A simple and fast alternative method to remove glycerol from chicken semen after cryopreservation.

A concentration of 11% of glycerol is the standard one for sperm cryopreservation in chickens, however, the presence of just 2% glycerol already causes severe fertility reduction, suggesting the necessity of removing glycerol before artificial insemination (AI). The major approach developed for this purpose is serial dilution followed by centrifugation (SDC), which demands special equipment (such as a refrigerate room) to maintain post-thaw semen at 4 °C, besides being time consuming. Therefore, we attempted to develop a simple method to remove glycerol from chicken frozen-thawed semen based on a colloidal gel, Percoll, which is ordinarily used to select motile and viable sperm in mammals as well as in fresh chicken semen. In this study, we used a Percoll based glycerol removal solution (GRS) containing sucrose to avoid frozen-thawed sperm suffering from osmotic stress. Subsequently, several conditions including GRS compositions (GRS A, B, C and D) and centrifugation temperatures (4 and 20 °C) were compared by their influence on sperm in vitro parameters. Afterwards, GRS A and D were selected for fertility evaluation, compared to conventional SDC method. Our results showed that the fertility with GRS A at both 4 and 20 °C were higher than GRS D (p < 0.05) and similar or even superior to the fertility obtained with SDC method. Altogether, our novel GRS protocol is a valuable method for chicken sperm cryobanking policy, supported by its notable results of fertility as well as saving 44% of time, with a simple equipment at flexible operation temperatures of 4 or 20 °C.

First insights on seminal extracellular vesicles in chickens of contrasted fertility.

Male subfertility causes are very varied and sometimes related to post-gonadic maturation disruption, involving seminal plasma constituents. Among them, extracellular vesicles are involved in key exchanges with sperm in mammals. However, in birds, the existence of seminal extracellular vesicles is still debated. The aim of the present work was first to clarify the putative presence of extracellular vesicles in the seminal plasma of chickens, secondly to characterize their size and protein markers in animals showing different fertility, and finally to make preliminary evaluations of their interactions with sperm. We successfully isolated extracellular vesicles from seminal plasma of males showing the highest differences in semen quality and fertility by using ultracentrifugation protocol (pool of 3 ejaculates/rooster, n =3/condition). Size characterization performed by electron microscopy revealed a high proportion of small extracellular vesicles (probably exosomes) in chicken seminal plasma. Smaller extracellular vesicles appeared more abundant in fertile than in subfertile roosters, with a mean diameter of 65.12 and 77.18 nm, respectively. Different protein markers of extracellular vesicles were found by western blotting (n = 6/condition). Among them, HSP90A was significantly more abundant in fertile than in subfertile males. In co-incubation experiments (n = 3/condition), extracellular vesicles enriched seminal fractions of fertile males showed a higher capacity to be incorporated into fertile than into subfertile sperm. Sperm viability and motility were impacted by the presence of extracellular vesicles from fertile males. In conclusion, we successfully demonstrated the presence of extracellular vesicles in chicken seminal plasma, with differential size, protein markers and putative incorporation capacity according to male fertility status.

Functional Aspects of Seminal Plasma in Bird Reproduction.

This review provides an updated overview of the seminal plasma composition, and the role of metabolic and protein components on the sperm function of avian species. In addition, the implication of seminal plasma on assisted reproductive techniques of birds was discussed. The semen of birds usually has exceptionally high sperm concentration with relatively little seminal plasma, but this contributes to very fast changes in sperm metabolism and function. The biochemical characteristics and physiological roles of the various seminal plasma components in birds (carbohydrates, lipids, amino acids, hormones, and proteins) are poorly understood. Seminal plasma content of proteins has an action on most cellular functions: metabolism, immunity, oxido-reduction regulation, proteolysis, apoptosis, ion homeostasis, and antimicrobial defenses. The variable amount of many proteins is related to a different fertility capacity of poultry sperm. The role of seminal plasma on semen conservation (chilling and freezing) remains largely a matter of speculation, as both inhibitory and stimulating effects have been found. Whereas the presence of seminal plasma did not seem to affect the sperm survival after freezing-thawing, DNA fragmentation is lower in the absence of seminal plasma. The molecular basis of the influence of seminal plasma on sperm cryo-resistance was also discussed in the present review.

The seminal acrosin-inhibitor ClTI1/SPINK2 is a fertility-associated marker in the chicken.

The seminal plasma is a very complex fluid, which surrounds sperm in semen. It contains numerous proteins including proteases and protease inhibitors that regulate proteolytic processes associated with protein activation and degradation. We previously identified a seminal protein, chicken liver trypsin inhibitor 1 (ClTI-1) over expressed in semen of roosters with high fertility, suggesting a role in male fertility. In the present study, we showed that ClTI-1 gene is actually SPINK2. Using normal healthy adult roosters, we showed that SPINK2 amount in seminal plasma was positively correlated with male fertility in chicken lines with highly contrasted genetic backgrounds (broiler and layer lines). Using affinity chromatography combined to mass spectrometry analysis and kinetic assays, we demonstrated for the first time that two chicken acrosin isoforms (acrosin and acrosin-like proteins) are the physiological serine protease targets of SPINK2 inhibitor. SPINK2 transcript was overexpressed all along the male tract, and the protein was present in the lumen as expected for secreted proteins. Altogether, these data emphasize the role of seminal SPINK2 Kazal-type inhibitor as an important actor of fertility in birds through its inhibitory action on acrosin isoforms proteins.

Intact Cell MALDI-TOF MS on Sperm: A Molecular Test For Male Fertility Diagnosis.

Currently, evaluation of sperm quality is primarily based on in vitro measures of sperm function such as motility, viability and/or acrosome reaction. However, results are often poorly correlated with fertility, and alternative diagnostic tools are therefore needed both in veterinary and human medicine. In a recent pilot study, we demonstrated that MS profiles from intact chicken sperm using MALDI-TOF profiles could detect significant differences between fertile/subfertile spermatozoa showing that such profiles could be useful for in vitro male fertility testing. In the present study, we performed larger standardized experimental procedures designed for the development of fertility- predictive mathematical models based on sperm cell MALDI-TOF MS profiles acquired through a fast, automated method. This intact cell MALDI-TOF MS-based method showed high diagnostic accuracy in identifying fertile/subfertile males in a large male population of known fertility from two distinct genetic lineages (meat and egg laying lines). We additionally identified 40% of the m/z peaks observed in sperm MS profiles through a top-down high-resolution protein identification analysis. This revealed that the MALDI-TOF MS spectra obtained from intact sperm cells contained a large proportion of protein degradation products, many implicated in important functional pathways in sperm such as energy metabolism, structure and movement. Proteins identified by our predictive model included diverse and important functional classes providing new insights into sperm function as it relates to fertility differences in this experimental system. Thus, in addition to the chicken model system developed here, with the use of appropriate models these methods should effectively translate to other animal taxa where similar tests for fertility are warranted.

Early steps of cryopreservation of day one honeybee (Apis mellifera) embryos treated with low-frequency sonophoresis.

Honeybees, major providers of pollination, are endangered in many areas. Embryo cryopreservation may be a very useful tool to maintain their genetic diversity. However, it is complex in insects, because embryos are chill sensitive and are surrounded by two protectant membranes, the chorion and vitelline. These membranes prevent penetration of cryoprotectant in the embryos. This study aimed to test different conditions of embryo preparation before cryopreservation, including low-frequency sonophoresis, a physical method of permeabilization, and passages through cryoprotectant solutions. Apis mellifera ligustica embryos were collected in artificial cell plugs 7.5 h after queens had been caged, in two different seasons (winter, spring) and were then incubated in vitro overnight (16.5 h). Embryos were individually sonicated and then incubated in three cryoprotectant baths (B1 = 10%, B2 = 20% and B3 = 40% of cryoprotectant) and quenched in liquid nitrogen. Artificial cell plugs and in vitro incubation device were efficient in producing future embryos hatching. Embryos stained ruby red with rhodamine B after sonophoresis treatment indicated that low-frequency ultrasound had permeabilized embryos. According to the treatment, different significant hatching rates were obtained after sonophoresis (up to 25%). After three cryoprotectant incubations, best hatching rates were obtained after 10 min in B1 and B2, and 40 s in B3. These results show that sonophoresis is an efficient tool to permeabilize the chorion and vitelline membrane of the day one honeybee embryo allowing a hatching rate of more than 20%. They also show that the season is an important variability factor.

Cryopreservation of Indian red jungle fowl (Gallus gallus murghi) semen with polyvinylpyrrolidone.

The Indian red jungle fowl is a sub-species of the genus Gallus native to South Asia; facing high risk of extinction in its native habitat. During cryopreservation, permeable cryoprotectants like glycerol are usually employed and we previously showed encouraging results with 20% glycerol. Because bird spermatozoa contain very little intracellular water, the possibility of replacing an internal cryoprotectant by an external one is opened. In the present study, we tested the replacement of internal cryoprotectant glycerol by the external cryoprotectant Polyvinylpyrrolidone (PVP). PVP is a non-permeable cryoprotectant and keeps the sperm in glassy state both in cooling and warming stages without making ice crystallization within the sperm cell. We evaluated the effect of various levels of polyvinylpyrrolidone (PVP) on Indian red jungle fowl semen quality and fertility outcomes. The qualifying semen ejaculates collected from eight mature cocks were pooled, divided into five aliquots, diluted (37 °C) with red fowl semen extender having PVP [0% (control) 4% (w/v), 6% (w/v), 8% (w/v) and 10% (w/v)]. Diluted semen was cryopreserved and stored in liquid nitrogen. The whole experiment was repeated/replicated for five times independently. Sperm motility, plasma membrane integrity, viability and acrosome integrity were recorded highest (P < 0.05) with 6% PVP at post-dilution, cooling, equilibration and freeze-thawing. Higher (P < 0.05) no. of fertile eggs, fertility, no. of hatched chicks, percent hatch and hatchability was recorded with 6% PVP compared to control. It is concluded that 6% PVP maintained better post-taw quality and fertility of Indian red jungle fowl spermatozoa than glycerol and can be used in routine practice avoiding the contraceptive effects of glycerol.

Central role of 5'-AMP-activated protein kinase in chicken sperm functions.

Avian gametes present specific features related to their internal long-term mode of fertilization. Among other central actors of energetic metabolism control, it has been suspected that 5'-AMP-activated protein kinase (AMPK) influences sperm functions and thus plays a key role in fertilization success. In the present work, we studied AMPK localization and function in chicken sperm incubated in vitro. Effects of the pharmacological AMPK activators (AICAR, metformin) and the AMPK inhibitor compound C were assessed by evaluating AMPKalpha (Thr(172)) phosphorylation (by Western blotting), semen quality (by viability, motility, and ability to perform acrosome reaction), and energetic metabolism indicators (lactate, ATP). Localization of AMPK in subcellular sperm compartments was evaluated by immunocytochemistry. Total AMPK was found in all compartments except for the nucleus, but the phosphorylated form phospho-Thr(172)-AMPK was essentially localized in the flagellum and acrosome. AMPK activators significantly improved AMPK phosphorylation, sperm motility (increased by 40% motile, 90% progressive, and 60% rapid sperm), acrosome reaction and lactate production (increased by 40%) and viability. The AMPK inhibitor significantly reduced AMPK phosphorylation and percentages of motility (decrease by 25%), progressive energy (decrease by 35%), and rapid sperm (decreased by 30%), acrosome reaction, lactate production, and ATP release. The two activators differed in their effect on ATP concentration: AICAR stimulated ATP formation, whereas metformin did not. Our results indicate that AMPK plays a key role in the regulation of chicken sperm functions and metabolism. This action differs from that suggested in mammals, mainly by its crucial involvement in the acrosome reaction process.

Exploring how sucrose-colloid selection improves the fertilizing ability of chicken sperm after cryopreservation with glycerol.

Currently, glycerol is the most effective cryoprotectant when combined with straw packaging for preserving chicken sperm. Glycerol, however, has toxic effects on sperm cells, which can reduce fertility when present in inseminated semen. Historically, the serial dilution (SD) method was developed to eliminate glycerol and mitigate its adverse effects. We have recently developed a new method for removing glycerol called sucrose-Percoll (SP), that can be performed at either 4°C (4°C-SP) or 20°C (20°C-SP). This SP protocol has been found to be simpler and faster to improve fertility compared to the traditional SD method. Nevertheless, the reasons for such effectiveness differences between glycerol removal procedures remained unclear and required more comprehensive understandings for future protocol developments. Here, we examined the effects of SP and SD protocols on the fertility duration. We also investigated the potential causes of varying effects of these methods by analyzing sperm quality parameters and sperm storage in the hen's reproductive tract. The fertility was significantly higher in 4°C-SP than 20°C-SP during the first 6 d after insemination, and also higher than sperm processed using SD. No difference was observed between 20°C-SP and SD between 7 and 13 d. However, a 2.7-time higher fertility was shown with 4°C-SP. In addition, the SP method demonstrated a 2-fold greater ability to remove glycerol than the SD method. Sperm centrifuged at 4°C-SP exhibited higher sperm storage compared to 20°C-SP and were higher than sperm treated with SD. Overall, our findings revealed that the differences in efficiencies between SP and SD methods were not related to in vitro sperm quality but resulted from a higher ability to remove glycerol, a higher storage capacity in the female reproductive tract, and a longer fertility ability. Since no impacts were observed in sperm cellular characteristics, further experiments are necessary to investigate the influences of glycerol removal treatments at the molecular level.

Is glycerol a good cryoprotectant for sperm cells? New exploration of its toxicity using avian model.

Glycerol is a cryoprotectant used widely for the cryopreservation of animal sperm, but it is linked to a decrease in fertility. The mechanism underlying the negative effects of glycerol remains unclear. Therefore, in this study, we aimed to gain a better understanding by using the chicken model. First, we investigated the impact of increasing the concentration of glycerol during insemination on hen fertility. Our findings revealed that 2% glycerol resulted in partial infertility, while 6% glycerol led to complete infertility. Subsequently, we examined the ability of sperm to colonize sperm storage tubules (SST) during in vivo insemination and in vitro incubation. The sperm used in the experiment were stained with Hoechst and contained 0, 2, or 6% glycerol. Furthermore, we conducted perivitelline membrane lysis tests and investigated sperm motility, mitochondrial function, ATP concentration, membrane integrity, and apoptosis after 60 min of incubation with different glycerol concentrations (0%, 1%, 2%, 6%, and 11%) at two temperatures to simulate pre-freezing (4 °C) and post-insemination (41 °C) conditions. Whereas 2% glycerol significantly reduced 50% of sperm containing SST, 6% glycerol completely inhibited SST colonization in vivo. On the other hand, in vitro incubation of sperm with SST revealed no effect of 2% glycerol, and 6% glycerol showed only a 17% reduction in sperm-filled SST. Moreover, glycerol reduced sperm-egg penetration rates and also affected sperm motility, bioenergetic metabolism, and cell death at 4 °C. These effects were observed when the concentration of glycerol exceeded 6%. Furthermore, at 41 °C, glycerol caused even greater damage, particularly in terms of reducing sperm motility. These data altogether reveal important effects of glycerol on sperm biology, sperm migration, SST colonization, and oocyte penetration. This suggests that glycerol plays a role in reducing fertility and presents opportunities for improving sperm cryopreservation.

Protein expression reveals a molecular sexual identity of avian primordial germ cells at pre-gonadal stages.

In poultry, in vitro propagated primordial germ cells (PGCs) represent an important tool for the cryopreservation of avian genetic resources. However, several studies have highlighted sexual differences exhibited by PGCs during in vitro propagation, which may compromise their reproductive capacities. To understand this phenomenon, we compared the proteome of pregonadal migratory male (ZZ) and female (ZW) chicken PGCs propagated in vitro by quantitative proteomic analysis using a GeLC-MS/MS strategy. Many proteins were found to be differentially abundant in chicken male and female PGCs indicating their early sexual identity. Many of the proteins more highly expressed in male PGCs were encoded by genes localised to the Z sex chromosome. This suggests that the known lack of dosage compensation of the transcription of Z-linked genes between sexes persists at the protein level in PGCs, and that this may be a key factor of their autonomous sex differentiation. We also found that globally, protein differences do not closely correlate with transcript differences indicating a selective translational mechanism in PGCs. Male and female PGC expressed protein sets were associated with differential biological processes and contained proteins known to be biologically relevant for male and female germ cell development, respectively. We also discovered that female PGCs have a higher capacity to uptake proteins from the cell culture medium than male PGCs. This study presents the first evidence of an early predetermined sex specific cell fate of chicken PGCs and their sexual molecular specificities which will enable the development of more precise sex-specific in vitro culture conditions for the preservation of avian genetic resources.

Intact cell MALDI-TOF mass spectrometry, a promising proteomic profiling method in farm animal clinical and reproduction research.

The objective of this review is to provide new insights into the possible use of a proteomic method known as Intact Cell Matrix-Assisted Laser Desorption-ionization Time-Of-Flight Mass Spectrometry (ICM-MS) in animal clinical research. Here, we give an overview of the basics of this technique, its advantages and disadvantages compared with other proteomic approaches, past applications and future perspectives. A special emphasis on its implementation in animal reproduction science is given, including examples of the reliable use of ICM-MS on fertility screening. In mammals, the ICM-MS profiles from pig epididymal spermatozoa reflect the proteome changes that they undergo during epididymal maturation and could be associated with the acquisition of fertilizing ability. In chicken, using adequate pre-processing and bioinformatics analysis tools, sperm ICM-MS profiles showed characteristic spectral features that allowed their classification according to their actual fertilizing ability. The association of ICM-MS and Top-down proteomic strategies allowed the identification of chicken fertility biomarkers candidates such as protein vitelline membrane outer layer protein 1 (VMO-1) and avian beta-defensin 10 (AvBD10). In female reproduction, a similar approach on ovarian follicular cells allowed the identification of specific markers of oocyte maturation in the oocyte and surrounding cumulus cells. Altogether, these results indicate that ICM-MS profiling could be a suitable approach for molecular phenotyping of male and female gametes.

Different concentrations of cysteamine, ergothioneine, and serine modulate quality and fertilizing ability of cryopreserved chicken sperm.

The aim of this study was to evaluate the effects of freezing diluents supplemented in three potential amines/amino acids, namely, antioxidant cysteamine (2-aminoethanethiol [AET]), ergothioneine (ERG), and serine (SER), in optimization of chicken sperm cryopreservation. The semen of 36 Pradu Hang Dum males, selected based on their motility vigor score, was frozen by a simple freezing method using nitrogen vapors and dimethylformamide (DMF). In a first experiment, a wide range of AET, ERG, and SER doses were tested. Semen quality was evaluated after incubation at 5°C or after cryopreservation in straws in the Blumberger Hahnen Sperma Verdünner (BHSV) diluent + DMF (6% v/v) with or without AET, ERG, or SER. The best targeted doses of AET, ERG, or SER were then selected for experiment 2 that was focused on cryopreserved semen. Frozen-thawed sperm quality was evaluated by different in vitro tests and by evaluation of fertility. Objective motility parameters were evaluated by computer-assisted sperm analysis. Membrane integrity, acrosome integrity, and mitochondria function were evaluated using appropriate dyes and flow cytometry. Lipid peroxide production was assessed by the thiobarbituric acid test (malondialdehyde production). Fertility obtained with frozen-thawed semen supplemented or not in AET, ERG, or SER was evaluated after artificial insemination of laying hens. ERG and AET decreased sperm lipid peroxidation and decreased fertility, even at low doses. The presence of 4 mmol of SER significantly decreased lipid peroxidation, increased the frozen-thawed sperm quality, and increased fertility after sperm cryopreservation (90% vs. control 84%, P < 0.05). In a third experiment, the use of 1 mmol of sucrose (the best result of our previous study) added to 4 mmol of SER-supplemented extender was tested. This addition allowed to the highest levels of fertility (93%). In conclusion, the addition of 4 mmol of SER in semen cryopreservation diluents decreases peroxidation and improves the efficiency of the process.

Effects of cholesterol content on activity of P-glycoproteins and membrane physical state, and consequences for anthelmintic resistance in the nematode Haemonchus contortus.

Eukaryote plasma membranes protect cells from chemical attack. Xenobiotics, taken up through passive diffusion, accumulate in the membranes, where they are captured by transporters, among which P-glycoproteins (Pgps). In nematodes such as Haemonchus contortus, eggshells and cuticles provide additional protective barriers against xenobiotics. Little is known about the role of these structures in the transport of chemical molecules. Pgps, members of the ABC transporter family, are present in eggshells and cuticles. Changes in the activity of these proteins have also been correlated with alterations in lipids, such as cholesterol content, in eggshells. However, the cellular mechanisms underlying these effects remain unclear. We show here that an experimental decrease in the cholesterol content of eggshells of Haemonchus contortus, with Methyl-beta-CycloDextrin (MßCD), results in an increase in membrane fluidity, favouring Pgp activity and leading to an increase in resistance to anthelmintics. This effect is modulated by the initial degree of anthelminthic resistance of the eggs. These results suggest that eggshell fluidity plays a major role in the modulation of Pgp activity. They confirm that Pgp activity is highly influenced by the local microenvironment, in particular sterols, as observed in some vertebrate models. Thus, eggshell barriers could play an active role in the transport of xenobiotics.

TITLE: Effets de la teneur en cholestérol sur l'activité des glycoprotéines P et sur l'état physique de la membrane, et conséquences pour la résistance aux anthelminthiques chez le nématode Haemonchus contortus. ABSTRACT: Les membranes plasmiques des eucaryotes protègent les cellules contre les attaques chimiques. Les xénobiotiques, absorbés par diffusion passive, s'accumulent dans les membranes où ils sont capturés par des transporteurs, parmi lesquels les glycoprotéines P (Pgp). Chez les nématodes, les coques des œufs et les cuticules constituent des barrières de protection supplémentaires contre les xénobiotiques. On en sait peu sur le rôle de ces structures dans le transport des molécules chimiques. Les Pgp, membres de la famille des transporteurs ABC, sont présents dans les coques et les cuticules. Des changements dans l'activité de ces protéines ont également été mis en corrélation avec des altérations des lipides, tels que la teneur en cholestérol, des coques des œufs. Cependant, les mécanismes cellulaires sous-jacents à ces effets restent flous. Nous montrons ici que la diminution expérimentale de la teneur en cholestérol des coques des œufs d'Haemonchus contortus, avec la méthyl-beta-cyclodextrine (MßCD), entraîne une augmentation de la fluidité membranaire favorisant l'activité des Pgp et une augmentation de la résistance aux anthelminthiques. Cet effet est modulé par le degré initial de résistance aux anthelminthiques des œufs. Ces résultats suggèrent que la fluidité de la coque joue un rôle majeur dans la modulation de l'activité des Pgp. Ils confirment que l'activité des Pgp est fortement influencée par le microenvironnement local, en particulier les stérols, comme observé dans certains modèles de vertébrés. Ainsi, les barrières de coques des oeufs pourraient jouer un rôle actif dans le transport des xénobiotiques.

Potential involvement of several signaling pathways in initiation of the chicken acrosome reaction.

Avian sperm biology has demonstrated specific features in preparation for fertilization. For example, capacitationlike processes and motility hyperactivation do not exist in the form described in mammals. The present study investigated the potential involvement of several signaling pathways, including protein kinase A (PKA), phosphatidylinositol 3 kinase (PIK3), mitogen-activated protein kinase 3/1 (MAPK3/1), and MAPK14 in the chicken acrosome reaction (AR). The presence in chicken spermatozoa of key proteins involved in these signaling pathways (i.e., cAMP-responsive element-binding protein [CREB], AKT, MAPK1, and MAPK14 and their respective phosphorylated forms) was detected using immunoblotting and localized by immunocytochemistry, mainly in the heads. The potential involvement of these pathways in the AR induced by inner perivitelline layer (IPVL) and Ca(2+) was then examined using specific inhibitors and phosphorylation status measurements. The effects of the specific inhibitors on motility were also measured. Phosphorylations of AKT, CREB, and MAPK1, but not MAPK14, were increased at the time of AR. Phosphorylation of AKT was increased in the presence of IPVL alone, whereas both IPVL and Ca(2+) were needed to increase CREB and MAPK1 phosphorylations. Inhibition of the three corresponding pathways blocked the increase in phosphorylation and significantly decreased AR. Inhibitions of the PKA and MAPK1 pathways also significantly decreased motility, whereas MAPK14 and PIK3 inhibition had no effect on motility. Our results suggest that the AR could be mediated by activation of the PKA, PIK3, and MAPK1 pathways through a sequential action involving, successively, PIK3 and then PKA and MAPK1 activations.

Identification of germinal disk region derived genes potentially involved in hen fertility.

Despite the regular decrease in fertility observed in hens, especially in "meat" lines, little is known about genes affecting fertility. We used the Affymetrix microarray to search for oocyte genes whose expression would vary in relation to fertility rate in both "laying" and "meat" line hens. We focused on oocyte genes because several of them have been found to be involved in fertility in other species. Based on microarray analysis, 54 and 84 genes were differentially expressed between germinal disc regions (GDR) of F1 maturation stage oocytes from hens exhibiting either high (100%) or low (from 22% to 80%) fertility rate from laying and meat lines respectively. Most of these differentially expressed genes were distributed between "laying" and "meat" lines indicating that mechanisms involved in the decrease in fertility rates in these two cases were independent. Real time RT-PCR performed on the same samples which were used for microarray confirmed in several cases differences in gene expression levels detected by microarray. Moreover the correlations between gene expression levels and fertility rates were evaluated for the 10 most interesting genes at different stages of follicular maturation and early embryo development on individual GDR samples from hens exhibiting different fertility rates. In total, we identified five genes whose expression levels correlated with fertility rate in accordance with findings of microarray analysis and real time RT-PCR: VWC2, CR407412, TAPA, FGL2, and TRAP6. The biological significance of these genes sheds light on potential mechanisms influencing fertility and could provide candidates for fertility markers in the hen.

New insights in the AMPK regulation in chicken spermatozoa: Role of direct AMPK activator and relationship between AMPK and PKA pathways.

Energy balance is an important feature for spermatozoa functions. The 5'-AMP-activated protein kinase (AMPK), a sensor of cell energy, has been implicated as a mediator between spermatozoa functions and energy balance. We recently identified and localized the AMPK protein in chicken spermatozoa and showed that its activation with non-specific activators significantly modified spermatozoa quality. The aim of the present study was to determine more directly the role of AMPK activation induced by the specific activator A-769662 and the interaction between AMPK and another pathway, the protein kinase A (PKA) signaling pathway in bird spermatozoa. The results showed that A-769662 induced at the low dose 50 µM an increase in spermatozoa motility, viability, and acrosome reaction through AMPK activation. Furthermore, phospho-Thr172-AMPK levels were greatly decreased by the PKA inhibitor H89 that also decreased spermatozoa quality. The inhibitory action of H89 was also efficient on A-769662 AMPK phosphorylation. We conclude that AMPK activity in bird spermatozoa is stimulated by low dose of A-769662 with consequent increase in spermatozoa quality, and that AMPK is upstream regulated by PKA pathway in this model.

Semen biotechnology optimization for successful fertilization in Japanese quail (Coturnix japonica).

Among the reproductive biotechnologies needed to improve Japanese quail conservation and valorization, optimized conditions of semen methodologies including sampling, treatment, and artificial insemination are a prerequisite. However, they have been poorly developed due to specific physiological and behavioral features of the species. The aim of the present study was to optimize them, from semen collection/treatment up to artificial insemination procedures. We studied different parameters including semen preparation (individual/pooled, presence of foam, type and pH of extender) and zootechnical parameters (depth of insemination in the female tract, number of sperm inseminated, insemination frequency). We showed that the separation of semen from individual males was required to optimize fertility, as a prerequisite for future semen cryopreservation. The deleterious effect of mixed foam extract addition on the fertility level was demonstrated. These results highlight parameters involved in male copulatory competitions and in sperm post copulation selection. Furthermore, we took into account extender effects and standardized the zootechnical conditions of insemination. The depth of intravaginal insemination (1 cm) was a key factor, but not the number of sperm inseminated (15-60 million sperm/female). Finally, artificial inseminations with optimized conditions led to successful fertility rates (up to 80%) and a duration of the fertile period equivalent to that obtained by natural mating.

Sucrose increases the quality and fertilizing ability of cryopreserved chicken sperms in contrast to raffinose.

Chicken semen conservation is an important tool for programs of genetic diversity management and of endangered breeds' conservation. However, the method still needs to be improved in order to be applied in a wide variety of environments and breeds. Our objective was to compare the effects of 2 external cryoprotectants saccharides (sucrose and raffinose) on the sperm freezability of a Thai local breed, Pradu Hang Dum, in which semen was frozen with a simple freezing method using nitrogen vapors and dimethyl formamide (DMF). Thirty-six males were selected on their motility vigor score for the experiments. In a first experiment, a large range of sucrose and raffinose doses were tested. Semen quality was evaluated after incubation at 5°C or after cryopreservation in straws in the saline Blumberger Hahnen Sperma Verdünner diluent + DMF (6% v/v) with or without sucrose/raffinose. The best targeted doses of sucrose and raffinose were then kept for experiment 2 that was focused on cryopreserved semen. In this experiment, semen quality was measured on frozen-thawed sperm: different objective motility data evaluated by computer-assisted sperm analysis (CASA), membrane integrity, acrosome integrity, mitochondria function evaluated using flow cytometry, lipid peroxide production assessed by the thiobarbituric acid test. Fertility obtained with frozen-thawed semen supplemented or not with sucrose or raffinose was also evaluated after artificial insemination of laying hens. The presence of sucrose at the osmotically inactive dose 1 mmol significantly increased the vigor motility, membrane integrity, acrosome integrity, and mitochondrial functions of frozen-thawed sperm (P < 0.05), and showed the highest levels of fertility after sperm cryopreservation (91% vs. control 86%, P < 0.001). Raffinose showed negative effects on in vitro semen quality from 1 to 100 mmol. Fertility was also negatively (P < 0.001) affected by raffinose (fertility rate 66 to 70%). We thus showed in the present study the high success of a simple chicken sperm cryopreservation method with an external cryoprotectant easily available and cheap, the sucrose, used at an osmotically inactive low concentration.

Chicken semen cryopreservation and use for the restoration of rare genetic resources.

For the past 50 yr, practices for ex situ preservation of endangered breeds have been extended. Semen and primordial germ cells, gonadic tissues have been frozen to create genetic stocks of chicken genetic diversity in cryobanks. Semen cryopreservation stays the preferred method since it is not invasive. Many protocols have been developed to cryopreserve chicken semen, but they give highly variable success rate. The aim of the present study was to standardize and prove the effectiveness of semen long-term storage for the restitution of lost families. We showed that semen straws stored for 18 yr in liquid nitrogen did not lose their fertilizing ability. We demonstrated the usefulness of cryopreserved semen stored in the French National Cryobank for the recovery of families of a subfertile experimental chicken line. In order to highlight the standardization of the cryopreserved method, different cryoprotectant protocols were also tested on a rare breed, freezing/thawing and insemination conditions were controlled. The best results were obtained using glycerol protocol, a sperm dilution of 1:4 (semen:extender). The insemination dose of 200 million sperm/female was as efficient as 400 million of sperm. Altogether, these results demonstrated the effectiveness of chicken semen long-term storage for the restoration of lost genetic resources and highlighted the importance of standardized chicken semen cryopreservation using procedures combining biophysical (cryoprotectants, freezing/thawing conditions) and zootechnical (artificial insemination) features.