2-Hydroxy-4-Methylselenobutanoic Acid (HMSeBA) Promote Follicle Development By Antioxidant Pathway in Gilts


 Background Dietary 2-hydroxy-4-methylselenobutanoic acid (HMSeBA) supplementation can exert antioxidant effects in poultry, pigs and weaned pigs. However, it is unknown whether HMSeBA could improve the development of follicle by anti-oxidize effects in gilt. This study was conducted to evaluate the effects of dietary HMSeBA supplementation on the follicle development in gilt. A total of 36 gilts were randomly fed the control diet (CON, negative control), Na2SeO3 diet containing 0.3 mg Se/kg (positive control) or the HMSeBA diet containing 0.3 mg Se/kg from weaning to the 19th day after the second estrus. In another study, the effect of HMSeBA on the cells viability, proliferation, release of 17βestradiol (E2 ) and antioxidant capacity were investigated in the mouse ovarian granulosa cells in vitro. Results Results showed that HMSeBA group increased the average daily body weight gain (ADG) and decreased the ratio of feed: gain during day 120 to 176 in gilts ( P < 0.05). The selenium (HMSeBA and Na 2 SeO 3 ) increased the weight of uterine at the third estrus. There was no effect of HMSeBA on the number of large follicles (diameter >5mm), but HMSeBA decreased the gene expression of growth differentiation factor-9 ( GDF-9 ) and bone morphogenetic protein-15 ( BMP-15 ) in cumulus-oocyte complexes (COCs). HMSeBA group increased the total selenium content in serum ( P < 0.05) and liver ( P < 0.01) and tended to increase the total selenium content in ovary ( P = 0.08). HMSeBA group decreased the malondialdehyde (MDA) concentration in the serum, liver and ovary ( P < 0.05), increased the total antioxidant capacity (T-AOC) in the liver, thioredoxin reductase (TrxR) in the ovary ( P < 0.05) and increased the activity of GPx in the serum, liver and ovary ( P < 0.05). Na 2 SeO 3 supplementation decreased MDA and increased the T-AOC in liver, increased the T-SOD and TrxR in the ovary compared with control. At the transcription level, HMSeBA group increased the glutathione peroxidase 2 ( GPx2 ) and TrxR1 ( P < 0.05) expression in the liver, and increased the GPx1 expression ( P < 0.05) in the ovary of gilts compared with Na2SeO3 treatment. Besides, HMSeBA group increased the expressions of superoxide dismutase 1 ( SOD1 ) and Thioredoxin l ( Trx1 ) in the liver. In vitro experiment, HMSeBA improved granulosa cells’ proliferation and E2 secretion ( P < 0.05). HMSeBA and Na 2 SeO 3 both increased the T-AOC and decreased MDA in granulosa cells in vitro. Meanwhile, HMSeBA increased T-SOD, GPx, glutathione reductase (GR) and TrxR activity in granulosa cells in vitro. In addition, HMSeBA up-regulated SOD2 and GPx1 gene expression in the granulosa cells in vitro.Conclusion These results demonstrate directly, HMSeBA was more conducive to absorption and storage of selenium in the liver and ovary in gilt, and beneficial to exert the effect of HMSeBA on the antioxidant function in the liver and ovary of gilt. Moreover, HMSeBA has stronger antioxidant capacity in granular cells in vitro , which is more conducive to promoting follicle development. Therefore, the new type of organic selenium, HMSeBA, could be potentially useful for the control of reproductive processes in gilt.

of gilts compared with Na2SeO3 treatment. Besides, HMSeBA group increased the expressions of

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Ovarian follicular development is the most important event in the gilt. Ovarian follicular 57 development is dependent on the proliferation and differentiation of the granulosa cells (GCs) [1]. 17β-58 estradiol (E2) is synthesized from androgen in granulose cell via the aromatization by cytochrome P450 59 aromatase. E2 represents one of the key ovarian hormones produced by the developing ovulatory follicle, 60 and is reflecting the differentiation of ovarian granulose cell. E2 is crucial for female reproduction, as 61 proved by the severe fertility defects when its synthesis or action are suppressed [2,3]. With the 62 metabolism of ovarian, the reactive oxygen species (ROS) and free radicals would be generated [4].

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Those metabolites must be neutralized locally to maintain tissue integrity and function. The antioxidants 64 such as vitamins C and the Se-dependent glutathione peroxidase (GPx) system would protect against 65 ROS and free radicals.

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Animals and diets 98 treatment groups as follows: 1) control diet (CON, negative control, basal diet, n = 6, 6 repeatments in 100 each treatment, 2 gilts/repeatment), 2) sodium selenite (Na2SeO3) supplemented diet (Na2SeO3, positive 101 control, basal diet + Na2SeO3 at 0.3 mg Se/kg, n = 6), 3) hydroxy-analogue of selenomethionine 102 supplemented diet (HMSeBA, Selisseo® 2% Se provided by Adisseo France, basal diet + HMSeBA at 103 0.3 mg Se/kg, n = 6). Control (CON) diet was corn-soybean meal-based, which was formulated according 104 to the nutrient requirements recommended by the National Research Council (NRC, 2012) except 105 selenium ( Table 1). The experiment was terminated at the 19th day after the second estrus. Gilts were 106 fed four times a day from weaning to 90 days of age (08:00; 12:00; 16:00; 20:00), and from 90 days to 107 slaughter, they were fed twice a day (08: 00; 16:00). Gilts were feed freely before 176 days of age, the 108 feed limited 2.5 kg/d/gilt from 176 days of age to slaughter. After 176 days of age, gilts were exposed to 109 a rotation of mature boars twice a day (08:00; 16:00). Estrous detection was carefully conducted by an  Gilts (n=5/treatment) were slaughtered on the morning of the 19th day after the second estrus after 114 fasting for 12 hours. Before slaughter, 10 mL blood sample was collected by acute jugular puncture to 115 obtain the serum. The serum was stored at -20 °C for further analysis.

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After the gilt was slaughtered, the liver and the reproductive tract were dissected. The liver sample 117 from the left side were collected from each gilt and rapidly frozen in liquid nitrogen then stored at -80°C.

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The right and left ovaries were separated from the uterine horns and weighed. The uterus was 119 subsequently trimmed of mesentery and weighed. All the surface antral follicles greater than 1 mm in 120 diameter were individually measured and recorded within different size categories (small: 1-5 mm in diameter and large: >5 mm in diameter). Meanwhile, the number of corpora lutea was recorded. Then, 122 the follicular content of left ovary was aspirated from all the follicles with >5 mm in diameter using a 123 10-mL syringe equipped with an 18-gauge needle. Following aspiration, cumulus-oocyte complexes 124 (COCs) were recovered from the aspirate using a dissecting microscope (40 × magnification). Then the 125 remaining follicular contents were centrifuged for 5 minutes at 2000 rpm. The supernatant (follicular 126 fluid) was collected and stored at -20°C until analysis was conducted.

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GCs were cultured with different concentration of Na2SeO3 or HMSeBA. After cultured 48hrs with 164 once media change, the media was collected. The spent media were assayed for the presence of estradiol by ELISA kit (R&D Systems Inc., Minneapolis, MN, USA) according to the manufacturer's protocol.

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Each experiment was carried out in triplicate in vitro experiment.

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The results are presented as mean ± SE. P < 0.05 was considered as a statistically significant difference.

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The average daily body weight gain (ADG) (P < 0.05) was greater in gilts receiving HMSeBA 205 during day 148 to 176 and day 120 to 176. The average daily feed intake (ADFI) of gilts in HMSeBA 206 group had lower tendency than that in Na2SeO3 and control groups (P = 0.05). The ratio of feed: gain was 207 lower in HMSeBA group than that in control group during day 148 to 176 and day 120 to 176. Body 208 weight at puberty was lower in the Na2SeO3 group than in the HMSeBA and control group (P < 0.05).
The age at puberty, backfat at puberty and duration of estrus cycle were not affected by selenium (Table   210 3).

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The selenium (HMSeBA and Na2SeO3) increased the weight of uterine and relative weight of uterine 213 (P < 0.05, table 4). There's no significant difference in the bodyweight at slaughter, ovaries weight, length 214 of uterine horn and internal organs.

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The number of small follicles (diameter, 1-5mm), large follicles (>5mm; these follicles will ovulate 216 in the estrus) and corpora lutea (No. of corpus luteum represents the number of ovulations at last estrus.)

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Dietary supplementation with HMSeBA significantly increased the total selenium content in serum 222 and liver (P < 0.05, Table 6) compared with control groups in gilts, while Na2SeO3 group increased the 223 total selenium content in serum and liver compared with control group in gilts too (P < 0.05). Dietary 224 supplementation with HMSeBA tended to increase the total selenium content in ovary compared with 225 control group in gilts (P = 0.08).

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There was no difference among the treatments in the concentration of T3 and T4 in the serum. Dietary

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Estradiol secretion is an important manifestation of the physiological function of granular cells.

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HMSeBA significantly increased the E2 concentration compared with the control group at 5 and 10 ng/mL (P < 0.05, Fig. 1b). However, adding different concentrations of Na2SeO3, there was no significant 254 difference in E2 concentration in granulosa cells.

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Compared with the control group, the total antioxidant capacity of cells in vitro was improved by 257 adding of inorganic selenium and organic selenium (HMSeBA, P <0.05, Fig. 2a). The addition of 258 HMSeBA at a concentration of 2.5 ng/mL increased the intracellular T-SOD content (P < 0.05, Fig. 2b).

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The addition of Na2SeO3 and HMSeBA at a concentration of 5 ng/mL increased the intracellular GPx 260 level (P < 0.05, Fig. 2c), compared with the control group. HMSeBA increased the GR concentration 261 compared with the control group at 2.5ng/mL (P < 0.05, Fig. 2d). HMSeBA at 2.5 ng/mL increased the 262 content of TrxR in the cells (P < 0.05) (Fig. 2e). Compared with the control group, Na2SeO3 and HMSeBA

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In the context of African swine fever, DLY gilts was used in this study. We found that the age and 285 weight of puberty in DLY gilts were higher than those in LY gilts (220-250d vs. 190-210d, 120-140kg   The relationship between FSH and GPx has been observed in rats [32] which have found that apoptosis 317 in preovulatory follicles induced by oxidative stress is prevented by FSH through stimulation of follicular 318 glutathione synthesis and suppression of ROS production. In present study, we found that HMSeBA increased the GPx activity in serum, liver and ovary, increased the T-AOC in the liver and the TrxR in