3.2.1 The effect of dietary lipid sources on growth and early survival of laboratory-held O. niloticus stocks at two ration levels
This part of the experiment aimed to produce stocks of male and female O. niloticus that had been subject to long-term food rationing that could be used as a stock of full and half rationed fish for use in subsequent experiments. Fish were rationed from first feeding (end of yolk sac – E.Y.S.) for several months until they could be used in subsequent physiological experiments. Owing to a lack of tank space, only two ration levels (full and half) could be studied in duplicate for 4 diets.
Newly hatched tilapia O. niloticus larvae were obtained from pure-bred stock held at Institute of Aquaculture, University of Stirling, Scotland. The feeding experiments
were conducted using 13 days post-fertilisation larval tilapia at the onset of exogenous feeding. Sixteen groups of 5 fry from each tank were sacrificed and weighed to the nearest 0.00lg. This data was used to calculate the average initial weight of fry per treatment tank. Initial high ration was set at 30% BW/day and initial low ration at 15% BW/day. These ration levels were 10 and 20% lower than those used by Coward (1997) and Macintosh and De Silva (1984) in a similar study of early growth and survival in T. zillii and O. mossambicus and O. niloticus x O. aureus hybrids, respectively. Ration levels were altered as necessary over the course of fry development. Alterations to rations were made when a large amount of excess (uneaten) food was observed in treatment tanks. On these occasions, fish in the high-ration tanks were fed to satiation 3 times during a 24 hour period; this providing an indication of the new high ration size to be allocated. Half ration groups were fed 50%
of the ration given to high ration groups.
Food rations for each treatment tank were weighed out in advance in 10 day quantities. Rations were initially provided by grinding the experimental pelleted feed to a suitable particle size (initially 250 -850µm). Particle and pellet size were gradually increased as fish grew (see Table 2.4 in Chapter 2). Daily rations were provided to each treatment group 3 times day-1. Faeces and excess food were siphoned from each tank twice daily. Any mortalities were removed as soon as discovered and rations altered accordingly. Every 10 days, 16 groups of at least 5 fry from each treatment group were sacrificed and weighed to the nearest 0.001g. The length was also measured individually utilising image analysis software (MRgrab 10.0.04 Carl zeiss vision GmbH, 2001) (see section 2.12.1) and the daily ration altered. The fish were then fixed in 10% NBF for gonadal development for later investigation which is not included in the thesis. Every 30 days, the true number of fish remaining in each
treatment tank was ascertained by counting manually. This was to avoid relying merely upon the presence of carcasses as an indicator of mortality. It was assumed that a degree of cannibalism could occur and would thus lead to over-estimations of population size in each tank. Once deemed large enough, fry were transferred into second (bigger) recirculating tanks (incorporating 50 x 38 x 41 cm). Rationing was maintained for a total of 18 months whereupon a series of physiological experiments were commenced. Thus growth data are only provided up to day 120 days here, the ration allocations are shown in (Table 2.7 in Chapter 2). Rationing of the 4 treatment groups was continued so as to provide a stock of rationed fish until terminating the spawning performance and oocyte recruitment sections of experiments.
3.2.2 Feeding procedure
Four diets, including the control with two rations (full and half ration) were used (Table 3.1). Diet 1 (D1) containing cod liver oil (CO) fed one ration only, diet 2 (D2) containing palm oil (PO), diet 3 (D3) containing a combination of palm and cod liver oil (PO & CO) (9:1 ratio) and diet 4 (D4) a commercial trout feed (Skretting UK) containing fish meal and fish oil as control.
Table 3.1 Experimental design
Fish in this investigation were maintained in 60 litre holding tanks (50 x 38 x 41 cm) in a recirculating system at the tropical aquarium at I.O.A. University of Stirling.
Stocks of O. niloticus were obtained from two replicates which had previously been fed with 3 experimental diets and a commercial trout diet (Skretting, UK) as control at full and half ration, respectively, for their entire life from onset of feeding until maturation (see section 3.2.2). Female broodfish were then collected randomly from their successive populations and measured (weight and total length) and tagged with Passive Integrated Transponder-PIT tags (Trovan, UK) under anaesthesia by immersion in 1:10,000 ethyl 4-aminobenzoate (Sigma, UK). The females were then placed into glass aquaria (114×45×42 cm) partitioned with Perspex dividers such that each individual had its own holding space. Fish were allowed to recover completely in clean aerated water prior to being placed to their respective glass tank (section 2.6).
Broodfish were fed according to the feeding protocol. Fish were fed three times daily
(900:1300:1700 hours) at a rate of 3 and 1.5% of BWday-1 as full and half ration, respectively with the experimental diets and a commercial pelleted trout feed (Skretting, UK) as a control until 3 successful batches of egg and larvae was obtained from each broodstock.
3.2.4 Spawning investigation
Fish were checked at two hourly intervals during the day for the evidence of spawning. In females undergoing ovulation and oviposition, the genital papilla were considerably swollen and extended. Fish also were observed for brooding behaviour which involved an over-extension of the lower jaw and females cleaning behaviour of a discrete area on the horizontal tank base. Once the indications of spawning were observed broodfish were netted and then anaesthetised, thereafter the genital papillae were blot-dried with absorbent tissue paper and the fish held dorsal side uppermost with the left hand holding the fish and the right hand carefully massaging the fish’s abdomen in such a manner that eggs were extruded from the ovary and collected into a clean plastic Petri-dish. Care was taken to avoid water contamination which would otherwise initiate water hardening and precocious closure of egg micropyles. Once eggs were obtained, sperm was collected and pooled from 2-3 anaesthetised males belonging to the same trial (diet and ration) by applying the same stripping techniques as detailed above after first emptying the fish’s urea bladder. Sperm released from the testes was collected directly from the genital papillae with the aid of disposable glass heamatocrit tubes (Camlab, U.K.). The females were then measured and weighed prior to returning them into the experimental glass tank and recovered in clean, aerated water and all data recorded.
Eggs were fertilised by adding the pooled sperm to the Petri-dish containing eggs.
Gametes were carefully but thoroughly mixed with a fine brush and a tiny volume of water. The mixture was then allowed to stand at ambient aquarium temperature for 4-5 minutes. Water was poured over the tip of the eggs with a 2 ml plastic pipette. After a further standing period of 4-5 minutes, water hardening occurred. In order to count the egg numbers, Petri-dishes containing the fertilised eggs were scanned using a scanner and the scanned pictures analysed using image analysis software (Section 2.21) to determine total fecundity according to Rana (1986) definition where total fecundity is the number of eggs in a freshly spawn batches. Fertilised eggs were then placed in round-bottomed plastic containers (Rana, 1986) supplied with clean, U.V.
sterilised water and left until hatch. The inter-spawning-interval (ISI) was determined (section 2.16) and all the data were recorded.
A sub-sample of 50 eggs per spawn was taken, prior to incubation and each egg individually measured to the nearest 0.1mm (see section 2.13) Since tilapia eggs are ellipsoid-shaped, it was important to measure both axes (long and short axes) in order to calculate egg diameter and volume (see section 2.13) according to the method of Coward and Bromage (1999a).
The fertilisation (%) and hatching rate (%) and inter-spawning-interval (ISI time elapsed between one spawn and the next) were also recorded.
After measuring the egg size, they were weighed and subsequently oven dried at 70ºC for 24h. Mean egg dried weight was determined to the nearest 0.1mg. The EW: BW ratio was also determined (Section 2.15) according to Coward and Bromage (1999a).
3.2.5 Larval quality
Larvae originating from each individual female were sacrificed by overdose of anaesthetic and weighed to the nearest 0.1mg at 10 days post-fertilisation. Their length was also measured to the nearest 0.1mm utilising MRGrab 1.0.0.4 (Carl Zeiss Vision GmbH, 2001) (see section 2.12.1).
3.2.6 Statistical analysis
Data were tested for normality and logarithmically transformed if necessary and statistical analyses were performed using SPSS for Windows (version 15) and Minitab (version 15) (section 2.25 in Chapter 2)