[Comparison of different methods for monitoring of acid base status in dairy cow herds]. / Methodenvergleich zur Überwachung des Säuren-Basen-Status in Milchviehherden.

OBJECT AND PURPOSE: Acid-base disorders in dairy herds can be diagnosed by determining urinary net base excretion (NBE). Modifications of this method are the differential NBE (dNBE) with determination of the urinary concentration-independent base-acid ratio (BAR) and the simplified NBE test with reduced urine volume (sNBE). The aim of this study was to compare these methods among themselves and as a pooled test, in their assessment of cow group acid base status as well as to derive recommendations for practical use. ANIMALS, MATERIAL, AND METHODS: The concentrations of NBE, dNBE, and sNBE were measured in urine samples derived from 855 German-Holstein cows in 127 cow groups at different stages of lactation. BAR was then calculated. dNBE and BAR were determined both individually per cow and as a pool of a group. Mixed linear models were used to examine the relationship between the mean of the individual animal values and the pool sample result of a group for these two parameters. In addition, all groups were evaluated with respect to acidotic or alkalotic load based on their single animal results of the respective methods, the mean values formed from them, and the measured pool result. By using the single animal BAR as reference, the sensitivity and specificity of the different methods were calculated. RESULTS: The calculated mean values of the individual measurements of dNBE and BAR differed from the measured value in the pool sample, especially in low and high measurement ranges. In the group assessment, NBE showed the best combined sensitivity and specificity for the detection of acid base disorders. The dNBE pool assay showed satisfactory specificity with respect to acidosis and alkalosis, while the sNBE on an individual animal basis and the BAR determination in the pool showed satisfactory sensitivity with respect to acidosis. CONCLUSIONS AND CLINICAL RELEVANCE: It was shown that NBE determined in individual animal samples is well suited for the assessment of acid base status of cow groups and can therefore be recommended for practical use. The determination of dNBE as well as BAR as a pool test is not sufficient for the detection of alkalotic load in cow groups but may help to confirm an existing acidotic load.

Evaluation of protocols to determine urine output and urinary urea nitrogen excretion in dairy cows with and without dietary salt supplementation.

Urine output and urinary urea-N excretion (UUNe) excretion are critical measures to accurately evaluate N metabolism in lactating dairy cows and environmental concerns related to manure N. The objectives of this study were: (1) to compare estimates of UUNe, urine output, and related variables from 3 pre-established measurement protocols (bladder catheterization, external collection cup, and spot sampling) and from dietary salt supplementation, (2) to study temporal variation in UUNe, urine output, and related variables as affected by measurement protocol, and (3) to evaluate urine specific gravity as a predictor of urine output. Twelve multiparous Holstein cows were used in a split-plot, Latin square design. Cows were randomly assigned to a diet (main plot) containing either 0.7% or 1.6% NaCl (DM basis) and then assigned to a sequence of 3 protocols (subplot) in a balanced 3 × 3 Latin square with 14-d period. For each protocol, measurements were conducted every 4 h for 3 consecutive days. Urine output was determined gravimetrically for bladder catheterization and external collection cup or based on measured cow BW, measured urinary creatinine concentration, and the assumed creatinine excretion of 29 mg/kg BW per day for spot sampling. Urine specific gravity was measured by refractometry. When averaged over a 3-d measurement period and compared with bladder catheterization, spot sampling underestimated urine output (6.8 kg/d; 20%) and UUNe (26 g/d; 13%) but exhibited greater concentration of urinary urea-N (+58 mg/dL; 10%). There were no differences in any measurements determined via bladder catheterization or external cup device protocols, except for urine output that tended to be 3.7 kg/d lower for collection cup compared with bladder catheterization. The 2 gravimetric protocols yielded lower urinary creatinine concentration than spot sampling (64.7 vs. 88.1 mg/dL) and lower creatinine excretion (25.3 mg/kg BW per day) than the value of 29 mg/kg BW per day generally assumed in the spot sampling protocol. Salt supplementation tended to increase urine output (+5.2 kg/d) and decrease urinary urea-N concentration (-93 mg/dL), urinary creatinine concentration (-9.5 mg/dL), milk protein concentration (-0.19 percentage unit) and milk protein yield (-70 g/d). There was greater temporal variation of urine output when measured via the collection cup compared with bladder catheterization in the first 2 d but not the third day of sampling, suggesting that an extended period of adaptation might have improved data quality of the collection cup protocol. The R2 of the linear regression to predict urine output with urine specific gravity was 67%, 73%, and 32% for bladder catheterization, collection cup, and spot sampling, respectively. In this study, spot sampling underestimated both urine output and UUNe, but UUNe determination did not differ between external collection cup and bladder catheterization. However, our data suggested the need to investigate the adaptation protocol, required days of measurements and the conversion of urine mass to urine volume to improve accuracy and precision of urine collection protocols.

Non-invasive metabolomics biomarkers of production efficiency and beef carcass quality traits.

The inter-cattle growth variations stem from the interaction of many metabolic processes making animal selection difficult. We hypothesized that growth could be predicted using metabolomics. Urinary biomarkers of cattle feed efficiency were explored using mass spectrometry-based untargeted and targeted metabolomics. Feed intake and weight-gain was measured in steers (n = 75) on forage-based growing rations (stage-1, 84 days) followed by high-concentrate finishing rations (stage-2, 84 days). Urine from days 0, 21, 42, 63, and 83 in each stage were analyzed from steers with the greater (n = 14) and least (n = 14) average-daily-gain (ADG) and comparable dry-matter-intake (DMI; within 0.32 SD of the mean). Steers were slaughtered after stage-2. Adjusted fat-thickness and carcass-yield-grade increased in greater-ADG-cattle selected in stage-1, but carcass traits did not differ between ADG-selected in stage-2. Overall 85 untargeted metabolites segregated greater- and least-ADG animals, with overlap across diets (both stages) and breed type, despite sampling time effects. Total 18-bile acids (BAs) and 5-steroids were quantified and associated with performance and carcass quality across ADG-classification depending on the stage. Stepwise logistic regression of urinary BA and steroids had > 90% accuracy identifying efficient-ADG-steers. Urine metabolomics provides new insight into the physiological mechanisms and potential biomarkers for feed efficiency.

Pattern of postruminal administration of l-tryptophan affects blood serotonin in cattle.

Serotonin (5-HT) has many important functions in both central and peripheral nervous systems. Although it has been demonstrated that manipulation of serotonin metabolism is possible in many species, there is limited information about l-tryptophan (TRP), a serotonin precursor, in cattle, and these provide conflicting results. Furthermore, there is no study evaluating how different patterns of intra-abomasal infusion of TRP impact circulating 5-HT. The objective of this study was to evaluate if intra-abomasal infusion patterns of TRP can affect circulating 5-HT and other metabolites from TRP metabolism in the plasma and serum and circulating glucose and insulin in cattle. Eight ruminally cannulated Holstein steers were used in a replicated 4 × 4 Latin square design. Each received intra-abomasal water infusion (control) or intra-abomasal TRP infusion (50 mg/kg BW) in 3 different patterns: a pulse infusion once a day (pulse once), pulse infusion twice a day (pulse twice), or continuous infusion (continuous). For continuous treatment, the TRP dose was diluted in tap water and infused by a peristaltic pump (300 mL/h). To equalize conditions, the other treatments had a water infusion (300 mL/h). The steers were fed every 2 h, and blood was collected from a jugular vein catheter every 4 h for 24 h after the initial infusion. Urine produced during the 24 h period was collected. Serum and plasma TRP, 5-HT and kynurenine, plasma glucose, and serum insulin concentrations were analyzed. Urine was analyzed for concentrations of 5-hydroxyindoleacetic acid. Both serum TRP and kynurenine were increased (P < 0.05) by all TRP infusion treatments, but concentrations in pulse dose treatments were greater than those in continuous infusion. Serum 5-HT increased (P < 0.05) with both pulse TRP infusion treatments; however, the continuous TRP infusion did not increase the serum 5-HT. Plasma 5-HT, glucose, and insulin had a tendency to increase with TRP pulse infusions. The urinary 5-hydroxyindoleacetic acid excretion was highest for pulse dose treatments. An acute supply of TRP in 1 or 2 daily doses increases serum 5-HT and increases circulating glucose and insulin in cattle. The TRP and kynurenine concentrations are similar in plasma and serum. However, the serum 5-HT concentration is more responsive to TRP administration than plasma.

Profile of bile acid subspecies is similar in blood and follicular fluid of cattle.

The composition of follicular fluid (FF) has an impact on the developmental capacity of the oocyte and the resulting embryo. FF is composed of blood plasma constituents which cross the blood follicular barrier and the secretory components of granulosa and theca cells. Moreover, it has been shown recently that follicular cells have the ability to synthesize bile acids (BAs). BAs are present in several fluids of mammals especially in bile, blood and urine. FF is an essential impacting factor on the oocyte quality and therefore resulting embryos. To achieve a better understanding of this subject, the presence and concentration of BAs were measured in fluid collected from bovine follicles, categorized according to their size, throughout two entire oestrus cycles and compared to those in blood and urine. The body fluids were collected during the same examination procedure and in total samples from four heifers were obtained. A broad spectrum of 11 BA derivatives was measured applying liquid chromatography-tandem mass spectrometry (LC-MS/MS). The simultaneous and direct quantification of BAs in different body fluids of cattle are reported. Within the follicular fluid, blood and urine, cholic acid and glycocholic acid are the dominant BA subspecies irrespective of the oestrus cycle stage. Moreover, BA concentrations in blood compared to those in the FF were similar. For the first time these results clearly highlight the presence of different BA subspecies in FF, blood and urine during the oestrus cycle in cattle.

Dietary supplementation of rumen-protected methionine decreases the nitrous oxide emissions of urine of beef cattle through decreasing urinary excretions of nitrogen and urea.

BACKGROUND: Two consecutive trials were carried out to study the effects of dietary supplementation of rumen-protected methionine (RPM) on nutrient digestibility, nitrogen (N) metabolism (Trial 1), and consequently the nitrous oxide (N2 O) emissions from urine in beef cattle (Trial 2). Eight 24-month-old castrated Simmental bulls with liveweights of 494 ± 28 kg, and four levels of dietary supplementation of RPM at 0, 10, 20, and 30 g head-1 d-1 , were allocated in a replicated 4 × 4 Latin square for Trial 1 and the N2 O emissions from the urine samples collected in Trial 1 were measured using a static incubation technique in Trial 2. RESULTS: Supplementation of RPM at 0, 10, 20, and 30 g head-1 d-1 to a basal ration deficient in methionine (Met) did not affect the apparent digestibility of dry matter, organic matter, neutral detergent fiber, or acid detergent fiber (P > 0.05), but decreased the urinary excretions of total N (P < 0.05) and urea (P < 0.001), increased the ratio of N retention / digested N (P < 0.05) in beef cattle, and decreased the estimated cattle urine N2 O-N emissions by 19.5%, 23.4%, and 32.6%, respectively (P < 0.001). CONCLUSION: Supplementation of RPM to Met-deficient rations was effective in improving the utilization rate of dietary N and decreasing the N2 O emissions from urine in beef cattle. © 2019 Society of Chemical Industry.

Feeding a diet with corn distillers grain with solubles to dairy cows alters manure characteristics and ammonia and hydrogen sulfide emissions from manure.

The objective of the experiment was to examine effects of a diet containing a high concentration (28.8% dry matter basis) of corn distillers grain with solubles on manure characteristics and NH3 and H2S emissions from dairy cow manure. Eighteen cows were blocked by parity and days in milk, and cows in each block were assigned to the following treatments: the control diet (CON) or CON with distillers grains with solubles at 28.8% (dry matter basis) replacing mainly soybean meal (DG). The experiment was conducted for 11 wk, and feces and urine from individual cows were collected over 3 d in wk 11 (a total of 8 spot samples per cow). Fecal or urine samples were composited by cow, and the composite feces and urine were analyzed for indigestible neutral detergent fiber and creatinine concentration, respectively, for individual cows to estimate total fecal and urine outputs. Immediately before the manure incubation, composited feces and urine were sampled to determine manure characteristics. Manure was reconstituted according to daily fecal and urine excretion estimated for individual cows. Individual manures were incubated using a continuous air flux multichamber system over 10 d to measure NH3 and H2S emissions. All data from 18 manures were analyzed using the Mixed procedure of SAS (SAS Institute Inc., Cary, NC). The ratio of feces to urine and the contents of manure total and volatile solids were not different among treatments. Urine from DG had lower pH and DG manure had lower N content and greater S content compared with CON. During the 10-d incubation, NH3 emission was considerably less for DG versus CON. The emission of H2S over 10 d for DG was greater compared with that for CON. After the incubation, manure pH and N and S concentrations were greater for DG versus CON. In conclusion, manure from cows fed a high-DG diet decreased urinary N contribution to manure N and lowered urine pH, which were the factors that caused the decrease in NH3 emission from DG manure. However, the DG diet increased dietary S concentration and increased S excretion in urine and feces. This increased H2S emission from DG manure during the 10-d manure incubation.

Release of urinary aquaporin-2-bearing extracellular vesicles is decreased in pregnant Japanese Black cattle.

Aquaporin-2 (AQP2), a vasopressin-regulated water channel, plays an important role in renal water homeostasis. It has been reported that the level of AQP2 in human urine is altered during pregnancy. However, little is known about the level of urinary AQP2 in pregnant cattle. In this study, we examined the level of AQP2-bearing extracellular vesicles (uEV-AQP2), which account for most urinary AQP2, in both heifers and cows during the gestational and postpartum periods. The level of uEV-AQP2 was significantly decreased during gestation in comparison with the other cattle examined. Similarly, the levels of EV marker proteins in uEVs, including tumor susceptibility gene 101 (TSG101) protein and apoptosis-linked gene 2-interacting protein X (ALIX), were significantly decreased during gestation. There were significant correlations between the levels of uEV-AQP2 and uEV-TSG101, or uEV-ALIX. Immunohistochemistry data from pregnant and non-pregnant cattle supported the notion that the level of uEV-AQP2 was decreased during gestation. These data indicate that the level of uEV-AQP2 is decreased in pregnant cattle, possibly through a decrease in both the number of EVs released into the urine and renal AQP2 expression.

16S rRNA gene profiling of bacterial communities mediating production of tsetse attractive phenols in mammalian urine.

Several types of odours are involved in the location of host animals by tsetse (Diptera: Glossinidae), a vector of animal African trypanosomiasis. Host animals' ageing urine has been shown to be the source of a phenolic blend attractive to the tsetse. Nevertheless, limited research has been performed on the microbial communities' role in the production of phenols. This study aimed at profiling bacterial communities mediating the production of tsetse attractive phenols in mammalian urine. Urine samples were collected from African buffalo (Syncerus caffer), cattle (Bos taurus) and eland (Taurotragus oryx) at Kongoni Game Valley Ranch and Kenyatta University in Kenya. Urine samples, of each animal species, were pooled and left open to age in ambient conditions. Bacteriological and phenols analyses were then carried out, at 4 days ageing intervals, for 24 days. Phenols analysis revealed nine volatile phenols: 4-cresol, ortho-cresol, 3-cresol, phenol, 3-ethylphenol, 3-propylphenol, 2-methyloxyphenol, 4-ethylphenol and 4-propylphenol. Eight out of 19 bacterial isolates from the ageing urine revealed the potential to mediate production of phenols. 16S rRNA gene characterisation of the isolates closely resembled Enterococcus faecalis KUB3006, Psychrobacter alimentarius PAMC 27887, Streptococcus agalactiae 2603V, Morganella morganii sub.sp. morganii KT, Micrococcus luteus NCTC2665, Planococcus massiliensis strain ES2, Ochrobactrum pituitosum AA2 and Enterococcus faecalis OGIRF. This study established that some of the phenols emitted from mammalian urine, which influence the tsetse's host-seeking behaviour, are well characterised by certain bacteria. These results may allow the development of biotechnological models in vector control that combines the use of these bacteria in the controlled release of semiochemicals.

Metabolomic studies as a tool for determining the post-mortem interval (PMI) in stillborn calves.

BACKGROUND: Perinatal mortality may vary between herds, but the cost of deaths are always higher than value of the calf. When diagnosing the cause of a calf's death it is important to determine when it occurred, before or after calving. Metabolomics is widely used to identify many human diseases, but quite rarely applied in veterinary science. The aim of this study was to compare the metabolic profiles of calves with different times of death and those of calves born alive. Into the study, twenty one healthy controls (singleton, normal assisted calving, born alive) and 75 stillborn (SB) calves (with a gestation length of ≥260 days, SB, or dead within 6 h of birth) were enrolled. Plasma and urine from SB and control calves were investigated by proton nuclear magnetic resonance based metabolomic methods. SB calves were divided into four PMI groups. One PMI group included calves that died after calving and the other groups - three comprised in utero deaths, based on pathophysiological changes (lung inflation, autolysis in internal organs, hemoglobin imbibition in the pleura and aortic arch). Partial Least Squares - Discriminant Analysis models based on plasma metabolites were calculated, reflecting assumed data clustering. RESULTS: Twenty six metabolites in plasma and 29 in urine changed significantly with PMI according to one way analysis of variance. Half the metabolites in plasma and the majority in urine increased with PMI. Six metabolites increased simultaneously in plasma and urine: acetate, sn-glycero-3-phosphocholine (GPC), leucine, valine, creatine, and alanine. CONCLUSIONS: Post-mortem changes in calves were associated with molecular variations in blood plasma and urine, showing the greatest differences for the group in which the post-mortem pathological changes were the most advanced. The results of the study show that evaluation of calf plasma or urine may be used as a diagnostic method for the determination of the PMI. Moreover, the metabolites, which unambiguously increased or decreased, can be used as potential biomarkers of PMI.

Comparison of urine and blood as a convenient and practical sample for estimating the contamination level of live cattle with radioactive cesium.

Blood and urine were compared to clarify which is a better sample for estimating the concentration of radioactive cesium in muscles of live cattle. The most probable concentration of 137 Cs in muscles was 21.0 times that in blood, and the error of this estimation was 28%. The concentration of 137 Cs in blood was estimated accurately using urine samples. The estimation error did not increase so much (33%), even when the concentration of 137 Cs in muscles was estimated using urine samples. On the other hand, the maximum volume of blood that can be collected with one syringe is 50 ml, whereas it is easy to collect 1,000 ml of urine. It took at least 360 min to confirm that a cow meets the legal standard by inspecting 50 ml of blood. However, with 1,000 ml of urine, a 20 min measurement time was sufficient for this purpose. This difference in the required measurement time is critically important for practical use. In addition, urine can be collected by farmers themselves, whereas the blood collection requires a veterinarian. Therefore, urine is a more convenient and practical sample for estimating the contamination level of live cattle with radioactive cesium.

Clinical utility of urine specific gravity, electrical conductivity, and color as on-farm methods for evaluating urine concentration in dairy cattle.

BACKGROUND: Urine concentration (UC) provides clinically useful information concerning hydration status and renal function of animals. OBJECTIVES: To characterize the clinical performance of urine specific gravity measured by optical refractometry (USG-R ) or Multistix-SG urine reagent dipstick (USG-D ), urine electrical conductivity using an OAKTON Con 6 conductivity handheld meter (UEC ), urine color (UColor ) using a custom-designed 8-point color chart, and urine creatinine concentration (UCreat ) for assessing UC in dairy cattle. ANIMALS: 20 periparturient Holstein-Friesian cows. METHODS: Urine was obtained by perineal stimulation or urethral catheterization and urine osmolality (UOsm , reference method), USG-R , USG-D , UEC , UColor , and UCreat determined. Diagnostic test performance was evaluated using Spearman's rho and logistic regression to determine the area under the receiver operating curve (AUC) and optimal cut point for diagnosing hypohydration (UOsm ≥800 mOsm/kg). P < .05 was considered significant. RESULTS: The best performing test for diagnosing hypohydration was USG-R (AUC = 0.90) at an optimal cut point ≥1.030. The second-best performing test was UEC (AUC = 0.82) at a cut point of ≥23.7 mS/cm, followed by UCreat (AUC = 0.76) at a cut point of ≥95.3 mg/dL, and UColor (AUC = 0.74) at a cut point of ≥4 on an 8-point scale. Urine specific gravity measured by dipstick performed poorly (AUC = 0.63). CONCLUSIONS AND CLINICAL IMPORTANCE: USG-R and UEC provide practical and sufficiently accurate methods for measuring UC in dairy cattle. Urine color had moderate clinical utility as a no-cost cow-side method for assessing UC, whereas dipstick refractometry is not recommended for assessing UC.

Urinalysis and determination of the urine protein-to-creatinine ratio reference interval in healthy cows.

BACKGROUND: There are no reference intervals for urinalysis in cattle. HYPOTHESIS/OBJECTIVES: Characterize the urine of healthy cows, establish urine protein-to-creatinine ratio (UPC) reference intervals, and test possible differences among dairy and beef cattle, age groups, or stage of lactation. ANIMALS: Seventy-seven dairy and 74 beef 2.5 to 17 year-old cows of different breeds housed mainly in free stall. METHODS: In this prospective study, urine specimens were collected by catheterization. Complete urinalysis was performed within 1 hour including specific gravity, dipstick evaluation, visual urine pH evaluation with 0.3 pH unit graded strips, and microscopic evaluation of the sediment. Urinary protein and creatinine concentrations and protein electrophoresis were determined on frozen aliquots. RESULTS: Overall reference intervals were 1.020 to 1.045 for USG, 7.0 to 8.7 for pH, and 0.04 to 0.25 for UPC; because of differences in creatinine concentration, UPC was lower in beef (0.04-0.14) than in dairy (0.05-0.25) cows and in the latter in dry than lactating cows. With dipstick evaluation, most analytes were absent except for blood, ketone, and protein in 24.7, 16.0, and 64.7% of cases, respectively. Microscopic evaluation revealed less than 3 red blood cells, leukocytes, and epithelial cells in 84, 99.3, and 100% cows, respectively. No band was observed at electrophoresis, except in 1 case at MW ~66 000. CONCLUSIONS AND CLINICAL IMPORTANCE: Creatininuria is higher in beef than dairy cows and proteinuria is likely more efficiently characterized by protein concentration than by UPC.

Resorcylic acid lactones in urine samples of slaughtered animals resulting from potential feed contamination with zearalenone.

Resorcylic Acid Lactones, including zeranol, anabolics listed in the group A4 of Directive 96/23/EC, are banned in Europe for use in animals since 1985. Zeranol, after administration to animals, is metabolized to taleranol and zearalanone. It can also naturally occur in the urine due to conversion of zearalenone that may be present in animal feed. In 2010-2017, in Poland, 7746 animal samples were tested for zeranol residues within the official monitoring program. In 13, zeranol was detected after screening. Re-examinations confirmed resorcylic acid lactones in six samples. The recommendations state that only the presence of zeranol and/or taleranol gives the basis for non-compliance. Confirmation should cover the entire profile of six resorcylic acid lactones. In case of detection, the relationship ratio should be verified. Following the proposed criteria, it could be concluded that zeranol detected in urine samples in Poland originated from contamination of feed with mycotoxin, not from illegal use.

Acid-base assessment of post-parturient German Holstein dairy cows from jugular venous blood and urine: A comparison of the strong ion approach and traditional blood gas analysis.

Evaluating acid-base status is important for monitoring dairy herd health. In a field study, we aimed to compare the acid-base status measured by net acid-base excretion (NABE) in urine with results of venous blood analysis in clinically healthy, but possibly metabolically burdened cows in their transition period. For this, we sampled blood from the jugular vein and urine from 145 German Holstein cows within 1 to 76 days post-partum. In blood, the metabolic parameters non-esterified fatty acids (NEFA) and ß-hydroxybutyrate (BHB), as well as numerous parameters of the acid-base status were measured. The traditional approach, based on bicarbonate concentration, base excess (BE) and anion gap (AG), was compared to the strong ion approach variables, e.g. acid total (Atot), measured strong ion difference (SIDm), strong ion gap (SIG), and unmeasured anions (XA), respectively. Results of both approaches were set against the outcome of urine analysis, i.e. the NABE, base-acid ratio and pH of urine, in a cluster analysis, which provided 7 moderately stable clusters. Evaluating and interpreting these 7 clusters offered novel insights into the pathophysiology of the acid-base equilibrium in fresh post-partum dairy cows. Especially in case of subclinical acid-base disorders, the parameters of the strong ion difference theory, particularly SIDm, Atot and SIG or XA, provided more in-depth information about acid-base status than the traditional parameters BE, bicarbonate or AG in blood. The acid-base status of fresh cows with protein aberrations in blood could be differentiated in a much better way using the strong ion approach than by traditional blood gas analysis or by the measurement of urinary excretion. Therefore, the strong ion approach seems to be a suitable supplement for monitoring acid-base balance in dairy cattle.

Validating and optimizing spot sampling of urine to estimate urine output with creatinine as a marker in dairy cows.

An experiment was conducted to validate and optimize the procedure of spot urine sampling with urinary creatinine as a marker to estimate urine outputs of dairy cows. Twelve lactating cows were used in a randomized complete block design. Cows were grouped and randomly assigned to 2 experimental diets: a corn silage-based diet and an alfalfa silage-based diet with supplemental potassium. The experiment lasted for 21 d and total collection (TC) of urine was conducted for the last 3 d. Twelve spot samples of urine from individual cows were collected over a 3-d period during TC to represent every 2-h sampling in a 24-h cycle. Creatinine excretion rate (mg/kg of body weight per d) was variable among cows from 16.7 to 34.5 with an average of 27.3. Creatinine concentrations of spot samples within cow were averaged to simulate urine samples obtained from various spot sampling frequencies (equally spaced 12, 6, 4, and 2 time points starting at feeding: 12TP, 6TP, 4TP, and 2TP, respectively). Large diurnal variation of urinary creatinine concentration was observed within cow. Creatinine concentration was greater (75 vs. 65 mg/dL) for 12TP compared with TC, resulting in underestimating (29.8 vs. 32.6 kg/d) urine outputs. When compared among 12TP, 6TP, 4TP, and 2TP, creatinine concentrations were different and urine outputs tended to be different for 2TP compared with 12TP, 6TP, and 4TP. In addition, despite underestimation of urine output, a regression analysis indicated strong linear relationships between 12TP, 6TP, or 4TP and TC, suggesting that this technique can successfully identify the differences in urine outputs altered by dietary treatments. However, 4TP failed to detect statistical differences in urine outputs between a corn silage-based diet and the alfalfa silage-based diet with supplemental potassium, indicating that a spot urine sampling frequency of at least 6 was required to identify dietary effects on urine outputs. According to the pattern of diurnal changes in urinary creatinine concentration, a spot sample at about 10 h after feeding may have potential to obtain a urine sample that is more representative (i.e., creatinine concentration) of TC urine compared with urine from multiple sampling frequencies. Overall, urinary creatinine as a marker with spot sampling of urine underestimated urine output. However, 12TP and 6TP were successful in identifying changes in urine outputs by dietary treatments.

The use of hair as a long-term indicator of low-dose ß2 agonist treatments in cattle: Implications for growth-promoting purposes monitoring.

The objective of this study was to assess the feasibility of using hair as a long-term indicator of cocktail (low-dose ß2 agonists) treatments in cattle. Six male Simmental cattle were treated with a mixture of low-dose clenbuterol, ractopamine, and salbutamol at dosages of 5.3, 223.3, and 50.0 µg/kg, respectively. The trial lasted for 112 days and included 28 days of treatment and 84 days of withdrawal. Plasma and urine samples taken during the treatment period contained the highest residues, with maximum concentrations of clenbuterol, ractopamine, and salbutamol in plasma of 1.49 ng/mL (Day 21), 43.78 (Day 14) ng/mL, and 8.07 ng/mL (Day 7), respectively, and in urine of 62.40 ng/mL (Day 28), 3995.77 ng/mL (Day 28), and 503.72 ng/mL (Day 1), respectively. On day 42 of withdrawal, the residues of all three ß2 agonists in plasma were below the limit of quantification (LOQ; 0.3 ng/mL for clenbuterol, and 0.5 ng/mL for ractopamine and salbutamol), and in urine samples were below or near the LOQ (the highest being ractopamine at 1.10 ng/mL). The highest concentrations of clenbuterol, ractopamine, and salbutamol in hair were 88.36, 1351.92, and 100.58 ng/g, respectively, on day 14 of withdrawal; and the residues were long-lasting, with 7.64, 28.55, and 8.77 ng/g, respectively, on day 84 of withdrawal. The results of this study demonstrate that hair could be utilized as a long-term indicator of the use of a combination of low-dose ß2 agonists in cattle, which could have implications for growth-promoting purposes monitoring.

Short communication: Milk urea nitrogen as a predictor of urinary nitrogen and urea nitrogen excretions of late-lactation dairy cows fed nitrogen-limiting diets.

Our objectives were to assess the relationships between milk urea N (MUN), serum urea N (SUN), urine N (UN), and urinary urea N (UUN) in late-lactation cows fed N-limiting diets and compare these relationships with those previously established. Data were from a pen-based study in which 128 Holstein cows had been assigned to 1 of 16 pens in a randomized complete block design to assess the effects of diets containing 16.2, 14.4, 13.1, and 11.8% crude protein (CP, dry matter basis) during a 12-wk period. At least half of the cows in each pen were randomly selected to collect pen-level samples of serum and urine in wk 3, 7, and 11, when wk in lactation averaged 35, 39, and 43, respectively. A mixed model was developed to study the relationship of MUN with SUN, UN, and UUN. Week of lactation did not affect the relation between MUN and SUN across dietary treatments. However, we found a week × MUN interaction, suggesting that between wk 35 and 43 of lactation, UN excretion decreased from 89 to 73 g/d (-17 g/d) when MUN was 6.0 mg/dL (11.8% dietary CP) but increased from 142 to 149 g/d (+7 g/d) when MUN was 13.3 mg/dL (16.2% dietary CP). These effects were essentially due to changes in UUN excretion, which declined from 54 to 37 g/d (-17 g/d) and increased from 112 to 117 g/d (+5 g/d) when MUN was 6.0 and 13.3 mg/dL, respectively. When MUN was 11.2 mg/dL (15% dietary CP), UN and UUN excretions remained constant over time. Based on root mean squared prediction error and the concordance correlation coefficient, these data did not conform to most previously published prediction equations because of both mean and slope biases. The discrepancy could have resulted from difference in study design (cow vs. pen as experimental unit), dietary treatments (energy vs. N-limiting diets), frequency of measurement and duration of adaptation period (single measurement after 1 to 3 wk of adaptation vs. repeated measurements over a 12-wk period), method for determining urine volume (total collection vs. spot sampling), and the assay used to measure MUN. However, our data captured changes in kidney physiology that warrant further studies of long-term renal adaptation to N-limiting diets.

Multiresidue Determination of the Anabolic-Agent Residues Steroids, Stilbenes, and Resorcylic Acid Lactones in Bovine Urine by GC-MS/MS with Microwave-Assisted Derivatization.

In this work, a GC-MS/MS method was developed for the determination of anabolic-agent residues in bovine urine. The optimized sample preparation was as follows: enzymatic hydrolysis by ß-glucuronidase-sulfatase enzyme from Helix pomatia for 16 h at 37.5 °C, liquid-liquid extraction with diethyl ether, solid-phase extraction with HLB and aminopropylsilane cartridges, and microwave-assisted derivatization using 25 µL of MSTFA/NH4I/ethanethiol and full microwave power for 2 min. The method was validated according to Decision 657/2002/EC, Codex Alimentarius, and Manual da Garantia da Qualidade Analítica guidelines. The acceptability criteria for quantitative analysis were met for α-ethinylestradiol, α-nandrolone, ß-estradiol, ß-zearalanol, ß-zearalenol, drostanolone, ethisterone, dienestrol, diethylstilbestrol, hexestrol, megestrol, methyltestosterone, and zearalenone. The analytes α-zearalenol, α-zearalanol, and norethandrolone were validated for qualitative analysis.

Co-exposure of the Mycotoxins Lolitrem B and Ergovaline in Steers Fed Perennial Ryegrass ( Lolium perenne) Straw: Metabolic Characterization of Excreta.

Past research showed a strong linear correlation between levels of the mycotoxins lolitrem B (LB, a tremorgen) and ergovaline (EV, an ergot alkaloid and potent vasoconstrictor) in perennial ryegrass (PRG) forage. The purpose of this study was to characterize the excretion of these two compounds in beef cattle consuming PRG straw and to utilize liquid chromatography-tandem mass spectrometry to investigate the metabolism of LB and EV in excreta. Four groups of steers ( n = 6/group) were fed endophyte-infected PRG for 64 days (2256/638, 1554/373, 1012/259, or 247/<100 µg/kg LB/EV). Concentrations of LB and EV in both PRG straw and feces showed a linear relationship to each other. Feces reflected a dose-response for both mycotoxins, with values increasing most rapidly through 21 days then plateauing. Urine contained no detectable level of either compound or the ergoline lysergic acid. Screening for metabolites showed oxidation and reduction biotransformations for both toxins, with additional conjugation products detected for ergovaline.