Comparison of blastoderm traits from 2 lines of broilers before and after egg storage and incubation.

We examined the impact of egg storage on the hatchability of eggs from 2 lines (A and B) of commercial broiler breeders with known differences in fertility (line B slightly higher) and hatchability following egg storage (line A slightly lower). Eggs from both lines were stored in a 16°C room for 3 to 4, 10 to 12, and 17 d, the blastoderms were isolated, evaluated, and statistically compared. No significant interactions (line × duration of storage) were observed in the following traits: blastoderm diameter, total and percentage of viable blastodermal cells, stage of blastoderm development, and embryo weight and stage of development after 7 d of incubation. However, significant differences were observed when comparing line averages across storage treatments (diameter and total number of blastodermal cells) and storage treatment averages across lines (percentage of viable cells, and embryo weight and stages following incubation). To ascertain the basis for the observed differences in fertility, perivitelline sperm-hole numbers were determined in eggs within 72 h of lay (3 to 4 d storage group). Eggs from line B had a significantly higher number of eggs having greater numbers of sperm holes than eggs from line A. It is concluded that no single blastoderm trait or combination of traits can be definitively associated with the inter-line differences in hatchability following storage of eggs. Alternatively, differences in PVL sperm-hole counts between lines may be associated with the reported differences in fertility between the 2 lines.

Oviductal expression of avidin, avidin-related protein-2, and progesterone receptor in turkey hens in relation to sperm storage: effects of oviduct tissue type, sperm presence, and turkey line.

The sperm storage tubules (SST) of the turkey hen, which are located in the uterovaginal junction (UVJ) of the oviduct, maintain viable sperm for up to 10 wk after a single insemination. The mechanisms of this in vivo sperm storage are poorly understood. Our objective was to evaluate mRNA and protein expression of avidin and 2 avidin-associated factors, avidin-related protein-2 (AVR2) and progesterone receptor, in the oviducts of 2 different lines to determine the extent to which they were sperm responsive and tissue specific. At 38 wk of age, Hybrid Grade Maker and Converter turkey hens were artificially inseminated with diluted semen (AI) or were sham-inseminated with extender alone (SI). Forty-eight hours after insemination, total RNA was extracted from the UVJ epithelium (containing SST) and vaginal epithelium (VGE) of SI and AI hens. Real time-polymerase chain reaction data showed a clear tissue region-specific effect on gene expression in the turkey hen oviduct, with much greater (P < 0.0001) expression in the UVJ compared with VGE region for avidin and AVR2 mRNA in both lines and for progesterone receptor mRNA in the Converter line. In contrast to real-time PCR data, in situ hybridization of SI and AI tissues showed that the presence of sperm increased avidin mRNA in the SST and UVJ surface epithelium in the Converter hens. Immunohistochemistry confirmed the presence of avidin protein in the epithelium of the UVJ in both lines; however, whereas avidin protein was localized in the SST of SI-Grade Maker hens, this protein was not detected in the SST of Converter hens. The upregulation of avidin and AVR2 mRNA within the sperm storage region indicates the involvement of avidin, and perhaps avidin analogs, in the sustained storage of sperm in the SST, possibly through the binding of biotin to avidin. The absence of avidin protein in the SST and VGE of Converter hens in the presence of increased mRNA may indicate a rapid turnover of protein.

Morphology of the turkey vagina with and without an egg mass in the uterus.

In this study we examined the gross anatomy of the uterus and vagina in turkeys in egg production. With no uterine egg mass, removal of the tunica serosa that enclosed the uterus revealed deep periodic in-folding of the muscularis transversely circumscribing the sac-like segment. When the connective tissue embracing the neutral buffered formalin fixed vagina was completely teased free, the exposed tubular segment was shaped as a counter-clockwise spiral or as a series of angular, random bends. The uterovaginal junction was flush with the uterine mucosa or projected slightly into the uterine lumen. With a uterine egg mass, the deep in-foldings of the uterus were abolished. The only alteration to the morphology of the vagina was that the uterovaginal junction appeared dilated and pressed into its juncture with the uterus. Whether an egg mass was present or not, uterovaginal junction folds that projected into the uterus possessed sperm storage tubules. An egg mass in the uterus compressed the uterovaginal junction folds, and its mucosa became contiguous with the uterine mucosa. Finally, from an evolutionary perspective, in the turkey and possibly other species possessing a nonintromittent phallus, vaginal pleomorphism may have been driven primarily by the need to accommodate the overall length of the vagina in a limited abdominal space and to a lesser extent on sexual selection.

Serotonin localization in the turkey vaginal but not sperm storage tubule epithelia.

Elucidating the cellular and molecular mechanisms regulating sperm selection and transport in the vagina of the hen had been the focus of a limited amount of research over the past decade. New observations indicate the presence of nonneuron endocrine cells in the epithelia lining the lumina of the turkey hen vagina and uterovaginal junction. Although no cells in the vagina or uterovaginal junction surface epithelia exhibited argentaffin staining, typical of cells containing neurosecretory granules, cells restricted to the vaginal and uterovaginal junction but not the sperm storage tubule epithelia were immunoreactive positive to serotonin. We speculate that if released into the vaginal lumen and submucosa, serotonin could augment cilia and sperm tail beat frequencies and facilitate smooth muscle contraction, respectively. If this is the response to sperm at insemination, it would represent the first evidence of a local control mechanism responding to sperm in the turkey vagina.

Turkey sperm reside in the tubular glands in the urodeum following artificial insemination.

A turkey hen in egg production requires 48 h after the last insemination to maximize the number of sperm in the uterovaginal junction sperm-storage tubules. Where the sperm that continue to fill the oviductal sperm-storage sites during this 48-h period reside remains unknown. Histological sections of the juncture of the vagina with the urodeum, the central compartment of the cloaca, revealed deep tubular glands containing periodic acid-Schiff-positive secretory material. When examined 36 h after the last artificial insemination, sperm were observed in the lumen of the tubular glands associated with the urodeum. We suggest that sperm reside in the tubular glands within the urodeum and are released in association with the secretory activity of the tubular glands. These sperm then may ascend the vagina to continue to populate the sperm-storage tubules. Alternatively, the sperm in the tubular glands of the urodeum may be evidence of spermatorrhea and have no functional role in the fertilization process.

Morphological and histochemical characterization of the seminiferous epithelial and Leydig cells of the turkey.

Unlike mammals, there is little fundamental information about spermatogenesis in birds. This study was undertaken to clarify the morphology, histochemistry, and lectin affinity of the seminiferous epithelial cells and Leydig cells in pre-pubertal (8- to 15-week old) and adult (40- to 44-week old) domestic turkeys. In adult turkeys, three types of spermatogonia were defined based on their chromatin distribution and nuclear morphology: the dark type A (A(d)); the pale type A (A(p)); and the type B. The A(d) is the least numerous and least conspicuous and consequently difficult to locate. Based on its spatial distribution and overall morphology, type A(d) spermatogonia were postulated to be the spermatogonia stem cells in the turkey. Antibodies to c-kit were localized to spermatogonia in the pre-pubertal and to a lesser extent in adult males. Peanut agglutinin (PNA) was specific for spermatocytes in the pre-pubertal males and spermatogonia and early spermatocytes in adult males. Wheat-germ agglutinin (WGA) highlighted Sertoli cells in both age groups. Bandeiraea simplicifolia I, soybean agglutinin, and winged-pea agglutinin staining were limited to the wall of the seminiferous tubule and some extra-tubular cell types. Concanavalin A staining was diffuse and not cell-specific and, therefore, could not be used to selectively identify a particular cell type. It was concluded that WGA and PNA could aid in identifying specific cell types in the seminiferous epithelium of testis from pre-pubertal and mature turkeys. Only Leydig cells were alkaline phosphatase reactive in the mature turkey testes. The information from this study is being used to adapt techniques for the isolation and partial purification developed for mammalian spermatogonia to avian spermatogonia and other specific cell types in the testes.

Effects of isolation and culture of turkey primary follicular oocytes on morphology and germinal vesicle integrity.

A novel approach to the production of transgenic poultry is to use primary follicular oocytes (PFOs). However, fundamental information regarding the impact of isolation and culture procedures on PFO integrity is lacking. This study describes the isolation and culture of PFOs from mature turkeys and the effects of these procedures on PFO morphology and germinal vesicle (GV) integrity. To isolate PFOs, ovarian cortex was incubated in trypsin-EDTA alone or further incubated in collagenase plus hyaluronidase (CH). About 200 to 500 PFOs, ranging in size from less than 100 microns in diameter to 1,000 microns, were recovered from each ovary. The culture of PFOs less than 100 microns in diameter for 4 h resulted in blebbing of the oolemma followed by extrusion of ooplasm. Primary follicular oocytes 100 to 250 microns in diameter survived culture for 24 h whereas larger PFOs survived for up to 7 d. Those PFOs with intact granulosa cell investments survived longer than those fully or partially denuded of granulosa cells with CH. Co-culture of PFOs (100 to 250 microns in diameter) on a monolayer of granulosa cells derived from mature, yellow-yolk follicles augmented PFO survival rates. The rate of GV breakdown was not influenced by the isolation or culture of the PFO. These data provide the basis for developing procedures for the in vitro maturation and in vitro fertilization of isolated PFOs.

Aquaporins are observed in the duct epithelia of the epididymal region of the large white turkey.

The cellular and molecular mechanisms regulating the reuptake of the testicular fluid supporting sperm exiting the testes in the bird are not known. The presence of aquaporins, proteins involved in transmembrane water transport, was investigated. Observations were limited to the ductuli efferentes, collecting ducts, and ductus epididymis. Interestingly all of these ducts were positive for aquaporins-2, -3, and -9 but not aquaporin-7. When positive, aquaporin was observed localized over the whole cell or the apical plasma membrane of the nonciliated cells and the apical plasma membrane and cilia of the ciliated cells. This study is the first to clearly demonstrate the presence of aquaporins-2, -3, and -9 in the epididymal region of any bird. We assume the aquaporins play a role in concentrating the sperm and in the promotion of sperm maturation in the epididymal region.

Comparative development of the turkey and chicken embryo from cleavage through hypoblast formation.

The development of the turkey and chicken embryo from the first cleavage division through hypoblast formation is described. The early development of the chicken embryo has been categorized into 14 stages. A similar staging sequence for the turkey was not proposed until 1993, when we described the early development of the turkey embryo, which was divided into 11 stages. Comparatively, differences in the temporal and spatial development of the turkey and chicken blastoderm were evident. Of significance is the observation that at oviposition the turkey is in Stage VII and characterized by the first signs of area pellucida formation. In contrast, the chicken embryo at oviposition is in Stage X and area pellucida formation is completed. Similarly, the hypoblast, which is already apparent in the Stage X chicken embryo, does not appear in the turkey embryo until the egg is incubated. Furthermore, the anterior-posterior (head-tail) axis in the early embryo is achieved prior to oviposition in the chicken but after the onset of incubation in the turkey. It is apparent that the turkey embryo is less mature than the chicken embryo at oviposition. Whether this distinction is related to differences between the hatchability of turkey and chicken eggs is not yet known.

Gross appearance of the turkey blastoderm at oviposition.

The blastoderm (fertilized ovum) and unfertilized germinal disc (UGD) of fresh laid eggs and eggs stored prior to incubation exhibit subtle but definable morphological variations. Such variations may lead to difficulty when attempting to determine true flock fertility based on the appearance of the blastoderm/UGD. The objectives of this study were to define and categorize such morphological variations and to determine whether sperm influence the frequency distribution of the different categories. Eleven categories of blastoderms were defined based on the relative density and appearance of the area alba, area pellucida, area opaca, and the periblast. The majority of the blastoderms were included in the first four categories. Unfertilized germinal discs were divided into six categories and were best differentiated from the blastoderms by the presence of vacuoles around its central dense area. They were also discernible from blastoderms based on their overall denser appearance. Differences in the frequency distribution of some of the UGD categories between virgin and inseminated hens may be due to the effect that supernumary sperm may have on the organization of the UGD (no fertilization but supernumary sperm present) or blastoderm (fertilized but failed to develop). It is recommended that before starting true fertility determinations during fresh egg breakouts, one should study the appearance of the UGD from virgin hens and then the blastoderm from inseminated hens. One then will learn to appreciate the subtle differences in shape and density of the blastoderm/UGD structural components.

Alkaline phosphatase reactivity in the vagina and uterovaginal junction sperm-storage tubules of turkeys in egg production: implications for sperm storage.

1. Currently there remains contradictory information on the localisation and possible role of alkaline phosphatase (AP) in the chicken and Japanese quail oviducts. 2. Using turkeys with a hard-shelled egg in their uteri, vaginal and uterovaginal junction mucosae were stretched and fixed as whole mounts prior to the histochemical localisation of AP activity. 3. Scattered AP reactive cells were observed in the vaginal and uterovaginal junction surface epithelia and intense AP reactivity of the sperm-storage tubule (SST) epithelium, localised to its apical border. 4. We suggest that such AP reactivity in hens in egg production may reflect cell differentiation and proliferation in the vagina and SST and possibly a mechanism for the transfer of lipid from the SST epithelia to resident sperm.

Net citrate production by isolated prostate epithelial cells.

The net production of citrate from exogenous substrates by rat ventral prostate was investigated. The preparation of isolated prostate epithelial cells was described. These cells were capable of oxidizing pyruvate (5 mmol/l) as a source of acetyl coenzyme A. The addition of aspartate + alpha-ketoglutarate (5 mmol/l) in the presence of pyruvate resulted in significant net production of citrate and excess oxalacetate. In the presence of aspartate and glutamate, the cells were capable of producing citrate without excessive oxalacetate production. Neither glucose alone nor glucose plus pyruvate resulted in net citrate production. The results demonstrated that aspartate could serve as a 4-carbon source of oxalacetate for citrate synthesis. Furthermore, the results indicate the intramitochondrial operation of a glutamate-aspartate-citrate pathway involving mitochondrial aspartate aminotransferase and glutamic dehydrogenase activities in prostate epithelial cells.

Evidence for the innervation of sperm storage tubules in the oviduct of the turkey (Meleagris gallopavo).

The presence of neural tissue and smooth muscle elements in the vicinity of the oviductal sperm storage tubules at the uterovaginal junction was assessed by several modes of light microscopy. Isolated neurones and small ganglia were identified in the uterovaginal junction of the turkey oviduct. The nerve cell bodies were observed in the tunica mucosa by bright field microscopy. Immunoreactivity against neurofilament antibody and recombinant fragment C of the tetanus toxin reacted with nerve fibres and the nuclei of neurones. Fluorescence microscopy and confocal laser scanning microscopy revealed that nerve fibres continued from the base of the tunica mucosa into the plicae. Axons appeared to terminate on, or run immediately adjacent to, individual sperm storage tubules. Neither phalloidin reacting with F-actin nor the monoclonal antibody against alpha-smooth muscle actin detected smooth muscle fibres in the tissue encapsulating individual sperm storage tubules. In contrast, F-actin was strongly localized in the apical region of the epithelial cells of the sperm storage tubule and in smooth muscle elements in the tunica mucosa and tunica muscularis. These observations present the first evidence for the innervation of the sperm storage tubules. It is suggested that a previously unrecognized neural factor may function in oviductal sperm storage in, and release of spermatozoa from, the sperm storage tubules of hens.

Testosterone stimulation of mitochondrial aspartate aminotransferase levels and biosynthesis in rat ventral prostate.

The effects of testosterone on mitochondrial aspartate aminotransferase (mAAT) synthesis in rat ventral prostate was investigated. Procedures for the isolation, purification and characterization of AAT isozymes were developed and described. Purified mAAT preparations contained no demonstrable contaminating proteins. Prostatic mAAT was characterized as a cationic protein with an estimated mol. wt of 120,000. Cytoplasmic AAT (cAAT) isozyme was identified as an anionic protein with an estimated mol. wt of 132,000. A cytosolic cationic isozyme, similar to mAAT, was also identified as pre-mAAT. Testosterone administration to castrated rats resulted in significant increases in leucine incorporation into mAAT, in the level of mAAT, and in mAAT activity. These effects of testosterone were observed within 2 h of administration. Conversely, testosterone administration had none of these effects on cAAT or on non-AAT protein pool. Testosterone treatment did appear to increase leucine incorporation into pre-mAAT. Testosterone treatment in organ cultures and in prostate epithelial cell cultures resulted in the same stimulatory effects on mAAT as observed in the in vivo studies. The hormone was effective at the physiological concentration of 2 X 10(-9) M. These results indicated that testosterone has a rapid and specific effect on the biosynthesis of mAAT. This continues to support our proposal that testosterone regulates prostate citrate production via a stimulatory effect on mAAT which results in increased mitochondrial synthesis of citrate from aspartate.

The effect of testosterone on citrate synthesis and citrate oxidation and a proposed mechanism for regulation of net citrate production in prostate.

Citrate oxidation by rat ventral prostate was reduced by castration and increased by testosterone administration. Similarly, the mitochondrial aconitase activity was decreased by castration; whereas cytosol aconitase was unaffected. The rate of citrate oxidation is extremely low in prostate. Castration also decreased mitochondrial aspartate aminotransferase activity while having no effect on the cytosol isoenzyme. Testosterone markedly stimulated the net production of citrate from aspartate plus glutamate by prostate mitochondria. These studies support the proposal that aspartate is a major source of oxalacetate for citrate production, and that a "glutamate-aspartate-citrate" pathway may be functional in prostate mitochondria. In addition, testosterone can regulate citrate production by a specific effect on mitochondrial aspartate aminotransferase activity. Testosterone also regulates the flux of citrate through the Krebs cycle, but this represents only a small proportion of the citrate accumulated. These conditions would be consistent with the function of prostate epithelium in accumulating and secreting citrate.