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

Tion of seminal plasma, 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 more (5006g for 20 min), with all the top rated 2/3 removed by aspiration, mixed, divided into aliquots, frozen and stored (270uC) until analysis. Right after thawing, all aliquots were spun in addition at 10,0006g for five min at 4uC along with the supernatants collected to make sure that all analyzed samples had been devoid of spermatozoa.Seminal Plasma Chemistry Analyses Materials and Techniques AnimalsSeminal plasma was collected from Asian elephant bulls (n = 21; 8 to 45 years) housed at ten institutions all through North America. Sixteen on the 21 bulls had previously sired calves and have been as a result recognized to become fertile by natural mating. The bulls had been managed beneath a protected get in touch with management program, housed in person enclosures with visual, olfactory, and/or controlled access to females, and given free access to water and common access to feed. All animal study protocols had been approved 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 applying a serum chemistry autoanalyzer (Roche Cobas Mira Chemistry Analyzer). Though ejaculates with definitive indicators of urine contamination had been excluded from this study, CRT and UUN levels have been also measured to recognize low levels of urine contamination. Magnesium (Mg2+) concentrations were measured by a colorimetric system CD28 Antagonist site utilizing a Hitachi Cobas C501 chemistry analyzer (performed at the Kansas State Veterinary Diagnostic Laboratory).Semen Collection and ProcessingSemen was collected applying the rectal massage technique as previously described [8]. Each and every ejaculate (n = 21 bulls; 205 ejaculates; 1?two ejaculate(s) per bull) was instantly evaluated for volume (ml), colour, percentages of total motile spermatozoa ( tMOT) and forward progressive motility ( pMOT), sperm concentration (6106 cells ml21), sperm morphology, osmolality, and pH. An aliquot (8 ml) was assessed subjectively for tMOT and pMOT ROR Species employing a phase contrast microscope (200X). Sperm concentration was determined employing a portable spectrophotometer (DVM Rapid TestTM, Worth Diagnostics) calibrated for measuring concentration of Asian elephant spermatozoa. Osmolality (mOsm) was determined using a vapor pressure 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 beneath oil immersion (1000X). Spermatozoa exhibiting standard morphology were categorized as `normal’ (Figure 1A), and spermatozoa exhibiting morphological abnormalities within the head (i.e. microcephalic, macrocephalic, bicephalic, misshaped, detached), mid-piece (i.e. bent necks, abnormal, bent, absent, proximal or distal cytoplasmicPLOS ON.