Bertrand Langlois

The History, Ethnology and Social Importance of Mare's Milk Consumption in Central Asia

Keywords: Central Asia, horse breeding, meat production, milk production, history, ethnology

The purpose of this paper is to make available for occidental lecturers a kind of synthesis of the information we accumulated over more than twenty years about horse breeding in Central Asia. It is also a trans-disciplinary approach mostly based on French bibliography and personal experiences. We will give a new perspective on the early time history of horse breeding to introduce the current breeding of horses in herds in Central Asia. We will continue with the description of the diet of the nomads and the central place taken by mare's milk in their culture. This induced rituals but also some medical aspects. These latter can now be partly explained with current modern studies. We will finally discuss on the prospects of horse milk products in the new globalised world.

Introduction

My work as a geneticist applied to horse production, combined with considerable cultural curiosity for anything connected to equines, has enabled me to consider subjects linked to the history of the techniques, ethnology and sociology of countries as distant from France as those in Central Asia. After an initial review of the bibliography [START_REF] Langlois | Croissance avant sevrage in Caractères quantitatifs chez le cheval: Aspects génétiques (growth before weaning in quantitative characters of the horse)[END_REF], my first mission in January 1984 to the Equine Research Centre of the ex-USSR [START_REF] Langlois | L'élevage du cheval en Union Soviétique (horse breeding in USSR)[END_REF] allowed me to become familiar with what was happening with horse production on the other side of the "iron curtain". In 1990, I participated in the expedition "Guillaume de Rubrouck" to Outer Mongolia, which involved six weeks on horseback visiting breeders in that region. We were the first French people to visit the country after those of the Croisière jaune. See the books of Kappler [START_REF] Kappler | Voyage Dans L'Empire Mongol 1253-1255 (William of Rubrouck, Travel in the Mongolian Empire 1253-1255[END_REF] and Jan [START_REF] Jan | Le réveil des Tartares. En Mongolie sur les traces de Guillaume de Rubrouck[END_REF]. In 2000 and 2001, I was lucky enough to be asked to complete two missions to the Hohhot Agricultural Corresponding author: Bertrand Langlois, M.Sc., senior researcher, research field: horse's quantitative genetics. E-mail: bertrand.langlois @jouy.inra.fr.

University, which honoured me with the title of Associate Professor [START_REF] Langlois | Rapport de mission en Chine, Mongolie intérieure du 3 au 12 août 2001 (Mission's Report in China[END_REF]. Not only that, but through its network of alumni, I was introduced into the horse farms of Inner Mongolia. And finally, in the autumn of 2007, I was able to complete a mission in Kyrgyzstan [START_REF] Langlois | Rapport de mission en Kirghizie du 28-10 au 06-11-2007[END_REF]. I thus feel qualified to put forward a technician's view of horse breeding in these regions, for many years totally unknown to Western equine experts, although it is now becoming increasingly well understood by young ethnologists, as shown by issue 40/2010 of EMSCAT journal and recent publication of the book by C. Ferret [START_REF] Ferret | Une Civilisation du Cheval, Les Usages de l'équidé de la Steppe à la Taïga (A Horse Civilization[END_REF].

A Look at History

V. Eisenmann [START_REF] Eisenmann | L'évolution des équidés (Evolution of the Equids)[END_REF] recently summarised the state of our knowledge on the wild horses known to exist at the end of the last glaciations, about 10,000 years ago, from their fossilised remains. It was these animals which were the subject of different attempts at domestication, now situated at a relatively late stage, between 4000 and 3500 BC. The two types of wild horse described at the historical period were Prjewalski's horse (Equus przewalskii) in Asia and the Tarpan (Equus ferus) in Europe. Although quite similar at first sight, with their massive shape and short nose revealing their adaptation to cold conditions [START_REF] Langlois | Inter-breed variation in the horse with regard to cold adaptation, a review[END_REF] and their short first phalanges, the Tarpan had shorter cannon bones and larger hooves than the Prjewalski, and its teeth also appeared to be less well-adapted to an abrasive diet. This suggests an Asiatic horse adapted to life on the steppes and a European horse adapted to forests. However, Equus gallicus, the Solutré horse (30000 BC) differed from these two others: the nose was greatly shortened, the cannons short, the first phalanges long and the hooves very broad. The same type of horse, Equus latipes, was present at Kostenski on the Don, but with even broader hooves. It is among these latter types that we should probably look for one of the origins of domestic horses, Equus caballus.

In Southern Europe, Equus antunesi has been known to be present for 100,000 years. Its limbs are svelte and its muzzle lengthened. According to the Luristan bronzes, it was also possible to identify horses in these mountains with very short limbs, living 4,000 years ago.

The first known horses (Equus scotti) were larger and heavier; their remains were found in Texas, dating from 0.7 million years ago. Those described in Europe, and particularly in Germany (Equus mosbachensis) go back half a million years. They probably migrated from North America via the Behring Straits, but it is not easy to determine that they were the origin of our modern working horses.

Indeed, the coexistence of man with horses does not probably date back to more than 100,000 years, when the onset of the last Ice Age favoured the development of steppe-like vegetation where horses became abundant and their hunting developed. Some hunting communities became subservient to their herds or reindeer or horses in Western Europe, or to bison and aurochs in Eastern Europe. With the advent of Homo sapiens sapiens, this dependence on a particular food source could not have been sustained over thousands of years without the development of a particular culture and considerable knowledge. We can still see evidence of this culture today in remarkable ancient wall paintings (30-10000 B.C.) I see no reason why it was not also been expressed in hunting techniques. In particular, I feel that the wealth of late paleolithic images representing horses with halters, underlying the hypothesis put forward by Edouard Piette which advanced the date of domestication beyond 10,000 years ago, should be reconsidered from this angle. Indeed, if we think that these attached horses were decoys to control their wild counterparts, then all the findings can be reconciled. Horses were not really domesticated before 4000 BC, as the first chariot burials in the Urals dated from the end of the 2nd and the beginning of 3rd millennium B.C. and the Botai site in Northern Kazakhstan (now considered as the first) only dates from between 3500 and 4000 BC. However, the proximity of hunters to their prey via the intermediary of decoys would have enabled man to observe that they could easily be mastered. Indeed, its natural behaviour meant that it could be domesticated, which is only the case for a few species. But what was the reason for this domestication? It is generally agreed that the main aim was to obtain a source of food, meat certainly, but also milk, as suggested by the excavations at the Botai site [START_REF] Outram | The earliest horse harnessing and milking[END_REF]. On reflection, a mare would serve as a much better decoy than a young male living naturally outside the herd, or a stallion that would maintain the cohesion of the herd. Fertilised by a wild stallion, the mare could then produce a foal. Her milking would therefore have preceded her consumption and, if this had been successful, might have delayed the latter. In this domestication process, the production of milk would therefore have taken precedence over that of meat. But it is pleasant to allow one's imagination free rein if one is not constrained by the little data available on this distant era.

Later on, it appears that horses were first of all harnessed in a pair to a chariot, following the model used for cattle [START_REF] Spruytte | Etudes expérimentales sur l'attelage: Contribution à l'histoire du cheval (Experimental Studies on Harnessing, a Contribution to the History of the Horse)[END_REF]. At least, that is what is suggested by archaeological remains. However, it is difficult to imagine [START_REF] Saurel | Histoire de l'équitation des origines à nos jours (History of Riding from Origin up to Day)[END_REF] that knowing how to harness them, man did not try to mount the animals, which seems to have been well established during the 1st millennium B.C. However, from various historical and linguistic cross-references, we do know that it was Indo-European populations which developed these techniques, from the steppes of Kazakhstan to the Iranian plateau, whence they spread to the Middle East, Europe and then Asia. These nomad peoples, long supposed to be illiterate, have left few traces, but we know that they were at the origin of the tree saddle, perhaps of the made of leather, or wooden stirrups [START_REF] Gagnazadou | Les étriers, Contribution à l'étude de leur diffusion de l'Asie vers les mondes iraniens et arabes (The stirrups contribution to the study of their diffusion from Asia to the Persian and Arabian world)[END_REF] and of the short, so-called Turkish composite bow, particularly suited to use by horsemen. Some, such as Lebedinski [START_REF] Lebedinski | Les Sarmates[END_REF], think that one of these horse-mounted peoples, the Sarmates and then the Alains, subsequently invented one-to-one combat with a lance and were thus the origin of the European military aristocracy that grew up at the end of the Roman Empire and during the Middle Ages. It was therefore these populations of nomadic livestock breeders from the steppes who instructed other, civilised, Assyrian, Greek, Egyptian and Gallo-Roman populations, and in the East a series of peoples that, for simplicity, can be qualified as Chinese, in the use of horses. According to Keegan [START_REF] Keegan | Histoire de la guerre[END_REF], their familiarity with the slaughter of animals also meant that they invented true warfare which no longer took the form of the intimidating rituals that had prevailed hitherto. We who believe ourselves as the descendents of Greeks, Romans or-more modestly-Gauls, usually forget that we owe these nomad peoples a great deal. Indeed, the barbarian invasions contributed much to revivifying this heritage.

The Raising of Horses in Herds in Central Asia

The principle underlying this type of animal husbandry is to exploit natural forage resources, scarce during some seasons and always widely dispersed, by means of wholly open air management. The breeds in these regions are well adapted to these grassland conditions; they can graze at great distance from water points and, during the winter, develop considerable resistance against the cold through their behaviour, coats and anatomy. In arid regions, mixed herds of fat-tailed sheep and Kashmiri goats are associated with the horses. When the grazing conditions improve, this basic combination makes more room for cattle. However, yaks and small Mongolian cows, whose droppings serve as fuel, are very unlike the animals that are familiar to us. In the Gobi region, where tufts of plants grow at least one metre apart, Bactrian camels are raised. It is in the culture of the nomads to rely on the body reserves of their animals, and they are loath to store goods for the winter, which they consider is the domain of sedentary farmers. They understand, and have always maintained, commercial relationships with the latter. But their methods differ; they prefer to maintain non-grazed zones for over-wintering, often found in mountain valleys that offer shelter against the winter winds. And indeed, given the size of their herds, it would be necessary for them to switch to an agropastoral system if they wanted to constitute winter reserves, but the ecological equilibrium of the steppes does not in most cases tolerate tilling. Under these harsh climates, this will inevitably lead to the disappearance of soil, which ends up in a sand storm over Beijing. It is not for nothing that particular attention is paid by Mongolians to causing the least damage to the soil (Boots with uprightly incurved toes, no stakes used to support the yurt, etc.). Under the influence of Chinese or Western advisors, the Mongols are now starting to introduce some hay cutting. This is not to feed their livestock during the winter, but to use in the event of problems to help ewes get through a difficult period so that breeding can start again the next year. Nomadic farming is indeed subject to constant crises that the Mongols refer to as "zhud". They make a distinction between three types:

The "too many hooves zhud" which refers to over-grazing can lead to epidemics of disease.

The "white" zhud that occurs after a period of thawing: the snow thaws and then turns into ice, thus preventing access to the land on foot.

The "black" zhud refers to drought that causes the disappearance of water sources.

The historical response of nomads to these difficulties is not that adopted by sedentary farmers, which is to confront them; rather, they disperse in a broad network of alliances of many types. If necessary, the nomad farmer will sacrifice some of his livestock, or in other words his fortune. He will take his absolute necessities with him, and move elsewhere. He mocks the advice of sedentary farmers, whom he qualifies as "green" zhud, considering that the remedies they suggest are often worse than the problems he is facing.

What is difficult for our mentality as sedentary farmers to grasp is that under this system, the path always seems to lead towards a catastrophe. At best, if all goes well, it is the "too many hooves zhud" that occurs. Under these conditions, domestic populations are subject to selection pressures that are very similar to those experienced by wild populations. This produces breeds that are very well adapted to their grassland conditions, and can either graze at great distance from a water source or, during winter, attain food sources that often lie 50 to 70 cm below a level of powdery snow in the coldest regions. In these horses, the hairs on the fetlock are like a brush. In addition, the aptitude of these animals to rapid fattening allows them to bear all periods of restriction satisfactorily. These consist not only in extreme summer heat and the drying of grasslands, but also extremely cold winters, with snowstorms and ice that only horses, of all the domestic animals, are able to break down if it is not too thick. These animals can therefore be used to graze very short on the soil (we have seen that the dentition of horses is well adapted to this, and they are also able to dig down for roots). They can also be raised in thickets, marshy zones, sparse forests and clearings. These inhospitable areas can thus be exploited by horses with a considerable aptitude for movement and which graze for about 14 hours in a 24-hour day, or as much time as cattle spend both grazing and ruminating [START_REF] Boy | Time-budgets of Camargue horses[END_REF][START_REF] Boyd | The 24-hour time budget of Przewalski horses[END_REF]. So horses are not only remarkable foragers, they also sort what they consume. For this reason, equine products (meat or milk) are considered to be of better quality than those from cool-muzzled animals: cattle, goats or camels. Only sheep, a warm-muzzled animal and thus close to man and consecrated like the horse in the ancient rituals of shamanism, escapes this discrimination.

Many years ago, Soviet animal production scientists considered that a kilogram of horse meat produced under these conditions cost half as much as beef and one-quarter less than sheep. The natural adaptation of horses to grassy steppes or forested environments is of course the reason for this, and the principal reason why horses have become the main domestic animals of the populations in these regions living in symbiosis with them. Since time immemorial, these peoples have used horses as a source of food and benefited from their great capacity for travelling; they can raise other species (sheep and cattle) but because of their nomadism, they are able to exploit vast areas with a low density of plant production. Furthermore, the horse gave these peoples an undeniable military superiority; in open country it enabled a reversal of the unfavourable demographic relationships that existed with hand-to-hand combat. Over time, the nomad confederations were able to threaten and even overwhelm powerful sedentary states. This dynamic of the steppes, oscillating between increasingly conquering federations and their dispersal, needs to be set against the ecological equilibrium of herds oscillating between development and "zhud". Indeed, some populations were dependent on their herds which, like wild animals, were subject to the selection pressures of the environments they colonised. One example of this is the taiga, the natural environment of reindeer, and some of the mountainous terrains which produce particular breeds. But in fact, total domestication was only achieved in the agricultural environments of the oases of Turkmenistan. Some horses were raised there on the products of farming, as a function of their end uses. This is the origin of what we now call the Warm-blooded breeds. In my opinion, the Akhal-Teke constitutes a sort of outlier of this phenomenon, which subsequently continued with Arab and English thoroughbreds. In the West, we are an extension of this approach insofar as our breeders are not subject to the constraints of natural selection because of agricultural production. We cannot therefore understand that the Mongols tried and rejected all efforts towards crossings with improved breeds, which would nonetheless have generated immediate and spectacular gains in productivity. In fact, these immediate gains were then cancelled out by the lack of adaptation of the animals to their production conditions, which could pollute their herds for several generations until the natural equilibrium was completely restored.

This review of production methods so distant from our own is nonetheless valuable, particularly in a period when the terms "environment" and "sustainable development" form part of the obligatory vocabulary of any discussion on the subject.

The Diet of the Centre Asiatic Nomads

White Food and Grey Food

For the administrative authorities, it is first of all the meat produced by a farm that counts; milk production is only seen as a supplementary benefit, and little account is taken of animals harnessed for work or ridden. The situation differs completely for the Turkic-Mongolian people. No account is taken of work accomplished ridden, as this is an everyday occurrence. It is nonetheless celebrated during festivals: naadam in Mongolia, equine games and badgé in Kirghizistan and Kazakhstan. But, above all, it is mare's milk that is valued by these cultures.

In summer, when their animals are lactating, the Mongols eat white food, i.e. dairy products. In winter their diet is made up of grey foods, i.e. boiled meat that they prefer to be very fatty. The most important white product is airag (in Mongolian), which is also called kumiss in Turkish languages. Airag is yoghurt made from fermented mare's milk. Because equine caseins are very short, they do not solidify as they coagulate but remain liquid. Its titre is between 2% and 3% of alcohol, obtained by the alcoholic fermentation of lactose. It is considered as both a food and a drink, and sometimes as a medicine. For the Mongols, winter is defined by an absence of airag, and summer by its abundance. There is however one exception, as they keep a small amount of airag frozen for the New Year festival. The collection of milk starts one month after foaling, so that the foal is assured of a sufficient supply to initiate its growth. Mares and foals are caught in the morning and attached to a rope staked in the ground so that without separating the mother and her foal, the latter is still prevented from suckling. Milking can then commence, at a rate of four or five times a day. One ethnographical point is that milking is performed from the left of the mare, while cows are milked from the right. The left is the male side, and both horses and airag were historically the exclusive domain of men.

Today, it is mainly the women who milk, but the men continue to participate. In the evening, the mare and her foal are released together so that they can graze. Indeed, efforts are made to ensure that they are not separated for more than 18 hours each day. If it is necessary to milk so many times a day, this is mainly because the milk produced by the mare is of an alveolar type, and the cisterns of the mammary gland hold very small quantities. In addition, there are marked variations in the ease of milking between mares. Some mares may need their own foals to be present for milk production to be triggered, while for others any foal will suffice, and others again can be milked alone. It is thus possible to collect about 2 litres of milk per day and per mare, or 300 to 400 litres per lactation, without recourse to supplementation. As a comparison, milking Cold-blooded mares receiving supplements in their diet can produce up to 3,000 litres per lactation, and milking can be mechanised, but this is not the point we are trying to make here. The milk collected in buckets-traditionally made of wood-is immediately transferred to a large container made of two cow skins sewn together using horsehair and camelhair threads, with the hide on the inside. A large wooden paddle or frother is placed in the container. The new milk is thus mixed with the ferment in the same way as in a yoghurt-maker. Each person entering the yurt is invited to stir the mixture vigorously, and is honoured to do so.

This skin container is situated in the yurt of the head of the family on the left when entering, i.e. on the masculine side. The airag is consumed constantly, with fresh milk replacing what has been drunk. It has a flavour situated between lemon juice and watery fresh milk. The Mongols can recognise numerous vintages from the quality of the ferments that give them their acidity, from that of the milk as a function of season and pastures, and the degree of advancement of the fermentation (weak-moderate-strong). However, this beverage cannot be stored for more than four days, after which the remains are distilled with a similar preparation of fermented cow's or yak's milk, the tarag, to produce arkhi, a milk alcohol with a titre of about 16°. To store airag or kumiss, the Russians have suggested adding sugar to prolong the alcoholic fermentation. This produces a sparkling drink that is more similar to milk and can be kept in small, encapsulated bottles. The Mongols think that the result bears no relation to airag and are totally uninterested in this idea.

Rituals

I. Bianquis [START_REF] Bianquis | Lait de jument et produits laitiers en milieu urbain en Mongolie: Du symbole à la réalité (Mares milk and milk products in the town in Mongolia: from symbol to reality)[END_REF][START_REF] Bianquis | Les alcools de lait en Mongolie, Rites, Croyance et lien social (Milk Alcohols in Mongolia, Rituals, Believe and Social Links[END_REF] recently reported on the different rituals involved linked to this product, and I agree with her principal findings.

On the steppe in summer, the farmers visit each other and airag gradually becomes their main food, supplying energy, proteins and vitamins. The average consumption of men can reach 10 litres a day. Some are proud to announce "25" litres a day, a sign of virility as a defiance of alcohol, and a manifestation of being a true Mongol.

Children are initiated in consuming airag at the very early age of around 8-9 months, so that later they will become true Mongols.

When airag is associated with hospitality rites or gifts to divinities, it is considered as arkhi, i.e. as an alcohol. In this case, each person drinks from the same cup, supplied by the host, on a white or blue stole (haddack). The gift is consecrated by flicking some of the precious liquid from the middle finger towards the four cardinal points, sometimes only to the ground and the sky, but other times both ways successively. The host then empties his cup. He fills it again and the guest is asked to respond by following the same ritual. The cup is then handed to the next guest, and so on.

Everyone drinks from the same cup, that of the host, making this a veritable communion ritual. This differs from the consumption of airag as a food, where each person drinks from his own, traditionally wooden, cup, which forms part of the essential utensils carried at all times by a Mongol, together with a knife, chopsticks and tobacco pouch. Airag can also be sprinkled on "Ovoo", piles of stones covered with fabric that are found throughout the steppe as offerings to the powers of nature that are venerated in Shamanism. Airag is also used to bless people during Buddhist rites. A special wooden spoon, rectangular in shape and sculpted to hold the liquid, is then used. Thus, on the occasion of departing for a voyage, the stirrups are blessed with a few drops of airag, and the rump of the horse is sprinkled with it to wish the rider luck.

Medicine

Because mare's milk is similar to human milk in terms of its composition, and because it is the principal source of vitamins in the high-protein diet of nomads and has always been free of tuberculosis, it benefits in Central Asia from a considerable reputation as a probiotic to treat numerous diseases of the digestive tract, liver and lungs. Its ability to lighten the skin when applied as a cosmetic or consumed via the digestive tract is considered by Chinese medicine as a symptom of good health and is taken as proof of its beneficial action. It should also be noted that historically, an ukase of the Russian Tsar banned all Cossacks from milking mares. This authoritarian measure was designed to ensure a better diet for foals, but in fact resulted in severe vitamin deficiencies in the soldiers and had to be abrogated a few months later. But mention is still made of it, and the health image of mare's milk is enhanced as a result. For this reason, from the Southern Urals in Bashkortostan, visited by the great Tolstoy himself, to Xilin Gol in Inner Mongolia, very active health cure tourism has developed. For example, this is the case in the town of Xilin Gol in Inner Mongolia, where hotels offer an abundant consumption of airag. Further away, in the East Ujim Qin district, I was able to visit a more medicalised dispensary for a mare's milk cure. The association of airag with the traditional Mongolian plant-based pharmacopoeia is apparently achieving success in the treatment of diabetes, allergies and skin diseases. In particular, diabetic ulcers can be treated by applications of mare's milk. 40% of the success of this treatment is attributed to mare's milk, and 60% to the pharmacopoeia. I am not qualified to pronounce upon these claims. In the dispensary I visited, about thirty mares were milked by hand four times a day to supply less than two litres of milk per mare. The milk collected here was put to ferment in stoneware salting vessels, half-buried in the floor of a yurt refrigerated by watering the floor. The ferments used had recently been renewed by supplies from Outer Mongolia. The airag was moved from the first vessel to a second, where it was filtered as a function of its degree of fermentation. From the second vessel, it was then distributed at regular times to those following the cure who, after consuming it, ensured that they protected themselves from the sun by placing one side of their del (robe) on their heads, apparently to prevent any allergic sensitization.

Current Modern Studies

Table 1 taken from Fox and Uniacke [START_REF] Fox | Chemical and physico-chemical properties of equid milk[END_REF], Martuzzi [START_REF] Martuzzi | Advances on equine milk and derivatives for human consumption[END_REF], and Salimei et al. [START_REF] Salimei | Ass's milk as a hypoallergenic food[END_REF], shows the comparative compositions of milks from different mammal species.

It can first of all be noted that equine milks, like human milk, are amongst the lightest and most aqueous. They also contain the highest lactose concentrations, and are less fatty (2%) (from which, 80% triglycerides, 5% phospholipids and 9% fatty acids, including a high proportion of medium-length and polyunsaturated fatty acids). This high proportion of fatty acids essential to human nutrition, combined with a low proportion of linoleic acid (0.1%) (Which is much lower than in the cow or ewe) means that mare's milk has a highly dietetic lipid fraction. And that is not all: the lipids are present in globules of 2 to 3 mm in diameter, stabilised by a membrane that is acquired in mammary secreting cells. Long mucine filaments (0.5 to 1 mm long) extend from the surface of these globules. They facilitate the adhesion of these fat globules to the intestinal epithelium, thus preventing the adhesion of bacteria and protecting the tissues to which they are bound from infections and tumours.

As for the protein fraction, a distinction must be made between whey and caseins which, by coagulating, enable the production of cheeses. In both humans and equines, the ratio of these two types is 1/1. The principal whey proteins are alpha-and beta-lactoglobulin. Lysozyme, which has antiseptic properties, is also found in large quantities, as well as immune globulins. The latter proteins may explain some of the milk's therapeutic properties because they intervene in tissue repair. The principal caseins are beta casein and, to a lesser extent, alpha-casein; however, a particular feature is that kappa-casein is almost absent. This explains the hypoallergenic properties of equine milk. It also means that equine milk curdles but does not form a gel under the action of the ferment; it remains liquid. It is impossible to make cheese with either mare's milk or she-ass milk.

Perspectives

The way in which this long tradition is likely to evolve raises another problem. Ex-USSR countries have already been confronted by the need to modernise this sector; to meet domestic demands and to supplement the production of meat so that the vast areas of land that are under-used for agricultural purposes can be exploited. In the first case, as the populations of Central Asia gradually moved towards large urban areas, they started to demand koumiss. It was thus decided to intensify and rationalise its production in regions closer to this new market demand. So during the 1980s, the regions of Moscow, Riazan, Tambov, Kirov, Minsk and Voroschilovgrad also started to produce kumiss in a more rational manner by equipping specialised farms that housed herds of about a hundred or more mares. In an intensive setting, milking mares receiving supplementation can, as we have seen, produce about 3,000 litres during six months of lactation. Milking is mechanised with adapted milking machines, milking parlours are organised to take account of the need not to wean foals so that the mares will properly produce their milk. Both mares and foals receive appropriately supplemented diets. Occasional experiments, performed without much enthusiasm, have also been carried out in Europe [START_REF] Doreau | Le lait de jument (Mare's milk)[END_REF]. In addition, the manufacture of kumiss has been standardised so that the final product is more similar to "mare's milk champagne" than to traditional kumiss. The milking procedures must of course comply with strict hygiene standards. The milk thus collected is filtered through four layers of gauze. It is left in containers that are plunged into a tank of running water, the temperature of which does not exceed 9 to 10°C. Milks from different milkings are not mixed. The ferment is cultured separately and only replenished with fresh milk. Milk collected during the day until 18:00 is thus stored until the next day at 11:00, when the manufacture of kumiss starts. The ferment is mixed with the milk until it reaches an acidity of 50-60 Terner degrees at a temperature of 26-28°C, which ensures the normal fermentation process. Immediately after the coagulation of caseins (which is relative given their nature), the mixture for the manufacture of kumiss is stirred using electric mixers at a rate of 430-480 rpm. This mixture is then left to mature for 1.5 to 2 hours, until an acidity of 65-70° Terner is reached. Fresh milk is then added until an acidity of 50-60° Terner is attained again. After that, it is mixed again for 20 minutes to achieve an acidity of 70° Terner. At that point, the milk is mixed for a further hour and then chilled to 17°C, before being placed in bottles that are sealed hermetically with capsules and placed in a cold room at 4°C for further maturation and chilling. After 24 hours, the kumiss is ready for sale. It can be kept chilled for 25 days. After a few days at ambient temperature, the koumiss bubbles like champagne.

It thus appears that the development of a productive mare's milk sector is possible, even in regions where it is not traditional. It can be based not only on demands from the medical sector (humanised milk, food for patients who have undergone digestive tract surgery, koumiss cures, cosmetology) but also on a demand from the general public which is certainly specific to certain cultures but has been seen to be able to win new markets. The major problem is therefore one of marketing, because the technical problems have been resolved, even if further improvements are still possible. In particular, meeting traditional market demands supposes the persistence of a certain extensification and local consumption via short market channels. At present, only a frozen product can circumvent this dilemma. Can other solutions, such as freeze-drying, be envisaged without changing the nature of the product? What would be our opinion of a frozen or freeze-dried wine? The problem is precisely the same. On the other hand, although airag can still be consumed in the steppes, it is more difficult to obtain supplies in towns, although this is possible through family or commercial channels. It is the globalisation and introduction into towns of products such as fruit juices or coca cola that are competing with airag, particularly among young people who aspire to a more modern lifestyle. Perhaps this will open the way to novel products based on mare's milk, but who can say?

Conclusion

For thousands of years, the consumption of mare's milk has played a central role in the identity and conviviality rites of the Mongols. Without exaggerating, it is possible to make a parallel with wine in France. This concept can certainly be extended to all the famous Turkic and Mongolian horse cultures, or about 30 million people.

How will this tradition evolve? That is very difficult to predict.

Technically, Russian animal production scientists had already shown that the intensification and standardisation of production were possible. Occasional experiments in Europe then confirmed these initial results. At that time the product was considered for its medical and/or cosmetological uses. It might now be possible to launch new products of interest to the general public, such as "mare's milk champagne" or fresh cheeses made using mixed milks. However, this can only be envisaged with the marketing support of major industrial groups. Indeed, it would be necessary to introduce an entirely novel product, which is a problematic proposition. However, the close similarity between mare's milk (or she-ass milk) and human milk is a strong marketing argument. The very low levels-or absence-of kappa caseins from these milks renders them hypoallergenic, so that their use can be recommended for infants with cow's milk intolerance. No other milk from a domestic animal benefits from this characteristic, but it also constitutes a drawback; because all its caseins are short, mare's milk cannot be used to make cheese unless it is mixed with milks from other species. Nevertheless, mare's milk (and she-ass milk) also displays other health benefits, the demonstration of which is now growing. This good "healthy" image could enable the launch of novel products. And we should add that, for an equivalent weight, a mare only produces a third of the methane generated by a cow [START_REF] Vermorel | Evaluation quantitative des émissions de méthane entérique par les animaux d'élevage en 2007 en France (Assessment of enteric methane emissions by farm animal in France in 2007)[END_REF]. This is a major advantage in terms of the greenhouse effect. The product could therefore develop its ecological image alongside its health benefits. However, it is still necessary to master the problems of lactose intolerance: indeed, these milks contain more than others, and although infants might digest them all without problems, the same does not apply in some adults, in whom the necessary enzyme is no longer expressed.

What will change for the 30 million people already consuming this milk? For those who remain breeders, it is likely that there will be no effect. Their lifestyle is balanced with the resources of their environment, and the system to which their culture belongs has remained stable for many centuries. On the other hand, those who migrate to towns to search for education or a more comfortable life are confronted by globalisation. While remaining attached to their culture and the products that can be supplied to them by family or commercial networks, they are starting to become accustomed to the products of the modern world. This situation can be seen as a battle where global products will gradually supplant those of local origin. However, in my opinion, new products arising from a local culture are likely to be able to hold their ground. Indeed, as proposed by Clausewitz [START_REF] Clausewitz | Vom Kriege (About the War)[END_REF], ultimately, and regardless of the defeats suffered, the final advantage always remains with the defender.

Table 1 -Comparative compositions of milks from domestic species compared to those of equids and human (%).

 1

	Species	Water	Caseins	Proteins	Whey	Fats	Lactose	Minerals
	Ewe	81.6	4.5		1.1	7.5	4.4	0.9
	Goat	86.8	2.8		0.5	4.5	4.4	1.0
	Sow	79.9	2.9		2.9	8.5	4.8	1.0
	Cow	87.3	3.9		2.6	3.6	4.8	0.8
	Mare	89.6	1.3		1.2	1.4	6.7	0.5
	She-ass	89.2	0.9		0.8	1.8	5.9	0.4
	Zebra	88.7	0.9		0.8	2.2	7.0	0.4
	Human	87.7	0.7		0.7	3.6	6.7	0.3


V Monteils

M Rey

‡ M Silberberg

# L Cauquil

S Combes

Modification of activities of the ruminal ecosystem and its bacterial and protozoan composition during repeated dietary changes in cows 1

Keywords: bacteria, cow, dietary disturbance, fermentation, protozoa, ruminal ecosystem

Dietary change alters the ruminal ecosystem and can be regarded as a disturbance. Studying the response to a disturbance can help us understand the behavior of the ecosystem. Our work is concerned with the response of the ruminal ecosystem (composition and activities) during the application of repeated dietary disturbances to 6 dry Holstein cows. For 2 mo, the cows received a hay-based diet [experimental period (EP) 0], followed by 3 EP of successive changes (EP 1, 2, and 3) comprised of 2 parts: the fi rst (10 d) with a corn silagebased diet and the second (25 d) with a hay-based diet. The measurements and samplings were done on the last days of EP 0 and of each part of EP 1 through 3, with the results of EP 0 used as covariables in the statistical models. The physicochemical measurements (pH and redox potential) and the fermentation variables (VFA, ammonia) were determined hourly between the morning and evening meals (n = 8 measurements/d). Samples of ruminal contents were taken 3 h after the morning meal to determine enzymatic activity [amylase, carboxymethyl cellulase (CMCase), and xylanase], to count the main protozoan genera and to quantify the bacteria by quantitative PCR, and to determine its structure by the capillary electrophoresis single-strand conforma-tion polymorphism process. The pH fell for the corn silage-based diet with the EP (P < 0.05) but not for the hay-based diet. The VFA concentration decreased for both diets with the EP (P < 0.001), with the primary changes in acetate and propionate. The ammonia concentration increased for the corn silage-based diet with the EP (P < 0.05), whereas for the hay-based diet the highest value was observed for EP 2 (P < 0.05). The total quantity of bacteria decreased between EP 1 and 3 (P < 0.05) for both diets. The structure of the bacterial community was not affected by the disturbances for the corn silage-based diet, whereas for the hay-based diet large differences were evident between EP 1 and 3 (P < 0.05) and 2 and 3 (P < 0.01). The number of protozoa increased over the EP, with a more marked effect for the corn silage-based diet (diet × EP interaction, P < 0.05). The specifi c amylase, CMCase, and xylanase activities decreased over the EP for both diets (P < 0.05). The dietary changes applied in our experiment involved strong modifi cations of the ruminal ecosystem and alterations of ruminal fermentation and enzymatic activities. These alterations were reinforced with the repetition of the dietary changes.

INTRODUCTION

A change in diet constitutes a disturbance of the ruminal ecosystem. The disturbance caused is associated with environmental changes for the microorganisms (e.g., nutrient availability, pH, digesta fl ow). Faced with a disturbance, the microbial composition might be resistant or altered with or without the capacity to return to the initial state (Allison and Martiny, 2008). Several hours after a dietary change, the composition of the bacterial community is modifi ed [START_REF] Tajima | Rumen bacterial community transition during adaptation to high-grain diet[END_REF][START_REF] Tajima | Phylogenetic analysis of archaeal 16S rRNA libraries from the rumen suggests the existence of a novel group of archaea not associated with known methanogens[END_REF], together with the physicochemical and fermentation measurements [START_REF] Goad | Ruminal microbial and fermentative changes associated with experimentally induced subacute acidosis in steers[END_REF]. Several days after, the ruminal ecosystem can exhibit a new state of equilibrium of fermentation measurements [START_REF] Michelland | Changes over time in the bacterial communities associated with fl uid and food particles and the ruminal parameters in the bovine rumen before and after a dietary change[END_REF].

One way of studying the ecosystem is to study its response to a disturbance. For digestive ecosystems, the most frequent disturbance is a change of diet. In the course of their lives, farm animals often experience dietary disturbances at critical periods, such as weaning, calving, dietary changes, turnout into pasture, etc. It also has been shown that the response of the ruminal ecosystem to a dietary disturbance depends on the initial conditions. For example, the decrease in ruminal pH after an acidogenic challenge was shown to depend on the nature of the diet ingested before the challenge and on the initial pH [START_REF] Dohme | Repeated ruminal acidosis challenges in lactating dairy cows at high and low risk for developing acidosis: Ruminal pH[END_REF]. A disturbance could therefore provoke a different response in an animal depending on the number of times the animal has experienced it in the course of its life (i.e., on its history). All these considerations led us to hypothesize that dietary disturbances alter the ruminal microorganism composition, the fermentation and physicochemical measurements, and the main enzymatic activities and that the repetition of these disturbances exacerbates the alterations. The main novelty of this study was the repetition of dietary changes with a medium-term approach associated and a detailed characterization of the ruminal responses.

MATERIALS AND METHODS

All animal housing and handling procedures were cared for in accordance with the guidelines for animal research of the French Ministry of Agriculture (France, 1988).

Animals, Diets, and Experimental Design

Six Holstein cows were fi tted with ruminal cannula (number 3 C, Bar Diamond, Parma, ID) 1 yr before the trial. The cows were neither gestating nor lactating throughout the trial. The animals were housed in individual 1.2 × 2.2 m pens of the experimental unit of UMR 1289 TANDEM (Poucharramet, France). At the beginning of the study, the mean BW of cows was 640 ± 78 kg.

The experimental design is depicted in Figure 1. There was an experimental period (EP) of 2 mo at the start of the trial (EP 0), during which the animals received a hay-based diet. Thereafter, 3 successive EP (EP 1, 2, and 3) constituted the remainder of the trial. Each of these 3 EP consisted of 2 parts: one period (10 d) when the animals received 8.5 kg DM/d of a corn silage-based diet (Table 1) and a second period (25 d) when they received 9.5 kg DM/d of the hay-based diet. The period of 10 d (corn silage-based diet) was thus considered as a disturbance period. A longer period (25 d, hay-based diet) was then chosen to allow the ruminal ecosystem to reach a novel equilibrium or to return to the previous state. This last period could also be considered as a disturbance period, taking into account the dietary change compared with the previous period. The length of both periods allowed time for adjustment after each dietary change and an approach of the medium-term effect of dietary changes. Indeed, the length of the hay-based diet period was longer than the corn silage-based diet period in part to take into account the slower digesta fl ow with the hay-based diet and the associated slower turnover of ruminal contents, as reported by [START_REF] Faichney | Transit des digesta dans le tube digestif des ruminants[END_REF].

Throughout the experiment, the cows were fed twice daily at 0900 and 1700 h in equal proportions with total mixed rations and with free access to fresh water. The composition of both diets met the recommended allowances of the animals [START_REF] Jarrige | Ruminant Nutrition: Recommended Allowances and Feed Tables[END_REF]) throughout the trial. The ani- 1 Contents (per kg of premix, DM basis): 90 g P, 90 g Ca, 100 g Mg, 18 g Na, 8 g Zn, 4 g Mn, 120 mg I, 100 mg Co, 30 mg Se, 1.6 g Cu, 800,000 IU vitamin A, 200,000 IU vitamin D3, 10 g vitamin E, 10 mg b carotene, 150 mg vitamin B1, and 2,000 mg vitamin C. For each data point and for each of the 6 cows: 8 observations were done for pH, redox potential, VFA and ammonia; and 1 observation was done for enzymatic activities, protozoan count, bacteria quantifi cation and structure mals were fed at 80% of their feed intake capacity according to [START_REF] Jarrige | Ruminant Nutrition: Recommended Allowances and Feed Tables[END_REF]. Individual intakes were measured daily by weighing the orts before the next meal. Dietary changes were made without any dietary transition so as to provoke a disturbance. The 6 dietary changes made during this study were all regarded as possible disturbances, both for the switch from the corn silage-based diet to the haybased diet and vice versa.

Samplings and Measurements

All samplings and measurements were made in the ventral sac of the rumen on the last day of EP 0 and of each part (the corn silage-and hay-based diets) of EP 1, 2, and 3 (Figure 1). The sampling times were retained to characterize the ruminal measurements after a time for adjustment to each disturbance, which was defi ned according to the dietary treatment as explained in the previous section.

The pH and the redox potential were measured with the ex vivo anaerobic method developed by [START_REF] Marden | A new device for measuring kinetics of ruminal pH and redox potential in dairy cattle[END_REF] and adapted by [START_REF] Julien | Effects of varying proportions of concentrates on ruminal reducing power and bacterial community structure in dry dairy cows fed hay-based diets[END_REF]. The device allowed for continuous sampling of ruminal fl uid in anaerobic conditions while maintaining the ruminal temperature and mixing. Each hour from the morning (T0) to the evening feeding (T8), pH and redox potential values were recorded with a digital pH meter (model 713; Metrohm, Herisau, Switzerland), a glass pH electrode (combined with Ag-AgCl reference; Metrohm), a redox potential platinum electrode (Ag/AgCl as reference; Metrohm), and a thermoelectrode (Pt100; Metrohm). The redox potential data were corrected by adding the potential of the reference hydrogen electrode (i.e., +199 mV; [START_REF] Nordstrom | Thermochemical redox equilibria of Zo Bell's solution[END_REF]. During each sampling, approximately 500 mL of ruminal contents were fi ltered through a 250-μm sieve. Three aliquots of the liquid fraction obtained (8 mL) were stored after adding 1 mL of mercuric chloride (2% wt/vol) and were immediately frozen at -20°C until VFA, NH 3, and lactic acid analyses.

To study the microbial community, an additional sample (1 L) of the ventral ruminal contents was collected on the same day 3 h after morning feeding. Filtration through a 1.6-mm metal sieve was done to make the sampling more homogeneous. Filtration through a 1.6-mm sieve was previously used to study the bacterial community structure and bacterial activity in ruminal contents (Michelland et al., 2009b;[START_REF] Privé | Temperature and duration of heating of sunfl ower oil affect ruminal biohydrogenation of linoleic acid in vitro[END_REF][START_REF] Monteils | Random changes in the heifer rumen in bacterial community structure, physico-chemical and fermentation parameters, and in vitro fi bre degradation[END_REF]. The samples were preserved at -80°C before DNA extraction and treatment. To study the protozoal community, 1 mL of ruminal fl uid (ruminal content fi ltered through a 250-μm sieve) was added to 1 mL of MFS solution (3.5% formaldehyde, 0.14 M NaCl, 0.92 mM methyl green) and stored away from light at room temperature until protozoa were enumerated.

The enzymatic activities were measured from 250 mL of ruminal contents sampled 3 h after the morning meal (same sample as for the microbial community determination). The microbial populations and solid and liquid fractions were isolated using the method of [START_REF] Martin | Postprandial variations in the activity of polysaccharide-degrading enzymes of fl uid-and particle-associated ruminal microbial populations[END_REF]. A 50-g aliquot of the total contents was mixed with 25 mL of 0.025 M 2-(N-morpholino) ethane-sulphonic acid (MES) buffer at pH 6.5 and stored at 4°C to determine the enzymatic activities of the total fraction. The rest of the sample was fi ltered through 250-μm mesh nylon cloth. A 40-mL aliquot of the liquid fraction collected was used to determine the enzymatic activities of the liquid rumen fraction. Part of the residue obtained after fi ltration (70 g) was washed for 5 min with agitation in an anaerobic saline solution kept at 39°C (pH 6.5; 5 mL/g) to eliminate the nonadhering microbial population according to [START_REF] Martin | Postprandial variations in the activity of polysaccharide-degrading enzymes of fl uid-and particle-associated ruminal microbial populations[END_REF]. After washing, the sample was fi ltered through a 100-μm mesh nylon cloth. The solid fraction was sampled (5 g) and suspended in 25 mL of MES buffer solution kept at 4°C to determine the enzymatic activity associated with the solid rumen fraction. All samples were immediately stored at -80°C until the enzymes were extracted. All withdrawn samples represented a maximum of 5 L/d for each cow (approximately 5% of the ruminal contents), which was considered a quantity that did not disturb ruminal function.

Analyses

Quantifi cation of VFA was performed by automated gas chromatography (5890 Series II with a fl ame ionization detector, Hewlett-Packard, Avondale, PA) according to [START_REF] Playne | Determination of ethanol, volatile fatty acids, lactic acid and succinic acid in fermentation liquids by gas chromatography[END_REF]. Ammonia concentration was determined with a colorimetric method by a continuous fl ow analyzer (SAN++, Skalar, Norcross, GA) as previously described by [START_REF] Krom | Spectrophotometric determination of ammonia[END_REF]. Total lactate (dllactate) was determined using a commercial kit (Boehringer Mannheim/R-Biopharm, St Didier au Mont d'Or, France). For DNA extraction, PCR amplifi cation and the capillary electrophoresis single-strand conformation polymorphism (CE-SSCP) processes were performed as described by Michelland et al. (2009b) for individual samples of ruminal contents. Total genomic DNA from approximately 0.2 g of sample was extracted and purifi ed with a QIAamp DNA Stool Mini kit (Qiagen Ltd, West Sussex, England) according to the manufacturer's instructions after thermal (from liquid N to 95°C) and mechanical lyses (bead beating using 400 mg of 0.1mm glass beads). Total bacteria were estimated from the number of copies of 16S rRNA gene per nanogram of DNA. To quantify the ruminal bacteria 16S rRNA gene, primers 520f and 799r2 were used as described previously [START_REF] Edwards | Characterization of the dynamics of initial bacterial colonization of nonconserved forage in the bovine rumen[END_REF]. Analysis was performed using the SYBR Premix Ex Taq kit (Applied Biosystems, Branchburg, NJ) on a StepOnePlus apparatus (Applied Biosystems) using absolute quantifi cation. Capillary electrophoresis single-stranded conformation polymorphism was performed from the V3 region of the 16S rRNA gene as described by Michelland et al. (2009a) using an ABI Prism 3100 Genetic (Applied Biosystems).

Ruminal fl uid added to MFS solution was diluted in an equal volume of PBS before protozoal enumeration under a microscope (400×) in a Neubaeur enumeration chamber [START_REF] Ranilla | Methane production and substrate degradation by rumen microbial communities containing single protozoal species in vitro[END_REF]. The enzymatic activities of the ruminal contents were determined for 3 fractions (i.e., total, solid, and liquid). The extraction of the enzymes and the measurement of the activities of amylase, carboxymethyl cellulase (CMCase), and xylanase were done using the method described by [START_REF] Martin | Postprandial variations in the activity of polysaccharide-degrading enzymes of fl uid-and particle-associated ruminal microbial populations[END_REF]. The protein contents of the enzymatic preparations were determined by the technique of Bradford (1976), with bovine serum albumin as standard (Sigma, Saint Quentin Fallavier, France). The specifi c activities were expressed as μmol•h -1 ⋅ mg protein -1 for the 3 fractions.

Calculations and Statistical Analyses

The measurements made at the end of EP 0 were used as covariables in all the statistical models used. The bacterial diversity was estimated using Simpson diversity index, which was calculated for each CE-SSCP profi le using -log10 Σ(a i )², with a i being the relative area under the ith peak [START_REF] Rosenzweig | Species Diversity in Space and Time[END_REF]. The time for adjustment after each dietary change allowed an approach of the medium-term effect of dietary changes and was not a studied factor.

All data were analyzed as repeated measures using a mixed model using the PASW Statistics 18 (SPSS, Inc.; IBM, 2011) software. Experimental period, time, diet (D, 2 levels: corn silage and hay), and fraction (F, 3 levels: total, solid, and liquid) were considered as fi xed factors, and the cow and the covariable were considered as random factors. The factors EP, diet, and fraction and their second-order interactions were tested.

For the statistical analysis of the physicochemical and fermentation measurements, protozoa counts, quantifi cation of total bacteria, and the diversity index, the model used was

Y ij = μ + D i + EP j + (D × EP) ij + ε ij ,
where Y is the dependent variable, μ is the overall mean, D i is the diet effect, EP j is the EP effect, (D × EP) ij is the interaction between diet and EP, and ε ij is the residual error.

For the statistical analyses of the enzymatic activi-ties, the fraction factor was added to the previous mixed model. The second statistical model was

Y ijk = μ + D i + EP j + F k + (D × EP) ij + (D × F) ik + (EP × F)jk + ε ijk ,
where Y is the dependent variable, μ is the overall mean, D i is the diet effect, EP j is the EP effect, Fk is the fraction effect, (D × EP) ij is the interaction between diet and EP, (D × F) ik is the interaction between diet and fraction, (EP × F) jk is the interaction between EP and fraction, and ε ijk is the residual error.

When the effects of factors were signifi cant at P < 0.05, the differences between the different levels of factors were tested by a Student's t test with Bonferonni's correction. The differences were declared signifi cant at P < 0.05.

The bacterial community structure was analyzed using the Statfi ngerprints package (Michelland et al., 2009a) and the R software (R Development Core Team, 2009). The overall statistical analysis of bacterial community structure was performed with a multivariate ANOVA (MANOVA). The effects were declared signifi cant at P < 0.05. The analysis of similarity of the community structure (ANOSIM) was based on the calculation of pairwise Euclidean distance between the CE-SSCP profi les to obtain a distance matrix with 10,000 Monte Carlo permutations. The data of ANOSIM represented the degree of similarity of the community structure (ANOSIM-R). The effects were declared not signifi cant at P > 0.05 whatever the value of ANOSIM-R. If P < 0.05, the value of ANOSIM-R indicated the degree of similarity between the groups: R > 0.75 for well-separated groups, 0.50 < R < 0.75 for separated but overlapping groups, 0.25 < R < 0.50 for separated but strongly overlapping groups, and R < 0.25 for nonseparated groups [START_REF] Ramette | Multivariate analyses in microbial ecology[END_REF].

RESULTS

Intake and Physicochemical and Fermentation Measurements

Throughout the trial, all food offered was eaten between each meal. The changes in pH with EP were different for the 2 diets (diet × EP interaction: P = 0.05; Table 2). The switch to the corn silage-based diet at EP 3 resulted in a lower pH than at EP 1 and 2 (mean pH = 6.27, 6.28, and 6.10 for the EP 1, 2 and 3, respectively, P < 0.05; Figure 2). For the hay-based diet there was no difference in pH between the 3 EP (mean pH = 6.58, 6.48, and 6.55 for EP 1, 2, and 3, respectively, P = 0.32). The redox potential did not differ between the 2 diets or between the EP (Table 2). The VFA concentration was greater for the corn silage-based diet than for the hay-based diet (P < 0.001; Table 2); it fell signifi cantly between each EP (P < 0.001). For the acetate content the diet × EP interaction was signifi cant (P < 0.05; Table 2). The acetate contents did not differ between EP 1 and 2 of the corn silage-based diet (mean acetate content = 51.5 and 53.8 mM for EP 1 and 2, respectively; Figure 3). Experimental period 3 of the corn silage-based diet had a lower acetate concentration than EP 2 (mean acetate = 53.8 and 43.6 mM for the EP 2 and 3, respectively, P < 0.05) but was not different from that of EP 1. For the hay-based diet, the acetate concentrations did not differ between EP 1 and 2, which were signifi cantly greater than that of EP 3 (mean acetate = 57.8, 50.8, and 31.4 mM for EP 1, 2, and 3, respectively, P < 0.001). For the propionate and butyrate concentrations the diet × EP interaction was not signifi cant. Concentrations of these 2 VFA were greater for the corn silage-based diet than for the hay-based diet (P < 0.001; Table 2). The successive EP led to lower and lower values of propionate and butyrate, with signifi cant differences between EP 1 and 3 (P < 0.05).

The ammonia concentration changed with the EP differently according to the nature of the diet (diet × EP interaction: P < 0.001; Table 2). Thus, the successive EP of the corn silage-based diet led to an increase in ammonia, with a signifi cantly greater value during EP 3 (mean ammonia = 59.4, 78.9, and 212.6 mg/L for EP 1, 2, and 3, respectively, P < 0.05; Figure 4). Experimental period 2 of the hay-based diet led to a greater ammonia concentration than during EP 1 and 3, for which it did not differ (mean ammonia = 56.0, 111.0, and 38.9 mg/L for EP 1, 2, and 3, respectively, P < 0.05).

The lactate concentration was typically under the detection threshold of the kit used for measurement. As a result, the data were not analyzed statistically (data not shown).

Microbiota

Quantifi cation of the Bacterial Community. The effects of the diet and the diet × EP interaction were not signifi cant for the total quantity of bacteria (Table 3), which fell between EP 1 and 2 (P = 0.020) and then increased slightly for EP 3, whose value was not different from the other 2. approach to the CE-SSCP profi les for studying the structure of the microbial community showed a diet × EP interaction (MANOVA, P = 0.002; data not shown). For the corn silage-based diet, the CE-SSCP profi les did not differ for the 3 EP of the treatment (P-value of ANOSIM-R > 0.05; Table 4). The CE-SSCP profi les obtained for EP 1 and 3 of the hay-based diet show large differences in the structures of the bacterial community (ANOSIM-R = 0.46 with P = 0.03; Table 4). Between EP 2 and 3 of the hay-based diet, the differences in the CE-SSCP profi les were very large and signifi cant (ANOSIM-R = 0.98 with P = 0.01; Table 4).

Structure of the Bacterial Community. The global

Diversity Index of the Bacterial Community. The estimation of Simpson diversity index showed different changes between the EP for the 2 diets (P = 0.036; Table 3). It fell between EP 1 and 2 of the corn silage-based diet, although not signifi cantly, and increased significantly (P = 0.007) between EP 2 and 3 (Figure 5). For this diet, the diversity indices of EP 1 and 3 were the same. The index increased signifi cantly between EP 1 and 2 of the hay-based diet (P = 0.018), whereas EP 3 of the hay-based diet was the same as for the fi rst 2.

Counting Protozoa. Counts of ciliated protozoa in the ruminal contents showed a diet × EP interaction for total protozoa (P = 0.026), the genera Entodinium (below and above 100 μm; P = 0.035 and <0.01, respectively), and Isotricha (P < 0.001; Table 3). The total number of protozoa increased signifi cantly between EP 1 and 3 of the corn silage-based diet (P = 0.022; Figure 6A). For the hay-based diet the total number of protozoa for EP 2 was more than that of EP 1 (P = 0.002), whereas that of EP 3 was the same as the other 2 EP. The number of Entodinium below 100 μm was greater for EP 2 and 3 of the corn silage-based diet than for EP 1 (P = 0.03; Figure 6B). For the hay-based diet, the number of Entodinium below 100 μm also increased, but the only signifi cant difference was between EP 1 and 3 (P = 0.025). The count of Entodinium above 100 μm was greater for EP 3 of the corn silage-based diet than for EP 1 (P = 0.022; Figure 6C). For the hay-based diet there was no difference in the counts of Entodinium above 100 μm among the 3 EP. The number of Isotricha increased between each EP of the corn silage-based diet (P < 0.01; Figure 6D), but there was no difference for the hay-based diet. The EP effect and the diet × EP interaction were not signifi cant for counts of Dasytricha. The count of the genus Dasytricha was greater for the hay-based diet than for the silage-based diet (P < 0.001; Table 3).

Specifi c Enzymatic Activities

The specifi c amylase and CMCase activities fell during the course of the 3 EP with signifi cant differences between (P = 0.017) EP 1 and 3 (Table 5). The xylanase activity also fell between the 3 EP, with a signifi cantly lower (P < 0.001) activity for EP 3. The diet × fraction interaction was signifi cant for the 3 activities (P < 0.01). The specifi c amylase activity was signifi cantly higher (P < 0.01) in the solid and total fractions for the corn silage-based diet than for the hay-based diet (Table 5). The specifi c CMCase activity was higher (P < 0.001) in the 3 fractions for the hay-based diet than for the corn silage-based diet, with a greater difference between the diets in the solid fraction. The specifi c xylanase activity was greater (P < 0.001) in the solid and total fractions for the hay-based diet than for the corn silage-based diet. No diet × EP or EP × fraction interaction was signifi cant for the specifi c amylase, CMCase, or xylanase activities (Table 5).

DISCUSSION

A change of diet led to a disturbance to the ruminal ecosystem, whatever the change applied (i.e., corn silage-to hay-based diet or vice versa). Dietary disturbance is a powerful tool to better understand the functioning of the ruminal ecosystem. Indeed, the response pattern to the perturbation [resistant (i.e., remaining unchanged) or altered, with or without the capacity to re-turn to the initial state] gives some insight into engineering the ruminal ecosystem toward an optimal outcome for the host, especially the digestive effi ciency. Very little published work treats the repetition of disturbances applied to a digestive ecosystem. Our work has the advantage of approaching the ruminal ecosystem under particular conditions of repeated disturbance more completely than was previously mentioned in the literature, with a study of microorganisms (bacteria and protozoa), their behavior (enzymatic activity), and the level of their activity (physicochemical and fermentation).

Experimental period 1 of the corn silage-based diet led to ruminal measurements typically observed for a diet based on corn silage, which is not particularly acidogenic, as confi rmed by the fairly high pH values. In our study, the intake of diet was not affected by EP, whereas a short-term decrease in intake has been reported after an acidogenic challenge (Blanch et al., 2009). When the corn silage-based diet was repeated, the ruminal pH fell and reached values under 6 for EP 3, as previously demonstrated by [START_REF] Dohme | Repeated ruminal acidosis challenges in lactating dairy cows at high and low risk for developing acidosis: Ruminal pH[END_REF]. The hay-based diet resulted in similar ruminal pH values for the 3 EP, despite the different previous pH levels (i.e., pH at the end of corn silage-based diet periods). The previous disturbance, as well as the number of the previous disturbances, seemed not to affect the ruminal pH, indicating for pH a capacity of the ecosystem to return to equilibrium. The redox potential value did not differ between the diets or among the EP. [START_REF] Julien | Effects of varying proportions of concentrates on ruminal reducing power and bacterial community structure in dry dairy cows fed hay-based diets[END_REF] showed decreasing values of redox potential for diets Table 5. Amylase, carboxymethyl cellulose, and xylanase specifi c activities of the ruminal content from 6 cows fed 3 EP of a corn silage-based diet followed by a hay-based diet according to the ruminal fraction 1 Specifi c activities, μmol• h -1 •mg protein with increasing fi ber content. It has been reported that this measurement needs a long time for adaptation to the diet before stabilizing [START_REF] Monteils | Mid to long term stability of ruminal physicochemistry in dairy cows fed a fi bre-or a starch-based diet[END_REF][START_REF] Monteils | Random changes in the heifer rumen in bacterial community structure, physico-chemical and fermentation parameters, and in vitro fi bre degradation[END_REF]. The brief length of the EP applied in our study between the disturbances could explain the absence of differences of redox potential between the 2 diets and among the EP. In our study, precautions were taken to consider differences of digesta fl ows for both diets by different times for adjustment before sampling. These precautions could have resulted in confusion of the effects of the diet and the time for adjustment. The total VFA, propionate, and butyrate concentrations decreased progressively with EP. The repetition of the disturbances did not allow the ruminal ecosystem to maintain its fermentation level. The negative effect of the repetition of the disturbances was more marked for the hay-based diet, with also a very large decrease in the acetate concentration. The decreases of amylase, CMCase, and xylanase enzymatic activities corroborated these data. The enzymatic activities were observed for whole microorganisms (bacteria, protozoa, and fungi) because the measure of the enzymatic activities showed the total activities without distinction. The repeated disturbances seemed to involve an alteration in the capacity of the ruminal microorganisms to ferment substrate; however, the ammonia concentration increased with EP for the corn silage-based diet. Considering all the fermentative data, this increase in ammonia could be attributed to decreased utilization by microorganisms rather than increased production. As noted previously, lactic acid concentration was typically under the detection threshold throughout the study. Thus, the repeated disturbances seemed to have no effect on this variable.

In our study the total quantity of bacteria fell together with the repeated disturbances and the decrease in VFA. This decrease could be the result of selection for species resistant to the applied disturbances. This idea might help to explain the decreases in enzymatic activity by modification of microorganisms and decreased utilization of ammonia for multiplication. The Simpson diversity index is an estimation of bacterial diversity from abundance and number of operational taxonomic units (OTU; [START_REF] Rosenzweig | Species Diversity in Space and Time[END_REF]. A change in this index indicated a change in the bacterial community (i.e., relative abundance of OTU or number of OTU or both). The succession of diet perturbations led to either modifi cation of the Simpson diversity index or the bacterial community structure depending of the dietary change. These changes might be attributed to the appearance or disappearance of OTU (as observed for the hay-based diet) or to changes in the relative abundance of certain OTU (as observed for the corn silagebased diet). All these changes in the bacterial community were counterproductive to ruminal function according to the fermentation measurements evaluated. In our study, the alteration in ruminal function could be linked to the decrease of abundance of certain OTU, probably the OTU that had great activity according to the strong decrease of fermentative measurements and enzymatic activities. Other explanations could be that the species that were resistant to the disturbances had altered fermentative capacities or that the disturbances involved an increase of the digestive fl ow and turnover, which implied a lesser multiplication of bacteria and decreased fermentative activity. In the present study, the bacteria number was estimated from the number of copies of the 16S rRNA gene. As a result, the distinction between live and dead bacteria could not be made, which could cause a misinterpretation of the bacteria density. The structure of the bacteria community was studied with the CE-SSCP method, which could be insuffi ciently precise to display the low differences observed for the corn silagebased diet.

The repetition of the corn silage-based diet involved an increase of the total protozoa, especially the populations of Entodinium below and above 100 μm and Isotricha. For the hay-based diet, the EP resulted in weak increases in the total protozoa and the Entodinium below 100 μm. The protozoa seemed to be more resistant to the applied disturbances than the bacteria, and multiplication still occurred, especially for the corn silage-based diet. Dasytricha also resisted the disturbances because of its content, which was similar throughout the study, but the absence of an increase with the disturbances resulted in supposed lesser appropriate conditions for this genus than for the others. The protozoa counts have been shown to be greater when animals were fed a diet richer in starch rather than in fi ber [START_REF] Ivan | Effects of Isotricha, Dasytricha, Entodinium, and total fauna on ruminal fermentation and duodenal fl ow in wethers fed different diets[END_REF]. The development of the protozoal population has been shown to accentuate the bacteria-protozoa competition for the use of substrates, notably starch [START_REF] Rowe | Quantitative effects of defaunation on rumen fermentation and digestion in sheep[END_REF], and to limit the development of amylolytic bacteria [START_REF] Fonty | L'écosystème microbien du réticulo-rumen[END_REF]. Entodinium and Dasytricha have been reported to degrade starch (Williams and Coleman, 1992), but to a lesser extent than bacteria [START_REF] Fonty | L'écosystème microbien du réticulo-rumen[END_REF]. For the hay-based diet, the bacteria-protozoa competition can be assumed to be weaker. Nevertheless, the amylase activity fell in the same way as for the corn silagebased diet. In our study, the measured amylase activity corresponded to the total activity of the microorganisms and did not allow distinguishing the part attributable to bacteria. The development of cellulolytic bacteria was shown to be favored by protozoa [START_REF] Fonty | L'écosystème microbien du réticulo-rumen[END_REF]; however, the fall in CMCase activity with the increase in the number of protozoa did not support this result. It seemed in our study that the decrease of bacterial density by a repetition of disturbances allowed an increase of the protozoan population. Nonetheless, the increase of protozoal density seemed not to be suffi cient to avoid an alteration in ruminal function.

In conclusion, dietary changes involved an altera-tion of the ruminal function with decreases in ruminal fermentative end products and major enzymatic activities and strong changes in the balance within the ruminal ecosystem. An accentuation of these alterations and changes was observed with the repetition of dietary changes. A logical consequence should be decreased performance, but this would need to be evaluated in additional experiments and over a longer term with growing or lactating animals. A more detailed study of the bacterial species could help us to better understand the changes in the observed functions.

LITERATURE CITED

Figure 1 .

 1 Figure 1. Experimental design of the trial. EP = experimental period.For each data point and for each of the 6 cows: 8 observations were done for pH, redox potential, VFA and ammonia; and 1 observation was done for enzymatic activities, protozoan count, bacteria quantifi cation and structure

Figure 2 .

 2 Figure 2. Ruminal pH measured after the morning feed and hourly for 8 h on the last day of the corn silage-based (squares) and hay-based diets (circles) from 6 cows fed 3 experimental periods (EP) of a corn silage-based diet followed by a hay-based diet (EP × diet P-value = 0.050). Bars = cow SD. a-c Overall means values across time that do not have a common superscript differ (P < 0.05) within the corn silage-based diet.

Figure 3 .

 3 Figure 3. Ruminal acetate concentration measured after the morning feed and every hour for 8 h on the last day of the corn silage-based (squares) and hay-based diets (circles) from 6 cows fed 3 experimental periods (EP) of a corn silage-based diet followed by a hay-based diet (EP × diet P-value = 0.035). Bars = cow SD. a-c Overall means values across time that do not have a common superscript differ (P < 0.05) within the corn silage-based diet. d-f Overall means values across time that do not have a common superscript differ (P < 0.05) within the hay-based diet.

Table 4 .

 4 Similarity analysis of the structure of the ruminal bacterial community from 6 cows fed 3 EP of a corn silage-based diet followed by a hay-based diet experimental period. ANOSIM-R = degree of similarity of the community structure by analysis of similarity of the profi les.

Figure 4 .

 4 Figure 4. Ruminal ammonia concentrations measured after the morning feed and hourly for 8 h on the last day of the corn silage-based (squares) and hay-based diets (circles) from 6 cows fed 3 experimental periods (EP) of a corn silage-based diet followed by a hay-based diet (EP × diet P-value < 0.001). Bars = cow SD. a,b Overall means values across time that do not have a common superscript differ (P < 0.05) within the corn silage-based diet. c,d Overall means values across time that do not have a common superscript differ (P < 0.05) within the hay-based diet.

Figure 5 .

 5 Figure 5. Simpson diversity index of the ruminal bacterial communities estimated from each individual capillary electrophoresis single-strand conformation polymorphism (CE-SSCP) profi les on the last day of the corn silage-based (squares) and hay-based diets (circles) from 6 cows fed 3 experimental periods (EP) of a corn silage-based diet followed by a hay-based diet (EP × diet P-value = 0.036). Bars = cow SD. a,b Index values that do not have a common superscript differ (P < 0.05) within the corn silage-based diet. c,d Index values that do not have a common superscript differ (P < 0.05) within the hay-based diet.

Figure 6 .

 6 Figure 6. (A) Total protozoa, (B) Entodinium below 100 μm, (C) Entodinium above 100 μm, and (D) Isotricha counts on the last day of the corn silagebased (squares) and hay-based diets (circles) from 6 cows fed 3 experimental periods (EP) of a corn silage-based diet followed by a hay-based diet (EP × diet P-value = 0.026 and 0.035, respectively, for total protozoal and Entodinium below 100 μm counts and <0.01 and <0.001, respectively, for Entodinium above 100 μm and Isotricha counts). Bars = cow SD. a-c Counts that do not have a common superscript differ (P < 0.05) within the corn silage-based diet. d,e Counts that do not have a common superscript differ (P < 0.05) within the hay-based diet.

Table 1 .

 1 Ingredient composition of the experimental diets fed to cows on corn silage-and hay-based diets (DM basis)

		Diet
	Item	Corn silage	Hay
	Ingredient, % DM
	Hay	-	90.4
	Soybean meal	8.6	7.5
	Corn silage	37.6	-
	Wheat meal	25.5	-
	Cracked corn	25.4	-
	Minerals 1	2.9	2.1
	Chemical composition
	DM, %	67.4	91.7
	OM, % DM	94.3	92.9
	NDF, % DM	21.3	25.8
	CP, % DM	13.0	10.8
	Starch, % DM	38.3	0.2
	NE l , Mcal/kg DM	1.58	1.12

Table 2 .

 2 Physicochemical and fermentative measurements of the ruminal content from 6 cows fed 3 EP of a corn silage-based diet followed by a hay-based diet 1 The largest value of the SEM is reported. Means of 144 observations for the diet data, and for 96 observations for the EP data.

		Diet		EP				P-value
	Item	Corn silage	Hay	1	2	3	SEM 2	Diet	EP	Diet × EP
	pH	6.22	6.54	6.42	6.38	6.32	0.35	<0.001	0.21	0.050
	Redox potential, mV -161	-163	-170	-160	-158	12	0.851	0.653	0.573
	VFA, mM	86.0	66.2	90.1 a	79.5 b	58.7 c	3.4	<0.001	<0.001	0.086
	Acetate, mM	49.7	46.7	54.7	52.4	37.5	1.8	0.097	0.002	0.035
	Propionate, mM	18.9	11.9	20.3 a	14.2 ab	11.7 b	1.7	<0.001	0.018	0.521
	Butyrate, mM	12.71	5.56	10.09 a	9.10 ab	7.41 b	0.84	<0.001	0.020	0.790
	NH 3 , mg/L	117	69	57.7	94.9	125.8	9.7	<0.001	0.002	<0.001
	a-c Within a row and within the EP category, means that do not have a common superscript differ; P < 0.05.
	1 EP = experimental period.

2

Table 3 .

 3 Ruminal total bacterial (cells × 105/ng DNA) and protozoa counts (cells × 104/mL) from 6 cows fed 3 EP of a corn silage-based diet followed by a hay-based diet 1 Largest value of the SEM is reported. Means of 144 observations for the Diet data, and for 96 observations for the EP data.

		Diet			EP				P-value
	Item	Corn silage	Hay	1	2	3	SEM 2	Diet	EP	Diet × EP
	Total bacteria	1.83	2.61	3.11 a	1.31 b	2. 23 ab	0.587	0.144	0.020	0.113
	Simpson diversity index	6.77	7.37	6.68	6.99	7.53	0.290	0.082	0.084	0.036
	Entodinium <100 μm	67.8	13.7	22.9	48.7	50.7	8.90	<0.001	<0.01	0.035
	Entodinium >100 μm	182	0.06	0.18	0.56	209	0.43	<0.001	<0.01	<0.01
	Isotricha	0.27	0.08	0.04	0.67	0.42	0.07	<0.01	<0.001	<0.001
	Dasytricha	0.24	319	149	171	184	0.48	<0.001	0.692	0.313
	Total protozoa	70.1	17.1	24.6	51.0	55.2	8.90	<0.001	<0.01	0.026
	a-c Within a row and within the EP category, means that do not have a common superscript differ; P < 0.05.
	1 EP = experimental period.

2

  Allison, S. D., and J. B. H. Martiny. 2008. Resistance, resilience, and redundancy in microbial communities. Proc. Natl. Acad. Sci. U. S. A. 105(Suppl. 1):11512-11519. Blanch, M., S. Calsamiglia, N. DiLorenzo, A. DiCostanzo, S. Muetzel, and R. J. Wallace. 2009. Physiological changes in rumen fermentation during acidosis induction and its control using a multivalent polyclonal antibody preparation in heifers. J. Anim. Sci. 87:1722-1730. Bradford, M. M. 1976. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of

at INRA Institut National de la Recherche Agronomique on December 20, 2012 www.journalofanimalscience.org Downloaded from

The authors thank the whole team who worked on this study during the experiments and by providing technical assistance in the laboratory. The Centre de Ressources Genotypage Sequençage platform of Toulouse, France, is acknowledged for technical assistance.

2 Present address: VetAgro Sup, F-63370 Lempdes,

France. 3 Corresponding author: valerie.monteils@vetagro-sup.fr Received June 1, 2011. Accepted August 15, 2012. at INRA Institut National de la Recherche Agronomique on December 20, 2012 www.journalofanimalscience.org Downloaded from

The largest value of the SEM is reported. Means of 144 observations for the Diet data, and for 96 observations for the EP data.

D × F = diet × fraction interaction; D × EP = diet × EP interaction; EP × F = EP × fraction interaction.

Carboxymethyl cellulose. at INRA Institut National de la Recherche Agronomique on December 20, 2012 www.journalofanimalscience.org Downloaded from