2011 (SBTE) 25th Annual Meeting Proceedings - International ...

Acta Scientiae Veterinariae, 2011. 39(Suppl 1): Abstracts - 25th Annual Meeting SBTE-Brazil. August 2011.
A203 EMBRYOLOGY, BIOLOGY OF DEVELOPMENT AND PHYSIOLOGY OF REPRODUCTION
SUPPLEMENTATION TION OF MEDIA FOR IN VITRO MATUR
TURATION TION OF OOCYTES OF RABBIT
ABBITS
Tássia Mangetti Gonçalves, , Juliano Rodrigues Sangalli, Marcos Roberto Chiaratti & Flávio Vieira Meirelles
FZEA- USP, PIRASSUNUNGA, SP, BRAZIL.
In vitro maturation is an important tool for biotechnologies. The importance of rabbits in this studies is due to the later embryonic
genome activation in this species, that turns it into an efficient model for dedifferentiate of somatic cell nuclei from many species (eg, pandas,
chickens, monkeys, cats and humans), supporting the development of interspecies nuclear transfer (WEN et al., 2005, Journal of Experimental
Zoology, 303:689-697). This study aimed to test supplementations of media for in vitro maturation of oocytes donated by immature rabbits using
a protocol commonly used with bovine oocytes. During in vitro maturation (IVM) of rabbit oocytes it was tested addition of 0.01% PVA or
0.003% BSA or control (no addition) in TCM 199 (10 mg/mL of FSH and 10 mg/mL of LH). After 20 h of IVM, oocytes denuded of cumulus
cells by gentle pipetting in 0.2% hyaluronidase were analyzed for the presence of first polar body (1st PB). Due to the low rate of 1st PB
extrusion, 1st PB-selected oocytes from the three experimental groups were activated together (5 mM ionomycin for 5 min and 2 mM 6-DMAP
for 4 h) (Tao et al., 2008, Journal of Animal Physiology and Animal Nutrition, 92:438-447). Presumptive zygotes were cultured in CR2aa in a
humidified atmosphere of 5% CO2 and 5% O2 for a maximum period of 4 to 5 days. Embryos were evaluated regarding their development to
morulae or blastocyst stages and the number of nuclei. Four independent experiments were carried out. The extrusion rate of 1st PB was
compared between groups using Tukey (P < 0.05). A significant increase in the rate of 1st PB extrusion was found when supplementation with
BSA (13.6%, 40/294) or PVA (12.6%, 34/270) was performed in comparison to no supplementation (6.3%; 21/333). When 1st PB oocytes
were activated, it was found that 69.5% (66/95) of them degenerated before first cleavage, 20% (19/95) blocked at 2-4 cell stage, 7.4% developed
into morulae, and 3.1% (3/95) reached the blastocyst stage. Thereafter, a positive effect of supplementation with BSA or PVA during maturation
was seen. However, the number of oocytes that extruded the 1st PB was very low. Furthermore, the rate of blastocyst formation was also very
low as well. These results suggest that the use of media commonly employed for bovine embryo production is not suitable for embryo production
using rabbit oocytes. Alternatively, the poor developmental rates found may have been caused by the use of oocytes from immature rabbits and,
therefore, with lower developmental potential.
Keywords: supplementation, rabbits, ivm.
A204 EMBRYOLOGY, BIOLOGY OF DEVELOPMENT AND PHYSIOLOGY OF REPRODUCTION
USE OF C57BL/6/EGFP MOUSE TESTICULAR CELLS TO VALIDA
ALIDATE
THE TECHNIQUE OF MICROINJECTION IN
EMBRYONIC CHIMERA PRODUCTION
Daniela Motta Souza, , Hugo Fernandes, Patrícia Villela Silva, Bruno Cazari, Pablo Diego Moço, Bruna Castilho Soto Campanha,
Isabele Picada Emanuelli & Marcelo Fábio Gouveia Nogueira
UNESP, ASSIS, SP, BRAZIL.
Among the techniques to produce chimeras, microinjection (MI) of embryonic stem cells (ESC) into blastocysts - or in the
perivitelline space (PVS.) of the embryos with 4-8 cells - is one of most popular. A well-established training model for this technique could be
very useful when ESC were not available and that was able to identify the injected cellular components and their subsequent aggregation within
the embryo. Hence, we aim to validate, in mice, a training model for MI in embryos (Swiss Webster, SW) using a pool of EGFP cells derived
from testis of C57BL/6/EGFP strain. Embryos were recovered from prepubertal females SW (n = 20) superstimulated and mated according to
Mancini et al. (2008; Transgenic Res 17:1015). As an alternative to the classical MI in blastocysts (Nagy et al., 2003; Manipulating the Mouse
Embryo 3rd edn., CSHL), in this study were used 4 to 8 cells embryos (collected at 2.5 days post coitum). Embryos from the same female were
randomly allocated to three groups: control (C, n = 17), embryos not subjected to MI; perforated (P, n = 15), embryos submitted to perforation
by micropipette, without cell injection; and microinjected (MI, n = 32), embryos perforated and submitted to PVS. injection with 6 to 8 cells from
EGFP testis. After manipulation, embryos from all groups underwent 24 h of in vitro culture (37°C, 5% CO 2
and saturated humidity). The
viability and quality of the embryos (according to the general and specific criteria; IETS Manual, 1998 and Nagy et al., 2003, respectively) and,
in group MI, the fluorescence of testicular cells, were evaluated pre and post-culture (bright field and under UV light for group MI). The results
were analyzed by X 2 test (total frequency observed) and ANOVA (considering the four replicates) with significance being considered when P
< 0.05. There was no difference among mortality rates of the groups (5.9, 26.7 and 25.0% for C, P and MI, respectively). The percentage of
embryos that have retained the quality, after 24 h of culture, was different (P < 0.01) among groups C, P and MI (94.1, 73.3 and 43.8%,
respectively). It was obtained one chimeric blastocyst in the MI group (3.1%, 1/32). Considering the proposed conditions, this model for training
of MI of EGFP testicular cells in the PVS. was feasible and practical to acquire skills, when ESC are available. The method allows easy
identification of injected and, eventually, aggregated cellular components. The source for EGFP testicular cells could be obese and senile males
(scheduled to be sacrificed) from local animal house or refrigerated testis sent from a distant animal facility.
Keywords: microinjection, mice, egfp testicular cells.
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