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

Acta Scientiae Veterinariae, 2011. 39(Suppl 1): Abstracts - 25th Annual Meeting SBTE-Brazil. August 2011.
A227 SUPPORTIVE BIOTECHNOLOGIES: CRYOPRESERVATION AND CRYOBIOLOGY, IMAGE ANALYSIS AND DIAGNOSIS, MOLECULAR BIOLOGY AND “OMICS”
COMP
OMPAR
ARATIVE ANALYSIS OF MEMBRANE INTEGRITY METHODS OF OVINE CRYOPRESER
OPRESERVED SEMEN
Aline Matos Arrais, Carla Sobrinho Paes de Carvalho, Bruno Pena Carvalho, Fernanda Queiroz Costa, Edgar Maurício Mogollon-Waltero & Angelo José
Burla Dias
UENF, CAMPOS DOS GOYTACAZES, RJ, BRAZIL.
Plasmatic membrane integrity is very important in assessing the viability of frozen sperm, due to the detrimental actions during
freezing. There are several techniques used for such analysis. Among the techniques used to evaluate the plasma membrane, the hypoosmotic
swelling test (HOST) and propidium iodide (PI) are the most used. The aim of this work was to conduct a comparative analysis of these methods
for the evaluation of cryopreserved ovine spermatozoa. Twenty straws of semen were used (10 with ethylene glycol - EG and 10 with glycerol
- Gc). The thawed semen was mixed with 2 mL of TALP and centrifuged. 10 µL of the pellet was removed, and added to the 300 µL of a solution
100 mOsm during 30 min to make the HOST. Sperm cells were evaluated in a phase contrast microscope and classified those sperm cells with
linear tails as injured, and those with coiled tail as intact. The remaining semen was resuspended in 1 mL TALP, with 150 µL removed. A total
of 3 µL of PI (10 µg / mL in PBS) was added to this volume . The evaluation was performed on epifluorescence microscope, with sperm cells
labeled in red classified as injured, and not labeled as intact. In both tests, 200 cells were analyzed for each treatment, in ten replicates. Data were
analyzed by the t test (LSD), with a significance level of 5%. The mean number of viable cells detected by the HOST was 135.4 ± 21.2 (Gc) and
140.6 ± 13.4 (EG) and was significantly different among the values obtained by staining with PI (Gc = 87.7 ± 23.0; EG = 114.7 ± 12.3). The
data differ from results reported by Brito et al. (2003, Theriogenology 60:1539-1551) for cattle semen, which found more intact sperm with IP
than with HOST. We concluded that the cryoprotectant used for freezing semen did not alter the pattern of response to the HOST and IP tests,
and there are differences among the methods for detection of plasmatic membrane integrity of ram semen, still needing to correlate these data with
fertility.
Keywords: sperm cell, membrane, cryopreservation.
A228 SUPPORTIVE BIOTECHNOLOGIES: CRYOPRESERVATION AND CRYOBIOLOGY, IMAGE ANALYSIS AND DIAGNOSIS, MOLECULAR BIOLOGY AND “OMICS”
PEPTIDE FINGERPRINTING ANALYSIS OF BOVINE EMBRYOS OF NELORE (
(BOS INDICUS) ) AND RED ANGUS (
(BOS
TAUR
URUS
US) ) RACES OBTAINED
THROUGH
IN VITRO FERTILIZA
TILIZATION
TION
R aquel Cellin Ro chetti 1 , Yeda Fumie
Wa tanabe 2 , Felip
elipe e Per
erecin
ecin 3 , Juliano Ro drigues Sangalli
4 , Flávio
V ieira Meir
eirelles
elles 5 , C hristina
Ramires Ferreira 6 , Marcos Nogueira Eberlin 7 , Fabio Cesar Gozzo 8 , Eduardo Jorge Pilau 9 , Ricardo Pimenta Bertolla 10 & Edson
Guimarães Lo Turco 11
1,10,11
UNIFESP, SÃO PAULO, SP, BRAZIL. 2 VITROGEN, CRAVINHOS, SP, BRAZIL. 3,4,5,7,8,9,10,11,12 USP/FZEA, PIRASSUNUNGA, SP, BRAZIL. 6,7,8,9 UNICAMP, CAMPINAS, SP, BRAZIL.
Fingerprinting studies performed by mass spectrometry (MS) using MALDI (Matrix – Assisted Laser Desorption/Ionization) are
becoming frequent because it turns the analysis faster and enables determination of specific biological characteristics. With the expansion of
livestock, studies related to molecular biomarkers of breeds have an important role in animal reproduction. In this context, an important feature
for improvement of reproductive response is to identify and define goups of breeds. This study aimed to introduce embryos peptide fingerprinting
technique for molecular identifications and develop applications for characterization of two bovine breeds for futures preimplantational diagnostics.
We used bovine embryos from Nellore (Group 1) and Red Angus (Group 2) breeds. Embryos were produced according to the protocol of
Vitrogen Ltda. (Ripamonte, P.; Genet. Mol. Res. Vol.4(4), pg. 726-33) located in Cravinhos, SP. For the study of proof of concept of this
methodology, five embryos from Group 1 and seven from Group 2 were used. Lipids and salts were removed from embryos through serial
washes in isopropanol (70 and 95%). Then, they were individually transferred to the MALDI plate (Waters) and covered with trypsin (20µg/mL)
during 12 h under moist chamber in 37°C. Afterwards, the samples were covered with matrix (sinapinic acid 15mg/mL) and the spectra was
acquired using the MALDI-QTOF (Synapt G1) in positive model with mass ranging from 800 to 3500 m/z. Statistical analysis was
performed online (www.metaboanalyst.ca). Data standardization was performed by z score. Ions were only considered different if they
showed a minimum of 4 - Fold Change and P = 0.05 regarding the Student-t test (Volcano). Considering the multivariate analysis, it was
performed regarding the principal component analysis (PCA) followed by the method of summation of partial least squares (PLS-DA). It was
observed 5300 ions from which 223 showed difference in the Volcano analysis. The first three PCs explained 78.2% of data variance (PC1
66.7%, PC2 7.8%, 3 PC3, 7%). These preliminary results showed that the analysis of peptide profile from individual embryos through
MALDI is fast, sensitive and it makes possible separation between zebu (Group 1) and taurus (Group 2) embryos. Thus, the peptide
fingerprinting may become a powerful tool to distinguish biological conditions, such as precocity, fertility, disease resistance, among others.
Keywords: peptide fingerprinting, embryos, mass spectrometry.
s450