Differences in growth of Trypanoplasma borreli in carp serum is dependent on transferrin genotype.

Kinetoplastid parasites require transferrin (Tf), being the main source of iron, for growth and multiplication. This group of parasites developed a unique receptor-mediated system for acquiring host Tf which bears no structural homology with the host transferrin receptor. Trypanoplasma borreli, a blood parasite of common carp, probably uses a similar mechanism to sequester iron from host transferrin. In this study, we demonstrate a critical role of Tf for parasite growth. For in vitro studies we isolated and purified Tf from carp homozygous for the D or G allele of Tf. We obtained Tf-depleted serum using specific antibodies to carp Tf and studied gene expression in vivo during T. borreli infection with Real Time-quantitative PCR. We demonstrate that T. borreli cannot survive in medium supplemented with Tf-depleted serum while reconstitution with Tf restores normal growth. The critical role of Tf for parasite survival was shown in incomplete medium (medium without serum): addition of purified Tf significantly increased parasite survival. We also demonstrate that Tf polymorphism has a significant impact on T. borreli multiplication. Cultured parasites die more quickly in an environment containing D-typed Tf, as compared to medium with G-typed Tf. Gene expression during T. borreli infection in carp did not show an acute phase response. We could, however, observe an increased transcription of Tf in the head kidney, which may be associated with an immunological function of the Tf protein.

Potassium ions in extender differentially influence the post-thaw sperm motility of salmonid fish.

Potassium ions are known to have an inhibitory effect on the sperm motility of salmonids. For this reason, the addition of K(+) to the extender is frequently applied. However, the effect of the addition of K(+) to the extender has not yet been tested. The aim of this study was to test the influence of potassium ion supplementation of the extender on the sperm motility parameters from five Salmonidae species (rainbow trout (Oncorhynchus mykiss), sex-reversed female rainbow trout, whitefish (Coregonus lavaretus), brown trout (Salmo trutta) and brook trout (Salvelinus fontinalis)). Semen samples were diluted in extender containing 0.18 M glucose in 9% methanol (GM) supplemented with 0, 20 or 40 mM potassium chloride. After thawing sperm were stored for 30, 60, 120 and 240 min at 4 °C. Our results demonstrated that the presence of potassium ions in the extender had a negative effect on percentage of motile sperm in four of the salmonid species. In contrast, potassium ions appeared to have a positive effect on percentage of post-thaw motile sperm in whitefish semen. However, this effect could be mimicked by changing the osmolality of the extender (which was achieved by increasing the glucose concentration to 0.22 M). The addition of potassium ions turned out to have no positive effect on post-thaw storage time. Our results suggest that osmolality, rather than potassium ions, seems to be essential for cryopreservation success of salmonids sperm. Further studies should focus on the effects of small changes in osmolality on the post-thaw quality of semen.

Isolation, characterisation and cDNA sequencing of a new form of parvalbumin from carp semen.

Parvalbumins (Pv) are calcium-binding proteins present mainly in the muscle and nervous system where they act as a Ca(2+) buffer. Our previous work demonstrated the presence of Pv-I in carp semen and indicated the presence of a second Pv (Pv-II). The purpose of the present work was to identify, purify and determine the full-length cDNA sequence of Pv-II from carp testis. Pv-II from seminal plasma was purified by ion-exchange chromatography (IEC) and preparative electrophoresis, while the Pv-II from spermatozoa was purified by IEC, gel filtration and preparative electrophoresis. The purified Pv-II was submitted to an analysis of molecular mass, isoelectric point (pI), amino-acid sequence and oligomerisation ability. The amino-acid sequence was used to construct primers and obtain the full-length cDNA sequence of seminal-specific Pv-II from carp testis. Analysis of the cDNA sequence indicated that carp-testis Pv-II was distinct from carp-muscle parvalbumins. Pv-II was distinct from Pv-I regarding sequence, molecular mass and pI. Both parvalbumins had the ability to form oligomers or to bind to other proteins. Carp seminal plasma had a protective effect against parvalbumin oligomerisation. Pv-II underwent post-translational modification such as n-acetylation and cysteinylation. The present study is the first to report the full-length cDNA sequence of parvalbumin from carp testis.