PARENT SESSION
BIOLOGY OF MALE AND FEMALE GAMETES
Harborside C
7:30 AM-10:00 AM

(190) INFLUENCE OF BICARBONATE, HEPES, AND MOPS BUFFERS ON CAT SPERM METABOLISM.

Aitken-Palmer, Copper¹, Wildt, David², Carpenter, James¹, Spindler, Rebecca², ¹ College of Veterinary Medicine, Manhattan, KS² Conservation and Research Center, Front Royal, VA

ABSTRACT- Media for sperm processing and evaluation often rely on NaHCO₃ or HEPES buffering systems. NaHCO₃ buffers uterine and oviductal fluids and is most physiologically appropriate for cells living under high CO₂ conditions. HEPES buffered medium maintains pH with atmospheric CO₂, but may alter cell physiology. This study compared NaHCO₃, HEPES, and an alternative buffer (MOPS) to test the hypothesis that these buffers allow prolonged assessment of viability in vitro without altering domestic cat sperm physiology (measured by sperm motility and metabolism). Sperm (15 ejaculates, 8 males) were washed in MTF (mouse tubal fluid medium) with NaHCO₃ (N-MTF), HEPES (H-MTF), or MOPS (M-MTF) and diluted to 8 × 10⁵ motile sperm/ml (> 500-l). Samples were incubated at 38°C, and aliquots assessed for sperm motility at 0, 2, 4, and 6-h. Additional 50-l samples at each time point sperm were removed, and the supernatant analyzed by microfluorescence for glucose, pyruvate, and lactate concentration to reflect sperm metabolism. Rate of sperm motility decline in vitro over time did not differ (P > 0.05) among treatments. Pyruvate was preferentially taken up over glucose (∼2 fold) and was not influenced by medium buffering system (N-MTF, 77.6 ±18.2; H-MTF: 91.7 ± 34.7; M-MTF: 96.5 ± 25.6 fmols/sperm/h). Sperm glucose uptake was greater (P < 0.05) in N-MTF (41.0 ± 13.7 fmols/sperm/h) and M-MTF (40.0 ± 9.6 fmols/sperm/h) than H-MTF (16.0 ± 4.7 fmols/sperm/h), suggesting that the latter altered sperm physiology. Sperm lactate production was less (P < 0.05) in M-MTF (47.1 ±35.9 fmols/sperm/h) compared to N-MTF (175.6 ±29.2 fmols/sperm/h) but not with H-MTF (135.8 ± 35.0 fmols/sperm/h). There was a positive correlation (r = 0.2775) between proportion of glucose and pyruvate metabolized to lactate and rate of sperm motility decline (P = 0.075). These results reveal that cat sperm (1) generally undergo oxidative metabolism and (2) experience increased glycolytic metabolism when maintained in mouse tubal fluid medium buffered by NaHCO₃ or HEPES, perhaps indicative of in vitro stress. These adverse effects, which may include altered pH and disrupted sperm physiology, may be reduced by MOPS buffering.

KEY WORDS: domestic cat, metabolism, mops, sperm