Magdalena Kristiawan
email: magdalena.kristiawan@univ-lr.fr

Vaclav Sobolik

Karim Allaf

Application of instantaneous auto-vaporization

Keywords: Ylang-Ylang (Cananga odorata Hook. fil. et Thomson, forma macrophylla), extraction, essential oil, steam distillation, Instantaneous Controlled Pressure Drop

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Introduction

The ylang-ylang tree, Cananga odorata Hook. fil. et Thomson, is a native of tropical Asia. Its essential oil is one of the most important perfume raw materials [START_REF] Stashenko | A study of the compositional variation of the essential oil of ylang-ylang (Cananga odorata Hook fil. et Thomson, forma genuina) during flower development[END_REF]. This oil is also used extensively in soap and detergents; aromatherapy; pharmaceutical industry; cosmetics and food flavors [START_REF] Buccellato | Ylang survey[END_REF][START_REF] Gaydou | Composition of the essential oil of ylang-ylang (Cananga odorata Hook. fil. et Thomson, forma genuina) from Madagascar[END_REF][START_REF]Colgate to offer aromatherapy under Palmolive[END_REF][START_REF] Lee | Biochemical mechanisms conferring cross-resistance to fumigant toxicities of essential oils in a chlorpyrifos-methyl resistant strain of Oryzaephilus surinamensis L. (Coleoptera: Silvanidae)[END_REF][START_REF] Valnet | Aromathérapie[END_REF].

Ylang oil is isolated by steam distillation and sometimes by solvent extraction from fresh mature flowers [START_REF] Buccellato | Ylang survey[END_REF][START_REF] Gaydou | Composition of the essential oil of ylang-ylang (Cananga odorata Hook. fil. et Thomson, forma genuina) from Madagascar[END_REF]. Low yield, losses of volatile compounds, long extraction times, toxic solvent residues, and degradation of unsaturated compounds, giving undesirable off-flavour compounds, due to heat may be encountered using these extraction methods [START_REF] Buccellato | Ylang survey[END_REF][START_REF] Assis | Comparison among different extraction methods (PFE, SFE, Sonication, Soxhlet) for the isolation of organic compounds from coal[END_REF][START_REF] Lehotay | Supercritical fluid extraction of pesticides in foods[END_REF][START_REF] Oszagyan | Supercritical fluid extraction of volatile compounds from lavandin and thyme[END_REF][START_REF] Sato | Fractional extraction with supercritical carbon dioxide for the removal of terpenes from Citrus oil[END_REF]. These shortcomings have led to the consideration of the use of supercritical fluids in essential oil extraction processes. CO 2 is the most commonly used supercritical fluid because of its modest critical conditions. Furthermore, the supercritical CO 2 extraction of aromatic plants gives high quality and solvent-free extracts [START_REF] Oszagyan | Supercritical fluid extraction of volatile compounds from lavandin and thyme[END_REF][START_REF] Sato | Fractional extraction with supercritical carbon dioxide for the removal of terpenes from Citrus oil[END_REF][START_REF] Anitescu | Isolation of coriander oil: comparison between steam distillation and supercritical CO 2 extraction[END_REF][START_REF] Reverchon | Supercritical fluid extraction and fractionation of essential oils and related products[END_REF][START_REF] Goto | Extraction of peppermint oil by supercritical carbon dioxide[END_REF][START_REF] Reverchon | Isolation of rosemary oil: comparison between hydrodistillation and supercritical CO 2 extraction[END_REF][START_REF] Reverchon | Supercritical CO 2 extraction of basil oil: characterization of products and process modelling[END_REF][START_REF] Gaspar | Modelling the extraction of essential oils with compressed carbon dioxide[END_REF]. However, its high cost related to specific equipments for high-pressure conditions (200 bar), requirement of CO 2 purity and CO 2 low dielectric constant (thus giving rise to a non-polar character that hinders or makes difficult the extraction of polar compounds) make important the searching for new extraction method [START_REF] Gaspar | Disruption of glandular trichomes with compressed CO 2 : alternative matrix pre-treatment for CO 2 extraction of essential oils[END_REF][START_REF] Luque De Castro | Towards more rational techniques for the isolation of valuable essential oils from plants[END_REF].

C3.

Pressure Drop (DIC) [START_REF] Allaf | Processus de traitement thermomécanique par détente instantanée contrôlée des fruits, jus et peaux d'agrumes[END_REF]. The DIC process is based on the thermo-mechanical effects induced by subjecting the raw material for a short period of time to a saturated steam pressure (about 1 to 6 bar according to the product), followed by an abrupt pressure drop towards vacuum (about 50 mbar). This abrupt pressure drop (∆P/∆t > 5 bar.s -1 ) provokes simultaneously auto vaporization of volatile compounds, instantaneous cooling of the products allowing to stop thermal degradation, swelling and eventually implies the rupture of cell walls, which enhances the global mass transfer.

The main objective of this work was to determine the optimum parameters of DIC process on the ylang oil extraction, and compare the results with oil obtained by steam distillation. We intend to make appropriate comparison in term of oil yield and composition.

Experimental

For evaluating DIC performance on essential oil extraction, we have realized the quantitative and qualitative comparative study between Steam Distillation (SD) and DIC extraction of dried ylang flowers. In this work, we have studied 3 parameters of DIC process:

(1) The processing steam pressure which varied from 2 bar to 6 bar; (2) The total processing time varying from 0.5 to 20 minutes and (3) The number of DIC cycles varying from 1 to 9.

Materials

Fully mature Ylang-Ylang flowers (Cananga odorata Hook. fil. et Thomson, forma macrophylla) were picked up at blossom in Lawang, East Java, Indonesia (October 2003). The flowers were air-dried to a final moisture content of 10.2 % dry basis and stored at room temperature prior to use

Extraction methods 2.2.1. DIC apparatus and procedure

The schematic diagram of the equipment used has been described in a previous study [START_REF] Rezzoug | Study of a new extraction process: Controlled instantaneous decompression. Application to the extraction of essential oil from rosemary leaves[END_REF]. This equipment is composed of three main elements:

-The autoclave (6L) where the flowers were placed and treated -The vacuum system which consists mainly of a vacuum tank (285 L) and a water ring vacuum pump -A pneumatic valve (100 mm spherical valve), which separates the processing vessel from the vacuum tank and can be opened in less than 200 ms to assure instantaneous pressure drop.

40 g of dried ylang-ylang flowers was treated in the autoclave in which a vacuum of 50 mbar was established by a brief connection with the vacuum tank. The initial vacuum treatment (Fig. 1a) facilitates the diffusion of steam into flowers. Consequently, the time necessary for heating the flowers to the temperature of the steam is reduced. Saturated steam is then injected into the autoclave at a fixed pressure level (Fig. 1b) and maintained for a determined time. An abrupt pressure drop follows this step (Fig. 1c). The equilibrium pressure after pressure drop depends on the operating pressure in the autoclave: the higher the operating pressure, the higher the equilibrium pressure. The vapour in the Ylang-Ylang flowers created by auto-vaporization after instantaneous pressure drop produces mechanical stresses which can deform or even break cells. This effect depends on the rheological properties of the plants which are function of humidity and temperature. Auto-vaporization as an adiabatic transformation induces instantaneous cooling of the residual product from 120-160°C (depending on the steam pressure) to about 30-35°C. The final temperature is proportional to the given final pressure. The next step is either the thermal treatment by steam followed by pressure drop or return to atmospheric pressure (Fig. 1d).

Figure 1. Several cycles of DIC treatment

If the DIC process is composed of several cycles, the treatment time is the total time during which a sample is exposed to the steam pressure. The treatment time of 1 cycle of DIC is then defined as the ratio of the treatment time to the number of DIC cycles.

After DIC treatment, the condensate (oil-in-water emulsion) was recovered from an extract container connected to the vacuum tank. The vacuum tank is equipped with cooling water jacket. Then this emulsion was subjected to liquid-liquid extraction step in order to isolate essential oil.

Steam distillation of flowers

A packed bed of 80 g of dried ylang-ylang flowers was put on a stainless steel grill fixed above 2 L of water in the extraction vessel. The steam, which is produced in the lower part of the apparatus by electric heating, passes through the ylang bed evaporates and carries the essential oil. These vapours are condensed and the water is recycled into the extraction vessel.

Liquid-liquid extraction of the aqueous extracts

Prior to analysis, the critical stage was the oil separation from the condensate obtained after the steam distillation and DIC extraction. The condensate was extracted three times with chloroform with addition NaCl to facilitate the emulsion breakdown. Sodium sulphate was added to remove moisture and the organic phase was then dried under vacuum in a rotaryevaporator at 30°C. Each dried sample was diluted with chloroform to 20 ml and stored in the dark at 4°C prior to yield and composition analysis by GC-MS.

Analytical methods

Extraction yield

The yield of DIC oil and steam distilled oil was based on dry plant mass:

% 100 (%) × = flowers dry of mass mass oil yield Oil (1)
where the oil mass was computed from the GC peak area of all volatiles molecules

Gas chromatography-mass spectrometry identification

The volatile molecules were analysed by gas chromatography coupled to mass spectrometry (GC-MS) A Varian computerized system comprising a 3900 gas chromatograph equipped by a fused-silica-capillary column with a non-polar stationary phase poly (dimethylsiloxane) CP-Sil 8 (30 m × 0.25 mm × 0.25 µm film thickness) was connected to a 2100T mass spectrometer. . The measurements were performed under the following conditions: carrier gas He; flow rate 1 mL.min -1 ; split 1:100; injection volume 0.1 µL; injection temperature 250°C; oven temperature progress from 60 to 170°C at 2.5°C.min -1 , from 170 to 250°C at 10 C.min -1 and holding at 250 C for 5 min; the ionisation mode was electronic impact at 70 eV. Mass spectra and reconstructed chromatograms were obtained by automatic scanning in the mass range m/z 30-400 a.m.u at 2.2 scan.s -1 . Identification of the components was achieved by a comparison of their mass spectral fragmentation patterns with those stored in the data bank (Varian NIST MS Database 1998 and Saturn libraries) and the literature [21; 22]. Quantitative analysis was performed by peak area measurement.

Scanning electron microscopy

Scanning electron micrographs of the untreated and DIC treated flowers were taken with a Jeol 5410 LV SEM with the aim to observe their structural changes. The oil yield increases with increasing pressure and total processing time (see Fig. 2). The increase is considerable during the first 10 min. The processing pressure has a significant effect on the oil yield while the effect of total processing time after 10 min was not significant. When the pressure increases from 2 to 6 bar for a total time of 10 min, the oil yield increases from 0.11% to 0.65%. However, oil yield obtained by DIC with 1 cycle is very low (less than 1 %) in comparison to yield of industrial steam distilled oil from fresh flowers (about 2.0-2.5%) [START_REF] Guenther | The essential oils[END_REF]. In order to increase oil yied, therefore the DIC extraction was carried out with several cycles and varying the processing time from 30 s to 20 min and pressure from 2 to 6 bar (see Fig. 3).

Results and discussion

Essential oil yield

(a) (b) Figure 3. Effect of pressure and total processing time of DIC with several cycles on oil yield

The yield obtained by DIC with several cycles (see Fig. 3) has the same tendency as that of 1 cycle. The oil yield increases essentially with increasing pressure and total processing time during the first 6 min. A closer look at Figs. 2 and3 shows that the increasing of number of cycles from 1 to 4 cycles at pressure 6 bar improve the oil yield three times; from 0.65% to 1.92%. Besides that, the increasing of processing pressure from 2 bar to 4 and 6 bar for 4 cycles improve the oil yield two times; from 0.52% to 1.20% and four times, from 0.52% to 1.92, respectively. Interestingly, Figs. 2 and 3 also show that the more and more number of cycles, the shorter total processing time required for achievement of maximum yield (4 cycles in 6 min vs. 1 cycle in 10 min). After 4 cycles, the extended total processing time greater than 6 min results only a small gain in oil yield. Therefore, the total processing time was maintained at 6 min for further study of DIC cycle and processing pressure. Oil yield as a function of the number of DIC cycles is shown in Fig. 4. The oil yield increases significantly with increasing processing pressure and DIC cycle. The oil yield achieved maximum after 8 cycles whatever the pressure used. It appears that the best result can be obtained at 6 bar with 8 cycles in 6 min with an oil yield of about 2.77% An overall evaluation of the data from Figs. 2, 3 and4 shows the most significant parameters of DIC process are the processing pressure and the number of cycles. The processing time is less significant. The effective time for each cycle is defined as the required time of heating steam for achievement of thermal equilibrium (the flowers temperature in the bulk is the same as that of steam) and rheological equilibrium (the achievement of flowers viscoelasticity required for swelling) [START_REF] Louka | Expansion ratio and color improvement of dried vegetables texturized by a new process "Controlled Sudden Decompression to the vacuum". Application to potatoes, carrots and onions[END_REF]. It seems that a heating time of 45 seconds for each cycle (8 cycles in 6 min) is sufficient to reach the equilibrium conditions in case of ylang flowers.

These facts confirm that the extraction mechanism of DIC is based only on the instantaneous auto-vaporization of liquid complex (water + volatile oil) and modification of internal structure at each cycle. Therefore, the mass transfer is not controlled by internal diffusion, which characterizes the conventional extraction process. In addition, the compounds-solid matrix interaction that leads to importance of auto-vaporization of these compounds from the modified structures needs to be taken into account when considering the overall extraction kinetics. This is the explication for S-shaped of yield-cycle plot in Fig. 4 for DIC with pressure of 6 bar. We observed a slow rate of rise at first cycle and then speeding up before dropping to zero at the last cycle. As the number of pressure drop (i.e. number of DIC cycle) increases, especially when high-pressure steam applied, the alveolation becomes more prominent (formation of wide, deep and interconnected-pores) and results in a higher breaking rate of cell walls. Such complex micro-alveolation structure allows auto-vaporization and liberation of imprisonedcompounds within endogenous site in the following pressure drop. Beside of the processing parameters, the internal structure modification depends also on the nature of flowers (physical, thermal stability, moisture content, etc).

A great advantage of the DIC method is its rapidity. The results of a comparative study of extraction kinetics of the steam distillation and the DIC process (6 bar and 8 cycles) are shown in Fig. 5. In case of DIC process, the increase of oil yield is considerable during the first 4 minutes. In contrast, the steam distillation required 12 h for isolating of 95% of essential oil. The yield of the DIC with of 8 cycles during a total processing time of 4 min (2.74%) at 6 bar was comparable to that obtained after 24 h by means of steam distillation (2.60%), which is the reference method in essential oil extraction. These results confirm a substantial saving of time, energy and plant material by using the DIC process. In conclusion, the process parameters of 6 bar, 8 cycles and 4 or 6 min can be regarded as optimum for Indonesian ylang oil extraction. Beside of the quantity aspect, the performance of an extraction method is appreciated according to high quality of oil produced. For this purpose, a comparison of chemical composition of ylang oil obtained by DIC and steam distillation methods was made. The results are summarized in next chapter. Oil yield (%) The gas chromatograms of ylang-ylang oil obtained from dried flowers by DIC and SD techniques are shown in Fig. 6. The chemical composition of these oils is presented in Table 1. From 67 peaks 58 components were identified, which constitute more than 90% of the volatile fraction.

Steam distillation Time (h)

Composition of essential oil

The components were grouped into compound families in order to better appreciate the effect of extraction method on the chemical composition of isolated oils (see Table 2). From Table 2, it can be seen that a substantially higher amount of oxygenated compounds and a lower amount of sesquiterpenes hydrocarbons are present in the ylang oil extracted by DIC in comparison with the steam distillation.

Value of ylang-ylang oils is determined by the content of oxygenated constituents and sesquiterpenes. The quality of ylang-ylang oil increases with the content of both light and heavy oxygenated compounds. The sesquiterpenic portion gives no more than a weak contribution to the typical ylang-ylang odour. As it is insoluble in alcohol, fine perfumery seeks the oils which are rich in oxygenated constituents. The sesquiterpenic oils are used in soap manufacture. According to Buccellato [START_REF] Buccellato | Ylang survey[END_REF], the oxygenated compounds (alcohols, esters, ethers, phenols and aldehydes) provide the medicinal, floral, spicy/balsamic and fruity note to the odour profile, whereas the sesquiterpenes contribute to the woody character only in ylang odour. It appears that the DIC process provided better quality of ylang oil than the steam distillation.

A lower content of sesquiterpenes in the DIC oil is probably due to the diminution of thermal degradation. The steam distillation uses a large quantity of water at a high temperature for a long time. Water is a polar solvent, which accelerates many reactions, especially via carbocation as intermediates. This results in hydrolytic, trans-esterification or oxidation reactions of the other compounds into sesquiterpenes.

The HOC and sesquiterpenes are the main components in the Indonesian ylang oil but their relative amounts differ according the extraction methods (see Table 1 and2).

Scanning electron microscopy

To prove the mechanical effect of DIC treatment, the scanning electron microscopy of untreated and DIC treated flowers was realized B-D). These micrographs proved the aptitude of DIC on the modification of internal structure. The higher the processing pressure and the more the DIC cycles, the higher the degree of alveolation which results in a higher breaking rate of cells walls. This facilitates liberation and vaporization of volatile molecules.

Conclusions

The essential oil extraction from ylang flowers is very efficient by using the DIC process. The maximum oil yield (2.77%) is obtained with eight cycles at a steam pressure of 6 bar absolute and total heating time between 4 and 6 min. In comparison with the steam distillation, the DIC oil contains higher content of light and heavy oxygenated components (73.92% vs. 59.19%). In contrast, its sesquiterpenes content is lower (13.29% vs. 39.75%). The DIC treatment increases the availability of the heavy oxygenated components in the flowers. The structure of the flowers is more alveolated at the higher pressure and number of DIC cycle. The DIC can be also used as a pretreatment technique for the improvement of the solvent extraction or others conventional extraction techniques of non-volatiles compounds.

Figure 2 .

 2 Figure 2. Effect of pressure and total processing time of DIC with 1 cycle on oil yield

Figure 4 .

 4 Figure 4. Effect of DIC cycle and processing pressure on oil yield

Figure 5 .

 5 Figure 5. Comparison of kinetics extraction between DIC at 6 bar, 8 cycles (a) and steam distillation (b)

Figure 6 .

 6 Figure 6. Comparison of gas chromatograms of Indonesian ylang-ylang oil obtained by DIC at 6 bar, 8 cycles, 4 min (above) and SD during 24 h (below).

Figure 7 .

 7 Figure 7. Scanning electron micrographs of untreated flowers (A) and DIC treated flowers at 2 bar, 1 cycle, 10 min. (B), 6 bar, 5 cycles, 4.4 min. (C), and 6 bar, 9 cycles, 10 min. (D).

17

 17

  A). The DIC treated flowers have a structure with larger alveoli with dimensions depending on the treatment conditions (see Figs.[START_REF] Gaspar | Disruption of glandular trichomes with compressed CO 2 : alternative matrix pre-treatment for CO 2 extraction of essential oils[END_REF]

  The need of more rational technique for obtaining high quality essential oils has led our laboratory to develop a new and alternative extraction process: Instantaneous Controlled 17th Int. Congress CHISA, CD ROM P3.32, Praha, Czech Republic, August 27-31 2006 Full text

Table 1 .

 1 The chemical composition of Indonesian ylang-ylang oil obtained by SD and DIC

	No Retention Compounds	Type	Steam distillation	DIC
		time (min)			24 h	6 bar, 8 cycles, 4 min
	1	7.682 Benzaldehyde	LOC	0.13%	1.21%
	2	8.151 5-Hepten-2-one	LOC	0.24%	0.24%
	3	10.13 Benzyl Alcohol	LOC	0.08%	5.51%
	4	11.652 cis-Linalool Oxide	LOC	0.12%	0.58%
	5	12.385 trans-Linalool Oxide	LOC	0.06%	0.28%
	6	13.029 Linalool	LOC	2.26%	1.40%
	7	13.186 3-Octyne-2-one	LOC	0.15%	0.20%
	8	16.038 Benzyl acetate	LOC	0.08%	0.30%
	9	19.855 beta-Citral	LOC	0.00%	0.13%
	10	20.53 trans-Geraniol	LOC	1.56%	2.40%
	11	21.391 Geranial	LOC	0.13%	0.28%
	12	25.281 alpha.-Cubebene	S	0.23%	0.24%
	13	25.64 Eugenol	LOC	0.26%	0.55%
	14	26.763 Copaene	S	1.32%	1.01%
	15	26.969 Geranyl acetate	LOC	0.23%	0.31%
	16	28.255 Methyl eugenol	LOC	0.53%	0.38%
	17	27.356 beta-Cubebene	S	0.21%	0.17%
	18	27.463 (-)-beta-Elemene	S	0.33%	0.00%
	19	29.218 beta-Caryophyllene	S	18.20%	9.85%
	20	29.468 Bicyclo[4.4.0]dec-1-ene	S	0.38%	0.18%
	21	30.394 n.i.	S	0.35%	0.14%
	22	30.852 alpha.-Caryophyllene	S	6.52%	3.82%
	23	31.035 alpha-Curcumene	S	0.71%	0.92%
	24	31.509 Epizonarene	S	0.30%	0.10%
	25	31.675 gamma-Muurolene	S	0.53%	0.37%
	26	31.961 1H-Cyclopenta[1,3cyclopropa[1,2]	S	2.05%	1.15%
	27	32.466 (+)-Epi-bicyclosesquiphellandrene	S	0.42%	0.21%
	28	32.834 alpha-Muurolene	S	0.59%	0.45%
	29	33.068 Isocaryophillene	S	4.25%	3.80%
	30	33.08 alpha.-Farnesene	S	-	0.03%
	31	33.279 beta-Bisabolene	S	0.09%	0.10%
	32	33.517 gamma-Cadinene	S	0.32%	0.15%
	33	33.804 beta-Cadinene	S	1.51%	1.00%
	34	33.948 Calamene + delta-Cadinene	S	0.91%	0.75%
	35	34.41 Naphthalene,1,2,3,4,4a,7-hexahydro-	S	0.17%	0.11%
	36	34.827 Cadala-1(10),3,8-triene	S	0.37%	0.30%
	37	35.274 n.i	.	HOC	1.07%	1.07%
	38	35.629 Longipinocarveol trans	HOC	0.61%	0.64%
	39	35.81 9-Methoxycalamenene	HOC	0.15%	0.19%
	40	36.043 n.i	.	HOC	0.09%	0.13%
	41	36.477 3-Hexen-1-ol	HOC	0.37%	0.28%
	42	36.955 Caryophyllene oxide	HOC	13.75%	20.14%
	43	37.263 Aristolenene epoxide	HOC	0.82%	0.65%
	44	37.546 n.i.	HOC	0.46%	0.40%
	45	38.143 12-Oxabicyclo[9.1.0]dodeca-3,7-diene HOC	4.01%	1.17%
	46	38.367 Tricyclo[5.2.2.0(1,6)]undecan-3-ol	HOC	1.00%	0.62%
	47	38.852 Cubenol	HOC	0.40%	0.29%
	48	39.027 Aromadendrene oxide-(1)	HOC	0.91%	0.51%
	49	39.15 Aromadendrene oxide-(2)	HOC	1.07%	0.83%
	50	39.359 Tetracyclo[6.3.2.0(2,5).0(1,8)tridecan-HOC	1.63%	1.64%

Table 2 .

 2 Yield and grouped compounds of ylang oil obtained by SD and DC

	Compounds	Percent in oil (%)
		Steam distillation	DIC
		24 h	6 bar, 8 cycles, 4 min
	Light Oxygenated Compounds	5.84%	13.77%
	Sesquiterpenes	39.75%	24.85%
	Heavy Oxygenated Compounds	53.35%	60.15%
	Oil yield (gr oil/100 gr DM)	2.60%	2.74%


domain: sdv sde sdv.bv

text:
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Detailed knowledge of the level of genetic diversity and patterns of population structure in cultivated alfalfa is needed for further progress in traditional and molecular breeding as well as for the management of genetic resources in this species. In this study, ten European cultivars were investigated : three cultivars (two varieties and one landrace) of Provence type, i.e. low dormancy cultivars mainly based on the subspecies M. sativa sativa, and seven cultivars (six varieties and one landrace of Flamande (or Flemish) type, i.e. more dormant cultivars introgressed with different parts of the subspecies M. sativa falcata. For each variety, 40 genotypes were examined using morphological traits and SSR markers.

Both morphological traits and molecular markers revealed a large within-cultivar diversity and a small but significant variation among cultivars. For morphological traits, a clear separation of cultivars of Flamande and Provence types was observed. Luzelle, a cultivar related to Flamande type with a high part of Falcata genome, was clearly separated from the rest of the cultivars. Among the other Flamande cultivars, we found no differentiation. Contrastingly, the three Provence cultivars were significantly differentiated for morphological traits. For molecular markers, we found significant differentiation between Luzelle and the other cultivars, as for morphological traits. Barmed, a non dormant cultivar bred from American and North-African genetic resources, was also separated from the other cultivars. No structure between Flamande and Provence types was detected using molecular markers. Finally, even if the structure among cultivars was different for morphological traits and molecular markers, a large within-cultivar was observed in both cases and Luzelle was separated from the other cultivars.

The history of alfalfa introduction in Europe (17 th Century), the reproductive mode of this species (allogamy), its genetic characteristics (autotetraploidy), the patterns of seed exchanges between farmers contribute to explain the large within-cultivar variation and the low differentiation among populations. Breeding and maintenance of genetic resources should exploit this situation for more efficiency.

Comparison

  Comparison of morphological traits and SSR markers to analyze genetic diversity of alfalfa cultivars Doris Herrmann, Sandrine S. Flajoulot, Philippe P. Barre, Christian C. Huyghe, Joelle J. Ronfort, Bernadette Julier To cite this version: Doris Herrmann, Sandrine S. Flajoulot, Philippe P. Barre, Christian C. Huyghe, Joelle J. Ronfort, et al.. Comparison of morphological traits and SSR markers to analyze genetic diversity of alfalfa cultivars. 41. North American Alfalfa Improvement Conference & 20. Trifolium Conference, National Alfalfa & Forage Alliance (NAFA). St. Paul, USA., Jun 2008, Dallas, United States. hal-02752049 DAPC, 34130 Mauguio, France. Email: bjulier@lusignan.inra.fr

Acknowledgements

We thank the ACVF (Association of French Breeders) section Alfalfa for contributing to genotyping and phenotyping. This work was supported by French Ministry of Agriculture (2005Agriculture ( -2008)). DH received a grant from Plant Breeding department of INRA.


Codrut ¸a Stoica
email: codruta.stoica@math.u-bordeaux1.fr

Keywords: Mathematics Subject Classification: 34D09 Evolution semiflow, evolution cocycle, skew-evolution semiflow, exponential dichotomy, exponential trichotomy

The paper emphasizes the properties of exponential dichotomy and exponential trichotomy for skew-evolution semiflows in Banach spaces, by means of evolution semiflows and evolution cocycles. The approach is from uniform point of view. Some characterizations which generalize classic results are also provided.

Preliminaries

The exponential dichotomy is one of the basic concepts in the theory of dynamical systems and plays an important role in the study of stable and instable manifolds. Various concepts of dichotomy were introduced and studied by S.N. Chow and H. Leiva in [START_REF] Chow | Existence and roughness of the exponential dichotomy for linear skew-product semiflows in Banach spaces[END_REF], M. Megan, A.L. Sasu and B. Sasu in [START_REF] Megan | Asymptotic Behaviour of Evolution Families[END_REF], P. Preda and C. Preda in [START_REF] Preda | Comportȃri asimptotice ale proceselor evolutive[END_REF]. A natural generalization of the notion of dichotomy is considered the trichotomy, which refers at a decomposition of the space at every moment into three closed subspaces: a stable subspace, an instable one and a center manifold. The trichotomy was introduced by R.J. Sacker and G.R. Sell in [START_REF] Sacker | Existence of dichotomies and invariant splittings for linear differential systems III[END_REF] and the exponential trichotomy by S. Elaydi and O. Hajek in [START_REF] Elaydi | Exponential trichotomy of differential systems[END_REF]. In recent years interesting results in the domain of trichotomy were obtained by L.H. Popescu in [START_REF] Popescu | Exponential dichotomy roughness on Banach spaces[END_REF], B. Sasu and A.L. Sasu in [START_REF] Sasu | Exponential trichotomy and p-admissibility for evolution families on the real line[END_REF] or L. Barreira and C. Vallis in [START_REF] Barreira | Smooth center manifolds for nonuniformly partially hyperbolic trajectories[END_REF].

A new concept of trichotomy, the null uniform exponential trichotomy for evolution operators was introduced in [START_REF] Megan | On null uniform exponential trichotomy of evolution operators in Hilbert spaces[END_REF]. The study has been continued by the definition of uniform exponential trichotomy by means of three projection families in [START_REF] Megan | On uniform exponential trichotomy of evolution operators in Banach spaces[END_REF]. The trichotomy is studied in the nonuniform setting for skew-evolution semiflows in [START_REF] Megan | Nonuniform trichotomy for skew-evolution semiflows in Banach spaces[END_REF] and [START_REF] Megan | Trichotomy for linear skew-product semiflows[END_REF] and for discrete time in [START_REF] Megan | Trichotomy for discrete skew-evolution semiflows in Banach spaces[END_REF].

In our paper we extend the asymptotic properties of exponential dichotomy and trichotomy for the newly introduced concept of skew-evolution semiflows, which can be considered generalization for evolution operators and skew-product semiflows.

Notations. Definitions. Examples

We consider (X, d) a metric space, V a Banach space and B(V ) the space of all bounded linear operators from V into itself. We denote the sets T = (t, t 0 ) ∈ R 2 , t ≥ t 0 ≥ 0 and Y = X × V . Let P : Y → Y be a projector given by P (x, v) = (x, P (x)v), where P (x) is a projection on Let X be the closure in

Y x = {x} × V , x ∈ X. Definition 2.1 A mapping ϕ : T × X → X is called evolution semiflow on X if following relations hold: (s 1 ) ϕ(t, t, x) = x, ∀(t, x) ∈ R + × X (s 2 ) ϕ(t, s, ϕ(s, t 0 , x)) = ϕ(t, t 0 , x), ∀t ≥ s ≥ t 0 ≥ 0, x ∈ X. Definition 2.2 A mapping Φ : T × X → B(V ) is called evolution cocycle over an evolution semiflow ϕ if: (c 1 ) Φ(t, t, x) = I, the identity operator on V , ∀(t, x) ∈ R + × X (c 2 ) Φ(t, s, ϕ(s, t 0 , x))Φ(s, t 0 , x) = Φ(t, t 0 , x), ∀t ≥ s ≥ t 0 ≥ 0, x ∈ X.
C(R + , R) of the set {f t , t ∈ R + }, where f t (τ ) = f (t + τ ), ∀τ ∈ R + .
Then the mapping

ϕ : T × X → X, ϕ(t, s, x) = x t-s is an evolution semiflow on X. Let us consider the Banach space V = R p , p ≥ 1, with the norm (v 1 , ..., v p ) = |v 1 | + ... + |v p |. The mapping Φ : T × X → B(V ), Φ(t, s, x)(v 1 , ..., v p ) = e t s x(τ -s)dτ v 1 , ..., e t s x(τ -s)dτ v p
is an evolution cocycle over ϕ and C = (ϕ, Φ) is a skew-evolution semiflow on Y . Definition 2.4 Two projector families {P k } k∈{1,2} are said to be compatible with a skew-evolution semiflow C = (ϕ, Φ) if (dc 1 ) P 1 (x) + P 2 (x) = I, P 1 (x)P 2 (x) = P 2 (x)P 1 (x) = 0 (dc 2 ) P k (ϕ(t, s, x))Φ(t, s, x)v = Φ(t, s, x)P k (x)v, k ∈ {1, 2} for all t ≥ s ≥ t 0 ≥ 0 and all (x, v) ∈ Y. Definition 2.5 A skew-evolution semiflow C = (ϕ, Φ) is uniformly exponentially dichotomic if there exist two projector families {P k } k∈{1,2} compatible with C and some constants

N 1 , N 2 ≥ 1, ν 1 , ν 2 > 0 such that (ued 1 ) e ν 1 (t-s) Φ(t, t 0 , x)P 1 (x)v ≤ N 1 Φ(s, t 0 , x)P 1 (x)v (ued 2 ) e ν 2 (t-s) Φ(s, t 0 , x)P 2 (x)v ≤ N 2 Φ(t, t 0 , x)P 2 (x)v for all t ≥ s ≥ t 0 ≥ 0 and all (x, v) ∈ Y.
Example 2.2 We consider X and the evolution semiflow ϕ as in Example 2.1.

Let V = R 2 with the norm (v 1 , v 2 ) = |v 1 | + |v 2 |. The mapping Φ : T × X → B(V ), Φ(t, s, x)(v) = v 1 e -2 t s x(τ -s)dτ , v 2 e 3 t s x(τ -s)dτ
is an evolution cocycle.

We consider the projectors

P 1 (x, v) = (v 1 , 0), P 2 (x, v) = (0, v 2 ). Then C = (ϕ, Φ) is a uniformly exponentially dichotomic skew-evolution semiflow with N 1 = N 2 = 1, ν 1 = 2, ν 2 = 3.
Definition 2.6 Three projector families {P k } k∈{1,2,3} are said to be compatible with a skew-evolution semiflow C = (ϕ, Φ) if (tc 1 ) P 1 (x) + P 2 (x) + P 3 (x) = I, P i (x)P j (x) = P j (x)P i (x) = 0, i, j ∈ {1, 2, 3}, i = j (tc 2 ) P k (ϕ(t, s, x))Φ(t, s, x)v = Φ(t, s, x)P k (x)v, k ∈ {1, 2, 3} for all t ≥ s ≥ t 0 ≥ 0 and all (x, v) ∈ Y. Definition 2.7 A skew-evolution semiflow C is uniformly exponentially trichotomic if there exist three projector families {P k } k∈{1,2,3} compatible with C and some constants

N 1 , N 2 , N 3 ≥ 1, ν 1 , ν 2 , ν 3 > 0 such that (uet 1 ) e ν 1 (t-s) Φ(t, t 0 , x)P 1 (x)v ≤ N 1 Φ(s, t 0 , x)P 1 (x)v (uet 2 ) e ν 2 (t-s) Φ(s, t 0 , x)P 2 (x)v ≤ N 2 Φ(t, t 0 , x)P 2 (x)v (uet 3 ) Φ(t, t 0 , x)P 3 (x)v ≤ N 3 e ν 3 (t-s) Φ(s, t 0 , x)P 3 (x)v Φ(s, t 0 , x)P 3 (x)v ≤ N 3 e ν 3 (t-s) Φ(t, t 0 , x)P 3 (x)v for all t ≥ s ≥ t 0 ≥ 0 and all (x, v) ∈ Y.
Example 2.3 Let us consider function a f , a metric space X and an evolution semiflow ϕ as in Example 2.1. Let µ > f (0).

We consider

V = R 3 with the norm (v 1 , v 2 , v 3 ) = |v 1 | + |v 2 | + |v 3 |. The mapping Φ : T × X → B(V ), Φ(t, s, x)(v) = = (e -µ(t-t 0 )+ t t 0 x(τ -t 0 )dτ v 1 , e t t 0 x(τ -t 0 )dτ v 2 , e -(t-t 0 )x(0)+ t t 0 x(τ -t 0 )dτ v 3 )
is an evolution cocycle.

We consider the projectors

P 1 (x, v) = (v 1 , 0, 0), P 2 (x, v) = (0, v 2 , 0), P 3 (x, v) = (0, 0, v 3 ).
Then C = (ϕ, Φ) is uniformly exponentially trichotomic with

N 1 = N 2 = N 3 = 1, ν 1 = µ -x(0), ν 2 = l, ν 3 = x(0).
Remark 2.1 For P 3 = 0 we obtain in Definition 2.7 the property of uniform exponential dichotomy.

Main results

To characterize the uniform exponential dichotomy we will consider the next theorem.

Theorem 3.1 A skew-evolution semiflow C = (ϕ, Φ) is uniformly exponentially dichotomic if and only if there exist two projector families {P k } k∈{1,2} compatible with C and a nondecreasing function

f : [0, ∞) → (1, ∞) with the property lim t→∞ f (t) = ∞ such that (i) f (t -s) Φ(t, t 0 , x)P 1 (x)v ≤ Φ(s, t 0 , x)P 1 (x)v (ii) f (t -s) Φ(s, t 0 , x)P 2 (x)v ≤ Φ(t, t 0 , x)P 2 (x)v for all t ≥ s ≥ t 0 ≥ 0 and all (x, v) ∈ Y.
Proof. Necessity. It is immediate if we consider f (t) = N -1 e νt , t ≥ 0, where N = max{N 1 , N 2 } and ν = min{ν 1 , ν 2 }, the constants N 1 , N 2 , ν 1 , ν 2 being given by Definition 2.5.

Sufficiency. We will show that (i) implies (ued 1 ). From the definition of function f there exists δ > 0 such that f (δ) > 1. We denote

ν = ln f (δ) δ > 0.
Let (t, s) ∈ T . There exist n ∈ N and r ∈ [0, δ) such that ts = nδ + r.

We have

e ν(t-s) Φ(t, t 0 , x)v ≤ f (δ)[f (δ)] n Φ(t, t 0 , x)v ≤ ≤ f (δ)[f (δ)] n-1 Φ(t -δ, t 0 , x)v ≤ ... ≤ f (δ) Φ(t -nδ, t 0 , x)v ≤
≤ f (δ)f (r) Φ(tnδ, t 0 , x)v ≤ f (δ) Φ(s, t 0 , x)v .

If we denote N = f (δ) > 1, (ued 1 ) follows.

By an analogous deduction we obtain that (ii) implies (ued 2 ).

Next result represent a characterization for the property of uniform exponential trichotomy.

... ≤ N n 3 Φ(tn, t 0 , x)P 3 (x)v ≤ N n+1 3 Φ(s, t 0 , x)P 3 (x)v = = N 3 e nν 3 Φ(s, t 0 , x)P 3 (x)v ≤ N 3 e ν 3 (t-s) Φ(s, t 0 , x)P 3 (x)v for all t ≥ s ≥ t 0 ≥ 0 and all (x, v) ∈ Y .

Similarly is obtained the second relation in (uet 3 ) from the corresponding relation in (iii).

Definition 2 . 3

 23 The mapping C : T × Y → Y defined by the relation C(t, s, x, v) = (ϕ(t, s, x), Φ(t, s, x)v), where Φ is an evolution cocycle over an evolution semiflow ϕ, is called skew-evolution semiflow on Y .Example 2.1 Let f : R + → R + be a decreasing function on [0, ∞) such that there exists lim t→∞ f (t) = l.

Theorem 3.2 Let C = (ϕ, Φ) be a skew-evolution semiflow with the property that for all (t 0 , x, v) ∈ R + ×Y the mapping s → Φ(s, t 0 , x)v is measurable. Then C is uniformly exponentially trichotomic if and only if there exist three projector families {P k } k∈{1,2,3} compatible with C, some constants N , M ≥ 1 and a nondecreasing function g : [0, ∞) → (1, ∞) with the property lim

Proof. Necessity. It can be easily verified. We obtain

ν 3 being given by Definition 2.7.

Sufficiency. We will prove that the relations in (i) imply (uet 1 ). We have

for all t ≥ s ≥ t 0 ≥ 0 and all (x, v) ∈ Y . We integrate the obtained relation on [s, t] and we have

We obtain, according to relations (3.1) and (3.2)

for all (t, s), (s, t 0 ) ∈ T and all (x, v) ∈ Y . If we denote

, u ≥ 0, similarly as in the proof of Theorem 3.1, we obtain (uet 1 ). An analogous deduction can be applied to prove that (ii) implies (uet 2 ).

To prove that the first relation in (iii) implies the first relation in (uet 3 ), we consider t ≥ s ≥ t 0 ≥ 0. We denote n = [ts]. We consider N 3 = g(1) > 1, ν 3 = ln N 3 > 0 and we obtain Φ(t, t 0 , x)P 3 (x)v ≤ N 3 Φ(t -1, t 0 , x)P 3 (x)v ≤ ...


G Loranger-Merciris

H Ozier-Lafontaine
email: harry.ozier-lafontaine@antilles.inra.fr

Ecological engineering: from concepts to applications Earthworm activities induces better resistance to plant-parasitic nematodes in banana plants

Keywords: Pontoscolex corethrurus, soil engineers, soil biophysical and chemical properties

 shown that the pathogenicity of nematode Radopholus similis was significantly reduced in the presence of earthworm Pontoscolex corethrurus. While a positive effect on the growth of banana was found, the processes involved were not clearly identified.

The objective of this work is to understand how earthworm Pontoscolex corethrurus activity affects the pathogenicity of the main parasitic nematodes of banana.

Indirect effects of earthworm activity via physical, chemical and biological modifications of the soil biotic and abiotic components are hypothesized as the main factor contributing to the decrease of nematodes pathogenicity by reducing root necrosis and increasing nutrient availability. This hypothesis was tested in controlled conditions in a greenhouse of the INRA research station in Guadeloupe (FWI). Four treatments : N-E-(absence of fauna); N-E+ (P. corethrurus alone); N+E-(phytoparasitic nematodes alone); N+E+ (earthworms plus nematodes) were setted-up and replicated five times. Each replication was composed by a banana plant cultivated in a pot containing 20 kg of soil. 337 days after the experiment set-up, shoot and root weight, shoot chemical contents, root necrosis extent and the populations of nematodes were determined. Soil samples were analysed for determination of total C and N and for nutrient contents (P2O5, NO3-and NH4+). Shrinkage curves of centimetric aggregates and casts were also calculated.

Results showed that: i) root necrosis induced by nematodes was significantly reduced (50%) in the presence of the earthworm P. corethrurus, whereas the population of nematodes did not decrease ii) the burrowing activity destroyed most of the initial structural pores (3-300µm) and increased the volume of finest structural pores diameter class (0.3-3µm). Earthworm aggregates could then shelter high microbial activity in the increased 0.3-3 µm pore class and probably became unfavourable to nematodes activity due to the decrease of larger pores of 3-300µm in diameter. This observation was corroborated by a greater phosphorus availability in earthworm casts, and a better plant phosphorus nutrition in the presence of earthworms that induced a better growth of banana plants.

These results confirm the indirect and positive effects enhanced by earthworm activity and mainly the availibilty of phosphorus via soil porosity transfer. The reduction of nematodes pathogenicity without a significant reduction of nematodes abundance, however, could not be clearly explained. [START_REF] Blouin | Belowground activities affect plant aboveground phenotype, inducing plant tolerance to parasites[END_REF] attested of similar effects in a rice experiment and hypothesized a systemic action of earthworm on plant growth driven by a modification of the expression of genes that code for tissue reparation. Complex interactions occurring in the soil biota-soil-plant continuum still remain a great challenge in the field of ecological engineering dedicated to the improvement agro-ecosystems performances.


Hélène G

Claude F-N

Role of the olfactory systems and importance of learning in the ewes' response to rams or their odors

In sheep, exposure of seasonally anestrous females to the male or its fleece results in activation of luteinizing hormone (LH) secretion and synchronized ovulation. The study of the neural pathways involved in this phenomenon, commonly named "male effect", show that the main olfactory system plays a critical role in the detection and the integration of the male odor. The accessory olfactory system participates in the perception of the ram odor but does not seem necessary for the endocrine response. According to the hypothesis that the neuroanatomical differences between the two olfactory systems could be associated with different functional roles, we investigated the importance of sexual experience and learning processes in the male effect. Our results showed that female responses depend on previous sexual experience. We also demonstrated that the LH response to male odor could result from an associative learning process. The aim of the present report was to summarize our current knowledge concerning the "male effect" and in particular to clarify the role of sexual experience and learning in the processes involved in this effect. ovine / male effect / LH / olfaction / experience

INTRODUCTION

Among the different factors involved in the control of reproduction, social interactions can greatly influence and modify the reproductive events, from sexual postures to endocrine regulations. Indeed, depending on the biological context, the effects of social interactions can result in inhibition of reproduction in some individuals of the group or, in stimulation and synchronization of neuroendocrinology and behavior. In rodents, housing together individuals in unisex groups results in modification of the estrous cycle of females [START_REF] Mcclintock | Estrous synchrony and its mediation by airborne chemical communication (Rattus norvegicus)[END_REF][START_REF] Van Der Lee | Spontaneous pseudopregnancy in mice[END_REF][START_REF] Whitten | Occurrence of anoestrus in mice caged in groups[END_REF] and testicular development in males [START_REF] Lawton | Inhibition of sexual maturation by a urinary pheromone in male prairie deer mice[END_REF]. Contacts between sexual partners can also modify the reproductive processes and affect most stages of reproduction: acceleration of pu-berty and testicular growth [START_REF] Vandenbergh | Effect of the presence of a male on the sexual maturation of female mice[END_REF][START_REF] Vandenbergh | The influence of the social environment on sexual maturation in male mice[END_REF], induction and synchronization of ovulation and estrus [START_REF] Whitten | Modification of the oestrous cycle of the mouse by external stimuli associated with the male[END_REF], blockage of pregnancy [START_REF] Bruce | A block to pregnancy in the mouse caused by proximity of strange males[END_REF]. It has been demonstrated that in most cases, the physiological response is initiated by a rapid change in hormonal concentrations (luteinizing hormone (LH) and prolactin in females, LH and testosterone in males) [START_REF] Macrides | Effects of exposure to vaginal odor and receptive females on plasma testosterone in the male hamster[END_REF][START_REF] Ryan | Changes in serum hormone levels associated with maleinduced ovulation in group-housed adult female mice[END_REF]. These phenomena have been extensively studied in rodents but they are not restricted to these species. In sheep and goats, the introduction of males in a flock of seasonally anoestrous females results in activation of the secretion of LH and synchronized ovulation [START_REF] Chemineau | Effect on oestrus and ovulation of exposing creole goats to the male at three times of the year[END_REF][START_REF] Martin | The physiological responses of anovulatory ewes to the introduction of rams -A review[END_REF]. This phenomenon, commonly called the "male effect", is frequently used in sheep and goat husbandry to advance and synchronize reproduction [START_REF] Thimonier | L'effet mâle chez les ovins : une technique actuelle de maîtrise de la reproduction[END_REF].

Article published by EDP Sciences and available at http://www.edpsciences.org/rnd or http://dx.doi.org/10.1051/rnd:2006021 Among the different sensory signals exchanged between the individuals, olfactory cues play a preponderant role in mammals since they can mimic most of the behavioral and physiological changes induced by interactions between conspecifics of the same sex or between sexual partners. It has been reported in many species that chemical signals serve as sexual attractants, and promote sexual arousal, appetitive and precopulatory postures and mating behavior (rat [START_REF] Brown | Odor preference and urinemarking scales in male and female rats: effects of gonadectomy and sexual experience on responses to conspecifics odors[END_REF], mouse [START_REF] Johnston | Endocrine control of female mouse odors that elicit luteinizing hormone surges and attraction in males[END_REF], hamster [START_REF] Petrulis | A reevaluation of dimethyl disulfide as a sex attractant in golden hamsters[END_REF], elephant [START_REF] Rasmussen | Purification, identification, concentration and bioactivity of (Z)-7dodecen-1-yl acetate: sex pheromone of the female Asian elephant, Elephas maximus[END_REF], rhesus macaque [START_REF] Michael | Primate sex pheromones of vaginal origin[END_REF]). These chemosignals can also modify the physiology and neuroendocrine state (mouse [START_REF] Bronson | Serum FSH, LH, and prolactin in adult ovariectomized mice bearing silastic implants of estradiol: responses to social cues[END_REF], rat [START_REF] Kamel | The influence of mating and related stimuli on plasma levels of luteinizing hormone, follicle stimulating hormone, prolactin, and testosterone in the male rat[END_REF], hamster [START_REF] Macrides | Effects of exposure to vaginal odor and receptive females on plasma testosterone in the male hamster[END_REF], prairie vole [START_REF] Dluzen | Male vole urine changes luteinizing hormone-releasing hormone and norepinephrine in female olfactory bulb[END_REF]). In sheep, ram fleece activates LH secretion in anestrous ewes [START_REF] Knight | Source of ram pheromones that stimulate ovulation in the ewe[END_REF].

However, in a flock or a group of ewes, not all the females exhibit an LH increase in response to the ram or its fleece. Several factors account for the variability of the response: the race, the period of the year, the male sexual activity, etc. Two arguments let us hypothesize that sexual experience can also be a critical factor, especially for the response to the ram odor. Firstly, preliminary data suggest that the male fleece is ineffective to activate the LH secretion in sexually naive ewes [START_REF] Cohen-Tannoudji | Effet du mâle sur la sécrétion gonadotrope (LH) de la brebis (Ovis aries L). Analyse de l'interaction olfactive[END_REF]. However, these results remain unclear and need confirmation. Our second argument came from our studies examining the neural pathways involved in the processes of the male effect. In mammals, olfactory signals are detected and integrated by two olfactory systems: the accessory and the main olfactory systems, which are anatomically and functionally separated and which possess their own receptors and central pathways. The accessory olfactory system is essentially connected to hypothalamic structures: the axons of the neuroreceptors of the vomeronasal organ (VNO) project to the accessory olfactory bulb (AOB) which in turn projects to the medial amygdala which has direct connections to the ventromedial hypothalamic nucleus and the preoptic area (rat [START_REF] Scalia | The differential projections of the olfactory bulb and accessory olfactory bulb in mammals[END_REF], hamster [START_REF] Kevetter | Connections of the corticomedial amygdala in the golden hamster. II. Efferents of the "olfactory amygdala[END_REF][START_REF] Kevetter | Connections of the corticomedial amygdala in the golden hamster. I. Efferents of the "vomeronasal amygdala[END_REF], sheep [START_REF] Jansen | Central connections of the ovine olfactory bulb formation identified using wheat germ agglutinin-conjugated horseradish peroxidase[END_REF]). On the contrary to the accessory system, the main olfactory system is linked to several cortical areas: the main olfactory bulb (MOB) projects to the cortical amygdala, to the piriform cortex which is connected via the thalamus to the orbitofrontal and insular cortices, and to the entorhinal cortex which is connected to the hippocampus [START_REF] Kevetter | Connections of the corticomedial amygdala in the golden hamster. II. Efferents of the "olfactory amygdala[END_REF][START_REF] Kevetter | Connections of the corticomedial amygdala in the golden hamster. I. Efferents of the "vomeronasal amygdala[END_REF][START_REF] Jansen | Central connections of the ovine olfactory bulb formation identified using wheat germ agglutinin-conjugated horseradish peroxidase[END_REF]. According to some authors, the neuroanatomical differences between the two olfactory systems could be associated with different functional roles [START_REF] Brennan | The vomeronasal system[END_REF][START_REF] Keverne | Non-olfactory chemoreceptors of the nose: recent advances in understanding the vomeronasal and trigeminal systems[END_REF][START_REF] Wysocki | The vomeronasal system: its role in learning and social behaviour[END_REF]. It has been postulated that the vomeronasal neural pathway that bypasses the cognitive regions of the brain, could be implicated in the detection and integration of chemochemicals eliciting preprogrammed and stereotyped responses. This has been verified in rodents since most chemosignals are detected by the accessory olfactory system and frequently induce stereotyped and automatic responses [START_REF] Keverne | Non-olfactory chemoreceptors of the nose: recent advances in understanding the vomeronasal and trigeminal systems[END_REF] (Tab. I). In contrast, the numerous cortical areas belonging to the main olfactory system could provide for elaborate treatment of the olfactory information, involving cognitive or learning mechanisms. According to our findings showing that the main olfactory system plays a critical role in the detection of the male odor, we hypothesized that sexual experience and learning can modulate the ewes' responses to the male effect.

The aim of the present report was to present the data that conduct us to this hypothesis and the procedures we used to assess the role of sexual experience and learning in the processes involved in the male effect.

DESCRIPTION OF THE MALE EFFECT

Endocrine response

Sheep are seasonal breeders and during the period of anestrus, ewes do not

Table I.

A comparison of the mechanisms of action of different mammalian pheromones acting between males and females. A part of this table has also been presented in [START_REF] Gelez | The "male effect" in sheep and goats: a review of the respective roles of the two olfactory systems[END_REF]. experience ovulation nor ovarian cycles. Exposure to a ram results in activation of the LH secretion, and a pulse of LH is observed within minutes whereas the secretion of FSH or prolactin does not undergo such rapid changes [START_REF] Martin | Increased plasma LH levels in seasonally anovular Merino ewes following the introduction of rams[END_REF][START_REF] Martin | Effect of the introduction of rams during the anoestrous season on the pulsatile secretion of LH in ovariectomized ewes[END_REF][START_REF] Poindron | Changes in gonadotrophins and prolactin levels in isolated (seasonally or lactationally) anovular ewes associated with ovulation caused by the introduction of rams[END_REF] (Fig. 1).

Species

If the contact with males is maintained, ewes experience a preovulatory surge of LH which takes place at around 36 h (ranging from 6 to 54 h, [START_REF] Oldham | Stimulation of the seasonally anovular Merinos ewes by rams. I. Time from introduction of the rams to the preovulatory surge and ovulation[END_REF]). Simultaneously, a rise in the concentration of FSH occurs [START_REF] Martin | The induction of oestrus and ovulation in seasonally anovular ewes by exposure to rams[END_REF]. The point of ovulation is observed around 48 h after the introduction of the male and is qualified as "silent" because it is not associated with estrous behavior [START_REF] Oldham | Stimulation of the seasonally anovular Merinos ewes by rams. I. Time from introduction of the rams to the preovulatory surge and ovulation[END_REF]. In half of the females, the develop-ment of the first corpus luteum is abnormal and regresses after 6-7 days, and the ewes re-cycle. Many of these ovulations are also silent [START_REF] Oldham | Stimulation of the seasonally anovular Merinon ewes by rams. II. Premature regression of ram-induced corpora lutea[END_REF]. Sexual behavior appears with subsequent ovulations and takes place in the form of two peaks of activity in the flock 18 and 25 days after the introduction of the male [START_REF] Oldham | Stimulation of the seasonally anovular Merinon ewes by rams. II. Premature regression of ram-induced corpora lutea[END_REF].

A direct contact with the male is not necessary to observe the increase of LH secretion since its effect can be in part mimicked by presentation of only its odor [START_REF] Knight | Source of ram pheromones that stimulate ovulation in the ewe[END_REF]. The active chemosignal is a mixture of compounds which have only been partially identified. The biological activity requires the simultaneous presence of compounds retained in both acid and neutral fractions [START_REF] Cohen-Tannoudji | Ram sexual pheromone: first approach of chemical identification[END_REF]. According to the definition of Karlson and Lüscher [START_REF] Karlson | Pheromones: a new term for a class of biologically active substances[END_REF] and Wilson and Bossert [START_REF] Wilson | Chemical Communication among Animals[END_REF], the ram odor may be classified as a primer pheromone, a substance secreted by an individual and eliciting a specific physiological change in a second individual of the same species. However, like most of the mammalian pheromones, the ram odor does not strictly adhere to all of the criteria defining a pheromone.

Behavioral response

The physiological changes induced by the male or its odor have been extensively studied. To know if these stimuli can also modify the females' behavior, we completed an experiment to observe in detail the behavior of ewes during exposure to the ram or its fleece. Preliminary observations were carried out to establish a precise ethogram consisting of a classification of all behaviors into six principal components: investigation of the stimulus, locomotor activity, indicators of stress, indicators of attention, feeding behavior, other activity [START_REF] Gelez | The "male effect" in sheep and goats: a review of the respective roles of the two olfactory systems[END_REF]. Then, we recorded all the behaviors displayed by three groups of anestrous ewes that were randomly exposed to female fleece, male fleece or rams.

Ear movements in sheep are associated with the "psychological state" of the animal. In a vigilant and attentive state, sheep keep their ears pricked as opposed to backwards. In the presence of the male or its fleece, most ewes keep their ears pricked whereas the proportion of ewes with ears backward is significantly higher in the presence of female fleece rather than male fleece or the male himself [START_REF] Gelez | The "male effect" in sheep and goats: a review of the respective roles of the two olfactory systems[END_REF]. Furthermore, ewes tend to look more often at the male fleece than the female fleece. These observations suggest that the ram or its odor can stimulate the ewes' attention. A link between behavioral and endocrine response remains possible, with only ewes that are vigilant or attentive during the presentation of these stimuli exhibiting endocrine responses.

The proportion of ewes urinating and the number of urinations were significantly higher during exposure to male or its fleece than during exposure to female fleece. Emission of urine only with male fleece and not with female fleece shows that ewes can discriminate between both odors, and that the male or its odor induces these behavioral changes. Similar observations are obtained in rodents. In mice, females urinate more frequently in the presence of the male or its odor than in the presence of female odor [START_REF] Maruniak | Urinary marking in female house mice: effects of ovarian steroids, sex experience and type of stimulus[END_REF]. In hamsters, male odor, on the contrary to female odor, increases vaginal marking behavior [START_REF] Johnston | The causation of two scentmarking behaviour patterns in female hamsters (Mesocricetus auratus)[END_REF]. By urinating, ewes may signal to males their physiological state since rams are able to distinguish between urine of receptive and non receptive females [START_REF] Blissitt | Discrimination between the odours of fresh oestrous and non-oestrous ewe urine by rams[END_REF].

Most ewes investigated at least once the two fleeces or the male but the total number of olfactory investigations did not differ between the three stimuli. This suggests that neither the male nor its fleece are attractive for ewes. In many species, odor from the opposite sex is attractive and can induce appetitive or copulatory behaviors (Tab. I). However, in most species, females are more attracted and interested by the male odor around the estrous period: rat [START_REF] Brown | Odor preference and urinemarking scales in male and female rats: effects of gonadectomy and sexual experience on responses to conspecifics odors[END_REF], dog [START_REF] Dunbar | Olfactory preferences in dogs: the response of male and female beagles to conspecific odors[END_REF], pig [START_REF] Signoret | Reproductive behaviour of pigs[END_REF], elephant [START_REF] Rasmussen | Frontalin: a chemical message of musth in Asian elephants (Elephas maximus)[END_REF]. In the context of the male effect, no ewes exhibited signs of receptivity. Our results show that male nudges were never followed by immobilizations or standing posture, characteristic of receptive behavior and acceptance of mating [START_REF] Fabre-Nys | Development and use of a method for quantifying female sexual behaviour in ewes[END_REF]. So, the ineffective attractiveness of the ram or its odor may be explained by the physiological state of ewes since they were in anestrus and they did not exhibit any sexual motivation.

In conclusion, our results show that the ram or its odor does not induce drastic behavioral changes, in particular they do not elicit strong attraction but seem to focus the attention of ewes and induce urine emission.

NEURAL PATHWAYS INVOLVED IN THE EFFECT OF THE MALE OR ITS ODOR

We used different procedures to assess the neural pathways involved in the effect of the male or its odor on the LH secretion of ewes. Firstly, to obtain a map of the brain areas activated by these stimuli, we used immunocytochemistry to examine the expression of the Fos protein, a marker of neuronal activation, in the brains of four groups of ewes exposed to different stimulations: male, male fleece, female fleece or the testing room (control group). In comparison with the control group, the male or its odor significantly increases Fos neuronal expression in the MOB and the AOB, anterior olfactory nucleus, cortical and basal amygdala, dentate gyrus, ventromedial nucleus of the hypothalamus, piriform and orbitofrontal cortices [START_REF] Gelez | Neural pathways involved in the endocrine response of anestrous ewes to the male or its odor[END_REF]. The MOB, the cortical amygdala and the dentate gyrus are specifically more activated by the male odor than the female odor. These results demonstrate that the structures belonging to the main olfactory system play a critical role in the detection and the integration of the male olfactory cue. The Fos neuronal expression detected in the AOB shows that the accessory olfactory system participates in the detection of the ram odor, even if its involvement seems limited.

Then, we adopted a functional approach and we tested the effects of lesion or inactivation of different neuronal relays belonging to the main or the accessory system. Our results confirm the respective role of the two olfactory systems. Indeed, destruction of the olfactory epithelium (by intranasal administration of zinc sulfate) or inactivation of the cortical amygdala (by retrodialysis infusion of the anaesthetic lidocaine) completely blocks the endocrine response to ram odor [START_REF] Gelez | The "male effect" in sheep and goats: a review of the respective roles of the two olfactory systems[END_REF][START_REF] Gelez | Inactivation of the olfactory amygdala prevents the endocrine response to male odour in anoestrus ewes[END_REF] (Tab. II). In contrast, lesion of the VNO or inactivation of the medial amygdala or the ventromedial nucleus of the hypothalamus does not impair the effect of ram odor on LH secretion [START_REF] Gelez | Inactivation of the olfactory amygdala prevents the endocrine response to male odour in anoestrus ewes[END_REF][START_REF] Cohen-Tannoudji | Non-involvement of the accessory olfactory system in the LH response of anoestrous ewes to male odour[END_REF]. These results show that the main olfactory system, on the contrary to the accessory system, is absolutely necessary for the detection and the integration of male olfactory cues. It is interesting to note that none of these surgical interventions affected the LH response to the male himself [START_REF] Bronson | Serum FSH, LH, and prolactin in adult ovariectomized mice bearing silastic implants of estradiol: responses to social cues[END_REF][START_REF] Gelez | The "male effect" in sheep and goats: a review of the respective roles of the two olfactory systems[END_REF] (Tab. II). These findings emphasize the role of sensory cues other than olfaction, but also suggest that other odors than those contained in the male fleece could account for a pheromonal activity and could be detected and processed by different neural pathways.

In rodents, pheromonal cues are frequently detected and processed by the vomeronasal neural pathway [START_REF] Keverne | Pheromonal influences on the endocrine regulation of reproduction[END_REF]. In the mouse and rat, sexual partner odor induces Fos activation only in the AOB and not the MOB [START_REF] Halem | Vomeronasal neuroepithelium and forebrain Fos responses to male pheromones in male and female mice[END_REF][START_REF] Kelliher | Neuronal Fos activation in olfactory bulb and forebrain of male rats having erections in the presence of inaccessible estrous females[END_REF]. In males and females, the destruction of the VNO blocks the endocrine changes provoked by urine of the opposite sex [START_REF] Beltramino | Release of LH in the female rat by olfactory stimuli. Effect of the removal of the vomeronasal organs or lesioning of the accessory olfactory bulbs[END_REF][START_REF] Coquelin | Pheromonally induced release of luteinizing hormone in male mice: involvement of the vomeronasal system[END_REF][START_REF] Wysocki | Male vomeronasal organ mediates female-induced testosterone surges in mice[END_REF]. There are few examples in which chemical signals of the communication between individuals are essentially detected by the main olfactory epithelium. This is the case for the mother pheromone inducing nipple search in newborn rabbits [START_REF] Hudson | Pheromonal release of suckling in rabbits does not depend on the vomeronasal organ[END_REF] or for the boar saliva eliciting standing posture in female pigs [START_REF] Dorries | Sensitivity and behavioral responses to the pheromone androsterone are not mediated by the vomeronasal organ in domestic pigs[END_REF]. In ferrets, nare occlusion eliminates heterosexual mate choice, suggesting a preponderant role of the main olfactory system in the processing of cues involved in the ferret mate recognition [START_REF] Kelliher | Nares occlusion eliminates heterosexual partner selection without disrupting coitus in ferrets of both sexes[END_REF]. However, all of these examples imply olfactory signals inducing behavioral responses, whereas primer pheromones are mainly detected by the VNO (Tab. I).

In summary, our results show that in sheep, on the contrary to other species, Table II. Summary of the main results showing the neural pathways involved in the male effect. Destruction of the main olfactory epithelium and inactivation of the cortical nucleus of amygdala blocks the endocrine response to the male odor. In contrast, destruction of the vomeronasal organ (VNO), section of the vomeronasal (VN) nerves or inactivation of the medial amygdala has no effect. None of these surgical procedures impair the endocrine response to the male.

Response to the male odor

Response the main olfactory system is mediating the detection of an olfactory message eliciting a physiological response. However, ram odor induces neuronal Fos responses in both the AOB and the MOB. This suggests that, even if the accessory system plays a minor role, it can participate in the detection and the integration of male olfactory cues. Similar data were obtained in male hamsters in which AOB and MOB neuronal Fos activation were observed in response to female vaginal secretions [START_REF] Fiber | Pheromones induce c-fos in limbic areas regulating male hamster mating behavior[END_REF]. In this species, both olfactory systems are involved in the detection of sexual chemosignals, especially after sexual experience [START_REF] Fewell | Experience facilitates vomeronasal and olfactory influence on Fos expression in medial preoptic area during pheromone exposure or mating in male hamsters[END_REF]. Similar implication of olfactory systems can occur in sheep.

ROLE OF LEARNING

The initial definition of pheromones requires that pheromones should elicit preprogrammed responses which are minimally influenced by experience [START_REF] Karlson | Pheromones: a new term for a class of biologically active substances[END_REF]. However, this term was initially defined in insects and its extension to mammals raises some problems [START_REF] Beauchamp | The pheromone concept in mammalian chemical communication: a critique[END_REF]. Indeed, in mammals, most behavioral responses elicited by sex-ual pheromones are facilitated by previous sexual experience. In many species, chemical signals serve as sexual attractants and promote appetitive and mating behaviors but these responses are preferentially expressed by sexually experienced individuals. In adult male rats, mice and dogs, sexual experience results in a preference for estrous over nonestrous female odors [START_REF] Carr | Responses of rats to sex odors[END_REF][START_REF] Doty | Attraction of beagles to conspecific urine, vaginal and anal sac secretion odors[END_REF][START_REF] Hayashi | Sex-attractant emitted by female mice[END_REF]. In mice, the presentation of female urine attracts and elicits vocalizations only in sexually experienced males [START_REF] Hayashi | Sex-attractant emitted by female mice[END_REF][START_REF] Dizinno | Ultrasonic vocalizations by male mice (Mus musculus) to female sex pheromone: experiential determinants[END_REF]. Similarly, the odor of estrous female rats produces erection only in sexually experienced males [START_REF] Sachs | Erection evoked in male rats by airborne scent from estrous females[END_REF]. In contrast, most chemosignals eliciting physiological changes are effective in naive individuals never exposed before to the emitter. In several species, male odors accelerate puberty in young females that have never been in contact with males, for example: mouse [START_REF] Colby | Regulatory effects of urinary pheromones on puberty in the mouse[END_REF], prairie vole [START_REF] Carter | Male-related pheromones and the activation of female reproduction in the prairie vole (Microtus ochrogaster)[END_REF], hamster [START_REF] Reasner | Acceleration of reproductive development in female Djungarian hamsters by adult males[END_REF], and cow [START_REF] Izard | The effects of bull urine on puberty and calving date in crossbred beef heifers[END_REF]. In hamsters, female vaginal secretions induce an increase of testosterone in sexually naive males [START_REF] Pfeiffer | Hormonal and behavioral responses of male hamsters to females and female odors: roles of olfaction, the vomeronasal system, and sexual experience[END_REF]. Naive male mice also reflexively release luteinizing hormone (LH) and testosterone pulses after encountering a female or her urinary pheromones [START_REF] Macrides | Strange females increase plasma testosterone levels in male mice[END_REF][START_REF] Maruniak | Gonadotropic responses of male mice to female urine[END_REF].

In rodents, pheromonal cues are essentially detected and integrated by the accessory olfactory system [START_REF] Brennan | The vomeronasal system[END_REF][START_REF] Keverne | Pheromonal influences on the endocrine regulation of reproduction[END_REF][START_REF] Keverne | The vomeronasal organ[END_REF]. Destruction of the VNO eliminates most responses to conspecific odors [START_REF] Wysocki | Consequences of removing the vomeronasal organ[END_REF]. However, in some cases, learning can compensate for the dramatic effects of the VNO lesion. In male hamsters, lesion of the VNO produces severe deficits in mating behavior in sexually naive males but have little effect in experienced males [START_REF] Meredith | Vomeronasal organ removal before sexual experience impairs male hamster mating behavior[END_REF]. Destruction of the VNO of male mice eliminates vocalizations in response to female urine but only in sexually naive individuals [START_REF] Bean | Olfactory and vomeronasal mediation of ultrasonic vocalizations in male mice[END_REF]. In contrast, concerning primer pheromones, the compensation of the lesion of the VNO by sexual experience does not appear possible [START_REF] Keverne | Non-olfactory chemoreceptors of the nose: recent advances in understanding the vomeronasal and trigeminal systems[END_REF]. For example, the destruction of the VNO impairs the surge of LH and testosterone elicited by female odor in both sexually naive and experienced males [START_REF] Coquelin | Pheromonally induced release of luteinizing hormone in male mice: involvement of the vomeronasal system[END_REF][START_REF] Wysocki | Male vomeronasal organ mediates female-induced testosterone surges in mice[END_REF]. In sheep, lesion of the VNO does affect the LH response to the male odor [START_REF] Cohen-Tannoudji | Non-involvement of the accessory olfactory system in the LH response of anoestrous ewes to male odour[END_REF]. However, this result has been obtained in sexually experienced ewes and it remains possible that learning processes can compensate for the VNO lesion. But before testing the effect of VNO lesion in sexually naive ewes, the ability of naive females to respond to the male odor had to be evaluated.

Another argument that conducts us to assess the role of sexual experience came from our data showing the neural pathways involved in the effect of the male or its odor. The neural pathway of the accessory system can be considered as direct since this system is essentially connected to hypothalamic structures and not to the cognitive centers of the brain. For this reason, it has been postulated that the chemosignals detected and integrated by the accessory system do not require learning mechanisms in order to be effective and would induce preprogrammed and stereotyped responses. In contrast, the numerous cortical areas belonging to the main olfactory system could provide for an elaborate treatment of the olfactory cues. The olfactory amygdala which is connected to several cortical areas, can access higher levels of olfactory processing than the vomeronasal amygdala which projects only to hypothalamic structures. The piriform and the entorhinal cortex, and the basolateral nucleus of the amygdala (which has cross connections with the cortical nucleus) are involved in memory and learning processes, including olfactory discrimination [START_REF] Datiche | Expression of Fos in the piriform cortex after acquisition of olfactory learning: an immunohistochemical study in the rat[END_REF][START_REF] Hess | Differential patterns of c-fos mRNA expression in amygdala during successive stages of odor discrimination learning[END_REF][START_REF] Tronel | Mapping of olfactory memory circuits: region-specific c-fos activation after odor-reward associative learning or after its retrieval[END_REF]. In our study, Fos neuronal activation detected in these structures after exposure to the male or its fleece suggests their involvement in the detection of the male odor. Furthermore, the results of our study testing the effect of lesion or inactivation of different relays belonging to the two olfactory systems demonstrate the critical role of the main olfactory system. This system would provide for elaborate cognitive processing of the information. Accordingly, we hypothesized that the response to the male or its odor could be modulated by previous experience and learning processes. To examine this question, we completed two experiments.

Role of sexual experience

The aim of this experiment was to compare the LH responses to the ram and its odor between sexually naive or experienced ewes. Our previous findings showed that age and sexual experience can differently affect ewe sexual behavior. Indeed, during their first interaction with males, sexually naive both young and adult females do not display proceptive behaviors whereas sexually naive adult ewes, on the contrary to naive young females, exhibit levels of receptivity similar to experienced adults [START_REF] Gelez | Role of experience in the neuroendocrine control of ewes' sexual behavior[END_REF]. These results prove the importance of dissociating the effect of these two factors. For this reason, we used 4 groups of females that differed by their sexual experience (sexually naive or experienced) and their age (young or adult). All the females were randomly exposed to female fleece, male fleece or males.

Response to male odor

Our results show that the male odor activates LH secretion in sexually experienced ewes both young and adult, but has no effect in most naive females [START_REF] Gelez | Importance of learning in the response of ewes to male odor[END_REF] (Fig. 2). The experienced ewes, regardless of their age, exhibited a significantly greater number of LH pulses during exposure to the male odor than during exposure to the female fleece. No significant increase of LH pulses was detected in naive females. The response latency was also significantly shorter for the experienced ewes. These findings demonstrate that sexual experience is a critical factor and facilitates the endocrine response to the ram odor. This markedly differs from other species. Indeed, in most mammals, the effects of olfactory cues on neuroendocrine secretions are independent of previous sexual experience and do not require learning mechanisms. The male odor accelerates sexual maturation in young females which have never been in contact with males (mouse [START_REF] Vandenbergh | Male odor accelerates female sexual maturation in mice[END_REF], prairie vole [START_REF] Carter | Male-related pheromones and the activation of female reproduction in the prairie vole (Microtus ochrogaster)[END_REF], hamster [START_REF] Reasner | Acceleration of reproductive development in female Djungarian hamsters by adult males[END_REF], cow [START_REF] Izard | The effects of bull urine on puberty and calving date in crossbred beef heifers[END_REF]). Sexually naive males exhibit a reflexive increase of testosterone during their first exposure to female urine or vaginal secretion (mouse [START_REF] Maruniak | Gonadotropic responses of male mice to female urine[END_REF], hamster [START_REF] Pfeiffer | Hormonal and behavioral responses of male hamsters to females and female odors: roles of olfaction, the vomeronasal system, and sexual experience[END_REF]). In sheep, on the contrary to rodents, it seems that the male odor needs to be learned to be effective. This suggests that sexual experience is required to recognize the ram odor and to give meaning to this olfactory signal. In contrast, exposure to the male himself results in activation of the LH secretion in all ewes, regardless of their experience or their age (Fig. 2). The male appears as a sufficiently powerful stimulus to modify the female LH secretion immediately, without prior exposure or sexual experience.

The analysis of the female behaviors reveals that both groups of experienced ewes investigated significantly more (number of olfactory investigations and number of times they "ate" the fleece) the male than the female fleece. These differences were not observed in the two groups of naive females, excepting that the naive adult ewes investigated more the male than the female fleece. These results suggest that the male odor does not possess innate attractive properties, on the contrary to some species (hamster [START_REF] Johnston | Sexual attraction function of golden hamster vaginal secretion[END_REF], mouse [START_REF] Jemiolo | Sociosexual olfactory preference in female mice: attractiveness of synthetic chemosignals[END_REF][START_REF] Moncho-Bogani | Attractive properties of sexual pheromones in mice: innate or learned?[END_REF]). Only the sexually experienced ewes urinated more in the presence of the ram fleece than the female fleece. This suggests that the emission of urine is not a pre-programmed response but rather a response to an identified stimulus. In ungulates, the emission of urine by females is frequent during sexual interactions and is commonly followed by male flehmen [START_REF] Banks | Some aspects of sexual behavior in domestic sheep, Bovis aries[END_REF][START_REF] Ladewig | Flehmen in male goats: role in sexual behavior[END_REF]. In most breeds, the females urinate more in response to male courtship when they were in anestrus rather than in estrus [START_REF] Bland | Correlation of flehmen by male sheep with female behaviour and oestrus[END_REF][START_REF] Stevens | Effect of ewe urine and vaginal secretions on ram investigative behaviour[END_REF]. In another breed of sheep, Romanov, and in goats, however, females urinated more in the presence of males when they were in estrus (Fabre-Nys, personal observation). This behavior can serve to attract males and stimulate their sexual arousal, and may be considered a sign of proceptivity similar to vaginal marking by female hamsters [START_REF] Johnston | Olfactory preferences, scent marking, and "proceptivity" in female hamsters[END_REF]. This same explanation is not applicable to our results since in the context of the male effect, ewes are in anestrus. The exact significance of female urine emission therefore remains unclear.

Response to male

In the presence of males, naive ewes exhibited an LH response similar to those of the experienced females and the LH response did not differ significantly among four groups of females. For all groups of females, the proportion of responsive ewes, the number of LH pulses and the maximum concentration of LH were significantly higher during exposure to males than during exposure to the female fleece. These results suggest that the male, on the contrary to its fleece alone, constitutes a sufficiently powerful stimulation that does not require prior sexual experience. These findings emphasize the importance of sensory information other than olfaction in the processes of the male effect. This has been previously demonstrated since bulbectomy does not impair the LH increase elicited by the male [START_REF] Cohen-Tannoudji | Non-pheromonal stimulation by the male of LH release in the anoestrous ewe[END_REF]. Furthermore, castrated rams or females treated with testosterone can induce ovulation in anestrous ewes [START_REF] Signoret | Effectiveness of testosterone treated wethers and ewes as teasers[END_REF]. Our results confirm these data since none of the interventions which eliminate the endocrine response to the male odor have an effect on the response to the male himself. When the male is presented, the destruction of the olfactory epithelium or the inactivation of the cortical amygdala does not disrupt the increase of LH [START_REF] Gelez | Inactivation of the olfactory amygdala prevents the endocrine response to male odour in anoestrus ewes[END_REF].

Associative olfactory learning

The results of the previous experiment show that the ram odor has to acquire a meaning for the females, in order to induce the endocrine response. During their first interactions with males, ewes could proceed to an associative olfactory learning between the male and its odor, to be able to subsequently respond to the odor alone. This olfactory associative learning can follow the rules of a pavlovian conditioning consisting of the association between an unconditioned (the male) and a conditioned stimuli (the ram odor). Afterwards, the conditioned cue could mimic the effect of the unconditioned stimulus. To assess this hypothesis, we tested the effectiveness of a neutral conditioned stimulus (lavender odor) previously associated with the male, to induce the endocrine and behavioral responses. During the breeding season, three groups of ewes were placed for two weeks with either males scented with lavender, unscented males or females scented with lavender. Then, during the anestrous period, all ewes were randomly exposed to unscented female fleece or female fleece scented with lavender. Our results show that the lavender odor induced an LH increase in a large proportion of ewes that had been previously in contact with males scented with lavender (Fig. 3). The females of this group showed a significantly greater number of LH pulses and a high LH maximum during exposure to the scented fleece than during exposure to the unscented fleece. The same effect was not observed in the two other groups of females (Fig. 3). The lavender odor also tended also to mimic the behavioral effect of the male odor. Indeed, only the ewes previously exposed to scented males tended to urinate more during exposure to the lavender scented fleece than in the presence of the unscented fleece.

Similar conditioning paradigms have been used to reproduce behavioral changes induced by chemosignals. For example, when mating occurs with females scented with an artificial odor, subsequent exposure to this odor induces vocalizations by male mice [START_REF] Nyby | Postpubertal experience establishes signal value of mammaliam sex odor[END_REF] and rats develop a preference for a sexual partner treated with this scent [START_REF] Kippin | The development of olfactory conditioned ejaculatory preferences in the male rat. I. Nature of the unconditioned stimulus[END_REF][START_REF] Kippin | The nature of the conditioned response mediating olfactory conditioned ejaculatory preference in the male rat[END_REF]. In contrast, conditioned endocrine responses are difficult to obtain [START_REF] Keverne | Non-olfactory chemoreceptors of the nose: recent advances in understanding the vomeronasal and trigeminal systems[END_REF], and this has only been demonstrated in one study. Graham and Desjardins [START_REF] Graham | Classical conditioning: induction of luteinizing hormone and testosterone secretion in anticipation of sexual activity[END_REF] showed that male rats exhibit an increase in circulating levels of LH and testosterone in response to an artificial odor (methylsalicylate) which had been previously paired with a receptive female during mating. In our experiment, the activation of LH secretion and the emission of urine in the response to the lavender odor suggest that the females have learned the association between the ram and the artificial odor. A similar associative olfactory learning between the sexual partner and its odor can occur during the first interactions with males, leading to assign a "meaning" to the male chemosignal.

CONCLUSION

Our results demonstrate the critical role of sexual experience in the endocrine response to the ram odor and suggest that olfactory associative learning mechanisms can occur during first interactions with males, giving the capability of ewes to subsequently recognize the male fleece. Our findings support the idea that processes of olfactory cues detected by the main olfactory system can involve cognitive or learning mechanisms. The response to the ram odor does not correspond to preprogrammed or reflex responses, but rather responses to a stimulus that had acquired a meaning. It is interesting to note that in other species, all the chemosignals that are integrated by the main olfactory system and require learning mechanisms, concern only behavioral responses (pig [START_REF] Dorries | Sensitivity and behavioral responses to the pheromone androsterone are not mediated by the vomeronasal organ in domestic pigs[END_REF], ferret [START_REF] Kelliher | Sex difference and testosterone modulation of pheromone-induced Neuronal Fos in the Ferret's main olfactory bulb and hypothalamus[END_REF]). In comparison, the ram odor constitutes an original model of action of a primer pheromone. This may correspond to an adaptative strategy of reproduction that would be advantageous for species living in variable conditions with seasonal breeding and high predator pressure.

58 +:

 58 Blocks the pheromonal effect. -: No effect.NT: Not tested.

Figure 1 .

 1 Figure 1. LH variations with blood samples collected every 10 min (top) and every 2 min (bottom) in anoestrous ewes after ram introduction. (Adapted from[START_REF] Martin | The physiological responses of anovulatory ewes to the introduction of rams -A review[END_REF] and Fabre-Nys and Martin, unpublished data. This figure has also been presented in[START_REF] Gelez | The "male effect" in sheep and goats: a review of the respective roles of the two olfactory systems[END_REF].)

Figure 2 .

 2 Figure 2. LH secretion in response to the ram and its odor in ewes that differed by their age (young or adult) or their sexual experience (sexually naive or experienced).

Figure 3 .

 3 Figure 3. LH secretion in response to a neutral odor (lavender odor) in ewes previously exposed to females scented with lavender, unscented males or males scented with lavender.

  Data are expressed in the number of females exhibiting an LH increase in response to the stimulus.

					to the male
	Structure	Control Lesion or inactivation Control Lesion or inactivation
	Main olfactory system
	Olfactory epithelium	10/13	3/13 *	9/13	11/13
	Cortical amygdala	6/9	0 /9 *	3/6	5 /6
	Accessory olfactory system
	VNO Electrocauterization	7/12	8/12		NT
	VN nerves section	7/10	7/10		NT
	Medial amygdala	6/10	6/10	5/9	6 /9

* Different from the control group. NT: Not tested.

ACKNOWLEDGEMENTS

The authors thank Didier Chesneau, Eric Archer and Raymond Campan for their help and their technical assistance.