Effects of oxygen concentration and oviductal epithelial tissue on the development of in vitro matured and fertilized bovine oocytes cultured in protein-free medium

Abstract.We investigated the effect of group culture on bovine embryo development, and also investigated the effect of embryo-culture conditioned medium on developmental competence of individually cultured bovine embryos. Slaughterhouse-derived bovine oocytes were matured and fertilized in vitro. The presumptive zygotes were cultured individually or cultured in groups of 2 to 5 embryos with a constant culture density (5 µl/embryo). After 7 days of culture, the rates of embryos developed to the blastocyst stage were significantly higher (P<0.05) in group cultures of more than 3 embryos/drop than for embryo culture of 1 or 2 embryos/drop. These results suggest a beneficial effect of group culture may be exerted by possible growth promoting factors secreted by embryos. In the next experiment, we investigated the effect of timing of fresh medium replacement on the development of embryos cultured in groups. The blastocyst formation rate was lower when culture medium was replaced freshly on days 2-4 after fertilization than on days 5-6. The blastocyst formation rates of single-cultured embryos were significantly (p<0.05) increased by the addition of conditioned medium derived from multiple-embryo culture. These results indicate that group culture promotes embryo development and that embryo culture-derived conditioned medium is effective for supporting development of single cultured embryos. Key words: Bovine, Conditioned medium, Development, Embryo, Single embryo (J. Reprod. Dev. 52: [137][138][139][140][141][142] 2006) ecently, ultrasound-guided ovum pick-up (OPU) has enabled repeat collection of oocytes from live cattle for genetic improvement [1][2][3]. However, the efficiency of embryo production by OPU is still low because of the limited number of oocytes collected and used for in vitro fertilization (IVF) and culture (IVC). In our field data, the blastocyst formation rate decreased when the number of in vitro fertilized oocytes collected from individual heifers was less than 3. Therefore, it is important to increase the efficiency of blastocyst production from small numbers of in vitro fertilized oocytes collected by OPU.Previous studies in mice [4, 5], sheep [6], and cattle [7][8][9] have demonstrated that group cultures promote embryo development compared with single-culture or co-culture with small numbers of embryos. In murine embryos, the blastocyst formation rate is promoted by an increase of embryo density in culture drops [10], suggesting that specific factor(s) are secreted from the embryos to influence embryo growth and development in an autocrine or paracrine manner. In the non-coculture system, embryo development is promoted by adding conditioned medium derived from bovine oviductal epithelial cells [11,12], buffalo/rat hepatocytes [13], bovine granulosa cells, or Vero

Section: Discussionmentioning

confidence: 99%

Effect of Group Culture and Embryo-culture Conditioned Medium on Development of Bovine Embryos

Fujita

Umeki

Shimura

et al. 2006

“…Embryos cultured in vitro are exposed to suboptimal culture conditions such as high oxygen tension (20%) which is one of the major differences from the in vivo environment [7]. It has been reported that low oxygen tension improves bovine embryo development, suggesting tha t low ox ygen co ncentra tio n r ed uc es the generation of free oxygen radicals [8][9][10][11][12][13]. Based on these reports, it is likely that oxygen radicals g en e r at e d un d e r h i g h o x yg e n t e n si on a r e diminished by active G6PD, and that the higher G6PD activity in female embryos is related to their higher developmental competence.…”

mentioning

confidence: 99%

Role of G6PD Activity on Sex Ratio and Developmental Competence of Bovine Embryos under Oxidative Stress.

Iwata¹,

Kimura

Hashimoto

et al. 2002

Abstract. Inactivation of the X-linked gene in the female embryo is one of the major events during mammalian early embryogenesis. Before this inactivation, enzyme activity encoded by the X-linked gene is different between male and female embryos. In the present study, we demonstrated a possibility that there may be a different response to specific culture conditions in vitro between male and female early embryo. Glucose-6-phosphate dehydrogenase (G6PD) is one of the X-linked enzymes and its activity is semi-quantitated by brilliant cresyl blue (BCB). In experiment 1, the relationship between semi-quantitated G6PD activity and the sex ratio of the embryos was examined. In experiment 2, the relationship between semi-quantitated G6PD activity and the developmental competence of embryos was examined. No relationship was found between G6PD activity and sex in 8-cell, 16-cell and blastocyst stage embryos. However, G6PD activity was high in female embryos and low in male embryos at the morula stage. When morula stage embryos were categorized by BCB and cultured under high oxygen tension (5% CO2, 95% air), the developmental competence of embryos having high G6PD activity was higher than those having low enzyme activity. However, when the categorized morula stage embryos were cultured under low oxygen tension (5% CO2, 5% O2, 90% N2), there was no difference in the developmental rates among the categories. These results indicate that G6PD activity is high in female morula stage embryos compared with that in male embryos, and that the activity of this enzyme strongly affects the developmental competence of morula stage embryo when they are cultured under high oxygen tension.

“…To decrease the effect of oxidative stress on embryos under atmospheric levels of oxygen, superoxide dismutase [25], 2-mercaptoethanol [26], cysteine, or cysteamine [11,12,26] have been successfully added to the culture medium. In contrast, other reports demonstrated that the potential of in vitro fertilized bovine embryos to develop into blastocysts was suppressed at low oxygen tension when embryos were co-cultured with bovine oviductal epithelial tissue or cells [5,6]. The low developmental potential of embryos under the co-culture system at the low oxygen tension was considered to be due to anaerobic conditions or degeneration of somatic cells.…”

Section: Discussionmentioning

confidence: 50%

“…Some studies reported beneficial effects [1][2][3], while others reported either no effect or adverse effects [4][5][6][7][8] of low oxygen tension on the developmental potential of preimplantation embryos. Although a few reports demonstrated that a reduced oxygen atmosphere provided a superior gas environment for the culture of porcine embryos [9,10], there are no reports in which the effect of oxygen tension on the developmental potential has been examined in porcine oocytes.…”

mentioning

confidence: 99%

Effect of Oxygen Tension on the Developmental Potential of Parthenogenetic Oocytes and Nuclear-Transferred Porcine Oocytes Receiving Fetal Fibroblast Cells.

Kawakami

Tani

Yin

et al. 2002

Abstract. The effects of oxygen tension (5% and 20%) during in vitro culture of oocytes and fetal fibroblast cells on the developmental potential of parthenogenetic and nuclear-transferred porcine oocytes were examined. The potential of parthenogenetic oocytes matured and cultured under different oxygen tensions to develop into blastocysts was not significantly different (5 to 16%). The proportions of enucleated oocytes receiving fetal fibroblast cells that developed into blastocysts were significantly lower when somatic cells were cultured under low oxygen tension (2 and 3%) than under high oxygen tension (7%). The effects of oxygen tension during culture of oocytes and somatic cells on the developmental potential of parthenogenetic and nuclear-transferred oocytes is discussed.