Aniline blue test for sperm protamination-a valuable add-on to the bull breeding soundness evaluation.

Bull breeding soundness evaluation (BBSE) is the most common procedure used to predict bull potential fertility. However, the use of traditional methods for semen evaluation can affect its reliability. The inclusion of additional advanced test in BBSE may increase its accuracy. This study aimed to investigate the correlation between the degree of sperm protamination and BBSE main parameters of scrotal circumference (SC), progressive motility (PM), morphologically normal sperm (NS), and different categories of morphological defects. In addition, to determine the correlation between the three methods used for protamine assessment, five Brangus bulls were subjected to the BBSE. Semen samples were collected via electro-ejaculation and evaluated using traditional methods. Three different methods were used to determine the degree of sperm protamination: aniline blue (AB) staining, chromomycin A3 staining with fluorescent microscope (CMA3-FLM), and CMA3 with flow cytometry (CMA3-FCM). Sperm protamine deficiency assessed using the three methods exhibited significant differences among bulls according to their classification by BBSE, and showed significant negative correlation with semen quality parameters of NS and PM. A significant positive correlation was found between AB positivity and morphological abnormalities. The three methods used for protamine assessment also revealed significant positive correlations. Among the three tests, AB staining was the cheapest and easiest test that offers an objective assessment method for sperm protamination. Hence, it can be concluded that the assessment of protamination using AB staining test might serve as an additional valuable parameter or a replacement whenever detail sperm motility and morphology analyses in conducting BBSE to predict bull fertility are not possible.

Clinical utility of plasma progesterone and blood and plasma glucose concentrations in predicting parturition in Holstein cows.

An accurate, practical, and low-cost method for predicting parturition is urgently needed in the dairy industry. The objective of this study was to evaluate changes in plasma progesterone concentration ([prog]) and glucose concentration in whole blood ([gluc]b) and plasma ([gluc]p) as predictors of parturition within 6, 12, and 24 h in primiparous and multiparous Holstein cows. Blood samples were obtained daily at approximately 0900 h from 34 primiparous and 72 multiparous Holstein cows in late gestation and the time of calving recorded to the nearest hour. Plasma [prog] was measured using an ELISA, and [gluc]b and [gluc]p using a low-cost point-of-care glucose meter. The optimal cut-point for predicting parturition was determined using binomial logistic regression with general estimating equations, because the data set consisted of repeated measures for each cow. Diagnostic test performance was evaluated by comparing the area under the receiver operating characteristic curve (AUC) and calculating the sensitivity, specificity, and κ at the optimal cut-point for predicting parturition. Plasma [prog] was the most accurate predictor of parturition within 24 h (AUC = 0.96) and 12 h (AUC = 0.93), whereas [gluc]b was the most accurate predictor of parturition within 6 h (primiparous, AUC = 0.96; multiparous, AUC = 0.86). We conclude that a decrease in plasma [prog] is currently the most accurate test for predicting calving within 24 h. Measurement of [gluc]b is a promising new test for the cow-side prediction of parturition in dairy cows due to its accuracy, practicality, and low cost.

Measurement of urine pH and net acid excretion and their association with urine calcium excretion in periparturient dairy cows.

Urine pH (UpH) and net acid excretion (NAE) are used to monitor the degree of systemic acidification and predict the magnitude of resultant hypercalciuria when feeding an acidogenic ration to control periparturient hypocalcemia in dairy cattle. The objectives of this study were to evaluate the diagnostic performance of urine dipstick and pH paper for measuring UpH, and to characterize the UpH-NAE relationship and the association of urine Ca concentration ([Ca]) with UpH and NAE. Urine samples (n = 1,116) were collected daily from 106 periparturient Holstein-Friesian cows fed an acidogenic ration during late gestation. Net acid excretion was measured by titration, and UpH was measured by a glass-electrode pH meter (reference method), Multistix-SG urine dipsticks (Siemens Medical Solutions Inc., Ann Arbor, MI), and Hydrion pH paper (Micro Essential Laboratory Inc., Brooklyn, NY). Diagnostic performance was evaluated using Spearman correlation coefficient (rs), Bland-Altman plots, and logistic regression. Urine pH measured by urine dipstick (rs = 0.94) and pH paper (rs = 0.96) were strongly associated with UpH. Method-comparison studies indicated that the urine dipstick measured an average of 0.28 pH units higher, and pH paper 0.10 pH units lower, than UpH. Urine [Ca] was more strongly associated with UpH (rs = -0.65) than NAE (rs = 0.52). Goals for controlling periparturient hypocalcemia under the study conditions were UpH <6.22 and <6.11, based on achieving urine [Ca] ≥5 mmol/L and estimated urinary Ca excretion ≥4 g/d, respectively. Urine pH was as accurate at predicting urine [Ca] as NAE when UpH >6.11. We conclude that pH paper is an accurate, practical, and low-cost cow-side test for measuring UpH and provides a clinically useful estimate of urine [Ca].

Changes in skeletal muscle thickness and echogenicity and plasma creatinine concentration as indicators of protein and intramuscular fat mobilization in periparturient dairy cows.

High-producing dairy cows experience a state of negative energy balance in the periparturient period that is partially addressed by increasing the rate of fat and protein mobilization. Previous studies have focused on the rate of fat mobilization, and consequently the rate of protein mobilization has not been well characterized. The objective of this study was therefore to determine the change in indicators of muscle mass during early lactation using ultrasonographic measurement of muscle thickness and changes in plasma creatinine concentration. The maximum thickness of the gluteus medius and longissimus dorsi muscles of 106 Holstein cows (34 primiparous, 72 multiparous) was determined ultrasonographically on d -3, 0, 3, 7, 14, 21, and 28 relative to the day of parturition. Plasma creatinine concentration was measured periodically during the same period. Mixed models analysis and Passing-Bablok regression were used to analyze the data. Gluteus medius thickness, longissimus dorsi loin thickness (LDLT), and longissimus dorsi thoracic thickness (LDTT) were decreased at 28 d postpartum compared with d 3 antepartum. Plasma creatinine concentration was weakly associated with gluteus medius thickness, LDLT, and LDTT (Spearman's rho = 0.31, 0.39, and 0.32, respectively). Plasma creatinine concentration in primiparous and multiparous cows at 28 d postpartum decreased by 0.24 and 0.30 mg/dL, respectively, compared with values 3 d antepartum. We concluded that ultrasonographic measurement of LDLT and LDTT and change in plasma creatinine concentration may provide practical methods for monitoring the rate of protein mobilization in periparturient dairy cows. Ultrasonographic examination of LDLT and LDTT therefore complements ultrasonographic measurement of backfat thickness and may be useful in the evaluation of energy reserve mobilization in periparturient dairy cows.

Clinical Utility of Plasma Fructosamine Concentration as a Hypoglycemic Biomarker during Early Lactation in Dairy Cattle.

BACKGROUND: Plasma fructosamine concentration ([FRA]) is a widely used long term hyperglycemic biomarker in humans and dogs, but its clinical usefulness as a hypoglycemic biomarker in dairy cattle is uncertain. OBJECTIVES: To evaluate the relationship between plasma [FRA] and glucose concentration ([gluc]) as well as indices of energy balance during early lactation in dairy cattle, and to characterize the influence of plasma total protein concentration ([TP]) and albumin concentration ([albumin]) on [FRA]. ANIMALS: Convenience sample comprising 103 periparturient Holstein-Friesian cattle. METHODS: Plasma [gluc], [TP], [albumin], and other clinicopathologic indices of energy status were determined periodically from Day 4 postpartum. Body condition score (BCS) was assessed, and backfat thickness (BFT) and longissimus dorsi muscle thickness (LDT) were measured ultrasonographically. Plasma [FRA] was measured at approximately 28 days postpartum. Associations between plasma [FRA] and study variables were evaluated using Spearman's rho and stepwise forward linear regression. Statistical significance was declared at P < 0.05. RESULTS: A positive association was detected between plasma [FRA] and mean plasma [gluc] from Days 4-28 postpartum (rs = +0.36, P < 0.001), and between plasma [FRA] and LDT (rs = +0.28, P = 0.007), BCS (rs = +0.23, P = 0.029), and BFT (rs = +0.21, P = 0.043). Multivariable regression identified a positive association between plasma [FRA] and mean plasma [gluc] and [albumin] from Days 4-28 postpartum. Correcting plasma [FRA] for [albumin] improved the association (rs = +0.46, P < 0.001) between plasma [FRA] and mean plasma [gluc]. CONCLUSIONS AND CLINICAL IMPORTANCE: Plasma [FRA] does not provide a clinically useful method for quantifying the magnitude of hypoglycemia or negative energy balance in dairy cows during early lactation.

Evaluation of 2 portable ion-selective electrode meters for determining whole blood, plasma, urine, milk, and abomasal fluid potassium concentrations in dairy cattle.

Two low-cost ion-selective electrode (ISE) handheld meters (CARDY C-131, LAQUAtwin B-731; Horiba Ltd., Albany, NY) have recently become available for measuring the potassium concentration ([K(+)]) in biological fluids. The primary objective of this study was to characterize the analytical performance of the ISE meters in measuring [K(+)] in bovine whole blood, plasma, urine, milk, and abomasal fluid. We completed 6 method comparison studies using 369 whole blood and plasma samples from 106 healthy periparturient Holstein-Friesian cows, 138 plasma samples from 27 periparturient Holstein-Friesian cows, 92 milk samples and 204 urine samples from 16 lactating Holstein-Friesian cows, and 94 abomasal fluid samples from 6 male Holstein-Friesian calves. Deming regression and Bland-Altman plots were used to characterize meter performance against reference methods (indirect ISE, Hitachi 911 and 917; inductively coupled plasma-optical emission spectroscopy). The CARDY ISE meter applied directly in plasma measured [K(+)] as being 7.3% lower than the indirect ISE reference method, consistent with the recommended adjustment of +7.5% when indirect ISE methods are used to analyze plasma. The LAQUAtwin ISE meter run in direct mode measured fat-free milk [K(+)] as being 3.6% lower than the indirect ISE reference method, consistent with a herd milk protein percentage of 3.4%. The LAQUAtwin ISE meter accurately measured abomasal fluid [K(+)] compared to the indirect ISE reference method. The LAQUAtwin ISE meter accurately measured urine [K(+)] compared to the indirect ISE reference method, but the median measured value for urine [K(+)] was 83% of the true value measured by inductively coupled plasma-optical emission spectroscopy. We conclude that the CARDY and LAQUAtwin ISE meters are practical, low-cost, rapid, accurate point-of-care instruments suitable for measuring [K(+)] in whole blood, plasma, milk, and abomasal fluid samples from cattle. Ion-selective electrode methodology is not suitable for measuring [K(+)] in bovine urine.

Evaluation of an Electrochemical Point-of-Care Meter for Measuring Glucose Concentration in Blood from Periparturient Dairy Cattle.

BACKGROUND: The Precision Xtra(®) meter is a promising low cost electrochemical point-of-care unit for measuring blood glucose concentration ([gluc]) in cattle blood. The meter uses an algorithm that assumes the intra-erythrocyte [gluc] equals the plasma [gluc] on a molal basis, and that the hematocrit is similar in humans and cattle. OBJECTIVES: The primary objective was to determine the accuracy of the meter for measuring plasma [gluc] in dairy cattle. Secondary objectives were to characterize the influence of hematocrit and sample temperature on the measured value for [gluc]. ANIMALS: A total of 106 periparturient Holstein-Friesian cattle. METHODS: Blood and plasma samples (1,109) were obtained and Deming regression and Bland-Altman plots were used to determine the accuracy of the meter against the reference method (plasma hexokinase assay). Multivariable regression and linear regression were used to determine the effect of hematocrit and sample temperature on the plasma [gluc] measured by the meter. RESULTS: Intra-erythrocyte [gluc] was 18% of plasma [gluc] on a molar basis. Sample temperature had a significant linear effect on plasma [gluc] as measured by the meter for 3/5 plasma samples when measured [gluc] > 160 mg/dL. CONCLUSIONS AND CLINICAL IMPORTANCE: The meter utilizes an algorithm that is optimized for human blood and is inaccurate when applied to bovine blood. Until a cattle-specific algorithm is developed, we recommend using plasma as the analyte instead of blood and calculating plasma [gluc] using the equation: [gluc] = 0.66 × [gluc]p-meter + 15, where [gluc]p-meter is the value reported by the meter. If blood is measured, then we recommend using the equation: [gluc] = 0.90 × [gluc]b-meter + 15.

Efficacy of oral potassium chloride administration in treating lactating dairy cows with experimentally induced hypokalemia, hypochloremia, and alkalemia.

Hypokalemia occurs commonly in lactating dairy cows. The objectives of this study were to determine (1) whether a 24-h oral KCl dose of 0.4 g/kg of body weight (BW) was effective and safe in hypokalemic cattle; (2) whether potassium was best administered as 2 large doses or multiple smaller doses over a 24-h period; and (3) the effect of oral KCl administration on plasma Mg concentration and urine Mg excretion in fasted lactating dairy cattle. Plasma K and Cl concentrations were decreased, and blood pH increased, in 15 lactating Holstein-Friesian cows by administering 2 intramuscular (i.m.) 10-mg injections of isoflupredone acetate 24h apart followed by 2 i.m. injections of furosemide (1mg/kg of BW) 8h apart and by decreasing feed intake. Cows were randomly assigned to 1 of 3 treatment groups with 5 cows/group: untreated control (group C); oral administration of KCl at 0.05 g/kg of BW 8 times at 3-h intervals (group K3); and oral administration of KCl at 0.2g/kg of BW twice at 12-h intervals (group K12). A 24-h KCl dose rate of 0.4 g/kg of BW increased plasma and milk K concentration and plasma Cl concentration, and corrected the metabolic alkalosis and alkalemia, with no clinically significant difference between 2 large doses (group K12) or multiple small doses (group K3) of KCl over 24 h. Oral KCl administration decreased peripheral fat mobilization in cattle with experimentally induced hypokalemia, as measured by changes in plasma nonesterified fatty acid concentration, and slightly augmented the fasting-induced decrease in plasma Mg concentration. Our findings support recommendations for a 24-h oral KCl dose of 0.4 g/kg of BW for treating moderately hypokalemic cattle. Additional Mg may need to be administered to inappetant lactating dairy cattle being treated with oral KCl to minimize K-induced decreases in magnesium absorption.