Wild Parr Research Articles

Precocial male maturation contributes to the introgression of farmed Atlantic salmon into wild populations

The escape of domesticated Atlantic salmon Salmo salar from aquaculture facilities represents a continued threat to the genetic and demographic stability of wild salmon stocks. Escaped farm-origin salmon have been shown to hybridize with wild conspecifics, yet the long-term genetic impacts are generally unknown. Theoretically, life history variation, specifically precocial maturation of male hybrids, could fast-track introgression, but evidence for this has been sparse. Here, we used empirical and experimental data to examine the role of precocious male maturation in introgression. We examined hybrid class composition using juvenile sampling and genetic assignment over a 5 yr period following an escape event in southern Newfoundland, a region with high rates of natural male precocial maturation. Initially following the escape, the proportion of first-generation (F1) hybrids was high and then decreased annually, contrasting the proportion of backcross wild individuals, which increased over time. The presence and temporal distribution of backcross wild individuals supports the hypothesis that high rates of precocial maturation can fast-track the long-term impacts of escaped farmed salmon on wild populations via interbreeding. This was experimentally tested using lab-created wild, farmed, and F1 parr released and recaptured from a Newfoundland river to assess levels of precocious male maturation. Observed rates of maturation of F1 hybrids were high and similar to wild parr (77 vs. 73%, respectively), indicating that most F1 hybrids mature precocially. Our results suggest that rates of precocial male maturation should be explicitly considered in risk assessments evaluating the genetic impacts of escaped farmed salmon on wild populations.

Artificial Rearing of Atlantic Salmon Juveniles for Supportive Breeding Programs Induces Long-Term Effects on Gut Microbiota after Stocking.

In supportive breeding programs for wild salmon populations, stocked parr experience higher mortality rates than wild ones. Among other aspects of phenotype, the gut microbiota of artificially raised parr differs from that of wild parr before stocking. Early steps of microbiota ontogeny are tightly dependent upon environmental conditions, both of which exert long-term effects on host physiology. Therefore, our objective was to assess to what extent the resilience capacity of the microbiota of stocked salmon may prevent taxonomic convergence with that of their wild congeners after two months in the same natural environment. Using the 16S SSU rRNA marker gene, we tested the general hypothesis that environmental conditions during the very first steps of microbiota ontogeny imprint a permanent effect on later stages of microbiota recruitment. Our results first showed that gut microbiota composition of stocked and wild parr from the same genetic population, and sharing the same environment, was dependent on the early rearing environment. In contrast, skin microbiota in stocked individuals converged to that of wild individuals. Taxonomic composition and co-occurrence network analyses suggest an impairment of wild bacteria recruitment and a higher instability for the gut microbiota of stocked parr. This study is the first to demonstrate the long-term effect of early microbiota ontogeny in artificial rearing for natural population conservation programs, raising the need to implement microbial ecology.

Estimating the relative fitness of escaped farmed salmon offspring in the wild and modelling the consequences of invasion for wild populations.

Throughout their native range, wild Atlantic salmon populations are threatened by hybridization and introgression with escapees from net‐pen salmon aquaculture. Although domestic–wild hybrid offspring have shown reduced fitness in laboratory and field experiments, consequential impacts on population abundance and genetic integrity remain difficult to predict in the field, in part because the strength of selection against domestic offspring is often unknown and context‐dependent. Here, we follow a single large escape event of farmed Atlantic salmon in southern Newfoundland and monitor changes in the in‐river proportions of hybrids and feral individuals over time using genetically based hybrid identification. Over a three‐year period following the escape, the overall proportion of wild parr increased consistently (total wild proportion of 71.6%, 75.1% and 87.5% each year, respectively), with subsequent declines in feral (genetically pure farmed individuals originating from escaped, farmed adults) and hybrid parr. We quantify the strength of selection against parr of aquaculture ancestry and explore the genetic and demographic consequences for populations in the region. Within‐cohort changes in the relative proportions of feral and F1 parr suggest reduced relative survival compared to wild individuals over the first (0.15 and 0.81 for feral and F1, respectively) and second years of life (0.26, 0.83). These relative survivorship estimates were used to inform an individual‐based salmon eco‐genetic model to project changes in adult abundance and overall allele frequency across three invasion scenarios ranging from short‐term to long‐term invasion and three relative survival scenarios. Modelling results indicate that total population abundance and time to recovery were greatly affected by relative survivorship and predict significant declines in wild population abundance under continued large escape events and calculated survivorship. Overall, this work demonstrates the importance of estimating the strength of selection against domestic offspring in the wild to predict the long‐term impact of farmed salmon escape events on wild populations.

Effects of acidic pH on wild, farmed, and F1 hybrid Atlantic salmon (Salmo salar) parr

Farmed escapees have the potential to introduce novel genes to wild salmon and alter locally adapted populations. We tested whether the acidic conditions found in rivers on Newfoundland’s south coast might differentially impact offspring of farmed Atlantic salmon (Salmo salar) in comparison to wild populations. We performed crosses with wild and farmed parents to obtain wild (W♂ × W♀), farmed (F♂ × F♀), and F1 hybrid (W♂ × F♀, F♂ × W♀) parr and conducted a common garden experiment at neutral or acidic pH. No differences were observed between pure wild crosses and F1 hybrids, suggesting that acidic waters do not differentially affect survival, growth, condition factor, and Na+/K+-ATPase activity of F1 hybrids. Trends in mortality show that pure farmed parr had lower survival than pure wild and F1 hybrids in low pH. Considering that production of F1 hybrids rather than pure farmed offspring is the most likely outcome in the wild, pure farmed parr survival may have little bearing on the prediction of genetic risks of farmed–wild interactions. There is no evidence to indicate that the survival of the F1 generation in acidic waters acts as a potential barrier against introgression.

Training the salmon\u2019s genes: influence of aerobic exercise, swimming performance and selection on gene expression in Atlantic salmon

BackgroundFarmed and wild Atlantic salmon are exposed to many infectious and non-infectious challenges that can cause mortality when they enter the sea. Exercise before transfer promotes growth, health and survival in the sea. Swimming performance in juveniles at the freshwater parr stage is positively associated with resistance to some diseases. Genetic variation is likely to affect response to exercise. In this study we map genetic differences associated with aerobic exercise, swimming performance and genetic origin.Eggs from the selectively bred Bolaks salmon and wild Lærdal River salmon strains were reared until parr in a common environment. Swimming performance was assessed by subjecting the fish to either continuous hard exercise or control conditions for 18 days. Heart was sampled for examination of gene expression using RNA-seq (~60 fish/treatment).ResultsLower expression of genes affecting immune function was found in domesticated than wild parr. Among wild parr under control exercise the expression of a large number of genes involved in general metabolism, stress and immune response was lower in superior swimmers suggesting that minimisation of energy expenditure during periods of low activity makes parr better able to sustain bursts of swimming for predator avoidance. A similar set of genes were down-regulated with training among wild parr with inferior swimming performance. These parr react to training in a way that their cardiac expression patterns become like the superior performing wild parr under control exercise conditions. Diversifying selection caused by breeding of domesticated stock, and adaptive pressures in wild stock, has affected the expression and frequency of single nucleotide polymorphisms (SNPs) for multiple functional groups of genes affecting diverse processes. SNPs associated with swimming performance in wild parr map to genes involved in energetic processes, coding for contractile filaments in the muscle and controlling cell proliferation.ConclusionsDomesticated parr have less phenotypic plasticity in response to training and lower expression of genes with functions affecting immune response. The genetic response to training is complex and depends on the background of parr and their swimming ability. Exercise should be tailored to the genetics and swimming performance of fish.

Assessing the outcomes of stocking hatchery-reared juveniles in the presence of wild Atlantic salmon

Stocking of hatchery-reared juvenile Atlantic Salmon Salmo salar is a common enhancement and recovery strategy across jurisdictions, yet its impact remains uncertain. We investigate the potential effectiveness of stocking in the Miramichi River system where approximately 150,000 first-feeding fry are stocked annually in reaches where wild juveniles are present. Local site and landscape level characteristics were used to fit predictive models for wild parr densities measured as Percent Habitat Saturation (PHS) using Partial Least Squares Regression (PLSR). The observed PHS of stocked sites was found to be significantly lower than predicted, suggesting stocking has not been effective in increasing the PHS in the studied stocked sites. The results suggest that habitat and other density dependent effects limit productive capacity in this system, and consequently, may limit the effectiveness of stocking in other systems with natural production.

Farmed Atlantic salmon Salmo salar L. parr may reduce early survival of wild fish.

The study examined the density-mediated effects on growth, survival and dispersal of wild and farmed Atlantic salmon Salmo salar offspring in the period immediately following emergence, using a substitutive design. In small confined stream channels, wild parr coexisting with farmed parr had a significantly poorer survival, than wild parr alone. Density did not affect this relationship. In larger unconfined stream channels, wild parr coexisting with farmed parr entered a downstream trap in higher numbers than wild parr in allopatry. The results suggests that during the earliest life stages, farmed S. salar can outcompete wild S. salar, resulting in a reduced survival of wild S. salar.

Evaluation of seasonal and daily changes of plasma thyroxine and cortisol levels in wild masu salmon Oncorhynchus masou, sampled by a Japanese fishing method.

A new fish sampling method was developed using a Japanese bait fishing rod (8-9 m carbon rod and a nylon line with a small fine wire single hook), which is considered to catch wild salmonid juveniles with low sampling stress. Using this method, seasonal and daily changes of plasma thyroxine (T4 ) and cortisol levels were examined in wild parr, pre-smolts and smolts of masu salmon Oncorhynchus masou in contiguous locations in a coastal river (Kesen River; 44 km) in northern Honshu Island, Japan, overlapping the period of smoltification and seaward migration from August to March. Plasma T4 and cortisol were low in 0+ and 1+ year parr caught in August and September. In March, some yearling (1+ year) fish, which were judged as pre-smolts, and smolts appeared mainly in mid and lower reaches, while parr (0+ and 1+ year parr) continued to appear in the upper and mid reaches. In March, 1+ year pre-smolts and smolts showed high plasma T4 levels while the levels of 1+ year parr were low. During March 2008-2010, plasma T4 levels of 1+ year pre-smolts and smolts had high levels from early to mid-March, whereas plasma cortisol levels of 1+ year smolts were low in early March and increased towards mid-March. Based on these data, plasma cortisol increases probably occur following the increases of plasma T4 levels to lead the 1+ year O. masou to the completion of smoltification and initiation of seaward migration.

Effects of rearing density and dietary fat content on burst‐swim performance and oxygen transport capacity in juvenile Atlantic salmon Salmo salar

The effects of hatchery rearing density (conventional or one third of conventional density) and feeding regime (high or reduced dietary fat levels) on burst-swim performance and oxygen transport capacity were studied in hatchery-reared Atlantic salmon Salmo salar, using wild fish as a reference group. There was no effect of rearing density or food regime on swimming performance in parr and smolts. The maximum swimming speed of wild parr was significantly higher than that of hatchery-reared conspecifics, while no such difference remained at the smolt stage. In smolts, relative ventricle mass was higher in wild S. salar compared with hatchery-reared fish. Moreover, wild S. salar had lower maximum oxygen consumption following a burst-swim challenge than hatchery fish. There were no effects of hatchery treatment on maximum oxygen consumption or relative ventricle mass. Haemoglobin and haematocrit levels, however, were lower in low-density fish than in fish reared at conventional density. Furthermore, dorsal-fin damage, an indicator of aggression, was similar in low-density reared and wild fish and lower than in S. salar reared at conventional density. Together, these results suggest that reduced rearing density is more important than reduced dietary fat levels in producing an S. salar smolt suitable for supplementary release.

Stable isotope marking of otoliths during vaccination: a novel method for mass-marking fish

Tagging or marking of fishes enables the collection of population-based information for ecological research, yet few techniques enable 100% mark detection success. We tested a new mass-marking technique: otolith marking with enriched stable isotopes delivered during vaccina- tion. Atlantic salmon (Salmo salar) parr were injected in either the abdominal cavity or muscle with a combination of enriched 137 Ba, 86 Sr and 26 Mg, using 1 of 3 carrier solutions (water, vaccine, vaccine mimic). Laser ablation inductively coupled plasma mass spectrometry of the otoliths indi- cated that 137 Ba and 86 Sr isotope enrichment treatments achieved 100% mark success, with 0 to 34% success for 26 Mg, compared to experimental controls. Mark strength was greater when enriched isotopes were injected into the abdominal cavity compared to muscle. Isotope markers did not affect fish condition or survival. Marks could be differentiated with 100% success from the background levels present in wild parr collected from 22 Norwegian rivers. Stable isotope mark- ing via vaccination with enriched stable isotopes is a mass-marking technique that, once opti- mised, could allow for cost-effective differentiation of wild and escaped farmed fish for each inde- pendent farming area.

Physical Injuries in Juvenile Atlantic Salmon, Slimy Sculpin, and Blacknose Dace Attributable to Electrofishing

AbstractJuvenile Atlantic salmon Salmo salar captured by electrofishing were X‐rayed and dissected to determine rates of electrofishing‐induced spinal injuries and hemorrhages. This study showed a low incidence of spinal injuries (11%) in electrofished wild parr captured alive and thereafter sacrificed. A negligible hemorrhage rate (2%) was observed. All of the spinal injuries observed were rated class 1 (minor injury), except for a class 2 injury in one parr. Larger, older wild parr (≥age 2) showed a higher incidence of spinal injuries (17%) than age‐1 parr (4%). Parr captured with two or more electrofishing sweeps had a spinal injury rate of 20%, compared with 6% when captured with a single electrofishing sweep. Among the mortalities, 8% of the parr had spinal injuries (class 1 except for one class 3) and 5% had hemorrhages. Spinal injury was observed in 6% of the mortalities of slimy sculpin Cottus cognatus and blacknose dace Rhinichthys atratulus; no hemorrhages were observed in these fish. This study showed that electrofishing produced a low incidence of injuries in juvenile Atlantic salmon, slimy sculpin, and blacknose dace. Nonetheless, caution is advised when conducting electrofishing surveys because sublethal effects other than physical injuries remain largely unknown.

Effect of hatchery environment on cranial morphology and developmental stability of Atlantic salmon (Salmo salarL.) from North-West Russia

Summary The study addresses the effect of hatchery rearing on morphological variation and developmental stability of Atlantic salmon parr from North-West Russia. Totally, we collected nine samples. Four wild samples were collected from each of the rivers Kola, Umba, Keret’ and Shuia. Five samples of hatchery-reared parr were the first-generation progeny of wild adults from these rivers reared separately at the four hatcheries (one hatchery was represented by two samples). Ten meristic and 48 morphometric cranial characters were analysed. We studied the morphological divergence between wild and hatchery fishes of the same river of origin. To analyze developmental stability we used fluctuating asymmetry (random deviations from perfect bilateral symmetry). It was found that hatchery-reared parr significantly differ from wild parr in both meristic characters and the shape of cranial bones. Different hatcheries caused similar effect on morphological variation in all populations. Fluctuating asymmetry in morphometric characters was significantly higher in hatchery fish than in wild from the Shuia River, indicating a higher level of developmental instability. However, wild parr from the Keret’ River had significantly higher fluctuating asymmetry than cultivated parr of the same origin, possible due to a high infection pressure of the parasite Gyrodactylus salaris Malmberg which has led to significant decline of the wild salmon population in this river, or from genetic changes caused by cultivation. The obtained results indicate a notable effect of hatchery environment on Atlantic salmon’s phenotype.

Seasonal foraging and piscivory by sympatric wild and hatchery-reared steelhead from an integrated hatchery program

We compared the diet of hatchery-reared steelhead produced from an integrated hatchery program as emigrating spring smolts and non-migrating hatchery residuals to their sympatric wild counterparts. Our results suggest that there is a potential for hatchery fish to affect wild steelhead populations due to dietary overlap and subyearling salmonid predation; however, relative ecological risk did not increase as steelhead delayed or forwent emigration. Predation by hatchery smolts was related to release timing, but not experience with native fish. Diet composition appears to be more strongly affected by seasonal and yearly differences in prey abundance and presence rather than differences in rearing environments. Hatchery and wild steelhead showed small but important foraging differences. Hatchery smolts did not consume as many salmonids as wild fish and hatchery residuals showed relatively stronger surface oriented feeding behavior than wild parr. Because most hatchery smolts emigrated shortly after release and the overall number of residuals in the study creek was low, we speculate that in this case there is low dietary and predatory-based risk of hatchery steelhead in Abernathy Creek negatively impacting wild salmonids.

Differences in pathogen resistance within and among cultured, conservation-dependent, and endangered populations of Atlantic salmon, Salmo salar L.

We report genetic differences for resistance to the pathogen Listonella anguillarum within and among one cultured and two wild Canadian populations of Atlantic salmon, Salmo salar, using a common-garden experimental protocol. Following exposure to the causative agent for vibriosis, parr originating from the endangered Stewiacke River population experienced significantly higher mortality than cultured parr, four generations removed from the Saint John River population, and wild parr from Tusket River. Pathogen resistance differed between sexes; males consistently experienced higher survival than females. There was no evidence that maturity influenced pathogen resistance in male parr. The population and sex differences in pathogen resistance documented here have implications for risk assessments of the demographic consequences of interbreeding between wild and farmed Atlantic salmon.

Cellular responses to temperature stress in steelhead trout (Onchorynchus mykiss) parr with different rearing histories

This study compared the heat-shock response and metabolic energy status in hatchery-raised and two groups of wild-caught steelhead trout parr collected from the Navarro River watershed, California. Wild parr were from coastal and inland sites with different thermal regimes. Fish were exposed in the laboratory to 25 ± 0.2°C for 2-h and the heat-shock response was assessed in muscle tissue via induction of heat-shock proteins (hsps) 63, 72, 78, and 89. Metabolic measurements included muscle phosphocreatine (PCr), ATP, ADP, and AMP, and hepatic glycogen. Inland and coastal fish overlapped considerably with regard to their hsp responses and energetic endpoints, but hatchery fish were distinct in the biochemical patterns they exhibited. Hsp expression levels after temperature shock were significantly lower in hatchery than in wild fish. Hatchery fish also had significantly lower hepatic glycogen and higher muscle ADP, ATP, and PCr concentrations than wild fish. Coastal and inland steelhead did not differ significantly with regard to peak hsp72 and hsp89 levels or to concentrations of energy metabolites. However, fish from the warm-water, inland site expressed significantly less hsp63, maintained higher basal levels of hsp72, and induced hsp89 more slowly than fish from the cold-water, coastal site. Discriminant function analysis revealed that hatchery fish can be distinguished from wild Navarro River fish with 84.9% certainty using the following function: f(x) = − 43.6 + 0.14(Gly) + 4.1(PCr) + 186.4(AMP) + 80.8(ADP) − 0.14(hsp63) + 0.005(hsp72). This study demonstrates that within a single species, rearing conditions or genetic variation can influence an organism’s disposition and cellular response to thermal stress. Extrapolation of results from laboratory studies on hatchery fish to wild fish may therefore not be possible, and caution must be used when interpreting hsp data obtained for wild fish with different thermal histories.

The feeding behaviour of cultured and wild Atlantic salmon, Salmo salar L., in the Louvenga River, Kola Peninsula, Russia

Abstract An underwater survey was conducted in the Louvenga River to investigate the behaviour and distribution of juvenile Atlantic salmon, Salmo salar L., and the adaptation of cultured fish to the natural environment. The food and feeding habits of 34 wild and 44 cultured parr released from the Kandalaksha hatchery were also studied. The cultured salmon fed mostly in the bottom 15 cm of the water column in current velocities of 0.2–0.35 m s−1. In contrast to wild fish, when cultured fish moved away from these areas and into areas with higher current velocities (average speeds of 0.52 m s−1) and lower drift density (2.66 particles m−3), they did not show a tendency to return to slower moving water. The diet of cultured parr was made up of benthic invertebrates (20%), terrestrial insects (32%), and drift items (33%), but these proportions were different in the diet of wild parr (2%, 24%, and 67%, respectively), with drift items predominating. The mean quantity of food per stomach indicated that the wild parr were feeding more actively than cultured parr. Invertebrates made up 3% of items in the drift, with the remaining 97% being exuvia of aquatic and terrestrial insects, algae, and various plant remains. Poor quality food items were found in 13% and 25% of the stomachs of wild and cultured parr, respectively, demonstrating that the cultured parr were less able to differentiate food items in the water column and made 20–30% more false feeding attempts than wild fish. The cultured parr were also more aggressive in terms of the frequency of aggressive interactions and reacted to each other at greater distances than wild fish. Thus, wild parr were able to optimize their feeding conditions by choosing habitats with preferred sizes of food items, higher densities of drift items, and current velocities that allowed them to maintain station and to feed more effectively. Conversely, cultured parr usually occupied suboptimal areas.

Cultured Atlantic salmon in nature: a review of their ecology and interaction with wild fish

AbstractWhen cultured Atlantic salmon are released into nature, they compete with wild fish for food, space, and breeding partners. As a result of morphological, physiological, ecological, and behavioural changes that occur in hatcheries, their comp etitive ability often differs from that of wild fish. These changes are partly phenotypic and partly genetic. Cultured juveniles' faster growth rate influences age and size at smolting and maturity, reproductive output, and longevity. Fast-growing parr tend to smolt younger, produce more but smaller eggs, attain maturity earlier, and die younger. Juvenile learning influences a number of behavioural traits, and differences in early experience appear to affect feeding and spawning success, migratory behaviour, and homing ability. Genetic change in hatcheries is chiefly the result of natural selection, with differential mortality among genotypes and broodstock selection based on production traits such as high adult body mass and fast growth rate. Experimental evidence has revealed that cultured parr's greater aggression often allows them to dominate wild parr, although smaller cultured parr can be subordinated if they co-occur in fast-flowing water and if wild smolts have established prior residence. During spawning, the fitness of wild salmon is superior to that of cultured conspecifics. Cultured males are inferior to wild males in intra-sexual competition, courting, and spawning; cultured females have greater egg retention, construct fewer nests, and are less efficient at covering their eggs in the substratum than their wild counterparts. In rivers, the early survival of cultured offspring is lower than that of their wild counterparts. The lifetime reproductive success of farmed fish has been estimated at 17% that of similar-sized wild salmon. As a result of ecological interaction and through density-dependent mechanisms, cultured fish may displace wild conspecifics to some extent, increase their mortality, and decrease their growth rate, adult size, reproductive output, biomass, and production.

Genetic variation in restored Atlantic salmon (Salmo salar L.) populations in the Ulla and Lérez rivers, Galicia, Spain

Abstract The populations of Atlantic salmon in the Ulla and Lérez rivers, located in Galicia in northwestern Spain, were close to extinction early in the 1990s. A restoration programme involving supportive breeding has been conducted since 1995, using a mixture of salmon populations from several Galician rivers. The programme utilizes progeny of adults returning to the rivers and wild parr reared in fresh water until maturity. Five microsatellite loci were used to compare genetic variability in the restored populations with that in populations before their collapse in the 1950s. DNA samples were obtained from scale collections (old samples) and from tissue samples of live fish caught in the rivers (modern samples). Average heterozygosities and allelic richness are very similar in modern and old samples. Populations inhabiting the Ulla and Lérez rivers today are more similar than they were in the past, possibly because they originated in the same stock mixture.

Divergence in smolt production from the stocking of 1-summer-old and 1-year-old Atlantic salmon parr in a northern Baltic river

Summary One-year-old and 1-summer-old salmon parr have been stocked in the Simojoki, a northern Baltic river, to protect the natural salmon stock from extinction. This enhancement practice was studied during a 5-year stocking period, when an average of 14.3 1-summer-old parr and 5.4 1-year-old parr was needed to produce one sea running smolt. The mean yield from stocking was 70 smolts per 1000 stocked 1-summer-old parr and 186 smolts per 1000 stocked 1-year-old parr. The mean cost of smolts produced by stocking 1-year-old and 1-summer-old parr was 1.2 and 2.6 times the cost of producing one 50 g smolt in the hatchery, respectively. There was a negative relationship between density of wild parr and length of smolts stocked as 1-summer-old parr in the river, but no relationship between wild parr density and length of smolts stocked as 1-year-old parr. This indicated that 1-summer-old parr were more susceptible than the older parr to competition with wild parr. Considering production costs and smolt yield, stocking of 1-year-old parr appeared to be more profitable than that of 1-summer-old parr in the enhancement of the endangered salmon stock.

Seasonal changes of liver glycogen content and condition factor of wild Atlantic salmon parr in a sub‐arctic river

Abstract – Mean liver glycogen content of 1–4‐year‐old Atlantic salmon parr varied from 0.5 to 9.5% (of fresh liver mass) and condition factor (CF) from 0.66 to 0.85 in the sub‐arctic River Teno system in northern Finland during 2 years of study. There were no significant differences in liver glycogen contents of different age groups as there were in CF. However, there were seasonal differences in liver glycogen content and CF of the wild parr. The lowest liver glycogen contents were observed in July and the highest in September and December whereas the lowest CFs were observed in May and the highest in July.