Clinical, ultrasonographic and pathological features following unilateral vasectomy in rams

Animal Reproduction Science 103 (2008) 52–68

Clinical, ultrasonographic and pathological features following unilateral vasectomy in rams Pagona G. Gouletsou ∗ , Apostolos D. Galatos, George C. Fthenakis Faculty of Veterinary Medicine, University of Thessaly, Karditsa, Greece Received 24 July 2006; received in revised form 6 November 2006; accepted 20 November 2006 Available online 30 November 2006

Abstract The effects of vasectomy on testes and related structures of animal species and men are largely disputable. These possible effects were studied in the ram, an established experimental animal model used to investigate genitalia pathophysiology. In each of five rams, vasectomy in the left spermatic cord was carried out; subsequently, the clinical and ultrasonographic features were monitored up to 12 months postoperatively. The rams were sequentially euthanatized 1, 3, 6, 9 and 12 months post-operatively; grossand histo-pathological examination of their testes and related structures were carried out. Four of the five rams developed sperm granulomas at the proximal to the testis end of vas deferens or/and at the tail of the epididymis; these were palpable from the first and the third month after vasectomy, respectively. Ultrasonographic findings on the vasectomy side were increased size and echogenicity of the epididymal tail, as well as anechoic areas, representing sperm granulomas, visible in the epididymal tail 1 week after vasectomy and in the proximal to the testis end of vas deferens 4 weeks after vasectomy. Gross pathological findings were limited on the vasectomy side and included adhesions between the parietal and the visceral vaginal tunic, enlarged and firm epididymal tail and presence of sperm granulomas at the epididymal tail or/and at the proximal to the testis end of vas deferens; the granulomas contained creamy material. Histopathological changes were observed mainly in the epididymal tails, consisting of a central mass of spermatozoa, surrounded by a layer of macrophages, surrounded in turn by loose vascular connective tissue rich in lymphocytes and plasma cells. With the exception of signs of mild hypospermatogenesis observed in one ram euthanatized 9 months after surgery, and of a slight increase in seminiferous tubule diameter and in seminiferous epithelium height in the rams euthanatized 6 and 9 months after surgery, which are both findings of no clinical importance, no clinical, ultrasonographic, gross- or other histo-pathological changes were observed in the testicular parenchyma during a 12-month post-operative period. These results demonstrate

∗ Corresponding author at: Faculty of Veterinary Medicine, University of Thessaly, P.O. Box 199, 43100 Karditsa, Greece. Tel.: +30 2441066071; fax: +30 2441066066. E-mail address: [email protected] (P.G. Gouletsou).

0378-4320/$ – see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.anireprosci.2006.11.016

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that vasectomy has little if any detrimental effect on the morphologic characteristics of the spermatogenesis in rams. © 2006 Elsevier B.V. All rights reserved. Keywords: Sheep-reproduction; Ram; Vasectomy; Ultrasonography; Animal model

1. Introduction The effects of vasectomy on testes and related structures of animal species and men are largely disputable. In the present study the ram, an experimental animal model that has already been used to investigate the physiology and pathophysiology of testes and related structures (Gouletsou et al., 2003, 2004, 2006), was selected. The ram model was chosen because of the size of its genitalia, which permit efficient clinical, ultrasonographic, gross- and histo-pathological examination. Furthermore, vasectomy in rams is an important aspect of reproductive management in sheep flocks. “Teaser rams” produced by this technique are widely used to induce the “ram effect” and to detect signs of oestrus in ewes for artificial insemination, embryo transfer or service by selected rams (Boundy and Cox, 1996). Until now few and contradictory reports have been published regarding the effects of vasectomy on testes and related structures of rams. Perera (1978) reported that spermatogenesis was affected after vasectomy in rams, with a sequential ebb-and-flow pattern of spermatogenic arrest, i.e. absence of elongated spermatids and spermatozoa in the seminiferous tubules, and hypospermatogenesis, i.e. lower than normal percentage of seminiferous tubules with elongated spermatids. Ball and Setchell (1983) found degenerative changes in testes of two out of four rams when they were euthanatized 51 and 83 days after surgery. Ahmad and Noakes (1995b) found occasional mild, patchy testicular degeneration on testes of two rams, 5 months after vasectomy. Finally, Osman (1980), who performed vasectomy in ram-lambs, did not find any testicular changes 1 year after surgery. There are also contradictory reports regarding the short- and long-term effects of vasectomy on testes and related structures of other animal species. Some authors have reported marked alterations in the histoarchitecture of testes after vasectomy in mice (Singh and Chakravarty, 2000), rats (Flickinger et al., 1987; Sarrat et al., 1996; Shiraishi et al., 2001), rabbits (Flickinger, 1975; Alexander and Tung, 1977), guinea pigs (Alexander, 1973; Tung and Alexander, 1977), monkeys (Lohiya et al., 1987) and dogs (Vare and Bansal, 1973, 1974). In contrast to these findings, other researchers found no changes in testes of rats (Flickinger, 1972; Heller and Rothchild, 1974; McDonald and Scothorne, 1988), hamsters (Lue et al., 1997), rabbits (Paufler and Foote, 1969), monkeys (Chapman et al., 1978; Hadley and Dym, 1983; Peng et al., 2002) and bucks (Batista et al., 2002). In studies investigating spermatogenesis after vasectomy in men, a wide spectrum of results, ranging from no effect to significant impairment has been reported (Gupta et al., 1975; Jenkins et al., 1979; Silber and Rodriguez-Rigau, 1981; Jarow et al., 1985; Matsuda et al., 1996; McDonald, 2000; Shiraishi et al., 2002; Raleigh et al., 2004; McVicar et al., 2005). In the present study we performed clinical and ultrasonographic evaluation of the genitalia, and correlated any changes observed with pathological findings in five vasectomized rams sequentially euthanatized for a period up to 1 year post-operatively.

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2. Materials and methods 2.1. Animals, husbandry and experimental design Five Karagouniko-breed 2–3-year-old rams were used. All the experimental animals were purchased from a Ministry of Agriculture sheep breeding station and were Brucella-free, as confirmed by regular bacteriological and serological tests. Throughout this study, they were housed in the premises of the Department of Obstetrics and Reproduction of the Faculty of Veterinary Medicine of the University of Thessaly, in Karditsa, Greece. They were fed twice-daily 1 kg of a commercial high-energy, high-protein diet, in small pellets, plus ad libitum high-quality hay. The genitalia of each ram were examined clinically and ultrasonographically at weekly intervals before and after surgery. The rams were sequentially euthanatized and their genitalia were examined. The experiment was carried out under a license obtained from the Greek Ministry of Agriculture. 2.2. Vasectomy technique The left spermatic cord was selected for vasectomy. The controlateral genital organs served as controls. Vasectomy was performed under general anaesthesia with the animal in dorsal recumbency, as described by Boundy and Cox (1996). Using aseptic technique a vertical incision 5 cm in length was made on the cranial surface of the neck of the scrotum, over the left spermatic cord, which was freed by blunt dissection and exteriorized. Particular care was taken to avoid any injury of the blood vessels. The vas deferens was identified, held firmly and an incision was made on the parietal vaginal tunic above it. A portion of the vas deferens was exteriorized and a piece of 4 cm in length was resected. Both cut ends of the vas deferens were ligated and the proximal end, which was 12–14 cm away from the epididymal tail, was anchored in the fatty tissue outside the vaginal tunic. Finally, the skin was sutured. 2.3. Pre- and post-vasectomy examinations 2.3.1. Clinical examination Clinical examination was carried out at weekly intervals starting 3 weeks before the vasectomy and up to 1 year post-operatively, by using a procedure already described in detail (Fthenakis et al., 2001; Gouletsou et al., 2004). Initially, the general health of the animals was assessed. Subsequently, the genitalia were examined, with the ram cast and restrained. The scrotal skin (cutis scroti) was observed. Each testis (testis) was palpated in order to assess its size, shape, consistency, resilience, pain reaction and free movement within the scrotum; the two testes were compared to each other. Each epididymis (epididymis) was examined as above, starting from the head (caput epididymidis) to the body (corpus epididymidis) to the tail (cauda epididymidis). Testis diameter and height, epididymal’s head width, edidymal’s body diameter and edidymal’s tail width and height were calculated over the scrotum by sliding calipers. Each spermatic cord (funiculus) was palpated as far as possible. 2.3.2. Ultrasonographic examination On the same occasions as above, the genitalia were examined ultrasonographically, by using an established procedure, already described in detail (Gouletsou et al., 2003). Ultrasonographic imaging of the testes and the epididymides was carried out with the ram in the standing position,

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by using an ultrasound scanner (AMI B7; Alliance Medical, Quebec, Canada) with a sector transducer, at the 6.0 MHz frequency. The testes were pulled downwards within the scrotum and maintained in that position. The examiner’s left hand was placed on the surface opposite to the one where the transducer was applied on, in order to stabilize them. Initially, the left testis and epididymis were imaged. The probe was placed on the caudal surface of the testis along its longitudinal axis (sagittal plane) and was moved from left to right to monitor the testicular parenchyma and the body of the epididymis; 40, 80 and 120 mm scanning depths were used. Then, the probe was moved upwards, in order to image the head of the epididymis and the pampiniform plexus (pampiniform plexus) and downwards to image the tail of the epididymis. In each testis, images in up to three fields/levels were obtained. The probe was then placed in a position perpendicular to the long axis and transverse sections of the testis, the epididymis and the spermatic cord were taken, starting from the upper part downwards. Again, 40, 80 and 120 mm scanning depths were used. The whole procedure was repeated after placing the probe on the left lateral surface of the testis. Subsequently, the whole procedure was repeated for the right testis. Finally, paired transverse sections of the genitalia were taken by moving the probe from the upper part of the genitalia downwards, by using 40–120 mm scanning depth. The testes, epididymides and spermatic cords were compared to each other. 2.4. Post-mortem examination Experimental animals were euthanatized 1 (ram 1), 3 (ram 2), 6 (ram 3), 9 (ram 4) and 12 (ram 5) months after surgery. A detailed post-mortem examination was carried out. The genitalia were removed for dissection. Initially, the scrotal skin and the parietal tunic (lamina parietalis) were dissected and the vaginal cavity (cavum vaginale) was observed. Then, the testes and epididymides were removed; they were observed, palpated and compared to each other and their dimensions were measured. Subsequently, they were dissected mid-sagittally and divided into a caudal and cranial half; the halves were subsequently dissected dorsally and 8-mm thick slices were cut. Any abnormal features were recorded. Sterile swabs were inserted by using aseptic technique, into each of the following parts of the right and left genitalia: vaginal cavity (cavum vaginale), epididymal head, epididymal body, epididymal tail and testicular parenchyma. Finally, tissue samples from the vasectomized and the control side were obtained for histological examination. These were fixed into Bouin’s solution and then cleared with alcohol 50%; they were embedded in paraffin, sectioned at 5 ␮m and stained with haematoxylin and eosin (H&E) and periodic acid-Schiff (PAS). In order to quantify any adverse changes on the testis as a consequence of vasectomy, morphometric analysis of seminiferous tubules was performed. The diameter of the seminiferous tubule, the diameter of the seminiferous tubular lumen and the height of the seminiferous tubule epithelium were measured at 400× magnification using an ocular micrometer calibrated with a stage micrometer. At least 80 tubular profiles with a round cross section, chosen randomly, were measured for each testis. Data are expressed as mean values ± standard deviation. The Mann–Whitney U-test was used to compare differences between left and right testis in the same animal. A p-value ≤0.05 was considered significant. Data were analysed using SAS (Statistical Analysis System, Statistical Software; Cary, NC, USA). Furthermore, in order to quantify the impact of vasectomy on spermatogenesis, one hundred round cross sections of seminiferous tubules from each testis were examined at 400× magnification and the percentage of tubules with elongated spermatids present in the tubular epithelium was determined (Lunstra and Schanbacher, 1988).

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2.5. Bacteriological examination In order to rule out the possibility that pathological features after vasectomy might have been the result of infection, the tissue and swab samples obtained during the post-mortem examination were cultured onto Columbia blood agar plates. Anaerobic or CO2 incubation of Petri dishes was carried out into an incubation jar, where appropriate gas generator envelopes (AnaeroGen 2.5 l or COGen, respectively; Oxoid, UK) were added and opened. The jar was sealed and re-opened 48 h later, checked for bacterial cultures and, if nothing had grown, was reincubated for up to further 48 h. For the identification of organisms standard techniques were used (Barrow and Feltham, 1993). 3. Results No clinical or ultrasonographic abnormal findings were recorded in the genitalia before vasectomy. Furthermore, no abnormal clinical, ultrasonographic and gross- and histo-pathological findings were recorded in the controlateral to vasectomy side. The abnormal features recorded in the vasectomized side of the genitalia are described here below. Furthermore, no bacteria were isolated from any sample cultured. 3.1. Clinical findings In all rams 1 week post-operatively the left testis diameter on the vasectomized side was mildly enlarged (5–10%) compared to the pre-vasectomy values and the testis was firm. Two weeks later the testis was found to be normal; no abnormal features were observed subsequently. From the first to the third post-operative week, the associated epididymal head width was enlarged (5–10%) compared to the pre-vasectomy values and was firmer on palpation; subsequently it appeared normal. Two to 3 weeks post-operatively, the epididymal body diameter was found enlarged (10–20%); subsequently it appeared normal. The main findings were at the epididymal tail. One week post-operatively the width and the height of the epididymal tail were enlarged (20–30%) compared to the pre-vasectomy values and the epididymal tail was firm; progressively it became even larger and firmer. One month after surgery palpation revealed a greatly enlarged in width and height (30–50%) and firm epididymal tail, which remained so until the end of the experiment, as well as nodular masses in the vas deferens. Two to 3 months post-operatively nodular masses were also palpated in the epididymal tail. 3.2. Ultrasonographic findings No ultrasonographic changes were observed in the testicular parenchyma of the vasectomized side; the echogenicity and the ultrasonographic size of the testicular parenchyma and the mediastinum testis appeared normal. During the first 2 post-operative months the epididymal head appeared larger and more echogenic; subsequently it appeared normal. The epididymal body, which normally cannot be imaged (Gouletsou et al., 2003) (Fig. 1b and d), was also visible during that period (Fig. 1a and c). The most significant ultrasonographic findings were observed in the epididymal tail. One week post-operatively this appeared enlarged and with increased echogenicity; anechoic areas were also visible therein (Fig. 1a). One month after vasectomy the anechoic areas were round,

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Fig. 1. Ultrasonographic findings 1 week after vasectomy. (a) Sagittal sonogram of the vasectomized testis (tp, testicular parenchyma) of ram 4: hypoechoic area with distinct borders (distance between cursors: 21.4 mm) within epididymal tail (et) and enlargement of epididymal tail and body (eb). (b) Sagittal sonogram of the non vasectomized testis of ram 4; normal appearance of epididymal tail (et). (c) Transverse sonogram of the vasectomized testis (tp) of ram 5: enlargement of epididymal body (eb) and vas deferens (distance between cursors: 16 mm), which normally cannot be imaged. (d) Transverse sonogram of the non vasectomized testis (tp) (s, scrotum).

with ill-defined borders, increasing in size until the end of the experiment (Fig. 2a and b). In two rams, anechoic masses with hyperechogenic walls were seen at the proximal end of vas deferens 1 month post-operatively; these appeared to grow by time. Enlarged, anechoic vas deferens with a hyperechogenic wall was identified medially to the epididymal body and to the pampiniform plexus since the first post-operative week (Fig. 1c). 3.3. Post-mortem findings In all rams loose adhesions between the parietal and visceral vaginal tunic, mainly located at the area of epididymal head or tail, were detected. In the ram euthanatized 1 month post-operatively, the epididymal tail was firm and the vas deferens distended, without any sign of a nodule. In the ram euthanatized 3 months after vasectomy, the epididymal tail at the vasectomized side was enlarged with a nodule containing creamy material and attached to a cyst (51 mm × 33 mm × 25 mm); the cyst contained yellowish watery fluid and a semi-solid creamy material (Fig. 3a). Another nodule (diameter: 28 mm) was seen at the proximal cut end of vas deferens (Fig. 3a). In the ram euthanatized 6 months post-operatively, the epididymal tail was larger and two nodular masses (diameter: 17.6 and 15.2 mm) containing creamy material were attached to the cut end of vas deferens. In the ram euthanatized 9 months post-operatively, the epididymal tail at the vasectomized side was enlarged and a nodule containing creamy material was observed;

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Fig. 2. Ultrasonographic findings of ram 5, 1 year after vasectomy. (a) Transverse sonogram of the epididymal tails: hypoechoic heterogeneous area with distinct wall (distance between cursors: 18.6 mm) and enlargement of the left epididymal tail (let) on the vasectomized side; normal appearance of the right epididymal tail (ret) on the non vasectomized side. (b) Sagittal sonogram of the epididymal tail on the vasectomized side: enlarged epididymal tail (et) with a hypoechoic area with distinct wall (distance between cursors: 16.4 mm) and a heterogeneous area with indistinct wall (arrowheads) (tp, testicular parenchyma).

a nodule (56 mm × 23 mm × 30 mm) containing semi-solid creamy material was also present at the cut end of vas deferens (Fig. 3b). In the fifth ram, the testis on the vasectomized side was less turgid than the controlateral and with a whitish appearance (Fig. 3c). The epididymal tail was much larger than the controlateral and contained two nodules (Fig. 3c and d). The dimensions of testes and epididymides of all rams are shown in Table 1. 3.4. Histopathological findings The germinal epithelium did not show any sign of degeneration, and spermatogenesis was not altered substantially in any animal. Spermatogonia, primary and secondary spermatocytes, spermatids and spermatozoa could be observed in the seminiferous tubules of all animals. The diameter of the seminiferous tubule, the diameter of the seminiferous tubular lumen and the percentage of seminiferous tubules with elongated spermatids present in the tubular epithelium are

Animal

Ram 1 (1 month po) left genitalia Ram 1 (1 month po) right genitalia Ram 2 (3 months po) left genitalia Ram 2 (3 months po) right genitalia Ram 3 (6 months po) left genitalia Ram 3 (6 months po) right genitalia Ram 4 (9 months po) left genitalia Ram 4 (9 months po) right genitalia Ram 5 (12 months po) left genitalia Ram 5 (12 months po) right genitalia po, post-operatively.

Testis

Epididymal head

Epididymal body

Epididymal tail

Dorsoventral

Mediolateral

Craniocaudal

Craniocaudal

Diameter

Mediolateral

Dorsoventral

Craniocaudal

78.8 76.9 100.2 105.8 88.06 88.62 89.2 95.7 89.2 91.8

63 62.3 79.5 81.5 73.18 73.62 68.2 71.7 75.8 77.8

45 45.4 55 53.38 49 47.6 47.1 46.2 51.2 52.3

28.4 26.8 37 36.41 34.05 32.67 36.8 37.46 31 33

10.6 9.7 9.3 8.9 8.88 8.9 11.5 12.3 11.2 9.5

32.5 31.8 39.5 33.38 26.68 23.87 42.2 33 55 30.7

26 25.4 28.5 25.3 27.6 23.4 42.6 33.6 65.15 37.8

21 21.2 23.47 22.04 26.6 19.03 28.5 22 37.8 19.7

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Table 1 Dimensions of testes and epididymides (mm)

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Fig. 3. Findings in the rams euthanatized 3 (ram 2) (a), 9 (ram 4) (b) and 12 (ram 5) (c and d) months after vasectomy. (a) The epididymal tail at the vasectomized (L) side is enlarged with a nodule (arrowhead) and attached to a cyst (51 mm × 33 mm × 25 mm) (black arrow); another nodule (diameter: 28 mm) is seen at the proximal cut end of vas deferens (white arrow). (b) The epididymal tail at the vasectomized (L) side is enlarged; a nodule (56 mm × 23 mm × 30 mm) (arrow) is also present at the cut end of vas deferens. (c) The testis on the vasectomized (L) side has a whitish appearance; the epididymal tail is much larger than the controlateral. (d) The epididymal tail on the vasectomized side has nodules containing creamy material.

shown in Table 2. No significant differences in the seminiferous tubule diameter or the seminiferous epithelium height were observed between the left and right testis in rams euthanatized 1, 3 and 12 months after vasectomy; however, seminiferous tubule diameter and seminiferous epithelium height were increased (p ≤ 0.05) in the left testis of rams euthanatized 6 and 9 months after vasectomy. No significant differences in the diameter of the seminiferous tubular lumen were observed between the left and right testis in all animals. The percentage of seminiferous tubules with elongated spermatids present in the tubular epithelium was similar between the left and right testis in all animals, and similar to the normal values, i.e. 65 ± 4% (Lunstra and Schanbacher, 1988), with the exception of the ram euthanatized 9 months after surgery. In the left testis of that animal a percentage of 48% was recorded, instead of 58% observed in the right testis.

Animal

No. of tubules measures/ram

Seminiferous tubule diameter (␮m)a

Seminiferous epithelium height (␮m)a

Seminiferous tubule lumen diameter (␮m)a

Percentage with elongated spermatids (%)

Ram 1 (1 month po) left testis Ram 1 (1 month po) right testis Ram 2 (3 months po) left testis Ram 2 (3 months po) right testis Ram 3 (6 months po) left testis Ram 3 (6 months po) right testis Ram 4 (9 months po) left testis Ram 4 (9 months po) right testis Ram 5 (12 months po) left testis Ram 5 (12 months po) right testis

86 86 100 97 81 84 80 102 80 80

191.7 (19.32) 189.53 (10.82) 197.5 (18.54) 200.64 (20.83) 202.16 (14.62) a 190.32 (13.49) a 196.4 (18.03) b 183.57 (13.95) b 198.59 (15.27) 200.78 (15.82)

64.8 (9.44) 66.71 (10.31) 73.25 (8.1) 75.77 (9.85) 69.68 (9.06) c 62.5 (8.42) c 66.87 (10.35) d 57.96 (8.57) d 69.84 (9.26) 68.28 (9.93)

53.2 (4.87) 54.58 (3.65) 51.32 (4.37) 49.06 (4.54) 62.7 (5.24) 65.39 (6.12) 62.22 (5.95) 64.56 (4.99) 59.81 (4.72) 62.87 (5.11)

57 67 63 64 70 57 48 58 68 71

po, post-operatively. Data in the same column with the same letters are different from each other (p ≤ 0.05). Comparisons are made between the left and right testis within the same ram. a Data are given as mean (S.D.).

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Table 2 Seminiferous tubule diameter, height of seminiferous tubule epithelium, diameter of seminiferous tubule lumen and percentage of seminiferous tubules with elongated spermatids

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Fig. 4. Histological findings in the epididymal tail on the vasectomized side of the ram euthanatized 3 months after surgery (ram 2): a central mass of degenerating spermatozoa (S) is surrounded by a layer of macrophages (M), surrounded in turn by loose vascular connective tissue with lymphocytes and plasma cells (C) (H&E stain, bar: 100 ␮m).

The lumen of the left epididymal duct at the epididymal head and body was larger than the right, whilst the epithelial height of the left epididymal duct was lower than the right in the rams euthanatized 9 and 12 months post-operatively. In the left epididymal tail, the tubular profiles were dilated comparing to the right side due to accumulation of spermatozoa. Rupture of the integrity of the epididymal duct at the left epididymal tail was evident in three of the five rams (rams euthanatized 3, 9 and 12 months after surgery). Sperm granulomas consisted of a central mass of degenerating spermatozoa, surrounded by a layer of macrophages, surrounded in turn by loose vascular connective tissue rich in lymphocytes and plasma cells (Fig. 4). Although a dilatation of the left epididymal ducts was also obvious in the other two rams, sperm granulomas were not observed in these animals. Sperm granulomas were also established at the proximal cut end of the left vas deferens in two rams. These were covered by connective tissue and contained spermatozoa, few macrophages, giant cells and eosinophilic debris. 4. Discussion 4.1. Effects of vasectomy There are only a few and rather contradictory studies on the effects of vasectomy on the genitalia of rams. Furthermore, they were performed either on ram-lambs (Osman, 1980) or on rams euthanatized soon (34–103 days) after surgery (Ball and Setchell, 1983) or on a small number (two animals) of rams (Ahmad and Noakes, 1995b). In contrast, the present study was performed on five adult rams monitored for up to 1 year post-operatively. As far as the histological examination is concerned, one ram was evaluated at each time point. However, clinical and ultrasonographical examinations were carried out every week to five animals till 1 month postoperatively, to four animals till 3 months post-operatively, to three animals till 6 months postoperatively, etc. Furthermore, as no animal showed seminiferous epithelium degeneration at any

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time point, one could speculate that if the same animal was euthanized earlier than the given time point it would not have shown seminiferous epithelium degeneration either. A large number of studies performed on various mammalian species, including men, indicated marked variations on the extent, nature and timing of post-operative changes of the testes; it is noteworthy that differences have been recorded even among strains of laboratory animal species. The changes described ranged from absence of detectable changes to autoimmune orchitis (McDonald, 2000). Absence of detectable changes of the seminiferous epithelium was reported by Paufler and Foote (1969), Flickinger (1972), Heller and Rothchild (1974), Chapman et al. (1978), Hadley and Dym (1983), McDonald and Scothorne (1988), Lue et al. (1997), Batista et al. (2002), and Peng et al. (2002). Mild changes of the seminiferous epithelium were reported by Alexander (1973), Gupta et al. (1975), Flickinger (1975), Bedford (1976), Matsuda et al. (1996), Aitken et al. (1999), Whyte et al. (2000), and Shiraishi et al. (2002), whilst severe changes were reported by Vare and Bansal (1973, 1974), Tung and Alexander (1977), Perera (1978), Jenkins et al. (1979), Silber and Rodriguez-Rigau (1981), Jarow et al. (1985), Flickinger et al. (1987), Lohiya et al. (1987), Matsuda et al. (1996), Sarrat et al. (1996), Singh and Chakravarty (2000), Shiraishi et al. (2001), Raleigh et al. (2004), and McVicar et al. (2005). Vasectomy has been reported to cause regressive changes in the seminiferous tubules in testes of several mammalian species (Singh and Chakravarty, 2000). It is not clear how the operation induces such effects, although several explanations have been proposed. According to Vare and Bansal (1974), the continuous production of spermatozoa by the testis, which cannot be drained, increases internal pressure of seminiferous tubules. The authors suggested that the atrophic and degenerative changes of the seminiferous tubules probably result from blood stasis, which, in turn, is caused by resistance of fibrous tunica albuginea to increase in internal pressure. Furthermore, blood stasis may limit oxygen and therefore injure the sensitive germinal epithelium. By time, as fluid is slowly absorbed, the pressure is reduced or released and the tubules are able to regenerate (Vare and Bansal, 1974). Other researchers discussed other pathways for development of the lesions. According to Plaut (1973) and to Heller and Rothchild (1974), vasectomy had no direct adverse effects on the testis; it was the procedural artifacts (e.g. infection or circulatory disturbances) that caused the testicular alterations recorded. Aydos et al. (1998) and Kolettis et al. (1999) suggested that oxidative stress and increased concentration of reactive oxygen species following vasectomy, induced the histological changes. Other authors suggested that the changes are the consequence of immunological response as sperm antibodies have been detected in the serum of vasectomized men (Newton, 1988; Jarow et al., 1994; McLachlan and Royce, 1996). The epididymis is the most likely site of their penetration, because of escape of soluble autoantigens (Flickinger et al., 1990; McLachlan and Royce, 1996). However, no association between presence of sperm antibodies in serum and structural changes within the testis has been established (Raleigh et al., 2004). The issue is of particular importance for men who undergo vasectomy operations, but occasionally wish to restore full function of their genital system. Actually, in men various histological changes have been reported after vasectomy. These include tubular dilatation (Matsuda et al., 1996), tubular wall thickening (Jarow et al., 1985), decreased number of germ cells (Matsuda et al., 1996), spermatids (Silber and Rodriguez-Rigau, 1981; Raleigh et al., 2004) or Sertoli cells (Matsuda et al., 1996), increased incidence of interstitial and peritubular fibrosis (Jarow et al., 1985; Shiraishi et al., 2002; Raleigh et al., 2004) and interstitial oedema (Jequier, 2000). Most of the histological changes seen, deteriorated with time (Matsuda et al., 1996; Raleigh et al., 2004).

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In our study, a mild increase was observed in seminiferous tubule diameter 6 and 9 months after surgery. However, since it was accompanied by an increase in seminiferous epithelium height, whilst the diameter of seminiferous lumen stayed the same, no evidence of dilatation of the seminiferous tubules due to higher hydrostatic pressure after vasectomy was obvious. Also, mild hypospermatogenesis was observed in the ram euthanatized 9 months after surgery, with fewer seminiferous tubules having elongated spermatids in the vasectomized side than in the control, i.e. 48% instead of 58%. The percentage of 48% is lower than the values found in the right testes in this experiment (range: 57–71%; mean: 63.4%) and the values reported by Lunstra and Schanbacher (1988), i.e. 65 ± 4%. In the rest of the animals the percentage of seminiferous tubules with elongated spermatids present in the left tubular epithelium was ranging from 57 to 70%. Furthermore, since no changes were observed 1, 3 and 12 months after surgery, and the seminiferous tubules in all rams showed spermatogenic activity with successive stages of transformation of spermatogonia to spermatozoa, our results demonstrate that vasectomy has little if any detrimental effect on the morphologic characteristics of the spermatogenesis. Our findings are in agreement with those of Osman (1980) and of McDonald and Scothorne (1988), who found no important alteration in the seminiferous epithelium after left unilateral vasectomy in rams and rats, respectively; however they are in contrast with the results of Perera (1978) and of Ahmad and Noakes (1995b), who found hypospermatogenesis and occasional mild, patchy testicular degeneration, respectively, on testes of rams. On the other hand, Ball and Setchell (1983) found degenerative changes in the testes of two out of four rams euthanatized 51 and 83 days after surgery, whilst the other two, euthanatized 34 and 103 days after vasectomy, showed no testicular abnormality. Factors responsible for the conflicting reports published by different investigators may be differences in species, breed, age, number of animals, vasectomy technique, vascular damage, infection, testicular size, and method for assessing testicular damage. It seems that the cause and significance of reports of testicular damage in mammalian testis following vasectomy is unclear and the mechanisms remain poorly understood (McDonald, 2000). Ball and Setchell (1983) suggested that the location of the sperm granulomas within the tubular genitalia might play a role in the testicular response to vasectomy. As they found testicular degeneration in two rams that had also sperm granulomas in the epididymal tail, whilst other two with no sperm granulomas in the epididymal tail showed no testicular abnormality, they proposed that the closer the granulomas were to the testis, the earlier the onset of the pathological changes, because fewer spermatozoa could be accommodated before pressure rose to a critical level. We also observed sperm granulomas in the epididymal tail of rams euthanatized 3, 9 and 12 months after surgery, but with no signs of testicular degeneration. With regard to epididymis, it has been suggested that accumulation of spermatozoa into the epididymal duct following obstruction distends the tubules and may cause cystic changes, rupture and formation of sperm granulomas (Ball and Setchell, 1983). Although, in some cases these sperm granulomas may develop as a consequence of poor surgical technique (Mayenco et al., 1996) or infection (Sargison et al., 1995), in the majority of cases complications occur regardless of the surgical procedure (Ball and Setchell, 1983). In our study, bacteriological examination ruled out the possibility of infection, which was also confirmed by the absence of inflammatory cell infiltration in the epididymis at sites remote from sperm granulomas. The most usual localization of sperm granulomas in rams is the epididymal tail or/and the cut end of the vas deferens (Perera, 1978; Osman, 1980; Ball and Setchell, 1983; Ahmad and Noakes, 1995b; Ahmad et al., 2000). Our results are in accord with those of previous authors. It is noteworthy that Karaka et al. (1999) reported the presence of naturally occurring sperm granulomas in the testis, the epididymal head and the epididymal tail; these were associated with testicular degeneration.

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4.2. Ultrasonographic findings Using the ram experimental model (Gouletsou et al., 2003), we have shown the value of ultrasonography in diagnosing experimental (Gouletsou et al., 2004) or spontaneous (Gouletsou et al., 2006) testicular and epididymal lesions. Other researchers also used ultrasonography to study experimentally induced testicular lesions caused by ligation of the spermatic cord (Ahmad and Noakes, 1995b), ligation of the testicular artery (Eilts et al., 1989), intratesticular injection of chlorhexidine gluconate solution (Ahmad and Noakes, 1995a), intratesticular injection of glycerol (Immegart and Threlfall, 2000) or testicular biopsy (DelVento et al., 1992) in animals. In the present study we detected lesions, which were subsequently confirmed during the post-mortem examination. The sequential ultrasonographic findings in the scrotal contents were consistent in all the experimental animals. The main ultrasonographic changes were observed in the epididymis and the vas deferens. Gross- and histo-pathological examination confirmed that the areas of decreased echogenicity corresponded to sperm granulomas. It seems that, at the early stage after vasectomy, ultrasonography offers the advantage of detecting sperm granulomas smaller than 1 cm in diameter and not yet palpable. Later on, ultrasonography can be used to compliment the clinical examination of the genitalia, thus increasing its accuracy, as it gives valuable information about the fine texture and the precise size of scrotal contents. Our findings are in general similar to those detected by Ahmad and Noakes (1995b) in two rams; however, these authors detected anechoic areas that turned to sperm granulomas at the epididymal tail at a later stage than we did, namely 4–6 weeks post-operatively. Also, Jessop and Ladds (1995) detected sperm granulomas in the epididymal tail of vasectomised rams 4 weeks after vasectomy and later than in the vas deferens. In our study, there was ultrasonographic evidence of granuloma development at the epididymal tail since 1 week after surgery. Batista et al. (2002) reported the presence of anechoic cavities in the testes 6–8 weeks post-operatively and assumed that they represented fluid between the testicular tunics, as a consequence of orchitis. However, they detected no signs of infection at the testicular level during that period of time. In our study neither fluid accumulation between the testicular tunics nor signs of inflammation were detected. Reddy et al. (2004) observed the same findings, i.e. enlargement of epididymis and presence of sperm granulomas. As far as the rams are concerned, the clinical examination of their genitalia is an integral part of preventive veterinary schemes in sheep flocks. Although Bruere and West (1993) consider that most abnormalities can be detected by means of this method, some concerns have been voiced recently about its accuracy (Beltsinger, 2001). Our findings point out that ultrasound scanning can be used to compliment the clinical examination of the genitalia of rams, thus increasing its accuracy. 5. Conclusions According to our study it seems that, following vasectomy in the ram, sperm granulomas at the epididymal tail and the cut end of vas deferens are the most frequent pathological findings. With the exception of signs of mild hypospermatogenesis observed in one ram euthanatized 9 months after surgery, and of a slight increase in seminiferous tubule diameter and in seminiferous epithelium height in the rams euthanatized 6 and 9 months after surgery, which are both findings of no clinical importance, no clinical, ultrasonographic, gross- or other histo-pathological changes were observed in the testicular parenchyma during a 12-month post-operative period. Our results indicate that vasectomy has little if any detrimental effect on the morphologic characteristics of

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the spermatogenesis of ram. Furthermore, they support the use of ultrasonography as a diagnostic means after vasectomy, as it is a reliably tool for examination of scrotal contents. References Ahmad, N., England, G.C.W., Noakes, D.E., 2000. Ultrasonography of spontaneous lesions of the genital system of three rams, and their influence on semen quality. Vet. Rec. 146, 10–15. Ahmad, N., Noakes, D.E., 1995a. A clinical and ultrasonographic study of induced testicular and epididymal lesions in goats and a ram. Anim. Rep. Sci. 39, 35–48. Ahmad, N., Noakes, D.E., 1995b. A clinical and ultrasonographic study of testes and related structures of goats and rams after unilateral vasectomy. Vet. Rec. 137, 112–117. Aitken, H., Kumarakuru, S., Orr, S., Reid, O., Bennett, N.K., McDonald, S.W., 1999. Effect of long-term vasectomy on seminiferous tubules in the guinea pig. Clin. Anat. 12, 250–263. Alexander, N.J., 1973. Autoimmune hypospermatogenesis in vasectomized guinea pig. Contraception 8, 147–164. Alexander, N.J., Tung, K.S.K., 1977. Immunological and morphological effects of vasectomy in the rabbit. Anat. Rec. 188, 330–350. Aydos, K., Kupeli, B., Soygur, T., Unsal, A., Erden, E., Tulunay, O., Kupeli, S., 1998. Analysis of the relationship between histologic alterations and the generation of reactive oxygen species in vasectomized rat testes. Urology 51, 510– 515. Ball, R.Y., Setchell, B.P., 1983. The passage of spermatozoa to regional lymph nodes in testicular lymph following vasectomy in rams and goats. J. Reprod. Fertil. 68, 145–153. Barrow, G.I., Feltham, R.K.A., 1993. Manual for the Identification of Medical Bacteria, 3rd ed. Cambridge University Press, Cambridge. Batista, M., Calero, P., Rodriguez, F., Gonzalez, F., Cabrera, F., Gracia, A., 2002. Structural changes in the testes and epididymides of bucks 16 weeks after bilateral vasectomy. Vet. Rec. 151, 740–741. Bedford, J.M., 1976. Adaptations of the male reproductive tract and the fate of spermatozoa following vasectomy in the rabbit, rhesus monkey, hamster and rat. Biol. Reprod. 14, 118–142. Beltsinger, H.J., 2001. Make sure the profit is in the bag! Breeding soundness evaluation of the ram. In: Proceedings of the 5th International Sheep Veterinary Congress, Stellenbosch, South Africa, pp. 250–251. Boundy, T., Cox, J., 1996. Vasectomy in the ram. In Pract. 18, 330–334. Bruere, A.N., West, D.W., 1993. The Sheep: Health, Disease & Production. Foundation for Veterinary Continuing Education of the New Zealand Veterinary Association, Palmerston North. Chapman, E.S., Heidger Jr., P.M., Harrison, R.M., Roberts, J.A., Domingue, G.J., Schlegel, J.U., 1978. Vasectomy in rhesus monkeys. IV. Electron microscopic studies of the seminiferous epithelium. Anat. Rec. 192, 41–54. DelVento, V.R., Amann, R.P., Trotter, G.W., Veeramachaneni, D.N.R., Squires, E.L., 1992. Ultrasonographic and quantitative histologic assessment of sequelae to testicular biopsy in stallions. Am. J. Vet. Res. 53, 2094–2101. Eilts, B.E., Pechman, R.D., Taylor, H.W., Usenik, E.A., 1989. Ultrasonographic evaluation of induced testicular lesions in male goats. Am. J. Vet. Res. 50, 1361–1364. Flickinger, C.J., 1972. Ultrastructure of the rat testis after vasectomy. Anat. Rec. 174, 477–494. Flickinger, C.J., 1975. Ultrastructure of the rabbit testis after vasectomy. Biol. Reprod. 13, 61–67. Flickinger, C.J., Herr, J.C., Caloras, D., Sisak, J.R., Howards, S.S., 1990. Inflammatory changes in the epididymis after vasectomy in the Lewis rat. Biol. Reprod. 43, 34–45. Flickinger, C.J., Herr, J.C., Howards, S.S., Caloras, D., Yarbro, E.S., Spell, D.R., Gallien, T.N., 1987. The influence of vasovasostomy on testicular alterations after vasectomy in Lewis rats. Anat. Rec. 217, 137–145. Fthenakis, G.C., Karagiannidis, A., Alexopoulos, C., Brozos, C., Saratsis, P., Kyriakis, S., 2001. Clinical and epidemiological findings during ram examination in 47 flocks in southern Greece. Prev. Vet. Med. 52, 43–52. Gouletsou, P.G., Amiridis, G.S., Cripps, P.J., Lainas, T., Deligiannis, K., Saratsis, P., Fthenakis, G.C., 2003. Ultrasonographic appearance of clinically healthy testes and epididymides of rams. Theriogenology 59, 1959–1972. Gouletsou, P.G., Fthenakis, G.C., Cripps, P.J., Papaioannou, N., Lainas, T., Psalla, D., Amiridis, G.S., 2004. Experimentally induced orchitis associated with Arcanobacterium pyogenes: clinical, ultrasonographic, seminological and pathological features. Theriogenology 62, 1307–1328. Gouletsou, P.G., Fthenakis, G.C., Tzora, A., Cripps, P.J., Saratsis, P., 2006. Isolation of Arcanobacterium pyogenes from the scrotal skin and the prepuce of healthy rams or from rams with testicular abnormalities. Small Rum. Res. 63, 177–182. Gupta, A.S., Kothari, L.K., Bapna, R.B., 1975. Surgical sterilization by vasectomy and its effects on the structure and function of the testis in man. Br. J. Surg. 62, 59–63.

P.G. Gouletsou et al. / Animal Reproduction Science 103 (2008) 52–68

67

Hadley, M.A., Dym, M., 1983. Spermatogenesis in vasectomized monkey: quantitative analyses. Anat. Rec. 205, 381–386. Heller, G.V., Rothchild, I., 1974. The influence of the surgical technique used for vasectomy on testis function in rats. J. Reprod. Fertil. 39, 81–84. Immegart, H.M., Threlfall, W.R., 2000. Evaluation of intratesticular injection of glycerol for nonsurgical sterilization of dogs. Am. J. Vet. Res. 61, 544–549. Jarow, J.P., Budin, R.E., Dym, M., Zirkin, B., Noren, S., Marshall, F.F., 1985. Quantitative pathologic changes in the human testis after vasectomy. A controlled study. N. Engl. J. Med. 3131, 1252–1256. Jarow, J.P., Goluboff, E.T., Chang, T.S., Marshall, F.F., 1994. Relationship between antisperm antibodies and testicular histological changes in humans after vasectomy. Urology 43, 521–524. Jenkins, I.L., Muir, V.Y., Blacklock, N.J., Turk, J.L., Hanley, H.G., 1979. Consequences of vasectomy: an immunological and histological study related to subsequent fertility. Br. J. Urol. 51, 406–410. Jequier, A.M., 2000. Vasectomy related infertility. In: Jequier, A.M. (Ed.), Male Infertility: A Guide for the Clinician. Blackwell Science, London, pp. 238–253. Jessop, T.S., Ladds, P.W., 1995. The immunopothology of unilateral vasectomy in the ram. Vet. Immunol. Immunopathol. 47, 123–133. Karaka, F., Aksoy, M., Kaya, A., Ataman, M.B., Tekeli, T., 1999. Spermatic granuloma in the ram: diagnosis by ultrasonography and semen characteristics. Vet. Radiol. Ultrasound 40, 402–406. Kolettis, P.N., Sharma, R.K., Pasqualotto, F.F., Nelson, D., Thomas, A.J., Agarwal, A., 1999. Effect of seminal oxidative stress on fertility after vasectomy. Fertil. Steril. 71, 249–255. Lohiya, N.K., Tiwari, S.N., Ansari, A.S., Watts, N., 1987. Long tern vasectomy effects on testis and accessory sex organ function in langur monkey. Acta Eur. Fertil. 18, 207–211. Lue, Y., Hikim, A.P., Wang, C., Bonavera, J., Baravarian, S., Leung, A., Swerdloff, R., 1997. Early effects of vasectomy on testicular structure and on germ cell and macrophage apoptosis in the hamster. J. Androl. 18, 166–173. Lunstra, D.D., Schanbacher, B.D., 1988. Testicular function and Leydig cell ultrastructure in long-term bilaterally cryptorchid rams. Biol. Reprod. 38, 211–220. Matsuda, T., Hiura, Y., Muguruma, K., Okuno, H., Horii, Y., Yoshida, O., 1996. Quantitative analysis of testicular histology in patients with vas deferens obstruction caused by childhood inguinal herniorrhaphy: comparison to vasectomized men. J. Urol. 155, 564–567. Mayenco, A.M., Garcia, P., Sanchez, M., 1996. Sperm granuloma in the dog: complication of vasectomy. J. Small Anim. Pract. 37, 392–393. McDonald, S.W., 2000. Cellular responses to vasectomy. Int. Rev. Cytol. 199, 295–339. McDonald, S.W., Scothorne, R.J., 1988. A quantitative study of the effects of vasectomy on spermatogenesis in rats. J. Anat. 159, 219–225. McLachlan, R.I., Royce, P., 1996. The medical consequences of vasectomy. Curr. Obstet. Gynaecol. 43, 34–45. McVicar, C.M., O’Neill, D.A., McClure, N., Clements, B., McCullough, S., Lewis, S.E.M., 2005. Effects of vasectomy on spermatogenesis and fertility outcome after testicular sperm extraction combined with ICSI. Hum. Reprod. 20, 2795–2800. Newton, R.A., 1988. IgG antisperm antibodies attached to sperm do not correlate with infertility after vasovasostomy. Microsurgery 9, 278–280. Osman, A.M., 1980. Long term effects of unilateral vasectomy in immature lambs. Zblatt. Vetmed. A 27, 392–407. Paufler, S.K., Foote, R.H., 1969. Spermatogenesis in the rabbit following ligation of the epididymis at different levels. Anat. Rec. 164, 339–348. Peng, B., Zhang, R.D., Dai, X.S., Deng, X.Z., Wan, Y., Yang, Z.W., 2002. Quantitative (stereological) study of the effects of vasectomy on spermatogenesis in rhesus monkeys (Macaca mulatta). Reproduction 124, 847–856. Perera, B.M., 1978. Changes in the structure and function of the testes and epididymides in vasectomized rams. Fertil. Steril. 29, 354–359. Plaut, S.M., 1973. Testicular morphology in rats vasectomized as adults. Science 181, 554–555. Raleigh, D., O’Donnell, L., Southwick, G.J., Kretser, D.M., McLachian, R.I., 2004. Stereological analysis of the human testis after vasectomy indicates impairment of spermatogenic efficiency with increasing obstructive interval. Fertil. Steril. 81, 1595–1603. Reddy, N.M., Gerscovich, E.O., Jain, K.A., Le-Petross, H.A.T., Brock, J.M., 2004. Vasectomy-related changes on sonographic examination of the scrotum. J. Clin. Ultrasound 32, 394–398. Sargison, N.D., Scott, P.R., Penny, C.D., Pirie, R.S., 1995. Spermatic granuloma in a vasectomized ram. Can. Vet. J. 36, 383–384. Sarrat, R., Whyte, J., Torres, A., Lostale, F., Diaz, M.P., 1996. Experimental vasectomy and testicular structure. Histol. Histopathol. 11, 1–6.

68

P.G. Gouletsou et al. / Animal Reproduction Science 103 (2008) 52–68

Shiraishi, K., Naito, K., Yoshida, K., 2001. Vasectomy impairs spermatogenesis through germ cell apoptosis mediated by p53-Bax pathway in rat. J. Urol. 166, 1565–1571. Shiraishi, K., Takihara, H., Naito, K., 2002. Influence of interstitial fibrosis on spermatogenesis after vasectomy and vasovasostomy. Contraception 65, 245–249. Silber, S.J., Rodriguez-Rigau, L.J., 1981. Quantitative analysis of testicular biopsy: determination of partial obstruction and prediction of sperm count after surgery for obstruction. Fertil. Steril. 36, 480–485. Singh, S.K., Chakravarty, S., 2000. Histologic changes in the mouse testis after bilateral vasectomy. As. J. Androl. 2, 115–120. Tung, K.S.K., Alexander, N.J., 1977. Immunopathologic studies on vasectomized puinea pig. Biol. Reprod. 17, 241–254. Vare, A.M., Bansal, P.C., 1973. Changes in the canine testis after bilateral vasectomy—an experimental study. Fertil. Steril. 24, 793–797. Vare, A.M., Bansal, P.C., 1974. The effects of ligation of cauda epididymidis of the dog testis. Fertil. Steril. 25, 256–260. Whyte, J., Sarrar, R., Cisneros, A.I., Whyte, A., Mazo, R., Torres, A., Lazaro, J., 2000. The vasectomized testis. Int. Surg. 85, 167–174.