IN VITRO EMBRYO CULTURE TECHNIQUE

Abstract: An in vitro culture technique of avian embryos up to blastoderm formation is described where a uterine embryo is abstracted from a hen either manually or by inducing premature oviposition with vasopressin or a prostaglandin. The embryo is then cultured in a culture medium (such as albumen) partially submerged, and in particular with the culture medium in line with the germinal disc but below the level of the albumen capsule.

Description: IN VITRO EMBRYO CULTURE TECHNIQUE This invention relates to an in vitro avian embryo culture technique, which is particularly suitable for application to poultry, particularly hens. The chick embryo, in the initial stages of its development from fertilisation to cleavage, has not been amenable to experimental intervention on account of the bulk, fragility and relative inaccessibility of the ovum. This problem has been discussed in recent reviews on the possible routes of transfer of exogenous genes into birds (Freeman and Messer, 1985; Crittenden and Salter, 1986) . Perry (1986a,b) has taken the alternative view and suggested that genetic manipulation of the avian ovum is practicable. The aim of devising a complete culture system for the chick embryo was to provide a means of rearing the manipulated ovum to maturity. Such a means is disclosed in EP-A-0295964, which relates among other things to a method for in vitro culture to an intermediate stage of embryonic development and beyond to hatch. Such a technique has potential applications not only in the genetic engineering of poultry, but also in the investigation of fundamental mechanisms of avian development and in the study of deleterious traits. Moreover, it affords a desirable alternative to surgical intervention in the laying hen. The chick embryo originates in the germinal disc, a small region of cytoplasm situated at the animal pole of the ovum (the familiar yolk) . During the first third of its development, the embryo remains floating at the surface of the yolk whilst the extra-embryonic membranes grow around the yolk and become vasσularised. In the remaining period of development, the embryo grows at the expense of the food reserves in the egg. For present purposes, the development of the chick may be treated as divided into three phases according to the changing requirements at successive stages from fertilisation to hatch. Phase I. Fertilisation to blastoderm formation. This phase takes place in the oviduct and terminates at oviposition. Gamete interaction occurs within 15 min after ovulation, and the first cleavage division some 4 h later (Perry, 1987) . In the following 20 h, subsequent divisions give rise to a simple sheet of cells overlying a subblastodermal cavity (Kochav, Ginsburg and Eyal-Giladi, 1980) . During its passage through the oviduct, the ovum is invested with albumen in the magnum, then with the shell membrane in the isthmus where cleavage commences. In the uterus, the albumen is doubled in volume by the absorption of uterine fluid (plumping fluid) , and finally the shell undergoes slow calcification. For hens laying in long sequences of one egg per day, oviposition is followed within 15-30 min by the next ovulation and the cycle is repeated. Phase II. Embryonic morphogenesis. This phase takes place in the first 3 days of incubation of the egg (stages 1-18, Hamburger and Hamilton [1951]). At stage 20, the embryo is 10mm in length, and the extra embryonic blastoderm extends around the yolk to its equator. Phase III. Embryonic growth. This phase takes place in the final 18 days of incubation of the egg (stages 18-45, Hamburger and Hamilton [1951]). Although the techniques disclosed in EP-A-0295964 can give excellent results, the preferred embodiment does have one disadvantage. This relates to the fact that the fertilised egg in the preferred embodiment of EP-A-0295964 is obtained surgically from the hen, which does not survive the treatment. It would be desirable if a technique could be devised in which the donor hen survived. This invention relates to such a technique. It has been discovered that uterine embryos, obtained under conditions which are non-fatal to the hen, may be cultured j_n vitro up to blastoderm formation. The embryos may then be cultured through Phases II and III as in EP-A-0295964. According to a first aspect of the present invention, there is provided a process for the _in vitro culture of an avian embryo up to blastoderm formation, the process comprising abstracting a uterine embryo from a hen and culturing the embryo partially submerged in culture medium. It is to be understood that the word "hen" means, unless the context requires otherwise, a female bird of appropriate maturity of any species or genus. The abstraction of the uterine embryo may be achieved by induced premature oviposition, for example with vasopressin, a prostaglandin (Hertelendy et al, 1974) or another appropriate hormone or other chemical or biological substance. Manual abstraction of the uterine embryo is another possibility (Eyal-Giladi and Kochav, 1976) as long as this can be achieved under conditions which enable the hen subsequently to be used as a source of embryos. The embryo may be abstracted at the early, mid or late stage of uterine development. It is preferred that the embryo be abstracted at about 6 to 9 hours, particularly 7.5 to 8 hours after oviposition of the previous egg if a hen is laying in a long sequence of one egg per day. An embryo abstracted in accordance with the teaching of this invention will generally contain from 16 to 128, often 16 to 64, cells per embryo. As a practical matter, the embryo as abstracted will generally be found to be surrounded by a capsule of dense albumen and a shell membrane. Prior to the culture in the medium, the shell membrane will generally be removed. The culture medium may be albumen (for example thin albumen from fresh eggs) , which may be diluted, for example with water or salt solution. It is preferred that three parts of thin albumen be diluted with one part of a salt solution. Ideal salt solutions are those that are similar in composition to the fluid absorbed by the albumen in the uterus. The culture time partially submerged in the medium is generally from 12 to 24 hours, for example about 18 hours. For optimal results, the culture medium should be generally in line with the germinal disc, which will generally be uppermost, but below the level of the albumen capsule. A process in accordance with this aspect of the invention preferably takes place in a closed container. The container may be made of an impermeable material such as glass, which may be sealed with a low gas permeability film such as SARAN WRAP (trade mark) . Any other material having the appropriate properties of SARAN WRAP may be used. Gas permeability may be measured (eg for carbon dioxide and/or water vapour) as described above. The 24 hour pH rise may be from 0.5 to 1.0, for example from 0.6 to 0.8 in the carbon dioxide permeability test. The water vapour permeability may be from 1.0 to 10, for example 2 to 5 mg/cm /24h. In general polyvinylidene chloride is a preferred material for the film. Embryos cultured by means of the present invention can subsequently be cultured through Phase II (embryonic morphogenesis) and Phase III (embryonic growth) as described in EP-A-0295964 , although it may be advantageous to allow an extra period of time in Phase III (for example about 12 hours) for embryos whose initial in vitro culture has been in accordance with this invention. It may also be advantageous to agitate or turn the eggs for a longer period (eg up to day 19) than was considered optimal in EP-A-0295964. In accordance with the teaching of EP-A-0295964, a process for the in vitro culture of an avian embryo during embryonic morphogenesis (Phase II) may comprise incubating an embryo in a culture medium in a liquid-filled closed liquid-impermeable container. The container may be partially gas permeable. The gas permeability may be provided by an eggshell (generally in combination with the inner shell membrane) and/or by an otherwise partially gas permeable seal, whose preferred characteristics are as for culture during embryonic growth (Phase III) as discussed below. It should be noted that the eggshell and inner shell membrane are partially gas permeable. The culture medium is preferably liquid albumen, which may be collected from freshly laid eggs. The container is for preference again part of an egg, but the preferred construction is somewhat different from that used for preference in Phase III. In Phase II, it is preferably the sharp end which is removed, for example by a 32 mm hole. This ensures the presence of an air cell or space between the outer shell membrane and the inner shell membrane; this appears to be advantageous as the air cell or space .expands during culturing to make up for the water lost by evaporation. If a seal is being used to close an egg of which the sharp end has been removed, the egg may be cultured in a generally horizontal position, with the seal then being to one side. The seal should be kept firmly in place against the shell of the egg. It is very much preferred that the cultured embryos be subjected to gentle to moderate agitation. Intermittent or continuous rocking, for example through an angle of 90° in hourly cycles, or other comparable intervals, is preferred. A Phase II culture process will generally begin at about 18 hours after uterine embryo abstraction (that is to say at about what would be the normal time of laying) and may last for 2 or more days, for example up to 8. However, it is preferred that a Phase II culture process be continued for in the order of 3 or 4 days before transferring the embryo to a Phase III culture process as described below. A process for the in vitro culture of avian embryos during the embryonic growth phase (Phase III) in accordance with the teaching of EP-A-0295964 may comprise incubating an embryo in a closed container, there being an air space above the embryo, the air space being separated from the external atmosphere by a partially gas permeable seal. The seal is preferably in the form of a film. The seal may be of plastics material , for example polyethylene. It has been found that commercially available cling-film forms an appropriate seal, particularly when used in two layers. Any other material having the appropriate properties of cling film may be used. The suitability of material for use as a seal may be measured indirectly by measuring carbon dioxide and/or water vapour permeability; carbon dioxide permeability can be measured by testing the rise in pH of egg albumen after 24 hours incubation at 38°C in otherwise impermeable containers. The albumen should be initially gassed with carbon dioxide to lower the pH 1.0 unit. A pH rise of from 0.5 to 1.5 is generally suitable, with the preferred range being 0.5 or 0.7 to 1.0 or 1.3, for example about 0.9. Water vapour permeability may be from 5 or 10 to 30 or 40 mg/cm 2 /24h. The container is preferably part of an egg, which will usually be chosen from the same species as is being cultured. It has been found particularly appropriate to remove the blunt end from a whole egg; a 40 mm diameter hole centred on the axis of the egg was found to be particularly suitable. The hole at the blunt end of the egg is sealed with the partially gas permeable seal. The seal may be made to adhere to the egg shell by means of albumen. Preferably the permeability characteristics are similar to those of a natural egg. Culture medium may be present in some embodiments of this aspect of the invention, particularly if prior culture stages have been in vitro, but the process can work without it, for example when the prior culture stages have been carried out naturally. The culture medium when present will usually comprise albumen, either undiluted or in a dilute form and desirable inter-uterine fluid. When hen eggs are used, it is preferred that the depth of the space between the embryo and the seal is from 5 to 15 mm, for example about 10 mm. It is preferred to agitate the incubated embryo gently, at least initially. Gentle agitation can be achieved by intermittent rocking, for example through an angle at 30°. Conventional incubation temperatures, for example about 38°C may be maintained. In vitro culture processes in accordance with this aspect of the invention are suitable for use from about day 4 (counting from fertilisation) up to hatch, which generally occurs at about day 22. However, for the final few days (for example from day 19) of embryonic life, it is preferred that there be no agitation. In addition, shortly before (for example 1 to 2 days before) the estimated hatch time it is preferred that the seal be perforated to allow a certain amount of air into the container. Further air may be allowed in later, for example by removing the seal and optionally covering the hole in the eggshell (when the egg forms the container) by a solid disc, which may be provided by a petri dish. The invention will now be illustrated by the following example. EXAMPLE White Leghorn hens at 30-51 weeks of age were artificially inseminated at weekly intervals. Uterine eggs were obtained by injecting 2 i.u. of vasopressin (Sigma) into the wing vein (Gilbert and Lake, 1963) at 7.5-8.Oh after oviposition of the preceding egg. For each hen, the treatment was given at intervals of 2 weeks for a period of 22 weeks. The embryos were cultured according to the procedures developed by Perry (1988) as as disclosed in EP-A-0295964. The shell membranes of the uterine eggs were removed and the eggs placed in glass jars with culture medium (10-15ml) that surrounded but did not cover the egg. The culture medium contained 3 parts of thin albumen, collected from freshly laid eggs, and 1 part of a salt solution (50mM KHC0 3 , 30mM NaHC0 3 , lOmM KC1, 2.5mM MgCl 2 .6H 2 0, 0.7mM CaCl 2 .2H 2 0, llmM glucose) that was similar in composition to the fluid absorbed by the albumen in the uterus. After sealing the jars with a plastic film (polyvinylidene chloride) , the preparations were incubated at 41°C for 18h. The contents of the jars were then transferred to recipient egg shells that were 3-4g heavier than the egg preceding the uterine egg. Thin albumen (l-2ml) was added to fill the shells if required,and the shells were sealed with cling film (polyethylene) . The reconstituted eggs were incubated at 37.6°C, R.H. 50-60%, rocking them through an angle of 90° at hourly intervals for 3 days. For the final 18 days of development, the embryos were transferred to large recipient shells by the method of Rowlett and Simkiss (1987) and the shells were sealed with cling film (polyethylene) . In these preparations an air space was provided above the embryo, and shells were selected in size to ensure that contact between the embryo and the seal was avoided during turning. The cultures were incubated at 37.6°C, R.H. 50-60%, rocking them through an angle of 30° at hourly intervals for 14 days. They were then moved to a hatcher and incubated at 37.5°C, R.H. 60-70%, in a stationary position. The cling film was perforated at 1-2 days before hatch to facilitate respiration, and the cling film replaced with a petri dish lid 6-12h before hatch. As controls, embryos obtained from freshly laid eggs were transferred to small, then large recipient egg shells, incubating them in the same conditions as for the uterine embryos. All the experimental procedures were carried out under sterile conditions. In this specification the first day of development of the uterine embryo is given as the day of ovulation. The results obtained were as follows. The rate of induced premature oviposition of the uterine eggs was 90.4% and individual differences were observed in response to vasopressin. The state of the eggs obtained was somewhat flaccid (15 eggs) to plumped (25 eggs) . The results for the development of uterine embryos are given in Table 1. Table 1 Percent viability of cultured blastodiscal embryos (uterine egg, day 0) and blastodermal embryos (laid egg, day 1) of the chick. Day Uterine egg Laid egg 0 n=40 1 •• n=32 2 •• 3 •• • 4 62.5 100.0 5 55.0 96.9 6 55.0 96.9 7 55.0 93.8 8 52.5 90.6 9 45.0 90.6 10 42.5 90.6 11 42.5 90.6 12 42.5 87.5 13 42.5 87.5 14 42.5 87.5 15 40.0 87.5 16 37.5 84.4 17 37.5 81.3 18 37.5 81.3 19 37.5 81.3 20 35.0 78.1 21 35.0 78.1 22 35.0 71.9 Hatch 22.5 62.5 At day 1, the blastodermal area was similar in appearance to embryos obtained from unincubated eggs and exhibited a growth ring formed of the area opaca and the area pellucida. The viability was about 60% at day 4 and, by day 19, it had declined to 37.5%. The beak became visible on day 20 and penetrated the chorioallantoic membrane on day 21 when active respiratory movements were observed. On day 22, the chicks emerged through the existing aperture of the recipient shells. Hatchability of the uterine embryos was 22.5%; high mortality was observed at day 4 and just before hatch. No differences in hatchability were noted between embryos cultured from the earlier stage (3/15) or the later stage (6/25) of uterine development. Of the nine chicks obtained, six were healthy and were reared to maturity. The 4 males and 2 females showed normal semen and egg production. the two pairs were artificially inseminated, and 13 and 14 eggs were obtained for 14 days. The fertility was 100% and 100%, and the hatchability was 100% and 92.9%, respectively in each pair. For embryos cultured from unincubated eggs, the viability was 100% on day 4, about 80% on day 18 and 62.5% at hatch. By comparison, the development of embryos from uterine eggs was slightly retarded, requiring an extra 12h to reach the hatching stage. The weight of the chicks was about 60% of the egg weight (Table 2) . Egg weight for uterine eggs was estimated from the weight of the preceding egg, and for unincubated eggs it was the weight of the donor egg. Table 2 Relationship between chick weight and egg weight for embryos cultured from uterine eggs and laid eggs Chick weight n Chick Egg Weight Weight Egg weight % Uterine egg 9 35.4+3.5 58.0+5.0* 61.2+4.4 Laid egg 14** 32.5+2.6 53.2+3.7 61.2+4.5 Mean +S.D. *: Previous egg weight **:Fourteen chicks were weighed out of 20 chicks REFERENCES Beadle, B.W. , Conrad, R.M. and Scott, H.M. (1938). Composition of the uterine secretion of the domestic fowl. Poultry Sci. 17: 498-504. Callebaut, M. (1983) . Autoradiographic demonstration of the penetration of albumen-derived material through the vitelline membrane into the egg yolk, exterior to the avian blastoderm. Poultry Sci. 62 : 1657-1659. Crittenden, L.B. and Salter, D.W. (1986). Gene insertion and long-term goals. Avian Diseases. 30: 43-46. Davidson, M. F. and Draper, M.H. (1969 ) . The accumulation of glucose in the white of the egg of the hen. J. Physiol . 202 : 119-120p. Dawes, C.H. (1975) . Acid-base relationships within the avian egg. 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Ph.D. Thesis, Univ. of Edinburgh. Lundy, H. (1969) A review of the effects of temperature, humidity, turning and gaseous environment in the incubator on the hatchability of the hen's egg. In: T.C. Carter and B.M. Freeman (Eds) The Fertility and Hatchability of the Hen's Egg, pp. 143-176 (Edinburgh, Oliver and Boyd) . Mindur, C. , Krawczyk, E. and Wezyk, S. (1985) Development of uterine chick embryos after storage at 5°C for 15 hours. British Poultry Science 26 527-529. New, D.A.T. (1956) . The formation of the sub-blastodermic fluid in the hens' egg. J. Embryol. exp. Morph. 4 : 221-227. New, D.A.T. (1957) . A critical period for the turning of hens' eggs. J. Embryol. exp. Morph. 5 : 293-299. New, D.A. T . ( 1966) . "The culture of vertebrate embryos" . London : Academic Press . Olsen , M. W. and Neher , B . H . ( 1948 ) The site of fertilization in the domestic fowl . Journal of Experimental Zoolocfy 109 355-366. Olszanska, B. , Szolajska, E . and Lassota, Z . ( 1984) . Effect of spatial position of uterine quail blastoderms cultured in vitro on bilateral symmetry formation. Roux' s Arch. Dev . Biol . 193 : 108-110. Ono, T. and Wakasugi, N. (1984) . Mineral content of quail embryos cultured in mineral-rich and mineral-free conditions. Poultry Sci. 63: 159-166. Perry, M.M. (1986a) . Embryo manipulation in poultry. Proc. XXVII. Br. Poultry Breeders' Roundtable. Perry, M.M. (1986b) . Embryo manipulation in poultry. Edinburgh Centre for Rural Economy. Annual report, 1985-86, 19-24. Perry, M.M. (1987) . Nuclear events from fertilisation to the early cleavage stages in the domestic fowl. (Gallus domesticus) . J. Anat. 150, 99-109. Perry, M.M. (1988) . A complete culture system for the chick embryo. Nature, 331, 70-72. Romanoff, A.L. (1943). Cultivation of the early chick embryo in vitro. Anat. Rec. 87 : 365-369. Rowlett, K. and Si kiss, K. (1985) . The surrogate egg. New Scientist. 1469 : 42-44. Rowlett, K. and Simkiss, K. (1987) . 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Claims: CLAIMS 1. A process for the in vitro culture of an avian embryo up to blastoderm formation, the process comprising abstracting a uterine embryo from a hen and culturing the embryo partially submerged in culture medium. 2. A process as claimed in claim 1 wherein the abstraction of the uterine embryo is achieved by induced premature oviposition. 3. A process as claimed in claim 2 wherein premature oviposition is induced with vasopressin or a prostaglandin. 4. A process as claimed in claim 1 wherein abstraction of the uterine embryo is by manual abstraction. 5. A process as claimed in claim 1 wherein the embryo is abstracted at from 6 to 9 hours after oviposition of the previous egg. 6. A process as claimed in claim 1 wherein the embryo abstracted contains from 16 to 128 cells. 7. A process as claimed in claim 1 wherein the culture medium is albumen. 8. A process as claimed in claim 1 wherein the albumen is diluted with water or salt solution. 9. A process as claimed in claim 1 wherein the uterine embryo is cultured in the medium for from 12 to 24 hours. 10. A process as claimed in claim 1 wherein the culture medium is generally in line with the germinal disc but below the level of the albumen capsule.

Inventors: PERRY MARGARET MARY, PERRY, MARGARET, MARY
Applicants: AGRICULTURAL & FOOD RES, THE AGRICULTURAL AND FOOD RESEARCH COUNCIL
Application year: 1990
Main classification: C12N5
IPC codes: C12N5