A Study of Morphological and Haemodynamic Determinants of Testicular Echotexture Characteristics in the Ram JENNIFER L. GIFFIN,* SARAH E. FRANKS,* JOSE RAFAEL RODRIGUEZ-SOSA, ANN HAHNEL,* AND P AWEL M. BARTLEWSKI* ,1 *Department of Biomedical Sciences, Ontario Veterinary College, University of Guelph, Guelph, Ontario, Canada N1G 2W1; and  Center for Animal Transgenesis and Germ Cell Research, New Bolton Center, School of Veterinary Medicine, University of Pennsylvania, Kennett Square, Pennsylvania 19104 The ultrasonographic image of an organ is a product of scattering and reflection of high-frequency ultrasound beams by discrete units of tissue. The number of acoustic tissue interfaces and vascularity affects the quantitative character- istics of grey-scale ultrasonographic images. This study was undertaken to examine the influences of scrotal/testicular integument and blood flow on testicular echotexture parameters in the ram. Serial ultrasonographic images were obtained during surgical castration of 7 Rideau Arcott rams aged 20–22 weeks. The first 2 sets of images were taken through the scrotum, prior to and after induction of anaesthesia. The third set was taken through the tunica vaginalis, the fourth set was obtained through the tunica albuginea, the fifth set was taken when the testicular cord and internal blood vessels were clamped, and the final set of images was recorded after allowing the blood to drain from dissected testicles (5 min). All images were then subjected to computerized image analyses and the testicles were pro- cessed for histology. The removal of the scrotal skin and tunica vaginalis both resulted in significant (P < 0.05) increments in numerical pixel values (NPVs) and pixel heterogeneity (standard deviation of pixel values) of the testicular parenchyma. There were no differences (P > 0.05) in testicular echotexture between images taken just before or after clamping the testicular cord vessels, or after draining. At all stages, NPVs were correlated (P  0.10) to the seminiferous tubule (ST) area and the ST lumen area, except for NPVs and the ST lumen area in images obtained through the tunica albuginea (P = 0.20). We concluded that: 1) attenuation of ultrasound waves by the scrotal skin and tunica vaginalis significantly altered testicular echotexture characteristics; 2) vascular blood flow did not affect the echotextural attributes of the rams’ testes; and 3) NPVs were a good indicator of ST microstructure in situ and ex vivo. Exp Biol Med 234:794–801, 2009 Key words: ultrasonography; computer-assisted image analysis; testis; seminiferous tubules; ram Introduction Scrotal ultrasonography is frequently performed to monitor male reproductive health. The use of this technique in both clinical and research settings offers many benefits as it allows for repeated and non-invasive visualization of the scrotal content in the same individuals (1–3). Recent studies in pre- and peripubertal bulls and rams have demonstrated that changes in ultrasonographic attributes of the testicular parenchyma are closely related to the histomorphological characteristics of seminiferous tubules (1) and the onset and efficiency of spermatogenesis (1, 2, 4–6). The use of grey-scale ultrasonographic imaging for microstructural and functional assessment of reproductive organs has greatly improved our understanding of the dynamic changes in testicular histophysiology (1, 5). Quantitative ultrasonogram analysis methods have greatly facilitated interpretation of ultrasonographic findings (7). The computer-assisted estimation of numerical pixel values (NPVs), expressed on the scale of 0 (‘‘absolute’’ black) to 255 (‘‘absolute’’ white), increases both the precision of results and the range of perceivable intensity variations that can be detected in the structures examined. NPVs are a measure of the echotexture or echogenicity of a tissue, which depends on its density and water/macromolecular content (7, 8). The ultrasonographic display of the tissue is a product of reflection and scattering of high-frequency ultrasound This study was funded by the Ontario Ministry of Agriculture, Food and Rural Affairs (PMB and AH), and the Natural Sciences and Engineering Research Council of Canada (AH). 1 To whom correspondence should be addressed at the Department of Biomedical Sciences, Ontario Veterinary College, University of Guelph, Guelph, ON, Canada N1G 2W1. E-mail: pmbart@uoguelph.ca Received December 9, 2008. Accepted March 27, 2009. 794 DOI: 10.3181/0812-RM-364 1535-3702/09/2347-0794$15.00 Copyright Ó 2009 by the Society for Experimental Biology and Medicine