Reproduction in Domestic Animals - Facultad de Ciencias Veterinarias
Reproduction in Domestic Animals - Facultad de Ciencias Veterinarias
Reproduction in Domestic Animals - Facultad de Ciencias Veterinarias
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16 t h International Congress on Animal <strong>Reproduction</strong><br />
Workshop Abstracts 15<br />
WS08-5<br />
The strict tim<strong>in</strong>g of reproductive capacity <strong>in</strong> roe buck – an<br />
i<strong>de</strong>al mo<strong>de</strong>l system for seasonally reproduc<strong>in</strong>g cervidae<br />
Schoen, J 1 *; Blottner, S 2<br />
1Institute of Veter<strong>in</strong>ary Biochemistry, Freie Universität Berl<strong>in</strong>, Germany;<br />
2Leibniz-Institute for Zoo- and Wildlife Research, Berl<strong>in</strong>, Germany<br />
The roe <strong>de</strong>er is a typical seasonal bree<strong>de</strong>r with a short sexually active<br />
period last<strong>in</strong>g from mid July to mid August. Activity of tissues which<br />
are relevant for reproduction is very strictly regulated compared to<br />
other cervids. Exogenous signals from the environment cause<br />
manifest cyclic changes <strong>in</strong> the endocr<strong>in</strong>e system of the animal, which<br />
result <strong>in</strong> dist<strong>in</strong>ct alterations of morphological and functional<br />
parameters with<strong>in</strong> all reproductive organs. In the male, these<br />
alterations concern sperm production with<strong>in</strong> the testis as well as<br />
structure and function of the epididymis and accessory sex glands. Of<br />
course they also greatly concern behavioural aspects, which we will<br />
however leave asi<strong>de</strong> here. Only at the time of rut, functionality of the<br />
reproductive tract is optimized so that sperm output of high quality<br />
and quantity can be ascerta<strong>in</strong>ed. Even <strong>in</strong> phases before and after the<br />
mat<strong>in</strong>g period, spermatozoa are produced to a certa<strong>in</strong> <strong>de</strong>gree, but they<br />
are morphologically and functionally of low quality, due to<br />
suboptimal conditions <strong>in</strong> the reproductive tract. The gonadotrop<strong>in</strong>s<br />
show cyclical releas<strong>in</strong>g patterns throughout the year with an LH peak<br />
at the beg<strong>in</strong>n<strong>in</strong>g of spermatogenesis activation <strong>in</strong> March. This causes<br />
modulations <strong>in</strong> the differentiation status of Leydig cells followed by<br />
an <strong>in</strong>crease of testosterone secretion. The endocr<strong>in</strong>e variations trigger<br />
the seasonal up and down of germ cell proliferation and<br />
differentiation <strong>in</strong> the testis, which is mirrored by extreme changes <strong>in</strong><br />
tubulus diameter and testis mass. In the epididymis remarkable<br />
alterations <strong>in</strong> the tissue composition of the duct as well as <strong>in</strong> its<br />
expression patterns are necessary to assure controlled maturation of<br />
spermatozoa dur<strong>in</strong>g the rut. Lastly, the cyclic changes <strong>in</strong> volume and<br />
exocr<strong>in</strong>e activity of the accessory glands cause a seasonally modified<br />
composite of the sem<strong>in</strong>al fluid. All of this leads to enormous<br />
variations <strong>in</strong> quality and quantity of sperm cells <strong>in</strong> the ejaculate.<br />
The variability of the reproductive capacity of the roe buck is based<br />
on the comb<strong>in</strong>ed actions and the subtle <strong>in</strong>terplay of the endocr<strong>in</strong>e<br />
system, germ<strong>in</strong>ative and somatic testicular cells, the epithelium of the<br />
epididymis and the accessory glands. This fragile <strong>in</strong>teraction is<br />
necessary to facilitate the optimal tim<strong>in</strong>g of male reproductive fitness<br />
towards rutt<strong>in</strong>g season. The non domesticated roe <strong>de</strong>er is an<br />
absolutely fasc<strong>in</strong>at<strong>in</strong>g mo<strong>de</strong>l that enables us to study the different<br />
levels of seasonally <strong>de</strong>term<strong>in</strong>ed regulation of reproduction with hardly<br />
any artificial <strong>in</strong>fluence.<br />
Workshop 09 - Cryopreservation of gametes and embryo<br />
Mo<strong>de</strong>rator: Sandor Cseh (Hungary)<br />
WS09-1<br />
Basic Cryobiology of Gametes and Embryos<br />
Leibo, SP<br />
Department of Biological Sciences, University of New Orleans; Audubon<br />
Center for Research of Endangered Species, New Orleans, LA U.S.A.<br />
The ability to cryopreserve spermatozoa, oocytes and cleavage-stage<br />
embryos is essential for the efficient husbandry of all mammalian<br />
species. Live offspr<strong>in</strong>g of more than fifty species have been produced<br />
by artificial <strong>in</strong>sem<strong>in</strong>ation of females or by <strong>in</strong> vitro fertilization of<br />
oocytes with cryopreserved spermatozoa. Live young of at least<br />
twenty-five species have resulted from transfer of cryopreserved<br />
embryos or oocytes. For some species, offspr<strong>in</strong>g have been produced<br />
from gametes or embryos that have been preserved <strong>in</strong> the frozen state<br />
for at least twenty-five years, lead<strong>in</strong>g to speculation that<br />
cryopreserved germplasm can probably be stored at least for<br />
millennia.<br />
Despite the diversity of species that have been successfully preserved,<br />
the basic pr<strong>in</strong>ciples from which the procedures to cryopreserve them<br />
have evolved are very similar. Gametes or embryos are suspen<strong>de</strong>d <strong>in</strong><br />
a solution of a cryoprotective additive (CPA) that causes the cells to<br />
un<strong>de</strong>rgo partial <strong>de</strong>hydration by loss of cell water. Then, the cells are<br />
either cooled to low subzero temperatures at high rates to “capture”<br />
the cells <strong>in</strong> the <strong>de</strong>hydrated state, or the cells are first cooled rather<br />
slowly to <strong>in</strong>termediate subzero temperatures caus<strong>in</strong>g <strong>de</strong>hydration <strong>in</strong><br />
response to the <strong>in</strong>creas<strong>in</strong>gly concentrated solution as extracellular<br />
water is removed <strong>in</strong> the form of ice. In both cases, the purpose is to<br />
prevent cell water from crystalliz<strong>in</strong>g with<strong>in</strong> the cell. After storage,<br />
cryopreserved cells are warmed to physiological temperatures and the<br />
CPA is removed. In general, if cells have been cryopreserved by<br />
be<strong>in</strong>g rapidly cooled, they are warmed rapidly; if preserved by slow<br />
cool<strong>in</strong>g, the cells are warmed relatively slowly.<br />
Literally, thousands of experimental reports have been published <strong>in</strong><br />
which <strong>de</strong>tails of methods to cryopreserve gametes and embryos are<br />
<strong>de</strong>scribed. Various low molecular weight compounds have been used<br />
successfully as cryoprotectants. Cool<strong>in</strong>g and warm<strong>in</strong>g rates rang<strong>in</strong>g<br />
from less than 0.5ºC/m<strong>in</strong>ute to more than 10,000ºC/m<strong>in</strong>ute have been<br />
used to cryopreserve germplasm. A wi<strong>de</strong> variety of <strong>de</strong>vices and<br />
conta<strong>in</strong>ers have been used to cryopreserve cells. In general, it appears<br />
that it is not the actual cool<strong>in</strong>g or warm<strong>in</strong>g procedure or the storage<br />
temperature that damages germplasm. Rather, most evi<strong>de</strong>nce<br />
<strong>in</strong>dicates that the key to successful cryopreservation is the<br />
composition of the suspend<strong>in</strong>g medium. Consequently, efforts<br />
cont<strong>in</strong>ue to <strong>de</strong>term<strong>in</strong>e the effects of such media on gametes and<br />
embryos and to seek better media for cell preservation.<br />
WS09-2<br />
Update on Vitrification as a novel Cryopreservation<br />
Technique for Oocytes and Embryos<br />
Liebermann, J<br />
Fertility Centers of Ill<strong>in</strong>ois, Chicago, IL, USA<br />
The impact of cryopreservation on the growth and improved<br />
efficiency of assisted reproduction <strong>in</strong> humans is becom<strong>in</strong>g<br />
<strong>in</strong>creas<strong>in</strong>gly appreciated, with close to a fifth of all births follow<strong>in</strong>g <strong>in</strong><br />
vitro fertilization and embryo transfer Worldwi<strong>de</strong> aris<strong>in</strong>g from<br />
cryopreservation of supernumerary embryos. As culture techniques<br />
and embryo quality improve, the ratio of fresh to cryopreserved<br />
embryo babies will approach a more equal ratio, <strong>in</strong>dicative of an<br />
<strong>in</strong>creased reliance on the use of embryos follow<strong>in</strong>g cryostorage. One<br />
of the most commonly discussed issues <strong>in</strong> cryobiology is whether<br />
slow-cool<strong>in</strong>g or rapid-cool<strong>in</strong>g protocols satisfy the fundamental<br />
cryobiological pr<strong>in</strong>ciples of reduc<strong>in</strong>g damage by ice crystal formation<br />
dur<strong>in</strong>g freez<strong>in</strong>g and thaw<strong>in</strong>g. One way to achieve this target is by<br />
attempt<strong>in</strong>g to establish a glassy or vitreous state, where<strong>in</strong> molecular<br />
translational motions are arrested without structural reorganization of<br />
the liquid. Such recent studies have opened the door to a potentially<br />
highly successful and consistent alternative to overcom<strong>in</strong>g the<br />
problems of slow-freez<strong>in</strong>g and thaw<strong>in</strong>g cells, named vitrification.<br />
Although <strong>in</strong>itially reported <strong>in</strong> 1985 as a successful cryopreservation<br />
approach for mouse embryos, vitrification has taken a backseat to the<br />
much more wi<strong>de</strong>ly adopted conventional freez<strong>in</strong>g technology applied<br />
to both gametes and embryos <strong>in</strong> animal and human assisted<br />
reproduction. Recent years have seen a resurgence of <strong>in</strong>terest <strong>in</strong> this<br />
ultra-rapid cryopreservation technology, as the limitations of slowrate<br />
freez<strong>in</strong>g have become more evi<strong>de</strong>nt <strong>in</strong> the cl<strong>in</strong>ical arena.<br />
Vitrification is a simple technology, potentially faster, and<br />
<strong>in</strong>expensive. Furthermore, vitrification has proven to be consistent<br />
and reliable when carried out properly, and it allows also more<br />
<strong>in</strong>dividual patient flexibility. Vitrification has so far been used for<br />
gametes and embryos (<strong>de</strong>velopmental days 1 to 5). However, its use<br />
has been more advantagous with chill-sensitive cells such as oocytes<br />
and blastocysts. Successful vitrification of human spermatozoa<br />
without cryoprotectants was recently <strong>de</strong>scribed. There is very few<br />
data available concern<strong>in</strong>g the vitrification of human ovarian tissue.<br />
Although some problems rema<strong>in</strong> to be fully addressed with<br />
vitrification as a rout<strong>in</strong>e cryopreservation technique, we believe that it<br />
shows much promise as a viable alternative to conventional freez<strong>in</strong>g<br />
technology. The issues with vitrification are well <strong>de</strong>f<strong>in</strong>ed and limited<br />
<strong>in</strong> number, and to our way of th<strong>in</strong>k<strong>in</strong>g easily surmountable.