Andrology laboratory review: Evaluation of sperm concentration

Brito, Leonardo F.C.; Althouse, Gary C.; Aurich, Christine; Chenoweth, Peter J.; Eilts, B.E.; Love, Charles C.; Luvoni, G.C.; Mitchell, Jere R.; Peter, Augustine T.; Pugh, David G.; Waberski, Dagmar

doi:10.1016/j.theriogenology.2016.01.002

Cited by 44 publications

(73 citation statements)

References 49 publications

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“…The commonly used methods for quantifying spermatozoa in canine semen include microscopy, to count spermatozoa in the chambers of a measurable volume; measuring of semen optical density using spectrophotometers; CASA; and fluorescent counting (Brito et al, ). Spermatozoa may be examined and counted using counting chambers, such as the 100‐µm‐deep haemocytometer.…”

Section: Introductionmentioning

confidence: 99%

Development of a fluorescent computer‐assisted spermatozoal quantification method and a comparison of results for manual counting with a haemocytometer and computer‐assisted semen analysis in dogs

Watts¹

2019

Reprod Domestic Animals

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The aim of this study was to develop and validate a novel, computer‐assisted spermatozoal quantification (CASQ) method of determining spermatozoal concentration in canine semen. In Experiment A, the spermatozoal concentration was measured (n = 28) with a haemocytometer using light microscopy, CASQ and computer‐assisted semen analysis (CASA; MMC sperm), following three independent dilutions. The limits of agreement between the haemocytometer and CASQ were −13.1% to 13.8% and −27.0% to 28.6% between the haemocytometer and CASA. The precision CVs (limits of agreement) were 5.7% (−7.8% to 8.9%) for the haemocytometer, 6.2% (−8.8% to 12.3%) for CASQ and 10.8% (−16.0% to 19.5%) for CASA. In Experiment B, spermatozoa were manually counted (n = 42) with the haemocytometer under fluorescent illumination using the CASQ sample. The limits of agreement between the CASQ and the haemocytometer were satisfactory (−4.6% to 4.6%) and the precision CVs (limits of agreement) were 6.2% (−9.0% to 11.4%) for the haemocytometer and 4.4% (−5.8% to 8.6%) for CASQ. The CASQ method was then clinically applied to compare the haemocytometer (light and fluorescent methods) with CASQ and CASA. Outlying data were removed. These studies demonstrated that CASQ was reliable and that the MMC sperm CASA was unreliable as methods for determining spermatozoal concentration in canine semen. Computer‐assisted spermatozoal quantification was also determined to be more precise than manual counting with the haemocytometer. Using the clinical protocol, the agreement between the haemocytometer and CASQ method was acceptable, but it was worse than in the experiments where duplicate samples and a larger volume of semen were analysed. The CASQ method may be a useful method to measure the membrane status of canine spermatozoa; however, further investigation is required. Counting spermatozoa using fluorescent microscopy and the haemocytometer may improve the efficiency of counting and the accuracy of the method.

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Section: Introductionmentioning

confidence: 99%

Development of a fluorescent computer‐assisted spermatozoal quantification method and a comparison of results for manual counting with a haemocytometer and computer‐assisted semen analysis in dogs

Watts¹

2019

Reprod Domestic Animals

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“…Despite our dependence on direct methods, spectrophotometry provides more accurate results when the equipment is properly calibrated and used correctly. It is therefore the most common method used to evaluate sperm concentration in animal semen processing centers (ANZAR et al, 2009;BRITO et al, 2016). Because it is a standardized method, it becomes the method of choice in research involving semen biotechnology (Purdy and Graham, 2004).…”

Section: Resultsmentioning

confidence: 99%

“…In addition to sperm cells, the samples may contain other cell types and soluble organic and inorganic compounds, which may alter the absorbance values obtained in the apparatus. The period between dilution and evaluation should also be standardized because the absorbance may change with time (BRITO et al, 2016). Another factor is the seminal characteristics between different species, as observed by Murgas et al (2010) and Vianna et al (2004) when comparing both methods.…”

Section: Hematocytometric Methodsmentioning

confidence: 99%

Comparative study between the hemocytometric and spectrophotometric methods for measuring the sperm concentration of young Nelore bulls

Cardeal

Alberton

Boscarato

et al. 2017

SCA

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Determination of sperm concentration using the Neubauer chamber (hemocytometric method) is a direct method for counting cells and also the most reliable. However, the process is time-consuming rendering it the least practical method when large numbers of ejaculates need to be processed. The spectrophotometer measures sperm concentrations as optical density and its main advantages are practicality and speed. This paper aimed to compare the results between evaluators using the hemocytometric method and the spectrophotometer for measuring sperm concentrations in young Nelore bulls. In total, 73 ejaculations from 20 young Nelore bulls were collected by electroejaculation. After soundness examination, 10 µL of the semen was diluted in 2 mL saline formaldehyde for measuring the sperm concentration per mL by the hemocytometric method (measured by three different evaluators) and the spectrophotometer method at 550 nm wavelength. No differences were detected in the results of sperm concentration measurements per mL among the evaluators using the hemocytometric method and the spectrophotometer (P > 0.05). The intraclass correlation was high (0.9), showing high replicability among the evaluator measurements. These results demonstrate that measurements performed using the spectrophotometer are reliable and can substitute the hemocytometric method in future for performing sperm concentration measurements in young Nelore bulls, thus improving and standardizing the techniques used in andrology laboratories. Key words: Absorbance. Andrology. Bos indicus. Neubauer chamber. Semen. ResumoA determinação da concentração espermática pela câmara de Neubauer (método hematocitométrico) é um método direto para contar células, e também é o mais confiável. Entretanto, o processo é demorado, o que o torna um método menos prático quando a quantidade de ejaculados a ser processado é muito grande. O espectrofotômetro é um dispositivo que mede a concentração de espermatozoides através

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“…The sperm concentration can be variable according to the age, season, frequenccy of semen collection or natural covering, different methods of semen collection, sexual stimulation, testicular size and sperm production capacity per testicular mass (Brito et al 2016). For this reason our value (317.9 ± 26 ×10 6 spermatozoa/mL) was less than reported by Morais et al (1994) (444.1±182,7 ×10 6 sperm/mL), but significantly higher when compared to the values found by Rota et al (2010), Canisso et al (2010) and Gloria et al (2011), 189.5±22,9×10 6 spermatozoa/mL; 187.7±89,8×10 6 spermatozoa/mL; 253.0 ±91,2 ×10 6 ; and 266 ± 67.2 ×10 6 spermatozoa/mL, respectively.…”

Section: Discussionmentioning

confidence: 99%

“…This evaluation was performed under an optical microscope in 200 ×magnification (CBRA 2013, Brito et al 2016). …”

Section: Raw Semenmentioning

confidence: 99%

Evaluation of hypo-osmotic swelling test and supra vital staining technique as indicators for donkey semen freezability

Oliveira¹,

Teixeira²,

Pignataro³

et al. 2017

PHK

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Summary: Reproductive biotechnologies such as cooling and freezing semen are employed in order to obtain genetic enhancement in domestic animal breeds. However, cryopreservation of donkey semen, as in several other domestic mammalian species, has not yet reached a standard procedure that provides satisfactory and repeatable results, as in bulls. Despite there are over 43 million donkeys worldwide, there are only few studies about donkey semen preservation. The aim of this study was to evaluate the predictive potential of hypo-osmotic test and the supra vital staining in raw semen to be used as indicators for donkey semen freezability. Ejaculates were collected twice per week from 10 fertile donkey stallions. Volume, appearance (color and density), concentration, total motility, morphology, sperm viability (supra vital staining -eosin-nigrosin-EN) and plasma membrane integrity (hypo-osmotic swelling test -HOST) were evaluated in raw semen. After dilution the samples were centrifugated and sperm pellets were resuspended in a freezing extender to a concentration of 50 ×10 6 cells/mL. Aliquots were packed into 0.5 mL straws and placed in the refrigerator (5°C) for 20 min. Subsequently these straws were kept above liquid nitrogen for 20 min, then plunged into nitrogen and stored in a holding tank. Post-thaw analyzes consisted in computerized analysis of sperm movement characteristics, sperm viability (EN), plasma membrane integrity (HOST and fluorescent probe) and acrosome membrane integrity using fluorescent probe. High correlation was found between EN (0.93) and HOST (0.69) in raw semen when compared with total motility after frozen-thawed semen. Low correlation was found between EN and HOST when compared with plasma and acrosomal membrane integrity in post-thawed with fluorescent probes. Since sperm motility is one of the key parameters to evaluate the quality of frozen semen, hypo-osmotic test and supra vital staining can be considered good predictive indicators in raw semen for donkey semen freezability.Keywords: Equus Asinus, cryopreservation, eosin-nigrosin, plasma membrane integrity, semen, reproduction Citation: Arruda de Oliveira R., Batista Silva Teixeira A., Almeida Pignataro T., Freitas M. L., Castro Alves Teixeira H., de Oliveira Carvalho J., Alves Nune Dode M., Pivato I., Budik S. (2017) Evaluation of Hypo-osmotic swelling test and supra vital staining technique as indicators for donkey semen freezability. Pferdeheilkunde 33,[159][160][161][162][163][164]

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Andrology laboratory review: Evaluation of sperm concentration

Cited by 44 publications

References 49 publications

Development of a fluorescent computer‐assisted spermatozoal quantification method and a comparison of results for manual counting with a haemocytometer and computer‐assisted semen analysis in dogs

Development of a fluorescent computer‐assisted spermatozoal quantification method and a comparison of results for manual counting with a haemocytometer and computer‐assisted semen analysis in dogs

Comparative study between the hemocytometric and spectrophotometric methods for measuring the sperm concentration of young Nelore bulls

Evaluation of hypo-osmotic swelling test and supra vital staining technique as indicators for donkey semen freezability

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