Source: UNIVERSITY OF FLORIDA submitted to NRP
THE INSULIN-LIKE GROWTH FACTOR SYSTEM AND STALLION INFERTILITY
Sponsoring Institution
National Institute of Food and Agriculture
Project Status
COMPLETE
Funding Source
ANIMAL HEALTH
Reporting Frequency
Annual
Accession No.
0186547
Grant No.
(N/A)
Cumulative Award Amt.
(N/A)
Proposal No.
(N/A)
Multistate No.
(N/A)
Project Start Date
Jan 1, 2000
Project End Date
Dec 31, 2005
Grant Year
(N/A)
Program Code
[(N/A)]- (N/A)
Recipient Organization
UNIVERSITY OF FLORIDA
G022 MCCARTY HALL
GAINESVILLE,FL 32611
Performing Department
COLLEGE OF VETERINARY MEDICINE
Non Technical Summary
Reduced fertility of breeding stallions causes significant economic loss in the equine industry. Inexplicable ("idiopathic") infertility is one of the most common diagnoses of reduced fertility in male mammalian species. The purpose of this project is to localize IGF I and II, IGF binding proteins and IGF receptors in the equine male genital tract and determine the effects of this system on equine fertility.
Animal Health Component
50%
Research Effort Categories
Basic
50%
Applied
50%
Developmental
(N/A)
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
3113810102025%
3113810100025%
3013810102025%
3013810100025%
Knowledge Area
311 - Animal Diseases; 301 - Reproductive Performance of Animals;

Subject Of Investigation
3810 - Horses, ponies, and mules;

Field Of Science
1000 - Biochemistry and biophysics; 1020 - Physiology;
Goals / Objectives
The objective of this project is to examine local factors, such as growth factors, that modulate testicular function. Specifically, insulin-like growth factor I (IGF-I), and insulin-like growth factor binding proteins (IGFBPs), identified in the testis and seminal plasma of other species, have been shown to have proliferative, differential, and metabolic effects on male germ cells. Deficiencies of IGF-I in other species have been associated with reduced fertility, sperm motility, and sperm morphology and concentration. We propose to characterize components of the IGF system in the equine male genital tract and examine the role of this system in equine infertility. Our long term goals are to localize IGF I and II, IGF binding proteins and IGF receptors in the equine male genital tract (seminal plasma, accessory sex glands, testes) and determine the effects of this system on fertility. Initially, we will assay equine seminal plasma for the presence of IGF I and II and the IGF binding proteins.
Project Methods
Ejaculates will be collected from horses of differing breeds but known fertility during the months of February through August (peak reproductive performance). All horses will be involved in on site breeding programs with complete breeding records. Ejaculates will be collected from each horse at sexual rest and at daily sperm output. Daily sperm output is defined as the number of sperm produced by an individual once the extra gonadal reserves have been evacuated. Semen will be collected from horses using a Missouri model artificial vagina and a live mare or phantom mount source. Concentration will be determined using a densimeter standardized for use with equine semen. Sperm motility will be videotaped on a negative phase contrast microscope fitted with a camera and connected to a video recorder. Videotapes of sperm motility will be interfaced with a computerized sperm motility analyzer system. A total of 400 cells will be evaluated and analyzed for motility. An aliquot of sperm will be preserved in buffered formal saline for morphologic evaluation. Two hundred cells will be assessed for morphologic abnormalities using a wet mount and differential interference contrast microscopy. One operator will perform all morphologic evaluations. Semen samples collected in the field will be centrifuged at 2000 x g for 10 minutes in a portable centrifuge at 20 C. The seminal plasma supernatant will be separated from the sperm pellet. Field samples will be maintained on ice until further processing. All samples will be centrifuged at 2000 x g for 30 minutes at 4 C. Centrifuged seminal plasma will be filtered , using 5.0 and 1.2 micron nylon syringe filters, in tandem, to remove any remaining sperm. Samples will be stored in 500 micro liter aliquots at -80 C until analyzed. The sperm pellet will be reserved at -80 C for additional analysis. Insulin-like growth factor I concentrations will be measured in seminal plasma samples using a standard radioimmunoassay that was validated for IGF-I in stallion seminal plasma by Dr. Simmen. Protein concentration will be determined for all samples using a commercially available protein assay kit. Prepared seminal plasma samples will be evaluated for presence of IGFBPs using Western ligand blotting and immunoprecipitation techniques validated for equine seminal plasma. Binding proteins identified using Western ligand blotting will be quantified by phosphorimaging. Logistic regression models will be used to investigate the effect of IGF-I, morphology, motility and concentration on pregnancy. In the logistic regression models, adjusted odds ratios and 95% confidence intervals will be reported. The significance level will be set at P less than 0.05.

Progress 01/01/00 to 12/31/05

Outputs
This project has been completed. Findings showed that Insulin-like growth factor-I, IGFBP-2 and IGFBP-5 were present in equine seminal plasma. Concentrations of IGF-I, IGF-I/protein, total IGF-I, IGFBP-2, IGFBP-2/protein and total IGFBP-2 were not significantly different (P > 0.13) in seminal plasma between stallions at either sexual rest (SR) or sexually active (SA). At SR, stallions having higher seminal plasma IGF-I had more total IGFBP-2/ejaculate (P < 0.01), morphologically normal sperm (P = 0.05) and higher first cycle pregnancy rates (P = 0.02). At SA, stallions having higher seminal plasma IGF-I had a lower number of cycles per pregnancy (P = 0.02). An association of seminal plasma IGF-I concentration with sperm motility, sperm morphology and pregnancy rates in bred mares suggests that IGF-I may play a role in sperm function.

Impacts
Identifying a relationship between insulin-like growth factors and associated binding proteins with fertility contributes to understanding factors in seminal plasma that contribute to subfertility in stallions. Breeding in horses is a multi-billion dollar industry. Male factor fertility issues in successful equine athletes has a significant economic impact. Better understanding of the physiology of factors in equine semen contributes to resolution of this problem.

Publications

  • Macpherson ML, Simmen RCM, Simmen FA, Hernandez J, Sheering BR, Varner DD, Loomis P, Cadario ME, Miller CD, Brinsko SP, Rigby S, Blanchard TL. Insulin-like growth factor-I and insulin-like growth factor binding proteins-2 and -5 in equine seminal plasma: association with sperm characteristics and fertility. Biology of Reproduction 2002;67:648-654.
  • Macpherson ML, Simmen R, Simmen F, Hernandez J, Sheerin B, Varner D, Cadario M, Miller C, Loomis P, Brinsko S, Rigby S, Blanchard T. Insulin-like growth factors and associated binding proteins in equine seminal plasma. Animal Reproduction Science 2001;68:347-348.
  • Macpherson ML, Simmen RCM, Simmen FA, Sheerin BR, Miller CD, Loomis P. Insulin-like Growth Factor-I and Associated Binding Proteins in Seminal Plasma of Stallions. Dorothy Russell Havemeyer Foundation Workshop on Advanced Current Topics in Stallion Veterinary Practice, Krakow, Poland, September, 2000:34.


Progress 10/01/01 to 10/01/02

Outputs
Results reported for last year were accepted for publication in the journal Biology of Reproduction. Current work is geared toward identifying the IGF-I receptor on sperm using immunofluorescent techniques. The receptor has been positively identified using these techniques. The manuscript is in progress.

Impacts
Identifying the IGF-I receptor on the head of equine sperm further validates the role of IGF-I in fertility of stallions.

Publications

  • Macpherson ML,Simmen RCM,Simmen FA,Hernandez J,Sheerin BR,Varner DD,Loomis P,Cadario ME,Miller CD,Brinsko SP,Rigby S,Blanchard TL.Insulin-like Growth Factor-I and Insulin-like Growth Factor Binding Proteins-2 and -5 in Equine Seminal Plasma: Association with Sperm Characteristics and Fertility. Biol Reprod 2002:67;648-654.


Progress 10/01/00 to 10/01/01

Outputs
Ejaculates were collected from stallions to 1) determine if insulin-like growth factor I (IGF-I) and insulin-like growth factor binding proteins (IGFBPs) were present in seminal plasma of stallions, 2)compare semen parameters (IGF proteins, sperm numbers, morphology and motility) from stallions at sexual rest and when sexually active, 3) compare semen parameters between stallions with high and low seminal plasma IGF-I concentrations, and 4) examine the relationship between seminal plasma IGF-I concentrations and fertility parameters of stallions. Ejaculates were collected from stallions at sexual rest (SR; n = 51) and when sexually active (SA; n = 46). Concentrations of IGF-I and IGFBP-2 in seminal plasma samples were determined by radioimmunoassay. Presence of IGFBPs in equine seminal plasma was verified using Western ligand blot and immunoprecipitation procedures. Insulin-like growth factor-I, IGFBP-2 and IGFBP-5 were present in equine seminal plasma. Concentrations of IGF-I, IGF-I/protein, total IGF-I, IGFBP-2, IGFBP-2/protein and total IGFBP-2 were not significantly different (P 0.13) in seminal plasma between stallions at either SR or SA. At SR, stallions having higher seminal plasma IGF-I had more total IGF-I/ejaculate (P < 0.01), total IGFBP-2/ejaculate (P < 0.01), morphologically normal sperm (P = 0.05) as well as higher first cycle pregnancy rates (P = 0.03). At SA, stallions having higher seminal plasma IGF-I had more total IGF-I (P < 0.01), higher pregnancy rates per season (P = 0.05) and lower number of cycles per pregnancy (P = 0.04). An association of seminal plasma IGF-I concentration with sperm motility, sperm morphology and pregnancy rates in bred mares suggests that IGF-I may play a role in sperm function.

Impacts
Information from this work suggests a role forIGF-I in equine fertility. Determining the involvement of the IGF system in male-factor infertility may lead to diagnostic and therapeutic tools.

Publications

  • Macpherson ML,Simmen RCM,Simmen FA,Hernandez J,Sheerin BR,Varner DD,Loomis P,Cadario ME,Miller CD,Brinsko SP,Rigby S,Blanchard TL.Insulin-like Growth Factor-I and Insulin-like Growth Factor Binding Proteins-2 and -5 in Equine Seminal Plasma: Association with Sperm Characteristics and Fertility. Biol Reprod, submitted November 2001.