Effect of pawpaw seed meal using various inclusion level on the gonad structure of Oreochromis Niloticus (gift)

This study investigates the effect of pawpaw seed meal using various level of inclusion in regulating procreation in Oreochromis niloticus (gift strain) via morphology (percentage defective sperm cells) as well as gonads using biomarkers. The pawpaw seed powder was compounded to a 2mm tilapia feed at 35% crude protein at 2, 4 and 6g per kg diet. A total of twenty (20) fingerlings (10 males and 10 females) with average weight of 20g were randomly allotted in triplicate into concrete ponds (1m x 1m x 1 0.8m) of 600 litres capacity with control. The Tilapia fish was nourished at 4% body weight per day in dual portions at 09.00 – 09.30 hours and 17.00 – 17.30 hours. The experiment lasted for 60 days. Minor tissue damage (testes) was observed in fingerlings fed PSM at a level of 2h/kg while disintegration of a lot of cells occurred at increased dietary levels of PSM (4g and 6g per kg diet) which resulted to lack of spermatids and oocytes in the fish testes. Hence, it is useful in battling difficulties of over-population of tilapia in fish pond. Aside infertility, there were no observable contrary effect from pawpaw seeds consumption. Therefore, safe usage of 6 g PSM per kg diet for sixty (60) days is recommended for economic efficiency of fish farms. Further research is required on reducing the antinutrient substances (carpaine) in pawpaw seed so that it can be safely use as an additive in feeds.


Introduction
In order to ease the diffi culties of malnutrition and starvation due to growing population, culturing of fi sh becomes an effective tool since fi sh is a valuable protein source alongside with several other qualities [1]. Fish contains wet body weight around 16-20% protein when related to 3.5% in milk, 6.6% in rice as well as wheat and 12% in egg. It has great nourishing values with good sense of taste and about 85-95% higher digestibility rate [2]. Fish protein signifi es a vital nutritive element in some countries that are densely populated where entire levels of intake of protein are very little (Food and Agriculture Organization, (FAO, 2004). According to FAO [3], the rapidly developing sector which has surpassed population development is termed aquaculture.
Among the most signifi cant set of cultured fi sh, tilapia is the second next to carp; in addition, it is the most extensively grown of any fi sh cultured on the earth [4]. Moreover, Burden [5] opines that Tilapia is cultured in no less than 85 nations on the globe. Tilapia is labelled ''aquatic chicken'' since it can be farmed commercially in an extensive variety of methods starting from the simple backyard methods to extremely intensive method or "factory farm" just like in poultry [6]. In majority of developing nations where tilapias are often cultured in cages, ponds, raceways, concrete tanks, as well as rice fi eld are as a result of its characteristics which makes it appropriate for culturing [7]. to alleviate this limitation include culturing of all-males which is mono-sex by means of exogenous hormone for sex reversal of sexually the same fi sh, predator usage, cage/ tank culture, sterilization, high stocking density, sporadic or selective cropping, and usage of slow growing tilapia species [6][7][8]. Regardless of all the techniques examined, hormone initiation before population of monosex appears to be the most encouraging and satisfactory method [9]. Fish sex hormonal control is achieved by applying a precise hormone to the fry prior to occurrence of sexual differentiation. Tilapia gonadal differentiation seems to take place within 8-25 days' post-hatch [10]. However, before usage, hormones have shortcomings of expensive technology, hatchery amenities as well as skilled workers are essential; likewise, hormones are costly and challenging to acquire [11].
On the other hand, there is need to investigate less expensive, suitable technology which does not pose a risk to human and environmental health in solving the diffi culties of uncontrolled tilapia breeding. The botanical name of pawpaw is Carica papaya and it is one of the economically signifi cant fruit trees in the Caricaceae family [6,12]. It contains folate, dietary fi ber and vitamin A, C and E. It is very rich in antioxidant nutrients. It has been discovered that Pawpaw Seed Meal (PSM) consists of phytochemicals which holds boundless potential being sex reversal, likewise a procreative obstruction agent in culturing of fi sh. Phytochemicals are chemical compounds such as fl avonoids, isofl avonoids and lignans amongst others which are natural steroid-like compounds derived from fruits, soy, cereals, tea and vegetables [13]. The usage of medicinal plants as enhancers of fertility as well as agents of sex reversal in fi sh has been gaining some awareness. A likely method to the usage of steroid hormones for sex reversal in tilapia might involve the use of plant extracts containing phytochemicals which are natural compounds characterized by estrogenic/androgenic activity. Moreover, with their effective immunostimulating and antioxidant properties, phytochemicals have been discovered to stimulate growth of fi sh [13]. Pawpaw (Carica papaya) has been used over the years to induce sterility in laboratory animals as well as therapeutic plants in rural communities to treat a lot of animal and human diseases [14,15]. Hence, this study was carried out to investigates the effects of the different inclusion levels of PSM on gonads of Genetically Improved Farmed Tilapia (GIFT).

Study area
This study was conducted at Durante Fish Industries Limited, Old Niger West Building, Challenge, Ibadan; South Western Nigeria.

Preparation of experimental diets
Ripe fruits of pawpaw, Carica papaya (East West Seeds Cinta breed) of honey dew diversity was obtained free of charge from Durante fi sh industries farm and dissected in order to take out the seeds afterwards sundried then pulverized into fi nely particle sizes of less than 250 μm. The particles were preserved in a dry, sterile container; 2mm skretting tilapia fi sh feed pellets were obtained from Durante Fish Industries limited with nutrient composition of 35% crude protein as well as 18.5MJ gross energy per kg feed was formulated as prepared diet by adding 2.0 g, 4.0g, 6.0g dosage treatment of PSM separately to 1 kg of 2mm pellet skretting tilapia fi sh feed Table 1.

Source and collection of test fi sh
A total of two hundred and forty (240) fi ngerlings of Oreochromis niloticus (GIFT) strain was obtained from a single spawn and were collected from Durante Fish Industries Limited, Old Niger West Building, Challenge, Ibadan, South Western Nigeria. They were acclimatized for a period of 14 days in the concrete tanks and the fi sh were nourished with a profi table feed. After acclimatization, a total of 20 fi sh (10 males and 10 females) of average weight of 20g were stocked in 12 concrete tanks (1m x 1m x 1 0.8m) each and was supplied with fresh water of 600 litres capacity (temperature of water 27 o C; hydrogen ion concentration (pH) 7.3; dissolved oxygen (DO) which ranges from 5.6-7.4 mg/l). Three (3) treatments was used and replicated three (3) times with control and replicates. The ponds were labelled control (0g), 2g, 4g and 6g respectively as used by Ugonna, et al. [16]. In addition, the fi sh was nourished at 4% body weight daily in two portions at 09.00 to 09.30hrs and 17.00 to 17.30hrs for period of 60 days; afterwards they were separated, grouped by sex then weighed.

Histopathological studies
Samples from testes were placed in formalin-saline solution in ratio 1:1 of 10% formalin plus 0.9% of sodium chloride for 24 hours. According to Luna [17], histological segments of 5μ diameter was prepared conforming to the standard procedures.

Statistical analysis
The collected data were accurately analysed via one-way Analysis of variance (ANOVA) as well as Duncan New Multiple Range (DNMR) post hoc test and mean differences at p <0.05 were signifi cantly considered.

Histology of testes [Magnifi cation 100X]
Plate 1 shows the histological unit of the testes in Oreochromis.niloticus which was fed on control diet of 0g PSM per kg revealed typical testicular tissue, numerous seminiferous tubules containing fairly numerous amounts of spermatogenic cells as well as normal distribution of sperm cell. There is observation of visible lesions. In Plate 2, tilapia  and also reduce pulse speed and weaken the nervous system. Moreover, Oyekunle and Omope [26] specifi ed that the precise device through which the elements lessen sperm count is unidentifi ed, nevertheless suggestions was made that the multiple papain might cross the barrier of the blood testes to apply dangerous impacts on seminiferous tubules control of the testes. A lot of studies have stated the existence of proteins in the sperm and semen. Additionally, prior study recounted the occurrence of serum proteins (lactoferrin, albumin) and glycoproteins (kinase, prostatic). The function of these proteins are to nurture the sperm cells. In 4g and 6g PSM, the amplifi ed fraction of fl awed sperm cell might be as a result of action of proteolytic of the proteases in pawpaw chymopapain and papain or in addition to the steroidal glycosides [27]. There is possibility that the semen proteins have been hydrolyzed by enzymes and compounds which makes them unobtainable for usage of the sperm cells, thus resulting to undernourishment plus fl aws perceived in the cells.

Sperm properties of Oreochromis niloticus
Study by Bucholtz, et al. [28] opine that development of gonad is an incessant procedure, nonetheless particular histological features could be used during reproductive stage to categorize phases of gonadal growth. Therefore, all these histological modifi cations in ovary and testis of the trial fi sh are as a result of dietary levels of PSM. The current fi ndings were in line to elucidate the achieved outcomes in both sexes (male as well as female) Nile tilapia [11,14,17]. This study used dosage of 2 to 8g per kg with period of exposure of 45 days which contrasts from the usage of prior studies. Likewise, Jegede [29] and Jegede [30] reported that Aloe vera latex as well as leaf of Hibiscus rosa-sinensis which are medicinal herbs cause related changes in the ovaries and testis of Nile tilapia; and likewise on laboratory animals like rats as well as rabbits [31,32].
Moreover, Farnsworth, et al. [33] opined that 5hydroxytryptamine is a lively substance which is accountable for the anti-implantation result of seed of papaya in female.
Also, papaya seeds extracts are competent of creating functional abnormalities of various mammalian organs/tissues plus systems possibly as a result of toxic impacts of benzylisothiocyanate. Additionally, Lucidi, et al. [34] stated that active growth of oocyte could infl uence steroidogenesis. Also, histological units which consist of atretic cavities might be as a result of decline in the level of oestrogen [35][36][37][38][39].
Findings in this study revealed that the acquired outcomes indicated that PSM high stages of 4 and 6 g per kg on extensive periods of exposure for 45 days in feeds of Nile tilapia fi sh showed positive infl uence to control the procreative procedure in Oreochromis niloticus which caused numerous histological fl uctuations in the testis which abridged fertility in males Nile tilapia. However, Ugonna, et al. [16] reported that masculinization was highest among the fi shes treated with 4g and 6g per kg which resulted in more than 85% males in these treatments. The inclusion level of PSM above 2g resulted in total non-appearance of female tilapia as well as percentage increase of sterile tilapia is encouraging for usage in commercial production.

Conclusion and recommendation
The study revealed that the damage which has been done on the tissues of the testes was negligible at decreased dietary  with basal feed which is enhanced with PSM indicated that seeds of pawpaw are an effi cient agent of inducing sterility as they were destructive to ovary and testes tissues.
Hence, it is suitable in contending diffi culties of overpopulation of tilapia in ponds. Other than infertility, there were no observable opposing reactions from the pawpaw seeds consumption. Moreso, further research is required on reducing the antinutrient substances (carpaine) in pawpaw seed so that it can be safely use as an additive in feeds.