Characteristics of anemic infants and their mothers
Mothers of the anemic infants tended to be older and more parous than those of controls, but differed significantly only in the percent of the group that had previously raised a live infant, which was larger for the IDA than for the control dams (). IDA infants showed a clear pattern of iron deficiency anemia and did not overlap with controls in hemoglobin measures (). Sex did not influence most hematological parameters but an interaction between sex and group was seen for MCV (F(1,98)=4.94, p=.03) with IDA males having significantly lower MCV than IDA female infants (p<.05, Tukey’s LSD). As intended by the matching procedure, the 2 groups did not differ in gender, age, or weight.
Comparison of dams’ backgrounds
Comparison of infant hematology.
There was no effect of IDA on plasma cortisol () using 2-factor (group, sex) RMANOVA (F(1,92)=.26, n.s.), nor was there a significant interaction between group and sex. Over all time points, the 2 sexes differed in plasma cortisol (F(1,92)=5.22, p=.025), with females having higher cortisol values than males. The analysis also demonstrated a significant effect of timepoint (F(3,90)=99.40, p<.0001). Contrasts between successive timepoints demonstrated a nonsignificant increase between the first and second timepoints (F(1,92)=3.78, p= .0548), and a strongly significant decrease between the second and third timepoints (after dexamethasone injection, F(1,92)=41.72, p<.0001) and a strongly significant increase between the third and fourth timepoints (after ACTH injection, F(1,92)=282.73, p<.0001). There were no significant interactions of timepoint with group (F(1,90)=1.63, p=.188) or sex (F(1,90)=2.32, p=.081), and the three way interaction was not significant (F(1,90)=2.38, p=.075).
Figure 1 Plasma cortisol response of control and IDA infants. The sample at 1100 h of Day 1 shows the initial response to separation from the home cage and placement in a novel environment, while the sample at 1600 h day 1 likely reflects adaptation to the novel (more ...)
Holding Cage observations (Day 1, Day 2)
Observations taken in the holding cage during a 5-min period near the beginning and end of the BBA were summarized as the duration of 3 activity states and the frequency of environmental exploration (). Examination of sex effects and group by sex interactions indicated that it was appropriate to examine the sexes separately for group effects on Day 1 (sex by group interactions, Day 1 F(1,87)=2.53–5.31, p=.04–.11; Day 2 F(1,86)=.01–1.72, p=.19–.94). IDA males differed from control males in having a greater duration of motor activity (F(1.87)=5.86, p=.020), a lower duration of hanging from top and sides of cage (F(1.87)=4.67, p=.037), a longer duration of sitting (F(1.87)=4.00, p=.053) and a higher incidence of exploratory activity (F(1.87)=10.92, p=.002) during the Day 1 observations, which were conducted within the first hour after the infants were separated from mother and transferred to the novel environment. Female IDA and control infants did not differ on these measures on Day 1. No effects of IDA were identified for these measures taken during the Day 2 observations, near the end of the 25-h session. Also, no sex or group by sex interactions were seen on Day 2.
Figure 2 Behavior in the Holding Cage situation. The duration for 3 states and the frequency for one behavior (environmental explore) during a 5-min period on Day 1 (top graph) and Day 2 (bottom graph) are compared between groups within sex. F=female, M=male. (more ...)
Examination of the composite indices of behavior that were derived from factor analyses (see above) indicated potential sex by group interactions on both days (Activity Day 1, F(1,86)=2.41, p=.12; Activity Day 2, F(1,85)=2.88, p=.09; Emotionality Day 1, F(1,86)=.35, p=.55; Emotionality Day 2, F(1,85)=6.99, p=.01). There were no effects of IDA on these composite indices for females (F(1,49)=.0005–2.06, p=.16–.98). Male IDA infants, by contrast, had higher z-scores than control males for the Activity factor for both Day 1 (F(1,37)=4.13, p=.049) and Day 2 (F(1,37)=4.32, p=.045) and lower z-scores for the Emotionality factor on Day 2 (F(1,37)=4.97, p=.03) ().
Composite z-scores derived from Holding Cage observations by exploratory and confirmatory factor analysis of a larger set of infants participating in the BBA. F=female, M=male. *p<.05 compared to like-sexed control.
Novel object contact
Contact with the novel objects was automatically recorded and summarized as the percent of 5-min periods in which the infants contacted the objects at least once. These scores were generally greater in the IDA infants than in controls over the 25-h period. Omitting the dark period of the light cycle, when infants contacted the object during less than 5% of the 5-min periods, contact averaged 70±3% of the 5-min periods for the IDA infants and 62±2% for controls (F(1,82)=4.09, p=.046) . This was due primarily to the difference in contact time with the second novel object on Day 2 (74±4% vs 61±3%, F(1,82)=6.83, p=.011); differences were not significant on Day 1. Groups did not differ in the number of 15-sec periods with contact within the 5-min periods, which averaged 3.0±0.2. There were no sex or sex by group effects on novel object contact.
Behavior of the infant monkeys showed definite changes during the 4 successively presented conditions of the human intruder tests (profile far, profile near, stare far, stare near). In particular, some behavioral states decreased in duration across conditions (sit, F(3,88)=23.87, p<.0001; crouch, F(3,88)=2.82, p=.04), while others increased (active, F(3,88)=42.70, p<.0001; hang, F(3,88)=9.49, p<.0001; stand, F(3,88)=18.99, p<.0001). There were no significant group or sex effects for these variables, but a group by sex interaction was indicated for “sit” (F(1,87)=5.82, p=.018). When sexes were analyzed separately for “sit”, a significant effect of group was found in the males (F(1,37)=7.83, p=.008), with IDA males showing less time in the “sit” behavior state than control males. Females did not differ in duration of “sit” (F(1,50)=.21, p=.65). IDA did not significantly influence the incidence of species-typical emotional behaviors (bark, scream, and coo vocalizations; lipsmack, fear grimace and threat facial expressions) recorded during the human intruder test. The number of infants displaying a vocalization or facial expression behavior at least once in the 1-min period increased significantly across the 4 conditions (profile far=6%, profile near=7%, stare far=30%, stare near=68%, χ2=127, df=3, p<.0001). The percent of the IDA group showing these behaviors was lower than the percent of control infants, but these differences were not statistically significant. The number of infants exhibiting body postures/movements indicative of distress (convulsive jerk, cling, scratch, self-bite, tooth grind) decreased across the first 3 conditions but increased again in the “stare near” condition. The number of IDA infants demonstrating these behaviors was also lower than but not statistically different from controls.
The individual species-typical emotional behavior occurring in the most infants (66%) in response to the human intruder was threat (see for description) under the “stare near” condition. This behavior was also highly sex dependent (males 65%, females 39%, χ2=5.50, df=1, p=.03) and demonstrated a sex-dependent effect of IDA. 21% of IDA males demonstrated threat as compared to 64% of control males (χ2=6.51, df=1, p=.01). In contrast, more IDA females demonstrated threat (35%) than control females (19%), although group differences were not significant.
Analysis was conducted on a distress index that was previously found to differ in response to the human intruder conditions depending on prenatal iron deprivation of nursery-reared infants (Golub et al. 2006
). The index was the sum of frequency of the following behaviors: cage shake, convulsive jerk, self-clasp, crouch, motor stereotypy, self-bite, scratch, tooth grind, screech vocalization. In the present data for mother-reared infants there was a significant effect of condition (RMANOVA, F(3,85)=12.65, p<.001) on the distress index. The frequency of distress-related behaviors increased from the least to the most challenging condition. Analysis by group of the contrast between successive conditions demonstrated that; (1) neither group increased their distress index from “profile far” to “profile near”, (2) the control group (but not the IDA group) increased from “profile near” to “stare far” (F(1,56)=4.87, p=.031) and (3) both groups increased from the “stare far” to the “stare near” conditions (control, F(1,56)=12.89, p=.0007; IDA, F(1,31)=6.55, p=.016).
Distributions of some temperament category scores (aggression, depressed) were skewed to the right with the majority of animals given the lowest score (1). These categories were scored as binary variables (1, >1). The other categories were analyzed by chi-square as the number of infants given each rating score. None of the temperament categories showed significant effects of group with both sexes combined. Three temperament categories demonstrated a significant effect of sex: active (χ2 =14.23 p=.027), nervous (χ2 =9.7 p=.044), tense (χ2 =12.60 p=.050). When the effect of group was examined separately for each sex for these three categories, IDA males were found to differ from control males on scoring of the active category (χ2 =13.19, p=.022) with more IDA males receiving lower scores. No group differences were identified for the factor clusters derived from multivariate analysis.