Reproduction in Domestic Animals - Facultad de Ciencias Veterinarias

16 t h International Congress on Animal Reproduction
142 Poster Abstracts
other dendrobatid frogs. But sacrificing endangered animals should be
avoided.
Sperm samples are obtained after gentle hormonal stimulation with
human chorionic gonadotropin (hCG) cloaca lavage and light
microscopic evaluation. Sperm cells are filiform with an arcuated and
21.1 µm long head and a single tail (35.0 µm length). Their acrosomal
complex is located at the anterior portion of the head and consists of
the acrosomal vesicle with low electron density, and the subjacent
electron-dense subacrosomal cone. The nucleus is circular in
transverse section (1.9 µm diameter), and conical in longitudinal
section. It is surrounded by several groups of mitochondria. The
highly condensed chromatin is electron-dense but shows numerous
electron-lucent inclusions. A short midpiece has a mitochondrial
collar with a proximal and a distal centriole. The latter gives rise to
the axoneme which alone forms the flagellum.
The sperm ultrastructure of D. auratus differs from that of other
Dendrobatidae because of the absence of a nuclear space and the
missing undulating membrane associated with an axial fibre. In
conclusion, it can be stated that the spermatozoa of D. auratus are the
first within the Dendrobatidae without accessory tail structures. This
tail conformation is analogue to these of Ranoidea and not
Bufonoidea. This observation is in contrary to Garda et al. (2002) and
Aguir-Jr. et al. (2004) who grouped the Dendrobatidae within the
Bufonoidea. Our findings indicate that the whole dendrobatid family
cannot be grouped within the Bufonoidea by means of the sperm tail
conformation.
Poster 12 - Neuroendocrine Control of Reproduction
P348
The preovulatory LH surge in the ewe appears to be
essentially timed by the hypothalamus
Ben Saïd, S 1 *; Clarke, IJ 2 ; Lomet, D 1 ; Caraty, A 1
1UMR Physiologie de la Reproduction et des Comportements, INRA-CNRS-
Université Tours/Haras Nationaux, 37380 Nouzilly, France ; 2 Department of
Physiology, Monash University 3800, Australia
The interval between the luteal regression and the preovulatory LH
surge is longer in high fecundity breeds like the Romanov (ROM),
than in breeds with lower fecundity, like the Ile de France (IF). This
difference in the surge onset persists when ovariectomized (OVX)
ewes of the two genotypes are challenged with an exogenous estradiol
(E) signal (Ben saïd et al, 2007). It has been suggested (Clarke, 1995)
that the preovulatory LH surge is the result of a coordinated positive
effect of E on the brain and pituitary and we hypothesized that a time
difference in the increase of pituitary sensitivity to E may exist
between the two breeds.
To test this hypothesis, we utilised the model of OVX Hypothalamo-
Pituitary-Disconnected (HPD) ewes receiving regular GnRH pulses
and monitored the effect of a preovulatory E signal on the LH
pituitary response in both genotypes during 2 successive artificial
cycles. Pituitary responsiveness was maintained with hourly i.v
injections of 250 ng GnRH (cycle 1) or 500 ng GnRH (cycle2),
throughout the experiment. After 12 days treatment with vaginal
progesterone implants, and 24 h after removal, a preovulatory E signal
was given (s.c. insertion of 2 x 3cm E implants) to the two genotypes
(4 ROM, 5 IF). LH secretion was monitored by sampling jugular
blood every 10 min for 30 h starting 4-5 h before E administration.
Before the E signal, and for the two GnRH doses, the amplitude of the
LH pulses was higher in ROM ewes compared to IF ewes (1.3 ± 0.2
ng/ml vs 0.5 ± 0.1 ng/ml for 250 ng/pulse (P< 0.05) and 2.1 ± 0.1
ng/ml vs 1.0 ± 0.0 ng/ml (P< 0.01) for 500 ng/pulse respectively,
values are mean ± SEM). Interestingly, around 10 h after E insertion
and for the two breeds, an increase in LH concentration occurs
resulting of both an increase of the basal level and of the GnRHinduced
pulses amplitude. Therefore, this E induced “LH discharge”
occurred earlier in the OVX-HPD-ROM ewes than in OVX-ROM
ewes treated by the same E signal.
In summary our results show that in the absence of hypothalamic
input, but with stable GnRH input, the increase in pituitary sensitivity
occurs earlier than in Hypothalamo-Pituitary-Intact animals and this
difference of timing is more pronounced in ROM ewes. The likely
explanation is that the latency to the onset of the LH surge is timed by
a negative feedback effect of E at the hypothalamic level which is
longer operative in ROM ewes. A possible role of other inhibitory
factors of hypothalamic or pituitary origin cannot be totally excluded.
P349
Transgenic domestic cloned kittens produced by
lentivector-mediated transgenesis
Gómez, MC 1 *, Pope, CE 1 , Kutner, R 2 , Ricks, DM 2 , Lyons, LA 3 , Truhe, M 3 ,
Dresser, BL 1 , Reiser, J 2
1Audubon Center for Research of Endangered Species, Audubon Nature
Institute, United States; 2 Department of Medicine, Gene Therapy Program,
Louisiana State University, United States; 3 School of Veterinary Medicine,
University of California Davis, United States
Introduction The domestic cat exhibits 90% homology to putative
genes of humans. Domestic cats carrying mutant human genes
associated with hereditary diseases would provide a powerful tool for
studying human disorders and developing gene therapy strategies. In
the present study, we evaluated 1) whether domestic cat cloned
blastocysts reconstructed with donor cells transduced with eGFPencoding
LV-vector carrying the human ubiquitin (hUbC) promoter
expressed the incorporated transgene and, 2) the in vivo viability of
transgenic cloned embryos after transfer into recipients.
Materials and methods High titer stocks of eGFP-encoding LVvectors
bearing hUbC promoter were generated for transduction of
donor cells. Domestic cat fetal fibroblasts (CFF) were infected
overnight with 82.000.000 IU/ml of a LV-UbC-eGFP vector stock.
Mature oocytes collected from cat donors were enucleated and a
single eGFP-positive CFF was introduced into the perivitelline space
of each oocyte. Fusion was induced by applying two electrical pulses
and fused couplets were activated 2 h later and placed in culture.
Transgene expression in cloned embryos was evaluated by observing
green fluorescence of blastomeres (Days 2, 5, and 8). To analyze LVvector
copies, qPCR quantification was performed on genomic DNA
derived from single blastocysts. A total of 186 transgenic cloned
embryos were transferred by laparoscopy to the oviduct of five
synchronous domestic cat recipients. The recipients were examined by
ultrasonography on day 22 to determine pregnancy status.
Results Cleavage rate (D2; 44/52=85%) and blastocyst development
(D8; 5/44=11%) of reconstructed embryos with hUbC promotercontaining
LV vectors was similar to those obtained previously with
hCMV-IE and hEF1 alpha promoter-containing LV vectors. On day 2,
32% of the cloned embryos expressed green fluorescence, and by day
5, the percentage of embryos expressing the eGFP transgene increased
to 41%. By day 8, all embryos displayed sustained transgene
expression and blastocysts (n=5) exhibited green fluorescence. In
blastocysts, each blastomere carried from 0.7 to 4 copies of the
provirus. Two (40%) recipient cats were pregnant when examined on
day 22. Five embryos (2.7%) had implant in two recipients. Three
fetuses were reabsorbed by day 39 and two near-term died in utero on
day 55 of gestation. The clonal status of the transgenic cloned kittens
was assessed by a standardize DNA identification panel for cats.
eGFP transgene expression was detectable by fluorescence imaging of
cloned kittens.
P350
Ontogeny of the daily rhythm in plasma melatonin
concentrations during postnatal development in wild and
domestic ewes
Gómez-Brunet, A 1 *; Santiago-Moreno, J 1 ; Chemineau, P 2 ; Malpaux, B 2 ;
Lopez-Sebastian, A 1
1Departamento de Reproducción Animal, SGIT-INIA, Madrid, Spain;
2Physiologie de la Reproduction et des Comportements, UMR INRA-CNRS-
Université de Tours-Haras Nationaux, Nouzilly, France
In seasonal reproductive species, melatonin, hormone synthesized and
released into the general circulation with a marked day-night rhythm,