Pangolins are threatened placental mammals distributed in Africa and Asia. Many efforts have been undertaken in the last century to maintain pangolins in captivity, but only a few of them succeeded in maintaining and keeping this species in a controlled environment. This study reports the first systematic breeding of the Critically Endangered Malayan pangolin (Manis javanica) in captivity. Our captive breeding approach successfully improved the reproductive rate for both wild and captive-born female pangolins. From 2016 to 2020, we had 33 wild pangolins and produced 49 captive-born offspring spanning three filial generations. The female offspring further bred 18 offspring, of which 14 (78%) were conceived during the first time of cohabitation with males, and four offspring were conceived during the second cohabitation event, suggesting that they may practice copulation-induced ovulation. We observed that captive-born female pangolins could reach sexual maturity at 7–9 months (n = 4), and male pangolins could mate and successfully fertilise females at nine months age (n = 1). We also observed a female pangolin conceiving on the eighth day after parturition (the fifth day after the death of its pup). Our captive pangolins had a female-biased sex ratio of 1:0.5 at birth, unlike other known captive-born mammals. Also, captive-born pangolins were generally more viable after successful weaning and had a similar gestation length (~185 days) to wild pangolins. Most importantly, we report the first self-sustaining captive population of Malayan pangolins, and this species has an efficient reproduction strategy. These advances provide more comprehensive information for people to understand pangolins, and have implications for conserving endangered Malayan pangolins and providing scientific guidance to the management of other pangolin species.

Pangolin is a mysterious animal in the Family Pholidota, Mammalia. Malayan pangolin (Manis javanica) is one of eight existing species and is listed in Manis. With the number of wild pangolins (Manis spp.) rapidly decreasing, captive breeding has become an important way to protect them from extinction. The research on mating behavior of pangolins is an important content to understand its reproductive characteristics and develop breeding management. From 2016 to 2022, a total of 360 mating events were observed in six males and 24 females through closed circuit television (CCTV) surveillance. The results show that males do not engage in complex courtship behavior before mating. In addition, we found that male pangolins adopted a ventrolateral mating position. Once males selected the side (left/right) of the female pangolin from which to approach to mate, they usually remained on the same side for subsequent mating, suggesting that male pangolins may have a preference in mating position. Finally, all mating events were observed at 1.72 ± 1.47 (n = 83, Mean ± SD) days after cohabitation and adjustment time before mating (from the male touching the female to intromission) took 4.98 ± 3.86 mins (n = 323). During mating, males hugged females and remained still for 47.37 ± 10.08 seconds (n = 323), which is the ejaculation and post-ejaculation quiescent time. Remarkably, we observed for the first time two peak mating times, 19:00 to 22:00 and 1:00 to 3:00, suggesting that they may have a preference for mating times. This study provides new insight into the mating behavior of M. javanica and contributes to the development of scientific conservation measures to improve the reproductive capacity of M. javanica.

Pangolins are one of nature's most fascinating species being scales covered and myrmecophagous diet, yet relatively little is known about the molecular basis. Here, we combine the multi-omics, evolution, and fundamental proteins feature analysis of both Chinese and Malayan pangolins, highlighting the molecular mechanism of both myrmecophagous diet and scale formation, representing a fascinating evolutionary strategy to occupy the unique ecological niches. In contrast to conserved organization of epidermal differentiation complex, pangolin has undergone large scale variation and gene loss events causing expression pattern and function conversion that contribute to cornified epithelium structures on stomach to adapt myrmecophagous diet. Our assemblies also enable us to discover large copies number of high glycine-tyrosine keratin–associated proteins (HGT-KRTAPs). In addition, highly homogenized tandem array, amino content, and the specific expression pattern further validate the strong connection between the molecular mechanism of scale hardness and HGT-KRTAPs.

This study tracked and recorded the weight changes of 13 captive Sunda pangolin cubs from lactation to maturity to explored the appropriate weaning time and reveal the rules of its weight growth. SPSS 25.0 was used to build a cubic equation model to fit the body weight change rules of 4 individuals who nonvoluntarily ingested artificial feed (NIAF) at 127 days after birth and 5 individuals who voluntarily ingested artificial feed (VIAF) at 86–108 days after birth. The body weight of NIAF cubs aged 0–120 days and VIAF cubs aged 0–150 days were estimated according to the fitting model. An independent sample T-test was performed on the mean body weight of the two groups during the late lactation period. The results showed that at 105 days after birth, the body weight of the VIAF group was significantly higher than that of the NIAF group (P = 0.049), and the body weight of the VIAF group was extremely significantly higher than that of the NIAF group at 114 days (P = 0.008); The peak cumulative body weight of the NIAF cubs during lactation appeared around 130 days of age (n = 3); The mortality rate was 66.7% (n = 3) after about 150 days if the feed was continuously consumed nonvoluntarily. It was concluded that the milk secretion period of the mother is about 0–5 months after giving birth; the weaning period of the cubs should be 4–5 months after birth. If the cubs don’t follow the mother to eat artificial feed for 3 months after birth, it can start be induced with artificial diet which adds termites, and the time point cannot be later than 130 days, otherwise it is not conducive to the survival of the cubs; When sexually mature, the body length and body weight of female cubs account for about 84% and 60% of the adult, respectively; the body maturity and body weight of female cubs tend to be stable about 15.3 months and 16.4 months, respectively. Finally, a special needle-shaped nipples and nursing patterns of female Sunda pangolins were also recorded in this study. These findings play an important role in guiding the nursing of captive Sunda pangolin cubs and other pangolin cubs. It is expected to improve the survival rate of the cubs by exploring the appropriate weaning time and the rules of weight growth. By scientifically planning the reproductive cycle of the female Sunda pangolins, our goal is to expand the population size and eventually release to the wild, meanwhile improving knowledge of this critically endangered species.

Ex-situ conservation and captive breeding are important measures for protecting endangered species. However, captive conditions inhibit reproduction in some wild animals, especially males. Under captive conditions, which differentially expressed microbiota and metabolites significantly influence or are key to reproductive performance? This study aimed to investigate the effects of differentially expressed microbiota and metabolites on reproductive performance, including male Malayan pangolins with normal reproductive behavior (NR, with natural mating behavior) and those with abnormal reproductive behavior (AR, without natural mating behavior). Five male Malayan pangolins with normal reproductive behavior and eight with abnormal reproductive behavior were divided into NR and AR groups. Fresh fecal samples were collected for metagenomic and metabolomic analysis. The results showed that Bacillota, Pseudomonadota, Bacteroidota, Actinomycetota, and Fusobacteriota were the dominant phyla. Escherichia, Lactobacillus, Lactococcus, Limosilactobacillus, and Ligilactobacillus were the dominant genera. The abundance of Absiella, Butyribacter, and Candidatus Scatovivens in the gut of the NR group was significantly higher than in the AR group (p < 0.05), while the abundance of Clostridium, Mycoplasmopsis, and Facklamia in the gut of the AR group was significantly higher than in the NR group. A total of 94 differentially expressed metabolites were identified, with Argininosuccinic acid and Cortol significantly upregulated in the AR group, and gamma-Aminobutyric acid and gamma-Glutamylglutamic acid significantly downregulated. These microbiota and metabolites can be screened as potential key biomarkers that might influence the reproductive performance of male pangolins. These findings provide important insights into the husbandry and management of captive pangolins and other endangered wild animals.

Ex situ conservation and captive breeding are important measures for conserving endangered species. However, the reproduction of some wild animals, especially males, is inhibited in captivity, but the underlying mechanism has not yet been elucidated. This study aimed to investigate the microbiota and their functions, metabolites, and their metabolic pathways impacting reproduction employing metagenomics and metabolomics analyses and using male Malayan pangolins with normal (with natural mating behavior) and abnormal (no natural mating behavior) reproduction as the research objects. The results showed that the relative abundance of Proteobacteria, Escherichia coli , and Shigella spp. was significantly higher in the abnormal reproduction (AR) group. However, the relative abundance of Firmicutes and Staphylococcus aureus was significantly higher in the normal reproduction (NR) group. Kyoto Encyclopedia of Genes and Genomes functional pathway enrichment analysis found that citrate cycle (TCA cycle, KO00020) and pyruvate metabolism (KO00620) were significantly enriched in pangolins with AR, whereas gonadotropin-releasing hormone secretion (KO04929) was significantly enriched in pangolins with NR. Metabolites such as tryptophan, arginine, and androgen were significantly enriched in pangolins with AR, whereas L-proline, taurine, choline, and spermidine were significantly enriched in pangolins with NR. Microbiota dysbiosis, energy metabolism disorder, deficiencies in key metabolic pathways and metabolites, and hormonal disturbances are all potential factors contributing to the inability of male Malayan pangolin to express natural reproductive behavior. This study provides evidence for AR of captive pangolins and offers important insights for the conservation of captive endangered species.