Study On Molecular Mechanisms Of The Effect Of Castration On Fat Deposition In Male Pigs

Gonad plays an important role in regulating growth and development of animals. Catration increases fat deposition in male pigs, and this influence growth performance and carcass trait of pigs. Until now, the molecular mechanisms underlying the effects of castration on fat deposition in male pigs remain unclear. This study was conducted to investigage the effects and mechanisms of castration on fat deposition in male pigs. The experiment mainely studied the differences of growth performance, meat quality, serum hormnes and lipid parameters, carcass fat content, back fat thickenss and adipose tissue morphology, and hormone related gene expression in the hypothalamus, pituitary, liver and adipose tissues between castrated and intact male pigs. Meanwhile, this study also analysized differential gene expression and identified their new alternative splicing and transcripts in subcutaneous adipose tissue from castrated and intact male pigs by using RNA-Seq method.Eighteen pairs of male piglets were selected from the breeding farm at Zhejiang Unversity according to a matched-pairs design. Each pair was composed of two male full siblings with similar body weight, and one of the piglets from each pair was randomly selected to be castrated at 35 days old. All pigs were slaughtered at 210 days old. Results from slaughter trail showed that:the castrated pigs had lower body weight, lean meat percentage, bone and skin percentage, but showed significantly higher carcass fat content. The castrates had 2.48kg higher carcass fat weight,0.28kg higher leaf fat weight,6.94% higher fat percentage and 23.02% higher average backfat thickness than those from intact male pigs. They also had larger adipocytes diameter of back subcutaneous fat than intact males (P< 0.05). Castration of male pigs led to decreased serum testosterone, GH, IGF-Ⅰand free fatty acids (FFA) levels (p< 0.05), and led to increased leptin, triglyceride (TG), total cholesterol (TC), high density lipoprotein (HDL-C) and low density lipoprotein (LDL-C) levels in serum (P < 0.05). However, castration had no significant effects on serum insulin, IGF-Ⅱand glucose levels in male pigs (p> 0.05). Castration decreased significantly hypothalamus GHRH (P< 0.05), pituitary GH and GHRHR (P< 0.05), liver GHR (P < 0.05), IGF-I and IGF-IR (P< 0.01), subcutaneous adipose tissue IGF-I and IGF-IR (P< 0.05) mRNA expression levels, but had no significant effects on adipose tissue GHR and leaf fat adipose tissue IGF-I and IGF-IR mRNA expression levels (P> 0.05).To further investigate the molecaular mechanisms of castration on regulating fat deposition in male pigs, we investigated castration-induced transcriptome changes in subcutaneous adipose tissue (3 pigs per treatment) from male pigs by using high-throughput deep sequencing technology (RNA-Seq). The results are as follows: a total of 7610 significantly differential expression genes were abtained from back fat adipose tissue in castrated male pigs. The number of genes that were more than 2 fold changes was 1919, and in which of them,850 were up-regualted and 1069 were down-regulated. Eight differentially expressed genes wich were closely related to fat metabolism were selected and quantified by qRT-PCR. Results illustrated the expression patterns of the eight genes were similar to those from RNA-Seq. Significant analysis of GO showed that castration could increase mRNA expression of fatty acid synthesis related genes (such as ACCα, SCD, ME1, LPIN1 and FABP3) and decrease mRNA expression of fatty acid oxidation and lipolysis related genes (such as HSL, ACOX3 and ACADM). Castration led to an increase of mRNA expression of inflammation and immune related genes like TNFSF9, IL4R, IL17RE, TLR4, MYD88 and MIF. Castration also changed mRNA expression of lipid metabolism related genes (such as LPL, ADIPOR2, APOE, APOL6 and LDLRAP1). In addition, we identified that a lot of genes had alternative splicing of transcripts, and about 7×105 splicing junctions were predicted in subcutaneous adipose tissue from castrated and intact male pigs. We also investigated whether splicing patterns for transcripts sharing the same transcriptin start site differ between castrated and intact male pigs'adipose tissue. The results showed that lots of genes (such as ACOX3* CEBPy,LDHD,ME1,PLIN2,APOA1,AQP3,FTO,PPARa and PPARy) were detected to spliced into different types of isoforms, and these isoforms had different expression levels between castrated and intact male pigs'adipose tissue. For example, after alternative splicing, PPARy got three types of transcriptional isoforms (A, B and C). Isoform A and B had higher mRNA expression leves in castrated male pigs. In contrast, isoform C had higher mRNA expression leves in intact male pigs. Moreover, total expression levels of PPARy were higher in castrated male pigs than intact male pigs.From these results, possible mechanisms of castration on regulating fat depositon in male pigs were that:1) Castration increased fat deposition in male pigs by reducing hypothalamus-pituitary-liver axis GH and IGF-I secretion and release.2) Castration increased fat deposition in male pigs by decreasing lipolysis related genes such as HSL, ACOX and ACADM, and increasing lipogenesis related genes such as PPARy, ACCα, SCD, ME1 and LPIN1.3) Castration increased activation of TLR4, and release of downstream inflammation and immune factors (TNFSF9, IL4R, IL17RE, and CD97) in MYD88 pathway, increased adipocyte inflammation, and then increased the differentiation and proliferation of adipocyte and fat deposition in male pigs.4) Castration increased fat deposition in male pigs by decreasing lipid metabolism related genes like ADIPOR2, APOE, APOL6 and LDLRAP1 in adopose tissue.