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15 pages, 2856 KB

Open AccessArticle

Imiquimod (R837), a TLR7-Specific Agonist, Regulates Boar Sperm Motility via PI3K/GSK3α/β/Hexokinase Pathway

by Weijing Zhang, Adedeji O. Adetunji, Wenxian Zeng, Eslam M. Bastawy, Lingjiang Min, Nengshui Ding and Zhendong Zhu

Biology 2025, 14(9), 1182; https://doi.org/10.3390/biology14091182 - 2 Sep 2025

Viewed by 522

Abstract

Mammalian sperm has a high metabolic activity and primarily relies on glycolysis and mitochondrial oxidative phosphorylation for energy supply. It has been reported that imiquimod (R837), a specific ligand of TLR7 protein, reduces the motility of sperm. However, the specific molecular mechanism by [...] Read more.

Mammalian sperm has a high metabolic activity and primarily relies on glycolysis and mitochondrial oxidative phosphorylation for energy supply. It has been reported that imiquimod (R837), a specific ligand of TLR7 protein, reduces the motility of sperm. However, the specific molecular mechanism by which TLR7 modulates boar sperm is unclear. In this study, the effect of R837, a Toll-like receptor 7 (TLR7) agonist, on boar sperm motility was investigated. Sperm samples were incubated with varying concentrations of R837 (0 to 0.8 µM) at different time points (30, 60, and 90 min). Findings reveal for the first time that TLR7 protein, a key component of the immune system’s Toll-like receptors, is predominantly localized in the middle section of the boar sperm tail, with a smaller concentration observed in the neck. Also, immunofluorescence (IF) revealed that approximately half of the boar sperm sample expressed TLR7. Furthermore, the TLR7 agonist influenced glycolytic hexokinase activity and mitochondrial function via the PI3K-GSK3β signaling pathway. It also selectively inhibited motility in the lower-layer sperm, while motility in the upper-layer sperm remained unaffected. Additionally, this study determined that incubation conditions for boar sperm with 0.2 μM R837 at 37 °C for 60 min yielded the most pronounced inhibition of forward motility in the lower layer of sperm, without compromising the integrity of the acrosome or plasma membrane. The present study reveals the crucial role of R837 in boar sperm motility and highlights TLR7 as an important protein that regulates boar sperm energy metabolism. Full article

(This article belongs to the Special Issue The Biology of Animal Reproduction)

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10 pages, 1778 KB

Open AccessArticle

Amplification of Ultra-Trace DNA from Early Sheep Embryos Based on qPCR: Establishing a Gender Identification System

by Peng Niu, Weikun Tao, Fei Huang, Xiaopeng Li, Xueyan Wang, Jie Wang, Qinghua Gao and Di Fang

Biology 2025, 14(9), 1144; https://doi.org/10.3390/biology14091144 - 29 Aug 2025

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Abstract

The ability to identify and control the sex of embryos holds significant commercial implications for livestock production. Due to the high costs and instability of detection methods, it is necessary to establish a time-saving, effective, simple, and reliable method to identify the sex [...] Read more.

The ability to identify and control the sex of embryos holds significant commercial implications for livestock production. Due to the high costs and instability of detection methods, it is necessary to establish a time-saving, effective, simple, and reliable method to identify the sex of mammalian embryos. This study designs primer probes based on the SRY gene sequence in the sex-determining region of the Y chromosome of sheep and combines qPCR to amplify both the SRY gene and the internal reference gene GAPDH as a control. By adjusting and optimizing the detection system with ultra-trace of genomic DNA (gDNA) from sheep blood and embryos, we sexed sheep embryos cultured in vitro. The results show that male embryos exhibit specific amplification bands for both the GAPDH and SRY genes, while female embryos only have amplification bands for the GAPDH gene. The sensitivity of the sheep embryo sex identification system established in this study can be as low as 5.64 pg of DNA, with an accuracy rate of 100% for embryo sex identification. Full article

(This article belongs to the Special Issue The Biology of Animal Reproduction)

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14 pages, 1680 KB

Open AccessArticle

Comparison of Superovulated Embryo Quality in Simmental Cattle Inseminated with 0 °C-Refrigerated and Liquid Nitrogen-Frozen Semen

by Jie-Ru Wang, Fei Huang, Peng Niu, Hong Cheng, Hui-Min Qu, Xiao-Peng Li, Xue-Yan Wang, Jie Wang, Jia-Jia Suo, Di Fang and Qing-Hua Gao

Biology 2025, 14(6), 658; https://doi.org/10.3390/biology14060658 - 6 Jun 2025

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Abstract

Semen quality plays a crucial role in bovine in vivo embryo production. This study aimed to compare the effects of 0 °C-refrigerated semen and liquid nitrogen-frozen semen on embryo quality in Simmental cattle. Semen collected from five bulls was equally divided into two [...] Read more.

Semen quality plays a crucial role in bovine in vivo embryo production. This study aimed to compare the effects of 0 °C-refrigerated semen and liquid nitrogen-frozen semen on embryo quality in Simmental cattle. Semen collected from five bulls was equally divided into two groups: one diluted with a 0 °C refrigeration solution and stored at 0 °C, and the other diluted with a cryopreservation solution and stored in liquid nitrogen for 24 h. We evaluated sperm motility, progressive motility (assessed via a computer-assisted sperm analyzer), acrosome integrity, and plasma membrane integrity in both groups. Fifty superovulated Simmental cows were artificially inseminated with semen from both groups. Embryos were non-surgically flushed on day seven, followed by BrdU proliferation staining and TUNEL apoptosis staining. Proliferation and apoptosis levels were quantified using marker genes. Results showed that 0 °C-refrigerated semen exhibited significantly higher sperm motility, progressive motility, acrosome integrity, and plasma membrane integrity compared to liquid nitrogen-frozen semen (p < 0.05). While total embryo numbers showed no significant difference between groups (p ≥ 0.05), embryos from 0 °C-refrigerated semen contained significantly more proliferative cells (p < 0.05) and fewer apoptotic cells (p < 0.05) than those from frozen semen. These findings demonstrate that 0 °C-refrigerated semen outperforms liquid nitrogen-frozen semen in both sperm quality parameters and resultant embryo quality. Full article

(This article belongs to the Special Issue The Biology of Animal Reproduction)

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20 pages, 6099 KB

Open AccessArticle

Structural Rearrangement of the Olfactory Epithelium in Male Baikal Yellowfin Sculpins Across the Reproductive Period

by Igor V. Klimenkov, Mikhail V. Pastukhov, Hung-Ming Chang, Ting-Yi Renn and Nikolay P. Sudakov

Biology 2025, 14(2), 179; https://doi.org/10.3390/biology14020179 - 10 Feb 2025

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Abstract

The morphological peculiarities of receptor neurons and support cells in the olfactory epithelium of male yellowfin sculpin (Cottocomephorus grewingkii; Dybowski, 1874) were studied during the pre-spawning, spawning (when males do not feed and have a higher sensitivity to female pheromones), and [...] Read more.

The morphological peculiarities of receptor neurons and support cells in the olfactory epithelium of male yellowfin sculpin (Cottocomephorus grewingkii; Dybowski, 1874) were studied during the pre-spawning, spawning (when males do not feed and have a higher sensitivity to female pheromones), and guarding (the fertilized eggs) periods. This study was performed using electron transmission and laser confocal microscopy. Structural changes in the fish olfactory epithelium are associated with the shift in olfactory signals from alimentary to pheromonal. These results expand our knowledge of the odorant-dependent plasticity of the periphery of the fish olfactory system. Full article

(This article belongs to the Special Issue The Biology of Animal Reproduction)

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17 pages, 6800 KB

Open AccessArticle

Deletion of ddx4 Ovary-Specific Transcript Causes Dysfunction of Meiosis and Derepress of DNA Transposons in Zebrafish Ovaries

by Yuanyuan Chen, Xing Lin, Jing Dai, Yifan Bai, Fei Liu and Daji Luo

Biology 2024, 13(12), 1055; https://doi.org/10.3390/biology13121055 - 16 Dec 2024

Cited by 1 | Viewed by 1657

Abstract

Alternative splicing of ddx4 (DEAD-box helicase 4), a key germline marker gene, has been reported to generate sex-specific transcripts in zebrafish gonads. The biological functions and regulatory mechanisms of the ddx4 ovary-specific transcript (ddx4-L) during oogenesis remain unclear. In this study, [...] Read more.

Alternative splicing of ddx4 (DEAD-box helicase 4), a key germline marker gene, has been reported to generate sex-specific transcripts in zebrafish gonads. The biological functions and regulatory mechanisms of the ddx4 ovary-specific transcript (ddx4-L) during oogenesis remain unclear. In this study, we found that ddx4-L mutants, in which ddx4-L was specifically deleted, had enlarged ovaries but laid fewer eggs, along with having a lower fertilization rate compared to WT controls. RNA-seq analysis was performed to detect the changes in gene expression between WT and ddx4-L mutant ovaries. A total of 524 upregulated and 610 downregulated DEGs were identified. GO and GSEA enrichment analyses showed that genes involved in fertilization and reproduction biological processes were significantly downregulated. More specifically, we observed a remarkable reduction in Sycp1, a core component of synaptonemal complex, in ddx4-L mutant ovaries at both the mRNA and protein levels. In addition, the expressions of transposon elements, as well as the events of alternative splicing, alternative polyadenylation, and RNA editing, were analyzed based on the RNA-seq data. We found that the deletion of ddx4-L resulted in derepression of DNA transposons in zebrafish ovaries, possibly causing genome instability. In conclusion, our work demonstrates that the ovary-specific ddx4 transcript plays important roles in oocyte meiosis and DNA transposon repression, which extends our understanding of the biological functions and regulatory mechanisms of sex-specific alternative splicing in zebrafish oogenesis and reproduction. Full article

(This article belongs to the Special Issue The Biology of Animal Reproduction)

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Review

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21 pages, 1693 KB

Open AccessReview

Selenium and Mammalian Uterine Health: A Comprehensive Review

by Ruoning Wu, Xiaohan Li, Zhaoming Li, Jilong Luo, Ziwei Zhang and Mengyao Guo

Biology 2025, 14(9), 1295; https://doi.org/10.3390/biology14091295 - 19 Sep 2025

Abstract

Selenium (Se) is an essential trace element for animals and plays an important role in maintaining various physiological functions. In recent years, more and more studies have focused on the relationship between selenium and mammalian uterus health. This review comprehensively summarizes the current [...] Read more.

Selenium (Se) is an essential trace element for animals and plays an important role in maintaining various physiological functions. In recent years, more and more studies have focused on the relationship between selenium and mammalian uterus health. This review comprehensively summarizes the current research progress in this field and introduces the physiological functions of selenium, including its antioxidant, anti-inflammatory, and immune regulatory functions. It also analyzed the role of selenium in uterine development, maintaining pregnancy, preventing and treating uterine diseases from multiple aspects. In addition, factors affecting animal selenium utilization and appropriate selenium supplementation strategies were also introduced. Understanding the relationship between selenium and animal uterine health is of great significance for improving animal reproductive performance, ensuring offspring health, and promoting sustainable development of the animal husbandry. Full article

(This article belongs to the Special Issue The Biology of Animal Reproduction)

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19 pages, 1456 KB

Open AccessReview

Gonadal Development and Its Influencing Factors in the Red Swamp Crayfish (Procambarus clarkii): A Review

by Yuchi Wang, Dengge Xu, Chao Guo, Tanglin Zhang, Jiashou Liu and Wei Li

Biology 2025, 14(9), 1138; https://doi.org/10.3390/biology14091138 - 27 Aug 2025

Viewed by 793

Abstract

The red swamp crayfish (Procambarus clarkii) is a crucial freshwater aquaculture species in China, and the reproductive capacity and farming efficiency are critically dependent on gonadal development. Consequently, the acceleration and synchronization of gonadal maturation constitutes fundamental prerequisites for intensive seedling [...] Read more.

The red swamp crayfish (Procambarus clarkii) is a crucial freshwater aquaculture species in China, and the reproductive capacity and farming efficiency are critically dependent on gonadal development. Consequently, the acceleration and synchronization of gonadal maturation constitutes fundamental prerequisites for intensive seedling breeding. This review systematically synthesizes current knowledge on gonadal development and its regulatory factors in P. clarkii, specifically addressing (1) histological characteristics, (2) developmental stages and cyclical patterns, and (3) key genes involved in gonadal regulation. We particularly focus on the combined effects of intrinsic and extrinsic factors—including temperature, photoperiod, nutrition, and endocrine regulation—on gonadal maturation. This comprehensive assessment offers both theoretical insights into gonadal development mechanisms and practical guidance for enhancing intensive seedling production in this economically vital species. Full article

(This article belongs to the Special Issue The Biology of Animal Reproduction)

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14 pages, 939 KB

Open AccessReview

Revisiting Male Fertility in Livestock: The Case of Bull Sperm RNA

by Rene A. Ramírez-Sosa, Francisco J. Jahuey-Martínez, Monserrath Felix-Portillo and José A. Martínez-Quintana

Biology 2025, 14(8), 969; https://doi.org/10.3390/biology14080969 - 1 Aug 2025

Viewed by 1712

Abstract

To achieve the goals of productivity and sustainability across diverse livestock systems, reproductive factors play a pivotal role. Historically, reproductive research has primarily focused on females, as they are responsible for maintaining pregnancy and delivering offspring following oocyte fertilization. However, since the early [...] Read more.

To achieve the goals of productivity and sustainability across diverse livestock systems, reproductive factors play a pivotal role. Historically, reproductive research has primarily focused on females, as they are responsible for maintaining pregnancy and delivering offspring following oocyte fertilization. However, since the early 2000s, the biological significance of sperm RNAs has been increasingly recognized in various livestock species. These RNAs contribute both genetically and epigenetically at the time of fertilization and during early embryonic development. Multiple types of sperm RNA have been identified in bovine, porcine, ovine, buffalo, and caprine spermatozoa. Notably, transcriptomic profiling has shown potential to differentiate between high- and low-fertility males, even when conventional semen quality values appear normal in both groups. This opens the possibility for more accurate identification of highly fertile sires. Nevertheless, a definitive marker or set of markers has yet to be established, likely due to the transcriptome’s sensitivity to environmental conditions and to the variability in evaluation methodologies. Therefore, global scientific efforts should aim to establish standardized, robust protocols, as sperm RNA represents a promising avenue for enhancing the sustainability of animal production systems. Full article

(This article belongs to the Special Issue The Biology of Animal Reproduction)

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20 pages, 3219 KB

Open AccessReview

The Role of TGF-β Signaling Pathway in Determining Small Ruminant Litter Size

by Ying Han, Guiling Cao, Wenting Chen, Changfa Wang and Muhammad Zahoor Khan

Biology 2025, 14(7), 786; https://doi.org/10.3390/biology14070786 - 29 Jun 2025

Viewed by 665

Abstract

The transforming growth factor-beta (TGF-β) superfamily plays a crucial role in regulating female reproductive traits, particularly litter size, in small ruminants, such as sheep and goats. This review comprehensively examines the molecular mechanisms through which TGF-β superfamily members—including bone morphogenetic proteins (BMPs [...] Read more.

The transforming growth factor-beta (TGF-β) superfamily plays a crucial role in regulating female reproductive traits, particularly litter size, in small ruminants, such as sheep and goats. This review comprehensively examines the molecular mechanisms through which TGF-β superfamily members—including bone morphogenetic proteins (BMPs), growth differentiation factor 9 (GDF9), inhibin (INHA and INHB), and associated signaling genes—influence ovarian follicular development, ovulation rate, and ultimately, litter size. We synthesize recent findings on polymorphisms in key genes, such as BMPR1B, BMP15, GDF9, inhibins and SMADs family genes, across diverse sheep and goat breeds worldwide. The manuscript highlights how specific mutations in these genes create an intricate signaling network that modulates granulosa cell proliferation, follicular sensitivity to FSH, and the prevention of dominant follicle selection. These molecular interactions result in increased ovulation rates and larger litter sizes in prolific breeds. The gene dosage effects observed in heterozygous versus homozygous mutation carriers further illuminate the complex nature of these reproductive regulations. This improved the understanding of the genetic basis for prolificacy provides valuable insights for marker-assisted selection strategies aimed at enhancing reproductive efficiency in small ruminant breeding programs, with significant implications for improving livestock productivity and economic outcomes. Full article

(This article belongs to the Special Issue The Biology of Animal Reproduction)

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