Salivary iodine concentrations (SIC) and urinary iodine concentrations are correlated. This study aimed to verify the use of SIC as a biomarker for estimating iodine intake in pregnant and lactating women and to diagnose abnormal thyroid function. A cross-sectional study was conducted in northern Xinjiang, China. Participants provided venous blood, random urine, saliva and milk samples. A total of 607 pregnant and 171 lactating women volunteered to participate in the study. The average daily iodine intake was calculated for each participant. Pregnant women were divided according to trimester. The median daily iodine intake was 436·41 μg/d in the first trimester, 425·83 μg/d in the second trimester and 430·56 μg/d in the third trimester. The average daily iodine intake in lactating women was 416·16 μg/d. Different indicators were used to diagnose excessive iodine intake (> 500 μg/d). Among pregnant women, SIC had an AUC of 0·62 (P < 0·01), sensitivity of 51·75 % and specificity of 65 %. Among lactating women, SIC had an AUC of 0·63 (P = 0·03), sensitivity of 43·52 % and specificity of 85 %. SIC was an effective biomarker for diagnosing abnormal thyroid function (P = 0·03). In conclusion, this study demonstrated that SIC is a reliable biomarker for evaluating both iodine nutrition status and abnormal thyroid function in pregnant and lactating women.

The effect of single dietary fibre (DF) on lowering uric acid (UA) level has been reported in the literature. However, the potential protective mechanism of DF against potassium oxybate-induced hyperuricaemia (HUA), as modelled by prophylactic administration, remains unclear. The data demonstrate that DF significantly decreased serum and cerebral tissue UA concentrations, inhibited xanthine oxidase expression and activity in the liver and reduced levels of creatinine and urea nitrogen in the serum. Additionally, it mitigated the deposition of amyloid-β in cerebral tissue. Correlation analysis showed that DF modulated the Toll-like receptor 4/NF-κB signalling pathway, attenuating oxidative stress and inflammatory responses in HUA mice. Additionally, DF helps to maintain the composition of the gut microbiota, reducing harmful Desulfovibrio and enriching beneficial Akkermansia and Ruminococcus populations. The results of the faecal metabolomics analysis indicate that DF facilitates the regulation of metabolic pathways involved in oxidative stress and inflammation. These pathways include pyrimidine metabolism, tryptophan metabolism, nucleotide metabolism and vitamin B6 metabolism. Additionally, the study found that DF has a preventive effect on anxiety-like behaviour induced by HUA. In summary, DF shows promise in mitigating HUA and cognitive deficits, primarily by modulating gut microbiota and metabolites.

Vitamin D deficiency is prevalent in patients with chronic spinal cord injury (SCI) and has been implicated as an aetiologic factor of osteoporosis and various skeletal and extra-skeletal issues in SCI patients. Few data were available regarding vitamin D status in patients with acute SCI or immediately assessed at hospital admission. This retrospective cross-sectional study evaluated vitamin D status in SCI patients at admission to a UK SCI centre in January–December 2017. A total of 196 eligible patients with serum 25(OH)D concentration records at admission were recruited. The results found that 24 % were vitamin D deficient (serum 25(OH)D < 25 nmol/l), 57 % of the patients had serum 25(OH)D < 50 nmol/l. The male patients, patients admitted in the winter–spring time (December–May), and patients with serum sodium < 135 mmol/l or with non-traumatic causes had a significant higher prevalence of vitamin D deficiency than their counterparts (28 % males v. 11⋅8 % females, P = 0⋅02; 30⋅2 % in winter–spring v. 12⋅9 % in summer–autumn, P = 0⋅007; 32⋅1 % non-traumatic v. 17⋅6 % traumatic SCI, P = 0⋅03; 38⋅9 % low serum sodium v. 18⋅8 % normal serum sodium, P = 0⋅010). There was a significant inverse association of serum 25(OH)D concentration with body mass index (BMI) (r = −0⋅311, P = 0⋅002), serum total cholesterol (r = −0⋅168, P = 0⋅04) and creatinine concentrations (r = −0⋅162, P = 0⋅02) that were also significant predictors of serum 25(OH)D concentration. Strategies for systematic screening and efficacy of vitamin D supplementation in SCI patients need to be implemented and further investigated to prevent the vitamin D deficiency-related chronic complications.

In medical research, continuous variables are often categorised into two or more groups before being included in the analysis; this practice often comes with a cost, such as loss of power in analysis, less reliable estimates, and can often leave residual confounding in the results. In this research report, we show this by way of estimates from a regression analysis looking at the association between acute kidney injury and post-operative mortality in a sample of 194 neonates who underwent the Norwood operation. Two models were developed, one using a continuous measure of renal function as the main explanatory variable and second using a categorised version of the same variable. A continuous measure of renal function is more likely to yield reliable estimates and also maintains more statistical power in the analysis to detect a relation between the exposure and outcome. It also reveals the true biological relationship between the exposure and outcome. Categorising a continuous variable may not only miss an important message, it can also get it wrong. Additionally, given a non-linear relationship is commonly encountered between the exposure and outcome variable, investigators are advised to retain a predictor with a linear term only when supported by data. All of this is particularly important in small data sets which account for the majority of clinical research studies.

The current trial investigates the effect of renal diet therapy and nutritional education on the estimated glomerular filtration rate (eGFR), blood pressure (BP) and depression among patients with chronic kidney disease (CKD). A total of 120 CKD patients (stages 3–4) (15<eGFR < 60) were randomised into an intensive nutrition intervention group (individualised renal diet therapy plus nutrition counselling: 0·75 g protein/kg/d and 30–35 kcal/kg/d with Na restriction) and a control group (routine and standard care) for 24 weeks. The primary outcome was the change in the eGFR. Secondary outcomes included changes in anthropometric measures, biochemistry (serum creatinine (Cr), uric acid, albumin, electrolytes, Ca, vitamin D, ferritin, blood urea nitrogen (BUN), and Hb), BP, nutritional status, depression and quality of life. The eGFR increased significantly in the intervention group compared with the control group (P < 0·001). Moreover, serum levels of Cr and the systolic and diastolic BP decreased significantly in the intervention group relative to the control group (P < 0·001, P < 0·001 and P = 0·020, respectively). The nutrition intervention also hindered the increase in the BUN level and the depression score (P = 0·045 and P = 0·028, respectively). Furthermore, the reduction in protein and Na intake was greater in the intervention group (P = 0·003 and P < 0·001, respectively). Nutritional treatment along with supportive education and counselling contributed to improvements in renal function, BP control and adherence to protein intake recommendations. A significant difference in the mean eGFR between the groups was also confirmed at the end of the study using ANCOVA (β = -5·06; 95 % CI (−8·203, −2·999)).

The fish paste product, fish balls ‘tsumire’, is a traditional type of Japanese food made from minced fish as well as imitation crab, kamaboko and hanpen. Although tsumire is known as a high-protein and low-fat food, there is a lack of scientific evidence on its health benefits. Hence, we aimed to investigate the effects of tsumire intake on organ weight and biomarker levels in Sprague–Dawley rats for 84 d as a preliminary study. Six-week-old male Sprague–Dawley rats were divided into two groups: group I, fed normal diets, and group II, fed normal diets with 5 % dried tsumire. Throughout the administration period, we monitored their body weight and food intake; at the end of this period, we measured their organ weight and analysed their blood biochemistry. No significant differences were observed with respect to body weight, food intake, organ weight and many biochemical parameters between the two groups. It was found that inorganic phosphorus and glucose levels were higher in group II rats than in group I rats. On the other hand, sodium, calcium, amylase and cholinesterase levels were significantly lower in group II than in group I. Interestingly, we found that the levels of aspartate aminotransferase, alanine transaminase, lactate dehydrogenase and leucine aminopeptidase in group II were significantly lower than in group I, and that other liver function parameters of group II tended to be lower than in group I. In conclusion, we consider that the Japanese traditional food, ‘tsumire,’ may be effective as a functional food for human health management worldwide.

Chronic tubulointerstitial nephropathy (CTIN) is one of the most common kidney diseases. However, treatment for CTIN has multiple limits. Adjuvant therapy through nutritional regulation has become a hot research topic at present. Icariin (ICA), an extraction of Chinese herbal medicine epimedium, has many pharmacological functions including anti-inflammation and tonifying kidney. Selenomethionine (SeMet) possesses the effects of antioxidant and lightening nephrotoxicity. However, little is known about the combined nephroprotection of them. This study was investigated to evaluate the joint effects of ICA and SeMet on CTIN and explore the mechanism. Based on a novel CTIN model developed in our previous study, mice were randomly divided into five groups (a: control; b: model; c: model + ICA; d: model + SeMet; e: model + ICA + SeMet). Renal tubule epithelial cells were treated with cyclosporine A and ochratoxin A without/with ICA or/and SeMet. The results showed that ICA or/and SeMet ameliorated CTIN by inhibiting the uptrends of blood urine nitrogen, serum creatinine, urine protein, urine gravity, histopathological damage degree and collagen I deposition. ICA or/and SeMet also increased cell proliferation and decreased apoptosis and the expression of transforming growth factor-beta 1 and α-smooth muscle actin. Emphatically, ICA and SeMet joint had better nephroprotection than alone in most indexes including fibrosis. Furthermore, ICA and SeMet joint decreased the activation of toll-like receptor 4 (TLR4)/NFκB pathway induced by CTIN. TLR4 overexpression counteracted the joint protection of ICA and SeMet. Therefore, ICA and SeMet in combination could protect against CTIN through blocking TLR4/NFκB pathway. The study will provide novel insights to explore an adjuvant therapeutic orientation.

This study applied causal criteria in directed acyclic graphs for handling covariates in associations for prognosis of severe coronavirus disease 2019 (COVID-19) cases. To identify non-specific blood tests and risk factors as predictors of hospitalisation due to COVID-19, one has to exclude noisy predictors by comparing the concordance statistics (area under the curve − AUC) for positive and negative cases of severe acute respiratory syndrome-coronavirus-2 (SARS-CoV-2). Predictors with significant AUC at negative stratum should be either controlled for their confounders or eliminated (when confounders are unavailable). Models were classified according to the difference of AUC between strata. The framework was applied to an open database with 5644 patients from Hospital Israelita Albert Einstein in Brazil with SARS-CoV-2 reverse transcription – polymerase chain reaction (RT-PCR) exam. C-reactive protein (CRP) was a noisy predictor: hospitalisation could have happened due to causes other than COVID-19 even when SARS-CoV-2 RT-PCR is positive and CRP is reactive, as most cases are asymptomatic to mild. Candidates of characteristic response from moderate-to-severe inflammation of COVID-19 were: combinations of eosinophils, monocytes and neutrophils, with age as risk factor; and creatinine, as risk factor, sharpens the odds ratio of the model with monocytes, neutrophils and age.

A well-balanced amino acid profile in the feedstuffs for livestock is essential to support adequate growth. This is well studied for monogastric species but still not well understood for ruminants and especially for the most unconventional species, like cervids. This study investigated the influence of ruminally protected lysine (Lys) and methionine (Met) supplementation during the fattening period, as well as two slaughter seasons (late autumn (LA) v. late winter (LW)), on the growth, carcass traits, body condition and blood plasma metabolites of fallow deer (Dama dama). Forty-five yearlings of fallow deer bucks were allocated into three groups, balanced by weight (40.2 ± 2.7 kg). The deer were pasture-fed and supplemented with barley, free-choice mineral premix, silage during the winter period, and varying levels of ruminally protected Lys and Met: no amino acids (Control), 9 g/day of Lys, and 9 g/day of Lys plus 3 g/day of Met (Lys+Met). Animals were slaughtered in two separate seasons, LA (six animals per group), and LW (nine animals per group). Animals culled in LA had higher average daily weight gain than LW (P = 0.002), due to the reduced growth during winter typical for seasonal cervids in temperate zone, mediated by the photoperiod. Dressing percentage was significantly higher in LW and in the Lys+Met group (P = 0.002). Body condition score (P = 0.024), kidney fat index (KFI) (P = 0.005), and internal fat (P < 0.001) increased significantly with Lys+Met supplementation. During LW, KFI (P = 0.004) and kidney fat (P = 0.001) were also significantly higher than in LA. Blood creatinine concentration increased significantly for deer receiving Lys (P = 0.002) and Lys+Met (P < 0.001). Also, triglycerides level increased in Lys group (P < 0.001). These findings highlight the effects of Lys and Met supplementation on the growth and internal fat storage for winter survival, suggesting a different use of the supplemented resource according to the season. Also, the observed effects on protein and fat metabolism of fallow deer may influence the production of farmed animals, and offer interesting insights about the physiology of the species.

The present study investigated the effects of glutamine (GLN) pretreatment on CD4+ T cell polarisation and remote kidney injury in mice with gut-derived polymicrobial sepsis. Mice were randomly assigned to three groups: normal control fed with American Institute of Nutrition (AIN)-93G diet and two sepsis groups provided with either AIN-93G-based diet or identical components, except part of casein was replaced by GLN. Mice were given their respective diets for 2 weeks. Then, mice in the sepsis groups were performed with caecal ligation and puncture and were killed 72 h after the surgery. Blood, spleens and kidneys were collected for further examination. The results showed that sepsis resulted in decreased circulating and splenic total T lymphocyte and CD4+ T cell percentages, whereas IL-4-, and forkhead box p3 (Foxp3)-expressing CD4+ T cells percentages were up-regulated. Compared with the sepsis control group, pretreatment with GLN maintained blood T and CD4+ T cells and reduced percentages of IL-4- and Foxp3-expressing CD4+ T cells. Also, a more pronounced activation and increased anti-apoptotic Bcl-2 gene expression of splenic CD4+ T cells were observed. Concomitant with the decreased plasma IL-6, keratinocyte-derived chemokine (KC) levels, the gene expression of KC, macrophage inflammatory protein-2 and renal injury biomarker kidney injury molecule-1 (Kim-1) were down-regulated when GLN was administered. These findings suggest that antecedent of GLN administration elicit a more balanced blood T helper cell polarisation, sustained T cell populations, prevented splenic CD4+ T cell apoptosis and attenuated kidney injury at late phase of polymicrobial sepsis. GLN may have benefits in subjects at risk of abdominal infection.

Serum vitamin D (25-hydroxyvitamin D (25OHD)) may influence serum parathyroid hormone (PTH) levels and bone mineral density (BMD). In the present study, we assessed serum 25OHD concentration and its association with PTH and BMD in urban males from Guiyang (N26.57°), the capital city of Guizhou province, Southwest China. We recruited 634 males aged >20 years from the Guiyang Health Measures Survey, and stratified them into three groups according to age: young (20–39 years), middle aged (40–59 years) and older (60–79 years). We measured serum concentrations of 25OHD, PTH levels and BMD of the lumbar spine (L1–L4), femoral neck and total hip. In addition, we also explored the relationship between 25OHD and lifestyle, socio-economic characteristics and medical history by applying covariance analysis and locally weighted regression plots. The results showed that serum 25OHD was <50 nmol/l in 59·3 % of the subjects and >75 nmol/l in 12·6 % of the subjects. Higher level of serum PTH was detected in relation to lower concentrations of serum 25OHD up to 50 nmol/l. A negative correlation between serum 25OHD and PTH concentrations was observed (r −0·207, P=0·003). Mean concentration of serum PTH increased gradually and plateaued while concentrations of serum 25OHD decreased to 50 nmol/l. Gradual increase in serum PTH was observed as 25OHD concentration was <25 nmol/l (P=0·004). BMD values at all sites were greater in the higher serum 25OHD concentration group. This study shows that low concentrations of serum 25OHD were common in males, and bone health was likely to be improved when serum 25OHD values were between 30 and 50 nmol/l.

The mechanisms by which different genotypes of Giardia duodenalis result in different symptoms remain unresolved. In particular, we lack detailed knowledge on which transport mechanisms (transcellular or paracellular) are affected by different Giardia isolates. Using horse radish peroxidase (HRP) and creatinine as transcellular and paracellular probes, respectively, we developed a robust assay that can be used with an Ussing chamber to investigate epithelial transport, as well as short-circuit current as an indicator of net ion transport. We investigated 2 Giardia isolates, both Assemblage A, one a lab-adapted strain and the other a field isolate. Results indicate that products from sonicated Giardia trophozoites increase both transcellular and paracellular transport. A non-significant increase in transepithelial electrical resistance (TEER) and short-circuit current were also noted. The paracellular transport was increased significantly more in the field isolate than in the lab-adapted strain. Our results indicate that while both transcellular and paracellular transport mechanisms may be increased following exposure of cells to Giardia trophozoite sonicate, perhaps by inducing non-specific increases in cellular traffic, it is important that in vitro studies of Giardia pathophysiology are conducted with different Giardia isolates, not just lab-attenuated strains.

This study presents an NMR-based metabonomic approach to elucidate the overall endogenous biochemical effects of a wholegrain diet. Two diets with similar levels of dietary fibre and macronutrients, but with contrasting levels of wholegrain ingredients, were prepared from wholegrain rye (wholegrain diet (WGD)) and non-wholegrain wheat (non-wholegrain diet (NWD)) and fed to four pigs in a crossover design. Plasma samples were collected after 7 d on each diet, and 1H NMR spectra were acquired on these. Partial least squares regression discriminant analysis (PLSDA) on spectra obtained for plasma samples revealed that the spectral region at 3·25 parts per million dominates the differentiation between the two diets, as the WGD is associated with higher spectral intensity in this region. Spiking experiments and LC–MS analyses of the plasma verified that this spectral difference could be ascribed to a significantly higher content of betaine in WGD plasma samples compared with NWD samples. In an identical study with the same diets, urine samples were collected, and1H NMR spectra were acquired on these. PLS-DA on spectra obtained for urine samples revealed changes in the intensities of spectral regions, which could be ascribed to differences in the content of betaine and creatine/creatinine between the two diets, and LC–MS analyses verified a significantly lower content of creatinine in WGD urine samples compared with NWD urine samples. In conclusion, using an explorative approach, the present studies disclosed biochemical effects of a wholegrain diet on plasma betaine content and excretion of betaine and creatinine.

Summary

Background and objective: A study was designed to utilise the resources of our computerised anaesthesia record keeping system to assess the attributable effects of increased preoperative creatinine (>1.3 mg dL−1) on outcome in patients undergoing non-cardiac surgery.

Methods: This retrospective study was based on data sets of 58 458 patients recorded with a computerised anaesthesia record keeping system over a period of 4 yr at a tertiary care university hospital. Cases were defined as patients with a preoperative creatinine >1.3 mg dL−1; controls (creatinine ≤1.3 mg dL−1) were selected and automatically matched according to several parameters (ASA physical status, high risk and urgency of surgery, age and gender) in a stepwise fashion. Main outcome measures were hospital mortality and the incidence of intraoperative cardiovascular events.

Results: Three-thousand-and-twenty-eight patients (5.2%) had preoperative creatinine values >1.3 mg dL−1. Matching was successful for 54.5% of the cases, leading to 1649 cases (mean creatinine 3.3 ± 2.2 mg dL−1) and 1649 controls (1.0 ± 0.2 mg dL−1). The crude mortality rates for the cases and matched controls were 2.2% (n = 36) and 0.9% (n = 15), respectively (P = 0.003). Intraoperative cardiovascular events were found in 30.1% of the patients (n = 496) and in 28.3% of the matched controls (n = 466; P = 0.25, power = 0.46). Using logistic regression analyses a significant association between preoperative increased creatinine and hospital mortality was found (odds ratio 2.62; 95% confidence interval [1.39; 4.93]).

Conclusions: An increased preoperative serum creatinine in patients undergoing non-cardiac surgery is associated with an increased perioperative risk, but not with a higher incidence of intraoperative cardiovascular events.

Correlated responses in physiological traits to divergent selection on components of efficient lean growth identified physiological traits for use as predictors of genetic merit and provided a biological explanation for the between-selection line differences in protein and lipid deposition. Responses (differences between high (H) and low (L) selection lines) in protein metabolism during ad-libitum feeding were associated with divergent selection for daily food intake (DFI) (reduced serum creatinine concentration (H = 1·30 v. L = 1·56, s.e.d. 0·08 mg/dl)) and for lean growth rate on an ad-libitum feeding regime (LGA) (increased serum urea (H = 48 v. L = 36, s.e.d. 4 mg/dl) and creatinine (H = 1·74 v. L = 1·45 mg/dl)) concentrations, but not with selection for lean growth on a restricted feeding regime (LGS). Following 24-h fasting, responses in lipid metabolism, in the form of higher serum non-esterified fatty acid concentrations, were detected with divergent selection for both LGA (399 v. 248, s.e.d. 66 µmol/l) and LGS (H = 361 v. L = 107 µmol/l). The high LGS line appeared to ‘preserve’ protein to a greater extent than the high LGA line and similarly there was greater maintenance of lipid depots by the low LGS line compared with the low LGA line. A tentative ranking of the two pairs of lean growth selection lines on the basis of ‘importance’ of protein deposition would be high LGS, high LGA, low LGA and low LGS.

Coheritabilities for serum creatinine concentration with predicted lysine balance and lysine required for protein deposition (-0·17 and 0·17, s.e. 0·08) indicated that serum creatinine concentration may usefully be included in breeding value prediction for lysine requirement and protein deposition to increase the accuracy of predicted genetic merit.

Fasting did not increase the coheritabilities for serum creatinine concentration, so inclusion of serum creatinine concentration in a selection criterion for dietary lysine requirement or protein deposition does not require withdrawal of food before blood sampling animals.

The effect of the physiological state and dietary protein level on urinary excretion of creatinine (C) and purine derivatives (PD) was studied in two experiments carried out with pregnant and lactating ewes to evaluate whether the PD/C ratio in urine can he confidently used as an index of PD excretion. In both experiments ewes were given ammonia-treated straw and concentrates including different levels of fish meal and the excretion in urine and milk and the plasma concentration of C, allantoin (AL), xanthine, hypoxanthine and uric acid was measured.

Creatinine excretion (in urine and milk) was higher in pregnant ewes than in those lactating (492 and 420 (s.e. 10.0) μmol/kg maternal live weight0.75) and no significant differences were found due to number of foetuses and dietary protein level. The coefficient of variation was 0·10 in both pregnancy and lactation and individual variation accounted for proportionately 0·78 and 0·93 of total variation. The AL/C ratio in urine was highly correlated with daily AL excretion (r = 0·90 and 0·78 in pregnant and lactating ewes, respectively). Changes in PD excretion with experimental treatments were mainly reflected in AL, as the main component (0-83) of total PD. Most of the variation in AL excretion was explained by differences in rumen fermentable organic matter intake (RFOMI) (R2 = 0·79) and AL excretion did not differ between treatments when expressed per kg of RFOMI. In contrast to this the ratio AL/digestible organic matter intake decreased with increasing levels of fish meal in the diet. Urinary PD excretion was better related to estimated PD kidney tubular load (r = 0·76) than to PD plasma concentration (r = 0·64).

The results suggest that creatinine excretion is scarcely affected by the number of foetuses in pregnancy and dietary protein level but if the AL/С in urine is used instead of total collection as an index of purines absorbed in the duodenum, differences in urinary creatinine excretion due to physiological state must be accounted for.

The potential of the spot urine sampling technique as an alternative to performing a total urine collection was evaluated. Twelve multiparous Holstein-Friesian cows were given two experimental diets in a complete change-over design using two 14-day experimental periods. Experimental diets were either silage offered ad libitum with 7 kg fresh weight concentrate supplement as a single meal (SF), or a complete diet formulated from the same ingredients with a similar foragexoncentrate ratio (CD). Total urine collections were performed every 2 h on days 11 and 14 of each experimental period. Subsamples of urine were stored at 20°C and subsequently analysed by high-performace liquid chromatography. Daily allantoin and purine derivative (PD) excretion were highly correlated (r = 0·995, no. = 48, P < 0·001). PD and creatinine excretion during each 2-h interval depended on time of collection (PD, P < 0·001 and creatinine, P < 0·05) and on cow (P < 0·01) but were unaffected by sampling day or treatment. Diurnal variations in the molar ratio ofPD or allantoin to creatinine (PD/c and Ale, respectively) followed similar diurnal patterns as observed for PD and allantoin excretion. The data were used to assess the error of prediction of daily mean PD/c or Ale ratios. Three spot sampling regimens (based on the collection of four 4-h samples, three 8-h samples or two 12-h samples) and also on either single or 2-day urine collections were evaluated. Collection of multiple samples within a day was more reliable than collecting fewer samples over several days. Prediction errors were greater for SF compared with CD. Even the most intensive sampling regimen did not allow an acceptable prediction of daily mean PDIc or Ale ratio, minimum r values for PDIc and Ale ratios were 0·098, 0·136 and 0·547, 0·579 for SF and CD, respectively. Furthermore, daily mean PDIc and Ale ratios proved poor predictors of daily PD and allantoin excretion (r values of 0·69 and 0·72, respectively). Total urine collection appears necessary to assess accurately daily PD excretion in dairy cows.