ADVANCES IN ARTIFICIAL INSEMINATION AND EMBRYO TRANSFER
IN SHEEP AND GOATS
Brian Buckrell DVM MSc Diplomate ACT
Reproductive technologies of AI and ET for sheep and goats are becoming more
commonplace but still not widely practiced in North America. Costs are high relative to
animal values, most procedures are invasive and results are generally inconsistent at best.
But, over the last decade, new information and new ideas, much of it suggested by our
improved understanding of ovarian activity through the use of transrectal imaging, is
being tested under research and field conditions .
Artificial Insemination
Laparoscopic AI
Frozen semen AI in sheep is still most effectively delivered using the laparoscopic
approach. It is faster, less labour intensive and, depending on the cost of semen,
generally less costly pre lamb born than alternatives. In the past few years little has
changed to improve results. But information on variation in timing of ovulation between
individuals, breeds and seasons made available through the use of of transrectal imaging
helps to explain the variation in results and encourages the use of GnRH administration
and/or teaser males to enhance LH release (see B. Buckrell, advances in the control of
reproduction in sheep and goat, in these proceedings).
Transcervical AI
Transcervical AI (TAI) has received considerable attention over the past 15 years. A
system that is practical, humane and consistently successful would revolutionize artificial
breeding of sheep and improve the systems presently used in goats.
The Guelph System1, first developed and tested since 1988, showed considerable early
promise under research and farm conditions. In the following decade over 400 units were
sold and others copied and sold similar products. Its widespread use and testing did show
that most sheep (about 70%) could be inseminated transcervically in a reasonable time
but success of penetration and pregnancy results were variable and unpredictable. Few
operators were patient enough to become competent and in many cases cervical injury
resulted or was suspected. Still, in capable hands it was shown to be a low cost, non-
invasive alternative to laparoscopic AI and is a system that can be used to pass the cervix
in about 70 percent of ewes presented producing pregnancy rates in the range of 40 to
50% (or about 35% overall) when using good quality frozen semen in adequate numbers
(generally 100x106).
The Gourley scope (Dennis Gourley, Elite Genetics, Iowa) was built on the initial
promise of the Guelph system by adding low cost industrial endoscopy to view the
passage of the insemination equipment into the cervix. The Gourley system was
expensive, suffered damage from rough handling and generally did not live up to its
initial promise and is no longer available.
280
Lewis and co workers 2 have spent many years examining the effect of exogenous
hormone administration on improving the ease of passage of transcervical insemination
equipment. Oxytocin administration facilitated passage of insemination equipment but
affected pregnancy rates negatively. They recommend its use only in training programs.
Recently the team developed a new system for TAI. Similar to the Guelph System,
animals were restrained in a foot trimming cradle, a speculum, assisted by a bovine AI
rod, was used to visualize the cervical os and a 17.5 cm semi flexible stainless steel tube
with a 4 mm brass bulb on the end was introduced through the cervical rings. The tube
has a proximal attachment for retro loading the AI gun. Their research shows promise in
that penetration did occur in a small group of ewes in less than 2 minutes each, embryo
fertilization resulted and no damage to sperm could be detected from use of the system.
Much testing remains to be done. In the early years of working with the Guelph System
our group learned that operator, breed, period since last lambing, age and fecundity all
affected the success of cervical penetration and the overall pregnancy rates and need to be
tested on any emerging systems.
Cervical AI
Cervical insemination is still the goal for practical on-farm AI programs. It is widely used
in Europe but with mixed success. In an attempt to answer many of the questions that
continue to face the AI industry, Irish workers 3 undertook a large project which
examined factors affecting the success of cervical AI programs using frozen semen in
sheep. Using 5 breeds and large ewe numbers in each trial, they determined that:
• there was no difference in pregnancy rate between ewes inseminated to a natural or
synchronised oestrus using vaginal progestagen and eCG
• the affect of operator (inseminator) was significant (p<0.05)
• there was an interaction between semen type (fresh or frozen) and oestrus type for
litter size reflecting the fact that the adverse effect of frozen-thawed semen on litter
size was greater in synchronised ewes
• a highly significant effect of ewe breed was shown on pregnancy rate with Finish
landrace ewes having the highest pregnancy rate and purebred Suffolk ewes having
the lowest pregnancy rate
• there was no effect of breed on either the interval from sponge removal to the LH
surge or on the interval from LH surge to ovulation.
• there was much less variation among ewes in the timing of ovulation in the case of
Finnish landrace breed than in any of the other breeds, possibly a factor in the
breed effect on pregnancy rate
• no breed effects were detected with respect to depth of penetration of the cervix at
AI or in the amount of mucus secretion
• cervical measurements were taken by breed but the differences in anatomical
measurements were not associated with corresponding differences in pregnancy
rate
• neither anatomical differences or the state of the cervix (mucus, penetrability) at the
time of AI were implicated in the breed differences in pregnancy rate
281
• ewes were bred at varying intervals following pessary removal but it was found that
timing of AI is not a likely explanation for the effect of ewe breed on pregnancy
rate following AI
• some ewes were double inseminated 6 h apart but the double insemination did not
greatly influence pregnancy rate.
Semen Processing
The Irish researchers {Boland MP, Byrne GP, et al. 2002 #14321}showed considerable
variation among rams in conception rate following insemination of frozen-thawed semen.
They used in vitro fertilization to evaluate the potential fertility of semen frozen from the
different rams. They concluded that the IVF technique has great and practical potential as
an effective means of assessing the fertilisation capacity of frozen-thawed semen and that
it may enable the identification of individual rams whose semen is better able to survive
the freeze-thaw process and yield high pregnancy rates.
Their results suggest that an IVF assay system maybe the only effective tool presently
available short of conducting an actual AI trial to evaluate rams brought into a semen
collection center. They calculated that if rams could have been effectively screened using
IVF in advance and the best 25% identified it would have raised pregnancy rate in their
project by 15% regardless of ewe breed. Results in Guelph on the ram effect on success
of in vitro fertilization support their findings 4.
It has been clearly shown that pregnancy rates increase the further frozen semen is
introduced through the cervical rings and into the uterus 5. Because of the difficulty in
developing a practical system for TAI attention has been paid to the effect of freeze-
thawing on sperm hoping that changes to processing and handling might improve the
success from vaginal and cervical insemination. It is know that a large proportion of the
frozen-thawed cells are killed by processing, others cells have reduced motility possibly
affecting cervical transit and undergo membrane changes similar to capacitation reducing
fertilizing life. In 1999, Maxwell6 described the beneficial effects they achieved from
adding antioxidants and seminal plasma to the thawed semen. The frozen thawed semen
treated with seminal plasma acted like fresh semen resulting in pregnancy rates from
cervical AI comparable to those normally achieved with laparoscopic AI. I had expected
that their initial findings with such exciting results might be supported by more work,
however, no further progress has been reported.
The Irish group {Boland MP, Byrne GP, et al. 2002 #14321}also examined the addition
of seminal plasma to frozen semen finding that including 10 or 20% seminal plasma in
the diluents increased the viability of spermatozoa by 10 to 15 percent over the standard
diluent. However, in a second study, in which 20% seminal plasma was incorporated in
the diluent, there was no significant effect on sperm viability or structural integrity of
spermatozoa compared with the standard diluent. Furthermore there was no evidence for
any beneficial effect on fertilization rate in the IVF assay. The jury is still out on the
value of adding seminal plasma. As with semen quality, seminal plasma may be an
individual ram factor, both the source of the plasma and the ram to which it is applied.
282
Chilled fresh-extended (liquid) semen continues to be an alternative to frozen semen for
sheep and goats. Properly processed, extended and managed, the semen remains viable
with acceptable fertility for up to a week – particularly if used laparoscopically or
transcervically. The addition of antioxidants was shown to extend the life of liquid semen
for up to 14 days if deposited intrauterine in both sheep and goats7. Systems used for
shipping chilled equine and canine semen should work quite well for sheep and goats.
Recent reviews covering fresh and frozen semen processing are available for each
species. 8 9. In Ontario, a small project using ram semen extended with a commercial tris
based extender (Triladyl – Minitub®) and the semen shipped in an Equitainer® at 50C
within 48 hrs to producers previously trained using disposable equipment to deposit the
semen vaginally (400x106 total sperm per dose) in synchronized ewes. The program
resulted in pregnancy rates ranging from 20 to 42 % in 6 trials. The program was simple
and at an acceptable cost per lamb born and there was considerable opportunity to
improve success through improved producer training.
In my opinion, regional sheep and goat organizations could benefit from developing
practical liquid semen insemination programs with respectable cost: benefit.
Advances in Embryo Transfer
The same problems, shared with cattle embryo programs, continue to limit results in
sheep and goat ET programs. Response to superovulation is highly variable and
unpredictable. A large proportion of embryos fail to be fertilized. Goat donors experience
premature failure of the corpora lutea with loss of transferable quality embryos. And most
procedures still require surgical intervention for best results.
Improvements in Superovulation
It is known that the ovulatory response to commercial FSH preparations used in
superovulation programs is related to the number of follicles (2–3 mm) present in the
ovaries, and that the final number of transferable embryos is negatively affected by the
presence of large follicle(s) (>6 mm) at the start of the FSH treatment 10 . Treatment in
sheep with FSH begun soon after ovulation (Day 0), has been shown to increase follicular
recruitment, ovulation rate, embryo quality and the number of transferable embryos
recovered over treatment begun when larger follicles were present 10. Day 0 protocols in
goats also showed improved production of follicles and transferable embryos.
Management of follicular wave emergence by exogenous gonadotrophin administration
has been explored as a method to reduce the variation in response to superovulation.
French workers 11 have spent a decade testing GnRH agonist (Busereline, 40 mg/day,
Receptal –Intervet USA) or antagonist (Antarelix, 0.5 mg/day, Teverelix -Europeptides
Argenteuil, France) combined with a progestagen treatment to suppress endogenous
gonadotropins and follicular development beyond 1–2 mm followed by exogenous
gonadotropins administered over 4 days to produce waves coordinated with the timing of
superovulation programs. The pre-treatment over a 2 week period suppresses large
follicles, doubles the number of small ones, and improves the response to timed FSH by
50%. As a uniform group of follicles is recruited by the administration of FSH,
synchronization of estrus occurs between 20 and 24 h after removal of the progestagen
283
sponge. An exogenous LH surge (3 mg of pLH) is administered i.v. 32–36 h after sponge
removal, allowing synchronization of ovulations 20–28 h later. This is followed by a
timed insemination 48–50 h after the end of progestagen treatment. The program has
produced more than 10 transferable embryos and seven lambs born per treated donor and
overcomes the problem of non-responding females (<5 ovulations). In the goat the
beneficial effect of this treatment on the ovulatory response was not realized as there was
an increase in the proportion of unfertilized ova and degenerated embryos.
Fertilization failure after superovulation
In sheep and goats, as in cattle ET programs, up to one third of potential embryos are
typically recovered unfertilized and the number increases as the response to
superovulation increases. In donors with more than 30 ovulations, a drop in both the
fertilization and transferable embryo rates is routinely observed 11 and has been
associated with sperm transport in through the cervix. Poor synchrony of ovulations after
sponge withdrawal is another possible cause after artificial breedings in ET programs.
Cognie reported good synchrony in LH surges and in the time at which ewes began to
superovulate (58 ± 6 h from progestagen removal) with a median time from first to last
ovulation of 6 h. However, in superovulated goats, the spread in ovulations was greater
(63 ± 9 hr) and they found that LH surges were observed between 24 and 64 h after
sponge removal and, the median time from first to last ovulation was 12 h. .
As stated in the first paper and reviewed by Cognie 6, to synchronize the time of
ovulation when frozen-thawed ram semen is used, an injection of GnRH 30–36 h after
sponge removal is recommended. In goats, the benefit from an injection of GnRH to
synchronize ovulation and to increase the production of transferable embryos remains
controversial. In a small-scale experiment, an increase in ovulation rate and in the
number of transferable embryos was obtained in goats treated with GnRH 24 and 48 h
after progestagen removal. In another trial, authors reported an increase in ovulation rate
in GnRH-treated goats, but with a low fertilization rate. The lower fertilization rate
observed in high-responding donors following vaginal or cervical insemination may be
attributed both to a disturbance in sperm transport and suboptimal ovum quality.
However, it continues to be shown that high fertilization rates can be achieved in sheep
by intrauterine insemination performed laparoscopically 48 h after sponge removal.
Premature Luteal Failure
Premature luteal regression in superovulated goats continues to be a problem. It is
reported to vary among breeds affecting from 10% (Alpine and Saanen breeds) to 32%
(Murciana breed) of treated females. Anyone offering ET services in goats will encounter
the problem. The reason is believed to be a premature release of uterine prostaglandins
which could be induced by the persistence of large oestrogenic follicles 3–4 days after
superovulation and results in poor embryo recovery or the recovery of poor-quality
embryos 12. A number of treatments continue to be used 11. The inhibition of synthesis of
prostaglandin with flunixine meglumine between the day of ovulation and the day of
embryo recovery has been shown to recude the occurrence and an increase of the number
of transferable embryos per treated goat . The induction of new ovulations 3.5 days after
estrus with hCG prevents premature luteal regression in superovulated and the
284
replacement of the source of vaginal progestagen can be used to carry the embryos to the
day of recovery.
Transcervical Embryo Transfer
Work at Guelph, using the Guelph transcervical AI equipment 13 showed the potential in
transcervical passage during mid diestrus using simple equipment. Embryo survival was
poor (16% of embryos transferred) but cervical passage was accomplished during early
diestrus and was shown to be easier the second of two attempts only minutes apart. Lewis
and coworkers 14 showed that the administration of estradiol the day before and oxytoxin
twenty minutes in advance allowed easier passage of instrumentation and did not
negatively affect luteal function or embryo development as compared to routine
laparoscopic procedures. This could prove to be very useful information and should
encourage more work on transcervical transfer of embryos in sheep and could improve
successes in goats.
Guelph Embryo Program Results
An industry-supported sheep improvement program at the University of Guelph (Ontario
Lamb Improvement Breeding Strategy) produced results in Table 1. The table
summarizes three years of embryo production, continuous throughout each year, using a
mix of maternal and sire breeds conducted in an attempt to increase the numbers of
offspring from industry-selected donors, many of which were aged or in poor health. The
program used traditional AI and ET, as well as juvenile and mature oocytes aspiration
with in vitro embryo production.
The in vivo embryo program used accepted protocols for superovulation and procedures
of surgical recovery and laparoscopic transfers. Table 1 compares the results from the
University program, using research facilities and staff, with results from a commercial
service (Small Ruminant Genetics, Georgetown, Ontario) where producers managed the
animals and administration of superovulation protocols etc.
Results are in line with what others routinely report. Of interest was the rejection rate of
recipients based on lack of a CL indicating failure of response to standard stimulation
protocol (all Rideau Arcott maternal breed, n=360). The rejection had a seasonal pattern
that was consistent over the three years of the program. From September through
February (traditional breeding season) the rate of rejection ranged, by month, from none
to 5%. From March through July, the rate ranged from a low of 11 % to a high of 27% in
June for all three years. Interesting, there was no difference between the seasons in the
pregnancy rates or embryo survival from recipients selected. The high numbers of
rejected recipients during the out-of-season period increased the costs of the program
during that period. Practitioners need to account for the increased numbers of recipients
required and the added costs to clients programs during the out of season period.
285
Table 1: Results of in vivo embryo transfer comparing research program with
commercial service
Commercial ET Service University Program
Embryo recovery procedures 132 330
CL count 12.2 11
Embryos recovered 9.3 8
Recovery rate (% of CLs) 77% 73%
Unfertilized oocytes 28% 32%
Donors failing to respond 4% 8%
Recipients rejected 12% 18%
Embryos/Transfer 2.2 2.9
Pregnancies established 76% 67%
Lambs born per flush 3.6 2.8
Embryo Survival 63% 52%
Males 52% 54%
Embryo Cryopreservation
Few advancements in the efficiency of cryopreservation of sheep and goat embryos have
been reported. Typical programs today employ programmable freezers using ethylene
glycol instead of glycerol and slow freezing rates. Martinez and Matkovic 15 compared
the effect of glycerol and ethylene glycol for sheep embryos. They evaluated the effect of
both cryoprotectant (glycerol vs ethylene glycol) and the effect of extending the thawing
process for ethylene glycol frozen embryos. They confirmed previous studies that showed
that ethylene glycol is superior to glycerol for sheep embryos and that this advantage was
very significant for embryos at the compact morula stage. They also found there was no
difference in the pregnancy rates (48%; 12/25) between thawing processes that removed
ethylene glycol in 10 minutes and a longer protocol that required 30 minutes.
Sheep and goat embryos are able to survive vitrification procedures and considerable
research has been reported using this system and with Cognie 11 suggesting that it may
provide an economical alternative to the current freezing. Vitrification does not require
any special equipment and, therefore, may be very well adapted to routine field use.
When fresh or vitrified sheep embryos recovered 7 days after estrus were transferred to
synchronized recipients (two embryos/recipient), the pregnancy rates (72% in both cases)
and the numbers of lambs born per embryo transferred were not different (60 and 50%),
respectively 16. These results with vitrified embryos were similar to those reported and
routinely achieved by more traditional slow freezing. However, using the same
vitrification and thawing methods with goat embryos, 11 have produced lower kidding
and embryonic survival rates after vitrification (48 and 39%, respectively) than after
conventional slow freezing (69 and 55%, respectively). Considerably more works needs
to be done testing vitrification under field conditions.
A good review paper17 covers recent developments in vitrification and compares the
success to traditional commercial freezing methods. In spite of the many systems used to
vitrify there is a growing improvement in results and in the simplification of
286
methodologies. And while results are improving, particularly for fresh embryos the
impracticality of handling embryos individually (generally about 3-4 minutes per straw)
vitrification may never be practical for practitioners where embryos are recovered in
large numbers from field ET programs.
A small trial in the University of Guelph program produced the results in Table 2.
Comparing the vitrification of in vivo produced embryos with those produced from
juvenile oocytes aspirated and produced in vitro.
Table 2. Results of a trail comparing survival of vitrified in vivo and in vitro produced
embryos.
in vitro produced embryos in vivo recovered embryos
Embryos 105 33
Recipients 35 16
Pregnancies (lambing) 14 (35%) 8 (50%)
Lambs Born 15 (14%) 9 (27%)
In vitro Embryo Production
In vitro embryo production continues to hold promise, possibly most particularly in small
ruminants, where invasive procedures are routinely used for embryo programs. The mass
production of low-cost embryos from abattoir ovaries, production from juvenile donors
and the production of offspring from diseased or aged animals at necropsy warrant ET
practitioners and providers of bovine IVF services to keep abreast of developments in
these species.
In general terms, based on survival as fresh or frozen transfers, it is still safe to say that in
vitro produced (IVP) embryos are inferior to those produced in vivo.
Oocytes are recovered from ovaries of slaughterhouse ewes/ does or aspirated from the
ovaries of anesthetised mature or juvenile donor females. The method of producing IVP
of embryos involves three steps: maturation of oocytes ( IVM), fertilization of the
matured secondary oocytes with frozen-thawed semen (IVF) and, culture of the putative
embryos (zygotes) for up to 1 week until formation of blastocysts that can be transferred
to recipients or cryopreserved for future use(IVC).
Cognie’s paper reviews the state of oocyte recovery 11. Abattoir-derived ovaries provide
a cheap and abundant source of oocytes, and collection by aspiration provides 1.5–2
cumulus–oocyte complexes of acceptable quality per adult sheep or goat ovary. Oocyte
recovery from live animals is accomplished by laparotomy or the laparoscopy-guided
“ovum pick up” (LOPU) technique. When LOPU is performed 24 h after the end of a
gonadotropin treatment, a mean of six oocytes per ewe are selected for IVF, resulting in
about 1.1 blastocysts. Two to three good-quality blastocysts, which resulted in about 1.5
lambs being born per ewe were reported but with a high degree of variation between
287
donors. Oocyte retrieval after repeated LOPU in unstimulated sheep and goats also
provided a high number of oocytes (4–6 per female/session). Cognie suggests that if the
quality of these oocytes for IVP is confirmed, this method could provide a way to
produce offspring from genetically valuable females without using hormones.
Baldassarre 18 and his group in Montreal are successfully and consistently using
laparoscopic ovum pick repeatedly on donor goats in their transgenic program.
Remarkable progress in producing embryos from 5- to 9-week-old lambs has been
reported after recovery of oocytes after gonadotropin treatment 19. However, only 19% of
cleaved prepubertal oocytes developed to the blastocyst stage compared to 65% for their
adult counterparts. Evidence suggests that prepubertal matured oocytes do not possess the
developmental potential of their adult counterparts. The beneficial effect of a single
treatment with estrogen and progesterone prior to gonadotropin on the developmental
capacity of lamb oocytes has been demonstrated. This could suggest that the acquisition
of oocyte competence for embryogenesis in the prepubertal female is progressive with
age and could be influenced by the hormonal environment of oocytes before IVM. The
fact that the number of oocytes recovered from the prepubertal female declines
significantly with increasing age of the donors seems to be balanced by the fact that their
development ability is improved when donors are older. Investigations into the control of
follicular growth in the juvenile female should allow the production of offspring from
juveniles to reduce the generation interval and increase the rate of genetic progress in
breeding schemes 16. Juvenile donors will have an important place in livestock-
improvement programs, especially when their embryos or oocytes can be efficiently
frozen.
The survival rate of fresh IVP goat embryos is similar to the survival rate obtained with
IVP sheep embryos which significantly lower than their in vivo counterparts (47%
versus 71%) 16.
Most programs recovering oocytes from mature or juvenile live donors are aspirating
targeted follicles on the ovarian surface. In the sheep IVP program at Guelph oocyte
recovery was done using a laparoscope-guided procedure, where the ovary was identified
using the laparoscope and elevated and exposed through a small midline incision (mini-
lap) 20-25. Aspiration was done systematically just under the surface of the exposed ovary
– not by targeting individual follicles. Average oocyte recovery from mature,
unstimulated donors was 8.5, from mature stimulated 9 and from stimulated juveniles
(between 8 and 12 weeks of age) 18. From mature donors, for every 100 good-quality
oocytes recovered 35 blastocysts were produced which yielded an average of 16 lambs
from fresh transfers or 13 from frozen-thawed transfers using slow freezing. An
interesting observation was that large numbers of oocytes could be recovered from
ovaries of diseased animals at necropsy. In one case of copper toxicity 5 donors yielded
an average of 27 oocytes producing 11 blastocysts and 3 lambs each. In a trial using 30
juvenile donors for repeat collections, up to three times at monthly intervals beginning at
8 weeks, it was shown that repeated aspirations vial mini-laparotomy had no affect on
future fertility or caused ovarian pathology 19.
288
References
1. Halbert GW, Dobson H, Walton JS, Sharpe P, Buckrell BC. Field evaluation of a
technique for transcervical intrauterine insemination of ewes.
Theriogenology. Vol. 33. 1990: 1231-44.
2. Meghan C, Wulster-Radcliffe, Lewis GS. Development of a new transcervical
artificial insemination method for sheep:effects of a new transcervical
artificial insemination catheter and traversing the cervix on semen quality
and fertility . Theriogenology 2002; 58:1361-71.
3. Boland MP, Byrne GP, Duffy P et al. AI for sheep using frozen-thawed semen.
Teagasc Project Report 2002; www.teagasc.ie/research/reports/sheep/4047.
4. Morrris LHA, Johnson WH, Leibo SP, Buckrell BC. Relationship between the
characteristics of frozen-thawed ram spermatozoa and in vitro embryo
production . Reprod Fert Dev 2001; 13:193-201.
5. Maxwell WMC, Hewitt LJ. A comparison of vaginal, cervical and intrauterine
insemination of sheep. Journal of Agricultural Science 1986; 106:191-3.
6. Maxwell WMC, Evans G, Mortimer ST, Gillan L, Gellatly ES, McPhie CA.
Normal fertility in ewes after cervical insemination wiht frozen-thawed
spermatozoa supplemented with seminal plasma. Reproducition Fertility and
Development 1999; 11:123-6.
7. Maxwell W.M.C., Stojanov T. Liquid storage of semen in the absence or presence
of some antioxidants. Reproduction, Fertility and Development 1996;
8:1013-20.
8. Leboeuf B, Restall B, Salamon S. Production and storage of goat semen for
artificial insemination . Animal Reproduction Science 2000; 62:113-41.
9. Salamon S, Maxwell WMC. Storage of ram semen. Animal Reproduction Science
2000; 62:77-111.
10. Rubianes E., Kmaid S., De Castro T., Carbajal B., Benquet N., Ponczak A.
Superovulation response to FSH treatments initiated at wave 1 embergence
or 4 days after CIDR insertion in ewes. Theriogenology 1997; 47:177.
11. Cognie Y, Baril G, Poulin N, Mermillod P. Current status of embryo technologies
in sheep and goat. Theriogenology 2003; 59:171-88.
12. Battye KM, Fairclough RJ, Cameron AWN, Trounson AO. Evidence for
prostaglandin involvement in early luteal regression of the superovulated
nanny goat (Capra hircus). Journal of Reproduction and Fertility. Vol. 84.
1988: 425-30.
13. Buckrell B.C., Buschbeck C., Gartley C.J., et al. A breeding trial using transcervical
289
technique for artificial insemination in sheep. 12th International congress
on animal reproduction. 3. The Hague, Netherlands: University of Utrecht,
Netherlands, 1992:1531-3.
14. Wulster-Radcliff BC, Costine BA, Lewis GS. Estradiol-oxytocin induced cervical
dilation in sheep:Applications to transcervical embryo transfer. J Animal Sci
1999; 77(2587-2593).
15. Martinez AG, Markovic M, Pessi HD, Baldassarre H. Cryopreservation of ovine
embryos:slow freezing and vitrification. Theriogenology 1996; 45(1):161.
16. Baril G, Traldi AL, Cognie Y, Leboeuf B, Beckers JF, Mermillod P. Sucessful
direct transfer of vitrified sheep embryos. Theriogenology 2001; (56):299-
305.
17. Vajta G. Vitrification of oocytes and embryos of domestic animals . Animal
Reproduction Science 2002; 60-61:357-64.
18. Baldassarre H, Wang B, Kafidi N, Keefer C, Lazaris A, Karatzas C.N. Advances in
the production and propagation of transgenic goats using laparoscopic ovum
pick-up and in vitro embryo production technologies. Theriogenology
2002; 57:275-84.
19. Walmsley S E. Development of a laparoscopic Assisted Method for oocyte
collection from mature and prepubertal ewes. DVSc Thesis, University of
Guelph 1999.
20. Walmsley SE , Pollard JW, Plante C, Songassen N, Buckrell BC. The effects of
age and repeated aspiration on the number of oocytes collected from
prepubertal Arcott lambs. Techniques for Gamete Manipulation and
Storage. 1996.
21. Walmsley SE, Buckrell BC, Songsasen N et al. A laparoscope-assisted method for
oocyte collection from mature and prepubertal ewes . Submitted to
Theriogenology 2003.
22. Walmsley SE, Buckrell BC, Buschbeck C, Rumph N, and Pollard JW. Rate of
abnormalities in lambs from in vitro produced embryos transferred on day 2
compared with day 6 post fertilization. Accepted for Publication
Theriogenology 2003.
23. Walmsley SE, Pollard JW, Buschbeck C, Rumph N, and Buckrell BC. Pregnancy
rates for IVP ovine embryos transferred on day 2 versus day 6. Proceedings
of the Satellite Symposium to the International Congress on Animal
Reproduction: Reproduction in Small Ruminants, Sandnes, Norway 2000;
69.
24. Buckrell BC, Walmsley SE, Randall AE, Plante C, and Pollard JW . Comparison of
in vivo and in vitro methods for embryo production in peripubertal ewes.
Theriogenology. 1999; 51:314.
290
