Breeding and Spawning of Fishes Role of Endocrine Gland PDF
Breeding and Spawning of Fishes Role of Endocrine Gland PDF
Breeding and Spawning of Fishes Role of Endocrine Gland PDF
E-ISSN: 2347-5129
P-ISSN: 2394-0506
(ICV-Poland) Impact Value: 5.62 Breeding and spawning of fishes: Role of endocrine
(GIF) Impact Factor: 0.549
IJFAS 2018; 6(4): 472-478 gland
© 2018 IJFAS
www.fisheriesjournal.com
Received: 27-05-2018 Shanthanagouda AH and Sachin O Khairnar
Accepted: 28-06-2018
Shanthanagouda AH Abstract
Department of Aquatic The fish pituitary is an endocrine gland of dual origin found on the ventral side of mid brain attached to it
Environment, College of by means of stalk. The pituitary gland produces and stores gonadotropin hormones (GtH), which play a
Fisheries Guru Angad Dev decisive role in ovulation and spermiation. Hypophysation is a technique whereby ripe fish brooders are
Veterinary and Animal Sciences stimulated by pituitary hormone introduction to spawn in captive condition. Injected pituitary gland
University, Ludhiana, Punjab, extract bypasses the brain-pituitary link, acting directly on the ovaries and testes, providing the surge in
India blood GtH levels that normally precede spawning. This technique has been developed for the production
of good quality spawn of certain fish species in captivity by manipulating its reproduction with control
Sachin O Khairnar timing and synchrony of egg production, while natural spawn collection yields some undesirable wild
Department of Aquaculture, species and is not profitable for culture. This paper explains general organisation and morphology of
College of Fisheries Guru Angad
pituitary gland and it’s the role in induce breeding of fishes.
Dev Veterinary and Animal
Sciences University, Ludhiana,
Punjab, India Keywords: Pituitary gland, gonadotropin, breeding, spawning, teleost
Introduction
Fish pituitary
The general organization and morphology of the pituitary gland has been described in a
number of teleost fishes. The pituitary is a major endocrine gland, a pea-sized body attached to
the base of the brain that is important in controlling growth, development and the functioning
of the other endocrine glands. It is situated on the ventral side of the brain. It is a small, soft,
whitish body whose size and shape vary with species. It is more or less round in carps; oval in
catla and rohu and pear-shaped in mrigal. It is located in a concave cavity called sella turcica
and enclosed by a membrane called durameter, which is attached to the brain by an
infundibular stalk (Fig. 1) [1]. It produces gonadotropic hormones (GtH) that are essential for
the maturation and spawning of fish. These gonadotropins include Follicle Stimulating
Hormone (FSH) and Luteinizing Hormone (LH) which are secreted and correlated with the
cycles of gonadal maturity.
As in higher vertebrates the pituitary or hypophysis of fishes consists of two distinct tissues
capable of producing hormones-adenohypophysis (anterior pituitary) which is derived from
the embryonic pouch, Rathke's pouch arising from the roof of the buccal cavity as an outward
evagination and the neurohypophysis (posterior pituitary) which originates from the downward
evagination from floor to third ventricle [2]. The adenohypophysis regulates gonadal functions
in fishes and is the site of synthesis, storage and release into circulation of several peptide and
hormones. The adenohypophysis is divided into the rostral (pro-adenohypophysis) and the
proximal (meso-adenohypophysis) pars distalis, and the pars intermedia (meta-
adenohypophysis) [3, 4].
The neurosecretory nuclei of the hypothalamus produce neurohormones control pituitary
hormone secretion, thus constituting the hypothalamo-hypophysial axis. The neurohypophysial
hormones are structurally and functionally related peptide hormone. Their main
representatives are oxytocin and vasopressin. The neurohypophysis interdigitates into all areas
Correspondence of adenohypophysis but major interdigitations are found in pars intermedia. The
Sachin O Khairnar neurosecretory materials from the neurons of preoptic nucleus are transported through the
Department of Aquaculture,
College of Fisheries Guru Angad
axons and stored in the axonal terminals ending at the junction between neurohypophysis and
Dev Veterinary and Animal adenohypophysis which is called as neuroadeno interface. This interface is highly vascular and
Sciences University, Ludhiana, it is the centre for storage and release of hormones.
Punjab, India
~ 472 ~
International Journal of Fisheries and Aquatic Studies
Fig 1: Fish pituitary gland and gonadotropins production. Note: GnRH-Gonadotropin Releasing Hormone, GtH-Gonadotropins. Blood enters
from the hypophyseal artery and exits to through the hypophyseal vein. Gonadotropins produced by gonadotropic cells stimulated by GnRH
from the hypothalamic nerve cells, is collected along the way.Figure adapted from Brian Harvey and Carolsfeld (and modified).
Biosynthesis of estrogen The entire process takes place in two different cells hence
In the estrogen biosynthesis pathway, a series of androgens referred as two cell hypothesis. The protein Cyp19a1 is the
(steroids) are formed in theca and leydig cells of females and only enzyme which plays a key role in the sex differentiation
[18, 19]
males respectively with the help of a number of enzymes . In addition, FSH is an important gonadotropin, since it
under the stimulation of LH. The androgens include stimulates the ovarian development and testicular
testosterone and androstenedione formed are then get diffused spermatogenesis during gametogenesis; and LH is mainly
into the granulosa cells in females and sertoli cells in males, constitutes for final gamete maturation (ovulation or
after which the granulosa/sertoli cells convert the final spermiation). In spite of many studies on FSH and LH in
steroids of the androgens (androstenedione or testosterone) to teleosts, their functions in reproduction still remain poorly
estrogens by an enzyme called P450 aromatase (P450 defined. This could be because of lack of studies on genetic
cytochrome Cyp19a1) under the influence of FSH (Fig. 2). approaches in adult teleosts [20].
~ 473 ~
International Journal of Fisheries and Aquatic Studies
Fig 2: Schematic diagram showing two-cell hypothesis. Note: StAR-Steroid Acute Regulatory Protein, P450scc-cholesterol side-chain cleavage,
LH-Luteinizing Hormone, LHR-Luteinizing Hormone Receptor, FSH-Follicle Stimulating Hormone, FSHR-Follicle Stimulating Hormone
Receptor. Adapted from: Lubzens et al. 2010 [21]; Schulz et al. 2010 [22] (and modified).
The LH produced by the pituitary gland binds with the LHR preservation of pituitary glands, preparation of pituitary
(LH receptors) on theca and leydig cells and stimulate extract and site, dose, method and time of injection [28].
androgen synthesis by the cholesterol which has entered with
the help of Star protein and in turn cause luteinisation in Identification of brooders for breeding purpose
females and promotes the production of testosterone in males. 1. Female brooder characters: Bulging belly, swollen pink
Whereas, FSH stimulates granulosa and sertoli cells for vent and eggs oozes out on pressing the belly
estrogen synthesis by binding to FSHR (FSH receptors) 2. Male brooder characters: White coloured liquid (Milt)
causing growth and maturation of ovarian follicles in females oozes out when belly pressed, Pectoral fins rough to
and spermatogenesis in the testes of males. However, the touch
process is not species specific; but, there is a great variability
in its effectiveness in different species. Brooders ratio
For every one female two males (weight of female=weight of
History of induce breeding in fishes males) are preferred for a successful. The 1:2 ratio is followed
The first attempt of induced breeding by using pituitary since males in carps are generally smaller to females and to
extract was done by B.A. Houssay (1930) in Argentina on a ensure complete fertilization of eggs produced by the female
viviparous fish [23]. He was successful in obtaining premature it is recommended to use two males per female and at the
birth of young fish. Subsequently, based on the lines of ideal temperature between 24-31 oC for breeding.
Houssay, Von Ihering and his team of Brazil, in 1934,
successfully induced bred a catfish with pituitary hormones Induced breeding using pituitary glands
and hence credit for the present day concept of induced Pituitary Gland Extract (PGE) of same or closely related
breeding of fish goes to Brazilians [24]. In India, the first species are normally injected. However, commercially
attempt on induced breeding was conducted by Hameed Khan available synthetic hormones e.g. Ovaprim and Ovatide are
in 1938 on mrigal, Cirrhinus mrigala by administration of also used for breeding purposes, but many of the farmers in
mammalian pituitary gland but eggs were not fertilized [25]. Punjab import the glands from West Bengal and the extract is
Later in 1955, H.L. Chaudhari succeeded in induced being injected during breeding and farmers are gaining better
spawning on small carp species, Esomus danricus by results.
administering the intra-peritoneal injection of catla pituitary
gland. The first success in induced breeding of Indian Major Selection of fish for pituitary gland
Carps (L. rohita and C. mrigala) was in the year 1957 by H.L For the purpose of induced breeding using pituitary gland,
Chaudhari and K. H. Alikunhi at Central Inland Fisheries mature fish (freshly killed or ice preserved) of either sex are
Research Institute, substation, Cuttack (Orissa) [26, 27]. Since selected of the same species (homoplastic) and or closely
then, the technique has been standardized and refined for the related but different species (heteroplastic). The fish which is
large-scale production of fish seed. used for collection of pituitary gland is referred as ‘donor
fish’. Among the carps in India, common carp is the most
Hypophysation in fishes commonly preferred donor fish due to its availability of
In order for the pituitary extract to be effective, donor fish mature fish round the year.
should be sexually mature whether male or female. Also, the
extraction of pituitary gland is preferred to be close to or Pituitary glands collection
within the spawning season. During breeding season (June- Fish pituitary gland was collected by dissecting and removing
August) mature brooders are selected from the brood stock a portion of the scalp as shown (Fig. 3) and the following
pond and injected with hormone injection for induced steps were followed [29].
breeding/hypophysation. The following process consists of The brain case (cranium) was removed obliquely using a
collection and identification of brooders, collection and butcher’s knife and the skull was removed.
~ 474 ~
International Journal of Fisheries and Aquatic Studies
The brain was exposed by removing blood, grey matter By taking care, the membrane was removed which covers
and fatty substance with foreceps and cotton. the gland and the fluid using cotton and then pituitary
The anterior end was then detached, i.e. optic gland was completely exposed.
and olfactory nerves of the brain using foreceps and Then carefully collected the pituitary gland by inserting
carefully lifted the entire brain up without disturbing the the blunt end of the forceps and transferred to a vial
pituitary gland and laid it back to expose the pituitary containing preservative, i.e. either absolute alcohol or
under the membrane. acetone.
3C. Make incisions behind the skull 3D. Remove gills for convenience
Preservation of pituitary glands place to avoid the sunlight. To increase the shelf life of the
There are three methods for preservation of fish pituitary glands it is advised to refrigerate. During washing and
glands according the facilities available with the farmers, storage, absolute alcohol dehydrates the glands, dissolves fats
which are as described below. and preserves it for a longer time (up to 2 years). Vials are
labelled for information includes date of collection, fish name,
A. Preservation in absolute alcohol maturity stage and other details if any.
Immediately after collection, the glands are washed and
transferred to fresh absolute alcohol in amber coloured glass B. Preservation in acetone
vials. Every after 24 hrs, pituitary glands are washed with Upon collection, glands are transferred to ice-chilled acetone
absolute alcohol and stored at room temperature in a dark and stored in a refrigerator for 2-3 days. During refrigeration,
~ 475 ~
International Journal of Fisheries and Aquatic Studies
acetone is changed 2-3 times and stored in a refrigerator. Like blotting paper. If acetone-dried glands are used, they can
absolute alcohol, acetone also has dehydrating and defattening directly be taken for maceration. Use one-third of the medium
effect and vials are labelled as explained above. for homogenization and the remaining two-thirds for rinsing
the homogenizer and the glass rod. In order to prepare the
C. Immediate freezing extract, initially glands are weighed and homogenized in
Immediately upon collection the glands are kept in freezer. distilled water or 0.3% physiological saline and then the
suspension is centrifuged at 5000rpm for 5-10 minutes.
Preparation of pituitary gland extract Maintain a dilution rate of 20-30 mg of pituitary in 1.0 mL of
Most often, pituitary gland extract (PGE) is prepared just the medium. After transferring the contents from the tissue
before the injection. Based on the weight of the brooders to be homogenizer to centrifuge tubes, centrifuge the extract at
injected, total quantity of glands required are calculated and 5000 rpm for 5-10 minutes (at the farm site hand centrifuge
used for extract preparation. About 10% extra glands could be used (Fig. 4D). Following which, supernatant is
are taken to compensate the wastage during the extraction collected using syringes for induce breeding.
procedure. The preserved glands are taken out and dried using
Site of injection ventral fin base, (ii) Below the dorsal fin base and above the
Pituitary gland extract injection is performed either lateral line, (iii) On the caudal peduncle just above the lateral
intramuscularly or intraperitonially. Intramuscular injection is line and (iv) Below the pectoral fin. While injecting care must
performed at different locations of the fish include (i) near the be taken not to prick through the scale (Fig. 5).
5A. Near the ventral fin base 5B. Below the dorsal fin base and above the lateral line
5C. On the caudal peduncle and above the lateral line 5D. Below the pectoral fin
Fig 5: Sites of injection on brooder fish for efficient breeding
~ 476 ~
International Journal of Fisheries and Aquatic Studies
~ 477 ~
International Journal of Fisheries and Aquatic Studies
~ 478 ~