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[EN] It is important to note that seasonality could affect ram reproductive parameters, and therefore, fertility results after artificial insemination. In this work, 1) we assessed fertility rates after cervical artificial insemination of 11,805 ewes at the beginning (June 21st to July 20th) and at the end (November 20th to December 21st) of the reproductive season in the Assaf breed for the last four years, and 2) we aimed to identify male factors influencing the different reproductive success obtained depending on the time at the mating season in which ovine artificial insemination was performed. For this purpose, we evaluated certain ram reproductive and ultrasonographical parameters as well as we performed a multiparametric and proteomic sperm analysis of 6–19 rams at two very distant points in the mating season (July as Early Breeding Season –EBS– and November as Late Breeding Season –LBS–). Rutinary assessments carried out in the ovine reproduction centers (testicular volume, libido, sperm production and mass motility) showed non-significant differences (P ≥ 0.05) between both studied times, as well as the ram ultrasonographic evaluation (Resistive and Pulsatility Index as Doppler parameters; and pixels mean gray level, and hypoechoic areas percentage and density as echotexture parameters). However, at level of sperm functionality, although sperm quality appeared non-significantly lower (P ≥ 0.05) in the EBS, we identified a significantly different (P < 0.05) sperm proteomic profile between the seasonality points. The following proteins were identified with the lowest abundance in the EBS with a fold change > 4, a P = 2.40e-07, and a q = 2.23e-06: Fibrous Sheath-Interacting Protein 2, Disintegrin and Metalloproteinase Domain-Containing Protein 20-like, Phosphoinositide-Specific Phospholipase C, Tektin 5, Armadillo Repeat-Containing Protein 12 Isoform X3, Solute Carrier Family 9B1, Radial Spoke Head Protein 3 Homolog, Pro-Interleukin-16, NADH Dehydrogenase [Ubiquinone] 1 Alpha Subcomplex Subunit 8, Testis, Prostate and Placenta-Expressed Protein, and Acyl Carrier Protein Mitochondrial. In conclusion, while our basic analyses on male and sperm quality showed similar results between the beginning and the end of the breeding season, on a proteomic level we detected a lower expression of sperm proteins linked to the energy metabolism, sperm-oocyte interactions, and flagellum structure in the EBS. Probably, this different protein expression could be related to the lower fertility rate of Assaf ewes after cervical artificial insemination at this time. More importantly, sperm proteins can be used as highly effective molecular markers in predicting sperm fertilization ability related to intraseasonal variations. SI

P. 184-192 Genome 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. SI

Tesis realizada en el laboratorio de Biología de la Reproducción del grupo de investigación SaBio, perteneciente al Instituto de Investigación en Recursos Cinegéticos IREC(UCLM-CSIC-JCCM)., El uso de técnicas de reproducción asistida en cérvidos durante los últimos años ha experimentado un importante avance no solo con propósitos conservacionistas (Jabbour et al. 1997; Pukazhenthi y Wildt 2003), sino también con propósitos económicos (carne, velvet y trofeos) cuya importancia es alta en algunos países como España, Nueva Zelanda, Australia o China (Asher et al. 2000). Esta importancia económica ha generado un interés creciente por parte de los criadores de ciervo a la hora de buscar asesoramiento de especialistas con el fin de desarrollar técnicas que les permitan optimizar sus rendimientos y producciones. Dentro de este contexto, existe una gran variedad de técnicas de reproducción asistida como la electroeyaculación, la sincronización del estro a través del uso de implantes de progestágenos, la inseminación artificial (IA), la transferencia de embriones o la fecundación in vitro (IVF) que han sido objeto de investigación en varias especies de cérvidos (Asher et al. 1999), siendo la más importante tanto para los criadores como para los investigadores el ciervo rojo (Cervus elaphus). Uno de los principales objetivos de la presente tesis doctoral ha sido evaluar el rendimiento y efectos que tiene el proceso de sex-sorting, mediante el uso del sistema (SX MoFlo, DakoCytomation Inc., Fort Collins, CO, USA) modificado para selección espermática, en eyaculados de ciervo rojo obtenidos mediante electroeyaculación. Dicho proceso nos permite separar y clasificar los espermatozoides de eyaculados en fracciones enriquecidas de espermatozoides Y- y espermatozoides X- con una pureza superior al 90%. Estudios previos en cérvidos (Gao et al. 2011) han demostrado que es posible obtener fracciones enriquecidas de espermatozoide Y- con capacidad fecundante y obtener descendencia mediante inseminación artificial en hembras superovuladas. Esta tecnología de sexado espermático es especialmente interesante en especies como el ciervo, en las cuales el interés productivo y en consecuencia el rendimiento económico se obtiene de uno de los sexos. Dado que en el ciervo rojo la principal producción es la cuerna, los machos son los que poseen un elevado valor económico. La posibilidad de elegir el sexo de la descendencia, supondría una enorme optimización de los medios de producción. Además, desde un punto de vista tanto ético como productivo nos permite evitar la eliminación masiva de hembras en las explotaciones., El autor ha disfrutado de una ayuda de formación de personal investigador de la Junta de Castilla y La Mancha (PRE123/2010) y los experimentos han sido financiados por el Ministerio de Economía y Competitividad por medio del proyecto del Plan Nacional AGL2010-21487. Además, el doctorando ha contado con financiación de la UCLM para realizar una estancia de 3 meses en el SLU de Uppsala (Suecia), lo que ha opermitido cumplir con los requisitos para acceder a la mención de doctorado internacional.

Póster presentado al 3rd World Congress of Reproduction, Oxidative stress & Antioxidants, celebrado en Paris (Francia) el 22 y 23 de mayo de 2014.-- et al., This work has been supported by the Spanish Ministry of Science and Innovation (Project AGL2010-21487).

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., Capacitation is a complex process that involves multiple changes in sperm physiology. One aspect of these changes is an increasing destabilization which prepares the membrane for the acrosome reaction and fusion with the oocyte. An important likely consequence of the changes in sperm plasma membrane function is a change in ion transport regulation, resulting in a change in intracellular ionic concentrations. We can monitor changes in sperm volume by flow cytometry. We have evaluated the effect of capacitation on volume of ram spermatozoa by means of CellLab QUANTA SC cytometer which can estimate cell volume due to the electrical resistance that produces spermatozoa as they passes through a capillary (Electronic Volume). Thawed sperm samples form 16 rams were incubated at 38.5°C and 5% CO2 in SOF-HEPES (287 mOsm) or SOF-HEPES with 10% of oestrum sheep serum (OSS; 295 mOsm). Sperm volume was analyzed before and after 30 min of incubation. Changes in the Electronic Volume were assessed in viable spermatozoa with intact acrosome (PI-/PNA-) and were shown as the rate of volume increase (DEV). Spermatozoa incubated with OSS showed a higher DEV than no-OSS incubation treatment (7.53 +/- 1.12% and 1.75 +/- 1.12%, respectively, p < 0.001). Our results show that capacitation causes an increase of sperm electronic volume of ram spermatozoa. Anyway, further studies should be carried out to determine the processes involved in this increase of volume.