Reproductive strategy and sperm cryopreservation of a spermcasting bivalve - the Australian flat oyster Ostrea angasi

Author: Md Hassan

Hassan, Md, 2017 Reproductive strategy and sperm cryopreservation of a spermcasting bivalve - the Australian flat oyster Ostrea angasi, Flinders University, School of Biological Sciences

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Spermcasting is a unique mode of reproduction in which males release gametes in water and females acquire male gametes and fertilize eggs inside the mantle cavity. Although spermcasting occurs in variety of sessile species, this spawning strategy is the least known among other reproductive modes in marine species. Some spermcasting bivalves are also important species in aquaculture but the breeding programs of these species are hindered due to the poor understanding of spermcast spawning and lack of techniques to control reproduction. This thesis aims to (a) elucidate adaptive strategies in the reproduction of a spermcasting species - the Australian flat oyster Ostrea angasi, (b) develop techniques for its sperm quantification, and (c) develop protocols sperm cryopreservation. Five experiments were conducted to achieve the thesis aims.

In Experiment 1, the structure and functional properties of male gametes were investigated to understand the reproductive strategy in O. angasi. The male gametes of this species are released in a spermatozeugma where a cluster of sperm is bounded by a gelatinous membrane. In seawater, individual sperm swim off the spermatozeugmata by dissociating the membrane. The sperm of O. angasi have one more mitochondrion than broadcasting oysters although their sperm dimensions are similar. The duration of spermatozeugmata dissociation and sperm motility varied among individuals of different masculine levels. The hermaphrodites with a large proportion of male gametes could maintain spermatozeugmata integrity and sperm motility longer than those with a small proportion of male gametes. Spermatozeugmata structure and functional properties have adaptive significance in gamete dispersal in seawater and fertilization success in the female mantle cavity.

In Experiment 2, adaptive strategies in gametogenesis, sex ratio and energy metabolism were studied to further understand the advantages of spermcast spawning in O. angasi. During gametogenesis, different stages of spermatozeugmata were found within a male or hermaphroditic oyster while oocytes mature simultaneously within a female or hermaphroditic oyster. The histological observation of partially spawned hermaphroditic oyster indicated that spermatozeugmata would be released before egg ovulation. In the sex ratio analysis of 2-3 years old O. angasi, the percentage of male, female, hermaphrodite and undifferentiated sexes was 41.3%, 5.8%, 46.7% and 6.2%, respectively. Glycogen was the main energy source for gametogenesis. The periods of energy storage versus energy utilization overlapped, and O. angasi displayed an intermediate energy metabolism strategy between conservative and opportunistic species. The patterns in gamete development, sex ratio and energy metabolism of O. angasi are of significance to reproductive and physiological adaptations in spermcasting species.

In Experiment 3, a rapid, cost-effective and reliable technique for estimation of sperm concentration was developed with spectrophotometry. A regression model of y = 1×10-8 x + 0.163; r² = 0.996 was generated at 350 nm wavelength. The model was validated by comparing sperm counts with the haemacytometer method. The spectrophotometric technique would increase the efficiency of sperm concentration determination and facilitate breeding programs and cryopreservation.

In Experiments 4 and 5, sperm cryopreservation protocols were developed by using programmable and non-programmable freezing methods. Sperm motility and plasma membrane integrity were used as indicators to assess sperm quality. The highest post-thaw sperm survival was achieved by the non-programmable freezing method, with the protocol including sperm equilibration with 15% ethylene glycol + 0.2 M trehalose for 20 min, package in 0.25 ml straws, exposure to liquid nitrogen vapor for 10 min at 8 cm above the liquid nitrogen surface, and storage in liquid nitrogen. This protocol would open a new option for effective cryopreservation to assist the development of controlled breeding and genetic improvement programs.

This thesis provides new knowledge to understand the reproductive strategy and physiological adaptation of the spermcasting oyster O. angasi. The regression model established with spectrophotometry provides a rapid method to quantify sperm concentration in this species. The newly-developed sperm cryopreservation protocol could overcome the seasonal constraints in sperm supply and provide a year-round superior sperm stock for breeding programs. The research outcomes of this thesis would enhance the capacity and efficiency for development of breeding programs in spermcasting molluscan species.

Keywords: Oyster, Reproduction, sperm, cryopreservation, spermcasting, mollusc, bivalve, Ostrea angasi

Subject: Biological Sciences thesis

Thesis type: Doctor of Philosophy
Completed: 2017
School: School of Biological Sciences
Supervisor: Jian Qin