Culture of Kenyan Goat (Capra hircus) Undifferentiated Spermatogonia in Feeder-Free Conditions.

The undifferentiated spermatogonial population in mammalian testes contains a spermatogonial stem cell (SSC) population that can regenerate continual spermatogenesis following transplantation. This capacity has the potential to be exploited as a surrogate sires breeding tool to achieve widespread dissemination of desirable genetics in livestock production. Because SSCs are relatively rare in testicular tissue, the ability to expand a population in vitro would be advantageous to provide large numbers for transplantation into surrogate recipient males. Here, we evaluated conditions that would support long-term in-vitro maintenance of undifferentiated spermatogonia from a goat breed that is endemic to Kenyan livestock production. Single-cell suspensions enriched for undifferentiated spermatogonia from pre-pubertal bucks were seeded on laminin-coated tissue culture plates and maintained in a commercial media based on serum-free composition. The serum-free media was conditioned on goat fetal fibroblasts and supplemented with a growth factor cocktail that included glial cell line-derived neurotrophic factor (GDNF), leukemia inhibitory factor (LIF), stromal cell-derived factor (SDF), and fibroblast growth factor (FGF) before use. Over 45 days, the primary cultures developed a cluster morphology indicative of in-vitro grown undifferentiated spermatogonia from other species and expressed the germ cell marker VASA, as well as the previously defined spermatogonial marker such as promyelocytic leukemia zinc finger (PLZF). Taken together, these findings provide a methodology for isolating the SSC containing undifferentiated spermatogonial population from goat testes and long-term maintenance in defined culture conditions.

Comparative Efficiency for in vitro Transfection of Goat Undifferentiated Spermatogonia Using Lipofectamine Reagents and Electroporation.

Introduction: Spermatogonial stem cells (SSC), also referred to as undifferentiated spermatogonia, are the germline stem cells responsible for continuous spermatogenesis throughout a male's life. They are, therefore, an ideal target for gene editing. Previously, SSC from animal testis have been isolated and transplanted to homologous recipients resulting in the successful reestablishment of donor-derived spermatogenesis. Methods: Enhanced green fluorescent protein (eGFP) gene transfection into goat SSC was evaluated using liposomal carriers and electroporation. The cells were isolated from the prepubertal Galla goats testis cultured in serum-free defined media and transfected with the eGFP gene. Green fluorescing of SSC colonies indicated transfection. Results: The use of lipofectamineTM stem reagent and lipofectamineTM 2000 carriers resulted in more SSC colonies expressing the eGFP gene (25.25% and 22.25%, respectively). Electroporation resulted in 15% ± 0.54 eGFP expressing SSC colonies. Furthermore, cell viability was higher in lipofectamine transfection (55% ± 0.21) as compared to electroporation (38% ± 0.14). Conclusion: These results indicated that lipofectamine was more effective in eGFP gene transfer into SSC. The successful transient transfection points to a possibility of transfecting transgenes into male germ cells in genetic engineering programs.

Culture of spermatogonial stem cells and use of surrogate sires as a breeding technology to propagate superior genetics in livestock production: A systematic review.

BACKGROUND AND AIM: Spermatogonial stem cells (SSCs) have previously been isolated from animals' testes, cultured in vitro, and successfully transplanted into compatible recipients. The SSC unique characteristic has potential for exploitation as a reproductive tool and this can be achieved through SSC intratesticular transplantation to surrogate sires. Here, we aimed at comprehensively analyzing published data on in vitro maintenance of SSC isolated from the testes of livestock animals and their applications. MATERIALS AND METHODS: The literature search was performed in PubMed, Science Direct, and Google Scholar electronic databases. Data screening was conducted using Rayyan Intelligent Systematic Review software (https://www.rayyan.ai/). Duplicate papers were excluded from the study. Abstracts were read and relevant full papers were reviewed for data extraction. RESULTS: From a total of 4786 full papers screened, data were extracted from 93 relevant papers. Of these, eight papers reported on long-term culture conditions (>1 month) for SSC in different livestock species, 22 papers on short-term cultures (5-15 days), 10 papers on transfection protocols, 18 papers on transplantation using different methods of preparation of livestock recipients, and five papers on donor-derived spermatogenesis. CONCLUSION: Optimization of SSC long-term culture systems has renewed the possibilities of utilization of these cells in gene-editing technologies to develop transgenic animals. Further, the development of genetically deficient recipients in the endogenous germline layer lends to a future possibility for the utilization of germ cell transplantation in livestock systems.

Morphometric and stereological methods for quantifying the coarse structural parameters of the ruminal tissues in sheep.

In ruminants, the rumen is the largest and most significant fore-stomach. Stereological analysis of important structural parameters that may be used to assess the functional capacity of the rumen is lacking. In the current investigation, five rams were used to demonstrate the methods for quantifying salient structural parameters related to rumen function. The sheep were euthanized with 20% sodium pentobarbital intravenously, the rumen was dissected out and divided into the various sacs for gross examination, and fixation by total immersion in 10% formalin. Macroscopic ruminal surface area was estimated using the point-associated area method. Volumes of the ruminal tissues were estimated by the volume displacement method, while volume densities of the components of the ruminal wall were estimated by point counting methods. Tissue blocks for histology were obtained by systematic random sampling and processed to obtain vertical sections for surface area and volume estimations. Papillary densities and numbers were estimated from horizontal sections. The volume of ruminal tissue was 536.54 ± 80.52 cm3, the macroscopic surface area was 1091 ± 115.75 cm2 with a papillary packing density of 84.64 ± 10.99 cm-2. Average absolute surface area was 4726.74 ± 628.56 cm2. The total number of ruminal papillae per rumen was 92,884.91 ± 6216.46. The methods documented here provide the possibility of doing a detailed stereological analysis of ruminal tissue in different experimental or even pathological conditions.