# Utilization of Urinary Neopterin Levels for Pregnancy Diagnosis in Mated Giant Pandas

## TL;DR

This study shows that measuring urinary neopterin levels can help determine if a giant panda is pregnant, especially after progesterone levels peak.

## Contribution

The study introduces a novel pregnancy diagnostic method using the neopterin ratio in giant pandas during the post-progesterone peak phase.

## Key findings

- Neopterin levels in pregnant pandas significantly increased during the post-progesterone peak phase compared to nonpregnant pandas.
- The neopterin ratio (S3/S2) of 1.71 ± 0.12 in parturient pandas reliably indicates successful pregnancy.
- The neopterin ratio can be used for on-site pregnancy diagnosis in giant pandas using commercially available assays.

## Abstract

This novel study aimed to investigate one of the most challenging aspects of giant panda pregnancy detection. We assessed urinary neopterin, a biomarker of cell-mediated immunity, in females that did and did not become pregnant following breeding. We found a significant difference in neopterin levels between pregnant and nonpregnant females during the last several weeks of the luteal phase, after the progesterone peak. To enable the research results to have practical predictive value for pregnancy diagnosis in mated giant pandas, we detected a significant difference in the neopterin ratio, calculated as the ratio of the mean neopterin level in the post-progesterone peak phase (progesterone decreases from its peak [inclusive] to approximately 200 ng/mg Cr) to that in the pre-progesterone peak phase (progesterone levels rise from 100 ng/mg Cr [inclusive] to its peak value) between the parturient group (ratio = 1.71 ± 0.12; n = 14) and the nonparturient group (ratio = 0.85 ± 0.04; n = 17). Specifically, the neopterin ratio of the parturient group (1.71 ± 0.12; n = 14) proved to be a robust indicator of pregnancy status. Neopterin assessment can be performed with commercially available assays, thereby making on-site pregnancy diagnosis practical at giant panda holding facilities.

Neopterin is a well-characterized marker of cell-mediated immunity. Here, we used neopterin as a biomarker to identify the cellular immunity status of breeding giant pandas throughout gestation. Urine samples were collected from captive adult female giant pandas that had entered estrus and were either naturally bred and/or artificially inseminated (AI) during the breeding seasons at the Chengdu Research Base of Giant Panda Breeding. This study was divided into two parts. In the first part, we examined the urinary neopterin and progesterone (Pg) profiles during pregnancy in parturient and nonparturient individuals. From the day of mating to the Pg peak, there was no correlation between neopterin and Pg in both parturient individuals (n = 3) and nonparturient individuals (n = 3). During the period when the Pg dropped from its peak to approximately 100 ng/mg Cr, the changes in neopterin and Pg showed a negative correlation in parturient individuals, while there was no correlation between the changes in neopterin and Pg in nonparturient individuals. Subsequently, urine samples were categorized into four phases based on the reproductive status of giant pandas: (1) Estrus phase (Es); (2) Primary Pg increase phase (S1), during which Pg levels rise from the day of mating to 100 ng/mg Cr; (3) Secondary Pg increase phase (S2), during which Pg levels rise from 100 ng/mg Cr (inclusive) to their peak value; and (4) Post-peak Pg decline phase (S3), during which Pg levels decrease from their peak (inclusive) until they drop to approximately 200 ng/mg Cr. Analysis revealed that differences between parturient and nonparturient females emerged in the S3 phase. Compared to nonparturient individuals, neopterin concentrations showed sustained elevation in parturient pandas during the S3 phase. The second part of the study, building on the findings of the first part, specifically focused on analyzing the urine samples of giant pandas during the S2 and S3 phases. Statistical analysis of an expanded sample of individuals (including those from the first part) revealed a significant difference in the neopterin mean S3/S2 ratio between the parturient group (mean S3/S2 = 1.71 ± 0.12; n = 14) and the nonparturient group (mean S3/S2 = 0.85 ± 0.04; n = 17). Correspondingly, parturient individuals gave birth 5 to 16 days after the end of the S3 phase (this time corresponds to the point when Pg levels decrease to approximately 200 ng/mg Cr). The results revealed the validity and applicability of using the neopterin mean S3/S2 ratio (1.71 ± 0.12) for diagnosing successful pregnancy in mated female giant pandas.