The following abstract presents results of a study conducted by a contract laboratory for the National Toxicology Program. The findings were not evaluated in accordance with the levels of evidence for reproductive or developmental criteria established by NTP in March 2009. The findings and conclusions for this study should not be construed to represent the views of NTP or the U.S. Government.
Three experiments were designed to test the influence of reduced feed consumption (and resultant weight loss) on reproductive performance of Swiss CD-1 mice. The ultimate goal was to assess the contribution of weight loss alone to diminished reproductive competence in the Reproductive Assessment by Continuous Breeding Protocol.
Experiment 1, a four-week pilot study in both sexes, determined the rations required to sustain body weight at 70, 80, and 90% of ad libitum controls (n=10/sex/group). After ration adjustment began on day 14, body weight targets were reached within two to five days for all groups of both sexes. However, males were more difficult to maintain on target and lost an additional 4 to 5% of body weight during the remainder of the experiment. Females were within 2% of target on day 28.
Experiments 2a (males) and 2b (females) were designed to determine the reproductive competence of adult Swiss CD-l mice during feed restriction. Target body weights were 70, 80, and 90% of ad libitum controls in both experiments. Twenty males or 20 females were assigned to each body weight group.
The fertility of males was tested twice during a 15-week period of feed restriction, at 8 and 15 weeks, and although target body weights were achieved (within 3%) by week 2, pregnancy rates of test females (nonrestricted virgins) were not significantly affected, even at the second test at 15 weeks when it was reduced by 21%. Lowered feed consumption did decrease the ability of males at 70% BWT to produce copulatory plugs (Mating Index) but, in spite of this, adequate number of litters were produced. The number of live pups per litter was reduced at 8 weeks for 70%, 80%, and 90% BWT groups, and the remainder was reduced at 15 weeks but was no longer significant, probably because at 15 weeks the number of litters per male was reduced instead. Pups delivered to all groups appeared to be in good health, and when adjusted for litter size, body weights were equivalent both at week 8 and week 15 mating.
Some reproductive and somatic organs of feed-restricted males were decreased by body weight loss. After adjustment for relative body size. the prostate was significantly smaller at 70% BWT, the seminal vesicles were smaller at 70 and 80% BWT, and the liver was relatively smaller in all groups compared to the ad lib controls. In contrast. the testicular and epididymal weights were conserved and were larger relative to body weight for all feed restricted groups. Sperm parameters (motility, morphology, and testicular spermatid head concentration) were unaffected by restricted feed at 70%, 80%. and 90% BWT, but sperm concentration was 30% lower in the cauda epididymis at 70% BWT. Occasional degenerative foci in seminiferous tubules were more frequently observed at 70% BWT, but intratesticular spermatid numbers were not reduced at any body weight. Intratesticular testosterone. on the other hand, was reduced by over 50% at 80% and 90% BWT, compared to the ad lib controls, and reduced over 85% for the 70% BWT group.
The fertility of feed-restricted female mice was tested after 17 weeks of feed restriction. and their estrual cyclicity was monitored at 8 and 15 weeks. After eight weeks of feed restriction, the percentage of time spent in the four stages of the estrous cycle at 70% BWT was altered--fewer animals exhibited pro-estrus and estrus, and more smears were unclear. After 15 weeks of feed restriction, the estrous cycle was longer at 70 and 80% BWT, and smears for the 70% BWT group exhibited an altered distribution among the stages of the cycle similar to that at eight weeks. Pregnancy rates following the 17-week fertility test were significantly different among groups. At 70% BWT, three dams delivered litters, but pups were found dead, while the 80, 90%. and ad lib groups delivered 6, 8, and 11 litters, respectively. The number of live pups per litter was decreased at 70, 80, and 90% BWT. Pups delivered to all groups appeared to be in good health, but when adjusted for litter size. body weights were not equivalent. Pups born to both 80 and 90% BWT dams were smaller.
Both reproductive and somatic organs of females were affected by feed restriction to a greater degree of severity than in males. After adjustment for relative body weight, both ovaries were significantly smaller at 70% BWT, the uterus was smaller at 70 and 80% BWT, and the liver was smaller in all feed-restricted groups. Histologically, the ovary and uterus were both hypoplastic for the 70% BWT'. For both Experiments 2a and 2b, clinical signs were consistent with malnutrition. In addition, both sexes had necrotic lesions at the tips of their tails. The severity (length) of the lesion and the incidence of the lesions were positively correlated with the amount of body weight loss.
Serum chemistries (in blood collected at necropsy) for both Experiments 2a and 2b were significantly altered by body weight loss. Albumin was increased at 70 and 80% BWT in males, while total protein and BUN were unaffected. In females, albumin and BUN were unaltered, but total protein was decreased at 70% BWT. Creatinine was decreased for all groups in both sexes. Alkaline phosphatase and bile acids were increased at 70% BWT for both sexes, and bile acids were increased at 80% BWT in males. Cholesterol and 5-nucleotidase were both decreased at 70% BWT in females, and cholesterol was decreased in 70% BWT for males and females; triglycerides were decreased at all reduced body weights for both sexes; 5- nucleotidase was increased in 70% BWT males. SDH, CPK, and ALT were normal at all feed-restricted levels for both sexes. Serum glucose was lower in males at 70, 80. and 90% BWT and in females at 70 and 80% BWT.
Thus, body weight loss following feed restriction for 15 to 17 weeks significantly altered reproductive competence. organ weights, and serum chemistries in both males and females. Reproductive competence in females was more severely affected than in males. The ability to conceive and deliver live young was severely compromised in the 70, 80, and 90% BWT females. Thus, in the absence of any potential toxicant, a 10% BWT loss in females can significantly reduce live litter numbers and litter size. A 10% BWT loss in males or females can reduce liver weight. A 10% BWT loss in males can cause decreased live pups per litter. A 20% BWT loss can reduce litter size and number, lengthen the estrus cycle, and reduce uterine weight In females, and may reduce live pups per litter, number of litters, and seminal vesicle function (weight) in males. A 30% BWT loss could lengthen the estrous cycle, reduce uterine and ovarian weight, disrupt successful delivery of litters in females, decrease seminal vesicle and prostate weight, and lower epididymal spermatozoa concentrations by 30% in males, and may decrease litter size or number in females mated to 30% BWT males. The weight of testis and cauda epididymis relative to body weight was conserved in all BWT groups, indicating that reduction in adjusted testicular weight would truly be a toxicant effect.
In summary. these data indicate that it is essential to carefully assess the contribution of diminished body weight or reduced body weight gain to diminished reproductive capacity when a potential reproductive toxicant is being evaluated.
NTIS # PB92174259