CONCENTRATIONS OF HYDROGEN IONS, OXYGEN, CARBON DIOXIDE AND BICARBONATE IN PORCINE FOLLICULAR FLUID J. F. KNUDSEN, L. J. LITKOWSKI, T. L. WILSON, H. D. GUTHRIEt AND S. K. BATTA Department of Physiology and * Division of Nephrology, University of Maryland School of Medicine, Baltimore, Maryland 21201, U.S.A. and \The United States Department of Agriculture, Beltsville, Maryland 20705, U.S.A.
(Received 26 June 1978) The chemical composition of human ovarian follicular fluid has been well characterized (Shalgi, Kraicer & Soferman, 1972; Edwards, 1974). However, compositional studies of porcine follicular fluid (PFF; Schuetz, 1974; Chang, Jones, Ellefson & Ryan, 1976), have not included acid\p=n-\basemeasurements (pH, oxygen and carbon dioxide tension (Po2, Pco2), concentration of bicarbonate HCO3\m=-\]). Recent studies of the nuclear maturation of porcine oocytes have demonstrated a rate of maturation of only 55\p=n-\80%for oocytes collected from ovaries obtained at the abattoir (Tsafriri & Channing, 1975) and it was suggested that maturing and non-maturing oocytes may have undergone nuclear degeneration. It has also been shown in monkeys that oocytes which appear mature when examined by light microscopy may in fact be degenerating (Batta, Stark Brackett, 1978). It is conceivable that these degenerative changes may be related to the method of collecting the ovaries and may be reflected in differences between the acid\p=n-\baseparameters of the tissue and values in vivo. The present studies compare the acid\p=n-\baseparameters of PFF from two sources : ovaries from a local abattoir and the ovaries of anaesthetized cyclic pigs. Porcine ovaries at random stages of the reproductive cycle were obtained from an abattoir and placed in 0-9% NaCl (unbuffered, pH 6-0) at room temperature. The total time interval between death and the recovery of PFF was 3-4 h. Follicular fluid was collected from large follicles (diameter 6-11 mm) by puncturing the thinnest part of the follicle with an 18-gauge needle connected to a 100 µ micropipette. The ends of the pipette were then sealed with clay and the pipette was placed on ice until required for analysis, up to 2 h later. (The acid-base parameters of human blood stored in this way do not change over a similar time interval: Siggaard-Andersen, 1961.) Follicular fluid was obtained similarly from anaesthetized (sodium pentobarbitone, 10 mg/kg) Duroc and Yorkshire sows (body weight about 200 kg) on day 18 of the oestrous cycle or at oestrus. All sows had completed at least one normal oestrous cycle before the experiment. Venous blood was obtained from the same anaesthetized pigs before laparotomy. Values of pH, Pco2 and Po2 were determined for PFF and venous blood with a BGA2 Radiometer Blood Micro System (Copenhagen) ; [HC03~] was calculated using the Henderson-Hasselbalch equation, corrected for the effect of pH on ' (Austin, Lacombe, Rand & Chatterjee, 1963). Results (means + s.e.m.) were tested for significance by Duncan's multiple range test (Steel «fe Torrie, 1960). The acid-base values for PFF reported here (Table 1) are the first such measurements for cyclic pigs. The values for venous blood are in agreement with the results reported by Cummings & Kaiser (1959) and do not differ significantly from the values for PFF obtained in vivo. In contrast, marked differences were found in the acid-base values for PFF collected from ovaries obtained at the abattoir: pH values were, on average, 0-59 units lower than those of PFF obtained in vivo, corresponding to a fourfold increase in [H+]. Furthermore, &
Table 1. The pH, concentration of hydrogen ions ([H+]), oxygen and carbon dioxide tensions (Po2, Pco2) and concentration of bicarbonate ([HC03~]) in porcine venous blood and follicular fluid (PFF) collected from live pigs or from ovaries obtained at the abattoir (means ± s.e.m. ; numbers of observations given in parentheses) pH
[H+] (nmol/1) 46+2(8) 40 + 2 (10)
Po8 (mmHg) 41+2(8) 51 + 5 (9)
Venous blood (four pigs) PFF obtained in vivo
7-36+0-04(8) 7-41+0-01 (11)
PFF obtained from abattoir ovaries (five pigs)
6-82+0-02* (11) 159 + 7* (11) 44+3 (11)
(four pigs) *
Pcoa (mmHg) 53 + 3(8) 45±2 (10)
[HC03-] (mmol/1) 28-6+0-7(8) 28-0 + 0-7 (10)
82+2* (11) 12-3 + 0-9* (11)
Significantly different (P
(Received 26 June 1978) The chemical composition of human ovarian follicular fluid has been well characterized (Shalgi, Kraicer & Soferman, 1972; Edwards, 1974). However, compositional studies of porcine follicular fluid (PFF; Schuetz, 1974; Chang, Jones, Ellefson & Ryan, 1976), have not included acid\p=n-\basemeasurements (pH, oxygen and carbon dioxide tension (Po2, Pco2), concentration of bicarbonate HCO3\m=-\]). Recent studies of the nuclear maturation of porcine oocytes have demonstrated a rate of maturation of only 55\p=n-\80%for oocytes collected from ovaries obtained at the abattoir (Tsafriri & Channing, 1975) and it was suggested that maturing and non-maturing oocytes may have undergone nuclear degeneration. It has also been shown in monkeys that oocytes which appear mature when examined by light microscopy may in fact be degenerating (Batta, Stark Brackett, 1978). It is conceivable that these degenerative changes may be related to the method of collecting the ovaries and may be reflected in differences between the acid\p=n-\baseparameters of the tissue and values in vivo. The present studies compare the acid\p=n-\baseparameters of PFF from two sources : ovaries from a local abattoir and the ovaries of anaesthetized cyclic pigs. Porcine ovaries at random stages of the reproductive cycle were obtained from an abattoir and placed in 0-9% NaCl (unbuffered, pH 6-0) at room temperature. The total time interval between death and the recovery of PFF was 3-4 h. Follicular fluid was collected from large follicles (diameter 6-11 mm) by puncturing the thinnest part of the follicle with an 18-gauge needle connected to a 100 µ micropipette. The ends of the pipette were then sealed with clay and the pipette was placed on ice until required for analysis, up to 2 h later. (The acid-base parameters of human blood stored in this way do not change over a similar time interval: Siggaard-Andersen, 1961.) Follicular fluid was obtained similarly from anaesthetized (sodium pentobarbitone, 10 mg/kg) Duroc and Yorkshire sows (body weight about 200 kg) on day 18 of the oestrous cycle or at oestrus. All sows had completed at least one normal oestrous cycle before the experiment. Venous blood was obtained from the same anaesthetized pigs before laparotomy. Values of pH, Pco2 and Po2 were determined for PFF and venous blood with a BGA2 Radiometer Blood Micro System (Copenhagen) ; [HC03~] was calculated using the Henderson-Hasselbalch equation, corrected for the effect of pH on ' (Austin, Lacombe, Rand & Chatterjee, 1963). Results (means + s.e.m.) were tested for significance by Duncan's multiple range test (Steel «fe Torrie, 1960). The acid-base values for PFF reported here (Table 1) are the first such measurements for cyclic pigs. The values for venous blood are in agreement with the results reported by Cummings & Kaiser (1959) and do not differ significantly from the values for PFF obtained in vivo. In contrast, marked differences were found in the acid-base values for PFF collected from ovaries obtained at the abattoir: pH values were, on average, 0-59 units lower than those of PFF obtained in vivo, corresponding to a fourfold increase in [H+]. Furthermore, &
Table 1. The pH, concentration of hydrogen ions ([H+]), oxygen and carbon dioxide tensions (Po2, Pco2) and concentration of bicarbonate ([HC03~]) in porcine venous blood and follicular fluid (PFF) collected from live pigs or from ovaries obtained at the abattoir (means ± s.e.m. ; numbers of observations given in parentheses) pH
[H+] (nmol/1) 46+2(8) 40 + 2 (10)
Po8 (mmHg) 41+2(8) 51 + 5 (9)
Venous blood (four pigs) PFF obtained in vivo
7-36+0-04(8) 7-41+0-01 (11)
PFF obtained from abattoir ovaries (five pigs)
6-82+0-02* (11) 159 + 7* (11) 44+3 (11)
(four pigs) *
Pcoa (mmHg) 53 + 3(8) 45±2 (10)
[HC03-] (mmol/1) 28-6+0-7(8) 28-0 + 0-7 (10)
82+2* (11) 12-3 + 0-9* (11)
Significantly different (P
