Full Text - Journal of Animal Science

A L P H A C H L O R O H Y D R I N : S T U D I E S ON T H E M E C H A N I S M OF ACTION
IN M A L E SWINE
L. A. Johnson and V. G. Pursel
U. S. Department o f Agriculture 1 2 3, Beltsville, Maryland 20705
Summary
A study was made of the possible causes of
infertility which results from feeding a-chlorohydrin to boars. When gilts were inseminated
via the oviducts, 37 of 54 ova recovered 2 days
later from control gilts were classed as fertilized. However, none of 49 ova were fertilized
after gilts were inseminated in the oviducts with
semen from boars treated with a-chlorohydrin
(5 mg/kg/day). When gilts were inseminated via
the oviducts with neat semen containing 1 or
10 mM a-chlorohydrin added in vitro, only two
of 111 ova were fertilized. The antifertility
action of a-chlorohydrin was very rapid, since
pregnancy did not result in any of 13 gilts
inseminated with semen collected either 7 or 31
hr. after boars were fed a-chlorohydrin. Of
eight gilts inseminated with epididymal semen
collected from boars previously treated with
a-chlorohydrin, none were pregnant.
These studies indicated that failure of sperm
cell transport in the gilt was not the primary
causative factor in a-chlorohydrin induced infertility of boar spermatozoa. Further, it was
determined that the epididymis is the site of
action of a-chlorohydrin. A probable mechanism for a-chlorohydrin induced infertility of
boar spermatozoa is discussed.
Introduction
The male antifertility agent 3-chloro 1,2
propanediol (a-chlorohydrin) caused infertility
when administered to the boar (Johnson and
Pursel, 1972). Its activity was reversible and
free from observable side effects. Alpha chlorohydrin also causes infertility in rams (Kreider
and Dutt, 1970) and in rats (Coppola, 1969;
Ericsson and Baker, 1970).
When either the boar or boar semen was
treated with a-chlorohydrin, sperm cell motility
was depressed through an inhibition of progressive movemefit (Johnson and Pursel, 1972).
Oxygen uptake was decreased in spermatozoa
from a-chlorohydrin treated rams (Krieder and
Dutt, 1972) and rats (Samojlik and Chang,
1970). However, a-chlorohydrin treated monkey sperm showed an increased oxygen uptake
Setty et al., 1970). The mechanism of action
has not been determined, although the epididymis has been shown to be the major site of
action in rats (Ericsson and Connor, 1969;
Turner, 1971; Crabo and Appelgren, 1972).
Among the mechanisms which have been suggested for a-chlorohydrin induced infertility in
the past is the failure of sperm cell transport in
the female (Erickson and Bennett, 1971).
This study was done to determine if the
treatment of boars or boar semen with
a-chlorohydrin results in the failure of sperm
cell transport in the female, and to determine
the approximate time span required for infertility to occur after feeding a-chlorohydrin
to the boar.
Experimental Procedure
1Animal Physiology and Genetics Institute, Agricultural Research Center-East, ARS.
2 The authors gratefuUy acknowledge the technical
assistance of E. M. Dirnen and L. L. Schulman, and
thank Ayerst Laboratories Inc., 685 Third Street, New
York, N. Y., for the AIMAX, PMS and HCG used in
the study.
3 Reference to commercial products or commercial
producers in this report does not constitute endorsement by the U.S. Department of Agriculture to the
exclusion of others that may also be suitable.
TrialI. Ten gilts were used, each of which
had expressed estrus at least twice. They were
fed a diet containing 1-(a-methylallylthiocarbam o y l ) - 2 - m e t h y l t h i o c a r b amoyl hydrazine
(AIMAX, ICI 33, 828, methallibure) at the rate
of 100 mg per gilt per day for 18 to 21
consecutive days. On the day after AIMAX was
withdrawn, each gilt was given a subcutaneous
injection of 1,000 IU Pregnant Mare's Serum
1207
JOURNAL OF ANIMAL SCIENCE,vol. 37, no. 5, 1973
1208
JOHNSON AND PURSEL
hr. after ovulation. The ova were flushed from
the oviducts and uteri with physiological saline
and examined as whole mount preparations by
phase-contrast microscopy. The ova were then
fixed and cleared for 48 hr. in 25% acetic
alcohol and stained with 1% orcein in 45%
acetic acid. Ova were examined for the presence
of a nucleus in each blastomere and for
numbers of accessory spermatozoa in the zona
pellucida using phase-contrast microscopy.
Trial 11. Eleven gilts were used in this trial.
The trial was conducted as trial I, with the
following differences. Semen was deposited in
the isthmus of the oviduct 4 hr. after expected
ovulation. Gilts were inseminated with semen
types 1, 2 and 3 as described in trial I; however,
some samples of type 3 semen contained
a-chlorohydrin at a final concentration of 1
mM rather than 10 raM. Gilts were slaughtered
between 46 and 52 hr. after ovulation and eggs
recovered and examined as in trial I.
Trial IlL Two boars were fed a-chlorohydfin
at the rate of 5 mg/kg of body weight/day.
Nineteen gilts were treated with AIMAX, PMS
and HCG as in trial I. Six of the 19 grits were
artifically inseminated with semen collected
from the boars before a-chlorohydrin was fed.
Seven of the gilts were inseminated with semen
collected 7 hr. (day 1) after initiation of
a-chlorohydrin treatment. The remaining six
gilts were inseminated with semen collected 31
hr. (day 2) after initial treatment, or 7 hr. after
the second dose of a-chlorohydrin was fed.
Semen was extended in Beltsville L1 extender
(BL1; Pursel, Johnson and Schulman, 1973). A
total of 6 x 109 spermatozoa in an 80 ml
volume were deposited intra-cervically in each
gilt using a spiral tip catheter (Melrose and
O'Hagan, 1961). The results of insemination
were determined by return of the gilts to estms,
or by slaughter at 25 days post-insemination.
Trial IV. To determine the fertility of
epididymal semen, boars were fed a-chlorohydrin (5 mg/kg body wt/day) for 5 days, then
slauglttered; caudal epididymal semen was recovered by slicing the tubules with a scapel.
The semen was extended and used for insemination (4 x 109 sperm in 80 ml BL1 per gilt) of
gilts previously treated with ALMA)(, PMS and
HCG as described in trial I. Epididymal semen
was obtained and used in the same manner
from two other boars not fed ot-chlorohydrin.
In addition, one boar in which a vas deferens
cannula had been surgically inserted (Johnson,
Pursel and Kraeling, 1971) was fed a-chlorohydrin (5 mg/kg wt/day). Both ejaculated and
Figure 1. Deposition of semen in the isthmus of the epididymal semen were collected from this boar
oviduct.
at the same time, both before and after
(PMS); 500 IU of Human Chorionic Gonadotrophin (HCG) was given intramuscularly 88
to 96 hr. later. Ovulation was calculated to
occur at 40 hr. after HCG injection.
Six hours before expected ovulation the gilts
were anesthesized with pentothal via the anterior vena cava. Closed circuit fluothane-oxygen-nitrous oxide anesthesia was used thereafter. Mid-ventral laparotomies were performed
and both oviducts were exposed. A blunt-tip
20-gauge needle, 4 cm long, was inserted into
the uterine lumen approximately 1 cm posterior to the utero-tubal junction and passed into
the isthmus of the oviduct (figure 1). Twenty
million motile sperm cells in a volume ofO.1 ml
were deposited in each oviduct.
Semen was collected as the sperm rich
fraction. Motility was estimated at 100X using
the light microscope. Gilts were inseminated
with the following types of semen: (1) neat
semen (+ extender) collected from boars
demonstrating normal fertility; (2) semen +
extender from a boar that had previously
demonstrated normal fertility and had been
treated for at least 5 consecutive days before
semen collection with a-chlorohydrin (Aldrich
Chemical Co., Milwaukee, Wisconsin) at the
rate of 5 mg/kg of body weight per day; (3)
neat semen as in (1), except that a-chlorohydrin was added to the semen to give a final
concentration of I0 mM. All semen was extended in Ringer-fructose prior to insemination
(Mann, 1964).
Gilts were slaughtered between 40 and 44
1209
ACTION OF ALPHA CHLOROHYDRIN IN BOARS
treatment, and used for insemination. A total
of 23 gilts were inseminated. Pregnancy was
determined as in trial III.
Results
~V
r,r
Trial I. Of 21 normal ova recovered from
Group 1 gilts, 12 were fertilized (table 1).
Seventeen ova were recovered from gilts slaughtered after insemination with semen from
~-chlorohydrin treated boars (Group 2). None
of the 17 ova were fertilized. A similar response
was obtained when ~-chlorohydrin was added
to semen (Group 3). Of 23 normal ova recovered, only two were fertilized, both from
the same gilt. Five abnormal ova were recovered, all from one gilt and all were fertilized.
The abnormal ova contained four or five
pronuclei or had irregular blastomeres. In the
two gilts, in which fertilization occurred, the
semen used had approximately 5 ruin. of
exposure to the a-chlorohydrin prior to deposition in the oviduct. In subsequent inseminations, a 20-min. exposure time was allowed. It
was quite possible that fertilization occurred
because of the lack of sufficient exposure time.
The number of accessory sperm cells were
estimated on each ovum. The ova from Group 1
had a greater number of spermatozoa per ovum
than the ova from other groups (table 1).
Recovery rates of ova in trial I were not
satisfactory. The unavoidable manipulation o f
the oviduct before ovulation may have interfered with the pickup of ova by the infundibulum,
Trial II. Of the 34 normal ova recovered in
Group 1, 25 were fertilized (table 1). Seven of
the unfertilized ova were from one gilt which
had no fertilized ova. Of the 110 normal ova
recovered fr0~ gilts inseminated with semen
from a-chlorohydrin treated boars or with
~-chlorohydrin treated semen (Groups 2 and 3),
none were fertilized. Two different a-chlorohydrin concentrations were added to semen (10
mM or 1 raM; Group 3), but fertilization was
not obtained in either case. Ova recovery rates
were much higher in this trial than in trial I,
probably due to the fact that inseminations
were performed after ovulation.
Trial 111. N o n e of 13 gilts were pregnant
after insemination with semen collected either
7 hr. after initial treatment with a-chlorohydrin
or 7 hr. after the second dose of a-chlorohydrin
(table 2). Inseminations were made within 1 hr.
o f collection semen. Four of six control gilts
were pregnant (4 of 6 vs. 0 of 13, P < .005 by
chi-square). Of the total of 19 gilts in this trial,
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1210
JOHNSON AND PURSEL
TABLE 2. PROPORTION OF GILTS PREGNANT
AFTER INSEMINATION WITH SEMEN
COLLECTED FROM BOARS BEFORE
AND AFTER TREATMENT
WITH o-CHLOROHYDRIN
Time of semen
collection
No. gilts
No. gilts
inseminated
pregnant
Before feeding
7 hr. after f e e d i n g
31 hr. after feedinga
6
7
6
4
0
0
aSemen collected 7 hr. after second a-chlorohydrin
feeding and 31 hr. after initial feeding
12 returned to estms and seven were slaughtered 25 days post-insemination.
Trial IV. None of eight gilts inseminated
with epididymal semen from boars treated with
ol-chlorohydrin were pregnant (table 3). Four of
the seven gilts inseminated with epididymal
semen from an untreated boar were pregnant,
based on recovery of 25-day-old embryos at
slaughter (4 of 7 vs. 0 of 8, P < .025
chi-square).
Discussion
The results of these experiments indicate (a)
that failure of sperm cell transport in the gilt
was not responsible for the antifertility activity
of a-chlorohydrin in boar spermatozoa (trials I
and II); (b) that a-chlorohydrin was rapidly
absorbed and rapidly transported to the site of
activity (trial III); (c) that the boar epididymis
was the site of ~-chlorohydrin activity (trial
IV). In addition, oviductal deposition of semen
resulted in a higher ovum recovery rate if
conducted after ovulation.
The results of these experiments lead us to
conclude that ~-ch/orohydrin acts at the level
of the cauda epididymis in the boar. This
conclusion is based on the evidence that cauda
TABLE 3. PROPORTION OF GILTS PREGNANT
A_FTER INSEMINATION WITH EJACULATED
OR EPIDIDYMAL SE_MEN FROM BOARS
FED a-CHLOROHYDRIN a
Ejaculated s e m e n E p i d i d y m a l s e m e n
T i m e o f se- No. in- No.
No. in- No.
m e n collec- s e m i n pregseminpregtion
ated
nant
ated
nant
Before
treatment
5
3
7
4
After
treatment
3
a5 mg/kg b o d y wt/day.
0
8
0
epididymal spermatozoa recovered from
a-cbAorohydrin treated boars do not have the
capacity to fertilize ova (trial IV). In addition,
the observed antifertility action takes place
within 7 hr. of a-chlorohydrin feeding (trial
III). Thus, the a-chlorohydrin reaching the
cauda epididymis acts upon the already mature
spermatozoa. Turner (1971) and Crabo and
Appelgren (1972) have reported similar results
with the rat, using somewhat different techniques. It also should be pointed out that the
boar, unlike many other mammals, has essentially no storage of spermatozoa in the ductus
deferens. Thus, it may be possible in some
species for cells to be affected in the ductus
deferens, but it is not possible in the boar.
Since the cauda epididymis is the environment in which the a-chlorohydrin acts, the next
question is how is a-chlorohydrin activity mediated? Based on these and other experiments we
suggest that a-chlorohydrin acts directly on the
sperm cell to cause infertility. Several findings
support this suggestion: (a) progressive sperm
cell motility is depressed both by treating the
boar or by treating ejaculated semen directly.
Data on the effect of a-chlorohydrin on motility was reported earlier (Johnson and Pursel,
1972) and the same effect on motility was seen
in this study. Alpha-chlorohydrin given to
male rabbits does not cause infertility or
depress sperm cell motility (Samojlik and
Chang, 1970); a similar non-effect response was
obtained when rabbit epididymal semen was
treated in vitro with ~-chlorohydrin in which
the response was based on motility patterns
alone (L. A. Johnson, unpublished). Another
finding (b) is that accessory gland secretions do
not carry sufficient (if any) (x-chlorohydrin to
cause infertility. This was shown when neat
boar semen was centrifuged, the plasma removed, and seminal plasma was added from a
vasectomized boar, which had been fed
a-chlorohydrin (25 mg/kg body wt/day) for 20
days. These spermatozoa exhibited both normal
motility patterns and normal fertility (L. A.
Johnson, unpublished). Kreider and Dutt
(1972) reported similar results with the ram.
Further (c) failure of sperm transport in the
female was not a causative factor in the
infertility of ~-chloryhydrin affected semen.
The results in trials I and II differ from those
reported by Erickson and Bennett (1971) for
the rat; they suggested that failure of sperm
transport in the female was the factor causing
infertility of rat spermatozoa. In their study,
spermatozoa were transferred from the uterus
of female rats 3 to 4 hours after mating to
a-chlorohydrin treated males, into the ovarian
ACTION OF ALPHA CHLOROHYDRIN IN BOARS
bursa o f unmated females, resulting in fertility.
In the present study, spermatozoa were deposited directly into the oviducts without prior
exposure to the uterine environment. The
differing results would seem to involve one o f
two factors. First, it could be that exposure to
the uterine environment might allow detoxification o f the spermatozoa from the effects of the
a-chlorohydrin, or secondly, that the mechanism of a-chlorohydrin antifertility in the rat
may be different from that in the boar.
The proportion of ova which could be
recovered after insemination into the oviducts
was somewhat lower than that which would be
obtained following intra-cervical insemination
techniques. Polge, Salamon and Wilmut (1970)
reported a similar problem in pigs inseminated
in the oviduct. Semen deposition 4 hr. after
ovulation appears to be preferred to 4 hr.
before ovulation, since a dramatic improvement
in ova recovery was experienced in trial II over
trial I. Results o f trial II showed a 77% recovery
rate, somewhat higher than the 58% reported
by Polge et al. (1970) in gilts inseminated 4 hr.
after ovulation.
We have suggested that a-chlorohydfin acts
directly on the boar sperm cell, causing infertility. In attempting to determine what specific
parts or constituents of the sperm cell are
affected we have ruled out certain aspects based
on the results o f preliminary experiments.
There were no significant morphological
changes observed in the affected sperm cells
when examined under the microscope; spermatozoa phospholipid content was unchanged;
pH of semen with a-chlorohydrin added was
not changed significantly; zinc content o f the
spermatozoa (a possible indication of plasma
membrane integrity) was unaffected by treatment (L. A. Johnson, unpublished data). It
seems possible that a-chlorohydrin may cause
infertility by blocking an enzyme site or sites on
the sperm cell.
1211
Literature Cited
Coppola, J. A. 1969. An extragonadal male anti-fertility agent. Life Sciences 8: Part I, 43.
Crabo, B. and L. E. Appelgren. 1972. Distributions of
14 C ~-chlorohydrin in mice and rats. J. Reprod.
Fertil 30:161.
Erickson, G. I. and J. P. Bennett. 1971. Mechanism of
antifertility activity of minimal effective dose
levels of c~-cMorohydrin in the male rat. Biol.
Repiod. 5:98 (Abstr.)
Ericsson, R. J. and N. D. Connor. 1969. Lesions of the
rat epididymis and subsequent sterility produced
by U-5897 (3-chloro-l,2-propanediol). Soc. Study
Reprod. 2nd Ann. Mtg. Davis, Calif. Sept. 8-10.
(Abstr.)
Ericsson, R. J. and V. F. Baker. 1970. Male antifertility compounds: Biological properties of U-5897
and U-15,646, I. Reprod. Fertil. 21:267.
Johnson, L. A., V. G. Pursel and R. R. Kraeling. 1971.
Cannulation of the Boar Vas Deferens. J. Anita.
Sci. 33:1159 (Abstr.).
Johnson, L. A. and V. G. Pursel. 1972. Reversible
Infertility in Male Swine fed c~-chlorohydrin. J.
Anim. Sci. 34:241.
Kreider, J. L. and R. H. Dutt. 1970. Induction of
temporary infertility in rams with an orally administered chlorohydrin. J. Anim. Sci. 31:95.
Kreider, J. L. and R. H. Dutt. 1972. Effect of
3-chloro-1, 2-propanedioi (U-5897) on accessory
gland secretions of rams. J. Anim. Sci. 35:246
(Abstr.)
Mann, T. 1964. "The Biochemistry of Semen and of
the Male Reproductive Tract." John Wiley & Co.
Melrose, D. R. and C. O'Hagan. 1961. Investigations
into the techniques of insemination in the pig.
Proc. IVth Inter. Congr. Anita. Reprod. and Art.
Insem. The Hague, 4:855.
Polge, C., S. Salamon and I. Wilmut. 1970. Fertilizing
capacity of frozen boar semen following surgical
inseminations. Vet. Rec. 87:424.
Pursel, V. G., L. A. Johnson and L. L. Schulman.
1973. Fertilizing Capacity of Boar Semen Stored at
15~ J. Anita. Sci. (In press).
Samojlik, E. and M. C. Chang. 1970. Anti.fertility
activity of 3-chloro-l,2-propanediol (U-5897) on
male rats. Biol. Reprod. 2:299.
Setty, B. S., A. B. Kar, S. K. Roy and S. R.
Chowdhury. 1970. Studies with sub-toxic doses of
a-chlorohydfin in the male monkey (Macaca Mulatta) Contraception 1: 279.
Turner, M. A. 1971. Effects of a-chlorohydrin upon
the fertility of spermatozoa of the cauda epididymis of the rat. J. Reprod. Fertil. 24: 267.