Study of the reproductive performance and the immunological response in zebrafish (Danio rerio) fed with bee pollen supplemented diets

Abstract

Bee pollen is a natural product collected by bees from plants and stored in the hive mixed mainly with salivary enzymes. It is a rich source of various nutrients, so it can be an excellent dietary supplement. This product has been described with many beneficial properties, such as antimicrobial, immunostimulating and antioxidants effects, which reflects in possible therapeutic features for different pathological situations. As diet can be associated with animal performance and reproduction, microbiota modulation and potentially factor for cancer, this study aimed to analyze if dietary bee pollen addition could influence zebrafish parameters. The zebrafish Danio rerio is a teleost fish from tropical freshwater, widely used as an experimental model to study complex vertebrate biology using many genetic approaches. The identification of mechanisms involved in physiological responses of fish submitted to supplemental treatment with bee pollen can provide important information for the recommendation of this product in the diet of also other species. This work studied bee pollen addition to the zebrafish diet, focusing its effect on reproduction, growth parameters, intestinal modulation and response against tumor development. Adult zebrafish were fed with different diets, three times a day, using commercial feed, brine shrimp and bee pollen. The fish received the diets for 60 days and throughout this period were tested weekly for: total number of eggs, egg production per female, embryo viability rate (72hpf), larval survival rate after exposure to Spring Viramae of Carp virus, larval survival rate after exposure to Salmonella enterica serovar Typhimurium and analysis of neutrophil recruitment after tail wound. After this, fish from each treatment were analyzed for weight gain and increased length, and the intestines were collected to assess the intestinal microbiota through metagenomics analysis, which enables the identification of bacterial diversity between samples. Also, serum amyloid A (saa) transcript levels from abdominal organs and from separated intestines were analyzed, as the protein encoded by this gene has effects on immune cells and some intestinal bacteria strongly induce its production. After 120 days of diet, fish remaining from each of the treatments were evaluated for tumor growth after melanoma allotransplantation procedure, a very aggressive form of skin cancer. Our results show that bee pollen failed to improve egg production and embryo viability. Instead, the offspring of breeders fed with bee pollen diets showed longer survival upon virus exposure and higher neutrophil migration to wounds. These results indicate that bee pollen can influence vertical immunity through important mechanisms related to offspring immunity in the early stages. Bee pollen diet also revealed different gut microbial abundance at family, genus and species level compared with fish from control group; and, unexpectedly, fish fed with bee pollen showed higher tumor growth rate and larger tumor size. Although some studies attribute bee pollen with antitumor activities, mostly in vitro experiments, our results show that this link should be questioned. Due to its variable composition, the effects caused by bee pollen ingestion need deeper investigation before recommendation, as it can also vary between different species and physiological states.