Inflammation disrupts the normal function of granulosa cells (GCs), which leads to ovarian dysfunction and fertility decline. Inflammatory conditions such as polycystic ovary syndrome (PCOS), primary ovarian insufficiency (POI), endometriosis, and age-related ovarian decline are often associated with chronic low-grade inflammation. Nicotinamide mononucleotide (NMN) is an important precursor of NAD⁺ and has gained attention for its potential to modulate cellular metabolism, redox homeostasis, and mitigate inflammation. This study investigated the protective roles of NMN against lipopolysaccharide LPS-mediated inflammation in GCs. The results of this experiment demonstrated that LPS had negative effects on GCs in term of reduced viability and proliferation rates and upregulated the production of pro-inflammatory cytokines, including interleukin-1 beta (IL-1β), interleukin-6 (IL-6), cyclooxygenase-2 (Cox-2), and tumor necrosis factor-alpha (TNF-α). Notably, the levels of NAD⁺ and NAD⁺/NADH ratio in GCs were reduced in response to inflammation. On the other hand, NMN supplementation restored the NAD⁺ levels and the NAD⁺/NADH ratio in GCs and significantly reduced the expression of pro-inflammatory markers at both mRNA and protein levels. It also enhanced cell viability and proliferation rates of GCs. Furthermore, NMN also reduced apoptosis rates in GCs by downregulating pro-apoptotic markers, including Caspase-3, Caspase-9, and Bax while upregulating anti-apoptotic marker Bcl-2. NMN supplementation significantly reduced reactive oxygen species ROS and improved steroidogenesis activity by restoring the estradiol (E2) and progesterone (P4) levels in LPS-treated GCs. Mechanistically, this study found that NMN suppressed the activation of the TLR4/NF-κB/MAPK signaling pathways in GCs, which regulates inflammatory processes. In conclusion, the findings of this study revealed that NMN has the potential to reduce LPS-mediated inflammatory changes in GCs by modulating NAD⁺ metabolism and inflammatory signaling pathways. NMN supplementation can be used as a potential therapeutic agent for ovarian inflammation and related fertility disorders.

Buffalo bull spermatozoa are rich in polyunsaturated fatty acids and are prone to lipid peroxidation. We hypothesised that lipid peroxidation in buffalo bull semen will increase with age and will affect the semen quality. The objective was to compare malondialdehyde (MDA) concentration and the quality of fresh and frozen-thawed semen between aged (13.6 ± 1.0 years; n = 3) and young (3.4 ± 0.3 years; n = 3) Nili-Ravi bulls. The concentration of MDA did not differ (p >.05) between aged vs. young bulls in fresh (2.3 ± 0.2 vs. 2.9 ± 0.7 nmol mL⁻¹), frozen-thawed (53.1 ± 2.8 vs. 48.4 ± 2.6 nmol mL⁻¹) semen and seminal plasma (5.71 ± 0.97 vs. 5.19 ± 1.36 nmol mL⁻¹), respectively. In fresh semen, sperm motility and total concentration did not differ (p > .05) between aged and young bulls. The volume of fresh semen increased (p > .05) while sperm viability and DNA integrity decreased (p < .05) in aged vs. young bulls. In frozen-thawed semen, sperm motility, viability and DNA integrity decreased (p < .05) in aged vs. young bulls. In frozen-thawed vs. fresh semen, MDA level increased within young (48.4 ± 2.6 vs. 2.3 ± 0.2 nmol mL⁻¹) and aged bulls (53.1 ± 2.8 vs. 2.9 ± 0.7 nmol mL⁻¹). Conversely, sperm motility and viability decreased (p < .05) within the age groups between fresh and frozen-thawed semen. In conclusion, (1) lipid peroxidation (MDA) does not increase due to age; however, it is negatively associated with semen quality and (2) cryopreservation steps up lipid peroxidation irrespective of age and deteriorates semen quality of Nili-Ravi bulls.

Orf is a highly contagious zoonotic disease of small ruminants caused by Parapoxvirus. Kisspeptin, encoded by the KISS1 gene with its cognate receptor GPR-54 is recognized as an upstream orchestrator in the hypothalamic-pituitary-gonadal axis. This study was designed to construct a DNA vaccine that produces a fused peptide composed of a major immunodominant protein of the orf virus (B2L) and kisspeptin-54, a neuropeptide with recognized roles in mammalian reproductive biology. The administration of this recombinant vaccine is shown to produce a significant antibody and cell-mediated immune response directed against B2L compared to the control group (p < 0.05). Furthermore, we found that rats inoculated with PBK-asd vaccine up-regulated antigen-mediated splenocyte proliferation and significantly raised antigen-specific tumor necrosis factor-alpha (TNFα-), interferon-gamma (IFN-ϒ) and interleukin (IL-2) compared to the control group (p < 0.05). This recombinant vaccine also stimulated antibody responses to kisspeptin and decreased serum luteinizing hormone and testosterone levels. Moreover, the current recombinant vaccine caused testicular atrophy and arrested spermatogenesis. It is concluded that this recombinant B2L and Kisspeptin-54 vaccine could be a promising approach for construction of bivalent orf virus and immunocastration vaccine. Furthermore, we concluded that the orf virus envelope protein (B2L) could be used as an immunomodulator for kisspeptin-54 to produce a strong antibody response.

Background: The microscopic process of postnatal testicular development in early-puberty animals is poorly understood. Therefore, in this study, 21 male Yiling goats with average ages of 0, 30, 60, 90, 120, 150 and 180 days old (each age group comprised three goats) were used to study the changes in organs, tissues and transcriptomes during postnatal testicle development to obtain a broad and deep insight into the dynamic process of testicular transition from growth to spermatogenesis in early-puberty animals.Results: The inflection point of testicular weight was at 119 days postpartum (dpp), and the testicular weight increased rapidly from 119 dpp to 150 dpp. Spermatozoa were observed in the testis at 90 dpp by using haematoxylin–eosin staining. We found from the transcriptome analysis of testes that the testicular development of Yiling goat from birth to 180 dpp experienced three stages, namely, growth, transition and spermatogenesis stages. The goats in the testicular growth stage (0–60 dpp) showed a high expression of growth-related genes in neurogenesis, angiogenesis and cell junction, and a low expression of spermatogenesis-related genes. The goats aged 60–120 dpp were in the transitional stage which had a gradually decreased growth-related gene transcription levels and increased spermatogenesis-related gene transcription levels. The goats aged 120–180 dpp were in the spermatogenesis stage. At this stage, highly expressed spermatogenesis-related genes, downregulated testicular growth- and immune-related genes and a shift in the focus of testicular development into spermatogenesis were observed. Additionally, we found several novel hub genes,which may play key roles in spermatogenesis, androgen synthesis and secretion, angiogenesis, cell junction and neurogenesis. Moreover, the results of this study were compared with previous studies on goat or other species, and some gene expression patterns shared in early-puberty mammals were discovered.Conclusions: The postnatal development of the testis undergoes a process of transition from organ growth to spermatogenesis. During this process, spermatogenesis-related genes are upregulated, whereas neurogenesis-, angiogenesis-, cell junction-, muscle- and immune-related genes are downregulated. In conclusion, the multipathway synergy promotes testicular transition from growth to spermatogenesis in early-puberty goats and may be a common rule shared by mammals.