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9 Pàg., 1. Birchen and Blue Leonesa are two endangered chicken breeds mainly raised in Curueño Valley in North Spain. The establishment of a germplasm bank to guarantee the preservation of these breeds is needed. However, cockerels from different breeder flocks can show variance in semen cryoresistance.2. The following work focused on the sperm characterisation and cryopreservation of Birchen and Blue Leonesa cockerels from four different breeders. A total of 30 semen pools were analysed. Besides conventional sperm analysis, including motility by computer-aided sperm analysis (CASA) and DNA fragmentation by TUNEL, the present study tested a double staining method (MitoTrackerTM Green FM/propidium iodide). This gave simultaneous assessment of plasma and acrosomal and mitochondrial membranes, which were previously validated by SYBR-14/PI, CASA, aniline blue and TUNEL.3. No significant differences were found among fresh semen variables between breeds and breeders. For post-thawed variables, significant differences (P, r, This research is part of a project that received funding from the European Union’s Horizon 2020 Research and Innovation Programme under grant agreement Nº 677353 IMAGE. Part of this research was funded by the project [RTA2017-00034-00-00].

International audience; 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. (C) 2019 Elsevier Inc. All rights reserved.

International audience; 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.

International audience; Sperm cryopreservation is an important strategy to conserve animal genetic diversity and transmit superior genetic background, facilitated by a non-invasive sampling and collection of large amounts of sperm. In chickens, various freezing protocols and cryoprotectants (CPAs) have been developed to freeze sperm, but each lab still debates and competes to disclose the most suitable freezing conditions. Many key points, such as semen donor selection, collection tips, diluent compositions, CPA selection, pre-freezing manipulation, semen packaging type, freezing and thawing rates, are all impactful to the efficiency of chicken sperm cryopreservation. Whereas glycerol is believed as the most efficient and the less toxic CPA, it also presents an unwanted contraceptive effect after insemination. Thus, one of the major developed strategies to conquer this issue is removing glycerol before insemination, but current protocols remain not efficient to completely abolish glycerol contraceptive effect. Novel methodologies will bring us a better understanding of the cellular and molecular mechanisms involved in sperm cryobiology to decipher the challenges of chicken sperm cryopreservation.

International audience; Recent reports showed a positive correlation between frozen-thawed rooster sperm DNA integrity and the concentrations of valine in seminal plasma. The present study evaluated the effect of supplementing valine to semen extender for freezing sperm of 2 endangered local Spanish chicken breeds with different sperm cryoresistance: Red Villafranquina (VF) showing low sperm DNA integrity after cryopreservation and Quail Castellana that shows higher DNA integrity. One pool of semen per breed was obtained twice a week for 10 wk (n = 40, 20 per breed). Each pool was divided into 2 fractions. One of these fractions was frozen in presence of valine as additive in the extender (concentration 10 mmol), whereas the other was used as control. The evaluation of the samples before and after freezing-thawing included motility (CASA-Mot system), viability (propidium iodide and SYBR-14), DNA integrity (terminal deoxynucleotidyl transferase dUTP nick end labeling), and fertility rate (percentage of eggs with blastoderm development after artificial insemination). Supplementation of valine increased several motility variables of fresh semen. In VF breed, valine increased percentage of progressive motile sperm (P = 0.025), curvilinear velocity (P = 0.033), straight-line velocity (P 5 0.040), and average path velocity (P = 0.033), whereas progressive motile sperm (P = 0.019), curvilinear velocity (P = 0.006), straight-line velocity (P = 0.003) and average path velocity (P = 0.004) were improved in the Quail Castellana breed. Valine addition increased the DNA integrity of cryopreserved semen (decreased post-thaw DNA fragmentation) in both breeds, with a significant effect (P = 0.002) in VF (36.3% VF-control vs 31%VF-valine). As expected, Quail Castellana cryopreserved sperm control showed higher fertility rate (34.4% 6 12.1) than VF cryopreserved sperm control (16.1% 1 6.2). Supplementing valine to the freezing extender doubled (P 5 0.026) the fertility rate of VF (32.6% 6 12.2) compared with the control (16.1% 1 6.2). In conclusion, supplementation of valine to chicken freezing extenders shows a positive effect on DNA fragmentation and fertilizing ability of frozen-thawed sperm, with a better response in a breed considered as the lowest freezer in our conservatory.

The in vitro sperm quality parameters (motility, M; viability, V; normal morphology, NM; plasma membrane integrity, PMI; mitochondrial function, MF) in Muscovy drakes (Cairina moschata) were evaluated by using microscopy and flow cytometry, the correlation among sperm quality parameters and results of artificial insemination was also assessed in present study. M, V and NM were detected by phase contrast microscopy assisted with eosinnigrosin staining, and PMI and MF were detected by using flow cytometry within appropriate fluorescence staining (SYBR-14/PI and R123/PI, respectively). Fertility (F), early embryonic mortality (EEM) and the survival embryo rate (SER) were assessed after the artificial insemination of Muscovy or Kaiya duck (Anas platyrhynchos) females. Sperm PMI and MF, the parameters detected by flow cytometry were positively correlated with sperm M, V, and NM, those were detected by phase contrast microscopy (P

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.

Semen cryopreservation allows crucial management of animal genetic diversity. However, the freeze-thaw process causes biochemical and physical alterations, impairing sperm energy-dependent functions. Currently, many chemicals are added to the media to enhance frozen-thawed sperm quality during artificial insemination. The aims of this study was to determine the effects of Metformin (Metf) on fresh chicken sperm motility and ability to perform acrosome reaction, and to evaluate Metf’s effects on the functions of cryopreserved sperm. Chicken semen was diluted and incubated at 35°C in media supplemented with or without different doses of 5’-AMP-Activated Protein Kinase (AMPK) activator, Metf (0,5 to 5 mM). We then looked for the concentration improving the most sperm quality to use it in the cryopreservation media used for chicken sperm. Our results show that 1 mM Metf is the concentration giving the best results regarding sperm quality. AMPKα phosphorylation, viability, acrosome reaction ability (AR), and various motility parameters, were negatively affected by the freeze-thaw process, and that Metf partially restored them. Sperm quality improved (mean increased by 23% for motility, by 10% for viability) as well as AMPKα phosphorylation (mean increased by 30%). Moreover, fluorescent intensity levels of phospho-AMPK were also stronger with Metf than in the control. These results show that the presence of Metf in fresh semen has a positive impact on the quality of sperm and helps reducing the gradual decline in sperm motility caused by cryopreservation by partially restoring several essential sperm functions, and thus leads to a better overall quality of cryopreserved sperm.

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.

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 (MβCD), 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.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.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 (MβCD), 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.

International audience; 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 (PICA) signaling pathway in bird spermatozoa. The results showed that A-769662 induced at the low dose 50 mu M an increase in spermatozoa motility, viability, and acrosome reaction through AMPK activation. Furthermore, phospho-Thr172-AMPK levels were greatly decreased by the PICA 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.

The population of red jungle fowl is declining and needs special attention for its conservation with suitable approaches. For ex situ in vitro conservation of Indian red jungle fowl, establishment of semen cryobank is an appropriate option, for which an extender with adequate retrieval capacity for functional spermatozoa is required. Therefore, studies were designed to evaluate a wide range of extenders for cryopreservation of Indian red jungle fowl (Gallus gallus murghi) sperm to achieve maximal post-thawed semen quality and fertility. For this purpose, semen from eight mature cocks were collected, initially evaluated (percent sperm motility, volume and concentration), pooled, assessed for motility, plasma membrane integrity, viability and acrosome integrity, and divided into six aliquots for dilution (1:5; 37 °C) in Beltsville poultry, red fowl extender, Lake, EK, Tselutin poultry and chicken semen extenders. Diluted semen was cooled from 37 °C to 4 °C @ −0.275 °C/min. Glycerol (20%) was added to chilled semen, equilibrated for 10 min, filled in 0.5 mL French straws, kept over LN2 vapours for 10 min and plunged into LN2 and stored at −196 °C. Percentages of motility, plasma membrane integrity, viability and acrosome integrity were higher (P < 0.05) in red fowl extender at 0, 2 and 4 h of incubation post-thaw. After cryopreservation and post-thawing at 37 °C the highest (P < 0.05) recovery rates and absolute livability index was also recorded in red fowl extender that was thus used for further artificial insemination of cooled-diluted (Liquid) and cryopreserved sperm. The no. of fertilized eggs (Liquid, 20.6 ± 0.4; Cryopreserved, 12.6 ± 0.5), percent fertility (86.7 ± 2.2; 57.2 ± 3.9), no. of hatched chicks (18.2 ± 0.8; 10.0 ± 0.3), percent hatch (76.5 ± 2.7; 45.3 ± 2.2) and hatchability of fertilized eggs (88.3 ± 3.4; 79.6 ± 3.4) were higher with sperm respectively freshly cooled-diluted or cryopreserved in red fowl extender. However, the rates obtained with frozen-thawed sperm were already successful for cryo-banking purpose and artificial insemination practice. In conclusion, we show the first fertility success obtained with cryopreserved Indian jungle fowl sperm. In addition, the red fowl extender is superior in maintaining the quality of Indian red jungle fowl cryopreserved sperm compared to Beltsville poultry, Lake, EK, Tselutin poultry and chicken semen extender.

The endogenous peptides and small proteins present in chicken sperm were identified in the context of the characterization of a fertility-diagnostic method based on the use of ICM-MS (Intact Cell Matrix-Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry). The interpretation and description of these data can be found in a research article, “Intact cell MALDI-TOF MS on sperm: a molecular test for male fertility diagnosis” (Soler et al., 2016) [1], and raw data derived from this analysis have been deposited to the ProteomeXchange Consortium via the PRIDE partner repository with the dataset identifier PRIDE: PXD002768. Here, we describe the inventory of all the molecular species identified, along with their biochemical features and functional analysis. This peptide/protein catalogue can be further employed as reference for other studies and reveal that the use of proteomics allows for a global evaluation of sperm cells functions. Keywords: Chicken, Sperm, Top-Down HRMS, Peptidome

The 5'-AMP-activated protein kinase, AMPK, is a key protein kinase in the metabolism of the cell that regulates many metabolic pathways. The involvement of cell metabolism in sperm ability to fertilize is well established, but only a few studies have focused on the role of AMPK in the control of male fertility. This article summarizes the known role of AMPK in this area. AMPK is involved in the regulation of sperm quality by its action on the proliferation of Sertoli cells. AMPK also directly controls the quality of sperm by its involvement in the regulation of motility and acrosome reaction. It is also involved in the management of lipid peroxidation and gametes antioxidant enzymes. Thus, AMPK appears as a key signaling protein for sperm and male fertility control.; Les spermatozoïdes ont besoin de très hauts niveaux d’énergie pour assurer leur mobilité et leur capacité fécondante. Peu d’études ont analysé le rôle de l’AMPK (5’adenosine monophosphate-activated protein kinase), une protéine kinase clé du métabolisme cellulaire, dans la fonctionnalité des gamètes mâles. Cet article présente une synthèse du rôle connu de cette kinase sur cet aspect. L’AMPK est impliquée dans la régulation de la qualité des spermatozoïdes par son action sur la prolifération des cellules somatiques testiculaires. Elle contrôle également directement la qualité des spermatozoïdes par son implication dans la régulation de leur mobilité et de leur capacité à réaliser la réaction acrosomique. Elle participe à la gestion du stress oxydatif en jouant un rôle dans la peroxydation lipidique et le contrôle des enzymes antioxydantes des gamètes. Ainsi, l’AMPK apparaît comme une protéine-clé de signalisation, à la fois pour les cellules somatiques testiculaires et pour les spermatozoïdes, et donc nécessaire à la fertilité masculine.

Intracellular cytoplasmic calcium ([Ca(2+) ]i ) has an important regulatory role in gamete functions. However, the biochemical components involved in Ca(2+) transport are still unknown in birds, an animal class that has lost functional sperm-specific CatSper channels. Here, we provide evidence for the presence and expression of various Ca(2+) channels in chicken sperm, including high voltage-activated channels (L and R types), the store-operated Ca(2+) channel (SOC) component Orai1, the transient receptor potential channel (TRPC1) and inositol-1,4,5-trisphosphate receptors (IP3 R1). L- and R-type channels were mainly localized in the acrosome and the midpiece, and T-type channels were not detected in chicken sperm. Orai1 was found in all compartments, but with a weak, diffuse signal in the flagellum. TRCP1 was mainly localized in the acrosome and the midpiece, but a weak diffuse signal was also observed in the nucleus and the flagellum. IP3 R1 was mainly detected in the nucleus. The L-type channel inhibitor nifedipine, the R-type channel inhibitor SNX-482 and the SOC inhibitors MRS-1845, 2-APB and YM-58483 decreased [Ca(2+) ]i sperm motility and acrosome reaction capability, with the SOC inhibitors inhibiting these functions most efficiently. Furthermore, we showed that Ca(2+) -mediated induction of AMP-activated protein kinase (AMPK) phosphorylation was blocked by SOC inhibition. Our identification of important regulators of Ca(2+) signaling in avian sperm suggests that SOCs play a predominant role in gamete function, whereas T-type channels may not be involved. In addition, Ca(2+) entry via SOCs appears to be the most likely pathway for AMPK activation and energy-requiring sperm functions such as motility and the acrosome reaction.

International audience; Aim: The study elucidates the impact of cryopreservation on lipid peroxidation (LPO), antioxidant potential, DNA integrity, and mitochondrial activity of Indian red jungle fowl sperm. Materials and Methods: Semen from eight mature cocks was pooled and diluted at 37 degrees C using a specific medium for red fowl species. The diluted semen samples were immediately refrigerated at 4 degrees C for 2 hours (0.275 degrees C min(-1)). Glycerol was then added to a final concentration of 20%, and the samples were equilibrated for 10 minutes at 5 degrees C before loading into 0.25 mL French straws. These straws were then frozen by placing them in liquid nitrogen vapor for 10 minutes and plunged into liquid nitrogen. Motility, viability, DNA integrity, antioxidant activity, and LPO were assessed before dilution (fresh semen), after equilibration (processed semen), and post-thawing (frozen semen). Results: Sperm motility, viability, DNA integrity, and mitochondrial activity decreased (p < 0.05) in processed and frozen semen compared with fresh semen. Nevertheless, the concentration of malondialdehyde (MDA) in sperm and seminal plasma was greater (p < 0.05) in frozen-thawed and processed semen compared with fresh semen. Multivariate regression analysis showed a negative impact of MDA concentration in sperm (R-2 = 0.90, Wilk's lambda = 0.003, p < 0.001) and seminal plasma (R-2 = 0.84, Wilk's lambda = 0.02, p < 0.001) on motility, viability, DNA integrity, and mitochondrial activity. Nonetheless, total antioxidant capacity (TAC) had a positive impact on sperm variables (R-2 = 0.82, Wilk's lambda = 0.096, p < 0.001). Conclusions: The decrease in motility, viability, DNA integrity, and mitochondrial activity of Indian red jungle fowl sperm was associated with an increase in LPO during cryopreservation. Furthermore, TAC was reduced during the freeze-thaw process, which was insufficient in protecting the sperm against high reactive oxygen species levels.

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International audience; 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 degrees C or after cryopreservation in straws in the saline Blumberger Hahnen Sperma Verdunner 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.

International audience; Birds have developed an original reproductive process that allows an efficient transmission of the genome of these flying vertebrates in highly changing environments. This original process includes the production by the heterogametic female (ZW) of telolecithal hard-shelled eggs by a most often unique female tract with an ovary containing a strict follicular hierarchy. The presence of sperm storage tubules in the oviduct allows quite long conservation of the sperm produced in high number by the two internal testis of the homogametic male (ZZ). These adaptations have been important for the development of poultry productions.

International audience; The aim of the present work was to examine the influence of access to pasture in an outdoor housing system on rooster sperm quality and response to cryopreservation and to examine the possible correlation between values for sperm quality variables and welfare indicators. Two groups of Black-barred Andaluza and Red-barred Vasca roosters were housed in an outdoor system, with one group given daily access to a grazing area containing plant species that typically grow on uncultivated Mediterranean land. Semen was collected once per week from each group, and the following sperm quality variables were assessed: sperm volume, appearance, concentration, motility, membrane integrity, acrosome integrity, and morphological abnormalities. In addition, two welfare indicators were examined: the heterophil/lymphocyte (H/L) ratio, and the duration of tonic immobility (TI). Ejaculates from the birds with access to pasture had higher percentages of sperm showing progressive motility (P = 0.019), and returned a higher motility index (P = 0.035). Unexpectedly, the H/L ratio was also higher in these birds. Virtually no differences were seen between the treatment groups with respect to sperm quality after freezing-thawing, although the semen of the Red-barred Vasca birds with access to pasture did show a higher percentage of progressive motility (P = 0.023) than the birds of the same breed with no such access. Significant correlations were detected between the H/L ratio and sperm motility (r = 0.420, P = 0.038), the sperm motility index (r = 0.526, P = 0.002), and progressive motility (r = 0.467, P = 0.003). No differences were seen between the treatment groups with respect to the duration of TI. In conclusion, access to pasture improved fresh sperm motility.

The experiment was designed to show the capacity of Indian Red Jungle Fowl ( Gallus gallus murghi) sperm to be cryopreserved. The changes in characteristics of Indian Red Jungle Fowl spermatozoa during different phases of cryopreservation with the glycerol cryoprotectant and straw packaging were recorded. Semen from eight mature cocks were pooled, diluted at 37 °C in a Tselutin poultry extender, cooled to 4 °C in 2 h (0.275 min−1) and equilibrated for 10 min after the addition of 11% glycerol. Cooled semen was filled in 0.5 mL French straws, kept over liquid nitrogen vapour for 10 min and plunged into liquid nitrogen. Frozen semen was thawed at 37 °C for 30 s. Sperm motility, plasma membrane plasticity, livability, and acrosomal integrity were assessed at the stage of pre-dilution (37 °C), post-dilution, cooling, equilibration and freeze-thawing. The experiment was repeated five times. Sperm motility did not change at post-dilution and cooling and averaged 83.3±3.1%. However, motility declined ( P

Lettre de la Cryobanque Nationale; Des avancées sur les techniques de cryopréservation de la semence et sur son utilisation en insémination animale

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. © 2017 Elsevier Inc.

The fertilizing capacity of pure, fresh avian semen may disappear in just half an hour, hindering its successful use in artificial insemination (AI) projects. Longer storage requires the use of infra-physiological temperatures and of semen diluents that help preserve the spermatozoa but that do not interfere with their fertilizing capacity. This study examines the effect on sperm quality of storing red-legged partridge sperm for 3 h at 5?C with 2 different semen extenders 1) a medium referred to as L&R-84, composed of sodium glutamate, glucose, magnesium acetate, potassium acetate, and polyvinylpyrrolidone, and 2) Lake 7.1 medium, composed of sodium glutamate, glucose, magnesium acetate, potassium citrate, and N,N-Bis(2-hydroxyethyl)taurine (BES). Extending with L&R-84 returned better curvilinear velocity (P < 0.01), straight-line velocity (P < 0.01), average path velocity (P < 0.01), linearity (P < 0.05), straightness (P < 0.05), and wobble (P < 0.05) values, while extending with the Lake 7.1 medium was associated with higher percentages (P < 0.001) of motile sperm. The fertility rate was higher (P < 0.05) when birds were inseminated with L&R-84-extended sperm than with Lake 7.1-extended sperm. The mean number of penetrations of perivitelline layer samples (taken from above the germinal disc) was also higher for the L&R-84-extended sperm (P < 0.05). These results show L&R-84 can be recommended as an extender for red-legged partridge semen to be stored for at least 3 h at 5?C. © 2017 Poultry Science Association Inc.

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La synthèse proposée ci-dessus provient d’un exercice visant à produire librement un point de vue collégial sur les recherches avicoles à venir. Elle a été élaborée à partir du travail d’un groupe pluridisciplinaire de chercheurs et de représentants des interprofessions et syndicats avicoles français. Elle est issue de deux réunions de la Commission Spécialisée Avicole de l’INRA (8/06/10 et 1/10/10) auxquelles ont participé: E. Blesbois (INRA PHASE), J. Champagne (ITAVI, CIP, SNAA), B. Coudurier (INRA, APA), J.B. Coulon (INRA PHASE), M. Duclos (INRA PHASE), N. Eterradossi (ANSES), X. Fernandez (INRA PHASE), C. Guérin-Dubiard (UMR Agrocampus-INRA CEPIA), H. Juin (INRA PHASE), E. Le Bihan (INRA GA), A.C. Lalmanach (INRA SA), F. Le Hazif (COOP de France), S. Letue (SYNALAF), P. Magdelaine (ITAVI), G. Martel (INRA SAD), G. Le Pottier (CIDEF), F. Pitel (INRA GA), P. Rault (SYSAAF), J.F. Vautherot (INRA SA), L. Zenner (ENV lyon), E. Zundel (INRA, SA). Des experts extérieurs à la commission ont également été sollicités : B. Dedieu (INRA SAD), A. Torre (INRA SAD).

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 Ca2+ was then examined using specific inhibitors and phosphorylation status measurements. The effects of the specific inhibitors on motility were also m...

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.

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.

Sperm require high levels of energy to ensure motility and acrosome reaction (AR) accomplishment. The AMP-activated protein kinase (AMPK) has been demonstrated to be strongly involved in the control of these properties. We address here the question of the potential role of calcium mobilization on AMPK activation and function in chicken sperm through the Ca(2+)/calmodulin-dependent protein kinase kinases (CaMKKs) mediated pathway. The presence of CaMKKs and their substrates CaMKI and CaMKIV was evaluated by western-blotting and indirect immunofluorescence. Sperm were incubated in presence or absence of extracellular Ca(2+), or of CaMKKs inhibitor (STO-609). Phosphorylations of AMPK, CaMKI, and CaMKIV, as well as sperm functions were evaluated. We demonstrate the presence of both CaMKKs (α and β), CaMKI and CaMKIV in chicken sperm. CaMKKα and CaMKI were localized in the acrosome, the midpiece, and at much lower fluorescence in the flagellum, whereas CaMKKβ was mostly localized in the flagellum and much less in the midpiece and the acrosome. CaMKIV was only present in the flagellum. The presence of extracellular calcium induced an increase in kinases phosphorylation and sperm activity. STO-609 reduced AMPK phosphorylation in the presence of extracellular Ca(2+) but not in its absence. STO-609 did not affect CaMKIV phosphorylation but decreased CaMKI phosphorylation and this inhibition was quicker in the presence of extracellular Ca(2+) than in its absence. STO-609 efficiently inhibited sperm motility and AR, both in the presence and absence of extracellular Ca(2+). Our results show for the first time the presence of CaMKKs (α and β) and one of its substrate, CaMKI in different subcellular compartments in germ cells, as well as the changes in the AMPK regulation pathway, sperm motility and AR related to Ca(2+) entry in sperm through the Ca(2+)/CaM/CaMKKs/CaMKI pathway. The Ca(2+)/CaMKKs/AMPK pathway is activated only under conditions of extracellular Ca(2+) entry in the cells.

International audience; 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.

The general decline in wild Iberian populations of the red-legged partridge (Alectoris rufa) has been accompanied by an increase in game-farm facilities producing hybrids with chukar partridges (Alectoris chukar). Genetic introgression from chukar partridges is thought to modify male red-legged partridge reproductive indicators. The aim of the present study was to determine the effects of such genetic introgression on seasonal reproductive patterns by comparing the sperm and plasma testosterone concentrations of males from pure red-legged and hybrid red-legged/chukar populations. Semen was collected twice monthly over a 12-mo period using a massage technique. Both types of bird showed a clear seasonal pattern of spermatogenic activity. The proportion of males ejaculating sperm was higher (P < 0.05) among the pure red-legged birds. The greatest sperm production was recorded in March to May among the pure birds and April to May among the hybrids. Reproductive activity in both groups decreased in June, to reach a minimum in August to December among the hybrids and in September to December among the pure birds. Spermatogenic activity resumed in January in both groups. The sperm concentration produced by the pure birds was smaller than that of the hybrids (P < 0.001), but the percentage of motile sperm was higher in the pure birds (P < 0.001). The sperm of the hybrids showed greater straight-line velocity (P < 0.05), linearity (P < 0.001), straightness (P < 0.001), sperm wobble (P < 0.05), and beat-cross frequency values (P < 0.001). The length and area of the sperm head were smaller in the pure birds (P < 0.05). The seasonal plasma testosterone concentration pattern followed a trend roughly parallel to the ejaculatory response. The present results suggest that genetic introgression influences the reproductive variables of the red-legged partridge. © 2015 Poultry Science Association Inc.

The Indian Red Jungle Fowl is a wild native gallus subspecies of Southern Asia. Semen has never been studied in this species. In order to better know the male reproductive capacities, experiments were conducted to study the semen characteristics, impact of ejaculate collection frequencies, and timing of collection on sperm quality parameters. Mean sperm concentration 800 million/mL, total sperm per ejaculate (0.015 billion), motility (63.5 %), live/total sperm (92.4 %), intact acrosome (75.5 %), and plasma membrane integrity (89.2 %) were recorded. Percentage of abnormal sperm (head, mid-piece, and tail) was 8.1 % and recovered mainly mid-piece abnormalities. The motile sperm percentage was positively correlated with intact acrosomes (r = 0.34) and plasma membrane integrity (r = 0.41). Total sperm per ejaculate (billion) was maximum at 72 h of collection followed by 24 and 48 h of collection. Daily and weekly sperm production (billion) was found maximum at 24 h of collection compared to 12, 48, and 72 h of collection. Sperm motility was higher at 24, 48, and 72 h of collection compared to 12 h of collection, but the number of live sperm were higher at 12 h of collection compared to 24, 48, and 72 h. Sperm concentration was better in the morning time, while the values for sperm viability and plasma membrane integrity were higher in the semen collected at evening time. In conclusion, the Indian Red Jungle Fowl shows a semen production quantitatively relatively low for the species as compared to domestic chicken and contrasted parameters of quality. The semen production is affected by the frequency of collection with an optimum for a daily collection preferentially held in the evening period. These results may now be used for artificial insemination and conservation program.

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.

Suite à un appel d’offre du Commissariat Général à la Stratégie et à la Prospective du gouvernement français, l’INRA a analysé les possibilités d’évolution des pratiques et des systèmes agricoles français vers des systèmes de production plus durables conciliant performances productives, économiques, environnementales et sociales. Une méthodologie d’aide à la conception et à l’évaluation de systèmes a été développée à cette occasion. Elle s’appuie sur un répertoire de plus de 200 pratiques agricoles élémentaires dont l’impact a été qualifié sur 35 indicateurs de performances, ainsi que sur une table de compatibilité entre pratiques élémentaires. Deux études de cas fondées sur des modalités différentes d’utilisation de cette méthodologie sont présentées : i) la conception pas à pas de système de production par incorporation progressive de pratiques élémentaires à partir d’une pratique d’entrée à effet majeur, pour accroître l’autonomie alimentaire en production bovine laitière en valorisant la prairie ; ii) l’évaluation à postériori des performances de systèmes en transcrivant sous forme de pratiques élémentaires des leviers d’action et marges de progrès identifiés à dire d’expert pour améliorer la compétitivité de la filière poulet de chair par la mise en place de deux systèmes de production très contrastés mais complémentaires. Les tendances d’évolution des performances des systèmes de production ainsi simulés ou reconstitués sont discutées. Dans les situations d’impact ambivalent d’une pratique sur une performance, le recours au descriptif détaillé des pratiques permet le plus souvent de réduire l’incertitude en prenant en compte les situations associées à chaque type d’impact., Following the bidding by the General Commission for Future Strategy of the French government, INRA has analyzed the different evolutions possible in French practices and agriculture systems towards a more sustainable production that manages to combine productive, economic, environmental and social performances. A method to help design and evaluating the systems was thus developed. This method is based on over 200 basic agricultural practices whose impact was qualified on 35 performance indicators as well as on a compatibility index between these elementary practices. Two studies based on different uses of this method are presented: i) the step by step conception of a production system by progressively incorporating elementary practices from one entry practice with major effect, to increase food autonomy in dairy cattle production by valorizing grasslands; ii) evaluating the system performances a posteriori by transcribing the action levers and progress margins identified by an expert panel into elementary practices in order to improve competition of the broiler industry through the development of two very different but complementary production systems. The tendencies of the simulated or rebuilt performance evolution of these production systems are discussed. When situations present an ambivalent impact of a practice on one performance, using the detailed description of these practices often allows reducing the uncertainty by considering the situations associated with each type of impact.

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.

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The changes in lipid composition of spermatozoa and seminal plasma and changes in motility, viability, and morphological integrity of spermatozoa were measured in turkey semen diluted in Beltsville poultry semen extender and stored for 48 h (4°C). The total phospholipid content of spermatozoa decreased during storage, while no quantitative decrease was observed in seminal plasma. More precisely, significant decreases in phosphatidylcholine, and to a lesser extent in sphingomyeline, phosphatidylserine, and phosphatidylinositol were observed in spermatozoa. The fatty acid profile of turkey spermatozoa partly reflected diet composition and had a high level of n-9 polyunsaturated fatty acids. Neither fatty acid profile nor free cholesterol were affected by storage. The lipid composition of seminal plasma was quite different from that observed in spermatozoa and was similar to the high density lipoprotein composition of chicken seminal plasma. In vitro storage did not significantly affect lipid classe...