Culture of bovine embryos in buffalo rat liver cell-conditioned media or with leukemia inhibitory factor.

Abstract

Experiments were designed to study development of bovine embryos in TCM-199 medium conditioned by preculture with buffalo rat liver (BRL) cells. Conditioned media were harvested after BRL cells were cultured until confluency (CON), or for an additional 2 d with the same cells but new medium (CON-N) or the same medium (CON-S). Glucose in TCM-199 was depleted by BRL cells to different concentrations depending on coculture procedures: CON = 3.94 mM, CON-N = 1.67 mM, and CON-S = 1.11 mM glucose. In Exp. 1, development of bovine zygotes in CON-S resulted in fewer blastocysts than development in CON (10 vs 28%, P < .05); CON-N was not different from CON (26% blastocysts). Experiment 2 examined effects of moving embryos to a fresh drop of different or identical conditioned medium after culture for 3 d. Initial culture in CON-N and final culture in CON resulted in a greater (P < .01) number of blastocysts compared with the control of CON followed by CON (32 vs 19% blastocysts). This was not entirely due to changing from low to high glucose because adding glucose to CON-N after 3 d yielded only 18% blastocysts. To test the hypothesis that beneficial effects of BRL cell-conditioned media may be due to secretion of leukemia inhibiting factor (LIF), LIF was added to B2, a more appropriate medium than Medium-199 for culturing bovine embryos without conditioning or coculture with BRL cells. In the absence of serum, the percentage of blastocysts per cleaved embryo (17 to 28%) was not improved with LIF; however, the mean number of cells per blastocyst was higher (P < .05) in treatments with LIF (65 to 74 cells) than without LIF (47 cells). In B2 medium + 10% fetal calf serum, LIF was of no benefit; development to blastocysts was good with or without LIF (43% of cleaved).

Effects of human versus mouse leukemia inhibitory factor on the in vitro development of bovine embryos

“…In the present study, embryonic development was impaired by hLIF, while the number of ICM cells was reduced by mLIF, in disagreement with the effects described by Yamanaka et al for bLIF [6,7]. While bLIF has been described to increase TE cell counts without affecting the ICM [6,7], hLIF has been noted to increase [8,9], decrease [12] or have no effect on the ICM (present work). In addition, mLIF was found here to have no effect on the number of TE cells yet to reduce the ICM.…”

“…The role of hLIF in bovine embryo culture has been controversial in previous studies [8][9][10][11] in which the effects of different supplements, such as serum, synthetic molecules and BSA were examined. In the present study, embryonic development was impaired by hLIF, while the number of ICM cells was reduced by mLIF, in disagreement with the effects described by Yamanaka et al for bLIF [6,7].…”

“…However, the use of hLIF has yielded controversial results as compared to bLIF, and mLIF has not been used for this purpose. When added from the morula stage, hLIF has been reported to have no effect on subsequent development [8,9]. During the course of blastocyst formation, hLIF has been found to stimulate expansion and hatching [8,10], but other authors report no comparable effect [11].…”

“…During the course of blastocyst formation, hLIF has been found to stimulate expansion and hatching [8,10], but other authors report no comparable effect [11]. It has also been described that the number of cells in the inner cell mass (ICM) and total cell numbers increase in the presence of hLIF, although this led to no improvement in pregnancy and survival rates to cryopreservation [8,9]. Recently, hLIF has been reported to have adverse effects on embryonic development kinetics, morphology, cell counts and expression of Oct4 and laminin, having no apparent influence on the subsequent formation of outgrowths [12].…”

Effects of glucose and protein sources on bovine embryo development in vitro

“…In medium conditioned by BRL cells, the addition of Ž . glucose after 3 days of culture did not affect blastocyst yield Funston et al, 1997 . In the presence of BSA, glucose tended to promote development, which is in contrast with Ž . the notable difference found in other studies Furnus et al, 1997 .…”