Pregnancy Test With Rabbit

Ovarian Manuplation and Ovulation

Ovarian function and early ambryonic development

Abstract as Intoduction:

Ovulation, its process and mechanism has been discussed (Wani, 1996). Here we shall discussed recent advances in ovulation process. They include ovarian angiogenesis and biochemical basis of corpur luteum formation, embryonic development blocks which hampered embryonic development beyond 8 cell stage in the past has been over comed. How it was achieved shall be disussed. New advance and terms used in literature like Genome actiation Gap-junction and blastocal formation shall be xaplained pre-implantation embryos and biochemical characteristics shall be discussed, however morphological events shall be depicted in some othe chapter , Embryo biosafety and early pregnancy factors has been reviewed in this chapter.

Use of anthelmintics like Benacil and tetramisole orally results in toxicity during pregnancy. The embryotoxicity was 100% during 9-18 days post coitus and abnormal lambs delivered by ewes dosed during pregnancy ranged from 34-57% (Grunzhinskas, 1984).

1.1 Ovarian Angiogenesis

Angiogenesis is a process of vascular growth. The ovarian angiogenesis increases new blood vessels in ovary. They supply nutrients and hormones to the follicles. Thus angiogenesis promotes follicular growth and corpusluteum formation (Tamanini & Ambrogi, 2004). The developing follicles have endothelial cells. They form a network in the theca layer. Growth factors mainly produced by granulose cells as VEGE (Vascular endothelial growth factor) and bFgF (basic fibroblast growth factor) act as stimuli for promoting follicular growth and antrum formation (Ziecik, 2004; xiao et al, 2001).

After ovulation, newly formed blood vessels cross basement membrane between theca and granulose cell layers and undergo a rapid growth to form corpus luteum. The luteal vascular growth varies and angiogenesis and angioregression determine corpusluteum growth and regression. Thus in future the improvement in angiogenic development of ovary may help in reproductive performance (Tamanini and Ambrogi, 2004).

These Hemodynamic changes in ovarian and corpora luteal walls was studied using colour Doppler ultrasonography. The flow of blood is assessed (Acosta and Miyamoto, 2004). This confirms our earlier studies using Laparoscopy for ovulation (Wani & Sahni, 1988, Wani, 1989 Sharief, 2000).

1.2 Embryo Block

Embryo blocks action of antibodies and becomes a homograph and not a heterograph, eight cell embryo block was very common block in mammalian embryonic development. Sheep and goat embryos did suffer from this block too. Development beyond 8 cell was diffcult in vitro. Use of special medias have overcomed this problem The 8-cell embryo block in development is overcomes by various growth factors incorporated in culture media. The HB-EGF (heparin-binding epidermal growth factor like growth factor) improves the development of 8-cell embryo to the blastocyst stage. This factor is helpful in preimplantation embryo development. (Tamada et al, 1999). embryo, makes direct contact with uterine lining only for a short period and thereby thrives on it indirectly through placenta via umbilical cord. It remains protected in an immunologically protected chamber the amnionic sac. Feotal development and biochemical constituents of foetal fluids and morphological development features observed in our studies are published . (Mufti, 1996, Mufti et al, 2000), Wani, 2004, 2005}. Embryo development can be viewed in web images too. Early embryo mesoderm cells called somites differntiatic into myoblasts-resulting into muscle and skeletal tissues. Various (MRF} Muscle Regulatory Factors in foetus determine the muscular growth and were professed to be used in future research. Their exploitation would bring muscular lambs at birth. (Wani 2001). Many advanced aspects of myoblast .Determination flow have been documented. Huskey, 2001. (Robert.J.Huskey, myoblast determination flow interent explorer- Huskey, R.J. 2001). Myoblast Determination flow Microsoft. Internet Explorer.

Tight junctions prevent ion and water movement between cells and Na/K pumps move into the centre of the blastocyst resulting in to osmotic pressure difflections and formation of blastocoel. Plasmogen and its activator results into enzymatic digestion of zona-pellucida during this process. (www.wisc.Edie/ansici-repro/Lec-18 or search Embryo block sheep Metacrawler- Microsoft Internet explorer ).

1.3 Genome activation

Activation of embryo is detected by pronuclear formation, cleavage and blastulation. Addition of vitamins during oocyte maturation helps subsequent blastulation and embryo viability (Bormann et al, 2003). Pregnancy associated glycoproteins (PAGS) are antigens synthesized in ruminant trophoblast. They enter maternal blood stream and thus signal early pregnancy (Wani, 2005).

Invitro growth of granulosa cell complexes isolated from cryopreserved ovine ovary has been reported (Newton et al, 1999). These cells have a capacity to produce immunereactive inhibin with FSH, (Compell et all, 1996). The ovine Booroola fecundity gene (Fec B) so widely known has been linked to markers from a region of human chromosome 4q. Thus linking research in ovine or caprine reproduction for improvements or control of reproduction in humans. The VEGF factors (Vascular endothelial growth factors) characterized and expressed in ovine corpus luteum could be of immense “healing touch” value if properly innovated to use in repairs of various body injuries. Ovine FSH has been of value in developing long-term contraceptive vaccine in bonnet (Redmer etal, 1996).

1.4 Gap-junction network

Mammalian oocytes are surrounded by follicular cells. A communication system exists between these oocytes and follicular cells. This system of communication is called Gap –Junction network. This system provides transfer of nutrients and signals between follicular cells and oocytes. The proteins of connexin family form these building blocks. Oogenesis, primodial follicle activation and subsequent ovulation is the result of these and some other proteins factors. They are e-kit receptor, transforming growth factor beta (TGF beta) and bone morphogenic proteins. Progressive morphological changes in the oocytes result into structural rearrangements. These generate energy. The increased energy facilitates oocyte development and ultimately determine its capacity and competence to dominate and ovulate (Fair, 2003).

Pregnancy recognition during day 12-22 in sheep and 15-21 in goats is made on the basis of interferon tau a member of type I interferon. It is the first protein secreted by trophoectoderm of the ruminant conceptus. It blocks the uterine luteolytic mechanism and is responsible for the sustenance of corpus Luteum during early pregnancy days. It acts on the uterine epithelial cells by suppressing their ability for transcription of gene for propogation or activation of oestrogen or oxytocin receptors. It also induces several proteins responsible for embryo implantation.

1.5 Per-implantation Embryo nutrition

The most important nutrients required by the developing mammalian preimplantation embryo are carbohydrates, anino acid which provide energy. It also prevent cellular stress. Quantification of embryo energy metabolism is thus necessary. An attempt in this direction has been shown in figures. (Jamada, 1998).

The method of assessing preimplantation embryo viability by the self-refrencing electophysiological technique is possible. This is an non-invasive measurement. The technique measures the gradients of calcium, potassium, oxygen, and hydrogen peroxide (Gardner, 1998).

Protein, prostaglandins and steroid synthesis in caruncular and intercaruncular endometrium in sheep has been estimated. It was determined that attachment occurs around day 15-16 which however was not confirmed by our recent studied (Wani et al 2996, 2007) (Findly et al,Comparing invitro and invivo embryonic development in ruminants is possible by use of immunocytochemistry, confocal laser scanning miscroscopy and fluorescence techniques. By combination of these techniques it has been possible to demonstrate that:

1. RNA gene activation is similar

2. Embryonic nuclear development is similar.

However, embryo produced through nuclear transfers are associated with increased apoptosis, in the inner cell mass of the blastocysts. Besides embryo produced in-vitro by nuclear transfers or otherwise are associated with mixoploidy. Mixoploidy means embryos with abnormal haploid or polyploidy cells besides normal diploid cells. (Cinobell et al, 2003).

Laparoscopy in sheep and goats has been used for ovulation detection, embryo aspiration and diagnosis of pregnancy. (Wani, 1982 Wani and Buchoo, 1990, 1997, 2000) Embryo recovered through laparoscopy in Boer goats have a of 62% and were economically viable. (Besenfelder et al, 1994)`

1.6 Embryo biosafety and disease risk

Scrapie, an ovine and caprine transmissible spongiform encephalopathy is widely spread among sheep populations in many European Countries. As it is known that susceptibility to scrapie is determined genetically, breeding programmes aiming at providing scrapie- resistant flocks have been established – (sipos et al, 2002).

The pregnant endometrium secreted a number of neutral –to-acidic proteins which constituted, in part, the histotroph. A vitamin A-transport protein, RBP was identified in cultures, conditioned by the endometrium at days 13-21 of pregnancy. The endometrium is the only source of retinol for embryonic tissues. The uterine RBP appears to transport retinol locally toward embryonic tissues. Secretion of RBP by caprine endometrium at days 13, 15 and 21 of pregnancy suggested that retinol played an important role in conceptus development during the periattachment period of early pregnancy – (Lue et al, 2002) (Young et al 2000, 2004).

Caprine oviduct epithelial cell (COEC) are commonly used in vitro goat embryo production protocols to stimulate early embryonic development. These feeder cells are usually collected from slaughterhouses from unknown serological status animals. In many regions of the world, issues derived from the resource may be contaminated with CEV (Caprine Arthritis Virus) CAEV (Caprine Arthritis Encephalitis Virus) and the use of such material in invitro fertilization systems may contribute to transmission of this pathogen to the cultured embryos and dissemination via embryo transfer (ET). The use in invitro fertilization systems of coculture feeder cells that support efficient replication of CAEV(Caprine Arthritis Encephalitis Virus) to high titers could represent a serious risk for permanent transmission of virus to the cultured embryos and to the surrogate dam involved – (Lamara et al, 2002).

1.7 Early pregnancy Factors

Pregnancy – associated glycoproteins (PAGs) are antigens synthesized in the superficial layers of the ruminant trophoblast. Initially, they were identified either as proteins differed into the maternal bloodstream (where they have applications in pregnancy diagnosis+PAG or as molecules binding to the LH receptor (PAG2). In this study, double radial immunodiffusion was used to test the ability of antisera raised against different PAG molecules (bovine, ovine and caprine) to react with placental extracts from non ruminants (rabbit, cat, mouse, pig and wild pig) and ruminants (cow, ewe and goat) Placental extracts from all nonrumiants tested except rabbit reacted with anti bovine. PAG2 (anti- boPAG2). Extracts of ruminant placentas react with different antisera, confirming the expression of various PAG molecules. According to the time at which the placentas were collected (early or middle pregnancy), the reaction differed as regards the thickness, position and number of precipitation lines, suggesting that PAG expression varies as pregnancy progresses. Bos indicus and Bos Taurus placental extracts exhibited different reactions with anti-boPAG2, a single precipitation line in the former case and two lines in the latter. This suggests differential expression of boPAG2related glycoproteins in these two subspecies – (EL-Amivi, et al, 2003).

References for detailed study

Acosta,T.J. and Miyamoto,A. 2004 Vascular control of ovarian function: ovulation, corpus luteum formation and regression. Animal Reproduction Science. 82/83: 127-140

Argov, N. and Sklan,D. 2004 Expression of MRNA of lipoprotein receptor related protein 8, low density lipoprotein receptor and very low density lipoprotein receptor in bovine ovarian cells during follicular development and corpus luteum formation and regression. Molecular Reproduction and Development. 68(2): 169-175

Bormann,C.L; Ongeri,E.M. and Krisher,R.L.2003 The effect of vitamins during maturation of caprine oocytes on subsequent developmental potential in vitro. Theriogenology. 59(5/6):1373-1380

Battye, K.M; A.J; O-Neill-Co. and Evans, G. 1991 Production of platelet-activating factor by the pre-implantation sheep embryo. Journal of Reproduction and fertility. 93(2):507-514

Buchoo, B.A and Wani, G.M.(1991). Induction of Oestrus in Corriedale ewes during anoestrus. Indian J.Anim sci 60(4):407-408.

Campbell,B.K; Telfer,E.E; Webb, R. and Baird, D.T. 2000 Ovarian autographs in sheep as a Molecular and Cellular Endocrinology.163:131-139

Campbell, B.K. and Baird, D.T. 2001 Inhibin A is a FsH-responsive marker of granulose cell differentiation which has both autocrine & paracrine actions in sheep. Journal of Endocrinology. 169: 333-345

Campbell,B.K; Webb, R; Dobson, H. and Dobers Ka, C. 2003 Reproduction in Domestic Ruminant. Symposium on Reproduction in Domestic Ruminants bheld in Crieff, Scotland – U.K. August 2003 PP 103-116. Reproduction Supplement Series. 61:103-116

Evans,A.C.2003 Characteristics of ovarian follicle development in domestic animals. Reprod. Domest. Anim. 38(4):240-6

El-Amri,B; Sousa,N.M.De; Mecif,K; Desbuleux,H; Banga-Mboko,H. and Beckers,J.F. 2003 Double radial immunodiffusion as a tool to identify pregnancy- associated glycoproteins in ruminant and nonruminant placentae. Theriogenology. 59(5/6):1291-1301

Fair,T. 2003 Follicular oocyte growth and acquisition of developmental competence. Animal Reproduction Science. 78(3/4): 203-216

Fakowska, Podstawka,M; Wrona,Z. and Krakowski,L. 2002 Biological role of interferon tau. Medycyna Weterynaryjna. 58(8):568-571

Findlay, J.K; Ackland, N; Burton, R.D; Davis, A.J; Walker, F.M.M; Walters, D.E. and Heap, R.B.1981 Protein, prostaglandin and steroid synthesis in caruncular and intercaruncular endometrium of sheep before implantation. Journal of Reproduction and Fertility. 62(2):361-377

Gardner,D.K.1998 Changes in requirements and utilization of nutrients during mammalian preimplantation embryo development and their significance in embryo culture. Theriogenology. 49(1):83-102

Gruzhinskas,V.A. 1984 Effect of the anthelmintics Benacil and tetramisole on prenatal and postnatal development (sheep, swine, cattle, horse). Veterinariya-Moscow, USSR. (11):61-62

Juengel,J.L; Nett,T.M; Anthony,R.V. and Niswender,G.D. 1997….on expression of mRNA encoding insulin-like growth factor 1 and growth hormone receptor in the ovine. J.Reprod.Fertil.

Kaloo, F.A (2002). MVSc Thesis, SKUAST-K.

Kaul, N.A (2002) MVSc Thesis, SKUAST-K.

Kawate,N; Morita,N; Tsuji,M; Tamada,H; Inaba,T. and Sawada,T.2000 Roles of pulsatile release of LH in the development and maintenance of corpus luteum function in the goat. Theriogenology. 54(7):1133-1143

Kanka, J; Nagai

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