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.

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.

Proteomic Changes Associated With Sperm Fertilizing Ability in Meat-Type Roosters.

The molecular basis of male fertility remains unclear, especially in chickens, where decades of genetic selection increased male fertility variability as a side effect. As transcription and translation are highly limited in sperm, proteins are key molecules defining their functionality, making proteomic approaches one of the most adequate methods to investigate sperm capacity. In this context, it is interesting to combine complementary proteomic approaches to maximize the identification of proteins related to sperm-fertilizing ability. In the present study, we aimed at identifying proteins related to fertility in meat-type roosters, showing fertility variability. Fertile roosters (fertility rates higher than 70% after artificial insemination) differed from subfertile roosters (fertility rates lower than 40%) in their sperm mass motility. Fertile and subfertile sperm protein contents were compared using two complementary label-free quantitative proteomic methods: Intact Cell MALDI-TOF-Mass Spectrometry and GeLC-MS/MS. Combining the two strategies, 57 proteins were identified as differentially abundant. Most of them were described for the first time as differentially abundant according to fertility in this species. These proteins were involved in various molecular pathways including flagellum integrity and movement, mitochondrial functions, sperm maturation, and storage in female tract as well as oocyte-sperm interaction. Collectively, our data improved our understanding of chicken sperm biology by revealing new actors involved in the complexity of male fertility that depends on multiple cell functions to reach optimal rates. This explains the inability of reductionist in vitro fertility testing in predicting male fertility and suggests that the use of a combination of markers is a promising approach.

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.

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.

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.

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.

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.

Qualitative and quantitative peptidomic and proteomic approaches to phenotyping chicken semen.

Understanding of the avian male gamete biology is essential to improve the conservation of genetic resources and performance in farming. In this study, the chicken semen peptidome/proteome and the molecular phenotype related to sperm quality were investigated. Spermatozoa (SPZ) and corresponding seminal plasma (SP) from 11 males with different fertilizing capacity were analyzed using three quantitative strategies (fluid and intact cells MALDI-MS, SDS-PAGE combined to LC-MS/MS with spectral counting and XIC methods). Individual MALDI profiling in combination with top-down MS allowed to characterize specific profiles per male and to identify 16 biomolecules (e.g.VMO1, AvBD10 and AvBD9 including polymorphism). Qualitative analysis identified 1165 proteins mainly involved in oxidoreduction mechanisms, energy processes, proteolysis and protein localization. Comparative analyses between the most and the least fertile males were performed. The enzymes involved in energy metabolism, respiratory chain or oxido-reduction activity were over-represented in SPZ of the most fertile males. The SP of the most and the least fertile males differed also on many proteins (e.g. ACE, AvBD10 and AvBD9, NEL precursor, acrosin). Thus proteomic is a "phenomic molecular tool" that may help to discriminate avian males on their reproductive capacity. The data have been deposited with ProteomeXchange (identifiers PXD000287 and PXD001254). BIOLOGICAL SIGNIFICANCE: This peptidomic and proteomic study i) characterized for the first time the semen protein composition of the main domestic avian species (Gallus gallus) by analysis of ejaculated spermatozoa and corresponding seminal plasma; ii) established a characteristic molecular phenotype distinguishing semen and males at an individual level; and iii) proposedthe first evidence of biomarkers related to fertility.

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.

Data for chicken semen proteome and label free quantitative analyses displaying sperm quality biomarkers.

Understanding of biology of the avian male gamete is essential to improve the conservation of genetic resources and performances in farming. In this study, the semen proteome of the main domestic avian species (Gallus gallus) and evaluation of the molecular phenotype related to sperm quality were investigated using GeLC-MS/MS approach and label-free quantitative proteomic based on Spectral Counting (SC) and extracted ion chromatograms (XIC) methods. Here we describe in details the peptide/protein inventory of chicken ejaculated spermatozoa (SPZ) and seminal plasma (SP). We also show differential analyses of chicken semen (SPZ and corresponding SP) from 11 males demonstrating different levels of fertilizing capacity and sperm motility. The interpretation and description of these data can be found in a research article published by Labas and colleagues in the Journal of Proteomics in 2014 [1]. This is a new resource for exploring the molecular mechanisms involved in fertilizing capacity and to reveal new sets of fertility biomarkers.

Relationships between sterol/phospholipid composition and xenobiotic transport in nematodes.

Therapeutic failure limits prophylaxis of nematode diseases and has been mainly attributed to mutations in cellular targets of anthelmintics. Besides these specific mechanisms, alterations of drug transport also occur in parasites resistant to anthelmintics and depend on both the presence of membrane pumps such as P-glycoprotein (Pgp) and on the lipid composition of membranes. We recently showed in the nematode Haemonchus contortus, using eggs as a model, that the total cholesterol (TC) concentration alters the transport of lipophilic molecules due to membrane pumps such as P-glycoprotein and the resistance to anthelmintics. The effect of TC may depend on the presence of other lipids interacting with TC. Therefore, we analysed the lipid composition and its relationship with Pgp and resistance to anthelmintics. Better correlations were found between Pgp and free cholesterol (FC) than with TC. We also showed that the relationships between lipid composition and resistance to anthelmintics or Pgp depended on the equilibrium between FC and phospholipids (PLs), mainly PLs known to be present primarily in either the external leaflets of cell membranes or the internal leaflets. The PLs phosphatidylcholine and phosphatidylethanolamine played the most significant role, but phosphatidic acid also influenced drug resistance.