Jean-Pierre Brillard

Isolation of Chicken Homolog of the FOXL2 Gene and Comparison of Its Expression Patterns With Those of Aromatase During Ovarian Development

Mutations in the forkhead transcription factor gene FOXL2 are involved in ovarian failure, which occurs in human BPES syndrome. This syndrome presents a sexually dimorphic expression, specific to the ovary in several vertebrates. We cloned the open reading frame of chicken FOXL2 (cFoxL2) and studied cFoxL2 expression in developing gonads and during adulthood to examine the role of FOXL2 in ovarian differentiation and function in birds. The spatial and temporal dynamics of cFoxL2 and aromatase expression were analyzed in parallel by using real‐time quantitative reverse transcriptase‐polymerase chain reaction and immunohistochemistry in attempt to investigate the possible role of cFoxL2 in the regulation of aromatase. The expression patterns of cFoxL2 and aromatase transcripts were highly correlated during the sex‐differentiation period (4.7–12.7 days of incubation). Aromatase and cFoxL2 proteins were colocalized in the medullar part of female gonads on embryonic day 14. Fourteen days after hatching, cFoxL2 protein was mainly detected in granulosa cells of developing follicles. In adult ovary follicular envelopes, apart from granulosa cells, cFoxL2 transcript and protein were detected at lower levels in theca cells where aromatase was present. A high level of cFoxL2 transcription was also observed in maturing and ovulated oocytes. Our results confirm that FoxL2 is an early regulator of ovarian development in birds and may be involved in aromatase transcription regulation. Developmental Dynamics 859–870, 2004.

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.

Comparison of assessment of fowl sperm viability by eosin-nigrosin and dual fluorescence (SYBR-14/PI).

The kinetics of fowl sperm viability/mortality following short-term and long-term in vitro storage were studied using 2 different staining methods: eosin/nigrosin (observed under light microscopy) and SYBR-14/PI (dual fluorescence). Based on data obtained at 0, 30 min and at 2, 4 and 24 h (T0, T30, T2, T4, and T24) after in vitro storage (4 degrees C, agitated) of fresh or frozen-thawed semen, the dual association SYBR-14/PI was more effective than eosin/nigrosin (P < 0.05) staining for the detection of sperm viability/mortality at early stages (30 min) in nonfrozen ejaculates stored above 0 degree C. In cryopreserved preparations, the 2 techniques were comparable for assessing viable spermatozoa immediately after thawing, but higher percentages (P < 0.05) of nonviable spermatozoa were detected by the SYBR-14/PI procedure for up to 4 h of in vitro storage post thawing (4 degrees C, agitated). Finally, comparable results were observed between the 2 techniques 24 h after beginning in vitro storage post thawing. It is concluded that the dual association SYBR-14/PI procedure is more effective (or, at least, more rapid) than eosin/nigrosin staining for the assessment of sperm viability/mortality in both fresh and cryopreserved samples of fowl semen. However, in the latter case, the thawing stage needs to be followed by a period of in vitro storage lasting at least 4 h to allow for easier discrimination between viable and nonviable populations of spermatozoa.

Phospholipid fatty acid composition, vitamin e content and susceptibility to lipid peroxidation of duck spermatozoa

Recent studies on chicken semen have suggested that the lipid and fatty acid composition of spermatozoa may be important determinants of fertility. Phospholipid fatty acid composition, vitamin E content and in vitro susceptibility to lipid peroxidation of duck spermatozoa were investigated using GC-MS and HPLC based methods. The total phospholipid fraction of duck spermatozoa was characterized by high proportions of the n-6 polyunsaturated fatty acids arachidonic (20:4n-6), docosatetraenoic (22:4n-6) and docosapentaenoic (22:5n-6) acids but a substantial proportion of the n-3 fatty acid docosahexaenoic (22:6n-3) acid was also present. Palmitic (16:0) and stearic (18:0) fatty acids were the major saturates in sperm phospholipids. Among the phospholipid classes, phosphatidylserine (PS) had the highest degree of unsaturation due to very high proportions of 22:6n-3, 22:5n-6, 22:4n-6 and 20:4n-6, comprising together more than 75% of total fatty acids in this fraction. Phosphatidylethanolamine (PE) also contained high proportions of these four C(20-22) polyunsaturates, which together formed 60% of total fatty acids in this phospholipid. Spermatozoa and seminal plasma of duck semen were characterized by unexpectedly low content of vitamin E, being more than 4-fold lower than in chicken semen. In duck semen the major proportion of the vitamin E (>70%) was located in the spermatozoa. The very high proportion of 22:6n-3 in PS and PE fractions of duck sperm lipids and the comparatively low levels of vitamin E could predispose semen to lipid peroxidation. Nevertheless the in vitro susceptibilities to Fe2+-stimulated lipid peroxidation of duck and chicken spermatozoa were very similar. The results of the study suggest that increased superoxide dismutase and glutathione peroxidase activity and increased antioxidant activity of seminal plasma may compensate for the low levels of vitamin E to help protect the membranes of duck spermatozoa, which exhibit a high degree of unsaturation from oxidative stress.

Qualitative aspects of sperm stock in males and females from Eupelmus orientalis and Dinarmus basalis (Hymenoptera: Chalcidoidea) as revealed by dual fluorescence

Abstract The quality of a sperm population can be characterized physiologically and its fecundity predicted by its viable : non‐viable sperm ratio. To improve the knowledge of reproductive strategies in two ectoparasitoid hymenopteran species, Eupelmus orientalis Crawford (Hymenoptera: Eupelmidae) and Dinarmus basalis Rondani (Hymenoptera: Pteromalidae), the assessment of sperm viability using the dual fluorescence staining procedure SYBR‐14 : propidium iodide was developed. The aim of the study was to provide a comparative test in vitro applicable to both sexes to study the evolution of sperm quality at various stages of the reproductive processes. The reliability of propidium iodide to detect non‐viable sperm (stained in red) was confirmed in both species on the basis of two stress tests (ethanol and Triton X‐100) but our study also revealed that propidium iodide concentrations must be adequately adjusted for each single species. This experiment also demonstrated the physiological heterogeneity of sperm populations in E. orientalis and D. basalis males and females. In both species, 40% of the sperm in the seminal vesicles was found to be non‐viable. By contrast with E. orientalis, the populations of non‐viable sperm estimated from the seminal vesicles of D. basalis were found to be strongly different from those observed in the spermatheca. From the present results, the population of viable sperm detected in the spermatheca of females from both species proved a reliable predictor of fertilization achieved in ovipositing females.

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); Dulbeccos 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.

Expression of adiponectin, chemerin and visfatin in plasma and different tissues during a laying season in turkeys

BackgroundIn mammals, adipose tissue is able to secrete various hormones called adipokines including adiponectin (ADP), chemerin (Chem) and visfatin (Visf) which are involved in controlling energy metabolism as well as reproductive functions. Visf receptor is still unknown whereas ADP and Chem mainly act through AdipoR1, AdipoR2 and CMKLR1 and GPR1 receptors, respectively. No studies have yet demonstrated the presence of these three adipokines in peripheral tissues, ovarian cells or turkey plasma. Here, we investigated the expression (mRNA and protein) of ADP, Chem, Visf and their receptors in peripheral tissues and ovarian cells (granulosa and theca cells) from hierarchical follicles. Furthermore, we determined the plasma profile of ADP, Visf and Chem at different physiological stages: start, peak and end of the laying period in Meleagris gallopavo turkeys. This data was correlated with the metabolic data (plasma glucose, triglycerides, cholesterol and phospholipids).MethodsTissue and ovarian cells mRNA and protein expression levels were determined by RT-qPCR and immunoblot, respectively. Plasma adipokines were measured by chicken ELISA and immunoblotting.ResultsIn turkeys, Chem is mainly expressed in the liver while ADP and Visf are mainly expressed in the abdominal adipose tissue and pectoral muscles,respectively. As in mammals, AdipoR1 and AdipoR2 expression levels (mRNA and protein) are highly present in muscle and liver, respectively, whereas the mRNA expression of CMKLR1 and GPR1 is ubiquitous. In ovarian cells, ADP, Visf, Chem and their receptors are more highly expressed in theca cells than in granulosa cells excepted for AdipoR1. Furthermore, we found that plasma levels of ADP, Chem and Visf were reduced at the end of the laying period compared to the start of this period. At the plasma levels, the levels of these adipokines are strongly negatively correlated with glucose and only plasma Chem is negatively correlated with cholesterol, triglycerides and phospholipids.ConclusionsIn turkeys, ADP, Visf and Chem and their receptors are expressed in peripheral tissues and ovarian cells. Plasma concentration of ADP, Visf and Chem decrease at the end of laying period and only plasma Chem is negatively correlated with levels of cholesterol, triglycerides and phospholipids levels during the entire laying period.

Sex ratios in mule duck embryos at various stages of incubation

Mule duck hatcheries have long reported varying degrees of unbalance in the sex ratio, with a preponderance of male mules at hatching. The aim of the present study was to assess the distributions of sex ratios at various stages of development in embryos originating from intra- and intergeneric crosses between parental lineages (Muscovy male x Muscovy female, Pekin male x Pekin female, Muscovy male x Pekin female or Mule, and Pekin male x Muscovy female or Hinny). In Experiment I, embryo sexing was performed on Days 1 and 5 of incubation (by multiplex PCR) and at hatching (by vent observation). The sex ratio was not significantly modified during the early stages of embryo development whatever the genetic origin (P>0.05, Days 1 and Day 5) but our results in mule and hinny ducklings confirmed the preponderance of males among normally hatched ducklings originating from the intergeneric lineage (58.9 and 55.4% males in mules and hinnies, respectively; P<0.05 in both cases). Sex ratio (vent sexing) in second grade (cull) ducklings revealed that 68% of these ducklings were females (P<0.05). In Experiment II, the distribution of sex ratio was also performed in mule duck eggs from 6 batches (400,000 eggs/batch) first examined for fertility (candling) on Day 18 of incubation. These results indicate that the percentage of males present in the population of normally hatched ducklings increases when fertility decreases. In addition, this experiment also revealed that 83.7-90.5% of viable male mule embryos develop up to hatching, compared to only 43.0-51.0% of female mule embryos. Given that a deviation in sex ratio during the first stages of incubation is unlikely (Experiment I), it is concluded that the skewed sex ratio of mule ducks at hatching is primarily due to increased late mortality in female mule embryos occurring between egg transfer and hatching. This mortality originated, at least in part, from the intergeneric origin of female mules, and was marked to a greater or lesser extent depending on the initial success of fertilization in a given batch, a possible indication that the initial quality of gametes may selectively exert its influence at the later stages of embryo development.

Duration of fertility and hatchability of the common duck (Anas platyrhynchos) in pure- or crossbreeding with Muscovy drakes (Cairina moschata)

A total of 540 common duck dams were used for a comparison of duration of fertility and hatchability between eggs issued from common dams inseminated with sperm (175 x 10(6) dose(-1)) from either common (pure-breeding or PB) or Muscovy (crossbreeding or CB) drakes. Artificial inseminations (AI) were performed at 3 periods of the reproductive season (27-35, 39-43 and 49-56 weeks) with 2 alternate inseminations/period at 3-week intervals (one with semen from common and the other from Muscovy). Fertility was estimated from egg candling while early embryo mortality (EEM), medium embryo mortality (MEM) and late embryo mortality (LEM) was estimated on Days 0-6 (PB+CB), Days 7-25 (PB) or Day 28 (CB) of incubation, and after, respectively. Overall fertility from Days 2-12 after AI was 61.1% in PB and 42.8% in CB. The maximum duration of fertility (time interval between AI and last fertile egg) was 8.1 days in PB versus 6.4 days in CB (p<0.05). The age of the dam influenced this interval, particularly in PB, with a longer duration at 40 weeks compared to 50 (p<0.05). On average, EEM represented 2.5% of fertile eggs while MEM accounted for 5% of surviving embryos on Day 6 and LEM, for 11.5% of hatched eggs. MEM was significantly higher in CB (6.3%) compared to PB (3.9%; p<0.05). Overall, an increase in EEM and MEM was observed in both types of eggs at and after 50 weeks of age. An increase in EEM (regardless of dams age) and in MEM (only in the oldest females) was observed with sperm storage duration. Sex ratio at hatching (49.2% males in PB vs. 53.0% in CB) was particularly unbalanced on the first fertile day (54.7% and 57.1%, respectively).

Timing associated with oviductal sperm storage and release after artificial insemination in domestic hens

Female birds store sperm in sperm storage tubules (SSTs) in the uterovaginal junction of their reproductive tract for days or weeks (depending on species) before fertilization. Sperm are transported from the SSTs to the infundibulum where fertilization occurs immediately after ovulation of each ovum. The timing of sperm release from the SSTs relative to ovulation is unknown for any bird. Here, we show that, after artificial insemination of domestic fowl Gallus domesticus, sperm are not accepted into any region of the oviduct before sexual maturity. Once hens reach maturity, there is a temporal shift in the distribution of sperm throughout the oviduct. Sperm are first accepted into and accumulate in the SSTs 6 to 8 days before ovulation but are at this point significantly less numerous in the infundibulum. From 1 to 6 days before ovulation, approximately 10-fold more sperm (235 × 10(3) sperm) populate the infundibulum than at 6 to 8 days before ovulation (26 × 10(3) sperm; P < 0.001). Our results suggest that the mechanisms underlying sperm acceptance and release in the oviduct are under fine temporal control, most likely mediated by female hormones.