BIOLOGY OF REPRODUCTION 54, 53-59 (1996)
Chromosomal Abnormalities in Bovine Embryos and Their Influence on Development'
Sheldon J. Kawarsky, 3 Parvathi K. Basrur,3 Robert B. Stubbings, 4 Peter J. Hansen,5 and W. Allan King 2' 3
Departmentof Biomedical Sciences,3 University of Guelph, Guelph, Ontario, CanadaNIG 2W1
Semex Canada,4 Guelph, Ontario, CanadaN1G 3Z2
Department of Dairy and Poultry Sciences,5 University of Florida, Gainesville, Florida32611
ABSTRACT
A study was undertaken to determine the relationship between chromosome composition and embryo development. Bovine cumulusoocyte complexes were matured invitro and exposed to semen from one of three different bulls, one of which was a 1/29 Robertsonian
translocation carrier. There were no significant differences among the three bulls intheir sperm penetration or inthe cleavage or developmental rates of resulting embryos, which were subjected to chromosome analysis on Day 2 (40-44 h postinsemination [hpil) and Day
5 (120-124 hpi) of development. No difference was detectable inthe growth rates of embryos of different chromosomal composition on
Day 2.On Day 5, atotal of 343 embryos were obtained from all three bulls, of which 158 embryos could be karyotyped and assessed for
cell numbers. Cell numbers for the Day 5 embryos showed that the mean numbers for the individual chromosome compositions (leastsquares means ± SEM) were 7.9 + 6.0 for haploids, 7.9 6.0 for polyploids, 16.8 4.3 for aneuploids, 23.4 4.0 for mixoploids, and
30.0 1.7 for diploids, indicating a significant reduction inthe growth rate of embryos with chromosomal abnormalities (p < 0.001). It
was concluded that development rates (as evidenced by cell numbers) were slowest inhaploid and polyploid embryos, intermediate in
aneuploid embryos, and fastest inmixoploid and diploid embryos.
INTRODUCTION
fect on bovine embryo development. With the recent advances in technology to produce embryos in vitro, the study
of preimplantation/preattachment embryos has become
more feasible, and it is possible to directly observe the impact of chromosomal abnormalities on embryo development. Bovine embryos derived by in vitro procedures are
similar to embryos produced in vivo in many respects, including rates of cleavage up to the eight-cell stage, protein
synthetic profiles [9], glucose metabolism [10], ultrastructure
[11], and rate of pregnancy after the transfer of selected blastocysts to recipients [8]. In vitro developmental rate, if identified as an indicator of chromosomal anomalies, may serve
as a tool to eliminate chromosomally abnormal embryos
from subsequent transfer. For this to be feasible, it is necessary to determine whether such embryos develop more
slowly than normal embryos and to identify the postinsemination interval at which developmentally advanced embryos tend to be of normal chromosome composition. The
present study was undertaken to examine the incidence and
types of chromosomal abnormalities detectable at specific
times of development of in vitro-derived bovine embryos
and to examine the relationship between these abnormalities and embryonic development.
Chromosomal abnormalities constitute a major cause of
embryonic loss in mammals [1, 2]. Incidence of chromosomal abnormalities in the embryos of domestic animals has
been estimated to be at 7-10% (for review see [2]). However, only a few chromosomally unbalanced individuals are
born alive in domestic animals or human beings [1, 2], indicating that most of these chromosomal errors cause embryonic or fetal loss. Chromosomal abnormalities present in
liveborns include those involving the sex chromosomes
(monosomies or trisomies), autosomal trisomies, or balanced structural rearrangements; all of these abnormalities
cause reproductive and health-related problems of varying
degrees [1, 21. Incidence of chromosomally abnormal embryos also declines as the age of the embryo increases, further indicating a progressive loss of abnormal embryos at
specific stages in early development [2, 3].
While various investigators have examined the incidence
of chromosomal abnormalities in bovine embryos produced in vitro [3-5], few have looked at these anomalies at
specific cell stages [3]. While it has been shown that the sex
chromosomal complement of bovine embryos can affect
the rate of development and lead to faster development of
the male embryos than of the female ones [6-8], it is not
clear when autosomal abnormalities begin to exert their ef-
MATERIALS AND METHODS
Accepted September 1, 1995.
Received July 12, 1995.
'Presented in part at the 27th annual meeting of the Society for the Study of Reproduction, July 1994, Ann Arbor, MI. This project was financially supported by the Natural Science
and Engineering Research Council, Agriculture Canada, the Canadian Association of Animal
Breeders, and the Ontario Ministry of Agriculture, Food and Rural Affairs.
2Correspondence: W. Allan King, Department of Biomedical Sciences, Ontario Veterinary College, University of Guelph, Guelph, Ontario, Canada NIG 2W1. FAX: (519) 7671450; e-mail: [email protected]
Embryo Production
Embryos were produced by in vitro fertilization as described by Xu et al. [8]. Briefly, cumulus-oocyte-complexes
(COCs) were obtained by slicing ovaries in a medium containing HEPES-buffered Ham's F-10 (Gibco Laboratories,
Burlington, ON) supplemented with 2.0% (v/v) steer serum,
53
54
KAWARSKY ET AL.
2.0 IU heparin/ml, 10 000 IU penicillin/ml, and 10 000 gg
streptomycin/ml (Gibco). The COCs were transferred into
50-pl droplets (10 COCs/droplet) of Tissue Culture Medium
(TCM) 199 with 25 mM HEPES (Gibco) supplemented with
10% steer serum under light mineral oil (centistokes < 33.5
at 40.0°C; Sigma, St. Louis, MO) and allowed to mature for
22-24 h. The expanded cumulus was removed mechanically by continuous pipetting in Tyrode's Albumin-LactatePyruvate-Hepes medium (TALP-Hepes; [12]). Motile spermatozoa were selected from frozen-thawed sperm by the
swim-up technique [12, 13]. Denuded oocytes were fertilized with 1 X 106 spermatozoa/ml for 18-20 h under light
mineral oil in 50 pl TALP-in vitro fertilization medium [12]
during coculture with bovine oviductal epithelial cell
(BOEC) explants. The BOEC explants form worm-like structures with active cilia [8]. Approximately 15-20 BOEC explants were added to each 50-gl culture droplet. After fertilization, presumptive zygotes were transferred to 50-gl
microdrops containing BOEC and in vitro culture (IVC) medium (TCM 199 supplemented with 10% steer serum), with
10 zygotes/50-pl droplet. Zygotes were cultured for two or
five days in vitro (day of insemination = 0). For embryos
cultured to Day 5, an additional 50 p1 of IVC medium was
added to the droplets 3 days after insemination. All incubations were carried out at 38.50C in a humidified atmosphere of 5.0% CO2 in air.
Experimental Design
Approximately 240-300 oocytes were collected on each
day of the experiment. The oocytes were divided evenly
into two main groups representing the duration of culture.
Each culture group was then further subdivided into three
subgroups that were simultaneously exposed to sperm samples from different bulls: a Holstein-Friesian (N), a Polled
Limousin (P), and a Swedish Red and White 1/29 Robertsonian translocation carrier (T). One culture group of embryos was examined on Day 2 (40-44 h postinsemination
[hpi]); the other was examined on Day 5 (120-124 hpi). This
experiment was replicated 12 times.
Sperm penetration (2 replicates only) was confirmed under the microscope by the presence of two or more pronuclei
(polyspermy by three or more pronuclei) and evidence of
sperm structures within the oocytes. Cleavage rates (Day 2
and Day 5 culture groups) were determined 40-44 hpi and
expressed as a percentage of the embryos exhibiting two or
more blastomeres out of the total number of oocytes used
for each bull. Embryo development (Day 5 only) beyond the
eight-cell stage was assessed at 120-124 hpi. Embryos were
assessed for development after fixation onto slides to count
the total number of nuclei, and the rate of development was
calculated as a percentage of the embryos that reached beyond the eight-cell stage out of the total number of oocytes
exposed to spermatozoa for each subgroup.
Chromosome Analysis
Embryos (Day 2 and Day 5) were prepared and examined
for their cytogenetic composition and the total number of
nuclei each embryo contained. Colcemid (Gibco) was added
to the culture drops to give a final concentration of 0.05 I1/
ml, and the drops were incubated for 5 h at 38.5°C in a humidified, 5.0% CO 2 in air environment. Each embryo was
treated with 1.0% (w/v) sodium citrate and fixed on slides
[14]. The slides were stained in a 4.0% (w/v) Giemsa solution
for 4 min, and the nuclei and metaphase spreads were
counted under a compound microscope at low power. The
chromosomes were examined at 1000 x under oil, and the
chromosome composition was determined for each embryo.
The categories for the chromosome composition of the embryos were classified as follows: haploid, polyploid, mixoploid (embryo with blastomeres of different ploidies), aneuploid, or diploid. Any embryo that did not fall into these
categories due to gross overspreading or clumped chromosomes was classified in the nonkaryotyped category.
StatisticalAnalysis
Statistical analyses were performed by least-squares
analysis of variance using the General Linear Models procedure (Tables 1 and 3) or by the Categorical Data Modelling (CATMOD) procedure (Table 2) of the Statistical Analysis System [15]. For categorical data analyzed by
least-squares analysis of variance, data analyzed were percentages calculated for each bull-replicate subclass. The
main effects in the model were bull and replicate, and,
where appropriate, chromosome composition and day
(Day 2 or 5). Data were analyzed three ways: 1) as a complete data set, 2) after nonkaryotyped embryos were excluded (to determine treatment effects in karyotyped embryos only), and 3) after nonkaryotyped and diploid
embryos were excluded (to determine treatment effects in
abnormal embryos only). Data were first analyzed with all
possible interactions in the model and then reanalyzed after
removing nonsignificant or nonestimable terms.
RESULTS
Penetration, Cleavage, and Development Rates
A total of 267 oocytes in two replicates were used to
determine the bovine oocyte penetration rates by bulls N,
T, and P (Table 1). Although polyspermy was observed,
there were no significant differences among the three bulls
tested. Cleavage and development rates beyond the 8-cell
stage were determined for all three bulls, with a total of 2489
oocytes in 12 replicates (Table 1). There were no significant
differences among the three bulls for any endpoints.
CHROMOSOMAL ABNORMALITIES AND DEVELOPMENT
55
TABLE 1. Penetration, cleavage, and development rates for bulls N,T, and pa
Penetration
Polyspermy
(%)
Bull N
Bull T
Bull P
SEM (%)
53/88 (60.2)
44/79 (55.7)
59/100 (59.0)
7.0
Development > 8-cellC
Cleavageb
(%)
(%)
2/53 (3.8)
3/44 (6.8)
3/59 (5.1)
1.9
(%)
346/833 (41.5)
350/840 (41.7)
330/816 (40.4)
3.3
63/ 39 5 d (15.9)
65/395 (16.4)
55/392 (14.0)
2.2
aNo effect of bull on any endpoint as determined by least-squares analysis of variance.
bNumber of embryos cleaved/number of oocytes (combined Day 2 and Day 5).
CNumber of embryos developed/number of oocytes (Day 5 only).
dOnly half of the oocytes were allowed to develop, therefore the number of oocytes is reduced.
Occurrenceof ChromosomalAbnormalities in Embryos
Embryonic Development and ChromosomalAbnormalities
The number and percentage of chromosomally abnormal embryos at Days 2 and 5 postinsemination are presented in Table 2. There were no effects of day or bull X
day on the incidence of abnormalities except that the frequency of mixoploids was greater (p < 0.01) on Day 5 (19
of 158, 12.0%) than on Day 2 (8 of 204, 3.9%). Mixoploidy
was seen only in embryos that had developed beyond the
8-cell stage. The most frequent types of mixoploidy seen
were diploid/triploids (10 of 158, 6.3%) and diploid/tetraploids (6 of 158, 3.8%). While mixoploidy was very common
on Day 5 (19 of 158, 12.0%), the most common abnormalities on both days were aneuploidy (53 of 362, 14.6%) and
polyploidy (39 of 362, 10.8%). Polyploid embryos consisted
mostly of triploids (28 of 362, 7.7%) and tetraploids (10 of
362, 2.8%).
The overall frequency of chromosomal abnormalities differed among embryos obtained with semen from different
bulls (p < 0.03). The frequency of abnormalities in embryos
was greatest for bull T (61 of 138, 44.2%), intermediate for
bull P (41 of 109, 37.6%), and least for bull N (34 of 115, 29.6%).
More embryos with aneuploidy were obtained with bull T (32
of 138, 23.2%) than with bulls N (10 of 115, 8.7%) or P (11 of
109, 10.1%) (effect of bull, p < 0.005). There were no differences in the frequency of other abnormalities among the embryos of the three bulls. Differences among bulls were similar
at Day 2 and Day 5; i.e., there were no bull X day effects.
There was no overall effect of bull on the rate of embryonic development as evidenced by cell number at Day 5
(least-squares mean
SEM: 15.4 + 1.7, 16.7 + 1.9, 16.3
2.0, for bulls N, T, and P, respectively). No difference was
detectable in the growth rates of embryos of different chromosome composition on Day 2. However, on Day 5 there
was a difference between chromosomal groups. The chromosome composition and cell number of embryos on Day 5
are summarized in Table 3. The frequency of chromosomally
normal (diploid) embryos increased as development progressed. The percentage of normal embryos (out of those
karyotyped) was 25.0% for the 2-8-cell stage, 53.3% for the
9-16-cell stage, 74.4% for the 17-31-cell stage, and 73.9%
for those embryos with 32 cells or more. Diploid embryos
(Fig. 1) were found at all cell stages but predominated among
those with higher cell numbers. Aneuploid embryos (Fig. 2)
were present at all cell stages but were most abundant among
embryos ranging from 2 to 31 cells, whereas haploid (Fig. 3)
and polyploid (Fig. 4) embryos were most frequent for embryos with cell numbers ranging from 2 to 16 cells. Mixoploid
embryos were absent from those in the early cleavage stages
(2 to 8 cells) but were among those with higher cell numbers.
As determined by least-squares ANOVA, the rate of development as evidenced by cell number for Day 5 embryos was
slowest for haploid (7.9 + 6.0 cells) and polyploid embryos
(7.9
6.0 cells), fastest for mixoploid (23.4
4.0 cells) and
TABLE 2. Number and percentage of chromosomally abnormal embryos on Day 2 and Day 5 produced by sperm from bulls N,T, and p.a
Day 2
Bull N
Bull T
Bull P
Total
Day 5
Bull N
Bull T
Bull P
Total
Haploid
(%)
Mixoploid b
(%)
Polyploid
(%)
Aneuploidc
(%)
3/62 (4.8)
2/81 (2.5)
4/61 (6.6)
9/204 (4.4)
2/62 (3.2)
1/81 (11.2
5/61 (8.2)
8/204 (3.9)
5/62 (8.1)
10/81 (12.3)
8/61 (13.1)
23/204 (11.3)
7/62 (11.3)
22/81 (27.2)
5/61 (8.2)
34/204 (16.7)
17/62 (27.4)
35/81 (43.2)
22/61 (36.1)
74/204 (36.3)
3/53 (5.7)
3/57 (5.3)
2/48 (4.2)
8/158 (5.1)
6/53 (11.3)
8/57 (14.0)
5/48 (10.4)
19/158 (12.0)
5/53 (9.4)
5/57 (8.8)
6/48 (12.5)
16/158 (10.1)
3/53 (5.7)
10/57 (17.5)
6/48 (12.5)
19/158 (12.0)
17/53 (32.1)
26/57 (45.6)
19/48 (39.6)
62/158 (39.2)
aNumber of embryos with specific chromosomal abnormality/number of embryos karyotyped.
bPercentage embryos classified as mixoploid affected by day (p < 0.01).
CPercentage embryos classified as aneuploid affected by bull (p < 0.005).
dPercentage of embryos classified as abnormal affected by bull (p < 0.03).
Total d
(%)
56
KAWARSKY ET AL.
TABLE 3. Chromosomal composition and cell numbers* for Day 5 embryos produced by sperm from bulls N, T, and P.
Number of embryos for each chromosomal composition
Cell stage
2-8
9-16
17-31
32Total
Cell number
Number of
embryos
Karyotyped
(%)
Diploid
(%karyotyped)
Haploid
Mixoploid
Polyploid
Aneuploid
160
73
54
56
343
24 (15.0)
45 (61.6)
43 (79.6)
46 (82.1)
158 (46.1)
6 (25.0)
24 (53.3)
32 (74.4)
34 (73.9)
96
30.0 + 1.7a
4
4
0
0
8
7.9 ± 6 .0 b
0
8
2
9
19
'
23.4 + 4.0aC
9
4
3
0
16
5
5
6
3
19
16.8 + 4.3bc
7.9 + 6 .0b
a'bCValues with different superscripts differ significantly as determined by pdiff procedure of SAS (p < 0.05). Overall effect of chromosomal type on cell number, p < 0.001.
*Least-squares means SEM.
diploid (30.0
1.7 cells) embryos, and intermediate for
aneuploid embryos (16.8 ± 4.3 cells).
DISCUSSION
The present study demonstrates that the chromosomal
status of the embryo influences in vitro developmental rate.
Development, as judged by total number of cells for Day 5,
was greatest in diploid and mixoploid embryos, least in
haploids and polyploids, and intermediate in aneuploids.
These developmental rates are unlikely to be an artifact of
the culture system used since consistent rates of cleavage,
development beyond the 8-cell stage, and incidence of
chromosomally normal embryos have been produced in
our laboratories with similar culture systems [8, 16-18].
Also, the percentage of chromosomally abnormal embryos
obtained with semen from the normal bull in this study falls
within the range (12-44%) reported in other studies using
semen from normal bulls [3-5, 19, 20]. The incidence of
chromosomal abnormalities in embryos produced by in vitro procedures is generally higher than that of embryos
formed in vivo [19, 20], probably in part because chromosomally abnormal embryos are less likely to be detected
among the latter because of their low mitotic index and/or
loss during earlier stages of embryonic development [1, 2].
Previous studies have shown that the sex of the embryo
has an effect on the developmental rate of mouse [21-23],
pig [24], and human embryos [251], with male embryos developing faster than female embryos. In cattle, male embryos cleave earlier than females [17], although the difference in developmental rate, as judged by cell numbers only,
becomes statistically significant between Days 5 and 8
[8, 17]. However, the effect of chromosome constitution as
a whole on embryonic development has not been fully explored in cattle. In the present study, haploid embryos were
noted to carry fewer cells than diploid embryos on Day 5
in vitro development. Lack of an entire set of chromosomes
may cause a reduction in developmental rate due to the
lower levels of gene products and the suboptimal nucleocytoplasmic ratio in the haploid embryos, which in turn may
increase the duration of the cell cycle and slow down development [26]. UJltrastructural evidence suggests that the
retarded development may have a metabolic basis. In cattle,
haploid parthenogenotes have been observed to possess
few microvilli and lipid droplets, suggesting poor uptake
and storage of nutrients. In addition to an acentrically located nucleus suggesting cytoskeletal disorganization [27],
some parthenogenotes were also noted to carry abnormal
golgi apparatus [27] indicative of an inability to process proteins [28]. Mouse haploid parthenogenotes have been seen
to develop more slowly in culture during the preimplantation period compared to fertilization-derived diploid embryos [29, 30].
Polyploid embryos were also noted to develop at a
slower rate than diploid embryos in the present study. Similarly, triploids and tetraploids among in vivo-derived bovine embryos possessed lower cell numbers relative to their
diploid counterparts [31]. Studies on murine embryos lead
to conflicting results: in one study, triploid embryos cleaved
more slowly than diploid [32-34], whereas other reports
indicate no difference in cleavage rates between these
groups [35-37]. The difference in the results may be due to
the different techniques used to induce polyploidy in these
studies. The possibility exists, however, that the duration of
the cell cycle is increased in polyploids because of the time
needed for the replication of larger numbers of chromosomes and that the prolonged cell cycle time may be responsible for the decrease in the developmental rate as evidenced by a lower cell number. Interestingly, mixoploids
in the present study exhibited rates of development similar
to those of the diploids on Day 5. Mixoploids with diploid/
triploid or diploid/tetraploid chromosome constitution can
arise through fusion of blastomeres in a growing embryo
[38] or through chromosome replications without subsequent karyokinesis during cleavage divisions [2]. The slower
rate of development in polyploid bovine embryos ([31],
FIG. 1. Metaphase spread and karyotype from diploid embryo showing normal
chromosome complement of 60.
FIG. 2. Metaphase spread and karyotype from trisomic embryo (trisomy 1)derived
from bull T. Note 1/29 chromosome (arrow) and two of normal chromosome 1.
FIG. 3. Haploid metaphase spread of 4-cell embryo. Note that chromosome complement is 30.
FIG. 4. Polyploid (tetraploid) metaphase spread of 2-cell embryo.
CHROMOSOMAL ABNORMALITIES AND DEVELOPMENT
57
58
KAWARSKY ET AL.
present study] indicates that diploid/polyploid mixoploids
also could eventually end up with a reduced cell number
later in development. It has been shown, however, that mixoploid embryos may produce live-born young if polyploid
cells enter the trophoblast lineage and participate in the formation of extraembryonic structures. Diploid/polyploid
mixoploidy has also been observed in the trophoblasts of
many domestic species after Day 10 in vivo [39-41].
The timing of the transcriptional burst in mammalian embryos is species-specific [42-44]. In cattle, there is a low
level of transcription at the 2-cell stage [18]; however, the
burst in activity occurs at the 8-cell stage, which is also the
point when development usually stops in vitro under suboptimal conditions [45, 46]. Given that transcription starts as
early as the 2-cell stage in in vitro-produced bovine embryos, it is conceivable that chromosome constitutions will
affect development even at this early cell stage. King et al.
[201 have shown that among in vivo-derived Day 2 embryos,
chromosomally abnormal ones are the least advanced in
development within each flush. Our results suggest that the
developmental rate of the embryo is also governed by its
ploidy, possibly as early as at the 2-cell stage.
In the present study, the rates of penetration, cleavage,
and development beyond the eight-cell stage were observed to be similar for all three bulls. This may be attributable to the in vitro fertilization system used, which may
have eliminated differences between bulls. However, it is
noteworthy that differences in these parameters have been
previously reported in embryo development [47-51]. In the
present study, bull-related differences were noted only in
the frequency of chromosomal abnormalities in the embryos produced. Bull T, a 1/29 Robertsonian translocation
carrier, was shown to produce more chromosomally abnormal embryos than bull N or bull P. This difference may
be attributable to the production of aneuploid embryos,
which, on a theoretical basis alone, should be much higher
for embryos produced by a translocation carrier than for
those from chromosomally normal bulls [52]. Carriers of this
type of translocation can produce gametes with six different
chromosome complements, only two of which could produce viable offspring while the other four types would produce chromosomally unbalanced embryos (either monosomic or trisomic involving chromosomes 1 and 29). Loss
of these embryos, occurring within the first or second week
of development [38, 53] has been postulated to be a major
factor leading to the reduction of fertility in 1/29 translocation carriers [54].
Fertility of 1/29 Robertsonian translocation carrier bulls
has been reported to be 7-20% lower than that of normal
bulls [55, 56]. As stated above, this reduction in fertility may
be attributable to the production of aneuploid embryos.
Aneuploid embryos produced by the three bulls (predominantly by bull T) had developmental rates intermediate to
the slowest (haploids and polyploids) and the fastest (dip-
loids and mixoploids). The slower rate of development of
aneuploid embryos relative to that of diploids may be due
to the repair mechanism that causes "developmental arrest"
as seen in human aneuploid blastomeres [571. This mechanism is postulated to lead to the elimination of genetic material either by exclusion from interphase nuclei followed
by its intracellular degradation or by extrusion of DNA and
formation of structures called pseudonuclei outside the cell
[58]. Alternatively, an imbalance in the expression of genes
due to the hypodiploid or hyperdiploid status of these embryos may be responsible for the reduced rate of embryonic
development compared to that of diploid embryos.
It has been estimated that the incidence of chromosomal
abnormalities in in vitro-derived bovine oocytes is approximately 15%, comprising 4.2% aneuploids and 10.7% diploids
[59], whereas the incidence of nondisjunction in bovine spermatogenesis is approximately 2.8% [60]. Therefore, a portion
of the abnormal embryos can arise from chromosomal abnormalities in the gametes, while errors in fertilization and
development account for the remainder. As our knowledge
of the factors controlling fertilization and in vitro culture systems improve, it may be possible to reduce the incidence of
chromosomal abnormalities in embryos. However, the total
elimination of chromosomal abnormalities would seem unlikely since some of these originate at meiosis.
In conclusion, results of this study have shown that chromosomal abnormalities are associated with developmental
delay in bovine embryos. It is possible that this delay originates at an early stage in embryonic development. Chromosomal abnormalities in bovine embryos affect growth in
vitro by delaying cleavage, which leads to lower than expected cell numbers at specific times in development. By
Day 5 in vitro, haploid and polyploid embryos exhibit the
slowest rate of development and mixoploid and diploid embryos the fastest, while aneuploid embryos grow at a rate
intermediate to those of these two groups. Our results also
suggest that eliminating embryos that are retarded in growth
on Day 5 in vitro, before sex-related developmental differences become apparent, may diminish the frequency of
chromosomally abnormal embryos; this in turn may reduce
the loss due to embryonic mortality when embryos are
transferred to recipients.
ACKNOWLEDGMENTS
The authors wish to thank Dr. A.D. Barth of the Department of Herd Medicine and
Theriogenology from the University of Saskatchewan for providing the semen from bull P;
United Breeders, Inc., Guelph, Ontario, for providing the semen from bull N; Lawrence Afflu
for collection of bovine tissues; and Dena L.Shepherd and Mary John for technical assistance.
REFERENCES
1. Jacobs PA. The role of chromosome abnormalities in reproductive failure. Reprod Nutr
Dev Suppl 1990; 1:63-74.
2. King WA. Chromosome abnormalities and pregnancy failure in domestic animals. In:
CHROMOSOMAL ABNORMALITIES AND DEVELOPMENT
McFeely RA (ed.), Advances in Veterinary Science and Comparative Medicine. San Diego,
CA: Academic Press; 1990: 34:229-250.
3. Iwasaki S, Hamano S, Kuwayama M, Yamashita M, Ushijima H, Nagaoka S, Nakahara T.
Developmental changes in the incidence of chromosome anomalies of bovine embryos
fertilized in vitro. J Exp Zool 1992; 261:79-85.
4. Iwasaki S, Shioya Y, Masuda H, Hanada A, Nakahara T. Incidence of chromosomal anomalies in early bovine embryos derived from in vitro fertilization. Gamete Res 1989; 22:8391.
5. Iwasaki S, Nakahara T. Incidence of embryos with chromosomal anomalies in the inner
cell mass among bovine blastocysts fertilized in vitro. Theriogenology 1990; 34:683-690.
6. Avery B, Bak A, Schmidt M. Differential cleavage rates and sex determination in bovine
embryo. Theriogenology 1989; 32:139-147.
7. Avery B, Madison V,Greve T. Sex and development in bovine in-vitro fertilized embryos.
Theriogenology 1991; 35:953-963.
8. Xu KP, Yadav BR, Rorie RW, Plante L,Betteridge KJ, King WA. Development and viability
of bovine embryos derived from oocytes matured and fertilized in vitro and co-cultured
with bovine oviductal epithelial cells. J Reprod Fertil 1992; 94:33-43.
9. Barnes F, Eyestone WH. Early cleavage and maternal zygotic transition in bovine embryos. Theriogenology 1990; 33:141-152.
10. Rieger D. Relationships between energy metabolism and development of early mammalian embryos. Theriogenology 1992; 37:75-93.
11. Plante L, King WA. Light and electron microscopic analysis of bovine embryos derived
by in vitro and in vivo fertilization. J Assist Reprod Genet 1994; 11:515-529.
12. Greve T, Xu KP, Callesen H, Hyttel P. In-vivo development of in-vitro fertilized bovine
oocytes matured in vivo and in-vitro.J In Vitro Fertil Embryo Transfer 1987; 4:281-285.
13. Parrish JJ, Susko-Parrish JL, Leibfried-Rutledge ML, Crister ES, Eyestone WH, First NL.
Bovine in vitro fertilization with frozen thawed semen. Theriogenology 1986; 25:591600.
14. King WA, Linares T, Gustavsson I, Bane A. A method for preparation of chromosomes
from bovine zygotes and blastocysts. Vet Sci Commun 1979; 3:51-56.
15. SAS. Statistical Analysis System: A User's Guide. Version 6, 4th edition, Cary, NC: Statistical Analysis System Institute, Inc.; 1989.
16. Plante L, King WA. Effect of time to first cleavage on hatching rate of bovine embryos
in vitro. Theriogenology 1992; 37:247.
17. Yadav BR, King WA, Betteridge KJ. Relationships between the completion of first cleavage and the chromosomal complement, sex, and development rates of bovine embryos
generated in vitro. Mol Reprod Dev 1993; 36:434-439.
3
18. Plante L, Plante C, Shepherd DL, King WA. Cleavage and H-uridine incorporation in
bovine embryos of high in vitro developmental potential. Mol Reprod Dev 1994; 39:375383.
19. King WA. Embryo-mediated pregnancy failure in cattle. Can VetJ 1991; 32:99-103.
20. King WA, Verini Supplizi A, Diop HEP, Bousquet D. Chromosomal analysis of embryos
produced by artificially inseminated superovulated cattle. Genet Sel Evol 1995; 27:189194.
21. Tsunoda Y,Tokunaga T, Sugie T.Altered sex ratio of live young after transfer of fast and
slow developing mouse embryos. Gamete Res 1985; 12:301-304.
22. Burgoyne PS. A Y-chromosomal effect on blastocyst cell number in mice. Development
1993; 117:341-345.
23. Zwingman T, Erickson RP, Boyer T, Ao A. Transcription of the sex-determining genes
Sryand Zfyin the mouse preimplantation embryo. Proc Natl Acad Sci USA 1993; 90:814817.
24. Cassar G, King WA, King GJ. Influence of sex on early growth of pig conceptuses. J
Reprod Fertil 1994; 101:317-320.
25. Pergament E, Fiddler M, Cho N, Johnson D, Holmgren WJ. Sexual differentiation and
preimplantation cell growth. Hum Reprod 1994; 9:1730-1732.
26. McGrath J, Solter D. Nuclearcytoplasmic interactions in the mouse embryo. J Embryol
Exp Morph Suppl 1986; 97:277-289.
27. Plante L.The preanachment bovine embryo: genome activity and ultrastructure. Guelph,
Canada: University of Guelph; 1993. Ph.D. Thesis.
28. Alberts B, Bray D, Lewis J, Raff M, Roberts K,Watson JD. Molecular Biology of the Cell.
2nd ed. New York: Garland Publishing, Inc; 1989: 451-458.
29. Kaufman MH, Sachs L. Complete preimplantation development in culture of parthenogenetic mouse embryos. J Embryol Exp Morphol 1976; 35:170-190.
30. Henery CC, Kaufman MH. Cleavage rate of haploid and diploid parthenogenetic mouse
embryos during the preimplantation period. Mol Reprod Dev 1992; 31:258-263.
31. King WA, Guay P, Picard L. A cytogenetical study of 7-day-old bovine embryos of poor
morphological quality. Genome 1987; 29:160-164.
59
32. Beatty RA, Fischberg M. Cell number in haploid, diploid and polyploid mouse embryos.
J Exp Biol 1951; 28:541-552.
33. Baranov VS. Spontaneous polyploidy in laboratory mice. Embryological and cytogenetic
analysis. Ontogenez 1976; 7:229-238.
34. Takagi N, Sasaki M.Digynic triploidy after superovulation in mice. Nature 1976; 264:278281.
35. Edwards RG. The number of cells and cleavages in haploid, diploid, polyploid, and other
heteroploid mouse embryos at 3/2 days gestation. J Exp Zool 1958; 138:189-207.
36. Henery CC, Kaufman MH. Cleavage rate of diandric triploid mouse embryos during the
preimplantation period. Mol Reprod Dev 1992; 32:251-258.
37. Henery CC, Kaufman MH. The cleavage rate of digynic triploid mouse embryos during
the preimplantation period. Mol Reprod Dev 1993; 34:272-279.
38. King WA, Linares T, Gustavsson I. Cytogenetics of preimplantation embryos sired by
bulls heterozygous for the 1/29 translocation. Hereditas 1981; 94:219-224.
39. Hare WCD, Singh EL,Betteridge KJ, Eaglesome MD, Randall GCB, Mitchell D, Bilton RJ,
Trounson AO. Chromosomal analysis of 159 bovine embryos collected 12 to 18 days
after estrus. CanJ Genet Cytol 1980; 22:615-626.
40. Long SE, Williams CV. A comparison of the chromosome complement of inner cell mass
and trophoblast cells in Day-10 pig embryos. J Reprod Fertil 1982; 66:645-648.
41. Murray JD, Moran C, Boland MP, Nancarrow CD, Sutton R, Hoskinson RM, Scaramuzzi
RJ. Polyploid cells in blastocysts and early fetuses from Australian Merino sheep. J Reprod Fertil 1986; 78:439-446.
42. Kopecny V. High-resolution autoradiographic studies of comparative nucleologenesis
and genome reactivation during early embryogenesis in pig, man and cattle. Reprod
Nutr Dev 1989; 29:589-600.
43. Telford NA, Watson A, Schultz GA. Transition from maternal to embryonic control in
early mammalian development: a comparison of several species. Mol Reprod Dev 1990;
26:90-100.
44. Schultz GA, Heyner S. Gene expression in pre-implantation mammalian embryos. Mutat
Res 1992; 296:17-31.
45. Camous S, Heyman Y, Meziou W, Menezo Y. Cleavage beyond the block stage and
survival after transfer of early bovine embryos cultured with trophoblastic vesicles. J
Reprod Fertil 1984; 72;479-485.
46. Frei RE, Schultz GA, Church RB. Qualitative and quantitative changes in protein synthesis
occur at the 8-16-cell stage of embryogenesis in the cow. J Reprod Fertil 1989; 86:637641.
47. Leibfried-Rutledge ML. Critser ES, ParrishJJ, First NL. In vitro maturation and fertilization
of bovine oocytes. Theriogenology 1989; 31:61-74.
48. Hillery FL, Parrish JJ, First NL. Bull specific effect on fertilization and embryo development in vitro. Theriogenology 1990; 33:249.
49. Shi DS, Lu KH, Gordon I. Effects of bulls on fertilization of bovine oocytes and their
subsequent development in vitro. Theriogenology 1990; 33:324.
50. Shamsuddin M, Larsson B. In vitro development of bovine embryos after fertilization
using semen from different donors. Reprod Domest Anim 1993; 28:77-84.
51. Lonergan P, Kommisrud E, Andresen O, Refsdal AO, Farstad W. Use of semen from a
bull heterozygous for the 1/29 translocation in an IVF program. Theriogenology 1994;
41:1379-1384.
52. Gustavsson I. Distribution and effects of the 1/29 Robertsonian translocation in cattle. J
Dairy Sci 1979; 62:825-835.
53. King WA, Linares T, Gustavsson I, Bane A. Presumptive translocation type trisomy in
embryos sired by bulls heterozygous for the 1/29 translocation. Hereditas 1980; 92:167169.
54. Popescu CP Cytogenetics study on embryos sired by a bull carrier of 1/29 translocation.
In: Proceedings, 4th Eur Colloq Cytogenet Domest Anim 1980; Uppsala, Sweden. 182186.
55. Dyrendahl I, Gustavsson I. Sexual functions, semen characteristics and fertility of bulls
carrying the 1/29 chromosome translocation. Hereditas 1979; 90:281-289.
56. Schmutz SM, MokerJS, Barth AD, Mapletoft RJ. Embryonic loss in superovulated cattle
caused by the 1/29 Robertsonian translocation. Theriogenology 1991; 35:705-714.
57. Tesafk J. Developmental control of human preimplantation embryos: a comparative
approach. J Vitro Fertil Embryo Transfer 1988; 5:347-362.
58. Tesaik J, Kopecny V, Plachot M, MandelbaumJ. Ultrastructural and autoradiographic observations on multinucleated blastomeres of human cleaving embryos obtained by in-vitro
fertilization. Hum Reprod 1987; 2:127-136.
59. Yadav BR, King WA, Xu KP, PollardJW, Plante L.Chromosome analysis of bovine oocytes
cultured in vitro. Genet Sel Evol 1991; 23:191-196.
60. Logue DN, Harvey MJA. Meiosis and spermatogenesis in bulls heterozygous for a presumptive 1/29 Robertsonian translocation. J Reprod Fertil 1978; 54:159-165.