Biotechnologies of reproduction applied to dairy cattle

production: Embryo transfer and IVF

D. Bousquet1, E. B. Burnside2, and B. J. Van Doormaal3

1LAlliance Boviteq inc., Saint-Hyacinthe, Qubec, Canada; 2The Alliance Semex, Guelph, Ontario, Canada;
3Canadian Dairy Network, Guelph, Ontario, Canada. Received 17 February 2003, accepted 30 July 2003.
Can. J. Anim. Sci. Downloaded from www.nrcresearchpress.com by 120.188.86.132 on 11/04/17

Bousquet, D., Burnside, E. B. and Van Doormaal, B. J. 2003. Biotechnologies of reproduction applied to dairy cattle produc-
tion: Embryo transfer and IVF. Can. J. Anim. Sci. 83: 403407. The objective of this paper is to review the utilization and out-
come of reproductive biotechnologies in dairy cattle. Embryo transfer and IVF have their respective limits that influence their
impact on a genetic improvement program. Embryo transfer is efficient and profitable in an artificial insemination breeding and
selection program and IVF has great potential. Development of markers highly correlated with various economic traits will pro-
gressively be added to the present schemes to make them even more powerful.

Key words: Reproductive technologies, embryo transfer, IVF, dairy cows, genetic

Bousquet, D., Burnside, E. B. et Van Doormaal, B. J. 2003. Biotechnologies de la reproduction appliques la production des
bovins laitiers : transfert dembryon et FIV. Can. J. Anim. Sci. 83: 403407. Le but de cet article est de prsenter les techniques
et rsultats de biotechnologies relies la reproduction. Linvestissement fait dans ces technologies doit tre proportionnel au
retour. En regard avec la situation actuelle, sur le plan technique, le transfert embryonnaire (TE) et la fcondation in vitro (FIV)
ont leurs limites respectives qui influencent limpact quils peuvent avoir sur les programmes damlioration gntique. Le trans-
fert embryonnaire est efficace et profitable au niveau de la reproduction par insmination artificielle et des programmes de slec-
For personal use only.

tion. De plus, les FIV ont un trs bon potentiel. Le dveloppement de marqueurs correspondants aux diffrents traits conomiques
sera progressivement ajout aux plans actuels pour les rendre encore plus puissants.

Mots cls: Technologies de la reproduction, transfert dembryon, FIV, vaches laitires, gntique
The reproductive biotechnologies discussed in the present
paper are related to the embryo. They have been developed
in parallel with our acquired knowledge regarding embryo
development. Although not as widely used as artificial
insemination, they have contributed substantially to the
improvement of the dairy cattle industry through the pro-
duction and reproduction of seedstock animals. Substantial
commercial application of embryo transfer started in the
early 1980s with the development of superovulatory regi-
mens and efficient cryopreservation techniques. More
recently (1990s), a new generation of embryo technology
has developed, based on in vitro embryo production with the
use of oocytes collected from live donors. The objectives of
the present paper are to discuss the benefits that those
embryo technologies have already given to the dairy indus-
try and their potential for the future.
EMBRYO TRANSFER (ET) AND IVF
AROUND THE WORLD
The International Embryo Transfer Society (IETS) has
retrieved data on ET over the past 10 yr and more recently data
Presented at the session Amliorer les productions animales
grce aux technologies reproductives. Improving Animal
Production with Reproductive Physiology during the
CSAS/ADSA/ASAS 2002 Joint Meeting, Qubec City, QC,
2025 July 2002.
1To whom correspondence should be addressed (daniel. bous-
quet@umontreal.ca).
403
on IVF activities. In 2000, throughout the world approximate-
ly 530 000 bovine in vivo derived embryos were transferred
(Thibier 2001). Since the early 1990s, annual growth in the
number of ET is a little more than 1%. In 2000, >110 000 cows
were flushed, with an average of 5.9 transferable embryos per
collection. Approximately 53% of the embryos were trans-
ferred frozen and the others fresh. In Canada and United States,
a total of 224 451 bovine in vivo derived embryos were trans-
ferred, with 74 and 37%, respectively, produced from dairy
cows. Although the utilization of ET is relatively low compared
to the use of AI, more than 95% of dairy bulls in AI centres are
produced through ET (The Semex Alliance, personal commu-
nication), clearly demonstrating the importance of this technol-
ogy to the dairy industry. With the advent of ET, every
high-ranking bull dam can contribute one or more sons for
progeny testing. This was not the case prior to availability of
ET technology.
International data retrieval of the utilization of IVF has
started only in recent years. In North America, 1741 IVF
embryos were transferred in 2000, corresponding to 1% of
the global total. The popularity of these embryos is much
higher in Europe, Asia and South America, where abbatoir-
Abbreviations: ET, embryo transfer; IVF, in vitro fertil-
ization; MAS, Marker Assisted Selection; MOET, multiple
ovulation and ET; OPU, Ovum Pick Up; PCR, polymerase
chain reaction
 404 CANADIAN JOURNAL OF ANIMAL SCIENCE

derived oocytes are predominantly used for IVF, as opposed Table 1. Embryo sexing results (using PCR) on biopsies collected from
to North America where antemortem oocyte retrieval is in vivo and in vitro produced embryosz
done on selected cows (Thibier 2001). In vivo In vitro
Number 879 3032
EMBRYO TRANSFER AND Males 429 (48.8%) 1696 (55.9%)
SELECTION INTENSITY Females 407 (46.3%) 1168 (38.5%)
New opportunities for selection programs accompanied the Unknown 43 (4.9%) 168 (5.6%)
introduction of ET technology. Although approximately Transferred fresh 326 1004
20% of the females do not respond to the superovulatory Pregnancies (day 30) 186 (57%) 532 (52.9%)
Pregnancies (day 60) 179/324 (55.2%) 487/994 (48.9%)
treatment and the number of transferable embryos varies zLAlliance
among donors and collections, this technology has helped to Boviteq Inc., September 1999 December 2001.
increase prolificacy of selected donor cows. From one calf
Can. J. Anim. Sci. Downloaded from www.nrcresearchpress.com by 120.188.86.132 on 11/04/17

per year, the average number has increased to >10 with ET. Table 2. Summary of embryo transfer activity in Canada for the year
This technology decreases generation intervals; at puberty, 2000z
the heifer can produce three calves. In contrast, with AI only Number
this productivity is usually obtained only when a cow reach- Donors Total 12 887
es 6 yr of age. Dairy 9 540 (74%)
Embryo sexing is conducted on biopsies collected from Embryos collected 83 549 (6.48/donor)
Embryos frozen 56 728 (4.4/donor)
embryos before transfer or freezing. The method used is Pregnancy rate
based on polymerase chain reaction (PCR) technology and Fresh embryos 14 652/23 579 (62.1%)
is readily applied under field conditions (Herr and Reed Frozen embryosy 12 715/22 072 (57.6%)
1991). Determination of the sex of the embryo can be effec- zReuben Mapletoft for CETA, CETAs Newsletter, 2001.
tive in >95% of the biopsies (Table 1). The pregnancy rate yEmbryos frozen in ethylene glycol.

obtained with biopsied embryos transferred fresh varies

according to the quality of the embryo and the use of heifers Table 3. Percentage of registered Holstein calves in Canada resulting
or cows as recipients [Table 1 and Darrow (2002)]. This from embryo transfer technology between 1985 and 2000z
For personal use only.

technique is effective in herds where replacement heifers
need to be produced within a short interval. Other groups
reported 90% success rate in embryo sexing, with a preg-
nancy rate ranging from 58 to 71% after transfer of fresh
biopsied embryos (Shea 1999).
Embryo freezing has had a substantial impact on the indus-
try. Using glycerol as cryoprotectant required removal of the
embryo from the straw and washing before transfer
(Miyamoto and Ishibashi 1978). However, the introduction of
more permeable cryoprotectants (e.g., ethylene glycol and
propylene glycol) eliminated the need for pretransfer embryo
washing, enabling direct transfer to recipients after thawing
(Voelkel and Hu 1992). The implementation of direct transfer
has greatly increased the utilization of ET. Data collected by
the Canadian Embryo Transfer Association (CETA) from its
members on the pregnancy rate obtained with embryos trans-
ferred directly to recipients in 2000 are shown in Table 2.
The main reason for using ET is to produce several prog-
eny from superior cows; this enhances the quality of replace-
ment heifers and bulls and thus intensifies genetic selection
and hastens genetic gain. Using ET, genetic progress for milk
production increased 20 to 30% (Nicholas and Smith 1983;
Colleau 1985; Meuwissen 1990; Nicholas 1996). In Canada,
the number of registered calves issued from ET increased
from 2.5 to 4% between 1985 and 2002 (Table 3).
Data on a total of 3172 donor cows for which at least one
ET recovery was done in 2001 were retrieved from Holstein
Canada and analysed to determine current trends in donor
cow selection (Van Doormaal 2002). Analysed from various
perspectives, all results indicated primary emphasis on over-
all type, both genetically and phenotypically. In terms of rel-
ative selection intensity at the genetic level, the emphasis on
conformation was approximately 2.5 times that placed on
Year Proportion of registrations (%)
1985 2.5
1990 3.4
1995 3.4
2000 4.0
2001 3.9
2002 4.1
zHolstein Canada, 2003.
yield traits (Fig. 1). It is interesting to note that the empha-
sis placed on this criterion has not changed over the past
10 yr. In 1991, 75.2% of donor cows were classified as
Excellent or Very Good and in 2001, this percentage
was 79.4%. On the other hand, fewer donor cows classified
as Good or lower were selected in 2001 (1.6%) compared
to 1991 (2.4%) (Van Doormaal 1991, 2002). Of the classi-
fied donor cows for 2001, 15.5% were Excellent while
another 63.9% were Very Good. These percentages are
very much higher than the proportions in the general
Canadian Holstein population (0.8 and 16.0%, respectively).
As a group, however, the average production perfor-
mance based on all donor cows for their most recent lacta-
tion is higher than breed average (Van Doormaal 2002),
indicating that on the whole the donors are above average
for production as well as for type, although some lower-pro-
ducing cows are being flushed (Table 4). Therefore, ET
technology is not being utilized to its full potential to
enhance genetic improvement.
THE APPLICATION OF EMBRYO TRANSFER IN
NUCLEUS PROGRAMS
The 1980s and early 1990s were characterized by extensive
research and development activity and some industrial
 BOUSQUET EMBRYO TRANSFER AND IVF 405
Can. J. Anim. Sci. Downloaded from www.nrcresearchpress.com by 120.188.86.132 on 11/04/17

Fig. 1. Relative genetic selection intensity for Holstein donor cows in 2001 compared to the active cow population (from Van Doormaal 2002).

activity in so-called Multiple Ovulation and ET (MOET) Table 4. Production data of Canadian Holstein cows selected as
nucleus programs. Early simulations (Nicholas and Smith donors in 2001 compared to the whole population in 2000z
1983) suggested rapid rates of improvement in closed nuclei Breed average in 2000 Donors in 2001
that would serve as a source of full-brother teams of sires
Milk Fat Protein Milk Fat Protein
and of AI proven sires. Later theoretical research (Leitch et
For personal use only.

Average
al. 1995) revealed inbreeding rates were accelerated in three 305-d (kg) 9 350 343 300 11 315 431 365
closed nuclei with reduced genetic variance and a slower zAdapted from Van Doormaal (2002).
rate of improvement. Attempts at MOET full-sib selection
(Lohuis et al. 1990, 1993) were not always in agreement
with theory, due to failure to get large enough full-sib fam- Table 5. In vitro embryo production results with oocytes collected
ilies in a short interval, thus reducing selection accuracy and from mature cows or prepubertal 6-mo-old heifersz
genetic progress. Industrial nucleus dairy breeding schemes Number (%) Mature donor cows Prepubertal heifers
have been attempted with varying results, from the early Donors 76 32
success of the Holland Genetics Scheme to closure of the Collection 316 32
Genus Nucleus Scheme, and abandonment of a major Oocytes 2 811 336
Cleaved 2 231 (81.9%) 256 (76%)
Nucleus Scheme proposed by Alta Genetics (due to finan- Embryos 1 290 (47.3%) 66 (20%)
cial challenges). Transfers (pregnancies)
Fresh 125 (52%)
IN VITRO EMBRYO PRODUCTION (IVF) zLAlliance Boviteq inc., September 1999 January 2002.
The production of embryos using IVF presents two advan-
tages over the conventional approach. First, it prolongs Woolliams (1989) clearly demonstrated the importance of
reproductive life of cows that do not respond to superovula- changing the bull at each embryo production in the conven-
tion treatment or produce only unfertilized ova. It is, there- tional method.
fore, an integral tool to keep selected cows within a breeding Second, IVF can be applied during different stages of
scheme part of a selection program. It was demonstrated that reproduction, including the prepubertal period and during
within 50 d, the number of pregnancies can be four times the first 3 mo of pregnancy. With the utilization of prepu-
higher than by using the conventional approach (Bousquet bertal heifers, it is possible to decrease the age of the donor
et al. 1999), thereby providing a means of increasing the female use, thereby contributing to a decreased generation
pressure of selection on bull-dams. Using the IVF technolo- interval. Using ultrasound-guided transvaginal collections,
gy appropriately, Leitch et al. (1995) found that the annual oocytes for IVF can be collected in heifers at 6 mo of age.
genetic progress was 10 to 30% superior to conventional In a study performed at LAlliance Boviteq on 32, 6-mo-old
ET, chiefly because IVF offers greater flexibility in sires for Holstein heifer calves, an average of two transferable
mating, thus reducing inbreeding. The implementation of embryos were produced per session (Table 5). The reduced
IVF technology allows the use of several bulls with one yield of transferable embryos compared to that obtained
donor, since the control of fertilisation is made in vitro. This with mature cows can be explained by the competency of
can be realised in one session of fertilisation as opposed to collected oocytes. Galli et al (2001) also found decreased
conventional ET, which is limited to one bull per session. developmental capacity of oocytes collected from 6-mo-old
 406 CANADIAN JOURNAL OF ANIMAL SCIENCE
Table 6. Estimated average number of calves produced per donor cow
per year after oocyte collection and IVF or using the conventional
methodz
IVM-IVF-IVC
Mature Prebubertal Conventional
donor cowsy heifers Donor cows
Frequency of collections 4/60 d 4/60 d 1/60 d
Collections/year 24 24 6 on them to keep only the ones that carry the desired genes.
Number of transferable 96 48 24
embryos/year
Pregnancy rate 53% 53% 60%
Number of calves 50 25 14
zAdapted from Bousquet et al.
Can. J. Anim. Sci. Downloaded from www.nrcresearchpress.com by 120.188.86.132 on 11/04/17
(1999).
yMature donor infertile cows.
calves compared to mature cows. In their study, 15 prepu-
bertal calves were used in 82 oocyte collection sessions. On
average, one transferable embryo per session was obtained.
Even if the developmental capacity of the oocytes collected
from calves is reduced compared to the ones from mature
donor cows (average of four transferable embryos), the
advantage still exists (Revel et al. 1995; Khatir et al. 1998).
New hormonal treatments and preparation of oocyte donor
animals are promising approaches to increase oocyte com-
petency. In a recent study, up to 80% of oocytes collected
For personal use only.
from 14- to 16-mo-old heifers produced blastocysts after
IVF (Blondin et al. 2002).
Data from LAlliance Boviteq and elsewhere give the
estimated potential of this technology compared to the con-
ventional approach (Table 6). Using Ovum Pick Up (OPU)
four times each 60-d period over 1 yr, 3.5 more calves will
be produced in the case of mature cows and two more calves
in prepubertal heifers. However, one might conclude that
OPU should not be performed on prepubertal calves; since
the progeny will be born before the performance of the
donor is known, the accuracy of selection could be reduced.
However, if a 6-mo-old-old heifer produces IVF embryos at
that age, her sons will reach puberty at the stage when she
has completed the first half of her 2-yr-old lactation.
The use of Marker Assisted Selection (MAS) has the
potential (if the markers are highly correlated with the
desired phenotype) to enhance the power of the present-day
breeding strategy (Georges 1998). Many genetic markers
linked with QTL (quantitative trait loci) affecting traits of
economic importance in livestock, including milk produc-
tion, conformation and health have been identified and
mapped during the past decade (Ashwell et al. 1998a, b;
Van Tassell et al. 2000). Aggrey et al. (1999) found that one
polymorphism of the bovine growth hormone receptor
(GHR) gene was associated with milk-related traits in
Holsteins. More recently, Plante et al. (2001) reported mark-
ers on chromosomes 20 and 26 affecting yield of milk fat
and protein in Canadian Holsteins. Burnside et al. (2002)
have identified four markers explaining significant protein
yield variation in a selective genotyping, daughter design,
for application in a commercial progeny-testing program.
There are several stages at which MAS can be applied to
accelerate genetic progress, notably, to designate from
which daughters of bull dams embryos will be selected.
Once the calves are born, MAS can be applied along with
the present selection approach to identify bulls that will be
reared for entry into the progeny test. Heifer calves can also
be selected using the same tools and placed in the juvenile
reproduction program. At 6 mo of age, their embryos pro-
duced with a selected bull can be biopsied and MAS applied
A recent paper described a procedure for multiple geno-
type analysis, including gender, kappa-casein, growth hor-
mone and prolactin from a single cell derived from bovine
preimplantation embryos using PCR (Chrenek et al. 2001).
The workers obtained an efficiency of genotyping by stan-
dard PCR from 88 to 91%. However, the efficiency of this
approach could be increased by using the PEP-PCR method
of DNA amplification (Garcia 2002).
CONCLUSION
Embryo technologies provide new possibilities for genetic
selection schemes. Some have already been implemented,
with ET being used on a regular basis for producing young
sires for progeny testing. The combined use of Ovum Pick
Up, followed by in vitro embryo production and marker
assisted selection, will undoubtedly be the next generation
of technologies applied in selection programs.
ACKNOWLEDGEMENTS
The authors gratefully acknowledge the professional and
technical staff of The Alliance Boviteq Inc. for their active
participation in producing the results presented in this article.
Aggrey, S. E., Yao, J., Sabour, M. P., Lin, C. Y., Zadworny, D.,
Hayes, J. F. and Kuhnlein, U. 1999. Markers within the regula-
tory region of the Growth hormone receptor gene and their associ-
ation with milk-related traits in Holsteins. J. Hered. 90: 148151.
Ashwell, M. S., VanRaden, P. M., Da Y., Rexroad, Jr., C. E.
and Miller, R. H. 1998a. Detection of putative loci affecting con-
formational type traits in an elite population of United States
Holsteins using microsatellite markers. J. Dairy Sci. 81:
11201125.
Ashwell, M. S., Da, Y., Van Tassell, C. P., VanRaden, P. M.,
Miller, R. H. and Rexroad, C. E. 1998b. Detection of putative
loci affecting milk production and composition, health, and type
traits in a United States Holsteins population. J. Dairy Sci. 81:
33093314.
Blondin, P., Bousquet, D., Twagiramungu, H., Barnes, F. and
Sirard, M. A. 2002. Manipulation of follicular development to
produce developmentally competent bovine oocytes. Biol. Reprod.
66: 3843.
Bousquet, D., Twagiramungu, H., Morin, N., Brisson, C.,
Carbonneau, G. and Durocher, J. 1999. In vitro embryo produc-
tion in the cow: an effective alternative to the conventional embryo
production approach. Theriogenology 51: 5970.
Burnside, E. B., Pan,Y., Jansen, G. B., Plante, Y., Vandervoort,
G. E. and Petitclerc, D. 2002. Confirmation of QTL for protein
yield and mammary system in a large half-sib family of Holsteins.
In Proceeding 7th World Congr. Genet. Appl. Livest. (in press).
Chrenek, P., Boulanger, L., Heyman, Y., Uhrin, P., Laurincik,
J., Bulla, J. and Renard, J. P. 2001. Sexing and multiple geno-
type analysis from a single cell of bovine embryo. Theriogenology
55: 10711081.

Colleau, J.-J. 1985. Efficacit gntique du transfert dembryons
dans les noyaux de slection chez les bovins laitiers. Gnt. Sl.
Evol. 17: 499538.
Darrow, M. D. 2002. Pregnancy rates of sexed female fresh and
frozen bovine embryos: a retrospective analysis. Theriogenology
57: 531 (Abstr.).
Galli, C., Crotti, G., Notari, C., Turini, P., Duchi, R. and
Lazzari, G. 2001. Embryo production by ovum pick up from live
donors. Theriogenology 55: 13411357.
Garcia, J. F. 2002. Practical considerations of embryo manipula-
tion: preimplantation genetic typing. Theriogenology 56:
13931399.
Georges, M. 1998. Perspectives for marker assisted selection in
Can. J. Anim. Sci. Downloaded from www.nrcresearchpress.com by 120.188.86.132 on 11/04/17
dairy cattle breeding. Proceedings of the European AI Vets 10th
meeting, Belgium. pp. 2529.
Herr, C. M. and Reed, K. C. 1991. Micromanipulation of bovine
embryos for sex determination. Theriogenology 35: 4554.
Khatir, H., Lonergan, P. and Mermillod, P. 1998. Kinetics of
nuclear maturation and protein profiles of oocytes from prepuber-
tal and adult cattle during in vitro maturation. Theriogenology 50:
917929.
Leitch, H. W., Smith, C., Burnside, E. B. and Quinton, M.
1995. Effects of female reproductive rate and mating design on
genetic response and inbreeding in closed nucleus dairy herds.
Anim. Sci. 60: 389400.
Lohuis, M., Smith, C. and Burnside, E. B. 1990. Embryo results
in a dispersed nucleus MOET/AI testing herd. Proceed. 4th World
Congr. Genet. Appl. Livest. 14: 221224.
Lohuis, M., Smith, C. and Dekkers, J. C. M. 1993. MOET
For personal use only.
results from a dispersed hybrid nucleus program in dairy cattle.
Anim. Prod. 57: 369378.
Meuwissen, T. H. E. 1990. The use of increased female reproduc-
tive rates in dairy cattle breeding schemes. Anim. Prod. 52: 2131.
Miyamoto, H. and Ishibashi, T. 1978. The protective action of
glycols against freezing damage of mouse and rat embryos. J.
Reprod. Fert. 54: 427432.
BOUSQUET EMBRYO TRANSFER AND IVF 407
Nicholas, F. W and Smith, C. 1983. Increased rates of genetic
change in dairy cattle by embryo transfer and splitting. Anim.
Prod. 36: 341353.
Nicholas, F. W. 1996. Genetic improvement through reproductive
technology. Anim. Reprod. Sci. 42: 205214.
Plante, Y., Gibson, J. P., Nadesalingam, J., Mehrabani-
Yeganeh, H., Lefebvre, S., Vandervoort, G. and Jansen, G. B.
2001. Detection of quantitative trait loci affecting milk production
traits on 10 chromosomes in Holstein cattle. J. Dairy Sci. 84:
15161524.
Revel, F., Mermillod, P., Peynot, N., Renard, J. P. and
Heyman, Y. 1995. Low developmental capacity of in vitro
matured and fertilized oocytes from calves compared with that of
cows. J. Reprod. Fertil. 103: 115120.
Shea, B. F. 1999. Determining the sex of bovine embryos using
polymerase chain reaction results: a six-year retrospective study.
Theriogenology 51: 841854.
Thibier, M. 2001. The animal embryo transfer industry in figures:
A report from the IETS data retrieval committee. IETS Newsletter,
December. pp. 1622.
Van Doormaal, B. J. 1991. To flush or not to flush? That is the
question. Holstein Journal September/October: 1214.
Van Doormaal, B. J. 2002. Study of embryo transfer Holstein
donor cows in 2001. [Online] Available: http://www.cdn.ca [5
August 2003].
Van Tassell, C. P., Ashwell, M. S. and Sonstegard, T. S. 2000.
Detection of putative loci affecting milk, health and conformation
traits in a US Holstein population using 105 microsatellite mark-
ers. J. Dairy Sci. 83: 18651872.
Voelkel, S. A. and Hu, Y. X. 1992. Direct transfer of frozen-
thawed bovine embryos. Theriogenology 37: 2337.
Woolliams J. A. 1989. Modification to MOET nucleus breeding
schemes to improve rates of genetic progress and decrease rates of
inbreeding in dairy cattle. Anim. Prod. 49: 114.
Can. J. Anim. Sci. Downloaded from www.nrcresearchpress.com by 120.188.86.132 on 11/04/17
For personal use only.