MOLECULAR REPRODUCTION AND DEVELOPMENT 33:357362 (1992)

Parthenogenetic Development of Bovine Oocytes Treated With Ethanol and Cytochalasin B After In Vitro Maturation YUTAKA FUKU1,l KEN SAWAI,' MAKOTO FURUDATE,2 NORIYUKI SATOF YASUAKI IWAZUMI,3 AND KAZUE OHSAK14 'Laboratory of Animal Genetics and Reproduction, Obihiro University of Agriculture and Veterinary Medicine, Obihiro, Japan; 'The Hokuren Livestock Experimental and Training Farm, Tokoro-gun, Hokkaido, Japan; 3The Kitami Branch of the Hokkaido Livestock Improvement Association, Tokoro-gun, Hokkaido, Japan; *Laboratory of Veterinary Obstetrics and Gynaecology, Obihiro University of Agriculture and Veterinary Medicine, Obihiro, Japan ABSTRACT The present study was conducted to investigate the effects of different culture durations (24-36 hr) on bovine oocyte maturation in vitro and the effect of the presence or absence of cumulus cells at the time of treatment to induce parthenogenetic activation (exposure to ethanol and cytochalasin B; CB) (experiment I). The effects of dosage (2.5or 5.0 pg/ml) and incubation time (2.5,5, or 10 hr) in CB (experiment II) on the subsequent development to the blastocyst stage in vitro was also investigated. In experiment I, cleavage and development to the blastocyst stage were not affected by the presence or absence of cumulus cells at the time of parthenogenetic activation. However, the 24-hr culture duration for in vitro maturation had a significantly lower rate of development to the blastocyst stage than the longer culture durations (27-36 hr). In experiment II, treatment with 5 pg/ml CB for 5 hr showed the highest percentage of development to blastocyst in the oocytes matured for both 27 and 30 hr. To determine the viability of the parthenogenetic embryos (morulae and blastocysts),four recipient heifers received two embryos each, and one heifer was found to be pregnant on day 35 following transfer. Although fetal heartbeat was not observed, the subsequent estrus was prolonged in all heifers. The present results demonstratedevelopment of in vitro-matured,parthenogenetically activated bovine embryos up to the preimplantation stage. o 1992 Wiley-Liss, Inc. Key Words: Cattle, Cumulus cells, Culture conditions INTRODUCTION Parthenogenetic activation of mammalian oocytes can be induced by various stimuli, such as exposure to ethanol (Cuthbertson, 1983; Kaufman, 1983; Nagai, 1987), ionophore (Ware et al., 19891, and electric current (Ware et al., 1989; Onodera and Tsunoda, 1989; Ozil, 1990). However, little information is available on the postactivation development in vitro of in vitromatured oocytes of farm animals. Using rabbit oocytes treated with electric stimulation in the presence of calcium ions (10 pM), Ozil (1990) obtained a high rate of development to the blastocyst stage (89%). The preg-

0 1992 WILEY-LISS, INC.

nancy rate to days 10-11 following transfer, was 29%. An age dependency for activation of in vitro-matured bovine oocytes has been reported previously (Nagai, 1987; Ware et al., 1989).To our knowledge, there are no reports for cattle on in vitro development to the blastocyst stage of parthenogenetically activated bovine oocytes after in vitro maturation and their viability after transfer to recipients. The aims of the present study were to examine the effects of culture duration for in vitro oocyte maturation and the presence or absence of cumulus cells a t the time of ethanol and cytochalasin B (CB) treatments on in vitro development to the blastocyst stage (experiment I) and the effects of dosage and incubation time for CB treatment on the in vitro development of in vitro-matured bovine oocytes (experiment 11).Viability of the parthenogenetic embryos (morula and blastocyst) derived from the present study was also evaluated by transfer to recipient heifers. MATERIALS AND METHODS Oocyte Maturation Ovaries were obtained from Holstein cows and heifers killed a t a local slaughterhouse and were transported in saline (9 g NaCl/liter) at 30-35°C to the laboratory within 1hr. The cumulus-oocyte complexes were collected from follicles 2-5 mm in diameter with a n 18-gauge needle attached to a 5-ml disposable syringe. Only oocytes with a n unexpanded cumulus oophorus and evenly granulated cytoplasm were washed three times with TCM 199 (Whittaker M.A. Bioproducts, Walkersville, MD; pH 7.4, Earle's salt with sodium bicarbonate and L-glutamine) supplemented with 0.3% (wiv) bovine serum albumin (BSA, fraction V, fatty acid-free; Sigma Chemical Co., St. Louis, MO) and 10 mM HEPES and were cultured in a four-well dish (Nunclon, Inter-Med., Copenhagen, Denmark) contain~~~~~~~~

Received April 23,1992; accepted June 10, 1992. Address reprint requests to Dr. Y. Fukui, Laboratory of Animal Genetics and Reproduction, Obihiro University of Agriculture and Veterinary Medicine, Hokkaido 080, Japan.

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ined for the number of nuclei by the method of Ushijima et al. (1988). Briefly, embryos were treated with 0.9% (viv) sodium citrate for 10-20 min and fixed with a solution of alcoho1:acetic acid:distilled water (3:2:1 by vol) a t 4°C for 1min. The fixed embryos were placed on slides with a small volume ofthe fixative, dried for 1hr, and stained with 10% ( w h ) Giemsa solution for 15 min. The nuclei numbers were determined under a phasecontrast microscope. Some of in vitro-developed embryos (morulae and blastocysts) were also examined for chromosome status following the method of Tateno et al. (1990). To evaluate the viability of embryos developed in vitro, eight embryos (morulae and blastocysts) were nonsurgically transferred into the uterus of four Holstein heifers (two embryos per heifer) on day 6 or 7 after Parthenogenetic Treatment induced estrus (day 0 was the day of estrus). Estrus was In experiment I, the oocytes cultured for five differ- induced by a n intramuscular injection of 250 kg prosent durations (24,27,30,33, or 36 hr) were treated with taglandin F,, (Estrumate, Sumitomo Chemical Co., Ja7% (viv) ethanol in synthetic oviduct fluid medium pan). Estrus behavior was observed every day following (SOFM) supplemented with 10% (viv) FCS and 25 mM transfer, and blood samples were taken approximately HEPES for 7 min at room temperature (Nagai, 1987) every 7 days to monitor plasma progesterone (P,) levels and 5 pg/ml CB (Sigma) in SOFM supplemented with prior to signs of estrus. Plasma P, concentrations were 10% (v/v) FCS but without HEPES for 5 h r a t 39°C measured by radioimmunoassay according to the under 5% CO, in air. The effect of the presence or ab- method of Makino (1973). Twenty-eight days after sence of cumulus cells surrounding the oocytes a t the transfer, pregnancy diagnosis was performed by a n ultime of ethanol and CB treatments was also examined. trasonic scanning instrument with B-mode ultrasound The cumulus cells were completely removed by re- frequency of 5.0 MH, (The Toshiba Sonolayer-L; model peated pipetting immediately before ethanol and CB SAL-32A). treatments in approximately one-half of the oocytes Statistical Analysis matured in vitro for each time point. The other oocytes were cultured within the cumulus cells. Following parIn experiment I, five different culture durations and thenogenetic activation, all oocytes were cultured in the effect of the presence or absence of cumulus cells on SOFM supplemented with 10% (v/v) FCS at 39°C for cleavage (two- to eight-cell stage) and development 6-8 days under a n atmosphere of 5% CO,, 5% 0,, 90% (blastocyst stage) rates were compared by analysis of N, (Fukui et al., 1991). variance using the Statistical Analysis System (SAS). In experiment 11, two dosages (2.5 and 5.0 pgiml) of The results of nuclei numbers were analyzed via StuCB and three incubation times (2.5,5, and 10 hr) were dent's t test on the logit using CATMOD procedures. In compared in oocytes matured for 27 or 30 hr. The cumu- experiment 11, averages of two or three replicate trials lus cells were partly removed by gentle pipetting imme- for each treatment were examined for statistical differdiately before ethanol and CB treatments in all oocytes ences using SAS. matured in this experiment. Parthenogenetic embryos were cultured to the blastocyst stage in vitro using RESULTS SOFM supplemented with 10% (viv) FCS for 6-8 days Experiment I under the same conditions as in experiment I. The results of cleavage and development to the blasTo investigate pronuclear formation of induced activation with ethanol and CB treatments, 63 activated tocyst stage of bovine oocytes cultured for five different and 56 nontreated (control) oocytes cultured for 30 h r in durations and the effect of the presence or absence of the maturation medium were fixed with acetic acid:al- cumulus cells a t the time of ethanol and CB treatments coho1 (1:3) after a n additional culture in SOFM supple- are shown in Table 1. The presence or absence of cumumented with 10% ( v k j FCS for 10 h r and stained with lus cells did not result in significant differences on ei1% (wiv) acetoorcein. Numbers of oocytes with two pro- ther cleavage or development to the blastocyst stage. nuclei without the second polar body were counted un- The five culture durations for oocyte maturation were der a phase-contrast microscope. not significantly different for cleavage to two- to eightcell stage; however, a 24-hr culture duration was signifDevelopment and Viability icantly inferior (P < 0.001) to the longer culture duraCleavage (two- to eight-cell stage) and development tions for development to the blastocyst stage (2.8-3.1% to the blastocyst stage were recorded at 3 and 8 days, and 5.2-10.9%, respectively). The numbers of nuclei of blastocysts (Fig. 1) are respectively, after the commencement of culture. Some blastocysts and expanding blastocysts were also exam- shown in Table 2. There was no significant difference in

ing 0.5 ml TCM 199 supplemented with 10% (viv) heatinactivated (56"C, 30 min) fetal calf serum (FCS) without HEPES. The medium was also supplemented with 2.5 pg bovine FSH-B-l/ml (U.S. Department of Agriculture), 5 pg bovine LH-B-5lml (U.S. National Hormone and Pituitary Program), 1 pg estradiol/ml (Sigma),and 1 x lo6 granulosa cells/ml. The granulosa cells were collected from antral follicles of about 10 mm in diameter by dissection following the method of Moor and Trounson (1977) and washed (500g, 5 min) and were prepared as described elsewhere (Fukui and Ono, 1989; Mochizuki e t al., 1991). Oocytes were statically cultured for 24,27,30,33, or 36 h r (experiment I) and for 27 or 30 h r (experiment 11)at 39°C under 5% CO, in air and 95% humidity.

PARTHENOGENETIC DEVELOPMENT OF BOVINE OOCYTES

359

TABLE 1. Effects of Maturation Culture Duration and the Presence or Absence of Cumulus Cells on Parthenogenetic Development of Bovine Oocytes

Culture duration (hr)

24 27 30 33 36

Presence ( + or absence (-1 of cumulus cells

No. oocytes cultured (No. trials)

+++++-

100 (3) 105 (3) 105 (5) 119 (6) 120 (6) 95 (6) 104 (2) 110 (2) 95 (3) 103 (3)

the mean number of nuclei per blastocyst among the four maturation culture time points (27,30,33, and 36 hr) nor the presence or absence of cumulus cells. The proportions of oocytes developing to blastocysts with 2 6 0 or 3100 nuclei were not affected by the maturation culture duration nor the presence or absence of cumulus cells. High-quality blastocysts, with 3 100 nuclei, were observed in oocytes matured for 27 and 33 hr.

Experiment I1 Effects of maturation culture duration (27 and 30 hr) and CB treatment (dosage 2.5 or 5.0 Fglml, incubation time 2.5, 5, or 10 hr) on development to the blastocyst stage are shown in Table 3. Analysis of variance showed that a 30 h r maturation culture duration was superior ( P < 0.001) to a 27 h r duration (6.1% and 3.6%, respectively). CB treatment for 5 h r was superior to 2.5and 10-hr incubations (8.1% vs. 1.8% and 2.9%, respectively; P < 0.001). Although the two dosages of CB had no significant effect on development, the best development to the blastocyst stage was obtained in the 27-hr (14.4%)and 30-hr (9.4%) maturation groups when activation was induced in the presence of 5 Fg/ml CB for 5 hr. This indicates that duration of exposure to CB may have more impact on further development of activated oocytes than duration of maturation. For pronuclear formation, four of 56 control oocytes (7.1%) had a female pronucleus, but no oocytes had two pronuclei without the second polar body. On the other hand, 34 of 63 oocytes (54.0%)treated with ethanol and CB had two pronuclei without the second polar body.

Viability Thirteen parthenogenetically developed embryos (morulae and blastocysts) were examined for chromosome analysis. All embryos had five to 10 metaphase plates, and 12 embryos had tetraploid and diploid chromosome sets, while only one embryo was diploid. Kryotyping was not performed in this study. To evaluate viability, two parthenogenetic embryos (morulae and blastocysts) were transferred to each recipient. The results of transfer to four recipient heifers are summarized in Table 4. Estrus was prolonged after transfer in all heifers. The plasma P, levels in two

Mean percentage % ( S E M ) of oocytes Cleaved Developed to (two-to eight-cell) blastocysts 30.5 t 17.3 29.7 2 14.3 22.2 t 5.2 18.4 t 4.4 24.3 t 5.3 46.1 2 5.5 28.2 -t 6.9 38.2 -t 14.5 38.1 2 1.1 35.1 t 5.0

2.8 2 2.0 3.1 2 1.6 8.8 2 5.5 8.8 -t 4.8 6.6 2 3.9 9.2 -t 4.4 6.7 t 0.3 10.9 t 5.5 6.7 t 3.6 5.2 t 1.5

heifers (Nos. 1 and 2) were maintained up to days 35 and 42, respectively, and they returned to estrus on days 41 and 48 after the previous estrus. One heifer (No. 2) was found to be pregnant, and fetal structures were observed in both uterine horns (Fig. 2). No heartbeat was observed; therefore, it was unclear whether the fetuses were alive a t the time of examination. The size of the pregnant uterus appeared to be smaller than in a normal day 35 pregnancy.

DISCUSSION The present results show that in vitro-matured bovine oocytes treated with ethanol and CB can develop to the blastocyst stage in vitro and can sustain pregnancy up to day 48 following transfer. In other studies with mice (Cuthbertson, 1983; Kubiak et al., 1991) and rabbits (Ozil, 1990), embryonic development following parthenogenetic activation until days 10-11 of pregnancy were reported. To our knowledge, this is the first report demonstrating that in vitro-developed embryos (morula and blastocyst) derived from parthenogenetic bovine oocytes have the ability to develop to the preimplantation stage. However, in our study, the heartbeat of the fetuses was not observed. The pregnant uterus appeared to be smaller than in a normal day 35 pregnancy. Ozil (1990) observed the smaller size of fetuses following transfer the parthenogenetic rabbit embryos a s compared with in vivo, control embryos, although the same author reported that 29% of eggs transferred had implanted and that 67% of those were living fetuses. In the present study, only one of four recipients was pregnant after transfer, but a higher conception rate (50-58%) has been reported with transfer in vitromatured and fertilized bovine embryos (Goto et al., 1988; Lu et al., 1990). In 13 parthenogenetically developed embryos (morula and blastocyst) examined for chromosome analysis, 12 embryos had tetraploid and diploid chromosome sets, while only one embryo was diploid, contrary to the results of pronuclear formation indicating that 54% of embryos examined were two pronuclei without the second polar body. This discrepancy may be due to a smaller number of embryos examined for chromosome analysis. Low viability and developmental ca-

360

Y. FUKUI ET AL.

PARTHENOGENETIC DEVELOPMENT OF BOVINE OOCYTES

361

TABLE 2. Number of Nuclei in Blastocysts Produced by Parthenogenetic Activation in Bovine Oocstes Matured In Vitro Culture duration (hr)

Presence (+) or absence ( -1 of cumulus cells

260 nuclei

0 0 16 9 7 2 6 7 2 3

9 1 4 0 3 4 0 2

+ + + +

24 27 30 33

-

+

36

No. blastocysts with 3100 nuclei

No. blastocysts examined

-

No. nucleihlastocyst Mean .t SE Min.-max.

-

-

3 0 0 0 2 1 0 0

-

* 6.9

70.1 37.4 2 56.7 2 30.5 & 84.3 64.1 37.0 65.0 2

* * *

4.7 9.2 0.5 21.6 12.6 4.0 8.1

39-118 23-7 1 26-88 30-3 1 37-165 30-105 33-41 52-80

TABLE 3. Effects of Culture Duration and Different Treatments With Cytochalasin B on Parthenogenetic Development to Blastocysts of In Vitro Bovine Oocytes Cvtochalasin B treatment (doses and times)

Culture Duration (hr) ~

~~~

27

2.5 hr ~

(0.0) 5187 (5.7) 51245 (2.0)

Total

Total

2.5 hr

51473 (1.1) 151253 (5.9) 201726 (2.8)

11125 (0.8) 2185 (2.4) 31210 (1.4)

5.0 pglml 5 hr 10 hr

Total Total

(%)

261395 (6.6) 201321 (6.2) 461716 (6.4)

311868 (3.6) 351574 (6.1) 6611442 (4.6)

~

01158"

30

2.5 pglml 5 hr 10 hr 512 16 (2.3) 4183 (4.8) 91299 (3.01

0199

(0.0) 6183 (7.2) 61182 (3.31

251174 (14.4) 131139 (9.4) 381313 (12.1)

0196

(0.0) 5197 (5.2) 51193 (2.6)

"No. of blastocysts developedlNo. of oocytes cultured (percent).

TABLE 4. Results of Transfer of Parthenogenetic Embryos (Morula/Blastocyst) Developed In Vitro Recipient heifers (No.)

Previous day of estrus

Plasma progesterone levels (nglml) Day of transfer

Oct 16

Oct 23

Nov 1

Nov 6

Nov 13

Nov 20

1

Oct9

Oct 16 (day 7Ia

1.9

7.6

7.3

6.1

5.0

-

2

Oct 9

Octl16

3.3

11.1

9.9

10.7

6.4

10.1

3.0

7.6

5.4

2.3

0.3

-

2.7

7.2

2.2

-

-

-

3 4

Oct 9 Oct 10

(day 7)

Octl16

(day 7)

Octll6

(day 6)

Day of estrus returned Nov 19 (day 41) Nov26 (day 48) Nov 13 (day 35) Nov 5 (day 26)

z

Pregnanc diagnosis -

+ -

"Days after the previous estrus. bExamined by an ultrasonic scanning method on day 35 (Nov 13).

pacity of tetraploid embryos have been reported for mouse (Snow, 1975) and cattle (Iwasaki et al., 1989). From the present trial of transfer with low viability of fetal formation, i t could be concluded, in agreement with Nagy et al. (1989), that paternally derived gene(s) might have a unique role in the development of tissues lacking parthenogenetic contribution. Further transfer trials of parthenogenetic bovine embryos are required Fig. 1. Blastocysts derived from a 7-day culture in SOFM supplemented with 10% (viv) FCS after in vitro maturation (33 hr in culture) and parthenogenetic activation with ethanol and CB (5 Fgirnl for 5 hr) treatments. Fig. 2. Ultrasonic scanning photographs showing day-35 pregnant uteri (left and right uteri) in one heifer (No. 2).

to determine the differentiation to living fetuses by isolating the embryos and by histological analysis. Attempts have been made to develop parthenogenetic oocytes matured in vitro in several animals (mouse: Onodera and Tsunoda, 1989; Didion et al., 1990; rabbit: Onodera and Tsunoda, 1989; pig: Prather et al., 1991). Onodera and Tsunoda (1989) reported that 32% of mouse and 25% of rabbit oocytes activated by electric stimulation developed to the blastocyst stage in vitro. To date, similar data in bovine oocytes matured in vitro have been found only through the eight-cell stage (Nagai, personal communication, 1992). In the present study, using ethanol and CB treatments, 2246% of parthenogenetic embryos cleaved to the two- to eight-cell stage and 5-1 1% developed t o the blastocyst

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Y. FUKUI ET AL.

stage when oocytes were matured for 27 to 36 hr. In comparison with those parthenogenetic embryos, the developmental capacity of in vitro-fertilized bovine embryos is higher in both cleavage ( 5 0 4 0 % )and blastocyst proportions (20-32%) (Choi et al., 1991; Fukui et al., 1991). It has been demonstrated that maturation age of the oocytes is a n important factor for parthenogenetic activation (Nagai, 1987; Ware et al., 1989; Didion et al., 1990). Nagai (1987) found that, in bovine oocytes cultured for 27-33 h r before ethanol treatment, 6068%of the oocytes were activated, as demonstrated by a female pronucleus(ei), whereas maturation for 24-26 h r resulted in a low activation rate (25-38%). However, diploid parthenogenesis (two pronuclei without the second polar body) were observed in only 7% of activated oocytes (Nagai, 1987). In the present study, using ethanol and CB, 54%of oocytes examined were diploid with two pronuclei. Ware et al. (1989) also described that both calcium ionophore and electric stimulation in bovine oocytes cultured for 26 and 30 hr resulted in a n activation response (pronuclear formation) similar to the ethanol activation reported by Nagai (1987). The present study found that cleavage was not affected by the culture durations (24-36 hr) of in vitro maturation but that development to the blastocyst stage was significantly reduced in oocytes cultured for 24 hr. The mechanism for this difference is still unknown. King et al. (1988) reported a high level (46%)of spontaneous activation of bovine oocytes matured in vitro for 24-27 hr, when the cumulus cells were partially removed. The present results have shown that the presence or absence of cumulus cells a t the time of induced activation is not a factor influencing subsequent development to the blastocyst stage. In conclusion, bovine oocytes matured in vitro and treated with ethanol and CB can develop to the blastocyst stage in vitro and embryos derived with the present method have the ability to establish pregnancy up to the preimplantation stage.

ACKNOWLEDGMENTS We thank Dr. T. Nagai for helpful discussions, Drs. K. Mikamo and H. Tateno for chromosomal analysis of embryos and valuable comments throughout the study, Dr. S. Raiti for the supply of bovine luteinizing hormone, and Dr. J. Bolt for the supply of bovine folliclestimulating hormone used for oocyte maturation. REFERENCES Choi YH, Fukui Y, Ono H (1991):Effects of media and the presence of bovine oviduct epithelial cells during in vitro fertilization on fertilizability and developmental capacity of bovine oocytes. Theriogenology 36363-873. Cuthbertoson KSR (1983): Parthenogenetic activation in vitro with ethanol and benzyl alcohol. J Exp Zoo1 226:311-314.

Didion BA, Martin MJ, Markert CL (1990): Parthenogenetic activation of mouse and pig oocytes matured in vitro. Theriogenology 33:1165-1175. Fukui Y, Ono H (1989): Effects of sera, hormones and granulosa cells added to culture medium for in-vitro maturation, fertilization, cleavage and development of bovine oocytes. J Reprod Fertil 86:501-506. Fukui Y, McGowan LT, James RW, Pugh PA, Tervit HR (1991):Factors affecting the in-vitro development t o blastocysts of bovine oocytes matured and fertilized in-vitro. J Reprod Fertil92:125-131. Goto K, Kajihara Y, Kosaka S, Koba M, Nakanishi Y, Ogawa K (1988): Pregnancies after co-culture of cumulus cells with bovine embryos derived from in-vitro fertilization of in-vitro matured follicular oocytes. J Reprod Fertil83:753-758. Iwasaki S, Kono T, Fukatsu H, Nakahara T (1989): Production of bovine tetraploid embryos by electrofusion and their developmental capacity in vitro. Gamete Res 24:261-267. Kaufman MH (1983): Early Mammalian Development: Parthenogenetic Studies, Cambridge: Cambridge University Press. King WA, Xu KP, Sirard MA, Greve T, Leclerc P, Lambert RD, Jacques P (1988): Cytogenetic study of parthenogenetically activated bovine oocytes matured in vivo and in vitro. Gamete Res 20:265-274. Kubiak J, Paldi A, Weber M, Maro B (1991): Genetically identical parthenogenetic mouse embryos produced by inhibition of the first meiotic cleavage with cytochalasin D. Development 111:763-769. Lu KH, Jiang HS, Wang WL, Gordon I (1990): Pregnancies established in cattle by transfer of fresh and frozen embryos derived from in vitro maturation and fertilization of oocytes and their subsequent culture in vitro. Theriogenology 33278 (abstract). Makino T (1973): Radioimmunoassay of plasma sex steroids. Fol Endocrinol Jpn 49:629-646 Fin Japanese I. Mochizuki H, Fukui Y, Ono H (1991):Effect of the number of granulosa cells added to culture medium for in vitro maturation, fertilization and development of bovine oocytes. Theriogenology 36:973986. Moor RM, Trounson A 0 (1977):Hormonal and follicular factors affecting maturation of sheep oocytes in vitro and their subsequent developmental capacity. J Reprod Fertil49:101-109. Nagai T (1987):Parthenogenetic activation of cattle follicular oocytes in vitro with ethanol. Gamete Res 16:243-249. Nagy A, Sass M, Markkula M (1989): Systematic non-uniform distribution of parthenogenetic cells in adult mouse chimaeras. Development 106:321-324. Onodera M, Tsunoda Y (1989): Parthenogenetic activation of mouse and rabbit eggs by electric stimulation in vitro. Gamete Res 22:277283. Ozil (1990): The parthenogenetic development of rabbit oocytes after repetitive pulsatile electrical stimulation. Development 109:117127. Prather RS, Eichen PA, Nicks DK, Peters MS (1991):Artificial activation of porcine oocytes matured in vitro. Mol Reprod Dev 28:405409. Snow MHL (1975):Embryonic development of tetraploid mice during the second half of gestation. J Embryo1 Exp Morphol34:707-721. Tateno H, Akaike M, Fukui Y, Kamiguchi Y, Mikamo K (1990): A method for chromosome analysis of ram spermatozoa using zonafree hamster oocytes. Theriogenology 34:845-852. Ushijima M, Okuda T, Nakayama A, Moji K, Ishida K, Murata H, Iguchi A, Etoh T (1988):Relationships between the cell number and quality of Day-8 bovine blastocysts. Proc 3rd Est Jpn SOCAnim Embryo Transfer 9:37-38 [in Japanese]. Ware CB, Barnes FL, Maiki-Laurila M, First NL (1989): Age dependence of bovine oocyte activation. Gamete Res 22:265-275.

Parthenogenetic development of bovine oocytes treated with ethanol and cytochalasin B after in vitro maturation.

The present study was conducted to investigate the effects of different culture durations (24-36 hr) on bovine oocyte maturation in vitro and the effe...
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