Impact of Temperature Variations on Breeding Behavior of Cirrhinus mrigala during Induced Spawning

Saeed Akram Khan1, Shahid Sherzada1*, Muhammad Ashraf1, Durr-e-Shehwar1, Sonia Iqbal1, Usman Atique1,3, Farzana Abbas1, Sohail Ahmad2 and Muhammad Sultan Haider4 1Department of Fisheries and Aquaculture, University of Veterinary and Animal Sciences, Lahore, Pakistan 2Department of Poultry Production, University of Veterinary and Animal Sciences, Lahore, Pakistan 3Department of Bioscience and Biotechnology, Chungnam National University, South Korea 4Department of Zoology, University of Lahore, Pakistan Article Information Received 10 May 2020 R vised 28 June 2021 Accepted 09 July 2021 Available online 10 September 2021


INTRODUCTION
C irrhinus mrigala is one of the most important Indian major carps and integral component of present semi-intensive fish polyculture system (Beyers and Rice, 2002). It is benthic feeder targeting detritus and left over food items from other surface feeders. It does not breed in captivity therefore it is induced to spawn by variety of fresh and synthetic hormones.
The induced breeding in hatcheries assisted in mass production of quality carp seed under controlled condition and assured timely supply of stocking material for culture farms (Mohapatra et al., 2016). Induced breeding is based on the principles of manipulating hormonal or O n l i n e

F i r s t A r t i c l e
is growth or breeding (Mills and Hurley, 1990). When temperature deviates from the normal, it stresses the fish and impairs physiological and behavioral activities (Beyers and Rice, 2002;Donaldson et al., 2008). For larval fishes, water temperature can influence growth rates (Leggett and Deblois., 1994), swimming performance and predator avoidance (Leggett and Deblois., 1994;Pepin, 1991), as well as metabolic rate (Blaxter, 1991) and cellular function (Somero and Hofmann, 1997). Large, abrupt changes in temperature can induce significant larval mortality which in turn has profound impacts on year-class strength (Planque and Fredou, 1999). Temperature effects can be positive or negative. Temperature induces growth, maturity, and decreases egg hatching rate if in acceptable range; if violates, it then disrupts its whole physiology and kills the fish consequently affecting recruitment (Pepin, 1991;Marimuthu et al., 2000). However, fish species which are capable of tolerating wide temperature ranges (eurythermal) behave quite differently and temperature effects are less prominent (Marimuthu et al., 2000). Present study was therefore, conducted to explore the optimum breeding temperature with maximum output in terms of release of eggs, rate of fertilization, hatching, embryological stages and survival of fry produced in C. mrigala in hatchery conditions.

Breeding trials
Healthy and mature fish were collected from pond complex of Department of Fisheries and Aquaculture, University of Veterinary and Animal Sciences Ravi Campus Pattoki, Punjab, Pakistan, and transferred to hatchery in brood stock holding tanks for conditioning. Four breeding trials were conducted at different temperatures during whole breeding seasons. Total of 36 brooders (24 males and 12 females) used in this study were divided into combination of two males and one female fish in each circular tank. Fish was induced to breed at four different temperatures viz. 26, 29, 32 and 34ºC. Other water quality parameters like dissolved oxygen, electrical conductivity and pH were also recorded (Table I). Prior to hormone administration fish were weighed and then the hormone dosage was calculated following standard injection rate of 0.4 ml/kg body weight to female and 0.2 ml to male. The males and females were released into respective circular spawning tanks maintained at temperatures of 26 ºC, 29 ºC, 32 ºC, and 34 ºC at different times of peak spawning season. During spawning process water flow was maintained at the rate of 23 liter/min to facilitate ovaprim best performance in inducing ovulation in female and release of sperms in male. In 26 o C group female fish started to release eggs after 8 h and 35 min in 26 ºC group, after 9½ h in 29 ºC group, after 9 h and 38 min in 10 h and 16 min in 34 ºC group after ovaprim injection.

Counting of eggs and hatchlings
Hatching rate was calculated from the number of eggs in a brood and the number of larvae hatched out (based on the method of Soundarapandian, 2008). The number of live ones was estimated volumetrically by taking 50 ml samples (Yen and Bart, 2008).

Examination of developmental stages of eggs
Eggs were collected at different time intervals from the incubation tank and each stage was comparatively observed to identify effects of varying temperatures. Egg diameter and yolk mass were measured with the help of eye piece micrometer.

Statistical analysis
Data were analyzed by using one-way ANOVA technique (F test, P≤0.05) to assess the impact of temperature (26, 29, 32 and 34 ºC) on reproduction, fecundity and hatching of C. mrigala. Significant treatment means were separated through Duncan's Multiple Range test with the help of SAS software (version 9.1). Following mathematical model was applied: Y ij = µ + τ i + ε ij where, Y ij is observation of dependent variable recorded on i th treatment group; µ is population mean; τ i is effect of i th treatment group (i = 1, 2, 3, 4) and ε ij is residual effect of j th observation on i th treatment group NID ~ 0, σ 2 .

Water quality parameters
The study indicated slight change in water quality parameters like dissolved oxygen, electrical conductivity and pH with increase in temperature; however, this change was not influential on breeding and larval development. Therefore, a non-significant influence of these parameters was observed on ovulation, fertilization and hatchability of Cirrhinus mrigala. These results were in line with a study conducted on Lake Malawi Tilapia (Chambo), Oreochromis karongae in which dissolved oxygen, electrical conductivity, pH and ammonia did not significantly influenced hatching period and the decrease in hatching period was attributed to temperature increase (Valeta et al., 2013). So, the temperature is an important factor determining egg and larval development as it influences metabolic rate (Blaxter, 1991;Kamler, 2008) and cellular function (Somero and Hofmann, 1997).

O n l i n e F i r s t A r t i c l e
Effects of temperature on fecundity During current study, the fish released eggs after 9.79 h of ovaprim administration in T 1 , 9.59 h in T 2 , 10.70 h in T 3, and 11.32 h in T 4 (Table II) which means that fishes kept at 29ºC took the minimum time to ovulate and when compared it with T 1 , T 3, and T 4 (Table II). Brooders in T 4 took maximum time (11 h 17 min) to ovulate. These observations were in close agreement with those of Nandeesha et al. (1990). Egg releasing time directly corresponded to rise in temperature but the opposite was true when temperature reduced from 29 ºC. Similar results were reported at temperature range of 29-32 ºC in which spawning was observed after a latency period of 5-6 h (Mohapatra et al., 2018). Maximum fecundity was observed in 29 ºC group, whereas minimum was observed in at 34 ºC; moreover, a slight variation in fecundity at 26 ºC and T 3 (31 ºC) was also noted. Current findings reveal that 29 ºC is an optimum temperature for maximum effectiveness of ovaprim and subsequent release of eggs which is evident by the number of eggs released which

O n l i n e F i r s t A r t i c l e
was the highest number among all the treatments (Table  II). These findings are very close to those of El-Gamal (2009) on breeding of common carp in which temperatures higher than optimum proportionately decreased fecundity. The Maximum number of eggs were observed in 29 o C group whereas the lowest were obtained in 34 o C. Similar findings were observed by Herzig and Winkler (1986) where maximum number of eggs (98503 eggs) were at 29 ºC and the lowest number of eggs (1937) were obtained at 34 ºC. These findings validate that exceeding temperature beyond optimum limit affect the eggs released.

Effects of temperature on fertilization rate
Current findings on C. mrigala further confirm that the highest fertilization rate was observed at 29 ºC and the lowest eggs got fertilized at 34 ºC (91.58 vs. 75.49%). Similarly, number of hatchlings produced were lower in 34 o C group while the higher were reported in 26 o C (91.3 vs. 63.48%). Similar fertilization rate of 95-100% and 90-98% hatching success was observed in Labeo rohita at a water temperature of 28-31 ºC using hormone Ovatide (Pandey et al., 2002). Fry recovered after yolk sac absorption followed the same pattern as observed in egg fertilization rate; however, spawn production was found to be lower as compared to the results by Mohapatra et al. (2018) in which spawning fecundity was found to be 0.13-0.182 and 0.125-0.158 million egg/ kg female body weight of Labeo rohita and C. mrigala, respectively. Similar results were observed in case of fertilization in which fertilized eggs (%) during spawning was found to be 90-100% for both species (Mohapatra et al., 2018). Spawn production per kg female body weight was found to be lower as compared to Mohapatra et al. (2016) in which spawn production per kg female body weight was found to be 0.109-0.14 million eggs/kg for C. mrigala and spawn survival (%) from fertilized egg ranged 87.3-94.74% in C. mrigala. Previously it has been observed that abrupt fluctuations in temperature negatively affected growth rates of larval fishes (Leskelä and Kucharczyk, 1995), metabolic rate (Blaxter, 1991) and cellular functions (Somero and Hofmann, 1997). These findings are quite in line with findings in current study which further confirmed that increase in temperature beyond optimum limit is not favorable to the developing egg. For the reasons it is evident from our finding and can also be suggested that 29 ºC is the optimum temperature for induced breeding of C. mrigala in Punjab Pakistan. Any sort of fluctuations in terms of increase or decrease in temperature pose a noteworthy impact on egg release, fertilization percentage and hatching of eggs while though it is slightly in contradiction to the findings of Das et al. (2006) who reported the highest hatching rate at 31 ºC in Labeo rohita which might be due to species differences. Table II also shows the effect of temperature on the early developmental stages specifically up till morula stage. Microscopic examination of egg development stages revealed denaturation in egg contents when temperature exceeded beyond optimal limit. Previous studies have demonstrated that temperature has direct effects on incubation of eggs (Kamler et al., 1998;Rechulicz et al., 2002). In present study, eggs incubated at 29ºC showed normal development pattern while increase in temperature to some extent fasten the embryonic development. Similar findings confirm our results that embryonic development responds positively during optimum temperature and if temperature deviates from the normal then it shows abnormal developments and sometimes resulting in even death of egg and/or hatchling by Kamler (2008) and Bermudes and Ritar (1999). Faster multiplication of cells decreased hatching time but decreased hatching rate indicating that 32-34 ºC is not favorable to normal embryonic development. Eggs kept at 27-29 ºC appeared more robust with healthier hatchlings than those kept at 32-34 ºC. It was observed that at high temperature time difference between different stages was reduced and a gap of 25 to 30 min was observed between some stages (Fig. 1). Higher temperatures also increased rupturing of the egg shells that sometimes led to premature hatching and consequently death of hatchlings which was very much evident in that batch where temperature remained over 32 ºC, and lot of larvae perished at different stages of development even when healthy larvae were transferred to this temperature they could not flourish and succumbed (Fig. 2). Effect of different temperatures was not limited to fertilization and hatching but when hatched out larvae were measured larvae developed at 29 ºC significantly longer to those grown up at 26 or 32 ºC.

CONCLUSION
Temperature has a great influence on fish breeding. Increase in temperature gradually enhances the breeding performance and embryonic development up to optimum value. In our study Exposure of eggs of C. mrigala to temperature exceeding 32ºC during early stages of development resulted in an abnormal development of the eggs. High temperature beyond optimum limit resulted in egg shell thinning and premature hatching of eggs which perished before the absorption of yolk sac. This study could provide useful insights into the role of temperature in induced spawning of fishes.