Preliminary Study on Sexual Maturation Pattern of Shenxian Pigs and Molecular Characteristics of Sexual Precocity in Boars

Comments 1:Introduction  

The Introduction requires conceptual tightening and clearer biological framing. The opening should be rewritten to avoid textbook-style definitions and instead introduce Shenxian pigs as a biologically distinctive model of early sexual maturation. The statement that sexual precocity is “desirable” should be reframed to indicate that it is potentially advantageous only under appropriate management, not inherently beneficial. The claim that Shenxian pigs reach sexual maturity at ~3 months must be biologically specified (e.g., first estrus, onset of spermatogenesis, or sexual behaviour). The literature review should be synthesized to define a clear knowledge gap for Shenxian pigs, rather than listing prior studies in other breeds. Finally, the rationale for the two-step transcriptomic design and DEG intersection strategy should be explicitly presented as a biological filtering approach to identify genes underlying the Shenxian precocity phenoty.

Response 1: Thank you very much for your suggestion. I have made revisions to the preface based on your suggestions and streamlined the content of the description. Revise the first and third paragraphs of the introduction in red font.

Comments 2: 2. Results and Analysis
2.1The results are clear and biologically informative, but the section would benefit from more analytical framing and less table-by-table narration. The three boar stages (mounting, penis protrusion, ejaculation) should be briefly linked to functional sexual maturity, and the seasonal differences should be interpreted in terms of reproductive physiology rather than only statistical significance. For sows, the key message is that the third estrus represents a stable and suitable mating stage, supported by both estrus dynamics and litter performance, and this should be stated more directly. The production and selection results (C1/C2 and X1/X2) should be framed as defining practical management thresholds (third estrus, ~5 months, ~60 kg, ~16 mm backfat), rather than mainly reporting P-values.

2.2This section is technically solid and the data quality is well documented, but the presentation should be more synthesis-driven and less procedural. The RNA-seq quality metrics, mapping statistics, PCA and correlation results clearly demonstrate good sample consistency and group separation, so these can be summarized more compactly. The DEG results, GO and KEGG enrichment should be framed more explicitly around testicular maturation biology (spermatogenesis, cilia/flagellum, proliferation and signaling pathways) rather than listed as catalogues of terms. The qPCR validation is appropriate and supports the RNA-seq findings, but it would be stronger if briefly linked to the biological roles of the validated genes in male reproductive maturation.
2.3This section provides a strong second-layer filter that complements the maturity comparison by directly addressing the hormonal basis of the Shenxian precocity phenotype. The RNA-seq quality, sample clustering and DEG statistics support the robustness of the comparison, so these technical results can be summarized more compactly. The key strength is that, unlike Section 2.2, the enrichment here clearly converges on steroid biosynthesis, hormone metabolism and endocrine regulation, which aligns well with the biological hypothesis of precocious sexual maturation. This conceptual shift should be highlighted more explicitly. The selection and qPCR validation of genes such as INSL3, CYP11B2 and SRD5A1 should be framed as identifying a hormonal regulatory signature distinguishing Shenxian pigs from crossbreds, rather than simply confirming DEG lists.
3.4 ?? - 2.4This section presents a clear integrative strategy by intersecting the two transcriptome datasets to prioritize genes linked to the Shenxian precocity phenotype. The approach is sound, but the rationale for narrowing from 39 overlapping upregulated genes to SRD5A1 and CYP11B2 should be stated more explicitly as based on hormonal pathway relevance. The biological interpretation is appropriate, but it would be stronger if briefly linked to known roles of these enzymes in androgen and steroid hormone biosynthesis and timing of puberty, rather than only listing their enzymatic functions.

Response 2: In section 2.1, the seasonal differences mainly highlight the impact of different temperatures on estrus, and we have simplified the content as shown in the red part of section 2.1. In sections 2.2 and 2.3, we simplified the description of the experimental process. We believe that your suggestion to verify gene function is very useful, but this experiment is a preliminary study, and we will conduct further research on a large population by increasing the sample size. Moreover, we have simplified the GO and KEGG analysis sections in sections 2.2 and 2.3, highlighting the differences between the two sequencing results. In section 2.4, we added the corresponding functions of each gene in the table and selected the genes most likely to affect precocious puberty through functional analysis. All the modifications mentioned above can be found in the red font section of 2Results and Analysis.

Comments 3:Discussion

3.3This section successfully identifies a distinct endocrine and metabolic signature distinguishing Shenxian pigs from crossbreds, centred on steroid biosynthesis, hormone regulation and oxidative balance. The choice of candidate genes (INSL3, CYP11B2, SRD5A1, ARSE, AIG1, GPX1, ATG4A, GUSB) is well supported by the enrichment results and literature. To strengthen the narrative, the discussion should be slightly tightened to emphasize that this gene set represents the hormonal and cellular regulation layer of precocity, complementing the sperm-maturation genes from Section 3.2, rather than presenting each gene largely in isolation.

Response 3: In section 3.3, we integrated the functions of all differentially expressed genes screened and summarized them, verifying the important role of this group of genes in hormone regulation. Please refer to the last paragraph in red font in section 3.3 for details.

Comments 4:Materials and Methods

The methodology is comprehensive and technically sound, covering animal phenotyping, sampling, RNA-seq and qPCR validation in sufficient detail for reproducibility. The overall experimental design is coherent and well aligned with the biological questions. To improve clarity, the section could be streamlined by separating more clearly the phenotyping/production part from the molecular transcriptomic workflow, and by slightly reducing the level of procedural detail for standard RNA-seq and qPCR steps. The grouping of animals and comparisons (S1/S2 and S/YS) is appropriate, but should be summarized more explicitly at the beginning of the section to help readers quickly understand the study structure.

Response 4: Thank you very much for your suggestion. We have rewritten section 4 on materials and methods based on the difference between phenotype experiments and molecular experiments, and simplified the experimental process. Please refer to sections 4.1 and 4.2 for details.

Comments 1: Conceptual ambiguity and sometimes incorrect use of fundamental reproductive biology terminology - The terms "sexual maturity," "sexual maturation," and "sexual precocity" are used interchangeably throughout the manuscript, although they represent distinct biological concepts.
In the Introduction and Discussion, early age at first mounting or estrus is automatically interpreted as sexual precocity, without proper physiological validation, for example, semen quality, hormonal profiling;

Response 1: Thank you for pointing this out. We agree with this comment.In the revised draft, we will strictly distinguish and use these terms. In this study, we used "significantly advanced age of first crossing/estrus" as a behavioral phenotype observation indicator suggesting the possibility of premature sexual development, rather than directly as the final diagnosis of "precocious puberty". We fully agree that the exact state of "sexual maturity" and whether it belongs to "precocious puberty" require comprehensive verification based on physiological indicators such as semen quality, reproductive organ development morphology, and hormone dynamics. We have revised the relevant statements in the discussion section, clarified the preliminary observational nature of this study, and emphasized the necessity of conducting further physiological validation in the future.

Comments 2: Insufficient sample size in the methodology - the experimental design raises serious concerns about the statistical representativeness of the study
  Transcriptome sequencing is performed on only 3 animals per group, which is insufficient to support a reliable analysis of differential gene expression.  The RNA-seq analysis is based on 3 animals per group, which is critically insufficient for reliable differential expression analysis. It is not clear.

Response 2: We deeply understand your concern about the representativeness of the sample size. Here, we hope to provide an explanation that takes into account the unique characteristics of our research subject and the widely accepted analytical practices in the current field. Objective reasons for limited sample size: The core material of this study, Shenxian pig, is a rare local pig germplasm resource in China and is currently in a strict conservation stage. The population itself is very limited, and individuals that can be used for scientific research are even more precious. We have made every effort in the experimental design to obtain samples that comply with ethical and conservation principles. Each group of 3 biological replicates (n=3) is the maximum sample size that can be achieved under current conditions and has biological independence. This does constitute a limitation of this study, but it is also a practical challenge that is often faced in conducting cutting-edge omics research using such precious genetic resources. The rationality and scientificity of RNA seq with 3 samples per group: Although larger sample sizes always provide higher statistical power, under specific conditions, RNA seq designs with n=3 samples per group are widely used in exploratory studies and are considered to provide valuable information. Widely accepted practice: Many high-quality studies, especially those involving large animals, rare samples, or preliminary screening stages, use a design with three biological replicates per group. This has become a common starting point for identifying differentially expressed genes (DEGs) between groups. Supplementing statistical methods and ensuring data quality: In order to maximize the reliability of results in limited samples, we have taken the following measures: strict quality control and standardization: We have conducted strict quality control on the raw sequencing data and used algorithms specifically designed for small sample count data such as DESeq2 for standardization and differential analysis. These algorithms adapt to situations with small sample sizes by accurately estimating dispersion. Significant threshold setting: We adopted a relatively strict differential expression threshold (usually | log2FoldChange |>1 and adjusted p-value<0.05) to reduce false positive rates and focus on the genes with the most significant changes. Subsequent validation and enrichment analysis: We consider RNA seq results as a screening tool for generating hypotheses. The differentially expressed gene sets discovered can be enriched and analyzed through pathways such as GO and KEGG, providing more stable and reliable insights from the perspective of biological functional pathways. This set based analysis method is relatively less sensitive to fluctuations in sample size. In addition, we validated key genes on independent samples using qRT PCR to increase their credibility. Clarify research positioning: In the revised manuscript, we will clarify more clearly that this study is a preliminary exploratory transcriptome analysis based on a valuable small population, aimed at screening candidate genes and pathways that may be related to sexual development timing, providing direction and targets for subsequent functional validation in larger populations or through model animals. The result should be interpreted as generating hypotheses rather than final conclusions.

Comments 3：The manuscript repeatedly suggests causal relationships between the identified differentially expressed genes, e.g., in section 3.4, SRD5A1 and CYP11B2 are described as "genes affecting premature sexual maturation." These same genes are then presented as key determinants of early sexual maturity without functional validation. Separately, biological terms/phases are again taken to mean the same stage of development. 

Although the topic is relevant, the manuscript in its current form does not meet the scientific standards for publication in an MDPI journal. Substantial revisions are required, focusing on statistical validity and improving the quality of the language.

Response 2:Your criticism of the possible over inference in our description of gene function is very insightful. We will explicitly emphasize that the differential expression of SRD5A1 and CYP11B2 genes only suggests their potential molecular association in the temporal regulation of sexual development in Shenxian pigs, and the direct causal relationship must be confirmed through subsequent studies such as gene editing, cell function experiments, or genetic association analysis in different populations.We have made modifications in section 3.4 and highlighted them in red font