配偶选择是动物在选择配偶时对具有优秀特征个体的倾向和偏好，对提高个体的繁殖成功率和适合度有关键作用。已发现肠道菌群变化能够影响果蝇和小鼠等模式动物的配偶选择行为，然而，肠道菌群是否影响大型哺乳动物的配偶选择行为，尚未见报道。大熊猫属季节性发情，且雌雄个体间具有强烈的选择性。圈养环境下，配偶选择偏好的正常表达能够提高雌性大熊猫的繁殖成功率和产仔率，但关于影响其配偶选择的因素和机制还有待研究。因此，本研究的目的是探索能否通过益生菌饲喂改善雌性大熊猫的肠道菌群组成，进而影响其配偶选择偏好。我们预测饲喂益生菌能够改善雌性大熊猫的肠道菌群组成，并提升雌性个体对优质雄性配偶的选择倾向。为验证该猜测，本研究以中国大熊猫保护研究中心的圈养雌性大熊猫为研究对象，使用扩增子测序技术，探索能否通过益生菌饲喂驱动肠道菌群（Gut microbiota, GM）组成和功能的变化。然后，通过气味选择实验，判别GM的改变是否对雌性配偶选择产生影响。主要结果如下：

1. 饲喂益生菌后雌性大熊猫肠道菌群的α和β多样性均未发生显著改变，但肠道菌群的组成和功能发生显著变化。肠道中有益菌（Muribaculaceae、Weissella cibaria）的丰度增加、致病菌或条件致病菌（Enterobacteriaceae、Campylobacter等）的丰度减少。肠道菌群的功能预测分析显示，饲喂益生菌后GM中与DNA修复和氨基酸代谢等重要功能相关的基因丰度显著升高（P < 0.05, t-test）。这表明益生菌能够改善圈养雌性大熊猫的肠道菌群组成和功能，有利于提升个体肠道健康。

2. 针对不同年龄雄性尿液气味的选择实验结果显示，圈养雌性大熊猫在饲喂益生菌后，表现出对壮年雄性尿液气味的显著偏好（P = 0.0095, t-test），同时对老年雄性尿液气味的探究频次（P = 0.0369, t-test）和探究时长（P = 0.0358, t-test）均显著降低。未饲喂益生菌的雌性在前后两次气味选择实验中，均未表现出对不同年龄雄性尿液气味的偏好，对不同年龄雄性尿液气味的探究频次和时长也未发生显著变化（P > 0.05）。因此，益生菌饲喂驱动的肠道菌群变化有助于促进圈养雌性大熊猫对优质雄性个体的配偶选择偏好。

3. 相关性分析结果显示，螺旋体门（Spirochaetota）和金黄杆菌属（Chryseobacterium）的相对丰度与圈养雌性大熊猫对壮年雄性尿液气味的探究频次（r = - 0.6910, p < 0.05, spearman）和探究时长（r = - 0.6609, p < 0.05, spearman）呈显著强负相关；放线菌属（Actinomyces）的相对丰度与圈养雌性大熊猫对老年雄性尿液气味的探究频次（r = -0.6848, P = 0.014, spearman）和探究时长（r = -0.5816, P = 0.0473, spearman）呈显著强负相关。

综上所述，理论上雌性个体竞争优质配偶的成本和收益取决于其自身条件，自身条件良好的雌性选择配偶会更具挑剔性。益生菌驱动的肠道菌群变化能够改善圈养雌性大熊猫的肠道健康状况，进而增强其对优质雄性个体的选择偏好。该结果印证了我们对肠道菌群改变雌性配偶选择偏好的猜想。本研究有助于我们更深入地理解大熊猫的配偶选择机制，为大熊猫的繁育和保护工作提供了新的思路，也为进一步研究动物的配偶选择行为提供了新的方向。

配偶选择是动物在选择配偶时对具有优秀特征个体的倾向和偏好，对提高个体的繁殖成功率和适合度有关键作用。已发现肠道菌群变化能够影响果蝇和小鼠等模式动物的配偶选择行为，然而，肠道菌群是否影响大型哺乳动物的配偶选择行为，尚未见报道。大熊猫属季节性发情，且雌雄个体间具有强烈的选择性。圈养环境下，配偶选择偏好的正常表达能够提高雌性大熊猫的繁殖成功率和产仔率，但关于影响其配偶选择的因素和机制还有待研究。因此，本研究的目的是探索能否通过益生菌饲喂改善雌性大熊猫的肠道菌群组成，进而影响其配偶选择偏好。我们预测饲喂益生菌能够改善雌性大熊猫的肠道菌群组成，并提升雌性个体对优质雄性配偶的选择倾向。为验证该猜测，本研究以中国大熊猫保护研究中心的圈养雌性大熊猫为研究对象，使用扩增子测序技术，探索能否通过益生菌饲喂驱动肠道菌群（Gut microbiota, GM）组成和功能的变化。然后，通过气味选择实验，判别GM的改变是否对雌性配偶选择产生影响。主要结果如下：

1. 饲喂益生菌后雌性大熊猫肠道菌群的α和β多样性均未发生显著改变，但肠道菌群的组成和功能发生显著变化。肠道中有益菌（Muribaculaceae、Weissella cibaria）的丰度增加、致病菌或条件致病菌（Enterobacteriaceae、Campylobacter等）的丰度减少。肠道菌群的功能预测分析显示，饲喂益生菌后GM中与DNA修复和氨基酸代谢等重要功能相关的基因丰度显著升高（P < 0.05, t-test）。这表明益生菌能够改善圈养雌性大熊猫的肠道菌群组成和功能，有利于提升个体肠道健康。

2. 针对不同年龄雄性尿液气味的选择实验结果显示，圈养雌性大熊猫在饲喂益生菌后，表现出对壮年雄性尿液气味的显著偏好（P = 0.0095, t-test），同时对老年雄性尿液气味的探究频次（P = 0.0369, t-test）和探究时长（P = 0.0358, t-test）均显著降低。未饲喂益生菌的雌性在前后两次气味选择实验中，均未表现出对不同年龄雄性尿液气味的偏好，对不同年龄雄性尿液气味的探究频次和时长也未发生显著变化（P > 0.05）。因此，益生菌饲喂驱动的肠道菌群变化有助于促进圈养雌性大熊猫对优质雄性个体的配偶选择偏好。

3. 相关性分析结果显示，螺旋体门（Spirochaetota）和金黄杆菌属（Chryseobacterium）的相对丰度与圈养雌性大熊猫对壮年雄性尿液气味的探究频次（r = - 0.6910, p < 0.05, spearman）和探究时长（r = - 0.6609, p < 0.05, spearman）呈显著强负相关；放线菌属（Actinomyces）的相对丰度与圈养雌性大熊猫对老年雄性尿液气味的探究频次（r = -0.6848, P = 0.014, spearman）和探究时长（r = -0.5816, P = 0.0473, spearman）呈显著强负相关。

综上所述，理论上雌性个体竞争优质配偶的成本和收益取决于其自身条件，自身条件良好的雌性选择配偶会更具挑剔性。益生菌驱动的肠道菌群变化能够改善圈养雌性大熊猫的肠道健康状况，进而增强其对优质雄性个体的选择偏好。该结果印证了我们对肠道菌群改变雌性配偶选择偏好的猜想。本研究有助于我们更深入地理解大熊猫的配偶选择机制，为大熊猫的繁育和保护工作提供了新的思路，也为进一步研究动物的配偶选择行为提供了新的方向。

Mate choice refers to the tendency and preference of animal individuals to choose mates with excellent characteristics, which plays a significant role in improving the reproductive success rate and fitness of individuals. Previous studies show that gut microbiota (GM) can influence the mate choice of model animals such as fruit flies and mice, however, it remains unclear whether GM changes can impact mate choice in large mammals. Giant pandas exhibit seasonal estrus and a strong mate choice between male and female individuals. Under captive conditions, the normal expression of mate preference can improve the reproductive success rate and birth rate of female giant pandas, but the factors and mechanisms that affect their mate choice remain to be studied. Therefore, the aim of this study is to explore whether the composition of the gut microbiota of female giant pandas can be improved through artificial probiotic feeding, thereby influencing their mate preference. We hypothesized that probiotic feeding can improve the composition of the gut microbiota of female giant pandas and enhance their preference for high-quality male mates. To test this hypothesis, this study used captive female giant pandas from China Conservation and Research Center for the Giant Panda as subjects to examine whether probiotic feeding could drive changes in the composition and function of the gut microbiota (GM) using amplicon sequencing technology. Subsequently, we determined whether changes in GM could affect females’ mate choice. The main results are as follows:

1. The alpha- and beta- diversity of the gut microbiota of those female pandas did not change significantly before and after probiotic feeding, yet the composition and function of the GM after probiotic feeding changed significantly in comparison with that of before feeding. Specifically, the abundance of beneficial bacteria (Muribaculaceae, Weissella cibaria) increased, while the abundance of pathogenic or opportunistic bacteria (Enterobacteriaceae, Campylobacter, etc.) decreased. Functional prediction analysis of the gut microbiota revealed that the gene abundance related to important functions such as DNA repair and amino acid metabolism in the GM significantly increased after probiotic feeding (P < 0.05, t-test). This indicates that probiotics can improve the composition and function of the gut microbiota of captive female giant pandas, which is beneficial for enhancing individual gut health.

2. The results of the odor preference test of captive female pandas between two males of different age show that female pandas being fed probiotic exhibited a significant more preference for the odor of prime age male urine than that of senior ones (P = 0.0095, t-test). Meanwhile, the frequency (P = 0.0369, t-test) and duration (P = 0.0358, t-test) of exploration for the odor of elder male urine were significantly reduced. In contrast, the females without being fed probiotics did not show a preference for the urine odor of males of different ages in both odor preference experiments, and there were no significant changes in the duration and frequency of exploration for the urine odor of males of different ages (P > 0.05, t-test). Therefore, changes in the gut microbiota driven by probiotic feeding can affect the mate preference of captive female giant pandas.

3. The results of correlation analysis showed that the relative abundances of Spirochaetota and Chryseobacterium were significantly and strongly negatively correlated with the frequency (r = -0.6910, P < 0.05, spearman) and duration (r = -0.6609, P < 0.05, spearman)of exploration for the odor of adult male urine by captive female giant pandas. Additionally the relative abundance of Actinomyces showed a significant and strong negative correlation with both the frequency (r = -0.6848, P = 0.014, spearman) and duration (r = -0.5816, P = 0.0473, spearman) of exploration for the urine odor of older male pandas by captive female giant pandas.

In summary, theoretically, the costs and benefits of female individuals competing for high-quality mates depend on their own conditions, and females with good conditions tend to be more selective in mate choice. Probiotic-driven changes in the gut microbiota can improve the gut health of captive female giant pandas, thereby enhancing their mate preference. This result confirmed our hypothesis that alterations in the gut microbiota can affect female mate preference. This study helps us to gain a deeper understanding of the mate choice mechanisms of giant pandas, providing new insights for their breeding and conservation efforts. It also offers a new direction for further exploring the mate choice behavior of animals.