Vle CONG. INTERN. REPROD. ANIM. INSEM. ARTIF., PARIS, 1968, VOL. li

HEXOKINASES IN SEMEN FROM THE BULL, BOAR AND RAM
R. A. P. HARRISON

A.R.C. Unit of Reproductive Physiology and Biochemistry,
Cambridge, England.

A very large number of investigations have been made on the
utilisation of sugars by mammalian spermatozoa for energy production
(1). The sugars are believed to be mainly metabolised via the
Embden-Meyerhof pathway, and their breakdown products then oxidised
via the Krebs cycle. The investigations have been carried out almost
exclusively however using whole cell suspensions, and very little
work has been done on the relevant enzymes in a cell-free suspension.
This work represents a preliminary quantitation and study of the
first enzyme in the glycolytic sequence, ATP-D-hexose-6-phospho-
transferase or hexokinase, in three species, the bull, the boar, and
the ram.

MATERIALS AND METHODS. Semen was collected by artificial vagina
from two Large White boars, four Friesian bulls, and ten Suffolk
rams; the boar ejaculates were used individually, the bull and ram
ejaculates were pooled. Three separate experiments using ram semen
and four separate experiments using bull and boar semen were carried
out.

The semen was spun at 2,000 g. for 10 mins. at room temperature,
and the serminal plasma removed. The sperm were then gently re-
suspended and respun in Ringer solution, and further resuspended and
respun in a medium consisting of 250 mM sucrose, 10 mM phosphate
buffer, 1 mM EDTA, 1 mM mercaptoethanol, and 10 mM glucose, all at
pH 7.0 (SPG). They were finally resuspended in SPG at three dif-
ferent sperm densities, measured in an absorptiometer, and samples
sonicated at 4° for 3 mins. to ensure total breakage. All further
manipulations were carried out in the cold. The homogenate was
spun at 800 g. for 10 mins. to remove the heads and any midpiece
fragments remaining, and the resulting supernatant was then further
spun at 100,000 g. for 60 mins. to precipitate all particulate
matter. The precipitates were resuspended in SPG and respun.

Hexokinase (HK) activity in the fractions was estimated at pH
7.6 at 37° using 10 mM glucose as the substrate, by a method very

(1) T. R. R, MANN, 1964. The Biochemistry of Semen and of the Male
Reproductive Tract. (Methuen).

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