Bioltechnologic Tools Goat as a Model to Evovle Embryo and Nuclear Trasfer Techniques

<h1>Bioltechnologic Tools Goat as a Model to Evovle Embryo and Nuclear Trasfer Techniques</h1>Author: <a title=" G.M. Wani" href="http://www.articlesbase.com/authors/-g.m.-wani/44585.htm"> G.M. Wani</a> . Introduction   Past century saw many techniques like micro-surgery of embryos, embryo co-culture, in vitro fertilization and production of monozygotic twins, chimeras besides cryo preservation and vetrification techniques for preserving goat embryos. The recent micro injection techniques made possible cloning and DNA transfer and production of animal clones like modern day “Dolley”. Various reproductive and genetic engineering techniques like DNA finger printing gene mapping and recumbent DNA technologies are in active use. Formation, growth and maturation of female gametes begins during embryonic stage and continues after birth evincing climax at ovulation (Wani, 2002) . The single cell released from ovarian follicle at ovulation in an “Egg”. In most of the mammals it is a “secondary oocyte”, when first polar body is already released at primary oocyte stage. In most of the mammals, the second meiotic division is arrested at metaphase and proceeds further only after sperm penetration. The number of primodial follicles in a mammalian dam far exceeds its production of offspring even after use of modern day techniques mentioned above. Biotechnology has been used for various purposes in biomedical sciences which stands reviewed Wani (1998), Wani (2003). 2. Augmentation of primodial germ cell The number of primordial germ cells in an ewe and goat is around 86,000 and cow nearly 1 lakh. Out of them hardly 100-200 grow to mature in life and only a few are fertilized and develop into a conceptus. The gap between potential pool of oocyte and those available for fertilization is large. Trapping this pool conventionally means using 0.2% of available genetic material for manipulation by known applicable techniques of superovulation, synchronization and assisted reproductive techniques reviewed and reported (Wani, 1998, 2003). The varied ovarian reaction, inadequate knowledge on atresia and mechanism of favourable status of maturing follicle and final selection of the particular oocyte for release and its fertilization make genetic selection much variable and distant relatively in terms of number of follicle which do degenerate at each atresia. Thus, the intricate mechanism of hormonal play in induction of ovulation or maturation of oocytes needs more awareness and thus signifies more research (Wani, et al 1997). In this context the neuroendocrine signal which triggers massive and sustained GnRH surge may be the switch inducing ovulation (Karsch et al., 1992). Few questions can be asked & needs answer in our todays review: Does recruitment of follicle have any relationship with preovulatory GnRH secreting neurons? Similarly, Is the post-dated GnRH surge, involving morphological changes in neuron (Karsch and Evans, 1996) related to morphological adjustment, recruitment for follicle maturation and its subsequent release at ovulation. The 40 fold increase in GnRH over and above baseline values during surge period thought to be dynamic switch control of ovulation in sheep needs further elucidation, which demands more studies on morphological aspects of follicular maturation, selection and ovulation, especially in the light of deterministic and permissive models of GnRH and LH surges vis-à-vis folliculogenesis and ovulation in goats (Karsch, 1997). Therefore, our innovative vision should be to remodel our techniques incorporating GnRH signals and oestradiol modulation for exploiting primodial germ cell pool to mature more oocytes in vivo or in vitro. This is what stem cell technology offers. If by 2150 A.D. we are able to use even 10% of available genetic primodial germ cell pool of an improved mammalian species, it would be a landmark in the biological discoveries. This would produce more from less and less numbers. This is what we all strive for. Keep the number of goats as minimal as possible. Increase their productivity many fold, so that we have enough milk, meat and fur available for growing population at minimum costs. This is what we call sustainable goat production system. 3 Embryo Production techniques Embryo production using techniques of synchronization, super-ovulation, embryo collection, embryo evaluation, grading culture, manipulation, preservation and transfer along with modern useful techniques of in vitro maturation of oocytes and in vitro fertilization, along with recent diagnostic and explorative use of ultra sounds and laparoscopy have been discussed. (Wani, 1984, 1992, 1995, 1998, 2002, 2003).  4 Embryo evaluation and grading techniques Various scales for grading mammalian embryos are reported (Wani, 1992) and are also detailed (Wani, 1996) However, what we need is a numerical grading mechanism for embryos as in sperms. The examination procedures of embryos shall be simplified for use in the field conditions in future.More intensified research on grading of embryos and oocytes are to be searched out. This is particularly important now, because of recent aging effect reported on “Dolley”. 5Determination of embryo viability Fluorescent technique for determining living embryo cells got a boost by revealation that staining with Fluorescin – diacetate (FDA) dye does not effect further development. Biochemical methods based on glucose uptake; oxygen uptake or enzymic releases have poor practical applicability. More refined techniques predicting embryo viability have to be searched, especially for use under field conditions. 6 Embryo transfer techniques in goats Surgical techniques (Wani, 1984) have been replaced with laparoscopic and trans-cervical non surgical methods (Hays, 1998). More simplification and search is needed to make whole set of techniques operative under farm conditions to produce future breeding sires from superior sire mothers (donors). More research on trans-cervical embryo transfers in recipients needs encouragement. A few innovative beginnings for ultrasonically guided laparoscopic collection of oocyte in being attempted and planned. 8Embryo manipulation techniques These techniques reviewed and improved by our colleagues in the developed countries need to be operative under field conditions in developing country where most of the goats are reared. (Wani, 1996). Manpower or expertise is available; funds are needed to intensify perfection of these techniques. .9 In vitro fertilization and prior sexing of embryos Some of the techniques already operative (Jufen et al., 1991; Wani, 1996). We have used in vitro fertilisation techniques in sheep with a unique distinction. We used semen from slaughtered rams for fertilization of oocytes recovered from slaughtered ewes (wani et al 2000, 1999, Wani 1996). This promises its potential use for regeneration of rare wild animals. The slaughter house pool shall provide valuable elite germ plasm even after their death. This is essential to conserve wild goat ancestors and best producers like Cherion, Ibex etc. .10 Cryo preservation and vetrification techniques Various culturing techniques like embryo co-culture, cryo preservation and vetrification needs more emphasis. This has been added with Cryopreservation of whole ovary and its use for oocyte maturation, fertilization and transfer. (Wani, 2003) 11 Ultrasonography and laparoscopy Prior indications of early pregnancy through ultrasonography, laparoscopy(Wani, 1981, 1982) have further improved and were reviewed (Wani, 1995). Early indication of pregnancy limits reproductive losses and promises efficient production systems (Wani,1995, Wani et al, 1998; Wani, 2005). New vistas in this arena have been opened up. In India our humble start in late seventies (Wani, 1981) has now advanced to detection of implantation of embryo too). We could moniter embryonic development and implantation through ultrasounds. The prospects of in vitro implantation and development of conceptus needs basic investigation. We tried to set up basic land marks in this regard. (Mufti et al 2000). The whole implantation concept has been rediscovered. A new invention defining the role of foetal and maternal compartments has been published. (Wani, et al, 2006, 2006a, 2006b, 2006c and 2007). A few new founded real photographs could be seen I the photogalary of this book. 12 Production of desired chimeras and clones Production of chimeras and clones having high yielding genes through aggregation of cells from two or more pre-implanation embryos may enhance Goat production in 21st century. The addition or modification of DNA related to production traits may help in production of high yielding goats replacing Dolly with “Trolleys of DNA chips” on sale like computer chips in future. New pre-implantation signals hinted (Heap et al., 1990) and reviewed (Wani, 1995) may open new progressive research phase involving more and more reproductive scientists to unveil the hidden truth (Al-Quran, Suratul Mominoon, 12-134) Inclusion of specific aminoacids in the culture media has helped to grow embryos beyond 8 cell stages. Now more advances in culturing ovine embryos by co-culture or group culture have been known. More search on rate of involution, development of concepts in vivo and in vitro implantation and transfer are some of the immediate research themes to be searched in near future (Wani, 1998, Wani 2002, Wani 2001). 13 Foetal maternal relationship Various biochemical and morphological investigation on foetal growth and development has been jointly explored by SKIMS and SKUAST (Mufti, 1996). This is an innovative search to unveil various mechanisms involving in vitro implanation and transformation of blastocyst into foetus in future. More funding is needed for investigative and innovative projects on foeto-maternal associations such as reported by Mufti et al (2000), wani et al 2007, 2006,abc. 14 DNA finger printing Genotype environment interactions are closely related to mammalian reproduction. Knowledge of genetic constitution and embryonic differentiation is a pre-requisite for breed conservation and improved selective breeding. Since, DNA of an individual is not directly influenced by environmental conditions except loci coding for T-cell receptors, antibodies and others; short term environmental influences like season, feeding and management may not influence. DNA sequences based on this central theme, an investigation was undertaken in collaboration with University of Hohenheim Germany. The study was financed by German Research Foundation. The project has ended, but its savings has enabled us to institute an fellowship to be called Gall, Geldermann, Wani fellowship for goat reproduction. Under this project, blood samples from Pashmina and Bakerwal Goats were analysed. DNA was extracted from leucocytes with phenol-chloroform. Different regions of the expressed DBR gene and the DBR pseudogene were amplified by PCR. Fragment lengths of the entire exon 2 and the microsatellite were analysed, using automatic DNA sequencer, Automatic Laser Fluorescence (ALF) and by Single Strained Confirmation Polymorphism References references Al-Quran, Surat Mominoon, 12:134. Young, L. E, 2002, Somatic cell nuclear transfer,419, 6907, 583-586 Anderson.G.B; Murray J.D and Medrano.J.F, 1996, Expression of a bovine k-CN cDNA in the mammary gland of transgenic mice utilizing a genomic milk Casey.D.C and White.K.L, 1990, Incorporation and expression of a neo gene after Cloning and sequence analysis of the cDNA for a protein from coccidioides Colman.A and Garner.I, 1995, The transgenic mammary gland as a bioreactor: Dugger.K.O; Villareal.K.M; Ngyuen.A; Zimmermann.C.R; Law.J.H and Galgiani.J.N, expectations and realizations, 6,107. Gall, C; Gelderman, H; Wani, G.M. 1997. Analysis of Goat Genetic Resources of goat breeds in Kashmir. Final report of research co-operation between SKUAST and University of Hohehuim, Germany . Gutierrez.A; Meade.H.M; Ditullio.P; Pollock.D; Harvey.M; Jimenez. Fores.R; Hays, B.L. 1998. Non-surgical embryo collection in the goats. Proceedings of 11th International Congress on Animal Reproduction and AI, Dublin 2: 165. Heap, R.B; Wang. M.W; Whyte, A and Taussing, M.J. 1991. Significant of peri-implantation signals. L. 17. Proceeding Int. Conf. On Frontiers in reproductive Physiology, Nov. 8-10, 1991 at AIIMS, New Delhi. Jufen, Q; Zhiming, H; Yong, Z and JianChen , W. 1991. Invitro capacitation of ejaculated spermatozoa and invitro fertilization in dairy goats. Theriogenoloty. 35(1):219. Karsch, F.J and Evans, N.P. 1996. Feedback actions of oestradiol on GnRH Secretion during follicular phase of the oestrus cycle. Acta. Neurobiol. Exp. 56:715-725. Karsch, F.J; Bowen, J.M; Caraty, A; Evans, N.P and Moenter, S.M. 197. Gonadotrophin-releasing harmone requirements for ovulation. Biology of reproduction, 56h:303-309. Karsch, F.J; Moenter S.M. and Caraty, A; Moenter, S.M. 1997.The neuroendoerine signal for ovulation.Animal Reproduction Science, 28:329-341. limitis with immunogenic potential, 218 (2),485-89 Maughan .P.J; Philip.R; Cho.M.J; Widholm.J.M and Vodkin.L.O, 1999, Biolistic Mufti, A.M. 1996. Studies on morphological characters and some metaolic consititutes at different stages of development of ovine foestus. MVSC Thesis, SKUAST, Srinagar. Mufti, A.M; Wani, G.M; Wani, N.A, and Khan, M.Z. 2000. Prenatal development of ovine foetus. Small Ruminant Research, 38: 87-89. Mufti, A.M; Wani, N.A; Mir, M.M; Khan, M.Z and Wani, G.M. 1997. Superovulatory response in corriedale ewes during different months of breeding season. Small Ruminant Research 25:181-84. Protein gene as an expression cassette, 5 (4), 271-79 transfer of Caprine hematopoietic cells using a modified retro virus, 68,2,449-53. Transformation, expression and inheritance of bovine beta-casein in soyabean Pawshe, C.H; Totey, S.M and Jain, S.K. 1994. Effect of the methods of recovery of goat Oocytes for invitro maturation and fertilization.Theriogenology, 42:117-125 Wani , G.M. 1996. Embryo biotechnology in sheep and goats. Valley Book House University Road, Srinagar. Wani, G.M. 1981. Ultrasonic pregnancy diagnosis in sheep and goats. World Review of Animal Production, 17:43-48 Wani, G.M. 1982. Laparoscopy in Farm animals . World Review of Anim. Production, 18:7-13. Wani, G.M. 1984. Investigations Caprine Embryo production and culture .Dr.med.Vet.Thesis vet Univ, Hannover, Germany. Wani, G.M. 1992. Advanced Techniques in Animal Reproduction. SKUAST Press, Shalimar No.75/250/12/92. Wani, G.M. 1995. Early diagnosis of pregnancy in surrogate mothers (Sheep and Goats).National Symposium on Embryo Transfer and Genetic Engineering Technologies in Small Ruminants; May 7-8, 1995, CIRG, Makhdoom, Farah, India. Wani, G.M. 1998. Role of assisted reproductive technologies in augumentation of sheep production. Lead paper presented on National eminar on “Strategies for maximizing production and efficiency in sheep”, held at CSWRI, Avikanagar, Rajasthan, April, 1998 PP.18-22. Wani, G.M. 2001. Enhancement of chevon Production through reproductive management proceedings IV National Seminar of Indian Society for Sheep and Goat Production and utilization (ISSGPU) Nov 9-11, 2001, Bharathiar University, Coimbatore, Tamilnadu, India. Wani, G.M. 2002. Research in Animal Reproduction-Vision 2050. Proceedings XVII Annual Convention of ISSAR and National Symposium, Nov.14-16, 2002, IVRI, Izatnagar U.P. Wani, G.M. 2003 a. Biotechnological advances in Goat Production. National Workshop on “Recent advances in Goat rearing”, MPKVK, Agric. Univ. Rahuri, 29-30 Dec, 2003. Wani, G.M. 2003. Goat Husbandry- multipurpose management. Encyclopedia of Dairy Sciences, edited by Roginishi Hubert, Euquary, J.W., Fax, P. Academic Press London Vol.2 PP 1259-1270. Wani, N.A, and Wani, G.M. 2003. Use of epididymal spermatozoa from slaughtered sheep for invitro fertilization of invitro matured Oocytes. Indian.J.Anim.Sci, 73:143-146. Wani, N.A; 1996. Invitro maturation and invitro gertilization of ovine Oocytes MVSc Thesis, SKUAST. Wani, N.A; Wani, G.M; Khan, M.Z and Salahuddin, S.2000. Effect of Oocyte harvesting techniques on invitro maturation and invitro fertilization in sheep. Small Ruminant Research. 36: 63-67. Wani, N.A; Wani, G.M; Khan, M.Z and Siddiqui, M.A. 1999. Effect of different factors on the recovery rate of Oocytes for invitro maturation and invitro-fertilization. Small Ruminant Research 34:71-76. Wani, N.A; Wani, G.M; Mir, M.M and Beg, M.A. 1998. Ultrasonic pregnancy diagnosis in Gaddi Goats. Small Ruminant Research 29:239-240. Wilmut,I; Beaujean.N; De-Sousa,P.A; Dinnyes.A; King.T.J; Paterson,L.A; Wells,D.NAbout the Author: Ph.D (Animal Reproduction / Gynaecology), Dr. Med. Vet (Animal Reproduction/ Production)Article Source: <a href="http://www.articlesbase.com/">ArticlesBase.com</a> - <a href="http://www.articlesbase.com/international-studies-articles/bioltechnologic-tools-goat-as-a-model-to-evovle-embryo-and-nuclear-trasfer-techniques-358358.html" title="Bioltechnologic Tools Goat as a Model to Evovle Embryo and Nuclear Trasfer Techniques">Bioltechnologic Tools Goat as a Model to Evovle Embryo and Nuclear Trasfer Techniques</a>

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Introduction

Past century saw many techniques like micro-surgery of embryos, embryo co-culture, in vitro fertilization and production of monozygotic twins, chimeras besides cryo preservation and vetrification techniques for preserving goat embryos. The recent micro injection techniques made possible cloning and DNA transfer and production of animal clones like modern day “Dolley”. Various reproductive and genetic engineering techniques like DNA finger printing gene mapping and recumbent DNA technologies are in active use.

Formation, growth and maturation of female gametes begins during embryonic stage and continues after birth evincing climax at ovulation (Wani, 2002) . The single cell released from ovarian follicle at ovulation in an “Egg”. In most of the mammals it is a “secondary oocyte”, when first polar body is already released at primary oocyte stage. In most of the mammals, the second meiotic division is arrested at metaphase and proceeds further only after sperm penetration. The number of primodial follicles in a mammalian dam far exceeds its production of offspring even after use of modern day techniques mentioned above.

Biotechnology has been used for various purposes in biomedical sciences which stands reviewed Wani (1998), Wani (2003).

2. Augmentation of primodial germ cell

The number of primordial germ cells in an ewe and goat is around 86,000 and cow nearly 1 lakh. Out of them hardly 100-200 grow to mature in life and only a few are fertilized and develop into a conceptus. The gap between potential pool of oocyte and those available for fertilization is large. Trapping this pool conventionally means using 0.2% of available genetic material for manipulation by known applicable techniques of superovulation, synchronization and assisted reproductive techniques reviewed and reported (Wani, 1998, 2003).

The varied ovarian reaction, inadequate knowledge on atresia and mechanism of favourable status of maturing follicle and final selection of the particular oocyte for release and its fertilization make genetic selection much variable and distant relatively in terms of number of follicle which do degenerate at each atresia. Thus, the intricate mechanism of hormonal play in induction of ovulation or maturation of oocytes needs more awareness and thus signifies more research (Wani, et al 1997).

In this context the neuroendocrine signal which triggers massive and sustained GnRH surge may be the switch inducing ovulation (Karsch et al., 1992). Few questions can be asked & needs answer in our todays review:

Does recruitment of follicle have any relationship with preovulatory GnRH secreting neurons? Similarly, Is the post-dated GnRH surge, involving morphological changes in neuron (Karsch and Evans, 1996) related to morphological adjustment, recruitment for follicle maturation and its subsequent release at ovulation. The 40 fold increase in GnRH over and above baseline values during surge period thought to be dynamic switch control of ovulation in sheep needs further elucidation, which demands more studies on morphological aspects of follicular maturation, selection and ovulation, especially in the light of deterministic and permissive models of GnRH and LH surges vis-à-vis folliculogenesis and ovulation in goats (Karsch, 1997).

Therefore, our innovative vision should be to remodel our techniques incorporating GnRH signals and oestradiol modulation for exploiting primodial germ cell pool to mature more oocytes in vivo or in vitro. This is what stem cell technology offers.

If by 2150 A.D. we are able to use even 10% of available genetic primodial germ cell pool of an improved mammalian species, it would be a landmark in the biological discoveries. This would produce more from less and less numbers. This is what we all strive for. Keep the number of goats as minimal as possible. Increase their productivity many fold, so that we have enough milk, meat and fur available for growing population at minimum costs. This is what we call sustainable goat production system.

3 Embryo Production techniques

Embryo production using techniques of synchronization, super-ovulation, embryo collection, embryo evaluation, grading culture, manipulation, preservation and transfer along with modern useful techniques of in vitro maturation of oocytes and in vitro fertilization, along with recent diagnostic and explorative use of ultra sounds and laparoscopy have been discussed. (Wani, 1984, 1992, 1995, 1998, 2002, 2003).

4 Embryo evaluation and grading techniques

Various scales for grading mammalian embryos are reported (Wani, 1992) and are also detailed (Wani, 1996) However, what we need is a numerical grading mechanism for embryos as in sperms. The examination procedures of embryos shall be simplified for use in the field conditions in future.More intensified research on grading of embryos and oocytes are to be searched out. This is particularly important now, because of recent aging effect reported on “Dolley”.

5Determination of embryo viability

Fluorescent technique for determining living embryo cells got a boost by revealation that staining with Fluorescin – diacetate (FDA) dye does not effect further development. Biochemical methods based on glucose uptake; oxygen uptake or enzymic releases have poor practical applicability. More refined techniques predicting embryo viability have to be searched, especially for use under field conditions.

6 Embryo transfer techniques in goats

Surgical techniques (Wani, 1984) have been replaced with laparoscopic and trans-cervical non surgical methods (Hays, 1998). More simplification and search is needed to make whole set of techniques operative under farm conditions to produce future breeding sires from superior sire mothers (donors). More research on trans-cervical embryo transfers in recipients needs encouragement. A few innovative beginnings for ultrasonically guided laparoscopic collection of oocyte in being attempted and planned.

8Embryo manipulation techniques

These techniques reviewed and improved by our colleagues in the developed countries need to be operative under field conditions in developing country where most of the goats are reared. (Wani, 1996). Manpower or expertise is available; funds are needed to intensify perfection of these techniques.
.9 In vitro fertilization and prior sexing of embryos

Some of the techniques already operative (Jufen et al., 1991; Wani, 1996). We have used in vitro fertilisation techniques in sheep with a unique distinction. We used semen from slaughtered rams for fertilization of oocytes recovered from slaughtered ewes (wani et al 2000, 1999, Wani 1996). This promises its potential use for regeneration of rare wild animals. The slaughter house pool shall provide valuable elite germ plasm even after their death. This is essential to conserve wild goat ancestors and best producers like Cherion, Ibex etc.

.10 Cryo preservation and vetrification techniques

Various culturing techniques like embryo co-culture, cryo preservation and vetrification needs more emphasis. This has been added with Cryopreservation of whole ovary and its use for oocyte maturation, fertilization and transfer. (Wani, 2003)

11 Ultrasonography and laparoscopy

Prior indications of early pregnancy through ultrasonography, laparoscopy(Wani, 1981, 1982) have further improved and were reviewed (Wani, 1995). Early indication of pregnancy limits reproductive losses and promises efficient production systems (Wani,1995, Wani et al, 1998; Wani, 2005). New vistas in this arena have been opened up. In India our humble start in late seventies (Wani, 1981) has now advanced to detection of implantation of embryo too). We could moniter embryonic development and implantation through ultrasounds. The prospects of in vitro implantation and development of conceptus needs basic investigation. We tried to set up basic land marks in this regard. (Mufti et al 2000). The whole implantation concept has been rediscovered. A new invention defining the role of foetal and maternal compartments has been published. (Wani, et al, 2006, 2006a, 2006b, 2006c and 2007). A few new founded real photographs could be seen I the photogalary of this book.

12 Production of desired chimeras and clones

Production of chimeras and clones having high yielding genes through aggregation of cells from two or more pre-implanation embryos may enhance Goat production in 21st century. The addition or modification of DNA related to production traits may help in production of high yielding goats replacing Dolly with “Trolleys of DNA chips” on sale like computer chips in future. New pre-implantation signals hinted (Heap et al., 1990) and reviewed (Wani, 1995) may open new progressive research phase involving more and more reproductive scientists to unveil the hidden truth (Al-Quran, Suratul Mominoon, 12-134)

Inclusion of specific aminoacids in the culture media has helped to grow embryos beyond 8 cell stages. Now more advances in culturing ovine embryos by co-culture or group culture have been known. More search on rate of involution, development of concepts in vivo and in vitro implantation and transfer are some of the immediate research themes to be searched in near future (Wani, 1998, Wani 2002, Wani 2001).

13 Foetal maternal relationship

Various biochemical and morphological investigation on foetal growth and development has been jointly explored by SKIMS and SKUAST (Mufti, 1996). This is an innovative search to unveil various mechanisms involving in vitro implanation and transformation of blastocyst into foetus in future. More funding is needed for investigative and innovative projects on foeto-maternal associations such as reported by Mufti et al (2000), wani et al 2007, 2006,abc.

14 DNA finger printing

Genotype environment interactions are closely related to mammalian reproduction. Knowledge of genetic constitution and embryonic differentiation is a pre-requisite for breed conservation and improved selective breeding. Since, DNA of an individual is not directly influenced by environmental conditions except loci coding for T-cell receptors, antibodies and others; short term environmental influences like season, feeding and management may not influence. DNA sequences based on this central theme, an investigation was undertaken in collaboration with University of Hohenheim Germany. The study was financed by German Research Foundation. The project has ended, but its savings has enabled us to institute an fellowship to be called Gall, Geldermann, Wani fellowship for goat reproduction. Under this project, blood samples from Pashmina and Bakerwal Goats were analysed.

DNA was extracted from leucocytes with phenol-chloroform. Different regions of the expressed DBR gene and the DBR pseudogene were amplified by PCR. Fragment lengths of the entire exon 2 and the microsatellite were analysed, using automatic DNA sequencer, Automatic Laser Fluorescence (ALF) and by Single Strained Confirmation Polymorphism References

references

Al-Quran, Surat Mominoon, 12:134. Young, L. E, 2002, Somatic cell nuclear transfer,419, 6907, 583-586

Anderson.G.B; Murray J.D and Medrano.J.F, 1996, Expression of a bovine k-CN cDNA in the mammary gland of transgenic mice utilizing a genomic milk

Casey.D.C and White.K.L, 1990, Incorporation and expression of a neo gene after Cloning and sequence analysis of the cDNA for a protein from coccidioides

Colman.A and Garner.I, 1995, The transgenic mammary gland as a bioreactor:

Dugger.K.O; Villareal.K.M; Ngyuen.A; Zimmermann.C.R; Law.J.H and Galgiani.J.N, expectations and realizations, 6,107.

Gall, C; Gelderman, H; Wani, G.M. 1997. Analysis of Goat Genetic Resources of goat breeds in Kashmir. Final report of research co-operation between SKUAST and University of Hohehuim, Germany .

Gutierrez.A; Meade.H.M; Ditullio.P; Pollock.D; Harvey.M; Jimenez. Fores.R;

Hays, B.L. 1998. Non-surgical embryo collection in the goats. Proceedings of 11th International Congress on Animal Reproduction and AI, Dublin 2: 165.

Heap, R.B; Wang. M.W; Whyte, A and Taussing, M.J. 1991. Significant of peri-implantation signals. L. 17. Proceeding Int. Conf. On Frontiers in reproductive Physiology, Nov. 8-10, 1991 at AIIMS, New Delhi.

Jufen, Q; Zhiming, H; Yong, Z and JianChen , W. 1991. Invitro capacitation of ejaculated spermatozoa and invitro fertilization in dairy goats. Theriogenoloty. 35(1):219.

Karsch, F.J and Evans, N.P. 1996. Feedback actions of oestradiol on GnRH Secretion during follicular phase of the oestrus cycle. Acta. Neurobiol. Exp. 56:715-725.

Karsch, F.J; Bowen, J.M; Caraty, A; Evans, N.P and Moenter, S.M. 197. Gonadotrophin-releasing harmone requirements for ovulation. Biology of reproduction, 56h:303-309.

Karsch, F.J; Moenter S.M. and Caraty, A; Moenter, S.M. 1997.The neuroendoerine signal for ovulation.Animal Reproduction Science, 28:329-341.

limitis with immunogenic potential, 218 (2),485-89

Maughan .P.J; Philip.R; Cho.M.J; Widholm.J.M and Vodkin.L.O, 1999, Biolistic

Mufti, A.M. 1996. Studies on morphological characters and some metaolic consititutes at different stages of development of ovine foestus. MVSC Thesis, SKUAST, Srinagar.

Mufti, A.M; Wani, G.M; Wani, N.A, and Khan, M.Z. 2000. Prenatal development of ovine foetus. Small Ruminant Research, 38: 87-89.

Mufti, A.M; Wani, N.A; Mir, M.M; Khan, M.Z and Wani, G.M. 1997. Superovulatory response in corriedale ewes during different months of breeding season. Small Ruminant Research 25:181-84.

Protein gene as an expression cassette, 5 (4), 271-79

transfer of Caprine hematopoietic cells using a modified retro virus, 68,2,449-53.

Transformation, expression and inheritance of bovine beta-casein in soyabean

Pawshe, C.H; Totey, S.M and Jain, S.K. 1994. Effect of the methods of recovery of goat Oocytes for invitro maturation and fertilization.Theriogenology, 42:117-125

Wani , G.M. 1996. Embryo biotechnology in sheep and goats. Valley Book House University Road, Srinagar.

Wani, G.M. 1981. Ultrasonic pregnancy diagnosis in sheep and goats. World Review of Animal Production, 17:43-48

Wani, G.M. 1982. Laparoscopy in Farm animals . World Review of Anim. Production, 18:7-13.

Wani, G.M. 1984. Investigations Caprine Embryo production and culture .Dr.med.Vet.Thesis vet Univ, Hannover, Germany.

Wani, G.M. 1992. Advanced Techniques in Animal Reproduction. SKUAST Press, Shalimar No.75/250/12/92.

Wani, G.M. 1995. Early diagnosis of pregnancy in surrogate mothers (Sheep and Goats).National Symposium on Embryo Transfer and Genetic Engineering Technologies in Small Ruminants; May 7-8, 1995, CIRG, Makhdoom, Farah, India.

Wani, G.M. 1998. Role of assisted reproductive technologies in augumentation of sheep production. Lead paper presented on National eminar on “Strategies for maximizing production and efficiency in sheep”, held at CSWRI, Avikanagar, Rajasthan, April, 1998 PP.18-22.

Wani, G.M. 2001. Enhancement of chevon Production through reproductive management proceedings IV National Seminar of Indian Society for Sheep and Goat Production and utilization (ISSGPU) Nov 9-11, 2001, Bharathiar University, Coimbatore, Tamilnadu, India.

Wani, G.M. 2002. Research in Animal Reproduction-Vision 2050. Proceedings XVII Annual Convention of ISSAR and National Symposium, Nov.14-16, 2002, IVRI, Izatnagar U.P.

Wani, G.M. 2003 a. Biotechnological advances in Goat Production. National Workshop on “Recent advances in Goat rearing”, MPKVK, Agric. Univ. Rahuri, 29-30 Dec, 2003.

Wani, G.M. 2003. Goat Husbandry- multipurpose management. Encyclopedia of Dairy Sciences, edited by Roginishi Hubert, Euquary, J.W., Fax, P. Academic Press London Vol.2 PP 1259-1270.

Wani, N.A, and Wani, G.M. 2003. Use of epididymal spermatozoa from slaughtered sheep for invitro fertilization of invitro matured Oocytes. Indian.J.Anim.Sci, 73:143-146.

Wani, N.A; 1996. Invitro maturation and invitro gertilization of ovine Oocytes MVSc Thesis, SKUAST.

Wani, N.A; Wani, G.M; Khan, M.Z and Salahuddin, S.2000. Effect of Oocyte harvesting techniques on invitro maturation and invitro fertilization in sheep. Small Ruminant Research. 36: 63-67.

Wani, N.A; Wani, G.M; Khan, M.Z and Siddiqui, M.A. 1999. Effect of different factors on the recovery rate of Oocytes for invitro maturation and invitro-fertilization. Small Ruminant Research 34:71-76.

Wani, N.A; Wani, G.M; Mir, M.M and Beg, M.A. 1998. Ultrasonic pregnancy diagnosis in Gaddi Goats. Small Ruminant Research 29:239-240.

Wilmut,I; Beaujean.N; De-Sousa,P.A; Dinnyes.A; King.T.J; Paterson,L.A; Wells,D.N

Ph.D (Animal Reproduction / Gynaecology), Dr. Med. Vet (Animal Reproduction/ Production)

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Article Tags: Embryo, Dna Fingerprinting, Biotechniques, Invitrofertilization, Nuclear Trsfers

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