Twelve neonatal Holstein bull calves (38.52 ± 5.87 kg) were fed milk replacer at a fixed or varied meal time to determine the effects on metabolic hormone secretion, average daily intake, growth, and energy metabolism. Body weights were measured every two weeks from birth to 8 weeks. Rumen fluid was collected every two weeks from week 2 through 8. Serial blood collections were conducted every two weeks from week 2 through 8. Blood was collected, beginning at 0530, at 0, 15, 30, 45, 60, 75, 90, 105, 120, 135, and 150 minutes. Plasma was analyzed for ghrelin, leptin, growth hormone (GH), and insulin-like growth factor-1 (IGF-1). Treatment did not affect body weight or average daily intake. Mean plasma ghrelin, leptin, GH, and IGF-1 concentrations were not affected by treatment. A treatment by week interaction was observed for plasma ghrelin concentrations. Plasma ghrelin concentrations were higher at weeks 2 and 4 in control calves. Plasma ghrelin concentrations decreased in all calves as they aged. A treatment by time interaction was observed for IGF-1, and a treatment by week by time interaction was observed for growth hormone and IGF-1. Growth hormone decreased as calves aged, while IGF-1 increased. There was no treatment effect or interactions of treatment and week on butyrate and propionate concentrations. However, both butyrate and propionate increased with age. Treatment and week effects were present for acetate, as well as a treatment by week interaction. Calves in the control group had a higher percentage of acetate. Acetate concentration increased in all calves as they aged. At weeks 4 and 8, intravenous glucose tolerance tests were performed to assess glucose clearance. A treatment effect was observed for glucose half life (T1/2), glucose clearance rate (k), and insulin. Glucose half- life was higher for calves in the control group, while the clearance rate was lower for the control group. Peak insulin concentrations were higher for calves in the treatment group. It is concluded that feeding time does not affect overall growth and feed intake, but does have an affect on the some of the regulatory mechanisms that control them.

Incomplete zona hatching or failure of the zona to rupture compromises post-transfer embryo viability and conceptus development. Assisted hatching prior to the transfer of frozen-thawed bovine embryos has been proposed as a means to increase recipient pregnancy rates. The objective of this study was to determine if laser-assisted hatching would improve in vivo-produced frozen-thawed bovine embryo hatching and pregnancy rates. In Experiment I,II and III frozen direct-transfer embryos received either two a or three symmetrical rents at either 40% or 80% through the outer zona surface using the Hamilton Thorne XYClone® (Hamilton Thorne Biosciences) diode laser at 90% power with a 600 µsecond pulse (Treatment A) or no zona renting (Treatment B). Embryo hatching rates combined were 51% of 86 embryos for Treatment A and 54% of 86 embryos for Treatment B. In Experiment IV, in vivo-produced nonsurgically collected direct transfer frozen-thawed Hereford embryos (n = 64) were utilized. In Experiment V, in vivo-produced nonsurgically collected glycerol frozen-thawed Brangus embryos (n = 46) were utilized. In Experiments IV and V, embryos received three symmetrical rents ~40% through outer zona surface at 90% power with a 600 µsecond pulse (Treatment A) or no zona renting (Treatment B). In Experiment IV, treatment did not affect pregnancy rates at 35 days or 60 days of gestation and were 41% and 28% for Treatment A and 44% and 41% for Treatment B, respectively. Likewise, there was no difference in calving rate for recipients confirmed pregnant at 60 days for Treatment A (89%) and Treatment B (77%). In Experiment V, pregnancy rates at 35 days and at 60 days of gestation were not affected by treatment and were 65% and 65% for Treatment A and 78% and 65% for Treatment B, respectively. Calving rates were not different for those recipients in Experiment V confirmed pregnant at 60 days for Treatment A (73%) and Treatment B (73%). In conclusion, laser-assisted hatching does not increase the number of in vivo-produced bovine embryos that hatch following in vitro culture or increase pregnancy rates of recipients receiving in vivo-produced frozen-thawed embryos.

The objectives of this research were 1) to determine the effect of replacing dried whey (DW) with milk chocolate product (MCP), dried whey permeate (DWP) with candy oats (CO), and spray-dried plasma protein (SDPP) with a novel swine nutritional supplement (SNS) on growth performance of weanling pigs, and 2) to determine the maximum level of supplemental L-Lys, along with DL-Met, L-Thr, and L-Trp that can be added in diets for 6- to 12 and 13- to 23-kg pigs. Three experiments were conducted to compare the feeding value of MCP (20% lactose and 60% sugars) and DW (70% lactose). Results from these experiments indicate that partial or total replacement of DW with MCP did not affect wk-1 feed intake or growth performance of weanling pigs. A similar experiment was conducted to compare the feeding value of CO (60% total sugars and 25% cooked oat-based cereals) and DWP (80% lactose). Results from this experiment indicate that a combination of DWP and CO increased wk-1 feed intake and growth performance of weanling pigs. Thus, MCP or CO could be considered as a formulation alternative to DW or DWP, respectively. Two experiments were conducted to determine the effect of replacing SDPP with SNS (concentrated plasma fraction). Results from these experiments indicate that the inclusion of SDPP or its replacement with SNS did not affect growth performance of weanling pigs. Eight experiments were conducted to determine the maximum level of supplemental L-Lys, along with DL-Met, L-Thr, and L-Trp that can be added in diets for 6- to 12-kg and 13- to 23-kg pigs without negatively affecting growth performance. Results from these experiments indicate that supplemental L-Lys levels of 0.198 and 0.298% or 0.331 and 0.423% can be added in diets for 6- to 12 or 13- to 23-kg pigs without negatively affecting G:F or ADG, respectively. The optimum SID Ile:Lys may not be greater than 0.55 in diets for 6- to 12-kg pigs containing low levels of red blood cells. The optimum SID Ile:Lys and SID Val:Lys may not be greater than 0.56 or 0.62, respectively, in corn-SBM diets for 13- to 23-kg pigs.

The addition of serum to embryo culture media may alter gene expression and trigger development of Large Offspring Syndrome. The objectives of this study were to determine gene expression levels in embryos cultured in the absence or presence of 5% calf serum and compare these expression patterns to in vivo derived embryos (IVD), and to determine the effects of serum on the length of day-14 embryos. Abattoir derived oocytes were fertilized and cultured in mSOFaa. At 72 hours post-insemination (hpi), embryos were randomly allocated into two treatments: mSOFaa without and with 5% calf serum. Embryos were then cultured to 168 hpi and blastocyst rates were assessed. In experiment 1, blastocysts from each treatment were pooled and stored at -80°C. In experiment 2, blastocysts (n=5-10) from each treatment were transferred into synchronized recipients, and were recovered 7 days post-transfer. Embryos were photographed, measured, and immediately stored at -80°C. Isolation of mRNA, reverse transcription and quantitative PCR were performed to determine transcript abundance for COX6A, IFNT1a, PLAC8, IGF2R and GAPDH for each sample. In both experiments, blastocyst development rates were higher in embryos cultured with serum compared to the no-serum treatment (14.9 and 7.4% respectively, P<0.001). In experiment 1, no differences were found in the expression of COX6A, IFNT1a, IGF2R and PLAC8; however upregulated expression of IGF2R, COX6A and IFNT1a were observed in some samples in both IVP treatments. In experiment 2, lengths of elongated embryos from the serum and no-serum culture treatments differed from the IVD treatment. Mean expression levels for COX6A, IFNT1a, PLAC8 and IGF2R did not differ across treatment groups. However, in the serum treatment 3 of 11 embryos over-expressed IFNT1a, 4 of 11 over-expressed IGF2R and 2 of 11 over-expressed PLAC8, over-expression being defined as two standard deviations above the mean of the IVD treatment for each respective gene. While mean expression levels were not affected by culture with serum under these conditions, very high expression of IFNT1a, IGF2R and PLAC8 in experiment 2 and IGF2R and IFNT1a in experiment 1 was observed in some embryos cultured with serum, but not in embryos cultured without serum or in in vivo derived embryos.

The role of glucocorticoids on intestinal closure in neonates has recently become an area of interest but a definitive mechanism remains to be identified. It is known that glucocorticoids enhance immunoglobulin absorption in dairy calves, but the role of maternal glucocorticoids at parturition is not clear. In the present experiment, we obtained plasma and milk samples from primiparous and multiparous Holstein cows (n=24) to measure cortisol at 72, 48 and 24 hours before and 3, 6, 12, 24 and 48 hours after spontaneous parturition. After parturition, calves (n=24) were immediately removed from their dams and a blood sample was taken from the calf before colostrum ingestion (3 hours postpartum). Calves were sampled at 6, 12, 24 and 48 hours postpartum to measure plasma cortisol concentration and determine the concentration of plasma immunoglobulin G (IgG). Milk cortisol concentration in cows was significantly lower compared with plasma (5.4 ng/ml vs. 10.2 ng/ml) (P<0.0001). Cortisol in plasma and milk at 24 hours prepartum was significantly lower compared with 3 hours postpartum (4.2 ng/ml vs. 11.2 ng/ml) (P<0.05). Calf plasma cortisol was significantly elevated at 3 hours postpartum (258.1 ng/ml) and declined thereafter to 60.6 ng/ml by 48 hours postpartum (P<0.0001). Calf IgG in plasma was significantly lower at 3 hours postpartum (134 mg/ml) compared with 12 and 24 hours postpartum (2,324 mg/ml and 3,015 mg/ml, respectively), indicating minimal concentrations of antibody in plasma before colostrum ingestion. Calves were divided into low (n=11) and high (n=9) cortisol groups based on mean plasma cortisol concentration in their dams. Although the difference in cortisol at 3, 6 and 12 hours postpartum between cows of low cortisol and high cortisol (20.5 ng/ml vs. 39.1 ng/ml, 1.6 ng/ml vs. 19.1 ng/ml and 2.9 ng/ml vs. 13.4 ng/ml, respectively) was significantly different (P<0.05), there was no significant difference between cortisol or IgG concentration in low cortisol compared with high cortisol calves. The present results indicate that maternal cortisol concentration at parturition does not influence calf cortisol concentration at birth or IgG concentration.

The differentiation potential of adult stem cells (ASC) has long been thought to be limited to cell lineages present in the organ from which they are derived; however, several studies have challenged this notion by demonstrating that some ASC exhibit a remarkably high degree of plasticity. Unlike terminally differentiated somatic cells, the less differentiated state of ASC can assume the functional phenotypes and expression profiles of cells unique to other tissues. The expansive repertoire of differentiation potential exhibited by ASC suggests these cells possess characteristics similar to pluripotent cells, including epigenomic regulatory pattern. Therefore, ASC may be better equipped for complete epigenetic reprogramming than terminally differentiated cells. The objective of Experiment 1 was to analyze bovine adipose-derived adult stem cells (ADAS) and fetal fibroblast (BFF) cells for the presence of the pluripotency-associated genes, Oct-4, Nanog, and Sox-2. Because the endogenous expression of these genes is believed to contribute to reprogramming efficiency, Experiment 2 sought to increase Oct-4, Nanog and Sox-2 expression levels in BFF cells through exposure to ADAS cell extracts. Transcripts for all three pluripotency-associated genes were detected in all BFF and ADAS cell samples at every passage analyzed; however, expression was quite low and highly variable between cell lines and passage numbers. Nevertheless, these findings support the notion that these cells are less differentiated than other somatic cells. This less differentiated state appears to sufficient for at least the partial reprogramming of BFF cells using ADAS cell extracts in a cell extract-based nuclear reprogramming system.

Streptococcus salivarius ssp. thermophilus and Lactobacillus delbrueckii ssp. bulgaricus are dairy cultures widely used in the dairy industry. Low sonication intensity condition is a non-destructive technique that uses sound waves to cause cavitation in aqueous solutions and may improve the permeability of membranes, speed up the transfer of substrates and promote cellular growth and propagation. The objective of this study was to determine the effect of low sonication intensities at different temperatures on acid tolerance, bile tolerance, protease activity and growth of the two dairy cultures. The cultures were freshly thawed and suspended in 0.1% peptone water and 18 ml of sample was sonicated using horn (diameter 13 mm) set at a maximum acoustic power output of 750 W, frequency 24 kHz. The treatments were four sonication intensities of 8.07, 14.68, 19.83 and 23.55 Watts/cm2 randomized at three different temperatures (4, 22 and 40°C) of inoculated peptone water before sonication. The energy input (1500 Joules) was kept constant in all treatments. Control samples did not receive any sonication treatment. Growth and bile tolerance of samples were determined hourly for 12 hours of incubation. Acid tolerance was determined for Streptococcus thermophilus every 5 minutes for 20 minutes of incubation and for Lactobacillus bulgaricus every minute for 5 minutes of incubation. Protease activity was determined at 0, 12 and 24 hours of incubation. The experimental design was a completely randomized design (CRD). Three replications were conducted for each experimental condition. Data were analyzed using Proc Mixed Model of Statistical Analysis System (SAS®). Differences of least square means were used to determine significant differences at P<0.05 for main effects (low sonication intensity, time and temperature) two way interaction effect (low sonication intensity * temperature and low sonication intensity * time) and three way interaction effects (low sonication intensity * time * temperature). Low sonication conditions include a) low sonication intensities, b) temperatures and c) times, all three of which played a role in influencing the desirable attributes of both microorganisms. Of all the low sonication intensities studied, 14.68 watts /cm2 had the best overall influence at certain time points for Streptococcus thermophilus improving its acid tolerance, bile tolerance and growth at 4°C, growth at 22°C, bile tolerance and growth at 40°C and improving the Lactobacillus bulgaricus bile tolerance and growth at 4°C, its acid tolerance and protease activity at 40°C. Low sonication intensity of 19.83 Watts/cm2 had the overall best influence at certain time points for acid tolerance of both microorganisms at 22°C. Low sonication intensity of 23.55 Watts/cm2 had the overall best influence at certain time points for protease activity of Streptococcus thermophilus at 40°C and Lactobacillus bulgaricus at 22°C. Some low sonication conditions improved certain characteristics of culture bacteria.

Equine embryo cryopreservation is unsuccessful in larger, more easily collected, day-7 embryos. It is imperative that methods to successfully cryopreserve large equine embryos or develop reliable methods to determine embryo size before collection. Therefore the objectives for this study were to quantify the amount of tritiated glycerol that would permeate various sizes of equine embryos and to determine if circulating progesterone concentration was correlated with in utero embryo size. Mean embryo diameter (± SEM) across treatments (1.4M and 3.4M tritiated glycerol) was 696.5µm ± 108.6µm and 925.9 µm ± 214.1µm, respectively and were not different (P=0.44). The percent permeation for 1.4M and 3.4M glycerol were not different (P=0.68). Embryos <400 µm in the 1.4M glycerol treatment group had higher (P=0.002) permeation than embryos >400 µm, 8.32% ± 3.85% and 0.35% ± 0.11%, respectively. Length of time, 60 or 120 minutes, did not affect amount of glycerol uptaken (P=0.26. Serum progesterone concentrations on day 7 post-ovulation were higher (P=0.009) for mares who produced two viable embryos from double ovulation (24.17±2.82ng/ml) compared with mares from which a single embryo (14.04±0.99ng/ml) was collected and control mares (13.53±1.80ng/ml). No differences (P=0.91) were detected in serum progesterone concentration on day 7 post-ovulation between mares from which a single embryo (14.04±0.99ng/ml) was collected and control mares (13.53±1.80ng/ml). Mares producing embryos >400µm tended to have higher (P=0.08) circulating progesterone concentrations than mares producing embryos <400µm. Serum progesterone concentrations day 7 post-ovulation in mares producing embryos >400µm and <400µm were not different (P=0.61 and P=0.68, respectively) than control mares. Single embryos <1000µm in diameter were correlated with circulating progesterone concentration day 7 post-ovulation (r=0.46, P=0.006). There was no significant correlation between embryo diameter, corpus luteum diameter, and serum progesterone concentration day 7 post-ovulation.This is the first study to quantify the amount of glycerol permeating into equine blastocysts and suggests that the capsule may be a barrier to cryoprotectant permeability. Maternal progesterone concentrations day 7 post-ovulation could be utilized in predicting embryo stage and size prior to collection for cryopreservation and in diagnosis of twin pregnancies as a result of double ovulation.

Seven experiments were performed to assess the use of glucose responses to insulin injections as a means of estimating insulin sensitivity in horses; to compare the insulin sensitivities of normal horses vs. those displaying hyperleptinemia; and to put this method into practical application. Experiment 3.1 examined dose-responses in mares of potentially different insulin sensitivities. Recombinant human insulin was injected at doses of 8, 20, 50, and 125 mU/kg BW, as needed, to estimate the dose of insulin causing a 50% decrease in glucose concentrations (ED50). Five mares each of low leptin concentrations (LL) and low BCS, LL and high BCS, and high leptin concentrations and high BCS, were studied. The ED50 was similar for LL mares, regardless of BCS, and was lower (P < 0.01) than for mares with high leptin concentrations. It was concluded that a dose of 50 mU/kg BW of recombinant human insulin could be used safely to start the dose-response curve; lower or higher doses could then be used to estimate ED50. Experiment 3.2 assessed the repeatability of the estimates for ED50 obtained in Exp. 3.1. Estimates obtained were highly correlated (R2 = 0.822) with those obtained in Exp. 3.1, with an average within-mare CV of 8.9%. The next five experiments studied the effects of 1) prior administration of epinephrine, 2) overnight feed deprivation versus hay or pasture consumption, 3) 10-d acclimatization to hay in a dry lot versus pasture grazing, 4) cinnamon extract supplementation, and 5) fish oil supplementation on insulin sensitivity. Epinephrine stimulated blood glucose (P < 0.05) and prevented the insulin-induced decrease in blood glucose in both sensitive and insensitive mares. Overnight feed deprivation decreased (P < 0.06) insulin sensitivity relative to overnight ad libitum access to hay, and both regimens resulted in reduced insulin sensitivity relative to overnight pasture availability. Ten days of hay consumption in a dry lot reduced (P < 0.05) insulin sensitivity in insensitive mares relative to pasture grazing. Supplementation with cinnamon extract or fish oil had no effect on insulin sensitivity of mares with known low insulin sensitivity under the conditions of these experiments.

Forty-two female Holstein calves were assigned to one of three treatments at d 2 of age to study the effects of adding resistant starch (RS) to the milk replacer on health and performance. Treatments were control (0g RS), 4g RS, or 8g RS mixed into the reconstituted milk replacer. Calves were housed in individual calf hutches and fed milk replacer once daily until d 42 of age. Water and an 18% crude protein calf starter were offered ad libitum beginning d 3 throughout the duration of the 56 d trial. Calves remained in their hutches until d 56 of age to determine immediate postweaning performance. Body weights (BW) were measured at birth and d 7, 14, 28, 42, and 56 d of age. Wither height (WH), hip height (HH), and hip width (HW) were measured on d 7, 14, 28, 42, and 56 d of age. Feed intake, body temperatures, and fecal scores were recorded once daily through d 56. On d 14, 28, 42, and 56, fecal samples were collected for analysis of pH and short chain fatty acids (SCFA), and blood was collected for analysis of plasma urea nitrogen (PUN) and total protein (TP). The PUN and TP were within normal ranges suggesting that there were no major metabolic problems. There was no effect (P>0.05) of treatment on BW, HH, HW, WH, or body temperatures. There was a treatment by week interaction (P<0.01) and a week effect (P<0.01) for grain intake, with all calves increasing intake throughout the duration of the study. There was a treatment by week interaction (P<0.01) for fecal scores. All calves had lower (P<0.01) fecal scores at the end of the study compared to the beginning. Fecal pH increased as calves aged (P<0.01). There was a treatment by week interaction (P<0.05) for propionate concentration in the feces. Propionate concentrations decreased (P<0.01) until weaning at week 6 for all treatments while calves consuming 4g RS had higher (P<0.05) concentrations compared to those consuming 8g RS over the entire trial. Acetate, butyrate, and total SCFA concentrations all decreased (P<0.01) for all calves until weaning at week 6. Incorporation of RS in the milk replacer of calves did show changes in fecal SCFA at 2 and 4 weeks of age. However, incorporation of RS in milk replacer had no overall treatment effects on health and performance of neonatal dairy calves.