Efficacy of quercetin in ameliorating hypoxia-induced hematological and histopathological alterations in rohu Labeo rohita.

Hypoxia, a major issue in aquatic ecosystems, in special reference to climate change, and exacerbated by anthropogenic activities. It is causing slow growth, disease outbreaks, and mortality in finfish and shellfish. Therefore, adaptation to lowering oxygen levels through supplementation of herbs or their extracts in diets is imperative. In this study, hypoxia was simulated in controlled conditions with quercetin-enriched diets. Quercetin is a plant pigment (flavonol) possessing anti-oxidant property and is present in vegetables, leaves, seeds, pulses, and fruits. The experiment was conducted on rohu Labeo rohita, which is most widely cultured in India. There were four treatments including T1 (Normoxia: > 5 ppm dissolved oxygen; DO2), T2 (hypoxia: 3-4 ppm DO2), T3 (hypoxia + 50 mg quercetin/kg diet), and T4 (hypoxia + 100 mg quercetin/kg diet). The study was conducted for 30 days, and water quality was measured regularly. The results revealed that the hematological parameters were negatively affected. The tissue micro-architecture illustrated the impairment through degeneration of neurons in the brain, increased pigmentation as melanosis in the kidney, increased thickness of primary lamellae in the gills, and dilatations of sinusoids in the liver in hypoxia groups, while quercetin-enriched diets improved the hematological and histomorphological parameters. The results confirm the utility of hematological and histopathological tools as biomarkers and reflect the possible threats of hypoxia on fish. In conclusion, quercetin in diets appeared to show resistance towards chronic hypoxia by restoring the structure and functions of the vital organs towards normalcy and could be recommended as a potential ameliorative agent.

Analysis of OCT4 and PGP9.5 gene expression in prenatal and postnatal buffalo (Bubalus bubalis) testes.

This study aimed to investigate and characterize the spermatogonial stem cells (SSCs) in buffaloes at different stages of development, including prenatal, neonatal, prepubertal, and adult testes. We sought a comprehensive understanding of these cells through a combination of histological, immunohistochemical, and ultrastructural analyses. Specifically, we examined changes in the expression of two potential SSC markers, OCT4 and PGP9.5, using immunohistochemistry. Additionally, we conducted a real-time quantitative polymerase chain reaction (RT-qPCR) to assess the relative gene expression of OCT4 and PGP9.5. The relative expression of the OCT4 gene was down-regulated in the adult testes compared to its expression during prepubertal and neonatal life. The relative expression of the PGP9.5 gene was up-regulated in the neonatal testes and down-regulated in the prepubertal and adult testes. The spermatogonia were round, oval-to-ellipsoidal cells lying over the basement membrane (BM) with a round-to-oval nucleus. Based on the immunoexpression of the putative SSC markers, OCT4 and PGP9.5, we concluded that the proportion of stem cells was highest during the neonatal stage, followed by the prepubertal and prenatal stages. This finding sheds light on the dynamics of spermatogonial stem cells in buffalo testes at different developmental stages, providing valuable insights into these cells' regulation and potential applications.

Enzyme histochemical characterization of orbital glands in fetuses of Indian buffalo (Bubalus bubalis).

Background: The orbital glands, viz. lacrimal gland, superficial and deep gland of third eyelid (LG, SGT and HG), are important for normal eye functions. These glands have different functions in various animals. The information about the enzyme histochemical nature of prenatal orbital glands in Indian buffalo seems to be unavailable. Therefore, the study was planned on orbital glands of six full term recently died fetuses from animals with dystocia. Methods: The frozen sections of all these glands were subjected to standard localization protocols for Alkaline Phosphatase (AKPase), Glucose 6 phosphatase (G-6-Pase), Lactate dehydrogenase (LDH), Succinate dehydrogenase (SDH), Glucose 6 phosphate dehydrogenase (G-6-PD), Nicotinamide Adenine Dinucleotide Hydrogen Diaphorase (NADHD), Nicotinamide Adenine Dinucleotide Phosphate Hydrogen diaphorase (NADPHD), Dihydroxy phenylalanine oxidase (DOPA-O), Tyrosinase, non-specific esterase (NSE) and Carbonic anhydrase (CAse). Results: The results revealed a mixed spectrum of reaction for the above enzymes in LG, SGT and HG which ranged from moderate (for LDH in SGT) to intense (for most of the enzymes in all three glands). However, DOPA-O, Tyrosinase and CAse did not show any reaction. From the present study, it can be postulated that the orbital glands of fetus have a high activity of metabolism as it has many developmental and functional activities which were mediated with the higher activity of the enzymes involved.

Antireproductive changes instigated by efficient drug delivery via papaya seed chloroform extract-based nanoparticles in male rat Bandicota bengalensis.

Anti-reproductive potential of papaya seed chloroform extract-based solid lipid nanoparticles (PSCEN) was investigated for the first time in lesser bandicoot rat, Bandicota bengalensis. Mature male rats (n = 30 per group) were fed bait (loose mixture of cracked wheat, powdered sugar, and groundnut oil in the ratio 88:10:2) containing two different concentrations of PSCEN (5% and 10%) in a bi-choice condition for 15 days with one group as vehicle control. The ingestion of active ingredient in 15 days treatment was significantly (P ≤ 0.05) higher by rats treated with 10% PSCEN (39.17-58.70 g/kg body weight) as compared to rats treated with 5% PSCEN (21.30-33.23 g/kg body weight). A dose dependent significant (P ≤ 0.05) decrease was observed in the level of testosterone, FSH, LH and GnRH in plasma of treated rats. A significant (P ≤ 0.05) decrease was also observed in level of total soluble proteins, total lipids, phospholipids and cholesterol in both plasma and testicular tissue, and level of 17ß-HSD and 3ß-HSD in testicular tissue indicating anti-reproductive effects of PSCEN treatment. There was observed significant (P ≤ 0.05) effect of treatment on histomorphology of testis and cauda epididymis in the form of reduced tubular diameter, germinal epithelial thickness, number of germ cells and dissociation of epithelial cycle in seminiferous tubules, and reduced tubular diameter, increased epithelial thickness, vacuolization, loose contact of principle cells and reduced number of spermatozoa in the cauda epididymal tubules. Maximum antifertility effect was observed with 10% PSCEN treatment, which was not reversed upto 105 days of treatment withdrawal indicating long-term efficacy. The current investigation suggests the use of PSCEN in the management of reproduction of B. bengalensis by exerting influence on testicular and cauda epididymal functions and biochemical parameters.

Biomarker-assisted assessment of aquatic health using the cosmopolitan common carp, Cyprinus carpio (L): a case study of bisphenol-A exposures.

Monitoring aquatic health from environmental pollutants is critical, none more so than bisphenol-A (BPA), a ubiquitous endocrine-disrupting chemical (EDC). The present study brings out the responses of selected transcripts, hormone levels, and tissue histomorphology in a widely distributed fish species Cyprinus carpio (Linn.), following exposure to environmentally relevant (10, 100 ng/L) and higher (1000 ng/L) concentration of BPA. The response of cyp19a1a, cyp19a1b, and c3 significantly decreased, while that of vtg increased in their respective tissue domains. The hematological parameters TEC, Hb, and Hct decreased significantly in contrast to TLC (p < 0.05) at all exposure concentrations, whereas none of the erythrocytic indices (MCV, MCH, and MCHC) was perturbed. The steroidogenic hormone levels, such as estradiol and progesterone, increased significantly with increasing BPA concentrations. In contrast, the testosterone and all the thyroid hormones (T3, T4, and TSH) were suppressed significantly (p < 0.05). At the histological level, the BPA induced chondrocyte proliferation, which was accompanied by hemorrhage of the gill lamellae, increased melanomacrophagic centers (MMCs), and degeneration of tubules and fluid accumulation in the kidney. In parallel, binucleated hepatocytes and inflammations were prominent in the liver. Collectively, the histomorphology confirmed induction of degenerative effects in all the tissues investigated, while the cyclic responses of biochemical markers suggest an ability to regulate the impacts. However, a chronic exposure could result in overriding the endemic reproductive pathways with potential population-level effects. In conclusion, the study identified multiple molecular, cellular, and physiological markers that could be employed to detect early signs of BPA and more broadly EDC exposures. These markers in combination with a wide distribution of C. carpio should allow comparative studies of pollutants at environmental concentrations.

Immunohistochemical localization of estrogen receptor alpha (ERα) in the oviduct of Indian buffalo during follicular and luteal phases of estrous cycle.

The localization and distribution of estrogen receptor alpha (ERα) in different segments of oviduct of buffalo during follicular and luteal phases of estrous cycle were investigated using immunohistochemistry. Tissue samples from the different segments of oviduct from 12 buffaloes (six each during follicular and luteal phases of estrous cycle) were collected from slaughter house after assessing the gross morphology of ovaries. In addition, blood samples were collected from the animals before slaughter to estimate levels of estrogen and progesterone hormones. The tissue distribution of estrogen receptor was determined by immunohistochemical technique using one-step polymer HRPO staining system. The estrogen receptor was localized in the lamina epithelialis, propria submucosa, tunica muscularis, and tunica serosa. The maximum localization was observed in the lamina epithelialis, where both ciliated and secretory cell types were positive for ERα. Percentage of positive cells varied during the follicular and luteal phases of estrous cycle. The lining epithelium of oviductal glands was also intensely positive for ERα. No immunostaining was observed in any tunic of the oviduct when the primary antibody was replaced by antibody diluent or buffer, and it served as negative control. The data showed that highest immune positive cells were observed in the ampulla region of the oviduct and these cells were lowest in the utero-tubal junction (p < 0.05). Infundibulum, ampulla, and isthmus showed a higher percentage of ERα-positive cells during follicular phase of estrous cycle as compared with those of the luteal phase of estrous cycle (p < 0.05). There was no significant difference in the percentage positive cells during the two phases of estrous cycle in the utero-tubal junction. Immunogold labeling with anti-ERα antibody confirmed the findings of immunohistochemical study at subcellular level. The higher expression during the follicular phase was directly correlated with the level of estrogen hormone.

Prenatal development of retina in buffalo (Bubalus bubalis).

The development of retina in Indian buffalo (Bubalus bubalis) has not been reported previously. The aim of the present study was therefore to report the major landmarks and the time course in the development of retina. Serial histological sections of Indian buffalo embryos and foetuses were used as group1 (<20.0 cm CVRL), group2 (>20.0 but <40.0 cm CVRL) and group3 (>40.0 cm CVRL). Age estimation was made on the basis of crown vertebral-rump length (CVRL), which ranged between 36 and 286 days (1.6-94.0 cm). The retina in Indian buffalo was developed in a similar manner to that of the other mammals with the principal differences in the time of occurrence of various layers of this nervous tunic. In 36 days (1.6 cm stage), the foetal retina was composed of pigmented layer and the layer of neuroblasts. Differentiation of layers was first observed in 47 days (4.0 cm CVRL) which became prominent in 52 days (5.1 cm stage). At 120 days (20.5 cm stage), the differentiation of inner plexiform layer and inner nuclear layer was evident. At 143 days (31.0 cm) foetal age, the faint line in neuroblastic layer was the first evidence of the future outer plexiform layer. In foetuses of group III, the retina was comprised of all 10 layers (eight cell layers and two membranes) viz. pigmented epithelium, layer of rods and cones, outer limiting membrane, outer nuclear layer, outer plexiform layer, inner nuclear layer, inner plexiform layer, ganglion cell layer, layer of nerve fibres and the inner limiting membrane.

Histomorphometrical Study on Regional Variation in Distribution of Sweat Glands in Buffalo Skin.

The study was conducted on skin of 24 buffaloes collected from slaughter house. The skin tissues were collected from dorsal, lateral, and ventral parts of head, neck, thorax, abdomen, and tail regions and fixed in 10% neutral buffered formalin. The tissues were processed for paraffin blocks preparation by acetone benzene schedule. The paraffin sections of 5-6 µm were cut with rotary microtome and stained with hematoxylin and eosin. The sweat glands in buffaloes were of saccular and simple coiled tubular type. Most of the sweat glands were associated with hair follicles and consisted of a coiled secretory portion (body) and a straight duct. The secretory portion was made up of glandular tubules, myoepithelium, and basement membrane. The duct portion had a narrow lumen and was lined by simple cuboidal epithelium. The glandular epithelium was simple squamous, simple cuboidal, or low columnar type depending upon their stage of secretary activity. Two types of sweat glands were observed, i.e., apocrine and merocrine. Large number of blood vessels and nerve fibers were observed in the vicinity of the sweat glands. In head, neck, and tail regions the maximum number of sweat glands/mm2 was observed in dorsal side which did not vary significantly (p<0.05) from lateral and ventral side. In abdomen region the number of sweat glands/mm2 was maximum on lateral region which varied significantly from ventral region (p<0.05). Overall, the maximum number of sweat glands/mm2 was in head region followed by abdomen, thorax, neck, and tail but without any significant (p<0.05) difference. Maximum sweat gland diameter was found in abdomen region followed by thorax, head, neck, and tail region.

Light and Electron Microscopic Studies on Prenatal Differentiation of Exocrine Pancreas in Buffalo.

The study was conducted on pancreas of 24 buffalo fetuses collected from abattoir and Veterinary clinics, GADVASU, Ludhiana. The buffalo fetuses were divided into three groups after measuring their CVRL, namely, group I (CVRL between 0 and 20 cm), group II (CVRL above 20 cm and up to 40 cm), and group III (CVRL above 40 cm) and their approximate age was calculated. The tissues were processed for light and ultrastructural studies. In group I, at 1.2 cm CVRL (34 days), the pancreas comprised tubules and solid nest of undifferentiated epithelial cells. At 7.5 cm CVRL (63 days) acinar cells with zymogen granules were observed. These acinar cells varied in shape from columnar to pyramidal. At 12.8 cm CVRL (86 days), parenchyma began to organize into lobes and lobules. The centroacinar cells were observed at 12.8 cm CVRL (86 days). In group II, at 28.3 cm CVRL (137 days), there was extensive branching of tubules that resulted in highly branched ductal tree connecting exocrine secretary units to the duct system. The interlobular and intralobular ducts were well observed at this age yet the intercalated ducts were not completely developed. In group III, exocrine pancreas showed a massive growth at 48 cm CVRL (182 days) with distinct pancreatic lobes and lobules. At 54 cm CVRL (195 days), well developed pancreatic architecture was seen with the presence of extensive development of exocrine part organized in lobes and lobules with interlobular and intralobular ducts whereas the intercalated ducts were observed in 80 cm CVRL (254 days).

Morphogenesis of Mammary Glands in Buffalo (Bubalus bubalis).

The present research was elucidated on the morphogenesis of mammary gland of buffalo during prenatal development. Total of 16 foetuses ranging from 1.2 cm (34 days) to 108 cm CVRL (curved crown rump length) (317 days) were used for study. The study revealed that mammary line was first observed at 1.2 cm CVRL (34 days), mammary hillock at 1.7 cm (37 days), and mammary bud at 2.6 cm CVRL (41 days) foetuses. Epidermal cone was found at 6.7 cm CVRL (58 days) whereas primary and secondary ducts were observed at 7.4 cm CVRL (62 days) and 15 cm CVRL (96 days), respectively. Connective tissue whorls were reported at 18.2 cm CVRL (110 days) and internal elastic lamina and muscle layers at 24.1 cm CVRL (129 days). Lobules were observed at 29.3 cm CVRL (140 days), rosette of furstenberg at 39.5 cm CVRL (163 days), and keratin plug at 45.5 cm CVRL (176 days) foetus. Primordia of sweat and sebaceous glands around hair follicle were seen at 21.2 cm CVRL (122 days) of foetal life. Differentiation of all the skin layers along with cornification was observed at 69 cm (229 days) in group III foetuses.

Embryonic Development of Heart in Indian Buffalo (Bubalus bubalis).

The present study was conducted on 35 buffalo foetuses from 0.9 cm CVRL (32 days) to 99.5 cm CVRL (298 days) to observe the morphogenesis and histogenesis of heart. The study revealed that, in 0.9 cm CVRL buffalo foetus, heart was unseptated and tubular which was clearly divided into common atrial chamber dorsally, primitive ventricles ventrally, primitive outflow tract with bulbous cordis region proximally, and aortic sac distally at 1.2 cm CVRL. Septum primum appeared at 1.9 cm CVRL whereas the truncal swellings and fold of interventricular septum appeared at 2.5 cm CVRL foetus. At 3.0 cm CVRL septum primum, endocardial cushions, septum secundum, and foramen ovale were observed. At 7.6 cm CVRL the endocardial cushions fused to form right and left atrioventricular openings and ventricular apex became pointed. Interventricular canal was obliterated and four-chambered heart was recognised along with atrioventricular valve, chordae tendineae, and papillary muscles in 8.7 cm CVRL (66 days) buffalo foetus. The endocardium as well as epicardium of the atria was thicker as compared to ventricle, whereas the myocardium of atria was thin as compared to ventricles in all the age groups. All the internal structures of heart were well differentiated from 50 cm CVRL onwards. The detailed structural components of buffalo heart during prenatal period have been discussed in the present paper.

Peroxidase(s) in environment protection.

Industrial discharges of untreated effluents into water bodies and emissions into air have deteriorated the quality of water and air, respectively. The huge amount of pollutants derived from industrial activities represents a threat for the environment and ecologic equilibrium. Phenols and halogenated phenols, polycyclic aromatic hydrocarbons (PAH), endocrine disruptive chemicals (EDC), pesticides, dioxins, polychlorinated biphenyls (PCB), industrial dyes, and other xenobiotics are among the most important pollutants. Peroxidases are enzymes that are able to transform a variety of compounds following a free radical mechanism, thereby yielding oxidized or polymerized products. The peroxidase transformation of these pollutants is accompanied by a reduction in their toxicity, due to loss of biological activity, reduction in the bioavailability, or the removal from aqueous phase, especially when the pollutant is found in water. The review describes the sources of peroxidases, the reactions catalyzed by them, and their applications in the management of pollutants in the environment.

Histogenesis of testicular parenchyma during prenatal life in buffalo / Histogénesis del parénquima testicular durante la vida intrauterina en el búfalo

The study was conducted on the testes of 18 buffalo foetii to reveal histogenesis and differentiation of different cells of testicular parenchyma. At 8.0 cm CVR (65 days) the seminiferous tubules were present at gonadal periphery and a network of polygonal mesenchymal cells was seen in the centre of testis. These tubules were surrounded by a distinct basement membrane and a single layer of peritubular cells at 10 cm CVR (74 days), which became double layered at 88.0 cm CVR (272 days). The testicular parenchyma at 12.0 cm CVR had two zones; outer zone having longitudinal tubules and inner zone having rounded tubules. But a reverse pattern of their arrangement was observed at 14.0 cm CVR (92 days). The pre-Sertoli cells were first observed in buffalo foetii of 8.0 cm CVR (65 days) in the periphery of seminiferous tubular epithelium whereas the gonocytes were demonstrable in the centre of tubules at 10.6 cm CVR (76 days). The fetal Leydig cells were also reported at 8.0 cm CVR (65 days) but at 14.0 cm CVR (92 days), the interstitium had considerably expanded due to the differentiation of mesenchymal cells into the Leydig cells.

El estudio fue realizado en los testículos de 18 fetos de búfalos, para revelar la histogénesis y diferenciación de las diferentes células de parénquima testicular. A los 8,0 cm de longitud corona-rabadilla (LCR) (65 días) los túbulos seminíferos estuvieron presentes en la periferia de la gónada y una red poligonal de células mesenquimales se observó en el centro del testículo. Estos túbulos estaban rodeados por una membrana basal y una sola capa de células peritubular a los 10 cm LCR (74 días), la cual se convirtió en una doble capa a los 88,0 cm LCR (272 días). El parénquima testicular a 12,0 cm LCR tenía dos zonas, zona exterior con túbulos longitudinales y zona interior con los túbulos redondeados transversalmente. Sin embargo, un patrón inverso en su disposición se observó a los 14,0 cm LCR (92 días). Las células pre-Sertoli se observaron primero en fetos de búfalos de 8,0 cm LCR (65 días) en la periferia del epitelio seminífero tubular, mientras que los gonocitos fueron visibles en el centro de los túbulos a 10,6 cm LCR (76 días). Las células de Leydig fetales también se observaron a los 8,0 cm LCR (65 días), pero a los 14,0 cm LCR (92 días), el intersticio tuvo una considerable expansión debido a la diferenciación de células mesenquimales en células de Leydig.

Toxic effect of formaldehyde on the respiratory organs of rabbits: a light and electron microscopic study.

In this study, the effect of direct exposure of formaldehyde in different concentration was observed in 14 rabbits aged 3-6 months and weighing 1100-1200 g. The animals were divided into two groups with six animals in each group, and two animals were kept as control. The animals of group I were exposed to 10% formalin for 12 weeks and those of group II to 40% formalin solution for 6 weeks. After completion of the experimental periods, the animals were killed and the tissue samples were collected from the nasal cavity, trachea and lungs in 10% neutral buffered formalin and Karnovsky's fixative to examine the histological and electron microscopic changes in the organs. The mucosal cells of nasal cavity showed loss of cilia and epithelial metaplasia was observed in places. There was vascular congestion and mild subepithelial odema. The tracheal epithelium was organized with hyperchromatic nuclei. There was subepithelial odema along with lymphomononuclear cellular infiltration. There was marked emphysema evident as bulla formation of air spaces due to rupture of interalveolar septum. An increased cellularity of alveolar wall was observed, resulting in its thickening. The epithelial lining of bronchioles showed loss of mucosal folds and hyperplasia of cells along with peribronchial lymphomononuclear cellular infiltration. Thickening of wall of blood vessels was evident. Congestion and haemorrhages were observed in places. It is concluded that the histopathological changes were more remarkable in the animals exposed to 40% formaldehyde for short duration than the animals exposed to 10% formaldehyde for longer duration with a more severe effect on the upper part of the respiratory tract than the lower one.

Anatomical study on true hermaphroditism in an Indian pig (Sus scrofa domesticus).

A pig was confirmed to be a true hermaphrodite on the basis of gross and histomorphological studies of the genital organs. The genitalia was consisted of left ovary, oviduct, two coiled uterine horns, body of uterus along with right testis and an epididymis. Vagina and vulva were absent but male urethra with prostate gland was present. Grossly the size of all the genital organs appeared to be normal. Histomorphologically, testis and epididymis were underdeveloped as there was no clearcut spermatogenia and sertoli cells but Leydig cells were normal. The ovary presents normal histological features with some portion of testicular tissue. Degeneration of uterine epithelium was observed along with normal endometrial glands.