Glycerol is not suitable for the cryopreservation of quail semen.

1. This study investigated the potential use of glycerol as a cryoprotectant for quail sperm cells. Its role in maintaining sperm fertilising ability in vivo and in vitro quality parameters, such as motility, was assessed.2. The data showed that the presence of glycerol in semen samples was associated with infertility, which suggested that removal prior to insemination is mandatory. Removal through serial dilution centrifugation was associated with fewer than 5% of motile sperm cells and resulted in no fertility.3. In conclusion, glycerol alone is not suitable for quail semen cryopreservation, and other approaches need to be investigated to develop cryobanking programmes for this species.

Avian sperm-borne RNAs: optimisation of a new isolation protocol.

1. Sperm-borne RNAs are involved in sperm and embryonic protein translation, the regulation of early development and the epigenetic inheritance of the paternal phenotype. Sperm-borne RNA purification protocols generally include a cell purification stage to discard contamination by somatic cells. In avian species, no protocol is currently available to isolate all the populations composing sperm-borne RNAs.2. This study evaluated the presence of somatic cells in semen samples of chickens and quails using visual examination after fluorescent nuclei staining. The efficiency of somatic cell lysis buffer (SCLB) on chicken liver cells and its impacts on chicken sperm cell integrity was explored. Three different approaches were tested to isolate RNA: two developed for mammalian sperm cells and a commercial kit for somatic cells. The efficiency and reliability of each approach was determined based on RNA quality and purity. Eventually, the presence of miRNA and mRNA in purified avian sperm-borne RNAs was investigated by RT-(q)PCR.3. No somatic cells were found in chicken and quail semen. The SCLB totally lysed chicken liver cells but also induced sperm cell necrosis. Consequently, this treatment wasn't performed on samples prior to RNA isolation. Among the tested RNA purification protocols, the commercial one was the least variable and isolated RNA with the highest purity levels. No DNA contamination was observed. Furthermore, the samples contained miRNA and mRNA already known as present in mammalian sperm cells (gga-miR-100-5p, gga-miR-191-5p, GAPDH and PLCZ1), but mRNAs associated with leucocytes (CD4) and Sertoli cells (SOX4, CLDN11) were not detected. This protocol was successfully applied to quail sperm cells.4. Altogether, the study reveals that it is unnecessary to pre-treat samples to remove somatic cell contamination before RNA purification and successfully describes an isolation protocol for sperm-borne RNAs, including small non-coding and long coding RNAs, in two distinct avian species highly valuable as biological models.

Freezing chicken semen: Influence of base medium osmolality, cryoprotectants, cryoprotectant concentration, and cooling rate on post-thaw sperm survival.

A classical chicken semen diluent (Lake's 7.1 diluent) was modified to have lowered osmolalities (ranging from 290 to 410 mOsm/kg). The modified medium with physiological osmolality of 325 mOsm/kg allowed cold storage of fresh semen for several days with very little loss of membrane integrity and motility, while high osmolalities inhibited motility. This modified medium was then used as base for freezing medium to test effects of the type and concentration of cryoprotective agent (CPA), and the cooling rate (CR). A number of CPAs (methylformamide, methylacetamide, dimethylformamide (DMF), dimethylacetamide (DMA), diethylformamide, and propylene glycol) were first compared by freezing semen with 0.6 mol/l of the respective CPA at a cooling rate of 250 °C/min. Post-thaw motility and membrane integrity were highest with DMA and DMF. Finally, in more detailed factorial experiments, semen from individual cocks or pooled semen was frozen using CRs of 4, 50, 250, and 440 °C/min and DMA concentrations ([DMA]) of 0.4, 0.6, 1.0, and 1.5 mol/l. Straws from each semen sample x treatment combination were divided for semen assessment at three different research groups for sperm motility, membrane integrity, kinked tails, and DNA fragmentation, using microscopy, computer assisted motility analysis, and flow cytometry. There were clear effects of both CR and [DMA] and their interaction. CRs 50 and 250 °C/min gave best post-thaw sperm performance. Higher DMA concentrations gave better post-thaw membrane integrity, but concentrations above 1.0 mol/l can decrease sperm velocity or even inhibit sperm motility. Therefore [DMA] may best be 0.6-1.0 mol/l at a CR of 50-250 °C/min.

An efficient method of guinea fowl sperm cryopreservation.

France is the only country that practices pedigree selection of guinea fowl for meat production. The increasing risk of line extinction for sanitary or breeding failure reasons makes clear the need for an efficient method of reproductive cell cryopreservation in this species. However, an efficient method of guinea fowl sperm freezing in secured packaging is still lacking. The aim of the present study was to develop such a method. Based on results previously obtained in chickens, different cryoprotectants and freezing/thawing processes were tested and then adapted to guinea fowl. Semen quality was measured by semen viability evaluation and then by fertility measured after intravaginal artificial insemination. The best results (70% fertility with frozen-thawed sperm) were obtained by the use of the permeant cryoprotectant agents dimethyl formamide combined with a freezing rate of 30°C/min. The initial insemination frequency also affected the fertility results: 2 consecutive days of inseminations were needed in the first week to ensure enough filling of the utero-vaginal glands of the guinea fowl hen and thus to get successive fertile eggs. Thereafter, a 2-wk insemination frequency was sufficient. This new method, combining biophysical (cryoprotectant agents, freeze/thaw rate) and zootechnical (artificial insemination frequency) features, is the first cryopreservation method successfully developed in secured packaging for guinea fowl sperm. This method is now available for the practice of gene bank conservation and male reproductive management.

A reappraisal of the factors involved in in vitro initiation of the acrosome reaction in chicken spermatozoa.

Chicken spermatozoa may remain in the female oviduct for a prolonged period before induction of the acrosome reaction on contact with the inner perivitelline layer (IPVL). By contrast, the acrosome reaction may be induced very rapidly in vitro in the presence of IPVL and Ca(2+). In the present study, we examined the extent to which the chicken acrosome reaction can be induced in media of various compositions in the presence or absence of IPVL and/or Ca(2+) and other factors known to be efficient in mammals. We also compared the efficacy of perivitelline layer (PL) taken at various states of oocyte maturation in initiating the reaction. The acrosome reaction was induced in less than 5 min in the presence of Ca(2+) and IPVL. Incubation of spermatozoa in different saline media (Beltsville poultry semen extender (BPSE); Dulbecco's modified eagle medium; NaCl-TES buffer) without IPVL showed a significant induction of acrosome reaction in BPSE supplemented with 5 mM Ca(2+) and in the three media after supplementation with Ca(2+) and Ca(2+) ionophore A23187. By contrast, the acrosome reaction was never induced without Ca(2+). BSA, NaHCO(3), and progesterone did not stimulate the acrosome reaction. Ca(2+) plus PL taken at various physiological states (follicle IPVL, ovulated IPVL, oviposited IPVL, and/or outer perivitelline layer) strongly stimulated the acrosome reaction, the latest states being the most efficient. Although PL induced the acrosome reaction in the presence of extracellular Ca(2+), it was not possible to induce hyperactivation in chicken spermatozoa. Taken together, these results emphasize the central role of Ca(2+) in the in vitro initiation of the acrosome reaction in chickens and show specific features of this induction in birds.

Predictors of success of semen cryopreservation in chickens.

Semen cryopreservation is very important for the ex situ management of genetic diversity in birds but it is rarely used. This is partly because of the highly variable success rates, and this emphasizes the need for predictors of semen freezability. This study evaluated the ability of semen quality tests to predict the success rates of semen cryopreservation in chickens and the relationships between each test. Individual variations of in vitro quality tests of semen were compared to the fertility obtained with fresh and cryopreserved semen. The in vitro semen quality tests represented viability, integrity, motility (percentage of viable and morphologically normal cells (PVN); mass motility (MMOT) and different motion parameters including percentage of motile spermatozoa (PMOT)) and biophysical tests (OSM, resistance to osmotic stress; membrane fluidity (FLUID)). Different in vitro tests were significantly correlated between each other for fresh (MMOT, PVN and FLUID, many criteria of objective motility) and cryopreserved semen (MMOT, different objective motility parameters, PVN). Fertility was significantly correlated with PVN for fresh semen and PVN and different objective motility criteria for cryopreserved semen. Membrane fluidity, followed by PVN, PMOT and MMOT, measured on fresh semen samples was positively correlated with fertility obtained with cryopreserved semen. The combination of the first three tests explained 85% of the variability of fertility observed with cryopreserved semen. In conclusion, we showed that different in vitro tests of semen quality are of predictive value for the success rate of semen cryopreservation in the chicken, the most accurate being membrane fluidity.

Semen cryopreservation for ex situ management of genetic diversity in chicken: creation of the French avian cryobank.

The need for semen preservation in domestic birds is a result of the reduction in genetic variability of domestic bird livestock and of the increasing risk of line extinction for health and safety reasons. Cryopreservation of embryos and primordial germ cells (PGC) is not routinely feasible in birds. The project therefore involved semen frozen in optimal safety and traceable conditions. Whole blood samples were also frozen to provide samples of analyses of genomes and health status. The feasibility of using ex situ conservation, i.e., collecting biological material to be stored outside the usual production area of the species (ex situ genetic stock), to preserve and manage rare breeds was tested with 4 subfertile populations: 3 rare experimental lines used for research into energy metabolism (R+), growth (Y33), and immunity (B4/B4), reared under known health status and the oldest endangered patrimonial French breed, the Gauloise dorée with an unknown health status. A general infrastructure was set up for the health screening and remediation of diseases, collection and storage of frozen cells and 2 sites were created for the storage of frozen samples. The screening and remediation of diseases of the Gauloise dorée, which was contaminated with various Salmonella and Mycoplasma strains, was achieved by successive treatment of parents, incubated eggs and young chicks with Baytril followed by Tiamulin. For each line, 474 to 994 semen straws have been frozen, thawed, and the semen evaluated. Insemination of frozen-thawed semen into females of the same genetic origin or of an egg-type commercial breed produced chicks in every case. For the most subfertile lines, insemination with egg-type females significantly increased the reproductive success. In conclusion, we report on the benefits of a semen and blood cryobanking complex for the management of endangered lines and strains of domestic birds. Current stocks made possible the restoration of more than 96% of the initial genome. This project also provided technical solutions to resolve some of the health problems frequently encountered for gene preservation in poultry.

Data on endogenous chicken sperm peptides and small proteins obtained through Top-Down High Resolution Mass Spectrometry.

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.

Fatty acid composition, glutathione peroxidase and superoxide dismutase activity and total antioxidant activity of avian semen.

This work demonstrates that spermatozoa from five avian species (chicken, turkey, guinea fowl, duck and goose) are all characterised by high proportions of polyunsaturated fatty acids, from 46 (turkey) to 55% (duck) of total. For each of the species, the most abundant fatty acids were arachidonic (20:4n-6) and docosatetraenoic (22:4n-6) acids, representing between 22 (turkey) and 40% (chicken) of total. Significant activities of the major isozymes of superoxide dismutase and glutathione peroxidase, which protect against the peroxidation associated with high degree of fatty acid unsaturation, were found in spermatozoa from all species. The seminal plasma also had these activities and showed additional mechanisms for protecting spermatozoa from peroxidation. In general terms, these lipid and enzyme proteins were similar between the five avian species and different from those reported for mammalian sperm.

Changes in lipid content of fowl spermatozoa after liquid storage at 2 to 5 degrees C.

Quantitative and qualitative changes may occur in the lipids of spermatozoa during in vitro storage of gametes and may indicate possible degradations occurring within the cells under these conditions. The aim of the present study was to investigate such changes. The motility, viability, morphological integrity and lipid content were measured in fowl semen stored for 48 h at 2 to 5 degrees C and diluted 1:1 in Beltsville Poultry Semen Extender (BPSE) under aerobic agitation. The total lipid content of spermatozoa decreased (P < 0.05) from 820 to 620 micrograms/10(9) cells over 48 h. There was no significant evolution in the total lipid content of seminal plasma (1000 to 850 micrograms/10(9) cells). The proportion of phospholipids in spermatozoa decreased from 75 to 60% of the total lipids. Among the phospholipids, the proportions of phosphatidylcholine, phosphatidylethanolamine and sphingomyelin decreased (P < 0.05) from 58, 13 and 10% at 0 h to 42, 10 and 9% at 48 h, respectively. In contrast, lysophosphatidylcholine, which was marginally represented at 0 h (2%), increased considerably (24%) at 48 h. During the same period, the proportion of motile spermatozoa decreased from 87.5 to 46% and the proportion of viable and morphologically normal cells decreased from 84 to 48%. These results indicate the occurrence of lipid lysis, peroxidation and/or endogenous metabolism able to modify the structure of the spermatozoal membranes and to alter their metabolism and fusion abilities.

Effects of vitamin E on fowl semen storage at 4 degrees C.

Vitamin E was assayed for either in chicken spermatozoa or seminal plasma. Effects of vitamin E on the motility and fertilizing ability of chicken semen stored for 24 hours at 4 degrees C were also studied. A mean of 0.25 mug vitamin E 10 (9) cells was found in spermatozoa and 0.074 mug in seminal plasma. When the medium for in vitro storage of semen was supplemented with vitamin E the motility of spermatozoa was not affected. However, vitamin E improved the fertilizing ability of semen stored for 24 hours at 4 degrees C, especially at the dose of 8 mug/ml of semen diluent.

Ability of chicken spermatozoa to undergo acrosome reaction after liquid storage or cryopreservation.

The effects of in vitro storage on the sperm's ability to undergo the acrosome reaction (AR) have never been studied in avian species despite its major importance for reproduction management. The ability of chicken sperm to undergo the AR was measured after liquid storage at 4 °C and after cryopreservation, and its relationship with other semen quality parameters, including viability, mass motility and objective motility parameters measured by computer semen analyser (CASA) was analysed in two different flocks. The percentage of intact acrosome-reacting spermatozoa (IAR) was dramatically decreased by 48 h liquid storage (loss of 2/3 among the spermatozoa initially able to undergo the AR) whereas motility, viability and morphological integrity were reduced by 10-15%. By contrast, cryopreservation did not affect the induction of AR in flock 1 (29% IAR) whereas it was strongly affected in flock 2 (7% IAR). Motility parameters, viability and morphology were considerably altered by freezing in every case (more that 50% loss). Positive correlations were found between the percentage of intact acrosome-reacting spermatozoa and viability, mass motility and many objective motility parameters. Our results showed that the sperm's ability to undergo the AR was much more affected than other sperm functions after storage at 4 °C, while cryopreservation only had an effect in semen with the lowest initial quality. These results raise questions regarding the specific features of chicken sperm biology that must be taken into account in the treatment of semen.

Cryoprotectant and freezing-process alter the ability of chicken sperm to acrosome react.

Sperm surviving after freezing-thawing is usually 40-50% of the initial population. Damage during this process affects both fertilizing ability and its duration in avian species. However, the effect of cryopreservation on the sperm ability to undergo the acrosome reaction, the initial event of fertilization, is still in question in birds. In this paper, the influence of cryoprotectant (glycerol and dimethylacetamide-DMA) and of two different cryopreservation processes (pellets or straws) on the ability of rooster sperm to acrosome react (AR measured with PNA-FITC) was studied. Motility parameters (CASA) and plasma membrane integrity (propidium iodide exclusion) were also measured. The addition of cryoprotectants (CPA) immediately provoked a dramatic decrease in the ability of sperm to undergo the acrosome reaction, glycerol being more harmful than DMA. The cryoprotectant removal was also harmful. The other parts of the freezing process further decreased the ability to AR. Motility was affected to a lesser extent by CPA presence although plasma membrane integrity was not altered. The DMA/rapid freezing procedure was the most harmful for plasma membrane integrity. Taken together, these results show that AR is more dramatically altered by CPA presence than motility and membrane integrity and CPA provokes a more pronounced effect than the freezing-thawing process especially in the case of using glycerol/slow freezing process.

Membrane fluidity and the ability of domestic bird spermatozoa to survive cryopreservation.

The ability to survive cryopreservation varies in spermatozoa from different bird species. Among the biological factors potentially responsible for such differences, species variations in membrane fluidity have a role in the restoration of the physiological state after freezing. Membrane fluidity may be assessed by measuring fluorescence polarization anisotropy with a fluorescent dye. Anistropy values are proportional to membrane rigidity and consequently inversely proportional to membrane fluidity. In the present study, polarization anisotropy of spermatozoa originating from species differing in the freezability of their semen (chicken, turkey and guinea fowl) was measured in addition to lipid composition (cholesterol/phospholipid ratio), sperm viability (membrane permeability to eosine) and morphological integrity before and after cryopreservation. The percentages of viable and normal spermatozoa in fresh sperm were highest in the chicken (87%), lowest in guinea fowl (64%), and intermediate in turkeys (69%). Anisotropy values were highest in guinea fowl (0.205), lowest in chickens (0.155), and intermediate in turkeys (0.180). As a consequence, membrane fluidity was highest in chickens and lowest in guinea fowl. Cryopreservation significantly decreased sperm viability and morphological integrity and increased anisotropy in all species but did not change the inter species hierarchy. Initial cholesterol/phospholipid ratios were lower in chickens than in guinea fowl, and intermediate in turkeys (0.25, 0.26 and 0.29, respectively). Cryopreservation induced a severe decrease in cholesterol/phospholipid ratios in turkeys and guinea fowl. Sperm membrane fluidity in chickens, turkeys and guinea fowl behaves as an indicator of sperm freezability in these species. Inter species differences for this parameter may be partly explained by differences in initial cholesterol/phospholipids content of spermatozoa. On the other hand, the rigidifying process induced by cryopreservation is not related to lipid damage by the same mechanisms.

Effect of dietary fat on the fatty acid composition and fertilizing ability of fowl semen.

Broiler breeder roosters received two diets, containing either 5% salmon oil (SO) or 5% corn oil (CO). The diets differed essentially in their polyunsaturated fatty acid (PUFA) composition, with n-6:n-3 fatty acid ratios of 41.6 in SO and 1.5 in CO. The effects of these diets on the fatty acid composition of spermatozoa and seminal plasma, and on fertility evaluated after artificial insemination were observed. Whatever the diet, the fatty acid composition of spermatozoa showed notable amounts of 20:4n-6 (5-9%) and 22:4n-6 (15-21%). These essential fatty acids were not detected in the diets and were synthesized from 18:2n-6, which was abundant in the diet (15-16%) but low in spermatozoa (2-3%). Spermatozoa were also very rich in saturated fatty acids (39%). There was a clear influence of dietary lipids on the spermatozoa fatty acid profile: the proportion of n-3 fatty acids in spermatozoa from males fed SO compared to CO was higher (9.6% vs. 4.3%) and that of n-6 fatty acids was lower (22.4% vs. 33.3%). The fatty acid composition of seminal plasma included a higher proportion of saturated fatty acids (49%) than the proportion in spermatozoa, whereas minor fatty acids (14:0, 16:1n-7, 16:1n-9, 22:5n-3) were not detected. The influence of dietary lipids on the seminal plasma fatty acid profile was the same as for the spermatozoa, especially in the PUFA profile. In addition, the SO diet gave significantly higher fertility rates (96%) than the CO diet (91.6%). These results clearly show that the lipid composition of the diet may modify the fatty acid composition of the semen and its fertilizing ability.