Radiographic characterisation of spinal curvature development in farmed New Zealand Chinook salmon Oncorhynchus tshawytscha throughout seawater production.

Spinal anomalies are a recognised source of downgrading in finfish aquaculture, but identifying their cause(s) is difficult and often requires extensive knowledge of the underlying pathology. Late-onset spinal curvatures (lordosis, kyphosis, scoliosis) can affect up to 40% of farmed New Zealand Chinook (king) salmon (Oncorhynchus tshawytscha) at harvest, but little is known about their pathogenesis. Curvature development was radiographically documented in two related cohorts of commercially-farmed Chinook salmon throughout seawater production to determine (1) the timing of radiographic onset and relationships between (2) the curvature types, (3) the spinal regions in which they develop and (4) their associations with co-existing vertebral body anomalies (vertebral compression, fusion and vertical shift). Onset of curvature varied between individuals, but initially occurred eight months post-seawater transfer. There were strong associations between the three curvature types and the four recognised spinal regions: lordosis was predominantly observed in regions (R)1 and R3, kyphosis in R2 and R4, manifesting as a distinct pattern of alternating lordosis and kyphosis from head to tail. This was subsequently accompanied by scoliosis, which primarily manifested in spinal regions R2 and R3, where most of the anaerobic musculature is concentrated. Co-existing vertebral body anomalies, of which vertebral compression and vertical shift were most common, appeared to arise either independent of curvature development or as secondary effects. Our results suggest that spinal curvature in farmed New Zealand Chinook salmon constitutes a late-onset, rapidly-developing lordosis-kyphosis-scoliosis (LKS) curvature complex with a possible neuromuscular origin.

Pathogenicity of the bacterium New Zealand rickettsia-like organism (NZ-RLO2) in Chinook salmon Oncorhynchus tshawytscha smolt.

Farmed New Zealand Chinook salmon Oncorhynchus tshawytscha Walbaum have been found to be infected by rickettsia-like organisms (NZ-RLO). While these Gram-negative intra-cellular bacteria are closely related to Piscirickettsia salmonis, a significant pathogen for farmed salmon globally, the pathogenicity of NZ-RLO is unknown. The aim of the present study was to determine if one strain, NZ-RLO2, causes disease in Chinook salmon. Post-smolt salmon were inoculated with NZ-RLO2 by intraperitoneal injection at high, medium and low doses and observed for 30 d. All fish in the high and medium dosed groups died by the end of the study and 63% of the low dose group died within 30 d of inoculation. Necropsy revealed the fish inoculated with NZ-RLO2 had internal multifocal haemorrhages. The most consistent histological finding in fish inoculated with NZ-RLO2 was neutrophilic and necrotizing pancreatitis and steatitis with intra-cytoplasmic organisms often visible within areas of inflammation. Other histological lesions included multifocal hepatic necrosis, haematopoietic cell necrosis and splenic and renal lymphoid depletion. The presence of NZ-RLO2 within the inoculated fish was confirmed by replication in cell culture and qPCR. The results suggest NZ-RLO2 can cause disease in Chinook salmon and therefore could be a significant pathogen in farmed Chinook salmon.

Prevalence of spinal abnormalities in Chinook salmon smolt and influence of early rearing temperature and growth rates.

Spinal abnormalities can be detected at harvest in around 40% of farmed Chinook salmon in New Zealand. However, whether these abnormalities are present in smolt is unknown. Radiographs of 3,736 smolt were taken immediately prior to transfer to sea water and evaluated for fusions, compressions, vertical shifts, and lordosis, kyphosis and/or scoliosis (LKS). The survey included smolt from two different chilling strategies that had been graded into slow- or fast-growing fish. Overall, 4.34% of Chinook salmon smolt had at least one spinal abnormality, similar to the rates of reported in Atlantic salmon smolt. The rate of abnormality was significantly higher in faster-growing fish. Fusions were most common with 2.68% of smolt affected. Smolt subjected to longer chilling times had lower rates of fusions. Compressions and vertical shifts were both observed in 1.31% of smolt. Although LKS is the most common abnormality of harvested fish, LKS was detected in just five smolt. The results suggest that some fusions in harvest fish have developed at the time of seawater transfer while LKS develops late in the production cycle. Overall, spinal abnormalities are uncommon in Chinook salmon smolt and may be influenced by chilling times and growth rates.

The external phenotype-skeleton link in post-hatch farmed Chinook salmon (Oncorhynchus tshawytscha).

Skeletal deformities in farmed fish are a recurrent problem. External malformations are easily recognized, but there is little information on how external malformations relate to malformations of the axial skeleton: the external phenotype-skeleton link. Here, this link is studied in post-hatch to first-feed life stages of Chinook salmon (Oncorhynchus tshawytscha) raised at 4, 8 and 12°C. Specimens were whole-mount-stained for cartilage and bone, and analysed by histology. In all temperature groups, externally normal specimens can have internal malformations, predominantly fused vertebral centra. Conversely, externally malformed fish usually display internal malformations. Externally curled animals typically have malformed haemal and neural arches. External malformations affecting a single region (tail malformation and bent neck) relate to malformed notochords and early fusion of fused vertebral centra. The frequencies of internal malformations in both externally normal and malformed specimens show a U-shaped response, with lowest frequency in 8°C specimens. The fused vertebral centra that occur in externally normal specimens represent a malformation that can be contained and could be carried through into harvest size animals. This study highlights the relationship between external phenotype and axial skeleton and may help to set the framework for the early identification of skeletal malformations on fish farms.

Vertebral column regionalisation in Chinook salmon, Oncorhynchus tshawytscha.

Teleost vertebral centra are often similar in size and shape, but vertebral-associated elements, i.e. neural arches, haemal arches and ribs, show regional differences. Here we examine how the presence, absence and specific anatomical and histological characters of vertebral centra-associated elements can be used to define vertebral column regions in juvenile Chinook salmon (Oncorhynchus tshawytscha). To investigate if the presence of regions within the vertebral column is independent of temperature, animals raised at 8 and 12 °C were studied at 1400 and 1530 degreedays, in the freshwater phase of the life cycle. Anatomy and composition of the skeletal tissues of the vertebral column were analysed using Alizarin red S whole-mount staining and histological sections. Six regions, termed I-VI, are recognised in the vertebral column of specimens of both temperature groups. Postcranial vertebrae (region I) carry neural arches and parapophyses but lack ribs. Abdominal vertebrae (region II) carry neural arches and ribs that articulate with parapophyses. Elastic- and fibrohyaline cartilage and Sharpey's fibres connect the bone of the parapophyses to the bone of the ribs. In the transitional region (III) vertebrae carry neural arches and parapophyses change stepwise into haemal arches. Ribs decrease in size, anterior to posterior. Vestigial ribs remain attached to the haemal arches with Sharpey's fibres. Caudal vertebrae (region IV) carry neural and haemal arches and spines. Basidorsals and basiventrals are small and surrounded by cancellous bone. Preural vertebrae (region V) carry neural and haemal arches with modified neural and haemal spines to support the caudal fin. Ural vertebrae (region VI) carry hypurals and epurals that represent modified haemal and neural arches and spines, respectively. The postcranial and transitional vertebrae and their respective characters are usually recognised, but should be considered as regions within the vertebral column of teleosts because of their distinctive morphological characters. While the number of vertebrae within each region can vary, each of the six regions is recognised in specimens of both temperature groups. This refined identification of regionalisation in the vertebral column of Chinook salmon can help to address evolutionary developmental and functional questions, and to support applied research into this farmed species.

Reproductive biology and spatiotemporal patterns of spawning in striped marlin Kajikia audax.

This study presents the first histology-based assessment of the reproductive dynamics of south-west Pacific striped marlin Kajikia audax. Maturity and reproductive status were assessed from histological sections of ovaries (n = 234) and testes (n = 243) of fish caught in commercial longline and recreational fisheries between 2006 and 2009. Spawning peaked in the Coral Sea during November and December at sea surface temperatures between 24.8 and 28.3° C. Lower jaw fork length (L(LJF)) at 50% maturity (L(LJF50)), a key variable for stock assessment, was estimated to be 2100 ± 102 mm (mean + s.e.) for females and 1668 ± 18 mm for males. Unlike large pelagic tunas Thunnus spp., the proportion of females increased with length and spawning fish formed multiple large-scale aggregations within a broad latitudinal band. This study provides a starting point for biological parameters needed for stock assessment and conservation of K. audax and introduces the multiple aggregation spawning concept as a reproductive mechanism to explain genetic heterogeneity observed in some highly migratory species.

Physiology, behaviour and welfare of fish during recreational fishing and after release.

This review investigates how recreational fishing affects the physiology, behaviour, and welfare of fish. Sentience and the capacity of fish to experience pain, suffering and fear are discussed, and practical recommendations for improving the treatment of fish during recreational fishing are provided. Handling procedures used in recreational fishing should match the environment where the fish is caught and the size and strength of the fish. Minimising the number of hooks on lures and baits, and using barbless hooks and circle hooks generally reduce rates of injury and the severity of tissue trauma. Capture time, handling time, and exposure to air play significant roles in the stress responses of fish and should be minimised by anglers. Keep-nets, gaffs, landing-nets, live-wells and other restraining devices should only be used when necessary because each device can prolong or intensify the negative influences of catching fish by hook-and-line. The use of fish as bait is discouraged unless they have been euthanised. Euthanasia of fish used as bait or fish captured recreationally should include a stunning blow to the head, followed by bleeding-out, or pithing the brain and spinal cord. Fish should be euthanised if they are bleeding, injured, deeply-hooked, foul-hooked, or severely exhausted, but local fisheries' regulations must be obeyed. Fish that are released after being caught may be subjected to additional factors that influence their welfare, such as elevated stress, barotrauma, suppressed feeding and growth, impaired reproductive function, increased potential for disease, infection, and delayed mortality.

11-Ketotestosterone induces silvering-related changes in immature female short-finned eels, Anguilla australis.

The developmental transition from a residential, immature 'yellow' eel to a migratory, maturing adult 'silver' eel is accompanied by many morphological changes that appear to be under endocrine control. High circulating levels of the teleost, and usually male-specific, androgen 11-ketotestosterone (11-KT) are found in migrating female short-finned eels, Anguilla australis. We examined the role of this steroid in silvering by implanting immature, female short-finned eels either with blank vehicles or with vehicles containing 11-KT. Six weeks after they had received the implants, eels treated with 11-KT had developed 'chisel-shaped' snouts and black pectoral fins with tapered ends, and the size of their eyes had increased significantly. 11-KT treated eels had a thicker dermis than control eels and an epidermis with fewer or no mucous cells. Ventricular mass at the end of the experiment was two-fold larger than in control eels. 11-KT treated eels also had larger livers and gonads. Ovaries contained predominantly cortical alveolus stage III oocytes, as opposed to the smaller gonads of control eels containing previtellogenic stage II oocytes. All of these changes correspond to changes during the developmental transition from yellow to silver eels in the wild. This demonstrates that silvering in eels is under endocrine control and that the presumed male-specific steroid 11-KT is capable of inducing silvering-related changes in a female teleost. We discuss how species-specific responses to 11-KT may differ depending on tissue-specific androgen receptor abundance and how a dual demand on liver function can explain the apparently positive effects of 11-KT on liver growth.

Mechanical efficiency of isolated in situ perfused hearts of the eel Anguilla australis.

Administration of exogenous Ach to isolated in situ saline-perfused hearts of Anguilla australis via the perfusate, resulted in reduction in cardiac frequency (F). This reduction in F became significant at Ach concentrations of 10(-11) M or greater. A weak inotropic effect of Ach at the lowest concentration tested (10(-13) M) was also observed. Maximum power output of preparations was 2.94+/-0.26 mW gVM(-1). The mechanical efficiency of A. australis hearts working at 25 and 50% of maximum power under different conditions of stroke volume (SV) and F was investigated. Cardiac frequency was manipulated using a combination of temperature and Ach administration. Stroke volume was manipulated by regulating input pressure of the perfusate supplying the preparations (pre-load). Values of MEF from preparations generating flow under conditions of low F/ high SV (treated with Ach 10(-7) M) were significantly greater than under conditions of high F/ low SV (untreated). The MEF of preparations appears to be related to inotropic state. The negative inotropy and increased mechanical efficiency produced by Ach appears to be the opposite of the so-called 'oxygen-wasting' effect produced by the positive inotropic agents, the catecholamines. This effect of Ach may be related to the dependence of teleost myocardium on extracellular Ca(2+) for excitation-contraction coupling during the cardiac cycle.

Temporal changes in the trot between the first and third horse inspections at a CCI*** 3-day event.

Kinematic parameters were measured from horses competing in a CCI*** 3-day event. The horses were filmed during the first and third horse inspection. This provided a repeat sample on 16 horses. The horses were filmed using a panning lateral S-VHS video camera (50 Hz). Spatial measurements were taken for mean +/- s.d. 5.66 +/- 0.92 strides for the first inspection and 5.05 +/- 1.27 for the third inspection. Within the calibration zone, data of the horses accelerating and obtaining a constant velocity were collected. The horses trotted with a higher mean velocity during the third inspection (0.26 +/- 0.05 m/s, P < 0.001). During the third inspection the horses trotted with a shorter stride length (0.193 +/- 0.03 m, P < 0.001) and stride duration (31 +/- 42 m/s, P < 0.001). The third inspection was characterised by a decrease in retraction percentage for both the forelimbs (3.69 +/- 2.39%, P < 0.001) and the hindlimbs (2.48 +/- 2.16%, P < 0.001). However, no significant difference was found between the 2 inspections for other temporal parameters when measured as a percentage of stride. It is proposed that the event horses trot with a decreased stride length and duration during the third horse inspection but maintain a consistent temporal relationship.

Linear and temporal stride characteristics of 3-day event horses at a CCI*** 3-day event horse inspection.

AIMS: To quantify the linear and temporal characteristics of the trot of 3-day event horses during the preliminary horse inspection of an advanced grade CCI*** 3-day event. METHODS: A cross sectional study was made of the kinematics of 24 3-day-event horses during the first horse inspection at a CCI*** level S-day event. Video footage was digitised and linear and temporal measurements were made. RESULTS: The horses trotted for an average of 10.44+/-1.55 strides. Spatial measurements were made on an average of 5.66+/-0.92 consecutive strides when the horses were within the calibration zone. The horses increased and then obtained a constant velocity within the calibration zone. Trotting on the asphalt track did not alter the relationships between stride length, stride duration and velocity when compared with previously published values. Horse-specific differences in stance and retraction percentages were identified. CONCLUSION: Horse specific differences were identified that may contribute to each horse's unique gait or kinematic fingerprint. It is proposed that the initiation of, and completion of, stance by the hind limb first may represent engagement of the hind quarters and be a response to dressage training.

A simple two-step method for the conversion of [3H]cortisol to [3H]-11-ketotestosterone.

Despite the existence of several protocols, problems appear to persist in the small scale chemical synthesis of radiolabeled 11-ketotestosterone from cortisol. We investigated the possibilities of using the mild oxidant pyridinium dichromate for the oxidative cleavage of the dihydroxyacetone side chain of cortisol and 17 beta-hydroxysteroid dehydrogenase for the subsequent reduction of the resulting 17-keto group. Our protocol has resulted in consistently high yields of both the intermediate, adrenosterone (70-80%), and the product, 11-ketotestosterone (up to 60%). This, taken together with the convenience and relatively low cost of our method, recommends the protocol for its use for the synthesis of [3H]-11-ketotestosterone for endocrine studies.

11-Ketotestosterone suppresses interrenal activity in rainbow trout (Oncorhynchus mykiss).

Interrenal activity in relation to sexual maturity in male rainbow trout, and the effects of in vivo treatment with 11-ketotestosterone (11-KT) were investigated. Silastic pellets containing either 11-KT or no steroid were implanted in maturing male and immature male and female rainbow trout for a total of 11 weeks. Two weeks before termination of the experiment, animals were subjected to crowding, and blood samples were taken 15-90 min after initial disturbance. At the termination of the experiment, head kidneys containing interrenal cells were removed and incubated in vitro with either human adrenocorticotropin 1-24 (ACTH) or with pregnenolone. 11-KT reduced the elevation of cortisol in response to confinement stress. Head kidney tissue of control and 11-KT-treated maturing males was significantly less responsive to ACTH and pregnenolone than tissue from immature males. Treatment of immature females with 11-KT similarly led to reductions in interrenal responsiveness. No difference in response was seen between tissue from control immature males and females. These data suggest that the attenuated stress response in maturing males is due, in part, to elevated 11-KT levels acting at one or more levels of the hypothalamus-pituitary-interrenal axis.

Agrobacterium-mediated transformation of five wild Solanum species using in vitro microtubers.

A genetic transformation method, using in vitro microtubers and Agrobacterium-mediated transformation has been developed for five wild Solanum species: S. verrucosum, S. hjertingii, S. papita, S. stoloniferum, S. demissum, which range in ploidy from diploid to hexaploid. A disarmed A. tumefaciens strain, C58 harbouring the co-integrate vector pGV3580::pKU2 with the genes of neomycin phosphotransferase (NPTII) and hygromycin phosphotransferase (HPTII) as selectable markers, was used. Selection of putative transformants was based on their ability to grow and produce roots on a medium containing 150 mg/l kanamycin. The transgenic nature of the putative transformants was confirmed by Polymerase Chain Reaction analysis and by NPTII dotblot assay to show the expression of the NPTII gene. Additionally, the transmission of transgenes, NPTII and HPTII in selfed-sexual progeny of some transgenic plants was also determined.

The effects of sympathomimetics on the cardiovascular system of sheep.

1. Sheep hearts have been used to study the effects of beta-adrenoceptor (beta-AR) agonists in order to better understand the effects of common asthma treatment drugs on heart rate, cardiac power output and cardiac pathology. Hearts have been examined both in vivo and in vitro. 2. In whole anaesthetized sheep, isoprenaline, fenoterol and salbutamol induced dose-dependent increases in heart rate. Hypokalaemia in response to salbutamol was accentuated in hypoxia. Many of these hearts showed significant myocardial lesions. Hypoxia alone caused no significant cardiac response. 3. As expected, the beta 1-AR agonist dobutamine caused dose-dependent increases in heart performance (heart rate and cardiac power output). Both responses were blocked by metoprolol and propranolol. The beta 2-AR agonist salbutamol caused dose-dependent increases in heart rate and although cardiac output increased, cardiac power output remained unchanged as a consequence of the fall in peripheral resistance. The heart rate changes were blocked by metoprolol. Importantly, propranolol blocked both the heart rate response and the fall in peripheral resistance. 4. Isolated atrial strips showed a right shift of their dose-response curve to isoprenaline in the presence of the highly selective beta 2-AR antagonist ICI 118,551 at concentrations above 1 x 10(-8) mol/L. 5. We conclude that the sheep heart shows many pharmacological characteristics of the human heart which makes it a good pharmacological model in addition to its being amenable to many common techniques available for humans.

Expression of reducing sugar accumulation in interspecific somatic hybrids of potato.

A somatic hybridisation programme was undertaken to evaluate the expression of reducing sugar accumulation in potato. Interspecific hybrids created between the Solanum tuberosum cultivar Record and the diploid species Solanum phureja were evaluated at the morphological and molecular levels. These analyses indicated that the protoplast regenerants were partial (asymmetric) hybrids which had undergone elimination of S. phureja chromosomes. Tubers of the parents exhibited significant differences for reducing sugar accumulation during cold storage with S. phureja having lower levels of glucose and fructose than Record. The somatic hybrids resembled the S. phureja parent in terms of reducing sugar accumulation demonstrating that low reducing sugar accumulation is dominant to high reducing sugar accumulation in these particular genotypes. These results are discussed in relation to the exploitation of asymmetric hybridisation for the production of potato genotypes for the potato processing industry.

Blood pressure and heart rate during tonic immobility in the black tipped reef shark,Carcharhinus melanoptera.

Tonic immobility was induced in black tipped reef sharks (Carcharhinus melanoptera) and heart rate and ventral aortic blood pressure recorded. Without branchial irrigation, tonic immobility was correlated with a significant depression in blood pressure and heart rate irrespective of the sharks being in air or in water. Tonic immobility with branchial irrigation resulted in a significant increase in blood pressure in sharks in air, but not in water. Heart rate was unchanged when the gills were irrigated. Intra-arterial injections of atropine abolished the bradycardia and blood pressure rise associated with tonic immobility. We conclude that, during tonic immobility, sharks are able to receive afferent information from the ventilatory system and make appropriate responses via the vagus nerve.

Cardiac performance of an isolated eel heart: effects of hypoxia and responses to coronary artery perfusion.

The pericardial sac containing the heart was removed from large (2.7-6.3 kg) long-finned eels (Anguilla dieffenbachii). Coronary arteries were cannulated in preparation for perfusion with eel Ringer or red cell suspensions. The hearts were maintained by Ringer perfusion while the performance of the heart was assessed. Responses of the hearts to increases in filling pressure and output pressure were recorded. Maximum cardiac output was 22.3 +/- 1.4 ml/min/kg body mass (mean +/- 1 SEM; N = 9). The highest cardiac power output was measured at maximum cardiac output and was 3.39 +/- 0.32 mW/g ventricle mass (mean +/- 1 SEM; N = 9). Eel hearts could sustain output pressures near 80 cm H2O, but cardiac output was reduced and cardiac power output was 1.89 +/- 0.24 mW/g ventricular mass (mean +/- 1 SEM; N = 9). Maximum cardiac output decreased by 14% when hearts pumped hypoxic Ringer with a PO2 of 11.5 torr. At high input pressures concomitant with high output pressures (80 cm H2O), cardiac power output decreased by 38% upon exposure to hypoxic Ringer. Coronary perfusion of hypoxic hearts with red cell suspensions (mean hematocrit 10.4%) at a rate of 2% of control cardiac output (0.20 ml/min/kg body mass) had no effect on maximum cardiac output. However, coronary perfusion enabled hypoxic hearts to maintain cardiac output when output pressure was raised to 80 cm H2O. Under conditions of high input pressure and high output pressure, power output increased by 20% compared to hypoxic hearts without coronary perfusion.(ABSTRACT TRUNCATED AT 250 WORDS)

Dimensional analysis of the ventricle of an in situ perfused trout heart using echocardiography.

The linear dimensions of the ventricle of an in situ perfused trout heart were measured with an ultrasound scanner. Using a pyramidal model, linear dimensions taken from real-time, two-dimensional, echotomographic images of the ventricle were used to calculate ventricular end-diastolic and end-systolic volumes and, by the difference between them, stroke volumes for various filling and output pressures. Stroke volumes calculated from the ultrasound measurements were significantly correlated with actual stroke volumes determined from cardiac output and heart rate and had a regression line slope close to one. Increases in stroke volume with greater filling pressures resulted from increased ventricular end-diastolic volume with constant end-systolic volume; end-systolic volumes were negligible. Maintenance of stroke volume with increasing output pressure was by increases in both end-diastolic and end-systolic volumes. Decreases in stroke volume with very high output pressures may result from convergence of end-systolic and end-diastolic volumes.