Spermatological parameters of wild and cultured beluga (Huso huso Linnaeus, 1758) and their effects on fertilization rate, hatching rate, larval growth and survival rates

[Research]

Spermatological parameters of wild and cultured beluga (Huso huso Linnaeus, 1758) and their effects on fertilization rate, hatching rate, larval growth and survival rates

Z. Madadi, H. Khara*

Department of Fishery, Lahijan Branch, Islamic Azad University, Lahijan, Iran

* Corresponding author’s E-mail: h.khara1974@yahoo.com

ABSTRACT

To compare the reproductive performance of the wild and cultured stocks of beluga, Huso huso males, some spermatological parameters were measured including: sperm motility, sperm density, spermatocrit and also fertilization rate, hatching rate and larval growth and survival rates. The semen samples were sampled from 11 wild spawners and 12 cultured brooders. A half of each semen sample was allocated for spermatological analysis and remaining were used for artificial propagation. In  wild beluga, sperm motility time  was 339.09 ± 23.11 s; spermatozoa motility rate  83.64 ± 2.01%; sperm density 15.22 ± 3.46 ×10⁹ and spermatocrit 5.88 ± 1.36% while  in cultured beluga, sperm motility time was 199.35 ± 29.02 s; spermatozoa motility rate 74.22 ± 2.4 %; sperm density 8.37 ± 0.24 ×10⁹ and spermatocrit 3.21 ± 0.29 %. The fertilization rate (%), hatching rate (%), larval growth (% g.day^-1).  and survival rate (%) in wild beluga were 61.72 ± 14.16, 43.33 ± 11.13, 12.28 ± 0.33 and 52.66 ± 7.77, while  in cultured beluga were 49.98 ± 6.55, 31.47 ± 8.49, 12.28 ± 0.62 and 57.37 ± 7.89 respectively. The spermatozoa motility (the ratio and duration of motility) were significantly different between wild and cultured brooders (P<0.05). The values of sperm density, spermatocrit, fertilization rate, hatching rate, larval growth and survival rate were not significantly different between wild and cultured beluga (P> 0.05).

INTRODUCTION

The Great sturgeon (or beluga), Huso huso Linnaeus, 1758 is the most valuable and unique sturgeon fish in the Caspian Sea due to the desirability of its meat and caviar throughout the world. During the last decades, the wild stocks of beluga have declined dramatically due to various reasons such as overfishing, water pollution and construction of dams and destruction of rivers (Kiabi et al. 1999). Thus, this sturgeon has been considered for biological conservation and also restocking program by means of artificial propagation in the southern basin of the Caspian Sea (Kiabi et al. 1999). Unfortunately, the efficiency of artificial propagation of wild stocks of beluga is adversely inconstant depending on brooder and gamete availability. To eliminate this problem, the production of cultured brooders of beluga in the hatchery conditions has been considered as a strategy to overcome the shortage of needed gametes for artificial breeding. On the other hand, the very long period is required to occur first sexual maturation in sturgeons especially beluga that makes their hatchery production very expensive. Furthermore, hatchery conditions are apparently different from wild environment and may affect the process of gonadal maturation and quality of gametes. For example, under culture condition, adult fishes are exposed to various types of stressors which may affect gonad development and gamete quality (Donaldson 1981; Barton & Iwama 1991; Pankhurst & Van Der Kraak 1997; Wendelaar Bonga 1997;  Iwama et al. 2005; Milla et al. 2009). Campbell et al. (1994) reported that chronic confinement of hatchery ponds not only disrupt the reproductive endocrinology of trout, but also result in reduced egg size and larval survival rate in rainbow trout, Oncorhynchus mykiss (Walbaum). In captive condition, the fertilization rate, gamete quality and spawning behavior of spotted grouper, Epinephelus akaara are influenced adversely (Okumura et al. 2002).

Due to these reasons, the efficiency of hatchery production of brooders of beluga should be investigated. Therefore, in the present study, we investigated the reproductive performance of the wild and cultured males of beluga, by means of spermatological parameters and also fertilization, hatching, larval growth and survival rates.

MATERIALS AND METHODS

This study was carried out in Shahid Marjani Artificial Sturgeon Propagation and Rearing Center, Gorgan, Iran. Two groups of beluga males were considered for the experiment: 1) cultured fish (n = 12, total weight (TW) = 23.00 ± 3.43kg and fork Length (FL) 151 ± 8.88 cm). These brooders were reared under captivity conditions in the hatchery for a period of 4 years. 2) The wild fish (n = 11, TW = 72 ± 7.98Kg;   FL = 193 ± 9.73 cm). These brooders had been captured in the southeastern part of the Caspian Sea during their upstream reproductive migration, and then transferred to the hatchery.  To induce ovulation in females, two dosages of pituitary extract including first dosage (20 mg dry pituitary/2 ml 0.7 % NaCl) followed by the final dosage (180-200 mg dry pituitary/3 ml 0.7 % NaCl) 12h later were injected intramuscularly. Also, to induce spermiation in males, 1 vial LHRH-A2 was injected coincident with final injection of females. After hormonal treatment, the brooders were checked out every 4 h one time to detect the final maturation. When the ovulation and spermiation occurred, the sperm quality of males was examined. To induce sperm motility, the semen samples was diluted  in ratio of 1 (semen) : 2000 (freshwater) followed by evaluating  sperm motility rate (%) immediately  and  sperm motility time after activation (%) under dark-field microscope coupled with a CCD video camera (SONY DXC-970MD, Japan) (Cosson et al. 2000). The video records were investigated by Adobe Premiere software and percentage of sperm motility was calculated in comparison with 12 captured pictures from 1s motility video. The duration of sperm motility was calculated after the onset of motility until all cells become immotile (Cosson et al. 2000).

Only forward-moving spermatozoa were classified as motile, while sperm cells simply vibrating or turning on their axes were considered as immotile (Aas et al. 1991). The sperm density was measured by haemocytometer counting chamber according to Hajirezaee et al. (2010a).  So that, milt was diluted 1000 times in a 0.7% NaCl solution. Then, a droplet of the diluted milt was placed on a haemocytometer slide (depth 0.1 mm) with a coverslip. After 10 min (a time to allows sperm sedimentation), the spermatozoa were counted in 16 individual cells of haemocytometer under a light microscopy (Nikon, Japan). At the end, the number of spermatozoa was calculated as follows:

Sperm density (ml^-1) = 1000 × number of counted sperm / [area (mm²) × chamber depth (mm) × dilution ratio.

Spermatocrit was determined by centrifuging semen for 10 min at 5000 rpm in a haematocrit centrifuge (D-78532, Tuttlingen Zentrifugen, Germany) according to Piironen (1985).

After egg collection from 3 wild females, the coelomic fluid was separated, the eggs were pooled and weighted and then divided into 23 trays (each tray containing 100 g pooled egg; 11 trays for wild fish and 12 trays for cultured ones).

For fertilization, the diluted semen with freshwater in a ratio of 1 ml (semen): 200 ml (freshwater) was added to each tray and then mixed for 4-5 min.

After fertilization, to eliminate the egg adhesion, the fertilized eggs were washed by 10% clay suspension for 40 min.

After washing, the eggs fertilized by sperm obtained from wild and cultured males, were incubated separately in Yushchenko incubators. 1 % Permanganate potassium solution was used against fungal contamination during incubation period of eggs. The fertilization rate (%) was calculated in the stage 4 blastomeres as follows:

Fertilization rate (%) = the number of fertilized eggs/total number of eggs × 100 

After about 6 days, the fertilized eggs were hatched. The hatching rate was calculated as follow:

Hatching rate (%) = the number of hatched eggs/total number of fertilized eggs × 100

The specific growth rate (SGR) and survival rate (SR) of alevins until active feeding and thereafter were calculated as follow:

SGR (%) = 100 × (ln W₂ - ln W₁)/days, where W₁ = Mean of primary weight, W₂ = Mean of final weight; SR (%) = (Total number of alive alevins / Total number of alevins) × 100

Statistical analysis

The SPSS software was used for data analysis. Because  the data [SR (%),  SGR (%),  fertilization rate (%),  hatching rate (%),  spermatocrit (%) and  sperm motility rate (%)]  did not have a normal distribution, proportional data were converted by angular transformation (arcsin √p).

The independent sample T-test was employed to compare the means between cultured and wild fish.

RESULTS

According to T-test analysis, there was significant differences between wild and cultured males of beluga in terms of the rate (%) and time of sperm motility (Fig. 1, P<0.05).  The motility rate (%) and sperm motility time of wild spermatozoa were significantly higher than spermatozoa taken from cultured beluga (P<0.05). The values of sperm density, spermatocrit (%) (Fig. 2, P> 0.05), fertilization rate (%), hatching rate (%) (Fig. 3, P> 0.05), SGR (%) and survival rate (%) (P> 0.05, Table 1) were similar in wild and cultured males.

Fig 1. Comparison of rate (%) and time of sperm motility between the sperm obtained from wild and cultured male beluga. Means with same superscripts are not significantly different (P> 0.05).

Fig 2. Comparison of sperm density and spermatocrit between the sperm obtained from wild and cultured male beluga, Huso huso. Means with same superscripts are not significantly different (P> 0.05).

Table 1. Comparison of larval growth and survival rates between the larvae obtained from the eggs fertilized with the sperm originating from wild and cultured male beluga, Huso huso. Means with same superscripts were not significantly different (P> 0.05).

* Survival rate ₁: survival rate of hatched alevins until active feeding; Survival rate _2: survival rate of hatched alevins after active feeding_; Survival rate _T: survival rate of hatched alevins over the course of the experiment; Specific growth rate ₁: Specific growth rate of hatched alevins until active feeding; Specific growth rate _2: Specific growth rate of hatched alevins after active feeding_; Specific growth rate _T: Specific growth rate of hatched alevins over the course of the experiment.

Fig 3. Comparison of fertilization and hatching rates between the sperm obtained from wild and cultured male beluga, Huso huso. Means with same superscripts are not significantly different (P> 0.05).