J. Anim Sci. 2008. 86:3224-3227. doi:10.2527/jas.2008-0846
© 2008 American Society of Animal Science
Technical note: Transfer of ovine embryos through a simplified mini-laparotomy technique1
Q. Y. Li,
H. Guan,
J. Hou,
X. R. An2 and
Y. F. Chen
State Key Laboratory for Agrobiotechnology and Department of Animal Physiology, College of Biological Science, China Agricultural University, Beijing, 100094, P. R. China
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Abstract
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The aim of this experiment was to examine whether a simplified mini-laparotomy technique is suitable for embryo transfer in ewes. In vitro produced blastocysts were transferred to the uterine horns of synchronous recipient ewes. Each recipient received 1 embryo by conventional laparotomy (n = 36), laparoscopy (n = 21), or by simplified mini-laparotomy (n = 33). Pregnancy rates for these 3 transfer techniques were 38.9, 47.6, 45.5%, respectively (P = 0.58). Of these techniques, the simplified mini-laparotomy was preferred because of benefits it provided in terms of savings in time and expense as well as reductions in surgical trauma and elimination of exteriorization of most of the reproductive system along with only one suture. It is likely that this technique will play a substantial role in adoption of embryo transfer by the sheep industry.
Key Words: embryo transfer • in vitro embryo production • laparoscopy • laparotomy • ovine
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INTRODUCTION
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Conventional transfer of ovine embryos has been done by mid-ventral laparotomy, and this procedure continues to be used throughout the world. However, it involves exteriorization of the reproductive tract, and this often leads to some degree of surgical trauma and formation of postoperative adhesions (Nellenschulte and Niemann, 1992
). In the 1980s, ovine embryos were first transferred by laparoscopy, a procedure that, relative to laparotomy, is quick, cheap, and less likely to cause adhesion formation (McKelvey and Robinson, 1984; Mutiga and Baker, 1984; Schiewe et al., 1984; McKelvey et al., 1985). This method has been widely accepted and developed further by other investigators (Nellenschulte and Niemann, 1992; Silva et al., 2003). A reliable, quick, and simple method for embryo transfer is needed, particularly in some developing countries where geographic location can limit ready access to technical expertise and facilities. To date, nonsurgical embryo transfer methods in ewes have not been sufficiently successful due to the specific anatomy of the ovine cervix (Halbert et al., 1990).
The objective of this investigation was to establish a simplified method of embryo transfer in ewes based on the methods of laparoscopy and laparotomy. This new approach should eliminate the disadvantages of conventional surgery (long operation time, more trauma to recipient ewes, adhesion formation) and laparoscopic transfer methods (expensive endoscope and associated facilities).
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MATERIALS AND METHODS
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All procedures were approved by the China Agricultural University Animal Care and Use Committee.
Preparation of Recipients
The study was conducted in May, 2006. All 99 recipient ewes were crossbreds (Mongolian x Merino) and were treated at the same time. Ewes were healthy and were maintained at a farm under natural day length conditions. Feed consisted of 70% alfalfa hay and 30% corn straw supplemented with 400 g of commercial concentrate per ewe daily. Animals had free access to water. Ewes were 2.1 ± 0.2 yr old, and the mean BW was 55 ± 2.4 kg. Recipient ewes were treated with controlled internal drug release devices (CIDR; 0.3 g of progesterone, Pharmacia and Upjohn Pty. Ltd., Rydalmere, New South Wales, Australia) for 12 d as well as receiving (i.m.) PMSG (400 IU per ewe; Pregnecol, Horizon Technology, North Ryde, New South Wales, Australia) at the time of CIDR withdrawal. Rams, fitted with harnesses and crayons, were introduced to the flock 24 h after PMSG administration. The occurrence of estrus was recorded every 12 h. Approximately 6.5 d later (d 0 = day of estrus), each ewe received 1 frozen-thawed embryo by 1 of 3 transfer methods (conventional laparotomy, laparoscopy, or mini-laparotomy).
Production of Embryos
Embryos (Dorset x Merino) were produced from oocyte-cumulus complexes aspirated from ovaries obtained at the slaughterhouse. The in vitro maturation, fertilization, and embryo culture procedures used were similar to those described by Walker et al. (1996). Putative zygotes were cultured for 5 d after which blastocyst stage embryos were frozen using the vitrification method of Vajta et al. (1998).
Embryo Transfer
Embryo transfer was accomplished under general anesthesia that was induced using i.v. thiopentone sodium (1 g/ewe; Jurox Pty. Ltd., Rutherford, New South Wales, Australia). Each recipient ewe was fixed in a surgery cradle held at an angle of 45 degrees to the horizontal for laparotomy and 75 degrees for both laparoscopy and mini-laparotomy. The area of the abdominal surface anterior to the udder was shaved and thoroughly disinfected. In conventional surgical transfer, 1 incision of about 5 cm in length was made on the abdominal wall approximately 8 cm anterior to the udder. One uterine horn was grasped between index and middle finger, and the tract was subsequently exteriorized to facilitate the counting of corpora lutea (CL). The uterus adjacent to the uterotubal junction was punctured using a blunt 18-gauge needle at a site free of visible blood vessels. Each embryo was suspended in 0.1 mL of medium in a siliconized glass pipette attached to a 1-mL syringe and then gently discharged into the uterine lumen. On withdrawal, the pipette was inspected to ensure that the embryo had been expelled. The uterine horn was repositioned into the abdominal cavity, and the incision was closed with 2 suture lines (including abdominal wall and skin).
For laparoscopic transfer, each anaesthetized recipient was fixed as described previously and 2 stab incisions of approximately 2 cm were made on either side of the midline some 8 to 10 cm anterior to the udder. The straight endoscope was inserted into the abdominal cavity via 1 incision, and the uterine grasping forceps was inserted via the second incision. Ovaries were located using the forceps, and the number of CL was counted. One uterine horn, ipsilateral to an ovary containing at least 1 CL, was grasped securely at the tip and exteriorized by drawing the grasping forceps outward from the abdominal cavity. The embryo was transferred to the uterine lumen in a manner similar to that described above. The uterine horn was gently returned to the abdominal cavity, and each stab incision was closed using a single suture.
For embryo transfer using the simplified mini-laparotomy method, the recipient ewe was restrained and 2 stab incisions, similar to those described for the transfer by laparoscopy, were made. The distance between the stab wounds was about 10 cm. The index finger of the left hand was inserted into the abdominal cavity via the left incision, and the index finger of the right hand was similarly inserted into the right side of the cavity. First, 1 uterine horn was located and positioned against the abdominal wall. Second, the 2 ovaries were found by moving the fingers along the uterine horn, and the number of CL was then counted by touch (Figure 1). If necessary, ovaries were also exteriorized by pushing each to the relevant incision point. Third, a loop of uterine horn was exteriorized by pushing to the nearest incision, and the embryo was transferred in the same way as described for laparoscopic transfer. After transfer, all recipients received (i.m.) 800,000 IU of penicillin and 1,000,000 IU of streptomycin (North China Pharmaceutical Factory, Shijiazhuang, P. R. China).
Determination of Pregnancy Status
Pregnancy status was determined by ultrasound scanning (50 s Tringa vet type-B ultrasonic instrument, Esaote Pie Medical, Maastricht, the Netherlands), which was conducted approximately 12 wk after the time of embryo transfer. Results were compared by chi-square analysis. Differences among groups were considered significant at P < 0.05.
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RESULTS
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Synchronization of Estrus of Treated Recipients
A total of 99 recipients were treated with CIDR and PMSG, and 90 (90.9%) were recorded as having been marked by the teaser rams within a 12-h interval after CIDR removal. Each of these marked ewes received 1 frozen/thawed blastocyst, and the pregnancy percentage was 43.3% (39/90).
Pregnancy Rate of Recipients
Pregnancy results for each of the 3 transfer groups are shown in Table 1. The percentages of ewes pregnant for the conventional laparotomy technique, the laparoscopic technique, and the simplified mini-laparotomy technique were 38.9% (14/36), 47.6% (10/21), and 45.5% (15/33), respectively (P = 0.58).
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Table 1. Pregnancy rates achieved after transfer of frozen-thawed blastocysts to recipient ewes by conventional laparotomy, laparoscopy, or mini-laparotomy
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DISCUSSION
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The purpose of this investigation was to develop a simple and inexpensive method for transfer of embryos on a large scale in the sheep industry of China. China is a country with more than 100,000,000 sheep, and substantial potential exists to improve the genetic status of the national flock through embryo transfer.
The mini-laparotomy technique proved to be as good as the other 2 techniques, and given the simplicity of the procedure, it is our preferred method of embryo transfer. It combines all the advantages of transfer by laparotomy and by laparoscopy as well as avoiding the disadvantages of these 2 techniques. Only 2 stab incisions are required, and each can be closed with a single suture. Suitability of the recipient for transfer can be determined by assessing the presence or absence of CL by touch; the ovaries can be exposed for closer inspection, although this is seldom necessary. In our hands, expensive equipment is not required, the procedure is relatively quick, and damage to the animal is reduced to a minimum due to its less time-consuming manipulation and small stab wound. Results demonstrate that this simplified transfer method is an excellent technique, and its application would be of particular value in natural grassland areas such as found in Inner Mongolia, where electricity supply can be a problem.
Transfer of frozen–thawed ovine embryos has resulted in pregnancy rates of approximately 30 to 75% (Willadsen, 1977; Tervit and Goold, 1984; Schiewe et al., 1990; Nellenschulte and Niemann, 1992). In this experiment, we achieved an overall pregnancy rate of 43.3% using frozen-thawed embryos produced using in vitro maturation and fertilization procedures. This is a satisfactory result given published data indicating that pregnancy rates following the transfer of frozen-thawed in vitro produced embryos is approximately 20 to 40% (Traldi et al., 1999; Walmsley et al., 1999; Isachenko et al., 2003).
In conclusion, transfer of sheep embryos using the simplified mini-laparotomy technique not only has the advantages of the laparoscopic technique (less operation time and minimal trauma to recipients) but also has the benefits of economy and convenience. The potential application of this technique to the sheep industry can be further exploited, including its use in intra-uterine insemination, tubal embryo transfer, and ova recovery from living ewes.
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Footnotes
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1 We are grateful to Simon Walker (South Australian Research and Development Institute) for revising the English version of the manuscript. This work was supported by a research grant from High-Tech Research & Development Program of China (2002AA206311). 
2 Corresponding author: xra{at}cau.edu.cn
Received for publication January 4, 2008.
Accepted for publication May 9, 2008.
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LITERATURE CITED
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Halbert, G. W., H. Dobson, L. S. Walton, and B. C. Buckrell. 1990. The structure of the cervical canal of the ewe. Theriogenology 33:977–992.[CrossRef][Medline]
Isachenko, V., J. L. Alabart, M. Dattena, F. Nawroth, P. Cappai, E. Isachenko, M. J. Cocero, J. Olivera, A. Roche, C. Accardo, A. Krivokharchenko, and J. Folch. 2003. New technology for vitrification and field (microscope-free) warming and transfer of small ruminant embryos. Theriogenology 59:1209–1218.[CrossRef][Medline]
McKelvey, W. A. C., and J. J. Robinson. 1984. Normal lambs born following transfer of embryos by laparoscopy. Vet. Rec. 115:230.[Medline]
McKelvey, W. A. C., J. J. Robinson, and R. P. A. Aitken. 1985. Simplified technique for the transfer of ovine embryos by laparoscopy. Vet. Rec. 117:492–494.[Abstract]
Mutiga, E. R., and A. A. Baker. 1984. Transfer of sheep embryos through a laparoscope. Vet. Rec. 114:401–402.[Medline]
Nellenschulte, E., and H. Niemann. 1992. Collection and transfer of ovine embryos by laparoscopy. Anim. Reprod. Sci. 27:293–304.[Medline]
Schiewe, M. C., M. Bush, L. S. Stuart, and D. E. Wildt. 1984. Laparoscopic embryo transfer in domestic sheep: A preliminary study. Theriogenology 22:675–682.[CrossRef]
Schiewe, M. C., W. F. Rall, L. D. Stuart, and D. E. Wildt. 1990. In situ straw dilution of ovine embryos following cryopreservation by conventional freezing or vitrification. Theriogenology 33:321 (Abstr.)[CrossRef]
Silva, J. C., L. L. Costa, R. Cidadao, and J. R. Silva. 2003. Plasma progesterone profiles, ovulation rate, donor embryo yield and recipient embryo survival in native Saloia sheep in the fall and spring breeding seasons. Theriogenology 60:521–532.[CrossRef][Medline]
Tervit, H. R., and P. G. Goold. 1984. Deep-freezing sheep embryos. Theriogenology 21:268 (Abstr.)[CrossRef]
Traldi, A. S., B. Leboeuf, Y. Cognie, N. Poulin, and P. Mermillod. 1999. Comparative results of in vitro and in vivo survival of vitrified in vitro produced goat and sheep embryos. Theriogenology 51:175 (Abstr.)[CrossRef]
Vajta, G., P. Holm, M. Kuwayama, P. J. Booth, H. Jacobsen, T. Greve, and H. Callensen. 1998. Open pulled straw (OPS) vitrification: A new way to reduce cryoinjuries of bovine ova and embryos. Mol. Reprod. Dev. 51:53–58.[CrossRef][Medline]
Walker, S. K., J. L. Hill, D. O. Kleemann, and C. D. Nancarrow. 1996. Development of ovine embryos in synthetic oviduct fluid containing amino acids at oviduct fluid concentrations. Biol. Reprod. 55:703–708.[Abstract]
Walmsley, S. E., J. W. Pollard, A. E. Randall, and L. H. A. Morris. 1999. Survival of in vitro produced ovine embryos of different developmental stages after vitrification in EFS-40. Theriogenology 51:177 (Abstr.)[CrossRef]
Willadsen, S. M. 1977. Factors affecting the survival of sheep embryos during freezing and thawing. Ciba Found. Symp. 52:175–201.[Medline]
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Abstract
INTRODUCTION
