Reproduction, Fertility and Development Reproduction, Fertility and Development Society
Vertebrate reproductive science and technology

Synchronisation of canine germinal vesicle stage oocytes prior to in vitro maturation alters the kinetics of nuclear progression during subsequent resumption of meiosis

Carol Hanna A , Suzanne Menges A , Duane Kraemer A and Charles R. Long A B

A Texas A and M University, College of Veterinary Medicine, Department of Physiology and Pharmacology, College Station, TX 77843-4466, USA.

B Corresponding author. Email:

Reproduction, Fertility and Development 20(5) 606-614
Submitted: 20 December 2007  Accepted: 7 April 2008   Published: 21 May 2008


Inhibition of meiosis before in vitro maturation (IVM) can improve meiotic competence in immature mammalian oocytes. Therefore, meiosis-inhibiting agents were evaluated singularly for the ability to arrest and synchronise germinal vesicle (GV) stage canine oocytes, and the most effective treatments were combined to improve meiotic resumption rates. Oocytes cultured in 2 ng mL–1 oestradiol (E2), 10 IU mL–1 eCG, or both (EG) for 72 h resulted in significantly fewer oocytes resuming meiosis in EG than the control, E2, or with eCG. Oocytes cultured in 50 or 100 μmol L–1 of butyrolactone 1 or roscovitine (ROS) for up to 48 h did not resume meiosis nor increase subsequent meiotic resumption rates following IVM. A combination of 50 μmol L–1 ROS and EG treatment for 48 h significantly increased the proportion of canine oocytes in meiotic arrest. More importantly, following 48 h of IVM, ROS+EG-treated oocytes demonstrated a dramatic increase in the ability to resume meiosis compared with the non-treated controls (51.3 ± 8.2% and 10.8 ± 4.5%, respectively; P < 0.05). These data indicate that chemical and biological meiotic inhibitors are effective at inducing GV arrest in canine oocytes. Furthermore, these inhibitors are reversible and beneficial to subsequent meiotic resumption in vitro.


Adona P. R., Pires P. R., Quetglas M. D., and Leal C. L. (2007). Prematuration of bovine oocytes with butyrolactone I: effects on meiosis progression, cytoskeleton, organelle distribution and embryo development. Anim. Reprod. Sci., in press.

Adona P. R.Pires P. R.Quetglas M. D.Schwarz K. R.Leal C. L.2008Nuclear maturation kinetics and in vitro embryo development of cattle oocytes prematured with butyrolactone I combined or not combined with roscovitine.Anim. Reprod. Sci.104389397doi:10.1016/J.ANIREPROSCI.2007.06.013Pubmed Abstract

Barretto L. S.Caiado Castro V. S.Garcia J. M.Mingoti G. Z.2007Role of roscovitine and IBMX on kinetics of nuclear and cytoplasmic maturation of bovine oocytes in vitro.Anim. Reprod. Sci.99202207doi:10.1016/J.ANIREPROSCI.2006.06.001Pubmed Abstract

Bolamba D.Russ K. D.Harper S. A.Sandler J. L.Durrant B. S.2006Effects of epidermal growth factor and hormones on granulosa expansion and nuclear maturation of dog oocytes in vitro.Theriogenology6510371047doi:10.1016/J.THERIOGENOLOGY.2005.06.017Pubmed Abstract

Choi Y. H.Love L. B.Varner D. D.Hinrichs K.2006Blastocyst development in equine oocytes with low meiotic competence after suppression of meiosis with roscovitine prior to in vitro maturation.Zygote1418doi:10.1017/S0967199406003534Pubmed Abstract

Cui X. S.Jin Y. X.Shen X. H.Lee J. Y.Lee H. S.Yin X. J.Kong I. K.Kim N. H.2006Epidermal growth factor enhances meiotic resumption of canine oocytes in the presence of BSA.Theriogenology66267274doi:10.1016/J.THERIOGENOLOGY.2005.11.011Pubmed Abstract

Farstad W.2000Assisted reproductive technology in canid species.Theriogenology53175186doi:10.1016/S0093-691X(99)00250-2Pubmed Abstract

Funahashi H.Cantley T. C.Day B. N.1997Synchronization of meiosis in porcine oocytes by exposure to dibutyryl cyclic adenosine monophosphate improves developmental competence following in vitro fertilization.Biol. Reprod.574953doi:10.1095/BIOLREPROD57.1.49Pubmed Abstract

Goodrowe K. L.Walker S. L.Ryckman D. P.Mastromonaco G. F.Hay M. A.Bateman H. L.Waddell W. T.2000Piecing together the puzzle of carnivore reproduction.Anim. Reprod. Sci.60–61389403doi:10.1016/S0378-4320(00)00084-1Pubmed Abstract

Gottelli D.Marino J.Sillero-Zubiri C.Funk S. M.2004The effect of the last glacial age on speciation and population genetic structure of the endangered Ethiopian wolf (Canis simensis).Mol. Ecol.1322752286doi:10.1111/J.1365-294X.2004.02226.XPubmed Abstract

Grupen C. G.Fung M.Armstrong D. T.2006Effects of milrinone and butyrolactone-I on porcine oocyte meiotic progression and developmental competence.Reprod. Fertil. Dev.18309317doi:10.1071/RD05125Pubmed Abstract

Hedrick P. W.Lee R. N.Buchanan C.2003Canine parvovirus enteritis, canine distemper, and major histocompatibility complex genetic variation in Mexican wolves.J. Wildl. Dis.39909913Pubmed Abstract

Hewitt D. A.England G. C. W.1998Incidence of oocyte nuclear maturation within the ovarian follicle of the bitch.Vet. Rec.143590591Pubmed Abstract

Hinrichs K.Williams K. A.1997Relationships among oocyte-cumulus morphology, follicular atresia, initial chromatin configuration, and oocyte meiotic competence in the horse.Biol. Reprod.57377384doi:10.1095/BIOLREPROD57.2.377Pubmed Abstract

Hoffmann U.Mostl E.2001Oestrogen concentrations in the faeces of bitches.J. Reprod. Fertil. Suppl.576770Pubmed Abstract

Hossein M. S.Kim M. K.Jang G.Oh H. J.Koo O.Kim J. J.Kang S. K.Lee B. C.Hwang W. S.2007Effects of thiol compounds on in vitro maturation of canine oocytes collected from different reproductive stages.Mol. Reprod. Dev.7412131220doi:10.1002/MRD.20674Pubmed Abstract

Kageyama S.Liu H.Kaneko N.Ooga M.Nagata M.Aoki F.2007Alterations in epigenetic modifications during oocyte growth in mice.Reproduction1338594doi:10.1530/REP-06-0025Pubmed Abstract

Kim M. K.Hossein M. S.Oh H. J.Fibrianto H. Y.Jang G.Kim H. J.Hong S. G.Park J. E.Kang S. K.Lee B. C.2007Glutathione content of in vivo- and in vitro-matured canine oocytes collected from different reproductive stages.J. Vet. Med. Sci.69627632doi:10.1292/JVMS.69.627Pubmed Abstract

Klein R.Schams D.Failing K.Hoffmann B.2003Investigations on the re-establishment of the positive feedback of oestradiol during anoestrus in the bitch.Reprod. Domest. Anim.381320doi:10.1046/J.1439-0531.2003.00389.XPubmed Abstract

Krischek C.Meinecke B.2001Roscovitine, a specific inhibitor of cyclin-dependent protein kinases, reversibly inhibits chromatin condensation during in vitro maturation of porcine oocytes.Zygote9309316Pubmed Abstract

Kubelka M.Motlik J.Schultz R. M.Pavlok A.2000Butyrolactone I reversibly inhibits meiotic maturation of bovine oocytes, without influencing chromosome condensation activity.Biol. Reprod.62292302doi:10.1095/BIOLREPROD62.2.292Pubmed Abstract

Kutzler M. A.2005Semen collection in the dog.Theriogenology64747754doi:10.1016/J.THERIOGENOLOGY.2005.05.023Pubmed Abstract

Kutzler M. A.2007Estrus induction and synchronization in canids and felids.Theriogenology68354374doi:10.1016/J.THERIOGENOLOGY.2007.04.014Pubmed Abstract

Lee H. S.Seo Y. I.Yin X. J.Cho S. G.Lee S. S.Kim N. H.Cho S. K.Kong I. K.2007Effect of follicle stimulation hormone and luteinizing hormone on cumulus cell expansion and in vitro nuclear maturation of canine oocytes.Reprod. Domest. Anim.42561565doi:10.1111/J.1439-0531.2006.00818.XPubmed Abstract

Liu Y.Sui H. S.Wang H. L.Yuan J. H.Luo M. J.Xia P.Tan J. H.2006Germinal vesicle chromatin configurations of bovine oocytes.Microsc. Res. Tech.69799807doi:10.1002/JEMT.20349Pubmed Abstract

Lonergan P.Dinnyes A.Fair T.Yang X.Boland M.2000Bovine oocyte and embryo development following meiotic inhibition with butyrolactone I.Mol. Reprod. Dev.57204209doi:10.1002/1098-2795(200010)57:2<204::AID-MRD12>3.0.CO;2-NPubmed Abstract

Luvoni G. C.Chigioni S.Allievi E.Macis D.2005Factors involved in vivo and in in vitro maturation of canine oocytes.Theriogenology634159doi:10.1016/J.THERIOGENOLOGY.2004.03.004Pubmed Abstract

Madgwick S.Jones K. T.2007How eggs arrest at metaphase II: MPF stabilisation plus APC/C inhibition equals Cytostatic Factor.Cell Div.24doi:10.1186/1747-1028-2-4Pubmed Abstract

Nickson D. A., Boyd J. S., Eckersall P. D., Ferguson J. M., Harvey M. J. A., and Renton J. P. (1993). Molecular biological methods for monitoring oocyte maturation and in vitro fertilization in bitches. J. Reprod. Fertil. 47(Suppl.), 231–240.

Oh H. J.Fibrianto Y. H.Kim M. K.Jang G.Hossein M. S.Kim H. J.Kang S. K.Lee B. C.Hwang W. S.2005Effects of canine serum collected from dogs at different estrous cycle stages on in vitro nuclear maturation of canine oocytes.Zygote13227232doi:10.1017/S0967199405003242Pubmed Abstract

Olson P. N.Bowen R. A.Behrendt M. D.Olson J. D.Nett T. M.1982Concentrations of reproductive hormones in canine serum throughout late anestrus, proestrus and estrus.Biol. Reprod.2711961206doi:10.1095/BIOLREPROD27.5.1196Pubmed Abstract

Otoi T.Fujii M.Tanaka M.Ooka A.Suzuki T.2000Canine oocyte diameter in relation to meiotic competence and sperm penetration.Theriogenology54535542doi:10.1016/S0093-691X(00)00368-XPubmed Abstract

Otoi T.Shimizu R.Naoi H.Wongsrikeao P.Agung B.Taniguchi M.2006Meiotic competence of canine oocytes embedded in collagen gel.Reprod. Domest. Anim.411721doi:10.1111/J.1439-0531.2006.00639.XPubmed Abstract

Otoi T.Shin T.Kraemer D. C.Westhusin M.2007Role of cumulus cells on in vitro maturation of canine oocytes.Reprod. Domest. Anim.42184189doi:10.1111/J.1439-0531.2006.00750.XPubmed Abstract

Ponderato N.Crotti G.Turini P.Duchi R.Galli C.Lazzari G.2002Embryonic and foetal development of bovine oocytes treated with a combination of butyrolactone I and roscovitine in an enriched medium prior to IVM and IVF.Mol. Reprod. Dev.62513518doi:10.1002/MRD.10134Pubmed Abstract

Rodrigues B. A.Rodrigues J. L.2003Meiotic response of in vitro-matured canine oocytes under different proteins and heterologous hormone supplementation.Reprod. Domest. Anim.385862doi:10.1046/J.1439-0531.2003.00404.XPubmed Abstract

Rodrigues B. A.Dos Santos L. C.Rodrigues J. L.2006The effect of hyaluronan concentrations in hST-supplemented TCM 199 on in vitro nuclear maturation of bitch cumulus-oocyte complexes.Theriogenology6616731676doi:10.1016/J.THERIOGENOLOGY.2006.01.015Pubmed Abstract

Silva A. R.Morato R. G.Silva L. D. M.2004The potential for gamete recovery from non-domestic canids and felids.Anim. Reprod. Sci.81159175doi:10.1016/J.ANIREPROSCI.2003.10.001Pubmed Abstract

Silva A. R.Cardoso R. C.Silva L. D. M.2006Influence of temperature during glycerol addition and post-thaw dilution on the quality of canine frozen semen.Reprod. Domest. Anim.417478doi:10.1111/J.1439-0531.2006.00630.XPubmed Abstract

Songsasen N.Wildt D. E.2005Size of the donor follicle, but not stage of reproductive cycle or seasonality, influences meiotic competency of selected domestic dog oocytes.Mol. Reprod. Dev.72113119doi:10.1002/MRD.20330Pubmed Abstract

Songsasen N.Yu I.Gomez M.Liebo S. P.2003Effects of meiosis-inhibiting agents and equine chorionic gonadotropin on nuclear maturation of canine oocytes.Mol. Reprod. Dev.65435445doi:10.1002/MRD.10321Pubmed Abstract

Songsasen N.Spindler R. E.Wildt D. E.2007Requirement for, and patterns of, pyruvate and glutamine metabolism in the domestic dog oocyte in vitro.Mol. Reprod. Dev.74870877doi:10.1002/MRD.20667Pubmed Abstract

Vanhoutte L.De Sutter P.Nogueira D.Gerris J.Dhont M.Van der Elst J.2007Nuclear and cytoplasmic maturation of in vitro-matured human oocytes after temporary nuclear arrest by phosphodiesterase 3-inhibitor.Hum. Reprod.2212391246doi:10.1093/HUMREP/DEM007

Vannucchi C. Oliveira C. M.Marques M. G.Assumpcao M. E. O. A.Visintin J. A.2006In vitro canine oocyte nuclear maturation in homologous oviductal cell co-culture with hormone- supplemented media.Theriogenology6616771681doi:10.1016/J.THERIOGENOLOGY.2006.01.008Pubmed Abstract

Verstegen J. P.Onclin K.Iguer-Ouada M.2005Long-term motility and fertility conservation of chilled canine semen using egg yolk added Tris-glucose extender in vitro and in vivo studies.Theriogenology64720733doi:10.1016/J.THERIOGENOLOGY.2005.05.035Pubmed Abstract

Ververidis H. N.Boscos C. M.Stefanakis A.Krambovitis E.2002Use of enzyme-immunoassay for oestradiol-17beta and progesterone quantification in canine serum.Anim. Reprod. Sci.695364doi:10.1016/S0378-4320(01)00171-3Pubmed Abstract

Weng Y. C.Sha S. W.Chiou C. M.Tang P. C.Yang J. H.Ju J. C.2007Butyrolactone I reversibly alters nuclear configuration, perioplasmic microtubules and development of porcine oocytes.Theriogenology67509519doi:10.1016/J.THERIOGENOLOGY.2006.08.017Pubmed Abstract

Wickramasinghe D.Ebert K. M.Albertini D. F.1991Meiotic competence acquisition is associated with the appearance of M-phase characteristics in growing mouse oocytes.Dev. Biol.143162172doi:10.1016/0012-1606(91)90063-9Pubmed Abstract

Willingham-Rocky L. A.Hinrichs K.Westhusin M. E.Kraemer D. C.2003Effects of stage of oestrous cycle and progesterone supplementation during culture on maturation of canine oocytes in vitro.Reproduction126501508doi:10.1530/REP.0.1260501Pubmed Abstract

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