- Email: [email protected]
Viability of bovine demi- and quarter-embryos after transfer
Theriogenology
38:107-l
13, 1992
VIABILITY OF BOVINE DEMI- AND QUARTER-EMBRYOS P.Bredbacka,’
M.Huhtinen,’
AFTER TRANSFER
J.Aalto* and V.Rainio3
‘Agricultural Research Centre, Department of Animal Breeding SF-31600 Jokioinen, Finland ‘East Breeding, P.O. Box 60, SF-76101 PieksZmZki, Finland 3University of Kuopio, Veterinary Research Station P.O. Box 6, SF-7021 1 Kuopio, Finland Received for publication: Accepted:
August April
2, 29,
2991 1992
ABSTRACT The viability of bovine demi- and quarter-embryos was investigated. Early compacting morullae were nonsurgically flushed from superovulated donor cows and were bisected by two micraneedles. One of the halves was then split further into two quarters. Each demi- and quarter-embryo was placed in an evacuated zona pellucida. One demi- or two quarter-embryos were transferred non-surgically into cow or heifer recipients. Viability was measured by ultrasound scanning of the fetuses on Days 35, 48 and 60 of pregnancy. The pregnancy rates at Day 60 were 46.2% (6/13) for heifers and 33.3% (4/12) for cows after the transfer of a single demi-embryo. The transfer of two quarter-embryos resulted in a pregnancy rate of 61.5% (8/13) for heifers and 8.3% (1112) for cows. Seven (53.8%) and four (33.3%) live fetuses were found on Day 60 following the transfer of demi-embryos into heifers and cows, respectively. The transfer of quarter-embryos resulted in 10 fetuses (38.5%) in the heifer recipients and only one fetus (4.2%) in the cow recipients. The results of this study suggest that heifers are more suitable than cows as recipients for quarter-embryos. Key words:
embryo transfer, demi-embryo,
quarter-embryo,
splitting
INTRODUCTION Identical twins have been produced by microsurgery of embryos in several mammalian species. In farm animals, the aim of embryo splitting is to generate more offspring from valualble donor females and to produce genetically identical animals for various research purpc’ses. In cattle, the splitting of nonsurgically recovered embryos has become a routine practice. Several investigators have been able to increase the number of pregnancies per donor cow by embryo bisection (l-8). With further splitting the advantage could become even greater, provided that the viability of each portion of the embryo remained at an acceptable level. Willadsen and Polge (9) produced monozygotic triplets, and their results suggested that approximately 25% of the normal number of cells is required to retain embryonic viability. These triplets were produced by quartering eight-cell embryos by blastomere separation, followed by agar embedding and culture in ligated sheep oviducts before transfer to heifer recipients. However, eight-cell stage embryos cannot be recovered nonsurgically. Under practical conditions further division into quarter embryos has not been considered feasible, although there is little published data to confirm this. In one study, the survival rate of embryos at Day 50, following micromanipulation at Days 5 to 6, agar embedding, culture in ligated sheep oviducts and selection of normally developed embryos prior to surgical transfer, was 41% for quarter-embryos and 75% for half embryos (10). Investigations in which nonsurgical
Copyright
0 1992 Butterworth-Heinemann
108
Theriogenology
transfer techniques have been used have either been less encouraging than those utilizing surgical transfer or were carried out with a small number of recipients (11-14). The aim of the present study was to evaluate the viability of bovine demi- and quarterembryos following nonsurgical transfer, and to assess the feasibility of transferring quarterembryos to increase the yield of offspring and to produce genetically identic animals. To our knowledge, this is the largest investigation on the viability of quarter-embryos in which flushing, micromanipulation and non-surgical transfer were all carried out on the same day. MATERIALS AND METHODS Ayrshire donor cows were given a single i.m. injection of 2,500 IU PMSG (Folligon; Intervet, Holland) between days 9 and 12 after estrus, followed by a single i.m. injection of 1.0 mg cloprostenol (Estrumat@; Coopers, Germany) to induce Iuteolysis. Cows were inseminated twice 12 hours apart beginning 2 to 9 hours after the start of standing estrus. Embryos were recovered nonsurgically 5.5 to 6.5 days post estrus. The stage of embryo development varied from the 16-cell stage to fully compacted morulae. Most of the embryos had about 32 cells and were precompacted morulae or in the process of compaction. Early compacting morulae with discernable blastomeres and with less than 10% dead cell mass were used in the experiment. Zona pellucidae were removed by brief exposure to Tyrode’s solution (PH 2.1). The embryos were then cultured for 20 to 80 minutes in Ca”/Mg’+ -free PBS with 1 g/l glucose and 0.1% BSA at room temperature to loosen cell contacts. Bisections were carried out in PBS + 0.4% BSA with two glass microneedles controlled by Leitz micromanipulators (Figure 1). One of the two equal sized demi-embryos was then further bisected into two quarter-embryos. Each demiand quarter-embryo was then placed in an empty zona pellucida (Figure 2). The zonae were obtained from in vitro-matured oocytes. One demi-embryo or two quarter-embryos were nonsurgically transferred into each recipient. This design ensured that embryos with genetic or other undetectable defects were represented in both the demi- and quarter-embryo groups. Synchronized Ayrshire heifers and lactating cows (_+1.25 days) were used as recipients. The cows were synchronized by inserting a progesteron-releasing intravaginal device (PRIDTU; Abbott, France) and a single i.m. injection of 0,625 mg cloprostenol the day before PRID
Figure 1.
Two quarter-embryos and one demi-embryo produced by splitting with microneedles.
Figure 2.Two quarter-embryos and one demi-embryo inserted into evacuated zona pellucidae.
Theriogenolog
109
y
removal. The heifer recipients were synchronized with two injections of cloprostenol given 13 days apart. Prior to transfer, selection of recipients was based on rectal findings. The viability of the embryos was measured by ultrasound scatming of fetuses on Days 35, 48 and 60 of pregnancy. The presence of a beating heart was used as the criterion for a live fetus. Progesterone concentrations were determined by radioimmunoassay from milk (cows) or serum (heifers) on Day 6 (the day of transfer) and on Days 13, 19 and 24 following standing estrus. The age of the donor cows ranged from 4 to 8 years. The recipient cows were 2 to 9 years of age and the heifers were 14 to 23 months old.
RESULTS A,total of 30 embryos was bisected into sets of one den&embryo and two quarter-embryos. Of these, two sets were excluded due to unequal splitting. Twenty-five of the remaining sets (including 25 demi-embryos and 50 quarter-embryos) were transferred into recipients. Pregnancy results are shown in Table 1. When heifers served as the recipients, pregnancy rates at Day 60 were 46.2% using one demi-embryo and 61.5% using two quarter-embryos. When cows were used as the recipients, 4 of 12 receiving one demi-embryo became pregnant, while only one of 12 cows receiving two quarter-embryos became pregnant. There was an overall difference (PCO.02, Fisher’s exact test) in Day 60 in the pregnancy rate between heifers (53.8%) and cows (20.8%).
Table 1. Pregnancy rates following embryo transfer of one demi-embryo or two quarterembryos into cow or heifer recipients
Recipient cow Heifer
Type of embryo transfer
(n)
one demi-embryo two quarter-embryos one demi-embryo two quarter-embryos
12 12 13 13
Recipients with high progesteronea (4%) 58.3b 16.7’ 61.5b 61.5b
Recipients pregnant at Day 35 Day 48 Day 60 (%) 41.7ba 16.7c 61.5b 61.5b
33.3b,C 33.3b.C 8.3’ 8.3c 53.gb 46.2b 61.5b 61.5b
* Recipients with progesterone levels above 15 nmol/l on Days 19 and 23. b+ Within columns, values with different superscripts differ (P < 0.05; Fisher’s exact test).
High progesterone concentrations (> 15 nmol/l) at both Days 19 and 24 were good predictors of pregnancy. Only two recipients with high progesterone levels were not found to be pregnant by ultrasonography on Day 35. Both cows had received a demi-embryo and both returned to estrus on Day 38, thus exhibiting a prolonged estrous cycle. Day-13 progesterone concentrations had no value in predicting pregnancy (data not shown). Two nonpregnant heifers into which one demi-embryo each had been transferred expressed low progesteron levels (<6 nmol4) on all days, including the day of transfer, when their levels were 2 and 3 rnnolli respectively. Fetal survival of quarter-embryos was higher in heifers than in cows (Table 2). Demiembryos were more viable than quarter-embryos when cows were used as recipients (Table 2).
110
On Day as did one spontaneous carried twin
Theriogenology
60, two of the heifers that received two quarter-embryos each carried twin fetuses, heifer into which one demi-embryo had been transferred. In this latter case a twinning had occurred during or after transfer. Yet another demi-embryo recipient fetuses on Day 35, but these died before Days 48 and 60, respectively.
The number of fetuses per whole embryo following splitting and transfer into heifers was 1.54 for quartered embryos on Day 60 of pregnancy. The corresponding number of fetuses for demi-embryos was 0.92 or 1.08, depending on whether or not the spontaneous twinning is excluded from the results. The number of fetuses per whole embryo after transfer into cows was 0.17 for quarter-embryos and 0.67 for demi-embryos. Pregnancies at Day 60 included three sets of identical triplets and two sets of identical twins. Table 2. Viability of quarter and half embryos after embryo transfer
Recipient cow Heifer
Type of embryos transferred (n) demi quarter demi quarter
12 24 13 26
Viable fetuses at Day 35 Day 48 Day 60 (%) 41.78 8.3b 69.2’ 38.5’
33.3a 4.2b 53.8’ 38.5*
33.3a 4.2b 53 8’9’ 38158
No. of fetuses per whole embryo at Day 60 0.67 0.17 1.08’ 1.54
a,b Within columns, values with different superscripts differ (P < 0.05; Fisher’s exact test). c Includes one spontaneous twinning of a demi-embryo.
DISCUSSION From the results in this study two conclusions can be drawn. First, the viability of quarterembryos is roughly half that of demi-embryos. Second, embryos with a significant decrease in cell mass appear to develop into fetuses at a lower rate after transfer into cows than into heifers. There are very few reports evaluating the viability of quarter-embryos. In our study, the viability of quarter-embryos in heifer recipients was approximately the same as that found by Willadsen et al. (lo), but demi-embryo viability was lower than in that study. However, two of the demi-embryo recipients in our study were, in retrospect, unsuitable as recipients since they appeared not to have been cycling. Methodologically, our experiment was also different in that the embryos were transferred nonsurgically and without selection for normal development after culture in ligated sheep oviducts. The results of both studies suggest that the viability of Day-5 or Day-6 quarter-embryos is approximately half that of the viability of demi-embryos of the same age. Quartering embryos may therefore be an effective way to increase the offspring from donor cows when the number of heifer recipients is not limited. Considering that about half of the transferable embryos from superovulated cows fulfill the criteria for microsurgery, thus assuming a yield of 1.5 fetuses per quartered embryo versus 0.7 to 0.8 fetuses per intact embryo, the overall increase would be at least 40%. Early morulae were micromanipulated in our study. There is reason to believe that the viability of quarter-embryos can be improved by splitting embryos at more developed stages. It has been reported that the splitting of late stage morulae and early stage blasocysts resulted in
Theriogenology
111
higher pregnancy rates than the splitting of early stage morulae (2). In another study, fetal survival rate was lower for demi-embryos derived from compact morulae or early stage blastocysts than for demi-embryos produced by bisection of blastocysts or advanced blastocysts (15). The results of a recent study suggest that bisection at late compaction may result in higher viability as compared with bisection at early compaction (as in this study) because more cells are allocated to the inner cell mass in subsequently developing blastocysts (16). Although these experiments were done with demi-embryos, similar results could be expected with quarterembryos. The practical advantage of using late compacted morulae or early stage blastocysts for splitting is that these embryonic stages do not require a zona pellucida (17,lg). In the present study, all embryos were at an early compacting stage and were also decompacted by Ca”/Mg+ +-free PBS prior to microsurgery and were therefore considered to need a protective zona pellucida. This may have contributed to better control of the experiment. On the other hand, the additional procedures may actually have decreased the viability of embryos due to more manipulation and prolonged in vitro exposure. One reason to use early instead of late stage compacting morulae was that cell damage could be monitored more accurately. Apprclximately 0 to 2 cells were lysed at each splitting. The cracked zona pellucida may have been responsible for the spontaneous splittings of the demi-embryos that resulted in twin pregnancies. This is not the first time we have observed such in vivfo splitting after manipulated embryos have been placed in evacuated zonae pellucidae. It may be that in the presence of a cracked zona pellucida a premature hatching occurs, during which the zona pellucida remains thick and stiff and is therefore able to cut the hatching embryo in twc’. In our present experiment, there was an effect of the recipients on the viability of the manipulated embryos. Since the recipient cows (ah of which were lactating) and heifers were kept on different farms, a possible influence of environmental factors cannot be ruled out. However, the advantage of using heifers rather than cows as recipients for demi-embryos has also been reported by other workers (5). No explanation for this phenomenon has been presented, but one possible cause is the larger reproductive tract of cows, which would result in a more remote dialogue between the corpus luteum and the more proximally transferred embryo. This communication might be suboptimal due to a reduced embryonic signal, which can be expected from embryos with a low viable cell mass. Another possibility for the lower pregnancy rates in cow recipients is that accurate selection of suitable cow recipients may be more difftcult due to the effects of calving. The selection of the recipients was based on rectal findings, with 23% (7 of 31) of the cow recipients and 21% (7 of 33) of the heifer recpients being discarded. Furthermore, all recipient cows were lactating, which may be a stress factor influencing fertility. Since cows are also needed as recipients, it would be valuable to improve the pregnancy rates after transfer of demi- and quarter-embryos to the same level as found in heifer recipients. If cows are less suitable as recipients due to suboptimal embryo-maternal communication, a solution for higher survival rates of demi- and quarter-embryos might be in co-transferring trophoblastic vesicles (19,20). More likely this would ensure a sufficient lutetrophic signal and thereby establish pregnancy. The transfer of twin embryos is often associated with problems in late pregnancy (6,9,21). Optimal development to term of quarter-embryos in heifer recipients may be achieved by the co-transfer of a single quarter-embryo with trophoblastic vesicles.
112
Theriogenology
In conclusion, the quartering of embryos may well be a way to increase the number of offspring in cattle. Identical triplets (and presumably also quadruplets) can be produced by embryo splitting, although at a relatively low frequency. Considering the potential economic value, the developmental potential of quarter-embryos calls for further research.
REFERENCES 1.
Ozil, J.P., Heyman, Y. and Renard, J.P. Production of monozygotic twins by micromanipulation and cervical transfer in the cow. Vet. Rec. 110: 126-127 (1982).
2.
Williams, T.J., Elsden, R.P. and Seidel, G.E., Jr. Effect of embryo age and stage on pregnancy rates from demi-embryos. Theriogenology 2: 276 abstr. (1984).
3.
Takeda, T., Hallowell, S.V., McCauley, A.D. and Hasler, J.F. Pregnancy rates with intact and split embryos transferred surgically and nonsurgically. Theriogenology 25: 204 abstr. (1986).
4.
Leibo, S.P. and Rail, W.F. Increase in production of pregnancies by bisection of bovine embryos. Theriogenology 3: 245 abstr. (1987).
5.
Arave, C.W., Bunch, T.D., Mickelsen, C.H. and Warnick, K. Factors affecting survivability of transferred whole and demi-embryos in a commercial dairy herd. Theriogenology 28: 373-382 (1987).
6.
Seike, N., Saeki, K., Utaka, K., Sakai, M., Takakura, R., Nagao, Y. and Kanagawa, H. Production of bovine identical twins via transfer of demi-embryos without zonae pellucidae. Theriogenology 2: 211-220 (1989).
7.
Kippax, IS., Christie, W.B. and Rowan, T.G. Effects of method of splitting, stage of development and presence or absence of zona pellucida on fetal survival in commercial bovine embryo transfer of bisected embryos. Theriogenology 35: 25-35 (1991).
8.
Gray, K.R., Bondioli, K.R. and Betts, C.L. The commercial application of embryo splitting in beef cattle. Theriogenology 35: 37-44 (1991).
9.
Willadsen, S.M. and Polge, C. Attempts to produce monozygotic quadruplets in cattle by blastomere separation. Vet. Rec. 108: 211-213 (1981)
10.
Willadsen, S.M., Lehn-Jensen, H., Fehilly, C.B. and Newcomb, R. The Production of monozygotic twins of preseIected parentage by micromanipulation of non-surgically collected cow embryos. Theriogenology 15: 23-29 (1981).
11.
Voelkel, S.A., Viker, S.D., Johnson, C.A., Hill, K.G., Humes, P.E. and Godke, R.A. Multiple embryo-transplant offspring produced from quartering a bovine embryo at the morula stage. Vet. Rec. 117: 528-530 (1985).
12.
Massip, A., Van Der Zwalmen, P. and Ectors, F. Developpement de quarts d’embryons bovins frais ou aprb vitrification. Ann. Med. Vet. 132: 483-487 (1988).
113
Theriogenology
13.
Tan, L., Li, L., Liao, H., Zhang, Y., Liu, R. and Yan, Z. The survival of quartered bovine embryos with and without zona pellucidae. Theriogenology 35: 279 abstr. (1991).
14.
Tao, T., Shie, J., Yanquing, H., Changheng, G., Wenzhu, M., Tiezhu, A. and The survival of zona-free bovine demi- and quarter embryos. Yuding, Z. Theriogenology 35: 281 abstr. (1991).
15.
McEvoy, T.G. and Sreenan, J.M. Effect of embryo quality and stage of development on the survival of zona pellucida-free cattle demi-embryos. Theriogenology 2: 1245 1253 (1990).
16.
Bredbacka, P., Bredbacka, K., Aalto, J. and Kukkola, H. Development of inner cell mass in intact and bisected cattIe morulae. Theriogenology 3: 188 abstr. (1991).
17.
Hoppe, R.W. and Bavister, B.D. Effect of removing the zona pellucida on development of hamster and bovine embryos in vitro and in vivo. Theriogenology 19: 391-404 (1983).
18.
Warfield, S.J., Seidel, G.E., Jr. and Elsden, R.P. Transfer of bovine demi-embryos with and without zonae pellucidae. Theriogenology 25: 212 abstr. (1986).
19.
Heyman, Y., Camous, S., Fevre, J., Meziou, W. and Martal, J. Maintenance of the corpus luteum after uterine transfer of trophoblastic vesicles to cyclic cows and ewes. J. Reprod. Fertil. 24: 533-540 (1984).
20.
Heyman, Y. Factors affecting the survival of whole and half-embryos cattle. Theriogenology 23: 63-75 (1985).
21.
Lambeth, V.A., Looney, C.R., Voelkel, S.A., Jackson, D., Hill, K. and Godke, R. Microsurgery on bovine embryos at me morula stage to produce monozygotic twin calves. Theriogenology 3: 85-95 (1983).
transferred
in
38:107-l
13, 1992
VIABILITY OF BOVINE DEMI- AND QUARTER-EMBRYOS P.Bredbacka,’
M.Huhtinen,’
AFTER TRANSFER
J.Aalto* and V.Rainio3
‘Agricultural Research Centre, Department of Animal Breeding SF-31600 Jokioinen, Finland ‘East Breeding, P.O. Box 60, SF-76101 PieksZmZki, Finland 3University of Kuopio, Veterinary Research Station P.O. Box 6, SF-7021 1 Kuopio, Finland Received for publication: Accepted:
August April
2, 29,
2991 1992
ABSTRACT The viability of bovine demi- and quarter-embryos was investigated. Early compacting morullae were nonsurgically flushed from superovulated donor cows and were bisected by two micraneedles. One of the halves was then split further into two quarters. Each demi- and quarter-embryo was placed in an evacuated zona pellucida. One demi- or two quarter-embryos were transferred non-surgically into cow or heifer recipients. Viability was measured by ultrasound scanning of the fetuses on Days 35, 48 and 60 of pregnancy. The pregnancy rates at Day 60 were 46.2% (6/13) for heifers and 33.3% (4/12) for cows after the transfer of a single demi-embryo. The transfer of two quarter-embryos resulted in a pregnancy rate of 61.5% (8/13) for heifers and 8.3% (1112) for cows. Seven (53.8%) and four (33.3%) live fetuses were found on Day 60 following the transfer of demi-embryos into heifers and cows, respectively. The transfer of quarter-embryos resulted in 10 fetuses (38.5%) in the heifer recipients and only one fetus (4.2%) in the cow recipients. The results of this study suggest that heifers are more suitable than cows as recipients for quarter-embryos. Key words:
embryo transfer, demi-embryo,
quarter-embryo,
splitting
INTRODUCTION Identical twins have been produced by microsurgery of embryos in several mammalian species. In farm animals, the aim of embryo splitting is to generate more offspring from valualble donor females and to produce genetically identical animals for various research purpc’ses. In cattle, the splitting of nonsurgically recovered embryos has become a routine practice. Several investigators have been able to increase the number of pregnancies per donor cow by embryo bisection (l-8). With further splitting the advantage could become even greater, provided that the viability of each portion of the embryo remained at an acceptable level. Willadsen and Polge (9) produced monozygotic triplets, and their results suggested that approximately 25% of the normal number of cells is required to retain embryonic viability. These triplets were produced by quartering eight-cell embryos by blastomere separation, followed by agar embedding and culture in ligated sheep oviducts before transfer to heifer recipients. However, eight-cell stage embryos cannot be recovered nonsurgically. Under practical conditions further division into quarter embryos has not been considered feasible, although there is little published data to confirm this. In one study, the survival rate of embryos at Day 50, following micromanipulation at Days 5 to 6, agar embedding, culture in ligated sheep oviducts and selection of normally developed embryos prior to surgical transfer, was 41% for quarter-embryos and 75% for half embryos (10). Investigations in which nonsurgical
Copyright
0 1992 Butterworth-Heinemann
108
Theriogenology
transfer techniques have been used have either been less encouraging than those utilizing surgical transfer or were carried out with a small number of recipients (11-14). The aim of the present study was to evaluate the viability of bovine demi- and quarterembryos following nonsurgical transfer, and to assess the feasibility of transferring quarterembryos to increase the yield of offspring and to produce genetically identic animals. To our knowledge, this is the largest investigation on the viability of quarter-embryos in which flushing, micromanipulation and non-surgical transfer were all carried out on the same day. MATERIALS AND METHODS Ayrshire donor cows were given a single i.m. injection of 2,500 IU PMSG (Folligon; Intervet, Holland) between days 9 and 12 after estrus, followed by a single i.m. injection of 1.0 mg cloprostenol (Estrumat@; Coopers, Germany) to induce Iuteolysis. Cows were inseminated twice 12 hours apart beginning 2 to 9 hours after the start of standing estrus. Embryos were recovered nonsurgically 5.5 to 6.5 days post estrus. The stage of embryo development varied from the 16-cell stage to fully compacted morulae. Most of the embryos had about 32 cells and were precompacted morulae or in the process of compaction. Early compacting morulae with discernable blastomeres and with less than 10% dead cell mass were used in the experiment. Zona pellucidae were removed by brief exposure to Tyrode’s solution (PH 2.1). The embryos were then cultured for 20 to 80 minutes in Ca”/Mg’+ -free PBS with 1 g/l glucose and 0.1% BSA at room temperature to loosen cell contacts. Bisections were carried out in PBS + 0.4% BSA with two glass microneedles controlled by Leitz micromanipulators (Figure 1). One of the two equal sized demi-embryos was then further bisected into two quarter-embryos. Each demiand quarter-embryo was then placed in an empty zona pellucida (Figure 2). The zonae were obtained from in vitro-matured oocytes. One demi-embryo or two quarter-embryos were nonsurgically transferred into each recipient. This design ensured that embryos with genetic or other undetectable defects were represented in both the demi- and quarter-embryo groups. Synchronized Ayrshire heifers and lactating cows (_+1.25 days) were used as recipients. The cows were synchronized by inserting a progesteron-releasing intravaginal device (PRIDTU; Abbott, France) and a single i.m. injection of 0,625 mg cloprostenol the day before PRID
Figure 1.
Two quarter-embryos and one demi-embryo produced by splitting with microneedles.
Figure 2.Two quarter-embryos and one demi-embryo inserted into evacuated zona pellucidae.
Theriogenolog
109
y
removal. The heifer recipients were synchronized with two injections of cloprostenol given 13 days apart. Prior to transfer, selection of recipients was based on rectal findings. The viability of the embryos was measured by ultrasound scatming of fetuses on Days 35, 48 and 60 of pregnancy. The presence of a beating heart was used as the criterion for a live fetus. Progesterone concentrations were determined by radioimmunoassay from milk (cows) or serum (heifers) on Day 6 (the day of transfer) and on Days 13, 19 and 24 following standing estrus. The age of the donor cows ranged from 4 to 8 years. The recipient cows were 2 to 9 years of age and the heifers were 14 to 23 months old.
RESULTS A,total of 30 embryos was bisected into sets of one den&embryo and two quarter-embryos. Of these, two sets were excluded due to unequal splitting. Twenty-five of the remaining sets (including 25 demi-embryos and 50 quarter-embryos) were transferred into recipients. Pregnancy results are shown in Table 1. When heifers served as the recipients, pregnancy rates at Day 60 were 46.2% using one demi-embryo and 61.5% using two quarter-embryos. When cows were used as the recipients, 4 of 12 receiving one demi-embryo became pregnant, while only one of 12 cows receiving two quarter-embryos became pregnant. There was an overall difference (PCO.02, Fisher’s exact test) in Day 60 in the pregnancy rate between heifers (53.8%) and cows (20.8%).
Table 1. Pregnancy rates following embryo transfer of one demi-embryo or two quarterembryos into cow or heifer recipients
Recipient cow Heifer
Type of embryo transfer
(n)
one demi-embryo two quarter-embryos one demi-embryo two quarter-embryos
12 12 13 13
Recipients with high progesteronea (4%) 58.3b 16.7’ 61.5b 61.5b
Recipients pregnant at Day 35 Day 48 Day 60 (%) 41.7ba 16.7c 61.5b 61.5b
33.3b,C 33.3b.C 8.3’ 8.3c 53.gb 46.2b 61.5b 61.5b
* Recipients with progesterone levels above 15 nmol/l on Days 19 and 23. b+ Within columns, values with different superscripts differ (P < 0.05; Fisher’s exact test).
High progesterone concentrations (> 15 nmol/l) at both Days 19 and 24 were good predictors of pregnancy. Only two recipients with high progesterone levels were not found to be pregnant by ultrasonography on Day 35. Both cows had received a demi-embryo and both returned to estrus on Day 38, thus exhibiting a prolonged estrous cycle. Day-13 progesterone concentrations had no value in predicting pregnancy (data not shown). Two nonpregnant heifers into which one demi-embryo each had been transferred expressed low progesteron levels (<6 nmol4) on all days, including the day of transfer, when their levels were 2 and 3 rnnolli respectively. Fetal survival of quarter-embryos was higher in heifers than in cows (Table 2). Demiembryos were more viable than quarter-embryos when cows were used as recipients (Table 2).
110
On Day as did one spontaneous carried twin
Theriogenology
60, two of the heifers that received two quarter-embryos each carried twin fetuses, heifer into which one demi-embryo had been transferred. In this latter case a twinning had occurred during or after transfer. Yet another demi-embryo recipient fetuses on Day 35, but these died before Days 48 and 60, respectively.
The number of fetuses per whole embryo following splitting and transfer into heifers was 1.54 for quartered embryos on Day 60 of pregnancy. The corresponding number of fetuses for demi-embryos was 0.92 or 1.08, depending on whether or not the spontaneous twinning is excluded from the results. The number of fetuses per whole embryo after transfer into cows was 0.17 for quarter-embryos and 0.67 for demi-embryos. Pregnancies at Day 60 included three sets of identical triplets and two sets of identical twins. Table 2. Viability of quarter and half embryos after embryo transfer
Recipient cow Heifer
Type of embryos transferred (n) demi quarter demi quarter
12 24 13 26
Viable fetuses at Day 35 Day 48 Day 60 (%) 41.78 8.3b 69.2’ 38.5’
33.3a 4.2b 53.8’ 38.5*
33.3a 4.2b 53 8’9’ 38158
No. of fetuses per whole embryo at Day 60 0.67 0.17 1.08’ 1.54
a,b Within columns, values with different superscripts differ (P < 0.05; Fisher’s exact test). c Includes one spontaneous twinning of a demi-embryo.
DISCUSSION From the results in this study two conclusions can be drawn. First, the viability of quarterembryos is roughly half that of demi-embryos. Second, embryos with a significant decrease in cell mass appear to develop into fetuses at a lower rate after transfer into cows than into heifers. There are very few reports evaluating the viability of quarter-embryos. In our study, the viability of quarter-embryos in heifer recipients was approximately the same as that found by Willadsen et al. (lo), but demi-embryo viability was lower than in that study. However, two of the demi-embryo recipients in our study were, in retrospect, unsuitable as recipients since they appeared not to have been cycling. Methodologically, our experiment was also different in that the embryos were transferred nonsurgically and without selection for normal development after culture in ligated sheep oviducts. The results of both studies suggest that the viability of Day-5 or Day-6 quarter-embryos is approximately half that of the viability of demi-embryos of the same age. Quartering embryos may therefore be an effective way to increase the offspring from donor cows when the number of heifer recipients is not limited. Considering that about half of the transferable embryos from superovulated cows fulfill the criteria for microsurgery, thus assuming a yield of 1.5 fetuses per quartered embryo versus 0.7 to 0.8 fetuses per intact embryo, the overall increase would be at least 40%. Early morulae were micromanipulated in our study. There is reason to believe that the viability of quarter-embryos can be improved by splitting embryos at more developed stages. It has been reported that the splitting of late stage morulae and early stage blasocysts resulted in
Theriogenology
111
higher pregnancy rates than the splitting of early stage morulae (2). In another study, fetal survival rate was lower for demi-embryos derived from compact morulae or early stage blastocysts than for demi-embryos produced by bisection of blastocysts or advanced blastocysts (15). The results of a recent study suggest that bisection at late compaction may result in higher viability as compared with bisection at early compaction (as in this study) because more cells are allocated to the inner cell mass in subsequently developing blastocysts (16). Although these experiments were done with demi-embryos, similar results could be expected with quarterembryos. The practical advantage of using late compacted morulae or early stage blastocysts for splitting is that these embryonic stages do not require a zona pellucida (17,lg). In the present study, all embryos were at an early compacting stage and were also decompacted by Ca”/Mg+ +-free PBS prior to microsurgery and were therefore considered to need a protective zona pellucida. This may have contributed to better control of the experiment. On the other hand, the additional procedures may actually have decreased the viability of embryos due to more manipulation and prolonged in vitro exposure. One reason to use early instead of late stage compacting morulae was that cell damage could be monitored more accurately. Apprclximately 0 to 2 cells were lysed at each splitting. The cracked zona pellucida may have been responsible for the spontaneous splittings of the demi-embryos that resulted in twin pregnancies. This is not the first time we have observed such in vivfo splitting after manipulated embryos have been placed in evacuated zonae pellucidae. It may be that in the presence of a cracked zona pellucida a premature hatching occurs, during which the zona pellucida remains thick and stiff and is therefore able to cut the hatching embryo in twc’. In our present experiment, there was an effect of the recipients on the viability of the manipulated embryos. Since the recipient cows (ah of which were lactating) and heifers were kept on different farms, a possible influence of environmental factors cannot be ruled out. However, the advantage of using heifers rather than cows as recipients for demi-embryos has also been reported by other workers (5). No explanation for this phenomenon has been presented, but one possible cause is the larger reproductive tract of cows, which would result in a more remote dialogue between the corpus luteum and the more proximally transferred embryo. This communication might be suboptimal due to a reduced embryonic signal, which can be expected from embryos with a low viable cell mass. Another possibility for the lower pregnancy rates in cow recipients is that accurate selection of suitable cow recipients may be more difftcult due to the effects of calving. The selection of the recipients was based on rectal findings, with 23% (7 of 31) of the cow recipients and 21% (7 of 33) of the heifer recpients being discarded. Furthermore, all recipient cows were lactating, which may be a stress factor influencing fertility. Since cows are also needed as recipients, it would be valuable to improve the pregnancy rates after transfer of demi- and quarter-embryos to the same level as found in heifer recipients. If cows are less suitable as recipients due to suboptimal embryo-maternal communication, a solution for higher survival rates of demi- and quarter-embryos might be in co-transferring trophoblastic vesicles (19,20). More likely this would ensure a sufficient lutetrophic signal and thereby establish pregnancy. The transfer of twin embryos is often associated with problems in late pregnancy (6,9,21). Optimal development to term of quarter-embryos in heifer recipients may be achieved by the co-transfer of a single quarter-embryo with trophoblastic vesicles.
112
Theriogenology
In conclusion, the quartering of embryos may well be a way to increase the number of offspring in cattle. Identical triplets (and presumably also quadruplets) can be produced by embryo splitting, although at a relatively low frequency. Considering the potential economic value, the developmental potential of quarter-embryos calls for further research.
REFERENCES 1.
Ozil, J.P., Heyman, Y. and Renard, J.P. Production of monozygotic twins by micromanipulation and cervical transfer in the cow. Vet. Rec. 110: 126-127 (1982).
2.
Williams, T.J., Elsden, R.P. and Seidel, G.E., Jr. Effect of embryo age and stage on pregnancy rates from demi-embryos. Theriogenology 2: 276 abstr. (1984).
3.
Takeda, T., Hallowell, S.V., McCauley, A.D. and Hasler, J.F. Pregnancy rates with intact and split embryos transferred surgically and nonsurgically. Theriogenology 25: 204 abstr. (1986).
4.
Leibo, S.P. and Rail, W.F. Increase in production of pregnancies by bisection of bovine embryos. Theriogenology 3: 245 abstr. (1987).
5.
Arave, C.W., Bunch, T.D., Mickelsen, C.H. and Warnick, K. Factors affecting survivability of transferred whole and demi-embryos in a commercial dairy herd. Theriogenology 28: 373-382 (1987).
6.
Seike, N., Saeki, K., Utaka, K., Sakai, M., Takakura, R., Nagao, Y. and Kanagawa, H. Production of bovine identical twins via transfer of demi-embryos without zonae pellucidae. Theriogenology 2: 211-220 (1989).
7.
Kippax, IS., Christie, W.B. and Rowan, T.G. Effects of method of splitting, stage of development and presence or absence of zona pellucida on fetal survival in commercial bovine embryo transfer of bisected embryos. Theriogenology 35: 25-35 (1991).
8.
Gray, K.R., Bondioli, K.R. and Betts, C.L. The commercial application of embryo splitting in beef cattle. Theriogenology 35: 37-44 (1991).
9.
Willadsen, S.M. and Polge, C. Attempts to produce monozygotic quadruplets in cattle by blastomere separation. Vet. Rec. 108: 211-213 (1981)
10.
Willadsen, S.M., Lehn-Jensen, H., Fehilly, C.B. and Newcomb, R. The Production of monozygotic twins of preseIected parentage by micromanipulation of non-surgically collected cow embryos. Theriogenology 15: 23-29 (1981).
11.
Voelkel, S.A., Viker, S.D., Johnson, C.A., Hill, K.G., Humes, P.E. and Godke, R.A. Multiple embryo-transplant offspring produced from quartering a bovine embryo at the morula stage. Vet. Rec. 117: 528-530 (1985).
12.
Massip, A., Van Der Zwalmen, P. and Ectors, F. Developpement de quarts d’embryons bovins frais ou aprb vitrification. Ann. Med. Vet. 132: 483-487 (1988).
113
Theriogenology
13.
Tan, L., Li, L., Liao, H., Zhang, Y., Liu, R. and Yan, Z. The survival of quartered bovine embryos with and without zona pellucidae. Theriogenology 35: 279 abstr. (1991).
14.
Tao, T., Shie, J., Yanquing, H., Changheng, G., Wenzhu, M., Tiezhu, A. and The survival of zona-free bovine demi- and quarter embryos. Yuding, Z. Theriogenology 35: 281 abstr. (1991).
15.
McEvoy, T.G. and Sreenan, J.M. Effect of embryo quality and stage of development on the survival of zona pellucida-free cattle demi-embryos. Theriogenology 2: 1245 1253 (1990).
16.
Bredbacka, P., Bredbacka, K., Aalto, J. and Kukkola, H. Development of inner cell mass in intact and bisected cattIe morulae. Theriogenology 3: 188 abstr. (1991).
17.
Hoppe, R.W. and Bavister, B.D. Effect of removing the zona pellucida on development of hamster and bovine embryos in vitro and in vivo. Theriogenology 19: 391-404 (1983).
18.
Warfield, S.J., Seidel, G.E., Jr. and Elsden, R.P. Transfer of bovine demi-embryos with and without zonae pellucidae. Theriogenology 25: 212 abstr. (1986).
19.
Heyman, Y., Camous, S., Fevre, J., Meziou, W. and Martal, J. Maintenance of the corpus luteum after uterine transfer of trophoblastic vesicles to cyclic cows and ewes. J. Reprod. Fertil. 24: 533-540 (1984).
20.
Heyman, Y. Factors affecting the survival of whole and half-embryos cattle. Theriogenology 23: 63-75 (1985).
21.
Lambeth, V.A., Looney, C.R., Voelkel, S.A., Jackson, D., Hill, K. and Godke, R. Microsurgery on bovine embryos at me morula stage to produce monozygotic twin calves. Theriogenology 3: 85-95 (1983).
transferred
in