THE JOURNAL OF EXPERIMENTAL ZOOLOGY 261:79-85 (1992)

Developmental Changes in the Incidence of Chromosome Anomalies of Bovine Embryos Fertilized In Vitro S. IWASAKI, S. HAMANO, M. KUWAYAMA, M. YAMASHITA, H. USHIJIMA, S. NAGAOKA, AND T. NAKAHARA NODAI Research Institute, Tokyo University of Agriculture, Setagaya-ku, Tokyo 156 (S.I., T.N.), Livestock Improvement Association of Japan, Inc., Shinagawa-ku, Tokyo 140 (S.H., M.K., M.Y., S.N.), and Chiba Prefectural Livestock Research Center, Inba-gun, Chiba 289-11 (H.U.), Japan In total, 196 two- to 32-cell bovine embryo and 104 blastocysts were obtained by the ABSTRACT in vitro fertilization of follicular oocytes matured in vitro, and 15 blastocysts fertilized in vivo were used. Chromosomal anomalies in these embryos and the inner cell mass (ICM) separated immunologically were investigated. Chromosomal anomalies were observed in 12.1%(5/41) of 2-cell embryos, 20.0-36.4% of 4- to 16-cell embryos, 7.1% (1/14) of 17- to 32-cell embryos, 44.2% (15/34) of blastocysts, and 18.6% (13/70) of ICM cells derived from in vitro fertilization. These anomalies were mainly 3N and 4N at 2-cell stage, 1N and 1N/2N at 4- to 32-cell stages, and 2N/4N in blastocysts and in their ICM cells. Chromosomal anomalies of blastocysts from in vivo fertilization and their ICM were observed in 20.0% (1/5)of blastocysts and 33.3% (319) of ICM cells and these compositions were mainly 2N/4N. These results indicate that the abnormalities a t early and blastocyst stages of embryos derived from in vitro fertilization were caused by abnormal fertilization in vitro and abnormal cleavage, respectively. Furthermore, a definite location of the chromosomal anomalies was observed in the trophectoderm of blastocysts derived from in vitro fertilization.

There have been many reports on the incidence of chromosomal anomalies in bovine embryos fertilized in vivo (Hare et al., '80; King et al., '79, '81;Popescu et al., '80; Abreu, '84) and in vitro (King et al., '85a; Iwasaki et al., '89a; Iwasaki and Nakahara, '90). Most of them dealt with preimplantation embryos, particularly the in vivo reports. Murray et al. ('86a) investigated the frequency of chromosomal anomalies in sheep embryos fertilized in vivo at various stages from l-cell to blastocyst, but the developmental changes of the anomalies were not discussed because of small sample size. In other reports, the incidence of chromosomal anomalies of early embryos were examined at a limited number of stages in cattle (King et al., '79, '81, '85a; Iwasaki et al., '89a) and in sheep (Long and Williams, '80). But little is known about the incidence and composition of chromosomal anomalies as they relate to developmental changes in bovine embryos. Recently, an in vitro culture system has been established for bovine embryos derived from in vitro matured follicular oocytes fertilized in vitro; such embryos can be cultured up to blastocyst stage (Xu et al., '87; Fukuda et al., '88; Goto et al., '88). Before 0 1992 WILEY-LISS, INC.

this, it had been difficult to study the developmental changes in morphology (e.g., ultrastructural analysis; Hyttel et al., '881, metabolic activities (e.g., measurement of the energy metabolism; Rieger and Guay, '881, and other biochemical traits (e.g.,glucose6-phosphate dehydrogenase activity; Iwasaki et al., '89b) using the early bovine embryos. The in vitro culture system made these studies using many early embryos at each stage possible. In the present study, the changes in chromosomal anomalies in bovine embryos derived from in-vitro fertilization from 2-cell to blastocyst stages and the location of their anomalies in the ICM or trophectoderm were investigated and compared with those from in vivo fertilization.

MATERIALS AND METHODS Preparation of bovine embryos In vitro maturation of follicular oocytes from ovaries of Japanese Black cattle and in vitro culture

Received May 24,1991; revision accepted June 6,1991.

80

S.IWASAKIET AL.

of embryos after in vitro fertilization were carried out by the method of Hanada ('85) with minor modification. Sperm were capacitated with caffeine and heparin using the method of Niwa et al. ('891, based on the method of Parrish et al. ('86), with minor modification. That is, sperm of a Japanese Black cattle bull was washed twice with 10 mM caffeinesodium benzoate (Sigma) and sperm concentration was adjusted t o 2 x 107/ml.Of this, 50 ~1 was taken and added to 50 p1 media containing 20 Fg/ml of sodium heparin (Novo Industry, Denmark) with 5 mg/ml of bovine serum albumin (BSA: Fraction V, Wako Jyunyaku Co., Japan). Final concentrations of sperm and heparin were at 1 x 107/mland at 10 pg/ml with 2.5 mg/ml ofBSA.Then, in vitro matured oocytes were immediately incubated with the treated sperm. After 5 h of sperm-ova incubation, oocytes were transferred to a development medium (25 mM HEPES buffered TCM 199 supplemented with 5% fetal bovine serum and antibiotics) and cocultured with a monolayer of cumulus cells until chromosome preparation. By the observation of oocytes at pronuclei stages, it was found that more than 95% of sperm could penetrate into the oocytes normally under this condition. Embryos of varying stages were obtained at the following times after the initiation of sperm-ovaincubation; two-cell: 24-28 h, four-cell: 40-44 h, eight-cell:48-53 h, 16-cell:72-76 h, 32-cell: 120-124 h, and blastocysts: 144-192 h. To examine the incidence of chromosomal anomalies in ICM cells of blastocysts derived from in vivo maturation, fertilization, and development,embryos fertilized in vivo were collected at day 7 after oestrus by nonsurgical flushing with Dulbecco's PBS including 0.3% BSA. The embryos at morulae stage were further cultured in vitro to blastocyst stage for 1day.

acid, methanol, and hypotonic solution (5:15:8) for 3-5 min. The second fixation was carried out by placing embryos in a mixture of acetic acid-methanol(1:3) for 30-40 min. The embryos were transferred on a glass slide and a small drop of the third fixative (acetic acid-methanol-distilled water, 3:3:1) was poured on the embryo, followed by drying by gentle blowing at the time of the initiation of breakage of the zona pellucida. Chromosome preparations were stained in 4% Giemsa (Merk) at pH 6.8 for 3.5 min and chromosome numbers were counted under a microscope with a magnification of x 1,000.

Separation of ICM cells and chromosome preparation The ICM cells were separated immunologically from trophectoderm cells by the method used in the previous work (Iwasaki et al., '90). Embryos were cultured in TCM 199 medium containing 0.4 pg/rnl of vinblastin sulfate (Sigma)for 6 h prior to the separation of ICM cells and were treated for 3-5 min with 0.5% pronase in phosphate-buffered saline (PBS)to remove the zona pellucida. They were then incubated with 10%anti-bovine spleen antiserum for 30 min at 385°C in 5% C02-95% air. Subsequently, the embryos were washed 5 times in PBS containing 0.3%BSA and then incubated with 10% guinea pig serum containing propidium iodide (PI, Sigma) at a final concentration of 10 pg/ml of complement solution for 30 min at 38.5"C. Finally, the embryos were washed in PBS with 0.3% BSA and transferred on a glass slide. The ICM cells were separated from trophectoderm cells using a glass pipette with thin tip elongated by a flame. The separated ICM cells were then observed under a Nikon fluorescent microscope with an excitation filter (365 nm) in combination with a barrier filter (420 nm) and confirmed no evidence of trophectoderm cells Chromosomepreparation and analysis fluorescing pink. Overall, ICM cells were successo f whole embryos fully separated at the rate of 82.4% (70/85). ICM Embryos were processed and chromosomes were cells were placed in 0.9% sodium citrate hypotonic prepared by the method described in the previous solution for 8 min at room temperature. Fixation study (Iwasaki et al., '89a, Iwasaki and Nakahara, and staining of chromosome preparation were car'90) with minor modification.That is, embryos were ried out as mentioned above. further cultured in TCM 199 medium containing Statistics 0.4 Fg/ml ofvinblastin (Sigma)for 12h (two-to16-cell embryos) or 6 h (morulae and blastocysts). SubseComparisons of results obtained in the present quently, embryos, except for blastocysts, were treated study were made by Student's t-test except for the for 40 sec with 0.5% Actinase E (Kaken Kagaku, comparison of the frequency of chromosomal anomJapan) to weaken the zona pellucida. They were then alies at various developmental stages, for which the placed in a hypotonic solution (0.9%sodium citrate) Chi-square test was used. for 20 rnin (two-to 16-cellembryos), 10 min (32-cell RESULTS embryos),or 5 min (blastocyst)at room temperature. Mitotic indexes at the 2- to 32-cell stages are sumThe first fixation was made by transferring the embryos to a fixative solution consisting of acetic marized in Table 1. The rates (93.0 and 95.2%) of

CHROMOSOMAL ANOMALIES IN BOVINE EMBRYOS

could be analyzed, and 94.7% (18/19) and 97.0% (64/66) of ICM cells could be analyzed at early and expanded blastocyst stages in embryos fertilized in vitro, respectively. Mitotic indexes were 10.3 and 10.9%in whole embryos and 12.0 and 10.8%in ICM cells of blastocysts at early and expanded stages, respectively. Mitotic index was 8.2% and 12.3% in whole and ICM cells of blastocysts fertilized in vivo, respectively. Chromosome composition of whole embryos and ICM cells in blastocysts are shown in Table 4 and Figure 1. In the embryos fertilized in vitro, chromosomal anomalies were observed in 44.4% (419) and 44.0% (11/25) of whole embryos at early and expanded blastocyst stages, respectively. In ICM cells, chromosomal anomalies were found in 1 (7.7%)and 12 (21.1%)embryos at early and expanded stages, respectively. The incidence of chromosomal anomalies in ICM cells was significantly lower than that in the whole embryos at blastocyst stage (18.6% vs 44.2%, P < 0.05). One (2.9%)was tetraploid and 14 (41.3%) were mosaics (2N/3N, 2N/4N, and 2N/ 3N/4N) in whole embryos and two (2.8%) were polyploid (3N and 4N), whereas 11 (15.7%) were mosaics (2N/3N and 2N/4N) in ICM cells. The chromosomes of mosaic embryos were mainly composed by 2N/4N. Chromosomal anomaly was observed in 20.0% (M)and 33.3% (3/9) of whole and ICM cells of blastocysts derived from in-vivo fertilization and their composition was mainly 2N/4N.

TABLE 1 . Mitotic index o f bovine embryos fertilized in vitro at each stage Stage of No. of No. Of metaphase embryo (-cell) Processed Analyzed (%I1 plates 2 3 4 5-7 8

9-16 17-32

57 4 21 17 45 36 16

81

Mitotic index

(%I2

53 (93.0)a.3 1.9 t 0.3 96.2 f 13.2"j3 100.0" 4(100.0)" 3.0 20 (95.2)" 3.9 2 0.2 98.7 t 5.6" 12 (70.6)a%b3.2 ? 1.8 53.0 & 28.7b 32 (71.1)a.b 3.0 f 1.9 37.5 f 23.4b3c 21 (58.3)b 3.2 k 2.2 25.5 t 17.1' 12 (81.3)a3b 5.5 t 1.4 25.5 t 7.4'

'No. of analyzed embryosino. of processed embryos. 'No. ofmetaphaseplatesitotal cell no. ofanalyzed embryo (mean t SD). 3Figures with different superscript letters were significantly different at the 5% level.

analyzed embryos to processed embryos at the 2-cell and 4-cell stages, respectively, were significantly higher t h a n those (25.5-53.0%) at the 5-32-cell stages. The mitotic index decreased gradually with progress of development from 98.7 to 25.5%. Chromosome composition of 2--32-cell embryos is shown in Table 2 and Figure 1. The incidence of chromosomal anomalies increased slightly with development from 12.1% at the 2-cell to 36.4% at the 9- to 16-cell stage, but there was no significant difference among the stages. At the 2-cell stage, three (7.3%) were 3N and 2 (4.9%)were4N. Haploid (7.1-18.2%) and haploid/diploid mosaic cells (7.1-15.0%) were most frequent, although 3N, 4N, 2N/3N, 2N/4N, and hypodiploid (2N-1) cells were also observed at lower frequencies (2.4-6.7%) at the 4--32-cell stages. This hypodiploid cell showed a 1/29 translocation (Fig. lc). Haploid cells bearing Y chromosomes were observed in four embryos, one each at 6-, 8-, 11-, and 13-cell stages. The mitotic indexes of whole embryos and ICM cells of early and expanded blastocysts derived from in vitro and in vivo fertilization are shown in Table 3. All of the whole blastocyst embryos processed

DISCUSSION A high mitotic index (96.2-100%) was observed in 2--4-cell embryos fertilized in vitro, although the index decreased thereafter. Murray et al. ('85) also reported that the 2--4-cell embryos gave a higher yield of usable chromosome spreads than 8-cell embryos in sheep. Comparing the lower mitotic index (71.3%, 399/560) at the 4-cell stage in our previous study (Iwasaki et al., '89a), the higher mitotic

TABLE 2. Chromosome composition of cells from early bovine embryos fertilized in vitro Stage (-cell) 2 3 4 5-7 8 9-16 17-32

No. of embryos Karyotyped Withanomaly(%)' 41 4 20 15 42 22 14

5 (12.1) l(25.0) 4 (20.0) 4 (26.7) 10 (23.8) 8 (36.4) 1 (7.1)

1 N 0 0 0 2(13.3) 5(11.9) 4(18.2) 1 (7.1)

1N/2N

Chromosome complement (%)' 3 N 4 N 2N/3N

0 0

3 (7.3) 0

3(15.0) 0 3 (7.1) 3(13.6)

0 0

0

l(2.4) l(4.5) 0

2(4.9) 0 0

l(6.7) 0 0 0

2N/4N

2N-13

0 0 0 l(6.7) 0 0

0

0 0

0

0

'No. of embryos with anomaly/No. of embryos karyotyped. (There is no significant difference among the stages) 'Percentage of embryos karyotyped. translocation.

l(25.0) 0 0 1 (2.4) 0

l(5.0) 0 0 0 0

82

S. IWASAKI ET AL.

Fig. 1. Metaphase spreads of normal and abnormal cells of at 4-cell stage, (d) A 3N (XXY) cell at %cell stage, (e)2N embryos at various stages: (a) A 1N (XI cell at 8-cell stage, (b) (XXY4N (XXXX)cells at blastocyst stage. (Long arrow: X, short A 2N (XY) cell at blastocyst stage, (c) A 2N-1(XX, t(1;29) cell arrow: Y, big arrowhead: t(1;29); 1000 x .)

CHROMOSOMAL ANOMALIES IN BOVINE EMBRYOS

83

TABLE 3. Mitotic index of whole embryos and ICM cells of bovine blastocysts fertilized in vitro and in vivo Stage of blastocyst

No. of embryos Processed Analyzed (%I1

Total cell number

No. of metaphase plates

Mitotic index

(%I2

Whole In vitro Early Expanded In vivo Expanded

25 59

25(100.0) 59(100.0)

85 & 16 111 f 40

8.6 f 3.0 11.8 5 5.3

10.3 5 3.3 10.9 f 5.4

5

5(100.0)

144 5 37

11.8 k 4.3

8.2 f 1.5

ICM In vitro Early Expanded In vivo Expanded

19 66

18 (94.7) 64 (97.0)

56 f 20 60 & 23

6.4 f 3.7 6.8 ? 3.8

12.0 k 4.8 11.8 5 6.7

10

10(100.0)

47 2 18

5.6 k 3.8

12.3 2 6.1

'No. of analyzed embryosho. of processed embryos. 'No. of metaphase plateskotal cell no. ofanalyzed embryo (mean t SD)

index observed in this study may be caused by the high rates (88.2%: 500/567 matured oocytes and 73.5%: 4171567) of embryo development to 2- and 4-cell stages, respectively (Hamano et al., unpublished data). Similarly, high cell numbers (56 and 60) of ICM were observed in early and expanded blastocysts fertilized in vitro, respectively. Preliminary data obtained by a differential fluorochrome staining technique showed that the blastocysts derived from the same in-vitro fertilization system possess about 30-45 ICM cells. The higher number of ICM cells may result from the use of higher quality embryos fertilized in vitro. Murry et al. ('86a) investigated the incidence of

chromosomal anomalies in sheep embryos at various cleavage stages from 1-cell to blastocyst. The frequency of their abnormalities was 6.25%, similar to the 6-11% incidence in 2-4-cell sheep embryos reported by Long and Williams ('80).However, developmental change in relation to anomalies was not discussed in detail. In the present study, no definite change with development was observed in the number of embryos with anomalies, but the chromosome composition varied among the stages. That is, only polyploid cells (3N and 4N) were observed in 2-cell embryos, although haploid or haploidldiploid mosaic cells were observed in 4-32cell embryos (Table 1).The polyploid cells were

TABLE 4 . Chromosome composition of whole embryos and ICM cells in bovine embryos fertilized in vitro Stage (-cell)

No. of embryos Karyotyped(%)l Withanomaly(%)'

3 N

4 N

Chromosome complement (%I3 2N13N 2Ni4N 2N/3N/4N

Whole In vitro Early Expanded Total In vivo Expanded

9 (36.0) 25 (42.4) 34 (40.5)

4(44.4) ll(44.0) X(44.2)

0 0 0

0 l(4.0) l(2.9)

l(11.1) 1 (4.0) 2 (5.9)

5(100.0)

l(20.0)

0

0

0

EM In vitro Early Expanded Total In vivo Expanded

13 (68.4) 57 (86.4) 70 (82.4)

1 (7.7) 12(21.1) 13(18.614

0 l(1.8) l(1.4)

0 l(1.8) l(1.4)

0

9 (90.0)

3(33.3)

0

0

'No. of embryos karyotypedho. of embryos processed. 'No. of embryos with anomalyino. of embryos karyotyped. 3Percentageof embryos karyotyped. 4Therewas a significant difference between whole embryos and ICM cells.

2(22.2) g(36.0) ll(32.4)

lNi2N

l(11.1) 0 1 (2.9)

0 0 0

l(20.0)

0

0

3 (5.3) 3 (4.3)

1 (7.7) 7(12.3) 8(11.4)

0 0 0

0 0 0

0

2(22.2)

0

l(11.1)

84

S.IWASAKI ET AL.

thought to be a result of polysperm penetration of ova or of the failure of polar body extrusion (King, '85b). The haploid cell was thought to be caused by a parthenogenesis. It has been suggested that the 1N/2N mosaic represented retained activity of the polar body in bovine embryos (King et al., '79) or was a product of polyspermy in sheep (Long and Williams, '80) and bovine embryos (King et al., '81). In the present study, arrested female nuclei were observed in the preparation of fixed oocytes at 18-20 h after the initiation of sperm-ova incubation in vitro, although the formation of the male pronucleus was normal. This fact suggests that the occurrence of cells bearing Y chromosome in the haploid cells may originate from the extra male pronucleus after penetration of Y-chromosome-bearingsperm and the incomplete formation of the female pronucleus (Iwasaki and Hamano, in press). However, the X-chromosome bearing cells were thought to be caused by gygenetic factors. The occurrence of haploid cells bearing Y chromosomes in 1N/2N (e.g., Y/XY or Y/XX)suggested that this anomaly might be caused by a dispermic egg (Long and Williams, '80), but there is also the possibility of fusion of parthenogenetic X cells or the arrest of cytokinetic division in the case of X/XX. In the present study, the incidence of chromosomal anomalies in whole blastocysts fertilized in vitro was high (32.4%),although only half this rate was observed in ICM cells. This tendency was not observed in the embryos fertilized in vivo because a small number of embryos using the sperm from same bull. Long and Williams ('82) showed that polyploid cells in whole day-10 pig embryos were primarily located in the trophoblast. In our results, the rate of embryos with anomaly in whole blastocysts was significantly higher than that in ICM cells and most of these were 2N/4N. Accordingly, it was suggested that the polyploid cells in whole embryos at blastocyst stage were located in the trophectoderm. The most frequently observed chromosomal anomalies were 2N/4N mixoploid in both whole blastocysts (73.3%)and ICM cells (61.5%)in the present study. Hare et al. ('80) also reported that 41.5%ofthe day 12-18 bovine blastocysts analyzed were 2N/4N mixoploids. Murray et al. ('86b) also reported a high proportion (51%) of day 13-14 embryos containing polyploid cells in sheep. The tetraploid cells may arise through the fusion of two diploid cells or from nuclear division without cytoplasmic division (Hare and Singh, '79).In the trophectoderm cells, 2N/4N cells may be caused by a binucleate cell. It has been reported that binucleate cells do not appear in the cow trophoblast until

day 17 of gestation (Greenstein et al., '58). From in vivo study, 2N/4N mixoploids were observed in the day 12-18 bovine blastocysts (Hare et al., 'SO). In the present study, however, 32.4%of blastocysts at 7-9 days after the initiation of sperm-ova incubation in vitro already showed 2N/4N mixoploids. There is no evidence that the time for appearance of binucleated trophoblast cells of blastocysts derived from in vitro fertilization is faster than that from in-vitro fertilization. This discrepancy should be clarified by a further study. Polyploid cells in diploid cells, including 6N and 8N, observed in blastocysts and fetuses should not be considered abnormal, as they appear t o be a normal part of the developmental processes leading to trophoblast formation and fetal differentiation (Murray et al., '86b). Consequently, the rates of true abnormal embryos were relatively low in the blastocysts derived from in-vitro fertilization in the present study. In addition, the sperm from one bull were used in this study. This may represent cytogenetics peculiar to this bull although calves derived from this sperm are all normal. In conclusion, there is no definite change in the incidence of anomalies of bovine embryos derived from in vitro fertilization with development at prior to the blastocyst stages and the anomalies observed were mainly caused by abnormal fertilization. In addition, it was suggested that the polyploid cells in whole embryos were located in the trophectoderm rather than the ICM.

ACKNOWLEDGMENTS This study was partly supported by the Hayashida Fund for Biotechnology Study of Tokyo University of Agriculture. LITERATURE CITED Abreu, F.G. de (1984) Cytogenetic analysis of early stage embryos from heifers and culled cows. In: Proceedings of the VIth European Colloquium of Cytogenetics in Domestic Animals, pp 416-421. Fukuda, Y., M. Ichikawa, K. Naito, and Y.Toyoda (1988) Normal development of bovine oocytes matured, fertilized, and cultured with cumulus cells in vitro. In: Proceedings of XIth International Congress of Animal Reproduction and Artificial Insemination, Dublin, Vol. 111, p. 327. Goto, K., Y. Kajihara, S. Kosaka, M. Koba, Y. Nakanishi, and K. Ogawa (1988) Pregnancies after co-culture of cumulus cells with bovine embryos derived from in-vitro fertilization of in-vitro matured follicular oocytes. J. Reprod. Fertil., 83: 753-758. Greenstein, J.S., R.W. Murray, and R.C. Foley (1958) Observations on the morphogenesis and histochemistry of the bovine pre-attachment placenta between 16 and 33 days of gestation. Anat. Rec., 132:321-341. Hanada, A. (1985) In vitro fertilization in bovine with special

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survival after transfer. Theriogenoloy,23: 161-174. King, W.A., D. Bouquet, T. Greve, and K.J. Betteridge (1985b) The pronuclei of bovine ova fertilized in vitro. Hereditas, 102:293-296. Long, S.E., and C. Williams (1980) Frequency of chromosomal abnormalities in early embryos of the domestic sheep (Ovis aries). J. Reprod. Fertil., 58:197-201. Long, S.E., and C. Williams (1982) A comparison of the chromosome complement of inner cell mass and trophoblast cells in Day-10 pig embryos. J. Reprod. Fertil., 66:645-648. Murray, J.D., M.P. Boland, and C. Moran (1986a) Frequency of chromosomal abnormalities in embryos from superovulated Merino ewes. J. Reprod. Fertil., 78:433-437. Murray, J.D., M.P. Boland, C. Moran, R. Sutton, C.D. Nancarrow, R.J. Scaramuzzi, and R.M. Hoskinson (1985) Occurrence of haploid and haploid/diploid mosaic embryos in untreated and andostenedine-immune Australian Merino sheep. J. Reprod. Fertil., 74:551-555. Murray, J.D., C. Moran, M.P. Boland, C.D. Nancarrow, R. Sutton C.D., R.M. Hoskinson, and R.J. Scaramuzzi (1986b) Polyploid cells in blastocysts and early fetuses from Australian Merino sheep. J. Reprod. Fertil., 74:439-446. Niwa, K., and 0.Ohgoda 0 (1988) Synergistic effect of caffeine and heparin on in-vitro fertilization of cattle oocytes matured in culture. Theriogenology,30:733-741. Parrish, J.J.,J.L. Susko-Parrish, M.L. Leibfried-Rutledge, E.S. Critser, W.H. Eyestone, and N.L. First (1986) Bovine in vitro fertilization with frozen-thawed semen. Theiogenology, 25: 591-600. Popescu, C.P. (1980) Cytogenetics study on embryos sired by a bull carrier of 1/29 translocation. In: IVth European Colloquium of Cytogenetics in Domestic Animals, pp. 182-186. Rieger, D., and P. Guay (1988) Measurement of the metabolism of energy substrates in individual bovine blastocysts. J. Reprod. Fertil., 83:585-591. Xu, K.P., T. Greve, H. Callesen, and P. Hyttel(1987) Pregnancy resulting from cattle oocytes matured and fertilized in vitro. J. Reprod. Fertil., 81:501-504.