TOXICOLOGY
AND APPLIED PHARMACOLOGY
Subacute
Toxicity RAYMOND
Departments
9, 293-299
(1966)
of Apholate B.
HERRICK
to the AND
MARTIN
of Animal Science and Entomology, College University of Hawaii, Honolulu, Hawaii Received
December
Mature
Chicken]
SHERMAN of Tropical 94822
Agriculture,
27, 1965
In a previous paper, Sherman and Herrick (1966) describe certain toxicologic and physiopathologic effects of feeding the alkylating agent apholate to young male chickens. Because of the suppressing effect of the chemosterilant on the immature gonad, it was of interest to follow up this study and investigate possible effects of feeding apholate on the reproductive performance of the mature chicken. This paper describes the toxicologic effects of feeding apholate to Single Comb White Leghorn cocks and hens. METHODS
The experimental animals used in this 14-week study were year-old Single Comb White Leghorn males and females. Four groups of birds were administered apholate in the feed at concentrations of 5000, 2500, 1000, and 500 ppm for a period of 7 weeks. These diets were prepared by dry-mixing an 8.8% apholate concentrate in sugar with a standard University of Hawaii laying ration. A fifth group of control birds receiving no apholate was placed on feed which contained sugar at the same level as the level of sugar in the 5000 ppm apholate mixture. All ‘rations were fed ad libitum, and fresh water was available at all times. To study possible recovery from treatment, surviving males and females from each treatment were placed on untreated feed ad libitum for a subsequent 7-week period. Feed consumption by group and individual body weights were recorded weekly. Each group of birds was composed of 6 males and 24 females except the group receiving 500 ppm of apholate, which was composed of 3 males and 12 females. All birds were confined in individual cages. Preliminary studies on fertility revealed that all males were 78-9370 fertile. Semen was collected twice each week by modifying slightly the method described by Burrows and Quinn (1937). A l-ml tuberculin syringe was used to measure the volume of semen obtained at each collection. The semen was either used for insemination or taken into the laboratory for quality evaluation. The interval between collection and microscopic observation was less than 30 minutes. Sperm motility was assessed by microscopic examination of a whole drop of undiluted semen. The rate of wave motion and the proportion of immotile spermatozoa 1 Presented in part at the annual meeting of the Poultry Science Association, St. Paul, Minnesota, August 4-7, 1964. Published with the approval of the Director of the Hawaii Agricdtural
Experiment
Station
as Technical
Paper 293
No.
782.
294
RAYMOND
B.
HERRICK
AND
MARTIN
SHERMAN
present were scored using ratings between 0 and 5 as described by Allen and Champion (1955). Sperm concentration was also determined by the method of Allen and Champion ( 1955). Hens were artificially inseminated each week with 0.05 ml of semen, All matings were pedigreed. Semen from each treated male was used to inseminate 4 hens receiving untreated feed, which served as testers. Semen collected from untreated males was used to inseminate the hens receiving apholate-treated rations. Females were inseminated with semen from the same male throughout the course of the experiment, or as long as the male was capable of ejaculating semen. Daily egg-production records were kept for all hens throughout the course of the experiment. All eggs were pedigreed daily and incubated weekly. The eggs were candled on days 8 and 18 of incubation, All infertile eggs and those with dead embryos were removed and broken out to determine the fertility or age at death. Abnormal embryos were recorded. All observations were made macroscopically. On day 18 of incubation, all eggs containing live embryos were basketed according to pedigree to make possible observations on chicks hatched from eggs laid by a particular dam. Hatchability (chicks/fertile eggs X 100) was recorded for each hatch. Total leukocyte and erythrocyte levels were determined by the procedures discussed earlier (Sherman and Herrick, 1966). Several survivors from each treatment group were autopsied after 7 weeks of treatment. The remaining birds were placed on untreated feed for an additional 7 weeks to trace recovery before they were autopsied. The data were analyzed by means of the t test (Snedecor, 1950). RESULTS
General signs of intoxication of adult chickens fed 5000 and 2500 ppm were the same as those described by Sherman and Herrick (1966) for the chick. All males receiving 5000 or 2500 ppm of apholate in the diet died within 3 weeks. Seventy-five per cent of the hens fed 5000 ppm of apholate and SOY, of those fed 2500 ppm of apholate in the diet died during the 14-week experimental period. In general, signs of intoxication were more severe among males. No signs of intoxication or mortality occurred among either males or females receiving 1000 or 500 ppm of apholate. Treated birds consumed less feed than controls. The degree of loss of appetite was related, in general, to the level of apholate in the ration. The loss of appetite is reflected in Table 1 by the weight loss among all treated birds. Weight loss was significant among both cocks and hens receiving 5000 and 2500 ppm of apholate in the diet as well as among hens fed 1000 ppm of apholate. Although feed consumption increased during the recovery period when surviving birds were transferred to untreated rations after 7 weeks, the hens failed to increase in body weight significantly except in the case of those hens on the lOOO-ppm level of apholate. Males fed treated feed at the level of 1000 ppm of apholate and then subsequently placed on untreated feed also regained lost weight. Apholate produced a significant effect on the reproductive performance of all treated males. A summary of semen characteristics (Table 2) indicates that fertility
TOXICITY
OF
APHOLATE
TO
OF COCKS
AND HENS
TABLE MEAN
BODY
WEIGHT
Apholate in diet
Pretreatment
(ppm)
THE
CHICKEN
295
1 ADMINISTERED
Week
APHOLATE-TREATED
1
Week
7
FEED
Week
(9)
14
(d
cd
(6)
2415
1859G
-11
2306
1906a 2446
-b
-b
2399
2370
2315
2566 -
2487
2462
2536
1633’= 17225
1194a 1352a
1369a
1755a
1624a
2070 2015
1998 2028
Males 5000 2500 1000 500 0
-11
Females 5000
2018
2500 1000
2077 1945
500
2101 1984
0 a Significantly b All
birds
less
than
the
tw-4
2011
P < 0.01.
dead.
CHARACTERISTICS Apholate in diet
control,
1699a 1915 -
OF SEMEN
TABLE 2 OF OXKS ADMINISTERED
APHOLATE-TREATED
FEED
Number Of cocks
Weeks Pretreatment
1
Mean
Fertility
2
3
7
(%I
5000
6
88
81
16
2500
6
86
64
13
-a -n
-n -a
1000 500
6
78 78 93
76 62
21 24
3 22
0
3 6
88
76
79
0 84
6
0.34 0.34
0.06 0.02
-a
-a
6 6
0.49 0.48
-a
-a
0.54
0.60
0.54
0.34
0.08
3 6
0.59 0.55
0.56 0.60
0.51 0.62
0.38 0.68
0.25
concentration
(IO”/mmJ)
2.07
1.48
0.34
1.44 2.26
0.20 1.14
-a -a
-n -a
2.13 2.07
0.28 0.60
0.01
2.62 1.79
0
Mean 5000 2500 1000 500 0
Mean
spermatozoa
5000 2500
6 6
1000
6
2.27 2.79
500
3 6
2.42 1.95
0
Mean 5000 2500 1000 500
dead. score
spermatozoa
4.8 4.7
3.7 3.3
6
4.6 4.7
4.8 4.0
4.6
4.5
of 5 based
on wave
motion
(ml)
motility
6 6 3 6
0 a All birds b Maximum
Volume
and
2.50
0.08 2.20
-0 -a
-0 -a
2.0 2.7
0.2 0.7
4.8
4.0
scoreb 2.0 1.5 3 .o 4.0 4.5
proportion
0.63
of immotile
spermatozoa.
296
RAYMOND
B.
HERRICK
AND
MARTIN
SHERMAN
was completely depressed and semen volume, sperm concentration, and sperm motility were severely restricted at the end of 7 weeks. Recovery of semen quality was slight during the subsequent 7-week period when the cocks previously fed 1000 ppm of apholate were fed untreated feed. Sufficient volumes of semen were not produced by these males during the recovery period to permit the insemination of tester hens; therefore, fertility estimates could not be obtained. The effect of apholate on testis size was pronounced as shown by the depressed testes weights of all treated males (Table 3). Decreased testis size did not appear TABLE EFFECT
Apholate in diet (rwm)
OF ADMINISTERING
Number of cocks
APHOLATE-TREATED
Mean body weighta
3
500
3 3
0
2352 2315 2383 14
1000 0
3 3
start
-c 142 -c 187 2
ZS66k 2536
after
17.12
after
91 2
a Mean & SE. 1, As per cent of body weight. c Significantly less than the control,
AND TESTICULAR
68
start
WEIGHTS
Mean testes weight”
(70,
OF COCKS
Mean testis tubule diameter (mm)
of experiment 2.35’ 3.20”
247
weeks
3 ON BODY
Mean testes weighta Cd
(d 7 weeks
1000
FEED
2 0.40 & 1.10
0.099 0.135
0.12 0.19
k
0.718
0.24
0.14
0.160
0.12
& 6.79
1.200
0.2 7
1.51
of experiment 4.10c 30.11
*
P < 0.01.
to be caused by inanition since the gonads of males receiving 1000 and 500 ppm of apholate were significantly depressed while body weight was not affected significantly. Microscopic examination of the testes of birds sacrificed 7 weeks after treatment revealed a decrease in tubule diameter and a degeneration of the cellular elements of the seminiferous tubules. Testis-tubule diameter of birds fed 1000 ppm of apholate averaged 0.12 mm (Table 3), and neither spermatocytes nor spermatids were observed. Only spermatogonia remained intact. Since the spermatogonia appeared to be unaffected in most of the tubules, the damage to the gonads would appear to be reversible. However, there was no cytologic evidence of gonad regeneration among birds after they were placed on untreated feed for 7 weeks. The mean tubule diameter remained 0.12 mm and there was no evidence of spermatogenesis. Moderate cellular degeneration occurred in the gonads of males receiving 500 ppm of apholate in the diet. Spermatogonia only were observed in some tubules while spermatogenesis was complete in others. The mean tubule diameter was 0.19 mm. Although the lower level of apholate did not produce the marked cellular degeneration noted among males fed 1000 ppm of apholate, the cytologic evidence supports impairment of normal reproductive performance. This is further supported by the marked decline in fertility and semen quality of these birds (Table 2). The gonads of the untreated control males were essentially normal. Tubule diameter averaged 0.27 mm and spermatozoa were observed in all tubules.
TOXICITY
OF
APHOLATE
TO
THE
297
CHICKEN
The depressing effect of apholate treatment on the reproductive performance of females paralleled that of the males. All treated hens became infertile within 4 weeks, and egg production ceased within 5 weeks (Table 4). None of these females returned to production during the 7-week recovery period, during which time they received untreated feed. MEAN
TABLE EGG PRODUCTION
HEN-DAY EGGS LAID
Apholate in diet (mm)
Number of hens
BY HENS
5000 1000 500 0
Pretreatment
2
1
production 18
0
0
0
0
70
33
0
83 76
40 62
23 42
0 0
0
24 12
0 12
24
68
15 57
1 68
65
0 0
0 0
0 0
9
0
0
46 86
0 82
0 84
60
61
0
1000 500
24 12
99
75
99 92
89 88
11 60
24
0
79
TABLE OF ADMINISTERiNG
Number of hens
APHOLATE-TREATED
Mean
body
5 FEED
weight=
ON BODY
AND OVARIAN
Mean
ovary
(!d 7 weeks
5000 2500 1000 500 0
weighta
(9)
after
start
WEIGHTS
OF HENS
Mean ovary weight” (%)
of experiment
1063c 1281c
f 99 -C 105
0.61c
i
0.06
0.058
0.95c
0.077
1677C 2063
k 134 3~ 112
2.03C 3.3oc
_’ 0.07 k 0.36 -c 0.31
0.162
2086
2
e
1.30
2.111
3~ 1.06 C 3.93
0.177 1.930
14 weeks
136
after
start
6
1957
-c
6
2161
rt 127
n Mean I!Z SE. b As per cent of body weight. C Significantly less than the control,
0 0
eggs (%)
58 61
1000 0
5
70
93 88
(wd
4
24 24
24 24
Apholate in diet
3
( YO1
5000 2 500
EFFECT
FEED
Weeks
Fertile
0
OF FERTILE
FED APHOLATE-TREATED
Hen-day 2500
4 AND PROPORTION
84
43.11
0.118
of experiment 3.64C 41.36
P < 0.01.
Treatment had a depressing effect on ovary size (Table 5). Ova were very small, and atretic follicles were present in the ovaries of many of the treated birds. The depressed gonad weight, like that exhibited among males, is apparently due to treatment, not to inanition. The body weight of females receiving the SOO-ppm level of apholate in the diet remained unaffected by treatment while the gonad weights were significantly depressed. Hatchability did not appear to be significantly affected by treatment. Approxi-
298
RAYMOND
B.
HERRICK
AND
MARTIN
SHERMAN
mately 70% of all fertile eggs hatched and all chicks were normal. No abnormaJ embryos were noted that appeared to be caused by treatment. A significant depression of the total leukocyte level occurred in the blood of all birds fed rations containing 1000 ppm or more of apholate (Table 6). This depression in number of circulating white blood cells occurred within 4 weeks after the initiation of treatment. TABLE MEAN
BLOOD
CELL
COUNTS
OF COCKS
Leukocytes Apholate in diet
(pw)
Number of bird+
6
HENS
AND
ADMINISTERED
(103/m&)
APHOLATE-TREATED
FEED
Erythrocytes
(lOe/mma)
Weeks Pretreatment
7
Weeks 14
Pretreatment
7
14
Males 1000
6
21.8
500
3 6
23.2 22.1
0
6.8 8.0 18.0
13.2
3.3
3.3 3.6
3.7 -
15.2
3.6 3.2
3.6
3.5
17.1 14.0
2.6 2.3
0.9 2.1
2.6 2.7
13.1
2.5 2.5
2.7 2.8
2.6 -
2.5
2.7
2.7
Females 5000
6
2500
6 6
1000 500
3 6
0
a Three
birds
in each group
18.4 20.8
3.2
19.2
5.6 8.8
19.4 19.0
11.6 19.6
were
sacrificed
19.4
after
‘I-week
blood
-
cell count.
Recovery of near normal leukocyte levels occurred among survivors of all treatments as shown by the increased numbers of leukocytes in the blood of treated birds after they were placed on untreated feed. Mean spleen weight was depressedonly among hens fed 5000 ppm of apholate in the diet for 7 weeks. Microscopic examination of stained sections of spleen tissue failed to reveal any marked diminution in the number of leukocytes or any significant contraction of the white pulp. Erythrocyte levels were affected only in the case of severe poisoning. A large depressionin erythrocyte numbers occurred among hens fed 5000 ppm of apholate for 7 weeks (Table 6). Recovery of normal erythrocyte levels occurred during the following ‘l-week recovery period. Appreciable changes in erythrocyte numbers did not occur among birds receiving lower levels of apholate. Femur sections from hens that had received 5000 ppm of apholate showedevidence of red bone marrow replacement with yellow marrow. Microscopic examination of femoral bone marrow smears showed increased numbers of immature erythrocytes among these birds. Bone morrow smears of birds receiving lower levels of apholate in the diet revealed red corpuscle development to be essentially normal. DISCUSSION
Apholate in the male chick caused arrest of gonad maturation (Sherman and Herrick, 1966), but in the mature male chicken actual degeneration of the gonads occurred. Apholate also caused testicular atrophy in the rat (Hayes, 1964). Hens fed apholate-treated feed ceasedegg production within 4 weeks and the gonads were highly involuted by 7 weeks. Although alkylating agents have demonstrated terato-
TOXICITY
OF APHOLATE
TO THE
CHICKEN
299
genie activity on the chick (Murphy et al., 1958), no teratogenic effects of apholate were observed in this study. Marked leukopenia occurred among mature birds fed apholate-treated feed. This has also been reported among chicks (Sherman and Herrick, 1966), sheep (Younger and Young, 1963), cattle (Khan, 1963), and rats (Hayes, 1964). SUMMARY Apholate administered to mature chickens at varying levels in the diet from 500 to 5000 ppm produced a significant depression in reproductive performance. Fertility was completely depressed in both cocks and hens. Among males, semen volume, sperm concentration, and sperm motility were greatly depressed. Testis weight of these birds was significantly depressed, and microscopic examination of these tissues revealed degeneration of the cellular elements of the seminiferous tubules. Hens fed apholate-treated diets ceased egg production, and the gonads became highly involuted. Marked leukopenia occurred among apholate-treated males and females. Appreciable changes were not found in erythrocyte numbers except in the case of severe poisoning. Males were more sensitive to high levels of apholate in the diet than females as indicated by the severity of signs of apholate intoxication and greater mortality. ACKNOWLEDGMENTS The authors wish to thank Miss Joan Takahama for her investigation was supported in part by U. S. Public Health Division of Environmental Engineering and Food Protection Medical Research, New Brunswick, New Jersey.
valuable Service and by
technical assistance. This Grant EF 197 from the the Squibb Institute for
REFERENCES ALLEN, C. J., and CHAMPION, L. R. (1955). Competitive fertilization in the fowl. Poultry Sci. 134, 1332-1342. BURROWS, W. H., and QUINN, J. P. (1937). The collection of spermatozoa from the domestic fowl and turkey. Poultry Sci. 16, 19-24. HAYES, W. J,, JR. (1964). The toxicology of chemosterilants. Bull. World Health Organ. 31, 721-736. KHAN, M. A. (1963). Toxicity of apholate to cattle. Can. J. Camp. Med. Vet. Sci. 27, 233-236. MURPHY, M. L., DEL MORO, A., and LACON, C. (1958). The comparative effects of five polyfunctional alkylating agents on the rat fetus, with additional notes on the chick embryo. Ann. N.Y. Acad. Sci. 69, 762-781. SHERMAN, M., and HERRICK, R. B. (1966). Acute and subacute toxicity of apholate to the chick and Japanese quail. Toxicol. AppZ. Pharmucol. 9, 279-292. SNEDECOR, G. W. (1950). Statistical Methods, 4th ed., pp. 62-64. Iowa State Univ. Press, Ames, Iowa. YOUNC~R, R. L., and YOUNG, J. E. (1963). Toxicologic studies and associated clinical and hematologic effects of apholate (an alkylating agent) in sheep-a preliminary report. Am. J. Vet. Res. 24 (lOl), 659-669.