Optimization of CASA-Mot Analysis of Donkey Sperm: Optimum Frame Rate and Values of Kinematic Variables for Different Counting Chamber and Fields

Gacem, Sabrina Ait; Catalán, Jaime; Valverde, Anthony; Soler, Carles; Miró, Jordi

doi:10.3390/ani10111993

Cited by 6 publications

(6 citation statements)

References 50 publications

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“…From the methodological point of view, the results showed in the present work were obtained using the optimal frame rate for both species and stallion breeds ( 10 , 11 ). Therefore, the subsequent subpopulation analyzed can be considered of high confidence.…”

Section: Discussionmentioning

confidence: 90%

“…The settings of the CASA system were those recommended by the manufacturer: particle area >4 and <75 μm 2 ; connectivity: 6; cutoff values were VAP ≥ 10 μm/s for a sperm cell to be considered as motile ( 10 , 11 ). The following sperm motility parameters were determined, sperm velocity: the curvilinear velocity (VCL), straight-line velocity (VSL), and average path velocity (VAP); and sperm movement trajectory: the frequency with which the actual track crossed the smoothed track in either direction [beat-cross frequency (BCF), hertz], and the maximum of the measured width of the head oscillation as the sperm cells swim (the amplitude of lateral head displacement [ALH]).…”

Section: Methodsmentioning

confidence: 99%

“…Improvement in CASA technology and especially the development of new software solutions and new high-performing cameras permitted the authors to analyze better the sperm trajectory (8,(10)(11)(12). Recent studies have suggested specific optimum video capture frame rates for a mathematically welltrack definition in various species (12,13).…”

Section: Introductionmentioning

confidence: 99%

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A New Approach of Sperm Motility Subpopulation Structure in Donkey and Horse

et al. 2021

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This study aimed to characterize the sperm kinematic values with high frames per second, to define the subpopulation structure of a horse and a donkey and compare them. A total of 57 fresh semen ejaculates (26 Spanish and 16 Arabian horse breeds and 10 donkeys) were collected and subsequently analyzed for kinematic parameters using the Computer-aided sperm motility analysis ISAS®v1.2 system and using a Spermtrack® 10-μm depth counting chamber. Sequences were recorded at 250 frames per second, and eight kinematic parameters were automatically evaluated. All kinematic parameters showed significant differences between a donkey and a horse and between horse breeds. All ejaculates evaluated showed excellent semen motility characteristics, with significantly higher values for all kinematic parameters for donkeys compared with horses except for beat-cross frequency. Donkey sperm was faster and linear than the horse. Regarding horse breeds differences, the Spanish horse had higher average path velocity, curvilinear velocity, and beat-cross frequency compared with the Arabian horse. Spanish horse sperm was rapid, but Arab horse was more linear. The principal component analysis showed three sperm subpopulations in the ejaculate of donkeys and horses with a significantly different motility characteristic between them. The dominant subpopulation for both donkey and horse was for rapid, straight, and linear with a high beat sperm (38.2 and 41.7%, respectively), whereas the lowest subpopulation was for the slowest and non-linear sperms. This, plus slight differences in the distribution of these subpopulations between Arabian and Spanish horses, were found. In conclusion, higher frames permitted to have a new interpretation of motile subpopulations with species and breed differences. More so, future works on donkey and horse breed spermatozoa should take into account differences between breeds that may interfere and alter the real analysis performed.

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Section: Discussionmentioning

confidence: 90%

Section: Methodsmentioning

confidence: 99%

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A New Approach of Sperm Motility Subpopulation Structure in Donkey and Horse

et al. 2021

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“…Sperm motility was evaluated by means of a CASA-Mot module of the ISAS v1.2 system (Proiser R + D, Valencia, Spain) equipped with a high-resolution digital camera model MQ003MG-CM (Proiser R + D, Valencia, Spain) capable of capturing up to 100 frames per second (fps). Before doing so, the content of each straw was diluted 1:2 (v:v) in a cooling extender based on skim milk prewarmed to 38 • C [53,54]. Briefly, 2 µL of each sperm sample was placed into a reusable Spermtrack10 chamber (Spk 10; Proiser R + D, Valencia, Spain), prewarmed to 38 • C. A minimum of 500 spermatozoa were counted per analysis under a 10× negative phase contrast microscope model UOP200i (Proiser R + D, Valencia, Spain).…”

Section: Sperm Motility Analysismentioning

confidence: 99%

Addition of Reduced Glutathione (GSH) to Freezing Medium Reduces Intracellular ROS Levels in Donkey Sperm

Yánez-Ortiz

Catalán

Delgado-Bermúdez

et al. 2021

Veterinary Sciences

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In donkeys, the use of frozen-thawed sperm for artificial insemination (AI) leads to low fertility rates. Furthermore, donkey sperm produce a large amount of reactive oxygen species (ROS), and post-AI inflammation induces the formation of neutrophil extracellular traps (NETosis), which further generates many more ROS. These high ROS levels may induce lipid peroxidation in the sperm plasma membrane, thus affecting its integrity. Enzymatic and non-enzymatic antioxidants, mainly found in the seminal plasma (SP), are responsible for maintaining the redox balance. However, this fluid is removed prior to cryopreservation, thereby exposing sperm cells to further oxidative stress. The exogenous addition of antioxidants to the freezing medium can reduce the detrimental effects caused by ROS generation. Therefore, the aim of this study was to evaluate how the addition of different reduced glutathione (GSH) concentrations (control, 2 mM, 4 mM, 6 mM, 8 mM, and 10 mM) to fresh sperm affect their cryotolerance. Total and progressive motility, kinematic parameters and motile sperm subpopulations were significantly (p < 0.05) different from the control in treatments containing 8 mM and 10 mM GSH, but not at lower concentrations. Plasma and acrosome membrane integrity, mitochondrial membrane potential (MMP) and intracellular superoxide levels (O2−) were not affected (p > 0.05) by any GSH concentration. Interestingly, however, the addition of 8 mM or 10 mM GSH reduced (p < 0.05) the percentages of viable sperm with high overall ROS levels compared to the control. In conclusion, frozen-thawed donkey sperm are able to tolerate high GSH concentrations, which differs from what has been observed in other species. This antioxidant capacity suggests that ROS could be important during post-AI and that the impact of using exogenous antioxidants like GSH to improve the sperm resilience to freeze-thawing is limited in this species.

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“…En el análisis cinemático, las trayectorias de las células pueden variar según la configuración de fotogramas por segundo (frame per second, fps) (Lu et al, 2014). De esta manera, las diferentes configuraciones de los sistemas CASA-Mot pueden condicionar los análisis cinéticos, ya sea por un inadecuado reconocimiento de la distancia en las trayectorias de las células espermáticas analizadas (Amann y Waberski, 2014) o por limitaciones técnicas de las cámaras digitales (Boryshpolets et al, 2013;Bompart et al, 2018;Caldeira et al, 2019;Gacem et al, 2020b). Con base en estos antecedentes, el objetivo del presente estudio fue determinar el efecto de la tasa de fotogramas sobre diferentes cámaras de recuento espermático para semen de verraco analizado con un sistema CASA-Mot, así como analizar la distribución de los espermatozoides a lo largo de los campos longitudinales de la cámara de recuento.…”

Section: Introductionunclassified

Condiciones óptimas del análisis CASA-Mot del semen de verraco: efecto de la tasa de fotogramas para diferentes cámaras y campos de recuento espermático

Barquero

Sevilla

Calderón-Calderón

et al. 2021

Rev. investig. vet. Perú

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El objetivo del estudio fue determinar el efecto de la tasa de fotogramas sobre diferentes profundidades de cámara de recuento espermático en semen de verraco y analizar la distribución de los espermatozoides en los campos longitudinales de la cámara de conteo. Se colectó una muestra de semen a 10 verracos Pietrain de 2-3 años. Se determinaron ocho parámetros de cinética espermática mediante el sistema CASA (Computer-assisted semen analysis)-Mot (ISAS®v1). Se utilizaron cámaras ISAS®D4C con profundidades de 10, 16 y 20 μm a una frecuencia de captura de imagen de 25, 40 y 60 fotogramas por segundo (fps). La profundidad de cámara afectó las variables de cinética espermática, obteniéndose valores más altos a 20 µm. La tasa de fotogramas (FR) presentó una relación positiva con los parámetros de cinética obteniéndose valores más altos a 60 fps, excepto para la amplitud del desplazamiento lateral de la cabeza (ALH) que presentó un comportamiento inverso. La variable más sensible al cambio en FR fue la velocidad curvilínea (VCL), donde el mejor nivel se observó con 80 fps. Se observó una disminución progresiva en los patrones cinemáticos conforme los espermatozoides avanzaban por los campos de las cámaras. Se concluye que el uso de diferentes cámaras de recuento y el incremento en el número total de fps genera cambios significativos en la estimación de parámetros de cinética espermática en el verraco.

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Optimization of CASA-Mot Analysis of Donkey Sperm: Optimum Frame Rate and Values of Kinematic Variables for Different Counting Chamber and Fields

Cited by 6 publications

References 50 publications

A New Approach of Sperm Motility Subpopulation Structure in Donkey and Horse

A New Approach of Sperm Motility Subpopulation Structure in Donkey and Horse

Addition of Reduced Glutathione (GSH) to Freezing Medium Reduces Intracellular ROS Levels in Donkey Sperm

Condiciones óptimas del análisis CASA-Mot del semen de verraco: efecto de la tasa de fotogramas para diferentes cámaras y campos de recuento espermático

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