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.

Immunoexpression of cytokine tumour necrosis factor-α suggesting its role in formation and regression of corpus luteum in Indian buffalo.

Tumour necrosis factor-α (TNF-α) is a cytokine that plays multiple important roles in corpus luteum (CL). Immunolocalization of expression of TNF-α in CL of buffalo was studied in different stages of its development and regression. Corpus luteum of healthy buffaloes (24) was collected from local slaughterhouses and categorized into early (stage I, 1-5 days, n = 6), mid (stage II, 6-11 days, n = 6), late luteal (stage III, 12-16 days, n = 6) and regressing phase (stage IV, 17-20 days, n = 6). In earliest phase of cyclic CL, per cent immunoexpression of TNF-α was significantly (p < .05) lower as compared to all phases with its expression being restricted to few developing luteal cells, usually in neutrophils. A significantly (p < .05) higher number of neutrophils with TNF-α immunoexpression were observed as compared to mid-luteal phase that indicated its role in initiation of angiogenesis at this stage. TNF-α immunoexpression almost doubled in mid-luteal phase, but the number of neutrophils exhibiting TNF-α was significantly (p < .05) lower with respect to all phases of CL. Immunoexpression percentage in late luteal phase increased sharply being significantly (p < .05) higher than earlier two phases of CL. In regressing phase, per cent immunostaining was maximum with highly significant (p < .05) difference as compared to all other stages, observed in all degrading luteal cells, abundant immune cells, that is neutrophils and macrophages which finally led to apoptosis and phagocytosis. Immunoexpression of TNF-α in early luteal phases served its role in initiation of angiogenesis, and its intense expression in regressing phase of CL suggested a shift in its role to apoptosis and structural luteal regression signifying both luteotropic and luteolytic roles in buffalo. This is probably the first study of its kind in buffaloes.

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.

Histomorphogenesis of sublingual salivary gland of Indian sheep.

The primordial anlage of sublingual gland was first noticed as a solid epithelial bud from oral epithelium at the 24th day of foetal development. The terminal buds were arranged in the form of clusters with undifferentiated epithelial cells and terminated in a bulb-like structure in the 30-day-old sheep foetus. On the 37th day, lumenization and branching of the main cord was noticed. The primary septa were observed from the 55th day onwards which resulted in the formation of lobulation on the 60th day. The capsule formation was initiated by aggregation of mesenchymal tissue on the 63rd day. On the 100th day, terminal tubules differentiated to form secretory end pieces. Tubular portions formed intercalated and striated ducts. Predominantly mucous type of acinar cells was seen from the 110th day onwards with myoepithelial cells. The number of lobules increased with increase in parenchyma from 130th day onwards. Micrometrical studies revealed that the mean diameter of acini, intercalated, striated and large ducts was increased with advancement of age and significant differences were observed between groups. Localization of acidic and neutral mucopolysaccharides were observed in mucous and goblet cells. Fine lipid droplets were observed in intralobular and interlobular connective tissue however, phospholipids were observed in cell membrane of acini and ducts. The current investigation provides microstructural standards for the organogenesis of the sublingual gland of miniature sheep and can lay the foundation for further studies in the morphological investigation of salivary gland development.

Developmental events in the mandibular salivary gland of Indian sheep (Ovis aries): Morphological and histochemical studies.

At 22nd day of fetal development, the primordial anlage of mandibular gland was first noticed as a solid epithelial bud from oral epithelium. The terminal buds were arranged in the form of clusters with undifferentiated epithelial cells and terminated in a bulb like structure in 28-day-old fetus. The lumenization and branching of the main cord was noticed at 35th day. The primary septa, which divide the glandular mass into lobes was observed from 53rd day onwards which resulted in the formation of distinct lobulation at 58th day. At 61st day, the capsule formation was initiated by the aggregation of mesenchymal tissue. The terminal tubules differentiated to form the secretory end pieces and the tubular portion leads to the formation of intercalated and striated ducts at 98th day. Predominantly mucous types of acinar cells were seen from 108th day onwards. The number of lobules increased with steep increase in parenchyma from 128th day onwards. Micrometrical studies revealed that the mean diameter of acinar cells and all ducts was increased with the advancement of age and the significant differences were observed between groups. Localization of acidic and neutral mucopolysaccharides was observed in mucous cells and goblet cells. RESEARCH HIGHLIGHTS: The primordial anlage of mandibular salivary gland was seen at 22nd day. Lobulation of gland was appeared at 53rd day of development, however; it was completed at 58th day. At 98th day, the terminal tubules differentiated to form the secretory end pieces. The parenchyma of the gland showed predominantly mucous type of cells from 108th day onwards. Myoepithelial cells were first appeared as flattened basal cells initially around the developing acinar cells at 132nd day. Localization of acidic as well as neutral mucopolysaccharides was observed in mucous cells and goblet cells. Fine lipid droplets were observed in intralobular as well as interlobular connective tissue, however; phospholipids were observed in the cell membrane of secretory cells and ducts.

Immunoexpression of vascular endothelial growth factor and vWF-regulating angiogenesis in cyclic corpus luteum of Indian buffalo.

The present study was conducted to localize the immunoexpression of VEGF-A (Vascular Endothelial Growth Factor) and von Willebrand factor (vWF) in corpora lutea of healthy buffaloes (24) collected from local slaughterhouses. CL collected were categorized into early (stage I, 1-5 days, n = 6), mid (stage II, 6-11 days, n = 6), late luteal phase (stage III, 12 to 16 days, n = 6) and regressing phase (stage IV, 17 to 20 days, n = 6). The percent positive immunostaining for VEGF-A was significantly (p < 0.05) higher in mid-luteal phase than the other three stages of CL. However, it was higher in early luteal phase as well indicated intense angiogenesis in both early and mid-luteal phases. The number of capillary endothelium expressing vWF was significantly (p < 0.05) highest in mid-luteal phase among all the phases. However, in late luteal phase, the percent area positive for VEGF-A immunostaining was reduced but it was significantly (p < 0.05) higher than corpus albicans phase. Thus, in regressing phase or corpus albicans, it was lowest and reduced considerably. However, in late luteal phase, the number of capillaries with vWF immunoexpression reduced significantly (p < 0.05) but it was lowest in corpus albicans phase. Therefore, the immunotaining pattern for VEGF-A and vWF concluded that there was a spositive linear correlation between the two, that is, as the VEGF-A expression was increased, the number of vWF positive capillaries also increased and vice versa. The VEGF-A expressed by the luteal parenchyma in different stages of development and regression of corpus luteum was thus observed to be involved in promoting the angiogenesis and luteal cell proliferation as supported by vWF expressed by endothelium of proliferating capillaries in buffalo corpus luteum throughout the estrous cycle.

Gross morphology and morphometry of native and decellularized heart valves of caprine: A comparative study.

The gross morphological examination of native caprine heart valves revealed distinctive structural characteristics of the caprine's cardiac anatomy. Four primary orifices were identified, each protected by thin, valve-like structures. Atrioventricular orifices featured tricuspid and bicuspid valves, while the aorta and pulmonary arteries were guarded by semilunar valves. Within the atrioventricular apparatus, distinct features were observed including the tricuspid valve's three leaflets and the bicuspid valve's anterior and posterior leaflets. Ultrasonography provided insights into valve thickness and chordae tendineae lengths. Morphometric studies compared leaflets/cusps within individual native valves, showcasing significant variations in dimensions. Comparative analysis between native and decellularized valves highlighted the effects of decellularization on leaflet thickness and chordae tendineae lengths. Decellularized valves exhibited reduced dimensions compared to native valves, indicating successful removal of cellular components. While some dimensions remained unchanged post-decellularization, significant reductions were observed in leaflet thicknesses and chordae tendineae lengths. Notably, semilunar valve cusps displayed varying responses to decellularization, with significant reductions in cusp lengths observed in the aortic valve, while the pulmonary valve exhibited more subtle changes. These findings underscore the importance of understanding structural alterations in heart valves post-decellularization, providing valuable insights for tissue engineering applications and regenerative medicine.

Histomorphological, immunohistochemical, and ultrastructural study on ontogeny of ileocaecal lymphoglandular complexes in prenatal and postnatal Indian buffalo: An innate mucosal immune barrier.

The present study was conducted on prenatal and postnatal development of lymphoglandular complexes (LGCs) in ileocaecal region of buffalo fetuses (n = 15) ranging from 11.5 cm curved crown rump length (CVRL) (80 days) to 100 cm CVRL (299 days) and neonatal buffalo calves (n = 10). The fetuses were categorized into three groups based on their CVRL. LGC formation was not evident in ileocaecal junction up to 32 cm CVRL (145 days). At 35 cm CVRL (152 days), diffuse lymphocytes were scattered around the base of glands that encircled them. At 54 cm CVRL (195 days), lymphoid aggregates were present in submucosa around deep submucosal glands and formed primordia of LGCs in ileocaeccal orifice region. At 100 cm CVRL (299 days), these complexes were completely visible grossly. The distinguished lymphoid nodules in submucosa were invaded by submucosal extensions of overlying mucosal glands to form a large clear complex of glands and lymphoid nodules called as "Lymphoglandular complex" at this stage. It is the first report of prenatal development of LGCs in large intestine of buffaloes. Abundant CD3+ T cells were observed towards periphery of LGC. In neonates, these complexes were uniform, few with demarcation into dark and light zones that is, germinal center formation. Lymphocytes interspersed in lamina propria were mainly CD3+ T lymphocytes. In conclusion, the development of LGCs in ileocaecal region started prenatally in terms of all its cellular components into completely developed and immunocompetent to generate mucosal immunity.

Histo-ontogenetic study of the parotid salivary gland of Indian buffalo.

The present study was aimed at elucidating the histogenesis of parotid gland of buffalo. The study was carried out on buffalo foetuses (n = 36), during different stages of prenatal life. The foetuses were categorised into three groups based on their curved crown rump length (CVRL). The primordial anlage of parotid salivary gland was evident at 40th day of development whereas the primary ducts, in the form of cords, were first observed at 81st day of prenatal life. The capsule formation as well as the lobulation of the gland was initiated at 127th day. At 141st day, the duct system of gland was completed. The terminal tubules attained the structure of acini at 167th day. The myoepithelial cells first appeared as flattened basal cells initially around the developing acinar cells at 167th day. The typical compound tubulo-acinar nature of the gland was first observed at 185th day. Purely serous acinar cells were seen from 185th day onwards. The micrometrical studies revealed that the mean diameter of acinar cells, intercalated ducts, striated ducts and large ducts increased with the advancement of age. The serous acinar cells were devoid of acidic as well as neutral mucopolysaccharides in prenatal age groups; however, large ducts with goblet cells exhibited positive reaction. Combined PAS-AB method revealed mixed reaction in acinar cells as well as in large ducts. Fine lipid droplets were observed in intralobular as well as interlobular connective tissue; however, phospholipids were observed in the cell membrane of secretory cells and ducts.

Enzyme histochemistry of cecal lymphoid tissue during prenatal period of buffalo.

AIM: This study was designed to elucidate the histoenzymic distribution of enzymes, i.e., phosphatases, oxidoreductases, dehydrogenases, and diaphorases in cecal lymphoid tissue during its development in the prenatal period. MATERIALS AND METHODS: The study was conducted on cecum of 15 buffalo fetuses ranging from 16 cm curved crown-rump length (CVRL) (100 days) to 100 cm CVRL (full term). The fetuses were categorized into three groups based on their CVRL. RESULTS: In Group I, the distribution of enzymes was uniformly weak in developing villi-like projections in cecum and completely absent from submucosa. In Group II, the enzymes showed a moderate to strong activity in epithelium lining tunica mucosa which progressively decreased as the fetus progresses toward late gestational age. However, the intense activity of enzymes was observed in developing lymphoid tissue in this group. In Group III, distribution of enzymes reduced in tunica mucosa of cecum with advancing age, whereas the intense activity was noticed in the developed lymphoid tissue complex. CONCLUSION: The distribution of enzymes was completely absent from submucosal region in cecum of Group I as there was no lymphoid tissue development at this age. In Group II, the enzymes showed a moderate to strong activity in epithelium lining tunica mucosa which progressively decreased toward late gestational age but an intense activity was observed in developing lymphoid tissue. In Group III, distribution of enzymes reduced in tunica mucosa with advancing age with intense activity noticed in the developed lymphoid tissue complex.

Ultrastructural changes in the sublingual salivary gland of prenatal buffalo (Bubalus bubalis).

AIM: The present study was aimed to elucidate ultrastructural changes in the development of sublingual salivary gland of buffalo during prenatal life. MATERIALS AND METHODS: The study was carried out on sublingual salivary gland of 36 buffalo fetuses ranging from 13.2 cm curved crown-rump length (CVRL) (88(th) day) to full term. The fetuses were categorized into three groups based on their CVRL. RESULTS: The cells lining the terminal tubules were undifferentiated with poorly developed cytoplasmic organelles but lacked secretory granules (SGs) at 13.2 cm CVRL (88(th) day). The SGs appeared first in the form of membrane-bound secretory vesicles with homogeneous electron-dense as well as electron-lucent contents at 21.2 cm CVRL (122(nd) day); however, mucous acinar cells contained electron-lucent granules, while serous secretory cells as well as serous demilunes showed electron-dense granules at 34 cm CVRL (150(th) day) of prenatal life. At 53.5 cm CVRL (194(th) day), both mucous and serous acini were differentiated by the density of SGs. CONCLUSION: The cytoplasm of acinar cells was filled with mitochondria, rough endoplasmic reticulum, and Golgi profiles in mid and late fetal age groups. The SGs were increased in number during the late fetal age group. The myoepithelial cells (MECs) were located at the base of the acinar cells as well as intercalated and striated ducts and were stellate in shape. The ultrastructure of MEC revealed a parallel stream of myofilaments in the cytoplasm and its processes. The mucous cells were predominantly present in the sublingual salivary gland and were pyramidal in shape.

Ileal and jejunal Peyer's patches in buffalo calves: Histomorphological comparison.

AIM: The present study was aimed to elucidate the histomorphology of ileal and jejunal Peyer's patches in the small intestine of buffalo calves and their structural comparison. MATERIALS AND METHODS: The study was conducted on neonatal (n=10) and pre-pubertal (n=10) buffalo calves. The age of the postnatal buffalo calves was estimated by their temporary and permanent dentition. RESULTS: The study revealed that several layers of oval to elongate elliptical lymphoid follicles were observed in submucosa on the anti-mesenteric side in the ileum of early neonatal calves. However, the follicles at this age, in jejunum were of all shapes present within one layer. The interfollicular space was occupied by the interfollicular tissue, which was diffuse and wider around jejunal lymphoid follicles as compared to ileal lymphoid follicles. However, toward the pubertal stage, the number of layers of lymphoid follicles was reduced in ileum due to involution while it remained similar in number in jejunum at this stage. CONCLUSION: The ileal Peyer's patches were found to have started involution more or less around reaching puberty, whereas the jejunal Peyer's patches appear to be functional throughout the lifespan of the animal.

In utero exposure of neonatal buffalo calves to pesticide residues and the alterations within their reproductive tract.

In utero exposure of neonates to pesticide residues could be damaging to the reproductive tract. Hence, the present study assessed the circulating concentrations of pesticide residues in buffalo and their neonatal calves as well as in the reproductive tract tissue samples of same calves. Also, histopathological alterations were revealed in the reproductive tract of calves. Pesticide residues were high (P<0.05) in the reproductive tract of calves (119.5 ± 20.2 ng/g, 35% positive) in comparison to their blood (32.1 ± 8.4 ng/ml, 15% positive) or blood of their dams (41.5 ± 8.3 ng/ml, 25% positive). The number of histopathological alterations were high (P<0.05) in the reproductive tract of a calf contaminated with high concentrations of pesticide residues (3.43 ± 1.29) in comparison to a tract positive for low residue concentrations (1.57 ± 0.60) or pesticide negative tract (0.28 ± 0.10). In conclusion, in utero exposure of neonatal buffalo calves to pesticide residues may be associated with damaging alterations in their reproductive tract.

Developmental changes in the parotid salivary gland of prenatal buffalo: an ultrastructural study / Cambios en el desarrollo de la glándula salival de la parótida de búfalo prenatal: un estudio ultraestructural

SUMMARY: The present study was undertaken to elucidate ultrastructural changes in development of parotid salivary gland of buffalo during different stages of prenatal life. The ultrastructural studies revealed that the cytoplasm of acinar cells was filled with mitochondria, rough endoplasmic reticulum and Golgi complex in mid and late foetal age groups. Medium electron dense secretory granules first appeared in the acinar cells of parotid gland at 30 cm CVRL (141st day). However, at 49.5 cm CVRL (185th day) two types of electron dense granules were identified on basis of granule density viz., dark and light. The dark electron dense granules were more in number, whereas light granules were comparatively less having electron lucent content within them was identified. The mean diameter of dark and light granules was measured about 0.45±0.1 µm and 0.30±0.1 µm, respectively, which showed that the dark granules were comparatively larger in size. The secretory granules were increased in number during the late foetal age group. The myoepithelial cells were located at the base of the acinar cells as well as intercalated and striated ducts, and were stellate in shape. The ultrastructure of myoepithelial cell revealed parallel stream of myofilaments in the cytoplasm and its processes. Lipofuscin pigments were also observed in between the acinar cells of parotid gland.

RESUMEN: El estudio se realizó para elucidar los cambios ultraestructurales en el desarrollo de la parótida del búfalo durante las diferentes etapas de la vida prenatal. Los estudios ultraestructurales revelaron que el citoplasma de las células acinares estaba saturado de mitocondrias, de retículo endoplasmático rugoso y Complexo golgiensis en las edades fetal media y tardía. Se observó un número mayor de gránulos oscuros densos de electrones, mientras que los gránulos ligeros fueron comparativamente menor en número con contenido de electrones. El diámetro medio de gránulos oscuros y ligeros se midió aproximadamente 0,45 / pm 0,1 / mu m y 0,30 / pm 0,1 / mu m, respectivamente, lo que mostró que los gránulos oscuros eran comparativamente mayores en tamaño. Los gránulos secretores aumentaron en número durante el último grupo de edad fetal. Las células mioepiteliales se localizaron en la base de las células acinares, así como en conductos intercalados y estriados, y tenían una forma estrellada. La ultraestructura de las células mioepiteliales reveló una corriente paralela de miofilamentos en el citoplasma y sus procesos. También se observaron pigmentos de lipofuscina entre las células acinares de la glándula parótida.

Lymphoglandular complexes in proximal colon of buffalo calves (Bubalus bubalis) / Complejos linfoglandulares en colon proximal de terneros de buffalo (Bubalus bubalis)

The present study was conducted on six healthy early neonatal and six prepubertal buffalo calves to study the location, gross morphology, histomorphology and histochemistry of lymphoglandular complexes in proximal colon. In very proximal part of colon of buffalo calves, an irregular oval mucosal lymphoid patch was found grossly as a proximal colon (PC) patch. Histologically, in proximal colon patch of early neonates (3-4 weeks), an extensive invasion of mucosal glands was observed towards lymphoid nodules that were present in submucosa. The structure as a whole thus formed a complex known as lymphoglandular complex (LGC). Large number of such complexes i.e., LGCs were observed in submucosa of proximal colon at this age. At some places, invasion of mucosal glands into lymphoid tissue was restricted to superficial layer of complexes, with the lymphoglandular complexes opening directly into the lumen but some were deep seated. However, by the age of 6 months in buffalo calves i.e., prepubertal period, LGCs were reduced and were present in single layer within the submucosa of the proximal colon. Moreover, some of LGCs were completely encapsulated by their own lamina muscularis mucosae. But some of the complexes still had their mucosal openings into lumen while others had lost their connection with tunica mucosa. Histochemically, the glands that were observed within LGCs contained mucosubstances, glycogen, mucopolysaccharides, and mucin. However, lipids were present around the lymphocytes observed towards the periphery of these LGCs.

El presente estudio se llevó a cabo en seis terneros de búfalo neonatos sanos y seis terneros prepuberales para estudiar la ubicación, morfología macroscópica, histomorfología e histoquímica de los complejos linfoglandulares en el colon proximal. Se observó en un área del colon proximal (AP) de los terneros de búfalo un óvalo linfoide de mucosa irregular en la parte más proximal de éste. Histológicamente, en el área proximal del colon de los terneros neonatos (3-4 semanas), se observó una invasión extensa de las glándulas mucosas hacia los nódulos linfáticos presentes en la submucosa. La estructura en su totalidad formaba un complejo conocido como complejo linfoglandular (CLG). A esta edad se observó un gran número de estos complejos es decir, se observaron CLGs en la submucosa del colon proximal. La invasión de las glándulas mucosas en el tejido linfoide, se limita a la capa superficial de los complejos, los complejos linfoglandulares distribuidos directamente en el lumen, sin embargo otros se encontraban arraigados de manera profunda. En búfalo a los 6 meses de edad, es decir en el período prepuberal, se observó un número reducido de CLGs presentes en una sola capa dentro de la submucosa del colon proximal. Por otra parte, algunos de CLGs estaban completamente encapsulados por su propia lamina muscularis mucosae. Algunos de los complejos mantenían abertura de las mucosas en el lumen, mientras que otros habían perdido su conexión con la mucosa. En análisis histoquímico, las glándulas que se observaron dentro del CLGs contenían mucosustancias, glucógeno, mucopolisacáridos y mucina. Sin embargo, se encontraron lípidos presentes alrededor de los linfocitos hacia la periferia de los CLGs.