Progress 09/01/08 to 08/31/11
Outputs
OUTPUTS: Male sub-fertility is a serious problem in many farm species. Current clinical tests have proven limited due to many other factors that are unrelated to overall fertility. There is emerging evidence that specific seminal plasma proteins (SP) associate with the sperm plasma membrane and facilitate sperm binding to epithelial cells in the oviduct. This creates a sperm reservoir that may influence fertility by maintaining sperm viability, suppressing motility and may be a useful predictor of male fertility. Therefore, identification of key SP proteins involved in sperm-oviduct binding may ultimately leads to tests that will allow limited resource farmers to predict, at an early age, the utility of goat sires used in animal production systems. In addition, this project will test for the first time whether goat sperm contain measurable amounts of mRNA that could represent activity of specific genes that are important to the health and function of the testis. PARTICIPANTS: D. Rucker and A. Faucette are African-American females that used research described above for their M.S. thesis and Ph.D. dissertation in Animal Science, respectively. TARGET AUDIENCES: Producers, student participants and breed organizations. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
Semen was collected monthly from Alpine (n=4) and Boar (n=4) bucks. Scrotal circumference and sperm concentration in the ejaculate was recorded. Scrotal circumference varied throughout the year (P<.05) and was greatest at the start of the breeding season for Alpine (29.9+/-0.98 cm) and Boer (32.7+/-0.98 cm) sires. Concentration of sperm in the ejaculate also varied throughout the year (P<.01). Highest concentrations were recorded during periods of sexual quiescence (April) in both Alpine (7.24+/- 0.74 x 109/ml) and Boer (6.41 plus/minus 0.74 x 109/ml) sires. Concentration of sperm was lowest during the breeding season in November/December for both Alpine (1.22+/-0.74 x 109/ml) and Boer (1.02+/-.074 x 109/ml) sires. Seminal plasma was separated from spermatozoa by centrifugation. Concentrations of proteins in seminal plasma gradually increased from August (Alpine - 43.10+/-16.96 mg/ml; Boer - 39.15+/-16.96 mg/ml) until the months of October - December when concentrations peaked sharply (P<.01) for Alpine and Boer sires (194.85+/-16.96 and 216.30 +/-23.98 mg/ml, respectively), then declined. 2D-SDS-PAGE revealed a complex profile of seminal plasma proteins. Major proteins were identified with approximate molecular weights of 139.0, 132.0, 96.7, 75.2, 61.0, 55.5, 41.6, 33.0, 22.0, 17.7, 16.9, 15.7 and 14.8 kDa. Of particular interest were a complex of low molecular weight (<20 kDa) acidic proteins. These abundant proteins were subjected to electrospray ionisation mass spectrometry. Mascot search results indicated the sequence of the 14.8 kDa protein was similar (Score = 127) to bodhesin-2 from Capra hircus. A second goal was to analyze alterations in gene expression in the goat testes during development and analyze alterations in gene products in sperm from mature bucks between breeding and non-breeding seasons. Testes were harvested from 5 Alpine bucks at 0, 2, 4, 6, and 8 mo of age. Northern blotting and in situ hybridization indicated that the largest changes in gene expression during testes development happen in the first 4 months in the goat. Sertoli cell marker Sex determining region Y-box 9 mRNA peaked at 2 mo of age then declined. Expression of Stimulated by Retinoic Acid gene and Protamine 1 genes was strongly upregulated in early and maturing germ cells, respectively. RNA from ejaculated sperm, collected during the breeding and non-breeding season, was interrogated for 44,000 gene products on Bovine Gene Expression Microarrays. 43 gene products were expressed 3-fold or more highly in peak breeding season, while concentrations of 12 mRNAs decreased 3-fold or more (P <0.01). 5-fold greater levels of glycerol kinase 2 mRNA in sperm from the peak breeding season and 6-fold lower levels of Sperm Adhesion Molecule 1 mRNA are being confirmed with real time PCR. Our expected output is to develop a simple accurate method that predicts the field fertility of fresh or frozen-thawed semen. Identification of key seminal plasma proteins that may be involved in sperm-oviduct binding may ultimately lead to tests that will allow limited resource farmers to predict, at an early age, the utility of goat sires used in animal production systems.
Publications
- Reevely, A.M., Tress, U., and Newton, G.R. 2008. Characterization of apical plasma membrane proteins carrying endometrial H-type 1 antigen. Proceedings of the Gordon Research Conference on Reproductive Tract Biology, Andover, New Hamphshire.
- Newton, G.R., Tress, U., and Ing, N.H. 2009. Alpha(1-2)fucosyltransferase gene expression by caprine endometrial tissues obtained during the estrous cycle and early pregnancy. Biol. Reprod. 81(Suppl. 1):319.
- Faucette, A.N., Forrest, D.N., Nuti, L.C., Newton, G.R. and Ing, N.H. 2011. Gene expression changes in goat testes during development and in sperm during the breeding and non-breeding seasons. J. Anim. Sci. 89 (Suppl. 2):W421.
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Progress 09/01/09 to 08/31/10
Outputs
OUTPUTS: Our working hypothesis is caprine seminal plasma contains specific proteins that are crucial for fertility. The identification of these proteins may lead to the development of a reliable, non-invasive marker for male fertility. To test this hypothesis, semen was collected monthly from Alpine (n=4) and Boar (n=4) bucks. Scrotal circumference and sperm concentration in the ejaculate was recorded. Scrotal circumference varied throughout the year and was greatest at the start of the breeding season for Alpine and Boer sires. Concentration of sperm in the ejaculate also varied throughout the year. Highest concentrations were recorded during periods of sexual quiescence (April) in both Alpine and Boer sires. Concentration of sperm was lowest during the breeding season in November/December for both Alpine and Boer sires. Weight of the total ejaculate collected each month did not differ. Seminal plasma was separated from spermatozoa by centrifugation and protein concentrations in seminal plasma were determined. Concentrations of proteins in seminal plasma gradually increased from August until the months of October - December when concentrations peaked sharply for Alpine and Boer sires and then declined. Isoelectric focusing and SDS-PAGE were used to characterize seminal plasma protein profiles in ejaculates. Analysis revealed a complex profile of seminal plasma proteins. Major proteins were identified with approximate molecular weights of 139.0, 132.0, 96.7, 75.2, 61.0, 55.5, 41.6, 33.0, 22.0, 17.7, 16.9, 15.7 and 14.8 kDa. Of particular interest were a complex of low molecular weight (less than 20 kDa) proteins with isoelectric points between pH 4 - 7. These abundant proteins were subjected to electrospray ionisation mass spectrometry. Mascot search results indicated the sequence of the 14.8 kDa protein was similar (Score = 127) to bodhesin-2 from Capra hircus. Bodhesin-2 belongs to a new family of animal lectins called spermadhesins and constitutes one of the main protein components of seminal plasma from pigs, cattle, horses, and sheep. Identification of key seminal plasma proteins may ultimately lead to tests that will predict, at an early age, the utility of sires used in animal production systems. PARTICIPANTS: L.C. Nuti, Ph.D. and G.R.Newton, Ph.D., Prairie View A&M University and N.H. Ing, Ph.D., Texas A&M University. TARGET AUDIENCES: Identification of key seminal plasma proteins that may be involved in spermâoviduct binding may ultimately lead to tests that will allow limited resource farmers to predict, at an early age, the utility of goat sires used in animal production systems. PROJECT MODIFICATIONS: The original objectives of the proposal are: 1) To quantify genetic diversity within and among 15 goat breeds located in Texas and the southeastern USA., and 2) To clarify the evolutionary genetic relationships among 15 goat breeds. The original Principle Investigator left the University on 10/1/09. Permission was received to narrow the focus to two breeds of goats, Alpine (dairy) and Boer (meat) breeds. Specific goals are look for markers for male fertility that can aid in selecting genetic resources for conservation and enhancement.
Impacts
Male sub-fertility is a serious problem in many farm species. Current clinical tests have proven limited due to many other factors that are unrelated to overall fertility. The biochemical composition of seminal plasma (SP) is complex, variable between species and likely influences several important biological processes. There is emerging evidence that specific SP associate with the sperm plasma membrane and facilitate sperm binding to epithelial cells in the oviduct. This creates a sperm reservoir that may influence fertility by maintaining sperm viability and suppressing motility. It has also been suggested that the capacity of sperm to bind oviductal explants in vitro might be a useful predictor of male fertility. Therefore, identification of key SP proteins involved in sperm-oviduct binding may ultimately leads to tests that will allow limited resource farmers to predict, at an early age, the utility of goat sires used in animal production systems.
Publications
- Baker, K.N., Horner, S.D., Rucker, D.K., Nuti, L.C. and Newton, G.R. 2010. The relationships between somatic cell count and bacteriology on milk quality and production in dairy goats. J. Anim. Sci. 88 (Suppl. 2):W44.
- Rucker, D., Baker, K., Tress, U., Nuti, L.C., and Newton, G.R. 2010. Characterization of seminal plasma proteins from dairy and meat goat sires. Biol. Reprod. 83 (Suppl. 1):532.
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Progress 09/01/08 to 08/31/09
Outputs
OUTPUTS: Major proteins were identified with approximate molecular weights of 139.0 132.0, 96.7, 75.2, 61, 55.5, 41.6, 33, 22, 17.7, 16.9, 15.7 and 14.9 kDa of particular interest were a complex of low molecular weight (<20 kDa) proteins with isoelectric points between pH 4.7. These proteins have physical characteristics that are strikingly similar to bovine seminal plasma proteins known to mediate sperm - oviduct interactions during estrus. MALI-TOF was used to identify these low molecular weight proteins. Analysis by the Protein Chemistry Laboratory at Texas A&M University is currently underway. PARTICIPANTS: Project participants include G.R. Newton and L.C. Nuti at Prairie View A&M University. They are responsible semen and tissue collections. They will conduct the proteomic analysis of seminal plasma proteins to identify potential markers for male fertility. N.H. Ing at Texas A&M University is a collaborator on the project. She is guiding the analysis on mRNA expression in sperm cells. Research Core facilities at Texas A&M University, including the Protein Chemistry Laboratory, are routinely used in these studies. Opportunities for minority student participation in research leading to the M.S. and Ph.D. degrees are provided by these studies. TARGET AUDIENCES: Scientific community and producers. PROJECT MODIFICATIONS: The original Project Director left the University on 10/1/08. The funding agency approved a change in the scope of work under a new Project Director (G.R. Newton). The original objective of the proposal indicated that "Genetic resources are the building blocks for all production systems. Conservation, maintenance, enhancement and access to these genetic resources will enable small goat producers to increase their profitability." One goal was to quantify genetic diversity within and among eight goat breeds located in Texas and the southeastern USA. Since the project was entering its fourth year in 2009, we propose to focus only on two breeds of goats, Alpine (dairy) and Boer (meat) breeds. Specifically we will look for markers for male fertility that can aid in selecting genetic resources for conservation and enhancement.
Impacts
Identification of key seminal plasma proteins that may be involved in sperm/oviduct binding may ultimately lead to tests that will allow limited resource farmers to predict, at an early age, the utility of goat sires used in animal production systems.
Publications
- Reevely, A.M., Tress, U., and Newton, G.R. 2008. Characterization of apical plasma membrane proteins carrying endometrial H-type 1 antigen. Proceedings of the Gordon Research Conference on Reproductive Tract Biology, Andover, New Hamphshire.
- Newton, G.R., Tress, U., and Ing, N.H. 2009. Alpha(1-2)fucosyltransferase gene expression by caprine endometrial tissues obtained during the estrous cycle and early pregnancy. Biol. Reprod. 81(Suppl. 1):319.
- Newton, G.R., Tress, U., Nuti, L.C., Johnson, B.M. and Rucker, D. 2009. Fertility Indices for Dairy and Meat Goat Sires. Proceedings of the Association of Research Directors, Inc. 15th Biennial Research Symposium. Abstract 184.
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