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Energy supply is crucial for testicular development. Nevertheless, the specific alterations in the energy metabolic pathways that affect testicular development have not been extensively investigated. This study aimed to investigate the variations in metabolites and alterations in energy metabolic pathways in the testes of Hu sheep with different developmental status at 6 months of age. Twelve rams with similar body weights but distinct testis developmental status were selected among 345 Hu sheep based on testis size and histomorphology results, and they were divided into the well-developed (L group, n = 6) and developmentally delayed group (S group, n = 6). A total of 660 metabolites were identified via widely targeted metabolic analysis. Among 148 differentially expressed metabolites, 78 were up-regulated and 70 were downregulated in the L group compared with the S group. Functional enrichment analysis indicated that a significant proportion of the identified differential metabolites was implicated in energy metabolism-related pathways. Moreover, the L group exhibited significantly higher expression levels of genes involved in glycolysis (GLTU8 and LDH), TCA (PDHA2, CS and IDH3G), gluconeogenesis (PCK1), pentose phosphate (G6PD), and fatty acid degradation (GK, ACSL1, FABP3, CPT1 and CTP2). The activity enzymes such as citrate synthase, pyruvate dehydrogenase, and lactate dehydrogenase also increased in the L group. In summary, this observation implied that the augmentation of energy metabolic pathways plays a crucial role in facilitating testicular development. The upregulation of energy metabolic pathways collectively facilitates the testicular development in Hu sheep., Competing Interests: Declaration of competing interest The authors declare that there are no potential conflicts of interest related to this study., (Copyright © 2024. Published by Elsevier Inc.)

The fatty acid content and the localization and expression of phospholipase A 2 (PLA 2 ) in the testis of Hu sheep were investigated. A total of 18 six-month-old Hu sheep were divided into small group (S, with left testis weight < 50 g), medium group (M, with left testis weight among 90-110 g), and large group (L, with left testis weight >160 g), which had six individuals each. The expression of PLA 2 in testicular tissues of different sizes was analyzed by immunohistochemistry, RT-qPCR, and Western blot. The fatty acid profile was detected by gas chromatography. Immunohistochemical labeling determined that PLA 2 protein was expressed in the Leydig and Sertoli cells of testis, and the immunohistochemical average optional density in the S group was significantly greater than the L group (P < 0.05). RT-qPCR and Western blot analysis showed that PLA 2 in the S group was greater than that in the L group (P < 0.05). Docosahexaenoic acid, ω-3 polyunsaturated fatty acid (PUFA), and total PUFA content in the testis of the L group were significantly less than those of the S and M groups (P < 0.01). This study showed that PLA 2 content in the S group was greater than that in the L group., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023. Published by Elsevier B.V.)

ADP-dependent glucokinase (ADPGK) plays an important role instead of hexokinase in regulating energy metabolism via the Embden-Meyerhof-Parnas Pathway. And energy provided via glycolysis promotes testis development and spermatogenesis. In this study, 466 Hu sheep were screened for mutations in the ADPGK gene to examine the association of the ADPGK gene polymorphisms with the testis traits and spermatogenesis. The NC&#95;056060.1: g.31295 C > T SNP was found in the 3'-UTR region, resulting in two genotypes CC and TC type with genotypic frequencies of 0.66 and 0.34, respectively. This mutation was significantly associated with testis weight, testis long circumference, testis short girth, epididymis weight, and sperm concentration ( p  < 0.05). Moreover, TC genotype individuals had an increased tendency in the expression of the ADPGK gene and had significant reproductive performance advantages compared with CC genotype individuals in the study. And compared with the small testes (<50 g), the ADPGK gene expression of big testes (>160 g) increased significantly. This indicates an association between the ADPGK gene and reproductive organ parameters and sperm count in selected Hu sheep breed, and this SNP may serve as an effective DNA molecular marker for marker-assisted selection in Hu sheep breeding programs.

Oxidative stress damages the structure of sperm and affects its fertility. Further maturation and storage of spermatozoa occur in the epididymis. Thus, a balanced environment between free-radical production and the antioxidant-enzyme system is important. This study aimed to investigate differences in antioxidative activity in epididymis between high and low fertility levels of Hu sheep. A total of 179 Hu ram lambs aged 56 days were fed up to 6 months in the same environment. After feeding trial, all rams were slaughtered. According to the epididymis weight, cauda sperm concentration, and histo-morphological observation, 18 individuals were selected and divided into low fertility (n = 9) and high fertility (n = 9) groups. The RNA sequencing results showed 1404 differentially expressed genes (DEGs), with 735 genes upregulated and 669 genes downregulated in the high fertility group compared with the low fertility group. Sixteen DEGs related to lipid metabolism and oxidative stress, including GPX3, GPX5, LRP1, PLCB1, CRISP2, BSPH1, and ACSBG1, were screened by qPCR, and results were correlated well with the sequencing data. Antioxidative indices such as total antioxidant capacity (21.29 ± 5.97 vs. 30.23 ± 4.56 μM/gprot) and glutathione peroxidase (39.75 ± 7.61 vs. 68.84 ± 12.51 U/mgprot) increased in the high fertility group. However, the relative expression of mtDNA and malondialdehyde concentration (0.75 ± 0.15 vs. 0.54 ± 0.10 nmol/mgprot) and the reactive oxygen species (ROS) content significantly decreased (P < 0.05). Therefore, epididymis from high-fertility group had strong antioxidative activity and protected spermatozoa from ROS., (Copyright © 2022 Elsevier Inc. All rights reserved.)

Grape seed tannin extract (GPE) from wine grape pomace has many effective anti-oxidative effects and is used as a promising natural feed additive in the animal feed industry. This study investigated the effect of GPE as a source of tannin on the antioxidant capacity and testis development in Hu lambs. Twenty-seven 3-month-old ram lambs were randomly assigned to three groups. For 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 with 0.36% GPE diet, and Group II (TAN2) was fed with 0.72% GPE diet. After an 81-day feeding trial, all lambs except the heaviest and lightest in each group were humanely slaughtered and investigated. Results showed that feeding GPE did not affect the body weight, average daily gain, dry matter intake, scrotal circumference, and testis index. Meanwhile, feeding with 0.36% GPE diet increased testis weight, testis volume, and epididymis weight (P ≤ 0.05) compared with those of the control, but no difference was found between TAN1 and TAN2 groups. Copper-zinc superoxide dismutase (Cu-ZnSOD), steroid acute regulatory protein (StAR), cholesterol side-chain cleavage enzyme (P450scc), follicle-stimulating hormone receptor (FSHR), elongation of very long chain fatty acid protein 2 (ELOVL2), fatty acid desaturase (FADS2), and proliferating cell nuclear antigen (PCNA) mRNA in TAN1 and TAN2 groups were significantly up-regulated (P < 0.05). GPE also markedly increased the antioxidant status of testis. Compared with the control group, the treatment groups showed significantly increased superoxide dismutase (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) and total antioxidant capacity (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). Consuming 0.72% GPE also changed the fatty acid profiles in testis with increased C15:1, C22:6n3, and total n-3 fatty acids (P < 0.05) but decreased C22:5n3 (P < 0.05). Therefore, feeding lambs with GPE stimulated testis seminiferous tubule development and increased the number of Sertoli cells (10.56 ± 0.44 in control, 14.10 ± 0.57 in TAN1 and 13.60 ± 0.42, P < 0.05), and seminiferous tubule diameter (109.30 ± 4.56 μm in control, 164.49 ± 5.37 μm in TAN1 and 146.56 ± 4.53 μm in TAN2, P < 0.05). These results suggested that feeding GPE in the early reproductive development stage of lambs upregulated the expression of antioxidative, steroidogenesis, and polyunsaturated fatty acid metabolism-related genes, changed the fatty acid profiles, increased the antioxidant capacity in lamb's testis, and contributed to testis development and spermatogenesis., Competing Interests: Declaration of competing interest The authors declare no conflict of interest., (Copyright © 2021 Elsevier Inc. All rights reserved.)