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In wild bonobos, during early lactation bone resorption is unexpected low, revealing unique adaptations.
1. Introduction
In humans, bone loss and osteoporosis, characterized by reduced bone density and increased fracture risk, profoundly impact health and impose substantial burdens on healthcare systems (Akkawi & Zmerly, Reference Akkawi and Zmerly2018; Salari, Reference Salari2014). These conditions disproportionately affect older women from well-studied populations of the global north (Sözen et al., Reference Sözen, Özışık and Çalık Başaran2017). In women, reduced bone mineral density (BMD) has been associated with aspects of reproduction, including short interbirth intervals and prolonged lactation periods (Akkawi & Zmerly, Reference Akkawi and Zmerly2018; Salari, Reference Salari2014; Stieglitz et al., Reference Stieglitz, Beheim, Trumble, Madimenos, Kaplan and Gurven2015). These stages involve complex physiological changes, yet much of what we know about bone metabolism is based on studies of modern humans, as well as some domesticated and wild mammal species living in captivity. Over the course of human evolution, reproductive strategies such as lactation patterns have changed (Sellen, Reference Sellen2007), potentially influencing bone metabolism in ways that are not yet fully understood. Likewise, non-human mammals living under controlled conditions may evolve metabolic traits that differ from wild conspecifics. Studying bone metabolism during these life stages in wild non-human primates provides valuable insights into evolutionary processes that may have shaped these mechanisms in humans.
In mammals, changes in calcium metabolism occur during pregnancy and lactation to meet foetal and offspring skeletal growth demands (Bowman & Miller, Reference Bowman and Miller2001; Kovacs, Reference Kovacs2011; Szulc et al., Reference Szulc, Naylor, Hoyle, Eastell and Leary2017). In humans, calcium transfer to the foetus involves increased intestinal absorption, modulation of renal calcium losses, and, to a lesser extent, resorption from maternal bones (Kaur et al., Reference Kaur, Godber, Lawson, Baker, Pearson and Hosking2003; Sowers, Reference Sowers1996). Lactation further intensifies these demands as calcium is secreted into milk (Wysolmerski, Reference Wysolmerski2003), inducing a temporary demineralization of the skeleton as the primary mechanism to fulfil calcium requirements (Kovacs, Reference Kovacs2011; Sowers et al., Reference Sowers, Hollis, Shapiro, Randolph, Janney, Zhang, Schork, Crutchfield and Russell-Aulet1996). A remarkable 10% reduction in bone mass has been observed in women who exclusively breastfeed (Kovacs, Reference Kovacs2011). However, studies have shown that this bone loss is largely reversible. Most women experience a full or near-full recovery of BMD after weaning and that lactation does not have long-term adverse effects on peak bone mass, bone density, or fracture risk (Augustine et al., Reference Augustine, Boudreau, Cauley, Majchel, Nagaraj, Roe, Sood, Stewart and Horwitz2023; Kovacs, Reference Kovacs2001; Kovacs & Kronenberg, Reference Kovacs and Kronenberg1997; Madimenos et al., Reference Madimenos, Snodgrass, Liebert, Cepon and Sugiyama2012; but see Affinito et al., Reference Affinito, Tommaselli, Di Carlo, Guida and Nappi1996; Brembeck et al., Reference Brembeck, Lorentzon, Ohlsson, Winkvist and Augustin2015). These results suggest that lactation-induced bone depletion in humans is a transient physiological process rather than a cumulative deficit.
Similar calcium demands and bone loss patterns during the postpartum period have been shown in captive non-human primates (e.g. Lees et al., Reference Lees, Jerome, Register and Carlson1998; Ott et al., Reference Ott, Lipkin and Newell‐Morris1999) and domesticated mammals (e.g. Liesegang et al., Reference Liesegang, Risteli and Wanner2007; Van Riet et al., Reference Van Riet, Millet, Liesegang, Nalon, Ampe, Tuyttens, Maes and Janssens2016). However, calcium demands bone resorption dynamics during pregnancy and lactation have yet to be studied in any wild-living mammal, leaving a critical gap in our understanding of the physiological costs of reproduction for females in natural living conditions. We address this gap through a non-invasive investigation of the physiological impact of pregnancy and lactation on bone resorption in wild-living bonobos (Pan paniscus), using a biomarker of bone turnover sampled from urine.
1.1. Bone turnover biomarkers
Urinary bone resorption biomarkers, such as C-terminal crosslinking telopeptide of Type I collagen (CTX-I), offer a non-invasive method to quantify bone turnover within the living skeleton and to assess how pregnancy and lactation, in particular, impact bone turnover (Liesegang et al., Reference Liesegang, Risteli and Wanner2007). Biomarkers such as CTX and NTX (N-terminal crosslinking telopeptide) are increasingly used for assessing bone turnover, as they are released into circulation in proportion to collagen degradation by osteoclasts (Herrmann & Seibel, Reference Herrmann and Seibel2008; de Ridder & Delemarre-van de Waal, Reference de Ridder and Delemarre-van de Waal1998; van der Sluis et al., Reference van der Sluis, Hop, van Leeuwen, Pols and de Muinck Keizer-schrama2002). This is largely because over 90% of the organic matrix of bone consists of type I collagen (Vasikaran, Reference Vasikaran2008; Yamaga et al., Reference Yamaga, Taga and Minaguchi1997), and collagen crosslinks such as NTX-I and CTX-I are direct degradation products of this collagen (Herrmann & Seibel, Reference Herrmann and Seibel2008; Rosen et al., Reference Rosen, Moses, Garber, Iloputaife, Ross, Lee and Greenspan2000). CTX is a small peptide fragment cleaved from type I collagen and thus can be measured in urine (Herrmann & Seibel, Reference Herrmann and Seibel2008; Urlacher et al., Reference Urlacher, Kim, Luan, Young and Adjetey2022). Therefore, the urinary excretion rate of crosslink-containing collagen fragments serves as an indicator of bone collagen degradation (Mora et al., Reference Mora, Prinster, Proverbio, Bellini, De Poli, Weber, Abbiati and Chiumello1998), offering a non-invasive method to evaluate bone resorption.
To our knowledge, only one other study has assessed bone turnover in a wild primate: Sandel et al. (Reference Sandel, Negrey, Arponen, Clark, Clift, Reddy and Ivaska2023) analysed urinary biomarkers, including osteocalcin (bone formation marker) and NTX (bone resorption marker), in a cross-sectional sample of wild chimpanzees (Ngogo, Kibale National Park, Uganda) to investigate skeletal growth during ontogeny. The study found that bone turnover biomarkers displayed a nonlinear relationship with age, with males showing distinct peaks in osteocalcin and NTX levels during adolescence (at 9.4 and 10.8 years, respectively), followed by a plateau at around 20 years in both sexes. Sandel et al. (Reference Sandel, Negrey, Arponen, Clark, Clift, Reddy and Ivaska2023) interpreted these findings as indicative of a potential adolescent growth spurt in chimpanzees, particularly in males, challenging the long-held assumption that such growth spurts are unique to humans. However, the study did not assess changes in bone turnover in females related to pregnancy or lactation.
1.2. Bone turnover during pregnancy and lactation in humans
During human pregnancy, maternal urinary CTX-I levels increase two- to threefold, reflecting increased bone resorption as the foetal skeleton develops (reviewed in Hannon & Eastell, Reference Hannon and Eastell2000). Similarly, bone turnover markers fluctuate during human lactation. This includes changes in bone formation markers such as amino-terminal telopeptides of procollagen type I and osteocalcin, as well as the bone resorption markers NTX and CTX-I (Carneiro et al., Reference Carneiro, Prebehalla, Tedesco, Sereika, Hugo, Hollis, Gundberg, Stewart and Horwitz2010; Møller et al., Reference Møller, Streym, Mosekilde, Heickendorff, Flyvbjerg, Frystyk, Jensen and Rejnmark2013; Prentice et al., Reference Prentice, Jarjou, Stirling, Buffenstein and Fairweather-Tait1998). Postpartum levels of these markers increase several-fold and remain elevated during lactation, often even exceeding levels observed in late pregnancy (Kovacs, Reference Kovacs2011; Yamaga et al., Reference Yamaga, Taga and Minaguchi1997).
Although studies have explored how pregnancy and lactation affect bone turnover markers in healthy women, most research has focused on populations in the global north (Augustine et al., Reference Augustine, Boudreau, Cauley, Majchel, Nagaraj, Roe, Sood, Stewart and Horwitz2023; Carneiro et al., Reference Carneiro, Prebehalla, Tedesco, Sereika, Hugo, Hollis, Gundberg, Stewart and Horwitz2010), which may not be universally representative. Research quantifying BMD in non-global north populations is more common. For example, BMD studies of the Shuar women of Amazonian Ecuador (Madimenos et al., Reference Madimenos, Snodgrass, Liebert, Cepon and Sugiyama2012) and cross-population comparisons of the Tsimane and Shuar (Madimenos et al., Reference Madimenos, Liebert, Cepon‐Robins, Urlacher, Josh Snodgrass, Sugiyama and Stieglitz2020) have provided valuable insights into the reproductive effects – and the variation of those effects – on skeletal health. Other studies have revealed differences in both baseline BMD and how BMD changes during pregnancy and lactation. For instance, compared with self-identified Caucasian mothers, African American mothers exhibit differences in baseline BMD and less lactation-induced bone loss at certain skeletal sites, such as the lumbar vertebrae and femoral neck (Augustine et al., Reference Augustine, Boudreau, Cauley, Majchel, Nagaraj, Roe, Sood, Stewart and Horwitz2023).
1.3. Bone turnover during pregnancy and lactation in non-human mammals
Differences in bone turnover patterns during pregnancy have also been documented across a variety of species. Similar to humans, domesticated animals exhibit increased serum CTX-I levels before parturition (Liesegang et al., Reference Liesegang, Eicher, Sassi, Risteli, Kraenzlin, Riond and Wanner2000, Reference Liesegang, Risteli and Wanner2006, Reference Liesegang, Risteli and Wanner2007; Van Riet et al., Reference Van Riet, Millet, Liesegang, Nalon, Ampe, Tuyttens, Maes and Janssens2016; Figure 1). However, studies of captive monkeys show varied patterns during pregnancy: southern pig-tailed macaques (Macaca nemestrina) exhibit significant changes in bone metabolism (Ott et al., Reference Ott, Lipkin and Newell‐Morris1999), whereasile long-tailed macaques (Macaca fascicularis) show no change in biomarker levels (Lees et al., Reference Lees, Jerome, Register and Carlson1998). Although biomarkers have not yet been quantified, a study measuring BMD directly in female rhesus macaques (Macaca mulatta) from Cayo Santiago showed that BMD increased with the number of offspring produced (Cerroni et al., Reference Cerroni, Tomlinson, Turnquist and Grynpas2003). Together, these findings illustrate the diversity of bone turnover responses during pregnancy and emphasize the importance of species-specific and ecological factors in shaping these patterns.
Figure 1. Schematic diagrams of the bone resorption marker CTX during pregnancy and lactation in domesticated (a) goats, (b) sheep, and (c) cows. Goat and sheep plots modified after Liesegang et al. (Reference Liesegang, Risteli and Wanner2006), showing the timing of birth and weaning of offspring. Cow plot modified after Liesegang et al. (Reference Liesegang, Eicher, Sassi, Risteli, Kraenzlin, Riond and Wanner2000), showing just the timing of birth, as data on weaning were not provided in the study. ap = ante partum; pp = postpartum.
Bone formation markers have also been investigated in various non-human mammals, revealing patterns similar to those found in humans. These patterns have been observed in sows (Van Riet et al., Reference Van Riet, Millet, Liesegang, Nalon, Ampe, Tuyttens, Maes and Janssens2016), goats and sheep (Liesegang et al., Reference Liesegang, Risteli and Wanner2006), mice (VanHouten & Wysolmerski, Reference VanHouten and Wysolmerski2003), little brown bats (Myotis lucifugus; Kwiecinski et al., Reference Kwiecinski, Krook and Wimsatt1987), and dogs (M. A. Miller et al., Reference Miller, Omura and Miller1989) (Figure 1). Captive monkeys (M. fascicularis, M. nemestrina, Chlorocebus aethiops, Callithrix jacchus) also show increased bone resorption and remodelling during lactation, with partial bone calcium depletion (Hiyaoka et al., Reference Hiyaoka, Yoshida, Cho and Yoshikawa1996; Lees et al., Reference Lees, Jerome, Register and Carlson1998; Ott et al., Reference Ott, Lipkin and Newell‐Morris1999; Power et al., Reference Power, Jerome, Bailey, Power, Oftedal, Layne and Tardif1999). These interspecies comparisons highlight the conserved physiological processes of bone metabolism during lactation across different mammalian species, but corresponding data for apes are currently lacking.
Despite the notable research, it is unclear to what extent these findings can be extrapolated to other modern human populations or to other, primarily wild-living, mammals. For example, in other non-global north human populations, including hunter-gatherers and pastoralists (Mays, Reference Mays2016), bone metabolism may be influenced by lifestyle factors, including nutrition, offspring feeding strategies, and reproductive histories (Mays, Reference Mays2016; Sellen, Reference Sellen2007).
Similarly, in other mammals, data are derived mainly from domesticated and captive animals; however, the activity levels, disease and contraception management practices, as well as the behaviours of these animals differ from their wild-living counterparts (Beaulieu, Reference Beaulieu2016; Calisi & Bentley, Reference Calisi and Bentley2009). Studies comparing captive and wild individuals have shown the profound impacts of the environment on physiological outcomes (Beaulieu, Reference Beaulieu2016; Turko et al., Reference Turko, Firth, Craig, Eliason, Raby and Borowiec2023). For example, captive conditions, such as temperature, light, and humidity, may introduce artificial stressors that can potentially skew the assessment of physiological parameters (Beaulieu, Reference Beaulieu2016). Factors such as parasite load, stressor, diet quality, quantity, and feeding schedule can all differ between captive and wild settings, further altering physiological outcomes (Turko et al., Reference Turko, Firth, Craig, Eliason, Raby and Borowiec2023). Among primates, wild-living baboons have lower thyroid hormone levels during pregnancy compared to the cycling phase, a pattern that contrasts with findings in captive primates (Gesquiere et al., Reference Gesquiere, Pugh, Alberts and Markham2018). The authors suggested that elevated thyroid hormone levels commonly observed during pregnancy in well-nourished women may not be applicable to undernourished populations (Gesquiere et al., Reference Gesquiere, Pugh, Alberts and Markham2018), highlighting the significant role of ecological factors in shaping physiological regulation. Moreover, activity patterns, muscle mass, or body weight can influence bone structure, resulting in differences between species or captive versus wild individuals of the same species in the baseline amount of bone structure (e.g. differences in cortical bone thickness or volume of trabecular bone) (Chirchir et al., Reference Chirchir, Ruff, Helgen and Potts2022; Harbers et al., Reference Harbers, Zanolli, Cazenave, Theil, Ortiz, Blanc, Locatelli, Schafberg, Lecompte, Baly, Laurens, Callou, Herrel, Puymerail and Cucchi2020; Pelletier et al., Reference Pelletier, Niinimäki and Salmi2021; Zack et al., Reference Zack, Smith and Angielczyk2022).
1.4. Maternal investment and bone resorption dynamics across species
Across species, maternal investment is highly variable, influencing calcium allocation patterns and potentially affecting bone resorption. In species with precocial young (e.g. cows, sheep), offspring are mobile shortly after birth and require less prolonged care. In contrast, primates have altricial young and exhibit extended maternal care, particularly through prolonged lactation, which maintains calcium demands over an extended period and may lead to sustained bone resorption. With their slow life history, primates, especially apes and humans, experience extended infant dependency, with maternal care and nutritional support spanning multiple seasons (Charnov & Berrigan, Reference Charnov and Berrigan1993; van Noordwijk et al., Reference van Noordwijk, Kuzawa and Van Schaik2013). However, humans differ from great apes in several aspects: human infants are born heavier, with more body fat; human milk is frequently supplemented with additional foods; infants receive their first solid foods prepared by caregivers; and the lactation period is shorter in most human populations, leading to an earlier weaning age compared with great apes (van Noordwijk et al., Reference van Noordwijk, Kuzawa and Van Schaik2013). In primates, the prolonged maternal investment exposes primates to fluctuating resource availability (van Noordwijk et al., Reference van Noordwijk, Kuzawa and Van Schaik2013), requiring mothers to balance their own reserves, including bone resorption, to meet offspring bone growth demands. This dynamic balancing of bone turnover is particularly relevant in species with long lactation periods, where environmental conditions and maternal energy balance may influence bone metabolism.
Bonobos exhibit an exceptionally prolonged period of maternal investment, characterized by long gestation, extended lactation, and extensive infant care. Pregnancy lasts approximately 240 days, similar to humans, but lactation is prolonged, typically lasting 4–5 years (Douglas, Reference Douglas2014; Jukic et al., Reference Jukic, Baird, Weinberg, McConnaughey and Wilcox2013; Oelze et al., Reference Oelze, Ott, Lee, O’Neal, Hohmann and Fruth2024; Stevens, Reference Stevens2020). During this time, offspring continue to rely on their mothers for both nutritional and social support, with milk remaining an important source even as solid food intake gradually increases (Oelze et al., Reference Oelze, Ott, Lee, O’Neal, Hohmann and Fruth2024). Additionally, bonobo infants are carried by their mothers for more than a year (Behringer et al., Reference Behringer, Berghänel, Deschner, Lee, Fruth and Hohmann2022; Lee, Hohmann, et al., Reference Lee, Hohmann, Lonsdorf, Fruth and Murray2020; Lee, Murray, et al., Reference Lee, Murray, Lonsdorf, Fruth, Stanton, Nichols and Hohmann2020). This maternal investment strategy differs markedly from that of domesticated species frequently studied in bone resorption research, such as pigs, goats, and sheep (Liesegang et al., Reference Liesegang, Risteli and Wanner2006; Van Riet et al., Reference Van Riet, Millet, Liesegang, Nalon, Ampe, Tuyttens, Maes and Janssens2016). In these species, weaning occurs within months (often earlier under artificial conditions), and offspring rapidly gain independence, reducing the duration of direct maternal care (Alley et al., Reference Alley, Fordham and Minot1995; Freitas-de-Melo et al., Reference Freitas-de-Melo, Orihuela, Hötzel and Ungerfeld2022). Compared to human populations in which bone resorption markers have been measured (see above), bonobos exhibit even longer lactation durations. This extended lactation in wild bonobos likely involves prolonged maternal calcium allocation, potentially resulting in a distinct bone resorption pattern compared to humans and domesticated mammals.
Here, we conducted the first investigation into maternal physiological costs during pregnancy and lactation on bone resorption in a wild-living animal, the bonobo. Investigating bone metabolism during pregnancy and lactation in bonobos can enhance our understanding of the evolutionary adaptations involved in maternal skeletal responses to reproduction. This can contribute to comparative insights into the mechanisms of bone turnover and calcium regulation in primates, with implications for human evolutionary biology. For biological validation, we first measured urinary CTX-I in two pregnant bonobos in a controlled zoo environment with a high and regular sampling frequency (n = 86 samples) and compared these data with published results from other captive animals and humans. Biological validation, in the context of our study, refers to demonstrating that urinary CTX-I measured in bonobos shows a pattern that corresponds to expected physiological changes, such as those observed during pregnancy and lactation in other mammals. This verifies that the assay detects biologically meaningful variation in urinary CTX-I levels, consistent with established knowledge of bone metabolism in other species, and ensures that these changes align with key life-history stages in bonobos. We then quantified changes in urinary CTX-I levels in pre-pregnant, pregnant, and lactating wild-living bonobos (n = 10 individuals, n = 384 samples, Table S1).
If the human and domesticated species pattern is typical for all mammals under diverse conditions, we predicted that urinary CTX-I levels, a marker of bone resorption, will progressively increase during pregnancy and peak in late gestation. Such a pattern would indicate increased bone turnover, consistent with findings in modern humans as well as captive and domesticated mammals. However, if wild mammals have evolved alternative strategies to meet calcium demands during pregnancy and lactation, urinary CTX-I levels might remain stable rather than increase. Additionally, we predicted higher urinary CTX-I levels in lactating compared to pregnant or cycling females, assuming lactation involves increased maternal bone resorption to meet calcium demands for milk production, as observed in other species. However, cultural factors in humans, such as diet and physical activity, may alter this pattern. If the timing, intensity, and duration of maternal investment influence bone resorption, as suggested in previous studies (see above), then we expected bone resorption to fluctuate throughout pregnancy and lactation in wild bonobos as mothers adjust to variation in resource availability throughout this extended period of investment.
Information on natural calcium resources in tropical forests is scarce; however, it is generally assumed that calcium content is low due to leaching (Gonçalves Bizuti et al., Reference Gonçalves Bizuti, Casagrande, Soares, Sartorio, Brugnaro and Gomes César2018). If this assumption applies for the conditions at LuiKotale, home to our wild study population, it can be argued that calcium content in the LuiKotale forest is limited, potentially constraining the capacity for bone remodelling and thereby necessitating physiological adjustments in females. During late pregnancy, when calcium demands are high, we might see an increase in urinary CTX-I levels. However, in contrast to domesticated or controlled environments, wild bonobos may exhibit a more moderate increase in bone resorption, reflecting adaptive strategies such as increased calcium absorption or retention. During lactation, CTX-I levels would likely be higher, as calcium demands for milk production peak. However, these levels might vary throughout lactation, reflecting the dynamic balancing act between conserving bone mass and meeting the energy and calcium demands of offspring bone growth. This balance would likely depend on the environmental conditions and the maternal energy balance.
2. Methods
2.1. Study species and subjects
Urine samples for this study originate from two distinct sources. The first data set comprises samples from two zoo female bonobos housed at Planckendael Zoo (Mechelen, Belgium), which were collected exclusively during their pregnancies as part of an earlier study (Heistermann et al., Reference Heistermann, Möhle, Vervaecke, van Elsacker and Keith Hodges1996). These samples were included in the present study for biological validation purposes, specifically to demonstrate that if a pregnancy-associated pattern in urinary CTX-I levels exists, it can be reliably detected in bonobos. Although lactation samples are unavailable for these individuals, the inclusion of these pregnancy samples is crucial for confirming detectable changes in CTX-I during pregnancy.
The second data set consists of samples collected from 10 wild female bonobos living in their natural habitat at LuiKotale, Democratic Republic of the Congo (DRC). These samples represent all three reproductive states – pre-pregnancy, pregnancy, and lactation – and were collected over a 10-year period.
Zoo-housed bonobos (biological validation)
Two multiparous female bonobos from Planckendael Zoo were housed in a single group, fed several times daily, and given ad libitum access to water. In total, 86 urine samples were collected from these individuals. For one female, 35 samples were collected in 1994. For the other female, 51 samples were collected between May 1992 and the birth of the offspring in February 1993. Both pregnancies were successful. Because these samples were collected as part of a validation study on the cycle and pregnancy of bonobos (Heistermann et al., Reference Heistermann, Möhle, Vervaecke, van Elsacker and Keith Hodges1996), there are no samples from the lactation period. However, due to the high sample density during pregnancy, we included these samples in the study to evaluate CTX-I levels under controlled conditions. Samples were collected on plastic sheets or from the floor with disposable plastic pipettes and transferred into 2 ml plastic vials. Urine was frozen immediately after collection. All samples were collected in the morning, at approximately the same time for each female.
Long-term stability studies on urinary CTX-I are lacking; however, bone resorption markers, including CTX-I, are generally stable when frozen (Szulc et al., Reference Szulc, Naylor, Hoyle, Eastell and Leary2017). Furthermore, plasma and serum CTX-I have been shown to remain unaffected by storage at −20°C for up to three years (Qvist et al., Reference Qvist, Munk, Hoyle and Christiansen2004). Although some of our samples were stored for over 30 years before analysis, CTX-I was consistently detected in our samples, and the observed patterns align with physiological expectations. This suggests that if degradation has occurred, it has not precluded reliable measurement.
Wild-living bonobos
Urine samples were collected from wild-living bonobos of the Bompusa West and East communities at LuiKotale, DRC. This bonobo population has never been provisioned with food and lives in an intact, natural forest habitat (Hohmann & Fruth, Reference Hohmann and Fruth2003). All subjects were individually known and habituated to human presence. Samples were collected opportunistically throughout the day, between 05:00 and 18:00, by capturing urine directly or pipetting urine from the vegetation, and were stored frozen.
Following the birth of an infant, we are confident that the urine samples collected belong to the mothers, rather than the infant, for the following reasons. First, urine from infant bonobos typically has a very low specific gravity, and samples with such low values were excluded from our analysis (<1.003; Sabbi et al., Reference Sabbi, Muller, Machanda, Otali, Fox, Wrangham and Emery Thompson2020). Second, in the context of another ongoing study, we measured sex steroids in the samples, which indicated that they came from reproductively active females.
A total of 384 urine samples from 10 female bonobos (on average 38 ± 13.7 samples per individual, range 22–68) were collected between 2011 and 2022. Samples were collected while females were cycling, pregnant, and lactating. All pregnancies included in our data set had a successful outcome. Nine of the 10 females included in the data set had one pregnancy; one female had two pregnancies. Therefore, in total, our data set encompassed 11 pre-pregnancy, pregnancy, and lactation periods from 10 females. Importantly, the sampling period for each female was limited to one or (in one case) two reproductive cycles and did not extend across the entire 10-year study period (for details regarding sample numbers, see Table S1). Five females were primiparous and five were multiparous. The females’ exact ages are unknown, because females are the dispersing sex in bonobos (Eriksson et al., Reference Eriksson, Siedel, Lukas, Kayser, Erler, Hashimoto, Hohmann, Boesch and Vigilant2006; Ishizuka et al., Reference Ishizuka, Toda and Furuichi2020). However, all primiparous females are estimated to be younger than 20 years of age and all multiparous females are older than 25 years.
Birthdates of all offspring were estimated based on three criteria: (1) the date of the female’s last observed encounter without offspring, (2) the date of the first observed encounter with new offspring, and (3) the developmental stage of the new born at first sighting. Estimates range from a few days up to a few weeks. We used the birthdate of each offspring as a reference point for timelines within females. All dates when a urine sample was collected from a given female were assigned a negative value (before pregnancy or during pregnancy) or a positive value (lactation) relative to the offspring’s birthdate (the number of days from the birthdate to the sample date). Samples were collected up to 765 days before and up to 667 days after a birth. As the average gestation length in bonobos is approximately 245 days (Stevens, Reference Stevens2020), we assumed −245 days from the birthdate to be the beginning of a pregnancy. All samples collected after the birthdate of an offspring (up to 667 days after birth) are from lactating females. However, the majority of urine samples during the lactation period were collected within the first 6 months after an offspring’s birth (for details, see Figure S1).
Our first data set included CTX-I levels during pregnancy and lactation. This data set encompassed 296 urine samples from 10 females. To achieve a more symmetrical distribution of our response variable, we log-transformed urinary CTX-I levels. The time of sample collection and the days relative to birth were mean-centred and standardized to two standard deviations.
Because we found an unexpected result at the beginning of the lactation period, we performed further analyses. To that end, we included the data collected pre-pregnancy to create a second data set (details in Table S1). Samples collected between −765 and −246 days relative to offspring birth were assigned to the pre-pregnancy period, samples collected between −245 and 0 days relative to offspring birth were assigned to the pregnancy period, and values greater than 0 were assigned to the lactation period. We calculated pregnancy trimesters by dividing the gestation length into thirds: samples collected on days 1–81 were assigned to the first trimester, those on days 82–162 to the second trimester, and samples on days 162–245 to the third trimester. Furthermore, we divided the lactation period into early lactation (from birth to 90 days after birth) and late lactation (more than 90 days after birth).
2.2. CTX-I measurement
Urine samples from LuiKotale were shipped to the Max Planck Institute for Evolutionary Anthropology (MPI EVA) in Leipzig, Germany. The samples were shipped frozen to the German Primate Center in Göttingen, Germany, for CTX-I measurement.
For CTX-I measurement, we used the commercial sandwich assay (two highly specific antibodies) Urine BETA CrossLaps® (CTX-I) ELISA (REF AC-05F1) from Immunodiagnostic Systems (IDS). The assay is designed for quantifying degradation products of C-terminal telopeptides of Type I collagen in human urine and was previously validated for use in bonobo urine (Behringer et al., Reference Behringer, Sonnweber, Hohmann, Stevens, Verspeek and Kivell2024). The assay description is in Supplemental Material S1. The results are expressed in ng/ml corrected for specific gravity. In all samples, specific gravity was measured using a digital hand refractometer to adjust for urine concentration, which is based on individual hydration status (Miller et al., Reference Miller, Brindle, Holman, Shofer, Klein, Soules and O’Conner2004).
2.3. Statistical analyses
We expected urinary CTX-I levels to vary in a non-linear fashion within and across different reproductive phases in female bonobos. Consequently, we ran generalized additive mixed effect models (GAMMs) as these allow for tracking complex, non-linear relationships (smooths) between the response variable and different predictors (Table S2). All GAMMs were specified with a Gaussian distribution and an identity link using the ‘gam’ function of the package ‘mgcv’ (Wood, Reference Wood2017). Smooth estimation was based on maximum likelihood estimation, and the smooth basis was set to penalized cubic regression splines. These and all other data preparations and statistical analyses were performed using R v. 4.2.3 (R Development Core Team, 2023) in RStudio v. 2023.06.1+524 (R Studio Team, 2020). The threshold for statistical significance was set at p = 0.05.
Our main model included log-transformed urinary CTX-I levels as the response variable. As CTX-I levels follow a circadian pattern (reviewed in Hannon & Eastell, Reference Hannon and Eastell2000), we included the time of sample collection (mean-centred and standardized to two standard deviations) to control for the effect of sample collection time on our response variable. Reproductive phases (pregnancy, lactation) and parity (primiparous, multiparous) were included as parametric predictor terms to account for the average effect of each level of the categorical predictors on the response variable, assuming a linear relationship (testing variance inflation factors using the ‘vif’ function of the ‘car’ package (Fox & Weisberg, Reference Fox and Weisberg2019) showed no issues with multicollinearity). Furthermore, we included smooth terms for the response variable across the days relative to birth (mean-centred and standardized). We specified these terms so that the model calculated different smooths across the days relative to birth for each reproductive phase and for each parity level. In other words, the model fit separate smooths across days during pregnancy (−245 to 0) and across days during lactation (1 to 667) and for primiparous and multiparous females. We performed a grid search (between 5 and 50) to find the best smoothing parameter values that minimize prediction error, thus optimizing the model’s performance while avoiding overfitting. Furthermore, we included a random smooth term for female identity to account for the repeated sampling nature of our data set and to allow for individual variation in the trajectories of the curves. As only one female was included in the data set with two parity states (primiparous and multiparous) and we expected different intercepts depending on parity, we included a separate intercept for each parity state for this female.
Model assumptions and model settings were checked using the ‘gam.check’ (package ‘mgcv’) and ‘acf_resid’ functions (detection of autocorrelation, package itsadug; van Rij et al., Reference van Rij, Wieling, Baayen and van Rijn2022). To compare models, we used the ‘compareML’ function (package ‘itsadug’). For plotting the smooths of log-transformed CTX-I across the days relative to the date of birth by reproductive phase (pregnant, lactating) and parity (primiparous, multiparous), we used the ‘plot_smooth’ function of the package ‘itsadug’.
To investigate differences in urinary CTX-I levels between the categorical reproductive phases variable (phases: before pregnancy, first trimester of pregnancy, second trimester, third trimester, and early and late lactation), we fitted a linear mixed model with a Gaussian error structure (Grafen & Hails, Reference Grafen and Hails2002). Log-transformed urinary CTX-I levels were the response variable. Time of sample collection (mean-centred and standardized to two standard deviations in the new data set) was entered as a control variable. We added a random intercept for the individual to control for repeated measures of females and to estimate the variance explained by interindividual differences. Furthermore, we included a predictor variable, ‘condition’, indicating whether a measurement was sampled from before pregnancy, from the first, second, or third trimester during pregnancy, or from after pregnancy (Table S2). Likelihood ratio tests were used to compare the full model to the null model (including only the random effect and the control variable time of sample collection; χ2 = 40.20, df = 5, p < 0.001). We visually inspected quantile–quantile plots and the distribution of residuals plotted against fitted values and found that model assumptions were met. Models were run using the ‘lme4’ package v1.1-34;15 (Bates et al., Reference Bates, Mächler, Bolker and Walker2015). Post-hoc tests comparing different reproductive phases of the variable condition were performed using the ‘emmeans’ package (Lenth, Reference Lenth2021).
3. Results
3.1. Urinary CTX-I levels increased during pregnancy in zoo bonobos – biological validation
In the zoo-housed bonobos, urinary CTX-I levels increased during pregnancy, approximately 3 months before parturition (Figure 2). In one female, levels increased by a factor of 4 during this period, whereas in the other female, levels increased by a factor of 10 (Figure 2, note y-axis scaling).
Figure 2. Urinary CTX-I levels in two zoo-housed female bonobos during pregnancy. SG = specific gravity. y-Axes display different scales to better visualize data within individuals. The light grey dashed line at −245 indicates the estimated day of conception based on the average gestation length in bonobos (Stevens, Reference Stevens2020). The black dashed line indicates the day of birth.
3.2. Urinary CTX-I levels during pregnancy and lactation in wild bonobos
The grid search revealed that specifying the smooth terms across days relative to offspring birth with eight basis functions minimized the cross-validation error while avoiding overfitting. Consequently, we ran our main model with the predictor variables reproductive phase (pregnant, lactation), parity (primiparous, multiparous), and smooths over days relative to birth for reproductive phase and parity (Table 1). This model differed significantly from the corresponding null model (χ2 = 6.50, df = 2, p = 0.002) and provided a better fit (AIC difference − 40). Model assumptions were met and autocorrelation of residuals was negligible.
Table 1. Results of the generalized additive mixed model on CTX-I levels across pregnancy and lactation in primiparous and multiparous female bonobos. For the parametric predictor variables, estimates (β), standard errors (SE), t-values and p-values are presented. For the smooth terms, effective degrees of freedom (edf), reference degrees of freedom (ref.Df) and F-statistic are provided
Overall, the model revealed that urinary CTX-I levels were higher in pregnant than in lactating females and that primiparae had higher levels than multiparae (Table 1, Figure 3). The smooth terms differed between reproductive phases. Throughout pregnancy, urinary CTX-I levels remained relatively constant, particularly in multiparous females. Primiparous females showed a slight increase in urinary CTX-I levels towards the end of the pregnancy, with the highest levels before giving birth. With the onset of lactation, females showed a marked decrease in urinary CTX-I levels that then increased within the first 6 months of lactation.
Figure 3. Smooths of log-transformed urinary CTX-I levels across days relative to offspring birth for pregnant (days −245 to 0) and lactating wild female bonobos (days 0 to 667). trajectories for primiparous (gold) and multiparous (purple) females are shown. Urinary CTX-I levels were corrected for specific gravity (SG) and then log-transformed. On the x-axis, the days relative to offspring birth are displayed. The light grey dashed line at −245 indicates the estimated day of conception based on the average gestation length in bonobos. The black dashed line at 0 indicates the day of birth.
As the decrease in urinary CTX-I levels at the beginning of the lactation period was unexpected (for comparison with other mammalian patterns, see Figure 1), we performed further analyses to understand whether urinary CTX-I levels returned to a baseline level. These analyses revealed that urinary CTX-I levels were lower in the early lactation state than in any other reproductive phases (Table 2, Figure 4, and Table S3 show means, standard deviations (SD), medians and ranges (lowest to highest value) of log-transformed CTX-I (ng/ml corr. SG) levels by reproductive state).
Figure 4. Differences in log-transformed urinary CTX-I levels by reproductive state in wild bonobos: before pregnancy (female is cycling, in orange), first trimester, second trimester, third trimester (pregnant in blue), early lactation (up to 90 days after offspring’s birth, in orange), and late lactation (>91 days after offspring’s birth, in orange). The central solid black line within each box represents the median. The top and bottom edges of the box represent the upper (75th percentile) and lower quartiles (25th percentile), respectively. The whiskers from the box indicate the range of data within 1.5 times the interquartile range from the quartiles. Individual data points are overlaid with a jitter effect, making them easier to distinguish and providing additional detail about the distribution of data points across conditions.
Table 2. Results of the post-hoc pairwise comparisons of urinary CTX-I levels during reproductive state (before pregnancy, first, seconf, and third trimester of pregnancy, early and late lactation) in female bonobos
4. Discussion
Our study represents the first measurement of a bone resorption marker during pregnancy and lactation in a wild mammal living under natural conditions, as well as in a great ape species with prolonged maternal care. By focusing on wild bonobos, we can consider these findings within a natural and evolutionarily relevant context that can be compared to previously published patterns in humans, captive primates, and domesticated mammals (Kovacs, Reference Kovacs2011; Liesegang et al., Reference Liesegang, Risteli and Wanner2006; Van Riet et al., Reference Van Riet, Millet, Liesegang, Nalon, Ampe, Tuyttens, Maes and Janssens2016). We predicted that urinary CTX-I levels, a marker of bone resorption, would increase during pregnancy and peak at the end, reflecting an increase in bone turnover following patterns found in, albeit limited, studies of humans and captive/domesticated mammals. This hypothesis was supported. Our investigation revealed an expected increase towards the end of pregnancy in zoo-housed and primiparous wild females. These findings biologically validate our measurements and further support the notion that pregnancy influences bone metabolism, likely as part of the broader physiological adaptations to meet foetal skeletal growth demands (Kovacs, Reference Kovacs2011).
In human studies, higher urinary CTX-I levels were found during lactation compared with pregnancy or cycling levels, based on the assumption that lactation involves increased maternal bone resorption to meet calcium demands for milk production (Kovacs, Reference Kovacs2011; Yamaga et al., Reference Yamaga, Taga and Minaguchi1997). In wild bonobos, urinary CTX-I levels did not further increase during lactation, as found in other domesticated or captive mammals (Liesegang et al., Reference Liesegang, Risteli and Wanner2007; Yamaga et al., Reference Yamaga, Taga and Minaguchi1997). Instead, CTX-I levels decreased, with levels in early lactation (the first 3 months) significantly lower than those during pregnancy and all other reproductive phases. This unexpected divergence challenges the assumption that lactation increases bone resorption to meet calcium demands and raises questions about the potential physiological or behavioural mechanisms that bonobos use to counteract bone loss and the utility of current comparative data from human and domesticated animal populations. We discuss each of these findings in more detail below.
We found increased urinary CTX-I levels at the end of pregnancy in zoo-housed bonobos and wild-living primiparous bonobos. This result aligns with human studies, where bone turnover markers, including serum CTX-I levels, are elevated in the third trimester (Cross et al., Reference Cross, Hillman, Allen, Krause and Vieira1995; Gallacher et al., Reference Gallacher, Fraser, Owens, Dryburgh, Logue, Jenkins, Kennedy and Boyle1994; Kaur et al., Reference Kaur, Godber, Lawson, Baker, Pearson and Hosking2003), and with findings in domesticated mammals, such as cows, where increased bone resorption occurs around parturition (Liesegang et al., Reference Liesegang, Eicher, Sassi, Risteli, Kraenzlin, Riond and Wanner2000). In the two zoo bonobos studied, urinary CTX-I levels increased 4- to 10-fold during pregnancy, indicating a pronounced rise in bone resorption. Comparatively, studies in humans report a two- to threefold increase in urinary CTX-I levels during pregnancy (Curtis et al., Reference Curtis, Parsons, Maslin, D’Angelo, Moon, Crozier, Gossiel, Bishop, Kennedy, Papageorghiou, Fraser, Gandhi, Prentice, Inskip, Godfrey, Schoenmakers, Javaid, Eastell, Cooper and Harvey2021; Hannon & Eastell, Reference Hannon and Eastell2000; Yamaga et al., Reference Yamaga, Taga and Minaguchi1997). Although the magnitude of the increase in bonobos is higher than the average reported for humans, it still falls within the broader range of variation observed across individuals and studies, for example domesticated animal studies (Liesegang et al., Reference Liesegang, Risteli and Wanner2006, Reference Liesegang, Risteli and Wanner2007). These findings suggest that bonobos exhibit physiological responses to pregnancy that are comparable to those seen in humans, albeit with some species-specific variability. Additionally, differences in normalization methods – such as the use of specific gravity (SG) in our study versus creatinine normalization in human studies – may contribute to differences in the observed fold increase in urinary CTX-I levels.
By contrast, urinary CTX-I levels in wild multiparous female bonobos remained constant throughout pregnancy. It is possible that the effect of bone resorption is only large enough to be detected in primiparous, often younger, females, as human bone mass density losses at the end of pregnancy are greater in adolescents than in adult women (Bowman & Miller, Reference Bowman and Miller2001). Zoo-housed bonobos experience an adolescent growth spurt, and afterwards bone growth slows around 13–15 years of age (Behringer et al., Reference Behringer, Sonnweber, Hohmann, Stevens, Verspeek and Kivell2024, Reference Behringer, Stevens, Kivell, Neufuss, Boesch and Hohmann2016; Berghaenel et al., Reference Berghaenel, Stevens, Hohmann, Deschner and Behringer2023). Although the exact ages of the primiparous wild females in our study remain unknown due to female dispersal, it is possible that some females were still growing, which could have influenced their bone resorption patterns during reproduction. This could also explain the comparable pattern in the two zoo-housed bonobos, as both were multiparous but under 16 years old when the samples were collected. If changes in CTX-I levels occur during pregnancy in older, multiparous females, they may be minor compared with primiparae and could fall within the large daily intra-individual variation (Behringer et al., Reference Behringer, Sonnweber, Hohmann, Stevens, Verspeek and Kivell2024). Previous non-human primate studies found that pregnancy reduced bone mass in primiparous pig-tailed macaques (Ott et al., Reference Ott, Lipkin and Newell‐Morris1999), in contrast to female long-tailed macaques in a breeding colony (Lees et al., Reference Lees, Jerome, Register and Carlson1998). Both studies speculated that the maternal skeleton might not be the primary source of calcium for the developing foetus and that calcium demands during pregnancy are primarily met by increased intestinal calcium absorption (Lees et al., Reference Lees, Jerome, Register and Carlson1998; Ott et al., Reference Ott, Lipkin and Newell‐Morris1999). In our study population, it is conceivable that multiparous females also cover the calcium needs of foetal growth through intestinal and renal calcium absorption. The assumption that calcium absorption may be pivotal in primate pregnancy is indirectly supported by research demonstrating increased feeding time (Papio anubis, Presbytis entellus, Pan troglodytes schweinfurthii) (Barton, Reference Barton1990; Koenig et al., Reference Koenig, Borries, Chalise and Winkler1997; Murray et al., Reference Murray, Lonsdorf, Eberly and Pusey2009) and/or improved food quality during pregnancy in natural conditions across several primate species (P. troglodytes schweinfurthii, Saimiri oerstedi, Varecia rubra) (Boinski, Reference Boinski1988; Murray et al., Reference Murray, Lonsdorf, Eberly and Pusey2009; Vasey, Reference Vasey2005). These nutritional changes, driven by increased energy needs, may allow pregnant females to cover specific nutritional demands of pregnancy that, in turn, limit calcium resorption from the skeleton. Such adaptive behavioural changes align with dietary patterns observed in human populations, such as the Aché and nomadic !Kung, who exhibit shifts in foraging strategies and food intake in response to reproductive and energetic demands (Hill & Hurtado, Reference Hill and Hurtado1996; Kolata, Reference Kolata1974). Therefore, human studies and our results suggest that pregnant females in natural conditions may be able to meet increasing calcium demands through adaptive changes to feeding duration or food quality, thereby increasing intestinal calcium availability and preventing bone loss and the associated increase of CTX-I levels.
In most human studies cited, it is not explicitly reported whether participants used supplements or avoided them (Augustine et al., Reference Augustine, Boudreau, Cauley, Majchel, Nagaraj, Roe, Sood, Stewart and Horwitz2023; Carneiro et al., Reference Carneiro, Prebehalla, Tedesco, Sereika, Hugo, Hollis, Gundberg, Stewart and Horwitz2010; Gallacher et al., Reference Gallacher, Fraser, Owens, Dryburgh, Logue, Jenkins, Kennedy and Boyle1994; Yamaga et al., Reference Yamaga, Taga and Minaguchi1997). However, the consistent observation of increased CTX-I levels during pregnancy in modern human populations suggests that either (1) many women may not receive or use supplementation or (2) that these increases occur even with supplementation. For example, studies investigating the effects of supplementation, such as vitamin D (Curtis et al., Reference Curtis, Parsons, Maslin, D’Angelo, Moon, Crozier, Gossiel, Bishop, Kennedy, Papageorghiou, Fraser, Gandhi, Prentice, Inskip, Godfrey, Schoenmakers, Javaid, Eastell, Cooper and Harvey2021; Krupa et al., Reference Krupa, Qamar, O’Callaghan, Onoyovwi, Al Mahmud, Ahmed, Gernand and Roth2024), have shown that supplementation moderates, but does not entirely prevent, the rise in CTX-I levels during pregnancy in humans. This finding may imply that single-nutrient supplementation is insufficient, as multiple nutritional factors relevant to bone homeostasis likely interact during pregnancy. Moreover, studies on calcium supplementation during pregnancy have yielded contradictory results. For example, one study reported that 1200 mg/day of calcium carbonate significantly reduced bone resorption in pregnant Mexican women and at one month postpartum (Ettinger et al., Reference Ettinger, Lamadrid-Figueroa, Mercado-García, Kordas, Wood, Peterson, Hu, Hernández-Avila and Téllez-Rojo2014), whereas another study observed the opposite effect in Gambian women (Jarjou et al., Reference Jarjou, Laskey, Sawo, Goldberg, Cole and Prentice2010). These discrepancies likely reflect differences in baseline nutritional status, dietary composition, and other factors affecting calcium absorption and bone turnover. Finally, the highly processed nature of modern diets in the global north, even when supplemented, may fail to meet the complex and dynamic nutritional demands of pregnancy. This limitation could explain why increased bone turnover is still observed during pregnancy despite access to supplementation in humans today.
By contrast to patterns found in previous studies of humans and other mammals, wild bonobo urinary CTX-I levels decreased, rather than increased, at the onset of lactation. Although wild bonobos showed an increase in CTX-I levels after 3 months postpartum, these levels never exceeded those measured during pregnancy. Urinary CTX-I levels during lactation were comparable between primiparae and multiparae, although primiparae exhibited higher overall CTX-I levels. Similarly, in goats and sheep, bone loss during second pregnancies and lactation was less pronounced than during the first reproductive cycle (Liesegang et al., Reference Liesegang, Risteli and Wanner2007). In line with our results, lower bone resorption markers have been observed in multiparous goats, sheep, and monkeys (Liesegang et al., Reference Liesegang, Risteli and Wanner2007; Ott et al., Reference Ott, Lipkin and Newell‐Morris1999).
The surprising decrease in urinary CTX-I levels during early lactation contradicts our expectation of substantial bone loss to meet the calcium requirements of a growing infant, as has been described in other mammals, including primates (Miller et al., Reference Miller, Omura and Miller1989; Lees et al., Reference Lees, Jerome, Register and Carlson1998; Liesegang et al., Reference Liesegang, Risteli and Wanner2007; Tojo et al., Reference Tojo, Kurabayashi, Honda, Yamamoto, Yahata, Takakuwa and Tanaka1998; Vajda et al., Reference Vajda, Kneissel, Muggenburg and Miller1999; Van Riet et al., Reference Van Riet, Millet, Liesegang, Nalon, Ampe, Tuyttens, Maes and Janssens2016; Watts et al., Reference Watts, Binkley, Owens, Al-Hendy, Puscheck, Shebley, Schlaff and Simon2021). Although urinary CTX-I levels in one human study declined throughout the course of lactation, the levels were nearly as high immediately after birth and three months later as they were during pregnancy (Kaji et al., Reference Kaji, Yasui, Suto, Mitani, Morine, Uemura, Maeda and Irahara2007; Figure 1). In our study, the female bonobos notably did not show high CTX-I levels in the first months following birth. This may suggest a potential inhibition of osteoclast activity. Several physiological and behavioural mechanisms might explain the decrease in bone resorption in wild bonobos during early lactation. One physiological hypothesis to explain this result is that bonobos conserve energy by reducing bone resorption during this energy-demanding reproductive phase. That lactation is such an energy demanding period is indicated by increased cortisol levels in bonobos (Nurmi et al., Reference Nurmi, Sonnweber, Schülke, Moscovice, Deschner and Hohmann2023) and other primate species (Cercopithecus mitis, Macaca assamensis) (Foerster et al., Reference Foerster, Cords and Monfort2012; Touitou et al., Reference Touitou, Heistermann, Schülke and Ostner2021), as cortisol is a key hormone involved in energy metabolism. Cortisol mobilizes glucose and other energy reserves to meet immediate physiological needs (Cooper et al., Reference Cooper, Seibel and Zhou2016). Bone remodelling is an energy-demanding process, and given that the skeleton is one of the largest organs in the mammalian body, it likely incurs high costs (Ducy, Reference Ducy2011). Reducing bone remodelling could be a strategy to conserve energy during the energy-intensive lactation period. The mechanism behind such a reduction in bone remodelling could be genetic: the downregulation of genes associated with bone resorption in hibernating bears has been shown to preserve bone density during long periods of inactivity (Goropashnaya et al., Reference Goropashnaya, Tøien, Ramaraj, Sundararajan, Schilkey, Barnes, Donahue and Fedorov2021), demonstrating that gene regulation can prevent bone loss during specific periods of the life cycle. Although this strategy may potentially affect calcium availability for breastmilk production if compensatory mechanisms are insufficient, the primary focus here is to explore the possibility that osteoclast activity may be genetically regulated as an adaptive energy-conservation strategy during lactation. Future studies of the mineral content of breastmilk in captive non-human primates may provide additional information to test this hypothesis (Hinde & Milligan, Reference Hinde and Milligan2011; Milligan, Reference Milligan2007). For example, whereas zoo-housed P. troglodytes produce milk with lower calcium content (0.021 g/100 ml) compared to humans (0.033 g/100 ml), Gorilla gorilla have similar levels (0.034 g/100 ml) (Milligan, Reference Milligan2007). However, no comparable data exist for bonobos, either in zoos or in the wild, and given ethical challenges, such data are unlikely to be collected in the future in any wild ape species.
Previous studies have shown that females can regain the bone mass lost during pregnancy and lactation, but this replenishment of skeletal reserves is possibly species-specific and can be affected by differences in the length of the lactation periods (Van Riet et al., Reference Van Riet, Millet, Liesegang, Nalon, Ampe, Tuyttens, Maes and Janssens2016). In human studies, where the average bone loss is 1–3% per lactation month (Kovacs, Reference Kovacs2005), pre-pregnancy levels of bone density typically recover within 6–12 months after weaning (Kovacs, Reference Kovacs2011). In monkeys (M. fascicularis, C. aethiops) with lactation periods ranging from one to several months, recovery from lactation-induced bone loss takes up to 10 months postpartum or longer (Hiyaoka et al., Reference Hiyaoka, Yoshida, Cho and Yoshikawa1996; Lees et al., Reference Lees, Jerome, Register and Carlson1998). Furthermore, in other primates (M. mulatta, Leontocebus fuscus), females struggle to recover bone minerals before subsequent pregnancies (Cerroni et al., Reference Cerroni, Tomlinson, Turnquist and Grynpas2003; Power, Reference Power1991). Our results suggest that bonobos may have a strategy for replenishing skeletal reserves that differs from what is known in other primates. Wild bonobos nurse their offspring for up to 4–5 years (Oelze et al., Reference Oelze, Ott, Lee, O’Neal, Hohmann and Fruth2024) and have an average interbirth interval of around 5.4 years (Tkaczynski et al., Reference Tkaczynski, Behringer, Ackermann, Fedurek, Fruth, Girard-Buttoz, Hobaiter, Lee, Löhrich, Preis, Samuni, Zommers, Zuberbühler, Deschner, Wittig, Hohmann and Crockford2020). Perhaps female bonobos, and possibly other wild primates, aim to minimize bone resorption at the beginning of this calcium-demanding period to ensure bone density is not depleted in order to reduce fracture-risk levels over longer lactation periods.
The decrease in bone resorption during early lactation in wild bonobos may also result from feeding adaptations. Increased calcium intake during lactation has been shown to improve maternal bone health by replacing maternal bone mass and reducing bone resorption in humans (reviewed in Thomas & Weisman, Reference Thomas and Weisman2006). Therefore, as suggested above in relation to stable CTX-I levels during late-stage pregnancy in multiparae, adapting feeding behaviour and dietary quality during lactation might explain the unique CTX-I patterns observed in wild bonobos. Field observations of several primate species indicate that lactating females increase their intake of high-energy foods, overall food energy, and time allocated to foraging, particularly when food quality is poor (e.g. Saimiri sciureus, Theropithecus gelada, Cebus capucinus, Callicebus cupreus) (Boinski, Reference Boinski1988; Dunbar et al., Reference Dunbar, Hannah-Stewart and Dunbar2002; McCabe & Fedigan, Reference McCabe and Fedigan2007; Tirado Herrera & Heymann, Reference Tirado Herrera and Heymann2004). Although there are currently no published data on food quality or quantity changes in wild bonobos during lactation, food items often consumed by male and female bonobos are high in calcium (see table 1 in Hohmann et al., Reference Hohmann, Ortmann, Remer and Fruth2019). If bonobos generally have a high-calcium diet, or if females increase their calcium intake during lactation, this can have physiological effects that limit bone resorption. Increased calcium absorption can lead to increased calcitonin release, which inhibits osteoclast activity (Taylor et al., Reference Taylor, Lewis and Balderstone1975; Woodrow et al., Reference Woodrow, Sharpe, Fudge, Hoff, Gagel and Kovacs2006). Additionally, high blood calcium levels prevent the secretion of parathyroid hormone, which stimulates osteoclast activity (Rejnmark & Ejlsmark-Svensson, Reference Rejnmark and Ejlsmark-Svensson2020). Both mechanisms would protect the maternal skeleton from excessive resorption during lactation. Other dietary adjustments, such as consuming antioxidant-rich foods containing vitamin E and polyphenols, have been shown to help combat oxidative stress and reduce bone resorption markers like CTX-I (Mottaghi & Nasri, Reference Mottaghi and Nasri2021). Further investigation is needed to determine whether any of these mechanisms are involved in wild bonobos during lactation. Importantly, however, zoo-housed females (or domesticated/captive mammals in general) lack the autonomy to display such behavioural adaptations due to environmental constraints or the absence of key ecological cues that might otherwise induce physiological or epigenetic plasticity. This raises the possibility that the inability to respond to changing metabolic demands through natural behavioural adjustments, rather than diet or activity levels alone, contributes to the observed differences in bone turnover between wild and captive individuals.
We speculate that the urinary CTX-I pattern in wild female bonobos reflects the unique dietary and environmental conditions of their habitat. These conditions differ from those of human populations in industrialized societies, as well as from those of domestic and laboratory animals, particularly in terms of food availability, activity patterns, and the duration of lactation. Our results highlight the value and importance of understanding the reproductive ecology of wild non-human primates living in their natural environments, particularly with regard to the costs of pregnancy and lactation on maternal bone loss.
This sentiment also applies to non-industrialized human groups, for which there is substantial variability in the duration of exclusive breastfeeding (Dupras et al., Reference Dupras, Schwarcz and Fairgrieve2001; Richards et al., Reference Richards, Mays and Fuller2002; Sellen, Reference Sellen2007) and diverse breastfeeding practices (Fink et al., Reference Fink, Fink, Wilson, Bennie, Carroll and Dick1992; Gray, Reference Gray1994; Kolata, Reference Kolata1974), yet nothing is known about the potential effects on bone density. For example, previous studies directly measuring bone structure from skeletal remains have shown that trabecular bone density (Chirchir et al., Reference Chirchir, Kivell, Ruff, Hublin, Carlson, Zipfel and Richmond2015, Reference Chirchir, Ruff, Junno and Potts2017; Ryan & Shaw, Reference Ryan and Shaw2015), cortical bone thickness, and cortical strength (Ruff & Larsen, Reference Ruff, Larsen, Carlson and Marchi2014; Shaw & Stock, Reference Shaw and Stock2013; Stock & Pfeiffer, Reference Stock and Pfeiffer2004) are higher in more active or mobile foraging populations compared to agriculturalists or recent humans. However, it is unclear if this increased skeletal robusticity is found in contemporary hunter-gatherer or pastoralist human groups with more active lifestyles and natural diets and, if so, if they experience the same loss in bone mass during reproductive phases as documented in global north populations. For instance, human foragers tend to avoid noncommunicable diseases and promote healthy ageing through high activity levels, but these benefits are counterbalanced by increased wear-and-tear, risk of injury, and energy constraints, especially during reproduction when energy is diverted toward pathogen defence and reproduction (Stieglitz, Reference Stieglitz2024).
Our results add to this complexity by demonstrating that the bone density costs associated with pregnancy and lactation, previously documented in industrialized humans and domesticated/captive mammals, are not representative of wild-living bonobos and perhaps other primates living in natural environments. Understanding these physiological mechanisms in wild bonobos could offer profound insights into natural strategies for bone preservation. Such knowledge could inform approaches to reduce osteoporosis in captive animals (Lennox & Goodship, Reference Lennox and Goodship2008) and women from industrialized societies, where modern lifestyles and reproductive patterns often disrupt traditional bone metabolism processes. By learning from the adaptive strategies of wild bonobos and other hominoids, we may identify more effective ways to mitigate bone density loss associated with reproduction and ageing in human populations.
5. Conclusions
Our study provides the first results of bone metabolic changes of wild bonobos during pregnancy and lactation, indicated by urinary CTX-I levels. We observed unique patterns in wild bonobos, with the lowest CTX-I levels during lactation. These levels are in contrast with those reported in human populations and captive mammals, highlighting the distinct adaptive strategies employed in natural environments. The decrease in urinary CTX-I levels during early lactation challenges existing assumptions about bone resorption dynamics in primates, suggesting potential behavioural and physiological adaptations to conserve bone mass under calcium-demanding conditions. These findings not only highlight unique adaptive strategies to conserve skeletal integrity under calcium-demanding conditions, but also offer evolutionary context for understanding the trade-offs between reproduction and skeletal health in primates, including humans and early hominins.
Our findings underscore the importance of studying non-human primates in their natural habitats to understand the full spectrum of reproductive costs on skeletal health. Further investigations into the dietary and behavioural mechanisms influencing bone health in wild bonobos could pave the way for innovative approaches to mitigate bone density loss associated with reproduction and ageing in humans.
Supplementary material
The supplementary material for this article can be found at https://doi.org/10.1017/ehs.2025.10013.
Acknowledgements
The authors express gratitude to the director, curator, and keepers of Zoo Planckendael. We thank the Institut Congolais pour la Conservation de la Nature (ICCN) for granting permission to work at LuiKotale and for issuing export permits. We acknowledge the support of the Institut National de Recherche Biomedicale (INRB) for facilitating sample export; and extend our appreciation to the communities of Lompole and Bekombo for facilitating research in their forests. We are grateful to all LuiKotale field assistants for their dedication in sample collection. Special thanks for long-term support go to (in alphabetical order) Meg Crofoot, Benedikt Grothe, Stomy Karhemere, Jason Kirby, José Kok, Richard McElreath, Jean-Jaques Muyembe, Zjef Pereboom, Mike Tomasello, and Martin Wikelski. We are also grateful to three reviewers whose constructive comments improved our manuscript.
Author contributions
V.B.: conceptualization, methodology, data curation, investigation, validation, funding acquisition, writing—original draft, writing—review and editing. R.S.: conceptualization, investigation, software, formal analysis, visualization, writing—original draft, writing—review and editing. B.F.: investigation, resources, funding acquisition, project administration, writing—review and editing. G.Hou.: conceptualization, investigation, writing—review and editing. P.H.D.: resources, funding acquisition, writing—review and editing. J.M.G.S.: resources, writing—review and editing. G.Hoh.: conceptualization, investigation, resources, funding acquisition, writing—original draft, writing—review and editing. T.L.K.: conceptualization, investigation, funding, writing—original draft, writing—review and editing.
Financial support
The following institutions supported analytical work: Deutsche Forschungs-gemeinschaft DFG BE 5511/4‐1, the Max Planck Institute (MPI) for Evolutionary Anthropology, and the German Primate Center. Funding for fieldwork came from the MPI for Evolutionary Anthropology, the MPI of Animal Behaviour, the Centre for Research and Conservation of the Royal Zoological Society of Antwerp, the Leakey Foundation, and the Ouwehand Zoo Foundation.
Competing interest
The authors declare no competing interests.
Research transparency and reproducibility
Source data for statistics and figures as well as used R code in the paper is permanently stored at GRO (“Replication Data for: Wild bonobos experience unusually low bone resorption during early lactation relative to humans and other mammals”, https://doi.org/10.25625/QSKFW4).
Data availability statement
Data are available at GRO.data, where the source data and R code are permanently stored.
Ethical statement
Ethical review and approval were not required for the animal study because all samples were collected non-invasively and with permission of the Institut Congolais pour la Conservation de la Nature (0683/ICCN/DG/ADG/014/ KV/2012). Permits for exporting the urine samples from the Democratic Republic of Congo were issued by the ICCN (0521/ICCN/DG/CWB/05/01/2014).
Open access
