Indigenous pig populations, including Bamei pigs (BM), Hezuo pigs (HZ), Huixian Qingni Black pigs (HX), and Minxian Black pigs (MX) in Gansu Province, live in a particular climate and a relatively closed geographical environment. These local pig breeds are characterized by excellent characteristics (e.g., cold tolerance, robust disease resistance, and superior meat quality). In the past few years, pig populations in Gansu Province have decreased significantly because of their poor lean meat percentage, high fat content, and slow growth rate. Maintaining the diversity of these four breeds can act as a source of new alleles to be incorporated into commercial breeds which are more susceptible to disease and less adaptable to changing conditions because of inbreeding. Genomic data analysis is adequate for determining the genetic diversity and livestock breeding population structure, even in local pig populations. However, the genetic diversity and population structure of the four native pig populations in Gansu Province are still unknown. Thus, we used “Zhongxin-I” porcine chip for the SNP detection of 102 individuals living on four pig conservation farms. A total of 57,466 SNPs were identified among the four pig breeds. The linkage disequilibrium (LD) plot showed that MX had the highest level of LD, followed by BM, HZ, and HX. The observed heterozygosity (Ho) in all four populations was higher than the expected heterozygosity (He). A principal component analysis (PCA) demonstrated that the four local pig populations were isolated. The identity displayed by the state matrix and G matrix heat map results indicated that small numbers of individuals among the four pig breeds had a high genetic distance and weak genetic relationships. The results of the population genetic structure of BM, HZ, HX, and MX pigs showed a slight genetic diversity loss. Our findings enabled us to better understand the genome characteristics of these four indigenous pig populations, which will provide novel insights for the future germplasm conservation and utilization of these indigenous pig populations.

Kisspeptin, a neuropeptide encoded by the Kiss1 gene, combines with its receptor Kiss1R to regulate the onset of puberty and male fertility by the hypothalamic–pituitary–gonadal axis. However, little is known regarding the expression signatures and molecular functions of Kiss1 in the testis. H&E staining revealed that well-arranged spermatogonia, spermatocytes, round and elongated spermatids, and spermatozoa, were observed in 4-, 6-, and 8-month-old testes compared to 1- and 3-month-old testes of Hezuo pigs; however, these were not observed in Landrance until 6 months. The diameter, perimeter, and cross-sectional area of seminiferous tubules and the perimeter and area of the tubular lumen increased gradually with age in both pigs. Still, Hezuo pigs grew faster than Landrance. The cloning results suggested that the Hezuo pigs’ Kiss1 CDS region is 417 bp in length, encodes 138 amino acids, and is highly conserved in the kisspeptin-10 region. qRT-PCR and Western blot indicated that the expression trends of Kiss1 mRNA and protein were essentially identical, with higher expression levels at post-pubertal stages. Immunohistochemistry demonstrated that the Kiss1 protein was mainly located in Leydig cells and post-pubertal spermatogenic cells, ranging from round spermatids to spermatozoa. These studies suggest that Kiss1 is an essential regulator in the onset of puberty and spermatogenesis of boars.

With the continuous advancement in reproductive biology, oocyte vitrification has become a critical technology for preserving female germplasm and protecting it from environmental disruptions. This technique also eliminates temporal and spatial constraints in animal embryo engineering research. However, during the vitrification of animal oocytes, exposure to low temperatures and high concentrations of cryoprotectants can cause various forms of damage, including cytoskeletal disruption, spindle abnormalities, mitochondrial dysfunction, apoptosis, oxidative stress and epigenetic modifications. These issues are now understood to severely restrict the subsequent developmental competence of oocytes, resulting in lower cleavage and blastocyst formation rates than those of fresh oocytes. Currently, the mechanisms of cryodamage in vitrified oocytes remain poorly understood, and standardized strategies to enhance vitrification efficiency have yet to be firmly established. This review provides a formal overview of the physiological factors underlying oocyte sensitivity to vitrification, alongside the mechanisms of cryodamage and the variables influencing post-thaw survival and reproductive success. It evaluates strategies for mitigating vitrification-induced stress, compares interspecies differences, and addresses current research limitations. By identifying future directions, this review offers new insights for optimizing mammalian oocyte cryopreservation techniques.

In vitro maturation (IVM) of oocytes is a critical initial step in mammalian in vitro production (IVP), and its quality directly influences the developmental competence of subsequent embryos. Oxidative stress is a major constraint on oocyte quality, which can be mitigated by exogenous antioxidants. In this study, porcine oocytes were matured in IVM medium supplemented with the antioxidant N-acetyl-L-cysteine (NAC; 0, 0.5, 1.5, and 4 mM) to evaluate its effects on maturation rate, antioxidant capacity, mitochondrial function, vitrification–warming survival rate, post-warming ROS levels, and early embryonic developmental rate. To elucidate the molecular mechanisms underlying the effects of NAC on porcine oocyte maturation, we conducted single-cell transcriptome sequencing. The results showed that, versus the control, 1.5 mM NAC substantially enhanced the IVM rate (p < 0.05), reduced ROS levels (p < 0.05), and increased mitochondrial activity, as assessed by MitoTracker, mitochondrial membrane potential (MMP), and ATP content (p < 0.05). These results suggest that 1.5 mM NAC relieves oxidative stress in oocytes and improves mitochondrial function. However, NAC addition showed no significant differences in vitrification–warming survival rate and post-warming ROS levels relative to the control group (p > 0.05). In addition, 1.5 mM NAC markedly improved the cleavage rate and blastocyst rate of porcine oocytes after in vitro fertilization (IVF, p < 0.05), and also enhanced the cleavage rate after parthenogenetic activation (PA, p < 0.05). Single-cell transcriptome sequencing revealed that, versus the control, differentially expressed genes (DEGs) identified after 1.5 mM NAC supplementation were mainly enriched in pathways related to oxidative phosphorylation (OXPHOS), spliceosome, and ubiquinone/terpenoid-quinone biosynthesis. Among these, the OXPHOS pathway showed the most significant enrichment, with upregulated expression of pathway-related genes such as COX6C, CYCS, and SDHD. The accuracy of the transcriptomic results was further validated by qPCR. In conclusion, supplementing with 1.5 mM NAC relieved oxidative stress, improved mitochondrial function, and thereby promoted oocyte maturation and improved oocyte quality, ultimately facilitating subsequent IVF early embryonic development.