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 />
142 Poster Abstracts<br />
other <strong>de</strong>ndrobatid frogs. But sacrific<strong>in</strong>g endangered animals should be<br />
avoi<strong>de</strong>d.<br />
Sperm samples are obta<strong>in</strong>ed after gentle hormonal stimulation with<br />
human chorionic gonadotrop<strong>in</strong> (hCG) cloaca lavage and light<br />
microscopic evaluation. Sperm cells are filiform with an arcuated and<br />
21.1 µm long head and a s<strong>in</strong>gle tail (35.0 µm length). Their acrosomal<br />
complex is located at the anterior portion of the head and consists of<br />
the acrosomal vesicle with low electron <strong>de</strong>nsity, and the subjacent<br />
electron-<strong>de</strong>nse subacrosomal cone. The nucleus is circular <strong>in</strong><br />
transverse section (1.9 µm diameter), and conical <strong>in</strong> longitud<strong>in</strong>al<br />
section. It is surroun<strong>de</strong>d by several groups of mitochondria. The<br />
highly con<strong>de</strong>nsed chromat<strong>in</strong> is electron-<strong>de</strong>nse but shows numerous<br />
electron-lucent <strong>in</strong>clusions. A short midpiece has a mitochondrial<br />
collar with a proximal and a distal centriole. The latter gives rise to<br />
the axoneme which alone forms the flagellum.<br />
The sperm ultrastructure of D. auratus differs from that of other<br />
Dendrobatidae because of the absence of a nuclear space and the<br />
miss<strong>in</strong>g undulat<strong>in</strong>g membrane associated with an axial fibre. In<br />
conclusion, it can be stated that the spermatozoa of D. auratus are the<br />
first with<strong>in</strong> the Dendrobatidae without accessory tail structures. This<br />
tail conformation is analogue to these of Ranoi<strong>de</strong>a and not<br />
Bufonoi<strong>de</strong>a. This observation is <strong>in</strong> contrary to Garda et al. (2002) and<br />
Aguir-Jr. et al. (2004) who grouped the Dendrobatidae with<strong>in</strong> the<br />
Bufonoi<strong>de</strong>a. Our f<strong>in</strong>d<strong>in</strong>gs <strong>in</strong>dicate that the whole <strong>de</strong>ndrobatid family<br />
cannot be grouped with<strong>in</strong> the Bufonoi<strong>de</strong>a by means of the sperm tail<br />
conformation.<br />
Poster 12 - Neuroendocr<strong>in</strong>e Control of <strong>Reproduction</strong><br />
P348<br />
The preovulatory LH surge <strong>in</strong> the ewe appears to be<br />
essentially timed by the hypothalamus<br />
Ben Saïd, S 1 *; Clarke, IJ 2 ; Lomet, D 1 ; Caraty, A 1<br />
1UMR Physiologie <strong>de</strong> la <strong>Reproduction</strong> et <strong>de</strong>s Comportements, INRA-CNRS-<br />
Université Tours/Haras Nationaux, 37380 Nouzilly, France ; 2 Department of<br />
Physiology, Monash University 3800, Australia<br />
The <strong>in</strong>terval between the luteal regression and the preovulatory LH<br />
surge is longer <strong>in</strong> high fecundity breeds like the Romanov (ROM),<br />
than <strong>in</strong> breeds with lower fecundity, like the Ile <strong>de</strong> France (IF). This<br />
difference <strong>in</strong> the surge onset persists when ovariectomized (OVX)<br />
ewes of the two genotypes are challenged with an exogenous estradiol<br />
(E) signal (Ben saïd et al, 2007). It has been suggested (Clarke, 1995)<br />
that the preovulatory LH surge is the result of a coord<strong>in</strong>ated positive<br />
effect of E on the bra<strong>in</strong> and pituitary and we hypothesized that a time<br />
difference <strong>in</strong> the <strong>in</strong>crease of pituitary sensitivity to E may exist<br />
between the two breeds.<br />
To test this hypothesis, we utilised the mo<strong>de</strong>l of OVX Hypothalamo-<br />
Pituitary-Disconnected (HPD) ewes receiv<strong>in</strong>g regular GnRH pulses<br />
and monitored the effect of a preovulatory E signal on the LH<br />
pituitary response <strong>in</strong> both genotypes dur<strong>in</strong>g 2 successive artificial<br />
cycles. Pituitary responsiveness was ma<strong>in</strong>ta<strong>in</strong>ed with hourly i.v<br />
<strong>in</strong>jections of 250 ng GnRH (cycle 1) or 500 ng GnRH (cycle2),<br />
throughout the experiment. After 12 days treatment with vag<strong>in</strong>al<br />
progesterone implants, and 24 h after removal, a preovulatory E signal<br />
was given (s.c. <strong>in</strong>sertion of 2 x 3cm E implants) to the two genotypes<br />
(4 ROM, 5 IF). LH secretion was monitored by sampl<strong>in</strong>g jugular<br />
blood every 10 m<strong>in</strong> for 30 h start<strong>in</strong>g 4-5 h before E adm<strong>in</strong>istration.<br />
Before the E signal, and for the two GnRH doses, the amplitu<strong>de</strong> of the<br />
LH pulses was higher <strong>in</strong> ROM ewes compared to IF ewes (1.3 ± 0.2<br />
ng/ml vs 0.5 ± 0.1 ng/ml for 250 ng/pulse (P< 0.05) and 2.1 ± 0.1<br />
ng/ml vs 1.0 ± 0.0 ng/ml (P< 0.01) for 500 ng/pulse respectively,<br />
values are mean ± SEM). Interest<strong>in</strong>gly, around 10 h after E <strong>in</strong>sertion<br />
and for the two breeds, an <strong>in</strong>crease <strong>in</strong> LH concentration occurs<br />
result<strong>in</strong>g of both an <strong>in</strong>crease of the basal level and of the GnRH<strong>in</strong>duced<br />
pulses amplitu<strong>de</strong>. Therefore, this E <strong>in</strong>duced “LH discharge”<br />
occurred earlier <strong>in</strong> the OVX-HPD-ROM ewes than <strong>in</strong> OVX-ROM<br />
ewes treated by the same E signal.<br />
In summary our results show that <strong>in</strong> the absence of hypothalamic<br />
<strong>in</strong>put, but with stable GnRH <strong>in</strong>put, the <strong>in</strong>crease <strong>in</strong> pituitary sensitivity<br />
occurs earlier than <strong>in</strong> Hypothalamo-Pituitary-Intact animals and this<br />
difference of tim<strong>in</strong>g is more pronounced <strong>in</strong> ROM ewes. The likely<br />
explanation is that the latency to the onset of the LH surge is timed by<br />
a negative feedback effect of E at the hypothalamic level which is<br />
longer operative <strong>in</strong> ROM ewes. A possible role of other <strong>in</strong>hibitory<br />
factors of hypothalamic or pituitary orig<strong>in</strong> cannot be totally exclu<strong>de</strong>d.<br />
P349<br />
Transgenic domestic cloned kittens produced by<br />
lentivector-mediated transgenesis<br />
Gómez, MC 1 *, Pope, CE 1 , Kutner, R 2 , Ricks, DM 2 , Lyons, LA 3 , Truhe, M 3 ,<br />
Dresser, BL 1 , Reiser, J 2<br />
1Audubon Center for Research of Endangered Species, Audubon Nature<br />
Institute, United States; 2 Department of Medic<strong>in</strong>e, Gene Therapy Program,<br />
Louisiana State University, United States; 3 School of Veter<strong>in</strong>ary Medic<strong>in</strong>e,<br />
University of California Davis, United States<br />
Introduction The domestic cat exhibits 90% homology to putative<br />
genes of humans. <strong>Domestic</strong> cats carry<strong>in</strong>g mutant human genes<br />
associated with hereditary diseases would provi<strong>de</strong> a powerful tool for<br />
study<strong>in</strong>g human disor<strong>de</strong>rs and <strong>de</strong>velop<strong>in</strong>g gene therapy strategies. In<br />
the present study, we evaluated 1) whether domestic cat cloned<br />
blastocysts reconstructed with donor cells transduced with eGFPencod<strong>in</strong>g<br />
LV-vector carry<strong>in</strong>g the human ubiquit<strong>in</strong> (hUbC) promoter<br />
expressed the <strong>in</strong>corporated transgene and, 2) the <strong>in</strong> vivo viability of<br />
transgenic cloned embryos after transfer <strong>in</strong>to recipients.<br />
Materials and methods High titer stocks of eGFP-encod<strong>in</strong>g LVvectors<br />
bear<strong>in</strong>g hUbC promoter were generated for transduction of<br />
donor cells. <strong>Domestic</strong> cat fetal fibroblasts (CFF) were <strong>in</strong>fected<br />
overnight with 82.000.000 IU/ml of a LV-UbC-eGFP vector stock.<br />
Mature oocytes collected from cat donors were enucleated and a<br />
s<strong>in</strong>gle eGFP-positive CFF was <strong>in</strong>troduced <strong>in</strong>to the perivitell<strong>in</strong>e space<br />
of each oocyte. Fusion was <strong>in</strong>duced by apply<strong>in</strong>g two electrical pulses<br />
and fused couplets were activated 2 h later and placed <strong>in</strong> culture.<br />
Transgene expression <strong>in</strong> cloned embryos was evaluated by observ<strong>in</strong>g<br />
green fluorescence of blastomeres (Days 2, 5, and 8). To analyze LVvector<br />
copies, qPCR quantification was performed on genomic DNA<br />
<strong>de</strong>rived from s<strong>in</strong>gle blastocysts. A total of 186 transgenic cloned<br />
embryos were transferred by laparoscopy to the oviduct of five<br />
synchronous domestic cat recipients. The recipients were exam<strong>in</strong>ed by<br />
ultrasonography on day 22 to <strong>de</strong>term<strong>in</strong>e pregnancy status.<br />
Results Cleavage rate (D2; 44/52=85%) and blastocyst <strong>de</strong>velopment<br />
(D8; 5/44=11%) of reconstructed embryos with hUbC promoterconta<strong>in</strong><strong>in</strong>g<br />
LV vectors was similar to those obta<strong>in</strong>ed previously with<br />
hCMV-IE and hEF1 alpha promoter-conta<strong>in</strong><strong>in</strong>g LV vectors. On day 2,<br />
32% of the cloned embryos expressed green fluorescence, and by day<br />
5, the percentage of embryos express<strong>in</strong>g the eGFP transgene <strong>in</strong>creased<br />
to 41%. By day 8, all embryos displayed susta<strong>in</strong>ed transgene<br />
expression and blastocysts (n=5) exhibited green fluorescence. In<br />
blastocysts, each blastomere carried from 0.7 to 4 copies of the<br />
provirus. Two (40%) recipient cats were pregnant when exam<strong>in</strong>ed on<br />
day 22. Five embryos (2.7%) had implant <strong>in</strong> two recipients. Three<br />
fetuses were reabsorbed by day 39 and two near-term died <strong>in</strong> utero on<br />
day 55 of gestation. The clonal status of the transgenic cloned kittens<br />
was assessed by a standardize DNA i<strong>de</strong>ntification panel for cats.<br />
eGFP transgene expression was <strong>de</strong>tectable by fluorescence imag<strong>in</strong>g of<br />
cloned kittens.<br />
P350<br />
Ontogeny of the daily rhythm <strong>in</strong> plasma melaton<strong>in</strong><br />
concentrations dur<strong>in</strong>g postnatal <strong>de</strong>velopment <strong>in</strong> wild and<br />
domestic ewes<br />
Gómez-Brunet, A 1 *; Santiago-Moreno, J 1 ; Chem<strong>in</strong>eau, P 2 ; Malpaux, B 2 ;<br />
Lopez-Sebastian, A 1<br />
1Departamento <strong>de</strong> Reproducción Animal, SGIT-INIA, Madrid, Spa<strong>in</strong>;<br />
2Physiologie <strong>de</strong> la <strong>Reproduction</strong> et <strong>de</strong>s Comportements, UMR INRA-CNRS-<br />
Université <strong>de</strong> Tours-Haras Nationaux, Nouzilly, France<br />
In seasonal reproductive species, melaton<strong>in</strong>, hormone synthesized and<br />
released <strong>in</strong>to the general circulation with a marked day-night rhythm,