Blood-borne extracellular vesicles (i.e. exosomes and microvesicles) carrying microRNAs (miRNAs) could make excellent biomarkers of disease and different physiologic states, including pregnancy status. We tested the hypothesis that circulating extracellular vesicle-derived miRNAs might differentiate the pregnancy status of cows that had maintained pregnancy to Day 30 from non-pregnant cows or from those that exhibited embryonic mortality between Days 17–30 of gestation. Cows were randomly assigned for artificial insemination with fertile semen (n= 36) or dead semen (n=8; control group) on Day 0 (day of estrus). Blood was collected from all animals on Day 0 and on Days 17 and 24 after artificial insemination. Cows receiving live sperm were retrospectively classified as pregnant on Day 30 (n=17) or exhibiting embryonic mortality between Days 17–30 (n=19). Extracellular vesicles from Day-17 and -24 samples were isolated from serum using ultra-centrifugation, and their presence was confirmed by nanoparticle tracking and Western blot analyses (for CD81) prior to RNA extraction. MicroRNA sequencing was performed on pregnant, embryonic-mortality, and control cows (n=4 per day), for a total of 24 independent reactions. In total, 214 miRNAs were identified in serum, 40 of which were novel. Based on differential abundance parameters, we identified 32 differentially abundant loci, representing 27 differentially abundant mature miRNA. At Days 17 and 24, specific miRNAs (e.g. miR-25, -16b and -3596) were identified that differentiated the pregnancy status. In summary, we identified several circulating extracellular vesicles derived miRNAs that differ in abundance between embryonic mortality and pregnant cows.

Fifteen mature beef bulls (BW = 800.4 ± 17.4 kg) were used in a 112-d experiment to evaluate effects of divergent planes of nutrition on novel measures of bull fertility. Bulls were ranked by BW and randomly assigned to one of two treatments: 1) managed on a positive plane of nutrition (POS, n = 8), or 2) managed on a negative plane of nutrition (NEG, n = 7). Bulls were individually fed a common diet adjusted biweekly to achieve targeted weight loss or gain of 12.5% of original BW. On d 112, electroejaculation was used to collect 2 ejaculates from each bull, which were combined, extended, and frozen. The Guava easyCyte 8HT Flow Cytometer was used to analyze cell membrane and acrosome integrity, mitochondrial energy potential, and oxidation status of frozen-thawed semen. Data were analyzed for effects of treatment with bull as the experimental unit using the MIXED procedure of SAS. By design, bull BW diverged (P < 0.0001) with POS bulls gaining 1.27 ± 0.08 kg/d, whereas NEG bulls lost 0.91 ± 0.08 kg/d. Treatment did not influence cell membrane integrity (P = 0.20), or proportion of live sperm with intact acrosome (P = 0.91). A greater (P = 0.04) proportion of sperm from POS bulls (35.1 ± 3.47%) were alive and stained reactive oxygen species positive compared with sperm from NEG bulls (23.8 ± 3.71%) indicating sperm from POS bulls was more prepared to withstand oxidative stressors. However, NEG bulls (27.2 ± 5.22) had a greater (P = 0.01) proportion of sperm with polarized mitochondrial energy potential compared with POS bulls (6.1 ± 4.89%), indicating greater energy for sustained motility. We conclude that plane of nutrition during spermatogenesis may impact sperm’s ability to withstand stressful environments encountered and to sustain motility in the female reproductive tract after cryopreservation.

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Beckwith-Wiedemann syndrome (BWS) is a loss-of-imprinting pediatric overgrowth syndrome that has an incidence of 1 in 13,700. Imprinted genes are expressed from only one of the two inherited parental chromosomes. These genes regulate the growth and development of the fetus and the placenta. The primary features of BWS include general overgrowth, enlarged tongue, abdominal wall defects, hypoglycemia, cardiac malformations, and embryonic tumors. Currently there are no animal models that recapitulate BWS. However, there is a similar overgrowth phenotype observed in ruminants as the result of embryo culture called large offspring syndrome (LOS). The phenotypes associated with LOS are increased birth weight, enlargement of internal organs, skeletal defects, hypoglycemia, and breathing difficulties. We propose that BWS and LOS are the result of similar loss-of-imprinting. In order to determine if the bovine will serve as a good model to study BWS we must first establish what the baseline gene expression and DNA methylation is in the bovine for the imprinted loci associated with BWS. Our study shows that genomic imprinting is conserved between human and bovine at the imprinted regions known to be misregulated in BWS. Future work will determine if LOS and BWS are epigenetically similar. If so, the bovine will serve as an appropriate animal model for studying the pediatric overgrowth syndrome BWS. Using the bovine as a model will allow researchers to understand the mechanisms that result in BWS and may provide therapeutic treatment options for these patients.