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

Acta Scientiae Veterinariae, 2011. 39(Suppl 1): Abstracts - 25th Annual Meeting SBTE-Brazil. August 2011.
A215 CLONING, TRANSGENESIS AND STEM CELLS
DEVEL
VELOPMENT OF BOVINE CLONE EMBRYOS USING FIBROBL
OBLAST
ASTS S CULTURED IN SOF SUPPLEMENTED WITH CONJUGA
ONJUGATED
LINOLEIC ACID (C-9,T-11)
Norton Klein 1 , Lain Uriel Ohlweiler 1 , Fábio Gallas Leivas 2 , Daniela dos Santos Brum 2 , Maicon Gaissler Lorena Pinto 1 , Fernanda Gemelli 1 , Nerissa Albino 1 ,
Laura Tais Nickelle Sasse
1 , Joana Cláudia Mezzalir
zzalira 1 & Alc
lceu Mezzalir
zzalira 1
1
LAB. DE REPRODUÇÃO ANIMAL PROF. ASSIS ROBERTO DE BEM, UNIVERSIDADE DO ESTADO DE SANTA CATARINA, (CAV/UDESC), LAGES, SC, BRAZIL. 2 BIOTECH, LAB. DE
BIOTECNOLOGIA DA REPRODUÇÃO, UNIVERSIDADE FEDERAL DO PAMPA, (UNIPAMPA), URUGUAIANA, RS, BRAZIL.
The standard protocol for cell culture is the one using DMEM-based media, whereas the standard medium for bovine embryo
culture is SOF. The culture of fibroblasts anticipates one step of the cloning procedure as it might enhance the further embryo culture, with cells
already adapted to SOF conditions. The Conjugated Linoleic Acid (CLA c-9, t-11) plays a role on reducing the anabolism of certain cell types,
then it might contribute for the reprogramming process. This study evaluated the development of clone embryos in which the donor (fibroblast)
cell was previously cultured or not with CLA prior to the reconstruction process. For that, cells on second passage were cultured in DMEM
added of 10% fetal calf serum (FCS) at 38.5ºC, in atmosphere containing 5% CO 2
and saturated humidity during 72 h. In a previous experiment,
we observed the viability of cells cultured in SOF. Six hours prior to cell fusion, DMEM medium was replaced by SOF added of 5% (G1) and
supplemented with 100 µM of c9, t11 CLA (Matreya, ref.001249) and 0.1% ethanol (G2). The SCNT cloning was performed with MII oocytes
as recipient cytoplasts according to Mezzalira et al. (2011, Cell. Reprog., 13, 65-76), being cleavage and blastocyst rates (D7), in 3 replications.
Data were analyzed through the Chi-square test (where P < 0.05 was considered significant). Group G2 presented better cleavage rate (94.5%,
104/110) in comparison to G1 (81.3%, 100/123), however blastocyst rates were not statistically different between G1 (31.7%) and G2 (36.4%).
Aiming to obtain at least one pregnancy for each treatment,as an additional evaluation step, D8 blastocysts were transferred, in pairs, to
synchronous recipient cows. Ultra-sound guided pregnancy diagnosis was performed on day 30, 60 and 90 after inovulation. Five cows were
transferred with G1 embryos and were detected pregnant (100%) on day 30, being rates respectively 40% at day 60 and 20% (n = 1) at day 90.
The only recipient cow transferred with fresh G2 embryos was detected pregnant on day 30 and maintains pregnancy up to day 80. In conclusion,
the use of CLA for cells treatment prior to reconstruction on SCNT enhances embryo development performance, at least during the first cell
divisions. Embryos produced in both experimental groups showed to be efficient on in vivo early development, but additional investigations are
needed in order to evaluate the nucleus donor treatment with CLA effect in the viability of viable offspring. [Financial support: UDESC, CNPQ
471330/2009-4].
Keywords: HMC, Reprogramming, c9 and t11 cla.
A216 CLONING, TRANSGENESIS AND STEM CELLS
PREGNANCY OF CLONED EMBRYOS FROM A FREEMARTIN COW OF AN ENDANGERED BREED
Joana Cláudia Mezzalira 1 , Maicon Gaissler Lorena Pinto 1 , Fabiano Carminatti Zago 2 , Alysson Macedo Silva 1 , Nerissa Albino 1 ,
Norton Klein 1 , Vilceu Bordignon 3 , Lain Uriel Ohlweiler 1 & Alceu Mezzalira 1
1
UDESC - UNIVERSIDADE DO ESTADO DE SANTA CATARINA, LAGES, SC, BRAZIL. 2 EPAGRI-EMPRESA DE PESQUISA AGROPECUÁRIA E EXTENSÃO RURAL DE SANTA CATARINA,
LAGES, SC, BRAZIL. 3 MCGILL UNIVERSITY, STE. ANNE DE BELLEVUE, QUEBEC, CANADÁ.
The Rouge Flamand (Flemish) cattle were introduced into Brazil in 1945. As a dual-purpose breed, it had easily adapted to the
regional climate conditions and several herds of Rouge Flamand cattle were established. However, during the last years the number of herds has
been considerably reduced due to specialized breeds import. There are about fifty remaining animals at the experimental station Epagri (Empresa
de Pesquisa Agropecuária e Extensão Rural do Estado de Santa Catarina) located in Lages, Santa Catarina, but they are threatened with extinction
due to imminent lack of genetic variability. One of these remaining animals is a freemartin cow, which is about 17 years old and extremely wellconformed,
though infertile. Over the last years, a number of animals of different species have been produced by somatic cell nuclear transfer
(SCNT) for different purposes. The main objective in this work is to use SCNT to rescue the genotype of a Rouge Flamand freemartin cow and
produce a fertile animal. Somatic cells were obtained from an ear skin biopsy through the explantation technique. Up to the third passage and after
obtaining at least 90% confluence, cells were cryopreserved in 0.25mL straws in DMEM + 10% fetal calf serum and 10% DMSO. The
handmade cloning steps were performed according to Mezzalira et al. (2011, Cell. Reprog, 13, 65-76). Cleavage and blastocyst rates were
respectively 87% (204/233) and 34% (80/233). Recipient cows of tricross European breeds were used as recipient females at day seven after
estrus and D8 blastocysts were transferred. Part of the SCNT blastocysts were vitrified/thawed according to Mezzalira et al. (2010, Reprod. Fert.
Dev, 22, 210). The remaining blastocysts were maintained fresh and were transferred in pairs to each recipient. From the six recipient cows that
received fresh embryos, 6 (100%) established pregnancy; 3 (50%) were pregnant up to 60 days and 2 (33%) are still pregnant at 80 and 180 days.
Vitrified/thawed embryos were transferred to two recipient cows, which were both confirmed pregnant but embryos died by day 45 after transfer.
For the first time, this study shows that SCNT embryos derived from a freemartin animal can develop into blastocyst and establish pregnancies
after it transfer to recipient cows.
Keywords: cell reprogramming, freemartin cattle, genetic biodiversity.
s444