Vitelline Membrane

Healthy, Salmonella-free, 26-week-old Shaoxing ducks were collected from the National Waterfowl Conservation Farm (Taizhou, Jiangsu, China). Separation and cultivation of dGCs was performed as previously described (Gilbert et al., 1977 (link)). Briefly, 10 to 15 adult prehierarchical follicles (small yellow follicles), which have thicker granulosa cell layer (Yang et al., 2019 (link)) of egg-laying ducks, were collected under aseptic conditions and rinsed with Ca²⁺- and Mg²⁺-free phosphate-buffered saline (PBS) to remove the yolks and vitelline membrane as fully as possible. Thereafter, the tissue was cut into 1–2 mm³-sized blocks, digested with 1 mg/ml collagenase (Type II; Sigma Chemical Company, St. Louis, MO, USA) at 37 °C for 5 min, and filtration through a 200-µm nylon filter. Filtered suspensions were centrifuged twice at 67 × g for 5 min. Pellets were washed with M199 media to remove the remaining collagenase and cell debris, resuspended in three mL 50% Percoll, and centrifuged at 421 × g for 15 min, after which the cell layer was aspirated. Granulosa cell suspensions were prepared by adding a pre-configured M199 media (5% fetal calf serum, two mmol/L L-glutamine, five µg/mL transferrin, 10 µg/mL insulin, 1.75 mM HEPES) and counted following staining with 0.1% trypan blue. Suspensions with a cell survival rate greater than 90% were used for experiments. Cells were used for follow-up experiments in disposable culture flasks after 24 h when the completely adherent. Cell purity was initially determined by hematoxylin and eosin (H&E) staining.

One day after injection of 5 ng Cx26 or Cx30 cRNA, oocytes were stripped off their vitelline membrane and paired with each other in agar wells. The next day, transjunctional conductances were measured by a dual-cell voltage clamp [21 (link)] before each ATP transfer experiment. Initial conductances were required to be between 5 and 50 μS so as to ensure a significant signal above the background while avoiding the possibility that cytoplasmic bridges could have formed between the oocytes.
Immediately following conductance measurements, one oocyte is injected with 32 nL of 1.25 mCi/mL ³⁵S-labeled ATP-γ-S (Perkin-Elmer). Precisely 60 min after injection, the acceptor and donor cells are separated by micro-dissection under a Stereo 10X microscope. The acceptor and donor cells are immediately removed separately from the agar well and lysed with a 0.1% SDS buffer, and each is placed in 20 mL scintillation vials (Research Products International, Mount Prospect, IL, USA). A total of 10 mL of UniverSol scintillation cocktail is added (MP Biomedical), and the vials are evenly shaken prior to scintillation counting using a Beckman Coulter LS6500 scintillation counter (Beckman Coulter Life Sciences, Indianapolis, IN, USA). Quantitative measurements of Alexa transfer through both Cx30 and 26 channels had been previously performed [22 (link)].

All egg quality measurements were conducted by laboratory personnel who were trained to operate all egg quality equipment. All egg quality parameters were performed at 27, 35, 51, 63, 75, and 87 weeks of age unless otherwise noted. Egg quality was conducted using six eggs per replicate. The quality parameters measured were shell strength, shell elasticity, vitelline membrane elasticity, vitelline membrane hardness, egg weight, albumen height, Haugh unit (HU), yolk color, egg solid percentage, and shell color. Shell strength and shell elasticity were determined using a texture analyzer (TA-HDplus) with a 250-load cell measuring in grams of force. Vitelline membrane strength was determined using the TA.XTplus Texture Analyzer (Stable Micro Systems, Surrey, UK) with a 1 mm blunt probe with a 5 kg load cell per the manufacturer’s instructions. Haugh unit and albumen height were analyzed using the TSS QCD System (Technical Services and Supplies, Dunnington, York, UK) [47 ]. HU was calculated using the following equation [47 ]:
Yolk color was also determined using the TSS QCD System yolk color scan. Yolk color scan was calibrated using the DSM Yolk Color Fan that determines the color density from lightest to darkest with a range of 1–15 [48 (link)]. Shell color was determined using refractometry of black, blue, and red wavelengths combined to provide a reflectance score from 83.3% (white) to 0% (black). Whole-egg solid analysis was performed only at weeks 35, 63, and 75. All six eggs from each cage were combined and mixed in a stomacher for 30 s. While eggs were being mixed, a metal pan was weighed and recorded. After mixing, the metal pan was filled with egg mixture, weighed, recorded, and placed in a drying oven until dry at 50 °C. The dry matter in the pans was taken out, weighed, and recorded. The solid percentage was calculated using the following formula:
where dw is the dry sample and pan weight, pw is the pan weight without sample, and ps is the pan and liquid sample weight.