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 />
8 Workshop Abstracts<br />
the number of nuclei, the number of blastomeres was higher <strong>in</strong><br />
polyspermic embryos due to early fragmentation. Further studies are<br />
nee<strong>de</strong>d to clarify the phenomenon of ploidy correction <strong>in</strong> polyspermic<br />
zygotes.<br />
Dur<strong>in</strong>g IVM, approximately 30% of oocytes fail to reach the M-II<br />
stage and about 60-78% of them are arrested at a certa<strong>in</strong> stage<br />
characterized by a metaphase plate without polar body. This stage is<br />
often referred as “metaphase-I (M-I) arrest”. Our previous studies<br />
showed, that unlike real M-I oocytes, “M-I arrested” oocytes obta<strong>in</strong><br />
cytoplasmic maturation and have the ability to <strong>de</strong>velop to the<br />
blastocyst stage result<strong>in</strong>g <strong>in</strong> polyploid embryos. Chromosome<br />
configurations of such oocytes are different from those of M-I oocytes<br />
at 33h IVM and similar to those of M-II oocytes <strong>de</strong>spite of their<br />
tetraploid status. It is suggested that <strong>in</strong> “M-I arrested” oocytes<br />
segregation of homologue chromosomes may occur dur<strong>in</strong>g IVM,<br />
however, the extrusion of the first polar body fails and a s<strong>in</strong>gle<br />
metaphase plate is formed aga<strong>in</strong>.<br />
In conclusion, <strong>de</strong>velopment to the blastocyst stage is not a perfect<br />
<strong>in</strong>dicator of embryo quality <strong>in</strong> porc<strong>in</strong>e IVP systems, s<strong>in</strong>ce polyploid<br />
embryos can <strong>de</strong>velop to blastocyst stage. Careful selection of M-II<br />
oocytes for IVF, regular monitor<strong>in</strong>g of polyspermy rates and further<br />
improvements of IVF systems are necessary to reduce polyploidy <strong>in</strong><br />
porc<strong>in</strong>e IVP systems.<br />
WS03-4<br />
Tyros<strong>in</strong>e k<strong>in</strong>ase receptors (TRK) and bov<strong>in</strong>e early<br />
embryonic <strong>de</strong>velopment<br />
Gómez, E*, Rodríguez, A; Caamaño, JN; Díez. C; Facal, N; Muñoz, M<br />
Genética y Reproducción, Serida, Gijón, Spa<strong>in</strong><br />
Neurotroph<strong>in</strong>s (NTs) mediate survival, growth and differentiation by<br />
b<strong>in</strong>d<strong>in</strong>g to two types of cell surface receptors, the anti-apoptotic Trk<br />
and the low aff<strong>in</strong>ity p75 neurotroph<strong>in</strong> receptor (p75 NTR ), also termed<br />
as p75 NGFR . Subtypes of Trk are NT-specific, such a way NGF b<strong>in</strong>ds<br />
to TrkA, NT3 to TrkC, and NT4 and BDNF to TrkB; on the contrary,<br />
p75 NTR is a common receptor for all NTs. Trk and p75 NTR receptors<br />
can form complexes, and this association enhances aff<strong>in</strong>ity for ligand<br />
b<strong>in</strong>d<strong>in</strong>g and neurotroph<strong>in</strong> discrim<strong>in</strong>ation.<br />
In previous work, we suggested a role for NTs dur<strong>in</strong>g early embryonic<br />
<strong>de</strong>velopment, as we <strong>de</strong>tected TrkA (a truncated isoform), TrkB and<br />
TrkC prote<strong>in</strong>s <strong>in</strong> immature oocytes, zygotes, 2-4 cell embryos,<br />
morulae, expan<strong>de</strong>d and hatched blastocysts, and the <strong>in</strong>ner cell mass of<br />
blastocysts. Us<strong>in</strong>g RT-PCR we found mRNA for TrkA, TrkC, p75 NTR<br />
and FGFr2 <strong>in</strong> all the embryonic stages <strong>de</strong>scribed above; mRNA levels<br />
ten<strong>de</strong>d to <strong>in</strong>crease dur<strong>in</strong>g blastulation, and sharply peaked <strong>in</strong> hatched<br />
blastocysts. However, s<strong>in</strong>gle (NGF, NT3, NT4 or BDNF, at 20 ng/mL<br />
each) or pooled NTs <strong>in</strong> embryo culture with the NT-cooperat<strong>in</strong>g<br />
factor bFGF (2 ng/mL), did not show relevant effects on <strong>de</strong>velopment<br />
and cell counts. Comparable results were obta<strong>in</strong>ed <strong>in</strong> a recent<br />
experiment <strong>in</strong> the absence of bFGF. bFGF was <strong>de</strong>trimental compared<br />
to controls without bFGF, while NT3, with or without bFGF,<br />
<strong>in</strong>creased cell numbers <strong>in</strong> the ICM as opposed to NT4 and pooled<br />
NTs. NT4 enhanced cell counts <strong>in</strong> the trophecto<strong>de</strong>rm as compared to<br />
controls with bFGF and pooled NTs only <strong>in</strong> the presence of bFGF.<br />
Dose-response studies and new experiments to progress <strong>in</strong> research<br />
with NTs and their receptors are <strong>in</strong> course. We have provi<strong>de</strong>d a novel<br />
<strong>de</strong>scription of Trk prote<strong>in</strong>s and TrkA, TrkC, p75 NTR and FGFr2<br />
mRNA expression, throughout mammalian embryonic <strong>de</strong>velopment.<br />
This research may help to <strong>de</strong>sign future research with NTs <strong>in</strong> bov<strong>in</strong>e<br />
embryo culture and embryonic stem cells <strong>de</strong>rivation and ma<strong>in</strong>tenance.<br />
Grant support: AGL2005–04479. Spanish-Hungarian bilateral project<br />
HH2005-0015 (TET E-20/2005). M. Muñoz is sponsored by FICYT.<br />
Workshop 04 - Imag<strong>in</strong>g techniques <strong>in</strong> reproduction<br />
Mo<strong>de</strong>rator: Gregg P Adams (Canada)<br />
WS04-1<br />
Use of 3-D and 4-D ultrasonography <strong>in</strong> veter<strong>in</strong>ary<br />
research<br />
Hil<strong>de</strong>brandt, TB 1 *, Drews, B 1 , Kurz, J 2 , Röllig, K 1 , Hermes, R 1 , Yang, S 3 ,<br />
Göritz, F 1<br />
1<strong>Reproduction</strong> Management, Leibniz Institute for Zoo & Wildlife Research,<br />
Germany; 2 Institute for Genetik & General Biology, University of Salzburg,<br />
Austria; 3 Yunnan Key Laboratory for Animal <strong>Reproduction</strong>, Kunm<strong>in</strong>g Institute<br />
of Zoology, CAS, Ch<strong>in</strong>a<br />
Three-dimensional-ultrasonography represents an <strong>in</strong><strong>de</strong>pen<strong>de</strong>nt (what<br />
is meant by <strong>in</strong><strong>de</strong>pen<strong>de</strong>nt) imag<strong>in</strong>g modality <strong>in</strong> human medic<strong>in</strong>e with<br />
a wi<strong>de</strong> range of applications, <strong>in</strong>clud<strong>in</strong>g prenatal diagnosis,<br />
gynecology, and oncology. Three-dimensional-ultrasonography<br />
allows morphometric measurements <strong>in</strong> a volume data set and, more<br />
recently, the analysis of data <strong>in</strong> tomographic ultrasound imag<strong>in</strong>g mo<strong>de</strong><br />
(TUI). The advantages of three-dimensional-ultrasonography have<br />
also been realized <strong>in</strong> animal research. Ultrasound equipment used <strong>in</strong><br />
animals requires additional components and animal-specific scanners<br />
for transrectal exam<strong>in</strong>ation. After substantial system modification<br />
transrectal three-dimensional-ultrasonography was applied <strong>in</strong>tensively<br />
to characterize embryo/fetal <strong>de</strong>velopment <strong>in</strong> Asian and African<br />
elephants. Three ultrasound systems were used dur<strong>in</strong>g this study: a<br />
stationary Voluson 530 and a 730 Expert, amd a portable Voluson I<br />
(General Electric Inc.). Growth curves for elephant embryo/fetal<br />
<strong>de</strong>velopment were established. In ren<strong>de</strong>r mo<strong>de</strong> the surface structures<br />
were imaged, and used to <strong>de</strong>pict the transition from the oval-shaped<br />
embryo to the complete fetus with its characteristic trunk. In <strong>in</strong>verseren<strong>de</strong>r<br />
mo<strong>de</strong>, the fluid-filled compartments were displayed separately.<br />
This mo<strong>de</strong> was also applied to imag<strong>in</strong>g of ovarian follicular<br />
<strong>de</strong>velopment <strong>in</strong> rhesus macaques dur<strong>in</strong>g superovulation. In<br />
conjunction with a scientific consultancy for National Geographic,<br />
additional <strong>in</strong>vestigations were performed <strong>in</strong> dogs, primates, hares and<br />
dolph<strong>in</strong>s. The ma<strong>in</strong> purpose was to i<strong>de</strong>ntify useful morphological<br />
parameters of the different <strong>de</strong>velopmental stages dur<strong>in</strong>g pregnancy,<br />
and exam<strong>in</strong>e <strong>in</strong>trauter<strong>in</strong>e fetal behavior. For the latter, fourdimensional<br />
ultrasound technology (3D <strong>in</strong> real-time) was applied. We<br />
found dramatic differences between species regard<strong>in</strong>g the level of<br />
fetal activity, <strong>in</strong>clud<strong>in</strong>g some <strong>in</strong>terest<strong>in</strong>g behavioral patterns observed<br />
from each species throughout their <strong>de</strong>velopment. Accord<strong>in</strong>g to these<br />
prelim<strong>in</strong>ary f<strong>in</strong>d<strong>in</strong>gs, we conclu<strong>de</strong> that <strong>in</strong>trauter<strong>in</strong>e behavior is an<br />
important element dur<strong>in</strong>g embryogenesis. There is also a strong<br />
<strong>in</strong>dication that <strong>in</strong>trauter<strong>in</strong>e behavior has parallels to known postnatal<br />
behavior <strong>in</strong> the different species. Four-dimensional ultrasonography<br />
is, a useful tool for i<strong>de</strong>ntify<strong>in</strong>g species-specific behavioral elements<br />
dur<strong>in</strong>g <strong>in</strong>trauter<strong>in</strong>e <strong>de</strong>velopment. The <strong>de</strong>f<strong>in</strong>ition of these key<br />
behaviors and the consequences of their presence or absence for the<br />
life history of an <strong>in</strong>dividual will improve our un<strong>de</strong>rstand<strong>in</strong>g of<br />
evolution and help <strong>in</strong> cl<strong>in</strong>ical diagnosis of <strong>de</strong>velopmental disor<strong>de</strong>rs.<br />
WS04-2<br />
In vivo imag<strong>in</strong>g of sperm transport and survival <strong>in</strong> the<br />
female genital tract us<strong>in</strong>g fiber confocal fluorescence<br />
microscopy<br />
Druart, X 1 *; Cognié J. 1 ; Baril, G. 1 ; Clément F. 2 ; Dacheux JL. 1 and Gatti JL. 1<br />
1UMR 6175 INRA, CNRS-Université <strong>de</strong> Tours-Haras Nationaux, Nouzilly,<br />
France ; 2 INRIA, Paris, France<br />
Introduction Fertility of ram sperm is reduced after 24 h of storage<br />
at 15°C <strong>in</strong> milk diluent <strong>de</strong>spite that the <strong>in</strong> vitro properties, such as<br />
viability and motility, are well preserved. This might be expla<strong>in</strong>ed by<br />
a <strong>de</strong>crease of sperm transport and survival of stored ram sperm <strong>in</strong> the<br />
female genital tract. We studied the <strong>in</strong>fluence of <strong>in</strong> vitro storage of<br />
ram sperm on <strong>in</strong> vivo transport <strong>in</strong> the female genital tract of the ewe<br />
us<strong>in</strong>g Fiber Confocal fluorescence Microscopy (FCM, Leica).