Direct but Not Indirect Methods Correlate the Percentages of Sperm With Altered Chromatin to the Intensity of Chromatin Damage

Although sperm chromatin damage, understood as damage to DNA or affectations in sperm protamination, has been proposed as a biomarker for sperm quality in both humans and livestock, the low incidence found in some animals raises concerns about its potential value. In this context, as separate methods measure different facets of chromatin damage, their comparison is of vital importance. This work aims at analyzing eight techniques assessing chromatin damage in pig sperm. With this purpose, cryopreserved sperm samples from 16 boars were evaluated through the following assays: TUNEL, TUNEL with decondensation, SCSA, alkaline and neutral sperm chromatin dispersion (SCD) tests, alkaline and neutral Comet assays, and chromomycin A3 test (CMA3). In all cases, the extent of chromatin damage and the percentage of sperm with fragmented DNA were determined. The degree of chromatin damage and the percentage of sperm with fragmented DNA were significantly correlated (p < 0.05) in direct methods (TUNEL, TUNEL with decondensation, and alkaline and neutral Comet) and CMA3, but not in the indirect ones (SCD and SCSA). Percentages of sperm with fragmented DNA determined by alkaline Comet were significantly (p < 0.05) correlated with TUNEL following decondensation and CMA3; those determined by neutral Comet were correlated with the percentage of High DNA Stainability (SCSA); those determined by SCSA were correlated with neutral and alkaline SCD; and those determined by neutral SCD were correlated with alkaline SCD. While, in pigs, percentages of sperm with fragmented DNA are directly related to the extent of chromatin damage when direct methods are used, this is not the case for indirect techniques. Thus, the results obtained herein differ from those reported for humans in which TUNEL, SCSA, alkaline SCD, and alkaline Comet were found to be correlated. These findings may shed some light on the interpretation of these tests and provide some clues for the standardization of chromatin damage methods.

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Diurnal variation of leukocyte counts affects the indirect estimation of reference intervals

LaboratoriumsMedizin ◽

10.1515/labmed-2020-0132 ◽

2021 ◽

Vol 0 (0) ◽

Author(s):

Antje Torge ◽

Rainer Haeckel ◽

Mustafa Özcürümez ◽

Alexander Krebs ◽

Ralf Junker

Keyword(s):

Diurnal Variation ◽

Indirect Method ◽

Venous Blood ◽

Direct Methods ◽

Reference Intervals ◽

Indirect Methods ◽

Indirect Estimation ◽

Late Afternoon ◽

Reference Limits ◽

Leukocyte Counts

Abstract It has been observed that the estimation of reference intervals of leukocytes in whole venous blood leads to higher upper reference limits (uRLs) with indirect methods than has been reported in the literature determined by direct approaches. This phenomenon was reinvestigated with a newer, more advanced indirect method, and could be confirmed. Furthermore, a diurnal variation was observed with lower values during the morning and higher values in the late afternoon and at night. This observation can explain why indirect approaches using samples collected during 24 h lead to higher uRLs than direct methods applied on samples collected presumably in the morning.

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The assessment of sperm DNA fragmentation in the saltwater crocodile (Crocodylus porosus)

Reproduction Fertility and Development ◽

10.1071/rd15300 ◽

2017 ◽

Vol 29 (3) ◽

pp. 630 ◽

Cited By ~ 8

Author(s):

S. D. Johnston ◽

C. López-Fernández ◽

F. Arroyo ◽

J. L. Fernández ◽

J. Gosálvez

Keyword(s):

Dna Damage ◽

Dna Fragmentation ◽

Sperm Chromatin ◽

Sperm Dna Fragmentation ◽

Fragmented Dna ◽

Sperm Dna ◽

Saltwater Crocodile ◽

Crocodylus Porosus ◽

Dispersion Test ◽

Sperm Nuclei

Herein we report a method of assessing DNA fragmentation in the saltwater crocodile using the sperm chromatin dispersion test (SCDt) after including frozen–thawed spermatozoa in a microgel (Halomax; Halotech DNA, Madrid, Spain). Following controlled protein depletion, which included a reducing agent, sperm nuclei with fragmented DNA showed a homogeneous and larger halo of chromatin dispersion with a corresponding reduced nucleoid core compared with sperm with non-fragmented DNA. The presence of DNA damage was confirmed directly by incorporation of modified nucleotides using in situ nick translation (ISNT) and indirectly by studying the correlation of the SCDt with the results of DNA damage visualisation using a two-tailed comet assay (r = 0.90; P = 0.037). Results of the SCDt immediately following thawing and after 5 h incubation at 37°C in order to induce a range of DNA damage revealed individual crocodile differences in both the baseline level of DNA damage and DNA longevity.

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Sperm cryopreservation of lane snapper Lutjanus synagris(Linnaeus, 1758)

Brazilian Journal of Biology ◽

10.1590/1519-6984.20613 ◽

2015 ◽

Vol 75 (3) ◽

pp. 662-669 ◽

Cited By ~ 2

Author(s):

EG Sanches ◽

IR Oliveira ◽

PCS Serralheiro ◽

VR Cerqueira

Keyword(s):

Sperm Motility ◽

Sperm Quality ◽

Equilibration Time ◽

Dilution Ratio ◽

Sperm Cryopreservation ◽

Ph Values ◽

Cryopreserved Sperm ◽

Time Density ◽

Motility Rate

AbstractThis study aims developing and evaluate a protocol of semen cryopreservation of the lane snapper Lutjanus synagris. Firstly, sperm motility rate, motility time, density and spermatocrit were appraised to characterize the sperm quality of the lane snapper. The effect of three extenders with distinct ionic compositions and pH values combined with seven concentrations of cryoprotector dimethylsulfoxide (0; 2.5; 5.0; 7.5; 10.0; 12.5 e 15.0%), five cooling rates (110, 90, 60, 45 e 30°C –min), nine equilibration time (1; 2,5; 5; 10; 15; 20; 25; 30 e 60 minutes) e five dilutions ratio (1:1; 1:3; 1:6; 1:10 e 1:20) on the sperm motility rate and motility time were analyzed. Fertilization test was accomplished to evaluate the viability of the cryopreserved sperm. The higher sperm motility rate and motility time (P<0.05) was achieved by combining extender with pH 8.2 with 10% concentration of dimethylsulfoxide and cooling rate 60°C –min, 1 minute of equilibration time and 1:3 (v/v) dilution ratio. The use of cryopreserved sperm presented fertilization rates >60% validating the present protocol for lane snapper. The cryoconserved sperm of lane snapper is a viable alternative, being possible to maintain appropriate sperm viability.

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Lectin-binding histochemistry of non-decalcified growth plate cartilage: a postembedment method for light microscopy of epon-embedded tissue.

Journal of Histochemistry & Cytochemistry ◽

10.1177/32.6.6373914 ◽

1984 ◽

Vol 32 (6) ◽

pp. 593-607 ◽

Cited By ~ 17

Author(s):

C E Farnum ◽

N J Wilsman

Keyword(s):

Growth Plate ◽

Morphological Analysis ◽

Lectin Binding ◽

Direct Methods ◽

Miniature Swine ◽

Double Labeling ◽

Indirect Methods ◽

Growth Plate Cartilage ◽

Disease States ◽

Intracellular Binding

A postembedment method for the localization of lectin-binding glycoconjugates was developed using Epon-embedded growth plate cartilage from Yucatan miniature swine. By testing a variety of etching, blocking, and incubation procedures, a standard protocol was developed for 1 micron thick sections that allowed visualization of both intracellular and extracellular glycoconjugates with affinity for wheat germ agglutinin and concanavalin A. Both fluorescent and peroxidase techniques were used, and comparisons were made between direct methods and indirect methods using the biotin-avidin bridging system. Differential extracellular lectin binding allowed visualization of interterritorial , territorial, and pericellular matrices. Double labeling experiments showed the precision with which intracellular binding could be localized to specific cytoplasmic compartments, with resolution of binding to the Golgi apparatus, endoplasmic reticulum, and nuclear membrane at the light microscopic level. This method allows the localization of both intracellular and extracellular lectin-binding glycoconjugates using fixation and embedment procedures that are compatible with simultaneous ultrastructural analysis. As such it should have applicability both to the morphological analysis of growth plate organization during normal endochondral ossification, as well as to the diagnostic pathology of matrix abnormalities in disease states of growing cartilage.

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Effects of Urbanization on Stream Ecosystems

Effects of Urbanization on Stream Ecosystems ◽

10.47886/9781888569735.ch5 ◽

2005 ◽

Keyword(s):

Water Quality ◽

Economic Valuation ◽

Consumer Surplus ◽

Direct Methods ◽

Benefit Transfer ◽

Quality Improvements ◽

Indirect Methods ◽

Benefit Estimation ◽

Cape Fear ◽

New Consumer

<em>Abstract.</em>—In this paper, we provide an introduction to water quality benefit estimation for noneconomists. Net water quality benefits are typically measured using the concept of consumer surplus, which is estimated using a number of economic valuation methodologies. These are divided into direct and indirect methods. Direct methods involve questioning survey respondents to determine their consumer surplus. Indirect methods use data from consumer market behavior to estimate economic values. When limited time or funding preclude costly data collection and the development of new consumer surplus estimates, the method of benefit transfer is used to tailor preexisting consumer surplus estimates to fit new policy situations. We provide an example of benefit transfer by estimating the value of water quality improvements for the Cape Fear River in North Carolina. Benefit transfer methods are used with three valuation approaches to estimate the benefits of water quality improvement.

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54 SINGLE LAYER CENTRIFUGATION BEFORE CRYOPRESERVATION IMPROVES BULL SPERM QUALITY

Reproduction Fertility and Development ◽

10.1071/rdv29n1ab54 ◽

2017 ◽

Vol 29 (1) ◽

pp. 134

Author(s):

T. Nongbua ◽

A. Utta ◽

N. Am-In ◽

J. Suwimonteerabutr ◽

A. Johannisson ◽

...

Keyword(s):

Membrane Potential ◽

Mitochondrial Membrane Potential ◽

Chromatin Structure ◽

Mitochondrial Membrane ◽

Linear Mixed Model ◽

Sperm Quality ◽

Single Layer ◽

Sperm Chromatin ◽

Sperm Viability ◽

Bull Sperm

Single layer centrifugation (SLC) with Bovicoll is a technique to enhance sperm quality. The purpose of this study was to investigate the effect of SLC before cryopreservation on bull sperm quality after thawing. Semen was collected from 8 bulls (American Brahman, n = 5 and Sahiwal, n = 3) at the North Eastern Bull Centre (KhonKaen, Thailand). The ejaculate was split: one part was prepared following the standard procedure at the bull centre (n = 88) as control. The other part was used for SLC with Bovicoll-B (Johannisson et al. 2016 Theriogenology 86, 140). The SLC-selected sperm samples were frozen using the same protocol as control (n = 88). After thawing at 37°C for 12 s, motility analysis was performed using the CEROS II® (Hamilton Thorne, Beverly, MA, USA); sperm chromatin structure, mitochondrial membrane potential, and sperm viability were assessed using a FC500 flow cytometer (Beckman Coulter, Brea, CA, USA). Treatment means were compared using the linear mixed model (Proc MIXED, SAS®, 9.3, SAS Institute Inc., Cary, NC, USA). Results are reported as least-squares means ± standard error. The sperm kinematics for SLC samples were higher than controls for progressive motility (26.37 ± 1.59%, 19.56 ± 1.59%), Linearity (LIN) (52.80 ± 0.87%, 44.94 ± 0.87%), Straightness (STR) (83.06% ± 0.59, 76.20 ± 0.59%), beat cross frequency (BCF) (29.25 ± 0.50 Hz, 24.35 ± 0.50 Hz) and wobble (WOB) (61.78 ± 0.63%, 57.40 ± 0.63%) (all P < 0.0001) respectively, whereas SLC-selected samples were lower than controls for slow motility (13.61 ± 0.71%, 15.56 ± 0.71%; P < 0.05), Amplitude of lateral head displacement (ALH) (4.88 ± 0.18 μm, 6.67 ± 0.18 μm), velocity average path, (VAP) (61.17 ± 1.93μ/s, 67.88 ± 1.93μ/s), and curvilinear velocity (VCL) (99.78 ± 3.77 μ/s, 122.91 ± 3.77 μ/s) (all P < 0.0001), respectively. Other parameters of sperm quality were not different between treatments, although there was considerable variation among individual bulls in sperm chromatin structure assay, mitochondrial membrane potential, and sperm viability. These results suggest that SLC can be used before cryopreservation to improve the kinematics of thawed bull sperm samples without adversely affecting other parameters of sperm quality.

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Direct and Indirect Methods for Calculating Thermal Emission From Layered Structures With Nonuniform Temperatures

Journal of Heat Transfer ◽

10.1115/1.4003543 ◽

2011 ◽

Vol 133 (7) ◽

Cited By ~ 29

Author(s):

L. P. Wang ◽

S. Basu ◽

Z. M. Zhang

Keyword(s):

Temperature Distribution ◽

Thermal Emission ◽

Local Density ◽

Direct Method ◽

Direct Methods ◽

Layered Structures ◽

Nonuniform Temperature ◽

Indirect Methods ◽

Nonuniform Temperature Distribution ◽

Direct And Indirect Methods

The determination of emissivity of layered structures is critical in many applications, such as radiation thermometry, microelectronics, radiative cooling, and energy harvesting. Two different approaches, i.e., the “indirect” and “direct” methods, are commonly used for computing the emissivity of an object. For an opaque surface at a uniform temperature, the indirect method involves calculating the spectral directional-hemispherical reflectance to deduce the spectral directional emissivity based on Kirchhoff’s law. On the other hand, a few studies have used a combination of Maxwell’s equations with the fluctuation-dissipation theorem to directly calculate the emissivity. The present study aims at unifying the direct and indirect methods for calculating the far-field thermal emission from layered structures with a nonuniform temperature distribution. Formulations for both methods are given to illustrate the equivalence between the indirect and the direct methods. Thermal emission from an asymmetric Fabry–Pérot resonance cavity with a nonuniform temperature distribution is taken as an example to show how to predict the intensity, emissivity, and the brightness temperature. The local density of states, however, can only be calculated using the direct method.

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Accurate Fundamental Stellar Parameters

Proceedings of the International Astronomical Union ◽

10.1017/s1743921310000591 ◽

2009 ◽

Vol 5 (S265) ◽

pp. 215-216

Author(s):

Hans Bruntt

Keyword(s):

Spectral Type ◽

Effective Temperature ◽

Direct Methods ◽

Space Missions ◽

Indirect Methods ◽

Fundamental Parameters ◽

Spectroscopic Analyses ◽

Solar Type ◽

Fundamental Stellar Parameters

AbstractWe combine results from interferometry, asteroseismology and spectroscopic analyses to determine accurate fundamental parameters (mass, radius and effective temperature) of 10 bright solar-type stars covering the H-R diagram from spectral type F5 to K1. Using “direct” techniques that are only weakly model-dependent we determine the mass, radius and effective temperature. We demonstrate that model-dependent or “indirect” methods can be reliably used even for relatively faint single stars for which direct methods are not applicable. This is important for the characterization of the targets of the CoRoT and Kepler space missions.

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A Comparative Study of Image Deblurring Techniques

Journal of Computational and Theoretical Nanoscience ◽

10.1166/jctn.2020.9282 ◽

2020 ◽

Vol 17 (9) ◽

pp. 4571-4579

Author(s):

Rajbir Singh ◽

Sumit Bansal

Keyword(s):

Comparative Study ◽

Image Restoration ◽

Digital Image ◽

Minimum Mean Square Error ◽

Direct Methods ◽

Structural Similarity ◽

Single Step ◽

Noise Model ◽

Image Deconvolution ◽

Indirect Methods

The method of recovering a true image from degraded one, to analyze that digital image and characteristics with no artifact errors is known as Image Restoration. These techniques are of two types: direct methods and indirect methods. Direct methods are those in which the results of image restoration are produced in one single step. Indirect methods are those in which the results of image restoration are produced after various steps. This method is termed as blind image deconvolution, when the known info is just the blurred digital image and no info about the (Point Spread Function) (PSF) or the degrading model. The target of the procedure is to recover both the latent (un-blurred) image and the blur kernel, simultaneously. In this paper, we presented a comprehensive research of image noise model,de-blurring methods, blur types, and a comparative study of various deblurring methods. We have implemented number experiments to study these methods according to their performance, (Peak Signal to Noise Ratio) PSNR, (structural similarity) SSIM, blur type, and (Minimum Mean Square Error) MMSE.

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