Advances in flow cytometry in basic and applied equine andrology

El objetivo de esta revisión es presentar las sondas actuales disponibles que evalúan diferentes compartimentos y funciones de los espermatozoides de los sementales, incluidos ensayos para investigar la funcionalidad de las membranas, el núcleo y las mitocondrias, y estudiar la señalización celular en esta célula en particular. También se presentarán los nuevos protocolos multiparamétricos para la evaluación de espermatozoides de sementales, recientemente desarrollados en el laboratorio de los autores. También se discutirá la aplicabilidad clínica potencial de las pruebas de diagnóstico basadas en citometría de flujo.The aim of this review is to present the current probes available that assess different compartments and functions of stallion spermatozoa, including assays to investigate the functionality of the membranes, nucleus and mitochondria, and to study cell signaling in this particular cell. New multi-parametric protocols for the assessment of stallion sperm, recently developed in the laboratory of the authors, will also be presented. The potential clinical applicability of diagnostic tests based on flow cytometry will also be discussed.Trabajo financiado por: Ministerio de Economía y Competitividad y Fondos FEDER. Ayuda AGL2013-43211-R Junta de Extremadura y Fondos FEDER. Ayuda GR 15029 Ministerio de Educación, Cultura y Deporte. Beca FPU13/03991, para Patricia Martín Muñoz Ministerio de Economía y Competitividad. Beca Juan de la Cierva” IJCI-2014-21671, Cristina Ortega FerrusolapeerReviewe

Autophagy and Apoptosis Have a Role in the Survival or Death of Stallion Spermatozoa during Conservation in Refrigeration

Apoptosis has been recognized as a cause of sperm death during cryopreservation and a cause of infertility in humans, however there is no data on its role in sperm death during conservation in refrigeration; autophagy has not been described to date in mature sperm. We investigated the role of apoptosis and autophagy during cooled storage of stallion spermatozoa. Samples from seven stallions were split; half of the ejaculate was processed by single layer centrifugation, while the other half was extended unprocessed, and stored at 5°C for five days. During the time of storage, sperm motility (CASA, daily) and membrane integrity (flow cytometry, daily) were evaluated. Apoptosis was evaluated on days 1, 3 and 5 (active caspase 3, increase in membrane permeability, phosphatidylserine translocation and mitochondrial membrane potential) using flow cytometry. Furthermore, LC3B processing was investigated by western blotting at the beginning and at the end of the period of storage. The decrease in sperm quality over the period of storage was to a large extent due to apoptosis; single layer centrifugation selected non-apoptotic spermatozoa, but there were no differences in sperm motility between selected and unselected sperm. A high percentage of spermatozoa showed active caspase 3 upon ejaculation, and during the period of storage there was an increase of apoptotic spermatozoa but no changes in the percentage of live sperm, revealed by the SYBR-14/PI assay, were observed. LC3B was differentially processed in sperm after single layer centrifugation compared with native sperm. In processed sperm more LC3B-II was present than in non-processed samples; furthermore, in non-processed sperm there was an increase in LC3B-II after five days of cooled storage. These results indicate that apoptosis plays a major role in the sperm death during storage in refrigeration and that autophagy plays a role in the survival of spermatozoa representing a new pro-survival mechanism in spermatozoa not previously described

Caspase 3 Activity and Lipoperoxidative Status in Raw Semen Predict the Outcome of Cryopreservation of Stallion Spermatozoa

[EN] Stallion-to-stallion variability in the quality of cryopreserved ejaculates postthaw affects the commercial acceptability of frozen semen and thus is a major constraint for the equine industry. In recent years, the molecular mechanisms associated with sperm damage during cryopreservation have become better understood. Identification of the freezability of the ejaculates before the freezing process is initiated will have a major impact on the equine industry. We studied three markers of oxidative stress in sperm, including 8-iso-PGF2alpha, 8-OH guanosine, and 4-hydroxynonenal (4-HNE); the presence of active caspase 3; and their changes after sperm cryopreservation. Although 4- HNE levels increased after cryopreservation (from 7% to 33%, P < 0.001), 8OH-guanosine and 8-ISO-PGF2alpha levels decreased after cryopreservation (from 130 to 35 arbitrary fluorescence units, P < 0.01, and from 1280 to 1233, P < 0.01, respectively). Postthaw sperm quality was classified as poor, average, or good using the 25th and 75th percentiles of all assays of sperm quality studied (motility, velocity, membrane functionality, and thiol content) as thresholds. Using these values, a sperm postthaw quality index was proposed. Receiver operating characteristic curves and the Youden J statistic were used to investigate the value of the measured parameters in fresh sperm as predictors of potential freezability. Using these techniques, we identified markers of bad freezers (percentages of caspase 3-positive dead sperm [area under the curve (AUC)= 0.820, P < 0.05] and percentages of caspase 3- and 4-HNEpositive sperm [AUC = 0.872, P < 0.05]) and good freezers (percentages of caspase 3-negative live sperm [AUC = 0.815, P < 0.05], percentages of live sperm with high thiol content [AUC = 0.907, P < 0.01], and percentages of 8-ISO-PGF2alphapositive sperm [AUC = 0.900, P < 0.01]. Moreover, we described for the first time the presence of 8-ISO-PGF2alpha in stallion spermatozoa and revealed the importance of considering different markers of oxidative stress.S

Dataset of the sperm proteome of stallions with different motility

This paper provides a detailed set of data on how the stal-lion sperm proteome differs among stallions with different sperm motilities, although within normal ranges. Findings distinguish proteins that may help to identify stallions of su-perior sperm motility. Sperm proteins were analyzed using a UHPLC/MS/MS system comprising of an Agilent 1290 in-finity series UHPLC coupled to an Agilent 6550 Q-TOF mass spectrometer (Agilent Technologies, Santa Clara, CA, USA). These data can be used to disclose potential targets to iden-tify good sperm samples and to study specific pathways in-volved in the regulation of sperm motility. This data article is linked to the paper "Proteins involved in mitochondrial metabolic functions and fertilization predominate in stallions with better motility Journal of Proteomics 247:104335 doi: 10.1016/j.jprot.2021.104335".(c) 2022 The Author(s). Published by Elsevier Inc.This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/

Improving sperm banking efficiency in endangered species through the use of a sperm selection method in brown bear (Ursus arctos) thawed sperm

[EN] Background: Sperm selection methods such as Single Layer Centrifugation (SLC) have been demonstrated to be a useful tool to improve the quality of sperm samples and therefore to increase the efficiency of other artificial reproductive techniques in several species. This procedure could help to improve the quality of genetic resource banks, which is essential for endangered species. In contrast, these sperm selection methods are optimized and focused on farm animals, where the recovery task is not as important as in endangered species because of their higher sperm availability. The aim of this study was to evaluate two centrifugation methods (300 x g/20 min and 600 x g/10 min) and three concentrations of SLC media (Androcoll-Bear -80, 65 and 50%) to optimise the procedure in order to recover as many sperm with the highest quality as possible. Sperm morphology could be important in the hydrodynamic relationship between the cell and centrifugation medium and thus the effect of sperm head morphometry on sperm yield and its hydrodynamic relationship were studied. Results: The samples selected with Androcoll-Bear 65% showed a very good yield (53.1 ± 2.9) although the yield from Androcoll-Bear 80% was lower (19.3 ± 3.3). The latter showed higher values of motility than the control immediately after post-thawing selection. However, both concentrations of colloid (65 and 80%) showed higher values of viable sperm and viable sperm with intact acrosome than the control. After an incubation of 2 h at 37 °C, the samples from Androcoll-Bear 80% had higher kinematics and proportion of viable sperm with intact acrosome. In the morphometric analysis, the sperm selected by the Androcoll-Bear 80% showed a head with a bigger area which was more elongated than the sperm from other treatments. Conclusions: We conclude that sperm selection with Androcoll-Bear at either 65% or 80% is a suitable technique that allows a sperm population with better quality than the initial sample to be obtained. We recommend the use of Androcoll-Bear 65% since the yield is better than Androcoll-Bear 80%. Our findings pave the way for further research on application of sperm selection techniques to sperm banking in the brown bear.SIThis work was supported in part by MINECO (CGL2013–48255-R) and Cantur S.A. Luis Anel-Lopez was supported by MINECO (CGL2013–48255-R). C ortega-Ferrusola is supported by a postdoctoral grant from “Ministerio de Economía y Competitividad “Juan de la Cierva” IJCI-2014-21671

Analysis of seminal plasma from brown bear (Ursus arctos) during the breeding season: Its relationship with testosterone levels

El plasma seminal (SP) juega un papel importante en la motilidad, la viabilidad y el mantenimiento de la capacidad de fertilización de los espermatozoides de mamíferos. Este estudio es el primero de los componentes SP del oso pardo (Ursus arctos) y tiene dos objetivos principales: 1) definir la composición SP en la eyaculación del oso y 2) identificar variaciones en la composición SP en relación con los niveles altos y bajos de testosterona en Suero durante la época de reproducción. Se obtuvieron cuarenta y ocho muestras de esperma de 30 osos pardos machos sexualmente maduros (Ursus arctos) mediante electroeyaculación, y se evaluaron sus niveles séricos de testosterona para clasificar a los animales en 2 grupos (niveles altos y bajos de testosterona, umbral 5 ng / dl). Se evaluaron las composiciones bioquímicas y de proteínas de las muestras de SP y se analizó la motilidad de los espermatozoides. Encontramos que la lactato deshidrogenasa fue significativamente mayor en las muestras con bajo contenido de testosterona en suero, mientras que las concentraciones de lipasa y Mg + fueron significativamente mayores en las muestras con alto contenido de testosterona en suero. En contraste, la motilidad de los espermatozoides no difirió significativamente (P> 0.05) entre los grupos de niveles de testosterona (motilidad total: 74.42.8% en el grupo de nivel alto vs. 77.1 ± 4.7% en el grupo de nivel bajo). Se construyó un modelo digital de referencia ya que no hay información para esta especie silvestre. Para ello, todas las imágenes de gel se agregaron en una imagen binaria multidimensional y se identificaron treinta y tres puntos como los puntos más repetidos. Se realizó un análisis de estas proteínas por equivalencia cualitativa (punto isoeléctrico y peso molecular) con datos publicados para un toro. La composición de la proteína SP se comparó entre osos con testosterona sérica alta y baja, y tres proteínas (aglutinante de esperma y dos enzimas no identificadas en el toro de referencia) mostraron diferencias cuantitativas significativas (P 0.05) between the testosterone level groups (total motility: 74.42.8% in the high-level group vs. 77.1±4.7% in the low-level group). A reference digital model was constructed since there is no information for this wild species. To do this, all gel images were added in a binary multidimensional image and thirty-three spots were identified as the most-repeated spots. An analysis of these proteins was done by qualitative equivalency (isoelectric point and molecular weight) with published data for a bull. SP protein composition was compared between bears with high and low serum testosterone, and three proteins (binder of sperm and two enzymes not identified in the reference bull) showed significant (P<0.05) quantitative differences. We conclude that male bears with high or low serum testosterone levels differs only in some properties of their SP, differences in enzyme LDIP2, energy source LACT2, one protein (similar to BSP1) and Mg ion were identified between these two groups. These data may inform the application of SP to improve bear semen extenders.• Ministerio de Economía y Competitividad. Subvencion parcial Proyecto CGL2013-48255-R • Cantur S.A. Subvencion parcial • Ministerio de Economía y Competitividad. Proyecto CGL2013-48255-R, para Luis Anel López • Ministerio de Economía y Competitividad. Beca Juan de la Cierva IJCI-2014-21671, para Cristina Ortega FerrusolapeerReviewe

Pulse Doppler ultrasound as a tool for the diagnosis of chronic testicular dysfunction in stallions

[EN] Testicular function is particularly susceptible to vascular insult, resulting in a negative impact on sperm production and quality of the ejaculate. A prompt diagnosis of testicular dysfunction enables implementation of appropriate treatment, hence improving fertility forecasts for stallions. The present research aims to: (1) assess if Doppler ultrasonography is a good tool to diagnose stallions with testicular dysfunction; (2) to study the relationship between Doppler parameters of the testicular artery and those of sperm quality assessed by flow cytometry and (3) to establish cut off values to differentiate fertile stallions from those with pathologies causing testicular dysfunction. A total of 10 stallions (n: 7 healthy stallions and n: 3 sub-fertile stallions) were used in this study. Two ejaculates per stallion were collected and preserved at 5ÊC in a commercial extender. The semen was evaluated at T0, T24 and T48h by flow cytometry. Integrity and viability of sperm (YoPro®-1/EthD-1), mitochondrial activity (MitoTracker® Deep Red FM) and the DNA fragmentation index (Sperm Chromatin Structure Assay) were assessed. Doppler parameters were measured at three different locations on the testicular artery (Supratesticular artery (SA); Capsular artery (CA) and Intratesticular artery (IA)). The Doppler parameters calculated were: Resistive Index (RI), Pulsatility Index (PI), Peak Systolic Velocity (PSV), End Diastolic Velocity (EDV), Time Average Maximum Velocity (TAMV), Total Arterial Blood Flow (TABF) and TABF rate. The capsular artery was the most reliable location to carry out spectral Doppler assessment, since blood flow parameters of this artery were most closely correlated with sperm quality parameters. Significant differences in all the Doppler parameters studied were observed between fertile and subfertile stallions (p0.05). The principal components analysis assay determined that fertile stallions are characterized by high EDV, TAMV, TABF and TABF rate values (high vascular perfusion). In contrast, subfertile stallions tend to present high values of PI and RI (high vascular resistance). The ROC curves revealed that the best Doppler parameters to predict sperm quality in stallions were: Doppler velocities (PSV, EDV and TAMV), the diameter of the capsular artery and TABF parameters (tissue perfusion parameters). Cut off values were established using a YoudenÂs Index to identify fertile stallions from stallions with testicular dysfunction. Spectral Doppler ultrasound is a good predictive tool for sperm quality since correlations were determined among Doppler parameters and markers of sperm quality. Doppler ultrasonography could be a valuable diagnostic tool for use by clinical practitioners for the diagnosis of stallions with testicular dysfunction and could be a viable alternative to invasive procedures traditionally used for diagnosis of sub-fertility disorders.SIC.O.F. is supported by a postdoctoral grant from “Ministerio de Economía y Competitividad “. "Juan de la Cierva” IJCI-2014-21671. The authors received financial support from: the Ministerio de Economía y Competitividad-FEDER, Madrid, Spain,grant AGL2013-43211-R;Junta de Extremadura-FEDER (GR 10010 and PCE1002). P.M.M. is supported by a predoctoral grant from the Ministerio de Educación, Cultura y Deporte, Madrid Spain FPU13/03991

Analysis of seminal plasma from brown bear (Ursus arctos) during the breeding season: Its relationship with testosterone levels

[EN] Seminal plasma (SP) plays an important role in the motility, viability and maintenance of the fertilizing capacity of mammalian spermatozoa. This study is the first on brown bear (Ursus arctos) SP components, and has two main objectives: 1) to define the SP composition in bear ejaculate and 2) to identify variations in SP composition in relation to high and low levels of testosterone in serum during the breeding season. Forty-eight sperm samples from 30 sexually mature male brown bears (Ursus arctos) were obtained by electroejaculation, and their serum testosterone levels were assessed to sort the animals into 2 groups (high and low testosterone levels, threshold 5 ng/dl). The biochemical and protein compositions of the SP samples were assessed, and sperm motility was analyzed. We found that lactate dehydrogenase was significantly higher in the low-serum-testosterone samples, while concentrations of lipase and Mg+ values were significantly higher in the high-serum-testosterone samples. In contrast, sperm motility did not significantly differ (P>0.05) between the testosterone level groups (total motility: 74.42.8% in the high-level group vs. 77.1±4.7% in the low-level group). A reference digital model was constructed since there is no information for this wild species. To do this, all gel images were added in a binary multidimensional image and thirty-three spots were identified as the most-repeated spots. An analysis of these proteins was done by qualitative equivalency (isoelectric point and molecular weight) with published data for a bull. SP protein composition was compared between bears with high and low serum testosterone, and three proteins (binder of sperm and two enzymes not identified in the reference bull) showed significant (P<0.05) quantitative differences. We conclude that male bears with high or low serum testosterone levels differs only in some properties of their SP, differences in enzyme LDIP2, energy source LACT2, one protein (similar to BSP1) and Mg ion were identified between these two groups. These data may inform the application of SP to improve bear semen extenders.SIThis work was supported in part by MINECO (CGL2013-48255-R) and Cantur S.A. Luis Anel-Lopez was supported by MINECO (CGL2013-48255-R). C. Ortega Ferrusola is supported by a postdoctoral grant from “Ministerio de Economía y Competitividad “Juan de la Cierva” IJCI-2014-21671

Improving sperm banking efficiency in endangered species through the use of a sperm selection method in brown bear (Ursus arctos) thawed sperm

ANTECEDENTES: Se ha demostrado que los métodos de selección de esperma, como la centrifugación de una sola capa (SLC), son una herramienta útil para mejorar la calidad de las muestras de esperma y, por lo tanto, para aumentar la eficiencia de otras técnicas de reproducción artificial en varias especies. Este procedimiento podría ayudar a mejorar la calidad de los bancos de recursos genéticos, que es esencial para las especies en peligro de extinción. En contraste, estos métodos de selección de espermatozoides están optimizados y enfocados en animales de granja, donde la tarea de recuperación no es tan importante como en las especies en peligro de extinción debido a su mayor disponibilidad de espermatozoides. El objetivo de este estudio fue evaluar dos métodos de centrifugación (300 xg / 20 min y 600 xg / 10 min) y tres concentraciones de medio SLC (Androcoll-Bear -80, 65 y 50%) para optimizar el procedimiento para recuperar La mayor cantidad de espermatozoides con la mayor calidad posible. La morfología del esperma podría ser importante en la relación hidrodinámica entre la célula y el medio de centrifugación y, por lo tanto, se estudió el efecto de la morfometría de la cabeza del esperma sobre el rendimiento del esperma y su relación hidrodinámica. RESULTADOS: Las muestras seleccionadas con Androcoll-Bear 65% mostraron un rendimiento muy bueno (53.1 ± 2.9), aunque el rendimiento de Androcoll-Bear 80% fue menor (19.3 ± 3.3). Este último mostró valores de motilidad más altos que el control inmediatamente después de la selección posterior a la descongelación. Sin embargo, ambas concentraciones de coloide (65 y 80%) mostraron valores más altos de esperma viable y de esperma viable con acrosoma intacto que el control. Después de una incubación de 2 horas a 37 ° C, las muestras de 80% de Androcoll-Bear tuvieron mayor cinemática y proporción de espermatozoides viables con acrosoma intacto. En el análisis morfométrico, el esperma seleccionado por el 80% de Androcoll-Bear mostró una cabeza con un área más grande que era más alargada que el esperma de otros tratamientos. CONCLUSIONES: Concluimos que la selección de espermatozoides con Androcoll-Bear al 65% u 80% es una técnica adecuada que permite obtener una población de espermatozoides con mejor calidad que la muestra inicial. Recomendamos el uso de Androcoll-Bear 65% ya que el rendimiento es mejor que Androcoll-Bear 80%. Nuestros hallazgos allanan el camino para futuras investigaciones sobre la aplicación de técnicas de selección de espermatozoides al banco de espermatozoides en el oso pardo.BACKGROUND: Sperm selection methods such as Single Layer Centrifugation (SLC) have been demonstrated to be a useful tool to improve the quality of sperm samples and therefore to increase the efficiency of other artificial reproductive techniques in several species. This procedure could help to improve the quality of genetic resource banks, which is essential for endangered species. In contrast, these sperm selection methods are optimized and focused on farm animals, where the recovery task is not as important as in endangered species because of their higher sperm availability. The aim of this study was to evaluate two centrifugation methods (300 x g/20 min and 600 x g/10 min) and three concentrations of SLC media (Androcoll-Bear −80, 65 and 50%) to optimise the procedure in order to recover as many sperm with the highest quality as possible. Sperm morphology could be important in the hydrodynamic relationship between the cell and centrifugation medium and thus the effect of sperm head morphometry on sperm yield and its hydrodynamic relationship were studied. RESULTS: The samples selected with Androcoll-Bear 65% showed a very good yield (53.1 ± 2.9) although the yield from Androcoll-Bear 80% was lower (19.3 ± 3.3). The latter showed higher values of motility than the control immediately after post-thawing selection. However, both concentrations of colloid (65 and 80%) showed higher values of viable sperm and viable sperm with intact acrosome than the control. After an incubation of 2 h at 37 °C, the samples from Androcoll-Bear 80% had higher kinematics and proportion of viable sperm with intact acrosome. In the morphometric analysis, the sperm selected by the Androcoll-Bear 80% showed a head with a bigger area which was more elongated than the sperm from other treatments. CONCLUSIONS: We conclude that sperm selection with Androcoll-Bear at either 65% or 80% is a suitable technique that allows a sperm population with better quality than the initial sample to be obtained. We recommend the use of Androcoll-Bear 65% since the yield is better than Androcoll-Bear 80%. Our findings pave the way for further research on application of sperm selection techniques to sperm banking in the brown bear.• Ministerio de Economía y Competitividad y Cantur S.A. • Ministerio de Economía y Competitividad. Proyecto CGL2013–48255-R, para Luis Anel López • Ministerio de Economía y Competitividad. Beca postdoctoral “Juan de la Cierva” IJCI-2014-21671, para Cristina Ortega FerrusolapeerReviewe

Morphological and ultrasonographic characterization of the three zones of supratesticular region of testicular artery in Assaf rams

[EN] To fully understand the histological, morphometrical and heamodynamic variations of different supratesticular artery regions, 20 mature and healthy Assaf rams were examined through ultrasound and morphological studies. The testicular artery images of the spermatic cord as shown by B-mode analysis indicated a tortuous pattern along its course toward the testis, although it tends to be less tortuous close to the inguinal ring. Doppler velocimetric values showed a progressive decline in flow velocity, in addition to pulsatility and vessel resistivity when entering the testis, where there were significant differences in the Doppler indices and velocities among the different regions. The peak systolic velocity, pulsatility index and resistive index were higher in the proximal supratesticular artery region, followed by middle and distal ones, while the end diastolic velocity was higher in the distal supratesticular region. The total arterial blood flow and total arterial blood flow rate reported a progressive and significant increase along the testicular cord until entering the testis. Histological examination revealed presence of vasa vasorum in the tunica adventitia, with their diameter is higher in the proximal supratesticular zone than middle and distal ones. Morphometrically, the thickness of the supratesticular artery wall showed a significant decline downward toward the testis; meanwhile, the outer arterial diameter and inner luminal diameter displayed a significant increase distally. The expression of alpha smooth muscle actin and vimentin was higher in the tunica media of the proximal supratesticular artery zone than in middle and distal ones.SIThe authors acknowledge the staff members and technicians of Comparative Anatomy and Pathology Department, and Animal Medicine and Surgery Department, Faculty of Veterinary Medicine, León University, Spain, and specially Professor P de Paz for their great help in the practical and laboratory parts of this study. Many thanks are extended to staff member of Anatomy and Embryology Department, Faculty of Veterinary Medicine, Sohag University, for their help with histological and morphometrical analyses. We are thankful and grateful for European Union for the financial support of this study through the project (ERASMUS+ KA107 2019/2020). This work was financially supported by the Junta de Castilla y León (LE253P18) and MINECO (AGL2017-83098-R) project and the University of León, and also by Sohag University, Egypt.This article was funded by Ministerio de Economía, Industria y Competitividad, Gobierno de España (AGL2017-83098-R) and Junta de Castilla y León (LE253P18)