Smz800n

Use a 1 ml pipette tip or a notch pipette to transfer organoids into a medium dish, add 50 μl of NIM medium to each organoid to prevent drying. Place virus on ice immediately after removing from −80°C freezer. Place the organoids under a microscope (Nikon SMZ800N). First, use a 4× microscope to find the location of the organoids, adjust the objective magnification according to the size of the organoids, and then use a micro syringe to start the injection. Each organoid was injected twice, 0.5 μl virus each time. After finishing the injection, add 50 μl of virus dilution solution (preparation of virus dilution solution: 500 μl NIM + 1:50 B27 + 1:2,000 Polybrene + 1:100 PS + 1:100 virus stock solution). After incubation for 24 h, the cells were transferred to T12.5 cell culture flasks, supplemented with 3 ml of NIM medium.

We collected 17 species of bird lice; 16 of these species were collected in the Australian Zoo Wildlife Hospital (AZWH) from euthanized birds and one species was from live birds (Australian white ibis, identified by D.P.) captured at the UniSC Fraser Coast campus (Table 1). Euthanized birds were initially identified by AZWH veterinary staff and verified by two of the authors (Y.D. and R.S.) based on morphology according to Pizzey et al. [24 ] and Menkhorst et al. [25 ]. We used the post-mortem-ruffling method to collect bird lice [26 (link)]. Ethyl acetate was used to treat euthanized birds if lice were still alive. Ethanol-acetate-soaked cotton balls were put in sealed bags containing euthanized birds, one bird per bag to avoid contamination. After 5 to 10 min, treated birds were placed on a clean A1-size white paper, with their feathers ruffled to get lice off bird feathers onto the white paper. Lice were kept in 2-mL collection tubes filled with 80% ethanol at −80 °C in a freezer until DNA extraction. Lice were checked under a microscope (Nikon SMZ 800N), imaged, and identified by morphological features and host records according to the world checklist of chewing lice [1 ].

Hatchlings dying in one month were sexed through histological analysis of gonads following Tokunaga [13 (link)]. Gonads were excised from dead hatchlings immediately once found and fixed in Bouin solution. After dehydrated with a serial change in alcohol, the specimens were embedded in paraffin and sliced to 5 μm-thick sections. The slices were stained with hematoxylin–eosin and observed under stereomicroscope (Nikon SMZ800N, Tokyo, Japan). Sexes of hatchings dying in one to two months were determined by observing the shape of gonads stained with hematoxylin–eosin under a stereomicroscope. Hatchings that died in two to four months were sexed in a similar way except staining. Living hatchlings were sexed by everting the hemipenes in males after four months [14 (link)] (Table S1).

The selected COCs were placed in groups of 30 for 10 min. in 500 µL 0.5% LB at RT. Stained COCs were washed three times in mPBS (PBS + 0.4% bovine saline albumin-BSA) and observed under a Nikon SMZ800N stereomicroscope. After washing, the COCs were divided into two qualitative subgroups based on LB staining. We focused on detection of damaged cytoplasmic membrane, which was characterized by green stained ooplasm. Unstained (LB-) COCs with 0% stained cumulus cells were classified as high-quality COCs and stained (LB+) COCs with green ooplasm were designated as COCs with poor quality. In the LB+ group percentage of stained cumulus cells was not considered as an important marker (Figure 1) (Dutta et al., 2016 (link)).

We used whisker HFs for the organ cultures. Whisker HFs of a 4-week-old Rex rabbit was isolated as has been previously described for mice [16 (link)]. Early or mid-anagen growth phase follicles were selected for culture, and the part of the hair shaft that extended over the epidermal surface was cut off. HFs were plated in 24-well plates with one follicle per well at 31°C saturated humidity air temperature with 5% CO₂ and 95% box in the general culture and cultured in one of the following basal media: Williams E medium (GIBCO, U.S.A.), penicillin–streptomycin (Solarbio, China), insulin (GIBCO, U.S.A.), hydrocortisone (Sigma, Germany), and L-glutamine (GIBCO, U.S.A.); basal medium containing AdWnt10b (Hanbio Co. LTD, China) and AdGFP (Hanbio Co. LTD, China) at ultimate titers of 10⁸; or basal medium containing XAV-939 (10 µmol/l)(Sigma, Germany) and AdWnt10b plus XAV-939. After 2 days, every culture was replaced with fresh media, and the length of the outgrowing hair shafts was determined by analyzing the digital images at 5 days of growth with stereo microscope (Nikon SMZ800N, Japan).