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Effect of removing the zona pellucida on development of hamster and bovine embryos in vitro and in vivo
THERIOGENOLOGY EFFECT OF REMOVING THE ZONA PELLUCIDA ON DEVELOPMENT OF HAMSTER AND BOVINE EMBRYOS --IN VITRO AND --IN VIVO Robert W. Hoppea and Barry D. Bavister Department of Veterinary Science, University of Wisconsin, Madison, Wisconsin 53706 aPresent address:
Dreamstreet Holsteins, Inc., Walton, N.Y. 13856
Box 209,
Received for publication: accepted:
September 3, 1982 November 30, 1982
ABSTRACT Uterine stage embryos collected from the hamster (8-cell) and cow (morula, early blastocyst) wre monitored for development -in vitro (embryo culture) and embryo transfer) following premature removal of the zona Removal of the zona pellucida did not significantly affect -in vitro development to the blastocyst stage of (1) 8-cell hamster embryos (zonae removed by a combined enzymic-mechanical procedure), (2) bovine morulae (zonae removed by mechanical means only) (3) early bovine blastocysts (zonae removed by the enzymic-mechanical technique). Zona-free hamster enbryos formed significantly fewer viable fetuses than did zona-intact embryos. The lower incidence of fetal development observed following transfer of zona-free 8-cell hamster embryos may have resulted in part from the formation of chimeras by fusion of these embryos -in utero. Such fusion was observed to occur in vitro between zona-free embryos placed in close proximity. The proEr=of pregnancies resulting from transfer of bovine blastocysts cultured from zona-free morulae was similar to that of zona-intact embryos. In this study w have demonstrated that (1) enzymic and mechanical procedures used to remove zonae pellucidae from uterine-stage hamster and bovine enbrvos do not adversely affect subseauent development of these embryos in vitro and in viva-and (2) zonae'pellucidae are not reauired for nfiivelo$&t these embryos. These findings have implications for microsurgery of mammalian embryos and for embryo transfer.
Acknowledgements: Supported by the Wisconsin Agricultural Experiment Station, College of Agricultural and Life Sciences, University of Wisconsin-Madison. We are indebted to: The Upjohn Co., Kalamazoo, Michigan for generous supplies of PGF2=, "Lutalyse"; American Breeders Service, De Forest, Wisconsin for supplying frozen semen; and IMV International Corp., Minneapolis, Minnesota for donating the equipment used for the bovine non-surgical embryo transfers.
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THERIOGENOLOGY INTRODUCTION Mammalian embryos from a number of species can survive and continue to develop following premature removal of the zona pellucida. Removal of the zona pellucida makes the pre-implantation embryo more accessible for microsurgical procedures such as bisection or manipulation of individual blastomeres. However, for this approach to be useful, it is essential to know how wall zona-free embryos develop -in vitro and in vivo compared with their zona-intact counterparts. -A variety of methods have been used to remove zonae pellucidae from embryos at all stages of developent from the zygote to blastocyst. Methods used have been chemical (1,2,3), mechanical (4,5), or a combination of the two (6,7). lhe ability of embryos artificially freed from their zonae pellucidae to continue cleavage and to form normal fetuses after transfer to recipients has varied widely, depending on species and stage of embryo used (2,6,8,9) and the method of zona removal (10,ll). Wlether the problem lies with the method of zona removal, with some need for the presence of the zona by the early embryo, with the transfer of zona-free embryos to recipients or some combination of these factors is not clear. The ability to obtain normal embryonic development following zona removal and microsurgery would be particularly useful in the cow, since procedures for embryo sexing and cloning could then be more easily developed. The present study was undertaken to achieve the following objectives: 1.
Evaluate procedures for zona pellucida removal, using uterinestage hamster embryos as a model system to be adapted for use in the cow.
2.
Assess embryo developmental potential in vitro and in vivo subsequent to removal of the zona by e?@iiiiind mexam methods, or by mechanical means alone.
3.
Compare survival rates -in vitro and -in vivo for zona-intact and zona-free hamster and cow embryos recovered from the uterus. MATERIALS AND METHODS
Animals Male and female hamsters ware housed under controlled temperature and photoperiod (14 light:10 dark, with li#ts on at 0600 h). Estrous cycles were monitored by daily observation of vaginal discharges (12). Holstein-Friesian heifers were used for the work on bovine embryos, Donors were not used as recipients. Recipients that were found to be non-pregnant 60 days following embryo transfer were reassigned to a different transfer experiment. These animals were kept at Bookhout Farm, Arlington, Wisconsin. This facility is maintained by the Agricultural Experiment Station of the University of Wisconsin-Madison
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College of Agricultural and Life Sciences. Cattle were housed in two free-stall pole barns which were open on the south side. One group consisted of open recipients and potential donors. Another group consisted of recently used donors and recipients into which an embryo had been transferred within the last 60 days. Superovulation and Insemination Superovulation of cycling hamsters was initiated the morning following estrus (estrus = day 0), using 15-20 I.U. of PMSG (Organon, West Orange, N.J.) injected intraperitoneally. Seventy-two hours later, lo-15 I.U. of HCG (Sigma, St. Louis, MO) was administered and the animals were bred that evening to intact males. Toward the end of the study, it was found that superovulated donor hamsters yielded embryos of similar quantity and quality regardless of tieetheror not they were given HCG to induce ovulation. All donor heifers were superovulated with FSH-P (Burns-Biotec, Omaha, NE) starting lo-12 days after estrus (estrus = day 0). Gonadotrophin injections were given twice daily for 5 days, for a total of 50 mgs. Seventy-two hours after the initial gonadotrophin injection, a luteolytic dose of PGF2 (25-30 mg) was administered. PGF2 was dinoprost tromethamine ?"Lutalyse", Upjohn Co., Kalamazoo, 141). Donors were inseminated with one straw of semen at 60 hours and again at 72 hours following PGF2a. Embryo Collection Uteri were excised from donor hamsters between 1500 and 1600 h on day 3 and flushed with l-2 ml of a Hepes-buffered, modified Tyrode's solution (TALP) containing glucose, lactate, pyruvate and 3 mg/ml Fraction V bovine serum albumin (BSA)(13). Flushing was accomplished with a 30 gauge needle attached to a 3 cc syringe. Flushings were collected in 35x10 mm plastic petri dishes (Falcon ~1008). Embryos were recovered and rinsed 2 or 3 times and held at room temperature in TALP-Hepes. Bovine embryos were recovered non-surgically from the uterine horns of,,donorheifers on day 6 or 7, utilizing a balloon-type catheter (Rusch, W. Germany) and a small-volume aspiration technique (14). The embryo recovery medium was sterile, modified Dulbecco's phosphatebuffered saline (15) containing 3 mg/ml BSA (Sigma), 100 I.U./ml of penicillin and 0.05 mg/ml of streptomycin. Uterine flushings (300-400 ml/horn) were collected in gas leveling bulbs (Corning Glass Works, Corning, NY) and transported back to the laboratory in a Styrofoam box that maintained the temperature of the medium at 25-30°C. The contents of the bulbs were transferred to sterile, siliconized glass evaporating dishes (Corning Glass Works). Embryos were removed from the dishes, rinsed 3 times in fresh medium and held at room temperature (22-24OC).
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THERIOGENOLOGY Removal of the Zona Pellucida Enaymic
and mechanical
The zonae pellucidae of hamster 8-cell and early blastocyst (day 7) cow embryos ware removed by first incubating then in a fresh, sterile solution of 2.5 mg/ml (hamster) or 5 mg/ml (cow) pronase (Sigma) prepared in TALP-Hepes medium. Embryos were continuously observed under a stereomicroscope until the zonae had noticeably thinned or ballooned or both. The time was recorded for partial digestion of the zona pellucida. The embryos were then pipetted in the rinsing solution (TALP-Hepes) until the partially-digested zonae were removed. Mechanical Bovine morulae (day 6) had their zonae pellucidae removed by the use of two glass microneedles, each mounted on a simple, hand-held micromanipulator (Figure 1). Left and right micromanipulators were attached to the glass plate of the stereomicroscope stage by a thin
EPOXY ADAPTOR
FOR
/
MICRONEEDLE MOVA sBLE -
GLASS
GLASS
SLIDE
ZPOXY
MICRONEEDLE (DETACHABLE)
Fig. 1. Hand-held glass micromanipulator. Each micromanipulator was fashioned out of 3 glass microscope slides according to a modification of a design outlined by Goldacre (16). The glass needles were pulled from 3 ~1 micropipettes (Drunmond Scientific, Broomall, PA) on a vertical pipette puller so that the shank diameter at the tip of the needle averaged 10-15 nrn. The needles were inserted and fixed with melted paraffin wax into the end of a bent piece of glass tubing cemented onto the movable vertical glass slide. The fixed vertical slide was attached perpendicularly to the base slide by a bead of epoxy glue. Stopcock grease was used between the moving glass surfaces.
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Fig. 2. Left and right micromanipulators in place on glass microscope base. All manipulations were performed under oil within a droplet of phosphate-buffered medium. layer of stopcock grease, facilitating movement in the horizontal plane (Figure 2). All manipulations were performed in small plastic petri dishes within a droplet of medium under oil. Hile one needle steadied the embryo, the other was used to make a series of connecting slits along the equator of its zona pellucida. Then, using both needles, the edges of the zona along the slit were everted and the embryo removed either by gravity or suction. Viability Assays In _- vitro
using
embryo culture
Eight-cell hamster embryos were cultured for 24 hours in a bicarbonate-buffered, modified Tyrode's solution (TALP: ref 17) containing 3 mg/ml BSA and 4 amino acids (13). Cow embryos collected on day 6 or 7 were cultured for 24 hours in a similar manner except that 15 mg/ml BSA was used, without amino acids. Embryos ware incubated in droplets containing 65-70 ul of culture medium placed under a layer of paraffin oil in 60 x 15 mm plastic petri dishes (Falcon 11007). The dishes containing the droplets were pre-equilibrated at 37°C in a waterjacketed incubator rhich automatically maintained a humidified, 5% CO2 in air mixture.
In vitro growth of both hamster and cow embryos was monitored for -development to the blastocyst stage every 18-24 hours. Embryos were examined within their culture droplets using an Olympus stereomicroscope
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THERIOGENOLOGY at 80-120X magnification. The developmental stage, condition, relative size and morphology of each embryo were all recorded. ~n -- vivo using embryo transfer Recipient hamsters (day 3 of pseudopregnancy) were anesthetized with Nembutal given intraperitoneally. A paralumbar incision was made just behind the last rib and the uterus retracted by means of a clamp on the ovarian fat pad. Embryos were introduced directly into the uterine lumen by means of a hand-drawn Pasteur pipette connected to a mouthtube. Numbers of viable and of resorbing fetuses were estimated at autopsy on day 10 of pregnancy. In a preliminary study designed to evaluate culture conditions, bovine embryos collected on day 7 (early blastocyst stage) were cultured for 24 hours, then transferred surgically via a standing flank laparotomy. Although the percentage of successful transfers was high (5/7 recipients or 71% became pregnant after transfer of single embryos), we desided that subsequent transfers would be performed non-surgically for practical reasons. A zona-intact or zona-free blastocyst tiich had been cultured from a morula was aspirated into a 0.25 ml plastic insemination straw so that the middle one-third of the straw containing the embryo was isolated by twD air bubbles from the remaining column of medium. At the transfer facility, the recipient heifer was palpated, given an epidural anesthetic, and the vulvar lips were washed and dried. The transfer gun, tiich holds the straw, was then manipulated per rectum through the cervix and into the uterine horn ipsilateral to the corpus luteum-bearing ovary (18). A single embryo was gently deposited 2-5 cm past the internal uterine bifurcation. Bovine embryo recipients were examined for pregnancy by rectal palpation at 50-60 days. Some pregnant heifers were reexamined as late as 240 days of gestation. Experimental design and statistical analysis Ranwter embryos Hamster embryos were collected from 4-6 superovulated donors per replicate, pooled, and randomly divided into two equal groups. Embryos in the treatment group had their zonae removed using the enzymicmechanical technique. Control embryos *re treated in the same way as the experimental group except that their zonae were not removed. Ten to 12 embryos were placed in each droplet of mediun. Within a culture dish, the number of droplets with zona-free embryos was equal to the number containing zona-intact embryos. Data collected over six replicates ware analyzed using a 3-way test for independence (19). Alternatively, hamster embryos were transferred to recipient females instead of being cultured. The double cervix of the hamster precludes embryo crossover between the uterine horns, permitting viability of control and treated embryos to be tested in the same recipient. From 9-11 (usually 10) zona-free embryos were transferred to one uterine horn of a recipient and the same number of zona-intact embryos to the other horn. Analysis was performed on these data using the t-test for paired samples (19).
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Cow
embryos
Bovine embryos were randomly assigned to a zona-intact control group or a zona-free experimental group. After manipulation and rinsing, one embryo (zona-intact or zona-free) was placed in each droplet of culture medium, and therefore, was considered a single data point. Embryos were observed for development to the blastocyst after 24 hours in culture. In a preliminary study, early blastocysts (day 7) were denuded by means of the enzymic-mechanical technique. Control and experimental embryos were cultured, but not transferred. For the main investigation, morulae (day 6) had their zonae removed using the mechanical technique. Zona-intact and zona-free embryos which developed to blastocysts after 24 hours in culture were transferred to recipients. Data generated in vitro and --in vivo were analyzed using the chi-squared test for indepe&n-@-(i9).
.’
Fig. 3A. Hamster zona-free, 8-cell embryos. by enzymic-mechanical procedure.
Zonae pellucidae removed
Fig. 38. Hamster zona-free, early blastocysts cultured (24 h) from zona-free 8-cell embryos.
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THERIOGENOLOGY RESULTS Hamster
In vitro Growth of &cell embryos to the blastocyst stage in culture was unaffected by a combined enzymic-mechanical technique used to remove the zona pellucida. Representative zona-free embryos before and after culture are shown in Figure 3. The results obtained by culturing hamster zona-intact and zona free embryos are shown in Table 1. Table 1.
Effect of removing the zona pellucida of hamster 8-cell embryos on subsequent development to blastocyst -in vitro. Status of embryo Zona-intact (control)
No. embryos cultured No. embryos developing into blastocysts (%)
149
Zona-free 158
68 (46%)
69 (44%)
Data are total of 6 replicates. There was no significant difference between the zona-intact group and the zona-free experimental group. About 15% of the denuded embryos formed chimeras, i.e., two embryos fused to form one large one. The time required for partial digestion of the zona pellucida in a 0.25% solution of pronase ranged from 5-10 seconds.
In --
0730
Table 2.
Development of zona-free 8-cell hamster anbryos following transfer to recipients. Status of embryo Zona-intact (control)
Zona-free
No. of embryos transferred
72
70
No. of viable fetuses formed at d 10 of gestation (X)
58 (81%)
27 (39%)a
Data are totals from 7 pregnant recipients. "Significantly lower than control value (p
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THERIOGENOLOGY Zona pellucida-free 8-cell hamster embryos were transferred to a total of 15 recipients. Seven of these animals became pregnant. Each recipient served as its own control since zona-free embryos were transferred to one uterine horn and zona-intact embryos to the contralateral horn. The number of viable fetuses formed following transfer of 8-cell embryos (Table 2) was significantly lowar in the zona-free group than in the zona-intact controls. Since the low incidence of fetal resorption (4-6% on both sides) did not affect the data analysis, it was not included in the total. Bovine In vitro _Table 3.
Development of early bovine blastocysts in culture following enzymic-mechanical removal of the zonae pellucidae. Status of embryo Zona-intact (control)
Zona-free
No. early blastocysts cultured
13
14
No. embryos developing into expanded blastocysts (X)
12 (92%)
13 (93%)
Using early bovine blastocysts, there was no significant loss of developmental ability in vitro following enzymic-mechanical zona removal (Tab1e 3). At least 7qOxtes were required for the zonae to noticeably soften in 0.5% pronase before complete removal was possible using a mouth-pipette. With this procedure, lo-1596of all attempts at zonaremoval resulted in such severe damage to the embryos that they had to be discarded. Table 4.
Development of bovine morulae in culture following mechanical removal of the zonae pellucidae. Status of embryos Zona-intact (control)
Zona-free
No. morulae cultured
25
29
No. of embryos developing into blastocysts (96)
21 (84%)
27 (93%)
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THERIOGENOLOGY There was no significant difference in blastocyst development between the zona-free group and the zona-intact group (Table 4). Very few embryos (5%) were severely damaged by the mechanical zona removal procedure. Figure 4 shows a bovine morula from tiich the zona was removed mechanically.
Fig. 4. Representative pellucida of upper (see Materials and one depicted here,
zona-intact and zona-free bovine morulae. Zona embryo was removed by the mechanical procedure Methods). Zona-free cow embryos, such as the are capable of full development.
In -- vi00 Table 5.
Development of zona-free, cultured bovine embryos following transfer to recipient heifers. Status of embryos Zona-intact (control)
No. of embryos transferred (1 embryo/recipient) No. of pregnant heifers at 60 d (S)
13
Zona-free 15
5 (39%)
5 (33%)
Table 5 shows the results of transferring zona-free and zona-intact bovine embryos to synchronized recipients. Zonae pellucidae were removed mechanically from morulae, Rich were then cultured to the blastocyst stage during the next 24 hours before non-surgical transfer.
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THERIOGENOLOGY The proportions of pregnancies developing after transfer of control and experimental embryos were not significantly different tien recipients were examined at 60 d. By 6-8 months after transfer of embryos, all 5 animals that received zona-free embryos are still pregnant. One of 5 recipients in the control group has aborted.
DISCUSSI.ON Zona
rmovat
and embryo culture
In our present study, uterine stage embryos of the hamster (8-cell) and cow (morulae and early blastocyst), following premature removal of the zonae pellucidae, survived as wall in vitro as zona-intact control embryos. These observations correlate alwith the results of a previous study using Muse embryos (6). The species of embryo used appears to be a major determinant in the selection of the most efficient method for removal of the zona pellucida. This is due to species differences in the reaction of the zona pellucida to proteolytic enzymes. In 0.25% pronase, the zonae pellucidae of mouse (Hoppe, unpublished data) and hamster embryos are completely solubilized in less than 1 minute. By contrast, the zonae of uterine stage cow embryos were only slightly softened after 7-10 minutes in 0.5% pronase. Complete dissolution of cow and sheep zonae pellucidae may take up to 30 minutes (2,lO). In our laboratory, zonae of cow embryos were not completely removed after 35 minutes exposure to 0.5% pronase. Activity of stock pronase may well vary between lots and manufacturers. Following zona removal by pronase and immediate transfer to recipients, Massey -et al. found that about one-third of bovine blastocysts established pregnancies (2). This is lower than the incidence of pregnancy one would expect after transfer of zona-intact embryos at the same developmental stage. A zona-intact control group was not included for comparison. The low incidence of pregnancy may have been due to the effects of prolonged exposure of the cow embryo to pronase. The degree of exposure to pronase has been found to critically affect continued development of denuded mouse blastocysts (6). For these reasons, a preliminary trial was conducted in our laboratory to test the efficacy of an enzymic-mechanical technique for removal of the zona pellucida from early bovine blastocysts. Although results obtained with culture of these embryos were encouraging (Table 3), the vigorous pipetting necessary to remove the pronase-softened zonae resulted in an unacceptable number of severely damaged embryos. Employing the simple glass micromanipulators described in Materials and Methods, a mechanical procedure for zona removal was devised. The perivitelline space of the bovine morula is sufficiently large to allow passage of the glass microneedles used to "slit" the zona pellucida without significant damage to the embryonic mass. Not only were fewer embryos severely damaged, but the morphological appearance of these zona-free embryos after 24 h in culture was much better than that of embryos subjected to the enzymic-mechanical procedure.
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THERIOGENOLOGY Transfer
of
aona-free
embryos
Significantly fewer fetuses resulted after transfer of zona-free, 8-cell hamster embryos compared with zona-intact controls (Table 2). The formation of chimeras in utero may aid in explaining this differdifferentiated cells (trophectoderm ence. Since the blastocysFcmns and inner cell mass), there is thought to be less chance of chimera formation between closely approximated embryos than hen relatively undifferentiated stages, such as the 8-cell, come into close contact (20). Bronson and McLaren did not find any significant difference in the proportion of pregnancies developing from transfer of zona-free and zona-intact mouse blastocysts to the uteri of pseudopregnant recipients (6). In our present study, about 15% of zona-free hamster 8-cell embryos routinely formed chimeras during culture. This problen is eliminated by the use of a monotocous species, such as the cow, in which only one embryo need be transferred to each recipient. In addition, the fact that a high percentage of bovine embryos transferred non-surgically will establish pregnancies makes the cow an attractive model species for studying the effects of zonae removal on subsequent embryonic development. The results obtained from transfer of cultured, zona-free and zonaintact, bovine embryos are encouraging (Table 5). The proportions of pregnant animals in both groups were the same (5/15 and 5/13, respectively), althou@ the total number of embryos transferred to date is small. In these experiments, zonae pellucidae ware, removed mechanically from morulae, tiich were then cultured to the blastocyst stage before embryo transfer was performed. If the denuded cow morula were transferred immediately after mechanical removal of the zona pellucida, our knowledge of pre-implantation embryology would benefit. Massey -et al. did not obtain pregnancies following immediate transfer of bovine morulae denuded in pronase (2). This is surprising in view of the successful results obtained in our present study with mechanically denuded morulae transferred at the blastocyst stage. This discrepancy could be due to different methods used for zona removal, to some inherent property of morula vs. blastocyst, to transfer technique, or some combination of these factors. Experiments are currently being undertaken in our laboratory to investigate ðer the bovine morula can survive following mechanical removal of the zona pellucida and immediate transfer to a synchronized recipient. In conjunction with this investigation, we are also testing the viability of zona-free, bisected morulae -in vitro and -in vivo. Preliminary data indicate that zona-free, bisected morulae can develop into early blastocysts after 24 hours of culture. A viable fetus (day 60 of gestation) has been obtained following transfer of a small number of these denuded, split, cultured embryos. These experiments indicate that bovine embryos, in spite of the difficulties in obtaining sufficient quantities, offer a promising mode? for studying mammalian embryonic development. We anticipate that basic information obtained from this model system may contribute to the evolution of new animal breeding technologies.
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THERIOCENOLOGY REFERENCES
1.
Brun, J.L. and Psychoyos, A. Dissolution of the rat zona pellucida by acidified media and blastocyst viability. J. Reprod. Fert. 30: 489-491 (1972).
2.
Massey, J.M., Anderson, J.G., Ellis, W.C., Sorensen, Jr, A.M. and Kraemer, O.C. Development of bovine embryos following enzymatic removal of the zona pellucida. Theriogenology, -17: 99 (abstract) (1982).
3.
Mintz, 6. Experimental study of the developing mammalian egg: removal of the zona pellucida. Science, 138: 594-595 (1962).
4.
Willadsen, S.M. The production of monozygotic twins of preselected parentage by micromanipulation of non-surgically collected cow embryos. Theriogenology, 15: 13-22 (1980).
5.
Gardner, R.L. Manipulations on the blastocyst. From: Schering Workshop on Intrinsic and Extrinsic factors in Early Mammalian Development, Advances in the Biosciences, Pergamon Press, N.Y., Vol. 2: 279-299 (1971).
6.
Bronson, R.A. and McLaren, A. Transfer to the mouse oviduct of eggs with and without the zona pellucida. J. Reprod. Fert. -22:
129-137 (1970).
7.
Modlinski, J.A. The role of the zona pellucida in the development of mouse eggs -in vivo. J. Embryol. exp. Morph. -23: 539-547 (1970).
8.
Rottmann, O.J. and Lampeter, W.W. Development of early mouse and rabbit embryos without zona pellucida. J. Reprod. Fert. -61: 303-306 (1981).
9.
Smithberg, M. The effect of different proteolytic enzymes on the zona pellucida of mouse ova. Anat. Rec. 117: 544 (abstract)(l953).
10.
Trounson, A.O. and Moore, N.W. The survival and development of sheep eggs following complete or partial removal of the zona pellucida. J. Reprod. Fert. -41: 97-105 (1974).
11.
Moor, R.M. and Cragle, R.G. The sheep egg: enzymatic removal of the zona pellucida and culture of eggs -in vitro. J. Reprod. Fert. -27: 401-409 (1971).
12.
Orsini, M.W. The external vaginal phenomena characterizing the stages of the estrous cycle, pregnancy, pseudopregnancy, lactation, and the anestrous hamster, Mesocricetus auratus Waterhouse. Proc. Animal Care Panel, Vol. 11(4): 193-206 (1961).
13.
Bavister, B.D., Leibfried, M.L. and Lieberman, G. Development of preimplantation embryos of the golden hamster in a defined culture medium. Biol. Reprod. (in press) (1983).
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14.
Rowe, R.F., Del Campo, M.R., Critser, J.K. and Ginther, 0.3. Embryo transfer in cattle: non-surgical collection techniques. J. Vet. Res. -41: 106-108 (1980).
15.
bittingham, D.G. Survival of mouse embryos after freezing and thawing. Nature 233: 125-126 (1971).
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Goldacre, R.J. (1954).
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Bavister, B.D. and Yanagimachi, R. The effects of sperm extracts and energy sources on the motility and acrosome reaction of hamster spermatozoa -in vitro. Biol. Reprod. -16: 228-237 (1977).
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Del Campo, M.R., Rowe, R.F., Lhaichareon, D. and Ginther, O.J. Effect of the relative locations of embryo and corpus luteum on embryo survival in cattle. J. Reprod., Nutrition and Develop. (in press)(1983).
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Sokal, R.R. and Rohlf, F.J. Biometry: the principles and practice of statistics in biological research. W.H. Freeman and Co. (1969).
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Tarkowski, A.K. Mouse chimeras developed from fused eggs. 190: 857-860 (1961).
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A simplified micromanipulator.
Pm.
Nature 173: 45
Nature
MARCH 1983VOL. 19 NO. 3
Dreamstreet Holsteins, Inc., Walton, N.Y. 13856
Box 209,
Received for publication: accepted:
September 3, 1982 November 30, 1982
ABSTRACT Uterine stage embryos collected from the hamster (8-cell) and cow (morula, early blastocyst) wre monitored for development -in vitro (embryo culture) and embryo transfer) following premature removal of the zona Removal of the zona pellucida did not significantly affect -in vitro development to the blastocyst stage of (1) 8-cell hamster embryos (zonae removed by a combined enzymic-mechanical procedure), (2) bovine morulae (zonae removed by mechanical means only) (3) early bovine blastocysts (zonae removed by the enzymic-mechanical technique). Zona-free hamster enbryos formed significantly fewer viable fetuses than did zona-intact embryos. The lower incidence of fetal development observed following transfer of zona-free 8-cell hamster embryos may have resulted in part from the formation of chimeras by fusion of these embryos -in utero. Such fusion was observed to occur in vitro between zona-free embryos placed in close proximity. The proEr=of pregnancies resulting from transfer of bovine blastocysts cultured from zona-free morulae was similar to that of zona-intact embryos. In this study w have demonstrated that (1) enzymic and mechanical procedures used to remove zonae pellucidae from uterine-stage hamster and bovine enbrvos do not adversely affect subseauent development of these embryos in vitro and in viva-and (2) zonae'pellucidae are not reauired for nfiivelo$&t these embryos. These findings have implications for microsurgery of mammalian embryos and for embryo transfer.
Acknowledgements: Supported by the Wisconsin Agricultural Experiment Station, College of Agricultural and Life Sciences, University of Wisconsin-Madison. We are indebted to: The Upjohn Co., Kalamazoo, Michigan for generous supplies of PGF2=, "Lutalyse"; American Breeders Service, De Forest, Wisconsin for supplying frozen semen; and IMV International Corp., Minneapolis, Minnesota for donating the equipment used for the bovine non-surgical embryo transfers.
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THERIOGENOLOGY INTRODUCTION Mammalian embryos from a number of species can survive and continue to develop following premature removal of the zona pellucida. Removal of the zona pellucida makes the pre-implantation embryo more accessible for microsurgical procedures such as bisection or manipulation of individual blastomeres. However, for this approach to be useful, it is essential to know how wall zona-free embryos develop -in vitro and in vivo compared with their zona-intact counterparts. -A variety of methods have been used to remove zonae pellucidae from embryos at all stages of developent from the zygote to blastocyst. Methods used have been chemical (1,2,3), mechanical (4,5), or a combination of the two (6,7). lhe ability of embryos artificially freed from their zonae pellucidae to continue cleavage and to form normal fetuses after transfer to recipients has varied widely, depending on species and stage of embryo used (2,6,8,9) and the method of zona removal (10,ll). Wlether the problem lies with the method of zona removal, with some need for the presence of the zona by the early embryo, with the transfer of zona-free embryos to recipients or some combination of these factors is not clear. The ability to obtain normal embryonic development following zona removal and microsurgery would be particularly useful in the cow, since procedures for embryo sexing and cloning could then be more easily developed. The present study was undertaken to achieve the following objectives: 1.
Evaluate procedures for zona pellucida removal, using uterinestage hamster embryos as a model system to be adapted for use in the cow.
2.
Assess embryo developmental potential in vitro and in vivo subsequent to removal of the zona by e?@iiiiind mexam methods, or by mechanical means alone.
3.
Compare survival rates -in vitro and -in vivo for zona-intact and zona-free hamster and cow embryos recovered from the uterus. MATERIALS AND METHODS
Animals Male and female hamsters ware housed under controlled temperature and photoperiod (14 light:10 dark, with li#ts on at 0600 h). Estrous cycles were monitored by daily observation of vaginal discharges (12). Holstein-Friesian heifers were used for the work on bovine embryos, Donors were not used as recipients. Recipients that were found to be non-pregnant 60 days following embryo transfer were reassigned to a different transfer experiment. These animals were kept at Bookhout Farm, Arlington, Wisconsin. This facility is maintained by the Agricultural Experiment Station of the University of Wisconsin-Madison
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College of Agricultural and Life Sciences. Cattle were housed in two free-stall pole barns which were open on the south side. One group consisted of open recipients and potential donors. Another group consisted of recently used donors and recipients into which an embryo had been transferred within the last 60 days. Superovulation and Insemination Superovulation of cycling hamsters was initiated the morning following estrus (estrus = day 0), using 15-20 I.U. of PMSG (Organon, West Orange, N.J.) injected intraperitoneally. Seventy-two hours later, lo-15 I.U. of HCG (Sigma, St. Louis, MO) was administered and the animals were bred that evening to intact males. Toward the end of the study, it was found that superovulated donor hamsters yielded embryos of similar quantity and quality regardless of tieetheror not they were given HCG to induce ovulation. All donor heifers were superovulated with FSH-P (Burns-Biotec, Omaha, NE) starting lo-12 days after estrus (estrus = day 0). Gonadotrophin injections were given twice daily for 5 days, for a total of 50 mgs. Seventy-two hours after the initial gonadotrophin injection, a luteolytic dose of PGF2 (25-30 mg) was administered. PGF2 was dinoprost tromethamine ?"Lutalyse", Upjohn Co., Kalamazoo, 141). Donors were inseminated with one straw of semen at 60 hours and again at 72 hours following PGF2a. Embryo Collection Uteri were excised from donor hamsters between 1500 and 1600 h on day 3 and flushed with l-2 ml of a Hepes-buffered, modified Tyrode's solution (TALP) containing glucose, lactate, pyruvate and 3 mg/ml Fraction V bovine serum albumin (BSA)(13). Flushing was accomplished with a 30 gauge needle attached to a 3 cc syringe. Flushings were collected in 35x10 mm plastic petri dishes (Falcon ~1008). Embryos were recovered and rinsed 2 or 3 times and held at room temperature in TALP-Hepes. Bovine embryos were recovered non-surgically from the uterine horns of,,donorheifers on day 6 or 7, utilizing a balloon-type catheter (Rusch, W. Germany) and a small-volume aspiration technique (14). The embryo recovery medium was sterile, modified Dulbecco's phosphatebuffered saline (15) containing 3 mg/ml BSA (Sigma), 100 I.U./ml of penicillin and 0.05 mg/ml of streptomycin. Uterine flushings (300-400 ml/horn) were collected in gas leveling bulbs (Corning Glass Works, Corning, NY) and transported back to the laboratory in a Styrofoam box that maintained the temperature of the medium at 25-30°C. The contents of the bulbs were transferred to sterile, siliconized glass evaporating dishes (Corning Glass Works). Embryos were removed from the dishes, rinsed 3 times in fresh medium and held at room temperature (22-24OC).
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THERIOGENOLOGY Removal of the Zona Pellucida Enaymic
and mechanical
The zonae pellucidae of hamster 8-cell and early blastocyst (day 7) cow embryos ware removed by first incubating then in a fresh, sterile solution of 2.5 mg/ml (hamster) or 5 mg/ml (cow) pronase (Sigma) prepared in TALP-Hepes medium. Embryos were continuously observed under a stereomicroscope until the zonae had noticeably thinned or ballooned or both. The time was recorded for partial digestion of the zona pellucida. The embryos were then pipetted in the rinsing solution (TALP-Hepes) until the partially-digested zonae were removed. Mechanical Bovine morulae (day 6) had their zonae pellucidae removed by the use of two glass microneedles, each mounted on a simple, hand-held micromanipulator (Figure 1). Left and right micromanipulators were attached to the glass plate of the stereomicroscope stage by a thin
EPOXY ADAPTOR
FOR
/
MICRONEEDLE MOVA sBLE -
GLASS
GLASS
SLIDE
ZPOXY
MICRONEEDLE (DETACHABLE)
Fig. 1. Hand-held glass micromanipulator. Each micromanipulator was fashioned out of 3 glass microscope slides according to a modification of a design outlined by Goldacre (16). The glass needles were pulled from 3 ~1 micropipettes (Drunmond Scientific, Broomall, PA) on a vertical pipette puller so that the shank diameter at the tip of the needle averaged 10-15 nrn. The needles were inserted and fixed with melted paraffin wax into the end of a bent piece of glass tubing cemented onto the movable vertical glass slide. The fixed vertical slide was attached perpendicularly to the base slide by a bead of epoxy glue. Stopcock grease was used between the moving glass surfaces.
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Fig. 2. Left and right micromanipulators in place on glass microscope base. All manipulations were performed under oil within a droplet of phosphate-buffered medium. layer of stopcock grease, facilitating movement in the horizontal plane (Figure 2). All manipulations were performed in small plastic petri dishes within a droplet of medium under oil. Hile one needle steadied the embryo, the other was used to make a series of connecting slits along the equator of its zona pellucida. Then, using both needles, the edges of the zona along the slit were everted and the embryo removed either by gravity or suction. Viability Assays In _- vitro
using
embryo culture
Eight-cell hamster embryos were cultured for 24 hours in a bicarbonate-buffered, modified Tyrode's solution (TALP: ref 17) containing 3 mg/ml BSA and 4 amino acids (13). Cow embryos collected on day 6 or 7 were cultured for 24 hours in a similar manner except that 15 mg/ml BSA was used, without amino acids. Embryos ware incubated in droplets containing 65-70 ul of culture medium placed under a layer of paraffin oil in 60 x 15 mm plastic petri dishes (Falcon 11007). The dishes containing the droplets were pre-equilibrated at 37°C in a waterjacketed incubator rhich automatically maintained a humidified, 5% CO2 in air mixture.
In vitro growth of both hamster and cow embryos was monitored for -development to the blastocyst stage every 18-24 hours. Embryos were examined within their culture droplets using an Olympus stereomicroscope
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THERIOGENOLOGY at 80-120X magnification. The developmental stage, condition, relative size and morphology of each embryo were all recorded. ~n -- vivo using embryo transfer Recipient hamsters (day 3 of pseudopregnancy) were anesthetized with Nembutal given intraperitoneally. A paralumbar incision was made just behind the last rib and the uterus retracted by means of a clamp on the ovarian fat pad. Embryos were introduced directly into the uterine lumen by means of a hand-drawn Pasteur pipette connected to a mouthtube. Numbers of viable and of resorbing fetuses were estimated at autopsy on day 10 of pregnancy. In a preliminary study designed to evaluate culture conditions, bovine embryos collected on day 7 (early blastocyst stage) were cultured for 24 hours, then transferred surgically via a standing flank laparotomy. Although the percentage of successful transfers was high (5/7 recipients or 71% became pregnant after transfer of single embryos), we desided that subsequent transfers would be performed non-surgically for practical reasons. A zona-intact or zona-free blastocyst tiich had been cultured from a morula was aspirated into a 0.25 ml plastic insemination straw so that the middle one-third of the straw containing the embryo was isolated by twD air bubbles from the remaining column of medium. At the transfer facility, the recipient heifer was palpated, given an epidural anesthetic, and the vulvar lips were washed and dried. The transfer gun, tiich holds the straw, was then manipulated per rectum through the cervix and into the uterine horn ipsilateral to the corpus luteum-bearing ovary (18). A single embryo was gently deposited 2-5 cm past the internal uterine bifurcation. Bovine embryo recipients were examined for pregnancy by rectal palpation at 50-60 days. Some pregnant heifers were reexamined as late as 240 days of gestation. Experimental design and statistical analysis Ranwter embryos Hamster embryos were collected from 4-6 superovulated donors per replicate, pooled, and randomly divided into two equal groups. Embryos in the treatment group had their zonae removed using the enzymicmechanical technique. Control embryos *re treated in the same way as the experimental group except that their zonae were not removed. Ten to 12 embryos were placed in each droplet of mediun. Within a culture dish, the number of droplets with zona-free embryos was equal to the number containing zona-intact embryos. Data collected over six replicates ware analyzed using a 3-way test for independence (19). Alternatively, hamster embryos were transferred to recipient females instead of being cultured. The double cervix of the hamster precludes embryo crossover between the uterine horns, permitting viability of control and treated embryos to be tested in the same recipient. From 9-11 (usually 10) zona-free embryos were transferred to one uterine horn of a recipient and the same number of zona-intact embryos to the other horn. Analysis was performed on these data using the t-test for paired samples (19).
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Cow
embryos
Bovine embryos were randomly assigned to a zona-intact control group or a zona-free experimental group. After manipulation and rinsing, one embryo (zona-intact or zona-free) was placed in each droplet of culture medium, and therefore, was considered a single data point. Embryos were observed for development to the blastocyst after 24 hours in culture. In a preliminary study, early blastocysts (day 7) were denuded by means of the enzymic-mechanical technique. Control and experimental embryos were cultured, but not transferred. For the main investigation, morulae (day 6) had their zonae removed using the mechanical technique. Zona-intact and zona-free embryos which developed to blastocysts after 24 hours in culture were transferred to recipients. Data generated in vitro and --in vivo were analyzed using the chi-squared test for indepe&n-@-(i9).
.’
Fig. 3A. Hamster zona-free, 8-cell embryos. by enzymic-mechanical procedure.
Zonae pellucidae removed
Fig. 38. Hamster zona-free, early blastocysts cultured (24 h) from zona-free 8-cell embryos.
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THERIOGENOLOGY RESULTS Hamster
In vitro Growth of &cell embryos to the blastocyst stage in culture was unaffected by a combined enzymic-mechanical technique used to remove the zona pellucida. Representative zona-free embryos before and after culture are shown in Figure 3. The results obtained by culturing hamster zona-intact and zona free embryos are shown in Table 1. Table 1.
Effect of removing the zona pellucida of hamster 8-cell embryos on subsequent development to blastocyst -in vitro. Status of embryo Zona-intact (control)
No. embryos cultured No. embryos developing into blastocysts (%)
149
Zona-free 158
68 (46%)
69 (44%)
Data are total of 6 replicates. There was no significant difference between the zona-intact group and the zona-free experimental group. About 15% of the denuded embryos formed chimeras, i.e., two embryos fused to form one large one. The time required for partial digestion of the zona pellucida in a 0.25% solution of pronase ranged from 5-10 seconds.
In --
0730
Table 2.
Development of zona-free 8-cell hamster anbryos following transfer to recipients. Status of embryo Zona-intact (control)
Zona-free
No. of embryos transferred
72
70
No. of viable fetuses formed at d 10 of gestation (X)
58 (81%)
27 (39%)a
Data are totals from 7 pregnant recipients. "Significantly lower than control value (p
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THERIOGENOLOGY Zona pellucida-free 8-cell hamster embryos were transferred to a total of 15 recipients. Seven of these animals became pregnant. Each recipient served as its own control since zona-free embryos were transferred to one uterine horn and zona-intact embryos to the contralateral horn. The number of viable fetuses formed following transfer of 8-cell embryos (Table 2) was significantly lowar in the zona-free group than in the zona-intact controls. Since the low incidence of fetal resorption (4-6% on both sides) did not affect the data analysis, it was not included in the total. Bovine In vitro _Table 3.
Development of early bovine blastocysts in culture following enzymic-mechanical removal of the zonae pellucidae. Status of embryo Zona-intact (control)
Zona-free
No. early blastocysts cultured
13
14
No. embryos developing into expanded blastocysts (X)
12 (92%)
13 (93%)
Using early bovine blastocysts, there was no significant loss of developmental ability in vitro following enzymic-mechanical zona removal (Tab1e 3). At least 7qOxtes were required for the zonae to noticeably soften in 0.5% pronase before complete removal was possible using a mouth-pipette. With this procedure, lo-1596of all attempts at zonaremoval resulted in such severe damage to the embryos that they had to be discarded. Table 4.
Development of bovine morulae in culture following mechanical removal of the zonae pellucidae. Status of embryos Zona-intact (control)
Zona-free
No. morulae cultured
25
29
No. of embryos developing into blastocysts (96)
21 (84%)
27 (93%)
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THERIOGENOLOGY There was no significant difference in blastocyst development between the zona-free group and the zona-intact group (Table 4). Very few embryos (5%) were severely damaged by the mechanical zona removal procedure. Figure 4 shows a bovine morula from tiich the zona was removed mechanically.
Fig. 4. Representative pellucida of upper (see Materials and one depicted here,
zona-intact and zona-free bovine morulae. Zona embryo was removed by the mechanical procedure Methods). Zona-free cow embryos, such as the are capable of full development.
In -- vi00 Table 5.
Development of zona-free, cultured bovine embryos following transfer to recipient heifers. Status of embryos Zona-intact (control)
No. of embryos transferred (1 embryo/recipient) No. of pregnant heifers at 60 d (S)
13
Zona-free 15
5 (39%)
5 (33%)
Table 5 shows the results of transferring zona-free and zona-intact bovine embryos to synchronized recipients. Zonae pellucidae were removed mechanically from morulae, Rich were then cultured to the blastocyst stage during the next 24 hours before non-surgical transfer.
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THERIOGENOLOGY The proportions of pregnancies developing after transfer of control and experimental embryos were not significantly different tien recipients were examined at 60 d. By 6-8 months after transfer of embryos, all 5 animals that received zona-free embryos are still pregnant. One of 5 recipients in the control group has aborted.
DISCUSSI.ON Zona
rmovat
and embryo culture
In our present study, uterine stage embryos of the hamster (8-cell) and cow (morulae and early blastocyst), following premature removal of the zonae pellucidae, survived as wall in vitro as zona-intact control embryos. These observations correlate alwith the results of a previous study using Muse embryos (6). The species of embryo used appears to be a major determinant in the selection of the most efficient method for removal of the zona pellucida. This is due to species differences in the reaction of the zona pellucida to proteolytic enzymes. In 0.25% pronase, the zonae pellucidae of mouse (Hoppe, unpublished data) and hamster embryos are completely solubilized in less than 1 minute. By contrast, the zonae of uterine stage cow embryos were only slightly softened after 7-10 minutes in 0.5% pronase. Complete dissolution of cow and sheep zonae pellucidae may take up to 30 minutes (2,lO). In our laboratory, zonae of cow embryos were not completely removed after 35 minutes exposure to 0.5% pronase. Activity of stock pronase may well vary between lots and manufacturers. Following zona removal by pronase and immediate transfer to recipients, Massey -et al. found that about one-third of bovine blastocysts established pregnancies (2). This is lower than the incidence of pregnancy one would expect after transfer of zona-intact embryos at the same developmental stage. A zona-intact control group was not included for comparison. The low incidence of pregnancy may have been due to the effects of prolonged exposure of the cow embryo to pronase. The degree of exposure to pronase has been found to critically affect continued development of denuded mouse blastocysts (6). For these reasons, a preliminary trial was conducted in our laboratory to test the efficacy of an enzymic-mechanical technique for removal of the zona pellucida from early bovine blastocysts. Although results obtained with culture of these embryos were encouraging (Table 3), the vigorous pipetting necessary to remove the pronase-softened zonae resulted in an unacceptable number of severely damaged embryos. Employing the simple glass micromanipulators described in Materials and Methods, a mechanical procedure for zona removal was devised. The perivitelline space of the bovine morula is sufficiently large to allow passage of the glass microneedles used to "slit" the zona pellucida without significant damage to the embryonic mass. Not only were fewer embryos severely damaged, but the morphological appearance of these zona-free embryos after 24 h in culture was much better than that of embryos subjected to the enzymic-mechanical procedure.
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THERIOGENOLOGY Transfer
of
aona-free
embryos
Significantly fewer fetuses resulted after transfer of zona-free, 8-cell hamster embryos compared with zona-intact controls (Table 2). The formation of chimeras in utero may aid in explaining this differdifferentiated cells (trophectoderm ence. Since the blastocysFcmns and inner cell mass), there is thought to be less chance of chimera formation between closely approximated embryos than hen relatively undifferentiated stages, such as the 8-cell, come into close contact (20). Bronson and McLaren did not find any significant difference in the proportion of pregnancies developing from transfer of zona-free and zona-intact mouse blastocysts to the uteri of pseudopregnant recipients (6). In our present study, about 15% of zona-free hamster 8-cell embryos routinely formed chimeras during culture. This problen is eliminated by the use of a monotocous species, such as the cow, in which only one embryo need be transferred to each recipient. In addition, the fact that a high percentage of bovine embryos transferred non-surgically will establish pregnancies makes the cow an attractive model species for studying the effects of zonae removal on subsequent embryonic development. The results obtained from transfer of cultured, zona-free and zonaintact, bovine embryos are encouraging (Table 5). The proportions of pregnant animals in both groups were the same (5/15 and 5/13, respectively), althou@ the total number of embryos transferred to date is small. In these experiments, zonae pellucidae ware, removed mechanically from morulae, tiich were then cultured to the blastocyst stage before embryo transfer was performed. If the denuded cow morula were transferred immediately after mechanical removal of the zona pellucida, our knowledge of pre-implantation embryology would benefit. Massey -et al. did not obtain pregnancies following immediate transfer of bovine morulae denuded in pronase (2). This is surprising in view of the successful results obtained in our present study with mechanically denuded morulae transferred at the blastocyst stage. This discrepancy could be due to different methods used for zona removal, to some inherent property of morula vs. blastocyst, to transfer technique, or some combination of these factors. Experiments are currently being undertaken in our laboratory to investigate ðer the bovine morula can survive following mechanical removal of the zona pellucida and immediate transfer to a synchronized recipient. In conjunction with this investigation, we are also testing the viability of zona-free, bisected morulae -in vitro and -in vivo. Preliminary data indicate that zona-free, bisected morulae can develop into early blastocysts after 24 hours of culture. A viable fetus (day 60 of gestation) has been obtained following transfer of a small number of these denuded, split, cultured embryos. These experiments indicate that bovine embryos, in spite of the difficulties in obtaining sufficient quantities, offer a promising mode? for studying mammalian embryonic development. We anticipate that basic information obtained from this model system may contribute to the evolution of new animal breeding technologies.
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THERIOCENOLOGY REFERENCES
1.
Brun, J.L. and Psychoyos, A. Dissolution of the rat zona pellucida by acidified media and blastocyst viability. J. Reprod. Fert. 30: 489-491 (1972).
2.
Massey, J.M., Anderson, J.G., Ellis, W.C., Sorensen, Jr, A.M. and Kraemer, O.C. Development of bovine embryos following enzymatic removal of the zona pellucida. Theriogenology, -17: 99 (abstract) (1982).
3.
Mintz, 6. Experimental study of the developing mammalian egg: removal of the zona pellucida. Science, 138: 594-595 (1962).
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Willadsen, S.M. The production of monozygotic twins of preselected parentage by micromanipulation of non-surgically collected cow embryos. Theriogenology, 15: 13-22 (1980).
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Gardner, R.L. Manipulations on the blastocyst. From: Schering Workshop on Intrinsic and Extrinsic factors in Early Mammalian Development, Advances in the Biosciences, Pergamon Press, N.Y., Vol. 2: 279-299 (1971).
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Bronson, R.A. and McLaren, A. Transfer to the mouse oviduct of eggs with and without the zona pellucida. J. Reprod. Fert. -22:
129-137 (1970).
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Modlinski, J.A. The role of the zona pellucida in the development of mouse eggs -in vivo. J. Embryol. exp. Morph. -23: 539-547 (1970).
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Rottmann, O.J. and Lampeter, W.W. Development of early mouse and rabbit embryos without zona pellucida. J. Reprod. Fert. -61: 303-306 (1981).
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Smithberg, M. The effect of different proteolytic enzymes on the zona pellucida of mouse ova. Anat. Rec. 117: 544 (abstract)(l953).
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Trounson, A.O. and Moore, N.W. The survival and development of sheep eggs following complete or partial removal of the zona pellucida. J. Reprod. Fert. -41: 97-105 (1974).
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Moor, R.M. and Cragle, R.G. The sheep egg: enzymatic removal of the zona pellucida and culture of eggs -in vitro. J. Reprod. Fert. -27: 401-409 (1971).
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Orsini, M.W. The external vaginal phenomena characterizing the stages of the estrous cycle, pregnancy, pseudopregnancy, lactation, and the anestrous hamster, Mesocricetus auratus Waterhouse. Proc. Animal Care Panel, Vol. 11(4): 193-206 (1961).
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Bavister, B.D., Leibfried, M.L. and Lieberman, G. Development of preimplantation embryos of the golden hamster in a defined culture medium. Biol. Reprod. (in press) (1983).
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Rowe, R.F., Del Campo, M.R., Critser, J.K. and Ginther, 0.3. Embryo transfer in cattle: non-surgical collection techniques. J. Vet. Res. -41: 106-108 (1980).
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bittingham, D.G. Survival of mouse embryos after freezing and thawing. Nature 233: 125-126 (1971).
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Goldacre, R.J. (1954).
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Bavister, B.D. and Yanagimachi, R. The effects of sperm extracts and energy sources on the motility and acrosome reaction of hamster spermatozoa -in vitro. Biol. Reprod. -16: 228-237 (1977).
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Del Campo, M.R., Rowe, R.F., Lhaichareon, D. and Ginther, O.J. Effect of the relative locations of embryo and corpus luteum on embryo survival in cattle. J. Reprod., Nutrition and Develop. (in press)(1983).
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Sokal, R.R. and Rohlf, F.J. Biometry: the principles and practice of statistics in biological research. W.H. Freeman and Co. (1969).
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Tarkowski, A.K. Mouse chimeras developed from fused eggs. 190: 857-860 (1961).
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