Efficiency of Two Enucleation Methods Connected to Handmade Cloning to Produce Transgenic Porcine Embryos

The purpose of our work was to establish an efficient-oriented enucleation method to produce transgenic embryos with handmade cloning (HMC). After 41–42 h oocytes maturation, the oocytes were further cultured with or without 0.4 μg/ml demecolcine for 45 min [chemically assisted handmade enucleation (CAHE) group vs polar body (PB) oriented handmade enucleation (OHE) group respectively]. After removal of the cumulus cells and partial digestion of the zona pellucida, oocytes with visible extrusion cones and/or polar bodies attached to the surface were subjected to oriented bisection. Putative cytoplasts without extrusion cones or PB were selected as recipients. Two cytoplasts were electrofused with one transgenic fibroblasts expressing green fluorescent protein (GFP), while non-transgenic fibroblasts were used as controls. Reconstructed embryos were cultured in Well of Wells (WOWs) with porcine zygote medium 3 (PZM-3) after activation. Cleavage and blastocyst rates were registered on day 2 and day 7 of in vitro culture respectively. Meanwhile, the total blastocyst cell number was counted on day 7. We found that the difference was only observed between blastocyst rates (38.6 ± 2% vs 48.1 ± 3%) of cloned embryos with GFP transgenic fibroblast cells after CAHE vs OHE. With adjusted time-lapse for zonae-free cloned embryos cultured in WOWs with PZM-3, it was obvious that in vitro developmental competence after CAHE was compromised when compared with the OHE method. OHE enucleation method seems to be a potential superior alternative method used for somatic cell nuclear transfer (SCNT) with transgenic fibroblast cells.     

Effect of an indwelling nasogastric tube on gastric emptying rates of liquids in horses

OBJECTIVE: To evaluate the effect of an indwelling nasogastric tube on gastric emptying of liquids in horses. ANIMALS: 9 healthy adult horses. PROCEDURE: A randomized block crossover design was used. For treatment group horses, a nasogastric tube was placed and 18 hours later, acetaminophen was administered; the nasogastric tube remained in place until the experiment was complete. For control group horses, a nasogastric tube was passed into t stomach, acetaminophen was administered, and the nasogastric tube was removed immediately. Serial blood samples were collected 15 minutes before and after administration of acetaminophen. Serum concentration of acetaminophen was determined by use of fluorescence polarization immunoassay. The variables, time to maximum acetaminophen concentration (Tmax) and the appearance constant for acetaminophen (Kapp), were determined. The values for Kapp and Tmax in horses with and without prolonged nasogastric tube placement were compared. RESULTS: No significant difference was found in Kapp between horses with and without prolonged nasogastric tube placement; the median difference in Kapp was 0.01 min(-1) (range, -0.48 to 0.80 min(-1). No significant difference was found in Tmax between horses with and without prolonged nasogastric tube placement; the median difference in Tmax was 5 minutes (range, -30 to 50 minutes). Reanalysis of data following the removal of possible outlier values from 1 horse resulted in a significant difference in Tmax between horses with and without prolonged nasogastric tube placement. CONCLUSIONS AND CLINICAL RELEVANCE: Although no clinically important impact of 18 hours of nasogastric intubation was found on gastric emptying in healthy was found among horses.     

Evaluation of the effects of penicillin G potassium and potassium chloride on the motility of the large intestine in horses

OBJECTIVE: To evaluate effects of IV administration of penicillin G potassium (KPEN) or potassium chloride (KCl) on defecation and myoelectric activity of the cecum and pelvic flexure of horses. ANIMALS: 5 healthy horses. PROCEDURE: Horses with 12 bipolar electrodes on the cecum and pelvic flexure received KPEN or KCl solution by IV bolus 4 hours apart. Each horse received the following: 2 X 10(7) U of KPEN (high-dose KPEN) followed by 34 mEq of KCl (high-dose KCl), 1 X 10(7) U of KPEN (low-dose KPEN) followed by 17 mEq of KCl (low-dose KCl), high-dose KCl followed by high-dose KPEN, and low-dose KCl followed by low-dose KPEN. Number of defecations and myoelectric activity were recorded for 60 minutes. The first three 5-minute segments and first four 15-minute segments of myoelectric activity were analyzed. RESULTS: Number of defecations during the first 15-minute segment was greater after high-dose KPEN treatment than after high-dose or low-dose KCl treatment. Compared with reference indexes, myoelectric activity was greater in the pelvic flexure for the first 5-minute segment after high-dose KCl treatment, in the cecum and pelvic flexure for the first 5-minute segment and in the pelvic flexure for the first 15-minute segment after low-dose KPEN treatment, and in the pelvic flexure for the first and second 5-minute segments and the first three 15-minute segments after high-dose KPEN treatment. CONCLUSIONS AND CLINICAL RELEVANCE: IV administration of KPEN stimulates defecation and myoelectric activity of the cecum and pelvic flexure in horses. Effects of KPEN may be beneficial during episodes of ileus.     

Familial and herd-level associations with paratuberculosis enzyme-linked immunosorbent assay status in beef cattle

A cross-sectional study was performed to determine the odds of having a positive paratuberculosis ELISA result if the dam was ELISA positive in Texas beef cattle, adjusted for individual and herd-level risk factors for seropositivity. Texas beef cattle (n = 2,621) were tested for paratuberculosis by using a commercial ELISA and microbiologic culture of feces for Mycobacterium avium subsp. paratuberculosis (MAP). Pedigree data were collected to identify dam-and sire-offspring pairs. Bayesian mixed-effects logistic regression was used to estimate the odds of seropositivity associated with age, dam ELISA status, sire ELISA status, herd size, herd history of clinical paratuberculosis, within-herd seroprevalence, within-herd fecal MAP prevalence, and within-herd fecal non-MAP Mycobacterium spp. prevalence. Herd of residence was included as a random effect to account for the correlation of observations within the same herd. Statistically probable associations were observed between ELISA status and herd fecal MAP prevalence [OR (odds ratio) 1.28 per 1% increase; P < 0.001] and herd seroprevalence (OR 1.21 per 1% increase; P < 0.001). The association with dam ELISA status was small (OR 1.35) and not highly probable (P = 0.69). Results indicate that use of dam ELISA status to make culling decisions in beef cattle may not improve the success of paratuberculosis control programs. Alternative strategies may be more effective for reducing the odds of seropositivity.     

Assessing familial aggregation of paratuberculosis in beef cattle of unknown pedigree

The objective of this study was to assess genetic similarity of beef cattle using microsatellite markers and to use this information to describe familial aggregation of paratuberculosis test results in Texas beef cattle. Paratuberculosis testing was performed on 2622 adult beef cattle using two commercially available serum ELISAs and radiometric fecal culture. Pedigree records were collected for registered purebred herds and herds with sufficiently detailed production records to identify parent-offspring pairs. Cases were defined as cattle with at least one positive paratuberculosis test result. Three controls were matched by herd of residence for each case. All parent-offspring pairs, cases, and controls were genotyped for 12 microsatellites. Bayesian analysis of allele frequency data was used to describe population substructure and assign individual cattle into groups of genetically similar cattle. The proportion of known parent-offspring pairs assigned to the same cluster was used to assess the validity of the approach to identify familial structure. Conditional logistic regression was used to describe the association between cluster assignment and paratuberculosis test-status matched by herd. Nine clusters of genetically similar individuals were identified and were supported by the proportion of parent-offspring pairs assigned to the same clusters. Increased odds of having at least one positive paratuberculosis test result were identified for two clusters compared to the cluster with the lowest proportion of positive paratuberculosis test results after conditioning on herd. The results of this study demonstrate that population substructure can be used to describe familial aggregation of paratuberculosis test results in beef cattle of unknown pedigree.     

Brown trout fry move inshore at night: a choice of water depth or velocity?

Abstract –  The instream positions of brown trout fry differ between daylight and darkness. According to field and laboratory observations, recently emerged 0+ brown trout use shallow and slow-flowing areas close to the bank at night and tend to move off-shore during daylight. In laboratory channels, we tested whether the use of habitats close to the river bank could be attributed to a choice of either water depth or velocity. In two complementary experiments, emerging brown trout alevins were given the choice of using shallow-slow or deep-swift habitats (experiment 1), and deep-slow or shallow-swift habitats (experiment 2). At night, a persistent preference for the shallow habitats was displayed, regardless of velocity. It was concluded that swim-up brown trout fry respond to shallowness rather than ambient low water velocity when selecting habitats close to the bank at night. The behavioural significance of this result and implications for river management are discussed.     

Bacterial microflora of normal and telangiectatic livers in cattle.

OBJECTIVE: To identify potential bacterial pathogens in normal and telangiectatic livers of mature cattle at slaughter and to identify consumer risk associated with hepatic telangiectasia. SAMPLE POPULATION: 50 normal livers and 50 severely telangiectatic livers. PROCEDURE: Normal and telangiectatic livers were collected at slaughter for aerobic and anaerobic bacterial culture. Isolates were identified, and patterns of isolation were analyzed. Histologic examination of all livers was performed. RESULTS: Human pathogens isolated from normal and telangiectatic livers included Escherichia coli O157:H7 and group-D streptococci. Most livers in both groups contained bacteria in low numbers; however, more normal livers yielded negative culture results. More group-D streptococci were isolated from the right lobes of telangiectatic livers than from the left lobes, and more gram-negative anaerobic bacteria were isolated from left lobes of telangiectatic livers than from right lobes. All telangiectatic lesions were free of fibrosis, active necrotizing processes, and inflammation. CONCLUSIONS AND CLINICAL RELEVANCE: The USDA regulation condemning telangiectatic livers is justified insofar as these livers contain more bacteria than normal livers do; however, normal livers contain similar species of microflora. Development of telangiectasia could not be linked to an infectious process. The finding of E coli O157:H7 in bovine livers suggests that information regarding bacterial content of other offal and muscle may identify sources of this and other potential foodborne pathogens and assist in establishing critical control points for the meat industry.     

Mitochondrial Activity, Distribution and Segregation in Bovine Oocytes and in Embryos Produced in Vitro

Contents Bovine oocytes and embryos produced in vitro were studied to determine the mitochondrial pattern of distribution, segregation and activity using DIOC 6 and Jc-1 fluorescence. The highest fluorescence level observed in mature oocytes was taken as 100% activity and six activity levels were estimated as follows: (1) 0%, (2) 1-15%, (3) 16-30%, (4) 31-50%, (5) 51-75% and (6) 76-100%. Three patterns of mitochondrial distribution were found: (1) diffused throughout the cytoplasm in oocytes and embryos, (2) pericytoplasmic in oocytes and embryos, and (3) perinuclear only in embryos. The segregation of mitochondria in blastomeres showed two distinct patterns: (1) symmetrical with an even mitochondrial population, and (2) asymmetrical with different numbers of mitochondria in each blastomere. In immature oocytes, mitochondrial activity was very low and the distribution was diffuse or negligible, while in mature oocytes the activity was high and the distribution was diffuse or pericytoplasmic. Competent embryos up to the 16-cell stage showed intermediate levels of activity (16-50%) but activity decreased thereafter up to the blastocyst stage. Non-competent embryos showed low levels of activity (1-15%) at all stages. These results suggest that mitochondria might play an important role during early development and that a minimum threshold of activity regulates the potential competence for reaching the blastocyst stage.