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

Acta Scientiae Veterinariae, 2011. 39(Suppl 1): Abstracts - 25th Annual Meeting SBTE-Brazil. August 2011.
A023 MALE REPRODUCTIVE PHYSIOLOGY AND SEMEN TECHNOLOGY
CORREL
ORRELATION AMONG OSMOTIC
TEST AND DIFFERENT PROCEDURES OF POST-THAW SPERM VIABILITY EVAL
ALUATION
Gabriel Felipe Oliveira de Menezes 1 , Rodrigo Freitas Bittencourt 1 , Léia Ribeiro Alves Santos 1 , Renata Cardoso Andrade 1 , Lais Oliveira Mascarenhas 1 ,
Alexandra Soares Rodrigues 2 , Priscila Assis Ferraz 2 & Antônio de Lisboa Ribeiro Filho 2
1
UNIME, SALVADOR, BA, BRAZIL. 2 UFBA, SALVADOR, BA, BRAZIL.
The evaluation of structural and functional integrity of sperm plasma membrane has been studied and improved with the goal of
increasing its reliability in assessing the potential fertility of the breeding (BITTENCOURT et al., 2005, Ciên Anim Bras, 6:213-218). This study
aimed to correlate the osmotic shock with the supravital test and fluorescent probes for evaluation of ovine semen thawed. For this experiment,
20 ovine frozen semen samples were used, from Santa Inês e Dorper breedS, with a straw of semen from each animal (n = 20). Straws were
thawed in a water bath at 38°C for 50 s, and then placed into 2 mL microtubes, previously warmed at 38°C. Aliquots were collected for the
supravital test dye eosin (EOSC); the sperm morphology was analyzed and the percentage of bent tails (BT) was calculated. For evaluation in
fluorescence microscopy it was employed the combination of fluorescent probes propidium iodide, FITC-PSA and JC-1, using the methodology
described by Celeghini et al. (2010, Arq Bras Med VetZoot, 62:536-543). The osmotic shock (OS) followed the dilution: One part semen to 10
(OS 10) parts of the final solution, deionized water (0 mOsmol). After incubation for 5 min at 37°C, samples were fixed with 10 µL of
formaldehyde buffered saline. The percentage of spermatozoa reactive to OS was determined by subtracting the percentage of spermatozoa with
OS-induced BT, by the sperm morphology obtained after thawing. These evaluations were observed by phase contrast microscopy with an
increase of 1000x immersion oil and made the 100 spermatozoa count. The statistical analysis was made in the package Statistical Analysis
System (SAS), version 5.0 (1996) with a significance level of 5%. The OS 10 was the only one to show positive and significant correlation with
EOSC (r = 0.8, P < 0.05). The absence of significant correlation between the OS and the fluorescent probes can be explained, since functional
activity in some situations cannot be directly associated with membrane integrity. The membrane can be intact but not functional; or superficially
injured, but with the functionality unchanged. Further studies should be made with fresh semen in an attempt to repeat the results of this study
to incorporate the OS into the routine evaluation of semen for breeding sheep.
Keywords: osmotic test, sperm, sheep.
A024 MALE REPRODUCTIVE PHYSIOLOGY AND SEMEN TECHNOLOGY
CANINE SEMEN CRYOPRESER
OPRESERVATION:
IDENTIFICATION TION OF CRITICAL POINTS
Cristina Fátima Lucio, , Fernanda Machado Regazzi, Liege Garcia Silva, Daniel Souza Ramos Angrimani, Marcílio Nichi, Camilla Motta
Mendes , Mayra Elena Assumpção & Camila Infantosi Vannucchi
DEPARTAMENTO DE REPRODUÇÃO ANIMAL - UNIVERSIDADE DE SÃO PAULO, SÃO PAULO, SP, BRAZIL.
Cryopreservation of canine semen is fundamental for the preservation of genetic material, as well as considered a biotechnique for
wild canids, with the domestic dog as a biological model. However, studies are still necessary to achieve the efficiency observed in other species.
The objective of this experiment was to verify seminal characteristics during the cryopreservation process, seeking to identify the critical step for
sperm viability. Eleven semen samples were cryopreserved in a two steps process, according to a protocol previously established (Thomassen,
R et al., 2006, Theriogenology 66, 1645-50). Aliquots of the fresh semen, the chilled (after 1 h at a 5 o C extender), after glycerolization (1 hour
after addition of 5% glycerol to the extender) and the thawed semen (30 s at 37°C) were evaluated for motility and vigor, computerized motility
analyses (CASA), percentage of live sperm by eosin/nigrosin staining, oxidative stress by TBARS concentration, mitochondrial activity by
DAB staining, mitochondrial membrane potential by JC1 probe and membrane integrity by FITC / PI probes using flow cytometry. Results
show that the cryopreservation protocol promotes decrease in subjective and computerized motility and vigor. However, the decrease in motility
was significantly more pronunciated in the sample that suffered exposure to the liquid nitrogen. The frozen-thawed sample showed decrease in
the percentage of progressive sperm motility and increase in slow and static sperm. The analysis of mitochondrial activity demonstrates that the
crioprotectant influx and the thawing process were deleterious, but an intense loss was observed for the thawed sample, corroborated by the
significant decrease in the mitochondrial membrane potential. The freezing-thawing process increases the association of the plasma and
acrosome membrane lesions, however, the cooling process promoted membrane stabilization with a low percentage of sperm injury. The
changes detected in the thawed semen can be explained in part by the oxidative stress. In conclusion, the decrease in temperature during cooling
and the change in osmolarity with the influx of glycerol in the spermatic cell did not cause deleterious changes. The crucial moment for the
viability of sperm during cryopreservation is the exposure to liquid nitrogen. Despite the intracellular ice formation, the intense production of
reactive oxygen species can be the explanation for these injurious factors. [FAPESP 09/52760-3].
Keywords: cryopreservation, semen, canine.
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