Comparison of two methods for obtaining spermatozoa from the cauda epididymis of Iberian red deer

P. 471-485We have compared two methods for salvaging epididymal sperm from post-mortem samples from Iberian red deer. Of each pair of testicles (29 samples), one cauda epididymis was processed by means of cuts (sperm was immediately diluted with extender) and the other was detached from the corpus and flushed from the vas deferens with 1 mL of extender. Sperm was processed for cryopreservation, and analyzed just after recovery, pre-freezing and post-thawing. Total spermatozoa recovered, contamination (concentration of epididymal cells and red blood cells (RBCs)) and quality (motility by CASA, and acrosomal status, viability and mitochondrial status by flow cytometry) were used to compare both methods. The number of recovered spermatozoa was similar for both methods. Contamination was higher for the cuts method, but when considering the final dilution before freezing, only RBCs concentration was significantly higher. Motility was similar just after extraction, but higher for both pre-frozen and post-thawed flushed sperm. Pre-freezing acrosomal status (P < 0.05) and viability (P < 0.1) were better for flushing; however post-thawing results were similar for the two methods. A clustering analysis using CASA data showed that the subpopulation pattern of motile sperm was different depending on the method, being better for flushing. With regard to yield, lower contamination (especially RBCs) and, in general, better quality results, flushing seems to be a more recommendable method for post-mortem sperm recovery. The cuts method may be more practical on certain occasions, but care must be taken in order to achieve rapid extension of the sample and to avoid contamination in order to improve sample condition.S

Sperm Subpopulations in Iberian Red Deer Epididymal Sperm and Their Changes Through the Cryopreservation Process

P. 316–327We have applied a statistical protocol based on principal component analysis, clustering methods, and discriminant analysis for the identification of sperm subpopulations in computer-assisted sperm analysis (CASA) data. Samples were obtained from the cauda epididymis of 11 Iberian red deer and cryopreserved following a standard protocol. Motility by CASA was analyzed just after sperm recovery, just before freezing, and after thawing, and eight motility descriptors for each individual spermatozoon were recorded. Sperm viability and acrosomal status were also assessed. Subpopulation analysis was performed in four sequential steps: principal component analysis using the eight motility descriptors; nonhierarchical clustering analysis (k-means) using the first two principal components; hierarchical clustering analysis (UPGMA); and selection of the final number of clusters. Three clusters were obtained for each motility analysis: slow and nonlinear; rapid and linear; and rapid, high ALH, nonlinear. We detected variations in the clusters between treatments (initial, prefreezing and postthawed). Indeed, motility increased and linearity decreased in the prefreezing analysis. A discriminant analysis isolated three descriptors that were used again in the same statistical analysis, giving four clusters that resembled the pattern found in the first classification. We also performed a clustering analysis of the males according to prefreezing/postthawed variation of total motility, viability, and acrosomal status. The proportion of the linear subpopulations in the prefreezing treatment, in both clustering analyses, correlated positively with postthawed viability recovery. Our results show that clustering analysis of CASA data gives useful and practical information that is not obtained by conventional sperm analysis.S

Reading prosody in Spanish dyslexics

Reading becomes expressive when word and text reading are quick, accurate and automatic. Recent studies have reported that skilled readers use greater pitch changes and fewer irrelevant pauses than poor readers. Given that developmental dyslexics have difficulty acquiring and automating the alphabetic code and developing orthographic representations of words, it is possible that their use of prosody when reading differs from that of typical readers. The goal of this study was to investigate whether the reading prosody of Spanish-speaking dyslexics differs from that of typical Spanish readers. Two experiments were performed. The first experiment involved 36 children (18 with dyslexia), and the second involved 46 adults (23 with dyslexia). Participants were asked to read aloud a text which included declarative, exclamatory and interrogative sentences. Data on pausing and reading rate (number of pauses, duration of pauses and utterances), pitch changes, intensity changes and syllable lengthening were extracted from the recordings. We found that dyslexic people read more slowly than typical readers and they also made more inappropriate and longer pauses, even as adults with considerable reading experience. We also observed that dyslexics differed from skilled readers in their use of some prosodic features, particularly pitch changes at the end of sentences. This is probably because they have trouble anticipating some structural features of prose, such as sentence ends.Proyecto de Investigación del Ministerio de Economía y Competitividad: PSI2012-31913 Programa Severo Ochoa (FICYT): BP14-03

Influencia del aprendizaje basado en equipos en la auto percepción de competencias docentes– fase cuantitativa

This study aims to investigate how the planning and implementation of didactic units based on the TBL contributes to improving the self-perception of professional skills of Secondary Education teachers. This article deals with the quantitative stage of alarger mixed design. This stage consists of an experimental design pre-experiment type with pre-test / post-test. The instrument used is a teacher self-perception questionnaire adapted from the professional competency model proposed byCampo (2016); participants are 55 Secondary Education teachers from the central region of Uruguay. A significant improvement is observed in the self-perception of all the competencies investigated: planning of teaching and learning processes, pedagogical use of technology, implementation of teaching strategies, communication and student interaction, evaluation of learning and reflection on one’s own teaching.En este estudio se pretende indagar de qué forma la planificación e instrumentación de unidades didácticas en la modalidad de ABE contribuye a la mejora de la autopercepción de las competencias profesionales de docentes de Educación Media. En este artículo, se incluye la etapa cuantitativa de un diseño mixto más amplio. Esta etapa consiste en un diseño experimental tipo preexperimento con preprueba / posprueba, utilizando un cuestionario de autopercepción docente, adaptación de la propuesta del modelo de competencias profesionales de Campo (2016). Se trabaja con 55 docentes de Educación Media de la región del centro de Uruguay y se constata una mejora significativa en la autopercepción de todas las competencias indagadas: planificación de los procesos de enseñanza y aprendizaje, uso pedagógico de la tecnología, implementación de estrategias de enseñanza, comunicación e interacción con los estudiantes, evaluación de los aprendizajes y reflexión sobre la propia docencia

Season effect on genitalia and epididymal sperm from Iberian red deer, roe deer and Cantabrian chamois

P. 1857-1875Seasonality deeply affects the physiology and behavior of many species, and must be taken into account when biological resource banks (BRBs) are established. We have studied the effect of seasonality on many reproductive parameters of free-ranging Iberian red deer, roe deer and Cantabrian chamois, living in Spain. Testicles from hunted animals were collected and sent to our laboratory at different times during the year. We recorded the weight and volume of testis, the weight of the epididymis and its separate parts (caput, corpus, and cauda), the weight of the sperm sample collected from the cauda epididymis, and several sperm parameters (sperm concentration, spermatozoa recovered, motility, HOS test reactivity, acrosomal status, and viability). We studied the data according to several periods, defined accordingly to each species. For red deer, we defined rut (mid-September to mid-October), post-rut (mid-October to mid-December), and non-breeding season (February). For roe deer, they were pre-rut (June), rut (July), post-rut (first fortnight of August), and non-breeding season (September). For chamois: non-breeding season (June to mid-September) and breeding season (October–November). The rut/breeding season yielded significantly higher numbers for almost all parameters. However, in the case of red deer, sperm quality was higher in the post-rut. For roe deer, testicular weight was similar in the pre-rut and in the rut, and sperm quality did not differ significantly between these two periods, although we noticed higher values in the rut. In the case of chamois, sperm quality did not differ significantly from the breeding season, but data distribution suggested that in the non-breeding season there are less males with sperm of good quality. On the whole, we find these results of interest for BRB planning. The best season to collect sperm in this species would be the breeding season. However, post-rut in red deer, pre-rut in roe deer, and non-breeding season in chamois could be used too, because of the acceptable sperm quality, despite the lower quantity salvaged. More in-depth research needs to be carried out on the quality of sperm salvaged at different times of the year in order to confirm these findings.S

Reduced glutathione and Trolox (vitamin E) as extender supplements in cryopreservation of red deer epididymal spermatozoa

P. 37-46The use of assisted reproductive techniques in cervids is increasing as the commercial use of these species increase. We have tested the suitability of the antioxidants Trolox and reduced glutathione (GSH) for freezing red deer epididymal spermatozoa, aiming at improving post-thawing quality. Samples from 19 stags were frozen in a TES-Tris-fructose extender (20% egg yolk, 8% glycerol), with 1 or 5 mM of antioxidant. Motility (CASA), lipoperoxidation (malondialdehyde –MDA– production), membrane status, mitochondrial activity, acrosomal status (flow cytometry) and chromatin status (SCSA: %DFI and %HDS; flow cytometry) were assessed after thawing and after 6 h at 39 °C. There were few differences between treatments after thawing, with Trolox reducing MDA production in a dose–response manner. After the incubation, sperm quality decreased and %DFI increased moderately, with no change for MDA. GSH improved motility, kinematic parameters and mitochondrial status, with a slight increase in %HDS. GSH 5 mM also increased moderately MDA production and %DFI, possibly due to enhanced metabolic activity and reducing power. Trolox maintained MDA low, but was detrimental to sperm quality. Trolox might not be appropriate for the cryopreservation of red deer epididymal spermatozoa, at least at the millimolar range. GSH results are promising, especially regarding motility improvement after the post-thawing incubation, and should be selected for future fertility trials.S

Improvement Strategies in Ovine Artificial Insemination

P. 30-42Artificial insemination in ram is scarcely widespread comparing with other domestic species. This has been due not only to fertility results being irregular and low but also because of the difficulty in the application of enhancements such as the use of frozen‐thawed sperm. Although there is a lot of information on the use of different options to improve these AI results (such as transcervical application, the use of thawed sperm, etc.) commercial programmes can be classified on two general categories: those using refrigerated semen (15°C) by superficial intracervical deposition (vaginal), and, more restricted, those using thawed sperm by intrauterine deposition (laparoscopy). In the present work, we have summarized our viewpoint on three general research lines for the improvement of AI results in sheep: semen preservation, AI procedures and semen assessment. Briefly, in ram it is necessary to develop a medium term methodology of sperm refrigeration (3–5 days), which would allow the distribution of sperm doses to a widespread area. Nevertheless, it is also necessary to develop an intrauterine transcervical AI technique, which allows thawed semen to be applied by vaginal insemination. Besides, the low predictive value of classic assessment techniques limits the ability to adjust the number of spermatozoa per dose according to its actual fertility

Evaluation of the qualitative and quantitative effectiveness of three media of centrifugation (Maxifreeze, Cushion Fluid Equine, and PureSperm 100) in preparation of fresh or frozen-thawed brown bear spermatozoa

P. 1119-1128Centrifugation is a crucial procedure in sperm cryopreservation protocols of brown bear (Ursus arctos), because the semen must be processed to increase sperm concentration and/or clean urine-contaminated samples. The efficacy of three media for centrifugation (Maxifreeze [IMV technologies, L'Aigle, France], Cushion Fluid Equine (Minitübe, Tiefenbach, Germany), and PureSperm [Nidacon, Gothenburg, Sweden]) on the quality of bear spermatozoa was evaluated. In experiment one, two cushioned media used for protecting against mechanical stress during centrifugation were analyzed. In experiment two, a density gradient based on PureSperm was assessed in relation to the maximum retrieval and the quality of fresh spermatozoa, and the freezability of the spermatozoa selected in this density gradient was studied in experiment three. Finally, the selection of frozen-thawed sperm using PureSperm was analyzed in experiment four. Our results indicate that the use of dense isotonic cushion solutions (Maxifreeze, Cushion Fluid Equine) in centrifugation did not improve the quality of recovered spermatozoa compared with standard centrifugation. However, a density gradient prepared with PureSperm improved the quality of spermatozoa in fresh semen and frozen-thawed semen, but the spermatozoa selected from the fresh sample with this density gradient did not show a better resistance to freezing with this density gradient in comparison with the control sample.S

Effect of length of time post-mortem on quality and freezing capacity of Cantabric chamois (Rupicapra pyrenaica parva) epididymal spermatozoa

P. 184-192Genome Resource Banks are keystones in the ex-situ conservation of wild species. Post-mortem (PM) collection of epididymal spermatozoa is an opportunistic and valuable source of germplasm, the time from the death of the animal limits its use. Seeking to improve germplasm preservation strategies for the chamois (Rupicapra sp.), the effect of PM time on epididymal sperm quality and freezability was studied using the Cantabrian chamois. Samples were classified according to PM collection time, up to 216 h (refrigerated), and cryopreserved (Tris-citric acid-fructose, 430 mOsm/kg, 15% egg yolk, 8% glycerol; freezing at −20 °C/min). Sperm quality was assessed after recovery and post-thawing (motility by CASA, HOS test, abnormal forms, cytoplasmic droplets, and viability and acrosomal damage by flow cytometry). The sperm mass pH and osmolality showed a positive correlation with time. Total sperm motility dropped after 2 days PM, with progressivity and sperm velocities remained similar up to 3 days PM. Sperm freezability was acceptable, with the post-thawing HOST, motility, progressivity, VAP, VCL, VSL and BCF negatively correlating with PM time. Overall, chamois epidydimal samples were not adequate for preservation after 6 days PM. Freezability capacity could make these spermatozoa suitable for specific ART even if kept refrigerated for several days PM.S

Extender osmolality, glycerol and egg yolk on the cryopreservation of epididymal spermatozoa for gamete banking of the Cantabric Chamois (Rupicapra pyrenaica parva)

P. 109-114Germplasm banking is a key technology enabling the ex-situ conservation of wild species. However, cryopreservation protocols must be tested to assure the applicability of the banked material. The objective of this study was defining a range of parameters for the composition of a semen extender for Cantabrian chamois epididymal spermatozoa (post-mortem collection). The freezing extender was based in a TES-Tris-fructose buffer, modifying its composition in three experiments: Osmolality of the buffer (320, 380 or 430 mOsm/kg, 8% glycerol, 15% egg yolk), glycerol (4 or 8%, 430 mOsm/kg, 15% egg yolk), egg yolk (10 or 15%, 430 mOsm/kg, 4% glycerol). Sperm was extended at 100 mill. spermatozoa/ml, cooled at 5 °C and frozen at −20 °C/min. Sperm quality was assessed pre and post-thawing (CASA, HOS test, abnormal forms, cytoplasmic droplets, and viability and acrosomal damage by flow cytometry). Freezability was good overall, with total motility of 65.5% ± 2.4 initial and 55.8% ± 2.4 post-thawing. The extenders affected the post-thaw sperm quality marginally. Whereas osmolalities and glycerol concentrations seemed not to differ, 430 mOsm/kg and 4% glycerol might be preferred. Egg yolk concentrations only differed on sperm velocity (VCL: 84.0 ± 6.7 μm/s in 10% vs. 70.7 ± 6.2 μm/s in 15%, P < 0.05). Our results suggest a good cryotolerance of chamois epididymal spermatozoa, with a preferred extender composition of hyperosmotic buffer, glycerol in the 4% range and lower egg yolk (10% range) than other ruminants.S