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IMPACT OF MODERN REPRODUCTIVE TECHNOLOGIES ON PRODUCTIVITY IN PIGS: A REVIEW

Chuck Okere, Ph.D.
Liasing Scientist - Research & Development
Genex Swine Group Inc. Regina, Saskatchewan
Introduction

The main objective of modern reproductive technologies in pig reproduction is to increase reproductive
efficiency and rates of genetic improvement. They also offer potential for greatly extending the
multiplication and transport of genetic materials and conserving unique genetic resources in reasonably
available forms for possible future use. The development and improvement of these technologies are
concentrating on gamete and embryo collection, sorting and preservation, in vitro production of embryos,
culturing, manipulation of embryos (splitting, nuclear transfer, production of chimeras, establishment
embryo stem cells, and gene transfer) and embryo transfer. Also, the development of these novel
technologies is facilitated by modern equipment for ultrasonography, microscopy, cryopreservation,
endoscopy, and flow cytometry, microinjectiors, micromanipulators and centrifugation.
This review will concentrate mainly on reproductive and molecular technologies that do not alter the
genome other than through the standard processes of selective breeding. The reason for not discussing
transgenic or genetic modification technologies in great length is because it is unlikely, in my opinion, that
these techniques will be used for swine improvement in the next ten years. This prediction is based both
on technological and consumer attitude.
Components of Sow and Boar Productivity

Herd reproductive performance depends on complex physiological pathways which determine male and
female reproductive performance such as age at sexual maturity, gamete production, libido, fertilization,
embryo, fetal and piglet survival. Main reproductive traits of interest for genetic improvement programs
are shown in Table 1.

Table 1. Main reproductive traits (Rothschild & Bidanel, 1998)

Reproductive function Sex Main components or predictive traits
Sexual maturity Male Age at first mating or sperm collection
Testes size
Female Age at first progesterone rise
Age at first estrous
Sexual behavior Male Ability to mount
Female Visible symptoms of estrous
Gamete production Male Sperm quantity and quality
Female Ovulation rate
Fertility Male Conception rate of mates
Female Conception rate
Weaning to estrus interval
Weaning to conception interval
Number of service per conception

This paper was presented at the 2001 Focus on the Future Conference, February 20-21, 2001
Red Deer, Alberta, Canada
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Prolificacy Male Litter size of mates

Female Fertilization rate
Number of embryos or fetuses
Embryo, fetus or prenatal survival rates
Uterine capacity and size
Number of piglets born or born alive
Mothering abilities Female Number of piglets weaned
Teat number
Milk production
Hormone levels Male Testosterone level
Female Progesterone level

Some Aspects of Genetics of Sow Reproduction

According Webb (2000) genetic improvement of any trait require two main tools:
Selection – which involves identifying the best animals to be the parents of the next generation. It
relies on the desired traits being inherited.
Crossbreeding - to produce a boost in performance that is often proportional to the genetic
dissimilarity of the breeds. It relies on heterosis (hybrid vigor).
Selection leads to permanent and cumulative changes, whereas heterosis must be regenerated at each
mating. Reproductive traits have the distinct disadvantage of low heritability, and can be measured only in
a small proportion of the adult population and also age dependent. Improvements by conventional
selection methods therefore deliver slow and measured response. Returns for investments are equally
small in comparison to growth and carcass traits, because these traits are shared over all members of the
2
litter (Webb, 1991b). Estimates of heritability (h ) for male and female reproductive traits in the pig are
shown in Table 2.
Table 2. Heritability estimate for reproductive traits in the pig.

2
Trait Mean h
Male traits
Testis weight 0.44
Semen volume 0.37
Sperm motility 0.17
Basal testosterone level 0.25
Libido 0.15

Female traits
Age at puberty 0.33
Standing reflex 0.29

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Ovulation rate 0.32

Prenatal survival rate 0.15
Age at first farrowing 0.13
Total born 0.11
Number born alive 0.09
Number weaned 0.07
Piglet survival to weaning 0.05
Litter birth weight 0.29
Litter weight at 21 days 0.17
Weaning to estrus interval 0.25
Farrowing interval 0.03
Sources: Nicholas, (1997), and Rothschild & Bidanel, (1998)
Any genetic improvement must be sought through increasing litter size, since much of the improvements
in reproductive efficiency have been brought about by increases in the number of litters produced per sow
per year, which is nearing the maximum possible. Litter size is the product of ovulation rate and prenatal
survival. Hence, a rapid improvement in the number of piglets born alive per litter could be made if both
ovulation rate and embryo/fetal survival could be improved simultaneously. Although ovulation rate is
moderately heritable (0.32), selection for that trait results in only small improvement in the number of
piglets born per litter since prenatal survival is lowly heritable (0.10) (Johnson et al. 1999). A major
limitation is therefore the high level of embryo mortality that occurs, with 30-40% of the fertilized eggs
failing to develop into embryos and survive to term.

Webb (1991a) identified alternative strategies to speed rate of improvement in litter size which include:

Hyperprolifics – Genes from a very small proportion of sows with consistently highest litter sizes
in multiplier herds are returned to the nucleus via AI or c-sections. However, the Hyperprolifics lag
behind the nucleus in lean growth.

Progeny testing – Large-scale national schemes offer the possibility of improving litter size by
progeny testing AI boars. In practice tens of thousands of sows will need to be mated by AI.

BLUP – Best linear unbiased prediction technique is a powerful tool for separating genetic and
non- genetic effects on performance and can now be used to predict genetic merit for litter size
from large numbers of relatives. This allows accurate selection of boars and gilts immediately
after performance test.

Modern Reproductive Technologies

Artificial insemination (AI)

More than 40 years ago AI in pigs reached a level of practicality but it has developed into an important
biotechnology tool in the porcine industry only in the last 20 years. Our major competitors in Europe lead
the world’s pork industry in AI use. Germany uses AI for over 40% of all inseminations, Norway uses AI
for 85% of all inseminations, Sweden for 50%, Denmark for 32%, and the Netherlands for 70% (Foxcroft,
1996). Canada usage is finally reaching levels that have been common in Europe for years. The effective
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use of AI in breeding programs is essential if Canadian producers wish to maintain a competitive edge in
the global market place. Leiding (2000) outlined the physiological potential of semen production in Tables
3 and 4. These show the importance of keeping the semen free from all relevant transmissible diseases.
Average sized semen production units collect about 100 ejaculates or more per day five times a week. In
the case of infection this will cause an immediate spreading of disease into several herds.

Table 3. Characteristics of ejaculates

Species Volume (cc) Sperm Conc./cc Sperm per
ejaculate
Boar 150-250 100-200 million 25-50 billion
Bull 3–5 1 billion 3-5 billion
Ram 1 1 billion 1 billion
Stallion 70 - 100 100 million 7-10 billion

Table 4. Calculation of semen doses per boar and year

One boar has a potential of producing 50 billion sperm cells per ejaculate.
From one ejaculate about 20 doses of semen can be produced.
Two ejaculates can be produced per week.
20 doses x 2 ejaculates/week x 52 weeks = 2080 doses per year
With 2080 doses of semen about 1000 sows can be inseminated.

Foxcroft (1996) observed that the purchase, quarantine and training of potential AI boars will become an
expensive investment if the eventual fertility of these boars proves to be unacceptable, because, at
present there are few, if any proven and reliable predictors of boar fertility. Additionally, if the trend for
using pooled semen for the AI of terminal line sows continues, the relative infertility of a given boar may
never be apparent from breeding herd records (Foxcroft, 1996).

Impact on productivity
The most practical obstacle to widespread use of AI in Canada remains problems of distance. Most
extenders in current usage give optimal conception rates for 72 hours; extenders need to be developed
which produce reliably conception rates for at least 7 days to overcome this problem. The way forward is
to encourage research aimed at developing appropriate technology for sex sorting of semen and
cryopreservation, since the current technology does not give acceptable conception rates and litter sizes
when frozen-thawed semen is used.
By using AI, genetically superior nucleus boars can be used extensively, particularly at the nucleus and
multiplier level. At the nucleus level, AI has made it possible to link several units to create a large ‘super
nucleus’, thereby increasing genetic gain at nucleus level and a decrease in genetic lag between nucleus
herds and the commercial population (Visscher et al. 2000). As well, AI is changing the market strategy of
pig breeding companies, from selling live boars to selling semen. As with other species, some of the
advantages using AI include:

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• Cost efficiency in not having to buy or maintain many boars.

• Better hygiene of semen doses compared to natural service.
• Better fertility, since each ejaculate is tested before use.
• Convenience of being able to breed any male to any female regardless of size, temperament, or age.
• AI provides a tool to enhance leanness, growth rate and other genetic improvements.
• AI techniques are relatively simple to learn.

Sex-sorted semen and cryopreservation

One current example that illustrates the importance of reproductive technology development is a process
that would allow producers to predetermine the sex of offspring. This process is based on the separation
of X- and Y- chromosomes bearing spermatozoa. Basically, three approaches have been used to try and
accomplish this with varying degrees of success. First, sperm cells have been separated on the basis of
DNA using flow-cytometry. Using this technique, sex ratios have been skewed in experimental and field
studies with rabbits (Johnson et al. 1989); Swine (Johnson, 1991, Rath et al. 1997 and Johnson et al.
1999); cattle (Seidel et al. 1997); and humans (Fugger et al. 1998). The shift in the sex ratio is usually
from the standard 1:1 to about 8 or 9:1 or vice versa.
The latest flow cytometry method is known The Beltsville Sperm Sexing Technology. Developed by Dr.
Larry Johnson of the USDA at Beltsville. This method involves treating sperm with a DNA-binding
Fluorochrome (dye), and then flow-cytometrically sorting with laser beam based on the amount of
fluorescence into separate X and Y populations that can subsequently be used for regular artificial
insemination or in vitro fertilization to produce sexed embryos for transfer. One limitation of cell-sorting
technology is that the process must be carried out one cell at a time. This makes the systems inherently
slow, because billions of sperm are need for conventional AI. Improvements in the Beltsville technology in
the last two years has lead to the development of a commercial high speed cell sorter which can produce
6
5 to 6 x 10 sperm/hour at 85 to 90% purity for X (female producing) or Y (male producing) sperm cells in
most livestock species.
Recently, studies (Long et al. 1998) and (Abeydeera et al. 1998) were conducted using sexed boar sperm
produced by the Beltsville technology and using oocytes matured in vitro for fertilization under in vitro
fertilization (IVF) conditions and results are as shown in Table 5. Continued progress in the development
of greater efficiency in sorting sperm into X and Y sperm populations offers great encouragement that
practical application of the sperm –sexing technology will occur within a few years.

Table 5. Farrowing results from gilts receiving embryos produced from sexed sperm.

No. of piglets
Treatment Litters Male Female Litter size
Sorted for X 6 1(3%) 33 (97%) 5.8
Control 3 12 (52%) 11(48%) 7.8

Sorted for Y 3 9 (100%) 0 3.0

The second approach involves isolating a protein from the surface of the X – or Y- bearing sperm that is
chromosome specific and thus sex specific. Recently, a University of Guelph spin-off company Gensel
Biotechnologies Inc. announced the identification of several sex-specific proteins on the surface of sperm
cells against which antibodies could be raised to enable the separation of male-producing sperm cells
from female producing sperm cells. So far, the plan is to prepare monoclonal antibodies that will be added
This paper was presented at the 2001 Focus on the Future Conference, February 20-21, 2001
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in solution to the semen. Sperm of the unwanted sex can be made to clump together and filtered off using
glass wool (Webb, 2000).
The third way is differential centrifugation. The rationale is that X sperm have a higher density than Y
sperm due to the X- chromosome being larger. Therefore, theoretically, one should be able to separate
these two populations of cells by differential centrifugation. To the best of my knowledge no reports exists
in literature that show success rates with this technique.

Potential impact on productivity

Webb (2000) suggested that the main benefits of semen sexing lies in improved feed efficiency and
carcass lean content. Compared to a castrate, a gilt has up to 15% better feed efficiency and 3% more
lean. An entire boar shows roughly the same advantage again over gilts. Also, argued that single sex
production also avoids the need for split –sex feeding. If successful the semen sexing technology will be
easy and cheap to apply to on-farm AI collection. It involves no genetic manipulation, and is safe and
acceptable to the consumer. In the short term sex determination probably represents the greatest single
potential step forward in pig production and is therefore well worth the effort (Webb, 2000).
More work needs to be done in field of semen cyropreservation. Concentrations of cyroprotectant, cooling
rate, size of straw and warming velocity are main factors influencing the post-thaw viability of sperm cells.
Freezing protocols show that glycerol, at a concentration of 3% could be used as an effective
cryoprotectant (Bwanga et al. 1991a). Main damage of the spermatozoal membrane was found to occur
0 0
during cooling to 5 C, whereas supercooling to –6 C did not cause further damage (Ortman and
0 -1 0
Martinez, 1994). Best spermatozoal quality is expected using a freezing rate of 30 C min and 1200 C
-1
min for thawing (Fiser et al.1993). Frozen (3% glycerol) thawed ejaculates showed a post-thawed
motility rate of 54.5% and 75.5% of the sperm cells showed normal apical ridges. After insemination,
fertilization rate was 75% (Bwanga et al. 1991b).

Embryo recovery, production, preservation and transfer technology

Porcine embryos or immature oocytes can be collected ex vivo from living donors by either surgical or
endoscopical flushing of uterine horns or from slaughtered animals. Other possibilities include in vitro
production of embryos (IVEP, e.g. from follicles collected from the slaughterhouse or from live animals),
non-surgical embryo transfer techniques, embryo storage and freezing techniques and cloning. These
techniques are described below.

Recovery procedures
An initial step in embryo recovery is the superovulation of donors, which usually results in about 30
collectable oocytes or embryos per donor. While both quantity and quality of embryos isolated from
prepuberal gilts are higher than from older animals, multiparous sows offer notable advantages as
embryo recipients in terms of pregnancy rates and embryo survival (Besenfelder et al. 1998). Prepuberal
gilts or synchronized donors (treated sows) are usually superovulated with a single injection of pregnant
mare’s serum gonadotrophin (PMSG).
Embryo recovery is routinely done by flushing the uterine horns surgically or after slaughter. The nature of
the sow’s cervix and the uterine horns appeared to make non-surgical recovery procedures less feasible.
For each technique, manipulation of the oviducts and the uterine horns must be kept to a minimum;
aseptic precautions must be taken at all stages of the recovery process. Recovery of oocytes from living
donors via endoscopy or the ultrasound-guided transvaginal aspiration known as ‘ovum pick up’ (OPU)
has been demonstrated by Besenfelder et al. (1997). Endoscopic embryo collection enables complete
embryo recovery minimizes manipulations and efforts and guarantees repeated use of donors. In
addition, collected embryos may be of known genetic merit, unlike slaughterhouse production. Using
techniques described above, within 6 days of ovulation, it is possible to achieve embryo recovery rates of
the order of 80-90%.

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In vitro embryo production (IVEP)

Since the early ‘70s great efforts have made to produce porcine embryos in vitro. The initial idea was to
produce a pool of pre-implantation stage embryos for other reproductive technologies. The production of
large number of in vitro produced porcine embryos includes numerous in vitro steps, which try to mimic
the in vivo development of embryos: oocyte maturation, fertilization and culture until transfer or
cryopreservation. The development of in vitro maturation and in vitro fertilization techniques remains a
key step in successful IVEP programs. In contrast to results in the cow and human, experiences to date
with in vitro fertilization in pigs have been much less successful. A review by Gordon (1997) reveled that
Japanese workers (Nagai et al.1984) first reported evidence of fertilization by IVF in pigs. Live births
were also reported in Cambridge after IVF of ovulated pig oocytes. Subsequent work by Mattioli et al
(1989) convincingly demonstrated that pig oocytes, matured and fertilized in vitro (IVM/IVF), could
undergo normal embryonic development. They reported blastocyst formation, establishment of
pregnancies and birth of live piglets. Also, researchers at the University of Guelph made important
contributions to our understanding of oocyte maturation protocols.

In vitro culture of early embryos

The requirements for embryo development in vitro can be broadly divided into nutritional and
environmental. It is now possible to produce pig embryos in the laboratory, which are suitable for transfer.
Rath et al. (1995) suggested that it is preferable to transfer porcine embryos derived from in vitro
fertilization at an early stage of development and not to culture them to the blastocyst stage before
transfer.

Recovery and In vitro maturation (IVM) of oocytes

Collection of oocytes by either follicular dissection or aspiration is possible. Although the collection of
oocytes by dissection is labor intensive, it ensures that oocytes are recovered from non-atretic follicles.
After collection, the selection of good quality cumulus-oocyte complexes (C-O-Cs) is important. Studies
also suggest that only maturation culture systems that involved the presence of abundant follicle wall
components were able to create conditions for competent oocyte maturation (Mattioli et al. 1988). A
review by Nagai (1994) noted that IVM of pig oocytes was possible in a simple medium (mTALP)
supplemented with pig follicular fluid and cystine.

In vitro Fertilization (IVF).

Hunter (1990) observed that the problem of IVF of pig oocytes demands proper understanding of the
factors contributing to the oviductal microenvironment during in vivo fertilization in pigs. However, the
most important problem in pig IVF is polyspermy (multiple sperm penetration). Funahashi and Day (1993)
showed that pre-fertilization incubation of boar sperm in suitable concentrations of pig follicular fluid could
reduce the incidence of polyspermy in an IVF system. Foxcroft et al. (1995) showed that boars all
significantly affected sperm penetration, monospermy and male pronuclear formation. Laurincik et al.
(1994) concluded that under IVF conditions, oocytes matured in vitro displayed lower penetration and
cleavage rates and asynchronous development and delayed cleavage.

Embryo preservation
In contrast to cattle, pig embryos are difficult to adapt to the cryopreservation conditions. It is becoming
clear that the sensitivity of pig embryo to cooling and freezing is probably the result of their high lipid
content (Gordon, 1997). Deep freezing protocols have been developed which tried to optimize the
cooling rates, temperatures, cryoprotectants (including nutrients and supplements) and the embryonic
stages. The three approaches to successful cyropreservation of porcine embryos are illustrated in Fig. 1.
Freezing of pre-hatching-stage pig blastocysts and their subsequent storage in liquid nitrogen was
reported by Kashiwazaki et al. (1990). Live piglets were born after transfer. In another study pregnancies
and live-born piglets were generated from early stage embryos (two-cell) which had been deep-frozen (-
0
196 C) after centrifugation and removal of cytoplasmic lipids (Nagashima et al. 1995). Because the
removal of cytoplasmic lipids by micromanipulations is labor intensive research efforts now target

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procedures that improves the ability of embryos to tolerate freezing by reducing their lipid content and /or
increasing the fluidity of the cell membranes.
Figure 1.0. Three approaches to the successful cryopreservation of pig embryos (Gordon, 1997).

Removing cytoplasmic lipids

from early embryos

Freezing of the hatching and

Vitrification
hatched blastocyst

Cryopreservation of pig
embryos

Recent work in Australia and the Netherlands indicate that vitrification may be an appropriate solution to
the problems of cryopreservation of pig embryos. Vitrification is the rapid cooling of a solution without the
formation of ice crystals. In this way embryos are not subjected to the physical damage otherwise
associated with crystal formation. Nagashima et al. (1996) and van der Lende (1998) demonstrated that
deplipidated two-and four-cell pig embryos could be successfully preserved by vitrification and that they
were capable of developing to blastocyst stage after. The next stage is to develop optimum vitrification
solution that is less toxic.

Embryo transfer methods and recipient management

The embryo transfer (ET) includes the collection or production of embryos (ex vivo or in vitro) from donor
pigs, the temporary culture and /or manipulation and reintroduction into a recipient animal (Besenfelder et
al. 1998). Embryo transfer in pigs usually involves surgical intervention. There are very few reports of
laparoscopic or transcervical transfers. Surgical transfer of porcine embryos into the oviduct and uterine
horns was developed to introduce new genetic materials into specific-pathogen –free pig herds (Cameron
et al. 1989). Reported pregnancy rates after surgical transfer range from 60 to 85%. In addition,
significant stress of the animals results from anesthesia, surgery, and manipulation of the genital tract.
Several attempts have been made to establish non-surgical or minimal invasive methods.
As with ruminants, exposure of the pig embryo to asynchronous uterus can be detrimental to further
development. Assessing synchrony requirements, Polge (1982) showed that there was no reduction in
pregnancy rates when donors were 1 day earlier than recipients but transfer to recipients more advanced
than donors did result in a decrease. Collection and transfer of one-cell stage and two- cell stage
embryos to oviducts of synchronized recipients can achieve acceptable embryo survival rates. The
difficulty at the stage of embryo development is in deciding whether they were fertilized or not.

Kim and Oguri (1990) examined various factors affecting pregnancy rates and litter size after surgical
transfers. They reported pregnancy rates of 75% and 90% and litter sizes of 5.3 and 6.1 after transfers to
recipients at 3 and 4 days after estrus, respectively. For transfer of embryos of 5 and 6 days of age, they
reported pregnancy rates of 66.7% and 100% and litter sizes of 6.5 and 5.6, respectively. It must be
remembered that embryo transfer in pigs is unlikely to maintain pregnancy unless recipient pigs receive at
least four embryos.
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Non-surgical embryo transfer

The cervix is a barrier for transferring porcine embryos non-surgically. Polge and Day (1968) reported
some of the first efforts in non-surgical embryo transfer in pigs and obtained viable conspectuses from
one gilt at d 17 after transfer. Li et al (1996) described a novel embryo transfer technology which enabled
embryos (four-cell to blastocyst stages) to be delivered into the lumen of the uterine horn of recipient gilts.
Five of 16 recipient became pregnant (31%) and farrowed an average of 6.2 ± 3.1 pigs per litter This
technology involves a four part transfer instruments: (I) a modified commercial AI spirette which is used to
produce a cervical lock; (ii) stainless steel tubing with a curved tip that can be manipulated over folds of
the cervix; (iii) a testing bar to ensure that the tip of part (ii) reaches the bifurcation of the uterus; and (iv)
a disposable tubing complex for embryo delivery. This report claimed that a technician can easily learn to
operate the instruments.

In the Netherlands, Hazeleger and Kemp (1999) and Dr. van der Lende and co-workers published a
technique for non-surgical embryo transfer that essentially mimics the process for artificial insemination.
They have developed a PVC rod that uses a very small amount flushing fluid (0.1 ml) to transport
embryos. The application instrument has a short 1 cm hook at it tip. After moving beyond the cervix it is
turned gently to reach the top of the uterine horns where the embryos are deposited. The report added
that transfer done in this way have taken no more that few minutes to complete, in sows at day 5 – 7 after
estrus. Their data showed 16 pregnancies from transferring 28-30 embryos of one donor sow into each of
27 recipients. Slaughter records on day 35 revealed an average litter size of 10.9 that they assume can
translate to 9.5 to 10 live born at farrowing.

Potential applications of embryo production and transfer technology

Movement of genetic resources in the swine industry currently relies predominantly on shipping of live
animals. The high cost of shipping plus the risk of disease transmission are some of the disadvantages of
transporting genetics via live animals (Li et al. 1996). Apart from the problems associated with
cyropreservation of boar semen, the use of AI can change only half the genetics of the offspring.
Shipping embryos might be a more cost- effective method for disseminating genetic material, but the high
cost of surgical collection and transfer of embryos in conjunction with relative inability to cryopreserve
porcine embryos has limited commercial application of this technology.
With improvements in non-surgical embryo transfer technology, shipping embryos instead of animals or
herds will become practical. Day (2000) suggested that embryos could be maintained in culture while
undergoing pre-implantation development for up 5 days during shipping and handling period, allowing
transport to even remote locations. This technology will benefit the swine industry by promoting national
and international sales via reducing surgery cost and the cost of shipping and handling.
Cryopreservation of pig embryos has important applications in modern reproductive technology. Embryos
of individual animals, lines and strains of high genetic value would be available at any time and in any
place, thus minimizing hygienic barriers. From a biosecurity viewpoint the health advantages are
enormous. PCR testing will be able to quickly differentiate infected vs. non-infected embryos. New
research shows that transferring early-stage embryos while the zona pellucida is still intact along with
embryo washing techniques can assure a disease-free embryo (except for viruses incorporated into the
pig genome) (Besenfelder et al.1998).
Recently the National Hog Farmer (45 (11) 2000) reported that embryo transfer technology has changed
a 280 purebred Duroc herd from an all-PRRS-positive herd to an-negative status. The report also added
that Drs. John Pollard and Marie-Claire Plante of Ontario Veterinary College, University of Guelph,
developed the technique and performed trials demonstrating the PRRS cycle could be stopped. In
addition, the technique greatly facilitates the performance of embryo transfer programs allowing the
transfer of frozen-thawed embryos at any time into appropriate recipients.
When large scale ET is adopted, the consequences for swine breeding programs is that superior females
could have as much influence on genetic progress as males through an increase in selection intensity.
Apart from the effect of increased selection intensity at the nucleus level, in vitro embryo production in

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conjunction with embryo preservation and transfer technology could alter the dissemination structure of
the industry (Visscher et al. 2000). These authors advocated the development of a few large-scale
‘embryo farms’ in which matured oocytes from superior nucleus sows are recovered and artificially
inseminated with semen from nucleus boars (from a different line). The embryos would be implanted into
recipients which sexually mature early and have large reproductive capacity, for example purebred
Meishan genotypes or other hyperprolific dam lines.
The piglets born from the recipients would be transported to commercial farms. The advantage of such a
scheme is that fewer animals are transported from nucleus to multiplier tiers, fewer sows are needed at
the multiplier level, and there is greater control over the multiplication process for the breeding
companies. They concluded that such a scheme might remove the need for a purebred multiplication tier,
and reduce crossbred tier in the industry, thereby reducing the genetic lag.
Embryo splitting, cloning and transgenic pigs

Embryo Splitting
A different and simpler cloning procedure, called embryo splitting, or artificial twining, was developed in
the 1980’s and was adopted by animal breeders. In this procedure, an early embryo is simply split into
individual cells or group of cells, as happens naturally with twins, triplets, and other multiple births. Each
cell or collection of cells develops into a new embryo, which is then implanted into a surrogate animal,
who carries it to full time. Although this technique permits the production of multiple clones, the clones are
derived from an embryo whose genetic potential are not completely known rather than from an adult
animal with known characteristics. This constitutes a serious limitation for practical applications of the
procedure.

Embryo splitting technology has been the subject of many reports in cattle, few studies have been
reported on embryo bisection in pigs. Cambridge workers (Polge et al.1982) described two sets of
identical twin pigs. Other studies by Reichelt and Niemann (1994) were more promising, showing that pig
morulae and blastocysts could be bisected relatively easily and yield large numbers of viable embryos.

Cloning in pigs
In 1952, Robert Briggs and Thomas King, developmental biologists developed a cloning method called
nuclear transfer, which was proposed in 1938 by the German scientist Hans Spemann. In this method,
the nucleus – the cellular structure that contains most of the genetic materials that controls growth and
development is removed from an egg cell (oocyte) of an organism, a procedure known as enucleation.
The nucleus from a body cell of another organism of the same species is then placed into the enucleated
egg cell to grow into an embryo. Because the embryo’s genes came from the body cell’s nucleus, the
embryo is genetically identical to the organism from which the body cell was obtained.
In 1997, scientists led by Ian Wilmut of the Roslin Institute near Edinburgh, Scotland reported they had
used mammary gland cells from an adult female sheep to create
a genetically identical lamb known as Dolly. This marked the first time researchers had produced a clone
using a specialized cell from an adult vertebrate. Variations of this technique pioneered by the Scottish
scientist have been used since 1999 to clone cattle and pigs.
In March 2000, The Scotland -based PPL Therapeutics announced that five healthy pigs were born as a
result of nuclear transfer (cloning) using adults cells. This is the first time cloned pigs have been
successfully produced from adult cells. The method used to produce the five female pigs, named Millie,
Christa, Alexis, Carrel and Dotcom, was different from that used to produce Dolly the sheep. Details are
described in Nature Biotechnology, 2000:18 (10), 1055-1059.
Japanese team lead by Akira Onishi announced in August 2000 the birth of Xena, a black-coated piglet
from a white- coated sow produced by cloned genetic material from fetal pig cells. She is named after the
field of research that scientists hope her birth might advance – xenotransplantation – the use of
genetically modified animal organs for transplant into humans. To create Xena, this team used a needle-
like pipette to inject genetic material from fetal pigskin into oocytes or egg cells that had been stripped of
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their own genetic material. Next, the team stimulated the injected eggs with an electrical pulse that
triggered them to develop into embryos. Those embryos were then transplanted into surrogate sows.
Xena was the one successful birth of 110 transplanted embryos. When researchers cloned Dolly the
sheep, they fused entire cells into empty host eggs. Researchers on Xena’s team believe their technique
of injecting only genetic material will allow better flexibility for genetic manipulation.

Transgenic pigs
Animals modified to carry genes from other species are called transgenic animals. The technique
producing transgenic animals is best illustrated by the recent work by Dr. Forsberg and colleagues at the
University of Guelph. This group has produced the ‘Enviropigs’ (Wayne, Jacques and Goldie) – named
after Canadian hockey players. Although outwardly they look like any other pigs of Yorkshire breed,
inside the nucleus of each cell of an Enviropig is a piece of DNA that contains a snippet of mouse and a
bit of bacteria genes. This composite gene was inserted into the nucleus of a one celled pig embryo with
a microscopic needle. These pigs are transgenic for the enzyme-phytase. The transgene will allow these
pigs to make phytase in the salivary gland, when swallowed with food, the phytase releases organic
phosphorus in the animal’s gut where it can be absorbed, thereby emitting less phosphate pollution.

Potential impact of cloning technology

The prospect of cloning offer the possibility to reduce the genetic lag between the nucleus, multiplier, and
the commercial tiers, since genetically superior or performance tested animals can be cloned and
supplied to the commercial farmer. A quote from the late Dr. Charlie Smith emphasizes this point “one
result would be that commercial animals could be genetically superior to the breeding animals. Thus, it
can be said that cloning could turn the conventional breeding on its head”
According to Van Vleck (1999) cloning, on first impression suggests a perfect way to improve
performance of livestock. Such impressions imply that breeders only need to find the perfect animal so
that no further effort to breed better animals is needed; when the perfect animal is found and cloned, the
traditional methods of breed improvement would be obsolete. He concluded that these perceptions are
generally incorrect for most important traits in farm animals.
Finally, can this technology be made inexpensive enough to compete with a mature technology, such as
AI or a niche technology such sexed semen or embryo transfer? There are complex technical, societal,
political and ethical determinants of its application that must be addressed.

Molecular Technologies
Most traits of economic importance are determined by the combined interaction of many genes with the
environment. One aim of molecular biotechnology is to identify the genes involved in the expression of
these traits. The use of such information in breeding programs would provide a measurement of the
genetic value of individuals, without looking at their phenotype or of their relatives’ phenotypes (Visscher
et al. 2000). Because DNA can be obtained from individuals of any sex or age, it means that these
individuals could be evaluated for late expression traits (carcass) early in life.
DNA technologies provide a major opportunity to advance productivity in the pig through their application
in:
• Characterizing and better understanding of animal genetic variation.
• Manipulating the variation within and between pig breeds to realize more rapid and better-targeted
gains in breeding value.
• Conserving genetic resources.

Gene markers and maps

Many genes control most traits of economic importance in the pigs, with each gene having a small effect.
In the pig, the DNA id distributed over 19 pairs of chromosomes and arranged into approximately 100,000
This paper was presented at the 2001 Focus on the Future Conference, February 20-21, 2001
Red Deer, Alberta, Canada
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genes. Once the sequence for a gene is known, its presence can be detected using a DNA test.
Numerous research efforts are directed towards identifying as many genes with useful effect as possible.
However, the precise function of most the genes detected so far is not clear. It is also possible that these
genes may be located on the chromosome near a gene that affects performance (growth rate) and for
which no known DNA test has been developed. Because of genetic linkage, the detected gene can show
association with growth rate, which is actually caused by its neighbor (Webb, 2000).
The DNA tested gene is known as a marker, because it marks a section of chromosome affecting
performance-affecting performance. The gene whose presence it detects is known as a quantitative trait
locus (QTL) (Webb, 2000). For markers as a selection tool, they must be fairly precisely localized on a
chromosome. A prerequisite for QTL scans is the genetic map. It is analogous to a road or geographical
map in that it provides references for the location of the genes. Research teams around the world are
collaborating in mapping the genome of the pig. The major contributors to this effort have been
international collaborative projects based in Europe (The PiGMap consortium), the Nordic Pig Gene
Mapping project and the USDA-MARC Linkage Maps project.

Marker Assisted Selection (MAS)

The fundamental premise of this program is that all genetically determined variation in productivity is
ultimately encoded in their DNA. If all the DNA sequence responsible for this differences in performance
could be identified, then superior animals to use as parents for the next generation could be chosen
simply by doing DNA tests. (Moran, 1999). Alternatively DNA markers tightly linked to and associated with
these casual genes could be used as proxies, if the casual genes had not been identified. The use of
DNA markers in this way has been termed marker-assisted selection (MAS). The potential of MAS is that
it can speed up the rate of improvement for traits like backfat thickness, where measurement of
performance is cheap and easy and conventional selection is efficient. It might also enable early
decisions on the choice of parents for intractable but important traits, only measurable post-slaughter, like
meat quality, or only expressed in one sex, like ovulation rate.

Marker Assisted Introgression (MAI)

A marker could be used to introduce favorable characteristics from a donor breed or line into a recipient
breed /line. This is done by breeding an F1 population and crossing it with the recipient line while
selecting for the desirable trait. After many generations of backcrossing, the genetic background of the
resulting line is primarily the same as the recipient but preserving the favorable trait of the donor. Let us
suppose for example that a single gene for lntramuscular fat is to be introduced from Duroc into the Large
White. An F1 cross of these breeds will be backcrossed to Large White over several generations, slowly
increasing the proportion of Large White while selecting for the desired trait. According to Visscher
(2000), there are several examples where MAI is already possible in commercial pig breeding. The
dominant white alleles (allele – one pair, or series of alternative forms of a gene that can occur at a given
locus on the same chromosome) which gives the pig white color, has been identified and can be
introgressed into a non-white line to create a recipient white line.

QTL mapping - candidate genes approach

These genes function in physiological processes, which suggest that allelic variants could cause variation
in a phenotype, related to that process. So, rather than searching at random for markers, the candidate
gene approach uses knowledge of physiology to identify likely QTLs with major effects (Webb, 2000). At
this stage, there is one prominent success in the use of DNA markers for reproductive trait in pigs - the
estrogen receptor gene (ESR) which affects litter size in some populations. This was arrived at via the
candidate gene approach rather than from gene mapping (Rothschild et. al 1997).

Using this approach, we at Genex Swine Group are currently involved in collaborative project with The
Guelph Molecular Supercentre, University of Guelph designed to identify quantitative trait loci affecting
female reproductive traits in a multigenerational Meishan-White composite swine population. This project
will identify regions of the pig genome that contains genes affecting various reproductive traits using a
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Red Deer, Alberta, Canada
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genome wide scan. The identified genomic regions for economically important traits like litter size will be
examined in our commercial swine populations to determine whether allelic variation at the quantitative
trait locus exists. Such specific information will significantly help us develop effective genetic improvement
programs for litter size in our traditional and prototype dam lines.

Combining technologies
Visscher et al. (2000) observed that the real impact of these technologies would come from combining
new reproductive technologies with current molecular biology techniques. The former will allow a rapid
turn over of generations, whereas the latter can provide selection, which does not need phenotypic
information when selection decisions are made.
Georges and Massey (1991) suggested speeding up genetic progress in cattle through velogenetics by
harvesting oocytes from calves in utero, thereby substantially reducing generation interval. The same
scheme could be adapted for pigs. One selection cycle starts with recovery and in vitro maturation from
suitable donors, followed by in vitro fertilization with sexed semen. Embryo transfer is carried out with
recipient with large uterine capacity (Chinese Meishan). After piglets are born, they can be selected
immediately using marker-assisted selection, and immature oocytes can be harvested from the selected
female piglets. The duration of one cycle of selection with this accelerated selection program is about 4-5
months.
Webb (2000) suggested that another possibility would be to have females from prolific dam lines serve as
surrogate mothers, which receive cloned and frozen embryos by non-surgical transfer. The slaughter
clones might receive transgenes for disease resistance and utilization of alternative feedstuffs.

Modern reproductive technologies: Potential impact and/or effective

Visscher (2000) summarized the likelihood of implementation and impact of various techniques in Table
4.
Table 6. Predicted impact of biotechnology techniques in terms of likelihood of effective use in the next 5
years and the estimation of the potential impact on genetic change (scores on scale 1 – 3, 1 = low, 3 =
high).

Technique Likelihood of effective use Potential impact

Sex sorted-semen 3 3
Gamete cyropreservation 3 3
Non-surgical embryo transfer 2 1
(ET)
In vitro embryo production 1 2
(IVEP)
Cloning 1 2
ET + IVEP + Cloning 1 3
Molecular biology techniques 3 2
Transgenes 0 0

An overview of problems and prospects of adopting some modern reproductive and /or biotechnology in
commercial swine breeding programs appears in Table 7.

This paper was presented at the 2001 Focus on the Future Conference, February 20-21, 2001
Red Deer, Alberta, Canada

Table 7. Opportunities and challenges of adopting new reproductive technologies in commercial swine
breeding programs (Adapted from Hammond & Leitch, 1998).
Techniques
Reproductive Techniques
Artificial Insemination (AI)
Embryo transfer (ET)
In vitro Embryo Production
(IVEP) & Oocyte Recovery
Cloning
Molecular Techniques
Marker Assisted Selection
(MAS)
Transgenics
This paper was presented at the 2001 Focus on the Future Conference, February 20-21, 2001
62
Opportunities Challenges
Enables dissemination of superior Requires high level
genetics management for collection,
processing of semen and use
Affords more controlled breeding
and mate selection and potentially Technology needs further
decreases inbreeding development in many areas
Potential for dissemination of Costly and logistically complex
genetics from superior dams, and
Technology not well refined for
transboundary transport of genetics
the pig
Passive immunity conferred on
Requires specialized training
offspring by recipient female
and expertise
Effective for genebanking and
regeneration of populations
Potential to reduce long generation Technology needs further
intervals by collecting ova from development
prepubertal females Requires specialized equipment
and training
Conservation of breeds at risk
Efficient use of semen in in vitro
fertilization as few sperm cells are
required
Enables dissemination of genetics
from superior females
Mass production of crossbreed Expensive
clones would enable optimum
Requires specialized equipment
combination of adapted local with
and training Technology needs
exotic genetics
further development
Identify marker genome sites to May require high level of
assist in selection of quantitative pedigree and descriptive
traits production information
Potentially circumvents expensive
performance recording
Measurement prior to expression –
age/sex
Transfer of useful genes to improve Potential consumer backlash
productivity
Red Deer, Alberta, Canada
 63

Conclusions.

The potential exists to improve different sow reproduction traits, such as age at puberty, oestrous
symptoms, ability to become pregnant, litter size, piglet survival and weight, milk production, maternal
behaviour and ability to show oestrus after weaning. In practice, however, considerable problems are
associated with genetic selection for most of these traits as they have low heritability values. The speed
at which genetic improvements can be achieved by traditional methods is slow. Many of the new
reproductive technologies offer possibilities for improving the rate of genetic progress. These include in
vitro embryo production (IVEP), non-surgical embryo transfer (ET), sperm sexing technology (SST), and
molecular biology techniques (QTL and MAS). The real impact on genetic progress will come from
combining new reproductive techniques with powerful molecular techniques. The former will allow a rapid
turnover of generations, whereas the latter can provide selection, which does not need phenotypic
information when the selection decisions are made.

Acknowledgements.

The author is indebted to Dr. John Cosgrove and members of the Genex Swine Group Research &
Development Team who contributed either directly or indirectly to the ideas contained in this paper. I also
wish to thank Linda Nelson for her constructive suggestions and sound editorial advice. The contents of
this publication do not necessarily reflect the views, policies or practices of Genex Swine Group Inc.
References

Abeydeera, LR., Johnson, LA., Welch, GR., Wang, WH., Boquest, AC., Cantley, TC., Rieke, A and Day,
BN. 1998. Birth of piglets preselected for gender following in vitro fertilization of in vitro matured pig
oocytes by X and Y chromosome bearing spermatozoa sorted by high-speed flow cytometry.
Theriogenelogy 50: 981-988.
Besenfelder, U., Model, J., Muller, M and Brem, G. 1997. Endoscopic embryo collection and transfer into
oviduct and uterus of pigs. Theriogenology 47: 1051-1060.
Besenfelder, U., Muller, M and Brem, G. 1998. Transgenics and modern reproductive technologies In:
Rothschild, MR and Ruvinsky, A. (ed.) The Genetics of the Pig. CAB International Wallingford,
UK, 345-374.
Bwanga, CO., Ekwall, H and Rodriguez Martinez, H. 1991. Cryopreservation of boar sperm: III.
Ultrastructure of boar spermatozoa frozen ultra-rapidly at various stages of conventional freezing
and thawing. Acta Veterinaria Scandinavica 32: 463-471
Bwanga, CO., Hofomo, PO., Grvle, IS., Einarsson, S and Rodriguez Martinez, H. 1991. In vivo fertilizing
capacity of deep-frozen boar semen package in plastic bags and maxi-straws. Zentralblatt der
Veterinarmedizinischen Akademie. 38: 281-286.
Cameron, RDA., Durack, M., Fogarty, R., Putra, DKH., and McVeigh, J. 1989. Practical experience with
commercial embryo transfer in pigs. Australian Veterinary Journal. 66: 314-318.
Day, BN. 2000. Reproductive biotechnologies: current status in porcine reproduction. Proceedings of the
th
14 International Congress on Animal Reproduction, Stockholm, Sweden, 2-6 July, 2000. PP
160-172.
Fiser, PS., Fairfull, RW., Hansen, C., Panich, PL., Shrestha, JNB and Underhill, L. 1993. The effect of
warming velocity on motility and acrosmal integrity of boar sperm as influenced by the rate of
freezing glycerol levels. Molecular Reproduction and Development 34: 190-195.
Foxcroft, GR. 1996. Current and future research in swine reproduction: Opportunities and challenges.
Proceedings of the National Workshop on Swine Research and Technology Transfer. January
12 & 13, 1996: 29-37.
Foxcroft, GR., Xu, X., Seth, PC., Harbison, DS and Cheung, AP. 1995. Semen dilution for assessment of
boar ejaculate quality in pigs IVM and IVF systems. Theriogenology. 35: 212-215.
Funahashi, H and Day, BN. 1993. Effects of follicular fluid at fertilization in vitro on sperm penetration in
pig oocytes. Journal of Reproduction and Fertility. 99: 97-103.

This paper was presented at the 2001 Focus on the Future Conference, February 20-21, 2001
Red Deer, Alberta, Canada
 64

Georges, M and Massey, JM. 1991. Velogenetics, or the synergistic use of marker assisted selection and
germ-line manipulation. . Theriogenology. 35: 151-159.
Gordon, I. 1997. Embryo transfer and associated techniques in pigs. In: Gordon, I. (ed.) Controlled
Reproduction in Pigs. CAB International Wallingford, UK, 183-217.
Hammond, K and Leitch, HW. 1998. Genetic resources and global program for their management. In:
Rothschild, MR and Ruvinsky, A. (ed.) The Genetics of the Pig. CAB International Wallingford,
UK, 405-425.
Hazeleger, W and Kemp, B. 1999. State of the art in pig embryo transfer. Theriogenology. 51: (1) 81-90.
Johnson, LA. 1991. Sex preselection in swine: Altered sex ratios in offspring following surgical
insemination of flow sorted X-and Y- bearing sperm. Reproduction in Domestic Animals. 26:309-
314.
Johnson, LA., Welch, GR and Rens, W. 1999. The Beltsville sperm sexing technology: High-speed sperm
sorting gives improved sperm output for in vitro fertilization and AI. Journal of Animal Science. 77
(Suppl.2):213-220.
Johnson, LA., Welch, GR., Rens, W and Dobrinsky, JR. 1998. Enhanced flow cytometric sorting of
mammalian sperm: High speed sorting and orienting nozzle for artificial insemination.
Theriogenelogy 49:361 (Abstr.)
Kashiwazaki, N., Ohatani, S., Miyamoto, K and Ogawa, S. 1991. Production of normal piglets from
0
hatched blastocysts frozen at –196 C. Veterinary Record. 16: 256-257.
Kim, HS and Oguri, N. 1990. Studies on embryo transfer in pigs. II. Factors affecting pregnancy rate and
litter size. Korean Journal of Animal Science. 32: 125-130.
Laurincik, J., Rath, D and Niemann, H. 1994. Differences in pronucleus formation and first cleavage
following in vitro fertilization between pig oocytes matured in vivo and in vitro. Journal of
Reproduction and Fertility. 102: 227-284.
Leiding, C. 2000. Prevention of disease transmission by use of semen in the porcine AI industry.
Livestock production Science, 62: 221-236.
Li, J., Rieke, A., Day, BN and prather, RS. 1996. Technical Note: Porcine non-surgical embryo transfer.
Journal of Animal Science. 74: 2263-2268.
Long, CR., Rath, D., Welch, GR., Schreier, LL., Dobrinsky, JR and Johnson, LA. 1998. In vitro production
of porcine embryos from semen sorted for sex with a high-speed cell sorter: comparison of two
fertilization media. Theriogenelogy 47: 363 (Abstr.)
Mattioli, M., Bacci, ML., Galeati., and Seren, E. 1988. Developmental competence of pig oocytes matured
and fertilized in vitro. Theriogenology. 31:1201-1207.
Nagai, T. 1994. Current status and perspectives in IVM-IVF of porcine oocytes. Theriogenology. 41:73-
78.
Nagai, T., Niwa, K and Iritani, A. 1984. Effect of sperm concentration during preincubation in a defined
medium on fertilization in vitro of pig follicular oocytes. Journal of Reproduction and Fertility. 70:
271-275.
Nagashima, H., Kashiwazaki, N., Ashman, RJ and Grupen, CG and Nottle, MB. 1995. Cyropreservation
of porcine embryos. Nature. 374: 416.
ST
Nicholas, FW. 1997. A review – pig genetics into the 21 century. Manipulating Pig Production VI,
Australasian Pig Science Association, Werribee, Victoria, and Australia.149-164.
Ortman, K and Rodriguez Martinez, H 1994. Membrane damage during dilution, cooling and freezing
thawing of boar spermatozoa packaged in plastic bags. Zentralblatt der Veterinarmedizinischen
Akademie. 41:37-41.
Polge, C and Day, BN. 1968. Pregnancy following nonsurgical egg transfer in pigs. Veterinary Record.
82: 712.
Polge, C. 1982. Embryo transplantation and preservation. In. Cole, DJA and Foxcroft, GR (eds) Control of
Pig Reproduction. Butterworth Scientific, London, 277-291.
Rath, D., Johnson, LA., Dobrinsky, JR., Welch, GR and Niemann, H. 1997. Production of piglets
preselected for sex following in vitro fertilization with X and Y chromosome -bearing spermatozoa
sorted by flow cytometry. Theriogenelogy 47: 795-800.
Rath, D., Niemann, H and Torries, CRL. 1995. In vitro development to blastocysts of early porcine
embryos produced in vivo. Theriogenology. 43: 913-926.

This paper was presented at the 2001 Focus on the Future Conference, February 20-21, 2001
Red Deer, Alberta, Canada
 65

Recihelt, B and Niemann, H. 1995. Inner cell mass and trophoblast cells in demi- and intact porcine
th
embryos. Proceedings of the 11 Meeting of the European Embryo transfer Association
(Hanover), p.230.
Rothschild, MF and Bidanel, JP. 1998. Biology and genetics of reproduction. In: Rothschild, MR and
Ruvinsky, A. (ed.) The Genetics of the Pig. CAB International Wallingford, UK, 313-343.
Rothschild, MF., Vaske, DA., Tuggle, CK.., Messer, LA., McLaren, DG., Short, TH., Eckardt, GR.,
Mileham, AJ and Plastow, GS. 1995. Estrogen receptor locus is a major gene for litter size in the
pig. In: Van Arendonk, JAM (ed.) Book of Abstracts of the European Association for Animal
Production, p. 53. (Abstr.)
Seidel, GE., Allen, CH., Johnson, LA.., Holland, MD., Brink, Z., Welch, GR., Graham, JE and Cattell, MB.
1997. Uterine insemination of heifers with very low numbers of unfrozen and sexed spermatozoa.
Theriogenelogy 48: 1255-1265.
Van der Lende, T. 1998. Optimism over embryos. PIG INTERNATIONAL. 45 (10): 27-28.
Van Vleck, LD 1999. Implications of cloning for breed improvement strategies: Are traditional methods of
animal improvement obsolete? Journal of Animal Science. 77 (Suppl.2): 111-121.
Visscher, P., Pong-Wong, R., Whittemore, C and Haley, C. 2000. Impact of biotechnology on
crossbreeding programmes in pigs. Livestock production Science, 62: 57-70.
Webb, AJ. 1991.New developments in the genetic improvement of lean growth. Proceedings of the
Fifteenth Annual Saskatchewan Pork Industry Symposium. November 13 and 14, 1991: 109-117.
Webb, AJ. 1991.Profit on the farm through genetics. Proceedings of the Fifteenth Annual Saskatchewan
Pork Industry Symposium. November 13 and 14, 1991: 1-11.

Webb, AJ. 2000. New technology for genetic improvement of livestock. Proceedings of the
Twenty-first Western Nutrition Conference, Winnipeg, Manitoba. September 28 and 29, 2000: 83-
96.

This paper was presented at the 2001 Focus on the Future Conference, February 20-21, 2001
Red Deer, Alberta, Canada