Production of sexed lambs after biopsy of ovine

blastocyts produced in vitro

Abstract Embryos were generated by in vitro fertilization of in vitro-matured oocytes, cultured

Resume Production d'agneaux de sexe determine apres biopsie de blastocytes ovins pro-
duits in vitro. Des embryons ont ete produits par fertilisation in vitro a la suite d'une maturation
in vitro d'oocytes. Ceux-ci ont ete cultives jusqu'au stage de blastocytes et le sexe a ete determine
par amplification en chalne par polymerase (ACP) d'echantillons obtenus par biopsie. Ils ont ete ensuite
transferes dans des porteuses synchronisees. Un diagnostic de gestation a ete pose chez 3 des 5 bre-
bis (60 %) et 4 agneaux de sexe pre-determine en sont nes.
(Traduit par docteur Andre Blouin)
Can Vet J 2000;41:398-400

Sexing embryos before transfer is of considerable eco- assess the possibility of producing lambs of predeter-
nomic advantage. For commercial embryo transfer, mined sex after the biopsy, sexing, and transfer to recip-
a sexing technique must be efficient, accurate, rapid, sim- ient ewes of in-vitro-produced embryos. The accuracy
ple, and without detrimental effects to the embryo. With of this procedure was to be verified by the subsequent
the extensive use of in vitro embryo production (IVEP) birth of sexed lambs.
technology, the possibility of establishing banks of The protocol for the IVEP was as previously described
sexed embryos may be the next landmark for the embryo (9). Briefly, ovaries obtained from slaughtered ewes
transfer industry. Sexing embryos by cytological meth- were sliced to obtain cumulus oocyte complexes (COCs),
ods was the first accurate method used (1), but the cur- which were then matured in tissue culture medium
rent technique of DNA amplification by polymerase (TCM)-199 (Sigma Chemical Company, St. Louis,
chain reaction (PCR) has improved the accuracy, effi- Missouri, USA) containing 10% fetal bovine serum
ciency, and speed of embryo sex detection (2,3). Micro- (HyClone, Logan, Utah, USA) for 24 h in 5% CO2 in air.
manipulation of bovine embryos by either splitting (4) After maturation, the cumulus cells were partially
or biopsy (5), combined with PCR-based sexing pro- removed by pipetting the COCs twice in HEPES-buffered
cedures, has resulted in high rates of survival of embryos synthetic oviductal fluid (HSOF) and twice in in vitro fer-
and birth of calves of known sex (5). tilization synthetic oviductal fluid (IVF-SOF). Oocytes
Cheng et al (6) reported IVEP in sheep as early as were then transferred (in groups of 25) into 500 pL of
1985. Since then, all major reproductive technologies, IVF-SOF, supplemented with 2% metestrous sheep
from controlled breeding to cloning (7), have been serum. Frozen-thawed sperm were subjected to Percoll
developed in sheep. However, techniques for sexing treatment to obtain motile sperm. Insemination was done
in vitro-produced ovine embryos have not been refined at the rate of 0.25 X 106 spermatozoa/mL in 500-mL
to suit commercial embryo transfer programs. Com- wells containing IVF-SOF with 25 oocytes. Oocytes
mercially available kits for field sexing of bovine were co-incubated with spermatozoa for 18 h in 5%
embryos by PCR may be used successfully for sheep C02, after which cumulus cells and sperm were removed
embryos; however, only very limited or preliminary by gentle pipetting. The presumptive zygotes were
reports are available (8). washed once in HSOF and twice in HSOF lacking
The present study was designed to evaluate a simple HEPES buffer, glutamine, and glucose (CSOF) before
method of sexing ovine embryos after they have been transfer to 500-pL wells of CSOF for culture in an
biopsied by using a microsurgical blade. The aim was to atmosphere of 5% CO2, 5% 02, and 90% N2. At approx-
imately 84 h postinsemination (hpi), all embryos were
Department of Biomedical Sciences (Kochhar, King) and transferred to fresh, C02-equilibrated CSOF, supple-
Department of Population Medicine (Buckrell, Pollard), mented with 4 mM of glucose, and cultured for a period
Ontario Veterinary College, University of Guelph, Guelph, of 162 hpi, when blastocysts were harvested.
Ontario N1G 2W1. Blastocysts or hatched blastocysts were randomly
Address correspondence and reprint requests to Dr. W.A. selected at 162 hpi for biopsy. The medium used for hold-
King. ing the embryos during manipulation was phosphate-
This study was funded by Ontario Ministry of Agriculture, Food buffered saline (PBS). The embryos were placed in
and Rural Affairs and Natural Sciences and Engineering 70-,uL drops of this medium and allowed to stick to
Research Council. Dr. H.P.S. Kochhar was a recipient of a the bottom of a 35-mm dish. The biopsy blade was
Commonwealth Scholarship. positioned above the embryo, taking care to exclude the
398 Can Vet J Volume 41, May 2000
 mately 48 h after eCG injection. On day 7 postestrus, the
ovaries were visualized with a laparoscope and the
presence of one or more corpora lutea (CL) was con-
sidered indicative of ovulation. Two male embryos
were transferred to the uterus ipsilateral to the CL in each
of 3 ewes and 3 female embryos in each of 2 ewes. When
600bp- - 447bp the ewes were subsequently examined for pregnancy 40 d
post transfer by transrectal ultrasonography using a
- 272bp B-mode, 5 MHz linear-array, real-time ultrasound probe
- 173bp (Alliance Medical Instruments, New York, USA), 2 recip-
ients of male and 1 recipient of female embryos were
1 2 3 4 5 6 7 diagnosed pregnant. One ewe gave birth to a live male
lamb (weight, 8 kg) on day 151 of gestation, while 2 ewes
Figure 1. Agarose electrophoresis gel showing 3 bands in male gave birth to stillborn female twins and a male singleton,
embryos (447, 272, and 173 base pairs, respectively) and respectively, on day 152 of gestation. All lambs were of
2 bands in female embryos (272 and 173 base pairs). Lane 1: the predicted sex. Postmortem examination of the still-
100 base pair molecular weight ladder; lanes 2 and 3: ampli- born lambs revealed no visible abnormalities.
fied genomic DNA from male and female sheep, respectively; Although sheep production has increased in Canada,
lanes 4 and 7: amplified DNA from female blastocysts; lanes the consumer market relies heavily upon importation to
5 and 6: amplified DNA from male blastocysts. meet the growing demand for lamb meat and mutton. In
vitro production and transfer of ovine embryos has
inner cell mass and include the trophectoderm. Approx- been recognized as one way in which to increase pro-
imately 16-30 cells (nearly one-third of the embryo) were ductivity of sheep flocks in Ontario (9). Ovine IVEP has
removed; embryos were then transferred back into been a consistent and efficient source of embryos for
20-pL droplets of CSOF while the biopsy was prepared transfer into recipients, resulting in live births (1 1). If
for sexing. Blastocysts collapsed following biopsy, but IVEP technology were used in combination with embryo
regained normal morphology after approximately 18-20 h sexing, the producer could have a choice of lamb gender
of culture. Hatching blastocysts were able to retain prior to embryo transfer. This would facilitate the estab-
their blastocoel and morphology when only the cells lishment of elite flocks and propagation of valuable
extruding from the crack in the zona pellucida were traits for enhanced production.
biopsied. Each biopsy was washed 3 times in PBS, The method of biopsy and sex determination of ovine
supplemented with 0.1% PVP (Sigma Chemical embryos that we used is simple and accurately and effi-
Company), and placed in 1.0 pL of medium in a 0.5-mL ciently predicts the sex of preimplantation embryos
Eppendorf tube. A lysis solution containing 5.6 ,uL of before transfer. The microblade used to biopsy the embryo
H20, 2.5 ,uL of 2 mM deoxynucleic triphosphates is easy to use and has proven to be effective with bovine
(dNTPs), 1.0 pL of 1OX PCR buffer (Roche Diagnostics, embryos (2). Factors influencing the outcome of the
Laval, Quebec), 0.75 mL of 50 mM of MgCl2, and 0.1 ,IL procedure include quality of the embryo and the size
of 20 jig/pL proteinase (Proteinase K, Roche Diag- of the biopsy, which must be large enough not to com-
nostics) was added to the tube. The cells were lysed and promise sexing efficiency but small enough not to com-
the amplification of DNA sequences was accomplished promise embryo viability. Lacaze et al (12) used as
by PCR, as described previously (3). Amplification few as 2 cells for sexing but could not show a significant
products were identified by endonuclease digestion of increase in pregnancy rate following transfer of these
the PCR product by using the enzyme Sacl (Roche embryos over embryos in which > 20 cells had been
Diagnostics). Fragments of DNA were resolved on 2% removed. We used morphologically normal, grade 1
agarose (GibcoBRL, Life Technologies, Grand Island, embryos and obtained a 60% pregnancy rate (3 of 5 ewes)
New York, USA) gels and viewed under ultraviolet light. and a 33% embryo survival rate (4 of 12 embryos) after
Male embryos elicited 3 bands of 447, 272, and 173 base transfer. These results are comparable with in vivo-
pairs (bp) and female embryos exhibited 2 bands produced bovine embryos, biopsied and sexed by the
at 272 and 173 bp (Figure 1). In total, 57 embryos were same technique (3).
subjected to biopsy, culture, and sexing in 3 replicates. In conclusion, this simple method of biopsy by
Fifty-three embryos survived the biopsy procedure, as microblade and sexing by PCR can be used effectively
evidenced by continued development and normal to determine the sex of ovine embryos before transfer.
morphology after 18-20 h of in vitro culture. The biop- To our knowledge, this is the first report in North
sied cells, sexed by PCR, revealed that 30 of the America of a live birth after biopsy, sex determination
53 embryos were male and 23 were female. by PCR, and transfer of sheep embryos produced by in
The estrous cycle of 5 ewes was synchronized by vitro fertilization.
using a previously described protocol (10). Briefly, 60 mg
medroxyprogesterone pessaries (Veramix Sponges,
Pharmacia and Upjohn, Orangeville, Ontario) were Acknowledgments
placed intravaginally on day 0. On day 12, ewes were The authors wish to thank Liz St. John for the help
injected with 400 IU of equine chorionic gonadotrophin with ovaries, Len Kuehner for help with biopsies, and
(eCG) (Equinex, Pharmacia and Upjohn) and the pes- Cathy, Pam, and Bob at Ponsonby Sheep Research
saries were removed. Estrus was observed approxi- Station for maintenance of the recipients. cvJ
Can V.t J Volume 41, May 2000 399
 References 8. Loi P, Ptak G, Dattena M, Ledda S, Naitana S, Cappai P. Embryo
transfer and related technologies in sheep reproduction. Proc
1. King WA. Sexing embryos by cytological methods. Theriogenology 14th Ann Meet Eur Embryo Transfer Assoc, Venice, 1998:
1984;21:7-17. 91-104.
2. Herr CM, Reed KC. Micromanipulation of bovine embryos for sex 9. Morris LHA. Effect of male in an ovine in vitro embryo pro-
determination. Theriogenology 1991;35:45-54. duction system [DVSc dissertation]. Guelph, Ontario. University
3. Bredbacka P, Peippo J. Sex diagnosis of ovine and bovine embryos of Guelph, 1998:48-54.
by enzymatic amplification and digestion of DNA from ZFY/ZFX 10. Mylne MJA, Hunton JR, Buckrell BC. Artificial insemination in
locus. Agric Sci Finl 1992;2:233-238. sheep. In: Youngquist RS, ed. Current Therapy in Large Animal
4. Ozil JP. Production of identical twins by bisection of blasto- Theriogenology. Philadelphia: WB Saunders, 1997:585-593.
cysts in the cow. J Reprod Fertil 1983;69:463-468. 11. Ledda S, Baglioli P, Calvia P, Leoni G, Naitana S. Meiotic pro-
5. Bredbacka P, Velmala R, Peippo J, Bredbacka K. Survival of gression and developmental competence of oocytes collected
biopsied and sexed bovine demi-embryos. Theriogenology from pre-pubertal and adult ewes. J Reprod Fertil 1997;109:73-78.
1994;43: 1023-1031. 12. Lacaze S, Lesclaux J, Coupet H. The sexing of bovine embryos in
6. Cheng WTK, Moor RM, Polge C. In vitro fertilization of pig south west of France II: influence of size of biopsy on sexing effi-
and sheep oocytes matured in vivo and in vitro. Theriogenology ciency and pregnancy rates [Abstract]. Proc 12th Ann Meet Eur
1986;25: 146. Embryo Transfer Assoc 1996:158.
7. Wilmut I, Schneike AE, McWhir J, Kind AJ, Campbell KHS.
Viable offspring derived from fetal and adult mammalian cells.
Nature 1997;385:810-813.

BOOK REVIEW COMPTE RENDU DE LIVRE

Colahan PT, Mayhew IG, Merritt AM, Moore JN. of the book deals with all the major body systems, dis-
Manual of Equine Medicine & Surgery. Mosby Inc. eases that affect these systems, clinical diagnosis of
1999. 514 pp. ISBN 0-8151-1741-8. these diseases, differential diagnoses, laboratory results
that support the diagnosis, and treatments. These chap-
This textbook is designed for the equine practitioner ters are very informative, concise, and easily under-
and contains complete reference pages to its parent stood, especially the sections on the ocular, cardiovascular,
text, Equine Medicine and Surgery, 5th ed., Colahan et al. respiratory, and musculoskeletal systems. Adequate
It is a portable, up-to-date, comprehensive reference page references are made to the parent text for a more
guide, which is a must for every equine practitioner, detailed description.
either out in the field or in a clinic situation. Its logical and I found this book very interesting and well done,
systematic approach to most of the common equine diseases although my clinical experience did not always support
and their treatment protocols makes it an invaluable tool. the literature. The book covers all the common, and
The first chapter deals with the more common clini- some not so common, diseases that can impact our
cal syndromes, their differential diagnoses, and their diag- equine friends and our industry. I am glad that a refer-
nostic approaches, all in alphabetical order. This diffi- ence manual like this has finally been written; I only wish
cult undertaking is done very well, although, in some that there had been such a book when I graduated,
cases, it is a little simplistic. The basic overall concept is instead of my lugging around some huge textbooks and
good, and I am sure that this chapter alone will be of all my notes from farm to farm. I did find that the print
great benefit to us all. The chapter on practice manage- size was a little small for us "aging fellers" with failing
ment is very informative, although I am not sure that it eyesight. I realize that if the print size was larger, the
has a place in a textbook on medicine and surgery. The book would end up being bigger and, therefore, not so
next few chapters deal with patient evaluation, in terms portable. That's something for those involved in inno-
of physical examination and prepurchase examination, vative technology to figure out, "big, yet small." Perhaps
which is well thought out and well constructed. The some illustrations in various areas (joint injection tech-
section on necropsy is rather boring and a little too niques and perineural blocking techniques in the chap-
long and detailed for a me; I prefer to send my cadavers ter on the musculoskeletal system) would be in order,
to a proper facility with a pathologist who knows what especially for the new graduate who will be called upon
he or she is doing. However, it is excellent, if you like to to perform some of these techniques and may need to
do field necropsies, although the information contained brush up on the landmarks. This is true also for "we older
therein is easily accessible from other sources. Principles fellows" who have become forgetful.
of critical care; neonate evaluation; and chemical In conclusion, I learned a lot from this manual and
restraint, anesthesia and surgery are very concise, and, believe it will be a very welcome tool for the equine prac-
for the most part, informative. In a few instances, clin- titioner; it certainly complements the currently available
ical experience has taught me otherwise. To cite a cou- texts in equine medicine and surgery, and current therapy.
ple of examples, detomidine, in my hands, is useful The concise and systematic format is quickly accessible
for front and hind leg procedures. I have found flu- and supplies enough detail for most of the major problems
nixin meglumine to be very effective in horses with encountered on a daily basis out in the field.
colic for its ability to block both pain and the effects of
endotoxemia. I have never found that it was less effec- Reviewed by John F.G. Atack, BA, DVM, and Linda M.
tive than phenylbutazone in blocking the inhibitory Berthiaume, DVM, Bellevue Equine Clinic, 5360 Hwy
effects of endotoxin on gastrointestinal motility. The rest 511, RR #4, Lanark, Ontario KOG IKO.
400 Can Vet J Volume 41, May 2000