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

Acta Scientiae Veterinariae, 2011. 39(Suppl 1): Abstracts - 25th Annual Meeting SBTE-Brazil. August 2011.
A017 MALE REPRODUCTIVE PHYSIOLOGY AND SEMEN TECHNOLOGY
ACTION OF NITRIC OXIDE ROUTES CGMP IN IN VITRO CAP
APACIT
CITATION TION OF CRYOPRESER
OPRESERVED BOVINE
SPERMATOZ
OZOA
Ana Carolina De Macedo Soares Leal 1 , Maria Clara Caldas-Bussiere 2 , Carla Sobrinho Paes De Carvalho 3 , Célia Raquel Quirino 4 &
Patricia Alves Pinho Machado Silva 5
1,2,3,4,5
UENF, NITEROI, RJ, BRAZIL. 2 UENF, CAMPOS DOS GOYTACAZES, RJ, BRAZIL.
The aim of the study was to evaluate the effects of nitric oxide (NO) in in vitro capacitation induced by heparin (H) of in
cryopreserved spermatozoa from 3 bulls, by adding L-arginine (L-arg, precursor synthesis NO), 8-bromo-cGMP (cGMP analog) and 1H-
(1,2,4) oxadiazole-(4,3-a) quinoxaline-1-one (ODQ, inhibitor of activity cGMP soluble). The control consisted of capacitated sperm in TALP
medium-sp plus H (10 mg/mL). It was added different concentrations of L-arg (1, 2.5, 5, 10 and 50mM), 5 and 10mM cGMP and 0.1mM ODQ
in medium capacitation (200mL). The capacitation and acrosome reaction were evaluated by chlortetracycline test (CTC) and the concentration
of nitrate and nitrite (NO3-/NO2-) in the medium capacitation by the Griess method after 4 h. It was performed 6 replicates of each animal. Those
data were analyzed by analysis of variance and means compared by the Tukey test, 5% probability. The addition of 1 and 10mM L-arg in the
medium capacitation increased the percentage (17.3% and 4.4%) of capacitated spermatozoa (P < 0.05) compared to control. The addition of
0,1mM ODQ decreased (11.1%) capacitation (P < 0.05) compared to control. The addition of 1mM L-arg + 0.1mM ODQ in the medium
capacitation reversed the inhibitory effect of ODQ. Treatment with cGMP (5 and 10mM) was used to evaluate whether L-arg had a similar role.
The addition of 5mM cGMP increased (11.3%) the capacitation and the addition of 10mM cGMP decresed (13.9%) the capacitation, but
increased (16,8%) the AR (P < 0.05). The addition of 5mM cGMP + 0.1mM ODQ partially reversed (97.4%) the inhibitory effect of ODQ (P
< 0.05). The addition of 10mM cGMP + 0.1mM ODQ inhibited capacitation and AR (P < 0,05). Increasing concentrations of L-arg showed a
dose-response effect in NO synthesis (P < 0.05). The addition of 0.1mM ODQ decreased (3.9%) NO synthesis (P < 0,05) compared to control.
The addition of 1mM L-arg + 0.1mM ODQ in medium capacitation reversed the inhibitory effect of ODQ (P < 0.05). The addition of 5 mM
cGMP increased (3.85%) the synthesis of NO compared to control but not differed from 1 mM L-arg. Yet the addition of 10 mM cGMP
increased (54.8%) NO synthesis compared with control and lower concentrations of L-arg (1-10 mM). These results indicate that: 1) NO is
involved in the control of capacitation routes cGMP, 2) the state of activity of cGMP regulates the synthesis of nitric oxide by feedback.
Keywords: nitric oxide, spermatozoa, bovine.
A018 MALE REPRODUCTIVE PHYSIOLOGY AND SEMEN TECHNOLOGY
TESTIS SIZE, SEMEN CRITERIA AND SEMINAL PLASMA PROTEINS OF RAMS SUBJECTED TO SCROTAL
INSULATION
Arlindo Alencar Moura 1 , David Ramos Da Rocha 1 , Carlos Eduardo Azevedo Souza 1 , Airton Alencar Araújo 2 ,
Maurício Fraga Van Tilburg 1 , Jorge Andre Matias Martins 1 , Veronica Gonzalez Cadavid 1 , Romulo Messias
Diogenes Lima 1 , Lucas Dos Santos Fonseca 1 & Aderson Martins Viana Neto 1
1
UNIVERSIDADE FEDERAL DO CEARA, FORTALEZA, CE, BRAZIL. 2 UNIVERSIDADE ESTADUAL DO CEARÁ, FORTALEZA, CE, BRAZIL.
N
We studied the effects of scrotal insulation on testis biometry, semen parameters and seminal plasma of White Morada Nova rams.
Six mature rams had their testes insulated for 8 days. Scrotal circumference (SC) and semen samples were taken 7 days before, during insulation
and weekly until semen samples were equivalent to pre-insulation values. From semen samples, seminal plasma was obtained by centrifugation
and subjected to 2-D electrophoresis. Commassie-stained gels were evaluated using PDQuest software (BioRad, USA). Variations in all
parameters were evaluated by analysis of variance and Tukey statistical test (P < 0.05). During insulation, rectal temperature was 38.2 ± 0.1 o C.
Testis temperature increased from 31.2 ± 0.2 o C, before insulation, to 35.2 ± 0.3 o C during insulation, returning to normal values 7 days after
withdrawal of the scrotal bags. Average SC before insulation was 30.0 ± 0.4 cm, decreasing progressively after insulation, reaching the smallest
size after 28 days (22.6 ± 0.6 cm), and returning to pre-insulation values after 56 days. Seminal parameters followed the same trend and 8 days
after the end of the insulation, there were no motile spem and, 8 days later, rams became azoospermic. The first sperm could be detected only 63
days after the end of insulation, and reached normal ranges only after 84 days. Before insulation, seminal plasma protein maps had, on average,
269 ± 25 spots, with 132 consistently present on all gels. However, 24 days after insulation, when seminal quality was severely compromised,
when most rams were azoospermic, only 107 ± 13 spots could be detected per gel, and 57 were present on every member of the matchset. Spots
remaining in seminal plasma samples after insulation had predominantly low molecular weight and, based on our previous results, were mostly
secreted by the accessory sex glands, corresponding to Ram Seminal Vesicles Proteins (RSVPs) 14 and 22 kDa and Bodhesin 2. In spite of the
large number of spots that were not expressed in the seminal plasma after insulation, 8 spots (28 and 70 kDa; pIs from 4.8 to 6.0) could not be
detected before insulation. Such proteins likely represent a response of the reproductive tract to thermal injury. In conclusion, extreme conditions
of heat stress, induced by scrotal insulation, caused drastic alterations in testis function and expresstion of seminal plasma proteins.
Keywords: proteomics, seminal plasma, scrotal insulation.
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