Applied Tropical Agriculture Volume 24, No 1, 144- 153, 2019 © A publication of the School of Agriculture and Agricultural Technology, The Federal University of Technology, Akure, Nigeria EFFECTS OF DIETARY SUPPLEMENTATION OF PIPER GUINEENSE FRUIT EXTRACT ON THE MILT QUALITY OF CLARIAS GARIEPINUS BROODSTOCK

*Ejete-Iroh, V. C., Adebayo, O. T. and Dada, A. A.

Department of Fisheries and Aquaculture Technology, The Federal University of Technology, Akure, Ondo State, Nigeria *Corresponding Author, Email: [email protected]

Abstract:

Clarias gariepinus is a major cultivable fish species in Nigeria. The method of its propagation and quality of seed produced is therefore crucial. In the present study, the effect of dietary supplementation of aqueous extract of Piper guineense on the milt quality of male catfish, C. gariepinus broodstocks was investigated. Seventy five (75) male C. gariepinus (mean weight ± 407.5 g) were fed with four diets supplemented with 0.6, 1.2, 1.8 and 2.4 g aqueous extract of P. guineense dry fruit extract / 100 g feed respectively (designated as D2, D3, D4 and D5) and the control diet D1 was not supplemented. The experiment was carried out for a period of 70 days and the broodstocks were fed at 3% body weight two times per day. Fish fed experimental diets showed significantly improved growth performance and reproductive indices over the fish in control treatment. Higher (P < 0.05) gonadosomatic index was recorded for the fish fed dietary P. guineense compared to fish fed on control diet. This result revealed that dietary supplementation of P. guineense fruit aqueous extract significantly improved gonadosomatic index, milt volume, milt count, motility duration, percentage motility, testosterone level and testicular histology of male C. gariepinus broodstocks, and it has a potential pro-fertility effects which can be exploited in fish seed production by hatchery operators.

Keywords: Piper guineense, milt quality, gonadosomatic index, Clarias gariepinus.

INTRODUCTION is because they are biodegradable and do not stay long in fish tissue, therefore, biologically safe to be consumed. The popularity of the African Catfish Clarias gariepinus Some medicinal plants are extensively used as (Burchell, 1822) in Nigeria has continued to increase aphrodisiac to relieve sexual dysfunction, or as fertility among fish consumers and this is due to its very high enhancing agents. They provide a boost of nutritional meat quality and taste especially in different processed value by improving sexual performance and libido forms such as smoked, fried, and dried form (Eyo and (Yakubu et al, 2007; Sumalatha et al., 2010). Most Ekanem, 2011). C. gariepinus remains the most potent herbal aphrodisiacs are available and have little cultivated species in Nigeria. This is due to several or no side effects on what? (Oyedeji and Bolarinwa, favorable characteristics that it exhibits. The 2013). Many authors have supplemented fish diets with characteristics include; fast growth rate, ability to strive different medicinal plants to improve growth under varying range of environmental conditions, performance, feed utilization, or enhance fertility. disease resistance, tolerates high stocking densities in Among them are; Kigera africana (Adeparusi et al., captivity, acceptability of formulated feed, high (2010), Garcinia kola (Dada and Ebhodage, 2011), fecundity, high market value, excellent taste, excellent Telfairia occidentalis (Dada and Ejete-Iroh, 2016), meat quality, and ease of artificial breeding, (Eyo et al., Mucuna pruriens (Dada and Ogunduyile, 2011), 2014). Due to the high demand of C. gariepinus in Stachytarpheta jamaicensis and Garcinia kola (Ekanem Nigerian market, there is need for high quality fish seed et al., 2017). Although culture of C. gariepinus seems to which has necessitated research into various ways of be successful in Nigeria, several farmers are still facing enhancing fertility to meet the growing demand. many challenges with fish seed propagation due to poor quality of broodstocks and fertility, which hinders the The use of plant extracts and phytochemicals in animal success of their fish production (Awom and Eyo, 2016). production system to boost growth and enhance fertility Many plant products are being tested for enhancement is gradually becoming popular worldwide. Plants of breeding of C. gariepinus in captivity; Piper products have been reported to have little or no side guineense is one of such plants. effects in both the cultured fish and the consumers. This

144 Ejete-Iroh, V. C., Adebayo, O. T. and Dada, A. A./ Applied Tropical Agriculture 24(1), 144-153, 2019 Piper guineense, (Schumach and Thonn, 2005), Experimental Fish commonly known as African black pepper or hot leave belongs to the family Piperaceae or Sapotaceae Seventy-five male C. gariepinus broodstocks with (Macmillian, 1984). In some parts of Nigeria, the seeds average weight (± 407.5 g) were obtained from a fish are consumed by women after child birth, to enhance farm in Akure, Ondo state. The broodstocks were uterine contraction for the expulsion of placenta and transported to the Research farm of the Department of other remains from the womb (Udoh et al., 1999), and Fisheries and Aquaculture Technology Research farm, also for the control of weight (Mba, 1994). Leaves of P. Federal University of Technology Akure and stocked. guineense are used traditionally for the treatment of The brood stocks were stocked at the density of 5 fish respiratory diseases and correction of female infertility per tank and conditioned for two weeks in 15 outdoor problems, and the seeds as an aphrodisiac (Noumi et al., concrete tanks (1 x 2 x 1.5m3). During the acclimation, 1998 and Mbongue et al. (2005)). Okoye and Ebeledike they were fed with commercial diet of 40% crude (2013), reported that the phytochemical analysis of the protein. leaves of P. guineense showed the presence of alkaloids, flavonoids, saponins, tannin, resins and essential oil. Experimental Procedure The proximate composition of P. guineense as reported by Nwankwo et al., (2014) revealed that the plant The experiment is a completely randomized design in contains crude protein, fat, carbohydrate, vitamins and which five treatments (diets) were applied in three minerals. Aqueous extract of P. guineense was reported replicates. The following treatments were used; the by Mbongue et al., (2005) to have a positive impact on control diet (D1) was without the P. guineense extract the male reproductive function because it stimulated the and the other diets were supplemented by 0.6, 1.2, 1.8 secretions of the testes, epididymis and seminal and 2.4g of P. guineense extract/kg feed respectively vesicles. Despite the fact that P. guineense leaves are and designated as D2, D3, D4 and D5. The experimental widely consumed by people in the Southern and Eastern diets containing aqueous extract of P. guineense dry Nigeria and other parts of West Africa and have other fruits at different graded levels were used to feed the fish ethno medical uses, there is limited or no information on twice daily (08.00-09.00 h and 17.00-18.00 h) at 3% their effect on fish reproductive index. This study is body weight for 70 days. therefore targeted at investigating the effect of the aqueous extract of P. guineense dry fruit on the Formulation of experimental diets reproductive performance and sex hormone (testosterone) of male C. gariepinus. The experimental diet was formulated according to the formulation determined for Africa catfish, C. gariepinus MATERIAL AND METHODS for 40% crude protein basal feed (Fagbenro and Adebayo, 2005) as given in the Table 1. All dietary Collection, authentication and preparation of Plant ingredients were ground into small particles size and materials weighed with a top load scale. Ingredients including protein sources, oil and vitamin premix were Fresh fruits of Piper guineense were purchased from a thoroughly mixed in a Hobbart A-2007 pelleting and local market in Ado-Ekiti, Ekiti State, Nigeria. mixing machine (Hobart Ltd London,) to obtain a Identification and authentication were done at the homogenous mass, cassava starch was added as a Department of Crops, Soil and Pest Management, binder. The aqueous extract of P. guineense was Federal University of Technology, Akure, Ondo State, measured and added to the ingredient's mixture Nigeria. The fruits of P. guineense were air dried and (according to the various inclusion levels) before ground into powdery form using electric blender extrusion. The ingredients mixture was pressed through (Marlex Electroline Excella). One- hundred grams of a mixer with 6mm die attached to the Hobart pelleting the dried powder were macerated in 200 ml of distilled machine. The pellets produced were dried and kept in a water for 12 hours at room temperature and was filtered cool and dry place until the start of experiment. with a muslin bag to obtain an extract concentration of 28 mg/ml.

145 Effects Of Dietary Supplementation Of Piper Guineense Fruit Table 1. Ingredient and Proximate Composition (g/kg) of Experimental diets fed to experimental fish Ingredients Experimental Diets Diet 1 Diet 2 Diet 3 Diet 4 Diet 5 Fishmeal (65%cp) 250 250 250 250 250 Soy bean (45%cp) 400 400 400 400 400 Yellow Maize 150 150 150 150 150 Blood meal (85%cp) 50 50 50 50 50 Fish oil 40 40 40 40 40 Vegetable Oil 60 60 60 60 60 Vit premix 30 30 30 30 30 Cassava starch 20 20 20 20 20 P. guineense extract 0 0.6 1.2 1.8 2.4 Proximate composition Moisture 4.48 5.26 5.45 6.19 6.89 Crude protein 39.24 39.91 40.36 40.45 40.95 Crude lipid 14.47 13.16 12.16 14.52 11.22 Crude fiber 2.13 2.13 2.12 2.13 2.13 Ash 13.75 19.36 16.22 22.68 19.24 NFE 25.93 20.18 23.69 13.81 19.57

V itamin premix**- (An Animal Care(R) Optimix Aqua Motility Duration product for catfish, containing the following per 5kg of premix: A = 20,000,000 I.U, D3 = 2,000,000 I.U, E = This was determined by placing 1µl of milt from each 200,000 mg, K3 = 10,000 mg, B2 =12,000 mg, B12 = male on a cavity microscope slide; a drop of distilled 9mg, B1 = 6,000 mg, B6 = 11,000 mg, C = 50,000mg, water was added and covered with a slip. The sperm folic acid = 2,000 mg, Niacin =80,000 mg, Calpan = activity was viewed on Olympus microscopic at 100 x 25,000mg, Biotin = 100 mg, x Zinc = 30,000mg, Copper magnification to see when all the sperm will stop = 5,000mg, Iron = 30,000 mg, Manganese = 50,000mg, moving (Mims, 1991). Iodine = 1,000mg, Selenium = 100mg, antioxidant = 125,000mg). Fish oil used; Biosea cod liver oil. Percentage Motility Vegetable oil; Mamador vegetable oil. Graded level of supplement as given by Mbongue et al., (2005). Each sample was estimated using light microscope at 400x magnification immediately after adding 20 µl Milt Volume distilled water as an activating solution. During spermatozoa activation, immotile sperm cell (ISC) was The sperm sacs were carefully removed by making an counted, and when the activation stopped, whole sperm opening with a sterile lancet around the abdominal cell (WSC) was counted using the method of Canyurt region. Small incisions were made into the lobes of the and Akan (2008). The motile sperm cells (MC) was then sperm sacs, the milt was squeezed out into a sterile petri calculated as dish and the milt volume was measured with a plastic MC = WSC – ISC syringe. % MC = MC/WSC x 100

146 Ejete-Iroh, V. C., Adebayo, O. T. and Dada, A. A./ Applied Tropical Agriculture 24(1), 144-153, 2019 Milt Count Statistical analysis Concentration of sperm was determined by counting the All data were recorded as mean ± standard deviation and number of spermatozoa in a sample diluted with were subjected to a one-way ANOVA to test the distilled water (100x) in Burker haemocytometer, at variance among the trial groups and the control using 400x magnification (Rainis et al., 2003). the Statistical Package for Social Sciences. (SPSS) 15 software for window. Duncan's multiple range tests was Blood sampling and testosterone determination used to compare differences among individual means Blood samples were taken from the caudal vein of (Zar, 1996). experimental fish using a sterile syringe with a drop of heparin in it. Blood samples collected were centrifuged RESULTS for 15 min with cooled centrifuge (4°C) to obtain serum. The water quality parameters measured varied as The serum samples were stored in the freezer at - 15°C follows: temperature; 26 to 26.50°C, dissolved oxygen; until the assays of hormone. The stored serum samples 6.11 - 6.61 mg/l, and hydrogen ion concentration (pH); were analyzed for testosterone (ng/ml) according to 9.60 - 9.77 as given for catfish (Viveen et al., 1986). Abraham (1977) by enzyme-linked immunosorbent assay (ELISA kits; Accu-Bind Elisa Microwells, Data on Table 2 shows result obtained from the growth Monobind Inc. Lake Forest, CA 9630, USA). The and reproductive performances of the male Clarias method was based on competitive binding, where an gariepinus brood stocks fed on supplementary diet of unknown amount of testosterone in the sample and a aqueous extract of P. guinieens. It was observed that defined amount of testosterone conjugated to there was normal feed intake for the experimental fish, horseradish peroxidase compete for the binding sites of which explains the reason for an increase in the weight testosterone antiserum coated to the well of the of the fish fed supplementary diets. There was microplate. After 1 hour of incubation in a shaker, the significant difference (P < 0.05) between the final microplate was washed four times and then the substrate weights of D1 (control) and D4 & D5, but there was no was added and the reaction was stopped by 2N significant difference between D4 and D5. The results hydrochloric acid. The serum testosterone showed that weight gain was highest in D5 followed by concentration of test samples was extrapolated from a D4, D3, and D2 while the lowest weight gain was in D1 calibration curve by correlating the absorbance of the (control). There was no significant difference in the sample at 550nm with the corresponding absorbance on weight of testes (P > 0.05) of D1, D2 and D3, but there the standard curve. was significant difference (P < 0.05) between D1, D4 and D5, with D5 having the highest value while D1 had Water quality parameters the lowest. It was observed that no significant difference Water quality parameters like temperature, pH, and (P > 0.05) occurred in the gonadosomatic index of D1, dissolved oxygen concentration were measured weekly D2, D3 and D4, but significant difference (P < 0.05) during the period of the study using mercury-in-glass occurred between D1 and D5. The milt motility showed thermometer, pH meter ( Hanna H198106 model) and significant difference (P < 0.05) between D1 and D3, D1 dissolved oxygen meter (JPP- 607 model) as described and D4 and between D1 and D5 but there was no by APHA (1987). significant difference (P > 0.05) between D1 and D2. There was no significant difference (p > 0.05) in milt Histology of the testis and ovary of fish fed the count of the fish fed D1 and the diets supplemented with experimental diets P. guineense, however, there was increase in milt count After 70 days of feeding, a total of ten males were of fish fed D3, D4 and D5. There was no significant randomly taken from all the five treatments. They were difference (P > 0.05) between the motility duration of dissected and the testes removed for sectioning and fish fed D1 and D2 and D5, but there was significant histological examination. The testes were placed in a difference (P < 0.05) between fish fed D1 and D3 and D4 formalin-solution made of equal volumes of 10% with D4 having the highest value. Percentage motility formalin and 0.9% sodium chloride solution for 24 across the different diets supplemented with aqueous hours (Bancroft and Cook, 1994)). Following extract of P. guineense fruit showed significant dehydration in increasing ethanol concentrations (50- difference (p < 0.05) when compared to D1. There were 99.9), the testes were cleared in xylene and embedded in increase in the percentage motility of milt in fish fed paraffin wax. Section of 10 ì was cut and stained in supplementary diet of P. guineense than the control fish. haematoxylin and eosin. The stained specimens were The result also showed increase in testosterone level of observed under a light microscope fitted with camera. the fish fed supplementary diets with the highest value Photograph of the stained specimens were taken for occurring in fish fed D4 (2.36). interpretation. 147 Effects Of Dietary Supplementation Of Piper Guineense Fruit Table 2: Reproductive performance of male Clarias gariepinus broodstocks fed supplemented diets of aqueous extract of Piper guineense fruit (mean ± S.E) Parameter D1 D2 D3 D 4 D5 Initial fish weight 417.33 ± 3.71c 406.67 ± 1.67ab 411.00 ± 2.08b 404.33 ± 2.33a 4 13.67 ± 1.86bc Final fish wei ght 520.37 ± 16.67a 543.30 ± 28.06a 560.33 ± 49.96a 610.0 ± 49.29b 63 2.47 ± 42.90b Weight Gain (g) 103.03± 13.31a 136.63 ± 27.38ab 149.33 ± 51.74ab 205.87 ± 52.05b 218 .77 ± 41.04c Weight of testes (g) 1.2 0 ± 0.52a 2.2 7 ± 0.63ab 2.03 ± 0.93ab 2.53 ± 0.18 b 3.87 ± 0.38c GSI (%) 0.24 ± 0.10a 0.4 1 ± 0.10ab 0.35 ± 0.15ab 0.45 ± 0.01ab 0.62 ± 0.90c Milt volume (ml) 0.43 ± 0.08a 0.6 0 ± 0.10ab 0.95 ± 0.13b 0.81 ± 0.22ab 0.99 ± 0.12b Miltcount(x104spzml) 28.13 ±0.80ab 25.1 7 ± 2.23a 26.63 ± 3.64b 37.13 ± 4.25b 31.53 ± 1.22ab Motility duration(sec) 1.17 ± 0.31a 1.9 0 ± 0.25ab 2.73 ± 0.24b 2.89 ± 0.41b 2.34 ± 0.56ab % Motility 36.29 ± 5.08a 58.3 5 ± 1.58b 57.75 ± 3.07b 61.58 ± 2.48b 69.86 ± 5.48b TT level (ng/ml) 0.43 ± 0.01a 0.4 7 ± 0.01a 1.35 ± 0.61a b 2.36 ± 0.21b 1.84 ± 0.77ab Mean in a given row with the same superscripts are not significantly different at P > 0.05

GSI = Gonado somatic index = Gonads weight (g) × 100/ fish weight TT = Testosterone Histology of C. gariepinus testes fed to the five experimental diets The histology of the testes of fish fed the experimental diets, showed normal distribution of the testicular cells.

The histology of the testes of fish fed the experimental diets, showed normal distribution of the testicular cells.

148 Ejete-Iroh, V. C., Adebayo, O. T. and Dada, A. A./ Applied Tropical Agriculture 24(1), 144-153, 2019

149 Effects Of Dietary Supplementation Of Piper Guineense Fruit

150 Ejete-Iroh, V. C., Adebayo, O. T. and Dada, A. A./ Applied Tropical Agriculture 24(1), 144-153, 2019 DISCUSSIONS similar to the result of Etuk and Muhammad (2009), who worked on fertility enhancing effects of aqueous The results obtained in this study show that C. stem bark extract of Lophira lanceolata in male rats, the gariepinus fed to the experimental diets responded result obtained from the testicular histology positively which is evident in the growth performance examination revealed increased spermatogenesis, and reproductive indices of the fish used. In this study, which was attributed to the increase in testosterone water parameters such as, temperature, pH, and level. The high milt count observed in the fish fed diets dissolved oxygen were within the acceptable range for supplemented with aqueous extract of P. guineense, fish culture as recommended by Boyd (1979) and Goos confirmed increased spermatogenesis seen in the and Richter (1996). This implies that the result obtained testicular histology of the fish, signifying a better in this study were not influenced by physio-chemical spermatogenetic activity of P. guineense extract. The parameters of the culture tanks. Crus-Casallas et al. improvement in the testicular histology as depicted in (2005), reported that viable sperm is an essential plates 2-5, could be due to antioxidative properties such component of any successful animal production and the as tannins and alkaloids present in the fruits Okoye and success of reproduction process is dependent on a Ebeledike, 2013). supply of high quality gamete. The results of this study have shown that P. guineense fruits extract has positive Conclusion effects on the milt quality of C. gariepinus such as milt count, motility duration, milt volume and percentage From the result of the present study, it could be deduced motility. There was significant difference (P < 0.05) in that Piper guineense can be used as fertility enhancer in weight gain of the test fish and in weight of testes at the male African catfish Clarias gariepinus broodstock inclusion levels of 1.80 g/kg (D4) and 2.40 g/kg (D5), since the results have shown that P. guineense aqueous when compared to the control. fruits extract up to the inclusion level of 2.4g/kg of feed have positive effects on the milt quality of C. gariepinus The increase in sperm motility, count, volume, and such as milt count, motility duration, milt volume and percentage motility in the fish fed diets with P. percentage motility. It also has positive effect on the guineense fruit extract when compare to the control testosterone and testicular histology of C. gariepinus could be attributed to P. guineense. This is in agreement broodstock. Therefore its use in broodstock production with the report of Mbongue et al. (2005), who reported should be encouraged as it will minimize the that P. guineense increased reproductive indices in male dependence on synthetic drugs as fertility enhancer. rats. Some active ingredients in P. guineense such as flavonoids, tannin have spermatogenic activities. The References inclusion of P. guineense in the diets therefore, provided a supply of additional nutrients to the test fish over the A b r a h a m , G . E . ( 1 9 7 7 ) . H a n d b o o k o f control. The flavonoids and vitamins in P. guineense Radioimmunoassay. New York, NY: Marcel extract maintain sperm morphology, sperm survival and Dekker. Pg? sperm function (Nwanna and Oboh, 2007). The Adeparusi, E.O., Dada, A. A., Alale, O. V. (2010). improvement in sperm count and testicular histology as Effects of Medicinal Plant (Kigelia Africana) shown in the present study could be due to anti oxidative on Sperm Quality of African Catfish Clarias properties of tannins and vitamin A present in P. gariepinus (Burchell, 1822) Broodstock. guineense (Nkang et al., 2003). The increase in milt Journal of Agriculture, 2(1): 193-199. quality in fish treated with P. guineense, could be as a APHA. (1987). Standard Method for the Examination result of the effect of vitamin A in the plant since vitamin Water Wastewater. 17 ed. Washinghton D.C. A protects the testis against lipid peroxidation, promotes pp. 1268. spermatogenesis and improves structural differentiation Awom, I. E. and Eyo, V. O. (2016). 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