Expressing hNF-LE397K results in abnormal gaiting in a transgenic model of CMT2E.

Charcot-Marie-Tooth disease (CMT) is the most commonly inherited peripheral neuropathy. CMT disease signs include distal limb neuropathy, abnormal gaiting, exacerbation of neuropathy, sensory defects and deafness. We generated a novel line of CMT2E mice expressing an hNF-L(E397K) transgene, which displayed muscle atrophy of the lower limbs without denervation, proximal reduction in large caliber axons and decreased nerve conduction velocity. In this study, we showed that hNF-L(E397K) mice developed abnormal gait of the hind limbs. The identification of severe gaiting defects in combination with previously observed muscle atrophy, reduced axon caliber and decreased nerve conduction velocity suggests that hNF-L(E397K) mice recapitulate many of clinical signs associated with CMT2E. Therefore, hNF-L(E397K) mice provide a context for potential therapeutic intervention.

Distal to proximal development of peripheral nerves requires the expression of neurofilament heavy.

At the initiation of radial growth, neurofilaments are likely to consist primarily of neurofilament light and medium as neurofilament heavy expression is developmentally delayed. To better understand the role of neurofilament heavy in structuring axons, axonal diameter and neurofilament organization were measured in proximal and distal segments of the sciatic nerve and along the entire length of the phrenic nerve. Deletion of neurofilament heavy reduced axonal diameters and neurofilament number in proximal nerve segments. However, neurofilament spacing was greater in proximal versus distal phrenic nerve segments. Taken together, these results suggest that loss of neurofilament heavy reduces radial growth in proximal axonal segments by reducing the accumulation of neurofilaments. As neurofilament heavy expression is developmentally delayed, these results suggest that without neurofilament heavy, the neurofilament network is established in a distal to proximal gradient perhaps to allow distal axonal segments to develop prior to proximal segments.

Variation of the neurofilament medium KSP repeat sub-domain across mammalian species: implications for altering axonal structure.

The evolution of larger mammals resulted in a corresponding increase in peripheral nerve length. To ensure optimal nervous system functionality and survival, nerve conduction velocities were likely to have increased to maintain the rate of signal propagation. Increases of conduction velocities may have required alterations in one of the two predominant properties that affect the speed of neuronal transmission: myelination or axonal diameter. A plausible mechanism to explain faster conduction velocities was a concomitant increase in axonal diameter with evolving axonal length. The carboxy terminal tail domain of the neurofilament medium subunit is a determinant of axonal diameter in large caliber myelinated axons. Sequence analysis of mammalian orthologs indicates that the neurofilament medium carboxy terminal tail contains a variable lysine-serine-proline (KSP) repeat sub-domain flanked by two highly conserved sub-domains. The number of KSP repeats within this region of neurofilament medium varies among species. Interestingly, the number of repeats does not change within a species, suggesting that selective pressure conserved the number of repeats within a species. Mapping KSP repeat numbers onto consensus phylogenetic trees reveals independent KSP expansion events across several mammalian clades. Linear regression analyses identified three subsets of mammals, one of which shows a positive correlation in the number of repeats with head-body length. For this subset of mammals, we hypothesize that variations in the number of KSP repeats within neurofilament medium carboxy terminal tail may have contributed to an increase in axonal caliber, increasing nerve conduction velocity as larger mammals evolved.

In vitro production of cattlexbuffalo hybrid embryos using cattle oocytes and African buffalo (Syncerus caffer caffer) epididymal sperm.

Interspecies hybridization of bovids occurs between domestic cattle and at least three other species; American bison (Bison bison), yak (Bos grunniens) and banteng (Bos banteng). Birth of a cattlexbuffalo (Bubalus bubalis) hybrid has reportedly occurred in Russia and in China, but these reports were not authenticated. Such hybrids could be important in improving livestock production and management of diseases that impede production in tropical Africa. This study investigated hybridization between cattle and its closest African wild bovid relative, the African buffalo (Syncerus caffer caffer). In an attempt to produce cattlexbuffalo hybrid embryos in vitro, matured cattle oocytes were subjected to a standard in vitro fertilization (IVF) procedure with either homologous cattle (n=1166 oocytes) or heterologous African buffalo (n=1202 oocytes) frozen-thawed epididymal sperm. After IVF, 67.2% of the oocytes inseminated with the homologous cattle sperm cleaved. In contrast, fertilization with buffalo sperm resulted in only a 4.6% cleavage rate. The cleavage intervals were also slower in hybrid embryos than in the IVF-derived cattle embryos. Of the cleaved homologous cattle embryos 52.2% progressed to the morula stage compared with 12.7% for the buffalo hybrid embryos. No hybrid embryos developed beyond the early morula stage, while 40.1% of the cleaved cattlexcattle embryos developed to the blastocyst stage. Transfer of buffalo hybrid IVF embryos to domestic cattle surrogates resulted in no pregnancies at 60 days post-transfer. This study indicates that interspecies fertilization of cattle oocytes with African buffalo epididymal sperm can occur in vitro, and that a barrier to hybridization occurs in the early stages of embryonic development. Chromosomal disparity is likely the cause of the fertilization abnormalities, abnormal development and subsequent arrest impairing the formation of hybrid embryos beyond the early morula stage. Transfer of the buffalo hybrid embryos did not rescue the embryos from development arrest.

Mechanisms of voltage-gated ion channel regulation: from gene expression to localization.

The ion channel milieu present in a neuron in large part determines the inherent excitability of a given cell and is responsible for the translation of sensory transduction and synaptic input to axonal output. Intrinsic excitability is a dynamic process subject to multiple levels of regulation from channel gene expression to post-translational modifications that influence channel activity. The goal of this review is to provide an overview of some of the mechanisms by which channels can be modified in order to influence neuronal output. We focus on four levels of regulation: channel gene transcription, alternative splicing of channel transcripts, post-translational modifications that alter channel kinetics (phosphorylation), and subcellular localization and trafficking of channel proteins.

Out-of-season breeding of milk goats--the effect of light treatment, melatonin and breed.

The purpose of this study was to evaluate the effectivity of melatonin in addition to light treatment (exposure to 2 hours of light during the night = a long-day photoperiod) to modify the breeding season of Saanen and cross-bred milk goats and to compare the difference between the breeds. Twenty-two Saanen and 22 cross-bred does were randomly divided into 3 treatment groups. Group 1 (controls) received no treatment, Group 2 received light treatment for 37 days and Group 3 received light treatment plus melatonin implants after the light treatment. After a further 35 days the 3 groups were brought together and a billy goat that had also been exposed to the extra light at night, had received a melatonin implant and had been isolated from the does during the treatment period, was introduced to the does for natural mating. Ultrasound scanning was used to diagnose pregnancy and all the pregnant goats kidded. Significantly more Saanen does compared to cross-bred does (P = 0.018) became pregnant and kidded after natural mating, when the group that received melatonin as well as light treatment was compared to the group that received light treatment only. Compared to light treatment only, the addition of melatonin to light treatment improved (P = 0.0028) conception after natural mating, in both the Saanen and the cross-bred does.

Nuclear transport: never-ending cycles of signals and receptors.

Proteins transported into and out of the nucleus require amino acid motifs called NLSs and NESs, respectively. The amino acid sequences of these signals vary considerably. A superfamily of transport receptors has been identified and each member contains three transport-related domains. Transport receptors bind to the signal sequences, either directly or through adapter proteins, to promote nucleocytoplasmic transport. The diversity of signals, receptors and adapter proteins suggests that there are many pathways for nuclear entry or exit. The direction of transport (into or out of the nucleus) is regulated in part by the small GTPase Ran as well as by intrinsic substrate motifs.

Attenuation of the slow component of delayed rectification, action potential prolongation, and triggered activity in mice expressing a dominant-negative Kv2 alpha subunit.

An in vivo experimental strategy, involving cardiac-specific expression of a mutant Kv 2.1 subunit that functions as a dominant negative, was exploited in studies focused on exploring the role of members of the Kv2 subfamily of pore-forming (alpha) subunits in the generation of functional voltage-gated K(+) channels in the mammalian heart. A mutant Kv2.1 alpha subunit (Kv2.1N216) was designed to produce a truncated protein containing the intracellular N terminus, the S1 membrane-spanning domain, and a portion of the S1/S2 loop. The truncated Kv2.1N216 was epitope tagged at the C terminus with the 8-amino acid FLAG peptide to generate Kv2. 1N216FLAG. No ionic currents are detected on expression of Kv2. 1N216FLAG in HEK-293 cells, although coexpression of this construct with wild-type Kv2.1 markedly reduced the amplitudes of Kv2. 1-induced currents. Using the alpha-myosin heavy chain promoter to direct cardiac specific expression of the transgene, 2 lines of Kv2. 1N216FLAG-expressing transgenic mice were generated. Electrophysiological recordings from ventricular myocytes isolated from these animals revealed that I(K, slow) is selectively reduced. The attenuation of I(K, slow) is accompanied by marked action potential prolongation, and, occasionally, spontaneous triggered activity (apparently induced by early afterdepolarizations) is observed. The time constant of inactivation of I(K, slow) in Kv2. 1N216FLAG-expressing cells (mean+/-SEM=830+/-103 ms; n=17) is accelerated compared with the time constant of I(K, slow) inactivation (mean+/-SEM=1147+/-57 ms; n=25) in nontransgenic cells. In addition, unlike I(K, slow) in wild-type cells, the component of I(K, slow) remaining in the Kv2.1N216FLAG-expressing cells is insensitive to 25 mmol/L tetraethylammonium. Taken together, these observations suggest that there are 2 distinct components of I(K, slow) in mouse ventricular myocytes and that Kv2 alpha subunits underlie the more slowly inactivating, tetraethylammonium-sensitive component of I(K, slow). In vivo telemetric recordings also reveal marked QT prolongation, consistent with a defect in ventricular repolarization, in Kv2.1N216FLAG-expressing transgenic mice.

Water fluoridation and the sudden infant death syndrome.

AIMS: To determine whether exposure to fluoridated water supplies prenatally or postnatally at the time of death increases the risk of sudden infant death syndrome (SIDS). METHODS: A nationwide, case-control study, with infant's water fluoridation status determined from census area unit information for mother's usual address at the time of the infant's birth, infant's usual address at the time of death / nominated sleep and address where infant died / was at nominated sleep. SIDS risk associated with fluoride exposure postnatally was assessed according to method of infant feeding (breast or reconstituted formula), for the two days prior to infant's death / nominated sleep. RESULTS: Infants exposed to fluoridated water supplies during pregnancy were not at increased risk for SIDS, adjusted odds ratio (OR) 1.19 (95% confidence interval (CI) 0.82, 1.74). For breast-fed infants at the time of death / nominated sleep, fluoridated water exposure was not associated with an increased risk for SIDS, adjusted OR 1.09 (95% CI 0.66, 1.79). Similarly, 'fluoridated' formula feeding, when compared with 'unfluoridated' formula feeding, showed no increased risk of SIDS, adjusted OR 1.25 (95% CI 0.73, 2.13). There was no evidence of an interaction between fluoridation and infant feeding for the last two days (chi2 = 0.171, df = 1, p = 0.68). CONCLUSION: Exposure to a fluoridated water supply prenatally or postnatally at the time of death did not affect the relative risk for SIDS.

Functional knockout of the transient outward current, long-QT syndrome, and cardiac remodeling in mice expressing a dominant-negative Kv4 alpha subunit.

A novel in vivo experimental strategy, involving cell type-specific expression of a dominant-negative K+ channel pore-forming alpha subunit, was developed and exploited to probe the molecular identity of the cardiac transient outward K+ current (I(to)). A point mutation (W to F) was introduced at position 362 in the pore region of Kv4.2 to produce a nonconducting mutant (Kv4.2W362F) subunit. Coexpression of Kv4.2W362F with Kv4.2 (or Kv4.3) attenuates the wild-type currents, and the effect is subfamily specific; ie, Kv4.2W362F does not affect heterologously expressed Kv1.4 currents. With the use of the alpha-myosin heavy chain promoter to direct cardiac-specific expression, several lines of Kv4.2W362F transgenic mice were generated. Electrophysiological recordings reveal that I(to) is selectively eliminated in ventricular myocytes isolated from transgenic mice expressing Kv4.2W362F, thereby demonstrating directly that the Kv 4 subfamily underlies I(to) in the mammalian heart. Functional knockout of I(to) leads to marked increases in action potential durations in ventricular myocytes and to prolongation of the QT interval in surface ECG recordings. In addition, a novel rapidly activating and inactivating K+ current, which is not detectable in myocytes from nontransgenic littermates, is evident in Kv4.2W362F-expressing ventricular cells. Importantly, these results demonstrate that electrical remodeling occurs in the heart when the expression of endogenous K- channels is altered.

In vitro maturation and fertilization of oocytes recovered from free-ranging Burchell's zebra (Equus burchelli) and Hartmann's zebra (Equus zebra hartmannae).

A noninvasive repeatable method to harvest oocytes for in vitro fertilization (IVF) could potentially be used to assist reproduction in endangered equid species. The objectives of this study were to evaluate a specific transvaginal ultrasound-guided oocyte recovery procedure for use in zebra mares and the general applicability of IVF procedures in zebra. Ovaries were collected from Burchell's zebra (Equus burchelli) and Hartmann's zebra (Equus zebra hartmannae) mares at routine culling for Expt. I. Of the 144 oocytes recovered from these ovaries, 70% were of excellent quality. No significant difference in oocyte quality was found between the two zebra species. Zona drilling was performed on in vitro-matured oocytes prior to IVF. Epididymal sperm from culled Burchell's zebra stallions were used for IVF. The sperm either were exposed to calcium ionophore or were not treated and served as a control. In vitro fertilized oocytes were then co-cultured with zebra granulosa cells (ZGC) or with bovine oviduct cells (BOC) for up to 8 days. Overall, a 38% cleavage rate was obtained with 16% of sperm-exposed oocytes developing to the morula or blastocyst stage. All of the embryos that developed to at least the morula stage were cultured on BOC; whereas, none of those cultured on ZGC reached the morula stage during the same interval. Cleavage rates of oocytes inseminated with ionophore-treated or with control sperm were not significantly different, suggesting that ionophore treatment of epididymal sperm for IVF in these zebra species may be redundant. In Expt. II, 10 transvaginal ultrasound-guided oocyte aspiration procedures on five captive Burchell's zebra mares recovered a total of 33 oocytes (6.6 oocytes/female) of which 94% were considered viable. This approach may be an attractive means of producing gametes for assisted reproduction in endangered species. Furthermore, results from this study indicate that IVF may become a means of producing offspring from zebra and other equid species in the future.

Developmental analysis reveals mismatches in the expression of K+ channel alpha subunits and voltage-gated K+ channel currents in rat ventricular myocytes.

In the experiments here, the developmental expression of the functional Ca(2+)-independent, depolarization-activated K+ channel currents, Ito and IK, and of the voltage-gated K+ channel (Kv) alpha subunits, Kv1.2, Kv1.4, Kv1.5, Kv2.1, and Kv4.2 in rat ventricular myocytes were examined quantitatively. Using the whole-cell patch clamp recording method, the properties and the densities of Ito and IK in ventricular myocytes isolated from postnatal day 5 (P5), 10 (P10), 15 (P15), 20 (P20), 25 (P25), 30 (P30), and adult (8-12 wk) rats were characterized and compared. These experiments revealed that mean Ito densities increase fourfold between birth and P30, whereas IK densities vary only slightly. Neither the time- nor the voltage-dependent properties of the currents vary measurably, suggesting that the subunits underlying functional Ito and IK channels are the same throughout postnatal development. In parallel experiments, the developmental expression of each of the voltage-gated K+ channel alpha subunits, Kv1.2, Kv1.4, Kv1.5, Kv2.1, and Kv4.2, was examined quantitatively at the mRNA and protein levels using subunit-specific probes. RNase protection assays revealed that Kv1.4 message levels are high at birth, increase between P0 and P10, and subsequently decrease to very low levels in adult rat ventricles. The decrease in message is accompanied by a marked reduction in Kv1.4 protein, consistent with our previous suggestion that Kv1.4 does not contribute to the formation of functional K+ channels in adult rat ventricular myocytes. In contrast to Kv1.4, the mRNA levels of Kv1.2, Kv1.5, Kv2.1, and Kv4.2 increase (three- to five-fold) between birth and adult. Western analyses, however, revealed that the expression patterns of these subunits proteins vary in distinct ways: Kv1.2 and Kv4.2, for example, increase between P5 and adult, whereas Kv1.5 remains constant and Kv2.1 decreases. Throughout development, therefore, there is a mismatch between the numbers of Kv alpha subunits expressed and the functional voltage-gated K+ channel currents distinguished electrophysiologically in rat ventricular myocytes. Alternative experimental approaches will be required to define directly the Kv alpha subunits that underlie functional voltage-gated K+ channels in these (and other) cells. In addition, the finding that Kv alpha subunit protein expression levels do not necessarily mirror mRNA levels suggests that caution should be exercised in attempting functional interpretations of observed changes in mRNA levels alone.

Myocardial potassium channels: electrophysiological and molecular diversity.

Myocardial K+ currents function to control resting membrane potentials, the heights and durations of action potentials, and refractoriness and automaticity. They are important targets for the actions of transmitters and hormones or drugs known, or postulated, to modulate cardiac functioning. A variety of K+ currents that subserve these functions have now been identified in myocardial cells isolated from different species, as well as in cells isolated from different regions of the heart in the same species. These currents include the voltage-gated K+ types, such as the transient outward (Ito) and delayed rectifier (IK) currents, as well as the inwardly rectifying currents, IK1, IK(ACh), and IK(ATP). The physiological and functional properties of the various K+ currents/channels expressed in different myocardial cell types are the focus of this review. Advances made in cloning K+ channel subunits of the Kv, eag, Kir, and IsK families are discussed, and progress made in identifying the K+ channel subunits expressed in the mammalian myocardium is summarized. The relationships between the various cloned K+ channel subunits and the functional K+ channels characterized electrophysiologically in myocardial cells are explored.

Differential expression of voltage-gated K+ channel subunits in adult rat heart. Relation to functional K+ channels?

Polyclonal antibodies against each of the K+ channel subunits (Kv1.2, Kv1.4, Kv1.5, Kv2.1, and Kv4.2) shown previously to be expressed in adult rat heart at the mRNA level were used to examine the distributions of these K+ channel subunits in adult rat atrial and ventricular membranes. Immunohistochemistry on isolated adult rat ventricular myocytes revealed strong labeling with the anti-Kv4.2 and anti-Kv1.2 antibodies. Although somewhat weaker (than with anti-Kv1.2 or anti-Kv4.2), positive staining was also observed with the anti-Kv1.5 and anti-Kv2.1 antibodies. Ventricular myocytes exposed to the anti-Kv1.4 antibody, in contrast, did not appear significantly different from background. Qualitatively similar results were obtained on isolated adult rat atrial myocytes. Western blots of atrial and ventricular membrane proteins confirmed the presence of Kv1.2, Kv1.5, Kv2.1, and Kv4.2 and revealed differences in the relative abundances of these subunits in the two membrane preparations. Kv4.2, for example, is more abundant in ventricular than in atrial membranes, whereas Kv1.2 and Kv2.1 are higher in atrial membranes; Kv1.5 levels are comparable in the two preparations. In contrast to these results, nothing was detected in Western blots of atrial or ventricular membrane proteins with the anti-Kv1.4 antibody at concentrations that revealed intense labeling of a 97-kD protein in adult rat brain membranes. A very faint band was detected at 97 kD in the atrial and ventricular preparations when the anti-Kv1.4 antibody was used at a 5- to 10-fold higher concentration. The simplest interpretation of these results is that Kv1.4 is not an abundant protein in adult rat atrial or ventricular myocytes. Therefore, it seems unlikely that Kv1.4 plays an important role in the formation of functional depolarization-activated K+ channels in these cells. The relation(s) between the (other four) K+ channel subunits and the depolarization-activated K+ channels identified electrophysiologically in adult rat atrial and ventricular myocytes is discussed in the present study.

Childhood asthma in four countries: a comparative survey.

BACKGROUND: Asthma mortality among young people varies widely between different countries. Geographical differences in asthma prevalence are also believed to exist, but evidence is sparse owing to the lack of multicentre surveys using common protocols. A survey was conducted of 12-year-old children living in defined areas of New Zealand, Wales, South Africa and Sweden, in order to see whether asthma prevalence and mortality rates in children show parallel differences. METHODS: Questionnaires enquiring about a history of asthma and respiratory symptoms were issued to the parent. The children performed a simple exercise challenge test. RESULTS: Information was obtained for 4353 children. A history of asthma at any time was reported for 16.8% of children in New Zealand, 12.0% in Wales, 11.5% in South Africa and 4.0% of Sweden, and a similar pattern was shown by several other indices of asthma (various relevant symptoms, inhaler use, response to exercise challenge, and asthma mortality at ages 5-19 years). In Sweden wheezing was negatively associated with pet ownership; elsewhere there was a positive (though non-significant) association. Cat ownership was highest in New Zealand and lowest in Sweden. CONCLUSIONS: The prevalence of asthma in children shows geographical variation which is parallel to that of asthma mortality, being high in New Zealand and low in Sweden. Differential exposure to animal allergens is a possible factor in this variation.

Breastfeeding and the risk of sudden infant death syndrome.

The New Zealand Cot Death Study, a multicentre case-control study, was set up to identify risk factors associated with sudden infant death syndrome (SIDS). In the 3 years of the study there were 485 infant deaths classified as SIDS in the study areas and 1800 infants who were randomly selected as controls. Data were collected by parent interviews and from obstetric notes. A full set of data for this analysis was available from 356 cases and 1529 control infants. The relationship between length of any breastfeeding and SIDS was examined: 92% of the controls were initially breastfed compared to 86% of the cases. As time went by, cases stopped breastfeeding sooner than controls: by 13 weeks, 67% controls were breastfed versus 49% cases. A reduced risk for SIDS in breastfed infants persisted during the first 6 months after controlling for confounding demographic, maternal and infant factors. Infants exclusively breastfed 'at discharge from the obstetric hospital' (odds ratio [OR] = 0.52, 95% confidence interval (CI): 0.35-0.71) and during the last 2 days (OR = 0.65, 95% CI: 0.46-0.91) had a significantly lower risk of SIDS than infants not breastfed after controlling for potential confounders. We have shown a substantial association of breastfeeding with a lowered risk for SIDS. This supports the need for more positive promotion and active community support to further enhance the level and length of exclusive breastfeeding.

Smoking and the sudden infant death syndrome.

OBJECTIVE: Maternal smoking has been shown to be a risk factor for sudden infant death syndrome (SIDS). The effect of smoking by the father and other household members has not previously been examined. METHODS: A large nationwide case-control study. Four hundred eighty-five SIDS deaths in the postneonatal age group were compared with 1800 control infants. RESULTS: Infants of mothers who smoked during pregnancy had a 4.09 (95% confidence interval [CI] = 3.28, 5.11) greater risk of death than infants of mothers who did not smoke. Infants of mothers who smoked postnatally also had an increased risk of SIDS compared with infants of nonsmokers and, furthermore, the risk increased with increasing levels of maternal smoking. Smoking by the father and other household members increased the risk (odds ratio [OR] = 2.41, 95% CI = 1.92, 3.02 and OR = 1.54, 95% CI = 1.20, 1.99, respectively). Smoking by the father increased the risk of SIDS if the mother smoked, but had no effect if she did not smoke. In analyses controlled for a wide range of potential confounders, smoking by the mother and father was still significantly associated with an increased risk of SIDS. CONCLUSION: Passive tobacco smoking is causally related to SIDS.

Dummies and the sudden infant death syndrome.

The association between dummy use and sudden infant death syndrome (SIDS) was investigated in 485 deaths due to SIDS in the postneonatal age group and compared with 1800 control infants. Parental interviews were completed in 87% of subjects. The prevalence of dummy use in New Zealand is low and varies within New Zealand. Dummy use in the two week period before death was less in cases of SIDS than in the last two weeks for controls (odds ratio (OR) 0.76, 95% confidence interval (CI) 0.57 to 1.02). Use of a dummy in the last sleep for cases of SIDS or in the nominated sleep for controls was significantly less in cases than controls (OR 0.44, 95% CI 0.26 to 0.73). The OR changed very little after controlling for a wide range of potential confounders. It is concluded that dummy use may protect against SIDS, but this observation needs to be repeated before dummies can be recommended for this purpose. If dummy sucking is protective then it is one of several factors that may explain the higher mortality from SIDS in New Zealand than in other countries, and may also explain in part the regional variation within New Zealand.

Birth of live calves after transfer of frozen-thawed bovine embryos fertilised in vitro.

Follicular oocytes were aspirated from bovine ovaries collected at a local abattoir. The cumulus-intact oocytes were matured, fertilised and subsequently cultured in vitro. Of 2297 oocytes exposed to in vitro procedures during a 30-day experimental period, 92 per cent matured, 83 per cent were fertilised, 73 per cent cleaved, 48 per cent developed to the morulae and 14 per cent developed to the expanded blastocyst stage. During this experimental period, 300 similar embryos fertilised in vitro were frozen at different post in vitro block developmental stages. After approximately one year of storage in liquid nitrogen, 98 of these embryos were thawed and cultured either for up to four hours or for two days in tissue culture medium-199. Culturing the embryos for up to four hours was not as successful for in vitro development as culturing for two days. Of the 40 embryos cultured for two days, 67 per cent of early blastocyst stage embryos developed to expanded blastocysts in vitro and 46 per cent of morula stage embryos developed to expanded blastocysts, whereas only 8 per cent of 16-cell stage embryos developed to expanded blastocysts. A 50 per cent pregnancy rate resulted when frozen-thawed embryos were co-cultured for two days before transfer compared with 20 per cent for frozen-thawed embryos cultured for up to four hours before transfer. Five calves were born after a normal gestation period with birthweights ranging from 37.3 to 54.5 kg.

Ethnic differences in mortality from sudden infant death syndrome in New Zealand.

OBJECTIVES: To examine the factors which might explain the higher mortality from sudden infant death syndrome in Maori infants (7.4/1000 live births in 1986 compared with 3.6 in non-Maori children). DESIGN: A large nationwide case control study. SETTING: New Zealand. 485 infants who died of sudden infant death syndrome were compared with 1800 control infants. There were 229 Maori and 240 non-Maori cases of sudden infant death syndrome (16 cases unassigned) and 353 Maori and 1410 non-Maori controls (37 unassigned). RESULTS: Maori infants had 3.81 times the risk (95% confidence interval 3.06 to 4.76) of sudden infant death syndrome compared with non-Maori infants. The risk factors for sudden infant death syndrome within groups were remarkably similar. When Maori and non-Maori controls were compared the prevalence of many of the known risk factors was higher in Maori infants. In particular, mothers were socioeconomically disadvantaged, younger, and more likely to smoke and their infants were of lower birth weight and more likely to share a bed with another person. Multivariate analysis controlling for potential confounders found that simply being Maori increased the risk of sudden infant death syndrome by only 1.37 (95% CI = 0.95 to 2.01), not statistically significantly different from 1. Population attributable risk was calculated for prone sleeping position, maternal smoking, not breast feeding, and infants sharing a bed with another person. In total these four risk factors accounted for 89% of deaths from sudden infant death syndrome in Maori infants and 79% in non-Maori infants. CONCLUSION: The high rate of sudden infant death syndrome among Maori infants is based largely on the high prevalence in the Maori population of the major risk factors. Other risk factors, not related to ethnicity, probably explain remaining differences between Maori and non-Maori children.