Immunostimulatory and antifertility effects of neem (Azadirachta indica) leaf extract on common carp (Cyprinus carpio Linnaeu)

Common carpis one of the most cultivated carp species throughout the world and originally native to temperate region of Asia, especially China. It is the most common cyprinid species and constitutes an important part of inland fish production and most commonly transplanted species of fish in the world. It is very much favored for cultivation in ponds either alone or in combination with other fishes, because of its growth rate, omnivorous habit, breeding in confined waters, hardy nature and easy adaptation to artificial feeds. However, in recent times, common carp is normally avoided in polyculture due to its frequent and prolific breeding under suitable conditions and it tolerates high temperature and turbidity and also established in most of natural inland waters, including rivers, lakes, streams, canals, wetlands and even village ponds of the country (Khan et al., 2016) and matures at the early age and in small sizes.
        
Moreover, application of antibiotics and other chemicals in an aquaculture system are quite expensive, besides being associated with negative impacts including development of antibiotic resistance, accumulation of residue in tissue/environment and immune-suppression, which not only affect the non-target species but also pose serious health hazards to the consumers (FAO/OIE/WHO 2006). Therefore, researches have diverted to find an alternative for antibiotic. In this regard several promising results were observed by the use of plants as dietary supplementation in fish. Meanwhile, neem had proved its immunostimulatory and antifertility activity in various fish (Obaroh and Nzeh, 2014; Kareem et al., 2015). But, in common carp this information is lacking and it is the need of industry.
 
In the current aquaculture practices, introduction of common carp is controversial, because of its prolific breeding and over consumption of feed by the offspring of common carp in polyculture practices, which leads to under productivity and economic losses to the farmers. To overcome this, several plant materials had been reported to possess properties that prevent conception when administered orally. Most of the herbs were observed to interfere with normal sperm production or motility and act as anti-implantation, abortifacient, anti-zygotic, anti-fertility, anti-androgenic agents, which suppress or inhibit reproduction (Obaroh and Nzeh, 2013). Earlier researchers have also used different plant meals and extracts to induce sterility in different fish species (Khalil et al., 2014). Among the various herbs, neem Azadirachta indica is one of the most widely available and used in India for various purposes.
        
A.indica is traditionally known as the ‘‘village pharmacy’’ or ‘‘village dispensary’’ in India (Biswas et al., 2002) and possesses antimicrobial, nematocidal, biopesticidal and immunomodulatory properties (Obaroh and Nzeh, 2013, Cavine, 2015). In fish, neem has been proved to increase the phagocytic activity, boost the antioxidant properties and reduce or stop prolific breeding in fish (MacArthur et al., 1995; Obaroh and Nzeh 2013). Further, it is also demonstrated that the gonadal maturation of tilapia was delayed by the ingestion of neem, A. indica (Jegede and Fagbenro, 2008). Keeping in view the above aspects, the current study was designed to evaluate the effects of dietary supplementations of A. indica extracts on growth performance, survival, feed efficiency, body indices, immunological and reproduction in common carp.

Water quality parameters such as water temperature (15.4-33.6°C), pH (8.98-9.40), dissolved oxygen (11.12-12.41 mg l^-1), total alkalinity (147-183 mg l^-1), total hardness (157-189 mg l^-1), ammonical nitrogen (0.07-0.16 mg l^-1), nitrite nitrogen (0.03-0.05 mg l^-1), nitrate nitrogen (1.44-1.87 mg l^-1) were well within the range in all the treatments recommended for carp culture and support optimum growth of fish throughout the culture period. For growth performance, NLE fed treatments (D2-D6) had significantly higher net weight gain (NWG) compared to control (D1).
        
The results for reproductive parameters in terms of GSI, relative fecundity and ova diameter were significantly lower in D4 (1.0 g NLE/kg, Table 1). Whereas, absolute fecundity (number of eggs/female) and somatic growth (result not shown) was significantly higher in D4 (1.0 g NLE/kg) and minimum in D6 (2.0g NLE/kg) (Table 1). The differences for all these parameters among the treatments were significant (P≤0.05).
 
  
 
The immunological parameters include phagocytic activity ratio and index of fish was significantly higher in all the NLE supplemented diets fed groups (D2 to D6) than control (D1). The difference among the treatments was significant being maximum in D4 for both the parameters. Collectively, both parameters showed a sigmoidal pattern of immune-modulatory responses to NLE (Fig 1A, B). Low NBT activities were recorded in D1 (control) and D2 (0.25g NLE/kg), whereas highest values were found for D5 (1.5g NLE/kg) and D4 (1g NLE/kg) treatments (Fig 1C). In CAT and SOD, the experimental groups were significantly higher than control; D3 and D4 doses raised the CAT, whereas D4 raised only SOD values among all the treatment groups (Fig 2A, B).
 

 

        
In addition, LPO was significantly (p≤0.05) lower (except D6) and GPx and GR responses in the experimental groups were significantly (p≤0.05) higher than control. LPO is significantly decreased on being fed with NLE up to 1.0 g kg^-1. GPx exhibited highly signifi­cant activity (P≤0.05) in 1.0g NLE/kg group (D4) compared to control and highest GR values were observed in D4 treatment and GR also exhibited significantly high response (P≤0.05) in 1.0 g NLE/kg (D4) compared to control (Fig 3A, B, C).
 

        
Several plant extracts are reported to stimulate appetite and promote weight gain, when these are administered to cultured fish (Harikrishnan et al., 2012). Besides, plant extracts have also been shown to improve digestibility and nutrients availability, resulting increased feed conversion and leading to a higher protein synthesis (Nya and Austin, 2009). Present study also showed improved SGR and lower FCR in common carp fed with NLE diet. Further, the result revealed an increase in weight gain with increase in NLE inclusion up to 1.0 g/kg diet and decrease in weight gain as the inclusion level increased to 2.0 g/kg diet. At the higher supplementation of NLE, negative effects observed might be due to the fact that high levels of NLE could reduce feed intake and also act as a phytoestrogen. It is also observed that the present study is in confirmation with the earlier reports in tilapia, silver carp and Asian sea bass (Talpura and Ikhwanuddin, 2013; Mona et al., 2015).
        
Plant extracts contain saponin a glycoside compound; one of the active photochemical substances responsible for the anti-fertility response (Lohiya and Goyal, 1992). These compounds normally act as anti-implantation, aborti-facient, anti-zygotic, anti-fertile, anti-androgenic or anti-spermatogenic agents and lead to the suppression or inhibition of reproduction (Cavine, 2015). Till date, there are no reports on the effects of these plant extracts, particularlyneem on the reproduction of C. carpio. In the current study it was observed that decreased GSI, relative fecundity and increased fish weight with increased levels of NLE up to 1.0 g/kg. Jegede et al., (2008) also reported similar results in neemfed Tilapia zilliiwhere relative fecundity decreased with increase in NLE and also reinvestigated gonad histology and observed alteration in the normal tissues of the testis and ovary. Obaroh and Nzeh (2013) and Kareem et al., (2015) reported that neem extract and leaves fed to Nile tilapia had significant effect on reproduction (no breeding). The earlier studies support the present findings of antifertility effect of neem in common carp. Perhaps, saponin content of neem could be responsible for the effect of reduced reproduction in common carp. Hence, the results obtained in the current study reveal that NLE (1.0 g/kg) could be useful in controlling the prolific breeding of common carp, and inhibition of its breeding capacity would make the fish to divert the reproductive energy for somatic growth.
        
The present findings of enhanced phagocytic activity in NLE supplement diets up to D4 are supported by the study conducted in silver carp, where phagocytic index significantly enhanced upon feeding with 2.0% NLE enriched diets from 1 to 4 weeks (Mona et al., 2015). Similar results were also recorded by Uthayakumar et al., (2014), where phagocytic activity was significantly enhanced in Channa straitus in a dose of 20 mg/kg of hexane soluble fractions of neem. Talpur and Ikhwanuddin (2013) also reported that neem extract showed maximum antibody titre and phagocytic activity. The present investigation also revealed higher phagocytic activity in treated groups, which indicates that addition of NLE improved the non-specific immunity of fish. The present and the earlier reports on neem and neem products evidence that the immune system was enhanced. It could be due to the presence of bioactive compounds and its broad- spectrum activity of immune stimulation, which reported to have various biological activities (Girish and Shankar, 2008).
        
Studies on NBT indicated that dietary administrations of herbs enhance the respiratory burst of phagocytes in fish (Nya and Austin, 2009). It is well accepted that fish phagocytes upon activation are able to generate superoxide anion and its reactive derivatives during the period of intense oxygen consumption. These reactive oxygen species are toxic for bacterial fish pathogens (Hardie et al., 2004) and widely recognized as an indicator of the status of macrophage and neutrophil activation. In the current study, higher respiratory activity was observed in D4 and D5 group and this is in agreement with Kumar et al., (2012) studies in gold fish. These events could have resulted in the formation of highly reactive compounds such as free radicals or oxy radicals that frequently react with cellular macromolecules, leading potentially to enzyme inactivation, lipid per-oxidation, DNA damage and cell death (Van der Oost et al., 2003). Further, catalase is the primary cellular enzymatic defence against H₂O₂, converting it into H₂O and O₂ and is critical for the process of scavenging free radicals (Dorval et al., 2003). Living organisms are protected from ROS by several defence mechanisms; include antioxidant enzymes such as superoxide dismutase, catalase, glutathione peroxidase and glutathione reductase and others.
        
In the present study, the treated groups showed high SOD and CAT activity. The high level of SOD and CAT enzymes activity in the treatment groups might have occurred due to the triggering of antioxidant enzymes by NLE and resulted in lower oxidative stress in the NLE treated groups.
        
The study revealed decreased Lipid peroxidation and elevated Glutathione peroxidase and Glutathione reductase in NLE supplemented fish. Similar trend was also reported by Chromcova et al., (2015) in the larvae of common carp exposed to neem azal and it is shown that both aqueous as well as 70% ethanolic neem leaf significantly reduce N-methyl-N’-nitrosoguanidine induced lipid peroxidation, by enhancing the activities of glutathionine dependent antioxidants as well as SOD and CAT.

To the best of our knowledge, this is the first research to check the efficacy of neem leaf extract on common carp gonadal maturation as well as implications on growth, immune and reproductive parameters. The study found that at certain levels of NLE (1.0 g/kg) improved the growth and delayed female gonadal maturation. However, it is yet unknown whether a blend of this extract could offer a wider range of benefits or have any synergistic properties and certainly the present study opens a window for further research directions. Such research, along with the findings of the current study, may demonstrate a range of effectiveness that could improve common carp (or any other teleost of similar nature) productivity using economically available resources.