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In vitro production and transfer of embryos has become a common practice within the dairy industry to efficiently breed superior animals and meet the consumption demand of the growing population. Cyropreservation is necessary for the application of commercialized embryo transfer, however, in vitro-produced embryos show morphological and physiological defects which negatively impact their ability to withstand cryopreservation in comparison to their in vivo counterparts. These artifacts result from culture conditions that cause stress to the embryo during development, leading to an accumulation of intracellular lipids, mitochondrial dysfunction, and ultimately poor ability to withstand freezing and thawing. The objective of these studies was to examine the effects of various metabolic regulators on the viability and cryotolerance of in vitro-produced embryos. Pilot studies revealed that evaluating early (stage 6) versus late (stage 7) blastocysts did not affect the trend seen in results, nor did culturing embryos in continuous versus sequential media. From the main experiment performed, it was concluded that a combination of metabolic regulators decreased lipid content, improved cryopreservation survival, and lowered the percentage of apoptotic cells present after thawing. Conditioned media increased the blastocyst percentage, but did not produce superior quality embryos as measured by cryotolerance. Research concerning the metabolic needs of the preimplantation embryo must continue to determine more relevant markers of embryo quality in vitro.

Quinoa is an important, highly nutritional grain crop in the Andean region of South America. DNA markers and linkage maps are important tools for the improvement of underdeveloped crops such as quinoa. The objectives of this study were to (i) develop a new set of SSR markers to augment the number of SSR markers available in quinoa, and (ii) construct a new genetic linkage map of quinoa based on SSRs using multiple recombinant-inbred line (RIL) populations. Here we report the development of 216 new polymorphic SSR markers from libraries enriched for GA, CAA, and AAT repeats, as well as 6 SSR markers developed from BAC-end sequences (BES-SSRs). Heterozygosity (H) values of the SSR markers ranged from 0.12 to 0.90, with an average value of 0.56. These new SSR and BES-SSR markers were analyzed on two RIL mapping populations (designated Population 1 and Population 40), each obtained by crossing Altiplano and coastal ecotypes of quinoa. Additional markers, including AFLPs, two 11S seed storage protein loci, a SNP, and the nucleolar organizing region (NOR), were also analyzed on one or both populations. Linkage maps were constructed for both populations. The Population 1 map contains 275 markers, including 200 SSR and 70 AFLP markers, as well as five additional markers. The map consists of 41 linkage groups (LGs) covering 913 cM. The Population 40 map contains 68 markers, including 62 SSR and six BES-SSR markers, and consists of 20 LGs covering 353 cM. Thirty-nine anchor markers common between both maps were used to combine 15 Population 1 LGs with 13 Population 40 LGs. The resulting integrated map consists of 13 LGs containing 140 SSR, 48 AFLP, four BES-SSR, one SNP, and one NOR marker spanning a total of 606 cM. A high level of segregation distortion was observed in both populations, indicating possible chromosomal regions associated with gametophytic factors or QTLs conferring a selective advantage under the particular growing conditions. As these maps are based primarily on easily-transferable SSR markers, they are particularly suitable for applications in the underdeveloped Andean regions where quinoa is grown.

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Enhancing the Proportions of Healthy Fatty Acids in Milk from Dairy Cows by Korie A.S. Nelson, Master of Science Utah State University, 2008 Major Professor: Randy Wiedmeier Department: Animal, Dairy, and Veterinary Sciences Twenty cows were used in a repeated measures, block design experiment for 9 wk to determine the effects of feeding partially ruminally inert calcium salts (Ca-salts) of fish oil (FO) and a general fatty acid (FA) supplement (EnerGII) at varying levels. The effects on cow health, milk components, composition of milk FA, and sensory evaluation of milk were evaluated. Cows in the 4 treatments were fed either a control diet of 57% forage and 43% concentrate mix with EnerGII fat supplement at 1.65% of diet DM (CTL) or EnerGII in basal diet was partially replaced with (a) 0.21% partially ruminally inert calcium salts (Ca-salts) of 71% fish oil (Ca-FO71) given at 0.41% DM (FH41); (b) 0.41% inert Ca-FO71 given at 0.83% DM (FH83); or (c) 0.83% inert Ca-FO 43% fish oil (Ca-FO43) given at 0.83% DM (FL). Cow health was not negatively affected by treatment diets. Treatment only significantly affected dry matter intake (DMI) and net energy of lactation (NEL), with FH83 having the lowest DMI. Week of trial significantly affected all milk components except protein percent, which did not change. Dry matter intake, milk yield, fat yield, fat percent, and protein yield demonstrated a net decrease over time. Lactose, solids, and somatic cell count all shared a net increase over time. iv Milk urea displayed no definitive trend over time. Content of conjugated linoleic acid (CLA) isomers C18:2 cis-9, trans-11 and trans-10, cis-12 combined over time was 0.54, 0.68, 1.18, and 0.82 g/100 g FA for CTL, FH41, FH83, and FL, respectively. Vaccenic acid (VA) C18:1 trans-11 content over time was 1.04, 1.51, 2.28, and 1.68 g/100 g FA; and total omega-3 FA content over time was 0.52, 0.76, 0.82, and 0.80 g/100 g FA for CTL, FH41, FH83, and FL, respectively. Eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) levels increased by as much as 6- and 2.7-fold, respectively, over CTL for the duration of the experiment. Although levels of EPA, DHA, VA, and CLA increased for treatments FH41, FH83, and FL over CTL, a trained sensory panel detected no difference in milk flavor between treatments with little or no intensity of off-flavors. Results suggest that feeding FO and EnerGII at varying levels enhanced CLA, VA, EPA, DHA, and total omega-3 FA in milk over the length of the experiment without negatively affecting cow health, milk composition, or flavor of milk.

Fireweed (Senecio madagascariensis) is a noxious and invasive weed affecting pastures in Hawaii, Australia, and South America. Fireweed contains compounds called pyrrolizidine alkaloids that are toxic to most grazing mammals. Toxic effects to cattle include irreversible damage to liver cells, hardening of the liver, and loss of liver function, which may lead to jaundice, swelling, and the accumulation of fluids in the stomach and other physiological malfunctions. External effects include rough appearance, diarrhea, low energy and dullness, photosensitization, and abnormal behavior, many of which can lead to death. Fireweed also can reduce pasture productivity by as much as 30-40%, particularly in Hawaii. As a result of these adverse effects on cattle and pasture production, people are seeking ways to manage fireweed and cattle. Condensed tannins, which are common in many forage legumes, bind with other molecules such as protein and alkaloids. Complementarities among secondary compounds such as condensed tannins and alkaloids can allow animals to consume more of plant material they would otherwise avoid due to toxicity; however, there is very little information on whether tannins actually protect animals from the toxic effect of alkaloids like the ones found in fireweed. The effects of condensed tannins on the toxicity of fireweed to cattle were examined in two stages of a research project that included studies in the lab (in vitro) and in live animals (in vivo). The in vitro studies showed that tannins bound pyrrolizidine alkaloids in cattle rumen fluid and binding was highest when mixed with tannins at 8%, with some effect at 12% as well, by weight of fireweed. The in vivo studies were inconclusive as animals from both the Fireweed and Fireweed-Tannin group demonstrated ill-effects brought on by the fireweed. The data gathered in the form of blood tests and liver tests did not identify a clear protective effect provided by having tannin in the diet, but the results likely were affected by the way we conducted the research, which involved dosing animals daily with large amounts of plant material. The findings also illustrate how strongly cattle avoid eating fireweed while grazing on pasture. In summary, management to reduce fireweed effects on cattle should focus on improving pasture diversity and resiliency, on multi-species grazing to take advantage of the innate resistance of sheep and goats to fireweed’s toxicity, and on ways to enable cattle to utilize fireweed.

Discovered in 1969 in California, enterovirus 71 (EV-71) is a serious cause of disease in young children. It is one of the major causative agents of hand, food, and mouth disease (HFMD), and can produce neurological complications, such as meningitis, encephalitis, and an acute flaccid paralysis. For serious cases, the fatality rate can be up to 26%, almost exclusively in young children. While the virus was initially discovered in the United States, it was soon detected in the Eastern hemisphere, causing outbreaks in Europe and Asia. The largest outbreak occurred in Taiwan in 2008, with approximately 490,000 cases and 128 fatalities. However, despite the seriousness of EV-71, there are currently no approved antiviral treatments. Physicians rely on supportive care and the off-label use of a purified antibody mixture, intravenous immunoglobulin, for treatment. Part of the difficulty in developing antivirals for EV-71 is a lack of drug testing in animal models. Animal testing is a crucial step in drug development, determining which compounds will progress to clinical trials in humans. However, viruses that cause disease in humans do not necessarily cause disease or the same type of disease in animals. As such, viruses often need to be adapted before they can cause disease in their animal hosts. Adaption isn’t always successful and can result in a virus that produces disease that is unlike that seen in humans. Furthermore, some animal models can produce disease only under a strict set of conditions, such as newborn mice. Sometimes these animal model conditions may be impractical for testing potential treatments. At the Institute for Antiviral Research (IAR), we developed an animal model for EV-71 in four-week-old AG129 mice. AG129 mice lack the alpha, beta, and gamma interferon receptors, making them immunocompromised. Being immunocompromised, these mice are more susceptible to infection, including infection from human viruses. In our model, EV-71 infection produces neurological signs, including a rear-limb paralysis (similar to the paralysis seen children with EV-71). The virus is also lethal in these animals, which provides an observable and consistent baseline for evaluating potential drugs. We assessed twenty-four potential treatments in our EV-71 model. Two compounds, STF434 and STF1019, provided 30% and 87% protection against mortality. Intravenous immunoglobulin was also examined and found to be about 50% protective against mortality, depending on the dose and time of administration. Intravenous immunoglobulin also reduced inflammatory modulators (cytokines) in the brain and spinal cord. We consider this to be highly relevant, given that inflammation is a serious component of EV-71 infection.