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Development of in vitro fertilized oocytes from pregnant and nonpregnant cows in oviductal epithelial and cumulus cell co-culture systems
Theriogenolog
y 38: 1077-I
084,1992
DEVELOPMENT OF IN VITRO FERTILIZED OOCYTES FROM PREGNANT AND NONPREGNANT COWS IN OVIDUCTAL EPITHELIAL AND CUMULUS CELL CO-CULTURE SYSTEMS E. Behboodi,’ G.B. Anderson’ and R.H. BonDurant2 Department of Animal Science’ and Department of Vet Med Reproduction2 University of California, Davis, CA 95616 Received for publication: February 25, 1992 Accepted: September 12, 1992 ABSTRACT The objectives of this study were 1) to measure cleavage, blastocyst formation, and blastocyst hatching after in vitro maturation (IVM), fertilization (IVF) and culture (IVC) of oocytes aspirated from pregnant versus nonpregnant cows, and 2) to compare embryo development in co-culture with bovine oviductal epithelial cells versus cumulus cells. No differences in cleavage (38 versus 40%), blastocyst formation (13 versus 13%), or blastocyst hatching (53 versus 51%) were observed for in vitro-matured, fertilized, and cultured oocytes from pregnant versus nonpregnant cows, respectively (P > 0.05), indicating that nonpregnant and early-pregnant cows are equally acceptable donors of oocytes for IVM/IVF/IVC procedures. Cleavage (36 versus 40%), blastocyst formation (11 versus 12%), and blastocyst hatching (50 versus 55 W) were not different for embryos co-cultured with oviductal epithelial cells versus cumulus cells (P > 0.05). Thus, equivalent embryo development can be obtained with co-culture systems commonly used for in vitro-derived bovine embryos. These results help to define variables that affect comparison of results across laboratories and that are relevant to the practical application of IVM/IVF/IVC procedures to cattle. Key words:
bovine, embryos, in vitro maturation,
in vitro fertilization
INTRODUCTION Procedures for oocyte in vitro maturation (IVM), fertilization (IVF) and culture (IVC) have become valuable techniques for the production of bovine embryos used in developmental studies and for use with embryo transfer to produce offspring from genetically superior females (1,2). Numerous variations in IVM/IVF/IVC procedures have been used by different laboratories; these variables complicate both the comparison of results obtained by different investigators and the selection of procedures for in vitro Acknowledgments This research was supported by funds from the California Dairy Foods Research Center and the California Milk Advisory Board. Semen was donated by Landmark Genetics, Hughson, CA. The assistance of undergraduate researchers Kirby Tran, Kathryn Makris, Sham Garabetian, and Carmine Bausone is acknowledged.
Copyright 0 1992 Butterworth-Heinemann
Thet-iogenology
generation of bovine embryos. Among variations in procedures used by different investigators are co-culture systems to support post-fertilization embryo development and pregnancy status of donor females. Oviductal epithelial cells (3) and cumulus cells (4) are commonly used in co-culture with in vitro-matured, in vitro-fertilized bovine oocytes to support development to the blastocyst stage. The birth of live calves has been reported from blastocysts that were co-cultured with cumulus cells (4) and oviductal epithelial cells (5), but information from a direct comparison of embryo development in these 2 coculture systems is not available. The harvesting of large numbers of bovine oocytes from ovaries obtained at slaughter often yields oocytes from both pregnant and nonpregnant females. Differences in follicular growth due to pregnancy status could affect oocyte quality. Furthermore, transvaginal collection of oocytes from cows during gestation has been proposed as a means by which to enhance reproduction in genetically superior females (6). This study was carried out to compare embryo development in co-culture with oviductal epithelial cells versus cumulus cells. We also examined oocyte maturation, fertilization, and development of oocytes from nonpregnant versus pregnant cows. Variable embryo development in media supplemented with different batches of estrous cow serum was detected by retrospective analysis of data collected during the course of this study. MATERIALS
AND METHODS
Collection of Oocytes Bovine ovaries were collected at NaCl) containing pecicillin (500 &ml) the laboratory at 25 C within 3 hours. with an 1&gauge needle from. follicles temperature between 25 to 30 C.
a local slaughterhouse into sterile saline (0.9 % and streptomycin (500 &ml) and transported to Immature oocytes were aspirated into a syringe having a diameter of 1 to 6 mm in a room with
Culture of Oocytes for In Vitro Maturation Oocytes were washed in Medium 199 (Sig_ma, St. Louis, MO) supplemented with 10% estrous cow serum (heat-inactivated at 56 C for 30 minutes) and 1 N/ml 178estradiol. They were then transferred (50 oocytes/dish) to a 60 x 15-mm organ tissue culture dish (Fisher, Pittsburgh, PA) containing 1 ml Medium 199 with 10% estrous cow serum and 1 &ml 170-estradiol, and the culture medium was covered with 0.5 ml sterile paraffin oil (Sigma). Oocytes were divided randomly among culture dishes_without oocyte quality grading or selection and were incubated for 24 hours at 39 C in a humidified atmosphere of 5% CO, and 95% air. To prepare estrous cow serum, blood was collected by jugular venipuncture from 4 estrous dairy cows. Sera were separated after clotting, filtered (0.22 ,um pore sire), heat-inactivated (56°C for 30 minutes) and pooled prior to storage at -20°C until use.
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Sperm Preparation A method described by Parrish et al. (7) was used to prepare bovine spermatozoa for IVF. Commercially distribute+ frozen semen from 3 dairy bulls (Landmark Genetics, Hughson, CA) was thawed in 37 C water and placed in 1 ml of Sperm-TALP medium (7) supplemented with 5 &ml heparin (Sigma); suspended thawed spermatozoa we5 then aliquoted into 5 tubes (0.2 ml/ tube) for each bull and incubated for 1 hour at 39 C in 5% CO, and 95% air for sperm swim-up (3). The swim-up spermato+ were washed once by centrifugation at 700 x g for 6 minutes and then incubated at 39 C in 5% CO, and 95% air for an additional 40 minutes before insemination. Co-Incubation
of Gocytes with Spermatozoa and Embryo Culture
The method used was similar to that described by Eyestone and First (8). Briefly, droplets (100 3 each) of Fert-TALP medium (7) were placed on the bottom of a 35 x lomm easy grip tissue culture dish (Fisher) and covered with 1 ml sterile paraffin oil. In vitro-matured oocytes were washed 3 times and transferred (50/droplet) to the fertilization me$um. Spermatozoa (3 x 104/oocyte) were co-incubated with oocytes for 18 hours at 39 C. Ova were then transferred to 1 ml of Medium 199 supplemented with 10% heatinactivated estrous cow serum and 1 &ml 17R-estradiol and co-cultured with either oviductal epi?elial cells or cumulus cells in a humidified atmosphere of 5% CO, and 95% air at 39 C. Embryos were examined daily under a dissecting microscope. For each culture plate the number of ova that cleaved to the 2-cell stage and the numbers of blastocysts that formed and hatched from the zona pellucida were recorded. Oviductal Epithelial Cell Preparation Oviducts were collected from cows and heifers at slaughter, and epithelial cells were stripped from the oviductal lumen as described by Eyestone and First (8). An effort was made to collect oviductal cells from animals that had recently ovulated, as indicated by the presence of a corpus hemorrhagicurn on one of the ovaries, and cells from 3 or 4 animals were pooled. Epithelial cells were cultured in a 60 x 15-mm organ tissue culture dish (Fisher) for 48 hours in Medium 199 supplemented with 10% estrous cow serum and 1 &ml 17Kestradiol. Clumps of oviductal cells were dissociated by gentle pipetting. Approximately 50 ~1 of the oviductal cell suspension were added to each l-ml drop of culture medium containing the embryos. A mixed oviductal population of ciliated vesicles and monolayer developed over time. Cumulus Cell Monolayer Preparation Cumulus cell cultures cumulus cells were removed by vortexing for 30 seconds. to 20 oocytes for each 60 x Medium 199 supplemented
were prepared as described by Goto et al. (4). Briefly, from randomly-selected oocytes 24 hours after fertilization Denuded oocytes were discarded. Cumulus cells from 15 15-mm organ tissue culture dish (Fisher) were cultured in with 10% estrous cow serum and 1 &ml l’l&estradiol.
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Theriogenology
Cumulus cells attached to the bottom of the culture dish and formed a monolayer after 72 to 96 hours. Embryos were cultured on this monolayer. Experiment 1: Cleavage and Blastocyst Formation Nonpregnant Cows
for Oocytes from Pregnant and
Ovaries from pregnant cows and heifers were collected from females between 40 days and 5 months gestation, as determined by fetal crown-rump length. Ovaries from nonpregnant cows were collected without regard to stage of the ovarian cycle. Oocytes from pregnant and nonpregnant females were fertilized with semen from the same bulls, and after fertilization were co-cultured with oviductal epithelial cells. Cleavage, blastocyst formation and blastocyst hatching were recorded. Experiment 2: Co-Culture with Cumulus Versus Oviductal Epithelial Cells Cumulus cell monolayers and oviductal epithelial cell suspensions were prepared as described above. In each replicate 30 to 70 in vitro-fertilized oocytes pooled from pregnant and nonpregnant cows were co-cultured with each cell type. The number of oocytes varied by replicate depending on oocyte yield, but equal numbers were allocated to each co-culture cell type per replicate. Cleavage, blastocyst formation and blastocyst hatching were all recorded. Effect of Batch of Estrous Cow Serum A retrospective analysis of embryo cleavage and blastocyst formation was carried out for 2 periods that reflected use of different batches of estrous cow serum, July to December, 1990 versus January to July, 1991. Identical procedures for IVM, IVF and IVC were used over the 2 periods, except for the source of estrous cow serum. For each period, the respective estrous cow serum was used as supplement for oocyte maturation and embryo culture media. Oocytes included in this analysis were the same ones used to test effects of pregnancy status of the donor female and type of co-culture system in Experiments 1 and 2. Statistical Analysis The cleavage rate was calculated as the percentage of inseminated ova that cleaved. The percentage of ova that formed blastocysts and the percentage of blastocysts that hatched were calculated using both the total number of oocytes cultured and the number of ova that continued to develop. Student’s t-test was used to assess treatment differences in cleavage, blastocyst formation and blastocyst hatching. RESULTS Approximately 3,000 oocytes from pregnant cows and nearly 4,000 oocytes from nonpregnant cows were collected and cultured. Data were analyzed from 24 replicates, each representing a separate day of ovary and oocyte collection. Table 1 shows the
Theriogenolog
1081
y
numbers and proportions of oocytes that cleaved and developed into blastocysts and hatched blastocysts. Percentages for cleavage, blastocyst formation and blastocyst hatching were not different for pregnant versus nonpregnant cows (P > 0.05); in fact, values for pregnant versus nonpregnant cows were nearly identical, regardless of whether calculated on the basis of the total number of oocytes cultured or on the number of embryos that continued to develop. The percentage of total cultured oocytes that developed into blastocysts and hatched blastocysts was 5 and 396, respectively, for pregnant and nonpregnant cows. Yield of oocytes from nonpregnant cows tended to be higher than that from pregnant females, possibly due to physical limitations imposed on follicular growth on the ovary containing the corpus luteum of pregnancy, but this difference could not be assessed accurately due to collection by different individuals over time.
Table 1. In vitro maturation and fertilization of oocytes from pregnant and nonpregnant cows
Replicates
Number cultured oocytes
Number cleaved loocytes
Number blastocysts /2&l embryos
Number hatched blastocysts
Pregnant
24
3007
Nonpregnant
24
3983
1148/3007 (38%) 162513983 (40%)
15211148 (13%) 21211625 (13%)
801152 (53%) 109/212 (51%)
status
Results of co-culture of fertilized oocytes with oviductal epithekl cells or cumulus cells are summarized below (Table 2). More than 1,000 oocytes from 19 replicates were collected and co-cultured with each of the 2 cell types. The percentage of ova that cleaved or developed into blastocysts or hatched blastocysts was not different for the 2 co-culture systems (P > 0.05). The percentage of the total number of cultured oocytes that developed into blastccysts and hatched blastocysts was 4 and 22, respectively, for co-culture with cumulus cells and 5 and 3% for co-culture with oviductal epithelial cells. When data were analyzed retrospectively by 2 periods during which different sources of estrous cow serum were used to supplement the maturation and culture media, the percentage of blastocysts that developed (19 versus 8%) P < 0.05) over oviductal cell monolayers in Experiment 2 was different. The same difference was observed for ova cultured over cumulus cell monolayers in Experiment 2 and in comparisons of oocytes from pregnant versus nonpregnant cows in Experiment 1 (data not shown). In no comparison between the 2 periods was there a difference in the percentage of oocytes that cleaved or blastocysts that hatched when matured and cultured in medium supplemented
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y
with the 2 serum batches (e.g., 41 versus 3996, P > 0.05, and 57 versus 5596, P > 0.05, respectively, for oocytes cultured with oviductal epithelial cells).
Table 2. In vitro co-culture of bovine embryos with cumulus cells and oviductal epithelial cells
Replicates
Number cultured oocytes
Number cleaved /oocytes
Number blastocysts /2-cell embryos
Number hatched blastocysts
Cumulus cells
19
1262
Oviductal cells
19
1036
45311262 (3696) 413/1063 (40%)
481453 (11%) 481413 (12%)
24148 (50%) 27148 (56%)
Co-culture
DISCUSSION Methods for IVM, IVF and IVC of bovine oocytes differ among laboratories (3,4,9). These differences can complicate comparisons of results across laboratories and make selection of a particular procedure for practical use of IVM/IVF/IVC more difficult. A common feature of IVC procedures is the co-culture of embryos with other cells. Such co-culture systems support in vitro development of zygotes to the blastocyst stage at a higher frequency than occurs in the absence of co-culture. Two common co-culture systems for in vitro-fertilized bovine oocytes use oviductal epithelial cells or cumulus cells (3,4). We have shown that under controlled conditions equivalent development to the blastocyst stage can be obtained with the 2 co-culture systems. Viability of cultured blastocysts was not measured in this study; however, the 2 co-culture systems were tested in parallel in each replicate, and blastocysts that developed with the 2 monolayers were available for side-by-side comparisons. No differences in blastocyst quality were apparent when evaluated under phase contrast microscopy by an experienced individual. Recently, Thibodeaux et al. (10) showed that the stage of the estrous cycle when oviductal epithelial cells are collected affects cell morphology and developmental patterns during primary culture. Additional studies are needed to determine if an effect is also seen on the support of embryo development in co-culture. In vitro cleavage rates, blastocyst formation, and blastocyst hatching rates of cocytes from pregnant and nonpregnant cows were nearly identical, suggesting that the quality of oocytes from early-pregnant cows is comparable to that of oocytes collected from nonpregnant cows for IVM, IVF and IVC procedures. These data document the usefulness of both pregnant and nonpregnant cows as a source of oocytes for developmental and other studies. Our results also support the use of oocytes collected
1083
Theriogenology
during early pregnancy to enhance reproduction of genetically superior cows. Repeated transvaginal aspiration of follicular oocytes from cyclic cows has been proposed as a competitive alternative to conventional superovulation/embryo transfer procedures (11,12). Transvaginal aspiration of oocytes may be useful when applied to early-pregnant cows, which are not candidates for conventional embryo transfer procedures (6). Overall, the rates of cleavage, blastocyst formation and blastocyst hatching were similar to those reported by Goto et al. (4), but slightly lower than those reported by Eyestone and First (8) and Lu et al. (9). These differences may be related to the selection of high-quality oocytes for IVM as practiced by the latter 2 groups. We randomly assigned all aspirated oocytes to treatments without quality grading or selection. For most bovine IVM and IVC systems, media are supplemented with serum, often estrous cow serum. We observed what may have been a serum batch effect on the proportion of embryos that developed to the blastocyst stage, but not on the proportion of oocytes that completed the first cleavage division. Since this difference was observed in a retrospective analysis of combined data from 2 experiments designed to study other effects, variables in addition to batch of estrous cow serum (e.g., seasonal effects) may have contributed to observed differences; however, the effect was consistent across the 2 separate experiments. Other investigators have reported results that suggest the effects of serum batch/source on oocyte maturation and the first cleavage division are minor (13,14). The effect of serum source on in vitro embryo development is well established (15). Thus, the source of estrous cow serum may influence embryo development without affecting oocyte maturation and cleavage. If differences we observed were indeed due to the effect of serum batch, our results corroborate both that the serum source can account for differences in the development observed in in vitro systems and that there is a need for a chemically-defined medium that adequately supports in vitro embryo development. REFERENCES 1.
Leibfried-Rutledge, M.L., Critser, E.S., Parrish, J.J. and First, N.L. maturation and fertilization of bovine oocytes. Theriogenology fi:61-74
In vitro (1989).
2.
Gordon, I. and Lu, K.H. Production of embryos in vitro and its impact on livestock production. Theriogenology %:77-87 (1990).
3.
Eyestone, W.H., Vignieri, J. and First, N.L. Co-culture of early bovine embryo with oviductal epithelium. Theriogenology Z288 abstr. (1987).
4.
Goto, K., Kajihara, Y., Kosaka, S., Koba, M., Nakanishsi, Y. and Ogawa, K. Pregnancies after co-culture of cumulus cells with bovine embryos derived from invitro fertilization of in-vitro matured follicular oocytes. J. Reprod. Fertil. B:753758 (1988).
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5.
Xu, K.P., Pollard, J. W., Rorie, R. W., Plante, L., King, W.A. and Betteridge, K.J. Pregnancy rate following transfer of bovine embryos produced by in vitro maturation, fertilization and co-culture. Theriogenology a!:35 abstr. (1990).
6.
Ryan, D.P., Blakewood, E.G., Swanson, W.F., Rodrigues, H. and Godke, R.A. The use of follicle stimulating hormone (FSH) to stimulate follicle development for in vitro fertilization during the first trimester of pregnancy in cattle. Theriogenology &j:315 abstr. (1990).
7.
Parrish, J.J., Susko-Parrish, J.L., Winer, M.A. and First, N.L. Capacitation of bovine spermatozoa by heparin. Biol. Reprod. j&1171-1180 (1988).
8.
Eyestone, W.H. and First, N.L. Co-culture of early cattle embryos to the blastocyst stage with oviductal tissue or in conditioned medium. J. Reprod. Fertil. &5:715-720 (1989).
9.
Lu, K.H., Gordon, I., Gallagher, M. and McGovern, M. Pregnancy established in cattle by transfer of embryos derived from in vitro fertilization of oocytes matured in vitro. Vet. Rec. 121:259-260 (1980).
10. Thibodeaux, J.K, Goodeaux, L.L., Roussel, J.D., Menezo, Y., Amborski, G.F., Moreau, J.D. and Godke, R.A. Effects of stage of the bovine oestrous cycle on invitro characteristics of uterine and oviductal epithelial cells. Human Reprod. &751760 (1991). 11. Pieterse, M.C., Vos, P.L.A.M., Kruip, Th.A.M., Wurth, Y.A., van Beneden, Th.H., Willemse, A.H. and Taveme, M.A.M. Transvaginal ultrasound guided follicular aspiration of bovine oocytes. Theriogenology Z: 19-24 (1991). 12. Pieterse, M.C., Vos, P.L.A.M., Kruip, Th.A.M., Willemse, A.H. and Taveme, M.A.M. Characteristics of bovine cycles during repeated transvaginal, ultrasoundguided puncturing of follicles for ovum pick-up. Theriogenology 3:401-413 (1991). 13. Younis, A.I., Brackett, B.G. and Fayrer-Hosken, R.A. hormones on bovine oocyte maturation and fertilization a: 189-201 (1989).
Influence of serum and in vitro. Gamete Res.
14. Sanbuissho, A. and Threlfall, W.R. The influence of serum and gonadotropins on in vitro maturation and fertilization of bovine oocytes. Theriogenology j&341-348 (1990). 15. Matsuoka, K., Sakata, S., Ichino, K., ShiMaya, Y. and Suzuki, T. Effect of superovulated cow serum for culture of bovine oocytes to the blastocyst stage. Theriogenology X:254 abstr. (1992).
y 38: 1077-I
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DEVELOPMENT OF IN VITRO FERTILIZED OOCYTES FROM PREGNANT AND NONPREGNANT COWS IN OVIDUCTAL EPITHELIAL AND CUMULUS CELL CO-CULTURE SYSTEMS E. Behboodi,’ G.B. Anderson’ and R.H. BonDurant2 Department of Animal Science’ and Department of Vet Med Reproduction2 University of California, Davis, CA 95616 Received for publication: February 25, 1992 Accepted: September 12, 1992 ABSTRACT The objectives of this study were 1) to measure cleavage, blastocyst formation, and blastocyst hatching after in vitro maturation (IVM), fertilization (IVF) and culture (IVC) of oocytes aspirated from pregnant versus nonpregnant cows, and 2) to compare embryo development in co-culture with bovine oviductal epithelial cells versus cumulus cells. No differences in cleavage (38 versus 40%), blastocyst formation (13 versus 13%), or blastocyst hatching (53 versus 51%) were observed for in vitro-matured, fertilized, and cultured oocytes from pregnant versus nonpregnant cows, respectively (P > 0.05), indicating that nonpregnant and early-pregnant cows are equally acceptable donors of oocytes for IVM/IVF/IVC procedures. Cleavage (36 versus 40%), blastocyst formation (11 versus 12%), and blastocyst hatching (50 versus 55 W) were not different for embryos co-cultured with oviductal epithelial cells versus cumulus cells (P > 0.05). Thus, equivalent embryo development can be obtained with co-culture systems commonly used for in vitro-derived bovine embryos. These results help to define variables that affect comparison of results across laboratories and that are relevant to the practical application of IVM/IVF/IVC procedures to cattle. Key words:
bovine, embryos, in vitro maturation,
in vitro fertilization
INTRODUCTION Procedures for oocyte in vitro maturation (IVM), fertilization (IVF) and culture (IVC) have become valuable techniques for the production of bovine embryos used in developmental studies and for use with embryo transfer to produce offspring from genetically superior females (1,2). Numerous variations in IVM/IVF/IVC procedures have been used by different laboratories; these variables complicate both the comparison of results obtained by different investigators and the selection of procedures for in vitro Acknowledgments This research was supported by funds from the California Dairy Foods Research Center and the California Milk Advisory Board. Semen was donated by Landmark Genetics, Hughson, CA. The assistance of undergraduate researchers Kirby Tran, Kathryn Makris, Sham Garabetian, and Carmine Bausone is acknowledged.
Copyright 0 1992 Butterworth-Heinemann
Thet-iogenology
generation of bovine embryos. Among variations in procedures used by different investigators are co-culture systems to support post-fertilization embryo development and pregnancy status of donor females. Oviductal epithelial cells (3) and cumulus cells (4) are commonly used in co-culture with in vitro-matured, in vitro-fertilized bovine oocytes to support development to the blastocyst stage. The birth of live calves has been reported from blastocysts that were co-cultured with cumulus cells (4) and oviductal epithelial cells (5), but information from a direct comparison of embryo development in these 2 coculture systems is not available. The harvesting of large numbers of bovine oocytes from ovaries obtained at slaughter often yields oocytes from both pregnant and nonpregnant females. Differences in follicular growth due to pregnancy status could affect oocyte quality. Furthermore, transvaginal collection of oocytes from cows during gestation has been proposed as a means by which to enhance reproduction in genetically superior females (6). This study was carried out to compare embryo development in co-culture with oviductal epithelial cells versus cumulus cells. We also examined oocyte maturation, fertilization, and development of oocytes from nonpregnant versus pregnant cows. Variable embryo development in media supplemented with different batches of estrous cow serum was detected by retrospective analysis of data collected during the course of this study. MATERIALS
AND METHODS
Collection of Oocytes Bovine ovaries were collected at NaCl) containing pecicillin (500 &ml) the laboratory at 25 C within 3 hours. with an 1&gauge needle from. follicles temperature between 25 to 30 C.
a local slaughterhouse into sterile saline (0.9 % and streptomycin (500 &ml) and transported to Immature oocytes were aspirated into a syringe having a diameter of 1 to 6 mm in a room with
Culture of Oocytes for In Vitro Maturation Oocytes were washed in Medium 199 (Sig_ma, St. Louis, MO) supplemented with 10% estrous cow serum (heat-inactivated at 56 C for 30 minutes) and 1 N/ml 178estradiol. They were then transferred (50 oocytes/dish) to a 60 x 15-mm organ tissue culture dish (Fisher, Pittsburgh, PA) containing 1 ml Medium 199 with 10% estrous cow serum and 1 &ml 170-estradiol, and the culture medium was covered with 0.5 ml sterile paraffin oil (Sigma). Oocytes were divided randomly among culture dishes_without oocyte quality grading or selection and were incubated for 24 hours at 39 C in a humidified atmosphere of 5% CO, and 95% air. To prepare estrous cow serum, blood was collected by jugular venipuncture from 4 estrous dairy cows. Sera were separated after clotting, filtered (0.22 ,um pore sire), heat-inactivated (56°C for 30 minutes) and pooled prior to storage at -20°C until use.
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Theriogenology
Sperm Preparation A method described by Parrish et al. (7) was used to prepare bovine spermatozoa for IVF. Commercially distribute+ frozen semen from 3 dairy bulls (Landmark Genetics, Hughson, CA) was thawed in 37 C water and placed in 1 ml of Sperm-TALP medium (7) supplemented with 5 &ml heparin (Sigma); suspended thawed spermatozoa we5 then aliquoted into 5 tubes (0.2 ml/ tube) for each bull and incubated for 1 hour at 39 C in 5% CO, and 95% air for sperm swim-up (3). The swim-up spermato+ were washed once by centrifugation at 700 x g for 6 minutes and then incubated at 39 C in 5% CO, and 95% air for an additional 40 minutes before insemination. Co-Incubation
of Gocytes with Spermatozoa and Embryo Culture
The method used was similar to that described by Eyestone and First (8). Briefly, droplets (100 3 each) of Fert-TALP medium (7) were placed on the bottom of a 35 x lomm easy grip tissue culture dish (Fisher) and covered with 1 ml sterile paraffin oil. In vitro-matured oocytes were washed 3 times and transferred (50/droplet) to the fertilization me$um. Spermatozoa (3 x 104/oocyte) were co-incubated with oocytes for 18 hours at 39 C. Ova were then transferred to 1 ml of Medium 199 supplemented with 10% heatinactivated estrous cow serum and 1 &ml 17R-estradiol and co-cultured with either oviductal epi?elial cells or cumulus cells in a humidified atmosphere of 5% CO, and 95% air at 39 C. Embryos were examined daily under a dissecting microscope. For each culture plate the number of ova that cleaved to the 2-cell stage and the numbers of blastocysts that formed and hatched from the zona pellucida were recorded. Oviductal Epithelial Cell Preparation Oviducts were collected from cows and heifers at slaughter, and epithelial cells were stripped from the oviductal lumen as described by Eyestone and First (8). An effort was made to collect oviductal cells from animals that had recently ovulated, as indicated by the presence of a corpus hemorrhagicurn on one of the ovaries, and cells from 3 or 4 animals were pooled. Epithelial cells were cultured in a 60 x 15-mm organ tissue culture dish (Fisher) for 48 hours in Medium 199 supplemented with 10% estrous cow serum and 1 &ml 17Kestradiol. Clumps of oviductal cells were dissociated by gentle pipetting. Approximately 50 ~1 of the oviductal cell suspension were added to each l-ml drop of culture medium containing the embryos. A mixed oviductal population of ciliated vesicles and monolayer developed over time. Cumulus Cell Monolayer Preparation Cumulus cell cultures cumulus cells were removed by vortexing for 30 seconds. to 20 oocytes for each 60 x Medium 199 supplemented
were prepared as described by Goto et al. (4). Briefly, from randomly-selected oocytes 24 hours after fertilization Denuded oocytes were discarded. Cumulus cells from 15 15-mm organ tissue culture dish (Fisher) were cultured in with 10% estrous cow serum and 1 &ml l’l&estradiol.
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Theriogenology
Cumulus cells attached to the bottom of the culture dish and formed a monolayer after 72 to 96 hours. Embryos were cultured on this monolayer. Experiment 1: Cleavage and Blastocyst Formation Nonpregnant Cows
for Oocytes from Pregnant and
Ovaries from pregnant cows and heifers were collected from females between 40 days and 5 months gestation, as determined by fetal crown-rump length. Ovaries from nonpregnant cows were collected without regard to stage of the ovarian cycle. Oocytes from pregnant and nonpregnant females were fertilized with semen from the same bulls, and after fertilization were co-cultured with oviductal epithelial cells. Cleavage, blastocyst formation and blastocyst hatching were recorded. Experiment 2: Co-Culture with Cumulus Versus Oviductal Epithelial Cells Cumulus cell monolayers and oviductal epithelial cell suspensions were prepared as described above. In each replicate 30 to 70 in vitro-fertilized oocytes pooled from pregnant and nonpregnant cows were co-cultured with each cell type. The number of oocytes varied by replicate depending on oocyte yield, but equal numbers were allocated to each co-culture cell type per replicate. Cleavage, blastocyst formation and blastocyst hatching were all recorded. Effect of Batch of Estrous Cow Serum A retrospective analysis of embryo cleavage and blastocyst formation was carried out for 2 periods that reflected use of different batches of estrous cow serum, July to December, 1990 versus January to July, 1991. Identical procedures for IVM, IVF and IVC were used over the 2 periods, except for the source of estrous cow serum. For each period, the respective estrous cow serum was used as supplement for oocyte maturation and embryo culture media. Oocytes included in this analysis were the same ones used to test effects of pregnancy status of the donor female and type of co-culture system in Experiments 1 and 2. Statistical Analysis The cleavage rate was calculated as the percentage of inseminated ova that cleaved. The percentage of ova that formed blastocysts and the percentage of blastocysts that hatched were calculated using both the total number of oocytes cultured and the number of ova that continued to develop. Student’s t-test was used to assess treatment differences in cleavage, blastocyst formation and blastocyst hatching. RESULTS Approximately 3,000 oocytes from pregnant cows and nearly 4,000 oocytes from nonpregnant cows were collected and cultured. Data were analyzed from 24 replicates, each representing a separate day of ovary and oocyte collection. Table 1 shows the
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numbers and proportions of oocytes that cleaved and developed into blastocysts and hatched blastocysts. Percentages for cleavage, blastocyst formation and blastocyst hatching were not different for pregnant versus nonpregnant cows (P > 0.05); in fact, values for pregnant versus nonpregnant cows were nearly identical, regardless of whether calculated on the basis of the total number of oocytes cultured or on the number of embryos that continued to develop. The percentage of total cultured oocytes that developed into blastocysts and hatched blastocysts was 5 and 396, respectively, for pregnant and nonpregnant cows. Yield of oocytes from nonpregnant cows tended to be higher than that from pregnant females, possibly due to physical limitations imposed on follicular growth on the ovary containing the corpus luteum of pregnancy, but this difference could not be assessed accurately due to collection by different individuals over time.
Table 1. In vitro maturation and fertilization of oocytes from pregnant and nonpregnant cows
Replicates
Number cultured oocytes
Number cleaved loocytes
Number blastocysts /2&l embryos
Number hatched blastocysts
Pregnant
24
3007
Nonpregnant
24
3983
1148/3007 (38%) 162513983 (40%)
15211148 (13%) 21211625 (13%)
801152 (53%) 109/212 (51%)
status
Results of co-culture of fertilized oocytes with oviductal epithekl cells or cumulus cells are summarized below (Table 2). More than 1,000 oocytes from 19 replicates were collected and co-cultured with each of the 2 cell types. The percentage of ova that cleaved or developed into blastocysts or hatched blastocysts was not different for the 2 co-culture systems (P > 0.05). The percentage of the total number of cultured oocytes that developed into blastccysts and hatched blastocysts was 4 and 22, respectively, for co-culture with cumulus cells and 5 and 3% for co-culture with oviductal epithelial cells. When data were analyzed retrospectively by 2 periods during which different sources of estrous cow serum were used to supplement the maturation and culture media, the percentage of blastocysts that developed (19 versus 8%) P < 0.05) over oviductal cell monolayers in Experiment 2 was different. The same difference was observed for ova cultured over cumulus cell monolayers in Experiment 2 and in comparisons of oocytes from pregnant versus nonpregnant cows in Experiment 1 (data not shown). In no comparison between the 2 periods was there a difference in the percentage of oocytes that cleaved or blastocysts that hatched when matured and cultured in medium supplemented
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y
with the 2 serum batches (e.g., 41 versus 3996, P > 0.05, and 57 versus 5596, P > 0.05, respectively, for oocytes cultured with oviductal epithelial cells).
Table 2. In vitro co-culture of bovine embryos with cumulus cells and oviductal epithelial cells
Replicates
Number cultured oocytes
Number cleaved /oocytes
Number blastocysts /2-cell embryos
Number hatched blastocysts
Cumulus cells
19
1262
Oviductal cells
19
1036
45311262 (3696) 413/1063 (40%)
481453 (11%) 481413 (12%)
24148 (50%) 27148 (56%)
Co-culture
DISCUSSION Methods for IVM, IVF and IVC of bovine oocytes differ among laboratories (3,4,9). These differences can complicate comparisons of results across laboratories and make selection of a particular procedure for practical use of IVM/IVF/IVC more difficult. A common feature of IVC procedures is the co-culture of embryos with other cells. Such co-culture systems support in vitro development of zygotes to the blastocyst stage at a higher frequency than occurs in the absence of co-culture. Two common co-culture systems for in vitro-fertilized bovine oocytes use oviductal epithelial cells or cumulus cells (3,4). We have shown that under controlled conditions equivalent development to the blastocyst stage can be obtained with the 2 co-culture systems. Viability of cultured blastocysts was not measured in this study; however, the 2 co-culture systems were tested in parallel in each replicate, and blastocysts that developed with the 2 monolayers were available for side-by-side comparisons. No differences in blastocyst quality were apparent when evaluated under phase contrast microscopy by an experienced individual. Recently, Thibodeaux et al. (10) showed that the stage of the estrous cycle when oviductal epithelial cells are collected affects cell morphology and developmental patterns during primary culture. Additional studies are needed to determine if an effect is also seen on the support of embryo development in co-culture. In vitro cleavage rates, blastocyst formation, and blastocyst hatching rates of cocytes from pregnant and nonpregnant cows were nearly identical, suggesting that the quality of oocytes from early-pregnant cows is comparable to that of oocytes collected from nonpregnant cows for IVM, IVF and IVC procedures. These data document the usefulness of both pregnant and nonpregnant cows as a source of oocytes for developmental and other studies. Our results also support the use of oocytes collected
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during early pregnancy to enhance reproduction of genetically superior cows. Repeated transvaginal aspiration of follicular oocytes from cyclic cows has been proposed as a competitive alternative to conventional superovulation/embryo transfer procedures (11,12). Transvaginal aspiration of oocytes may be useful when applied to early-pregnant cows, which are not candidates for conventional embryo transfer procedures (6). Overall, the rates of cleavage, blastocyst formation and blastocyst hatching were similar to those reported by Goto et al. (4), but slightly lower than those reported by Eyestone and First (8) and Lu et al. (9). These differences may be related to the selection of high-quality oocytes for IVM as practiced by the latter 2 groups. We randomly assigned all aspirated oocytes to treatments without quality grading or selection. For most bovine IVM and IVC systems, media are supplemented with serum, often estrous cow serum. We observed what may have been a serum batch effect on the proportion of embryos that developed to the blastocyst stage, but not on the proportion of oocytes that completed the first cleavage division. Since this difference was observed in a retrospective analysis of combined data from 2 experiments designed to study other effects, variables in addition to batch of estrous cow serum (e.g., seasonal effects) may have contributed to observed differences; however, the effect was consistent across the 2 separate experiments. Other investigators have reported results that suggest the effects of serum batch/source on oocyte maturation and the first cleavage division are minor (13,14). The effect of serum source on in vitro embryo development is well established (15). Thus, the source of estrous cow serum may influence embryo development without affecting oocyte maturation and cleavage. If differences we observed were indeed due to the effect of serum batch, our results corroborate both that the serum source can account for differences in the development observed in in vitro systems and that there is a need for a chemically-defined medium that adequately supports in vitro embryo development. REFERENCES 1.
Leibfried-Rutledge, M.L., Critser, E.S., Parrish, J.J. and First, N.L. maturation and fertilization of bovine oocytes. Theriogenology fi:61-74
In vitro (1989).
2.
Gordon, I. and Lu, K.H. Production of embryos in vitro and its impact on livestock production. Theriogenology %:77-87 (1990).
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Eyestone, W.H., Vignieri, J. and First, N.L. Co-culture of early bovine embryo with oviductal epithelium. Theriogenology Z288 abstr. (1987).
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Goto, K., Kajihara, Y., Kosaka, S., Koba, M., Nakanishsi, Y. and Ogawa, K. Pregnancies after co-culture of cumulus cells with bovine embryos derived from invitro fertilization of in-vitro matured follicular oocytes. J. Reprod. Fertil. B:753758 (1988).
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Xu, K.P., Pollard, J. W., Rorie, R. W., Plante, L., King, W.A. and Betteridge, K.J. Pregnancy rate following transfer of bovine embryos produced by in vitro maturation, fertilization and co-culture. Theriogenology a!:35 abstr. (1990).
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Ryan, D.P., Blakewood, E.G., Swanson, W.F., Rodrigues, H. and Godke, R.A. The use of follicle stimulating hormone (FSH) to stimulate follicle development for in vitro fertilization during the first trimester of pregnancy in cattle. Theriogenology &j:315 abstr. (1990).
7.
Parrish, J.J., Susko-Parrish, J.L., Winer, M.A. and First, N.L. Capacitation of bovine spermatozoa by heparin. Biol. Reprod. j&1171-1180 (1988).
8.
Eyestone, W.H. and First, N.L. Co-culture of early cattle embryos to the blastocyst stage with oviductal tissue or in conditioned medium. J. Reprod. Fertil. &5:715-720 (1989).
9.
Lu, K.H., Gordon, I., Gallagher, M. and McGovern, M. Pregnancy established in cattle by transfer of embryos derived from in vitro fertilization of oocytes matured in vitro. Vet. Rec. 121:259-260 (1980).
10. Thibodeaux, J.K, Goodeaux, L.L., Roussel, J.D., Menezo, Y., Amborski, G.F., Moreau, J.D. and Godke, R.A. Effects of stage of the bovine oestrous cycle on invitro characteristics of uterine and oviductal epithelial cells. Human Reprod. &751760 (1991). 11. Pieterse, M.C., Vos, P.L.A.M., Kruip, Th.A.M., Wurth, Y.A., van Beneden, Th.H., Willemse, A.H. and Taveme, M.A.M. Transvaginal ultrasound guided follicular aspiration of bovine oocytes. Theriogenology Z: 19-24 (1991). 12. Pieterse, M.C., Vos, P.L.A.M., Kruip, Th.A.M., Willemse, A.H. and Taveme, M.A.M. Characteristics of bovine cycles during repeated transvaginal, ultrasoundguided puncturing of follicles for ovum pick-up. Theriogenology 3:401-413 (1991). 13. Younis, A.I., Brackett, B.G. and Fayrer-Hosken, R.A. hormones on bovine oocyte maturation and fertilization a: 189-201 (1989).
Influence of serum and in vitro. Gamete Res.
14. Sanbuissho, A. and Threlfall, W.R. The influence of serum and gonadotropins on in vitro maturation and fertilization of bovine oocytes. Theriogenology j&341-348 (1990). 15. Matsuoka, K., Sakata, S., Ichino, K., ShiMaya, Y. and Suzuki, T. Effect of superovulated cow serum for culture of bovine oocytes to the blastocyst stage. Theriogenology X:254 abstr. (1992).