Ma et al. BMC Veterinary Research (2017) 13:344 DOI 10.1186/s12917-017-1231-0 RESEARCHARTICLE Open Access Isolation and identification of culturable fungi from the genitals and semen of healthy giant pandas (Ailuropoda melanoleuca) Xiaoping Ma1,2†, Changcheng Li2†, Jiafa Hou1* and Yu Gu3* Abstract Background: In order to better understand the possible role of fungi in giant panda reproduction and overall health, it is important to provide a baseline for the normal fungal composition in the reproductive system. Using morphology and internal transcribed spacer (ITS) sequence analysis, we systematically isolated and identified fungal species from the vagina, foreskin, and semen of 21 (11 males and 10 females) healthy giant pandas to understand the normal fungal flora of the genital tracts. Results: A total of 76 fungal strains were obtained, representing 42 genera and 60 species. Among them 47 fungal strains were obtained from vaginal samples, 24 from foreskins, and 5 from semen samples. Several fungal strains were isolated from more than one sample. More fungal species were isolated from females from males. The predominant genera were Aspergillus, Trichosporon,andPenicillium,followedby Candida, Cladosporium, Sordariomycetes,andDiaporthe. The average number of strains in the female vagina was significantly higher than in the foreskin and semen of male. Conclusions: A total of 60 fungal species (belonging to 42 genera) were identified in the giant panda’s genital tract. Some of the species were commonly shared in both males and females. These findings provide novel information on the fungal community in the reproductive tracts of giant pandas. Keywords: Culturable fungi, Giant panda (Ailuropoda melanoleuca), Vagina, Foreskin, Semen Background Microorganisms are responsible for diseases that can dir- The giant panda is one of the rarest endangered ani- ectly or indirectly affect reproductive success in animals. mals [1]. In recent years, breeding giant pandas in Fungi are one of the most important microorganisms that captivity has become necessary for conservation and are known to cause abortion in cattle [3] and horses [4]. improving their reproductive rate. Although the cap- Fungal infections are becoming more frequent in tive giant panda population has increased, its genetic humans due to the widespread use of antibiotics and diversity is far lower than that of the wild population immunosuppressors [5]. In 1929, a study confirmed [2], and some captive female giant pandas are unable the existence of fungi in the human vagina [6]. to carry cubs to term or are subject to secondary Torulopsis and Candida spp. were later identified infertility. from vaginal samples [7]. Studies have confirmed the presence of fungi in the reproductive organs of animals [8–10]. To date, few * Correspondence: [email protected]; [email protected] studies have been performed on microorganisms of †Equal contributors the genitourinary tract of the giant panda [11], and 1 College of Veterinary Medicine, Nanjing Agricultural University, Nanjing fungal infections have been identified as a potential 210095, China 3College of Life Sciences, Sichuan Agricultural University, Ya’an 625014, China threat to giant panda fertility [12]. Low breeding rates Full list of author information is available at the end of the article © The Author(s). 2017 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Ma et al. BMC Veterinary Research (2017) 13:344 Page 2 of 9 strongly affect the population of giant pandas. There is a None of the animals had previous genital infections noted close relationship between sterility and vaginal dysbiosis, in their files, and detailed physical examinations found no which is characterized by the imbalance of vaginal micro- evidence of vaginitis or balanoposthitis. Pandas with a flora due to various disorders and conditions. This high- recent history of disease or animals treated with antifungal lights the importance of an improved understanding of drugs during the last 6 months were excluded from this the normal vaginal flora of giant pandas. study. In this study, we investigated the fungal compositions in the vaginas, foreskins, and semen of 21 healthy giant Sampling procedure pandas living in a semi-captive, semi-closed breeding en- Samples were collected during estrus when the female vironment. Our goal is to provide a baseline information pandas were ready for artificial insemination from on the normal fungal flora in the reproductive system to February to May of 2013. All personnel involved in better understand the possible roles of fungi in the sampling wore sterile protective clothing, hats, masks, reproduction and overall health of giant pandas. and latex gloves. Ketamine was used for anesthesia in giant pandas, and isoflurane was administered via Methods anesthetic machine to maintain anesthesia. Samples Animals were collected from either the bottoms of vaginas The giant pandas used in this study lived in a semi- (cervical orifice) or foreskins with sterilized cotton captive, semi-closed breeding environment at the swabs when the pandas were supine after anesthesia. Chengdu Research Base of Giant Panda Breeding The vulvas, foreskins, and surrounding areas were (Sichuan, China). The pandas were fed bamboo shoots rinsed three times with sterilized, warm physiological and steamed corn bread. They were allowed to drink saline solution, then wiped with a disposable sterile water ad libitum. towel. Samples were collected using guarded swabs by Samples were collected from 10 female during estrus and inserting a sterile cervical canal into the cervical. 11 male pandas. Details of the samples are listed in Table 1. Sterile cotton swabs were then inserted to the vagina Table 1 List of samples taken from giant pandas Name Sex Sample Age Sample date Note A female vaginal secretion 11 yr. 5mo. 2013.4 Pre-procreated B female vaginal secretion 5 yr. 2013.3 First artificial breeding C female vaginal secretion 5 yr. 7mo. 2013.2 First artificial breeding D female vaginal secretion 12 yr. 2013.4 First artificial breeding E female vaginal secretion 6 yr. 9mo. 2013.5 First artificial breeding F female vaginal secretion 7 yr. 8mo. 2013.3 First artificial breeding G female vaginal secretion 5 yr. 7mo. 2013.4 First artificial breeding H female vaginal secretion – 2013.3 First artificial breeding I female vaginal secretion 18 yr. 8mo. 2013.2 Not pregnant for 5 years J female vaginal secretion 10 yr. 7mo. 2013.4 Not pregnant for 2 years K male prepuce inclusions 10 yr. 3mo. 2013.3 L male prepuce inclusions 14 yr. 7mo. 2013.4 M male prepuce inclusions 10 yr. 7mo. 2013.5 N male prepuce inclusions 10 yr. 7mo. 2013.4 O male prepuce inclusions 10 yr. 6mo. 2013.4 and semen P male semen 8 yr. 6mo. 2013.5 Q male prepuce inclusions 7-10 yr 2013.3 R male prepuce inclusions 8 yr 2013.4 S male prepuce inclusions 7 yr.8mo 2013.2 T male prepuce inclusions 8 yr. 2013.4 U male prepuce inclusions and semen 8 yr. 7mo 2013.4 Note: Because panda H was captured in the wild, its age is not known Ma et al. BMC Veterinary Research (2017) 13:344 Page 3 of 9 bottoms (cervical orifice) through the cervical canal PCR products (8 μl) were examined using 1.0% agarose to collect the cervix outflow. The semen from three gel electrophoresis containing 0.5 mg/ml of ethidium male giant pandas was collected through electroejacu- bromide. A 300–600 bp fragment of the rDNA was lation after general anesthesia, with sterilized cotton produced. The PCR products were subjected to DNA se- swabs using a sterilized special semen collection cup. quencing performed by Invitrogen (Shanghai Invitrogen Special care was taken to ensure that the swab did Biotechnology Company, Shanghai, China). not come into contact with other parts of the body. Partial gene sequences of the isolates were submitted All samples were quickly placed in sterilized plastic to GenBank Accession and the numbers are listed in sample bags, transported into laboratory on ice within Table 2. A sequence similarity search was performed 2 h, and then immediately inoculated under a BSL-2 using BLAST (https://www.ncbi.nlm.nih.gov/nuccore/). safety cabinet. A total of 23 samples were subjected Isolates were identified on the selection of the most to fungal analysis (Table 1). similar sequences using BLAST. The phylogenetic tree was constructed by Mega 6.0 software with neighbor- Fungal culture and identification joining method. Fungal culture Samples were streak inoculated aerobically onto Sabouraud dextrose agar (MOLTOX, Inc., Boone, NC) Statistical analysis containing 2% (v/v) sterile olive oil, malt extract agar, Data were analysized by IBM SPSS 20.0 software for or yeast extract peptone dextrose agar. All media Windows. The independent-samples test of nonparamet- were supplemented with antibiotics (Chloramphenicol ric tests or one-way ANOVA were used. Differences 0.005% (m/v)). showing p-values less than 0.05 were considered statisti- Cultural examination was carried out in a BSL-2 cally significant. safety cabinet of a bioclean room. Sterilized sealing film was used to cover each plate. Blank plates were used as control to ensure no microorganisms were Results from aerial contamination. Each sample was plated in Morphological identification 3 culture plates with 3 control plates. All culture In this study, a total of 23 samples from vaginas, dishes were inoculated and stored at 25 °C for 7– foreskins, and semen were collected from 21 healthy 30 days before being considered negative.