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. 2024 Aug 19;10(5):e1583. doi: 10.1002/vms3.1583

Potential contribution of alpha‐fetoprotein level to biomarker of pregnancy outcome in Asian elephants

Fanwen Zeng 1, Mian Huang 1, Kang Huang 1, Jiaqi Sa 1, Shouquan Zhang 2,✉, Xuanjiao Chen 1,✉
PMCID: PMC11332394  PMID: 39158971

Abstract

Alpha‐fetoprotein (AFP) is a structural serum glycoprotein that plays vital roles in reproduction and mammalian development. Analysis of serum prolactin (PRL) is considered one of the useful methods for diagnosing pregnancy in Asian elephants. However, the expression profiles of AFP in pregnant and nonpregnant Asian elephants remain unclear, nor is the relationship with PRL. In this study, serum seven gonadal hormones and AFP in three pregnant and seven nonpregnant Asian elephants were analysed by via radioimmunoassay (RIA) and enzyme‐linked immunosorbent (ELISA) assay. We found that the mean (±SD) concentration of prolactin (PRL) in pregnant (136.782 ± 30.987 ng/mL) elephants was significantly higher than that in nonpregnant elephants (52.803 ± 21.070 ng/mL; p ≤ 0.0005). The mean (±SD) concentration of AFP in pregnant elephants (11.598 ± 0.824 ng/mL) was significantly higher than that in nonpregnant elephants (7.200 ± 2.283 ng/mL; p ≤ 0.05). Furthermore, the AFP concentration was positively correlated with the PRL concentration in the 10 Asian elephants studied. In conclusion, our findings suggest that serum AFP concentration is a potential biomarker of pregnancy outcomes in Asian elephants.

Keywords: alpha‐fetoprotein, Asian elephants, biomarker, pregnancy outcomes, prolactin

Alpha‐fetoprotein (AFP) is a structural serum glycoprotein that plays vital roles in reproduction and mammalian development. Analysis of serum prolactin (PRL) is considered one of the useful methods for diagnosing pregnancy in Asian elephants. We found the AFP concentration was positively correlated with the PRL concentration in the 10 Asian elephants studied. Therefore, we suggest that the serum AFP concentration is a potential biomarker of pregnancy outcomes in Asian elephants.

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1. INTRODUCTION

Asian elephant (Elephas maximus) populations are susceptible to losses as a consequence of habitat fragmentation, poaching driven by illegal ivory trade, and the resultant pressure factors on the remaining familial groups (Archie & Chiyo, 2012; Brown, 2019). The Asian elephant is listed as an Endangered species by the International Union for the Conservation of Nature (IUCN) (Arrollo et al., 2009; Baillie et al., 2004; Williams et al., 2019). The reproductive status of Asian elephants has been a matter of concern for decades (Hermes et al., 2007; Toin et al., 2020). Establishing wild animal in situ protected areas and creating ex situ captive breeding populations are effective methods that have played an essential role in the conservation of Asian elephants (Ortega, 2009). However, many captive elephant populations are not self‐sustaining, partly because of health and reproductive issues (Brown, 2015; Fernandez et al., 2021; Thitaram et al., 2008). Captive Asian elephants have difficulty producing offspring and have even shown pseudopregnancy, which can limit managed breeding success (Lueders et al., 2018). Therefore, accurate diagnosis of pregnancy outcomes is essential for improving reproductive efficiency.

Pregnancy in Asian elephants can be determined by measuring circulating progesterone and its metabolite concentrations. It need to be monitored continuously at least once to twice a week for 4 months (Brown, 2014; Hodges, 1998). However, a sustained increase in serum and urinary progestogen levels is sometimes associated with a prolonged luteal phase, independent of pregnancy (Lueders et al., 2018). The secretion of prolactin (PRL) shows remarkable changes during gestation in many mammals (Edgerton & Hafs, 1973; Reimers et al., 1978; Sherwood, 1971). PRL is mainly secreted by the anterior pituitary gland, and its main role is to develop the mammary gland and stimulate milk production (Bartke, 1971; Taya & Sasamoto, 1981). In Asian elephants, monitoring serum PRL is a useful method to diagnose pregnancy (Carden et al., 1998; Towiboon et al., 2022). PRL in the placenta of Asian elephants rises rapidly during 4−7 months of pregnancy, increasing up to 100‐fold from baseline (Brown & Lehnhardt, 1995; Yamamoto et al., 2012). During pregnancy in Asian elephants, PRL concentrations peak at 11–14 months and remain high until birth (Brown, 2000; Hodges, 1998). Unlike progestogen tests, which require a sample once or twice a week to diagnose pregnancy, a single measurement of serum prolactin levels after 7 months of gestation is a reliable pregnancy test. Pregnancy testing is the key to preventing obstructed labour or stillbirth problems, and measuring prolactin levels is generally a good method to improve the reproductive success of captive Asian elephants (Carden et al., 1998; Towiboon et al., 2022).

Alpha‐fetoprotein (AFP) is a serum glycoprotein discovered in 1956 with a molecular weight of approximately 68 kDa and is a characteristic protein throughout fetal development period in many species (Bergstrand & Czar, 1956; Deutsch, 1991; Glowska‐Ciemny et al., 2022). AFP synthesis begins with embryonic haematopoiesis in the yolk sac during the initial embryonic development. Subsequently, AFP is synthesised predominantly in the liver and is gradually replaced by albumin shortly after birth (Abelev, 1989; Koga et al., 1974). AFP is considered an essential protein for mammalian pregnancy because it suppresses the mother's immune response to the developing embryo (Rizzo et al., 2019). At 29 days after conception, human fetal serum showed measurable AFP concentrations, and pregnancy was the only normal situation (Crandall, 1981). Thus, AFP in human maternal serum can be used as a biomarker of pregnancy outcomes (Rizzo et al., 2019).

Given the limited information on pregnancy outcomes in Asian elephants, the present study aimed to explore the possible role of AFP levels as a predictor of pregnancy outcomes in Asian elephants. In addition, the relationship between AFP and PRL in the reproductive phase was determined. The ultimate goal was to understand the physical condition of elephants and apply this information for the development of more successful breeding programs.

2. MATERIALS AND METHODS

2.1. Study animals

All the experimental protocols were performed in accordance with the Guidelines for Animal Welfare of China.

Three pregnant Asian elephants (E1, E2, E3; aged from 15 to 37 years, average 23.00 ± 12.17 years) and seven nonpregnant Asian elephants (E4, E5, E6, E7, E8, E9, E10; aged from 9 to 53 years, average 30.43 ± 14.27 years) from the zoos in southern China, including Guangzhou, Shenzhen and Dongguan were studied (Table 1). All elephants were fed Napier grass (Pennisetum purpureum Schumach), fresh vegetables, and fruits (bananas, apples and carrots) three times daily, and were provided with mixed hay and water at all times. They interacted extensively with their keepers because they are trained and live in a free‐contact management system. Blood samples (10 mL; n = 15) were obtained from elephant ear veins using a 7# intravenous infusion needle and 10 mL syringe between February 2023 and July 2023. Blood was allowed to coagulate at room temperature (RT) for 30 min and centrifuged at 1600 × g for 10 min at 4°C to obtain serum, which was stored at –80°C and analysed within 2 weeks of collection.

TABLE 1.

Sampling and reproductive status of 10 Asian elephants in this study.

Name Age (year) Sampling month Mating date Reproductive state
E1 37 May 2023 March 2022 Pregnant
E2 17 May 2023 December 2021 Pregnant
E3 15 May 2023 December 2022 Pregnant
E4 9 May 2023 None Nonpregnant
E5 53 May 2023 None Nonpregnant
E6 30 February 2023 October 2022 Nonpregnant
E7 34 June 2023 December 2022 Nonpregnant
E8 32 June 2023 None Nonpregnant
E9 38 June 2023 None Nonpregnant
E10 17 July 2023 October 2021 Nonpregnant
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2.2. Hormone analysis

Seven gonadal hormones, including follicle‐stimulating hormone (FSH), luteinising hormone (LH), PRL, progesterone (P), testosterone (T), estradiol (E2), and human chorionic gonadotropin (HCG), were measured via radioimmunoassay (RIA) using commercially available kits from Beijing North Institute of Biotechnology Co., Ltd (BNIBT; Beijing, China) according to the manufacturer's protocol and as previous described (Taya, 1985; Yamamoto et al., 2010; Yamamoto et al., 2012). In brief, the PRL were determined by a heterologous RlA using an anti‐human PRL rabbit sera and the progesterone were determined by a double‐antibody RIA using 125I labelled radioligands. The amount of serum used for the assays was 200 µL; the assays were performed in triplicate. Linearity was also demonstrated by the correlations between the dilution curves of the serum that were approximately parallel to the standard curve. The inter‐ and intra‐assay coefficients of variation were less than 10% for all assays.

2.3. AFP enzyme‐linked immunosorbent (ELISA) and procedure

Serum AFP concentrations were measured using a commercial sandwich AFP enzyme‐linked immunosorbent (ELISA) assay Kit (BNIBT) according to the manufacturer's protocol. Serum samples were diluted in the sample buffer at a 1:10 dilution ratio. Subsequently, 100 µL of blank, controls, standards and diluted samples were added to a 96‐well plate and incubated for 30 min at 37°C. Three technical replicates were performed for each group. After washing three times with washing buffer, horseradish peroxidase (HRP)‐labelled streptavidin was added to each well followed by incubation for 1 h at 37°C. The plate was washed again and incubated with a tetramethylbenzidine (TMB)‐substrate for 15 min at RT. The reaction was terminated by adding sulphuric acid solution. The absorbance was immediately determined using an ELISA reader (Multiskan FC, Massachusetts, USA) at 450 nm.

2.4. Statistical analyses

Raw data were analysed using Excel 2013 software (Microsoft, Redmond, WA, USA). Statistical Package for the Social Sciences (SPSS version 18.0, SPSS, Chicago, IL, USA) software (IBM, Armonk, NY, USA) and GraphPad Prism software 8.0 (San Diego, CA, USA) were used to perform statistical analyses of all experimental results. The statistical significance of differences in mean values was analysed using two‐way ANOVA or unpaired Student's t‐test. Data were expressed as mean ± SD, and results with p values ≤ 0.05 were considered statistically significant.

3. RESULTS

Elephants E1–E3 were pregnant at the beginning of the study (1st to 22th month) and exhibited clinical signs of pregnancy, such as breast and belly bulging, while the other elephants did not show clinical signs of pregnancy. Serum gonadal hormone concentrations of individual elephants are summarised in Table 2. Pregnancy in Asian elephants can be determined by measuring circulating progesterone and its metabolite concentrations, which need to be monitored continuously at least once to twice a week for 4 months. However, in the current study, we did not measure the circulating serum hormone levels for several weeks in Asian elephants; therefore, progesterone could not be used to determine pregnancy status of the Asian elephants in this study. Hence, PRL concentrations were used. The mean (±SD) concentration of PRL in E1–E3 (range, 107.359−169.127 ng/mL; mean, 136.782 ± 30.987 ng/mL) was significantly higher than that in E4–E10 (range, 19.442−76.796 ng/mL; mean, 52.803 ± 21.070 ng/mL; p ≤ 0.0005) (Figure 1). There were no significant differences in the concentrations of other gonadal hormones between the two groups.

TABLE 2.

Summary of mean values of FSH, LH, PRL, P, T, E2 and HCG of the 10 Asian elephants in this study.

Hormone E1‐E3 E4‐E10 p
FSH (mIU/mL) 1.354 ± 0.336a 1.168 ± 0.177a 0.2712
LH (mIU/mL) 9.248 ± 2.487a 7.6017 ± 2.622a 0.3836
PRL (µIU/mL) 136.782 ± 30.987a 52.803 ± 21.070b 0.0009
P (ng/mL) 0.014 ± 0.007a 0.099 ± 0.202a 0.5016
T (ng/mL) 0.115 ± 0.182a 0.030 ± 0.050a 0.2578
E2 (pg/mL) 12.665 ± 2.124a 9.492 ± 4.844a 0.3189
HCG (mIU/mL) 4.736 ± 2.181a 5.962 ± 3.603a 0.6053
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Note: Values are expressed as mean ± SD of the mean; saliency analysis using the Student's t‐test; different labelled letters represent significant differences (p ≤ 0.0005).

FSH, follicle stimulating hormone; LH, luteinising hormone; PRL, prolactin; P, progesterone; T, testosterone; E2, estradiol; HCG, human chorionic gonadotropin.

FIGURE 1.

FIGURE 1

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Mean (±SD) concentrations of serum AFP. Mean AFP levels in E1–E3 were significantly higher than those in E4–E10 (p ≤ 0.05).

Mean serum AFP concentrations in this study are shown in Figure 1. The mean (±SD) concentration of AFP in E1–E3 (range, 11.778−12.426 ng/mL; mean, 11.598 ± 0.824 ng/mL) was significantly higher than that in E4–E10 (range, 3.814−10.69 ng/mL; mean, 7.200 ± 2.283 ng/mL; p ≤ 0.05). In addition, serum AFP and PRL concentrations were positively correlated (r = 0.6640, p = 0.0445; Figure 2), suggesting the potential use of AFP for pregnancy diagnosis in Asian elephants.

FIGURE 2.

FIGURE 2

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Correlation between AFP and PRL concentrations. Detected levels of serum concentration AFP concentration (X‐axis) and serum PRL concentration (Y‐axis) in Asian elephants.

4. DISCUSSION

Accurate pregnancy detection is a key factor in improving fertility success in Asian elephants, allowing managers to adapt management strategies to prevent obstructed labour or miscarriage (Kajaysri & Nokkaew, 2014; Rasmussen & Schulte, 1998). AFP has been proven to be a good indicator of fetal well‐being in human medicine for decades (Vincze et al., 2015). Although this molecule is present in most mammalian species, pregnancy outcomes in Asian elephants have not been reported. To the best of our knowledge, this is the first study to confirm the presence of AFP in Asian elephants and its association with reproduction. In this study, the relationships between serum AFP and PRL concentrations and gestational state were demonstrated in Asian elephants.

Hormone analysis demonstrated that PRL in E1–E3 (136.782 ± 30.987 ng/mL) was significantly higher than that in E4–E10 (52.803 ± 21.070 ng/mL), suggesting the possibility of pregnancy in E1–E3. E1–E3 gave birth to calves on the expected date, which confirmed the clinical manifestation of pregnancy; thus, it can be stated that PRL plays an important role in determining pregnancy outcomes. Serum prolactin analysis is a useful tool for monitoring the reproductive status of Asian elephant females (Carden et al., 1998; McNeilly et al., 1983). Serum prolactin concentrations in Asian elephants are significantly higher throughout gestation than during estrus, similar to the observations of this study (And & Lehnhardt, 1997; Brown & Lehnhardt, 1995; Towiboon et al., 2022). In elephants, the biological actions of PRL are primarily used to support pregnancy and fetal development, and are related with lactation and reproduction (Yamamoto et al., 2012). During pregnancy, PRL secretion is significantly increased, facilitating mammary gland development, maintaining lactation, promoting luteal formation, and maintaining progesterone secretion, which may be related to the secretion activity of pituitary prolactin cells stimulated by estrogen (Yamamoto et al., 2012). In cycling elephants, prolactin concentration did not differ between the follicular phase and the luteal phase (Yamamoto et al., 2010). However, during the follicular phase of the female African elephant, prolactin secretion is sometimes increased (Brown et al., 2004; Yamamoto et al., 2010). This can be due to physiological problems, one of which is an abnormal ovarian cycle, and it may be linked to hyperprolactinaemia (Prado et al., 2019; Prado‐Oviedo et al., 2013). In this study, our data showed nonpregnant Asian elephants did not exhibit elevated prolactin (Table 2).

AFP is expressed at high levels during embryonic development and is therefore considered essential for mammalian development (Bader et al., 2004; Gabant et al., 2002). We found that pregnant elephants had higher AFP concentrations than nonpregnant elephants (Figure 1), which is consistent with the observations reported in other mammalian studies (Abelev, 1989; Bériot et al., 2014; Mizejewski, 2003; Yamada et al., 1995). In mammals, AFP concentrations rapidly increase in early pregnancy, reach a peak, plateau, and finally a decline towards the end of the term (Bériot et al., 2014; Smith et al., 1979; Vincze et al., 2016). Unfortunately, the Asian elephants in this study were not evaluated for postnatal AFP, which also guides future research. After conception, AFP can be detected in human fetal serum at a concentration of 1–5 mg/mL, whereas in mouse serum AFP concentration is approximately 2 mg/mL (Soltani, 1979). Our results found that AFP concentrations in E10 was close to, but still lower than the AFP concentrations in pregnant females. No abnormalities were found on routine blood examination nor were there any indications of a tumour that may produce AFP.  Finally, there was no evidence of pseudopregnancy identified in this elephant. In this study, the threshold of serum AFP concentration in pregnant and nonpregnant Asian elephants could not be definitively determined. However, our results indicated that pregnant Asian elephants have higher AFP concentrations than nonpregnant elephants. Therefore, determination of specific concentrations requires additional verification experiments that need to be carried out for validation, and more Asian elephants need to be tested. In addition, we hope that subsequent experiments will be able to measure the early, middle and late phases of pregnancy AFP is rapidly rising to confirm the pregnancy. The results of the present study also revealed that the serum AFP concentration was positively correlated with the serum PRL concentration. In previous study, a strong positive correlation between human maternal AFP levels and gestational age was observed in normal pregnancy (Gupta et al., 1987). We were speculated that AFP levels in pregnant Asian elephants may also increase with gestational age. Additionally, prolactin also increases rapidly during pregnancy in Asian elephants. Hence, it is not surprising that AFP was positively correlated with PRL.

5. CONCLUSION

The findings of this study established that serum AFP concentration is a reliable biomarker of pregnancy outcomes in Asian elephants. Serum AFP and PRL concentrations were positively correlated, confirming the role of AFP in supporting the diagnosis of pregnancy in Asian elephants.

AUTHOR CONTRIBUTIONS

Fanwen Zeng: Writing – review & editing. Mian Huang: Validation & Investigation. Jiaqi Sa: Formal analysis & Visualisation. Kang Huang: Software; Validation. Shouquan Zhang: Methodology. Xuanjiao Chen: Supervision; Project administration. All authors have read and agreed to the published version of the manuscript.

CONFLICT OF INTEREST STATEMENT

No potential conflict of interest was reported by the authors.

ETHICAL STATEMENT

None.

PEER REVIEW

The peer review history for this article is available at https://publons.com/publon/10.1002/vms3.1583.

ACKNOWLEDGEMENTS

The authors thank the Guangzhou Zoo research project (grant numbers: YL202401), which supported this work.

Zeng, F. , Huang, M. , Huang, K. , Sa, J. , Zhang, S. , & Chen, X. (2024). Potential contribution of alpha‐fetoprotein level to biomarker of pregnancy outcome in Asian elephants. Veterinary Medicine and Science, 10, e1583. 10.1002/vms3.1583

Contributor Information

Shouquan Zhang, Email: sqzhang@scau.edu.cn.

Xuanjiao Chen, Email: 13631363846@139.com.

DATA AVAILABILITY STATEMENT

None.

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