American Journal of Primatology 69:503–518 (2007)

RESEARCH ARTICLE Growth and Developmental Outcomes of Three High-Risk Infant Rhesus Macaques (Macaca mulatta) AMANDA M. DETTMER1, LISA A. HOUSER1, GERALD C. RUPPENTHAL1 SAVERIO CAPUANO1,2, AND LAURA HEWITSON1– 3 1 Pittsburgh Development Center at Magee-Women’s Research Institute, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania 2 Department of Obstetrics, Gynecology and Reproductive Sciences, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania 3 Department of Environmental and Occupational Health, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania

Infants classified as ‘‘high risk’’ are born with a greater chance of developing medical complications at birth, and may have cognitive and other developmental complications later in life. Very few reports exist regarding the survival and outcome of such infants in primate colonies. Here we present early growth and developmental data on three high-risk infant rhesus macaques (one female and two males) that were born either with intrauterine growth restriction (IUGR; born below the 1st birth weight percentile for gestational age) or extremely prematurely (at gestational days 128 and 140; mean full-term gestation 5 164 days). We compared the outcome of these infants with that of healthy controls born at term and found no gross developmental delays in these infants with respect to growth, neonatal reflex and motor skill development, early cognitive development, or social behavior. Neurological and cognitive assessments were compared in terms of both postnatal and gestational age. The survival of these infants was dependent on a 24-hr staffed nursery and a fluid protocol that catered to each high-risk infant’s individual needs. When such measures are implemented, infants such as these have a good chance of survival and can serve as excellent models for high-risk human babies and their subsequent development. Am. J. Primatol. 69:503–518, 2007. c 2007 Wiley-Liss, Inc. Key words: primates; Macaca mulatta; behavior; development; socialization INTRODUCTION Both human and nonhuman primate (NHP) premature and low-birth-weight infants are born with a higher risk of medical complications and developmental Contract grant sponsor: NIH; Contract grant number: HD12912; ES12359. Correspondence to: Laura Hewitson, Pittsburgh Development Center, 204 Craft Ave., Pittsburgh, PA 15213. E-mail: [email protected]

Received 5 October 2005; revised 8 May 2006; revision accepted 8 May 2006 DOI 10.1002/ajp.20349 Published online 9 February 2007 in Wiley InterScience (www.interscience.wiley.com).

r 2007 Wiley-Liss, Inc.

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delays than term infants. Such medical complications may include hyaline membrane disease [Lynch, 2004; Pruitt et al., 1979; Revak et al., 1996; Yoder et al., 2000] and other respiratory complications [Bharkoo et al., 2000], necrotizing enterocolitis [Loh et al., 2001; Yeh et al., 2004], and anemia [Armony-Sivan et al., 2004], all of which require neonatal intensive care. Clinically, the outcome of premature and low-birth-weight neonates varies depending on the severity of conditions at birth, and may result in growth retardation [Cooke et al., 2004], neurodevelopmental abnormalities [Spinillo et al., 2004], and increased morbidity and mortality [Garite et al., 2004]. A convincing body of data shows that preterm human infants suffer high rates of cognitive and behavioral deficits later in life [Bhutta et al., 2002; Hack et al., 2002; Walther et al., 2000]. Similar deficits are reported for high-risk NHP neonates [Davenport et al., 1973; Gunderson et al., 1989]. Once the infant’s condition has stabilized, delays in the onset of object concept permanence and motor skills, such as walking and hand-eye movements [Ruppenthal et al., 1983], and attaining self-feeding [Sackett et al., 1975], are often observed. In addition, high-risk infant monkeys that are left with their mothers in a colony have a high mortality rate (495%), whereas in a 24-hr staffed neonatal intensive-care unit (NICU) over 80% of such infants survive [Sackett et al., 1975]. Without roundthe-clock intensive care, premature primate infants may also show cognitive deficits [Schrier et al., 1983, 1990]. Thus, the importance of a primate NICU is underscored for researchers studying the development and behavior of infant primates. Information concerning nursery rearing of NHPs is readily available, including some reports on how to improve the odds of survival for high-risk infants [Champoux et al., 2002; Ruppenthal, 1979; Ruppenthal & Sackett, 2005; Sackett et al., 1975, 2002; Worlein & Sackett, 1997]. Both immature physiology and behavioral developmental status can impact success or failure. Occasionally infants that have been predicted to succumb to severe defects survive because of the intensive care they receive, and even develop quite normally (unpublished data). While this is an interesting phenomenon in itself, because these medically high-risk infants are a good model for human perinates and have been increasingly used to study prematurity over the past several years [Sackett, 1980; Sarkadi-Nagy et al., 2003], it is imperative to learn how to improve survivorship and reduce morbidity in this population. Here we present perinatal data on the growth, development, and behavior of one severely low-birth-weight infant and two extremely premature rhesus infants, which, after stabilization and with 24-hr intensive care, we predicted would show no gross differences compared to healthy and full-term controls. MATERIALS AND METHODS Subjects The subjects included three high-risk infant rhesus macaques and 14 control infants. The first infant, AB, was a male twin conceived by intracytoplasmic sperm injection (ICSI) [Hewitson et al., 1999], an established method of assisted reproduction that has been used clinically since 1992. Two embryos conceived by ICSI were transferred into the oviduct of a surrogate female. Both embryos implanted, but the second fetus, a female, was stillborn with a normal birth weight. AB was born at a normal gestation of 153 days with a birth weight of 174 g, placing him below the 1st birth weight percentile for his gestational age. He was diagnosed at birth with intrauterine growth restriction (IUGR). The

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normal gestational age at birth for rhesus macaques is 16476.7 days [Mahoney et al., 1979]. The average birth weight for rhesus macaque males is 490760 g [Catchpole & Van Wagenen, 1956]. The two other high-risk infants were naturally conceived and born prematurely. A male, AC, was born at gestational day 128, and a female, AE, was born at gestational day 140. The infants were classified according to protocols established at the Infant Primate Research Laboratory (IPRL) in Seattle [Ruppenthal & Sackett, 2005], where prematurity is defined as a delivery resulting in more than 2 standard deviations (SDs) from the mean gestational age for full-term macaque infants, and IUGR refers to a delivery resulting in a birth weight under the lowest 10th percentile for gestational age. Husbandry Pregnancies were estimated via timed mating and confirmed through ultrasound. In addition to receiving daily health checks, the dams were examined monthly by ultrasound once pregnancy was established. Serial ultrasound examinations were performed beginning 21 days post-embryo transfer on dam 001, AB’s surrogate mother. At the first examination it was noted that two fetuses were present, and that measurements of the yolk sacs and gestational sacs were within normal limits for published rhesus macaque values. Measurements of biparietal diameter and femur length also remained within published values throughout the first and second trimesters. It was not noted until late in the third trimester (predicted gestation day 141) that the male fetus (AB) was growing at a slower rate than the female fetus (stillborn). This discrepancy was attributed to competition for maternal resources associated with a twin pregnancy, and no other evidence of fetal or placental abnormalities was noted. Furthermore, no evidence of impending premature parturition was noted from ultrasound evaluation or manual palpation of the dam’s cervix prior to the premature births of AC and AE. All infants were delivered naturally and reared in a 24-hr staffed nursery according to procedures outlined by Ruppenthal and Sackett [1992]. All infants were housed in an incubator until they were able to thermoregulate (98–1011F). Diurnal measurements were recorded every 1–4 hr to assess the respiration, heart rate, and body temperature of each subject until they attained thermoregulatory capability and a stable breathing status. Weights were recorded daily for each infant. Immediately following birth, supplemental oxygen, subcutaneous fluids, and/or antibiotic treatments were administered as needed. AB received ampicillin (100 mg/kg) twice a day for 7 days and gentamicin (5 mg/kg) four times over 7 days. AC received ampicillin twice a day for 5 days and gentamicin three times over 5 days. The infants were fed cow’s milk formula (Enfamil with Lipil; Mead and Johnson, Evansville, IN) containing docosahexaenoic acid (DHA) and arachidonic acid (ARA), which have been shown to improve visual function in preterm infants [Neuringer et al., 1988; Reisbick et al., 1997]. Changes in the feeding regimens were dictated virtually on a feeding-by-feeding basis. Attempts were made to ensure that 1) if residual fluid remained in the stomach prior to the subsequent feeding (as measured by a gavage feeding tube), the next feeding was adjusted accordingly; 2) if the remaining formula in the stomach appeared to be undigested, as indicated by volume or a curdled consistency, at the next feeding the volume was reduced or diluted with glucose/electrolytes; and if bile was present in residual fluid, clear fluids were administered in a subsequent feeding or until bile was absorbed; and 3) if it appeared that no digestion had occurred, only subcutaneous fluids were administered to maintain hydration until the gut became motile.

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We have found from previous experience that these precautions are paramount for reducing morbidity [Ruppenthal & Sackett, 2005]. Aspiration pneumonia was the leading cause of morbidity in the IPRL until gavage feeding was implemented. Due to a lack of coordinated suck-and-swallow reflex, which is common in premature humans and NHPs and can lead to aspiration [Ruppenthal & Sackett, 2005], each infant required feeding by a 3.5-French gastric gavage feeding tube for several days following birth. The sucking reflex is present as early as 120 gestational days, but coordination and successful feeding requires either a gestational age of 4150 days [Ruppenthal & Sackett, 2005] or postnatal development (unpublished observation). AB and AE were gavage-fed for 9 and 8 days, respectively, and AC was gavage-fed for 20 days. Once the infants were stable and thermoregulatory, they were moved to single-cage housing with a mobile hanging cloth surrogate. These cages allowed for visual, auditory, and olfactory contact with conspecifics. Tactile contact occurred in daily play cage socialization sessions that lasted at least 30 min per day.

Growth Measures The crown–rump length (CRL) of AB and AC was estimated at birth with the use of a ruler. As calipers and measuring tape became available, formal anthropometrics were recorded for AE. Weights were recorded daily for each infant.

Neonatal Reflexes and Sensorimotor Skills Nursery assessments based on the Brazelton assessment scale, which was originally developed for human infants [Brazelton, 1984], were performed from birth through 30 days of age once the subject was stable enough for testing. In some instances the test occurred inside the incubator. Tests were performed three times a week and measured basic motor reflexes, survival reflexes, and sensorimotor skills. The criterion for each measure was reached once the infant displayed the highest possible score.

Object Concept Permanence Based on methods studied by Piaget [1954] and modified by Ruppenthal and Sackett [1992] and Ruppenthal et al. [2004], a visible displacement task measuring emerging cognition was given to each infant three times per week once the infant was stably housed in a single cage. Testing normally begins at postnatal day 14, which is when AE began testing. AB began testing at day 16, and AC did not begin testing until day 56 due to the length of his stay in the incubator. The four stages of object permanence testing were as follows: 1) plain reach (the infant was required to reach out an pick up a toy in plain view), 2) hiding behind a screen (the infant had to retrieve the toy after it was covered by an opaque screen), 3) hiding in a well (the infant had to retrieve the toy after it was placed in a well and covered with an opaque lid), and 4) A-not-B (an advanced well task). The criterion for plain reach was three out of five correct responses in one test session. For the last three stages the criterion was eight out of 10 correct responses over two consecutive test days.

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Social Behavior After they emerged from the incubators, the infants received daily socialization for at least 30 min per day in a play cage (59’’ wide  27’’ deep  32’’ high) equipped with toys, as well as chains and rings for climbing. Each infant was part of a social pair that was formally observed in the play cage three times per week. AB was paired with AC, and AE was paired with a male infant that was not included in this study. Five-minute focal animal sampling was employed, and the infants were observed randomly across each session. TABLE I. Crown-Rump Lengths (CRL) of High-Risk Infants as Compared to Reported CRLs for Normal Infants [Catchpole & Van Wagenen, 1956] Mean (male) AB SD from mean ACa SD from mean Mean (female) AE SD from mean a

196712 mm 125 5.92 135 5.05 194710 mm 147.5 4.65

Measured on day 8.

Po0.05.

Fig. 1. Infant AB at birth, with a CRL of approximately 125 mm (ruler in image shown in inches).

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A four-digit, mutually exclusive, and exhaustive coding scheme was used to record behaviors [Sackett et al., 1973]. Social and nonsocial interactions, including passive, exploration, play, affiliative, aggressive, and fear/withdrawal behaviors, were recorded. Data Collection and Analyses Growth and object permanence data were collected by a single observer. Neonatal assessment and social data were collected by multiple observers. Interrater reliability for social behavior was achieved by computerized calculation of a kappa score (k) of .80 or above for each of five consecutive randomized focal animal sessions. Reliability for nursery assessments was achieved when observers obtained an 89% agreement on three consecutive randomized assessments. Because of the small sample size of this study, one-sample t-tests were employed for weight, object concept, and social data. Descriptive summaries were generated for nursery assessments due to the multitude of measures recorded, which, given our small sample size, may invalidate the t-tests. A P-value of o0.05 was considered statistically significant. Weight data were analyzed for mean weight per month through the first 6 months of life. Nursery assessment and object concept data were analyzed for both postnatal age and gestational age at criterion. Because of their premature status and extended stays in the incubators, the high-risk infants often commenced testing or reached criterion at later postnatal ages. Many reflexes and some motor skills are developed by birth, and the adjustment for gestational ages put all of the subjects (including controls) on the same absolute scale. Social data were analyzed for mean total duration and frequency across all observational sessions for the first 4 months of life. Because

Fig. 2. Average monthly weights of high-risk macaque infants and controls.

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the literature reports mixed results in comparisons of human ICSI and naturally conceved(NC) infants [Dhont et al., 1997; Hansen et al., 2002], for all measures AB was compared with five ICSI control infants born at term with a normal weight. AC and AE were compared with nine NC controls born at term with a normal weight. Sex differences were not examined, since they are generally not seen in these early measures. SPSS statistical software was used for all analyses. RESULTS Growth Measures

Anthropometrics Rough estimates of CRL were recorded using a ruler at or near the time of birth. The CRLs of the high-risk infants were significantly smaller than reported infant CRLs at birth [Catchpole & Van Wagenen, 1956] (Po0.05; Table I). Particularly alarming was AB’s CRL at birth, which measured only 125 mm (Fig. 1). The normal male CRL at birth is 196 mm [Catchpole & Van Wagenen, 1956]. Weight Average weights were compared throughout the first 6 months of life. Birth weight data were not available for NC animals. ICSI animals were heavier than NC animals across all 6 months, consistent with in vitro reproduction of cows and sheep [Young et al., 1998]. All high-risk infants were born with a significantly

Fig. 3. Basic motor skills: postnatal age at criterion (in days).

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lower birth weight than controls (Po0.05). AB and AC remained at a significantly lower weight than their respective controls throughout all 6 months (Po0.05); however, by month 6 AC’s weight was approaching normal for NCs (Fig. 2). AE weighed significantly less than the NC controls throughout months 1 and 2 (Po0.05), but by month 6 she weighed significantly more than the NC controls (Po0.05). Neonatal Reflexes and Sensorimotor Skills The results for nursery assessments varied widely. Brief descriptive summaries are presented for both postnatal age and gestational age at criterion for three groups of measures: basic motor reflexes, survival reflexes, and sensorimotor skills.

Postnatal age at criterion Basic motor skills included grasping, clasping, and placing of the hands and feet. AB reached criterion later than ICSI controls on all measures. AC reached

Fig. 4. Survival reflexes: postnatal age at criterion (in days).

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criterion later than NC controls on all measures, and AE reached criterion later only on placing (Fig. 3). Survival reflexes included rooting and snout orientations toward a stimulus, as well as sucking and startle reflexes. AC had delayed performances on rooting, snout, and suck, and AB had delayed responses on rooting and snout (Fig. 4). Sensorimotor skills included auditory orientation toward a stimulus, and visual orientation toward (and following) a stimulus (both near and far). AC showed delayed responses on visual orientation (near and far), and extremely delayed responses to visual following (near and far). AB showed delayed responses on visual orientation and following (near only; Fig. 5).

Gestational age at criterion When adjusted for gestational age, many of the discrepancies seen for postnatal day of age were either reversed or had disappeared. For basic motor skills, AC reached criterion at the same time as or sooner than controls for all measures. AE reached criterion sooner than controls on clasping and grasping. AB’s delays for grasping and clasping disappeared, but he still took longer to place his hands and feet than ICSI controls (Fig. 6). For survival reflexes, one noticeable change occurred: whereas postnatally AC reached criterion later than controls on all measures, gestationally he reached criterion at, or earlier than, controls. AE also reached criterion sooner by gestational age than controls on snout and startle. A similar reversal also occurred for AB, who reached criterion sooner than ICSI controls on suck (but still later on root; Fig. 7). The most noticeable change in results occurred for sensorimotor skills. For postnatal age at criterion, AC was much delayed in visual orientation and

Fig. 5. Sensorimotor reflexes: postnatal age at criterion (in days).

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Fig. 6. Basic motor skills: gestational age at criterion (in days).

following (near and far), but for gestational age he reached criterion much sooner on visual orientation (both near and far) and following (far). He was within normal limits for visual following (near). AE reached criterion much sooner than controls on visual orientation (near and far) and following (near). AB’s delays were still seen, although they were drastically reduced (Fig. 8).

Object Concept Permanence

Postnatal age at criterion AB reached criterion later than ICSI controls on plain reach and full screen, but showed no difference on the full-well task. He did not complete A-not-B due to consistent balking at the task and aggression toward the testers. AC completed plain reach later than NC controls; however, he began testing at a much later age due to his long stay in the incubator. On all other tasks, he reached criterion at an earlier age than controls. AE did not differ from NC controls on plain reach.

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Fig. 7. Survival reflexes: gestational age at criterion (in days).

For all other tasks, she reached criterion at a much earlier age than controls (Po.05 for all results; Fig. 9).

Gestational age at criterion Similar to other nursery assessment results, most significant postnatal age discrepancies in object concept permanence disappeared when adjusted for gestational age. AE reached criterion at an earlier gestational age than controls for plain reach and full screen (Po.05; Fig. 10). Because AE’s gestational age at birth was 24 days earlier than normal, it is logical to expect that she would reach criterion sooner than controls. Also notable is AC’s performance on plain reach: for postnatal age he reached criterion later than controls, but for gestational age there was no difference. Social Behavior All of the infants displayed species-typical repertoires, including high frequencies and durations of positive behaviors (explore, play, and sexual

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Fig. 8. Sensorimotor reflexes: gestational age at criterion (in days).

behaviors) and low occurrences of negative behaviors (fear/withdrawal and rock/ huddle/self-clasp) and aggression. AB and AE both showed a higher mean total duration of positive behavior and a lower mean total duration of negative behavior, as well as a higher mean total frequency of positive behaviors and a lower mean total frequency of negative behaviors than their respective controls (Po.05). AC displayed a higher mean total frequency of passive behavior than NC controls (Po.05; Table II). DISCUSSION These high-risk infants showed no gross developmental abnormalities compared to control animals. Any differences observed between the groups were generally observed at or shortly after the time of birth, but most of these disparities soon disappeared. The two infants that were born prematurely (AC and AE) did not experience symptoms of hyaline membrane disease, which is caused by a lack of surfactant in the lungs and is a normal occurrence in rhesus macaques born at such low gestational ages [Pruitt et al., 1979]. All three highrisk infants displayed species-typical behaviors. While some disparities were observed in neonatal reflexes and motor skills, these differences were not permanent, and abnormal performances were not observed on later cognitive tasks when adjusted for gestational age. Although information on the survival of extremely high-risk macaque infants is not widely available in the literature, these infants represent some of the most extreme cases of high-risk birth and survival in the laboratory setting. More importantly, all of the infants continue to thrive now as juveniles. Their survival is probably due to a combination of factors. Although in utero development occurs

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Fig. 9. Postnatal age at criterion (in days) for four stages of object permanence in high-risk and control infants.

at fixed biological time points, there is some individual variation in this development. Such variation may explain why the most premature infant, AC, survived and did not suffer from respiratory distress, while another infant born in our laboratory at a few gestational days past AC’s experienced multiple episodes of apnea, required resuscitation numerous times, and did not survive past 12 hr. Another factor contributing to the successful outcome of these infants is our 24-hr staffed nursery. Without round-the-clock intensive care, these infants surely would not have survived, or would have shown definite cognitive deficits [Schrier et al., 1983, 1990]. The necessity of such a facility is stressed for any investigator wishing to study infant primate development and behavior, especially since high-risk infants (such as the three discussed here) can serve as excellent models for human prematurity. Finally, our success with these infants was often dependent on a fluid protocol–one that followed procedures established over the last 40 years [Ruppenthal & Sackett, 1992] but was altered daily according to each animal’s individual needs (i.e., food and oxygen requirements). It is imperative to remain flexible in these protocols to ensure maximal survival of high-risk primate infants.

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Fig. 10. Gestational age at criterion (in days) for four stages of object permanence in high-risk and control infants.

TABLE II. Mean Total Duration (sec) and Mean Total Frequency of Social Behaviors Across all 300-Second Observational Sessions for the First Four Months of Life Measure Mean total duration (sec)

Mean total frequency

Behavior ICSI/control Passive Positive Negative Aggressive Passive Positive Negative Aggressive

56.22 180.04 40.47 0.06 6.85 17.31 4.03 0.02

AB

NC/control

46.10 249.74 3.75 0.37 4.62 34.8 0.53 0.08

65.78 167.02 41.73 0 5.69 16.07 3.71 0

AC

AE

85.17 35.87 165.84 263.85 47.74 0.27 1.22 0 4.65 11.05 28.41 28.4 3.48 0.06 0.43 0

Po0.05.

The fact that all three of these infants, especially AB and AC, survived is remarkable in itself. Historically, rhesus macaque colonies have not seen infants delivered prior to 130 days’ gestation survive [Shaughnessy et al., 1978].

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