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

Acta Scientiae Veterinariae, 2011. 39(Suppl 1): Abstracts - 25th Annual Meeting SBTE-Brazil. August 2011.
A161 EMBRYOLOGY, BIOLOGY OF DEVELOPMENT AND PHYSIOLOGY OF REPRODUCTION
USE OF β-MER
-MERCAPT
APTOETHANOL FOR IN VITRO MATUR
TURATION TION AND CULTURE OF OVINE EMBRYOS
Jorgea Pradieé 1 , Liziane Lemos Vianna 1 , Alexander Oliveira Gonçalves 1 , Elisa Caroline da Silva Santos 1 , Rafael Gianela Mondadori 1 , Thomaz Lucia Jr. 1 ,
Arnaldo Diniz Vieira 1 & Ligia Margareth Cantarelli Pegoraro 2
1
UNIVERSIDADE FEDERAL DE PELOTAS, PELOTAS, RS, BRAZIL. 2 EMBRAPA, PELOTAS, RS, BRAZIL.
One of the factors limiting the success of ovine IVP is the formation of reactive oxygen species (ROS) resulting from cell
metabolism. The action of the ROS can be physiologically inhibited by antioxidants such as glutathione (GSH). As GSH’s concentration is
reduced during most steps of the IVP, the media used for IVP are commonly supplemented with antioxidant substances. This study evaluated
the development of ovine embryos after supplementation of the media for IVM and IVC with 20 µM β-mercaptoethanol (βME), a GSH
precursor. Ovaries from pubertal sheep were collected in slaughterhouse and transported to the laboratory in a saline antibiotic solution at 30ºC.
Cumulus-oocytes complexes (COC) were recovered and selected for IVM, which was conducted during 22-24 h, using the 199 TCM medium
including estradiol, FSH, LH, pyruvate, estrus sheep serum and antibiotics. For this experiment 6 replicates were performed. Further comparisons
were done between the IVM medium with (n = 328) or without (n = 320) βME. Sperm selection was conducted though swim-up method using
fresh sperm and tris-citric acid-glucose medium. The IVF was done with sperm concentration of 1 x 106/mL in SOF medium with 2% estrus
sheep serum. Thereafter, presumptive zygotes were denuded and cultured for 8 days in SOFaa medium and 2.5% FCS with or without âME.
The IVM, IVF and IVC were conducted in an incubator with 5% CO2 and saturated humidity, at 39ºC. The rates of embryo development at D2
(cleavage) and D8 (blastocyst) were compared between treatments through the chi-square test. Although cleavage rates were the same (70%) for
medium with or without âME, the blastocyst formation rate was greater (P < 0.001) for the control (17%) than for the βME-supplemented
medium (5%). Therefore, supplementation of IVM and IVC medium with âME at the tested concentration did not benefit cleavage rates and was
associated with reduced blastocyst rate. Further studies are still needed to investigate potential benefits of supplementation with âME at distinct
concentration for the development of ovine embryos.
Keywords: sheep, embryo, antioxidant.
A162 EMBRYOLOGY, BIOLOGY OF DEVELOPMENT AND PHYSIOLOGY OF REPRODUCTION
ADDITION OF NERVE GROW TH FACT
CTOR ON MATUR
TURATION TION AND DEVEL
VELOPMENT MEDIA FOR IN VITRO
PRODUCTION OF SHEEP EMBRYOS
M. Vilar
ilariño
iño 1 , M. Crisp
ispo 2 , P.C.
dos San
antos-N
os-Net
eto 1 , R. Wijma
1 , N. Bar
arrer
era 1 , A. de León
2 , L. Barb
arbeit
eito 2 & A. Menchac
enchaca 1
1
FUNDACIÓN IRAUY, MONTEVIDEO, URUGUAY. 2 INSTITUTO PASTEUR DE MONTEVIDEO, MONTEVIDEO, URUGUAY.
Nerve Growth Factor (NGF) is a neurotrophic factor that promotes survival and proliferation in different types of non-neuronal
cells. In the present study we assessed whether NGF could improve the in vitro embryo production in sheep. A total of 1342 cumulus oocyte
complexes (COCs) covered by at least two layers of granulosa cells and homogeneous cytoplasm were selected for in vitro maturation (IVM)
after aspiration from ovine ovaries obtained in a local slaughterhouse. The selected COCs were incubated in groups of 25-30 in 100 µL drops
of TCM199 supplemented with 10% estrous ovine serum, 10 µg/mL FSH, 10 µg/mL LH, 100 ìM cysteamine and antibiotics; covered with
embryo tested mineral oil for 24 h, at 39°C and 5% CO2 in air. For in vitro fertilization (IVF), matured oocytes (25-30/100 µL drop) were
incubated for 22 h in fertilization medium with 1x106 frozen-thawed spermatozoa selected by swim-up method. The fertilization medium
consisted of synthetic oviductal fluid (SOF) supplemented with 2% estrous ovine serum, 10 ìg/mL heparin and 10 ìg/mL hypotaurine. In vitro
development (IVD) was performed in groups of 25-30 zygotes in 100 ìL drops of SOFaa BSA covered with mineral oil at 39°C and 5% O2,
5%CO2, 90% N2. NGF was evaluated using 0, 100 or 1000 ng/mL during IVM (0 ng/mL, n = 226; 100 ng/mL, n = 264; and 1000 ng/mL, n
= 286), or IVD (0 ng/mL, n = 191; 100 ng/mL, n = 189 and 1000 ng/mL, n = 186). Cleavage rate (2 cell embryos/oocytes) and development/
rate (morula and blastocysts/oocytes) were recorded after 48 h and 6 d in culture after fertilization, respectively. Statistical analysis was performed
by logistic regression. For IVM, no differences were found in the cleavage rate (46%, 104/226; 54.5%, 144/264; and 50%, 143/286), the
development rate (12.4%, 28/226; 17.8%, 47/264; and 13.6%, 39/286), and the developed/cleaved rate (26.9%, 28/104; 32.6%, 47/144; and
27.3%, 39/143) for 0, 100 and 1000 ng/mL NGF, respectively. When NGF was used during IVD, no differences were found in the development
rate (41.4%, 79/191; 45.5%, 86/189; and 46.8%, 87/186) and developed/cleaved rate (53.7%, 79/147; 57%, 86/151; and 56.5%, 87/154) for 0,
100 and 1000 ng/mL NGF, respectively. These results suggest that the addition of NGF in the maturation or development media does not affect
the number of in vitro produced sheep embryos.
Keywords: ngf, in vitro production, embryos sheep.
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