MINERAL PROFILES OF OVARIAN ANTRAL FOLLICULAR FLUID IN BUFFALOES DURING FOLLICULAR DEVELOPMENT*

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1 Indian J. Anim. Res., 41 (2): 87-93, 2007 MINERAL PROFILES OF OVARIAN ANTRAL FOLLICULAR FLUID IN BUFFALOES DURING FOLLICULAR DEVELOPMENT* G.P. Kalmath and J.P. Ravindra** Department of Veterinary Physiology, University of Agricultural Sciences, Hebbal, Bangalore , India ABSTRACT Mineral profiles (sodium, potassium, calcium and magnesium) of ovarian antral follicular fluid in buffaloes 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 sodium, potassium, calcium and magnesium using clinical analyzer (photometer, BT-224 Biotechnica instruments). While sodium and calcium concentrations increased with advancement in follicular size, potassium concentration showed a reverse trend. Magnesium concentration did not show definite trend with respect to the follicle size. There were variations in the concentrations of different mineral constituents (sodium, potassium, calcium and magnesium) during different months with different ambient temperature. It was concluded that the concentration of different mineral constituents significantly differed with size of the follicle and also with respect to the different months of the year. INTRODUCTION The ovarian follicular fluid provides suitable microenvironment for the development, growth and maturation of the oocyte and is vital for the maintenance of fertility in the female. Being routinely harvested with the oocytes during in vitro maturation, fertilization and culture techniques, follicular fluid could be used as an effective media supplement for in vitro maturation as it influences the ability of in vitro-matured oocyte to acquire developmental competence (Sirard et al., 1995). The follicular fluid composition varies greatly depending on the stage of follicular development and exerts variable effects on oocyte development (Wise, 1987). This necessitates the characterization of follicular fluid at different stages of follicular development so that fluid from follicle at appropriate stage of development can be used to establish optimal environment for maturation of viable oocyte, which could improve the efficiency of in vitro fertilization. Buffaloes are known to be 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 the profiles of sodium, potassium, calcium and magnesium, and estradiol-17β in the follicular fluid of different size follicles during different months of the year. 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 * Part of M.V.Sc. Thesis of the first author submitted to U.A.S., Bangalore , India. ** Present address: National Institute of Animal Nutrition and Physiology, Adugodi, Bangalore

2 88 INDIAN JOURNAL OF ANIMAL RESEARCH collected in each collection and such collections 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 (Kulkarni, 1988) in to three size groups, small (<6 mm), medium (6-10 mm) and large (11-16 mm) follicles. 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 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 mineral profiles Follicular fluid was analyzed for sodium, potassium, calcium and magnesium concentrations using clinical analyzer (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. Assay of follicular fluid for Estradiol 17-β Representative follicular fluid samples (six) each showing lowest and highest concentration of follicular fluid sodium and potassium 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. (1997). Statistical analysis The follicular fluid biochemical (acid phosphatase and alkaline phosphatase) constituents in different size follicles during different months of the year (Table 1, 2, 3 and 4) were analyzed by Two-way ANOVA (Snedecor and Cochran, 1994). The concentration of estradiol 17β in different size follicle was analyzed by One-way ANOVA (Snedecor and Cochran, 1994). The sodium and potassium concentrations were compared with the functional activity (estradiol 17-b) of the follicle by Pearson s correlation (Snedecor and Cochran, 1994). RESULTS AND DISCUSSION Concentration of follicular fluid estradiol 17- β in different size follicles The follicular fluid estradiol 17-β concentration significantly (P<0.05) varied with the size of the follicle, with large follicle group showing significantly higher estradiol 17-β concentration ( ng/ml) when compared to small (5.753 ng/ml) and medium ( ng/ml) group (Table 5). Follicular fluid sodium The mean sodium concentration (meq/ L) in the follicular fluid of buffalo follicles at different stages of development and during different periods of the year are presented in Table 1. The variation observed in the mean concentration of the follicular fluid sodium among the different categories was statistically significant (P<0.05). Sodium concentration was lowest (244.20±6.5 meq/l) in the small follicle group and highest (281.77±7.40 meq/l) in the large sized follicles with follicular fluid of medium sized follicles showing intermediate concentration (263.41±8.11 meq/l). The follicular fluid sodium was highest (288.7 meq/ L) during the month of February ( C) and lowest (225.4 meq/l) during July ( C). The variation in the sodium concentration among the different periods of the year was statistically significant (P<0.05). However, the

3 Vol. 41, No. 2, Table 1. Mean concentration (meq/l; Mean ± S. E.) of follicular fluid sodium in different size buffalo follicles during different months of the year (n = 4) December ± ± ± ± 10.4 a January ± ± ± ± 10.7 a February ± ± ± ± 09.8 b March ± ± ± ± 16.3 ab April ± ± ± ± 11.9 a June ± ± ± ± 10.0 ab July ± ± ± ± 15.7 a August ± ± ± ± 08.8 a Mean ± S. E ± 06.5 a ± 08.1 b ± 07.4 c Note: Two-way ANOVA; Month effect: P<0.05; Size effect: P<0.05; Interaction effect: P>0.05; Table 2. Mean concentration (meq/l; Mean ± S. E.) of the follicular fluid potassium in different size buffalo follicles during different months of the year (n = 4) December 6.92 ± ± ± ± 0.22 c January 5.95 ± ± ± ± 0.31 b February 5.51 ± ± ± ± 0.11 a March 5.73 ± ± ± ± 0.10 b April 6.55 ± ± ± ± 0.17 c June 6.49 ± ± ± ± 0.18 b July 6.00 ± ± ± ± 0.15 b August 6.21 ± ± ± ± 0.19 b Mean ± S. E ± 0.10 a 5.80 ± 0.11 b 5.31 ± 0.11 c Note: Two-way ANOVA; Month effect: P<0.05; Size effect: P<0.05; Interaction effect: P>0.05; Table 3. Mean concentration (mg/dl; Mean ± S. E.) of follicular fluid calcium in different size follicles of buffaloes during different months of the year (n = 4) December ± ± ± ± 0.12 a January ± ± ± ± 0.21 b February ± ± ± ± 0.14 b March ± ± ± ± 0.18 b April ± ± ± ± 0.19 a June ± ± ± ± 0.14 a July ± ± ± ± 0.09 a August ± ± ± ± 0.06 a Mean ±S. E ± 0.13 a ± 0.11 b ± 0.09 b Note: Two-way ANOVA; Month effect: P<0.05; Size effect: P< 0.05; Interaction effect: P>0.05; observed negative correlation between the follicular fluid sodium and ambient temperature (r=-0.24, p=0.568) was not significant (P>0.05). The follicular fluid sodium showed significant positive correlation with estradiol 17-b (r=+0.672, p=0.017). The increased concentration of follicular fluid sodium observed in the present study is in accordance with the earlier reports in cattle (Wise, 1987) and in buffaloes (Kaur

4 90 INDIAN JOURNAL OF ANIMAL RESEARCH Table 4. Mean concentration (meq/dl; Mean ± S. E.) of follicular fluid magnesium in different size follicles of buffaloes during different months of the year (n = 4) December 2.22 ± ± ± ± 0.03 b January 2.13 ± ± ± ± 0.02 a February 2.15 ± ± ± ± 0.01 bc March 2.15 ± ± ± ± 0.01 bc April 2.17 ± ± ± ± 0.01 d June 2.33 ± ± ± ± 0.02 e July 2.12 ± ± ± ± 0.02 bc August 2.13 ± ± ± ± 0.02 c Mean ± S. E ± 0.02 a 2.11 ± 0.02 b 2.13 ± 0.01 c Note: Two-way ANOVA; Month effect: P<0.05; Size effect: P<0.05; Interaction effect:p>0.05; Table 5. Estradiol 17-β concentration (ng/ml; Mean ± S. E.) in the follicular fluid of different sized follicles Group N Concentration Small ± 0.23 a Medium ± 4.11 b Large ± 4.46 c Note: One-way ANOVA; Size effect: P<0.05; Mean values with different superscripts with in a column were significantly different (P<0.05). Table 6. Relative concentration of estradiol 17-β in follicular fluid samples with highest and lowest concentration of sodium and potassium Sodium Estradiol 17-β Potassium Estradiol 17-β (meq/l) (ng/ml) (meq/l) (ng/ml) et al., 1997). With the advancement of follicles from early antral to ovulatory stage, a tremendous enlargement in the follicle dimension is largely due to the movement of water from blood to antrum, a process that needs osmotic gradient across the follicular wall (Sharma et al., 1995). Thus, increased sodium concentration in large antral follicles could facilitate the osmosis. The significant positive correlation between follicular fluid sodium and estradiol 17-β, observed in the present study (Table 6), indicates that the increased steroidogenic (estradiol 17-β) activity of the developing follicle could be the factor that attributed to the increased concentration of sodium in large follicles, as the 19-hydroxyandrostenedione (a major intermediate of estrogen synthesis) has

5 Vol. 41, No. 2, potent sodium retention action (Sekihara, 1983). There was indeed a significant positive correlation between follicular fluid sodium and estradiol 17-β (Table 6). The increase in steroidogenic activity and thus increase in sodium concentration results in expansion of antral follicle leading to final stage of follicular growth. Follicular fluid potassium The mean concentrations (meq/l) of the follicular fluid potassium in the three categories of buffalo follicles during different months of the year are shown in Table 2. The mean concentrations of potassium between the adjacent categories was found to be statistically significant (P<0.05). The follicular fluid potassium showed declining trend with growth of the follicle as evidenced by its concentration in small (6.170 meq/l), medium (5.803 meq/l) and large (5.308 meq/l) groups of follicles. The follicular fluid potassium levels were higher (6.25 meq/l) during the month of December and lower (5.25 meq/l) during the month of February when the temperatures were C and C respectively. Even though the variation in the potassium concentration among the different periods of the year was statistically significant (P<0.05) the positive correlation (r=+0.035, p=0.934) between follicular fluid potassium and the ambient temperature was insignificant (P>0.05). The results of the present study are in agreement with the results of Schuetz and Anisowicz (1974), Chang et al. (1976) and Knudsen et al. (1979) in pigs. The follicular fluid potassium concentration also had significant negative correlation with follicular fluid estradiol 17-β (Table 6). The decreased concentration of follicular fluid potassium with follicular development could be due to the increased utilization of glucose by developing follicle, a process that leads to transfer of potassium ions from extra cellular sites to intracellular sites (Chang et al., 1976). This increased utilization of glucose could be in terms of steroidogenic activity of the growing follicle. The increase in sodium concentration and decrease in potassium concentration with the follicular development observed in the present study may assist in oocyte maturation as reported by Powers and Biggers (1976), in mice. Follicular fluid calcium The mean follicular fluid calcium concentration (mg/dl) in the follicular fluid of different follicle groups of buffaloes during different months of the year is given in the Table 3. With respect to the size and period, follicular fluid calcium showed significant (P<0.05) variation. Follicular fluid calcium concentration in large follicle (11.86 mg/dl) and medium follicle (11.68 mg/dl) groups was significantly higher when compared with that of small follicle (11.40 mg/dl) group. The follicular fluid calcium concentration was highest (12.45 mg/dl) during the month of February and lowest (11.10 mg/dl) during the month of August when the ambient temperatures were C and C respectively. Pearson s correlation showed insignificant (P>0.05) negative correlation (r=-0.412, p=0.31) between follicular fluid calcium concentration and ambient temperature Increased follicular fluid calcium with the follicular development observed in the present study is in accordance with the findings of Yamada et al. (1998) and Wise (1987) in cattle and Eissa (1996) and Kaur et al. (1997) in buffalo. This increased calcium has a role in steroidogenic capability of growing follicle and in that it might play an important role in the gonadotropin regulation of ovarian steroidogenesis, (Wise, 1987). This increased calcium in large follicle could also help in the process of ovulation, as it is a potent activator of thrombin formation, which helps in the plasmin s proteolytic activity through converting fibrinogen into fibrin, a primary substrate for

6 92 INDIAN JOURNAL OF ANIMAL RESEARCH plasmin s proteolytic activity (Yamada and Gentry, 1995). This proteolytic activity helps in ovulation by weakening the follicular wall (Espey, 1994). Thus the increased concentration of calcium with follicular development (increase in diameter) may be related to steroidogenic capability of the growing follicle and in final stages of development calcium also had a role to play in ovulation. Follicular fluid magnesium Mean follicular fluid magnesium concentration (meq/l) of buffalo follicles of different size group during different months of the year is shown in the Table 4. The changes in the follicular fluid magnesium concentration was found to be significant (P<0.05) among the follicles of different diameter and among the different months of the year. The concentration of magnesium was highest (2.18 meq/l) in small follicles and lowest (2.11 meq/l) in medium sized follicles and intermediate (2.13 meq/l) in large follicles. The magnesium concentration was highest (2.24 meq/l) during the month of June ( C) and was lowest (2.07 meq/l) during the month of January ( C). The positive correlation (r=+0.585, p=0.123) between the follicular fluid magnesium and the ambient temperature was insignificant (P>0.05). Similar results are also reported by Yamada et al. (1998) and Wise (1987) in cattle. The growth of the follicles in early stages of development (small follicle) is by proliferation (Monniaux, 1987). The higher concentration of follicular fluid magnesium in smaller follicles could help in the mitosis of follicular cells through formation of thrombin, a potent mitogen (Forbes, 1987). This is possible as magnesium can substitute (Prendergast and Mann, 1977) for calcium in thrombin formation under condition of low Ca/Mg ratio that exists in small follicles. As magnesium is antagonistic to calcium (Yamada et al., 1998), the decreased magnesium with follicular development could facilitate the calcium action in large follicle. In the present study even though the mineral profiles showed significant (P<0.05) variation during different periods of the year, the ambient temperature seemed to have no bearing on the ionic composition of the antral follicles, as there was no trend in the variation of the ionic concentrations with the monthly average mean temperature. But this variation could be due to the influence of the ambient temperature on the size to which the follicle grows. From the present study it was concluded that follicular fluid mineral profile varied significantly, both with respect to size and with respect to different months of the year. The ambient temperature seems to have indirect influence on these constituents through its effect on growth of the follicle. 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 Chang, S.C.S. et al. (1976). Biol. Reprod., 15: Eissa, H.M. (1996). Br. Vet. J., 152: Espey, L.L. (1994). Biol. Reprod., 50: Faulker, W.R. and Meitis, S. (1982). Selected Methods for the Small Clinical Chemistry Laboratory. Washington, D.C., pp Forbes, J.M. (1987). In Proceedings of Nutrition Society, 46(2):

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