Ploidy instability of embryogenic cucumber (Cucumis sativus L.) callus culture

Embryogenic callus cultures were established from immature cucumber(Cucumis sativus L.) embryos on E20A (Dumas de Vaulxet al. 1981) or MS (Murashige and Skoog 1962) media supplemented with 6-benzylaminopurine (BAP), α-naphthylacetic acid (NAA) and/or 2,4-dichlorophenoxyacetic acid (2,4-D). Regeneration of plants was observed after a transfer to culture media either without growth regulators or supplemented with kinetin and NAA. Flow cytometry was employed to estimate DNA ploidy levels. Most of cell nuclei in young leaf tissues were found in G₁ phase with 2C DNA content. Callus cultures were mixoploid with DNA content ranging from 2C to 32C. The frequency of polyploid cells was increasing with the age of culture and the polyploidization was accompanied by a gradual loss of regeneration ability. Plants regenerated from callus cultures were classified as diploid (57 %), tetraploid (18 %), octoploid (4 %) and mixoploid (2n/4n, 4 %) and (4n/8n, 17 %). The results of this study confirmed a close link between the polyploidization and the loss of totipotencyin vitro. Tetraploid plants obtained in this study have a potential to be used in interspecific crosses where their tetraploid status could help in overcoming existing breeding barriers due to differences in chromosome number.     

Comparison of superovulatory response of mature outbred mice treated with FSH or PMSG and developmental potential of embryos produced

A superovulatory treatment for mice based on FSH administration was compared with a standard one based on PMSG. Our aim was to determine if a mean number of embryos recovered per donor could be increased and if in vitro or in vivo viability was affected by the hormonal treatment used. Thus, female Swiss mice were subjected to 2 superovulatory treatments, and the 1-cell and 2-cell stage embryos were cultured in 2 different media to the blastocyst stage or were transferred to pseudopregnant recipients. The data show that despite a lower mating percentage (52% with FSH vs 66% with PMSG), the FSH-treated mice provided twice the number of total embryos (53.4 vs 24.5) with a similar percentage of morphologically normal embryos (74% for FSH vs 69% for PMSG). We also found that in vitro culture results can be influenced by the source of gonadotropins depending on the culture medium used. A culture medium such as CZB which prevents the 2-cell block, provided the same developmental rates regardless of hormonal treatment used. However, with M-16 medium, which does not prevent this blockage, only 39% of the 2-cell FSH-derived embryos and 49% of the PMSG-derived 2-cell embryos developed into blastocysts (P<0.05). FSH-derived embryos resulted in a higher percentage of pregnant recipients (73 vs 56%) than PMSG-derived embryos, but the number of alive fetuses and the number of implantations per pregnant recipient was affected only by the kind of culture system used before transfer. The results show that FSH can provide very good superovulatory response in mice, thus reducing the number of donors needed for a given experiment and providing embryos of at least the same quality as those derived from the standard PMSG treatment.     

Frequency of sex chromosomal mosaicism in bovine embryos and its effects on sexing using a single blastomere by PCR

Assessment of nuclear status is important when a biopsied single blastomere is used for embryo sexing. In this study we investigated the nuclear status of blastomeres derived from 8- to 16-cell stage in vitro fertilised bovine embryos to determine the representativeness of a single blastomere for embryo sexing. In 24 embryos analysed, the agreement in sex determination between a biopsied single blastomere and a matched blastocyst by polymerase chain reaction (PCR) was 83.3%. To clarify the discrepancies, karyotypes of blastomeres in 8- to 16-cell stage bovine embryos were analysed. We applied vinblastine sulfate at various concentrations and for different exposure times for metaphase plate induction in 8- to 16-cell stage bovine embryos. The 1.0 mg/ml vinblastine sulfate treatment for 15 h was selected as the most effective condition for induction of a metaphase plate (> 45%). Among 22 embryos under these conditions, only 8 of 10 that had a normal diploid chromosome complement showed a sex chromosomal composition of XX or XY (36.4%) and 2 diploid embryos showed mosaicism of the opposite sex of XX and XY in blastomeres of the embryo (9.1%). One haploid embryo contained only one X-chromosome (4.5%). Four of another 11 embryos with a mixoploid chromosomal complement contained a haploid blastomere with a wrong sex chromosome (18.2%). In conclusion, assessment of nuclear status of 8- to 16-cell stage bovine embryos revealed that morphologically normal embryos had a considerable proportion of mixoploid blastomeres and sex chromosomal mosaicism; these could be the cause of discrepancies in the sex between biopsied single blastomeres and matched blastocysts by PCR.     

Cytological studies on mixoploidy and embryonic development in mosaic silkworms induced by low temperature treatment

Mosaicism in silkworms is easily inducible by applying a cold shock to the hybrid eggs between two strains with different markers at the early developmental stage. When BF₁ eggs from the cross between a tetraploid female (which had been induced from the cross between a red egg and a white egg strain) and a male of the red egg strain were exposed to a cold shock, hexaploid eggs and two types of mosaic eggs were obtained: black-black (BB-) mosaic eggs, which possessed two kinds of black serosa cells with different-sized nuclei, and black-red (BR-) mosaic eggs. Cytological studies on the embryos from these mosaic eggs revealed that the BB-mosaic type was a 3n/6n mixoploid, while the BR-mosaic type was mixoploid with n/2n/3n, n/3n, or 2n/6n constitution. The hatchability of BB- and BR-mosaic eggs was considerably lower than that of either n/2n or 2n/4n mixoploid eggs obtained by a cold treatment of diploid eggs. Since abnormal development was observed at a high frequency in the embryos from BB- and BR-mosaic eggs at the blastokinesis stage, it is speculated that the coexistence of hexaploid or triploid cells may be a factor in the induction of such abnormality in early embryogenesis.     

The effect of donor cell serum starvation and oocyte activation compounds on the development of somatic cell cloned embryos

Two experiments, one comparing nuclear transfer (NT) embryo activation compounds, the other donor cell treatments, were conducted with a goal of identifying factors that improve the in vitro development of cloned bovine embryos. In experiment 1, 539 NT embryos were produced by combining serum starved bovine fetal fibroblasts with enucleated in vitro matured oocytes, activated with ionomycin, then randomly allocated to be incubated for 4 hours in either Butyrolactone-I (BL-I) or 6-dimethylaminopurine (DMAP). There was no significant difference in development to blastocyst or compact morula of fused embryos at Day 6.5 between BL-I and DMAP activated embryos (22.4% vs. 20.2%; p = 0.18). Karyotyping of 20 blastocysts and compact morula from each group determined that 65% of BL1 and 63% of DMAP embryos were diploid with the remainder mixoploid (2n + 4n). In Experiment 2, the development of 389 NT embryos reconstructed from either serum starved or serum fed fetal fibroblasts was assessed. More Day 7 blastocysts and compact morula developed in the serum starved group (34.5% vs. 18.8%; p = 0.008). To verify the viability of BL-I activated embryos, 10 blastocytes from experiment 2 were transferred into 4 recipient cows. Two morphologically normal fetuses, genetically identical to the original fetal cell line, were surgically recovered at day 45 of gestation. In summary, serum starvation of bovine fetal fibroblasts prior to NT significantly improved development to blastocyst. Additionally, we have shown that BL-I is a novel alternative compound for use in combination with ionomycin to activate NT embryos.     

Chromosome aberrations in in vitro-produced bovine embryos at days 2-5 post-insemination

Availability of embryos of high quality is required to obtain satisfactory embryonic developmental rates and normal calves following transfer of in vitro-produced (IVP) bovine embryos. One relevant quality parameter is the frequency of chromosome aberrations, which can be evaluated using multicolor fluorescent in situ hybridization (FISH) with chromosome 6- and chromosome 7-specific probes in cattle. In this study, interphase nuclei (n = 3805) were analyzed from 426 bovine IVP embryos. We found that 73%, 72%, 81%, and 58% of the embryos from Days 2, 3, 4, and 5 post-insemination (pi), respectively, displayed a normal diploid chromosome number in all cells. When looking at the types of chromosome aberrations, the percentages of mixoploidy at Days 2, 3, 4, and 5 pi were 22%, 15%, 16%, and 42%, respectively, whereas the percentages of polyploidy (i.e., all nuclei in an embryo were analyzed and were polyploid) were 5%, 13%, 3%, and 0%, respectively. In conclusion, numerical chromosome aberrations were detected as early as Day 2 pi. The development of polyploid embryos is slow and is apparently arrested during the third cell cycle, whereas the mixoploid embryos seem to continue development.     

安徽产石蒜两个居群的核型研究

观察了石蒜(Lycoris radinta)两个不同居群植物的染色体数目和核型,发现野生石蒜在一个植株的不同根尖细胞里,存在两种倍性的细胞,如生于宣城敬亭山的居群既有正常三倍体：2n=33=18st 15T,属于“4A”核型;还有异常二倍体：2n=20 1B=2st 18T 1B,属于“4B”核型;生于芜湖的居群核型为：2n=20 1B=lm 9T 4t 6st 1B和2n=20 1B=1M 9T 10st 1B,属于“3B”和“3C”核型。     

Embryo production from superovulated sheep inseminated with sex-sorted ram spermatozoa

An experiment was undertaken to assess the fertilizing capacity of sex-sorted, frozen-thawed ram spermatozoa, artificially inseminated into superovulated ewes, and the quality and survivability of the resultant pre-sexed embryos. Synchronized (intravaginal progestagen pessary and GnRH) donors were superovulated using PMSG and repeat ovarian stimulation with FSH before insemination. Ewes (n=67) were inseminated with either 30x10(6) or 15x10(6) motile non-sorted (control) or 15x10(6) motile sex-sorted (sorted) frozen-thawed spermatozoa (control: C30 or C15; sorted: S15, respectively) and the resultant embryos transferred immediately into synchronized recipients (n=160). The percentage of transferable embryos, pregnancy rate and embryo survival were similar (P>0.05) across all treatments. Oocyte cleavage rate was higher for ewes inseminated with S15 (172/230; 74.8%; P<0.05) than for C15 (97/151; 64.2%) or C30 (89/141; 63.1%) spermatozoa. Of the lambs resulting from embryos produced with sex-sorted spermatozoa, 86/93 (92.5%) were born of the predicted sex. This study demonstrated for the first time that pre-sexed offspring derived from superovulated sheep can be produced following transfer of embryos. Furthermore, sex-sorting by flow cytometry did not compromise the in vivo fertilizing capacity of ram spermatozoa in superovulated sheep, nor did it affect the quality or survivability of the resultant embryos.     

Preimplantation embryo development and serum steroid levels in immature rats induced to ovulate or superovulate with pregnant mares' serum gonadotropin injection or follicle-stimulating hormone infusions

Follicular stimulation protocols using pregnant mares' serum gonadotropin (PMSG) or a follicle-stimulating hormone (FSH) preparation were compared to evaluate the yield and quality of embryos obtained from immature rats. Rats received a superovulatory dose of PMSG (401U), a nonsuperovulatory dose of the same gonadotrophin (4 IU), or a continu ous s.c. infusion over a 72-h period with a purified FSH preparation containing an opti mum ratio of luteinizing hormone (LH): FSH (FSH-hCG). The females were caged with fertile males on the evening of the 3rd day of gonadotropin treatment and scored for the occurrence of mating on the next morning; subgroups were killed on days 1–4 of preg-nancy. High fertilization rates were observed in rats treated with 4 IU PMSG (84.1%) and in rats infused with FSH-hCG (91.0%); however, a much lower fertilization rate was observed following treatment with 40 IU PMSG (41.5%). From median ovulation rates of 9 and 79 in rats treated with 4 IU PMSG and in rats infused with FSH-hCG, medians of 8 and 69 embryos, respectively, were recovered from reproductive tracts flushed on day 4 of pregnancy, from which 75% were morulae or blastocysts; in contrast, from a median ovu lation rate of 42.5, a median of only 12 embryos was recovered on day 3 of pregnancy following superovulation with 40 IU PMSG of which 80% were degenerate ova. Serum steroid profiles during the first 4 days of pregnancy differed significantly among treatment groups, the major differences being in substantially elevated levels of estradiol and andro-gens on days 1–3 in rats receiving the high (40 IU) dose of PMSG. Levels of these steroids in rats superovulated with the FSH-hCG infusion regimen were only marginally elevated above levels observed in rats treated with the low (4 IU) nonsuperovulatory dose of PMSG. Consistent with high ovulation rates, serum progesterone levels rose to considera bly higher levels during the period in both superovulated groups than in animals receiving the low, nonsuperovulatory dose of PMSG. This work describes a novel method to superovulate rate (FSH-hCG) leading to high yields of normally developing embryos at all preimplantation stages and illustrates the close association between high yield of emyryos and low levels of circulating andorgens and estradiol-17β during the preimplantation period.     

Embryo losses in Rasa Aragonesa ewes actively immunized against androstenedione or passively immunized against testosterone

Two-day-old embryos from untreated ewes were transferred to the oviducts of ewes actively immunized against androstenedione (n=26, Group A), passively immunized against testosterone (n=19, Group B) or left untreated (n=25, Group C). Donor ewes superovulated after treatment with follicle-stimulating hormone and fluorogestone acetate (FGA). Recipient ewes were treated with FGA and pregnant mare serum gonadotropin (PMSG, 300 I.U.). Group A received two injections of Fecundin at a 4-wk interval. FGA sponges were inserted when the second injection was given. Group B was treated with antitestosterone antiserum (35 ml) at sponge withdrawal. Each recipient received two morphologically viable embryos 52 to 62 h after the onset of estrus. Antibody titre at embryo transfer and progesterone concentration on Days 2, 4, 6, and 12 after estrus were determined. Fertility was lower in Group A when compared to Group C (42.3 vs 84.1%; P<0.01) while that of Group B (63.2%) did not differ from those of Groups A and C. In immunized groups, most of the embryo losses occurring were complete (both embryos were lost), resulting in a decreased fertility, while in the untreated group embryo losses were mainly partial (only one embryo was lost), hence lowering prolificacy. Fertility in immunized groups changed according to the antibody titre reached. Ewes from Groups A and B with higher antibody titres displayed lower fertility than control ewes. On Days 4 and 12 of the cycle, Group A plasma progesterone concentrations positively correlated with antibody titres and were higher with respect to those of Group C (P<0.05). Progesterone levels in Group B were similar to those of Group C. These results indicate that ewes reaching higher antibody levels had more embryo losses, attributable to the adverse influences of the oviductal and/or uterine environment on embryo development.     

Use of PMSG antiserum in superovulated cattle?

Two Pregnant Mare Serum Gonadotrophin (PMSG) antisera were tested in 174 dairy cows that were superovulated with PMSG and were then given prostaglandin at 60 hours after PMSG. At 48 hours after injection of prostaglandin, the cows were given either PMSG antiserum (monoclonal (n=56) or polyclonal (n=57)), or saline as control (n=61). Ova (n=1,206) were recovered either nonsurgically or after slaughter. Of these, 757 were evaluated morphologically to be transferable embryos. A proportion of these embryos (n=295 from 52 flushed donors) were transferred to synchronized recipients and the pregnancy results were recorded. The reproductive function of 37 flushed donors was followed for 6 months after superovulation. No significant effect of the PMSG antisera could be demonstrated in any of the parameters studied (i.e., ovulation rate, number of follicles at collection, total yield of ova, fertilization rate, number of transferable embryos, pregnancy results after transfer of embryos, or period required by the donor cows for restitution of reproductive function after superovulation and recovery). It is concluded that use of PMSG antiserum did not improve the embryo yield in terms of the number and quality of transferable embryos or enhance normalization of reproductive function of the donor in the 6-month period after superovulation. Therefore, in an embryo transfer operation, the routine use of PMSG antiserum in a PMSG superovulation regimen in cattle is not recommended.     

Chromosomal abnormalities in Day-6, in vitro-produced pig embryos

A cytogenetic study was undertaken to quantify, by chromosomal karyotyping, the incidence and type of chromosomal abnormalities present in Day-6 in vitro-produced (IVP) porcine embryos. Morphologically normal Day-6 blastocysts (n=318) were fixed and grouped into six classes according to the number of total cells (from < or =20 to 61-70). Of 248 embryos suitable for analysis, 97 (39.1%) displayed chromosomal abnormalities. The abnormalities included haploidy (9.3%), polyploidy (71.1%) and mixoploidy (19.6%). Within polyploid embryos, triploidy and tetraploidy showed the highest incidence (56.5 and 27.5%, respectively); among mixoploid embryos, diploid-triploid embryos (2n/3n) were prevalent (36.8%). Overall, the mean cell number was 34.3 +/- 12.1 and the mitotic index was 8.6 +/- 6.1. Chromosomally abnormal embryos had fewer (P<0.01) total cells compared to normal (2n) embryos (31.8 +/- 1.3 versus 35.9 +/- 1.0). In addition, the incidence of polyploidy decreased as the number of cells increased, while that of mixoploidy did not differ. These data indicate that polyploidy affects a large percentage of IVP porcine embryos capable of developing to blastocysts and the incidence of chromosomal abnormalities is much higher than that reported previously in in vivo embryos in this species. Given the ability of morphologically normal embryos with an abnormal chromosome complement to undergo preimplantation development in vitro, and the inability to identify blastocysts with abnormal karyotype without cytogenetic analysis, careful consideration should be given to factors affecting ploidy of IVP embryos, especially the incidence of polyspermic fertilization, when evaluating criteria of a porcine in vitro embryo production scheme.     

Incidence of chromosomal mosaicism in human embryos at different developmental stages analyzed by fluorescence in situ hybridization

Chromosomal mosaicism has been reported in in vitro-cultured embryos at early cleavage stages, as well as in morulae and blastocysts. We have assessed the incidence and pattern of mosaicism during in vitro development of human embryos from early-cleavage stages to morula and blastocyst. Fifty spare embryos were fixed for fluorescence in situ hybridization (FISH) analysis for chromosomes X, Y, 13, 18, and 21 on days 2 or 3 (4- to 10-cell stage) (n = 16), on day 4 (morula stage) (n = 14), on day 5 (pre-expanded blastocyst) (n = 5), and the expanded blastocyst stages (n = 15). Blocked embryos (no cleavage observed within the last 24 hr) were not included. A total of 2367 cells were analyzed. Four early-cleavage stage embryos were found uniformly diploid; all of the others were mosaic for the chromosomes analyzed (mean diploid nuclei 48.3% +/- 28.7). All of the embryos at more advanced developmental stages, except one fully normal morula, had mosaic chromosome constitutions, with an increase in the percentage of diploid cells in morulae, pre-expanded, and expanded blastocysts, respectively (mean diploid nuclei 78.6% +/- 11.7, 66.0% +/- 20.8, 79.6% +/- 12.8), in comparison with earlier stages. Hypotheses about the origin of mosaicism and embryo regulation mechanisms will be discussed.     

Embryo transfer in fallow deer (Dama dama): superovulation, embryo recovery and laparoscopic transfer of fresh and cryopreserved embryos

Multiple ovulation-embryo transfer (MOET) protocols for farmed fallow deer (Dama dama ) were investigated in a series of 3 experiments. A total of 37 donors, of either European (D.d. dama ; n = 30) or Mesopotamian hybrid (D.d. mesopotamica x D.d. dama ; n =7) genotype, each received an intravaginal silastic device containing 0.3 g progesterone (CIDR((R))-type G device) for 14 d and injections of 0.5 units ovine FSH (8 x 0.06 unit injections from Days 10 to 14 of device insertion) and 100 IU PMSG (either with the first or last FSH injection). All donors received laparoscopic intrauterine inseminations of fresh semen (50 x 10(6) spermatozoa) from a Mesopotamian sire 36 h after withdrawal of CIDR((R)) devices. Embryos were recovered by laparotomy on Day 6 (Day 0 = estrus). Mean ovulation rates for the 3 experiments were 8.1, 9.8 and 7.0, with no effect of PMSG timing (P>0.10). However, embryo recovery rates, albeit low throughout the study (29.6%), were significantly improved with later PMSG administration (33.9 vs 20.1%; P<0.05). Hybrid and European donors performed in a similar manner. A range of embryo development stages was recovered throughout the study. In 2 experiments laparoscopic transfer of embryos to 48 recipient does treated previously with intravaginal CIDR((R)) devices for 14 d yielded a total pregnancy rate of 37.5%. In the experiment with fresh embryos, the use of clenbuterol to reduce uterine turgidity resulted in a higher proportion of does conceiving (3 4 ; 75%) compared with that of the untreated does (0 6 , 0%; P<0.05). In the second experiment, in which all the does routinely received clenbuterol, 10 19 (53%) and 5 19 (26%) does conceived following the transfer of fresh and cryopreserved embryos, respectively (P<0.05). While the overall efficiency of the MOET program was low (equivalent of 0.9 to 1.0 surrogate pregnancies per donor), improvements in the recovery rate of transferable embryos have considerable potential for genetic improvement of farm stock and captive propagation of endangered Mesopotamian fallow deer through maternal surrogacy programs.     

Effect of estradiol supplementation on superovulation in Swamp buffalo

The effect of estradiol-17beta (E(2)) supplementation on superovulation with (PMSG) or (FSH) was investigated in Swamp buffalo. Sixty-eight buffalo were treated in seven groups. Group 1 served as control and was superovulated by standard PMSG or FSH treatment used in routine bovine embryo transfer protocols. Group 2 was superovulated by standard PMSG regimen plus two injections of E(2) at a 48 h interval beginning one day before the onset of gonadotropin treatment (short-term supplementation) for a total dosage of 2.5 mg E(2); Groups 3 and 4 received the same regimen as Group 2, but in doses of 5.0 and 7.5 mg E(2), respectively. Group 5 received the standard FSH regimen (40% LH). Group 6 received short-term E(2) (7.5 mg) supplementation of FSH-p. Group 7 was superovulated by standard FSH regimen (40% LH) plus three injections of E(2) at 48-72 h intervals beginning five days before the onset of gonadotropin treatment (long-term supplementation) for a total dosage of 7.5 mg E(2). The number of corpora lutea (CL) and follicles >/= 8 mm in diameter were recorded by palpation per rectum and after slaughter. The mean numbers of CL and follicles were 0.99, 5.8, 8.0, 10.6, 4.0, 3.9, 8.1 and 0.25, 6.8, 6.2, 6.2, 1.6, 0.0, 4.1 for Groups 1, 2, 3, 4, 5, 6, 7, respectively. In Group 7, the rates of nonsurgical and postmortem embryo recovery were 46 and 90.4%, respectively and 54.4% of the collected ova were fertilized. These results indicate the possibility of producing viable embryos in buffalo by using E(2) supplementation for the gonadotropin treatment.     

Superovulation treatments and embryo transfer in Angora goats

A high incidence of early luteal regression after PMSG superovulation was associated with low recovery of embryos from reproductive tracts of Angora goats flushed later than Day 5 after onset of oestrus. Embryos were successfully recovered (mean 7.9/female) by flushing on Days 2-5. Mean ovulation rate after an FSH regimen (16.1 +/- 0.8) was significantly higher than that after a single injection of PMSG (10.8 +/- 1.2). Fertilization rate and survival of embryos following transfer to naturally synchronized recipient feral goats did not differ between the two gonadotrophin regimens: the mean number of kids born to 47 donors treated with FSH (7.5 +/- 0.6) was significantly greater than that to 28 donors treated with PMSG (4.8 +/- 0.6). Irrespective of hormonal treatment, the numbers of embryos recovered and of kids born were correlated with ovulation rate (r = 0.82, P less than 0.001 for both). Embryo survival was influenced by ovulation rate in recipients, with 52%, 63% and 75% of transferred embryos being carried to term by recipients with 1,2 and 3 CL, respectively (P less than 0.01). More embryos survived (65%) when 2 embryos were transferred to each recipient than when 1 (51%) or 3 (48%) were transferred. In recipients receiving 2 embryos, survival was significantly improved by transfer of both embryos to the same oviduct (70%) than when one was transferred to each oviduct (62%). The percentage survival of embryos was optimal when oestrus of recipients was synchronized within +/- 1 day of oestrus in donors.     

Bovine parthenogenesis is characterized by abnormal chromosomal complements: Implications for maternal and paternal co-dependence during early bovine development

The present study was conducted to examine the karyotypes of parthenogenetic bovine embryos arising from the application of standard oocyte activation and diploidization methods. Bovine cumulus–oocyte complexes were collected and matured in vitro for 24 hr prior to oocyte activation with either 5 μM ionomycin or 7% ethanol for 5 min. Groups of activated oocytes were further treated with 5 μg/ml cytochalasin D or 1.9 mM 6-dimethylaminopurine (DMAP) for 6 hr. Cleavage varied significantly (P < .05) among the treatment groups with 68.0% of the ethanol- and DMAP-treated oocytes dividing. Blastocyst development did not vary with 18.4 ± 2.5% of all treated oocytes progressing to this stage. Blastocyst development did not occur in groups subjected to oocyte activation alone. Blastocysts displayed haploid (2.3%), diploid (11.4%), tetraploid (40.9%), octaploid (4.5%), and mixoploid chromosomal complements (40.9%). Two-cell stage parthenogenotes resulting from ethanol or ionomycin treatment alone displayed haploid (66.7%), diploid (16.7%), tetraploid (4.2%), and mixoploid (12.5%) complements. Our results demonstrate that diploid bovine parthenogenotes arising from these procedures are a minority, with the majority of parthenogenotes displaying polyploid and mixoploid chromosomal complements. The events contributing to these abnormal chromosomal complements occur as early as completion of the first cell cycle, possibly linking these events with the absence of a paternally supplied centrosome. Dev. Genet. 21:160–166, 1997. © 1997 Wiley-Liss, Inc.     

Successful in vitro culture of early cleavage stage embryos recovered from superovulated red deer (Cervus elaphus)

Three separate embryo culture systems were evaluated for their ability to support development of early cleavage stage red deer (Cervus elaphus ) embryos: ligated sheep oviducts (Treatment A); cervine oviduct epithelial monolayer in TCM 199 + 10% deer serum (Treatment B); synthetic oviduct fluid + 20% human serum at 7% O(2) atmosphere (Treatment Q. In addition, 2 superovulation protocols were compared for their efficacy in producing early cleavage stage embryos. Twenty red deer (2 to 7 yr old) were synchronized in April with intravaginal CIDR devices for 12 d. All animals received a total of 0.4 units of ovine FSH administered in 8 equal doses, 12 h apart, beginning 72 h before removal of CIDR devices. The deer additionally received 200 IU PMSG, either with the first FSH injection (Group 1, n = 10) or with the last FSH injection (Group 2, n = 10). Hinds were placed with fertile stags following withdrawal of CIDR devices. Ova were collected by surgical recovery 63 h post CIDR removal. At the time of collection, animals in Group 2 had a significantly greater mean (+/- SEM) ovulation rate (11.2 +/- 2.4 vs 5.3 +/- 2.4), with more animals responding to treatment (>1 ovulation), than the animals in Group 1 (10/10 vs 4/10). Late in the breeding season (June), 10 additional red deer (Group 3, Experiment 2) were superovulated using the same protocol as for the deer in Group 2, with ova collection advanced by 24 h. Mean (+/- SEM) ovulation rate was 6.4 +/- 1.2 with 9 10 animals responding. Ova recovery did not differ among the groups (range 73 to 87%). Superovulation treatment did not affect cultured embryo development to the morula/blastocyst stage. Furthermore, there was no difference among the 3 culture systems in their support of development either to the morula (range 50 to 58%) or to the blastocyst (range 22 to 26%) stage. After laparoscopic transfer of 4 morula/blastocyst embryos to recipient red deer (2 from Treatment B and 2 from Treatment C) 2 live calves were born from embryos cultured in Treatment B.     

Interspecific transfer of IVM IVF-derived red sheep (Ovis orientalis gmelini ) embryos to domestic sheep (Ovis aries )

Two embryo production methodologies were investigated to generate Red sheep embryos for use in an interspecific embryo transfer program. In Experiment 1, 4 multiparous female Red sheep (Ovis orientalis gmelini ) were implanted with CIDR type G devices for 11 d. Forty-eight hours prior to CIDR removal, a total of 22.5 mg bid of FSH-P was administered over a 3-d period. Laparoscopic embryo collection was performed 5 d post breeding, and embryos were transferred to domestic recipient ewes (Ovis aries and Ovis orientalis musimon ). In Experiment 2, 7 nulliparous female Red sheep were implanted with CIDR devices and injected with 200 IU of PMSG and 25 mg of FSH-P on the 8th day of implant insertion. At 60 to 70 h post PMSG/FSH-P treatment, follicular oocytes were aspirated laparoscopically. The recovered oocytes were matured in M199 (with fetal calf serum, FSH, LH, penicillin and streptomycin) at 39 degrees C in a humidified atmosphere containing 5% CO(2). At 24 h oocytes were fertilized with frozen-thawed semen at a concentration of 1.6 x 10(6) sperm/ml. The ova/embryos were placed in CR2 or BOEC culture medium at 20-22 h post IVF. Following 3 to 4 d in culture, embryos were transferred laparoscopically to the uterine horn of synchronized recipients. In Experiment 1, 4 embryos and 6 UFO were collected from 2 embryo donors, respectively. Two embryos were transferred with the aid of a laparoscope to each of 2 Rambouillet recipients, one of which gave birth to a healthy Red sheep lamb at 158 d of gestation. In Experiment2, a total of 62 oocytes was collected from 7 oocyte donors; 16 developed to the 16- to 32-cell stage and were transferred to 8 recipients. Three of these IVM-IVF embryos were transferred laparoscopically to 2 Mouflon recipients, resulting in no pregnancies. Thirteen IVM-IVF embryos were transferred to 6 Rambouillet recipients. Each of these gave birth to a single healthy Red sheep lamb. Gestation lengths of the 3 IVM-IVF lambs ranged from 152 to 162 d. This research demonstrates that when using compatible species IVM-IVF technology in conjunction with interspecific ET can lead to the production of live offspring and can be used to propagate exotic ovine species.     

Comparative studies on the superovulatory effect of PMSG and FSH in water buffalo (Bubalus bubalis )

A total of thirty-eight lactating water buffalo cows were treated in four experiments simultaneously either with FSH (first group) or PMSG(second group). To the first group (half of the animals), a total dose of 40 mg FSH-P at 12-hr intervals was given i.m. within a 4-day period. The second group was treated i.m. with 3000 IU PMSG (Gestyl). Forty-eight hours after initiation of the superovulatory treatment all buffaloes were given 500 ug Cloprostenol. Fi seen buffaloes from the FSH-treated group (78.9%) and 17 from the second group (89.5%) came into heat at average PGF 2 alpha/standing heat intervals of 42.8+/-1.48 and 44.8+/-2.31, respectively. Superovulatory treatment resulted in meath number of 4.3+/-0.87 and 1.9+/-0.50 CL and 0.5+/-0.24 and 2.2+/-0.82 follicles for the first and second group. Twenty-five eggs were recovered after non-surgical flushing from 8 of 13 flushes in the first group and all except one were fertilized and classified as good embryos. Twelve eggs were recovered from 4 of 11 flushes in the second group and 11 of the eggs were fertilized and 10 of them classified as good ones.