Plasma leptin concentrations in horses have been shown to vary widely. However, many factors may perturb these measurements. Previous research has documented a link between hyperinsulinemia and hyperleptinemia in horses. This condition has been speculated to be associated with components of equine metabolic syndrome, such as insulin resistance and laminitis. However, many of the methods accepted in diagnosing these metabolic abnormalities are expensive and often difficult to perform. Thus, the objectives of the experiments in this thesis were 1) to determine if it is possible to differentiate hyperleptinemic horses from normal ones by hormone manipulation with dexamethasone, a glucocorticoid analog; 2) to evaluate intravenous injection combinations of insulin and glucose to assess insulin sensitivity; and 3) to assess insulin sensitivity in hyperleptinemic and normal horses using two concentrations of exogenous insulin. In the first experiment, it was determined that a single injection of dexamethasone did not enhance the ability to differentiate between hyperleptinemic and normal horses. In the second experiment, the combined intravenous insulin and glucose tests proved to be very simple and inexpensive. However, repeatability among estimates on the same horses was not strong enough to be conclusive or predictive in assessing insulin sensitivity in horses. In the third experiment, a single injection of exogenous insulin at 0.02 IU/kg of body weight proved to be a consistent and efficacious method of differentiating hyperleptinemic from normal horses. Glucose concentrations in hyperleptinemic horses decreased very little (14%) at this insulin dosage compared to those in normal horses that dropped (P < 0.01) 46% from a pre-injection average of 85 to 46 mg/dL 40 min after injection. It was concluded that a low-level insulin injection coupled with routine plasma leptin estimates constitute a simple method of identifying hyperleptinemic horses.

Transgene expression in stably transgenic organisms is affected by many factors, including the copy number of the transgene in the genome, and by interactions between the transgene and flanking DNA sequences. Transgene copy number has also been shown to effect genetic stability in transgenic plants. Two commonly used methods for transfecting cells are liposome mediated transfection and electroporation. Little is known about the mean transgene copy number or variability of the copy number with these techniques. Quantitative PCR (Q-PCR) has been shown to be an effective method for determining transgene copy number. The objective of this study was to determine transgene copy number after liposome mediated transfection and electroporation. The mean transgene copy number and variability between individual integration events have been determined. Q-PCR conditions were optimized for primer annealing temperature and concentration when amplifying a region of the plasmid hEFGFP used for transfection. The quantitative nature of the Q-PCR reaction was confirmed by amplifying 10-fold dilutions of the plasmid and plotting the threshold cycle (CT) value against the log of the plasmid concentration. A correlation coefficient of 1.00 and a calculated PCR efficiency of 93.3% were obtained from this analysis. Caprine fibroblasts were transfected by electroporation or FuGENE® HD reagent with either a circular or linearized hEFGFP plasmid and plated at low density in medium containing Geneticin®. After 10 days of culture, single cell colonies were isolated and expanded. When cultures reached 1-2 million cells, genomic DNA was isolated. Transgene copy number was determined by amplifying genomic DNA from individual clones representing 1x105 cells with Q-PCR. Transgene copy number was calculated by comparing CT values to a standard curve. The transgene copy number for electroporation circular was 2.7  0.75 (n=32) and 1.3  0.65 (n=19) when using a linear DNA construct. FuGENE® HD using a circular plasmid construct generated a gene copy number of 0.5  0.11 (n=14) and 0.64  0.13 (n=16) for the linear plasmid construct. There were significant differences when comparing electroporation circular to all other treatments, however, there were no differences when comparing electroporation linear, FuGENE® HD circular and FuGENE® HD linear to each other.

Pulsed electric field processing represents a promising non-thermal technology which involves the application of pulses of high voltage (20-80 kV/cm) for less than one second to fluid foods placed between two electrodes. During the application of certain PEF conditions microbial inactivation occurs by causing a pore formation and destruction of the cell membranes. Lactobacillus acidophilus is an important probiotic bacterium used for the production of fermented dairy products. The objective of this study was to study the influence of pulsed electric field (PEF) on the growth characteristics, bile tolerance, acid tolerance, and protease activity of Lactobacillus acidophilus LA-K. Freshly thawed Lactobacillus acidophilus LA-K was suspended in peptone and treated in a pilot plant PEF system (OSU-4M). The treatments were pulse width (3, 6 and 9 µs), pulse period (10,000; 20,000 and 30,000 µs), voltage (5, 15 and 25 kV/cm) and flow rate (10, 60 and 110 mL/min). Control was run through PEF system at 60 mL/min without receiving any pulsed electric field condition. Growth and bile tolerance on control and treatment samples were determined hourly throughout 16 hours of incubation. Acid tolerance was determined at 0, 5, 10 and 15 minutes of incubation. Protease activity was determined at 0, 12 and 24 hours of incubation. The experimental design was a repeated measure design. Data were analyzed using the PROC GLM of the Statistical Analysis Systems (SAS). Differences of least square means were used to determine significant differences at P < 0.05 for main effects (pulse width, pulse period, voltage, flow rate) and interaction effects (pulse width * time, pulse period * time, voltage * time, and flow rate * time). Bipolar pulse width and pulse period significantly lowered acid tolerance and bile tolerance as well as slowed log stage growth of Lactobacillus acidophilus LA-K. Voltage significantly influenced acid tolerance, bile tolerance and growth of Lactobacillus acidophilus LA-K. Bipolar pulse width, pulse period and voltage did not influenced protease activity of Lactobacillus acidophilus LA-K. Flow rate significantly influenced bile tolerance and growth of Lactobacillus acidophilus LA-K. Flow rate did not significantly influenced acid tolerance and protease activity of Lactobacillus acidophilus LA-K.

Considerable research has been conducted comparing the effect of continuous and some form of rotational stocking method on animal and forage performance, but most research utilizes put-and-take stocking rates. The purpose of this research was to determine the effect of 12-paddock rotational stocking method (ROT) or continuous stocking method (CONT) of winter wheat (Triticum aestivum) and annual ryegrass (Lolium multiflorum) pastures on beef heifer and forage performance while maintaining a continuous stocking rate of 3.7 heifers/ha. Two experiments were conducted and analyzed separately due to confounding year and location. Heifer ADG and WG/ha tended to be increased by ROT compared to CONT in Exp 1 (P = 0.10). Differing from Exp 1, heifer ADG and WG/ha was not affected by stocking method in experiment 2 (P = 0.36). Forage mass (Exp 1) was increased by ROT compared to CONT in a time by treatment manner (P < 0.005). Forage mass (Exp 1) was increased by ROT compared to CONT in the final four sampling periods. Forage mass below 500 kg DM/ha in CONT pastures (Exp 1) may have limited heifer DMI in the final two sampling periods. Similar to Exp 1, forage mass (Exp 2) was increased by ROT when compared to CONT in the final two sampling periods (P < 0.05). However, CONT forage mass was not limited in Exp 2. Forage CP (Exp 1) was increased in ROT compared to CONT in a time x treatment manner (P < 0.05). Forage IVTD, NDF, and ADF (Exp 1) were not affected by stocking method, but were influenced by time. Forage nutritive value in Exp 2 was increased by CONT compared to ROT on d 113 in a time x treatment manner (P < 0.05). The results of this research indicate that ROT does increase forage mass on winter wheat and annual ryegrass pastures and may sustain higher stocking rates compared to CONT. However, greater heifer ADG under ROT only occurred when CONT forage mass was limited. The use of ROT compared to CONT under continuous stocking rates of 3.7 heifers/ha does not consistently increase heifer ADG or WG/ha.

Breeding beef heifers to produce calves at 2 years of age could be a profitable management decision. The current practices have proved inefficient, which has justified further research in the area. The objective of this study was to examine the effects of repeated progestogen priming on the attainment of puberty by prepubertal heifers, to evaluate the potential additive effect of progestogen priming plus sodium monensin on the attainment of puberty by prepubertal heifers, to examine subsequent reproductive performance, including the response to synchronization of the estrous cycle, first-service pregnancy rates and overall pregnancy rates and to examine the effects of repeated progestogen priming on follicular dynamics of prepubertal beef heifers. Data were collected and analyzed for birth weight, adjusted weaning weight, postweaning gain/loss in weight and daily gain throughout the experiment. Also the average frame score, average breeding weight, average breeding body condition score, average change in body condition score, average number of animals that showed estrus behavior prior to the breeding season, average number of animals that responded to synchronization, average number of corpora lutea formed during the treatment periods, reproductive tract score at breeding as well as the change in reproductive tract score, follicle populations, change in ovary scores throughout the experiment, pregnancy percentage and the average fetal age at pregnancy determination were evaluated. In this study, treatment groups fed sodium monensin (Rumensin and MGA+Rumensin groups) had significant (P<0.05) advancements in fertility and maturational status when compared with Control animals. The MGA group did excel in follicle development when compared with the Control group, but more heifers became pregnant from artificial insemination combined with natural mating in the Rumensin groups than in the Control group. In conclusion, these experiments provide evidence for the positive effect of sodium monensin on puberty attainment and increased overall pregnancy rates when using artificial insemination in conjunction with natural mating. However, the additive effect of melengestrol acetate, if any, is yet to be defined.

Due to necessary storage of hatching eggs in commercial hatcheries, the embryo is being regressed. End of lay broiler breeder eggs have the poorest hatchability and are most affected by pre incubation storage. Warming these eggs is the only way the embryo can develop. The objective of this study was to study the effects of daily warming of end of lay broiler breeder hatching eggs during the storage period on embryo mortality and hatchability. Six trials were conducted, three trials warmed for three of the four day storage period and three trials warmed for five of the six days storage period. There were six warming treatments for each trial; 0, 30, 60, 90, 120, and 150 minutes daily. All eggs were from Hubbard Classic broiler breeders 50-58 weeks of age. Hatch time was recorded for trials 3 and 6. Two hundred and eighty eight randomly selected males were used in trial 5 to study the effects of daily warming on chick growth. They were fed a starter broiler diet and grown for 13 days in a Petersime starter battery. In all hatchability trials percent fertility, percent fertile hatchability, percent total hatchability, percent early dead, percent mid-dead, percent late dead, percent pips, and percent total embryonic mortality was not affected (P>0.05) by any of the daily warming treatments. Hatch time and chick growth were not affected by any of the daily warming treatments. Eggs could be warmed for as much as 150 minutes daily during storage without affecting hatchability which contradicts to management procedures. These results suggest that it is unnecessary for refrigerated trucks to transport eggs from the farm to the hatchery.

A study was conducted to determine effects of differing fat and protein levels in commercial milk replacers on the growth performance and rumen development of neonatal Holstein calves. Fifty-three calves (23 male; 30 female) were assigned to one of three dietary treatments which included 3 milk replacers (20/20; 25/15; 28/20) fed once per day at 10% of birth weight through weaning. Calves were offered a 20% CP calf starter and water free choice beginning on day 5. All calves were weaned on day 42. Body weights were measured at birth and weekly through weaning. Additionally, hip height, wither height, and body length were measured weekly. Feed intake and fecal scores were recorded twice daily. Beginning on day 7 and continuing weekly through weaning, blood samples were collected prior to morning feeding for analysis of IGF-I and on days 14, 28, 42, and 56 for PUN and â-hydroxybutyrate concentrations. On days 28 and 56 rumen fluid was collected for analysis of pH and VFA to evaluate possible differences in rumen development. Calves fed 28/20 milk replacer had greater (P < 0.05) daily starter intake, body weights, PUN, and â-hydroxybutyrate concentrations when compared to calves fed 20/20. There was also a treatment by week interaction (P < 0.05) for starter intake, with calves on 28/20 milk replacer consuming the greatest amounts after week 3 and maintaining the greater starter intake through week 8. A treatment over time effect was observed for fecal scores (P < 0.05). No other differences were observed (P > 0.05) in fecal scores among any treatments. Average wither heights were greater (P < 0.05) in calves fed 28/20 milk replacer. There were no effects (P > 0.05) of treatment on IGF-I concentrations, rumen pH, or concentrations of VFA. Acetate, as a percent of total VFA, tended (P < 0.10) to be greater in calves fed 28/20 milk replacer. These data indicate that increasing the dietary protein content of milk replacer without reducing fat content improved growth in young dairy calves without compromising health or rumen development.

Three oryx species and nine of eighteen antelope species across three tribes are considered endangered by the IUCN. Though the common eland (Taurotragus oryx) is not endangered, it lends itself well to the adaption of domestic assisted reproductive techniques due to its large size and calm temperament (Hansen et al., 1985). For these techniques to be used efficiently however, the details of the estrous cycle need to be defined within this and other species. Previously, Nowak (1999) proposed the eland estrous cycle to be ~21 days. In addition to cycle length, specifics of the estrous cycle have yet to be defined and are the objectives of this project. The overall objective is the characterization of the common eland estrous cycle. Specific objectives were to (1) observe behavior peri-estrus (2) determine ovulatory follicle size and (3) produce hormone profiles. The study animals consisted of two groups of four eland females (n=8) housed as a single bachelorette herd at ACRES. Each group was administered both of two commercial estrus synchronization regimens: Lutalyse® protocol (Regimen 1) and altrenogest protocol (Regimen 2). Regimen 1 received PGF2á injections on day 0 and day 11. Regimen 2 received altrenogest for 7 days and given PGF2á on day 7. After regimen administration intensive sample collections were performed around time of expected estrus and then repeated at expected time of subsequent natural estrus: blood samples were taken every 12 hours and ultrasonography was performed every 24 hours until the disappearance of a large follicle. HeatWatch® patches were applied to detect mounting behavior. Mounts were recorded least often during late morning (0600 to 1200). Dominant and ovulatory follicle size was determined to be 7 to 10 mm in diameter and estrous cycle duration was 21 ± 1.6 days. A total of seven of 30 possible ovulations (23.3%) was detected across both regimens. Homosexual mounting behavior was recorded, though all mounts were 2 seconds or less. Although further work needs to be conducted to confirm, the parameters defined here should help in the application of reproductive techniques to nondomestic ungulates.

Nuclear transfer (NT) results in low success rates and research has reported problems with the welfare of cloned animals produced. Evidence suggests the use of a less differentiated donor cell, such as a stem cell, may lead to an increase in the efficiency of the NT procedure. Stem cell characteristics must be clearly defined in order to isolate, identify and optimally culture potential donor cells. Stem cell characteristics such as self-renewal and differentiation have not been identified in stem cells from porcine adipose tissue, an easily attainable tissue resource. The phenotypic profile of cell surface proteins and chromosomal stability of porcine adipose tissue-derived stem cells (pASCs) through in vitro culture have not been described. Also, research has not identified epigenetic modifications in adult somatic mesenchymal stem cells (MSCs) from porcine adipose tissue. The objective of Project 1 was to develop a protocol for the isolation and culture of pASCs and to determine stem cell-like characteristics. The objective of Project 2 was to characterize the surface protein phenotype of undifferentiated porcine ASCs and to determine the chromosomal stability through in vitro culture. The objective of Project 3 was to analyze pASCs and compare them to fetal porcine fibroblasts (FPF) for gene expression profiles of chromatin remodeling proteins, global methylation and acetylation patterns and methylated lysine residues within promoter regions of developmentally important transcription factors. Improved culture conditions and defined cellular characteristics of these pASCs have been identified. pASCs can self-renew and differentiate into multiple tissue lineages. Porcine ASCs do not express the stem cell surface markers analyzed at similar levels compared with those of human ASCs reported in the literature. Also, in vitro-cultured porcine ASCs used as donor cells for NT should be chosen from early population doublings (PDs) due to increased levels of aneuploidy at later PDs. Further characterization of porcine ASCs in this manner could assist the isolation and purification of a population of MSCs from the easily obtainable tissue of adipose that can later be tested as donor cells in nuclear transfer.

Gastrointestinal nematode (GIN) parasitism is a problem for small ruminant producers. Haemonchus contortus is the most detrimental because of substantial economic losses. Over the past decades, chemical anthelmintics have been used to control GINs. Recently, GINs have developed resistance to most anthelmintics, and now reliance on anthelmintics is not possible. Alternative control methods are needed. Also, the use of fewer chemicals in agricultural products has called for alternative methods to be less synthetic and more organic. One promising alternative method is the feeding of condensed tannin (CT) containing forages as either fresh forage or dried products such as hay, ground hay, and pellets. Studies have shown that grazing or feeding sericea lespedeza (SL), a forage plant high in CTs, as whole plant resulted in some level of controlling H. contortus infection. The leaf has the highest CT content; so this study was conducted to determine which percentage of SL leaf meal was the most effective at controlling H. contortus infection in lambs and to determine any effect on reducing GIN larval development in the feces. Thirty-two cross bred lambs were randomly allocated into 4 groups with 8 animals each for a 5 week confinement trial. One group served as the control and received no SL. The other 3 groups received diets containing 25, 50, and 75% SL. Each week, feces and blood were collected to monitor infection level based on fecal egg count (FEC), blood packed cell volume (PCV), and enumeration and identification of larvae from fecal cultures. Fecal egg count decreased significantly (P < 0.05) over time for all 4 groups; however, there were no significant differences between groups. There was a trend on regression analysis for the control group FEC to be significantly (P < 0.001) greater during the trial. Reduction in FEC for the 3 treatment groups was 37.44-67.28% subsequent to week 1. There was no effect on PCV or nematode larvae population distribution and viability in feces. The antiparasitic effect of SL fed as leaf meal was not conclusive in this trial, but there was some evidence that suggested further evaluation is warranted.