Since the cloning of Dolly there has been little change in the efficiency of nuclear transfer (NT). Research is beginning to investigate the characteristics of donor cells. Adiposetissue is an abundant source of adult-derived cells that have displayed stemness in-vitro(Gimble et al., 2003). The overall goal of this research was to define the in-vitro characteristicsof bovine adipose-derived adult stem cells (ADAS) for the use in NT. Isolation methods weredetermined by a 3 x 3 factorial design. 1 g of subcutaneous fat was collected and subjected to0.10%, 0.25% or 0.50% collagenase type I solution for 1, 2 and 3 h. Nucleated cells werecounted using heochst stain. There was no significant difference (P>0.05) in number of nucleated cells released during the incubation period or collagenase concentrations. Viable cells were determined by those that remained adherent 24 h post plating. Incubation in 0.25% collagenase for 2 h had the consistently highest percentage of viable cells (45%). The lifespan and growth characteristics were determined by in a 2 x 2 factorial experiment of DMEM or DMEM:F12 supplemented or not supplemented with growth factors. DMEM with growth factors supplementation was significantly shorter lifespan (P>0.05) than DMEM:F12. The averagelifespan was ~30 population doublings (PDs), with 1 cell cycle every two days until passage 8(P8). Two bovine ADAS cell lines were differentiated into adipocytes, chondrocytes and osteoblasts at middle and late passages along side of adult derived skin fibroblasts. Differentiation was confirmed by histological staining resulting in early passage ADAS cells staining more intensely compared to late passage ADAS cells and skin fibroblasts. Global levels of DNA methylation and histone acetylation were analyzed from P1 to P6 in ADAS and skin fibroblasts from three animals. There was no significant difference (P>0.05) between cell types for DNA methylation or histone acetylation. The percentage of cleaved and developing blastocyst embryos from the ADAS cells (62% and 8%) and skin fibroblasts cells (42% and 8%)were not different (P>0.05). Interspecies nuclear transfer utilized eland ADAS cells into enucleated bovine oocytes. A total of 3 interspecies embryos (1%) developed to blastocyst.

The purpose of this research was to determine the state and regional mineral status of Louisiana forages and beef cattle. Louisiana beef cattle operations (n = 25) were sampled and divided into seven geographical regions, including the northwest (NW), northeast (NE), central (CE), southwest (SW), south central (SC), Florida parishes (FP) and southeast (SE) regions. Over a two year period, water and soil samples were collected from each operation annually, forage samples were collected quarterly in Aug to Sep, Nov to Dec, Feb to Mar and May to June and bovine serum samples were collected twice annually in the fall and spring seasons. The highest (P < 0.05) average regional water K and S concentrations were observed in the SE region and water Ca and Mg concentrations were the highest (P < 0.05) in the NE, CE and SE regions. However, all water mineral concentrations, with exception of Na, were lower than the reported upper desired levels considered safe for livestock consumption (Socha et al., 2003). Similar to water, soil Ca, Mg and K concentrations in our study, were higher (P < 0.05) in the SE compared to all other regions. Soil Cu concentrations were below critical levels in the CE region and all soil Zn concentrations, except the SE region, were lower than reported critical levels indicating soil deficiency. The average forage concentration for each mineral were: Ca (0.42%), P (0.28%), Mg (0.21%), K (1.83%), Na (0.10%), S (0.32%), Cu (8.12 ppm), Fe (323.46 ppm), Mn (254.85 ppm) and Zn (41.29 ppm). In addition, only mean forage Cu concentrations were lower than minimum requirements and regional forage K (NW region), Mg (FP region), Na (CE region) and S (NW and SE regions) concentrations were higher (P < 0.05) than other regions. The average regional serum K concentration in the NE region was higher (P < 0.05) than all other regions. Average bovine serum mineral concentrations in Louisiana were: Ca (9.02 mg/100 ml), P (13.62 mg/100 ml), Mg (1.92 mg/100 ml), K (21.66 mg/100 ml), Na (303.30 mg/100 ml), S (103.31 mg/100 ml), Cu (0.63 µg/ml), Fe (7.44 µg/ml), Zn (1.28 µg/ml), Mn (8.08 ng/ml) and Se (64.48 ng/ml). Furthermore, of these minerals, serum Mg, Na, Cu and Mn concentrations were lower than critical levels, indicative of deficiency.

A study was conducted to determine the effects of varying levels of cottonseed hulls on growth and metabolic indications of rumen development of dairy calves. Sixty-four Holstein calves (Heifers, n=40; Bulls, n=24) were randomly assigned to one of four dietary treatments which included calf starters containing no cottonseed hulls (control; C), 10% cottonseed hulls (10% CSH), 15% cottonseed hulls (15% CSH), or 20% cottonseed hulls (20% CSH). Calves were fed their respective treatments beginning on day 6 until day 56 of age. Body weights were measured at birth and biweekly thereafter until day 56 of age. Wither and hip heights were measured beginning on day 14 and biweekly thereafter until day 56 of age. Feed intake and fecal scores were recorded twice daily through day 56. On days 14, 28, 42, and 56, rumen fluid was collected for analysis of pH, volatile fatty acids (VFA), and ammonia (NH3), and blood was collected for analysis of plasma urea nitrogen (PUN) and â-hydroxybutyrate (BHBA). There was no treatment effect on average daily starter intake, body weight, wither height, and hip height but a treatment effect on fecal scores. Calves consuming CSH had higher rumen pH than C. Rumen pH also decreased as calves aged. There was no treatment effect on rumen acetate, propionate, butyrate, and total VFA concentrations. A treatment effect on NH3 concentrations was observed, and NH3 concentrations decreased over time. There was no treatment effect on BHBA, but a main effect of sex was observed in which the males had greater BHBA levels. There was no treatment effect on PUN concentrations, but a main effect of sex was observed with females having greater PUN concentrations. Overall, incorporating cottonseed hulls into a calf starter showed no significant effect on growth and rumen development in Holstein dairy calves.

This study investigated the effects of semen extender constituents and cryopreservation on bovine spermatozoal mitochondrial function. Three yearling Holstein bulls were used. Two ejaculates per bull were collected and pooled on a weekly basis for five weeks and extended in four treatments: 1) sodium citrate egg yolk extender with antibiotics (lincomycin, spectinomycin, gentamicin and tylosin); 2) ¡°1¡± with glycerol; 3) ¡°2¡± without antibiotics; and 4) ¡°1¡± without antibiotics. Each was divided into portions for analyses before freezing and after cryopreservation. The pre-freeze and thawed semen were transferred to a 37¡ãC water bath, the same assays were performed. In experiment 1, resazurin reduction (RD) was measured spectrophotometrically at sequential 25 minute intervals for 125 minutes. In experiment 2, specific activities of cytochrome c oxidase (CytoCox) and citrate synthase (CS) were measured spectrophotometrically immediately post-thaw and after 125 minutes of incubation. In experiment 3, ATP was measured using luciferin-luciferase assay simultaneously with RD. Total and progressive motilities (TM and PM), progressive (PV), curvilinear (VCL) and pathway (VAP) velocities were measured simultaneously with RD, ATP content and CytoCox and CS using computer assisted semen analysis system (CASA). In experiment 4, the NADH dehydrogenase (ND1) gene of mtDNA was sequenced before and after cryopreservation using PCR. Data were analyzed by least square methods; mean differences were delineated by Tukey¡¯s test. In experiment 1, RD differed among treatments (P<0.05). Cryopreservation decreased (P<0.05) RD, TM, PM, PV, VAP and VCL. Resazurin reduction correlated with PM (r=0.45, P<0.05) and TM (r=0.2, P<0.05). In experiment 2, incubation time and incubation with Triton X100 were sources of variation in CytoCox and CS specific activities (P>0.05). Only CS from spermatozoa incubated with Triton X100 correlated with RD (r=0.22, P<0.05). CytoCox and CS did not correlate with motility parameters. In experiment 3, spermatozoal ATP was not different (P>0.05) among treatments. However, cryopreservation decreased (P<0.05) ATP. Spermatozoal ATP correlated with motility parameters (r¡Ý0.65) and RD (r=-0.30) (P<0.05). In experiment 4, the frequency of amino acid change was higher (P<0.05) post-thaw in the treatment containing only antibiotics. Cryopreservation, more than extender constituents impacted mitochondrial function of bovine spermatozoa.

Gastrointestinal nematode parasites cause extensive damage to small ruminants, and Haemonchus contortus is a major concern to production. In the past, small ruminants were dewormed at regular intervals and control methods were based primarily on the use of anthelmintics. At present, anthelmintic resistance has been reported worldwide and has developed into a serious problem for small ruminant management programs. In view of this, alternate control methods are needed. One alternative method is the use of copper oxide wire particles (COWP). Three independent trials were conducted during Spring, Summer, and Fall of 2008. Two trials evaluated the effect of COWP in food pellets in reducing H. contortus infection in crossbred ewes and Suffolk lambs. The third trial compared the effect of Copasure® COWP with that of an industrial-grade COWP. Trials consisted of similar protocols where ewes and lambs were allocated into groups based on fecal egg count (FEC). Copasure® COWP in feed pellets were fed to ewes at mid-lactation when FEC increased to over 1000 eggs per gram, and to individual lambs when FAMACHA© score was 4/5. Ewe/lamb infection was monitored weekly by FEC and blood packed cell volume. Results of Trial 1 indicated that Copasure® COWP in feed pellets was effective in reducing the peri-parturient rise in ewes, based on FEC. Trial 2 indicated that Copasure® COWP in feed pellets was just as good as the levamisole/albendazole treatment when the FAMACHA System© was used to determine when to treat lambs. Trial 3 indicated that Copasure® COWP effectively reduced parasite infection while the industrial-grade COWP did not. The results from these trials demonstrated that the use of Copasure® COWP in feed pellets reduced H. contortus infection and may be useful alone or when used with other control methods.

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.