Transcriptomic analysis of mechanism underlying the effect of induced molting on semen quality and reproductive performance in aged Houdan roosters

Abstract

The reproductive performance of breeder roosters has significant economic importance in the poultry industry. Breeder roosters have severely reduced semen quality with age and will be at risk of culling in the following years. In order to extend the use of breeder roosters, we drew on the induced molting model of hens and selected 35 Houdan roosters aged 50 wk for induced molting. By comparing the body weight, testicular weight, semen quality, and reproductive performance before and after induced molting, we found that induced molting could restore the body weight and testicular weight to the levels before molting (P > 0.05). At the same time, it significantly improved sperm motility (P < 0.05) and also improved reproductive performance such as fertilization rate and hatching rate. To further reveal the mechanism underlying the effects of induced molting on semen quality and reproductive performance in aged Houdan roosters, we collected testes from 3 periods: 1 d before fasting (F0), 15 d after fasting (F15), and 32 d after recovery feeding (R32) for transcriptome sequencing analysis. A total of 5,671 genes were detected in F0, F15, and R32, and trend analysis of the 5,671 differential genes showed 2 significant trends (profile 5 and profile 2). KEGG enrichment analysis of the genes in the 2 profiles, revealed significantly enriched pathway regulation of actin cytoskeleton. In the regulation of actin cytoskeleton pathway, we found a protein kinase gene (SRC) and a senescence gene (ROCK2). SRC was highly expressed at F15, leading to the phosphorylation of key substrates, which in turn disrupted the Sertoli cell spermatid connection and the spermiogenesis process, resulting in no mature spermatozoa produced from F15, SRC expression was inhibited at R32, the expression level was reduced, and mature spermatozoa reappeared. The senescence gene ROCK2 was highly expressed at F15 compared to F0 and R32, which may have been responsible for inducing senescence atrophy in the testes.

Keywords: Houdan rooster; induced molting; regulation of actin cytoskeleton; semen quality.

Figure 1

Changes in appearance, weight, and weight of testes in different periods. (A) Photographs of testes in different periods. (B) Changes of testis weight in different periods. (C) Changes of chicken weight in different periods. (D) The change of weight loss rate of chickens in different periods. Figure 1 Photographs were taken in 90 mm cell culture dishes. If the same letter label is not used, there is a difference (P < 0.05).

Figure 2

Effect of induced molting on semen quality and reproductive performance of aged Houdan roosters. (A) Changes of sperm density in different periods. (B) Changes of sperm motility in different periods. (C) Comparison of fertilization rate, stillbirth rate and hatching healthy chick rate between molting Houdan rooster group and nonmolting Houdan rooster group. If the same letter label is not used, there is a difference (P < 0.05).

Figure 3

The effect of induced molting on the morphology and function of testis tissue of aged Houdan roosters. (A) Slices of cock testes in different blue arrow indicate the diameter of seminiferous tubules. (B) Diameter of seminiferous tubules at different stages. Mature sperm (MS). If the periods of starvation, 4 pictures in the upper part of Figure A are 10× photographic results (scale: 200 µm), and 4 pictures in the lower part are 40× photographic results (scale: 50 µm), the same letter label is not used, there is a difference (P < 0.05).

Figure 4

Differentially expressed genes in F0, F15, and R32. (A) The number of differentially expressed genes in F0, F15, and R32, the X-axis represents 3 periods (F0, F15, and R32). Blue represents transcripts that were significant upregulated, and red indicates that those transcripts were significantly downregulated. The parameters P-value < 0.05 and Log FC ≥ 1 were used as the thresholds to judge the significance of gene expression differences. (B) The scatter plot of the differentially expressed genes in “F0 vs. F15.” (C) The scatter plot of the differentially expressed genes in “F15 vs. R32.” (D) The scatter plot of the differentially expressed genes in “F0 vs. R32.” In Figures B–D, blue and red scatter plot represent upregulated genes and downregulated, respectively. Gray scatter plot represents that the genes were not differential expressed.

Figure 5

Trend analysis of DEGs enrichment across the F0, F15, and R32. (A) Trend analysis of differentially expressed genes. The 5,671 DEGs from F0, F15, and R32 were clustered into 8 profiles. (B) DEGs GO annotation analysis in profile 5. (C) DEGs GO annotation analysis in profile 2. (D) Top 20 KEGG pathway enrichment of DEGs in profile 5, the bubble size represents the number of DEGs, and the bubble color represents the Q-value. (E) Top 20 KEGG pathway enrichment of DEGs in profile 2, the bubble size represents the number of DEGs, and the bubble color represents the Q-value.

Figure 6

Expression comparison of 9 genes by qRT-PCR and RNA-Seq in 3 periods F0, F15, and R32. (A) ACTB; (B) SRC; (C) PAK1; (D) IQGAP2; (E) GSN; (F) PTK3CD; (G) FOXO1; (H) ROCK2; (I) SOX9. The black color represents the qRT-PCR analyses of the gene expression levels. The gray color represents the transcriptome sequencing analyses (RPKM) of the gene expression levels.