Wistar rats. The following groups received the respective treatments for 60 days: control animals, non-ovariectomized rats that received an atherogenic diet (NEAD), ovariectomized rats that received an atherogenic diet (NOAD), non-ovariectomized rats that received an atherogenic diet and oral Nω-nitro- Atherosclerosis is a multifactorial condition that is associated with chronic inflammatory lesions in the arterial wall. One of the main triggers of atherosclerosis is dyslipidemia, but a wide range of extra-dietary factors may also be involved, including hypertension, smoking, and a sedentary lifestyle ,2,3. OneIn addition to vascular injury itself, dyslipidemias are also involved in a wide range of diseases. Several other organs may be affected by lipid imbalance, among which the heart, kidneys, and liver are notable. The reduction of renal and hepatic function is an important factor that exacerbates subjacent cardiovascular disease and increases its morbidity and mortality . Moreove−/−) and LDL receptor homozygous knockout (LDLR−/−) models [Therapeutic approaches for the treatment of dyslipidemia and atherosclerosis have changed radically in recent decades, mainly through a better understanding of the pathophysiological basis of the disease . This ad) models . Despite) models . Therefol-arginine methyl ester hydrochloride (l-NAME), phenylephrine (Phe), sodium nitroprusside, acetylcholine, NaCl, KCl, CaCl2, MgSO4, NaHCO3, KH2PO4, dextrose, ethylenediaminetetraacetic acid, cholesterol, cholecalciferol, colic acid, and methimazole were purchased from Sigma-Aldrich . All of the other reagents were obtained in analytical grade.The following drugs, salts, and solutions were used: ketamine hydrochloride , xylazine hydrochloride , and heparin . Nω-nitro-Twelve week old female Wistar rats, weighing 220–250 g, were housed in plastic cages with environmental enrichment at 22 ± 2 °C and 55% ± 10% humidity under a 12 h/12 h light/dark cycle. The animals had ad libitum access to food and water. All of the experimental procedures were approved by the Institutional Ethics Committee of Federal University of Grande Dourados  and conducted in agreement with the Brazilian Legal Framework on the Scientific Use of Animals.Female rats were intraperitoneally anesthetized with 100 mg/kg ketamine plus 20 mg/kg xylazine. The animals then underwent laparotomy, and the two ovaries were gently removed. The non-ovariectomized groups (including the control group) underwent laparotomy, but the ovaries were not removed. After the surgical procedure, the animals were hydrated with saline solution  and received the anti-inflammatory drug ketoprofen  and antibiotic enrofloxacin .n = 8/group): normotensive and non-ovariectomized rats that received an standard diet (control rats), normotensive and non-ovariectomized rats that received an atherogenic diet (NEAD), normotensive and ovariectomized rats that received an atherogenic diet (NOAD), normotensive and non-ovariectomized rats that received an atherogenic diet and oral l-NAME , and normotensive and ovariectomized rats that received an atherogenic diet and oral l-NAME . The atherogenic diet had the following composition: 64.4% standard diet, 0.5% cholesterol, 0.1% sodium cholate, 5% sucrose, 5% lard, 5% hydrogenated fat, and 20% egg powder. Animals in the NEAD, NOAD, LEAD, and LOAD groups received oral methimazole (2 mg/kg) daily and four intraperitoneal injections (every 2 days) of 175,000 IU/kg cholecalciferol at each dose [The animals were randomly allocated to the following groups (ach dose . All of These experiments were performed on the first day after beginning the treatments and at the end of the experiment period (60 days). All of the animals were placed in metabolic cages for 24 h with food and water ad libitum. Twenty-four hour urine volume, pH, and density were measured. An aliquot was collected for electrolyte  and creatinine determination.At the end of the experiment period, the animals were intramuscularly anesthetized with 100 mg/kg ketamine plus 20 mg/kg xylazine. After anesthesia, all of the rats underwent electrocardiography (ECG) according to the methodology of Romão et al. . Using fAfter ECG recording and while under deep anesthesia, systolic blood pressure (SBP) and mean arterial pressure (MAP) were determined by the direct method according to Gasparotto Junior et al. . A subcu2, 1.2 mM MgSO4, 25.0 mM NaHCO3, 1.2 mM KH2PO4, 11.1 mM dextrose, and 0.03 mM ethylenediaminetetraacetic acid) that was aerated with 95% O2 and 5% CO2 at a constant flow rate of 4 mL/min. Changes in perfusion pressure  were captured by a pressure transducer that was connected to the PowerLab system and Chart 7.1 software . After a stabilization time of 30 min, tissue integrity was assessed with a bolus injection of KCl (120 mmol). Different doses of Phe were then administered in the MVB  and kidney  preparations. Angiotensin II (AngII) was administered only in the kidney preparations . After a new stabilization time of 30 min, the tissues were continuously perfused with PSS plus 3 µM Phe to induce a prolonged increase in perfusion pressure. After stabilization of the contractile process, vascular reactivity to acetylcholine  and sodium nitroprusside  was evaluated. A 10 min period elapsed between each drug administration. At the end of the experiments, the animals were euthanized by deep anesthesia in a saturation chamber (isoflurane with >20% saturation).After the direct blood pressure measurements, the mesenteric vascular bed (MVB) and left kidney were isolated and prepared for perfusion according to previously described methods . The reng for 10 min. Alanine aminotransferase (ALT), aspartate aminotransferase (AST), indirect bilirubin, direct bilirubin, total bilirubin, total protein, albumin, globulin, the albumin/globulin ratio, creatinine, urea, triglycerides (TG), total cholesterol (TC), high-density lipoprotein cholesterol (HDL-C), alkaline phosphatase, γ-glutamyl transpeptidase (GGT), sodium, calcium, magnesium, chloride, and potassium levels were measured using an automated biochemical analyzer (Roche Cobas Integra 400 plus). Low-density lipoprotein cholesterol (LDL-C) and very-low-density lipoprotein cholesterol (VLDL-C) were calculated according to the Friedwald formula [Immediately before euthanasia, blood samples were collected from the previously cannulated left carotid artery. Serum was obtained by centrifugation at 1500×  formula .After euthanasia, the heart, right kidney, and liver were cleaned, weighed, and longitudinally sectioned. The relative organ weight was determined as the following: Relative Weight (RW)% = absolute organ weight × 100/body weight.g for 20 min at 4 °C. Different dilutions were used for each tissue. The following enzymes were evaluated in the supernatant: catalase , and right kidney were homogenized in phosphate buffer (pH 6.5) and centrifuged at 5269× rding to ). Lipoperding to . The resrding to .Parts of the left ventricle, right kidney, liver, subclavian artery, and right carotid artery were placed in 10% buffered formalin. The samples were then dehydrated in alcohol, cleared with xylene, and embedded in paraffin. The samples were sectioned (5 mm) and stained with hematoxylin-eosin and orcein (arteries) and examined under a light microscope. Right and left ventricles and interventricular septum were measured. The intima layer of the subclavian and carotid artery was also measured. We assembled several blades with the entire arterial branch. All neointimal formations were identified per slide. For each atherosclerotic formation, six lines of division were established. For each neointimal formation, we performed six measurements, and one mean for each slide was calculated. When all of the slides were evaluated, a new mean for each animal was calculated. The area of the microscope was adjusted using an ocular micrometer with a 100× objective to determine the size of the field per square micrometer (µm²). All images were obtained and evaluated using Motic Images Plus 2.0 software.n = 8 animals per group. Values of p < 0.05 were considered statistically significant.Statistical analyses were performed using analysis of variance (ANOVA) followed by Bonferroni’s post hoc test. The results are expressed as the mean ± standard error of the mean (SEM) of Different urinary parameters in the various groups over 60 days of treatment are presented in No significant electrocardiographic changes were observed among the different experimental groups .Blood pressure and heart rate data in the different groups are presented in Biochemical parameters in the various experimental groups over 60 days of treatment are presented in Oxidized LDL, MDA, and NT levels were significantly elevated in all of the experimental groups compared with control-rats. The NEAD, LEAD, and LOAD groups exhibited significant increases in sVCAM-1, sICAM-1, IL-6, and IL-1β levels compared with the control. As expected, estradiol levels were significantly lower in ovariectomized animals.Phe, ACh, and SNP reactivity in the isolated and perfused MVB in the various groups are presented in Renal vascular reactivity in the various groups over 60 days of treatment is presented in The initial and final body weight and relative weight of the heart and right kidney did not show any statistically significant changes compared to the different experimental groups . The relThe levels of SOD, CAT, and LPO in cardiac, renal, and arterial tissues are presented in The interventricular septum and intimal layer of the subclavian and carotid arteries in the NEAD group exhibited significant thickening compared with all of the other experimental groups . The rigRepresentative histological images of the liver, left ventricle, kidney, and arteries (subclavian and carotid) in the various experimental groups are shown in Animal models cannot fully reproduce all stages of the development of atherosclerosis in humans. However, in many cases, they can predict important pathophysiological mechanisms of the disease and assist in the identification of new therapeutic targets . Non-genl-NAME to reduce the body supply of NO, an important vasodilator and antioxidant and antiplatelet agent [l-NAME in the proposed model. We included l-NAME to increase LDL oxidation, a limiting step in atheromatous plaque formation. Additionally, NO is an important endothelial mediator that controls vascular tone, and NO inhibition can induce endothelial dysfunction that precedes atherosclerotic lesions [Despite the fact that females are known to have specific cardiovascular characteristics, the treatments that are specifically directed toward this gender are nonexistent . Additioet agent . No stud lesions .In rodents, a standard HFD alone is incapable of inducing more advanced stages of atherosclerosis. Data have shown that an HFD leads only to earlier events of the disease, such as slight thickening of the intimal layer of the arteries . Thus, wAlthough all the models proposed in this study have induced relevant pathophysiological changes, including dyslipidemia and steatosis, one fact caught our attention. Only the NEAD group showed increased LDL-C and significant atherosclerotic lesions. Several data have shown that estrogen promotes natural protection to the endothelium and is related to the decrease of serum cholesterol, especially LDL-C. This natural protection occurs through the regulation of anti-atherogenic agents, such as nitric oxide and indirect effects on the liver . In humal-NAME raised blood pressure only in the LOAD group. This is likely attributable to the fact that non-ovariectomized females (LEAD group) had high levels of circulating estradiol, which likely contributed to the observed cardioprotective effects [l-NAME-treated groups, with even more pronounced effects in ovariectomized animals (LOAD group). This was mainly attributable to the antioxidant effects of estradiol, which exerts protective effects on endothelial function even in the absence of NO [Another significant difference among the present experimental models involved cardiovascular hemodynamics. Interestingly,  effects ,42. As ece of NO . AlthougSome of the data on cardiac architecture were also interesting. Although we did not detect any significant electrocardiographic alterations, we also observed important cardiac structural changes, including fibrosis and hypertrophy, especially in the NOAD group. Hypertension is a well-known risk factor for the development of ventricular hypertrophy. In the present study, only the LOAD group exhibited a significant increase in SBP. Nevertheless, no trace of cardiac muscle hypertrophy was found in this group. We believe this was attributable to the slight elevation of blood pressure and the time of exposure to these hemodynamic changes . It is ul-NAME administration. The administration of l-NAME in the proposed models did not improve the atherogenic profile and actually aggravated renal function. Concentric inflammatory areas in the kidneys were commonly observed among the histological findings in these animals. l-NAME may reduce renal function by causing less dilation of the efferent arteriole and reducing glomerular perfusion pressure and filtration rate [l-NAME had a greater impact on renal function than on the development of the atherosclerotic process.In general, renal function in the experimental groups was significantly influenced by ion rate . Thus, il-NAME may be a possible reason, but further studies are needed to examine this possibility. Second, the use of more effective methodologies to raise blood pressure could have been employed. The use of spontaneously hypertensive rats or animals with renovascular hypertensive may be promising, which would simulate conditions that are closer to the human condition. Nevertheless, the present findings reveal a new non-genetic rat model of atherosclerotic disease that may contribute to further research on the prevention, staging, and regression of atherosclerosis and its comorbidities.The present study had two important limitations. First, during execution of the protocols, we could not determine why only the NEAD group exhibited higher LDL levels and lower HDL levels if the diet that was offered to the animals was the same. The presence of estradiol and absence of The present study identified a viable alternative to induce atherogenesis and hepato-renal impairment in female rats. Although the absence of estradiol and inhibition of NO synthesis did not directly affect hepatic changes or atherosclerotic plaque formation, renal function appeared to be highly dependent on NO levels.


This study was aimed at describing calf comfort and determining the individual and pen level factors that affect comfort status  of smallholder dairy farms in Meru County, Kenya.A cross-sectional study was carried out on 52 calves that were up to 1 year old in 38 dairy farms (mean±standard deviation: Herd size=1.71±0.7 milking cows and milk production=6.7±3.1 L/day) in Meru, Kenya, in 2017, with the intention to describe their comfort and determine the factors associated with leg hygiene as a critical parameter for calf comfort assessment. Calves’ biodata, health status, and leg hygiene were assessed, along with pen characteristics such as area, hygiene, and knee impact and knee wetness scores, while a questionnaire was administered to the farmers to gather information regarding calf housing management practices in the farm.2. Approximately 75% of the calves (39/52) were kept in pens, and the rest were reared outdoors. For 39 calves kept indoors, 26% (10/39) of them had wooden or concrete floors while 74% (29/39) had dirt floors. Nearly two-thirds (62%) of indoor calves (26/39) were reared in pens with bedding, and 23% (9/39) and 33% (13/39) of the calves reared indoors were kept in pens displaying a failed knee impact test and failed knee wetness test. Indoor housed calves had an increased probability of having dirty calf legs (cleanliness score of >2.5) by 8.6 times (p=0.031), compared to outdoor-housed calves. In the final multivariable logistic regression model of 39 calves in pens, concrete or wood floors , poor body condition  and use of bedding  appeared to be positively correlated with dirtiness of calf legs, compared to dirt floors, good body condition, and no bedding, respectively.The calves had a mean body weight of 85.2±32.8 kg and average daily weight gain of 0.50±0.45 kg per day. 71% of calves had a good body condition score (≥2.5), and the mean space allowance per calf was 2.52±1.56 mOverall, some calf comfort aspects were covered for the majority of calves examined, but 69% of the pens were categorized as dirty, especially those with wooden or concrete floors and poor bedding management. Smallholder dairy farmers in Kenya should be trained on calf housing management to improve calf comfort and productivity. Animal welfare has become a global concern in livestock, companion, and wild animals . To mainIn Kenya, dairy farming is a significant contributor to the gross domestic product . Dairy cThis study was aimed at describing calf comfort and determining the individual and pen level factors that affected calf comfort  of smallholder dairy farms in Meru County, Kenya.The study was approved by the Research Ethics Board and the Animal Care Committee of the University of Prince Edward Island, the Naari Dairy Farmers Cooperative Society (NDFCS), and Farmers Helping Farmers (FHF), a partnering non-governmental organization with over 35 years of experience working with Kenyan farmers and farm groups.The study was explained orally to all participants, and signatures for informed consent were obtained from all the participants in the study.This research was a cross-sectional study carried out in 38 dairy farms in the Naari region of Meru County in Kenya between May and August 2017. The full list of active farmers in the NDFCS was used as the initial sampling frame that was limited to 200 farms using simple random sampling. The following inclusion criteria were used to further identify 100 farms from the 200 farms for ongoing monitoring for the project: Zero-grazing units for the lactating cows and up to three cows per farm. Farms without calves and farms with calves older than 1 year were excluded from the study, leaving 52 calves, and 38 farms for the study.The 52 calves were assessed for their: Biodata, welfare, health status, and management.For biodata, the weight was measured in kg using a dairy cow heart girth weight tape. The height was measured using a height stick with a level that was placed at the withers. All calves were hybrids; thus, the breeds were described as cross-bred if the calves were visibly and predominantly Friesian, Guernsey, Ayrshire or Jersey, and indigenous if they were visibly and predominantly Zebu, Boran, or other local breeds. The body condition score was described using the 5-score chart that ranged from 1 (very thin) to 5 (excessively fat), with quarter-point increments .For calf welfare data, the leg hygiene of the calves was assessed using a 5-score system  that incFor the calves kept in pens: Data on pen dimensions, floor type, and roof adequacy, type of bedding, pen hygiene, knee wetness, and knee impact test scores were collected. The pen dimensions were measured in cm. The knee impact test  was used to determine how soft the stall surface was and was categorized into three possible levels: Normal, marginal, and hard. If the floor was soft and did not cause any level of discomfort on the knees, the floor was categorized as normal which indicated a passing grade on the knee impact test. If the floor was somewhat uncomfortable on the knees, such as a cement floor with a modest amount of bedding or a dirt floor that was compacted, then it was classified as marginal. If the floor caused extreme discomfort on the knees on impact, the floor was classified as hard and this indicated failure of the knee impact test.The degree of wetness on the floor surface was assessed using the knee wetness test, which was categorized as normal if the knee was completely dry after about 10-15 s of knee contact on the floor, marginal if the knee had some noticeable moisture, and wet if the knee was completely wet after the contact with the floor. The knee wetness and impact tests have been used elsewhere to assess floor conditions for cattle . We inclThe pen hygiene score reflected the leg hygiene score where the categories included: (1) Very clean, (2) clean, (3) fair, (4) dirty, and (5) very dirty. The adequacy of the roof (yes or no) was determined based on parameters such as the cover of the entire pen and the allowance of water to enter or not to enter into the pen through holes.The health status of the calves was determined by conducting a physical examination of the calf that included but not limited to; heart rate, respiratory rate, color of mucous membranes, palpation of superficial lymph nodes, rumen movements (where applicable), skin condition, joints and feet examination, and examination of the umbilicus. Any abnormalities observed were recorded and treated on the farm level.A questionnaire was administered to the 38 farmers face-to-face by the investigator using the native language of Kimeru to gather information on the housing management status of the pre-weaned and weaned calves.® 14.2 statistical software  for analysis. Descriptive statistics, such as means, standard deviations, and ranges, were determined for continuous variables, while proportions were calculated for binary and categorical variables.Collected data were entered manually into Microsoft Excel 2013 , cleaned, coded, and imported to StataAverage daily weight gain (ADG) was estimated by dividing the difference between body weight during the study and estimated birth weight by the age of the calves in days. The average birth weight was estimated as an average from birth weights reported in smallholder farms in Africa -38. CompUnivariable logistic regressions were used to determine unconditional associations of variables with calf cleanliness. This outcome of interest was generated by dichotomizing the leg hygiene score, with scores lower or equal to 2.5 categorized as clean, while scores >2.5 were categorized as dirty. This parameter was chosen for regression analyses because calf cleanliness represents an overall comfort parameter, and it had sufficient variability for regression analyses.Variables with a p<0.2, on univariable analyses, were eligible to be fit in a multivariable logistic regression model to determine factors associated with calf dirtiness, while controlling for possible confounding effects. Since there were 52 calves in total but only 39 calves reared in pens, two multivariable models were used, one for each of the calf-level variables and one for the respective pen-level variables. Variables eligible for these separate models were also utilized to build a combined model of both calf-level and pen-level variables. These multivariable logistic regression models were fit through backward elimination, retaining variables with p<0.1 due to the small sample size. The final combined logistic regression model was assessed for two-way interactions, independence, linearity, goodness of fit, influential observations, and predictive ability.The age of calves ranged from 1 week to 12 months with an average of 5.2±3.1 months, with more details provided in Lameness, carpal lesions, and hock lesions were absent in all the examined calves, while a neck lesion was seen in one female 10 months old calf.2, with the number of calves per pen ranging from 1 to 3, with a mean of 1.4±0.5 calves per pen. The available pen area per calf averaged 2.52±1.56 m2, with a range of 1.02–7.64 m2. While the calves in the smallholder dairy farms were reared in housing systems that were quite different from each other across farms in the study, all the calf pens assessed had an appropriate roof. For the 39 calves kept in pens, 23% and 33% of them had a failed knee impact and knee wetness test, respectively, with 62% of pens having bedding and 26% of pen floors being wooden or concrete. The overall mean pen hygiene score was 2.9±0.9, while the mean leg hygiene score was 2.3±1.1. 65% (34/52) of all the calves observed were categorized as clean, with a leg hygiene score ≤2.5. For the 39 pens, 23 of them (59%) were categorized as clean.75% of the calves (39/52) were reared in pens on 28 separate farms . The ave2=7.90; p=0.019).On univariable analyses, variables with a p<0.2 included: Age, breed, BCS, and the housing variable, all which were eligible to be fit into the preliminary multivariable logistic regression model of calf-related variables associated with calf cleanliness among the 52 calves. Type of floor, type of bedding and pen hygiene score were variables with a p<0.2 on univariable analyses, and therefore eligible to be fit into the preliminary multivariable logistic regression model of pen-level variables associated with calf cleanliness among the 39 calves in pens.For the multivariable logistic regression of calf-related variables, body condition score had a slight correlation with age (r=0.20). The probability of calves being categorized as dirty was higher in older calves (2.5–5.5 months) in comparison to younger calves . Cross-bred calves had higher odds of having a dirty score compared to indigenous calves , while indoor-reared calves were dirtier than outdoor-reared calves . The multivariable logistic regression model of pen-related variables indicated that the odds of calves being dirty were 6.8 times higher in calves kept in groups compared to the odds of calves housed individually (p=0.097). Calves in pens categorized as dirty had 6.4 times higher odds of being categorized as dirty (p=0.088) compared to calves in pens categorized as clean. The odds of calves having a dirty score (>2.5) were higher in pens with bedding than calves in pens without bedding (p=0.025). In addition, use of sawdust or wood shavings as bedding increased the odds of calves being categorized as dirty by 13.2 times versus no bedding at all (p=0.017).2=3.34; p=0.503).When combining the calf- and pen-related factors in the multivariable logistic regression model for calf leg hygiene in the 39 calves raised in pens, concrete or wood floors, poor body condition and use of bedding were risk factors associated with calves categorized as dirty . There wThis is the first assessment of calf comfort among smallholder dairy farms in tropical countries and provides useful information for guiding extension officers and animal health professionals on how to assess basic parameters of calf welfare, without impairing the 5 domains of welfare recently proposed . This stThere is limited information on calf- and pen-related factors associated with calf comfort in smallholder dairy farms in Kenya. However, leg hygiene scores of the calf can be used as indicators of calf comfort.In the calf-related multivariable logistic regression model, indoor calves (75%) housed in pens were dirtier than outdoor-reared calves, and this could be a result of poor management practices, such as delays in dirty bedding and/or manure removal. The 13 calves kept outside were either tethered on a long rope or grazing in a field, and therefore their frequent movement during the day, and the availability of more space for lying, standing and movement explain the low probability of having dirty leg hygiene scores (OR=0.12) in comparison to indoor calves. A survey of calf management practices in Quebec, Canada showed that inappropriate calf housing systems such as tie-stalls (13.9%) and attachment against a wall (5.7%) were risk factors for poor calf welfare . The out2 available for each calf in our study was higher than that recommended (1.4 m2) for calves with an average weight of up to 160 kg in Germany [2, while individual calves had 3.1±1.7 m2), in addition to poor manure and bedding management practices in group-housed calves.The average lying space of 2.5 m Germany . Calves A higher body condition score can be attributed to sufficient feed intake, good health status, and subsequently larger body weight which was observed in calves with body condition scores >2.5 (p=0.019). Calves with higher body condition scores (>2.25) were also less likely to have dirty leg scores which could be due to better management practices by the farmers, such as the regular removal of manure. A recent study stipulated that changes in an animal’s BCS may reflect the adjustment of its welfare status, but this relationship is also affected by other factors .The estimated average daily gain (0.5 kg) was higher than the mean daily gain reported in a study carried out in the Nakuru region of Kenya , and thiIn the pen-related (and combined) multivariable logistic regression model, calves kept in pens with wooden or concrete floors had significantly higher odds of being dirty  which coAvailability of any bedding in the pens increased the odds of a dirty leg hygiene score of the calves which could be due to the delayed removal of dirty wet bedding and the insufficient or delayed addition of new bedding in the pens. The higher odds of calves being dirty in pens bedded with sawdust and wood shavings could be due to low availability and the high cost of acquiring new sawdust and wood shavings in comparison to crop waste that is readily available at the farms. Other studies have shown that the type of bedding used in rearing calves can have an effect on calf cleanliness .As expected, calf cleanliness was strongly related to the pen hygiene, since a dirty score for the pen was closely related with a large portion of the pen being wet, a fact that increases the likelihood of the calves being dirty. We also speculate that poor moisture absorbency of crop waste may explain the lower pen hygiene level in crop-waste bedded pens compared to sawdust or wood shavings bedded pens . Crop waOverall, some welfare aspects of the examined calves were adequate, with nearly adequate pen sizes, sufficient roofs, and knee impact, and knee wetness tests. Moreover, 65% of calves had relatively high leg hygiene levels. However, there was room for improvement, particularly in overall pen hygiene where 69% of the pens were categorized as dirty. Rearing calves in pens with sawdust and wood shavings were negatively associated with the calf leg hygiene, providing a reminder that the addition of clean, dry bedding should be accompanied with removal of the previous wet bedding. The cleanliness of the calves was also associated with the type of floor in the pens, being worse in concrete or wooden floors, likely because they cannot absorb moisture. If possible, calves should be reared on surfaces that allow moisture to drain away, such as sand or sandy dirt, which makes it easier to keep calves dry with less labor . If calves are reared on wood or concrete floors, they require manure removal and dry bedding addition/management at least daily, if not twice daily. These recommendations would ensure that calves would be kept in pens with clean, soft, and dry bedding to optimize their performance. Farmers should be trained on the importance of good housing management practices of the calves to enhance their growth and welfare. Further, research should be carried out to determine the solid feed intake, growth rate and diseases of dairy calves in Kenya.The body condition score could be a good indicator of good welfare of the calves since it was associated with leg hygiene and is an indicator of nutritional management. Further, research should be carried out to determine relationships between calf welfare parameters, body condition score, feed intake, growth rate, and diseases of dairy calves in Kenya.The small sample size for calf-related factors (52) and pen-related factors (39) limited the number of significant relationships and associations observed. The small number of farms (38) also may have limited the variation in calf cleanliness. Furthermore, the cross-sectional nature of this study provides us with data on housing, calf comfort, and hygiene, however without information on previous housing, welfare, and hygiene levels. Other calf welfare traits, such as the five domains of welfare, and detailed calf management practices (including feeding) would also be useful parameters to measure. A larger cohort study of more calves on more farms measuring the 5 domains of welfare would add perspective to the observations in our study.EKK, JVL, GKG, and SLM were involved in the study design, data analysis, and writing of the manuscript. All authors read and approved the final manuscript.


Based on quantitative immunoblot analyses, in both groups a reduction of two key regulators of mitochondrial biogenesis/remodeling and activity, namely PGC-1α and Sirt3, was revealed during bed-rest, with a subsequent up-regulation after rehabilitation, indicating an involvement of PGC-1α-Sirt3 axis in response to the treatments. A difference was observed comparing the young and elderly subjects as, for both proteins, the abundance in the elderly was more affected by immobility and less responsive to exercise. The expression levels of TOM20 and Citrate Synthase, assayed as markers of outer mitochondrial membrane and mitochondrial mass, showed a noticeable sensitivity in the elderly group, where they were affected by bed-rest and rehabilitation recalling the pattern of PGC-1α. TOM20 and CS remained unchanged in young subjects. Single OXPHOS complexes showed peculiar patterns, which were in some cases dissimilar from PGC-1α, and suggest different influences on protein biogenesis and degradation. Overall, exercise was capable to counteract the effect of immobility, when present, except for complex V, which was markedly downregulated by bed-rest, but remained unaffected after rehabilitation, maybe as result of greater extent of degradation processes over biogenesis. Phosphorylation extent of AMPK, and its upstream activator LKB1, did not change after bed-rest and rehabilitation in either young or elderly subjects, suggesting that the activation of energy-sensing LKB1-AMPK signaling pathway was “missed” due to its transient nature, or was not triggered under our conditions. Our study demonstrates that, as far as the expression of various proteins related to mitochondrial biogenesis/remodeling, adaptations to bed-rest and rehabilitation in the two populations were different. The impact of bed-rest was greater in the elderly subjects, where the pattern (decrease after bed rest and recovery following rehabilitation) was accompanied by changes of mitochondrial mass. Modifications of protein abundance were matched with data obtained from gene expression analyses of four public human datasets focusing on related genes.The aim of the study was to evaluate the expression levels of proteins related to mitochondrial biogenesis regulation and bioenergetics in  Skeletal muscle is a very plastic tissue that responds and adapts quickly to inactivity or exercise. Around the fourth decade of life, skeletal muscle mass and functional performance, including oxidative metabolism, inevitably decline . Such dein vivo following 10 days  was greater in E compared to Y, as well as the rehabilitation was slower and/or less complete in E.We discussed in detail in a recent publication  previousThere is still debate about which mechanisms are involved in the loss of muscle mass and function after inactivity. During the last few years interest has arisen around the role played by skeletal muscle mitochondrial function and biogenesis following inactivity and aging , as wellThe aim of the present study was to evaluate the changes occurred as response to inactivity and rehabilitation by E people, compared to Y, in the expression levels of the proteins above mentioned, playing a key role for mitochondrial biogenesis and function. In no previous studies, the expression levels of such proteins were determined in Y and E subjects undergoing the same duration of inactivity in bed followed by the same rehabilitation protocol, as in the present study. We hypothesized a pattern of changes in the protein expression similar to that described for systemic variables directly related to mitochondrial function (muscle mass and peak aerobic power) in the same subjects exposed to the same environmental stimuli, i.e., a more pronounced decrease during bed-rest in E vs. Y and a slower/incomplete rehabilitation.in silico analyses of public human gene expression datasets, focusing in particular on mitochondria-related genes involved in skeletal muscle responses to immobility and rehabilitation in young adult individuals, or demonstrated to change in association with aging. Combining data from bioinformatics analyses of gene expression with those of protein abundance from our bed-rest study encouraged us to hypothesize possible molecular mechanisms implicated in the effects observed in Y and E subjects.Furthermore, with the aim to validate our protein expression data, we performed some Twenty-three healthy men, of which 7 young  and 16 elderly subjects  were recruited for the study. All participants underwent medical examination and routine blood and urine analysis. Basic anthropometric parameters of the two groups and exclusion criteria are reported previously in The study was conducted in concomitance with the study of vastus lateralis muscle of each subject: one before starting bed-rest for BDC, one after the bed-rest period (BR14), the last after the rehabilitation protocol (R+14) and specifically 4–5 days after the final training session during the period where the subjects completed the in vivo performance tests.Three different biopsies were taken from vastus lateralis muscle. Biopsy was done after anesthesia of the skin, subcutaneous fat tissue, and muscle fascia with 2 ml of lidocaine (2%). A small incision was then made to penetrate skin and fascia, and the tissue sample was harvested with a purpose-built rongeur . The samples put in cryopreservation solution were immediately frozen in liquid nitrogen, and stored at -80°C until the analyses , in a suspension 1:4 w/v with PBS containing 0.32 M sucrose, P8340 – Sigma protease inhibitors (1:50 v/v) and 10 mM NaF + 1 mM Nahibitors . Samplesry assay  using BS® CRL-1446TM) were obtained by resuspension of cellular pellet (106 cells/ml) in RIPA buffer , containing protease (P8340 - Sigma) and phosphatase (10 mM NaF and 1 mM Na3VO4) inhibitors. After 30 min of incubation at 4°C, the samples were centrifuged at 14,000 ×g for 15 min at 4°C. Extracts were used as internal standards (IS) for quantification of immunoblot results.Extracts of H9c2 cell line , GAPDH 1:10000 , CS 1:10000 , PGC-1α 1:5000 , Sirt3 1:1000 , LKB1 and p-LKB1Ser482 1:1000  and TOM20 1:7000 . We also probed the membranes with AbCam Ab Cocktail (ab 110413) vs. OXPHOS complexes (1:5000) for complex I , complex II (succinate dehydrogenase [ubiquinone] iron-sulfur subunit B – SDHB), complex III (Cytochrome b-c1 complex subunit 2 – UQCRC2), complex V (CV subunit α – ATP5A1). For complex IV a single antibody against cytochrome c oxidase subunit 4 – COXIV  was used due to its greater efficiency. Thereafter, the membranes were incubated in the presence of the proper secondary antibody  conjugated with horseradish peroxidase (HRP).Separation of sample’s different proteins was obtained by electrophoresis in denaturing conditions (SDS-PAGE), following Laemmli’s method  with TriFor quantification purposes, each gel was loaded with 11 samples along with molecular weight markers  and IS (prepared as described above) in order to normalize the results from the single gels with those of replicates or different samples. 20 μg of proteins were loaded for each sample and 40 μg of IS. Samples from both Y and E subjects were assayed together matching the conditions .The protein bands were visualized by an enhanced chemiluminescence method using ChemiDoc (Bio-Rad Laboratories) and quantified with ImageQuant TL program . Quantification was made based on “Adjusted Volume Intensity,” i.e., the volume given by the sum of the intensities of the pixels inside the boundary volume corrected for the background. The intensity of each band was normalized on total bands revealed by Coomassie-staining of PVDF membrane, considered as appropriate loading and transferring control . Each saIn separate experiments, the single subjects were also analyzed individually by loading each gel with samples of the three investigated conditions . Gels were loaded with scalar amounts  of Y or E samples, to verify the linearity of the band intensity vs. the loaded protein amount. As an example, see representative immunoblot images denoted as (4) of t-test. Comparisons among the different conditions within each group were achieved using paired t-test. Bonferroni correction for multiple comparisons was applied.Continuous variables were summarized as mean ± standard deviation. Data were tested for normal distribution using Shapiro–Wilk test. Equality of variance was assessed using Levene test. The significance of differences of expression levels of analyzed proteins between groups (Y vs. E) throughout different conditions (BDC or BR 14 or R+14) was explored using the Linear Mixed Models for Longitudinal Data. Comparisons between groups for each condition were performed using 1  of a relationship between two variables. Genes with a significant negative strong correlation were selected, correlation coefficient < -0.6, Benjamini–Hochberg corrected p < 0.05 . Rule ofAll participants were able to comply with the study protocol. No dropouts and no medical complications occurred see .Data about the expression of PGC-1α, a master regulator of mitochondrial biogenesis and structural/functional integrity, are given in The expression levels of Sirt3, the most characterized sirtuin in mitochondria, declined during bed-rest following a pattern similar to that of PGC-1α , in accoThe effects of bed-rest and rehabilitation on OXPHOS complexes protein expression are shown in In order to estimate mitochondrial mass we determined the expression levels of TOM20, a component of the translocase of the outer mitochondrial membrane, and CS, an enzyme of the mitochondrial matrix. The patterns of TOM20  and CS  protein The expression levels of the key glycolytic enzyme GAPDH are shown in Thr172/AMPK ratio. Intriguingly, no significant changes were observed in the ratio  was significantly up- and down-regulated by immobility and rehabilitation, respectively, although just below the threshold chosen to define the top DEGs  undergoing disuse atrophy due to shorter (5 days) immobilization attained using a short leg cast with the ankle in a neutral position.We further analyzed the GEO dataset GSE8872 , includip < 0.05). Notably, among the few genes resulted to be significantly upregulated there are UCP3 , showing a ctrl/immobility FC = -1.51, p = 0.0055, as well as MT1X and MT2A, codifying for metallothioneins 1X and 2A .From DEG analysis, performed with the aim to see if there were changes in mitochondria-related genes’ expression as an early response to muscle inactivity, resulted some interesting data, which we considered relevant for our bed-rest study , a gene encoding for the channel-forming subunit of the translocase of the outer mitochondrial membrane, which might be indicative of reduction of mitochondrial mass/biogenesis in elderly people. Conversely, negligible changes were observed for all OXPHOS complexes, while for PPARGC1A the decline was just below the threshold selected . Finally, though of different extent, the downregulation of gene expression for TMEM70  and COA7 , is also interesting with respect to our bed-rest study, as such genes are required for assembly of mitochondrial ATP synthase complex V and respiratory chain complex I and complex IV, respectively.From DGE analysis of GSE9103 , and the same trend was observed in the young controls  (2 uptake (V’O2peak), a variable estimating maximal aerobic power, which should be related to mitochondrial function, was at baseline about 30% lower in E vs. Y (2 delivery) are more relevant in this respect  and elderly (E) subjects undergoing 14 days of profound inactivity (bed-rest), followed by 14 days of rehabilitation by a multimodal exercise program with an aerobic phase consisting in high-intensity intervals training. More specifically, we intended to compare the changes of the expression levels of key proteins related to the regulation of mitochondrial energy metabolism with those of “systemic” variables of functional evaluation, determined in the same subjects and recently published . The stu= 0.052) . We expe= 0.052)  was that= 0.052) . What ob E vs. Y . In othe respect . In the +-dependent protein deacetylase localized solely inside mitochondria  cochondria . In addichondria . In linechondria .The changes in the expression of such proteins observed in the present study were paralleled by systemic changes , with thTaken as a whole, our data document that the various OXPHOS complexes show diverse patterns of expression following inactivity and rehabilitation, each of them very similar in both E and Y. The patterns of respiratory chain complexes CII, CIII, and CIV, similar to that described for PGC-1α, are in line with studies using a protocol of 2 weeks of one-leg immobilization . The disAs for the peculiar behavior exhibited by CV in the experimental conditions of the present study , it should be considered that CV is recognized to be finely regulated at post-transcriptional level and to be expressed in large excess with respect to the working molecules. In this context, CV is reported to be a main target of Sirt3, undergoing a deacetylation-mediated activation in several models , and speThe patterns of the expression levels of the mitochondrial matrix protein CS, and of the outer mitochondrial membrane protein TOM20, both usually recognized as reliable mitochondrial mass markers, reveal a marked difference between E and Y subjects, as in these latter no changes were observed. Based on this behavior apparently conflicting with the pattern of PGC-1α, we may hypothesize specific effects on protein turnover as occurring in Y, rather than modulation of mitochondrial biogenesis. Regardless of the mechanism involved in the effects observed, the data of CS and TOM20 expression levels, taken as a whole, are in line with a less pronounced susceptibility to immobility of Y, with respect to E.The glycolytic marker GAPDH increased during bed-rest and decreased after rehabilitation in both groups, with a more pronounced effect in Y, suggesting an up-regulation of glycolytic metabolism during bed-rest, possibly as a compensatory response to the mitochondrial impairment, and a subsequent return to a more oxidative metabolism following rehabilitation. These results are part of the still open debate on glycolytic and oxidative metabolism in muscle atrophy and inactivity. In fact, there is not agreement on this topic in literature, likely due to dissimilar protocols applied by diverse authors. In accordance with our data are various bed-rest studies documenting an increased reliance on glycolysis . Convers2+/calmodulin-dependent protein kinase kinases (CaMKKs), also key activators of AMPK, were documented to be not considerably expressed in skeletal muscle  was likely present. This is not difficult to conceive. Indeed, skeletal muscle energy turnover in resting conditions is very low, and the energy charge only rarely challenged . In addiOn the other hand, the lack of activation of LKB1-AMPK axis by the rehabilitation intervention could be considered, at a first sight, rather unexpected. According to 2+-dependent signaling and was due to diminished intracellular Ca2+ levels, which might be counteracted by exercise  not linked to LKB1-AMPK axis, among the multiple signaling pathways appearing to converge on regulation of PGC-1α . One migexercise . If this reports .Several studies have tried to comprehend through gene expression analyses the molecular mechanisms involved in skeletal muscle responses to immobility and rehabilitation in humans, as well as associated with aging.Overall, data emerged from our analysis of GSE24215 gene expression dataset, focused on mitochondria- and OXPHOS-related genes  in young adult populations, are in accordance with the results of protein abundance obtained for the young group of our bed-rest study. This support two main messages: (i) decline of the steady-state levels of mitochondria-related proteins in atrophic muscle and recovery after rehabilitation for most of them, (ii) mitochondrial remodeling rather than biogenesis at the basis of mitochondria modulation. Nevertheless, with regard to some discrepancies observed, it should be noted that difference between mRNA and protein stability might be diverse under different conditions. Moreover, the data of gene expression are not always related to the same protein subunits which were analyzed by immunoblot for the single OXPHOS complexes. In the case of complex I, intriguingly, despite NDUFB3 and NDUFB10 gene expression was downregulated by immobility, the absence of changes for NDUFB8, a nuclear DNA-encoded subunit integral to the assembly of complex I, is in accordance with our immunoblot data.Interestingly, from DEG analysis of GSE24215 dataset we also observed an up- and down-regulation by immobility and exercise training of gene expression for FBXO32 (Atrogin-1) and FBXO2, two essential components of ubiquitin-proteasome pathway. FBXO32 (Atrogin-1) is a specific constituent of muscle playing a critical role in mediating the loss of muscle protein . IntriguOn the other hand, one must consider also that data from diverse studies should be compared with caution, due to the multiple protocols of immobility and rehabilitative exercise training operated in different laboratories.medial gastrocnemius muscle and comparison between the two datasets was based on similarity to vastus lateralis as concerns fiber composition (about 50% fast twitch and 50% slow twitch fibers). The combined data suggest that the immobility effects on mitochondria-related genes appeared to augment with the timespan of the immobility and/or severity on the protocol (leg cast immobilization vs. bed-rest). This finding may be considered in line with our previous reports documenting an impaired mitochondrial respiration, evaluated ex vivo in vastus lateralis muscle, after prolonged bed-rest conditions (21 days)  for a number of genes (such as some muscle proteins as MYH3 and MYL12A), including, however, only few mitochondrial OXPHOS subunits. For some of such genes , a downregulation of the expression resulted also from GSE24215 analysis along with a number of connected genes. GSE8872 data are from 21 days) .Of note, very few genes from GSE8872 resulted to be significantly upregulated by immobility, among which there is UCP3. An upregulation of muscle UCP3 protein was demonstrated as well, but following prolonged muscle unloading . In accovastus lateralis muscle after 48 h of knee immobilization. The authors suggested that this may play a role in the initiation of the atrophy program, and inferred that the atrophy program in humans might be denoted by an early transcriptional response for metallothioneins, maybe as consequence of elevated levels of metals and ROS generated in immobilization. Searching in GSE24215 dataset, collecting data from prolonged immobility conditions similar to our bed-rest study, we observed no changes in metallothioneins gene expression in line with the hypothesis that upregulation should be transitory.From another point of view, the finding that MT1X, MT2A appeared also among the few genes, which resulted as significantly upregulated from our analysis of GSE8872 dataset is worthy of note, though not directly linked to mitochondria-related proteins investigated in our bed-rest study. Indeed, metallothioneins are a group of genes associated with muscle atrophy in humans  and theiUnfortunately, there was not enough tissue remaining from our bed-rest study with which to perform additional assays, but future directions should include measurements for the expression levels of UCP3 and metallothioneins proteins following prolonged immobility, to evaluate their possible involvement in muscle disuse atrophy.In summary, combining data from the analysis of gene expression of two different datasets with those of protein abundance from our bed-rest study support the idea that immobility and exercise can affect mitochondria-related protein expression levels by both gene expression regulation and protein degradative pathways.vastus lateralis relevant for our bed-rest study and obtained some evidence for gene expression downregulation. Specifically, to explain the decline observed for TOM40 and PGC-1α, although it was lower for this latter, it should be postulated that a reduced physical activity by the aged people examined had a crucial role. Indeed, in elderly sedentary subjects exercise was reported to restore PGC-1α protein levels to the ones of young people  we focused in particular on certain genes/proteins in g people . FurtherIn conclusion, data from our analyses of the effects of aging on expression of a number of mitochondria-related genes relevant in the context of the proteins analyzed in our bed-rest study prompt us to highlight that immobility should be more critical for mitochondrial efficiency and energy production in case of people more aged than our elderly subjects. Indeed, we might infer that the trend of the effects documented by our bed-rest study should go on with age.The limited number of subjects enrolled in our study may weaken its outcomes. Nevertheless, this is a consequence of logistical limitations, which are intrinsic to this type of studies. Specifically, this is a very complex study performed with two populations of subjects (Y and E) and three muscle biopsies per subject, one of them obtained after 14 days of bed-rest and another one after 14 days of supervised exercise program. From a statistical standpoint, limitations are mainly related to the uneven sample size of the groups (7 Y and 16 E subjects) and particularly to the more restricted size of Y group that impact on statistical power to detect differences at some points.In addition, only limited amounts of tissue specimens from the muscle biopsies were available for our experiments, due to the numerous participants to the bed-rest campaign where our study was comprised. This reduced the number of proteins we decided to assay by quantitative immunoblot analyses in order to achieve accurate quantifications. Most important in this respect, we did not determine the expression levels of key components of ubiquitin-proteasome pathway despite our hypothesis that a reduction of mitochondrial-related proteins in atrophic muscle might be ascribed, at least in part, to regulation of the expression of such pathway. Our hypothesis is validated based on our gene expression data mining from human public datasets, as well as on solid literature.vastus lateralis muscle, our study confirms a crucial role of mitochondrial biogenesis/remodeling in muscle plasticity following inactivity and exercise rehabilitation. The heterogeneous patterns of the expression levels observed for some proteins are indicative of different responses to the treatments by the respective degradative ways compared to the biogenesis regulatory pathways. Furthermore, our study provide evidence that responses to bed-rest causing atrophy, as well as adaptations to rehabilitation, in E and Y populations were of different extent and qualitative diverse. Namely, the impact of bed-rest of most proteins was greater, and the rehabilitation recovery was less complete in the elderly subjects, where the changes observed were associated with modifications of mitochondrial mass. Results on protein expression levels are reinforced by data obtained from in silico analyses of four public human gene expression datasets, focusing on mitochondria-related genes affected in skeletal muscle responses to disuse and rehabilitation of adult individuals, or declined in association with aging.In summary, based on the expression levels of key proteins related to mitochondrial biogenesis regulation and bioenergetics in Alibegovic, A, C., Sonne, M.‘P., Højbjerre, L., Bork-Jensen, J., Jacobsen, S., Nilsson, E., Frch, K., Hiscock, N., Mortensen, B., Friedrichsen, M., Stallknecht, B., Dela, F., Vaag, A. Sep 20, 2010. Insulin resistance induced by physical inactivity is associated with multiple transcriptional changes in skeletal muscle in young men. GEO – Gene Expression Omnibus, version May 15, 2017 [GSE24215].Asmann, Y. W., Nair, K. S., Lanza, I. R., Short, D. K., Short, K. R., Bigelow, M. L., Joyner, M. J. submission date sep 19, 2007. Skeletal Muscle Transcript Profiles in Trained or Sedentary Young and Old Subjects. GEO – Gene Expression Omnibus, version Oct 29, 2018, [GSE9103] last update date Dec 10 2018.Phillips, B. E., Timmons, J. A. Jun 12, 2013. Impact of resistance exercise on human skeletal muscle gene expression – ageing. GEO – Gene Expression Omnibus, version Oct 29, 2018 [GSE47881].Yiwen, C. Aug 24, 2007. Transcriptional pathways associated with skeletal muscle disuse atrophy in humans. GEO – Gene Expression Omnibus, version Jul 08, 2016 [GSE8872].This study was performed in accordance with the ethical standards of the 1964 Declaration of Helsinki and was approved by the National Ethical Committee of the Slovenian Ministry of Health on April 17, 2012 under the acronym: IR-aging 1200.BG and IM designed the research. DS, BŠ, RadP, and JR organized the bed-rest campaign. AB and BM performed the experiments, prepared figures, and references’ list. AB and MC analyzed the data. RafP performed the gene expression analyses. MI performed the statistical analysis. AB, MC, and IM interpreted the results of experiments. IM, MC, AB, and BG edited and revised the manuscript. All authors approved the final version of manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


AQP0–AQP12, have been identified in mammals, which regulate various important biological functions in kidney, brain, lung, digestive system, eye, and skin. Consequently, dysfunction of AQPs is involved in a wide variety of disorders. AQPs are also present in the heart, even with a specific distribution pattern in cardiomyocytes, but whether their presence is essential for proper (electro)physiological cardiac function has not intensively been studied. This review summarizes recent findings and highlights the involvement of AQPs in normal and pathological cardiac function. We conclude that AQPs are at least implicated in proper cardiac water homeostasis and energy balance as well as heart failure and arsenic cardiotoxicity. However, this review also demonstrates that many effects of cardiac AQPs, especially on excitation-contraction coupling processes, are virtually unexplored.Mammalian aquaporins (AQPs) are transmembrane channels expressed in a large variety of cells and tissues throughout the body. They are known as water channels, but they also facilitate the transport of small solutes, gasses, and monovalent cations. To date, 13 different AQPs, encoded by the genes  Aquaporins (AQPs) constitute a major and diverse transmembrane channel family found in most living organisms ,2,3. The2 and CO2. AQP channels are voltage-independent and do not show gating properties. To date, 13 AQPs are known in mammals , and they are divided into three subfamilies based on their pore selectivity. AQPs are relatively small membrane proteins that constitute a monomer containing six transmembrane spanning domains, intracellular C- and N-termini, and a central water pore ,8,9,10. AQP knock-out transgenic mice. We conclude that AQPs are not only important for cardiac water homeostasis, but may also affect cardiac excitation-contraction coupling—either directly or indirectly via dysfunction of other organs—due to their permeability to glycerol, arsenite, and other small, neutral solutes as well as to interactions with various ion channel proteins and connexins. However, despite the great progress in the determination of the characteristics and functions of AQPs in the heart, various effects of cardiac AQPs seem still unknown.The mammalian AQPs are present in many cell types and organs, for example in kidney, brain, lung, digestive system, eye, and skin . It is gAQP1 knock-out resulted in increased AQPs 4, 7, and 11 expression in male, but not female, hearts. More details about the dependence of findings on AQP expression in the heart on the techniques and species of the underlying studies, can be found in the reviews of Egan et al. i) to extracellular ([X]o) ionic concentration i transient [2+]i cycling in cardiomyocytes  channels, resulting in the start of the [Ca2+]i transient. This so-called Ca2+-induced Ca2+ release is importantly modulated by the organization of T-tubuli, with co-localization of ICa,L and RyR2 channels, and the open probability of RyR2 channels. The [Ca2+]i transient amplitude depends on the Ca2+ content of the SR. The [Ca2+]i transient decline is importantly due to Ca2+ reuptake into the SR through activity of SR Ca2+ ATPase (SERCA) and extrusion of Ca2+ into the interstitium via the sarcolemmal Na+/Ca2+ exchanger (NCX). The SERCA and NCX activity compete during the [Ca2+]i transient. Thus, changes in SERCA function indirectly affect NCX activity and vice versa, which may result in altered SR Ca2+ content and [Ca2+]i transients. The diastolic Ca2+ concentration is regulated by the [Ca2+]i transient decline, and leak of RyR2 channels. Thus, changes in [Ca2+]i cycling may affect cardiac contractility i and increased risk for cardiac arrhythmias and failure [AQP4 KO compared to wild-type mice suggesting that the AQP4 KO-induced [Ca2+]i changes may also result in altered structural remodeling [2+]i levels, systolic [Ca2+]i levels are also increased in cells isolated from AQP4 KO mice i cycling, structural changes may also affect cardiac contractility i cycling, all important for proper cardiac excitation. It is, therefore, highly susceptible that AQPs are important determinants of both proper cardiac electrophysiology and arrhythmogenesis, but detailed studies are required to test this hypothesis. AQP dysfunction may reduce blood pressure and maximal cardiac output, which may be due to reduced cardiac contractility consequent to changes in [Ca2+]i cycling and cardiomyocyte and heart morphology. Further research is required to elucidate the exact mechanism of the reduced pump function of the heart. AQPs, especially AQP7, which is a member of the aquaglyceroporin subfamily and is abundantly expressed in the human heart, seem to have a role in cardiac energy balance. AQP7 acts as a glycerol facilitator and glycerol is a substrate for energy production. AQP mutations and variants may have a direct or indirect effect on cardiac function, while some pathophysiological conditions, e.g., arsenic toxicity and CHF, may be due to the dysfunction of AQPs. Furthermore, the use of specific medications, like lithium in case of bipolar disorders, may result in dysfunction of AQPs and improper cardiac functioning. Despite the impact of AQPs on proper cardiac water homeostasis, energy balance, heart failure, and arsenic cardiotoxicity, and their potential effects on cardiac excitation-contraction coupling, the cardiac phenotype of loss of function of specific AQPs is not always immediately clear. In this respect, it is worth noting that this is likely due to the presence of other water-specific AQPs, which may take over cardiac water transport function [The multiple essential roles that AQPs play in cardiac water homeostasis are evident from the changes in myocardial edema in pathophysiological conditions. The effects of AQPs on cardiac electrophysiology are largely unexplored. However, as discussed above, AQPs have tight protein–protein interactions with various membrane ion channels and connexins as well as effects on [Cafunction . In fact


Comparisons between groups were made separately for seven days of the week, five weekdays and two weekend days without and with an adjustment for 2000 kcal of energy. Relative differences  in mean diet nutritional values between groups were calculated, where RD (%) = (RS − nRS) × 100/nRS, and were verified with a two-tailed t-test. In the seven-day diet of the RS group, in comparison to the nRS group, a lower daily intake of sucrose (by 22%) and MUFA (by 8%) and a higher content of most nutrients (by 7–38%) was found. No significant differences were observed in energy intake between RS and nRS women over the course of seven days, including weekdays and the weekend. Daily intake for the weekend, in comparison to weekdays, in RC and nRC women was higher with respect to energy , sucrose , and most nutrients . However, after the adjustment for 2000 kcal of energy, the daily intake of most nutrients in the weekend was lower , than during weekdays. The intention to restrict sucrose has improved diet quality by decreasing sucrose intake and increasing the content of most nutrients, but had no effect on energy intake throughout the week. The quality of the women’s diet during the weekend was compromised, regardless of restricting or not restricting sugar. Encouragement to restrict sugar intake throughout the week and control the food intake during the weekend may be an effective strategy for young women to maintain a healthy diet.Restricted sugar intake is an important part of a healthy lifestyle and may contribute to the prevention of diet-related diseases. The aim of the study was to investigate whether declared sugar restriction is reflected in actual sucrose intake and diet quality of young non-dieting women, with differentiation between the weekend and weekdays. A convenient sample of 192 non-dieting women aged 20–30 years old was recruited. The sample was divided into two groups based on each woman’s declaration: “restricting sugar” (RS,  Sugar consumption is increasing globally, including in Poland ,2. Over Excessive sugar intake can directly and indirectly contribute to cardiovascular diseases and type 2 diabetes . The dirLastly, the reduction of sugar intake in a young women’s diet is of particular importance to the health of their children, starting from the early stages of life. The emerging evidence links maternal free-sugar intake in pregnancy with an increased risk of atopy and atopic asthma  and poorTherefore, health messages encouraging people to “cut down on sugar” has been actively promoted lately through media and public health interventions ,16. One Another question, which to our knowledge has not been answered to date, is whether the declared sugar restriction is effective throughout the week. It has been previously reported that during the weekend, energy and sugar intake is higher and diet quality lower, in a population of healthy adults ,19. HoweThis study aimed to investigate whether young, non-dieting women who declared restricting sugar intake had lower sucrose intake and a better quality diet than women who do not restrict sugar and whether this restriction is effective throughout the week.The study was approved by the Bioethics Committee of the Faculty of Medical Sciences, University of Warmia and Mazury in Olsztyn on 17 June 2010, Resolution No. 20/2010. A written agreement to participate in the study was required. All data was collected by well-trained researchers during one-to-one appointments.The data for this cross-sectional study were collected from December 2013 to March 2014. During the first contact (first visit), subjects were screened with regard to inclusion/exclusion criteria. Each subject who met the criteria and agreed to take part in the study by providing informed consent was invited to meet the researcher on two further, consecutive occasions. During the second visit, templates of food records were distributed to respondents and a detailed explanation from trained researchers regarding the accurate completion was provided. During the third visit, records in food diaries were verified and anthropometric measurements were taken see .The participants were a convenient sample of female volunteers recruited through advertising at the university, local work places, and public spaces. The recruitment was carried out in the rural and urban area of western-central Poland. The inclusion criteria were: age (18–30 years), female gender, and answering the question: “do you cut down on sugar in your habitual diet?” (yes or no). It was clearly explained to respondents that reducing sugar intake is thought to be a part of habitual diet only and not through dieting. The exclusion criteria were: chronic diseases affecting dietary habits or dieting due to obesity, diabetes, celiac disease, or other diseases. n = 117) and “not-restricting sugar” . During dietary data verification, 52 participants (RS/nRS 30/22) were excluded due to misreported dietary intakes, e.g., unrecorded meals or omitted days in food records or an incomplete record of food amount; significantly more respondents were excluded from RS than nRS group (p = 0.0461). To verify food records, no cut-offs with regard to energy intake were applied. A further 14 participants (11/3) were excluded due to a presence of chronic diseases such as diabetes, hyperthyroidism, stomach ulcer, celiac disease, bulimia, and endocrinological disorders. In total 66 participants (41/25) were excluded from the screened sample. The final study sample consisted of 192 non-dieting women (76/116) aged 20–30 years  to detect a 20% difference in sucrose intake between two groups, including recoding error or missing data of 20%. An adequacy of sample size was checked for the data under study, and the post-hoc statistical power was calculated. When compared, the mean difference in daily sucrose intake between women restricting and not restricting sugar intake (76/116), assuming 5% significance level, the statistical power was 73.5%. Thus, we have found that the sample size was sufficient.An estimated food record method was applied, covering seven consecutive days, five weekdays (Monday–Friday) and two weekend days (Saturday–Sunday) . Respondhttp://energia.waw.pl/) with an implemented food database. The food database was composed of 1178 products: 962 from the official database of foods commonly consumed in Poland [To resolve any doubts , all food records and completed questionnaires were verified by researchers during interviews with respondents (third visit) . The amon Poland  and 216 n Poland ) of ethnn Poland . Plate wSocioeconomic variables were collected using standard questions. A closed-question questionnaire was used. The questions included: place of residence , level of education , and current occupation . 2) was calculated [2), normal weight (18.5 to 24.9 kg/m2), overweight (25 to 29.9 kg/m2), and obesity (≥30 kg/m2) using WHO standards [The measurements of body weight (kg) and height (cm) were taken between 8 and 12 a.m. during one-to-one appointments by trained staff, according to the International Standards for Anthropometric Assessment guidelines by International Society for the Advancement of Kinanthropometry (ISAK) . Professlculated . The BMIt-test (continuous data) or χ2 test  [I cut down on sugar”), nRS—"not restricting sugar” (women who declared “I don’t cut down on sugar”), “2 weekend days”—mean daily intake calculated for two weekend days, “5 weekdays”—mean daily intake calculated for five weekdays. A p-value < 0.05 was considered significant. The statistical analysis was carried out using STATISTICA software .Continuous data are presented as means and 95% confidence intervals (95%CI), and categorical variables as sample percentages (%). The differences between groups were verified using a two-tailed al data) . VariablThe mean age of the study participants was 24.6 years old. The majority of women had a regular weight (71%), 22% were overweight or obese, and 7% were underweight. Most women lived in urbanized areas (92%), declared a secondary level of education (60%), and worked in the office environment (33%). No differences between RS and nRS groups were found regarding age, BMI, body fat, and socioeconomic variables .Mean values of the daily dietary intake are presented in Sucrose intake was significantly lower in the RS group  . The crude model revealed that diets of RS women over the course of seven days, five week days, and two weekend days (when compared to the nRS women) was significantly lower in MUFA  and higher in β-carotene , fiber , calcium , magnesium , vitamin B2 , phosphorus , vitamin C , folic acid , copper , B12 , zinc , iron , potassium , protein . After the adjustment for 2000 kcal of energy, the diet of RS women was still higher in most nutrients than nRS women, although fewer differences were statistically confirmed . Mean values of daily dietary intake are presented in the In the crude model, daily dietary intake of most nutrients was higher at the weekend than during the weekdays in both RS and nRS groups. At the weekend, in comparison to weekdays, higher daily intake of MUFA , PUFA (39 and 42%), fat (by 39 and 35%), SFA (by 38 and 30%), phosphorus (by 33 and 17%), vitamin B1 (by 32 and 31%), carbohydrates (by 32 and 30%), vitamin E (by 31 and 39%), calcium (by 26 and 32%), cholesterol (by 26 and 16%), zinc (by 25% in both groups), sodium (by 25 and 22%), niacin (by 23 and 27%), magnesium (by 23 and 24%), iron (by 23% in both groups), vitamin B2 (by 23 and 17%), folic acid (by 22 and 19%), vitamin B6 (by 21 and 25%), copper (by 20 and 24%), fibre (by 20 and 22%), potassium (by 17 and 24%) was revealed. Also, in RS women, daily intake of retinol (by 98%) and vitamin D (by 34%) was higher at the weekend than weekdays .After the adjustment for 2000 kcal, the daily intake of most nutrients was lower at the weekend than during the weekdays in both groups of women. In the RS group, the decrease ranged from 6 to 30% and regarded the following nutrients (in the descending order): β-carotene, potassium, vitamin C, copper, fiber, magnesium, iron, vitamin B1, vitamin B2, niacin, zinc, sodium, calcium, and protein. In the nRS group the decrease ranged from 3 to 27% and regarded (in the descending order): retinol, vitamin B12, vitamin D, phosphorus, vitamin C, vitamin B2, cholesterol, folic acid, fiber, sodium, iron, potassium, magnesium, copper, vitamin B6, zinc, niacin, protein, and carbohydrates .Our study revealed that young, non-dieting women who declared restricting sugar intake consumed less sucrose and more nutrients over the course of seven days than women who did not intend to self-regulate sugar intake. No differences were observed in energy intake between the groups. At the weekend, women from both groups consumed significantly more energy, sucrose, and other nutrients than during the weekdays. However, after the adjustment for 2000 kcal, the intake of most nutrient in both groups was lower than during weekdays, indicating that the weekend diet was abundant in calories and of low quality.Over the course of seven days, women who declared cutting down on sugar consumed less sucrose  than women who did not attempt to regulate sugar intake, although no difference in energy intake between the groups was observed. Interestingly, 1/3 of women who declared restricting sugar had sucrose intake above the 10% of daily energy, suggesting that the perception of their own dietary behavior was not coherent with actions in this subset. High sucrose intake in women who restricted sugar intake from our study may have been a result of either low levels of the adherence to the intention, or was caused by low awareness about “hidden” sugars in products not typically associated with high sugar content, such as low-fat dairy, salad dressings, sauces, instant soups, ready meals, etc. It has been reported, that intention (directly) and personally-endorse motives (indirectly) predict sugar intake . PerhapsNo differences in energy intake between women restricting and not restricting sugar observed in our study can be explained by the “seesaw” effect. The term was originally used by Drewnowski et al.  to descrOver the course of seven days, women who restricted sugar intake had a better diet quality than those with no restriction. Considering that the sample included only non-dieting participants, sugar restriction was most likely health-oriented. Our findings are consistent with previous reports. People with strong healthy eating motivation display healthier lifestyles than those with weak motivations . An inveThe diet quality of women from both groups was compromised during the weekend and contained significantly more energy . The over-indulgence during the weekend is a finding well-established in the literature. A study by An  found thIn our study, an increased intake of most nutrients was reported during the weekend in both groups. However, the adjustment for 2000 kcal of energy intake proved that it was most likely a result of overeating since the decrease in relative differences was observed in the intake of majority of nutrients . In the “restricting sugar” group, the greatest decrease observed regarding β-carotene intake. Considering that fruit and vegetables are primary dietary sources of this pro-vitamin, it can be assumed that foods from this group were less frequently consumed at the weekend. In the “not restricting sugar” group, after the adjustment for 2000 kcal, the greatest decrease was observed in terms of retinol, vitamin A, and vitamin B12 intake, which suggested a lower intake of animal-originated foods at the weekend. These foods could have been substituted with alcohol. Due to software limitations, the percentage of energy from alcohol intake was not possible to extract from our database. However, a parallel analysis of the food frequency consumption (data not shown) revealed that alcohol intake in our study sample corresponded with a frequency of “few times per month” to “once a week”. Hence, the increased intake of alcohol on one of the weekend days could not be overruled. Another explanation for the increased calorie load could be attributed to a higher intake of processed plant-foods  consumed while watching television or socializing at the weekend.Although the quality of diet was lower at the weekend in both groups of women, it has to be stressed that the “restricting sugar” group had still a better nutrient profile than the “not restricting sugar” group. After the adjustment for 2000 kcal, during the weekend, women who restricted sugar had higher intakes of fiber (by 30%), protein (by 9%), vitamins (by up to 52%), and minerals (by up to 27%) than the “not restricting sugar” group. This indicates that the weekend diet of women who declared cutting down on sugar was slightly more favorable, despite the overindulgence.Although the research has reached its aims, we are aware that it may have some limitations. First, as the focus of the study was on sucrose intake, other potential restrictions have not been investigated. It cannot be ruled out that other modifications were present in women’s diet, e.g., restricting salt or fat and/or cholesterol intake ,37. The Second, since data were collected only during winter months, our study has not considered the seasonality of food consumption. Although seasonal variations can be significant in some specific groups ,39, it hThird, we are aware of limitations related to food recording as a method chosen to assess dietary intake, in particular the risk of underreporting. In our study, the mean reported energy intake was 1601 kcal/day. The daily resting energy expenditure of women in our study sample calculated with the Mifflin–St Jeor equation  was 1381Fourth, although the minimum sample size criteria were met in this study, our investigations have only been on a small scale. Perhaps a larger sample size would reveal more significant differences between the groups, allowing stronger conclusions. Concerning might also be a significantly higher number of women who were excluded from the RS group, due to misreporting. It could be that women in the RS group were more prone to omit inputs in the food dairies more frequently than women from the nRS, due to concerns of how they will be perceived if they input foods that were incoherent with the sugar intake declaration. However, the exclusion of these women was necessary to minimize the risk of bias in this group. Finally, our results are limited to young women and may be not applicable to men.The intention to cut down on sugar has improved the quality of women’s diets by decreasing sucrose intake and increasing the nutrients content, but no impact on energy intake was shown. Irrespective of restricting or not restricting sugar intake, weekend diets were characterized by high energy , sucrose, and nutrient content. However, when adjusted for 2000 kcal, diet quality in both groups was lower than during weekdays. To improve overall diet quality, two recommendations can be made. First, to encourage young women to restrict sugar intake in their habitual diet, and second, to monitor diet quality during the weekend.


PET data were analysed with statistical parametric mapping. We found an electrode insertional effect in cortical areas, while DBS resulted in a more widespread metabolic pattern. The consequences of simultaneous electrode and DBS factors revealed a combination of both effects. Therefore, the insertion metabolic effects differed from the stimulation ones, which should be considered when assessing DBS protocols.Deep brain stimulation (DBS) is a neurosurgery technique widely used in movement disorders, although its mechanism of action remains unclear. In fact, apart from the stimulation itself, the mechanical insertion of the electrode may play a crucial role. Here we aimed to distinguish between the insertional and the DBS effects on brain glucose metabolism. To this end, electrodes were implanted targeting the medial prefrontal cortex in five adult male Wistar rats. Positron Emission Tomography (PET) studies were performed before surgery (D0) and seven (D7) and nine days (D9) after that. DBS was applied during the  Indeed, certain symptomatology improvement has been related to the mere insertion of the electrodes in the treatment of epilepsy fluoro-D-glucose  was intravenously injected and, after 45 min of uptake, animals were scanned for 40 min. Images were reconstructed using a 2D-OSEM algorithm, with a spatial resolution of 1.45 mm Full Width Half Maximum (FWHM), a voxel size of 0.3875 x 0.3875 x 0.775 mm3, and an energy window of 400-700 keV. Decay and dead-time corrections were applied.Animals were scanned using a small-animal PET/CT scanner , under anaesthesia with isoflurane . 2-Deoxy-2-[μm. Images were reconstructed using an FDK algorithm (isotropic voxel size of 0.124 mm) [CT studies were acquired with the same scanner, using the following parameters: 340 mA, 40 kV, 360 projections, 8 shots, and pixel size of 200 .124 mm) .The MRI study was acquired with a 7-Tesla Biospec 70/20 scanner . A T2-weighted spin-echo sequence was acquired with TE=33 ms, TR=3732 ms, and a slice thickness of 0.8 mm (34 slices). The matrix size was 256 × 256 pixels with a FOV of 3.5 × 3.5 cm2.PET images postprocessing and voxel value normalization were performed following the protocols previously described by our group , 15. Brihttp://www.fil.ion.ucl.ac.uk/spm/software/spm12/) by means of paired T-tests, setting a significance threshold of p<0.005 uncorrected (voxel-level significance), but cluster-based corrected in order to avoid type II errors [The statistical analysis consisted on a voxel-wise analysis of PET data using SPM12 , the stimulation , and the combination of both the insertion and the stimulation , on brain metabolism.In this sense, we assume that the metabolic differences we show in the study II are almost completely due to the acute effect of the high-frequency electrical stimulation. Although the microlesional effect related to the electrode insertion is highly variable between subjects , the stiThe presence of the electrodes  led to a reduced FDG uptake in parietal association (PtA), primary somatosensory (S1), and visual cortices .The electrical stimulation  led to a decreased FDG uptake in brainstem (Bstm), amygdaloid nuclei , and hypothalamus (HTh), together with an increased metabolism in caudate-putamen (CPu), piriform (Pir), S1, and auditory cortex (Au) .The combination of both effects  showed a decreased FDG uptake in Bstm, red nucleus (RMC), and periaqueductal grey matter (PAG) and higher metabolism in secondary somatosensory (S2), insular (I), and Pir cortices .First, we describe an insertional effect on brain glucose metabolism in sensory areas that are connected to mPFC . Second,The insertion effect could appear in response to the microlesion induced by the electrode in the mPFC  and the Therefore, the wide variability showed in relation to the clinical and physiological consequences of the electrode insertion could be highly dependent on several factors (e.g., the health state of the subjects, the DBS target selected, the number of microelectrode recording trajectories performed during the surgery , the timOur work is subjected to several limitations. On the one hand, the small sample size selected, which responds to the aim of providing preliminary evidences that we considered to be important for understanding the DBS mechanism of action through a metabolic perspective. Therefore, we applied strict statistical thresholds in order to show more accurate results, which lead us to discard potentially important effects that do not reach statistical significance. In fact, the electrode insertion has been proved to produce lower metabolic changes than subthalamotomy or stimulation , 20, whiOn the other hand, we have only included healthy animals in our study, which does not allow us to extrapolate the observed changes to a disease model due to the differences related to a diseased brain. Nevertheless, we aimed to describe the metabolic consequences of electrode insertion and electrical stimulation, excluding any other intervening factors, in order to isolate those effects and improve their metabolic characterization.In conclusion, our study highlights the importance of the design of appropriate protocols, particularly in neuroimaging, emphasizing the value of scanning the same subject with/without DBS, for a full understanding of the DBS mechanism of action and its clinical consequences. This will allow taking advantage of the electrodes and the stimulation consequences in order to optimize the DBS protocols for achieving the desired therapeutic effects.