Leibovitz l 15 medium

C57Bl/6 mice (n = 82, 4 weeks old) were obtained from Charles River Laboratories (USA). The ovaries were removed through small dorsolateral skin incisions and were placed either in Leibovitz L-15 medium (Lonza, Verviers, Belgium) supplemented with 10% Fetal Bovine Serum (FBS; Thermo Fisher Scientific, Gibco, Waltham, Mass., USA) (transport solution for slow freezing (SF)) or in Dulbecco’s phosphate-buffered solution (DPBS; Thermo Fisher Scientific, Gibco, Waltham, Mass., USA) supplemented with 20% FBS (transport solution for vitrification (VT)). Leibovitz L-15 is the conventional medium for the SF procedure, whereas there is no consensus for ovarian tissue VT. Therefore, we used Youm’s VT procedure^{30 (link)}. In order to cryopreserve only the ovary, adjacent tissues taken at the time of ovariectomy were removed under a binocular using a scalpel. The Animal Ethics Committee of the University of Liège approved this study (# 1547) and all experiments were performed in accordance with relevant guidelines and regulations.

Ovarian cortex from sheep was obtained and prepared as described previously [16 (link)]. The Animal Ethics Committee of the University of Liège approved the use of sheep ovarian tissue obtained from the Ovine Research Center (University of Namur). Briefly, after euthanasia, ovaries were transferred and kept at 4°C until processing in Leibovitz L-15 medium (Lonza, Verviers, Belgium, BE12-700 F) supplemented with 10% normal sheep serum (Hormonology Laboratory, Marloie, Belgium). In the laboratory, the medulla was removed and the cortex was cut into strips (5 × 5 × 1 mm) before equilibration during 30 min at 4°C in cryopreservative medium containing Leibovitz L-15 medium supplemented with 10% normal sheep serum, 10% dimethylsulfoxide (1.5 M) (Sigma-Aldrich, Bornem, Belgium) and 0.1 M sucrose.

Five fish cell lines previously described were used in this study: E-11 cell line (a clone of the SSN-1 cell line), GF-1 (grouper fin 1), SAF-1 (Sparus aurata fin 1), DLB-1 (Dicentrarchus labrax brain 1), and SaB-1 (Sparus aurata brain 1).
The NNV strains used in this study were the following: SGWak97 and SJNag93 belonging to the RGNNV and SJNNV genotypes (RGNNV and SJNNV hereafter, respectively) [20 (link)]; and SpSsIAusc160.03, a reassortant RGNNV/SJNNV strain isolated from diseased farmed sole [10 (link)], hereafter RG/SJ). The viruses were propagated in semiconfluent E-11 cells with Leibovitz L-15 medium (Lonza, Basilea, Switzerland) containing 5% fetal bovine serum (FBS, Lonza, Basilea, Switzerland) at 25 °C and stored at −80 °C, as previously described [35 (link)]. Viral strains were titrated in triplicate by the endpoint dilution method on 96-well plates and expressed as 50% tissue culture infective dose (TCID₅₀) according to the method described in [40 (link)].

Tissue samples from neurosurgical operations were divided into three parts (approximately 8 mm³ each) and preserved firstly in one cryotube (Greiner Bio One) for snap-freezing for subsequent protein extraction and stored in a −80 degree freezer. Another cryotube was similarly stored for subsequent RNA extraction. The third sample was placed in Leibovitz L-15 Medium (Lonza, Inc) and transported on ice.

The Animal Ethics Committee of the University of Namur approved the use of sheep ovarian tissue. Eight ovaries harvested from four ewes (5 ½, 7, 6 and 9 months old) were obtained from the Ovine Research Center (University of Namur). After euthanasia, the ovaries were immersed in Leibovitz L-15 medium (Lonza, Verviers, Belgium, BE12-700F) supplemented with 10% normal sheep serum and transported at 4°C to the laboratory within 1 h. For each ovary, the medulla was removed, and the cortex was cut into small pieces (2.5×2.5×1 mm). The cortical fragments were fixed in 4% paraformaldehyde, embedded in paraffin and fully cut into 5-µm-thick serial sections stained with hematoxylin and eosin (H&E) (Tribune Stainer HCS 33).

For all experiments, bilateral oophorectomy was performed on the mice under gas anesthesia (Isoflurane, Dechra, Northwich, UK), and the ovaries were placed in Leibovitz L-15 medium (Lonza, Verviers, Belgium) supplemented with 10% Fetal Bovine Serum (FBS; Thermo Fisher Scientific, Gibco, Waltham, MA, USA), a transport solution. The oviduct and fat tissue taken during oophorectomy were removed from the ovaries under a binocular microscope using a scalpel. Fresh ovaries were then directly autotransplanted, and ovaries that were going to be slow-frozen were prepared accordingly.

The Atlantic salmon kidney cell line (ASK) [58 (link)] were kindly provided by Knut Falk (Norwegian Veterinary Institute). Cells were routinely split once a week (1:2) and routinely cultivated at 20°C in Leibovitz L-15 medium supplemented with 4 mM L-glutamine (both reagents from Lonza Biowhittaker, Verviers, Belgium), 10% fetal bovine serum, 40 μM 2-mercaptoethanol (both from Gibco, Life Technologies, Bleiswijk, The Netherlands) and gentamicin (50 μg/ml—Lonza Biowhittaker, Walkersville, USA). Cells were placed at 15°C one week before the experiment started, for acclimatization, and for all the duration of the experimental period. No experimental animals were used in this study.