Effect of Hormonal Supplementation Periods and In Vitro Maturation Media on Developmental Competence of Pig Oocytes

Ano: 2010 | Volume: 38 | Número: 4

Autores: Alecssandra Sobreira de Lima, Mariana Groke Marques, Anibal Ballarotti Nascimento, Mayra Elena Ortiz D’Avila Assumpção, José Antonio Visitin

Palavras-chave: swine, fertilization, polispermy, in vitro maturation, maturation media, hormonal supplementation

Resumo Inglês:

Background: The oocyte ability to undergo successful fertilization, cleavage and embryonic development depends on meiotic
maturation and developmental competence acquisition. In vitro maturation (IVM) protocols currently use eCG, hCG or a combination
of both, the effect of these gonadotrophins during IVM and subsequent embryonic development is still controversial. Several
media have been used for IVM of porcine oocytes: TCM199, Whitten’s and NCSU23 have also been shown to support pig
oocyte IVM. This study was designed to determine the effect of hormonal supplementation period and maturation media during
in vitro maturation of pig oocytes (1) and subsequent embryonic development (2).
Materials, Methods & Results: Oocytes with intact cumulus oophurus layers and homogeneous cytoplasm were collected
from prebubertal gilts. IVM was subjected in NCSU23, TCM199 or Whitten’s media supplemented with 10 IU/mL eCG and
10 IU/mL hCG for the first 24 or 48 h of IVM. In each replicate the oocytes were fixed every 4 h from 32 to 48 h IVM or the past
48 h after IVM, oocytes were fertilized in vitro in mTBM medium for six hours and cultured in NCSU23 medium for nine days.
Cleavage, blastocyst and hatching rates were evaluated at 48 h (day 2), 168 h (day 7) and 216 h (day 9), respectively.
The addition of eCG and hCG during the first 24 h IVM increased the proportion of oocytes that reached MII stage at 44 h of
maturation in NCSU23 medium. This effect was also observed in Whitten medium at 44 and 48 h (P <0.05). However, it was
not observed in the TCM199 medium. No effect of maturation medium on oocyte nuclear maturation (P >0.05) was observed
in oocytes matured in the presence of eCG and hCG during the first 24 h IVM or during 48 h IVM. A progressive increase of
maturation indexes was observed on oocytes matured with hormonal supplementation in Whitten media for 24 h. Higher
indexes were obtained at 44 and 48 h. When NCSU23 media was used, no difference after 36 h of maturation was observed. The
same result was observed in TCM199. A progressive increase of maturation indexes was observed on oocytes matured with
hormonal supplementation for 48 h in Whitten media. Higher indexes were obtained in 36 and 40 h. When NCSU23 or
TCM199 were used, no difference was observed. No effect of IVM media on the percentage of fertilized oocytes and polyspermic
oocytes or number of spermatozoa per fertilized oocytes was observed. Also, no effect of IVM media on cleavage and
blastocyst rates was seen. However, the proportion of hatched blastocysts was lower in NCSU23 compared to Whitten or
TCM199.
Discussion: Similar results were reported by Marques et al. [13], that it no differences between hormonal supplementation for
22 or 44 h were observed. Therefore, more studies are needed to elucidate the role of these hormones in nuclear in vitro
maturation in pig oocytes.
In conclusion, no effect of maturation media on meiotic progression was observed. However, the proportion of oocytes that
reached metaphase II (MII) stage was higher when eCG + hCG were added for 24 h than 48 h mainly at the 44 h of maturation.
In addition, no differences were observed in cleavage and blastocyst rates of the cultured embryos. However, embryos cultured
in NCSU23 showed lower rates of hatching compared to other media. These results indicated no effect of maturation media on
the fertilization and embryonic development even in the presence of cysteine, PFF and EGF, except for hatched embryos that
these rates were lower in NCSU23.