Development of bovine embryos produced in vitro from the one-cell to the blastocyst stage in serum-free oviduct-conditioned medium was investigated for 8 days consecutively by time-lapse cinematography. Three movies were analysed (130 embryos). The following observations were made. (1) Development under cine-recording conditions was similar to that in a classical incubator. (2) The highest proportion of embryos at the two-cell, three–four-cell, five–eight-cell, 9–16-cell, morula and blastocyst stages were recorded at 34, 46, 61, 115, 149 and 192 h after insemination, respectively. Cleavage asynchrony between blastomeres within individual embryos started at the two-cell stage. (3) The duration of the first three cell cycles was 35 h, 14 h and 11–62 h, respectively. (4) Detailed analysis of 13 embryos revealed that developmental arrest ('Lag-phase') occurred at the four-cell (1 of 13), five-cell (2 of 13), six-cell (3 of 13), seven-cell (3 of 13) or eight-cell stage (4 of 13); this phase lasted about 59 h. Embryos arrested at the eight-cell stage developed into morula–blastocysts (3 of 4) at a higher rate than did those arrested at earlier stages (2 of 9). (5) The faster the embryos cleaved into early stages (two-cell, three–four-cell and five–eight-cell), the higher the probability that they developed into morula–blastocyst: 70% of the embryos reaching the two-cell stage before 30–31 h after insemination developed into morula–blastocyst. We suggest that the lag-phase as well as the link between early cleavage kinetics and further development could be related to the transcriptional activity of the embryo at about the 5–8-cell stage.
B. Grisart, A. Massip and F. Dessy
A. Massip, P. Mermillod, C. Wils and F. Dessy
Blastocysts derived from bovine zygotes fertilized and matured in vitro and cultured for 7 days in conditioned medium were frozen in 1.36 mol glycerol l−1 and 0.25 mol sucrose l−1. In vitro survival after thawing was unaffected by dilution rate in 0.25 mol sucrose l−1. The proportion of blastocysts that re-expanded after 24 h was 81% (70 of 86) and 47% (33 of 70) hatched. Seven pregnancies beyond 2 months resulted from transfer of 21 blastocysts to 19 recipients. Total embryonic loss was 46.2%, of which 31% occurred between days 21 and 35. In vitro survival after thawing was influenced by culture conditions, the best being culture with oviduct epithelial cells, where 55–82% of blastocysts re-expanded, of which 41–54% hatched. Conditioned medium also supported re-expansion, but low hatching (6%), whereas M199 plus fetal calf serum allowed only limited re-expansion (19–40%). This behaviour was not a consequence of freezing. It is suggested that blastocysts produced in vitro have reduced metabolic activity leading to high embryonic loss before or just at the time of implantation and that oviduct cells create a favourable environment after thawing, allowing hatching in vitro.
P. Mermillod, C. Wils, A. Massip and F. Dessy
Summary. On four occasions ovaries from a total of 35 cows were collected separately at the abattoir where they had been killed. The age of 20 of these cows was recorded. Oocytes from these ovaries were collected separately and were submitted to in vitro maturation, in vitro fertilization and in vitro culture procedures. Ovaries of 34 randomly chosen cows were pooled and treated as the control. Ova from individual cows were cultured in 10 μl droplets and those from pooled ovaries were cultured in groups of 50 in 50 μl droplets of oviductal cell-conditioned medium. The 35 cows treated individually supplied 493 oocytes (mean 14·1 oocytes per cow) with high individual variation (sd = 10·0; range = 0–38) and 47 expanded blastocysts (9·5% of oocytes; mean 1·3 blastocysts per cow; range = 0–6). Among these cows, 16 produced one or more blastocysts. Considerable variation in average development rates was detected over the four replicate experiments (11·3, 4·0, 9·0 and 13·5%). The 34 cows treated as the control supplied 397 oocytes (mean 11·7 oocytes per cow) and 44 expanded blastocysts (11·1% of oocytes; mean 1·3 blastocysts per cow) with high variations between replicates (11·1, 4·0 and 18·1%). No difference was observed between individual and pooled ovaries regarding either the number of oocytes, the rate of blastocyst formation, or the number of blastocysts per cow. No effect of age was detected.
The conclusion was that culture of zygotes in small groups does not impair bovine embryo development in vitro but that high individual variation in oocyte number and the rate of embryonic development may explain the variable results observed in this study between replicate experiments and in general in bovine IVF. These variations will impair the prediction of blastocyst production from individual cows of high genetic value.
Keywords: in vitro fertilization; cow; embryo; blastocyst; breeding; oviduct; conditioned medium
D. Rieger, B. Grisart, E. Semple, A. Van Langendonckt, K. J. Betteridge and F. Dessy
The objective of this study was to compare the development and metabolic activity of cattle embryos co-cultured with bovine oviductal cells or cultured in serum-free medium previously conditioned by bovine oviductal cells. Zygotes were produced by in vitro fertilization of oocytes from bovine ovaries obtained from an abattoir. Development to the four-cell stage occurred by 48 h after fertilization in both culture systems, but co-cultured embryos reached the 16-cell stage by 96 h, whereas those cultured in conditioned medium did not do so until 24 h later. Similarly, the morula and blastocyst stages were reached 24 h earlier in co-culture than in conditioned medium. There were significantly more cells in the blastocysts from co-culture (96.8 ± 6.1 versus 56.7 ± 3.3; P ≤ 0.0001). The metabolism of glutamine did not differ between embryos cultured in the two systems, but the metabolism of glucose was significantly greater in embryos cultured in conditioned medium. The first significant increase in glucose metabolism occurred between the four-cell and the 16-cell stages in embryos cultured in conditioned medium, but occurred between the 16-cell and morula stages in the co-cultured embryos, such that the glucose metabolism was significantly greater at the 16-cell stage in embryos cultured in conditioned medium compared with co-cultured embryos (6.5 ±1.0 versus 1.5 ± 0.4 pmol per embryo per 3 h, P ≤ 0.0001). The concentration of glucose was significantly less, and that of lactate significantly greater, in co-culture medium than in conditioned medium. The results suggest that the activity of enzymes involved in glucose transport or metabolism in the early cattle embryo can be affected by the prior culture conditions, and that a high rate of glucose metabolism may be unfavourable for development.
A. Van Langendonckt, I. Donnay, N. Schuurbiers, P. Auquier, C. Carolan, A. Massip and F. Dessy
The effects of fetal calf serum (FCS) or serum fractions on the development of bovine embryos was investigated. Bovine zygotes were produced in vitro and were cultured in a semi-defined culture medium (mSOF). In the first experiment, blastocysts produced in mSOF supplemented with 10% whole heat-treated FCS or desalted FCS appeared about 1 day earlier, their proportion was significantly (P < 0.05) higher (whole: 30%, desalted: 29%) and they had significantly (P < 0.05) more cells at day 8 (119 cells, 127 cells) than did blastocysts produced in mSOF without any supplement (16%, 98 cells) or mSOF supplemented with a glucose concentration equivalent to that of serum (15%, 88 cells). Our results indicate that high molecular mass components (>5 kDa) of serum are responsible for the effects of FCS on the kinetics of development, on the percentage of blastocysts obtained and the total number of cells in blastocysts. A further analysis using time-lapse microcinematography showed that the acceleration of development induced by serum occurred between the 9–16-cell and morula stages. Finally, in an experiment designed to analyse by microcinematography the effect of the addition of FCS using semen from a different bull to inseminate the oocytes, a different batch of serum and adding mSOF at a different time (42 h after insemination), acceleration was similarly observed between these two stages. Our microcinematographic studies demonstrate that the addition of FCS at two developmental stages (three–four-cell and five–eight-cell) before the 8–16-cell stage accelerates development just after this critical blocking stage.