Lation [5] and artificial vagina [7]. Nevertheless, high proportion of ejaculates obtained via rectal massage represented a sperm-rich fraction with minimal seminal plasma contribution and might not represent a `true ejaculate’. Very good motility ejaculates were characterized by larger volume and decrease sperm concentration, thus indicating much better top quality ejaculates with higher percentages of sperm motility have been diluted with larger amounts of seminal plasma. Thus, although the rectal massage technique is sensible and represents a reasonably protected alternative for semen collection from captive managed bull elephants, an option strategy that mimics organic ejaculatory responses including contributions from the accessory sex glands may perhaps be warranted to improve semen collection efficiencies in elephants. As observed herein and within a earlier study [3], a high proportion of ejaculates obtained by means of the rectal massage techniqueFigure two. One-dimensional gel electrophoresis of Asian elephant seminal plasma proteins. Every column represents protein profiles from seminal plasma samples obtained from distinctive ejaculates. E1, E2, and E3 Beta-secretase supplier denote person Asian elephant bulls. G1, G2, G3, G4, G5, and G6 denote seminal plasma samples obtained from ejaculates exhibiting great motility ( 65 tMOT). P1, P2, P3, P4, and P5 denote seminal plasma samples obtained from ejaculates exhibiting poor motility (#10 tMOT). MW: molecular weight marker. Band A was detected in 85 of seminal plasma samples from great motility ejaculates (a representative gel is shown). Within this figure, protein bands were visualized with Coomassie staining. doi:ten.1371/journal.pone.0071033.gPLOS A single | plosone.orgLactotransferrin in Elephant Seminal PlasmaFigure 3. Protein separation and peptide mass sequencing of Asian elephant seminal plasma. Seminal plasma proteins were separated by two-dimensional gel electrophoresis. Chosen spots were excised and submitted for mass spectrometry (MALDI-TOF) for protein identification by de novo sequencing of MALDI-PSD tandem mass spectra. Panel A: 2D gel of seminal plasma proteins from an ejaculate exhibiting Xanthine Oxidase Inhibitor list excellent sperm motility ( 65 tMOT). The same sample from Panel A can also be shown in Figure two, Lane G5. Cored spots from within the train of this protein (circled; corresponding with band A in Figure two) were submitted for mass spectrometric evaluation. Panel B: Example of a 2D gel of seminal plasma proteins from an ejaculate exhibiting poor sperm motility (#10 tMOT). Panel C: Mass spectrum benefits from MALDI-TOF. Panel D: Peptide mass sequencing identified the cored proteins as getting homology to lactotransferrin (i = isoleucine or leucine). doi:10.1371/journal.pone.0071033.gPLOS 1 | plosone.orgLactotransferrin in Elephant Seminal PlasmaFigure 4. Amino acid sequence of predicted African elephant (Loxodonta africana) lactotransferrin determined from the 29 Mammals sequencing project ( Alignment of tryptic peptide sequences determined by MALDI-PSD sequencing of SPITC derivitized peptides shown italicized. Residues marked as “i” represent either isoleucine (I) or leucine (L), that are isobaric. Asparagine 546 has probably been deamidated to its corresponding aspartic acid. doi:10.1371/journal.pone.0071033.gconsistently contained spermatozoa with poor motility. Nonetheless, top quality ejaculates have also been obtained using the same rectal massage strategy [3,4]. This suggests that maybe other variables, such as seasonality and/or circulatin.