Recipient Organization
UNIVERSITY OF GEORGIA
200 D.W. BROOKS DR
ATHENS,GA 30602-5016
Performing Department
Poultry Science
Non Technical Summary
In the commercial layer industry, approximately 50% of chicks are culled immediately after hatch, because they are male. In the US, approximately 226 million male chicks were culled in 2002 alone. On the surface, this practice appears wasteful, however due to their slow growth rate and inferior meat characteristics when compared to broilers, male chicks from layer breeders are just not economical to maintain. If the poultry industry could manipulate layer hens such that a majority of chicks were female, productivity would dramatically increase, and thriftless killing of male chicks would decline. While many attempts have been made to estimate embryo sex and eliminate male eggs prior to hatch, it would be even more fruitful if hens produced more female-bearing eggs from the start. For decades, researchers have documented significant skews in the production of male versus female offspring in a variety of species. Birds have demonstrated a particular aptitude towards manipulating offspring sex, some species producing up to 77% females in response to environmental cues. Such patterns have mainly engaged the interest of ecologists and, to date, the commercial value of these findings has gone untapped. A full understanding of the mechanisms responsible for the control of offspring sex by hens could allow for the purposeful manipulation of chick sex ratios by breeders. Such a method employed within the layer industry could save millions of industry dollars and millions of animal lives as well. Recent studies in four avian species show that females with high levels of stress hormones skew offspring sex ratios significantly towards females. Such an effect would be extremely useful in an industry context, however there are clear concerns with (1) using a steroid-based hormonal treatment on hens and (2) long-term disruptions that have been documented as a side-effect of chronic treatments with stress hormones. Our objective is to determine the mechanism by which corticosterone is acting on offspring sex ratios with an ultimate goal of developing a non-hormonal treatment by which offspring sex can be purposefully controlled.
Animal Health Component
0%
Research Effort Categories
Basic
80%
Applied
0%
Developmental
20%
Goals / Objectives
The major goals of this project are:1. To determine the mechanism by which corticosterone influences offspring sex in birds2. To determine whether testosterone is also a mediator of offspring sex ratios in birds3. To target when during the sex determination process sex ratio adjustments are taking place.
Project Methods
in Objective: To determine the roles of maternal stress and corticosterone inoffspring sex ratio adjustment in Leghorn chickensHypothesis 1: Long-term elevation of CORT triggers skews in the offspring sex ratio.EXPERIMENT 1a: Effects of chronic stress on offspring sex ratios: The chronic stressor chosen for this experiment is a repeated restraint stress that takes place three times daily for 2 weeks. White Leghorn hens (Hy-line W36) in this experiment will be randomly distributed into one of two treatment groups (n = 100 per group): (1) the chronic stress group, which will be repeatedly restrained by wrapping tightly in a cloth such that the wings are pinned to the body for 10 minute periods three times per day for 2 weeks, and (2) a control group where hens remain unhandled and undisturbed (except for feeding periods) for the same two week period. Stress treatments for all 100 hens per group will take place 2, 4, and 6h after ovulation so as not to interfere with ovulatory processes of the following follicle. Blood samples will be taken from stressed birds following the completion of each stress challenge and from a separate set of undisturbed hens at the same times during the day for comparison. Blood samples will also be taken from a subset of the stressed and control birds at 4 and 6h prior to ovulation (when birds will not be undergoing a stress response but should exhibit elevated baseline CORT levels indicative of chronic stress). CORT as well as testosterone will be measured in blood samples. Finally, CORT concentrations will be measured in the yolk of each collected egg as well. For the duration of this experiment, all 200 hens will be artificially inseminated biweekly using pooled semen from 10 roosters. Fertilized eggs will be collected from stressed and control birds 7d and 14d following the onset of the stress treatments. Eggs will be incubated for 2d and embryos will be separated and sexed using molecular techniques. Sex ratios and yolk CORT as well as circulating plasma concentrations of CORT and testosterone will be analyzed in relation to the two treatment groups, as well as in relation to the position of the egg in the clutch cycle.EXPERIMENT 1b: Effects of chronic exogenous CORT elevation on offspring sex ratios: White Leghorn hens (Hyline 36) in this experiment will be randomly distributed to one of two treatment groups (n = 100 in each): (1) A CORT implant treatment group, which will receive a 1-inch silastic implant containing crystalline CORT (Sigma-Aldrich Co., St. Louis, MO) or (2) a control group receiving an empty control implant. Females will be artificially inseminated using pooled semen from 10 roosters biweekly for 4 weeks. Oviposition will be monitored daily, laying time recorded, and fertilized eggs will be collected 4, 7, and 13d following implantation and incubated for 2d to allow minimal embryonic development. Embryos will be isolated and sexed using PCR-based molecular techniques. Concentrations of CORT and testosterone will be measured in blood samples taken 4h after ovulation as well as 4 and 6h prior to ovulation in females from both treatment groups on days 3, 6, and 12 (to correspond with ovulation of collected eggs above). CORT concentrations will be measured in the yolk of each collected egg as well. Hypothesis 2: CORT influences sex ratios by decreasing the rate of follicular growth.EXPERIMENT 2: Effects of CORT exposure and maternal stress history on follicular growth as it relates to offspring sex ratio:This experiment will be conducted on yolks collected from 400 eggs (100 per treatment group) during Experiments 1a and 1b, in which birds experienced chronic stress and/or chronic CORT elevation. This is because the majority of follicular growth, characterized by the rapid yolk deposition phase stops 24-48h prior to ovulation. Because all eggs for all experiments will be collected after only 2d of incubation, the yolks should still be relatively intact. Yolks will be frozen upon collection sectioned into a 2mm slice.Follicular growth will be calculated by staining yolk rings according to methods described in Young and Badyaev [2004]. This method provides a measure of follicular growth rate during the period of rapid yolk deposition as well as a total time of follicular growth based on the number of yolk rings.To verify that the 2d period of incubation does not change the yolk ring layering, we will initially compare subsamples of freshly collected yolks under the chronic stress conditions to those of yolks that have undergone initial embryonic development.Growth rates and periods from chronically stressed and CORT treated birds will be compared to those from control birds, and growth rates will also be compared with the sexes of embryos collected from the same eggs.Sexes will be coded as 0 for female and 1 for male, and sexes will be analyzed in relation to follicular growth rate using a logistic regression. This experiment will determine whether chronic stress and CORT implants depress follicular growth, and whether follicular growth relates to offspring sex in this species.Hypothesis 3: CORT acts directly at the time of meiotic segregation to skew offspring sex ratiosEXPERIMENT 3a: Treatment with a long-lasting CORT agonist during meiotic segregation:We plan to utilize the long-lasting CORT agonist, 3,5-dihydroxyphenylglycine (DHPG), injected 6h prior to ovulation to determine whether a longer-lasting physiological CORT elevation "catches" the critical window necessary to induce changes in offspring sex ratios. 120 birds will receive either (1) CORT agonist injection or (2) a control injection 6h prior to ovulation, which corresponds to about 2h before meiotic segregation resumes and completes.Oocytes ovulated 6h following injection will take 24h to pass through the oviduct and will thus be collected 29h after injection, incubated, and embryos will be molecularly sexed to determine offspring sex ratios.From a separate set of females (n = 20 per group) blood samples will be collected 1h and 4h following injections to determine the effects of the CORT agonist on circulating CORT and testosterone concentrations.EXPERIMENT 3b: Amelioration of CORT-induced sex ratios skews by acute exposure to a CORT antagonist: To approach the same question in another way, we will implant birds with either a CORT or control implant (n=60 per group, similar to expt. 1 above) and treat half of those birds with RU-486, a glucocorticoid receptor antagonist, at the time of meiotic segregation. Injections of RU-486 will take place 5h prior to ovulation, which corresponds to 1h prior to the completion of chromosome segregation.Eggs will be collected and sexed as described above, and blood samples will also be collected from a separate set of hens as described above.Hypothesis 4: CORT acts through testosterone or another unidentified mediator to influence processes of sex chromosome segregationEXPERIMENT 4: Treatment with Chronic CORT and Testosterone:During this experiment, we will chronically elevate CORT concentrations using a CORT implant as described above, and in a subset of these implanted birds, we will also add a testosterone implant to prevent the CORT-related depression of testosterone concentrations that would normally occur. Birds will receive one of the following (n = 60 per group): (1) 2 1-inch silastic implants (CORT and T)(crystalline CORT and Testosterone obtained from Sigma-Aldrich Co., St. Louis, MO) (2) 2 blank implants (BL), (3)1 T and 1 BL, or 4) 1 CORT and 1 BL. Eggs will be collected 7d and 14d after introduction of the implants. Eggs will be incubated for 8d and embryos will be molecularly sexed as described above. Blood samples will be collected from a separate set of hens 2d, 7d, 10d, and 14d following introduction of the implants to monitor CORT and testosterone concentrations throughout the experiment.