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OBJECTIVE: To characterize the patterns of placental separation during the third stage of labor. METHODS: Continuous real-time ultrasound was performed during the third stage of labor in 101 normal deliveries. The sequence of placental separation was recorded for determining whether the process was multiphasic, the site from which separation commenced and the mode of its progression. RESULTS: Separation in 97 cases was multiphasic. Monophasic separation in which all parts of the placenta appeared to separate simultaneously occurred in two cases only. Pathological prolongation of the third stage precluded determination of separation in two cases. Ninety-two cases had a uterine wall placenta (anterior or posterior); the separation commenced at one pole and progressed sequentially towards the opposite side in 89 of them. The process started at the lower pole (down-up separation) in 83/92 cases (90.2%) and began from the upper pole (up-down separation) in only 6/92 cases (6.5%). Nine cases had a fundal placenta; of these the separation was also multiphasic but began sequentially from either the anterior or posterior pole, or simultaneously from both, in 8 (88.9%) cases so that the fundal part was separated last (bipolar separation). CONCLUSIONS: Placental separation is usually an orderly multiphasic phenomenon that begins mostly from the lower pole of the placenta and propagates sequentially upwards. Fundal placentae, however, separate first at their poles with the fundal part being separated last. Recognition of the sequence of events and understanding of the mechanism of placental separation may aid in detecting cases prone to third-stage complications and in managing pathological ones.
80 infants, toddlers, and preschoolers were observed before, during, and after separations from their mothers, who were attending conferences (M duration = 4 days). Half the sample was separated only once and the other half experienced 3 separations across a 6-month period. The study was designed: (a) to determine how separations affect children's behavior when there are no changes in the family constellation as there typically are during other separations, such as the birth of a new child, and (b) to determine the effects of repeated separations. Fewer changes in sleep and play behaviors suggested that this type of separation was less stressful than separations for the birth of another child. Nonetheless, the separations were still stressful, but principally for the single-separation group. In that group, changes were noted in both play and sleep behaviors. Following reunion, their sleep behaviors and more sophisticated play behaviors returned to baseline. However, activity level and the more insecure behaviors, such as wandering aimlessly, watching other children play, and interacting with their teachers, remained elevated following the mother's return. The multiple-separation group, in contrast, showed only 1 behavior change during their third separation, i.e., reduced interactions with their peers during the separation period, which returned to baseline following reunion. Repeated-measures analyses of the first and third separations of the multiple-separations group suggested that only the first separation was stressful. Thus, the infants and children in this study seemed to adapt to repeated separations.
Mononuclear leukocytes (MNC) were separated from heparinized and EDTA-treated whole bovine blood by centrifugation after mixing with a commercial colloidal silica preparation (Sepracell-MN (S-MN]. Cell yields and lymphocyte blast transformation (LBT) to pokeweed mitogen (PWM), phytohaemagglutinin (PHA), concanavalin A (Con A), and Brucella abortus antigens were tested against MNC obtained from heparinized whole blood using Ficoll-Hypaque (FH). Separation with S-MN was more rapid and less labor intensive than separation with FH. There was a higher average total yield of MNC but a lower percentage of monocytes in the FH- than in the S-MN-separated MNC. In mitogen-induced LBT assays, MNC responded comparably to each mitogen regardless of the separation technique or anticoagulant used, and a cell concentration effect was demonstrated. In general, FH-separated MNC responded greater to PWM than did S-MN/EDTA separated MNC, but S-MN/heparin separated MNC had the greatest LBT responses to PWM. Overall, S-MN/EDTA separated MNC had the greatest responses to PHA, and responses to Con A were variable among experiments with respect to the separation technique. In antigen-induced LBT assays, two B. abortus antigens were used: a heat-killed strain S1119 (HKA) and a gamma-irradiated strain 19 (gamma BA). The LBT responses of three steers vaccinated with live B. abortus strain 19 were compared with three nonvaccinated steers in three separate experiments. Using HKA, FH separation resulted in an overall greater LBT response for vaccinates than nonvaccinates and a greater differential between responses of vaccinates and nonvaccinates than did S-MN derived MNC regardless of the anticoagulant used. Using gamma BA, FH produced the most responsive MNC in one experiment and S-MN/heparin produced the most responsive MNC in the other. At the highest cell concentration tested, FH-separated MNC had the greatest LBT responses for vaccinated calves, but differences between S-MN- and FH-separated MNC responses were not significantly different (P greater than 0.05). In conclusion, S-MN is a rapid and simple technique for separation of MNC from bovine blood. The technique produces an adequate cell population for mitogen-induced LBT studies; however, FH-separated MNC were generally more responsive in the B. abortus-induced LBT assay.
Separations of 12 compound classes, polychlorinated biphenyls (PCBs), diphenyl ethers (PCDEs), naphthalenes (PCNs), dibenzothiophenes (PCDTs), dibenzo-p-dioxins (PCDDs), dibenzofurans (PCDFs), terphenyls (PCTs) and alkanes (PCAs), toxaphene, organohalogenated pesticides (OCPs), and polybrominated biphenyls (PBBs) and diphenyl ethers (PBDEs) by comprehensive two-dimensional gas chromatography were evaluated. Five column combinations, DB-1 x 007-210, DB-1 x HT-8, DB-1 x LC-50, DB-1 x 007-65HT and DB-1 x VF-23ms were used to study, primarily, group-type separations, but attention was devoted also to within-class separation, especially for those classes which were not addressed in much detail before, the PCNs, OCPs, PBBs and PCTs. The DB-1 x 007-210 column set did not offer any extra separation compared to one-dimensional GC. For the DB-1 x HT-8 column combination, the useful principle of congener separation on the basis of number of halogen substituents in a molecule was confirmed (PCBs, toxaphene) and extended (PCTs, PBDEs). No practically useful group-type separation was observed for this column combination. The DB-1 x LC-50 set provides group separation based on planarity: planar compounds such as PCDDs, PCDFs, PCDTs and PCNs are much more retained than, and therefore separated from, non-planar analytes. Within the classes of PCBs, PBBs and PCTs highly useful separation of planar from non-planar compounds was also observed. The DB-1 x 007-65HT column set effectively separates PCAs and PBDEs from all other compound classes, and provides a good separation of brominated and chlorinated analogue classes from each other. This column set was the most efficient one for within-class separation of OCPs and PCNs. Finally, DB-1 x VF-23ms yields excellent within-class separations, especially of non-aromatic compounds, viz. OCPs, toxaphene and PCAs. No group separation was observed here. The applicability of the approach was demonstrated for a sediment extract and a dust extract. In the sediment extract, PCDDs, PCDFs, PCAs and PCNs were identified and their efficient separation was achieved. In the dust sample, separation of PCAs and PBDEs was achieved and several new PBDE congeners were identified.
The objectives of these experiments were to characterize separation of frozen-thawed bovine spermatozoa on a Percoll gradient and then to compare sperm separation by either a swim-up or Percoll gradient procedure for the ability of spermatozoa to fertilize oocytes in vitro. The Percoll gradient was a 45 and 90% discontinuous gradient. Initial experiments found that centrifugation of semen on the Percoll gradient for 15 min at 700 g was sufficient to obtain optimal recovery of motile spermatozoa. Most of the nonmotile spermatozoa were recovered at the interface of the 45 and 90% Percoll layers, while the motile spermatozoa were primarily in the sperm pellet at the bottom of the gradient. When frozen-thawed semen from each of 7 bulls was separated by swimup, a mean +/- SEM of 9% +/- 1 of the motile spermatozoa were recovered after the procedure. In contrast, more spermatozoa were recovered after Percoll gradient separation (P < 0.05), with 40% +/- 4 of the motile spermatozoa recovered. The effect of separation procedure on in vitro fertilization found swim-up separated spermatozoa penetrated a mean +/- SEM of 74% +/- 5 of the oocytes, while fewer oocytes were penetrated by Percoll separated spermatozoa at 52% +/- 8 (P < 0.05). There was no effect of the separation procedure on the rates of polyspermy as measured by sperm/penetrated ova, with a mean +/- SEM of 1.25 +/-.09 for swim-up separated spermatozoa and 1.14 +/-.07 for Percoll separated spermatozoa (P>0.05). A carry over of Percoll into the fertilization medium with the Percoll separated spermatozoa was found not the cause for the decreased penetration of oocytes by these spermatozoa. In 2 of 3 bulls tested, the decreased penetration of oocytes by Percoll separated spermatozoa could be overcome by increasing the sperm concentration during fertilization from 1 x 10(6) to 5 x 10(6)/ml. When development of embryos fertilized by either swim-up or Percoll separated spermatozoa was compared for the semen from 2 bulls, a difference in cleavage rate was found in favor of swim-up separated spermatozoa (P < 0.05), but there was no effect of separation procedure on development (Day 7) to the morula + blastocyst or blastocyst stage (P>0.05). The disadvantages of the Percoll procedure could easily be overcome and the procedure was faster and yielded a six-fold greater recovery of motile spermatozoa than the swim-up method.
Rhesus mother-infant pairs were housed in a playpen apparatus beginning just before the birth of four male infants. The infants were separated from their mothers four times beginning at a mean age of 218 days. In Type A separations (I and IV) the infants were removed and housed away from their familiar environment in a protected setting; in Type B separations (II and III) the infants remained in the familiar setting and mothers were removed. One pair was separated every 2 weeks for 6 days; for a particular infant, a mean of 8 weeks intervened between each of the separations. On the basis of infant behavior during separation. Type B separations appeared to have a more deleterious effect on the infant: infants did not show the typical behavioral signs of depression under Type A housing conditions, whereas, under Type B conditions, infants expressed the typical depressive reaction to separation. However, comparisons of pre- and postseparation behaviors in the mother-infant pairs indicated that Type A separations were more perturbing. Increases in ventral-ventral contact between mothers and infants were greater following Type A separations and increases in time at nipple occurred only after Type A separations; infant grooming by mother increased only after the first, a Type A, separation. Type B separations may have affected mothers more severely in that reciprocity between maternal cradling and infant clinging was greater following Type B separations than following Type A separations when infants clung significantly more often than mother cradled.
Studies were conducted to produce faster, simpler, more rugged protocols for separating wheat proteins by high performance capillary electrophoresis (HPCE). Three areas were targeted for improvement: initial capillary equilibration procedures, buffer composition, and post-separation rinsing procedures. For the initial equilibration of capillaries, a brief rinse with a hydroxypropylmethylcellulose (HPMC) solution was the most critical factor for successful separation of wheat proteins. To reduce separation time and maintain resolution, beta-alanine and glycine were each used in place of sodium phosphate as buffer ions. Two isoelectric buffers, aspartic acid and iminodiacetic acid (IDA) were also tested. Each of these four buffer systems generated substantially lower currents, and provided faster separations, than sodium phosphate-based buffers. Finally, post-separation rinsing procedures were re-examined with the goal of reducing the time necessary to rinse the capillary after each separation. A critical factor in achieving this goal was removal of albumins and globulins prior to separation. These proteins bind to the capillary wall and cause rising baselines and excessive peak tailing. Once these proteins were removed, capillaries could be rinsed with buffer for only 2 min between separations. Capillary equilibration procedures were shortened from 90 min to 30 min. Likewise, separation times were reduced by approximately 40% (25 min to 15 min) by using glycine in place of sodium phosphate in the separation buffer. Finally, post-separation times were reduced by 80% (10 min to 2 min). Overall, these factors resulted in a reduction in total separation time of 50% (35 to 17 min) and maintained high resolution separations and good run-to-run repeatability.
This review describes the existence of a phenomenon, sequential separation of centromeres, in mitotic cells of various species including both animals and plants. Critical observations at metaanaphase show that the centromeres of chromosomes in a given genome do not separate into two sister units randomly, but that there is a genetically controlled, nonrandom, species-specific sequence which is independent of the length of the chromosome or the position of the centromere. A stricter control appears to exist for late-separating than for early-separating chromosomes. At early stages of metaanaphase several chromosomes initiate onset of separation simultaneously or in rapid succession, but late-separating chromosomes are better defined in their sequential position. The effect of Colcemid on the sequence of separation is minimal. It is proposed that aneuploidy in humans and other organisms may result from out-of-phase separation of a given chromosome. With the exception of chromosome No. 16, it appears that very early- or very late-separating centromeres are involved in human trisomies more often than those in between. Perhaps one function of centromeric heterochromatin is the control of centromere separation. The amount of such chromatin shows a positive correlation with the timing of separation of the centromeres. Superimposed upon this quantitative influence is the qualitative aspect, as discussed for various genomes. This suggestion explains a lack of extremely large quantities of heterochromatin near the centromere. Its existence in the form of homogeneously staining regions distal to the centromere, as in some cancer cells or in sex chromosomes, seemingly has no influence on the separation of centromeres. A brief discussion of centromere separation errors in human disease is provided, and suggestions for further studies are made.
In maternally separated rats, variable chronic stress decreased the emotional reactivity and provoked a state of hypoactivity of the hypothalamic-pituitary-adrenal system at 3 months old but increased its activity after the open field test. The anterodorsal thalami nuclei control of the endocrine response under stress conditions was not manifested however its seems activate grooming behavior. The development of behavioral and endocrine response to stress is influenced by early postnatal environment. On the other hand, the anterodorsal thalami nuclei exert an inhibitory influence on the hypothalamic-pituitary-adrenal system under basal and stressful conditions. The aim of this work is to determine the magnitude of behavioral and hypothalamic-pituitary-adrenal responses to variable chronic stress in adult female rats with anterodorsal thalami nuclei lesions, previously isolated for 4.5 h daily during the first 3 weeks of life. The groups were: non-maternally separated sham and lesioned, maternally separated sham and lesioned with variable chronic stress with and without open field test. At 3 months old, under variable chronic stress, maternal separation provoked an increase in ambulation in sham and lesioned animals (P<0.01) but this parameter was not modified by lesion in either non-maternally separated or maternally separated groups. Neither the lesion nor the maternal separation changed the defecation and rearing parameter. Grooming behavior was lower in maternally separated lesioned rats (P<0.05). Under variable chronic stress maternal separation decreased adrenocorticotrophin hormone in comparison with non-maternally separated (P<0.001) and the lesion did not alter this response. Regarding corticosterone concentrations, maternal separation did not affect this hormone under variable chronic stress conditions and after the open field test there was an increase of this in both non-maternally separated and maternally separated sham and lesioned (P<0.001).
Eleven pairs of symmetrically conjoined twins underwent surgical separation at the Queen Sirikit National Institute of Child Health. Six were omphalopagus, 4 were thoracopagus and 1 was pygopagus. Eight were female and 3 were male. Three pairs were separated on emergency or semi-emergency bases, and the remaining 8 pairs were separated electively at an older age. Of the 3 pairs who had early emergency separations, one pair, whose combined birth weight was only 2,500 g, underwent emergency separation at the age of 44 days after the death of one twin. The second twin also expired one hour after the separation. In the remaining 2 pairs, early separation was done because of the deterioration of one twin due to complex cardiac anomalies. In both cases, the infants with cardiac anomalies expired but the others survived the separation satisfactorily. In one pair of thoracopagus conjoined twins, one twin had cyanotic cardiac anomalies. They were electively separated at the age of 2 years and 9 months. The twin with cardiac anomalies expired 2 hours after surgery, but the other survived the separation satisfactorily. In the remaining 7 pairs who underwent elective separations, both twins of each pair survived the separation satisfactorily. However, one twin expired unexpectedly 10 days after the separation.
OBJECTIVES: To determine potential risk factors and behaviors associated with separation anxiety and develop a practical index to help in the diagnosis of separation anxiety in dogs. DESIGN: Case-control study. ANIMALS: 200 dogs with separation anxiety and 200 control dogs with other behavior problems. PROCEDURES: Medical records were reviewed for signalment, history of behavior problems, home environment, management, potentially associated behaviors, and concurrent problems. RESULTS: Dogs from a home with a single adult human were approximately 2.5 times as likely to have separation anxiety as dogs from multiple owner homes, and sexually intact dogs were a third as likely to have separation anxiety as neutered dogs. Several factors associated with hyperattachment to the owner were significantly associated with separation anxiety. Spoiling activities, sex of the dog, and the presence of other pets in the home were not associated with separation anxiety. CONCLUSIONS AND CLINICAL RELEVANCE: Results do not support the theory that early separation from the dam leads to future development of separation anxiety. Hyperattachment to the owner was significantly associated with separation anxiety; extreme following of the owner, departure cue anxiety, and excessive greeting may help clinicians distinguish between canine separation anxiety and other separation-related problems.
The following methods were evaluated for their ability to separate motile cryopreserved sperm from semen after thawing: single washing, Percoll separation followed by a single washing, and Sephadex column separation. For Sephadex separation, washing, and Percoll separation, percent recovery of motile sperm was 65%, 76%, and 28%, and motility was 81%, 39%, and 60%, respectively. Percoll separation and washing were the best methods for removing seminal constituents, but sperm velocity and linearity were lower after Percoll separation and washing than after Sephadex separation. During 3 hours of incubation, there was an additional decrease in the motility, viability (exclusion of supravital dye), velocity, linearity, and intact acrosomes of Percoll-separated sperm, indicating that Percoll separation may not be suitable for cryopreserved sperm. Motile, washed sperm also had lower velocities and higher spontaneous acrosome reactions than Sephadex-separated sperm, but velocity and linearity were maintained during incubation. When semen was separated with Sephadex followed by washing, motility was well maintained (84%). The Sephadex method is a promising technique for selecting and concentrating motile cryopreserved sperm.