Progress 10/01/03 to 09/30/08
Outputs
OUTPUTS: Several years ago, while processing reproductive tracts from roosters, we found many small stones in the reproductive tract, especially in the tubules carrying sperm. We also found that fertility and serum testosterone concentrations were low in roosters with stones. Our initial hypothesis was that the stones were the result of a high calcium diet because the roosters ate the same diet as laying hens. Extensive testing of diets with different amounts of calcium and vitamin D had no effect on the number of stones in the tract. Next, we tested the hypothesis that the stones were the result of roosters being immunized prepubertally against the avian infectious bronchitis virus (AIBV). This virus has a tropism for ciliated cells such as those found in the trachea, the reproductive tract, and kidney. Specific pathogen free (SPF) eggs were hatched and roosters were immunized at 2, 6, 10, and 14 weeks of age with the live attenuated AIBV according to the commercial schedule or not immunized (controls). At 26 weeks of age, which is about 10 weeks after roosters start producing sperm, 80% of roosters immunized with AIBV had stones, decreased daily sperm production, and lower serum testosterone concentrations. In a second study, we tested if immunization with a killed AIBV would also cause epididymal stones in adult roosters. We found that the killed AIBV triggered a rapid and robust antibody response and also epididymal stones. Next we did an in vitro study to test the ability of AIBV to enter the ciliated cells of the epididymis and undergo replication. Using 3"RACE we showed that AIBV entered and replicated in the epididymal region. To understand the etiology of the stones, we examined the roosters at 12, 16, 20, and 24 weeks of age to determine when stones first appeared following immunization with AIBV. Numerous stones were being formed in the epididymis as early as week 20. We did find that the formation of stones following AIBV immunization did not occur in roosters immunized with the Newcastle virus, another common avian pathogen. Early studies indicated that fertility was significantly reduced, following either natural mating or artificial insemination, as indicated by the number of eggs hatched when sperm were obtained from roosters with stones. Extensive studies were done to determine the cause for this reduced fertility. Sperm concentrations, sperm viability, sperm motility, sperm quality and fertilizing ability, acrosome integrity, in vivo sperm fertility, and serum testosterone concentrations were examined among roosters immunized against AIBV compared with non-immunized roosters. We found that pre-pubertal vaccination against AIBV had a limited effect on these different endpoints. Using a commercial kit to measure antibody titers against AIBV, we found AIBV antibodies in the blood serum as expected but also in the seminal fluid. Western blot analysis indicated that these AIBV present in blood serum and seminal plasma reacted with sperm membranes. Further analysis using 2-D gel analyses and sequencing of the proteins bands of interest showed that AIBV antibodies reacted with several proteins unique to the sperm membrane. PARTICIPANTS: Nothing significant to report during this reporting period. TARGET AUDIENCES: Nothing significant to report during this reporting period. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
The formation of stones in the rooster reproductive tract is an international problem. The presence of stones has a significant negative impact on fertility. In the broiler industry, hatchability is 83%. A 1% increase in hatchability/year would increase profits $36-38 M. Therefore any management change that could increase fertility even slightly will have a significant impact on the financial gains of the poultry industry. For this reason, the findings from our research on the role of AIBV in causing reproductive impairment in the rooster are of significant value to the poultry industry and to the production of other animals that are immunized. Our findings do not only apply to poultry but even have implications for humans. There are a number of outcomes from our research. 1) Immunization with either the live attenuated or killed AIBV causes stones in the reproductive tract of the rooster and has a negative impact on fertility. 2) Stones are formed in young roosters, even within four weeks after roosters have started to produce sperm and cause erosion of the epithelial cells which are responsible for the transfer of ions, fluids, and proteins in and out of the reproductive tract. The correct ion concentration and osmolarity of the epididymal fluid are necessary for proper sperm maturation. The AIBV can enter and replicate in the ciliated cells of the reproductive tract; however, this replication is not the primary cause for the formation of epididymal stones. Sperm produced by roosters with epididymal sperm are impaired so that fertility is reduced whether under natural mating circumstances or the use of artificial insemination. In response to immunization with the AIBV, anti-AIBV antibodies are found in the blood serum but also in the seminal plasma. These anti-AIBV antibodies bind to sperm membranes and cause clumping of sperm. The binding of anti-AIBV antibodies to sperm membranes indicates that AIBV shares one or more common epitopes with proteins in sperm membranes. Clumps of sperm, as indicated by the staining for actin, were visible as calcification of the clumps was occurring. To eliminate the formation of epididymal stones, it is necessary to identify the common epitopes shared by AIBV and sperm membrane proteins and then synthesize fragments of AIBV which will still induce immunity against this virus but will not result in the production of epididymal stones and significant reduction of fertility.
Publications
- Bahr, J.M., Dalponte, M., Janssen, S.J. and Nakai, M. 2006. Ion transporters for fluid resorption in the rooster (Gallus domesticus) epididymal region. Animal Reproduction Science 95 331-337.
- Boltz, C.R., Belton, R.A., Boltz, D.A., Dirks, A., Bunick, D., Scherba, G. and Bahr, J.M. 2007. Vaccination of roosters against avian infectious bronchitis virus results in autoimmunity to sperm (Submitted).
- Janssen, S.J., Kirby, J.D., Hess, R.A., Rhoads. M., Bailey, K., Bunick, D., Wang, H. and Bahr, J.M. 2000. Characterization of a new poultry disease: Epididymal calcium stones in diverse rooster populations. Poult Sci 79:568-574.
- Boltz, D.A., Nakai, M. and Bahr, J.M. 2004. Avian infectious bronchitis virus: A possible cause of reduced fertility in the rooster. Avian Diseases 48:909-915.
- Jackson, U., Boltz, D.A., Nakai, M., Bunick, D., Scherba, G. and Bahr, J.M. 2006. Prepubertal exposure to the avian infectious bronchitis virus induces epididymal stones in the rooster after puberty. Journal of Poultry Science 43:280-285.
- Jackson, U.H. 2004. Avian infectious bronchitis virus (AIBV) vaccine: Possible cause of epididymal region stone formation. M.S. thesis, University of Illinois.
- Boltz, D.A. 2005. Avian infectious bronchitis virus: A tropism for the rooster reproductive tract and effects on reproductive performance. Ph.D. dissertation, University of Illinois.
- Zimmerman, C.R. 2005. Vaccination of roosters against avian infectious bronchitis virus (AIBV): Elucidating the problem of reduced fertility by sperm quality analysis and detection of sperm autoimmunity. M.S. thesis, University of Illinois.
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Progress 01/01/07 to 12/31/07
Outputs
OUTPUTS: We have previously reported that roosters vaccinated with the live attenuated avian infectious bronchitis virus (AIBV) have decreased serum testosterone concentrations, epididymal stones, and reduced fertility. The objectives of our research were to determine: 1) if the epididymal stones were caused by anti-AIBV antibodies binding to sperm membranes, and 2) if the formation of epididymal stones was due specifically to immunization against AIBV or could also be caused by immunization against another common viral disease, Newcastle disease. To accomplish the first objective, specific pathogen free Leghorn roosters were divided into 3 groups that received one of the following treatments: no vaccination (NONVAC), vaccination with the killed virus (KVAC), or vaccination with the live attenuated virus (LVAC). Semen was collected from NONVAC, sperm obtained and sperm membrane proteins were isolated. Sperm membrane proteins were run on a western blot. Blood serum and epididymal
fluid obtained from the three groups of roosters as well as blood serum obtained from NONVAC and LVAC hens were used as the source of the primary antibody. Also testis and the reproductive tracts from NONVAC roosters were removed, fixed and histological sections prepared. Immunohistochemistry was performed by reacting the sections with blood serum from NONVAC and LVAC. To accomplish the second objective, 16 specific free roosters were divided into two groups: NONVAC and Newcastle virus vaccinated. Blood was taken every other week starting at week 16 and semen was collected weekly starting at week 18. Roosters were euthanized at 30 weeks of age to check for presence of epididymal stones.
PARTICIPANTS: David Boltz, graduate student, Claire Zimmerman (Boltz), graduate student, Joseph Esch, undergraduate student, and Jessica Sweet, undergraduate student.
TARGET AUDIENCES: Poultry scientists, industry, and vaccine manufacturers.
Impacts
The performance of western blots using sperm membrane proteins from NONVAC reacted with blood serum or epididymal fluid from the NONVAC, LVAC, and KVAC roosters and blood serum from NONVAC and LVAC hens indicated that there were antibodies in the blood serum and epididymal fluid of LVAC, KVAC and LVAC hens that bound to sperm membrane proteins. These results were confirmed with immunohistochemistry. Vaccination with the Newcastle virus did induce a robust antibody response in the roosters as indicated by the measurement of the Newcastle viral antibodies in the blood. However, vaccination with this virus, in contrast to immunization with AIBV, did not cause epididymal stones nor any change in number of sperm ejaculated. These results clearly show that immunization with AIBV causes the production of antibodies which are present in the blood serum and the epididymal fluid and which bind to sperm membranes. Previous results showed the clumping of sperm in the reproductive
tract as early as 20 weeks of age. The clumping of sperm is the precursor to stone formation which also induces an inflammation of the region, as indicated by the infiltration of immune cells. The binding of AIBV antibodies to sperm membranes appear to be specific for AIBV because immunization with the Newcastle virus did not cause stone formation. Apparently AIBV antibodies and sperm membranes share several common epitopes, also referred to as molecular mimicry. The importance of these findings is that new vaccines need to be developed which will provide protection against AIBV but will not cause the formation of antibodies which will bind to sperm membranes.
Publications
- Boltz, D.A., Zimmerman, C.R., Nakai, M., Bunick, D., Scherba, G. and Bahr, J.M. 2006. Epididymal stone formation and decreased sperm production in roosters vaccinated with a killed strain of avian infectious bronchitis virus. Avian Diseases 50:594-598.
- Boltz, C.R., Boltz, D.A., Bunick, D., Scherba, G. and Bahr, J.M. 2007. Vaccination against the avian infectious bronchitis virus affects sperm concentration, sperm quality, and blood testosterone concentrations in cockerels. British Poultry Science 48; 617-624.
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Progress 01/01/06 to 12/31/06
Outputs
Specific pathogen free Leghorn roosters were divided into 3 groups that received one of the following treatments: no vaccination (NONVAC), vaccination with the killed virus (KVAC), or vaccination with the live attenuated virus (LVAC). Semen was collected daily from wks 17-27 and assayed for sperm concentration, viability, motility, and fertility. Blood plasma was also collected for testosterone radioimmunoassay. At week 16, the LVAC group had a significantly reduced testosterone concentration (1.75 +/- 0.4 ng/mL) compared to KVAC (3.50 +/- 0.7 ng/mL), but not when compared to NONVAC (2.66 +/- 0.5 ng/mL). At week 19, the LVAC treatment group had a significantly decreased average sperm concentration (2.9 +/- 0.2 billion sperm/mL) compared to both the NONVAC (3.7 +/- 0.1 billion sperm/mL) and KVAC (3.4 +/- 0.3 billion sperm/mL) groups. At week 20, the NONVAC group had significantly more percent viable sperm (97.9 +/- 0.3) than both the KVAC (92.4 +/- 0.6) and LVAC (91.1
+/- 0.7) groups. Also at week 20, the LVAC treatment group had significantly decreased metabolic ability to reduce the tetrazolium dye MTT (0.11 +/- 0.01) when compared to both NONVAC (0.15 +/- 0.01) and KVAC (0.15 +/- 0.01) groups. At week 22, the NONVAC group had significantly less percent viable sperm detected by flow cytometry (95.6 +/- 0.3) than both the KVAC (97.4 +/- 0.2) and LVAC (96.6 +/- 0.6) treatment groups, which correlates to the results of the MTT reduction assay for that week. MTT reduction results for week 22 indicated that the NONVAC group had significantly reduced sperm metabolic activity (0.08 +/1 0.01) when compared to both KVAC (0.14 +/- 0.01) and LVAC (0.17 +/- 0.02). Also at week 22, the NONVAC group had statistically less acrosome reacted sperm (3.45 +/- 0.27) than both the KVAC (5.08 +/- 0.49) and LVAC (5.61 +/- 0.76) groups. At week 25, KVAC had significantly less acrosome damage (4.06 +/- 0.19) than both the NONVAC (8.95 +/- 1.92) and LVAC (6.77 +/- 0.64)
groups. At week 27, KVAC had significantly less acrosome damage (2.5 +/- 0.14) than the NONVAC group (6.19 +/- 1.1), but did not statistically differ from the LVAC group (4.32 +/- 0.64). There were no significant differences detected in sperm motility through Accudenz or in numbers of sperm holes in the perivitelline membranes of eggs following insemination with semen from 27 week old roosters across treatment groups. In this study we primarily found differences in sperm viability and acrosome integrity in the KVAC and LVAC groups during wks 19-22, which correlates to the period when epididymal stones were beginning to form, based on previous work. Interestingly, these changes in sperm quality were not consistent throughout the subsequent weeks in the study. These unexpected findings may be explained by the young age of the birds or the frequency of ejaculations, because daily ejaculations promote sperm viability, motility, and increased sperm concentrations in semen. Therefore
exposure to either the live or killed AIBV can have deleterious effects on the sperm. We also determined if roosters were making sperm antibodies which could account for the clumping of sperm and subsequent stone formation.
Impacts
These data confirm our previous data that immunization of roosters against the avian infectious bronchitis virus, whether a live attenuated or killed virus, epididymal stones are formed and sperm quality is affected. Furthermore, it appears that the AIBV and sperm share some common epitopes so that when roosters make antibodies against AIBV these antibodies are also recognizing sperm membranes. To prevent the formation of epididymal stones, it will be necessary for immunization to use some part of the AIBV which does not share common epitopes with sperm membrane proteins.
Publications
- Boltz, D.A., Nakai, M. and Bahr, J.M. 2004. Avian infectious bronchitis virus: A possible cause of reduced fertility in the rooster. Avian Diseases 48:909-915.
- Jackson, U., Boltz, D.A., Nakai, M., Bunick, D., Scherba, G. and Bahr, J.M. 2006. Prepubertal exposure to the avian infectious bronchitis virus induces epididymal stones in the rooster after puberty. Journal of Poultry Science 43:280-285.
- Boltz, D.A., Zimmerman, C.R., Nakai, M., Bunick, D., Scherba, G. and Bahr, J.M. 2006. Epididymal stone formation and decreased sperm production in roosters vaccinated with a killed strain of avian infectious bronchitis virus. Avian Diseases (In Press).
- Bahr, J.M., Dalponte, M., Janssen, S.J. and Nakai, M. 2006. Ion transporters for fluid resorption in the rooster (Gallus domesticus) epididymal region. Animal Reproduction Science 95 331-337.
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Progress 01/01/05 to 12/31/05
Outputs
Specific pathogen-free Leghorn roosters were divided into 3 groups that received one of the following treatments: no vaccination (NONVAC), vaccination with the killed virus (KVAC), or vaccination with the live attenuated virus (LVAC). Semen was collected daily from wks 17-27 and assayed for sperm concentration, viability, motility, and fertility. Blood plasma was also collected for testosterone radioimmunoassay. At week 16, the LVAC group had a significantly reduced testosterone concentration (1.75 + or - 0.4 ng/mL) compared to KVAC (3.50 + or - 0.7 ng/mL), but not when compared to NONVAC (2.66 + or - 0.5 ng/mL). At week 19, the LVAC treatment group had a significantly decreased average sperm concentration (2.9 + or - 0.2 billion sperm/mL) compared to both the NONVAC (3.7 + or - 0.1 billion sperm/mL) and KVAC (3.4 + or - 0.3 billion sperm/mL) groups. At week 20, the NONVAC group had a significantly higher percentage of viable sperm (97.9 + or - 0.3) than both the KVAC
(92.4 + or - 0.6) and LVAC (91.1 + or - 0.7) groups. Also at week 20, the LVAC treatment group had significantly decreased metabolic ability to reduce the tetrazolium dye MTT (0.11 + or - 0.01) when compared to both NONVAC (0.15 + or - 0.01) and KVAC (0.15 + or - 0.01) groups. At week 22, the NONVAC group had significantly less viable sperm detected by flow cytometry (95.6 + or - 0.3) than both the KVAC (97.4 + or - 0.2) and LVAC (96.6 + or - 0.6) treatment groups, which correlates to the results of the MTT reduction assay for that week. MTT reduction results for week 22 indicated that the NONVAC group had significantly reduced sperm metabolic activity (0.08 + or - 0.01) when compared to both KVAC (0.14 + or - 0.01) and LVAC (0.17 + or - 0.02). Also at week 22, the NONVAC group had statistically less acrosome reacted sperm (3.45 + or - 0.27) than both the KVAC (5.08 + or - 0.49) and LVAC (5.61 + or - 0.76) groups. At week 25, KVAC had significantly less acrosome damage (4.06 + or -
0.19) than both the NONVAC (8.95 + or - 1.92) and LVAC (6.77 + or - 0.64) groups. At week 27, KVAC had significantly less acrosome damage (2.5 + or - 0.14) than the NONVAC group (6.19 + or - 1.1), but did not statistically differ from the LVAC group (4.32 + or - 0.64). There were no significant differences detected in sperm motility through Accudenz or in numbers of sperm holes in the perivitelline membranes of eggs following insemination with semen from 27 week old roosters across treatment groups. In this study we primarily found differences in sperm viability and acrosome integrity in the KVAC and LVAC groups during wks 19-22, which correlates to the period when epididymal stones were beginning to form, based on previous work. Interestingly, these changes in sperm quality were not consistent throughout the subsequent weeks in the study. These unexpected findings may be explained by the young age of the birds or the frequency of ejaculations, because daily ejaculations promote sperm
viability, motility, and increased sperm concentrations in semen. In conclusion, the finding that the exposure to a live attenuated or killed vaccine can affect sperm quality is significant.
Impacts
These data confirm our previous data that during immunization of roosters against the avian infectious bronchitis virus, whether a live attenuated or killed virus, epididymal stones are formed and sperm quality is affected.
Publications
- Zimmerman, C.R., Boltz, D.A. and Bahr, J.M. 2005. Exposure to the avian infectious bronchitis virus: A model to study viral effects on sperm function. Biology of Reproduction, Abst. # 194, p. 125.
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Progress 01/01/04 to 12/31/04
Outputs
Several years ago we discovered stones in the reproductive tract (epididymal region) of roosters of the laying and broiler breeder strains. These stones, composed of calcium carbonate, are associated with a decrease in testosterone concentrations and sperm production and a 35% reduction in fertility. These stones have been found in roosters throughout the United States, Brazil and Japan. It was suggested that immunization with the live attenuated avian infectious bronchitis virus (AIBV) may be the cause. To test this hypothesis, we have used specific pathogen free (SPF) roosters. At 2,6,10, 14 weeks after hatching, one group of roosters was immunized with the live AIBV whereas the other group of roosters was not immunized. In experiment 1, roosters were euthanized at 12, 16, 20 and 26 weeks of age and the epididymal region was prepared for histology. No stones were present in the epididymal region of roosters at 12 and 16 weeks of age whereas stones were forming and
were present in the epididymal region of roosters at 20 and 26 weeks of age, respectively. Further analysis of the stones indicated that they contained sperm. It appears that as the stones form, the core of stone is cellular debris around which calcium is deposited. In experiment 2, we used control roosters (not immunized) and roosters immunized with the live attenuated AIBV at 2,6,10, and 16 weeks of age and seminal plasma was collected from adult roosters and analyzed for amino acids and proteins by 1D SDS-PAGE and 2D gels. The concentrations of a number of amino acids, specifically tyrosine, arginine, and histidine in seminal plasma were lower in the seminal plasma of roosters with epididymal stones. Results from the 1D SDS-PAGE indicated the absence of a 36 kDa and 100 kDa in seminal plasma collected from roosters with epididymal stones. Results from 2 D gels indicated also a difference in proteins in seminal plasma from roosters with stones compared to roosters without stones. In
experiment 3, we characterized differences in sperm concentration, morphology, sperm storage and fertilization between vaccinated and non-vaccinated roosters. Semen samples for vaccinated roosters contained less sperm than semen samples from non-vaccinated roosters. Many sperm from vaccinated roosters retained the cytoplasmic droplet. Examination of the perivitelline membrane for sperm holes as an indication of the number of sperm that attempted to the egg indicated that fewer sperm from vaccinated roosters attempted to or were able to fertilize eggs compared the sperm from non-vaccinated roosters. Collectively these results indicate that immunization of roosters with the live attenuated AIBV causes stones in the epididymal region, alters the protein and amino acid concentrations of the seminal plasma and decrease fertility.
Impacts
The purpose of this research is to increase profits for the poultry industry. Currently the hatchability of eggs of broiler breeders (chickens sold in grocery stores) is 83%. Just a 1% increase in hatchability would increase the profits of the broiler breeder industry $36 million annually. Hatchability could be increased 1% or more if the fertility of roosters was improved. The current practice of vaccinating roosters with the live attenuated avian infectious bronchitis virus which causes stones in the reproductive tract and reduces fertility 35% needs to be changed to improve profit for the poultry industry.
Publications
- Boltz, D.A., Nakai, M. and Bahr, J.M. 2004. Avian infectious bronchitis virus: A possible cause of reduced fertility in the rooster. Avian Diseases 48:909-915.
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Progress 01/01/03 to 12/31/03
Outputs
A recent discovery in our lab was the detection of epididymal stones in the rooster reproductive tract. These stones, composed of calcium carbonate, were shown to be associated with a decrease in testosterone concentrations, sperm production, and fertility. The reduction in fertility was 30% in birds with epididymal stones. Epididymal stones were shown to be present in a number of strains across the United States and were also reported in Japan and Brazil. The cause of the epididymal stones is unknown. It was proposed that the cause of the epididymals stones is avian infectious bronchitis virus (AIBV). The current project of determining the effects of vaccination with live attenuated AIBV on the development of epididymal stones is still under investigation. We have found that vaccination of roosters with AIBV results in the formation of epididymal stones whereas roosters not vaccinated did not develop stones. We also plan to determine if the virus is capable of
infecting tissues in culture. Further studies will be done to determine if the AIBV can infect the reproductive tract in vivo. These finding are significant because AIBV is routinely vaccinated for in the industry and if the vaccination is the cause of these epididymal stones then vaccination poses a significant problem to the industry. The fact that the vaccine AIBV is capable of infecting the reproductive tract is also a significant finding. AIBV has been shown to infect non-respiratory tissue such as the oviduct. If the virus infects the oviduct, the oviductal tissue can be damaged and results in poor laying efficiency. A similar problem may be occurring in the male reproductive tract. If the virus is capable of infecting the rooster reproductive tract then it could be a direct cause of the stone formation resulting in decreased fertility. We are also investigating the decrease in testosterone and its effects on epididymal stone formation. Testosterone has been shown to be
decreased in birds that were vaccinated and had stones. It is possible that the formation of the epididymal stones is caused by the decrease in testosterone. The decrease in testosterone may alter the normal functions of the rooster reproductive tract resulting in stone formation. Recent data indicate that sperm are clumping in the epididymal tract of vaccinated roosters but not in non-vaccinated birds. Seminal plasma of vaccinated birds contains larger amounts of proteins than seminal plasma from non-vaccinated roosters.
Impacts
We have shown that vaccination with a virus can result in a decrease in fertility.
Publications
- Boltz, D. 2002. Avian infectious bronchitis virus: A possible cause of reduced fertility in the rooster. M.S. Thesis, University of IL, Urbana-Champaign.
- Boltz, D., Nakai, M. and Bahr, J. 2003. Avian infectious bronchitis virus: A possible cause of reduced fertility in the rooster. Avian Diseases (Submitted).
- Boltz, D. and Bahr, J. 2003 Avian infectious bronchitis virus targets the epididymal region of the rooster reproductive tract potentially reducing fertility. XXII World's Poultry Congress (Submitted).
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