Comparative Analysis of Biochemical Constituents between Ovarian Follicular Fluid and Serum in Ganjam Goat (Capra hircus)

Ganjam goat is one among the recognized breed of India. It is found in the Southern part of Ganjam district of Odisha and bordering region of Andhra Pradesh, mostly used for meat purpose. This breed has been a major source of livelihood of Golla tribe since generations and therefore also known as Golla goats. Reproduction is most essential component in the life of any animal species as it is needed for the propagation and continuity of the species (Pineda, 2003). The ovarian Folliculogenesis is most important physiological event that significantly affects the success of animal breeding. It is an ordered sequence of development and variation steps including both somatic and germ cells in an ovarian follicle and represented the production of oocyte composition which indicated fertilization and embryonic development (Ana and Adrain, 2012; Tripathi et al., 2015). Follicular fluid is in part exudates of serum and is also partially composed of locally produced substances, which are related to the metabolic activity of the follicular cells (Gerard et al., 2002). This metabolic activity, together with the barrier properties of follicular wall (Bagavandoss et al., 1983) is changing significantly during the growth phase of follicles (Wise, 1987; Gosden et al., 1988). Therefore a different biochemical composition of the follicular fluid in different sized follicles can be expected. Follicular fluid composition has been under intensive investigation in recent times in a bid to increase knowledge of follicular development, oocyte maturation and follicular atresia (Mishra et al., 2003). The constituents of follicular fluids are considered as a regulating factor in follicular development and steroidegenesis (Thakur et al., 2003). Follicular fluid provides an important environment for maturation of oocytes in ovary and the concentration of different biochemical components of the follicular fluid is major driving force for the physiological, biochemical and metabolic aspects of the nuclear and cytoplasmic maturation of the oocyte and the release of the egg from the ruptured follicle and fertilization (Hafez and Hafez, 2008).

The knowledge of the biochemical composition of follicular fluid can also provide useful information about the requirements, growth and maturation of oocyte and which may be used as a provisional guide for formulating suitable culture media for in vitro cell culture and maturation in a particular species. Serum biochemical parameters assume special significance in animals due to their role in body homeostasis, thus providing vital clues on the body’s response to disease and production. Reference values for the biochemical parameters required on each situation to eliminate bias due to the possible influences of variants like breed, age, sex, environment etc. (Vasava et al., 2016). There is great variation among goat breeds concerning their serum biochemical profile (Tambuwal et al., 2002; Tibbo et al., 2004).
       
Considering all these vital role of follicular fluid and serum biochemical constituents the present study was undertaken with the objective of elucidating the concentration of certain biochemical components namely glucose, total cholesterol, total protein, albumin, globulin, acid phosphatase (ACP), alanine transaminase (ALT) and aspartate aminotransferase (AST) in relation to follicle size and a comparision was made with that of serum. No attempt till date has been made to investigate and determine specifically these comparative studies in Ganjam goat. Such an investigation would provide the necessary data that may improve the culture conditions of oocytes and follicular cells like theca, granulosa and cumulus cells in particular breed.

Experimental period
 
Experiment was conducted in Department of Veterinary Physiology, College of Veterinary Science and Animal Husbandry, O.U.A.T., Bhubaneswar, from February to June, 2018.
 
Sample collection
 
For serum biochemical profile analysis 5ml of blood was collected in clot activator vials in order to aid in the process of clotting from the jugular vein just prior to slaughter. Then it was centrifuged at 3000 RPM for 30 minutes, clear non hemolysed supernatant serum was harvested and stored in deep freezer at (-20°C) for further use.
       
Ovaries (n=100) were collected from slaughter houses and transported to laboratory in chilled (4°C) normal saline. No pre-slaughter information regarding reproductive status of these goats was available. Ovaries were washed properly 2 to 3 times with normal saline solution to make them free from cellular debris and foreign materials.

Collection and storage of follicular fluid
 
A hand illuminated lens and a vernier caliper were used to measure the surface diameter of growing follicles present on ovaries. These growing follicles of ovaries were classified in to three categories on the basis of diameter as small (<3 mm); medium (3-5 mm) and large (>5 mm) (Crozet et al., 1995). Follicular fluid was aspirated from all the three categories of follicles separately using a tuberculin syringe. Fluid was centrifuged at 2000 rpm for 5 minutes and the supernatant was stored at -20°C for further analysis.
 
Biochemical analysis of follicular fluid
 
Serum biochemical parameters like glucose, cholesterol, total protein, albumin, globulin, acid phosphatase, ALT and AST were estimated as per the instruction given in commercial kits (Coral clinical system) using clinical analyzer (Systronic-635).
 
Statistical analysis
 
All the parameters for each test sample were compared for obtaining the mean value with standard error (S.E.). Then they were analyzed by Duncan method (One- way- ANOVA) and the significance (P-value) was recorded at 5% (P<0.05) level. The complete statistical analyses were done with the help of Statistical Package for Social Scientist (SPSS) Version 22, Windows 10.0.

As mentioned in Table 1, the result showed that there were no significant differences in glucose concentrations among small, medium and large follicles whereas level of glucose in serum was significantly (P<0.05) higher than the levels in small and medium follicles. The results demonstrated that glucose concentration increases as follicle size increase. These findings were in agreement with findings of Thakur et al., (2003); Nandi et al., (2007); Herrick et al., (2006). Glucose metabolism is less intense in larger follicles as compared with smaller ones, resulting in lower consumption of glucose from fluid of large follicles (Rufai et al., 2013). Perhaps this is because the large follicles can filter and reserve the high concentrations of glucose from blood for utilization in their development to the mature graffian follicle (Leroy et al., 2004). Higher concentration of glucose in serum as compared to all size of follicles implies that the principal source of follicular fluid glucose is blood and very little glucose, if any is synthesized locally by granulose cells of follicle (Arshad et al., 2005).
 

 
The cholesterol value among different follicular size revealed that concentration of cholesterol in large follicles was significantly (P<0.05) higher than that of small and medium sized follicles (Table 1). Furthermore serum cholesterol level was found to be significantly (P<0.05) higher than that of small and medium sized follicles. Increase trend of cholesterol concentration with follicular size was in agreement with findings of (Bordoloi et al., 2000; Thakur et al., 2003; Mishra et al., 2003) in goat and (Brantmeier et al., 1987) in cattle. Cholesterol was the precursor for steroid synthesis and the follicular fluid contained only high-density lipoprotein, therefore, the avascular granulosa cells of the follicles totally depended on the cholesterol from high density lipoprotein, which was derived from the blood plasma by crossing the basement membrane of granulosa cells (Mishra et al., 2003). As the production of steroids increased, the follicle’s level of cholesterol also increased (Wise, 1987). Leroy et al., (2004) also noted that serum concentrations of cholesterol were significantly (P<0.05) higher than in small and medium sized follicles.

As shown in Table 1, the total protein concentration of serum was significantly (P<0.05) higher compared to all three classes of follicular fluids. Among the follicular fluids of different size there was no significant difference found in small, medium and large sized follicle. In the present investigation non-significant increase in total protein of large follicles was in agreement with findings of (Arshad et al., 2005; AbdEllah et al., 2010). Leroy et al., (2004) observed that serum contents of total proteins were significantly higher (P<0.05) than in small, medium and large follicles. According to Wise (1987), the high correlation between total protein contents in follicular fluid and serum suggests that a substantial part of the protein contents in follicular fluid originates from the serum. The serum albumin value registered a significant increase (p<0.05) than medium and large size follicles (Table 1). The value of same in follicular fluid revealed that concentration was gradually decreased from small to large follicle but no significant differences were observed between different sized follicles. The decreased level of albumin in follicular fluid as follicle size increased in this study was in accordance with (Albomohsen et al., 2011) who suggested that the higher levels of albumin in small follicles suggest an active inward transport of albumin compound from blood into follicles, which may be required for various physiological functions such as growth and maturation of follicles. Average value presented in Table 1 showed the mean globulin concentration in small, medium and large sized follicles were found to be non-significant. A significant (P<0.05) difference in globulin was observed between FF and serum which was in accordance with observation made by Arshad et al., (2005). Globulin though present in small quantity in follicular fluid, might be necessary for protecting the follicle from external environments. Globulin has a significant importance in the body due to its immunity producing activity.
       
Average value of acid phosphatase is presented in Table 1. Result showed that acid phosphatase value of small sized follicular fluid was significantly (P<0.05) higher than serum and two other follicular classes. The present values are similar to those reported by Seema et al., (2014). Higher level in small follicles was attributed to limited ability of small follicles to respond to gonadotropins stimulation and few small follicles may undergo atresia. It could play a regulatory role in endocrine functions that control growth and nutrition of oocyte in developing follicles (Henderson and Cupps, 1990). As shown in Table 1, ALT concentrations were lower in all follicle sizes than in serum and the difference was significant (P<0.05) between serum, medium and large follicle. The result obtained by Rahman et al., (2008) in Camel is collaborated with the present finding who stated that higher activity of ALT in small follicles indicate the higher requirement of glutamine during the early stages of growth. A significant (P<0.05) difference was observed between AST values of serum to that of different sized follicles whereas difference between three follicular classes was non-significant (Table 1). The finding of AST distribution in current study was in agreement with Chang et al., (1976) in porcine. The AST catalyses reversible biotransformationreactions between aspartic and glutamic acid. High concentration of this enzyme in medium follicle further supports the view that glutamic acid is much more essential during the early stages of follicular growth (Rahman et al., 2008). Literatures in respect of ALT and AST values in follicular fluid of goat are scanty. The present study will provide the reference for the same.

Based on the findings of the present study it can be concluded that follicular fluid of glucose, cholesterol, total protein and globulin were higher in large follicles than in small follicles. Moreover, serum levels of most of biochemical constituents were higher than their levels in different sizes of follicular fluid. This present investigation of comparative analysis of biochemical constituents between serum and ovarian follicles in Ganjam goat of Odisha will provide clues to researchers who work on reproductive physiology in different breeds of goat. Insights into the follicular fluid composition provide a useful indication of the requirements for oocyte and follicular cell growth in vitro and may be used as a guide for the formulations of cell culture conditions.

We thank the Dean, CVSc and AH, OUAT for providing the facilities to conduct the present study.