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 again (5006g for 20 min), using the top rated 2/3 removed by aspiration, mixed, divided into aliquots, frozen and stored (270uC) till evaluation. After thawing, all aliquots were spun in addition at 10,0006g for 5 min at 4uC and the supernatants collected to make sure that all analyzed samples were devoid of spermatozoa.Seminal Plasma Chemistry Analyses Components and Solutions AnimalsSeminal plasma was collected from Asian elephant bulls (n = 21; eight to 45 years) housed at 10 institutions throughout North America. Sixteen in the 21 bulls had previously sired calves and were for that reason identified to become fertile by all-natural mating. The bulls were managed under a protected make contact with management program, housed in individual enclosures with visual, olfactory, and/or controlled Farnesyl Transferase custom synthesis access to females, and offered absolutely free access to water and regular access to feed. All animal study 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 working with a serum chemistry autoNOD2 list Analyzer (Roche Cobas Mira Chemistry Analyzer). Despite the fact that ejaculates with definitive signs of urine contamination have been excluded from this study, CRT and UUN levels were also measured to identify low levels of urine contamination. Magnesium (Mg2+) concentrations were measured by a colorimetric strategy using a Hitachi Cobas C501 chemistry analyzer (performed at the Kansas State Veterinary Diagnostic Laboratory).Semen Collection and ProcessingSemen was collected utilizing the rectal massage approach as previously described [8]. Each and every ejaculate (n = 21 bulls; 205 ejaculates; 1?two ejaculate(s) per bull) was right away 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 (eight ml) was assessed subjectively for tMOT and pMOT employing a phase contrast microscope (200X). Sperm concentration was determined working with a portable spectrophotometer (DVM Fast TestTM, Worth Diagnostics) calibrated for measuring concentration of Asian elephant spermatozoa. Osmolality (mOsm) was determined employing a vapor stress osmometer (VAPRO, Wescor Inc.) and pH was determined utilizing a hand held pH meter (Twin pH, Horiba Ltd.). Sperm morphology was evaluated using Spermac stain (Conception Technologies) as previously described [3]. For morphological assessment, a minimum of 200 spermatozoa have been assessed individually working with bright-field microscopy below 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.