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

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1 Journal of Reproduction and Development, Vol. 52, No. 1, 2006 Technical Note Effect of Group Culture and Embryo-culture Conditioned Medium on Development of Bovine Embryos Tatsuo FUJITA 1), Hidenobu UMEKI 1), Hideaki SHIMURA 1), Kouji KUGUMIYA 1) and Kazuho SHIGA 1) 1) Livestock Research Institute of Oita Prefectural Agriculture, Forestry and Fisheries, Research Center, Kuju, Oita , Japan 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: , 2006) ecently, ultrasound-guided ovum pick-up (OPU) has enabled repeat collection of oocytes from live cattle for genetic improvement [1 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 Accepted for publication: October 11, 2005 Published online: November 17, 2005 Correspondence: T. Fujita ( fujita-tatsuo@pref.oita.lg.jp) oocytes collected by OPU. Previous studies in mice [4, 5], sheep [6], and cattle [7 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

2 138 FUJITA et al. cells [14]. Therefore, some growth factors secreted from co-culture cells should be contained in the conditioned media and should promote growth of preimplantation embryos. However, the effect of embryo culture-derived conditioned medium on preimplantation development is still unkown. Recent study has suggested that the density of cultured embryos is important for development. In the present study, we investigated the effect of embryo density on the development of bovine embryos in group culture, and the effect of embryocultured conditioned medium on developmental competence of individually cultured bovine embryos. Materials and Methods Oocyte collection and in-vitro maturation Bovine ovaries were obtained at a slaughterhouse and transported to the laboratory in sterile 0.85% NaCl solution containing 200 IU/ml penicillin (Meiji Seika Kaisha, Ltd. Tokyo, Japan) and 0.2 mg/ml streptomycin (Meiji Seika Kaisha Ltd.) at 30 C. All visible follicles (2 5 mm in diameter) on the ovarian surface were aspirated using a 5-ml syringe with an 18-gauge needle. Cumulus-oocyte complexes (COCs) were selected and washed twice with Dulbecco s Phosphate Buffered Saline (Gibco, Gland Island, NY) supplemented with 20% calf serum (CS; Gibco). COCs were transferred into a 100 µl drop of TCM- 199 (Gibco, Gland Island, NY) containing 25 mm HEPES and 10% (v/v) fetal calf serum (FCS; Sigma) under liquid paraffin (Bristol-Myers Squibb, Princeton, NJ) pre-equilibrated in a CO 2 incubator. Then COCs were transferred in 100 µl drops of TCM-199 containing antibiotics, sodium pyruvate (5.5 mg/ml; Sigma), and 10% FCS. The oocytes were then matured for h at 38.5 C under 5% CO 2 and 95% air in a humid atmosphere. Sperm preparation and in vitro fertilization To thaw frozen semen, straws were dropped into water at 37 C for 1 min and then the spermatozoa were washed twice by centrifugation (1,000 g for 2 min) with 20 volumes of Brackett and Oliphant (BO) solution [16] without bovine serum albumin (BSA). The sperm pellet was suspended in BO solution containing heparin (10 µg/ml; Novo Nordisk A/S, Denmark), caffeine (2.5 mm; Sigma), and BSA (10 mg/ml; Sigma) at a concentration of cells/ml. After h of maturation, oocytes were selected and washed three times in BO solution with heparin, caffeine, and BSA. Then oocytes were transferred into 100 µl drops of sperm suspension (20 oocytes per drop) under liquid paraffin and incubated for 5 h at 38.5 C under 5% CO 2 and 95% air in a humid atmosphere. In vitro culture After 5 h of exposure to sperm, the presumptive zygotes were washed and the adhering cumulus cells were removed with a capillary pipette in culture medium (mcr1aa) consisting of CR1aa [17] supplemented with 5% FCS and 25 mg/ml bovine linoleic acid albumin (Sigma). Then, zygotes showing the first polar body were selected and used for the experiments. Experiment 1: Effect of the number of cultured embryos on their development One, 2, 3, 4, and 5 presumptive zygotes were cultured in 5, 10, 15, 20, and 25 µl drops of culture media, respectively (1 zygote/5 µl culture drop) for 7 9 days at 38.5 C under 5% CO 2 and 95% air in a humid atmosphere. Experiment 2: Effect of timing of medium replacement on embryo development After in vitro fertilization (day 0), 5 drops of 400 µl mcr1aa containing presumptive zygotes were prepared for medium replacement on days 2, 3, 4, 5, and 6, respectively. Medium replacement was performed initially by removing 350 µl of the medium from culture drops followed by adding the same volume of fresh medium on days 2, 3, 4, 5, and 6, respectively. Experiment 3: Effect of embryo culture-derived conditioned medium on development of single embryos A group culture drop (50 55 zygotes/400 µl) and single culture drops (1 zygote/5 µl) were prepared simultaneously. On days 2, 3, and 4 after fertilization, 5 µl of conditioned medium derived from the group culture drop was added and mixed to the single culture drops using micropipette tips. Thereafter 5 µl of mixed medium was removed from the single culture drops, and pooled as another drop. The pooled medium was then completely returned to the group culture drop at

3 EFFECT OF BOVINE EMBRYO-CULTURE CONDITIONED MEDIUM 139 once. Half of the single-embryo cultured drops were cultured without the addition of conditioned medium as a control. The rates of cleavage on day 2 and blastocyst formation on days 7 9 after fertilization were recorded. Degenerated or non-fertilized embryos were excluded from the records. Statistical analysis of differences was carried out by chi-square test. Results Experiment 1 Table 1 shows the effect of embryo number on development of bovine embryos at the same cell density (5 µl medium/embryo). The rates of cleavage, blastocyst formation and blastocyst formation/cleavage were 66.7, 13.3, and 20%, respectively, when a single embryo was cultured in a 5 µl drop of mcr1aa. There were no significant differences in cleavage rates among the groups. However, the rate of blastocyst formation increased as the number of embryos in the drop increased. Significant increases in blastocyst formation (P<0.05) were observed in the 5 embryos/drop group compared with the 1 and 2 embryos/drop groups. Experiment 2 Table 2a shows the effect of timing of medium change on embryo development. Medium replacement was performed between day 2 and day 6. Although no significant differences were observed in the rates of cleavage and blastocyst formation between any medium-replacement interval, the rate of blastocyst formation (blastocyst/cleaved) was significantly decreased (P<0.05) when the medium was replaced on days 2 4 compared with on days 5 6 (Table 2b). Table 1. Effect of the number of embryos cultured at constant density on embryo development Number of Droplet Number of Number of Number of embryos volume (µl) cleaved (%) blastcysts (%) blastcysts/cleaved (%) /45 (66.7) 6/45 (13.3) a 6/30 (20.0) a /49 (63.3) 7/49 (14.3) ab 7/31 (22.6) a /50 (58.0) 12/50 (24.0) ab 12/29 (41.4) ab /50 (64.0) 13/50 (26.0) ab 13/32 (40.6) ab /49 (63.3) 16/49 (32.7) b 16/31 (51.6) b a,b Values with different superscripts within a column are significantly different (P<0.05, chi-square test). Table 2a. Effect of timing of medium change on development of group-cultured embryos Day of medium Number of Number of Number of change 1) cleaved (%) blastcysts (%) blastcysts/cleaved (%) 2 46/53 (86.8) 20/53 (37.7) 20/46 (43.5) 3 45/54 (83.3) 22/54 (40.7) 22/45 (48.9) 4 42/52 (80.8) 20/52 (38.5) 20/42 (47.6) 5 40/50 (80.0) 25/50 (50.0) 25/40 (62.5) 6 38/51 (74.5) 24/51 (47.1) 24/38 (63.2) 1) 350 µl of 400 µl of the medium was changed once. Table 2b. Effect of timing of medium change on development of group-cultured embryos Day of medium Number of Number of Number of change 1) cleaved (%) blastcysts (%) blastcysts/cleaved (%) /159 (83.6) 62/159 (39.0) 62/133 (46.6) a /101 (77.2) 49/101 (48.5) 49/78 (62.8) b 1) The results in Table 2a were aggregated for days 2 4 and days 5 6. a,b Values differ significantly (P<0.05, chi-square test).

4 140 FUJITA et al. Table 3. Effect of embryo culture-derived conditioned medium on development of single-cultured embryos Single or Addition of Number of Number of Number of group culture conditioned medium Replicates cleaved (%) blastcysts (%) blastcysts/cleaved (%) Single 1) 3 82/114 (71.9) 15/114 (13.2) a 15/82 (18.3) a Single + 3) 3 85/112 (75.9) 31/112 (27.7) b 31/85 (36.5) b Group 2) (Supplier) 3 128/155 (82.6) 68/155 (43.9) c 68/128(53.1) c 1) Single embryos were cultured in 5 µl droplets. 2) Fifty to 55 embryos were cultured in a 400 µl drop. 3) Five µl of group cultured medium was added to single embryo cultured drops between the 2nd-4th days after fertilization, and then 5 µl of mixed medium was removed. a-c Values with different superscripts within a column are significantly different (P<0.001). Experiment 3 The blastocyst formation rate was significantly (P<0.001) increased by the addition of embryo culture-derived conditioned medium (Table 3). There was no difference between the rate of cleavage for the embryos in single culture and those in the conditioned medium supplemented group. The rate of blastocyst formation was significantly higher (P<0.001) for group culture (50 55 embryos) than for single- and conditioned medium supplemented single-culture. Discussion The present study suggested that the number of cultured embryos in a culture drop is important for supporting embryo development, and that embryo culture derived-conditioned medium promotes the development of single-cultured embryos. In the initial experiment, an increase in the number of cultured embryos promoted the blastocyst formation rate, even with a constant volume of culture medium per embryo. A similar finding was observed in a previous study we conducted, which showed that the blastocyst formation rate of OPU-IVF embryos increases with the increase in number of embryos per drop (unpublished data). These results suggest that group culture is efficient for culture of small numbers of embryos. The present study also suggests the possibility that a specific factor(s) derived from cultured embryos in the medium supports embryo development. However, no experiments were conducted in the present study to clarify the optimal embryo-culture density. Several reports have suggested optimal culture densities between 1 and 13 µl/embryo [8, 9, 15]. We used a culture density of 5 8 µl/embryo in the present study according to our usual OPU-IVF and culture protocol. Therefore, the culture density of our study does not differ from previous reports. Previous studies on mice [4, 5] and sheep [6] have indicated that higher numbers of embryos in a culture drop support embryo development. According to Lane and Gardner [4], the blastocyst formation rate increases when the embryo density increases. One of these factors may be associated with the blood platelet-activating factor called embryo-derived platelet-activating factor (EPAF). Stoddart et al. [5] confirmed that EPAF, which is secreted from mouse embryos, stimulates embryo development into blastocysts. Moreover, according to Gardner et al. [6], some other factors are probably secreted by the embryos to stimulate the development of sheep embryos. Furthermore, Ferry et al. [8] reported that culture drops with a larger number of embryos in a constant culture volume tend to promote development of bovine embryos. Experiment 2 elucidated the timing of secretion or accumulation of the postulated embryo-derived growth promoting factor in the culture medium. The results of medium replacement between days 2 and 6 post-ivf demonstrated that there were no differences in the blastocyst formation rate among the respective medium replacement intervals. However, there was a significant decrease in blastocyst formation when medium replacement was carried out before day 4 post IVF compared with after day 5 post IVF. The blastocyst formation rate after day 5 post IVF was equivalent to the rate typically obtained in our laboratory (unpublished data). This finding suggests that embryo-secreted factors released or adequately accumulated in culture medium are important for embryo development until day 4 post IVF. One of the reasons for the low tendency of embryo development when the medium was changed before day 4 post IVF may be the dilution or

5 EFFECT OF BOVINE EMBRYO-CULTURE CONDITIONED MEDIUM 141 decrease of such factors. In cases of in-vitro culture of mammalian embryos, termination of embryo development occurs at a specific developmental stage. This phenomenon is known as the in-vitro block. In the case of cattle, developmental arrest occurs at the 8- to 16-cell stage on days 2 3, called the 8-cell block [18]. Therefore, we believe that the blastocyt development rate was not substantially influenced by the medium replacements on days 5 6 post IVF because the 8- cell block barrier had previously invalidated this procedure. Several methods have been reported for overcoming the 8-cell block, such as co-culture systems with granulosa [19 21], oviduct epithelial cells [11, 12], trophoblastic tissues [22], addition of growth factors [23 27], glucose deprivation [28], culture under low oxygen concentration [29, 30], addition of reducing agents [13], and cytokines [32, 33]. Particularly in culture systems that replace coculture with somatic cells, methods employing a conditioned medium of bovine oviduct epithelial cells [11, 12], buffalo rat hepatocytes [13], bovine granulosa cells, and vero cells [14] have been documented. However, attempts to promote embryo development by adding embryo culturederived conditioned medium have not been reported. The blastocyst formation rates of single-cultured embryos were significantly increased by addition of embryo culture-derived conditioned medium. In this experiment, the amount of culture medium actually replaced was half the volume of a single culture drop. Possible inhibitory effects of reusing culture medium have been suggested by Ferry et al. [8] because of the lack of certain components in the medium, such as glutamine. In the present study, we used a culture medium containing bovine calf serum for preparation of the conditioned medium. A recent study has enabled culture of bovine embryos using a defined medium [14]. Preparation of embryo culture-derived conditioned medium with a defined medium may support embryo development and may also lead to elucidation of the factor(s) secreted by embryos. However, it is still necessary to note that it may possible to decrease the toxic factors in the culture medium by embryonic metabolism to provide suitable conditions for the embryos. In conclusion, the number of embryos in culture medium at a constant embryo density is critical for developmental competence, and embryo culturederived conditioned medium promotes singleculture embryo development. The present method enables genetic improvement of cattle by increasing the competence of transferable embryos derived from OPU-IVF techniques. Acknowledgments The authors thank Dr. M. Takahashi of the National Agricultural Research Center for Kyushu Okinawa Region for critical reading of the manuscript, and the Oita Prefecture Meat Inspection Office and Oita Meat Trading Center Ltd. for their kind help in collecting bovine ovaries. References 1. Pieterse MC, Kappen KA, Kruip TAM, Taverne MAM. Aspiration of bovine oocytes during transvaginal ultrasound scanning of the ovaries. Theriogenology 1988; 30: Pieterse MC, Vos PLAM, Kruip TAM, Wurth YA, Beneden TH, Willemse AH, Taverne MAM. Transvaginal ultrasound guided follicular aspiration of bovine oocytes. Theriogenology 1991; 35: Meintjes M, Bellow MS, Brossard JR, Paul JB, Godke RA. Transvaginal aspiration of bovine oocytes from hormone-treated pregnant beef cattle for IVF. Theriogenology 1993; 39: 266 (abstr). 4. Lane M, Gardner DK. Effect of incubation volume and embryo density on the development and viability of mouse embryos in vitro. Hum Reprod 1992; 7: Stoddart NR, Wild AE, Fleming TP. Stimulation of development in vitro by platelet-activating factor receptor ligands released by mouse preimplantation embryos. J Reprod Fertil 1996; 108: Gardner DK, Lane M, Spitzer A, Batt PA. Enhanced rates of cleavaged and development for sheep zygotes cultured to the blastocyst stage in the absence of serum and somatic cells: amino acids, vitamins, and culturing embryos in groups stimulate development. Biol Reprod 1994; 50: Palma GA, Clement-Stegewald A, Berg U, Brem G. Role of the embryo number in the development of in

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