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

Alfadel F, Yimer N, Hiew MWH

Trop Anim Health Prod, 2023 Feb 10;55(2):76.
PMID: 36764981 DOI: 10.1007/s11250-023-03490-x

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.
Evaluation of the analytical performance of a portable ion-selective electrode meter for measuring whole-blood, plasma, milk, abomasal-fluid, and urine sodium concentrations in cattle

Megahed AA, Hiew M, Grünberg W, Trefz FM, Constable PD

J Dairy Sci, 2019 Aug;102(8):7435-7444.
PMID: 31202658 DOI: 10.3168/jds.2018-16198

A portable ion-selective electrode (ISE) meter (LAQUAtwin B-722; Horiba Instruments Inc., Irvine, CA) is available for measuring the sodium ion concentration ([Na]) in biological fluids. The objective of this study was to characterize the analytical performance of the ISE meter in measuring [Na] in whole-blood, plasma, milk, abomasal fluid, and urine samples from cattle. Method comparison studies were performed using whole-blood and plasma samples from 106 sick calves and 11 sick cows admitted to a veterinary teaching hospital, 80 milk and 206 urine samples from 16 lactating Holstein-Friesian cows with experimentally induced free water, electrolyte, and acid-base imbalances, and 67 abomasal fluid samples from 7 healthy male Holstein-Friesian calves fed fresh milk with or without an oral electrolyte solution. Deming regression and Bland-Altman plots were used to determine the accuracy of the meter against reference methods. The meter used in direct mode on undiluted samples measured whole-blood [Na] 9.7 mmol/L (7.3%) lower than a direct ISE reference method and plasma [Na] 16.7 mmol/L (12.7%) lower than an indirect ISE reference method. The meter run in direct mode measured milk [Na] 3.1 mmol/L lower and abomasal fluid [Na] 9.0% lower than indirect ISE reference methods. The meter run in indirect mode on diluted samples accurately measured urine [Na] compared with an indirect ISE reference method. We conclude that, after adjustment for the bias determined from Bland-Altman plots, the LAQUAtwin ISE meter provides a clinically useful and low-cost cow-side instrument for measuring [Na] in whole blood, plasma, milk, and abomasal fluid.
Measurement of urine pH and net acid excretion and their association with urine calcium excretion in periparturient dairy cows

Constable PD, Megahed AA, Hiew MWH

J Dairy Sci, 2019 Dec;102(12):11370-11383.
PMID: 31548071 DOI: 10.3168/jds.2019-16805

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

Megahed AA, Hiew MWH, Ragland D, Constable PD

J Dairy Sci, 2019 Jun;102(6):5550-5565.
PMID: 30954258 DOI: 10.3168/jds.2018-15063

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 progesterone and blood and plasma glucose concentrations in predicting parturition in Holstein cows

Hiew MWH, Megahed AA, Horstman LA, Constable PD

J Dairy Sci, 2020 Jun;103(6):5575-5590.
PMID: 32307156 DOI: 10.3168/jds.2019-17800

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

Megahed AA, Hiew MWH, Constable PD

J. Vet. Intern. Med., 2018 Mar;32(2):846-852.
PMID: 29424465 DOI: 10.1111/jvim.15049

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
Fulltext A preliminary study on the effects of E-Z Mixin® and EquiPlus® extenders supplemented with Edible Bird's Nest on the quality of chilled Arabian stallion semen

Al-Khaldi K, Yimer N, Al-Bulushi S, Haron AW, Hiew M, Babji AS

Anim Reprod, 2021 Jun 21;18(2):e20200027.
PMID: 34221142 DOI: 10.1590/1984-3143-AR2020-0027

The aim of this study was to evaluate the effects of adding different concentrations of edible bird's nest (EBN) which is secreted by swiftlet birds (Aerodramus fuciphagus), into EquiPlus® and E-Z Mixin® extenders on the quality of chilled Arabian stallion semen at various storage times (0, 24 and 48 h). Ten ejaculates were collected from five stallions, and diluted using the two extenders containing 0% (control), 0.12%, 0.24% and 0.24% of EBN + seminal plasma (SP). All the diluted semen samples were then cooled and stored at 5 °C, and examined at 0, 24 and 48 h. Sperm kinetic parameters were assessed using computer assisted sperm analysis (CASA) and viability were assessed using Hoechst33342/PI stain. In both extenders, total motility (TM) and progressive motility (PM) were significantly higher at 0.12% and 0.24% compared to 0.24% + SP at 24 and 48 h. At 0.12%, E-Z mixin® treated semen had significantly higher TM and PM than EquiPlus® at 24 and 48 h. At 0.12% and 0.24%, average path velocity (VAP), straight-line velocity (VSL) and curvilinear velocity (VCL) were significantly higher in E-Z mixin® treated semen compared to EquiPlus® at 24 and 48 h. Comparisons between the two extender types at different concentrations of EBN showed no significant difference in lateral head amplitude (ALH), linearity (LIN), straightness (STR), beat cross frequency (BCF) and viability, irrespective of the storage time. The percentage of viable was significantly higher in E-Z mixin® than EquiPlus® at 0 and 48 h in control and 0.12%. Supplementation of the E-Z mixin® extender with 0.12% and 0.24% EBN concentrations in the absence of SP provided better CASA parameters such as TM, PM, VAP, VSL, and VCL at 24 and 48 h storage time. In conclusion, the results of this study indicated that chilled semen from Arabian stallion that was extended using E-Z mixin® and supplemented with 0.12% and 0.24% EBN concentrations performed better and yielded superior results in sperm kinetic parameters and % viable compared to EquiPlus® at 24 and 48 h storage time.
Effect of indomethacin on embryo implantation and histomorphology of uterus, ovary, kidney, and liver of rats

Amir M, Yimer N, Hiew M, Yusoff SM, Hussen B, Quddus A

Saudi J Biol Sci, 2023 Dec;30(12):103837.
PMID: 37964780 DOI: 10.1016/j.sjbs.2023.103837

BACKGROUND: This study aimed to determine the effects of Indomethacin (IMC) treatment on embryo implantation and histomorphology of uterus, ovary, and other vital organs and its effective dosage in establishing embryo implantation dysfunction model in Sprague-Dawley (SD) rats.
MATERIALS AND METHODS: The experiments were performed on 24 (6 × 4 groups) adult female SD rats aged 12 weeks old. G1 was the control group and received a normal diet with normal saline. However, on pregnancy days 3 (Pd3) and 4 (Pd4), G2, G3, and G4 were given normal saline and subcutaneously administered IMC twice daily at different doses of 4.33, 4.66 and 5.00 mg/kg body weight, respectively. The rats were euthanized on day 8 of pregnancy (Pd8). The uterus was excised and examined for signs of pregnancy, followed by tissue samples from liver, kidney, and ovary (for histomorphological examination using haematoxylin and eosin stain).
RESULTS: All IMC treatment doses disrupted the implantation process and caused a significant reduction in embryo development. Analysis for histopathological changes revealed that IMC doses above 4.33 mg/kg body weight caused more adverse reproductive health effects in rats. Vasoconstriction and micro vascularization were detected in the liver, while degenerative Bowman's capsules and inflammatory cells were observed in kidney sections from IMC-treated rats.
CONCLUSION: IMC therapy interfered with implantation and embryo development in rats, resulting in significant uterine vasoconstriction and atrophy, 4.33 mg/kg bwt dose appeared to be optimum to establish embryo implantation dysfunction in SD rats.