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Parthenogenetic development of bovine oocytes activated by different methods
212
Theriogenology
PARTHENOGENETIC
DEVELOPMENT OF BOVINE OOCYTES ACTIVATED DIFFERENT METHODS
BY
M. Stojkovic’, V. Zakhartchenko’, G. Brem* and E. Wolf’ ‘Department of Molecular Animal Breeding and Genetics, 2BFZF Hackerstr. 27, D-85784 OberschleiBheim, Germany The objective of this study was to compare the cleavage rate of bovine oocytes matured in vitro for 22 h (fresh oocytes) or cultured for additional 18 h (aged oocytes) activated by ethanol or ethanol followed by cycloheximide and cytochalasin B treatment. Cycloheximide induces pronuclear development and cytochalasin B has been used for the diploidization of bovine oocytes activated parthenogenetically by blockage of extrusion of the second polar body (Presicce GA & Yang X, Mol Reprod Dev 1994, 38:380-385). Oocytes were collected from slaughterhouse ovaries and matured in TCM 199 supplemented with 10% estrous cow serum (MPM) and 10 mg/ml FSH. Expanded cumulus cells were removed by 1 mglml hyaluronidase. Only oocytes with visible polar body were activated by incubation in 7% ethanol for 5 min (activation A) or the same treatment followed by incubation in 5 mg/ml cycloheximide, 5 mg/ml cytochalasin B in MPM for 5 h (activation B). Activated oocytes were washed and cultured in CR1 medium supplemented with 10% estrous cow serum. For chromosomal analysis, Day 7 blastocysts were cultured in MPM supplemented with 0.5 mg/ml colchicine for 12 h and suspended in 1% sodium citrate for 20 min. Blastocysts were fixed in methanol:acetic acid (1:l) for 5 min and stained with Hoechst 33342 (4 mglml). Chromosomes were counted under a fluorescent microscope. The cleavage rates of activated oocytes on Day 2, Day 4 and Day 7 are presented in Table 1. Table 1. Cleavage rates of fresh or aged bovine oocytes after activation with ethanol (A) or ethanol followed by cycloheximide and cytochalasin B treatment (B). 2-cell n (%)
>8-cell n (%)
Blastocysts n (%)
Activation
Type of oocytes
No. of oocytes
A
fresh
185
58
(31)a
11
(6)a
B
fresh
169
116
(69)b
94
(56)b
38
A
aged
187
75
(40)a
37
(20)c
6
B
aged
161
111
(69)b
82
(51)b
31
6
(6)a (22)b (Qa (19)b
Proportions marked by different superscripts differ significantly (chi2-test: PeO.01). Both fresh and aged oocytes activated by treatment than oocytes activated by treatment A. A single stimulation the mature oocytes. Blastocysts produced by treatment mixoploid (2N/4N; 36%). This may have been caused by which inhibits cytokinesis.
B developed at a higher rate could not effectively activate B were diploid (2N; 64%) or the cytochalasin B treatment
Theriogenology
PARTHENOGENETIC
DEVELOPMENT OF BOVINE OOCYTES ACTIVATED DIFFERENT METHODS
BY
M. Stojkovic’, V. Zakhartchenko’, G. Brem* and E. Wolf’ ‘Department of Molecular Animal Breeding and Genetics, 2BFZF Hackerstr. 27, D-85784 OberschleiBheim, Germany The objective of this study was to compare the cleavage rate of bovine oocytes matured in vitro for 22 h (fresh oocytes) or cultured for additional 18 h (aged oocytes) activated by ethanol or ethanol followed by cycloheximide and cytochalasin B treatment. Cycloheximide induces pronuclear development and cytochalasin B has been used for the diploidization of bovine oocytes activated parthenogenetically by blockage of extrusion of the second polar body (Presicce GA & Yang X, Mol Reprod Dev 1994, 38:380-385). Oocytes were collected from slaughterhouse ovaries and matured in TCM 199 supplemented with 10% estrous cow serum (MPM) and 10 mg/ml FSH. Expanded cumulus cells were removed by 1 mglml hyaluronidase. Only oocytes with visible polar body were activated by incubation in 7% ethanol for 5 min (activation A) or the same treatment followed by incubation in 5 mg/ml cycloheximide, 5 mg/ml cytochalasin B in MPM for 5 h (activation B). Activated oocytes were washed and cultured in CR1 medium supplemented with 10% estrous cow serum. For chromosomal analysis, Day 7 blastocysts were cultured in MPM supplemented with 0.5 mg/ml colchicine for 12 h and suspended in 1% sodium citrate for 20 min. Blastocysts were fixed in methanol:acetic acid (1:l) for 5 min and stained with Hoechst 33342 (4 mglml). Chromosomes were counted under a fluorescent microscope. The cleavage rates of activated oocytes on Day 2, Day 4 and Day 7 are presented in Table 1. Table 1. Cleavage rates of fresh or aged bovine oocytes after activation with ethanol (A) or ethanol followed by cycloheximide and cytochalasin B treatment (B). 2-cell n (%)
>8-cell n (%)
Blastocysts n (%)
Activation
Type of oocytes
No. of oocytes
A
fresh
185
58
(31)a
11
(6)a
B
fresh
169
116
(69)b
94
(56)b
38
A
aged
187
75
(40)a
37
(20)c
6
B
aged
161
111
(69)b
82
(51)b
31
6
(6)a (22)b (Qa (19)b
Proportions marked by different superscripts differ significantly (chi2-test: PeO.01). Both fresh and aged oocytes activated by treatment than oocytes activated by treatment A. A single stimulation the mature oocytes. Blastocysts produced by treatment mixoploid (2N/4N; 36%). This may have been caused by which inhibits cytokinesis.
B developed at a higher rate could not effectively activate B were diploid (2N; 64%) or the cytochalasin B treatment