The cryopreservation potential and ultrastructure of Agulhas sole Austroglossus pectoralis spermatozoa
- Authors: Markovina, Michael Zeljan
- Date: 2008
- Subjects: Spermatozoa , Spermatozoa -- Cryopreservation , Aquaculture , Fishes -- Breeding , Soleidae , Flatfishes , Agulhas Current (South Africa)
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:5251 , http://hdl.handle.net/10962/d1005094 , Spermatozoa , Spermatozoa -- Cryopreservation , Aquaculture , Fishes -- Breeding , Soleidae , Flatfishes , Agulhas Current (South Africa)
- Description: As the estimated market demand for the Agulhas sole Austroglossus pectoralis exceeds the annual catch from trawlers, this species is a potential aquaculture candidate. Broodstock conditioning and gamete preservation is part of research and development aiming at establishing a breeding protocol for a new aquaculture species. Based on a literature review of the morphology of pleuronectiform spermatozoa, this study was designed firstly, to contribute to the field of spermatozoan morphology by describing the ultrastructure of A. pectoralis spermatozoa. This was followed by an experiment to cryopreserve mature spermatozoa to provide baseline data for future studies on this and related species. The testis of A. pectoralis was a paired structure encased in a membrane, the tunica albuginea. The primary testis was located on the dorsal surface of the rib cage and the secondary testis on the ventral side. The testis was of an unrestricted spermatogonial type, based upon observations of spermatogonia along the entire length of the lobule. Mature spermatozoa of A. pectoralis had an acrosome-free ovoid head 1.68 ± 1.6μm in length and 1.7 ± 1.6μm in diameter, a short mid-piece of 0.5 ± 0.1μm in length, containing 7 irregularly shaped mitochondria forming a ring-like structure at the base of the nucleus. The flagellae were 47.4 ± 4.8μm in length, most with two plasma membrane lateral fin-like projections. However, some flagellae had either zero or three lateral fin projections. Cross-sections of the flagellae showed an axenome with a 9+2 microtubule configuration. The proximal and distal centriols were coaxal, situated deep within the nuclear fossa. The structure of A. pectoralis spermatozoa conformed to the type 1 ect-aquasperm, also found in externally fertilizing species. This type has been suggested to be the plesiomorphic form in Neopterigians. Finally, this study contributed to a cryopreservation protocol for A. pectoralis spermatozoa by testing the two cryoprotectants dimethyl sulphoxide (DMSO) and glycerol. Glycerol, at a concentration of 10%, offered better cryoprotection than DMSO. This was established using flow cytometry analysis of post-thaw nuclear membrane integrity after 64 days of storage in liquid nitrogen. The toxicity of DMSO to isolated cellular proteins may have resulted in DMSO-treated sperm having the highest percent (35.2% ± 3.2%) of non-viable cells compared with 23.0% ± 2.5% and 27.8% ± 3.4% for glycerol and the control, respectively. The presence of sucrose in the Modified Mounib Medium extender solution may explain why 45.5% ± 5% of the sperm cells were potentially viable in the control treatment. Initially, the white margined sole Dagatichthys marginatus (Soleidae) was selected as the most suitable candidate for flatfish aquaculture in South Africa. Thus, the aim of this study was to investigate the cryogenic potential and ultrastructure of D. marginatus spermatozoa. However, due to a skewed sex ratio, there were not enough males available to study this species. A skewed sex ratio is common amongst soleids, thus, the need to develop effective cryopreservation methods and to develop an understanding of sperm morphology so that the best time for cryopreservation can be chosen. In conclusion, this first description of spermatozan morphology of A. pectoralis contributed to our understanding of soleid sperm ultrastructure. In addition, a comparison of testis appearance between fish sampled just prior to spawning season and fish with mature sperm provided information on the spawning season of this species. The findings from the cryopreservation experiment suggested that glycerol was a feasible cryoprotectant for this species when sperm was prepared under field conditions.
- Full Text:
- Date Issued: 2008
- Authors: Markovina, Michael Zeljan
- Date: 2008
- Subjects: Spermatozoa , Spermatozoa -- Cryopreservation , Aquaculture , Fishes -- Breeding , Soleidae , Flatfishes , Agulhas Current (South Africa)
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:5251 , http://hdl.handle.net/10962/d1005094 , Spermatozoa , Spermatozoa -- Cryopreservation , Aquaculture , Fishes -- Breeding , Soleidae , Flatfishes , Agulhas Current (South Africa)
- Description: As the estimated market demand for the Agulhas sole Austroglossus pectoralis exceeds the annual catch from trawlers, this species is a potential aquaculture candidate. Broodstock conditioning and gamete preservation is part of research and development aiming at establishing a breeding protocol for a new aquaculture species. Based on a literature review of the morphology of pleuronectiform spermatozoa, this study was designed firstly, to contribute to the field of spermatozoan morphology by describing the ultrastructure of A. pectoralis spermatozoa. This was followed by an experiment to cryopreserve mature spermatozoa to provide baseline data for future studies on this and related species. The testis of A. pectoralis was a paired structure encased in a membrane, the tunica albuginea. The primary testis was located on the dorsal surface of the rib cage and the secondary testis on the ventral side. The testis was of an unrestricted spermatogonial type, based upon observations of spermatogonia along the entire length of the lobule. Mature spermatozoa of A. pectoralis had an acrosome-free ovoid head 1.68 ± 1.6μm in length and 1.7 ± 1.6μm in diameter, a short mid-piece of 0.5 ± 0.1μm in length, containing 7 irregularly shaped mitochondria forming a ring-like structure at the base of the nucleus. The flagellae were 47.4 ± 4.8μm in length, most with two plasma membrane lateral fin-like projections. However, some flagellae had either zero or three lateral fin projections. Cross-sections of the flagellae showed an axenome with a 9+2 microtubule configuration. The proximal and distal centriols were coaxal, situated deep within the nuclear fossa. The structure of A. pectoralis spermatozoa conformed to the type 1 ect-aquasperm, also found in externally fertilizing species. This type has been suggested to be the plesiomorphic form in Neopterigians. Finally, this study contributed to a cryopreservation protocol for A. pectoralis spermatozoa by testing the two cryoprotectants dimethyl sulphoxide (DMSO) and glycerol. Glycerol, at a concentration of 10%, offered better cryoprotection than DMSO. This was established using flow cytometry analysis of post-thaw nuclear membrane integrity after 64 days of storage in liquid nitrogen. The toxicity of DMSO to isolated cellular proteins may have resulted in DMSO-treated sperm having the highest percent (35.2% ± 3.2%) of non-viable cells compared with 23.0% ± 2.5% and 27.8% ± 3.4% for glycerol and the control, respectively. The presence of sucrose in the Modified Mounib Medium extender solution may explain why 45.5% ± 5% of the sperm cells were potentially viable in the control treatment. Initially, the white margined sole Dagatichthys marginatus (Soleidae) was selected as the most suitable candidate for flatfish aquaculture in South Africa. Thus, the aim of this study was to investigate the cryogenic potential and ultrastructure of D. marginatus spermatozoa. However, due to a skewed sex ratio, there were not enough males available to study this species. A skewed sex ratio is common amongst soleids, thus, the need to develop effective cryopreservation methods and to develop an understanding of sperm morphology so that the best time for cryopreservation can be chosen. In conclusion, this first description of spermatozan morphology of A. pectoralis contributed to our understanding of soleid sperm ultrastructure. In addition, a comparison of testis appearance between fish sampled just prior to spawning season and fish with mature sperm provided information on the spawning season of this species. The findings from the cryopreservation experiment suggested that glycerol was a feasible cryoprotectant for this species when sperm was prepared under field conditions.
- Full Text:
- Date Issued: 2008
The biotechnology of effluent-grown Spirulina, and application in aquaculture nutrition
- Authors: Maart, Brenton Ashley
- Date: 1993
- Subjects: Aquaculture , Spirulina , Algae -- Biotechnology , Fishes -- Feeding and feeds
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:4050 , http://hdl.handle.net/10962/d1004111 , Aquaculture , Spirulina , Algae -- Biotechnology , Fishes -- Feeding and feeds
- Description: The biotechnology of production and utilisation of the cyanobacterium Spirulina has been well documented. Research has centred mainly on application in human and animal nutrition, and has been motivated by the high protein, vitamin, fatty acid and growth factor contents. The main obstacle in realising the full potential of this feed source has been the high production costs associated with its mass culture in defined media. The observation of blooms of Spirulina in tannery effluent evaporation ponds in Wellington, South Africa, prompted this investigation into the harvesting, and nutritional and toxicological evaluation of this potentially low-cost production system, with the ultimate aim of using the product in aquaculture rations. An investigation of the chemical gradient along the evaporation cascade showed a positive correlation between the prevailing chemical conditions and the dominant species populations. A standing crop of 9.5 tonnes/ha of Spirulina was found to be present in the latter alkaline ponds, characterised by relatively lower organic and sulphur contents. Initial harvesting of the biomass was achieved by the design, construction and implementation of a small-scale screen harvest, which yielded a 25 kg (dry weight) crop. A scale-up model was then designed, and implemented in a technical scale harvest, yielding a crop of 250 kg (dry weight). Both these harvests utilised the bloom of surface-autoflocculated biomass. Concentrated cell slurries were sun-dried on muslin beds, and milled to a coarse powder. An evaluation of the harvest revealed a chemical content similar to other published reports of defined media cultures, with the exception of the protein and amino acid contents. The observed lower levels of the latter two are almost certainly due to the sun-drying method employed, known to reduce the protein content due to thermal denaturation. Legislation demands the strict toxicological evaluation of new protein sources, and because of the effluent-nature of the growth medium of this source of Spirulina, its viability lies only in the application as an animal feed or supplement. A range of toxicological tests were chosen that were targeted to elucidate the possible toxicological constraints of this effluentgrown source of protein in animal nutrition. The nucleic acid and pesticide contents of the harvested biomass were within the prescribed safety ranges. Atomic absorption showed minimal accumulation of minerals and heavy metals from the effluent. A bioassay with the brine shrimp Anemia salina showed that the biomass contained no toxicologically active water-soluble components. A short term feeding trial with new-born chicks showed that supplementation with Spirulina had no effect on the growth rates and feed conversion ratios of the different feeding groups. Pathological analyses showed that the liver was the only target organ to elicit a change in response to supplementation of the diets with Spirulina. A general decrease in liver weight was noted, with Cu, Ca, Fe and Zn being significantly accumulated. A histopathological examination however, showed no cellular and functional aberration from the control animals. The toxicological analyses gave the preliminary safe go-ahead for the evaluation of effluent-grown Spirulina in aquaculture nutrition. The South African abalone Haliotis midae, and the rainbow trout Oncorhynchus mykiss were chosen as representative species of edible cultured organisms. The technology for the culture of the perlemoen abalone is being established in South Africa, with the main area of research being the development of an artificial diet for high density culture. A 40 day growth trial demonstrated that lower concentrations of Spirulina supplemented to an agar-based fishmeal diet resulted in growth rates and feed conversion ratios similar to the control fishmeal and purified-casein diets, and thus has application potential in the nutrition of this high-cost marine delicacy. The aquaculture technology of freshwater rainbow trout is already well established. An eight week feeding trial with various concentrations of Spirulina showed that this effluent-grown protein source can partially replace fishmeal in semi-purified diets. Fish fed Spirulina did not exhibit decisive manifestations of toxicity, as determined in a histopathological study. In addition, Spirulina supplementation resulted in enhanced colouration of the skin and flesh, which may have implications in the aesthetic marketing of this sought-after table fish. The primary aim of this preliminary investigation thus concerned the determination of the biotechnological potential of this effluent-source of Spirulina. A technology transfer from the economically unfeasible defined-media culture was implemented. This project is ultimately aimed as a contribution towards the treatment of tannery wastewater, by the removal of contaminants from the effluent in the form of organic biomass.
- Full Text:
- Date Issued: 1993
- Authors: Maart, Brenton Ashley
- Date: 1993
- Subjects: Aquaculture , Spirulina , Algae -- Biotechnology , Fishes -- Feeding and feeds
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:4050 , http://hdl.handle.net/10962/d1004111 , Aquaculture , Spirulina , Algae -- Biotechnology , Fishes -- Feeding and feeds
- Description: The biotechnology of production and utilisation of the cyanobacterium Spirulina has been well documented. Research has centred mainly on application in human and animal nutrition, and has been motivated by the high protein, vitamin, fatty acid and growth factor contents. The main obstacle in realising the full potential of this feed source has been the high production costs associated with its mass culture in defined media. The observation of blooms of Spirulina in tannery effluent evaporation ponds in Wellington, South Africa, prompted this investigation into the harvesting, and nutritional and toxicological evaluation of this potentially low-cost production system, with the ultimate aim of using the product in aquaculture rations. An investigation of the chemical gradient along the evaporation cascade showed a positive correlation between the prevailing chemical conditions and the dominant species populations. A standing crop of 9.5 tonnes/ha of Spirulina was found to be present in the latter alkaline ponds, characterised by relatively lower organic and sulphur contents. Initial harvesting of the biomass was achieved by the design, construction and implementation of a small-scale screen harvest, which yielded a 25 kg (dry weight) crop. A scale-up model was then designed, and implemented in a technical scale harvest, yielding a crop of 250 kg (dry weight). Both these harvests utilised the bloom of surface-autoflocculated biomass. Concentrated cell slurries were sun-dried on muslin beds, and milled to a coarse powder. An evaluation of the harvest revealed a chemical content similar to other published reports of defined media cultures, with the exception of the protein and amino acid contents. The observed lower levels of the latter two are almost certainly due to the sun-drying method employed, known to reduce the protein content due to thermal denaturation. Legislation demands the strict toxicological evaluation of new protein sources, and because of the effluent-nature of the growth medium of this source of Spirulina, its viability lies only in the application as an animal feed or supplement. A range of toxicological tests were chosen that were targeted to elucidate the possible toxicological constraints of this effluentgrown source of protein in animal nutrition. The nucleic acid and pesticide contents of the harvested biomass were within the prescribed safety ranges. Atomic absorption showed minimal accumulation of minerals and heavy metals from the effluent. A bioassay with the brine shrimp Anemia salina showed that the biomass contained no toxicologically active water-soluble components. A short term feeding trial with new-born chicks showed that supplementation with Spirulina had no effect on the growth rates and feed conversion ratios of the different feeding groups. Pathological analyses showed that the liver was the only target organ to elicit a change in response to supplementation of the diets with Spirulina. A general decrease in liver weight was noted, with Cu, Ca, Fe and Zn being significantly accumulated. A histopathological examination however, showed no cellular and functional aberration from the control animals. The toxicological analyses gave the preliminary safe go-ahead for the evaluation of effluent-grown Spirulina in aquaculture nutrition. The South African abalone Haliotis midae, and the rainbow trout Oncorhynchus mykiss were chosen as representative species of edible cultured organisms. The technology for the culture of the perlemoen abalone is being established in South Africa, with the main area of research being the development of an artificial diet for high density culture. A 40 day growth trial demonstrated that lower concentrations of Spirulina supplemented to an agar-based fishmeal diet resulted in growth rates and feed conversion ratios similar to the control fishmeal and purified-casein diets, and thus has application potential in the nutrition of this high-cost marine delicacy. The aquaculture technology of freshwater rainbow trout is already well established. An eight week feeding trial with various concentrations of Spirulina showed that this effluent-grown protein source can partially replace fishmeal in semi-purified diets. Fish fed Spirulina did not exhibit decisive manifestations of toxicity, as determined in a histopathological study. In addition, Spirulina supplementation resulted in enhanced colouration of the skin and flesh, which may have implications in the aesthetic marketing of this sought-after table fish. The primary aim of this preliminary investigation thus concerned the determination of the biotechnological potential of this effluent-source of Spirulina. A technology transfer from the economically unfeasible defined-media culture was implemented. This project is ultimately aimed as a contribution towards the treatment of tannery wastewater, by the removal of contaminants from the effluent in the form of organic biomass.
- Full Text:
- Date Issued: 1993
The chemistry of a new water-recirculation aquaculture system with emphasis on the influence of ozone on water quality
- Authors: Stobart, Michael David
- Date: 1992
- Subjects: Water quality -- Environmental aspects -- Testing , Ozone -- Physiological effect , Aquatic ecology , Aquaculture
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:5244 , http://hdl.handle.net/10962/d1005087 , Water quality -- Environmental aspects -- Testing , Ozone -- Physiological effect , Aquatic ecology , Aquaculture
- Description: Water quality changes that occur during establishment and maintenance of nitrification in two identical recirculating aquaculture systems containing rainbow trout are described. The time taken for the nitrification process to become established was 40-45 days. Mortality of fish attributed to elevated nitrite concentrations were recorded during the initial conditioning phase of the systems. Un-ionised ammonia concentrations did not attain lethal levels during this period. Nitrate concentrations accumulated slowly throughout the study, while the pH and alkalinity of the water decreased with progressive nitrification. Levels of carbon dioxide, calcium, dissolved and suspended solids remained relatively stable until the carrying capacity of the systems was increased, upon which they increased rapidly and general water quality deteriorated. Permanently elevated concentrations of ammonia and nitrite served as an indication that the carrying capacity of the systems had been exceeded. The use of ozone as a water enhancement treatment in aquaculture systems during one- and six-hour applications was also considered in this study. Ozonation significantly reduced nitrite levels at · low concentrations (0,1 - 0,15 mg/l), although they returned to pre-treatment levels within a few hours of cessation of the treatment. The formation of an unstable, intermediate product that reforms as nitrite in the absence of ozone, rather than the complete oxidation of nitrite to nitrate, is proposed. Ozonation also resulted in decreased dissolved solids, and improved the clarity and odour of the water. Ozone had no effect on ammonia concentrations (at pH > 7,0), or on nitrate or calcium levels, and did not conclusively increase the redox potential of the water. Residual ozone concentrations up to 0,04 mg/l in a sixhour treatment had no harmful effects on rainbow trout, and there were no signs of gas-bubble disease arising from supersaturation of the water with oxygen. Activated carbon filters were effective at removing residual ozone from the water after treatment. Treatment of the water with oxygen alone had no effect on nitrite concentrations.
- Full Text:
- Date Issued: 1992
- Authors: Stobart, Michael David
- Date: 1992
- Subjects: Water quality -- Environmental aspects -- Testing , Ozone -- Physiological effect , Aquatic ecology , Aquaculture
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:5244 , http://hdl.handle.net/10962/d1005087 , Water quality -- Environmental aspects -- Testing , Ozone -- Physiological effect , Aquatic ecology , Aquaculture
- Description: Water quality changes that occur during establishment and maintenance of nitrification in two identical recirculating aquaculture systems containing rainbow trout are described. The time taken for the nitrification process to become established was 40-45 days. Mortality of fish attributed to elevated nitrite concentrations were recorded during the initial conditioning phase of the systems. Un-ionised ammonia concentrations did not attain lethal levels during this period. Nitrate concentrations accumulated slowly throughout the study, while the pH and alkalinity of the water decreased with progressive nitrification. Levels of carbon dioxide, calcium, dissolved and suspended solids remained relatively stable until the carrying capacity of the systems was increased, upon which they increased rapidly and general water quality deteriorated. Permanently elevated concentrations of ammonia and nitrite served as an indication that the carrying capacity of the systems had been exceeded. The use of ozone as a water enhancement treatment in aquaculture systems during one- and six-hour applications was also considered in this study. Ozonation significantly reduced nitrite levels at · low concentrations (0,1 - 0,15 mg/l), although they returned to pre-treatment levels within a few hours of cessation of the treatment. The formation of an unstable, intermediate product that reforms as nitrite in the absence of ozone, rather than the complete oxidation of nitrite to nitrate, is proposed. Ozonation also resulted in decreased dissolved solids, and improved the clarity and odour of the water. Ozone had no effect on ammonia concentrations (at pH > 7,0), or on nitrate or calcium levels, and did not conclusively increase the redox potential of the water. Residual ozone concentrations up to 0,04 mg/l in a sixhour treatment had no harmful effects on rainbow trout, and there were no signs of gas-bubble disease arising from supersaturation of the water with oxygen. Activated carbon filters were effective at removing residual ozone from the water after treatment. Treatment of the water with oxygen alone had no effect on nitrite concentrations.
- Full Text:
- Date Issued: 1992
An investigation into the dietary requirements of Oreochromis Mossambicus fry and the formulation and preparation of a dry food for use in aquaculture
- Authors: Thorpe, Peter Stuart
- Date: 1989
- Subjects: Cichlids , Tilapia -- Nutrition , Aquaculture
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:5184 , http://hdl.handle.net/10962/d1001960 , Cichlids , Tilapia -- Nutrition , Aquaculture
- Description: The need for developing a dry feed which satisfied the nutrient requirements of Oreochromis mossambicus fry was identified. The spawning and grading techniques which resulted in a higher fecundity and ensured uniformity within and between samples of fry are discussed. Preparation techniques were developed which met the physical requirements of fry feeds. An analysis of the flesh and yolk sac fry for amino acid composition was undertaken. The initial test diet was based on these results, as well as on the natural feeding ecology of the species. Feeding trials were undertaken and growth responses monitored to determine the optimum levels of the various dietary components. A feed was developed which gave superior growth to that obtained with natural food organisms. This feed consisted of Torula yeast (47%), Weider Super Protein (44.39%), Vitamin supplement (0.45% - multivitamin), additional vitamin C supplement (0.16%), Spirulina (5%), and methionine supplement (3%). An optimum particle size range for O. mossambicus fry (between 5 & 25mm) was established at 125-200um. The fry should be fed the following feed ratios depending on age: 30.4% body weight/day up to day 5, 30.6% body weight/day up to day 10 and 25.1% body weight/day up to day 15. An optimum feeding frequency of 8/10hr day was recommended. A feed conversion ratio of 1.24:1 and a protein efficiency ratio of 0.682:1 was obtained. Digestable energy of the feed was determined at 16.1 kj/g feed. The established dry feed is suitable for use in commercial hatcheries, and as a formulation for further research on the intensive rearing of O. mossambicus fry.
- Full Text:
- Date Issued: 1989
- Authors: Thorpe, Peter Stuart
- Date: 1989
- Subjects: Cichlids , Tilapia -- Nutrition , Aquaculture
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:5184 , http://hdl.handle.net/10962/d1001960 , Cichlids , Tilapia -- Nutrition , Aquaculture
- Description: The need for developing a dry feed which satisfied the nutrient requirements of Oreochromis mossambicus fry was identified. The spawning and grading techniques which resulted in a higher fecundity and ensured uniformity within and between samples of fry are discussed. Preparation techniques were developed which met the physical requirements of fry feeds. An analysis of the flesh and yolk sac fry for amino acid composition was undertaken. The initial test diet was based on these results, as well as on the natural feeding ecology of the species. Feeding trials were undertaken and growth responses monitored to determine the optimum levels of the various dietary components. A feed was developed which gave superior growth to that obtained with natural food organisms. This feed consisted of Torula yeast (47%), Weider Super Protein (44.39%), Vitamin supplement (0.45% - multivitamin), additional vitamin C supplement (0.16%), Spirulina (5%), and methionine supplement (3%). An optimum particle size range for O. mossambicus fry (between 5 & 25mm) was established at 125-200um. The fry should be fed the following feed ratios depending on age: 30.4% body weight/day up to day 5, 30.6% body weight/day up to day 10 and 25.1% body weight/day up to day 15. An optimum feeding frequency of 8/10hr day was recommended. A feed conversion ratio of 1.24:1 and a protein efficiency ratio of 0.682:1 was obtained. Digestable energy of the feed was determined at 16.1 kj/g feed. The established dry feed is suitable for use in commercial hatcheries, and as a formulation for further research on the intensive rearing of O. mossambicus fry.
- Full Text:
- Date Issued: 1989
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