Chromosomal analysis of embryos produced by artificially inseminated superovulated cattle

Analyse chromosomique chez des embryons provenant de vaches super-ovulees et inseminees artificiellement. Afin de determiner la garniture chromosomique d'embryons bovins aux stades de pre-morula, 30 genisses Holstein ont ete superovulees et inseminees. Les embryons furent recoltes aux jours 2, 3 ou 4 et soumis a une analyse chromosomique. Sur un total de 298 embryons/ovules recoltes, 101 montraient une ou plusieurs cellules en metaphase et 60 de ceux-ci ont pu etre examines pour leur caryotype. Huit embryons analyses presentaient une garniture chromosomique anormale : 3 triploides, 1 tetraploide et 4 mixoploides. Il est conclu que les anomalies se produisent au moment de la fertilisation ou juste apres.

Huit embryons analysés présentaient une garniture chromosomique anormale : 3 triploïdes, INTRODUCTION Under intense agricultural management superovulation is used to induce multiple ovulation in cattle to provide genetically valuable embryos for collection and transfer. However, only about 60% of the embryos that are collected from superovulated cattle have normal morphology and are considered suitable for transfer (Schneider Jr, et al, 1980;Schiewe et al, 1987;Lopez Gatius et al, 1988). In humans, chromosomal abnormalities in the embryo or fetus are the most frequent causes of malformations and pregnancy failure (Jacobs et al, 1978). Chromosomal analysis of cattle embryos at the morula and blastocyst stages, when embryo transfer is usually performed, has revealed abnormalities that are thought to compromise development (King, 1991). These abnormalities include aneuploidy, mixoploidy and polyploidy (for review see King, 1990). Prior to the morula and blastocyst stages very few observations on the chromosomal constitution of embryos produced by superovulated cattle have been documented (King and Picard, 1985;Murray et al, 1985). Hence, little is known of the situation close to the time of fertilization.
The objective of this study was to determine the chromosomal complement of pre-morula stage embryos produced by superovulated cattle. Some of the embryos reported here were included in a preliminary report published in abstract form (Verini Supplizi et al, 1988).

MATERIALS AND METHODS
Embryos were produced by superovulated Holstein heifers inseminated once or twice with semen from a highly fertile Holstein bull during the 24 h following onset of behavioural estrus. Superovulation was induced by treatment with folliclestimulating hormone (FSH-p; Burns-Biotech Laboratory, Oakland, CA, USA) and cloprostenol (Estrumate: ICI Pharms, Mississauga, ON, Canada) as previously described (King et al, 1987). Females were checked for signs of behavioural estrus twice daily. The first detection of behavioural estrus was designated day zero.
Embryos were collected by post-mortem retrograde flush of the oviducts on day 2 (n = 6), 3 (n = 23) and 4 (n = 1). Only one oviduct from 11 of the heifers was available for use in this study. For all collections the flushing medium was Dulbecco's phosphate-buffered saline (PBS, pH 7.2) supplemented with 2% fetal calf serum (FCS) and antibiotics (100 iu penicillin, 100 pg streptomycin/ml). Once the embryos were located in the flushing medium they were washed in PBS containing 10% FCS and antibiotics and transferred to Hams F10 containing 20% FCS and antibiotics and colcemide (0.05 pg/ml medium; Sigma, Saint Louis, MO, USA).
The embryos were incubated in this medium for 4-8 h and then fixed individually on slides as previously described (King et al, 1979). Slides were then stained with Giemsa and examined for cell number and chromosome composition. Fertilization was evaluated after fixation and was considered to have occurred if any of the following were observed: mitotic chromosomes; 2 or more pronuclei/nuclei; or 2 or more blastomeres. Ova that presented meiotic chromosomes or lacked nuclei were considered unfertilized.

RESULTS
Flushing the reproductive tracts of the 30 females yielded a total of 298 embryos/ova. The mean rate of fertilization (percent of total recovery) was 83.2%. In all 101 embryos (33.9%) had one or more cells in metaphase. The karyotype of 60 of these embryos (59.4%) could be determined while 41 had metaphase spreads that were either incomplete or of insufficient quality for analysis. Of the 60 karyotyped embryos, 52 (86.6%) were found to be diploid (60XX or 60XY) and 8 (13.3%) were other than diploid (table I). The abnormal complements included 3 triploids, embryos had cleaved to 2-cell stage although 4 nuclei (2 haploid and 2 diploid) were present in each. The fourth mixoploid had not cleaved but contained a haploid and a diploid nucleus (fig 1). The diploid nucleus in this embryo contained 62 chromosomes. In all cases the abnormal embryos were among the least developmentally advanced embryos, estimated on the basis of cell number, within the flush of the donor female from which they originated (table I).

DISCUSSION
Chromosome abnormalities have been observed in the embryos of most domestic animals. In sheep and pigs, a frequency of chromosomally abnormal embryos of 10.4 and 6.6%, respectively, has been reported (for review see King, 1990 (1987) found a higher rate of chromosomally abnormal embryos on day 7 among morphologically abnormal embryos with low cell numbers than among morphologically normal ones. In the present study the abnormal embryos were among the least developmentally advanced within individual donors (table I).
If indeed chromosomally abnormal embryos have a slower rate of development and hence a lower cell number, the present observations suggest that development may begin to slow down as early as day 2.
Chromosome analysis of in vitro-produced bovine embryos has shown abnormalities in 12.1% of embryos at the 2-cell stage, 20.0-36.4% at 4to 16-cell stage and 44.2% at blastocyst (Iwasaki et al, 1989). Kawarsky (1994) has noted a frequency of abnormalities of 27.4% on day 2 (1-8 cells) and 32.1% on day 5 (8-cell stage to morula) in vitro. As with the in vivo studies, both of these in vitro studies suggest an accumulation of chromosomally abnormal embryos over the first week of development. Unfortunately the limitation of in vitro culture prevents monitoring development beyond the blastocyst stage into the second week of development to determine if there is an elimination of abnormal embryos as the embryo begins to elongate.
All 8 abnormal embryos were either 1 or 2 cells suggesting that the abnormality occurred at or close to the time of fertilization before completion of the first cell cycle. The 3 triploids and 1 tetraploid were 2-cell embryos. Unfortunately, the exact origin of the extra haploid set(s) of chromosomes could not be determined. In humans, triploid fetuses originate from dispermic fertilization (66%), diploid sperm (24%) or diploid oocytes (10%; Jacobs et al, 1978). Tetraploid embryos are less common and mechanisms leading to their production are not well studied. They could, however, arise by combinations of the mechanisms proposed for triploids as well as by failure of cytokinesis at first cleavage or by endoreduplication of the pronuclei. In cattle all of these pathways are possible since polyspermic fertilization, diploid sperm, diploid oocytes and endoreduplication have been reported Iwasaki et al, 1989;Yadav et al, 1991;Kawarsky, 1994). In pigs it has been reported that the incidence of polyploidy arises due to ageing of the oocyte when insemination is delayed (Bomsel-Helmreich, 1961). However, this has not been confirmed in cattle.
All 4 of the mixoploid embryos exhibited haploid nuclei (2 in the 3 two-cell embryos and 1 in the 1-cell embryo). The presence of a Y chromosome in the haploid cells in 2 of the embryos suggests that the oocytes leading to these embryos were fertilized by 2 spermatozoa. The X-chromosome bearing haploid cells may have originated from a spermatozoon, a binucleated oocyte or a polar body. The fate of the haploid cells is not known. King and Picard (1985) and Iwasaki and Hamano (1991) described morula and pre-morula with haploid cells. However, older embryos with such cells have not been reported. It is possible that these nuclei die, become quiescent or are somehow eliminated from the embryo. It is also possible that they eventually fuse with their diploid cohorts as diploid-triploid mixoploids have been reported in blastocysts and elongated blastocysts (Hare et al, 1980;King et al, 1987).
It was concluded that roughly 13% of day 2-4 embryos from superovulated cattle that could be cytogenetically analyzed were chromosomally abnormal. The abnormalities most likely arose at or soon after fertilization due to fertilization by a second spermatozoon or failure of polar body extrusion.

ACKNOWLEDGMENTS
The financial support of Natural Sciences and Engineering Research Council of Canada and the Canadian Association of Animal Breeders is appreciated. Scholarships from the government of Italy (AVS) and the Canadian International Development Agency (HEPD) are gratefully acknowledged.