Perivitelline Layer Research Articles

Genotyping blastoderms of avian eggs.

Undeveloped eggs occur frequently in birds and are often considered infertile, and discarded. However, the majority of undeveloped eggs may in fact have been fertilised and embryos might have died at an early stage. Such eggs contain valuable information, for example about offspring sex and paternity, and level of inbreeding. Obtaining such information may also give insight into the patterns and causes of early embryo mortality. Here we describe a simple technique for removing embryo cells from the blastoderm to obtain DNA to genotype the offspring and unequivocally ascertain fertilisation status, while retaining the overlying perivitelline layer (PVL) for sperm counts over the entire membrane. We tested this method on freshly collected eggs (high-quality material), as well as on eggs from abandoned clutches and unhatched eggs (potentially deteriorated material) of blue tits (Cyanistes caeruleus). We sampled a total of 707 eggs from a wild population of blue tits, extracted DNA from the eggs' blastoderm using a Qiagen kit, and genotyped the samples with 14 polymorphic microsatellite markers, plus one sexing marker. Overall, we successfully genotyped 97% of all eggs. Our study is the most extensive dataset of genotyped undeveloped eggs to date and demonstrates that one can reliably genotype undeveloped fertile eggs as well as retain the PVL for observations of sperm and embryo cells.

Capacitation and Acrosomal Reaction Parameters of Fresh and Cryopreserved Rooster Semen

Background: Semen cryopreservation induces alterations in spermatozoa and changes in the plasma membrane, limiting the acrosome reaction and fertilizing ability. The research aim was to study the sperm capacitation indicators and acrosome reaction capacity in rooster semen, comparing fresh semen and post-thaw semen. Methods: Fifty ejaculates were obtained from five roosters through dorsal-ventral massage. Semen was diluted and preserved fresh or cryopreserved in Beltsville Poultry Semen Extender. In both groups of preserved semen, basic seminal parameters, capacitation and acrosome reaction were assessed. The latter were evaluated both with and without a perivitelline layer as the acrosome reaction inducer, using chlortetracycline staining. Result: The results revealed a higher percentage of sperm progressive motility in fresh semen (85.2±1.5%) compared with thawed semen (44.8±1.7%; P less than 0.05). The percentages were similar when comparing sperm with an acrosome reaction in fresh semen (10.6±1.1%) to sperm incubated without a perivitelline layer (13.2±1.7%; P greater than 0.05). However, when incubated with a perivitelline layer for 40 minutes, fresh semen spermatozoa exhibited a higher percentage of acrosome reaction (59.2±2.1%) compared with thawed semen incubated with a perivitelline layer (40.6±2.3%; P less than 0.05).

Polymyxin B neutralizes endotoxin and improves the quality of chicken semen during liquid storage

Despite its importance in gamete utilization for livestock production, poultry semen cryopreservation in a liquid state, is limited in the poultry industry due to a significant decline in sperm viability and functionality during liquid storage. Lipopolysaccharide (LPS) is released from gram-negative bacteria and impairs sperm function in mammals. Using exogeneous LPS, we show this endotoxin compromises sperm viability and function, including motility and penetrability to the inner peri-vitelline layer (IPVL) during liquid storage at 4 °C. This outcome was supported by LPS quantification showing an extreme increase in the first 24 h of storage. Polymyxin B (PMB) is an LPS neutralizer previously shown to improve fertility in boar semen, thus we explored the effect of PMB on chicken semen quality during liquid storage. Sperm viability and penetrability tests showed that PMB completely abolishes the deleterious effect by LPS. However, co-addition of PMB with penicillin G (PenG), an antibiotic against gram positive bacteria, reduces IPVL-penetrability while improving sperm viability post-storage. Furthermore, artificial insemination trials showed that PMB addition improves semen fertility at the post liquid storage. Our results show that chicken semen quality during liquid storage is impaired by bacterial LPS, but improved by PMB addition due to cancelled endotoxic effects, which offers a new approach for prolonged fertility of poultry semen storage in a liquid state.

Quantitative mRNA and Protein Analyses of ZPB1, ZPB2, and ZPC in Different Genetic Strains of Broiler Breeders

The avian inner perivitelline layer (IPVL), containing the zona pellucida (ZP) family of proteins, surrounds the ovulated ovum. Sperm binding at the germinal disc (GD) region of the IPVL initiates fertilization in avian species, and the amount of sperm binding at the GD reflects female fertility. RT-PCR and quantitative Western blot analyses were used to determine differences in ZP protein mRNA expression and protein concentration between GD and nongerminal disc (NGD) regions among four genetic strains of broiler breeders. Both the mRNA expression and protein concentration of chicken ZPB2 was greater in the GD region, compared to the NGD region, and ZPB2 protein was more abundant in the small prehierarchical follicles. Chicken ZPC mRNA, but not ZPC protein, was greater in the NGD region compared to the GD region, and hepatically expressed ZPB1 protein was more concentrated in the NGD region. Testosterone increased the expression of both ZPC mRNA and protein in cultured granulosa cells from prehierarchical follicles. The current research is the first to investigate the relative amount of ZP proteins between the GD region and NGD region in chicken IPVL. The results provide further evidence that ZPB2 may be critical for avian sperm binding at the GD region of the IPVL.

Quantitative Protein Analysis of ZPB2, ZPB1 and ZPC in the Germinal Disc and a Non-Germinal Disc Region of the Inner Perivitelline Layer in Two Genetic Lines of Turkey Hens That Differ in Fertility.

Simple SummaryAt ovulation, the avian egg is only surrounded by one coat of protein containing zona pellucida (ZP) proteins. These proteins serve as key components for binding sperm and initiating fertilization. In birds, most of the sperm bind around the germinal disc region of the ovulated egg, and the number of sperm that bind to this region is associated with improved fertility. Previous research has reported differences in mRNA expression of these ZP proteins between the germinal disc and non-germinal disc regions and between two lines of turkey hens (E-line, high fertility and F-line, low fertility). Until now, differences in the ZP protein abundance in this layer have not been directly measured. In the current study, the protein abundance of the ZP proteins was evaluated, using quantitative Western analyses, between genetic lines (E-line and F-line) and location on the egg (germinal disc and non-germinal disc). Several differences in protein abundance, and discrepancies with mRNA expression, are reported. These findings are the first to examine quantitative differences in the protein composition of this important protein layer and provide insight into potential protein markers that may be used in the poultry industry for improving fertility in breeding stock.The avian inner perivitelline layer (IPVL), containing the zona pellucida (ZP) family of proteins, surrounds the ovulated ovum. In mammalian species, ZP proteins serve as key component(s) in binding sperm and initiating the acrosome reaction. Sperm binding at the germinal disc (GD) region of the IPVL initiates fertilization in avian species, and the amount of sperm binding at the GD reflects female fertility. The current research determined whether reported differences in mRNA expression in two genetic lines of turkey hens (E, high fertility and F, low fertility) translated to the protein level. ZPB2 in the IPVL is greater in the GD region compared with the nongerminal disc (NGD) region, as indicated by both mRNA and protein expression. However, protein expressions of ZPB1 and ZPC in the IPVL of E- and F-line turkey hens was in contrast to previously reported mRNA expression. The results indicate that the mRNA expression of ZP proteins at their site of synthesis in E- and F-line hens often does not directly correlate with the IPVL abundance of these proteins. The greater protein concentration of ZPB2 in the GD region compared with the NGD regions suggests that this protein may be critical for sperm binding at the GD region.

Multifaceted roles of the egg perivitelline layer in avian reproduction: Functional insights from the proteomes of chicken egg inner and outer sublayers

The avian egg perivitelline layer (PL) is a proteinaceous structure that encloses the egg yolk. It consists of the inner and the outer perivitelline layers (IPL and OPL, respectively) that are assumed to play distinct roles in bird reproduction. To gain insight into their respective function, we analyzed the proteome of IPL and OPL in chicken unfertilized eggs after mechanical separation, using a GeLC-MS/MS strategy. Of the 412 proteins identified, 173 proteins were uniquely recovered in IPL and 98 proteins in OPL, while 141 proteins were identified in both sublayers. Genes coding the most abundant proteins were shown to be expressed either in the liver/ovary (IPL formation) or in the oviduct (OPL formation), but rarely in both. The presence of oviduct-specific proteins (including LYZ, VMO1, AvBD11, PTN, OVAL and LOC10175704) in IPL strongly suggests that they participate in the physical association of IPL to OPL, whose tight attachment was further evidenced by analyses of IPL/OPL interfaces (by scanning electron microscopy). Functional annotation of identified proteins revealed functions associated with fertilization and early development for IPL, while OPL would rather participate in egg defense and embryogenesis. Collectively, our data highlight the complementary functions of IPL and OPL that are major determinants of bird reproductive success. SignificanceThe present study unveils for the first time the individual proteomes of the two sublayers composing the chicken egg perivitelline layer (PL), which allowed to assign their respective putative biological roles in avian reproduction. The combination of proteomics with gene expression and ultrastructural analyses provides insightful data on the structure and biochemistry of the avian PL. The functional annotation of PL proteins highlights the multifaceted biological functions of this structure in reproduction including fertilization, embryonic development, and antimicrobial protection. This work will stimulate further research to validate predicted functions and to compare the physiology and the functional specificities of PL in egg-laying species.

Transcriptome and Proteome Analysis Revealed Key Pathways Regulating Final Stage of Oocyte Maturation of the Turkey (Meleagris gallopavo).

In birds, the zona pellucida (ZP) matrix that surrounds the ovulated oocyte—called the inner perivitelline layer—is involved in sperm–zona interaction and successful fertilization. To identify the important genes and proteins connected with the final step of egg development, next-generation sequencing and two-dimensional electrophoresis, combined with mass spectrometry, were used for the analysis of mature oocytes at the F1 developmental stage. A total of 8161 genes and 228 proteins were annotated. Six subfamilies of genes, with codes ZP, ZP1–4, ZPD, and ZPAX, were identified, with the dominant expression of ZPD. The main expression site for ZP1 was the liver; however, granulosa cells may also participate in local ZP1 secretion. A ubiquitination system was identified in mature oocytes, where ZP1 was found to be the main ubiquitinated protein. Analysis of transcripts classified in estrogen receptor (ESR) signaling indicated the presence of ESR1 and ESR2, as well as a set of estrogen-dependent genes involved in both genomic and nongenomic mechanisms for the regulation of gene expression by estrogen. Oxidative phosphorylation was found to be a possible source of adenosine triphosphate, and the nuclear factor erythroid 2-related factor 2 signaling pathway could be involved in the response against oxidative stress. Oocyte–granulosa cell communication by tight, adherens, and gap junctions seems to be essential for the final step of oocyte maturation.

Sperm numbers on the perivitelline layers of blue tit eggs are repeatable within a clutch, but independent of the occurrence of extra‐pair paternity

In many socially monogamous bird species, females produce offspring sired by males other than their social partner. A large body of research has aimed to elucidate the evolutionary causes and consequences of such extra‐pair paternity, but relatively little is known about the underlying behaviour. The number of sperm on the egg's perivitelline layers (PVL) is related to recent copulation activity and may thus give some insight into the female's mating behaviour. We used a simple technique that allowed us to remove embryonic cells from the blastoderm for DNA extraction whilst keeping the PVL intact for sperm counts. Using 243 eggs from 99 blue tits Cyanistes caeruleus, we show that PVL sperm numbers were repeatable within clutches (r = 0.40 [95% CI: 0.25–0.53]). However, neither overall sperm numbers nor changes in sperm numbers across the laying sequence differed between clutches that contained extra‐pair sired eggs and those that did not. Our results, therefore, provide no evidence that females with and without extra‐pair young differ in their copulation activity.

Functional difference of ATP-generating pathways in rooster sperm (Gallus gallus domesticus)

Adenosine triphosphate (ATP) production via glycolysis and oxidative phosphorylation is essential for the maintenance of flagellar motility in sperm; however, the primary energy production pathways supporting fertilization vary among species. Inconsistency in thought exists regarding which pathways maintain ATP production and sperm motility in poultry. Glycolysis and mitochondrial oxidation contribute to flagellar motion in chicken sperm, but the relative dependence on these pathways for motility and penetrability into the inner perivitelline layer remains unclear. In the present study, there was use of various inhibitors and energy substrates to evaluate the relative contribution of anaerobic glycolysis and mitochondrial oxidation to chicken sperm flagellar motility, ATP production, and penetrating capacity through the perivitelline layer. Although both pathways contributed to these processes to varying extent, glucose was the primary substrate for sperm penetration into the inner perivitelline layer in chickens. Furthermore, results from metabolic stress analyses indicated that there was less perivitelline penetrability in response to pyruvate that was not due to changes in reactive oxygen species or intracellular pH. Overall, results from the present study indicate glycolysis and mitochondrial oxidation pathways have distinct functions in the flagellar motility and penetrability of the perivitelline membrane by rooster sperm. There, therefore, are new insights as a result of findings in the present study into the energy production system of sperm through which there is utilization of extracellular metabolic substrates for maintaining sperm fertilization capacity.

Characterization and biological role of cysteine-rich venom protein belonging to CRISPs from turkey seminal plasma†.

Turkey semen contains cysteine-rich secretory proteins (CRISPs) that belong to the dominant seminal plasma proteins. We aimed to isolate and characterize CRISP from turkey seminal plasma and evaluate its possible involvement in yellow semen syndrome (YSS). YSS, which is well characterized, causes reduced fertility and hatchability. The protein was purified using hydrophobic interaction, gel filtration, and reverse phase chromatography. It then was subjected to identification by mass spectrometry, analysis of physicochemical properties, and specific antibody production. The biological function of the isolated protein was tested and included its effects on sperm motility and migration and sperm-egg interactions. Sperm motility was measured with the CASA system using Hobson Sperm Tracker. The reproductive tract of turkey toms was analyzed for gene expression; immunohistochemistry was used for protein localization in the male reproductive tract, spermatozoa, and inner perivitelline layer. The isolated protein was identified as cysteine-rich venom protein-like isoform X2 (CRVP X2; XP_010706464.1) and contained feature motifs of CRISP family proteins. Turkey CRVP X2 was present in both spermatozoa and seminal plasma. The extensive secretion of CRVP X2 by the epithelial cells of the epididymis and ductus deferens suggests its involvement in post-testicular sperm maturation. The internally localized CRVP X2 in the proximal part of the sperm tail might be responsible for stimulation of sperm motility. CRVP X2 on the sperm head might be involved in several events prior to fusion and may also participate in gamete fusion itself. Although the mechanisms by which CRVP X2 mediates fertilization are still unknown, the involvement of complementary sites cannot be excluded. The disturbance of CRVP X2 expression can serve as an etiologic factor of YSS in the turkey. This study expands the understanding of the detailed mechanism of fertilization in birds by clarifying the specific role of CRVP X2.

Mechanical Separation and Protein Solubilization of the Outer and Inner Perivitelline Sublayers from Hen's Eggs

The perivitelline layer that surrounds the egg yolk plays a fundamental role in fertilization, in egg defense, and in the development of the avian embryo. It is formed by two proteinaceous sublayers that are tightly associated and formed by distinct female reproductive organs. Both structures are assumed to have their own functional specificities, which remain to be defined. To characterize the function of proteins composing each sublayer, the first challenge is to establish the conditions that would allow for the mechanical separation of these two intricate layers, while limiting any structural damage. The second step is to optimize the experimental conditions to facilitate protein solubilization from these two sublayers, for subsequent biochemical analyses. The efficiency of this approach is assessed by analyzing the protein profile of each sublayer by Sodium Dodecyl Sulfate-Poly-Acrylamide Gel Electrophoresis (SDS-PAGE), which is expected to be distinct between the two structures. This two-step procedure remains simple; it requires classical biochemical equipment and reagents; and is compatible with further in-depth proteomics. It may also be transposed to other avian eggs for comparative biology, knowing that the structure and the composition of the perivitelline layer has been shown to have species-specific features. In addition, the non-denaturing conditions developed for sublayers separation (step 1) allow their structural analyses by scanning and transmission electron microscopy. It may also constitute the initial step for subsequent protein purification to analyze their respective biological activities and 3D structure, or to perform further immunohistochemical or functional analyses. Such studies would help to decipher the physiological function of these two sublayers, whose structural and functional integrities are determinant criteria of the reproductive success.

Mechanical Separation and Protein Solubilization of the Outer and Inner Perivitelline Sublayers from Hen's Eggs.

The perivitelline layer that surrounds the egg yolk plays a fundamental role in fertilization, in egg defense, and in the development of the avian embryo. It is formed by two proteinaceous sublayers that are tightly associated and formed by distinct female reproductive organs. Both structures are assumed to have their own functional specificities, which remain to be defined. To characterize the function of proteins composing each sublayer, the first challenge is to establish the conditions that would allow for the mechanical separation of these two intricate layers, while limiting any structural damage. The second step is to optimize the experimental conditions to facilitate protein solubilization from these two sublayers, for subsequent biochemical analyses. The efficiency of this approach is assessed by analyzing the protein profile of each sublayer by Sodium Dodecyl Sulfate-Poly-Acrylamide Gel Electrophoresis (SDS-PAGE), which is expected to be distinct between the two structures. This two-step procedure remains simple; it requires classical biochemical equipment and reagents; and is compatible with further in-depth proteomics. It may also be transposed to other avian eggs for comparative biology, knowing that the structure and the composition of the perivitelline layer has been shown to have species-specific features. In addition, the non-denaturing conditions developed for sublayers separation (step 1) allow their structural analyses by scanning and transmission electron microscopy. It may also constitute the initial step for subsequent protein purification to analyze their respective biological activities and 3D structure, or to perform further immunohistochemical or functional analyses. Such studies would help to decipher the physiological function of these two sublayers, whose structural and functional integrities are determinant criteria of the reproductive success.

In vitro sperm storage with poultry oviductal secretions.

In the hen oviduct, tubules have been identified that preserve the sperm, maintaining viability for up to 15 weeks. This study aimed to evaluate the physiological status of rooster sperm when preserved in vitro with uterus vaginal junction secretions (UVJS). Males and females of the Rhode Island breed were used. Sperm aliquots were prepared using Lake extender and Lake extender with UVJS (10.00%, 30.00%, 60.00%, and 90.00%). Subsequently, a basic sperm evaluation was performed and sperm physiological status was determined through the presence and distribution of Ca2+ and its acrosomal reaction capability via perivitelline layer (PVL) co-incubation. It was observed that motility was decreased in sperm preserved with UVJS at 6 and 24 hr) compared to 40 min and fresh semen. The sperm decapacitation percentage was increased when preserved with UVJS at 40 min, 6 and 24 hr compared to fresh semen. The acrosomal reaction was increased in sperm co-incubated with PVL, even when preserved with UVJS. It was concluded that UVJS induced physiological changes in sperm by inducing a decapacitation process, which increased sperm viability when preserved in vitro.

Reproductive coordination breeds success: the importance of the partnership in avian sperm biology

Previous experience with a partner can improve reproductive coordination between a pair and increase offspring production. We paired inexperienced zebra finches and investigated how a pairs’ experience and their reproductive success together (i.e., whether they were successful or unsuccessful at rearing chicks) related to the number of sperm reaching the ovum, sperm motile performance, and hatching success. In contrast to unsuccessful pairs, successful pairs increased their relative hatching rates over sequential breeding attempts, with pairs hatching 100% of eggs after successfully fledging their previous clutch. Across the study, hatching failure was primarily due to early embryo death. Further, the number of sperm reaching the perivitelline layer (PVL) significantly decreased after fledging chicks in successful pairs, and overall, less sperm was found on the PVL in successful pairs compared with unsuccessful pairs. Across breeding attempts, males in successful pairs also exhibited a significant decline in sperm swimming speed, whereas it increased over breeding attempts in unsuccessful pairs. Our results support the idea of an optimal level of supernumerary sperm on the avian egg. However, our data suggest that there are likely to be interactions between the quality of a partnership and male sperm traits that may contribute to fitness in socially monogamous birds and that have been largely neglected to date. Significance statement Breeding experience with a partner can improve physiological and behavioural coordination and the reproductive success of a pair. In the zebra finch, we assessed whether experience and reproductive success altered sperm swimming speed, the number of sperm reaching the ovum, and the number of chicks hatching. These three measures are potentially correlated, given that birds require multiple sperm to fuse with the ovum for fertilization and development to occur, and the idea that sperm swimming speed impacts a sperm’s success at fertilizing ova. However, too many or too few sperm can lead to hatching failure, so there will be an optimal number of sperm to ensure fertilization and healthy development. We found that only pairs that successfully reared chicks increased hatching success in subsequent clutches and decreased the amount of sperm reaching the ovum. Additionally, with more experience, the swimming speed of the top 10% of their sperm decreased. These findings are important because they link partnership quality to sperm biology.

Cryopreservation of turkey spermatozoa without permeant cryoprotectants

In avian species, cryopreservation of semen is necessary for developing sperm cryobanks. It is very difficult, however to cryopreserve turkey sperm and have sperm be viable after thawing. Glycerol, the commonly used sperm cryoprotectant in many species, is toxic to sperm of avian species. The aim of this study was to evaluate whether the non-permeating dextran was effective for the cryopreservation and maintenance of turkey spermatozoa viability after thawing, avoiding the use of permeating cryoprotectants. Turkey sperm were diluted with a medium supplemented with 11% glycerol or dextran with a 1,000 molecular weight (MW), dextran with a 10,000 MW, or dextran with a 20,000 MW each at a 2%, 5%, or 10% concentration. Sperm kinetic characteristics, membrane and acrosome integrity (AI), and the capacity of spermatozoa to interact with the autologous perivitelline layer were evaluated after equilibration and cryopreservation. Results indicate that with use of glycerol and the 1,000 MW dextran there was lesser sperm viability after both equilibration and cryopreservation, compared with use of the 10,000 or 20,000 MW dextran compounds. There was a greater cryoprotective effect with the 10,000 and 20,000 MW dextran compounds at the 10% concentration with spermatozoa maintaining a greater functionality and capacity to interact with the autologous perivitelline layer. In conclusion, the results of this study indicate turkey spermatozoa could be effectively cryopreserved in extender without the use of glycerol as a penetrating cryoprotectant but with the use of the 10,000 or 20,000 MW dextran compounds at a 10% extender concentration.

Oocyte-sperm interactions in the fertilization of birds

A thorough understanding of the mechanisms leading to the interaction between the sperm and the ovum in the process of fertilization in birds can facilitate more effective programming and control of the reproduction of these animals in breeding farms. In addition, it may allow the introduction of extracorporeal fertilization techniques, which may be important in the creation of transgenic animals and the reproduction of endangered species. In birds, the process of fertilization is not well known. It is conditioned by a series of interactions between mature reproductive cells. Oocytes are formed in the ovarian follicles of the left ovary. After ovulation, an ovum in the metaphase of the second meiotic division enters the oviduct along with the inner perivitelline layer (IPVL). It gets fertilized in this infundibulum. Male gametes are formed in paired testes located in the abdominal cavity. Sperm cells in the female reproductive tract do not require capacitation and are already fully capable of fertilization. As a result of internal insemination, male reproductive cells enter the oviduct. In this organ, they are selected and stored in the primary and secondary sperm storage tubules of the mucous membrane. They are released in batches shortly before ovulation. After reaching the oocyte, the sperm binds to the IPVL. This induces an acrosomal reaction that allows the male reproductive cells to penetrate to the surface of the oocyte, especially at the germinal pole. Next, as a result of physiological polyspermy, many sperm cells reach the ooplasm where they form haploid male pronucleus. This phenomenon is necessary to activate an polylecithal egg and produce a haploid female pronucleus. In the final stage, the female pronucleus merges with the single male pronucleus, which leads to the formation of a diploid zygote. The excess male pronuclei present in ooplasm are broken down by endonucleases (DNases). Understanding the mechanisms leading to the interaction between sperm and oocyte in birds may allow for more accurate programming and breeding of these animals in poultry farms and the introduction of extracorporeal fertilization techniques. In addition, it could be useful for the reproduction of endangered bird species

Differential sperm-egg interactions in experimental pairings between two subspecies and their hybrids in a passerine bird.

Speciation research has largely overlooked reproductive barriers acting between copulation and the formation of the zygote (i.e., postmating, prezygotic [PMPZ] barriers), especially in internally fertilizing vertebrates. Nonetheless, it is becoming clear that PMPZ reproductive barriers can play a role in the formation and maintenance of species boundaries. We investigated sperm‐egg interactions in the recently diverged subspecies pairs of the long‐tailed finch, Poephila acuticauda acuticauda and P. a. hecki, to explore potential PMPZ barriers. Specifically, we compared the number of sperm reaching the perivitelline layer (PVL) of the ova, and hence the site of fertilization, in both intra‐ and inter‐subspecies pairings and pairings of F1 hybrid adults with one parental subspecies. Although we found no difference in PVL sperm number among intra‐ and inter‐subspecific pairs, a significantly lower number of sperm reached the site of fertilization in a backcross pairing. As low numbers of PVL sperm appear to be associated with low fertilization success in birds, our findings offer insight into the potential role of postcopulatory processes in limiting gene flow between the subspecies and may help explain the relatively narrow hybrid zone that exists in the wild in this species. Though further work is needed to gain a comprehensive understanding of the morphological, physiological, and molecular mechanisms underlying our results, our study supports the role of PMPZ reproductive barriers in avian speciation, even in recently diverged taxa, that may not yet be fully genetically incompatible.

Comparison of the Efficacy of Hydrogen Peroxide and Salt for Control of Fungal Infections on Brown Trout (Salmo trutta) Eggs

Background: Fungal infections can cause serious problems infecting fish eggs, especially unfertilized or dead eggs. In the past, this problem was solved by using very effective chemicals such as malachite green and formalin. But, due to its toxicity and carcinogenicity, malachite green was banned for use in fish intended for human consumption. Formalin also has been banned in most countries. Chemicals and drugs recommended for use to treat fungal infections are hydrogen peroxide, salt, potassium permanganate etc. The goal of the present study was to determine and compare the efficacy of antifungal effects of hydrogen peroxide and sodium chloride on brown trout eggs.Materials, Methods & Results: The experiment was conducted in the brown trout hatchery, Šipovo, Bosnia and Herzegovina. The experimental groups contained 500 and 1000 mg/L of hydrogen peroxide with 15 and 30 min of exposition; 1 and 2.5% of sodium chloride with 15 and 30 min of exposition and a negative control group (no chemical treatment). The treatment concentrations were calculated and prepared from hydrogen peroxide of 35% active ingredient, and sodium chloride (sterilized) of 100% active ingredient. Eggs for the study were spawned from 11 females and 4 males. The first treatment was performed on the fourth day, and each next treatment was performed at 3-day intervals. Six treatments were administered until the 19th day after the fertilization. The treatment of the eggs was provided until the eggs reached the eyed stage. The effectiveness of the chemical treatments was measured by a hatch rate. There was a significant difference between all treated groups and the negative control group (P < 0.05). Hydrogen peroxide with a concentration of 500 mg/L for 30 min was the most effective and demonstrated a higher hatching rate (75.7%). Sodium chloride treatments resulted in statistically significantly lower hatching rates than hydrogen peroxide treatments. The hatching rate in salt treatment with a concentration of 2.5% for 30 min was 27.3% lower than in hydrogen peroxide treatment with a concentration of 500 mg/L for 30 min.Discussion: Hydrogen peroxide is an effective antifungal, antibacterial and antiviral compound, and according to the Food and Drug Administration (FDA), hydrogen peroxide and salt are approved and classified as a low regulatory priority for the control of oomycetes on all species and life stages of fish. It is considered to be a very environmentally compatible chemical because it does not produce any toxic bioproducts when it decomposes. Hydrogen peroxide stood out as the best candidate substance for fungal control. The fact that the treatment with hydrogen peroxide at a concentration of 500 mg/L for 30 min was more effective than treatments at a concentration of 1000 mg/L can be explained by temperature dependency and treatment frequency of this chemical. Salt was not such an effective fungicide as hydrogen peroxide. When using salt, toxicity to the eggs should also be considered. Salt solutions may cause egg deaths at levels of 2.5% or higher. It is possible that high salinities have an inhibitory effect on the movement of fish embryo due to the high osmotic impact on the perivitelline layer. Thus, hydrogen peroxide has proven to be efficient, inexpensive, easy to use and environmentally safe in preventing fungal infections on brown trout eggs.

D-Aspartate amends reproductive performance of aged roosters by changing gene expression and testicular histology.

Male broiler breeders (n=32) of 55 weeks of age were administered four different doses of capsulated d-aspartate (DA; 0, 100, 200 or 300mgkg-1day-1, p.o. (DA0, DA100, DA200 and DA300 respectively)) for 12 successive weeks to assess reproductive performance, blood testosterone, testicular histology and transcript levels of steroidogenic acute regulatory protein (StAR), cholesterol side-chain cleavage enzyme (P450scc), androgen receptor (AR), LH receptor (LHR), 3β-hydroxysteroid dehydrogenase (3BHSD), proliferating cell nuclear antigen (PCNA), glutamate ionotropic receptor NMDA type subunit 1 (GRIN1) and glutamate ionotropic receptor NMDA type subunit 2B (GRIN2B). Blood samples and ejaculates were collected, and bodyweight was recorded weekly for 10 weeks. AI was performed weekly for the last 2 weeks to determine the number of sperm penetration holes in the perivitelline layer, fertility and hatchability. Testes histology and transcript levels were evaluated in the 12th week. Bodyweight, numbers of Leydig cells and blood vessels, testis index and levels of sperm abnormalities were not affected (P>0.05) by the treatment. However, sperm total and forward motility, plasma membrane integrity and functionality of sperm, ejaculate volume, testosterone concentration and fertility were higher (P<0.05) in both the DA200 and DA300 groups compared with the other groups. In the DA100 and DA200 groups, sperm concentration, number of spermatogonia, thickness of the seminiferous epithelium and the diameter of tubules were significantly higher (P<0.05) than the other DA-treated groups. The number of penetration holes, hatchability and malondialdehyde concentration were higher in the DA200, all DA-treated and DA300 groups respectively compared with the control and other treatment groups. Except for P450scc, AR, LHR and PCNA transcript levels in the DA300 groups, the relative expression of the genes evaluated improved significantly in the other DA-treated groups. Based on these experimental findings, it is concluded that DA improves reproductive performance of aged roosters.

Characterization of the functions and proteomes associated with membrane rafts in chicken sperm.

Cellular membranes are heterogeneous, and this has a great impact on cellular function. Despite the central role of membrane functions in multiple cellular processes in sperm, their molecular mechanisms are poorly understood. Membrane rafts are specific membrane domains enriched in cholesterol, ganglioside GM1, and functional proteins, and they are involved in the regulation of a variety of cellular functions. Studies of the functional characterization of membrane rafts in mammalian sperm have demonstrated roles in sperm-egg binding and the acrosomal reaction. Recently, our biochemical and cell biological studies showed that membrane rafts are present and might play functional roles in chicken sperm. In this study, we isolated membrane rafts from chicken sperm as a detergent-resistant membranes (DRM) floating on a density gradient in the presence of 1% Triton X-100, and characterized the function and proteomes associated with these domains. Biochemical comparison of the DRM between fresh and cryopreserved sperm demonstrated that cryopreservation induces cholesterol loss specifically from membrane rafts, indicating the functional connection with reduced post-thaw fertility in chicken sperm. Furthermore, using an avidin-biotin system, we found that sperm DRM is highly enriched in a 60 KDa single protein able to bind to the inner perivitelline layer. To identify possible roles of membrane rafts, quantitative proteomics, combined with a stable isotope dimethyl labeling approach, identified 82 proteins exclusively or relatively more associated with membrane rafts. Our results demonstrate the functional distinctions between membrane domains and provide compelling evidence that membrane rafts are involved in various cellular pathways inherent to chicken sperm.