Fertility Of Birds Research Articles

New insights into sperm rheotaxis, agglutination and bundle formation in Sharkasi chickens based on an in vitro study

Fertility in birds is dependent on their ability to store adequate populations of viable sperm for extended durations in sperm storage tubules (SSTs). The exact mechanisms by which sperm enter, reside, and egress from the SSTs are still controversial. Sharkasi chicken sperm showed a high tendency to agglutinate, forming motile thread-like bundles comprising many cells. Since it is difficult to observe sperm motility and behavior inside the opaque oviduct, we employed a microfluidic device with a microchannel cross-section resembling close to that of sperm glands allowing for the study of sperm agglutination and motility behavior. This study discusses how sperm bundles are formed, how they move, and what role they may have in extending sperm residency inside the SSTs. We investigated sperm velocity and rheotaxis behavior when a fluid flow was generated inside a microfluidic channel by hydrostatic pressure (flow velocity = 33 µm/s). Spermatozoa tended to swim against the flow (positive rheotaxis) and sperm bundles had significantly lower velocity compared to lonesome sperm. Sperm bundles were observed to swim in a spiral-like motion and to grow in length and thickness as more lonesome sperm are recruited. Sperm bundles were observed approaching and adhering to the sidewalls of the microfluidic channels to avoid being swept with fluid flow velocity > 33 µm/s. Scanning and transmission electron microscopy revealed that sperm bundles were supported by a copious dense substance. The findings show the distinct motility of Sharkasi chicken sperm, as well as sperm's capacity to agglutinate and form motile bundles, which provides a better understanding of long-term sperm storage in the SSTs.

Physiological factors influencing female fertility in birds.

Fertility is fundamental to reproductive success, but not all copulation attempts result in a fertilized embryo. Fertilization failure is especially costly for females, but we still lack a clear understanding of the causes of variation in female fertility across taxa. Birds make a useful model system for fertility research, partly because their large eggs are easily studied outside of the female's body, but also because of the wealth of data available on the reproductive productivity of commercial birds. Here, we review the factors contributing to female infertility in birds, providing evidence that female fertility traits are understudied relative to male fertility traits, and that avian fertility research has been dominated by studies focused on Galliformes and captive (relative to wild) populations. We then discuss the key stages of the female reproductive cycle where fertility may be compromised, and make recommendations for future research. We particularly emphasize that studies must differentiate between infertility and embryo mortality as causes of hatching failure, and that non-breeding individuals should be monitored more routinely where possible. This review lays the groundwork for developing a clearer understanding of the causes of female infertility, with important consequences for multiple fields including reproductive science, conservation and commercial breeding.

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.

Ратич Іриней Борисович — доктор сільськогосподарських наук, член-кореспондент НААН

Irynei Ratych is a doctor of agricultural sciences, associate member of NAAS. The main attention was paid to the study of artificial sources of ultraviolet radiation radiation impact on protein metabolism processes in skin and blood of young cattle. One of the stages of research work dealt with studies of metabolism processes and fertility in birds in connection with protein, amino acidic and mineral nutrition of different age, breed and fertility groups of birds. When researching metabolism of mineral sulfur in bird organism, he for the first time conducted a complex study of biochemical mechanisms of exogenous suplhate impact on bird organism with regard for the amount of protein, sulfur-content amino acids and total sulfur in diet by methods of radiology, biochemistry, physiology and nutrition. Theoretical foundations and practical recommendations have been developed on improving efficiency of sodium sulphate use in feed of birds of different age, type and productivity direction.

The seminal acrosin-inhibitor ClTI1/SPINK2 is a fertility-associated marker in the chicken.

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.

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

Experimental heatwaves negatively impact sperm quality in the zebra finch.

For sexually reproducing species, functionally competent sperm are critical to reproduction. While high atmospheric temperatures are known to influence the timing of breeding, incubation and reproductive success in birds, the effect of temperature on sperm quality remains largely unexplored. Here, we experimentally investigated the impact of ecologically relevant extreme temperatures on cloacal temperature and sperm morphology and motility in zebra finches Taeniopygia guttata We periodically sampled males exposed to 30°C or 40°C temperatures daily for 14 consecutive days. Following a 12-day (23°C) recovery period, birds were again exposed to heat, but under the alternate treatment (e.g. birds initially exposed to 40°C were exposed to 30°C). Elevated temperatures led to an increase in cloacal temperature and a reduction in the proportion of sperm with normal morphology; these effects were most notable under 40°C conditions, and were influenced by the duration of heat exposure and prior exposure to high temperature. Our findings highlight the potential role of temperature in determining male fertility in birds, and perhaps also in constraining the timing of avian breeding. Given the increased frequency of heatwaves in a warming world, our results suggest the need for further work on climatic influences on sperm quality and male fertility.

Sperm-Egg Interaction during Fertilization in Birds.

Fertilization in animals that employ sexual reproduction is an indispensable event for the production of the next generation. A significant advancement in our understanding of the molecular mechanisms of sperm-egg interaction in mammalian species was achieved in the last few decades. However, the same level of knowledge has not been accumulated for birds because of egg size and the difficulty in mimicking the physiological polyspermy that takes place during normal fertilization. In this review, we summarize the current understanding of sperm-egg interaction mechanism during fertilization in birds, especially focusing on sperm-egg binding, sperm acrosome reaction and the authentic sperm protease required for the hole formation on the perivitelline membrane. We explain that the zona pellucida proteins (ZP1 and ZP3) in the perivitelline membrane play important roles in sperm-egg binding, induction of the acrosome reaction as well as sperm penetration by digestion of sperm protease. We anticipate that a deeper understanding of avian fertilization will open up new avenues to create powerful tools for a myriad of applications in the poultry industries including the production of transgenic and cloned birds.

Optimisation of total RNA isolation method from bird sperm

The aim of the current study was to determine the optimal RNA extraction in sperm, to support further molecular applications determining the biological roles of sperm in chicken reproduction. Generally, the major issue with RNA isolation is low quantity of RNA is sperm cells. The recent research done on sperm indicates that RNA isolation protocols are specific to the species. RNA extractions from chicken sperm samples (n=47) were respectively performed using TRIzol ® method in combination with PureLink RNA Mini Kit (TRIzol ® Plus RNA Purification System, Invitrogen) and column-based RNA isolation method (PureLink TM RNA Mini Kit, Invitrogen). Both methods were used according to the manufacturer’s protocol but with small modifications. DNase treatment (PureLink ® DNase Set, Invitrogen) was performed in order to eliminate contaminating genomic DNA from samples. To test the quantity and quality the isolated total RNA was checked by the NanoDrop (Thermo Scientific, Wilmington, DE). In general, higher yields were obtained by pure column based isolation when compared to combined TRIzol ® and column based approach. The mean of the yields of total RNA samples ranged from 36.93 ± 2.8 ng (TRIzol ® ) to 381.77 ± 191.3 ng (PureLink TM ). All RNA samples had spectrophotometry values between 1.40 – 1.45 (TRIzol ® ); 1.78 – 1.90 (PureLink TM ) for absorbance ratios A260/A280, and between 0.52 – 1.21 (TRIzol ® ); 1.38 – 2.23 (PureLink TM ) for ratios A260/A230. These results demonstrate that the most suitable protocol of total RNA extraction from bird sperm is column-based RNA isolation method and should be further tested in studies on fertility in birds.

Sperm proteasome degrades egg envelope glycoprotein ZP1 during fertilization of Japanese quail (Coturnix japonica)

At the time of fertilization, the extracellular matrix surrounding avian oocytes, termed the perivitelline membrane (pvm), is hydrolyzed by a sperm-borne protease, although the actual protease that is responsible for the digestion of the pvm remains to be identified. Here, we show evidence that the ubiquitin-proteasome system is functional in the fertilization of Japanese quail. The activities for the induction of the acrosome reaction and binding to ZP3 as revealed by ligand blotting of purified serum ZP1 are similar to those of pvm ZP1. Western blot analysis of purified ZP1 and ZP3 by the use of the anti-ubiquitin antibody showed that only pvm ZP1 was reactive to the antibody. In vitro penetration assay of the sperm on the pvm indicated that fragments of ZP1 and intact ZP3 were released from the pvm. Western blot analysis using the anti-20S proteasome antibody and ultrastructural analysis showed that immunoreactive proteasome was localized in the acrosomal region of the sperm. Inclusion of specific proteasome inhibitor MG132 in the incubation mixture, or depletion of extracellular ATP by the addition of apyrase, efficiently suppressed the sperm perforation of the pvm. These results demonstrate for the first time that the sperm proteasome is important for fertilization in birds and that the extracellular ubiquitination of ZP1 might occur during its transport via blood circulation.

Reproductive Endocrine Disruption by Environmental Xenobiotics that Modulate the Estrogen-Signaling Pathway, Particularly Tetrachlorodibenzo-p-dioxin (TCDD)

Environmental pollutants are known to exert endocrine-disrupting effects on several hormonal axes of animals, including reproduction and development. Many of these xenobiotics modulate the estrogen-receptor signaling pathway(s) in agonistic or antagonistic ways. Some of the compounds are themselves estrogenic (so-called xenoestrogens, environmental estrogens, or ecoestrogens), and are classified as synthetic estrogens, phyto- or fungal estrogens, alkylphenol ethoxylates; certain non-coplanar polyclorinated biphenyls (PCBs), etc. Other molecules are anti-androgenic, e.g., p, p'-dichloro-diphenyl-dichloroethylene (DDE); while still others disrupt estrogen function in a manner that is dose, species and tissue specific, e.g., certain co-planar PCBs and dioxin-like molecules (e.g., tetrachlorodibenzo-p-dioxin [TCDD]). Exposure to some compounds has been correlated with skewing of sex ratios in aquatic species, and feminization and demasculinization of male animals; declines in human sperm counts; and overall diminution in fertility of birds, fish, and mammals. This review will cover these various xenobiotics and evaluate them for their estrogen-modulating effects; and then further concentrate on TCDD specifically. Dioxins are produced as by-products of herbicide overuse, from paper bleaching, plastics manufacture, and waste incineration. TCDD has been correlated with altered fecundity and endometriosis in monkeys, and with certain cancers in experimental animals and humans. TCDD is also correlated with reproductive deficits in many laboratory species. In summary, we believe that some of the reproductive deficits from endocrine-disrupting xenobiotics may be attributable to the modulation of the estrogen-signaling pathway, in positive and negative manners, depending on dose, species, and tissue specificity.

Binding of spermatozoa to the perivitelline layer in the presence of a protease inhibitor

The extracellular investment of oocytes in a number of species contains species-specific receptors to which spermatozoa bind as a prelude to fertilization; however, little is known about the nature and distribution of sperm receptors in avian oocytes. In order to elucidate the early step of fertilization in birds, we observed the binding of spermatozoa to the perivitelline layer (PL) of quail ova. When the PL obtained from the largest follicles were incubated in vitro with spermatozoa, perforations were observed. The presence of trypsin inhibitors during incubation inhibited the sperm-induced perforations of the PL and binding of spermatozoa to the PL could be observed. The number of spermatozoa bound to the PL increased in the ovum from more mature follicles, and concentrated binding of spermatozoa to the PL overlying the germinal disc region was observed in the largest follicle. The number of spermatozoa bound to the PL overlying the germinal disc region decreased in the oviposited eggs. These results demonstrate that sperm receptors exist in the PL over the germinal disc in the mature follicle.

Predietors of fertilization in the Japanese quail, Coturnix japonica

To better understand the relationship between mating behaviour, insemination and fertilization in birds, 233 mating trials were conducted with Japanese quail to determine the percentage of eggs laid that were fertile following a single confirmed or putative insemination. Fertilization success of single inseminations was highly variable. Insemination did not guarantee fertilization of any eggs; following one-third of the trials, no fertile eggs were laid. Only 3% of inseminations fertilized as many as eight eggs, making it unlikely that a single insemination could fertilize an entire clutch in this species. Forced copulations had the same fertilization success as other copulations, and thus could be a successful male reproductive strategy in this species. Voiding of foamy material containing sperm by the female shortly after copulation did not reduce fertilization success in any consistently significant manner. Fertilization success was unaffected by the presence of a hard-shelled egg in the uterus at the time of insemination. Trials in which the female ran from the male's initial approach resulted in fewer fertile eggs following insemination; that is, this behaviour predicted low fertilization success. Fertilization success was highly correlated and thus predictable if the same female was mated again with the same male, but was uncorrelated if she was mated with a different male or if the same male was mated with different females. These two predictors of fertilization success (female behaviour and male-female combination) are interpreted as evidence for female control of paternity via sperm selection (post-copulatory female choice).

Fertility and testicular fatty acid composition in the chicken as influenced by vitamin E and ethoxyquin.

AbstractThe fatty acid composition of testicular lipids has been determined and related to fertility data from groups of dubbed White Leghorn cockerels after a 50‐week feeding period on rations containing 10% safflower oil or coconut oil. Supplements of ethoxyquin ord‐α‐tocopherol acetate maintained fertility in birds raised on rations containing safflower oils. This response was associated with higher proportions of 22∶4 ω6 and lower proportions of 18∶2 ω6 in testicular lipids. Testes size was quite variable in the unsupplemented group with changes in fatty acid composition being more pronounced in the smaller testes. A multiple regression was calculated using data from those birds on the safflower oil ration. With a correlation ratio of 0.90 fertility was expressed as a function of testes size, semen concentration and the proportions of 18∶2 ω6, 20∶4 ω6 and 22∶4 ω6 in testicular lipids. Despite the low intake of linoleate significant levels of polyunsaturated fatty acids were maintained in testicular lipids of birds fed the coconut oil rations. The major changes in fatty acid composition of testicular lipids produced by this variable was a decrease in the proportion of 18∶2 ω6 and an increase in the proportion of 18∶1.

The Effect of Reserpine on the Metabolism of Turkey Semen

SEVERAL workers have reported that reserpine produced a significant reduction in fertility when administered to pigeons, pheasants and domestic turkeys. Khazan et al. (1960) reported that a severe atrophy of the testes resulted when reserpine was fed to male pigeons. Hewitt and Reynolds (1957) found that reserpine reduced egg production, fertility and hatchability in pheasant breeders. Studies reported by Rudolph et al. (1962) and Casey et al. (1963) indicated that reserpine administered in the feed resulted in a significant reduction in the fertility of breeder turkeys.A differential effect of reserpine, with respect to sex, in mature turkeys on fertility was reported by Jones et al. (1966). They reported that the reduction in fertility was greater in the female than in the male, although a reduction was observed in both sexes.Although reports have been published on the general effects of reserpine on fertility in birds, no work has been …