Lecture 5.induced breeding mcm
- 2. 1. Basis of Induced breeding 2. Induced reproduction 3. Historical background 4. The fish Pituitary 5. Hormones of pituitary related with development of gonads and spawning 6. Induced breeding in carps by hypophysation a. Brood stock management b. Collection of pituitary gland c. Preservation and storage of gland d. Prep and preservation of extract e. Determination of pituitary dosage f. Ponds for induced breeding and breeding happas g. Injections of pituitary extract to breeders h. Spawning and fertilization i. Stripping or artificial insemination
- 3. I) Basis for induced breeding • Reproduction in fish-regulated by environmental stimuli. Environmental stimuli Brain hypothalamus Pituitary gland Gonadotropic hormones Sex steroidal hormones By sensory receptors stimulates Releasing hormones Gonads Regulation of secondary sexual chtrs, nuptial coloration, breeding behavior I) Provision of appropriate environmental stimuli II) Administration of hormones for maturation and release of gonads
- 4. II) Induced reproduction • Under culture conditions, the required environmental factors may not be available or persist for sufficient length of time for spontaneous maturation to occur. • This led to Induced reproduction or Hypophysation technique, where pituitary homogenates are injected into fish. • This stimulates gonadotropin surge, by-passing environmental variables. • Inducement of spawning in fishes that do not ordinarily breed under conditions of confinement.
- 5. III) Historical background • In India, first attempt to induce spawn of carps by administering mammalian pituitary extract by made by Hamid Khan (1938) but without success. • Chaudhuri and Alikunhi (1957) succeeded in making Labeo rohita, C. mrigala, C. reba, L. bata and Puntius sarana to breed by injecting them with fish pituitary extract. • Chaudhuri (1959) successfully carried out cross breeding of 12 different sps by artificial insemination.
- 6. III) Fish pituitary • Pituitary gland or hypophysis-situated on ventral side of brain in concavity of cranium called sella turcicia and covered by duramater. Pituitary has 2 parts: • Neurohypophysis (nervous part) • Adenophypophysis (glandular region) Pro-adenohypophysis (ant lobe, ant glandular region) meso-adenohypophysis (middle lobe, middle glandular region) Meta-adenohypophysis (posterior glandular region)
- 7. IV) Hormones of pituitary related with development of gonads and spawning Adenohypophyseal hormones: • GH • Somatotropic hormone • TSH • GTH • ACTH • Prolactin • Melanocyte stimulating hormone Neurohypophyseal hormones: • Oxytocin • Vassopressin • ADH
- 8. • GTH-play imp role in regulating development of gonads and spawning. • Gonads release: Androgen, estrogen and progesterone. • Further environmental factors like temp, rain influence the gonads and other endocrine system
- 9. VI) Induced breeding in carps by hypophysation 1. Brood stock Management • The brood pond • Characteristic of ripe brood fish and their selection for stocking • Stocking rate • Caring the brooders • Manuring and maintenance of water quality in brood-stocking ponds • Supplementary feeding • Rate of feeding
- 10. 2. Collection of pituitary gland 3. Preservation and storage of gland 4.Preparation and preservation of pituitary extract 5. Determination of Pituitary dosage 6.Ponds for induced breeding and breeding Happas 7. Injections of pituitary extract to the breeders 8.Spawning and fertilization 9.Stripping or artificial insemination
- 11. The primary requirement of carp breeding is an adequate stock of good breeders. The excellent brood fish culture is the foremost essential material basis and key factor for successful artificial propagation. Brood stock: Group of mature individuals used in aquaculture for breeding purposes a. The brood Pond Ponds of moderate size (0.5 to 1 ha) & depth (1.5- 2.0 m), preferably rectangular, flat bottom. Moderate water temp (27-32°C). Exposure of 6-8 hrs light in a day at least for 2-3 months. Inductive for rapid gonadal growth of carps
- 12. b. Characteristics of ripe brood fish and their selection for stocking • Smooth pectoral fin. •Abdomen is soft and bulging. •Pinkish vent. •The vent of fully ripe female is slightly elevated. •Rough pectoral fin. • Abdomen is smooth and not bulging. •Vent not pinkish. •Whitish secretion called ‘Milt’ come out on pressing the vent. Spawner Milter
- 14. • Selection of female is very important. • A simple catheter ca be used to find out if the fish is having eggs or not and shape and size of eggs suggest that the female is quite ripe. • Grass carp- red colored eggs • silver carp- blue • Color varies with various ecological zones and also with the age of female. Healthy, disease free breeders of catla, rohu, mrigal, silver carp, grass carp and common carp of 2-4 yrs and 1-5 kgs are normally selected
- 15. c. Stocking rate (sex ratio) • Sex composition of breeders is normally maintained as 1:1. • Males and females are kept separately in brood pond so that it increases the affinity of mating of the pair during breeding experiment. Stocking is done 1500-2000 kg/ha with species composition of • 40 surface feeders (Catla 30-20 and silver carp 10-20)+ • 30 column feeders (rohu 20 and grass carp 10)+ • 30 bottom feeders (mrigal 20 and common carp 10).
- 16. d. Caring the brooders • Fortnightly or weekly seining- for exercise, general health checkup and gonadal maturation. • Frequent replacing breeders from one pond to another- for food utilization. • In case of infection- bath in 10 ppm KMno4 e. Manuring and maintenance of water quality in brood-stocking ponds •Initial manuring of brood pond- 3 weeks prior to stocking with cattle dung or compost manure. •7 days Before fertilization- lime treatment •1/8th to 1/4th water change frequently.
- 17. Seining
- 18. f. Supplementary feeding • Supplementary feed given daily. • a complete feed is a proportionate vegetable and animal protein, carbohydrate and fat. Mixture is prepared from: half cooked rice and pulse (1:1, 50%)+ oil cake (20%)+ cattle dung (20%)+ Fish meal/silk worm pupae/ cooked slaughter house refuse (10%). (Rohu, catla, mrigal, common carp) 1-2% diet of BW before spawning and 3-4% diet after spawning. No feed is given for one week before actual breeding. Grass carp-fresh aquatic vegetation- Hydrilla, Vallisneria, Utricularia. Silver carp- Spirulina, Oscillotoria, Chlorella
- 19. g. Feeding rate • Complete stoppage of feed before spawning: for proper defattening and physical tuning of breeder. • Excess feeding may result in heavy accumulation of mesenteric fat inhibiting the process of smooth ovulation.
- 20. Collection of pituitary gland IMC-serve as Donor fishes. However, Common carp is most preferable because of its easy raising. The glands from fresh and fully ripe donors are used. Suitable period: breeding season.
- 21. Preservation and storage of glands Brazilian Method Acetone dried method Methods
- 22. Brazilian method of preservation in Abs alcohol Glands are removed into absolute alcohol Transferred to air- tight phials After 24 hrs- Transferred to other air- tight phials containing fresh alcohol Phials are stored in dark air tight bottle containing ab Al at room temp in desssicator containing anhydrous CaCl2 in cool place or kept inside refrigerator • Pituitary banks are maintained. • Since pituitary hormones are water soluble, hence Brazilian method of preservation in absolute alcohol is suitable. Gland remain effective for 5 years
- 23. Acetone-dried method Glands are removed into acetone and put into refrigerator (acetone changed 2- 3 times) After 36 hrs of storage, the phials are taken out and kept in room temp. Glands are removed and kept on filter paper to dry Glands are stored inside air-tight sterile phials and kept inside dessicator containg anhydrous CaCl2 which is then placed inside a refrigerator Acetone dried glands retain potency for 6 months to 10 years
- 24. Preparation and preservation of pituitary extract • Gland register- serially labeled phials, details of phial no. no. and wt of gland and donor sp’s Extract: Required quantity of gland → homogenized in tissue homogenizer →macerated in Physiological saline (0.3% NaCl) Intraperitoneal injection: no need of centrifugation Intramuscular injection: suspension if centrifuged at 1000 rpm for 5 mins
- 25. Preservation Glycerine preservation method 1) 1/3 of total volume of extract is made up by adding DW. 2) Solution is kept in Refrigerator for 24 hrs 3) Glycerine is added to make up 2/3 of volume (glycerine : water, 2:1) 4) Suspension again stored in refrigerator for 24 hrs and afterwards centrifuged. 5) Supernatent is filtered and stored in air tight bottles inside refrigerator 6) Sealed in ampoules (labeled giving details of hormones conc., date of prep)
- 26. Dose Calculation of doses and dilution: I. 1-3 whole glands from ripe donor to nearly same wt as that of recipient fish II. For IMC 2 doses: 2-3 mg/kg BW and 5-8 mg/kg/BW III. Exotic carps: 3-4 mg/kg/BW, 7-10 mg/kg BW Weight of spawner Provocative dose First injection Final dose Second injection 0.5- 1 kg 0.5 ml 1 ml 1- 2 kg 0.75 ml 1.5 ml Above 2 kg 1 ml 2 ml
- 27. Ponds for induced breeding and the breeding hapas • Brood stock • Brood stock ponds (No blooms, predaceous insects & fishes. • In these ponds only breeding hapas are fixed, inside which the injected brood fishes of both sexes are released to spawn. BREEDING HAPA Box shaped cloth container, like an inverted net with a cover. (fine-meshed). Hapa should have fine thin cloth such that ovarian eggs do not pass out when released by fish
- 29. Dimensions of hapas a) 4 kg fish: 3.6X1.8X1.0 m b) 1.5-4 kg: 2.4X1.2X1.0 m c) Under 1.5 kg: 1.8X1.0X1.0 m It has an opening on wider side It is fixed with the help of bamboo poles When fixed, bottom of hapa should not touch muddy pond bottom at least 15-20 cm of hapa should remain above water level.
- 30. Injection of pituitary extracts to breeders • Hypodermic syringe (2ml) with 0.1 ml graduations is used. • Thickness and length of needle depends upon size of recipient fish. • Generally BDH no. 22 needle- 1-3 kg wt no. 19 needle – larger fishes no. 24 needle – smaller fishes Mode of injection: Intraperitoneal or intramuscular
- 31. • Intraperitoneal: injected at the base of pelvic fin. Fish may die due to damage of internal organ. Practiced in US. • Tip of needle is inserted under a scale and is pushed through abdominal wall into body cavity and then directed parallel to ventral surface
- 32. Intramuscular: popular in Brazil and India. Administered in caudal peduncle. While injecting the needle is first inserted under a scale parallel to body and muscle is pierced through quickly at 45°, so that no damage is done to scale
- 33. • In case of single injection, this could be done in late in the evening and spawning could be over by early hours of morning. • Both injected males and females are kept together in breeding hapa for spawning. • In breeding hapa for C.carpio, hanging fibrous roots of water hyacinth are kept because eggs of common carp are sticky.
- 34. Spawning and fertilization • After few hrs of injection, the brood fishes spawn naturally in breeding hapa. • Sex play is observed between male and female after final injection. • IMC- spawn within 6-8 hrs • In case of Chinese carps, stripping is done for better fertilization
- 35. Stripping or artificial insemination • Eggs and milt are forcible extruded out by applying pressure on the belly of read to breed breeders kept in hapas. Methods Dry method and Wet method Dry method: After 3-4 hr of second injection, abdomen is pressed and eggs and milt are collected in tray and mixed with spoon. After few mins fertilized eggs are transferred into breeding hapa. Wet method: physiological saline is used to receive milt first and eggs later
- 37. Thank you