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The CRISPR/Cas9 genome editing tool has the potential to improve the livestock breeding industry by allowing for the introduction of desirable traits. Although an efficient and targeted tool, the CRISPR/Cas9 system can have some drawbacks, including off-target mutations and mosaicism, particularly when used in developing embryos. Here, we introduced genome editing reagents into single-cell bovine embryos to compare the effect of Cas9 mRNA and protein on the mutation efficiency, level of mosaicism, and evaluate potential off-target mutations utilizing next generation sequencing. We designed guide-RNAs targeting three loci (POLLED, H11, and ZFX) in the bovine genome and saw a significantly higher rate of mutation in embryos injected with Cas9 protein (84.2%) vs. Cas9 mRNA (68.5%). In addition, the level of mosaicism was higher in embryos injected with Cas9 mRNA (100%) compared to those injected with Cas9 protein (94.2%), with little to no unintended off-target mutations detected. This study demonstrated that the use of gRNA/Cas9 ribonucleoprotein complex resulted in a high editing efficiency at three different loci in bovine embryos and decreased levels of mosaicism relative to Cas9 mRNA. Additional optimization will be required to further reduce mosaicism to levels that make single-step embryo editing in cattle commercially feasible.

Simple Summary: In the abdominal testes of most mammals, with the exception of elephants and a few animal species, spermatogenesis is characterised by an arrest of spermatogenesis due to heat stress. However, the mechanisms underlying normal spermatogenesis in the elephant abdominal testis remain unknown. We hypothesised that comparing the expression of heat stress tolerance molecules of elephant testes with that of other animals and investigating the mechanism of spermatogenesis in elephants might provide clues to overcome the causes of heat stress-induced spermatogenesis dysfunction. The results showed that the elephant's body cells, other than the testes, have a similar mechanism to those of other mammals but that the spermatogenic cells have a different mechanism, with different molecular immunoexpression under heat stress in the abdomen. These findings suggest that these molecules and their relationships may be important in future investigations of the mechanisms that overcome heat stress to maintain spermatogenesis in elephants. The abdominal testes of Asian elephants show normal spermatogenesis. Heat shock in cryptorchid testes elevates heat shock factor (HSF) expression, leading to germ cell apoptosis, while increased heat shock proteins (HSPs) levels provide protection. To investigate how heat shock affects elephant spermatogenic cells, focusing on heat shock-related molecules and the cell death mechanism, immunohistochemistry and TUNEL staining were employed to assess the immunoexpression of several heat shock-related molecules and the status of apoptosis in elephant fibroblasts (EF) induced by heat shock stimulus. Additionally, the immunoexpression of heat shock-related molecules and cell proliferation status in the elephant spermatogenic cells. Our finding indicated that heat shock-induced HSF1 immunoexpression in EF leads to apoptosis mediated by T-cell death-associated gene 51 (TDAG51) while also upregulating HSP70 to protect damaged cells. In elephant spermatogenic cells, immunostaining revealed a predominance of proliferating cell nuclear antigen (PCNA)-positive cells with minimal TDAG51- and TUNEL-positive cells, suggesting active proliferation and apoptosis suppression during normal spermatogenesis in the abdominal testis. Interestingly, spermatogonia co-immunoexpressed HSF1 and HSP90, potentially reducing apoptosis through protective mechanisms different from those observed in other mammals. Spermatogenic cells did not show immunolocalisation of HSP70, and hence, it may not contribute to protecting the spermatogonia from heat shock because the transcriptional activity of HSF1 is suppressed by HSP90A binding. This study provides insight into the specific heat shock response and defence mechanisms in elephant spermatogenic cells and may contribute to our understanding of species-specific adaptation to environmental stresses of the testis. [ABSTRACT FROM AUTHOR]

We evaluated the quality and fertilizing ability of frozen–thawed porcine sperm that were selected using a commercially available device (MIGLIS, Menicon Life Science) consisting of three parts: an outer lid, an inner lid, and a tube. Firstly, to determine an adequate concentration of caffeine for separation, frozen–thawed sperm were incubated with different concentrations of caffeine (0, 1, 2.5, 5, and 10 mM) in a MIGLIS device. To determine the appropriate incubation time for separating sperm in the MIGLIS device, frozen–thawed sperm were incubated with 2.5 mM caffeine for 5, 10, 15, or 20 min. To evaluate the fertilization and embryo development of oocytes fertilized with frozen–thawed sperm separated into two regions (outer and inner) in the MIGLIS device, the separated sperm from the three boars was used to fertilize in vitro‐matured oocytes and cultured in vitro for 7 days. Sperm quality parameters of sperm collected from the inner tube after incubation with 2.5 mM caffeine were superior to sperm incubated without caffeine. Moreover, sperm collected from the inner tube after incubation for 10 min had a higher progressive motility. The rate of blastocyst produced from spermatozoa collected from the inner tube after incubation with 2.5 mM caffeine for 10 min significantly increased compared to that produced from spermatozoa from the outer tube, regardless of the boar. In conclusion, sperm sorting using the MIGLIS device may be useful for separating high‐quality sperm after incubation with 2.5 mM caffeine for 10 min to improve blastocyst formation. [ABSTRACT FROM AUTHOR]

Background: Porcine tissues display a great potential as donor tissues in xenotransplantation, including cell therapy. Cryopreserving clinical grade porcine tissue and using it as a source for establishing therapeutic cells should be advantageous for transportation and scheduled manufacturing of MSCs. Of note, we previously performed encapsulated porcine islet transplantation for the treatment of unstable type 1 diabetes mellitus in the clinical setting. It has been reported that co‐transplantation of islets and Mesenchymal stem cells (MSCs) enhanced efficacy. We assume that co‐transplantation of porcine islets and porcine islet‐derived MSCs could improve the efficacy of clinical islet xenotransplantation. Methods: MSCs were established from fresh and cryopreserved non‐clinical grade neonatal porcine islets and bone marrow (termed non‐clinical grade npISLET‐MSCs and npBM‐MSCs, respectively), as well as from cryopreserved clinical grade neonatal porcine islets (termed clinical grade npISLET‐MSCs). Subsequently, the cell proliferation rate and diameter, surface marker expression, adipogenesis, osteogenesis, and colony‐forming efficiency of the MSCs were assessed. Results: Cell proliferation rate and diameter did not differ between clinical grade and non‐clinical grade npISLET‐MSCs. However, non‐clinical grade npBM‐MSCs were significantly shorter and smaller than both npISLET‐MSCs (p < 0.05). MSC markers (CD29, CD44, and CD90) were strongly expressed in clinical grade npISLET‐MSCs and non‐clinical grade npISLET‐MSCs and npBM‐MSCs. The expression of MSC‐negative markers CD31, CD34, and SLA‐DR was low in all MSCs. Clinical grade npISLET‐MSCs derived from adipose and osteoid tissues were positive for Oil Red and alkaline phosphatase staining. The results of colony‐forming assay were not significantly different between clinical grade npISLET‐MSCs and non‐clinical grade npBM‐MSCs. Conclusion: The method described herein was successful in of developing clinical grade npISLET‐MSCs from cryopreserved islets. Cryopreserved clinical grade porcine islets could be an excellent stable source of MSCs for cell therapy. [ABSTRACT FROM AUTHOR]

Sterilization of the culture medium using ultraviolet (UV)‐C reduces the potential adverse effects of microorganisms and allows for long‐term use. In the present study, we investigated the effects of a medium directly irradiated with UV‐C prior to in vitro culture on the development and quality of porcine in vitro‐fertilized embryos and the free amino acid composition of the culture media. The culture media (porcine zygote medium [PZM‐5] and porcine blastocyst medium [PBM]) were irradiated with UV‐C at 228 and 260 nm for 1 and 3 days, respectively. Next, the culture media were irradiated with UV‐C at 228 nm for 3, 7, or 14 days. After in vitro fertilization, the embryos were cultured in the UV‐C‐irradiated media for 7 days. Free amino acid levels in culture media irradiated with 228 and 260 nm UV‐C for 3 days were analysed. The blastocyst formation rate of embryos cultured in media irradiated with 260 nm UV‐C for 3 days was significantly lower than that of embryos cultured in non‐irradiated control media. However, 228 nm UV‐C irradiation for up to 14 days did not affect blastocyst formation rates and quality in the resulting blastocysts. Moreover, 260 nm UV‐C irradiation significantly increased the taurine concentration in both culture media and decreased methionine concentration in the PBM. In conclusion, UV‐C irradiation at 228 nm before in vitro culture had no detrimental effects on embryonic development. However, 260 nm UV‐C irradiation decreased embryo development and altered the composition of free amino acids in the medium. [ABSTRACT FROM AUTHOR]

This study examined the effects of ergothioneine (EGT) supplementation as an antioxidant on the quality of boar spermatozoa when using liquid and frozen preservation methods. In the first experiment, boar semen was preserved in an extender supplemented with 0, 50, 100 and 200 µM EGT, at 15 °C, part of the samples for one and another part for three weeks. In comparison with the control (without EGT), EGT supplementation at 100 µM significantly increased the percentage of total motility of spermatozoa that were preserved as a liquid both for one and three weeks (P < 0.05). EGT supplementation did not affect the quality of preserved spermatozoa, irrespective of the EGT concentration. In the second experiment, semen was frozen and thawed in the freezing extender supplemented with 0, 50, 100 and 200 µM EGT. In comparison with the control, the 100 µM EGT supplementation significantly increased the percentages of total and progressive motility of frozen-thawed spermatozoa (P < 0.05). EGT (100 µM) supplementation did not affect the viability, the plasma membrane integrity, or the acrosomal integrity of frozen-thawed spermatozoa. These findings indicate that supplementing extenders with 100 µM EGT may improve the motility of boar sperm in both liquid and freezing preservation methods. [ABSTRACT FROM AUTHOR]