Water hardness inuenced reproductive potential in two freshwater sh species; Poecilia reticulata and Betta splendens

Objective: Hardness of water in the form of CaCO3 affect reproductive potential in various sh species, differently. The study evaluates the effect of water hardness on growth and reproduction of two aquarium shes, Poecilia reticulata (Ovo-viviparous sp.) and Betta splendens (Oviparous sp.) by growing them under 150 (control), 320, 540 and 900 ppm CaCO3 levels in semi natural aquaria. Results: Growth increased with increasing water hardness, reporting a signicant growth of P. reticulata (p=0.005) at 900ppm. Similarly, the reproductive potential of P. reticulata was improved signicantly, recording the highest fecundity (16.22 ± 3.90) and Gonadosomatic Index (GSI-2.48 ± 0.597) at 900ppm. However, in B. splendens water hardness adversely affected the reproduction by reporting a signicantly low hatchability and disturbed bubble nests at 900ppm, compared to the largest bubble nest formed at the control (108.58 ± 16.19 cm2). Thus, study revealed differential effects of water hardness on reproductive potential of the test species, by increasing the potential of P. reticulata while decreasing that of B. splendens. Though larval survival was affected in both species, larval growth was improved signicantly in P. reticulata at 900ppm level. Understanding reproductive potential of aquarium shes in natural waters is crucial for their propagation purposes. .


Introduction
Water hardness i.e. a measure of Calcium (Ca2+), Magnesium (Mg2+) and/or Iron (Fe2+) in water, is crucial for the growth, reproduction and embryo development of sh [1][2][3][4]. It affects the growth and reproduction of oviparous and ovoviviparous shes differently due to variations in their requirement of CaCO3 in the reproduction [1,[5][6][7][8]. Considering the importance of hardness in the early life stage processes such as hatchability, larval growth and survival of eggs, it is recommended to maintain the water hardness above 20 mg/L CaCO3 [9,10].
Water hardness in Vavuniya District, of the Dry Zone of Sri Lanka reports remarkably high values, 100 -1000 mg/L [11]. In Vavuniya, ornamental sh trade is mainly depended on ground water. Thus, evaluating the effect of higher hardness on sh health is of prime economic and ecological importance.
Freshwater species, Poecillia reticulata (Guppy) and Betta splendens (Siamese ghting sh) were ovoviviparous and oviparous shes, respectively and were popular verities in the aquarium sh trade. P.reticulta produces eggs that are hatched within the body (ovoviviparous, live bearers) and the hatchlings are born alive while B.splendens lay eggs in a foam nest (oviparous, egg layers) for external fertilization. Both species, can be easily reared and bred under laboratory conditions. The study anticipates to evaluate the effect of water hardness on two aquarium sh species; P.reticulata (Guppy) and B.splendens (Siamese ghting sh) as representatives for ovo-viviparous & oviparous shes by measuring the growth performance of adults and larvae, and reproductive potential in terms of gonadosomatic Index (GSI), fecundity, fertility, hatchability and larval survival under semi-natural aquaria conditions.

Preparation of Aquaria
Glass aquaria of the size of 25x13x12 cm3 (24 tanks), simulating natural pond environment were used for the exposure. Being an aggressive sh, male B.splendens were planned to keep in 20 separate cubic aquaria (12x12x12 cm3) at a rate of each male per cube.
The experiment composed of control (tap borne water 150 ppm CaCO3) and three treatment setups; 320, 540 and 900 ppm prepared by adding analytical grade CaCO3 to aged tap water. The hardness was determined by EDTA titration [12] and the treatment setups were screened weekly to maintain the hardness. All the experiments were conducted as per the guidelines given by the research review panel of the Department of Bioscience, University of Jaffna.

Introduction of shes to Aquaria & Maintenance
Ovo-viviparous sh as P.reticulata and oviparous shes as B.splendens were chosen for the experiment.
The virgin shes of both species (males and females) were purchased from a nearby aquarium in Vavuniya and were transferred to the laboratory. Sexing was done by examining external morphology, where male shes of both species possessed narrow and bright colored bodies and colorful caudal ns compared to round bellies and short caudal ns of the female shes, displaying sexual dimorphism.
Initial weight and standard length were measured, reporting 0.48753 ± 0.00833 g/ 2.9844 ± 0.0522 cm for P.reticulata and 1.2667 ± 0.0721 g/ 3.4969 ± 0.0120 cm for B.splendens. Then the shes were acclimatized to aquarium condition (20 min. in each setup) and introduced to the experimental setups, 150 ppm (control), 320, 540 and 900 ppm CaCO3 and reared for 1½ month. This series was selected to cover the hardness range, 100-1000 ppm in Vavuniya [8].In each experimental setup 15 females were introduced to 5 males of B.splendens, separately. To avoid aggression, each male of B.splendens was kept separately in the smaller aquaria. For, P.reticulata, 25 females were introduced to 5 males. The exposure was conducted with three replicates.
Feeding (5% of the total body weight) were done twice a day at ad libitum with commercial sh pellets. The aquaria were maintained to keep the temperature 25-27 ºC and pH 6.5-7.5 by replacing the media with newly prepared hard water on weekly basis. Debris was siphoned out. Mild aeration level was maintained as the shes are air breathers.

Determination of growth performance
Weight and length of adult sh were measured, and the length weight relationship was analyzed , using W= a TLb: (Log W = log a + b log TL) [13] to obtain the linear regression.

Determination of reproductive potential
Two gravid females from each setup (N= 12 per each species) were randomly tested for fecundity (the number of ripening eggs found in the female just prior to spawning) and GSI (the ratio of sh gonad weight to body weight). Euthanizing was conducted with 0.02% MS222 solution.
Reproductive potential of B.splendens To estimate the bubble nest size of B.splendens, three sets of clean breeding aquaria (size-60x30x30 cm3) were prepared without arti cial bottom stones and aeration. A oating plant leaf was put on the air water interface to facilitate the nest formation. After adding the male in to the breeding aquarium, a clean glass cube containing female sh was placed near it to stimulate the nest building. Then the bubble nests built by male B.splendens were measured (diameter) by a ruler [14].
Hatchability of B.splendens was estimated after a successful courtship with a gravid female where repeated introductions of the females were required. Without disturbing the bubble nest, the number of eggs released was counted. After mating female was removed, and male was allowed for 24 -48 hour for pre hatched parental care. Next, the hatched larvae were counted, and the hatchability was determined, as the number of larvae hatched over the total number eggs [15].
Reproductive potential of P. reticulata Fertility of P.reticulata was determined by counting the intra-follicular embryos inside the femaleby sacri cing few shes (N=6) at the 21 st day after mating [16]. Breeding tank of P.reticulata was formed with the arti cial aquarium stones and Vallisneria to provide hiding place for young ones. After female broods release young ones, they were separated from their parents and counted.
Larval survival rate was considered after one week of exposure to the hardness treatments by counting the number of surviving one weeks-old larvae divided by the total number of hatched larvae / released young ones [15]. The larval growth performance (N=12) was estimated in every 10 days interval by measuring the length gain.

Statistical analyses
Normality of the data set was tested before applying the statistics with SPSS 20.0 (IBM, USA). One-way ANOVA and Tukey pairwise comparison were used to analyse the differences of weight, standard length, fecundity and Gonadosomatic Index, Laval growth (length) of the shes. The linear regression analysis was used to nd the length weight relationship (LWR). In LWR linear regression analysis, the slope of regression lines explicit the exponent coe cient value 'b'. Signi cant variation in the estimates of 'b' for the sh species was examined from the expected value (ideal value 'b' = 3) was tested by t-test [17,18].
Students t-test was applied to analyze the variation i.e. derived by dividing the difference between 'b' and '3' by standard error of 'b' [19].

Results
Growth performance of exposed shes Growth improved with the increasing water hardness by showing signi cantly high weight values, particularly above 540 ppm treatments (p<0.05), reaching 80% and 40% weight increments, respectively for P.reticulata (N=30) and B.splendens (N=20) in the highest hardness level (900ppm). Similarly, length values of the both species also increased with higher hardness levels, though only 14-17 % increments were recorded.
When the growth pattern was estimated P.reticulata showed isometric growth at 540 and 900 ppm levels while B.splendens showed isometric growth in all hardness levels. (Table 1).
According to Shim and Ho [20], dissolved Calcium is essential for growth of live bearer (ovoviviparous) sh especially P.reticulata as they are native to hard waters. They also found that rearing P.reticulata in extreme water hardness (2500ppm) showed 10 times higherweight gain than in soft water (167ppm). In the same way, James and Sampath [21] found that Xiphophorus helleri (live bearer) reared in 1018ppm hardness level exhibited better growth performance compared to 76ppm level. Water hardness in uenced growth performances of P.reticulata were further reiterated by weight and length relationship, which revealed isometric growth occurred only above 540ppm level. On the other hand, increasing water hardness showed no apparent effect on B.splendens resulting isometric growth in all hardness levels. Similarly, common snook Centropomus undecimalis and largemouth bass Micropterus salmoides exposed to higher hardness showed no apparent growth performances compared to their counter parts [3,4].
Reproduction of P.reticulata was enhanced showing faster sexual maturation, with higher hardness conditions in compliance with James and Sampath [21] and Stratton [22], who reported higher and faster sexual maturation of X. helleri, in exceptionally high hardness medium. In line with this observation, Shim and Ho, [20] suggested that dissolved calcium is essential for sexual maturation of P.reticulata. As an oviparous sh, B.splendens showed slightly lowered reproductive potential under higher hardness. This is reiterated by previous work, Ratinam [8] who found suppressed gonadal development and maturity of Pterophyllum scalare and aborted maturation in Barbus conchonius and Barbus letrazona beyond 120 ppm hardness. Further, in compliance with previous studies, it was observed that high calcium in hard water deposit on the surface of the eggs of B.splendens, blocking the water absorbed into the perivitelline [7,23] leading to dehydration and shrinking of the eggs [6]. Thus, being an oviparous sh B.spledens is suitable to the soft water than the hard water environment.
Increasing hardness caused high mortality in larvae of both species. Newborn are unable to tolerate the adverse environmental factors like extreme hard water [24] due to the stress condition in the physiology created by excess amount of calcium. Numerous studies carried on various sh species; Clarias gariepinus, Atlantic salmon (Salmo salar), rainbow trout (Oncorhynchus mykiss), and brown trout (Salvelinus fontinails), Rutilus frisii kutum (kutum), reiterated this nding [6,7,25,26]. Thus, soft water is preferable for larval rearing for both P.reticulata and B. splendens.
Hence, it may conclude that P.reticulata requires more calcium for the growth and reproduction than B.splendens which grow and reproduce well in soft water environment.

Limitations
This study was not intended to describe mechanism/s of action of CaCO3 in mediating growth and reproductive alteration of P.reticulata and B.splendens.