Dietary grape seed tannin extract supplementation stimulated testis development, changed fatty acid proles and increased antioxidant capacity in testis in Hu lambs in pre-puberty


 Background

Grape seed tannin extract (GPE) that is from wine grape pomace has many effective anti-oxidative effects and is used as a promising natural feed additive in the animal feed industry. In this study, we investigated the effect of GPE as a source of tannin on the antioxidant capacity and testis development in Hu lamb.
Method:

Twenty-seven 3-month-old ram lambs were randomly assigned to three groups. Within each treatment group, nine lambs were allocated to nine pens (one lamb per pen). The lambs in the control group were fed a control diet without GPE for 61 days from D21 to D80. Group I (TAN1) was fed a 0.36% GPE diet. Group II (TAN2) was fed a 0.72% GPE diet. After 81-day feeding trial, all lambs except the heaviest and lightest in each group were humanely slaughtered and investigated.
Results

Feeding GPE did not affect the body weight, average daily gain, dry matter intake, scrotal circumference and testis index, whereas feeding 0.36% GPE diet increased testis weight, testis volume, and epididymis weight (P < 0.05) compared with the control. Cu-ZnSOD, P450scc, P450arom, FSHR, FADS2, ELOVL2 and PCNA mRNA in TAN1 and TAN2 groups were significantly up-regulated (P < 0.05). GPE also markedly increased antioxidant status of testis. Compared with the control group, GPE significantly increased the SOD activity (314.23 ± 18.64 U/mg prot in control, 505.22 ± 63.47 U/mg prot in TAN1 and 587.88 ± 55.94 U/mg prot in TAN2, P < 0.05), T-AOC (98.23 ± 18.99 U/g prot in control, 202.15 ± 34.19 U/g prot in TAN1 and 189.57 ± 18.95 U/g prot in TAN2, P < 0.05). GPE also increased NEFAs levels (158.36 ± 26.32 mmol/kg prot in control, 205.15 ± 14.51 mmol/kg prot in TNA1 and 250.06 ± 33.82 mmol/kg prot in TAN2 P = 0.08). But, consuming GPE downregulated testicular total cholesterol concentration (3.89 ± 0.57 mmol/kg prot in TAN1, 4.21 ± 1.04 mmol/kg prot in TAN2 and 7.20 ± 1.46 mmol/kg prot, P = 0.09). Consuming 0.72% GPE also changed fatty acid profiles in testis with increased C15:1, C22:6n3 and total n-3 fatty acids (P < 0.05). Therefore, feeding lambs with 0.72% GPE stimulated testis seminiferous tubule development, increased the number of Sertoli cells (10.56 ± 0.44 vs. 13.60 ± 0.42, P < 0.05), and seminiferous tubule diameter (146.56 ± 4.53 µm vs. 109.3 ± 4.56 µm, P < 0.05).
Conclusion

All these results suggested that feeding GPE in the early reproductive development stage of lambs upregulated the expression of antioxidative, steroidogenesis, and PUFA metabolism-related genes; changed the fatty acids profiles, increased the antioxidant capacity in lamb’s testis; and contributed to testis development and spermatogenesis.


Introduction
Based on data from the international Organization of Vine and Wine, China is the second in a list of vineyard surface area of major vine-growing countries (2019). Wine grape pomace (WGP)is the major byproduct of the red wine industry and consists mainly of stems, skins and seeds. WPG is treated as a waste due to the lack of alternative uses with economic bene ts for a long time. Importantly, WGP and it extract have even more effective anti-oxidative effects than vitamin C and Vitamin E [1,2], and thus can be used as a promising natural feed additive in the animal industry. It is estimated that only 3% of the global WGP is used as animal feed. The low usage of WGP in livestock production is due to its high content of monomeric phenolic compounds, mainly tannin which is associated with reduced digestibility through the inhibition of cellulolytic and proteolytic enzymes [3,4]. In pursuit of sustainable and economically viable ruminant livestock systems, many farmers are under increasing pressure to maximize the use of available agricultural by-products-based diets for their livestock without compromising the performance. In livestock husbandry, tannin has been studied for it deterrent effects on herbivores, pathogens, parasites, or toxicity [5][6][7]. Tannin also affected feed utilization and especially protected protein from fermentation including a shift in N excretion from urine to feces, and reduced ruminate methanogenesis [8,9].
China's lamb production areas are mainly located in the West and Northwest, and are characterized by fragile ecology [10]. The Hexi/Gansu Corridor is one of the main wine producing areas and the lamb production area, suggesting the possible use of WGP in the lamb industry. China highly values ecological and environmental protection. Guided by the conviction that lucid waters and lush mountains are invaluable assets, the country advocates harmonious coexistence between humans and nature, and sticks to the path of green and sustainable development, which includes the requirement of pen-raising for sheep.
However, oxidative stress is induced in lambs raised in pens, which decreases testes weight and spermatogenesis, impairs epididymis sperm quality [11]. Most of previously studies about dietary supplements on testis antioxidant capacity or spermatogenesis were in adult rams [11] Interestingly, grape seed tannin extract (GPE) attenuates oxidative stress, and enhances sperm motility in rats [12]. Thus, the current study aimed to evaluate the effect of GPE on reproduction hormone synthesis, testis development, fatty acid pro les and testis antioxidative in Hu ram lambs after feeding GPE diets from pre-puberty.

Animals and feeding trial
Chinese Hu sheep is famous for their high fecundity and early sexual maturity with reached puberty at 120 days of age. In this study, twenty-seven-3-month-old Hu lambs (average body weight of (22.9 ± 1.3 kg) were housed in individual pens (1 m ×1.5 m) and randomly divided into three groups (n = 9). Lambs in control group were fed diets without GPE, while in TAN1 and TAN2 received diets contained 0.36% and 0.72% GPE. The GPE was purchased from the Shanghai Jiaoyuan Co. Ltd. (Shanghai China), and purity quotient of GPE was up to 95%,condensed tannin was 56.5%. The diets (Table 1) were formulated as diet pellets with 6 mm in diameter. The entire experimental period lasted for 81 days with the adaptation period from D0 to D20 and normal commencing trial period from D21 to D80. All rams were fed thrice a day at 8:00, 14:00 and 19:00 under free food intake and with libitum access to fresh water and multi-mutrient blocks. The daily feed intake was calculated from D21 to D30 and D71 to D80. All rams were weighted at the D21 and D80.
The trial was performed at Minqin Zhongtian Sheep Industry Co., Ltd. (Minqin, China) from August to November.
This study was conducted in strict accordance with the recommendations from the Guide for the Animal Care and Use Committee of Lanzhou University.
No lamb was harmed during the feeding trial.

Sample collection
Peripheral blood samples were collected at D81 and centrifuged at 1000 g at 4°C for 30 min to isolated serum. All lambs except the heaviest and lightest in each group were humanely slaughtered by a licensed slaughter man as previously described at the Minqin Zhongtian Sheep Industry Co. Ltd [13]. Their body, testis, epididymis weight and testis volume was evaluated as previously described [13]. After that, tissues from left testis without tunica albuginea were collected and stored using nitrogen and 10% formalin, respectively.

RNA isolation and cDNA synthesis
Total RNA was isolated using TRIzol reagent in accordance with the manufacturer's instruction. The RNA purity and integrity were detected by using NanoDrop 2000 spectrophotometer (Thermo Fisher, Waltham, USA) and formaldehyde denaturing gel electrophoresis. The total RNA (2.5 µg) with A260/280 ratio between 1.8 to 2.0 and A260/230 ratio higher than 2.0 was reverse-transcribed to cDNA by using Trans-Script one-step gDNA removal and cDNA synthesis super-mix kit (TransGen Biotech, Beijing, China) at 42°C for 15 min and 85°C for 5 s following the manufacturer's instructions. The cDNA was diluted 1:10 with nuclease-free water and used for quantitative gene expression.

Western blotting analysis
Total protein was isolated from testis using IP cells lysis buffer. The protein concentrations were estimated by BCA. Approximately 30 µg total protein was separated by SDS-PAGE on a polyacrylamide gel and then transferred onto PVDF membranes with a pore size of 0.45 µm. Then, membranes were blocked 2 h at room temperature with 5% fat-free milk, and incubated with primary antibodies 2.6 Testosterone, estradiol and LH levels LH in the blood serum was detecting using Sheep Luteinizing Hormone (LH) ELISA Kit (CUSABIO, Wuhan, China) with detection reange 2-75 mIU/mL, and intra-assay and inter-assay coe cient of variations (CVs) were both ≤ 15%, according to the instruction. Testosterone and estradiol were detected by using an automated chemiluminescent microparticle immunoassay with Architect 2nd generation testosterone kit and Architect estradiol assay kit on Architect i4000SR system (Abbott Park, USA) as we previously mentioned [13].

Lipid extraction and fatty acid analysis
Testis lipids were extracted from 0.5 g of tissue by homogenization in chloroform/methanol (2:1, v/v). Trans-methylation of these samples was performed using the Metcalf method [14]. Fatty acids methyl esters were separated and quanti ed by TRACE 1300 gas chromatograph (Thermo Fisher, Waltham, USA) equipped with a ame ionization detector and a 100 m × 0.25 mm × 0.25 µm (HP-88, Agilent Technologies) fused silica capillary column. Nitrogen was used as carrier gas and temperature programming was from 50°C to 175°C at 13°C/min, and held there for 27 min, and then temperatureprogrammed at 3°C/min to 215°C, and held there for 27 min. The injector and detector temperatures were set at 240°C. Peak identi cation was based on the elution pro le of known FAME chromatographic standards (fatty acid methyl esters, C4-C24, Nuchek Prep, Elysian, MN, USA) and previously reported [15,16]. Relative quanti cation was normalized with the sum of all the detected species and shown as a percentage of total species [17].

Biochemical assays
Testicular tissues were weighted and homogenized in ice-cold PBS to create a 1:10 suspension with repeated freezing and thawing. After centrifuged at 10000 g for 5 min at 4°C, the supernatants concentrations were determined using BCA method.
Total antioxidant capacity (T-AOC) assay kit, superoxide dismutase (SOD) assay kit, total cholesterol assay kit, total triglyceride assay kit and non-esteri ed free fatty acids (NEFAs) assay kit (Nanjing Jiancheng, Nanjing, China) were used to measuring T-AOC, SOD, cholesterol, triglyceride and NEFAs on Epoch Microplate Reader (Bio-Tek Instruments, Winooski, VT, USA) according to the manufacturer's protocols.
The biochemical concentrations were normalized with total protein in testicular tissues suspension.

Testicular histology
Testicular histology was analysed as previously described [13]. Brie y, testicular tissues (5 mm×5 mm× 5 mm) were xed, dehydrated and embedded. Then sectioned at 5 µm thicknesses, and stained with hematoxylin and eosin (H&E). Histological evaluation was performed by light microscopy and images were captured using the Scopeimage 9.0 software (Ningbo yongxin, Ningbo, China). Diameter of seminiferous tubules and the number of Sertoli cells were analysed. For each tissue, 6 to10 seminiferous tubules were elevated.

Statistical analysis
The experimental results were presented as the group mean ± standard error of mean. The data was evaluated with SPSS program, version 13.0 (SPSS, Chicago, USA). Normal parameters were analysed by One-sample Kolmogorov-Smirnov test. Homogeneity of variances were analysed by Leven's test. Signi cant differences were analysed by one-way ANOVA and Welch (unequal variances). Multiple comparisons between groups were compared with LSD and Dunnett T3 (unequal variances). P values < 0.05 was considered statistically signi cant.

Effect of GPE on testicular development and histology
No signi cant differences in the dry matter intake, body weight, average daily gain, scrotal circumference and testis index were observed among groups after consuming GPE (P > 0.05). Compared with the control group, supplementation with 0.72% GPE signi cantly increased the testis weight, 0.36% GPE signi cantly increased testis weight, testis volume, and epididymis weight (P < 0.05) ( Table 3). Histological studies showed extensive changes evident by germinal epithelium in the control and GPE treatment groups. The thickness of cellular wall of tubules and spermatogenic cells in TAN1 and TAN2 groups were signi cantly increased with four or more layers of different stages of germ cells in their seminiferous epithelium. The lumen of seminiferous tubules became larger with diameter 164.49 ± 5.37 µm in TAN1, 146.56 ± 4.53 µm in TAN2 and 109.3 ± 4.56 µm in the control group (P < 0.05). The elongated spermatozoids were also observed in the lumina of seminiferous after GPE treatment (Fig. 1). The number of Sertoli cells per seminiferous tubule cross-section was also increased after feeding GPE (14.10 ± 0.57 in TAN1 and 13.60 ± 0.42 in TAN2) compared with the control group (10.56 ± 0.44, P < 0.05).

Effect of GPE on hormone levels
The average concentrations of plasma T, E2 and LH were not affected after GPE treatment (Table 5).
3.5 Effect of GPE on testis fatty acid.
As observed for the fatty acids pro le, GPE treatment altered the fatty acids composition in testes.
Supplementation 0.72% GPE signi cantly increased C15:1, C22:6n3 and total n-3 fatty acids in comparison to the control, but decreased C22:5n3 (P < 0.05). C20:3n3 in TAN1 group was also much more than in the control group.

Discussion
The testes, as the endocrine glands that are part of the male reproductive system, and their main functions are to secrete androgens and produce sperm. There is a large amount of ROS produced during spermatogenesis. ROS and lipid peroxidation reactions play an important role in the metabolism.
However, it could cause metabolic dysfunction once the ROS balance is disrupted. ROS could attack PUFA in the cell membrane, disrupted the structure of the cell membrane and induced various diseases [18]. Oxidase such as SOD and catalase play an important role in the antioxidant defence system. With the increase of ecological and environmental protection efforts, China's sheep industry is developing towards house feeding, but captive raising ROS levels of testicular tissue is not conducive to ram sperm production [11]. Therefore, improving the antioxidant capacity of male animals and protecting the structural and functional integrity of the testes are essential for improving the quality of male semen.
Grape residue contains tannic acid and has strong antioxidant capacity. Grape seed tannin can capture free radicals in the body and inhibit the production of free radicals [19]. It has functions of bacteriostasis, improving rumen fermentation of ruminants and intestinal microbial environment, lowering blood lipids, etc., protecting the health of the body, and is a valuable food antioxidant [5][6][7][8][9].
T-AOC can comprehensively evaluate the strength of the antioxidant defense system in animals and effectively translate the compensatory capacity of the body to external stimuli. The supplementation of GPE in the present study did not affect the body weight, the average daily gain,but increased T-AOC, SOD concentration in testis, up-regulated GPX3 and Cu-ZnSOD expression. These results were consisted with Zhao J et al [11]. This could enhance antioxidant capacity in testis, and prevent oxidant stress happening.
Polyphenols also regulate expression of genes related to lipogenesis and lipocatabolic in liver [20].
Related research revealed that tannin could reduce blood lipids and blood sugar [21]. Lipid in testis contains high a ratio of PUFA. The PUFA composition and pro le is associated with testis development and spermatogenesis [22]. Fatty acid from liposynthesis and absorb from food could be metabolism to SFA, MUFA and PUFA through fatty acid synthetase, desaturase and elongase. These enzymes could be regulated by metabolite, hormone, transcription factor or competitive substances of rate-limiting enzyme [13]. The composition of PUFA is vulnerable to the diet. In the present study, GPE stimulated FADS2 and ELOVL2 expression, and contribute to EPA and DHA synthesis. Previously studies indicated that decreasing of DHA in sperm membrane was associated with decreasing fertility [23,24]. Fortunately, the concentration of DHA in this study was upregulated after consuming GPE. DHA in testicular may contribute to spermatogenesis and sperm quality [24]. The sperm motility, membrane uidity is associated with n-3 PUFA in sperm [23][24][25]. PUFA in the sperm head and tile affects the sperm capacitation and interaction between sperm and uterine surface environment [26].
StAR activation is the rst stage in steroid hormone synthesis, which modulates the translocation of cholesterol from outer to inner mitochondrial membrane. In the present study, GPE signi cantly upregulated the expression of StAR, P540scc and P450arom, and stimulate testis development with testis weight, diameter of seminiferous tubule and number of Sertoli cells increased. The sperm production was determined by adult testis size and the number of Sertoli cells [27]. In this study, GPE signi cantly increased testis and epididymis weight, stimulated spermatogenesis with increased number of Sertoli cells, and up-regulated PCNA mRNA expression in testis. However, the GPE did not increase the levels of LH, testosterone and estradiol in plasma in the present study. These results may be due to the discrepancy between the hormone levels in plasma and genes expression induced by the fatty acid pro le changed by GPE. Seminar result was also reported in pig feeding Soybean oil and hydrogenated soybean oil from aged one-month to 18-month. Soybean oil inhibited testosterone and estradiol, however, it increased sterogenesis related genes expression [28].

Availability of data and materials
The datasets used/or analysed during the current study are available from the corresponding author on reasonable request.

Ethics approval and consent to participate
This study was conducted in strict accordance with the recommendations from the Guide for the Animal Care and Use Committee of Lanzhou University.

Consent for publication
All of the authors have approved the nal version of the manuscript and agreed with this submission to the Journal of Animal Science and Biotechnology. were signi cantly upregulated in TAN1 and TAN2 groups. The relative expression of GSTA1 (glutathione S-transferase A1), GPX3 (glutathione peroxidase3) and StAR (steroid acute regulatory protein) genes were also signi cantly upregulated in TAN1 group. Values were shown as means ± SEM. *P < 0.05 and **P < 0.01 compared with the control group.

Figure 3
Histological images of testes from control and GPE groups. Hematoxylin and eosin (H&E) staining, 100 × (Green row: multiple changes were presented in a further magni cation (400 ×) of the green bordered box.). Testes from the GPE groups showed a compact and regular arrangement of germ cells and elongated spermatozoids in their seminiferous epithelium.