Human embryogenesis

Human embryogenesis is the process of cell division and cellular differentiation of the human
embryo during early prenatal development. It spans from the moment of fertilization to the end
of the 8th week of gestational age, whereafter the embryo is called a fetus.

From one cell to blastocyst

A human develops from a single cell called a zygote, which results from the fusion of two
reproductive cells; an ovum (egg) being fertilized by a single spermatozoon (sperm). The cell is
surrounded by a strong membrane of glycoproteins called the zona pellucida which the
successful sperm has managed to penetrate.

The zygote undergoes cleavage, increasing the number of cells within the zona pellucida. After
the 8-cell stage, embryos undergo what is called compactation, where the cells bind tightly to
each other, forming a compact sphere. After compactation, the embryo is in the morula stage (16
cells). Cavitation occurs next, where the outermost layer of cells - the trophoblast - secrete
water into the morula. As a consequence of this when the number of cells reaches 40 to 150, a
central, fluid-filled cavity (blastocoel) has been formed. The zona pellucida begins to
degenerate, allowing the embryo to increase its volume. This stage in the developing embryo,
reached after four to six days, is the blastocyst (akin to the blastula stage), and lasts
approximately until the implantation in the uterus, and is referred to as the preimplantation
phase of development.

Each cell of the preimplantation embryo is totipotent. That is, each cell has the potential to form
all of the different cell types in the developing embryo. This totipotency means that some cells
can be removed from the preimplantation embryo and the remaining cells will compensate for
their absence. This has allowed the development of a technique known as preimplantation
genetic diagnosis (PGD), whereby a small number of cells from the preimplantation embryo
created by IVF, can be removed by biopsy and subjected to genetic diagnosis. This allows
embryos that are not affected by defined genetic diseases to be selected and then transferred to
the mother's uterus.

Blastocyst differentiation

Blastocyst with an inner cell mass and trophoblast.

The blastocyst is characterized by a group of cells, called the inner cell mass (also called
embryoblast) and the mentioned trophoblast (the outer cells).
The inner cell mass gives rise to the embryo proper, the amnion, yolk sac and allantois, while
the trophoblast will eventually form the placenta. The blastocyst can be thought of as a ball of a
(mostly single) layer of trophoblast cells, with the inner cell mass attached to this ball's inner
wall. The embryo plus its membranes is called the conceptus. By this stage the conceptus is in
the uterus. The zona pellucida ultimately disappears completely, allowing the blastocyst to
invade the endometrium, performing implantation.

Implantation

The trophoblast then differentiates into two distinct layers: the inner is the cytotrophoblast
consisting of cuboidal cells that are the source of dividing cells, and the outer is the
syncytiotrophoblast.

The syncytiotrophoblast implants the blastocyst in the endometrium (innermost epithelial lining)
of the uterus by forming finger-like projections called chorionic villi that make their way into
the uterus, and spaces called lacunae that fill up with the mother's blood. This is assisted by
hydrolytic enzymes that erode the epithelium. The syncytiotrophoblast also produces human
chorionic gonadotropin (hCG), a hormone that "notifies" the mother's body that she is pregnant,
preventing menstruation by sustaining the function of the corpus luteum. The villi begin to
branch, and contain blood vessels of the fetus that allow gas exchange between mother and
child.

Inner cell mass differentiation

While the syncytiotrophoblast starts to penetrate into the wall of the uterus, the inner cell mass
(embryoblast) also develops.

The embryoblast forms a bilaminar (two layered) embryo, composed of the epiblast and the
hypoblast. The epiblast is adjacent to the trophoblast and made of columnar cells; the hypoblast
is closest to the blastocyst cavity, and made of cuboidal cells. The epiblast, now called primitive
ectoderm will perform gastrulation, approximately at day 16 after fertilization. In this process, it
gives rise to all three germ layers of the embryo: ectoderm, mesoderm, and endoderm. The
hypoblast, or primitive endoderm, will give rise to extraembryonic structures only, such as the
lining of the primary yolk sac.

Cavity formation
By separating from the trophoblast, the epiblast forms a new cavity, the amniotic cavity. This is
lined by the amnionic membrane, with cells that come from the epiblast (called amnioblasts).
Some hypoblast cells migrate along the inner cytotrophoblast lining of the blastocoel, secreting
an extracellular matrix along the way. These hypoblast cells and extracellular matrix are called
Heuser's membrane (or exocoelomic membrane), and the blastocoel is now called the primary
yolk sac (or exocoelomic cavity).
Cytotrophoblast cells and cells of Heuser's membrane continue secreting extracellular matrix
between them. This matrix is called the extraembryonic reticulum. Cells of the epiblast migrate
along the outer edges of this reticulum and form the extraembryonic mesoderm, which makes it
difficult to maintain the extraembryonic reticulum. Soon pockets form in the reticulum, which
ultimately coalesce to form the chorionic cavity or extraembryonic coelom.

Another layer of cells leaves the hypoblast and migrates along the inside of the primary yolk
sac. The primary yolk sac is pushed to the opposite side of the embryo (the abembryonic pole),
while a new cavity forms, the secondary or definitive yolk sac. The remnants of the primary yolk
sac are called exocoelomic vesicles.

Susceptibility
Toxic exposures during the first two weeks following fertilization (second and third weeks of
gestational age) may cause prenatal death but do not cause developmental defects. Instead, the
body performs a miscarriage. On the other hand, subsequent toxic exposures in the embryonic
period often cause major congenital malformations, since the precursors of the major organ
systems are developing.

The embryogenesis (or embryogénie ) human indicates the development process of the
human Embryon since the stage of Zygote until the birth.

Stages of embryogenesis
The Fecundation

The zygote is born from the union of a Gamète female (a Ovocyte), and of a male gamète, (a
Spermatozoïde). The gamètes are produced by the Méiose germinal cells. The male gamète is
thus haploid: it contains only a Chromatide of each Chromosome of the germinal cell which
generated it; while the gamète female contains a chromosome with two chromatides (it will
finish its meiosis only if it there has fecundation, and will then become haploid).

The chromatides homologous with the male and the female are linked in the zygote. This one
and its descent thus contains “mixed” chromosomes. Moreover, the Gène S of those can cross
(what one calls “Crossing-over”) while passing from one chromatide to another to ensure an
optimal variation of the following generation. Consequently, the zygote contains all the
necessary informations to be transformed into living organism, by a complex process of
segmentation and cellular differentiation.

The segmentation: egg with the morula

The fertilized egg will undergo a series of cellular divisions during its migration in the
Fallopian tube. This process bears the name of segmentation. This segmentation is total and
asynchronous. It initially divides the zygote into 2 cells girls, then 3 (largest blastomère is
divided into first), then 6 and 8 and so on for quickly leading to a cellular mass bearing the
name of Morula. The process of segmentation bears also the name of cleavage. This term is
perfectly evocative since the fertilized egg does not increase, or little, of volume during
these first successive divisions. The first cellular divisions, until stage 4 to 8 cells, do not
objectify important morphological differences between the cells girls. Starting from stage 8
to 16 cells, the compaction will initiate the first events of embryonic differentiation. The
compaction generates a new distribution of the cells of future the morula: a) the peripheral
cells will undergo a polarization and are divided into a layer which surrounds all surface of
fertilized egg. These polarized cells constitute the primitive trophoblaste b) the internal
cells and initially nonpolarized gather to constitute the mass of the embryoblaste. At the end
of the fourth day after fecundation, the morula starts to grow hollow of a cavity with liquid
contents (future Blastocèle).

Cavitation

The trophoblastic cells secrete a fluid which will push the cellular cluster in a corner of the
sphere. The Chorion, trophoblastic cell external coming from cavitation, makes it possible to
receive the oxygen of the mother. It secretes hormones which order with the uterus to
accommodate the fetus. Certain substances control the maternal immunizing response so
that there is no rejection of the embryo (as it is the case of a grafted body).

The blastulation

At the mankind, the Zygote (or egg) is of type Oligolécithe: the Vitellus, in minor amount, is
dispersed there uniformly in all the Cytoplasme (consult the article on the Zygote for more
details).

At the first stage of the blastulation, the zygote is divided into blastomères by Mitose. As for
all the zygotes oligolécithes, the process is of type holoblastic and radiate. The first division
is done according to a southernmost plan passing by the poles of the zygote. Then, one of
both blastomères resulting also divides according to a southernmost plan. The other
blastomère, on the other hand, divides according to an equatorial plan. Progressively of
successive divisions the cells, remaining bound, form the Morula. The external cells of this
one form the Trophoblaste which will constitute the surface layer of the Placenta. The
internal cells are bound with a pole of the morula and form the cellular mass interns as well
as a cavity, the Blastocèle.

At this stage, the morula became Blastula or Blastocyste.

The gastrulation

Introduction
The process of Gastrulation sets up the cellular structures which generate the embryo and
support the development of it.

For this purpose, the cells of the blastocyste are different, move and are rearranged to form:
 • embryonic fabrics (germinal layers);
• the fabrics extraembryonnaires (structures of support).

At the mankind, the gastrulation is of the type “immigration”. An embryonic disc is formed
on the level of the embryonic button. The individual cells migrate through the disc with
concomitant differentiation in ecto-, endo- and mésoderme. This process is applicable to all
the mammals, like their ancestors the reptiles and the birds.

The embryo didermic and feeder functions

With the apical pole of the blastocyste an internal cluster of cells is formed, whether one
calls the embryoblaste or embryonic button. It is there that the genesis of the embryo starts.

The feeder function of the peripheral cells of the blastocyste is specified. They constitute
the trophoblaste which will be divided into syncytiotrophoblaste (multicellular cytoplasm)
outside, and in cytotrophoblaste inside.

The embryoblaste is divided into hypoblaste and épiblaste. Between this one and the
cytotrophoblaste digs the amniotic cavity. The épiblaste and the hypoblaste form the
embryonic disc together didermic.

Cells of the épiblaste are different in amnioblastes and recover the interior wall of the
amniotic cavity.

In the syncytiotrophoblaste vacuoles open which are connected between them to form
capillaries which extend in uterine fabrics to constitute the maternal sines. The vacuoles
form gaps which fill of blood. These vessels are at the base of utéro-placental circulation.

First migration of cells coming from the épiblaste along the internal wall of the
cytotrophoblaste to form there a fine membrane (endoderm extraembryonnaire, also called
“membrane of Heuser”) which transforms the blastocèle into primary education vitelline bag
(in J-11) .

Appearance of the mésoderme and growth of the amnion

The reticulum extraembryonnaire (a fabric acellulaire) appears between the membrane of
Heuser and the cytotrophoblaste. It opens there vacuoles which amalgamate by forming the
cavity extraembryonnaire which fills of liquid. It is supposed that the reticulum is generated
by the membrane.

Mesodermic cells, which would be epiblastic cells differentiated coming from the caudal part
of the embryonic disc, migrate through the reticulum and while being fixed the walls of the
cavity extraembryonnaire recover. This mésoderme extraembryonnaire is called "
Somatopleure " on the level of the cytotrophoblaste, amniotic cavity and embryonic pedicle
and " Splanchnopleure " on the level of the endoderm extraembryonnaire of the primary
education and secondary vitelline bags.

A new migration of endodermal cells extraembryonnaires pushes back the cells of the
preceding one. The primary education vitelline bag becomes secondary vitelline bag while
retracting under the push of the cavity extraembryonnaire and leaving a remainder which
will reabsorb quickly.

The cavity extraembryonnaire reabsorbs under the effect of the growth of the amniotic
cavity. The cœlomic liquid is lost and the somatopleures extraembryonnaires amnion and
cavity extraembryonnaire amalgamate.

The tridermic embryo

Cells of the convergent épiblaste towards the dorsal longitudinal axis of the embryonic disc
and form a pad called there “primitive line”. From this one, the epiblastic cells are inserted
and disperse in the disc, transforming the pad into furrow. The epiblastic cells know triple
destiny:

• Some will fit between the cells of the hypoblaste by forming final embryonic
endoderm
• others stop between the épiblaste and the hypoblaste and is different in mésoderme
embryonic final
• Those which remained in the épiblaste become the final embryonic ectoderm.

At this stage, the embryo is tridermic. It includes/understands an ectoderm, an endoderm

and a mésoderme, all having been formed starting from the épiblaste.

The structuring of the embryonic disc

The appearance of the primitive line defines not only the bilateral symmetry of the disc, but
also the caudal-crâniale orientation of its axis. The “node of Hensen”, a depression located
about the middle of the axis, finishes the primitive line.

At the caudal end of the embryonic disc, a zone of the ectoderm remained stuck to
endoderm: it is the cloacal membrane, the counterpart of the blastopore of the invaginated
gastrulas. In the same way, a membrane called pharyngienne (or bucco-pharyngienne) is
formed towards the crâniale end: its rupture will put the primitive mouth in contact with the
Pharynx.

The neurulation
At the beginning of the process of neurulation it there with the installation of the Chorde.
This one starts with a proliferation of mesodermic cells with height of the node of Hensen.
Then, the cells migrate towards the pole crânial and form a hollow tube, the process (or
tubes) notochordal, in the axis of the disc, between the ectoderm and endoderm. Initially,
the tube notochordal amalgamates with endoderm forming the plate chordale thus. Later, it
separates from endoderm and becomes thus a full roller: the chorde itself.

The presence of the chorde induces the formation of the plate neurale starting from the
overlying ectoderm. This one develops in width with the level of the pole crânial and will
produce the Cerveau there. Towards the caudal pole, it takes the shape of a gutter whose
edges, while being closed again, are at the origin of the Spinal-cord.

In short:
 • Formation of the prosencéphale, the mésencéphale and the rhombencéphale
• complete Invagination of the gutter neurale and insulation of the tube neural
• Extension of the tube neural in direction of the poles of the disc
• Appearance of the peaks neurales whose migratory cells will form many fabrics of the
organization

The metamerisation
Meanwhile, the mésoderme intraembryonniare is different. The para-axial mésoderme,
located on both sides chorde, is compacted by forming 44 pairs of Somite S, to start with the
pole crânial. During the process of individualization (the metamerisation), those remain
connected to the mésoderme by the intermediate blade. The side blade separates in two
layers: the somatopleure which recovers the ectoderm and the splanchnopleure which
recovers endoderm.

It is starting from the celebrities that constitute themselves:

• the sclérotome which is at the origin of the formation of the vertebrae and the
formative mésenchyme of the intervertebral discs
• the dermatomes which, while diffusing, form the derm of the neck and of the trunk
• the myotomes which produce the muscles

The arteriovenous system is set up, allowing the first exchanges between the embryo and the
mother:

• a circulation extraembryonnaire on the level of trophoblastic villosities

• a primitive circulation intraembryonnaire

Delimitation of the embryo

The delimitation and the shape of the body of the embryo are induced by a differential
growth between the amnion and the vitelline bag. This one developing practically more, the
amnion overflows it on all the sides

Laterally, the amnion recovers the embryonic disc: the tube neural, the chorde, the
mésoderme intraembryonnaire and part of vitelline which will become the primitive
intestine. The edges of the various layers meeting at the base of the disc, those amalgamate
and close the body of the embryo which becomes three-dimensional. However, a channel
remains temporarily between the intestine and the vitelline bag. Longitudinally, the embryo
adopts a convex form.
 Embryo Culture
If you and your partner are about to undergo IVF treatment, or if you are considering pursuing the IVF
process, than it is important to know as much as you can about the steps involved. IVF is a very
delicate process, and it is essential that all steps are performed correctly in order for it to be
successful. One of the most important parts of IVF is the embryo culture stage. It is during this stage
that an embryo will be formed and nurtured until it is ready to be transferred into your uterus.

What is embryo Culture:

Embryo culture is the term used to describe the process immediately follow egg retrieval. It is during
the culture process that your eggs and your partner?s sperm will be combined in order to produce a
fertilized egg (known as an zygote). Once a zygote has been formed, the culture process will continue
in order to encourage the growth of the zygote into an embryo. Lasting from 2 to 5 days, the embryo
culture process is vital to the success of any IVF procedure. Without accurate and controlled embryo
culture, IVF transfer may not be successful.

Fertilization:
Immediately following your IVF retrieval, any aspirated follicular fluid will be transported to your
fertility clinic ?s laboratory. Here, your follicular fluid will be examined under a microscope, in order to
identify all eggs that are present. Each egg and it?s surrounding cells will then be washed in a special
medium, in order to remove any toxins and impurities. These eggs will then be transferred, in
separate dishes, to a special incubator containing carbon dioxide. The eggs will remain in this
incubator until fertilization is ready to take place. This usually happens between two and six hours
after egg retrieval, depending upon the maturity of the eggs.

When the eggs are matured, they will each be combined with some of your partner?s sperm. His
sperm will have been washed and divided up into specific amounts. Typically, no more than 100,000
sperm per milliliter are used during the fertilization procedure. The sperm and egg will be combined in
a dish that contains special culture medium. This culture medium, made up of protein, salt, and
antibiotics, is designed to help the embryo during the first days of division. The dish is then placed
back inside of the incubator.

Monitoring:
Your developing embryos will be monitored carefully by an embryologist , a person who specializes in
embryo development for IVF and other fertility treatments . After 18 hours of development, your
embryologist will make the first check on your embryos. By this stage, your embryos will still be single
cells. However, they will contain two clear bubbles (known as pronuclei) inside. These pronuclei are
evidence that the embryo contains genetic material from both you and your male partner. Embryos
without pronuclei are discarded.

Your embryos will then be left to develop for another 24 hours. At this point, embryos will be
monitored for cell division. Most embryos have developed into two or four-cell embryos at this point.
Some laboratories will allow embryos to continue culturing, while other labs will proceed with embryo
transfer at this point. Depending upon your particular health needs, you may or may not choose to
have a transfer done at this point.

Embryos can be cultured for a various lengths of time, depending upon the reproductive history of you
and your partner. Embryos can be cultured for:
• Two Days: Embryos that are cultured for two days are generally transferred at the two or four-cell
stage. This type of transfer is beneficial for couples who have a low number of embryos available
for transfer, or who have embryos that are developing poorly.
• Three Days: Embryos that are cultured for three days are usually transferred at the six to eight
cell stage. Many laboratories prefer to culture embryos until this stage because it allows for
increased monitoring. Embryos cultured for three days can be checked by the embryologist for
gene activation and cleavage, which improves the likelihood of transferring a viable embryo.
• Five Days: Embryos that are cultured for five days are transferred at the blastocyst stage.
Blastocysts consist of 12 to 16 cells and are well on their way to be ready for implantation into the
uterus Many labs opt to transfer at the blastocyst stage, particularly if you have had repeated
miscarriages or IVF failures.

The environments in which your embryos are cultured are of the utmost importance when it comes to
completing the culture stage successfully. Some essential components in IVF culture environments
include:

• Culture Medium: All developing embryos are cultured is a special medium, designed to help
them develop and grow. There are generally two types of culture mediums used by laboratories:
one is for initial embryo development (up to 3 days) and the second if for later development (up to
blastocyst stage).
• Temperature: Embryos need to be cultured at a specific temperature to ensure survival. The
temperature inside of the embryonic incubators is maintained at 37 degrees Celsius. This is the
same temperature that is found inside of your fallopian tubes.
 Biotechnology
Biotechnology is truly multidisciplinary in nature and it encompasses several disciplines
of basic sciences and engineering. The science disciplines from which biotechnology
draws heavily are; microbiology, Chemistry, biochemistry, genetics, molecular biology,
immunology, cell and tissue culture and physiology. On the engineering side, it leans
heavily on process chemical and biochemical engineering since large scale cultivation of
microorganisms and cells , their down stream processing etc. are based on them.

Here are the some of the Key Contribution / Application of Biotechnology to the Human
Welfare.

A. Medical Biotechnology

• Monoclaonal antibodies (used for disease eg. hepatitis B and other viral
disease, cancer etc.)
• DNA probes ( used for disease diagnosis, eg Kala azar, sleeping sickness
, malaria etc)
• Recombinant Vaccine ( Human hepatitis vaccine ( the one by Shantha
Biotech in India)
• Production of valuable drugs like Human insulin, human interferon,
human and bovine growth hormones etc.
• Gene therapy to cure genetic diseases eg. Cystic fibrosis
• Babies of specified sex through artificial insemination ( by separation of
X/ Y Chromosomes)
• Identification of parents / Criminals using DNA or autoantibody
fingerprinting( In India CDED, Hyderabad id the center where this work
is going on and the Dr. Lalji Singh is the person who had promote these
technique in India and currently is Director CCMB, Hyderabad.

B. Plant Biotechnology

• Gene Transfers ( genetic Engineering ) for insect resistance , protection

against viruses , herbicide resistance , storage protein improvements etc. (
i.e. production of Transgenic Plants through Genetic Engineering )
• Molecular markers eg RFLPs and RAPDs for linkage mapping and
mapping of quantitative trait loci.
• Germplasm conservation through storage in Liquid nitrogen or by slow
growth
 • Rapid clonal multiplication through meristem culture eg of many fruit
and forest trees, such as teak.
• Rapid isolation of homozygous line by chromosome doubling of haploids
produced through anther culture / interspecific hybridization / ovary
culture.( Very successful in variety development in China eg in rice and
wheat)

C. Animal Biotechnology
• Hormone –induced super ovulation and / or embryo splitting in farm
animals, involves embryo transfer and, in many cases in vitro
fertilization. ( For rapid multiplication of animals of superior genotypes)
• Production of transgenic animals for increased milk , growth rate,
resistance , resistance to disease etc. and production of some valuable
proteins in milk / urine / blood.

D. Environmental Biotechnology

• Efficient sewage treatment , deodorization of human excreta

• Degradation of petroleum and management of oil spills
• Detoxification of wastes and industrial effluents
• Biocontrol of plant deisese and insect pests by using viruses, bacteria,
amoebae, fungi etc.

E. Industrial Biotechnology
• Production of useful compounds eg. Ethanol, lactic acid, glycerine , citric
acid , gluconic acid , acetone etc.( produced by microorganisms , mainly
bacteria , from less useful substrates)
• Production of antibiotics eg penicillin, streptomycin. Erythromycin,
mitomycin, cycloheximide etc. (produced by fungi, bacteria and
actinomycetes as secondary metabolites.)
• Fuel produced from cheap, less useful and abundant substrates e.g.
Sugarcane biogases, wood etc.

Mineral Extraction through leaching from low grade ores, eg, copper , uranium etc.
Biotechnology of Reproduction and Farm Animals Welfare

Introduction

Based on the progress in scientific knowledge of endocrinology, reproductive physiology, cell biology
and embryology during the last fifty years new biotechniques have been developed for and introduced
into animal breeding and husbandry. Among them are oestrussynchronisation/induction, artificial
insemination, multiple ovulation induction and embryo transfer (MOET), in vitro embryo production
(IVP) and cloning by nuclear transfer (NT). The aims of these reproductive technologies were initially
to speed up the genetic improvements of farm animals by the increase of offspring of selected males
and females and the reduction of the generation intervals. The technique of cloning by nuclear
transfer is mainly applied for experimental purposes, with the prospect of a more practical
implementation in the near future, with the aims of the enhancement of the uniformity of herds for
an easier management or for the multiplication of transgenic animals after gene-targeting.

Parallel to these developments public concern about new biotechnologies has grown, and call now,
anno 2000, scientists to account. Although concerns of the general public may be related to a variety
of reasons (i.e.: fear for food quality, the aversion for the attidude of playing God, or the feeling that
something happens which cannot be controlled and will lead to an unwanted society), an important
part of societal concerns seems to involve questions about the welfare of the animals, even though
solid information on the consequences of new biotechnologies for animal welfare is generally lacking.
Also scientists directly involved in the development of new biotechnologies have recognized the
importance of animal welfare, but, perhaps understandably, their prime concern is technological
progress. Thus, animal welfare is often treated as an ethical or public perception issue rather than as
a biological one, and, so far, scientist in the field of farm animal biotechnology generally fail to specify
concrete steps to monitor and prevent possibly adverse effects of the treatments imposed.

In this paper we evaluate the subsequent technologies and judge them on their effects on animal
welfare by use of biologically measurable parameters. We argue that the welfare of farm animals
requires special attention. We would like to suggest that the introduction of new biotechniques into
farm animal husbandry should be accompanied by a study of health and welfare with the help of a
comprehensive welfare protocol for the benefit of a sustainable animal production.

In this paper, we provide evidence demonstrating that new biotechnologies may have profound and
negative effects on essential biological functions and systems in the animals involved, and, hence,
may detrimentally affect animal welfare. Therefore, we argue that within the context of farm animal
biotechnologies, animal welfare should receive special attention. We would like to suggest that the
introduction of new biotechnologies into farm animal husbandry should be accompanied by systematic
and scientifically valid studies into the effects on animal welfare, with the help of a comprehensive
welfare protocol.

Sustainable production

Incompatible with sustainable production is in our opinion each situation in which the animal cannot
reproduce anymore without help of one of the modern techniques. Since the new techniques are
aimed to accelerate the selection for some traits, the results of a particular breeding programme can
be incompatible with sustainable production. For example, the selection of turkeys for meet
production has lead to large and overweighted cocks in such a way that fertilisation of the hens can

only be done by A.I.. The double muscled belgium blue cows cannot reproduce without the help of
the caesarian section. Transfer of embryos of meat cows into dairy cows may lead to more dystocia,
etc. Not the techniques themselves are by definition incompatible with animal welfare but the goals
for what these techniques are used.

Incompatible with sustainable production is also the situation in which the public concerns about
animal welfare in connection with the modern reproductive biotechnologies leads to abjuration of the
farm products.

Artificial insemination

The most universally adopted (zoo)technique in cattle seems to trigger little concerns. Nobody has
thought that A.I. may already be deviated from the natural situation. However, palpation of the
genital tract per rectum as well as the artificial insemination into the uterine cavity cause an increase
in plasma cortisol levels as demonstrated by Nakao et al., (1994). When genetically valuable bulls will
not mount on a dummy or on living animals (lack of libido) they often are subjected to
electrostimulation to induce an ejaculation. This procedure leads to a strong release of ACTH,
followed by a rise in cortisol levels (Colenbrander et al., personal communication). The question can
be raised whether a rising cortisol level is an indication of stress and seriously affected welfare. But in
many other species of small farm animals including sheep, there is a trend towards the use of more
invasive insemination procedures, like intrauterine insemination via laparoscopy or laparotomy with
minimal anaesthesia. It is argued by breeder organisations that with experienced operators the stress
is minimal. They found little evidence that a single laparoscopic procedure affects stress sensitive
physiological events like the timing of ovulation (Walker et al., 1986). Notwithstanding these opinions,
objective measurements have to be developed to judge the degree of stress.

Oestrussynchronisation and oestrusinduction

Oestrussynchronisation has been developed in the early sixties of the last century. It started with
methods based on the artificial replacement of a corpus luteum by progestagens. Withdrawal of the
progestagen device, either a sponge (sheep), a PRID or ear implant (cattle) at one moment for the
whole herd resulted in the synchronous oestrus. The techniques were aimed to help a large scaled
introduction of A.I. in the sheep industry and in the extensive beef industry. The fertility of the herd
in the synchronized oestrus was compromised and lowered by 10 to 15%. Research to understand
what was going on revealed that synchronisation of the oestrus does not mean a concurrently
synchronisation of the genital tract (Kruip 1972; 1973). The results lead to the assumption that 15%
reduction of the fertility is due to low fertilisation during the synchronised oestrus and/or early
embryonic death. Whether this is compatible with animal welfare and sustainable production has to
be discussed. Till the moment that cryopreservation of embryos was introduced, the oestrus
synchronisation was used in embryo transfer for the synchronisation of donors and recipients.
Oestrusinduction is based mainly on the same treatments with the same drugs. In many cases it will
be used as a medication of animals suffering of sub- or even anoestrus. But even then one should
asked whether that treatment is more or less a symptom contest. Multiple ovulation and embryo
transfer

Both multiple ovulation induction and embryo transfer are generally accepted technologies. However,
the transfer of embryos after multiple ovulation has given increase in embryonic death, larger calves
with longer gestation times, and more dystocia (van Wagtendonk-de Leeuw et al. 2000).

Although, at present, the mechanisms underlying these effects are unknown, multiple ovulation
induction has been found to be associated with a number of disturbances which might be linked to
these abnormalities. Firstly, multiple ovulation induction can lead to an asynchrony in the
development of the preovulatory follicle and the meiotic process of the oocyte (deLoos 1992; Hyttel
et al. 1986). Secondly, the growth of a cohort follicles results in abnormally high oestradiol-17b (E2)
before and in high progesterone (P4) levels after ovulation, respectively. It has been demonstrated
that high levels of E2 affect the microtubular organisation, meiosis and extrusion of polar bodies
(Kruip et al. 1988). Earlier and higher P4 levels may affect the uterine environment and result in

asynchrony between the developmental stage of the embryo and the uterine environment at the
moment of transfer. P4 treatment during the first 3 days of pregnancy has been shown to promote
the development of advanced embryos in the uterus (Wilmut et al. 1981; Klemann et al.1994).
Transfer of advanced embryos, i.e. longer embryos with more cells, increases the rate of
abnormalities in resulting calves (Lazzari et al., personal communication). Moreover, in a study of
Dorland et al. (1993), a high percentages of mixoploid embryos was detected, as well as one totally
haploid and one completely triploid embryo, after superovulation. These abnormal configurations of
nuclear chromosomes cannot be detected by examining the morphology of the embryos. The
embryos look good and will be transfered. According to Karwasky et al. (1996), 27- 45% of the
embryos after multiple ovulation induction have an abnormal karyogram and are expected to die
shortly before or after attachment.

The welfare of oocyte/embryo donors and recipients

In addition to well-documented and clearly undesirable side-effects on the offspring, it is conceivable

that MOET, IVP and NT, may also negatively affect the welfare of the oocyte donor or the recipients of
embryos. An obvious source for reduced welfare of the recipients of transfered embryos is the
enhanced probability of dystocia after MOET, IVP and NT.

It might be speculated that, because of a considerable increase of the size of the ovaries, multiple
ovulation induction in cattle is associated with pain, especially during manual palpation. Although the
needle puncture through the vaginal wall of the oocyte donor is invasive, repeated puncture for OPU
was not accompanied by adhesions or any pathological changes in the tissue (Kruip et al.1994).

An other question is whether the flushing of the uterus is always without any measurable negative
effect? For the large species like cattle and horse the flushing can be done non-surgically, but for
other species like sheep and goats, the collection of embryos has to be done surgically. In the pig,
embryo collection can only be performed after surgical shortening of the uterus. Those animals can
not reproduce anymore naturally. In the face of these facts, published data on negative effects on
animal welfare in this particular situations are currently lacking.

Ovum Pick-up & Embryo production in vitro

A very good alternative for superovulation induction and embryo flushing is the recently developed
series of technologies like non-surgical transvaginal ultrasound guided ovum pick-up (OPU) followed
by in vitro maturation (IVM) and fertilisation (IVF) and embryo culture (IVC) up to the blastocyst
stage (Pieterse et al. 1988; Kruip et al. 1991; Kruip et al. 1994).

There is a large body of evidence demonstrating that, in comparison with in vivo produced controls,
the size and weight of IVP calves is higher (30% over 50 kg), the gestation time of IVP calves longer,
the % dystocia and the incidence of caesarean sections is much higher as well as the % abortions
and perinatal death (Behboodi et al., 1995; Kruip and DenDaas 1997; Van Wagtendonk-de Leeuw et
al.1998; 2000). In general the calves are less active and vital (Reinders et al. 1995). In addition, the
% of hydroallantois and congenital malformations, including abnormal limbs and spinal cords, is
increased in IVP calves and lambs. Taken together, these problems are defined as the large offspring
syndrome (LOS)(Young et al., 1998). Farin & Farin (1995) and Sinclair et al. (1997) found a
differential growth of different organs (liver, heart, kidneys and adrenal gland) after IVP. Postnatally
some IVP calves have obvious anomalies (deRoos et al. 2000).

Epidemiological studies in humans provided evidence for the association between prenatal life and
adult diseases or susceptibility to diseases (Barker 1995). Why should that be different in animals.
Birth weight is one of the first parameters to pay attention to for the judgement of normal
development. Although an embryo/fetus will develop according to its genome, the activity of the
genome can be influenced epigenetically by environmental factors. Along that way the lifetime, the
health of organs, malformations and anomalies, abnormal karyograms, gestation length, birth weight
and neonatal problems can be introduced. Most of them by inproper differentiation of the mesoderm,
ectoderm and entoderm. One can suggest that these differentiation, driven by the expression of
developmentally important genes, should have been programmed already in the ovary in the oocyte
or in the genital tract or in culture in the embryo. This programming occurs on special critical periods
of embryogenesis (Wilson et al. 1995). Changes in the timing of the expression of these genes induce
malformations later in development and might explain some birth defects. Good examples of these
are the HOX genes. Many data are available to support the relevance of this statement (Boerjan et al.
2000). Compaction and blastulation are affected by culture conditions. The same can be said about
the differentiation of the inner cell mass. Differentiation means onset of gene expression. Recent
studies have collected good data to support this concept (Wrenzicky et al. 2000; Young et al. 2000)
of disturbed gene expressions. Special genes that are affected by culture conditions are the imprinted
genes (Young 2000). Demethylation of the IGF2rec gene in in vitro produced mice embryos lead to
abnormalities like oversized offspring (Lau et al. 1997).

The most important question is now: how can the IVP culture protocol or transfer protocols affect the
molecular and morphogenetic processes govering the phylotypic period? The hypothesis and possible
answer is: "Just by inadequate conditions either in the culture or later in the uterine environment" as
pronounced by many speakers during the IETS satellite symposium "Embryonic origin of animal
health and welfare" (Dieleman et al. 2000). IVP embryos misses in their first cleavages obviously the
interaction with the mother or comparable environment. Serum seems to be one factor that has this
effect on the genome or on the mitochondria, changing the intra cellular metabolism.

The use of immature oocytes coming from atretic follicles might be an other reason for the problems
observed sofar. It has been speculate (Kruip et al. 2000) that the imprinting is lost due to atresia of
the follicle or during the maturation in vitro. If true, the selection of COCs is for that reason very
important to avoid or to reduce the % problems. In fact, the selection of the COC is the first act that
should and can be controlled much better in order to prevent problems as LOS.

Cloning by nuclear transfer

Similar to MOET and IVP, NT has also been shown to induce LOS symptoms in resulting offspring,
including high rates of peri- and postnatal death (Willadsen 1991; Garry et al. 1996; Kato et al.
1998; Chavatte-Palmer et al. 2000; McCreath et al. 2000). The occurrence of LOS symptoms after NT
may be related to in vitro embryo manipulations also used in MOET and IVP, for example IVC or
embryo transfer, but also to factors specifically associated with NT. Firstly, donor nuclei transferred to
enucleated oocytes have to go through the process of genetic reprogramming, which is the
transformation from the pattern of gene expression that is characteristic of the donor cell to one that
is appropriate for early embryonic development. This process may be incomplete and result in
inappropriate patterns of gene expression. Secondly, NT involves the exposure of reconstructed
oocytes to various environmental stimuli intended to facilitate fusion between nucleus and recipient
cytoplasm, for example electric shock or treatment with protein inhibitors (Brower 1998; Campbell
1999; Colman 1998; 2000). Such stimuli may disrupt epigenetic modifications of imprinted genes.
The relative contributions of in vitro embryo culture or nuclear transfer to the induction of
abnormality remain to be determined (Wilmut et al. 1998; Hill et al. 1999; Young and Fairburn 2000).
Theoretically, any procedure at any stage of the sequential process of in vitro embryo production and
manipulation (e.g., in vitro maturation of oocytes or in vitro fertilization) may influence embryo
development and characteristics of offspring (Van Wagtendonk-de Leeuw et al. 2000).

Welfare protocol

We conclude that there are convincing arguments to support the idea that treatments, applied in farm
animal biotechnology, in their effects on animal welfare are by no means biologically neutral. On the
contrary, several treatments appear to directly threaten the animal's pre- and postnatal survival.
Therefore, we believe that within the context of farm animal biotechnologies, animal welfare should
receive special attention.

We propose to systematically monitor welfare of animals involved in, or produced by, new
biotechnologies, and to evaluate potential risks of these technologies, on the basis of a
comprehensive welfare protocol. This protocol would have to specify: 1) which treatment groups to
compare, and the number of subjects needed, 2) which parameters to monitor, and 3) at which
stages of farm animal's life. Although a (limited) number of studies specifically addressing side-effects
of in vitro reproductive technologies or transgenesis have been reported (Walker et al. 1996; Van
Reenen and Blokhuis 1997; De Sousa et al. 2000), currently such a welfare protocol does not exist.
Some initial ideas are considered below.

Ad 1) One of the main goals of the implementation of a welfare protocol would be the accurate and
unbiased estimation of the essential treatment effects, i.e. of MOET, IVP or NT. An essential condition
in this respect would be the inclusion of adequate control groups, in sufficient numbers, e.g.: in vivo
produced offspring with the same genetic background as the in vitro produced treatment group. With
respect to the evaluation of effects of NT, the experimental design should allow for separating effects
of inadequate reprogramming of the transferred nucleus/genome as much as possible from effects of
reproductive technologies used in the process of generating cloned animals. This will require specific
breeding steps (Smith et al. 1987; Gibson 1998).

The sensitivity of a welfare protocol in terms of the ability to detect relevant effects if they do exist, is
greatly influenced by the number of animals investigated. It will be easy to reliably identify in vitro
embryo manipulations with extremely unfavourable effects, but large numbers of animals will be
needed to detect smaller, but still biologically relevant, harmful effects of nuclear transfer.

Ad 2) Relevant parameters and biological functions that are clearly associated with welfare are:
clinical symptoms of health or disease, measures of growth and fertility, measures of
immunoresistance and behavioural measures. Parameters for a welfare protocol could also be
provided by immunotoxicological (Luster et al. 1994; Van Loveren et al. 1995, 1998) or
pharmacological disciplines (Martinod 1995).

We propose to formulate, for each of the important livestock species involved infarm animal
biotechnology, a basic set of welfare parameters, encompassing a cross-section of the most essential
parameters and biological functions, the scope of which could then be adjusted, either extended or
reduced, according to the specific properties of the treatment under observation. For example, in a
study on the welfare of offspring of a transgenic bull carrying a human lactoferrin transgene designed
to express in udder tissue of lactating transgenic females, next to parameters concerning growth,
general health, behaviour, reproduction and immunocompetence, specific measures on milk
production, characteristics of milk and udder health were included in the protocol (Van Reenen and
Blokhuis, 1973;1997). Protocols used for the evaluation of in vitro reproductive technologies should
involve observations appropriate for detecting LOS symptoms, such as specific measures of neonatal
vitality and viability, or ultrasound measurements to investigate disproportionate organ development
(Garry et al. 1996; Van Wagtendonk-de Leeuw et al. 2000). In addition to these parameters some
molecular biological indices should be incorporated in the protocol, especially those that would enable
to assess and reduce the risks of impaired welfare. A promising concept considers gene expression
profiles in preimplantation embryos of imprinted genes (Niemann and Wrenzycki 2000).

3) Stages of life. The stages of life of a farm animal at which to monitor welfare aspects should, in
our view, at least include: (a) gestation and birth, (b) the developmental phase from birth to puberty,
and (c) a representative period of adult life, including the stage of (re)productive performance.
Investigations up to senescence could have scientific value, but need not necessarily be relevant for
all farm animals since their productive lives usually represent only part of the entire possible life-
span.

Conclusion
MOET, including synchronisation and induction of oestrus and AI, as well as IVP, NT may have
undesirable and sometimes serious consequences for farm animal welfare. We suggest that
(potential) risks of biotechnologies for farm animal welfare should be comprehensively and
systematically assessed. This type of research should be multidisciplinary, should be logically
integrated into ongoing research programmes, and and should make use of appropriate and
scientifically valid experimental designs and protocols. Results obtained accordingly allow for
developing and using the safest biotechnological methods and procedures, and, thereby, enable
technological progress which is ethically justified, and beneficial for socieity in general as well as the
scientific and agricultural community.