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Animal Reproduction Science journal homepage: www.elsevier.com/locate/anireprosci
Seasonal changes in some oxidant and antioxidant parameters during folliculogenesis in Egyptian buffalo夽
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Heba F. Hozyen a,∗ , Hodallah H. Ahmed b , G.E.S. Essawy b , S.I.A. Shalaby a a b
Dept. Reproduction and A. I., NRC, Egypt Dept. Physiology, Cairo University, Egypt
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Article history: Received 21 January 2014 Received in revised form 1 October 2014 Accepted 5 October 2014 Available online xxx
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Keywords: Seasonal change Follicular size Lipid peroxidation SOD TAC Buffalo
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1. Introduction
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Knowledge regarding oxidant and antioxidant status in follicular fluid remains limited and its studying in vivo should enhance our understanding of the impact of them on fertility and contribute to optimization of in vitro maturation conditions. The present study was conducted on follicular fluid and serum samples obtained from 708 buffaloes. They were examined for Malondialdehyde (MDA) as indicator of lipid peroxidation as well as superoxide dismutase (SOD) and total antioxidant capacity (TAC) as antioxidant markers. The obtained results revealed that MDA levels and SOD activity in follicular fluid decreased significantly as follicle size increased, while TAC increased significantly with the increase in follicular size. Whereas MDA level was significantly higher in summer, the TAC was significantly higher in spring. Moreover, MDA levels and SOD activities were significantly higher in the follicular fluid from different size follicles during the luteal phase than follicular phase. MDA levels in medium follicles in luteal phase and small follicles in follicular and luteal phases were significantly higher in summer than other seasons. Serum MDA levels were significantly increased in summer. In addition, MDA levels, SOD activities and TAC in serum were significantly higher during luteal phase than follicular phase in summer. TAC levels were significantly higher in follicular fluid than serum, while MDA was significantly lower in follicular fluid than serum. In conclusion, the present study revealed that oxidants/antioxidants balance may play a role in normal follicular development and oxidative stress that occur in summer could be related to reproductive seasonality in buffalo. © 2014 Published by Elsevier B.V.
Buffalo is an important world-wide species in terms of milk and meat production (Terzano et al., 2012). Within the ovarian follicle, the developing oocyte is surrounded
夽 This paper is part of the special issue entitled: 4th Mammalian Embryo Genomics meeting, October 2013, Quebec City. ∗ Corresponding author. Animal Reproduction and AI dept., Veterinary Division, National Research Centre, Dokki, Giza, Egypt. Tahrir street, Dokki, Giza Postal code: 12622. Tel.: +20 1221271525. E-mail address: [email protected] (H. F. Hozyen).
by follicular fluid which is mainly derived from blood besides the locally produced substances (Nandi et al., 2008). Metabolic activity of follicular fluid, together with the ‘barrier’ properties of the follicular wall, is changing significantly during the growth and expansion of each follicle (Khan et al., 2011) and with the phase of estrous cycle (Kor and Moradi, 2013). Follicular fluid microenvironment can be regarded as a biological window providing a valuable insight into the process of normal follicular development as well as the pathogenesis of some reproductive problems in buffaloes (El-Shahat and Kandil, 2012). Heat stress has been of major concern in reducing buffaloes’
http://dx.doi.org/10.1016/j.anireprosci.2014.10.005 0378-4320/© 2014 Published by Elsevier B.V.
Please cite this article in press as: F. Hozyen, H., et al., Seasonal changes in some oxidant and antioxidant parameters during folliculogenesis in Egyptian buffalo. Anim. Reprod. Sci. (2014), http://dx.doi.org/10.1016/j.anireprosci.2014.10.005
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productivity in tropical and sub-tropical areas (Silanikove et al., 1997). According to Nour El-Din (2013), summer tempera41 tures in Egypt are extremely high, reaching 38 ◦ C to 43 ◦ C. 42 Based on the historical records over a period of twelve 43 years (1999–2010), the subtropical climate in Cairo is 44 characterized by hot summer season (June–August) with 45 averages 23 ◦ C–35 ◦ C of minimum and maximum temper46 atures and 74% mean temperature humidity index (THI%). 47 The optimum conditions for buffaloes as suggested by 48 ◦ 49Q2 Payne (1990) are 13–18 C and in terms of the mean temperature–humidity index (THI); values of THI > 72 is 50 considered as stressful and THI > 78 is considered very 51 severe heat stress to this animal. 52 The ovarian follicles contain their own potential sources 53 of reactive oxygen species (ROS) which are potent stim54 ulators of lipid peroxidation (Filaire and Toumib, 2012). 55 However, ROS must be continuously inactivated by antiox56 idants to keep the oxidant/antioxidant balance to maintain 57 normal cell function (Basini et al., 2008). Moreover, lipid 58 peroxidation is most often induced by superoxide radical 59 (O− 2 ) and its damage is mainly inhibited by SOD which 60 is an enzyme that contributes to the first line of antiox61 idant pathway as it removes the O− 2 , repairs cells and 62 reduces the damage done to them by superoxide, the 63 most common free radical in the body (Ayres et al., 1998). 64 Total antioxidant capacity (TAC) covers both enzymatic and 65 non-enzymatic antioxidants (Gupta et al., 2011) and its 66 measuring considers the cumulative effect of all antiox67 idants present in plasma and body fluids (Sharma et al., 68 2013). 69 El-Sayed et al. (2010) reported that the antioxidant 70 status can be considered as one determinant of repro71 ductive function in bovine. The balance between ROS and 72 antioxidants may have an important role in reproductive 73 processes such as follicular development (Zhang et al., 74 2006). Megahed et al. (2008) stated that heat stress may 75 affect fertility of animals through increased production of 76 free radicals and according to Lasota et al. (2009), high 77 levels of SOD are needed to neutralize increased lipid per78 oxidation in follicles that occurs in summer. The current 79 study aimed to investigate the pattern of lipid peroxidation 80 and two relevant antioxidant markers in follicular fluid of 81 different size ovarian follicles and serum of buffalo consid82 ering the effect of seasonal changes and phase of estrous 83 cycle. 84 39
2.2. Experimental design
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2. Materials and methods
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2.1. Ovaries
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Ovaries were collected from 708 non-pregnant female buffaloes (Egyptian breed) in good health and with clinically normal reproductive tracts from local slaughterhouse located near to Cairo (Bahtim abattoir, Al-Qaliobia, Egypt). Immediately after slaughtering, both ovaries from each animal were collected in plastic bags containing 0.9% NaCl and transported in ice tank to be inspected at the laboratory.
Follicles were collected over one year during different seasons. The averages of minimum and maximum temperatures in summer (June–August) were 22 ◦ C–35 ◦ C with 56.0% mean relative humidity (RH), in autumn (September–November) 18 ◦ C–28 ◦ C with 60.3% RH, in winter (December–February) 10 ◦ C–20 ◦ C, with 58.0% RH, in spring (March–May) 15 ◦ C–28 ◦ C with 48.7% RH. The stage of estrous cycle (follicular or luteal) was identified according to the presence or absence of the corpus luteum on the ovary according to Jaglan et al. (2010) and Mondal et al. (2004). Follicular diameter was measured using a caliper and follicles were divided into three categories: small (≥3 mm), medium (4–9 mm) and large (≥10 mm) according to Dominguez (1995). Ovaries with cystic follicles as well as the morphologically atretic follicles, identified macroscopically by their opaque wall (Ali et al., 2008) were excluded from the study. 2.3. Sampling 2.3.1. Follicular fluid The contents of the ovarian follicles of different size (small, medium and large) were aspirated using a 10 ml syringe attached to an 18 gauge needle and centrifuged at 3000 rpm for 10 min for separation of the fluid from the cell fraction. Follicular fluids from each group from each pair of the ovaries were pooled in one sample for each individual buffalo. No sample pooling was needed for the large size category. Collected follicular fluid samples were kept at −20 ◦ C until analysis. 2.3.2. Blood Samples were collected during slaughtering for serum separation after centrifugation at 3000 rpm for 10 min. Collected serum samples were kept at −20 ◦ C until analysis. 2.4. Measured parameters 2.4.1. MDA MDA level was determined colorimetrically according to the method of Ohkawa et al. (1979) using kits purchased from Biodiagnostic, Egypt. 2.4.2. SOD SOD activity was estimated kinetically using SOD assay kits (Biodiagnostic, Egypt) according to Nishikimi et al. (1972). 2.4.3. TAC TAC level was determined colorimetrically according to the method of Koracevic et al. (2001) using kits purchased from Biodiagnostic, Egypt. 2.5. Statistical analysis The differences among different follicular sizes and seasons of the year were analyzed statistically by oneway ANOVA. The differences between follicular and luteal phases as well as between the average concentrations of
Please cite this article in press as: F. Hozyen, H., et al., Seasonal changes in some oxidant and antioxidant parameters during folliculogenesis in Egyptian buffalo. Anim. Reprod. Sci. (2014), http://dx.doi.org/10.1016/j.anireprosci.2014.10.005
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Table 1 Effect of follicular size, season of the year and the phase of the estrous cycle on Malondialdehyde (MDA) concentrations (nmol/mL) in the follicular fluid of buffaloes. Follicle size
Estrous phase
Summer
Autumn
Winter
Spring
Mean at different phases
Mean in different size follicles
Large
Follicular Luteal Follicular Luteal Follicular Luteal 29.93b ± 1.03
21.54aA ± 1.48 26.54aA ± 2.10 26.29aA ± 1.97 33.82bB ± 1.21 33.21bA ± 0.66 38.57bB ± 0.40 27.20ab ± 1.23
18.47aA ± 1.11 21.93aA ± 1.74 25.06aA ± 1.26 28.48abA ± 2.22 31.57abA ± 2.01 38.25bB ± 2.53 25.11a ± 1.08
17.19aA ± 1.76 25.02aB ± 2.56 24.50aA ± 0.14 25.72aA ± 1.92 27.27aA ± 1.48 34.31aB ± 2.13 24.98a ± 0.90
19.49aA ± 1.59 21.75aA ± 1.12 22.98aA ± 1.47 28.57abB ± 1.93 28.09aA ± 1.38 34.40aB ± 1.25
19.28A ± 0.75 23.93B ± 1.023 24.52A ± 0.78 29.45B ± 1.013 29.82A ± 0.86 37.06B ± 0.98
21.64A ± 0.69
Medium Small Mean at different seasons
27.32B ± 0.74 33.04C ± 0.81
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.
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different measured parameters in follicular fluid and serum were analyzed by independent samples t-test using SPSS 16.0 for windows. Means were compared by the least significance difference at 5% level of probability.
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3.1. Effect of follicular size, season of the year and phase of the estrous cycle on Malondialdehyde (MDA) concentrations (nmol/mL) in the follicular fluid of buffaloes Table 1 shows that the overall mean of MDA level was indirectly proportional with follicle size; it was significantly higher (P < 0.01) in small follicles vs medium and large follicles as well as in medium follicles vs large ones. In the same time, the overall mean of MDA level in buffalo follicular fluid was significantly higher (P < 0.05) in summer than winter and spring. Moreover, the overall mean of MDA level in follicles obtained during luteal phase was significantly higher (P < 0.05) than that obtained during follicular phase. Meanwhile, the effect of season on different size follicles clarifies that in summer MDA level was significantly higher (P < 0.05) in the follicular fluid of small and medium follicles obtained during luteal phase as well as in small follicles obtained during follicular phase. However, no significant changes were observed in MDA level in
large follicles obtained during follicular and luteal phases throughout the seasons of the year. 3.2. Effect of follicular size, season of the year and phase of 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 SOD activity in buffalo follicular fluid increased as follicle size decreased. It was significantly higher (P < 0.01) in small follicles than medium and large follicles as well as in medium follicles than large ones. No significant changes were detected in the overall mean of SOD activity during different seasons of the year. Meanwhile, SOD activity of different size follicles was significantly higher (P < 0.05) in follicular fluid obtained during luteal phase as compared to that obtained during follicular phase. 3.3. Effect of follicular size, season of the year and phase of 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 between the follicle size and TAC; as it was significantly higher (P < 0.01) in large follicles than medium and small follicles as well as in medium follicles than small ones. The overall mean of TAC in buffalo follicular fluid obtained
Table 2 Effect of follicular size, season of the year and the phase of the estrous cycle on superoxide dismutase (SOD) activity (U/mL) in the follicular fluid of buffaloes. Follicle size
Estrous phase
Summer
Autumn
Winter
Spring
Mean at different phases
Mean in different size follicles
Large
Follicular Luteal Follicular Luteal Follicular Luteal 43.92a ± 1.13
36.88aA ± 2.62 40.06aA ± 2.60 41.60aA ± 1.45 44.66aA ± 1.72 47.78aA ± 2.95 53.52aA ± 1.54 44.52a ± 1.00
37.48aA ± 0.93 44.93aB ± 2.48 40.90aA ± 1.21 45.60aA ± 1.44 47.82aA ± 2.42 49.75aA ± 3.51 45.46a ± 1.29
37.01aA ± 2.35 43.57aB ± 2.09 43.96aA ± 1.82 46.80aA ± 2.67 48.93aA ± 2.49 52.73aA ± 4.61 44.75a ± 1.39
38.47aA ± 1.60 40.68aA ± 3.15 41.49aA ± 2.12 46.80aA ± 2.20 48.25aA ± 3.97 53.58aA ± 4.50
37.47A ± 0.97 42.28B ± 1.28 41.94A ± 0.81 45.87B ± 0.96 48.16A ± 1.42 52.40B ± 1.80
39.88A ± 0.85
Medium Small Mean at different seasons
43.96B ± 0.67 50.18C ± 1.16
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.
Please cite this article in press as: F. Hozyen, H., et al., Seasonal changes in some oxidant and antioxidant parameters during folliculogenesis in Egyptian buffalo. Anim. Reprod. Sci. (2014), http://dx.doi.org/10.1016/j.anireprosci.2014.10.005
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Table 3 Effect 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. Follicle size
Estrous phase
Summer
Autumn
Winter
Spring
Mean at different phases
Mean in different size follicles
Large
Follicular Luteal Follicular Luteal Follicular Luteal 0.76a ± 0.022
0.87aA ± 0.033 0.91aA ± 0.013 0.74aA ± 0.033 0.75aA ± 0.035 0.60aA ± 0.031 0.62aA ± 0.031 0.77a ± 0.031
0.92aA ± 0.072 0.94aA ± 0.051 0.77abA ± 0.015 0.77aA ± 0.061 0.63aA ± 0.039 0.66aA ± 0.012 0.78ab ± 0.023
0.91aA ± 0.016 0.94aA ± 0.045 0.76abA ± 0.026 0.78aA ± 0.054 0.64aA ± 0.008 0.66aA ± 0.025 0.85b ± 0.038
0.94aA ± 0.051 0.94aA ± 0.056 0.85bA ± 0.051 0.87aA ± 0.055 0.72aA ± 0.061 0.74aA ± 0.008
0.91A ± 0.025 0.93A ± 0.022 0.78A ± 0.018 0.80A ± 0.028 0.65A ± 0.024 0.67A ± 0.029
0.92C ± 0.017
Medium Small Mean at different seasons
0.79B ± 0.016 0.66A ± 0.019
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 4 Effect of season of the year and the phase of the estrous cycle on Malondialdehyde (MDA) concentrations, superoxide dismutase (SOD) activity and total antioxidant capacity (TAC) in the serum of buffaloes. Parameters
Estrous phase
Summer
Autumn
Winter
Spring
MDA
FollicularLuteal
SOD
Overall mean FollicularLuteal
TAC
Overall mean FollicularLuteal
33.79A ± 0.86 37.20B ± 1.79 35.49b ± 1.07 40.73A ± 1.34 46.94B ± 0.87 43.84a ± 1.15 0.63A ± 0.05 0.74B ± 0.013 0.69a ± 0.031
33.50A ± 1.01 34.61A ± 1.07 34.06ab ±0.71 40.68A ± 3.11 45.85A ± 3.12 43.26a ± 2.23 0.69A ± 0.02 0.66A ± 0.043 0.68a ± 0.024
30.52A ± 1.25 33.53A ± 1.18 32.13a ± 0.92 43.81A ± 0.88 46.19A ± 1.78 45.00a ± 1.01 0.65A ± 0.025 0.63A ± 0.024 0.64a ± 0.017
31.92A ± 1.16 34.41A ± 1.94 33.17ab ± 1.09 40.76A ± 1.39 43.82A ± 1.47 42.65a ± 1.10 0.65A ± 0.04 0.64A ± 0.012 0.65a ± 0.021
Overall mean
Data are presented as means ± SE N = 8 per group. Means having different superscripts in the same column (A, B) within different parameters differ significantly. Means having different superscripts (a, b) within the overall mean of row are significantly different.
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during spring was significantly higher than that obtained during summer and autumn (P < 0.05). No statistical significance was recorded in TAC between different size follicles obtained during luteal and follicular phases. 3.4. Effect of season of the year and the phase of the estrous cycle on Malondialdehyde (MDA) concentrations, superoxide dismutase (SOD) activity and total antioxidant capacity (TAC) in the serum of buffaloes Table 4 clarifies that the overall mean of MDA level in buffalo serum was significantly higher (P < 0.05) in summer than winter. However, no significant changes were reported in the overall means of serum SOD activity and TAC during different seasons of the year. Meanwhile, in summer MDA, SOD and TAC in buffalo serum were significantly higher (P < 0.05) in luteal phase than follicular phase. 3.5. Average malondialdehyde (MDA) concentrations (nmol/mL), superoxide dismutase (SOD) activity (U/mL) and total antioxidant capacity (TAC, mM/L) in follicular fluid and serum of buffaloes As shown in Table 5, the overall mean of follicular fluid MDA level (26.92 ± 0.55) in buffalo was significantly (P < 0.05) lower than overall mean of serum MDA (33.72 ± 0.49). On the contrary, the overall mean of TAC increased significantly (P < 0.05) in follicular fluid
(0.79 ± 0.02) than serum (0.67 ± 0.02). However, there were no significant changes between follicular and serum SOD. 4. Discussion
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The results of the present study revealed that the overall means of MDA concentration and SOD activity in buffalo follicular fluid were inversely proportional with the follicle size. It was significantly higher in small follicles vs large and medium follicles and in medium follicles vs large ones. On the other hand, the TAC in buffalo follicular fluid was directly proportional with the follicle size, i.e., significantly higher in large follicles than medium and small follicles and in medium follicles than small ones. The obtained results Table 5 Average malondialdehyde (MDA) concentrations (nmol/ml), superoxide dismutase (SOD) activity (U/ml) and total antioxidant capacity (TAC, mM/L) in follicular fluid and serum of buffaloes.
Follicular fluid (n = 192) Serum (n = 64)
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MDA (nmol/ml)
SOD (U/ml)
TAC (mM/L)
26.92A ± 0.55
44.64 ± 0.60
0.79A ± 0.02
33.72B ± 0.49
43.70 ± 0.76
0.67B ± 0.02
Data are presented as means ± SE. Means having different superscripts in the same column (A, B) differ significantly between follicular fluid and serum.
Please cite this article in press as: F. Hozyen, H., et al., Seasonal changes in some oxidant and antioxidant parameters during folliculogenesis in Egyptian buffalo. Anim. Reprod. Sci. (2014), http://dx.doi.org/10.1016/j.anireprosci.2014.10.005
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are in agreement with Combelles et al. (2010) in bovine who reported that SOD activities were significantly higher in follicular fluid from small follicles than medium follicles which in turn possessed elevated MDA concentrations when compared to large follicles. In the same respect, ElShahat et al. (2013) reported that lipid peroxidation was found to be higher in the small follicles than in large and medium follicles. On the other hand, Lasota et al. (2009) did not find any significant changes in total SOD activity in porcine follicular fluid obtained from large and small follicles. While El-Shahat and Kandil (2012) did not record any significant changes in MDA concentration between different size follicles in buffaloes. Ayres et al. (1998) reported that lipid peroxidation is most often induced by O− 2 and SOD catalyzes the reaction that converts O− 2 to H2 O2 and molecular oxygen. Meanwhile, the previous authors added that E2 offer protection from lipid peroxidation by inhibiting the formation of O− 2 and was also found to interfere with the oxidative chain propagation leading to lipid peroxidation. Moreover, Agarwal et al. (2003) further added that follicles through their developmental journey become a dominant source of ROS which when overproduced in cells and tissues cause oxidative stress. In the same respect, Circu and Aw (2010) have established the relationship between oxidative stress and cell fate; as the cumulative damage from ROS and oxidative stress eventually leads to cell death. The elevated levels of lipid peroxidation in small follicles reported in the present study were associated with high activity of the free radical scavenger SOD indicating that lipid peroxidation is tightly controlled by each follicle throughout folliculogenesis and one probable mechanism for this control is enzymatic reduction by SOD (Hennet et al., 2013). Moreover, the higher levels of TAC reported in large follicles in the current study may have a role in the defense systems against oxidants, which implies (1) systems that prevent ROS generation, (2) antioxidant systems that inactivate oxidants and (3) systems that are able to limit the deleterious effects of oxidants by allowing the repair of oxidative damage (Cheeseman and Slater, 1993). The increased level of TAC in large follicles may be involved in maintaining low levels of ROS which is necessary to keep normal cell function (Gupta et al., 2011). High ambient temperature is a major limitation on buffalo and can result in impairment of production and reproduction performance (Marai and Haeeb, 2010). Regarding the effect of season on oxidant and antioxidant parameters in the follicular fluid and serum of buffalo, the current study clarifies that the overall mean of MDA level in the follicular fluid was significantly higher in summer than winter and spring, while the overall mean of TAC was significantly higher in spring than summer and autumn. The overall mean of serum MDA level in the present study was significantly higher in summer season than winter with no significant changes in the overall mean of SOD and TAC throughout the four seasons. MDA was reported to reflect the increase in lipid peroxidation after exposure to ROS (Killic et al., 2003). The increased lipid peroxidation and decreased TAC observed in the present study in summer could be related to the fact that heat stress during summer is one of the main reasons for oxidative stress resulting
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from increased production of free radicals and a decrease in antioxidant defense (Trevisan et al., 2001 and Williams et al., 2002). Although buffalo is a polyestrous animal, yet it exhibits a distinct seasonal variation as its breeding frequency is highest during winter, decreases in autumn and spring and is lowest during summer (Rensis and Scaramuzzi, 2003). In the same time, increased environmental temperature during summer was reported to be associated with increased intensity of production of ROS (Lasota et al., 2009). According to Megahed et al. (2008), heat stress associated with elevated MDA level means that fertility of buffaloes may be affected in summer through an increased production of free radicals. Similar changes in serum MDA levels were observed by Megahed et al. (2008) in buffalo and Chandra and Aggarwal (2009) in cows. Moreover, Megahed et al. (2008) found that in Egyptian buffaloes SOD activities in serum were significantly lower in the summer than winter. On the other hand, Lallawmkimi (2009) reported significantly higher SOD levels during summer compared to winter in Murrah buffaloes. Regarding the effect of estrous phase, the present study indicated that follicular fluid MDA concentration in medium follicles obtained during luteal phase and small follicles obtained during luteal and follicular phases was significantly higher in summer than other seasons. Moreover, MDA, SOD and TAC in buffalo serum were significantly higher during luteal phase than follicular phase in summer. El-Shahat and Kandil (2012) attributed the increase in MDA during luteal phase in medium and small follicles to the increase in lipid peroxidation within the plasma membrane of luteal cells, which may be associated with the observed loss of gonadotrophin receptors, decreased cAMP formation and decreased steroidogenic ability of the CL in the regression phase. In the current work, the overall means of MDA level in buffalo follicular fluid was significantly lower than that of serum. On the contrary, the overall mean of TAC was significantly higher in follicular fluid than serum with no significant changes between the overall means of follicular and serum SOD activities. Jozwik et al. (1999) reported that lipid peroxidation level in women follicular fluid is significantly lower than in blood and they attributed this gradient between follicular fluid and blood to the lower rate of initiation of peroxidation in the follicular fluid, suggestive of the presence of efficient antioxidant defense systems in the direct milieu of the oocyte. Elevated levels of TAC in follicular fluid than in serum probably may reflect the antioxidant activity of granulosa cells (Revelli et al., 2009). In conclusion the present study indicated that (a) lipid peroxidation increased in buffalo follicular fluid from small follicles and during luteal phase, (b) SOD represents the dominant antioxidant defense in small follicles and (c) high temperature during summer season associated with increased oxidative stress in both follicular fluid and serum could be related to reproductive seasonality in buffalo. Conflicts of Interest All authors declare that they do not have any conflicts of interest.
Please cite this article in press as: F. Hozyen, H., et al., Seasonal changes in some oxidant and antioxidant parameters during folliculogenesis in Egyptian buffalo. Anim. Reprod. Sci. (2014), http://dx.doi.org/10.1016/j.anireprosci.2014.10.005
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Uncited references Ganaie et al., 2013 and Machatkova et al., 2004. References Agarwal, A., Saleh, R.A., Badaiwy, M.A., 2003. Role of reactive oxygen species in the pathophysiology of human reproduction. Fertil. Steril. 79, 829–843. Ali, S., Ahmad, N., Akhtar, N., Rahman, Z.U., Noakes, D.E., 2008. Metabolite contents of blood serum and fluid from small and large sized follicles in dromedary camels during the peak and the low breeding seasons. Anim. Reprod. Sci. 108, 446–456. Ayres, S., Abplanalp, W., Liu, J.H., Ravi Subbiah, M.T., 1998. Mechanisms involved in the protective effect of estradiol-17b on lipid peroxidation and DNA damage. Am. J. Physiol. Endocrinol. Metab. 274, 1002–1008. Basini, G., Simona, B., Santini, S.E., Grasselli, F., 2008. Reactive oxygen species and anti-oxidant defences in swine follicular fluids. Reprod. Fertil. Develop. 20, 269–274. Chandra, G., Aggarwal, A., 2009. Effect of dl-␣-Tocopherol acetate on calving induced oxidative stress in periparturient crossbred cows during summer and winter seasons. Indian J. Anim. Nutr. 26, 204–210. Cheeseman, K.H., Slater, T.F., 1993. An introduction to free radical biochemistry. Brit. Med. Bull. 49, 481–493. Circu, M.L., Aw, T.Y., 2010. Reactive oxygen species, cellular redox systems, and apoptosis. Free Radic. Biol. Med. 48, 749–762. Combelles, C.M., Holick, E.A., Paolella, L.J., Walker, D.C., Wu, Q., 2010. Profiling of SOD isoenzymes in compartments of the developing bovine antral follicle. Reproduction 139, 871–881. Dominguez, M.M., 1995. Effect of body condition, reproductive status and breed on follicular population and oocyte quality in cows. Theriogenology 25, 800–805. El-Sayed, M.M., Mahmoud, M.A., El- Nahas, H.A., El-Toumy, S.A., El-Wakil, E.A., Ghareeb, M.A., 2010. Bio-guided isolation and structure elucidation of antioxidant compounds from the leaves of Ficus Sycomorus. Pharmacology online 3, 317–332. El-Shahat, K.H., Kandil, M., 2012. Antioxidant capacity of follicular fluid in relation to follicular size and stage of estrous cycle in buffaloes. Theriogenology 77, 1513–1518. El-Shahat, K.H., Abo-El Maaty, A.M., Moawad, A.R., 2013. Follicular fluid composition in relation to follicular size in pregnant and non-pregnant dromedary camels (Camelus dromedaries). Anim. Reprod. 10, 16–23. Filaire, E., Toumib, H., 2012. Reactive oxygen species and exercise on bone metabolism: friend or enemy? (Review). Joint Bone Spine 79, 341–346. Ganaie, A.H., Shanker, G., Bumla, N.A., Ghasura, R.S., Mir, N.A., 2013. Biochemical and physiological changes during thermal stress in bovines. J. Vet. Sci. Technol. 4, 1–6. Gupta, S., Choi, A., Yu, H.Y., Czerniak, S.M., Holick, E.A., Paolella, L.J., Agarwal, A., Combelles, C.M., 2011. Fluctuations in total antioxidant capacity, catalase activity, and hydrogen peroxide levels of follicular fluid during bovine folliculogenesis. Reprod. Fertil. Dev. 23, 673–680. Hennet, M.L., Yu, H.Y., Combelles, C.M., 2013. Follicular fluid hydrogen peroxide and lipid hydroperoxide in bovine antral follicles of various size, atresia, and dominance status. J. Assist. Reprod. Genet. 30, 333–340. Jaglan, P., Das, G.K., Sunil Kumar, B.V., Kumar, R., Khan, F.A., Meur, S.K., 2010. Cyclical changes in collagen concentration in relation to growth and development of buffalo corpus luteum. Vet. Res. Commun. 34, 511–518. Jozwik, M., Wolczynski, S., Szamatowicz, M., 1999. Oxidative stress markers in preovulatory follicular fluid in humans. Mol. Hum. Reprod. 5, 409–413. Khan, F.A., Das, G.K., Pande, M., Mir, R.A., Shankar, U., 2011. Changes in biochemical composition of follicular fluid during reproductive acyclicity in water buffalo (Bubalus bubalis). Anim. Reprod. Sci. 127, 38–42.
Killic, E., Yazar, S., Saraymen, R., HOzbilge, H., 2003. Serum Malondialdehyde level in patients infected with Ascaris lumbricoides. World J. Gastroentrol. 9, 2332–2334. Kor, N.M., Moradi, K., 2013. A review of biochemical metabolites concentration and hormonal composition of ovarian follicular fluid in domestic animals. Annu. Rev. Res. Biol. 3, 246–255. Koracevic, D., Koracevic, G., Djordjevic, V.S., Andrejevic, S., Cosic, V.V., 2001. Method for the measurement of antioxidant activity in human fluids. J. Clin. Pathol. 54, 356–361. Lallawmkimi, C.M., 2009. Impact of Thermal Stress and Vitamin-E Supplementation on Heat Shock Protein 72 and Antioxidant Enzymes in Murrah Buffaloes. NDRI deemed University, Karnal (Haryana), India, Ph.D. Thesis. Lasota, B., Błaszczyk, B., Stankiewicz, T., Udała, J., Szewczyk, M., MatusiakBielska, D., 2009. Superoxide dismutase and glutathione peroxidase activity in porcine follicular fluid in relation to follicle size, birth status of gilts, ovarian location and year season. Acta Sci. Pol. Zootechnica 8, 19–30. Machatkova, M., Krausova, K., Jokesova, E., Tomanek, M., 2004. Developmental competence of bovine oocytes: effects of follicle size and the phase of follicular wave on in vitro embryo production. Theriogenology 61, 329–335. Marai, I.F., Haeeb, A.A., 2010. Buffalo’s biological functions as affected by heat stress. Livest. Sci. 127, 89–109. Megahed, G.A., Anwar, M.M., Wasfi, S.I., Hammadeh, M.F., 2008. Influence of heat stress on the cortisol and oxidant-antioxidants balance during oestrous phase in buffalo-cows (Bubalus Bubalis): themo-protective role of antioxidant treatment. Repod. Domest. Anim. 43, 672–677. Mondal, S., Kumar, V., Reddy, I.J., Khub, S., 2004. Progesterone and nucleic acid content of buffalo corpus luteum in relation to stages of estrus cycle. Indian J. Anim. Sci. 74, 710–712. Nandi, S., Girish Kumar, V., Manjunatha, B.M., Ramesh, H.S., Gupta, P.S., 2008. Follicular fluid concentrations of glucose, lactate and pyruvate in buffalo and sheep, and their effects on cultured oocytes, granulosa and cumulus cells. Theriogenology 69, 186–196. Nishikimi, M., Rao, A., Yagi, K., 1972. The occurrence of superoxide anion in the reaction of reduced phenazine methosulphate and molecular oxygen. Biochem. Biophys. Res. Commun. 46, 849–854. Nour El-Din, M. M. 2013. Proposed climate change adaptation strategy for the ministry of water resources & irrigation in Egypt. Joint programme for climate change risk management in Egypt. Ohkawa, H., Ohishi, W., Yagi, K., 1979. Assay for lipid peroxides in animal tissues by thiobarbituric acid reaction. Anal. Biochem. 95, 351–358. Rensis, F.D., Scaramuzzi, R.J., 2003. Heat stress and seasonal effects on reproduction in the dairy cow. Theriogenology 60, 1139–1151. Revelli, A., Piane, L.D., Casano, S., Molinari, E., Massobrio, M., Rinaudo, P., 2009. Follicular fluid content and oocyte quality: from single biochemical markers to metabolomics. Reprod. Biol. Endocrinol. 7, 40–52. Sharma, R.K., Reynolds, N., Rakhit, M., Agarwal, A., 2013. Methods for Detection of ROS in the Female Reproductive System. In: Agarwal, A., Aziz, N., Rizk, B. (Eds.), Studies on Women’s Health, Oxidative Stress in Applied Basic Research and Clinical Practice. Springer Science and Business Media, New York, pp. 33–60. Silanikove, N., Maltz, E., Halevi, A., Shinder, D., 1997. Metabolism of water, sodium, potassium and chloride by high yielding dairy cows at the onset of lactation. J. Dairy Sci. 80, 949–956. Terzano, G.M., Barile, V.L., Borghese, A., 2012. Overview on reproductive endocrine aspects in buffalo. J. Buffalo Sci. 1, 126–138. Trevisan, M., Browne, R., Ram, M., Muti, P., Freudenheim, J., Carosella, A.M., Armstrong, D., 2001. Correlates of markers of oxidative status in the general population. Am. J. Epidemiol. 154, 348–356. Williams, C.A., Kronfeld, D.S., Hess, T.M., Saker, K.E., Waldron, J.N., Crandell, K.M., Hoffman, R.M., Harris, P.A., 2002. Antioxidant supplementation and subsequent oxidative stress of horses during an 80-km endurance race. J. Anim. Sci. 82, 588–594. Zhang, X., Li, X.H., Ma, X., Wang, Z.H., Lu, S., Guo, Y.L., 2006. Redox-induced apoptosis of human oocytes in resting follicles in vitro. J. Soc. Gynecol. Invest. 13, 451–458.
Please cite this article in press as: F. Hozyen, H., et al., Seasonal changes in some oxidant and antioxidant parameters during folliculogenesis in Egyptian buffalo. Anim. Reprod. Sci. (2014), http://dx.doi.org/10.1016/j.anireprosci.2014.10.005
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Contents lists available at ScienceDirect
Animal Reproduction Science journal homepage: www.elsevier.com/locate/anireprosci
Seasonal changes in some oxidant and antioxidant parameters during folliculogenesis in Egyptian buffalo夽
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Heba F. Hozyen a,∗ , Hodallah H. Ahmed b , G.E.S. Essawy b , S.I.A. Shalaby a a b
Dept. Reproduction and A. I., NRC, Egypt Dept. Physiology, Cairo University, Egypt
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Article history: Received 21 January 2014 Received in revised form 1 October 2014 Accepted 5 October 2014 Available online xxx
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Keywords: Seasonal change Follicular size Lipid peroxidation SOD TAC Buffalo
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1. Introduction
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Knowledge regarding oxidant and antioxidant status in follicular fluid remains limited and its studying in vivo should enhance our understanding of the impact of them on fertility and contribute to optimization of in vitro maturation conditions. The present study was conducted on follicular fluid and serum samples obtained from 708 buffaloes. They were examined for Malondialdehyde (MDA) as indicator of lipid peroxidation as well as superoxide dismutase (SOD) and total antioxidant capacity (TAC) as antioxidant markers. The obtained results revealed that MDA levels and SOD activity in follicular fluid decreased significantly as follicle size increased, while TAC increased significantly with the increase in follicular size. Whereas MDA level was significantly higher in summer, the TAC was significantly higher in spring. Moreover, MDA levels and SOD activities were significantly higher in the follicular fluid from different size follicles during the luteal phase than follicular phase. MDA levels in medium follicles in luteal phase and small follicles in follicular and luteal phases were significantly higher in summer than other seasons. Serum MDA levels were significantly increased in summer. In addition, MDA levels, SOD activities and TAC in serum were significantly higher during luteal phase than follicular phase in summer. TAC levels were significantly higher in follicular fluid than serum, while MDA was significantly lower in follicular fluid than serum. In conclusion, the present study revealed that oxidants/antioxidants balance may play a role in normal follicular development and oxidative stress that occur in summer could be related to reproductive seasonality in buffalo. © 2014 Published by Elsevier B.V.
Buffalo is an important world-wide species in terms of milk and meat production (Terzano et al., 2012). Within the ovarian follicle, the developing oocyte is surrounded
夽 This paper is part of the special issue entitled: 4th Mammalian Embryo Genomics meeting, October 2013, Quebec City. ∗ Corresponding author. Animal Reproduction and AI dept., Veterinary Division, National Research Centre, Dokki, Giza, Egypt. Tahrir street, Dokki, Giza Postal code: 12622. Tel.: +20 1221271525. E-mail address: [email protected] (H. F. Hozyen).
by follicular fluid which is mainly derived from blood besides the locally produced substances (Nandi et al., 2008). Metabolic activity of follicular fluid, together with the ‘barrier’ properties of the follicular wall, is changing significantly during the growth and expansion of each follicle (Khan et al., 2011) and with the phase of estrous cycle (Kor and Moradi, 2013). Follicular fluid microenvironment can be regarded as a biological window providing a valuable insight into the process of normal follicular development as well as the pathogenesis of some reproductive problems in buffaloes (El-Shahat and Kandil, 2012). Heat stress has been of major concern in reducing buffaloes’
http://dx.doi.org/10.1016/j.anireprosci.2014.10.005 0378-4320/© 2014 Published by Elsevier B.V.
Please cite this article in press as: F. Hozyen, H., et al., Seasonal changes in some oxidant and antioxidant parameters during folliculogenesis in Egyptian buffalo. Anim. Reprod. Sci. (2014), http://dx.doi.org/10.1016/j.anireprosci.2014.10.005
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productivity in tropical and sub-tropical areas (Silanikove et al., 1997). According to Nour El-Din (2013), summer tempera41 tures in Egypt are extremely high, reaching 38 ◦ C to 43 ◦ C. 42 Based on the historical records over a period of twelve 43 years (1999–2010), the subtropical climate in Cairo is 44 characterized by hot summer season (June–August) with 45 averages 23 ◦ C–35 ◦ C of minimum and maximum temper46 atures and 74% mean temperature humidity index (THI%). 47 The optimum conditions for buffaloes as suggested by 48 ◦ 49Q2 Payne (1990) are 13–18 C and in terms of the mean temperature–humidity index (THI); values of THI > 72 is 50 considered as stressful and THI > 78 is considered very 51 severe heat stress to this animal. 52 The ovarian follicles contain their own potential sources 53 of reactive oxygen species (ROS) which are potent stim54 ulators of lipid peroxidation (Filaire and Toumib, 2012). 55 However, ROS must be continuously inactivated by antiox56 idants to keep the oxidant/antioxidant balance to maintain 57 normal cell function (Basini et al., 2008). Moreover, lipid 58 peroxidation is most often induced by superoxide radical 59 (O− 2 ) and its damage is mainly inhibited by SOD which 60 is an enzyme that contributes to the first line of antiox61 idant pathway as it removes the O− 2 , repairs cells and 62 reduces the damage done to them by superoxide, the 63 most common free radical in the body (Ayres et al., 1998). 64 Total antioxidant capacity (TAC) covers both enzymatic and 65 non-enzymatic antioxidants (Gupta et al., 2011) and its 66 measuring considers the cumulative effect of all antiox67 idants present in plasma and body fluids (Sharma et al., 68 2013). 69 El-Sayed et al. (2010) reported that the antioxidant 70 status can be considered as one determinant of repro71 ductive function in bovine. The balance between ROS and 72 antioxidants may have an important role in reproductive 73 processes such as follicular development (Zhang et al., 74 2006). Megahed et al. (2008) stated that heat stress may 75 affect fertility of animals through increased production of 76 free radicals and according to Lasota et al. (2009), high 77 levels of SOD are needed to neutralize increased lipid per78 oxidation in follicles that occurs in summer. The current 79 study aimed to investigate the pattern of lipid peroxidation 80 and two relevant antioxidant markers in follicular fluid of 81 different size ovarian follicles and serum of buffalo consid82 ering the effect of seasonal changes and phase of estrous 83 cycle. 84 39
2.2. Experimental design
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2. Materials and methods
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2.1. Ovaries
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Ovaries were collected from 708 non-pregnant female buffaloes (Egyptian breed) in good health and with clinically normal reproductive tracts from local slaughterhouse located near to Cairo (Bahtim abattoir, Al-Qaliobia, Egypt). Immediately after slaughtering, both ovaries from each animal were collected in plastic bags containing 0.9% NaCl and transported in ice tank to be inspected at the laboratory.
Follicles were collected over one year during different seasons. The averages of minimum and maximum temperatures in summer (June–August) were 22 ◦ C–35 ◦ C with 56.0% mean relative humidity (RH), in autumn (September–November) 18 ◦ C–28 ◦ C with 60.3% RH, in winter (December–February) 10 ◦ C–20 ◦ C, with 58.0% RH, in spring (March–May) 15 ◦ C–28 ◦ C with 48.7% RH. The stage of estrous cycle (follicular or luteal) was identified according to the presence or absence of the corpus luteum on the ovary according to Jaglan et al. (2010) and Mondal et al. (2004). Follicular diameter was measured using a caliper and follicles were divided into three categories: small (≥3 mm), medium (4–9 mm) and large (≥10 mm) according to Dominguez (1995). Ovaries with cystic follicles as well as the morphologically atretic follicles, identified macroscopically by their opaque wall (Ali et al., 2008) were excluded from the study. 2.3. Sampling 2.3.1. Follicular fluid The contents of the ovarian follicles of different size (small, medium and large) were aspirated using a 10 ml syringe attached to an 18 gauge needle and centrifuged at 3000 rpm for 10 min for separation of the fluid from the cell fraction. Follicular fluids from each group from each pair of the ovaries were pooled in one sample for each individual buffalo. No sample pooling was needed for the large size category. Collected follicular fluid samples were kept at −20 ◦ C until analysis. 2.3.2. Blood Samples were collected during slaughtering for serum separation after centrifugation at 3000 rpm for 10 min. Collected serum samples were kept at −20 ◦ C until analysis. 2.4. Measured parameters 2.4.1. MDA MDA level was determined colorimetrically according to the method of Ohkawa et al. (1979) using kits purchased from Biodiagnostic, Egypt. 2.4.2. SOD SOD activity was estimated kinetically using SOD assay kits (Biodiagnostic, Egypt) according to Nishikimi et al. (1972). 2.4.3. TAC TAC level was determined colorimetrically according to the method of Koracevic et al. (2001) using kits purchased from Biodiagnostic, Egypt. 2.5. Statistical analysis The differences among different follicular sizes and seasons of the year were analyzed statistically by oneway ANOVA. The differences between follicular and luteal phases as well as between the average concentrations of
Please cite this article in press as: F. Hozyen, H., et al., Seasonal changes in some oxidant and antioxidant parameters during folliculogenesis in Egyptian buffalo. Anim. Reprod. Sci. (2014), http://dx.doi.org/10.1016/j.anireprosci.2014.10.005
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Table 1 Effect of follicular size, season of the year and the phase of the estrous cycle on Malondialdehyde (MDA) concentrations (nmol/mL) in the follicular fluid of buffaloes. Follicle size
Estrous phase
Summer
Autumn
Winter
Spring
Mean at different phases
Mean in different size follicles
Large
Follicular Luteal Follicular Luteal Follicular Luteal 29.93b ± 1.03
21.54aA ± 1.48 26.54aA ± 2.10 26.29aA ± 1.97 33.82bB ± 1.21 33.21bA ± 0.66 38.57bB ± 0.40 27.20ab ± 1.23
18.47aA ± 1.11 21.93aA ± 1.74 25.06aA ± 1.26 28.48abA ± 2.22 31.57abA ± 2.01 38.25bB ± 2.53 25.11a ± 1.08
17.19aA ± 1.76 25.02aB ± 2.56 24.50aA ± 0.14 25.72aA ± 1.92 27.27aA ± 1.48 34.31aB ± 2.13 24.98a ± 0.90
19.49aA ± 1.59 21.75aA ± 1.12 22.98aA ± 1.47 28.57abB ± 1.93 28.09aA ± 1.38 34.40aB ± 1.25
19.28A ± 0.75 23.93B ± 1.023 24.52A ± 0.78 29.45B ± 1.013 29.82A ± 0.86 37.06B ± 0.98
21.64A ± 0.69
Medium Small Mean at different seasons
27.32B ± 0.74 33.04C ± 0.81
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.
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different measured parameters in follicular fluid and serum were analyzed by independent samples t-test using SPSS 16.0 for windows. Means were compared by the least significance difference at 5% level of probability.
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3.1. Effect of follicular size, season of the year and phase of the estrous cycle on Malondialdehyde (MDA) concentrations (nmol/mL) in the follicular fluid of buffaloes Table 1 shows that the overall mean of MDA level was indirectly proportional with follicle size; it was significantly higher (P < 0.01) in small follicles vs medium and large follicles as well as in medium follicles vs large ones. In the same time, the overall mean of MDA level in buffalo follicular fluid was significantly higher (P < 0.05) in summer than winter and spring. Moreover, the overall mean of MDA level in follicles obtained during luteal phase was significantly higher (P < 0.05) than that obtained during follicular phase. Meanwhile, the effect of season on different size follicles clarifies that in summer MDA level was significantly higher (P < 0.05) in the follicular fluid of small and medium follicles obtained during luteal phase as well as in small follicles obtained during follicular phase. However, no significant changes were observed in MDA level in
large follicles obtained during follicular and luteal phases throughout the seasons of the year. 3.2. Effect of follicular size, season of the year and phase of 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 SOD activity in buffalo follicular fluid increased as follicle size decreased. It was significantly higher (P < 0.01) in small follicles than medium and large follicles as well as in medium follicles than large ones. No significant changes were detected in the overall mean of SOD activity during different seasons of the year. Meanwhile, SOD activity of different size follicles was significantly higher (P < 0.05) in follicular fluid obtained during luteal phase as compared to that obtained during follicular phase. 3.3. Effect of follicular size, season of the year and phase of 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 between the follicle size and TAC; as it was significantly higher (P < 0.01) in large follicles than medium and small follicles as well as in medium follicles than small ones. The overall mean of TAC in buffalo follicular fluid obtained
Table 2 Effect of follicular size, season of the year and the phase of the estrous cycle on superoxide dismutase (SOD) activity (U/mL) in the follicular fluid of buffaloes. Follicle size
Estrous phase
Summer
Autumn
Winter
Spring
Mean at different phases
Mean in different size follicles
Large
Follicular Luteal Follicular Luteal Follicular Luteal 43.92a ± 1.13
36.88aA ± 2.62 40.06aA ± 2.60 41.60aA ± 1.45 44.66aA ± 1.72 47.78aA ± 2.95 53.52aA ± 1.54 44.52a ± 1.00
37.48aA ± 0.93 44.93aB ± 2.48 40.90aA ± 1.21 45.60aA ± 1.44 47.82aA ± 2.42 49.75aA ± 3.51 45.46a ± 1.29
37.01aA ± 2.35 43.57aB ± 2.09 43.96aA ± 1.82 46.80aA ± 2.67 48.93aA ± 2.49 52.73aA ± 4.61 44.75a ± 1.39
38.47aA ± 1.60 40.68aA ± 3.15 41.49aA ± 2.12 46.80aA ± 2.20 48.25aA ± 3.97 53.58aA ± 4.50
37.47A ± 0.97 42.28B ± 1.28 41.94A ± 0.81 45.87B ± 0.96 48.16A ± 1.42 52.40B ± 1.80
39.88A ± 0.85
Medium Small Mean at different seasons
43.96B ± 0.67 50.18C ± 1.16
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.
Please cite this article in press as: F. Hozyen, H., et al., Seasonal changes in some oxidant and antioxidant parameters during folliculogenesis in Egyptian buffalo. Anim. Reprod. Sci. (2014), http://dx.doi.org/10.1016/j.anireprosci.2014.10.005
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Table 3 Effect 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. Follicle size
Estrous phase
Summer
Autumn
Winter
Spring
Mean at different phases
Mean in different size follicles
Large
Follicular Luteal Follicular Luteal Follicular Luteal 0.76a ± 0.022
0.87aA ± 0.033 0.91aA ± 0.013 0.74aA ± 0.033 0.75aA ± 0.035 0.60aA ± 0.031 0.62aA ± 0.031 0.77a ± 0.031
0.92aA ± 0.072 0.94aA ± 0.051 0.77abA ± 0.015 0.77aA ± 0.061 0.63aA ± 0.039 0.66aA ± 0.012 0.78ab ± 0.023
0.91aA ± 0.016 0.94aA ± 0.045 0.76abA ± 0.026 0.78aA ± 0.054 0.64aA ± 0.008 0.66aA ± 0.025 0.85b ± 0.038
0.94aA ± 0.051 0.94aA ± 0.056 0.85bA ± 0.051 0.87aA ± 0.055 0.72aA ± 0.061 0.74aA ± 0.008
0.91A ± 0.025 0.93A ± 0.022 0.78A ± 0.018 0.80A ± 0.028 0.65A ± 0.024 0.67A ± 0.029
0.92C ± 0.017
Medium Small Mean at different seasons
0.79B ± 0.016 0.66A ± 0.019
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 4 Effect of season of the year and the phase of the estrous cycle on Malondialdehyde (MDA) concentrations, superoxide dismutase (SOD) activity and total antioxidant capacity (TAC) in the serum of buffaloes. Parameters
Estrous phase
Summer
Autumn
Winter
Spring
MDA
FollicularLuteal
SOD
Overall mean FollicularLuteal
TAC
Overall mean FollicularLuteal
33.79A ± 0.86 37.20B ± 1.79 35.49b ± 1.07 40.73A ± 1.34 46.94B ± 0.87 43.84a ± 1.15 0.63A ± 0.05 0.74B ± 0.013 0.69a ± 0.031
33.50A ± 1.01 34.61A ± 1.07 34.06ab ±0.71 40.68A ± 3.11 45.85A ± 3.12 43.26a ± 2.23 0.69A ± 0.02 0.66A ± 0.043 0.68a ± 0.024
30.52A ± 1.25 33.53A ± 1.18 32.13a ± 0.92 43.81A ± 0.88 46.19A ± 1.78 45.00a ± 1.01 0.65A ± 0.025 0.63A ± 0.024 0.64a ± 0.017
31.92A ± 1.16 34.41A ± 1.94 33.17ab ± 1.09 40.76A ± 1.39 43.82A ± 1.47 42.65a ± 1.10 0.65A ± 0.04 0.64A ± 0.012 0.65a ± 0.021
Overall mean
Data are presented as means ± SE N = 8 per group. Means having different superscripts in the same column (A, B) within different parameters differ significantly. Means having different superscripts (a, b) within the overall mean of row are significantly different.
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during spring was significantly higher than that obtained during summer and autumn (P < 0.05). No statistical significance was recorded in TAC between different size follicles obtained during luteal and follicular phases. 3.4. Effect of season of the year and the phase of the estrous cycle on Malondialdehyde (MDA) concentrations, superoxide dismutase (SOD) activity and total antioxidant capacity (TAC) in the serum of buffaloes Table 4 clarifies that the overall mean of MDA level in buffalo serum was significantly higher (P < 0.05) in summer than winter. However, no significant changes were reported in the overall means of serum SOD activity and TAC during different seasons of the year. Meanwhile, in summer MDA, SOD and TAC in buffalo serum were significantly higher (P < 0.05) in luteal phase than follicular phase. 3.5. Average malondialdehyde (MDA) concentrations (nmol/mL), superoxide dismutase (SOD) activity (U/mL) and total antioxidant capacity (TAC, mM/L) in follicular fluid and serum of buffaloes As shown in Table 5, the overall mean of follicular fluid MDA level (26.92 ± 0.55) in buffalo was significantly (P < 0.05) lower than overall mean of serum MDA (33.72 ± 0.49). On the contrary, the overall mean of TAC increased significantly (P < 0.05) in follicular fluid
(0.79 ± 0.02) than serum (0.67 ± 0.02). However, there were no significant changes between follicular and serum SOD. 4. Discussion
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The results of the present study revealed that the overall means of MDA concentration and SOD activity in buffalo follicular fluid were inversely proportional with the follicle size. It was significantly higher in small follicles vs large and medium follicles and in medium follicles vs large ones. On the other hand, the TAC in buffalo follicular fluid was directly proportional with the follicle size, i.e., significantly higher in large follicles than medium and small follicles and in medium follicles than small ones. The obtained results Table 5 Average malondialdehyde (MDA) concentrations (nmol/ml), superoxide dismutase (SOD) activity (U/ml) and total antioxidant capacity (TAC, mM/L) in follicular fluid and serum of buffaloes.
Follicular fluid (n = 192) Serum (n = 64)
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MDA (nmol/ml)
SOD (U/ml)
TAC (mM/L)
26.92A ± 0.55
44.64 ± 0.60
0.79A ± 0.02
33.72B ± 0.49
43.70 ± 0.76
0.67B ± 0.02
Data are presented as means ± SE. Means having different superscripts in the same column (A, B) differ significantly between follicular fluid and serum.
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are in agreement with Combelles et al. (2010) in bovine who reported that SOD activities were significantly higher in follicular fluid from small follicles than medium follicles which in turn possessed elevated MDA concentrations when compared to large follicles. In the same respect, ElShahat et al. (2013) reported that lipid peroxidation was found to be higher in the small follicles than in large and medium follicles. On the other hand, Lasota et al. (2009) did not find any significant changes in total SOD activity in porcine follicular fluid obtained from large and small follicles. While El-Shahat and Kandil (2012) did not record any significant changes in MDA concentration between different size follicles in buffaloes. Ayres et al. (1998) reported that lipid peroxidation is most often induced by O− 2 and SOD catalyzes the reaction that converts O− 2 to H2 O2 and molecular oxygen. Meanwhile, the previous authors added that E2 offer protection from lipid peroxidation by inhibiting the formation of O− 2 and was also found to interfere with the oxidative chain propagation leading to lipid peroxidation. Moreover, Agarwal et al. (2003) further added that follicles through their developmental journey become a dominant source of ROS which when overproduced in cells and tissues cause oxidative stress. In the same respect, Circu and Aw (2010) have established the relationship between oxidative stress and cell fate; as the cumulative damage from ROS and oxidative stress eventually leads to cell death. The elevated levels of lipid peroxidation in small follicles reported in the present study were associated with high activity of the free radical scavenger SOD indicating that lipid peroxidation is tightly controlled by each follicle throughout folliculogenesis and one probable mechanism for this control is enzymatic reduction by SOD (Hennet et al., 2013). Moreover, the higher levels of TAC reported in large follicles in the current study may have a role in the defense systems against oxidants, which implies (1) systems that prevent ROS generation, (2) antioxidant systems that inactivate oxidants and (3) systems that are able to limit the deleterious effects of oxidants by allowing the repair of oxidative damage (Cheeseman and Slater, 1993). The increased level of TAC in large follicles may be involved in maintaining low levels of ROS which is necessary to keep normal cell function (Gupta et al., 2011). High ambient temperature is a major limitation on buffalo and can result in impairment of production and reproduction performance (Marai and Haeeb, 2010). Regarding the effect of season on oxidant and antioxidant parameters in the follicular fluid and serum of buffalo, the current study clarifies that the overall mean of MDA level in the follicular fluid was significantly higher in summer than winter and spring, while the overall mean of TAC was significantly higher in spring than summer and autumn. The overall mean of serum MDA level in the present study was significantly higher in summer season than winter with no significant changes in the overall mean of SOD and TAC throughout the four seasons. MDA was reported to reflect the increase in lipid peroxidation after exposure to ROS (Killic et al., 2003). The increased lipid peroxidation and decreased TAC observed in the present study in summer could be related to the fact that heat stress during summer is one of the main reasons for oxidative stress resulting
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from increased production of free radicals and a decrease in antioxidant defense (Trevisan et al., 2001 and Williams et al., 2002). Although buffalo is a polyestrous animal, yet it exhibits a distinct seasonal variation as its breeding frequency is highest during winter, decreases in autumn and spring and is lowest during summer (Rensis and Scaramuzzi, 2003). In the same time, increased environmental temperature during summer was reported to be associated with increased intensity of production of ROS (Lasota et al., 2009). According to Megahed et al. (2008), heat stress associated with elevated MDA level means that fertility of buffaloes may be affected in summer through an increased production of free radicals. Similar changes in serum MDA levels were observed by Megahed et al. (2008) in buffalo and Chandra and Aggarwal (2009) in cows. Moreover, Megahed et al. (2008) found that in Egyptian buffaloes SOD activities in serum were significantly lower in the summer than winter. On the other hand, Lallawmkimi (2009) reported significantly higher SOD levels during summer compared to winter in Murrah buffaloes. Regarding the effect of estrous phase, the present study indicated that follicular fluid MDA concentration in medium follicles obtained during luteal phase and small follicles obtained during luteal and follicular phases was significantly higher in summer than other seasons. Moreover, MDA, SOD and TAC in buffalo serum were significantly higher during luteal phase than follicular phase in summer. El-Shahat and Kandil (2012) attributed the increase in MDA during luteal phase in medium and small follicles to the increase in lipid peroxidation within the plasma membrane of luteal cells, which may be associated with the observed loss of gonadotrophin receptors, decreased cAMP formation and decreased steroidogenic ability of the CL in the regression phase. In the current work, the overall means of MDA level in buffalo follicular fluid was significantly lower than that of serum. On the contrary, the overall mean of TAC was significantly higher in follicular fluid than serum with no significant changes between the overall means of follicular and serum SOD activities. Jozwik et al. (1999) reported that lipid peroxidation level in women follicular fluid is significantly lower than in blood and they attributed this gradient between follicular fluid and blood to the lower rate of initiation of peroxidation in the follicular fluid, suggestive of the presence of efficient antioxidant defense systems in the direct milieu of the oocyte. Elevated levels of TAC in follicular fluid than in serum probably may reflect the antioxidant activity of granulosa cells (Revelli et al., 2009). In conclusion the present study indicated that (a) lipid peroxidation increased in buffalo follicular fluid from small follicles and during luteal phase, (b) SOD represents the dominant antioxidant defense in small follicles and (c) high temperature during summer season associated with increased oxidative stress in both follicular fluid and serum could be related to reproductive seasonality in buffalo. Conflicts of Interest All authors declare that they do not have any conflicts of interest.
Please cite this article in press as: F. Hozyen, H., et al., Seasonal changes in some oxidant and antioxidant parameters during folliculogenesis in Egyptian buffalo. Anim. Reprod. Sci. (2014), http://dx.doi.org/10.1016/j.anireprosci.2014.10.005
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