| During the semen production of Italian Mediterranean bulls,it was found that the semen quality of some bulls was not high,resulting in decreased sperm motility and increased deformity rate.To understand the reasons for the difference in semen quality of bulls,this study tested the physiological body condition,semen quality and seminal biochemical indexes of bulls.At the same time,non-targeted metabolomics and quantitative proteomics techniques were used to analyze the seminal plasma of bulls with different viability.The seminal plasma was analyzed for metabolites and protein levels to clarify the changes in the biochemistry,metabolism and protein levels of seminal plasma with different viability.The results provided some references for further understanding the molecular mechanisms affecting buffalo semen quality.1.Analysis of semen quality and determination of physiological and biochemical indexes of Italian Mediterranean bulls with different viabilityFirstly,the sperm motility of the bulls was tested and divided into normal motility and low motility groups.Physiological parameters,semen quality parameters and sperm motility parameters of bulls were detected.The content of seminal plasma hormone,related enzymes,metal elements and antioxidant indexes were analyzed.The results showed:(1)The sperm motility of the normal motility group was 0.68±0.01,and that of the low motility group was0.23 ± 0.09.(2)Compared with the low motility group,the body weight,respiratory rate and rectal temperature of the normal vitality group bulls were not significantly different(P>0.05),indicating that the physiological body conditions of the two groups were basically the same.(3)The normal motility group had relatively complete acrosome membrane and plasma membrane,and the malformation rate was low.All the motility indexes of normal motility sperm were better than those of low motility sperm.(4)Compared with the low motility group,the testosterone hormone level,fructose content,and some enzyme contents and activities in the seminal plasma of bulls of the normal motility group were increased,while the triglyceride content was extremely significantly decreased(P<0.01).The level of some important metal elements were increased.Seminal plasma had a strong antioxidant capacity.2.Study on non-targeted metabolome of seminal plasma of Italian Mediterranean bulls with different viabilityIn order to further understand the differences in the metabolic levels of the seminal plasma of different motility,liquid chromatography-mass spectrometry(LC-MS)was used to screen out the significant difference metabolites between the groups,and then the metabolic pathways involved in the difference metabolites were enriched and analyzed to find out the potential targeted metabolic pathways in the seminal plasma of different motility bulls.The results showed that a total of 1750 metabolites were identified,and 37 differential metabolites were screened out(VIP>1 and P-value<0.05).Metabolic pathway analysis showed that differential metabolites were mainly involved in 28 metabolic pathways,including amino acid biosynthesis and catabolism,glutathione metabolism,TCA cycle,fatty acid degradation and so on.3.Study on the proteomics of Italian Mediterranean bulls seminal plasma with different viabilityThe i TRAQ technology was used to detect different motility seminal plasma proteins,and the changes of protein levels were analyzed.The molecular mechanism of the formation of different motility was discussed in combination with non-targeted metabolomics.The results showed that a total of 2298 proteins were identified,of which 2179 proteins were quantified.Compared with the low motility group,the number of differentially expressed proteins was188(including 107 up-regulated and 81 down-regulated).The metabolic pathways of differential proteins were concentrated in energy metabolism,fatty acid metabolism and some amino acid metabolism related pathways.Six different proteins were selected for Western Blot analysis,and the results showed that they were consistent with the proteomics results.In summary,there are differences in seminal plasma metabolism and protein levels in different motility Italian Mediterranean bulls,and the differences in metabolites and proteins are mainly related to energy metabolism,fatty acid metabolism,and amino acid metabolism. |
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