The domestic chicken: Causes and consequences of an egg a day.

The domestic laying chicken has been intensely selected to be a persistent ovulator. That is, the tendency for broodiness has been nearly eliminated and, given the appropriate lighting and nutrition, many strains of laying hens produce an egg on almost every day. The regulatory mechanisms involved in coordination of neuroendocrine and ovarian events have been well studied and described. In spite of this, there has been little attention focused on the oocyte itself. Recent findings in mammals have indicated that the oocyte produces several oocyte-specific factors, including growth differentiation factor 9 (GDF9) and bone morphogenetic factor 15 (BMP15), which influence the surrounding cells and follicular development. Our studies indicate that GDF9 is present in the hen oocyte and influences granulosa cell proliferation. Additionally, Bmp15 mRNA is most abundant in oocytes of small follicles and stimulates an increase in follicle stimulating hormone (FSH) receptor mRNA in granulosa cells. BMP15 also enhances yolk uptake in growing follicles by decreasing tight junctions between granulosa cells. These studies indicate that the oocyte likely contributes to follicle development. Commercial laying hens also spontaneously develop ovarian cancer at a high rate, and susceptibility to this disease has been associated with ovulatory events in women. Studies have shown that ovulation, or events associated with ovulation, increase the prevalence of ovarian cancer in hens. Inhibition of ovulation in hens through a hormonal strategy mimicking oral contraceptives results in a decrease of ovarian cancer incidence. Recent studies in women have suggested that some ovarian tumors may arise from the distal oviduct. Gene expression profiles in very early stage tumors from hens show a high expression of oviduct-related genes, supporting the possibility of oviduct origin for some ovarian tumors. Genetic selection for high productivity in commercial laying hens has generated an efficient and valuable food source as well as an important animal model for human ovarian cancer.

Expression of anti-Mullerian hormone in hens selected for different ovulation rates.

In hens, the granulosa layer is the primary source of anti-Mullerian hormone (AMH), as it is in mammals. Small follicles express the greatest amount of Amh mRNA with less in the larger follicles. Laying hens have a distinct ovarian hierarchy of follicles while broiler breeder hens often have excessive follicle growth with a disrupted hierarchy. The objective of Experiment 1 was to examine Amh expression in two strains of hens differing in ovulatory efficiency. Amh expression was greater (P<0.01) in broiler breeder hens (n=6) as compared with laying hens (n=6). Experiment 2 was designed to examine whether alterations in follicular development due to diet, within the broiler breeder hens, were correlated with changes in the expression of Amh. Restricted feeding (RF) in broiler breeder hens promotes optimal follicular development. Egg production in broiler breeder hens on full feed (FF; n=8) was 78% that of hens on RF (n=9). The number of large follicles (P<0.05), total ovarian weight (P<0.01), and Amh mRNA expression were greater in FF hens as compared with RF hens (P<0.01). There was no difference in FSH receptor expression between the two groups. A direct nutritional effect was not supported because culture of granulosa cells with varying concentrations of glucose and insulin showed no effect on granulosa Amh expression. Finally, testis-conditioned medium resulted in a dose-related increase in granulosa cell proliferation, which could be inhibited by preincubation with AMH antibody. AMH may enhance granulosa cell proliferation through an autocrine or paracrine mechanism although excessive AMH may inhibit optimal follicle selection.

Dietary aspirin decreases the stage of ovarian cancer in the hen.

OBJECTIVE: We aimed to determine the effects of dietary aspirin treatment on ovarian cancer incidence and progression in the hen as a model for the human disease. METHODS: Hens were fed a standard layer diet (control) or the same diet containing 0.1% aspirin for 1 year. Liver prostaglandin E(2) (PGE(2)) was measured using an enzyme immunoassay. Incidence and stage of ovarian cancer were determined through necropsy and immunohistochemical analysis of ovarian sections for each hen. RESULTS: Aspirin treatment decreased liver PGE(2) in treated hens as compared to control hens. Treatment with aspirin did not decrease ovarian cancer incidence. Significantly more control hens developed late stage ovarian cancer than early stage, while the same was not true for aspirin-treated hens. Hens that developed ovarian cancer, even early ovarian cancer, produced significantly fewer eggs in the year prior to diagnosis than hens without ovarian cancer. CONCLUSIONS: Aspirin treatment may inhibit the progression of ovarian cancer in the hen and egg production may be used to identify hens with early stages of the disease.

VEGF expression and the effect of NSAIDs on ascites cell proliferation in the hen model of ovarian cancer.

OBJECTIVES: We aimed to determine the expression of vascular endothelial growth factor (VEGF) and the effect of nonsteroidal anti-inflammatory drugs (NSAIDs) on the proliferation of cells isolated from ascites in the hen model of ovarian cancer. METHODS: Ovarian tumor and normal ovary were collected from hens and ascites cells were isolated from hens with ovarian cancer. Quantitative real-time PCR was used to quantify mRNA expression. Immunohistochemical and/or Western blot analyses were used to localize protein expression in ovarian tumors, normal ovaries, and ascites cells. Cells were treated with a nonspecific, COX-1-specific, or COX-2-specific NSAID and proliferation was determined. RESULTS: VEGF mRNA was increased in ascites cells and there was a trend for a correlation between VEGF mRNA in ascites cells and ascites volume. VEGF protein was localized to theca cells of normal ovaries, in glandular areas of tumors, and to the cytoplasm of ascites cells. Aspirin and a COX-1-specific inhibitor decreased the proliferation of ascites cells, whereas a COX-2-specific inhibitor did not. CONCLUSIONS: VEGF may play a role in ovarian cancer progression in the hen and the proliferation of ascites cells can be decreased by targeting the COX-1 but not COX-2 pathway.

Expression and regulation of anti-mullerian hormone in an oviparous species, the hen.

Anti-mullerian hormone (AMH) has a critical role in regression of the mullerian duct system during development in male mammalian and avian species and in regression of the right oviduct in female avian species. AMH in adult female birds has not been investigated. Chicken-specific cDNA primers were used to isolate Amh by RT-PCR. This probe was used in Northern blot analysis to identify a 2.8-kb band with expression in total ovarian RNA and in granulosa cell RNA. Quantitative real-time PCR was used to assess Amh expression in follicles of different maturity (1, 3, 5, and 6-12 mm and the largest F1 follicle; n = 4-6 of each size). There was an increased amount of Amh mRNA in the granulosa layer of the smaller follicles and a lower amount in the granulosa layer of the larger follicles (P < 0.01). There was no difference in granulosa Amh expression between the germinal disc and non-germinal disc region of 6- to 12-mm follicles, although expression differed with follicle size (P < 0.01). To examine hormone regulation of Amh, granulosa cells (from 6- to 8-mm follicles) were cultured with various concentrations of estradiol (E(2)) and progesterone (P(4)), and Amh mRNA was assessed. Neither E(2) nor P(4) influenced Amh mRNA accumulation. Granulosa cells were also cultured in the presence of oocyte-conditioned medium (OCM), which decreased Amh mRNA expression in a dose-related manner (P < 0.05); FSH receptor expression was not affected. Heat treatment of OCM abolished the effect, but growth differentiation factor 9 antiserum did not block the suppression. Immunohistochemistry confirmed that the granulosa layer was the predominant source of AMH in the small follicles of the hen and indicated that AMH was present early in follicle development, with expression in very small follicles (approximately 150 mum).

Use of genetic strains of chickens in studies of ovarian cancer.

Ovarian cancer is a deadly disease often diagnosed late in development when there is little chance for a successful recovery. Although ovarian cancer is a rare occurrence in most animals, the domestic hen has been shown to spontaneously develop the disease with an age-related incidence. Two strains of hens derived from a similar genetic background and maintained at Cornell University have been shown to differ in the incidence of the disease. At 2 yr of age, the C strain hens have a greater incidence of ovarian neoplasms than do K strain hens. Interestingly, levels of plasma estradiol are elevated in the C strain compared with K strain hens. In addition, plasma immunoreactive inhibin is lower in the C strain than in the K strain. Finally, mRNA expression of the alpha-subunit of inhibin in the granulosa cell layer of the large yellow follicles is lower in the C strain compared with the K strain hens. Further studies using these as well as other strains of hens may be useful in learning more about the etiology of this disease.

Expression and function of growth differentiation factor-9 in an oviparous species, Gallus domesticus.

Many studies have indicated a critical role for the oocyte growth factor, growth differentiation factor-9 (GDF9), in mammalian follicle development, but no information has been available concerning oviparous species. We cloned a cDNA for chicken GDF9 (162 base pairs) and used it in Northern blot analysis to identify a transcript at 1.7 kilobase in RNA isolated from the ovary of the hen. We also sequenced two full-length clones from a normalized chicken reproductive tract cDNA library. The cDNA clone for chicken GDF9 encodes a protein of approximately 449 amino acids and all six cysteine residues, and three of the four glycosylation sites are conserved with respect to mammalian GDF9. Chicken GDF9 is approximately 65% similar in the full-length cDNA sequence and 80% similar in amino acid sequence at the C-terminal region to GDF9 from several mammals. Quantitative polymerase chain reaction analysis (n = 5) indicated that GDF9 mRNA is greatest in follicles < 1 mm in size compared with larger follicles or granulosa layers isolated from larger follicles. Immunocytochemical analysis showed strong expression of GDF9 in hen oocytes. In yolk-filled oocytes, the GDF9 was localized at the periphery of the oocyte. Finally, oocyte-conditioned medium (from < 1-mm oocytes) resulted in a 2-fold increase in granulosa cell proliferation, which could be inhibited by preincubation of the conditioned medium with GDF9 antibody. These data suggest that GDF9 is present in the hen oocyte and that this factor is capable of enhancing granulosa cell proliferation, as has been demonstrated in mammals.

Allocation of inner cell mass and trophectoderm cells to the preimplantation blastocyst of the domestic ferret, Mustela putorius furo.

The growth of ferret preimplantation blastocysts in vivo, collected between 156 and 240 hr post coitum, was investigated. A technique, combining immunosurgery and differential fluorochrome staining, was used to discriminate between inner cell mass (ICM) and trophectoderm (TE) cells. Using the stains propidium iodide and bisbenzimide (Hoechst 33342), the ICM was stained blue and the TE was stained pink. The ICM and TE counts for 90 blastocysts, respectively, averaged 25 and 63 at 156 hr and increased exponentially to 2077 and 4137 at 240 hr. The Box-Cox procedure was used for choosing a transformation that minimized the error sum of squares for a linear regression of Y (cell count) on X (time in hr). Logarithmic transformations of the ICM, TE and total cell count gave a good fit, but the following equations obtained by the Box-Cox procedure provided the best fit, where Y is cell count and X is time in hours. For inner cell mass: Y = [(176.06 + 2.45X)/-899.44 + 1]-3.33; trophectoderm: Y = [(301.38 + 14.48X)/-6863.42 + 1]-10; and total: Y = [(2266.97 + 17.0X)/-7837.21 + 1]-5. The R2 values were 0.73, 0.84, and 0.84, respectively. The exponential growth of the ferret embryo during the time interval that measurements were made fits the general pattern described for other mammalian embryos. This report is the first to characterize the pattern of cell allocation and growth in preimplantation blastocysts of the ferret, and the first such report for a carnivore. The pattern of in vivo development provides a standard for judging the quality of in vitro produced and matured ferret embryos and, concomitantly, a means to evaluate culture systems.

Development of rabbit zygotes into blastocysts in defined protein-free medium and offspring born following culture and embryo transfer.

We report here on an improved, completely defined culture system for producing embryos in vitro which mimics development in vivo. This system avoids the confounding effects of the many unknowns introduced by the multivariate components of the serum or by unknowns attached to bovine serum albumin (BSA). Zygotes were obtained from superovulated rabbits and cultured in modified defined RPMI 1640:Dulbecco's MEM, 1:1 (RD) medium. The effect of a novel and potentially ideal antioxidant, tempol, was tested (20 to 0.001 mM) but found to be either toxic or ineffective. In the presence of 20% O(2), 600 units of Superoxide dismutase or 2.5 mM of taurine increased embryo hatching after 72 h of culture in RD medium to 75 and 76%, respectively, compared with 46% in the control (P < 0.05). The need for antioxidants was reduced with 5% O(2). The beneficial effects of RD medium were demonstrated when 60 zygotes cultured for 48 h to the early blastocyst stage in this medium were transferred and resulted in 30 young (50%) compared with 35/60 (58%) young from uncultured control transfers. Only 12% of the young were obtained from slower developing morulae. Thus, high viability was established for rapidly growing embryos in culture, but fewer slow growing embryos survived after transfer. A further comparison of embryos cultured in RD medium with a high potassium simple, optimized, defined medium (KSOM), revealed that both yielded results approaching those of direct transfer without culture. Simple defined media may also be useful for the culture of embryos of other species.

Effects of gas atmosphere, platelet-derived growth factor and leukemia inhibitory factor on cell numbers of rabbit embryos cultured in a protein-free medium.

While culture conditions for embryos have improved, development in vitro is not equivalent to in vivo growth. Platelet-derived growth factor (PDGF), mouse (mLIF) or human (hLIF) leukemia inhibitory factor and 10% fetal bovine serum (FBS) added to protein-free culture medium, as well as two gas atmospheres were evaluated for their effect on rabbit embryo development. Adding PDGF, mLIF or hLIF to the culture medium did not result in detectable differences in total cell number for blastocysts. The culture of rabbit embryos under a gas atmosphere of 10% CO2:5% O2:85% N2 resulted in improved total cell numbers (P < 0.01) for blastocysts compared to those developing under 5% CO2:95% air (230 vs 159, respectively). Supplementing RD (RPMI-1640 and Dulbecco's MEM, 1:1) medium with 10% FBS improved the number of total cells (264 vs 155) and inner cell mass cells (71 vs 47). These results indicate that when defined culture conditions promote a high proportion of two-cell embryos developing into blastocysts, the addition of certain growth factors did not have a detectable beneficial effect, while 10% FBS improved culture conditions.

Ethylene glycol monomethyl ether effects on health and reproduction in male rabbits.

Male Dutch rabbits were weighed and randomly assigned within each weight group to five groups of six animals each (plus one more in the highest dose group). They received 0, 12.5, 25.0, 37.5, or 50.0 mg of ethylene glycol monomethyl ether (EGME) per kg of body weight in the drinking water 5 d/week for 12 weeks. Feed and water consumption were monitored daily and body weight weekly. All animals consumed the water and feed, maintained body weight, and were in good health throughout the experiment. Semen was collected twice weekly for 12 weeks, and 96% of the ejaculates were obtained. By weeks 6 and 9, most males in groups receiving 50.0 or 37.5 mg of EGME per kg were oligospermic. Only minor changes in other characteristics of sperm obtained from treated animals were found, as measured by computer-assisted sperm analysis. Fertility of the males still producing sufficient sperm during week 12 to use for insemination was tested with 96 does producing 2839 oocytes, and fertility of treated males (41%) was not lower (P > 0.05) than 47% in controls. At necropsy, all vital organs were grossly normal, with no notable histopathology. However, the groups of animals receiving 37.5 and 50 mg of EGME per kg of body weight produced fewer sperm and had smaller testes than controls (P < 0.05). Although all rabbits appeared grossly normal, there was a marked disruption of spermatogenesis as ingestion of EGME increased above 25 mg/kg of body weight. Rabbit testes appear to be more sensitive to EGME than testes of rats or mice.

Nuclear transfer of putative rabbit embryonic stem cells leads to normal blastocyst development.

Rabbit embryonic stem-like cells, characterized by embryoid body formation and differentiation into cell types representative of all three germ layers, were studied for their ability to promote early embryonic development after nuclear transfer. After culture of the reconstructed embryos, 23% (n = 35) developed successfully into morulae or blastocysts, compared with 34% (n = 62) for cloned embryos derived from nuclear transfer with embryonic blastomeres. The cloned embryos from the embryonic stem-like cells appeared normal, with an average of 26% inner cell mass cells, similar to that of control non-manipulated embryos (25%) or cloned embryos from blastomeres (25%). Thus, nuclear transfer of rabbit embryonic stem-like cells leads to early embryonic development that is indistinguishable from blastomere fusion. These results have implications for the development of gene targeting in a species (rabbit) that may be a more suitable model for studying certain human diseases. In addition, this technique may be applicable to other species from which putative embryonic stem cells have been derived, particularly agriculturally important animals.

Rabbit blastocyst: allocation of cells to the inner cell mass and trophectoderm.

The proportion of total cells in the blastocyst allocated to the inner cell mass (ICM) and trophectoderm (TE) is important for future development and may be sensitive indicator to evaluate culture conditions. The number of cells and their distribution within the two primary cell lineages were determined for the rabbit embryo developing in vivo after superovulation or nonsuperovulation or embryo transfer and compared with embryos developing in vitro. Comparisons were made with cultured embryos or embryos grown in vivo until 3.5, 4.0, and 4.5 days of age. Embryos from superovulated rabbits developed in vivo for 3.5, 4.0, and 4.5 days, respectively, had 361, 758, and 902 total cells (P < 0.05), and in nonsuperovulated rabbits 130, 414, and 905 total cells (P < 0.05), with increasing proportions of ICM cells over time (P < 0.05). One-cell embryos recovered from superovulated females and transferred to nonsuperovulated recipients developed more slowly with 70, 299, and 550 total cells after 3.5, 4.0, and 4.5 days of culture (P < 0.05), respectively. The proportion of ICM cells increased with age of the embryo. Corresponding values for one-cell embryos cultured in vitro resulted in 70, 299, and 550 total cells (P < 0.05). However, in vitro culture of morula-stage embryos in the presence of fetal bovine serum for 24 hr did not delay growth. In addition, the proportions of ICM/total cells were 0.17, 0.25, and 0.29 for embryos developing in vitro at 3.5, 4.0, and 4.5 days respectively, similar to those for embryos developing in vivo at each of the three recovery times.(ABSTRACT TRUNCATED AT 250 WORDS)

Pluripotency of cultured rabbit inner cell mass cells detected by isozyme analysis and eye pigmentation of fetuses following injection into blastocysts or morulae.

Pluripotency of isolated rabbit inner cell masses (ICMs) and cultured (3 days) inner cell mass (ICM) cells was tested by injecting these donor cells into day 3.5 blastocysts (experiment 1) or day 3 morulae (experiment 2) to produce chimeric embryos. Injected (n = 107) and noninjected (n = 103) embryos were transferred to the opposite uterine horns of the same recipient females. Chimerism was determined by adenosine deaminase (ADA) isozyme analysis on fetal tissue and by eye pigmentation at midgestation. In experiment 1, 53% and 64%, respectively, of blastocysts injected with ICMs or cultured ICM cells developed to midgestation, compared with 52% and 48% for controls. Of these fetuses, four (31%) and one (6%), respectively, had ADA chimerism. In experiment 2, 38% and 62%, respectively, of the morulae injected with ICMs or cultured ICM cells developed to midgestation, compared with 46% and 56% for control morulae. Six (43%) chimeric fetuses from morulae injected with ICMs were detected by ADA analysis, but 12 (86%) chimeric fetuses were detected by eye pigmentation, indicating that eye pigmentation was a more sensitive marker for chimerism than our ADA assay. None of the 14 fetuses recovered after injecting morulae with cultured ICM cells were chimeric with either marker. No chimeras developed from control embryos. These studies demonstrate 1) that pregnancy rates are not compromised by injection of blastocysts or morulae with ICMs or cultured ICM cells, 2) that chimeric rabbit fetuses can be produced by injecting ICMs into either blastocysts or morulae, and 3) that cultured ICM cells can contribute to embryonic development when injected into blastocysts.

Staining procedure to detect viability and the true acrosome reaction in spermatozoa of various species.

A simple dual stain procedure (DS) for simultaneously determining sperm viability and acrosomal status is described. The DS includes the use of vital stain trypan blue to detect live and dead spermatozoa and Giemsa to detect the presence or absence of an acrosome. For staining, spermatozoa are washed, incubated with trypan blue, washed, dried onto slides, and subjected to Giemsa. Dead spermatozoa stain blue in the postacrosomal region while live spermatozoa remain unstained. The acrosome stains light purple-dark pink while acrosome-free sperm remain unstained. This staining pattern enables differentiation of spermatozoa which have undergone a true acrosome reaction (TAR) from those which have undergone a false acrosome reaction (FAR). Incubation of bull, boar, ram, and stallion spermatozoa for 60 minutes at 37 degrees C in the presence of calcium ionophore A23187 increased the proportion of spermatozoa undergoing a TAR in all species except the stallion. Incubation of bull spermatozoa for up to 24 hours at 37 degrees C resulted in a decrease over time in the percentage of live acrosome-intact spermatozoa and a simultaneous increase in the percentage of spermatozoa categorized as having undergone a TAR and FAR. The DS could be a useful technique in evaluating sperm viability and acrosomal status in fertilization and clinical studies.

Bilateral removal of the cauda epididymides in the neonatal pig as a technique for creating teaser boars.

Five pairs of crossbred littermate boars were used to assess the efficacy of bilateral removal of the cauda epididymides at an early age as a technique for creating teaser boars. The cauda epididymides were surgically removed in one of each litter pair; the other of the pair served as an intact control. Boars subjected to removal of the epididymides (Epid) were rendered sterile by the technique. The Epid-treated and control untreated littermate boars had similar levels of sexual aggression and libido, as measured by behavioral characteristics at semen collection. The Epid-treated boars showed a slight, but not significant, reduction in ejaculate volume. Upon slaughter at 273 d of age, Epid and control boars had similar weights for the accessory sexual organs and penis and similar penile lengths. The Epid-treated boars displayed enlarged caput epididymides and granulomata. It is suggested that bilateral removal of the cauda epididymides in the neonatal pig may prove a worthwhile alternative to the traditional vasectomy procedure to create teaser boars.

Neonatal lamb losses due to feral pig predation.

An investigation into causes of low lamb marking percentages was made on a property in north-west New South Wales from 1971 to 1975. Investigations revealed that from 11 to 70% of the ewes in lamb were losing all their lambs. Observations suggested that feral pig predation was a factor in the perinatal loss. In 1975, 2 groups of ewes were placed in adjoining paddocks prior to lambing. Feral pigs were excluded from one paddock for most of lambing by means of an electric fence. In this paddock, 117% of lambs were marked compared with 80% in the adjoining paddock. It was estimated that in 1975 over 600 lambs were killed by feral pigs from 1,422 ewes lambing in paddocks with feral pigs. The problems involved in the diagnosis of feral pig predation are discussed.

Serological evidence of inter-epidemic infection of feral pigs in New South Wales with Murray Valley encephalitis virus.

The sera of 617 feral pigs, collected from three widely separated areas of northern and central New South Wales, were examined for antibody to Murray Valley encephalitis (MVE) virus and to Ross River virus. Haemagglutination-inhibition (HI) antibody was detected to MVE in 58% of sera and to Ross River virus in 15% of sera. Neutralization tests suggested that the MVE HI antibody resulted from infection with MVE virus in the summers of 1971-1972 and 1972-1973 when the virus was not known to be active in New South Wales. These same tests suggested that more than one flavivirus infected the feral pigs in the summer of 1973-1974 and that Kunjin virus was active in the summer of 1975-1976.