Indian J. Anim. Res., 41 (2): 106-110, 2007 i.exeenzymatic PROFILES OF ACID AND ALKALINE PHOSPHATASES IN OVARIAN ANTRAL FOLLICULAR FLUID OF BUFFALOES* G.P. Kalmath and J.P. Ravindra 1 ** Department of Veterinary Physiology, Veterinary College, Bangalore - 560 024, India ABSTRACT Enzymatic profiles of alkaline and acid phosphatases in the buffalo ovarian antral follicular fluid were studied using ovaries procured from Civil Slaughterhouse, Bangalore. Antral follicular fluid aspirated from small (<6 mm), medium (6-10 mm) and large (11-16 mm) follicles was analyzed for phosphatase activity using clinical analyzer (photometer, BT-224 Biotechnica instruments). The acid phosphatase activity decreased with advancement in follicular size reaching the lowest value in large follicle group. Similar trend was observed with respect to the alkaline phosphatase activity. The acid and alkaline phosphatase activities were highest during the hotter months of April and June when the monthly average mean temperatures were (27.20 0 C) and (25.85 0 C) respectively. It was lowest during the cooler months of December and January, when the ambient temperatures were (21.20 0 C) and (20.95 0 C) respectively. However during July and August, the activity was intermediate. From the study it was concluded that the enzyme activity decreased with the follicular development and it increased with the increase in ambient temperature. INTRODUCTION The ovarian follicular fluid can be routinely harvested with the oocytes during in vitro maturation; fertilization and culture techniques. The follicular fluid could be used as an effective media supplement for in vitro maturation as it influences the ability of in vitromatured oocyte to acquire developmental competence (Sirard et al., 1995). The follicular fluid composition could vary depending on the stage of follicular development and may exert variable effects on oocyte development. Characterization of follicular fluid from follicles at different stages of development helps in understanding the regulation and requirements of follicle growth. Also, the fluid from follicle at an appropriate stage of development can be used to establish optimal environment for maturation of viable oocyte. Buffaloes are sensitive to ambient temperature and their reproductive activity varies according to the season (Pandey and Razada, 1979). Hence the present study was undertaken with the objective of elucidating profiles of acid and alkaline phosphatases, and estradiol 17-β in the follicular fluid of different size follicles during different months of the year. Profiles of minerals in the follicular fluid have been described in the accompanying paper. MATERIAL AND METHODS Collection of ovaries: The buffalo ovaries from apparently healthy animals were collected during different months of the year (December, 1998 to August, 1999) from civil slaughterhouse Bangalore. Based on rainfall, temperature and humidity the department of meteorology, Government of India has categorized the seasons as: December to February - winter March to June - summer The July and August months were considered to look into the effect of transition from summer activity to winter activity of the ovarian antral follicles. Ten ovaries were collected in each collection and such collections * Part of M.V.Sc. Thesis of the first author submitted to the University of Agricultural sciences, Bangalore - 560 024. 1 Present address: Principal Scientist (Animal Physiology), N.I.A.N.P., Adugodi, Bangalore - 560 030, India. **Corresponding Author.
Vol. 41, No. 2, 2007 107 were repeated thrice a week. The collected ovaries were transported with in an hour to the laboratory in plastic bag containing ice cold 0.92% NaCl (w/v). Collection of antral follicular fluid: The antral follicles were classified in to three size groups, small (<6 mm), medium (6-10 mm) and large (11-16 mm) follicles as followed in the study on buffaloes by Kulkarni 1988. Antral follicular fluid from different group of follicles was collected by aspiration using sterile tuberculin syringe fitted with needle. Follicular fluid was transferred to polythene microcentrifuge tubes (Tarsons). Fluid from follicles of similar size was pooled to have an adequate volume for analysis. The pooled samples were centrifuged at 1500 rpm at 5 0 C for 15 min, to make them cell free. The cellfree samples were then frozen quickly and stored at -20 0 C until used, for further analysis. Follicular fluid aspirated from follicles of thirty ovaries collected over a period of one week was pooled according to their group to have weekly samples. This yielded one sample in each group per week and four samples per month. Analysis of follicular fluid for enzyme profiles: Follicular fluid acid and alkaline phosphatase activities were analyzed using Autoanalyzer (photometer, BT-224 Biotechnica instruments). Procedures and reagent kit developed, according to Tietz (1976), Faulker and Meites (1982), Henry (1984), and supplied by Rashmi Diagnostics Limited, Bangalore were used for the analysis. Analysis of follicular fluid for Estradiol 17-β: Representative follicular fluid samples (six) each showing lowest and highest activity of follicular fluid alkaline phosphatase were selected from different size groups (small, medium and large) and used for estradiol-17β assay. Follicular fluid was diluted to 1:100 in PBS. The assay procedure followed was, as described by Raghava et al. (1992). Statistical analysis: The follicular fluid biochemical (acid phosphatase and alkaline phosphatase) constituents in different size follicles during different months of the year (Table 1 and 2) were analyzed by Two-way ANOVA (Snedecor and Cochron, 1994). The phosphatase activity was compared with the functional activity (estradiol 17-β) using Pearson s correlation (Snedecor and Cochron 1994). RESULTS AND DISCUSSION The mean follicular fluid acid phosphatase activity (U/L) and alkaline phosphatase activity (IU/L) with respect to different size of the follicle and different months of the year is given in the Table 1 and 2. The follicular fluid acid phosphatase activity varied significantly (P< 0.05) among follicles of different size and also among different months of the year. It was highest (12.55 U/L) in small follicles and decreased with the advancement of follicle size as indicated by the lowered activity (11.36 U/L) in the medium sized follicles and least activity (10.15 U/L) in large follicles. Acid phosphatase activity was high during the month of April (13.84 U/ L) and was low in the month January (9.25 U/ L) when the temperatures were 27.20 0 C and 20.95 0 C respectively. On Pearson s correlation the acid phosphatase activity showed significant (P<0.05) positive (r = + 0.833, p = 0.01) correlation with the ambient temperature. Follicular fluid alkaline phosphatase activity changed significantly (P<0.05) between follicles of different diameters with small follicles showing the highest activity (315. IU/ L), large follicles showing the lowest activity (208.9 IU/L) and the medium follicles with intermediate activity (249.1 IU/L). Alkaline phosphatase activity also changed significantly (P<0.05) with respect to the different months of the year. Activity was highest during April (292.30 IU/L) and lowest during December (217.63 IU/L) when the ambient temperatures were 27.20 0 C and 21.20 0 C respectively. Significant positive correlation (r = +0.869,
108 INDIAN JOURNAL OF ANIMAL RESEARCH Table 1. Mean follicular fluid acid phosphatase activity (U/L; Mean±S.E.) in different size antral follicles of buffaloes during different months of the year (n = 4) Month Small Medium Large Mean±S.E. December 10.78±2.01 09.84±2.46 09.64±2.21 10.08±1.17 acd January 09.84±0.74 09.28±1.40 08.63±1.53 09.25±0.68 a February 11.77±1.01 11.19±2.01 10.65±0.62 11.20±0.72 cd March 14.33±2.66 13.45±2.15 12.07±1.86 13.28±1.20 e April 15.09±2.22 13.68±1.26 11.26±1.23 13.84±0.95 e June 15.57±1.41 12.76±0.46 09.57±0.94 12.63±0.91 cd July 11.88±1.12 10.16±1.38 08.80±1.02 10.28±0.72 c August 11.15±1.40 10.49±0.81 09.12±1.18 10.25±0.66 c Mean±S.E. 12.55±0.64 a 11.36±0.58 a 10.15±0.48 b Note: Two-way ANOVA; Month effect: P<0.05; Size effect: P<0.05; Interaction effect P>0.05; Mean values with different superscripts with in a row or column differ significantly (P<0.05). Table 2. Mean follicular fluid alkaline phosphatase activity (IU/L; Mean±S. E.) in different size antral follicles of buffaloes during different months of the year (n = 4) Month Small Medium Large Mean±S.E. December 256.7±37.2 212.3±22.2 183.9±27.1 217.7±17.8 a January 305.6±26.8 230.0±24.4 187.9±41.5 228.7±22.1 ab February 337.1±62.5 243.0±22.2 200.7±28.4 249.5±27. 7ab March 336.0±46.6 248.9±40.7 215.0±28.9 266.7±25.5 ab April 367.9±45.7 288.1±26.1 221.0±14.5 292.3±24.4 b June 329.9±44.5 312.1±33.1 219.4±30.4 287.1±24.0 b July 351.1±73.9 291.1±59.2 292.6±54.4 286.6±31.7 b August 298.5±06.0 278.7±30.6 185.6±26.1 232.9±18.9 ab Mean±S.E. 315.1±14.5 a 249.1±11.1 b 208.9±10.3 c Note: Two-way ANOVA; Month effect: P<0.05; Size effect: P<0.05; Interaction effect: P>0.05; Mean values with different superscripts within a row or column differ significantly (P<0.05). p = 0.005) between alkaline phosphatase activity and the ambient temperature was also observed on Pearson correlation. As the follicular fluid alkaline phosphatase activity varied from 504.2IU/L to 108.3IU/L from small to large group of follicles, the follicular fluid estradiol 17-b concentration increased from 4.8 ng/ml to 40 ng/ml from small to large group of follicles respectively (Table 3). The follicular fluid estradiol 17-b showed significant (P<0.05) negative correlation (r = -0.821, p = 0.001) with the alkaline phosphatase activity. In the present study both size and ambient temperature had significant (P<0.05) effect on acid and alkaline phosphatase activity (Table 1 and 2). The results are in agreement with the earlier reports in bovine (Lobel and Levy 1968; VanKampen 1978; Henderson and Cupps 1990) in buffalo (Madan et al., 1998) in pigs (McGaughey 1975; Chang et al., 1976) and in women (Caucing et al., 1972). The higher phosphatase activity in the initial stages of the follicular development, could be due to, progesterone and androgen dominant environment that exists in the small follicle, in that, higher concentration of progesterone and androgen could be conducive to the phosphatase activity. This is supported by the positive correlation of follicular fluid alkaline phosphatase activity with fluid progesterone and androgens (androstenedione, dehydroxyepiandrostenedione/testosterone) in cattle (Wise, 1987), and higher acid phosphatase activity in theca interna than in
Table 3. Relative concentration of estradiol 17-β in follicular fluid samples with highest and lowest activity of alkaline phosphatase Samples with low Estradiol 17-β Samples with high Estradiol 17-β Alkaline phosphatase (ng/ml) Alkaline phosphatase (ng/ml) (IU/L) activity (IU/L) activity 108.3 16.0 413.7 05.4 108.3 24.0 419.8 05.6 111.6 40.0 465.7 04.8 119.3 34.0 468.1 05.4 142.6 12.0 483.7 06.6 147.7 26.0 504.2 06.4 M±Sem 122.97±7.33 25.33±4.31 459.2±14.57 5.76±0.33 granulosa cells of the mature follicles in goats (Singh and Rajya 1982). The decreased follicular fluid acid and alkaline phosphatase activity with the development of the follicle in the present study could be due to shift in the follicular hormonal milieu from androgen dominant to estrogen dominant, with the development of follicle. This is supported by the significant negative correlation (r = -0.821, p = 0.001) between alkaline phosphatase activity and follicular fluid estradiol 17-β concentration, observed in the present study. Higher concentration of estradiol -17β in larger follicle than smaller follicle (Palta et al., 1998; Atheya and Totey, 2002) is due to the acquisition of LH receptors by granulose cells in the presence of estradiol -17β and FSH (Ireland and Roche, 1983). This increase in concentration of estradiol -17β with increase in size of the follicle (Wise, 1987; Mekkawy et al., 1988) could exert inhibitory effect on phosphatase activity. Thus, follicular growth may be associated with the decreased activity of the phosphatases. The significant (P<0.05) difference in the acid and alkaline phosphatase activity with Vol. 41, No. 2, 2007 109 respect to different months (ambient temperature) of the year observed in the present study shows that the ambient temperature had an influence on the antral follicular phosphatase activity and that could be through its influence on the size to which a follicle grows. To conclude, follicular fluid phosphatase activity decreased with the increase in size and steroidogenic activity of the antral follicle and increased with the increase in the ambient temperature. The ambient temperature also had an influence on the antral follicular phosphatase activity and that could be through its influence on the size to which a follicle grows. ACKNOWLEDGMENT Financial support provided by Indian Council of Agricultural Research is gratefully acknowledged, as is the cooperation of Dr. S.G. Ramachandra of I. I. Sc., Bangalore during RIA of Estradiol-17β. The first author was a Junior Research Fellow under the project Ovarian Antral Follicular Dynamics in Buffaloes. REFERENCES Atheya, U.K. and Totey, S.M. (2002). Buffalo J., 18(1): 137-141. Caucing, H. et al. Gynecol. Invest., 3: 215. Chang, S.C.S. et al. (1976). Biol. Reprod., 15: 321-28. Chauhan, M.S. et al. (1997). Theriogenology, 48: 461-69. Faulker, W.R. and Meitis, S. (1982). Selected Methods for the Small Clinical Chemistry Laboratory. Washington, D.C., pp. 125. Henderson, K.A. and Cupps, P.T. (1990). J. Anim. Sci., 68: 1363-69.
110 INDIAN JOURNAL OF ANIMAL RESEARCH Henry, J.B. (1984). Clinical Diagnosis and Management. 17 th Ed. W.B. Saunders Co., Philadelphia, pp. 157. Ireland, J.J. and Roche, J.F. (1983). Endocrinology, 112: 150-56. Kulkarni, B.A. (1988). In II world buffalo congress, Vol. III: 233-38. Lobel, B.L. and Levy, E. (1968). Endocrinol. Suppl., 132: 7. Madan, M.C. et al. (1998). J. Anim. Reprod., 19(2): 160. McGaughey, R.W. (1975). Biol. Reprod., 13: 147. Mekkawy, M.Y. et al. (1988). Alexandria J. Vet. Sci., 4: 391-96. Palta, P. et al. (1998). Indian J. Anim. Sci., 68(5): 444-447. Pandey, M.D. and Razada, B.C. (1979). In: Proceedings of the FAO seminar on Buffalo Reproduction and AI (Karnal), pp. 235-245. Raghava, R.V. et al. (1997). Buffalo J., 13: 237-42. Singh, N. and Rajya, B.S. (1982). Indian J. Anim. Hlth., 6: 1-3. Sirard, M.A. et al. (1995). Theriogenology, 44(1): 85-94. Snedecor, G.W. and Cochran, W.G. (1994). Statistical Methods. 8 th Ed., Affiliated East West Press, New Delhi. Tietz, N.W. (1976). Fundamentals of Clinical Chemistry, Philadelphia, W.B. Saunders Co., pp. 614-874. Van Kempen, H. (1978). M.S. Thesis. University of California, Davis. Wise, T. (1987). J. Anim. Sci., 64: 1153-1169.