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

Acta Scientiae Veterinariae, 2011. 39(Suppl 1): Abstracts - 25th Annual Meeting SBTE-Brazil. August 2011.
A231 SUPPORTIVE BIOTECHNOLOGIES: CRYOPRESERVATION AND CRYOBIOLOGY, IMAGE ANALYSIS AND DIAGNOSIS, MOLECULAR BIOLOGY AND “OMICS”
ENDOMETRIAL BIOPSY IN MARES IN HEAT REPEATER:
IMMUNOHISTOCHEMIC
OCHEMICAL ANALYSIS OF T LYMPHOCY
YMPHOCYTES AND
MACROPHAGES
Franciele Basso Fernandes Silva, , Joana Fernandes Eigenheer Moreira, Juliana da Silva Leite & Ana Maria Reis Ferreira
UFF, NITEROI, RJ, BRAZIL.
Heat repeaters mares bring large economic losses, so the endometrial biopsy has been a major procedure in the evaluation of
uterine health in horses, besides being easy, safe, cheap and with minimum distress to the animal (SNIDER et al. 2011, Theriogenology 75,
1567–1581; EIGENHEER-MOREIRA et al. 2007, Pesquisa Veterinária Brasileira 27, 506-512). The aim of this study was to evaluate the
presence of T lymphocytes and macrophages in the endometrium of heat repeaters mares during estrus and diestrus. Endometrial biopsies
were performed in ten heat repeaters mares, five in estrus and five in diestrus. For the control group were selected and examined four mares
sound. After collection, samples were preserved in 10% buffered formaldehyde and subsequently subjected to routine histologic processing
to perform the immunohistochemistry (IHC). The IHC technique for identification of T lymphocytes and macrophages was performed with
antibody rabbit polyclonal anti-CD3 (A 0452, DAKO, Denmark) at 1:200 dilution and mouse antibody monoclonal anti-antigen myeloid/
histiocytic IgG1 Clone MAC 387 kappa (M0747, DAKO, Denmark) at dilution 1:400, respectively. For analysis of the results was made
count of immunostained cells in five random fields and the average number of cells per field in two groups and control group was compared
to significance level α = 0.05, through the Student’s t statistic for independent groups. The control group had at the time of diestrus, an average
of 9.1 immunostained cells for antibody anti-CD3 and 8.1 to antibody anti-antigen myeloid/histiocytic at the time of estrus an average of 13.2
and 1.6 respectively. Heat repeaters mares assessed during estrus showed a mean of 20.4 cells immunostained for the antibody anti-CD3 and
10.0 for the antibody anti-antigen myeloid/histiocytic and animals assessed during the diestrus had an average of 11.3 and 8.3, respectively.
When compared with the control group, the group of heat repeaters mares in diestro showed no statistically significant difference for T
lymphocytes (Student’s t-test: t =0.434; g.l.=6; p-value = 0.680) and macrophages (Student’s t-test: t = 0.061; g.l. = 6; p-value = 0.953),
however the group observed on estrus time showed statistically significant difference for macrophages (Student’s t test: t = 2.814; g.l. = 4;
value-p = 0.048) which was not repeated for T lymphocytes (Student’s t-test: t = 2.068; g.l. = 4; p-value = 0.107). This way, we can conclude
that heat repeaters mares in estrus period have increased infiltration of macrophages than observed in healthy mares.
Keywords: endometrial biopsy, cd3, mac 387.
A232 SUPPORTIVE BIOTECHNOLOGIES: CRYOPRESERVATION AND CRYOBIOLOGY, IMAGE ANALYSIS AND DIAGNOSIS, MOLECULAR BIOLOGY AND “OMICS”
MICRO-FTIR BIOCHEMICAL CHARACTERIZA
CTERIZATION TION OF BULL SPERMATOZ
OZOA IRRADIA
ADIATED BY LOW POWER LASER
Thiago Revers ers Dreyer
er 1 , Adr
driano Felip
elipe e Siqueir
iqueira 2 , Taciana Deprá Magr
agrini
3 , Mayr
yra Elena Assumpção
4 , Her
erculano Silv
ilva Mar
artinho
5 & Mar
arcella Pec
ecor
ora
Milazzotto 6
1,3,5,6
UNIVERSIDADE FEDERAL DO ABC, SANTO ANDRÉ, SP, BRAZIL. 2,4 UNIVERSIDADE DE SÃO PAULO, SÃO PAULO, SP, BRAZIL.
Low level laser therapy increases ATP production and consequently the energy supply to the cell. In sperm cells it could reflect
in increased motility, acrosomal reaction and consequently in the fertilizing potential. The present study aimed the biochemical characterization,
by Fourier Transform infrared micro-spectroscopy, of bovine sperm cells irradiated by low power laser under different fluences. Semen
straws were thawed in a water bath at 37°C for 30 s, washed and diluted in TALP medium to a concentration of 7x10 6 spermatozoa/mL.
Samples were placed in Petri dishes for irradiation and during incubation times. Irradiation were done with a diode laser in the red region (ë
=633nm) with fluences of 30, 150 and 300mJ.cm -2 (obtained due to sample irradiation with laser power of 5mW for 1, 5 and 10 min,
respectively); 45, 230 and 450mJ.cm -2 (7.5mW for 1, 5 and 10 min); and of 60, 300 and 600mJ.cm -2 (10mW for 1, 5 and 10 min). Micro-FTIR
analyses were held with samples air dried in a platinum slide after irradiation and 30 min after irradiation. Spectra were acquired in a Varian 620-
IR FTIR Spectrochemical Imaging Microscope system, in the range of frequencies 400-4000 cm -1 . For statistical analysis, acquired spectra
were divided in three different regions, in the range 900-1300cm -1 (nucleic acids region), 1300-1800cm -1 (proteins and phospholipids) and
2820-3000 cm -1 (lipids). The spectra were baseline corrected and normalized. Statistical analysis were held by principal component analysis
(PCA) and by t-student test (P < 0.05) from intensities of the spectral frequencies. Treatments with higher fluencies (300 and 600mJ.cm -2 )
resulted in low intensity DNA bands (1083 cm -1 ), which could be due to the negative effect of higher fluences in the molecule integrity. For the
fluence of 300 mJ.cm -2 , spectral intensities showed no statistically significant deviation between the used laser powers. Phospholipids
(1740cm -1 ), proteins (1655cm -1 ) and lipids (2852cm -1 ) bands were higher for 600mJ.cm -2 fluence compared to 30, 45 and 60mJ.cm -2 , which
showed no statistically significant deviation by the applied methodology. Based on these data, we can conclude that irradiation with fluence
of 600mJ.cm -2 lead to alterations of the structure of cellular molecules which may be reflected in sperm metabolism. [Acknowledgment:
FAPESP, UFABC, Capes, Departamento de Reprodução Animal da Faculdade de Medicina Veterinária e Zootecnia da Universidade de São
Paulo].
Keywords: micro-ftir, low power laser irradiation, bovine.
s452