Pacific abalone (Haliotis discus hannai) is a highly commercial seafood in Southeast Asia. The aim of the present study was to improve the sperm cryopreservation technique for this valuable species using an antifreeze protein III (AFPIII). Post-thaw sperm quality parameters including motility, acrosome integrity (AI), plasma membrane integrity (PMI), mitochondrial membrane potential (MMP), DNA integrity, fertility, hatchability, and mRNA abundance level of heat shock protein 90 (HSP90) were determined to ensure improvement of the cryopreservation technique. Post-thaw motility of sperm cryopreserved with AFPIII at 10 µg/mL combined with 8% dimethyl sulfoxide (DMSO) (61.3 ± 2.7%), 8% ethylene glycol (EG) (54.3 ± 3.3%), 6% propylene glycol (PG) (36.6 ± 2.6%), or 2% glycerol (GLY) (51.7 ± 3.0%) was significantly improved than that of sperm cryopreserved without AFPIII. Post-thaw motility of sperm cryopreserved with 2% MeOH and 1 µg/mL of AFPIII was also improved than that of sperm cryopreserved without AFPIII. A combination of 10 µg/mL AFPIII with 8% DMSO resulted in the highest post-thaw motility, showing AI of 60.1 ± 3.9%, PMI of 67.2 ± 4.0%, and MMP of 59.1 ± 4.3%. DNA integrity of sperm cryopreserved using 10 µg/mL AFPIII combined with 8% DMSO was not significantly (p > 0.05) different from that of fresh sperm. Cryopreservation using a combination of AFPIII with 8% DMSO improved fertilization and hatching rates of sperm compared to that of cryopreservation without supplementation of 10 µg/mL AFPIII. Sperm cryopreserved using AFPIII showed higher mRNA abundance levels of HSP90 than those cryopreserved without AFPIII. Results of the present study suggest that 10 µg/mL AFPIII combined with 8% DMSO can be used for large scale cryopreservation of Pacific abalone sperm and for hatchery production.

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Axonemal protein 66.0 is one of the major axonemal dynein protein involved in activation of sperm motility. The axonemal protein 66.0 gene was successfully cloned and characterized from testis tissue in the Pacific abalone, Haliotis discus hannai. The cloned gene was named Hdh-Axp66.0 based on the predicted molecular mass. The full-length cDNA of Hdh-Axp66.0 was 2023 bp, with a coding region of 1694 bp; it encoded 564 deduced amino acids with a predicted molecular mass of 65.42 kDa and an isoelectric point of 8.68. In silico analysis prediction suggested this gene has two cAMP-dependent protein kinase phosphorylation sites, seven predicted protein kinase C phosphorylation sites, three N-linked glycosylation sites, a tyrosine kinase phosphorylation motif, three coiled-coil domains, and several Ca 2+ binding sites, which revealed the possible involvement of Hdh-Axp66.0 in activation of sperm motility. Tissue distribution analysis indicated that Hdh-Axp66.0 was distributed in testis, heart, gill and mantle tissues. However, quantitative real-time PCR (qPCR) revealed that Hdh-Axp66.0 mRNA was highly expressed in testis tissue. Further, highly elevated relative mRNA expression was observed in testis tissue during the ripening stage compared with different testicular developmental stages. These results suggest the possible involvement of Hdh-Axp66.0 in testicular development and reproduction.

The research investigated the impacts of inclusion of column feeder rohu (Labeo rohita) on growth and production in freshwater prawn-carp-mola polyculture system for a period of 172 days. Four stocking densities of Rohu were maintained as 500, 1,000, 1,500 and 2,000 ha -1 in treatment R 500 , R 1000 , R 1500 and R 2000 , respectively in triplicates. All ponds each 120 m 2 were stocked with juvenile freshwater prawn (Macrobrachium rosenbergii), silver carp (Hypophthalmichthys molitrix), catla (Catla catla) and small fish mola (Amblypharyngodon mola) at the fixed stocking densities of 20,000, 1,500, 1,000 and 20,000 ha -1 , respectively. Prawns were fed with pelleted feed twice daily started with 10% and gradually reduced to 3% of body weight and continued throughout the study period. All fish were fed with mixture of soaked rice bran and mustard oilcake (2:1) at the rate of 3% of the body weight daily. All the water quality parameters and chlorophyll-a were measured. The density of rohu significantly (P<0.05) influenced the survival rate, growth and production of freshwater prawn. Catla and Mola production were affected adversely with increasing rohu density. The production of rohu increased with increasing density although the individual weight decreased. The combined production of all finfish was significantly lower in R 0 whereas, the combined production of all species including prawn did not differ significantly (P<0.05) among the treatments. The treatments R 0 and R 500 fetched higher net profit without significant difference between them. Therefore, inclusion of rohu at a density of 500 ha -1 may be recommended for prawn-carp-mola polyculture.

Temperature has crucial effects on gonadal development and reproduction of abalone. To understand the impact of thermal stress on molecular and physiological processes triggering the regulation of reproduction, changes in the mRNA expression of neuroendocrine genes encoding two abalone gonadotropin-releasing hormone (Hdh-GnRH, Hdh-GnRH-like peptide), GnRH receptor (Hdh-GnRH-R), Hdh-APGWamide, serotonin receptor (5-HThdh), and a heat shock protein HSP70 were examined in ganglia and testis of male Pacific abalone (Haliotis discus hannai). Abalone were exposed to low water temperature (LWT) and high water temperature (HWT) in early and peak breeding seasons for 7 days. Then, gonadosomatic index (GSI) was calculated, relative gene expression was measured by qRT-PCR, and levels of testosterone in hemolymph were also measured by ELISA during the peak breeding season. GSI did not show any significant changes during the early breeding season. However, it was significantly decreased in LWT- or HWT-exposed abalone compared to the normal water temperature (NWT) group during the peak breeding season. In the early breeding season, changes of mRNA expression of all five genes were significant between LWT and HWT groups on day-7. In the peak breeding season, compared to the NWT group, the mRNA expressions of different genes were significantly decreased in different tissues both in LWT and HWT groups of abalone, such as Hdh-GnRH-like peptide in the cerebral ganglion (CG) and testis; Hdh-GnRH in the pleuropedal ganglion (PPG) and branchial ganglion (BG); Hdh-GnRH-R in the CG, PPG, and testis; and Hdh-APGWamide in the PPG and testis. Interestingly, the expression of 5-HThdh was significantly increased in the HWT group but decreased in the LWT group. Expression of HSP70 was significantly increased in the testis after exposure to HWT. Hemolymph levels of testosterone were significantly decreased in the HWT group compared to those in the NWT group. Altogether, these results denote that thermal stress has a repressive effect on gonadal maturation and reproduction by regulating the expression of Hdh-GnRH-like peptide, Hdh-GnRH, Hdh-GnRH-R, Hdh-APGWamide, 5-HThdh, and HSP70 genes and levels of hemolymph testosterone.