Future Research in Reproduction - Vision 2100

Abstract

Endocrinological advances would need more research on hormone synergism, agonistic and antagonistic actions. Follicular atresia vis-in-vis intrinsic GnRH system would need more experimentation. The deterministic and permissive modes of GnRH action would be applied to reduce hyper stimulation of ovaries. The understanding of dominant follicular dynamics, shall help to harvest Follicular pool in thousands rather than in hundred at present. New biological sources of hormone from fish and other animals should find more clinical trials to popularize cheap and biological rather than chemical hormonal sources.

Sensitive disease diagnostics using RIA, REA, hormone detection kits, ultrasonography, laparoscopy, DNA finger printing, immunological and other psychometric techniques needs consideration. Gap junction and development of conceptus, foetal biological research will need emphasis by 2050. In vitro fertilization and embryo culture shall be replaced with in vitro implantation and in vitro foetal development. Foetomuscular development and foeto-maternal interactions will be more vivid and clear. Reproductive care and disease prevention shall need herbal drug stimulants. Such bio-plant stimulants and medicaments need innovative research attempts. Corpora luteal bio-sensitivity and its use in augmentation of reproduction needs trials. Fertility enhancement needs identification of desired fertility objectives. The problems and constraints in animal’s reproduction have to be addressed by using modern reproductive technique to improve our traditional reproductive management.

*Part of Prof.C.R. Sane Oration Lecture delivered by the author at XVII Annual Convention of ISSAR and National Symposium on Reproductive Technologies for Augumentation of Fertility to be held at Indian Veterinary Research Institute, Izatnagar on November 14-16, 2002.

Introduction

The use of artificial insemination (AI), embryo transfer (ET) have helped in elite gene propogation. "Dolley" the super sheep is known and many more genetic clones are in offering. Today our concern is bio-safety of germplasm and the animals products. With high risk of new disease emergence, all researchers are warned to use their materials with caution. The "mad cow disease" and heavy economic losses in U.K. and Europe are eye openers for all of us. The future of gene technology lies in bio-safety and fulfillment of international zoo-sanitation regulations. Disease free zones need more emphasis than producing high yielding livestock from disease infested area. Thus safe and quality animals material (germplasm) and products is the slogan for tomorrow. Zoo-sanitation certifications will demand more trained veterinarians and other para vet staff. This necessities review of human resource development too.

For production and utilization of elite gene pool of animals, we need to know available resources, both biological and material. For example, increasing meat/milk or fibre production from our small ruminants we need to know them better. We must analyse their gene pool both qualitatively and quantitatively as was done in goats (Gall el al., 1997) using DNA techniques. Once we know merits and demerits of our gene pool, could we preserve them or utilize them for further. The methods and postulations in this direction can be reviewed as -

Endocrinological advances:

Ovarian oogenic and endocrine functions are interlocked via Hypothalamo-Hypophysial Gonadal Axis (HHGA). The cyclicity and its behavioral overt and convert phases are intrinsic to these ovarian functions. They have been thus rightly named as " the Zeitgeber or biological clock (Baird, 1987). The existence of feedback loops of ovarian steroids with respective gonadotrphin is a biphasic phenomenon, where higher concentrations in blood have inhibitory or suppressive effects, low concentrations have stimulatory effects (Barrac Lough, 1973), Many reproductive mechanisms hang on changes in these ovarian steroids such as labour pains (Csapo, 1981), ovulatory failures (Snchez-criado et al., 1990). Interestingly these hormones interact to modify or sometimes antagonize each other. For example P4 appears to be potent antagonist of E2 at a combined ratio of 1:1 (Heap and Flint, 1987). The synergism, agonistic and antagonistic actions of these hormone combinations are the basis for all reproduction manipulations viz Control of oestrus, ovulation, conception, gestation, fertility, infertility, contraception and or abortions. Fish and sheep ovarian extracts were tested to find out species sensitivity. It seems it is possible to tap huge fish ovarian extracts for intra and interpolation in mammalian ovary (Najar, 1999). Coming years will see more attempts to use such resources to avoid side effects from chemical and synthetic analogues of hormones.

New hormonal roles- follicular atresia:

GnRH – regulates gonadotrpin release from pituitary, but its continuous and prolonged use in natural form or as its potent agonist induces an inhibitory effect on ovarian steroidogenesis, follicular development and ovulation (Kledzik et al., 1978; Srivastawa et al., 1995). GnRH receptor gene encoding is expressed in the rat ovary (Kaiser et al., 1992). A new concept prevails today which postulates that an intrinisic GnRH system competes with receptor. Thus its biological response exist within the ovary. The follicular atresia is brought by this intrinsic GnRH system (IGS) (Erickson et al., 1994). A new vision of autocrine and paracrine secretion of ovary vis-in-vis GnRH becomes evident. Future modulations in correcting reproductive failure involving hormone therapy needs rational understanding of all endocrine, autorine and related control mechanisms. Ovarectomized ewes when implanted with E2 and P4 implants were maintained in an artifical follicular phase. This speaks of an endocrine model compatible to a natural sequencing hormones of desired levels (Krash and evans, 1996).

Intrinisic GnRH system (IGS)

Two theoretical methods called deterministic and permissive models have been put forth to explain role of GnRH (Karsch et al., 1977).

Deterministic model

Deterministic model envisages that a surge-like increase in GnRH is needed to induce preovulatory LH surge. This model befits animals whose LH release and ovulation is either time bound (Copulation) or signal dependant (cervical signal, rabbit and neural signal Central circadian pacemaker in rata refers).

Permissive models

Permissive models explain GnRH in relation to ovulatory LH sruge. Here feed back of steroids exert their influence on anterior pituitary gland. This is supported by evidences that LH surge continue in situations where GnRH secretion is apparently abolished. For example in monkeys with hypothalamic lesions, or in women with hypothalamic amenorrhea or congenital GnRH deficiency, normal LH surges occur (Martin et al., 1990).

Hormone Agonists/antagonists

Hormone antagonist or antihormone are those compounds or elements which prevent an endogenous hormones from exerting its biological effect(s) (Vanlook, 1988), These antihormone need to be safe i.e. free from side effects. The hormone antagonists in principle acts in 3 ways.

i) Inhibition of hormone synthesis
ii) Neutralising hormone action by specific antibodies.
iii) Blocking the receptors of the hormone.

Use of antagonists, agonists have demonstrated their role in ovulation control. Their low histamine-releasing activity (Fraser, 1981) support their wider use which needs further extensive studies. More details are reviewed (Najar, 1999).

Hormones-biological sources

The synergism, agonistic or antignostic actions of steroid and other reproductive hormones are the basis of reproductive manipulations like ovulation and fertility control, conception, gestation, fertility, infertility and contraception (Wani, 2002b). Fish and sheep ovarian extracts were tested for interclass sensitivity. It was revealed that intra and interpolation in mammalian ovaries is possible using these materials (Najar, 1999). Future attempts to utilize these cheap and easily available biological materials for ovarian manipulation may reduce the use of high cost chemical and synthetic analogues (Najar, 1999).

The biggest problem in ovarian manipulation faced in the past was due to varied superovulatory response (wani, 1984). The follicular atresia was postulated to reduce this constraint if induced adequately. As our understanding of (IGS) intrinisic GnRH system was poor then, we could not use it to overcome our problems. Now we know follicular atresia is related to this IGS (Erickson et al., 1994) and therefore, our later manipulations incorporated GnRH with comparably better success in ovarian manipulations. A new vision of autocrine and paracrine secretions of ovarian GnRH became the subject of discussion. Estrogen alone can induce ovulation but for CNS (Central Nervious System) priming progesterone combination in desired ratio is essential (Evans et al., 1995). Thus future modeulations in correcting reproductive failures needs a proper understanding of hormonal inter and intra polation with physical, chemical, biological and neuroendocrine secretions. Pheromones and other psycoemotional factors too need an analysis.

In our experiments on infertility (anoestrus) in cows of Kashmir (District Budgam) we were puzzled to see better response with low doses (300 ug) of GnRH than the higher dose of 400 ug (Shah, 1996) . We were unable to explain our results until the recent studies on permissive and deterministic models of GnRH action came to light (Karsch and Evans, 1996,Karsch et al., 1997).

Herbal-biostinulators

Pheromones, sire introduction effects, sensory reflexus and vast other stimulants increase receptivity as well as fertility in domestic animals. Sprays and herbal cure products will be in great demand to increase or decrease fertility in coming years. The use of stimulants or repellents as sprays and scents may be in need. Herbal cures for various reproductive wastage needs further research. The use of herbal products, fruits, nuts and medicaments in ancient Kashmiri culture after delivery, need to e looked at. The Cichorium spp. Saffron, Cofula anthemodies, Prunella vulgaris, Artemisa and other products in use during post delivery period needs scientific analysis.

The ovarian sensitivity decrease soon after partuition and its resurgence after 14 days in cows, 40 odd days in women and specific periods in other animals, reveal a lot which needs exploration to phase out various sequences of reproduction.

Corpora luteal biosensitivity.

Corpus luteum growth and development is the first earliest pregnancy signal along with maternal recognition factors discussed (Wani, 1996). The corpus luteum development and receptivity to PGF2 alpha is now known. The ascending and descending corpora lutea are insensitive to PGF2 alpha. What makes them insensitive and what resurges their sensitivity need exploration. May be in future, CL insensitivity might have direct effects on survival of the conceptus. Does conceptus make CL more sensitive to PGF2 alpha? Or in other words, does the gravid CL and non-gravid CL behave similarly or differently to PGF2 alpha. Many such questions vis-à-vis the ovarian morphology, uterine environment and behavioral patterns need to be studied and co-related to develop a manipulative and curative safe system. In some species CL or pregnancy is necessary till delivery, in others it is needed only till placenta develops. Its complete genesis and biostimulation and bioregulatory roles are obscure and needs further research.

Dominant follicle dynamics

The dominant follicle dynamics may be in focus for ovulation control. Dominant follicle or would be bride at each ovulation appears around day 7 of cycle in women. It occurs much earlier on day- 1 to 0 in ruminants (cow, sheep,goats). This time point may be the focus to try future selective interferences in ovulation control. Antagonist use during this phase has shown decreasing levels of estrogens in serum thereby indirectly hinting at dominant follicle alteration, as it is one of chief source of this hormone during this phase (Fraser, 1987). Dominant follicle in its sojourn to freedom and release at ovulation seems to attain some specific qualities. Why? It alone releases itself of bondage’s in ovary (ovulation) and rest of its competitors are hindered through the process as atretic follicles. What make this dominant follicle to attain these qualities among a vast pool of follicles needs further study. In coming 50 years one shall possibly understand and interlock a pre-determined messenger system triggering dominance in one or few follicles destined to become the new offspring of that species. This system when decoded shall offer new opportunities in controlling ovulation both for contraception (women) or augmentation (animals). New genepool of dominant follicles from dead high yielding animal ovaries would help to propagate and multiply the elite.

Over stimulatory effect during superovulation could be avoided by use of antagonists as dominant follicles seem to evade suppression, thus follicular selection will prevent excessive stimulation and cystic follicles. Follicular fluid constituents containing occyte awaiting for biochemical signals to resume (meiosis ) growth and development to ensure timely and healthy ovulation need more exploration (Lenton et al., 1988). The specific signals triggering occyte release might help in in vitro occyte maturation success rate.

Development of conceptus-in vitro foetus

The lean meat production needs more studies on development of conceptus. We know that prenatal development undergoes in three stages. The early pregnancy signals skeletal & nervous tissue growth, on priority. During mid-gestation the hind and fore-limbs develop. During late gestation muscular development along with peripheral extremities including hair develop. In order to increase lean meat production, research in needed to investigate relations between hard and soft tissues. Maternal and foetal interactions needs a probe. Some basic studies (Mufti, 1996) indicated prospects of manipulating lean meat production (Wani, 2001, 2002 a) by bio-stimulators during late gestation, which may help, the production of muscular lambs/kids at birth, besides, increasing birth weight. More details have been described (Wani, 2001, 2002a) Fig.1-5). More studies in future are needed to reveal the exact mechanism involved in critical transition of power control from maternal to embryonic genome. Application of recent techniques of reverse transcription, PCR and others may reveal the details (Schultz et al., 1990).

Slaughter house follicle pool

A good number of our endangered species die in wild climatic conditions are hard and harsh. Under temperate climatic conditions dead bodies are entrapped in snow for long. It gives a hope to revive their follicular pool. Our studies on resumption of life using follicles from slaughtered sheep and fertilizing them with epididymal semen was an pioneering attempt in this direction (Wani, 1996) (Fig 6-8). The protein content of unfertilized ovum is around 100 mg in rabbit, 27 mg in mouse (Brinister, 1967, 1971). The small genetic material needs extensive analysis to unveil vast informations in its codans and anticodans. Further ovum maturation or resumption of meiosis is initiated during birth or shortly before birth and thus the pool of follicles remains fixed at birth. Therefore, foetal ovary has to be the subject of further manipulations especially in view of its immunological insensitivity (Guraya, 1985).

Gamete biosafety

Bacterial counts in semen in unprecedented numbers effect semen fertility (Kher, and Dholokia, 1985), The contaminational hazards have increased due to environmental pollution, water and air contamination. Above all the development of resistance of known bateria and other micro-organism to antibiotics (Gupta and Maurya, 1993). We have examined the semen of about 40 bulls maintained at the Frozen semen bank, Rambirbag, Kashmir for bacterial loads in semen and tested antibiotic sensitivity (Koul, 2002).

Presence of bacteria in large numbers in semen, effects its quality and fertility (Kher and Dholokia, 1985). The source of contamination of semen vary from inflammatory foci and lesions on penile apparatus, contaminations on equipments, appliances, diluters, extenders air and laboratory environment. These contamination hazards have increased with environmental pollution. Many bacteria having developed resistance of routine antibiotics used (Gupta and Maurya 1993) in semen extenders has made inseminations more critical and vulnerable. More studies in this direction are needed to develop a bio-safe and infection free germplasm pool of frozen and fresh semen in future.

Reproductive physiology

Reproduction is a complex phenomenon, starting with oogenesis as early as 135th day post mating. By 50-100th day post conception (mating) sheep and goat conceptus in-utero shows follicular growth. This follicular growth and development upto primary oocycte stage in some mammals ceases at birth (Wani, 1996). The further growth and maturation of primary occytes is triggered at puberty i.e. at the onset of first sexual activity. The release of ova i.e. ovulation follows. During this proves of folliculogeneis and ovulation, followed by fertilization and development of conceptus, the male and female gametes undergo various morphological and physical changes. They pass through various barriers, reservoirs, under the influence of bio-physical, hormonal,morpho-chemical mechanisms (Wani, 1996, Wani and Buchoo, 1990, Wani, 1984a and b, Wani et al.,1986). More in-depth studies on barrier mechanism vis-à-vis morphological changes during different hormonal milieu is needed.

Foetal muscular development

Genetic,hormonal and environmental factors influence foetal growth and development. The proper in-utero maturation and growth of foetus signify proper care and management of gravid nannies. The pre-natal development of foetus is exhibited at birth by birth weight. The head and skeletal tissues grow and mature earlier, followed by fore and hind limbs. The muscular development of foetus occurs during late gestation. Thus, during early pregnancy nervous and skeletal tissues are having priority for nutritive requirements in-utero(Musti, 1996). Maternal nutrition is important for growth and development of meat in off spring in-utero. Muscular growth stimulants may thus help production of quality meat lambs or kinds in future.

Foeto-maternal interaction

The role of endometrial stromal cells during pre-implantation stage needs further research. Some studies on ultrastructure of endometrium (Brinsfield et al., 1974) are still not sufficient to answer the questions. What is the role of immune cells and endometrial cells in building of the immunological barrier to protect the foetus. Foetal biology is abarren field of research in the world. After few initial steps by Dr. Moris Yound in UK and D. Srivastava in India, only a scattered information is available on the subject (Wani and Buchoo, 1990; Mufti, 1996). Much is still to be unveiled by 2050. The placental hormone influence mammary development and milk production of the dam. This needs further research by 2050. Foetal membranes serve as Chief source of nutritive exchanges. The arrangement of cotyledons, caruncles, positioning of single and twin conceptus, indicate complex mechanisms with common roots. (Mufti et al., 2000) These mechanisms of foeto-maternal interactions and relationships emphasize proper foetal number diagnosis and corresponding materno-nutritional management strategies.

Small ruminant reproduction

There is a need of ovulation, pregnancy and foetal number detection by methods described (Wani, 1981, 1982; Wani and Sahni, 1980, 1981; Wani and Buchoo, 1991, 1993). These reports demonstrated successfully results ofr ovulation detection, multiple ovulation predictions, early pregnancy and foetal numbers. The induction of multiple ovulation, in-vitro fertilizations, embryo collections, embryo culture, conservation, micromanipulation, splitting, cloning and sexing are some of the reproductive high-tech applications used to increase reproduction rates (Wani 1996; Wani et al., 1990; Wani et al., 2000 a,b; Buchoo et al., 2000). These techniques have but a little application under field conditions in small ruminants especially under extensive management system. However, their use has already revolutionized propagation of elite germplasm and can be used under buck mother farms to produce high quality sires.

The futuristic attempts in this direction should include in vitro post implantation and foetal development experiments. These future manipulations at organogenesis level may help to induce desired quality changes in offspring so that dressing percentages could be increased from present 40-50 per cent to 55-60 per cent or more.

Reproductive management

The reproductive management strategies to enhance meat production could safely be divided into two kinds:

i. Traditional management: Animal are still managed under zero- input/ migratory /tribal or extensive management system. The application of above described methods will be limited. They may indirectly help in this system by better sire availability.

ii. Modern management: Zero-grazing, stall-fed or semi-intensive, commercial goat farming system may use the above methods more and more to produce high quality germplasm. They may use variety of improved reproductive technique or reported (Wani,1996; Wani et al., 200a; Wani et al., 1998; Mufti et al., 1997; Wani and Sahani, 1981; Wani et al., 1986, 1987, 1988; Wani and Buchoo, 1991; Buchoo and Wani, 1991; Wani, 1989, Wani, 1995; Wani, 1984a and b; Wani and Sahani, 1980a).

Enhancement of fertility

Desired fertility objectives have been discussed (Wani, 2001). Increased prolificacy, early rebreeding, safe/clean parturition, set-time animal crop, elite gene introduction are some of the desired fertility enhancement goals. The constraints in achieving desired objectives in small ruminant livestock sector are the problems of inbreeding, reproductive losses (Wani, et al., 1980; 1981; 1994), pre-pubertal mating, indiscriminate or illicit mating, venereal diseases, nutritional and other stresses. These above referred factors reduce fertility status of our ruminants especially under transhumane migratory systems.

These constraints mentioned could be overcomed by adopting enhancement strategies (Wani, 2001), which include exchange of sires by tribal, Gujjar, Bakarwals and Chopans to reduce inbreeding. This could be ensured through legislature or forced castrations and introduction of sires from outside the flock. The pregnancy detection methods described (Wani, 1981) and later on used extensive in J&K under farm condition (Wani et al., 1998; Wani, 1989) needs adoption as a routine in each flock for better care of offspring and dam.

Nutritional bio-conversion

The nutritional status of dams before mating could be improved by grazing them in pastures having 4-5 cm sward height herbage, yielding 1500kgs and above of dry matter per hectare . Such pasturing shall increase ovulation. Reduced sward height pastures needs supplementation and introduction of nutritional block at least 4-5 weeks before mating. Thus proper feeding 100 days prior to mating and parturition may help in healthy and heavy weight lamb/kid crop.

The pasture nutrient analysis and its supplementation as per requirement may help to avoid losses due to such deficiencies as cobalt, iron, phosphorus etc. which effect herbage and through herbage the animal health. The sire fertility test and use of disease free semen is the foremost requirement towards increased productivity from small ruminants.

The nutrition plays important role in maintaining fertility status of animals. With decrease in land for fodder cultivation, alternate sources and technologies to utilize agricultural waste for animal food is needed. The biotechnological intervention in converting crop residues and other agri-horticulture waste into animal feed needs further research.

Conclusion

Critical gaps identified are poor-zoo-sanitary conditions and sufficient human resources. Both these area need emphasis. Development and application of accurate, sensitive and timely disease diagnosis and surveillance techniques especially in the field of animal reproduction should invite more research proposals in future. This will ensure biosafe livestocks and germplasm. Gene propogation of elite germplasm should ensure increase in per animal production. This would means more production from less livestock, thereby relieving soil, water and environmental pressures.

Endocrinological advances should aims at replacing chemical hormones with biological hormones, which could be cost-effective and without side effects. The use of fish, sheep and other ovarian extracts need further research. Follicular atresia, intrinsic GnRH system, hormone agonists and antagonists should attract more funds and research attempts. The use of herbal stimulators and medicaments in caring and prevention of reproductive diseases and incapacities needs immediate attention. More innovative approaches using these indigenously available materials need to be prioritized.

Copora lutea biosensitivity, development of conceptus, follicular dynamics, gamete safety procedures, foeto-maternal interaction, stromal cell studies would be future areas of frontline researches in animal reproduction.

A policy in view of majority 85% of our livestock production systems being small holder enterprises. Research and extension establishments and NGO in animal reproduction needs reorganization to serve as a protective armour around small producers and farm women.

Blending of advanced technology available as embryo transfer, AI, blastomer splitting, DNA micro-injections, DNA finger prints, gene mapping, DNA hybridization, ovulation control, in vitro fertilization, foetal monitoring, ultrasonography with traditional modes of livestock production system needs further innovative attempts. This should aim to further fortify our cost-efficient livestock productivity.

Hitherto untapped areas of wild and zoo animal conservation, protection and propagation, pet animal care, wildlife retrieval and referral clinics are the need of the hour. Nutritional requirement during foetal life, prenatal and neonatal life needs more emphasis along with care and management of dams during mating, pregnancy and parturition. Increased prolificacy, early rebreeding, set-time parturitions are some of the desired fertility enhancements goals. The problems of inbreeding, reproductive losses, pre-pubertal and illicit mating, nutritional stresses could be overcome by adopting fertility enhancement procedures like sire exchanges, pregnancy detection, better nutrition before mating, during pregnancy and after parturition.

Extension and information technology missions for animal reproduction are to be intensified especially in rural India. Scientist Industry interaction, technology interaction under rural sectors and linkages between research, extension, NGO and farmers needs priority by 2050.

Gross appearance of twin pregnancy
Plate VIII Intact ambiotic Sac in single ovine foetus

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Wani G.M. and Buchoo, B.A. 1991. detection of induced ovulation by laparoscopy in ewe lambs. Indian Journal of Animal reproduction, 12(1):76-77.
Wani, G.M. and Sahni, K.L. 1980. Ultrasonic pregnancy diagnosis in sheep and goats. 2nd All Indian Symposium on Animal Reproduction, Banglore, pp. 1-5.
Wani, G.m. gelderman, H. and Hahn, J. 1986. Brunstsychronization bei Ziege. Zuchthygiene, 21: 1-2.
Wani, G.M., Gelderman, H. and Hahn, J. 1990. Superovulations during early luteal phase in goats. World Review of Animal Production, 29(2):41-43.
Wani, G.M., Risam, K.S. and Nowshahri, M.A. 1988. Effect of oestrus synchronization on lambing in corriedale ewes. Indian Journal of Animal Science, 58(7):800-801.
Wani, G.M. 1989.Plasma progesterone concentrations during oestrus cycle in goats. Indian Journal of Animal Reproduction, 19(1):44-46.
Wani, G.M. Sadat Rehman and Shah Masood 2000. Higher Education and Human resource Development: Knowledge Perspectives in Current Millennium. Paper presented at the Invernational Conference on Higher Education for Human development, organized by Association of Indian Universities at New Delhi on February6 22-24, 2000.pp.239-247.
Wani, G.M., Sinha, N.K. and Khan, B.U. 1987. Oestrus synchronization with progestagens in Muzzaffarnagri ewes. Indian journal of Animal Science. 57(12)
Wani, G.M., Sinha, N.K. and Sahni, K.L, 1980. Haematic type of foetal mummification Muzaffernagri ewes. Indian Veterinary Medical Journal, 4:46-47.
Wani, G.M, Sinha, N.K. and Sahni, K.L. 1980. Note on gestational oestrus in the Muzzafernagri ewes. Indian Journal of Animal Science, 50(11):1009.
Wani, G.M and Sahni, K.L. 1980. An ultrasonic technique for detecting pregnancy in sheep and goats. Veterinary Research Bulletin, 3:35-37.
Wani, G.M., Sinha, N.k. and Sahni, K.l. 1980. Note on seasonality of breeding behaviour in Jamunapari goats, Indian Journal of Animal Science, 50:1153-55.
Wani, G.M., Sinha, N.k. and Sahni, K.L. 1981. Incidence of abortionsand stillbirths in Muzzaffarnagri and crossbred ewes. Indian Veterinary Journal, 58:558-561.
Wani, G.M., Tripathi, S.S. and Saxana, V.B. 1980. Sialoproteins in the cervicaql mucus of repeat breeding cows during oestrus. Indian Veterinary Medical Journal, 4:49-53.
Wani, N.A., Wani, G.M., Mir, M.M. and Beg, M.A. 1998. Ultrasonic pregnancy diagnosisin Gaddi goats. Small Ruminant Research, 29: 239-240.
Wani, G.M. (2002b). Advances in animal Reproduction in: Proceedings of NationalSeminar on "Recent Research Trends in Life Sciences" , (August 26-28, 2002) organized by PG Department of Envirornmental Science and Centre of Research for Development, The University of Kashmir, Srinagar- 190006.

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G.M. Wani
Sher-e-Kashmir University of Agricultural Sciences and Technology of Kashmir Post Box 461, GPO, Srinagar, Kashmir - 190001
Email: Wanimohyuddin@yahoo.com