Gas chromatographic-mass spectrometric analysis of chemical volatiles in buffalo (Bubalus bubalis) urine.

Isolation of active fraction and characterization of chemosignals from urine have been attempted in several mammalian species in the recent years. The objective of this study was to identify the urinary volatiles across various reproductive stages of buffalo cow, namely, estrus, diestrus, and pregnancy, and in bull, by chemical extraction followed by gas chromatography-linked mass spectrometry (GC-MS). Urine samples were collected from six buffalo cows at two different phases of estrous cycle, namely, estrus and diestrus. Besides, urinary samples were collected from five pregnant buffalo cows (60-75 days after artificial insemination (AI)) and six adult bulls. Thin-layer chromatography was performed as a preliminary test for qualitative comparison of different compounds extracted by organic solvents. Identification of the urinary compounds was carried out in a gas chromatograph (Perkin Elmer, Autosystem XL) linked to a mass spectrometer (Turbomass). The results of GC-MS analysis indicated the presence of 21 compounds with varying molecular weights and retention time, which were further categorized as diestrus-specific, pregnancy-specific, and bull-specific urinary compounds. No compound, however, could be identified as estrus-specific. We concluded that qualitative differences do exist in estrus, diestrus, and pregnant buffalo cow urine and in bull urine, as evidenced by GC-MS.

Influence of induced heat stress on HSP70 in buffalo lymphocytes.

Heat stress in farm animals, such as cattle and buffalo during summer and post-summer seasons is a problem for livestock producers. The effect of heat stress becomes pronounced when heat stress is accompanied with ambient humidity impairing the immune status, growth, production and reproductive performance of animals. Increase in HSP70 levels from cell cultures in presence of different stressors often does not reflect the physiological adaptability of animals governing thermal regulation. In this study we directly compared the effect of different heat stress conditions with the immune status and HSP70 expression patterns from buffalo lymphocytes both in vivo and in vitro. Murrah buffalo calves were exposed to induced heat stress with two experimental treatments: hot-dry (42 °C with existing relative humidity) or hot humid (35 °C with 70% relative humidity) condition in psychometric chamber, 4 h daily for 12 days and compared with control animals maintained in an experimental shed under natural conditions. There was >200-fold increase in serum-HSP70 levels in both heat stress conditions compared with control. Furthermore, the immune status of the calves failed to activate the level of HSP70 expression in serum lymphocytes. Lymphocytes cultured in vitro at higher temperature exert 2.5-fold increase in HSP70 concentration. This study is the first of its kind to demonstrate more complex expression pattern of buffalo serum-HSP70 level as a thermo adaptive response compared with in vitro treated cells. Results from this study indicate that serum-HSP70 levels could be used as a sensitive biomarker for heat stress management in large farm animals.

Cyclical changes in collagen concentration in relation to growth and development of buffalo corpus luteum.

In the present study, changes in luteal fresh weight and concentration of collagen in cyclic buffalo corpus luteum were investigated at 4 stages of its growth and development/regression. The collagen concentration was determined by estimating hydroxyproline, a collagen specific amino acid present in luteal tissues. The mean fresh weight increased (P < 0.001) over the luteal phase, reached maximum at late-luteal stage and decreased (P < 0.001) subsequently at follicular stage. The weight of the mature CL was 2.5 times heavier than the CL haemorrhagicum and regressing CL. Results showed that cyclic buffalo CL contains collagen at all 4 stages of development during oestrous cycle. The collagen in luteal tissues constitutes about 0.9% to 1.2% of luteal fresh weight with the highest content appearing in mature tissue. The concentration of collagen increased (P < 0.001) with the stages of CL development over the luteal phase and the highest concentration was recorded at follicular phase with the decline of luteal weight. The total content of collagen per CL also showed the same trend during luteal phase but decreased at follicular phase with the loss of luteal tissues. In conclusion, collagen concentration in cyclic buffalo CL changes with the growth and development of CL across the oestrous cycle. The synthesis of collagen is faster between early- to mid-luteal stage than mid- to late-luteal stage.

Ascorbic acid, catalase and chlorpromazine reduce cryopreservation-induced damages to crossbred bull spermatozoa.

The present study evaluated the effectiveness of ascorbic acid, catalase, chlorpromazine and their combinations in reducing the cryodamages to crossbred bull (Bos taurus x Bos indicus) spermatozoa. A total of 32 ejaculates (eight each from four bulls) were diluted in Tris-citric acid-fructose-egg yolk-glycerol extender. Each ejaculate was split into six parts (five treatment and one control). Treatment groups included 10 mm ascorbic acid, 0.1 mm chlorpromazine, 200 IU/ml catalase, 10 mm ascorbic acid + 0.1 mm chlorpromazine or 200 IU/ml catalase + 0.1 mm chlorpromazine in the extender. Fluorescent probes (Fluorescein isothiocyanate--Pisum sativum agglutinin + Propidium iodide) were used for the assessment of spermatozoa viability and acrosomal status. The proportion of acrosome intact live (AIL), acrosome intact dead, acrosome reacted live and acrosome reacted dead sperm was assessed in fresh, equilibrated and frozen-thawed semen. The functional status of the sperm was assessed using hypo-osmotic sperm swelling test (HOSST). Activities of acrosin and hyaluronidase enzyme were also determined. Lipid peroxidation level was assayed based on the melonaldehyde (MDA) production. In cryopreserved semen, the values of AIL spermatozoa, HOSST response, hyaluronidase and acrosin activity were reduced by 53%, 47%, 34% and 54%, respectively from their initial values in fresh semen. However, MDA level was threefold higher in the frozen-thawed sperm compared with fresh sperm. Significant (p < 0.05) improvement in motility, viability, HOSST response, retention of hyaluonidase and acrosin and reduction in MDA was recorded in ascorbic acid, catalase, ascorbic acid + chlorpromazine and catalase + chlorpromazine incorporated groups. The percentage of AIL sperm was significantly (p < 0.05) higher in ascorbic acid, catalase and ascorbic acid + chlorpromazine incorporated groups compared with the control. Chlorpromazine alone did not improve the post-thaw semen quality but when combined with either ascorbic acid or catalase, improvement in semen quality was noticed. It was inferred that incorporation of ascorbic acid, catalase and ascorbic acid + chlorpromazine in semen extender improved the post-thaw semen quality in crossbred bulls.

Survival and developmental competence of buffalo preantral follicles using three-dimensional collagen gel culture system.

The aim of the present study was to develop a three-dimensional (3D) collagen gel culture system for the in vitro growth and survival of buffalo preantral follicles with or without growth factors. Buffalo ovaries were collected from a local abattoir and preantral follicles were isolated through microdissection. Isolated preantral follicles were put either in collagen gel coated culture dish or embedded in a microdrop of collagen gel. The culture medium was TCM-199 fortified with fetal calf serum (10%), insulin transferin selenium solution (ITS, 1%), epidermal growth factor (EGF, 20 ng/ml) and follicle stimulating hormone (FSH, 0.5 microg/ml). Follicles were divided into three groups and cultured in the medium described above (group a, control), with addition of insulin like growth factor (IGF-I, 100 ng/ml, group b), or with addition of IGF-I and basic fibroblast growth factor (bFGF, 10 ng/ml, group c). Preantral follicles were incubated at 38.5 degrees C in 5% CO(2) and maximum humidity. Culture medium was replenished after every 72 h and spent medium was stored at -30 degrees C for hormone analysis. We found that the extracellular matrix of collagen gel maintained follicle viability and growth by providing surface interaction and increasing attachment of follicles. Preantral follicles embedded in collagen gel droplets had better antrum formation and development as compared to the whole surface coated culture method. Follicles cultured with IGF-I on collagen gel matrix showed a significantly (P<0.05) higher survival rate and larger mean diameter of follicles on day 10 of culture with improved growth and mucification as compared to the control group. However, follicles cultured in the combination of IGF-I with bFGF had decreased survival rate and smaller mean follicles diameter than the IGF-I group (b). Progesterone (P(4)) accumulation was greater on day 9 of culture in follicles cultured in IGF-I as compared to control; whereas, P(4) was markedly decreased in the combination of IGF-I with bFGF. Follicles of the control group could survive for up to 10-15 days before degenerating, but follicles cultured with growth factors were able to survive up to 20 days and showed signs of early antrum formation. In summary, we have shown that collagen gel was a novel and efficacious 3D microenvironment for the extended culture of buffalo preantral follicles. Supplementation of culture medium with growth factors was found to be essential for antrum formation.

Ascorbic acid in buffalo ovary in relation to oestrous cycle.

Concentration of ascorbic acid was determined in different parts of buffalo ovary at four different stages of oestrous cycle viz. early luteal, mid luteal, late luteal and follicular. The stages were decided from the physical and morphological examinations of corpora lutea. The ovary was dissected in three components viz. corpus luteum, follicular fluid and ovarian stromal tissue for ascorbic acid assay. Corpus luteum showed significant change in concentration of ascorbic acid with the advancement of oestrous cycle, value being highest in late- luteal stage. Follicular fluid and ovarian stromal tissue did not show significant changes in ascorbic acid at any stage of the oestrous cycle. Small follicles, irrespective of the stage of oestrous cycle had, however, significantly higher ascorbic acid content than large follicles.

Effect of buffalo follicular fluid treatment on follicle population and ovulation rate in guinea pigs.

A number of workers have studied the effect of follicular fluid (FF) on the secretion of follicular stimulating hormone (FSH) but little is known about its potential as a regulator of ovarian activity, including ovulation rate. This paper describes the effect of charcoal treated-buffalo follicular fluid (buFF) treatment on follicular growth and ovulation rate in guinea pigs. Eighteen guinea pigs in three groups of 6 each were given 0.2 ml buFF at 12 hr interval for 3 days at different stages of estrous cycle viz., early-luteal, mid-luteal or follicular phase. One control group received equal volume of saline. Estrus was monitored every morning and evening by inspection of the opening of vaginal membrane and its cytology. All animals were sacrificed at 24 hr after the onset of estrus. Both the ovaries were dissected out, weighed and number of ovulation points recorded. One ovary from each animal was processed for histological examination to determine the population of healthy and atretic follicles. In early-luteal and follicular phase-treated animals the onset of estrus was delayed (P < 0.01) and ovulation rate was not affected. However, estrus occurred at normal when the treatment was initiated at midluteal stage and 50% animals failed to ovulate in this group. The total follicle population at metestrus increased significantly in all treated animals because of increase in number of follicles of size class II (400 to < 600 microns diam.). Atresia was also declined due to treatment. These results demonstrated that the buFF contained some inhibitory substances that delayed the onset of estrus in guinea pigs.

Effect of passive immunization with buffalo follicular fluid antisera on ovarian activity in guinea pigs.

Ovarian activity and follicular populations were studied in guinea pigs (Cavia porcellus) following administration of antisera against buffalo follicular fluid (buFF). Antibodies were raised in rabbits and the titre tested by immunodiffusion assay. Fourteen guinea pigs cycling normally were randomized into two groups. Animals in Group I (n = 8) were treated (i.p.) with 0.5 ml antisera and in Group II (control, n = 6) with the same volume of normal rabbit serum at 12 h intervals on the 10th and 11th day of their oestrous cycle. They were sacrificed 24 h after onset of estrus when ovulation points were counted and ovaries processed for microscopical examination. Treatment with buFF-antisera increased ovulation rate (3.6 vs. 2.0; p < 0.01) but had no significant effect on the total number of follicles. However, the treatment reduced the percentages of atretic follicles in all size classes. These results indicated that the administration of a buFF-antisera produced in the rabbits increased ovulation rate in guinea pigs by reducing the incidence of atresia.

Characteristics of proteoglycans of buffalo ovarian follicular fluid during maturation of follicles.

The proteoglycans (PGs) and glycosaminoglycans (GAGs) of buffalo ovarian follicular fluid (FF) have been studied in small (2-4.9 mm), medium (5-9.9 mm) and large (> or = 10 mm) follicles. GAGs in different categories of follicles were isolated, assayed and analysed. On the basis of hexosamine analysis, glucosamine accounted for all the free GAGs in FF of small and medium follicles. No free GAG was found in large follicles. The concentration of GAGs in the form of PGs decreased significantly with follicular maturation. Qualitative analysis of GAGs from PGs showed higher galactosamine than glucosamine. The ratios of GalNH2:GluNH2 and neutral sugars were highest in small follicles followed by medium and large follicles. On the other hand, the percentage of sialic acid in GAGs was highest in large follicles followed by medium and small follicles. The fractionation of PGs by gel filtration indicated the presence of two types of PGs in buffalo ovarian FF. Difference in distribution of two types of PGs in small and large follicles was also noted.

Rapid photometric assay of growth of Mycoplasma mycoides subsp. capri.

A new spectrophotometric technique for evaluation of early growth in liquid culture of Mycoplasma mycoides subsp. capri has been developed. As turbidity does not appear until after incubation to 18 h the method utilizes the change in absorbance of the medium at 550 nm to monitor growth. The change in absorbance of the medium (which contains phenol red) occurs when the pH changes due to microbial growth. For measurement of growth at later stages when turbidity is proportional to number of colony forming units, two other wavelengths (450 nm and 700 nm) have been suggested.

A new technique for localization of cellulase in situ using silver nitrate.

A new staining technique has been developed for the histochemical localization of cellulase in plant tissues by light microscopy. The products of cellulolysis are reducing sugars which can reduce the salts of heavy metals under appropriate conditions. The present technique relies on the deposition of black silver oxide due to reduction of alkaline silver nitrate to detect cellulase in tissues.

Anionic activation of human salivary amylase.

In all earlier studies on alpha-amylase, the influence of different ions were studied in phosphate buffer. The present report shows the effect of different ions individually with Tris and amino acid. Though it has been claimed recently that sodium ion is an activator of alpha-amylase, this study reconfirms that sodium ion does not activate human salivary amylase.