Echographic and thermographic examination of bull testis

1. Echographic and thermographic examination of bull testis György Gábor, DVM, Ph.D senior scientist Res. Inst. For Animal Breeding and Nutr. HERCEGHALOM, HUNGARY

2. Introduction • The aim of these examinations: • to determine if ultrasonic echotexture and endocrine status is associated with semen production of bulls • to determine the change in scrotal surface temperature (SST) after GnRH treatment • to select those young bulls which are certainly not useful for the AI before the semen examination

3. Material and Methods • Experimental animals: H-F AI bulls 6-79 months of age in several examinations between 1994 and 1997 • Morphological examinations: scrotal circumference (SC): Coulter scrotal tape scrotal width (SCW) and and length (SCL) 1. digitalization of the video image: capture card 2. evaluation of the video image: Testigabsas software

4. Coulter Scrotal Tape

5. Evaluation of the video image (Testigabsas software)

6. Examination of echotexture 1. • The ultrasonic examination of the testes was done with a B-mode scanner (Pie Medical, Model 450) with a 7.5 MHz linear-array transducer. Gain was held constant for all examinations. • The transducer was aligned at the center of each testis and perpendicular to the vertical axis. A custom, electromechanical device for holding the probe was used to assure that the same pressure was applied by the probe against tissues upon which liberal amounts of ultrasound coupling gel had been applied. • At approximately 0.76 kg/cm2 pressure, an indicator lamp lighted as a spring-loaded switch was compressed. At that instance the ultrasound-image was frozen on the scanner console and the image was captured and stored.

7. The custom electromechanical device

8. Examination of echotexture 2.(Testigabsas software) • Image analysis was used for the evaluation of ultrasonic images. The ultrasonic images of left and right testes are aligned and recorded on the left panel. After making a distance-calibration an examination area (box) was created (6.5 x 1.0 cm, area: 6.5 cm2). The box was moved to the surface of the testicular tissue just under the scrotal skin and fat (white area). The average gray level (termed echotexture) and a histogram of the different gray levels (right panel) of the examined area were presented immediately. The bar below the histogram is of gray levels with zero on the left (dark) and 64 on the right (light). All of the nominal values were saved into a data file.

9. Examination of the scrotal surface temperature (SST) • An im injection of 100 μg GnRH was administered. The SST was measured before and 45 min. after treatment. An AGEMA 880 LWB infrared thermography camera was used. • Thermograms were stored on a computer, and subsequently analyzed with Irwin2 image analysis software. The SST at the top of the scrotum (SSTTOP) and the SST at the bottom of scrotum (SSTBOT) were measured, using a line one pixel high and the width of the scrotum.

10. Semen collection and evaluation • Collection by artificial vagina, twice a week. • Evaluation: • the total number of spermatozoa per ejaculate (TNS), the percentage of the live and motile spermatozoa by • HTM motility analyzer or • visual analysis

11. Endocrine measures • Serum testosterone concentration Blood samples were collected just before and 90 min after GnRH treatment. Samples were centrifuged and plasma frozen pending determination of plasma testosterone concentration by 3H radioimmunoassay. The sensitivity of the assay was 0.3 nmol and the intra- and inter-assay coefficients of variation were 7.1% and 11.7%, respectively.

12. RESULTS • Morphological examinations - SC is a highly repeatable measure r=0.75-0.95 P<0.0001 - examination of the video image has a lower repeatability SCL: r=0.25-0.54 SCW: r=0.42-0.88 CONCLUSION: using of SC is recommended

13. Testicular echotexture 1 • Relatively high repeatability in both, young and older bulls r=0.53-0.86; P<0.01-0.001 • Good correlation with the total number of spermatozoa per ejaculate in young bulls r= between -0.29 and -0.70 P= between 0.001 and 0.11

14. Testicular echotexture 2

15. Testicular echotexture 3 • A lower echotexture suggests a greater seminiferous tubule lumenal space and fluid content within the examined area of the testicular parenchyma. Older bulls expected to have greater seminiferous tubule activity, have a lower echotexture. Amount of blood content could also play a role. • CONCLUSION: Echotexture probably useful on practical level to predict semen production in young (12-36 months) bulls. The highly significant negative correlation between echotexture and TSN supports this concept. However in older bulls (60-79 months), there was no correlation with TSN and echotexture.

16. Change of SST after GnRH challenge 1 Thermal (SST, oC) data of 20 older bulls measured with 30 days interval Ambient 10 oC 20 oC 26 oC Correlation 1 2 3 1-2 2-3 1-3 TOP 1 33.7 ± .1 34.8 ± .2 36.4 ± .1 .28 .49b .21 TOP 2 35.1 ± .1 35.1 ± .2 36.3 ± .2 .39 .59b .35 TOP D 1.44 ± .1 .40 ± .1 -.10 ± .2 -.09 .49b .08 BOT 1 30.4 ± .2 30.8 ± .2 33.1 ± .2 .31 .56b .35 BOT 2 32.2 ± .2 31.5 ± .2 33.4 ± .2 .44a .67c .29 BOT D 1.80 ± .2 .70 ± .2 .30 ± .2 .30 .43a .38 GRAD 1 3.3 ± .2 3.9 ± .3 3.3 ± .3 .38 .56b .32 GRAD 2 2.9 ± .2 3.6 ± .2 2.9 ± .2 .43a .66b .40 GRAD D -.37 ± .1 -.3 ± .2 -.36 ± .2 .42a .53b .29

17. Change of SST after GnRH challenge 2

18. Change of SST after GnRH challenge 3 • SST increased following GnRH treatment. • SST measurements (after GnRH treatment) in young bulls were of some value in predicting the number of spermatozoa that could be collected. • However the ambient temperature has a greater effect on basal (pre-treatment) SST than SST following GnRH. These results support the combination of infrared thermography of SST and the GnRH challenge in order to decrease the effect of the ambient temperature.

19. The original aims were: • to determine if ultrasonic echotexture and endocrine status are associated with semen production of bulls • to determine the change in scrotal surface temperature (SST) after GnRH treatment • to select those young bulls which are certainly not useful for the AI before the semen examination

20. Replies 1 • Yes, in young bulls echotexture can be further optimized to improve the predictability of number of spermatozoa collected. • Yes, significant regression equations were derived for the total number of spermatozoa in older bulls; plasma testosterone concentrations were the significant independent variables: Experiment 1 Y = 5.72 + 0.076 Testosterone D Experiment 2 Y = 10.37 - 0.201 Initial Testosterone Experiment 3 Y = 11.30 - 0.094GnRH induced T.

21. Replies 1(cont.)

22. Replies 2 • SST increased following GnRH treatment both in young and older bull groups. • SST measurements (after GnRH treatment) were of some value in predicting the number of spermatozoa that could be collected. • BUT: The ambient temperature has a great effect on SST. Combination of infrared thermography of SST and the GnRH challenge could help to decrease the effect of the ambient temperature.

23. Final conclusions • SC, ET, and SST represent the scrotal and testicular characteristics referring to semen and endocrine measures • An andrological data base for males (bulls) may help to find those markers which are the most typical for the selection of those young bulls before the semen examination which are certainly not useful for the AI.

24. The future…. • New devices or improvement of the old ones?

25. Electronic tonometer

26. A custom thermograph

27. My right hand with the Computherm 1000