Jan 3, 1996 - FSH concentrations increase to the time of puberty (12,17,23,28,29). ... at 1500 x g for 15 min the serum was harvested and stored at -20°C until analysis. ... was fixed in Bouin's solution and then prepared for histological sectioning. ..... 6*2bc. 45*5a. 45*6a. 4*1a abc Values in columns with no common.
ELSEVIER
CHANGES IN CIRCULATING HORMONE CONCENTRATIONS, TESTES HISTOLOGY AND TESTES ULTRASONOGRAPHY DURING SEXUAL MATURATION IN BEEF BULLS A.C.O. Evans,la
R.A. Piersoq2 A Garcia,’ Li&l. McDougall,’ and N.C Rawlings
F. Hrudka 3
‘Departments of Veterinary Physiological Sciences and 3Veterinary Anatomy Western College of Veterinary Medicine, Saskatoon, Saskatchewan, Canada 2Department of Obstetrics and Gynecology, Royal University Hospital University of Saskatchewan, Saskatoon, Canada Received
for publication. Accepted.
tune
13,
January
1995 3,
1996
ABSTRACT Nine groups of bull calves (n = 5 to 6 per group) were castrated every 5 wk from 5 to 45 wk of age, and the stages of spermatogenesis were identified histologically. Prior to castration, the testes of each calf were examined by ultrasonography, and the pixel intensities of the parenchyma were quantitated. Testis ultrasonograms were also recorded every 2 wk from 10 bull calves between 2 and 40 wk of age. Blood samples were collected at weekly intervals until castration. There was an early transient rise in circulating LH concentrations between 4 and 25 wk of age, while circulating FSH concentrations were high initially but decreased between 14 and 30 wk of age. Circulating testosterone concentrations increased gradually from 6 to 35 wk of age and then rapidly to 42 wk of age. There was a progressive increase in the more mature cell types during spermatogenesis as the animals aged, with the most dramatic changes occurring between 15 and 45 wk of age. Outer seminiferous tubule diameter increased between 10 and 45 wk of age, with the most rapid increase occurring from 30 wk of age. Inner tubule diameter increased between 30 and 35 wk of age. The echogenicity of the testes (as determined by ultrasonography) increased between 20 and 40 wk of age. From these data we conclude that testis echogenicity increased during the most active phase of growth of the seminiferous tubules as more mature germ cells were produced. Cessation of the early rise in gonadotrophin secretion immediately preceded this active phase of testicular development. Testosterone secretion rose markedly with the production of mature spermatozoa. Key words.
puberty, bulls, hormones,
testes. ultrasonography
Acknowledgements We thank S. Cook for technical assistance, F. Schumann for castrating the calves, W. Kerr and staff for care of the animals, and the NIDDK and USDA for providing purified hormones. A.C.O.Evans was supported by a University of Saskatchewan graduate scholarship. This research was funded by Natural Sciences and Engineering Research Council of Canada. aPresent Address: T9025, VRT, Cornell University, Ithaca, NY 14853, USA. bCorrespondence and reprint requests. Fax (306) 966 7376
Theriogenology 46:345-357. 1996 0 1996 by Eisevier Science Inc.
0093-691X/96/$15.00 PII SOO93-691X(96)00190-9
Theriogenology
346 R’JTRODUCTION
Puberty in the bull has been defined as the age at which an ejaculate contains 50 million spermatozoa with a minimum motility of 10% (30). Such an ejaculate is considered to be able to lead to a pregnancy; however, the number of sperm cells per ejaculate and cell motility increase greatly beyond these values as the bull matures (16). Endocrine and testicular development in the bull calf have been described in several studies (6,13,16,17), but the factors regulating the rate of sexual maturation in bull calves remain unclear. Serum concentrations of LH and FSH are low after birth and then show a transient rise between 10 and 20 wk of age. From about 24 wk of age, mean LH and FSH concentrations increase to the time of puberty (12,17,23,28,29). Circulating testosterone concentrations are low until about 15 to 20 wk of age, at which time they increase until puberty (12,16,24,25). Changes in scrotal circumference ( 16) and histology of the testes (1,6) are well documented for the period of sexual maturation in bulls. Histological examination has shown that the establishment of spermatogenesis is a progressive process that takes place over a number of weeks (1,6). In developing Holstein bulls, spermatogonia predominate as the most mature cell type present in seminiferous tubules at 16 wk of age. These are replaced by spermatocytes (cells in meiosis) at 24 wk of age, and then by differentiating and mature spermatozoa at 32 wk of age (6). Noninvasive methods for characterizing spermatogenesis in the testes are limited to measurements of scrotal circumference and semen evaluation. While scrotal circumference measurements are a good indication of testicular mass during the period of sexual maturation (6), semen is produced only during the later weeks of the prepubertal period. Diagnostic ultrasonography is a noninvasive technique that allows for the assessment of shape, size and internal structures of body organs, Using ultrasonography, the normal anatomy of the bull (2,20), boar (3), buck (8) and dog (9,10,21) testes have been described. While gross pathological testicular lesions have been diagnosed using ultrasonography in humans (15). goats (8) and dogs (lo), no correlations between ultrasonographic findings and semen quality have been found in boars (3) humans (18), bulls (7) or dogs (10). Ultrasonographic imaging is based on the relative density (fluid content) of the tissues being examined (14) that is, the intrinsic ability of tissues to reflect or transmit high frequency acoustic pressure waves. The microanatomic organization of tissues and relative fluid components of the organs are the primary determinants of echotexture. In a simplistic model, the greater the fluid content, the less echogenic the tissue. During the period of sexual maturation, the cellular content of the seminiferous tubules changes, and lumination of the seminiferous tubules occurs (6) with the onset of fluid secretion. Ultrasonography may allow us to characterize these testicular changes during the period of sexual maturation. The aim of the present study was to examine the temporal relationships between changes in circulating hormones, testes histology and testes ultrasonographic images during the period of sexual maturation in bulls. Our specific objectives were to see if ultrasonographic imaging could detect important changes in testicular development and to see if alterations in circulating concentrations of LH, FSH and testosterone were associated with these changes.
Theriogenology
347 MATERIALS AND METHODS
Animals and Blood Collection Forty-seven spring born bull calves (30 Hereford x Hereford and 17 Hereford x Charolais, born 29 March f 9 d (mean * SD)) were divided into 9 groups each consisting of 5 or 6 calves each. Each group contained 1 to 2 Hereford x Charolais bull calves. The calves were nursed at pasture until 27 wk of age, after which time they were kept in a paddock and provided with a ground concentrate “grower” ration, hay and water ad libitum. Five calves were weighed and castrated at 5, 10, 20, 25, 35, 40 and 45 wk of age, respectively, while 6 calves were weighed and castrated at 15 and 30 wk of age. The testes of each animal were examined by ultrasonography immediately prior to castration. In addition, the testes of the 10 bull calves castrated at 40 and 45 wk of age (5 at each age) were also examined by ultrasonography every 2 wk from 2 to 40 wk of age, and scrotal circumference was measured every 2 wk from 28 to 40 wk of age. Blood samples were taken from all calves (jugular venipuncture) from 2 wk of age until castration. The samples were taken weekly to 24 wk of age and then once every 2 wk until 45 wk of age. Blood samples were allowed to clot at room temperature for 18 to 24 h. After centritigation at 1500 x g for 15 min the serum was harvested and stored at -20°C until analysis. The housing facilities provided and all the procedures conducted were in accordance with the regulations of the Canadian Council for Animal Care. Castration and Histological Examination Calves were castrated by a routine method using an emasculator. After castration, the epididymis was dissected from each testis and the testes were weighed. A portion of the parenchyma was fixed in Bouin’s solution and then prepared for histological sectioning. Sections (5-urn thick) were stained using the hematoxylin periodic acid-Schiff method (H-PAS) and mounted for examination under a light microscope. For each section, the inner (luminal) and outer seminiferous tubule diameters were measured using a calibrated eyepiece micrometer at either x 100 or x 400 magnification. Measurements were made on 15 different round tubules selected at random from each testis, and mean diameters were calculated for each animal. The histological sections were also analyzed for the most mature stages of spermatogenesis. In the cross-sections of 20 seminiferous tubules (seminal chords) per testis, the most mature cell type in the process of spermatogenesis was determined according to a scheme using 8 groups of cells. These groups, based on the descriptions of Curtis and Amann (6) were 1) seminal chords with no germ cells; 2) seminal chords with gonadocytes and prespermatogonia; 3) spermatogonia; 4) cells in leptotene or zygotene stages of meiosis; 5) cells in pachytene or diplotene stages of meiosis, or secondary spermatocytes; 6) round spermatids (r); 7) elongating spermatids (el) and 8) mature sperm cells. All histological determinations were made by a single operator within a 2-wk period and were done in random order without knowledge of the age at which the animals were castrated.
348
Theriogenology
Ultrasound Examinations and Echo-Texture Determinations Immediately prior to castration the testis of each calf was examined by ultrasonography. The testis of the 10 calves that were castrated at 40 and 45 wk of age were examined every 2 wk from 2 to 40 wk of age, The ultrasonographic examinations were done using a 5 MHZ linear-array transducer connected to a B-mode scanner (Aloka SSD-500, Overseas Monitor Corporation Ltd., Richmond, BC, Canada). Mineral oil was used as the contact medium to eliminate air between the transducer and the scrotum. Each testis was viewed vertically and horizontally, resulting in longitudinal and cross-sectional images, respectively. Care was taken to view each testis through its widest dimension. Each image was recorded using a high resolution Super-VHS VCR (Panasonic AG-1970, Professional Communications Systems Ltd., Saskatoon, Sask, Canada) for later analysis. Images were acquired from the videotape as a 640 x 480 pixel image using a commercial image acquisition board (Coreco OC-TCX, Infrascan, Richmond, BC). Both the longitudinal and cross-section images from each testis were digitally captured from the video tapes. Grey scale values of the testicular parenchyma of each testis were determined from the digital images and expressed in pixel intensity units. The total range of the pixel intensities was from 0 to 255, where a value of 0 represented black and a value of 255 represented white. The pixel intensity analyses were determined images using software developed for this purpose and optimized for ultrasonographic (SYNERGYNEIO, R.A. Pierson, University of Saskatchewan; 20). Mean pixel intensities were determined in five 15mm diameter circles placed at random around the parenchyma of the testis in each image. A mean pixel intensity was calculated for each animal based on 2 images per testis and the 2 testes (twenty 15 mm areas per animal in total). The images were analyzed in random order and without knowledge of the age at which the animal was examined by ultrasonography. Radioimmunoassay All serum samples were analyzed for concentrations of LH and FSH by validated radioimmunoassay (11,26). The sensitivity of the LH assay was 0.1 @ml (defined as the lowest concentrations of unlabelled LH capable of displacing iodinated LH from the first antibody; P
