MD Ricardo Carbajal
email: ricardo.carbajal@aphp.fr

Mathie Lorrot

Yael Levy

Emmanuel Grimprel

Thibault Lecarpentier

Sebastien Heritier

Judith Faivre

Aurélie Schnuriger

Pauline Parisot

Eléonore Blondiaux

Solene Loschi

Simon Rivière

Julia Guilbert

Anne- Sophie Romain

Pierre-Louis Leger

Romain Guedj

Multisystem inflammatory syndrome in children rose and fell with the first wave of the COVID-19 pandemic in France

à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

KEY NOTES

 France saw a strong lagged correlation between the rise and fall of multisystem inflammatory syndrome (MIS-C) in children and the first wave of COVID-19.

 The study also compared seven children with MIS-C, who were all positive for the virus that causes COVID-19, and 40 with Kawasaki disease who did not have the virus.

 The authors concluded that MIS-C patients clearly demonstrated a different clinical picture to patients with Kawasaki disease.

KEY WORDS

COVID-19, Kawasaki disease, multisystem inflammatory syndrome, pandemic, PIMS Background More than one million people have died since the start of the COVID-19 pandemic (1), but children have been relatively spared, both in terms of the incidence of the disease and the severity. Patients under 20 years of age have been reported to represent less than 2.0% of patients (2). However, on 27 April 2020 the UK National Health Service issued an alert after a small number of children presented with a inflammatory syndrome related to COVID-19. These children were extremely ill and had similar features to atypical Kawasaki disease (KD), toxic shock syndrome (3), and KD shock syndrome [START_REF] Riphagen | Hyperinflammatory shock in children during COVID-19 pandemic[END_REF]. The UK Royal College of Paediatrics and Child Health defined these cases as paediatric multisystem inflammatory syndrome temporally associated with COVID-19 (PIMS-TS) (3). The US Centers for Disease Control and Prevention immediately defined it as multisystem inflammatory syndrome in children (MIS-C) [START_REF]Information for Healthcare Providers about Multisystem Inflammatory Syndrome in Children (MIS-C). Case Definition for MIS-C[END_REF] and the World Health Organization (WHO) named it multisystem inflammatory syndrome in children and adolescents, temporally related to COVID-19 [START_REF] Health Organization | Multisystem inflammatory syndrome in children and adolescents temporally related to COVID-19[END_REF]. In May and June 2020, reports from Italy [START_REF] Verdoni | An outbreak of severe Kawasaki-like disease at the Italian epicentre of the SARS-CoV-2 epidemic: an observational cohort study[END_REF], the UK [START_REF] Riphagen | Hyperinflammatory shock in children during COVID-19 pandemic[END_REF][START_REF] Whittaker | Clinical Characteristics of 58 Children With a Pediatric Inflammatory Multisystem Syndrome Temporally Associated With SARS-CoV-2[END_REF], France [START_REF] Toubiana | Kawasaki-like multisystem inflammatory syndrome in children during the covid-19 pandemic in Paris, France: prospective observational study[END_REF][START_REF] Pouletty | Paediatric multisystem inflammatory syndrome temporally associated with SARS-CoV-2 mimicking Kawasaki disease (Kawa-COVID-19): a multicentre cohort[END_REF], Spain [START_REF] Cabrero-Hernandez | Severe SARS-CoV-2 Infection in Children With Suspected Acute Abdomen: A Case Series From a Tertiary Hospital in Spain[END_REF], Switzerland [START_REF] Belhadjer | Acute heart failure in multisystem inflammatory syndrome in children (MIS-C) in the context of global SARS-CoV-2 pandemic[END_REF] and the US [START_REF] Cheung | Multisystem Inflammatory Syndrome Related to COVID-19 in Previously Healthy Children and Adolescents in New York City[END_REF][START_REF] Kaushik | Multisystem Inflammatory Syndrome in Children (MIS-C) Associated with SARS-CoV-2 Infection: A Multi-institutional Study from New York City[END_REF][START_REF] Feldstein | Multisystem Inflammatory Syndrome in U.S. Children and Adolescents[END_REF][START_REF] Dufort | Multisystem Inflammatory Syndrome in Children in New York State[END_REF]) described groups of children fulfilling these descriptions. These reports used different terminologies: MIS-C, PIMS-TS, Kawasaki-like disease or a combination of these.

The announcement of this syndrome has raised important global concerns in the public and, in particular, among parents.

It has not been clearly demonstrated that this inflammatory condition is caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) that results in COVID-19. However, the temporal associations described in different countries, and the confirmation of the SARS-CoV-2 infection in most patients presenting with this inflammatory condition, provides a strong argument that they are linked [START_REF] Mccrindle | SARS-CoV-2-Related Inflammatory Multisystem Syndrome in Children: Different or Shared Etiology and Pathophysiology as Kawasaki Disease?[END_REF][START_REF] Son | Pediatric inflammatory syndrome temporally related to covid-19[END_REF]. The reports from different countries have described some differences, but they have also pointed out some similarities between these patients and patients with KD. Relatively few patients have been reported to date and that is why more data is needed on this syndrome, for a deeper insight into this condition and to better understand the local kinetics of the evolution of the COVID-19 pandemic and the occurrence of MIS-C.

We observed an outbreak of MIS-C in our paediatric university hospital in Paris, as in many other centres, during the first wave of the COVID-19 pandemic. This study had two aims.

The first was to describe the kinetics of the MIS-C cases and the COVID-19 pandemic in France, in order to determine whether they were related and what influence the pandemic had on MIS-C cases. The second was to describe the clinical, laboratory, echocardiographic and imaging characteristics of the children involved in this MIS-C outbreak, who were all tested positive for SARS-CoV-2, to patients with KD.

METHODS

Patients and definitions

We retrospectively reviewed the records of all patients up to 18 years of age who had been admitted to the University Pediatric Hospital Armand Trousseau in Paris with a diagnosis of KD or Kawasaki-like disease from 1 January 2018 to 15 July 2020. The paediatric emergency department in this tertiary paediatric hospital is visited by 55,000 patients a year.

Children and adolescents fulfilling the MIS-C (5), PIMS-TS (3), or WHO (6) definitions of an inflammatory multisystem syndrome were also included. KD is an acute vasculitis of unknown origin that presents as a febrile illness and predominantly affects children under five years of age. Diagnosis was based on the clinical criteria described in the 2017 guidelines issued by the American Heart Association [START_REF] Mccrindle | Diagnosis, Treatment, and Long-Term Management of Kawasaki Disease: A Scientific Statement for Health Professionals From the American Heart Association[END_REF]. Patients who meet the case definition are said to have complete KD, namely typical or classic KD, whereas those who do not have sufficient principal clinical findings may be diagnosed with incomplete or atypical KD [START_REF] Mccrindle | Diagnosis, Treatment, and Long-Term Management of Kawasaki Disease: A Scientific Statement for Health Professionals From the American Heart Association[END_REF].

The diagnosis of classic KD is based on fever for five or more days, together with at least four of the five principal clinical features. These are: a) oral changes, including erythema, cracked lips, a 'strawberry tongue' and diffuse erythema of the oropharyngeal mucosa, b) a bilateral bulbar conjunctival infection, c) erythematous rash, d) erythema and oedema of the hands and feet and e) cervical lymphadenopathy of at least 1.5cm in diameter. If coronary artery abnormalities are detected, the diagnosis of KD is considered to be confirmed in most cases [START_REF] Mccrindle | Diagnosis, Treatment, and Long-Term Management of Kawasaki Disease: A Scientific Statement for Health Professionals From the American Heart Association[END_REF]. Other clinical, laboratory, and echocardiographic findings can support the diagnosis of incomplete KD if a patient's clinical presentation suggests KD, but their clinical features do not meet the epidemiological case definition. KD shock syndrome has been defined by Kanegaye et al (20) as: initiation of volume expansion and infusion of vasoactive agents or transfer to intensive care due to systolic hypotension for age or a decrease of at least 20% in systolic blood pressure from baseline or clinical signs of poor perfusion. MIS-C cases have been defined in three ways by the US Centers for Disease Control and Prevention (5) First, a patient under the age of 21 years presenting with fever, laboratory evidence of inflammation, evidence of clinically severe illness requiring hospitalisation and the involvement of two or more organs, namely cardiac, renal, respiratory, haematological, gastrointestinal, dermatological or neurological. Second, no alternative plausible diagnoses.

Third, being positive for current or recent SARS-CoV-2 infections or COVID-19 exposure in the four weeks before the onset of symptoms. Some of these patients may fulfil all, or some, of the criteria for Kawasaki disease [START_REF]Information for Healthcare Providers about Multisystem Inflammatory Syndrome in Children (MIS-C). Case Definition for MIS-C[END_REF]. The comparisons of the MIS-C, PIMS-TS and WHO definitions are shown in Table S1.

Data collected

Data on patient demographics, clinical, laboratory, echocardiographic, imaging, therapeutic, and outcomes were collected for patients seen from 1 January 2018 to 15 July 2020. We collected data using a standardized form from both the hospital's electronic health record system and paper health records. For patients seen from 1 April 2020 to 15 July 2020, we collected data on SARS-CoV-2 infections and close COVID-19 contacts and determined if patients fulfilled the MIS-C (5), PIMS-TS (3), WHO (6) definitions or the classic or incomplete KD criteria [START_REF] Mccrindle | Diagnosis, Treatment, and Long-Term Management of Kawasaki Disease: A Scientific Statement for Health Professionals From the American Heart Association[END_REF]. Since data collection on race and ethnicity are not allowed in France, these were not available. Although the above definitions are very similar, we chose to use the definition for MIC-S in our hospital because we felt that the link suggested with a SARS-CoV-2 infection was more straightforward, Table S1.

Treatment

As MIS-C is a new syndrome, patients who were affected were treated according to our hospital protocol for KD. The first-line treatment was intravenous immunoglobulins at 2 g/kg as a single infusion given over 10 to 12 hours. If apyrexia was not obtained within 36 hours, or fever reappeared less than seven days after initial treatment, the treating physician could decide on a second dose of intravenous immunoglobulins and, or, the use of corticosteroids [START_REF] Mccrindle | Diagnosis, Treatment, and Long-Term Management of Kawasaki Disease: A Scientific Statement for Health Professionals From the American Heart Association[END_REF].

Detecting SARS-CoV-2

We identified SARS-CoV-2 ribonucleic acid using reverse-transcriptase polymerase chain reaction (RT-PCR) on nasopharyngeal swabs or stool samples. Samples were obtained within the first 48 hours of admission. We used the Allplex nCoV assay (Seegene Inc, Seoul, South Korea) and the Bosphore v2 nCoV assay (Anatolia Geneworks, Istanbul, Turkey). We identified SARS-Cov-2 IgG antibodies using the Alinity-i SARS-CoV-2 IgG (Abbott Molecular, Illinois, USA). Serology was performed during hospitalisation for patients admitted from 1 April to 15 July 2020. Positive RT-PCR or serology results defined patients with a proven SARS-CoV-2 infection.

Comparing MIS-C and KD patients

All the MIS-C patients had the SARS-CoV-2 infection. They were compared to KD patients, including those who did not have the viral infection during the pandemic phase of the study.

Evolution of the pandemic and occurrence of MIS-C

To determine the relationship between the occurrence of MIS-C and the evolution of the COVID-19 pandemic in France, which had 66.5 million inhabitants in January 2020 [START_REF] Insee | Bilan démographique[END_REF], we obtained official French COVID-19 data from Public Health France. The cumulative confirmed COVID-19 cases for the whole country were collected and for every week from the beginning of the epidemic, as well as the mean daily number of new cases. These data were used to construct chronological curves that were analysed according to the occurrence of

Statistical analysis

The characteristics of the two groups were compared using the Student t-test for continuous variables and the chi-square test or Fisher's exact test, as necessary, for categorical variables. The strength of the relationship between the number of new COVID-19 cases and the number of MIS-C cases was tested with Spearman's Rho correlation coefficient. A linear regression model was used to measure the extent of the explained variance. Analyses were performed with SPSS, version 18 (SPSS Inc, Chicago, USA). A p value of <0.05 was considered statistically significant.

Ethical approval

This study was approved by the Ethics Committee for Research of the Sorbonne University.

Parents were informed that their children's data would be anonymously used and could ask for them to be excluded from the analysis; none of them did so.

RESULTS

The study comprised 47 patients admitted to the hospital from 1 January 2018 to 15 July 2020. These included seven patients diagnosed with MIS-C and 40 diagnosed with KD.

Description of the MIS-C outbreak

Between 13 April and 15 July 2020, seven children diagnosed with MIS-C and 10 children diagnosed with typical or atypical KD were admitted to our hospital. The combined incidence of MIS-C and typical and atypical KD during that period was 5.7 patients per month. In comparison, 30 children diagnosed with typical or atypical KD were admitted from January 2018 to March 2020, with a mean incidence of 1.1 patients per month. Figure 1,panel A shows all the children with typical or atypical KD and the children with MIS-C, admitted by month, and their need for a PICU stay from 1 January 2018 to 15 July 1 2020. The main characteristics of the seven MIS-C patients are shown in Table 1. There were two males aged 0.5 and 12.6 years and five females aged 5.8, 6.0, 7.5, 8.3 and 11.3 years.

All seven MIS-C patients tested positive for the SARS-CoV-2 infection: one had a positive RT-PCR from a nasopharyngeal swab and all seven had positive immunoglobulin G (IgG) serology. A known COVID-19 contact was found in five of these seven children and these are detailed in Table 2. Two of the seven the children with MIS-C also fulfilled the criteria for classic KD. The use of the PIMS-TS and WHO definitions yielded the same results as the MIS-C definition. All seven MIS-C patients were admitted to the PICU: the two boys stayed for two days and the five girls for three to 12 days. None of them had coronary aneurisms. An abnormal echocardiography was found in six of the seven MIS-C patients, mainly due to a low ejection fraction or mitral regurgitation. All seven children received intravenous immunoglobulins and four also received corticosteroids. The outcomes were favourable in all seven children (Table 1). 3). Compared to the children with KD, the children with MIS-C were older, and a lower proportion were under five years of age (14.3% versus 85.0%, p<0.001). The MIS-C patients were admitted to the PICU more frequently (100% versus 10.0%, p<0.001) and had higher frequencies of abdominal pain (71.4% versus 12.5%, p=0.003), myocardial dysfunction (85.7% versus 5.4%, p<0.001) and KD shock syndrome (85.7% versus 5.0%, p<0.001). The patients with MIC-C also had a higher mean and standard deviation of C-reactive protein (339 ±131 versus 153 ±87, p<0.001) and a lower mean and standard deviation of serum sodium (130 ±4.2 versus 134 ±3.0, p=0.011). Figure 2 shows the correlation between the new weekly ED COVID-19 cases reported for the Paris region from March to July 2020 and the weekly number of children presenting with MIS-C and confirmed SARS-CoV-2 who were admitted to our hospital. For the correlation analysis, the weekly numbers of MIS-C cases were plotted against the new weekly ED COVID-19 cases for the region at four weekly time points before admission. The Spearman's Rho correlation coefficient for the same week that the children with MIS-C were admitted was 0.367 (p=0.102). We also looked at the one to four weeks before admission and they were: 0.465 (p=0.039), 0.577 (p=0.01), 0.705 (p=0.001) and 0.724 (p=0.001), respectively. The Rsquared measures were highest (0.806) for the correlation between the weekly number of children with MIS-C and the weekly new ED COVID-19 cases reported in the Paris region three weeks before.

Comparison of MIS-C patients and KD patients

Evolution of the MIS-C outbreak related to COVID-19 in France

Figure S1 shows the COVID-19 pandemic evolution in France from March to July 2020. This includes the official cumulative confirmed cases and new daily cases in France, as well as the 10 KD virus negative cases and the MIS-C virus positive cases during this period, displayed by when they were first seen in our hospital. We also plotted the weekly numbers of MIS-C cases against the new daily official COVID-19 cases for the whole of France at four weekly time points before those admissions (Figure S2). The results were almost identical to those obtained for the COVID-19 cases in the Paris region.

DISCUSSION

This study showed a strong lagged correlation between the rise and fall of the number of children and adolescents with MIS-C and the rise and fall of the first wave of the COVID-19 pandemic in France. To the best of our knowledge, this has not been reported before. The correlation was highest for the number of new COVID-19 cases reported three to four weeks before the occurrence of MIS-C cases. The correlations were similar when we used the number of new ED cases in the Paris region or the total number of new official cases for the whole of France. All seven of the MIS-C patients tested positive for SARS-CoV-2. Compared to children with KD, MIS-C patients were significantly older and were more likely to be admitted to the PICU. They also had a higher frequency of abdominal pain, myocardial dysfunction and KD shock syndrome, a higher C-reactive protein and lower serum sodium.

During the period of the MIS-C outbreak, 10 children diagnosed with KD were admitted. They did not meet the MIS-C criteria and did not test positive for SARS-CoV-2.

To date, SARS-CoV-2 has not been definitely proved to cause MIS-C. However, MIS-C also appeared during outbreaks of COVID-19 in Europe (7-9) and the United States [START_REF] Cheung | Multisystem Inflammatory Syndrome Related to COVID-19 in Previously Healthy Children and Adolescents in New York City[END_REF][START_REF] Kaushik | Multisystem Inflammatory Syndrome in Children (MIS-C) Associated with SARS-CoV-2 Infection: A Multi-institutional Study from New York City[END_REF][START_REF] Feldstein | Multisystem Inflammatory Syndrome in U.S. Children and Adolescents[END_REF][START_REF] Dufort | Multisystem Inflammatory Syndrome in Children in New York State[END_REF] and this link has been highly suggested [START_REF] Rowley | Understanding SARS-CoV-2-related multisystem inflammatory syndrome in children[END_REF]. The strong lagged correlation between the rise and fall of MIS-C cases, and the rise and fall of the first wave of the COVID-19 pandemic in France, reported in this paper further supports this association. If MIS-C is related to SARS-CoV-2, the delay between the peak of the virus in the general population and the MIS-C cases suggests a post-infectious phenomenon, rather than a condition resulting from an acute infection [START_REF] Son | Pediatric inflammatory syndrome temporally related to covid-19[END_REF]. Our results are consistent with previous reports (7-9, 16) of MIS-C cases. These have indicated an absence of COVID-19 symptoms before an MISC-C diagnosis, often negative RT-PCR results, very frequent positive antibodies, frequent family exposure and the development of MIS-C after a time lag of three to six weeks. These elements further suggest that SARS-CoV-2 may be acting as either a trigger or as an immune-modulating factor [START_REF] Mccrindle | SARS-CoV-2-Related Inflammatory Multisystem Syndrome in Children: Different or Shared Etiology and Pathophysiology as Kawasaki Disease?[END_REF]. All seven of the MIS-C patients in our series tested positive for IgG antibodies, while only one tested positive for nasopharyngeal RT-PCR. It could be argued that the presence of antibodies to SARS-CoV-2 does not, in itself, imply a post infectious process linked to MIS-C. This is because positive antibody tests could also become more and more common in children as the COVID-19 spreads in the population. However, this is not a likely explanation for the findings in our series. A cross-sectional prospective study conducted in the Paris region during approximately the same period as our study found that only 10.5% of 605 asymptomatic or pauci-symptomatic children aged 0-15 years tested positive for SARS-CoV-2 serology [START_REF] Cohen | Assessment of spread of SARS-CoV-2 by RT-PCR and concomitant serology in children in a region heavily affected by COVID-19 pandemic[END_REF]. It is notable that 87.3% of those who tested positive in that study had confirmed or suspected COVID-19 contacts [START_REF] Cohen | Assessment of spread of SARS-CoV-2 by RT-PCR and concomitant serology in children in a region heavily affected by COVID-19 pandemic[END_REF].

We do not know the role played by lockdowns on the MIS-C outbreaks. It has been reported that many children are infected by their parents, so it is possible that these restrictions may have enhanced close contact and eventually increase the viral load from adults to susceptible children.

While MIS-C has been reported in several countries in Europe and in the USA, it has not been reported in China or Japan [START_REF] Rowley | Understanding SARS-CoV-2-related multisystem inflammatory syndrome in children[END_REF] where KD is more frequent. A series of KD patients in a paediatric Japanese hospital, published online in this journal in August 2020, found no increase in the incidence of KD or changes in its clinical features during the local COVID-19 pandemic (24). It could be that MIS-C is such a rare condition that it has only been observed in countries with a very large number of COVID-19 cases, such as Italy, Spain, France, the UK and the United States [START_REF] Rowley | Understanding SARS-CoV-2-related multisystem inflammatory syndrome in children[END_REF]. The fact that the MIS-C cases in our report occurred during a shorter period than the first wave of the COVID-19 pandemic in France, and that the cases correlated with the peak of this pandemic, is consistent with this hypothesis. Figure S1 suggests that MIS-C cases appeared when the official number of new confirmed COVID-19 cases was around 4,500 per day in France. In reality, the real number of new cases would have been much higher, because the testing rate during the earlier stages of the pandemic was relatively low.

A clear and consensual definition of this new multisystem inflammatory syndrome that is temporally associated with COVID-19 is needed. The syndrome is currently referred to as PIMS-TS or just PIMS, MIS-C, multisystem inflammatory syndrome in children and adolescents temporally related to COVID-19, Kawasaki-like disease or atypical KD. In our series, the use of the MIS-C, PIMS-TS and WHO definitions yielded the same results.

Although this inflammatory condition shares multiple clinical and laboratory features with KD, some important differences exist [START_REF] Mccrindle | SARS-CoV-2-Related Inflammatory Multisystem Syndrome in Children: Different or Shared Etiology and Pathophysiology as Kawasaki Disease?[END_REF]. Cheung et al reported the clinical characteristics of 17 patients with PIMS: eight who met the criteria for typical KD and five for incomplete KD [START_REF] Cheung | Multisystem Inflammatory Syndrome Related to COVID-19 in Previously Healthy Children and Adolescents in New York City[END_REF].

Dufort et al reported that 36% of the 99 MIS-C patients they studied also had a diagnosis of KD [START_REF] Dufort | Multisystem Inflammatory Syndrome in Children in New York State[END_REF]. The current case definition of MIS-C is very broad and would be met by children with many infectious and inflammatory conditions. In our series, only children who tested positive for SARS-CoV-2 fulfilled the MIS-C criteria. We suggest that an MIS-C diagnosis should only be given to children who test positive for SARS-CoV-2, as these infected patients have a clearly different clinical picture, as confirmed by Dufort et al [START_REF] Dufort | Multisystem Inflammatory Syndrome in Children in New York State[END_REF].

We did not collect racial or ethnic data, as this is not permitted in France. Previous case series have suggested that MIS-C primarily affects people of African or Caribbean ancestry [START_REF] Whittaker | Clinical Characteristics of 58 Children With a Pediatric Inflammatory Multisystem Syndrome Temporally Associated With SARS-CoV-2[END_REF][START_REF] Toubiana | Kawasaki-like multisystem inflammatory syndrome in children during the covid-19 pandemic in Paris, France: prospective observational study[END_REF]. However, one case series from New York reported that twelve out of seventeen patients they studied were white [START_REF] Cheung | Multisystem Inflammatory Syndrome Related to COVID-19 in Previously Healthy Children and Adolescents in New York City[END_REF]. Although predisposing genetic factors have been described for KD, caution is necessary when interpreting the current racial or ethnic data for MIS-C. An alternative explanation may be that there is a higher prevalence of SARS-CoV-2 in some groups, with the spread of infection driven by decreased opportunities for social distancing because of dense housing. Some groups with a higher prevalence of SARS-CoV-2 are also more likely to be essential key workers or have to work for economic reasons (25).

Social disparities have been reported for COVID-19

This study had some limitations. Its retrospective design meant that there was less data on inflammatory investigations in the 30 children with KD admitted during the pre COVID-19 pandemic, which precluded a larger comparison with MIS-C patients. Second, this was a single centre study with few patients and the results may have been due to local variations.

Third, the data on the MIS-C outbreak was only collected for three months, with a short follow-up period. However, the decline of the observed outbreak was as sharp as its rise and no new cases of MIS-C were seen during the last two months of the study period. Fourth, although the correlation between the rise and fall of the MIS-C outbreak and the COVID-19 pandemic was very strong, it does not provide formal proof of causality. Fifth, correlations should be interpreted with caution because of the small sample size.

CONCLUSION

The rise and fall of the COVID-19 epidemic was associated with the rise and fall of an outbreak of MIS-C in children and adolescents in France. The fall in the MIS-C cases as the first wave of the COVID-19 pandemic reduced in intensity in France further supports their association. Although MIS-C has been relatively rare, clinicians should be aware of this new clinical presentation, as more cases are likely if the second wave of COVID-19 epidemic has the same impact as the first one. Because the number of patients by MIS-C is relatively low, it is very important to carry out multicentre studies, so that we can develop a better understanding of this new condition. *Please note. As France was about to enter its second lockdown at the time of editing, I have described the period of the study as the first wave*

24.

Iio K, Uda K, Hataya H, Yasui F, Honda T, Sanada T, et al. Kawasaki disease or Kawasaki-like disease: influence of SARS-CoV-2 infections in Japan. Acta Paediatr. 2020. Epub 2020/08/18. 25.

Martinez DA, Hinson JS, Klein EY, Irvin NA, Saheed M, Page KR, et al. SARS-CoV-2 Positivity Rate for Latinos in the Baltimore-Washington, DC Region. JAMA. 2020(June 18).

The tables are quite big and I have made some suggestions about how they could take up a bit less room. cases that were reported in the region during the same week (black regression line, Rho 0.367, p=0.102), one week before (green, Rho 0.465, p=0.039), two weeks before (blue, Rho 0.577, p=0.01), three weeks before (red, Rho 0.705, p=0.001) and four weeks before (orange, Rho 0.724, p=0.001). The R-squared measure was highest for the correlation between the weekly MIS-C and weekly new COVID-19 cases reported three weeks before. Rho, Spearman's Rho correlation coefficient.

  MIS-C.In order to test the influence of the number of new COVID-19 cases in our region on the occurrence of MIS-C cases, we obtained data on COVID-19 visits to emergency departments (ED) from the Oscour surveillance network. This surveillance network automatically collects anonymous, real-time, demographic, and diagnostic data from 86% of all French emergency departments. These COVID-19 data, available at Public Health France, enabled us to calculate the daily and weekly number of COVID-19 ED visits of patients of all ages in the whole Paris region from the beginning of the pandemic and correlate them to the occurrence of MIS-C cases in children in our hospital.

From 1

 1 January 2018 to 15 July 2020, we identified 40 children who presented with typical or atypical KD, including 10 who presented during the COVID-19 lockdown. None of them tested positive for SARS-Cov-2. We compared their patient demographics, clinical, laboratory, echocardiographic, therapeutic and outcomes with the seven children with MIS-C, who all tested positive for SARS-CoV-2 (Table

Figure 1 ,

 1 Figure 1, panel B shows the evolution of the daily number of new cases of COVID-19

Figure 1 .

 1 Figure 1. Panel A. Monthly occurrence of KD cases and MIS-C in our paediatric university hospital in Paris from January 2018 to July 2020. Red dashed bars indicate cases admitted to the paediatric intensive care unit (PICU). Panel B. Daily number of new cases of COVID-19 reported for the Paris region from March to July 2020 and the weekly number of KD, and MIS-C cases who tested positive for SARS-CoV-2, displayed by the week of occurrence, admitted to the same hospital (bars). N numbers within the bars indicate the number of patients.

Figure 2 .

 2 Figure 2. Correlation between new weekly COVID-19 cases reported for the Paris region from March to July 2020 and weekly number of children presenting with MIS-C and SARS-CoV-2 to our paediatric university hospital. The figure shows correlations between the weekly number of MIS-C cases and weekly COVID-19cases that were reported in the region during the same week (black regression line, Rho 0.367, p=0.102), one week before (green, Rho 0.465, p=0.039), two weeks before (blue, Rho 0.577, p=0.01), three weeks before (red, Rho 0.705, p=0.001) and four weeks before (orange, Rho 0.724, p=0.001). The R-squared measure was highest for the correlation between the weekly MIS-C and weekly new COVID-19 cases reported three weeks before. Rho, Spearman's Rho correlation coefficient.

Figure 1 .

 1 Figure 1. Panel A. Monthly occurrence of Kawasaki disease cases and MIS-C in a Pediatric University Hospital in Paris from January 2018 through July 2020. Red dashed bars indicate the cases admitted to the PICU. Panel B. Daily number of new cases of COVID-19 seen the Emergency Departments of the sentinel surveillance network for the region of Paris from March to July 2020 as well as the weekly number of Kawasaki disease and MIS-C cases with a SARS-CoV-2 infection, displayed by week of occurrence, admitted to a Parisian University Pediatric Hospital (bars); numbers within the bars indicate the number of patients. PICU= Pediatric Intensive Care Unit.

Table 1 .

 1 Description of the seven children with MIS-C children who presented to the paediatric university hospital during the first wave of the COVID-19 Pandemic

		Patient 1 Patient 2 Patient 3 Patient 4	Patient 5 Patient 6	Patient 7
	Date of hospitalisation	13 April	19 April	19 April	20 April	20 April	28 April	5 May
	Age (years)	8.3	12.6	5.8	11.3	0.5	6.0	7.5
	Sex	Female	Male	Female	Female	Male	Female	Female
	SARS-CoV-2 infection	Yes	Yes	Yes	Yes	Yes	Yes	Yes
	RT-PCR (nasal swab/stool)	Neg/ND	Neg/Neg	Pos/ND	Neg/Neg	Neg/ND	Neg/ND	Neg/ND
	Serology (IgG)	Pos	Pos	ND	Pos	Pos	Pos	Pos
	COVID-19 contact 1	No	Yes	No	Yes	Yes	Yes	Yes
	Other respiratory viruses 2	No	No	ND	No	No	No	No
	PICU stay in days	3	2	8	12	2	7	8
	Met PIMS-TS definition	Yes	Yes	Yes	Yes	Yes	Yes	Yes
	Met WHO definition	Yes	Yes	Yes	Yes	Yes	Yes	Yes
	Kawasaki major criteria 3
	Type of presentation	Classic	Incomple	Incomple	Incomple	Incomple	Classic	Incomple
			te	te	te	te		te
	Number of major criteria	4	3	3	1	3	5	2
	Extremity changes	Yes	No	Yes	Yes	No	Yes	No
	Rash	Yes	Yes	Yes	No	Yes	Yes	Yes
	Conjunctivitis	Yes	Yes	No	No	Yes	Yes	No
	Oral changes	Yes	Yes	Yes	No	Yes	Yes	Yes
	Cervical lymphadenopathy	No	No	No	No	No	Yes	No
	Days of fever at diagnosis	5	6	7	4	7	5	6
	Other signs and symptoms
	Abdominal pain	Yes	Yes	Yes	Yes	No	No	Yes
	Other	Vomiting	Vomiting	Vomiting	Vomiting Hydrocel	Cervical	Vomiting
		, stiff	, diarrhea	,		e,	pain	,
		neck		diarrhoea		irritabilit		diarrhoea
				, sterile		y
				pyuria
	Echocardiography	Abnorma l	Abnorma l	Abnorma l Normal No Abnorma l Yes No	No	Yes
	Electrocardiography	Normal	Normal	ST	ST	Normal	Normal	Normal
				segment	segment
				elevation	depressio
					n
	Myocardial dysfunction	Yes	Yes	Yes	Yes	No	Yes	Yes
	KDSS	Yes	Yes	Yes	Yes	Yes	Yes	Yes
	Chest X-ray	Normal	Normal	Right	Cardiome	Bilateral	Cardiome	Cardiome
				Pleural	galy	alveolar	galy	galy,
				effusion,		opacities		bilateral
				Cardiome				perihilar
				galy				alveolar
								opacities
	Others imaging findings	Pleural	Peritonea	Peritonea	Peritonea	ND	Pleural	ND
		effusion	l	l effusion	l and		effusion,
			effusion,		pleural		retrophar
			terminal		effusion.		yngeal
			ileitis		Typical		abscess

Table 2 .

 2 Description of close contacts with COVID-19 in seven children with MIS-C during the two months before the onset of their disease

	Pa	Lockdown	Contact with confirmed or	Symp	Day 1	Day 1	Contac
	tie	respected 1	suspected COVID-19	toms	Fever	Hospi	to
	nt					betwe		tal	COVI
	No					en			D-19
	.					conta			(days)
						ct and
						MIS_
						C
						diagn
						osis
			Yes/No	Day 1 of	Setting
				contact
	1	Yes	No	NA	NA	none	8	13
							April	April
	2	Yes	Yes	7 March	adult,	none	13	19	37
					party		April	April
	3	Yes	No	NA	NA	???	14	19
							April	April
	4	Yes	Yes	17 March grandfa	none	18	20	32
					ther,		April	April
					househ
					old
	5	Yes	Yes	17 March mother 2	cough	15	20	34
					,	,	April	April
					househ	laryng
					old	itis on
						1
						April
	6	Yes	Yes	30 March	adult	none	23	28	24
					cousin,		April	April
					househ
					old
	7	Yes	Yes	5 March	father,	none	30	5 May	58
					househ		April
					old
	MIS-C=Multisystem Inflammatory Syndrome in Children
	1 France's lockdown started on 17 March 2020.

2

Child's mother continued to go to work and got COVID-19

Table 3

 3 Comparison between 40 typical or atypical Kawasaki disease patients and seven MIS-C patients with a SARS-CoV-2 infection Please be consistent about number of decimal places. SD) unless indicated otherwise MIS-C=Multisystem Inflammatory Syndrome in Children. CRP=C-reactive protein. KDSS=Kawasaki disease shock syndrome. IVIG=Intravenous immunoglobulins a American Heart Association criteria(19) b Left ventricle ejection fraction c The highest value before diagnosis during hospital stay is reported unless indicated otherwise d All 47 patients received IVIG e Missing data on electrocardiography for 16 patients

		Kawasaki disease	MIS-C with	P value
		(no SARS-CoV-2	SARS-CoV-2
		infection)	infection
		N=40	N=7
	Age, years	2.3 (2.0)	7.5 (4.0)	0.013
	Age, younger than 5 years, n (%)	34 (85.0%)	1 (14.3%)	0.001
	Sex, male, n (%)	26 (65.0%)	3 (42.9%)	0.403
	PICU stay, n (%)	4 (10.0%)	7 (100.0%)	<0.001
	Kawasaki major criteria a
	>4, n (%)	17 (42.5%)	2 (28.6%)	0.685
	Number of major criteria	3.2 (1.2)	2.9 (1.5)	0.503
	Other symptoms
	Abdominal pain, n (%)	5 (12.5%)	5 (71.4%)	0.003
	Aneurisms or dilated coronaries, n (%)	7 (17.5%)	0 (0.0%)	0.573
	Ejection fraction b below 40%, n (%)	1 (2.5%)	4 (57.1%)	0.001
	Mitral regurgitation, n (%)	10 (25.0%)	2 (28.6%)	1
	Pericardial effusion, n (%)	11 (27.0%)	1 (14.3%)	0.659
	Abnormal electrocardiography, n (%)	0 (0.0%) e	4 (57.1%)	0.001
	Myocardial dysfunction, n (%)	2 (5.0%)	6 (85.7%)	<0.001
	KDSS, n (%)	1 (2.5%)	6 (85.7%)	<0.001
	Laboratory values c
	White blood cell count, /µL	14 873 (4 583)	11 444 (7 903)	0.303
	Neutrophils count, /µL	9 572 (4 948)	9 783 (7 205)	0.925
	Lymphocyte count, /µL	3 790 (2149)	866 (434)	<0.001
	Haemoglobin, g/dL	10.0 (1.1)	9.1 (2.2)	0.322
	Platelet count, /µL	398 175 (170 885)	175 857 (69 247)	<0.001
	CRP, mg/L	153 (87)	339 (131)	<0.001
	CRP, >250mg/L, n (%)	8 (20.0%)	6 (85.7%)	0.002
	ALT, U/L	56 (67)	23 (16)	0.206
	AST, U/L	53 (76)	55 (37)	0.951
	Albumin (lowest), g/L	28.1 (4.5)	20.2 (2.2)	<0.001
	Sodium (lowest), mEq/L	134 (3)	130 (4.2)	0.011
	Sodium below 132 mEq/L, n (%)	8 (20.0%)	4 (57.1%)	0.06
	Creatinine, µmol/L	23.2 (8.5)	83 (68.0)	0.059
	Treatment
	Inotropic support, n (%)	1 (2.5%)	5 (71.4%)	<0.001
	IVIG (number of doses) d	1.2 (0.4)	1.6 (0.5)	0.041
	Corticosteroids, n (%)	7 (17.5%)	4 (57.1%)	0.042
	Favorable outcome, n (%)	10 (100.0%)	7(100.0%)	NA
	Values are means (

Pediatric Hematology and Oncology Department, APHP Hôpital Armand Trousseau -5 Pediatric Anesthesiology Department, APHP Hôpital Armand Trousseau 6 Virology Department, APHP Hôpital Armand Trousseau -Sorbonne Université 7 Pediatric Cardiology Unit, APHP Hôpital Armand Trousseau -Sorbonne Université 8 Pediatric Imaging Department, APHP Hôpital Armand Trousseau -Sorbonne Université

ABBREVIATIONS

Table S1. Comparison of MIS-C, PIMS-TS and WHO definitions for a SARS-CoV-2 Related Multisystem Inflammatory Syndrome in Children

Case Definition for Multisystem Inflammatory Syndrome in Children (MIS-C) 1 • An individual aged <21 years presenting with fever a , laboratory evidence of inflammation b , and evidence of clinically severe illness requiring hospitalization, with multisystem (>2) organ involvement (cardiac, renal, respiratory, hematologic, gastrointestinal, dermatologic or neurological); AND • No alternative plausible diagnoses; AND • Positive for current or recent SARS-CoV-2 infection by RT-PCR, serology, or antigen test; or COVID-19 exposure within the 4 weeks prior to the onset of symptoms a Fever >38.0°C for ≥24 hours, or report of subjective fever lasting ≥24 hours b Including, but not limited to, one or more of the following: an elevated C-reactive protein (CRP), erythrocyte sedimentation rate (ESR), fibrinogen, procalcitonin, d-dimer, ferritin, lactic acid dehydrogenase (LDH), or interleukin 6 (IL-6), elevated neutrophils, reduced lymphocytes and low albumin.

Additional comments • Some individuals may fulfill full or partial criteria for Kawasaki disease but should be reported if they meet the case definition for MIS-C • Consider MIS-C in any pediatric death with evidence of SARS-CoV-2 infection

Paediatric multisystem inflammatory syndrome temporally associated with COVID-19 (PIMS-TS) 2

Case definition: 1. A child presenting with persistent fever, inflammation (neutrophilia, elevated CRP and lymphopaenia) and evidence of single or multi-organ dysfunction (shock, cardiac, respiratory, renal, gastrointestinal or neurological disorder) with additional features a . This may include children fulfilling full or partial criteria for Kawasaki disease. 2. Exclusion of any other microbial cause, including bacterial sepsis, staphylococcal or streptococcal shock syndromes, infections associated with myocarditis such as enterovirus (waiting for results of these investigations should not delay seeking expert advice). 3. SARS-CoV-2 PCR testing may be positive or negative a Clinical. All patients: Persistent fever >38.5°C. Most patients: Oxygen requirement, Hypotension. Some patients: abdominal pain, confusion, conjunctivitis, cough, diarrhea, headache, lymphadenopathy, mucus membrane changes, neck swelling, rash, respiratory symptoms, sore throat, swollen hands and feet, syncope, vomiting Multisystem inflammatory syndrome in children and adolescents with COVID-19 3 The figure shows correlations between the weekly number of MIS-C cases and the new daily COVID-19 cases that were reported in France during the same week (black regression line, Spearman's Rho correlation coefficient 0.307, p=0.164), one week before (green, Spearman's Rho correlation coefficient 0.397, p=0.075), two weeks before (blue, Spearman's Rho correlation coefficient 0.554, p=0.011), three weeks before (red, Spearman's Rho correlation coefficient 0.692, p=0.001), and four weeks before (orange, Spearman's Rho correlation coefficient 0.711, p=0.001). The R-Square was highest for the correlation between the weekly number of MIS-C cases and the daily new COVID-19 cases reported in France three weeks before.

By the World Health Organization

Preliminary case definition


A Huerre

M.-C Jullien

O Theodoly

M.-P Valignat

Absolute 3D Reconstruction of Thin Films Topography in microfluidic channels by Interference Reflection Microscopy

The travel of droplets, bubbles, vesicles, capsules, living cells or small organisms in microchannels is a hallmark in microfluidics applications. A full description of the dynamics of such objects requires a quantitative understanding of the complex hydrodynamic and interfacial interactions between objects and channel walls. In this paper, we present an interferometric method that allows absolute topographic reconstruction of the interspace between an object and channel walls for objects confined in microfluidic channels. Wide field microscopic imaging in reflection interference contrast mode (RICM) is directly performed at the bottom wall of microfluidic chips. Importantly, we show that the reflections at both the lower and upper surface of the microchannel have to be considered in the quantitative analysis of the optical signal. More precisely, the contribution of the reflection at the upper surface is weighted depending on the light coherence length and channel height. Using several wavelengths and illumination apertures, our method allows reconstructing the topography of thin films on channel walls in a range of 0-500 nm, with a precision as accurate as 2 nm for the thinnest films. A complete description of the protocol is examplified for oil in water droplets travelling in channels of height 10-400 µm at a speed up to 5 mm.s -1 .

Introduction

Many microfluidics applications manipulate objects in confined channels where strong interactions occur between objects and channel walls. Typical examples are found in digital microfluidics where microreactors 1,2 (droplets, vesicles or capsules) or living entities (single cells 3-7 or small organisms 8,9 ) are manipulated at high throughput rate in microcircuits. Object-wall interactions play a central role in experimentations with confined items because they strongly influence the shape of the objects, their velocity and the stress that they encounter. Unfortunately, interfacial interactions remain difficult to control and characterise because they result from complex and entangled phenomena, such as the hydrodynamics of thin lubrication films, the deformability of the objects, the composition and rheological properties of the interfaces, or the disjoining pressures between objects and walls. Hence, the characterisation of the contact zone between an object and channel walls is an important issue for both fundamental and applicative reasons, but few studies have investigated these issues especially in microfluidics systems. Reflection interference contrast microscopy (RICM) has proven to be a powerful technique to characterise the thickness of the contact zone between an object and a flat surface [10][11][12][13][14][15][16][17][18][19] in static and dynamic conditions but has hardly been used in microfluidics. RICM is a priori well adapted to perform measurements in microfluidics chips with flat channel walls, which is typically the case of devices microfabricated by conventional photolithography routines 7,20 , specific issues must be considered due to confinement. RICM imaging of objects in microchannels may indeed result from a combination of contributions, mainly the reflection at the lower but also the upper channel surfaces of the channel, the stray signal from the side walls of the channel, as well as the signal refracted by objects edges or diffused by the materials constitutive of the chip. Recovering thickness information from experimental RICM images in microfluidics channels requires therefore specific attention and modelling. In this paper, we present a novel normalisation and analysis method adapted for RICM experimentation in microfluidic channels that takes into account multiple reflections in channels and allows accurate and absolute measurements of thin film thickness at the walls of microfluidic channels.

Principles of interferometric microscopy measurement

Interferometric measurements are performed here with a wide field microscope in epi-illumination. An image focused on the contact region between an object and the lower wall of a microfluidic channel is taken in RICM, i.e. with a crossed Please do not adjust margins Please do not adjust margins polariser/analyser cube and an antiflex objective (Figure 1).

ARTICLE

Interferometric microscopy measurement between two semi-infinite media 2.1 Modelling of optical signal

In RICM, the total intensity reflected by a sample results from multiple reflections and interferences in the thin layers composing the reflecting interface (light is coherent for thin films with thickness of hundreds of nanometers). With convergent illumination, the total theoretical intensity is obtained by integration over the incident light cone of half aperture  0 :

Eq. 2 where is the reflected intensity for light with incidence  i calculated by taking into account the Fresnel coefficients of the sample. At the interface between two semi-infinite media i and j of index n i and n j (Figure 2-a), the parallel and perpendicular Fresnel coefficients are:

Eq. 3

Eq. 4

where  i and  j are the incident and transmitted angles linked by the Descartes-Snell's law:

Eq. 5

and the total intensity is given by 18 :

Eq. 6

For comparison with experiments, calculated intensities are finally normalised into a theoretical signal S th using the same normalisation procedure as for experimental intensities:

Eq. 7

where I th1 , and are the theoretical intensities at respectively the glass/fluid (n i = n glass and n j = n fluid ), glass/PDMS (n i = n glass and n j = n PDMS ) and glass/water (n i = n glass and n j = n water ) interfaces.

Validation of the optical model using glass/water and glass/PDMS interfaces

We consider the case of a simple interface between a glass plate and an infinite medium (Figure 2-a The agreement between theoretical and experimental normalised signals for all media and INA conditions is excellent without adjusting parameter. These data therefore validate the correctness of our absolute calculation and the robustness of data normalisation/calibration using intensities at the PDMS and water interfaces with glass.

2.3 Effect of field stop openingImaging collects reflected signal at the bottom and top contact zone between thr doplet and chanells lower and upper walls.

Figure 3 shows that raw reflected intensities depend markedly on the opening of the field stop, even for a simple interface between two infinite media (here glass/mineral oil). However, the normalised signals using eq.1 and the I PDMS and I W signals measured at each corresponding field openings are similar for all three conditions of field opening. This shows that the normalisation method corrects efficiently for the effects induced by changes in illumination induced by field stop conditions.

Absolute measurements by interferometric microscopy in closed microfluidic channels

In this section, we examine the effect of the reflection on the upper wall of a channel on the collected light intensity.

Optical model

We refine here the optical model to take into account the effect of the upper wall (ceiling) of the channel. Let us consider a microfluidic channel of height H (Figure 2-b). The reflecting zone is now composed of two parallel interfaces separated by a distance H >> λ. Light in this thick layer loses its coherence, so that no interference occurs between the beams reflected on the upper and lower interfaces of medium 1. The resulting reflected signal in the channel is the sum of the intensities of the beam reflected at the 0-1 interface on one hand and of the beam reflected at the 1-3 interface on the other hand. Since this latter beam crosses twice the 0-1 interface, it is twice attenuated by the transmission factor . The reflected intensity in a microfluidic channel can finally be written as:

Eq. 9

where the second term represents the contribution of the ceiling reflection. The total intensity for a cone of light with incidence  0 , is given by: Eq. 10 and the corresponding normalised signal by: Eq.11

Experimental validation of the ceiling reflection contribution

Let us consider a channel of height H = 25 µm with glass substrate and PDMS ceiling filled with a simple fluid (Figure 4 3 that calculations without reflection at the ceiling do not match with experimental data, and conversely that calculations with reflection at the ceiling agree quite well with experimental data. This demonstrates the necessity to consider the reflection across the channel to perform correct RICM measurements in microfluidics.

It appears clearly in Table

Weighting of the ceiling contribution

We have checked that the ceiling contribution in RICM signal is significant for channels of height H = 25 µm but one expects that this contribution of the ceiling to diminish with channels of larger height and to eventually vanish for very thick channels. In order to establish the range of channel heights where the ceiling contributes to the RICM signal, we Please do not adjust margins Please do not adjust margins performed a series of measurements in channels of heights ranging from 10 to 400 µm (Figure 4). Channels were filled with mineral oil, INA was fixed at 0.31, and pictures were taken at three different wavelengths,  = 450 ± 25 nm, 546 ± 10 nm and 610 ± 10 nm. In order to assess the contribution of the reflection at the ceiling in the calculations, we introduced a ceiling parameter p as follows :

Eq. 12 where p varies between 0 and 1, and p = 0 corresponds to a negligible reflection at the ceiling, whereas p = 1 to a ceiling reflection that is fully recorded in the experimental image. The adjustable parameter p is determined for each channel height so that theoretical values with Eq. 12 match the experimental data in the corresponding channels.

Absolute measurements by interferometric microscopy in closed microfluidic channels containing a droplet 4.1 Optical model

We now consider a droplet squeezed between the upper and lower walls of a channel, with a lubrication film of thickness d 1 between droplet and walls on both sides (Figure 2-c). Let us first consider the case of a thin film of thickness d 1 separating the substrate from an infinite medium. The reflecting zone is composed of two parallel interfaces separated by a distance d 1 . The Fresnel coefficients of this stacking made of medium 0 (impinging, semi-infinite), medium 1 (thin film of thickness d 1 ) and medium 2 (emerging, semi-infinite) can be calculated using the Drude formula 21 for the -p and the -s polarisation:

Eq. 13

where and I th4 ( 0 ), the total intensity for a cone of light with incidence  0 , is given by: Eq.14

For the case of the squeezed object, one has to take into account the reflection on the upper wall of a microfluidic channel of height H (Figure 2-c). The confined object can be considered as a layer of medium 2 with a macroscopic thickness H, across which light loses its coherence like in the aforementioned case of a channel filled with a simple fluid. No interference occurs between the beams reflected on the upper and lower interface of medium 2 and the resulting signal reflected in the channel is the sum of the intensities from the beam reflected at the 0-1-2 stacking on one hand and the beam reflected at the 2-1-3 stacking on the other hand. Since this latter beam is also attenuated twice by the transmission factor due to its double crossing of the 0-1-2 stacking, the intensity reflected in the channel can finally be written as:

Eq. 15

Example of application

We now present an application of the method to reconstruct the topography of a water lubrication film separating a moving oil droplet from the walls of a confining microfluidic channel made of a glass substrate bonded to a PDMS chip.

Experimental

Glass coverslides used as substrates were cleaned in a Pyranha solutions (Sulfuric acid 95% / Hydrogen peroxide 50% -2/3, 1/3 w/w) for 5 minutes, rinsed in three different water baths and dried with nitrogen gas. Microchannels were moulded using PDMS (Sylgard 184 Dow Corning) with a 1/10 w/w crosslinker concentration, cured for at least 2 hours at 70 degrees, cleaned with sequential washings with Isopropanol MilliQ water -Decon 5% -Ethanol -MilliQ Water and dried with nitrogen. Glass coverslips and PDMS chips were finally bonded with an air plasma. Oil-in-water droplets of radius R = 55 ± 5 µm were generated at a T-junction in the microfluidic system 10,11 and driven in a Hele-Shaw cell of height H = 25 µm.

The droplets were composed of mineral oil (SIGMA 8042-47-5), whereas the external phase was an aqueous solution of deionized water with Sodium Dodecyl Sulfate (SDS) at 1.42 mM (0.6 times the critical micellar concentration or CMC). The surface tension between the two fluids was  = 1.6.10 -2 N/m at 0.6 CMC, and the viscosities were equal to µ f = 10 -3 Pa.s for the aqueous solution and µ d = 25.10 -3 Pa.s for the mineral oil. The water flow rate was tuned using a Nemesys syringe pump leading to droplet speed of 5 mm.s -1 . Image of the contact zones between travelling droplets and glass substrate (Figure 6-left) was recorded with a high-speed camera (Photron Fastcam SA4). In order to compare theoretical and experimental values, we calculated an error function versus thickness following:

Inversion of a normalised intensity image into absolute thickness map

Eq. 11

The minimum of the error function presented in Figure 5-c allows straightforward determination of the best fit between experimental data and theoretical predictions for the 6 couples of and INA conditions. For the thin film of aqueous solution separating the static drop and channels walls of Figure 5-a , the best fit on Figure 5-c yields a thickness of 25 ± 2 nm.

Topographic reconstruction of a dynamic interface

The method allows also reconstruction of the topography of thin films with complex and dynamical shapes. Let us consider the contact zone of a droplet of 50 µm diameter travelling in a 25 µm high channel at a velocity of 500 µm.s-1 (Figure 6-a). The reconstructed topography (Figure 6-b) reveals that hydrodynamic effects imprint important structural changes in lubrication film topography. The lubrication film that is perfectly flat for resting drop (Figure 5-a) adopts a complex 3D catamaran-like structure for drop moving at 500 µm.s -1 . The central zone is roughly 150 nm thick, whereas the two elongated and narrow side wings are twice thinner. Such analysis procedures on dynamic thin films that yield information with unprecedented in-and out of-plane spatial resolution shall open new avenues in the characterisation and fundamental understanding of the movement of micro-objects in confined geometries.

Conclusion

We present here a refinement of RICM measurement method that allows absolute measurement of thin films in microfluidic channels. The method takes into account the reflection at the ceiling of channels and allows absolute 3D reconstruction of thin films topography with an accuracy about ± 2nm. For fundamental and applicative perspectives, this method will be of special interest for the dynamics in microchannels of bubbles, droplets, vesicles, capsules, as well as living cells and small organisms.

Journal Name 2 |

 2 J. Name., 2012, 00, 1-3 This journal is © The Royal Society of Chemistry 20xx

  b).Table 3 reports the experimental signals S and the theoretical values obtained for different fluids either by not taking (S th1 ) or by taking (S th2 ) into account the reflection at the PDMS ceiling.

Table 3 :

 3 Comparison of the theoretical contrasts for different fluids calculated with a single reflection at the bottom of the channel and with double reflection at bottom and top channel walls. Values correspond to the mean of six different conditions (two INA and three openings of field stop) and the associated error is the quadratic mean of individual errors. Calculations and measurements correspond to a wavelength of = 546 nm and a numerical aperture INA = 0.82 (or INA = 0.31 that gives the same result for simple interfaces).

Figure 5 -

 5 Figure 5-a presents the RICM picture of a static oil droplet confined in a 25 µm high channel. The contact zone presents an homogeneous brightness indicating an homogeneously flat film. To determine its thickness, we computed theoretical normalized signals for a confined droplet with ceiling coefficient p = 1, three different wavelengths and two different

FiguresFigure 1 :

 1 Figures

Figure 2 :

 2 Figure 2 : Schematics of the optical configurations corresponding to the systems considerared in our experiments. (a) A simple interface between two infinte medium, a glass substrate and a fluid of known refractive index (b) a microfluidic channel filles with a fluid of known refractiveindex. Imaging collects reflected signal at the bottom glass water and upper water/PDMS interfaces. (c) A confined oil droplet travelling in a microfluidic channel filled with an immiscible fluid. Imaging collects reflected signal at the bottom and top contact zone between thr doplet and chanells lower and upper

Figure 3 :

 3 Figure 3 : Image normalization and illumination conditions. (Top) RICM images of a microfluidic channel filled with mineral oil taken three different openings of the field stop for a focus at the Glass/Oil interface. (Bottom) Profiles of raw intensity (blue line) taken at the centre of images (yellow line in and normalised signal using Equation 1 (red crosses) in the middle of the images.

Figure 4 :

 4 Figure 4 : Contribution of microfluidic channel ceiling to optical reflected signal. Adjustable ceiling parameter p as a function of the channel's height H. Ceiling reflection is fully recorded for H< 30 µm and negligible for H > 150-200 µm.

Figure 5 :

 5 Figure 5 : Conversion of intensity value into absolute thickness. (a) Raw picture of a the contact zone between an oil droplet and the wall of a confining microfluidic channels filled with water. A thin water film separates the oil droplet from the wall . (b) Normalised signal as a function of water film thickness. The dashed curves correspond to an INA of 0.31, the plain ones to an INA of 0.66. Red, green and blue colors correspond respectively to wavelengths of 4500, 5460 and 6100 nm. (c) Error function ΔS (Equation 8) as a function of the film thickness for one experimental measurement with the 3 wavelengths and the 2 INA. The minimum (red dot) gives access to thickness of the water film.

Figure 6 :

 6 Figure 6 : 3D reconstruction of the topography of the contact zone between a moving droplet and microfluidic channel wall. (Left) Raw image of the droplet contact zone with the lower channel interface of glass. (Right) Reconstruction of the droplet interface using our method of RICM.

  Combination of crossed polarisers and quarter wavelength plate in the antiflex objective decreases stray light and increases the signal to noise ratio of images. We used a Zeiss

	Observer inverted microscope (Carl Zeiss, Jena, Germany)
	equipped with a Zeiss Neofluar 63/1.25 antiflex objective, a
	crossed-polarisers cube, a CCD camera (Coolsnap HQ2;
	Photometrics), and adjustable field and aperture stops. The
	illumination numerical aperture (INA) was determined for each
	given aperture opening by measuring the diameter of the
	illumination cone on a screen positioned at different heights.
	Accessible INA values were comprised between 0.31 and 0.82.
	The source was an X-cite 120Q lamp (Exfo, Mississauga,
	Canada) coupled to a narrow bandpass filter (λ = 546 nm ± 12
	nm). In order to correct for the inhomogeneities of the
	illumination, raw images were divided by the image of a
	homogeneous zone (e.g. substrate/solution interface, Figure
	2.a) normalised to an average value of 1. The light intensities I f
	of these flattened images were then converted into
	normalised signal S using the intensities measured for the
	substrate/solution and substrate/chip interfaces. To clarify our
	purpose, we consider in the following of our discussion the
	case	of	microfluidic	devices	fabricated	with
	Polydimethylsiloxane (PDMS) chips assembled on a glass slide,
	which corresponds to our experimental conditions. Obviously,
	the method is not restricted to chips made of PDMS and glass,
	and for other types of chips, one would just have to consider
	the correct refractive indices of the materials constituting the
	chips in the optical modelling. The reference light intensities
	measured at the glass/water and glass/PDMS interfaces are
	thereafter called I	w and I PDMS and the experimental normalised
	signal was calculated as:
	Eq. 1
	Data normalisation along Equation 1 allows correction of
	background noise and of day-to-day variations of source
	intensity. Moreover, the use of I PDMS and I	w as references is
	convenient for microfluidics because they are directly
	accessible on the chip with exactly the same optical conditions
	as for the measurements in channels. The markedly different
	brightness's of I PDMS and I	w is an asset for a good precision of
	the normalisation procedure. Alternative solutions to get
	reference signals could be channels filled with different fluids.
	In the following, I will refer to raw and flattened intensities and
	S to normalised signals.

Table 1

 1 ). Six different media have been tested, namely PDMS elastomer (Sylgard 184 Dow Corning) sealed on a glass substrate, Millipore water, isopropanol (VWR 20839.297), n-hexadecane (Fischer scientific/270/05), mineral oil (SIGMA 8042-47-5) and immersion oil for fluorescence microscopy (Zeiss Immersol TM 518 F). The refractive indices were measured by ellipsometry for PDMS and with a refractometer (Atago PAL-RI) for liquids (see Table1). Fluids used and their optical indices. Isopro. stands for isopropanol, Imm. Oil for Zeiss immersion oil for microscopy, and min. oil for mineral oil. of the aperture stop (INA = 0.31 and 0.82). We report on Table2the average signals of images normalised using Eq. 1 together with the experimental error estimated using Eq. 8:

	Water	PDMS	Isopro.	Imm. oil	Hexadecane	Min.
						oil
	1.332	1.41	1.375	1.518	1.434	1.467

RICM images of the glass/media interfaces were taken under monochromatic illumination at a wavelength of  = 546 ± 5 nm, with minimal opening of the field stop and two different Please do not adjust margins Please do not adjust margins openings

Table 2 :

 2 Experimental and theoretical signals S and Sth1 for different fluids at different INA.

  One can see on Figure4that p is constant and equal to 1 for channels of height H below 25 µm. Between H = 25 and 140 µm, p decreases monotonously. Eventually, p vanishes for H > 150 µm. Ceiling reflection is therefore fully recorded in experimental data for H < 25 µm, partially recorded for 25 < H < 150 µm and negligible for H > 150 µm. A correct procedure to perform RICM measurement in microfluidic channel requires therefore an adequate modelling of ceiling reflection but also an empirical determination of the fraction of ceiling reflection that is effectively collected in the conditions of experimental acquisition.

Acknowledgements

This work was supported by CNRS, IPGG (Equipex ANR-10-EQPX-34), ESPCI, ANR under the contract 13-BS09-0011-01, Labex INFORM and INSERM.

Confined object travelling in a microfluidic channel

Interference microscopy at the object/channel interface

Absolute 3D topography of object in the contact zone


REPRODUCTIVE PERFORMANCE OF A MATERNAL RABBIT CROSS: FAUVE-DE-BOURGOGNE × INRA-1777

Keywords: Genetic resources, crossbreed, local breed, biodiversity, Oryctolagus cuniculus

Here we compared the reproductive performance of the French rabbit breed Fauve-de-Bourgogne (Fauve), the maternal rabbit line INRA-1777 (INRA), and their cross (Crossed). We followed the life of female rabbits of the three genotypes from 70 days of life until the weaning of litters produced from the third artificial insemination (AI) attempt. They had the same age, were house in the same room, were manage under a reproductive rhythm of 42 days and received antibiotic free diets. Crossed females were heavier at first AI than Fauve females (4.2 vs 3.9 kg; P<0.05), INRA females being between both genotypes (4.1 kg). Between the first AI attempt and the birth of the first litters, we observed the presence of Pasteurella spp. This pathogen impaired the prolificacy of all genotypes and caused mortality of females around parturitions from the second AI attempts. Most losses were from the INRA genotype (four diagnosed deaths for INRA, one for Crossed and none for Fauve females). In brief, the overall losses of Fauve, INRA and Crossed females were two, fourteen and four females, respectively. The high female survival (91%), together with an acceptable reproductive performance (7.5 newborn kits, kit survival during lactation of 80% and weaning weight of kits of 871 g) of Crossed females, indicates that this maternal cross combine the alleged rusticity of the Fauve-de-Bourgogne breed with the reproductive potential of INRA maternal line.

INTRODUCTION

Genetic selection of purebred rabbit lines tends to erode the genetic diversity. First because most rabbit selection programs used a limited genetic background as the base population (New Zealand White for maternal lines and California for paternal lines), and secondly because selection herds are managed in closed population with no introduction of foreign individuals (Garreau et al., 2015a). The limitation of such strategy is a reduction of the effective population size, the accumulation of inbreeding and a degraded fitness in the selection herds [START_REF] Pekkala | The effect of inbreeding rate on fitness, inbreeding depression and heterosis over a range of inbreeding coefficients[END_REF]. Intensive farming systems assembles particular conditions -like high stock-densities and frequently use of antibiotics -favouring the dissemination and resistance of pathogens [START_REF] Pulkkinen | Intensive fish farming and the evolution of pathogen virulence: the case of columnaris disease in Finland[END_REF]. According to [START_REF] Hamilton | Sexual reproduction as an adaptation to resist parasites (a review)[END_REF], to resist numerous pathogens, hosts species needs to preserve an array of genotypes, and one strategy to boost the genetic diversity is to cross inbreed-divergent genotypes together. For the rabbit, crossbreds are already present in every commercial farm, being the product of crossing two maternal lines mainly selected for prolificacy (Garreau et al., 2015a). However, as the inclusion of functional trait and the establishment of robust lines is recent in the history of rabbit selection (Sánchez et al. 2008;[START_REF] Blasco | Selection for environmental variance of litter size in rabbits[END_REF], it could be argued that the available purebred lines are genetically close and too similar to produce a 'true' outbred individual joining functional and productive traits. In this sense, the cross between genotypes with different origin and selection history may boost the genetic diversity of rabbit populations. One possibility is to cross high selected lines with local breeds that have been kept and selected for different purposes. The Fauve-de-Bourgogne is a medium size breed, historically used as a backyard animal for meat, now kept and selected for beauty contests by amateur farmers. Fauve-de-Bourgogne farmers report few losses and characterizes this breed as being rustic. The reproductive potential of this breed is very low (fertility rate below 50% and 4.5 newborn kits), but the average daily gain (31.2 g/day) and the survival of growing kits (83%) in an antibiotic 'free' environment are acceptable (Savietto et al., 2020). The alleged rusticity of Fauve-de-Bourgogne animals (farmer's observations), and the acceptable growth rate and survival of growing kits make this local breed a potential candidate to be cross with high prolific lines to produce outbred maternal females joining functional and productive traits.

Here we evaluate the reproductive performances and the survival of a maternal rabbit cross between Fauve-de-Bourgogne males and the INRA-1777 line compared to the purebreds giving origin to this cross.

MATERIALS AND METHODS

The French Committee no. 115 for Ethics, Science and Animal Health approved all procedures.

Purebreds and Crossbred Animals

Fauve-de-Bourgogne (hereinafter called Fauve) female rabbits (n = 23) were daughters of 9 males and 13 females gathered from five different farms located in the French regions of Alsace, Lorraine and Bourgogne. Fourteen Fauve female rabbits were born on January 2019 and 9 on March 2019. INRA-1777 (called INRA) female rabbits (n = 48) were all born on March 2019 from 15 males and 32 females rabbits of the generation 53 of selection for prolificacy. The crossbred Fauve × INRA female rabbits (called Crossed; n = 43) were all born on March 2019 from the natural matting of six Fauve male and eight INRA female rabbits.

Housing, Diets and Reproductive Rhythm

Each female rabbits lived indoors in wire cages (W×H×L: 46×60×90 cm) equipped with a plastic mattress, a drinker a food dispenser and a non-treated pine-tree stick (W×H×L: 3×3×10 cm), replaced when needed and used for environmental enrichment purpose. Daylight was set to 16/8h light/dark. The indoor temperature could vary between 15 and 28 °C. All females received from weaning (35 days) to 126 days old a commercial diet for growing rabbits containing 11.3 MJ of DE per kg of DM and 15.4% of CP on a DM basis. From 126 old onwards they received an experimental diet containing 12.4 MJ per kg of DM and 17.0% of CP on a DM basis. The 14 Fauve female rabbits born on January 2019 received the commercial diet for growing rabbits from 70 up to 168 days old and then the experimental maternal diet onwards. Diets contained no antibiotics. Females were first artificially inseminated (AI) at age 145 days (14 older Fauve female rabbits 42 days later), being re-inseminated 11 days post-partum date (reproductive cycle of 42 days). Fauve and Crossed female rabbits were AI with fresh semen of Fauve males and INRA females with fresh semen of INRA males. The day after birth and for each AI attempt, females producing milk (visually checked) adopted the alive kits to a number close to the average number of newborn kits of each genotype. We performed cross fostering among genotypes on half of the litters from INRA and Crossed female rabbits. Fauve kits were in all cases adopted by Fauve female rabbits.

Measurements

We weighted females at 1st AI insemination, checked their fertility rate at each AI attempt and assessed their survival rate between 1st and 3rd AI attempt. At each parturition date, we counted the number of newborn and stillbirth kits and weighted the newborn kits. We counted and weighted the alive kits just before and after cross fostering (the day after birth), at 18 days after birth and at weaning. We then calculated the survival during lactation based on the number of nursed kits.

Statistical Analysis

We used the R-software, version 3.6.0 (R core Team, 2019) to analyse our data. We used a logistic regression model to analyse binomial data (fertility rate, female rabbit survival at 3rd AI and kits survival during lactation) and a linear model to analyse continuous data (female live weight at 1st AI, the number and weight of litters and kits). All models included the genotype, the AI attempt and their interaction as dependent variables, except for female live weight at 1st AI and survival between 1st and 3rd AI. For these two variables, only genotype entered the statistical model as a dependent variable. Model for live weight at 1st AI also included age as a co-variable to take into account the age differences of Fauve females.

RESULTS AND DISCUSSION

The population size decreased with time. resistant to streptomycin and amoxicillin. Losses of the INRA female rabbits between the 2nd and the 3rd AI attempt, involved two additional deaths for no specific reason and the culling of four animals (three with a poor general state and one with an internal abscess). Losses of the Crossed females involved two deaths. The low prolificacy of all genotypes at parturitions of the 1st AI attempt indicates the presence of Pasteurella spp between the 1st AI attempt and the 1st parturition. Although it is difficult to precise the number of infected animals that actually tolerated/resisted this pathogen, Crossed females seem to better respond to the Pasteurella spp than INRA females, an heritage of its Fauve ancestors and the heterotic effect (survival rate in the same conditions of 91.3%).

Main reproductive traits are in [START_REF] Bolet | Evaluation of the reproductive performance of eight rabbit breeds on experimental farms[END_REF]. For the INRA line the results are below those of the first 12 generations of selection (10.4 newborn kits; Garreau et al., 2015b), but are close to the values observed recently at our selection nucleus (on average of 8.4 newborn kits from 5968 litters produced between January 1st, 2015 and January 1st, 2020). Number of weaned kits is a variable directly related to the number of kits raised after cross fostering, an input linked to our cross fostering practice, and the maternal ability of females affecting kits survival. On average (all cross fostering from AI attempts 1 to 3), Fauve, INRA and Crossed females raised litter of 5.7, 9.2 and 7.3 kits, having a kit survival during lactation of 71, 76 and 80%. As a result, Fauve, INRA and Crossed females weaned 4.2, 7.1 and 5.9 kits in the whole period. The low relative maternal ability of Fauve females (weight of Fauve kits at 18 days was 15% inferior respect to INRA kits) may explain the high nest mortality of Fauve litters compared to the other genotypes. Live weight of kits at birth, mid-lactation (18 days) and at weaning (35 days) reflects the female capacity to transfer the obtained resources to its progeny. Except for kits from the litters of the 1st AI attempt (no differences between genotypes), Fauve females gave birth to lighter kits (54.1 g on average) than INRA and Crossed females (65.3 and 61.5 g,respectively). At mid-lactation, Crossed kits were heavier than INRA and Fauve kits (335,311 and 263g,respectively;P<0.05), except for kits from the 1st AI attempt (INRA kits were the heaviest ones: 355 g on average; P<0.05). INRA kits from the 1st AI attempt were heavier than both Fauve and Crossed kits (1040, 873 and 693 g, respectively; P<0.05). The trend changed in litters produced from the 2nd and the 3rd AI attempt. INRA and Crossed kits had a similar weaning weight, while Fauve kits were always lighter.

CONCLUSIONS

The maternal cross between Fauve-de-Bourgogne males and INRA-1777 females has a litter size of 1.6 newborn kits less than the current generation of the INRA-1777 line. They have, however, a similar fertility rate and kit survival during lactation. Crossed females appear to resist/tolerate better the presence of Pasteurella spp, having an adult survival rates at 3rd AI attempt 20 points of percentage higher than females of the INRA-1777 line. This genotype appear to combine the benefits of both ancestors, the alleged rusticity of Fauve-de-Bourgogne breed and the reproductive performance of INRA-1777 maternal line.

Table 1 :

 1 At 1st AI attempt Fauve, INRA and Crossed females were 23, 48 and 43. At 2nd AI attempt, the figures were 21, 45 and 42 animals, and at 3rd AI attempt Fauve were 21, INRA 34 and Crossed females 39. Female survival was 20 points of percentage higher for Crossed (P=0.061) respect to INRA females (Table 1). Most losses occurred around second parturition, when we found four INRA and one Crossed individuals death. The necropsy revealed the presence of Pasteurella spp Survival at third parturition and main reproductive traits of Fauve, INRA and Crossed (Fauve × INRA) females at/from the first, second and third artificial insemination (AI) attempts. Means (SE).

					abc Means of a trait at a given AI
					attempt followed by distinct letters
					differs at P<0.05. *Fauve: Fauve-
					de-Bourgogne; INRA: the INRA-
	Traits	Fauve	Genotypes* INRA	Crossed	1777 maternal line; Crossed: Crossbreed between Fauve-de-Bourgogne males and INRA-1777
	Female survival at third AI (%)	91.3 (5.9)	70.8 (6.6)	90.7 (4.4)	females.
	Fertility rate at (%)
	First AI attempt	43.5 a (10.3)	79.2 b (5.9)	65.1 ab (7.3)
	Second AI attempt	61.9 (10.6)	60.0 (7.3)	71.4 (7.0)
	Third AI attempt	23.8 a (9.3)	61.8 b (8.3)	69.2 b (7.4)
	Overall	42.2 a (6.5)	67.7 b (4.3)	68.6 b (4.2)
	Newborn kits from (n)
	First AI attempt	2.6 a (1.2)	6.3 b (0.6)	6.0 b (0.7)
	Second AI attempt	5.3 a (1.0)	9.6 b (0.7)	8.2 ab (0.7)
	Third AI attempt	6.3 a (1.6)	10.7 b (0.8)	7.3 a (0.7)
	Overall	4.7 a (0.8)	8.8 c (0.4)	7.2 b (0.4)
	Weaned kits from (n)
	First AI attempt	3.7 (1.2)	5.1 (0.4)	5.1 (0.4)
	Second AI attempt	4.6 a (1.2)	8.4 b (0.5)	7.1 b (0.5)
	Third AI attempt	4.3 a (1.2)	7.9 b (0.6)	5.6 ab (0.5)
	Overall	4.2 a (0.6)	7.1 c (0.3)	5.9 b (0.3)
	Kits survival 0 to 35 days (%)
	First AI attempt	71.4 (9.9)	81.7 (2.6)	85.5 (2.7)
	Second AI attempt	73.0 (6.6)	72.7 (2.8)	79.3 (2.6)
	Third AI attempt	68.0 (9.3)	70.6 (3.2)	73.2 (3.3)
	Overall	70.9 (4.9)	75.4 (1.7)	79.8 (1.7)
	Weight of newborn kits from (g)
	First AI attempt	60.9 (3.6)	69.8 (2.2)	63.7 (2.1)
	Second AI attempt	52.3 a (3.1)	63.1 b (2.2)	58.5 ab (2.1)
	Third AI attempt	49.0 a (4.9)	63.0 b (2.4)	62.4 b (2.1)
	Overall	54.1 a (2.3)	65.3 b (1.2)	61.5 b (1.2)
	Weight of kits at 18 days from (g)
	First AI attempt	262.7 a (23.2)	354.4 c (7.6)	322.8 b (8.8)
	Second AI attempt	279.9 ab (15.4) 280.6 a (10.4)	319.8 b (9.1)
	Third AI attempt	246.5 a (26.7)	280.6 a (11.2)	362.5 b (9.7)
	Overall	263.1 a (12.9)	310.9 b (5.7)	335.0 c (5.3)
	Weight of weaned kits from (n)
	First AI attempt	692.5 a (40.5) 1039.5 c (13.6) 872.6 b (14.3)
	Second AI attempt	738.1 a (31.2)	843.4 b (14.3) 796.0 ab (13.4)
	Third AI attempt	634.4 a (47.3)	913.1 b (15.5)	943.1 b (14.8)
	Overall	688.3 a (28.3)	932.0 b (11.0)	870.6 c (10.9)

Table 1 .

 1 At 1st AI attempt, Fauve were lighter than INRA and Crossed females (3877 a , 4108 ab and 4209 b g; P<0.05). Fertility of Fauve females at 1st and at 3rd AI attempts was Crossed females produced 6.0, 8.2 and 7.3 newborn kits at 1st, 2nd and 3rd AI attempts, with an overall result of 7.2 kits, a value between Fauve and INRA females. Results for Fauve are in line to that reported by

	low respect to INRA and
	Crossed females. The overall
	figures for Fauve, INRA and
	Crossed females were 42, 68
	and 69%, respectively. Fauve
	females also produced a
	reduced number of newborn
	kits than INRA and Crossed. At
	1st, 2nd and 3rd AI attempts
	Fauve produced 2.6, 5.3, and
	6.3 newborn kits while INRA
	produced 6.3, 9.6 and 10.7 kits,
	resulting in and overall result of
	4.7 and 8.8 kits for Fauve and
	INRA	females.

ACKNOWLEDGEMENTS

Authors acknowledges the Department PHASE of INRAE institute for the economic support.