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ARTICLE IN PRESSG ModelNIREP 5076 1–6

Animal Reproduction Science xxx (2014) xxx–xxx

Contents lists available at ScienceDirect

Animal Reproduction Science

jou rn al hom epage : w ww.elsev ier .com/ locate /an i r eprosc i

easonal changes in some oxidant and antioxidantarameters during folliculogenesis in Egyptian buffalo�

eba F. Hozyena,∗, Hodallah H. Ahmedb, G.E.S. Essawyb, S.I.A. Shalabya

Dept. Reproduction and A. I., NRC, EgyptDept. Physiology, Cairo University, Egypt

r t i c l e i n f o

rticle history:eceived 21 January 2014eceived in revised form 1 October 2014ccepted 5 October 2014vailable online xxx

eywords:easonal changeollicular sizeipid peroxidationODACuffalo

a b s t r a c t

Knowledge regarding oxidant and antioxidant status in follicular fluid remains limited andits studying in vivo should enhance our understanding of the impact of them on fertilityand contribute to optimization of in vitro maturation conditions. The present study wasconducted on follicular fluid and serum samples obtained from 708 buffaloes. They wereexamined for Malondialdehyde (MDA) as indicator of lipid peroxidation as well as super-oxide dismutase (SOD) and total antioxidant capacity (TAC) as antioxidant markers. Theobtained results revealed that MDA levels and SOD activity in follicular fluid decreased sig-nificantly as follicle size increased, while TAC increased significantly with the increase infollicular size. Whereas MDA level was significantly higher in summer, the TAC was signif-icantly higher in spring. Moreover, MDA levels and SOD activities were significantly higherin the follicular fluid from different size follicles during the luteal phase than follicularphase. MDA levels in medium follicles in luteal phase and small follicles in follicular andluteal phases were significantly higher in summer than other seasons. Serum MDA levelswere significantly increased in summer. In addition, MDA levels, SOD activities and TAC inserum were significantly higher during luteal phase than follicular phase in summer. TAC

levels were significantly higher in follicular fluid than serum, while MDA was significantlylower in follicular fluid than serum. In conclusion, the present study revealed that oxi-dants/antioxidants balance may play a role in normal follicular development and oxidativestress that occur in summer could be related to reproductive seasonality in buffalo.

© 2014 Published by Elsevier B.V.

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. Introduction

Please cite this article in press as: F. Hozyen, H., et al., Seasonduring folliculogenesis in Egyptian buffalo. Anim. Reprod. Sci. (20

Buffalo is an important world-wide species in terms ofilk and meat production (Terzano et al., 2012). Within

he ovarian follicle, the developing oocyte is surrounded

� This paper is part of the special issue entitled: 4th Mammalian Embryoenomics meeting, October 2013, Quebec City.∗ Corresponding author. Animal Reproduction and AI dept., Veterinaryivision, National Research Centre, Dokki, Giza, Egypt. Tahrir street, Dokki,iza Postal code: 12622. Tel.: +20 1221271525.

E-mail address: Drheba23@yahoo.com (H. F. Hozyen).

http://dx.doi.org/10.1016/j.anireprosci.2014.10.005378-4320/© 2014 Published by Elsevier B.V.

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by follicular fluid which is mainly derived from bloodbesides the locally produced substances (Nandi et al.,2008). Metabolic activity of follicular fluid, together withthe ‘barrier’ properties of the follicular wall, is changingsignificantly during the growth and expansion of each fol-licle (Khan et al., 2011) and with the phase of estrouscycle (Kor and Moradi, 2013). Follicular fluid microenviron-ment can be regarded as a biological window providing a

al changes in some oxidant and antioxidant parameters14), http://dx.doi.org/10.1016/j.anireprosci.2014.10.005

valuable insight into the process of normal follicular devel-opment as well as the pathogenesis of some reproductiveproblems in buffaloes (El-Shahat and Kandil, 2012). Heatstress has been of major concern in reducing buffaloes’

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productivity in tropical and sub-tropical areas (Silanikoveet al., 1997).

According to Nour El-Din (2013), summer tempera-tures in Egypt are extremely high, reaching 38 ◦C to 43 ◦C.Based on the historical records over a period of twelveyears (1999–2010), the subtropical climate in Cairo ischaracterized by hot summer season (June–August) withaverages 23 ◦C–35 ◦C of minimum and maximum temper-atures and 74% mean temperature humidity index (THI%).The optimum conditions for buffaloes as suggested byPayne (1990) are 13–18 ◦C and in terms of the meantemperature–humidity index (THI); values of THI > 72 isconsidered as stressful and THI > 78 is considered verysevere heat stress to this animal.

The ovarian follicles contain their own potential sourcesof reactive oxygen species (ROS) which are potent stim-ulators of lipid peroxidation (Filaire and Toumib, 2012).However, ROS must be continuously inactivated by antiox-idants to keep the oxidant/antioxidant balance to maintainnormal cell function (Basini et al., 2008). Moreover, lipidperoxidation is most often induced by superoxide radical(O−

2) and its damage is mainly inhibited by SOD whichis an enzyme that contributes to the first line of antiox-idant pathway as it removes the O−

2, repairs cells andreduces the damage done to them by superoxide, themost common free radical in the body (Ayres et al., 1998).Total antioxidant capacity (TAC) covers both enzymatic andnon-enzymatic antioxidants (Gupta et al., 2011) and itsmeasuring considers the cumulative effect of all antiox-idants present in plasma and body fluids (Sharma et al.,2013).

El-Sayed et al. (2010) reported that the antioxidantstatus can be considered as one determinant of repro-ductive function in bovine. The balance between ROS andantioxidants may have an important role in reproductiveprocesses such as follicular development (Zhang et al.,2006). Megahed et al. (2008) stated that heat stress mayaffect fertility of animals through increased production offree radicals and according to Lasota et al. (2009), highlevels of SOD are needed to neutralize increased lipid per-oxidation in follicles that occurs in summer. The currentstudy aimed to investigate the pattern of lipid peroxidationand two relevant antioxidant markers in follicular fluid ofdifferent size ovarian follicles and serum of buffalo consid-ering the effect of seasonal changes and phase of estrouscycle.

2. Materials and methods

2.1. Ovaries

Ovaries were collected from 708 non-pregnant femalebuffaloes (Egyptian breed) in good health and with clini-cally normal reproductive tracts from local slaughterhouselocated near to Cairo (Bahtim abattoir, Al-Qaliobia, Egypt).

Please cite this article in press as: F. Hozyen, H., et al., Seasonduring folliculogenesis in Egyptian buffalo. Anim. Reprod. Sci. (20

Immediately after slaughtering, both ovaries from eachanimal were collected in plastic bags containing 0.9%NaCl and transported in ice tank to be inspected at thelaboratory.

PRESSn Science xxx (2014) xxx–xxx

2.2. Experimental design

Follicles were collected over one year during differ-ent seasons. The averages of minimum and maximumtemperatures in summer (June–August) were 22 ◦C–35 ◦Cwith 56.0% mean relative humidity (RH), in autumn(September–November) 18 ◦C–28 ◦C with 60.3% RH, in win-ter (December–February) 10 ◦C–20 ◦C, with 58.0% RH, inspring (March–May) 15 ◦C–28 ◦C with 48.7% RH. The stageof estrous cycle (follicular or luteal) was identified accord-ing to the presence or absence of the corpus luteum on theovary according to Jaglan et al. (2010) and Mondal et al.(2004). Follicular diameter was measured using a caliperand follicles were divided into three categories: small(≥3 mm), medium (4–9 mm) and large (≥10 mm) accordingto Dominguez (1995). Ovaries with cystic follicles as well asthe morphologically atretic follicles, identified macroscop-ically by their opaque wall (Ali et al., 2008) were excludedfrom the study.

2.3. Sampling

2.3.1. Follicular fluidThe contents of the ovarian follicles of different size

(small, medium and large) were aspirated using a 10 mlsyringe attached to an 18 gauge needle and centrifuged at3000 rpm for 10 min for separation of the fluid from the cellfraction. Follicular fluids from each group from each pair ofthe ovaries were pooled in one sample for each individ-ual buffalo. No sample pooling was needed for the largesize category. Collected follicular fluid samples were keptat −20 ◦C until analysis.

2.3.2. BloodSamples were collected during slaughtering for serum

separation after centrifugation at 3000 rpm for 10 min. Col-lected serum samples were kept at −20 ◦C until analysis.

2.4. Measured parameters

2.4.1. MDAMDA level was determined colorimetrically according

to the method of Ohkawa et al. (1979) using kits purchasedfrom Biodiagnostic, Egypt.

2.4.2. SODSOD activity was estimated kinetically using SOD assay

kits (Biodiagnostic, Egypt) according to Nishikimi et al.(1972).

2.4.3. TACTAC level was determined colorimetrically according to

the method of Koracevic et al. (2001) using kits purchasedfrom Biodiagnostic, Egypt.

2.5. Statistical analysis

al changes in some oxidant and antioxidant parameters14), http://dx.doi.org/10.1016/j.anireprosci.2014.10.005

The differences among different follicular sizes andseasons of the year were analyzed statistically by one-way ANOVA. The differences between follicular and lutealphases as well as between the average concentrations of

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Table 1Effect of follicular size, season of the year and the phase of the estrous cycle on Malondialdehyde (MDA) concentrations (nmol/mL) in the follicular fluid ofbuffaloes.

Folliclesize

Estrousphase

Summer Autumn Winter Spring Mean at differentphases

Mean in different sizefollicles

Large FollicularLuteal

21.54aA ± 1.4826.54aA ± 2.10

18.47aA ± 1.1121.93aA ± 1.74

17.19aA ± 1.7625.02aB ± 2.56

19.49aA ± 1.5921.75aA ± 1.12

19.28A ± 0.7523.93B ± 1.023

21.64A ± 0.69

Medium FollicularLuteal

26.29aA ± 1.9733.82bB ± 1.21

25.06aA ± 1.2628.48abA ± 2.22

24.50aA ± 0.1425.72aA ± 1.92

22.98aA ± 1.4728.57abB ± 1.93

24.52A ± 0.7829.45B ± 1.013

27.32B ± 0.74

Small FollicularLuteal

33.21bA ± 0.6638.57bB ± 0.40

31.57abA ± 2.0138.25bB ± 2.53

27.27aA ± 1.4834.31aB ± 2.13

28.09aA ± 1.3834.40aB ± 1.25

29.82A ± 0.8637.06B ± 0.98

33.04C ± 0.81

Mean atdifferentseasons

29.93b ± 1.03 27.20ab ± 1.23 25.11a ± 1.08 24.98a ± 0.90

Data are presented as means ± SE N = 8 per group.M cantly dM h follicuM n are si

dw1n

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DMMM

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eans having different superscripts (a, b) within the same raw are signifieans having different superscripts in the same column (A, B) within eaceans having different superscripts (A, B, C) within the size overall colum

ifferent measured parameters in follicular fluid and serumere analyzed by independent samples t-test using SPSS

6.0 for windows. Means were compared by the least sig-ificance difference at 5% level of probability.

. Results

.1. Effect of follicular size, season of the year and phasef the estrous cycle on Malondialdehyde (MDA)oncentrations (nmol/mL) in the follicular fluid ofuffaloes

Table 1 shows that the overall mean of MDA level wasndirectly proportional with follicle size; it was signifi-antly higher (P < 0.01) in small follicles vs medium andarge follicles as well as in medium follicles vs large ones.n the same time, the overall mean of MDA level in buffaloollicular fluid was significantly higher (P < 0.05) in sum-

er than winter and spring. Moreover, the overall meanf MDA level in follicles obtained during luteal phase wasignificantly higher (P < 0.05) than that obtained during fol-icular phase. Meanwhile, the effect of season on differentize follicles clarifies that in summer MDA level was sig-

Please cite this article in press as: F. Hozyen, H., et al., Seasonduring folliculogenesis in Egyptian buffalo. Anim. Reprod. Sci. (20

ificantly higher (P < 0.05) in the follicular fluid of smallnd medium follicles obtained during luteal phase as wells in small follicles obtained during follicular phase. How-ver, no significant changes were observed in MDA level in

able 2ffect of follicular size, season of the year and the phase of the estrous cycle on supe

Folliclesize

Estrousphase

Summer Autumn Winter

Large FollicularLuteal

36.88aA ± 2.6240.06aA ± 2.60

37.48aA ± 0.9344.93aB ± 2.48

37.01aA ±43.57aB ±

Medium FollicularLuteal

41.60aA ± 1.4544.66aA ± 1.72

40.90aA ± 1.2145.60aA ± 1.44

43.96aA ±46.80aA ±

Small FollicularLuteal

47.78aA ± 2.9553.52aA ± 1.54

47.82aA ± 2.4249.75aA ± 3.51

48.93aA ±52.73aA ±

Mean atdifferentseasons

43.92a ± 1.13 44.52a ± 1.00 45.46a ± 1.29 44.75a ± 1

ata are presented as means ± SE N = 8 per group.eans having different superscripts (a, b) within the same raw are significantly deans having different superscripts in the same column (A, B) within each follicueans having different superscripts (A, B, C) within the size overall column are si

ifferent.lar size differ significantly between follicular and luteal phases.gnificantly different.

large follicles obtained during follicular and luteal phasesthroughout the seasons of the year.

3.2. Effect of follicular size, season of the year and phaseof the estrous cycle on superoxide dismutase (SOD)activity (U/mL) in the follicular fluid of buffaloes

It is shown from Table 2 that the overall mean of SODactivity in buffalo follicular fluid increased as follicle sizedecreased. It was significantly higher (P < 0.01) in smallfollicles than medium and large follicles as well as inmedium follicles than large ones. No significant changeswere detected in the overall mean of SOD activity duringdifferent seasons of the year. Meanwhile, SOD activity ofdifferent size follicles was significantly higher (P < 0.05) infollicular fluid obtained during luteal phase as compared tothat obtained during follicular phase.

3.3. Effect of follicular size, season of the year and phaseof the estrous cycle on total antioxidant capacity (TAC)(mM/L) in the follicular fluid of buffaloes

It is clear from Table 3 that there was a direct relation

al changes in some oxidant and antioxidant parameters14), http://dx.doi.org/10.1016/j.anireprosci.2014.10.005

between the follicle size and TAC; as it was significantlyhigher (P < 0.01) in large follicles than medium and smallfollicles as well as in medium follicles than small ones.The overall mean of TAC in buffalo follicular fluid obtained

roxide dismutase (SOD) activity (U/mL) in the follicular fluid of buffaloes.

Spring Mean at differentphases

Mean in different sizefollicles

2.35 2.09

38.47aA ± 1.6040.68aA ± 3.15

37.47A ± 0.9742.28B ± 1.28

39.88A ± 0.85

1.82 2.67

41.49aA ± 2.1246.80aA ± 2.20

41.94A ± 0.8145.87B ± 0.96

43.96B ± 0.67

2.49 4.61

48.25aA ± 3.9753.58aA ± 4.50

48.16A ± 1.4252.40B ± 1.80

50.18C ± 1.16

.39

ifferent.lar size differ significantly between follicular and luteal phases.gnificantly different.

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Table 3Effect of follicular size, season of the year and the phase of the estrous cycle on total antioxidant capacity (TAC) (mM/L) in the follicular fluid of buffaloes.

Folliclesize

Estrousphase

Summer Autumn Winter Spring Mean at differentphases

Mean in different sizefollicles

Large FollicularLuteal

0.87aA ± 0.0330.91aA ± 0.013

0.92aA ± 0.0720.94aA ± 0.051

0.91aA ± 0.0160.94aA ± 0.045

0.94aA ± 0.0510.94aA ± 0.056

0.91A ± 0.0250.93A ± 0.022

0.92C ± 0.017

Medium FollicularLuteal

0.74aA ± 0.0330.75aA ± 0.035

0.77abA ± 0.0150.77aA ± 0.061

0.76abA ± 0.0260.78aA ± 0.054

0.85bA ± 0.0510.87aA ± 0.055

0.78A ± 0.0180.80A ± 0.028

0.79B ± 0.016

Small FollicularLuteal

0.60aA ± 0.0310.62aA ± 0.031

0.63aA ± 0.0390.66aA ± 0.012

0.64aA ± 0.0080.66aA ± 0.025

0.72aA ± 0.0610.74aA ± 0.008

0.65A ± 0.0240.67A ± 0.029

0.66A ± 0.019

Mean atdifferentseasons

0.76a ± 0.022 0.77a ± 0.031 0.78ab ± 0.023 0.85b ± 0.038

Data are presented as means ± SE N = 8 per group.Means having different superscripts (a, b) within the same raw are significantly different.Means having different superscripts in the same column (A, B) within each follicular size differ significantly between follicular and luteal phases.Means having different superscripts (A, B, C) within the size overall column are significantly different.

Table 4Effect of season of the year and the phase of the estrous cycle on Malondialdehyde (MDA) concentrations, superoxide dismutase (SOD) activity and totalantioxidant capacity (TAC) in the serum of buffaloes.

Parameters Estrous phase Summer Autumn Winter Spring

MDA FollicularLuteal 33.79A ± 0.8637.20B ± 1.79

33.50A ± 1.0134.61A ± 1.07

30.52A ± 1.2533.53A ± 1.18

31.92A ± 1.1634.41A ± 1.94

Overall mean 35.49b ± 1.07 34.06ab ±0.71 32.13a ± 0.92 33.17ab ± 1.09SOD FollicularLuteal 40.73A ± 1.34

46.94B ± 0.8740.68A ± 3.1145.85A ± 3.12

43.81A ± 0.8846.19A ± 1.78

40.76A ± 1.3943.82A ± 1.47

Overall mean 43.84a ± 1.15 43.26a ± 2.23 45.00a ± 1.01 42.65a ± 1.10TAC FollicularLuteal 0.63A ± 0.05

0.74B ± 0.0130.69A ± 0.020.66A ± 0.043

0.65A ± 0.0250.63A ± 0.024

0.65A ± 0.040.64A ± 0.012

Overall mean 0.69a ± 0.031 0.68a ± 0.024 0.64a ± 0.017 0.65a ± 0.021

erent pa are sign

On the other hand, the TAC in buffalo follicular fluid wasdirectly proportional with the follicle size, i.e., significantlyhigher in large follicles than medium and small follicles andin medium follicles than small ones. The obtained results

Table 5Average malondialdehyde (MDA) concentrations (nmol/ml), superoxidedismutase (SOD) activity (U/ml) and total antioxidant capacity (TAC,mM/L) in follicular fluid and serum of buffaloes.

MDA(nmol/ml)

SOD (U/ml) TAC (mM/L)

Follicular fluid(n = 192)

26.92A ± 0.55 44.64 ± 0.60 0.79A ± 0.02

Serum 33.72B ± 0.49 43.70 ± 0.76 0.67B ± 0.02

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Data are presented as means ± SE N = 8 per group.Means having different superscripts in the same column (A, B) within diffMeans having different superscripts (a, b) within the overall mean of row

during spring was significantly higher than that obtainedduring summer and autumn (P < 0.05). No statistical signif-icance was recorded in TAC between different size folliclesobtained during luteal and follicular phases.

3.4. Effect of season of the year and the phase of theestrous cycle on Malondialdehyde (MDA) concentrations,superoxide dismutase (SOD) activity and totalantioxidant capacity (TAC) in the serum of buffaloes

Table 4 clarifies that the overall mean of MDA level inbuffalo serum was significantly higher (P < 0.05) in sum-mer than winter. However, no significant changes werereported in the overall means of serum SOD activity andTAC during different seasons of the year. Meanwhile, insummer MDA, SOD and TAC in buffalo serum were sig-nificantly higher (P < 0.05) in luteal phase than follicularphase.

3.5. Average malondialdehyde (MDA) concentrations(nmol/mL), superoxide dismutase (SOD) activity (U/mL)and total antioxidant capacity (TAC, mM/L) in follicularfluid and serum of buffaloes

As shown in Table 5, the overall mean of follicular

Please cite this article in press as: F. Hozyen, H., et al., Seasonduring folliculogenesis in Egyptian buffalo. Anim. Reprod. Sci. (20

fluid MDA level (26.92 ± 0.55) in buffalo was signifi-cantly (P < 0.05) lower than overall mean of serum MDA(33.72 ± 0.49). On the contrary, the overall mean ofTAC increased significantly (P < 0.05) in follicular fluid

rameters differ significantly.ificantly different.

(0.79 ± 0.02) than serum (0.67 ± 0.02). However, therewere no significant changes between follicular and serumSOD.

4. Discussion

The results of the present study revealed that the over-all means of MDA concentration and SOD activity in buffalofollicular fluid were inversely proportional with the folli-cle size. It was significantly higher in small follicles vs largeand medium follicles and in medium follicles vs large ones.

al changes in some oxidant and antioxidant parameters14), http://dx.doi.org/10.1016/j.anireprosci.2014.10.005

(n = 64)

Data are presented as means ± SE.Means having different superscripts in the same column (A, B) differ sig-nificantly between follicular fluid and serum.

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re in agreement with Combelles et al. (2010) in bovineho reported that SOD activities were significantly higher

n follicular fluid from small follicles than medium folli-les which in turn possessed elevated MDA concentrationshen compared to large follicles. In the same respect, El-

hahat et al. (2013) reported that lipid peroxidation wasound to be higher in the small follicles than in large and

edium follicles. On the other hand, Lasota et al. (2009)id not find any significant changes in total SOD activity inorcine follicular fluid obtained from large and small folli-les. While El-Shahat and Kandil (2012) did not record anyignificant changes in MDA concentration between differ-nt size follicles in buffaloes.

Ayres et al. (1998) reported that lipid peroxidation isost often induced by O−

2 and SOD catalyzes the reac-ion that converts O−

2 to H2O2 and molecular oxygen.eanwhile, the previous authors added that E2 offer pro-

ection from lipid peroxidation by inhibiting the formationf O−

2 and was also found to interfere with the oxidativehain propagation leading to lipid peroxidation. Moreover,garwal et al. (2003) further added that follicles through

heir developmental journey become a dominant source ofOS which when overproduced in cells and tissues causexidative stress. In the same respect, Circu and Aw (2010)ave established the relationship between oxidative stressnd cell fate; as the cumulative damage from ROS andxidative stress eventually leads to cell death.

The elevated levels of lipid peroxidation in small fol-icles reported in the present study were associated withigh activity of the free radical scavenger SOD indicatinghat lipid peroxidation is tightly controlled by each folliclehroughout folliculogenesis and one probable mechanismor this control is enzymatic reduction by SOD (Hennett al., 2013). Moreover, the higher levels of TAC reportedn large follicles in the current study may have a role inhe defense systems against oxidants, which implies (1)ystems that prevent ROS generation, (2) antioxidant sys-ems that inactivate oxidants and (3) systems that are ableo limit the deleterious effects of oxidants by allowing theepair of oxidative damage (Cheeseman and Slater, 1993).he increased level of TAC in large follicles may be involvedn maintaining low levels of ROS which is necessary to keepormal cell function (Gupta et al., 2011).

High ambient temperature is a major limitation onuffalo and can result in impairment of productionnd reproduction performance (Marai and Haeeb, 2010).egarding the effect of season on oxidant and antioxidantarameters in the follicular fluid and serum of buffalo, theurrent study clarifies that the overall mean of MDA level inhe follicular fluid was significantly higher in summer thaninter and spring, while the overall mean of TAC was sig-ificantly higher in spring than summer and autumn. Theverall mean of serum MDA level in the present study wasignificantly higher in summer season than winter with noignificant changes in the overall mean of SOD and TAChroughout the four seasons. MDA was reported to reflecthe increase in lipid peroxidation after exposure to ROS

Please cite this article in press as: F. Hozyen, H., et al., Seasonduring folliculogenesis in Egyptian buffalo. Anim. Reprod. Sci. (20

Killic et al., 2003). The increased lipid peroxidation andecreased TAC observed in the present study in summerould be related to the fact that heat stress during summers one of the main reasons for oxidative stress resulting

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from increased production of free radicals and a decreasein antioxidant defense (Trevisan et al., 2001 and Williamset al., 2002).

Although buffalo is a polyestrous animal, yet it exhibitsa distinct seasonal variation as its breeding frequency ishighest during winter, decreases in autumn and spring andis lowest during summer (Rensis and Scaramuzzi, 2003). Inthe same time, increased environmental temperature dur-ing summer was reported to be associated with increasedintensity of production of ROS (Lasota et al., 2009). Accord-ing to Megahed et al. (2008), heat stress associated withelevated MDA level means that fertility of buffaloes maybe affected in summer through an increased production offree radicals. Similar changes in serum MDA levels wereobserved by Megahed et al. (2008) in buffalo and Chandraand Aggarwal (2009) in cows. Moreover, Megahed et al.(2008) found that in Egyptian buffaloes SOD activities inserum were significantly lower in the summer than winter.On the other hand, Lallawmkimi (2009) reported signif-icantly higher SOD levels during summer compared towinter in Murrah buffaloes.

Regarding the effect of estrous phase, the presentstudy indicated that follicular fluid MDA concentration inmedium follicles obtained during luteal phase and smallfollicles obtained during luteal and follicular phases wassignificantly higher in summer than other seasons. More-over, MDA, SOD and TAC in buffalo serum were significantlyhigher during luteal phase than follicular phase in summer.El-Shahat and Kandil (2012) attributed the increase in MDAduring luteal phase in medium and small follicles to theincrease in lipid peroxidation within the plasma membraneof luteal cells, which may be associated with the observedloss of gonadotrophin receptors, decreased cAMP forma-tion and decreased steroidogenic ability of the CL in theregression phase.

In the current work, the overall means of MDA levelin buffalo follicular fluid was significantly lower than thatof serum. On the contrary, the overall mean of TAC wassignificantly higher in follicular fluid than serum with nosignificant changes between the overall means of follicularand serum SOD activities. Jozwik et al. (1999) reported thatlipid peroxidation level in women follicular fluid is signifi-cantly lower than in blood and they attributed this gradientbetween follicular fluid and blood to the lower rate of ini-tiation of peroxidation in the follicular fluid, suggestive ofthe presence of efficient antioxidant defense systems in thedirect milieu of the oocyte. Elevated levels of TAC in follicu-lar fluid than in serum probably may reflect the antioxidantactivity of granulosa cells (Revelli et al., 2009).

In conclusion the present study indicated that (a) lipidperoxidation increased in buffalo follicular fluid from smallfollicles and during luteal phase, (b) SOD represents thedominant antioxidant defense in small follicles and (c)high temperature during summer season associated withincreased oxidative stress in both follicular fluid and serumcould be related to reproductive seasonality in buffalo.

al changes in some oxidant and antioxidant parameters14), http://dx.doi.org/10.1016/j.anireprosci.2014.10.005

Conflicts of Interest

All authors declare that they do not have any conflictsof interest.

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Uncited references

Ganaie et al., 2013 and Machatkova et al., 2004.

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