J Vet Diagn Invest 8:91-95 (1996)
Chromosomal aneuploidy associated with spontaneous
abortions and neonatal losses in cattle
S. M. Schmutz, J. S. Moker, E. G. Clark, J. P. Orr
Abstract. Pericardial sac samples from 77 bovine aborted fetuses and stillborn calves were submitted for
tissue culture; cells from 55 of these samples were grown successfully in culture. Six of the 55 karyotyped fetuses
(10.7%) had an abnormal chromosome complement, in 3 of which (5.5%) the abnormality was probably the
cause of death. This level of abnormality is relatively high when one considers that most fetuses were > 8 months
gestational age. Approximately 5-7% of human stillbirths and 50% of first-trimester aborted fetuses have
chromosome anomalies. If a similar situation exists in cattle, as suggested by these data, chromosome abnormalities may be a major cause of early fetal loss in cattle. Most chromosomally abnormal fetuses had multiple
malformations, which suggests that the diagnostic use of chromosome analysis is most cost effective for malformed fetuses and newborns. Twins were present in a higher proportion of these fetuses than expected based
on their incidence among liveborn cattle.
Fetal loss, especially late gestation loss, remains a
major economic concern for the cattle rancher and a
frequent enigma to the pathologist. The cause of many
cases of abortion remains undiagnosed even after a
complete necropsy and histopathologic and microbiologic examination. In 1 study, 15 3-7% (95/2,544) of
aborted fetuses had some phenotypic anomaly. In another study,8 9.2% of 931 necropsied specimens had 1
or more gross anomalies, whereas in yet another20 only
1.7% (10/595) of the necropsied aborted fetuses had
anomalies. The cause of these gross abnormalities was
undetermined in most cases, but some were probably
due to chromosome abnormalities such as aneuploidy,
as was postulated some time ago22 but has rarely been
investigated.
A few studies have been done on cattle embryos and
early fetuses. In 225 oocytes from slaughtered heifers,
23% had chromosomal anomalies.16 Out of 12 embryos 12-16 days old, there was 1 case of tetraploidy.21
When 224 fetuses between 50 and 180 days old were
karyotyped, no cases of aneuploidy were found.12
Fewer chromosomal studies have been conducted
on aborted and stillborn cattle. In 5 bovine early neonates that died, no anomalies, either phenotypic or
chromosomal, were found.3 In a chromosome study,
of 18 aborted fetuses and stillborn cattle, 4 showed
aneuploidy.8 That study was expanded, and the results
are reported in this paper.
Materials and methods
Sampling. An announcement about this study and a call
for submissions were placed in a newsletter sent to all veterinarians and pathologists in Saskatchewan in fall 1990 and
again in 1991 at the onset on the abortion season. In January
1991, a letter was also sent to the 83 producers with bulls in
the Saskatoon Record of Performance Test Station asking
for submissions. This letter was sent to encourage submission
of malformed fetuses; farmers and ranchers often presume
the reason for the abortions in these cases is evident and
therefore they do not submit these fetuses.
Tissue culture. A small piece of pericardium was collected
aseptically from each “fresh” fetus or stillbirth submitted to
the Western College of Veterinary Medicine for necropsy.
(“Fresh” in this context means not frozen and dead <24 hr,
which is necessary to predict reasonable success for culturing
dividing cells necessary for chromosome analysis.) Each sample was set up in replicate tissue cultures.a
Karyotyping. All tissue cultures that grew were harvested
8
for chromosome analysis. Slides were made, and microscopic analysis conducted. Chromosomes were Giemsa
stained for the detection of numerical abnormalities. Photomicrographs were taken. GTG banding (G banding with
trypsinization and Giemsa staining) of chromosomes28 was
done, when possible, to study abnormal cells in more detail.
Cells from animals with chromosome abnormalities were
frozen for future study.
Results
Chromosome analysis or karyotyping was attempted
on 77 bovine fetuses and stillborn calves during this
study: 46 in 1991 and 31 in 1992. Several breeds of
both beef and dairy cattle were represented among the
67 fetuses for which breed was reported (Table 1). Age
was reported for 61 of the fetuses and ranged from 6
months gestation to 7 days postpartum, with the vast
From the Departments of Animal and Poultry Science (Schmutz,
Moker) and Veterinary Pathology (Clark, Orr), Western College of
Veterinary Medicine, University of Saskatchewan, Saskatoon, SK
S7N 0W0, Canada.
Received for publication April 23, 1994.
91
92
Schmutz et al.
number does not include 2 other animals (nos. 5, 6;
majority being near term (Fig. 1). Thirty-three of the
77 animals submitted had phenotypic abnormalities
at necropsy, and 28 of these were karyotyped.
Karyotyping was completed on 55 (7 1%) of the 77
samples. Six of the 55 karyotyped animals (10.7%) had
an abnormal chromosome complement (Table 2).
Twenty-two of the specimens were not karyotyped because the cells from them did not grow. Cells can fail
to grow for a variety of reasons: autolysis of tissues,
contamination, and too long a period from death to
receipt. Culture failure is typically near this level in
such studies in humans.1 Contamination was minimal
because all samples were carefully collected. However,
autolysis and late receipt were problems. When abnormal chromosomes were found, the age of the dam
was supplied by the owner/rancher. Of the 55 successfully karyotyped animals, 19 were males, 34 were
females, and 2 were chimeras.
Twelve fetuses were the result of reported twin pregnancies, representing 8 different sets of twins. This
Figure 1. Histogram depicting the ages of the aborted fetuses and
stillborn calves submitted for tissue culture, those karyotyped, and
those with malformations.
Table 2) in which the chromosome results suggested
twinning but that were not reported by the owners as
twins.
Table 3 summarizes the number of fetuses and stillborn calves with congenital anomalies out of the total
submitted to the Western College of Veterinary Medicine over the course of this and the previous study.
The first chromosomally abnormal animal was a
newborn Hereford with a cleft palate, hydrocephalus,
a cardiac interventricular septal defect, and arthrogryposis. Trisomy of chromosome 21 (i.e., an extra chromosome 21) was found in all cells (Fig. 2). The dam
was only 4 years old, and the producer agreed to keep
her in the breeding herd because the risk of recurrence
is thought to be very low based on human risks of
nondisjunction.13
Animal no. 2 was a newborn heifer calf of a Simmental bull-heifer twin pair. She had no structural
anomalies, which is rather surprising because she carried an extra unidentified chromosome in a significant
proportion of her cells (=a mosaic trisomy). Apparently, her normal cells dictated her phenotype and may
have been the predominant type in other tissues. Her
bull twin had a normal male karyotype.
Animal no. 3 was a newborn crossbred Hereford bull
calf. This trisomic calf had hydrocephalus, scoliosis,
arthrogryposis, mild hydronephrosis, and anal atresia.
An extra chromosome that was not identified was present in all cells.
Animal no. 4 was reported as a female Angus newborn calf, although she appeared to have been a Charolais cross. She had multiple malformations, including
hypoplasia of palpebral fissures, cleft palate, kyphoscoliosis, and arthrogryposis. She had trisomy 22 in all of
her cells (Fig. 3).
Animals nos. 5 and 6 were both chimeras, a mixture
of cells from 2 different origins as indicated by the
presence of X and Y chromosomes in 1 cell type and
2 X chromosomes in the other. This is the typical
karyotype of a freemartin and her bull twin, 11,25 which
is not usually a cause of fetal death. Neither chimeric
animal was reported as having been born as a twin.
Bovine fetal cytogenetics
Table 3. Number of fetuses/stillborn calves with congenital
anomalies relative to the total number necropsied at the Western
College of Veterinary Medicine over the time period of this and the
previous8 study.
Animal no. 5 was a newborn of a crossbred cow sired
by a Longhorn bull. The female cell line carried the
1;29 translocation (i.e., the calf was 60,XY/59,XX,t).
The dam of this fetus had a normal karyotype. The
blood lymphocytes of the sire did not grow, so no
karyotype was determined. Moldy feed was used in
this herd, suggestive perhaps of a mycotic abortion, as
was documented in a later case submitted from this
same herd. Fetal tissues were also positive for bovine
viral diarrhea virus by direct immunofluorescence.
Animal no. 6 was a Limousin newborn calf with male/
female chimerism and no other chromosome abnormalities. The cause of abortion remained undiagnosed.
Discussion
This study would not have detected structural errors
that typically lead to malformation and/or abortion,
such as deletions and duplications, because chromosomes were only banded in cases where numerical errors were found. Therefore, only numerical errors and
1 Robertsonian translocation were found.
No breed predisposition to numerical chromosome
errors was seen nor was it expected; nondisjunction,
which is the failure of homologous chromosomes paired
during meiosis to separate to form part of the haploid
complement of gametes, is believed to be the primary
cause of such errors.4 Only fetuses late in gestation or
newborn stillbirths were submitted (Fig. 1); thus, the
ages of these fetuses could be a bias in producer detection and submission, or alternatively abortion in
cattle, rather than resorbtion, may only occur in later
gestation (Kirkbride, personal communication).
Three of these 6 chromosomally abnormal calves
(nos. 1, 3, 4), probably aborted as a result of the extra
chromosome or trisomy detected (5.5% of the total)
(Table 2). A few cases of autosomal trisomy have been
reported in livebom but malformed calves,10,14,18,14,30
and most of these appear to be trisomy 17. No other
trisomy has been reported as viable in cattle. Likewise
most trisomic human fetuses abort, although trisomy
13, 18, and 21 are viable.5,6,7 It is more difficult to
predict viability of mosaic trisomy, such as in animal
no. 2.
Figure 2. Karyotype of animal no. 1, a Hereford newborn with
trisomy 21.
The sex ratio of the karyotyped calves was significantly skewed towards females. This skewness may be
an artifact of submissions or more an effect of small
sample size. In humans, there are subtle sex ratio discrepancies in some malformations, such as neural tube
defects.’
The dams of these abnormal calves were all relatively young animals, several calving for the first time.
In humans4 and in a previous study of bovine fetuses,8
there is a tendency for extra chromosomes to occur
more frequently in older dams. The present data neither support nor refute this observation because the
risk of chromosome error should be considered in pro-
Figure 3. Karyotype of animal no. 4, an Angus cross newborn
with trisomy 22.
94
Schmutz et al.
portion to all individuals of a particular age group that
gave birth, and this assessment was not possible here.
This number of lethal cytogenetic anomalies, 3 of
55 (5.5%), is lower than the 4 of 18 (22%) previously
found8 but may indicate a more significant problem
than expected because this study included fetuses without structural abnormalities and the previous study
included only anomalous fetuses. If we include only
the phenotypically abnormal fetuses from this study,
the rate increases to 3/28 (10.7%). Both fetal studies
included chimeric cases.
Although twinning is typically considered an increased risk factor during gestation in cattle2 and humans, 17 there may be an additional message in these
data. One worker (J. Hall) has postulated that twinning
may sometimes be the result of the normal cells in the
embryo rejecting the “mutant” cell mass and that if
this rejection happens before day 14 a second fetus
could form from these “mutant” cells.23 A review of
33 amorphic masses that were twins to normal bovine
calves suggests that these are a possible outcome of
monozygous twinning. 32 An alternative hypothesis for
twinning when 1 twin has a cytogenetic anomaly and
the other does not has also been suggested.26 Three of
the 6 chromosomally anomalous calves in this study
were twins (Table 2). Incidence of twinning was 14/77
(18%) in the entire sample studied and has been estimated at 0.5-4% in liveborn calves.2 This high proportion of twins with cytogenetic anomalies can therefore not be explained simply by increased abortion
among twins but lends some support to Hall’s hypothesis. Human twins have an 18-fold increased risk
of death and a 50% greater risk of malformation than
do singletons. 17 Because this increased risk of malformation was found among same-sex twins, monozygosity may be a contributing factor.17 All 3 of the twins
with chromosomal anomalies in this study were opposite-sex twins, but chromosomal chimerism from
placental anastomosis is predominantly a bovine
problem11 and is rarely reported in other species. Because most such chimeric twins in cattle share DNA
fingerprints, 25 they may genetically act more like
monozygotic twins than do chimeras of other species.
Lymphocyte chimerism and chimerism in fibroblasts
grown from pericardium and skin were both found,
which suggests the exchange of cells is not limited to
blood between twins.
Although the phenotypes associated with specific
aneuploidies in humans are well documented,27 it is
premature to attempt such correlations from these few
bovine cases. In this and a previous study,8 we were
unable to identify the extra chromosome in all cases.
The only trisomy identified twice was trisomy 21, once
in the current study and once in the previous study.8
Both cases had 2 malformations in common, cleft palate and cardiac interventricular septal defect, but each
also had other malformations not shared. A 2-day-old
calf, which presumably died of heart malformations,
with trisomy (putatively trisomy 21) has been reported 18 However cleft palate and ventral septal defect
should not be assumed to mean that trisomy 21 is
present; 2 of the 4 chromosomally abnormal calves in
this study and 3 of the 5 in the previous study8 had
cleft palate. This is also a frequent anomaly in chromosomally abnormal mice.31 Likewise, 1 of the 4 cases
in this study and 2 of the 5 cases previously reported8
had a ventricular septal defect. Trisomy 17 has previously been reported a few times10,14,24 (although not
in this study) and may be an example of a documentable phenotype-chromosome association. Extreme
brachygnathia appears to be the salient feature. A
3-week-old trisomic calf with a ventricular septal defect, patent foramen ovale, and umbilical hernia has
been described.30
Several generalizations can be made based upon these
findings. Fetuses and newborns with extra chromosomes usually have multiple malformations, as is the
case in humans.1 These malformations are most likely
a result of the chromosome problem, as is the ultimate
death of the animal. This is also true in other mammals, such as humans and mice.4 All of the chromosomally abnormal animals in this study were newborns, not fetuses. This is most likely a bias of the
samples submitted rather than what is actually happening in nature. In fact, the level of abnormality found
in this study may be the “tip of the iceberg” if we can
assume cattle would show a trend similar to that in
humans, where 50% of abortions at 8-16 weeks gestation,6 6-12% of abortions after 20 weeks,9 and 5-7%
of stillbirths1,19,29 are due to cytogenetic causes. This
speculation is consistent with the finding that only
“small” chromosome trisomies were found in this and
a previous study,8 and no cases of total monosomy
were found. All monosomies and trisomies of “large”
chromosomes are probably lethal early in gestation in
cattle, as they are in humans,6,7 presumably because of
the large number of genes involved. If we can further
extrapolate between humans and cattle, the risk of recurrence of aneuploid fetuses is very low,13 and therefore ranchers should be advised that they need not cull
dams or sires of chromosomally anomalous offspring.
Acknowledgements
We thank Kathy Caspell and Lois Ridgeway for their help
in collecting samples for tissue culture from the appropriate
fetuses, and we thank the other pathologists at WCVM who
contributed specimens. Funding for this work came from the
Saskatchewan Agricultural Development Fund.
Bovine fetal cytogenetics
Sources and manufacturers
a. GIBCO/BRL, Mississaugua, ON, Canada.
References
1. Angell RR, Sanidson A, Bain AD: 1984, Chromosome variation in perinatal mortality: a survey of 500 cases. J Med Genet
21:39-44.
2. Bearden HJ, Fuquay J: 1992, Applied animal reproduction.
Prentice-Hall, Englewood Cliffs, NJ.
3. Berepubo NA, Long SE: 1983, A study of the relationship
between chromosome anomalies and reproductive wastage in
domestic animals. Theriogenology 20:176-191.
4. Bond DJ, Chandley AC: 1983, Aneuploidy. Oxford University
Press, Toronto, ON, Canada.
5. Boué A, Gropp A, Boué J: 1985, Cytogenetics of pregnancy
wastage. Adv Hum Genet 14:1-50.
6. Byrne J, Warburton D, Kline J, et al.: 1985, Morphology of
early fetal deaths and their chromosomal characteristics. Teratology 32:297-315.
7. Chandley AC: 1981, The origin of chromosomal aberations in
man and their potential for survival and reproduction in the
adult human population. Ann Genet 24:5-11.
8. Coates JW, Schmutz SM, Rousseaux CG: 1988, A survey of
malformed aborted bovine fetuses, stillbirths and nonviable neonates for abnormal karyotypes. Can J Vet Res 52:258-263.
9. Creasy MR, Alberman ER: 1976, A cytogenetic study of human
spontaneous abortions using banding techniques. Hum Genet
31:977-196.
10. Dunn HO, Johnson RH: 1972, A 61 ,XY cell line in a calf with
extreme brachygnathia. J Dairy Sci 55:524-526.
11. Dunn HO, McEntee K, Hall CE, et al.: 1979, Cytogenetic and
reproductive studies of bulls born co-twin with freemartins. J
Reprod Fertil 57:21-30.
12. Fechheimer NS, Harper RL: 1980, Karyological examination
of bovine fetuses collected at an abattoir. Eur Colloq Cytogenet
Domest Anim 4:194-199.
13. Hassold TJ: 1986, Chromosome abnormalities in human reproductive wastage. Trends Genet 2:105-110.
14. Herzog von A, Hoehn H: 1968, Autosomale Trisomie bei einem Kalb mit Brachygnathis inferior und Ascites congenitus.
Dtsch Tierärztl Wochenschr 75:604-605.
95
15. Kirkbride CA, Bicknell EJ, Reed DE, et al.: 1973, A diagnostic
survey of bovine abortion and stillbirth in the northern plains
states. J Am Vet Med Assoc 162:556-560.
16. Koenig LF, Eldridge FE, Harris N: 1983, A cytogenetic analysis
of bovine oocytes in vitro. J Dairy Sci 66(suppl. 1):253.
17. Layde PM, Erickson JD, Falek A, McCarthy BJ: 1980, Congenital malformations in twins. Am J Hum Genet 32:69-78.
18. Long SE: 1984, Autosomal trisomy in a calf. Vet Rec 115: 1617.
19. Machin GA, Crolla JA: 1974, Chromosome constitution of 500
infants dying during the perinatal period. Humangenetik 23:
183-198.
20. Maxie G: 1986, Etiologic agent or condition associated with
abortion in cattle, VLS, Guelph, 1983-1985. Can Vet J 27:A6.
21. McFeeley RA, Rajakoski E: 1968, Chromosome studies in early
embryos of the cow. Proc Int Congr Anim Reprod AI 2:905907.
22. Miller RB: 1977, A summary of some of the pathogenetic mechanisms involved in bovine abortion. Can Vet J 18:87-95.
23. Morel1 V: 1993, Double take. Equinox, Nexus, June, p. 30.
24. Mori M, Sasaki M, Makino S, et al.: 1969, Autosomal trisomy
in a malformed new born calf. Proc Jpn Acad 45:955-959.
25. Plante Y, Schmutz SM, Lang KDM, Moker JS: 1992, Detection
of leucochimaerism in bovine twins by DNA fingerprinting.
Anim Genet 23:295-302.
26. Rogers JG, Voullaire L, Gold H: 1982, Monozygotic twins
discordant for trisomy 21. Am J Med Genet 11:143-146.
27. Schinzel A: 1984, Catalogue of unbalanced chromosome aberrations in man. Walter de Gruyter, Berlin, Germany.
28. Seabright M: 1971, A rapid banding technique for human chromosomes. Lancet 2:971-972.
29. Sutherland GR, Cartner RF, Bauld R, et al.: 1978, Chromosome studies at pediatric necropsy. Ann Hum Genet 42:173181.
30. Tschudi von P, Ueltschi G, Martig J, Ktipfer U: 1975, Autosomale Trisomie als Ursache eines hohen Ventrikkelseptumdefekts bei einem Kalb Simmentalrasse. Schweiz Arch Tierheilkd 117:335-340.
31. Vekemans M, Trasler T: 1987, Liability to cleft palate in trisomy 19 mouse embryos. J Craniofacial Genet Dev Biol Suppl
2:235-240.
32. Young GB: 1952. Acardius amorphus monsters in cattle. Vet
Rec 64:206-208.