Derivation of Canine Induced Pluripotent Stem Cells

Dogs and humans have many inherited genetic diseases in common and conditions that are increasingly prevalent in humans also occur naturally in dogs. The use of dogs for the experimental and clinical testing of stem cell and regenerative medicine products would benefit canine health and welfare and provide relevant animal models for the translation of therapies to the human field. Induced pluripotent stem cells (iPSCs) have the capacity to turn into all cells of the body and therefore have the potential to provide cells for therapeutic use and for disease modelling. The objective of this study was to derive and characterize iPSCs from karyotypically abnormal adult canine cells. Aneuploid adipose‐derived mesenchymal stromal cells (AdMSCs) from an adult female Weimeraner were re‐programmed into iPSCs via overexpression of four human pluripotency factors (Oct 4, Sox2, Klf4 and c‐myc) using retroviral vectors. The iPSCs showed similarity to human ESCs with regard to morphology, pluripotency marker expression and the ability to differentiate into derivatives of all three germ layers in vitro (endoderm, ectoderm and mesoderm). The iPSCs also demonstrated silencing of the viral transgenes and re‐activation of the silent X chromosome, suggesting full reprogramming had occurred. The levels of aneuploidy observed in the AdMSCs were maintained in the iPSCs. This finding demonstrates the potential for generating canine induced pluripotent stem cells for use as disease models in addition to regenerative medicine and pharmaceutical testing.     

Effect of Roscovitine‐Treated Donor Cells on Development of Porcine Cloned Embryos

Synchronization of the donor cell cycle is an important factor for successful animal cloning by nuclear transfer. To improve the efficiency of porcine cloning, in the present report, we evaluated effects of contact inhibition, serum starvation and roscovitine treatment of donor cells on in vitro and in vivo developmental potency of cloned porcine embryos. Fibroblasts derived from a porcine foetus at day 30 of gestation were isolated and cultured to 70% confluency. Then, cells were either cultured to 100% confluency for contact inhibition, or cultured in 0.5% serum for 72 h for serum starvation or with 15 μm roscovitine for 24 h. Cells were most effectively synchronized at G0/G1 in the serum starvation group (87.5%) compared with the contact inhibition and roscovitine treatment groups (76.3% and 79.9% respectively p < 0.05). However, after somatic cell nuclear transfer followed by in vitro culture, the serum starvation group showed a significantly lower blastocyst formation rate (5.6%) compared with the contact inhibition and roscovitine treatment groups (11.6% and 20.0% respectively). Differential expression of apoptosis‐related genes and the level of apoptosis in each treatment group explain the variation in developmental competence among the groups. Significantly higher level of apoptosis was observed in the serum starvation group. On the other hand, the roscovitine treatment group shows the lowest level of apoptosis and the best in vitro development among the groups. Cloned embryos derived from roscovitine‐treated donor cells were transferred to surrogate pigs. Three healthy live piglets were produced. In conclusion, we suggest that roscovitine treatment of donor cells improves development of cloned porcine embryos and can raise the efficiency of cloned piglet production.     

Clinical,Ultrasonographic and Pathological Findings in a Bull with Segmental Aplasia of the Mesonephric Duct

On assessment for use in an AI stud, a 12‐month‐old bull was found to produce low volume ejaculates with 41% of the sperm having morphological abnormalities. No left epididymal tail was palpable and the head of the epididymis on the left was twice the size compared with the right. Ultrasound examination showed the left testis to contain a large central area of decreased echogenicity, which could be followed proximally to a 15‐mm echolucent lesion at the site of the epididymal head. Postmortem examination revealed a 15‐mm diameter cyst in the region of the left epididymal head, and absence of the body and tail of the epididymis. The mediastinum testis of the left testis was dilated, corresponding to the area of decreased echogenicity observed on ultrasonography. No left seminal vesicle was present and the ampulla was significantly smaller than the same structure on the right. Histological examination revealed incomplete or absent spermatogenesis involving the majority of seminiferous tubules in the left testis, and a small proportion of those of the right testis. The cystic structure at the site of the left epididymal head was lined by irregular, sometimes attenuated, epithelium and contained sparse spermatozoa. This case demonstrates the adverse impact, which segmental aplasia of the mesonephric duct had on the testicular and epididymal function of a bull, and highlights the importance of careful clinical assessment in its diagnosis.     

The Effect of Pasture Nitrate Concentration and Concentrate Intake after Turnout on Embryo Growth and Viability in the Lactating Dairy Cow

This study investigated the effects on embryo growth and survival rate of feeding heavily‐fertilised spring grass, containing high levels of quickly‐degradable nitrogen, to pregnant cows. Forty‐eight lactating Holstein cows between 2 and 8 weeks pregnant were turned‐out, after a one‐week transition period onto high‐ or low‐nitrate pasture and fed a high‐ or low‐concentrate supplement. Cows grazing the High nitrate pasture had significantly higher milk and plasma urea concentrations than cows grazing the Control pasture, while cows which were fed less concentrate had a notably higher plasma ammonia. However, there was no evidence that an increased quickly‐degradable nitrogen (QDN) intake from pasture affected embryo survival or growth from 20 days onwards. This suggests that the impact of turnout on fertility mainly affects ovulation, fertilisation and/or the early embryo.     

A Review of Methods Used in the Laboratory Diagnosis of Fireblight1)

Most well–known laboratory methods which can be used in the diagnosis of fireblight, as well as more recent developments in identification, have been discussed. They vary from the use of simple culture media to the use of immunofluorescent microscopy. The use of any one method alone is not advised, but by combining methods, accurate and rapid diagnosis is possible. In order to minimize diagnostic errors, especially among less experienced workers, the use of 5 % sucrose–nutrient agar for bacterial isolation followed by serological control is recommended. For further information reference is given particularly to the procedures described by Lelliott at the first EPPO Conference on Fireblight held in 1967 at Canterbury.     

我国兽用生物制品质量现状及改进措施

总结了近年来我国兽用生物制品质量监督抽检结果,对产品质量现状进行了分析,并就如何提高兽用生物制品质量提出了应对措施。     

Monitoring the fate of autologous and allogeneic mesenchymal progenitor cells injected into the superficial digital flexor tendon of horses: preliminary study

Autologous mesenchymal progenitor cells (MPCs) purified from bone marrow aspirates are being used in the treatment of superficial digital flexor tendon (SDFT) injuries in the horse with promising results. In this study the fate of autologous and allogeneic MPCs following injection into the SDFT was monitored by stable transfection of MPCs with green fluorescent protein (GFP). Small lesions were created manually in one forelimb SDFT of 2 horses and injected with autologous MPCs, allogeneic MPCs or bone marrow supernatant alone. Post mortem examinations performed after 10 or 34 days revealed GFP labelled cells located mainly within injected lesions, but with a small proportion integrated into the crimp pattern of adjacent healthy areas of tendon. Furthermore, there was no visible cell mediated immune response to allogeneic MPCs in either of the host horses.