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    Tervit

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    Suhail Magray
    This document summarizes research on culturing sheep and cattle embryos in vitro. Key findings include: - Sheep 8-cell embryos developed best to morulae in synthetic oviduct fluid with 5% oxygen over 3 days of culture. - Cultured sheep embryos transferred to recipients developed to normal 13-14 day conceptuses, though survival was lower for 1-cell and 2-4 cell embryos. - Cattle 1-cell embryos were retarded in culture compared to in vivo development, while 8-cell embryos developed at a similar rate to in vivo in culture.

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    Tervit

    Uploaded by

    Suhail Magray
    21 views5 pages
    This document summarizes research on culturing sheep and cattle embryos in vitro. Key findings include: - Sheep 8-cell embryos developed best to morulae in synthetic oviduct fluid with 5% oxygen over 3 days of culture. - Cultured sheep embryos transferred to recipients developed to normal 13-14 day conceptuses, though survival was lower for 1-cell and 2-4 cell embryos. - Cattle 1-cell embryos were retarded in culture compared to in vivo development, while 8-cell embryos developed at a similar rate to in vivo in culture.

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    This document summarizes research on culturing sheep and cattle embryos in vitro. Key findings include: - Sheep 8-cell embryos developed best to morulae in synthetic oviduct fluid with 5% oxygen over 3 days of culture. - Cultured sheep embryos transferred to recipients developed to normal 13-14 day conceptuses, though survival was lower for 1-cell and 2-4 cell embryos. - Cattle 1-cell embryos were retarded in culture compared to in vivo development, while 8-cell embryos developed at a similar rate to in vivo in culture.

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    © All Rights Reserved
    Download as PDF, TXT or read online from Scribd
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    21 views5 pages

    Tervit

    Uploaded by

    Suhail Magray
    This document summarizes research on culturing sheep and cattle embryos in vitro. Key findings include: - Sheep 8-cell embryos developed best to morulae in synthetic oviduct fluid with 5% oxygen over 3 days of culture. - Cultured sheep embryos transferred to recipients developed to normal 13-14 day conceptuses, though survival was lower for 1-cell and 2-4 cell embryos. - Cattle 1-cell embryos were retarded in culture compared to in vivo development, while 8-cell embryos developed at a similar rate to in vivo in culture.

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    of 5

    SUCCESSFUL CULTURE IN VITRO OF SHEEP

    AND CATTLE OVA


    H. R. TERVIT, D. G. WHITTINGHAM and L. E. A. ROWSON
    Agricultural Research Council, Unit of Reproductive Physiology and Biochemistry,
    307 Huntingdon Road, Cambridge CB3 0JQ, and
    *Physiological Laboratory, University of Cambridge
    (Received 15th April 1972, accepted 9th May 1972)
    Fertilized sheep and cattle ova have not been reported to develop readily
    during culture in vitro. Up to 60% of sheep morulae develop normally during
    culture (Moor & Cragle, 1971) but earlier cleavage stages undergo limited
    development (Hancock, 1963; Kraemer, 1966; Tervit & McDonald, 1969;
    Moore, 1970) and it has been suggested that there is a block to development in
    vitro at the eight- to twelve-cell stage (Wintenberger, Dauzier & Thibault, 1953).
    Only the early cleavage stages of cattle ova have been cultured and these have
    not been reported to develop beyond the twenty-four-cell stage in vitro (Thibault,
    1966; Brinster, 1968; Sreenan, 1968; Sreenan, Scanlon & Gordon, 1968). This
    communication describes the successful culture of one-cell to eight-cell sheep
    ova and one-cell and eight-cell cattle ova to the morula and blastocyst stages
    and reports a high embryo survival after transfer of cultured ova to recipient
    animals.
    Welsh mountain ewes were induced to superovulate and were mated at the

    ensuing oestrus. They were subjected to laparotomy 48 to 72 hr after the onset


    of oestrus and the embryos were flushed from the reproductive tract in a modi¬
    fied Dulbecco's phosphate-buffered salt solution (PBS) supplemented with 0-33
    mM-sodium pyruvate, 5-56 mM-glucose, 4 mg/ml bovine serum albumin and
    100 U/ml penicillin (sodium salt). This medium was similar to that used
    successfully for the freeze-preservation of mouse embryos (Whittingham, 1971a)
    and had an osmolarity of 290 mosmol and a pH of 7-2. The ova were held at
    37° C in this medium until they were placed in culture. Cattle ova were flushed
    from the reproductive tracts of superovulated donors with TCM-199 by the
    technique described by Rowson, Moor & Lawson (1969) and were held at 37° C
    in this medium before being placed in culture.
    Preliminary experiments examined the development of eight-cell sheep ova
    in a series of biological and chemically defined media. The embryos were
    cultured in droplets of medium under light-weight liquid paraffin in an atmos¬
    phere of 5% C02 in air (Brinster, 1963). The results showed that ova cleaved
    most readily in a medium with a composition based on the biochemical analysis
    of sheep oviduct fluid (Restall & Wales, 1966). In this medium, which will be
    referred to as synthetic oviduct fluid (SOF), eight-cell ova cleaved to an average
    of twenty-four cells as measured by the number of nuclei in each embryo after
    3 days in culture. The medium contained 107-70 mM-NaCl, 7-16 mM-KCl,
    493
    494 H. R. Tervit et al.
    119 mM-KH2P04, 1-71 mM-CaCl2, 0-49 mM-MgCl2, 2507 mM-NaHC03, 3-30
    mM-Na lactate, 0-33 mM-Na pyruvate, 1-50 mM-glucose, 32 mg bovine serum
    albumin (1 ml), 100 units penicillin (sodium salt)/ml and 50 µg streptomycin/
    ml. The osmolarity was 270 mosmol and the pH 7-2 to 7-4. The medium was
    prepared in 13 ml vols from stock solutions using the routine described by
    Whittingham (1971b). After millipore (0-45 µ ) filtration, it was stored in
    air-tight test-tubes at 5° C.
    Since oxygen concentration has been shown to be an important factor for the
    normal development of cultured mouse embryos (Whitten, 1971), sheep
    embryos were cultured in SOF in the presence of varying oxygen concentrations
    in an attempt to further their development in vitro. The embryos were cultured
    in 6-ml glass test-tubes using the method described by Biggers, Whitten &
    Whittingham (1971). One ml of SOF was equilibrated, in the tubes, by gassing
    for approximately 1 min with one of the following gas mixtures: 5% C02/95%
    N2; 5% C02/5% O2/90% N2; 5% CO2/10% 02/85% N2; or 5% C02 in air,
    and the gassed tubes were sealed and placed in an incubator at 37° C overnight.
    The sheep ova collected in modified PBS were washed in 2 ml SOF, previously
    gassed with 5% C02 in 95% N2 and were then placed into equilibrated test-
    tubes which were regassed with the appropriate gas mixture, corked and
    incubated at 37° C. Every 2nd day, the cultures were regassed. As a control,
    ova were cultured in droplets of medium under liquid paraffin in an atmosphere
    of 5% C02 in air. After culture, the embryos were either fixed, stained and
    examined for the number of nuclei present or held in the supplemented PBS
    until transfer to recipient animals.
    The development of sheep ova in various oxygen concentrations is summarized
    in Table 1. Initially, ten eight-cell ova were cultured for 3 days at each oxygen
    level and were stained to examine for the number of nuclei present. Over a
    period of 3 days, the eight-cell sheep ovum would be expected to develop in vivo
    to a late morula. Of the stained ova, those cultured in 5 % oxygen gave the
    most comparable development in vitro. Ova cultured in 10% oxygen, although
    cleaving readily, were less developed after 3 days culture. Before culture, ova
    were washed in SOF exposed to air, and it is possible that extra oxygen was
    transferred with the ova to the test-tubes. This addition of oxygen may explain
    the development of ova during 'anaerobic' culture. Alternatively, either the
    eight-cell ovum may have sufficient energy reserves to support at least one
    cleavage in the absence of oxygen or some embryos may have been recovered
    just before mitosis. High concentrations of oxygen inhibited development of ova
    cultured both in test-tubes and under paraffin oil. This effect is similar to that
    reported for mouse embryo culture (Whitten, 1971). Under conditions in vivo,
    the one-cell and two- to four-cell sheep ovum would be expected to develop to
    a blastocyst over 6 and 5 days, respectively, and attempts were made to culture
    these early stages in 5 % oxygen. After culture for 5 or 6 days, the one-cell ova
    were slightly retarded and the two- to four-cell ova rather more retarded, with
    most of the latter developing to morulae.
    Cultured sheep ova were transferred to recipients whose oestrous cycles were
    closely synchronized ( ± 24 hr) with the stage of development of the ova. One
    uterine horn of each recipient was ligated anterior to the uterine body so that
    Culture of sheep and cattle ova in vitro 495

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    496 H. R. Tervit et al.
    ova from different treatments transferred to the two uterine horns could not
    migrate between horns. The recipients were slaughtered by Day 14 to ensure
    that embryos in the contralateral horn were not lost through any uterine
    luteolytic effect due to the death of embryos in the uterine horn ipsilateral to
    the corpus luteum. After transfer ofthirteen cultured eight-cell ova, eleven were
    recovered as viable conceptuses on Day 13 or 14. The conceptuses were measured
    for total length and length of embryonic disc and were found to be normal in
    appearance and to have developed to a stage comparable with non-cultured
    13- and 14-day conceptuses (Rowson & Moor, 1966). A lower proportion of
    cultured one-cell and two- to four-cell ova were recovered as viable embryos
    and, as expected, ova showing the greatest development during culture survived
    most readily on retransfer to recipient ewes. The lower survival of cultured
    one-cell and two- to four-cell ova, relative to eight-cell ova, may be due to the
    increased length of culture of these ova and not to the cleavage stage at com¬
    mencement. In a further examination of the viability of the cultured ovum,
    embryos have been transferred to recipients which have been allowed to proceed
    to term.
    The cattle ova recovered in TCM-199 were washed in 2 ml of pregassed SOF
    before being placed into test-tubes containing SOF equilibrated with 5% 02.
    The development of ova during culture is shown in Table 1. The one-cell cattle
    ovum would be expected to develop in vivo to a late morula or early blastocyst
    over 6 days. Most cultured ova were found to be retarded. Eight-cell ova would
    be expected to develop in vivo to late morulae and blastocysts in 4 days. Cultured
    ova were found to develop at a similar rate. Four of these cultured eight-cell
    ova were transferred to synchronized recipients. One ovum was transferred to
    each uterine horn of two recipients and of these recipients, one exhibited oestrus
    17 days after transfer and one was diagnosed as carrying twin embryos when
    palpated per rectum on Day 35 of the cycle. Again, those ova which achieved
    the greatest development during culture survived most readily on transfer to
    the recipient.
    The results show that a combination of satisfactory medium and gas phase
    has allowed sheep and cattle ova to be cultured from the early cleavage stages
    to viable morulae and blastocysts and has allowed the sheep ovum to develop
    through the supposed 'block to development'.
    H.R.T. is a recipient of a New Zealand Department of Scientific and
    Industrial Research Fellowship and D.G.W. a Beit Memorial Fellow.

    REFERENCES
    Biggers, J. D., Whitten, W. K. & Whittingham, D. G. (1971) The culture of mouse embryos in vitro. In:
    Methods of Mammalian Embryology, p. 86. Ed. J. C. Daniel, Jr. Freeman, San Francisco.
    Brinster, R. L. (1963) A method for in vitro cultivation of mouse ova from two-cell to blastocyst.
    Expl Cell Res. 32, 205.
    Brinster, R. L. (1968) In vitro culture of mammalian embryos. 7. Anim. Sci. 27, Suppl. 1, 1.
    Hancock, J. L. (1963) Survival in vitro of sheep eggs. Anim. Prod. 5, 237.
    Kraemer, D. C. (1966) In vitro fertilisation and culture of ovine ova. Diss. Abstr. 27, 285.
    Moor, R. M. & Cragle, R. G. (1971) The sheep egg: enzymatic removal of the zona pellucida and
    culture of eggs in vitro. J. Reprod. Fert. 27, 401.
    Moore, N. W. (1970) Preliminary studies on in vitro culture of fertilised sheep ova. Aust. J. biol. Set. 23,
    721.
    Culture of sheep and cattle ova in vitro 497
    Restall, . J. & Wales, R. G. (1966) The Fallopian tube of the sheep. III. The chemical composition
    of the fluid from the Fallopian tube. Aust. J. biol. Sci. 19, 687.
    Rowson, L. E. A. & Moor, R. M. (1966) The development of the sheep conceptus during the first
    fourteen days. 7. Anat. 100, 777.
    Rowson, L. . ., Moor, R. M. & Lawson, R. A. S. (1969) Fertility following egg transfer in the cow:
    effect of method, medium and synchronisation of oestrus. 7. Reprod. Fert. 18, 517.
    Sreenan, J. (1968) In vivo and in vitro culture of cattle eggs. VIe Cong. Intern. Reprod. Anim. Insem.
    Artif., Paris, 1, 577.
    Sreenan, J., Scanlon, P. & Gordon, I. (1968) Culture of fertilised cattle eggs. 7. agrie. Sci., Camb. 70,
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    Tervit, H. R. & McDonald, M. F. (1969) Culture and transplantation of sheep ova. N-Z- Jl agrie.
    Res. 12, 313.
    Thibault, C. (1966) La culture in vitro de l'œuf de vache. Annls Biol. anim. Biochim. Biophys. 6, 159.
    Whitten, W. K. (1971) Nutrient requirementsfor the culture of'pre-implantation embryos in vitro. In: Advances
    in the Biosciences, Vol. 6, p. 129. Ed. G. Raspé. Pergamon Press, Oxford.
    Whittinoham, D. G. (1971a) Survival of mouse embryos after freezing and thawing. Nature, Lond. 233,
    125.
    Whittingham, D. G. (1971b) Culture of mouse ova. J. Reprod. Fert. Suppl. 14, 7.
    Winterberger, S., Dauzier, L. & Thibault, C. (1953) Le développement in vitro de l'œuf de la
    brebis et de celui de la chèvre. C.r. Séanc. Soc. Biol. 147, 1971.

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