PLoS ONE plos plosone PLOS ONE 1932-6203 Public Library of Science San Francisco, CA USA 10.1371/journal.pone.0226280 PONE-D-19-19251 Research Article Biology and life sciencesOrganismsEukaryotaAnimalsVertebratesAmniotesMammalsDogs Biology and life sciencesEvolutionary biologyPopulation genetics Biology and life sciencesGeneticsPopulation genetics Biology and life sciencesPopulation biologyPopulation genetics Research and analysis methodsImaging techniquesMorphometry Biology and life sciencesEvolutionary biologyPopulation geneticsGenetic polymorphism Biology and life sciencesGeneticsPopulation geneticsGenetic polymorphism Biology and life sciencesPopulation biologyPopulation geneticsGenetic polymorphism Biology and life sciencesOrganismsEukaryotaAnimalsAnimal typesPets and companion animals Biology and life sciencesZoologyAnimal typesPets and companion animals Biology and life sciencesGeneticsGenetic lociAlleles Medicine and health sciencesDiagnostic medicineAuscultation Biology and life sciencesGeneticsHeredityHeterozygosity Breeding French bulldogs so that they breathe well—A long way to go Brachysephalic syndrome in French bulldogs Ravn-Mølby Eva-Marie Formal analysis Investigation Writing – original draft Writing – review & editing 1 http://orcid.org/0000-0003-4744-8841 Sindahl Line Formal analysis Investigation Writing – original draft Writing – review & editing 1 http://orcid.org/0000-0003-2417-0787 Nielsen Søren Saxmose Formal analysis Validation Writing – review & editing 1 Bruun Camilla S. Supervision Writing – review & editing 1 http://orcid.org/0000-0003-0397-3273 Sandøe Peter Supervision Writing – review & editing 1 2 http://orcid.org/0000-0002-3563-7648 Fredholm Merete Conceptualization Funding acquisition Methodology Supervision Writing – original draft Writing – review & editing 1 * Department of Veterinary and Animal Sciences, University of Copenhagen, Frederiksberg C, Denmark Department of Food and Resource Economics, University of Copenhagen, Frederiksberg C, Denmark Loor Juan J. Editor University of Illinois, UNITED STATES

The authors have declared that no competing interests exist.

 * E-mail: mf@sund.ku.dk 16 12 2019 2019 14 12 e0226280 9 7 2019 23 11 2019 2019 Ravn-Mølby et al This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Brachycephalic syndrome (BS) is a pathophysiological disorder caused by excessive soft tissue within the upper airways of short-nosed dog breeds, causing obstruction of the nasal, pharyngeal and laryngeal lumen, resulting in severe respiratory distress. As the prevalence of BS appears to be high among some of the affected breeds, there is an urgent need for breeding efforts to improve the health status of those dogs. In the present study, we evaluated correlations between morphometric and other phenotypic characteristics and BS in a population of 69 French bulldogs from Denmark to identify parameters that could serve as a basis for breeding against BS. Furthermore, the genetic variation was monitored to determine whether it would be possible to breed based on these characteristics without simultaneously causing a critical reduction in genetic variation. Six phenotypic characteristics were correlated with the Brachycephalic Syndrome Functional (BSF) score. Among the morphometric risk factors, nostril stenosis (NS) and neck girth (NG) had the highest impact on the BSF score, accounting for 32% and 4% of the variation, respectively. The genetic variation in the population was comparable to other pure breeds, i.e. estimated and observed heterozygosity was 0.60 and the average inbreeding coefficient was 0.01. If only dogs with Grades 1 and 2 NS (no or only mild NS) were selected for breeding the mean BSF score would be reduced significantly. However, it would result in the exclusion of 81% of the population for breeding and this is not prudent. Excluding only dogs with severe stenosis (Grade 4) would exclude 50% of the population without any adverse impact on genetic variation within the population. Although exclusion of dogs with Grade 4 would result in an apparent reduction in the mean BSF score, this reduction is not significant. As NS accounts for 32% of the variation in BSF score, a possible long term strategy to reduce the prevalence of the BS in French bulldogs would seem to be a selection scheme that first excluded dogs with the most severe NS from breeding, gradually moving towards selecting dogs with lower NS grades. According to our findings there is no viable short term solution for reducing the prevalence of BS in the French bulldog population.

 We wish to thank the Danish Kennel Club for financial support provided to MF for the project. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Data Availability All relevant data are within the paper and its Supporting Information files. Background

Brachycephalic syndrome (BS) constitutes a major health and welfare problem in several short-nosed breeds as it causes breathing difficulties, heat and exercise intolerance, sleep-disordered breathing, cyanosis and collapse in the affected individuals [1]. In line with the increased popularity of brachycephalic breeds and the demand for hyper-types, the problem seems to be continuously growing. Surgery and medical management can alleviate the symptoms of BS at the individual level, but prophylaxis through breeding is the only available solution at population level. As such, there is an urgent need for breeding efforts to improve the health status of dog breeds affected by BS.

This study aimed to elucidate the possibility of establishing a breeding strategy that could reduce BS in French bulldogs. In order to establish a breeding strategy, two conditions must be met: 1) effective selection criteria must be established, and 2) the proportion of the population deemed phenotypically acceptable for breeding must have sufficient genetic variation. While information on genetic variation can be readily obtained at population level, identification of appropriate selection criteria poses a bigger challenge.

Because health problems related to BS primarily occur due to anatomic abnormalities in the upper airways, quantitative assessment of the anatomical deviation using computer tomography (CT) and laryngoscopy seems to be an obvious way to grade BS. However, no published studies have evaluated whether objective measures of the problematic anatomic abnormalities reflect the degree of functional deterioration. It is the functional impact of the patho-anatomy that affects the daily life of the individual dog and which therefore should be in focus. There are several other reasons for substituting assessment of the anatomical deviations with functional assessment of BS: Firstly CT and laryngoscopy require generalized anesthesia, which poses an increased risk in brachycephalic dogs, and secondly, CT and laryngoscopy are expensive to carry out, which might withhold some breeders from having them performed. Therefore, in the current study, functional assessment is used to identify phenotypic risk factors that may be used as part of an effective breeding strategy.

As BS has evolved in line with intensified breeding for shorter muzzles in brachycephalic breeds, muzzle length has been recognized as the determining factor for BS, despite a lack of evidence relating to the scaling effect [2, 3]. While Packer and others [3] found the risk of BS increased steeply in a non-linear manner concordant with a decrease in the craniofacial ratio (CFR), Liu and others [2] found only a weak association between CFR and the risk of BS in French bulldogs. A short muzzle in itself therefore does not seem to be a major risk factor for BS when evaluated within the range of lengths represented within the French bulldog population. Accordingly, recent studies have identified various other risk factors including sex, body condition score (BCS) and external morphometric features including neck girth (NG) and degree of nostril stenosis (NS) [2, 3].

Although the degree of external NS is not a direct expression of the degree of intranasal stenosis, the inner nasal vestibule is often concomitantly reduced due to a relatively large inner wing of nasal cartilage [4, 5]. While moderate-to-severe stenotic nostrils have been found to increase the mean BS index by only 16% across brachycephalic breeds, it has been shown that the risk of BS is increased by about 20 times in French bulldogs with moderate-to-severely stenotic nares [2, 6].

Exercise testing in the form of a walk test, measured as distance walked during a 6-minute period, has proved effective in capturing differences in physical capacity of dogs with chronic heart failure, mild to moderate pulmonary disease, centronuclear myopathy and in dogs undergoing weight loss [7, 8, 9, 10, 11, 12]. Likewise, it has been shown that the severity of BS in English Bulldogs is negatively correlated with the 6MWT distance and lately, the potential of exercise testing and laryngeal/tracheal auscultation as a valuable tool for grading of BS has been established [13, 14].

In the present study we use the 6-minute walking test (6MWT), auscultation and dB recordings of upper airway noise to establish Brachycephalic Syndrome Functional scores (BSF scores). We evaluate the correlation between the BSF scores and morphometric and other phenotypic parameters (including parameters that might confound the results), in order to identify potential conformational risk factors applicable as tools in a breeding strategy. To investigate whether it would be possible to implement a selection scheme aimed at reducing the risk of BS at population level without creating a genetic bottleneck, the genetic variation in the total study population was evaluated and compared to the genetic variation in subpopulations selected based on morphometric traits highly correlated with low-level BS.

 Methods

The study was performed at the University of Copenhagen, Department of Veterinary and Animal Science during March and April 2018. The dogs were tested in a quiet, air- and temperature-regulated (21–22°C) corridor within the department.

The study protocol was approved by the Research Ethics Committee for Science and Health at the University of Copenhagen (Ref 504-0027/18-5000).

Animal material

A total of 79 French bulldogs were recruited via the social media platform Facebook during March 2018. A public recruitment poster was posted on the first author’s personal wall and was shared 269 times by public dog groups, on private walls, and on veterinary pages. A total of 90 dogs unrelated at the parental level were included on first-come, first-served basis. Both FCI-registered and non-registered dogs were accepted for the study. A waiting list was established for all subsequent inquiries. Of the 90 dogs, 11 did not arrive for their appointment or their owners cancelled too late to invite the next on the waiting list. Consequently, the test population comprised 79 dogs. The dogs were 1–5 years of age and had no history of upper airway surgery or any detectable orthopedic, cardiovascular, neurological or pulmonary disease. Intake of medication that could potentially affect the physical capacity and clinical evaluation (e.g. steroids and nonsteroidal anti-inflammatory drugs) within the past 6 weeks was also a reason for exclusion. Informed consent forms were signed by all dog owners before the clinical study was performed.

 Clinical study

The clinical part of the study aimed to (i) register morphometric characteristics that could be correlated to BS, as well as other phenotypic characteristics that could be confounders for the correlations discovered (13 parameters in total), and (ii) use the BSF score as a proxy for degree of BS.

The morphometric and phenotypic characteristics included height (H), neck length (NL), neck girth (NG), the neck-length-to-neck-girth ratio (NLGR), muzzle length (ML), cranial length (CL), craniofacial ratio (CFR), nostril stenosis (NS), BCS and body weight (W). Details on how these parameters were established are provided in Table 1 and Fig 1. Measurements of the skull, neck and height were performed in accordance with [15], while the degree of NS was graded in accordance with [2]. The BCS scoring system used was the 9-point body condition scoring system [16]. The same two investigators scored and agreed on all BCS as well as nostril gradings (intra-observer error was not taken into account), and the latter were subsequently reviewed and confirmed using the rostro-caudal photos taken of nares. One investigator took all the morphometric measurements of the participating dogs to avoid any observer-related systematic error [2]. In addition to the morphometric measurements, the sex, neuter status and age were noted for each participating dog, based on owner information. All morphometric and phenotypic recordings are provided in S1 Table.

10.1371/journal.pone.0226280.g001 Representative examples of the four NS grades described in <xref ref-type="table" rid="pone.0226280.t001">Table 1</xref>.

From left to right: Grade 1, Grade 2, Grade 3, Grade 4.

 10.1371/journal.pone.0226280.t001 Description of measurements of morphometric and phenotypic characteristics.
PARAMETER DESCRIPTION
Nostril senosis (NS)
Grade 1 Open nostrils: Nostrils are wide open.
Grade 2 Mildly stenotic nostrils: Slightly narrowed nostrils where the lateral nostril wall does not touch the medial nostril wall.
Grade 3 Moderately stenotic nostrils: The lateral nostril wall touches the medial nostril wall at the dorsal part of the nostril and the nostrils are only open at the bottom. The nostril wings were not able to move dorsolaterally immediately after the exercise test, and there may be nasal flaring.
Grade 4 Severely stenotic nostrils: Nostrils are almost closed. The dog may switch from nasal to oral breathing after stress or very gentle exercise such as playing.
Height (H) The linear distance from the ground to the dorsocranial angle of the scapula (withers). Measured when the dog is in an upright, stacked position.
Muzzle length (ML) The distance along the skull from the rostral end of the planum nasale to the dorsal plane between the punctae lacrimale of the left and right eye (the stop).
Cranial length (CL) The distance along the skull from the stop to the external occipital protuberance.
Neck length (NL) The distance along the body from the external occipital protuberance to the dorsal plane of the withers. This is measured along the median plane of the dog.
Neck girth (NG) The circumference of the neck at the median distance between the external occipital protuberance and the withers.
Neck girth-to-length ratio (NGLR) The ratio of neck girth to neck length: NGLR = NGNL
Cranio-facial ratio (CFR) The ratio of cranial length to muzzle length: CFR = CLML
Body Condition Score (BCS) [16]

The BSF score was used as a proxy for degree of BS. This score is a continuous variable calculated on the basis of 16 assessments of upper airway noise and the presence of respiratory distress at rest during and after a submaximal exercise test and after a 15-minute recovery period. Information relating to 1) respiratory distress 2) audible upper airway noise during auscultation of the a) lungs and b) trachea/larynx, 3) the presence of audible upper airway noise without a stethoscope, and 4) decibel recordings was registered prior to and immediately after a 6-minute walk test (6MWT) and after a 15-minute recovery period. The decibel recordings were done using the app “Sound Meter” version 1.1 by Examobile on an iPhone 8, where 0–30 dB was considered baseline (background noise), while 30–40 dB yielded a score of 1, and 40–50 dB a score of 2. The presence of audible upper respiratory noise without a stethoscope was also registered during exercise. The presence of respiratory distress as well as intermittent noise returned a score of 1, while continuous abnormal upper airway noise yielded a score of 2. The scores were summed to generate a total score for each dog ranging from 0–32 (see Table 2 for details on the calculation scheme). All scores for the individual dogs are presented in S2 Table.

10.1371/journal.pone.0226280.t002 Illustration of the scoring system used to calculate the Brachycephalic Syndrome Functional score (BSF score).
Score0 Score1 Score2
Pre-exercise Presence of respiratory distress No Yes
Presence of audible upper airway noise during auscultation of the lungs No Yes, intermittent Yes, continuously
Presence of audible upper airway noise during auscultation of trachea/larynx No Yes, intermittent Yes, continuously
Presence of upper airway noise audible without stethoscope No Yes, intermittent Yes, continuously
Decibel recordings 20–30 Hz 30–40 Hz 40–50 Hz
During 6MWT Presence of upper airway noise audible without stethoscope No Yes, intermittent Yes, continuously
Post-exercise Presence of respiratory distress No Yes
Presence of audible upper airway noise during auscultation of the lungs No Yes, intermittent Yes, continuously
Presence of audible upper airway noise during auscultation of trachea/larynx No Yes, intermittent Yes, continuously
Presence of upper airway noise audible without stethoscope No Yes, intermittent Yes, continuously
Decibel recordings 20–30 Hz 30–40 Hz 40–50 Hz
Recovery Presence of respiratory distress No Yes
Presence of audible upper airway noise during auscultation of the lungs No Yes, intermittent Yes, continuously
Presence of audible upper airway noise during auscultation of trachea/larynx No Yes, intermittent Yes, continuously
Presence of upper airway noise audible without stethoscope No Yes, intermittent Yes, continuously
Decibel recordings 20–30 Hz 30–40 Hz 40–50 Hz
Data processing and statistical analysis

The Spearman Correlation test was used to assess the correlation between non-parametric numeric variables, while the Pearson Correlation test was performed on the variables deemed to follow a normal distribution. The Wilcoxon rank-sum test was used for comparison of the ranks of non-parametric variables with two levels, whereas the Kruskal-Wallis test by Ranks was performed on the nostril grade as it had >2 levels. An unpaired two-tailed t-test without homogeneity of variance was used to compare means for data deemed to follow a normal distribution, and multivariable linear regression analysis was performed with the BSF score as the outcome and including parameters that were both relevant for breeding and significantly correlated with the BSF score as input variables: degree of NS (with three levels: 1–2, 3 and 4, NG (cm), muzzle length (cm), CFR. A backwards stepwise model selection based on Akaike’s Information Criterion was used to determine the model with the best fit. The Wilcoxon rank-sum test was used to compare non-parametric parameters. Statistical analyses were performed in R v. 3.5.1 [17] using a significance level of 0.05.

 Genotyping and evaluation of genetic variation

Buccal swabs were collected from all dogs for genetic testing. DNA was extracted from the swabs using the WizardR Genomic DNA Purification Kit (Promega, Madison, USA) for purification of DNA. Microsatellite genotyping was performed using the Canine Genotypes TM panel 1.1, F-860S/L (Finnzymes Diagnostics, Finnzymes Oy, Keilaranta 16 A, 02150 Espoo, Finland) comprising 18 microsatellites distributed on 18 of the 19 canine autosomes. Breed detection was carried out in all the non-FCI registered dogs using the Wisdom PanelTM 4.0. Allele frequencies (f(A)), numbers of alleles (AA), effective alleles (AAE), observed and expected heterozygosity (HO and HE, respectively) and inbreeding coefficients (F) were calculated using Microsoft Excel. AAE is defined as the average number of equally frequent alleles (effective alleles) needed to achieve the average level of gene diversity (AE=11−HE). HE was calculated using the formula: HE=1−∑i=1nf(A)i2 where f(A)i is the frequency of the ith of n alleles, while HO is the heterozygosity present in the population directly derived from the actual genotype dataset. F is the population inbreeding coefficient (F=HE−HOHE).

For the sake of investigating possibilities for selection, the population was hypothetically divided into three subpopulations. The genetic variation parameters and the BSF scores were compared between the subpopulation of dogs with NS1 and NS2 (NS1-2), the subpopulation of dogs with NS1, NS2, and NS3 (NS1-3), and the total study population (NS1-4). This was achieved by performing an unpaired two-tailed t-test without homogeneity of variance for normally distributed variables and a Wilcoxon rank-sum test for non-parametric variables.

 Results

A total of 79 French bulldogs participated in the study. On the day of testing, 7 dogs were excluded due to: orthopedic (n = 1) or behavior (n = 4) problems, being under one year of age (n = 1) or having an elevated core temperature (n = 1). After breed determination and revision of pedigrees, three dogs were excluded due to being of mixed breeds. Consequently, the total number included in the analyses was 69 dogs.

Evaluation of the clinical parameters

The BSF scores of the total study population were distributed with a mean value of 14.12 ± 7.22 standard deviations. The scores ranged from 0 to 29 out of 32, with only 3 dogs out of all 69 given a score of 0 (Fig 2).

10.1371/journal.pone.0226280.g002 The distribution of BSF scores within the study population.

The correlations between the BSF score and the 14 clinical parameters are presented in Table 3, which shows the mean and median for normally and non-normally distributed parameters, respectively. As can be seen, the population included in the analyses was almost equally distributed in terms of sex at 43% male and 57% female, with a total of 83% of the dogs being intact. The median age was 2.5 years, with the youngest dog tested at exactly 12 months of age and the oldest dog participating one month before turning six years old. The mean weight ± SD was 12.47 ± 2.25 kg, where 62.3% of dogs were overweight (BCS > 5/9).

10.1371/journal.pone.0226280.t003 Correlations between BSF score and morphometric and phenotypic parameters in 69 French bulldogs.
Mean ± SD (min-max) r (95% CI) p
NL (cm) 11.17 ± 1.89 (7–17) -0.15 (-0.37; 0.093) 0.23p
NLGR* 0.31 ± 0.050 (0.19–0.44) -0.31 (-0.51; -0.080) 0.0094p
CL (cm) 13.38 ± 1.07 (10–15.5) 0.17 (-0.065; 0.39) 0.15p
ML* (cm) 2.43 ± 0.54 (1.50–4) -0.26 (-0.47; -0.024) 0.032p
CFR* 0.18 ± 0.040 (0.10–0.30) -0.31 (-0.51; -0.080) 0.0092p
W (kg) 12.47 ± 2.25 (7–18.80) 0.10 (-0.14; 0.33) 0.39p
6MWT distance 511 ± 113 (270–752.80) -0.52 (-0.67; -0.32) 0.0000056p
Median (min-max) Ϸ p
NG* (cm) 36.57 (31–44) 0.29 0.015s
H (cm) 33.32 (29–39) -0.16 0.20s
Age (months) 31.87 (12–71) -0.084 0.49s
NS*Grade 1Grade 2Grade 3Grade 4 3 (1–4) 0.00008k
1.4%
17.4%
31.9%
49.3%
Neuter status NeuteredIntact 17.4% 0.25w
82.6%
Sex* MaleFemale 43.5% 0.017w
56.5%
BCS* Normal Overweight 37.7% 0.037w
62.3%

NL = neck length; NLGR = Neck length to girth ratio; CL = cranial length; ML = muzzle length; CFR = Craniofacial ratio; W = weight; 6MWT = 6-minute walking test; NG = neck girth; H = height; NS = nostril stenosis; BCS = body condition score

*Variables that are statistically correlated with the BSF score at a significance level of 0.05

r = Pearson Correlation coefficient and associated test (p)

Ϸ = Spearman Correlation coefficient and associated test (p)

k = Kruskal-Wallis test

w = Wilcoxon rank-sum test

NLGR, ML, CFR, NG, sex, NS, being overweight and distance covered during 6MWT were all correlated with the BSF score. No significant correlation was found with NL, CL, W, H, age or neuter status. The multivariable regression analysis was carried out using the five significantly correlated morphometric parameters (CFR, ML, NG, NGLR and NS). The analysis showed that only NG and NS contributed to the model with the best fit (Table 4). The model suggested that NS had the highest impact on the BSF score with 10.59 added to the score if the dog presented with severely stenotic nostrils (Grade 4) instead of open (Grade 1) or mildly stenotic (Grade 2) nostrils, or an additional 8.45 if the nostrils were moderately stenotic (Grade 3). NS alone could explain 32% of the variation in BSF scores, while NG added 0.50 BSF scores per cm, and could explain 4%.

10.1371/journal.pone.0226280.t004 Results of the multivariable linear regression analysis of BSF score and morphometric parameters in 69 French bulldogs.
Estimate SE P
Intercept -11.93 8.09 0.15
Nostril grade    1–2                3                4 0 0 Reference
+8.45 2.05 0.0001
+10.59 1.91 <0.0001
Neck girth (cm) +0.50 0.22 0.030

Based on the multivariable regression analysis, two subpopulations (NS1-2 and NS1-3) were investigated in order to assess the impact of a potential NS-based breeding strategy. The NS1-2 population comprised 13 individuals with Grade 1 and 2 NS, corresponding to 19% of the total study population. The NS1-3 population also included individuals with Grade 3 NS, increasing the number of dogs to 35, or 51% of the total study population. The mean BSF score of the NS1-2 population was significantly lower than that of the total study population, i.e., 5.62 ± 6.98 (p = 0.0009), whereas the NS1-3 population had a slightly and non-significantly lower mean BSF score of 11.40 ± 7.07 (p = 0.070).

 Evaluation of genetic variation

All but two samples were successfully genotyped, and a total of 95 alleles were detected across the 18 microsatellite loci of the total study population. The two samples that were not successfully genotyped missed the genotype of only one microsatellite locus each (AHTh260 and FH2848). The remaining 17 microsatellite loci in these samples were included in the study. The genotyping data is presented in S3 Table.

Frequencies of the individual alleles detected in the 18 microsatellites together with the genetic parameters used to evaluate genetic variation in the total population (NS1-4) and the two subpopulations (NS1-3, and NS1-2) are presented in Table 5.

10.1371/journal.pone.0226280.t005 Genetic parameters.
Microsatellites Alleles and allele frequencies AA AAE HO HE F
AHT121 96 98 100 102 104 106 108 112
NS1-4 0.09 0.02 0.30 0.07 0.38 0.04 0.08 0.01 8 3.91 0.77 0.74 -0.035
NS1-3 0.13 0.03 0.29 0.11 0.36 0.04 0.04 7 4.05 0.74 0.75 0.018
NS1-2 0.23 0.04 0.27 0.08 0.35 0.04 6 3.88 0.77 0.74 -0.038
AHT137 131 133 135 137 141 147 149 151 153
NS1-4 0.10 0.34 0.28 0.07 0.01 0.07 0.01 0.10 0.02 9 4.46 0.71 0.78 0.085
NS1-3 0.09 0.33 0.27 0.06   0.09 0.01 0.14 0.01 8 4.52 0.71 0.78 0.088
NS1-2 0.04 0.31 0.27 0.08   0.12   0.15 0.04 7 4.64 0.69 0.78 0.121
AHTh171 219 225 231 233 235
NS1-4 0.31 0.49 0.01 0.19 0.01 5 2.68 0.65 0.63 -0.036
NS1-3 0.36 0.43 0.01 0.20 4 2.82 0.66 0.65 -0.023
NS1-2 0.31 0.46   0.23 3 2.77 0.62 0.64 0.030
AHTh260 238 244 246 248 250 252
NS1-4 0.63 0.01 0.05 0.15 0.10 0.06 6 2.30 0.57 0.56 -0.0099
NS1-3 0.69   0.07 0.10 0.13 0.01 5 1.97 0.49 0.49 0.0041
NS1-2 0.69   0.04 0.12 0.15   4 1.94 0.46 0.49 0.052
AHTk211 87 91 95
NS1-4 0.62 0.01 0.38 3 1.89 0.45 0.47 0.045
NS1-3 0.63 0.01 0.36 3 1.90 0.46 0.47 0.028
NS1-2 0.69 0.04 0.27 3 1.82 0.46 0.45 -0.024
AHTk253 286 288 290 292
NS1-4 0.22 0.14 0.19 0.44 4 3.36 0.67 0.70 0.046
NS1-3 0.20 0.14 0.26 0.40 4 3.48 0.77 0.71 -0.080
NS1-2 0.15 0.19 0.35 0.31 4 3.61 0.77 0.72 -0.065
CXX279 116 118 120 124 126 131
NS1-4 0.01 0.04 0.17 0.54 0.22 0.02 6 2.70 0.62 0.63 0.014
NS1-3 0.01 0.01 0.20 0.53 0.21 0.03 6 2.73 0.63 0.63 0.0062
NS1-2     0.23 0.42 0.27 0.08 4 3.24 0.77 0.69 -0.11
FH2054 148 152 156 160 164 168 172 176
NS1-4 0.15 0.05 0.45 0.02 0.02 0.04 0.26 0.01 8 3.36 0.65 0.70 0.075
NS1-3 0.19 0.09 0.39 0.03 0.04 0.04 0.23   7 3.95 0.77 0.75 -0.031
NS1-2 0.19 0.12 0.35   0.08 0.04 0.23   6 4.28 0.77 0.77 -0.0051
FH2848 230 236 238 240 244
NS1-4 0.02 0.04 0.26 0.60 0.08 5 2.29 0.56 0.56 0.0071
NS1-3 0.03 0.07 0.29 0.54 0.07 5 2.59 0.69 0.61 -0.12
NS1-2 0.04 0.08 0.31 0.46 0.12 5 3.029 0.77 0.67 -0.15
INRA21 95 97 99 101 105
NS1-4 0.83 0.05 0.07 0.01 0.04 5 1.43 0.33 0.30 -0.093
NS1-3 0.86 0.04 0.04   0.06 4 1.34 0.29 0.25 -0.14
NS1-2 0.88 0.04 0.04   0.04 4 1.28 0.31 0.22 -0.40
INU005 110 122 124 126 130
NS1-4 0.04 0.01 0.38 0.30 0.25 5 3.35 0.67 0.70 0.045
NS1-3 0.03 0.01 0.49 0.29 0.19 5 2.77 0.69 0.64 -0.080
NS1-2 0.08   0.38 0.31 0.23 4 3.34 0.85 0.70 -0.21
INU030 144 150 152 154 156
NS1-4 0.01 0.70 0.28 0.01 0.01 5 1.76 0.43 0.43 0.0030
NS1-3 0.01 0.69 0.29   0.01 4 1.78 0.49 0.44 -0.11
NS1-2   0.85 0.12   0.04 3 1.35 0.31 0.26 -0.19
INU055 208 210 212 218 220
NS1-4 0.01 0.42 0.01 0.55 0.01 5 2.087 0.52 0.52 0.0015
NS1-3 0.01 0.44 0.01 0.51 0.01 5 2.20 0.51 0.55 0.066
NS1-2 0.04 0.42   0.54   3 2.13 0.77 0.53 -0.45
REN16C04 202 204 206
NS1-4 0.64 0.13 0.22 3 2.11 0.46 0.53 0.12
NS1-3 0.66 0.10 0.24 3 1.99 0.49 0.50 0.014
NS1-2 0.54 0.15 0.31 3 2.44 0.54 0.59 0.084
REN169D01 212 216 218
.NS1-4 0.32 0.36 0.33 3 2.93 0.68 0.66 -0.032
NS1-3 0.37 0.27 0.36 3 2.95 0.63 0.66 0.046
NS1-2 0.54 0.27 0.19 3 2.50 0.69 0.60 -0.151
REN169O18 156 164 166 168 170 172
NS1-4 0.09 0.01 0.06 0.31 0.49 0.04 6 2.86 0.71 0.65 -0.092
NS1-3 0.06 0.01 0.03 0.40 0.46 0.04 6 2.65 0.57 0.62 0.084
NS1-2 0.08     0.38 0.46 0.08 4 2.71 0.54 0.63 0.15
REN247M23 266 268 270 272
NS1-4 0.01 0.51 0.10 0.37 4 2.46 0.54 0.59 0.089
NS1-3   0.43 0.16 0.41 3 2.64 0.57 0.62 0.083
NS1-2   0.46 0.04 0.50 3 2.16 0.46 0.54 0.14
REN54P11 222 226 228 234 236
NS1-4 0.07 0.13 0.61 0.04 0.16 5 2.37 0.61 0.58 -0.054
NS1-3 0.10 0.10 0.56 0.03 0.21 5 2.64 0.71 0.62 -0.14
NS1-2 0.12 0.04 0.50   0.35 4 2.57 0.77 0.61 -0.26

NS1-4 = total population; NS1-3 = dogs with nostril score 1, 2 and 3; NS1-2 = dogs with nostril score 1 and 2; AA = number of alleles; AAE = number of effective alleles; HO = observed heterozygosity; HE = expected heterozygosity; F = inbreeding coefficient.

Polymorphism was found in all autosomal loci with a median of five alleles per locus and with the absolute number of alleles per locus ranging from three (AHTk211, REN16C04, REN169D01) to nine (AHT137). In total, 92% of the 95 alleles present in the total study population were represented in the NS1-3 population, while 77% were represented in the 13 dogs that made up the NS1-2 population. As seen in Table 5, the two subpopulations are comparable to the total population with respect to the number of effective alleles (AAE) and the observed (HO) and expected heterozygosity (HE). However, the number of alleles (AA) is significantly lower in the NS1-2 population compared to the total population. Nevertheless, since there are no deviations in HO and HE in the three populations, the coefficient of inbreeding (F) is close to zero (0.010 ± 0.061, -0.017 ± 0.080 and -0.083 ± 0.18). It should, however, be noted that because of the small sample size in NS1-2 (only 13 dogs), the standard deviation for F is high.

10.1371/journal.pone.0226280.t006 Summary of genetic data in the total population and subpopulations.
Total study population p NS1-3 p NS1-2
AA/locus Median (min-max) 5 (3–9) 0.019w* 5 (3–8) 0.44w 4 (3–7)
AAE/locus Mean ±SD (min-max) 2.68 ± 0.78 (1.43–4.46) 0.79t 2.72 ± 0.81 (1.34–4.52) 0.89t 2.76 ± 0.94 (1.28–4.64)
HO Mean ±SD (min-max) 0.59 ± 0.12 (0.33–0.77) 0.42t 0.60 ± 0.13 (0.29–0.77) 0.72t 0.63 ± 0.17 (0.31–0.85)
HE Mean ±SD (min-max) 0.60 ± 0.12 (0.30–0.77) 0.90t 0.60 ± 0.13 (0.25–0.78) 0.99t 0.59 ± 0.16 (0.22–0.78)
F Mean ±SD (min-max) 0.010 ± 0.061 (-0.093–0.12) 0.046t* -0.017 ± 0.080 (-0.14–0.88) 0.26t -0.083 ± 0.18 (-0.45–0.14)

AA = number of alleles; AAE = number of effective alleles; HO = observed heterozygosity; HE = expected heterozygosity; F = Inbreeding coefficient; w = Wilcoxon rank sum test, t = t-test; p = the p-value for the test of difference in means in the two subpopulations NS1-2 and NS1-3, compared to NS1-4. Variables that are significantly different when compared to NS1-4 at a significance level of 0.05 are marked with *.

 Discussion

Due to the severe health and welfare problems experienced by brachycephalic dogs [1, 5] with exaggerated phenotypes, several studies have been performed to evaluate the severity of BS based on exercise tests [13, 18]. In the present study we have introduced the calculation of BSF scores, on a continuous scale, that rely on recordings of upper airway noise and decibel recordings before, during, and after exercise to assess the degree of BS. Some of the correlations between BSF score and morphometric and phenotypic variables (Table 3) are substantial, e.g. walking distance and neck length to girth ratio, whereas others are minor e.g. weight and cranial length. Generally the correlations make sense, because weight in itself would rarely be expected to have a major impact as it is an unspecific parameter, whereas walking distance is a patho-physiologic adverse outcome. Although BSF scores cannot be considered as a definitive diagnostic tool they have been shown to reflect the severity of BS. To lend support to the functional characterization, efforts have also been made to identify the phenotypic characteristics that present the highest risk, to aid in selection for healthier conformation. Since the acute and chronic respiratory distress inherent in dogs with BS appears to increase in line with a decreasing muzzle length, it can be assumed that ML and CFR are risk factors. There is, however, contradicting evidence about the impact of these conformational features on the development of the syndrome. In agreement with another study on French bulldogs [2], we found no significant effect of ML, whereas NS explained 32% of the variation in BSF scores. Our study also corroborates several other studies that indicate NG and BCS are further risk factors for BS [2, 3, 6]. NG and BCS explained 4% and 8% of the variation in BSF scores in our French bulldog population, respectively.

As a result of selection for specific conformational and/or performance traits, all modern breed dogs exhibit reduced genetic diversity. It is therefore of utmost importance that we take genetic variation into consideration when contemplating selection schemes. The average number of alleles per locus (AA) and the average effective alleles per locus (AAE) identified in the French bulldogs was similar to other pure breeds that have been monitored for diversity [19], as well as English bulldogs [20]. However, the genetic diversity seems to be higher in French bulldogs than English bulldogs since only 2 of the 18 loci (11%) were approaching fixation in our population (a single allele with frequency >70), while the corresponding number was 7 out of 33 loci (21%) in English bulldogs. In addition, while most of the loci (58%) had one or two alleles that dominated in the English bulldog population, the corresponding figure was 44% in the French bulldog population. The only significant difference between the total population and the two subpopulations (NS1-3 and NS1-2) was the reduction in the average number of alleles in the NS1-2 subpopulation. This is not remarkable since this subpopulation was made up of only 13 dogs. Due to sampling errors in this small subpopulation, the genetic parameters are not very reliable, which is most notably reflected in the very high mean SD for F (see Table 6).

Our study unambiguously shows that the NS score has a large impact on the functional ability of French bulldogs. This single parameter results in an additional 10.59 and 8.45 score points in dogs with open or mildly stenotic nares, respectively. It follows that dogs with open nares are more likely to lead a healthier life compared to dogs with stenotic nares. Hence, selecting only dogs with open nares (NS1-2) for breeding would seem to be an obvious means to improve functional ability and, in turn, health and welfare at the population level, and would in fact comply with the FCI standard for the breed, which states that the nose should ‘allow normal nasal breathing’[21].

Nevertheless, since this would exclude 81% of the population from breeding, this is obviously not a prudent solution. The average level of genetic diversity in the French bulldog population is comparable to many other pure dog breeds, implying that selection should be performed cautiously while also taking other health aspects into consideration. The breed has a high prevalence of, for instance, dystocia [22] and thoracic vertebral malformations (hemivertebrae) [23]–both of which are severe problems that must be considered during selection. A lenient way to ensure that the prevalence of BS is reduced among French bulldogs would be to avoid using dogs with severely stenotic nares for breeding. This would lead to progress, albeit at a very slow pace since dogs with moderately stenotic nares would have a negative impact on the progress. However, slow progress must be accepted to avoid a devastating reduction in genetic diversity within the breed.

In spite of the limited number of animals included in the present study, we have confirmed that NS has a considerable impact on BS evaluated based on the BSF score. The main shortcoming of this and other studies where the BSF score has been used to reflect the severity of BS is that the BSF score is not a definitive diagnostic evaluation. One possible way to validate the diagnostic value of the score is to compare the score with results from laryngoscopic evaluation of the problematic anatomic abnormalities combined with diagnostic imaging such as CT. This would require general anesthesia and was not performed due to the considerable risk posed to brachycephalic dogs, as mentioned previously. Nevertheless, lending support to the diagnostic value of the BSF score, the study by Riggs and co-workers [14] show that the association between subjectively determined functional grades (BSF score) and objectively determined BS indices, as determined by whole body barometric plethysmography, is high.

 Supporting information Morphometric and phenotypic registrations.

(XLSX)

 Registration of upper airway noise and presence of respiratory distress at rest, during and after a submaximal exercise test and after a 15-minute recovery period.

(XLSX)

 Microsatellite genotyping results.

(XLSX)

The authors wish to thank Charlotte Bjørner Larsen for excellent technical assistant.

 References Roedler F.S., Pohl S., Oechtering G.U. How does severe brachycephaly affect dog’s lives? Results of a structured preoperative owner questionnaire. The Veterinary Journal. 2013; 198, 606–610. doi: 10.1016/j.tvjl.2013.09.009 24176279 Liu N.-C., Troconis E.L., Kalmar L., Price D.J., Wright H.E., Adams V.J., Sargan D. R., Ladlow J.F. Conformational risk factors of brachycephalic obstructive airway syndrome (BOAS) in pugs, French bulldogs, and bulldogs. PLoS ONE. 2017; 12 (8), 1–24. Packer R.M.A., Hendricks A., Tivers M.S., Burn C. Impact of facial conformation on canine health: Brachycephalic obstructive airway syndrome. PLoS ONE. 2015; 10 (10), 1–21. Meola S.D. Brachycephalic airway syndrome. Topics in Companion Animal Medicine. 2013; 28 (3), 91–6. doi: 10.1053/j.tcam.2013.06.004 24182996 Packer R.M.A., Tivers M.S. Strategies for the management and prevention of conformation related respiratory disorders in brachycephalic dogs. Veterinary Medicine. 2015; Research and Reports, 6, 219–232. doi: 10.2147/VMRR.S60475 30101109 Liu N.-C., Adams V.J., Kalmar L., Ladlow J.F., Sargan D.R. Whole-body barometric plethysmography characterizes upper airway obstruction in 3 brachycephalic breeds of dogs. Journal of Veterinary Internal Medicine. 2016; 30 (3), 853–65. doi: 10.1111/jvim.13933 27159898 Agudelo C.F., Schanilec P. Evaluation of the functional capacity in dogs with naturally acquired heart disease. Veterinarni Medicina. 2013; 58 (5), 264–270. Boddy K.N., Roche B.M., Schwartz D.S., Nakayama T., Hamlin R.L., 2004: Evaluation of the six-minute walk test in dogs. American Journal of Veterinary Research, 65 (3), 311–313. doi: 10.2460/ajvr.2004.65.311 15027678 Swimmer RA and Rozanski EA. Evaluation of the 6-Minute Walk Test in Pet Dogs. J Vet Intern Med 2011; 25:405–406. doi: 10.1111/j.1939-1676.2011.0689.x 21352372 Lilja-Maula L., Laurila H.P., Syrjä P., Lappalainen A.K., Krafft E., Clercx C., Rajamäki M.M. Long-term outcome and use of 6-minute walk test in West Highland white terriers with idiopathic pulmonary fibrosis. Journal of Veterinary Internal Medicine. 2014; 28, 379–385. doi: 10.1111/jvim.12281 24417616 Cerda-Gonzalez S., Talarico L., Todhunter R. Noninvasive Assesment og Neuromoscular Disease in Dogs: Use of the 6-minute Walk Test to Assess Submaximal Exercise Tolerance in Dogs with Centronuclear Myopahty. Journal of Veterinary Internal Medicine. 2016; 30, 808–812. doi: 10.1111/jvim.13939 27012153 Manens J., Ricci J., Damoiseaux C., Gault S., Contiero B., Diez M., Clercx C. Effect of body weight loss on cardiopulmonary function assessed by 6-minute walk test and arterial blood gas analysis in obese dogs. Journal of Veterinary Internal Medicine. 2014; 28, 371–378. doi: 10.1111/jvim.12260 24351032 Lilja-Maula L., Lappalainen A.K., Hyytiäinen H.K., Kuusela E., Kaimio M., Schildt K., Mölsä S., Morelius M., Rajamäki M.M. Comparison of submaximal exercise test results and severity of brachycephalic obstructive airway syndrome in English bulldogs. The Veterinary Journal. 2017; 219, 22–26. doi: 10.1016/j.tvjl.2016.11.019 28093105 Riggs J, Liu N-C, Sutton DR, Sargan D, Ladlow JF. Validation of exercise testing and laryngeal auscultation for grading brachycephalic obstructive airway syndrome in pugs, French bulldogs, and English bulldogs by using whole-body barometric plethysmography. Veterinary Surgery. 2019; 48:488–496. doi: 10.1111/vsu.13159 30666670 Sutter N.B., Mosher D.S., Gray M.M., Ostrander E.A. Morphometrics within dog breeds are highly reproducible and dispute Rensch’s rule. Mammalian Genome. 2008; 19 (10–12), 713–723. doi: 10.1007/s00335-008-9153-6 19020935 www.aaha.org/public_documents/professional/guidelines/weightmgmt_bodyconditionscoring.pdf Solway S, Brooks D, Lacasse Y, Thomas S. A qualitative systematic overview of the measurement properties of functional walk tests used in the cardiorespiratory domain. Chest. 2001 Jan; 119(1):256–70. doi: 10.1378/chest.119.1.256 11157613 Aromaa M, Lilja-Maula L, Rajamäki MM. Assesment of welfare and brachycephalic obstructive airway syndrome signs in young, breeding age French Bulldogs and Pugs, using owner questionnaire, physical examination and walk tests. Animal Welfare. 2019; 28:287–298. Pedersen N., Liu H., Theilen G., Sacks B. The effects of dog breed development on genetic diversity and the relative influences of performance and conformation breeding. Journal of Animal Breeding and Genetics. 2012; 130, 236–248. doi: 10.1111/jbg.12017 23679949 Pedersen N.C., Pooch A.S., Liu H. A genetic assessment of the English bulldog. Canine Genetics and Epidemiology. 2016; 6 (3), 1–16. http://www.fci.be/Nomenclature/Standards/101g09-en.pdf Evans KM, Adams VJ. Proportion of litters of purebred dogs born by caesarean section. J Small Anim Pract. 2010; 51(2): 113–118. doi: 10.1111/j.1748-5827.2009.00902.x 20136998 Ryana R, Gutierrez-Quintanab R, ter Haara G, De Decker S. Prevalence of thoracic vertebral malformations in French bulldogs, Pugs and English bulldogs with and without associated neurological deficits. The Veterinary Journal. 2017; 221, 25–29. doi: 10.1016/j.tvjl.2017.01.018 28283076 10.1371/journal.pone.0226280.r001 Decision Letter 0 Loor Juan J Academic Editor 2019 Juan J Loor This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Submission Version 0

12 Aug 2019

PONE-D-19-19251

Breeding French bulldogs that are able to breathe - possible but a long way to go

PLOS ONE

Dear Dr. Fredholm,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

We would appreciate receiving your revised manuscript by Sep 26 2019 11:59PM. When you are ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter.

To enhance the reproducibility of your results, we recommend that if applicable you deposit your laboratory protocols in protocols.io, where a protocol can be assigned its own identifier (DOI) such that it can be cited independently in the future. For instructions see: http://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols

Please include the following items when submitting your revised manuscript:

A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). This letter should be uploaded as separate file and labeled 'Response to Reviewers'.

A marked-up copy of your manuscript that highlights changes made to the original version. This file should be uploaded as separate file and labeled 'Revised Manuscript with Track Changes'.

An unmarked version of your revised paper without tracked changes. This file should be uploaded as separate file and labeled 'Manuscript'.

Please note while forming your response, if your article is accepted, you may have the opportunity to make the peer review history publicly available. The record will include editor decision letters (with reviews) and your responses to reviewer comments. If eligible, we will contact you to opt in or out.

We look forward to receiving your revised manuscript.

Kind regards,

Juan J Loor

Academic Editor

PLOS ONE

Journal requirements:

When submitting your revision, we need you to address these additional requirements.

1. Please ensure that your manuscript meets PLOS ONE's style requirements, including those for file naming. The PLOS ONE style templates can be found at

http://www.journals.plos.org/plosone/s/file?id=wjVg/PLOSOne_formatting_sample_main_body.pdf and http://www.journals.plos.org/plosone/s/file?id=ba62/PLOSOne_formatting_sample_title_authors_affiliations.pdf

2. Thank you for stating the following in the Acknowledgments Section of your manuscript: "We also wish to thank the Danish Kennel Club for financial support to the project."

We note that you have provided funding information that is not currently declared in your Funding Statement. However, funding information should not appear in the Acknowledgments section or other areas of your manuscript. We will only publish funding information present in the Funding Statement section of the online submission form.

Please remove any funding-related text from the manuscript and let us know how you would like to update your Funding Statement. Currently, your Funding Statement reads as follows:

 "No"

[Note: HTML markup is below. Please do not edit.]

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Yes

Reviewer #2: No

**********

2. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #2: Yes

**********

3. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

Reviewer #2: Yes

**********

4. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: Yes

Reviewer #2: Yes

**********

5. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: Abstract: the authors are definite that NS is the only way to improve BOAS by breeding selection- what about functional grading or genetic testing to gain estimated breeding values?

Also are you sure that NS is a single gene trait- ie progeny from NS1/2 will not be NS3/4? if so please cite the references/ evidence.

54. Not necessarily, anatomical lesions do not always equate to disease severity, particularly as BOAS is a dynamic disease and static pictures may not reveal the extent of disease. Are you talking about direct visualisation? or rhinoscopy? or CT? and if so what criteria are you using to grade disease severity from anatomical lesions?

61 BS increased steeply between breeds...

116 Intra-observer error checked? tricky measurements....

125 Decibel recording- was this based on previous data/ pilot studies or published studies?

What about open mouth vs nasal breathing? ie dogs with severe nasal obstruction may mouth breathe throughout exercise and sound better than dogs with mild/moderate nasal obstruction that nasal breathe throughout exercise- how do you correct for this?

129 I would argue that respiratory distress plus intermittent laryngeal noise is a more severe presentation than a continuous nasal stertor.

This is an interesting study (I need a second reviewer to check the genetics) but the functional grading is not without flaws that should be discussed.

Reviewer #2: The submission entitled “Breeding French bulldogs that able to breathe – possible but a long way to go” addresses and an incredibly important topic and the authors present some valuable findings however there are many concerns with the manuscript in its current form. The authors should be lauded for their efforts!

Major Concerns:

The title is inappropriate. French bulldogs are able to breathe in their current physiological form. The title implies they cannot which is a misnomer. Their work addresses the view that it would be possible to breed with intention for improved nares structure plus other attributes to reduce the incidence of brachycephalic syndrome.

The presentation in its current form is disingenuous. The authors are really developing a model to characterize overall brachycephalic syndrome (BS) and the contributory factors. The authors then use that descriptive rubric to model the effects on the French bulldog population if selection using that rubric were employed. Several main concerns center on this. The authors present the brachycephalic syndrome functional (BSF) score as approved methodology, yet this manuscript is just describing the BSF. Importantly the BSF has not been validated to demonstrate that the BSF score accurately predicts a dogs phenotype in a replicate population.

The authors should be commended for assessing whether implementation of the BSF score in breeding would create genetic bottlenecks that would be harmful for the breed population. However, the abstract implies, as written, that the breeding selection was done using the BSF when in fact it was a hypothetical modeling of selection not actual implementation. This distinction must be clearly stated in the abstract, in the methods, in the results, and in the discussion.

Furthermore, the authors come to the conclusion that genetic variability within the population would be limited based upon data from 18 microsatellites. This is a limitation of the study and needs to be discussed. It is also curious why the authors only used those 18 microsatellites—yes they are on a panel that can be used but the rationale as to why these 18 are sufficient to reflect breed population diversity must be discussed. And how these msats are distributed across the genome.

In the discussion, the authors state “Our study unambiguously shows that the NS score has a large impact on the health and welfare of French bulldogs”. Their data demonstrate that the BSF score based upon the measurements taken on a sample of French bulldogs correlated with the measurements that were used to form the BSF score. The BSF score includes physiological parameters of heart rate, body temperature, etc—these features are key to include in the main text. To find the components, they are listed in the supplemental material. The manuscript would be much more powerful if the elements of the BSF were included. The authors need to assess welfare and health using their BSF score and the NS on a validation population before making such a statement. If the authors did this and demonstrated that functional welfare and health parameters are predicted by their BSF score that would be a huge advancement in the field and for the brachycephalic dogs. Unfortunately, that is not what is presented in the current manuscript.

Line 18: should be “those” not these

Line 28 should be no or only mild NS

Line 31-32 “Although it results in apparent reduction…” unclear antecedent. To what is “it” referring?

Line 35-36 it is feasible but not prudent to undertake removing all dogs with the severest form of BS.

Lines 73-76 need to be rewritten to reflect that this is a model development paper and that the BSF scoring system does not currently exist. OR the authors must provide a reference for the validation of this tool.

Line 105, Line 119. See comment for lines 73-75. The scoring system does not yet exist and the authors are modeling such a scoring system in this manuscript. That needs to be more explicitly stated and better defined. If however, that BSF currently exists and is in use, the authors need to reference that and demonstrate its validity.

Line 125 reference 9 is a human 6MWT reference based upon bipedal locomotion. The authors will need to mention why this is a valid application for a quadruped dog.

Line 185. The dogs have biological sex (male or female) not gender, which is a social, construct. Change gender to sex throughout the paper.

Line 257 needs referencing.

Line 260 needs referencing.

**********

6. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: No

Reviewer #2: No

[NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files to be viewed.]

While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email us at figures@plos.org. Please note that Supporting Information files do not need this step.

 10.1371/journal.pone.0226280.r002 Author response to Decision Letter 0 Submission Version 1

20 Sep 2019

Comments to the Author

Reviewer #1: Abstract: the authors are definite that NS is the only way to improve BOAS by breeding selection- what about functional grading or genetic testing to gain estimated breeding values?

We agree with the reviewer and have therefore modified the statement in the abstract to indicate that selection based on NS score is not the only possible selection scheme (l. 39 – l. 40)

Also are you sure that NS is a single gene trait- ie progeny from NS1/2 will not be NS3/4? if so please cite the references/ evidence.

We concede that NS is not a single gene trait - it is a trait for which the heritability has not been established. However, since the NS accounts for 32% of the variation in the BSF score it appears to be a good proxy for the health status.

54. Not necessarily, anatomical lesions do not always equate to disease severity, particularly as BOAS is a dynamic disease and static pictures may not reveal the extent of disease. Are you talking about direct visualisation? or rhinoscopy? or CT? and if so what criteria are you using to grade disease severity from anatomical lesions?

We agree with the reviewer that BOAS/BS is a dynamic disease. We have explained this in more detail in l. 60 – l. 67. Although there is no ‘golden standard’ for assessing the degree of BOAS/BS it is generally accepted that exercise testing and laryngeal and tracheal auscultation can be used for grading of BS. In l. 85 - l. 89 we have explained this in more detail and included relevant references.

61 BS increased steeply between breeds...

We are not sure what the reviewer is alluding to here. However, BS cannot “increase” between breeds, because “breed” is a categorical variable.

116 Intra-observer error checked? tricky measurements....

We agree with the reviewer: The parameters with the highest likelihood of intra-observer error were NS and BCS. We have stated that intra-observer error was not taken into account (l. 127 - 128)

125 Decibel recording- was this based on previous data/ pilot studies or published studies?

What about open mouth vs nasal breathing? ie dogs with severe nasal obstruction may mouth breathe throughout exercise and sound better than dogs with mild/moderate nasal obstruction that nasal breathe throughout exercise- how do you correct for this?

Decibel recordings were included in an attempt to strengthen the objectivity of the evaluation of ‘upper airway noice’. To our knowledge this has not been done before. The recordings were made before exercise, right after exercise, and after a 15 minute resting period without taking NS or other factors into consideration. As can be seen in S2 Table (columns J, S and AB), there is a very high correlation with respect to the dogs in which these recordings were positive across recording time points. Decibel recordings are one of several observations to evaluate the degree of BS. In combination with other assessments of airway noise and registrations of respiratory distress it contributes information to the combined BSF score.

129 I would argue that respiratory distress plus intermittent laryngeal noise is a more severe presentation than a continuous nasal stertor.

This is an interesting study (I need a second reviewer to check the genetics) but the functional grading is not without flaws that should be discussed.

Thank you for the kind words and the careful review of our study. We have explained the BSF scores were established, on a continuous scale, based on recordings of upper airway noise and decibel recordings in the discussion, and acknowledged that the grading is not a definitive diagnostic tool (l. 276– l. 281).

Reviewer #2: The submission entitled “Breeding French bulldogs that able to breathe – possible but a long way to go” addresses and an incredibly important topic and the authors present some valuable findings however there are many concerns with the manuscript in its current form. The authors should be lauded for their efforts!

Thank you for the kind words and for the careful review of our paper.

Major Concerns:

The title is inappropriate. French bulldogs are able to breathe in their current physiological form. The title implies they cannot which is a misnomer. Their work addresses the view that it would be possible to breed with intention for improved nares structure plus other attributes to reduce the incidence of brachycephalic syndrome.

We agree that the title was misleading. The title is now: Breeding French bulldogs with unobstructed breathing - possible but a long way to go

The presentation in its current form is disingenuous. The authors are really developing a model to characterize overall brachycephalic syndrome (BS) and the contributory factors. The authors then use that descriptive rubric to model the effects on the French bulldog population if selection using that rubric were employed. Several main concerns center on this. The authors present the brachycephalic syndrome functional (BSF) score as approved methodology, yet this manuscript is just describing the BSF. Importantly the BSF has not been validated to demonstrate that the BSF score accurately predicts a dogs phenotype in a replicate population.

We agree with the reviewer and have acknowledged this by changing the wording in the final part of the discussion (l. 324 – l. 334) explaining that validation is needed.

The authors should be commended for assessing whether implementation of the BSF score in breeding would create genetic bottlenecks that would be harmful for the breed population. However, the abstract implies, as written, that the breeding selection was done using the BSF when in fact it was a hypothetical modeling of selection not actual implementation. This distinction must be clearly stated in the abstract, in the methods, in the results, and in the discussion.

We concede that the way we have described the subpopulations could lead to misunderstandings. We have now made it clear that the subpopulations are hypothetical populations (l. 30-33 and l. 180 - 181).

Furthermore, the authors come to the conclusion that genetic variability within the population would be limited based upon data from 18 microsatellites. This is a limitation of the study and needs to be discussed. It is also curious why the authors only used those 18 microsatellites—yes they are on a panel that can be used but the rationale as to why these 18 are sufficient to reflect breed population diversity must be discussed. And how these msats are distributed across the genome.

We have included information on the distribution of the 18 microsatellites genotyped in this study (distributed on 18 of the 19 canine autosomes) (see l. 170 -171). We concede that it is a low number of markers, still microsatellites are highly polymorphic, neutral to selection and an equivalent number of markers have been used (successfully) in other population studies in dogs (https://doi.org/10.1371/journal.pone.0221418

https://doi.org/10.1017/S1751731117003573).

In the discussion, the authors state “Our study unambiguously shows that the NS score has a large impact on the health and welfare of French bulldogs”. Their data demonstrate that the BSF score based upon the measurements taken on a sample of French bulldogs correlated with the measurements that were used to form the BSF score. The BSF score includes physiological parameters of heart rate, body temperature, etc—these features are key to include in the main text. To find the components, they are listed in the supplemental material. The manuscript would be much more powerful if the elements of the BSF were included.

Thank you for pointing this out to us. We have now included S2 Table as Table 2 in the manuscript in order to ensure that the readers can see immediately how the BSF scores are established.

The authors need to assess welfare and health using their BSF score and the NS on a validation population before making such a statement. If the authors did this and demonstrated that functional welfare and health parameters are predicted by their BSF score that would be a huge advancement in the field and for the brachycephalic dogs. Unfortunately, that is not what is presented in the current manuscript.

We accept this point which was also made by the other referee. We have acknowledged this by changing the wording in the final part of the discussion (l. 324 – l. 334) explaining that validation is needed.

Line 18: should be “those” not these

Has been corrected (l. 20)

Line 28 should be no or only mild NS

Has been corrected (l. 31)

Line 31-32 Although it results in apparent reduction…” unclear antecedent. To what is “it” referring?

Has been explained (‘it’ refers to exclusion of dogs with Grade 4) (l. 35)

Line 35-36 it is feasible but not prudent to undertake removing all dogs with the severest form of BS.

Has been corrected (‘feasible’ substituted with ‘prudent’) – l. 39.

Lines 73-76 need to be rewritten to reflect that this is a model development paper and that the BSF scoring system does not currently exist. OR the authors must provide a reference for the validation of this tool.

Relevant references have been included. See line 85.

Line 105, Line 119. See comment for lines 73-75. The scoring system does not yet exist and the authors are modeling such a scoring system in this manuscript. That needs to be more explicitly stated and better defined. If however, that BSF currently exists and is in use, the authors need to reference that and demonstrate its validity.

See our response to the previous point.

Line 125 reference 9 is a human 6MWT reference based upon bipedal locomotion. The authors will need to mention why this is a valid application for a quadruped dog.

Thank you for pointing this out. The mentioned reference has been substituted with a more relevant reference.

Line 185. The dogs have biological sex (male or female) not gender, which is a social, construct. Change gender to sex throughout the paper.

Has been corrected

Line 257 needs referencing.

Reference 1 and 2 have been included (l. 275)

Line 260 needs referencing.

Reference 9 and 10 have been included (l. 277)

Submitted filename: Response to Reviewers.docx

 10.1371/journal.pone.0226280.r003 Decision Letter 1 Loor Juan J Academic Editor 2019 Juan J Loor This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Submission Version 1

21 Oct 2019

PONE-D-19-19251R1

Breeding French bulldogs with unobstructed breathing - possible but a long way to go

PLOS ONE

Dear Dr. Fredholm,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

We would appreciate receiving your revised manuscript by Dec 05 2019 11:59PM. When you are ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter.

To enhance the reproducibility of your results, we recommend that if applicable you deposit your laboratory protocols in protocols.io, where a protocol can be assigned its own identifier (DOI) such that it can be cited independently in the future. For instructions see: http://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols

Please include the following items when submitting your revised manuscript:

A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). This letter should be uploaded as separate file and labeled 'Response to Reviewers'.

A marked-up copy of your manuscript that highlights changes made to the original version. This file should be uploaded as separate file and labeled 'Revised Manuscript with Track Changes'.

An unmarked version of your revised paper without tracked changes. This file should be uploaded as separate file and labeled 'Manuscript'.

Please note while forming your response, if your article is accepted, you may have the opportunity to make the peer review history publicly available. The record will include editor decision letters (with reviews) and your responses to reviewer comments. If eligible, we will contact you to opt in or out.

We look forward to receiving your revised manuscript.

Kind regards,

Juan J Loor

Academic Editor

PLOS ONE

[Note: HTML markup is below. Please do not edit.]

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #1: (No Response)

Reviewer #2: (No Response)

**********

2. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Partly

Reviewer #2: Yes

**********

3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #2: Yes

**********

4. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

Reviewer #2: Yes

**********

5. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: Yes

Reviewer #2: Yes

**********

6. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: the insistence that brachycephalic obstructive airway syndrome is best evaluated by direct visual examination misses the facts that a. this is a functional disease b. there is no current scheme to score CT/ endoscopy lesions with disease severity. Please adjust lines 59 and 319 -321 accordingly.

I think the authors are stating that there is a relationship between bfs and bs but they do not clearly define bs. From the study it seems that bs is a composite of various external conformational measurements. If this is the case can this be clearly stated.

Can the authors address the weak r results

Line 277. this study does not 'establish' - there is no validation of the bfs. preferable to use 'introduce' to avoid over confidence in an as yet unproven system. This is particularly the case as a new (and interesting) decibel recording system has been used which needs further investigation before it is advocated as a grading scheme

Reviewer #2: The title, although improved, remains in need of editing. The intent of the title is to convey that through concerted selection practices targeting BS can reduce the prevalence of BS. The current title of “Breeding French bulldogs with unobstructed breathing – possible but a long way to go” while catchy is still not informative. Of course it is possible to breed French bulldogs with unobstructed breathing. That however is not the intent of the manuscript. The manuscript is demonstrating that although French bulldogs with unobstructed breathing do exist, they are not that prevalent in the population the authors used and to increase the proportion of French bulldogs in a population that do not have BS it will take concerted selection and time in order to maintain genetic diversity. A more accurate title would be something along the lines of that conveys the concept that concerted selection can reduce obstructed breathing in the French bulldog but will take time.

Line 78 “Exercise testing is used both in humans [7, 8] and dogs [9] to measure reduced functional ability.” Please define what is meant by “functional ability”.

Line 164 should be microsatellites.

Line 268-269. The authors use reference 9 to illustrate the effect of BS on health and welfare. However the study detailed in reference 9 did not evaluate brachycephalic dogs. Thus this statement still is in need of references to demonstrate that “several studies….”

Line 268-269: Additionally the sentence needs editing as it appears something is missing: “..several studies have been performed to evaluate the severity of BS.” It would appear that the authors are tying together severity of BS and an impact on health or function.

Line 272: should be reflect the severity of BS (not reflect upon).

Line 272-273: comma needed after characterization “To lend support to the functional characterization, efforts have also been made to identify the…”

Please check all references. Line 286 reference for pure breed genetic variation is the R program for analysis

**********

7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: No

Reviewer #2: No

[NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files to be viewed.]

While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email us at figures@plos.org. Please note that Supporting Information files do not need this step.

 10.1371/journal.pone.0226280.r004 Author response to Decision Letter 1 Submission Version 2

15 Nov 2019

6. Review Comments to the Author

Reviewer #1: the insistence that brachycephalic obstructive airway syndrome is best evaluated by direct visual examination misses the facts that a. this is a functional disease b. there is no current scheme to score CT/ endoscopy lesions with disease severity. Please adjust lines 59 and 319 -321 accordingly.

I think the authors are stating that there is a relationship between bfs and bs but they do not clearly define bs. From the study it seems that bs is a composite of various external conformational measurements. If this is the case can this be clearly stated.

Thank you for this comment. We agree that some key definitions and explanations were missing:

In line 16-19 we have explained more precisely what BS is (ie. Brachycephalic syndrome (BS) is a pathophysiological disorder caused by excessive soft tissue within the upper airways of short-nosed dog breeds, causing obstruction of the nasal, pharyngeal and laryngeal lumen and thereby giving rise to various symptoms such as respiratory distress). In line 61 to 71 we have clarified why a functional assessment is a reasonable proxy measure for BS.

Can the authors address the weak r results

We have addressed the r results further in line 310-314

Line 277. this study does not 'establish' - there is no validation of the bfs. preferable to use 'introduce' to avoid over confidence in an as yet unproven system. This is particularly the case as a new (and interesting) decibel recording system has been used which needs further investigation before it is advocated as a grading scheme

We have rephrased this sentence (line 307-308): In the present study we have introduced the calculation of BSF scores, on a continuous scale, that rely on recordings of upper airway noise and decibel recordings before, during, and after exercise to assess the degree of BS

Reviewer #2: The title, although improved, remains in need of editing. The intent of the title is to convey that through concerted selection practices targeting BS can reduce the prevalence of BS. The current title of “Breeding French bulldogs with unobstructed breathing – possible but a long way to go” while catchy is still not informative. Of course it is possible to breed French bulldogs with unobstructed breathing. That however is not the intent of the manuscript. The manuscript is demonstrating that although French bulldogs with unobstructed breathing do exist, they are not that prevalent in the population the authors used and to increase the proportion of French bulldogs in a population that do not have BS it will take concerted selection and time in order to maintain genetic diversity. A more accurate title would be something along the lines of that conveys the concept that concerted selection can reduce obstructed breathing in the French bulldog but will take time.

We have changed the title so that it now reads: Breeding French bulldogs so that they breathe well - a long way to go.

Line 78 “Exercise testing is used both in humans [7, 8] and dogs [9] to measure reduced functional ability.” Please define what is meant by “functional ability”.

Exercise testing has been explained in more detail and with more appropriate references (see Line 104-112)

Line 164 should be microsatellites.

Spelling has been corrected (line 200)

Line 268-269. The authors use reference 9 to illustrate the effect of BS on health and welfare. However the study detailed in reference 9 did not evaluate brachycephalic dogs. Thus this statement still is in need of references to demonstrate that “several studies….”

We apologize for not providing sufficient documentation for this statement. Three more appropriate references have been provided (line 305 and 307)

Line 268-269: Additionally the sentence needs editing as it appears something is missing: “..several studies have been performed to evaluate the severity of BS.” It would appear that the authors are tying together severity of BS and an impact on health or function.

We have included ‘based on exercise tests’(line 307)to make the statement more comprehensible.

Line 272: should be reflect the severity of BS (not reflect upon).

We have removed ‘upon’ as suggested

Line 272-273: comma needed after characterization “To lend support to the functional characterization, efforts have also been made to identify the…”

Thank you: we have included ‘,’ as suggested (line 316)

Submitted filename: Response to Reviewers.docx

 10.1371/journal.pone.0226280.r005 Decision Letter 2 Loor Juan J Academic Editor 2019 Juan J Loor This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Submission Version 2

25 Nov 2019

Breeding French bulldogs with unobstructed breathing - possible but a long way to go

PONE-D-19-19251R2

Dear Dr. Fredholm,

We are pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it complies with all outstanding technical requirements.

Within one week, you will receive an e-mail containing information on the amendments required prior to publication. When all required modifications have been addressed, you will receive a formal acceptance letter and your manuscript will proceed to our production department and be scheduled for publication.

Shortly after the formal acceptance letter is sent, an invoice for payment will follow. To ensure an efficient production and billing process, please log into Editorial Manager at https://www.editorialmanager.com/pone/, click the "Update My Information" link at the top of the page, and update your user information. If you have any billing related questions, please contact our Author Billing department directly at authorbilling@plos.org.

If your institution or institutions have a press office, please notify them about your upcoming paper to enable them to help maximize its impact. If they will be preparing press materials for this manuscript, you must inform our press team as soon as possible and no later than 48 hours after receiving the formal acceptance. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact onepress@plos.org.

With kind regards,

Juan J Loor

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

Reviewers' comments:

 10.1371/journal.pone.0226280.r006 Acceptance letter Loor Juan J Academic Editor 2019 Juan J Loor This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

6 Dec 2019

PONE-D-19-19251R2

Breeding French bulldogs so that they breathe well - a long way to go

Dear Dr. Fredholm:

I am pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department.

If your institution or institutions have a press office, please notify them about your upcoming paper at this point, to enable them to help maximize its impact. If they will be preparing press materials for this manuscript, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information please contact onepress@plos.org.

For any other questions or concerns, please email plosone@plos.org.

Thank you for submitting your work to PLOS ONE.

With kind regards,

PLOS ONE Editorial Office Staff

on behalf of

Dr. Juan J Loor

Academic Editor

PLOS ONE