GnRH Immunocastration in Male Xizang Sheep: Impacts on Rumen Microbiome and Metabolite Profiles for Enhanced Health and Productivity

Abstract

Castration is a prevalent and indispensable practice in sheep husbandry, aiding in enhancing meat quality, mitigating aggressive behavior, and managing unwanted reproduction. Nevertheless, the conventional surgical castration procedure poses several challenges, including heightened stress and pain, detrimental impacts on animal welfare, and diminished economic efficacy in farming operations. Consequently, immunocastration methods, serving as substitutes for surgical castration, are progressively finding application in livestock. The rumen, an essential and distinctive digestive and absorptive organ in ruminants, has been associated with enhanced meat quality and productive performance following castration in previous research studies, albeit fewer investigations have explored the potential impacts of GnRH immunization on the rumen's internal milieu in sheep post-de-escalation. Hence, the present study delved into evaluating the impact of GnRH immunocastration on the rumen microbiome and metabolomics in male Xizang sheep. This was achieved through the establishment of a GnRH immunocastration animal model and the collection of rumen fluid for microbiological and comprehensive metabolomics investigations. The outcomes of this investigation unveiled that the impact of GnRH immunocastration on body weight gain was more pronounced during the achievement of the castration objective. In addition, the Firmicutes-to-Bacteroidota ratio in the immune male (IM) group exceeded that of the control group (EM), suggesting that GnRH immunodeficiency may enhance the digestion and absorption of feed in male Xizang sheep. At the taxonomic level, the elevated presence of Prevotella and Quinella bacteria in the IM group compared to the EM group indicated that castration influenced a segment of the rumen microbiota in male Xizang sheep, thereby bolstering the digestive and metabolic efficacy of the rumen concerning nutrient utilization, particularly in the breakdown and absorption of proteins, carbohydrates, and lipids, ultimately expediting the fattening process and weight gain in male Xizang sheep following castration. Moreover, analysis of ruminal fluid metabolomics revealed that GnRH immunization had notable impacts on certain metabolites in the ruminal fluid of male Xizang sheep, with metabolites like 5-hydroxyindole acetic acid and 3-hydroxyindole acetic acid showing significant downregulation in the IM group compared to the EM group, while niacin and tyramine exhibited significant upregulation. These findings indicate a profound influence of GnRH immunization on the maintenance of ruminal equilibrium and ruminal health (including the health of ruminal epithelial cells). This study validates that GnRH immunocastration not only achieves the objectives of castration but also enhances ruminal health in male Xizang sheep, thus laying a foundational theoretical basis for the application and dissemination of GnRH immunocastration technology.

Keywords: GnRH immunocastration; male Xizang sheep; microorganisms; rumen.

Figure 1

Changes in serum testosterone and body weight in three groups of male Xizang sheep (5 sheep in each group). (A) Serum testosterone levels in three groups of male Xizang sheep on day 112 of the official trial period. (B) Changes in body weights of male Xizang sheep in the three groups during the formal test period (day 1 was the time of the first GnRH vaccination in the IM group and day 56 was the time of the second GnRH vaccination in the IM group). ** denotes highly significant difference; ns denotes non-significant difference; different letters denote significant difference (p < 0.05); the same letter denotes non-significant difference (p > 0.05).

Figure 2

Effect of GnRH immunization on rumen microbial diversity in male Xizang sheep (EM VS IM, 5 sheep in each group). (A) ASV analysis of microorganisms. (B) Principal component analysis (PCoA) of microorganisms. (C) Chao1 exponential analysis of microorganisms (ns indicates that the difference is not significant). (D) Shannon exponential analysis of microorganisms (ns indicates that the difference is not significant).

Figure 3

Effect of GnRH immunocastration on rumen microbial composition in male Xizang sheep (EM VS IM, 5 sheep in each group). (A) Percentage of rumen microbial abundance at portal level. (B) Percentage of rumen microbial abundance at genus level. (C) Analysis of rumen microbial LEfSe (LDA effect size).

Figure 4

Effect of GnRH immunocastration on metabolite composition of rumen fluid in male Xizang sheep (EM VS IM, 5 sheep in each group). (A) OPLS-DA analysis of rumen metabolome. (B) volcano plot analysis of rumen metabolome. (C) Ring plot of rumen differential metabolite class composition. (D) Bar plot of rumen metabolite differential multiplicity.

Figure 5

KEGG enrichment analysis of rumen fluid differential metabolites. (A) Differential metabolite pathway categorization diagram. (B) Differential metabolite pathway enrichment plot. (C) Differential metabolite abundance score plot.

Figure 6

Association analysis: (A) Association analysis of top 10 phylum level microbiota with top 20 differential metabolites. (B) Association analysis of top 10 genus level microbiota with top 20 differential metabolites. * Significant correlations are indicated.