2.1. Fish and Sampling

Rainbow trout specimens show similar symptoms of whirling movements, blackening of the body, a curved tail, the presence of a cyst before the anal fin on the lateral line, hemorrhage on the gills, and skeletal deformities (Figure 1) were taken from three farms of Himachal Pradesh and one farm of Uttarakhand, India (Figure 2). The sampling period spanned three years, from 2017 to 2019. The percentage prevalence of deformity was calculated from each farm on the basis of the total number of skeletal deformed fish observed to the total sample collected from each population and expressed as a percentage [28]. During these three years, approximately 424 randomly collected fish samples (Table 1) showing signs of deformity and black caudal peduncle were brought to the fish parasitology and mycology laboratory of the ICAR-Directorate of Coldwater Fisheries Research (DCFR), Bhimtal, for postmortem analysis. Furthermore, the possible causes and impacts of skeletal anomalies were investigated via various approaches.

2.2. Physiological Parameters of the Water

The physicochemical parameters of water from rainbow trout raceways, such as temperature, pH, dissolved oxygen (DO), phosphate (PO₄), nitrogen–ammonia (NH₃N), nitrate-nitrogen(NO₃N), and chlorides, were recorded during this period. The temperature (°C) and pH were measured via a digital meter (Hanna, USA). The DO concentration (mg L⁻¹) was measured via a multiparameter probe (Oakton DO 6+ Dissolved Oxygen Meters, Coleparmer, USA). PO₄, NH₃N, NO₃N, and chloride contents were estimated via commercially available kits (Himedia, India) following the manufacturer’s protocol.

2.3. Histopathology

The moribund trouts were anesthetized with clove oil (40 μL/L) until they remained motionless and were fixed in Bouin’s fluid. For analysis of the cellular pathology of various tissues in deformed fishes, tissue samples (caudal fin, skin and muscle, visceral mass, spleen, kidney, gill, head, and brain) were processed for histopathological analysis. The protocol for histology followed that of Sharma et al. [29]. Briefly, samples were processed in a series of alcohols (70%, 85%, 95%, and 100%), xylene, and finally embedded in paraffin blocks. The skin and muscle tissues were cross-sectioned at a thickness of 4 μm and stained with hematoxylin and eosin (Sigma). Histopathological observations were made with an upright microscope (Nikon Eclipse Ci) with a digitally attached camera (Nikon Ds Fi2) to record pathological changes through microphotography.

2.4. Parasite Investigation

Skin scrapings and squash smear preparations of mucus from the fins, gills, brain, head kidney, liver, and intestine of moribund fishes were mounted on glass slides with a drop of saline solution for parasite observation under a microscope as suggested by Karlsbakk et al. [30].

2.5. Whole-Mount Study

The moribund trouts were anesthetized with clove oil (40 μL/L) until they remained motionless and were fixed in 10% formalin. Whole-mount staining of bone and cartilage was performed with a modified protocol of the alcian blue technique by Dingerkus and Uhler [31] for skeletal anomalies. The fish were first fixed in 10% formalin and washed with distilled water for 48 h. The staining technique was started by placing the fish in 10 mg alcian blue, 80 mL ethyl alcohol, and 20 mL glacial acetic acid for up to 48 h. Subsequently, the alcian blue stain was removed, and further processing was carried out with 96%, 75%, 40%, and 15% graded ethyl alcohol for 3 h and an enzyme solution of 30 mL saturated aqueous sodium borate, 7 mL distilled water, and 1 gm trypsin for 72 h. The process was again continued with 0.5% aqueous potassium hydroxide (KOH) with alizarin red S for 24 h and 0.5% KOH–glycerine series (3 : 1, 1 : 1, 1 : 3) to pure glycerin. A few drops of thymol were added to the glycerin and hydrogen peroxide (H₂O₂) solutions for the final stage.

3.1. Percentage Prevalence of Deformity and Clinical Signs

During the three years of sampling period, mortalities were observed in rainbow trout fry aged 90–110 days, with an average weight of 2 ± 1.2 g. Approximately 8–15 trout fry die daily in outdoor raceways, and an approximately 13%–15% prevalence of skeletal anomalies is reported during the sampling years (Table 1). Notably, the symptoms manifested in the initial week of June and persisted until the final week of September. Fish were lethargic, and there was low acceptance to feed. At present, only a limited percentage of farms can consistently produce 100% of nondeformed fish. Earlier records on vertebra deformity rates in wild fish have shown a range of 3%–43% in salmonids [32, 33].

The affected fish displayed a black tail, which was the first sign of spinal instability. The appearance of curvy caudal region, frenzied tail-chasing motion was evident from the samples. According to Chong [34], abnormal whirling behavior and blackening probably happen due to damage to the sympathetic nerves of the spinal cord, which are located caudal to the site of spinal cartilage damage and regulate the melanin pigmentation.

3.2. Parasitological Investigations

Skeletal anomalies may arise due to parasite infection (Myxobolus sp.), elevated incubation temperatures [18, 35–37], or other possible reasons. Since, whirling movement and mortality were associated with the deformity, it was first suspected to be myxozoan infection. However, the myxozoan parasites were absent in all the tested samples in microscopy examination. The absence of Myxobolus spores in various tissues led to the hypothesis that abiotic, nutritional factors could be possible reasons for these skeletal anomalies.

The farmed rainbow trout in both states were fed a balanced diet (50% fish meal, 15% soybean meal, 15% wheat flour, 10% linseed oil, 8% yeast extract, and 2% vitamin and mineral mixture) and an appropriate ration size; hence, suspecting/conspiring the feed or nutrients as a factor responsible for triggering the deformities was unsubstantiated.

3.3. Physicochemical Parameters

The physicochemical parameters of the trout raceways were as follows: water temperature, 3.3°C–23.70°C (Figure 3); pH, 8.0–9.5; DO, 5.8–10.7 ppm; PO_4, 0.08–0.95 ppm; NH₃N, 0.136–0.72 ppm; NO₃N, 0.02–0.747 ppm; and chlorides, 1.63–7.44 ppm. Rainbow trout spawn from autumn to winter months (November to January) in both states of India [2]. The optimum incubation period for rainbow trout is 370° days. The temperature recorded from all the farms and hatcheries ranged from 4°C to 8°C from December to January, and the water temperature of the sampled rainbow trout farms ranged from 17°C to 23.7°C from June–July. The ideal temperature range for incubating rainbow trout eggs is 7°C–12°C; lower incubation temperatures, such as 4°C-5°C, and more than 12°C–14°C have a detrimental impact on the early stages of development [38].

3.4. Histopathological and Whole-Mount Investigations

Histopathology and whole-mount and molecular biology are essential laboratory tools for assessing vertebral anomalies in fish [20, 36]. Histology was performed for different tissues, such as the stomach, liver, pyloric caeca, and pancreas (Figure 4), of which the pancreas was the most infected organ where degeneration of acinar cells was observed in addition to vacuolation. The stomach, pyloric caeca, and intestine were found to be normal, with intact epithelium, glands, and muscle layers (both inner circular and outer longitudinal). The ultrastructure of the spleen was normal; however, some smears of pigmented macrophages were observed. Yellowish to brown pigmented macrophage aggregates were found in the pancreatic tissue, and the space of degenerated acinar cells was replaced by fibrosis. Macrophage aggregates in internal organs have been linked to a range of phenomena, including infectious diseases and parasite infestations [39], and heat stress [40].

Furthermore, inflammatory leukocytes infiltrated the liver, and hepatocytes exhibited some degree of lipid infiltration. Lipid accumulation in the livers of captive fish is likely a consequence of the fluctuating water temperature. Indeed, some studies reported that temperature was an important factor that modulated the liver lipid accumulation in Atlantic salmon and steel head trout [41–43].

Rainbow trout stained with alcian blue and alizarin red presented anomalies in caudal regions (Supporting File). A similar result was observed in cultured Sole, Solea sengalenis [20], rainbow trout, O. mykiss [44], and Salmo gairdneri [45]; Atlantic halibut, Hippoglossus hippoglosus [19]; and European seabass, Dicentrarchus labrax [46], at the early stages of development. In salmonid fishes, high temperatures during both incubation and rearing are the principal factors that negatively impact skeletal development [47]. During an experimental larval rearing of rainbow trout under three temperatures, the range of deformities was between 2% and 8% when the temperature was below 13°C, and substantial larval losses were reported, with 13%–20% deformed stock when the temperature was above 13°C–14.5°C [48]. The phenomenon, how favorable environmental conditions change the skeletal phenotype, including the induction of skeletal deformities, was portrayed by Martini et al. in 2020. Moreover, in both geographic locations, the diurnal water temperature fluctuates rapidly, which could lead to deformities in rainbow trout fry and fingerlings.

In nutshell, the skeletal deformities, whirling behavior and mortality were associated with the major pancreatic anomalies, abnormal build up of lipid in liver with no signs of parasites presence. An observation was made by authors in [49, 50] during an outbreak of pancreatic disease in Atlantic salmon from Ireland, observing whirling movement and mortality due to skeletal myopathy associated with pancreatic acinar cell degeneration and liver cell necrosis. McLoughlin et al. made similar observations in 1995, 1996, and 2002 during an outbreak of pancreatic disease in Atlantic salmon from Ireland, observing whirling movement and mortality due to skeletal myopathy associated with pancreatic acinar cell degeneration and liver cell necrosis.