Indigofera cryptantha-induced pigmenturia in cattle in South Africa.

Two field cases of reddish-black pigmenturia occurred where cattle grazed on an established Cenchrus ciliaris (blue buffalo grass) pasture in South Africa. The pasture was noticeably invaded by Indigofera cryptantha, which was heavily grazed. Apart from the discolored urine, no other clinical abnormalities were detected. Urinalysis revealed hemoglobinuria, proteinuria and an alkaline pH. When the animals were immediately removed from the infested pasture, they made an uneventful recovery. However, a bull died when one of the herds could not be removed from the I. cryptantha-infested pasture. Macroscopically, the kidneys were dark red in color and the urinary bladder contained the dark pigmented urine. Microscopically, the renal tubules contained eosinophilic, granular pigment casts in the lumen. In addition, many renal tubular epithelial cells were attenuated with granular cytoplasm and were detached from the basement membranes. Chemical analysis was performed on dried, milled plant material and two urine samples collected during the field investigations. Qualitative UPLC-UV-qTOF/MS analysis revealed the presence of indican (indoxyl-ß-glucoside) in the stems, leaves and pods of I. cryptantha and indoxyl sulfate was identified, and confirmed with an analytical standard, in the urine samples. It is proposed that following ingestion of I. cryptantha, indican will be hydrolysed in the liver to indoxyl and conjugated with sulfate. Indoxyl sulfate will then be excreted in relatively high concentrations in the urine. In the alkaline urine, two indoxyl molecules might dimerize to form leucoindigo with subsequent oxidation to indigo, thus, contributing to the dark pigmentation of the urine. It is also possible that indoxyl sulfate contributed to the renal failure and death of the bull. Although I. suffruticosa-induced hemoglobinuria has been described in Brazil, this is the first report of I. cryptantha-induced pigmenturia in cattle in South Africa.

Effect of di(n-butyl) phthalate on the blood-testis barrier during puberty onset.

Di(n-butyl) phthalate (DBP) is considered a substance of serious concern because of its reproductive toxicity and endocrine-disrupting properties. Exposure to DBP causes morphological and functional changes in the male reproductive system of birds and mammals. However, there are no detailed reports on the effects of DBP on the Sertoli cell and junctional complexes of the blood-testis barrier (BTB) in birds. The present study investigated dose-related ultrastructural changes in Sertoli cells and junctional complexes of the BTB in adult Japanese quail (Coturnix coturnix japonica) exposed to DBP prior to puberty. A total of 25 Japanese quail were used for the study. Exposure to DBP doses of 50, 200 and 400 mg DBP/kg/d caused dose-related ultrastructural changes in junctional complexes including dilation and separation, while disruption of cytoplasmic membranes and mitochondria was observed in Sertoli cells. There was a significant difference in the sum of vacuoles, vacuole diameter, nuclear width, nuclear length, nuclear area, sum of damaged spherical mitochondria, width of elongated mitochondria and the sum of damaged elongated mitochondria among the five treatment groups (p Ë‚ 0.05). Prepubertal exposure to DBP at doses of 50, 200 and 400 mg DBP/kg/d for 30 days led to adverse effects in the adult male Japanese quail reproductive system by inducing structural changes in the Sertoli cells and junctional complexes. Such changes might disrupt the BTB and potentially interfere with spermatogenesis. Results indicated that the Sertoli cell is sensitive to DBP exposure and might be an important cellular target for DBP-induced testicular toxicity.

Regulation of spermatogenic cell apoptosis by the pro-apoptotic proteins in the testicular tissues of mammalian and avian species.

Apoptosis of germ cells is an important feature of spermatogenesis, as this process allows the removal of excess germ cells from testicular tissue. This is crucial to control the number of germ cells that can be supported and nourished by the Sertoli cells. It has been established that up to 75 % of germ cells are lost during the development of spermatogonia. In this process, germ cells with defective genes are removed. Also, apoptosis regulates homeostasis of testicular tissue by maintaining a balance between germ cell proliferation and cell death. This is necessary as it guarantees normal spermatogenesis. Apoptosis also occurs during maturation divisions of spermatocytes and spermatids but albeit to a lesser extent. Several factors, known pro-apoptotic proteins, play a critical role in the process of apoptosis. The most vital pro-apoptotic proteins are caspase-3, B-cells lymphoma 2 (Bcl2), truncated BH3 interacting death domain (tBID), tumor suppressor protein (p53), and Bcl-2 associated protein (BAX). Execution of apoptosis may be triggered by either an extrinsic or an intrinsic pathway. The extrinsic pathway is initiated by death receptors and death ligands. Death receptors trigger pro-apoptotic proteins such as caspase-3 for the execution of apoptosis. The intrinsic pathway, on the other hand, is triggered by nutrient deprivation, stress, or DNA damage, which in turn activates Bcl2 families of pro-apoptotic proteins that foster apoptosis. The present review focuses on pro-apoptotic proteins and their mechanisms of action, with special emphasis on their involvement in germ cell apoptosis in the testicular tissues of mammalian and avian species.

Immunohistochemical Changes in the Testicular Excurrent Duct System of Healthy, Male Japanese Quail (Coturnix coturnix japonica) Observed at 4, 6-7, 12, and 52 Weeks of Age.

The immunolocalization of the cytoskeletal and the extracellular matrix proteins was investigated in the testicular excurrent duct system of healthy Japanese quail at 4, 6−7, 12 and 52 weeks of age. TdT dUTP Nick End Labeling (TUNEL) assay was used to assess apoptotic cell formation. The epithelia of the testicular excurrent duct system in birds of all age groups displayed various immunolabeling intensities and localization of cytokeratin 5 and beta-tubulin, while α-SMA was observed in epithelia only of 4-week-old birds. In all age groups, vimentin immunostaining was observed in the rete testes and efferent ductular epithelia, but not in the epididymal duct unit. The periductal smooth muscle cells of the excurrent duct system displayed variably intense immunopositivity with cytokeratin 5, desmin, fibronectin, α-SMA, and beta-tubulin. Furthermore, beta-tubulin and vimentin immunolabeled endothelial cells and fibroblasts with various intensities, while fibronectin immunostained extracellular matrices surrounding these cells. TUNEL-positive apoptotic cells were observed in the rete testes and efferent ductular epithelia, with increased frequency (p < 0.001) in 52-week-old birds. The study serves as a baseline normal for this region in healthy birds at 4, 6−7, 12, and 52 weeks of age, for comparison in future similar immunohistochemical studies involving environmental toxins affecting this region.

Junctional complexes of the blood-testis barrier in the Japanese quail (Coturnix Coturnix japonica).

This study investigated the developmental changes in the adherens junctions, gap junctions, as well as tight junctions forming the blood-testis barrier (BTB) in Japanese quail (Coturnix Coturnix japonica) testis. Testicular tissue from pre-pubertal, pubertal, adult, and aged Japanese quail were examined by immunohistochemistry and transmission electron microscopy (TEM). The tight junction proteins claudin-3, claudin-11, occludin and zonula occludens-1 (ZO-1), were generally localised in the cytoplasm of Sertoli cells, spermatogonia, and spermatocytes of pre-pubertal, pubertal, some adult birds. The adherens junction protein E-cadherin had a similar distribution pattern. During pre-pubertal development, the gap junction protein connexin-43 (Cx43) was only localised between Leydig cells in the testicular interstitium. However, TEM revealed the presence of gap junctions between cells of the seminiferous epithelium as early as the pre-pubertal stage. Furthermore, TEM confirmed the presence of tight and adherens junctions in the seminiferous epithelia of all age groups. The findings of this study document age-related differences in the immunolocalisation and intensity of the junctional proteins and the ultrastructure of the junctional complexes forming the BTB in quail testes. Additionally, the junctional complexes forming the BTB in the Japanese quail are well established prior to puberty. This study provides baseline information for the future evaluation of pathological changes in the BTB of avian species at different developmental stages.

Ontogeny of the testicular excurrent duct system of male Japanese quail (Coturnix japonica): A histological, ultrastructural, and histometric study.

The testicular excurrent duct system undergoes several physiological and morphological changes during the reproductive stage or breeding season in mammals, birds, and reptiles. Studies on normal age-related histomorphological changes in the excurrent duct system of Japanese quails (Coturnix japonica) remain unreported, despite the extensive use of this bird as an avian model in research studies. The current study investigated the histological, ultrastructural, and histometric changes in the testicular excurrent duct system of the Japanese quail during three reproductive stages, namely prepubertal, pubertal, and adult. Simple squamous to low cuboidal cells formed the epithelia of the rete testis in prepubertal and pubertal birds, while in adult birds the lining was low cuboidal to cuboidal. In pubertal and adult birds, the nonciliated Type I epithelial cells of the proximal efferent duct displayed a subapical endocytotic apparatus comprising coated pits, coated apical tubules, and endosomes. There was a significant increase (p ≤ .001) in epithelial heights of all ducts of the excurrent duct system in the mature, sexually active, adult birds when compared to the other age groups. The luminal and tubular diameters, and the cross-sectional areas of efferent ducts and the epididymal duct unit increased significantly (p ≤ .001) with age. It is concluded that the morphology and morphometry of the excurrent ducts of the testis of the Japanese quail change as birds mature.

Structural observations on spermatogenic cells of Japanese quail (Coturnix coturnix japonica) pre-pubertally exposed to dibutyl phthalate: A light and transmission electron microscopy study.

Exposure to dibutyl phthalate (DBP) induces testicular damage in mammals. However, studies on the effects of DBP on spermatogenic cells in birds are grossly lacking. Therefore, this study was designed to determine the effects of the pre-pubertal exposure to DBP on the histology and ultrastructure of spermatogenic cells in the testis of adult Japanese quail (Coturnix coturnix japonica). The birds were randomly divided into five dosage groups at the age of 4 weeks. The control group received a corn oil vehicle only (a dose of 1 mL/kg body weight), while the other four experimental groups received a daily dosage of 10, 50, 200, 400 mg/kg body weight of DBP (dissolved in corn oil), respectively with the aid of gastric lavage, for 30 days. Testicular samples were processed and examined by light microscopy and transmission electron microscopy. Histopathological evaluation revealed vacuole formation, germ cell degenerations, and the absence of spermatogenic cell series. Ultrastructurally, chromatin clumps in spermatocyte and degenerated spermatogonia with ruptured nuclear membranes resting on the distorted basement membranes were observed. Others were intracytoplasmic vacuoles in round spermatids and fragments of dense apoptotic bodies. In conclusion, the findings of the present study reveal that spermatogenic cells of Japanese quails seem to be more sensitive to DBP-induced degeneration compared to mammalian species studied. The Japanese quail could be used to monitor environmental contamination with low doses of DBP.

Use of rectal tissue for reconstruction of an anogenital cleft in a juvenile dog with recurrent rectal prolapse.

CASE DESCRIPTION: A 4-month-old 4.2 kg sexually intact female mixed-breed dog was evaluated for rectal and vesicular tenesmus, intermittent rectal prolapse, fecal incontinence, and an anogenital cleft. CLINICAL FINDINGS: Rectal prolapse and an anogenital cleft were confirmed on physical examination. Results of a CBC and serum biochemical analysis were within respective reference ranges, and abdominal ultrasonography revealed no abnormalities. Urinalysis revealed evidence of a urinary tract infection. TREATMENT AND OUTCOME: An H-perineoplasty was performed and the prolapse was reduced. The repair partially dehisced and was repaired (with concurrent reduction of a recurrent rectal prolapse) but dehisced again. There was limited tissue available for additional reconstruction. In another surgical procedure, the rectum was allowed to prolapse, the most dorsal 40% of the prolapsed rectal tissue was resected, and the rectal tissue margin and skin in this region were apposed. The remaining rectal tissue flap was folded ventrally, and the lateral margins of the aborad aspect were sutured to the dorsolateral vestibular mucosa. In a subsequent surgery, 2.5 to 3 cm of the rectal tissue flap was excised. The remainder was used to create ventral margins for the rectum and vestibular mucosa. The perineal skin between the anus and dorsal vulvar commissure was closed. The patient experienced mild cutaneous partial dehiscence of the repair that healed by second intention. Over an 18-month follow-up period, some fecal incontinence persisted, but straining resolved and urinary tract infection did not recur. CLINICAL RELEVANCE: For the dog of this report, the use of rectal mucosa in surgical repair of an anogenital cleft provided an acceptable clinical outcome.

Age-related histomorphometric and ultrastructural changes in the Sertoli cells of Japanese quail (Coturnix Coturnix japonica).

Prepared sections from pre-pubertal, pubertal, adult, and aged Japanese quail testes were examined by light microscopy and transmission electron microscopy (TEM) and measurements of seminiferous tubular diameter (STD), luminal diameter (SLD), epithelial height (SEH) and cross-sectional area of the seminiferous tubules (AST) were taken using an image analyzer. Apoptotic Sertoli cells with features such as cell shrinkage and chromatin condensation were observed in pre-pubertal and aged quail. There was a significant difference between the mean Sertoli cell number (SCN), SLD, SEH, STD and AST among the four age groups (P < 0.001). The highest SCN (mean ± standard error) was recorded in the adult (30.53 ± 0.42), with the aged group displaying the lowest mean (11.80 ± 0.27) SCN. Spearman's rho correlation coefficients demonstrated a strong relationship between the SCN and SEH in the pubertal (ρ=0.915; P < 0.001), adult (ρ=0.878; P < 0.001), and aged (ρ=0.858; P < 0.001) groups, while a significant moderate correlation was observed in the pre-pubertal (ρ=0.606; P < 0.001) group. There were significant moderate correlations between the SCN and STD in the pre-pubertal (ρ=0.445; P < 0.001), pubertal (ρ=0.653; P < 0.001), adult (ρ=0.440; P < 0.001), and aged (ρ=0.514; P < 0.001) groups. Furthermore, significant moderate correlations were estimated between the SCN and AST in the pre-pubertal (ρ=0.453; P < 0.001), pubertal (ρ=0.661; P < 0.001), adult (ρ=0.393; P = 0.001), and aged (ρ=0.498; P < 0.001) groups. This study provides baseline data on the morphology and development of the Sertoli cell, as well as testicular morphometry in avian species during the pre-pubertal, pubertal, adult, and aged stages using the Japanese quail as a model.

Dose-related morphological changes in the epididymal region of sexually active adult male Japanese quail treated with di-n-butyl phthalate (DBP) commencing during the pre-pubertal stage.

Di-n-butyl phthalate (DBP) is widely used as a plasticizer in personal care and medical products and is known to induce toxicity in the male reproductive organs in both mammals and birds. In this study, there was investigation of the effects of DBP on the epithelium of the rete testis, proximal, and distal efferent ductules and epididymal duct of adult Japanese quail (Coturnix japonica) following treatment with varying doses during the pre-pubertal and peri-pubertal periods. Pre-pubertal quail (n = 25) 4 weeks post-hatching were dosed orally with 10, 50, 200 and 400 mg DBP/kg/d, for 30 days and control birds were administered corn-oil only (n = 5 per group). Histo-metrically, there was lesser (P <  0.001) epithelial heights of the rete testis and efferent ductules in all quail DBP-treated groups, but not in the epididymal duct epithelium. There were no morphological change effects as a result of DBP treatments in the rete testis epithelium, while there were epithelial cytoplasmic vacuoles detected in the distal efferent ductule and epididymal duct of birds treated with 50, 200 and 400 mg DPB/kg/d. There were several lesions, including degenerative changes, cytoplasmic vacuoles, apoptosis and autophagy in the epithelium of the proximal efferent ductule in quail treated with 200 and 400 mg DBP/kg/d. Overall, the results indicate that treatment with DBP during the pre-pubertal period induced dose-dependent histometric and morphological changes in the epithelium of the epididymal region. It is concluded that the proximal efferent ductule was a highly sensitive component of the epididymal tissues of Japanese quail following treatment with DBP during the pre-pubertal period.

Apoptosis of germ cells in the normal testis of the Japanese quail (Coturnix coturnix japonica).

It has been established that excess germ cells in normal and in pathological conditions are removed from testicular tissue by the mechanism of apoptosis. Studies on germ cell apoptosis in avian species are grossly lacking, and there are only a few reports on induced germ cell degenerations in the testis tissue of birds. This study was designed to investigate the process of apoptosis of germ cells in the Japanese quail (Coturnix coturnix japonica). Germ cell degenerations were investigated in birds of all age groups, namely pre-pubertal, pubertal, adult, and aged. Apoptosis of germ cells in the quails, as shown by hematoxylin & eosin (H&E), TdT dUTP Nick End Labeling (TUNEL) assay and electron microscopy, was similar to that observed in previous studies of germ cells and somatic cells of mammalian species. The observed morphological features of these apoptotic cells ranged from irregular plasma and nuclear membranes in the early stage of apoptosis to rupture of the nuclear membrane, condensation of nuclear material, as well as fragments of apoptotic bodies, in later stages of apoptosis. In the TUNEL-positive cell counts, there was a significant difference between the mean cell counts for the four age groups (P < 0.05). Post hoc analysis revealed a highly significant difference in the aged group relative to the pubertal and adult age groups, while the cell counts of the pre-pubertal group were significantly higher than those of the pubertal group. However, there was no significant difference between cell counts of the pre-pubertal and the adult, and between the pre-pubertal and the aged groups.