Helcio Raymundo
email: helcioru@uol.com.br

João G M Reis

Passenger Transport Drawbacks: An Analysis of its "Disutilities" Applying the AHP Approach in a Case Study in Tokyo, Japan

Keywords: urban mobility, transportation disadvantages, decision-making

Passenger transport is a key player in urban mobility. However, it imposes some disadvantages, such as: time wasting, cost of fares and other costs, insecurity, discomfort and damage to the environment. These disadvantages herein called disutilities affect passenger choices and therefore it is necessary to consider them encompassing all modes of transportation. In this study, an analysis of these disutilities was conducted with the purpose of measuring its drawbacks. To this end, a case study in the Greater Tokyo Area (Japan) was carried out and assessed using the Analytic Hierarchy Process. The results showed that from 0,000 to 1,000 automobiles produce the highest level of disutility (0.182) compared with seven other modes of transportation.

Introduction

People usually move from one place to another to meet their needs, as human activities occur in different areas of the city [START_REF] Hanson | The Geography of Urban Transportation[END_REF] [START_REF] Schatzki | The Timespace of Human Activity: On performance[END_REF]. Therefore, Passenger Transport (PT) is the main provider of access to work, leisure, education, etc. [START_REF] Carson | Reducing social exclusion by improving transport-assessing the problems and appraising the options[END_REF]. PT can be divided twofold: (i) private, such as automobiles and motorcycles; and (ii) public, where passengers pay a fare to ride, like on buses and trains. Even with PT being important for the economy, it imposes some disadvantages to the passengers and the society, given that it wastes time, costs money, is unsafe, uncomfortable and harms the environment, consuming non-renewable energy and urban space [START_REF] Mackinnon | Transport and Economic Development[END_REF] [START_REF] Raymundo | Knowledge Management in Public Transportation: Experiences in Brazilian Bus Companies[END_REF]. These disadvantages, called disutilities, influence customer choices and transportation takes the character of a service of negative consumption, in other words, something that everyone needs, but nobody wants [6] [7]. Thus, there is not PT mode that does not present some issues. All modes of transport may present drawbacks; hence, it is important to minimize their levels of disutility [START_REF]Advances in the Human Side of Service Engineering: Proceedings of the AHFE 2016 International Conference on The Human Side of Service Engineering[END_REF].

The purpose of this study is to evaluate the levels of disutility in PT modes and classify them. To this end, a case study was conducted in the Greater Tokyo Area, Japan. This area was chosen due to the data access, high concentration of population and availability of all transportation modes. The evaluation was made applying the Analytic Hierarchy Process (AHP) approach.

Methodology

The levels of disutilities can be measured or evaluated by specialists. The weights of each disutility are submitted to the AHP to calculate the "Total the Disutility" of each mode of transportation.

Analytic Hierarchy Process (AHP)

AHP is a structured technique for organizing and analyzing complex decisions based on mathematics and psychology, developed by Thomas L. Saaty in the 1970's. It is a multi-criterial decision-making approach that measures and establishes priority scales based on specialists' judgments about a given subject via the means of peer comparisons [START_REF] Saaty | An Innovative Orders of Magnitude Approach to AHP-based Multi-Criteria Decision Making: Prioritizing Divergent Intangible Humane Acts[END_REF]. AHP is a methodology to calibrate the numeric scale for the measurement of quantitative as well as qualitative performances [START_REF] Sureshchandar | A Framework for Evaluating the Criticality of Software Metrics: An Analytic Hierarchy Process (AHP) Approach[END_REF], and it has been used to solve several logistical, strategic and transport engineering problems. AHP applications consist of breaking the problem into steps [START_REF] Saaty | Decision Making with the Analytic Hierarchy Process[END_REF]: (i) define it; (ii) structure the decision hierarchy; (iii) construct a set of pairwise comparison matrices; and (iv) use the priorities obtained to weigh them.

In the present case, AHP is a means to determine the level of disutility of the PT modes, considering a displacement between two points. The criteria considered for the decision-making is related to "Passengers" and "Society" and the sub-criteria related to the disutilities is outlined in Section 3. To set the weights adopted, bibliographical references were selected, as can be seen in Table 1.

Table 1. Bibliographical references for weights adopted in pairwise comparisons

[12] X X X X X X X Vasconcellos [13] X X X X X ANTP [14] X X X Gibson et al. [15]
X

AHP Framework

The AHP method utilizes peer comparison between each one of the criteria, sub-criteria and alternatives, considering levels from a nominal scale [START_REF] Deng | Supplier Selection Using AHP Methodology Extended by D Numbers[END_REF], shown in Table 2, and, in this case, considering the bibliographical references previously cited in Table 1. These comparisons are established to determine the levels of importance of each criterion, comparing, for example, criterion "i" with criterion "j" [START_REF] Vasconcellos | Corporación Andina de Fomento: Desarrollo urbano y movilidad en América Latina[END_REF] (Eq. 1).

𝐴 = ( 1 𝑎12 … 𝑎1𝑛 𝑎21 1 … 𝑎2𝑛 … … … … 𝑎𝑛1 𝑎𝑛2 … 1 ) (1)
Thus, AHP correlates several criteria and performs by peer comparison, identifying inconsistencies in the analyses. If the specialist specifies that b > a and a > c, therefore it is expected that b > c, otherwise an inconsistency is pointed out by the software. Thus, AHP displays an inconsistency index ("II") [17] [18], mathematically expressed by (Eq. 2). The maximum inconsistency allowed to guarantee the reliability of the chosen decision is II < 0.10. In the case of "II" being greater or equal to 0.10, it is necessary to adjust the comparisons before proceeding to the criteria analysis (Eq. 2):

𝐼𝐼 = 𝜆𝑚𝑎𝑥 𝑛 𝑛 1 (2)
where: "λmax" = auto maximum value; and "n" = matrix dimension or order of the matrix.

Then, the "consistency ratio" ("CR") can be obtained by (Eq. 3) [START_REF] Costa | Introdução ao Método de Análise Hierárquica: Análise Multicritério no Auxílio à Decisão[END_REF] [START_REF] Castro | Políticas Públicas e Priorização na Escolha de Modais de Transporte para Escoamento da Produção Agrícola Sul-Mato-Grossense[END_REF]:

𝐶𝑅 = 𝐼𝐼 𝑅𝐼 (3)
where "CR" corresponds to the "consistency ratio", related to the answers given by specialists; "II" represents the inconsistency index and "RI" is the "random index", calculated to square matrices of "n" order by the "Oak Ridge National Laboratory" -ORNL, being 1 = 0.00; 2 = 0.00; 3 = 0.58; 4 =0.90; 5 = 1.12; 6 = 1.24; and 7 = 1.32.

To determine the weights of each criterion, sub-criterion and alternatives, changes between the options should be compared, considering the preferred scenario and the less recommended one. Whenever possible, averages should override the judgments of specialists to avoid errors by the subjectivity of responses. To reduce the inconsistencies in the model, we opted to work with quantitative comparisons between the weights, using collected data from the case study and specialists' opinions [START_REF] Chun-Yu | An Improved Analytic Hierarchy Process and Application in Grain Production[END_REF].

Decision Tree

A decision tree was adopted to solve the proposed problem [START_REF] Tan | Introduction to Data Mining. Classification: Basic Concepts, Decision Trees and Model Evaluation[END_REF], considering two criteria and five sub-criteria. A second layer of 11 sub-criteria was considered from the five sub-criteria. Peer comparisons were performed considering Table 1.

The decision tree (Figure 1) identifies the objective, criteria and respective sub-criteria (in two layers), indicating the alternatives considered. Once the decision model variables were determined, the data was inputted in the Expert Choice® version 11 (2014), generating the results shown in Section 4.

Case Study

The case study refers to an experiment from Narita Airport to Tokyo Central Station, for the following PT modes: Automobile (own car); Taxi; Bus (Ordinary); Bus (Limousine Service); two Train options (Narita Skyaccess / Oedo Line and Narita Express), the first of them is a train with several stops and a compulsory transfer from one line to another, and the other is an express train, with few stops and no transfers; Cycling, Walking and Motorcycle, totalizing nine modes. Due to the AHP software limitation to eight alternatives, it was necessary to blend Bus (Ordinary) and Bus (Limousine Service) into a single alternative, simply called "Bus". However, this was a minor change that did not invalidate the analysis.

To perform the peer comparisons of the AHP method, the weight values of the disutilies per each mode of transportation were defined according to the bibliographical references shown in Table 1 and in transport specialist opinions. The purpose was created an overview of method application. Future studies intents to compare the judgments of specialist with the system users. Some restrictions of the AHP method, such as limited judgment scales and the consideration of which criteria are independent of each other, did not invalidate the results obtained.

3

Results and Discussion

The main objective of the study to measure the "Total Disutility". The results showed that an Automobile produces the highest level of disutility (0.182), followed by Bus, Taxi, Motorcycle, Trains, Walking, Cycling and Express Trains (0.083), as can be seen in Table 3. Apart from Bus, our result is close to that found in other studies, like Vasconcellos [START_REF] Vasconcellos | Corporación Andina de Fomento: Desarrollo urbano y movilidad en América Latina[END_REF]. EMPLASA [START_REF]Empresa Metropolitana de Planejamento da Grande São Paulo: Manual PAITIP: Programa de Ação Imediata de Transporte Integrado de Passageiros[END_REF] could explain the second position occupied by Bus. Additionally, the criterion "Passenger" has a higher weight (0.667), compared with the criterion "Society" (0.333). In the case of the criterion "Passenger", Automobile (0.169) produces the highest disutility and the lowest is related to Express Trains (0.092). Concerning "Society, the highest disutility is represented by Bus (0.277) and the lowest by "Walking" (0.026).

Sub-criterion (two layers) and Alternatives

The results corresponding to each sub-criterion and the alternatives weights are represented in Tables 4 and5 and discussed in the sub-items 3.2.1 to 3.2.5. The results are in general compatible with what is (currently) observed in the Greater Tokyo Area, concerning the level of supply of transportation modes (quality and quantity), the demand requirements and the equilibrium between these elements [START_REF] Raymundo | Measuring Passenger Transport Quality by Disutilities[END_REF].

3.1.1

Total Time of Displacement -Walking is the mode with the highest level of disutility caused by its very long travel time. In second place is Trains (long waiting time, travel time and transfer time). Cycling comes in third place (long travel time), followed by Bus. The lowest levels are represented by Motorcycle, Automobile and Taxi, while Express Trains play an intermediate role. Our result is compatible with what is observed in Tokyo, where Motorcycle, Automobile and Taxi perform almost the same average speed [START_REF] Raymundo | Measuring Passenger Transport Quality by Disutilities[END_REF].

3.1.2

Cost -Automobile shows the highest level of "Cost" due to its operating cost, followed by Walking (high time cost), Taxi, Trains and Cycling. In an intermediate position, there are the Express Trains and Motorcycle, while the lowest level is represented by Bus. Fares, and consequently their subsidies, influenced the performance of the public modes, determining their competitiveness. Vasconcellos [START_REF] Vasconcellos | Corporación Andina de Fomento: Desarrollo urbano y movilidad en América Latina[END_REF] shows Automobile with a very high cost, while public transportation has the lowest values.

3.1.3

Insecurity -Motorcycle represents the highest level of "Insecurity", followed by Automobile, Taxi, Walking, Cycling, Bus, Trains and Express Trains. Even in Japan, non-motorized modes (Walking and Cycling) are subjected to a high risk and are relatively less protected from traffic threats shared between Automobile, Motorcycle, Taxi and Bus, not to mention trucks. The data collected by Raymundo and Reis [START_REF] Raymundo | Measuring Passenger Transport Quality by Disutilities[END_REF] related to Tokyo confirms our result, where Motorcycle is the most unsafe, followed by Automobile, Walking and Cycling, Trains and Bus.

3.1.4

Discomfort -A trade-off occurs due to Trains showing the highest level of "Discomfort" and Express Trains the lowest. In Trains, compared to Express Trains, passengers in Tokyo face worse conditions concerning infrastructure (terminals and stations) and worse conditions in vehicle comfort. From the higher levels to the lower ones, we have Bus, Motorcycle, Cycling, Automobile and Walking. ANTP [START_REF]Transportes Públicos: Avaliando a qualidade da mobilidade urbana: aplicação de metodologia experimental[END_REF] reached similar conclusions when analyzing the discomfort of PT modes in São Paulo, Brazil.

3.1.5

Negative Impacts on Communities -The highest position is occupied by Bus, followed by Automobile, Taxi, Motorcycle, Trains and Express Trains, while the lowest level is represented by Walking and Cycling. Bus has the highest position in "Environmental Impacts" because most of them are still diesel powered, polluting much more than the other modes. Gibson et al. [START_REF] Gibson | Update of the Handbook on External Costs of Transport, Report for the European Commission: DG MOVE[END_REF], studying most of cities in the European Union countries, arrived at a similar result to ours.

Conclusions

The measurement of disutilities of the selected PT modes in the Greater Tokyo Area and the establishment of priorities among them allowed us to identify the reasons that influence the performance of these transportation systems.

When it is known that, on a scale from 0,000 to 1,000, where the values are complementary, the Automobile produces the highest level of disutility (0.182), followed by Bus (0.148), Taxi (0,140), Motorcycle (0.125), Trains (0.119), Walking (0.107), Cycling (0.098) and Express Trains (0.083), it is not difficult to suppose that similar situations are happening in most metropolitan areas of the world, requiring the minimization of total disutility produced by PT.

The results show that it is worthwhile for public transportation to invest in better trains, subsidize fares and substitute the bus fleet by vehicles that pollute less. For private transportation, it is worth protecting walking and cycling from the shared traffic threats. Finally, it is important to highlight in a more transparent way the harmful effects of automobiles, taxis and motorcycles. From this, it could be possible to improve them and then to integrate them to the non-motorized individual modes and to the public and collective modes in a more effective manner.

Fig. 1 .

 1 Fig. 1. Decision tree Case Study

Table 2 .

 2 Peer comparison nominal scale (Source: Adapt[START_REF] Saaty | Decision Making with the Analytic Hierarchy Process[END_REF])

	Scale	Meaning
	1	Equal importance
	3	Moderate importance
	5	Strong importance
	7	Very strong importance
	9	Extreme importance
	2, 4, 6 and 8	Intermediate values

Table 3 .

 3 General Result

	Objective and Crite-	Alternative Weights (bold highest disutility -italic lowest disutility)
	ria	Automo-	Bus	Trains	Walk-	Cy-	Taxi	Exp.	Motorcy-
		bile			ing	cling		Trains	cle
	TOTAL DISUTILITY	0.182	0.148	0.119	0.107	0.098	0.140	0.083	0.125
	Passenger	0.169	0.097	0.130	0.138	0.122	0.123	0.092	0.129
	Society	0.216	0.277	0.093	0.026	0.029	0.185	0.060	0.114

Table 4 .

 4 Sub-criteria (first layer) and alternatives weights

	Sub-criteria (first	Alternative weights (bold highest disutility -italic lowest disutility)
	layer)	Automo-	Bus	Trains	Walk-	Cy-	Taxi	Exp.	Motorcy-
		bile			ing	cling		Trains	cle
	Time of Displacement	0.077	0.136	0.156	0.196	0.147	0.081	0.141	0.066
	Cost	0.184	0.076	0.121	0.152	0.121	0.129	0.119	0.091
	Insecurity	0.224	0.067	0.050	0.135	0.102	0.143	0.044	0,229
	Discomfort	0.116	0.172	0.255	0.059	0.117	0.106	0.046	0.129
	Negative Inpacts on	0.216	0.277	0.099	0.026	0.029	0.185	0.060	0.114
	Communities

Table 5 .

 5 Sub-criteria (second layer) and alternatives weights

		Sub-	Alternative weights (bold highest disutility -italic lowest disutility)
	Sub-criteria	criteria
	(first layer)	(second	Automobile	Bus	Trains	Walking	Cy-	Taxi	Exp.	Motorcy-
		layer)					cling		Trains	cle
		Access	0.207	0.142	0.086	0.015	0.179	0.122	0.062	0.187
	Time of Dis-	Waiting	0.022	0.286	0.309	0.022	0.022	0.139	0.177	0.022
	placement	Travel	0.027	0.053	0.077	0.461	0.238	0.022	0.095	0.027
		Transfer	0.177	0.066	0.487	0.066	0.066	0.066	0.066	0.066
		Vehicles	0.262	0.086	0.092	0.020	0.020	0.301	0.074	0.145
	Cost
		Other)	0.158	0,073	0.130	0.195	0.162	0.074	0.134	0.074
	Insecurity	Accidents	0.224	0.067	0.050	0.135	0.102	0.143	0.044	0.236
		Infrastruc-
			0.094	0.068	0.232	0.204	0.133	0.094	0.037	0.137
	Discomfort	ture
		Vehicles	0.124	0.211	0.263	0.031	0.084	0.110	0.050	0.126
		Infrastruc-
			0.284	0.144	0.080	0.030	0.040	0.233	0.066	0.123
	Negative	ture
	Impacts on	Environ-
	Communities	mental Im-	0.190	0.328	0.098	0.025	0.025	0.167	0.058	0.110
		pacts


Frederick Sauermann
email: f.sauermann@wzl.rwth-aachen.de

Felix Basse
email: f.basse@wzl.rwth-aachen.de

Günther Schuh
email: g.schuh@wzl.rwth-aachen.de

Bastian Franzkoch
email: bastian.franzkoch@ortlinghaus.com

Jan-Philipp Prote
email: j.prote@wzl.rwth-aachen.de

Melanie Luckert
email: m.luckert@wzl.rwth-aachen.de

Bastian Franzkoch²

Analysis of the Potential Benefits of Digital Assembly Instructions for Single and Small Batch Production

Keywords: Assembly, Digitization, Learning effects

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

Introduction

Individualization of products and reduction of lead-times within a production are two of a set of challenges for modern manufacturing companies. Still, high quality remains a crucial factor. Mass production of homogeneous products is increasingly replaced by more customer oriented manufacturing. [START_REF]Mass Customization: Challenges and Solutions[END_REF] On the one hand, industrial production still tries to keep the costs low but on the other hand producers seek to serve the needs of the customers by supplying a high variety of products [START_REF]Leitfaden zur Baukastengestaltung: Ergebnisse des Forschungsprojekts Gestaltung innovativer Baukasten-und Wertschöpfungsstrukturen (GiBWert)[END_REF]. A big challenge for producers is to chase both goals without losing track of one of them [START_REF] Alptekinoğlu | Leadtime-variety tradeoff in product differentiation[END_REF]. In order to cope with these challenges, former manufacturing processes have to be revised as a whole [START_REF] Schuh | Fast Forward Factory[END_REF]. The principles of Industry 4.0 are commonly seen as one of the most promising answers to the demands of a new efficient production [START_REF]Industrie 4.0 kompakt. Competence Book[END_REF].

Research in this area is not comprehensive yet, but the following two studies have been carried out. HÖFFLER and LEUTNER give a summary of the research that was done in the field of digital assembly instructions [START_REF] Höffler | Instructional animation versus static pictures: A meta-analysis[END_REF]. Having analyzed 24 studies with in total 76 comparisons of the instructional effectiveness of animations and static pictures, they conclude that there is a "substantial overall advantage of animations over static pic-tures". How animations have been designed seems to play a major role for the effectiveness of an instructional animation. [START_REF] Michas | Learning a procedural task[END_REF] After this summary, CURRAN et al. conducted a study similar to the one in this paper. However, they did not exclude learning effects in favor of digital instructions from analog ones as their study participants assembled the products with both an analog and subsequently with a digital instruction. [START_REF]Collaborative Product and Service Life Cycle Management for a Sustainable World[END_REF] Especially the findings from HÖFFLER and LEUTNER emphasize the relevance of the regarded research question. In order to analyze the benefits of Industry 4.0 at a relatively basic level, a study was conducted researching the effects of using digital assembly instructions in comparison to conventional, paper-based ones in terms of productivity, quality and learning rate. In this study that will be presented in this paper, 24 test persons were asked to assemble penholders using either a digital or an analog assembly instruction and simulating a small batch production. The results have to be seen with caution, considering the number of test persons. Though this study serves as a first pointer for further research, the results suggest high benefits of implementing digital assembly instructions especially for single and small batch production. In the following chapter, the materials and methods used in the study are described.

Materials and Methods

The study was conducted at assembly stations in the Demonstration Factory Aachen (DFA), in which a transformable production is depicted in a real production environment and in which production systematic and logistical questions are researched on the basis of empirical data. The test persons were students of RWTH Aachen University who also work as student assistants in the Laboratory for Machine Tools and Production Engineering (WZL). None of the test persons knew the test before. In the following, the detailed execution of the tests is described and the underlying hypotheses presented.

Description of test execution

In total 24 students took part in the experiment on ten different dates in the DFA, assembling ten penholders per date using either a digital or an analog assembly instruction. On average, four test persons participated at each date. In order to get background information of the participants on age, field of studies and prior knowledge a questionnaire was handed out. Concerning prior knowledge the participants were asked for a self-assessment compared to their colleagues in terms of experience in assembling, experience in handling CAD programs, abilities in engineering drawing and overall technical abilities each on scale from one (very low) to five (very high). After completing the questionnaire, the test persons had to assemble a penholder by using either a digital assembly instruction or an analog one. A penholder consists of eight steel sheets that are to be assembled with different types of screws in several sizes combined with washers as shown in Fig. 1. The test persons needed to use Phillips and Allen screwdrivers in different sizes. They were told that they would assemble different versions of the penholder in each of the ten rounds. The exact number of variants was not known to the test persons. In fact, there have only been four different versions that differed in the orientation of the components and the selection of screws.

The paper-based instructions looked like the one shown in Fig. 1. The instructions for the different variants were collected in folders at the assembly stations. When starting the time, each test person got the number of the version to assemble at a desk between the assembly stations. Having got the number, the test person walked to his/her assembly station, looked up the matching instruction and began to assemble the penholder using the components lying on the stations in several signed boxes. After completion, the test person brought the fully assembled penholder back to the starting point, checking the time again. Subsequently, the penholder was inspected on defects.

The test persons with the digital assembly instructions were handed a laminated sheet of paper with a Radio Frequency Identification (RFID) tag on its back, which is used to communicate with a sensor above the assembly station. Placing this sheet of paper on the sensor, the digital instruction was automatically loaded on a touch screen according to the matching version. The digital instructions consisted of step-by-step animations with a list of c-parts and highlighted the right parts to assemble in a special color on a tablet screen. By touching the screen, the test persons could switch between the assembly steps and were able to turn the images in three dimensions.

Fig. 1. Example of a variant of the penholder

The group of participants consisted of 24 people being on average 22.2 years old. 21 test persons were male and three female. Half of the test persons were provided with digital assembly instructions and the other half got paper-based ones.

The participants were composed of students from the fields of mechanical engineering (25 %), industrial engineering (50 %) and scientific programming (25 %). They were asked to give a self-assessment about several aspects on a scale from 1 (very low) to 5 (very high). With regard to the assessment of the technical skills, the values of the mechanical engineering students were with 2.83 just slightly higher than those of the industrial engineering students with 2.67. The students of scientific programming assessed their skills with a value of 1.64 substantially lower. The allocation of the analog and digital instructions to the group of participants was done randomly. However, the self-assessment of the technical skills in the group with the digital instructions was on average 35 % higher than in the group using the analog one, which all scientific programmer were part of.

Hypotheses of the benefits

Before the study was carried out, three qualitative hypotheses were proposed for the expected results with regard to the improvement of the benefits in terms of (1) productivity, (2) quality and (3) learning rate (see Fig. 2). The first hypothesis states that the productivity of assembling a variant with a digital assembly instruction is higher than with an analog one with regard to lead-time. The second hypothesis is that the number of errors per assembled penholder is lower when the penholder is assembled using a digital assembly instruction. As a third hypothesis it was formulated that assemblers are quicker to learn how to assemble the penholders when using a digital assembly instruction, being quantified by using the learning rate. The learning rate describes the inverse relationship between costs per unit and number of units being produced. In the study, a proportional relationship between lead-time and costs per unit is assumed. Even though the quality of the products produce it is not represented in this figure, it still has to be kept in mind when assessing the learning rate.

3

Results and Discussion

In the following, the results will be presented. Subsequently, the findings based on the results will be discussed.

Results

Before analyzing the data in depth, the sample is checked on outlier. Analyzing the defects it becomes obvious that there is one outlier in the group of participants using the digital assembly instruction. While the assemblers in the group of study participants

Learning rate

Analog Assembly Instruction Digital Assembly Instruction Point of measurement using the analog instruction were a homogeneous group with a standard deviation of 1.19 with regard to quality, the group of participants using the digital instruction had one member who made disproportionately many mistakes resulting in a standard deviation of 2.6 for this group. Excluding this participant for the analysis of the data, the standard deviation drops from 2.6 to 1.24, which is close to the 1.19 of the other group. Hence, it makes sense from a statistical point of view to analyze the data excluding the outlier. The following results have arisen concerning the three hypotheses of productivity, quality and learning rate after excluding the outlier in the analysis.

The study confirms the expected outcome of a higher productivity when using a digital assembly instruction compared to the analog one (see Fig. 3). Assembling the first penholder, the study participants with the analog instruction needed on average 31.2 % more time (in total 12 minutes and 51 seconds) than the participants did with the digital one (9 minutes and 48 seconds). Even though the difference became smaller with the number of penholders assembled, the results for the digital assembly instruction were for the last five points of measurement on average still 14.9 % better. Overall, the productivity of the analog assembly instruction was 17.0 % worse, meaning that assemblers needed on average 01:01 minutes more time per piece.

Fig. 3. Development of lead time

Before going into the analysis of the results, it is important to understand the performance indicator used to describe the quality. Instead of using the simple number of defects per unit, the defects are weighted based on their severity. For example, using a wrong screw (e.g. different length) is less important for a penholder than a wrong position of the objects on the main plate (e.g. the container). Hence, the defects are weighted with factors from one to three.

Analyzing the plain data, the outcome was that the weighted number of defects was 96 defects for the analog assembly instruction while using a digital one resulted in 76 defects. Overall, the numbers mean on average 0.8 defects per assembled penholder for the analog assembly instruction and 0.69 for the digital one, which leads to the conclusion that the hypotheses can be confirmed as well. The outlier of weighted defects in the second point of measurement of the digital instruction can be explained by a high number of mistakes made by one test person. Comparing these figures, the study shows that the former group made on average 15.7 % more defects per penholder than the group with a digital assembly instruction. Additionally, it can be seen in the study that in case of using a digital instruction the last 80 % of the assembled penholders had only 6.6 % of the total number of defects, whereas the analog instruction had in the same timeframe 26 % of the total defects of the analog one. Directly comparing the absolute numbers, the group of assemblers with the analog instruction made five times more defects in this timeframe. For the first 20 % of the assembled products the ratio between the two kinds of assembly instruction is 1:1. These findings can be explained by the hypothesis, that a digital assembly instruction helps assemblers to understand the way of assembling the product better and more sustainably than the analog one.

Fig. 4. Weighted defects

While the first two hypotheses can be confirmed, the last hypothesis concerning the learning effects has to be rejected. As can be seen in Fig. 3, the learning rate of assemblers using the digital assembly instruction was 20.9 % whereas the assemblers with an analog instruction had a learning rate of 23.9 %. This means that for every doubling of the number of penholders having been produced the costs per unit fell on average on 79.1 % of the initial value of the assembly costs when using a digital instruction. Hence, the analog assembly instruction seems to be about three percentage points better with regard to the learning effects. However, taking quality in consideration when assessing this figure, the advantage of analog instructions declines.

Discussion

The results confirm two of the three hypotheses, if the outlier in the analysis of the quality is excluded. Still, it has to be pointed out that the number of participants in the study was small. Conducting the study with a higher number, the influence of the outlier on the results would have been less significant. With the number of participants being small, the results have to be seen critically. Especially the fact that the number of weighted defects of the last 80 % of assembled products account only for 17.4 % of the total number of defects and with 89.1 % of all assembled products being flawless, indicates that the wealth of variants of the penholder was too low for the participants. On the other hand, those numbers can show that a digital assembly instruction helps to improve quality significantly compared to an analog one, when assembling similar products in high quantity.

Concerning the productivity, the results indicate that a digital assembly instruction has a significant potential of reducing the lead-time per unit. In particular, the study shows that the potential is even more promising, the more variants are assembled with a small number of totally assembled products as the difference in the lead-time is at the beginning substantially higher. This finding is especially interesting for companies with small lot sizes, i.e. companies in the single and small batch size industry, and companies having a high employee turnover. Considering current developments of shorter product life cycles, this advantage of the digital assembly instruction is even more promising even for other industries.

As stated in chapter 2.1, all scientific programmers assembled the penholder using an analog instruction. With their self-assessment being as low as 1.64 compared to the 2.67 and 2.83 respectively, one might expect worse results for this group of participants. However, this is only the case in terms of quality but not in terms of lead-time. Having assembled almost twice as much defects (64.6 % of the total amount of defects in the group of participants using the analog instruction), they were as fast as the other part of the group with just one second less of the overall time needed. If the digital assembly instruction truly helps to understand the way of assembling products better and more sustainably as stated in chapter 3.1, needs to be further researched.

Having a look at the test execution, it has to be stated that there was a difference of the position of the assembly instruction. Whereas the analog one lied on the assembly table next to the components that were to be assembled, the screen for the digital one hung in front of the assemblers. Moving down the screen to the position of the analog instruction could lead to even better results of the digitally instructed products.

Conclusion

In times of an increasing amount of variants and shorter product life cycles, a study was conducted in order to analyze the potential of digital assembly instructions compared to analog ones in the closed environment of the DFA. Besides researching the consequences on lead-time and learning effects, the study observed the quality of the assembled products, in order to prevent a potential loss of quality in favor of lead-time.

As expected and formulated in the hypotheses, the study proved that digital assembly instructions lead to a rise in the productivity of the assemblers compared to their colleagues with analog instructions. Excluding one outlier from the test data of the digital assembly instruction, the study proved furthermore that a digital assembly instruction helps assemblers to assemble products not only faster but also with less defects in both short-and long-term perspective. In terms of learning effects, the analog assembly instruction showed better results, which is caused by high initial lead-times compared to the ones that used digital one. Hence, it is to further research under which conditions digital assembly instructions should be used, taking into consideration, that deriving digital instructions can be expensive in the single and small batch industry, it is to analyze how a critical number of products per variant can be determined to assess the economical suitability. By an increasing complexity of products, this question becomes even more relevant considering the advantages in terms of efficiency and quality.

With the number of participants being small, further research needs to be done to validate the results. In cooperation with the German mid-size company Ortlinghaus-Werke GmbH this will be done in their assembly lines in the plant Wermelskirchen.

Fig. 2 .

 2 Fig. 2. Hypotheses

  Regression line of analog assembly instructionRegression line of digital assembly instruction

Acknowledgements

The project, on which this report is based, is supported and realized by "Digital in NRW. The Competence Centre for SME in North Rhine-Westphalia". The Competence Centre is part of the funding initiative "SME 4.0 -Digital Production and Work Processes", which is funded as part of the research priority on "SME-Digital -Strategies for the Digital Transformation of Business Processes" by the German Federal Ministry of Economics and Energy.


Felix Optehostert
email: felix.optehostert@fir.rwth-aachen.de

Daniela Müller
email: daniela.mueller@fir.rwth-aachen.de

Philipp Jussen
email: philipp.jussen@fir.rwth-aachen.de

Dispositioning Strategies of Maintenance Tasks in Offshore Wind Farms

Keywords: Tool Asset Management, Operation and Maintenance, Disposition, Offshore Wind Farms

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

Introduction

Improving the profitability of offshore wind farms is essential for a successful and sustainable energy turnaround. However, in relation to other energy sources offshore wind farms are still not as competitive. This becomes obvious when comparing the levelized cost of energy (LCOE), a key performance indicator for profitability of energy sources (Figure 1. Levelized Cost of Energy [START_REF]Was kostet Offshore-Windenergie wirklich?: Eine makroökonomische Betrachtung[END_REF]). The LCOE depicts the costs of power production respective to the expected operating life of a power plant. This calculation considers the total lifetime cost, such as discounted capital costs, overheads and expenses for fuel divided by the lifetime discounted electricity outcome. As it can be seen in Figure 1 the LCOE for offshore wind farms exceed the LCOE for any other energy source.

To achieve a competitive advantage, the LCOE must be reduced. There are defined levers to do so, such as optimizing the O&M processes and organization, which consumes 28% of the [overall] lifecycle costs [START_REF] Berger | Offshore wind toward 2020: On the pathway to cost competitiveness[END_REF]. Consequently, O&M is a key value driver and crucial for improving the profitability of offshore wind farms. For example, a reduction of 10% O&M costs leads to an advance of 4% of earnings before interest and taxes (EBIT). An improvement of 1% of availability would lead to an increase of the EBIT by 2% [START_REF] Berger | Offshore wind toward 2020: On the pathway to cost competitiveness[END_REF].

Reducing O&M costs by optimizing the disposition efficiency is the overall goal of this study. Typologies of the disposition of maintenance tasks in offshore wind farms will be introduced. These typologies help to design the maintenance organization and determine the task and resource planning strategies. Effectively, it is used to design a software that plans and dispositions the tasks and resources in offshore wind farms. This software tool was developed as a prototype in the research project DispoOffshore, funded by the German Federal Ministry for Economics.

2

Maintenance in Offshore Wind Farms

The maintenance of offshore wind farms requires different standards and is more challenging than onshore maintenance. Those challenges and standards must be considered when designing disposition strategies for the maintenance operations.

Offshore wind farms are not easy to access, as they are placed several kilometers off shore and from the service hub. Therefore, vessels have to drop off the technicians by performing precise maneuvers. Additionally there is no access to the wind turbines when, due to bad sight or huge wave heights from the weather conditions, vessels cannot safely travel [START_REF] Cv | Offshore Windenergie: Studie zur Rentabilität von Offshore-Windparks in der Deutschen Nord-und Ostsee[END_REF]. The efficiency of a daily working plan is therefore dependent on the ideal allocation of maintenance tasks to service technicians and vessels. This assignment determines whether the routes of the vessels are optimal and whether the appropriate technician with the required qualifications will finish all necessary tasks. If this assignment is not performed in an optimum fashion, the vessels will have insufficient routes and too few tasks for the estimated working hours each day. These considerations apply to designing the working plan of each day.

Previous work in this field suggests different approaches on planning and control of tasks and resources. In relation to this paper, methods of production economics and service management were examined. The Production Planning and Control Model (PPC-model) addresses the termination of problems and allocations of production services and resources [START_REF] Schuh | Aachener PPS-Modell[END_REF]. This approach minimizes processing time needed to plan more tasks with limited resources (here: workers). To deal with the problem of combined tours and personnel planning within short time windows, ZÄPFEL and BÖGL came up with multi-period vehicle routing and crew scheduling with outsourcing options. The strategy considers different vehicles with variable capacities and outsourcing options for tours and external staff [START_REF] Zäpfel | Multi-period vehicle routing and crew scheduling with outsourcing options[END_REF]. The Product Service System (PSS) capacity planning, by KRUG, schedules the performance of PSS close to optimum. One part of this concept is the prediction of future service dates and the warrant resources [START_REF] Krug | Framework zur strategischen Kapazitätsplanung hybrider Leistungsbündel[END_REF]. IRAWAN developed a strategy to optimize maintenance routing and scheduling for offshore wind farms. They also proposed a solution method based on a Dantzig-Wolfe decomposition of the problem, which strived to find the optimal route that minimizes maintenance costs [START_REF] Irawan | Optimisation of maintenance routing and scheduling for offshore wind farms[END_REF]. STÅLHANE evolved a method for the similar purpose of optimizing the routing and scheduling of vessels to perform maintenance at offshore wind farms. On the other hand, two alternative models of optimization were assessed: an arc-flow and a pathflow [START_REF] Stålhane | Optimization of routing and scheduling of vessels to perform maintenance at offshore wind farms[END_REF].

None of the previously mentioned approaches meets all requirements for the dispositioning in offshore wind farms on an operational level. The research by IRAWAN and STÅLHANE are most relevant to this paper. However, they do not take into consideration the staff's qualification, organizational circumstances nor a prioritization of the tasks, as an important part of the disposition on an operational level.

Disposition Strategies of Maintenance Tasks and Resources in Offshore Wind Farms

Based on the special requirements of offshore wind farms, a morphological analysis of the disposition of maintenance tasks in offshore wind farms is introduced. This analysis can be used to design the maintenance processes and disposition strategies for the offshore wind farm's maintenance organization.

The Morphological Analysis

The morphological analysis (or box) was the chosen technique to identify and evaluate the imposed requirements of offshore wind farms. As there are complex restrictions for the maintenance organization of offshore wind farms, it is hard to identify a solution which meets all requirements. The morphological box depicts all the requirements while helping to determine the best disposition strategy.

The morphological analysis was first introduced by ZWICKY, showing the totality of conceivable solutions for a problem. First, the problem is defined, analyzed, and generalized to partial aspects. These partial aspects are classified with independent parameters, which can apply to all possible solutions. After this, the parameters are specified with their characteristics. All parameters must be depicted in the morphological box, the dimensions of which equal the characteristics. All possible solutions have to be assessed and evaluated without prejudice before the final decision can be made [START_REF] Zwicky | Entdecken, Erfinden, Forschen im morphologischen Weltbild[END_REF].

The morphological analysis is suitable to describe complex structures by organizing the tokens of dispositioning. By doing so, the parameters must fulfill the following criteria: purpose significance, ability to ascertain, differentiability, integrity, ability to quantify, and independence from one another [START_REF] Ley | Entwicklung von Entscheidungshilfen zur Integration der Fertigungshilfmitteldisposition in EDV-gestützte Produktionsplanungs-und -steuerungssysteme[END_REF][START_REF] Wf | Entwicklung von Anforderungen und Gestaltungsvorschlägen zur Integration von flexiblen Fertigungszellen in den Informationsfluß der PPS[END_REF][12]. In the following, a morphological analysis for the dispositioning strategies in offshore wind farms is introduced.

Morphological Analysis of Wind Farm Specific Requirements

The morphological analysis was performed throughout interviews and surveys with experts in the field of dispositioning tasks for offshore wind farms. The results of these expert interviews were partitioned into 4 categories, which cover 26 parameters. These parameters can apply to a variety of feasible solutions, and each are specified with 2 to 7 characteristics.

These categories involve the "Sequence of dispositioning steps", "Prioritizing and optimizing", "Assignment team and means of transport" and "Weather", all described in the following.

The category "Sequence of dispositioning steps" contains all of the steps needed to assign tasks to the respective qualified person or resource, assemble the teams, and assign the means of transport (Figure 2). The chronology of these steps can vary and therefore influences the outcome of the disposition strategy. The allotted number determines on which position, within the heuristic, the respective assignment step would be.

Fig. 2. Sequence of dispositioning steps

The category "Prioritizing and optimizing" determines whether the tasks were preselected during preparation, whether the tasks were allocated manually, automatically, or semiautomatically, and whether the tasks are divided into subtasks ad recombined to task bunches (Figure 3). Furthermore, parameters for the prioritization of tasks found in the task repository and the optimization of criterion for the algorithm are incorporated in this category. Either the due dates, locations, first-in-first-out principal or their urgency, which can be related to opportunity costs by not operating wind turbines, can prioritize tasks. This prioritization influences the order of the tasks in the repository. To receive the optimal task disposition, the algorithm needs a criterion to optimize. The criteria meet targets, such as making full use of the ships' or teams' workload, utilizing Categories Characteristics Parameters shorter routes, lowering costs, minimizing the preparation time, the shortest overall duration or maximizing the quantity of disposed tasks.

Fig. 3. Prioritizing maintenance tasks

The category "Assignment teams and means of transport" determines all aspects that are needed for the team allocation. Figure 4 shows alternative options regarding the means of transport as a part of this category. This selection can be made manually, semi-or full-automatically, but there are also different options within the means of transport themselves. Either a helicopter or a ship can be used. Although the helicopter requires less time to reach the wind farm, it is more expensive than a ship transfer. However, this is only necessary for urgent repairs during weather conditions the ships cannot handle.

Fig. 4. Team assembling and means of transport

The last category, "Weather", displays if certain weather characteristics are considered in the distribution (Figure 5). This covers the wind direction, wave height, restricted sight (due to fog or night time), and tide.

Yes

Disposition Strategy Development

A disposition strategy is an approach to make fast disposition decisions with justifiable expenditure for the purpose of the striven for targets [START_REF] Hellingrath | Modellierungsrahmen für Dispositionsstrategien in GNL[END_REF]. Based on the morphological analysis, described in the previous section, different disposition strategies for offshore wind farms can be designed. This can be achieved by choosing a specific path through the morphological box. The tasks are defined by several attributes, such as the assumed time the technicians need to accomplish the task, the due date to make sure that the guarantee does not expire, and the priority measured by urgency such as troubleshooting over regular maintenance and rectification of defects to inspections. A list of required qualifications confines the variety of teams and means of transport. In addition, lists with needed material and tools, as well as the location of the task, whether it is on a wind energy plant and the platform, are part of a task's characterization. Changes in characteristics of a parameter lead to different disposition strategies. The relevant characteristics depend on the operational procedures for maintenance of the considered wind farm.

Two examples: As there are only six food boxes available per wind turbine the maximum number of people on a turbine is set to six. These boxes contain supplies for workers in case recovery is not possible at that time. This needs to be considered when assigning tasks to the technician teams on the same wind turbine.

Additionally, altering the choice for means of transport has a significant effect on the disposition too, as displayed in Figure 6. Using a ship offers two different opportunities of usage. A so-called "taxi service", which does not restrain the teams to the use of only one ship, allows the team to use any vessel in the wind farm on demand. Using a so-called "shuttle service" links the teams to a certain ship which has a predefined route through the wind farm. Figure 6 illustrates the alternation between a "shuttle service" to a "taxi service". These modifications have a decisive effect on the disposition software, effects on the time the teams spend on one ship and how many ships are in use.

It becomes obvious that by altering the boundary conditions and characteristics, the disposition strategy changes.

Conclusion

Introduced was a morphological analysis, which enables a systemic approach to design the disposition strategy of offshore wind farms. This morphological analysis was acquired by interviews and surveys with experts in the field of dispositioning tasks for offshore wind farms and based on the special requirements of offshore wind farms regarding the maintenance organization, planning, and control. The results show 4 parameter categories, which cover 26 parameters. These parameters can apply to a variety of feasible solutions and are specified with each 2 to 7 characteristics. Based on the described morphological analysis, different disposition strategies for offshore wind farms can be designed. The consequences of choosing different characteristics were shown in examples. The work presented is the basis for designing a software-based dispositioning tool for usage in offshore wind farms. This software tool will help to increase the effectiveness of the disposition in offshore wind farms by maximizing the number of finished maintenance tasks per day and minimizing the time technicians stay on the wind turbine and the ships. The next steps include the validation of this software tool, in total, during live operation in a wind farm. Further research should be conducted in regards to digitalize and connect the whole data chain, from automatic generation of maintenance tasks to automatic control and disposition of maintenance resources.

Fig. 1 .

 1 Fig.1. LOCE[START_REF]Was kostet Offshore-Windenergie wirklich?: Eine makroökonomische Betrachtung[END_REF]

  Fig. 5. Weather

Fig. 6 .

 6 Fig. 6. Means of transport


S Fahiminiya

S Roche

N Ge´rard

Improvement of 2D-PAGE Resolution of Human, Porcine and Canine Follicular Fluid: Comparison of Two Immunodepletion Columns

Follicular fluid provides the microenvironment within which somatic cells proliferate and differentiate, and the oocyte matures. It contains a number of soluble factors implicated in various stages of follicular development, most of them being functionally unknown. The presence of several high-abundance proteins, mainly originating from the blood circulation, is a major challenge of follicular fluid proteomic analysis, as these proteins can mask or decrease the visualization of follicle-specific proteins. In this study, we evaluated the efficiency of two immunodepletion columns (ProteomeLabÔ IgY-HSA and MARS-6) on follicular fluids of human, porcine and canine prior to 2D-PAGE. Our results showed that both columns were suitable to remove some of the high-abundance proteins present in human and canine follicular fluid. In conclusion, we demonstrated that the immunodepletion strategy enables the detection of new protein spots, increases resolution and highly improves the intensity of low-abundance proteins by 2D-PAGE.

Introduction

Follicular fluid (FF) accumulates in the antrum of growing follicles. It provides the microenvironment within which somatic cells proliferate and differentiate, and the oocyte matures. Previous proteomic studies on follicular fluid of various species [START_REF] Cabrera | Protein electrophoretic patterns of bovine and porcine ovarian follicular fluid[END_REF][START_REF] Angelucci | Proteomeanalysisof humanfollicular fluid[END_REF][START_REF] Fahiminiya | Steroid hormones content and proteomic analysis of canine follicular fluid during the preovulatory period[END_REF] demonstrated that it contains several high-abundance proteins (albumin, immunoglobulins, alpha-1-antitrypsin, transferrin and haptoglobin), which constitute approximately 85-90% of the total protein mass [START_REF] Jacobs | Utilizing human blood plasma for proteomic biomarker discovery[END_REF]. The presence of these high-abundance proteins in follicular fluid can preclude the identification, or even the detection, of follicle-specific proteins (low-abundance proteins) which locally produced and secreted by ovarian cells into the follicular fluid [START_REF] Fortune | Follicular development: the role of the follicular microenvironment in selection of the dominant follicle[END_REF]. Detection and identification of these low-abundance proteins like growth or differentiation factors in follicular fluid would help to better understand ovarian follicular growth and oocyte development in mammals. Therefore, an efficient approach to overcome this challenge might be to remove some high-abundance plasma proteins from follicular fluid prior to its proteomic analysis. Recently, the application of immunodepletion columns has been raised to remove up to 20 high-abundance proteins from human biological fluids. To our knowledge, no pre-fractionation method was developed specially for follicular fluid. In this study, we evaluated the efficiency of two immunodepletion columns (ProteomeLabÔ IgY-HSA and MARS-6) to remove high-abundance proteins from human (HFF), porcine (PFF) and canine (CFF) follicular fluids prior to 2D-PAGE analysis. These two columns are based on either immunoglobulin G (IgG) in mammals [START_REF] Martosella | Reversed-phase high-performance liquid chromatographic prefractionationofimmunodepleted humanserum proteins to enhance mass spectrometry identification of lower-abundant proteins[END_REF] or immunoglobulin yolk (IgY) in chickens [START_REF] Zhang | The use of gene-specific IgY antibodies for drug target discovery[END_REF][START_REF] Huang | Immunoaffinity separation of plasma proteins by IgY microbeads: meeting the needs of proteomic sample preparation and analysis[END_REF][START_REF] Linke | Rat plasma proteomics: effects of abundant protein depletion on proteomic anal-ysis[END_REF]). Indeed, the immunodepletion of follicular fluid allowed us to visualize several peptides and proteins on 2D gels, previously masked by high-abundance proteins.

Material and Methods

Porcine ovaries were collected at the slaughterhouse, and the follicular fluids were punctured from ovaries with a 20-G needle. Canine follicular fluids were aspirated from ovaries collected by ovariectomy [START_REF] Fahiminiya | Steroid hormones content and proteomic analysis of canine follicular fluid during the preovulatory period[END_REF]. Human follicular fluids were collected according to [START_REF] Guerif | Overnight incubation improves selection of frozen-thawed blastocysts for transfer: preliminary study using supernumerary embryos[END_REF] method. In each species, the follicular fluid was collected from three different ovaries. After collection, follicular fluid samples were centrifuged for 10 min at 1500 • g and stored at )80°C till immunodepletion step.

Two immunodepletion spin columns were used to remove high-abundance plasma proteins from the follicular fluids of human, porcine and canine species. As far as, we know these two immunodepletion methods has no side effect on the protein structure. All steps were performed at room temperature as recommended by the manufacturers. The depleted fractions were concentrated by 10-kDa molecular mass cut-off centrifugal concentrators (Microcon Ò ; Millipore, Bedford, MA, USA), and the pH of fractions was neutralized by 50 mM Tris pH 8.8. Names and properties of the two immunodepletion columns are presented in Table 1.

All the chemicals and materials used for proteomic analysis were purchased from Bio-Rad (Marnes-la-Coquette, France), unless otherwise indicated. The protein assay and 2D-PAGE analysis were performed as previously described [START_REF] Fahiminiya | Steroid hormones content and proteomic analysis of canine follicular fluid during the preovulatory period[END_REF]. Briefly, the total protein content of crude and depleted follicular fluids was determined using DC (detergent compatible) assay with bovine serum albumin as standard (Pierce, Rockford, IL, USA) and statistically analysed by the non-parametric Kruskal-Wallis test. For analytical separations, 2D-PAGE was performed on three individual samples in each species: 100 lg of proteins was loaded upon ReadyStripÔ IPG (11-cm IPG strips, pH range 3-10) that were used for isoelectric focusing (IEF). After IEF, the strips were equilibrated with SDS-PAGE equilibration buffer containing DTT (2%) and iodoacetamide (2.5%). Equilibrated IPG strips were subjected to SDS-PAGE (10%). Electrophoresis was run at 10 mA⁄gel for 45 min and 200 V until the dye front reached the bottom of the gels (5-6 h). Gels were stained with silver nitrate and were scanned at 16-bit resolution by ImageScanner (Amersham Pharmacia Biotech, GE Healthcare Europe GmBH, Orsay, France). Resulting TIFF images were analysed with Progenesis work station software (version 2008; Nonlinear Dynamics Ltd, Newcastle upon Tyne, UK).

Results and Discussion

The major challenge of follicular fluid proteomic analysis is its high similarity with plasma, in which a few proteins (high abundance) constitute more than 80% of the total protein content. One efficient methodology prior to its proteomic analysis could be the depletion of high-abundance proteins, to visualize, and consequently identify some lower-abundance ones. The latter may be most probably produced locally by follicle cells. To our knowledge, no method has been developed to deplete follicular fluid before its proteomic analysis. Here, we compared the efficiency of two immunodepletion columns in three species, with the aim to determine whether the removal of high-abundant proteins could improve the resolution of 2D protein patterns of follicular fluid.

Depletion efficiency and reproducibility of Proteom-eLabÔ IgY-HSA and MARS-6 on HFF, PFF and CFF were determined after the measurement of the total protein content (Table 2) and 2D-PAGE analysis (Fig. 1). The mean of total protein concentration of crude follicular fluids varied among species, the higher concentration being observed for PFF and the lower for HFF. In addition, both columns displayed the higher depletion efficiency (87.27 ± 2.56% and 81.81 ± 3.65%, respectively) for HFF. This can be explained by the fact that the antigens used in the immunodepletion columns are generated against human proteins.

Crude and depleted fractions of HFF, CFF and PFF were resolved by 2D-PAGE, to visualize the efficiency of the two immunodepletion columns tested (Fig. 1). The 2D-PAGE proteins profiles of crude follicular fluid of human, porcine and canine showed some similarities (Fig. 1, column A). Of note is the fact that some highabundance plasma proteins (especially albumin and immunoglobulins) were visualized as intense and large spots on the 2D-PAGE, which most probably mask lowabundance proteins. The total numbers of protein spots detected on the 2D-PAGE of crude PFF (794) was higher than on the crude HFF (592) and crude CFF (627). Although the total number of spots revealed by 2D-PAGE has the same trend as the total protein concentration measured in the three species (PFF > CFF > HFF), the reason of this difference among species remains unknown. It may be due to the higher quantity of some proteins or the presence of some species-specific peptidergic factors.

The 2D-PAGE protein profiles of depleted follicular fluids with ProteomeLabÔ IgY-HSA showed that albumin was completely removed from HFF and CFF, but partially from PFF (Fig. 1, column B). This result is consistent with the depletion efficiency percentages that we measured for this column. The reason why albumin is completely removed from CFF, but not from PFF, is not clear. One possible hypothesis could be explained by a higher similarity of antigenic sites of albumin between human and canine, than between human and porcine. With the aim to evaluate the capacity of ProteomeLabÔ IgY-HSA to bind non-specific proteins, 2D-PAGE was performed on the bound fraction of follicular fluid. We observed that only albumin was removed from follicular fluid of all species and no additional protein spot was visualized on 2D gels (data not shown).

The 2D-PAGE protein profiles of depleted follicular fluids with MARS-6 showed that albumin was removed completely from HFF and CFF, but again partially from PFF (Fig. 1,column C). In contrast, we observed that both heavy and light chains of immunoglobulins were completely depleted in all three species. This result was in accordance with the depletion efficiencies (HFF and CFF > PFF). Figure 1C clearly showed that HFF depleted with MARS-6 exhibit many more protein (+254) spots in entire pH interval as compared with crude samples. Again, this can be explained by the specificity of this immunodepletion column against human proteins. Concerning CFF, some proteins showed a higher intensity after depletion by MARS-6, compared with crude follicular fluid, but no new proteins were revealed. Finally, the effective removal of immunoglobulins from PFF did not exhibit any new proteins after depletion by MARS-6. Taken together, our data suggest that immunodepletion of the six most highly abundant plasma proteins using the MARS-6 column is more effective at reducing the dynamic range of follicular fluid proteins than the ProteomeLabÔ IgY-HSA column, and results in the visualization of more medium-and low-abundance follicular fluid proteins by 2D-PAGE.

Conclusion

This study provides the first comprehensive investigation of commercially available immunodepletion columns, to improve the protein profiling of follicular fluid derived from human, porcine and canine. In this study, we showed that both immunodepletion columns were more effective to remove high-abundance proteins especially albumin from HFF and CFF, compared with PFF. In addition, new proteins were revealed in HFF 2D-PAGE after depletion by MARS-6. Based on our results, we conclude that both columns are efficient and reproducible methods that can be used in combination with 2D-PAGE and ⁄ or mass spectrometry before proteomic analysis of human and CFF.

Fig. 1 .

 1 Fig.1. Representative 2D-PAGE of crude and depleted follicular fluids from human (HFF), porcine (PFF), and canine (CFF). Proteins samples (100 lg) were applied to non-linear pH 3-10 IPG strips in the first dimension and then separated on a 10% SDS-PAGE gel in the second dimension. Proteins were visualized by staining with silver nitrate. The positions of some of high-abundance plasma protein are marked in crude follicular fluid 2D-PAGE: ALB, albumin; A1AT, alpha-1-antitrypsin; TF, transferrin; HP, haptoglobin; APOA1, apolipoprotein A-I; IGHC, immunoglobulin gamma heavy chain; IGLC, immunoglobulin gamma light chain. The '+' on the images shows the appearance of new proteins or increase the intensity of some proteins after immunodepletion (S.Fahiminiya and N. Ge´rard, personal communications)

Table 1 .

 1 Major characteristics of the evaluated immunodepletion columns

			Multiple affinity removal
		ProteomeLabÔ	spin cartridge -human 6
	Binding specificity	IgY-HSA	(MARS-6)
	Albumin	+	+
	Alpha-1-Antitrypsin	)	+
	Haptoglobulin	)	+
	IgA	)	+
	IgG	)	+
	Transferrin	)	+
	Loading capacity in ll	1 0	1 4
	Method for immunodepletion		Spin columns

Table 2 .

 2 Total protein concentration and the percentage of depletion efficiency in crude and depleted FF samples

				Depletion efficiency (%; mean ± SEM)
		Total protein concentration (mg ⁄ ml; mean ± SEM)	ProteomeLabÔ IgY-HSA	Multiple affinity removal spin cartridge -human 6 (MARS-6)
	HFF	81.85 ± 16.45	87.27 ± 2.56	81.81 ± 3.66
	CFF	85.00 ± 9.00	83.13 ± 1.74	78.30 ± 6.50
	PFF	91.40 ± 2.10	84.98 ± 1.02	72.90 ± 11.10

CFF, canine follicular fluid; HFF, human follicular fluid; PFF, porcine follicular fluid; IgY, immunoglobulin yolk. All values are based on three independent experiments. Differences are not significant. The depletion efficiency was calculated with following formula: ðProtein amount ðlgÞ in crude FF À protein amount ðlgÞ in depleted FF Â 100%Þ= ðprotein amount ðlgÞ in crude FFÞ:

Ó 2011 Blackwell Verlag GmbH

Acknowledgements

The authors thank Dr Dominique Roye`re, Dr Joe¨lle Dupont and Dr Karine Reynaud for providing human and canine follicular fluids, respectively. Pascal Papillier is acknowledged for collecting porcine ovaries from slaughterhouse. SF and NG thank Dr Sylvain Lehmann for accepting them in his laboratory during the test of immunodepletion columns. This research programme was financially supported by INRA, France. Somayyeh Fahiminiya was PhD student supported by a fellowship from INRA.

Conflict of interests

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

Author contributions

SF carried out immunodepletion, 2D-PAGE analysis of HFF, PFF and CFF, computerized image analysis and drafted the manuscript. SR helped for immunodepletion of follicular fluid samples. NG designed and supervised the study, participated to the immunodepletion of follicular fluid samples and revised the manuscript. All authors read and approved the final manuscript.


Typological and historical-comparative perspectives on tone and vowel length in Ryukyuan

A B b. disyllables J. 2.1 2.2 2.3 2.4 2.5 R. A B C c. trisyllables J. 3.1 3.2 3.4 3.5 3.6 3.7 R. A B C D?

2 The heart of the problem

(Hattori , 1979a,b,b): a. several dialects have unexpected long vowels, i.e. not deriving from monophtongization b. correlation between those vowels and tone classes c. C-class dissyllables: CVːCV :: CVĆV

The symbol "μ" stands for a mora. The level pattern (High? Mid?) of Shuri is left unmarked. I apologize if any error happened in the conversion process, but this likely has no influence on the conclusions presented here. Also, my use of "tone" is rather liberal, and it can broadly refer to "pitch" or "melody". a. vowel length goes back to proto-Japonic, and tonogenesis occured several times independently in the Japanese archipelago (Hattori 1979a,b;[START_REF] Shimabukuro | A reconstruction of the accentual history of the Japanese and Ryukyuan languages[END_REF][START_REF] Vovin | Long vowels in proto-Japanese[END_REF]) b. vowel length goes back (at least?) to proto-Northern Ryukyuan [START_REF] Lawrence | Kita Ryūkyū sogo no meishi onchō: Shiron[END_REF][START_REF] Lawrence | Historical reanalysis in the Nakijin dialect noun accentuation system[END_REF][START_REF] Matsumori | Ryūkyū ni okeru 2-onsetsugo dai 4•5-rui no gotō chōboin o meguru mondai: Kita Ryūkyū sogo no akusento saiken ni mukete[END_REF][START_REF] Matsumori | Ryūkyū akusento no rekishiteki keisei katei: Ruibetsu goi 2-hakugo no tokui na gōryū no shikata o tegakari ni[END_REF]) c. vowel length is a secondary development from a *HL pattern (Boer 2010: 208-246;Hirayama et al. 1966: 173;[START_REF] Kindaichi | Akusento kara mita Ryūkyūgo shohōgen no keitō[END_REF][START_REF] Kindaichi | Nihongo sogo no akusento to Ryūkyū hōgen[END_REF][START_REF] Pellard | Once again on long vowels and register in Proto-Japonic[END_REF][START_REF] Pellard | Kita-Ryūkyū shohōgen ni mirareru chōboin to onchō no taiō kankei ni tsuite: Okinawago chūnanbu hōgen ni okeru C-keiretsu no gotō chōboin wo chūshin ni[END_REF]) d. vowel length is a secondary development from *HL in pNR (Thorpe 1983: 128-152) 3 Typology of tone-length historical interactions

(

( 32 )

 32 Such words imply that: a. B & C cannot have been distinguished by vowel length only b. otherwise there could not be any B-class word with an initial long vowel c. why do 'hundred' , 'turtle' , 'pigeon' keep their long vowel? 10 C-class words without initial long vowels (33) Some C-class dissyllables: a. shift to class A in Asama & Okazen and class B in Kin b. have a HL/F pattern in Nakijin, Ie or Wadomari c. all are cases of dissyllables reduced to monosyllables, without initial long vowels d. suggest earlier *HL (or at least *RːL), and not *RːH or *LːH (34) Okazen: Falling reduced dissyllables (not †LːH, not †Rː) a. *kage > kàːɡ ́'shadow' but b. *keu > k'jûː 'today' c. *naka > nâː 'middle' d. *u(w)a > w'âː 'pig' (35) Wadomari: some reduced dissyllables point to earlier *HL a. *kage > hàɡí hàɡí 'shadow' but b. *keu > çûː çûː 'today' c. *ko(w)e > ɸúì ɸúì 'voice' (36) *uwa (C) 'pig': Asama Okazen Kametsu Wadomari Ie Nakijin Kin Shuri w'áː w'âː w'áː w'áː w'âː w'âː wǎː w'aː a. vowel length should have prevented contraction b. should appear as †ʔuːwa in Asama, Okazen, Kin and Shuri 11 More internal reconstruction (37) C-Class dissyllables have split reflexes in Asama and Okazen according to the presence or absence of initial glottalization Asama Okazen a. *kage kàːɡ ́kàːɡ ́'shadow' but b. *iki ʔíːkì ʔíːkí 'breath' c. *usu ʔúːs ̀ʔúːs ́'mortar' (38) Possible reconstructions: a. *LːH | *RːH > HːL | LːH in Asama??? b. *LːH | *RːH > HːH | LːH in Okazen??? c. I suggest *HL > RːL > Asama LːH | HːL, Okazen LːH | HːH (39) B-class and particles: Asama, Okazen Kanami, Yonama LRː, LLː-H LRː, LRː-L a. in some dialects, rightward tone spread occurs onto a following particle, resulting in an "unaccented" pattern b. but other dialects have a fall between the noun and the particle c. *LH(-L) is the most likely origin d. problematic for if B-class was "unaccented", with a final H (e.g. Thorpe 1983) vowel length in C-class dissyllables is likely a secondary development b. B-& C-class dissyllables must have been distinguished by their tone pattern c. we can probably dispense with reconstructing vowel length, but tone patterns are definitely required d. comparative and internal evidence point to earlier *HL (41) General correspondence for B-& C-class words between: a. the location of the "rising accent" in Yoron b. the location of the fall (accent?) in Kametsu c. the location of the vowel length in Asama & Okazen, with H shifted one mora to the right d. the location of the last Low in Wadomari & Kin (i.

  If vowel length is original: a. why does Shuri preserve it in C-class dissyllables only? b. why does Nakijin loose it in C-class dissyllables only? c. why does Kin loose it in A-class dissyllabic nouns and B-& C-class trisyllables only? and what about the length in the first syllable of B-class dissyllables? d. vowel length is clearly correlated with both tone class (i.e.

	3.2 Tone > length (27) Shuri: prosodically lengthened vowels often shorten in com-	b. HL pattern in Amami Ōshima, Kikai, Yoron, Tokunoshima,
	pounds, but not etymologically long ones	North Okinawa (and Southern Ryukyuan)
	(10) Czech (Slavic,Kortlandt 1975: 19, but see Kapović 2005): a. juː 'night' + naːka 'middle' → junaka 'midnight'	c. short vowels everywhere else
	a. *vòl'ā [vǒlʲaː] > vůle [ˈvuːlɛ] 'will' b. *kòžā [kǒʒaː] > kůže [ˈkuːʒɛ] 'skin' b. naːbi 'pan' + keː 'spoon' → nabiɡeː 'ladle' c. tɕaː 'tea' + doːɡu 'tool' → tɕadoːɡu 'tea set' d. ʔáɡì 'frying' + toːɸu 'tofu' → ʔáɡídòːɸù 'fried tofu' (11) Noto Japanese (Hirako 2015): Bessho Hannoura (28) Asama: shift of the location of length between derived words	(20) Argument from parsimony: a. vowel shortening: at least 4 times independently b. vowel lengthening: around 3 times independently
	2.5 *LF > M͡ LːH(-L) LːH *àmê > a᷆ ːmé àːmé a. k'úːɾù 'black' (n.) ↔ k'ùɾùːháŋ 'black' (adj.) 'rain' b. s ːɾù 'white' (n.) ↔ s ɾùːháŋ 'white' (adj.)	6 Distribution of vowel length
	(12) Iwate Japanese, Kunohe dialect (Uwano 1996b): 2.4 ∪ 2.5 CV [-high] CV [+high] *HL > RːL | LːH | M͡ LːH *mátù > mǎːdzɯ̀| màːdzɯ́| ma᷆ ːdzɯ4 More problems and solutions (21) C-class is well-attested for nouns only (29) Asama: length alternations within verb paradigms a. no reason for vowel length to be restricted to nouns Conclusive ↔ Converb b. verbs and adjectives commonly display fewer tonal or ac-a. k'ìjùí ↔ k'íːtɕ ̀'cut' centual patterns than nouns c. restriction naturally explained is length is secondary b. nàjùí ↔ nàːt ́'become'
	(13) Some dialects have long vowels in classes other than C Asama Kametsu Wadomari Nakijin Kin Shuri A 'cow' ʔús ː ʔùs (-H) ʔùɕí ɸùɕíː ʔúɕí ʔúɕì B 'island' s mǎː s má(-L) ɕìmǎː ɕìmǎː ɕíːmǎː ɕima C 'pan' nàːb ́náb ̀nàbí nábì nàːbí naːbi (14) Some dialects have long vowels in trisyllables too Asama Kame. Wadom. Nakijin Kin Shuri A 'smoke' k bùːs ́k búɕí çìbúɕí kìbúːɕì kíbúɕí kíbúɕì B 'mirror' kàɡàmǐː kàɡàmí hàɡànǐː kàɡàːmíː káɡámǐː kaɡaŋ C 'knife' kàtàːná kàtánà hàtàná hàt'àːná kàtàná katana (15) Thorpe (1983): A /μ/ *H /μμ/ *HHˑ /μμμ/ *HHˑH ? 9 B-class words with initial long vowels (30) pNR (pR?) *pjaaku (B) 'hundred' Asama Wadomari Yoron Nakijin Ie Kin Shuri ɕàːkǔː çàːkúː pjàːkú pjàːkú: ' t'jàːɸú háːkǔː çaːku (31) Proto-Okinawan *poːtu (B) 'pigeon' and *kaːme (B) 'turtle' Ie Nakijin Kin Shuri a. *pot'u pòːt'ú pòːt'úː hóːtùí hoːtu b. *kaːme hàːmí hàːmíː kàːmǐː kaːmiː	(22) to an
	B /μ/ *R /μμ/ *LHˑ, *LLˑ-H /μμμ/ *LLHˑ, *LLLˑ-H ?	accent in the case of high vowels
	C a. no explanation for the development of length /μμ/ *LˑH(-L) /μμμ/ *LHˑL ?	a. /(cu-ci)k'a/ b. /(hici)-(ci * k'a)/ [çìtɕ'ìtɕ'ík'àː] 'five bundles' [tɕ'ùtɕ'ìːk'á] 'one bundle'
	b. C-class becomes characterized by second position from the
	left instead of penultimate	(24) Sani (Uwano 1996a, 2000): CVːCV > CVĆV, but no such corre-
		Hu Lamet spondence for C-class words!
		a. *V [-high] > V́jám ja̤ m 'to die' a. *poːki > pók'ì 'broom'
		b. *Vː [-high] > V̀jàm ja̤ ːm 'to cry' but b. *iki (C) > ʔìk'í 'breath' (Shuri ʔiːtɕi)
	LRː > HːL > RːL > HL > LH (18) Lawrence's (2016) pNR reconstruction: C *LːH > FːH	cf. c. *ame (B) > ʔàw ́'rain' (7) Cantonese (Sinitic): bipartition of the "Upper Entering" (Tone 7, 陰入) tone class according to vowel length a. 七 cat¹ [tsʰɐt˥] 'seven' (25) Shodon: unexpected vowel losses and lengthening if we follow Lawrence's (2016) reconstruction A ?*kubiː k'ûp 'neck' b. 八 baat³ [paːt˧] 'eight' ?*putuːka ɸút.kà '2nd day'
	A *HHː, *HHːL 2nd vowel long, first 2 moras High B *LRː, *LLRː final vowel long, final mora High C *RːH, *LRːH penultimate vowel long, final 2 moras High	3.1.2 Short > Low, Long > High (8) Cheyenne (Algonquian, Frantz 1972; Leman 1981): B ?*kagamiː káɡǎm 'mirror' ?*omoteː ʔùmôːt ́'face' C ?*kataːna kát.nà 'knife'
	b. length not predictable from a simple generalisation a. tone and vowel length are independent	a. *weθkweni > /hèʔè/ 'liver' b. *eθkweˑwa > /hèʔé/ 'woman' 8 Vowel length and morphology
	c. natural system? 5 Geography	(9) Sani Ryukyuan (Amami, Uwano 1996a, 2000): (26) Asama: prosodically lengthened vowels often shorten in com-pounds, but not etymologically long ones a. *poːki > pók'ì 'broom' b. *toːra > tórà 'straw bag' a. háː-'red' + ɕàːtá 'sugar' → háːdʑàtà 'brown sugar'
	(19) C-class dissyllables a. initial length in 3 areas: Tokunoshima, North Okinawa, Cen-tral & South Okinawa	b. t ː c. dòːɡú 'tool' + hákúː 'box' → dòːɡùbáːkù 'toobox' 'hand' + mùːtú 'origin' → t mùtǔː 'at hand' d. s mǎː 'island' + tòːɸú 'tofu' → s màdòːɸú 'local tofu'

3.1 Length > tone

3.1.1 Short > High, Long > Low (6) Hu (Mon-Khmer, Svantesson 2001): c. thus unable to account for quadrisyllables like *sutomete C 'morning' (Asama s tùm ːt , Nakijin ɕìtìmítì) (16) Uwano (1996b: 32): C *LːH > M͡ LːH > FˑH > HL (17) Matsumori (1996): tone and vowel length are independent B *LRː, LL-H > HRː, HL-H > HH > LH > LH > tone pattern) and length, which are independently required 7 Lengthening and shortening in Northern Ryukyuan (23) Iambic lengthening in Nakijin

[START_REF] Lawrence | Nakijin phonology: Feet and extrametricality in a Japanese dialect[END_REF][START_REF] Ogawa | Nakijin hōgen akusento no shosō[END_REF]

vowels are lengthened when they are the head of an iambic foot (i.e. in even-numbered syllables), and not adjacent

  Explanation for lengthening: a. delayed rise, i.e. rightward H shift b. Yoron: "the accented location is often realized as Rising […] or Mid […], with a following High" (Uwano 1999: 183-182) c. i.e. /μμ/ → HL | RL | RF d. contour tones are longer e. cf. also *HL > RːL | LːH | M͡ LːH in Kunohe (12) (45) Remaining questions: a. distinctive rise ("ascending kernel")? fall ("lowering kernel")? b. A-class: L*H⁺ (Kam. & Wad.)? M(M)L (Yoron) ? H(H)L (Shuri?) (46) Relationship with Japanese: a. rejecting vowel length ⇒ 8 dissyllabic classes in pJ b. more than in any modern variety c. we need to consider the possibility that some Japanese classes might be due to secondary splits

	(43) Tentative pNR reconstruction (close to Matsumori's 2001 &
	2009 suggestions)
	A unaccented	μ μμ μμμ
	B final accent	μ́μμ́μμμĆ
	penultimate accent μμ μμμ
	(D initial accent	μμμ )
	(44)
						e, H occurs
			one mora later than in Yoron & Kametsu)
			e. vowel lengthening is concomitant with tone change, i.e. the
			last syllable before tone change is lengthened
			f. but A-class is special
		(42)	Yoron	Kametsu	Asama Wadomari Kin
			1 A μː M͡ Lː μː Hː	Hː	μː Hː	μː Hː
			B μː́Rː	μː Fː	Rː	μː Rː	μː Rː
			2 A μμ ML μμ LH(-H) HHː μμ LH(-H) μμ HH
			B μμ́LF μμ́LH(-L) LRː	μμ̀LRː	μμ̀HːRː
			C μμ HL μμ HL	LːH	μμ LH(-L) μμ LːH
			3 A μμμ MML μμμ LHH(-H) HHːL μμμ LHH μμμ HHH
			B μμμ́LLF μμμ́LLH(-L) LLRː μμμ̀LLRː μμμ̀HHRː
			C μμμ LHL μμμ LHL	LLːH μμμ LLH μμμ LLH