Reproduction in Domestic Animals - Facultad de Ciencias Veterinarias

16 t h International Congress on Animal Reproduction
Workshop Abstracts 15
WS08-5
The strict timing of reproductive capacity in roe buck – an
ideal model system for seasonally reproducing cervidae
Schoen, J 1 *; Blottner, S 2
1Institute of Veterinary Biochemistry, Freie Universität Berlin, Germany;
2Leibniz-Institute for Zoo- and Wildlife Research, Berlin, Germany
The roe deer is a typical seasonal breeder with a short sexually active
period lasting from mid July to mid August. Activity of tissues which
are relevant for reproduction is very strictly regulated compared to
other cervids. Exogenous signals from the environment cause
manifest cyclic changes in the endocrine system of the animal, which
result in distinct alterations of morphological and functional
parameters within all reproductive organs. In the male, these
alterations concern sperm production within the testis as well as
structure and function of the epididymis and accessory sex glands. Of
course they also greatly concern behavioural aspects, which we will
however leave aside here. Only at the time of rut, functionality of the
reproductive tract is optimized so that sperm output of high quality
and quantity can be ascertained. Even in phases before and after the
mating period, spermatozoa are produced to a certain degree, but they
are morphologically and functionally of low quality, due to
suboptimal conditions in the reproductive tract. The gonadotropins
show cyclical releasing patterns throughout the year with an LH peak
at the beginning of spermatogenesis activation in March. This causes
modulations in the differentiation status of Leydig cells followed by
an increase of testosterone secretion. The endocrine variations trigger
the seasonal up and down of germ cell proliferation and
differentiation in the testis, which is mirrored by extreme changes in
tubulus diameter and testis mass. In the epididymis remarkable
alterations in the tissue composition of the duct as well as in its
expression patterns are necessary to assure controlled maturation of
spermatozoa during the rut. Lastly, the cyclic changes in volume and
exocrine activity of the accessory glands cause a seasonally modified
composite of the seminal fluid. All of this leads to enormous
variations in quality and quantity of sperm cells in the ejaculate.
The variability of the reproductive capacity of the roe buck is based
on the combined actions and the subtle interplay of the endocrine
system, germinative and somatic testicular cells, the epithelium of the
epididymis and the accessory glands. This fragile interaction is
necessary to facilitate the optimal timing of male reproductive fitness
towards rutting season. The non domesticated roe deer is an
absolutely fascinating model that enables us to study the different
levels of seasonally determined regulation of reproduction with hardly
any artificial influence.
Workshop 09 - Cryopreservation of gametes and embryo
Moderator: Sandor Cseh (Hungary)
WS09-1
Basic Cryobiology of Gametes and Embryos
Leibo, SP
Department of Biological Sciences, University of New Orleans; Audubon
Center for Research of Endangered Species, New Orleans, LA U.S.A.
The ability to cryopreserve spermatozoa, oocytes and cleavage-stage
embryos is essential for the efficient husbandry of all mammalian
species. Live offspring of more than fifty species have been produced
by artificial insemination of females or by in vitro fertilization of
oocytes with cryopreserved spermatozoa. Live young of at least
twenty-five species have resulted from transfer of cryopreserved
embryos or oocytes. For some species, offspring have been produced
from gametes or embryos that have been preserved in the frozen state
for at least twenty-five years, leading to speculation that
cryopreserved germplasm can probably be stored at least for
millennia.
Despite the diversity of species that have been successfully preserved,
the basic principles from which the procedures to cryopreserve them
have evolved are very similar. Gametes or embryos are suspended in
a solution of a cryoprotective additive (CPA) that causes the cells to
undergo partial dehydration by loss of cell water. Then, the cells are
either cooled to low subzero temperatures at high rates to “capture”
the cells in the dehydrated state, or the cells are first cooled rather
slowly to intermediate subzero temperatures causing dehydration in
response to the increasingly concentrated solution as extracellular
water is removed in the form of ice. In both cases, the purpose is to
prevent cell water from crystallizing within the cell. After storage,
cryopreserved cells are warmed to physiological temperatures and the
CPA is removed. In general, if cells have been cryopreserved by
being rapidly cooled, they are warmed rapidly; if preserved by slow
cooling, the cells are warmed relatively slowly.
Literally, thousands of experimental reports have been published in
which details of methods to cryopreserve gametes and embryos are
described. Various low molecular weight compounds have been used
successfully as cryoprotectants. Cooling and warming rates ranging
from less than 0.5ºC/minute to more than 10,000ºC/minute have been
used to cryopreserve germplasm. A wide variety of devices and
containers have been used to cryopreserve cells. In general, it appears
that it is not the actual cooling or warming procedure or the storage
temperature that damages germplasm. Rather, most evidence
indicates that the key to successful cryopreservation is the
composition of the suspending medium. Consequently, efforts
continue to determine the effects of such media on gametes and
embryos and to seek better media for cell preservation.
WS09-2
Update on Vitrification as a novel Cryopreservation
Technique for Oocytes and Embryos
Liebermann, J
Fertility Centers of Illinois, Chicago, IL, USA
The impact of cryopreservation on the growth and improved
efficiency of assisted reproduction in humans is becoming
increasingly appreciated, with close to a fifth of all births following in
vitro fertilization and embryo transfer Worldwide arising from
cryopreservation of supernumerary embryos. As culture techniques
and embryo quality improve, the ratio of fresh to cryopreserved
embryo babies will approach a more equal ratio, indicative of an
increased reliance on the use of embryos following cryostorage. One
of the most commonly discussed issues in cryobiology is whether
slow-cooling or rapid-cooling protocols satisfy the fundamental
cryobiological principles of reducing damage by ice crystal formation
during freezing and thawing. One way to achieve this target is by
attempting to establish a glassy or vitreous state, wherein molecular
translational motions are arrested without structural reorganization of
the liquid. Such recent studies have opened the door to a potentially
highly successful and consistent alternative to overcoming the
problems of slow-freezing and thawing cells, named vitrification.
Although initially reported in 1985 as a successful cryopreservation
approach for mouse embryos, vitrification has taken a backseat to the
much more widely adopted conventional freezing technology applied
to both gametes and embryos in animal and human assisted
reproduction. Recent years have seen a resurgence of interest in this
ultra-rapid cryopreservation technology, as the limitations of slowrate
freezing have become more evident in the clinical arena.
Vitrification is a simple technology, potentially faster, and
inexpensive. Furthermore, vitrification has proven to be consistent
and reliable when carried out properly, and it allows also more
individual patient flexibility. Vitrification has so far been used for
gametes and embryos (developmental days 1 to 5). However, its use
has been more advantagous with chill-sensitive cells such as oocytes
and blastocysts. Successful vitrification of human spermatozoa
without cryoprotectants was recently described. There is very few
data available concerning the vitrification of human ovarian tissue.
Although some problems remain to be fully addressed with
vitrification as a routine cryopreservation technique, we believe that it
shows much promise as a viable alternative to conventional freezing
technology. The issues with vitrification are well defined and limited
in number, and to our way of thinking easily surmountable.