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Antioxidant Status of Cyclic and Acyclic Goats: Screening of Blood and Follicular Fluid

Ashiq Hussain Bhat Sudershan Kumar Imtiyaz Ahmad Reshi Rameez Ali Dar Megha Shriram Kose Anavil Bharadhawaz
Vol 7(3), 34-39
DOI- http://dx.doi.org/10.5455/ijlr.20170215041736

Present study was designed with a dual aim of ascertaining the follicular population of goat ovaries and to obtain total antioxidant status by screening the blood collected before slaughter and follicular fluid collected from ovaries after slaughter. Samples (blood and reproductive tracts) of the goats were collected from a local abattoir located in Jammu city. The reproductive tracts were divided into two groups viz. cyclic and acyclic depending on the presence or absence of corpus luteum or corpus haemorrhagicum and size of surface follicles on the ovary. The study revealed a significant (P < 0.05) difference between cyclic and acyclic goats when compared on the basis of number of medium and large surface follicles, however, the number of small follicles didn’t differ significantly (P > 0.05). Activity of superoxide dismutase, catalase, lipid peroxidase and glutathione-s-transferase revealed a considerable although non-significant (P > 0.05) difference between cyclic and acyclic goats in follicular fluid indicating a role of these enzymatic antioxidants in the maintenance of cyclicity in goats.


Keywords : Follicular Population Antioxidant Status Ovaries Cyclicity Goats

Introduction

Goat, a polyestrous animal, exhibits marked seasonality in breeding behaviour. In India, breeds of goats exhibit a trend of seasonal anoestrous (acyclicity) in summer months, particularly March to June. This acyclicity is a major constraint to augment fertility in this species. Follicular population tends to vary with the reproductive status of the animal, stage of estrous cycle and presence or absence of corpus luteum on the ovaries (Abdoon and Kandil, 2001). Follicular fluid, a mixed secretion of follicles and plasma, testifies its potential importance in ovarian physiology. This small reservoir of fluid reflects the biochemical and endocrinological activity of the follicles and thus serves as a guide to its growth and differentiation (Deshpande and Pathak, 2010). High level of ROS (Reactive Oxygen Species), due to an increased production of oxidant species and/or a decreased efficacy of antioxidant system, can lead to oxidative stress, which affect multiple physiological processes in reproduction and fertility, from oocyte maturation to fertilization, embryo development and pregnancy (Agarwal et al., 2006). Several studies indicate that follicular atresia in mammalian species due to the accumulation of toxic metabolites often results from oxidative stress. It has been suggested that ROS under moderate concentrations play a role in signal transduction processes involved in growth and protection from apoptosis. Conversely, increase of ROS levels is primarily responsible for the alteration of macromolecules, such as lipids, proteins and nucleic acids, which lead to significant damage of cell structures and thereby cause oxidative stress (Behrman et al., 2001). To prevent damage due to ROS, cells possess a number of antioxidants.

Antioxidants, in the mammalian body, protect the key cell components by neutralizing the damaging effects of free radicals, which are natural by-products of cell metabolism (Ames et al., 1993). Antioxidants play an important role in prevention of many diseases in animals including reproductive disorders like tubal infertility, unexplained infertility, polycystic ovarian diseases, abortion etc. In addition to it, low levels of total antioxidant capacity have a key role in male infertility (Fingerova et al., 2007). So the measurement of total antioxidant status can be an important diagnostic tool in the evaluation of many such diseases in animals. Total antioxidant status refers to a full spectrum of antioxidant activity against various reactive oxygen/nitrogen radicals and compose of enzymatic (e.g., superoxide dismutase, catalase, and glutathione peroxidase) and non-enzymatic (e.g., ascorbate, urate, vitamin E, pyruvate, taurine and hypotaurine) components (Fingerova et al., 2007).

Materials and Methods

Collection of Blood and Reproductive Tracts

Blood (before slaughter) and reproductive tracts (after slaughter) of the goats were collected from a local abattior located in the Jammu city. Blood was collected from the goats before slaughter in 10ml centrifuge tubes (containing heparin as anticoagulant) by jugular venipuncture. Immediately after slaughter, reproductive tracts of goats with no apparent clinical abnormalities were collected and transported to the laboratory in Phosphate Buffered Saline in an ice box within 30 minutes.

Classification of Reproductive Tracts

In the laboratory reproductive tracts were classified into two groups; Group I- Ovaries with a corpus hemorrhagicum (CH), a large corpus luteum (CL) and > 5 mm follicle(s) in diameter or a regressing CL with follicle(s) > 6 mm in diameter were classified as active and the animals as cyclic. Group II- Ovaries without a CL or CH or the presence of a regressed CL with follicles < 5mm in diameter, such ovaries were classified as inactive and the animals as non-cyclic (8). Follicles were divided into three groups of small (< 2 mm), medium (2-4 mm) and large (> 4 mm) depending on their diameter (9).

Collection of Follicular Fluid

Follicular fluid was aspirated by using a separate hypodermic tuberculin syringe for each ovary and was pooled irrespective of their size. It was collected by applying slight pressure to avoid additional traumatisation of follicles. Aspirated fluid was centrifuged at 3000 rpm for 15 minutes to remove cellular debris. The sample was kept at -200C till further use.

Processing of Samples

The blood was washed with normal saline and centrifuged thrice to get the haemolysate (1% and 33%). In 1% haemolysate, activity of superoxide dismutase (SOD), catalase (CAT) and glutathione-s-transferace (GST) was measured while as 33% haemolysate was used for measuring the activity of lipid peroxidase (LPO). The activity of SOD, CAT, GST and LPO in erythrocyte lysate and follicular fluid was determined by the methods of (Marklund and Marklund, 1974), (Aebi et al., 1983), (Hafeman et al., 1974) and (Ohkawa et al., 1979), respectively.

Statistical Analysis

The data generated was subjected to paired t-test as per method described by Snedecor and Cochran (Snedecor et al., 1994). The results of the analyses are summarized in Tables 1, 2 and 3.

Results and Discussion

In the present study 48 ovaries (24 each) of cyclic and acyclic goats were processed and findings revealed that the average number of small sized surface follicles didn’t differ significantly between cyclic and acyclic however, the number of medium and large sized surface follicles were significantly higher (P < 0.05) in cyclic as compared to acyclic goats (Table 1). These results are consistent with the earlier reports of Andurkar et al., 2004 and Sontakke et al., 2009. However, Lohan et al., 2004 reported lesser number of small follicles and greater number of large follicles in cyclic as compared to acyclic buffaloes. Moreover, the possible reason for higher number of medium and large sized follicles in cyclic goats may be because of the continuous grazing on green forages available in the meadows around Jammu city. Green forages are rich in β-carotene which is important for the growth of follicles. On the other hand, decreased number of medium and large sized follicles in acyclic goats may be due to the increased rate of premature atresia of small follicles in them.

Table 1: Average number of small, medium and large size surface follicles in cyclic and acyclic goats (Mean ± S.E)

S. No. Follicle Size Cyclic (n=24) Acyclic (n=24) t-value
1 Small (< 2mm) 5.71 ± 0.59 4.83 ± 0.43 1.21NS
2 Medium (2-4 mm) 3.74 ± 0.44 1.04 ± 0.18 5.47*
3 Large (> 4mm) 1.54 ± 0.20 0.37 ± 0.10 5.24*

* Indicates significant values at P < 0.05; NS Indicates non-significant values at P > 0.05

Activity of SOD (U/mg Protein), CAT (U/mg Hb), and GST (µM/l) in follicular fluid was found higher although non-significantly (P > 0.05) in cyclic as compared to acyclic goats while reverse was found true for LPO (nmol MDA/mg of Protein), indicating a negative correlation of LPO with SOD, CAT and GST (Table 2). Slightly decreased activity of these enzymes in acyclic goats can be correlated to the lesser number of surface follicles present on the ovaries of these goats owing to the fact that ROS are involved in the initiation of apoptosis in follicles, influencing folliculogenesis and steriodogenesis (Devine et al., 2012). It has been observed that SOD, CAT and GST play a major role in maintaining low levels of oxidative stress (Behrman et al., 2001).

Table 2: Antioxidant status of follicular fluid in cyclic and acyclic goats (Mean ± S.E)

S. No. Variable Cyclic (n=10) Acyclic (n=10) t- value
1 SOD (U/mg Protein) 4.33 ± 0.03 4.25 ± 0.04 1.61NS
2 CAT (U/mg Hb) 4.79 ± 0.04 4.66 ± 0.04 2.15NS
3 LPO (nmol MDA/mg of Protein) 1.38 ± 0.02 1.39 ± 0.04 -0.07NS
4 GST (µM/l) 0.57 ± 0.02 0.54 ± 0.06 1.15NS

NS indicates non-significant values at P > 0.05

When compared in blood, the difference in the activity of SOD, CAT, LPO and GST between cyclic and acyclic goats was minimal (Table 3), indicating a bigger role of antioxidant mechanism at ovarian level. Similar results have been reported in humans by Jozwik et al., 1999, where the follicular fluid was found more active than serum in terms of these marker enzymes. They also concluded that this efficient antioxidant defense mechanism in the direct milieu of the oocyte and is able to protect antral follicles against apoptosis. Therefore, some correlation can be drawn between the higher number of surface follicles and antioxidant status of cyclic goats as compared to acyclic ones.

Table 3: Antioxidant status of blood in cyclic and acyclic goats (Mean ± S.E)

S. No. Variable Cyclic (n=10) Acyclic (n=10) t- value
1 SOD (U/mg Protein) 3.42 ± 0.08 3.40 ± 0.05 -0.352NS
2 CAT (U/mg Hb) 4.18 ± 0.06 4.12 ± 0.11 -0.57NS
3 LPO (nmol MDA/mg of Protein) 1.59 ± 0.05 1.62 ± 0.02 -0.45NS
4 GST (µM/l) 0.44 ± 0.02 0.49 ± 0.01 1.54NS

NS indicates non-significant values at P > 0.05

Conclusion

The present study suggests that follicular population tends to be higher in ovaries with better antioxidant capacity. Follicular population also varies with the cyclic nature of the ovaries and higher numbers of follicles are present on ovaries of cyclic goats as compared to acyclic ones.

References

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