Introduction
The use of aquaculture has grown over the years with efforts channeled towards bridging the gap between demand and supply of aquatic products by culturing aquatic animals in captivity. Today the success of many aquaculture operations depends largely on the availability of a steady supply of larvae for rearing to market size (Rottmann et al., Reference Rottmann, Shireman and Chapman1991). According to Adewolu & Adeoti (Reference Adewolu and Adeoti2010), one of the major problems identified that continually hinders the promotion and development of the aquaculture industries is the scarcity of fish fingerlings of a desired cultured species. Wild supply is fast depleting, hence this source is not capable of supplying the needed amount of fish seed for culture (FAO, 1996). Artificial propagation of the fish remains the only means to provide a more steady supply of desired fish species of good quality.
Many hormones have been researched for the purpose of controlled maturation and spawning of cultured fishes. Some are of natural or synthetic origin. The efficacy of human chorionic gonadotropin (HCG), acetone-dried carp pituitary extract, deoxycorticosterone acetate (DoCA), and Ovaprim®, fresh common carp pituitary gland (PG) has been reported by Adebayo & Popoola (Reference Adebayo and Popoola2008). Breeding performance of fish using pituitary of African catfish Clarias gariepinus (Burchell, 1822) (Olufeagba et al., Reference Olufeagba, Aluko and Omotosho1998, Olufeagba et al., Reference Olufeagba, Aluko, Omotosho and Raji1999), Nile Tilapia Oreochromis niloticus (Linnaeus, 1758) and many other non-pisci pituitary hormones (e.g. frog) (Nwokoye et al., Reference Nwokoye, Nwuba and Eyo2007) have also been well documented. However, little information is known about the effect of preservation of natural pituitary extract on the spawning performance of the fish.
African catfish C. gariepinus is one of the most popularly cultured and consumed fish in many parts of Africa, Asia and Europe (FAO, 1996). The artificial breeding pattern (i.e. sacrificing the male) and domestic processing method (decapitating the fish) make it a very cheap and steady source of acquiring natural inducing hormone (pituitary extract) for breeding (Fagbenro et al., Reference Fagbenro, Salami and Sydenham1993, Adebayo & Popoola, Reference Adebayo and Popoola2008). Considering the continuous increase in the price of synthetic hormones used in induced breeding, it is inevitable that cheaper means of artificially propagating cultured fishes need to be found. Hence, this study was designed to investigate the spawning performance of Clarias gariepinus induced to spawn using preserved (24 h preservation in ethanol) and fresh pituitary extract of Clarias gariepinus. Their performances were also compared with commonly use synthetic hormone Ovaprim®.
Materials and Methods
Thirty broodstocks (mean weight of 1.3 kg) of reproductive age (above 1 year comprising of 20 males and 10 females) were obtained from the University of Agriculture Makurdi Fisheries Research Farm, in Benue state, Nigeria. They were acclimatized for 2 weeks in concrete tanks and fed 45% crude protein Coppens® diet (Helmond, North Brabant. The Netherlands). Six male broodstocks were initially tranquilized with 150 mg/l solution of tricane methane sulphonate (MS222) (Wagner et al., Reference Wagner, Jeppsen, Arndt, Routledge and Bradwisch1997) and decapitated to remove the PG. This was done by cutting off the head of the male and the lower jaw. The ventral side of the brain was lacerated to expose the PG. The PGs were then collected with a pair of tweezers and placed in separate Petri dishes. This was preserved in 96% ethanol; 24 h after, another set of six male broodstock were decapitated to get the fresh PG (kept in empty Petri dishes). Both fresh and preserved PG were weighed, lacerated and diluted appropriately in 0.9 g/l saline solution to obtain a 1 g ml −1 solution of the prepared hormone. This was administered to the broodstock at a rate of 1 ml kg −1 of the female broodstock weight according to the method used by Nwokoye et al. (Reference Nwokoye, Nwuba and Eyo2007) and Efe et al. (Reference Efe, Ebere and Nenibarini2015). Ovaprim® synthetic hormone was used as the control and administered at a rate of 0.5 ml kg −1 of fish. In total, nine females were injected (three female per treatments).
The females were conditioned in different tanks according to the hormone administration it received. Latency period was about 9 h 30 min, at this time; the fish had started releasing their egg in bits in the holding chamber. Eggs from each female were then stripped into separate bowls according to the hormone administered and the eggs gently mixed for 10 s with the aid of a rinsed chicken feather. The mass of egg stripped from each fish was calculated by estimating the differences between the weight of fish before and after stripping. Weighing was done using a sensitive weighing balance. The total number of eggs spawned per fish was determined by multiplying the weight of striped eggs by the number of eggs in 1 g (using the relation of 700 eggs in a 1 g mass of egg reported by Tiamiyu et al., Reference Tiamiyu, Okomoda, Oyeniyi and Aperegh2015). A small portion of the eggs (15–25 egg range) from all the treatment was also isolated and measured under a photomicroscope fitted with a Dino eye to determine egg size before fertilization. Six of the remaining male were then tranquilized according to the methods of Wagner et al. (Reference Wagner, Jeppsen, Arndt, Routledge and Bradwisch1997) and sacrificed to obtain the testis used for fertilization. To remove any error as a result of variation in male broodstock quality, each testis were macerated into the same bowl and the sperm content mixed using a feather. An equal volume of milt was obtained by using a new syringe to suck the sperm from the bowl. This was then used to fertilize the eggs according to the hormone used to induce ovulation. Rinsed chicken feathers were then used to mix the eggs and sperm uniformly for 1 min, after which a small quantity of water (100 ml) was added and the content mixed again for another minute. The excess water, as well as the excess sperm, was then decanted out of the small bowl leaving behind the fertilized eggs. For the sake of accessing the breeding performance, triplicate representative sample of eggs (equal mass of 10 g amounting to 7000 eggs using the relation of 700 eggs in 1 g mass of egg reported by Tiamiyu et al., Reference Tiamiyu, Okomoda, Oyeniyi and Aperegh2015 for the same species) were spawned on nine nylon mesh substrate suspended over continuously oxygenated water in nine 50 l bowls. The bowls were tagged appropriately in triplicate according to the inducing hormone used. The fertilized eggs were distributed evenly over the nylon substrate with the aid of the feather used for mixing. The size of fertilized eggs was also recorded under a photomicroscope.
The time taken for the small portion of the eggs initially separated (to determine unfertilized egg size) to become opaque (dead eggs) was noted to estimate fertilization rate. Ella (Reference Ella1987) method was used to estimate percentage fertilization according to the formulae stated below.
$$\begin{equation*}
{\rm hence},{\rm{ }}\%{\rm{}}\ {\rm fertilization} = \ \frac{{N - b}}{N} \times 100
\end{equation*}$$
where (N) represents the total number of eggs striped, (b) number of bad eggs and was obtained from the relationship below:
$$\begin{equation*}
b = \frac{y}{x} \times N
\end{equation*}$$
where (x) is the total number of eggs spawned (7000 eggs) and (y) is the number of bad eggs counted. Hence, the number of good eggs (g) is denoted using: N − b.
The hatching rate of each cross was evaluated per crosses. The number of hatched larvae was determined using the ratio of hatch fry to total incubated egg number and expressing it as a percentage as shown below. Hatching rate was calculated as:
$$\begin{equation*}
\frac{{no.\,of\,hatched\,larvae}}{{total\,no.\,of\,spawned\,eggs}} \times 100
\end{equation*}$$
After hatching and yolk absorption of the larvae on the third day, 300 of the hatched fry from each treatment were stocked in triplicate glass aquariums (according to each treatment) and fed with shell-free Artemia ad libitum. Growth parameters of the hatchlings were observed for 15 days. Fish were bulk weighed every 3 days using a sensitive weighing balance and mean weights was obtained by dividing bulk weight by the number of fish that survive. Survival after been fed for the 15 days was recorded. Growth parameters were determined as follows:
-
1. Mean weight gained = W 2 − W 1
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2. Growth rate (g/d) =
$\frac{{{W_2} - {W_1}}}{{{t_{2 - {t_1}}}}}$
where W1 = initial weight (g)
W2 = final weight (g)
t2 − t1 = duration between W2 and W1 (days)
-
3. Specific growth rate (%/day) =
$\frac{{lo{g_e}( {{W_2}} ) - {\rm{\ }}lo{g_e}( {{W_1}} )}}{{{t_{2{\rm{\ }} - {\rm{\ }}{t_1}}}}}$
-
4. % survival rate =
$\frac{{{\rm{total\ number\ of\ fish\ - \ mortality}}}}{{{\rm{\ total\ number\ of\ fish}}}}{\rm{ \times 100}}$
Note, ‘Total number of fish’ herein denotes the total number of hatch fry (for % survival at first feeding) or total stocked larvae (for % survival after 15 days feeding trial).
-
5. % Deformity =
$\frac{{{\rm{total\ number\ of\ deformed\ larvae}}}}{{{\rm{\ total\ number\ of\ hatched\ larvae}}}}{\rm{ \times 100}}$
Note: deformed larvae were determined by direct observation and counting as appropriate. Generally, the criteria used to determine deformity were the absence of straight body, distinct head distinguished from the yolk and truncated or wavy body parts.
Descriptive statistics were analysed using mini tab 14 computer software followed by one-way analysis of variance (ANOVA), and when significant differences were observed data were separated using Fisher's least significant difference.
Results
The result of the hatching success of C. gariepinus are presented in Table 1. This study reveals that preservation led to significant weight loss (8.10%) in preserved PG compared with fresh ones (0.51%). There was no difference between the weight of the female and the striped eggs as a result of the different hormone. Similarly, fecundity was statistically the same (P > 0.05) across the treatments with synthetic (Ovaprim®) and natural hormone (PG). However, egg size before fertilization was higher in Ovaprim® induced females (0.69 mm) and lowest in fresh PG-induced females (0.59). The same trend was observed after fertilization. Furthermore, females induced with preserved pituitary had similar egg size compared with both Ovaprim® (0.81) and fresh PG (0.80) induced females. Fertilization was observed higher in the females induced with natural hormones (97.5% and 95.50% respectively for preserved and fresh pituitary). While the reverse was the case with hatchability as the study observed at a significantly higher percentage in Ovaprim® (64.52%) compared with females induced with preserved PG (59.74%). The least hatchability of 51.39% was observed with the application of fresh PG. Survival at the end of exogenous feeding was generally higher (94.5–96.1%) and was statistically the same across the treatments.
Characteristics and breeding performance of Clarias gariepinus broodstock induced using Ovaprim®, preserved and fresh pituitary extract
Means in the same row with different superscript letters differ significantly.
Early growth performance of fry from the different treatments fed with shell-free Artemia cyst ad libitum is shown in Table 2. There was no difference in the final weight of fry spawned with preserved PG and Ovaprim® (104 mg) while least weight was recorded in the fresh pituitary, the same trend was observed in weight gain and growth rate (90 and 94% for fresh PG and preserved PG/Ovaprim® respectively). However, there were no significant differences in the survival and percentage deformity of fish induced either with natural or synthetic hormone.
Growth performance of Clarias gariepinus fry from broodstock induced with different hormones
Means in the same row with different superscript letters differ significantly.
The results of water quality parameters as presented in this study for all treatment were all statistically the same, temperature ranged from 28.85 to 27°C, total dissolved solids ranged from 116.6 to 118.05 ppt, dissolved oxygen varied from 4.60 to 4.70, pH was between 5.71 and 5.73, conductivity also was between 232.5 and 240.0 (Table 3).
Water quality parameters of the experimental units
Means in the same column do not differ significantly.
Discussion
Despite decades of research on the induced breeding practices of cyprinids (common carp, grass carp, Chinese carps and Indian minor carps), catfish and sturgeon fishes using pituitary extracts (Horváth et al., Reference Horváth, Tamas and Seagrave2002, Solomon et al., Reference Solomon, Tiamiyu, Adamu and Okomoda2015), no study has been conducted to evaluate the effect of preservation on the efficacy of natural hormone on breeding performance. There were no differences in broodstock characteristics such as weight, fecundity, and mass of egg striped in the present study. Efe et al. (Reference Efe, Ebere and Nenibarini2015) earlier opined that fecundity is an important index in determining the reproductive capacity of fish and a measure of the efficiency of the inducing agent. Ataguba et al. (Reference Ataguba, Solomon and Onwuka2012) had also revealed that the aim of synthetic hormone administration for induced breeding is to ripen eggs in the ovaries of the fish and this is evident in the ease of stripping when gently pressed. Hence, the insignificant value of fecundity recorded in this study could be an indication of the equivalent efficiency of the hormones under study. The values of fecundity in this study are higher than that reported by Nwokoye et al. (Reference Nwokoye, Nwuba and Eyo2007) for Heterobranchus bidorsalis Geoffroy Saint-Hilaire, 1809 using homoplastic (9522) and Ovaprim® (11,349.23). Also, the values are higher that the reported works of Haniffa & Sridhar (Reference Haniffa and Sridhar2002) for Heteropneustes fossilis (Bloch, 1794) spawned using the combination of HCG and Ovaprim® (6336 ± 800 eggs). The findings of this study, however, are comparable with the works of Tiamiyu et al. (Reference Tiamiyu, Okomoda, Oyeniyi and Aperegh2015) and Solomon et al. (Reference Solomon, Tiamiyu and Ihundu2011).
Hatchability was better in Ovaprim® induced females than those induced with natural hormones (PG). Ovaprim® has been reported to be highly effective in many freshwater and marine water species (Rowland, Reference Rowland1983, Solomon et al., Reference Solomon, Tiamiyu, Adamu and Okomoda2015). Today, it is probably the most popular synthetic hormone used among fish farmers to induce ovulation in cultured species (Solomon et al., Reference Solomon, Tiamiyu and Ihundu2011). Because of increasing cost, research has been focused on finding an alternative as well as adding suitable diluents to induce ovulation at a minimal cost. Solomon et al. (Reference Solomon, Tiamiyu and Ihundu2011); (Reference Solomon, Tiamiyu, Adamu and Okomoda2015) had earlier reported ovulation in C. gariepinus and common carp Cyprinus carpio Linnaeus, 1758 administered diluted Ovaprim® hormones (normal saline as diluents) up to 900% (1:9) and 500% (1:5) respectively. However, the report of Tiamiyu et al. (Reference Tiamiyu, Okomoda, Oyeniyi and Aperegh2015) suggests that optimum dilution of Ovaprim® using coconut water and saline water as diluents were 400% (1:4) and 100% (1:1) respectively at a homogenized latency period of 10 h. The result of the present study is similar to these reported works, hence, preservation of natural hormone may be a better choice for the fish farmer to reduce the cost of breeding than diluting Ovaprim® with diluents. Although, the breeding cost was not estimated in this study, however, the breeding pattern and domestic processing method of the African catfish would enable the acquisition of natural inducing hormone at no cost.
Hatchability in this study is higher in value but similar in trend to the observations of Olaniyi & Akinbola (Reference Olaniyi and Akinbola2013) for Clarias gariepinus induced with Ovaprim and catfish PG (46.3 and 25.9% respectively). Saidin (Reference Saidin, Macleen, Diz and Hosillos1986) had also reported low hatching rate of between 10–45% for Clarias macrocephalus Günther, 1864. However, this study falls within the range of 51–73% reported by Adebayo & Popoola, (Reference Adebayo and Popoola2008) using different hormone treatments. The better hatchability recorded in Ovaprim® may be because it leads to the ovulation of large egg size compared with the natural hormone. Egg size has been previously reported to have significant positive correlation with many breeding traits such as hatchability (Ataguba et al., Reference Ataguba, Okomoda and Onwuka2013), larval length and survival (Buckley et al., Reference Buckley, Smigielski, Halavik, Caldorone, Burns and Laurence1991 and Rideout et al., Reference Rideout, Tippel and Litvak2005). Hempel (Reference Hempel1979) had also reported that larger eggs provide more energy for larvae development which is explained by a larger yolk sac. Similar observation to the present study was reported by Olaniyi & Akinbola (Reference Olaniyi and Akinbola2013) as they noted that yolk absorption was faster in catfish pituitary induced and spawned fry compared with those induced and spawned with Ovaprim®, probably due to differences in egg size. However, the superior performance of preserved PG over fresh PG may be attributed to water removal and contraction of PG leading to possibly higher concentration of PG extract injected to the fish. This is justified by the fact that preserved PG in ethanol are sometimes referred to as acetone-dried pituitary extract (Fagbenro et al., Reference Fagbenro, Salami and Sydenham1993) and this study recorded significant weight loss of PG. Efe et al. (Reference Efe, Ebere and Nenibarini2015) have previously opined that hatching success is hormonal dose dependent. Solomon et al. (Reference Solomon, Tiamiyu, Adamu and Okomoda2015) and Tiamiyu et al. (Reference Tiamiyu, Okomoda, Oyeniyi and Aperegh2015) had also established facts that suggest that reduction in the efficacy of the hormone as a result of dilution significantly affect performance in for C. carpio and C. gariepinus respectively. This is similarly to the findings of Haniffa & Sridhar (Reference Haniffa and Sridhar2002) using variable hormonal doses. Although the doses used in this study were standardized by volume, the acetone preservation procedure that led to drying and moisture loss (high % weight loss of PG) would have interfered with the concentration of preserved PG administered, hence, better performance.
The results of many previous studies on induced breeding with non-piscine sources of pituitary extract are in line with the findings of this study. Nwadukwe (Reference Nwadukwe1993) had reported the efficacy of frog PG in oocyte maturation and spawning success of Heterobranchus longifilis Valenciennes, 1840 (hatchability of 63.08%). Fagbenro et al. (Reference Fagbenro, Salami and Sydenham1993), however, reported insignificant spawning performance of Clarias isheriensis (Sydenham, 1980) when administered acetone-dried pituitary extracts obtained from common toad Bufo regularis, African bullfrog, Rana adspersa, and chicken, Gallus domesticus, as replacement for piscine–sourced pituitary extracts. The variation in values recorded in these study compared with the present study are likely due to the efficacy of the hormone used for induces breeding as well as the species of fish induced.
The result of the growth performance revealed comparatively same performance using preserved PG or Ovaprim® (P > 0.05). This was, however, higher than values recorded for fresh PG (P < 0.05). It is well known that Ovaprim® is a mixture of the analogue of salmon gonadotropin releasing hormone (sGnRHa) and a dopamine antagonist domperidone (Goudie et al., Reference Goudie, Simco, Davis and Parker1992; Hill et al., Reference Hill, Kilgore, Pouder, Powell, Watson and Yanong2009), hence, it could be rightly described as a product of processed and preserved natural hormone for a long period of time. However, the mechanism of how preservation enhances the efficacy of hormone in inducing ovulation is beyond the scope of this study, hence, the need for more research. According to Ajah (Reference Ajah2007), fry survival rate depends on several factors such as feed availability, pH, temperature, dissolved oxygen, ammonia, nitrite, nitrate, etc. However, Efe et al. (Reference Efe, Ebere and Nenibarini2015) reported that fry survival could also be as a result of differences in the type of hormone administration. Contrary to this observation, this study has demonstrated that differences in hormonal administration may not have a significant effect on the survival rate of fish. The contradictions of the two study may be as a result of differences in the rearing period of the study, more so water quality parameters obtained in this study were all within the recommended range reported by Boyd (Reference Boyd1981, Reference Boyd1997), and hence were not considered to have affected the outcome of this research. Although deformity has been linked to type of hormonal administration in previous study (Nwokoye et al., Reference Nwokoye, Nwuba and Eyo2007, Ayoola et al., Reference Ayoola, Kuton and Chukwu2012, Efe et al., Reference Efe, Ebere and Nenibarini2015), the extremely low levels of deformity observed in this research are indications that deformity may necessarily not be caused as a result of differences in hormonal administration but by other factors one of which may be inbreeding depression of broodstock. This is justified by the observations of deformities in the control and the insignificant values of deformity recorded across the treatment. This hypothesis is subject to verification in future studies.
Conclusion
This study has demonstrated the superior performance of preserved PG over fresh PG, it could also be inferred that inducing catfish with ethanol-preserved PG gave a similar performance as that of commercially sold expensive synthetic hormone Ovaprim®. Hence, the use of preserved PG can be exploited in the commercial production of catfish fry. Future study can focus on the effect of prolonged preservation time on the efficacy of the hormone. Short-term preservation, as well as long-term cryopreservation protocols, can be developed to ensure utilization of testis obtained from fish whose pituitary has been extracted. This will further cut the cost of catfish production and may lead to a new breeding system that could be described as a closed spawning system (as both inducing hormone and testis are obtained from the same fish).
