Effect of claw blocks on the healing duration and lesion severities of claw lesions in lame cows in Western Thailand.

Background and Aim: Lameness is a major complication in dairy cattle affecting health and milk production. Several factors are found to contribute to this condition and specific treatments are required, including the process of claw trimming. The elevation of the claw, such as with the application of a claw block, was reported to be beneficial in the more severe cases. This study aimed to determine the efficiency of a claw block on claw lesions of lame cows in dairy farms in Western Thailand. Materials and Methods: Locomotion scores of 376 dairy cows were determined by a veterinarian using a scale of 1-5 (1 = normal; 5 = severely lame) at the time of the visit. Cows with a score of 3 or greater were defined as clinically lame. In total, 134 clinically lame cows from 11 dairy farms in the Kanchanaburi and Ratchaburi provinces were included in the analysis. Claw lesions included a white line abscess, bruised sole, sole ulcer, sole abscess, white line separate, and double soles. Wooden or rubber claw blocks were applied to the unaffected claw of the same hoof as the injured claw of 116 cows, which were classified as the treatment cases, and 18 cows were left untreated and classified as the control cases. Each cow was checked on every week of the healing process for 2 months unless the cow was culled earlier. Survival analysis was based on the Kaplan-Meier estimator and Cox Proportional Hazard regression. Results: The median healing time for lame cows with and without claw blocks was 21 and 36 days, respectively. After adjusting for the lesion severity and type, the lame cows with and without a claw block had hazard ratios of 2.16 and 3.08, respectively. The healing times between the four lesion types in cows with a claw block were not significantly different. The healing time was longer in lame cows, with a severity score of 4. Conclusion: The results from this study reveal that the treatment of lame cows with claw blocks promoted the healing capacity of claw lesions after claw trimming.

Discrimination of functional hepatocytes derived from mesenchymal stem cells using FTIR microspectroscopy.

Functional hepatocytes differentiated in vitro from mesenchymal stem cells (MSCs) need to be fully characterized before they could be applied as a therapy to treat liver disease. Here, we employed Fourier Transform Infrared (FTIR) microspectroscopy to investigate the characteristics of hepatocyte-like cells derived from rat bone marrow mesenchymal stem cells (rBM-MSCs) by detecting changes in macromolecular composition occurring during the hepatogenesis process. Partial Least Squares Discriminant Analysis (PLS-DA) enabled us to discriminate undifferentiated rBM-MSCs, and early, mid-stage and late stage rBM-MSCs derived hepatocytes by their characteristic FTIR "spectroscopic signatures". The predominant spectroscopic changes responsible for this discrimination were changes in FTIR absorbance bands at: 3012 cm(-1) (cis C[double bond, length as m-dash]C stretch from unsaturated lipids), 2952 cm(-1) (ν(as)CH(3) from lipids), 2854 cm(-1) (ν(s)CH(2) from lipids) and 1722 cm(-1) (C[double bond, length as m-dash]O stretching from lipids), which were associated with triglyceride and unsaturated fatty acid accumulation in the hepatocyte-like cells occurring during differentiation. Based on these findings, rBM-MSCs derived hepatocytes are characterized by high lipid content which facilitates a means of identifying hepatocytes from their stem cells progenitors by using FTIR microspectroscopy. Other complex changes in spectral bands assigned to proteins and nucleic acids were observed during hepatocyte differentiation indicating that mRNA translation was taking place producing proteins related to the formation of the new hepatocyte-like phenotype, which was corroborated by immunohistochemistry. The results show FTIR microspectroscopy combined with bioinformatic modeling constitutes a powerful new phenotypic-based methodology for monitoring and characterization of the process of stem cell differentiation leading to the formation of hepatocytes, providing complementary information to existing methodologies such as immunohistochemistry and gene analysis, but having advantages of being reagent-free and non-destructive of the sample.

Full-term development of gaur-bovine interspecies somatic cell nuclear transfer embryos: effect of trichostatin A treatment.

Trichostatin A (TSA) has previously been used in somatic cell nuclear transfer (SCNT) to improve the cloning efficiency in several species, which led our team to investigate the effects of TSA on the full-term development of bovine SCNT and gaur-bovine interspecies SCNT (gaur iSCNT; gaur somatic cells as donors and bovine oocytes as recipients) embryos. Treatment with 50 nM TSA for 10 h after fusion had no positive effects on the rates of fusion, cleavage, or the development to eight-cell or morula stages in both bovine SCNT and gaur iSCNT embryos. However, TSA treatment significantly enhanced the blastocyst formation rate in bovine SCNT embryos (44 vs. 32-34% in the TSA-treated and TSA-untreated groups, respectively), but had no effects on gaur iSCNT embryos. The fresh blastocysts derived from bovine SCNT and gaur iSCNT embryos (fresh groups), as well as vitrified bovine SCNT blastocysts (vitrified group), were transferred to bovine recipients. We found that TSA treatment increased the pregnancy rates only in recipients receiving fresh bovine SCNT embryos. In recipients receiving TSA-treated bovine SCNT embryos, three cloned calves from the fresh group and twin cloned calves from the vitrified group were delivered; however, no calf was born from the TSA-untreated bovine SCNT embryos. In contrast, one gaur iSCNT calf was born from a recipient receiving blastocysts from the TSA-untreated group. In summary, TSA improved the preimplantation development and pregnancy rates of bovine SCNT embryos, but did not have any beneficial effect on gaur iSCNT embryos. However, one gaur iSCNT calf reached full-term development.

Development of intergeneric and intrageneric somatic cell nuclear transfer (SCNT) cat embryos and the determination of telomere length in cloned offspring.

Somatic cell nuclear transfer (SCNT) holds potential as a useful tool for agricultural and biomedical applications. In vitro development of marbled cat intergeneric SCNT reconstructed into domestic cat cytoplast revealed that cloned, marbled cat embryo development was blocked at the morula stage. No pregnancies resulted from the transfer of one- to eight-cell stage embryos into domestic cat surrogate mothers. This suggested that abnormalities occurred in the cloned marbled cat embryos, which may be associated with incomplete reprogramming during early embryo development. Two pregnancies were established in surrogate mothers that received cloned domestic cat embryos, but SCNT offspring developed abnormally. Some specific phenotypes that were observed included incomplete abdominal wall disclosure, improper fetal development. In addition, some of the fetuses were mummified or stillbirths. The two live births died within 5 days. Telomere lengths of cloned kittens as determined by qualtitative polymerase chain reaction (qPCR) were inconclusive: some were found to be shorter, longer, or the same as donor control cells. Our findings support the hypothesis that telomere lengths do not govern the health of these cloned animals. A lack of complete reprogramming may lead to developmental failure and the abnormalities observed in cloned offspring.

Spectroscopic signature of mouse embryonic stem cell-derived hepatocytes using synchrotron Fourier transform infrared microspectroscopy.

Stem cell-based therapy for liver regeneration has been proposed to overcome the persistent shortage in the supply of suitable donor organs. A requirement for this to succeed is to find a rapid method to detect functional hepatocytes, differentiated from embryonic stem cells. We propose Fourier transform infrared (FTIR) microspectroscopy as a versatile method to identify the early and last stages of the differentiation process leading to the formation of hepatocytes. Using synchrotron-FTIR microspectroscopy, the means of identifying hepatocytes at the single-cell level is possible and explored. Principal component analysis and subsequent partial least-squares (PLS) discriminant analysis is applied to distinguish endoderm induction from hepatic progenitor cells and matured hepatocyte-like cells. The data are well modeled by PLS with endoderm induction, hepatic progenitor cells, and mature hepatocyte-like cells able to be discriminated with very high sensitivity and specificity. This method provides a practical tool to monitor endoderm induction and has the potential to be applied for quality control of cell differentiation leading to hepatocyte formation.

The effects of manipulation medium, culture system and recipient cytoplast on in vitro development of intraspecies and intergeneric felid embryos.

The aim of this study was to investigate if reconstructed felid embryos obtained by intraspecies or intergeneric cloning can develop in vitro. Fibroblast cells (f) from a domestic cat (DCf), marbled cat (MCf) and bovine (Bf) were used as donor cells, and oocytes (o) from domestic cats (DCo) and bovine (Bo) were used as recipient cytoplasts. There were two intraspecies (donor cell + recipient cytoplast: DCf + DCo and Bf + Bo) and three intergeneric (MCf + DCo, DCf + Bo and MCf + Bo) cloning groups in the study. In Experiment 1, the effects of manipulation media, modified TCM-199 (199H) or Emcare holding medium (EHM), on in vitro development of DCf + DCo embryos were investigated. The blastocyst formation rate (BFR) of the embryos manipulated in EHM (33.3%) was higher (P<0.05) compared with those manipulated in 199H (18.1%). In Experiment 2, DCf + DCo and MCf + DCo embryos were cocultured with or without domestic cat oviductal epithelium cells. Irrespective of coculture, the same BFR was obtained for DCf + DCo embryos (44.4 vs. 38.0%), while MCf + DCo embryos could not develop beyond the morula stage. In experiment 3, although the development of MCf + DCo and DCf + Bo embryos was arrested at the morula stage, 8.6% of MCf + Bo embryos were able to develop to the blastocyst stage. These results demonstrated that EHM was superior to 199H as an embryo manipulation medium and that the DCo and Bo could support the early embryonic development of intergeneric cloned marbled cat embryos up to the morula stage. However, postimplantation development still needs to be investigated.