Embryo recovery and transfer for the production of transgenic
goats from Korean native strain,
Capra hircus aegagrus
C.S. Lee
a, N.Z. Fang
a,b, D.B. Koo
a, Y.S. Lee
a, G.D. Zheng
a,c, K.B. Oh
a,
W.S. Youn
a, Y.M. Han
a, S.J. Kim
a, J.H. Lim
d, S.T. Shin
d,
S.W. Jin
e, K.S. Lee
e, J.H. Ko
f, J.S. Koo
f, C.S. Park
g,
K.S. Lee
g, O.J. Yoo
f, K.K. Lee
a,*aKorea Research Institute of Bioscience and Biotechnology, Taejon, South Korea bDepartment of Animal Science, College of Agriculture, Yanbian University, Yanbian, China
cDepartment of Anatomy, College of Medicine, Yanbian University, Yanbian, China dCollege of Veterinary Medicine, Chungnam National University, Taejon, South Korea
eHanmi Pharmaceutical Company, Seoul, South Korea
fDepartment of Biological Sciences, BioMedical Research Center, KAIST, Taejon, South Korea gDepartment of Animal Science, College of Agriculture, Chungnam National University, Taejon, South Korea
Accepted 28 October 1999
Abstract
During the breeding season in Korea (September 1997 to April 1998), a goat embryo recovery and transfer program using a Korean native strain (Capra hircus aegagrus) was performed for the production of transgenic goats. Donors were synchronized with norgestomet implants and superovulated by a combined treatment with FSH and hCG. The treatment regime induced a consistent and ef®cient superovulation rate of 90% from donors with an ovulation rate of 12.10.5. 50.5% of the recovered oocytes/embryos were fertilized and most of them were at the 1-cell stage. After DNA microinjection, a total of 188 embryos were immediately transferred to naturally cycling or hormonally synchronized recipients with two or three embryos per animal. There was a tendency for the pregnancy rate of naturally cycling recipients to be higher (38.9%) than that of hormonally synchronized recipients (25.7%). When the recipients were classi®ed into a two or three embryo-received group, the embryo viability was markedly decreased from 58.3% in two received group to 35.3% in three embryo-received group, without an increase in the kidding rate. This resulted from a high occurrence of abortions or stillbirths from the multiple-pregnant recipients which had received three embryos. This indicated that the transfer of two embryos per recipient is highly recommendable for an optimal embryo survival in Korean goats. Altogether, 188 embryos were transferred to 71 recipients and two transgenic Korean goats were produced from 25 offspring.#2000 Elsevier Science B.V. All rights reserved.
Keywords:Transgenic; Korean goat; Pregnancy; Embryo transfer; Superovulation
*Corresponding author. Tel.:82-42-860-4420; fax:82-42-860-4608.
E-mail address: leekk@kribb4680.kribb.re.kr (K.K. Lee)
1. Introduction
Production of valuable proteins of pharmaceutical interest in the milk of transgenic farm animals has become an attractive alternative to microbial and animal cell bioreactors. The generation of transgenic large ruminants (cattle) is, however, prohibitively expensive because of the long gestation period, small litter size and high maintenance costs of this ruminant livestock species. For these reasons, the use of dairy goats as a production animal is a possibility. There have been several reports on the production of trans-genic goats (Denman et al., 1991; Ebert et al., 1991; Ebert and Schindler, 1993; Gootwine et al., 1997) and the feasibility of the large-scale production for the industrial application has been con®rmed (Ebert et al., 1994; Edmunds et al., 1998).
In view of the high cost of livestock transgenesis, optimizing the yield of microinjectable embryos from donors and obtaining high pregnancy rates in recipients are of major concern. In previous reports (Lee et al., 1997; Youn et al., 1997), the hormonal treatment regime and embryo recovery time was determined for the ef®cient production of microin-jectable 1-cell embryos from Korean native goats. A microinjection procedure was also established to sup-port subsequent development to the blastocyst stage in vitro. In this trial, the aim was to determine factors affecting pregnancy rate and embryo viability of DNA-injected embryos following transfer, and to examine the overall ef®ciency of the production of transgenic goats from the Korean native strain (Capra hircus aegagrus). Firstly the production of transgenic Korean goats containing a goatb-casein/human col-ony-stimulating factor (hG-CSF) fusion gene is reported on.
2. Materials and methods
2.1. Synchronization and embryo collection
This experiment was performed for 8 months, from September (1997) to April, in Korea. Nulliparous and 2 to 3-year-old pluriparous Korean native goats were used as donors and recipients. The estrous cycle of donors was synchronized by ear implantation of 6 mg norgestomet implants (Synchromate-B, Rhone
Mer-ieux, Inc., Athens, GA) for 14 days. Superovulation was induced by a combined injection of FSH and hCG. A total of 5.6 mg of FSH (Ovagen, Immuno-Chemical Products, New Zealand) was given to goats over a 4-day period as injections, twice daily, starting 2.5 4-days before implant removal and continuing to 1 day after removal. A single injection of hCG (100 IU) was given at the time of last FSH injection for the induction of ovulation. Within 24 h following implant removal, the donors demonstrated estrus and were mated to bucks of proven fertility every 6 h (two or three times). 68 h following implant removal, embryos were surgically recovered. Donors were fasted 24 h prior to surgery. A low dose of xylazine hydrochloride (Rompun, Bayer Korea, Korea; 0.12 mg xylazine hydrochloride/kg body weight) was intramuscularly injected as a pre-anesthetic agent. After a subcutaneous injection of lidocaine (Kwangmyung co., Korea; 0.1 g/animal) for local anesthesia, a mid-ventral incision was made and the reproductive tract was exteriorized. Ovaries were observed for fresh ovulation sites to serve as an estimate of the number of embryos expected. The oviducts were ¯ushed in a retrograde fashion with sterile phosphate-buffered saline as previously reported (Lee et al., 1997).
2.2. Embryo manipulation and microinjection
After ¯ushing, the number of oocytes/embryos was evaluated for each donor under a stereomicroscope. Except cleaved embryos, 1-cell oocytes/embryos were brie¯y centrifuged (10,000g, 7 min) to visualize pronuclei to con®rm fertilization and injecting of DNA. Microinjection was carried out in a TL-Hepes medium (Hagen et al., 1991), using only fertilized 1-cell embryos. The 3.7 kb BssHII/KpnI fragment of pGbc-hGCSF, in which hG-CSF (human granulocyte-colony stimulating factor) gene was fused to the promoter sequences of goat b-casein gene, was injected into one of the pronuclei of the 1-cell embryos. Following microinjection, embryos were placed into modi®ed synthetic oviduct ¯uid (m-SOF) medium (Takahashi and First, 1992) supplemen-ted with 10% FBS and cultured for 1 or 2 h in a humidi®ed (38.58C) 5% CO2incubator until transfer.
2.3. Embryo transfer
Two treatment groups of recipients were subjected to the embryo transfer experiment to establish an ef®cient production system from DNA-injected goat embryos. One group (induced group) was prepared by norgestomet ear implantation for 14 days and single injection (400 IU) of PMSG 48 h prior to implant removal. This treatment induced a synchronized ovu-lation in 78% (35/45) of the treated animals. Another group (natural group) was a naturally cycling group of recipients, in which estrus was detected by subjecting them to vasectomized bucks. Recipients, showing estrus synchrony with donors within a 12 h period, were used. Two or three embryos were surgically transferred into one oviduct ipsilateral to the ovulated ovary, using a syringe connected to a sterile poly-ethylene tube, which was inserted into the oviduct lumen via the ®mbria. The number of ovulation sites on both ovaries of recipients was recorded during embryo transfer. Pregnancy was diagnosed by trans-abdominal ultrasound scanning about 45 days follow-ing embryo transfer.
When donors and non-pregnant recipients were repeatedly used, an interval of 2 months was kept between successive surgery. Does were used up to three times as donors as long as no adhesions occurred.
2.4. Identi®cation of transgenic goats
Transgenic goats were screened by the Southern blot analysis, using genomic DNA extracted from the ear tissue of 1-week-old kids (Sambrook et al., 1989). Puri®ed genomic DNA (10mg) was digested with
HindIII, which cuts once within the transgene, trans-ferred to Nylon membrane (Boehringer Mannheim, Germany), and hybridized to32P-labeledHindIII/NaeI fragment (1.4 kb) of hG-CSF gene which spanned from exon I to exon IV of the gene.
2.5. Statistical analysis
A one-way analysis of variance and independentt -test were carried out for the comparison of ovulation rates. The pregnancy rate and embryo viability were compared with Chi-square analysis. Difference of p< 0.05 was considered signi®cant according to PC-based program (dBSTAT1.0, Information Publish-ing Group, Korea).
3. Results
3.1. Superovulation and embryo recovery
The response to superovulation and the results of embryo collection were presented at 2 months inter-vals (Table 1). A total of 174 does were superovulated and 89.7% responded with a mean of 12 ovulations per doe. Although the ovulation rate obtained in the Nov± Dec period was signi®cantly lower than that of the Sep±Oct and Jan±Feb periods, this difference could not be solely attributed to seasonality. There was individual variance in age, body weight, and operation number, which might have a signi®cant effect on the superovulation rate. Altogether 1298 oocytes/embryos were recovered with a mean recovery rate of 69.3%. Approximately half (49.5%) of the oocytes were
Table 1
Developmental state of recovered embryos from superovulated Korean goatsa Month No. of donors Ovulation
rateb
(meanSEM)
Oocytes/embryos recovered (% ovulated)
Unfertilized oocytes (% recovered)
Fertilized embryos (% recovered)
Treated Responding (% treated)
1-cell 2-cell 4-cell
Sep±Oct 37 33 (89.2) 13.1c0.9 285 (66.1) 144 (50.5) 105 (36.8) 36 (12.6)
Nov±Dec 42 38 (90.5) 9.8d1.0 257 (69.3) 126 (49.2) 99 (38.7) 31 (12.1) 1 (0.4)
Jan±Feb 37 32 (86.5) 13.9c1.2 303 (68.1) 160 (52.8) 123 (40.6) 20 (6.6) Mar±Apr 58 53 (91.4) 11.8c,d 0.8 453 (72.5) 212 (46.8) 212 (46.8) 29 (6.4)
Total 174 156 (89.7) 12.00.5 1298 (69.3) 642 (49.5) 539 (41.5) 116 (8.9) 1 (0.08)
unfertilized. Most of the fertilized embryos were at the 1-cell stage. The presence of pronuclei and occurrence of cleavage during in vitro culture separated fertilized 1-cell embryos from unfertilized oocytes. Although some 1-cell embryos did not present pronuclei due to their early development, these were classi®ed as fer-tilized 1-cell embryos, because they had developed beyond the 2- or 4-cell stage during the 1-day culture. Cumulatively, superovulation was successfully induced by a combined treatment with FSH and hCG after synchronization with norgestomet implants. The microinjectable 1-cell fertilized embryos were a signi®cant proportion of the recovered oocytes/ embryos.
3.2. Microinjection and embryo transfer
The zygote embryos clearly showing two pronuclei after a brief centrifugation were microinjected and then immediately transferred into the oviducts of the recipients.
The pregnancy and embryo survival rates of DNA-injected embryos were compared in the two recipient treatment groups, namely, the hormonally synchro-nized (induced) and naturally cycling (natural) reci-pients (Table 2). Although there was no signi®cant difference, the pregnancy rate of the natural recipients (38.9%) was higher than that of induced recipients
(25.7%). The pregnancy rate in this report was com-parable to the results of previous reports (Ebert and Schindler, 1993; Gootwine et al., 1997). However, as different numbers of embryos per recipient were transferred, the effects of different numbers of trans-ferred embryos on pregnancy and embryo survival were also examined by classifying the recipients according to the number of transferred embryos (Table 3).
Although the pregnancy rate of the group which received three embryos (37%) was somewhat higher than the 24% for the group which received two embryos, embryo viability was considerably reduced from 58.3% of the group which received two embryos to 35.3%. The decrease in embryo viability in the group which received three embryos, was evidently linked to a higher frequency of abortions and still-births in the recipients (Table 4). The occurrence of abortions and stillbirths was clearly evident in the recipients with multiple pregnancies, especially in recipients carrying three fetuses. Abortions occurred in half (2/4) at mid-pregnancy, and two fetuses were stillborn from one of the recipients. Thus only one recipient carrying three fetuses successfully produced three offspring.
The overall ef®ciency for the production of trans-genic Korean goats is summarized in Table 5. Approximately 50% of fertilized 1-cell embryos were
Table 2
Effects of different states of recipients on pregnancy and embryo survivala
No. of
aMeans followed by different superscripts differ signi®cantly. bMean number of transferred embryos per recipient.
cPercentage of embryos born to viable young from transferred embryos to the pregnant recipients.
Table 3
Effects of different number of transferred embryos on pregnancy and embryo survival No. of
No. of viable young (kidding rate)
% of embryo survivala
2 25 6 (24.0) 7 (1.2) 58.3
3 46 17 (37.0) 18 (1.1) 35.3
successfully injected. Of the injected embryos, almost all embryos (97.4%) were recovered after injection. 71% of the recovered embryos could be surgically transferred to the oviducts of 71 recipients because of a limitation in the prepared recipients. Altogether 23 recipients were pregnant and produced 25 offspring. Southern blot analysis ®nally identi®ed two transgenic goats in this study (Table 5). An example of Southern analysis, which de®ned that one of the transgenic goats contains about 10 copies of intact transgene, is shown in Fig. 1.
4. Discussion
In this report, factors affecting the pregnancy rate and embryo viability of DNA-injected embryos were evaluated after embryo transfer and the overall ef®ciency for the production of transgenic goats from the Korean native strain (Capra hircus aegagrus) examined.
For a superovulation response of the Korean goats, a single hCG injection was given to the donors together with a multiple injection of FSH (Ovagen). This treatment regime produced comparable results to other trials (Gootwine et al., 1997; Selgrath et al., 1990). In these trials, FSH alone was successfully used for superovulation (Ebert et al., 1994; Gootwine et al., 1997; Selgrath et al., 1990). However hCG together with FSH was used for the induction of an ef®cient and consistent ovulation rate from the Korean goats. Donors respond poorly to the FSH treatment alone (35% of donor goats) regardless of the dose and treatment regime (Youn et al., 1997). Some research-ers also use GnRH (Krisher et al., 1994), LH (Baril et al., 1996) or hCG (Majumdar et al., 1997) to help control the time of ovulation. Although these treat-ments increased the ovulation rate in the animals, they were not essential because FSH or PMSG treatment alone is suf®cient in inducing ovulation. This phe-nomenon may be due to the physiological difference between Korean native goats and other breeds. Since Ovagen has an extremely low LH activity, and it was
Table 4
Kidding pro®le of three-embryo-received recipients No. of fetus No. of pregnant
Recipient
Pregnant state (No. of recipients) No. of viable young Abortion Stillbirth Normal
1 9 ± ± 9 9
2 4 1 1 (1)a 2 5
3 4 2 1 (2)a 1 4
aNumber of stillborn fetuses in the recipient was parenthesized.
Table 5
Overall ef®ciency for the production of transgenic Korean goats No. of embryos
injected
No. of embryos recovered (% injected)
No. of embryos transferred (% survived)
No. of offspring (% transferred)
No. of transgenic (% of offspring) 272 265 (97.4) 188 (70.9) 25 (13.3) 2 (8)
demonstrated that highly puri®ed FSH preparations yield poor ovulation rates in cows (Donaldson and Ward, 1986), different preparations of FSH for sim-plifying the treatment regime in Korean goats need to be tested.
Although the pregnancy rate was not signi®cantly different between the naturally cycling recipients and hormonally synchronized recipients, the possibility could not be ruled out that the higher pregnancy rate in naturally cycling recipients re¯ects a better implan-tation environment for the recipients. Increased early embryonic loss and failure of implantation in super-ovulated recipients was established in several mam-mals and the phenomenon was speculated to be linked to the hostile maternal endocrine environment (Moon et al., 1990). In this experiment, a single injection of PMSG (400 IU) was given to prepare the induced recipients. Although this dose was known to be non-superovulatory (Ebert et al., 1991), the ovulation rate of induced recipients (5.29) was signi®cantly (p< 0.05) higher than that of naturally cycling reci-pients (1.78) (Table 2). Therefore, it is conceivable that the higher ovulation rate in induced recipients may detrimentally effect the pregnancy rate. It has been also reported that plasma progesterone concen-tration was correlated to the ovulation rate by mon-itoring plasma progesterone concentrations of recipients (Gootwine et al., 1997). Since, however, no relationship could be found between progesterone concentrations and pregnancy rates, a further trial is needed to clarify the relationship between ovulation rates and pregnancy rates in Korea goats.
A high frequency in the occurrence of abortions and stillbirths in recipients carrying three fetuses was demonstrated in this report. This may not be due to defective fetal development caused from in vitro manipulation, but due to the recipients' condition, as the aborted or stillborn fetuses were morphologi-cally normal. This inverse relationship between num-ber of transferred embryos and embryo viability was reported in other transgenic studies (Gootwine et al., 1997; Niemann et al., 1994). Nevertheless, it was reported that a gradual increase of kidding rate depended on the number of transferred embryos. However, in this experiment, the kidding rate was not increased in the recipients which received three embryos (Table 3), because of a high frequency of fetal loss from multiple-pregnant recipients. The mean
kidding rate of Korean native goats was reported to be 1.7 (Song, 1997), which is slightly lower than other strains used in the other transgenic studies (Gordon, 1997). This may partially explain the frequent loss of fetuses in the multiple-pregnant recipients of Korean goats. In addition, the possibility of the seasonal effect on abortion cannot be ruled out as all abortions occurred in the hot and rainy summer. In one multi-ple-pregnant goat kidding three young successfully (Table 4), pregnancy was maintained from autumn to next early spring, an optimal period for breeding in Korean goats. Thus it is speculative that multiple kidding more than three young is hardly possible at hot summer. Therefore two embryos per recipient is highly recommendable in Korean goats.The overall ef®ciency for the production of microinjectable 1-cell embryos and transgenic goats in this report can be evaluated as a reasonable result, when compared to other reports (Ebert and Schindler, 1993; Gootwine et al., 1997; Selgrath et al., 1990). However, the ef®ciency in the production of transgenic goats is one of the critical factors in applying this technology to the production of pharmaceutical protein from transgenic animals. Therefore it is necessary to improve the fertilization rate of donors and pregnancy rate of recipients in Korean native goats. From this viewpoint, recently developed nuclear transplantation technology (Baguisi et al., 1999; Cibelli et al., 1998; Schnieke et al., 1997) will give a better chance in this ®eld. This study was supported by grants from MOST (HS2400) and from MAFF-SGRP (AG520M), Repub-lic of Korea.
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