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. 2025 Aug 18;11(5):e70558. doi: 10.1002/vms3.70558

The Elecsys AMH Assay Is a Suitable Method to Detect Gonadal Tissue in Male Alpacas and Llamas

Sait Sendag 1,2,✉, Henrik Wagner 2, Ali Osman Turgut 3, Davut Koca 1, Gerhard Schuler 2, Axel Wehrend 2
PMCID: PMC12359723  PMID: 40824174

ABSTRACT

Objective

Anti‐Müllerian hormone (AMH) has become an important hormonal parameter for the detection of gonadal tissue and for the diagnosis of gonadal functions and pathologies. To our knowledge, there is currently no homologous test for AMH measurements in South American camelids (SACs). Therefore, the objective of the present study was to determine serum AMH concentrations in postpubertal male alpacas and, for the first time, in llamas, using the Elecsys AMH assay kit that has not previously been tested in these species. To obtain indications of the specificity of this method in SAC, measurements were carried out in male gelding in which concentrations below the detection limit were to be expected.

Methods

In this context, 37 blood samples collected by jugular venipuncture from 21 alpacas and 16 llamas were used. The obtained blood was centrifuged at 3000 g for 20 min, and the serum was stored in Eppendorf tubes at −20°C until AMH concentrations were measurement. The measurement of AMH levels was conducted in a commercial diagnostic laboratory (Laboklin, Bad Kissingen, Germany) using the electrochemiluminescence immunoassay kit Elecsys AMH run on the fully automated Cobas e 601 analyser (Roche Diagnostics Deutschland GmbH, Mannheim). The AMH test had a minimum detection limit of 0.01 ng/mL and a maximum detection limit of 23 ng/mL. The intra‐assay coefficient of variation is between 2.7% and 3.3%.

Results

Blood serum AMH levels ranged between 4.10 and 22 ng/mL (median: 9.80 ng/mL) and 1.79 and 10.05 ng/mL (median: 4.00) in intact alpacas (age: 6.30 ± 2.71 years; n = 10) and llamas (age: 5.50 ± 4.34; n = 8), respectively, and were significantly different between samples obtained from the two species (p < 0.05). Correlation analyses regarding an age dependence of AMH concentrations yielded negative correlation coefficients for both species but non‐significant p values (alpaca: r = −0.165, p = 0.649; llama: r = −0.547, p = 0.160). In alpaca (n = 11) and llama geldings (n = 8), blood serum AMH levels were below 0.01 ng/mL (p < 0.001). These results prove that the antibodies used in the Elecsys AMH assay significantly and specifically cross‐react with SAC AMH.

Conclusions

In gelding llamas and alpacas, AMH concentrations were below the limit of detection (<0.01 ng/mL), which was significantly lower compared to intact animals (p < 0.001). The Elecsys AMH assay is therefore considered a suitable method for detecting gonadal tissue in SAC.

Keywords: anti‐Müllerian hormone, AMH assay, South American camelids

Serum AMH values were different and statistically significant between the two species (p < 0.05).

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1. Introduction

Anti‐Müllerian hormone (AMH), also known as Müllerian‐inhibiting substance (MIS), is a 140 kDa glycoprotein consisting of 553–557 amino acids that belongs to the transforming growth factor‐beta (TGF‐β) family. Its primary physiological role in mammals occurs during embryogenesis, wherein males AMH is synthesized by foetal Sertoli cells at the time of testicular differentiation and induces regression of the Müllerian ducts that in females form the base for the development of the oviducts, uterus and upper part of the vagina (Teixeira et al. 2001; Knight and Glister 2006). Prepubertal AMH concentrations are significantly higher than postpubertal levels in bulls (Rota et al. 2002), humans (Lee et al. 1996) and mice (Rey and Josso 1996), even though measured concentrations differ between species (Claes et al. 2013). The germ cell number in adult males depends on the number of Sertoli cells produced during perinatal development. The AMH concentrations measurable in the systemic circulation are exclusively originating from expression in Sertoli cells. In this way, AMH may be considered a functional marker of Sertoli cells. Therefore, in addition to the diagnosis of abdominal cryptorchidism, recently AMH has received increasing attention in research on disorders related to male fertility such as Sertoli cell tumours (Xu et al. 2019).

In females, AMH is only expressed after the end of the critical phase of sex differentiation, with the granulosa cells of early developing follicles being the relevant site of AMH expression. Accordingly, systemic AMH levels have been shown to correlate with antral follicle count, which is a major predictor of the age‐related decline in female fertility due to ovarian reserve reduction (Rico et al. 2012). Currently, AMH is regarded as a biomarker for reproductive function also in females and has been determined in different species as a marker for ovarian follicle reserve or in suspected cases of incomplete castration or ovarian pathologies such as granulosa cell tumours (Scarlet et al. 2018; Nelissen and Miller 2022; Koca et al. 2023; Koca, Nak, et al. 2024; Koca, Aktar, et al. 2024; Turgut and Koca 2024a; Turgut and Koca 2024b; Chumsria et al. 2024).

Llamas and alpacas, the two domesticated South American camel (SAC) species, have become increasingly widespread in Central Europe in recent years (Wagner et al. 2023). This development was accompanied by a significant increase in the need for care and medical treatment when keeping and breeding this species. However, little is known about the physiological concentrations of AMH in healthy male alpacas and llamas. Accordingly, reliable reference blood values of AMH in these animals have not yet been defined. To the best of our knowledge, to date, there is no sufficiently validated AMH assay kit developed for SACs. The Elecsys electrochemiluminescence immunoassay AMH (Roche, Germany) was originally developed for blood AMH measurements in women (Deeks 2015; Jacobs et al. 2019). However, this AMH test kit has so far been successfully used for the determination of blood AMH levels in various domestic animal species such as cattle, sheep (Rico et al. 2012; Koca et al. 2023; Koca, Nak, et al. 2024; Koca, Aktar et al. 2024; Turgut and Koca 2024a), dromedary (Seyedasgari et al. 2024), cat (Gözer et al. 2023) and dog (Walter et al. 2019), suggesting that the antiserum applied exhibits a significant cross‐reactivity in a wide range of species. However, a literature search found no evidence of previous use in SAC.

Therefore, the objective of the present study was to determine serum AMH concentrations in postpubertal male alpacas and, for the first time, in llamas, using the Elecsys AMH assay kit that has not previously been tested in these species. To obtain indications of the specificity of this method in SAC, measurements were carried out in male gelding in which concentrations below the detection limit were to be expected. Another aim of our study will be to compare blood AMH levels in alpaca and llamas, with the results of this comparison helping to inform our understanding of the differences between these two species.

2. Materials and Methods

2.1. Animals

In this study, 37 blood samples from 21 alpacas and 16 llamas were used. The animals were kept on private farms in the region of Hesse (50° 35′ N—8° 40′ E, Germany). The altitude of the city where animals are bred is 155–159 m. The coldest month in this city is January, with −0.1°C, and the warmest month is July, with 18.2°C. Early spring is usually dry.

2.2. Experimental Design

A total of 37 adult male SACs were used in the study. Of these, 21 were alpacas and 16 were llamas. To prove the validity of the Elecsys AMH assay, animals were divided into intact and gelding subgroups within each species. In this context, 21 alpacas were divided into two subgroups: 10 intact animals and 11 geldings. Similarly, 16 llamas were categorised as 8 intact and 8 gelding animals. The experimental groups in the study were formed according to the intact and gelding status and age of the alpacas and llamas, as summarized in Table 1.

TABLE 1.

Overview of the animals examined.

Species Body condition score Gonadal (physiological) status n Age (years) (mean ± SD)
Alpaca 2.5–3.5 Intact 10 6.30 ± 2.71
Gelding 11 8.27 ± 3.55
Llama 2.5–3.5 Intact 8 5.50 ± 4.34
Gelding 8 11.88 ± 4.39
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2.3. Blood Sampling

Blood samples were collected from the animals by jugular venipuncture (August 2024). The animals were visited (between 09:00 and 15:00 h) at the farms where they were bred. The animals were kept standing and held by a person. Blood was collected using a non‐vacuum syringe. The obtained blood was centrifuged at 3000 g for 20 min, and the serum was stored in Eppendorf tubes at −20°C until AMH concentrations were measurement.

2.4. AMH Analysis

The measurement of AMH levels was conducted in a commercial diagnostic laboratory (Laboklin, Bad Kissingen, Germany), using the electrochemiluminescence immunoassay kit Elecsys AMH, which runs on the fully automated Cobas e 601 analyser (Roche Diagnostics Deutschland GmbH, Mannheim). Before the primary trial, the samples underwent method validation by the manufacturer's instructions. The calibration and standard curves were carefully examined using precisely assigned values. The assay showed an analytical sensitivity of 10 pg/mL. The coefficients of variance within and between assays were 0.5%–1.4% and 0.7%–1.9%, respectively (Koca et al. 2023; Koca, Nak, et al. 2024; Turgut and Koca 2024a). The detailed methodology for determining AMH for this test kit is available in the specific commercial brochure (Elecsys AMH cobas; 2025‐02, V 2.0 English).

2.5. Statistical Analysis

Minitab (version 21.4.1, LLC, Pennsylvania, USA) was used for the statistical analysis. Descriptive statistics were computed to summarize the distribution of serum AMH levels in intact llamas and alpacas separately. An independent sample t‐test was used to compare the serum AMH concentrations and the age of intact llamas and alpacas, respectively. Pearson correlation analysis was performed to evaluate the relationship between age and serum AMH levels of intact animals. The statistical significance level was defined as p < 0.05.

3. Results

In gelding llamas and alpacas, AMH concentrations were below the limit of detection (<0.01 ng/mL), which was significantly lower compared to intact animals (p < 0.001). Conversely, serum AMH levels ranged between 1.79–10.05 and 4.10–22 ng/mL in intact llamas and alpacas, respectively. Median AMH concentrations were 9.80 ng/mL in alpacas and 4.00 ng/mL in llamas (Figure 1). This difference was statistically significant (p = 0.018). Correlation analyses (Figure 2) regarding an age dependence of AMH concentrations yielded negative correlation coefficients for both species but non‐significant p values (alpaca: r = −0.165, p = 0.649; llama: r = −0.547, p = 0.160).

FIGURE 1.

FIGURE 1

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Serum AMH concentrations in intact male (a) alpacas and (b) llamas (p < 0.05). AMH, anti‐Müllerian hormone.

FIGURE 2.

FIGURE 2

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Presentation of AMH concentrations of intact alpacas and llamas as a function of the age of the sampled animals, including the results of linear regression analyses. AMH concentrations yielded negative correlation coefficients for both species but non‐significant p values (alpaca: r = −0.165, p = 0.649; llama: r = −0.547, p = 0.160).

4. Discussion

The objective of this study was to determine serum AMH concentrations in postpubertal male alpacas and, for the first time, in llamas using the Elecsys AMH assay kit, which has not been previously tested in these species. To obtain indications of the specificity of this method in SAC, measurements were performed in gelding males, in which concentrations below the detection limit were expected.

In endocrinological diagnostics, veterinarians are often faced with the situation that for target molecules with a species‐specific structure and no homologous measurement methods are (commercially) available. Sometimes suitable heterologous methods are available in such cases. However, before using a heterologous assay for the first time in a ‘new’ target species, sufficient validation must be carried out, as applicability in such cases is not automatic and the likelihood of generating erroneous data should not be underestimated. In the most favourable case, the measured values (largely) correspond to the actual concentrations. To answer biological or clinical questions, however, it can also be useful if a heterologous measurement method does not provide correct measured values, but these correlate closely with the actual concentrations. In these cases, the interpretation can be based on method‐specific reference values, or the measured values can be mathematically transformed accordingly so that established reference values can be used for the interpretation (Hussein et al. 2022; Längerer et al. 2025). To the best of our knowledge, there is currently no homologous assay available to measure AMH in SAC and there are only a small number of reports on measurements using heterologous assays, including only one study reporting AMH measurements in males (Ciccarelli et al. 2018).

To our knowledge, this is the first study that reports on using the commercially available heterologous AMH immunoassay Elecsys, originally developed for use in humans, to assess circulating AMH levels in male intact and gelding alpacas and llamas. The pronounced difference in concentrations between intact and castrated animals and the fact that the concentrations in castrated animals were below the detection limit indicate that AMH in male SAC is specifically detected by the Elecsys method. Thus, this method is at least suitable for the diagnosis of abdominal cryptorchidism in alpacas and llamas. The extent to which the values measured in intact animals are correct cannot currently be estimated with certainty, because neither homologous methods for comparative measurements nor authentic SAC AMH standards for spiking experiments are available.

A very surprising observation was the pronounced and statistically significant species difference given the very close phylogenetic relationship between the two target species. This difference cannot be explained by a different age distribution of the sampled animals, which was comparable in both species (p > 0.05). One possible cause would be the higher incidence of testicular pathologies in one of the two species. The frequent occurrence of testicular anomalies has been described in SAC (Tibary and Vaughan 2006). Even though no detailed andrological examination was carried out when the blood samples were taken, there were no indications of this. Finally, a purely random effect cannot be ruled out. Further measurements on a larger number of animals are required for clarification.

In male SAC, a literature search revealed only one study in which AMH concentrations were determined (Ciccarelli et al. 2018). These studies were carried out on 6‐, 7‐, and 8‐month‐old prepubertal and adult alpacas aged between 5 and 9 years (n = 5 for each age group), with samples being taken in the adult group before and 24 h after castration. The ELISA kit used (AMH ELISA test kit KMH‐01, Preventia Diagnostics Inc.) was not described in detail about the reagents used, and no information on the test kit or the manufacturer could be found on the Internet at present. The sharp drop in AMH concentrations in the postpubertal stallions within 24 h after castration demonstrated that this assay specifically detected AMH in alpacas. However, with presurgical concentrations between 0.57 and 1.59 ng/mL (mean: 0.94 ng/mL), the measured values were only about one‐tenth of the values measured by us, whereby the age of the animals was comparable between the two studies (Ciccarelli et al. 2018): 5–9 years; our study: 3–10 years. The cause of the very different measured values between the two studies is unclear. Possibly, methodological factors such as a different degree of cross‐reaction of the antisera used with SAC AMH or matrix effects contributed to this difference. In a very recent study (Seyedasgari et al. 2024), in which female dromedary camels were used in a multiple ovulation embryo transfer program, the serum AMH values before and after synchronisation were examined with the Elecsys assay used in our study. These researchers found the mean peripheral AMH concentration in synchronised and non‐synchronised camels to be 1.46 ± 0.15 and 0.95 ± 0.09 ng/mL, respectively. In conclusion, this study (Seyedasgari et al. 2024) demonstrated that highly consistent AMH values in dromedary camels are a reliable predictor of superovulation response and outcome in the same animals.

As a result, this study was able to compare serum AMH ranges in intact and gelding adult alpacas and llamas. Similar studies to be carried out in the future in intact alpacas and llamas of the same age, nutrition and care conditions will be able to support our results and contribute to the determination of reference AMH values in these animals.

5. Conclusion

In gelding llamas and alpacas, AMH concentrations were below the limit of detection (<0.01 ng/mL), which was significantly lower compared to intact animals (p < 0.001). Our results indicate that the Elecsys AMH method, which is available in many commercial laboratories, is at least a reliable hormonal detection method for the presence of testicular tissue in male SAC.

Author Contributions

Sait Sendag: conceptualization, methodology, validation, investigation, writing – original draft, editing. Henrik Wagner: methodology, investigation, validation. Ali Osman Turgut: statistical analysis, writing – review, editing. Davut Koca: conceptualization, methodology, writing – review, editing. Gerhard Schuler: conceptualization, validation, writing – original draft, editing, supervision. Axel Wehrend: conceptualization, validation, writing – review, editing, supervision.

Ethics Statement

The Justus Liebig University Giessen's animal welfare office obtained approval from the local ethical authorities to utilize the blood samples (internal correspondence number or IRB number: kTV 11‐2018).

Conflicts of Interest

The authors declare no conflicts of interest.

Acknowledgements

The authors gratefully acknowledge the skilled technical assistance in the laboratory provided by Sabine Feller and Julia Blad‐Stahl.

Open access funding enabled and organized by Projekt DEAL.

Sendag, S. , Wagner H., Turgut A. O., Koca D., Schuler G., and Wehrend A.. 2025. “The Elecsys AMH Assay Is a Suitable Method to Detect Gonadal Tissue in Male Alpacas and Llamas.” Veterinary Medicine and Science 11, no. 5: 11, e70558. 10.1002/vms3.70558

Funding: The authors received no specific funding for this work.

Data Availability Statement

The data presented in this study are available on request from the corresponding author.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Data Availability Statement

The data presented in this study are available on request from the corresponding author.

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