| Boar fertility not only affects the economic value of boar studs, but also has a good effect on reproductive performance of sow in pig farms. The major constrain factor of boar fertility has been the poor sperm motility and sperm morphology which are influenced by environmental factors, with diet being one of the most important modifying agents. Among dietary factors, numerous experimental data indicate a positive effect of fish-derived n-3 polyunsaturated fatty acids(PUFAs) on boar semen quality and fertility. However, the contradicting results reported are likely due to variations in the quantities and sources of oil duration of supplementation, and excess fish oil supplementation may also have negative effects such as increased lipid peroxidation. As a fat-soluble antioxidant, vitamin E(VE) was included in premix to alleviate or reverse the negative impact of sperm lipid peroxidation of PUFAs addition. But it is unclear which component improves semen quality due to the untied supplementation with n-3 PUFAs and VE.In this study, we evaluated the effect of n-6:n-3 dietary ratio and VE supplementation on semen quality, sperm fatty acid composition and antioxidant status in boars. Then, we furtherly confirmed the effect of the effective n-6:n-3 dietary ratio and VE supplementation on sperm production, motility and morphology characteristics and libido, and investigated the effects of dietary n-6:n-3 ratio and VE on the physical and functional properties of sperm membrane and antioxidant status as well as the possible mechanism of dietary n-6:n-3 ratio and VE in improving boar fertility. It is hopeful that the present study would provide theoretical guidance and technical support for inclusion of dietary n-3PUFA and VE into boar diets. The main contents and results were as follows:In the first part, the effect of dietary n-6:n-3 fatty acid ratio and VE on semen quality, fatty acid composition and antioxidant status in boars were investigated. Forty-eight healthy and mature Landrace boars were individually housed in pens and randomly distributed according to age(24.30 ± 1.78 month) and semen quality into six dietary groups(n = 8). The isoenergetic and isonitrogenous basal diet which was based on mainly raw material of corn-soybean meal was formulated. The experiment followed a 3 × 2 factorial design. Throughout the entire experimental period, boars were fed 2.5 kg of a basal diet top-dressed with 60 g of the oil(60g soybean oil, 15 g fish oil and 45 g soybean oil, 60 g fish oil) per day to achieve n-6:n-3 dietary ratio of 14.4:1, 6.6:1 and 2.2:1(high-, medium- and low- ratio). The second factor was VE inclusion levels: 200 and 400 mg/kg VE(low- and high- dose). At the completion of a 14-week period, semen parameters were evaluated. At 0 and 8 weeks, sperm fatty acid composition and antioxidant status in the serum, sperm and seminal plasma were determinated. The main results are as follows:1) The dietary n-6:n-3 ratio had significant effect on sperm fatty acid composition. Compared with high ratio group, the medium ratio and low ratio groups very effectively increased docosahexaenoic acid(DHA) and n-3 PUFA and decreased docosapentaenoic acid(DPA) and n-6:n-3 ratio in spermatozoa(P < 0.05) at week 8. There was no treatment effect on total PUFA(P > 0.05).2) The dietary n-6:n-3 ratio and VE had significant effect on antioxidant status of serum, sperm and seminal plasma. The medium ratio group significantly enhanced the superoxide dismutase(SOD, 88.2 vs 66.2, 72.6 U/m L) and total antioxidant capacity(TAC, 5.8 vs 4.7, 4.7 U/m L)(P < 0.05) in seminal plasma than other two groups at week 8. There were no differences between high and medium ratio group in malondialdehye(MDA) of sperm(3.1 vs 3.2 nmol/108 sperm cells, P > 0.05) and serum(2.7 vs 3.2 nmol/m L, P > 0.05). High-dose VE supplementation increased sperm SOD, TAC and SOD of seminal plasma and serum(P < 0.05) and decreased sperm MDA(3.1 vs 3.7 nmol/108 sperm cells)(P < 0.05) than low-dose VE supplementation at week 8.3) The dietary n-6:n-3 ratio and VE had significant effect on progressive sperm motility. The medium ratio group resulted in the grestest progressive sperm motility(P < 0.05) than the high and low ratio groups at week 8, 10, 12 and 14. High-dose VE supplementation increased the progressive sperm motility(P < 0.05) than low-dose VE supplementation at week 8, 10, 12 and 14.In the second part, we investigated the effects of dietary n-6:n-3 ratio and VE on sperm production, sperm motility characteristics, sperm morphology, liquid-stored sperm motility and boar libido. A total of forty healthy and mature Duroc boars were selected at 591.5 ± 9.2 d of age and individually housed in pens. The average body weight was 272.9 ± 47.6 kg at initiation of the experiment. They were randomly distributed according to age, weight and semen quality into four dietary groups(n = 10). The isoenergetic and isonitrogenous basal diet which was based on mainly raw material of corn-soybean meal was formulated. The experiment followed a 2 × 2 factorial design. Throughout the entire experimental period, boars were fed 2.5 kg of a basal diet top-dressed with 60 g of the oil(60g soybean oil, 15 g fish oil and 45 g soybean oil) per day to achieve n-6:n-3 dietary ratio of 14.4:1 and 6.6:1(high- and low- ratio). The second factor was VE inclusion levels: 200 and 400 mg/kg VE(low- and high- dose). At the completion of a 20-week period, boar libido was recorded, fresh semen was collected from each boar and each ejaculate was assessed for sperm production, sperm motility characteristics and sperm morphology. Semen was diluted in Beltsville Thaw Solution(BTS) extender and stored at 17 oC to assess sperm motility at 12, 36, 72 and 120 h of storage. The main results are as follows:1) There was no significant effect of dietary n-6:n-3 and VE on semen volume, sperm concentration, total sperm cells and libido at 0 and 8 weeks(P > 0.05).2) At 12, 16 and 20 weeks, the low ratio group increased the total motility, sperm progressive motility, straight-line velocity(VSL), average path velocity(VAP) and curvilinear velocity(VCL) of fresh semen than high ratio group(P < 0.05). High-dose VE supplementation increased total motility, sperm progressive motility, VSL and VCL of fresh semen at week 12 and 16(P < 0.05) than low-dose group.3) The low ratio group yielded better normal spermatozoa, accordingly, decreased the abnormal midpiece than higher ratio group at week 12, 16 and 20(P < 0.05). The high-dose VE supplementation positively affected normal sperm morphology(at week 16 and 20), and decreased the proximal cytoplasmic droplet(at week 12, 16 and 20) than low-dose VE supplementation(P < 0.05).4) There was no significant effect of dietary n-6:n-3 ratio on sperm motility of liquid stored semen(P > 0.05); high-dose VE supplementation significantly(P < 0.05) increased sperm progressive motility of liquid stored semen at week 12(12h and 36h) and week 20(12h, 36 h, 72 h and 120h) compared with low-dose VE supplementation.In the third part, this study was conducted to investigated the effects of dietary n-6:n-3 ratio and VE on the physical and functional properties of sperm membrane and antioxidant status as well as the mechanism of dietary n-6:n-3 ratio and VE in improving boar fertility. The third part had the same experimental animal, diet and design with the second part. The serum, sperm and seminal plasma were collected at 0, 8, 12, 16 and 20 weeks to monitor structural membrane integrity, function membrane integrity, membrane fluidity, mitochondrial membrane potential(MMP) and reactive oxygen species(ROS) level, sperm fatty acid composition, sperm α-tocopherol content, SOD, glutathione peroxidase(GSH-Px), MDA and 8-hydroxydeoxyguanosine(8-OHd G) in sperm as wellas the SOD, GSH-Px and MDA content in serum and seminal plasma and seminal TAC. The main results are as follows:1) Increased DHA and total n-3 PUFA in boar sperm was founded at the expense of DPA and total n-6 PUFA in low ratio group compared with high ratio group at week 12 and 16(P < 0.05).2) At 0 and 8 week, sperm membrane integrity parameters and membrane fluidity did not differ among treatment groups. The structural membrane integrity and membrane fluidity are significantly enhanced in boars consuming 6.6:1 diets at week 12 and 16; the MMP and the functional membrane integrity are significantly enhanced in boars consuming 6.6:1 diets at 12 and 16 week, respectively(P < 0.05). The MMP is significantly enhanced in boars consuming 400mg/kg VE on week 12 and 16(P < 0.05).3) At week 12, 16 and/or 20 week, low ratio group and high-dose VE supplementation enhanced the SOD, GSH-Px and reduced the MDA in serum(P < 0.05), and enhanced the SOD, prevented the 8-OHd G in sperm(P < 0.05). In addition, high-dose VE supplementation significantly enhanced GSH-Px and reduced ROS, 8-OHd G in sperm(P < 0.05), and the SOD, GSH-Px and TAC of seminal plasma are enhanced(P < 0.05) and MDA content in seminal plasma is reduced in boars consuming 400mg/kg VE(P < 0.05). Although there were no significant differences for the sperm α-tocopherol content between two ratio treatments, the α-tocopherol content enhanced while the levels of VE in diet increased(P < 0.05).Present study demonstrated that the adjustment of n-6:n-3 ratio(6.6) improved the sperm motility and sperm morphology, and supply of VE(400 mg/kg) successfully improved sperm motility and sperm morphology. Feeding boars with 6.6 n-6:n-3 ratio diets influenced the sperm fatty acid composition, enhanced the physical and functional properties of sperm membrane, and improved sperm motility and morphlogy; supplementing 400 mg/kg VE to boar diets enhanced antioxidant status in serum, sperm and seminal plasma, prevented sperm peroxidation damage, directly leading to the improvement of motility and morphology parameters. In conclusion, there were significant effect of dietary n-6:n-3 ratio and VE on boar fertility. |
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