Effects of co-culture and embryo number on the in vitro development of bovine embryos.

It is generally accepted that culturing embryos in groups or with somatic cells improves both the yield and quality of the blastocysts obtained. The aims of this study were 1) to compare the yield and quality of the embryos obtained after culture in several number conditions and in several culture systems and 2) to assess the effect of co-culture started at various stages of embryo development. Under cell-free culture conditions (modified synthetic oviduct fluid [mSOF] supplemented with 10% fetal calf serum [FCS] 48 h post insemination, the rate of Day 10 blastocysts was lower when embryos were cultured in small groups (1 to 6 per drop) than in large groups (4 versus 23% ; P < 0.01). There was no group effect when embryos were co-cultured either with Buffalo rat liver (BRL) cells in TCM 199, or in a culture system allowing the progressive development of cumulus cells in mSOF, even if co-culture started at 66 or 114 h post insemination. However, embryos cultured singly had lower cell numbers than embryos cultured in large groups when co-culture started at 114 h post insemination. This suggests that 1) somatic cells improve the development of singly cultured bovine embryos up to the blastocyst stage after the 9-16 cell stage; 2) co-culture affects blastocyst cell number of singly cultured embryos by acting roughly between the 5-8 and the 9-16 cell stage; and 3) cooperation between embryos could replace the effect of co-culture either on the yield of blastocysts or on blastocyst cell number. Blastocysts appeared significantly earlier in co-culture with cumulus cells in mSOF than in co-culture with BRL cells in TCM 199 (detection of the blastocysts: 7.3 +/- 0.1 d post insemination with cumulus cells versus 8.1 +/- 0.1 d with BRL cells; P < 0.001) and had a significant higher number of cells (143 +/- 9 versus 85 +/- 11; P < 0.001). This system thus seems suitable for the culture of small numbers of embryos resulting from in vitro maturation and fertilization of oocytes from individual donor cows.

Effect of estrus-associated glycoprotein and tissue inhibitor of metalloproteinase-1 secreted by oviduct cells on in vitro bovine embryo development.

The effect of two glycoproteins (estrus-associated glycoprotein [EGP] and tissue inhibitor of metalloproteinase [TIMP-1]) secreted by bovine oviduct cells on in vitro bovine embryo development was assessed. A first set of experiments was conducted to determine whether the embryotrophic activity of the bovine oviduct-conditioned medium (BOCM) was correlated with the presence of EGP or TIMP-1. EGP and TIMP-1 were detected in BOCM, supporting the development of 22% zygotes to the blastocyst stage, as well as in BOCM yielding a low blastocyst rate (3-4% blastocysts). These glycoproteins do not seem to be necessary for bovine embryo development up to the blastocyst stage in our BOCM. In a second experiment, zygotes were cultured in modified synthetic oviduct fluid (mSOF) supplemented with different concentrations (0.5, 5, 50, and 500 micrograms/ml) of purified bovine EGP. In the third experiment, since purified bovine TIMP-1 was not available, zygotes were cultured in BOCM depleted of TIMP-1 by immunoprecipitation treatment. Adding EGP to mSOF, or removing TIMP-1 from BOCM, did not affect bovine embryo development up to the blastocyst stage, or mean number of cells per blastocyst after 8 days of culture. The results indicate that, under our culture conditions, EGP and TIMP-1 do not play an important role in sustaining bovine embryo development, and do not influence blastocyst quality, assessed in terms of total number of cells per embryo.

Correlations between chemical parameters, mitogenic activity and embryotrophic activity of bovine oviduct-conditioned medium.

To establish parameters predicting the quality of bovine oviduct epithelial cell-conditioned media, we compared media conditioned by oviduct cells from cows at Day 2 (n = 3) and Day 15 (n = 3) of the estrous cycle. In addition, we tested the influence of time of conditioning. Media were evaluated for their embryotrophic activity using a cumulus cell co-culture system as a control. The same media were tested for their mitogenic activity on NIH 3T3 cells and for chemical parameters, including total protein, and de novo synthesized protein as well as for concentrations of glucose, lactate and ammonium. Analysis of variance did not reveal a significant effect by stage of the estrous cycle on the embryotrophic activity of conditioned media. However, there was a significant effect by time of conditioning on the proportion of 5- to 8-cell embryos (P < 0.01) and of blastocysts and hatched blastocysts (P < 0.05). None of the conditioned media (19 to 31% blastocysts) was superior to the cumulus cell co-culture system (32% blastocysts). In the conditioned media, the proportion of 5- to 8-cell embryos correlated positively with mitogenic activity on 3T3 cells (r = 0.64; P < 0.05), whereas the proportion of blastocysts was not significantly correlated with this parameter. In summary, our results provide evidence for an effect of time of conditioning on embryotrophic activity of oviduct epithelial cell-conditioned media. The fact that mitogens for NIH 3T3 cells affect the proportion of 5- to 8-cell embryos but not of blastocysts suggests different culture requirements for early and late preimplantation stage development of bovine embryos.

Effect of high molecular weight factors present in bovine oviduct-conditioned medium on in vitro bovine embryo development.

To investigate the presence of embryotrophic factors in bovine oviduct-conditioned medium (BOCM), the high molecular weight fraction (> 10 KDa) from BOCM was added to 3 chemically defined embryo culture media (TCM199, DMEM/F12 and modified synthetic oviduct fluid [mSOF]). Zygotes were obtained by in vitro maturation and fertilization of oocytes. Conditioning of TCM199 with oviduct cells increased both cleavage to the 5- to 8-cell stage (59 vs 37%) and further development to the blastocyst stage (19 vs 4%). The low molecular weight fraction (< 10 KDa) of BOCM maintained development to the 5- to 8-cell stage but did not allow development to the blastocyst stage. Adding the high molecular weight fraction to the inactive low molecular weight fraction restored bovine embryo development up to the blastocyst stage. This embryotrophic effect of the high molecular weight fraction was not observed when this fraction was added to TCM199 or DMEM/F12 medium. Whereas adding this fraction to mSOF medium significantly (P<0.05) increased embryo development up to the blastocyst stage (36%) in comparison with that of mSOF (15%) or BOCM (14%). These results show that BOCM contains high molecular weight factors promoting embryo development up to the blastocyst stage. Some chemically defined media mask the effect of these embryotrophic factors.

Three year results of in vitro production of bovine embryos in serum-poor bovine oviduct conditioned medium. An overview.

This paper presents a synthesis of 3 year results of in vitro production of bovine embryos in medium previously conditioned by bovine oviduct epithelial cells. In Louvain-la-Neuve, Belgium, a total of 18356 oocytes were matured and inseminated in vitro: 13967 (76%) had cleaved at 3 days post-insemination and 3593 (26%) became blastocysts using this culture system. Our data show that conditioned medium can be stored frozen for up to 3 years without significant loss of activity and is resistant to lyophilization. One single batch of conditioned medium was tested within the same period in four different laboratories and yielded variable results: 27 and 37% blastocysts/cleaved embryos in two of them and only 7 and 0% in the two others whereas in each case more than 30% blastocysts were obtained with the local reference co-culture system. In one laboratory, the batch of oil used to overlay the culture drops had a detrimental effect on the blastocyst rate in conditioned medium but not in co-culture.

Characterization of bovine oviduct epithelial cell monolayers cultured under serum-free conditions.

We have developed a culture system for early bovine embryos in serum-free media conditioned by oviduct cell monolayers. A gentle mechanical procedure for oviduct cell isolation has been applied for this purpose avoiding the use of proteolytic enzymes. The aim of the present study was to identify the cell types present in the monolayers and to examine their fate in primary culture in serum-free or in serum-containing media by means of electronmicroscopical, immunocytochemical, and biochemical analyses. The cell dissociation procedure yielded two cell populations: ciliary cells and secretory cells that gradually dedifferentiate during culture. These cells formed a confluent monolayer after 6 d of culture in Tissue Culture Medium 199 medium supplemented with 10% fetal calf serum. Confluent cells displayed a typical epithelial cell morphology as assessed by phase contrast and electron microscopy and all the cells contained cytokeratin filaments as determined by immunocytochemistry. The overall histoarchitecture of the monolayer was preserved after washing and further culture for 7 d in serum-free medium. However, some degenerative signs indicate that the serum-free culture should not be extended for more than 7 d. Confluent oviduct cells also maintained their metabolic and protein secretory activity when deprived of serum. Total protein content in the culture supernatant linearly increased as a function of time and numerous peaks were detected after separation of proteins by high performance ion exchange chromatography. Protein elution patterns were reproducible and most of the proteins present in the culture medium were neosynthesized as determined by the incorporation of radiolabeled amino acids into nondialyzable proteins.

In vitro development of bovine embryos in Buffalo rat liver- or bovine oviduct-conditioned medium.

A culture system for bovine embryos was developed using Buffalo rat liver cell (BRL) line-conditioned medium without serum. Zygotes, obtained by in vitro maturation and fertilization of oocytes, were cultured either in unconditioned medium (TCM 199 or DMEM/F12) or in the same medium conditioned by bovine oviduct or BRL cells. No serum was added during conditioning or during embryo culture. The DMEM/F12 medium was superior to TCM 199 for development of bovine embryos to the 5 to 8-cell stage: on average between 50 and 57% of the embryos reached this stage after 2 d of culture in DMEM/F12 or in conditioned medium, while 36% reached this stage in TCM 199. Further development to the blastocyst stage was enhanced by conditioning. The highest percentage of blastocysts was achieved in DMEM/F12 medium conditioned with BRL cells (30%). The yield of blastocysts was similar in TCM 199 and in DMEM/F12 media conditioned with bovine oviduct cells (22 versus 20%), but after conditioning with BRL cells, DMEM/F12 medium yielded a higher percentage of blastocysts than TCM 199 (30 versus 18%). This might be explained by the fact that viability of BRL cells was better in DMEM/F12 medium than in TCM 199 when serum was omitted. Blastocysts produced in BRL-conditioned medium had a higher number of cells than blastocysts obtained in bovine oviduct-conditioned medium, and their transfer to recipients led to pregnancies and birth of calves. In conclusion, culture of bovine embryos in DMEM/F12 medium conditioned with BRL cells without serum led to the development of good-quality blastocysts and is thus a promising method for producing embryos for the study of potential embryotrophic factors. The use of rat liver cell lines guarantees against bovine viruses and allows for better production of embryos.

Characterization of the embryotrophic activity of exogenous protein-free oviduct-conditioned medium used in culture of cattle embryos.

Bovine zygotes, obtained after in vitro maturation and fertilization of oocytes from slaughtered cow ovaries, were cultured in droplets of nonconditioned or conditioned medium on bovine oviduct cell monolayers. The media tested were Medium 199 alone and Medium 199 supplemented with 10% fetal calf serum (FCS). Oviduct conditioning increased both early cleavage and development to blastocysts. Only the effect on early cleavage was mimicked by FCS. The blastocysts obtained in serum-free conditioned medium (SFCM) appeared morphologically normal and had the same cell number as those produced in conditioned medium containing serum. Their hatching rates did not differ. Transfer of 16 blastocysts developed in SFCM to 16 synchronized recipients resulted in five pregnancies (31%), indicating good embryonal viability. Boiling of SFCM resulted in a total loss of activity, while heating at 56 degrees C for 30 min had no deleterious effect. A 10-kDa ultrafiltration of SFCM removed the blastocyst development-supporting activity from the filtrate but not the early cleavage-favoring activity. This allows us to conclude that at least two different factors are present in SFCM: one of low molecular mass (< approximately 10 kDa), needed to obtain the 5-8 cell stage and mimicked by FCS, and another of higher molecular mass allowing embryos to develop from the 8-cell to blastocyst stage.