Human milk: From complex tailored nutrition to bioactive impact on child cognition and behavior.

Human milk is a highly complex liquid food tailor-made to match an infant's needs. Beyond documented positive effects of breastfeeding on infant and maternal health, there is increasing evidence that milk constituents also impact child neurodevelopment. Non-nutrient milk bioactives would contribute to the (long-term) development of child cognition and behavior, a process termed 'Lactocrine Programming'. In this review we discuss the current state of the field on human milk composition and its links with child cognitive and behavioral development. To promote state-of-the-art methodologies and designs that facilitate data pooling and meta-analytic endeavors, we present detailed recommendations and best practices for future studies. Finally, we determine important scientific gaps that need to be filled to advance the field, and discuss innovative directions for future research. Unveiling the mechanisms underlying the links between human milk and child cognition and behavior will deepen our understanding of the broad functions of this complex liquid food, as well as provide necessary information for designing future interventions.

Are hair cortisol levels dependent on hair growth rate? A pilot study in rhesus macaques.

Research incorporating the analysis of glucocorticoids, specifically cortisol, in hair samples has exploded over the past 10-15 years, yet factors contributing to the accumulation of cortisol in hair are not yet fully characterized. In particular, it is not clear whether cortisol accumulation in hair is dependent on hair growth rate, a possibility raised by prior rodent studies reporting glucocorticoid-mediated inhibition of hair growth. Using rhesus macaque monkeys (Macaca mulatta), an extensively studied nonhuman primate species, the present pilot study evaluated the hypothesis that hair cortisol accumulation is inversely related to hair growth rate (i.e., slower hair growth leading to elevated cortisol levels). Hair samples were collected from 19 adult female macaques and 17 infants (9 males) 3 months apart using a shave-reshave procedure from the same site below the posterior vertex of the scalp. The second hair samples were measured to the nearest millimeter (mm) for growth rate over the previous 3 months and assayed for hair cortisol concentrations (HCCs) using enzyme immunoassay. Because of the possibility of age-related differences in hair growth rate, correlational analyses were performed separately for adults and infants to determine whether HCC values were associated with growth rate in each age group. These analyses revealed that neither group displayed a significant correlation of HCCs with hair growth. The results additionally showed that overall, adults had a faster hair growth rate than infants and, as expected from previous studies, had lower HCCs than infants. Our results suggest that higher HCCs within the non-stress range do not result from cortisol-mediated inhibition of hair growth. Moreover, similarities between humans and macaque monkeys in both HPA axis regulation and hair growth rates argue that these findings are relevant for human hair cortisol studies. Extrapolation to other species in which the features of hair growth and the relevant regulatory mechanisms are less well understood should be done with caution.

Nonverbal face-to-face interactions in macaques and humans: A translational pilot study.

Human and nonhuman primate mother-infant dyads engage in face-to-face interactions critical for optimal infant development. In semi-free-ranging rhesus macaques (Macaca mulatta), maternal primiparity and infant sex influence the expression of nonverbal face-to-face mother-infant interactions. However, whether similar patterns of variation exist in laboratory-housed macaques or human mothers is not well understood. Comparing both species would yield information regarding the translational validity of macaques to humans in this important social/developmental domain. In this pilot study, we first compared semi-free-ranging (n = 39) and laboratory-housed (n = 20) macaques, finding that laboratory-housed dyads, first-time mothers, and mothers of sons engaged in higher rates of face-to-face interactions regardless of housing. After translating the nonhuman primate coding scheme for use in a small but diverse group of human mother-infant dyads (N = 27; 44.4% African American, 18.5% American Indian, 7.4% Asian/Asian American, and 29.6% White), we found that, like macaques, human mothers of sons engaged in more face-to-face interactions; however, experienced (vs. first-time) mothers engaged in more interactions. Macaques and humans also engaged in species-specific interactions with their infants. We conclude that components of caregiver-infant nonverbal face-to-face interactions are translatable across human and nonhuman primate species and represent an exciting avenue for future caregiving work.

Dominance rank predicts social network position across developmental stages in rhesus monkeys.

Social network analysis is increasingly common in studying complex interactions among individuals. Across a range of primates, high-ranking adults are generally more socially connected, which results in better fitness outcomes. However, it still remains unclear whether this relationship between social network position and dominance rank emerges in infancy and whether, in species with a social transmission of dominance rank, social network positions are driven by the presence of the mother. To fill this gap, we first explored whether dominance ranks were related to social network position, measured via eigenvector centrality, in infants, juveniles, and adults in a troop of semi-free-ranging rhesus macaques (Macaca mulatta). We then examined relationships between dominance rank and eigenvector centrality in a peer-only group of yearlings who were reared with their mothers in either a rich, socially complex environment of multigenerational (MG) kin support or a unigenerational group of mothers and their infants from birth through 8 months. In Experiment 1, we found that mother's network position predicted offspring network position and that dominants across all age categories were more central in affiliative networks (social contact, social grooming, and social play). Experiment 2 showed that high-ranking yearlings in a peer-only group were more central only in the social contact network. Moreover, yearlings reared in a socially complex environment of MG kin support were more central. Our findings suggest that the relationship between dominance rank and social network position begins early in life, and that complex early social environments can promote later social competency. Our data add to the growing body of evidence that the presence/absence of the mother and kin influence how dominance rank affects social network position. These findings have important implications for the role of caregivers in the social status of developing primates, which ultimately ties to health and fitness outcomes.

Impacts of early social experience on cognitive development in infant rhesus macaques.

Although much is known about the influences of early life experiences on the neurobiology and behavior of macaque models of child development, there is scant literature on cognitive development with respect to early rearing. Here, we examined the effects of rearing condition on affective reactivity and cognitive development in infant rhesus macaques. Infants were pseudo-randomly assigned to one of the two rearing conditions: nursery reared (NR, N = 32; 16 peer-reared, 16 surrogate-peer-reared) or mother-peer-reared (MPR, N = 7). During the first month of life, infants were administered the Primate Neonatal Neurobehavioral Assessment (PNNA). Beginning at 4 months old, infants were tested on cognitive tasks that assessed reward association, cognitive flexibility, and impulsivity. We found no gross cognitive differences between MPR and NR infants. However, MPR infants were more reactive than NR infants on the PNNA. Additionally, reactivity on the PNNA correlated with impulsivity, such that infants who were more reactive at 1 month of age completed fewer trials correctly on this task at 8-10 months. These findings are the first to directly compare cognitive development in MPR and NR infants, and add to the existing literature elucidating the influences of early social experience on temperament and development.

Interindividual differences in neonatal sociality and emotionality predict juvenile social status in rhesus monkeys.

In humans, socioeconomic status (SES) has profound outcomes on socio-emotional development and health. However, while much is known about the consequences of SES, little research has examined the predictors of SES due to the longitudinal nature of such studies. We sought to explore whether interindividual differences in neonatal sociality, temperament, and early social experiences predicted juvenile social status in rhesus monkeys (Macaca mulatta), as a proxy for SES in humans. We performed neonatal imitation tests in infants' first week of life and emotional reactivity assessments at 2 and 4 weeks of age. We examined whether these traits, as well as the rearing environment in the first 8 months of life (with the mother or with same-aged peers only) and maternal social status predicted juvenile (2-3 years old) social status following the formation of peer social groups at 8 months. We found that infants who exhibited higher rates of neonatal imitation and newborn emotional reactivity achieved higher social status as juveniles, as did infants who were reared with their mothers, compared to infants reared with peers. Maternal social status was only associated with juvenile status for infant dyads reared in the same maternal group, indicating that relative social relationships were transferred through social experience. These results suggest that neonatal imitation and emotional reactivity may reflect ingrained predispositions toward sociality that predict later outcomes, and that nonnormative social experiences can alter socio-developmental trajectories. Our results indicate that neonatal characteristics and early social experiences predict later social outcomes in adolescence, including gradients of social stratification.

A descriptive analysis of gut microbiota composition in differentially reared infant rhesus monkeys (Macaca mulatta) across the first 6 months of life.

The gastrointestinal microbiome is recognized as a critical component in host immune function, physiology, and behavior. Early life experiences that alter diet and social contact also influence these outcomes. Despite the growing number of studies in this area, no studies to date have examined the contribution of early life experiences on the gut microbiome in infants across development. Such studies are important for understanding the biological and environmental factors that contribute to optimal gut microbial colonization and subsequent health. We studied infant rhesus monkeys (Macaca mulatta) across the first 6 months of life that were pseudo-randomly assigned to one of two different rearing conditions at birth: mother-peer-reared (MPR), in which infants were reared in social groups with many other adults and peers and nursed on their mothers, or nursery-reared (NR), in which infants were reared by human caregivers, fed formula, and given daily social contact with peers. We analyzed the microbiome from rectal swabs (total N = 97; MPR = 43, NR = 54) taken on the day of birth and at postnatal Days 14, 30, 90, and 180 using 16S rRNA gene sequencing. Bacterial composition differences were evident as early as 14 days, with MPR infants exhibiting a lower abundance of Bifidobacterium and a higher abundance of Bacteroides than NR infants. The most marked differences were observed at 90 days, when Bifidobacterium, Lactobacillus, Streptococcus, Bacteroides, Clostridium, and Prevotella differed across rearing groups. By Day 180, no differences in the relative abundances of the bacteria of interest were observed. These novel findings in developing primate neonates indicate that the early social environment as well as diet influence gut microbiota composition very early in life. These results also lay the groundwork for mechanistic studies examining the effects of early experiences on gut microbiota across development with the ultimate goal of understanding the clinical significance of developmental changes.

Cortisol in Neonatal Mother's Milk Predicts Later Infant Social and Cognitive Functioning in Rhesus Monkeys.

Milk provides not only the building blocks for somatic development but also the hormonal signals that contribute to the biopsychological organization of the infant. Among mammals, glucocorticoids (GCs) in mother's milk have been associated with infant temperament. This study extended prior work to investigate rhesus monkey (Macaca mulatta) mother-infant dyads (N = 34) from birth through 8 months postpartum. Regression analysis revealed that cortisol concentrations in milk during the neonatal period predicted impulsivity on a cognitive task, but not global social behaviors, months later. During this time period, sex-differentiated social behavior emerged. For female infants, milk cortisol concentrations predicted total frequency of play. Collectively, these findings support and extend the "lactational programming" hypothesis on the impact of maternal-origin hormones ingested via milk.

Sex and rank affect how infant rhesus macaques look at faces.

We investigated how differences in infant sex and mothers' dominance status affect infant rhesus macaques' (Macaca mulatta) interest in visually exploring emotional facial expressions. Thirty-eight infants were presented with animated avatars of macaque facial expressions during the first month of life. Sons of high-ranking mothers looked more at faces, especially the eye region, than sons of low-ranking mothers, but no difference in looking duration was found for daughters. Males looked significantly more at eyes than females, but this effect was reversed in infants who were reared without mothers in a primate nursery facility. In addition, in mother-infant interactions, mothers of sons were more likely to gaze at their infant's face compared to mothers of daughters. Combined with previous research indicating that rhesus macaque mothers interact differently with infants based on their own rank and infant's sex, these results support the view that social experiences shape early face preferences in rhesus macaques.

The Signature of Maternal Social Rank in Placenta Deoxyribonucleic Acid Methylation Profiles in Rhesus Monkeys.

The effects of social status on human health can be modeled in captive cohorts of nonhuman primates. This study shows that maternal social rank is associated with broad changes in DNA methylation in placentae of rhesus monkeys (N = 10). Differentially methylated genes between social ranks are enriched in signaling pathways playing major roles in placenta physiology. Moreover, the authors found significant overlaps with genes whose expression was previously associated with social rank in adult rhesus monkeys (Tung et al., 2012) and whose methylation was associated with perinatal stress in newborn humans and rhesus monkeys (Nieratschker et al., 2014). These results are consistent with the hypothesis that system-wide epigenetic changes in multiple tissues are involved in long-term adaptations to the social environment.

The well-being of laboratory non-human primates.

The well-being of non-human primates in captivity is of joint concern to scientists, veterinarians, colony managers, caretakers, and researchers working with non-human primates in biomedical research. With increased regulatory, accreditation, and research focus on optimizing the use of social housing for laboratory primates, as well as the advent of techniques to assess indices of chronic stress and related measures of well-being, there is no better time to present the most current advances in the field of non-human primate behavioral management. The collective body of research presented here was inspired in part by a 2014 symposium entitled, "Chronic Hormones and Demographic Variables: Center-Wide Studies on Non-Human Primate Well-Being" held at the American Society of Primatologists' 37th Annual Meeting in Decatur, GA. By aiming to target readership with scientific and/or management oversight of captive primate behavioral management programs, this special issue provides badly-needed guidance for implementing social housing programs in a research environment and leverages collaboration across multiple facilities to address key components of behavioral management, explore refinements in how well-being can be measured, and identify the interrelationships between varying indices. Am. J. Primatol. 79:e22520, 2017. © 2016 Wiley Periodicals, Inc.

Rhesus macaques (Macaca mulatta) with self-injurious behavior show less behavioral anxiety during the human intruder test.

Self-injurious behavior (SIB) has been linked to anxiety in the human literature, but relatively few studies have explored this link in rhesus macaques. A widely used behavioral assessment of anxiety, the human intruder test (HIT), employs the mildly stressful stimulus of an unfamiliar experimenter to assess anxious behavior in macaques. The HIT was conducted on 59 (20 male) laboratory housed rhesus macaques, 30 with a record of SIB (10 male). If monkeys with SIB have a more anxious phenotype, they should show stronger reactions to the HIT. However, contrary to our predictions, monkeys with SIB did not show higher levels of anxious behavior compared to controls. They spent significantly less time showing anxious behavior and displayed little aggression in response to the stare of the intruder. SIB and control monkeys did not differ in a range score (number of unique behaviors expressed per phase); however, SIB monkeys had a lower change score (total number of behaviors expressed including repetitions) than controls. In general, monkeys that paced regardless of SIB status, showed a reduction in pacing when the intruder entered the room. Possible explanations for the failure of SIB monkeys to show increased anxiety in the HIT include greater exposure of SIB monkeys to unfamiliar humans because of their condition, evidence for a subtype of SIB which is not anxiety related, and/or the presence of comorbid depressive-like symptoms. Am. J. Primatol. 79:e22569, 2017. © 2016 Wiley Periodicals, Inc.

Differential relationships between chronic hormone profiles in pregnancy and maternal investment in rhesus monkey mothers with hair loss in the neonatal period.

Hair loss is commonly used as an indicator of well being in primate facilities, yet it has been shown to also occur in otherwise healthy pregnant and postpartum females. There is significant variability in the incidence of hair loss during these important developmental periods, reasons for which remain unclear. We studied female rhesus monkeys (Macaca mulatta, n = 47) with and without hair loss in pregnancy/postpartum. We hypothesized that, similar to previously published reports, pregnancy would result in an increased likelihood of hair loss, and that hair loss would be correlated with higher hair cortisol concentrations (HCCs). We further hypothesized that hair loss among pregnant females is related to differential maternal investment. We studied a subset of monkeys (n = 26) from mid-to-late pregnancy through peak lactation, some of which exhibited hair loss in the perinatal period (n = 15), and some of which did not (n = 11). We examined fetal measurements, infant birth weight, infant growth rate, and milk yield volume (MYV) in the first 30 days as indices of investment. We found that pregnant monkeys showed a greater incidence of hair loss across the study year (χ2(2) = 6.55, P = 0.038), and that mothers with hair loss had significantly higher HCCs in pregnancy than those without (F(2,28) = 3.8, P = 0.017, ηp2 = 0.21). HCCs in pregnancy were correlated with severity of hair loss in the neonatal period (r(37) = 0.42, P = 0.008). Moreover, HCCs in pregnancy were positively correlated with infant birth weight (r(12) = 0.56, P = 0.038), infant growth rate (r(12) = 0.64, P = 0.014), and MYV (r(11) = 0.85, P < 0.001) for alopecic but not non-alopecic mothers. These mothers did not differ in fetal measurements, infant birth weight/growth rate, or MYV. Our results suggest that hair loss in some monkeys, especially during the birthing season, may be a signal of greater physiological stress during pregnancy and differential investment by mothers to their offspring. Am. J. Primatol. 79:e22489, 2017. © 2015 Wiley Periodicals, Inc.

Rank acquisition in rhesus macaque yearlings following permanent maternal separation: The importance of the social and physical environment.

Rank acquisition is a developmental milestone for young primates, but the processes by which primate yearlings attain social rank in the absence of the mother remain unclear. We studied 18 maternally reared yearling rhesus macaques (Macaca mulatta) that differed in their social and physical rearing environments. We found that early social experience and maternal rank, but not individual traits (weight, sex, age), predicted dominance acquisition in the new peer-only social group. Yearlings also used coalitions to reinforce the hierarchy, and social affiliation (play and grooming) was likely a product, rather than a determinant, of rank acquisition. Following relocation to a familiar environment, significant rank changes occurred indicating that familiarity with a physical environment was salient in rank acquisition. Our results add to the growing body of literature emphasizing the role of the social and physical environment on behavioral development, namely social asymmetries among peers.

Early life adversity alters normal sex-dependent developmental dynamics of DNA methylation.

Studies in rodents, nonhuman primates, and humans suggest that epigenetic processes mediate between early life experiences and adult phenotype. However, the normal evolution of epigenetic programs during child development, the effect of sex, and the impact of early life adversity on these trajectories are not well understood. This study mapped the genome-wide DNA methylation changes in CD3+ T lymphocytes from rhesus monkeys from postnatal day 14 through 2 years of age in both males and females and determined the impact of maternal deprivation on the DNA methylation profile. We show here that DNA methylation profiles evolve from birth to adolescence and are sex dependent. DNA methylation changes accompany imposed weaning, attenuating the difference between males and females. Maternal separation at birth alters the normal evolution of DNA methylation profiles and targets genes that are also affected by a later stage maternal separation, that is, weaning. Our results suggest that early life events dynamically interfere with the normal developmental evolution of the DNA methylation profile and that these changes are highly effected by sex.

First-time rhesus monkey mothers, and mothers of sons, preferentially engage in face-to-face interactions with their infants.

Face-to-face interactions between mothers and infants occur in both human and non-human primates, but there is large variability in the occurrence of these behaviors and the reason for this variability remains largely unexplored. Other types of maternal investment have been shown to be dependent on infant sex (e.g. milk production and maternal responsiveness) and maternal experience (e.g. symmetrical communication). Thus, we sought to determine whether variability in face-to-face interactions, that is, mutual gazing (MG), which are hypothesized to be important for later socio-cognitive development, could be explained by these variables. We studied 28 semi-free ranging rhesus monkey (Macaca mulatta) mother-infant dyads (6 primiparous; 12 male infants) born and reared at the Laboratory of Comparative Ethology field station at the NIH Animal Center in Poolesville, MD, across the first 90 postnatal days. Infant sex (i.e. male) was a significant predictor of maternal grooming (ß ± SE = 0.359 ± 0.164, Z = 2.19, P = 0.029) whereas both parity (i.e. first time mothers) and infant sex (i.e. male) significantly predicted MG (parity: ß ± SE = -0.735 ± 0.223, Z = -3.30, P < 0.001; infant sex: ß ± SE = 0.436 ± 0.201, Z = 2.17, P = 0.029). Separation from the mother (outside of arm's reach) was not influenced by parity or infant sex. Together with existing literature, these findings point toward differential maternal investment for sons versus daughters. Mothers may be investing differentially in sons, behaviorally, to ensure their future social competence and thus later reproductive success. Collectively, our findings add to the literature that is beginning to identify early life experiences that may lead to sex differences in neurological and behavioral development.

Reproductive consequences of a matrilineal overthrow in rhesus monkeys.

Matrilineal overthrows in macaque societies are rare but devastating events, often resulting in severe morbidity, mortality, and loss of individual and group fitness. The handful of documented macaque overthrows provides scant evidence to reveal the severity or longevity of reproductive consequences resulting from such violent events. We analyzed archival records from semi-free ranging rhesus monkeys, Macaca mulatta, across 6 years (55 ≤ N ≤ 107, from 2007 to 2012) during which time a matrilineal overthrow occurred (in 2009) to test the hypothesis that extremely violent interactions such as a matrilineal overthrow would significantly reduce reproductive fitness for the involved matrilines and for the troop collectively. The matrilineal overthrow resulted in a significant increase in infant loss for the population from the previous year (χ(2) = 8.117, df = 1, P = 0.004), as evidenced by the fact that in 2009, but not in other years, the proportion of infants lost was greater than the proportion of viable infants (χ(2) = 4.55, df = 1, P = 0.03). Moreover, the deposed matriline suffered 100% infant loss in 2009, a significant change from the previous year (χ(2) = 7.87, df = 1, P = 0.005) while the attacking matriline suffered 50% infant loss (also a significant change from the previous year; χ(2) = 4.44, df = 1, P = 0.035), with the uninvolved, lowest-ranking matriline showing no change in infant loss from the previous year (χ(2) = 0.008, df = 1, P = 0.93). The deposed matriline did not produce viable offspring again until 3 years later. We further found that rates of severe fighting (as indicated by the number of fight wounds requiring medical treatment) were positively correlated with infant loss across the 6 years of the study (r[s] = 0.943, P = 0.005). Our data indicate that extreme periods of intra-group conflict, such as the matrilineal overthrow, have marked short-term consequences for individual fitness, and may be extreme examples of the long-term influences that group violence exerts on the mean fitness within a primate troop.

Development of a cognitive testing apparatus for socially housed mother-peer-reared infant rhesus monkeys.

Though cognitive testing of infant monkeys has been practiced for the past 40 years, these assessments have been limited primarily to nursery-reared infants due to the confounds of separating mother-reared infants for assessments. Here, we describe a pilot study in which we developed a cognitive testing apparatus for socially housed, mother-peer-reared rhesus macaques under 1 year of age (Macaca mulatta) that allowed the infants to freely return to their mothers for contact comfort. Infants aged 151.2 ± 18.3 days (mean ± SEM; n = 5) were trained and tested on an object detour reach task. Infants completed training in 5.0 ± 0.2 days, and completed testing in 6.2 ± 0.9 days. Across 4 days of testing, infants improved to nearly errorless performance (Friedman test: χ(2) = 13.27, df = 3, p = 0.004) and learned to do the task more quickly (Friedman test: χ(2) = 11.69, df = 3, p = 0.009). These are the first cognitive data in group-housed, mother-peer-reared rhesus monkeys under 1 year of age, and they underscore the utility of this apparatus for studying cognitive development in a normative population of infant monkeys.

The integrative biology of reproductive functioning in nonhuman primates.

At the 34th annual meeting of the American Society of Primatologists in 2011, the society organized an interdisciplinary symposium entitled, "Reproductive Function & Dysfunction in Nonhuman Primates." The articles in this special section, excluding this introduction, represent the findings presented by four of the five speakers in that symposium. The data presented highlight the myriad factors that contribute to primate reproductive function and dysfunction, including hormones, genes, maternal variance, environmental factors, social relationships, and strategic interactions. Collectively, these articles emphasize the integrative nature of primate reproductive function, and highlight the importance of the nonhuman primate as a model for human reproductive function and dysfunction in humans.