Influence of Caffeine and Chondroitin Sulfate on Swine Sperm Capacitation and In Vitro Embryo Production

Ano: 2011 | Volume: 39 | Número: 2

Autores: Viviane Purri de Oliveira, Mariana Groke Marques, Renata Simões, Mayra Elena Ortiz D’Avila Assumpção, José Antonio Visintin

Palavras-chave: sperm, pi/cfda, coomassie blue g, chondroitin sulfate, caffeine, swine

Resumo Inglês:

Background: The establishment of an in vitro production (IVP) of embryo in swine allows the generation of embryos with the
same quality as in vivo produced embryos with less costs and time. In order to achieve successful fertilization under normal
circumstances in vivo, mammalian spermatozoa must first undergo capacitation and then acrosome reaction. The purpose of this
study was compared the efficacious of IP/CFDA fluorescence and Coomassie Blue G (CB) staining to detect capacitated sperm
cells in refrigerated and fresh semen. Morever, it was investigated the efficacious of caffeine and chondroitin sulphate to
promote in vitro sperm capacitation and in vitro embryo produced (IVP) of swine embryos.
Materials, Methods & Results: A sperm-rich fraction from ejaculate was obtained using the gloved-hand method and the gelfree
fraction was separated using sterile gauze. The semen was diluted in BTS at a final concentration of 1.5 x 108 cells/mL. The
sperm suspension was incubated for 2 h at 25°C, refrigerated and maintained for 1 h at 15-18°C (refrigerated group) or used
immediately (fresh group). Sperm capacitation was assessed by IP/CFDA fluorescence and CB staining for both fresh and
refrigerated semen. For PI/CFDA evaluation, a final solution containing 1.7 mM formaldehyde, 7.3 mM PI and 20 mM CFDA in 950
μL saline was prepared. In the dark, 40 μL PI/CFDA final solution was added to 10 μL semen and after 8 min, slides were analyzed
on epifluorescence microscopy. For CB evaluation, sperm cells were fixed in 4% paraformaldehyde for 10 min and centrifuged
twice at 320 x g in ammonium acetate pH 9 for 8 min. A smear was made and stained with 2.75 mg/mL CB in solution containing
12.5% methanol, 25% glacial acetic acid and 62.5% water, for 2 min. The smear was washed in running water, air dried and sealed
with Permount®, diluted 2:1 in xilol to avoid staining oxidation. Our results showed that refrigeration did not affect sperm
capacitation and comparing staining methods, the PI/CFDA combination was more efficient to detect capacitated sperm, when
compared to CB staining. In experiment 2, we evaluated the effect of different incubation time (1 - 5 h) with chondroitin sulfate and
caffeine on sperm capacitation. For in vitro fertilization, oocytes were obtained from slaughterhouse ovaries. Oocytes with a
thick and intact cumulus oophurus layer and cytoplasm with homogenous granules were selected for in vitro maturation for 44
h. According to the results of experiment 2, it was used for in vitro fertilization refrigerated semen was capacitated with 50 μg/mL
chondroitin sulfate for 2 h or capacitated with 5 μg/mL caffeine for 3 h. Six hours after insemination, cumulus oophorus cells were
mechanically removed and oocytes were washed and incubated in microdrops of culture medium. Embryo development after
fertilization with sperm capacitated with caffeine or chondroitin sulfate was evaluated on days 3, 5 and 7 of culture. No differences
were observed in days 3 or 5 of in vitro culture. However, it was observed an increase on blastocyst rate on Day 7 of culture when
caffeine was used as the capacitor agent.
Discussion: Molecular basis of sperm capacitation is still poor understood. Sperm capacitation can occur in vitro spontaneously
in defined media without addition of biological fluids. We observed that sperm capacitation increased as incubation period
enlarged and it was observed using Coomassie blue G and PI/CFDA for fresh semen and for refrigerated semen. It can be
concluded that the cooling of semen did not change their pattern of sperm capacitation and this is best assessed by IP/CFDA
than by CB. In addition to the use of caffeine in sperm capacitation produces more blastocysts than the chondroitin sulfate after
in vitro fertilization.