Withania coagulans fruit extract: a possible useful additive in ameliorating growth and immunity of Labeo rohita (Hamilton, 1822)

Abstract This study aimed to assess the Withania coagulans fruit extract's effects on growth and haematological parameters of Labeo rohita. Healthy fish (n = 120) were divided in four groups. Experimental diet was prepared with different extraction of W. coagulans (0%, 1%, 1.5% and 2%). Diet was provided for eight weeks to all groups except the control group which fed on basal diet. The 1.5% and 2% W. coagulans extracts lead improvement in feed conversion ratio and specific fish growth rate; haematological parameters in fish group fed with 2% of W. coagulans showed increased in WBC, RBC, Hb, MCHC, PCV. The values of plasma proteins and lysozyme activity were higher in the experimental group compared control group. Fish fed with W. coagulans extracts showed increased of immunity and less mortality respect to others. It suggested that W. coagulans could be considered valuable support for the improve growth and immunity in Labeo rohita. Graphical Abstract


Introduction
Labeo rohita is one of the most important carp among the three major Indian carps. Due to its polyculture ability, it has been cultured with Catla catla and Cirrhinus mrigalan (FAO 2020). Stressful aquaculture conditions can reduce the fish growth and making them highly prone to different pathogenic and mechanical infections (Galina et al. 2009;Xueqin et al. 2012). Many studies suggest the medicinal herbs as immune-stimulants in different optimal doses, frequency and duration have proved to a promising role in enhancing the immunity of the fish Bricknell and Dalmo 2005;Bowden 2008;Magnad ottir 2006). Among these plant extractions, Withania coagulan, which belongs to the family Solanaceae, is well known for their many pharmaceutical properties such as anti-diabetic, anti-microbial, anti-fungal, anti-oxidant etc. (Kataria et al. 2013). Further, Studies indicate a higher amount of magnesium, calcium, potassium in W. coagulans (Ullah et al. 2013). further, whole plant contains carbohydrates, lipids, and protein but fruit contains ash (4.21%), carbohydrates (60.14%), lipids (5%), and protein (4.65%). The fruits of the W. coagulans are composed of milk coagulating enzymes, essential oil, two esterase, free amino acids such as 'Proline, tyrosine, valine, hydroxyproline, glycine, cysteine, asparagine, glutamic, and aspartic acids' (Hameed and Hussain 2015;Naz et al. 2009).
In view of such consideration, the aim of the current study was to evaluate the effect of different concentrations of Withania coagulans fruits administered with diet on growth, immunity and haematological parameters of Labeo rohita.

Results and discussion
Researchers from around the world focused their attention on the improvement of haematological profile of fish, promoting growth and increasing immunity. In this regard, many natural and synthetic feed additives are used but the natural (mostly plant derivatives) are economical and trust worthy as compared to synthetic due to different reason like it is eco-friendly (biodegradable) and harmless to humans (biocompatible) (Ajiboye et al. 2012;Immanuel et al. 2009). The findings gathered from the current survey revealed that the FCR showed lower values in the fish group fed on 2% followed by 1.5% than the control group and group 2 (1%), while the SGR was found higher in fish fed on 1.5% and 2% extraction of W. coagulans in the diet (table  S2). Ojha et al. (2014) conducted a study on diabetic rats while treating with W. coagulans and they observed that the rat restored the weight of the body, function of the kidney and also lower the hyperglycemia. Many other medicinal plant additives known to promote the growth of fishes have been tested. In particular, it has been found that extract of 0.5% and 1% of Aloe vera was effective (P < 0.05) for Amphilophus labiatu weight gaining, FCR and SGR (Alishahi et al. 2010). A study carried out on fingerlings of Clarias gariepinus showed a positive effect of 0.5% garlic (Allium sativum) extract on total length and weight of fish and an increase in the WBC, Hb, PCV and RBC values of fish (Nwabueze 2012). Regarding the effect of W. coagulans on haematological parameters measured in L. rohita, the findings herein obtained showed that the WBC, RBC, PCV, MCV, MCHC, MCH, Hb values were higher in fish groups fed on W. coagulans. Moreover, the plasma protein and the value of lysozyme activity were significantly increased in fish fed on an experimental diet as compared to control fish. However, fish fed with 2% of W. coagulans showed a better response of blood parameters than other groups (table S3). A previous study conducted to assess the effect of W. coagulans extracts on Cyprinus carpio fed for 56 days reported that the growth parameter and immunity of this fish species was improved; in particular, a positive impact of diet supplemented with W. coagulans was reported on WBC, RBC, Hb, MCV and Hct (Tasa 2019). Other authors reported that the Garginia cambogia extract can enhance the WBC counts (Dada and Ikuerowo 2009). According to Ndong and Fall (2011), the WBC counts were increased in hybrid tilapia fed with a diet supplemented with garlic at 0.5% and 1%. Also, Bazari et al. (2017) introducing Aloe vera extract in the diet composition of Siberian sturgeon (Acipenser baerii) observed an increase of WBC, RBC and MCV values. Sahu et al. (2007) described that RBC count result increase in the fingerlings of L. rohita fed on Magnifera indica kermel, and this intensification of RBC values has been suggested as a sign of enhanced cellular immunity. In line with this hypothesis, the current study showed that the W. coagulans improved the leukocytes population by increasing the monocytes, lymphocytes and neutrophils values in all experimental groups (table S3), suggesting a boosting of immunity because of the well-known role that these cell play in antifungal, antibacterial and demolishing of other foreign particles. The lymphocytes level increases the immune system and they are perfect markers of immunity and used for the treatment of xenobiotic material (Banaee et al. 2008). Besides this Akrami et al. (2015) reported that in beluga the basal diet with onion powder of 1% increased the lysozyme activity.
After challenging with the virulent strain of the bacteria it was found that there was very low mortality in the fish groups that were fed on the herbal extract as compared to the control group that was fed only on basal diet. Furthermore, precisely, group 3 that were fed on 2% of the W. coagulans shows a far better result as compared to the other treated groups of the experiment (table S4 and figure S1). Previously some other researcher also reared some fishes on the herbal extract such as Soltanian and Fereidouni (2016) evaluate the immunomodulatory properties of Henna (Lawsonia inermis), injected intraperitoneal (i.p.) with different concentration and found that Disease resistance against Aeromonas hydrophila has also been improved following herbal treatment. Another study carried out in Pakistan by a group of researchers concludes that ginger and garlic extract if corporate in the feed of the fish can enhance their immunity against the parasite and bacterial ).

Experimental
See Supplementary materials for materials and methods (preparation of Withania coagulans fruits extract, fish sampling, sampling of blood, haematological analysis, immunological and growth analysis), analysis of data and tables.

Conclusion
According to the results obtained in the current study, it could be stated that the use of Withania coagulans extracts as a dietary supplement in fish is effective and can be used to improve fish health and production. However, the diet supplemented with 1.5% and 2% of W. coagulans extract showed a greater positive impact on fish growth performance and haematological parameters which in turn enhance the fish immunity against infection. Specifically, the L. rohita specimens fed with a diet supplemented by W. coagulans extract improved the leukocytes population by increasing the monocytes, lymphocytes and neutrophils values suggesting a boosting of immunity.

Funding
The author(s) reported there is no funding associated with the work featured in this article.

Data availability statement
The datasets generated during and/or analyzed during the current study are available from the corresponding author on reasonable request.