OBJECTIVES: Equine tendinopathies are challenging because of the poor healing capacity of tendons commonly resulting in high re-injury rates. Within the tendon, different regions - tendon proper (TP) and peritenon (PERI) - contribute to the tendon matrix in differing capacities during injury and aging. Aged tendons have decreased repair potential; the underlying transcriptional and epigenetic changes that occur in the TP and PERI regions are not well understood. The objective of this study was to assess TP and PERI regional differences in adolescent, midlife, and geriatric horses using RNA sequencing and DNA methylation techniques. RESULTS: Differences existed between TP and PERI regions of equine superficial digital flexor tendons by age as evidenced by RNASeq and DNA methylation. Cluster analysis indicated that regional distinctions existed regardless of age. Genes such as DCN, COMP, FN1, and LOX maintained elevated TP expression while genes such as GSN and AHNAK were abundant in PERI. Increased gene activity was present in adolescent and geriatric populations but decreased during midlife. Regional differences in DNA methylation were also noted. Notably, when evaluating all ages of TP against PERI, five genes (HAND2, CHD9, RASL11B, ADGRD1, and COL14A1) had regions of differential methylation as well as differential gene expression.

Background

Tendon injuries amount to one of the leading causes of career-ending injuries in horses due to the inability for tendon to completely repair and the high reinjury potential. As a result, novel therapeutics are necessary to improve repair with the goal of decreasing leg lameness and potential reinjury. Small leucine-rich repeat proteoglycans (SLRPs), a class of regulatory molecules responsible for collagen organization and maturation, may be one such therapeutic to improve tendon repair. Before SLRP supplementation can occur in vivo, proper evaluation of the effect of these molecules in vitro needs to be assessed. The objective of this study was to evaluate the effectiveness of purified bovine biglycan or decorin on tendon proper and peritenon cell populations in three-dimensional tendon constructs.

Methods

Equine tendon proper or peritenon cell seeded fibrin three-dimensional constructs were supplemented with biglycan or decorin at two concentrations (5 nM or 25 nM). The functionality and ultrastructural morphology of the constructs were assessed using biomechanics, collagen content analysis, transmission electron microscopy (TEM), and gene expression by real time - quantitative polymerase chain reaction (RT-qPCR).

Results

SLRP supplementation affected both tendon proper and peritenon cells-seeded constructs. With additional SLRPs, material and tensile properties of constructs strengthened, though ultrastructural analyses indicated production of similar-sized or smaller fibrils. Overall expression of tendon markers was bolstered more in peritenon cells supplemented with either SLRP, while supplementation of SLRPs to TP cell-derived constructs demonstrated fewer changes in tendon and extracellular matrix markers. Moreover, relative to non-supplemented tendon proper cell-seeded constructs, SLRP supplementation of the peritenon cells showed increases in mechanical strength, material properties, and collagen content.

Conclusions

The SLRP-supplemented peritenon cells produced constructs with greater mechanical and material properties than tendon proper seeded constructs, as well as increased expression of matrix assembly molecules. These findings provide evidence that SLRPs should be further investigated for their potential to improve tendon formation in engineered grafts or post-injury.

A healthy gastrointestinal (GI) tract with a properly established microbiota is necessary for a foal to develop into a healthy weanling. A foals health can be critically impacted by aberrations in the microbiome such as with diarrhea which can cause great morbidity and mortality in foals. In this study, we hypothesized that gut establishment in the foal transitioning from a diet of milk to a diet of grain, forage, and pasture would be detectable through analyses of the fecal microbiotas. Fecal samples from 37 sets of foals and mares were collected at multiple time points ranging from birth to weaning. Bacterial DNA was isolated from the samples, and the V4 domain of bacterial 16S rRNA genes were amplified via polymerase chain reaction. Next generation sequencing was then performed on the resulting amplicons, and analyses were performed to characterize the microbiome as well as the relative abundance of microbiota present. We found that bacterial population compositions followed a pattern throughout the early life of the foal in an age-dependent manner. As foals transitioned from milk consumption to a forage and grain diet, there were recognizable changes in fecal microbial compositions from initial populations predominant in the ability to metabolize milk to populations capable of utilizing fibrous plant material. We were also able to recognize differences in microbial populations amongst diarrheic foals as well as microbial population differences associated with differences in management styles between facilities. Future efforts will gauge the effects of lesser abundant bacterial populations that could also be essential to GI health, as well as to determine how associations between microbial population profiles and animal management practices can be used to inform strategies for improving upon the health and growth of horses overall.

A healthy gastrointestinal (GI) tract with a properly established microbiota is necessary for a foal to develop into a healthy weanling. A foal's health can be critically impacted by aberrations in the microbiome such as with diarrhea which can cause great morbidity and mortality in foals. In this study, we hypothesized that gut establishment in the foal transitioning from a diet of milk to a diet of grain, forage, and pasture would be detectable through analyses of the fecal microbiotas. Fecal samples from 37 sets of foals and mares were collected at multiple time points ranging from birth to weaning. Bacterial DNA was isolated from the samples, and the V4 domain of bacterial 16S rRNA genes were amplified via polymerase chain reaction. Next generation sequencing was then performed on the resulting amplicons, and analyses were performed to characterize the microbiome as well as the relative abundance of microbiota present. We found that bacterial population compositions followed a pattern throughout the early life of the foal in an age-dependent manner. As foals transitioned from milk consumption to a forage and grain diet, there were recognizable changes in fecal microbial compositions from initial populations predominant in the ability to metabolize milk to populations capable of utilizing fibrous plant material. We were also able to recognize differences in microbial populations amongst diarrheic foals as well as microbial population differences associated with differences in management styles between facilities. Future efforts will gauge the effects of lesser abundant bacterial populations that could also be essential to GI health, as well as to determine how associations between microbial population profiles and animal management practices can be used to inform strategies for improving upon the health and growth of horses overall.

Significant improvements in genetics, nutrition, and food efficiency have had a great impact on the rapid growth of broilers, notably with increases in muscle mass. However, with rapid growth, the broiler industry has been negatively impacted by the increased incidence of myopathies, including white striping. White striping affects the pectoralis major muscle of broilers, particularly the larger breasts of rapidly growing modern commercial broiler lines. In this study, we documented the growth process of commercial broiler chickens from hatching to market weight at 6 wk. Gross pathology and histopathology analyses were performed on pectoralis major muscle collected weekly from birds culled from 1 to 6 wk. The severity of both gross and histologic pathologies in the breast muscle increased over time. White striping was initially observed at week 2, with a rise in the incidence and severity through the sixth week. Mild histopathology was noted in week 2, characterized by macrophage infiltration and limited phagocytosis of the muscle. Muscle condition deteriorated with age and weight gain, with more prevalent macrophages, phagocytosis, and interstitial fibroblasts. By week 5 and 6, there was severe myopathy including regions of obliterated muscle tissue. Linear regression models show a positive correlation between white striping, gross pathology, and histopathology relative to weight and age.

Conventional broiler diets include synthetic methionine to optimize fast muscle growth. Recently, a conventional synthetic methionine-rich diet was compared to alternative diet regimens providing natural sources of methionine. Broilers fed diets with natural methionine sources grew at a slightly slower rate. From this study, we hypothesized that the difference in a growth rate would be reflected in features of the breast muscle from broilers fed the alternative diet. We hypothesized that white striping of pectoralis major muscle would be reduced in slower growing broilers fed the alternative diet regimen with natural methionine. We also hypothesized that there would be associated differences in gene expression for cell differentiation and pathology markers. Broilers fed a conventional corn/soy diet regimen with synthetic methionine were compared to those fed roasted cowpea and sunflower seed meal (60% corn/soy, 20% sunflower seed meal, and 20% roasted cowpea) and no synthetic methionine. Overall broiler growth, muscle gene expression, and muscle collagen content data were compared. Expression analyses of combinations of MYOD1, PPARG, COL1A2, TRIM63, SOD1, PTGS2, and CD36 genes were used to examine differentiation and inflammation in the pectoralis muscles. The group fed an alternative diet gained less weight than those fed the control diet in the starter and grower phases but not in the finisher phase. Ultimately, the conventional diet resulted in a greater final weight for the broilers. However, mean white striping scores for the pectoralis major muscles were greater in the conventional control diet regimen. Gene expression results indicated greater expression of PPARG, PTGS2, and CD36 in the muscle of broilers fed the control diet. These data associate white striping with fat deposition and inflammation. Thus, whether due to differences in feed intake, growth rate, or actual compositional differences, the alternative diet with natural methionine sources seemed to curtail amounts of white striping in broiler muscle. More studies are necessary to further discern the effect of growth rate and natural methionine sources on white striping.