Evaluation of glucose and lactate production by canine luteal cells in early cyclic and gestational diestrus

There is great similarity in several aspects between the cyclic and pregnant canine luteal phase. In order to investigate the metabolic pathways of luteal cells in early cyclic and gestational diestrus, we aimed to analyze glucose and lactate production by these cells cultured in vitro. Ovariohysterectomy was performed in 6 females, 3 in early gestational diestrus and 3 in initial cyclic diestrus. Corpora lutea (CL) were enzymatically digested in a solution containing 5 ml DMEM (Dulbecco’s Modified Eagle’s Medium - high glucose, Sigma -Aldrich, Germany) and 0.0075g collagenase type 1 (Collagenase from Clostridium histolyticum Type I, Sigma -Aldrich, Germany). After one hour at 37.5°C and vortexing every 15 minutes, the cell suspension was filtered (Filter with 70 μm, BD Falcon®, USA), centrifuged 3 times (340G, 220G and 100G respectively at 20°C for 10 min) and resuspended in 12 ml DMEM supplemented with antibiotic, antifungal, L-glutamine (Sigma -Aldrich, USA) and fetal serum bovine (Sigma -Aldrich, EUA). The solution containing luteal cells (experimental solution) was distributed in 24 well plates and incubated in controlled atmosphere (containing 5% CO2 and 95% air). After 24 hours, wells were washed with HBSS (14,175,079; Gibco BRL) and medium replaced. A plate containing only culture medium was used as control (control solution). Experimental and control solution samples were collected 36 (moment 1), 48 (moment 2) and 60 (moment 3) hours after the start of culture, stored in plastic tubes and kept in a freezer at -80o C. Glucose and lactate levels were assessed using VITROS Chemistry Products Calibrator kits (Products Vitro Chemistry, United Kingdom). The statistical analysis was performed using ANOVA (p u003c0.05) in SAS PROC GLM. We observed that glucose consumption and lactate production increased during in vitro culture in both gestational luteal cells and cyclic diestrus, but glucose consumption and lactate production in gestational CL was greater than cyclic CL at initial (glucose in moment 1, p=0.0328 and lactate in moment 2, p=0.0221) and final (glucose and lactate in moment 3, p=0.0085 and p=0.0009, respectively) culture stages. According with these results, we believe that there is no difference in the metabolic pathways used by pregnant and cyclic luteal cells. However, at initial diestrus, energy metabolism appears to be greater in pregnant than cyclic CL.