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Effects of culture systems on development of in vitro fertilized bovine ova into blastocysts
THERIOGENOLOGY
EFFECTS OF CULTURE SYSTEMS ON DEVELOPMENT OF IN VITRO FERTILIZED BOVINE OVA INTO BLASTOCYSTS 2 Y. Aoyagi, l,a Y. Fukui, Y. Iwazumi,'M.
Urakawa'
and H. Ono
2
1 Hokkaido Livestock Improvement Association Inc., Tokorogun, Hokkaido 099-14, Japan 2 Department of Meat Animal Reproduction Obihiro University of Agriculture and Veterinary Medicine Obihiro 080, Japan Received
for publication: April 22, 1990 Accepted: August 22, 1990 ABSTRACT
We examined the effects of co-culture with oviductal epithelial cells, cumulus cells, trophoblastic vesicles or amniotic sac cells on the development of bovine eight-cell embryos derived from in vitro maturation and fertilization into blastocysts. Frozen-thawed spermatozoa were treated with caffeine plus Ca-ionophore A23187 for capacitation and were then co-incubated for 4 h with oocytes matured in vitro. Ova resulting from this in vitro fertilization were cultured in HEPES-buffered TCM-199 + 10% fetal calf serum(FCS) for 68 h and then removed from the cumulus cell mass. The eightcell embryos were cultured using four co-culture systems either without cells(controls) or within rabbit oviducts. The co-culture of oviductal epithelial cells, trophoblastic vesicles or amniotic sac cells significantly (P
amniotic vesicles
cell
INTRODUCTION Techniques of bovine in vitro fertilization have been progressing rapidly and are valuable not only for embryo Acknowledgment:We wish to thank Dr. G. E. Seidel of Corolado State University,USA, and Dr. T. Takeda of Zen-Noh, Japan, for reading this manuscript and making valuable cements. aPresent address:Central Research Institute for Feed and Live stock, Zen-Noh 1708-2 Tsukuriya, Tsukuba, Ibaraki 30033, Japan
OCTOBER 1990 VOL 34 NO. 4
749
transfer programs, but also for use in nuclear transplantation and gene injection. Newborn calves have been produced by the transfer of embryos derived from in vitro fertilization and using rabbit(l,2) or sheep(3,4) oviducts. However, despite the use of various culture media and methods, development of early bovine embryos is frequently arrested at the 8- to 16-cell stage(5,6). However, it has been found that ovine oviductal cells can provide an enviroment compatible with normal embryonic development(7). Eyestone et a1.(8) and Heyman et al.(g) have reported a co-culture system using bovine oviductal cells or bovine trophoblastic vesicles in which early bovine embryos that were recovered from superovulated cattle developed beyond the 16-cell stage. In addition, recent studies by Kajihara et al.(lO) and Lu et al.(ll) also have shown that co-culture of in vitro matured and fertilized oocytes with bovine cumulus cells or bovine oviductal cells enhanced development to the morula or blastocyst stage. The objective of our study was to evaluate the effects of co-culture systems using oviductal epithelial cells, cumulus cells, trophoblastic vesicles or amniotic sac cells on the development of in vitro fertilized bovine eight-cell enbryos into blastocysts. MATERIALS
AND METHODS
Bovine ovaries were collected mainly from Holstein cows and heifers at a slaughterhouse. Ovaries were transported to our laboratory in sterile saline(38'C) for 1 to 2 h. Cumulus-oocyte complexes were collected from follicles 2 to 5 mm in diameter with an 18-g needle attached to a IOml syringe. Healthy oocytes with a dense layer of cumulus cells were used for the experiments. The medium used for oocyte maturation was TCM-199(Gibco Laboratories, Grand Island, NY, USA) supplemented with 10% fetal calf serum(FCS;Gibco), 25 mM HEPES, 100 &g/ml streptomycine, lOOI.U./ml penicillin G, 10 ug/ml LH (P-LH, Schering Corporation, Kenilworth, NJ, USA) and estradiol (1 pg/ml E2:ova hormone Benzoate, Teikoku Zoki Co.,Tokyo, Japan). Thirty to 40 oocytes were placed in each well(fourwell petri dish, Nunclon, Inter-Med.,Roskilde, Denmark) with the medium (500 pl) covered by liquid paraffin(Nakarai chemicals Co.,Kyoto, Japan) and cultured for 22 to 24 h in a CO 2 incubator (390C,5%C02 in air). Sperm capacitation was similar to the method described by Hanada(1) and Aoyagi et a1.(12), Frozen semen(a Japanese Black bull) in a 0.5-ml straw(5xlO sperm per straw) was used. After thawing at 37OC, 1.5 ml of semen was suspended
OCTOBER 1990 VOL 34 NO. 4
THERIOGENOLOGY in 5 ml of synthetic medium (BO medium;l3) containing 10 mM caffeine and washed twice by centrifugation for 5 min at 45079. Sperm concentration was adjusted to approximately 7 x10 /ml, and the sperm suspensions were diluted twofold with BO medium containing 2% bovine serum albumin (BSA; Fraction V, Sigma Co., Louis,Mo,USA) immediately after the treatment for 30 set with 0.1 pM calcium ionohore (A23187 free-acid), which was first dissolved with ethanol and dimethyl sulphoxide(3:l). The diluted sperm suspensions were preincubated in drops (80 to 100 @/drop) covered by liquid paraffin for 2.5 h in a CO2 incubator(39'C, 5% CO2 in air). The cultured oocytes were added to the semen suspension drops which had been preincubated, and gametes were cultured together for 4 h in a CO2 incubator. All ova inseminated in vitro were continuously cultured in wells containing TCM-199+10%FCS (25 mM HEPES, lOOpg/ml streptomycin and 100 I.U./ml penicilin G) for 68 h in a CO2 incubator. The ova were then removed from the cumulus cell mass by pipetting and examined for cleavage stage. Six culture systems were evaluated in our study, and eight-cell ova(Figure 1; 414 embryos/l721 inseminated oocytes:24.9%) were cultured or transferred as follows: In Group l(controls), eight-cell ova were placed in a well with 880 ~1 of TCM-199 plus 10% FCS without any other cells. In Group 2, eight-cell ova were co-cultured with oviduct epithelial cells, which were obtained at a slaughterhouse, by flushing oviducts of cows with Dulbecco's phosphate buffered saline(PBS) and 10% FCS and washing with TCM-199 plus 10% FCS;880 pl of the cell suspension was placed in each well for 3 d before co-culture(l4). In Group 3, eight-cell ova were co-cultured with cumulus cells which were removed by pipetting at 68 h after insemination. The ova were placed in a well with cumulus cells in 880@ of TCM-199 plus 10% FCS(lO,lS). Co-culture of Group 4 eight-cell ova was performed with trophoblastic vesicles. Bovine trophoblastic vesicles were prepared from Day-13 blastocysts recovered from superovulated cows. Each embryo was cut into several fragments and the embryonic disc was removed. The trophoblastic tissue was cultured for 24 h in TCM-199 and 10% FCS in a incubator(39"C, 5% CO in air). The diameters of co2 trophoblastic vesicles af 5er culture were approximately 1 to 2 mm. These trophoblastic vesicles were frozen and stored in liquid nitrogen until co-culture (9). After thawing and three-step sucrose dilution of the cryoprotectant, six trophoblastic vesicles were placed in a well with 880 pl of TCM-199 plus 10% FCS. In Group 5, eight-cell
OCTOBER 1990 VOL 34 NO. 4
ova were co-cultured
with
751
THERIOGENOLOGY amniotic sac cells. Bovine amniotic sacs at 60 to 120 d of pregnancy were obtained from a slaughter house. The fetal age was estimated from the crown-rump length. Each amniotic sac was cut into small fragments (1x1 mm) and were kept in PBS plus 10% FCS; six fragments were placed in a well(880 pl of TCM-199 plus 10% FCS) for 5 to 12 d before co-culture. Five to 15 ova per well were cultured in vitro(in all five culture systems) for the medium was changed every 2 d. Group 6 eight-cell ova were cultured in vivo in rabbit oviducts. Japanese white rabbits (4 to 5 kg body weight) were pretreated with 100 1-U. human chorionic gonadotropin (hCG) to induce pseudopregnancy. Then, 2 to 5 d later, rabbits were laparotomized under local anaesthesia with 2% xylocaine for embryo transfer into the ampula, about 2 to 3 cm from the infundibulum. Rabbits were killed 4 d later, and the dissected oviducts were flushed with PBS supplemented with 10% FCS. After recovery from the rabbit oviducts, ova were examined for the presence of blastocysts, and the remaining embryos were further cultured in wells of TCM-199 plus 10% FCS for 24 h. After culture or recovery, the embryos were classified morphologically. Normal blastocysts(good or excellent quality) were frozen. The freezing procedure has been described by Takeda et a1.(16). After equilibration in PBS + 0.4% BSA + 10% glycerol for 5 min, each blastocyst was aspirated into a plastic straw (0.25 ml). After cooling to -5.1°C at 1_5'Cfmin, seeding was induced and the straws were cooled at 0.5°C/min to -32.5OC before being plunged into liquid nitrogen. Rapid thawing was achived by placing the straw from -196'C into a 35'C water bath for 30 set immediately after air-thawing for 5 sec. A three step dilution in a 0.3 M of sucrose solution was used to remove the cryoprotectant. A total of 22 Holstein heifers and cows served as recipients. Transfers were performed at Day 7 or 8 of the estrous cycle nonsurgically using the modified Cassou gun. After transfer, each recipient was checked daily for return to estrus. Palpation per rectum or ultra-sound diagnosis was performed repeatedly in each recipient at Days 35 to 40 and against Days 60 to 70 to confirm pregnancy. Chi-square tests were used to analyze the effect of culture systems on rates of development into blastocysts and proportions of freezable embryos. RESULTS The proportions of embryos developing into blastocysts did not differ significantly among co-culture systems using trophoblastic vesicles, amniotic sac cells or oviduct
752
OCXOBER 19W VOL 34 NO. 4
THERIOGENOLOGY epithelial cells, and ranged from 39.0 to 50.7%. The percentage of development in the control culture(l.98) was significantly(P
OCTOBER 1990 VOL 34 NO. 4
753
THERIOGENOLOGY
Figure 1.
Higher than the eight-cell stage embryo cultured for 3 days after in vitro fertilization and in vitro maturation(200x).
Figure 2.
Blastocyst stage embryo developed from an eight-cell stage embryo by co-culture with trophoblastic vesicles for 4 days(200x).
754
OCTOBER 1990 VOL 34 NO. 4
THERIOGENOLOGY
x r-
.
0 II? ln
m
m w
m w
OCTOBER 1990 VOL 34 NO. 4
P
2
(c,
r
6
! Q
$
Pregnancies
(Days)
Returned to estrus or palpated open (Days)
Birth weight
in this study
Gestation period
produced Sex of calves
46
291
47kg
male
male,female 289 40,25kg 22 23 60 20 .--__-------_----_-_ .- ----_---------------male 291 58kg 24 25 21 male,male 285 35,34kg Frozen 2 569 25 R996 Frozen 2 male Frozen 287 46kg R997 2 ~~-~~~-__~~---__--~~--__---------_---_----_----__--_----_---_---_----_---__--_--Frozen 23 Rabbit 2 350 oviducts
Frozen 2 R891 Frozen 2 SA2 2 647 Trophoblastic Frozen Frozen 2 605 vesicles Frozen 2 568 Frozen 2 KY1 Frozen 1 KY2 ~~-__~---___-------_---------_---------Frozen 2 R20 AV3 Amniotic Frozen 2 Frozen 2 412 570 sac cells
R395
25 Fresh 1 Control --~~~~-__~~--~~--_~~---_-----_---_----__-_---~~---_----_---~---------_---_----_-19 Fresh 2 R4 17 Fresh 2 R472 Oviduct 41 Frozen 2 DMI epithelial 287 36kg male Frozen 2 489 cells ~~-~~~--___----_---_---_-----__--___--__-~---__---_---_~--__---~----_--------~--_ 294 56kg male Fresh 1 R42 Cumulus 18 Fresh 1 R48 cells
No.of transferred blastocysts
after transfer of blastocysts
Recipient Culture Fresh/ number system frozen embryos
Table 2.
THERIOGENOLOGY
The recovery rate of ova from rabbit oviducts was 76.3%. Eyestone et a1.(19) and Aoyagi et a1.(12) have reported that about 30% of ova were not recovered from sheep or from rabbit oviducts. Culture of embryos in vivo in sheep or rabbit oviducts resulted in some loss of embryos, and there were differences in the development rate of embryos among hosts. However, these problems were not connected to in vitro procedures. Development rates of embryos by co-culture with trophoblastic vesicles, amniotic sac cells, or oviductal epithelial cells were higher than in the other culture systems. After 4 d of co-culture with trophoblastic vesicles, 50.7% of the in vitro fertilized eight-cell ova developed into blastocysts as compared to 46% of the one to eight-cell bovine in vivo embryos using same co-culture(9). Our results have demonstrated that co-culture of ova with oviductal epithelial cells, trophoblastic vesicles or amniotic sac cells is a useful method for advancing development of in vitro fertilized bovine ova into blastocysts and for producing normal calves using frozen-thawed embryos derived from in vitro maturation and fertilization. The results of our study have indicated that bovine embryos needed for transplantation or other research purposes can be produced simply and economically by in vitro procedures.
REFFERENCES 1.
Hanada, A. In vitro fertilization in cattle, with particular reference to sperm capacitation by ionophore A23187. Jpn. J. Anim. Reprod. -31:56-61(19851.
2.
Sirard, M.A. and Lambert, R.D. Birth of calves after in vitro fertilization using laparoscopy and rabbit oviduct Vet. Rec. 119:167-169(1986). incubation of zygotes.
3.
Parrish, J.J., Susuko-Parrish, J.L., Leibfried-Rutledge, M.L., Crister, E.S., Eyestone, W.H. and First, N.L. Bovine in vitro fertilization with frozen-thawed semen. Theriogenology 21:591-600(1986).
4.
Lu, K.H., Gordon, I., Gallagher, M. and McGovern, H. Pregnancy established in cattle by transfer of embryos derived from in-vitro fertilization of oocytes matured Vet. Rec. 121:259-260(1987). in-vitro.
5.
Eyestone, W.H. and First, N.L. A study of the 8- to 16cell developmental block in bovine embryos cultured in Theriogenology -25:152 abstr.(1986). vitro.
6.
Bavister, B.D. Role of oviductal secretions in Theriogenology embryonic growth in vivo and in vitro. 29:143-154(1988). -
OCTOBER 1990 VOL 34 NO. 4
757
THERIOGENOLOGY 7.
Stimulation of early Gandolfi, F. and Moor, R.M. embryonic development in the sheep by co-culture with oviduct epithelial cells. J. Reprod. Fertil. -81:23-28(1987).
8.
Eyestone, W.H., Vignieri, J. and First, N.L. Co-culture of early bovine embryos with oviductal epithelium. Theriogenology -27:228 abstr.(1987).
9.
Heyman, Y., Menezo, Y., Chesn&, P., Camous, S. and Garnier, V. In vitro cleavage of bovine and ovine early embryos: improved development using co-culture with trophoblastic vesicles. Theriogenology -27:59-68 (1987).
10.
Kajihara, Y., Goto, K., Kosaka, S., Nakanishi, Y. and Ogawa, K. In vitro fertilization of bovine follicular oocytes and their development upto hatched blastocysts in vitro. Jpn. J. Anim. Reprod. -33:173-179(1987).
11.
Lu, K.H., Gordon, I., Chen, H.B., Gallagher, M. and McGovern, H. Birth of twins after transfer of cattle embryos produced by in vitro techniques. Vet. Rec. 122:539-540(1988).
12.
Aoyagi, Y., Fujii, K., Iwazumi, Y., Furudate, M., Fukui, Y. and Ono, H. Effects of two treatments on semen from different bulls on in vitro fertilization results of bovine oocytes. Theriogenology -30:973-985 (1988).
13.
Brackett, B.G. and Oliphant, G. Capacitation of rabbit spermatozoa in vitro. Biol. Reprod. -12:260-274(1975).
14.
Fukui, Y. and Ono, H. In vitro development to blastocysts of in vitro matured and fertilized bovine oocytes. Vet. Rec. 122:282 abstr.(1988).
15.
Goto, K., Kajihara, Y., Kosaka, S., Koba, M., Nakanishi, Y. and Ogawa, K. Pregnancies after co-culture of cumulus cells with bovine embryos derived from in-vitro fertilization of in vitro matured follicular oocytes. J. Reprod. Fertil. -83:753-758 (1988).
16.
Takeda, T., Elsden, R.P. and Seidel, G.E., Jr. Survival of cryopreserved bovine embryos cooled at 0.5 or 1°C/minute. Theriogenology -23:232 abstr.(1985).
17.
Kuzan, F.B. and Wright, R.W., Jr. Observations on the development of bovine morulae on various cellular and noncellular substrata. J. Anim. Sci. -54:811-816(1982).
758
OCTOBER 1990 VOL 34 NO. 4
THERIOGENOLOGY 18.
Eyestone, W.H. and First, N.L. Co-culture of early cattle embryos to the blastocyst stage with oviductal J. Reprod. Fertil. tissue or in conditioned medium. -85:715-720(1989).
19.
Eyestone, W-H., Northey, D.L. and Leibfried-Rutledge, Culture of l-cell bovine embryos in the sheep M.L. oviduct. Biol. Reprod. -32:lOO abstr.(1985).
OCTOBER 1990 VOL 34 NO. 4
759
EFFECTS OF CULTURE SYSTEMS ON DEVELOPMENT OF IN VITRO FERTILIZED BOVINE OVA INTO BLASTOCYSTS 2 Y. Aoyagi, l,a Y. Fukui, Y. Iwazumi,'M.
Urakawa'
and H. Ono
2
1 Hokkaido Livestock Improvement Association Inc., Tokorogun, Hokkaido 099-14, Japan 2 Department of Meat Animal Reproduction Obihiro University of Agriculture and Veterinary Medicine Obihiro 080, Japan Received
for publication: April 22, 1990 Accepted: August 22, 1990 ABSTRACT
We examined the effects of co-culture with oviductal epithelial cells, cumulus cells, trophoblastic vesicles or amniotic sac cells on the development of bovine eight-cell embryos derived from in vitro maturation and fertilization into blastocysts. Frozen-thawed spermatozoa were treated with caffeine plus Ca-ionophore A23187 for capacitation and were then co-incubated for 4 h with oocytes matured in vitro. Ova resulting from this in vitro fertilization were cultured in HEPES-buffered TCM-199 + 10% fetal calf serum(FCS) for 68 h and then removed from the cumulus cell mass. The eightcell embryos were cultured using four co-culture systems either without cells(controls) or within rabbit oviducts. The co-culture of oviductal epithelial cells, trophoblastic vesicles or amniotic sac cells significantly (P
amniotic vesicles
cell
INTRODUCTION Techniques of bovine in vitro fertilization have been progressing rapidly and are valuable not only for embryo Acknowledgment:We wish to thank Dr. G. E. Seidel of Corolado State University,USA, and Dr. T. Takeda of Zen-Noh, Japan, for reading this manuscript and making valuable cements. aPresent address:Central Research Institute for Feed and Live stock, Zen-Noh 1708-2 Tsukuriya, Tsukuba, Ibaraki 30033, Japan
OCTOBER 1990 VOL 34 NO. 4
749
transfer programs, but also for use in nuclear transplantation and gene injection. Newborn calves have been produced by the transfer of embryos derived from in vitro fertilization and using rabbit(l,2) or sheep(3,4) oviducts. However, despite the use of various culture media and methods, development of early bovine embryos is frequently arrested at the 8- to 16-cell stage(5,6). However, it has been found that ovine oviductal cells can provide an enviroment compatible with normal embryonic development(7). Eyestone et a1.(8) and Heyman et al.(g) have reported a co-culture system using bovine oviductal cells or bovine trophoblastic vesicles in which early bovine embryos that were recovered from superovulated cattle developed beyond the 16-cell stage. In addition, recent studies by Kajihara et al.(lO) and Lu et al.(ll) also have shown that co-culture of in vitro matured and fertilized oocytes with bovine cumulus cells or bovine oviductal cells enhanced development to the morula or blastocyst stage. The objective of our study was to evaluate the effects of co-culture systems using oviductal epithelial cells, cumulus cells, trophoblastic vesicles or amniotic sac cells on the development of in vitro fertilized bovine eight-cell enbryos into blastocysts. MATERIALS
AND METHODS
Bovine ovaries were collected mainly from Holstein cows and heifers at a slaughterhouse. Ovaries were transported to our laboratory in sterile saline(38'C) for 1 to 2 h. Cumulus-oocyte complexes were collected from follicles 2 to 5 mm in diameter with an 18-g needle attached to a IOml syringe. Healthy oocytes with a dense layer of cumulus cells were used for the experiments. The medium used for oocyte maturation was TCM-199(Gibco Laboratories, Grand Island, NY, USA) supplemented with 10% fetal calf serum(FCS;Gibco), 25 mM HEPES, 100 &g/ml streptomycine, lOOI.U./ml penicillin G, 10 ug/ml LH (P-LH, Schering Corporation, Kenilworth, NJ, USA) and estradiol (1 pg/ml E2:ova hormone Benzoate, Teikoku Zoki Co.,Tokyo, Japan). Thirty to 40 oocytes were placed in each well(fourwell petri dish, Nunclon, Inter-Med.,Roskilde, Denmark) with the medium (500 pl) covered by liquid paraffin(Nakarai chemicals Co.,Kyoto, Japan) and cultured for 22 to 24 h in a CO 2 incubator (390C,5%C02 in air). Sperm capacitation was similar to the method described by Hanada(1) and Aoyagi et a1.(12), Frozen semen(a Japanese Black bull) in a 0.5-ml straw(5xlO sperm per straw) was used. After thawing at 37OC, 1.5 ml of semen was suspended
OCTOBER 1990 VOL 34 NO. 4
THERIOGENOLOGY in 5 ml of synthetic medium (BO medium;l3) containing 10 mM caffeine and washed twice by centrifugation for 5 min at 45079. Sperm concentration was adjusted to approximately 7 x10 /ml, and the sperm suspensions were diluted twofold with BO medium containing 2% bovine serum albumin (BSA; Fraction V, Sigma Co., Louis,Mo,USA) immediately after the treatment for 30 set with 0.1 pM calcium ionohore (A23187 free-acid), which was first dissolved with ethanol and dimethyl sulphoxide(3:l). The diluted sperm suspensions were preincubated in drops (80 to 100 @/drop) covered by liquid paraffin for 2.5 h in a CO2 incubator(39'C, 5% CO2 in air). The cultured oocytes were added to the semen suspension drops which had been preincubated, and gametes were cultured together for 4 h in a CO2 incubator. All ova inseminated in vitro were continuously cultured in wells containing TCM-199+10%FCS (25 mM HEPES, lOOpg/ml streptomycin and 100 I.U./ml penicilin G) for 68 h in a CO2 incubator. The ova were then removed from the cumulus cell mass by pipetting and examined for cleavage stage. Six culture systems were evaluated in our study, and eight-cell ova(Figure 1; 414 embryos/l721 inseminated oocytes:24.9%) were cultured or transferred as follows: In Group l(controls), eight-cell ova were placed in a well with 880 ~1 of TCM-199 plus 10% FCS without any other cells. In Group 2, eight-cell ova were co-cultured with oviduct epithelial cells, which were obtained at a slaughterhouse, by flushing oviducts of cows with Dulbecco's phosphate buffered saline(PBS) and 10% FCS and washing with TCM-199 plus 10% FCS;880 pl of the cell suspension was placed in each well for 3 d before co-culture(l4). In Group 3, eight-cell ova were co-cultured with cumulus cells which were removed by pipetting at 68 h after insemination. The ova were placed in a well with cumulus cells in 880@ of TCM-199 plus 10% FCS(lO,lS). Co-culture of Group 4 eight-cell ova was performed with trophoblastic vesicles. Bovine trophoblastic vesicles were prepared from Day-13 blastocysts recovered from superovulated cows. Each embryo was cut into several fragments and the embryonic disc was removed. The trophoblastic tissue was cultured for 24 h in TCM-199 and 10% FCS in a incubator(39"C, 5% CO in air). The diameters of co2 trophoblastic vesicles af 5er culture were approximately 1 to 2 mm. These trophoblastic vesicles were frozen and stored in liquid nitrogen until co-culture (9). After thawing and three-step sucrose dilution of the cryoprotectant, six trophoblastic vesicles were placed in a well with 880 pl of TCM-199 plus 10% FCS. In Group 5, eight-cell
OCTOBER 1990 VOL 34 NO. 4
ova were co-cultured
with
751
THERIOGENOLOGY amniotic sac cells. Bovine amniotic sacs at 60 to 120 d of pregnancy were obtained from a slaughter house. The fetal age was estimated from the crown-rump length. Each amniotic sac was cut into small fragments (1x1 mm) and were kept in PBS plus 10% FCS; six fragments were placed in a well(880 pl of TCM-199 plus 10% FCS) for 5 to 12 d before co-culture. Five to 15 ova per well were cultured in vitro(in all five culture systems) for the medium was changed every 2 d. Group 6 eight-cell ova were cultured in vivo in rabbit oviducts. Japanese white rabbits (4 to 5 kg body weight) were pretreated with 100 1-U. human chorionic gonadotropin (hCG) to induce pseudopregnancy. Then, 2 to 5 d later, rabbits were laparotomized under local anaesthesia with 2% xylocaine for embryo transfer into the ampula, about 2 to 3 cm from the infundibulum. Rabbits were killed 4 d later, and the dissected oviducts were flushed with PBS supplemented with 10% FCS. After recovery from the rabbit oviducts, ova were examined for the presence of blastocysts, and the remaining embryos were further cultured in wells of TCM-199 plus 10% FCS for 24 h. After culture or recovery, the embryos were classified morphologically. Normal blastocysts(good or excellent quality) were frozen. The freezing procedure has been described by Takeda et a1.(16). After equilibration in PBS + 0.4% BSA + 10% glycerol for 5 min, each blastocyst was aspirated into a plastic straw (0.25 ml). After cooling to -5.1°C at 1_5'Cfmin, seeding was induced and the straws were cooled at 0.5°C/min to -32.5OC before being plunged into liquid nitrogen. Rapid thawing was achived by placing the straw from -196'C into a 35'C water bath for 30 set immediately after air-thawing for 5 sec. A three step dilution in a 0.3 M of sucrose solution was used to remove the cryoprotectant. A total of 22 Holstein heifers and cows served as recipients. Transfers were performed at Day 7 or 8 of the estrous cycle nonsurgically using the modified Cassou gun. After transfer, each recipient was checked daily for return to estrus. Palpation per rectum or ultra-sound diagnosis was performed repeatedly in each recipient at Days 35 to 40 and against Days 60 to 70 to confirm pregnancy. Chi-square tests were used to analyze the effect of culture systems on rates of development into blastocysts and proportions of freezable embryos. RESULTS The proportions of embryos developing into blastocysts did not differ significantly among co-culture systems using trophoblastic vesicles, amniotic sac cells or oviduct
752
OCXOBER 19W VOL 34 NO. 4
THERIOGENOLOGY epithelial cells, and ranged from 39.0 to 50.7%. The percentage of development in the control culture(l.98) was significantly(P
OCTOBER 1990 VOL 34 NO. 4
753
THERIOGENOLOGY
Figure 1.
Higher than the eight-cell stage embryo cultured for 3 days after in vitro fertilization and in vitro maturation(200x).
Figure 2.
Blastocyst stage embryo developed from an eight-cell stage embryo by co-culture with trophoblastic vesicles for 4 days(200x).
754
OCTOBER 1990 VOL 34 NO. 4
THERIOGENOLOGY
x r-
.
0 II? ln
m
m w
m w
OCTOBER 1990 VOL 34 NO. 4
P
2
(c,
r
6
! Q
$
Pregnancies
(Days)
Returned to estrus or palpated open (Days)
Birth weight
in this study
Gestation period
produced Sex of calves
46
291
47kg
male
male,female 289 40,25kg 22 23 60 20 .--__-------_----_-_ .- ----_---------------male 291 58kg 24 25 21 male,male 285 35,34kg Frozen 2 569 25 R996 Frozen 2 male Frozen 287 46kg R997 2 ~~-~~~-__~~---__--~~--__---------_---_----_----__--_----_---_---_----_---__--_--Frozen 23 Rabbit 2 350 oviducts
Frozen 2 R891 Frozen 2 SA2 2 647 Trophoblastic Frozen Frozen 2 605 vesicles Frozen 2 568 Frozen 2 KY1 Frozen 1 KY2 ~~-__~---___-------_---------_---------Frozen 2 R20 AV3 Amniotic Frozen 2 Frozen 2 412 570 sac cells
R395
25 Fresh 1 Control --~~~~-__~~--~~--_~~---_-----_---_----__-_---~~---_----_---~---------_---_----_-19 Fresh 2 R4 17 Fresh 2 R472 Oviduct 41 Frozen 2 DMI epithelial 287 36kg male Frozen 2 489 cells ~~-~~~--___----_---_---_-----__--___--__-~---__---_---_~--__---~----_--------~--_ 294 56kg male Fresh 1 R42 Cumulus 18 Fresh 1 R48 cells
No.of transferred blastocysts
after transfer of blastocysts
Recipient Culture Fresh/ number system frozen embryos
Table 2.
THERIOGENOLOGY
The recovery rate of ova from rabbit oviducts was 76.3%. Eyestone et a1.(19) and Aoyagi et a1.(12) have reported that about 30% of ova were not recovered from sheep or from rabbit oviducts. Culture of embryos in vivo in sheep or rabbit oviducts resulted in some loss of embryos, and there were differences in the development rate of embryos among hosts. However, these problems were not connected to in vitro procedures. Development rates of embryos by co-culture with trophoblastic vesicles, amniotic sac cells, or oviductal epithelial cells were higher than in the other culture systems. After 4 d of co-culture with trophoblastic vesicles, 50.7% of the in vitro fertilized eight-cell ova developed into blastocysts as compared to 46% of the one to eight-cell bovine in vivo embryos using same co-culture(9). Our results have demonstrated that co-culture of ova with oviductal epithelial cells, trophoblastic vesicles or amniotic sac cells is a useful method for advancing development of in vitro fertilized bovine ova into blastocysts and for producing normal calves using frozen-thawed embryos derived from in vitro maturation and fertilization. The results of our study have indicated that bovine embryos needed for transplantation or other research purposes can be produced simply and economically by in vitro procedures.
REFFERENCES 1.
Hanada, A. In vitro fertilization in cattle, with particular reference to sperm capacitation by ionophore A23187. Jpn. J. Anim. Reprod. -31:56-61(19851.
2.
Sirard, M.A. and Lambert, R.D. Birth of calves after in vitro fertilization using laparoscopy and rabbit oviduct Vet. Rec. 119:167-169(1986). incubation of zygotes.
3.
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