Caroline Massot
email: massot.caroline@ghicl.net

Cécile Donze

Marc-Alexandre Guyot

Sebastien Leteneur

Sébastien Leteneur

Dr C Massot Orcid

Low back pain in patients with multiple sclerosis: A systematic review and the prevalence in a French multiple sclerosis population

Keywords: Multiple Sclerosis, Low back pain, Pain, Musculoskeletal pain

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

Introduction

With a prevalence ranging from 5% to 22% [START_REF] Dagenais | A systematic review of low back pain cost of illness studies in the United States and internationally[END_REF], spinal pain is a common cause of pain in the general population and is one of the largest public health problems. Low back pain (LBP) is the most common cause of spinal pain and its occurrence was approximately 84% of the population during their lifetime. The types of LBP are defined by the duration of pain: acute, subacute and chronic. Acute LBP is pain lasting less than 4 weeks, and subacute pain is pain lasting 4 to 12 weeks. Chronic low back pain (CLBP) is defined as continuous pain in the low back region that persists for more than 12 weeks without significant improvement in the intensity of pain. Five to 10% of LBP patients develop CLBP [START_REF] Meucci | Prevalence of chronic low back pain: systematic review[END_REF]. The prevalence of CLBP varies from 4,2% to 23% in the general population [START_REF] Meucci | Prevalence of chronic low back pain: systematic review[END_REF]. CLBP is a leading cause of activity limitations and work absence, leading to a major cost burden for the society [START_REF] Golob | Low back pain[END_REF][START_REF] Depont | Medical and non-medical direct costs of chronic low back pain in patients consulting primary care physicians in France[END_REF]. The causes of CLBP are multidimensional and complex. LBP is generally due to spinal instability [START_REF] Ghamkhar | Trunk muscles activation pattern during walking in subjects with and without chronic low back pain: a systematic review[END_REF].

Then, pain induces adaptative but altered muscle activation patterns [START_REF] Ghamkhar | Trunk muscles activation pattern during walking in subjects with and without chronic low back pain: a systematic review[END_REF], altered lumbosacral proprioceptive acuity [START_REF] Radebold | Impaired postural control of the lumbar spine is associated with delayed muscle response times in patients with chronic idiopathic low back pain[END_REF], and restricted range of motion [START_REF] Laird | Comparing lumbo-pelvic kinematics in people with and without back pain: a systematic review and meta-analysis[END_REF], which increase the risk of chronic or recurrent LBP [START_REF] Panjabi | The stabilizing system of the spine. Part I. Function, dysfunction, adaptation, and enhancement[END_REF][START_REF] Panjabi | The stabilizing system of the spine. Part II. Neutral zone and instability hypothesis[END_REF]. These modifications in the neuromuscular activity and biomechanics of the spine affect an individual's postural stability and gait. For example, CLBP alone can induce a forward trunk inclination [START_REF] Bourigua | Chronic low back pain sufferers exhibit freezing-like behaviors when asked to move their trunk as fast as possible[END_REF], which alters the kinetic and kinematic characteristics of an individual's gait and gait initiation pattern [START_REF] Leteneur | Trunk's natural inclination influences stance limb kinetics, but not body kinematics, during gait initiation in able men[END_REF]. During a dynamic task or gait, CLBP patients reduce their trunk velocity and range of motion to decrease the forces applied on the lower back [START_REF] Bourigua | Chronic low back pain sufferers exhibit freezing-like behaviors when asked to move their trunk as fast as possible[END_REF]. These changes in posture decrease an individual's adaptability during movement, leading to freezing-like behavior [START_REF] Bourigua | Chronic low back pain sufferers exhibit freezing-like behaviors when asked to move their trunk as fast as possible[END_REF]. In addition, during a dynamic challenging task, CLBP patients do not adapt their base of support [START_REF] Bourigua | Chronic low back pain sufferers exhibit freezing-like behaviors when asked to move their trunk as fast as possible[END_REF]. These changes in adaptative strategies increase an individual's risk of physical deconditioning.

Multiple sclerosis (MS) is a chronic inflammatory disease of the central nervous system that induces motor and sensory deficits, visual impairments and gait and postural balance disorders. Pain is a common symptom in patients with MS, considering 29 to 86% of MS patients report pain [START_REF] O'connor | Pain associated with multiple sclerosis: Systematic review and proposed classification[END_REF] as one of the most distressing symptoms [START_REF] Kalia | Severity of chronic pain and its relationship to quality of life in multiple sclerosis[END_REF]. Several authors have proposed pain classification systems for individuals with MS [START_REF] O'connor | Pain associated with multiple sclerosis: Systematic review and proposed classification[END_REF][START_REF] Solaro | Pain and multiple sclerosis: pathophysiology and treatment[END_REF]. Even though neuropathic pain is the most commonly reported type of pain in individuals with MS [START_REF] O'connor | Pain associated with multiple sclerosis: Systematic review and proposed classification[END_REF], the presence of the MS disease predisposes patients to develop nociceptive or musculoskeletal pain, especially back pain [START_REF] O'connor | Pain associated with multiple sclerosis: systematic review and proposed classification[END_REF], which are less often reported. The prevalence of back pain and its predisposing factors in the MS population are not well known [START_REF] Massot | Back Pain and Musculoskeletal Disorders in Multiple Sclerosis[END_REF]. Nevertheless, muscle trunk weakness, somatosensory disorders, asymmetric posture and gait impairments due to MS can induce back pain [START_REF] Massot | Back Pain and Musculoskeletal Disorders in Multiple Sclerosis[END_REF]. In addition to the presence of MS, the presence of LBP can exacerbate the functional difficulties of these patients, such as postural instability and gait impairments.

Independent of the assessment of MS-specific impairments, the functional evaluation of LBP involves several clinical tests, but there is no gold standard [START_REF] Salvioli | Movement Control Impairment and Low Back Pain: State of the Art of Diagnostic Framing[END_REF]. The use of motion capture systems allows the characterization of balance, gait and movement disorders in LBP patients, but there is limited and inconsistent evidence of kinematic/kinetic measures that can be used clinically [START_REF] Papi | Is there evidence to use kinematic/kinetic measures clinically in low back pain patients? A systematic review[END_REF]. For example, Delpierre et al. proposed an index based on 10 discrete variables extracted from the range of motion, but it is a complex and time-consuming assessment for clinical practice [START_REF] Delpierre | An index to quantify deviations from normal trunk mobility: Clinical correlation and initial test of validity[END_REF]. Alternatively, in daily practice, clinical questionnaires are often used to assess the impact of LBP, but they do not reflect the real capacity of patients in daily life [START_REF] Lin | Relationship between physical activity and disability in low back pain: a systematic review and metaanalysis[END_REF].

To the best of our knowledge, the functional impact of LBP on daily life activities in the MS population has rarely been evaluated.

The first objective of this study was to conduct a systematic review of the literature to determine the impact of LBP on the functional outcomes of MS patients. To complete this review, the second objective was to record the types of LBP in a large sample of MS patients by administering a French national survey to examine the functional impact and specific care of LBP in MS patients.

Methods

Systematic review

The systematic review was performed in accordance with the PRISMA guidelines (http://www.prisma-statement.org.org/). A systematic search was conducted during December 2019 in the following databases: PubMed, Web of Science and the Cochrane Library. The following keywords and MeSH headings were used for the search: Multiple Sclerosis AND back pain OR musculoskeletal pain OR lumbar pain. To supplement the literature search, the reference lists of the selected articles were searched, and Google Scholar searches were conducted. Only articles in the English language were analyzed. There were no limitations regarding the year of publication.

The population of interest was MS patients with a definite diagnosis of MS or clinical isolated syndrome in addition to back pain, especially LBP. The outcomes of interest included the prevalence of back pain and LBP, severity of pain, type of pain, duration of pain and impact on quality of life or activities in daily life.

The eligibility assessment was performed by screening the titles and abstracts of the articles (Figure 1). Due to methodological heterogeneity in the studies, there was a lack of comparative data, and no meta-analyses could be performed. The data are reported descriptively. The following data were extracted: the study design, population characteristics, prevalence of back pain and LBP, severity of pain, pain score and impact on daily activities or quality of life (Tables 12).

The Newcastle-Ottawa Scale for case control studies was used to assess the quality of the case control studies [START_REF] Al-Smadi | A pilot investigation of the hypoalgesic effects of transcutaneous electrical nerve stimulation upon low back pain in people with multiple sclerosis[END_REF][START_REF] Warke | Efficacy of transcutaneous electrical nerve stimulation (tens) for chronic low-back pain in a multiple sclerosis population: a randomized, placebo-controlled clinical trial[END_REF]. Each study could obtain a maximum of nine stars: 4 stars for the selection of the study groups, 2 stars for the comparability of the groups, and 3 stars for the exposure or outcome of interest. Two reviewers used the Newcastle-Ottawa Scale checklist to rate the methodological quality of the studies. Any disagreements in the ratings were resolved through discussions.

Survey

The survey assessed the prevalence of LBP in a French MS population and identified limitations in daily living. MS patients were recruited online with social media applications, such as Carenity (Paris, France), and PARC SeP (Plateforme d'Accompagnement de Ressources et de Coordination pour la Sclérose en Plaques et la maladie de Parkinson; Lille, France) which is a regional care network for MS and Parkinson patients from January to May 2018. The recruitment with the PARC SeP was done in thanks to their Facebook homepage, with a link to the online questionnaire. There was no other approach to recruit patients. The inclusion criterion was MS patients older than 18 years old. The project was reviewed and approved by the local ethical committee.

The questionnaire took approximately 10 minutes to complete (Appendix 1). All data were self-reported. The first part of the questionnaire concerned the general assessment of the patient and his or her case of MS: sociodemographic data; information on the history of MS, rheumatologic disease or spine surgery; and whether the individual used a walking aid or wheelchair were recorded. Subsequently, if patients reported LBP during this first part of the questionnaire, supplementary questions about LBP were asked to distinguish current LBP from a history of LBP.

-For patients with current LBP, the questions concerned the duration of LBP (i.e., < 4 weeks, 4-12 weeks and > 12 weeks), treatments (i.e., physiotherapy, medical treatment, or none), type of emergence (i.e., suddenly or progressively), radiculopathy (i.e., if pain spread into the lower limbs), and pain occurrences (i.e., at rest, during sitting posture, standing position, at walk or at rest and during specific postures).

Neuropathic pain was evaluated with the "Douleur Neuropathique 4" questionnaire (DN4). DN4 is the French questionnaire that has been validated to evaluate the symptoms of neuropathic pain in patients with LBP and MS [START_REF] Attal | The neuropathic components of chronic low back pain: a prospective multicenter study using the DN4 Questionnaire[END_REF][START_REF] Solaro | The prevalence of pain in multiple sclerosis: a multicenter cross-sectional study[END_REF]. The DN4 result is positive if the score is greater than or equal to 4, indicating the presence of neuropathic pain. A visual analog scale was used to assess the intensity of all types of pain (from 0 to 10). The EIFEL (Echelle d'Incapacité Fonctionnelle pour l'Evaluation des Lombalgies) questionnaire is the French adaptation of the Roland and Morris Disability Questionnaire. The EIFEL questionnaire was used to evaluate the influence of LBP, independently of the aspects of MS, on the patients' quality of life and their limitations on daily activities [START_REF] Coste | French validation of a disability rating scale for the evaluation of low back pain (EIFEL questionnaire)[END_REF]. The EIFEL questionnaire was self-administered.

-The patients with a history of LBP had specific questions on the duration of LBP, frequency of LBP episodes, and whether they received treatments.

The population was divided into the following 3 groups according their responses to the first part of the survey: no LBP MS patients, current LBP MS patients and history of LBP MS patients.

All data analyses were performed using R software (version 3.4.2). For each group, descriptive statistics were used to evaluate means, medians, and interquartile ranges for continuous variables and frequencies for noncontinuous measures. For group comparisons, the Chi-squared test with Fisher's test correction and the Kruskal-Wallis test or Mann-Whitney test were used when the application compliance condition was not respected. The significance level was adjusted using the Bonferroni method where needed (p<0.016) for the comparison of the 3 groups. Then, for all the tests, the significance level was set to be p<0.05.

Results

Systematic review

After a systematic search of the databases, 924 articles were retrieved. Fourteen of the articles were included: 12 articles reported back pain in MS patients (transversal studies), and 2 articles evaluated the effect of transcutaneous electrical nerve stimulation on LBP in MS patients (case control studies). Figure 1 shows the process of study inclusion. The characteristics of the population, methods of pain evaluation, prevalence of back pain and LBP and the impact on daily living for the 12 transversal studies are reported in Table 1, and those for the 2 case control studies are reported in Table 2.

Characteristics of the MS population

These studies evaluated 5780 patients, including 4001 females and 1356 males. Three studies did not report the sex of the MS patients [START_REF] Al-Smadi | A pilot investigation of the hypoalgesic effects of transcutaneous electrical nerve stimulation upon low back pain in people with multiple sclerosis[END_REF][START_REF] De | Pain syndromes in multiple sclerosis[END_REF][START_REF] Stenager | Acute and chronic pain syndromes in multiple sclerosis[END_REF]. A total of 3565 patients had remitting relapsing MS, 682 had secondary progressive MS, and 224 patients had primary progressive MS. Only one study evaluated LBP in patients with clinical isolated syndrome [START_REF] Seixas | Pain in portuguese patients with multiple sclerosis[END_REF]. Five studies did not report the form of MS [START_REF] Al-Smadi | A pilot investigation of the hypoalgesic effects of transcutaneous electrical nerve stimulation upon low back pain in people with multiple sclerosis[END_REF][START_REF] Warke | Efficacy of transcutaneous electrical nerve stimulation (tens) for chronic low-back pain in a multiple sclerosis population: a randomized, placebo-controlled clinical trial[END_REF][START_REF] De | Pain syndromes in multiple sclerosis[END_REF][START_REF] Shayestehazar | A Survey of Severity and Distribution of Musculoskeletal Pain in Multiple Sclerosis Patients; a Cross-Sectional Study[END_REF][START_REF] Marck | Prevalence of Comorbidities, Overweight and Obesity in an International Sample of People with Multiple Sclerosis and Associations with Modifiable Lifestyle Factors[END_REF]. The patients were between 18 and 80 years old. Three studies evaluated patients with a low EDSS score (EDSS<4) [START_REF] Solaro | The prevalence of pain in multiple sclerosis: a multicenter cross-sectional study[END_REF][START_REF] Seixas | Pain in portuguese patients with multiple sclerosis[END_REF][START_REF] Boneschi | Lifetime and actual prevalence of pain and headache in multiple sclerosis[END_REF]. Two studies did not report the EDSS score of the MS patients [START_REF] De | Pain syndromes in multiple sclerosis[END_REF][START_REF] Shayestehazar | A Survey of Severity and Distribution of Musculoskeletal Pain in Multiple Sclerosis Patients; a Cross-Sectional Study[END_REF].

Evaluation of back pain

Most of the studies evaluated back pain with interviews [START_REF] Massot | Back Pain and Musculoskeletal Disorders in Multiple Sclerosis[END_REF][START_REF] Solaro | The prevalence of pain in multiple sclerosis: a multicenter cross-sectional study[END_REF][START_REF] De | Pain syndromes in multiple sclerosis[END_REF][START_REF] Stenager | Acute and chronic pain syndromes in multiple sclerosis[END_REF][START_REF] Seixas | Pain in portuguese patients with multiple sclerosis[END_REF][START_REF] Shayestehazar | A Survey of Severity and Distribution of Musculoskeletal Pain in Multiple Sclerosis Patients; a Cross-Sectional Study[END_REF][START_REF] Boneschi | Lifetime and actual prevalence of pain and headache in multiple sclerosis[END_REF][START_REF] Vidović | Pain syndromes in multiple sclerosis patients -patient experience at Lipik Special Hospital for Medical Rehabilitation[END_REF][START_REF] Svendsen | Pain in patients with multiple sclerosis: a population-based study[END_REF][START_REF] Kahraman | Frequency, type, distribution of pain and related factors in persons with multiple sclerosis[END_REF][START_REF] Łabuz-Roszak | Prevalence of pain in patients with multiple sclerosis and its association with anxiety, depressive symptoms and quality of life[END_REF], clinical exams [START_REF] Massot | Back Pain and Musculoskeletal Disorders in Multiple Sclerosis[END_REF][START_REF] Stenager | Acute and chronic pain syndromes in multiple sclerosis[END_REF][START_REF] Seixas | Pain in portuguese patients with multiple sclerosis[END_REF][START_REF] Vidović | Pain syndromes in multiple sclerosis patients -patient experience at Lipik Special Hospital for Medical Rehabilitation[END_REF][START_REF] Svendsen | Pain in patients with multiple sclerosis: a population-based study[END_REF] or an online survey [START_REF] Marck | Prevalence of Comorbidities, Overweight and Obesity in an International Sample of People with Multiple Sclerosis and Associations with Modifiable Lifestyle Factors[END_REF]. Back pain was evaluated by several different types of rating scales: a self-administered comorbidity questionnaire, a numeric rating scale, the Nordic Musculoskeletal Questionnaire, the painDETECT Questionnaire, the visual analog scale, the McGill pain questionnaire and the Brief Pain Inventory. When the quality of life of the MS patients was assessed, it was again assessed by several different scales: the healthrelated quality of life questionnaire, Multiple Sclerosis International Quality of Life, European Quality of Life-5 Dimensions. Only Marck et al. 2016 reported the specific impact of LBP on quality of life or on the ability to perform activities of daily living [START_REF] Marck | Prevalence of Comorbidities, Overweight and Obesity in an International Sample of People with Multiple Sclerosis and Associations with Modifiable Lifestyle Factors[END_REF].

Prevalence, risk factors, type of back pain and LBP, duration of pain in MS patients and impact on the ability to perform activities of daily living

The prevalence of back pain in MS patients varied from 8,6 to 50% [START_REF] Solaro | The prevalence of pain in multiple sclerosis: a multicenter cross-sectional study[END_REF][START_REF] De | Pain syndromes in multiple sclerosis[END_REF][START_REF] Stenager | Acute and chronic pain syndromes in multiple sclerosis[END_REF][START_REF] Seixas | Pain in portuguese patients with multiple sclerosis[END_REF][START_REF] Shayestehazar | A Survey of Severity and Distribution of Musculoskeletal Pain in Multiple Sclerosis Patients; a Cross-Sectional Study[END_REF][START_REF] Marck | Prevalence of Comorbidities, Overweight and Obesity in an International Sample of People with Multiple Sclerosis and Associations with Modifiable Lifestyle Factors[END_REF][START_REF] Boneschi | Lifetime and actual prevalence of pain and headache in multiple sclerosis[END_REF][START_REF] Vidović | Pain syndromes in multiple sclerosis patients -patient experience at Lipik Special Hospital for Medical Rehabilitation[END_REF][START_REF] Kahraman | Frequency, type, distribution of pain and related factors in persons with multiple sclerosis[END_REF][START_REF] Łabuz-Roszak | Prevalence of pain in patients with multiple sclerosis and its association with anxiety, depressive symptoms and quality of life[END_REF]. Back pain was the most common cause of comorbidities and the most limiting factor for daily activities in MS patients [START_REF] Marck | Prevalence of Comorbidities, Overweight and Obesity in an International Sample of People with Multiple Sclerosis and Associations with Modifiable Lifestyle Factors[END_REF]. The prevalence of LBP varied from 41,6 to 52,4% [START_REF] Massot | Back Pain and Musculoskeletal Disorders in Multiple Sclerosis[END_REF][START_REF] Vidović | Pain syndromes in multiple sclerosis patients -patient experience at Lipik Special Hospital for Medical Rehabilitation[END_REF][START_REF] Kahraman | Frequency, type, distribution of pain and related factors in persons with multiple sclerosis[END_REF].

Few studies have suggested that back pain and LBP are common in patients with an early stage of the disease [START_REF] Marck | Prevalence of Comorbidities, Overweight and Obesity in an International Sample of People with Multiple Sclerosis and Associations with Modifiable Lifestyle Factors[END_REF][START_REF] Kahraman | Frequency, type, distribution of pain and related factors in persons with multiple sclerosis[END_REF]. Massot et al. suggested that female MS patients with progressive form, visual disorders or a high EDSS score are at risk of developing LBP. Only three studies assessed back pain intensity, and they reported the intensity to be moderate [START_REF] Shayestehazar | A Survey of Severity and Distribution of Musculoskeletal Pain in Multiple Sclerosis Patients; a Cross-Sectional Study[END_REF][START_REF] Vidović | Pain syndromes in multiple sclerosis patients -patient experience at Lipik Special Hospital for Medical Rehabilitation[END_REF][START_REF] Łabuz-Roszak | Prevalence of pain in patients with multiple sclerosis and its association with anxiety, depressive symptoms and quality of life[END_REF]. No studies evaluated the intensity or duration of pain in MS patients with LBP.

Treatment of LBP

Only transcutaneous electrical nerve stimulation treatment was evaluated [START_REF] Al-Smadi | A pilot investigation of the hypoalgesic effects of transcutaneous electrical nerve stimulation upon low back pain in people with multiple sclerosis[END_REF][START_REF] Warke | Efficacy of transcutaneous electrical nerve stimulation (tens) for chronic low-back pain in a multiple sclerosis population: a randomized, placebo-controlled clinical trial[END_REF]. This technique did not lead to significant improvement in the LBP of the MS patients (Table 2).

Quality assessment of studies

According to the Ottawa scale checklist, the 2 case-control studies were of moderate quality.

A detailed overview of the quality of the studies is provided in Table 3. The total score was 6 stars out of a possible nine stars.

Results of the survey

Sociodemographic results

Two hundred thirty-seven MS patients participated in the study, and 234 questionnaires were completed (return rate of approximately 99%). The sociodemographic and medical data are shown in Table 4. One hundred and seventy-eight patients presented with LBP (i.e., a prevalence of approximately 76.1%), 41 had a history of LBP (17.5%), and 15 patients did not report having LBP (6.4%).

Patients with current LBP

Regarding the duration, 69.6% of patients had chronic LBP, 19.9% suffered from subacute LBP and 17.4% had acute LBP. No difference in the sociodemographic factors was found between CLBP patients and subacute or acute LBP patients.

A total of 74.7% of patients reported that LBP began progressively, 11.2% disclaimed that the pain began suddenly, whereas 14% of patients did not know when the pain started. A total of 45.5% of patients reported a trigger factor for LBP, 33.7% reported no trigger factor and 20.8% did not know whether LBP began with a trigger factor.

Only 73.6% of patients reported having LBP to a doctor, and 63.3% of these patients had complementary exams. CLBP patients more often reported to their doctor than patients with acute LBP (p=0.002), and they also had more complementary exams than patients with subacute LBP (p=0.006) or with acute LBP (p=0.028). To treat their LBP, 52.7% of patients underwent medical treatments, 72.5% underwent physiotherapy treatment and 28.2% underwent osteopathy treatment. No difference was found between groups treated with physiotherapy.

The pain evaluation results are presented in Table 5. Regarding the type of pain, the intensity of pain in all MS patients with LBP (independent of the duration) was not different between patients with neuropathic pain and those with nonneuropathic pain. For all MS patients with LBP, a total of 48.3% of patients reported pain spreading into the lower limb. For these patients, 24.4% had neuropathic pain with a positive DN4 result. For 52.3% of patients, the pain occurred only during physical activity, such as walking or transfer to a wheelchair. A total of 32.7% of patients reported that pain occurred both during physical activity and at rest, whereas 14,9% declared that the pain appeared at rest only. Regarding the duration of LBP, the pain intensity was not significantly different among the 3 LBP groups (acute, subacute or CLBP). Particularly for the patients with the CLBP who were the most represented patients, patients with a positive DN4 result did not have more severe pain than those without a positive DN4 result. Compared to the subacute and acute LBP patients, CLBP patients presented an increased intensity of pain during physical activity (p<0.001) and during the day (p<0.001).

The median EIFEL score was 5 ± 4,7 for the MS patients with LBP. Patients with CLBP had more limitations of activities in daily life than patients with acute LBP (p=0.03). LBP led to absence from work in 31% of MS patients, and there was no difference between the CLBP patients and patients with subacute or acute LBP in the rate of absence from work.

Independent of the duration of LBP, 36,5% of patients used one or more walking aids for locomotion: 30.3% used a stick, 18% used a manual wheelchair, 8.4% used an electric wheelchair and 0.6% a walker.

Patients with a history of LBP.

Eighty-five percent of patients with a history of LBP claimed having more than one episode of LBP during the last year. A total of 65.8% of these patients had a consultation with a doctor for LBP, and 63% of them had complementary exams. Regarding the treatment, 43,9% of patients underwent physiotherapy treatment, 36,6% were administered drugs, and 24,4% underwent osteopathy treatment.

Discussion

This article is the first to review the literature and report the impacts of LBP on functional outcomes in MS patients. Finally, to address the lack of information in the literature, the sociodemographic data, characteristics, medical care and limitations in daily life activities in the French MS population were collected from the national survey. In MS patients, the prevalence of back pain and LBP varied from 8.6 to 50% [START_REF] Marck | Prevalence of Comorbidities, Overweight and Obesity in an International Sample of People with Multiple Sclerosis and Associations with Modifiable Lifestyle Factors[END_REF][START_REF] Boneschi | Lifetime and actual prevalence of pain and headache in multiple sclerosis[END_REF][START_REF] Łabuz-Roszak | Prevalence of pain in patients with multiple sclerosis and its association with anxiety, depressive symptoms and quality of life[END_REF] and from 41.6 to 52.4% [START_REF] Massot | Back Pain and Musculoskeletal Disorders in Multiple Sclerosis[END_REF][START_REF] Vidović | Pain syndromes in multiple sclerosis patients -patient experience at Lipik Special Hospital for Medical Rehabilitation[END_REF], respectively. LBP is present even in patients with an EDSS score of < 4 [START_REF] Solaro | The prevalence of pain in multiple sclerosis: a multicenter cross-sectional study[END_REF][START_REF] Seixas | Pain in portuguese patients with multiple sclerosis[END_REF][START_REF] Boneschi | Lifetime and actual prevalence of pain and headache in multiple sclerosis[END_REF], and its prevalence is at least twice as high as that in the general population [START_REF] Meucci | Prevalence of chronic low back pain: systematic review[END_REF].

The present survey reports a higher prevalence (76.1%), with 17.5% of patients suffering from recurrent episodes of LBP. This difference may be explained in part by the method of recruiting MS patients for the survey. Due to their own interest in the study, patients concerned about LBP or having had a history of LBP might be over-represented in this study.

Although LBP is the leading cause of comorbidities and limitations in activities in daily life in MS patients, the etiologies, history, types, severities and treatments of LBP are only rarely evaluated [START_REF] Shayestehazar | A Survey of Severity and Distribution of Musculoskeletal Pain in Multiple Sclerosis Patients; a Cross-Sectional Study[END_REF][START_REF] Marck | Prevalence of Comorbidities, Overweight and Obesity in an International Sample of People with Multiple Sclerosis and Associations with Modifiable Lifestyle Factors[END_REF]. No specific studies on back pain in MS patients were found in the systematic review, and LBP was only described in studies about pain in MS patients.

Although several classifications of pain have been proposed, neuropathic and musculoskeletal pains remain the most commonly evaluated types of pain. This dichotomic evaluation appears to be insufficiently relevant for LBP in MS patients and cannot explain the cause of LBP. Indeed, in our survey, 32.7% of MS patients with LBP presented with both neuropathic and nociceptive pain, whereas 24.4% of patients had only neuropathic pain with a positive DN4 result. This high prevalence of neuropathic pain in LBP patients is consistent with the high prevalence of neuropathic pain and low back lesions in the MS population [START_REF] O'connor | Pain associated with multiple sclerosis: Systematic review and proposed classification[END_REF][START_REF] Baron | Neuropathic low back pain in clinical practice[END_REF].

Moreover, common causes of LBP, such as degenerated discs, the compression of nerve roots or inflammation, can also induce neuropathic pain.

The physiopathology of LBP related to MS is not known. Several risk factors have been suggested in a few studies [START_REF] Massot | Back Pain and Musculoskeletal Disorders in Multiple Sclerosis[END_REF][START_REF] Shayestehazar | A Survey of Severity and Distribution of Musculoskeletal Pain in Multiple Sclerosis Patients; a Cross-Sectional Study[END_REF], but none of them have been assessed. The authors suggested that lower limb weakness, spasticity, somatosensory disorders, asymmetric posture and walking impairments can induce LBP [START_REF] O'connor | Pain associated with multiple sclerosis: Systematic review and proposed classification[END_REF][START_REF] Solaro | The prevalence of pain in multiple sclerosis: a multicenter cross-sectional study[END_REF][START_REF] Brola | Symptomatology and pathogenesis of different types of pain in multiple sclerosis[END_REF]. Nevertheless, these factors could not explain the high prevalence of LBP observed at the early stage of MS. However, the authors suggested that somatosensory disorders in MS patients induce altered balance control of the trunk, which can eventually lead to spine instability and LBP [START_REF] Huisinga | Coherence analysis of trunk and leg acceleration reveals altered postural sway strategy during standing in persons with multiple sclerosis[END_REF]. In addition, Ketelhut et al.

(2015) reported modifications in trunk muscle activity during walking in MS patients with a low EDSS score [START_REF] Ketelhut | Core muscle characteristics during walking of patients with multiple sclerosis[END_REF]. Lateral flexor, external, and internal obliques and the rectus abdominis were less activated on the most affected side during walking, suggesting an early weakness in trunk muscles leading to compensation strategies. This trunk muscle weakness worsened with the EDSS score and led to an increase in trunk sway during upright standing or during walking [START_REF] Huisinga | Coherence analysis of trunk and leg acceleration reveals altered postural sway strategy during standing in persons with multiple sclerosis[END_REF][START_REF] Corporaal | Balance control in multiple sclerosis: correlations of trunk sway during stance and gait tests with disease severity[END_REF]. This type of weakness can also affect the posture of the lumbar spine. Since lumbar lordosis plays an important role in shock attenuation during gait [START_REF] Castillo | Shock attenuation in the human lumbar spine during walking and running[END_REF], the presence of both posture and lumbar lordosis disorders in MS patients decrease these individuals' capacities to attenuate shock. In addition, from the early stage of MS, patients are less able to absorb repetitive impacts at foot contact during gait [START_REF] Lizama | The use of laboratory gait analysis for understanding gait deterioration in people with multiple sclerosis[END_REF], which can also worsen LBP.

In the general population, LBP, especially CLBP, reduces walking speed, stride length and coordination between trunk segments [START_REF] Lamoth | Effects of chronic low back pain on trunk coordination and back muscle activity during walking: changes in motor control[END_REF]. During gait, CLBP patients modify their pattern of trunk muscular activity and reduce the acceleration and movements of the trunk to decrease the forces applied to the lumbar spine and avoid painful sensations [START_REF] Bourigua | Chronic low back pain sufferers exhibit freezing-like behaviors when asked to move their trunk as fast as possible[END_REF][START_REF] Lamoth | Effects of chronic low back pain on trunk coordination and back muscle activity during walking: changes in motor control[END_REF]. These modifications in trunk coordination impair the ability of patients to deal with unexpected perturbations [START_REF] Lamoth | Effects of attention on the control of locomotion in individuals with chronic low back pain[END_REF]. Moreover, patients adopt a less adaptive posture, a freezing-like posture, during trunk movements [START_REF] Bourigua | Chronic low back pain sufferers exhibit freezing-like behaviors when asked to move their trunk as fast as possible[END_REF]. For MS patients, these specific modifications due to LBP add up to the MS symptoms and worsen these patients' general disability due to postural and gait disorders. The convergence between LBP and MS appears as a vicious circle since worsening of one's gait and the progression of postural disorders with the progression of the disease can increase the risk of LBP [START_REF] Bourigua | Chronic low back pain sufferers exhibit freezing-like behaviors when asked to move their trunk as fast as possible[END_REF].

Regarding the modifications that LBP can induce in addition to MS impairments, the early diagnosis and treatment of LBP appears to be important but challenging for the care of MS patients. Concerning the diagnosis, this systematic review showed that a specific clinical test or tool to evaluate LBP in MS is missing. Concerning the clinical care of LBP in MS patients, according to the survey results, most patients talked about LBP with their doctor if the pain persisted for more than 12 weeks (73,6%), and most of them received physiotherapy (72,5%) or medical treatments (52,7%). These results were consistent with the European guidelines for the management of LBP [START_REF] Henrotin | Recommandations européennes pour la prise en charge des patients souffrant de lombalgie aiguë et chronique[END_REF]. Nevertheless, the present literature review and the subsequent survey suggested that LBP associated with MS depends on physiopathological mechanisms that are different than those reported in the general population. A better understanding of the physiopathological mechanisms may allow us to propose a prevention strategy and specific care for LBP in MS patients, especially at the early stage of the disease. Moreover, this article highlights the importance of developing specific tools to evaluate LBP and its specific impact on MS.

Study limitations

Several limitations were identified in our review and survey. Due to the limited number of articles in the literature, there were insufficient data to perform a meta-analysis, and the interpretation of the results is also limited. Only papers in the English language were included. Regarding the survey, a questionnaire was used for the French MS population, and it was available online only. All data were self-reported, and it is therefore not possible to assess the accuracy or reliability of the data. In addition, since patients who filled out our questionnaire were interested in LBP, there may be an overestimation of the prevalence of LBP. The survey did not address questions about the onset of LBP, about the timing or order of the diagnoses of MS and LBP, or about the efficiency of LBP management.

Conclusion

The prevalence of LBP is 2 or 3 times higher in MS patients, even in those at an early stage of the disease, than in the general population. Mixed types of LBP (neuropathic and nociceptive) and LBP of moderate intensity are present in these patients, but the limitations in the ability to perform activities of daily living are significant. Altered muscle activation patterns and somatosensory deficits in the trunk can induce LBP, and LBP alone can induce balance and gait disorders, which worsen the patients' physical function. This vicious circle occurs even at the early stage of the disease. Specific management of LBP should be proposed to MS patients as soon as possible before the patients develop CLBP. All groups showed a decrease in pain and a decrease in the impact of back pain on daily living after treatment. No significant difference was found between groups.

Warke 2006 [START_REF] Warke | Efficacy of transcutaneous electrical nerve stimulation (tens) for chronic low-back pain in a multiple sclerosis population: a randomized, placebo-controlled clinical trial[END_REF] 90 MS patients (69 females, 21 males) with CLBP allocated into 3 groups. No significant differences were detected in the demographic data between groups.

Randomized placebo controlled study 3 groups of patients :

-30 patients with low frequency TENS (4 Hz,200 µs) -30 patients with high frequency TENS (110 Hz, 200 µs) -30 patients with placebo TENS Patients were assessed at the beginning of the study, at 6 weeks, at 10 weeks and at 32 weeks. Patients had to apply TENS on the lumbar spine at least twice daily for 45 minutes for 6 weeks. Outcome measures were the VAS for average LBP, McGill Questionnaire Visual analog scale for the worst LBP and back and leg spasms, the Roland Morris Disability Questionnaire, Barthel Index, Rivermead mobility Index, Multiple Sclerosis Quality of life-54 Instrument results.

VAS and McGill questionnaire scores decreased in the 3 groups during treatment period, but no significant difference was found across groups. The high frequency TENS group showed a decrease in the worst LBP and back and leg spasms, but the results were not significant. No significant differences were found in the other outcomes.

Abbreviations: CLBP: chronic low back pain; MS: multiple sclerosis; TENS: transcutaneous electrical nerve stimulation; VAS: Visual Analog Scale

Table 1 :

 1 The prevalence, severity of pain, and limitations in activities in daily living due to back pain in MS patients that were reported in the included studies

	Study	Population	Outcomes measures	Results for back pain
	Kahraman et	223 MS patients: 150 females, 73 males	Interviews and questionnaire.	LBP was the most common type of pain:
	al. 2019 [34]	93% RRMS, 5.6% SPMS, 1.4%	The Nordic Musculoskeletal	52.4%
		progressive-relapsing MS.	Questionnaire was used to evaluate	MS patients with musculoskeletal pain had
		124 MS patients presented	back pain.	a lower EDSS score than did MS patients
		musculoskeletal pain: 88 females and 36	The painDETECT Questionnaire was	with neuropathic pain.
		males, mean age 34 years [27-42],	used to evaluate neuropathic pain.
		median EDSS score: 1.5 [1.0-2.5],	The Modified Fatigue Impact Scale, the
		mean disease duration: 5 [2.5-11]	Beck Depression Inventory, the
			Epworth Sleepiness Scale and the
		47 MS patients presented with	Multiple Sclerosis International Quality
		neuropathic pain: 32 females and 15	of Life were used.
		males, mean age 35 [29-42], median
		EDSS score: 2.0 [1.5-2.75], mean disease
		duration: 4.25 [2-8.25]
		52 patients had no pain: 30 females and
		22 males, mean age 34 [25.25-41.75],
		median EDSS score: 1.5 [0-2.5], mean
		disease duration: 4 [0.8-9.5]
	Labuz-Roszak	144 MS patients: 110 females, 34 males	Interviews and questionnaires	50% of patients presented with back pain.
	et al. 2019	Mean age 41±12	Pain severity was measured with the	The intensity was moderate to severe, with
	[35]	Median EDSS score: 3.2±1.9	Visual Analogic Scale	a score of 5.6±2.4 for back pain.
		Mean disease duration: 10.3±8.6	Quality of life was assessed by the
		79.2% RRMS	European Quality of life-5 Dimensions.
		20.8% SPMS	The depressive symptoms were
			evaluated with the Hospital Anxiety
			and Depression Scale, and the EDSS

Abbreviations: EDSS: Expanded Disability Status Scale; PPMS: primary progressive multiple sclerosis; RRMS: remittent recurrent multiple sclerosis; SPMS: secondary progressive multiple sclerosis

Table 2 :

 2 The treatments for low back pain in MS patients that were reported in the studies

	Study Population	Protocols and Outcomes measures	Results
	Al-	15 MS patients with	Randomized placebo controlled study
	Smaidi	CLBP were allocated	3 groups of patients :
	2003	into 3 groups.	-5 patients with low frequency TENS (4 Hz,200 µs)
	[21]		-5 patients with high frequency TENS (110 Hz, 200 µs)
			-5 patients with placebo TENS
			Patients were assessed at the beginning of the study, at 6 weeks,
			and at 10 weeks.
			Patients had to apply TENS on the lumbar spine at least twice
			daily for 45 minutes for 6 weeks.
			Outcome measures were the Roland Morris Disability
			Questionnaire, Short Form-36, McGill Questionnaire, and VAS
			scores.

Table 3 :

 3 Methodological evaluation of the studies performed with the Newcastle Ottawa Scale

		Selection				Comparability	Exposure		Total
		Criteria 1 Criteria 2 Criteria 3 Criteria 4	Criteria 1	Criteria 2 Criteria 1 Criteria 2 Criteria 3
	Warke et al.	X	X	X	X	X		X	6
	2006 [22]
	Al-Smaidi et	X	X	X		X	X	X	6
	al. 2003 [21]

Table 4 :

 4 Sociode

	mographi
	c data of
	the MS
	patients

Table 5 :

 5 Pain evaluation in patients with current LBP performed with the Visual Analog Scale

		Score
	At the evaluation	5 ±2.2
	During the day	5 ±1.9
	Upon waking up	5 ±2.8
	At night	4 ±2.7

Acknowledgements

This work was supported by the ELSAT2020 project, the European Union with the European Regional Development Fund, the French state, the Région Hauts-de-France Council, Carenity and ParcSep networks: authors gratefully acknowledge the support of these institutions.

Declaration of conflicting interests

The authors report no conflicts of interest.


G M Bragança

A S Alcântara-Neto

R I T P Batista

F Z Brandão

V J F Freitas

Pascal Mermillod

J M G Souza-Fabjan

A S Alcantara-Neto

Oviduct fluid during IVF moderately modulates polyspermy in in vitro-produced goat embryos during the non-breeding season

Keywords: anestrus, caprine, IVP, monospermy, oviduct proteins

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

Introduction

In most mammalian, gamete transport, final sperm capacitation, fertilization, and early embryo development are reproductive events that occur in the oviduct lumen [START_REF] Maillo | Oviductal response to gametes and early embryos in mammals[END_REF]. In this microenvironment, gametes and early embryos are immersed in the oviduct fluid (OF), which is a mixture of plasma exudate, epithelial cell secretion, and follicular fluid released during ovulation [START_REF] Segura-Aguilar | The uterine tubal fluid: secretion, composition and biological effects[END_REF]. In addition to metabolic components such as glucose, lactate, pyruvate, and amino acids, OF contains a wide variety of proteins which contribute to the success of early reproductive events [START_REF] Avilés | Oviductal secretions: Will they be key factors for the future ARTs?[END_REF]. Extracellular vesicles (EVs), including microvesicles and exosomes, are present in OF and play a fundamental role in the dialogue of oviduct epithelial cells with gametes/embryos [START_REF] Almiñana | Oviduct extracellular vesicles protein content and their role during oviduct-embryo cross-talk[END_REF][START_REF] Almiñana | Extracellular Vesicles in the Oviduct : Progress , Challenges and Implications for the Reproductive Success[END_REF]. Analyses of transcripts from oviduct epithelial cells [START_REF] Bauersachs | Monitoring gene expression changes in bovine oviduct epithelial cells during the oestrous cycle[END_REF] and protein composition [START_REF] Lamy | Steroid hormones in bovine oviductal fluid during the estrous cycle[END_REF] suggest that the composition of OF is dynamic and changes according to the concentration of sex steroid hormones such as 17β-estradiol (E2) and progesterone (P4), during the estrous cycle.

In sheep expressing spontaneous estrus, 624 proteins were identified in the OF, among which 280 were found to be differentially expressed throughout the estrous cycle, with 64 (23%) being more abundant at estrus and 17 (6%) at luteal phase [START_REF] Soleilhavoup | Proteomes of the Female Genital Tract During the Oestrous Cycle[END_REF]. Among the proteins in abundance at estrus, the oviduct glycoprotein-1 (OVGP1) has a role during the fertilization and interacts with heat shock protein (HSP) family members, such as HSPA8 and HSPA1A, and in the zona pellucida (ZP) hardening of the oocyte [START_REF] Mondéjar | Identification of Potential Oviductal Factors Responsible for Zona Pellucida Hardening and Monospermy During Fertilization in Mammals1[END_REF]. HSPA8 sperm exposure enhanced the sperm survival in boars and bulls and improved monospermy in pigs [START_REF] Elliott | Effects of HSPA8, an evolutionarily conserved oviductal protein, on boar and bull spermatozoa[END_REF]. Annexins (i.e., ANXA1 and ANXA5) are oviduct sperm-binding proteins involved with the sperm reservoir formation. This mechanism keeps the sperm viable near the site of fertilization, controlling the number of sperm cells arriving around the oocyte, and may intrinsically reduce the risk of polyspermy [START_REF] Almiñana | Oviduct extracellular vesicles protein content and their role during oviduct-embryo cross-talk[END_REF][START_REF] Ignotz | Annexins Are Candidate Oviductal Receptors for Bovine Sperm Surface Proteins and Thus May Serve to Hold Bovine Sperm in the Oviductal[END_REF]. Myosin-9 (MYH9) has been identified as an important sperm-interacting protein with a possible role in sperm capacitation around fertilization. It acts by forming complexes with other proteins of the OF (e.g., OVGP1) before interacting with the sperm surface [START_REF] Lamy | Identification by proteomics of oviductal sperm-interacting proteins[END_REF].

In IVF, oocyte polyspermic penetration is a common problem observed in several species, including goats [START_REF] Souza-Fabjan | In vitro embryo production in goats: Slaughterhouse and laparoscopic ovum pick upderived oocytes have different kinetics and requirements regarding maturation media[END_REF][START_REF] Batista | Combination of oviduct fluid and heparin to improve monospermic zygotes production during porcine in vitro fertilization[END_REF][START_REF] Ferraz | Improved bovine embryo production in an oviduct-on-a-chip system: prevention of poly-spermic fertilization and parthenogenic activation[END_REF][START_REF] Gasparrini | Influence of the duration of in vitro maturation and gamete co-incubation on the efficiency of in vitro embryo development in Italian Mediterranean buffalo (Bubalus bubalis)[END_REF]. It leads to embryo development failure and consequently a reduction in IVF efficiency. High rates of polyspermy have been observed in prepuberal (⁓48%) [START_REF] Rodríguez-Gonzáles | Selection of prepubertal goat oocytes using the brilliant cresyl blue test[END_REF] and adult goats (∼34%) [START_REF] Souza-Fabjan | In vitro embryo production in goats: Slaughterhouse and laparoscopic ovum pick upderived oocytes have different kinetics and requirements regarding maturation media[END_REF]. Studies of different species have already demonstrated that IVF medium supplementations or gametes treatment with OF or EVs extracted from OF could promote gametes viability, fertilization rate, and embryo production [START_REF] Angélico | Bovine oviductal fluid ( bOF ) collected in the follicular or luteal phase of the estrous cycle exerts similar effects on ram sperm kinematics and acrosome reactivity in[END_REF][START_REF] Alcântara-Neto | Oviduct fluid extracellular vesicles regulate polyspermy during porcine in vitro fertilisation[END_REF][START_REF] Pradeep | Purification , sequence characterization and effect of goat oviduct-specific glycoprotein on in vitro embryo development[END_REF]. One of the benefits of using OF in in vitro systems is that it enhances monospermic penetrations, possibly through inducing ZP hardening [START_REF] Mondéjar | Identification of Potential Oviductal Factors Responsible for Zona Pellucida Hardening and Monospermy During Fertilization in Mammals1[END_REF][START_REF] Batista | Combination of oviduct fluid and heparin to improve monospermic zygotes production during porcine in vitro fertilization[END_REF][START_REF] Coy | Oviductspecific glycoprotein and heparin modulate sperm-zona pellucida interaction during fertilization and contribute to the control of polyspermy[END_REF]. An improvement of cleavage and blastocyst rates was also reported using OVGP1 purified from OF in a goat IVF protocol [START_REF] Pradeep | Purification , sequence characterization and effect of goat oviduct-specific glycoprotein on in vitro embryo development[END_REF]. OF effects on gametes were observed when using heterologous co-incubation systems, such as ram sperm/bovine OF [START_REF] Angélico | Bovine oviductal fluid ( bOF ) collected in the follicular or luteal phase of the estrous cycle exerts similar effects on ram sperm kinematics and acrosome reactivity in[END_REF] and oocytes from several species (cows, sheep, goats, pigs, rats, rabbits, and humans) with OF from different species [START_REF] Avile | The human is an exception to the evolutionarily-conserved phenomenon of pre-fertilization zona pellucida resistance to proteolysis induced by[END_REF].

There is no information about the OF proteome in goats, and only OVGP1 has been reported in the literature [START_REF] Pradeep | Purification , sequence characterization and effect of goat oviduct-specific glycoprotein on in vitro embryo development[END_REF][START_REF] Abe | Immunological detection and characterization of an estrus-associated antigen in the goat oviduct[END_REF][START_REF] Gandolfi | Similarity of an Oviduct-specific Glycoprotein between Different Species[END_REF]. Proteome studies in bovine [START_REF] Lamy | Regulation of the bovine oviductal fluid proteome[END_REF] and ovine [START_REF] Soleilhavoup | Proteomes of the Female Genital Tract During the Oestrous Cycle[END_REF] showed some similarities of OF protein expression profile throughout the estrous cycle. The follicular phase (late or pre-ovulatory) is related with high E2 concentration, which is the major modulator of the OF proteins involved in the gamete interactions and fertilization [START_REF] Almiñana | Extracellular Vesicles in the Oviduct : Progress , Challenges and Implications for the Reproductive Success[END_REF][START_REF] Lamy | Steroid hormones in bovine oviductal fluid during the estrous cycle[END_REF][START_REF] Almiñana | Deciphering the oviductal extracellular vesicles content across the estrous cycle : implications for the gametes-oviduct interactions and the environment of the potential embryo[END_REF].

In small ruminants under temperate climate conditions, the seasonal anestrus compromises natural reproduction, although follicular waves and steroid production are maintained [START_REF] Evans | Follicular waves are associated with transient fluctuations in FSH but not oestradiol or inhibin-A concentrations in anoestrous ewes[END_REF].

Thus, based on this evidence, a study that determines the protein profile and role of OF from anestrous females seems sound. Therefore, the present study was designed to i) determine the presence of proteins involved in the fertilization process (OVGP1, ANXA1, ANAX5, HSPA8, HSPA1A, and MYH9) in the OF of goats in anestrus; and ii) assess the functional effect of this fluid during IVF on polyspermy and embryo development.

Materials and Methods

The study was performed at Unité de Physiologie de la Reproduction et des Comportements in Nouzilly, France (47 o 22'N and 00 o 41'E), during the non-breeding season (June to September) [START_REF] Chemineau | Seasonality of estrus and ovulation is not modified by subjecting female Alpine goats to a tropical photoperiod[END_REF][START_REF] Chemineau | Seasonality of Reproduction in Mammals: Intimate Regulatory Mechanisms and Practical Implications[END_REF]. Chemicals were purchased from the Sigma Chemical Co. (Saint Louis, MO, USA), except where otherwise indicated.

Experimental design

The goat oviducts and ovaries used during the study were obtained at a local slaughterhouse. In June, the flushing containing OF was centrifuged, aliquoted, and stored at -80 ºC. From early July to early September, a sample was used to determine the presence of important proteins by western blotting [START_REF] Almiñana | Oviduct extracellular vesicles protein content and their role during oviduct-embryo cross-talk[END_REF]. Over six runs 1,576 COCs were used and submitted to in vitro maturation (IVM), followed by co-culture with three different sperm concentrations (1.0, 2.0, and 4.0 x 10 6 cells/mL) in IVF medium supplemented with 10% oviduct flushing, corresponding to ± 10% OF (OF1, OF2, and OF4) or without (CTRL1, CTRL2, and CTRL4). After IVM, the COCs were allocated to the following groups: CTRL1 (n=259), CTRL2 (n=270), and CTRL4 (n=254); and OF1 (n=261), OF2 (n=266), and OF4 (n=266) for the IVF. After IVF, a part of the presumptive zygotes (n=628) was denuded, fixed, stained, and used to evaluate polyspermy, while the others (n=914) were cultured until day 8 to evaluate development.

OF recovery

Genital tracts and the attached ovaries were transported to the laboratory in individual bottles (dry) in a thermos box at 30 ºC within 2-3 h of collection. The estrous cycle phase was assessed by ovarian morphology, according to Camp et al. [START_REF] Camp | Ovarian Activity During Normal and Abnormal Length Estrous CyCles in the Goat[END_REF]. The absence of corpus luteum (CL) and preovulatory follicles, characterizing acyclicity, was observed throughout the experiment. Consequently, all the oviducts and ovaries used were classified as being in seasonal anestrous. Oviducts were dissected free of surrounding tissues: A needle coupled to a 1 mL syringe was inserted into the infundibulum and 500 µL of IVF medium were slowly and carefully injected into the oviduct lumen. A manual descendent (from ampulla to isthmus) pressure was applied to collect the flushing. The oviductal flushing of the pair of oviducts from each female was recovered in a falcon tube of 15 mL and centrifuged at 300 × g for 15 min at room temperature to remove the cells. The supernatant was transferred into a new tube (FALCON white lid) and centrifuged at 12,000 × g for 15 min at 4 ºC to remove cellular debris and apoptotic bodies. Clarified OF was stored at -80 ºC until use. Before use, a pool of samples from three females was prepared, aliquoted, and frozen for use throughout the experiment.

Western blotting

Proteins were selected based on the previous identification of oviduct proteins with known reproductive roles in bovine [START_REF] Almiñana | Oviduct extracellular vesicles protein content and their role during oviduct-embryo cross-talk[END_REF][START_REF] Mh | Bovine oviductal and uterine fluid support in vitro embryo development[END_REF] and porcine [START_REF] Batista | Combination of oviduct fluid and heparin to improve monospermic zygotes production during porcine in vitro fertilization[END_REF][START_REF] Alcântara-Neto | Oviduct fluid extracellular vesicles regulate polyspermy during porcine in vitro fertilisation[END_REF] species. Therefore, bovine and porcine OF were used as positive control in western blots (WB). For each species, an OF pool from three animals was used. OF samples were diluted in reducing Laemmli loading buffer (2x buffer; composition: 125 mM Tris, 20% glycerol, 4% sodium dodecyl sulfate, and 10% tris (2-carboxyethyl)phosphine), followed by vortexing, heating in a water bath at 95 ºC for 5 min and being centrifuged at 12,000 g for 5 min. Proteins were separated by sodium dodecyl sulfate polyacrylamide using a 10% polyacrylamide gel containing 40 μg of proteins per lane at 180 V for 45 min [START_REF] Laemmli | Cleavage of structural proteins during the assembly of the head of bacteriophage[END_REF]. After separation, proteins were transferred onto nitrocellulose membranes (Bio-Rad, 1704271) over 30 min up to 1.0 A and 25 V using semi-liquid Transblot ® Turbo™ System (Bio-Rad). After transfer, membranes were washed with distilled water and blocked with Tris-buffered saline solution containing Tween 20 (0.5% w/v; TBST), supplemented with lyophilized low-fat milk (5% w/v; TBST-milk) for 2 h at room temperature. Then, membranes were incubated with primary antibodies overnight at 4 °C, washed four times in TBST, and incubated with IRDye ® Fluorescent secondary antibodies for 45 min at 37 °C in the dark, both under gentle agitation. The protein bands were revealed with UV light exposure, and images were digitalized using Odyssey CLx Near-Infrared Fluorescence Imaging System (LICOR), in triplicate. The primary antibodies used for immunoblotting detection were anti-oviduct-specific glycoprotein (OVGP1; Santa Cruz Biotechnology, sc-377267); anti-annexin A1 and A5 (ANXA1 and 5; Santa Cruz Biotechnology); anti-heat-shock protein 70 (HSP1A1; Stressgen, SPA-810); anti-myosin heavy chain 9 (MYH9-H40, Santa Cruz Biotechnology, sc-98978); and Anti-Hsc70 (HSPA8; Abcam, ab65170). Secondary antibodies were IRDye ® 800CW anti-Rabbit IgG and IRDye ® 680RD anti-Mouse IgG (1:10000; LICOR).

Cumulus-oocyte complexes (COCs) recovery

Slaughterhouse ovaries from adult goats were collected and transported to the laboratory in a thermos box containing saline solution (0.9% NaCl) at 30 ºC within 4 h of collection. Ovaries were washed with fresh saline solution at 30 ºC and the stage of estrous cycle was classified. Oocytes were aspirated from all visible follicles between 2 and 6 mm in diameter with an 18-ga short bevel needle connected to a Falcon tube under controlled vacuum (30 mm Hg). The collection tubes had been previously filled with 3 mL of HEPESbuffered TCM 199 supplemented with 10 IU/mL heparin (Choay; Glaxo Wellcome Production, Notre Dame de Bondeville, France), 4 µg/mL gentamicin (G1272), and 1 mg/mL BSA (A 9647).

In vitro maturation

COCs were isolated under stereomicroscope (Nikon Corporation, Japan) and screened by morphology. Only COCs surrounded by at least one complete layer of unexpanded cumulus cells and finely granulated oocyte cytoplasm were used for IVM; the rest were discarded [START_REF] Souza-Fabjan | Assessment of the reproductive parameters, laparoscopic oocyte recovery and the first embryos produced in vitro from endangered Canindé goats (Capra hircus)[END_REF]. COCs were washed four times in four-well petri dishes (Nunc, Roskilde, Denmark) containing 500 µL of TCM 199 (M4530) supplemented with gentamicin (4 µg/mL). Then, 40 to 50 COC groups were transferred into another four-well petri dish containing 500 µL of TCM199 supplemented with 10 ng/mL EGF and 100 µM cysteamine, and incubated for 22 h at 38.8 ºC in a humidified atmosphere of 5% CO2 in air.

Semen preparation and in vitro fertilization

For each replicate used in the experiments, two straws of frozen semen (frozen at the same time) from the same batch and from the same two bucks were thawed (37 ºC for 30 s) and pooled. Motile sperm were separated by centrifugation (15 min at 700 × g) on 3 mL of Percoll (Pharmacia, Uppsala, Sweden), discontinuous density gradient (45/90%). The sperm pellet was washed by centrifugation (100 × g for 5 min) in 600 µL of IVF-Hepes medium that consisted of synthetic oviduct fluid [SOF; 107.7 mM NaCI, 7.16 mM KCl, 1.19 mM KH2PO4, 1.71 mM CaC12, 0.49 mM MgCl2, 25.07 mM NaHCO3, 3.3 mM Na lactate, 0.3 mM Na pyruvate, 1.3 µg/mL phenol red, 1 mM glutamine, 3% essential (M-6766), 1% nonessential (B-7145) amino acids, and 0.3% BSA fraction V; pH = 7.3; 280 m0sm] supplemented with 2.4 mg/mL Hepes (H3375), and 4 µg/mL gentamicin. The sperm pellet was resuspended in IVF medium (described below).

After maturation, the COCs were washed once in 500 µL of IVF medium and transferred into the fertilization well containing 250 µL of medium. Sperm suspension was diluted in IVF medium to reach final concentrations of 1.0, 2.0, or 4.0 × 10 6 cells/mL by adding 250 µL to IVF wells. The IVF medium consisted in SOF (already described) containing 10% of heat inactivated estrus sheep serum (ESS), 5 µg/mL heparin (Calbiochem 375095) and 4 µg/mL gentamicin (control groups; CTRL1, CTRL2 and CTRL4) or this same medium supplemented with 10% of OF (OF groups; OF1, OF2, and OF4). Sperm and COCs (40 to 50/well) were co-incubated for 18 h at 38.8 ºC in a humidified atmosphere of 5% CO2 in air.

Assessment of fertilization

The presumptive zygotes reserved for evaluation of polyspermy (approximately 40% of the initial number of inseminated COCs) from each group were transferred within a 10 µL droplet onto a grease-free slide and dried at room temperature. They were then fixed in 100% ethanol for at least 3 h. After drying, structures were stained in 1 µg/mL Hoechst 33342 fluorochrome (stains all nuclei) in Vectashield mounting medium (Vector Labs, Burlingame, CA, USA), and overlaid with a cover slip sealed with nail varnish. Slides were stored at 4 ºC until they were examined using epifluorescence microscopy to evaluate fertilization. Zygotes with three or more pronuclei (or decondensing sperm heads) were considered polyspermic.

In vitro development (IVD)

After IVF, the zygotes were placed into 15 mL Falcon tubes containing 2 mL of washing medium (SOF; 2.4 mg/mL Hepes and 2 µL/mL BSA) and vortexed for 2 min to remove cumulus cells. The presumptive zygotes were recovered in 35 mm petri plates, washed four times in culture medium (SOF; supplemented with 3 mg/mL BSA), and reserved for evaluation of polyspermy or transferred in groups of maximum 25 into four-well petri dishes containing 25 µL (1 µL/structure) drops of culture medium covered with 700 µL mineral oil. The presumptive zygotes were cultured for 8 days at 38.8 ºC in a humidified atmosphere of 5% O2, 5% CO2, and 90% N2. At 48 h post-insemination (PI), the drops were supplemented with 10% fetal calf serum (FCS). On day 8, all blastocysts were washed, spotted onto microscopy slides, fixed in ethanol 100% for 3 h, dried, and stained with Hoechst (1 µg/mL) to count their total cell number under epifluorescence microscope.

Statistical analyses

The efficiency of development was evaluated (1) as the percentage of cleaved embryos 2 days PI and the percentage of blastocysts at 8 days PI, expressed (2) on the basis of the number of oocytes entering into IVD or (3) on the basis of the number of cleaved embryos at day 2. The following end points were assessed: penetration rate (penetrated zygotes/total oocytes x 100); monospermy rate (monospermic/penetrated zygotes x 100); and IVF efficiency (monospermic zygotes/total oocytes x 100). Data were tested for normality using the Kolmogorov-Smirnov test. Variables were compared by one-way ANOVA followed by Tukey test. Differences were considered significant when P < 0.05 and tendency when P < 0.10. Data are shown in mean ± S.E.M.

Results

Western blotting

The analysis of candidate proteins confirmed the presence of the six proteins (OVGP1, ANXA1, ANXA5, HSPA1A, HSPA8, and MYH9) associated with reproductive functions in the OF obtained from goats in anestrus. The size of each protein observed in the goat sample is compatible with the sizes observed in the bovine and swine samples used as positive controls (Fig. 1).

Effect of OF on IVF outcomes

When comparing OF with CTRL at the same sperm concentration (OF1 vs. CTRL1, OF2 vs. CTRL2, and OF4 vs. CTRL4, respectively), the fertilization parameters, such as penetration rate (Fig. 2A, P = 0.32, P = 0.83, P = 0.62), monospermy rate (Fig. 2B, P = 0.14, P = 0.08, P = 0.18), and IVF efficiency (Fig. 2C, P = 0.11, P = 0.55, P = 0.15), were similar.

The increase in sperm concentration had no effect on the penetration rate (Fig. 2A) either for control (P = 0.82, CTRL1: 67 ± 11; CTRL2: 69 ± 16, and CTRL4: 72 ± 15%) or OF groups (P = 0.74, OF1: 74 ± 13; OF2: 71 ± 14, and OF4: 67 ± 17%). Regardless of the concentration used, when data were pooled, gametes exposure to OF during IVF improved (P = 0.04) monospermy and tended (P = 0.06) to enhance IVF efficiency in terms of production of normally fertilized zygotes from total oocytes (Fig. 2D). In addition, IVF efficiency was greater (P = 0.002) in OF1 than OF4 [60 ± 13 vs 37 ± 5%), with no effect (P = 0.11) in the control groups [CTRL1: 45 ± 16; CTRL2: 46 ± 13, and CTRL4: 31 ± 9%) (Fig. 2C). The monospermy rate decreased as sperm concentration was increased, in both OF and CTRL (Fig. 2B).

Embryo development

Gametes exposure to OF during IVF did not affect any parameter related to developmental competence, inferred by rates of cleavage (P = 0.63, P = 0.61, P = 0.94), blastocysts in relation to oocytes (P = 0.75, P = 0.85, P = 0.94), blastocysts in relation to cleaved embryos (P = 0.26, P = 0.86, P = 0.92) and hatched blastocysts in relation to blastocysts (P = 0.99, P = 0.83, P = 0.89), as well as the total number of cells per blastocyst (P = 0.94, P = 0.83, P = 0.89, Table 1). When comparing OF1 to OF4, there was a tendency (P = 0.08) to favor OF1 in the blastocyst/cleaved oocytes parameter. When the data were pooled regardless of the treatment, average cleavage was 72 ± 2.6%, blastocyst 37 ± 3.0%, blastocyst in relation to cleaved 51 ± 4.8%, hatched 62 ± 1.2%, and total number of cells per blastocyst 174 ± 1.8. Goat blastocysts are shown in Fig. 3.

Discussion

To mimic physiological reproductive conditions in vivo, co-culture of gametes/embryos with oviduct epithelial cells, EVs (microvesicles and exosomes), and purified or integral OF proteins may represent the best models of in vitro systems [START_REF] Maillo | Oviductal response to gametes and early embryos in mammals[END_REF].

Interestingly, in this study we demonstrated that the main proteins (OVGP1, ANXA1, ANAX5, HSPA8, HSPA1A, and MYH9) involved in the fertilization process are present in the OF of anestrus goats. However, a moderate effect only was observed on fertilization parameters evaluated after the OF exposure of the gametes during IVF. Even though no differences were found when treatments using the same sperm concentrations (OF1 vs. CTRL1, OF2 vs. CTRL2, and OF4 vs. CTRL4) were compared, when data were pooled regardless of concentrations, the OF exposure significantly improved monospermy and tended to enhance IVF efficiency in terms of production of normally fertilized zygotes.

A comparison of the extreme concentrations within the same treatment (CTRL1 vs CTRL4 and OF1 vs OF4) revealed that penetration rate was not affected; however, a monospermy decrease was observed when 4 x 10 6 sperm/mL was used in both (CTRL4 and OF4). These data partly diverge from those previously described in prepubertal goats [START_REF] Palomo | The influence of sperm concentration, length of the gamete co-culture and the evolution of different sperm parameters on the in vitro fertilization of prepubertal goat oocytes[END_REF],

where penetration rate was enhanced when 4 x 10 6 sperm/mL was used, but polyspermy was also increased. For adult goats, the standard sperm concentration used for IVF is between 1 and 2 x 10 6 sperm/mL [START_REF] Souza-Fabjan | In vitro embryo production in goats: Slaughterhouse and laparoscopic ovum pick upderived oocytes have different kinetics and requirements regarding maturation media[END_REF][START_REF] Leoni | In vitro production and cryotolerance of prepubertal and adult goat blastocysts obtained from oocytes collected by laparoscopic oocyte-pick-up (LOPU) after FSH treatment[END_REF], but 4 x 10 6 sperm/mL has also been reported in the literature, aiming to enhance oocyte penetration [START_REF] Pradeep | Purification , sequence characterization and effect of goat oviduct-specific glycoprotein on in vitro embryo development[END_REF][START_REF] Han | Interactive effects of low temperature and roscovitine (ROS) on meiotic resumption and developmental potential of goat oocytes[END_REF]. However, our data demonstrate that penetration was not affected by concentration and thus 1 x 10 6 sperm/mL could be recommended for adult goat IVF.

Importantly, IVF efficiency was greater in OF1 than in OF4. Conversely, no effect was observed on the control (CTRL1 vs CTRL4). We believe that the effect observed in OF groups could be due to an enhancement of sperm quality. In this way, we recently demonstrated that bovine OF was able to enhance ram sperm motility for up to 4 h as well as rate of acrosome reaction after long (18-24 h) incubation periods without affecting sperm viability [START_REF] Angélico | Bovine oviductal fluid ( bOF ) collected in the follicular or luteal phase of the estrous cycle exerts similar effects on ram sperm kinematics and acrosome reactivity in[END_REF]. In addition, there was a tendency to favor OF1 in the rate of blastocyst in relation to the cleaved structures. This parameter is often associated with the quality of the oocyte and its capacity to sustain embryonic events until the maternal to zygotic transition, which occurs at 8-16 cells in goats [START_REF] Ling | Identification of lncRNAs by RNA Sequencing Analysis During in Vivo Pre-Implantation Developmental Transformation in the Goat[END_REF]. However, in the current study, the oocytes were pooled at the beginning of the experiment, before the treatments were applied, and it is unlikely that this affected this parameter in any way. One aspect that may certainly not be overlooked is the possibility that the development of the polyspermic embryos was blocked before they reached the blastocyst stage [START_REF] Mizushima | Fertilization 2: polyspermic fertilization[END_REF]. When data were pooled regardless of sperm concentration, the OF total average (OF1+OF2+OF4) promoted a greater monospermy rate than the CTRL total average and tended to increase IVF efficiency. Overall, these data reinforce our hypothesis that the association of 10% OF and 1.0 x 10 6 cells/mL sperm during IVF is the best combination to increase monospermy, obviously reducing polyspermy, improving IVF efficiency, and resulting in a lower number of embryos being blocked. Based on these data, we suggest that when OF is present, the IVF must be performed using a lower sperm concentration.

It is important to highlight that there were no significant differences in the comparison between treatments using the same sperm concentrations (OF1 vs. CTRL1, OF2 vs. CTRL2, and OF4 vs. CTRL4). It should be emphasized that the OF was collected from anestrus goats.

Considering the high influence of the season on small ruminant reproductive systems due to hormonal alterations [START_REF] Bartlewski | Ovarian antral follicular dynamics and their associations with peripheral concentrations of gonadotropins and ovarian steroids in anoestrous Finnish Landrace ewes[END_REF], we suppose that this may have affected the overall protein concentrations and their role in the polyspermy modulation. The OF depends on the concentration of ovarian steroid hormones, which modulate the physiological and reproductive events that occur in the oviduct lumen [START_REF] Camp | Ovarian Activity During Normal and Abnormal Length Estrous CyCles in the Goat[END_REF]. During the estrous cycle, fluctuations of E2 and P4 concentrations induce changes in the epithelium of the oviduct and secretory function [START_REF] Segura-Aguilar | The uterine tubal fluid: secretion, composition and biological effects[END_REF][START_REF] Avilés | Oviductal secretions: Will they be key factors for the future ARTs?[END_REF], which alter the proteomic and metabolomic profile of OF [START_REF] Almiñana | Oviduct extracellular vesicles protein content and their role during oviduct-embryo cross-talk[END_REF][START_REF] Chemineau | Seasonality of Reproduction in Mammals: Intimate Regulatory Mechanisms and Practical Implications[END_REF] to provide an optimal microenvironment for fertilization and early development. The practically constant concentrations of E2 during anestrus [START_REF] Evans | Follicular waves are associated with transient fluctuations in FSH but not oestradiol or inhibin-A concentrations in anoestrous ewes[END_REF] may have modified the profile of proteins involved in the fertilization process, which were capable of exerting only a moderate control of polyspermy in the present study.

According to previous studies, throughout the estrous cycle the OF proteins OVGP1, HSPA8, HSPA1A, and MYH9 were most abundant at estrus in sheep showing a spontaneous cycle [START_REF] Soleilhavoup | Proteomes of the Female Genital Tract During the Oestrous Cycle[END_REF]. OVGP1 and HSPA8 were also detected at luteal phase but in much lower quantities than during estrus [START_REF] Soleilhavoup | Proteomes of the Female Genital Tract During the Oestrous Cycle[END_REF]. In bovine, at ipsilateral to ovulation side, ANXA1 and MYH9 are most abundant in the pre-and post-ovulatory periods, respectively. OVGP1 is overabundant in both pre-and post-ovulatory stages compared with mid-luteal, HSPA8 is more abundant in the preovulatory stage, and there is no significant fluctuation in HSPA1A during the estrous cycle phases [START_REF] Lamy | Regulation of the bovine oviductal fluid proteome[END_REF]. In heifers with induced estrus (synchronization by Ovsynch ® protocol plus buserelin application) HSPA8, OVGP1, MHY9, and ANXA1 were among the 20 most abundant proteins [START_REF] Papp | A novel approach to study the bovine oviductal fl uid proteome using transvaginal endoscopy[END_REF].

OVGP1 has been proposed as the main player in monospermy regulation, and ZP hardening could be the mechanism involved [START_REF] Lamy | Steroid hormones in bovine oviductal fluid during the estrous cycle[END_REF][START_REF] Souza-Fabjan | In vitro embryo production in goats: Slaughterhouse and laparoscopic ovum pick upderived oocytes have different kinetics and requirements regarding maturation media[END_REF]. While in aquatic organisms a rapid depolarization of the oocyte plasma membrane prevents polyspermy within a few seconds, a similar mechanism is still controversial in mammals even though a primary block to polyspermy, before the release of cortical granules, is also suspected. In mice and humans, the oocyte Juno membrane protein has been shown to play a role in polyspermy regulation before the cortical granule reaction [START_REF] Bianchi | Juno is the egg Izumo receptor and is essential for mammalian fertilization[END_REF]. Shortly after fertilization, Juno is released in the perivitelline space and binds to the Izumo partner of additional sperms, preventing their fusion to the oocyte membrane [START_REF] Bianchi | Izumo meets Juno: Preventing polyspermy in fertilization[END_REF]. Juno and Izumo orthologs are found in most mammalian species, but their role in domestic species polyspermy regulation is yet to be explored.

However, it has been shown that anti-Izumo antibodies can reduce fertilization in pigs [START_REF] Tanihara | Roles of the zona pellucida and functional exposure of the sperm-egg fusion factor "IZUMO" during in vitro fertilization in pigs[END_REF].

Nevertheless, the possible effect of OF on Juno-Izumo interactions needs to be investigated.

There is evidence in the literature that monospermy modulation in goats may be different to that in other species. Possibly, the OVGP1 has a concentration-dependent effect in goats, or it is only active in a short temporal window near ovulation, as occurs in swine [START_REF] Avilés | Oviductal secretions: Will they be key factors for the future ARTs?[END_REF].

Besides OVGP1, MYH9 participates in the gamete-oviduct interaction process; OVGP1 may bind to spermatozoa and oocytes through the partner MYH9 by a non-glycosylated Nterminal conserved region [START_REF] Kadam | Identification and characterization of oviductal glycoprotein-binding protein partner on gametes : epitopic similarity to non-muscle myosin IIA[END_REF]. It has been proposed that monospermic fertilization may be enhanced by the functional effects of OVGP1 and MYH9 via EVs, binding to the ZP, perhaps through modification of its carbohydrate and protein composition [START_REF] Ignotz | Annexins Are Candidate Oviductal Receptors for Bovine Sperm Surface Proteins and Thus May Serve to Hold Bovine Sperm in the Oviductal[END_REF]. Additionally, EVs could bind to spermatozoa by annexins (e.g., ANXA1 and ANXA5), preventing a massive spermatozoa arrival at the oocyte and fertilization by apoptotic spermatozoa, leading to improvements in IVF efficiency [START_REF] Ignotz | Annexins Are Candidate Oviductal Receptors for Bovine Sperm Surface Proteins and Thus May Serve to Hold Bovine Sperm in the Oviductal[END_REF]. In addition, OVGP1 and ANXA1 are amongst the most abundant embryo-interacting proteins from OF in 4-6 cells and morula stage embryos [START_REF] Banliat | Identification of 56 Proteins Involved in Embryo -Maternal Interactions in the Bovine Oviduct[END_REF]. HSPA8 increases sperm viability in vitro and has been associated with the maintenance of sperm survival around ovulation [START_REF] Elliott | Effects of HSPA8, an evolutionarily conserved oviductal protein, on boar and bull spermatozoa[END_REF]. HSPA1A is an EVs marker, an exosomal-specific protein present in 89% of proteomic studies [START_REF] Almiñana | Oviduct extracellular vesicles protein content and their role during oviduct-embryo cross-talk[END_REF][START_REF] Mathivanan | Exosomes : Extracellular organelles important in intercellular communication[END_REF], which confirms the presence of EVs in goats' OF used in our study. A study demonstrated that at adult oviduct, the percentage of HSPA1A positive cells tended to decrease during estrus and increase during diestrus, and was associated with the rise of the percentage of estrogen receptor-positive cells.

HSPA1A is an oviduct protein that is modulated differentially between oviduct regions, being more abundant in highly steroid-responsive regions, such as the infundibulum and ampulla. Moreover, HSPA1A-positive cells were found to be more abundant during rats in early pregnancy than in non-pregnant rats, suggesting they play a role in early embryo development [START_REF] Mariani | Constitutive expression of heat shock proteins hsp25 and hsp70 in the rat oviduct during neonatal development , the oestrous cycle and early pregnancy[END_REF].

Data in the literature support our speculation that the moderate OF effect seen in our study could be associated with the low steroid levels during the non-breeding season, which decreased the concentration of some OF proteins. The constant level of E2, with rare fluctuations in small ruminant females in anestrus [START_REF] Evans | Follicular waves are associated with transient fluctuations in FSH but not oestradiol or inhibin-A concentrations in anoestrous ewes[END_REF][START_REF] Bartlewski | Ovarian antral follicular dynamics and their associations with peripheral concentrations of gonadotropins and ovarian steroids in anoestrous Finnish Landrace ewes[END_REF], may indicate a moderate role of OF in monospermy modulation. Therefore, future studies aiming to compare the IVF effect of OF throughout the estrous cycle at breeding season might be of interest. In addition, the characterization of the protein profile of OF at different stages of the estrous cycle may be important for the identification of additional proteins that enhance the beneficial effect of OF on monospermic fertilization. Another possibility would be to supplement goat IVF medium with heterologous OF from a non-seasonal species (e.g., cows) as an alternative to reduce polyspermy in goat IVF.

In conclusion, the main proteins (OVGP1, ANXA1, ANAX5, HSPA8, HSPA1A, and MYH9) involved in the fertilization process are present in the OF of anestrus goats.

Moreover, the OF supplementation in IVF during gametes co-culture may moderately modulate polyspermy incidence and enhance IVF efficiency, especially when 1.0 x 10 6 sperm is used.

A

  ,B differ within column between treatments (OF vs. CTRL) in the same sperm concentration a,b differ within column among sperm concentrations (1 vs 2 vs 4) in the same treatment C,D differ within column between the total averages (Total OF vs Total CTRL) * OF1, OF2 and OF4: IVF medium supplemented with 10% of OF and sperm concentration used at IVF was 1.0, 2.0, and 4.0 × 10 6 cell/mL ** CTRL1, CTRL2, and CTRL4: no supplementation with OF, and sperm concentration used at IVF was 1.0, 2.0, and 4.0 × 10 6 cell/mL *** Total average for OF (OF1, OF2, and OF4) and CTRL (CTRL1, CTRL2, and CTRL4) groups # Bl: blastocyst, Hbl: hatched blastocyst, and Total cells: number of blastomeres per blastocyst (n=22/group in three runs).

Figure captions Fig. 1 .

 captions1 Figure captions

Fig. 2 .

 2 Fig. 2. Effect of goat oviduct fluid (OF) supplementation or not (Control: CTRL) at IVF with three sperm concentrations: 1.0 x 10 6 (OF1 and CTRL1), 2.0 x 10 6 (OF2 and CTRL2), and 4.0 x 10 6 (OF4 and CTRL4) on the goat IVF parameters. (A,D) Penetration rate; (B,D)

Fig. 3 .

 3 Fig. 3. (A) Goat embryos at day 7 of culture produced from oocytes recovered in the non-

Table 1 .

 1 Effect of adult anestrous goat oviduct fluid (OF) supplemented at IVF on in vitro embryo production (IVP) system, considering different sperm concentrations at IVF. Mean ± S.E.M.

	Treatment	n	Cleavage (%) Bl (%) #	Bl/cleaved (%) # Hbl/total bl (%) # Total cells (n) #
	OF1 *	148	65 ± 7.1	41 ± 4.7	65 ± 8.1	64 ± 3.7	174 ± 11.5
	CTRL1 **	154	71 ± 6.6	38 ± 6.5	54 ± 5.8	65 ± 8.6	175 ± 15.0
	OF2 *	155	77 ± 1.7	39 ± 5.8	50 ± 7.6	59 ± 7.9	181 ± 11.6
	CTRL2 **	156	72 ± 4.7	38 ± 4.3	52 ± 4.7	65 ± 8.7	177 ± 14.9
	OF4 *	152	75 ± 6.5	33 ± 3.5	44 ± 1.9	59 ± 8.5	168 ± 12.1
	CTRL4 **	149	74 ± 4.3	32 ± 5.7	43 ± 7.1	61 ± 13.9	170 ± 14.4
	Total OF ***	455	72 ± 3.3	38 ± 2.7	53 ± 4.2	61 ± 3.9	174 ± 3.8
	Total CTRL *** 459	72 ± 2.9	36 ± 3.1	50 ± 3.4	63 ± 5.8	174 ± 2.1

"n" represents the number of structures after IVF submitted to IVD

Acknowledgments G.M. Bragança received a scholarship from CAPES/EMBRAPA (Brasília, Brazil, 88882.156906/2017-01). The authors thank the CAPES/COFECUB bilateral framework for their financial support in collaboration (88881.142966/2017-01) with Universidade Federal Fluminense, Universidade Estadual do Ceará, and Institut National de la Recherche Agronomique. F.Z. Brandão, V.J.F. Freitas, and JMG Souza-Fabjan are CNPq fellows, and J.M.G. Souza-Fabjan is a FAPERJ fellow.

Conflict of interest

None of the authors have any conflict of interest to declare.