Barbiturate euthanasia solution-induced tissue artifact in nonhuman primates.

BACKGROUND: Barbiturate euthanasia solutions are a humane and approved means of euthanasia. Overdosing causes significant tissue damage in a variety of laboratory animals. METHODS: One hundred seventeen non-human primates (NHP) representing 7 species including 12 fetuses euthanized for humane and research reasons by various vascular routes with Euthasol, Sodium Pentobarbital, Fatal Plus, Beuthanasia D, or Euthanasia 5 were evaluated for euthanasia-induced tissue damage. Lungs and livers were histologically graded for hemolysis, vascular damage, edema, and necrosis. Severity of tissue damage was analyzed for differences on the basis of agent, age, sex, dose, and injection route. RESULTS: Severity of tissue damage was directly related to dose and the intracardiac injection route, but did not differ by species, sex, and agent used. CONCLUSIONS: When the recommended dose of agent was used, tissue damage was generally reduced, minimal, or undetectable. Barbiturate-induced artifacts in NHPs are essentially the same as in other laboratory species.

Antibody response of chickens to sheep red blood cells: crosses among divergently selected lines and relaxed sublines.

Crosses were made among lines of chickens that had undergone 30 generations of selection for high or low antibody response 5 d after an intravenous injection with SRBC, and between sublines in which selection was relaxed in generation 24. Antibody responses at 5, 10, and 14 d after injection were measured in the 4 lines and in reciprocal crosses among them. Divergence between the high and low lines selected for SRBC antibody was immediate and increased during selection. Although significant in both cases, separation of the relaxed subline from its respective selected line was greater in the high than the low line. Five-day SRBC titers of the relaxed lines and the crosses were intermediate to the high and low selected lines, with the direction and magnitude of heterosis being line dependent. A high proportion of chickens from low line mating combinations did not have detectable antibody titers at 10 and 14 d postinoculation with SRBC, precluding statistical analysis of these data. Results are discussed in the context of intra- and interlocus effects on the selected trait.

Cloning and characterization of the bovine class 1 and class 2 insulin-like growth factor-I mRNAs.

Insulin-like growth factor-I (IGF-I) is an important regulator of growth, development, and metabolism, and is the primary mediator of the growth-promoting activity of growth hormone (GH) in animals. In several species, the IGF-I polypeptide is generated from IGF-I mRNA containing either exon 1 (class 1 IGF-I mRNA) or exon 2 (class 2 IGF-I mRNA) as the leader exon. The objectives of this study were to identify class 1 and class 2 IGF-I mRNAs in cattle and to compare their expression in different tissues, their response to GH, and their translational efficiency. Three class 1 IGF-I cDNAs corresponding to three different transcription start sites in exon 1 and one class 2 IGF-I cDNA were identified from adult cattle liver using 5'-rapid amplification of cDNA ends (5'-RACE). Both classes of IGF-I mRNAs were expressed in a variety of tissues, with the highest level in liver; class 1 IGF-I mRNA was more abundant than class 2 IGF-I mRNA in all tissues. Six hours after a single intramuscular injection of 500 mg of recombinant bovine GH, class 1 and class 2 IGF-I mRNAs in steer liver were increased by 29% (P=0.07) and 62% (P<0.05), respectively. The luciferase reporter mRNA fused to a class 1 IGF-I 5'-untranslated region (5'-UTR) was translated four times more efficiently in vitro than the luciferase reporter mRNA fused to a class 2 IGF-I 5'-UTR (P<0.05). These results indicate that the IGF-I gene in cattle is transcribed as class 1 and class 2 IGF-I mRNAs and that the two classes of IGF-I mRNAs may be regulated differentially at both transcriptional and translational levels.

Heterosis of growth and reproductive traits in fowl.

Long-term divergent selection for high (HH) and low (LL) BW at 56 d of age in White Plymouth Rock chickens resulted in deviations from a typical chronological timeline for reproductive development. Line HH chickens of the 42nd generation of selection (S(42)) were heavier at 28, 56, 168, and 266 d of age, as well as at sexual maturity than those from line LL of the same generation, and commenced egg production at a younger age (188 d vs. 217 d). Once the LL chickens reached sexual maturity, 99.5% of their ovulations resulted in normal oviposits, in contrast to 94.6% for HH females. Heterosis, as measured by differences of F(1) progeny from the parental lines, can have a positive or negative sign. Although negative heterosis does not imply inferiority, results may be confusing because, depending on the trait, positive or negative can biologically be the phenotype that is preferred. Heterosis was calculated using coefficients of variation and means for reciprocal crosses from S(41) parents of the selected lines. Growth and reproductive traits were measured in this experiment. In almost all cases, heterosis calculated using coefficients of variation had a narrower range of magnitudes.

Performance of pureline broiler breeders fed two levels of vitamin E.

Reported in this paper is an experiment designed to evaluate responses of two commercial broiler dam purelines (A and B) continuously fed 10 or 300 IU of vitamin E/kg from 168 to 441 d of age. Prior to Day 168, all pullets were fed diets containing 10 IU of vitamin E/kg. During the early laying period, percentage hen-day ovulations and percentage hen-day normal egg production were similar for both lines and diets. During the latter part of the laying cycle, there were differences between lines for these traits (A > B), as well as for BW and egg weight for which line differences were reversed (A < B). Also during this period, percentage hen-day ovulations and percentage hen-day normal egg production differed between diets (300 > 10 IU/kg). These differences between diets were consistent with the greater number of females that entered lay and higher hen-housed egg production of the 300- than 10-IU/kg group. Although during the laying cycle all females received a fixed amount of feed, BW gains were greater for Line B than A and for the 300- than the 10-IU/kg level of vitamin E. Heterophil (H):lymphocyte (L) ratios, percentage livability, and relative asymmetries of shank length and diameter were similar among groups. Pullets from Line B and those fed the higher level of vitamin E exhibited more fear than their counterparts. Head shaking did not differ between vitamin E levels; however, there was a line-by-time of day interaction for this behavior. The 30-fold difference in dietary vitamin E was reflected by a 15-fold difference in plasma vitamin E levels in both lines. For vitamin E level in the yolk, however, there was a line-by-diet interaction. The interaction resulted from no difference between lines at the 10 IU/kg level and differences of 10- and 6+-fold in Lines B and A at the 300 IU/ kg level, respectively. Overall, responses to continuous feeding of vitamin E at these levels were influenced by genetic stock, age, duration of feeding, and measurement criteria.

Effect of route of inoculation on humoral immune response of White Leghorn chickens selected for high or low antibody response to sheep red blood cells.

Effects of route of SRBC inoculation and antigen dosage on primary and secondary antibody response of White Leghorn lines selected for high (HA) or low (LA) 5-d antibody response to a single i.v. inoculation with 0.1 mL of a 0.25% suspension of SRBC were studied in two trials. In the first trial, chicks from parents of generation S24 of each line were randomly assigned to one of four treatments. At 35 d of age, they were inoculated into the brachial vein with 0.1 mL of 0.25% suspension of SRBC or into the breast muscle with 0.1 mL of 0.25, 2.50, or 25.00% SRBC. Plasma SRBC antibody was measured 3, 6, 10, and 20 d later. In the second trial, chicks from parents of generation S25 of each line were randomly assigned to treatment groups. At 28 d of age they were inoculated with 0.1 mL of 0.25% SRBC into the brachial vein, 0.1 mL of 25.00% SRBC into the thigh (T-L) or breast muscle (B-L), or 0.5 mL of 25.00% SRBC into the thigh (T-H) or breast muscle (B-H). Twenty-one days later, chicks (except five per group) were given a booster inoculation of 0.1 mL of 25.00% SRBC into the thigh muscle. Six and 10 d after each inoculation, plasma SRBC antibody, IgG, and IgM titers were measured. The SRBC antibody titers after primary i.v. inoculation with SRBC were always higher for HA than LA chicks. When inoculations were i.m., differences between lines varied with dosage. Low dosages inoculated into the breast failed to induce line differences consistently, whereas at higher dosages, titers were greater for HA than LA chicks regardless of inoculation site. For Line LA, inoculation into the thigh elicited higher titers than inoculations into the breast. Antibody titers to the booster inoculation of SRBC were similar for the lines.

Antibody responses of hens fed vitamin E and passively acquired antibodies of their chicks.

Antibody responses of hens and their progeny were studied in commercial broiler nuclear lines. Starting at 168 days of age, individually housed pullets from lines A and B were fed a 16% crude protein and 2752 kcal metabolizable energy/kg mash diet supplemented with either 10 or 300 IU/kg of vitamin E fed as dl-alpha-tocopherol acetate. Fifty-eight days later (226 days of age), 12 hens per line-vitamin E subclass were inoculated i.v. with 0.1 ml of a 2.5% suspension of sheep red blood cells (SRBC). Plasma antibody titers were measured 6, 20, 40, 54, 70, and 88 days after inoculation. Hens from both lines were artificially mated to males from line C, and progeny from eggs collected 9-15, 25-30, and 65-70 days after inoculation were tested for antibodies to SRBC. Hens were reinoculated i.v. with 0.1 ml of 0.25% SRBC 88 days after the first inoculation, and their antibody levels were measured 3, 6, and 20 days later. Eggs laid 10-13 days after reinoculation were incubated, and antibody titers of chicks were measured at hatch. Antibody response of hens to an initial inoculation of SRBC was line-diet-time after inoculation specific. In line A, titers were greater for hens fed the lower than the higher vitamin E diet, whereas diet had no effect on the antibody levels in line B. Line effects (A > B) were observed on days 6 and 20 after inoculation but not thereafter. After the second inoculation, dietary vitamin E level had no effect on antibody levels of hens within lines, whereas a between-line difference (A > B) was observed for the lower but not the higher level of dietary vitamin E. Although there was no difference between diets for antibody transferred to progeny by line B, there was a difference (lower > higher) for line A. After reinoculation of their dams, antibody titers of chicks from line A, but not line B, reached levels similar to those after the first inoculation. Antibody levels were higher for chicks at hatch than in 16-day embryos or 10 days posthatch. The results of this research suggest genetic variation in response to immune stimulation by dietary vitamin E.

Vitamin E and immune responses of broiler pureline chickens.

Immunological responses of cockerels fed diets containing either 10 or 300 mg/kg of vitamin E were measured in three commercial broiler nuclear lines designated as A, B, and C. All cockerels were fed the 10 mg/kg diet to 91 d of age, at which time half were continued on that diet and the other half were fed a diet supplemented to contain 300 mg/kg of vitamin E. Sixteen days after feeding the diets, cutaneous basophil hypersensitivity (CBH), which is an in vivo, cell-mediated immune response, was determined as the increase in toe-web skin thickness 24 h after an injection with phytohemagglutinin-P (PHA-P). Twenty-two days after feeding the diets, heterophils and lymphocytes were enumerated. At this time cockerels were also inoculated intravenously with 0.1 mL of 0.25% SRBC. The injection with SRBC was repeated 27 d later. Six and 20 d after each injection, antibody titers were determined. Mercaptoethanol-resistant (IgG) and mercaptoethanol-sensitive (IgM) antibodies were also measured in the secondary titers. The ratio of heterophils to lymphocytes increased, and CBH response was depressed by feeding the higher vitamin E diet. For cockerels of Lines A and B, the higher level of vitamin E maintained primary and secondary SRBC antibody levels, whereas for Line C, levels were depressed. The effect of the higher vitamin E diet on IgG and IgM was also stock-dependent. The enhanced heterophil:lymphocyte ratio from feeding vitamin E suggests an improved phagocytic ability of the immune system. Relative asymmetries for toe-web skin thickness and shank length were similar for the three lines.

Hematocrit values in weight-selected and relaxed lines of White Rock chickens.

Hematocrits (PCV) were measured at 29 and 106 d of age (PCV1 and PCV2, respectively) in male and female White Plymouth Rocks. Four lines were used, two of which had undergone 40 generations of divergent selection for 8-wk BW (HWS, LWS), and two respective sublines (HWR, LWR), in which selection had been relaxed for five generations. At both ages, males and females did not differ for PCV in lines HWR, LWR, and LWS. For line HWS there was an age by sex interaction that resulted from an age effect for males but not for females, and from a sex effect at each age. At both ages, PCV was higher for the HW than the LW lines. Initially, there was no difference between the selected and their respective relaxed lines, but by 106 d, HWR chickens had a higher PCV than HWS chickens. In lines HWR and LWR, PCV increased with age. There was a negative correlation in HWS males for PCV1 with 28 and 56 d BW. The HWR males also had a negative correlation for PCV1 with BW at 28 d, but not between PCV2 and BW. The correlation for PCV1 with PCV2 was high and positive for HWR males and females.

Mode of inheritance of unselected traits in lines of chickens selected for high or low antibody response to sheep red blood cells. 1. Resistance to marble spleen disease virus and juvenile body weight.

Two lines of White Leghorns that had undergone long-term selection for high (HH) or low (LL) antibody response to sheep red blood cell antigen(s) formed the nuclear lines for this experiment. Matings were made in a full diallel cross to produce in a single hatch from age-contemporary breeders the parental lines, reciprocal F1 and F2 crosses, and backcrosses for 16 progeny types. For males and females, there were parental line differences in BW to 42 d of age, after which there was decline between lines for males. Differences in BW between reciprocal F1 crosses and maternal heterosis declined with age, primarily reflecting dissipation of effects of egg weight. Heterosis of BW was dependent on the particular F1 cross and recombination effects were not important. At 50 d of age chicks were inoculated with either a 1 or 10% suspension of spleen extract from chickens infected with marble spleen disease virus (MSDV). A third group served as uninjected controls. Response to MSDV was evaluated by spleen weight 6 d after inoculation. Spleen weights relative to BW of control chicks were heavier for the HH than LL line with evidence from the crosses of sexlinkage and negative heterosis. Line LL chicks were more resistant to MSDV than Line HH chicks was F1 crosses intermediate to and different from either parental line with no evidence of heterosis.

Mode of inheritance of unselected traits in lines of chickens selected for high or low antibody response to sheep red blood cells. 2. Heterophils, lymphocytes, and hematocrits.

The nuclear lines for this experiment were White Leghorns that had undergone long-term selection for high (HH) or low (LL) antibody response to sheep red blood cell antigen(s). Sixteen progeny types consisting of parental lines, reciprocal F1 and F2 crosses, and backcrosses were produced in a single hatch from age-contemporary parents. At 30 d of age, blood was obtained from a random sample of 10 males per progeny type (n = 160) and slides prepared for subsequent determination of number of heterophils and lymphocytes. Twelve days later, blood was collected from random samples of 10 males and 10 females per progeny type (n = 320) for measuring hematocrits. There were no differences between parental lines for heterophils, lymphocytes, or the heterophil:lymphocyte ratio. Reciprocal effects were evident in the F1 crosses and directional heterosis was present in one cross but not the other. Neither maternal heterosis nor recombination effects were significant for either heterophils or lymphocytes. Although hematocrits were similar for males and females and parental lines, sex-linked and recombination effects appeared to be important.

Role of myelin basic protein and proteolipid protein genes in multiple sclerosis: single strand conformation polymorphism analysis of the human sequences.

Susceptibility to multiple sclerosis (MS) is widely held to have a strong genetic component. While the identities of genes conferring susceptibility are currently unknown, possible candidates include those genes coding for proteins which function in central nervous system (CNS) myelin. Two such genes are the human myelin basic protein (MBP) and proteolipid protein (PLP) genes, whose products make up approximately 80% of the total protein in CNS myelin. The association of a variable number tandem repeat (VNTR) 5' to the human MBP gene with MS has been the subject of conflicting reports. Here we test the hypothesis that mutations in the human MBP and PLP genes might be associated with MS by examining the entire expressed sequence of both genes by single strand conformation polymorphism (SSCP) analysis, using a panel of 71 MS patients and 71 controls. We have also re-examined the VNTR region in patients and controls. Three base changes were found in the human PLP gene and nine base changes in the human MBP gene; these were essentially equally distributed between patients and controls. No preferential distribution of various alleles of the VNTR between patients and controls was found. Although intronic and regulatory regions have not been examined, it would appear unlikely that mutations in these genes coding for the two major CNS myelin proteins contribute significantly to genetic susceptibility to MS.

Analysis of polymorphisms of the 2',3'-cyclic nucleotide-3'-phosphodiesterase gene in patients with multiple sclerosis.

Susceptibility to multiple sclerosis (MS) is widely held to have a genetic component. Possible candidate genes conferring this susceptibility include those coding for proteins specific to central nervous system (CNS) myelin. 2',3'-cyclic nucleotide-3'-phosphodiesterase (CNPase) is an enzyme found at high concentrations in CNS myelin, however its function is unknown. The amino acid sequence of CNPase shows a C-terminal motif characteristic of proteins involved in signal transduction pathways, suggesting a key role in myelin function. We have analysed the entire expressed sequence of the human CNPase gene in patients with multiple sclerosis and in healthy controls using single strand conformation polymorphism (SSCP) analysis. Nine previously undescribed mutations were detected, most of these occurred with equal frequency in both groups. However, a T-->C transition at nucleotide position 4306 in the region of the gene coding for the 3' untranslated region of the mature mRNA was found in a homozygous form in two out of 54 patients but in none of 100 controls. While the significance of this is unclear, it would appear unlikely that mutations in the expressed regions of the human CPNase gene contribute to genetic susceptibility to MS in the majority of sufferers.

Single strand conformation analysis of two genes contained within the first intron of the neurofibromatosis type I gene in patients with multiple sclerosis.

Single strand conformation analysis has become the most widely used technique to screen large numbers of DNA samples for unknown mutations which may contribute to genetic susceptibility to disease. The method relies on the electrophoretic migration of a single strand of nucleic acid in a polyacrylamide gel being dependent on its conformation which is in turn dependent on its sequence. We have examined two closely related genes present in the first intron of the neurofibromatosis type I gene--the oligodendrocyte myelin glycoprotein (OMGP) gene and the ecotropic viral integration 2A (EVI2A) gene--in 36 patients with multiple sclerosis (MS) and 36 healthy controls. A single mutation was found in the OMGP gene which resulted in an amino-acid change of glycine to aspartic acid. This occurred in identical proportions (16.6%) in MS patients and controls. Two rare mutations were found in the EVI2A gene, one resulting in an arginine substituting for a glutamine (one control and one patient), the other in the replacement of a glycine with serine (one control only). A third common polymorphism was seen in 5'-untranslated region of the EVI2A gene, with 65% of patients and controls showing a T-->C transition in either a heterozygous or a homozygous form. This makes it unlikely that either of these genes is involved in genetic susceptibility to MS, but regions of these genes outside of the exonic sequences have not been examined.

Multiple sclerosis in island populations: prevalence in the Bailiwicks of Guernsey and Jersey.

The aim of this study was to establish for the first time the prevalence of multiple sclerosis in the Bailiwicks of Guernsey and Jersey, as representing the most southerly part of the British Isles. All patients with multiple sclerosis in the Channel Islands resident on prevalence day were identified by contacting all medical practices, Multiple Sclerosis, and Action Research for Multiple Sclerosis societies by letter and visits. The crude overall prevalence rates were 113/100,000 (95% confidence interval (95% CI) 90.3-135.7) and 86.7/100,000 (95% CI 63.3-110.0) for the Bailiwicks of Jersey and Guernsey respectively. When standardised to the age and sex structure of a previously reported Northern Ireland population the standardised prevalence ratios were 120.2/100,000 (95% CI 96.0-144.3) for Jersey and 95.6/100,000 (95% CI 69.9-121.3) for the Bailiwick of Guernsey. When compared with recent studies in the northern United Kingdom the prevalence rates for multiple sclerosis in the Channel Islands lend some support to the proposed latitudinal gradient in the British Isles although the standardised prevalence ratio in the Bailiwick of Jersey is similar to those found in recent studies of southern Britain. The standardised prevalence rates of probable and definite multiple sclerosis for the male populations were 37.3/100,000 (95% CI 17.9-56.7) for the Bailiwick of Guernsey and 45.5/100,000 (95% CI 26.3-64.7) for the Bailiwick of Jersey whereas the standardised prevalence rates for the female populations were 97.5/100,000 (95% CI 73.9-143.5) and 139.5/100,000 (95% CI 112.6-181.2) respectively. Thus there is a striking and unexplained 43% higher prevalence of probable and definite multiple sclerosis in the female population of Jersey compared with that of the Bailiwick of Guernsey. This seems to be due to an unusually low prevalence of the disease among the female population of the Bailiwick of Guernsey compared with that of the United Kingdom mainland.

Ectopic ureter with seminal vesicle insertion in an infant with tracheoesophageal fistula and possible adult polycystic kidney disease.

We report on a male newborn with a tracheoesophageal fistula and possible adult polycystic kidney disease as well as a single ectopic ureter with seminal vesicle insertion. There is an association of tracheoesophageal fistula, hindgut and sacral anomalies with ureteral ectopia but adult polycystic kidney disease has not been described. The diagnostic modalities currently available are discussed, as well as the implications of the association with other anomalies.

Ureterolithotomy for juxtavesicular stone.

Stones within the distal 8 cm. of the ureter often can be approached more readily through the posterior wall of the bladder than extravesically. The method and its indications are described.

Surgical management of infantile (ectopic) ureteroceles: report of a modified approach.

Since 1970, our experience in managing 22 patients (24 renal units) with infantile (ectopic) ureteroceles suggests that no single procedure is universally applicable. Certain general principles, however, guide management strategy. First, preservation of the upper pole moiety is successful if there is functioning renal parenchyma on IVP (9 cases with one failure). Second, when extirpation is required, total ureterectomy-ureterocelectomy is unnecessary (13 cases with no reoperations specifically for stump extirpation). Third, urinary tract infections are fairly common in the first postoperative year (4 of 16 patients), being more common in children with common sheath ("double barrel") reimplantations. This review of our experience often showed the primary procedure to be long and difficult with significant blood loss. Although it did not occur in this series, devascularization of the lower pole ureter remains a danger. Therefore, a two-stage procedure was developed to obviate these problems. In the last 4 patients (5 renal units) in this series, the ureteroceles were first incised endoscopically. Six weeks later, reimplantation or upper pole nephrectomy-partial ureterectomy was performed. Adequate early decompression of the ureterocele resulted in less operative time and blood loss (average 27.5 cc for upper pole nephrectomy) at the subsequent procedure. We now prefer this method, especially in very young or compromised patients, although the follow-up time is inadequate to recommend it generally.

Cecil urethroplasty with concurrent scrotoplasty for repair of hypospadias.

During 13 years about 8 per cent of 234 patients operated upon for hypospadias presented surgical problems of reconstruction of the urethra from the urethral meatus proximal to the penoscrotal junction. These cases commonly are referred to as perineal or penoscrotal hypospadias. The problem occurs when the urethral groove fails to develop adequately and the scrotal folds have failed to rotate caudally. This hypospadias is in contrast to penoscrotal or distal hypospadias, when the urethral groove is developed but fusion is incomplete. In these patients the Cecil procedure has been used but modified to extend the urethra from behind the penoscrotal junction to the glans at the second stage of the urethroplasty. The modification consists of rotating the scrotum caudally from its high location. The tension and acute angulation that contributed to frequent complications previously associated with such attempts have been avoided and results are satisfactory.