Effect of surgical restriction of growth of the testicular artery on testis size and histology in bulls.

The growth of the testicular artery was restricted on one side in young bulls and subsequent testicular development was monitored. After the animals had been killed, the testes were studied histologically and compared with testes from hypoplastic bulls. The growth rate of testes from the experimental side was significantly lower than that of testes from the sham-operated side over a period of 578 days. At death, the experimental testes had a mean (+/- SD) mass of 76 (+/- 41) g compared with 220 (+/- 31) g for the control testes. The sham-operated testes accounted for 0.071 (+/- 0.008)% of live body mass compared with 0.025 (+/- 0.014)% for the experimental testes. The seminiferous tubules in the sham-operated testes had a mean diameter (+/- SD) of 211 (+/- 25) microns, whereas those of the artery-restricted testes and hypoplastic testes were significantly smaller (152 (+/- 37) and 145 (+/- 45) microns, respectively). In the artery-restricted and hypoplastic testes, the interstitial tissue accounted for a significantly greater proportion of the testicular tissue than in the sham-operated testes and spermatogenesis was either totally absent or present in only a small proportion of tubules. It is suggested that the artery-restricted testes could be used as a model for testicular hypoplasia.     

Effect of surgery and efferent duct ligation on testicular blood flow and testicular steroidogenesis in the rat

A moderate reduction in testicular blood flow was observed in both testes 24 h after unilateral efferent duct ligation without any corresponding change in testosterone secretion as indicated in the peripheral blood, in testicular venous blood, or in testicular tissue fluid. At 21 days a pronounced unilateral decrease in blood flow was associated with the extensive degeneration of tubules in the testis on the ligated side. These changes were also associated with decreased testosterone secretion by the testis on the ligated side, although Leydig cell function was not abolished since testosterone in the tissue increased rather than decreased. It is therefore concluded that testicular blood flow may play an important role in the changes of testosterone secretion that follow unilateral efferent duct ligation.     

Effect of uni- and bilateral cryptorchidism on testicular inhibin and testosterone secretion in rats

The effect of uni- and bilateral cryptorchidism on testicular inhibin and testosterone secretion and their relationships to gonadotropins were studied in rats. Mature Wistar male rats weighing approximately 300 g were made either uni- or bilaterally cryptorchid. Testicular inhibin and testosterone content and plasma levels of LH and FSH were examined 2 weeks later. A similar remarkable decrease in testicular inhibin content was found in uni- and bilaterally cryptorchid testes. On the other hand, the testicular testosterone content was significantly decreased only in unilaterally cryptorchid testis with an inverse increase in the contralateral testis. Plasma testosterone levels were normal and plasma LH and FSH increased significantly in both of the cryptorchid groups. These results showed that cryptorchidism impairs both Sertoli and Leydig cell functions. While testosterone production was compensated by increased LH for 2 weeks, neither inhibin secretion nor storage changed in cryptorchid or contralateral testes during the same period.     

Testosterone, luteinizing hormone, follicle stimulating hormone, and prolactin response to unilateral castration in prepubertal Holstein bulls

Hemicastration of Holstein bulls at 3 months of age resulted in increased (P<0.005) testicular weitht and testis sperm cell content at 330 days after treatment, but did not alter sperm cell concentration in the remaining hypertrophied testis. Radioimmuroassay of blood hormones at 1, 6, 12, and 24 weeks after treatment revealed that unilateral castration did not alter (P>0.1) basal levels or GnRH response profiles of either LH or testosterone compared to intact bulls. Hemicastration caused FSH to be elevated (P<0.01) compared to intact bulls at all sampling periods in both unstimulated and GnRH stimulated bulls. Prolactin varied with season and was greater (P<0.001) in hemicastrated bulls than in intact bulls at 1 and 6 weeks after treatment. Results indicate that unilateral castration at 3 months of age caused testicular hypertrophy of both steroidogenic and gametogenic function and this phenomena may be triggered by increased FSH or prolactin secretion, or both. Further, results indicate different testicular regulation mechanisms exist for pituitary LH and FSH release in bulls.     

Effects of hemicastration and unilateral vasectomy on the remaining gonad and on the FSH,LH and testosterone blood concentration in bulls

The effects of unilateral castration and vasectomy on the weight and microscopic appearance of the contralateral testis and on the blood levels of testosterone, LH and FSH, were studied in German Fleckvieh bulls. Testicular weights were higher in hemicastrated bulls (P < 0.01) and unilaterally vasectomized bulls (P < 0.05) when compared to controls, 377 ± 45g (x ± s, N = 4 and 281 ± 12g, N = 4 vs 226 ± 38g, N = 3, respectively.Testosterone concentrations were higher during the weeks 14 to 22 after surgery in both treated groups. LH levels were not different from controls, but FSH levels increased significantly (P < 0.01) two weeks after hemicastration and unilateral vasectomy.Different factors appear to regulate the LH and the FSH concentrations in bulls. The increase of FSH after hemicastration may indicate a reduced production of inhibin or an inhibin-like substance from the testes, and a similar increase after unilateral vasectomy suggests that this substance may be resorbed distal to the testes.     

Testosterone response of cryptorchid and hypophysectomized rats to human chorionic gonadotrophin (hCG) stimulation

The testosterone responses to a single injection of hCG (100 i.u.) in hypophysectomized (hypox.), cryptorchid or sham-operated rats were followed over a 5-day period. In sham-operated rats, hCG induced a biphasic rise in serum testosterone, peaks being observed at 2 and 72 h. Reduced testis weights, elevated FSH and LH levels and reduced serum testosterone levels were found after 4 weeks of cryptorchidism, but hCG stimulation resulted in a normal 2 h peak in serum testosterone. However, the secondary rise at 72 h in cryptorchid rats was significantly lower than sham-operated rats. Reduced testis weight and undetectable serum FSH and LH levels together with decreased testosterone levels were found 4 weeks after hypophysectomy. Serum testosterone levels rose 2 h after hCG in comparison to hypox. controls but this peak was significantly reduced compared with sham-operated rats. The second rise in serum testosterone began on day 2, peaking on day 4 at levels comparable to that seen in sham-operated rats after hCG. The in vitro basal and hCG stimulated secretion of testosterone by cryptorchid testes was greater than that secreted by normal rat testes (518.0 +/- 45.9 and 3337.6 +/- 304.1 pmol per testis per 4 h compared with 223.6 +/- 24.9 and 1312.9 +/- 141.4 pmol per testis per 4 h for normal rat testes). In cryptorchid animals a single injection of 100 i.u. hCG resulted in a pattern of in vitro refractoriness similar to normal rats, lasting from 12 h to 2 days, during which testosterone secretion was reduced to near basal levels. The in vitro basal and hCG-stimulated secretion of testosterone by hypox. rat testes was severely diminished compared with normal rat testes. The temporal pattern of in vitro secretion of testosterone from hypox. rat testes mimicked the in vivo serum testosterone pattern seen in these animals. This study demonstrates important differences in the in vivo and in vitro testosterone response to hCG after testicular damage.     

Disturbed testicular descent in the rat after prenatal exposure to the antiandrogen flutamide.

Exposure of rats in utero to the anti-androgen flutamide resulted in feminization of the external genitalia that was noticeable at birth. This exposure also resulted in a high degree of cryptorchidism during adulthood. In most affected animals, testes were lying in 'ovary position' close to the caudal pole of the ipsilateral kidney. Cryptorchidism occurred despite normal prenatal development of the gubernacular cones and the transformation of these structures, postnatally, into muscular cremaster sacs. Inter- and intralitter variation in the response to prenatal exposure to flutamide was observed as well as intra-individual variation. Cryptorchidism frequently occurred unilaterally with right side cryptorchidism predominating. Cryptorchidism occurred in association with marked suppression of the growth of the ipsilateral epididymis and deferent duct. The possibility is considered that the poor development or absence of these structures contributes to cryptorchidism. Intra-individual variation supports the concept of the local nature of the influence of testis hormones in stabilization and further differentiation of the ipsilateral Wolffian duct derivatives. Cryptorchidism was enhanced when rats were treated postnatally with testosterone or oestradiol. The effect of testosterone was unexpected in view of the generally held hypothesis that androgens enhance testis descent. The effect of oestradiol was as expected: other animal models have been described in which induction of cryptorchidism by oestradiol occurs. Additional treatment with oestradiol caused further suppression of growth of the epididymis and deferent duct. The response to prenatal exposure to flutamide was not altered by further injections of flutamide postnatally. Such injections were without effect in males not exposed to flutamide prenatally except for minor, but statistically significant, testicular enlargement during adulthood. A model is thus presented that describes cryptorchidism as an endogenous developmental disorder.     

Morphological study on the hypoplastic testis with aplasia of the epididymis, ductus deferens, and gland of the ductus deferens in the TW inbred rat

In the TW inbred rat, about 50% of the males show bilateral or unilateral testicular hypoplasia with aplasia of the ipsilateral epididymis, ductus deferens and gland of the ductus deferens. To investigate the pathogenesis of the testicular abnormality in the TW rats, the weight and morphology of the testes on the aplastic and normal side were studied between one week and one year of age. The weight of the testes on the affected side was greater than those on the normal side at four and five weeks. However, it rose to a plateau at six weeks and then remained at about one half to one third the weight of a normal testis. As for the testicular histology, there were no obvious changes from one day to three weeks of age. The diameter of the seminiferous tubules became larger and the number of germ cells decreased at four and five weeks. At six weeks, degeneration and loss of germ cells were observed and many multinucleated giant cells appeared. Thereafter, the loss of germ cells became more severe, and they eventually disappeared with increasing age, but Sertoli's cells continued to exist. In the interstitial area, edematous changes and proliferation of Leydig's cells were observed. The efferent duct of another strain, with normal testes, was ligated at three weeks of age, and changes of the testis after the operation were examined to investigate whether or not these anomalies of the TW strain were due to the absence of the accessories, which may block the excretion of the testicular fluid.(ABSTRACT TRUNCATED AT 250 WORDS)     

Clinical, morphological and endocrinological aspects of cryptorchidism in the horse

The authors analysed clinical, histological and hormonal data obtained from 205 cryptorchid horses. The majority of the unilaterally and bilaterally retained testes were located in the inguinal canal; however, the ratio of inguinal vs abdominal retention appeared to decrease with advancing age. In unilateral cryptorchidism, a pronounced preference was noted for left abdominal retention, whereas for inguinal cryptorchids, the retained testes occurred equally on both sides. Right inguinal retention was found to decrease with advancing age. Histology of cryptorchid testes revealed apparently normal Leydig cells and arrested spermatogenesis. Plasma testosterone concentrations were similar in normal stallions and unilateral cryptorchids, even in those which had the scrotal testis removed. Plasma oestradiol-17beta levels were lower in unilateral cryptorchids than in stallions.     

Functional arterio-venous anastomoses between the testicular artery and the pampiniform plexus in the spermatic cord of rams

Blood flow to the testis, haemoglobin oxygen saturation and testosterone concentration in arterial and venous testicular blood vessels were studied in Texel rams in the breeding and non-breeding season. Blood flow in the proximal and distal testicular artery was measured electromagnetically. The mean flow in the proximal testicular artery was 18.5 ml/min and in the distal testicular artery 7.5 ml/min, and there was no detectable seasonal influence. Haemoglobin oxygen saturation and testosterone concentration were measured in the saphenous artery and vein, the distal testicular artery and vein, and in the proximal testicular vein. The haemoglobin oxygen saturation in the proximal testicular vein was significantly higher than in the distal testicular vein in both seasons. The mean testosterone concentration was significantly lower in the proximal testicular vein than in the distal testicular vein in both seasons. Based on haemoglobin oxygen saturation and testosterone data, it was calculated that between 28 and 46% of the testicular arterial blood was bypassing the testis and was directly flowing through arterio-venous anastomoses towards the pampiniform plexus in the spermatic cord of conscious rams. In anaesthetized rams 55 and 64% of the blood was flowing directly from the testicular artery to the pampiniform plexus based on blood flow data. Transfer of testosterone and oxygen by passive diffusion from the testicular artery to the pampiniform plexus and vice versa in the spermatic cord was not detected.     

Testicular dysfunction is responsible for low sperm quality in Belgian Blue bulls

In a previous study, we demonstrated that Belgian Blue (BB) bulls have a higher prevalence of small scrota and poorer semen morphology compared to the Holstein Friesian (HF) breed in Belgium. The present study tested the hypothesis that the underlying reason for these BB traits negative to fertility was testicular degeneration, associated with an eventual hypoplastic background. At culling, sperm quality and testicular histology of BB bulls were assessed and compared to that of HF bulls. Besides semen quality being generally poorer in the BB breed, significantly more degenerative changes were encountered in BB compared to HF testicles (degeneration index: 37.7+/-11.9 versus 29.3+/-9.9 for BB and HF bulls, respectively; P=0.053). These results correlated to the percentage of normal spermatozoa (r=-0.44; P=0.024) and primary abnormalities (r=0.38; P=0.053). Moreover, the relative amount of collagen fibers present in the testicular interstitial connective tissue was correlated with % normal sperm (r=-0.47; P=0.017), primary defects (0.48; P=0.014), and the degeneration results (r=0.63; P<0.001). The % testicular interstitial collagen fibers differed significantly between breeds (10.6+/-4.0% for the BB versus 7.6+/-1.9% for the HF bulls; P=0.016). This increased amount of connective tissue in BB testes might hypothetically be responsible for the poorer sperm quality. This condition can be defined as a mild form of testicular hypoplasia, and might, in turn, be responsible for the higher sensitivity to testicular degeneration, which is encountered in the BB breed.     

Compensatory testosterone secretion in unilaterally orchidectomized rats

Blood and testis samples were taken from rats 3 weeks after unilateral (sinistral) orchidectomy or sham operation to study the regulation of circulating testosterone. Although plasma testosterone concentrations did not differ, the concentration of testicular testosterone was twofold greater in orchidectomized rats than in sham operated controls. At autopsy, weights of right testes as well as Leydig cell number of orchidectomized and control rats were similar. These observations indicate that, after unilateral orchidectomy, compensatory hypersecretion is not related to compensatory testicular hypertrophy.     

A comparison of Leydig cell function after unilateral and bilateral cryptorchidism and efferent-duct-ligation

Surgical induction of cryptorchidism or ligation of the efferent ducts disrupts spermatogenesis. The response of Leydig cells to disrupted gametogenesis was studied in vitro in tissue and collagenase dispersed Leydig cells obtained from the testes of rats that were made unilaterally or bilaterally cryptorchid or had been efferent-duct-ligated. Four wks after surgery, androgen secretion per mg of tissue or per Leydig cell in response to maximal luteinizing hormone (LH) stimulation was greater in tissue from damaged than from sham-operated testes. It was concluded that disruption of spermatogenesis resulted in Leydig cells that were hyperresponsive to LH stimulation in vitro. Unilateral lesions produced different responsiveness of Leydig cells from the testes ipsilateral and contralateral to the lesion, supporting the hypothesis that intragonadal modulation of Leydig cells function occurs when the function of seminiferous tubules is impaired. Stimulated androgen production of Leydig cells from the contralateral nonligated testis did not differ from that of the sham-operated controls. With unilateral cryptorchidism, which is accompanied by an increase in the temperature of the operated testis, Leydig cells from the scrotal testis were also hyperresponsive compared to those from sham-operated controls. This suggests a possible intergonadal influence of aspermatogenesis caused by cryptorchidism.     

Contribution of the scrotum,testes, and testicular artery to scrotal/testicular thermoregulation in bulls at two ambient temperatures

The objective of this study was to determine the contribution of the scrotum, testes, and the testicular artery to scrotal/testicular thermoregulation in bulls at two ambient temperatures. Crossbred beef bulls, 1.5 years of age, were placed in controlled environment chambers at ambient temperatures of 15°C (n = 5) or 25°C (n = 6). The distal lateral aspects and entire ventral part of the scrotum was incised under caudal epidural anaesthesia (xylazine, 0.07 mg kg⁻¹). Both testes were withdrawn from the scrotum and then replaced and maintained by clamping the scrotal incisions with towel clamps. One testis was randomly chosen to be the exposed testis and was withdrawn prior to temperature measurements. Surface and internal temperatures were measured with infrared thermography and needle thermocouples, respectively. Temperature gradients (°C; difference in temperature from top to bottom at 15 and at 25°C) were: scrotal surface (with testis), 1.5 and 1.3; scrotal surface (without testis), 2.1 and 1.6; surface of exposed testis, −0.6 and 0.0; sub-tunic of exposed testis, −2.2 and −0.6; intratesticular (covered testis), 0.0 and 0.4; and intratesticular (exposed testis), −1.3 and 0.4. The scrotum markedly affects testicular temperature but the testes have limited influence on scrotal surface temperature. The bovine scrotum and testes have opposing temperature gradients that complement one another, resulting in a relatively uniform intratesticular temperature. These temperature gradients are attributed in part to the testicular artery, which goes from the top of the testis to the bottom, divides into several branches and ramifies dorsally and laterally before entering the testicular parenchyma. Intra-arterial temperatures (measured with needle thermocouples) were lower (P < 0.05) where the artery entered the testis than at both the bottom and top of the testis for both the covered (31.7, 33.4 and 34.3°C) and exposed testis (29.6, 32.0 and 32.5°C) at an ambient temperature of 15°C. Temperature differences were similar, but less pronounced, at 25°C (covered testis, 34.8, 36.3 and 36.5°C; exposed testis, 32.4, 33.5, 33.9°C). Results supported the hypothesis that blood within the testicular artery has a similar temperature at the top of the testis (just ventral to the testicular vascular cone) compared with the bottom, but subsequently cools before entering the testicular parenchyma.     

Testicular development, histology, and hormone profiles in three yearling angus bulls with spermatogenic arrest

This article discusses the interactions between testis criteria and hormone profiles in Angus bulls with spermatogenic arrest. From 2 to 12 months (mo), testis diameter and hormone concentrations (basal and GnRH-stimulated) were evaluated in 27 bulls. At 12 mo, testes were excised. The z statistical test was used to determine whether parameters in three infertile bulls were different (P < 0.05) from those in 24 bulls with normal spermatouenesis. Bull 1 had Sertoli cell-only syndrome and Bull 2 had 90% of the tubules without germ cells and only A1 spermatogonia in the remaining. In Bull 3, germ cells did not advance beyond the primary spermatocyte stage. At 12 mo, testes of Bull 1 (99 g), Bull 2 (105 g) and Bull 3 (32 g) weighed less than those of normal bulls (251.5 +/- 56 g). Sertoli cell numbers/testis in Bull 1 (3.8 x 10(9)) and Bull 2 (4.3 x 10(9)) were not different from those in normal bulls (4.9 +/- 0.3 x 10(9)), but were reduced in Bull 3 (1.6 x 10(9)). The number of Leydig cells per gram of testis parenchyma was higher in Bull 1 (5.4 x 10(7)), Bull 2 (7.3 x 10(7)) and Bull 3 (19 x 10(7)) than in normal bulls (3.6 +/- 0.2 x 10(7)). In Bulls 1 and 2, basal and GnRH-stimulated LH, FSH, testosterone (T), androstenedione (delta4A) and estradiol 17-beta (E2) were within normal ranges at most ages. However, basal FSH and LH were greater in Bull 3 than in normal bulls, probably the causes for higher Leydig cell density. Also in the same animal, GnRH induced lower responses in LH and FSH, consequence of low basal T and E2 at some ages. Basal and GnRH-stimulated delta4A in Bull 3 were greater than in normal bulls after 6 mo, indicating impairment of Leydig cell differentiation. Deficiency in hormone secretion did not appear to be the cause of infertility, which points toward impaired gonadal responses or secretion of intratesticular factors, or genetic defects. Moreover, infertile animals may not always show pronounced changes in hormone secretion, but evaluation of testis growth around puberty can help identify those animals that do not have proper gonadal development.     

Testosterone reserve capacity in prepubertal and juvenile testes after sugery for undescended testis]

Tests testosterone reserve capacity of 6--15 year-old boys was estimated after operative correction of testicular maldescensus by a maximal stimulation test. Subnormal plasma testosterone levels were found in only 2 out of 14 patients with bilateral and 4 with unilateral orchidopexy. Prepubertal boys with unilateral anorchia had normal basal testosterone values and a normal testosterone rise after stimulation. In prepubertal boys with bilateral testes atrophy there was observed a diminished rise after stimulation. The basal testosterone levels were normal. The testosterone basal levels of pubertal boys with unilateral anorchia or bilateral atrophy were subnormal and the stimuation of testosterone production was reduced. The testicular volume of patients without atrophy or anorchia after orchidopexy was normal in prepuberty. During puberta a progressive relative decrease of the testicular volume was observed as compared to normal development. In conclusion, the results demonstrate that the endocrine function in most patients with unilateral or bilateral orchidopexy is in the normal range--a regular puberty can be expected.     

Propranolol inhibits the compensatory increase in androgen secretion after unilateral orchidectomy in rats

Adrenergic antagonists were administered to rats by intratesticular injection at the time of unilateral orchidectomy and 5 h before autopsy, 24 h after surgery. Injections of the beta-receptor antagonist DL-propranolol (0.5 or 1.0 mg/injection) significantly inhibited the increase in the concentration of androgens in testicular vein plasma or interstitial fluid that occurred in unilaterally orchidectomized animals injected with vehicle. DL-Propranolol injections in animals with both testes did not reduce testicular or peripheral androgen concentrations or their increase after hCG administration. Injections of the less potent isomer (+)-propranolol or the alpha-receptor antagonist phentolamine did not inhibit the response to unilateral orchidectomy. It is concluded that the compensatory increase in androgen secretion induced by unilateral orchidectomy is, at least in part, the result of beta-adrenergic stimulation of steroidogenesis.     

Germ Cell Weakness as a Cause of Testicular Hypoplasia in Bulls

Sporadic cases of testicular hypoplasia were earlier found in bulls of the Swedish Red and White breed. An accumulation of cases have occurred since 1970 in sons of 2 outstanding progenytested bull sires, 2 F and 27 U, which had a common father, 545 B. The history and clinical examination of affected bulls varied. Some had azoospermia and very small testes at a young age, while others could be normal in all respect when they were young but had a short reproductive life and had to be culled at about 3 years of age. Most of the affected bulls were between these 2 extremes. The histologic examination showed principally different degrees of testicular degeneration. There were always some germ cells left in all affected seminiferous tubules indicating that there was not a lack of germ cells causing the hypoplasia. Germ cell weakness is obviously a hereditary condition. The sires 545 B, 2 F and 27 U had a relatively low fertility. In their pedigree were several bulls known to have had a low fertility. No sons of 2 F and only a few sons of 27 U were used for A.I. services and at present only few cases of testicular hypoplasia are seen.     

Effect of hemicastration on the level of testicular steroids and growth in bulls and boars

Ten, bull calves of the Norwegian Red breed were hemicastrated at the age of 1 1 2 -3 months . Ten, normal bull calves of similar age served as controls. No significant differences were found in plasma testosterone levels or in weight between the two groups during the ensuing seven-month test period. Eight, male pigs were hemicastrated at 1-2 months of age. Eight, normal male pigs served as controls. Plasma testosterone, androstenone, and body weight were measured fortnightly in all pigs until the age of 6-7 months. Androstenone in adipose tissue was measured from 4-5 months of age. No significant differences were found between normal and hemicastrated animals in any monthly interval. However, when combining the measurement at 5-6 and 6-7 months of age for plasma testosterone and 5alpha-androstenone and 5alpha-androstenone in fat, the normal pigs had significantly higher values than the hemicastrates (p<0.05). The weight of the single testis from the hemicastrated pigs at slaughter nearly equalled the combined weight of both testes from the controls. Thus, hemicastration did not appear to have any significant effect on the level of testicular steroids in plasma in bulls or growth rate in bulls and boars, but did have a slight effect on testicular steroids in plasma in pigs at 5-7 months of age.     

Computer analysis of video and ultrasonographic images for evaluation of bull testes

The objectives of this study were to determine relationships between scrotal size (SC; estimated from a video image) and testicular size, and between ultrasonographic echotexture of the testis and seminiferous tubule area in bulls. Video images of the scrotum of 49 Holstein-Friesian (H-F) bulls were recorded and digitized. Scrotal width and length were measured with custom software. After slaughter, scrotums (containing testes) were excised, SC and testicular height, width and volume were measured, and the testes were examined ultrasonographically. Correlations between SC and testicular width or volume (r = 0.86, P < 0.001 and r = 0.84, P < 0.001, respectively) were much higher than those between scrotal width and testicular width or volume (r = 0.23, P < 0.11 and r = 0.28, P < 0.06). Histological examination of the testes was performed in 31 of the bulls. Ultrasonographic echotexture of the testes (determined with custom software) was highly correlated (r = -0.5, P < 0.005) with seminiferous tubule area. Although SC was superior to video imaging for estimating testicular size, ultrasonographic imaging of the testes has considerable potential for the evaluation of testicular function in bulls.