A New Cell Line from the Brain of Red Hybrid Tilapia (Oreochromis spp.) for Tilapia Lake Virus Propagation.

Tilapia lake virus (TiLV) presents a substantial threat to global tilapia production. Despite the development of numerous cell lines for TiLV isolation and propagation, none have been specifically derived from red hybrid tilapia (Oreochromis spp.). In this study, we successfully established a new cell line, RHTiB, from the red hybrid tilapia brain. RHTiB cells were cultured for 1.5 years through over 50 passages and demonstrated optimal growth at 25 °C in Leibovitz-15 medium supplemented with 10% fetal bovine serum at pH 7.4. Morphologically, RHTiB cells displayed a fibroblast-like appearance, and cytochrome oxidase I gene sequencing confirmed their origin from Oreochromis spp. Mycoplasma contamination testing yielded negative results. The revival rate of the cells post-cryopreservation was observed to be between 75 and 80% after 30 days. Chromosomal analysis at the 25th passage revealed a diploid count of 22 pairs (2n = 44). While no visible cytopathic effects were observed, both immunofluorescence microscopy and RT-qPCR analysis demonstrated successful TiLV propagation in the RHTiB cell line, with a maximum TiLV concentration of 107.82 ± 0.22 viral copies/400 ng cDNA after 9 days of incubation. The establishment of this species-specific cell line represents a valuable advancement in the diagnostic and isolation tools for viral diseases potentially impacting red hybrid tilapia.

Antibody fragments targeting the extracellular domain of follicular stimulating hormone receptor for contraception in male dogs and cats.

The increased LH levels resulting from the absence of negative feedback after castration has been linked to long-term health issues. A need exists for an alternative contraceptive agent that functions without interfering the LH pathways. This study aimed to develop antibody fragments against the follicular-stimulating hormone receptor (anti-FSHr) using phage-display technology and evaluate its effects on Sertoli cell functions. Phage clones against the extracellular domain of dog and cat FSHr selected from an antibody fragment phagemid library were analyzed for binding kinetics by surface plasmon resonance. Sertoli cells were isolated from testes of adult animals (five dogs and five cats). Efficacy test was performed by treating Sertoli cell cultures (SCCs) with anti-FSHr antibody fragments compared with untreated in triplicates. Expressions of androgen binding protein (ABP), inhibin subunit beta B (IHBB) and vascular endothelial growth factor A (VEGFA) mRNA in SCCs were quantified by RT-qPCR. The results demonstrated that the molecular weight of the purified dog and cat anti-FSHr antibody fragment was 25 kDa and 15 kDa, respectively. Based on protein molecular weight, the antibody fragment of dogs and cats was therefore, so-called single-chain variable fragments (scFv) and nanobody (nb), respectively. The binding affinity with dissociation constant (KD) was 2.32 × 10-7 M and 2.83 × 10-9 M for dog and cat anti-FSHr antibody fragments, respectively. The cross-binding kinetic interactions between the dog anti-FSHr scFv and the cat ECD of FSHr could not be fitted to the curves to determine the binding kinetics. However, the cross-binding affinity KD between the cat anti-FSHr nb and the dog ECD FSHr was 1.75 × 10-4 M. The mRNA expression of ABP, IHBB and VEGFA in SCCs was less (P < 0.05) in both dogs (12.26, 4.07 and 5.11 folds, respectively) and cats (39.53, 14.07 and 20.29 folds, respectively) treated with anti-FSHr antibody fragments, indicating the Sertoli cell functions were suppressed. In conclusion, this study demonstrated the establishment of species-specific antibody fragments against FSHr in SCCs for dogs and cats. The fragment proteins illustrate potential to be developed as non-surgical contraceptive agent targeting FSHr in companion animals.

De novo reconstruction of a functional in vivo-like equine endometrium using collagen-based tissue engineering.

To better understand molecular aspects of equine endometrial function, there is a need for advanced in vitro culture systems that more closely imitate the intricate 3-dimensional (3D) in vivo endometrial structure than current techniques. However, development of a 3D in vitro model of this complex tissue is challenging. This study aimed to develop an in vitro 3D endometrial tissue (3D-ET) with an epithelial cell phenotype optimized by treatment with a Rho-associated protein kinase (ROCK) inhibitor. Equine endometrial epithelial (eECs) and mesenchymal stromal (eMSCs) cells were isolated separately, and eECs cultured in various concentrations of Rock inhibitor (0, 5, 10 µmol) in epithelial medium (EC-medium) containing 10% knock-out serum replacement (KSR). The optimal concentration of Rock inhibitor for enhancing eEC proliferation and viability was 10 µM. However, 10 µM Rock inhibitor in the 10% KSR EC-medium was able to maintain mucin1 (Muc1) gene expression for only a short period. In contrast, fetal bovine serum (FBS) was able to maintain Muc1 gene expression for longer culture durations. An in vitro 3D-ET was successfully constructed using a collagen-based scaffold to support the eECs and eMSCs. The 3D-ET closely mimicked in vivo endometrium by displaying gland-like eEC-derived structures positive for the endometrial gland marker, Fork headbox A2 (FOXA2), and by mimicking the 3D morphology of the stromal compartment. In addition, the 3D-ET expressed the secretory protein MUC1 on its glandular epithelial surface and responded to LPS challenge by upregulating the expression of the interleukin-6 (IL6) and prostaglandin F synthase (PGFS) genes (P < 0.01), along with an increase in their secretory products, IL-6 (P < 0.01) and prostaglandin F2alpha (PGF2α) (P < 0.001) respectively. In the future, this culture system can be used to study both normal physiology and pathological processes of the equine endometrium.