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The submarine incubation system, a new tool for in vitro embryo culture: A technique report
ELSEVIER
THE
SUBMARINE
INCUBATION CULTURE: G. Vajta,’
‘Embryo
SYSTEM, A NEW TOOL FOR A TECHNIQUE REPORT
P. Holm,
r T. Greve*
IN VITRO
EMBRYO
and H. Callesen’
Technology
Center, Danish Institute of Agricultural Sciences DK-8830 Tjele, Denmark ‘Department nf Clinical Studies, Sectinn of Reproduction Royal Veterinary and Agricultural University DK-1870 Frederiksberg C, Denmark Received
for- publication: Accepted:
March 13, 1997 July 2, 1997
ABSTRACT ln vitro culture ot sensitive structures such as oocytes and preimplantation embryos requires a specific, stable environment (temperature, gas atmosphere and humidity levels). Most availahlc carbon dioxide thcrmostatcs arc not adequate for this purpose. as their large interior area is undivided and frequent opening of the doors required by the daily work disturbs equilibrium of the cultures kept inside. A new approach for- overcoming this problem is described here. Tissue culture dishes containing embryos are individually wrapped in laminated foil hags nearly impermeable to carbon dioxide, oxygen and nitrogen and are filled with the desired gas mixture, then heat-scaled and submerged into a circulating temperature-adjusted water hath for the required culturc period (up to 7 d). In this way, all wrapped culture dishes function as individual incubators (“submarines”). The advantages of this system are stahility of temperature, humidity and gas mixture: quick recovery of these parameters after opening: flexrhrlity in using different gas mixtures; safety: cost efficiency: reduced contamination risks: few prohlems with cleaning; easy transport. When transparent foil is used for wrapping, frcqucnt microscopic observations arc also possible without disturbing the gas atmosphere and humidity. Slightly elevated atmospheric pressure inside the foil bags (between 1 and 9 water cm) has no apparent deleterious effects on embryo development. Expired. sterile filtered human lung air containing 4%> carbon dioxide and 16 to 17% oxygen expired into the bags was also found to sustain bovine embryo development. Thus, the culture system is also suitable for use under field conditions. To prove the efficiency of this incuhatinn system. in vitro matured and fertilized bovine zygotes were cultured in TCM 199 and calf serum on a granulosa cell monolayer using expired lung air in the submerged foil bags. The cumulative result of 25 identical replicates was 517 blastocysts from 1,052 oocytes (49%) at 8 d after fertilization. The Submarine Incubation System provides an altcrnativc method for in vitro embryo production and/or culture of other sensitive tissues or cells. 0 1997 by Elsewer Science
Key
words:
lnc
incubator.
Themgenology48.1379-1385.1997 0 1997 by Elsev~er Sctence
foil hag, in vitro
Inc
culture,
embryo.
transportation 0093-691w97/$17.00 PII s0093-691x(97)00379-8
Theriogenology INTRODUCTION In vitro culture of cells or tissues requires the establishment of a physical, chemical and biological environment similar to that surrounding the cells or tissues in vivo. Various tissue culture media and dishes have been developed to fulfill the biological and chemical requirements, and sophisticated thermostats are offered to keep gas phases, humidity and temperature at optimum levels. However, most of these incubators have only one large or incompletely divided storage area, so opening of the door disturbs the environment of all the cultures inside. In spite of the latest technology compensating for these factors, such small disturbances may have a cumulative negative effect on cultures, with decreasing development and viability as a possible consequence. Certain tissues or cells such as mammalian oocytes and preimplantation embryos arc very sensitive to environmental changes. On the other hand, in vitro production of these embryos requires frequent opening of the thermostats for changing the medium, adding spermatozoa and transferring embryos from one dish to another. Laboratories where at least one in vitro fertilization program is started each day minimize these problems by purchasing a number of carbon dioxide thermostats, by strictly regulating the time and duration of openings, and by maintaining the temperature of the whole laboratory near to the physiological body temperature (37 to 39°C). However, from a financial and/or technical point of view, these solutions are difficult for most laboratories to resolve. In this study we report a simple new incubation method which can be established in commercially available water baths and which fulfills all the requirements for in vitro embryo production. We call it the Submarine Incubation System. MATERIALS
AND METHODS
In Vitro Embryo Production Except where indicated, all chemicals were obtained from Sigma Chemical Company (St. Louis, MO 63178 . USA). Maturation of bovine oocytes, fertilization and culture of embryos has been described in detail elsewhere (5). Briefly, oocytes were aspirated from slaughterhouse-derived ovaries, matured in 4-well dishes (Nunc, DK-4000 Roskilde, Denmark) for 24 h in bicarbonate buffered TCM-199 medium supplemented with 15% calf serum, eCG and hCG. Insemination (Day 0) was performed using frozen-thawed, Percoll-selected spermatozoa. After 30 h, zygotes were vortexed, then cultured on the granulosa cell monolayer formed spontaneously in the maturation dishes in 400 pl TCM-199 supplemented with 5% (10% from Day 4, except in Experiment 3) calf serum. The cultures were evaluated on Day 7 and 8 after insemination. For maturation and fertilization, a double-wall K-system box with continuous gas flow and heated by an external circulator (Henning Knudsen Engineering, DK-3460 Birkerod, Denmark) was used. Embryo culture was performed in the Submarine Incubation System as described below. Except where otherwise indicated, evaluations were made on Day 8 by stereomicroscopy, determining the blastocystioocyte ratio.
Theriogenology
1381
The Submarine Incubation System A 10 x 1 l-cm bag was prepared from laminated foil (S12MY PETP Metal/75MY PE Guld, SFK, DK-2650 Hvidovre, Denmark; oxygen and carbon dioxide permeability max. 0.005 g/m2/24h/l atm, 23°C. 5/950/c RH, DIN 53122) with the use of a heat-adjustable, 12-mm wide sealing iron (Comecta, Selecta s.a., SP-08630 Abrera, Barcelona, Spain). After fertilization, uncovered 4-well dishes containing the oocytes were inserted, and the bags were completely sealed. Using a 19-g injection needle, a hole was made in each bag in the proximity of one of the comers and distant from the culture dish. A tube was attached to the injection needle, and the bag was completely filled with a sterile-filtered, preheated and humidified gas mixture at a flow rate of approximately 500 ccm/min. The needle was then removed, and the gas was pushed out through the hole with moderate mechanical pressure. The bag was filled and emptied twice more to ensure an almost total gas exchange in the bag before the hole was heat-sealed. The bag was then placed on a shelf of a plastic test tube rack (Cat. no. 5970, Nalgene, Rochester, NY 14602-0365, USA) and submerged into a 38.7”C circulated water bath (DT OB-22, Heto, DK-3450 Allerfld, Denmark). An 800-ml tissue culture flask (Nunc, DK-4000 Roskilde, Denmark) filled with water was used as a weight to prevent floating (Figure 1). The water level was adjusted so that the water covered the upper shelf of the rack. Thus, the bags containing the dishes were pressed up against the shelves by the water and were subjected either to 1 cm (upper shelf) or 5 cm (lower shelf) of water pressure.
Figure 1. Schematic drawing of the Submarine Incubation System. Foil bags (1) containing the four-well dishes and filled with the required gas mixture are placed on the shelves of a test tube rack (2) and submerged into a circulating water bath (3) with an 800 g weight (4). Experiment 1 Industriallv nroduced gas mixture vs exnired lung air for atmosnheric gas. In Experiment la (6 replicates) embryo culture from Day 1 to Day 8 was performed in 10 x 1 l-cm laminated foil bags filled either with a mixture of 4% carbon dioxide in air (+lO%, AGA A/S, DK-7000 Fredericia, Denmark; group A) or with expired lung air from one person (group B). In Experiment lb, expired lung air of the same person was used to fill the foil bags in 25 successive identical replicates.
1382
Theriogenology
Experiment 2 Effect of increased pressure on embrvo develoument. In 3 replicates, a special deep container and two test-tube racks placed on top of each other were used so that the 10 x 1 l-cm laminated foil bags containing the embryo cultures were submerged either 1, 5,9 or 13 cm under the water level from Day 1 to Day 4 of culture. From Day 4 to Day 8, all the bags were immersed to 1 cm under the water level. Experiment 3 Transportable incubator. To adjust the size of the 4-well dish to the transportable incubator, the outer frame was cut, and the remaining 4.5 x 4.5 x l.O-cm part of the dish with the 4 wells was used for the whole experiment. In 3 replicates, embryo cultures were performed in 6.5 x 7.0cm laminated foil sacks filled with expired air, then heat-sealed and submerged into a Minitiib transportable incubator (Ref. no. 19180/0000, Minitiib GMBH, D-84184 Tiefenbach, Germany) previously sealed with glue to become waterproof, then filled with water. The cultures were left undisturbed from Day 1 until evaluation (Day 8). Experiment 4 Transparent foil for freauent microscouic investipation. In 4 replicates, embryo culture was perfonned in 10 x 1 l-cm transparent laminated foil bags (Camclear Polyester Laminate 12/45, Anti-Fog, Rexam Metallising, IP24 3QY Thetford, UK; oxygen barrier < 5.0 cc/m2/24h, 23°C 5O%RH, carbon dioxide barrier < 12.0 cc/m2/24h) filled with expired air. The inner bottom surface of the foil was moistened by sterile paraffin oil before loading to keep the foil attached to the bottom of the dish in order to increase optical clarity. During the 7 d of the culture, the bags were taken out of the water bath 5 times, dried and placed on the heated stage of an inverted microscope. They were then observed, photographed and returned to the water bath. Each investigation was performed in less than 5 min. Statistical Evaluation Data were analyzed by the Chi-square test, and PcO.05 was considered to be statistically significant. RESULTS Experiment 1 In Experiment la, the cumulative rate of development of the 6 replicates was 136 blastocysts I294 oocytes (46%) and 134 blastocysts I285 oocytes (47%) in 4% carbon dioxide in air and expired lung air, respectively (P>O.O5). In Experiment lb, the cumulative rate of 25 replicates was 5 17 blastocysts / 1,052 oocytes (49%, range: 40 to 55%).
Theriogenology
1383
Experiment 2 Submerging the foil bags under increased pressure, i.e., under 13 cm water, did not impair the developmental rate: the cumulative results of 3 replicates were 45, 49, 44 and 38% (P>O.O5; 58/129, 691142, 571130 and 55/146 blastocysts / oocytes, respectively) in the groups submerged from Days 1 to 4 at 1,5,9 or 13 cm below the water surface. Experiment 3 The cumulative developmental rate of the 3 replicates performed in the transportable incubator was 58 blastocysts / 139 oocytes (42%, range: 37 to 44%). Experiment 4 The cumulative developmental rate of the 4 replicates performed in the transparent foil was 80 blastocysts / 178 oocytes (45%, range: 39 to 51%). DISCUSSION The simple, new incubation system presented here can be established in most commercially available water baths. The advantages and disadvantages of this system are summarized below: In contrast with most carbon dioxide incubators, heat is conducted in the Submarine Incubation System by circulating water instead of gas. Most water bath circulators work with an accuracy of O.Ol-O.O5”C, which can not be achieved by gas flow. Other advantages of our system are the quick recovery rate of temperature without the danger of overheating the cultures, and the possibility of rapid, accurate changes of temperature, i.e., for temporary cooling in certain embryo manipulation procedures (2). The preheated and humidified gas is promptly injected into the foil bags, and this atmosphere is maintained for the whole period of culture. Although direct investigations were not performed, the 60 to 80 ml gas mixture was adequate to cover the requirements of 100 preimplantation stage bovine embryos cultured in 4 x 400 ul medium for a period of 3 d. Moreover, in Experiment 3, acceptable development of embryos cultured in 2 x 400 ul medium and 20 to 30 ml gas mixture for 6 d was also observed. Each bag may be filled with the required gas mixture, thus providing considerable flexibility to the system. As shown in Experiment 2, the elevated atmospheric pressure in the bag (up to 13 water cm) caused by the hydrostatic pressure of water did not impair early embryo development. Stability, safety and cost-efficiency of this systemaremarked. During incubation, the only source of trouble was the water bath circulator, which is generally regarded as a trouble-free piece of laboratory equipment. Infections that occasionally occur in primary cultures can be localized and eliminated without the risk of propagating the infectious agent and without the need to stop the whole incubator. In our experience, regular use of water bath desinfectants has precluded contamination of water even when the same water was used for more than 5 mo. Although the inside of the foil bags were not sterilized, infection of the cultures was not detected
1384
Theriogenology
during the whole experimental period. The price and maintenance costs of the Submarine Incubation System are much lower than those of a carbon dioxide incubator. Previous experiments have demonstrated the beneficial effects of slightly elevated pH levels on bovine embryo development in vitro that are produced either by increased bicarbonate concentration in the medium or decreased carbon dioxide concentration in the atmosphere (6, Vajta et al., unpublished observations). Expired human lung air contains 4% carbon dioxide and 16 to 17% oxygen, with an accuracy comparable to those of the generally used industriallyproduced gas bottles (approximately &lo%), may be sterilized by filtration; moreover. it comes both preheated and humidified. The foil bag system is uniquely suitable for filling with expired air for embryo culture, since it is easy and requires a low volume. In Experiment 1, the blastocyst rates achieved either with the expired lung air or with gas bottle-derived 4% carbon dioxide in air were similar, and in a large series of replicates performed with expired lung air, high and stable embryo development rates were obtained. A transportable version of the Submarine Incubation System was also developed. Although the heat-accuracy of the modified Minitiib thermostat was lower than that of the circulating baths, an acceptable rate of embryo development was achieved using the transportable model for up to 6 d of incubation. Moreover, these experiments were performed with the use of expired lung air, which makes the system suitable to establish and maintain appropriate culture conditions under field situations. The use of a transparent foil facilitated optical follow-up of the embryos without disturbing their atmospheric environment. In Experiment 4, repeated microscopic observations did not result in the impairment of development of the embryos, and the quality of the microscopic view was satisfactory for embryo evaluation. To date, the only registered disadvantage of the Submarine Incubation System is that 1 to 2 min of extra work are required for each culture dish. Compared with the amount of work required to produce bovine embryo cultures, this extra effort seems minimal. Since the Submarine Incubation System was first introduced into our laboratory in April, 1996, it has been used successfully both for routine embryo production and for experiments dealing with the effects of the different gas mixtures on embryo development. This system is a reliable and cost-efficient way to culture sensitive tissues or cells, such as ova and preimplantation embryos. The developmental rates of in-vitro produced bovine embryos achieved with this system have been among the highest yet reported (1, 3, 4). The versatility of this system (easy transport, on-sight use, microscopic follow up) offers a wide variety of applications in embryology and tissue culture work.
Theriogenology
1385
REFERENCES 1. Brackett BG, Zuelke KA. Analysis of factors involved in the in vitro production of bovine embryos. Theriogenology 1993; 39: 43-64. 2. Chesne 0, Heyman Y, Peynot N, Renard JP: Nuclear transfer in cattle; birth of cloned calves and estimations of blastomere totipotency in morulae used as a source of nuclei. Life Sci 1993; 316:481-491. 3. Farin CE, Hasler JF, Martus NS. Comparison of Menezo B2 and TCM 199 media for in vitro production of bovine blastocysts. Theriogenology 1995; 43: 2lOabstr. 4. Hernandez-Lendezma JJ, Villanueva C, Sikes JD, Roberts RM. Comparison of co-culture and conditioned medium on expansion and hatching of in-vitro derived bovine blastocysts. Theriogenology 1995; 43: 233abstr. 5. Vajta G, Holm P, Greve, T Callesen H. Overall efficiency of in-vitro embryo production and vitrification in cattle. Theriogenology 1996; 45: 683-689. 6. Vajta G, Holm P, Greve T, Callesen H. Effect of pH of culture medium on in vitro development of bovine embryos. Theriogenology 1997; 47: 286abstr.
THE
SUBMARINE
INCUBATION CULTURE: G. Vajta,’
‘Embryo
SYSTEM, A NEW TOOL FOR A TECHNIQUE REPORT
P. Holm,
r T. Greve*
IN VITRO
EMBRYO
and H. Callesen’
Technology
Center, Danish Institute of Agricultural Sciences DK-8830 Tjele, Denmark ‘Department nf Clinical Studies, Sectinn of Reproduction Royal Veterinary and Agricultural University DK-1870 Frederiksberg C, Denmark Received
for- publication: Accepted:
March 13, 1997 July 2, 1997
ABSTRACT ln vitro culture ot sensitive structures such as oocytes and preimplantation embryos requires a specific, stable environment (temperature, gas atmosphere and humidity levels). Most availahlc carbon dioxide thcrmostatcs arc not adequate for this purpose. as their large interior area is undivided and frequent opening of the doors required by the daily work disturbs equilibrium of the cultures kept inside. A new approach for- overcoming this problem is described here. Tissue culture dishes containing embryos are individually wrapped in laminated foil hags nearly impermeable to carbon dioxide, oxygen and nitrogen and are filled with the desired gas mixture, then heat-scaled and submerged into a circulating temperature-adjusted water hath for the required culturc period (up to 7 d). In this way, all wrapped culture dishes function as individual incubators (“submarines”). The advantages of this system are stahility of temperature, humidity and gas mixture: quick recovery of these parameters after opening: flexrhrlity in using different gas mixtures; safety: cost efficiency: reduced contamination risks: few prohlems with cleaning; easy transport. When transparent foil is used for wrapping, frcqucnt microscopic observations arc also possible without disturbing the gas atmosphere and humidity. Slightly elevated atmospheric pressure inside the foil bags (between 1 and 9 water cm) has no apparent deleterious effects on embryo development. Expired. sterile filtered human lung air containing 4%> carbon dioxide and 16 to 17% oxygen expired into the bags was also found to sustain bovine embryo development. Thus, the culture system is also suitable for use under field conditions. To prove the efficiency of this incuhatinn system. in vitro matured and fertilized bovine zygotes were cultured in TCM 199 and calf serum on a granulosa cell monolayer using expired lung air in the submerged foil bags. The cumulative result of 25 identical replicates was 517 blastocysts from 1,052 oocytes (49%) at 8 d after fertilization. The Submarine Incubation System provides an altcrnativc method for in vitro embryo production and/or culture of other sensitive tissues or cells. 0 1997 by Elsewer Science
Key
words:
lnc
incubator.
Themgenology48.1379-1385.1997 0 1997 by Elsev~er Sctence
foil hag, in vitro
Inc
culture,
embryo.
transportation 0093-691w97/$17.00 PII s0093-691x(97)00379-8
Theriogenology INTRODUCTION In vitro culture of cells or tissues requires the establishment of a physical, chemical and biological environment similar to that surrounding the cells or tissues in vivo. Various tissue culture media and dishes have been developed to fulfill the biological and chemical requirements, and sophisticated thermostats are offered to keep gas phases, humidity and temperature at optimum levels. However, most of these incubators have only one large or incompletely divided storage area, so opening of the door disturbs the environment of all the cultures inside. In spite of the latest technology compensating for these factors, such small disturbances may have a cumulative negative effect on cultures, with decreasing development and viability as a possible consequence. Certain tissues or cells such as mammalian oocytes and preimplantation embryos arc very sensitive to environmental changes. On the other hand, in vitro production of these embryos requires frequent opening of the thermostats for changing the medium, adding spermatozoa and transferring embryos from one dish to another. Laboratories where at least one in vitro fertilization program is started each day minimize these problems by purchasing a number of carbon dioxide thermostats, by strictly regulating the time and duration of openings, and by maintaining the temperature of the whole laboratory near to the physiological body temperature (37 to 39°C). However, from a financial and/or technical point of view, these solutions are difficult for most laboratories to resolve. In this study we report a simple new incubation method which can be established in commercially available water baths and which fulfills all the requirements for in vitro embryo production. We call it the Submarine Incubation System. MATERIALS
AND METHODS
In Vitro Embryo Production Except where indicated, all chemicals were obtained from Sigma Chemical Company (St. Louis, MO 63178 . USA). Maturation of bovine oocytes, fertilization and culture of embryos has been described in detail elsewhere (5). Briefly, oocytes were aspirated from slaughterhouse-derived ovaries, matured in 4-well dishes (Nunc, DK-4000 Roskilde, Denmark) for 24 h in bicarbonate buffered TCM-199 medium supplemented with 15% calf serum, eCG and hCG. Insemination (Day 0) was performed using frozen-thawed, Percoll-selected spermatozoa. After 30 h, zygotes were vortexed, then cultured on the granulosa cell monolayer formed spontaneously in the maturation dishes in 400 pl TCM-199 supplemented with 5% (10% from Day 4, except in Experiment 3) calf serum. The cultures were evaluated on Day 7 and 8 after insemination. For maturation and fertilization, a double-wall K-system box with continuous gas flow and heated by an external circulator (Henning Knudsen Engineering, DK-3460 Birkerod, Denmark) was used. Embryo culture was performed in the Submarine Incubation System as described below. Except where otherwise indicated, evaluations were made on Day 8 by stereomicroscopy, determining the blastocystioocyte ratio.
Theriogenology
1381
The Submarine Incubation System A 10 x 1 l-cm bag was prepared from laminated foil (S12MY PETP Metal/75MY PE Guld, SFK, DK-2650 Hvidovre, Denmark; oxygen and carbon dioxide permeability max. 0.005 g/m2/24h/l atm, 23°C. 5/950/c RH, DIN 53122) with the use of a heat-adjustable, 12-mm wide sealing iron (Comecta, Selecta s.a., SP-08630 Abrera, Barcelona, Spain). After fertilization, uncovered 4-well dishes containing the oocytes were inserted, and the bags were completely sealed. Using a 19-g injection needle, a hole was made in each bag in the proximity of one of the comers and distant from the culture dish. A tube was attached to the injection needle, and the bag was completely filled with a sterile-filtered, preheated and humidified gas mixture at a flow rate of approximately 500 ccm/min. The needle was then removed, and the gas was pushed out through the hole with moderate mechanical pressure. The bag was filled and emptied twice more to ensure an almost total gas exchange in the bag before the hole was heat-sealed. The bag was then placed on a shelf of a plastic test tube rack (Cat. no. 5970, Nalgene, Rochester, NY 14602-0365, USA) and submerged into a 38.7”C circulated water bath (DT OB-22, Heto, DK-3450 Allerfld, Denmark). An 800-ml tissue culture flask (Nunc, DK-4000 Roskilde, Denmark) filled with water was used as a weight to prevent floating (Figure 1). The water level was adjusted so that the water covered the upper shelf of the rack. Thus, the bags containing the dishes were pressed up against the shelves by the water and were subjected either to 1 cm (upper shelf) or 5 cm (lower shelf) of water pressure.
Figure 1. Schematic drawing of the Submarine Incubation System. Foil bags (1) containing the four-well dishes and filled with the required gas mixture are placed on the shelves of a test tube rack (2) and submerged into a circulating water bath (3) with an 800 g weight (4). Experiment 1 Industriallv nroduced gas mixture vs exnired lung air for atmosnheric gas. In Experiment la (6 replicates) embryo culture from Day 1 to Day 8 was performed in 10 x 1 l-cm laminated foil bags filled either with a mixture of 4% carbon dioxide in air (+lO%, AGA A/S, DK-7000 Fredericia, Denmark; group A) or with expired lung air from one person (group B). In Experiment lb, expired lung air of the same person was used to fill the foil bags in 25 successive identical replicates.
1382
Theriogenology
Experiment 2 Effect of increased pressure on embrvo develoument. In 3 replicates, a special deep container and two test-tube racks placed on top of each other were used so that the 10 x 1 l-cm laminated foil bags containing the embryo cultures were submerged either 1, 5,9 or 13 cm under the water level from Day 1 to Day 4 of culture. From Day 4 to Day 8, all the bags were immersed to 1 cm under the water level. Experiment 3 Transportable incubator. To adjust the size of the 4-well dish to the transportable incubator, the outer frame was cut, and the remaining 4.5 x 4.5 x l.O-cm part of the dish with the 4 wells was used for the whole experiment. In 3 replicates, embryo cultures were performed in 6.5 x 7.0cm laminated foil sacks filled with expired air, then heat-sealed and submerged into a Minitiib transportable incubator (Ref. no. 19180/0000, Minitiib GMBH, D-84184 Tiefenbach, Germany) previously sealed with glue to become waterproof, then filled with water. The cultures were left undisturbed from Day 1 until evaluation (Day 8). Experiment 4 Transparent foil for freauent microscouic investipation. In 4 replicates, embryo culture was perfonned in 10 x 1 l-cm transparent laminated foil bags (Camclear Polyester Laminate 12/45, Anti-Fog, Rexam Metallising, IP24 3QY Thetford, UK; oxygen barrier < 5.0 cc/m2/24h, 23°C 5O%RH, carbon dioxide barrier < 12.0 cc/m2/24h) filled with expired air. The inner bottom surface of the foil was moistened by sterile paraffin oil before loading to keep the foil attached to the bottom of the dish in order to increase optical clarity. During the 7 d of the culture, the bags were taken out of the water bath 5 times, dried and placed on the heated stage of an inverted microscope. They were then observed, photographed and returned to the water bath. Each investigation was performed in less than 5 min. Statistical Evaluation Data were analyzed by the Chi-square test, and PcO.05 was considered to be statistically significant. RESULTS Experiment 1 In Experiment la, the cumulative rate of development of the 6 replicates was 136 blastocysts I294 oocytes (46%) and 134 blastocysts I285 oocytes (47%) in 4% carbon dioxide in air and expired lung air, respectively (P>O.O5). In Experiment lb, the cumulative rate of 25 replicates was 5 17 blastocysts / 1,052 oocytes (49%, range: 40 to 55%).
Theriogenology
1383
Experiment 2 Submerging the foil bags under increased pressure, i.e., under 13 cm water, did not impair the developmental rate: the cumulative results of 3 replicates were 45, 49, 44 and 38% (P>O.O5; 58/129, 691142, 571130 and 55/146 blastocysts / oocytes, respectively) in the groups submerged from Days 1 to 4 at 1,5,9 or 13 cm below the water surface. Experiment 3 The cumulative developmental rate of the 3 replicates performed in the transportable incubator was 58 blastocysts / 139 oocytes (42%, range: 37 to 44%). Experiment 4 The cumulative developmental rate of the 4 replicates performed in the transparent foil was 80 blastocysts / 178 oocytes (45%, range: 39 to 51%). DISCUSSION The simple, new incubation system presented here can be established in most commercially available water baths. The advantages and disadvantages of this system are summarized below: In contrast with most carbon dioxide incubators, heat is conducted in the Submarine Incubation System by circulating water instead of gas. Most water bath circulators work with an accuracy of O.Ol-O.O5”C, which can not be achieved by gas flow. Other advantages of our system are the quick recovery rate of temperature without the danger of overheating the cultures, and the possibility of rapid, accurate changes of temperature, i.e., for temporary cooling in certain embryo manipulation procedures (2). The preheated and humidified gas is promptly injected into the foil bags, and this atmosphere is maintained for the whole period of culture. Although direct investigations were not performed, the 60 to 80 ml gas mixture was adequate to cover the requirements of 100 preimplantation stage bovine embryos cultured in 4 x 400 ul medium for a period of 3 d. Moreover, in Experiment 3, acceptable development of embryos cultured in 2 x 400 ul medium and 20 to 30 ml gas mixture for 6 d was also observed. Each bag may be filled with the required gas mixture, thus providing considerable flexibility to the system. As shown in Experiment 2, the elevated atmospheric pressure in the bag (up to 13 water cm) caused by the hydrostatic pressure of water did not impair early embryo development. Stability, safety and cost-efficiency of this systemaremarked. During incubation, the only source of trouble was the water bath circulator, which is generally regarded as a trouble-free piece of laboratory equipment. Infections that occasionally occur in primary cultures can be localized and eliminated without the risk of propagating the infectious agent and without the need to stop the whole incubator. In our experience, regular use of water bath desinfectants has precluded contamination of water even when the same water was used for more than 5 mo. Although the inside of the foil bags were not sterilized, infection of the cultures was not detected
1384
Theriogenology
during the whole experimental period. The price and maintenance costs of the Submarine Incubation System are much lower than those of a carbon dioxide incubator. Previous experiments have demonstrated the beneficial effects of slightly elevated pH levels on bovine embryo development in vitro that are produced either by increased bicarbonate concentration in the medium or decreased carbon dioxide concentration in the atmosphere (6, Vajta et al., unpublished observations). Expired human lung air contains 4% carbon dioxide and 16 to 17% oxygen, with an accuracy comparable to those of the generally used industriallyproduced gas bottles (approximately &lo%), may be sterilized by filtration; moreover. it comes both preheated and humidified. The foil bag system is uniquely suitable for filling with expired air for embryo culture, since it is easy and requires a low volume. In Experiment 1, the blastocyst rates achieved either with the expired lung air or with gas bottle-derived 4% carbon dioxide in air were similar, and in a large series of replicates performed with expired lung air, high and stable embryo development rates were obtained. A transportable version of the Submarine Incubation System was also developed. Although the heat-accuracy of the modified Minitiib thermostat was lower than that of the circulating baths, an acceptable rate of embryo development was achieved using the transportable model for up to 6 d of incubation. Moreover, these experiments were performed with the use of expired lung air, which makes the system suitable to establish and maintain appropriate culture conditions under field situations. The use of a transparent foil facilitated optical follow-up of the embryos without disturbing their atmospheric environment. In Experiment 4, repeated microscopic observations did not result in the impairment of development of the embryos, and the quality of the microscopic view was satisfactory for embryo evaluation. To date, the only registered disadvantage of the Submarine Incubation System is that 1 to 2 min of extra work are required for each culture dish. Compared with the amount of work required to produce bovine embryo cultures, this extra effort seems minimal. Since the Submarine Incubation System was first introduced into our laboratory in April, 1996, it has been used successfully both for routine embryo production and for experiments dealing with the effects of the different gas mixtures on embryo development. This system is a reliable and cost-efficient way to culture sensitive tissues or cells, such as ova and preimplantation embryos. The developmental rates of in-vitro produced bovine embryos achieved with this system have been among the highest yet reported (1, 3, 4). The versatility of this system (easy transport, on-sight use, microscopic follow up) offers a wide variety of applications in embryology and tissue culture work.
Theriogenology
1385
REFERENCES 1. Brackett BG, Zuelke KA. Analysis of factors involved in the in vitro production of bovine embryos. Theriogenology 1993; 39: 43-64. 2. Chesne 0, Heyman Y, Peynot N, Renard JP: Nuclear transfer in cattle; birth of cloned calves and estimations of blastomere totipotency in morulae used as a source of nuclei. Life Sci 1993; 316:481-491. 3. Farin CE, Hasler JF, Martus NS. Comparison of Menezo B2 and TCM 199 media for in vitro production of bovine blastocysts. Theriogenology 1995; 43: 2lOabstr. 4. Hernandez-Lendezma JJ, Villanueva C, Sikes JD, Roberts RM. Comparison of co-culture and conditioned medium on expansion and hatching of in-vitro derived bovine blastocysts. Theriogenology 1995; 43: 233abstr. 5. Vajta G, Holm P, Greve, T Callesen H. Overall efficiency of in-vitro embryo production and vitrification in cattle. Theriogenology 1996; 45: 683-689. 6. Vajta G, Holm P, Greve T, Callesen H. Effect of pH of culture medium on in vitro development of bovine embryos. Theriogenology 1997; 47: 286abstr.