European Journal of Experimental Biology Open Access

Keywords

Dietary fat, ovary, histology, Channa striatus, Saul

Introduction

The striped murrel, Channa striatus, is a highly priced freshwater fish of the Indian sub-continent and south-east Asia. They are in great demand as food fish due to their good flavour [1], few spines, medicinal importance [2–6] and air-breathing nature [4] that facilitate high density culture and easy transportation in live state to the retail markets. Murrel adapts well to low oxygen condition and hence can be cultured in high stocking densities. The culture of C. striatus is widely popular in Thailand and on limited scale in India, Philippines and Taiwan [4, 7-9] due to non-availability of seed. The striped murrel breeds in ponds and rivers a little prior to or with the onset of monsoons [7, 10, 12] and their spawning season extends to the last monsoons [13]. It is a batchs pawner and breeds two or three times in a season, the breeding season extends from February–March to October–November and lay floating egg in a nest made up of leafy vegetables [13]. Both parents guard the eggs and fry [11]. Parmeshwaran and Murugesan [14] attempted captive breeding of striped murrel in India when they injected carppituitary glands to both male and female with varying success. Marimuthu et al. [15-17] successfully carried out spawning of striped murrel in a net enclosure fixed in a pond and in cement tanks using Ovatide (sGnRH).

The maturation in this fish starts with the development of ovarian follicle as an aggregate of ova and epithelial cells. These follicles start as oogonia, or mother cells, which are regularly generated in the germinal epithelium. The oogonia are composed of cells which forms and produce oocytes. The epithelial cells grow as the ovum grows and are distinctly detached from each other by a gradually thickening hyaline capsule. These are main source for nourishing the ovum and secreting the yolk. In many species, several generations of ovum may be recorded in different developmental stages at a time.

The basic information on dietary requirements of nutrient components such as protein and energy is a prerequisite for the formulation of balanced diet for the fishes particularly during maturation season. The carnivorous fishes particularly diets rich in proteins and lipids at the time of attaining maturity which need to be met out from artificial diets when cultured in captive conditions. India has huge potential for the production of cheaper plant sources e.g. de-oiled cakes like linseed oil cake etc. (rich in essential fatty acid,, omega-3 HUFA) which can be utilized as source of lipid in carnivorous fish nutrition not only for growth purposes but also for gonadal maturation through dietary manipulations. Recycling of agro-based by-products, like mustard oil cake, linseed oil cake etc. can be used in place of animal oils as source of lipid and EFA. Thus, the fatty acid composition of these plant origin are good source of HUFA which can be utilized for carnivore fishes nutrition as a supplement. in place of animal lipid source.. Sarowar et al. [18] have studied the impacts of different diets on growth and survival of C. striatus grow-outs. Influence of dietary lipid/protein ratio requirement has been studied in C. striatus [19]. The present study was taken up to evaluate the impact of dietary lipids on the ovarian growth by utilizing various agro-based dietary lipid ingredients by the striped murrel, C. striatus. The parameter for study were the estimation of fecundity, maturation condition of ovary and histology.

Materials and Methods

Procurement of Test Fish

Wild striped murrels, C. striatus (Avg. weight 388–407 g, Photo-1) were collected from different sites of Unnao and Barabanki districts of Uttar Pradesh, India and were stocked in ponds at NBFGR farm facility 3 months prior to experiments. They were fed with laboratory made feed(Table-1) ad libitum during acclimation period.

Feed Preparation and Feeding

Six type of feeds were formulated having similar feed ingredients in same quantities as given in Table-1 excepting different source of fat namely low level of highly unsaturated fatty acid (L3HUF, F1); high level of highly unsaturated fatty acid (H3HUF, F2); mustard oil (MUSOL, F3); linseed oil (LINOL, F4); mixed oil (MIXOL, F5); saturated fat (SATOL, F6) and a control (NATFO, F7) comprising of natural food stuffs (Table 2). F1, contained 0.5% n-3 fatty acid and 7.5% saturated oil; F2, 1.0% n-3 fatty acid and 7.0% saturated oil; F3, 8.0% mustard oil; F4, 8.0% linseed oil; F5, 4.0% mustard oil and 4% linseed oil; F6, 8% saturated oils. (Table-2). In order to evaluate the effect of different oil sources on the ovary of C. striatus, the experiment was conducted in indoor condition in 14 (7 types of feed, 2 replicates) rectangular plastic pools of 1200litre capacity, each filled-up with 800 litre borewell water stocked with 10 juveniles having an initial average weight 273.6+90g to 325.4+41g. The tanks were provided aeration from a portable aerator round the clock. During the experiment, the fishes were fed twice a day at 10:00 and 17:00 hours ad libitum per day. Rearing tanks were cleaned every second day and about half of the water was replaced with fresh bore-well water to reduce the nitrogenous waste accumulated as debris and faecal matters.

Histological study

After 12-weeks of feeding trials with seven feed combinations (Table-2), the fishes were sacrificed. The ovary from control(F7,NATFO) and experimental fishes (feed with different fats (F1 to F6) were excised and fixed in 4 % formaldehyde and processed by standard histological techniques ([20] i.e., kept in aqueous Bouin's fluid for 24-hr and washed for 8-hr in running tap water. The organs were routinely processed (dehydrated in ethanol series, embedded in paraffin, serially sectioned at 6μ). Sections of the ovary tissue were stained with Haematoxylin and Eosin (HE). Histological slides were observed under microscope (Labomed, Model: Digi 2) for assessment of the maturity condition.

Results and Discussion

The experimental feeds had an impact on ovary condition and the fecundity of C. striatus. The size of ovary with different types of feeds and the fecundity is given in Figure 1-7 and Table-3. The fecundity was recorded as 53796+3452 with F-1, 66576+2756 with F-2, 10893+4531 with F-3, 52497+2587 with F-4, 9440+5276 with F-5, 4720+2329 with F-6 and 7703+2368 with F-7 . The H/E sections of ovary of control fish fed with natural feedstuffs (NATFO, F7) showed normal architecture of ovary tissue with normal follicles and granulation (Figure 8). F-1, fed fishes showing normal follicles with granulation (Figure 9); F-2, showing normal follicles with dense granulation(Figure 10); F-3, fed fishes showing normal follicles with granulation and spacing between follicles(Figure 11); F-4, showing huge numbers of follicles with granulation(Figure 12); F-5, showing huge numbers of follicles with granulation and different stages of ovary(Figure 13) and F-6, fed fishes showing huge numbers of follicles with granulation and spaces(Figure 14). Ovarian tissues of C. striatus fed with natural food (NATFO, F7) showing normal follicles. The results exhibited comparatively massive changes in the ovary tissue by having larger number of follicles with granulation and with linseed oil and mixed oil enriched feeds. The results also exhibited increased granulation and numbers of follicles and better effects towards fecundity, granulation etc., after addition of various dietary fats in the feed in comparison to control.

In all the treatments, the most of the cellular features were the presence of well-organized architecture of the ovary and cellular structures which indicated that all types of oils tested in the study were not harmful rather beneficial to striped murrel, C. striatus at 8% supplementation. Hence these fat ingredients may be used either alone or in combination in the diet of this species which did not create significant changes in ovary structure but also immensely improved fecundity.

Figure-8 Ovary of C. striatus fed with Natural feed (NATFO, F7, control) showing normal follicles(H/E 40X)

Figure-9 Ovary of C. striatus fed with low unsaturated fatty acid (L3HUF) ( F1) showing normal follicles with granulation (H/E 40X)

Figure-10 Ovary of C. striatus fed with high unsaturated fatty acid (H3HUF) ( F2) showing normal follicles with dense granulation (H/E 40X)

Figure-11 Ovary of C. striatus fed fishes with F-3 (MUSOL) showing normal follicles with granulation and spacing between follicles (H/E 40X)

Figure-12 Ovary of C. striatus fed with linseed oil (LINOL, F4) showing huge nos. of follicles with granulation (H/E 40X)

Figure-13 Ovary of C. striatus fed with mixed oil (MIXOL, F5) showing huge nos. of follicles with granulation and different stages of ovary (H/E 40X)

Figure-14 Ovary of C. striatus fed with saturated oil (SATOL, F6) F-6, showing huge nos. of follicles with granulation and spaces (H/E 40X)

A few workers have attempted captive breeding of striped murrels with varying success. Alikunhi [11] and Parameshwaran and Murugesan [14] attempted breeding of this species by using pituitary gland. Haniffa et al. [21], Selvaraj and Francis [22] induced bred this fish using sGnRH and HCG respectively. Haniffa et al. [23]also made a comparative study on induced breeding of this fish using pituitary gland, HCG, LHRH-a & Pimozide andovaprim. Marimuthu et al. [15] used Ovatide for breeding in hapa inside the pond. In teleosts, ovarian development and the ultimate production of mature eggs is a highly complex process, timed and modulated by various environmental and endocrine pathways such that young’s are produced only at times when fry survival is optimal – usually when food availability is at its highest. Ovarian development in teleosts is broadly divided into seven distinct developmental stages based upon the biochemical properties and histological morphology of the nucleus, cytoplasm and follicular layer: oogonial proliferation, oogenesis, folliculogenesis, cortical alveolar formation, vitellogenesis, final maturation and ovulation [24,25]. Even within an individual oocyte however, there are likely to be periods in which these distinct phases overlap [26].Many authors observed the effect of pesticide on ovary. Also, Kumar and Pant [27] found that 2-4 month exposure of an Indian teleost to lead caused disappearance of oocytes in the ovaries. In addition, Magar and Bias [28] investigated histopathological effects of sublethal concentration of insecticide which widely used in agriculture in the ovary of fresh water teleost, Channa punctatus. They observed complete loss of normal configuration of ovary, necrosis, elongated ovarian follicles, and fragmented ova with abnormal shapes.

Dayal and co-workers [29] have reported influence of different sources of dietary lipid on the growth, feed efficiency and survival of snakehead C.striatus grow-out. The ovarian morphology in the present study resembles basically that described in the bass, Ecetrarcuslabrax [30] and that of bullhead catfish; Ictalorusnebulosus[31]. A characteristic Balbiani’s body as that described by Beams and Kessel[32] and Mayer etal.[30] was clearly demonstrated in medium-sized follicles. Exogenous factors known to play regulatory roles in the reproductive physiology of tilapia include light intensity [33-36], water temperature [37-39], lunar cycles [40], maternal size and age[41-43], food ration size [44-46],dietary protein level [47-53] and dietary lipid level [54,55]. In other teleports that spawn pelagic eggs, especially marine perciformes, full-grown oocytes contain oil droplets which occupy up to half or more of ooplasm volume [56. These oil droplets coalesce into one or two large oil globules during maturation (see later discussion). In contrast with the Vg-associated lipids, oil globules contain mainly neutral lipids such as triglycerides and wax or steryl esters. These neutral lipids are rich in monoun saturated fatty acids that, in fishes, preferentially serve as metabolic energy reserves[57]. Thus, it appears that vitellogenin transports mainly structural lipids and essential fatty acids into growing oocytes to support embryo growth, whereas neutral lipids are taken up by other means and stored as isolated oil inclusions for meeting the energy demands during embryogenesis. In overall, this study showed that C. striatus performed optimally on diets containing LINOL, MUSOL, H3HUF and MIXOL with respect to gonadal maturation. It can be deduced that out of six types of fats used in the present study, the essential fatty acid (H3HUF, F2) is considered best as a feed substitute in the artificial diets. The other fats have shown very mild to moderate level of changes in the ovarian tissue at 8% addition in the diet in a 12-week trial. Therefore, these fats may be used in combination with other fats to cut down the feed price and without effecting the survival and growth of C. striatus. Therefore, it is recommended that various fats used in the present study in to the diet of C. striatus could be used for improved growth performance and better nutrient utilization in the development of ovarian tissue. However, further study aimed at increasing the concentration of the long chain polyunsaturated fatty acid in the fillet.

Conclusion

The observations, in the present study, suggest that manipulation with different dietary fat sources in the feed has direct relation with fecundity and cellular level modifications in the ovary of C. striatus brood fish.

Acknowledgements

Authors are highly grateful to the Director, NBFGR, Lucknow for providing facilities to conduct this research work.

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