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

Acta Scientiae Veterinariae, 2011. 39(Suppl 1): Abstracts - 25th Annual Meeting SBTE-Brazil. August 2011.
A025 MALE REPRODUCTIVE PHYSIOLOGY AND SEMEN TECHNOLOGY
DETECTION OF PATHOGENS IN BOVINE SEMEN USING FLUORESCENT MULTIPLEX PCR
Francisc
ancisca a Elda Fer
erreir
eira Dias
1 , Tania
Vasc
asconc
oncelos Cavalc
alcan
ante 2 , Ana Kelen Felip
elipe e Lima 3 , Car
aris Mar
aroni Nunes
4 , Juliano Franc
anco De Sousa
5 & José Fer
ernando
Garcia 6
1,2,3
ESCOLA DE MEDICINAVETERINÁRIA E ZOOTECNIA, UFT, ARAGUAÍNA, TO, BRAZIL. 4,6 LABORATÓRIO DE BIOQUÍMICA E BIOLOGIA MOLECULAR ANIMAL, UNESP, ARAÇATUBA,
SP, BRASIL. 5 LABORATÓRIO BRIO GENÉTICA E BIOTECNOLOGIA LTDA, ARAGUAÍNA, TO, BRAZIL.
The use of cryoprotectants in semen allows the survival of most infectious agents found there, contaminating herds using artificial
insemination (AMIN, 2003; Veterinary Journal 166: 86-92).Thus, the PCR is a tool that has been used with the purpose of simultaneously
detecting multiple infectious agents causing similar clinical syndromes and/or share similar epidemiological features (MARKOULATOS et al.,
2002; J. Clin. Lab. Analysis 16: 47-51). This study evaluated the use of PCR combined with capillary electrophoresis for detection of pathogenic
bacteria in bovine semen. Doses of semen free of pathogens were infected with decreasing concentrations of bacteria obtained by serial dilutions
in base 10, to obtain samples containing from 1 to 10-7 bacterias/mL from the initial concentration of Lepstospira interrogans serovar pomona
(108 bact/mL), Brucella abortus (2.8 x 108 bact/mL), Campylobacter fetus (1.5 x 105 bact/mL) and Haemophilus somnus (1.5 x 105 bact/mL).
The samples were subjected to DNA extraction by the method of phenol/chloroform, and the extracted DNA was amplified by multiplex
fluorescent PCR-using species-specific primers for each bacterium (each pair labeled with fluorescent substance or HEX FAN) for amplification
of DNA fragments of 193 bp (B. abortus), 330 bp (L. pomona), 400 bp (H. somnus) and 415 bp (C. fetus). After multiplex PCR reaction,
visualization of the fragments by electrophoresis on 8% polyacrylamide gel was stained with Silver Nitrate, and by capillary electrophoresis with
automated analysis of DNA fragments. Could be detected simultaneously, in a single reaction, fragments of 193, 330, 400 and 415 base pairs
of B. abortus, L. pomona, H. somnus and C. fetus, respectively. Using analysis in gel and capillary electrophoresis was detected in a dilution of
10-2 and 10-3 times the initial concentration of each bacterium, respectively. Thus, capillary electrophoresis can be an alternative to PCR detection
of pathogenic bacteria in semen and seems to be an effective and rapid method of pathogens detection, when compared to traditional electrophoretic
system, which might be an excellent tool to quality health control of centers in artificial insemination and embryo transfer.
Keywords: cattle semen, multiplex-pcr, pathogenic bacteria.
A026 MALE REPRODUCTIVE PHYSIOLOGY AND SEMEN TECHNOLOGY
DIFFERENT PROTOC
OCOLS OLS OF THAWING RAM SEMEN CRYOPRESER
OPRESERVED IN EXTENDER CONT
ONTAINING LOW-DENSIT
W-DENSITY
LIPOPROTEIN
Luís Cláudio Oliveira Moura 1 , Maira Corona Da Silva 2 , Ana Cláudia Dumont Oliveira 3 , Mariana Machado Neves 4 , Paola Pereira Das
Neves Snoeck 5 & Marc Roger Jean Marie Henry 6
1,2,5
UESC, ILHÉUS, BA, BRASIL. 3,6 UFMG, BELO HORIZONTE, MG, BRAZIL. 4 UFV, VIÇOSA, MG, BRAZIL.
Artificial insemination is an important tool in genetic improvement of livestock species, although there are limitations on the use of
cryopreserved ram semen, due to its low fertility compared to fresh semen. Several factors can affect the membrane integrity and fertilizing ability
of sheep spermatozoa, including the velocity and temperature of thawing. The objective of the following experiment was to evaluate the influence
of thawing time and temperature on the viability of cryopreserved spermatozoa in extender with low-density lipoprotein (LDL). Three Santa Inês
rams were used as donors of semen. Five ejaculates per animal were collected. After fresh semen analysis, the ejaculates were fractionated and
diluted in a control extender (E1-15% egg yolk) and extenders with different concentrations of LDL (E2-10% and E3-20%), in order to obtain
400x106 sperm/mL. Samples were cooled from room temperature to 5°C using an average cooling rate of –0.25°C/min in TK4000 ® machine
and then maintained in equilibrium (2 h at 5°C), after that, frozen at 4cm in nitrogen vapor and stored in cryogenic cylinder. Thawed samples (38°/
30s and 50°C/15s) were subjected to computer analysis (CASA) for evaluation of motility immediately after thawing and after three hours of
incubation at 38°C. The membrane integrity was evaluated by the hypoosmotic test (HOST) and by fluorescent probes. No significant
differences (P > 0.05) between extenders in evaluations of motility immediately after thawing at 38°C were observed. The motility of E1 (74,0%)
(38°C) was better (P < 0.05) than E1, E2 and E3 (49.2%; 47.2%; 45.7%, respectively) (50°C). The treatments did not differ in evaluations of
motility at the end of the heat resistance test. E2 (38.1%) and E3 (39.9%) thawed at 50°C had lower (P < 0.05) percentage of cells with functional
integrity than sperm diluted in E1 (57.2%) and E3 (52.6%) thawed at 38°C. E1 (47.8%) thawed at 50°C preserved better the structural integrity
of sperm membranes (P < 0.05) than other treatments thawed at 38°C. It is possible to affirm that thawing at 50°C was more efficient than 38°C
to preserve the structural integrity of sperm membranes when extender formulated with egg yolk was used. Also, it was demonstrated that the
protocol of thawing at 38°C proved to be the most appropriate to preserve motility and functional integrity of plasma membrane in samples
diluted in extender containing 20% of LDL.
Keywords: santa inês, spermatozoa, extender.
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