Behavioral and Physiological Differences in Female Rabbits at Different Stages of the Estrous Cycle.

Estrus involves a series of complex physiological signs and changes in behavior before ovulation, which play a crucial role in animal reproduction. However, there have been few studies that evaluate behaviors during the different stages of estrus cycle in female rabbits. Therefore, more detailed information is needed on distinguishing the various stages of the estrous cycle. This study explored the behavioral and physiological differences at various estrous cycle stages in female New Zealand White rabbits. The continuous recording method was employed to record the daily behaviors of twenty postpartum female rabbits during the estrous cycle. Compared with the diestrus stage, the duration of foraging and drinking behavior in estrus decreased significantly, and the frequency of grooming and biting behaviors increased (p < 0.05). Differences in reproductive hormone levels (FSH, LH, P4, and E2) and follicle development were measured at each stage via ELISA and HE staining. The FSH and LH levels showed an increasing trend and then decreased, with the lowest being in late estrus (p < 0.05). The P4 level was the lowest in estrus (p < 0.05), and E2 showed a gradually increasing trend. There was no significant difference in the number of primordial follicles at each stage, but the number of primary follicles in estrus was significantly higher than at the other stages (p < 0.05). To further understand the molecular regulation mechanism of the estrous cycle in female rabbits, we analyzed the ovarian transcription patterns of female rabbits in diestrus (D group) and estrus (E group) employing RNA-seq. A total of 967 differentially expressed genes (DEGs) were screened from the ovaries of female rabbits between the diestrus and estrus groups. A KEGG analysis of DEGs enriched in the estrogen signaling pathway, aldosterone synthesis, and secretion pathway, such as CYP19A1 and IGF1R, was performed. The rabbits' behavior, related physiological hormones, and molecular regulation also differed at different estrous cycle stages. The results provide recommendations for the adequate management practices of postpartum re-estrus and breeding female rabbits.

Semen quality and seminal plasma metabolites in male rabbits (Oryctolagus cuniculus) under heat stress.

Heat stress causes infertility in male rabbits in summer. This study was conducted to determine the effects of heat stress on semen quality and seminal plasma metabolites of male rabbits. To achieve these objectives, the temperature and humidity index (THI) was used to determine the stress state of male rabbits during different months, thereby the rabbits were divided into heat stress and no heat stress groups. The quality of the semen and the biochemical indices of seminal plasma were then analyzed. Next the plasma metabolites of rabbits in both groups were evaluated using the ultra-high performance liquid chromatography-mass spectroscopy (UPLC-MS)/MS technique. Our results showed that the THI value of the rabbit housing in May was 20.94 (no heat stress). The THI value of the housing in August was 29.10 (heat stress group, n = 10). Compared with the non-heat stress group, the sperm motility, density, and pH in the heat stress group (n = 10) were significantly decreased (P < 0.01); the semen volume decreased significantly (P < 0.05); and the sperm malformation rate increased significantly (P < 0.01). The number of grade A sperm significantly decreased, while the numbers of B and C grade sperm significantly increased (P < 0.01). The total sperm output (TSO), total motile sperm (TMS), and total functional sperm fraction (TFSF) decreased significantly (P < 0.01). Heat stress protein 70 (HSP70) and acid phosphatase (ACP) in the seminal plasma of rabbits in the heat stress group (n = 20) were significantly increased (P < 0.01). Seminal plasma testosterone (T), α-glucosidase (α-Glu), and fructose decreased significantly (P < 0.01). The concentrations of Mg2+ (P < 0.05), Na+ (P < 0.01), and K+ (P < 0.01) in metal ions were significantly decreased. These findings indicated that heat stress severely affected the quality of the male rabbit semen. Furthermore, UPLC-MS/MS technology was used to analyze the seminal plasma samples of rabbits in the heat stress group and non-heat stress group (n = 9 for each group). In total, 346 metabolites were identified, with variable importance in project (VIP) > 1.0, fold change (FC) > 1.5 or < 0.667, and P < 0.05 as the threshold. A total of 71 differential metabolites were matched, including stearic acid, betaine, arachidonic acid, L-malic acid, and indole. The Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis of differential metabolites revealed 51 metabolic pathways, including synthesis and degradation of ketones, serine and threonine metabolism, tryptophan metabolism, and the citric acid cycle. Our study has shown that the sperm motility, sperm pH value, and sperm density of male rabbits decreased significantly under heat stress, and the sperm malformation rate increased significantly. Furthermore, the quality of semen was shown to deteriorate and the energy metabolism pathway was disturbed. These findings provide a theoretical reference for alleviating the adaptive heat stress in male rabbits.

NRAS promotes the proliferation of melanocytes to increase melanin deposition in Rex rabbits.

Melanocytes play a major role in the formation of mammalian fur color and are regulated by several genes. Despite playing the pivotal role in the study of melanoma, the mechanistic role of NRAS (neuroblastoma RAS viral oncogene homolog) in the formation of mammalian epidermal color is still elusive. First of all, the expression levels of NRAS mRNA and protein in the dorsal skin of different colored Rex rabbits were detected by qRT-PCR and Western blot. Then, the subcellular localization of NRAS was identified in melanocytes by indirect immunofluorescence. Next, the expression of NRAS was overexpressed and knocked down in melanocytes, and its efficiency was verified by qRT-PCR and Western blot. Subsequently, NaOH, CCK-8, and Annexin V-FITC were used to verify the changes in melanin content, proliferation, and apoptosis in melanocytes. Finally, we analyzed the regulation of NRAS on other genes (MITF, TYR, DCT, PMEL, and CREB) that affect melanin production. In silico studies showed NRAS as a stable and hydrophilic protein, and it is localized in the cytoplasm and nucleus of melanocytes. The mRNA and protein expression levels of NRAS were significantly different in skin of different colored Rex rabbits, and the highest level was found in black skin (P < 0.01). Moreover, the NRAS demonstrated impact on the proliferation, apoptosis, and melanin production of melanocytes (P < 0.05), and the strong correlation of NRAS with melanin-related genes was evidently observed (P < 0.05). Our results suggested that NRAS can be used as a gene that regulates melanin production and controls melanocyte proliferation and apoptosis, providing a new theoretical basis for studying the mechanism of mammalian fur color formation.

miR-129-5p Participates in Hair Follicle Growth by Targeting HOXC13 in Rabbit.

Mammalian hair formation is critically determined by the growth of hair follicles (HF). MiRNAs are crucial in the periodic development of hair follicles; they maintain epidermal homeostasis by targeting genes and influencing the activity of signaling pathways and related regulators. Our study discovered miR-129-5p to be overexpressed in the skin of Angora rabbits during catagen, and was negatively correlated with HOXC13 expression (Pearson's R = −0.313, p < 0.05). The dual-Luciferase reporter gene detection system and Western blotting confirmed that miR-129-5p targeted HOXC13. In addition, miR-129-5p overexpression was found to significantly inhibit the expression of hair follicle development-related genes (HFDRGs), such as BCL2, WNT2, CCND1, and LEF1 (p < 0.01), and promoted the expression of SFRP2, TGF-ß1, and FGF2 (p < 0.01), which was the same as the knockdown of HOXC13. In contrast, the knockout of miR-129-5p was the opposite, and it demonstrated similar results to the overexpression of HOXC13. CCK8 and flow cytometry demonstrated that miR-129-5p mimics significantly promoted the apoptosis of dermal papilla cells (DPCs) and inhibited proliferation (p < 0.01), while the inhibitor was found to reduce the apoptosis of DPCs and promote proliferation (p < 0.01). These results showed that miR-129-5p can participate in the periodic development of HF by targeting HOXC13, and it can induce apoptosis and inhibit proliferation of DPCs. These results will help to understand the role and mechanism of miR-129-5p in the periodic development of HF, and will provide support for subsequent studies, not only providing a theoretical basis for genetically improving the quality of hair in animals in the future, but also a new theory and method for diagnosing and treating hair loss in humans.