The reproductive potential of earthworm Eudrilus eugeniae is influenced by the sugarcane bagasse, cow dung, and elephant dung as dietary supplements. The aim of the present work is to determine the earthworm reproduction through the following reproductive parameters, viz., hatching of cocoons, sperm count, and fatty acid profile in seminal vesicle of sperm cells analyzed for the first time. Thus, the gas chromatography mass spectroscopy (GCMS) technique is used to identify the bioactive fatty acids present in the sperm cells of earthworm, and there is a lack of information on its fatty acid profile. Interestingly in all the experimental treatments, the presence of essential saturated and polyunsaturated fatty acids, viz., palmitic acid, linoleic acid, octadecanoic acid methyl ester, oleic acid, and cholesterol compounds, was recorded. The results revealed that the earthworm feed in T3 (50\% sugarcane bagasse + 25\% cow dung + 25\% elephant dung) showed maximum hatching of cocoon, sperm count, and also the presence of higher percentage of cholestanoid (60.84\%) and fatty aldehyde (14.57\%) compounds, and it indicates higher quality of suitable substrate for earthworm reproduction. In the T2 treatment (50\% sugarcane bagasse + 50\% elephant dung), observed maximum percentage of fatty alcohol (5.4\%) and fatty acid methyl ester (12.88\%) compounds due to these dietary fatty acids, which enhanced the sperm production and also have a good impact on earthworm reproduction. Polyunsaturated and saturated fatty acids 23.39\% are found in T1 treatment (50\% sugarcane bagasse + 50\% cow dung), and due to the presence of cow dung, it undergoes higher degree of saturation in the absorbed fatty acids. Different fatty acid compositions observed in seminal vesicle of treated earthworms and its combined mixture of both ruminant and monogastric animal dung were metabolized in the earthworm system and stored as fatty acids in the earthworm sperm cells due to the various substrates as a food source. Hence, based on the present finding, we found that the reproductive rate was higher in T3 treatment, followed by T2 and T1.