Sucrose is known to play an important role in the cryopreservation of sperm and female gonads; however, its effect on the cryopreservation of pig spermatogonial stem cells (pSSCs) has not been tested.  The aim of this work was to study the effect of sucrose during pSSC cryopreservation and to find the most effective concentration in freezing medium.  pSSCs were cryopreserved with freezing media containing different concentrations of sucrose (70, 140, 210, and 280 mmol L^–1) and a control group without sucrose.  The survival rates, plasma membrane integrity, and mitochondrial membrane potential of thawed cells were detected by trypan blue (TB) staining, SYBR-14/propidium iodide (PI) dual staining, and JC-1 staining, respectively.  All the staining results showed an obvious increase in cell survival in the sucrose-treated groups as compared to that in the control group, with the exception of 280 mmol L^–1 sucrose.  Moreover, the 210 mmol L^–1 sucrose group yielded the highest survival rate among all the groups (P<0.05).  The results of SYBR-14/PI dual staining and JC-1 staining were consistent with those of TB staining as above described.  Quantitative real-time PCR (qRT-PCR) indicated that the mRNA levels of three apoptosis-promoting genes (BAX, APAF1 and CASPASE9) were significantly higher in thawed cells than in cells before freezing (P<0.05).  Moreover, the mRNA level of one anti-apoptotic gene (XIAP) was significantly lower in thawed cells than in cells before freezing (P<0.05).  When comparing the mRNA expression of apoptosis-related genes in thawed cells, the mRNA level of the anti-apoptotic genes in the control group was significantly lower than that in the sucrose-treated
groups (P<0.05).  Western blot analyses showed that the expression levels of cleaved CASPASE9, CASPASE3 and PARP-1 in the sucrose-treated groups were lower than those in the control group and were the lowest in the 210 mmol L^–1 sucrose group.  Both qRT-PCR and Western blot analyses suggested that sucrose inhibited cell apoptosis during freezing and thawing.  Briefly, sucrose promoted pSSCs survival after freezing and thawing, especially at a concentration of 210 mmol L^–1, which possibly assisted pSSC dehydration and inhibited cell apoptosis.  These findings hold great promise for further studies of the regulatory mechanism of proliferation and differentiation of pSSCs. 

Previous studies have shown that octamer-binding transcription factor 4 (Oct4) plays a significant role in early embryonic development of mammalian animals, and different Oct4 expression levels induce multi-lineage differentiation which are regulated by DNA methylation. To explore the relationship between the methylation pattern of Oct4 gene exon 1 and embryonic development, in this work, five different tissues (heart, liver, lung, cerebrum and cerebellum) from three germ layers were chosen from low age (50–60 d) and advanced age (60–70 d) of fetal cattle and the differences between tissues or ages were analyzed, respectively. The result showed that the DNA methylation level of Oct4 gene exon 1 was significant different (P<0.01) between any two of three germ layers in low age (<60 d), but kept steady of advanced age (P>0.05) (>60 d), suggesting that 60-d post coital was an important boundary for embryonic development. In addition, in ectoderm (cerebrum and cerebellum), there was no significant methylation difference of Oct4 gene exon 1 between low age and advanced age (P>0.05), but the result of endoderm (liver and lung) and mesoderm (heart) were on the contrary (P<0.01), which indicated the development of ectoderm was earlier than endoderm and mesoderm. The methylation differences from the 3rd, 5th and 9th CpG-dinucleotide loci of Oct4 gene exon 1 were significantly different between each two of three germ layers (P<0.05), indicating that these three loci may have important influence on bovine embryonic development. This study showed that bovine germ layers differentiation was significantly related to the DNA methylation status of Oct4 gene exon 1. This work firstly identified the DNA methylation profile of bovine Oct4 gene exon 1 and its association with germ layers development in fetus and adult of cattle. Moreover, the work also provided epigenetic information for further studying bovine embryonic development and cellular reprogramming.

Paired-like homeodomain transcription factor 1 (PITX1) plays an important role in pituitary development by indirectly regulating the expression of the GH and PRL genes, and therefore PITX1 gene is regarded as a potential candidate gene for building the relationship between the gene polymorphism and milk traits. The aim of this study was to explore the novel genetic variant in PITX1 gene and its effect on milk performance in dairy goats. Herein, a novel genetic variation (NW_00314033: g.201G>A or IVS1+41G>A) located at nt41 position of the first intron of the goat PITX1 gene was reported at the P1 locus, which can be genotyped by the Msp I PCR-RFLP. In the Msp I PCR-RFLP analyis, the GG variant was a major genotype, and the A variant was a minor allele in Guanzhong dairy goats which was at Hardy-Weinberg disequilibrium (chi-square c2=140, P<0.01). The establishment of associations between different genotypes and milk performance was performed in the analyzed population. A total of three significant associations of the polymorphism with average milk fat content (%) (P=0.045), morning milk fat content (%) (P=0.049), and afternoon milk fat content (%) (P=0.050), were found, respectively. A significant relationship between the polymorphism and average total solid content (P=0.029) was also detected. This novel single nucleotide polymorphism (SNP) extended the spectrum of genetic variation of the goat PITX1 gene, and its significant association with milk performance would benefit from the application of DNA markers related to improving milk performance through marker-assisted selection (MAS) in dairy goats.