Is it possible to store spotted wolffish (Anarhichas minor) sperm by refrigeration?

Spotted wolffish Anarhichas minor reproduction in captivity is dependent on in vitro fertilization. However, it is often challenging to acquire sufficient fresh sperm to fertilize the eggs that are obtained. In this study, we evaluate the possibility to store spotted wolffish sperm by refrigeration. Spotted wolffish sperm has the particularity that is already motile on stripping, and currently it is not possible to immobilize and reactivate. Thus, sperm refrigeration protocols should focus in extending this motility period that usually lasts up to 2 days. In a first experiment, we evaluated the possibility that the motility period of the sperm was limited by contamination with urine. The urea concentration in the sperm obtained both by stripping (17.10 ± 1.98 mg/dL) and directly from the testis (12.59 ± 2.37 mg/dL) was similar (p > 0.05), which indicate that the sperm collection method used avoid contamination with urine. Afterwards, we tested the possibility that the sperm motility period was limited by energy stores. The ATP concentration (initial value 5.65 ± 0.86 nmol/109 cells) remained stable (p = 0.099) during 30 h after sperm collection, and similar values (p = 0.329) were recorded at end of sperm storage in both diluted (3.88 ± 1.35 nmol/109 cells) and undiluted samples (4.76 ± 1.08 nmol/109). This indicates that the low intracellular ATP consumption, derived from the slow sperm motility, can probably be compensated rapidly enough by mitochondrial synthesis of ATP in the spotted wolffish sperm. In both experiments, diluted sperm kept higher percentage of motile cells during the storage time.

Safety and immunogenicity of a hexavalent DTwP-IPV-HB-PRP∼T vaccine versus separate DTwP-HB-PRP∼T and IPV vaccines in healthy infants in India.

Background: Multivalent vaccines containing whole-cell pertussis (wP) antigens combined with established diphtheria (D), tetanus (T), hepatitis B (HB), Haemophilus influenzae type b (Hib), and inactivated poliomyelitis (IPV) antigens allow the provision of a high-quality, affordable DTwP-IPV-HB-PRP∼T vaccine. Methods: Phase I/II, randomized, active-controlled, open-label study in healthy toddlers (Cohort I) and infants (Cohort II). Toddlers in Cohort I who had completed primary series D, T, P, HB, Hib, and polio vaccination received a booster dose of DTwP-IPV-HB-PRP∼T (N = 30) or DTwP-HB-PRP∼T + IPV (N = 15) vaccines at 15-18 months of age. After satisfactory review of safety data in Cohort I, infants in Cohort II received DTwP-IPV-HB-PRP∼T (N = 100) or DTwP-HB-PRP∼T + IPV (N = 50) at 6-8, 10-12, and 14-16 weeks of age. All infants in Cohort II had received previous oral polio and HB vaccines per country recommendations. Results: Booster and primary series vaccinations were well tolerated with no clinically significant differences between vaccine groups. Most adverse events were mild and resolved spontaneously; there were no vaccine-related serious adverse events and no deaths. In both vaccine groups, anti-D, anti-T, anti-HB, anti-Hib, and anti-polio 1, 2, and 3 seroprotection was 100% post-booster and post-primary series. For the pertussis antigens, booster response rate was > 86% in both groups. For the primary series, vaccine response rate was slightly higher for DTwP-IPV-HB-PRP∼T than DTwP-HB-PRP∼T + IPV for anti-PT (80.2% and 70.8%) and anti-FHA (81.3% and 68.8%), slightly lower for anti-PRN (72.5% and 81.3%), and similar in each group for anti-FIM (95.6% and 97.9%). Conclusions: This study demonstrated a good safety and immunogenicity profile of the hexavalent DTwP-IPV-HB-PRP∼T vaccine for infant primary series vaccination at 6-8, 10-12, and 14-16 weeks of age and booster vaccination at 15-18 months of age and supported progression to the next development phase.

Antibody persistence following administration of a hexavalent DTwP-IPV-HB-PRP∼T vaccine versus separate DTwP-HB-PRP∼T and IPV vaccines at 12-24 months of age and safety and immunogenicity of a booster dose of DTwP-IPV-HB-PRP∼T in healthy infants in India.

Background: The combination of whole-cell pertussis (wP) antigens with established diphtheria (D), tetanus (T), hepatitis B (HB), Haemophilus influenzae type b (Hib), and inactivated poliomyelitis (IPV) antigens provides a high-quality DTwP-IPV-HB-PRP∼T vaccine. This study evaluated a DTwP-IPV-HB-PRP∼T booster coadministered with measles, mumps, and rubella (MMR) vaccine. Methods: Phase II, open-label, randomized study. Healthy toddlers who had previously completed a DTwP-IPV-HB-PRP∼T or separate DTwP-HB-PRP∼T and IPV primary vaccination series received a DTwP-IPV-HB-PRP∼T booster vaccine at 12-24 months of age. All participants had also received 1 or 2 doses of measles-containing vaccine between primary vaccination and enrolment (N = 100 and N = 6, respectively). Those who had received 1 prior measles-containing vaccine received an MMR dose either concomitantly (N = 50) or 28 days after (N = 50) the DTwP-IPV-HB-PRP∼T booster. Immunogenicity was evaluated using validated assays and safety by parental reports. Results: Pre-booster vaccination, 100.0% participants showed antibody persistence after DTwP-IPV-HB-PRP∼T or DTwP-HB-PRP∼T and IPV for anti-T (≥0.01 IU/mL), anti-Hib (≥0.15 µg/mL), and anti-polio 3 (≥8 1/dil) and at least 95.8% of participants for anti-D (≥0.01 IU/mL), anti-HB (≥10 mIU/mL), and anti-polio 1 and 2 (≥8 1/dil). For the pertussis antigens, pre-booster antibody persistence (≥2 EU/mL) ranged from 88.6 to 88.7% (anti-PT), 91.4-98.6% (anti-FHA), 69.0-74.3% (anti-PRN), and 97.1-97.2% (anti-FIM). For the booster response, seroprotection based on either the primary series or measles-containing vaccination regimen was 100.0% for anti-D and anti-T (≥0.01 IU/mL and ≥0.10 IU/mL), anti-HB (≥10 mIU/mL and ≥100 mIU/mL), anti-Hib (≥0.15 µg/mL and ≥1 µg/mL) and anti-polio 1, 2, and 3 (≥8 1/dil), and for the pertussis antigens booster response ranged from 88.6 to 91.8% (anti-PT), 91.1-95.9% (anti-FHA), 88.6-93.9% (anti-PRN), and 95.9-98.6% (anti-FIM). There were no safety concerns in any group. Conclusions: This study showed good antibody persistence of the DTwP-IPV-HB-PRP∼T vaccine and good immunogenicity and safety of a booster dose given with MMR in the second year of life.Clinical Trials Registry India Number: CTRI/2018/04/013375.

Effect of Doublesynch and Estradoublesynch protocols on estrus induction, conception rate, plasma progesterone, protein, and cholesterol profile in anestrus Gir heifers.

AIM: This study aimed to evaluate the efficacy of Doublesynch and Estradoublesynch protocols on estrus induction, conception rates, plasma progesterone, protein, and cholesterol profile in anestrus Gir heifers. MATERIALS AND METHODS: In this study, 50 pubertal anestrus Gir heifers were selected from the field and farm conditions. The heifers were dewormed (injection ivermectin, 100 mg, s/c) and supplemented with minerals and vitamins (injection organic phosphorus 800 mg and injection Vitamin AD3E and Biotin 10 ml i/m) and multi-mineral bolus at 1 bolus daily for 7 days. The heifers were randomly divided into three groups: Doublesynch (n=20), Estradoublesynch (n=20), and control (n=10). The animals were monitored for estrus response, estrus interval, behavioral signs, and conception rates after induced/first, second, and third cycle post-treatment. Blood samples were obtained on day 0, day 9, day 12, and on day 12 post-artificial insemination (AI) for determination of plasma progesterone, protein, and cholesterol profile. RESULTS: The estrus response rate between Doublesynch and Estradoublesynch protocols was similar between treated heifers (85% and 95%). The interval from the second prostaglandin F2α (PGF2α) injection to estrus induction did not differ between the groups (63.87±4.19 vs. 58.27±3.83 h). The conception rates following induced estrus (20% vs. 30%), at the second cycle (23.07% vs. 16.66%), at the third cycle (22.22% vs. 30.00%), and the overall conception rate (45% and 55%) within 27.89±5.75 and 26.45±5.48 days were the same across the treatment groups. The mean plasma progesterone concentrations were significantly (p<0.01) higher on day 9 (second PGF2α injection) and day 12 post-AI compared to day 0 (first PGF2α injection) and the day of fixed-timed artificial insemination. The concentrations were also significantly (p<0.05) higher in conceived than non-conceived heifers on day 9 of treatment and day 12 post-AI in both the protocols. The mean plasma cholesterol concentrations were significantly higher during peak follicular and luteal phases compared to the initial anestrus phase in both the protocols. The values were also higher in non-conceived than conceived animals in both the protocols. The plasma protein profile was not influenced by the sampling days or conceived and non-conceived status. CONCLUSION: The results showed that both Doublesynch and Estradoublesynch protocols resulted in similar estrus induction and conception rates with modulation of plasma progesterone and cholesterol profile in anestrus Gir heifers.

Leucine kinetics during a brief fast in diabetes in pregnancy.

The effect of diabetes in pregnancy on leucine turnover and oxidation was examined in 12 insulin-dependent diabetic (IDDM) subjects and 12 gestationally diabetic (GDM) subjects during the third trimester of pregnancy. The data were compared with those in normal pregnant women studied during the same time period and reported previously. Eight of the IDDM subjects were on continuous subcutaneous insulin infusion (insulin pump), and four were on conventional twice-daily insulin treatment. Of the GDM group, seven were on insulin therapy and five were on dietary management. Leucine kinetics were quantified using [1-13C]leucine tracer in combination with respiratory calorimetry and measurement of lean body mass using the H2[18O] dilution method. In addition, glucose kinetics were measured in insulin-treated subjects using [6,6(2)H2]glucose tracer. Despite rigorous metabolic control, fasting plasma glucose (IDDM 5.5 +/- 1.9 mmol/L [P < .05], GDM 4.7 +/- 1.3 [P < .01], controls 3.6 +/- .6, mean +/- SD) and hemoglobin A1 ([HbA1] IDDM 7.9 +/- 1.9%, GDM 7.5% +/- 2.1%) levels were higher in diabetic subjects. Although total insulin levels were higher in insulin-treated diabetic subjects, free-insulin concentrations were similar in all groups. Rates of excretion of urinary urea nitrogen and respiratory quotients were also similar. The rate of glucose turnover was lower in insulin-treated subjects compared with normals. Leucine flux, a measure of the rate of protein breakdown, and leucine oxidation were higher in IDDM and insulin-treated GDM subjects. The rate of leucine oxidation was increased in conventionally managed IDDM and insulin-treated GDM subjects.(ABSTRACT TRUNCATED AT 250 WORDS)

Hydrolytic degradation of 2,6-dichlorobenzylthiopseudourea hydrochloride.

Degradation of 2,6-dichlorobenzylthiopseudourea hydrochloride was followed in basic medium (pH 7.5) to isolate and characterize all possible degradation products. IR, Raman, and NMR spectroscopy, TLC, and elemental analysis were used to identify the products. Degradation of base-hydrolyzed 2,6-dichlorobenzylthiopseudourea hydrochloride produced 2,6-dichlorobenzylthiol and cyanamide and was followed by oxidation (air) to produce bis(2,6-dichlorobenzyl) disulfide, dimerization to give cyanoguanidine, and hydrolysis to yield urea. The kinetics of hydrolysis at 22.5 degrees (pH 7.0 and 7.5) and at 37 degrees (pH 7.0) revealed a pseudo-first-order reaction with respect to the substrate. Apparent first-order rate constants and energy of activation, entropy of activation, and half-life values were determined.

Modified USP assay for simultaneous determination of aspirin and nonaspirin salicylates in aspirin and buffered aspirin tablets.

Modified USP procedures are described for the simultaneous determination of nonaspirin salicylates and aspirin in aspirin and buffered aspirin tablets. The existing USP procedures are not stability indicating for intact aspirin when significant levels of nonaspirin salicylates are present, as is often the case in short-term, high temperature stability programs. The modified procedures yeld considerably shorter analysis times and stability-indicating assays for intact aspirin without the need for sophisticated equipment other than that presently required by USP XIX.

GLC assay for fenclorac.

A rapid stability-indicating GLC method is described for the determination of fenclorac as the diethylamine salt in dosage formulations. The procedure is also applied in detecting and quantitating the weight percent of 3-chloro-4-cyclohexylphenylglycolic acid and alpha-chloro-4-cyclohexylphenylacetic acid impurities associated with the synthesis of the fenclorac drug substance. The former compound, in addition to being an impurity, is also the degradation product of fenclorac. The procedure involves preparations of the silyl derivatives, addition of an internal standard (triphenylethylene), and use of a hydrogen flame-ionization detector. The column, with a phenyl methyl silicone liquid phase and operated at about 195degrees, is well suited for the separation. The silyl derivatives were characterized by GLC-mass spectrometry.

Characterization of impurities in sulfasalazine.

The chemical structures of four impurities isolated from sulfasalazine were determined. Three impurities are the by-products of the reaction process in drug synthesis, i.e., during diazotization of sulfapyridine and during coupling with salicylic acid. Only one contaminant was identified as a starting material, sulfapyridine, in the drug synthesis. The four impurities were characterized as 2-[[p-(2-pyridylsulfamoyl)-phenyl]azo]hydroxybenzene (I), 3-[[P-(2-pyridylsulfamoyl) phenyl]-azo]salicylic acid (II), 5-[[p-[4-(2-pyridylanilino)]-N-phenyl]azo]salicylic acid (III), and sulfapyridine (IV). Compounds I-III are novel molecules, and IV is the precursor of sulfasalazine. The isolation of the impurities was accomplished by TLC and liquid extraction procedures. The methods used to characterize the impurities were a combination of IR, UV, and NMR spectroscopy, mass spectrometry, and TLC. For I and III, comparisons also were made with the synthesized materials to supplement the evaluation.

Solid-state acetylation of codeine phosphate by aspirin.

A nonsolvolytic (solid-state) acetylation of codeine phosphate in the presence of aspirin to yield acetylcodeine phosphate is reported. GLC assays for the simultaneous determination of aspirin and salicylic acid and codeine and acetylcodeine are described. The apparent heat of activation for codeine phosphate is estimated, and the possible reaction mechanisms are discussed.

Kinetics of hydrolysis of fenclorac.

The kinetics of hydrolysis of fenclorac were studied to determine its stability in aqueous solution at different pH's and temperatures. For this study, a stability-specific liquid chromatographic assay method was developed to separate fenclorac from its hydrolysis product, alpha-hydroxy-3-chloro-4-cyclohexylbenzeneacetic acid. The k-pH profile in the 0-12 pH range in various buffer solutions shows that fenclorac is stable in its undissociated form in strongly acidic media and is unstable in neutral and alkaline media. The instability of fenclorac in aqueous solution is proportional to the degree of ionization of the carboxyl group in the 1-4 pH range and is independent of pH above 4. The rate-determining step in the mechanism of hydrolysis of fenclorac involves ionization of the carbon-chlorine bond. The ionization is catalyzed by an intramolecular necleophilic attack on the alpha-carbon by the dissociated carboxyl group, resulting in the formation of an unstable intermediate, a three-membered ring lactone. This unstable intermediate rapidly hydrolyzes to the final hydrolysis product. This mechanism is supported by experimental evidence such as the medium effect, positive salt effect, common ion effect, and substituent effect. Arrhenius parameters for the hydrolysis of fenclorac and its 3-nitro substituted analog were obtained.

Novel method of derivatization of an amidinourea (lidamidine) for GLC analysis.

A new quantitative GLC method for analysis of lidamidine hydrochloride (I) was developed. The method was based on derivatization of I to 1-(2,6-dimethylphenyl)-4-methylamino-dihydro-1,3,5-triazin-2-one (II) using dimethylformamide dimethylacetal reagent. Compound II was synthesized and characterized by IR, NMR, mass spectrometry, and elemental analysis. The assigned structure was in agreement with characterization analyses. Cyclization of I to a triazinone using dimethylformamide dimethylacetal reagent presented a new route for the preparation of II.

Simultaneous GLC analysis of aspirin and nonaspirin salicylates in pharmaceutical tablet formulations.

The analysis of aspirin and nonaspirin salicylates in buffered and plain tablet formulations employing nearly nonaqueous extraction is described. The results obtained compare favorably with those obtained from USP procedures. A simultaneous assay for aspirin and nonaspirin salicylates in buffered tablets involves the use of an acidified chromatographic siliceous earth column for the separation of the aspirin and nonaspirin salicylates from various buffers or antacids. The methods described here have definite advantages over USP XX procedures, and the buffered aspirin tablet procedures also is adaptable to aspirin formulations containing codeine, acetaminophen, propoxyphene, caffeine, and many antihistamines.

Pharmacodynamic effects of levonorgestrel (LNG) administered either orally or subdermally to early postpartum lactating mothers on the urinary levels of follicle stimulating hormone (FSH), luteinizing hormone (LH) and testosterone (T) in their breast-fed male infants.

Pharmacodynamic effect of levonorgestrel (LNG) present in small amounts in infant's circulation has not yet been studied adequately. In our present study, nine women were taking oral minipills (LNG 30 micrograms daily) and 10 were using subdermal implants, Norplant(R)-2, during early postpartum period from four weeks to 15 weeks. These were healthy lactating women in age group 20 to 35 yrs, who had full-term normal delivery of male infants. Daily 4-hour urine samples (from 8 AM to 12 noon) were collected from four weeks onwards to 15 weeks for estimations by radioimmunoassays of follicle stimulating hormone (FSH), luteinizing hormone (LH) and testosterone (T) levels. No significant differences (P greater than 0.05) were found between geometric means of the total area under curve at weekly intervals for FSH, LH and T hormones between the male infants from control group (n=10) when compared with oral minipill or Norplant(R)-2 users. These results are reassuring for future sexual growth and development of children who are exposed to contraceptive steroids during their infancy; however, they do require further confirmation by long-term epidemiological studies incorporating monitoring and surveillance of such children.

ICMR task force study on hormonal contraception. Transfer of levonorgestrel (LNG) administered through different drug delivery systems from the maternal circulation into the newborn infant's circulation via breast milk.

The transfer of levonorgestrel (LNG) from the maternal plasma via breast milk to the infant was studied in 38 fully lactating and breast-feeding women at 4-6 weeks postpartum, for a duration of 28 days. These volunteers were provided with LNG contraceptive treatment delivered through three, different routes of drug delivery system: (i) intrauterine devices impregnated with LNG (LNG-IUD); (ii) subdermal implant (Norplant (R)-2); and (iii) minipills (LNG 30 micrograms daily). On the first day after either the LNG-IUD (n = 14 women) or Norplant (R)-2 (n = 14 women) insertion, the maternal blood and breast milk samples were collected at 2, 4 and 8 hourly intervals. This was followed by daily collection of these samples as well as infant's blood from days 2 to 4 and thereafter on days 7, 14 and 28. For infant's blood samples from LNG minipill users (n = 10 women), only a single 4-hour sample was collected on the first day and no samples were collected on days 3 and 4. The rest of the schedule for collection of maternal blood and breast milk as well as infant's blood samples were the same in minipill users as for the other two treatment groups. The study revealed a lower LNG percentage transfer from maternal sera to breast milk--11.8 +/- 2, 7 +/- 2 and 8 +/- 1 and relatively higher percentage LNG transfer from breast milk to infant's sera--75 +/- 17, 68 +/- 20 and 32 +/- 3, in LNG-IUD, Norplant (R)-2 and minipill users, respectively. Therefore, LNG contraceptive steroid is transferred into the infant's circulation, the biological significance of which remains to be established.

Episodic release of luteinizing hormone and testosterone in Surti and Marwari bucks younger than one year of age.

Studies on the episodic release of luteinizing hormone (LH) and testosterone in Surti (n=2) and Marwari (n=2) bucks younger than one year of age (6 to 8 months) were carried out by collecting blood plasma during the breeding season. The studies revealed that definite pulsatile releases of LH and testosterone occur in both breeds of bucks. The overall number of LH and testosterone pluses over a 24 hour period was 9.1+/-1.00 and 7.5+/-0.28, respectively. The peak, basal and mean LH and testosterone concentrations did not show significant differences between the two breeds. The duration and interval of LH and testosterone pulses differed during light and dark hours. The time interval between LH peak followed by the testosterone peak was significantly (P<0.05) longer during the night than the day hours for both the breeds. The physiological basis of the findings are discussed.

Value of radionuclide angiogram for the diagnosis of Ebstein's anomaly.

A radionuclide angiogram in a patient with Ebstein's anomaly showed a very dilated right atrium with a minimally enlarged right ventricle and a normal sized left ventricle. This study led to the establishment of a definitive diagnosis by means of an echocardiogram. Late close of the tricuspid valve was demonstrated. In addition prolapse of the tricuspid and mitral valves was documented. A tricuspid valve prolapse has never been reported in this condition.

Cutaneous fistula communicating with the hip in a patient with a painful total hip prosthesis. Demonstration by radionuclide arthrography.

Radionuclide arthrography is becoming increasingly useful in the evaluation of femoral component loosening in patients with a painful total hip prosthesis. Additional potential advantages of radionuclide arthrography include detection of abnormal communications with the hip, such as bursae, abscess cavities, and fistulas. A case of cutaneous fistula communicating with the hip in a patient with loosening and infection of the femoral component of the total hip prosthesis that is clearly demonstrated by radionuclide arthrography is presented.

Obstruction of the iliac veins due to markedly distended urinary bladder demonstrated by radionuclide venography. A case report.

An unusual case of obstruction of the iliac veins by a markedly distended urinary bladder (UB) is presented. Initial radionuclide venography (RNV) was positive, revealing obstruction of the iliac veins associated with marked dilatation of the UB. After the UB was emptied, RNV was negative.

Demographic data of the neonatal intensive care population of a county hospital in Texas.

Morbidity and mortality associated with neonatal intensive care affect strongly the socioeconomic aspect of the health-care system. A retrospective study of the neonatal intensive care population at a county hospital in Texas showed that most deaths were related to nontreatable causes. Prematurity was a major cause both of increased mortality and morbidity. Although improved management of a premature infant with hyaline membrane disease using artificial surfactant improved survival, this treatment did not change the morbidity. The incidence of morbidity was related directly to the degree of prematurity. Illicit drug use also had a direct correlation with infants who had low birth weights. Overall, the mortality and morbidity data were consistent with incidence data reported nationally.