834 RESEARCH NOTES

TABLE 3.—Effect of nitrate on performance of laying quail (Exp. 4)

Nitrate No. Hen-day prod.' Feed cons. 2 Water cons. 2 Mort. 1

(p.p.m.) bird/rep. (%) (gm./bird-day) (ml./bird-day) (%)
0 2 7 66^2 20J 643 1A~
660 2 8 69.6 18.5 57.0 4.2
1320 2 6 70.3 20.3 64.1 6.3
2640 2 6 72.5 21.7 63.9 6.3
3960 2 6 68.9 22.8 57.1 12.5
'77-day production period.
2
Data for first 4 wk. of production.

the first week. Poults reacted similarly in a to water containing less than 3960 p.p.m.

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previous study (Adams et al., 1969).
Levels of nitrate had a significant but
inconsistent effect on weight gain (Table 2).
Neither feed and water consumption during
growing or laying phases nor hen-day egg
production (Tables 2 and 3) were significantly
affected by levels of nitrate used. General
appearance of the birds during the experiment
was good. Although 3960 p.p.m. nitrate in-
creased mortality of quail during both growing
and laying phases, those that survived that
dosage laid as well as the control birds. My
results suggest that Japanese quail are tolerant
nitrate.
REFERENCES
Adams, A. W., F. J. Emerick and C. W. Carlson,
1966. Effects of nitrate and nitrite in the drinking
water on chicks, poults, and laying hens. Poultry
Sci. 45: 1215-1222.
Adams, A. W., J. L. West and A. J. Kahrs, 1969.
Some effects on turkeys of nitrate in the drinking
water. Poultry Sci. 48: 1222-1229.
Kienholz, E. W., 1968. Sodium nitrate as a growth
promoting stimulant in market turkeys. Report to
National Turkey Federation, January 9, 1968.

A TRYPSIN-LIKE ENZYME IN ACROSOMAL EXTRACTS OF CHICKEN, TURKEY

AND QUAIL SPERMATOZOA

B. B. LANGFORD AND B. HOWARTH, JR.

Department of Poultry Science, University of Georgia, Athens, Georgia 30602
(Received for publication September 17, 1973)

ABSTRACT The presence of an active trypsin-like enzyme in the acrosomes of chicken,

turkey and quail spermatozoa was evaluated. Pooled semen samples (0.5 ml.) were collected
from each species and the sperm washed several times with modified Ringer's solution.
Acrosomal extracts were prepared and assayed for trypsin-like enzyme by the presence of
esterase activity using TAME (p-toluensulfonyl-L-arginine methyl ester) as a substrate and
measuring the change in optical density per minute at 247 m\x. Esterase activity of acrosomal
extracts obtained from recently ejaculated spermatozoa of the chicken, turkey and quail indicate
that an active trypsin-like enzyme is present in the acrosomes of spermatozoa from all three
of these species.
POULTRY SCIENCE 53: 834-837, 1974

S PERM penetration of the investments

surrounding mammalian ova is aided by
hydrolytic enzymes contained within the
sperm acrosome. One of these enzymes is
a trypsin-like enzyme (TLE) or protease
which has been demonstrated by Stambaugh
and Buckley (1969) to be active in the dis-
solution of the zona pellucida of rabbit ova.
Multamaki and Niemi (1969) also reported
a zona pellucida dissolving protease in
 RESEARCH NOTES
acrosomal extracts from bull spermatozoa.
Chicken spermatozoa have likewise been
shown to contain high trypsin-like enzymatic
activity (Buruiani, 1956; Ho and Meizel,
1970). Polakoski (1972) showed that the high
trypsin-like activity was present in the acro-
somes (acrosomal extracts) of cock sper-
matozoa. Furthermore, the necessity of an
active acrosomal trypsin-like enzyme for di-
gestion and penetration of the vitelline mem-
brane of the hen's ovum has been demon-
strated both in vitro (Howarth and Digby,
1973) and in vivo (Palmer and Howarth, 1973).
TLE has not been demonstrated in the turkey
or quail. The purpose of this study was to
determine if the acrosome of the turkey and
quail contain an active TLE.
MATERIALS AND METHODS
Pooled semen samples were collected by
abdominal massage from Athens Ran-
dombred cocks and Large White toms (Bur-
rows and Quinn, 1937). Pooled Japanese quail
semen was obtained using the method of
Marks and Lepore (1965). Three aliquots (0.5
ml.) of spermatozoa from each pooled semen
sample were used in evaluating acrosomal
TLE activity for each species. Prior to re-
moving the acrosomes each semen sample
was washed several times with modified
Ringer's solution (Olsen and Neher, 1948)
to remove the seminal plasma.
Acrosomal extracts were prepared from
each aliquot using the method described by
Polakoski et al. (1972). Acrosomes of the
washed spermatozoa were removed by treat-
ment with 0.075% Triton X-100 and 0.075%
TABLE 1.—Esterase activity
Species AO.D./min
Chicken .053
Turkey .063
Quail .025
'Species differences in esterase activity, as measured by change in O.D. per 1 x 10'2 spermatozoa,
were not significant.
835
Hyamine during a 90 minute incubation period
at 37° C. The sperm-acrosome mixture was
then centrifuged twice for 15 minutes at 1500
g. to remove the ruptured acrosomes and
cellular debris. The supernatant containing
the acrosomal protein was treated with equal
volumes of cold ethanol for 24 hours at 4° C.
The ethanol was removed from the resulting
precipitate by dializing against modified
Ringer's solution (Hartree and Srivastava,
1965).
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The acrosomal extracts were assayed for
trypsin-like enzyme by the presence of es-
terase activity toward TAME (p-toluensul-
fonyl-L-arginine methyl ester) (Hummel,
1959). Esterase activity was determined by
measuring the change in optical density
(O.D.) per minute at 247 mix. (Walsch, 1970).
A unit of measure was arbitrarily defined
as the change in O.D. per minute per 1 x
10 l2 spermatozoa.
RESULTS AND DISCUSSION
The results of this study indicate that
chicken, turkey and quail acrosomal extracts
contain an active TLE capable of esterase
activity using TAME as a substrate (Table
1). Species differences in esterase activity
by acrosomal extracts were not significant.
TAME was chosen as the substrate for this
study because it exhibits greater enzyme
sensitivity and specificity (chymotrypsin does
not hydrolyze TAME) than other substrates
(Hummel, 1959).
Acrosomal TLE activity has been reported
in epididymal rabbit spermatozoa (Stambaugh
and Buckley, 1968), but Zaneveld et al. (1968)
of,acrosomal extracts toward TAME
A O . D . / m i n / l x 10'2
cells/0.5 ml. spermatozoa'
2.25 x 10'2 .0236
4.23 x 1012 .0149
1.33 x 1012 .0195
836 RESEARCH NOTES
observed very little TLE activity in ejaculated
rabbit spermatozoa. However, Zaneveld et
al. (1969) demonstrated that TLE activity was
again present in capacitated rabbit spermato-
zoa. These observations led the latter authors
to postulate that a seminal plasma trypsin
inhibitor is added to sperm during ejaculation
and is removed during capacitation.
Polakoski (1971) noted that the seminal
plasma of most mammalian species as well
as the fowl contain trypsin inhibitors. When
a proteolytic enzyme and its inhibitor interact,
an inactive enzyme-inhibitor complex is
formed (Laskowski and Laskowski, 1954).
A TLE-TLE inhibitor complex has been
demonstrated in the ejaculated spermatozoa
of several mammalian species but has not
been demonstrated in chicken spermatozoa
(Zaneveld and Polakoski, 1971; Polakoski,
1971). This study demonstrated the presence
of an active TLE in ejaculated chicken,
turkey and quail spermatozoa supporting the
observation that chicken (Howarth, 1970) and
turkey (Howarth and Palmer, 1972) sper-
matozoa do not require capacitation.
The need for an active acrosomal TLE for
penetration of the vitelline membrane of the
hen's ovum by cock spermatozoa has been
demonstrated both in vitro (Howarth and
Digby, 1973) and in vivo (Palmer and
Howarth, 1973). The presence of an active
TLE in turkey and quail acrosomes suggests
that a trypsin-like enzyme may likewise be
involved in sperm penetration of the turkey
and quail ovum.
REFERENCES
Burrows, W. H., and J. P. Quinn, 1937. Collection
of spermatozoa from domestic fowl and turkey.
Poultry Sci. 16: 19-24.
Buruiana, L. M., 1956. Sur l'activite hyaluronidasique
et trypsinique du sperme. Naturwissenschafte, 43:
523.
Hartree, E. F., and P. N. Srivastava, 1965. Chemical
composition of the acrosomes of ram spermatozoa.
J. Reprod. Fertil. 9: 47-60.
Ho, J. J. L., and S. Meizel, 1970. Electrophoretic
detection of multiple forms of trypsin-like activity
in spermatozoa of the domestic fowl. J. Reprod.
Fertil. 23: 177-179.
Howarth, B., Jr., 1970. An examination for sperm
capacitation in the fowl. Biol. Reprod. 3: 338-341.
Howarth, B., Jr., and S. T. Digby, 1973. Evidence
for the penetration of the vitelline membrane of
the hen's ovum by a trypsin-like acrosomal enzyme.
J. Reprod. Fertil. 33: 123-125.
Howarth, B., Jr., and M. B. Palmer, 1972. An exami-
nation of the need for sperm capacitation in the
turkey, Meleagris Gallopavo. J. Reprod. Fertil. 28:
443-445.
Downloaded from http://ps.oxfordjournals.org/ at University of Rhode Island on February 8, 2015
Hummel, B. C. W., 1959. A modified spectropho-
tometry determination of chymotrypsin, trypsin and
thrombin. Can. J. Biochem. Physiol. 37: 1393-1399.
Laskowski, M., and M. Laskowski, Jr., 1954. Natural-
ly occurring trypsin inhibitors. Advances in Protein
Chemistry, IX: 203-242.
Marks, H. L., and P. D. Lepore, 1965. A procedure
for artificial insemination of Japanese quail. Poultry
Sci. 44: 1001-1003.
Multamaki, S., and M. Niemi, 1969. Zona pellucida
dissolving protease in an acrosomal preparation of
the bull spermatozoa. Scand. J. Clin. Lab. Invest.
23: 108.
Olsen, M. W., and B. H. Neher, 1948. The site of
fertilization in the domestic fowl. J. Exp. Zool. 109:
355-366.
Palmer, M. B., and B. Howarth, Jr., 1973. The
requirement of a trypsin-like acrosomal enzyme for
fertilization in the domestic fowl. J. Reprod. Fertil.
(In Press).
Polakoski, K. L., 1971. Enzymes and enzyme inhibi-
tors involved in animal fertilization. M.S. Thesis,
Department of Biochemistry, University of Georgia.
Polakoski, K. L., 1972. Properties and function of
enzymes and inhibitors involved in mammalian fer-
tilization. Ph.D. Dissertation, Department of Bio-
chemistry, University of Georgia.
Polakoski, K. L., L. J. D. Zaneveld and W. L.
Williams, 1972. Purification of a proteolytic enzyme
from rabbit acrosomes. Biol. Reprod. 6: 23-29.
Stambaugh, R., and J. Buckley, 1968. Zona pellucida
dissolution enzymes of the rabbit sperm head.
Science, 161: 585-586.
Stambaugh, R., and J. Buckley, 1969. Identification
and subcellular localization of the enzymes effecting
penetration of the zona pellucida by rabbit sper-
matozoa. J. Reprod. Fertil. 19: 423-432.
Walsch, K. A., 1970. Trypsinogenes and trypsins of
various species. In: Methods in Enzymology, Vol.
XIX, edited by G. E. Perlmann and L. Loramd.
1970. Academic Press, New York and London, pp.
42-43.
 RESEARCH N O T E S 837

Zaneveld, L. J. D., R. A. McRorie and W. L. Williams,
1968. A sperm enzyme that removes the corona
radiata from ova and its inhibition by decapacitation
factor. Fedn. Proc. Fedn. Am. Soc. Exp. Biol. 27:
567.
Zaneveld, L. J. D., and K. L. Polakoski, 1971.
Characterization of sperm acrosomal hyaluronidase
and acrosin. Fourth Annual Meeting of the Society
for the Study of Reproduction, pp. 4-5.
Zaneveld, L. J. D., P. N. Srivastava and W. L.
Williams, 1969. Relationship of a trypsin-like en-
zyme in rabbit spermatozoa to capacitation. J.
Reprod. Fertil. 20: 337-339.

FEEDING ROASTED CORN TO BROILERS, ROASTERS A N D LAYING H E N S

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R . L . ADAMS
Department of Animal Sciences, Purdue University, West Lafayette, Indiana 47907
(Received for publication September 19, 1973)

ABSTRACT Broiler type cockerels were fed rations containing either dry roasted or normal
corn to 13 weeks of age. The roasted corn ration had no significant effect on weight gain
or feed efficiency at 4, 8 or 13 weeks of age. The roasted corn had no significant effect
on carcass finish at 8 weeks or at 13 weeks.
Fifty-two week old White Leghorns were fed rations containing either dry roasted or normal
corn for 16 weeks. The roasted corn had no significant effect on egg production, feed efficiency
or egg weight.
There is no apparent benefit from the feeding of dry roasted corn to broilers, roasters or
layers.
POULTRY SCIENCE 53: 837-839, 1974

S INCE 1965 various types of processed

corn have been fed to cattle and poultry.
The feeding of dry roasted corn to cattle has
been reported to improve both weight gain
and feed efficiency (Perry et al., 1970; Cun-
ningham and Perry, 1972). The results of
feeding processed corn to poultry has been
inconsistent. Deyoe et al. (1967) obtained no
significant differences in weight gain or feed
efficiency with broilers when expanded corn
replaced 0, 25, or 100% of the normal corn
in the diet. Significantly poorer growth was
obtained when 75% of the normal corn was
replaced. Adams and Naber (1969) found
small but non-significant growth and feed
efficiency reductions from the feeding of
expanded corn (steam heated at 140° C.)
Sloan et al. (1971) observed no differences
in growth, but reported non-significant im-
provements in feed efficiency from the feed-
ing of expanded-extruded corn. No reports
were found on the feeding of processed corn
to layers.
The objectives of this study were to study
the effects of feeding roasted corn to broilers,
roasters, and layers.
EXPERIMENTAL PROCEDURE
The roasted corn used in these experiments
was roasted in a Roast-A Tron.1 The machine
employs a revolving cylinder within a jacket.
The cylinder has fins on the outside which
lift the grain through jets of flame which point
downward from the top of the jacket. Expo-
sure time to the infrared dry heat is controlled
by adjusting the incline of the grain containing
jacket. The corn was roasted to an internal
temperature of 150° C.
Broilers and Roasters. Day old male broiler
chicks were randomly assigned to ten floor
1. Unit manufactured and supplied by Mix Mill,
Bluff ton, Indiana.
Journal paper no. 5253 from Purdue University
Agricultural Experiment Station.