Lines of research:
- To improve knowledge on the reproductive physiology of wild species and the response of their spermatozoa and gonadal tissue to cryopreservation processes (conventional freezing, ultra-rapid freezing, vitrification). For this aim, a basic approach (endocrine, cellular, molecular) is carried out taking into account a comparative spermatology and cryobiology perspective, including wild and domestic species as experimental models.
- Development of new semen extenders, cryopreservation procedures and assisted reproduction techniques for its application in threatened and game wild species, as well as in threatened domestic breeds.
- Assisted reproductive techniques (creation of germplasm and tissue banks, artificial insemination, collection and transfer of embryos, etc.) applied to stakeholders involved with the conservation of biodiversity and rural environment sustainability: zoos, natural parks, national parks, game reserves, game farms, livestock associations, companies.
To develop these research works we have 4 laboratories:
1. Spermatology & cryoconservation in wild species laboratory.
The objectives are focused to improve the knowledge of sperm cryoresistance and to develop new cryopreservation procedures for sperm and gonad tissue of wild species. The aims include the investigation of the role of endocrine status on sperm cryoresistance, the characterization of gametes, the development of species-specific assisted reproduction technologies, creation of genome resource banking,. The laboratory facilities have adequate equipment for spermatology studies, sperm analysis and cryopreservation: spectrophotometers, light microscopes, fluorescence microscopies, phase contrast microscopies connected to image analysis equipment (Motic Image Advanced 3.0 CASA, Sperm Class Analizet, SCA® v. 4.0), SQS2 (Sperm quality system), precision balances, lab mixers, pH meters, oscillating agitator, cryogenic showcases, freezers, liquid nitrogen tanks, conventional centrifuges, micro-centrifuges, plate-centrifuges, refrigerated centrifuges, thermal plates, water baths, ultraviolet sterilization systems, cell counting systems, defroster system for pellets (75ºC), CO2 incubator, biological freezer unit, laminar flow cabinet, etc. We have capacity to establish small laboratories in game reserves and mountain areas, and to capture wild animals in the field (teleanesthesia equipment - darts, blowguns, CO2-powered pistol, -, pulseoximetry monitoring, anesthesia equipment ) for recovery of sperm samples by electroejaculation (Electroeyaculator Lane Pulsator IIIZ; P-T Electronics, Sandy, OR, USA ) and transrectal ultrasound-guided massage of sexual glands - TUMASG method- (Prosound 2, Aloka).
2. Histology and immunohistochemistry laboratory applied in animal reproduction.
The objective is to support areas related to reproductive physiology and cryobiology, in domestic and wild species. Histological analysis of reproductive structures and spermatogenic activity evaluation are carried out, from samples of different species that are sent from natural parks, national parks, game reserves and zoos with which we collaborate. Also, studies of protein expression in spermatozoa and epididymal tissue are implemented according to species, gametes origin and endocrine state, using Western Blot, immunocytochemistry and immunohistochemistry techniques. The laboratory facilities have basic equipment for histological studies: freezers, refrigerators, precision balances, pH meters, hot plates, lab mixers, conventional centrifuges, slides and cassettes filing cabinets, hood for gases extraction emitted during tissues preparation and fixation, paraffin inclusion center (LEICA, HistoCore Arcadia H), cooling plate (LEICA, HistoCore Arcadia C), rotary microtome (LEICA, Mod. RM2125 RTS), with roughing system, manual advancement and retraction system, two histological baths for paraffin inclusion (SELECTA, Mod. Termofin), which allows its fusion and conservation in liquid state for tissue sections visualization, Moticam Motic Images Plus 3.0 camera (Image analysis software), phase contrast optical microscope (LEITZ, Mod. Laborlux S) for observation and analysis of histological images using different stains.
3. Reproductive endocrinology laboratory (RIA and ELISA).
Focuses on the set-up and development of radioimmunoassay techniques (RIA) for the determination of steroid (progesterone, testosterone and cortisol), protein (prolactin) and glycoprotein (LH) hormones in different biological fluids, mainly in blood plasma. Its application in the field of Reproductive Physiology allows us to address scientific and technological research on topics that include various reproductive traits in domestic and wild small ruminants (sheep, goats).The specific objectives are primarily focused on:
- Determining the neuroendocrine mechanisms that control the annual reproductive cycle in these species and determine their interaction with the environment (photoperiod)
- Implementation of reproductive control techniques in accordance with new perspectives and legislation of the European Union.
4. Spermatology in Animal Production.
Researchers: Manuel Álvarez Rodríguez, Eduardo de Mercado de la Peña y Helena Nieto Cristóbal.
This laboratory focuses its activity on cellular and molecular biology techniques for reproduction in livestock species, particularly the porcine model. One of the main objectives of this laboratory is to determine if spermatozoa react to
in vitro capacitation before and after being cryopreserved. In addition, it seeks to develop the most suitable defined composition media to mimic the
in vivo state of the process. The changes studied could be related to genetic information (RNA and protein expression) or epigenetic information (histone composition, non-coding RNA) and may constitute biomarkers of sperm status. In addition, seminal plasma (with its load of exosomes and eventual battery of small RNAs) has a clear effect on gene expression (at the immunomodulatory level) throughout the female genital tract, including sperm storage sites, and these changes that can be long-lasting and affect physiology, behavior, and fertility:
is this an evolutionarily conserved mechanism? The techniques used in this laboratory include isolating exosomes and mapping small RNA profiles (miRNA, piRNA, Y-RNA) in seminal plasma as potential molecular biomarkers for non-invasive evaluation of male fertility. Finally, the establishment of a valid
in vitro model to test gene expression changes in sperm reservoir epithelial cells could lead to a deeper understanding of the immune responses of these specific cells upon exposure to fluid components. seminal, which so far have been performed
in vivo. By definition, the laboratory is of high comparative relevance, reduces the use of experimental animals and addresses animal welfare issues towards the establishment of 3R principles.
An example of techniques used in the laboratory, for sample handling and analysis, includes qPCR, NGS, Western Blotting, cryobiology, in vitro culture, etc.