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Plasma NT‐proBNP and Cell‐Free DNA Concentrations after Prolonged Strenuous Exercise in Working Farm Dogs

Background Plasma N‐terminal pro–B‐type natriuretic peptide (NT‐proBNP) concentration is increased in dogs with myocardial dysfunction, and cell‐free DNA (cfDNA) increases in numerous disease states. In humans, both of these biomarkers can be altered after endurance exercise. Objective To investigate the effect of prolonged strenuous exercise on circulating NT‐proBNP and cfDNA concentrations in working farm dogs. Animals Six healthy, privately owned working farm dogs (4 Huntaways and 2 heading dogs) from the same hill country farm in New Zealand. Methods Prospective, nonrandomised cohort study. Venous blood samples were collected before and after the dogs worked over 4 days. Plasma NT‐proBNP concentrations were measured by a commercially available ELISA assay and cfDNA concentrations were determined by fluorometry without prior DNA extraction. Results The baseline (before work, Day 1) median plasma NT‐proBNP concentration was 664 pmol/L. A linear mixed‐effects model showed that work increased plasma NT‐proBNP concentrations by 101 ± 9% (P < 0.001), but with each consecutive day of work, NT‐proBNP concentrations declined by 16 ± 4% (P < 0.001). The baseline median plasma cfDNA concentration was 653 ng/mL, and plasma cfDNA concentrations increased by 138 ± 45 ng/mL after work (P = 0.004). Conclusions and Clinical Importance The plasma concentration of NT‐proBNP in healthy Huntaways and heading dogs after work can exceed the upper limit of the reference range. Results in dogs sampled on the day of prolonged strenuous exercise should be interpreted with caution. Plasma concentrations of cfDNA also increase with exercise, but further studies are needed to establish reference ranges in healthy dogs.

T he natriuretic peptides play a regulatory role in cardiovascular function through their endocrine and paracrine actions. B-type (or brain) natriuretic peptide (BNP) is primarily produced by the myocardium, and increased synthesis and secretion of BNP occurs in response to stimuli such as cardiomyocyte stretch and ischemia. 1 Circulating BNP or its breakdown products, including NT-proBNP, can be used as a biomarker to aid in the diagnosis and prognosis of cardiac disease in humans, dogs, and cats. In particular, plasma NT-proBNP concentrations have been shown to have high accuracy in differentiating cardiac and noncardiac disease in humans, dogs, and cats presenting with dyspnoea. [2][3][4][5] An ELISA that detects the N-terminal fragment of BNP (NT-proBNP) in plasma has been validated for use in dogs. 6,a Previous studies have shown that there is a significant variation in natriuretic peptides between healthy dogs of different breeds, 7 as well as a high degree of week-to-week variability in individual dogs. 8 Extracardiac factors such as renal function can also influence circulating NT-proBNP concentrations. 9 In humans, prolonged strenuous exercise significantly increases plasma NT-proBNP concentrations above the upper limit of the reference range in endurance athletes. 10 Similar studies have not been performed in dogs, although 5-minute period of submaximal exercise in healthy dogs did not significantly affect plasma NT-proBNP concentrations. 11 Prolonged strenuous exercise in people causes transient cardiac dysfunction (known as cardiac fatigue) 12 and might even induce subclinical myocardial necrosis, 13 leading to increases in plasma NT-proBNP concentrations. Skeletal muscle fiber damage can also occur with endurance exercise, leading to increases in biomarkers such as serum creatine kinase (CK) and aspartate aminotransferase (AST) activities. 14 Cell-free DNA (cfDNA) is another biomarker that has been proposed as a novel indicator of exerciseinduced muscle injury and overtraining. 15,16 Cell-free DNA consists of double-stranded fragments of DNA in plasma that are not bound to cells. In the past, cfDNA was thought to originate largely from cells undergoing apoptosis or necrosis, but in recent years, it has been suggested that DNA might also be released from living cells. 17,18 Plasma concentrations of cfDNA are increased in various disease states in dogs, 19 including mammary tumors, 20,21 lymphoid neoplasia, 22 pulmonary thromboembolism, 23 sarcomas, trauma, and sepsis. 24 However, as for NT-proBNP, the effect of prolonged strenuous exercise on plasma cfDNA concentrations in dogs is unknown.
The influence of exercise on the interpretation of circulating NT-proBNP and cfDNA concentrations in dogs is particularly important when considering the diagnostic use of these biomarkers in working dogs. In New Zealand, working dogs on sheep farms routinely work 2-8 hours per day, multiple days in a row. Furthermore, a common breed of working dog, the Huntaway, is predisposed to the development of dilated cardiomyopathy. 25 If a working dog develops exercise intolerance, fatigue, or dyspnoea during work, veterinarians might use plasma NT-proBNP concentrations to differentiate cardiac from noncardiac disease. However, if plasma NT-proBNP concentrations increase with prolonged periods of work, this could result in the misdiagnosis of cardiac disease. The aim of this study was to determine whether repeated periods of prolonged strenuous exercise influences circulating plasma NT-proBNP and cfDNA concentrations in healthy working dogs.

Animals
The study protocol was approved by the Massey University Animal Ethics Committee. The study population consisted of 6 unrelated adult working farm dogs belonging to the same shepherd on a large sheep and beef property near Taihape, in the Rangitikei region of New Zealand. Healthy adult dogs primarily used for work with livestock were considered eligible for inclusion. Exclusion criteria included auscultation of a heart murmur or arrhythmia (with the exception of sinus arrhythmia), or the presence of any injury or disease process which would prevent the dog from performing a full day of work. The study population included 4 Huntaways and 2 Heading dogs, with a mean age of 4 years (range 1.25-7 years) and equal numbers of males and females (3 of each). Sampling took place in January 2015 and for the 2 weeks before sampling, the dogs had been rested and largely confined to kennels. They had not performed any stock work during this 2week period and were only let out of the kennels for a short duration each day when they were allowed to walk around in the immediate vicinity of the kennels. Each dog had an electromechanical activity monitor b mounted on their collar on the first day, and the time of starting and finishing work was recorded daily. The owner of the dogs was contacted approximately 6 and 12 months after the collection of samples to provide follow-up on the ongoing health of the dogs.

Sample Collection and Analysis
On Day 1, a complete physical examination was performed on all dogs before starting farm work. A venous blood sample (3 mL) was collected from the cephalic vein of each dog and submitted to a commercial veterinary diagnostic laboratory c for a complete blood count and serum biochemistry panel. Venous blood samples (1.5-2 mL) for NT-proBNP and cfDNA analysis were collected immediately before the dogs starting work and within 30-90 minutes of the dogs finishing work each day. It was not possible to sample dogs immediately after exercise as they were usually working in remote locations on the farm. At the time of sample collection, cardiac auscultation was also performed on each dog, and heart rate was recorded. Blood samples were collected by needle and syringe and then transferred immediately into tubes containing ethylenediaminetetraacetic acid (EDTA). Within 60 minutes of collection, samples were centrifuged at 5000 9 g for 15 minutes to separate plasma. The plasma samples were frozen at À20°C for up to 4 days before analysis at a veterinary diagnostic laboratory c for NT-proBNP concentrations by the Cardiopet ELISA, as well as CK and AST activities.
After the initial laboratory analysis, plasma samples were refrozen for transport to Massey University and then thawed for determination of cfDNA concentration. A total of 20 lL of each plasma sample was used for cfDNA quantification with the Qubit dsDNA HS Assay Kit and Qubit 2.0. d The Qubit assay uses a fluorescent dye selective for double-stranded DNA, and the amount of fluorescence recorded is proportional to the amount of double-stranded DNA in the sample. Before each run, the Qubit 2.0 was calibrated with the standards provided, and the concentration of cfDNA in each sample was calculated by the manufacturer's dilution algorithm. All plasma samples from the dogs were run as a single batch. A suggested reference range of 512-654 ng/ mL for plasma cfDNA concentration (mean AE 2 standard deviations) was calculated using data from 24 healthy dogs presented in a recent paper by Burnett et al., 19 which used the same analytical methods.

Statistical Analysis
Statistical analyses were performed by commercially available software. e Exploratory data analysis and Shapiro-Wilk tests revealed that plasma cfDNA concentrations were normally distributed, whereas plasma NT-proBNP concentrations and CK and AST activities were not, so log transformations of these variables were performed to achieve normality. Linear mixed-effects models by a restricted maximum likelihood approach were used for statistical analysis. The dogs were considered a random effect, whereas work (before or after) and day were considered as fixed effects. Duplicate models were run with day as a categorical variable and a numeric variable to enable trends between days and overall trends to be analyzed. Separate models were fitted for each variable (NT-proBNP, cfDNA, CK, and AST). Coefficient values and standard errors from models of log-transformed variables were back transformed using the formula (exp value À 1) 9 100 to give the percentage change in the dependent variable per day or with work. For plasma NT-proBNP concentrations, the estimated standard deviation of the between-day component (intercept) and the within-day component (residual) was used to calculate the intraclass correlation. Pearson's product-moment correlations were used to explore correlations between after work variables, including plasma NT-proBNP concentrations and CK and AST activities. The recorded heart rates of all dogs before and after work across all sampling points were averaged, and the means compared by a paired t-test.

Results
All dogs were normal on physical examination at the start of the study. One dog was kicked by a cow on day 3 and sustained a traumatic tibial fracture and was therefore excluded from further analysis from Day 3 onwards. All other dogs remained in good health and continued working for the duration of the study and over the following 12 months. Each day during the sampling period, the dogs performed stock work for a total of 10-12 hours, including short periods of rest. Stock work primarily involved mustering ewes and cattle on steep hill country and over rough terrain. Because of technical difficulties, a complete set of activity data was only collected from one dog. Activity counts for this dog were as follows: 26,396 (Day 1); 45,056 (Day 2); 37,616 (Day 3); and 30,344 (Day 4). Counts approximate the number of footfalls by the dog, and as all dogs worked as a team, for the same duration and over the same terrain, it is suggested that these counts are representative of all dogs in the study.
The overall mean of the after work heart rates (taken within 30-90 minutes of finishing work) was 114 beats per minute, with a standard deviation of 13. This is significantly higher than the before work mean of 93 beats per minute, with a standard deviation of 11. A paired t-test showed this difference in means was statistically significant, with a T value of À7.47 and P < 0.001. No cardiac murmurs were auscultated, and the femoral pulse was strong and synchronous in all dogs at each time point. Three of the dogs exhibited an increased heart rate on inspiration relative to expiration, consistent with physiological sinus arrhythmias. No pathological arrhythmias were noted.
As illustrated in Figure 1A, plasma NT-proBNP concentrations were consistently higher after work than before work on the same day and this frequently resulted in values above the suggested reference range in dogs (<900 pmol/L). Two male Huntaways had plasma NT-proBNP concentrations above this range on Day 1 before work, which increased further after work on Day 1, but normalized in subsequent before work samples. A linear mixed-effects model with day as a categorical fixed effect showed that plasma NT-proBNP concentrations increased by 101 AE 9% with work, (P < 0.001), and Days 2, 3, and 4 were significantly different to Day 1 (P < 0.001). The intraclass correlation was 0.79, indicating a large amount of variation between days compared to the variation within each day. As seen in Table 1, the model with Day as a numeric variable gives a negative, statistically significant value (À0.176775, P < 0.001) for Day, and back transformation of this value shows that plasma NT-proBNP concentrations decrease by 16 AE 4% with each repeated day of work. There was no correlation between heart rate and plasma NT-proBNP concentrations.
A linear mixed-effects model estimated that plasma cfDNA concentrations increased by 138.1 AE 45.2 ng/mL with work samples, as shown by the fixed effect value in Table 1 (P = 0.004). As seen in Figure 1B, there was a large amount of variation in plasma cfDNA concentrations between dogs at each sampling point, compared to the between-day variation (intraclass correlation of 0.085). Plasma cfDNA concentration did not change significantly over repeated days of work (P = 0.076). One sample (Dog 3, before work Day 3) was considered an outlier at 1930 ng/mL and was excluded from statistical analysis as prior, and subsequent samples from the same dog were considerably lower and more consistent with cfDNA concentrations recorded in the other dogs. Plasma CK and AST activities were also significantly increased after work (Table 1 and Figures 1C-D), and the largest increases were seen on Day 2 and 3 when the highest activity counts were recorded.

Discussion
Results of the present study demonstrate that prolonged strenuous exercise in previously rested dogs significantly increases circulating NT-proBNP concentrations, but the impact of exertion lessens over consecutive days of work. In Huntaways and Heading dogs, if sampling for plasma NT-proBNP concentration is performed on a day when the dogs have been working, the results can exceed the upper limit of the reference range in the absence of any clinical evidence of cardiac dysfunction. If sampling is delayed until the following day (before further work), plasma NT-proBNP concentration normalizes in the majority of cases and results might more accurately reflect the true likelihood of cardiac disease in an individual dog. Plasma cfDNA is a new biomarker in dogs and lacks reference ranges established using large-scale studies, but the results of this research show that plasma cfDNA concentrations also increase with physical work, despite marked variation between individual dogs.
The transient increases observed in circulating NT-proBNP concentrations with endurance exercise in dogs in the present study are similar to what has been described in humans participating in endurance exercise events such as marathons, ultramarathons, and professional cycling events. 13,26,27 In humans, the duration of exercise, but not the intensity, is associated the with peak plasma NT-proBNP concentrations after exercise. 10,28,29 Because of the small sample size and the practical limitations of dogs working in remote areas on the farm, it was not possible to quantify exercise intensity in the present study. In regard to the total amount of exercise daily, the absolute activity counts recorded from one of the dogs are not in themselves quantitatively informative, but they show differences in activity between the days. As the dogs in this study worked as a team of dogs, it is suggested that the monitored dog acted as an indicator for the relative activity of all the dogs. The highest increases in after work plasma NT-proBNP concentrations were seen on Day 1 and declined over subsequent days, whereas the highest activity counts were seen on Day 2, followed by Day 3. This suggests that increases in plasma NT-proBNP concentrations in dogs after exercise are not solely determined by the total amount of physical activity performed. The exact mechanism responsible for increases in plasma NT-proBNP concentrations seen after endurance exercise is unclear, although prolonged exertion might induce reversible diastolic left ventricular dysfunction, 26 or decreased myocardial contractility, 12 referred to as cardiac fatigue. Increases in specific biomarkers of cardiomyocyte necrosis, such as plasma cardiac troponin T concentrations, have been found to be significantly correlated with increases in plasma NT-proBNP concentrations after exercise, indicating that subclinical myocardial injury and necrosis, 13 or cardiomyocyte membrane leakage, 30 might also contribute to elevated plasma NT-proBNP concentrations with endurance exercise.
There is also the possibility that the changes in plasma NT-proBNP concentrations seen after work and over consecutive days in the present study are a result of altered clearance of the peptide, rather than changes in expression and release. NT-proBNP is thought to be removed from circulation by organs with high blood flow rates, including the kidneys and liver, compared to the biologically active carboxy-terminal of BNP, which is also cleared by active receptor-mediated binding. 1 During endurance exercise, the glomerular filtration rate usually decreases, 31 which could reduce renal clearance of NT-proBNP and increase plasma concentrations of the peptide, similar to what is seen in dogs with renal dysfunction. 9 However, the glomerular filtration rates of the dogs in the present study were not assessed.
Of particular interest in the present study, the magnitude of increases in NT-proBNP concentrations seen with exercise declined over the 4-day study period. In humans, BNP might play a role in myocardial adaption to endurance training. 30 Healthy male Army recruits had increased resting plasma BNP concentrations and increased left ventricular mass after a 10-week endurance training program, 32 whereas endurance athletes with a longer history of training did not have elevated plasma BNP and NT-proBNP concentrations at rest, 33,34 suggesting that with prolonged endurance training, the myocardium might adapt to prolonged periods of exertion. The dogs in the present study had a long history of endurance training as they had been working for 6-12 hours daily (including short periods of rest) in the months before the study. However, in the 2 weeks immediately before the sampling period, the dogs had been rested and largely confined to the kennels, and it is not known what effect this might have had on any prior myocardial adaption to endurance exercise. To understand the significance and physiology of this apparent trend for repeated exercise to have a diminished effect on plasma NT-proBNP concentrations in dogs, further studies are needed, involving larger numbers of working dogs over a longer sampling period, with differing levels of prior work.
Two of the dogs included in the present study, both male Huntaways, had plasma NT-proBNP concentrations on the first day that would usually be associated with an increased likelihood of cardiac disease (>900 pmol/L). These values increased further with work, but normalized over the course of the sampling period, and over the following year neither dog developed any clinical signs of cardiac dysfunction. Because of the small sample size in the present study, it might be coincidental that the 2 dogs with elevated NT-proBNP concentrations initially were both male Huntaways, whereas the 2 female Huntaways and the heading dogs had resting values within the reference range. However, studies in retired racing greyhounds have found that plasma NT-proBNP concentrations in this breed at rest are significantly higher than non-Greyhound controls, with 11 of 24 healthy greyhounds having NT-proBNP concentrations above the upper limit of the reference range. 35 A similar situation might exist in Huntaways, and there might also be an influence of sex on plasma NT-proBNP concentrations at rest, although in humans, females tend to have higher concentrations than males. 36 The high before work NT-proBNP concentrations on Day 1 could also be related to the dogs' anticipation of imminent work after a fortnight of rest. All the dogs were restless and excited on Day 1 before work, and epinephrine and norepinephrine can induce BNP synthesis in human cardiomyocytes in vitro. 37 It might therefore be possible that activation of the sympathetic nervous system in these dogs contributed to elevated plasma NT-proBNP concentrations in Day 1 before work samples. A larger sample size would be needed to further investigate any breed or sex-specific effect on circulating NT-proBNP concentrations in working Huntaway dogs, and sampling without a lengthy period of inactivity before the start of the study could help to minimize the influence of circulating catecholamines.
Similar to NT-proBNP, the concentrations and activities of other biomarkers measured in the present study (CK, AST, and cfDNA) were increased in plasma after work. The changes in plasma CK and AST activities are consistent with previous reports in dogs and humans, 14,38 as both cardiac and skeletal muscle can release CK and AST into circulation when there is muscle damage or increased muscle membrane permeability. 39 Transient increases in plasma cfDNA concentrations with exercise have been reported previously in humans and have been proposed as a marker of overtraining and exerciseinduced inflammation in muscle. 15,16 The origin of cfDNA released during exercise is the subject of much debate, but hemopoietic cells might be the main source. 40 There was no significant correlation between plasma cfDNA concentrations and either CK or AST activities, which suggests that muscle leakage alone is unlikely to be an explanation for the increases seen in plasma cfDNA concentrations with work on Days 1, 3, and 4. On Day 2 of the study, the before and after work plasma cfDNA concentrations were similar, and the interval between finishing work and sampling was 90 minutes (compared to 30-45 minutes on the other days), because of the dogs working in a remote area of the farm. In people competing in a half-marathon event, plasma cfDNA concentrations returned to baseline values within 2 hours of finishing the race, 41 so it is possible that the similarity between before and after work values is because of the relative delay in sample collection. The small sample size and wide individual variation in plasma cfDNA concentrations in the present study mean that it is not possible to make any conclusions regarding the influence of exercise duration, intensity, and prior training on plasma cfDNA concentrations in dogs. Furthermore, most of the plasma cfDNA concentrations measured in these working dogs were higher than what was reported in a previous study by the same analytical technique. 19 In the previous study, most of the control dogs were colony-housed, relatively inactive dogs, so it might be that the higher values are reflective of prior endurance training or subclinical soft tissue injury. However, further studies are needed to determine a reference range for plasma cfDNA concentrations in working farm dogs.
The main limitations of this prospective study were the low number of dogs enrolled, the short duration of the study, and the lack of echocardiography. To minimize variation in animal husbandry practices and in the duration, intensity, and environment of work, all recruited dogs belonged to the same shepherd, which limited the size of the study population. Breed differences have been documented in plasma NT-proBNP concentrations in dogs, 7 but the small study size precluded analysis of any breed-related effects. The duration of the present study (4 days) was based on the farming operation activities and the number of consecutive days of high-intensity work that the dogs were required for. This limited the detection of longer term trends in plasma NT-proBNP, cfDNA, CK, and AST with continued endurance exercise. The local veterinary clinic did not have the equipment or expertise to facilitate echocardiography of the dogs, and transporting them to the nearest veterinary hospital with these imaging capabilities was unfortunately not practical. Therefore, assessments regarding the cardiac health of the dogs had to be based on through clinical examinations and the dogs' continued ability to undertake demanding physical work daily without any difficulty.
In conclusion, the present study demonstrates that plasma NT-proBNP concentrations in healthy Huntaways and heading dogs increase with prolonged hill country work and can exceed the upper limit of the reference range. If a working dog presents with clinical signs suggestive of cardiorespiratory disease during or following a day of work, clinicians should consider delaying sampling for plasma NT-proBNP concentrations until the following day to overcome any confounding effect of recent exercise. Further work is needed to determine the effect of prior training, the intensity and duration of activity, and possible breedrelated differences in Huntaways.


extfieldsofstudy: Biology Medicine

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Characterisation of InlA truncation in Listeria monocytogenes isolates from farm animals and human cases in the province of Quebec

The introduction of Listeria monocytogenes into the food production chain is a concern, with numerous grouped cases of listeriosis associated with milk-derived or pork-derived products have been documented. Management of this zoonotic pathogen considers all strains as an equal risk. Recently, a new perspective for characterisation of strain virulence was introduced with the discovery of the unaltered sequence of InlA as a determinant of strain virulence; this has also been reported as an infrequent finding among so-called environmental strains, that is, strains isolated from food or from surfaces in food industries. The aim of this study was to differentiate L monocytogenes strains isolated from animal cases versus those from human cases and to differentiate clinical strains from environmental ones using a Caenorhabditis elegans virulence testing model. In Quebec in 2013/2014, the surveillance of L monocytogenes clinical isolates registered a total of 20 strains of animal origin and 16 pulsed-field gel electrophoresis types isolated from human cases. The mixed PCR multiplex agglutination protocol used for geno-serotyping clearly discriminated genogroup IVB strains from bovine and human origins. The presence of a premature stop codon single nucleotide polymorphism in the inlA gene sequence in clinical strains and the identical behaviour of particular strains in the C elegans model are discussed in this paper from the perspective of industrial management of L monocytogenes risk.

INTRODUCTION
Listeria monocytogenes is a great concern both for industrial and public health stakeholders. These bacteria cause listeriosis, a foodborne disease acquired through the consumption of contaminated ready-to-eat products, including milk or meat products. Pork meat has been associated with largescale outbreaks in the past (de valk and others 2001) and is considered a risk factor, particularly for sensitive populations such as pregnant women, children and the elderly. In the last few years, the increase in human cases (not associated with intensified surveillance), mainly for the elderly, has increased the concern surrounding management of L monocytogenes in food in Canada and Europe (Lomonaco and others 2015). One critical question to answer is whether the virulence of L monocytogenes has increased. The determinants of virulence have been studied, and there is a large amount of data that allow for discrimination of strains from lineages III or IV from lineages I and II (Ragon and others 2008). Lineages I and II are considered to be the most worrisome because of their potential to cause outbreaks or sporadic listeriosis. Among such strains, discrimination based on pathogenic properties still represents a challenge. A few years ago, the very promising concept of virulence determination through inlA gene sequencing was introduced (Van Stelten and others 2010, Van Stelten and others 2011). Published studies revealed the presence of a premature stop codon for internalin A translation. Because this stop codon inactivated this determinant of the first step of pathogenicity in the digestive tract, a key determinant for virulence differentiation of strains appeared to have been discovered. In Quebec, human listeriosis surveillance works hand-in-hand with food surveillance. Few strains isolated from clinical cases in farms (animal primary productions) are available, so we took the opportunity that arose to test virulent strains isolated from animals or humans with clinical signs of listeriosis. After we performed the serovar determination and comparison according to strain origin, we wanted to confirm if the inlA sequences were complete in these strains and to test their behaviour in a Caenorhabditis elegans infection model, a very attractive but controversial model of pathogenicity testing for L monocytogenes ( InlA integrity and behaviour of L monocytogenes strain in C elegans has actually been described. Some strains isolated during a previous study involving pig primary production (Larivière-Gauthier and others 2014) were added to the study's strain collection because only some of them showed stop codon in their inlA sequences and because these strains were (based on pulsotype comparison) frequently or not associated with clinical human cases. These additional strains were expected to show opposite virulence potentials. The aim of this study was to analyse inlA sequences from strains isolated from clinical forms of human or animal listerioses in Quebec and to compare the behaviour of some selected strains in a C elegans virulence model.

MATERIALS AND METHODS Strain collections
Strains were collected from L monocytogenes surveillance in 2013/2014: 20 strains were obtained from animal disease surveillance (laboratory of the Ministry of Agriculture, Fisheries and Food, Quebec (MAPAQ)), among which 13 were included in this study because they satisfied the required 'clinical' criteria (ie, isolated from organs after animal necropsy, excluding isolation from milk and silage) and 16 strains isolated from clinical cases in humans were provided by the Provincial Public Health Laboratory (public health surveillance). The collection was completed with four strains of pig origin isolated in a previous project (Larivière-Gauthier and others 2014). These strains showed a single nucleotide polymorphism (SNP) premature stop codon in the inlA sequence (Pulso1 and Pulso9 strains) or an integrated inlA sequence (Pulso4 and Pulso6 strains). Strains Pulso6 and Pulso9 showed 100% similarity between pulsed-field gel electrophoresis (PFGE) banding patterns with strains recovered from clinical human cases; strains Pulso1 and Pulso4 have not previously been identified in clinical human surveillance. These added strains were expected to show opposite virulence potentials.

InlA sequencing
The inlA gene sequences were determined after overlapping amplification (Ragon and others 2008) using the Sanger method at the Centre d'Innovation Génome Québec (Applied Biosystems 3730xl DNA analyzer). InlA gene sequencing was extensively analysed, and the occurrence and position of SNP before in silico translation of the sequences were compared. Sequences were aligned and screened for mutations causing a premature stop codon or amino acid deletion using ClustalX 2.1 software, with inlA L monocytogenes EGD-e (NCBI: NC_003210.1) as reference.

C elegans virulence model
Bacterial strains (Fig 1) were cultured 24 hours at 37°C in BHI (Brain Heart Infusion), acid shocked (0.1 N HCl pH 4.5, one hour) then washed and suspended in phosphate-buffered saline (PBS) (concentration, see Fig  1) before being used in trials. Strain N2 worms, maintained for five days on nematode-growing medium plates and inoculated with Escherichia coli OP50 feeding strain at 25°C, were harvested (non-synchronised culture), washed three times in PBS, then inoculated in 96-well plates in PBS and maintained for five more days at 30°C with daily addition of fresh bacteria.
Observation of worm viability (based on worm locomotion and/or pharyngeal pumping and assessed under an inverted phase contrast microscope according to Thomsen and others (2006) allowed determination of a living worm count of 15 mature worms and 30 L1/L2 larvae in each of the tested conditions. The distribution of number of living worms counts (n=8) were compared with control conditions (Mann-Whitney U test; SPSS Statistics V.17, Licenced U de Montreal, α=0.05)

RESULTS
All 16 of the strains of human origin were considered clinical because they were isolated from hospitalised patients. From the 20 strains provided by MAPAQ, only 13 strains strictly associated with clinical listeriosis in animals were included. It should be noted that we excluded strains isolated from milk tanks and silage, all belonging to the 1/2a serovar. Although serotyping revealed that the greater proportion of clinical strains (regardless of origin) belonged to the serovar 1/2a (18/29, 62%), there were still some strains (nine out of 29 with similar proportions from humans and animals, that is, 5/16 and 4/13, respectively) that belonged to the IVB serogroup (a serogroup that contains 4b and 4a/ab serovars). Focusing on this IVB group, a clear distinction appeared where only 4b serovar strains were found in human surveillance, whereas strains shown in ruminant clinical cases all belonged to 4a/4ab serovars (table presented in Fig 2). No SNP occurred on the sequence coding for the InlA LPXTG domain. Moreover, inlA sequencing showed that a majority of strains had a complete sequence (table presented in Fig 2). But it should be noted that two out of the 16 strains isolated from clinical listeriosis cases had a premature stop codon; the mutations were expected to induce truncations of InlA at 700 and 762 amino acid (aa) positions, respectively. One of these-which showed the longest InlA size but lost LPXTG domain-was used in the C elegans virulence model, as well as another strain presenting a complete sequence (tables presented in Figs 1,2). Four more strains (different PFGE type) from porcine origin were added; some had been recurrently detected in production (and food) but had never been associated with human cases (table presented in Fig 1; Pulso1 and Pulso4 strains). The two others were kept because of their 100% homology with strains involved in clinical human cases (sporadically for strain Pulso9 or recurrently for strain Pulso6; table presented in Fig  1). After five days, unfed C elegans worms were almost all dead. Using E coli OP 50 as a control, viability was maintained at 74% after five days, both in larvae and in mature worm forms. By comparison, L monocytogenes lowered the viability of the worms and significantly so for the larvae forms (Mann-Whitney U test, P<0.05). No differences in virulence behaviour were revealed using this model to compare the L monocytogenes strains (Fig 3 and Fig 4). The strain that was expected to be the least virulent (Pulso1) showed the largest decrease in value for worm viability for both forms (50% and 55%, respectively, for larvae and mature worms) (table presented in Fig 1).

DISCUSSION
L monocytogenes virulence determination is a current research concern, and many authors have contributed to the advancement of this endeavour. A quite recent proposition (relevant when considering the capital role of InlA in pathogenicity) is that the truncation of InlA, induced by the presence of a premature stop codon in inlA, is associated with lower virulence properties (Nightingale and others 2008). Moreover, a great proportion of truncated forms were shown in strains isolated from food or food-related environments but not in L monocytogenes strains involved in clinical human cases (Van Stelten and others 2010). Discussion of the relevance of this virulence determinant arose when it was demonstrated that some truncated forms can express virulence in animal models (Van Stelten and others 2011, Holch and others 2013). Our analyses confirm that truncated forms can be found in clinical human cases in Quebec. The truncation positions, induced by SNP stop codon (700 and 762 aa) were previously reported (Kovacevic and others 2013). For the 700 aa truncation position, the entire anchorage region (between 700 and 800 aa), including the determinant proline in the LPXTG motif, was not translated. So, at least for the strain harbouring the 700 aa truncated InlA, pathogenicity was expected to be extremely low. The clinical origin of such strains led us to question the dose of ingested inoculum. In studies that demonstrated, in experimental conditions, a maintenance of virulence for strains harbouring truncated InlA, high doses of inoculum were used (Van Stelten and others 2011, Holch and others 2013). High-dose ingestion of L monocytogenes could be observed in outbreaks, giving an opportunity for a strain harbouring InlA truncated form to cause disease. We suggest that inlA analysis alone is not sufficient to characterise the virulence potential of L monocytogenes strains.
Looking for a complementary test that avoids labour intensive and ethically questioned experimentations  1). Bars: SD. confirmed the worm model as able to compare virulence for food-derived strains (Karthikeyan and others 2015). Our results, when incubating worms at 30°C with acid-shocked L monocytogenes (to promote prfAdriven virulence factors in the pathogen), confirmed a decrease of viability of worms in contact with L monocytogenes (in a lesser proportion than Salmonella Typhimurium). Contrary to the work of Karthikeyan and others (2015), the worms did not show any morphological changes when they were submitted to our L monocytogenes strain inoculations. The loss of viability was solely based on immobility and loss of pharyngeal pumping activity, in accordance with Neuhaus and others (2013). In our conditions, neither on both the mature form nor on the young larvae, L monocytogenes showed differences in virulence regardless of origin (environmental or clinical) and in their inlA integrity.

CONCLUSION
Using complete serotyping, differences in IVB geno-serogroup between clinical strains from bovine and human origins were shown, with serovar 4b found only in human clinical case strains. The virulence analysis tools used in this study (inlA sequencing and C elegans model) on both confirmed virulent and presumed less virulent strains could not univocally established the public health risk associated with L monocytogenes strains. No pig clinical case strains were provided by the MAPAQ laboratory, but serovar 4b strains were previously found in the collection of strains from healthy pigs in primary production (Larivière-Gauthier and others 2014). As calls for farm to fork strain traceability continue to be made, accurate surveillance is needed to enlarge the collection of strains in animal surveillance, particularly from pig production. It should be mentioned that we did receive all the collected L monocytogenes strains isolated in the MAPAQ laboratory, and we observed that they mainly originated from bovine clinical surveillance (where L monocytogenes present economical relevance).


extfieldsofstudy:
Psychology
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A norm knockout method on indirect reciprocity to reveal indispensable norms

Although various norms for reciprocity-based cooperation have been suggested that are evolutionarily stable against invasion from free riders, the process of alternation of norms and the role of diversified norms remain unclear in the evolution of cooperation. We clarify the co-evolutionary dynamics of norms and cooperation in indirect reciprocity and also identify the indispensable norms for the evolution of cooperation. Inspired by the gene knockout method, a genetic engineering technique, we developed the norm knockout method and clarified the norms necessary for the establishment of cooperation. The results of numerical investigations revealed that the majority of norms gradually transitioned to tolerant norms after defectors are eliminated by strict norms. Furthermore, no cooperation emerges when specific norms that are intolerant to defectors are knocked out.

Reciprocity is a fundamental mechanism that underlies all cooperative societies. Theoretically it is well known that direct reciprocity, typified by the ''I'll help you if you help me" attitude, promotes cooperative regimes 1,2 . However, in recent societies that have high relational mobility, indirect reciprocity such as ''I'll help you and somebody else will help me" plays a more important role in promoting cooperation. Indirect reciprocity has therefore been the focus of much research in the interdisciplinary fields in recent decades [3][4][5][6][7] .
Many theoretical studies on indirect reciprocity have explored norms that become evolutionarily stable against defection and the invasion of free riders, and several typical norms have been proposed [8][9][10][11] . These approaches have clarified the robust norms that can maintain the cooperative regime. The norms in the studies on the indirect reciprocity are regarded as assessment rules that label the other's action as either Good or Bad. They include tolerant norms that assess cooperative behaviors toward defectors as good 4 and strict norms that assess such behaviors as bad 12 . Other theoretical studies analysing the global dynamics of norms assume that at most a few robust norms are shared in the population [13][14][15] .
Their approaches have clarified the robustness of the norms against invasion of other norms including free riders when the norms are acceptable in the population. However, little is known about a process by which gradual changes toward cooperation occur as new norms emerge and compete, which is to say, the co-evolutionary process of norm-diversity and cooperation. A study on the indirect reciprocity has dealt with co-existing different norms and has analysed their frequencies in the population as a consequence of a dynamical process 16 . In the study, each individual keeps a private image of everyone else and errors of perception and implementation are included in a limited strategy space. Although they have considered some action rules and assessment rules, all possible norms in indirect reciprocity have not been studied all-together. How cooperation evolves cannot be fully understood unless the evolution of norms is also considered.
It is thus a challenging task to theoretically understand how cooperation can be formed even under a collection of norms in a social system. How is the co-existence of cooperation and diversity possible at all? Are there any indispensable norms needed to facilitate the evolution of cooperation in the melting pot of norms, even though some norms never become dominant? Do norms that could be accepted as a result of the co-evolutionary process have common aspects? These questions can be addressed only if all possible norms are considered, and the combination of norms governing a group can evolve.
Here we explore the dynamics of co-evolution of cooperation by using different social norms. The process of the evolution of norms has a transition from stricter to tolerant norms. Additionally, we find a set of norms that Scientific RepoRts | 7:44146 | DOI: 10.1038/srep44146 seem not to have an impact on promoting cooperation, but are fundamental to allow a transition to a cooperative regime from a defective regime.

Results
Agent-based simulations 17,18 are an optimal tool to tackle the challenge outlined above. See Methods for the details of our agent-based model described by the ODD protocol 19 . Using an evolutionary game theoretical framework and constructing an interaction model based on players' private rules and local information, we model a giving game to elucidate the dynamics of the evolution of cooperation amid the coexistence of diverse norms (Fig. 1). We conducted numerical simulations of all 16 possible norm combinations that could react to the four combinations of assessment criteria to clarify the dynamics of the evolution of cooperation from the melting pot of diverse norms. Figure 2A shows time-series graphs of each norm's population and cooperation ratio. As shown, the majority undergo an alternation from strict to tolerant norms, mostly in the order of SH → SJ → ST. Figure 3A shows the transition in the norm with the greatest population ratio. In many cases, the majority transitioned from the state where strict SH 11 was the majority to SJ 5,12 . Afterwards, the majority norm changed to tolerant ST 4,20,21 and ALLG. In contrast, as shown in Figs 2B and 3B, in an environment with errors, alternation from strict norms to tolerant norms was observed. However, the likelihood of going through SJ decreased. Alternation paths through IS 22,23 , which could not be seen in an environment without errors 24 , increased. It is important to note here that similar paths toward cooperation are observed when only ALLB-individuals are initially assumed. New norms are created during the evolutionary process at the same time cooperation evolves. This indicates that cooperation and diversity of norms jointly evolve in the model.
Why does the alternation of norms emerge? For one thing, in states in which defection is dominant, ALLB (BBBB) and SH (GBBB) coexist and jointly form the majority. However, BGBB and IS (GGBB) continue to exist as the minority. The characteristic of these groups is having the evaluation rule of **BB. Evaluation rule **BB assesses donors that took D as B, regardless of the evaluation of the recipient. In states in which defection is dominant, those who adopt **BB strategies consider many partners as B. As a result, cooperation does not occur for the most part. The ALLB and SH norms thus survive because they do not lower their own cost. On the other hand, after cooperation is achieved, ALLG (GGGG), ST (GGBG), IS (GGBB), and GGGB coexist. The common characteristic of these norms is having the evaluation rule of GG**. Thus, reciprocally cooperating norms survive. Because SJ (GBBG), which becomes the majority temporarily when the cooperation ratio rises in an environment without errors, does not belong to either group, it cannot stably exist. Also, it is rare that SJ makes up the majority temporarily in an environment with errors. Meanwhile, because IS belongs to both norm groups with **BB and GG**, IS can constantly exist.
We discover several norms that are indispensable to the evolution of cooperation. Reputation-based cooperation cannot emerge without indispensable norms. To elucidate indispensable norms for the evolution of cooperation, we propose a novel analysis using the norm knockout method. This method enables us to determine which norms are indispensable for the evolution of cooperation. The norm knockout method is inspired by the targeted gene knockout technique used in genetic engineering 25 . Gene knockout, a genetic technique in which one of an organism's genes is made inoperative, is used to research genes whose sequences are known but whose functions are not well-understood. Researchers infer the gene's function from differences between the knockout animal and a normal animal. For simulating evolution, we utilized a method that removed only one particular norm from the population to understand whether that norm is an indispensable one that plays a critical role in the evolution of cooperation. Figure 4 shows the cooperation ratio when a particular norm is knocked out. Regardless of whether there is an error, if SH or IS is knocked out, cooperation does not evolve at all. We define indispensable norms in the When SH and ALLB coexist, cooperation does not emerge. When ALLB is completely driven out by SH, SJ invades and the cooperation ratio abruptly rises. At the same time, SH is driven out by SJ. After cooperation is completely achieved, SJ permits the invasion of ST, and also coexists with other tolerant norms (IS, ST, GGGB, and ALLG). Finally, strategies whose norm is expressed as GG** (in other words, norms that constantly cooperate if cooperation has been selected in the past by the recipient) coexist. In (B), both errors in perception and implementation were introduced, and simulation similar to a was run (b = 5, c = 1, N = 500, R = 500, G = 1000, p = 0.001, q = 0.001). As in (A), when SH and ALLB coexist, cooperation does not emerge. However, cooperation is achieved without going through SJ. evolution of cooperation as the norms that, when knocked out, have an average cooperation ratio of less than 0.1 after 1,000 generations. In an environment with no errors, SH and IS are indispensable norms. In an environment with errors, SH, IS, and ST are indispensable norms.
When an indispensable norm is knocked out, cooperation does not evolve. When cooperation evolves, alternation from strict norms to tolerant norms was observed, as shown in Figs 2 and 3. To analyse whether alternation also occurs when a norm is knocked out, the population ratio of norms when typical norms are knocked out is displayed as time-series graphs (see Fig. 5). Figure 5 shows the results in the cases where SH or IS were knocked out. We discovered that the first condition for the necessary process when cooperation evolves is whether SH can antagonize ALLB. No norm that resists the invasion of ALLB appears in a society in which SH does not exist. Also, in a society in which IS does not exist, SH cannot antagonize ALLB. We found that IS is a norm indispensable for SH to resist ALLB.

Discussion
Our model offered two major findings on the evolution of cooperation on indirect reciprocity. On the one hand, the most essential contribution is the discovery of indispensable norms by the norm knockout method. By using the norm knockout method, we were able to elucidate the existence of norms indispensable for the evolution of cooperation from a melting pot of norms. Regardless of the existence of errors, SH and IS were indispensable norms. In addition, in an environment with errors, ST is an indispensable norm. Interestingly, SH and IS are reconciled to the minorities after the cooperative regime emerges while they temporarily become major norms in the process of dynamics. We call such minority norms required for the evolution of cooperation "unsung hero norms". The results clearly illustrate the two roles of norms: one to catalyse a cooperative regime and the other to maintain the regime. Norms having the GG** for the evaluation rule play the latter role.
On the other hand, we discovered alternation of norms. Recent analysis of evolutionary stability against the invasion of free riders could identify neither superiority among norms nor the process on the path to cooperation.  Among studies on indirect reciprocity, ours is the first exhaustive theoretical analysis on all possible norms, although several studies have addressed the comparison of two types of reciprocal norms 14,15,26 . Others analyse the alternation of norms in direct reciprocity [27][28][29] . We find the alternation of the norms and also discover the indispensable norms that are required to foster indirect reciprocity.
An empirical study 30 supports the co-existence of various norms in the cooperative regime and indicates that the ST norm plays a more important role in human cooperation than SJ, which is consistent with our simulation. This is because we show that the SJ norm cannot survive in the cooperative regime, while the ST one can. Our approach may provide deep insight on the evolution of cooperation because several norms absolutely play an essential role in order to evolve cooperation even though, on the surface, it seems as though they are not directly leading to the evolution of the cooperation.
The present work considers a single action rule (cooperate with Good, defect with Bad) to stress the role of multiple assessment rules. However, the other papers stress the role of multiple co-existing action rules 13,[31][32][33] . Integrating the multiple assessment and action rules may be a useful extension of this paper. We analyse what happens when one norm is absent from the population; however, we have not analysed all the indispensable combinations of norms yet. Extending the norm knockout method to combinations of norms may also be a useful extension of this paper.

Methods
In this section, we describe the details of our agent-based model that uses the norm knockout method. The following model description follows the ODD protocol 19 . Purpose. The aim of the model is to understand the dynamics of norms during the evolution of cooperation, and to find indispensable norms without which cooperative societies could never emerge. In particular, we reveal the effect of these indispensable norms on indirect reciprocity using a new methodology we call the "norm knockout method". We utilize the giving game framework 34 for simulation.

Entities, state variables, and scales.
The entities in the model are agents who play as donor and recipient in the giving game with no spatial structures. The donor chooses cooperation or defection with a recipient using an image that the donor has to the recipient. An image is either Good or Bad. If a donor's image to a recipient is Good, the donor cooperates with the recipient. If the image is Bad, the donor defects. The group size of the model is N. Each agent has it's own norm and a list of images to other agents. The agent also has a probability of errors and a payoff of the game.
The norm of an agent is denoted as one of four possible "assessment combinations", and there are two possible "alleles (G/B)" at the "locus" for each of the four assessment combinations. The first locus of the gene represents an assessment rule to an agent who cooperates with a Good recipient. The second locus represents an assessment rule to an agent who cooperates with a Bad recipient. The third locus represents an assessment rule to an agent who defects with a Good recipient. The fourth locus represents an assessment rule to an agent who defects with a Bad recipient. Incidentally, all agents evaluate themselves as Good. For instance, ALLG always assesses others as Good, and thus, its "genotype" is GGGG using the above mentioned definition of the four loci. Similarly, ALLB is described as BBBB, IS as GGBB, ST as GGBG, and SJ as GBBG.
Each agent has two different types of errors: one, the probability that the agent's updating of its evaluation of others, Good/Bad, is inverted (errors in perception), described as p, and two, the probability to perform an action differently from the one prescribed by its action rule (errors in implementation), described as q. The evolution process of norms involves adopting a genetic algorithm 35 . State variables and initialization in the simulation are shown in Table 1.
Process overview and scheduling. Our simulation runs throughout G generations. A generation consists of R rounds. The agents play the giving game R times as donor in each generation. At the end of a generation, they evolve their own norms using accumulated payoffs that are obtained in the generation. One round has two phases: (A) a phase to play giving games and (B) a phase to update images. After all agents play giving games in phase (A), all agents update their images in phase (B). In phase (A), each agent becomes a donor and each donor randomly chooses a recipient from N − 1 players excluding itself. The donor chooses whether to give benefits to the recipient or not. At that time, the action of the donor is inverted with the probability q. The donor who cooperates pays cost c and the recipient receives benefit b (b > c > 0). In phase (B), each agent (set to i) evaluates and updates an image to the other agent (set to j). The new image to j from i's viewpoint depends on j's action (C/D) as a donor in the last round and depends on the image to k from the viewpoint of i (G/B), where k was a recipient of j in the last round. At that time, the image to j is inverted with the probability p. In the first round in a generation, j's action (C/D) is regarded as random.
After R rounds of the giving game are played in every generation, agents evolve their norm. The evolution process of norms involves adopting the genetic algorithm 35 . Because each locus of norm has an independent meaning for assessment to others, the adaptive process should contain a combination of these elements rather than a string of norms. We have modeled a process of updating norms not as a string of norms but rather as four different assessment rules, which enables the norms to be interpreted as different situations depending on the norm genotype. The first, second, third, and fourth loci represent the assessment for pro-social behavior, tolerant behavior, anti-social behavior, and justified defection (punishment), respectively. Each agent randomly selects two agents from N agents (including itself) to become its parents. For choosing parents, we adopt a roulette selection method. This roulette selection sets a probability distribution of all agents as Π i = (U i − U min ) 2 /∑ j (U j − U min ) 2 , where U i denotes the agent i's accumulated payoff in a generation given by U i = bW − cV, with W being the number of donations i received in the generation and V the number of donations i gave. U min means a minimum value Scientific RepoRts | 7:44146 | DOI: 10.1038/srep44146 of the accumulated payoffs among all. Finally, each agent updates its norm using a uniform crossover technique. With mutation rate m, each locus is inverted for maintaining the diversity of the norm space.
Design concepts. Basic principles. An agent-based simulation is utilized to study indirect reciprocity. We explore how different combinations of norms interact to produce an evolutionary progression towards cooperation.
Emergence. A cooperative regime in the situation of social dilemma emerges from interactions among agents who have various social norms.
Adaptation. The agents of the model play the giving game using their images to others. The agents update their images to others using their norms every round. They evolve their own norms using accumulated payoffs that are obtained in the generation. A norm that can obtain a higher payoff can increase the population through the generation.
Objectives. The objective of all agents is to maximize their own payoff. To maximize payoff, they change their own norm at the end of each generation.
Learning. The agents change their norms in each generation using a genetic algorithm. The fitness of each agent is calculated from the accumulated payoff in the generation. To select the parents of the agent, the model utilizes a roulette selection method.
Interaction. The interaction between the agents is one to one interaction. The giving game consists of the donor and the recipient. There are no spatial structures in the society.
Stochasticity. The interaction between agents is a stochastic process because interaction partners are chosen randomly from the society. At the start of the simulation, each agent is randomly assigned a norm of all 16 norms.
Observation. Three indexes are used for observation: average cooperation ratio in the society, the transition of norms with the greatest populations, and population ratio of each norm.
Initialization. At the start of simulation, the norm of each agent is chosen randomly from all 16 possible norm combinations. In the first round of each generation, the evaluation of all agents is initialized as Good 11,23,36 and payoff of the agents is initialized as 0.
Input data. After initialization, the model does not include any external inputs, i.e., the number of agents (N), error ratio (p, q), benefit (b), and cost (c) are constant.

Submodels.
The norm knockout method. The norm knockout method is implemented as follows. When we knock out a particular norm, that norm is removed in the first round of each generation. Concretely, if the norm of an agent evolves into a norm that is knocked out as a result of the adopting process, the norm of the agent is changed to one of the other 15 norms randomly. In other words, the norm that is knocked out will never exist at all in the society.


Dose Dependent Treatment with Boric Acid Induces More Changes in the Sperm Cells of Endangered Anatolian Trout Salmo Rizeensis

The aim of this study was to test the usefulness of boric acid for endangered Anatolian trout Salmo rizeensis sperm. Activation media was supplemented with boric acid (0.5, 1, 2, 3, 4 and 5 mM). Sperm motility and duration were determined in sperm samples. In addition, fertility and hatching rate were examined. Our data indicated that addition of boric acid (3 mM) to activation media was increased the percentage and duration of motile sperm, fertility and hatching rate in endangered Anatolian trout (S. rizeensis). On the other hand, the motility rate decreased with increasing the concentration of boric acid (p<0.05). In particular, a remarkable increase was observed after concentration 3 mM. Consequently, sperm quality was affected by quantitative changes different concentrations of boric acid and the best results were obtained at concentration 3 mM. Research Article

Introduction
Salmo trutta are the most important Salmonid fi sh species owing to its aquaculture potential, economic value and wide consumer demand [1]. Salmo trutta forms inhabit in the upper streams of rivers and occur in North Africa, Europe, West Asia and Anatolia [2,3]. In addition, it is an important potential species for recreational fi shery. Recently, S. t. macrostigma ecotype has been described by Turan et al. [4], as S. rizeensis [5]. In particular, populations of the species are affected by natural hybridization, the local devastation in water sources through habitat fragmentation and modifi cation, water eutrophication and contaminations, environmental instability and global warming [6][7][8][9][10]. Sperm motility is the essential functional parameter for successful fertilization in fi sh [11,12]. Sperm cells in most fi sh species immotile in seminal fl uid and require to release into the water in order to trigger motility and become metabolically active [12,13]. Therefore, characteristics of activation media are crucial in terms of initiation and progression of sperm motility [12].
Trace elements have a crucial role for the male reproductive process. Boric acid (H 3 BO 3 ) is a bioactive benefi cial element [14], and widely used in glass, ceramic, detergent, plastic, agricultural, textile, metallurgy, nuclear and medicine industries owing to its excellent characteristics and it is produced from boron ore and salt lake brine [15][16][17]. The greatest majority of boric acid is produced by Eti Maden Works (Turkey) in the world [18]. Increasing production does not meet the demand due to using as extensive [19,20]. Several studies about the nutritional benefi ts [14,19,21,22], metabolic functions [23][24][25][26], U shaped dose responses on growth of embryonic fi sh and frogs [27,28], and therapeutic applications [29][30][31], toxic effect on male reproduction system of different species (e.g. rat, rodent, dog, human) [32][33][34][35], of boron and its compounds have been published in the latest available literature. As far as the authors of this work are aware, no attempt has been made to use of boric acid in sperm activation medium of fi sh species. In this context, the aim of this study was to examine effect of activation media supplemented with boric acid (0.5 mM; 1 mM; 2 mM; 3 mM; 4 mM; 5 mM) on sperm quality and fertility of endangered trout S. rizeensis.

Materials and Methods
Six mature endangered Anatolian trout males (1388.00±0.55 g, 44.52±2.62 cm as mean±SD) were captured from Uzungöl Stream, Trabzon, Turkey for sperm collection. F spermatocrit ish were anesthetized in 50 mg/L Benzoacaine, sperm samples were collected through abdominal massage and special care was taken to prevent contamination (e.g. blood, feces or urine).

Results
Sperm parameters (mean ± SD) are presented in table 1.
Effect of boric acid on the percentage and duration of motile sperm for S. rizeensis is shown in fi gure 1. The results of the present study indicated that differences in the percentage and duration of motile sperm of S. rizeensis sperm were signifi cant among the treatments (p<0.05). Highest motility rate (80.00%) and duration of motility (60 s) were at concentration 3 mM.
After concentration 3 mM, a remarkable decrease was observed in motility rate and duration. Effect of supplementation of boric acid on fertility and hatching rate for S. rizeensis is presented in fi gure 2. The trials in this study indicated that differences in the fertility and hatching rate were signifi cant among the treatments (p<0.05). Highest fertility (92.17%) and hatching rate (82.09%) were at concentration 3 mM. In particular, a signifi cant decrease was observed in fertility and hatching rate after concentration 3 mM.
In this study, we demonstrated the usefulness of boric acid in different activation mediums for S. rizeensis sperm. Using 3 mM boric acid in activation media resulted in high sperm motility rate and duration, fertility and hatching rate of S.
rizeensis. This may be due to the fact that boric acid is involved in a number of metabolic processes and interact with critical biological substances, including pyridoxine, polysaccharides, dehydroascorbic acid, ribofl avin, and the pyridine nucleotides [38][39][40]23]. However, motility rate and duration decreased with increasing concentration of boric acid. In particular, a    In conclusion, based on these results, boric acid was used effi ciently for activation media in S. rizeensis sperm. Our study provides new insights related to use of boric acid on fi sh sperm quality. The knowledge of effects of boric acid and its mechanism of action might be helpful for both research and commercial use. Further studies would be needed to evaluation the precise mechanisms of boric acid response.

Funding
This work was funded by Karadeniz Technical University.
Authors like to express deep thanks to continuous help of Aziz KUTLUYER and Devrim ALTUNTAŞ.