Reproduction in Domestic Animals - Facultad de Ciencias Veterinarias

16 t h International Congress on Animal Reproduction
Poster Abstracts 197
Kg) of zoo (ZOO SAFARI-Fasano-BR). Ovaries were kept in
physiological saline and maintained at 35°C before oocyte recovery.
Ovary were placed in phosphate buffered saline (PBS) supplemented
with 50 micrograms/ml of gentamicin. Each ovary was sliced
repeatedly with scalpel blade to release cumulus-oocyte complex
(COCs) in a 90 mm culture dish containing PBS at 37°C. COCs were
immediately fixed in formalin (2%) and they remain over-night at 4°C
temperature. Subsequently oocyte were collect and fixed on slide two
each. Only one oocyte was collect by leopard ovaries. Oocytes was
stained with Hoechst 33258 and observed at fluorescence microscope
(E600 Nikon, 365 nm excitation) for assess the meiotic status.
Evaluation of oocytes diameter was done by stereomicroscope (Nikon
ECLIPSE 50 I).
Results Cat oocyte external diameter (100 micron on average) was
smaller than in leopard (230 micron on average), however the
nucleus-cytoplasm ratio is approximately the same 0,13 micron (14,17
micron GV diameter Vs 109 micron oocyte diameter for cat’s oocyte)
vs 0,17 micron (50,13 micron GV diameter Vs 229,65 micron oocyte
diameter for leopard oocytes).
Conclusions Though the effective size of leopard oocytes is greater
than cat’s oocytes the proportion of oocytes share are keeped. Is only
the first study that shown the size of oocyte in leopard, and other
research needed. Knowledge of morphology and size is important for
subsequent in vitro maturation (IVM) an in vitro fertilization (IVF)
for maintenance of genetic diversity in dangerous species.
P514
Correlation analyses of a porcine in vitro production
system for prediction of blastocyst rates
Petersen, KM*, Avery, B; Schmidt, M; Bogh, IB
Veterinary Obstetrics and R, Faculty of Life Sciences, University of
Copenhagen, Denmark
The aim was to find the best predictor for blastocyst formation in a
standard porcine IVP system. For that purpose blastocyst rates were
compared with 2-pronuclear-, polyspermy- and cleavage rates, where
rates were calculated over total number of IVF oocytes, and the data
subjected to correlation analyses (GraphPad Prism 3), under the
assumption that when two variables vary together, there is a
correlation between them. The correlation coefficient r, which ranges
from – 1 to + 1, quantifies the direction and magnitude of correlation,
and how well X and Y vary together. The best way to interpret the
value of r is to calculate r 2 , which ranges from zero to one, and is the
fraction of the variance in the two variables that is shared. For
example, if r 2 =0.25 then 25 % of the variance in X can be explained
by variation in Y. Linear regression assumes that the data are linear,
and finds the line that best predicts Y from X, according to the
equation Y = αX + B. An r 2 value of 0 means that there is no linear
relationship between X and Y, where an r 2 value of 1 indicates a
perfect correlation. The oocytes were collected from slaughterhouse
sow ovaries, IVM was performed for 44 h in TCM-199 with EGF,
hCG, eCG and 10 % ECS, IVF for 24 h in a Krebs-Ringer bicarbonate
based solution with 2 mM caffeine and 0.6 % BSA, supplemented
with washed semen, originating from fresh ejaculates in extender, and
IVC for a week in PZM with 5 % ECS. IVM and IVF incubations
were done under 5 % CO 2 in 95 % air, and IVC in 5 % CO 2 , 5 % O 2 ,
and 90 % N2 at 38.5 º C. Blastocyst and cleavage rates were assessed
at day 6 post insemination, penetration rates after fixation 24 h post
IVF in acid methanol and subsequent Orcein staining. In conclusion,
none of the combinations showed perfect correlation; however the
most precise predictor for blastocyst formation was the 2-pronuclear
rates, which showed a significant correlation, whereas the correlations
for polyspermy or cleavage were very weak (low r 2 and high P). The
weak correlation for the cleavage rate is probably due to the fact that
cleaved embryos are a mix of 2-PN and polyspermic oocytes, which
pulls in opposite directions with regard to blastocyst formation.
P515
Influence of oocyte and embryo transport interval time on
efficiency and commercial profitability of a large bovine
in vitro embryo production scale
Rodrigues, JL 1 *, Queiroz, LM 2 , Feltrin, C 1 , Peixer, M 2 , Malard, P 2 , Santana,
G 2 , Xavier, M 2 , Rodrigues, B 1
1Laboratory of Embryology and Biotechnics of Reprod, Faculty of Veterinary
Medicine, Federal University of Rio Grande do Sul, Brazil; 2 Animal
Biotechnology, BIO, Brazil
Introduction During the last 5 years the extraordinary increase in the
number of Nelore females submitted to ultrasound guided follicular
aspiration (ovum pick up - OPU) in Brazil, has leading to the
development of different estrategies to overcome logistical barriers
and achieve profitable pregnancy rates after transfer of IVF embryos.
The objective of this experiment was to evaluate the influence of
interval time transport on viability of oocytes and embryos: Group 1
(G1) 1 to 2 h; Group 2 (G2) 3 to 5 h; Group 3(G3) 6 to 9 h and Group
4 (G4) 10 to 16 h.
Materials and Methods Oocytes were collected by OPU (zero time),
washed in modified PBS solution, and loaded into plastic cryovials
containing modified TCM199 HEPES maintained at 38°C to provide
oocyte in vitro maturation (IVM) during the transport period to the
laboratory. At arrival to the laboratory oocytes were transferred to
modified TCM199 and cultured at 39ºC in a 100% humidified
atmosphere containing 5% CO2. The in vitro fertilization procedure
was initiated 24 hours after OPU, and during the next 24 hours the
oocytes were incubated with the spermatozoa in the same conditions
as described above. After that, the presumptive zygotes were
transferred to modified SOFaa medium and then cultured for 7 days at
the same IVM conditions under atmosphere containing 5% CO2, 5%
O2 and 90 N2 %. On day 7 the embryos were evaluated,
morphologically classified and loaded in 0,25 ml straws containing
modified SOFaa HEPES supplemented with BSA and kept at 38°C.
The embryos were transferred into previously synchronized recipients
and pregnancy diagnosis was performed by ultrasound 60 days later.
Statistical analysis was performed by the Chi-square test (P