Effect of different media additives on capacitation of frozen-thawed ram spermatozoa as a potential replacement for estrous sheep serum

Capacitation is a key process through which spermatozoa acquire their fertilizing ability. This event is required for the successful application of assisted reproductive technologies such as IVF. The aim of the present study was to investigate the effect of using a synthetic oviductal fluid medium supplemented with either heparin–hypotaurine alone, in combination with progesterone (P4), 17β-estradiol (E2), or BSA, or just β-cyclodextrin, in replacement for estrous sheep serum (ESS) for ram sperm capacitation. After incubation in the corresponding media for 15 (time 0) or 60 minutes, sperm function was evaluated by computerized sperm motility analysis and flow cytometry (plasma membrane status and fluidity). Treatments rendering the best results in regards to sperm function parameters related to capacitation were used for an IVF test. Herein, neither heparin–hypotaurine (alone), or in combination with P4, or E2, nor β-cyclodextrin induced capacitation-related changes in frozen–thawed ram spermatozoa. Only the medium supplemented with heparin–hypotaurine–BSA was able to induce changes compatible with in vitro capacitation relating to sperm motility pattern and plasma membrane fluidity, comparable to those in ESS-containing medium. Both media yielded sperm parameter values that differed (P < 0.05) from those obtained in the rest of the media tested. However, after the IVF trial, BSA was unable to support cleavage rates (21.80%) comparable to those obtained with ESS (52.60%; P < 0.05). We conclude that heparin–hypotaurine, P4, E2, β-cyclodextrin, or BSA is not suitable for replacing ESS in capacitation and fertilization media for ram spermatozoa.M. Ramón was supported by the Research Recruitment Program from the National Institute for Agricultural and Food Research program.Peer Reviewe

Taking advantage of the use of supervised learning methods for characterization of sperm population structure related with freezability in the Iberian red deer

P. 1661-1672Using Iberian red deer as a model, this study presents a supervised learning method, the Support Vector Machines (SVM), to characterize sperm population structure related with freezability. Male freezability was assessed by evaluating motility, membrane status and mitochondrial membrane potential of sperm after a freezing-thawing procedure. The SVM model was generated using sperm motility information captured by computer-assisted sperm analysis (CASA) from thawed semen, belonging to six stags with marked differences on their freezability. A total of 1369 sperm tracks were recorded for seven kinematic parameters and assigned to four motility patterns based on them: weak motile, progressive, transitional and hyperactivated-like. Then, these data were split in two sets: the training set, used to train the SVM model, and the testing set, used to examine how the SVM method and three other unsupervised methods, a non-hierarchical, a hierarchical and a multistep clustering procedures, performed the sperm classification into subpopulations. The SVM was revealed as the most accurate method in the characterization of sperm subpopulations, showing all the sperm subpopulations obtained in this way high significant correlations with those sperm parameters used to characterize freezability of males. Given its superiority, the SVM method was used to characterize the sperm motile subpopulations in Iberian red deer. Sperm motile data from frozen–thawed semen belonging to 25 stags were recorded and loaded into the SVM model. The sperm population structure revealed that those males showing poor freezability were characterized by high percentages of sperm with a weak motility pattern. In opposite, males showing good freezability were characterized by higher percentages of sperm with a progressive and hyperactivated-like motility pattern and lower percentages of sperm with a weak motile pattern. We also identified a sperm subpopulation with a transitional motility pattern. This subpopulation increased as the freezability of males improved, and may be used as indicative of overall sperm motility.S

Sperm Cell Population Dynamics in Ram Semen during the Cryopreservation Process

[EN] Background: Sperm cryopreservation has become an indispensable tool in biology. Initially, studies were aimed towards the development of efficient freezing protocols in different species that would allow for an efficient storage of semen samples for long periods of time, ensuring its viability. Nowadays, it is widely known that an important individual component exists in the cryoresistance of semen, and efforts are aimed at identifying those sperm characteristics that may allow us to predict this cryoresistance. This knowledge would lead, ultimately, to the design of optimized freezing protocols for the sperm characteristics of each male. Methodology/Principal Findings: We have evaluated the changes that occur in the sperm head dimensions throughout the cryopreservation process. We have found three different patterns of response, each of one related to a different sperm quality at thawing. We have been able to characterize males based on these patterns. For each male, its pattern remained constant among different ejaculates. This latter would imply that males always respond in the same way to freezing, giving even more importance to this sperm feature. Conclusions/Significance: Changes in the sperm head during cryopreservation process have resulted useful to identify the ability of semen of males for freezing. We suggest that analyses of these response patterns would represent an important tool to characterize the cryoresistance of males when implemented within breeding programs. We also propose follow-up experiments to examine the outcomes of the use of different freezing protocols depending on the pattern of response of males.SIThis work was supported by the Education and Science Council of Castilla-La Mancha (PCC08-0105-8042). Manuel Ramón was supported by the DOC- INIA program. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript

Conocimiento, conducta y actitud ante el Código europeo contra el cáncer por parte de los profesionales sanitarios de atención primaria

Objetivo: Determinar si los profesionales de atención primaria (AP) conocen el Código europeo contra el cáncer (CECC) y si ello se relaciona con que practiquen sus recomendaciones. Diseño: Observacional descriptivo. Emplazamiento: Centros de AP españoles. Participantes: Profesionales de AP. Intervenciones: Cuestionario autoadministrado. Mediciones principales: Factores de riesgo y protectores del cáncer, pruebas de cribado y conocimiento del CECC. Resultados: Respondieron 1.734 profesionales (10, 5%), edad media de 48, 5 años (DT: 9, 2), el 71, 5% mujeres. El 50, 0% desconoce el CECC; los tutores (OR = 1, 61), adscritos al PAPPS (OR = 1, 51) y que llevan más tiempo trabajando en AP (OR = 2, 62) son los que más lo conocen. El 7, 2% fuma y el 79, 1% bebe alcohol. El 64, 1% presenta normopeso y el 19% realiza actividad física. Evita exponerse al sol el 52, 7% y usa protectores el 53, 8%. El 85, 2% nunca se ha realizado un cribado colorrectal, el 11% nunca una citología y el 12% una mamografía. Existe relación entre conocer el CECC y el tabaquismo (p < 0, 001), sobrepeso/obesidad (p = 0, 024), actividad física (p = 0, 003), ingesta de carnes (p < 0, 001), mamografía (p < 0, 001), citología (p = 0, 022), PSA (p = 0, 045), así como el fomento de sus consejos entre los pacientes. Conclusiones: El CECC es desconocido por la mitad de los profesionales de AP. La práctica de sus recomendaciones es desigual, siendo la prevalencia de tabaquismo baja, y la protección solar una conducta poco extendida. Los que lo conocen son los que más practican sus recomendaciones. Objective: To decide if Primary Care (PC) professionals know the European Code against Cancer (CECC) and if this relates to practice the recommendations with themselves and with their patients. Design: descriptive, observational study. Setting: Spanish Health Centers. Participants: Health professionals. Interventions: self-administered questionnaire. Main measurements: cancer protective and risk factors, screening for colorectal, breast, cervix and prostate cancer, level of knowledge of the ECAC. Results: 1734 participants (10.5%), mean age 47.4 years (SD: 8.6), 71.5% female. 50.0% do not know the CECC; tutors (OR = 1.61), assigned to the PAPPS (OR = 1.51) and who have been working in AP for more time (OR = 2.62) are the ones who know it most. 7.2% smoke and 79.1% drink alcohol. 64.1% presented normal weight and 19% performed physical activity. They avoid exposing themselves to the sun 52.7% and use protectors 53.8%. 85.2% have never had a colorectal screening, 11% never had a cytology, and 12% had a mammogram. There is a relationship between knowing the CECC and smoking (P <.001), overweight/obesity (P =.024), physical activity (P =.003), meat intake (P <.001), mammography (P <.001), cytology (P =.022), PSA (P =.045), as well as the promotion of their advice among patients. Conclusions: The CECC is unknown by half of the AP professionals. The practice of its recommendations is uneven, with the prevalence of low smoking, and sun protection a little extended behavior. Those who know him are the ones who most practice his recommendations

Taking advantage of the use of supervised learning methods for characterization of sperm population structure related with freezability in the iberian red deer

Using Iberian red deer as model, this study presents a supervised learning method, the Support Vector Machines (SVM), to characterize sperm population structure related with freezability. Male freezability was assessed by evaluating motility, membrane status and mitochondrial membrane potential of sperm after a freezing-thawing procedure. The SVM model was generated using sperm motility information captured by computer-assisted sperm analysis (CASA) from thawed semen, belonging to 6 stags with marked differences on their freezability. A total of 1369 sperm tracks were recorded for seven kinematic parameters and assigned to four motility patterns based on them: weak motile, progressive, transitional and hyperactivated-like. Then, this data were split in two sets: the training set, used to train the SVM model, and the testing set, used to examine how the SVM method and three other unsupervised methods, a non-hierarchical, a ierarchical and a multi-step clustering procedures, performed the sperm classification into subpopulations. The SVM was revealed as the most accurate method in the characterization of sperm subpopulations, showing all the sperm subpopulations obtained in this way high significant correlations with those sperm parameters used to characterize freezability of males. Given its superiority, the SVM method was used to characterize the sperm motile subpopulations in Iberian red deer. Sperm motile data from frozen thawed semen belonging to 25 stags were recorded and loaded into the SVM model. The sperm population structure revealed that those males showing por freezability were characterized by high percentages of sperm with a weak motility pattern. In opposite, males showing good freezability were characterized by higher percentages of sperm with a progressive and hyperactivated-like motility pattern and lower percentages of sperm with a weak motile pattern. We also identified a sperm subpopulation with a transitional motility pattern. This subpopulation increased as the freezability of males improved, and may be used as indicative of overall sperm motility

Advances in sperm cryopreservation of samples collected by vaginal artificial and electroejaculation from Blanca-Celtiberica goat breed

Blanca-Celtibérica goat is an endangered autochthonous breed from Spain. To prevent the disappearance of these autochthonous breeds and in order to maintain the genetic diversity, the Food and Agriculture Organization of the United Nations (FAO) recommends the preservation of these breeds by in situ and ex situ conservation programs (FAO, 2010). The creation of Genetic Resource Banks (GRB) is a measure of ex situ conservation and allows us the storage of semen, oocytes and embryos indefinitely, being an essential tool in order to preserve the genetic diversity of species. The most widespread application in the development of GRB has been the semen collection and its freezing. This requires the knowledge of reproductive physiology as well as the suitable assisted reproductive technologies for each species. In the case of the Blanca-Celtibérica goat breed, the geographical location in addition to extensive production systems and the lack of buck center make semen collection by routine techniques not feasible. Thus, when animals can not be trained to semen collection by artificial vagina, there are other alternatives such as the electroejaculation, being possible to obtain semen of males living in the countryside. Besides the semen collection, the conservation of semen samples is also a key aspect. Up to date, although several studies have been carried out on sperm cryopreservation in caprine, most of them have used samples collected by artificial vagina. However, it is known that the semen collection method influences on the sperm production and composition of seminal plasma (Marco-Jiménez et al., 2005; Marco-Jiménez et al., 2008) as well as the cryoresistance of sperm samples (Álvarez et al., 2012). Another problem during the buck semen cryopreservation is the low seminal quality found after thawing when freezing extenders based on egg yolk or skim milk are used. Some components of egg yolk and skim milk have negative interactions with seminal plasma as a result of the production of toxic compounds to the spermatozoa. This effect could be more pronounced on sperm samples collected by electroejaculation since the volume of seminal plasma is higher than in samples obtained by artificial vagina. With this background, the general aim of this Doctoral Thesis has been to develop an optimal method in order to collect and cryopreserve buck semen of Blanca-Celtibérica breed which allow us to storage seminal doses to create a Germoplasm Bank.La autora ha disfrutado de la concesión de una beca para la realización del Doctorado subvencionada por el Instituto Nacional de Investigación y Tecnología Agraria y Alimentaria (INIA). Los diversos experimentos que forman parte de esta tesis han sido parcialmente financiados por el proyecto RZ2008-00009-C02 concedido por el INIA.Peer Reviewe

Taking advantage of the use of supervised learning methods for characterization of sperm population structure related with freezability in the Iberian red deer

Using Iberian red deer as a model, this study presents a supervised learning method, the Support Vector Machines (SVM), to characterize sperm population structure related with freezability. Male freezability was assessed by evaluating motility, membrane status and mitochondrial membrane potential of sperm after a freezing-thawing procedure. The SVM model was generated using sperm motility information captured by computer-assisted sperm analysis (CASA) from thawed semen, belonging to six stags with marked differences on their freezability. A total of 1369 sperm tracks were recorded for seven kinematic parameters and assigned to four motility patterns based on them: weak motile, progressive, transitional and hyperactivated-like. Then, these data were split in two sets: the training set, used to train the SVM model, and the testing set, used to examine how the SVM method and three other unsupervised methods, a non-hierarchical, a hierarchical and a multistep clustering procedures, performed the sperm classification into subpopulations. The SVM was revealed as the most accurate method in the characterization of sperm subpopulations, showing all the sperm subpopulations obtained in this way high significant correlations with those sperm parameters used to characterize freezability of males. Given its superiority, the SVM method was used to characterize the sperm motile subpopulations in Iberian red deer. Sperm motile data from frozen-thawed semen belonging to 25 stags were recorded and loaded into the SVM model. The sperm population structure revealed that those males showing poor freezability were characterized by high percentages of sperm with a weak motility pattern. In opposite, males showing good freezability were characterized by higher percentages of sperm with a progressive and hyperactivated-like motility pattern and lower percentages of sperm with a weak motile pattern. We also identified a sperm subpopulation with a transitional motility pattern. This subpopulation increased as the freezability of males improved, and may be used as indicative of overall sperm motility.This study was supported in part by the Spanish Ministry of Science and Innovation (Project AGL2007-60 271/GAN), and by the Education and Science Council of the Junta de Castilla y León, Spain (Project LE019A10-2). Felipe Martínez-Pastor was supported by the Ramón y Cajal program (Ministry of Science and Innovation, Spain).Peer Reviewe

Understanding sperm heterogeneity: Biological and practical implications

Sperm are the most diverse cell type known. This diversity is thought to reflect adaptation to conditions under which sperm function as a way to ensure the survival of sperm in fertilization environments and to maximize fertilizing capacity thereof. The existence of morphological diversity among species is widely assumed, although this diversity seems less clear as we go deeper (between males, between ejaculates from the same male and even within the same ejaculate), with different theories addressing this heterogeneity. Moreover, the development of assisted reproductive techniques (ART) has led to changes in the physiological conditions in which sperm fertilize, which could lead, ultimately, to a selection towards more favourable sperm design. Regardless of the origin of this diversity, when studying the relationship between shape and function of sperm, it is advisable to assess the degree of heterogeneity of sperm and takes into account to be more likely to identify those morphological characteristics determining the fertile ability of sperm. Otherwise, these relationships could be hidden as a result of considering an average shape not representative of morphological characteristics of sperm. In addition, the knowledge of this morphological diversity in terms of changes arising from modifications in the sperm environment and mechanisms that generate these changes could be useful for understanding the reproductive capacity of males but also in enhancing their fertile ability.Manuel Ramón was supported by DOC-INIA programme.Peer Reviewe

Individual variation in sperm cryopreservation in the Blanca-Celtibérica goat breed

Resumen del póster presentado al 12th International Congress of the Spanish Association of Animal Reproduction, celebrado en Alicante del 16 al 18 de octubre de 2014.This study addressed the inter-male variation in the resistance of spermatozoa to the cryopreservation process in the Blanca-Celtibérica goat breed. Semen from six males (age >1.5 years) was collected and cryopreserved. Five replicates were performed. To evaluate sperm cryoresistance, percentage of sperm motility (SM; 0–100%) and sperm quality (SQ; 0–5) parameters were subjectively assessed after collection (AC), and in pre-freezing (PF) and post-thawing (PT) stages. Values of SM and SQ decreased (p ≤ 0.05) through the cryopreservation process. Inter-male variability for SM and SQ were higher (p ≤ 0.05) in PF stage for both parameters (SM standard deviations were 1.79, 15.06 and 3.19% for AC, PF and PT; SQ standard deviations were 0.03, 0.73 and 0.49 for AC, PF and PT). Thus, the major differences in sperm cryoresistance among males were observed in pre-freezing stage and may partly determine the sperm quality observed after thawing. The sperm motility changes that occur prior to freezing could predict the different semen cryoability of goat males.Peer Reviewe