Elisabeth Blesbois
email: elisabeth.blesbois@tours.inra.fr

Biological Features of the Avian Male Gamete and their Application to Biotechnology of Conservation

Keywords: bird, fertilization, sperm, storage

Male gamete biology shows specific features in birds that contribute to the reproductive strategy of flying animals adapted to highly variable environmental conditions. In this paper, we review the main characteristics of bird sperm, their selection and storage in the highly specialized female oviduct, specific features of polyspermic fertilization, and the consequences of these biological adaptations on semen biotechnology. Among other features, the storage of sperm in specific oviducal glands is a key factor to increase reproductive "freedom" but is also a critical point for the success of semen in vitro storage.

Introduction

Birds have developed original reproductive processes which are very efficient for the adaptive strategies of flying animals to highly variable environmental conditions. Oviparity has evolved to the production of telolecithal, hardshelled eggs, enabling the autonomous development of the embryo in a highly protected micro-environment. The highly specialized egg production results in the formation of a single oviduct in the adult female of most bird species. The oviduct shares two main functions: 1) the storage and selection of sperm prior to the polyspermic fertilization of the oocyte in the infundibulum; and 2) the constitution of the outer vitelline membrane, albumen and shell of the egg that contributes to the autonomy of the embryo development. The oviduct is also the site of zygote formation and divisions up to stage 30-50000 cells when the embryo genome is activated at the time of oviposition. Among other features, the presence of sperm storage tubules (SSTs, see review in Bakst et al., 1994, Blesbois andBrillard, 2007) in the uterovaginal junction (UVJ) dedicated to the prolonged storage of sperm provides "adaptive freedom" to the female to sustain fertilizing potential even in the absence of males. Sperm are produced by the two intra-abdominal testis of the males at body temperature (41-43°), a fact that may be related to the short interval of time required for spermatogenesis (reviewed by [START_REF] Aire | Spermatogenesis and testicular Cycles[END_REF]. For example, in the chicken, guinea-fowl, duck and turkey, the duration of spermatogenesis is approx-imately 14 days which provides males with extended potential to produce large quantities of gametes within a given period of time. Following their evacuation from the rete testis, avian sperm are rapidly transported through the epididymis to the vas deferens, where they are stored for a limited period of time (e.g. 2-3 days in the chicken, [START_REF] Williams | Variations in the plasma levels of luteinizing hormone and androstenedione and their relationship with the adult daily sperm output in cockerels raised under different photoschedules[END_REF] prior to being exported at ejaculation. Unlike in mammalian species, there is no sexual accessory gland in the vas deferens of birds. The rapidity and low specialization of the transport of bird sperm in the male tract is in contrast with the high complexity of their selection and storage in the female (from 10 days storage in the quail up to 70d in the turkey) (reviewed by [START_REF] Aire | Spermatogenesis and testicular Cycles[END_REF]. Sperm storage in the female tract has been used to develop original reproductive adaptation. Thus, the choice of sires with high reproductive potential based on the preferential selection and storage of their sperm has probably developed in wild species [START_REF] Birkhead | Reproductive isolation in birds: postcopulatory prezygotic barriers[END_REF]. In domestic species, the development of genealogic selection in the poultry industry has been followed by the standardization of egg and meat production. However the control of reproductive performances is still not optimal and the production, quality and efficiency of storage of the male gamete have not been widely taken into account. This has now resulted in conventional breeding in early decreases in reproductive performance and in major welfare problems (reviewed by [START_REF] Hammerstedt | Symposium summary and challenges for future[END_REF][START_REF] Brillard | Natural mating in broiler breeders: present and future concerns[END_REF][START_REF] Jong | Major welfare issues in broiler breeders[END_REF].

The process of in vivo sperm storage in the oviduct is also a challenge to the development of appropriate methodologies to preserve sperm in vitro, as avian sperm must still undergo the normal processes of selection and prolonged storage in the SSTs following in vitro storage. Procedures to store poultry semen in vitro in a liquid state were first developed to facilitate the management of breeder flocks subjected to artificial insemination (AI) and also to transport genetic material to distant locations without the need for setting up of local breeder male facilities. Cryopreservation has been considered to be an alternative strategy to limit the erosion of genetic diversity and is also of major interest for the preservation of endangered breeds as it greatly facilitates the development of international programs to assess genetic biodiversity. Recent advances in cryopreservation technology for poultry semen have resulted in the emergence of cryobanking which is now being developed in an increasing number of countries (reviewed by [START_REF] Blesbois | Current status in avian semen cryopreservation[END_REF][START_REF] Blesbois | Strategy of avian germplasm cryobanking in France[END_REF][START_REF] Blesbois | Freezing avian semen[END_REF].

In the present review we will develop major aspects of specific features of bird sperm biology and their selection for fertilization, and the consequences on reproduction strategies and on reproductive biotechnology.

The Male Gamete at Ejaculation

Throughout the reproductive cycle, the key aim of male gamete production in birds is to produce "the highest number of gametes for the lowest reproductive cost". Following the rapid spermatogenesis, spermatozoa remain for a short time (1-3 days) in the male tract without annex gland. The signs of sperm maturation are much less developed than in mammals. Unlike mammals, chicken spermatozoa already possess most of their motility capacity and a significant part of their fertilizing capacity when they leave the testis [START_REF] Howarth | Fertilizing ability of cock spermatozoa from the testis, epididymis and vas deferens following intramagnal insemination[END_REF][START_REF] Ashizawa | Effects of temperature on the immobilization and the initiation of motility of spermatozoa in the reproductive tract of the domestic fowl[END_REF]. These capacities are then increased in the epididymis before transit in the deferent duct. At the time of ejaculation the number of spermatozoa per ejaculate is usually very high (0.5-1 billion in the chicken; 2 billion in the turkey) but concentrated in small volumes (less than 0.5 ml). In many species, the phallus is "vestigial" or has totally disappeared as in all the galliform order.

Bird spermatozoa show characteristics that are common to all the amniote species and also specific adaptations to their complex system of internal fertilization. These thin gametes (Fig. 1) possess a head, an intermediate piece and a long flagellum (reviewed by [START_REF] Aire | Spermatogenesis and testicular Cycles[END_REF]. The head contains a small acrosome (2.5 μm long and 0.5 μm wide in the chicken) and the nucleus (6×0.5 μm in the chicken). The acrosome includes the acrosome vesicle that contains different hydrolytic enzymes such as acrosin, involved in the digestion of the inner perivitelline layer (IPVL) that surrounds the oocyte and the perforatorim, a rigid actin-based structure with an unknown role in birds. The nucleus, surrounded by its double nuclear membrane, is extremely condensed, a protection to the genome during the very long passage in the oviduct before the fusion with the oocyte. In the Passeriformes, a helicoidal sheath surrounds the head. The intermediate piece contains a proximal and a distal centriole and also a highly variable number of mitochondria (approximately 30 mitochondria in the chicken, 150 in the Japanese quail) that may extend some distance into the flagellum depending on the species. It is the "motor" of the energetic metabolism of these gametes that do not contain significant intracellular reserves but must be very motile to access the female SST and then the oocyte and must also stay alive for a number of weeks in the female tract (Fig. 2). The flagellum is the longest part of the cell (70-90 μm in the chicken). It starts at the level of the distal centriole and contains an axoneme with one central doublet and nine peripheral microtubule doublets, mainly constituted of tubulin. In most birds (except Passeriformes), the axoneme is surrounded by a fibrous sheath in most of the flagellum. At ejaculation spermatozoa are diluted in seminal plasma that comes from diffuse secretions and excretions of the male tract (reviewed by [START_REF] Etches | The male[END_REF]. This biological medium presents a neutral (or slightly basic) pH and its osmolarity is sustained by a high content of amino acids such as glutamate and different proteins, the most abundant being a serum-like albumin [START_REF] Blesbois E And Caffin | Serum like" albumin of fowl seminal plasma and effects of albumin on fowl spermatozoa stored at 4 degrees C[END_REF]. It contains high levels of cholesterol and phospholipids originating from numerous lipidic and lipoproteic (high density lipoproteins, very high density lipoproteins) vesicules [START_REF] Blesbois | Effects of high-density lipoproteins on storage at 4 degrees C of fowl spermatozoa[END_REF][START_REF] Douard | Changes in turkey semen lipids during liquid in vitro storage[END_REF]. Seminal plasma stimulates sperm motility and is useful at the time of ejaculation. It is not a good medium of sperm storage despite the presence high molecular weight fractions that sustain fertility, and is eliminated before the sperm reach the SST [START_REF] Blesbois | Effect of different fractions of seminal plasma on the fertilizing ability of fowl spermatozoa stored in vitro[END_REF].

In Vivo Storage and Selection of Sperm in the Female Tract

At mating, sperm are either deposited into the cloaca ("cloacal kiss") in species such as chickens and turkeys, or introduced into the mid-vagina in species with a retractable penis (ostrich, duck, goose). Sperm are then subjected to a drastic process of vaginal selection prior to being stored in the SSTs. Only 1 % of the initial sperm population reaches the utero-vaginal junction (UVJ) [START_REF] Brillard | Quantification of spermatozoa in the sperm-storage tubules of turkey hens and its relation to sperm numbers in the perivitelline layer of eggs[END_REF]. As only 1% of the sub-population of pre-selected sperm can be further recovered in the inner perivitelline layer (IPVL), it can be postulated that the overall population of pre-selected sperm transported to the site of fertilization does not exceed 1/1, 000 th of the initial population deposited in the vagina (reviewed by [START_REF] Blesbois | Specific features of in vivo and in vitro sperm storage in birds[END_REF]. Thus the female oviduct would act as a succession of biological filters hind-ering the migration and storage of low quality or damaged sperm. The first filter is the access to the UVJ-SSTs. Despite the existence of a secondary site of storage of spermatozoa in the infundibulum, it is generally recognized that UVJ-SSTs are the main sperm storage site in poultry as most sperm stored for prolonged periods of time are located at this site. These sperm storage tubules are cylindrical, generally non-branched structures found in the folds of the upper vaginal mucosa. Their numbers are species-dependent, ranging from less than 200-500 in passerines [START_REF] Briskie | Patterns of sperm storage in relation to sperm competition in passerine birds[END_REF] to 20, 000 in the turkey [START_REF] Goodrich-Smith | A technique for studying the utero-vaginal sperm storage tubules[END_REF] and may be genetically selected to increase the storage length [START_REF] Brillard | Physiological responses of hens divergently selected on the number of chicks obtained from a single insemination[END_REF]. It has long been accepted that sperm that reach the SSTs must be motile (reviewed by [START_REF] Bakst | Fate of fluorescent stained sperm following insemination[END_REF]. Sperm with high motility show higher rates of success after heterospermic insemination than sperm with low motility, and their advantage increases with the time of storage in the SSTs (Pizzari et al., 2008). Motility has also been suggested to continue during the stay in the SSTs where sperm have to maintain position against a fluid current generated by the SST epithelial cells, and motility would also be involved in their release [START_REF] Froman | Deduction of a Model for Sperm Storage in the Oviduct of the Domestic Fowl (Gallus domesticus)[END_REF]. Immunological status of sperm and oviduct are also important for sperm selection. [START_REF] Steele | Evidence for a species-specific barrier to sperm transport within the vagina of chicken hens[END_REF] showed that immunoglobulin identification of sperm could be involved in the access to SSTs. More recently, a series of studies suggested a potential role of cytokines in the elimination of "undesirable" sperm in the vagina and the immune protection of transforming growth factor-βs in the SSTs [START_REF] Das | Expression of transforming growth factor β-isoforms and their receptors in utero-vaginal junction of hen oviduct in presence or absence of resident sperm with reference to sperm storage[END_REF]2009).

Contribution of Sperm to the Fertilization Process

The initial event of fertilization in birds is the fixation of numerous sperm on the IPVL that surrounds the oocyte at ovulation. The fixation is followed by the acrosome reaction (AR), an exocytosis process involving the fusion between the sperm plasma membrane and the outer acrosomal membrane (Okamura and Nishiyama, 1978a). N-Glycanes fractions of the IPVL ZP proteins and specific arrangement of ZP3 fractions would be involved in the sperm fixation (Horrock et al., 2000;Han et al., 2010;[START_REF] Wassarman | Egg's ZP3 structure speaks volumes[END_REF]. But the specificity is low and other perivitelline layer fractions i.e. the outer perivitelline are also efficient [START_REF] Lemoine | A reappraisal of factors involved in the in vitro induction of acrosome reaction in chickens[END_REF]. The fixation of sperm to the perivitelline layer induces the activation of different signaling pathways (sperm PI3-kinase followed by PKA and MAPK1) and leads to acrosome reaction [START_REF] Lemoine | Signaling pathways involved in the chicken acrosome reaction[END_REF]. The AR leads to the liberation of proteolytic acrosomic enzymes such as acrosin that help the digestion of the IPVL and also the mobilization of intra acrosomal calcium that could be involved in different processes including the oocyte activation. Calcium plays a central role in the AR. Extra-cellular calcium is an absolute requirement for AR and is found in abundance in the oviduct. Specific "Calcium storage structures" have also been described in the infundibulum of chicken and quail [START_REF] Rabbani | Sperm-egg interaction is mediated by a sperm-associated body in quail[END_REF]2007). Calcium is also involved as second messenger in the successive steps of the AR in all animal species with internal fertilization and its liberation from the intra-acrosomic stones would also be involved [START_REF] Rabbani | Characterization of the sperm-associated body and its role in the fertilization of the chicken Gallus domesticus[END_REF].

Many sperm may undergo AR and then penetrate the bird's oocyte after the fusion between the inner membrane of the acrosome and the plasma membrane of the oocyte (Okamura and Nishiyama, 1978b). They preferentially penetrate near to but not on the germinal disc and then evolve to the formation of male pronuclei (reviewed by [START_REF] Stepinska | Fertilization. In: Reproductive Biology and Phylogeny of Birds[END_REF]. The pronuclei formation involves profound changes in the sperm nuclei, including chromatin decondensation, disappearance and then neo formation of the nuclear membrane. However, only one male pronucleus is involved in the final syngamy. The role of polyspermy in birds is still in question. One hypothesis involves the amount of phospholipase C zeta (PLCζ) produced by sperm that is needed to activate the oocyte. Indeed, the presence of PLCζ in chicken sperm has been proven [START_REF] Coward | Phospholipase Cζ, the trigger of egg activation in mammals, is present in non-mammalian species[END_REF]. The injection of chicken PLCζ RNA into mice oocytes induced egg activation [START_REF] Dong | Xenopus and chicken sperm contain a cytosolic soluble protein factor which can trigger calcium oscillations in mouse eggs[END_REF]. The injection of quail sperm and PLCζ RNA or of higher concentration of PLCζ RNA alone has proven its efficiency to induce the formation of the quail female pronucleus [START_REF] Mizushima | Developmental enhancement of intracytoplasmic sperm injection (ICSI)-generated quail embryos by phospholipase Cζ cRNA[END_REF]2009). The existence of a positive dose effect of PLCζ RNA on the rate of early embryo development lead to suggest that polyspermy could help to increase the rate of PLCζ liberated in the oocyte and needed for an optimal activation of the egg. This could be an adaptive strategy related to the very large size of the telolecithal oocyte. A higher chance of activation of the oocyte would be given by polyspermy.

Biotechnology of Conservation

The general and the specific features of the biology of sperm in birds have been taken as a basis to develop biotechnologies that may be used to extend the period of time between the production of the male gametes and their use in the formation of zygotes. Most of these technologies derive from the use of ejaculated sperm. One exception is the transfer and storage of spermatogonial cells (Trefil et al., 2006;2010).

Extending the time between production and utilization of the male gamete permit two main types of action:

1) Manage the diffusion of the initial genetic potential of the males; 2) Include an in vitro incubation that may be used profitably to modify the property of sperm, including changes in their genetic potential. Until very recently the first method met with some success in birds but not the second. Although very recent results [START_REF] Mizushima | Novel method of Gene transfer in Birds: intracytoplasmic sperm injection for green fluorescent protein expression in quail blastoderms[END_REF] suggest that ICSI of sperm treated with triton X-100 could be successful in the quail, suggesting future applications for gene transfer, this approach is as yet barely used. The very efficient DNAse of bird oocytes has been suggested to rapidly eliminate "foreign" materials and is suspected to be involved in the poor success of these methods [START_REF] Stepinska | Fertilization. In: Reproductive Biology and Phylogeny of Birds[END_REF]. Thus we will now focus this chapter on the more widely used methods of semen storage. Semen in vitro storage must be done very carefully in birds because every small defect in the in vitro procedures will be exacerbated during the long process of semen in vivo storage in the female oviduct. The sperm that have been previously stored in vitro make up part of the populations that may be rapidly eliminated in the oviduct. Four main factors are important to ensure the success of in vitro storage. (a)

The in vitro media must share physical properties compatible with cell life. (b) As bird sperm are cells without intra-cellular reserves, the sperm energetic metabolism is one of the main sources of problems during and after in vitro storage. The course of intracellular metabolism also includes the management of free radicals and peroxides. (c) The plasticity of the sperm membrane to resist osmotic and temperature changes induced by in vitro storage is an absolute requirement to retain the biological properties of sperm (d) The last crucial question is the maintenance of the capacity of the Journal of Poultry Science,49 (3) sperm to undergo the acrosome reaction because birds are species in which the AR may be induced very rapidly in vitro and does not need highly specific media. This means that AR would be inhibited by in vitro storage media.

These factors are important for liquid semen storage at temperatures above 0℃ and also for cryopreservation. However, the dependence on energetic metabolism will be greater with liquid storage and the resistance of the plasma membrane to osmotic stress will be the main problem with cryopreservation (reviewed by [START_REF] Blesbois | Specific features of in vivo and in vitro sperm storage in birds[END_REF].

(a) Among other characteristics, semen diluents must provide relatively constant physico-chemical stability to sperm during in vitro storage. Iso-osmotic and nearly neutral pH conditions must be ensured. This is generally achieved by mixing balanced salt solutions in the osmolarity range 320-450 milliosmoles and the 6.8-7.4 pH range. Decreases in pH during storage may indicate an increase in sperm glycolysis by products such as lactic acid, while an increase in pH has been reported to be related to increasing numbers of dead sperm.

(b) At the time of ejaculation, avian sperm contain very few intra-cellular energy reserves, but repeated studies have demonstrated that they can access various types of metabolite "fuels" present in the extra-cellular medium to ensure their basal metabolism and motility. The energy metabolites available to sperm vary between species, but they generally consist mainly of sugars (mainly fructose or glucose) and fatty acids found in seminal plasma and the oviduct, including SSTs. As a consequence, energy substrates may be added to diluents in order to prolong sperm viability and activity in vitro. At the same time, sperm membranes must be protected against free radicals and peroxides. The addition of antioxidants such as vitamin E and selenium to semen diluents has been found effective to counteract the action of free radicals [START_REF] Blesbois | Effects of vitamin E on fowl semen storage at 4 degrees C[END_REF][START_REF] Dimitrov | Effect of organic selenium on Turkey semen quality during liquid storage[END_REF]. Peroxidation occurs more rapidly under quite high storage temperatures (15 to 41℃, [START_REF] Donoghue | Effects of water and lipid-soluble antioxidants on turkey sperm viability, membrane integrity and motility during liquid storage[END_REF][START_REF] Douard | Impact of composition changes in the storage medium on lipids and quality of turkey spermatozoa[END_REF], and when the males are aging [START_REF] Douard | Reproductive period affects lipid composition and quality of fresh and stored spermatozoa in turkeys[END_REF]. However, the fertilizing potential of sperm kept in vitro at body-like temperatures (41℃ for chicken) cannot be maintained over many minutes. In order to improve in vitro storage conditions, sperm metabolism must be decreased. This is generally achieved by reducing the temperature.

(c) The general principle guiding the choice of in vitro storage temperature conditions for avian semen is "the longer the duration of storage, the lower the temperature". However the sperm plasma membranes are very rich polyunsaturated fatty acids (PUFAs) fractions that present certain specificity in birds. They contain mainly n-6 PUFAs and always contain n-9 PUFAs even after dietary changes in chickens and turkeys [START_REF] Blesbois | Effect of dietary fat on thefatty acid composition and on fertilizing ability of fowl semen[END_REF][START_REF] Douard | Changes in turkey semen lipids during liquid in vitro storage[END_REF]2003;[START_REF] Brillard | Natural mating in broiler breeders: present and future concerns[END_REF]. They are highly susceptible to lipid peroxidation that may be limited by anti-oxidant supplementation (see b). They are also rapidly submitted to local phase transitions during storage at "infra-physiological" temperatures that may alter their membrane fluidity and plasticity. Membrane fluidity and resistance to osmotic stress induced by temperature changes are species-specific [START_REF] Blanco | Species variation in osmotic, cryoprotectant, and cooling rate tolerance in poultry, eagle, and falcon spermatozoa[END_REF][START_REF] Blesbois | Membrane fluidity and the ability to survive cryopreservation in domestic bird spermatozoa[END_REF]. Membrane fluidity also depends on the sperm phospholipid composition and on the phospholipids/cholesterol ratios. They may be modified to a certain extent by in vivo (diet, place in the reproduction cycle) or in vitro (cholesterol or phospholipid supply or depletion) conditions (reviewed by [START_REF] Blesbois | Specificity of fatty acids in domestic birds spermatozoa[END_REF] and are affected by freezing (Fig. 3). Membrane fluidity of fresh semen added to motility and viability may predict 85% of the variability of fertility obtained with frozen-thawed semen (Table 2). In addition, a progressive decrease in temperature (e.g. 0.5℃/min) helps to prevent adverse cold shock effects. However, even under optimal conditions of liquid storage, significant losses of the main plasma membrane phospholipids are observed in the chicken and the turkey [START_REF] Blesbois | Changes in lipid content of fowl spermatozoa after liquid storage at 2 to 5 degrees C[END_REF][START_REF] Douard | Changes in turkey semen lipids during liquid in vitro storage[END_REF]. The endogenous phospholipases have been suspected to play a part in the degradation process [START_REF] Douard | Impact of composition changes in the storage medium on lipids and quality of turkey spermatozoa[END_REF].

Sperm plasma membranes are submitted to specific osmotic stress during cryopreservation. During the freezing steps, the cells are subjected to successive reductions in volume due to dehydration and internal cryoprotectant action. Then during the thawing process, swelling of the sperm with rehydration and expulsion of the cryoprotectant from the intra-cellular fraction, and also a transient increase in intra-cellular ice particle size induces very high tensions in all cell membranes. The nuclear membrane seems quite well resistant, but mitochondrial and plasma membranes are highly sensitive to these physical stress. The membrane injuries that occur during the freeze-thaw process lead to the death of more than 50% of sperm, to membrane rigidifying, metabolic defects with mobility alteration and ATP decrease and changes in the plasma membrane glycocalyx [START_REF] Long | Avian semen cryopreservation: what are the biological challenges?[END_REF][START_REF] Blesbois | Predictors of semen cryopreservation in chickens[END_REF]Peleaz and Long, 2008). Most of these changes do not seem to show localization in their action on the plasma membrane, but the acrosomal cap could show specific reactivity.

(d) Acrosome reaction shares specific conditions in birds when compared to mammals. Specific features lead to huge differences in the sensitivity of bird spermatozoa to in vitro storage. In most mammalian species, acrosome reaction is preceded by sperm capacitation with major changes in the plasma membrane (calcium entry, cholesterol removal, changes in phospholipids and glycocalyx) that prepare sperm for AR. In vitro storage show "capacitation-like "effects that may be very useful for in vitro fertilization in mammals. In the absence of capacitation in birds, in vitro storage alters the capacity of chicken sperm to undergo the acrosome reaction [START_REF] Lemoine | Capacity of chicken spermatozoa to undergo the acrosome reaction after in vitro storage[END_REF]. During liquid storage, the alteration of AR capacity is more rapid and much stronger that the alteration of the other sperm functions such as viability and motility. We may thus now suggest that the capacity to undergo the AR is a prerequisite for the success of liquid storage of bird sperm.

The acrosome reaction is also frequently altered by the freezing process in birds. But conversely to liquid storage, AR capacity seems to be only secondarily affected by cryopreservation. Indeed, in high quality semen, AR is much less altered than motility and viability [START_REF] Lemoine | Capacity of chicken spermatozoa to undergo the acrosome reaction after in vitro storage[END_REF]. Finally, when present, the alteration of AR is irreversibly induced by the first contact with the internal cryoprotectant (CPAs) [START_REF] Mocé | Cryoprotectant and Freezing-Process alter the Ability of Chicken Sperm to Acrosome React[END_REF]. We may thus suggest that the interaction with the CPAs is critical for maintaining the capacity of sperm to undergo AR after cryopreservation. Consequently, AR induction would be a key test when deciding how to add CPAs prior to the freezing process.

All these elements show that bird AR shares very specific process that may help to emphasize the differences occurring in sperm quality changes during liquid and freezing semen storage. However the nature of the changes is different in liquid storage and in cryopreservation and these two storage methods have very different applications: liquid storage is extensively used in the practice of artificial insemination while semen freezing is used in the conservation of genetic diversity.

Liquid Storage and Artificial Insemination

Artificial insemination (AI) technology needs preservation of sperm viability and fertilizing potential under in vitro conditions over short or extended periods of time. AI practice in the poultry industry expanded for selection and multiplication purposes over the second half of the twentieth century but its justification for the production of broiler-type chicks remains species-specific. For example, the turkey industry switched from natural mating to AI in the 1960s because of the strong negative phenotypic correlation between breast widths and mating potential (reviewed by [START_REF] Krueger | Tom mangement-past, present and future[END_REF]. AI has also become increasingly popular to produce guinea fowl chicks because the hens are not suited to trap nesting conditions. AI has also become extensively used to produce mule ducklings (male Muscovy X female Pekin), a consequence of the repeated problems of mating behavior encountered in heterospecific breeder flocks. AI is now widespread in the poultry industry for selection purposes, but it has also become increasingly popular to produce broiler chicks in regions with low labor costs. In all these cases, semen is recommended to be stored for very few hours to avoid a decrease in fertility (6-24 h in the chicken). However, as previously described, the length of storage will depend on the storage conditions and on the physiological state of the animals [START_REF] Blesbois | Specific features of in vivo and in vitro sperm storage in birds[END_REF].

Semen Freezing and Cryobanking

The extensive use of semen freezing for current reproduction practice remains unrealistic, partly due to the high cost of employing frozen compared to fresh semen and partly due to the difficulty of obtaining freeze/thaw avian semen without significant loss of fertilizing potential. As previously described, in the case of sperm treated for cryopreservation, damage caused to plasma membranes results in a dramatic increase in sperm rejection prior to storage in the SSTs, thus preventing subsequent fertilization (reviewed by [START_REF] Blesbois | Specific features of in vivo and in vitro sperm storage in birds[END_REF]. Such impairment of fertility parameters may be partly compensated by increasing the number of sperm deposited at each insemination and also by increasing the frequency of insemination (Table 1; [START_REF] Seigneurin | Proc. 13 European Poultry Conference (EPC 2010[END_REF]. However, even under optimal conditions, high intra-line and inter-line (Table 3) variability does exist, mainly related to the initial semen quality and female fertility (Blesbois et al., 2007;[START_REF] Blesbois | Predictors of semen cryopreservation in chickens[END_REF]. Surprisingly, embryo mortality following insemination with frozenthawed spermatozoa is generally below that observed with fresh semen, at least in the chicken [START_REF] Seigneurin | Effects of the freezing rate on viability and fertility of frozen-thawed fowl spermatozoa[END_REF]. This indicates that freeze/thaw procedures may 'emphasize' the natural process of sperm selection exerted by the lower oviduct prior to their acceptance in the SSTs.

With species-specific zootechnical adaptations, semen freezing has successfully been used in chickens, geese, and pekin and muscovy ducks (reviewed by [START_REF] Blesbois | Freezing avian semen[END_REF]. Taking as models the methods developed in domestic birds (diluent, cryoprotectant, freezing and thawing rate), sperm freezing is also now under development in a small number of wild species including wild pheasianides, Barbary partridge and Griffon vultures, although fertility results are still lacking [START_REF] Jalme | Cryopreservation of semen from endangered pheasants: the first step towards cryobank for endangered avian species[END_REF][START_REF] Madeddu | Ejaculate collection efficiency and post-thaw semen quality in wild-caught Griffon vultures from the Sardinian population[END_REF][START_REF] Madeddu | Differences in semen freezability and intracellular ATP content between the rooster and the Barbary partridge[END_REF]. The successes obtained in domestic species open up the possibility of creating national programs that include the storage of genetic diversity in the form of sperm. The main advantage of the use of semen for this purpose is the non-invasive procedure developed to collect, treat and transfer the cells to the female through AI. Thus, despite the fact that semen will still share only half of the total genome, it still remains the main cell type to be used for breed reconstruction, creation of synthetic breeds, gene introgression and use for live-cryo-aided schemes. In most national cryobanking programs, sperm cells are completed by the additions in the cryobanks of alternative diploid cells with reproductive potential such as Blastodermal and primordial or gonadic germ cells (reviewed by [START_REF] Blesbois | Freezing avian semen[END_REF].

However the progress made in the last 15 years in this area concerns a small number of domestic or wild bird species. This still has to be developed in others such as turkeys, guinea fowl and quails.

Conclusion

Bird sperm show original adaptations to their complex system of internal fertilization. The rapid and rather unspecific fabrication of mature sperm in the adult male contrasts with the highly specific process to which sperm are submitted in the oviduct, including highly efficient selection and long storage in specific glands. The polyspermic fertilization of the telolecithal egg in birds also shares specific features that will affect the induction of the acrosome reaction and penetration of the ovum, the selection of the male pronucleus to be involved in the syngamy and the action of egg DNAses.

These specific features contribute to the "adaptive freedom" of bird reproductive systems. They have also been successfully used to optimize the management of males in the poultry industry and to develop methods of conservation of genetic resources in national programs of cryobanking.

New basic knowledge of birds' semen biology are however needed to understand what are the main factors involved in the selection of the sperm to be used for fertilization and consequently lead to zygote formation. Application of sperm biology to methods of semen storage should also be extended

Fig. 1 .

 1 Fig. 1. Chicken sperm observed with optical microscope. Chicken sperm observed under optical microscope on an eosin-nigrosin smear (photos I. Grasseau, INRA, France): left=one viable and morphologically normal sperm; 2a right=abnormal sperm (coiled or bent flagella, one sperm with intermediate piece permeable to eosin with arrow).

Fig. 2 .

 2 Fig.2. Specific features of the male gamete in the female oviduct in birds. 2a: Scheme of the ovary and oviduct of the hen ovary (adapted bySauveur, 1988 from Romanoff and Romanoff, 1949). 2b: Two sperm crossing the hen inner perivitelline membrane in the same hole (from[START_REF] Bakst | Fate of fluorescent stained sperm following insemination[END_REF]. 2c: dissociated hen sperm storage tubules full of sperm marked with Hoescht 33258 (optical microscopy, Brillard J. P. INRA, France).

Fig. 3 .

 3 Fig. 3. Membrane fluidity, lipids, ability to cryopreservation (Blesboiset al., 2005). Anisotropy in the opposite of fluidity: the higher the anisotropy (reprnsent rigidity) and the lower the fluidity. Thus sperm fluidity is higher in chicken than in turkey and guinea fowl and the highest rigidity is found in guinea fowl (p≤0.05). Freeze-thaw process increases significantly the sperm rigidity.

Table 1 . Specific parameters of freezing semen handling and insemination depending on species (Seigneurin and Blesbois, 2010).

 1

		Blesbois: Avian Male Gamete, Fertilization and Storage	145
		Chicken	Muscovy Duck	Pekin Duck	Goose	Guinea Fowl
	Dilution rate (% semen)	25-40	65	35	70	35
	AI Dose (×10 6 sperm)	400	300	300	40	200
	AI Frequency (/week)	2	2	2	3	2

Table 3 . Examples of fertility obtained with cryopreserved semen from chicken (Blesbois et al., 2007).

 3 Blesbois: Avian Male Gamete, Fertilization and Storage 147

	Genetic origin	Homologous females	Commercial females
	Gauloise dorée breed	21% b	39% a
	R+experimental line (high heat production line)	14% c	25% b
	B4 experimental line (subfertile
	line with specific histocompati-	7% d	43% a
	bility haplotype)
	a, b, c, d represent significant differences (P≤0.05).

Journal of PoultryScience, 49 (3)


Aubert, Didier

Sorbonne Nouvelle University didier.aubert@sorbonne-nouvelle.fr +33 (0)6. 88.46.15.27 Fernando Brito. "Judío" (Baca, Sinaloa. April 3, 2015).

This photograph belongs to a series of portraits made by news photographer and documentarist Fernando Brito in his native state of Sinaloa, Mexico. i Along the Zuaque (now Fuerte) river, which flows down from the Sierra Madre Occidental into the Gulf of California, Yoremem (plural of "Yoreme," or Mayo Indians) have maintained religious rituals combining indigenous cosmology with seventeenth-century Jesuit influence. These complex ceremonies play a vital role in sustaining native culture's claim to identity and relevance, in spite of a five-hundred-year-long confrontation with missionary, colonial, imperial and capitalistic forces, which have profoundly affected the region and its people through violence, cultural assimilation, and the exploitation of natural resources.

Under pressure from mining interests, large-scale agribusiness, hydroelectric infrastructure projects, and more recently the extension of the drug-related economy, the Yoremem's relation to their environment has been dramatically transformed. While outsiders (yoris) have been steadily increasing in numbers and influence, migration and relocation have removed indigenous communities from their original land. As a consequence, it has been suggested that the Mayos' territory has almost "ceased to be an element of identity," ii and the cultivation of unique cultural practices now defines their cohesion as "those who respect traditions" (which is what "yoremem" actually means). In spite of their Christian trappings, their ceremonies actually reflect "the competition for resources, both natural and symbolical," iii as they are celebrated-with numerous local variations-in nineteen "ceremonial centers" scattered among the northern hills of Sinaloa. These places are connected by the river and, for in some cases, by their history as former Jesuit missions. Among the oldest ones is Baca (located in the municipality of Choix), iv where this photograph was taken by Fernando Brito in collaboration with anthropologist Héctor Parra Zurita as part of a series documenting participants in Easter ceremonies Although the set up chosen by Brito and Parra seems to follow well-established anthropological and (less frequently) documentary models (frontal pose, similar background, uniform lighting), the sitters can also be seen as embodying a form of "native survivance." Performing for the camera, largely on their own terms, they are not simply "documented" as passive tokens of native culture. They also manifest "an active sense of presence over historical absence," v which is deeply rooted in the ongoing tradition they are carrying forward.

Participation in these rituals, which don't involve Catholic clergy, is predicated on binding social commitments and vows, and the distribution of organizational roles as well as ceremonial duties. This engagement largely trumps ethnic identity: these days, "mestizos" are allowed to participate in Baca's all-important Lent and Easter ceremonies, which last seven weeks. Tradition is therefore understood as the adherence to local specificities of the ritual, and it defines the community in opposition to European/mestizo forms of Catholicism, the devotion to popular saints such as Sinaloa's Jesus Malverde, and event recent inroads made by evangelical movements. vi Posing for Brito in front of the church's main altar honoring Saint Michael the Archangel (the patron Saint of Baca's ceremonial center), various participants of the Easter rituals such as musicians, dancers, young Marias and this so-called judío conjure up a powerful and at times unsettling image of the syncretic religious tradition of Sinaloa's native people. vii The background is unequivocally Catholic in its imagery. Ex-votos and candles were left on the altar, in the hope of obtaining blessings from the Saint: traditionally, membership in one of the orders that organize and enact the ceremonial (cofradías) results from a pledge made in exchange for such a request. Flanking Saint Michael are Mexico's very own Virgen of Guadalupe, along with Saint Jude of Thaddeus, carrying his Jesus medallion and wearing a characteristic green sash: throughout Mexico, the patron saint of desperate causes is especially popular among the poorest and most vulnerable sections of the population. viii A news photographer by training, Brito first devoted a classically composed and visually compelling photo essay to the various stages the ceremony, both inside and outside the Church, as processions and dances take place for over a month before Easter. Yet the altar series illustrated here, which he produced alongside more conventional forms of reportage, adds urgency and depth to this collective portrait through the complex interplay of performance and personality, historical traditions and contemporary upheaval.

Judíos such as the one pictured here (the neighboring Yaqui people, in Sonora, often call similar figures "fariseos") are key characters in the ceremonial cycle. They appear on Ash Friday and perform different tasks until Holy Saturday. For several weeks, they wander the region gathering alms that will help defray the cost of the ceremonies, pay the musicians and dancers, and buy fireworks or decorations for the church. Although they are not allowed to speak, they make their presence heard because of the rattling tenábaris made of moth cocoons stuffed with tiny pebbles, which can be seen around this young man's ankles. Most judíos pictured by Brito in this series wear other characteristic elements such as scarves, or towels wrapped around their shoulders. Some play the tambourine or carry hand rattles that allow them to mimic the ceremonial music played by official musicians and dancers. These grotesque, humorous figures are also emblems of wickedness, roaming the hills and villages to find Christ and bring him to the cross (the sandals most of them wear seem to refer to those worn by Roman legionnaires). This photograph, along with the rest of the series, was taken on Good Friday: on the following day, judíos remove their masks, set them on fire, and receive baptism to recover their human form. ix The disorder they wrought on the community is thus ultimately defeated. x Brito's studio-like set up pretends to record the ritual's material culture in a pseudoethnographic manner (the full-length portrait remotely echoes nineteenth-century anthropological imagery), while suggesting simultaneously the complexity of Sinaloa's present situation. All the men and children pictured in this series are fully "in character" yet momentarily removed from the ritual itself: after obtaining the authorization from Choix's indigenous governor, Brito and Parra called in each participant who was invited to act out their individualized interpretation of the ritual. Some are dancing and one is sitting on the floor, thus evading the visual constraints of standardized formats. Other portraits show the smiling faces of musicians or the serious look of young Marias in white dresses. For the judíos, the photographic encounter becomes an occasion to extend and possibly enhance the ceremonial performance-both as an affirmation of identity and defiance toward yori intrusions. The traditional deer-or goat-leather mask most of them are wearing (evoking the "cult of the wilderness" in pre-Jesuit cosmogonies), seems to be staring back at the viewer.

The judíos follow a military-like hierarchy, and their silent, slightly menacing presence reminds us of times when their dances were seen by Mexican officials and the press as forebodings of rebellion. xi In this specific portrait, the viewer's attention is drawn to this young man's modern-day, mass-produced hoghead mask, and to the (toy) gun tucked in his belt. Asked about the picture, and whether the weapon replaces the spears carried by judíos and fariseos in other ceremonial centers, Brito offered the following details: Before [the judíos] brought whips but then they fought [so they] decided not to use "violence."

The boy himself chose the outfit with the weapon, each judío disguises himself, and they make fun of their reality. At the time… the government was building new gas pipelines that passed through that area and some of the judíos were dressed in their work clothes… others wore the mask of [drug trafficker] "El Chapo" Guzmán, and another time they wore a Donald Trump mask." xii In pragmatic terms, costumes bought in stores are simply cheaper than traditional masks, which are destined to be burned to ashes after the ceremony. Yet Brito's account confirms that they also enhance the carnival-like dimension of the masquerade, in response to the overwhelming social, economic and political forces engulfing the region. The photographer's interest in Baca is also predicated on the fact that it is located in a particularly remote area, plagued by poverty, which has become an appealing location for drug production. xiii This is yet another cause for the displacement of indigenous populations. xiv Through jarring figures such as this one, the series conjures up a sense of disquiet which offsets the ever-present risk of a picturesque form of folclorismo. xv This creature's defiant stance is no quaint play acting of ancient superstitions waiting to be transformed into tourist attractions. The gun, along with the hoghead-often used in drug-related executions to dishonor corpses-remind the viewer of Brito's better-known crime scene pictures in the streets of Culiacán, his hometown. Even Jude of Thaddeus's presence is a visual reminder of narcoculture, which has integrated him as one of its patron saints. Thus Brito's work is not "salvage ethnography" trying to preserve what "must inevitably be blown away by the winds of modernism," to quote one of the earliest comprehensive studies of Mayo culture. xvi Neither is it the assimilation of native religion as part of an idealized and exotic Mexicanidad. The attempt to find an ad-hoc visual representation for yoreme figures pays tribute to the foundational value of the community's ritual as crucial to its survival and cohesion. This striking character may also reflect "Yoreme conduct as a new survival strategy in today's global age," xvii by incorporating elements of modernity that threaten this local culture's very existence: this precarious balancing act is enacted in the confident, and at time defiant poses seen in some of Brito's photographs. Mocking and threatening authority, standing his ground, yet eager to recover his humanity as soon as the circle of violence and disruption ends, this young judío is no mere ghost from the past.

i Born in 1975, Fernando Brito began his career as a photographer for Culiacán's local newspapers La I and El Debate, as well as providing pictures about cartel-related violence to international newsmedia. His series Your Steps Were Lost in the Landscape earned him a World Press Photo award in 2011, and was exhibited widely in Europe and the United States. He still lives in Culiacán, where he works as a freelance photographer.

ii Victor Joel Santos Ramírez, "La población indígena de Sinaloa, 1910de Sinaloa, -2000," ," Curtis (1868Curtis ( -1952)), Vizenor also acknowledges their contemporary appeal, as they "create a sense of native presence," Vizenor, Native Liberty, 193. vi López Aceves, Harriss Clare and Moctezuma Zamarrón, "Autoridad y religión," 221-231. vii I'm using the word "syncretic" in a broad sense, although some anthropologists consider the term to be misleading in the case of Yoreme culture. See Alejandro Figueroa, Por la tierra y por los santos: identidad y persistencia cultural entre yaquis y mayos (México: Consejo Nacional para la Cultura y las Artes, 1994), 242-243 and David Yetman and Thomas R. Van Devender, Mayo Ethnobotany: Land, History, and Traditional

  in Historia Temática de Sinaloa. Tomo I. Régión, población y salud. ed. Rigoberto Arturo Román Alarcón y Rafael Valdez Aguilar (Culiacán: Instituto Sinaloense de Cultura, 2015), 212. José Luis Moctezuma Zamarrón, Hugo Eduardo López Aceves, and Claudia Jean Harriss Clare, "Sistemas productivos y movilidad indígena entre Yaquis, Mayos y Guarijós," in Movilidad Migratoria de la Población Indígena de México, Volume II, ed. Margarita Nolasco and Miguel Ángel Rubio (México: Instituto Nacional de Antropología e Historia, 2012), 427-456. iii Hugo Eduardo López Aceves, Claudia Jean Harriss Clare and José Luis Moctezuma Zamarrón, "Autoridad y religión en el noroeste de México: los sistemas normativos entre Yaquis, Mayos y Guarijós," in Los Dioses, El Evangelio y el Costumbre: Ensayos de pluralidad en las regiones indígenas de México, Volume IV, ed. Ella F. Quintal et al. (México: Instituto Nacional de Antropología e Historia, 2010),183. iv Hugo Eduardo López Aceves, "Los Mayos de Sinaloa: esbozo etnográfico y regional," Cuicuilco, 14: 39 (2007): 19. v Vizenor, Gerald, Native Liberty: Natural Reason and Cultural Survivance (Lincoln, NE: University of Nebraska Press, 2009), 1. Although critical of the use of photography by would-be ethnographers such as Edward

  Knowledge in Northwest Mexico (Berkeley: University of California Press, 2002), 22. viii A comparable photographic essay focusing on Saint Jude Thaddeus' following in Mexico is Sofia Jaramillo's "Mexicans Seek Help from the Saint of Lost Causes", apimagesblog.com, August 6, 2015, https://apimagesblog.com/blog/2015/08/06/mexicans-seek-help-from-saint-of-lost-causes ix A step-by-step account of the equivalent ceremony in the Mayo River valley, where the part of the judíos is played by Pariserom, can be found in N. Ross Crumrine, The Easter Ceremonial in the Socio-Cultural Identity of Mayos, Sonora, Mexico, Phd Dissertation, University of Arizona, 1968, 16-26. x Crumrine, The Easter Ceremonial, 40. xi Fidel Camacho Ibarra, "While the Deer Rises with the Dawn," Artes de México, 117 (2015), 69. See also Helena Simonett, "Narrativity and Selfhood in Mayo-Yoreme Mortuary Rituals," The World of Music, 51: 2 (2009): 53-54. xii Email from Fernando Brito, August 14, 2019. Translated by the author. Used with Fernando Brito's permission. xiii López Aceves, "Los Mayos de Sinaloa," 20. https://www.sinembargo.mx/17-11-2015/1554070 xiv "Choix está en guerra, y hay muertos, desplazados; el Cártel de Sinaloa se fracturó: Ejecito," Sin Embargo, November 17, 2015, https://www.sinembargo.mx/17-11-2015/1554070; Comisión estatal de los derechos humanos de Sinaloa, Informe especial sobre los desplazamientos internos de personas en Sinaloa, April 2013, 119, https://www.cedhsinaloa.org.mx/index.php/recomendaciones/recomendaciones-generales/909-no-10-1/file. For an in-depth, and courageous exposé of the dangerous connections between transnational mining companies and drug cartels, see Sibely Cañedo, Marcos Vizcarra, and Sheila Arias, ://www.connectas.org/especiales/desplazados-por-yugo-narco-minero-sinaloa/ xv Helena Simonett, "Re-localized Rap and its Representation of the Hombre digno," in Transnational Encounters: Music and Performance at the U.S.-Mexico Border, ed. Alejandro L. Madrid (New York: Oxford University Press, 2011), 132. xvi Ralph Beals, The Contemporary Culture of the Cáhita Indians, Bureau of American Ethnology, Bulletin 142 (1945), 91. xvii Simonett, "Relocalized Rap," 132.

	"Desplazados	por	el	yugo	narco-minero	en	Sinaloa,"	Connectas,	s.d.

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