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Summary: This study is a comparative analysis of the biochemical, hormonal, and mineral compositions of follicular fluid in preovulatory and cystic follicles of water buffalo (Bubalus bubalis). In total, reproductive tracts from 215 buffalo along with intact ovaries were collected randomly from an abattoir. The incidence of cystic conditions found in this study was 3.72% (8/215), involving the right ovary in 62.5% of instances and the left ovary in 37.5% of instances during the non-breeding season. Follicular fluid was aspirated from preovulatory follicles (12–15 mm diameter, oestrogen-active, follicular phase or stage IV corpus luteum on one of the two ovaries, n = 10) and cystic follicles (at least 20 mm diameter, no corpus luteum on any one of the two ovaries, n = 8). The follicular fluid samples were assayed for biochemical components (uric acid, creatinine, blood urea nitrogen, cholesterol, total protein, glucose, ascorbic acid, and alkaline phosphatase), hormones (progesterone, estradiol, and insulin), and minerals (calcium, magnesium, phosphorus, copper, zinc, and cobalt). Cystic follicles had greater (P < 0.05) concentrations of creatinine, blood urea nitrogen, cholesterol, progesterone, copper, zinc, and cobalt, and lesser (P < 0.05) concentrations of uric acid, glucose, ascorbic acid, estradiol, insulin, calcium, magnesium, and phosphorus compared with preovulatory follicles. These results indicated the marked differences in follicular fluid composition between preovulatory and cystic follicles in buffalo. Some of the changes were indicative of oxidative stress and disturbed steroidogenesis, two important mechanisms shown to be associated with cystic ovarian disease in various species. Further studies are warranted to investigate whether these differences are directly or indirectly involved in the formation of cystic follicles or are mere manifestations of the condition. [ABSTRACT FROM AUTHOR]

Abstract: Ovarian acyclicity is one of the most important causes of infertility in water buffalo. Recent studies have indicated alterations in the composition of follicular fluid during the condition. The aim of this study was to determine the changes in follicular fluid concentrations of estradiol, progesterone and insulin during ovarian acyclicity in water buffalo. Ovaries were collected from 50 acyclic and 95 cyclic (control) buffaloes and follicular fluid was aspirated from small (5.0–6.9mm), medium (7.0–9.9mm) and large (≥10.0mm) sized follicles. Estradiol concentration was lower (P <0.0001) in acyclic (1.4±0.09ng/ml) than in cyclic (3.3±0.18ng/ml) buffaloes. Regardless of the ovarian cyclic status, there was an increase (P <0.01) in estradiol concentration with the increase in follicle size; the mean concentrations were 2.4±0.16ng/ml, 2.8±0.29ng/ml and 3.5±0.41ng/ml in small, medium and large follicles, respectively. A higher (P <0.001) progesterone concentration was recorded in acyclic (24.3±2.61ng/ml) compared to the cyclic (7.6±0.79ng/ml) group. Furthermore, acyclic buffaloes had a lower (P <0.05) concentration of insulin in the follicular fluid than that of cyclic buffaloes (15.2±1.55μIU/ml versus 25.9±2.78μIU/ml, respectively). In conclusion, acyclic buffaloes have lower concentrations of estradiol and insulin concurrent with higher concentrations of progesterone in the follicular fluid. These hormonal changes in the follicular microenvironment are possibly a manifestation of the disturbances in the normal follicular development leading to anovulation and anestrus in acyclic buffaloes. [Copyright &y& Elsevier]

Abstract: This study describes the changes in biochemical composition of follicular fluid during reproductive acyclicity in buffalo. A total of 73 pairs of ovaries collected from 26 reproductively acyclic and 47 reproductively cyclic buffaloes were used in the investigation. Ovarian follicles were classified into small (5.0–6.9mm), medium (7.0–9.9mm) and large (≥10.0mm) sized categories depending upon their diameter. Follicular fluid was aspirated, processed and assayed for glucose, cholesterol, total protein, acid phosphatase and alkaline phosphatase. Glucose concentration was lesser in reproductively acyclic compared to cyclic buffaloes (19.3±2.59mg/dl compared to 32.6±2.60mg/dl; P <0.05), mainly due to difference in concentration between small sized follicles (12.4±2.59mg/dl compared to 28.0±3.32mg/dl; P <0.05). Cholesterol concentration was also lesser in reproductively acyclic compared to cyclic buffaloes (32.2±2.14mg/dl compared to 35.5±2.16mg/dl; P <0.05) and this was related to the lesser concentration found in large follicles (13.8±3.45mg/dl compared to 37.2±4.10mg/dl; P <0.001). Total protein and acid phosphatase levels were not affected by either the reproductive cyclicity status or the follicular size (4.9±1.07g/dl to 6.0±0.28g/dl and 1.2±0.17U/dl to 2.5±1.22U/dl, respectively). An increased alkaline phosphatase activity was, however, observed in reproductively acyclic compared to cyclic buffaloes (27.5±3.08U/dl compared to 14.0±1.09U/dl; P <0.0001). In conclusion, results of the present study indicate an alteration in the biochemical composition of follicular fluid during reproductive acyclicity in buffalo. The findings provide further support to the notion that poor nutrition is an important factor triggering reproductive acyclicity in buffalo. [Copyright &y& Elsevier]

Abstract: The objective of this study was to evaluate the changes in follicular fluid nitric oxide (NO) and ascorbic acid (AA) concentrations with varying follicle size and functional status, and stage of estrous cycle in buffalo. The main effect of follicle size on NO concentration and size-by-status interaction were statistically significant (P <0.05). Small follicles had a higher (P <0.05) NO concentration compared to medium and large follicles. Further, estrogen-active (EA) small follicles showed increased (P <0.05) NO concentrations than the corresponding estrogen-inactive (EI) follicles. Within EA category, higher (P <0.01) concentrations were recorded in small compared to medium and large follicles. There was no significant main effect of stage (P >0.1) on NO concentration but the stage-by-size interaction was significant (P <0.05) with medium follicles showing a higher (P <0.05) concentration during late luteal stage compared to the mid luteal stage. During early and mid luteal stages, higher (P <0.05) NO concentrations were recorded in small than in medium follicles. A significant (P <0.01) main effect of size on AA concentration was observed with higher values in medium than in small and large follicles. Size-by-status interaction for AA approached statistical significance (P <0.06) with higher (P <0.05) concentrations recorded in medium than in large EA follicles. The main effect of stage on AA concentration was, however, non-significant (P >0.1) but the stage-by-size interaction approached statistical significance (P <0.06) with lower (P <0.05) levels recorded in large compared to medium size follicles during the follicular stage. In conclusion, the present study demonstrates that the concentrations of follicular fluid NO and AA vary according to the follicle size, functional status and stage of estrous cycle suggesting their possible role in process of follicular development during estrous cycle in buffalo. [Copyright &y& Elsevier]

Abstract: The objective of this study was to examine the follicular fluid biochemical and hormonal changes associated with ovarian follicular cysts in buffalo. Follicular fluid was aspirated from eight cysts and eight preovulatory follicles, and subjected to biochemical and hormonal analyses. Cysts were characterized by a greater (P <0.01) concentration of nitric oxide and lesser concentrations of ascorbic acid and glucose than that of preovulatory follicles (P <0.01 and P <0.05, respectively). Furthermore, follicular cysts had greater concentrations of progesterone (P <0.001), triiodothyronine (T3) and cortisol (P <0.05) and lesser concentrations of insulin (P <0.001) than preovulatory follicles. The results indicate follicular cysts in buffalo have an altered biochemical and hormonal composition. The alterations include increases in nitric oxide, progesterone, cortisol and T3 concentrations with a concurrent reduction in ascorbic acid, insulin and glucose concentrations. The study suggests that greater progesterone concentrations possibly inhibit the onset of LH surge resulting in formation of follicular cysts in buffalo. In addition, it implies the plausible role of intra-ovarian regulators such as nitric oxide, ascorbic acid and insulin in development of the condition. [Copyright &y& Elsevier]

This study investigated the expression and localization of insulin-like growth factor (IGF) system at different stages of buffalo CL and the role of IGF-I in stimulating vascular endothelial growth factor (VEGF) and progesterone (P4) production in cultured luteal cells. The mRNA expression of IGF system, VEGF, steroidogenic acute regulatory protein, P450scc, and hydroxysteroid dehydrogenase (HSD) was investigated by quantitative real-time polymerase chain reaction (PCR). Protein expression of IGF was demonstrated by Western blot and localization by immunohistochemistry. Progesterone and VEGF production was assayed using RIA and ELISA. A relatively high mRNA expression of IGF-I and IGF-II in early, mid- and late luteal phases with immunoreactivity mostly restricted to cytoplasm of large luteal cells indicates their autocrine role, whereas very weak immunoreactivity in endothelial cells during the mid-luteal phase indicates their paracrine role. Insulin-like growth factor receptors, IGF-IR and IGF-IIR, were restricted to large luteal cells with high mRNA and protein expressions in the mid-luteal phase. The significantly higher expression of insulin-like growth factor binding protein (IGFBP)-1, -3, -5, and -6 in the early or mid-luteal phase suggested their stimulatory role, whereas that of IGFBP-2 and -4 in mid-, late, and regressive luteal stages implied their inhibitory role. The mRNA expressions of key steroidogenic factors and VEGF were significantly higher (P < 0.05) when the culture medium was supplemented with 100 ng/mL of IGF-I for 72 hours. Moreover, IGF-I at a dose of 100 ng/mL increased P4 and VEGF production (P < 0.05). It can be concluded that IGF family members via their autocrine and paracrine effect play significant roles in promoting angiogenesis through the production of VEGF in luteal cells and steroid synthesis through the production of key steroidogenic factors. [ABSTRACT FROM AUTHOR]

Abstract: Evidence obtained during recent years provided has insight into the regulation of corpus luteum (CL) development, function, and regression by locally produced ghrelin. The present study was carried out to evaluate the expression and localization of ghrelin and its receptor (GHS-R1a) in bubaline CL during different stages of the estrous cycle and investigate the role of ghrelin on progesterone (P4) production along with messenger RNA (mRNA) expression of P4 synthesis intermediates. The mRNA and protein expression of ghrelin and GHS-R1a was significantly greater in mid- and late luteal phases. Both factors were localized in luteal cells, exclusively in the cytoplasm. Immunoreactivity of ghrelin and GHS-R1a was greater during mid- and late luteal phases. Luteal cells were cultured in vitro and treated with ghrelin each at 1, 10, and 100 ng/mL concentrations for 48 h after obtaining 75% to 80% confluence. At a dose of 1 ng/mL, there was no significant difference in P4 secretion between control and treatment group. At 10 and 100 ng/mL, there was a decrease (P < 0.05) in P4 concentration, cytochrome P45011A1 (CYP11A1), and 3-beta-hydroxysteroid dehydrogenase mRNA expression and localization. There was no difference in mRNA expression of steroidogenic acute regulatory protein between control and treatment group. In summary, the present study provided evidence that ghrelin and its receptor are expressed in bubaline CL and are localized exclusively in the cell cytoplasm and ghrelin has an inhibitory effect on P4 production in buffalo. [Copyright &y& Elsevier]

Highlights: [•] LH, IGF1 and EGF treatment increased expression of VEGF in the cultured GCs. [•] These factors increased PCNA expression and decreased BAX expression in cultured GC. [•] To conclude these three factors (a) have cytoprotective effect. [•] And (b) stimulate VEGF production from granulosa cells of bubaline ovarian follicles. [Copyright &y& Elsevier]

Abstract: The objective of the present study was to characterize the temporal patterns of gene expression for vascular endothelial growth factors (VEGF) and VEGF receptors during ovarian follicular growth, development and maturation in buffalo (Bubalus bubalis). Follicles were classified into four groups according to size and the concentration of estradiol-17β (E2) in follicular fluid (FF): Group I (small), 4–6mm diameter, E2 >0.5ng/ml of FF; Group II (medium), 7–9mm, E2 =0.5–5ng/ml; Group III (large), 10–13mm, E2 =5–40ng/ml; Group IV(pre-ovulatory), >13mm, E2 >180ng/ml). The mRNAs for FSH receptor (FSHR), LH receptor (LHR) and aromatase (CYP19A1) in theca interna and granulosa layers were also determined, further defining the maturational state of each group. The relative expression of VEGF isoforms (120, 164, and 188 amino acid forms), as determined by quantitative real-time PCR (qRT-PCR), increased during follicular development in both the granulosa (P <0.05) and theca layers. Relative amounts of VEGF receptors (VEGFR-1 and VEGFR-2) were least in granulosa cell (GC) and theca interna cell (TI) layers of Gp-I follicles. The amount of VEGFR-2 transcripts increased in the granulosa layer throughout development, reaching a maximum in Gp-IV follicles (P <0.05). The relative amount of VEGF isoforms and receptors in follicle lysates, as determined by western blotting, increased throughout follicular maturation to maximum amounts in pre-ovulatory follicles. Immunohistochemistry revealed a clear localization of VEGF isoforms and receptors in both steroidogenic cell types (GC and TI) and of VEGF receptors in the vascular endothelial cells of the thecal blood vessels. The most intense immunofluorescence was evident in pre-ovulatory follicles compared to other smaller follicles. These data provide evidence that the VEGF may contribute to the extensive capillary proliferation associated with the increase in size, selection, and maturation of the pre-ovulatory follicle. This may facilitate follicle maturation by enhancing the supply of nutrients, hormones, and other essential blood-borne signals to the follicle. VEGF may also promote maturation of follicles through recently recognized, non-angiogenic mechanisms. [Copyright &y& Elsevier]