Childhood neighborhood context and adult substance use problems: the role of socio-economic status at the age of 30 years.

OBJECTIVES: To examine whether the (a) childhood neighborhood context predicts alcohol use disorder, nicotine dependence, and cannabis use disorder symptoms at the age of 39 years; and (b) socio-economic status during young adulthood mediates these relationships. Gender differences were also examined. STUDY DESIGN: The Seattle Social Development Project is a prospective longitudinal study of 808 individuals followed up from ages 10 to 39 years in Seattle, Washington, United States. The sample was gender balanced (51% were men). METHODS: Alcohol, nicotine, and cannabis use disorder symptoms were assessed using the Diagnostic and Statistical Manual of Mental Disorders IV-based Diagnostic Interview Schedule. Childhood neighborhood data consisted of 10 neighborhood-level variables from the 1990 national census, which were consolidated using principal component analyses. Two components with eigenvalues greater than 1 were extracted-neighborhood disadvantage and neighborhood stability. Educational attainment and employment status represented socio-economic status during young adulthood. Covariates included baseline symptoms of psychopathology, baseline substance use, gender, ethnicity, and childhood socio-economic status at the family level. Negative binomial regression was used as the primary modeling strategy. Six models for each outcome measure were estimated. The first three models examined associations between two neighborhood components and each substance use outcome measure. Next, we tested the second research question by adding unemployment and college graduate indicators at the age of 30 years as potential mediators underlying the link between the childhood neighborhood context and three substance use measures. RESULTS: Study findings revealed that childhood neighborhood stability significantly reduced alcohol and cannabis use disorder symptoms nearly 3 decades later. Path analyses suggested that socio-economic status during the transition to adulthood did not influence these relationships but rather had independent effects on problematic nicotine and cannabis use. Furthermore, the effects of childhood neighborhood factors on problematic nicotine use were stronger for men. CONCLUSIONS: Neighborhood characteristics during childhood may be important factors for alcohol and cannabis use disorder symptoms among adults and nicotine dependence disorder symptoms among men. Prevention efforts that address community stability and disadvantage can and should start in childhood, with a focus on intervention targets that might gain salience later in life to discourage the development and persistence of problematic substance use in adulthood.

Preventing adolescent health-risk behaviors by strengthening protection during childhood.

OBJECTIVE: To examine the long-term effects of an intervention combining teacher training, parent education, and social competence training for children during the elementary grades on adolescent health-risk behaviors at age 18 years. DESIGN: Nonrandomized controlled trial with follow-up 6 years after intervention. SETTING: Public elementary schools serving high-crime areas in Seattle, Wash. PARTICIPANTS: Of the fifth-grade students enrolled in participating schools, 643 (76%) were given written parental consent for the longitudinal study and 598 (93%) were followed up and interviewed at age 18 years. INTERVENTIONS: A full intervention provided in grades 1 through 6 of 5 days of in-service training for teachers each intervention year, developmentally appropriate parenting classes offered to parents when children were in grades 1 through 3 and 5 through 6, and developmentally adjusted social competence training for children in grades 1 and 6. A late intervention, provided in grades 5 and 6 only, paralleled the full intervention at these grades. MAIN OUTCOME MEASURES: Self-reported violent and nonviolent crime, substance use, sexual activity, pregnancy, bonding to school, school achievement, grade repetition and school dropout, suspension and/or expulsion, and school misbehavior; delinquency charges from court records; grade point average; California Achievement Test scores: and disciplinary action reports from school records. RESULTS: Fewer students receiving full intervention than control students reported violent delinquent acts (48.3% vs 59.7%; P=.04), heavy drinking (15.4% vs 25.6%; P=.04), sexual intercourse (72.1% vs 83.0%; P=.02), having multiple sex partners (49.7% vs 61.5%; P=.04), and pregnancy or causing pregnancy (17.1% vs 26.4%; P=.06) by age 18 years. The full intervention student group reported more commitment (P=.03) and attachment (P=.006) to school, better academic achievement (P=.01), and less school misbehavior (P=.02) than control students. Late intervention in grades 5 and 6 only did not significantly affect health-risk behaviors in adolescence. CONCLUSIONS: A package of interventions with teachers, parents, and children provided throughout the elementary grades can have enduring effects in reducing violent behavior, heavy drinking, and sexual intercourse by age 18 years among multiethnic urban children. Results are consistent with the theoretical model guiding the intervention and support efforts to reduce health-risk behaviors through universal interventions in selected communities or schools serving high-crime neighborhoods.

The Work Preference Inventory: assessing intrinsic and extrinsic motivational orientations.

The Work Preference Inventory (WPI) is designed to assess individual differences in intrinsic and extrinsic motivational orientations. Both the college student and the working adult versions aim to capture the major elements of intrinsic motivation (self-determination, competence, task involvement, curiosity, enjoyment, and interest) and extrinsic motivation (concerns with competition, evaluation, recognition, money or other tangible incentives, and constraint by others). The instrument is scored on two primary scales, each subdivided into 2 secondary scales. The WPI has meaningful factor structures, adequate internal consistency, good short-term test-retest reliability, and good longer term stability. Moreover, WPI scores are related in meaningful ways to other questionnaire and behavioral measures of motivation, as well as personality characteristics, attitudes, and behaviors.

Basilar membrane motion in relation to two-tone suppression.

It is proposed that two-tone suppression of rate responses in auditory-nerve fibres by a low-side suppressor cannot be explained in terms of basilar membrane motion. In a model, the amplitude of the mechanical response, either to the tone at characteristic frequency (CF), or to the CF tone combined with a second, lower frequency tone (a suppressor), is taken as the effective stimulus to inner hair cells (IHC), the voltage response of which is considered responsible for excitatory drive to auditory-nerve fibres. Many empirical mechanical and physiological effects are simulated accurately by the model, particularly phenomena observed in two-tone experiments using low-side suppressor tones, that authors have described as two-tone suppression. It is argued in this paper, however, that such phenomena strictly do not constitute suppression in the cochlear response and provide no explanation for rate suppression in nerve fibres. According to the model presented here and consistent with experimental data, suppression of the spike response to a CF tone in an auditory-nerve fibre by a low-side suppressor cannot be explained in terms of the mechanics of the BM. Conclusions by others that experiments support a mechanical explanation for low-side rate suppression are shown to be questionable. It is concluded that low-side suppression of neural responses is explicable only in terms of a non-mechanical factor derived from the response to the low frequency tone, that depresses responsiveness in fibres at the CF location. Adherence to the model of low-side neural rate suppression depending on reduced net mechanical response of the BM is contrary to experimental evidence; furthermore it overlooks a profound influence additional to synaptic drive, that is implied in the shaping of responses in auditory-nerve fibres.

Time course of rate responses to two-tone stimuli in auditory nerve fibres in the guinea pig.

Peri-stimulus time histograms (PSTHs) were constructed from responses of auditory nerve fibres in anaesthetized guinea pigs. Acoustic stimuli consisted of pure tones, presented either as tone bursts, or in two-tone combinations in which a gated test tone was superimposed on a continuous excitatory tone at characteristic frequency (CF). The majority of the sample of fibres displayed two-tone rate suppression (2TRS). The suppression was either a monotonic or a non-monotonic function of the level of the superimposed test tone. Monotonic suppression of CF-driven rate occurred only for test tones at frequencies higher than CF, presented at levels up to the maximum available (approx. 100 dB SPL). For test tones below CF, 2TRS initially increased, then reverted towards excitation for higher levels of the test tone. Three levels were identified in non-monotonic, two-tone rate functions; (1) the threshold for rate suppression, (2) the maximally suppressing level and (3) the level (referred to as the balance point) at which average firing rate was restored to the background, CF-driven rate. PSTHs for two-tone responses obtained for test tone levels between the maximally-suppressing level and the balance point typically showed brief decrements (notches) in spike rate, at the onset and following the offset of the test tone. The latency, depth and duration of notches, however, depended on the level of the test tone, in a different manner for onset and offset. In some cases, without overt rate excitation above the probe-driven rate, the offset notch became more pronounced and of extended duration with increased level of the test tone, suggestive of adaptation to the test tone. Two-tone responses, in which rate exceeded the background, CF-driven rate, in general were preceded by a reduced onset notch and were followed by a longer-lasting depression of the background spike rate, typical of post-excitatory depression. Relative to responses obtained to the test tones presented alone, excitatory two-tone responses were of lower rate and were delayed by the onset notch. Onset notches sometimes preceded rate excitation in responses to single tones. Some features of the time course of rate suppression and excitation displayed in PSTHs for responses to one and two-tone stimuli seem inconsistent with current models of 2TRS.

Two-tone suppression, excitation and the after effect in rate responses in auditory nerve fibres in the cat.

Responses were recorded from single, auditory nerve fibres in the anaesthetized cat. Acoustic stimuli consisted of two tones, one of which was at characteristic frequency (CF), the other (the suppressor) was at considerably lower frequency. Tones were presented in simultaneous and sequential configurations. For simultaneous presentations, well-known response properties were observed. The rising limb of the two-tone rate-intensity function closely matched that of the appropriately adapted response to the suppressor tone presented alone. Also, whether strongly suppressed relative to CF-driven rate, or equal to CF-driven rate, rate responses to the two-tone stimuli persisted unchanged when the CF tone was terminated and the suppressor tone continued alone. These results support the hypothesis that the suppressor tone has dual influences, suppressive and excitatory, that are distinct and additive. Peristimulus response histograms confirm in the cat that depression and slow recovery of sensitivity to CF may follow termination of the suppressor tone, as reported for the guinea pig [Hill, K.G. and Palmer, A.R. (1991) Hear. Res. 55, 167-176]. This delay in recovery of normal sensitivity to CF appeared to be directly related to the amount of excitation of the fibre that is attributable to the suppressor tone. A similar, delayed re-establishment of sensitivity also occurred in the response to a tone at CF, presented immediately following excitation by a suppressor tone. However, no delay occurred in the onset of response to the suppressor when preceded by the CF tone.(ABSTRACT TRUNCATED AT 250 WORDS)

Excitation and suppression of primary auditory fibres in the pigeon.

Spike potentials were recorded from single fibres in the auditory nerve of the pigeon. In fibres with recognizable responses to sound, spontaneous activity and properties of responses to tonal stimuli were studied in quiet background conditions. Mean spontaneous rate in the sample of fibres was 35 spikes/s. Tuning of spike response to tones was manifest as a single peak in rate at each sound pressure level (SPL) in the frequency-intensity plane. The majority of fibres showed only excitation of spike rate above spontaneous rate. Post stimulus time histograms (PSTs) in such cases were typical of excitatory responses, previously described in birds and mammals showing pronounced adaptation and post-stimulus suppression of spike rate. In most cases of excitation-only responses, however, slopes of rate functions depended on stimulus frequency. Close to characteristic frequency (CF), slopes tended to decrease with increasing SPL, whereas away from CF, slopes tended to increase with SPL. In a minority of excitation-only responses, slopes of rate functions were parallel. In some fibres, tones adjacent to the response area caused overt suppression of spontaneous firing. For these fibres, the slopes of rate functions were more-strongly frequency-dependent, being negative at low SPL when rate suppression occurred. Suppression of spontaneous activity at low SPL was non-monotonic and quite different from suppression of spike rate at stimulus intensities above rat saturation. In PSTs of suppressed spontaneous activity, rebound occurred at the termination of the tone. The results clarify previous observations of suppression of primary auditory responses in birds. We conclude that responses in the majority of auditory fibres in the pigeon are the product of opposing excitatory and suppressive influences in the cochlea, generated by single tones in quite.

Induced suppression in spike responses to tone-on-noise stimuli in the auditory nerve of the pigeon.

Spike potentials were recorded from single, afferent fibres in the pigeon auditory nerve. Pure-tone stimuli were presented in quiet and in combination with wide band noise. Presented alone, tones produced tuned response areas; noise generally drove spike rate to well above the spontaneous rate measured in quiet. When presented in combination with noise, tones up to 75 dB SPL at frequencies far from the fibre's response area had no effect on the noise-driven spike rate. As the tone frequency was shifted towards the response area, from above or below CF, suppression of the noise-driven spike rate became stronger until the tone reached the edge of the response area. Suppression of the noise-driven rate was directly proportional to the level of the tone. Within the area of response to the tone, tone-driven spike rates generally were unchanged or variably decreased (occasionally slightly increased) by tone-on-noise stimulation, depending on the relation of the tone frequency to CF and the level of the tone relative to that of the noise. Tuning properties were unaffected. It is suggested that in the pigeon, the suppression of driven spike rate during presentation of combination stimuli, which is common to all fibres, depends on the same mechanism as the suppression of spontaneous firing by tones that is observed in a proportion of fibres (Temchin, A.N. (1988), J. Comp. Physiol. A 163, 99-115; Hill et al., (1989) Hear. Res. 39, 37-48).

Temporal synchronization in the primary auditory response in the pigeon.

Spike potentials were recorded from single fibres in the auditory nerve of the pigeon. In responses elicited by tonal stimuli, the timing of each spike relative to stimulus waveform was measured and period histograms were constructed. Phase locking of spikes was estimated in terms of a synchronicity index obtained by vector addition within the period histogram. A second measure of synchrony in the spike responses was obtained, that of temporal dispersion. For a population of fibres, vector strength of phase locking decreased for frequencies above 1 kHz, as reported for several other species. Temporal dispersion, however, also decreased with frequency, indicating enhanced temporal synchrony as frequency increased within the bandwidth of phase locking. The upper frequency limit of phase locking appears to depend on irreducible jitter of biological origin in the timing of spikes. For individual fibres, the bandwidth of synchronization of spikes consistently exceeds the response area, covering in addition the areas of suppression adjacent to the response area. Spike trains suppressed by a tonal stimulus become synchronized to that stimulus. Phase angles of synchronized responses systematically change as a function of tone level, when tone frequency is above or below CF, as reported for other avian species. Synchronicity and phase angle intensity functions are quite independent of spike rate intensity functions.

A model proposing synaptic and extra-synaptic influences on the responses of cochlear nerve fibres.

A unique property of sensory coding in the vertebrate auditory system is the existence of the classical form of excitatory centre-inhibitory surround in relative spike rate along the stimulus frequency dimension, in addition to a representation of temporal fine structure of high frequency periodic stimuli in the discharge pattern of primary afferent spike trains. We present a model which designates three factors that influence rate and temporal synchrony in spike responses; an excitatory factor, a suppressive factor and a synchronizing factor. The model proposes that an essential integration of bioelectric signals occurs in the primary afferent fibre. It is presumed that mean spike rate depends on mean level of membrane depolarization and synchronization depends on periodic modulation of membrane potential at the spike initiating zone. In the model, the excitatory factor is synaptically-mediated, excitatory post-synaptic potential (e.p.s.p.); the suppressive factor is negative DC polarization of the fibre membrane and the synchronizing factor is AC modulation of the fibre membrane potential. It is proposed that both the negatively-polarizing and high-frequency modulating signals are derived from extracellular current flow in the cochlea.

Auditory brainstem response in tammar wallaby (Macropus eugenii).

Auditory brainstem responses (ABR) elicited by click and tonal stimuli were recorded from the tammar wallaby (Macropus eugenii), a marsupial mammal. The morphology, threshold, amplitude, and latency of ABRs recorded in the tammar wallaby are similar to those of other marsupials and mammals used in auditory research, including humans. Thresholds determined by an algorithm employing cross-correlation and by conventional visual detection methods were comparable. The findings from this study indicate that tammar wallaby is a suitable model for auditory research and that algorithms employing cross-correlation are useful for detection of the ABR waveform.

Development of auditory function in the tammar wallaby Macropus eugenii.

Auditory brainstem responses (ABRs) were evoked in developing wallabies by click and tone burst stimuli delivered by bone conduction and air conduction, at progressive stages of post-natal (pouch) life. ABRs were recorded through the onset of auditory responses (95-110 days), the opening of the external ear canal (125-130 days) and the maturation of ABR thresholds and latencies to values corresponding to those in adults ( > 180 days). ABRs were evoked in response to bone-conducted clicks some days prior to the age at which an acoustically evoked response was first observed (around 95 days of pouch life). ABRs could be evoked by bone-conducted and intense air-conducted stimuli prior to opening of the ear canal. A trend of decreasing threshold and latency with age was observed for both modes of stimulation. The morphology of the ABR became more complex, according to both increased age and increased stimulus intensity. The ABR waveforms indicated relatively greater mechanosensitivity to bone-conducted stimuli than to air-conducted stimuli, prior to opening of the ear canal. Following opening of the ear canal, thresholds to air-conducted clicks and tones were substantially reduced and decreased further over the next 10-20 days, while thresholds to bone-conducted clicks continued slowly to decrease. Thresholds to tone bursts in the centre frequency range (4-12 kHz) remained less than those for low (0.5-1.5 kHz) and higher (16 kHz) frequencies. Latencies of an identified peak in ABR waveforms characteristically decreased with age (at constant stimulus intensity) and with stimulus intensity (for a given age). ABR waveforms obtained at progressive ages, but judged to be at corresponding sensation levels, underwent maturational changes, independent of conductive aspects of the wallabies' hearing, for 2-3 weeks after opening of the ear canal.

Developmental risk factors for youth violence.

PURPOSE: To replicate earlier research findings on risk factors for youth violence and to explore the effects on violent behavior of constructs shown to increase risk for other problem behaviors, within a developmental frame. METHODS: Data were from the Seattle Social Development Project (SSDP), a prospective study involving a panel of youths followed since 1985. Potential risk factors for violence at age 18 years were measured at ages 10, 14, and 16 years. Bivariate relationships involving risk factor constructs in the individual, family, school, peer and community domains and violence were examined at each age to assess changes in their strength of prediction over time. Attention was also given to the additive strength of increasing numbers of risk factors in the prediction of violence at age 18 years. A final set of analyses explored the extent to which youths were correctly classified as having committed a violent act (or not) at age 18 years on the basis of their overall level of risk at ages 10, 14, and 16 years. RESULTS: At each age, risk factors strongly related to later violence were distributed among the five domains. Ten of 15 risk factors constructs measured at age 10 years were significantly predictive of violence at age 18 years. Twenty of 25 constructs measured at age 14 years and 19 of 21 constructs measured at age 16 years were significantly predictive of later violence. Many constructs predicted violence from more than one developmental point. Hyperactivity (parent rating), low academic performance, peer delinquency, and availability of drugs in the neighborhood predicted violence from ages 10, 14, and 16 years. Analyses of the additive effects of risk factors revealed that youths exposed to multiple risks were notably more likely than others to engage in later violence. The odds for violence of youths exposed to more than five risk factors compared to the odds for violence of youths exposed to fewer than two risk factors at each age were seven times greater at age 10 years, 10 times greater at age 14 years, and nearly 11 times greater at age 16 years. However, despite information gained from all significant risk factors, the overall accuracy in predicting youths who would go on to commit violent acts was limited. CONCLUSIONS: Findings from the study have important implications for preventive intervention programs. Prevention efforts must be comprehensive and developmentally sensitive, responding to large groups or populations exposed to multiple risks.

Developmental pathways to alcohol abuse and dependence in young adulthood.

OBJECTIVE: To determine if people who were diagnosed with alcohol abuse or dependence (AAD) at age 21 had different developmental patterns of alcohol use in adolescence than non-AAD individuals. METHOD: An ethnically diverse urban sample of 808 children was surveyed at age 10 in 1985 and followed prospectively to age 21 in 1996. AAD at age 21 was assessed following DSM-IV criteria. Latent Transition Analysis (LTA) was used to identify four statuses of alcohol use (nonuse, initiation only, current use only, heavy episodic drinking), as well as transition probabilities between these four statuses from elementary school to middle school and from middle school to high school among the AAD and non-AAD group. RESULTS: The prevalence of alcohol use statuses during elementary school was similar in the two groups. Differences in alcohol use emerged in middle school and became more pronounced in high school. In middle school, AAD individuals were more likely to have initiated or been current drinkers than non-AAD individuals. However, the two groups did not differ in the prevalence of heavy episodic drinking in middle school. In high school, most AAD individuals were in the heavy episodic drinking status (54%), while most non-AAD individuals were in the initiation only (33%) or current use only (34%) statuses. CONCLUSIONS: These findings suggest preventive intervention targets for different developmental periods. Alcohol abuse and dependence at age 21 may be prevented by delaying alcohol initiation, by reducing current use in middle school and by reducing heavy episodic drinking in high school.

Childhood and adolescent predictors of alcohol abuse and dependence in young adulthood.

OBJECTIVE: To provide a comprehensive examination of childhood and adolescent predictors of alcohol abuse and dependence at age 21, theoretically guided by the social development model. METHOD: Data were taken from an ethnically diverse urban sample of 808 students (51% male), surveyed at age 10 and followed prospectively to age 21 in 1996. Potential predictors of alcohol abuse and dependence at age 21 were measured at ages 10, 14 and 16. Relationships between these predictors and alcohol abuse and dependence were examined at each age, to assess changes in their patterns of prediction over time. RESULTS: Strong bonding to school, close parental monitoring of children and clearly defined family rules for behavior, appropriate parental rewards for good behaviors, high level of refusal skills and strong belief in the moral order predicted a lower risk for alcohol abuse and dependence at age 21. Of these, strong bonding to school consistently predicted lower alcohol abuse and dependence from all three ages (10, 14 and 16). By contrast, youths who had a higher risk of alcohol abuse and dependence at age 21 engaged in more problem behaviors, had more opportunities to be involved with antisocial individuals and spent more time with and were more bonded to those individuals, viewed fewer negative consequences from antisocial behaviors and held more favorable views on alcohol use. Of these, prior problem behaviors and antisocial opportunities and involvements at ages 10, 14 and 16 consistently predicted alcohol abuse and dependence at age 21. CONCLUSIONS: These important malleable predictors, identifiable as early as age 10, provide potential intervention targets for the prevention of alcohol abuse and dependence in early adulthood.

Exploring the effects of age of alcohol use initiation and psychosocial risk factors on subsequent alcohol misuse.

OBJECTIVE: This study examines whether the age of initiation of alcohol use mediates the effects of other variables that predict alcohol misuse among adolescents and also whether the age of initiation of alcohol use accounts for known gender differences in the severity of alcohol misuse. METHOD: Data were taken from an ethnically diverse sample of 808 (412 male) students who were recruited in grade 5 at age 10-11 and followed prospectively on an annual basis for the next 7 years to age 17-18. State-of-the-art missing data methodology was used to address nonresponse due to noninitiation of alcohol use. Structural equation modeling was used to examine hypotheses for the prediction of alcohol misuse. RESULTS: A younger age of alcohol initiation was strongly related to a higher level of alcohol misuse at age 17-18 and fully mediated the effects of parent drinking, proactive parenting, school bonding, peer alcohol initiation and ethnicity, all measured at age 10-11, and perceived harmfulness of alcohol use, measured at age 10-11 and age 11-12. However, age of alcohol initiation did not fully account for gender differences in the level of alcohol misuse at age 17-18. To further examine the role of gender, interactions between gender and school bonding, and gender and friend's alcohol initiation, were evaluated. However, neither of the interaction terms had direct effects on either age of initiation or level of alcohol-related problems. CONCLUSIONS: Most measured risk factors for alcohol misuse were mediated through age of alcohol initiation. Only gender differences in alcohol misuse at age 17-18 were not mediated by age of alcohol initiation. Variables associated with these differences require further study. The results of this study indicate the importance of prevention strategies to delay the age of initiation of alcohol use.

The social predictors of adolescent alcohol misuse: a test of the social development model.

OBJECTIVE: This study was conducted to investigate the ability of the social development model (SDM) to predict alcohol misuse at age 16 and to investigate the ability of the SDM to mediate the effects of alcohol use at age 14 on alcohol misuse at age 16. METHOD: The sample of 807 (411 males) is from the longitudinal panel of the Seattle Social Development Project which, in 1985, surveyed all consenting fifth-grade students from 18 elementary schools serving high-crime neighborhoods in Seattle, Washington. Alcohol use was measured at age 14, predictors of alcohol misuse were measured at age 15 and alcohol misuse was measured at age 16. Structural equation modeling was used to examine the fit of the model to the data. RESULTS: All factor loadings were highly significant and the measurement model achieved a good fit with the data (Comparative Fit Index [CFI] = 0.93). A second-order structural model fit the data well (CFI = 0.91) and also explained 45% of the variance in alcohol misuse at age 16. The SDM partially and significantly mediated the direct effect of age-14 alcohol use on age-16 alcohol misuse. CONCLUSIONS: The risk and protective processes specified by the SDM serve as potential targets for the prevention or reduction of adolescent alcohol misuse.

Use of a uterine-cell monolayer culture system for micromanipulated bovine embryos.

The objective of this study was to evaluate the growth of micromanipulated bovine embryos in two in vitro culture systems. Sixty ova (day 7 from estrus) were collected in Dulbecco's phosphate-buffered saline (PBS), with 2% fetal calf serum, and transferred to a PBS holding medium containing 10% fetal calf serum to prepare for micromanipulation. Forty embryos (morula to expanded blastocyst stages) were selected for embryo splitting using a modified microsurgery procedure. Thirty-nine of these embryos were successfully bisected into demi-embryos (DE) and the halves allotted by post-manipulation quality grades into one of two treatment groups (Trt). DE in Trt A were cultured in Ham's F-10 medium with 10% FCS (HF-10) while the remaining DE halves from each embryo were cocultured in HF-10 on a monolayer of endometrial fibroblasts (8 x 10(4) viable fibroblast cells plated three days prior to culture) in Trt B. Embryo development, recorded at 12-hour intervals, was evaluated by a split-plot analysis of variance. Results indicated that embryo viability decreased (P<0.001) over time in culture. Overall viability was greater (P<0.001) for DE in Trt B than in Trt A, with a significant (P<0.05) Trt x Time interaction, indicating that embryo viability decreased more rapidly across time in HF-10 than in the monolayer coculture system. The percentage of DE developing at 12, 24, 36, 48, 60 and 72 hours in culture was: 44%, 41%, 33%, 28%, 21% and 18% for Trt A and 69%, 69%, 69%, 67%, 62% and 62% for Trt B. Fourteen of the DE in Trt B attached to fibroblast monolayer and initiated trophoblastic outgrowth and four additional DE remained viable for up to 17.5 days in vitro as intact blastocysts. These findings are the first reported that demonstrate that the zona-free bovine DE will develop during in vitro culture. Also, the bovine endometrial fibroblast monolayer system proved to be excellent for both short term (

Transferable embryo recovery rates following different insemination schedules in superovulated beef cattle.

The objective of this study was to evaluate the transferable embryo recovery rates from superovulated donor cattle after different artificial insemination (AI) schedules. Sixty mixed-breed crossbred females were administered follicle stimulating hormone (FSH) and prostaglandin F(2)alpha (PGF(2)alpha) to induce a superovulatory response. At standing estrus, donor females were randomly allotted to one of five treatment groups for AI. Donors were inseminated with two units of high-quality or low-quality frozen semen at 12, 24, 36, or 48 h after the onset of estrus in treatment Groups I, II, III, and IV, respectively, or inseminated with two units at 12, 24, 36, and 48 h (eight units/donor) in control Group V. Donor females inseminated once at either 12 or 24 h after the onset of estrus did not differ from donors inseminated in Group V in overall fertilization and transferable embryo recovery rates. The highest fertilization rate (89.5%) and transferable embryo recovery rate (74.9%) per donor resulted when AI was performed with high-quality semen at 24 h after the onset of estrus. These findings indicate that repeated insemination of superovulated beef cattle is not necessary to attain optimal fertilization rates and production of transferable quality embryos in beef cattle.

Microsurgery on bovine embryos at the morula stage to produce monozygotic twin calves.

Mature Brangus donor cows were superovulated with follicle stim-ulating hormone administered twice daily in intramuscular injections. On day 6.5 to 7 post-estrus, embryos were collected non-surgically using a phosphate-buffered saline medium. A total of 37 ova was collected, of which 28 were advanced morulae and early blastocysts. Twenty of these embryos were selected for micromanipulation with a radial-type Leitz micromanipulator. While the embryos were in a holding medium containing 10% fetal calf serum, three glass microinstruments were used to open the zona pellucida, remove the mass of blastomeres and bisect the embryo on a vertical plane. Halved embryos were inserted into bovine zonae and placed either as single half-embryos or twin half-embryos in 0.25 ml French straws with fresh holding medium. The micromanipulated embryos (demi-embryos) were then non-surgically transplanted, either as a single demi-embryo or as a twin demi-embryo pair, into the uterine horn of day 6.5 to 8 recipient beef females ipsilateral to the existing corpus luteum. Of the 14 micromanipulated embryos that were transplanted to recipients, pregnancy rates were 16.6% for the single demi-embryos and 62.5% for the twin demi-embryos. No pregnancies resulted from bisected blastocysts.