Figure 1.
Asian elephant sperm morphology.
A) Normal; B) Proximal cytoplasmic droplet; C) Abnormal mid-piece; D) Tightly coiled tail; E) Bent tail with cytoplasmic droplet; F) Spermac staining (solid arrow: Spermac positive; dotted arrow: Spermac negative). Magnification 1000X.
Table 1.
Characteristics of Asian elephant ejaculates and comparison of semen traits between good and poor motility ejaculates.
Table 2.
Summary statistics for seminal plasma components1.
Table 3.
Pearson product-moment coefficients between conventional semen parameters and seminal plasma components1.
Figure 2.
One-dimensional gel electrophoresis of Asian elephant seminal plasma proteins.
Each column represents protein profiles from seminal plasma samples obtained from different ejaculates. E1, E2, and E3 denote individual Asian elephant bulls. G1, G2, G3, G4, G5, and G6 denote seminal plasma samples obtained from ejaculates exhibiting good 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 good motility ejaculates (a representative gel is shown). In this figure, protein bands were visualized with Coomassie staining.
Figure 3.
Protein separation and peptide mass sequencing of Asian elephant seminal plasma.
Seminal plasma proteins were separated by two-dimensional gel electrophoresis. Selected 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 good sperm motility (≥65% tMOT). The same sample from Panel A is also shown in Figure 2, Lane G5. Cored spots from within the train of this protein (circled; corresponding with band A in Figure 2) were submitted for mass spectrometric analysis. Panel B: Example of a 2D gel of seminal plasma proteins from an ejaculate exhibiting poor sperm motility (≤10% tMOT). Panel C: Mass spectrum results from MALDI-TOF. Panel D: Peptide mass sequencing identified the cored proteins as having homology to lactotransferrin (i = isoleucine or leucine).
Figure 4.
Amino acid sequence of predicted African elephant (Loxodonta africana) lactotransferrin determined from the 29 Mammals sequencing project
(http://www.broadinstitute.org). 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), which are isobaric. Asparagine 546 has most likely been deamidated to its corresponding aspartic acid.
Figure 5.
Immunodetection of lactotransferrin in elephant seminal plasma samples.
Panels A, B, and C represent several immunoblots following 1D SDS-PAGE protein separation. E1-E6 denote individual Asian elephant bulls. G1 through G11 represent seminal plasma samples obtained from ejaculates exhibiting good motility (≥65% tMOT). P1 through P17 represent seminal plasma samples obtained from ejaculates exhibiting poor motility (≤10% tMOT). M1 represents a seminal plasma sample obtained from ejaculates exhibiting moderate motility (45% tMOT). CON: liver control. Approximately 85% of the seminal plasma samples from ejaculates exhibiting good motility were positive for the presence of lactotransferrin. Conversely, lactotransferrin was undetected in over 90% of seminal plasma samples from ejaculates exhibiting poor motility.