Biotechnological approaches of embryo production for productive and reproductive improvement of farm Animals
Abstract
Cloning is considered as the most recent technology for genetic improvement in farm animals It is useful to produce many copies of embryos by allowing genotypes of highest genetic merit to be introduced directly into commercial herds in farm animals. Cloning by embryo splitting using micromanipulation can be used to produce a maximal number of embryos for transfer. Transfer of splitted embryos, which are derived either from in vivo or IVMFC embryo (quarter embryo or demi-embryo), resulted in a live offsprings. Transfer of fresh demi-embryos has higher pregnancy rate and delivery of offsprinsg than frozen-thawed demi-embryos. At the same time, demi-embryos have higher survival rate than quarter embryos
Cloning technique in summary is carried out by using micromanipulator and inverted microscope as follow: (1) The recipient oocytes matured (22-24 h) in a maturation medium (TCM-199) and checked for the extrusion of first polar body. (2) Enucleation of the first polar body with minimal amount of cytoplasm which near to the polar body (ensure the metaphase chromosomes are taken). (3) Then the enucleated oocytes are returned to culture for additional 18- 20 hr with addition of cytochalsin B in culture media to gain activation competence. There are many types of donor cells i.e. blastomeres, embryonic stem cell (ICM) and somatic cell (fetal skin fibroblast, fetal muscle cell, mammary gland epithelial cells, cumulus cell and granulosa cell). A nucleated donor cell in G1 or S phase (static interphase) introduced into the perivittline space of the recipient oocytes and fused into the ooplasm. Fusion of donor nuclei to the ooplasm of recipient oocytes can be undertaken using either electrofusion (DC or AC) or chemical (Ca ionophore A23187, cytochalsin B or ethanol) while, its preferable to use two methods together, chemical followed by electrofusion. The enucleated recipient oocyte cytoplasm has the ability to reprogramme the donor nuclei and reconstructed embryo begins its development of the newly fertilized oocyte. Reprogramming is a process where synthesis of nuclear and ribosomal RNA is blocked in donor nuclei after fusion whatever the cellular cycle stage of the transferred nucleus and the cytoplasm condition of cytoplast controls results this inhibition and the reconstituted embryo commences development as thought it is recently fertilized oocyte. There are several factors influences efficiency of cloning procedures. In recipient cells, maturation in vivo (superovulation & recovery) gives better result in NT embryo development than in vitro maturation. Removal of minimal amounts of cytoplasm (during enucleation of polar body) from recipient cells increase the number of NT blastocyst cell. The best time for enucleation of polar body is 22-24 hr maturation. The best donor cell for NT is somatic cell (fetal fibroblast cell) and culture for 5 days before NT. The donor cell must be in G1 or S phase to induce hypomethylation of DNA. The donor cell should be cultured in serum starvation media (0.5% fetal calf serum) and chromatine has a minor influence on chromosome constitution of manipulated embryos. Therefore, cell cycle can be synchronized between donor cells (in G1 by using Demecolcine or Nocodazol) then fused with the oocyte cytoplasm leading to the highest rate of oocytes developed to the blastocyst stage. Fusion by using electrofusion direct current (DC) followed by alternative current (AC) is sufficient to fusion of donor cell-recipient cell cytoplasm.
Production of chimeric phenotypic depends on the cloning technique. Chimeras produced in domestic animals either by the blastocyst injection techniques (injection of ICM) or aggregation of bisected embryos (8-16 cell stage) within the zona pellucida and then cultured for further development to blastocyst. The predominant sex of chimeric offspring is male
The advantage of cloning is the production of unlimited number of young from an original high genetic-quality embryo (multiple copies). NT embryos have high level of gene expression (transcription of growth factors). Moreover, somatic nuclear transfer produces transgenic livestock and accelerates the time consuming for progeny testing.
The disadvantages of cloning technology are, transfer of NT embryo produce low pregnancy rate (36-42%), the percentage of live offspring does not exceed 1-3% of the transferred NT embryo, fetal oversize and increase peri- and postnatal mortality are found in offspring derived from nuclear transfer embryos.
Animals that integrate the isolated or modify genes (recombinant DNA) into their genome are known as transgenesis. Application of transgenic livestock has been commercial for agricultural traits and biochemical purposes. Therefore, the gene microinjection technology has economical importance because disease resistance, growth rate, feed conversion efficiency and milk yield are controlled by numbers of genes. Gene transfer in dairy industry play a role in modifications of milk proteins to provide novel forms of cheese, improve milk nutritional value for protein intake of humans and reducing milk saturated fat content to keep human health. Another interesting application is that it could enhance disease resistance. The strategy for disease resistant transgensis is to target expression of appropriate gene to the mammary gland and then we could harvest the human antithrombin III, α-antitrypsin, tissue plasminogen activator, α-glucosidase and lactoferrin are currently in advanced clinical trails.
Gene transfer technique can be done by microinjection of the desired gene into male pronucleui of a recently fertilized oocytes, germinal vesicle gene injection at immature oocyte or cytoplasmic gene injection at different stages of the cell cycle of oocytes and zygotes. Sperm cell- mediated gene transfer, through binding of DNA to the equatorial and post acrosomal region of the sperm head. Thus binding is reversible and may occur by interaction of the nucleic acid with specific protein components. Optimal binding of the foreign gene occurs when both sperm motility and DNA concentrations are high. Binding of DNA in capacitated sperm was more effective than uncapacitated sperm. Using of sperm-mediated gene transfer in in vitro fertilization was valuable in production of transgenic offspring. Gene transfer using embryonic stem cells, germ cell or somatic cells. These allow the direct modification of endogenous genes (gene targeting). These cells carrying new DNA are introduce in the embryos to produce chimeric embryos, which transferred to give transgenic animals. Using in vitro maturation/ fertilization and culture (IVMFC) derived embryos, form transgenic animal production. In vitro system is used to produce transgenic embryos of transferable quality. The propagation of transgenic trait can be accomplished through in vitro production techniques by using semen from transgenic bull for in vitro system.
Controlling and pre-selecting the sex of livestock has advantages in improving animal production. The economic importance of animals production depends on female sex for calving and milk production. Recent and accurate methods for embryo sexing depend on detection of special DNA sequences present in Y-chromosome. (1) Using of Y-specific DNA probes, for in situ hybridization (radioactive isotopes labeling probe, FISH) detection of slides by fluorescent, is safe, fast and its microscope accuracy is 95% in embryo sexing of most domestic animals, while it is expensive in its application. (2) Polymrase chain reaction (PCR), it depends on identification of particular DNA sequence in a minute amount (1-3 blastomeres, embryo biopsy) with specific amplification of sequence by embryo sexing primers. Detection of specific DNA bands of male sex by using gel electrophoresis which stained with Ethedium bromide and exposed to ultraviolet light. This method is accurate 95% in biopsy embryos with minute cell number, fast and can be done at the farm. (3) Sexing by male-specific antigen, it depends on the detection of H-Y antigen by specific antibodies. This method with 80-90% accuracy while, it arrests embryo viability. (4) X-chromosome linked enzyme, it depends on coloremitric assay of glucose phosphate dehydrogenase (G6PD) or hypoxanthine phosphoribosyl transferase (HPRT) which twice higher level in female embryo than male embryos. This method is not accurate in separation of sexes; and (5) Sexing according to embryo development, this method depends on the monitoring the rate of embryo development to morula and expanded blastocyst. The male embryos generally faster in development to more advanced stage in the first 8 days after IVF than female embryos. This method is not accurate in embryos sexing and we can’t depend on it.
Gamete micromanipualtion assist fertilization under conditions of reduced sperm number or when sperm motility is compromised or then non-existent in semen of valuable male animal. There are four main approaches for gamete micromanipulation: (1) Intracytoplasmic sperm injection (ICSI), Injection of single sperm (immobile or killed sperm) by using injecting micropipette deep into matured oocyte cytoplasm. Then activation using calcium ionophore A23187, ethanol 7% or electro-activation to activate extrusion of 2nd polar body. ICSI is the best method which can be used for fertilization and its success is very high to produce offspring after transfer and it is practically when sperm in low numbers. (2) Insertion through the zona (SUZI) and the placement of more than one sperm into the perivittiline space of matured oocyte by using injecting micropipette. Fertilization rate by this method is generally low. (3) Zona drilling and partial zona dissection; tearing of holes in the zona by using acid Tyrods (pH 2.6), was sprayed from a manipulating pipette over the area of the zona (mature oocyte) being drilled. This method is not practical in domestic animals. (4) Zona thinning by partial digestion with pronase and capable to be safe and enhancing fertilization at low sperm concentration. This method enhances fertilization and it acts effectively as sperm receptor and induces of acrosomal reaction (AR).
Gene expression in preimplantation embryos, in vitro produced embryo has great potentially sever effects on fetal, prenatal and postnatal viability. Culture medium, which lead to gene expression of IGF I, IGF II, and GH, Apolipoprotein E, pyruvate-kinase-3 and protein phosphate-gamma improves the embryo production in vitro. Therefore, the favorable in vitro culture medium provides gene expression pattern closer to that of in vivo derived embryos. While, the development regulating antigen (TEC 1, 2 and 3) expression in blastocyst are similar in in vitro produced embryos cultured in TCM-199+ SOF+ serum or BSA, and that of in vivo derived embryos. CX43 gene (from canex family) is responsible for gap junction for maintenance compaction transcription is found in the blastocyst of in vivo embryos while, there is no mRNA for CX43 in in vitro derived embryos. Morula of in vivo embryo has cytokine leukemia inhibitory factor gene (LIF mRNA) and its receptors (LR-B) are never expressed in in vivo derived morula or blastocyst. While, its expression is clear in in vitro derived embryos, which, may be lead to abnormal development in ICM and trophectoderm of the blastocyst and it may be the possible cause of large calf syndrome in the in vitro produced embryos. The heat shock protein gene (HSP 70.1 mRNA) is highly expressed in the blastocyst derived from in vitro culture medium (serum enriched medium) than in vivo derived embryo.
In conclusion, the application of reproductive biotechnology (cloning, gene transfer, embryo sexing, ICSI and gene expression) can improve the productive and genetic potentials in farm animals by disseminating the genetic and productive merits of the highly producing animals to Egyptian livestock.
Introduction
It is clearly evident that there is continuos increase in population (67 millions) in Egypt, which needs an increase in animal production for self-sufficiency in meat production. At the same time, farm animals are generally considered slow breeding and suffer from several infertility problems. These problems could be due to genetic, nutritional, environmental or due to the traditional breeding systems. Applications of embryo micromanipulation techniques such as cloning, gene transfer, embryo sexing and gene expression in in vitro produced (IVP) embryos are important for improving the productive and reproductive potentials of Egyptian farm animals. Nuclear transfer significantly enhances the dissemination of genetic improvement from selected herds and produces many copies of the selected animals. So, manipulation of genetic constitution of farm animals is very important for improving their productivity. In this respect, the development of recombinant DNA technology have enable the molecular biologist to isolate and modify genes in such a way as to allow novel constructs to be introduced into the animal genome. Animals that integrate this recombinant DNA into their genome are known as transgenic animals.
Furthermore, farmers mainly depend on females for the production of offspring and milk, and on males for meat production. In this case, determination of embryo sex and implanting embryo of known sex would be more beneficial economically.
In addition, micromanipulation of gametes could be of great value in identifying the problems during IVP programs, and consequently enhances fertilization rate. Intracytoplasmic sperm injection (ICSI) may assist fertilization by using sperm from superior bull, even if it is suffers from infertility due to reduced sperm number or sperm motility.
Also, there is a great need to characterize the specific mechanisms controlling early mammalian development in order to improve success of embryo culture and for assessing the health of early mammalian embryos. Culture media used for in vitro embryo culture must provide a mRNA expression pattern closer to that of in vivo derived embryos to improve the viability of preimplanted embryos.
This state of art is undertaken to highlight the importance of the applications of the new biotechnological approaches of embryo production (cloning, gene transfer, embryo sexing, ICSI and gene expression) for productive and reproductive improvement of farm animals.
During the last two decades great attention was paid to the application of biotechnology in modifying the genetic and productive performance of farm animals. These approaches include cloning, gene transfer, embryo sexing, sperm microinjection (ICSI) and improving gene experssion in the developed embryos.
I. Cloning and its potential impact in cattle production
There has been a certain fascination with the possibility of producing copies, or clones of outstanding beef or dairy animals in large number. Cloning could be useful in dairy animals by allowing genotypes of the highest genetic merit to be introduced directly into commercial herds, Also, cloning in animal can employed to produce genetically similar multiple calves in short time
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1. Cloning by embryo splitting and quartering
1.1. Use of in vivo drived embryo
Work at Cambridge with two-cell sheep embryos was the first to show that each blastomeres has the potential to develop into normally organized blastocyst (Willadsen and Fehilly, 1983). The developed technique resulted in the production of identical twins in both cattle and other farm animals. Ectors (1996) reported the production of calves from embryos containing only a quarter of their original cells mass. The technology had moved to splitting sheep and cattle embryos at early stages of development. For cattle, this meant that embryos could now be flushed from a superovulated donor cow and split at the late morula/ orearly blastocyst stage, without the surgical intervention previously required with early tubal embryos. This permitted the number of pregnancies obtained from a given collection of embryos to be markedly increased.
Recently, the need for rapid biopsy technique to provide cells for embryo sexing in cattle helped to improve the effectiveness of the splitting process (Agca et al., 1998). Pregnancy rates with demi-embryos produced from excellent/ good quality embryos can be expected to be 50% and above (Nibart, 1992). Where two demi-embryos are transferred, then pregnancy rates were comparable to those in recipients receiving intact embryos (Holm et al., 1998). In Japan, Utsumi and Iritani (1990), developed a special metal microblade for splitting blastocysts and obtained a 40% pregnancy rate after transfer of pairs of demi-embryos into the ipsilateral horns of recipient Holstein- Friesians.
Attempts to produce calves from embryos containing only a quarter of their original cells mass have reported previously by Bredbacka et al. (1992). The general experience is that the survival rate of such quarter embryos is only half that found with demi-embryos. In sheep, Zhu et al. (2001) described work in which five lambs were produced from single blastomeres taken from an eight-cell embryo, when the blastomeres were aggregated with blastomeres taken from earlier-stage embryos.
Vivanco et al. (1991) used new techniques of electrostatically Anchoring goat embryos to the base of the culture dish, thus eliminating the need for a holding pipette, and therefore cutting down the time of micromanipulation, more than 50% of demi embryo resulted in live offspring. Nowshari and Holtz (1993) split goat embryos with a simple and efficient procedure and froze demi-embryos without zona pellucida. Nine does becoming pregnant from transfer of fresh demi-embryos, eight produced offspring (five twins and three singles). For frozen-thawed demi-embryo, six became pregnant and two produced offspring, both singles.
1. 2. Use of IVMFC drived embryos
For those who may be attempting to produce the maximal number of embryos for transfer, the linking of oocyte recovery from the live cow, IVF and embryo manipulation may have certain attractions. When in vitro matured, fertilized and cultured (IVMFC) embryos are available, there may be some advantages in micromanipulating these embryos at an early cleavage stage (8-16 cells), rather than applying the conventional splitting technique at the later, blastocyst stage. Peura et al. (1999) showed that removal of half the total number of blastomeres from 2-16-cell IVMF embryos did not adversely affect development to the blastocyst stage. Workers also showed that multiple calves may be produced from single blastomeres isolated from four-cell IVMF embryos. There was some evidence that pregnancy rates could be increased if these quarter-embryos were co-transferred with fresh trophoblastic vesicles. The facility to split embryos generating identical twins was developed and embryo was offered as a commercial service. Whereas direct transfer of embryos yielded around 60 calves per 100 embryos, transfer of split embryos typically produced 105 calves per 100 embryos. The limitation of only being able to produce two identical animals was lifted when embryo nuclear transfer was successfully achieved using blastomese (Westhusin et al. , 2001).The first commercial use of nuclear transfer was short lived partly because of the low efficiency and the birth of unusually large offspring (Wilson et al ., 1995 ) This early work on embryo manipulation led eventually to the nuclear transfer technology already produced Dolly ewes in 1997 (Wilmut et al., 1997)
2. Developments in large-scale cloning technology
2.1. Cloning procedure
Loi et al. (1998) showed that it was possible to produce multiples (clones) by fusing a whole nucleated blastomeres from a donor sheep embryo into an enucleated recipient oocyte, opened up an important new area of exploration in embryology and developmental biology. A simple slit was first made in the ZP of sheep secondary oocyte at a point close to the first polar body. Enucleating the oocyte was undertaken by aspirating the polar body and part of the ooplasm; using the same pipette, a nucleated blastomere was then introduced into the perivitelline space of the oocyte and fused into the ooplasm. Fusion of the blastomere and cytoplast was achieved by various means, including electrofusion. In this alignment of the cell and cytoplast was achieved by alternating current (AC) and pulses of direct current (DC) used to cause fusion (Shin et al., 2001) or by using a cytoskeletal inhibitor (cytochalasin B), it was possible to remove the metaphase II chromosomes without piercing the ooplasm membrane. Evidence was found of an increased rate of development in cattle NT embryo treated with cytochalasin B post fusion (Shiga et al., 1999).
The enucleated recipient oocyte (a cytoplasm) appears to have the ability to reprogramme the donor nucleus and the reconstituted embryo commences development as though it is a recently fertilized oocyte (Zakhartchenko et al., 1999). Nuclear reprogramming may involve the uptake of regulatory proteins by the transferred nucleus and it is believed that such proteins are released into the oocyte cytoplasm when the metaphase II chromosomes are being removed in the enucleation process (Betts et al., 2001). Kang et al. (2001) concluded that the somatic nucleus is remodeled in such a way as to resemble and behave like a pronucleus in a zygote. This is accomplished by the exchange of proteins between the nucleus and cytoplasm.
2.2. Factors known to influence efficiency of cloning procedures
2.2.1. Recipient oocytes
2.2.1.1. Source of recipient oocytes
The early cloning reports in cattle used, as recipient cells, metaphase II bovine oocytes matured in vivo after surgical collection from superovulated cattle (Wethusin et al., 1991). When in vitro oocyte were employed, results were poorer than with those matured in vivo (Chesne et al., 1991). However, subsequent reports on in vitro or in vivo comparisons demonstrated convincingly the efficacy of IVM bovine oocytes for cloning when selected on the basis of certain parameters (Barnes et al., 1993). These include the duration of maturation and subsequent culture, the selection of oocytes on the basis of follicle size (>3 mm) and the presence of a polar body.
2.2.1.2. Quality of cytoplasm in recipient oocyte
In cattle and sheep, NT requires removal of a substantial amount of ooplasm from recipient oocytes to ensure that the metaphase chromosomes are taken out. Northey et al. (1991) showed that removing half the cytoplasm in bovine oocyte prior to fertilization led to a corresponding decrease in the total number of cells in the blastocyst. The same process would presumably occur in cloned embryos. This reduced cell number may contribute to decreased viability in cloned embryos. According to Zakhartchenko et al. (1997), oocytes only having a minimal amount of cytoplasm removed had 30% more cells than the NT embryos obtained with half of the normal amount of cytoplasm. Greising et al. (1994) found evidence of a higher developmental capacity in cloned embryos derived from recipients consisting of two cytoplasts rather than one (38% blastocyts vs. 14%); the larger available cytoplasmic mass may have been a contributory factor in this. Also, the transfer of embryonic nuclei (with some embryonic cytoplasm) into recipient cells containing a large amount of foreign cytoplasm means that members of a given clone may not share a common cytoplasm; there is the potential for mitochondrial genes to differ. A report by Plante et al. (1992) describes a unique cytoplasmic marker, which can be used to identify each individual animal within a clone.
2.2.2.3. Time of enucleation of recipient oocyte
The general consensus of reports suggests that oocytes aging beyond the time for nuclear maturation (i.e. 24-26 h) is a general requirement for activation competence of IVM (metaphase II) cattle oocytes (Bordignon and Smith, 1998). Rao et al. (1993) concluded that there was an optimum stage between 32 and 40 h of age for recipient oocytes used for NT. Moreover, Baran et al. (2002) reported the least success with 36-48 h matured cultured oocytes in comparison with normally matured (24-26 h). The selection of oocytes showing the first polar body at 24 h is described in the report of Barnes et al. (1993); such selection may effectively remove oocytes that have not shown a normal rate of meiosis. The same authors note that the size of ovarian follicle from which the oocyte is derived influences its subsequent developmental potential when used for cloning. Workers at Cornell attempted to capitalize on the better enucleation rate of young oocytes (22-24 h) and the better activation of the aged oocytes (30-40 h); they developed a technique involving enucleation at 22-24 h and performed NT after incubating the cytoplast to a maturation age of 30-40 h (Shiga et al., 1999) then oocytes returned to culture for an additional 18-20 h to gain activation competence.
2.2.2. Source of donor NT
2.2.2.1. Embryonic cells
All blastomeres of a single bovine embryo are genetically identical. The genome of each blastomere is derived from replication through mitosis of the original single diploid genome constructed by the fusion of the male and female pronuclei. The use of donor embryos at advanced rather than at early cleavage stages has the advantage of larger numbers of blastomeres to work with. Mohamed Nour and Takahashi (2000) reported development of NT embryos to the blastocyst stage after fusion of nucleated blastomeres from four-cell to 32-cell donor embryos. Fusion of blastomeres to enucleated oocytes in goats (nuclear transplantation) produced about a 25% success rate for blastocyst stages developing to offspring (First, 1992).
Subsequently, Peura et al. (1999) used 16-64-cell cattle embryos as nucleus without observing differences either in the fusion frequency or in the developmental potential of the transplanted embryos. Also, Baran et al. (2002) used 16-cell-stage bovine embryos and showed that their nuclei could be reprogrammed within the cytoplasm of IVM oocytes. Van Stekelenburg-Hamers et al. (1994) have shown that advanced nuclei from bovine embryos up to the 50-cell stage can support normal development after use in NTs; they found that nuclei from such embryos reverted to the equivalent of an earlier developmental stage when transferred to ooplasm.
On the other hand, Ectors et al. (1996) has shown that bovine embryos undergo a process of polarization at about the 32-cell stage, which is just prior to compaction. They found that, after NT, polarized blastomeres gave a much lower rate of development to the morula/blastocyst stage than did non-polarized blastomeres (27% vs. 3%). The same authors found that prior to polarization, microvilli are evenly distributed over the entire surface of the blastomeres and organelles are distributed throughout the cytoplasm; after polarization, microvilli are only to be found at the apical surface of the blastomeres. Inner cell mass (ICM) cells (Keefer et al., 1993), could be used as donor nuclei. Their study showed that these cells are pluripotent and after NT can give rise to embryos capable of normal embryonic and fetal development. It would be possible to use stem cells as donors of nuclei for transfer to enucleated oocytes; the number of clones would then become practically limitless. The same authors observed that cell lines were derived from immunologically isolated ICM cells; NT clones were produced by fusing the embryo stem (ES-like) cells into enucleated oocytes using polyethylene glycol as the fusion agent.
2.2.2.2. Somatic cells
Somatic nuclear transfer holds the greatest promise for significant improvements in the generation of transgenic livestock (Kilby et al., 1993). This advance depended upon coordination of cell cycle in donor and recipient cells (Campbell et al., 1996a). Now there were offspring in three species (sheep, cattle and goat) followed the first induction of quiescence (G0) donor somatic cells (Campbell et al., 1996b)
An alternative approach has been pioneered by Chesne et al., (1993). They discovered that, after aging of the bovine oocyte at reduced temperature, the level of maturation promoting factor (MPF) is reduced. As a result, nuclear envelope breakdown does not occur after nuclear transfer. In these circumstances, it is expected that the transferred nucleus itself will determine whether DNA replication takes place. Successful cloning by using fetal fibroblasts cells as a donor from sheep (Loi et al., 1998), cattle (Heyman et al., 2002 ) and Buffalo (Kitiyanant et al., 2001) and using adult mammary epithelial cells from cattle (Zakhartchenko et al., 1999). Cumulus cells (Alberio et al., 2001) and granulosa cells (Arats et al., 2001). The previous experiments suggests that cloning using fetal cells is more efficient than using adult cells (Betts et al, 2001). Preparation of donor (Karyoplasts) somatic cells (fetal skin fibroblast or muscle fibroblasts or mammary epithelial cells) were dispersed in Hank’s solution supplemented with collagenase type –I (Shiga et al, 1999). Then separated cells were cultured in Dulbecco’s modified Eagles medium (D-MEM) supplemented with 10 % fetal bovine serum (FBS) at 39 °C in atmosphere of 5% CO2 in air cells were passage at least 4 times and for 5 day prior to nuclear transfer. Then the donor cells were cultured in D-MEM supplemented with 0.5 FBS (serum starvation, to induce quiescence, (G0) or 10 % FBS (serum enriched) then transfers the donor cells to the inoculated recipient oocytes.
2.2.3. Effect of donor and recipient cell stage
In cattle cloning research, a number of reports have now drawn attention to the effect of donor cell cycle stage on the development of embryos after NT. For high rates of development, the donor cell should be in the G1 phase (Hill et al., 2001); the short duration of that phase led Collas et al. (1992) to develop a method for synchronizing sheep blastomeres in G1. Those workers found that the morphology of prematurely condensed G1 and early S chromatin has a minor influence on chromosome constitution and hypomethylation of DNA of manipulated embryos (Jones et al., 2001). The morphology of late S chromatin, however, can effect the chromosome constitution in embryos and may account for reduced development of nuclear transplant embryos when late-S-phase donor nuclei are employed. There are certain agents that can be employed to bring blastomeres to the most appropriate stage of the cell cycle. Demecolcine and nocodazole are agents that can induce cell arrest at metaphase by depolarizing microtubules. Bovine blastomeres were synchronized using demecolcine or nocodazole in studies reported by Kitiyanant et al. (2001); those synchronized to the midportion of the cell cycle and fused to aged (42 h) oocytes developed to the blastocyst stage at higher rates than non-synchronized blastomeres. Nocodazole was also employed by Techakumphu et al. (1993) to induce temporary metaphase arrest without impairing the viability of the blastomeres in NT studies. The development of bovine NT embryos was improved by fusion of the donor blastomeres into activated oocyte cytoplasm in the studies of Stice and Keefer (1992). It was apparent that, when donor blastomeres of unknown cell cycle stage were used, activation of enucleated oocytes prior to fusion resulted in enhanced development of NT embryos. Cell cycle stage synchrony between donor nucleus and recipient oocyte is crucial for the development of the NT embryos (Kasinothan et al., 2001). These workers found that S-phase nuclei transferred into S-phase cytoplasm resulted in embryos with normal chromosome content and high rates of development to the blastocyst stage.
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2.2.4. Blastomeres-cytoplast fusion and oocyte activation
Fusion of donor nuclei to recipient oocyte can be undertaken using chemical methods (Ca ionophore A 23187, cytochalsin B and ethanol) or electrofusion. In both sheep (Loi et al., 1998) and cattle (Shiga et al., 1999) electrofusion appears to be much more efficient than using the chemical methods. A report by Long et al. (1991) deals with the effect of DC voltage and pulse duration on electrofusion and cell lysis in NT in cattle.
The karyoplast-cytoplast fusion in cattle is usually carried out in a chamber attached to the electrofusion apparatus and in a non-electrolyte fusion solution. The fusion chamber is a microscope slide with electrodes on its surface set at a gap varying from 0.2- to 1.0 mm. Prior to fusion, oocytes are either aligned mechanically or electrically using AC and fusion is induced by DC (Kano et al., 1993).
Both fusion of the donor blastomere to the recipient oocyte and activation of the oocyte are crucial steps in NT. Both events are usually initiated by formation of pores, or at least a destabilization of the cell membranes that mediate cell fusion, and by an influx of Ca2+ into the cell, oocyte activation. Robl et al. (1992) suggests that the most response after sperm-oocyte fusion. In this, transient increases in Ca2+ results in cortical granule exocytosis and initiates resumption of meiosis, with consequent extrusion of the second polar body. The synergistic effect of a calcium ionophore (A23187) and protein inhibitor (cycloheximide) was found to be highly effective, regardless of the age of the oocytes (Shin et al., 2001). Kitiyanant et al. (2001) concluded that electrical pulse alone is sufficient to activate aged oocytes.
2.3. Reprogramming of transplanted nuclei
There are an increased number of animal species that have been successfully cloned from the somatic and differentiated cells. We still only know a little bit what happens to the nucleus of the somatic cell after it is transferred to the cytoplasm of an oocyte, how the nucleus is reprogrammed and how much of this reprogramming is necessary for successful development of a newly created cloned individual (Fulka et al., 1998). The same authors found that the time required to progress from the zygote to the blastocyst stage was similar for fertilized embryos and nuclear transfer embryos (8-cell stage donor nuclei). Therefore, the donor nuclei were reprogrammed, since they reverted to the same morphological and temporal developmental pattern as the zygote.
2.3.1. Protein synthesis level
The developmentally regulated antigen TEC-3 that is expressed during bovine preimplantation development on morula and blastocyst. TEC-3 is absent from unfertilized and fertilized oocytes, and from all stages before the 32-cell stage. The TEC-3 antigen, present on blastomeres of the morula stage embryo, disappeared after fusion was expressed again when the nuclear transfer embryos developed to the morula and blastocyst stage (Van Stekelenburg-Hamers et al., 1994).
2.3.2. Nuclear RNA synthesis level
Synthesis of nuclear and ribosomal RNA is blocked after fusion whatever the cellular cycle stage of the transferred nucleus; the cytoplasm condition of cytoplast controls results this inhibition. In normal bovine embryogenesis, the embryo relies upon maternally derived RNA transcripts up to the 8-cell stage, at which time major zygotic gene activation begins (Telford et al., 1990). Transcription of the embryonic genome in nuclear transplant embryos begins either sooner than with normal development. (Smith et al., 1996), or in the same state as with normal development, but it runs through more intensively (Kanka et al., 1996). Expression analysis of individual genes showed difference in the blastocyst state for nuclear transplant and normal embryos stage (Westhusin et al., 1995).
3. Production of chimeric phenotypic
Attempts to produce chimeras in domestic animals derived from surgically flushed embryos have been reported in sheep (Role et al., 1989) and cattle (Picard et al., 1990). The blastocyst injection technique (Picard et al ,1990) or aggregation of bisected embryos with the zona pellucida intact (Cibelli et al.,1998a) was used in cattle to produce chimeric calves.
Mammalian chimeras have been valuable in the study of physiology, behaviour and developmental biology of embryos. There may, however, be ways of using bovine chimeras to influence the sex ratio in calves. When the first experimental chimeras were formed in mammals, little was known of cell mingling during development and even less about the cellular basis of sex determination (McLaren, 1984). Fehilly et al. (1984) reported that 26/36 of chimeric lambs were male at birth. Studies reported by Landa and Riha (1992) involved the desegregation of 16-cell bovine embryos and the construction of 14-cell aggregations; two male and one female calves were born after transfer of such chimeras. The aggregation of eight-cell IVF cattle embryos (Holstein-Japanese Red) has been reported by Boediono et al. (1993) in Japan, with the majority of aggregated embryos progressing to the blastocyst stage and produced chimeric bull. It may be possible to use such technology to produce chimeric beef embryos, in the expectation that the sex ratio would favor the male.
4. Advantages of cloning technology
4.1. Producing unlimited number of identical young
The ultimate goal for a commercial embryo-cloning project is likely to be the production of an unlimited number of young from an original high-genetic-quality embryo. Data reported by Roelen et al. (1998) indicated that multiple-generation embryos had a lower fusion rate and in some case a lower developmental rate than NT embryos from original embryos.
4.2. Gene expression in cloned embryos
One of the valuable and still scarce pieces of direct evidence on reprogramming is the analysis of gene expression patterns in cattle blastocyst produced in vivo and in vitro by nuclear transfer (Roelen et al, 1998). Nuclear transfer blastocysts possessed both a high level of transcripts for the IGF-I receptor and growth factors IGF-II and TGF-alpha as well as a low level of transcripts for growth factor IGF-I in comparison with in vivo and in vitro produced blastocysts. Transcripts for the growth factor bFGF were detected in nuclear transfer blastocysts but not in in vivo and in vitro blastocysts. Clearly, the patterns of gene expression in nuclear transfer embryos are differed from those of normal embryos.
4.3. Marriage of nuclear transfer and advanced molecular tools
Recent reports on the generation of transgenic sheep and cattle (Schnieke et al., 1997) via somatic nuclear transfer inspired great exceptions about this elegant approach to improve the generation of transgenic livestock. Fetal fibroblasts were transferred in vitro , screened for transgene integration and then transferred into enucleated oocytes. After fusion of both components and activation of the reconstituted nuclear transfer complexes, blastocyst was transferred to synchronized recipients and gave rise to transgenic offspring.
5. Disadvantages of cloning technology
5.1. Pregnancy rates and calf birth weight with cloned embryos
It is clear that a major limiting factor in the commercial application of NT procedures is the pregnancy rate achieved with cloned embryos. Using ovulated oocytes as recipient’s cells and blastomeres from 16-64-cell embryos as donor cells, pregnancy rate of 36% were achieved by 463 cloned embryos transferred to recipient cattle (Zakhartchenko et al., 2001). A total of 302 transferred were involved in studies reported by Huang et al. (2000); 42.4% of recipients were pregnant at 35 days.
5.2. Early embryo death
Factors affecting the success of nuclear transfer are poorly defined and the percentage of live offspring does not exceed 1-3% of the transferred reconstituted embryos (Wilmut et al., 1997; Wakayama et al., 1998). A better understanding of the underlying fundamental molecular process, such as cell cycle compatibility between cytoplasm and donor nucleus (Campbell et al., 1996a), cell cycle synchronization of the donor cell (Kues et al., 2000), reprogramming and the relevance of differentiation vs. totipotency is urgently needed. In addition, methods have to be established that allow reliable determination of the capacity of a given nuclear transfer embryo to develop into a normal offspring. Currently, an increased peri- and postnatal mortality is found in offspring derived from nuclear transfer embryos (Kato et al. 1998).
5.3. Fetal oversize
Several exceptionally large calves (up to 155 lb.) occurred among the 100 or so birth reported by Willadsen et al. (1991). There is no mention of instances of fetal oversize in the calving reports of Sinclair et al. (1999). On the other hand, Kang et al. (2002) stated that 20-30% of cloned calves is larger than normal (up to twice normal size). The current procedures may result in adverse effects in later embryonic life (Ozil, 1992). Much further evidence is required before an accurate assessment of the seriousness of the large-calf syndrome can be made.
In sheep, data have been reported that the IVC of embryos from the zygote stage for either 3 or 5 days can resulted in extended gestation length and increased in birth weight and lamb mortality (Walker et al., 1992); it is suggested that such anomalies may be due to inappropriate activation of the embryonic genome induced by deficiencies in the culture medium
Conclusion
The ability to produce offspring by nuclear transfer from cultured cells (somatic cells) has provided new opportunities for genetic modification. Both gene addition and precise genetic modification have been achieved. Cloning by nuclear transfer can accelerate the time consuming for transgenic production by prescreening of donor cells for the optimal expression of the desired trait in vitro and 100% transgenic offspring.
II. Production of transgenic animals
The application of genetic engineering in animals is certainly likely to be a factor in expanding the range of options available in the twenty-first century to meet global food supplies (Niemann and Kues, 2000).
1. Application of transgenic Livestock
1.1.Transgenic animals with agriculture traits
Economically important traits in cattle, such as disease resistance, growth rate, feed conversion efficiency and milk yield, are controlled by certain numbers of genes ( Nottle et al., 1999), and it will be some time before these are identified and become amenable to control. In the meantime, considerable progress can be made in the use of those genes that are available for enhancing the productive potentials of farm animals (Damak et al., 1996). Transgenic sheep carrying a keratin IGF-I constructed gene show expression in their skin and the clear flace was about 6.2% greater in transgenic vs. non-transgenic animals (Damak et al., 1996). Zawada et al (1998) has discussed the options and prospects for altering milk composition in dairy cattle. They have reported the initiation of a research programme at New Zealand to develop transgenic cattle with modified milk composition. In countries where the dairy industry plays a major role in the agricultural economy, modifications of milk proteins, as to provide novel forms of cheese may eventually be one commercial possibility, using gene transfer. Milk contributes significantly to the protein intake of humans in the developing countries and it may be possible to improve its nutritional value as a food by reducing its lactose and BLG content where the population intolerant. In the developed countries, modification of milk content to give a reduced saturated fat content may be a further way in which the new technology can be employed to the advantages of human health and welfare.
1.2. Transgenic animals in biomedicine
The use of transgenic animals for the production of human proteins of biomedical importance is one area in which considerable research emphasis is currently concentrated. The strategy is to target expression of an appropriate gene to the mammary gland and then harvest the product from the milk (Murray et al., 1999). Another interesting application could be enhanced disease resistant (Muller and Brem, 1994). Several products such as human antithrombin III,α-antitrypsin,tissue plasminogen activator ( tPA), α-glucoseidase and lactoferrin are currently in advanced clinical trials (Ziomek ,1998; Meade et al. ,1999). Among the female sheep produced was one (Tracy) capable of producing more than 30 gl –1 of human plasma α1-antitrypsin in her milk (Meade et al., 1999); obtaining therapeutic proteins in this way has the potential to markedly reduce costs of production.
A dairy cow is capable of producing 1 kg or more of endogenous protein daily and the milk can obviously be taken from the animal in the conventional way without compromising welfare. Dairy cattle offer the advantage of producing markedly higher yields of milk than can be obtained from sheep or goats; animals welfare considerations in mammalian species that are quite unaccustomed to routine milking should not be overlooked. Eyestone (1999) had produced transgenic dairy cattle containing the bovine casein-human lactoferrin coding sequences. In the USA, Ebert et al. (1991) has reported studies on the production of transgenic goats; one of these secreted human tissue-type plasminogen activator in her milk at a concentration of 3µg ml-1 and transmitted the gene to some of her progeny.
2. Gene transfer technique
2.1. Embryo microinjection
2.1.1. Pronuclei gene injection
Microinjection of several hundred copies of a gene constructed into the pronuclei of the recently fertilized oocyte was the method successfully used in cattle for the production of transgenic animals (Chauhan et al., 1999). The microinjection method takes advantage of the unique DNA processing events that apparently occur in the pronuclei, especially the male structure; the pronucleus provides the specialized nuclear environment for the incorporation of DNA sequences and for their inclusion in a functional chromosomal region. A total of 66 microinjected bovine zygotes being transferred to the oviducts of recipient cattle (Wall, 1996); 11 recipients calved to produce 21 young, although no calf proved to be transgenic. The ease with which nuclear microinjection can be varies with species, it is essential to employ procedures which allow visualization of the pronuclei, such as the use of differential interference-contrast and phase contrast microscopy (Kay et al., 1991), staining (Minhas et al., 1984) or centrifugation (Thomas and Seidel, 1991). Once injected, the development of the embryo to the transferable stage is usually required. In contrary, Jura et al. (1992) found that coculture with bovine oviductal epithelial cells (BOEC) was an effective method.
A reduced developmental potential of zygotes after microinjection of DNA has been recorded in cattle (Lemme et al., 1994), sheep (Rexroad et al., 1990) and goat (Selgrath et al., 1990). Handling of oocytes, mimicking gene transfer manipulation conditions, was not in itself responsible for such decreased developmental potential; microinjection of DNA conducted at the mid to late stage did, however, decrease embryo development.
2.1.2. Germinal vesicle gene injection
Although the visualization of pronuclei in bovine oocytes can present difficulties, in most morphologically normal oocytes, the germinal vesicle (GV) should be evident. Jura et al. (1990) attempted to microinject the GV of such oocytes with a buffer solution and then mature the cells. Also, Gange et al. (1991) reported some evidence to suggest that this injection approach may have merit as a method of gene transfer.
2.1.3. Cytoplasmic gene injection
Several authors have examined the possibility of using direct cytoplasmic injection of DNA. Galli et al. (1991), working with cattle, sheep, administered a DNA repetitive sequence at different stages of the cell cycle of oocytes and zygotes and analyzed its metabolic fate; it is apparent that DNA metabolism varies among the farm ungulates and at different cell stages. Powell et al. (1992) also deals with the frequency of integrating DNA into the sheep genome by cytoplasmic injection in a report.
2.2.Sperm cell-mediated gene transfer
It is apparent that exogenous DNA, as well as other negatively charged molecules, specially bind to the equatorial and post acrosomal region of the sperm head and that such binding is largely reversible and may occur by the interaction of the nucleic acid with specific protein components (Gandolfi, 1998). Optimal binding of the foreign gene occurs when both sperm motility and DNA concentrations are high (Squires, 1999).
Castro et al. (1990) showed that the sperm of bull, buffalo, ram and goat were all able to bind foreign DNA; the authors suggested that such a capacity might be a general feature of all living sperm cells. Moreover, Peterson et al. (1990) reported on the adsorption of DNA to the sperm of bulls, rams and fallow deer. Capacitated ram sperm were found to be more effective in binding DNA than uncapacitated sperm. Rieth et al. (2000) used electroporation to test the ability of bull sperm to carry foreign DNA into IVM bovine oocytes; they showed that foreign DNA was stable captured by the sperm and carried into the oocytes at fertilization. The authors concluded that sperm mediated DNA transfer may be the means of providing transgenic cattle embryos by a less traumatic procedure than microinjection. Also, Atkinson et al. (1991) were able to demonstrate that bull sperm could bind and internalize foreign DNA; they concluded that the inability of many laboratories to validate this method of gene transfer was due to the relatively low frequency with which sperm took up the DNA. At the same time, Italian researchers continue their efforts in cattle to better define the conditions required for effective sperm-mediated gene transfer using an AI procedure (Lavitrano et al., 1992; Lauria and Gandolfi, 1993).
2.3. Gene transfer using embryonic stem (ES) cells, germ cells or somatic cells
The main advantage in using ES cells is that their use allows the directed modification of endogenous genes, known as ‘gene targeting’. It is possible, by way of such gene targeting and subsequent germ-line transmission from chimeras, to make almost any desired change to the genome (Arat et al., 2001). As noted by Tani et al. (2000), application of similar technology to farm livestock can only follow a clear demonstration of germ-line competence of stem cells from these animals.
In the stem cell approach, cells are initially isolated from the inner cell mass (ICM) of the blastocyst and grown in culture. This provided large numbers of cells for use in gene transfer. The stem cells are subsequently introduced into blastocysts to produce chimeric embryos. Some of the cells may get incorporated into the gonads, resulting in germ cells carrying the new DNA (Keefer et al., 1993).
The greatest disadvantage of using ES cells in the conventional manner for the transfer of foreign DNA in cattle is the fact that the calves born after the injection of stem cells will be chimeras. In this species, it could as noted by Wilmut et al. (1992b) be a matter of 6 years before calves are born carrying the transfer genes. There are many researchers believe that the ES cell route is likely to become the method of choice for genetic manipulation in farm animals (Nortarianni and Lauria, 1992). It may well be possible to combine NT technology with gene transfer into ES cells to produce non-chimeric transgenic cattle, or the use of genetically modified ovine or bovine and caprine donor cells in nuclear transfer (Cibellli et al., 1998b; Baguisi et al., 1999).
Lavoir et al. (1994) have succeeded in isolating and identifying female germ cells from bovine gonadal cell suspension between days 35 and 130 of gestation; the optimal period to isolate such cells appears to be between days 50 and 70. The future challenge in transgenic farm animal production is the isolation and handling of primary cell culture, either from somatic or embryonic origin (Schnieke et al., 1997; Kuese et al., 1998).
2.4. IVMF derived embryos for transgenic cattle production.
The future challenge in transgenic farm animals production is the isolation and handling of primary cell culture, either from somatic or embryonic origin (Kuese et al., 1998). The production of transgenic cattle by way of superovulating and breeding donors, with subsequent recovery of recently fertilized oocytes for DNA microinjection, involves procedures that are both labour-intensive and extremely costly (Tervit, 1990). In view of the very substantial costs involved in acquiring and maintaining recipient cattle over the 9-month gestation period, the elimination of calves other than those likely to express the foreign gene is an important consideration. According to Thomas et al. (1993), the prohibitive expense is a factor contributing to the lack of literature dealing with the production of transgenic animal and for the limited success reported in the few studies that have been conducted. The same authors deal with studies in which an in vitro system was used exclusively to produce transgenic bovine embryos of transferable quality. The propagation of transgenic trait in a given cattle population can be accomplished through in vitro production techniques by using semen from a transgenic bull for in vitro fertilization and collecting oocytes by means of ultrasound from transgenic female founder animals and their subsequent used in IVF (Eyestone, 1999).
Conclusion
Attention must be paid to the application of genetic engineering in farm animals to produce transgenic animals. Gene transfer has economic importance either in animal production or biomedical trait. The common and available gene transfer techniques are gene microinjection in the male pronuclei of recently fertilized oocytes or semen cell mediated gene transfer. These methods produced low percentage of transgenic animals. While, production of chimeric embryos in in-vitro system by using embryo stem cell or somatic cells which isolated from superior genetic animals produce 100 % transgenic animal.
III. Embryo sexing procedures
Controlling and predicting the sex of livestock has considerable advantage in animal production.
The first success with gender preselection in cattle was a calf born on Christmas Day in 1975 in Canada after embryo sexing. Many of sexed calves have born since that time, almost all of them involving embryo sexing (Agca, et al., 1998; Chen, et al., 1999; Rho et al., 2001).
.
1.Use of Y-specific DNA probes
The presence or absence of elements normally located on the Y chromosome determines the sexual differentiation of the bovine embryo. Detection of the DNA sequences present on the Y chromosome may be the basis for a sexing procedure, if the sequences are repetitive and male-specific. The cloning of repeated sequences specific for the bovine Y chromosome has been possible and these DNA fragments have been employed in sexing attempts (Agca et al, 1998). The use of a biontinylated (fluorescent labeling) Y-specific probe enables the sexing of bovine embryos within 30h in the report of Chrenek et al. (2001). This technique was subsequently used by Bousquet et al. (1999); pregnancy rates in recipients cattle in exceed of 50% was recorded by those authors. Rho et al. (2001) described the use of DNA probe technology in determining the sex of cattle embryos from a biopsy sample; although accurate, their method involved an 8-day delay and the use of radioactive isotopes (³H) for the detection of DNA hybridization. Moreover, Dual fluorescent in situ hybridization (FISH), is recent technique availability of DNA probes for chromosome-specific repeated DNA sequences has made DNA-DNA in situ hybridization a feasible approach for detecting Y chromosome (Hassanane et al, 1999); sexing was possible using either tritiated or the biotinylated Y probe. In situ hybridization can be employed to identify sex chromosome in interphase and metaphase nuclei (chromosomes occur in a specific number and morphology and can be individually identified in most domestic animals).
2. Polymerase chain reaction (PCR)
Some functional genes are known to be present on a particular regions of the mammalian Y chromosome, most of the chromosome is composed of non-functional repeated sequence. The PCR method is capable of identifying a particular repeated DNA sequence in a minute amount of sample material by specific amplification of the sequence. The adventage of the PCR technique (Chen et al., 1999) led to a marked improvement in cattle embryo sexing methodology and it has been successfully applied to sexing in Buffalo (Appa Rao and Totey, 1999). According to Ohh (1996), the method was highly accurate and could be carried out on the farm within 3h, and it was described as the rapid Y-chromosome detecting (YCD) assay. The YCD assay involves removal of a small number of blastomeres (two to ten), requiring according to the authors, less than 2 min, and the subsequent amplification of the male-specific DNA sequences. A study by Hilgers and Herr (1993) examined reagents used in the culture and storage of bovine embryos for sexing; they concluded that it is advisable to using products derived from animal blood and tissue (e.g. serum preparations).
Bovine embryo sexing is now routinely used on farms in France, using the DNA probe in combination with PCR technique (Marquant-Le Guienne and Humblot, 1998). On average, five to eight cells are taken for sampling; it is recorded that 95% of biopsied embryos can be sexed with accuracy. A marked reduction in the percentage of recipients required to produce heifer replacements in the dairy herd is one of the several advantages claimed for this sexing procedure (Nibart et al., 1993). A mean pregnancy rate of 58% after the transfer of biopsied and sexed embryos is a given by Agca et al. (1998); the same authors show that only high-grade embryos can be successfully frozen after sexing.
Near-normal pregnancy rate after cervical transfer of biopsied bovine embryos was reported by Holm et al. (1998) from their studies in France. In Germany, Roschlau et al. (1992) described their experiences in sexing more than 2500 cattle embryos as part of ET activities; no adverse effect on pregnancy rate was recorded with fresh embryos but a 10% reduction occurred when embryos were frozen. Ohh et al. (1996) showed that removing one cell from the 16-32-cell stage IVMFC-derived bovine embryo did not adversely affect its subsequent development in vitro. A further report from the same group showed that the sexing procedure had no significant effect on pregnancy rate (Machaty et al., 1993).
3. Sexing by male-specific antigen
In the immunological detection of a male-specific antigen on the bovine embryo, it is believed that the histocompatability antigen (H-Y antigen) is expressed at the late morula stage. Several workers have recorded their attempts at sexing by identifying this male-specific antigen. In Dublin, workers with sheep, Ware (1986) employed the H-Y sexing procedure with limited success; one problem was apparently reduced viability of the sheep embryos after sexing. The bovine embryos are exposed to H-Y antibodies, which bind to those that are male; a second antibody, usually with fluorescent label, is then employed to permit identification under the microscope. A major disadvantage of this procedure is that the accuracy of sexing does not appear to exceed 80-90% (Van Vliet et al., 1989).
Attempts to sex bovine embryos with such antibodies, including monoclonal and poly-clonal antibodies (Hossepian de Lima et al., 1993) or the use them to inhibit blastocoele formation is described (Utsumi et al., 1993). When morula- stage embryos are cultured in the presence of this antibody, the development of male embryos is arrested whereas female embryos continue to develop to blastocyst stage (Utsumi and Iritani, 1993)
4. X-chromosome-linked enzymes
The availability of effective procedures for the IVC of IVF-derived cattle embryos may provide novel opportunities for sexing the early cattle embryo. The early cleavage period is the only time in the life of the embryo when both X sex chromosomes are active. The products coded for by the active genes on the X chromosome (which produce enzyme) are secreted in double the usual amounts and such differences may be detected by suitably sensitive assay procedures. The process of X chromosome inactivation in the bovine embryo has been identified by Fuente et al. (1993) to occur at early blastocyst stage and to increase progressively as the blastocyst elongates.
A non-invasive method of sexing embryos, based on a colorimetric assay for glucose-6-phosphate dehydrogenase (G6PD) activity has been reports in cattle (Gutierrez-Adan et al., 2001) and hypoanthine phosphoribosyl transferase (HPRT) (Monk et al, 1990). They are x-linked enzyme which there is quantitative difference of approximately 1:2 between male and female cells of embryos; enzymatic activity was not sufficiently different to permit accurate separation of the sexes.
5. Sex of early embryonic development
Some early evidence of sexed-dependent developmental changes in cattle embryos produced in vivo was provided by workers in Denmark (Avery et al., 1992). However, when cattle embryos are recovered from superovulated donor cattle, the impact of the sex effect differences may be masked by the fact that ovulations probably occur over a period of several hours (Callesen et al., 1992). An early-ovulated oocyte fertilized by an X-bearing sperm (female embryo) might therefore be further developed than a later-ovulated oocyte fertilized by an Y-bearing sperm (male embryo). With IVF-produced embryos, such sources of variation would not be expected.
Studies reported by Tiffin et al. (1991) showed that total glucose metabolism in male cattle embryo was twice that in female embryos and increased between the morula and expanded blastocyst stage. Such differences are related to sex-developmental changes. Apparent relationship between sex and developmental rate may be employed as a method for non-invasive sexing of IVMFC-derived bovine embryos. Results demonstrated that male embryos generally develop to a more advanced stage than female in the first 8 days after IVF (Xu et al., 1991b). As early as 3 days after IVF, the fastest-developing embryos were males (Marquant-Leguienne et al., 1992). There is even data showing that the male predominance of bovine IVMFC-derived embryos is evident as early as the two-cell stage (Marquant-Le Guienne and Humblot, 1998). Further report by Xu et al. (1992b) recorded a pregnancy rate of 63% after ET and provided evidence of the normality of their sexual dimorphism system.
Conclusion
The quest to know the sex of the offspring before implanting an embryo of known sex would be more beneficial economically. This is especially true in domestic animals such as cattle and buffalo where productivity and economic return for the farmer are mainly dependent on the female sex. Also, the selection of male embryos from superior genetic superovulated dams may increase the genetic gain in national breeding programs. Moreover, problems like free martinism could also be controlled if sexed embryos are used.
The best two methods we can use it in embryo sexing are Fluorescence in Situ Hybridization (FISH) and Polymerase Chain Reaction (PCR). They are accurate (95%), rapid and they need only 1-3 cells for investigation, which means high survival rate and normal development of the embryos after biopsy. While, FISH technique is more expensive than PCR technique
IV. Gamete Micromanipulation
Intracytoplasmic sperm microinjection (ICSI) and related technology could be used for the propagation of domestic and exotic species. A bull could have excellent characteristics but prove infertile in AI or IVF and so ICSI could be used to rescue the genetic traits. There are four main approaches to gamete micromanipulation, which may assist fertilization under conditions of reduced sperm number or when sperm motility is compromised or even non-existent.
1. Intracytoplasmic sperm injection (ICSI)
The injection of single sperm into the oocyte cytoplasm is attractive because it ensures monospermic fertilization. In vitro matured bovine oocytes were injected with freeze thawed killed sperm and calves resulted from the transfer (Reyes et al., 2001). Then exogenously activated using calcium ionophore. Animal lamb was born after the injection of male sperm into an inactivated sheep oocyte (Catt , 1996).
Sperm were ‘swim up’ from a fresh ejaculate, which had been diluted 20-fold, suspended in 10% polyvinylpyrrolidone (360000 mol. wt.) in medium to retard the motility enabling capture and to give the necessary operating pressures. A single motile sperm was immobilized by swiping the injection pipette across the tail (Wei and Fukui, 1999). The immobilized sperm is aspirated tail first into the injection pipette and maneuvered to the tip. The denuded matured oocyte is held in a wide bore holding pipette and the injection pipette introduced deep into the cytoplasm. Backpressure is then exerted and the sperm and plasmalemma moves up the injection pipette (Katayose et al., 1999). Pressure is continued until the plasmalemma rupture and the cytoplasm moves past the sperm. The cytoplasm and the sperm are returned into the oocyte and injection pipette withdrawn.
In all the species tested oocyte exogenous activation is not mandatory for at least early fertilization events to take place. In previous studies where sperm have been killed before injection, exogenous activation was invariably used (Hwang et al., 2000).
Results reported by Keefer et al. (1991) indicated that, BOs are not sufficiently stimulated by the sperm injection procedure to complete meiosis. In the work of Goto et al. (1990) and subsequently, BOs were deliberately activated by a 10-min period of exposure to calcium ionophore (A23187). In the studies of Wei and Fukui (1999), a 5 min were based on results reported by Ware et al. (1989), who examined the effect of various dose levels of A 23187 on BO activation. Ethanol (7% for 7 min) has also been employed to activate oocytes in the work reported by Li et al. (1999).
The examination of oocytes after sperm injection indicated that the early fertilization events were normal and that the injected sperm was forming a pronucleus. It was possible to produce live births from ovine ICSI the efficiencies of the procedures were low, a problem found with all domestic species ICSI (Catt et al., 1994). Pregnancies can true potential of in vitro derived embryos is largely unknown. Of practical interest are the results of Catt et al. (1994) showing higher fertilization rates with sperm injection than with SUZI. Yang and Anderson (1992) succeeded in achieving fertilization with sperm from bull suffering from the ‘stump tail’ infertility defect. Such results make it clear that sperm injection can be superior to IVF in certain instances of bull infertility or subfertility. However, the real practical significance of ICSI in cattle may eventually be in terms of devising computer-controlled fertilization system which make optimal use of sperm, practically sperm in low numbers that may have been sexed by flow cytometry or other techniques (Fishel et al. (1993).
2. Insertion through the zona (subzonal insemination or SUZI)
The placement of more than one sperm into the perivitelline space has been termed subzonal insemination or SUZI. With increasingly more sophisticated injection pipettes and technique, the safe insertion of sperm became commonplace. SUZI was used after the advent of partial zona dissection to attempt to control polyspermia. The injection pipette is inserted through the zona, using the holding pipette as a backstop, in the region of the first polar body. Careful control of the pressure inside the injection needle ensures the correct numbers of sperm are deposited. On withdrawal of the pipette the zona seals itself, preventing the sperm from escaping. A limited number of reports dealing SUZI in cattle have appeared in Germany (Schwiderski et al., 1990), the USA (Heuwieser et al., 1991); the technique was regarded as useful for studying sperm-oocyte interactions, but fertilization rates were generally low.
3. Zona drilling and partial zona dissection
Tearing or drilling of holes in the zona has been used in an attempt to bypass the zona barrier. Acidic solution (Acid Tyrodes pH 2.6) was sprayed from a manipulating pipette over the area of the zona being drilled. The acid solution etched through the zona forming a hole. The oocyte is secured onto a holding pipette and a manipulating pipette inserted through the zona, tangential to the oocyte, to pierce the other side of the zona. The oocyte is released from the holding pipette and the manipulating pipette rubbed against the holding pipette, wearing the zona away (Cohen, 1992). This method allows some control over the size of the hole created (Tucker et al., 1996). A larger hole may cause precocious escape of the embryo from the zona and abnormal hatching may occur with a hole is too small. The techniques of zona opening have not found any practical uses in domestic.
4. Zona thinning procedures
There may be occasions when it would be useful to achieve fertilization of IVM bovine ocytes (BOs) with lower than usual sperm doses. Mordel et al. (1992) demonstrated that zona thinning by partial digestion with pronase to be safe and capable of enhancing fertilization at very low sperm concentrations. The ability of zona pellucida (ZP) to function as a receptor is attributed to the presence of certain oligosaccharides (Miller et al., 1992); pronase, which digests protein, does not affect carbohydrate structure. The data of Mordel et al. (1992) suggest that pronase treatment may be used to enhance fertilization efficiency at very low sperm concentrations; ZP continues to act effectively under such conditions, as both sperm receptor and inducer of acrosome reaction (AR).
Under ordinary in vivo and in vitro conditions, the ZP of the cow oocytes is regarded as essential for normal fertilization. It induces the AR of bull sperm and serves to block polyspermic fertilization. However, studies with IVM BOs, using pronase, have clearly shown that the ZP is not indispensable for IVM and IVF (Xu et al., 1991a); although the percentage of ZP-free zygotes that became blastocysts was lower than usual, the embryos were capable of giving rise to pregnancies on transfer to recipient cattle.
Conclusion
Intracytoplasmic sperm injection (ICSI) is practic for enhancing fertilization in IVF and production of trangenic animals (sperm cell- mediated gene transfer). It increases the in vitro embryo production (IVP) in domastic animals. It enhances fertilization by using sperm of superior genetic animal even if it is suffering from infertility (few sperm number, compromised sperm motility or stump tail)
V. Expression of developmentally important genes in preimplantation embryos
IVP may have profound effects on the genomic activity of preimplantation embryos with potentially severe effects on fetal, prenatal and postnatal viability (Wrenzycki et al., 1998a). With the advent of sensitive reverse Transcriptase Polymerase Chain Reaction (RT-PCR) procedures (Matale et al., 2001), it is now possible to gain insight into the transcriptional activity of individual oocytes and embryos by determining the relative abundance of transcripts from various developmentally important genes. Messenger RNA phenotyping is a useful tool to assess the normality of in vitro –produced embryos and to optimize in vitro culture conditions. The selection of genes representing a large number of essential physiological functions in preimplantation development seems important. Primary candidates for studying imprinted genes are apolipoprotein E, pyruvate-kinase-3, protein phosphate-gamma, as well as IGF-II and IGF-IIr. All these are known to exert essential function in early differentiation and cell cycle (Mann et al., 1999; Prelle et al, 2001).
Comparative aspects of embryonic gene expression in embryos derived in vitro or in vivo
Studies have focussed on comparative aspects of gene expression in bovine in vitro derived versus in vivo embryos. No differences were found in the mRNA expression of a set of translation initiating factors genes in bovine embryos grown in either TCM 199 supplemented with serum plus epithelial cell co-culture under 5% CO2 in SOF (Synthetic Oviductal Fluid) supplemented with citrate under 10% CO2 atmosphere (De Sousa et al., 1998). In vivo and IVP bovine blastocysts expressed the surface antigen proteins (Western Blot) TEC-1, -2-3, -4 (developmental regulating antigen in a similar manner (Rizos et al., 2002).
To mature oocytes, studies have employed the standard culture medium TCM-199 supplemented with hormones (FSH, LH, Estradiol 17-B) and estrus cow serum. Non-surgical transfer of embryos grown in this medium resulted in pregnancy rates of 50% (Eckert and Neimann, 1995). Cx43 transcripts were detected in bovine morulae and blastocysts collected in vivo from superovulated donors whereas IVP blastocysts and hatched blastocysts grown in either this medium or TCM 199 plus BSA contained no mRNA for Cx43 (Wrenzycki et al., 1998b). Cx43 is a member of the connexin family and encodes for gap junctions that are crucial for maintenance of compaction. The cytokine leukemia inhibitory factor (LIF) plays an essential role during early differentiation and implantation in bovine. The receptors consist of two dimerizing subunits, glycoprotein 130 (gp 130) and LIF receptor-B (LR-B). Authors found that transcripts for the bLIF ligand and the two-receptor subunits were abundantly amplified from cumulus cells. While gp 130 and LR-B mRNA were consistently detected throughout bovine embryo development in vitro from the immature oocytes to the hatched blastocyst, bLIF transcripts could not be amplified from immature oocytes. The gene was only occasionally detected from the matured oocytes up to the 16-cell stage as well as in blastocysts depending on embryo batch, was never found in morulae. In contrast, in vivo derived bovine embryos never expressed any bLIF-mRNA, and LR-B-mRNA could not be identified at the morula to blastocyst transition while gp 130 transcripts were observed from the morula up to the hatched blastocyst stage (Eckert and Neimann, 1995). These observations indicate a severe perturbation of the mRNA expression patterns of the specific LIF/LIF-receptor system in bovine embryos grown in vitro. This could potentially lead to abnormal development of the ICM and trophectoderm in the blastocyst. However, it is still not clear that the bovine LIF/LIF-receptor system plays a similar essential role in preimplatation development and implantation (Eckert and Neimann, 1998). This relationship needs to be investigated further especially as it could be relevant to the large calf syndrome
A relative abundance of a set of genes involved in compaction and activation was investigated in embryos produced in vitro either in serum enriched or defined medium (Wrenzycki et al., 1999). Transcription of the majority of these genes was increased in the defined medium starting at the 8- to 16-cell stage of development corresponding with the burst of bovine embryonic genomic activity (Telford et al., 1990). Under protein-free condition the production of higher mRNA levels is critical for further development, especially for the first step of differentiation occurring at the morula/blastocyst stage. However, the possibility cannot ruled out that a restraining serum effects is beneficial as the blastocyst formation rate was significantly higher in serum-enriched medium than in PVA-supplemented medium. Only heat shock protein 70.1 (Hsp 70.1) mRNA was significantly higher in serum-generated embryos in comparison with PVA-derived counterparts. Hsp has been shown to be unregulated in IVP blastocysts following heat stress (Kawarsky and King, 1998). It was also observed that culture in either serum-enriched or serum-free medium had marked effects on the ultrastructure of the blastocysts. Embryos cultured in serum-free medium possessed large amounts of lipids and fewer lysosome-like vesicles than those cultured in serum-free medium (Abe et al., 1999). The mRNA-expression levels of bovine morulae and blastocysts derived in TCM 199 supplemented with either serum, BSA or PVA were compared with their in vivo counterparts. The relative abundance of glutamine-1 mRNA was significantly elevated in morulae derived in vivo versus in vitro counterparts. In vivo derived blastocysts had significantly reduced levels of Hsp70.1 mRNA compared with IVP blastocysts cultured in serum-enriched medium (Wrenzycki et al., 1998b). Preliminary data show that culture in SOF-medium supplemented with either serum or BSA results in different expression patterns than culture in TCM medium. Although SOF-medium provided an expression pattern closer to that of in vivo embryos, there were still considerable differences in the in vitro derived embryos.
Conclusion
The tissue culture media used in in vitro embryo production effects on the genomic activity of preimplantation embryos with potentially severe effects on fetal, prenatal and post natal viability.
Therefore, We must use suitable medium (i.e. TCM + SOF +serum) which provided gene expression pattern closer to that of in vivo derived embryos. Moreover, messenger RNA phenotyping (PCR) is a useful tool to assess the normality of in-vitro produced embryos and to optimize in-vitro culture conditions .The best medium is the medium which lead to expression of genes which is essential for early embryonic development and normal cell cycle (i.e. Apolipoprotein E , pyruvate – Kinase-3, protein phosphate-gamma, IGF II, GH ) as indication to improvement in embryo production.
General conclusion
In Egypt, low reproductive potential in farm animals is one of the great factors hindering the national economy. This could be due to nutritional, management, genetic factors as well as the traditional breeding programss. To solve this problem, we should apply the modern biotechnological approaches to improve its genetic, productive and reproductive potentials.
The first step is to select herds from the Egyptian farm animals on the basis of their genetic, productive and reproductive characters. These animals can be used as a nucleus for spreading the high genetic performance throughout the country. Application of cloning technique to produce many copies from these selected animals. Also, it can be used for the production of chimera animals. Moreover, gene microinjection (gene responsible for high milk yield, disease resistance, growth rate) in the male pronucleus of in vitro fertilized oocyte can accelerate the time consuming in genetic improvement of livestock. Controlling the embryo sex (by fluorescent in situ hybridization and PCR) in the early implanted embryos has commercial application to serves the dairy farms by transferring female embryos only for milk yield and offspring production. While, male embryos can be used for meat production. So, it will save money and time consume in farms.
In addition, through biotechnology we can overcome problems of in vitro fertilization by using intracytoplasmic sperm microinjection. Also, ICSI can be used for fertilization of oocytes with sperm from valuable bulls that suffering from low sperm number or have low motility. ICSI of sperm cell mediated gene transfer or previously sexed sperm have valuable outcome in genetic improvement in livestock.
Furthermore, attention should be paid to improve the viability of the preimplanted embryos. Using a suitable culture media, leads to good gene expression close to that found in in vivo derived embryos.
The development of biotechnology center is crucial to improve the Egyptian farm animals. This center will manipulate the genetic character and accelerate its improvement. Production of good quality embryos from valuable animals and the development of Embryos Bank will facilitate the dissemination of these good productive characters in a commercial aspect.
References
Abe, H., Yamashita, S., Itoh, T., Satoh, T. and Hoshi, H. (1999): Ultrastructure of bovine embryos developed from in vitro –matured and fertilized oocytes: Comparative morphological evaluation of embryos cultured either in serum-free medium or in serum-supplemented medium. Mol. Reprod. Dev., 53: 325-335.
Agca, Y., Monson, R.L., Northey, D.L., Peschel, D.E., Schnefer, D.M. and Rutlegde, J.J. (1998): Normal calves from transfer of biopsed, sexed and vitrified IVP bovine embryos. Theriogenol., 50 (1): 129-145.
Alberio, R., Brero, A. and Motlik, J., Cremer, T., Wolf, E. and Zakhartchenko, V. (2001): Remodeling of donor nuclei, DNA-synthesis and ploidy of bovine cumulus cell nuclear transfer embryos: effect of activation protocol. Mol. Reprod. Dev., 59 (4): 31-379.
Appa Rao, K.B. and Totey, S.M. (1999): Cloning and sequencing of buffalo male-specific repetitive DNA: sexing of in vitro developed buffalo embryos using multiplex and nested polymerase chain reaction. Theriogenol. 51 (4): 785-797.
Arat, S., Rzucidlo, S.J., Gibbons, J., Miyoshi, K. and Stice, S.L. (2001): Production of transgenic bovine embryos transfer of transfected granulosa cells into enucleated oocytes. Mol. Reprod. Dev., 60 (1): 20-26.
Atkinson, P.W., Hines, E.R., Beaton, S., Matthaei, K.I., Reed, K.c. and Bradley, M.P. (1991): Association of exogenous DNA with cattle and insect spermatozoa in vitro. Mol. Reprod. Dev., 29: 1-5.
Avery, B., Jorgensen, C.B., Madison, V., and Greve, T. (1992): Morphological development and sex of bovine in vitro –fertilized embryos. Mol. Reprod. Dev., 32: 265-270.
Baguisi, A., Behboodi, E., Melican, D., Poolock, J.S., Destrempes, M.M., Cammuso, C., Williamsm J.L., Nims, S.D., Porter, C.A., Midura, P., P., Palaccios, M.J., Ayres, S.L., Denniston, R.S., Hayes, M.L., Ziomoek, C.A., Meade, H.M., Godke, R.A., Gavin, W.G., Overstrom, E.W. and Echelard, Y. (1999): Production of goat by somatic cell nuclear transfer. Nat. Biotechnol., 17: 456-461.
Baran, V., Vignon, X., Bourhis, L., Renerd, J.P. and Flechon, J.E. (2002): Nuclear changes in bovine nucleo transferred embryos. Biol. Reprod., 66 (2): 534-543.
Barnes, F., Endoebrock, M., Looney, C., Powell, R., Westhusin, M. and Bondioli, K., (1993): Embryo cloning in cattle: the use of in vitro matured oocytes. J. Reprod. Fert., 97: 317-320.
Betts, D., Bor Jignon, V., Hill, J., Wigner, O., Westhusin, M., Smith, L. and King, W. (2001): Reprogramming of telomerase activity and rebuilding of telomere length in cloned cattle. Proc. Nat. Acad. Sci., USA 98 (3): 1077-1082.
Boediono, A., Takagi, M., Saha, S. and Suzuki, T. (1993): Chimeric blastocyst production using IVF bovine embryos (Holstein-Japanese Red) without zona pellucida. Theriogenol., 39: 191.
Bordignon, V. and Smith, L.C. (1998): Telophase enucleation an improved method to prepare recipient cytoplasts for use in bovine nuclear transfer. Mol. Reprod. Dev. 49 (1): 29-36.
Bousquet, D., Twagiramungu, H., Morin, N., Brisson, C., Carbonean, G. and Durocher, J. (1999): In vitro embryo production in the cow: an effective alternative to the conventional embryo production approach. Theriogenol., 51 (1): 59-70.
Bredbacka, P., Hinhtinen, M., Aalto, J., and Rainio, V. (1992): Viability of bovine demi-and quarter-embryos after transfer. Theriogenol., 38: 107-113.
Callesen, H., Bak, A. and Greve, T. (1992): Prediction of sex embryos from superovulated cattle based on developmental stages at recovery. Proc. 8th Conference of the European Embryo Transfer Association (Lyon), 134.
Campbell, K.H., Loi, P., Otaegui, P.J. and Wilmut, L. (1996a): cell cycle co-ordination in embryocloning by nuclear transfer. Rev. Reprod., 1: 40-46.
Campbell, K.H., McWhir, J., itchie, W.A., Wilmut, I. (1996b): Sheep cloned by nuclear transfer from a cultured cell line. Nature 380: 64-66.
Catt, J.W., Krzyminksa, W., Tilia, L., Csehi, E., Ryan, J.P., Pike, I. and O’neill, C. (1994): Subzonal insertion of multiple sperm is a treatment for male factor in fertility. Fert. Steril. 61: 118-124.
Catt, W. (1996): Intracytoplasmic sperm injection (ICSI) and related technology . Animal Reproduction science 42,239-250.
Chauhan, M.S., Nadir, S., Bailey, T.L., Pryor, A.W.,Buchers, P., Notter, D.R., Velaander, W.H. and Gwazdauskas, F.c. (1999): Bovine follicular dynamics, oocyte recovery and development of oocytes microinjected with a green fluorescent protein construct. J. Dairy Sci., 82 (5): 918-926.
Castro, F.O., Hernandez, O., Uliver, C., Solano, R., Milanes, C., Agulilar, A., de Armes, R., Herrera, L. and la Fuente, J. (1990): Introduction of foreign DNA into the spermatozoa of farm animals. Theriogenol., 34: 1099-1110.
Chen, C.M., Hu, C.L., Wang, C.H., Hung, C.M., Wu, H.K., Choo, K.B. Cheng, W.T. (1999): Gender determination in single bovine blastomeres by polymerase chain reaction amplification of sex-specific polymorphic fragments in the amelogenin gene. Mol. Reprod. Dev. 54 (3): 209-214.
Chesne, P., Peynot, N., Desmedt, V., Rao, V.H. and Renard, J.P. (1991): Effect of in vitro or in vivo maturation of cattle oocytes on cleavage rate following nuclear transfer. Proc. of the 7th Meeting of the European Embryo Transfer Association (Cambridge): 136.
Chesne, P., Heyman, Y., Peynot, N. and Renard, J.P. (1993): Nuclear transfer in cattl: birth of cloned cattle and estimation of blastomere totipotency in morulae and source of nuclei. C. R. Acad. Sci., 316: 487-491.
Chrenek, P., Boulanger, L., Heymany, Y., Uhrin, P., Laurinicik, J., Bulla, J. and Renard, J.P. (2001): Sexing and multiple genotype analysis from a single cell of bovine embryo. Theriogenol., 55 (5): 1071-1081.
Cibelli, J., Stice, S.L., Golueke, P.J., Kana, J.J., Jerry, J., Blackwell, C., Ponce, de Leon, F.A. and Robl, J.M. (1998a): Transgenic bovine chimeric offspring produced from somatic cell derived stem cells. Nat. Biotechnol., 16 (7): 642-646.
Cibelli, J., Stice, S.L., Golueke, P.J., Kana, J.J., Jerry, J., Blackwell, C., Ponce, de Leon, F.A. and Robl, J.M. (1998b): Cloned calves produced from non-quiescent fetal fibroblasts. Science 280: 1256-1258.
Cohen, J. (1992): Review of clinical microsurgical fertilization: In: J Cohen, H.E., Matter, B.E., Talansky, A. and Grifo J. (ed). Micromanipulation of Human Gametes and Embryos. Raven Press New York, pp: 163-190.
Collas, P., Balise, J.J. and Robl, J.M. (1992): Influence of cell cycle stage of the donor nucleus on development of nuclear transplant rabbit embryos. Biol. Reprod., 46: 492-500.
Damak, S., Su, H., Jay, N.P. and Bullock, D.W. (1996): Improved wool production in transgenic sheep expressing insulin-like growth factor 1. J. Biotechnol., 14: 185-188.
De Sousa, P.A., Watson, A.J. and Schultz, R.M. (1998): Transient expression of a translation initiation factor is conservatively associated with embryonic gene activation in murine and bovine embryo. Biol. Reprod., 59: 969-977.
Ebert, K.M., Selgrath, J.P., Di Tullio, P., Denman, J., Smith, T.E., Memon, M.A., Schnidler, J.E., Monastersky, G.M., Vitale, J.A. and Gordon, K. (1991): Transgenic production of a variant of human tissue-type plasminogen activator in goat milk; generation of transonic goats and analysis of expression. Bio. Technol., 9: 835-843.
Ector, F.J. (1996): Cloning nuclear transfer in bovine species results and perspectives. Bull. Mem. Acad. R. Med. Belg. 151 (12): 493-499.
Eckert, J. and Niemann, H. (1995): In vitro maturation, fertilization and culture to blastocysts of bovine oocytes in protein-free media. Theriogenol., 43: 1211-1225.
Eckert, J. and Niemann, H. (1998): mRNA expression of leukaemia factor (LIF) and its receptor subunits glycoprotein 130 and LIF-receptor-B in bovine embryos derived in vitro or in vivo. Mol. Reprod. Dev., 4: 957-965.
Eyestone, W.H. (1999): Production and breeding of transgenic cattle using in vitro embryo production technology. Theriogenol. 51: 509-517.
Fehilly, C.B., Willadsin, S.M. and Tucker, E.M. (1984): Experimental chimaerism in sheep. J. Reprod. Fert., 70: 347-351.
Fishel, S., Timson, J., Green, S., Hall, J., Dowell, K. and Klentzeris, L. (1993): Micromanipulation. Reproductive Medicine Review 2:199-222.
First, N.L. (1992): Animal biotechnologies: Potential impact on animal products and their production. In J.F. MacDonald (Ed) Animal Biotechnology: Opportunities and Challenges pp.37-47.
Fuente, R.de la ,Plante, L. and King, W.A.(1993): X chromosome inactivation in the preattachment bovine embryo .Theriogenology 39,20.
Fulka, J., First, N.L., Loi, P. and Moor, R.M. (1998): Cloning by somatic cell nuclear transfer. Bio Assay 20: 847-851.
Gandolfi, F. (1998): spermatozoa DNA binding and transgenic animals. Transgenic Res., 7: 147-155.
Gange, M.B., Pothier, F. and Sirard, M.A. (1991): Electroporation of bovine spermatozoa to carry foreign DNA in oocytes. Mol. Reprod. Dev., 29: 6-15.
Galli, A., Basetti, M., Balduzzi, D., Martignoni, M.m Bornaghi, V. and Maffii, M. (1991): Frozen bovine semen quality and bovine cervical mucus penetration test. Theriolgenol., 35: 837-844.
Goto, K., Kinoshita, A., Takuma, Y. and Ogawa, K. (1990): Fertilization by sperm injection in cattle. Theriogenol., 33: 238.
Greising, T., Monghan, P., Healy, A., Gallagher, M., Wahid, H. and Gordon, I. (1994): The influence of cytolpalsmic composition and quality of cytoplasm in recipient oocytes on the developmental capacity of bovine nuclear transfer embryos. Theriogenol., 41: 208.
Hassanane, H., Kovars, A., Laurent, O., Lindblad, K., Gustavsson, I. (1999): Simultaneous detection of X- and Y bearing spermatozoa by double colour fluorescence in situ hybridization. Mol. Reprod. Vev., 53: 407-412.
Heuwieser, W., Yang, X., Jiang, S. and Foote, R.H. (1991): Fetilization of bovine oocytes by microinjection of spermatozoa into the perivitelline space. Theriogenol., 35: 213.
Heyman, Y., Chavatte-Palmer, P., Bourihis, D., Camous, S., Viganon, X. and renard, J.P. (2002): Frequencey and occurrence of late-gestation losses from cattle cloned embryos. Biol. Reprod., 66 (1): 6-13.
Hilgers, L.J. and Herr, C. (1993): DNA contamination of reagents used in embryo transfer and culture. Theriogenol., 40: 023-932.
Hill, J.R., Winger, Q.A., Burghardt, R.C. and Westhusin, M.E. (2001): bovine nuclear transfer embryo development using cells derived from a cloned fetus. Anim. Reprod. Sci., 67 (1-2): 17-260.
Holm, P., Shukri, N.N., Vajta, G., Booth, P., Bendixen, C. and Callesen, H. (1998): Developmental kinetics of the first cell cycles of bovine in vitro viability and sex. Theriogenol., 50 (8): 1285-1299.
Hossepian de Lima, V.F.M. Moreira-Filho, CA., De Bem, A.R. and Jorge, W. (1993): Sex determination of murine and bovine embryos using cytotoxicity and immunofluorescence assay. Theriolgenol., 39: 1343-1352.
Huang, S.z., Huang, Y., Chen, M.J., Chen, W.Y., Huang, Z., Li, J.C., Ren, Z.R. and Zeng, Y.T. (2000): A study of transgenic IVF cattle integrated with human serum albumin gene. Yi Chuan Xue Bao 27: 573-79.
Hwang, S., Lee, E., Yoon, J., Yoon, B.K., Lee, J. and Choi, D. (2000): Effects of electric stimulation on bovine oocytes activation and embryo development in intracytoplasmic sperm injection. Procedure. J. Assist. Reprod. Genet. 17 (6): 310-3114.
Jones, K.L., Hill, J., Shin, T.Y., Lui, L. and Westhusin, M. (2001): DNA hypomethylation of karyoplasts for bovine nuclear transplantation. Mol. Reprod. Dev., 60 (2): 208-213.
Jura, J., Smorag, Z., Kastka, L. and Bak, M. (1990): In vitro maturation of bovine oocytes following buffer microinjection into germinal vesicle or cytoplasm. Theriogenol., 33: 263.
Jura, Kopchick, J.J., Chen, W.Y, Modliinski, J.A. and Smorag, Z. (1992): Production of transferable bovine embryos from exogenous DNA microinjected zygotes and 2-cell embryos. Proc. 11th International Congress on animal Reproduction (The Hague), 2: 697-699.
Kang, Y.K., Koo, D.B., Park, J.S., Choi, Y.H., Chung, A.S., Lee, K.K., Han, Y.M. (2001): Aberrant methylation of donor genome in cloned bovine embryos. Nat. Genet 28 (2): 173-177.
Kang, Y.K., Park, J.S ,Koo, D.B., Choi, Y.H., Kim, S.U, Lee, K.K., Han, Y.M. (2002): Limited demethylation leaves mosaic-type methylation states in cloned bovine preimplantation embryo. EMBO J. 21: 1092-1100.
Kanka, J., Hoza, K.P., Heyman, Y., Chesne, P., Degrolard, J., Renard, J.P. and Flechon, J.E. (1996): Transcriptional activity and nuclear ultrastructure of embryonic rabbit nuclei after transcription to enucleated oocytes. Mol. Reprod. Dev., 43 2): 135-144.
Kansinothan, P., Knott, J.G., Moreira, P.N., Burnuside, A.S., Jerry, D.J. and Robl, J.M. (2001): Effect of fibroblast donor cell age and cell cycle on development. Bio. Reprod., 64 (5): 1487-1493.
Katayose, H., Yanagida, K., Shinoki, T., Kawahara, T., Horiuchi, T. and Sato, A. (1999): Efficient injection of bull spermatozoa into oocytes using a Piezo driven pipette. Theriogenol., 52: 1215-1224.
Kato, Y., Ytani, T., sotomaru, Y., Kurokawa, K., Kato, J., Doguchi, H., Yasue, H., Tsunoda, Y. (1998): Eight calves cloned from somatic cells of a single adult. Science 282: 2095-2098.
Kawarsky,S.J.and King ,W.A. (1998): Expression and localization of Hsp70 in the bovine embryo. Thriogenology, 49 ,273 abst.
Kay, G.W., Hawk, H.W., Waterman, R.A. and Wall, R.J. (1991): Identification of pronuclei in in vitro fertilized cow embryos. Biol. Reprod., 44 (Suppl. 1): 74 (abst. 87)
Keefer, C.L., Stice, S.L. and Maki-Laurila, M. (1991): Cleavage development of bovine oocytes fertilized by sperm injection. Mol. Reprod. Dev., 25: 281-285.
Keefer, C.L., Koppang, R., Parprocki, A.M., Golueke, P., Stice, S., Maki-Laurila, M. and Matthews, L. (1993): Bovine inner cell mass (ICM) cells as donor nuclei in the production of nuclear transfer embryos. Theriogenol., 39: 242.
Kilby, N.J., Snaith, M.R. and Murray, J.A.H. (1993): Site-specific recombinase: tools for genome engenerring. Trends Genet., 9: 413-421.
Kitiyanant, Y., Saikhun, J., Chaisalee, B., White, K.L. and Pauvasuthipaisit, K. (2001): Somatic cell cloning in Buffalo (Bubalus bubalis): Effects of Interspecies Cytoplasmic Recipients and activation procedures. Cloning Stem Cells 3 (3): 97-104.
Kono, T., Sotomoru, Y., Aono, F., Takahashi, T., Ogihara, K., Sekizawa, F., Arai, T. and Nakahara, T. (1993): Effect of ooplast activation on the development of nuclear transferred bovine embryos. Theriogenol., 39: 248.
Kues, W.A., Anger, M., Carnwath, J.W., Paul, D., Motlik, J., Niemann, H. (2000): Cell cycle synchronization of procien fetal fibroblasts: effects of serum deprivation and reversible cell cycle inhibitors. Biol. Reprod., 62: 412-419.
Landa, V. and Riha, J. (1992): Directed aggregation chimaeras constructed from individual blastomeres of bovine 16-cell stage embryos. Proc. 12th International Congress on animal Reproduction (The Hague) 2: 703-313.
Lauria, A. and Gandolfi, F. (1993): Recent advances in sperm mediated gene transfer. Mol. Reprod. Dev., 36: 255-257.
Lavitrano, M., French, D., Zani, M., Frati, L. and Spadafora, C. (1992): The interaction between exogenous DNA and sperm cells. Mol. Reprod. Dev., 31: 161-169.
Lavoir, M.C., Basrur, P.K. and Betteridge, K.J. (1994): Isolation and identification of germ cells from fetal bovine ovaries. Mol. Reprod. Dev., 37: 413-424.
Lemme, E., Eckert, J., Carnwath, J.W. and Niemann, H. (1994): Expression of 6WTK-LacZ gene construct in in vitro produced bovine embryos following microinjection into pronuclei or cytoplasm. Theriogenol., 41: 236 (Abst.).
Li, X., Tamano, K., Oian, X.O., Funauchi, K., Furudate, M. and Minato, Y. (1999): Oocyte activation and parthenogenetic development of bovine oocytes following intracytoplasmic sperm injection. Zygote 7: 233-237.
Loi, P., Ptak, G., Dattena, M., Ledda, S., Naitana, S. and Cappai, P. (1998): Embryo transfer and related technologies in sheep reproduction. Reprod. Nutr. Dev., 38: 615-628.
Long, C., Levanduski, M., Duplantis, S. and westhusin, M. (1991): Nuclear transfer in the bovine embryo: the effect of DC voltage and pulse duration on electrofusion and cell lysis. Theriogenol., 35: 232.
Machaty,Z.,Paldi,A.Csaki,T.,Varga,Z.,Kiss,I,Barandi,.and Vajta,G.(1993): Biopsy and sex determination by PCR of IVF bovine embryos. Journal of Reproduction and fertility ,98,467-470.
Mann, M., Latham, K.E. and Varmuza, S. (1999): Identification of genes showing altered expression in preimplantation and early post implantation parthenogenetic embryos. Dev. Biol., 17: 223-232.
Marquant-Leguienne, B. and Humblot, P. (1998): Practical measures to improve in vitro blastocyst production in the bovine. Theriogenol., 49: 3-11.
Marquant-Leguienne, B., Humbolt, P., Guillon, N., Girard, O. Thibier, M. (1992): In vitro fertilization as a tool to evaluate fertility in the bovine. Proc. 12th International Congress on Animal Reproduction (The Hague) 2: 662-664.
Matale, D. R., De Sousa, P.A., Westhusin, M.E. and Watson, A.J. (2001): Sensitivity of bovine blastocyst gene expression patterns to culture environments assessed by differential display RT-PCR. Reprod., 122 (5): 687-693.
McLaren, A. (1984): Chimeras and sexual differentiation. In: Chimeras in Developmental Biology. Academic Press, London, pp.381-399.
Meade, H.M., Echelard, Y., Ziomek, C.A., Young, M.W., Harvey, M., Cole, E.S., Groeet, S., Smith, T.E. and Curling, J.M. (1999): Expression of recombinant protein in the milk of transgenic animals. In: Fernandiez. J.M.., Hoeffler, J.P. (Eds). Gene Expression System. Academic Press, San Diego, USA, pp. 399-427.
Miller, G.F., Gliedt, D.L., Lester, T.D., Pierson, J.N., Rakes, J.M. and Rorie, R.W. (1992): Addition of bovine oviductal epithelial cells (BOEC) and/or penicillamine, hypoaurine and epinephrine (PHE) to bovine in vitro fertilization (IVF medium increases the subsequent embryo cleavage rate. Theriogenol., 37: 259.
Müller, M. and Brem, G. (1994): Transgenic strategies to increase disease resistance livestock. Reprod. Fertil., Dev., 6: 605-613.
Murray, J.D. (1999): Genetic modification of animals in the next centry. Theriogenol., 51: 149-159.
Minhas, B.S.m Capehart, J.S., Bowen, M.J., Womack, J.E., McCrady, J.D., Harms, P.G., Wagner, T.E. and Kraemer, D.C. (1984): Visualization of pronuclei in living bovine zygotes. Biol Reprod., 30: 687-691.
Mohamed Nour, M.S. and Takahashi, Y. (2000): In vitro developmental potential of bovine nuclear transfer embryos derived from primary cultured cumulus cells. J. Vet. Med. Sci., 62 (3): 339-3442.
Monk, M., Handyside, A., Muggleton,-Harris, A. and Whittingham, D. (1990): Preimplantation sexing and diagnosis of hypoxanthine phosphoribosyl transferase deficiency in mice by biochemical microassay. Am. J., Med. Genet., 35 (2): 201-205.
Mordel, N., Ohad, S., Zentner, B., Schnker, J.G., Gordon, J. and Laufer, N. (1992): Enhancing I vitro fertilization of mouse oocytes by partial zona pellucida digestion. J. Assisted Reproduction and Genetics 9: 128-132.
Nibart, M. (1992): Practical application of two advanced biotechnologies to bovine embryo transfer: splitting and sexing. In: Lauria, A. and Gadolfi, F. (Eds) Embryonic Development and Manipulation in Animal Production. Portland Press, London, pp. 215-224.
Nibart, M., Thurad, J.M., Esposito, L., Herpe, P., Hascoet, J. Marrec, C., Lebrun, D. and Rohou, A. (1993): Bovine embryo sexing and modelization in an attempt to produce female calves by embryo transfer. Proc. 9th Congress of the European Embryo transfer Association (Lyon), 244.
Niemann, H. and Kues, W.A. (2000): Transgenig livestock premises and promises. Anim. Reprod. Sci., 60-61: 277-293.
Northey, D.L., Nuttleman, P.R. and Rosenkrans, G.F. (1991): Removal of bovine oocyte cytoplasm prior to fertilization reduces cell number in embryos. Biol. Reprod., 44 (Suppl. 1), 156 (Abst. 413).
Nortarianni, E. and Laurie, S. (1992): Embryonic stem cells from domestic animals: establishment and potential applications. In: Lauria, A. and Gadolfi, F. (eds) Embryonic Development and Manipulation in Animal Production. Portland Press, London, pp. 175-182.
Nottle, M.B., Nagashima, H., Verma, P.J., Du, Z.T., Grupen, C.G., McLlatrickk, S.M., Ashman, R.J., Hading, M.P., Giannakis, C., Wigley, P.J., Lyons, L.G., Harrison, D.T., Luxford, B.G., Campbell, R.J., Crawford, R.J. and Robins, a.J. (1999): Production and analysis of transgenic pigs containing a metallothionein porcine growth hormone gene construct. In: Murray, J.D., Andreson, G.B., Oberbaauer, A.M., McGloughlin, M.M. (Eds). Transgenic Animals in Agriculture. CABI Publ., New York USA, pp. 145-156.
Nowshari, M.A. and Holtz, W. (1993): Transfer of split goat embryos without zona pellucidae either fresh or after freezing. J. Anim. Sci., 71: 3403.
Ohh, B.K., Hwang, K.C., Lee., W.Y., Lee, B.C., Hwang, W.S. and Han, J.Y. (1996): Simple and rapid sex determination of preimplantatied bovine embryos with male specific repetitive sequences. Korean J. Anim. Sci., 38: 43-51.
Ozil, J.P. (1992): Embryo cloning. Human Reproduction 7 (Suppl.2), 73.
Peterson,A.J., Schofield, K.M. ,McLaughlin, R.R.(1990): Adsorption of DNA to the sperm of rams ,bulls and fallow bucks. Proc. the New Zealand Society of Animal Production 50:407-410.
Peura, T.T., Lane, M.W., Vjta, G. and Trounson, A.Q. (1999): Cloning of bovine embryos from vitrified donor blastomeres. J. Reprod. Fertil., 116 (1): 95-101.
Picard, L., Chartain, I., King, W.A. and Betteridge, K.J. (1990): Product of chimaeric bovine embryos and calves by aggregation of inner cell masses with morulae. Mol. Reprod. Dev., 27: 295-304.
Plante, Y., Scmutz, S.M. and Lang, K. D.M. (1992): Restriction fragment length polymorphism in the mitochondrial DNA of cloned cattle. Theriogenol., 38: 897-904.
Powell, D.J., Galli, C. and Moor, R. M. (1992): The fate of DNA injected into mammalian oocytes and zygotes at different stages of the cell cycle. J. Reprod. Fert., 95: 211-220.
Prelle, K., Kovic, M., Boxhammer, K., Motilk, J., Edward, D., Arnold, G.J. and Wolf, E. (2001): Insulin-like factor I (IGF-I) and long R (3) IGF-I differently affect development and messenger ribonucleic acid abundance for IGF-binding proteins and type I IGF receptors in in vitro produced bovine embryos. Endocrinol., 142 (5): 1702.
Rao, V.H., Chesne, P., Renard, J.P. and Heyman, Y. (1993): Influence of age of in vitro matured oocytes and electrical stimulation’s on the development of cattle nuclear transfer embryo. Theriogenol., 39: 292.
Rexroad, C.E., Jr, Powell, A.M., Rohan, R. and Wall, R.J. (1990): Evaluation of co-culture as a method for selecting viable microinjected sheep embryos for transfer. Animal Biotechnology 1: 1-10.
Reyes, R., Flores-Alonso, J.C., Rodriguez-Herrnandez, H.M., Merchant-Larios, H.M. and Delgado, N.M. (2001): Arch. Androl. 47: 23-29.
Rho, G.J., Kang, t.Y., Kochhar, H.P., Hahnel, A.C. and Betteridge, K.J. (2001): Effect of blastomere sex and fluroscent labelling on the development of bovine chimeric embryos reconstructed at the four-cell stage. Mol. Reprod. Dev., 60 (2): 202-207.
Rieth, A., Pothier, F., Sirard, M.A. (2000): Electroporation of bovine spermatozoa to carry DNA containing highly repetitive sequemces into oocytes and detection of homologous recombination events. Mol. Reprod. Dev., 57 (4): 338-345.
Rizos, D., Lonergan, p., Bolnad, M.P., Arroyo-Garcia, R., Pintado, B., de la Fuente, J. and Gutierrez-Adan, A. (2002): Analysis of differential messenger RNA expression between bovine blastocysts produced in different culture systems: Implications for blastocyst quality. Biol. Reprod., 66 (3): 589-585.
Roelen, B.a., Van Eijk, M.J., Van Rooijen, M.A.,, Bevers, M.M., Larson, J.H., Lewis, H.A., Mummery, C.L. (1998): Molecular cloning, genetic mapping, and developmental expression of a bovine transforming growth factor beta (TGF-beta) type I receptor. Mol. Reprod. Dev., 49 (1): 1-9.
Role, R.W., Pool, S.H., Prichard, J.F, Betteridge, K. and Godke, R.A. (1989): Production of chimeric blastocysts comprising sheep ICM and goat trohoblast for intergenetic transfer. J.Anim. Sci., 67: 401-402.
Roschlau,D. Roschlau ,K, Roselius ,R.,Dexne, U. Michaelis ,U.Strehl,R. and Unicki ,P.(1992): Experiences in sexing of bovine embryoscommercial programmes. Proceedings of the Eighth Conference of the European Embryo transfere Association (lyon),204.
Schnieke, A.E., Kind, A.J., Ritchie, W.A., Mycock, K., Scott, A.R., Ritchie, M., Wilmut, I., Colman, A. and Campbell, K.H.S. (1997): Human factor IX transgenic sheep produced by transfer of nuclei from transfected fetal fibroblasts.Animal Reproduction Science 278: 2038—2139 .
Schwiderski, H., Kauffold, P., Jura, J. and Duschinski, U. (1990): Effect of microinjection of motile bovine spermatozoa into the perivitelline space of oocytes. Reprod. Domestic Animals 25(6): 277-282.
Selgrath, J.P., Memon, M.A., Smith, T.E. and Ebert K.M. (1990): Collection and transfer of microinjectable embryos from dairy goats. Theriogenol., 34: 1195-1205.
Shiga, K., Fujita, T., Hirose, K., Sosae, Y. and Nagai, T. (1999): Production of calves by transfer of nuclei from cultured somatic cells obtained from Japanese black bulls. Theriogenol., 52 (3): 527-535.
Shin, S.J., Lee, B.C., Bark, J.I., Lim, J.m. and Hwand, W.S. (2001): A separate procedure of fusion and activation in an early development of bovine reconstructed oocytes. Theriogenol., 55 (8): 1697-1704.
Sinclair, K.D., McEvoy, T.G., Maxfield, E.K., Maltin, C.A., Young, L.E., Wilmut, I., Broadbent, P.J. and Robinson, J.J. (1999): Aberrant fetal growth and development after in vitro culture of sheep zygotes. J. Reprod. Fertil., 116: 177-186.
Smith, S.D., Saloy, E., Kanaka, J., Holm, P. and Callesen, H. (1996): Influence of recipient cytoplasm cell stage on transcription in bovine nucleus transfer embryo. Mol. Reprod. Dev., 45 (4): 444-450.
Squires, E.J. (1999): Status of sperm-mediated delivery methods for gene transfer. In: Murray, J.D., Andreson, G.B., Oberbaauer, A.M., McGloughlin, M.M. (Eds). Transgenic Animals in Agriculture. CABI Publ., New York USA, pp. 87-96.
Stice, S.L. and Keefer, C. (1992): Improved developmental rates for bovine nucleus transfer embryos using cold shock activated oocytes. Biol. Reproduction 46 (supple 1), 166 (abst.462).
Tani, T, Kato,Y, Tsunoda,Y.(2000): Developmental potential of cumulus cell derived culture frozen in a quiescent state after nucleus transfer. Theriogenology ,53(8) 1623-1629.
Techakumphu, M., Adenot, P., Chesne, P. and Rao, V. H. (1993): Viability of bovine blastomeres after metaphase arrest with Nocodazole. Theriogenology ,39 : 328.
Telford, N.A., Watson A.J. and Schultz, G. A. (1990): Transition from maternal to embryonic control in early mammalian development: a comparison of several species. Mol. Reprod. Dev., 26: 90-100.
Tervit, H. R. (1990): Potential use of embryo technology in gene transfer in dairy cattle. Proc. the 23rd International Dairy Congress ( Monterarl)1: 566-578.
Thomas, W.K. and Seidel, G.E. (1991): In vitro methods for producing transgenic bovine embryos. J. of Animal Science 69 (supple.1) 401-402.
Thomas, W.K., Schnieke, A. and Seidel, G.E. (1993): methods for producing transgenic bovine embryos from in vitro matured and fertilized oocytes. Theriogenol., 40: 679-688.
Tiffin, G. J., Rieger, D., Betteridge, K.J., Yadave, B.R. and King, W .A. (1991): Glucose and glutamine metabolism in pre–attachment cattle embryos in relation to sex and stage of development. J. Reprod . Fert.., 93: 125-132.
Tucker, M.J., Morton, P.C., Wright, G., Ingargcola, P.E., Sweitzer, C.L., Elsner, C.W., Mitchall-Leaf, D.E. and Massey, J. (1996): Enhancement of out come from intracytoplasmic sperm injection does co-culture or assisted hatching improve implantation rate. Human Reprod. 11: 2434-2437.
Utsumi, K. and Iritani , A. (1990) : Production of cattle identical twins by splitting blastocysts using a metal microblade. Theriogenol., 33: 341.
Utsumi,K. and Iritani , A. ( 1993): Embryo sexing by male specific antibody and by PCR using male specific (SRY) primer . Mol. Reprod Dev., 36:238-241.
Utsumi, K, Hayashi, M., Takaura , R., Utaka , K. and Tritani, A (1993): Embryo sex selection by a rat of male – specific antibody and the cytogenetic and developmental confirmation in cattle embryo. Mol. Reprod. Dev., 34: 25-32.
Van Stekelenburg-Hamers, A.E.P., Rebel, H.G., Van Izen, W.G., De Loos, F.A.M., Dorst, M. Mummery, C.L., Weima, S.M. and Trounson, A.O. (1994): Stage-specific appearance of the mouse antigen TEC-3 in normal and nuclear transfer bovine embryos: re-expression after nuclear transfer. Mol. Reprod. Dev., 37: 27-33.
Van Vliet, R.A., Verrinder Gibbins, A.M. and Walton, J.S. (1989): Livestock embryo sexing: a review of current methods, with emphasis on Y-specific DNA probes. Theriogenol., 39: 421-438.
Vivanco, H.W., Rangel, R., Lynch, P. and Rhodes, A. (1991): Large- scale commercial application of bisection of sheep embryos. Theriogenol., 35: 292.
Wakayama, T., Perry, A.C.F, Zuccotti, M., Johanson, K.R. and Yanagimachi, R. (1998): Full-term development of mice from enucleated oocytes injected with cumulus cell nuclei. Nature 394: 369-374.
Walker, S. K., Heart, T.M. and Seamark, R.F. (1992): In vitro culture of sheep embryos without co-culture: successes and perspective. Theriogenol., 37: 111-126.
Wall, R.J. (1996): Transgenic livestock: progress and prospects for the future. Theriogenol., 45: 57-68.
Ware, C. B. (1986): Studies in embryo micromanipulation and freezing, .PhD thesis, National University of Ireland, Dublin.
Ware, C. B., Barnes, F.L., Maiki-Laurila, M. and First, N.L. (1989): Age dependence of bovine oocyte activation. Gamete Research 22, 265-275.
Wei, H. and Fukui, Y. (1999): Effects of bull, sperm type and pretreatment on male pronuclear formation after cytoplasic sperm injection in cattle. Reprod. Fertil. Dev., 11: 59-65.
Westhusin, M.E., Arcellana-Panlilo, M., Harvey, M., Jones, K and Schultz, G.A. (1995): Gene expression in cloned bovine embryos application of molecular biology to reproduction. IETS Satellite Symposium, Catgary.
Westhusin, M.E., Long, C.R. and Shin, T. (2001): Cloning to reproduce desired genotype. Theriogenol., 55:; 35-49.
Westhusin, M.E., Pryor, J .H. and Bondioli, K. R. (1991): Nuclear transfer in the bovine embryo: a comparison of 5-days, 6- day, frozen-thawed and nuclear transfer donor embryos. Mol. Reprod. Dev., 28: 119-123.
Willadsen, S.M. and Fehilly, C.B. (1983): The developmental potential and regulatory capacity of blastomeres from two, four and eight –cell sheep embryos. In: Beiier, H.M. and Linder, H.R. (Eds), Fertility of the Human Egg In vitro. Springer-Velarg, Berlin, pp. 353-4357.
Willadsen, S.M., Janzen, R.E., McAlister, R.J., Shea, B.F., Hamilton, G. and McDermand, D. (1991): The viability of late morulae and blastocyst produced by nuclear transplantation in cattle. Theriogenol., 35: 161-170.
Wilmut, I. and Campbell, K.H.S. (1998): Quiecence in nuclear transfer. Science 218: 1611.
Wilmut, I., Haley, C.S. and Wooliams, J.A. (1992): Impact of biotechnology on animal breeding. Anim. Reprod. Sci., 28: 149-162.
Wilmut, I., Schieke, A.E., Whir, J. (1997): Viable offspring derived from fetal and adult mammalian cells. Nature London 385: 810-813.
Wilson, J.M., Williams , J.D., Dondioli,K.R. (1995): Camparison of birth weight and growth characteristics of bovine nuclear transfer (cloning),embryo transfer and natural mating .Animal Reproduction Science,Champaign, II,38.
Wrenzycki, C., Herramnn, D., Carnwath, J.W., Niemann, H. (1998a): Expression of RNA from developmentally important genes in preimplantation bovine embryos produced in TCM supplemented with BSA. J. Reprod. Fertil., 112: 387-398.
Wrenzycki, C., Herramnn, D., Lemme, E., Korsawe, K., Carnwath, J.W., Niemann, H. (1998b): Determination of the relative abundance of various developmentally important gene transcripts in bovine embryos generated in vitro or in vivo using a semi-quantitative RT-PCR assay. In IETS Satellite Workshop Proc. “Embryo development in vitro current challenges and future concepts”: 14-15.
Wrenzycki, C., Herramnn, D., Carnwath, J.W., Niemann, H. (1999): Alterations in the relative abundance of gene transcripts in preimplantation bovine embryos cultured in medium supplemented with either serum or PVA. Mol. Reprod. Dev., 53: 8-18.
Xu, K.P., Loskutoff, N.M. and Betteridge, K.J. (1991a): Pregnancies resulting from bovine embryo derived from in vitro culture of zona-free zygotes produced by in vitro maturation and fertilization of follicular oocytes. Theriogenol., 35: 296.
Xu, K.P., Yadav, B.R., King, W.AA. and Betteridge, K.J. (1991b): Sex related difference in the development of bovine embryos produced in vitro . Biol. Reprod., (Suppl.1) 97 (Abst. 177).
Xu, K.P., Yadav, B.R., King, W.AA. and Betteridge, K.J. (!992): Sex-related differences in developmental rates of bovine embryos produced and cultured in vitro. Mol. Reprod. Dev., 11: 249-252.
Yang, X. and Andreson, G.B. (1992): Micromanipulation of mammalian embryos: principles, progress and future possibilities. Theriogenol., 33: 354.
Zakhartchenko, v., Alberio, R., Kovic, M.m Prelle, Schernthaner, W., Stojkovic, M., Wenigerkind, H., Wanke, R., Duchler, M., Steinborn, R., Muller, M., Brem, G. and Wolf, E. (1999): Adult cloning in cattle: potential of nuclei from a permanent cell line and from primary culture. Mol. Reprod. Dev., 54 (3): 264-272.
Zakhartchenko, V., Muller, S., Alberio, R., Schernthaner, Stojkovic, M., Wenigerkind, H., Wanke, R., W., Muller, M., Wolf, E. and Brem, G. (2001): Nuclear transfer in cattle with non-transfected and transfected fetal or cloned transgenic fetal and postnatal fibroblasts. Mol. Reprod. Dev., 60 (3): 362-369.
Zakhartchenko, V., Stojkovic, M., Brem, G. and Wolf, E. (1997): Karyoplast-cytoplast volume ratio in bovine nuclear transfer embryo: effect on development potential. Mol. Reprod. Dev., 48 (3): 332-338.
Zawada, W.M., Cibelli, J.B., Chei, P.K., Clarkson, E.D., Golueke, P.J., Witta, S.E., Beil, K.P., Kane, j., Abel ponce de Leon, F., Jerry, D.J., Robl, J.M., Freed, C.R. and Stice, S.L. (1998): Somatic cell cloned transgenic bovine neurons for transplantation in parkinson rats. Nat. Med. 4: 569-574.
Zhu, S.E., Zeng, S.M., Yu, W.L., Li, S.J., Zhang, Z.C., Chen, Y.F. (2001): Vitrification of in vivo and in vitro produced ovine blastocysts. Anim. Biotechnol., 12 (2): 193-203.
Ziomek, C.A. (1998): Commercialization of proteins produced in the mammary gland. Theriogenol. 49: 139-144.