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ABSTRACTDNA methylation has proven to be the most promising age-predictive biomarker in mammals resulting in the emergence of ‘epigenetic clocks’ that describe the relationship between methylation levels and age. Using Targeted bisulfite Sequencing, we evaluated blood DNA-methylation data from 96 domesticated cows (Bos Taurus) of which 88 were adults and 8 were calves. This allowed us to measure DNA methylation across three thousand regions in the genome that were conserved across mammals. The significant association of age with the changes in DNA methylation enabled us to construct an epigenetic clock that predicts the age of cows to within nine months. We also investigated whether factors exist that moderate the association between epigenetic age and actual age and found that milk production levels significantly increase the rate of epigenetic ageing, suggesting that the stress of excessive milk production might be accelerating epigenetic ageing in cows.

Decimal reduction time (D-value) was calculated for six non-O157 Shiga toxin–producing Escherichia coli(STEC) in a laboratory medium and ground beef. For the laboratory medium, an overnight culture of each strain of STEC was divided into 10-mL sample bags and heated in a water bath for a specific time on the basis of the temperatures. Survival curves were generated by plotting the surviving bacterial population against time, and a linear-log primary model was used to estimate the D-values from survival curves. The z-values (the temperature raised to reduce the D-value by one-tenth) were calculated by plotting the log D-values against temperature. Similarly, for ground beef, six fat contents, 5, 10, 15, 20, 25, and 30% of ground beef were formulated for this study. Inoculated meat was divided into 5-g pouches and submerged in a water bath set at specific temperatures (55, 60, 65, 68, and 71.1°C). The average D-value for these strains in a laboratory medium was 17.96 min at 55°C, which reduced significantly (P< 0.05) to 1.58 min at 60°C, and then further reduced (P< 0.05) to 0.46 min at 65°C. In ground beef, a negative correlation (P< 0.05) between fat content of ground beef and D-values was observed at 55°C. However, at temperatures greater than 60°C, there was no impact (P> 0.05) of fat content of ground beef on the thermal resistance of non-O157 STECs. Irrespective of the fat content of ground beef, the D-values ranged from 15.93 to 11.69, 1.15 to 1.12, and 0.14 to 0.09 min and 0.05 at 55, 60, 65, and 68°C, respectively. The data generated from this study can be helpful for the meat industry to develop predictive models for thermal inactivation of non-O157 STECs in ground beef with varying fat content.

Spermatozoon motility is an important factor in successful artificial reproductive technologies. Successful reproduction requires properly developed spermatozoa with adequate forward, progressive motility that allows for transport through the female reproductive tract. Motility is driven by production of ATP; however, cryopreservation is known to have damaging effects on spermatozoa. Mitochondria utilize oxidative phosphorylation to synthesize ATP through an electrochemical proton motive force that is composed primarily of mitochondrial membrane potential (Δψm). The mitochondrial membrane potential can be measured using a fluorescent, carbocyanine JC-1 dye (5,5’,6,6’-tetrachloro-1,1’,3,3’-tetraethylbenzimi-dazolylcarbocyanine iodide). The objective of this study was to examine correlations between the mitochondrial membrane potential and motility score. Cryopreserved semen samples from three bulls were donated to Tarleton State University for research purposes. Samples were thawed, placed under a phase contrast microscope, and analyzed by three individuals with varying levels of training for rough motility. These samples were then prepared with JC-1 dye according to manufacturer’s instructions. Two microliters of dye were added to a stock preparation of each sample. Samples were examined on a FACS Calibur flow cytometer at University of Texas Southwestern Core in Dallas, TX. Flow cytometry analysis was performed using FlowJo V10.7. Statistical analysis was performed using SAS v9.4. The Pearson correlation coefficient showed a strong, positive correlation (r = 0.90) between the mitochondrial membrane potential and motility (P = 0.28), thus indicating as the mitochondrial membrane potential increases, so does the rough motility score. These data represent a subset of a population that demonstrates the need of further research on the ability for spermatozoa to produce ATP and the correlation in forward, progressive motility. This research can provide a foundation in which future researchers may develop an assay that allows for testing of mitochondrial membrane potential by producers to select bulls with greater breeding potential.

Sperm cryopreservation is an important tool for assisted reproductive techniques, nonetheless there are major factors associated with reduced longevity and poor survivability of cryopreserved sperm in the female reproductive tract. Several studies have focused on molecular markers associated with apoptosis, mitochondrial integrity and oxidative stress in cryopreserved sperm. Research indicates miRNA 26a-5p and miRNA-574 exhibit anti-inflammatory attributes in some cells, by downregulating the effects on specific genes associated with inflammation and apoptosis. The aim of this study was to evaluate the quality of bovine semen, including a variety of breeds, post-thaw viability through kinetic screening using Computer Assisted Semen Evaluation (CASA). Cryopreserved semen samples from 5 bulls were thawed in a water bath (37.5°C for 35 s), then aliquoted in eppendorf tubes and initial CASA (Version 14 CEROS) measurements of total motility, progressive motility, and kinematic factors were assessed. Semen samples were placed in a heat block at 37.5 C for incubation for three 30-minute periods (30-minutes, 60-minutes, and 90-minutes) to measure thermoresistance and longevity to extrinsic factors influencing oxidative stress. After incubation periods 80 µL of each semen sample was screened on CASA. Data were analyzed using a Student-T in GraphPad Prism. Data are presented as LSMEANS and P-values ≤ 0.05 were considered significant and tendencies were considered at a P > 0.05. Semen samples were clustered into subpopulation threshold-based on sperm velocity and linearity at each time point. Levels of miRNA-574 and miRNA-26a-5p were quantified using qPCR. No significant differences of miRNA-574 or miRNA-26a-5p were detected in semen at post-thaw. Incubation of semen for 30-minutes resulted in total motility decrease (P = 0.02) and progressive motility tended to decline (P = 0.06). None of the semen was viable past the first 30-minute period. These data suggest that there are limited differences in thermoresistance in sperm in a rapid stress test. However, further research needs to be conducted to evaluate the cells on aspects of viability and overall function.

Developmental competence of oocytes is progressively obtained, both as females reach puberty and as follicular diameter increases. The poor quality of oocytes, and the subsequent embryos derived from them, in prepubertal females and from small follicle sizes suggests that essential processes during cytoplasmic maturation of the oocyte have not been fulfilled. The objective of this experiment was to identify differentially expressed genes in oocytes derived from gilts or sows and from large or small follicle sizes that correlate with developmental competence. Oocytes were collected from 2–6 mm (sows and gilts) and 8–14 mm follicles (sows) and matured in vitro for 42–44 hrs in defined TCM199 supplemented with 0.5 mM cysteamine, 0.5 mM citric acid, 100 ng/ml epidermal growth factor, 0.05% ITS (v/v), 0.01% recombumin (v/v), 0.1 U/ml porcine LH and FSH and 2 mg/ml fetuin. Mature oocytes were identified by the presence of the first polar body in a Hepes buffered medium supplemented with 5% sucrose, lysed in pools of 150 and the poly A+ RNA extracted. Samples were processed through two cycles of linear amplification and hybridized to Affymetrix Porcine Genome Arrays. Microarray data was sorted and filtered by gene and by treatment to remove genes that were classified as having two absent calls per treatment. The data was then normalized and analyzed using a one-way analysis of variance. Level of significance was calculated using the Benjamini and Hochberg False Discovery Rate (FDR) and Bonferroni correction of α < 0.0167. A total of 11,600 genes were identified as present in the sow 2–6 mm and sow 8–14 mm treatment groups, with over 3500 genes (30.5%) being differentially expressed. Genes differentially expressed in oocytes derived from sow 2–6 and 8–14 mm follicles include genes involved in regulation of oxidative stress (glutathione S-transferase and peroxiredoxin 5), growth factors (growth differentiation factor 9 and insulin-like growth factor 2), and metabolism (pyruvate caboxylase). Over 12,200 genes were present in the sow 2–6 mm and gilt 2–6 mm treatment groups but only 1500 genes (12.9%) were differentially expressed. Oocytes derived from sow 2–6 mm and gilt 2–6 mm follicles differentially expressed growth factors (bone morphogenic protein 15), genes involved in programmed cell death (anti-apoptotic bcl), regulation of oxidative stress (gluthathione S-transferase and peroxiredoxin 1 and 4), metabolism (phosphoenolpyruvate carboxykinase 2) and DNA methyltransferases (Dnmt 1). Gene expression is significantly affected by maternal age as puberty approaches, as well as follicle size, and may be the basis for differences observed in developmental competence of porcine oocytes. (poster)