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Developmental competence of pig oocytes matured and fertilized in vitro
THERIOGENOLOGY
DEVELOPMENTALCOMPETENCEOF FIG OOCYTES MATURED AND FERTILIZED IN VITRO M.
Mattioli.M.L. Bacci, G. Galeati and E. Seren Istitutodi FisiologiaVeterinaria Via Belmeloro812 40126 Bologna Italy
Received for publication:October 7, 1988 Accepted: ApriZ 13, 1989 ABSTRACT Pig follicles3 to 6 mm in diameter were everted and matured for 44 h. The oocytes were then collected and exposed to capacitatedboar sperm purifiedby centrifugation in a two step (65 and 70%) Percoll gradient. Of 110 ova fixed 14 h after in vitro fertilization,78% were penetrated and 47% were monospermic.Next, 681 oocytes were cultured in vitro for 44 h after in vitro fertilization and the 266 embryos which had reached the two- to four-cellstage were transferredinto the oviducts of 12 synchronized recipient gilts. Four days later, 211 embryos (7921 were recovered by uterine flushing. 40.7% of these were at the blastocyst stage, and 20% were at the morula stage. In a final experiment,four out of eight gilts which had received 40 to 50 two- to four-cellembryos,were diagnosedpregnant 30 and 37 d after in vitro fertilization.One sow farrowed nine live piglets and one stillborn, two pregnancies were in progress,while one sow returned to estrus 47 d after in vitro fertilization. These results demonstrate that pig oocytes matured and fertilizedin vitro can develop to the blastocyststage and establisha normal pregnancy resulting in the birth of live piglets. Key words: pig, in vitro fertilization,oocyte maturation, developmentalcompetence INTRODUCTION In the last few years much information has been obtained concerning the mechanisms controlling gamete maturationand interaction.This has enabled successful in vitro fertilizationof in vitro matured oocytes. In domestic animals, this goal has been reached in cows (l-3) and in sheep (41, although the low number of offspring indicates that the techniqueneeds further improvement.In the case Acknowledgements: This research was supportedby M.P.I. 60% and C.N.R. Gruppo CoordinamentoVeterinaria.
JUNE 1969 VOL. 31 NO. 6
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THERIOGENOLOGY
of the pig, piglets were born after in vitro fertilization of oocytes matured in vivo (51, whereas the in vitro maturationof pig oocytes has thus far often resulted in ova showing developmental abnormalities after fertilization. The absence or incompleteformationof a male pronucleus is the most frequently reported deviation from normal development(6-81. We have recently shown that if pig oocytes are matured with the whole wall of the everted follicle, they become capable of forming normal male pronucleiand become more fertilizable (9). However, a common defect observed in cows and sheep, resultingfrom the fertilizationof oocytes that have not reached complete maturationin vitro, is represented by the failure of oocytes to progress to organizedblastocyst-stage embryos (16, 111. Our experimentwas thus designed to test the developmentalcompetenceof pig oocytes matured in vitro and fertilizedin vitro. MATERIALSAND METHODS Oocyte Maturation Ovaries were collected at a local abatoir from prepubertalgilts with an approximateweight of 80 to 100 kg. Preliminary experimentsdid not show any significant differencein the developmentalcompetence of pig oocytes obtained from mature or immature gilts. Dissection of ovaries was carried out at 20W in Dulbecco's phosphate buffered saline supplementedwith bovine serum albumin (BSA) 0.47.(w/v),pyruvate 0.36 mM, glucose 5.5 mM and kanamycin 70 pg/ml (flushing medium). After isolation, healthy follicles (selectedon the basis of their bright translucent appearance,good vascularizationand regular granulosa)with a diameterof 3 to 6 mm were opened and turned inside out. In this way the granulosalayer as well as the cumulus oocyte complex were directly in contact with the culture medium (91. Everted follicleswere then matured in modified TCM 199 at 39°C as previouslydescribed (9). After 40 to 44 h of culture,the oocytes were collectedand exposed to capacitatedboar sperm. Sperm Preparationand Capacitation Immediately after collection, the boar sperm was diluted in a commercial-type Modena extender (Semen Italy, Modenal to a final concentration of 4 x 107 sperm/ml and incubatedfor 24 to 48 h at lb°C in a system providing gentle agitationfor 10 min every other hours. Before use, an aliquot of diluted semen was centrifugedat 1000 x g for 3 min, the pellet was resuspended in fertilization medium (TCM 199 supplementedwith pyruvate 1 mM, glucose 3.05 mM, calcium lactate 8.76 mM and fetal calf serum 127.1, and layered onto a two step Percoll gradient (65 to 70%). After
1202
JUNE 1969VOL. 31 NO. 6
THERIOGENOLOGY
centrifugationat 2000 x g, for 15 min at 20*C the sperm concentrated between 65% and 70% Percoll layers were collectedand used directly for in vitro fertilization. In this purified fraction the percentageof motile sperm always exceeded 90%, while abnormal spermatozoa (those with a protoplasmicdroplet or bent tail) comprisedless than 5% of the sperm. Ten to fifteen oocytes,after mechanical removal of expanded cumuli with a finely drawn pipette, were incubatedin 2 ml of fertilizationmedium containing1 x 10' sperm/ml.Cultureswere carried out at 390C in an atmosphere of 5% CO2 in air. Six to eight hours after exposureto sperm, the oocytes were removed from the fertilizationdishes and washed by gently pipetting through a pipette with a bore of approximately150 pm. Excessive adherent spermatozoa were removed by this procedure in order to reduce the incidence of polispermy (12). The oocytes were then cultured in Brinster'smedium to enhance early embryo development (12). Oocytes matured and fertilizedin vitro were then used for the followingtrials. Trial 1. Oocytes tn = 110) were collected 14 h after exposureto sperm and fixed in acetic alcohol (l:3 v/v). Forty-eighthours later, the eggs were stained with Lacmoid to evaluate the penetration rate and the incidence of polispermyby phase contrastmicroscopy. Trial 2. Oocytes tn 681) were cultured for a total of 44 h after exposureto sperm, and the embryos reaching the two- to four-cellstage were transferredinto the oviduct of synchronizedrecipientgilts (about ten embryos per oviduct) through a mid-ventralincisionunder general anesthesia. Estrus inductionin prepubertalgilts (average weight 80 kg) was carried out by administrationof pregnant mare serum gonadotropinCPMSGf (1250 IU i.m.1 followed 56 h later by human chorionicgonadotropin(hCG) (750 IU). The hCG was given 40 to 44 h before the programedtime for in vitro fertilization. Four days later the gilts were again anesthetizedand their uterine horns were flushed . q
Trial 3. Two- to four -cell embryos (n = 380) obtained as describedabove, were transferredinto the oviducts of eight recipientgilts. The animals were kept in individual crates, and pregnancydiagnosiswas carried out 30 and 37 d after in vitro fertilization by means of a Kontron ultrasoundscanner (ModelSIGMA 10). RESULTS Microscopic examination in Trial 1 of 110 oocytes collected14 h after exposure to capacitated boar sperm demonstrateda fertilizationrate of 782. Of these ova, 47% were monospermic, while the overall mean number of
JUNE 1969 VOL. 31 NO. 6
1203
THERIOGENOLOGY
sperm/penetrated oocyte was
1.67
~0.30
(x
+SEMI.
In Trial 2, the first two-cellembryos were observed 20 to 30 h after in vitro fertilizationand by 44 h, 266 out of 681 oocytes (39%) reached the two- to four-cellstage (Table 11. These embryos, classifiedas morphologically normal, judged by the dimensionof the blastomersand the uniform distributionof the dark cytoplasm,were transferredinto 12 recipientgilts. Four days later, a total of 211 embryos (73%) were recoveredby uterine flushing. As shown in Table 1, 41% of recovered embryos had reached the blastocyst stage. A small proportionof these had hatched. Twenty percent of recoveredembryos were at the morula stage, while the remainingembryos were degenerated and/or fragmented. Table 1. Embryonicdevelopmental stages from pig oocytes matured and fertilizedin vitro No. of oocytes exposed to sperm
681
No. of two- to four-cellembryos after 48 h (% of oocytes exposed to sperm1
266 (39)
No. embryos recovered4 d after transfer (% of transferredembryos)
211
(791
No. of morulae (% of recoveredembryos)
42 (20)
No. of blastocysts (% of recoveredembryos)
86 (41)
No. of hatched blastocysts (% of total blastocystsl
11 (13)
In Trial 3, four out of eight recipient gilts were diagnosedpregnant 30 and 37 d after in vitro fertilization. One of these farrowednine live piglets and one stillborn. Pregnancywas in progress in two animals, while one gilt aborted and returned to estrus 47 d after in vitro fertilization. DISCUSSION The data reported in this paper demonstratethat pig oocytes matured and fertilizedin vitro can undergo normal embryonic development. For the first time, blastocyst formation,establishmentof pregnancies, and birth of live piglets were obtainedusing this technique.The methodology involves two distinct processeswhich need to be considered
1204
JUNE 1969 VOL. 31 NO. 6
THERIOGENOLOGY
separately:in vitro maturationand in vitro fertilization. Concerning oocyte maturation,our results demonstrate the suitability of the culture condition used. We have recently shown that the follicle wall during maturation is necessaryfor the oocyte to undergo normal fertilization (9); the data presentedhere indicatethat complex ooplasm maturation,essentialfor early embryo development(II)). was also achieved in our in vitro fertilization system. A similar follicle cell requirement for full maturation of sheep and cow oocytes has already been demonstrated (10, 11). The process by which this somatic tissue influences oocyte maturationhas been discussedelsewhere (141. The methodologyused for in vitro fertilizationin our study resulted in a high level of fertilization, suggesting that the sperm treatment was suitable for inducing capacitation.However, about half of the penetrated eggs were polispermic. This cannot be ascribed to inadequate maturationof the oocytes since the same situationof high polispermyhas been observed after in vitro fertilizationof pig oocytes matured in vivo (5, 12. 13). This ineffective block to polispermy could be dependent on the cortical granule substancesecretedafter fertilizationin vitro that does not diffuse in the perivitelline space, as occurs in vivo (13). The absence of an adequate cellular environment such as that of the oviduct may be at the root of this problem. Further research is thus required to optimize the in vitro fertilization system, taking into consideration parameterssuch as sperm concentrationand the volume of the medium in which the oocytes and sperm are coincubated. Early embryo developmentafter in vitro fertilization was slightlyretarded when compared with that of in vivo fertilization(151: pronucleidid not appear earlier than 10 h after in vitro fertilization(91, and the first cleavage did not take place until1 about 30 h after in vitro fertilization,with a delay of about 10 h. However, 6 d after in vitro fertilization, the embryos were perfectly synchronizedwith the in vivo process, as the blastocysts had just started to hatch (15). In conclusion,the possibilityof obtaininga large quantity of embryos capable of normal developmentrepresents an importantstep both for the study of gamete interaction and in future applications of biotechnologysuch as the introductionof foreign genes in early stage embryos and in nuclear transplantation.
JUNE 1969 VOL. 31 NO. 6
1205
THERIOGENOLOGY
REFERENCES 1. Lu, K.H., Gordon, I., Gallangher,M. and MC Govern, H. pregnancyestablishedin cattle by transferof embryos derived from in vitro fertilizationof oocytes matured in vitro. Vet. Rec. 121:259-260(19871. 2. Xu, K.P., Greve, T., Callasen, H. and Hyttel, P. pregnancyresulting from cattle oocytes matured and fertilized in vitro. J. Reprod. Fertil. 81:501-504 (19871. and Toyoda, Y. 3. Fukuda. Y., Ichikawa, M., Naito, K. Normal developmentof bovine oocytes matured, fertilized and cultured with cumulus cells in vitro. 11th Int. Congr. Anim. Reprod. Artif. Insem. ;1:327abstr. (1988). 4. Crozet, N., Hunecew, D., Desmedt. V., Theron, M.C., Szollosi,D., Torres, S. and Sevellec, C. In vitro fertilizationwith normal developmentin sheep. Gamete Res. 16:159-170 (1987). 5.
Cheng, N.T.K., Moor, R.M. and Polge, C. In vitro fertilizationof pig and sheep oocytes matured in vivo and in vitro. Theriogenologyz146 (1986).
6. Motlik, J. and Fulka, J. Fertilizationof pig follicular oocytes cultivated in vitro. J. Reprod. Fertil. 36:235-237 (1974). 7. Nagai, T., Niwa, K. and Iritani, A. Effect of sperm concentrationduring preincubation in a defined medium on fertilizationin vitro of pig follicularoocytes. J. Reprod. Fertil. m271-275 (1984). 8. Toyoda, Y., Itagaki, Y., Minato, Y. and Fukuda, Y. Fertilizationof pig eggs matured in vivo and in vitro. Proc. 10th Int. Congr. Anim. Reprod. Artif. Insem.&395 abstr. (1984). 9. Mattioli,H., Galeati, G. and Seren, E. Effect of folliclesomatic cells during pig oocyte maturation on egg penetrabilityand male pronucleusformation. Gamete Res. 28:177-184(19881. 10. Staigmiller,R.B. and Moor, R.M. Effect of follicle cells on the maturationand developmentalcompetence of ovine oocytes matured outside the follicle.Gamete Res. 5L:221-229 (1984).
1206
JUNE 1989VOL. 31 NO. 6
THERIOGENOLOGY 11.
Leibfried-Rutledge,M.L., Cristier. E.S., Eyestone, W.H., Northey, D.L. and First, N.L. Development potentialof bovine oocytes matured in vitro or in vivo. Biol. Reprod.-376-383 (19871.
12. Cheng, W.T.H. In Vitro Fertilization of Farm Animal Oocytes.ph. D. Thesis. Instituteof Animal physiology, Cambridge,1985. 13. Cran, D.G., Cheng, W.T.K. The corticalreactionof pig oocytes during in vivo and in vitro fertilization. Gamete Res. 13:241-251 (19861. 14. Mattioli,M., Galeati, G., Bacci, H.L. and Seren, E. Follicularfactors influence oocyte fertilizability by modulating intercellular cooperation between cumulus cells and oocyte. Gamete Res. 21_:223-232 (19881. 15. Hunter, R.H.F. Chronologicaland cytologicaldetails of fertilizationand early embryonicdevelopment in the domestic pig, Sus scrofa. Anat. Rec. 178:169-186(1974).
JUNE 1969VOL.
31 NO. 6
1207
DEVELOPMENTALCOMPETENCEOF FIG OOCYTES MATURED AND FERTILIZED IN VITRO M.
Mattioli.M.L. Bacci, G. Galeati and E. Seren Istitutodi FisiologiaVeterinaria Via Belmeloro812 40126 Bologna Italy
Received for publication:October 7, 1988 Accepted: ApriZ 13, 1989 ABSTRACT Pig follicles3 to 6 mm in diameter were everted and matured for 44 h. The oocytes were then collected and exposed to capacitatedboar sperm purifiedby centrifugation in a two step (65 and 70%) Percoll gradient. Of 110 ova fixed 14 h after in vitro fertilization,78% were penetrated and 47% were monospermic.Next, 681 oocytes were cultured in vitro for 44 h after in vitro fertilization and the 266 embryos which had reached the two- to four-cellstage were transferredinto the oviducts of 12 synchronized recipient gilts. Four days later, 211 embryos (7921 were recovered by uterine flushing. 40.7% of these were at the blastocyst stage, and 20% were at the morula stage. In a final experiment,four out of eight gilts which had received 40 to 50 two- to four-cellembryos,were diagnosedpregnant 30 and 37 d after in vitro fertilization.One sow farrowed nine live piglets and one stillborn, two pregnancies were in progress,while one sow returned to estrus 47 d after in vitro fertilization. These results demonstrate that pig oocytes matured and fertilizedin vitro can develop to the blastocyststage and establisha normal pregnancy resulting in the birth of live piglets. Key words: pig, in vitro fertilization,oocyte maturation, developmentalcompetence INTRODUCTION In the last few years much information has been obtained concerning the mechanisms controlling gamete maturationand interaction.This has enabled successful in vitro fertilizationof in vitro matured oocytes. In domestic animals, this goal has been reached in cows (l-3) and in sheep (41, although the low number of offspring indicates that the techniqueneeds further improvement.In the case Acknowledgements: This research was supportedby M.P.I. 60% and C.N.R. Gruppo CoordinamentoVeterinaria.
JUNE 1969 VOL. 31 NO. 6
1201
THERIOGENOLOGY
of the pig, piglets were born after in vitro fertilization of oocytes matured in vivo (51, whereas the in vitro maturationof pig oocytes has thus far often resulted in ova showing developmental abnormalities after fertilization. The absence or incompleteformationof a male pronucleus is the most frequently reported deviation from normal development(6-81. We have recently shown that if pig oocytes are matured with the whole wall of the everted follicle, they become capable of forming normal male pronucleiand become more fertilizable (9). However, a common defect observed in cows and sheep, resultingfrom the fertilizationof oocytes that have not reached complete maturationin vitro, is represented by the failure of oocytes to progress to organizedblastocyst-stage embryos (16, 111. Our experimentwas thus designed to test the developmentalcompetenceof pig oocytes matured in vitro and fertilizedin vitro. MATERIALSAND METHODS Oocyte Maturation Ovaries were collected at a local abatoir from prepubertalgilts with an approximateweight of 80 to 100 kg. Preliminary experimentsdid not show any significant differencein the developmentalcompetence of pig oocytes obtained from mature or immature gilts. Dissection of ovaries was carried out at 20W in Dulbecco's phosphate buffered saline supplementedwith bovine serum albumin (BSA) 0.47.(w/v),pyruvate 0.36 mM, glucose 5.5 mM and kanamycin 70 pg/ml (flushing medium). After isolation, healthy follicles (selectedon the basis of their bright translucent appearance,good vascularizationand regular granulosa)with a diameterof 3 to 6 mm were opened and turned inside out. In this way the granulosalayer as well as the cumulus oocyte complex were directly in contact with the culture medium (91. Everted follicleswere then matured in modified TCM 199 at 39°C as previouslydescribed (9). After 40 to 44 h of culture,the oocytes were collectedand exposed to capacitatedboar sperm. Sperm Preparationand Capacitation Immediately after collection, the boar sperm was diluted in a commercial-type Modena extender (Semen Italy, Modenal to a final concentration of 4 x 107 sperm/ml and incubatedfor 24 to 48 h at lb°C in a system providing gentle agitationfor 10 min every other hours. Before use, an aliquot of diluted semen was centrifugedat 1000 x g for 3 min, the pellet was resuspended in fertilization medium (TCM 199 supplementedwith pyruvate 1 mM, glucose 3.05 mM, calcium lactate 8.76 mM and fetal calf serum 127.1, and layered onto a two step Percoll gradient (65 to 70%). After
1202
JUNE 1969VOL. 31 NO. 6
THERIOGENOLOGY
centrifugationat 2000 x g, for 15 min at 20*C the sperm concentrated between 65% and 70% Percoll layers were collectedand used directly for in vitro fertilization. In this purified fraction the percentageof motile sperm always exceeded 90%, while abnormal spermatozoa (those with a protoplasmicdroplet or bent tail) comprisedless than 5% of the sperm. Ten to fifteen oocytes,after mechanical removal of expanded cumuli with a finely drawn pipette, were incubatedin 2 ml of fertilizationmedium containing1 x 10' sperm/ml.Cultureswere carried out at 390C in an atmosphere of 5% CO2 in air. Six to eight hours after exposureto sperm, the oocytes were removed from the fertilizationdishes and washed by gently pipetting through a pipette with a bore of approximately150 pm. Excessive adherent spermatozoa were removed by this procedure in order to reduce the incidence of polispermy (12). The oocytes were then cultured in Brinster'smedium to enhance early embryo development (12). Oocytes matured and fertilizedin vitro were then used for the followingtrials. Trial 1. Oocytes tn = 110) were collected 14 h after exposureto sperm and fixed in acetic alcohol (l:3 v/v). Forty-eighthours later, the eggs were stained with Lacmoid to evaluate the penetration rate and the incidence of polispermyby phase contrastmicroscopy. Trial 2. Oocytes tn 681) were cultured for a total of 44 h after exposureto sperm, and the embryos reaching the two- to four-cellstage were transferredinto the oviduct of synchronizedrecipientgilts (about ten embryos per oviduct) through a mid-ventralincisionunder general anesthesia. Estrus inductionin prepubertalgilts (average weight 80 kg) was carried out by administrationof pregnant mare serum gonadotropinCPMSGf (1250 IU i.m.1 followed 56 h later by human chorionicgonadotropin(hCG) (750 IU). The hCG was given 40 to 44 h before the programedtime for in vitro fertilization. Four days later the gilts were again anesthetizedand their uterine horns were flushed . q
Trial 3. Two- to four -cell embryos (n = 380) obtained as describedabove, were transferredinto the oviducts of eight recipientgilts. The animals were kept in individual crates, and pregnancydiagnosiswas carried out 30 and 37 d after in vitro fertilization by means of a Kontron ultrasoundscanner (ModelSIGMA 10). RESULTS Microscopic examination in Trial 1 of 110 oocytes collected14 h after exposure to capacitated boar sperm demonstrateda fertilizationrate of 782. Of these ova, 47% were monospermic, while the overall mean number of
JUNE 1969 VOL. 31 NO. 6
1203
THERIOGENOLOGY
sperm/penetrated oocyte was
1.67
~0.30
(x
+SEMI.
In Trial 2, the first two-cellembryos were observed 20 to 30 h after in vitro fertilizationand by 44 h, 266 out of 681 oocytes (39%) reached the two- to four-cellstage (Table 11. These embryos, classifiedas morphologically normal, judged by the dimensionof the blastomersand the uniform distributionof the dark cytoplasm,were transferredinto 12 recipientgilts. Four days later, a total of 211 embryos (73%) were recoveredby uterine flushing. As shown in Table 1, 41% of recovered embryos had reached the blastocyst stage. A small proportionof these had hatched. Twenty percent of recoveredembryos were at the morula stage, while the remainingembryos were degenerated and/or fragmented. Table 1. Embryonicdevelopmental stages from pig oocytes matured and fertilizedin vitro No. of oocytes exposed to sperm
681
No. of two- to four-cellembryos after 48 h (% of oocytes exposed to sperm1
266 (39)
No. embryos recovered4 d after transfer (% of transferredembryos)
211
(791
No. of morulae (% of recoveredembryos)
42 (20)
No. of blastocysts (% of recoveredembryos)
86 (41)
No. of hatched blastocysts (% of total blastocystsl
11 (13)
In Trial 3, four out of eight recipient gilts were diagnosedpregnant 30 and 37 d after in vitro fertilization. One of these farrowednine live piglets and one stillborn. Pregnancywas in progress in two animals, while one gilt aborted and returned to estrus 47 d after in vitro fertilization. DISCUSSION The data reported in this paper demonstratethat pig oocytes matured and fertilizedin vitro can undergo normal embryonic development. For the first time, blastocyst formation,establishmentof pregnancies, and birth of live piglets were obtainedusing this technique.The methodology involves two distinct processeswhich need to be considered
1204
JUNE 1969 VOL. 31 NO. 6
THERIOGENOLOGY
separately:in vitro maturationand in vitro fertilization. Concerning oocyte maturation,our results demonstrate the suitability of the culture condition used. We have recently shown that the follicle wall during maturation is necessaryfor the oocyte to undergo normal fertilization (9); the data presentedhere indicatethat complex ooplasm maturation,essentialfor early embryo development(II)). was also achieved in our in vitro fertilization system. A similar follicle cell requirement for full maturation of sheep and cow oocytes has already been demonstrated (10, 11). The process by which this somatic tissue influences oocyte maturationhas been discussedelsewhere (141. The methodologyused for in vitro fertilizationin our study resulted in a high level of fertilization, suggesting that the sperm treatment was suitable for inducing capacitation.However, about half of the penetrated eggs were polispermic. This cannot be ascribed to inadequate maturationof the oocytes since the same situationof high polispermyhas been observed after in vitro fertilizationof pig oocytes matured in vivo (5, 12. 13). This ineffective block to polispermy could be dependent on the cortical granule substancesecretedafter fertilizationin vitro that does not diffuse in the perivitelline space, as occurs in vivo (13). The absence of an adequate cellular environment such as that of the oviduct may be at the root of this problem. Further research is thus required to optimize the in vitro fertilization system, taking into consideration parameterssuch as sperm concentrationand the volume of the medium in which the oocytes and sperm are coincubated. Early embryo developmentafter in vitro fertilization was slightlyretarded when compared with that of in vivo fertilization(151: pronucleidid not appear earlier than 10 h after in vitro fertilization(91, and the first cleavage did not take place until1 about 30 h after in vitro fertilization,with a delay of about 10 h. However, 6 d after in vitro fertilization, the embryos were perfectly synchronizedwith the in vivo process, as the blastocysts had just started to hatch (15). In conclusion,the possibilityof obtaininga large quantity of embryos capable of normal developmentrepresents an importantstep both for the study of gamete interaction and in future applications of biotechnologysuch as the introductionof foreign genes in early stage embryos and in nuclear transplantation.
JUNE 1969 VOL. 31 NO. 6
1205
THERIOGENOLOGY
REFERENCES 1. Lu, K.H., Gordon, I., Gallangher,M. and MC Govern, H. pregnancyestablishedin cattle by transferof embryos derived from in vitro fertilizationof oocytes matured in vitro. Vet. Rec. 121:259-260(19871. 2. Xu, K.P., Greve, T., Callasen, H. and Hyttel, P. pregnancyresulting from cattle oocytes matured and fertilized in vitro. J. Reprod. Fertil. 81:501-504 (19871. and Toyoda, Y. 3. Fukuda. Y., Ichikawa, M., Naito, K. Normal developmentof bovine oocytes matured, fertilized and cultured with cumulus cells in vitro. 11th Int. Congr. Anim. Reprod. Artif. Insem. ;1:327abstr. (1988). 4. Crozet, N., Hunecew, D., Desmedt. V., Theron, M.C., Szollosi,D., Torres, S. and Sevellec, C. In vitro fertilizationwith normal developmentin sheep. Gamete Res. 16:159-170 (1987). 5.
Cheng, N.T.K., Moor, R.M. and Polge, C. In vitro fertilizationof pig and sheep oocytes matured in vivo and in vitro. Theriogenologyz146 (1986).
6. Motlik, J. and Fulka, J. Fertilizationof pig follicular oocytes cultivated in vitro. J. Reprod. Fertil. 36:235-237 (1974). 7. Nagai, T., Niwa, K. and Iritani, A. Effect of sperm concentrationduring preincubation in a defined medium on fertilizationin vitro of pig follicularoocytes. J. Reprod. Fertil. m271-275 (1984). 8. Toyoda, Y., Itagaki, Y., Minato, Y. and Fukuda, Y. Fertilizationof pig eggs matured in vivo and in vitro. Proc. 10th Int. Congr. Anim. Reprod. Artif. Insem.&395 abstr. (1984). 9. Mattioli,H., Galeati, G. and Seren, E. Effect of folliclesomatic cells during pig oocyte maturation on egg penetrabilityand male pronucleusformation. Gamete Res. 28:177-184(19881. 10. Staigmiller,R.B. and Moor, R.M. Effect of follicle cells on the maturationand developmentalcompetence of ovine oocytes matured outside the follicle.Gamete Res. 5L:221-229 (1984).
1206
JUNE 1989VOL. 31 NO. 6
THERIOGENOLOGY 11.
Leibfried-Rutledge,M.L., Cristier. E.S., Eyestone, W.H., Northey, D.L. and First, N.L. Development potentialof bovine oocytes matured in vitro or in vivo. Biol. Reprod.-376-383 (19871.
12. Cheng, W.T.H. In Vitro Fertilization of Farm Animal Oocytes.ph. D. Thesis. Instituteof Animal physiology, Cambridge,1985. 13. Cran, D.G., Cheng, W.T.K. The corticalreactionof pig oocytes during in vivo and in vitro fertilization. Gamete Res. 13:241-251 (19861. 14. Mattioli,M., Galeati, G., Bacci, H.L. and Seren, E. Follicularfactors influence oocyte fertilizability by modulating intercellular cooperation between cumulus cells and oocyte. Gamete Res. 21_:223-232 (19881. 15. Hunter, R.H.F. Chronologicaland cytologicaldetails of fertilizationand early embryonicdevelopment in the domestic pig, Sus scrofa. Anat. Rec. 178:169-186(1974).
JUNE 1969VOL.
31 NO. 6
1207