R.K. Splan

Detection of Prosecretory Mitogen Lacritin in Nonprimate Tears Primarily as a C-Terminal-Like Fragment

PURPOSE Lacritin is a human tear glycoprotein that promotes basal tear protein secretion in cultured rat lacrimal acinar cells and proliferation of subconfluent human corneal epithelial cells. When topically added to rabbit eyes, lacritin promotes basal tearing. Despite these activities on several species, lacritins presence in nonprimate tears or other tissues has not been explored. Here we probed for lacritin in normal horse tears. METHODS Sequences were collected from the Ensembl genomic alignment of human LACRT gene with high-quality draft horse genome (EquCab2.0) and analyzed. Normal horse tears were collected and assayed by Western blotting, ELISA, and mass spectrometry. Newly generated rabbit antibodies, respectively, against N- and C-terminal regions of human lacritin were employed. RESULTS Identity was 75% and 45%, respectively, at nucleotide and protein levels. Structural features were conserved, including a C-terminal amphipathic α-helix. Anti-C-terminal antibodies strongly detected a ∼13 kDa band in horse tears that was validated by mass spectrometry. In human tears, the same antibody detected uncleaved lacritin (∼24 kDa) strongly and C-terminal fragments of ∼13 and ∼11 kDa weakly. Anti-N-terminal antibodies were slightly reactive with a ∼24 kDa horse antigen and showed no reaction with the anti-C-terminal-reactive ∼13 kDa species. Similar respective levels of horse C-terminal versus N-terminal immunoreactivity were apparent by ELISA. CONCLUSIONS Lacritin is present in horse tears, largely as a C-terminal fragment homologous to the mitogenic and bactericidal region in human lacritin, suggesting potential benefit in corneal wound repair.

Genetic variability of the Norwegian Fjord horse in North America

Pedigrees of a reference population of 1 659 North American Norwegian Fjord horses were traced to founders and analysed for coefficients of inbreeding and genetic variability. Effective population size was 207.8 and there were 641 total founders. Pedigree completeness was close to 100 percent for 6 generations, with 9.8 average complete generation equivalents. The average inbreeding coefficient was 3.2 percent for the entire pedigree and 1.6 percent for pedigrees traced back five generations. Average inbreeding coefficients by year of birth increased until 1983, before decreasing and then stabilizing through 2009. Effective number of founders, ancestors and genomes were 96, 30.0 and 12.7, respectively. Low effective number of founders and ancestors indicate that genetic diversity has been lost in the development of the breed in North America. However, registry-enforced breeding strategies have contributed to lower inbreeding coefficients in the current generation.

Temporal variables of the canter of the Tennessee Walking Horse

Temporal stride characteristics of the canter were compared between performance-shod (PS) and light-shod (LS) Tennessee Walking Horses, which generally differ in training and shoeing methods. Four consecutive strides for ten PS and ten LS horses were filmed (30 Hz), and frame-by-frame analysis performed to determine stride duration and individual limb stance duration. Also analysed was the percentage of stride duration devoted to single, bipedal or tripedal limb support. Footfall sequence for PS was trailing hind (TrH), leading hind (LdH), trailing fore (TrF) and leading fore (LdF), whereas footfall sequence for LS horses varied, with the majority of LS performing a sequence of TrH‐TrF‐LdH ‐LdF. Stride duration was greater for PS. As a percentage of stride duration, PS demonstrated greater duration of TrF and LdF, while LS demonstrated greater LdH duration. Hind and fore single limb support, and tripedal support, were greater for LS; however, PS utilized bipedal support to a larger extent than LS. Hind or forelimb bipedal support was demonstrated only in PS, while only LS demonstrated lateral bipedal support. Thus, while both performance- and light-shod Tennessee Walking Horses perform the canter, temporal variables for this gait differ dramatically between the two groups.

Characterization and conservation of the American Milking Devon

The American Milking Devon Association was established in 1978 to record and preserve a unique type of cattle originally developed in the United States in the 17 th century from imported English and contemporary American stocks. Phenotypically, it exhibits a spectrum of red colour, with light-coloured curved horns and medium body size. The breed is one of the few remaining triple-purpose breeds in the United States, and has been selected by breeders since colonial times for excellent temperament and powerful draught ability, along with desirable dairy and beef characteristics. It is well adapted to low-input management schemes and harsh environments. The American Milking Devon is currently found on the ‘critical’ list of the American Livestock Breeds Conservancy, indicating that there are fewer than 200 new registrations annually. Since the inception of the American Milking Devon Association, a small group of dedicated breeders has done much to preserve these cattle. However, new management tools are now being introduced to assist breeders and allow conservation of genetic diversity while retaining the original breed traits in terms of size, colour, beef and dairy production, and character and working ability.

AN ALTERNATIVE FOR MIXED MODEL ANALYSES OF LARGE, MESSY DATA SETS (MTDFREML)

Portable Fortran based programs (MTDFREML) were developed using a derivative-free algorithm to obtain REML estimates of (co )variance components. Computations are based on Hendersons mixed model equations for multiple-trait models with missing observations on some traits and incorporation of relationships among relatives. Many fixed and random factors are allowed with number oflevels dependent on computer memory. Data sets with more than 40,000 genetic effects have been analyzed. Options allow solving MME at convergence. Constraints are automatically imposed. Expectations, standard errors of contrasts of solutions for fixed effects and prediction error variances of solutions for random effects can be obtained. Dimensions can be changed to match data with computer capability. A Fortran compiler is necessary. No fee is charged but the University of Waterloo must certify a license has been obtained for sparse matrix subroutines (SP ARSP AK) used in the program. As an example, birth weights of 4891 progeny of389 sires nested within 12 breeds and of2893 dams nested within 3 breeds of dam were analyzed to estimate components of variance due to sires and dams and to estimate differences among breeds of sires. For MTDFREML the analysis was trivial but for PROC MIXED the analysis was impossible unless dams were dropped from the model.

EMPIRICAL ESTIMATES OF POWER FOR BINOMIAL DATA WITH MIXED MODELS

Observations on return to estrus from anestrus postpartum beef cows were used as the basis for a simulation study to develop a method to determine numbers of locations and animals per treatment per location to achieve a specified power of test. Estimates of among location and total variance were obtained by REML from the data set and then used to generate simulated data for the binomial trait. Each combination of several pre-determined factors was replicated 1000 times. Pre-determined factors were number of locations, number of animals per treatment per location, desired detectable difference due to treatment, alpha-probability level and ratio of among location to total variance. Two methods were used to test for treatment differences. In Method 1, simulated data were analyzed using a mixed model with the variance components used for the simulation based on estimates from the postpartum cow data. For Method 2, variance components were re-estimated from each replicate of the simulated data and used in the mixed model equations. The number of significant differences due to treatment was counted for the 1000 replicates. The fraction of replicates with significant differences is an empirical estimate of the power of the test. The comparison of power of test between the two methods indicates Method 2 may be preferable for empirical estimation of power of test.

Possible Role of MicroRNA in Equine Insulin Resistance: A Pilot Study

&NA; MicroRNAs (miRNAs) are a class of small endogenous single‐stranded noncoding RNA molecules that have important roles in several biological processes. Research in human and laboratory animals has shown that miRNAs can regulate genes associated with type 2 diabetes mellitus and metabolic syndrome, and that the levels of specific miRNAs circulating in the bloodstream can serve as potential biomarkers for the diagnosis and prognosis of these diseases. We hypothesized that insulin‐resistant (IR) horses would have a different circulating miRNA profile than those that are healthy. Fifteen nonpregnant mares housed at the Virginia Tech Middelburg Agricultural Research and Extension Center were evaluated for insulin sensitivity, with the frequent sampling intravenous glucose tolerance test. Selected mares, representing the most insulin‐sensitive (IS, n = 3) and IR (n = 3) states, and paired for age, weight, and body condition, underwent miRNA profiling. Serum samples were collected, miRNA extracted, and microarray analysis performed to investigate the presence and relative amount of 340 equine miRNAs. Confirmation by quantitative real‐time polymerase chain reaction revealed that miRNA was present in the serum of all animals. Results demonstrated different miRNA profiles between groups: Six miRNAs were expressed only in IS mares, five miRNAs were found to have lower quantity in IR mares relative to the IS ones, and three miRNAs were higher quantity in IR mares relative to the IS ones. The novel results of this preliminary study suggest potential new tools that could be developed for the diagnosis and treatment of metabolic syndrome in horses. HighlightsThe use of microRNA as bio markers for insulin resistance in horses.Comparison of circulating microRNA profile in three insulin‐resistant and insulin‐sensitive horses characterized by a gold standard test.Evidence of different microRNA profiles in insulin‐resistant versus insulin‐sensitive horses.MicroRNAs found to have relation with insulin‐related pathways.

Estimates of parameters between direct and maternal genetic effects for weaning weight and genetic effects for carcass traits in crossbred cattle

Estimates of heritabilities and genetic correlations were obtained from weaning weight records of 23,681 crossbred steers and heifers, and carcass data of 4,094 crossbred steers using REML applied to animal models. Direct and maternal heritabilities for weaning weight were 0.14 and 0.19, respectively. The genetic correlation between direct and maternal weaning weight was negative (-0.18). Heritabilities for carcass traits of steers were moderate to large (0.34 to 0.60). Genetic correlations between direct genetic effects for weaning weight and carcass traits were small, except with hot carcass weight (0.70), ribeye area (0.29) and adjusted fat thickness (0.26). Genetic correlations of maternal genetic effects for weaning weight with direct genetic effects for carcass traits were: hot carcass weight (0.61), retail product percentage (-0.33), fat percentage (0.33), ribeye area (0.29), marbling score (0.28), and adjusted fat thickness (0.25). These results indicate that maternal genetic effects for weaning weight may be correlated with genetics for propensity to fatten in steers. Selection for only direct weaning weight would be expected to increase carcass weight and ribeye area and slightly decrease marbling and retail product percentage. Selection for either increased maternal or direct weaning weight would be expected to result in increased carcass weight, ribeye area, and fat thickness, but would not be expected to affect tenderness.