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:

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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:
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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:

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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:
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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:
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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:

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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
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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

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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

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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
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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 lifespan. 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.

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