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Cold culture of different stage mouse embryos in bicarbonated and bicarbonate-free media
THERIOGENOLOGY
COLD CULTURE OF DIFFERENT STAGE MOUSE EMBRYPS MEDIA BICARBONATED AND BICARBONATE-FREE " C. M. Herr and R. Wright, Jr. Department of Animal Sciences Washington State University Pullman, WA 99164-6332 Received
for publication: Accepted:
August April
5, 11,
IN
1987 1988
ABSTRACT Mouse embryos of different stages of development were cultured to expanded blastocysts following storage (1 to 8 d) at 4'C in the presence or absence of HCO;. The effect of oxygen tension on the cold storage of one- and two-cell mouse embryos at 4°C was evaluated by 37°C culture and transfer to pseudopregnant recipients. Survival at 4'C of early, one- to four-cell mouse embryos was improved with HCO; in the medium. The presence of HCO- was not of benefit for morulae or blastocyst survival following cold storage. Reducing the oxygen atmosphere from 20 to 5% 0 improved survival of one-cell mouse embryos stored at 4°C. The surviva? of two- and four-cell embryos, morulae and blastocysts at 4°C was similar in 90% N 5% CO and 5% CO in air, but it was significantly poorer in air alon g: The gollapse of'morulae and blastocysts during cold storage up to 5 d was reduced with HCO; in the storage medium. Blastocysts stored for 6 d at 4'C failed to iurvive following immediate transfer to pseudopregnant recipients. Blastocyst survival was improved compared to controls (direct transfer of unstored blastocysts to recipients) when cultured for 36 h at 37°C following 6 d of cold storage. This result suggests that cold-stored mouse blastocysts may require a metabolic period of readjustment to survive following transfer to synchronized recipients. Key words:
mouse,
embryo,
cold
culture,
storage
INTRODUCTION Storage of developmentally arrested early mammalian embryos could facilitate research in embryo culture, in vitro fertilization and gene transfer. The latter, modification of a mammalian genome with foreign genes (gene transfer) is possible by microinjection of genes into the pronucleus of a zygote (1). In several species, transportation and microinjection may require that embryos be maintained in vitro for hours (2). Farrel and Bavister (3) observed that significantly fewer two-cell hamster embryos developed in utero after only 20 min of exposure to collection media compared with those transferred directly. Freezing early embryos twice, before and after microinjection, would be too traumatic embryc for survival. Few studies have investigated short-term storage of early embryos (4).
1
Research supported by the College of Agriculture and Home Economics, Research Center, Washington State University, Project No. 0313. BScientific Paper No. 7834. Reprint requests.
JULY 1988 VOL. 30 NO. 1
THERIOGENOLOGY
Embryos of suspended several mammalian experience development when held below normal body t&!&?&re (cold culture). This has been observed in the mouse (5), rabbit (6), sheep (7) and cow (8). Rabbit embryos are particularly tolerant of this treatment; offspring have resulted from embryos held in cold storage for 2 wk (9). Early stage bovine (10) and mouse (11) embryos have poor survival rates at low temperatures, but their survival rates improve at later stages. The early mouse embryo requires bicarbonate ions HCO- to develop in vitro. Attempts to replace bicarbonate with organic (12) St- inorganic (13) buffers have failed to support in vitro development. No studies have directly examined the HCO; and 02 tension requirement of early mouse embryos in cold culture. The purpose of this study was to evaluate the effect of HCO- in cold culture media on the viability of preimplantation stage mouse es bryos. MATERIALS Media
AND METHODS
Preparation
Whitten's medium (WM) was used for embryo collection, cold culture Whitten's Medium was pH adjusted to 7.3 under and culture at 37°C (14). the appropriate gaseous atmosphere and supplemented with sodium lactate (0.242%), dextrose (O.l%), crystalline bovine serum albumin (Sigma), Both cold culture fraction 5 (0.4%), and sodium pyruvate (28mg/l). medium without sodium bicarbonate and the collection medium were buffered acid (hepes). with IOmM N-Z-hydroxyethyl piperazine -N'-2 ethanesulphonic No antibiotics or antimycotics were present in the media. Media were prepared from a 10X stock solution containing all salts and glucose (but no buffer or calcium lactate). Reagents with the lowest listed heavy metal contaminants were selected. _ The stock solution was filtered (0.2 ml one h after oreoaration and stored at 4°C. All media were prepared from the same 'stock solution. Media were filtered immediately after preparation, stored in sealed containers at 4'C (under a 10% CO2 atmosphere when containing HCO-) and used within 1 wk of preparation. Whitten's medium stored for 3 lao is equal to freshly prepared medium in its abilit to support one-cell mouse embryo development to blastocyst suggesting that any decline in embryo survival with stage (15jl additional ;ime in cold culture would not be due to media breakdown. Embryo
Collection
Embryos were collected from naturally bred Swiss Webster X C57/6J hybrid females after pairing with Swiss Webster males (24 to 72 h after mating). Mid- to late-cycle, one-cell embryos were collected by teasing apart-the ampullary region of the oviducts. Embryos were treated with hvaluronidase (300 units/cc) to remove surroundina cumulus cells (16). Embryos were outside the.treatment atmosphere for n'o longer than 20‘min. Late two-cell, early four-cell, morulae and blastocyst stage embryos were collected by oviductal or uterine flushes. Morulae were pooled separately from early blastocysts, and equal numbers of each were allocated to the treatments. Cold Culture Embryos
were
placed
in 3 ml of media
in 5-cm diameter
petri
dishes.
JULY 1988 VOL. 30 NO. 1
THERIOGENOLOGY
Dishes were transferred to 4-l gas tight jars gassed with the appropriate A bicarbonated solution with phenol humidified atmosphere and sealed. red (pH indicator) in the jars was used for verification of jar seal Refrigeration was conducted in a light-proof cabinet located integrity. in a 10 x 20-ft cold room at 4°C. Culture
at 37°C
collection (control) or cold culture, embryos were Following transferred to microdrops of Whitten's Medium under paraffin oil and After 5% 0 and 90% N atmosphere. cultured at 37°C in a 5% CO culture, embryos were scored f8; survlY?ors, i.e. ex $ anded blastocysts of normal appearance. Embryo
Transfer
Recipients for embryo transfer were prepared by breeding females Six to eight embryos were transferred to each with vasectomized males. uterine horn. Morulae and blastocysts (Day 4 embryos) were transferred Seventeen days after transfer to recipients, to Day 3 recipients. fetuses were surgically removed and counted. Experimental
Design
The experiment was designed to evaluate the Embryos. and oxygen tension on cold culture of one-cell embryos. assigned to cold culture under either Pooled Of embryo effect Hco? were arbitrarily air; 5% CO and air or 5% CO,,, 5% 0 and 90% N atmosphere. In separate co& cultured ?or 1, 2, 3 or 4 d. 2X repeate 8 trials, embryos k-e One-cell
Two-cell Embryos. The experiment was designed to evaluate the -effect of HCO on two-cell embryos in cold culture. Pooled embryos were arbitrarily ajsigned to cold culture with or without HCO-. In separate 2X repeated trials, embryos were cold cultured for 3, 5 0~6 d. Four-cell Embryos. as for two-cell embryos, 6 d.
This experiment was performed in the same manner except embryos were cold cultured for 3, 4, 5 or
Morulae and Blastocysts. This experiment was performed in the same manner as for two-cell embryos, except embryos were cold cultured for 7 or 8 d. In addition, the effect on HCO- on the collapse of blastocysts in cold culture was investigated. M?luse blastocysts collapse with greater frequency as time in cold culture increases (1_1!. Blastocysts were cold cultured in the presence or absence of HCO for 5 or 8 d. Immediately after removal from cold culture, embryos w?!re examined for blastocoele formation. Embryo Transfer. Embryo transfer was used to evaluate the ability of embryos to complete development subsequent to cold culture. Two-cell embryos cultured at 37°C to morulae or blastocysts were compared to two-cell embryos that had been cold-cultured 4 d (with HCO;) and then cultured to morulae at 37°C. Blastocysts blastocysts cold
JULY
transferred directly to recipients were compared cultured for 6 d and cultured at 37°C for 36 h
1988 VOL.
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1
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161
THERIOGENOLOGY
blastocysts transferred directly after cold culture (no in vitro 37°C culture). The direct transfer treatment (no cold culture) was a composite of results obtained in the laboratory. Statistical Analysis of Data Embryos directly cultured at 37°C (culture studies) or directly transferred (transfer study) served as controls. Data were binomial with embryos either succeeding or failing to develop to expanded blastocysts of normal appearance (culture studies) or embryos either succeeding or failing to develop to late-term fetuses (transfer study). Statistical analysis was performed by pair comparison of the binomial populations with a two-tailed statistical test (17). RESULTS One-Cell Embyros No significant difference in survival was observed between control and one-cell embryos cold cultured for 1 d under 5% CO ;c~~akkda~5]t~0%W2 atmosphere (Table 1). Survival under this atmosphere $i Influence of cold culture atmosphere on survival of one-cell mouse embryos ). Total embryos Percentage" Days in Cold culture" survival per treatment atmosphere cold-culture
Table 1.
Oa
air
93d
: 1
5% ::,:"g; ;;::90% N2
5; 5od 89
0 2 2 2
air 5% co . 5% CD2, 5; :;: 90% N2
92d Oe 4qf 62f
0 3 z
fl :
air 5% CO , air 5% co2, 3% 02, 90% N2
air 5% CO , air 5% C02, 3% 02, 90% N2
97d 0; 34f 45 90;
:e Oe
45 123 121 123 40 62 :z 40 46 114 113
t: 66 67
iZero days in cold culture represents directly cultured controls. Embryos cultured in air were maintained in hepes buffered Whitten's medium. 'Percentage survival is the proportion (X100) of the total embryos per treatment developing to expanded blastocysts in subsequent 37°C culture. Values with different letters within lines are significantly different (P>O.O5).
162
JULY 1988 VOL. 30 NO. 1
THERIOGENOLOGY
5% 0 and additional days in cold culture to 0% after 4 d. The 5% CO all 90% N atmosphere provided the highest one-cell embryo sur oval o Survival in the 5% CO and in air atmosphere atmos Jh eres tested. compared favorably to air alone but was poorer ?han in 5% COP, 5% 02 and 90% N2.
3’
Two-cell
3
Embryos
No significant difference was observed between embryos cold cultured for 3 d in the 5%CO 5% 0 and 90% N atmosphere and in the 5% CO2 and Th&efore,'the embryo P in the remaining time trials were air atmosphere. There was no significant cold cultured in either air or 5% CO and air. controls and embryos cold difference in survival between u r?stored cultured for 3 d in 5% CO2 and air_, but embryo survival after 3 d in the Embryos air atmosphere (absence of HCO ) was significantly lower. cold cultured for 5 and 6 d also &owed a greater survival rate with HCO; in the medium (Table 2). Table
2.
Influence of mouse embryos
Days in cold culture Oa 3 3 3
cold-culture
Cold cultureb atmosphere
air 5% CO2, air 5% CO2, 5% 2, 90% N2
atmosphere
z
air 5% CO2, air
0 6 6
air 5% CO,, air
survival
of
two-cell
PercentageC survival
Total embryos per treatment
100;
40 96 96 95
;;d 93d 97d
0
on
0: 88
40 118 121 41 55 132
Efero days in cold cultur: represents directly cultured controls. Embryos cultured in air were maintained in hepes buffered Whitten's medium. 'Percentage survival is the proportion (X100) of the total embryos per treatment developing to expanded blastocysts in subsequent 37°C culture. Values with different letters within lines are significantly different (P>O.O5). Four-cell
Embryos
Four-cell embryos had a significantly higher survival rate in HCONo significant difference wa 2 than in HCO; free medium (Table 3). observed between embryos cold cultured 3 d with HCO- and the unstored as controls. Early four-cell embryos tended to collaps i and appeared two-cell embryos with increased time in cold culture. This did not, however, impair their ability to develop into blastocysts.
JULY
1988 VOL. 30 NO. 1
THERIOGENOLOGY
Table 3.
Influence of cold culture atmosphere on survival of four-cell mouse embyros
Days in cold culture Oa
Cold cultureb atmosphere
;
air 5% CO,, air
: 4
air 5% CO,, air
0
5 5
air 5% CO,, air
0 6 6
air 5% COP, air
PercentageC survival 100; gide 100," zd 9gd I'f 76
100d 0; 48
Total embryos per treatment ;; 70 40 78 76
:; 76
%: 48
:Zero days in cold-culture represents directly cultured controls. Embryos cultured in air were maintained in hepes buffered Whitten's medium. 'Percentage survival is the proportion (X100) of the total embryos per treatment developing to expanded blastocysts in subsequent 37°C culture. Values with different letters within lines are significantly d,ifferent(P70.05). Table 4.
Influence of cold-culture atmosphere on survival of morulae and early blastocysts
Days ina cold culture 0 7 7
Cold cultureb atmosphere
Total embryos per treatment
air 5% COP, air
1;: I77
air 5% CO,, air
40 115 113
0
8 8
PercentageC survival
iZero days in cold culture represents directly cultured controls. Embryos cultured in air were maintained in hepes buffered Whitten's medium. 'Percentage survival is the proportion (X100) of the total embryos per treatment developing to expanded blastocysts in subsequent 37*C culture. Values with different letters within lines are significantly different (P>O.O5).
164
JULY 1988 VOL. 30 NO. 1
THERIOGENOLOGY
Two-cell embryos which were directly cultured at 37°C to _. ,_.morulae and then transferred to recipients yielded greater proportions (although not significant) of late-term fetuses than two-cell embryos cold cultured for 4 d before in vitro culture to morulae and transfer to recipients. DISCUSSION In this study, mouse embryos of different preimplantation stages were cultured to exoanded blastocvsts followinq culture at 4'C in the presence or absence of HCO-. Also"investigatedwas the effect of oxygen tension on the cold culturz of one- and two-cell embryos. The ability of cold cultured embryos to complete development was evaluated by transfer of two-cell (cultured in vitro at 37°C to morulae) and blastocyst stage embryos to pseudopregnant females. Every effort was made to maintain the correct CO - containing HCO- concentration ([H&l) ’ only atmosphere over the media. indirectly related to the amount af sodium bicarbonate %dded"to the medium, but is directly proportional to the concentration of CO in the culture atmosphere and the pH of the medium (18,19). As CO is ?-eleased the FH increases. into a culture atmosphere containing no (or low) CO The increased pH has been shcwn to result in embryo &mage (8 and 18). One-cell embryo survival subsequent to cold-culture was affected by the oxygen tension in the cold-culture atmosphere. Embryos cold cultured 5% 02 and 90% N survived in greater numbers than embryos under 5% CO cultured un?ier 5% CO and a Pr. This observation is consistent with observations of WhitteG (19) for one-cell mouse embryos cultured at 37°C. Whitten's hypothesis was that one-cell mouse embryos are particularly sensitive to oxidative damage. In our study, survival of two-cell embryos cold cultured in a 5% CO2 and air atmosphere was not signficantly different from the survival rate in 5% CO2, 5% 02 and 90% N2. Survival of early (one- to four-cell) embryos subsequent to cold culture was increased with the addition of HCO- to the cold culture media. This observation is consistent with prev?ous studies on HCOrequirements of early mouse embryos in 37'C culture (18). Two-cell mous.3 embryos at 37°C in the absence of HCO- for only 2 h have diminished ability to develop to blastocysts subsjquent to culture when compared with embryos maintained the entire time with HCO; (20). In the same study, eight-cell mouse embryos were cultured for 8 h at 37°C in the absence of HCO; before a significant decrease in viability was observed. In our study, morula and blastocyst survival was not significantly affected by HCOj in the cold culture media. The importance of HCO- to the cold culture of mouse embryos may be Mouse embryos incorporate CO related to energy metabaism. into acetate, malate, citrate and ketoglutarate, presumably via p5 ruvate carboxylase-mediated CO2 condensation with pyruvate to form oxaloacetate (13). Embryos cultured at low temperature also metabolize carbohydrates although at a reduced rate (21). Therefore, the parameters necessary for energy metabolism at 37"C, i.e. HC03, may be necessary at reduced temperatures as well.
JULY 1988 VOL. 30 NO. 1
THERIOGENOLOGY
Morulae
and Blastocysts
Morula and blastocyst survival did not increase with HCO- in the No significant difference w3 s found cold culture medium (Table 4). between treatments for embryos stored in the presence or absence of Collapse of cold cultured blastocysts was HCOfor 7 and 8 d. sig l+lficantly reduced with HCO; after 5 d in cold-culture but not after 8 d (Table 5). Table
5.
Effect of bicarbonate in cold culture the blastocoele of mouse blastocysts Cold cultureb atmosphere
Oays in cold culture 5 5
5% co
PercentageC survival
on the collapse
of
Total embryos per treatment
&$ir
5% CO
, gir E.ir
:
media
68 72
16d 13d
aEmbryos cultured in air were maintained in hepes buffered Whitten's bmedium. Values with different letters within lines differ significantly (P70.05). Embryo
Transfer
to Recipients
None of the blastocysts that were directly transferred to recipients In contrast, after 6 d of cold culture developed into late-term fetuses. blastocysts cold-cultured for 6 d, followed by 36 h of in vitro culture prior to transfer, developed into late-term fetuses at the same rate as unstored controls (Table 6). Table
6.
Development of untreated embryos
Days in cold-culture 0 4 0 6 6
Stage duringa cold-culture 2-cell 2-cell blastocyst blastocyst blastocyst
late-term
Stage whenb transferred morula morula blastocyst blastocyst blastocystC
fetuses
from
cold
Percentage developing to late-term fetuses 21 8 22 0 19
cultured
Total embryos transferred 180 180 240 180 180
"For embryos stored for 0 d, stage of embryo during cold culture brepresents stage at collection. If stage at cold-culture differs from stage at transfer, embryos were cultured at 37°C to stage at transfer. 'In this treatment, embryos were cultured for 36 h at 37°C subsequent cold culture and prior to transfer.
166
and
to
JULY 1988 VOL. 30 NO. 1
THERIOGENOLOGY
It has been su gested that cold culture maintains embryos in arrested development ?9). Although embryos held at low temperatures do not divide, cold cultured embryos are not equivalent to embryos of the In our study, the same stage that have not been cold cultured. blastocysts (Day 4 embryos) transferred directly to Day 3 recipients subsequent to cold culture for 6 d produced no late-term fetuses, whereas cold cultured (6 d) blastocysts transferred to Day 3 recipients after 36 h in culture at 37°C developed into late-term fetuses at proportions Rabbit embryos cold cultured for 2 wk and similar to unstored controls. transferred to recipients developed to offspring later than embryos of the same stage that were directly transferred (9). It has been suggested that the development of cold cultured embryos continues to progress at a Our study, however, found slower rate compared to unstored controls. that a readjustment period is required for blastocysts after cold culture. The results of this study suggest that cold-cultured embryos should be treated like earlier stage embryos regarding recipient synchrony for transfer. In addition, the presence of HCO- in the medium used for cold culture benefits early stage embryo surviva P. REFERENCES Brinster, R., Chen H.Y., Trumbauer, M.E., Yagle, M.K., and Palmiter, Factors affecting the efficiency of introducting foreign DNA R.D. Proc. Natl. Acad. Sci. into mice by microinjecting eggs. g:4438-4442 (1985).
1.
Hammer, R.E., Pursel, V.G., Rexroad, C.E. Jr., Wall, R.J., Bolt, Ebert, K.M., Palmiter, R.D., and Brinster, R.L. Production of transgenic rabbits, sheep and pigs by microinjection. Nature. 315:680-683 (1985).
2.
D.J.,
3.
Farrel, P.S. and Bavister, B.D. Short-term exposure to two-cell hamster embryos to collection media is detrimental to viability. Biol. Reprod. -31:109-114 (1984).
4.
Maurer, R.R. Can. J. Anim.
5.
Whittingham, P.G. and Wales, R.G. in vitro. Adv. Biosci. 6:128-141 --
6.
Hafez,
E.S.E.
Storage of mammalian oocytes Sci. 56:131-145 (1976).
Storage
Storage (1971).
of fertilized
ova.
and
embryos:
of two-cell
A review.
mouse
embryos
Int. J. Fert. g: 459-466
(1963). 7.
Moore, M.W. and Bilton, R.S. The stora e of fertilized 5°C. Aust. J. Biol. Sci. g:1421-1427 9 1973).
8.
Trounson, A.O., Willadsen, S.M., Rowson, L.E.A., and Newcomb, R. The storage room temperatures of cow eggs at and at low J. Reprod. Fertil. %:173-178 (1976). temperatures.
9.
Hughes, embryos
and Anderson, G.B. M.A. Short-term storage at 4°C. Theriogenology g:311-319 (1983).
JULY 1988 VOL. 30 NO. 1
sheep ova at
of
rabbit
THERIOGENOLOGY
10.
Lindner, G.M., Anderson, G.B., BonDurant, R.H. and Cupps, P.T. Survival of bovine embryos stored at 4°C. Theriogenology g:311-319 (1983).
11.
Herr, C. Factors Influencing Survival of Preimplantation Stage Mouse Embryos Maintained in Developmental Arrest In Vitro. Ph.D. Thesis. Washington State University, Pullman, WA, 7987
12.
Brinster, R.L. -In vitro culture of the embryo. In Sherman, A.I. (ed.), Pathways to Conception. Charles C. Thomas,Springfield, IL, 1971. pp. 245-277.
13. Quinn, P. and Wales, R.G. Growth and metabolism of preimplantation mouse embryos cultured in phosphate-buffered medium. J. Reprod. Fertil. E:289-300 (1973). 14.
the culture Whitten, W.K. Nutrient requirements for preimplantation embryos -in vitro. Adv. Biosci. 6:128-141 (1971).
of
15.
Herr, C. and Wright, R.W., Jr. Storage life of Whitten's embryo culture medium. Theriogenology g:158 abstr. (1986).
16.
Rafferty, K.A. Methods in Experimental Embryology of the Mouse. The John Hopkins Press, Baltimore and London, 1970.
17. Mendenhall, W. Introduction to Probability and Statistics. Roxbury Press, North Scituate, MA, 1979. 18.
Rrinster, R.L. Studies on the development of mouse embryos in vitro. IV. Interaction of energy sources. J. Reprod. Fertir m7 (1965).
19.
Whitten, W.K. Effect of oxygen on cleavage of mouse eggs -in vitro. Sot. Study Reprod. 2nd Ann. Meeting. Davis, CA. Biol. Reprod. Abstr. 52 (1969).
20.
Quinn, P. and Wales, R.G. Fixation of carbon dioxide by preimplantation mouse embryos ti vitro and the activities of enzymes involved in the process. Aust.J.01. Sci. -24:1277-1290 (1971).
21.
Anderson, G.B. and Foote, R.H.. Effects of low temperature storage upon subsequent energy metabolism of rabbit embryos. Exp. Cell Res. g:302-306 (1974).
1372
168
JULY 1988 VOL. 30 NO. 1
COLD CULTURE OF DIFFERENT STAGE MOUSE EMBRYPS MEDIA BICARBONATED AND BICARBONATE-FREE " C. M. Herr and R. Wright, Jr. Department of Animal Sciences Washington State University Pullman, WA 99164-6332 Received
for publication: Accepted:
August April
5, 11,
IN
1987 1988
ABSTRACT Mouse embryos of different stages of development were cultured to expanded blastocysts following storage (1 to 8 d) at 4'C in the presence or absence of HCO;. The effect of oxygen tension on the cold storage of one- and two-cell mouse embryos at 4°C was evaluated by 37°C culture and transfer to pseudopregnant recipients. Survival at 4'C of early, one- to four-cell mouse embryos was improved with HCO; in the medium. The presence of HCO- was not of benefit for morulae or blastocyst survival following cold storage. Reducing the oxygen atmosphere from 20 to 5% 0 improved survival of one-cell mouse embryos stored at 4°C. The surviva? of two- and four-cell embryos, morulae and blastocysts at 4°C was similar in 90% N 5% CO and 5% CO in air, but it was significantly poorer in air alon g: The gollapse of'morulae and blastocysts during cold storage up to 5 d was reduced with HCO; in the storage medium. Blastocysts stored for 6 d at 4'C failed to iurvive following immediate transfer to pseudopregnant recipients. Blastocyst survival was improved compared to controls (direct transfer of unstored blastocysts to recipients) when cultured for 36 h at 37°C following 6 d of cold storage. This result suggests that cold-stored mouse blastocysts may require a metabolic period of readjustment to survive following transfer to synchronized recipients. Key words:
mouse,
embryo,
cold
culture,
storage
INTRODUCTION Storage of developmentally arrested early mammalian embryos could facilitate research in embryo culture, in vitro fertilization and gene transfer. The latter, modification of a mammalian genome with foreign genes (gene transfer) is possible by microinjection of genes into the pronucleus of a zygote (1). In several species, transportation and microinjection may require that embryos be maintained in vitro for hours (2). Farrel and Bavister (3) observed that significantly fewer two-cell hamster embryos developed in utero after only 20 min of exposure to collection media compared with those transferred directly. Freezing early embryos twice, before and after microinjection, would be too traumatic embryc for survival. Few studies have investigated short-term storage of early embryos (4).
1
Research supported by the College of Agriculture and Home Economics, Research Center, Washington State University, Project No. 0313. BScientific Paper No. 7834. Reprint requests.
JULY 1988 VOL. 30 NO. 1
THERIOGENOLOGY
Embryos of suspended several mammalian experience development when held below normal body t&!&?&re (cold culture). This has been observed in the mouse (5), rabbit (6), sheep (7) and cow (8). Rabbit embryos are particularly tolerant of this treatment; offspring have resulted from embryos held in cold storage for 2 wk (9). Early stage bovine (10) and mouse (11) embryos have poor survival rates at low temperatures, but their survival rates improve at later stages. The early mouse embryo requires bicarbonate ions HCO- to develop in vitro. Attempts to replace bicarbonate with organic (12) St- inorganic (13) buffers have failed to support in vitro development. No studies have directly examined the HCO; and 02 tension requirement of early mouse embryos in cold culture. The purpose of this study was to evaluate the effect of HCO- in cold culture media on the viability of preimplantation stage mouse es bryos. MATERIALS Media
AND METHODS
Preparation
Whitten's medium (WM) was used for embryo collection, cold culture Whitten's Medium was pH adjusted to 7.3 under and culture at 37°C (14). the appropriate gaseous atmosphere and supplemented with sodium lactate (0.242%), dextrose (O.l%), crystalline bovine serum albumin (Sigma), Both cold culture fraction 5 (0.4%), and sodium pyruvate (28mg/l). medium without sodium bicarbonate and the collection medium were buffered acid (hepes). with IOmM N-Z-hydroxyethyl piperazine -N'-2 ethanesulphonic No antibiotics or antimycotics were present in the media. Media were prepared from a 10X stock solution containing all salts and glucose (but no buffer or calcium lactate). Reagents with the lowest listed heavy metal contaminants were selected. _ The stock solution was filtered (0.2 ml one h after oreoaration and stored at 4°C. All media were prepared from the same 'stock solution. Media were filtered immediately after preparation, stored in sealed containers at 4'C (under a 10% CO2 atmosphere when containing HCO-) and used within 1 wk of preparation. Whitten's medium stored for 3 lao is equal to freshly prepared medium in its abilit to support one-cell mouse embryo development to blastocyst suggesting that any decline in embryo survival with stage (15jl additional ;ime in cold culture would not be due to media breakdown. Embryo
Collection
Embryos were collected from naturally bred Swiss Webster X C57/6J hybrid females after pairing with Swiss Webster males (24 to 72 h after mating). Mid- to late-cycle, one-cell embryos were collected by teasing apart-the ampullary region of the oviducts. Embryos were treated with hvaluronidase (300 units/cc) to remove surroundina cumulus cells (16). Embryos were outside the.treatment atmosphere for n'o longer than 20‘min. Late two-cell, early four-cell, morulae and blastocyst stage embryos were collected by oviductal or uterine flushes. Morulae were pooled separately from early blastocysts, and equal numbers of each were allocated to the treatments. Cold Culture Embryos
were
placed
in 3 ml of media
in 5-cm diameter
petri
dishes.
JULY 1988 VOL. 30 NO. 1
THERIOGENOLOGY
Dishes were transferred to 4-l gas tight jars gassed with the appropriate A bicarbonated solution with phenol humidified atmosphere and sealed. red (pH indicator) in the jars was used for verification of jar seal Refrigeration was conducted in a light-proof cabinet located integrity. in a 10 x 20-ft cold room at 4°C. Culture
at 37°C
collection (control) or cold culture, embryos were Following transferred to microdrops of Whitten's Medium under paraffin oil and After 5% 0 and 90% N atmosphere. cultured at 37°C in a 5% CO culture, embryos were scored f8; survlY?ors, i.e. ex $ anded blastocysts of normal appearance. Embryo
Transfer
Recipients for embryo transfer were prepared by breeding females Six to eight embryos were transferred to each with vasectomized males. uterine horn. Morulae and blastocysts (Day 4 embryos) were transferred Seventeen days after transfer to recipients, to Day 3 recipients. fetuses were surgically removed and counted. Experimental
Design
The experiment was designed to evaluate the Embryos. and oxygen tension on cold culture of one-cell embryos. assigned to cold culture under either Pooled Of embryo effect Hco? were arbitrarily air; 5% CO and air or 5% CO,,, 5% 0 and 90% N atmosphere. In separate co& cultured ?or 1, 2, 3 or 4 d. 2X repeate 8 trials, embryos k-e One-cell
Two-cell Embryos. The experiment was designed to evaluate the -effect of HCO on two-cell embryos in cold culture. Pooled embryos were arbitrarily ajsigned to cold culture with or without HCO-. In separate 2X repeated trials, embryos were cold cultured for 3, 5 0~6 d. Four-cell Embryos. as for two-cell embryos, 6 d.
This experiment was performed in the same manner except embryos were cold cultured for 3, 4, 5 or
Morulae and Blastocysts. This experiment was performed in the same manner as for two-cell embryos, except embryos were cold cultured for 7 or 8 d. In addition, the effect on HCO- on the collapse of blastocysts in cold culture was investigated. M?luse blastocysts collapse with greater frequency as time in cold culture increases (1_1!. Blastocysts were cold cultured in the presence or absence of HCO for 5 or 8 d. Immediately after removal from cold culture, embryos w?!re examined for blastocoele formation. Embryo Transfer. Embryo transfer was used to evaluate the ability of embryos to complete development subsequent to cold culture. Two-cell embryos cultured at 37°C to morulae or blastocysts were compared to two-cell embryos that had been cold-cultured 4 d (with HCO;) and then cultured to morulae at 37°C. Blastocysts blastocysts cold
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blastocysts transferred directly after cold culture (no in vitro 37°C culture). The direct transfer treatment (no cold culture) was a composite of results obtained in the laboratory. Statistical Analysis of Data Embryos directly cultured at 37°C (culture studies) or directly transferred (transfer study) served as controls. Data were binomial with embryos either succeeding or failing to develop to expanded blastocysts of normal appearance (culture studies) or embryos either succeeding or failing to develop to late-term fetuses (transfer study). Statistical analysis was performed by pair comparison of the binomial populations with a two-tailed statistical test (17). RESULTS One-Cell Embyros No significant difference in survival was observed between control and one-cell embryos cold cultured for 1 d under 5% CO ;c~~akkda~5]t~0%W2 atmosphere (Table 1). Survival under this atmosphere $i Influence of cold culture atmosphere on survival of one-cell mouse embryos ). Total embryos Percentage" Days in Cold culture" survival per treatment atmosphere cold-culture
Table 1.
Oa
air
93d
: 1
5% ::,:"g; ;;::90% N2
5; 5od 89
0 2 2 2
air 5% co . 5% CD2, 5; :;: 90% N2
92d Oe 4qf 62f
0 3 z
fl :
air 5% CO , air 5% co2, 3% 02, 90% N2
air 5% CO , air 5% C02, 3% 02, 90% N2
97d 0; 34f 45 90;
:e Oe
45 123 121 123 40 62 :z 40 46 114 113
t: 66 67
iZero days in cold culture represents directly cultured controls. Embryos cultured in air were maintained in hepes buffered Whitten's medium. 'Percentage survival is the proportion (X100) of the total embryos per treatment developing to expanded blastocysts in subsequent 37°C culture. Values with different letters within lines are significantly different (P>O.O5).
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5% 0 and additional days in cold culture to 0% after 4 d. The 5% CO all 90% N atmosphere provided the highest one-cell embryo sur oval o Survival in the 5% CO and in air atmosphere atmos Jh eres tested. compared favorably to air alone but was poorer ?han in 5% COP, 5% 02 and 90% N2.
3’
Two-cell
3
Embryos
No significant difference was observed between embryos cold cultured for 3 d in the 5%CO 5% 0 and 90% N atmosphere and in the 5% CO2 and Th&efore,'the embryo P in the remaining time trials were air atmosphere. There was no significant cold cultured in either air or 5% CO and air. controls and embryos cold difference in survival between u r?stored cultured for 3 d in 5% CO2 and air_, but embryo survival after 3 d in the Embryos air atmosphere (absence of HCO ) was significantly lower. cold cultured for 5 and 6 d also &owed a greater survival rate with HCO; in the medium (Table 2). Table
2.
Influence of mouse embryos
Days in cold culture Oa 3 3 3
cold-culture
Cold cultureb atmosphere
air 5% CO2, air 5% CO2, 5% 2, 90% N2
atmosphere
z
air 5% CO2, air
0 6 6
air 5% CO,, air
survival
of
two-cell
PercentageC survival
Total embryos per treatment
100;
40 96 96 95
;;d 93d 97d
0
on
0: 88
40 118 121 41 55 132
Efero days in cold cultur: represents directly cultured controls. Embryos cultured in air were maintained in hepes buffered Whitten's medium. 'Percentage survival is the proportion (X100) of the total embryos per treatment developing to expanded blastocysts in subsequent 37°C culture. Values with different letters within lines are significantly different (P>O.O5). Four-cell
Embryos
Four-cell embryos had a significantly higher survival rate in HCONo significant difference wa 2 than in HCO; free medium (Table 3). observed between embryos cold cultured 3 d with HCO- and the unstored as controls. Early four-cell embryos tended to collaps i and appeared two-cell embryos with increased time in cold culture. This did not, however, impair their ability to develop into blastocysts.
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Table 3.
Influence of cold culture atmosphere on survival of four-cell mouse embyros
Days in cold culture Oa
Cold cultureb atmosphere
;
air 5% CO,, air
: 4
air 5% CO,, air
0
5 5
air 5% CO,, air
0 6 6
air 5% COP, air
PercentageC survival 100; gide 100," zd 9gd I'f 76
100d 0; 48
Total embryos per treatment ;; 70 40 78 76
:; 76
%: 48
:Zero days in cold-culture represents directly cultured controls. Embryos cultured in air were maintained in hepes buffered Whitten's medium. 'Percentage survival is the proportion (X100) of the total embryos per treatment developing to expanded blastocysts in subsequent 37°C culture. Values with different letters within lines are significantly d,ifferent(P70.05). Table 4.
Influence of cold-culture atmosphere on survival of morulae and early blastocysts
Days ina cold culture 0 7 7
Cold cultureb atmosphere
Total embryos per treatment
air 5% COP, air
1;: I77
air 5% CO,, air
40 115 113
0
8 8
PercentageC survival
iZero days in cold culture represents directly cultured controls. Embryos cultured in air were maintained in hepes buffered Whitten's medium. 'Percentage survival is the proportion (X100) of the total embryos per treatment developing to expanded blastocysts in subsequent 37*C culture. Values with different letters within lines are significantly different (P>O.O5).
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Two-cell embryos which were directly cultured at 37°C to _. ,_.morulae and then transferred to recipients yielded greater proportions (although not significant) of late-term fetuses than two-cell embryos cold cultured for 4 d before in vitro culture to morulae and transfer to recipients. DISCUSSION In this study, mouse embryos of different preimplantation stages were cultured to exoanded blastocvsts followinq culture at 4'C in the presence or absence of HCO-. Also"investigatedwas the effect of oxygen tension on the cold culturz of one- and two-cell embryos. The ability of cold cultured embryos to complete development was evaluated by transfer of two-cell (cultured in vitro at 37°C to morulae) and blastocyst stage embryos to pseudopregnant females. Every effort was made to maintain the correct CO - containing HCO- concentration ([H&l) ’ only atmosphere over the media. indirectly related to the amount af sodium bicarbonate %dded"to the medium, but is directly proportional to the concentration of CO in the culture atmosphere and the pH of the medium (18,19). As CO is ?-eleased the FH increases. into a culture atmosphere containing no (or low) CO The increased pH has been shcwn to result in embryo &mage (8 and 18). One-cell embryo survival subsequent to cold-culture was affected by the oxygen tension in the cold-culture atmosphere. Embryos cold cultured 5% 02 and 90% N survived in greater numbers than embryos under 5% CO cultured un?ier 5% CO and a Pr. This observation is consistent with observations of WhitteG (19) for one-cell mouse embryos cultured at 37°C. Whitten's hypothesis was that one-cell mouse embryos are particularly sensitive to oxidative damage. In our study, survival of two-cell embryos cold cultured in a 5% CO2 and air atmosphere was not signficantly different from the survival rate in 5% CO2, 5% 02 and 90% N2. Survival of early (one- to four-cell) embryos subsequent to cold culture was increased with the addition of HCO- to the cold culture media. This observation is consistent with prev?ous studies on HCOrequirements of early mouse embryos in 37'C culture (18). Two-cell mous.3 embryos at 37°C in the absence of HCO- for only 2 h have diminished ability to develop to blastocysts subsjquent to culture when compared with embryos maintained the entire time with HCO; (20). In the same study, eight-cell mouse embryos were cultured for 8 h at 37°C in the absence of HCO; before a significant decrease in viability was observed. In our study, morula and blastocyst survival was not significantly affected by HCOj in the cold culture media. The importance of HCO- to the cold culture of mouse embryos may be Mouse embryos incorporate CO related to energy metabaism. into acetate, malate, citrate and ketoglutarate, presumably via p5 ruvate carboxylase-mediated CO2 condensation with pyruvate to form oxaloacetate (13). Embryos cultured at low temperature also metabolize carbohydrates although at a reduced rate (21). Therefore, the parameters necessary for energy metabolism at 37"C, i.e. HC03, may be necessary at reduced temperatures as well.
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Morulae
and Blastocysts
Morula and blastocyst survival did not increase with HCO- in the No significant difference w3 s found cold culture medium (Table 4). between treatments for embryos stored in the presence or absence of Collapse of cold cultured blastocysts was HCOfor 7 and 8 d. sig l+lficantly reduced with HCO; after 5 d in cold-culture but not after 8 d (Table 5). Table
5.
Effect of bicarbonate in cold culture the blastocoele of mouse blastocysts Cold cultureb atmosphere
Oays in cold culture 5 5
5% co
PercentageC survival
on the collapse
of
Total embryos per treatment
&$ir
5% CO
, gir E.ir
:
media
68 72
16d 13d
aEmbryos cultured in air were maintained in hepes buffered Whitten's bmedium. Values with different letters within lines differ significantly (P70.05). Embryo
Transfer
to Recipients
None of the blastocysts that were directly transferred to recipients In contrast, after 6 d of cold culture developed into late-term fetuses. blastocysts cold-cultured for 6 d, followed by 36 h of in vitro culture prior to transfer, developed into late-term fetuses at the same rate as unstored controls (Table 6). Table
6.
Development of untreated embryos
Days in cold-culture 0 4 0 6 6
Stage duringa cold-culture 2-cell 2-cell blastocyst blastocyst blastocyst
late-term
Stage whenb transferred morula morula blastocyst blastocyst blastocystC
fetuses
from
cold
Percentage developing to late-term fetuses 21 8 22 0 19
cultured
Total embryos transferred 180 180 240 180 180
"For embryos stored for 0 d, stage of embryo during cold culture brepresents stage at collection. If stage at cold-culture differs from stage at transfer, embryos were cultured at 37°C to stage at transfer. 'In this treatment, embryos were cultured for 36 h at 37°C subsequent cold culture and prior to transfer.
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It has been su gested that cold culture maintains embryos in arrested development ?9). Although embryos held at low temperatures do not divide, cold cultured embryos are not equivalent to embryos of the In our study, the same stage that have not been cold cultured. blastocysts (Day 4 embryos) transferred directly to Day 3 recipients subsequent to cold culture for 6 d produced no late-term fetuses, whereas cold cultured (6 d) blastocysts transferred to Day 3 recipients after 36 h in culture at 37°C developed into late-term fetuses at proportions Rabbit embryos cold cultured for 2 wk and similar to unstored controls. transferred to recipients developed to offspring later than embryos of the same stage that were directly transferred (9). It has been suggested that the development of cold cultured embryos continues to progress at a Our study, however, found slower rate compared to unstored controls. that a readjustment period is required for blastocysts after cold culture. The results of this study suggest that cold-cultured embryos should be treated like earlier stage embryos regarding recipient synchrony for transfer. In addition, the presence of HCO- in the medium used for cold culture benefits early stage embryo surviva P. REFERENCES Brinster, R., Chen H.Y., Trumbauer, M.E., Yagle, M.K., and Palmiter, Factors affecting the efficiency of introducting foreign DNA R.D. Proc. Natl. Acad. Sci. into mice by microinjecting eggs. g:4438-4442 (1985).
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Hammer, R.E., Pursel, V.G., Rexroad, C.E. Jr., Wall, R.J., Bolt, Ebert, K.M., Palmiter, R.D., and Brinster, R.L. Production of transgenic rabbits, sheep and pigs by microinjection. Nature. 315:680-683 (1985).
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Farrel, P.S. and Bavister, B.D. Short-term exposure to two-cell hamster embryos to collection media is detrimental to viability. Biol. Reprod. -31:109-114 (1984).
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Maurer, R.R. Can. J. Anim.
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Whittingham, P.G. and Wales, R.G. in vitro. Adv. Biosci. 6:128-141 --
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Hafez,
E.S.E.
Storage of mammalian oocytes Sci. 56:131-145 (1976).
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A review.
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embryos
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(1963). 7.
Moore, M.W. and Bilton, R.S. The stora e of fertilized 5°C. Aust. J. Biol. Sci. g:1421-1427 9 1973).
8.
Trounson, A.O., Willadsen, S.M., Rowson, L.E.A., and Newcomb, R. The storage room temperatures of cow eggs at and at low J. Reprod. Fertil. %:173-178 (1976). temperatures.
9.
Hughes, embryos
and Anderson, G.B. M.A. Short-term storage at 4°C. Theriogenology g:311-319 (1983).
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Lindner, G.M., Anderson, G.B., BonDurant, R.H. and Cupps, P.T. Survival of bovine embryos stored at 4°C. Theriogenology g:311-319 (1983).
11.
Herr, C. Factors Influencing Survival of Preimplantation Stage Mouse Embryos Maintained in Developmental Arrest In Vitro. Ph.D. Thesis. Washington State University, Pullman, WA, 7987
12.
Brinster, R.L. -In vitro culture of the embryo. In Sherman, A.I. (ed.), Pathways to Conception. Charles C. Thomas,Springfield, IL, 1971. pp. 245-277.
13. Quinn, P. and Wales, R.G. Growth and metabolism of preimplantation mouse embryos cultured in phosphate-buffered medium. J. Reprod. Fertil. E:289-300 (1973). 14.
the culture Whitten, W.K. Nutrient requirements for preimplantation embryos -in vitro. Adv. Biosci. 6:128-141 (1971).
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Herr, C. and Wright, R.W., Jr. Storage life of Whitten's embryo culture medium. Theriogenology g:158 abstr. (1986).
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Rafferty, K.A. Methods in Experimental Embryology of the Mouse. The John Hopkins Press, Baltimore and London, 1970.
17. Mendenhall, W. Introduction to Probability and Statistics. Roxbury Press, North Scituate, MA, 1979. 18.
Rrinster, R.L. Studies on the development of mouse embryos in vitro. IV. Interaction of energy sources. J. Reprod. Fertir m7 (1965).
19.
Whitten, W.K. Effect of oxygen on cleavage of mouse eggs -in vitro. Sot. Study Reprod. 2nd Ann. Meeting. Davis, CA. Biol. Reprod. Abstr. 52 (1969).
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Quinn, P. and Wales, R.G. Fixation of carbon dioxide by preimplantation mouse embryos ti vitro and the activities of enzymes involved in the process. Aust.J.01. Sci. -24:1277-1290 (1971).
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Anderson, G.B. and Foote, R.H.. Effects of low temperature storage upon subsequent energy metabolism of rabbit embryos. Exp. Cell Res. g:302-306 (1974).
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