Biology and Physiology of Reproduction (BIOFREZ)

Strategic Area:
Introduction:

The overall objective of the group of Biology and Physiology of Reproduction is the increase and improvement of the reproductive success in wild and domestic animal species, focussed on the ovine species.

Currently, the group consists of 15 researchers working at laboratories in the Faculty of Veterinary Medicine at University of Zaragoza, in the Departments of Biochemistry and Molecular and Cell Biology, and Animal Production, both integrated into the IUCA. The group is co-directed by Professors José Alfonso Abecia Martínez, José Álvaro Cebrián Pérez, Fernando Forcada Miranda and Mª Teresa Muiño Blanco.

The group's work deals with basic and applied research in the areas of Cellular and Molecular Biology and Animal Production. The specific objectives of the group cover different aspects of Cell and Molecular Biology and Animal Production related to the following topics: reproductive management that directly affects the fertilizing capacity of semen doses, embryonic development and fertility, and therefore, the efficiency and performance of farms.

The main objectives are:

  • Selection of semen doses with high fertilizing capacity
  • Sperm cryopreservation protocols
  • Preparation of semen doses "sex-directed"
  • Capacitation and acrosome reaction mechanisms
  • "In vitro" fertilization development
  • Undernutrition effect on reproductive performance of sheep
  • Optimization of superovulatory treatments in recovering of endangered breeds
  • Effect of undernutrition on reproductive yields of sheep embryo donors undergoing superovulation treatments, and recipients of embryos in MOET programs
  • Electronic Animal Identification

Our group maintains several research projects of national, regional and international institutions. We collaborate in the development of national and regional plans within the livestock area, in particular the Improvement of Animal Production Technology, and in turn, within the objective of Improving Reproductive Technologies in livestock species. Our studies are focused on basics, technology and methodology of breeding sheep that are potentially transferable to the sector. For this purpose, we maintain close cooperation with the CENSYRA (Local Government) and associations of producers in Aragon with significance in the ovine sector, such as the “Asociación Nacional de Criadores de Ovino de la raza Rasa aragonesa (ANGRA) y Asociación Cooperativa Pastores

Research Lines:
  • Ovine semen cryopreservation and molecular basis of membrane changes associated with cryopreservation and sperm maturation.

    The aim of this line is to develop an efficient method for freezing ram semen. We are interested in the sperm subpopulations freezability for the management of gene banks resources. This research line takes into account aspects of cell damage that are not reflected in the classical parameters of semen quality, such as apoptosis, mitochondrial functionality and others. Furthermore, we are trying to clone proteins of 14, 20 and 22 kD from seminal plasma with capacity to protect and repair the cold-shock damage due to cryopreservation. Also,the selection and isolation of semen doses enriched in specific characteristics, particularly in sperm carrying the Y or X chromosome. 
The group is developing other molecular studies leading to the control of sperm maturation, capacitation and acrosome reaction, and the process of apoptosis.

  • Physiological basis of the reproductive efficiency improvement.

    Ovine species issued as a model for the improvement of farm animal reproductive efficiency. We study the possibilities of getting an adequate fertility and prolificacy at each mating or insemination, as a result of optimal evaluation and control of both sexual activity and the growth and follicular development, determining the ovulation rate. 
It attempts to optimize the influence of controllable factors at farm level on these parameters, both environmental (melatonin) and nutritional and social conditions. 
We also analyse the influence of these factors on the mechanisms that determine embryo viability at the stage of free life of the embryo, studying steroid receptor and oxytocin endometrial and oviductal concentrations, and endometrial plasma levels of prostaglandins and metabolic hormones. 
Furthermore we also analyse the production of interferon tau by the embryo in the moments before implantation.

Background:

The group has obtained interesting results since 1993, presenting a wealth of about 60 publications in SCI journals in the last 5 years, and the realization of 9 Doctoral Theses. A total of 25 national and international research grants have been achieved during this time. We maintain an active partnership with many other groups of researchers from different countries. This aspect is reflected in the realization of common projects and by the continued presence in our laboratory of researchers from different centres.

The Group's expertise is focused on two lines: cellular and molecular sperm biology, and physiology of reproduction. In turn, these are reflected in the following aspects that are the basis of undertaken projects: sperm quality, selection of sperm dosesenriched in specific characteristics, cryopreservation protocols, the effect of melatonin to improve the ovulation rate and litter size, the effect of undernutrition on the embryonic development, the use of embryo transfer for the recovery of populations and the hormonal response linked to the reproductive processes.

Facilities and equipment:
  • Laboratory RESOURCES (2012)

    • Unit of cell separation: “Centrifugal Countercurrent distribution”
    • Flow Cytometer
    • Fluorescence Microscopy
    • Fluorimeter, and Absorbance Luminometer
    • UV densitometer and infrared evaluation "ODYSSEY"
    • Laboratory for IVF and IVM processes
    • CASA analysis system
    • Ram farm
  • METHODOLOGY

    • Methods, Systems to assess semen quality:
      • Scan-CASA motility
      • Cell viability determination
      • Level of Apoptosis: phosphatidylserine reversion, caspases activity, DNA fragmentation (TUNEL) and mitochondrial integrity
      • Mitochondrial and Plasma membrane integrity and function
      • Capacitation state-CTC grade
      • Breathing Sperm: Oximeter
      • Zona binding assay (ZBA)
      • IVF
      • Antioxidant activity: SOD, GPx, GRH, catalase, etc..
      • Sexing of sperm subpopulations
    • Methods, Systems Separation of sperm subpopulations:
      • Swim-up
      • Countercurrent distribution ◦ centrifugation
      • gradient centrifugation: Sucrose cleaning, cleaning Percoll.
      • Filtering.
    • Methods, Systems analysis of seminal plasma components
      • Gel filtration chromatography
      • Electrophoresis
      • Fluorometry
      • Rating by densitometry "ODISSEY"
      • Western-Blot
      • Team 1 and 2D electrophoresis
      • Protein purification
      • Electroelution. Dialysis.
    • Methods, Systems study of sperm surface
      • Isolation of membrane proteins
      • Immunocytochemistry
      • Isolation of rafts
      • Preparation of polyclonal antibodies
      • Availability of analytical flow cytometer
    • Methods, Systems sperm freezing
      • Programmable thermostatic bath
      • Computerized cryopreservation
    • Methods of production-systems-embryo collection
      • Intrauterine artificial insemination in sheep and ectocervical
      • Superovulation techniques in vivo embryo production
      • Embryo recovery by laparotomy
      • Evaluation of ovulation rate by laparoscopy, and transrectal ultrasound
      • Conservation embryos by vitrification
      • Embryo transfer technology
    • Methods, Systems Maturation and Fertilization "in vitro"
      • IVM, IVF and embryo culture in vitro
      • Sexing of embryos
Technological Offer/Services:
  • Semen quality.
  • Spermiogram.
  • Sire selection.
  • Analysing of hormones (progesterone, testosterone, prostaglandins, cortisol).
  • Advising farmers associations on various aspects of sheep reproduction: organization of reproductive planning of an operation, artificial insemination, hormone use, nutritional management, etc..
  • Advising of using melatonin to improve sheep reproduction.
  • Assistance in ovulatory synchronization, embryo collection and embryo transfer
Contact:

Professor José A. Cebrián
Head of Group
pcebrian@unizar.es

Dept. of Biochemistry and Molecular and Cellular Biology

Faculty of Veterinary-University of Zaragoza