Dietary effect of Cissus populnea and Securidaca longepedunculata aqueous leave extracts on reproductive, haematological and biochemical parameters of African catfish, Clarias gariepinus (Burchell, 1822) broodstocks

This study evaluated gametes quality, haematological and biochemical parameters of Clarias gariepinus brood stocks fed with varying concentrations of Cissus populnea (CP) and Securidaca longepedunculata (SL). Fish were fed with Diet 1 (control, 0 ml plant extract), Diet 2 (0.5 ml/kg CP), Diet 3 (1.0 ml/kg CP), Diet 4 (0.5 ml/kg SL), Diet 5 (1.0 ml/kg SL) and Diet 6 (0.5 ml/kg CP + 0.5 ml/kg SL) for 90 days. The results of haematocrit and red blood cell of treated groups were relatively similar to the control. The white blood cell and haemoglobin values were lower than the control group while the mean corpuscular volume, mean corpuscular haemoglobin were significantly difference across treatments however; an elevated glucose level was recorded in the treated groups. Also, the mean platelet volume and platelet distribution width were relatively similar across treatments. The serum testosterone, progesterone and estrogen were found to be higher in fish fed diet 2 (0.5ml/kg CP). C. populnea at 0.5 ml/kg significantly (p<0.05) improved eggs weight, fecundity, gonadosomatic index, % fertilization and hatchability. Male brood stocks fed diet 2 (0.5 ml/kg CP) recorded the highest values for sperm motility (56.67 ± 0.33%), milt count and volume (820.33 ± 0.33×106/ml and 1.80 ± 0.05 ml respectively) across all groups. Similarly, fish fed diet 2 (0.5ml/kg CP) had the highest values for eggs weight (283.7±102.4g), fecundity (168,286 ± 57157), gonadosomatic index (32.59 ± 2.72), fertilization (62 ± 20.4%) and hatchability (62.92 ± 19.75%).The dietary supplementation of 0.5 ml/kg C. populnea extract highly enhanced the reproductive profiles of male and female C gariepinus brood stocks.


INTRODUCTION
Traditional knowledge to solve health problems of mankind and animals exists in all countries of the world (Rukangira, 2001), with history dating back to 3000 BC (Sofowora, 1982).
Despite the increasing availability of conventional pharmacological therapies for the management of fertility related abnormalities in males and females, herbal remedies have continued to increase the repertoire of available options (Dada and Ajilore, 2009). In addition, herbal medicines due to their antioxidant and antimicrobial activities are capable of improving sexual dysfunction and fertility (Zhang et al., 2011). Furthermore, food plants such as garlic (Malviya et al., 2011) and Garcinia kola (Ralebona et al., 2012), have shown to improve erectile dysfunction and sexual performance in experimental animals.
Securidaca longepedunculata is a plant commonly found in the tropical area of Africa (Ndamitso et al., 2013). In Nigeria, it is known locally as Uwar maganigunar in Hausa (North), Ipeta in Yourba (South west) and Ezeogwu in Ibo (South east). Phytochemical studies of the root and bark of this plant has led to the isolation of flavonoids and xanthones (Rakuambo et al., 2004) and its antibacterial activities on selected pathogens (Ndamitso et al., 2013). While, Cissus populnea (Guill) belongs to the family Vitaceae and native to the tropical West Africa. In Nigeria, the vernacular names include Ogbolo (South west Nigeria), and Dafarara (North). Extract from the plant have been credited with antibacterial properties and its effect on flutamide-induced testicular toxicities in pre-pubertal rats (Oremosu et al., 2013).
African catfish, Clarias gariepinus is one of the popular cultured species worldwide (Muchlisin et al., 2010) including Nigeria. The gonadal development of fishes is affected by various factors such as genetics, brood fish condition, environmental variability and nutrition (Muchlisin, 2005;Abidin, 2006;Muchlisin, 2014). Hence, the objective of the present study was to investigate the dietary effects of C. populnea and S. longepedunculata extracts on the reproductive functions, haematological and biochemical profiles of male and female Clarias gariepinus brood stocks. tanks were covered with net to prevent the fish from escaping and fed the experimental feeds for a period of 90 days. At the end of feeding trial, the fish were fasted for 24 hour prior to the day of blood samples collection. 1.5% CP Extract 0 ml 5 ml 10 ml 0 ml 0 ml 0 ml SL Extract 0 ml 0 ml 0 ml 5 ml 10 ml 0 ml CP + SL (5ml + 5ml) 0 ml 0 ml 0 ml 0 ml 0 ml 10 ml

Collection of blood samples for haematological and hormonal analysis
Blood samples were collected via the caudal vein puncture as described by Kori-Siakpere et al. (2005) into labeled ethylenediaminetetraacetic (EDTA) bottles and sterile plain sample bottles. Fasting blood glucose was analyzed using a drop of blood from each of the fish with Accu-check glucometer. Haematological parameters were analyzed using semi-automated haematology analyzer (Mindray). Samples of blood in the plain bottles were spun at 3000 rpm to collect the serum that was used for hormonal analysis. The hormonal assay for testosterone, progesterone and estrogen were carried out at the reproductive endocrinology laboratory, Department of Physiology, Faculty of Basic Medical Sciences, University of Lagos using appropriate Elisa kits according to the manufacturer's procedures.

Sperm quality analysis
Milt production and quality were determined at the end of the experiment; twelve male fish were randomly selected with two males from each diet group. The sperm sacs were carefully removed by making an opening with a sterile lancet around the abdominal region. Small incisions were made into the lobes of the sperm sacs, the milt was squeezed out into a sterile petri dish and the volume of the milt was measured with a plastic syringe. Milt was diluted with seawater at a ratio of 1:1000 according to Protocol (2009), and a drop was placed on a clean grease-free glass slide. A cover slip was gently placed on the slide to avoid air bubbles and viewed under the microscope at x40 and x100 objective lenses for sperm motility which was score over a percentage. Sperm concentration was determined by counting the number of spermatozoa in each square of Burker hemocytometer, under x40 and x100 objective lenses (Rainis et al., 2003).

Fecundity estimation
This was done using gravimetric method as described by Akinwande et al. (2012). The eggs were carefully weighed after removing excess water on filter paper and counted. The number of eggs per 1 g weight was determined and used to calculate the total number of eggs.

Artificial fertilization, incubation and hatching
The female fish were injected with the ovaprim hormone (Syndel Laboratories Ltd Canada) at 0.5 ml per kilogram of body weight for a latency period of 12 hours. Three hundred eggs collected by hand stripping of female fish from each dietary treatment were gently mixed with milt from the male fish for artificial fertilization. They were incubated in a continuously aerated glass trough, translucent eggs containing embryonic eyes at the time of polar cap formation (about 20 min after fertilization prior to the 2-cell stage of first cleavage) were considered fertilized and counted to estimate percentage fertilization while opaque eggs were considered unfertilized.
Percentage fertilization was calculated as described by Britz and Hecht (1998) as follows: The mean number of hatchlings in each mating combination was obtained by direct counting of un-hatched eggs as well as the number of hatchlings in the incubation troughs. Percentage hatchability and gonadosomatic index were also calculated as described by Britz and Hecht (1998) as follow:

Statistical analysis
All values were recorded as mean standard deviation and subjected to one-way analysis of variance (ANOVA) using SPSS 15 for window software package. Significant means were subjected to a multiple comparison test (Tukey) for post hoc comparison at P < 0.05 level.

RESULTS
White blood cell count recorded highest values in the fish fed control diet both in the male and female catfish however, the value of control diet (222.40 ± 7.70 x 10 9 /L) differed significantly (p<0.05) from diets 3 (177.22 ± 2.55x 10 9 /L) and 5 (183.05 ± 1.920 x 10 9 /L) in male catfish. Although, no significant differences were recorded in the values of RBC, HCT and HBG across diets in male catfish, the values of these parameters were low when compared with control diet except RBC and HCT in diet 2. There were no significant differences (p>0.05) recorded in the values of RBC, HCT and HBG across diets with the exception of diets 3 and 5 though, the values of these parameters were low when compared with the control group in female cat fish (Tables 2  and 3).
The blood glucose recorded a non significant increase across the treated groups when compared with the control groups in both the male and female catfish. Diets 5 and 1 recorded the highest (99.21 ± 11.93 mg/L) and lowest (68.50 ± 1.50 mg/L) values respectively in male catfish while diets 5 and 1 recorded the highest (99.21 ± 17.25 mg/L) and lowest (60.00 ± 2.00mg/L) values respectively in female catfish. The haematocrit levels (57.28 ± 5.89 and 57.90 ± 0.10) for male and female brood stocks respectively were high in the fish fed diet 2 and the control group respectively; these values were relatively similar across all treatments. The mean corpuscular haemoglobin concentration (MCHC) of female catfish was not significantly difference (p>0.05) across treatments whereas significant difference (p<0.05) was recorded with male catfish with the exception of treatments 2 and 6). Also, no significant difference was recorded in the values of MCH across diets in both male and female catfish (Tables 2 and 3).
The inclusion effects of C. populnea plant extract brought about a significant increase in egg weight but on the other hand the inclusion of S. longepedunculata caused a significant reduction in egg weight at the two different concentrations of the plant extract while the egg weight of fish on diet 6 did not significantly different from those on control diet ( Table 6). The result of fecundity did not show significance difference (p>0.05) amongst diets 1, 2 and 3 (fish fed C. populnea diets), but a significant reduction in fecundity was recorded in fish fed the S. longepedunculata diets. Likewise, the gonadosomatic index values except with diet 6 did not significantly different (p>0.05) from the control. Better percentage fertilization was recorded with fish fed diets 2, 3, 4 and 6 with the exception of fish fed 1.0mg/kg feed of S. longepedunculata. Similar to the male reproductive parameters, diet 2 had the highest values for eggs weight (283.7±102.4g), fecundity (168,286 ± 57157), gonadosomatic index (32.59 ± 2.72), fertilization (62 ± 20.4%) and hatchability (62.92 ± 19.75%) while the lowest values (181.7 ± 4.6g, 81,499 ± 13463, 18.44 ± 8.77, 50 ± 12.7%, and 50.63 ± 10.10% respectively) for these parameters were recorded in fish fed diets 4 and 5.Hachability was only significantly higher in fish fed 0.5ml/kg CP diet and diet 6 (0.5ml CP and 0.5ml SL).

DISCUSSION
Haematological characteristics help fish biologists to interpret physiological responses by fish and deviation from normal response may indicate a disturbance in the physiological process (Dienye and Olumuji, 2014). The reduction in White blood cells count (WBC) observed in the present study suggests that both S. longepedunculata and C. populnea extracts have anti-microbial properties. Aguoru et al. (2014) reported the presence of alkaloids, flavonoids, saponins and anthroquinone in C. populnea and each of these phytochemicals is known to play a crucial role in the immune system stimulation and in the function of organs related to blood cell formation such as thymus, spleen, and bone marrow as reported by Jeong and Lee (1998). The relatively similar red blood cell (RBC) count values across treatments when compared with the control group in both male and female brood stocks may be due to better cellular immunity as reported by earlier studies (Ojha et al., 2014). This was also corroborated by Sahu et al. (2007) who reported that increased RBC counts in Labeo rohita fingerlings fed with Mangifera indica was an indication of enhanced cellular immunity.
The Haematocrit (HCT) values of brood stocks treated with extracts were similar to that of control which indicated that the extracts did not affect oxygen carrying capacity of the blood. The decrease in the haemoglobin values with increase in the concentration of C. populnea and S. longepedunculata extracts was similar to the report of Ojha et al. (2014) who studied the effect of ethanolic extract of Mucuna pruriens on growth, metabolism and immunity of Labeo rohita fingerlings. The result was also confirmed by the study of Omoniyi et al. (2002) who evaluated the effect of lethal and sublethal concentrations of tobacco (Nicotiana tabacum) leaf dust extract on weight and haematological changes in Clarias gariepinus.
Red cells indices; MCV, MCH and MCHC are particularly important for the diagnosis of anemia and lead poison in humans and most animals (Ahilan et al., 2004). The Significant difference (p<0.05) recorded in the values of MCHC in male catfish could be as a result of defense reaction (Ahilan et al., 2004). MCV in all the inclusions levels tested were normocytic, implying that the extracts cannot cause microcytic anemia in fish. The mean platelet volume (MPV) and PDW (Platelet Distribution width) values in both extracts at different inclusion levels tested were relatively similar to the control suggesting that the extracts of C. populnea and S. longepedunculata did not affect the coagulation pathways.
The sperm count and results showed that the performance of C. populnea at the dose of 0.5ml increased compared to other treatments including control. Extracts from the stem of C. populnea are believed to improve fertility in men with low sperm count (Ojekale et al., 2006) implying that the herb can also improve sperm counts if used in fish. The sperm motility and counts of fish fed diet 2 (0.5ml CP) was the highest as observed by previous study of Soladoye and Chukwuma, (2012) who reported that saponins and flavonoids, which are secondary metabolites together with tannins and steroids that are present in the stem back of the C. populnea possessed potentials for fertility enhancement. The result is similar to Dada and Aguda (2015), who used extract of African walnut (Tetracarpidium conophorum) to achieve positive results on the reproductive indices in male African catfish (C. gariepinus) brood stock. Furthermore, Adeparusi (2010) used Kigelia africana fruits at the dose of 100g/kg to achieve significant increase in sperm count, sperm motility, and fertilization ability in C. gariepinus brood stock. This result is corroborated by Ojekale et al. (2015), who used oral administration of C. populnea extract over a 64 day period to achieve a four-fold increase in sperm count in the test rats.
The testicular histology showed better packed spermatozoa in group of rats treated with C. populnea, suggesting that oral administration of C. populnea aqueous extract improves spermatogenesis in male rats (Ojekale et al., 2015). On the other hand, S. longepedunculata inclusion also improves spermatogenesis in low concentration but at high dose, there was low sperm count and low motility which could be as a result of toxicity of the extract (Akah and Nwambie, 1994). This also agreed with the finding of Dandekar et al. (2002) that S. longepedunculata contains some compounds that have negative effect on animal reproductive parameters. The possible mechanisms for the anti-gonad action of S. longepedunculata extract could be by exerting a direct inhibitory action on the testis which affects androgen biosynthesis pathways and the pituitary gland, thereby causing changes in gonadotrophin concentrations and subsequent spermatogenic impairment or changing the concentration of neurotransmitters (Sarkar et al., 2000).
The elevated values of sex hormones; testosterone, progesterone and estrogen as well as milt volume, milt count and motility in the 0.5ml/kg of feed inclusion of C. populnea was evident in high percentage fertilization and hatchability obtained. This was similar to the result of Sharma et al. (2009) who reported that two doses i.e. 50 and 100 mg/kg of aqueous extract of Anacyclus pyrethrum on administration in albino rats showed pronounced anabolic and spermatogenic effect in animals of respective groups.
This result was also in agreement with Ogbeche et al. (2002), that androgen is the most effective stimulator for spermatogenesis. Many researchers in recent time have authenticated the efficacy of plant extracts to increase sex hormones in animals. Fertility enhancing effects of aqueous stem bark extract of Lophira lanceolata in male rats was carried out by Etuk and Muhammad (2009), the result obtained from the testicular histology examination revealed increased spermatogenesis, which was attributed to the increase in testosterone level. Oremosu et al. (2013) investigated the effects of C. populnea and Panax ginseng on flutamide-induced testicular toxicities in pre-pubertal rats. The result revealed significant increase in the serum testosterone and estrogen level, suggesting that C. populnea can ameliorate the injury caused by toxicity.
Fish on diet 2 (0.5ml/kg CP) had the highest fecundity count compared with other treatments while fish on diet 4 (0.5ml/kg SL) showed the lowest fecundity count. The reduction could be attributed to the concentration of toxic substances in the leaves of the plant. This result was corroborated by Ajiboye et al. (2012) who investigated the effect of aqueous extract of S .longependunculata root on redox homoestasis of rat liver and kidney. Their result showed that the administration of aqueous extract of S. longepedunculata root at all doses produced a significant (p<0.05) decrease in the activity of superoxide dismutase (SOD) in the liver and kidney of treated animals. Dapar et al. (2007) also discovered that S. longepedunculata extract has histological damage on heart, liver, kidney and lungs of rats, suggesting the presence of toxic constituents. This result also revealed that the leaves extract of C. populnea is very useful as fertility enhancer in C. gariepinus brood stocks management since almost all the fertility variables in this study were markedly increased.

CONCLUSIONS
The present results showed that the inclusion of C. populnea up to 0.5ml/kg will enhance fertility without having any deleterious effect on the health of the fish. Hence, it could be included in the fish feeds, but with cautious because higher concentration can adversely affect the fish. In view of the above the leave extract of C. populnea up to 0.5ml/kg could be incorporated in C. gariepinus brood stock diet as fertility enhancer rather than using synthetic drugs that may not be easily affordable and may also have residual effect on the organism. This study also revealed that S. longepedunculata showed negative effects on the reproductive performance of C. gariepinus at both low and high concentrations.