Tion of seminal plasma, each and every ejaculate (92 ejaculates; 11 bulls; 1?7 ejaculate(s) per

Tion of seminal plasma, each and every ejaculate (92 ejaculates; 11 bulls; 1?7 ejaculate(s) per bull) was initially centrifuged (2006g for five min) to pellet spermatozoa and cellular debris. The seminal plasma supernatant was removed and centrifuged once again (5006g for 20 min), with all the prime 2/3 removed by aspiration, mixed, divided into aliquots, frozen and stored (270uC) till analysis. Right after thawing, all aliquots have been spun furthermore at 10,0006g for five min at 4uC and the supernatants collected to ensure that all analyzed samples have been devoid of spermatozoa.Seminal Plasma Chemistry Analyses Materials and Approaches AnimalsSeminal plasma was collected from Asian elephant bulls (n = 21; 8 to 45 years) housed at 10 institutions all through North America. Sixteen in the 21 bulls had previously sired calves and have been for that reason identified to become fertile by natural mating. The bulls have been managed under a protected contact management system, housed in individual enclosures with visual, olfactory, and/or controlled access to females, and offered absolutely free access to water and normal access to feed. All animal investigation protocols were authorized by the Smithsonian Conservation Biology Institute’s Institutional Animal Care and Use Committee. Seminal plasma electrolytes (Na+: sodium; P32: phosphorus; K+: potassium; Ca2+: calcium; Cl2: chloride; HCO32: bicarbonate), enzymes (LDH: lactate dehydrogenase; CPK: creatine phosphokinase; AST: aspartate aminotransferase; ALT: alanine aminotransferase; AP: alkaline phosphatase), proteins (TP: total protein; ALB: albumin), sugars (GLU: glucose), cholesterol (CHO), creatinine (CRT), and urine urea nitrogen (UUN) had been determined using a serum chemistry autoanalyzer (Roche Cobas Mira Chemistry Analyzer). Although ejaculates with definitive indicators of urine contamination were excluded from this study, CRT and UUN levels were also measured to determine low levels of urine contamination. Magnesium (Mg2+) concentrations were measured by a colorimetric system p38γ review applying a Hitachi Cobas C501 chemistry analyzer (performed in the Kansas State Veterinary Diagnostic Laboratory).Semen Collection and ProcessingSemen was collected applying the rectal massage technique as previously described [8]. Every ejaculate (n = 21 bulls; 205 ejaculates; 1?two ejaculate(s) per bull) was promptly evaluated for volume (ml), color, percentages of total motile spermatozoa ( tMOT) and forward progressive motility ( pMOT), sperm concentration (6106 cells ml21), sperm morphology, osmolality, and pH. An aliquot (eight ml) was assessed subjectively for tMOT and pMOT employing a phase contrast microscope (200X). Sperm concentration was determined applying a transportable spectrophotometer (DVM Rapid TestTM, Worth Diagnostics) calibrated for measuring concentration of Asian elephant spermatozoa. T-type calcium channel site osmolality (mOsm) was determined making use of a vapor stress osmometer (VAPRO, Wescor Inc.) and pH was determined making use of a hand held pH meter (Twin pH, Horiba Ltd.). Sperm morphology was evaluated utilizing Spermac stain (Conception Technologies) as previously described [3]. For morphological assessment, a minimum of 200 spermatozoa have been assessed individually applying bright-field microscopy under oil immersion (1000X). Spermatozoa exhibiting standard morphology have been categorized as `normal’ (Figure 1A), and spermatozoa exhibiting morphological abnormalities in the head (i.e. microcephalic, macrocephalic, bicephalic, misshaped, detached), mid-piece (i.e. bent necks, abnormal, bent, absent, proximal or distal cytoplasmicPLOS ON.