Multiplexed efficient on-chip sample preparation and sensitive amplification-free detection of Ebola virus.

An automated microfluidic sample preparation multiplexer (SPM) has been developed and evaluated for Ebola virus detection. Metered air bubbles controlled by microvalves are used to improve bead-solution mixing thereby enhancing the hybridization of the target Ebola virus RNA with capture probes bound to the beads. The method uses thermally stable 4-formyl benzamide functionalized (4FB) magnetic beads rather than streptavidin coated beads with a high density of capture probes to improve the target capture efficiency. Exploiting an on-chip concentration protocol in the SPM and the single molecule detection capability of the antiresonant reflecting optical waveguide (ARROW) biosensor chip, a detection limit of 0.021pfu/mL for clinical samples is achieved without target amplification. This RNA target capture efficiency is two orders of magnitude higher than previous results using streptavidin beads and the limit of detection (LOD) improves 10×. The wide dynamic range of this technique covers the whole clinically applicable concentration range. In addition, the current sample preparation time is ~1h which is eight times faster than previous work. This multiplexed, miniaturized sample preparation microdevice establishes a key technology that intended to develop next generation point-of-care (POC) detection system.

Optofluidic analysis system for amplification-free, direct detection of Ebola infection.

The massive outbreak of highly lethal Ebola hemorrhagic fever in West Africa illustrates the urgent need for diagnostic instruments that can identify and quantify infections rapidly, accurately, and with low complexity. Here, we report on-chip sample preparation, amplification-free detection and quantification of Ebola virus on clinical samples using hybrid optofluidic integration. Sample preparation and target preconcentration are implemented on a PDMS-based microfluidic chip (automaton), followed by single nucleic acid fluorescence detection in liquid-core optical waveguides on a silicon chip in under ten minutes. We demonstrate excellent specificity, a limit of detection of 0.2 pfu/mL and a dynamic range of thirteen orders of magnitude, far outperforming other amplification-free methods. This chip-scale approach and reduced complexity compared to gold standard RT-PCR methods is ideal for portable instruments that can provide immediate diagnosis and continued monitoring of infectious diseases at the point-of-care.

Effects of dietary enrichment with a marine oil-based n-3 LCPUFA supplement in sows with predicted birth weight phenotypes on growth performance and carcass quality of offspring.

Effects of a marine oil-based n-3 LCPUFA supplement (mLCPUFA) fed from weaning until the end of the next lactation to sows with a predicted low litter birth weight (LBW) phenotype on growth performance and carcass quality of litters born to these sows were studied, based on the hypothesis that LBW litters would benefit most from mLCPUFA supplementation. Sows were allocated to be fed either standard corn/soybean meal-based gestation and lactation diets (CON), or the same diets enriched with 0.5% of the mLCPUFA supplement at the expense of corn. The growth performance from birth until slaughter of the litters with the lowest average birth weight in each treatment (n=24 per treatment) is reported in this paper. At weaning, each litter was split between two nursery pens with three to six pigs per pen. At the end of the 5-week nursery period, two barrows and two gilts from each litter that had individual birth weights closest to their litter average birth weight, were moved to experimental grow-finish pens (barn A), where they were housed as two pigs per pen, sorted by sex within litter. Remaining pigs in each litter were moved to another grow-finish barn (barn B) and kept in mixed-sex pens of up to 10 littermates. After 8 weeks, one of the two pigs in each pen in barn A was relocated to the pens holding their respective littermates in barn B. The remaining barrows and gilts were individually housed in the pens in barn A until slaughter. Maternal mLCPUFA supplementation increased docosahexaenoic acid (DHA) concentration in the brain, liver and Semitendinosus muscle of stillborn pigs (P<0.01), did not affect eicosapentaenoic acid and DHA concentrations in sow serum at the end of lactation, and did not affect average daily gain, average daily feed intake or feed utilization efficiency of the offspring. BW was higher (P<0.01) in the second half of the grow-finish phase in pigs from mLCPUFA sows compared with controls in barn A, where space and competition for feed was minimal, but not barn B. Carcass quality was not affected by treatment for pigs from barn A, but maternal mLCPUFA supplementation negatively affected carcass quality in pigs from barn B. Collectively, these results suggest that nutritional supplementation of sows can have lasting effects on litter development, but that feeding mLCPUFA to sows during gestation and lactation was not effective in improving growth rates or carcass quality of LBW litters.

Effects of dietary enrichment with a marine oil-based n-3 LCPUFA supplement in sows with predicted birth weight phenotypes on birth litter quality and growth performance to weaning.

The effects of a marine oil-based n-3 long-chain polyunsaturated fatty acid (mLCPUFA) supplement fed to the sow from weaning, through the rebreeding period, during gestation and until end of lactation on litter characteristics from birth until weaning were studied in sows with known litter birth weight phenotypes. It was hypothesized that low birth weight (LBW) litters would benefit more from mLCPUFA supplementation than high birth weight litters. A total of 163 sows (mean parity=4.9 ± 0.9) were rebred after weaning. Sows were pair-matched by parity and litter average birth weight of the previous three litters. Within pairs, sows were allocated to be fed either standard corn/soyabean meal-based gestation and lactation diets (CON), or the same diets enriched with 0.5% of the mLCPUFA supplement at the expense of corn. Each litter between 9 and 16 total pigs born was classified as LBW or medium/high average birth weight (MHBW) litter and there was a significant correlation (P<0.001) between litter average birth weight of the current and previous litters within sows (r=0.49). Sow serum was harvested at day 113 of gestation for determination of immunoglobulin G (IgG) concentrations. The number of pigs born total and alive were lower (P=0.01) in mLCPUFA than CON sows, whereas the number of stillborn and mummified pigs were similar between treatments. Number of stillborns (trend) and mummies (P<0.01) were higher in LBW than MHBW litters. Tissue weights and brain : tissue weight ratios were similar between treatments, but LBW litters had decreased tissue weights and increased brain : tissue weight ratios compared with MHBW litters. Placental weight was lower (P=0.01) in LBW than MHBW litters, but was not different between treatments. Average and total litter weight at day 1 was similar between treatments. mLCPUFA increased weaning weight (P=0.08) and average daily gain (P<0.05) in MHBW litters, but not in LBW litters. Pre-weaning mortality was similar between treatments, but was higher (P<0.01) in LBW than MHBW litters. IgG concentration in sow serum was similar between treatments and litter birth weight categories. In conclusion, litter birth weight phenotype was repeatable within sows and LBW litters showed the benchmarks of intra-uterine growth retardation (lower placental weight and brain sparing effects). As maternal mLCPUFA supplementation decreased litter size overall, only improved litter growth rate until weaning in MHBW litters, and did not affect pre-weaning mortality, maternal mLCPUFA supplementation was not an effective strategy in our study for mitigating negative effects of a LBW litter phenotype.

Integration of programmable microfluidics and on-chip fluorescence detection for biosensing applications.

We describe the integration of an actively controlled programmable microfluidic sample processor with on-chip optical fluorescence detection to create a single, hybrid sensor system. An array of lifting gate microvalves (automaton) is fabricated with soft lithography, which is reconfigurably joined to a liquid-core, anti-resonant reflecting optical waveguide (ARROW) silicon chip fabricated with conventional microfabrication. In the automaton, various sample handling steps such as mixing, transporting, splitting, isolating, and storing are achieved rapidly and precisely to detect viral nucleic acid targets, while the optofluidic chip provides single particle detection sensitivity using integrated optics. Specifically, an assay for detection of viral nucleic acid targets is implemented. Labeled target nucleic acids are first captured and isolated on magnetic microbeads in the automaton, followed by optical detection of single beads on the ARROW chip. The combination of automated microfluidic sample preparation and highly sensitive optical detection opens possibilities for portable instruments for point-of-use analysis of minute, low concentration biological samples.

Consequences of a low litter birth weight phenotype for postnatal lean growth performance and neonatal testicular morphology in the pig.

The consequences of a low litter average birth weight phenotype for postnatal growth performance and carcass quality of all progeny, and testicular development in male offspring, were investigated. Using data from 25 sows with one, and 223 sows with two consecutive farrowing events, individual birth weight (BW) was measured and each litter between 9 and 16 total pigs born was classified as low (LBW), medium (MBW) or high (HBW) birth weight: low and high BW being defined as >1 standard deviation below or above, respectively, the population mean for each litter size. Litter average BW was repeatable within sows. At castration, testicular tissue was collected from 40 male pigs in LBW and HBW litters with individual BW close to their litter average BW and used for histomorphometric analysis. LBW piglets had a lower absolute number of germ cells, Sertoli cells and Leydig cells in their testes and a higher brain : testis weight ratio than HBW piglets. Overall, LBW litters had lower placental weight and higher brain : liver, brain : intestine and brain : Semitendinosus muscle weight ratios than MBW and HBW litters. In the nursery and grow-finish (GF) phase, pigs were kept in pens by BW classification (9 HBW, 17 MBW and 10 LBW pens) with 13 males and 13 females per pen. Average daily gain tended to be lower in LBW than HBW litters in lactation (P = 0.06) and throughout the nursery and GF phases (P < 0.01), resulting in an increasing difference in body weight between LBW, MBW and HBW litters (P < 0.05). Average daily feed intake was lower (P < 0.001) in LBW than HBW litters in the nursery and GF phases. Feed utilization efficiency (feed/gain) was similar for LBW and HBW litters in the nursery, but was lower (P < 0.001) in HBW than LBW litters in the GF phase. By design, slaughter weight was similar between BW classifications; however, LBW litters needed 9 more days to reach the same slaughter weight than HBW litters (P < 0.001). BW classification did not affect carcass composition traits. In conclusion, LBW litters showed benchmarks of intrauterine growth retardation, LBW had a negative impact on testicular development and germ and somatic cell populations, and was associated with decreased postnatal growth during all phases of production; however, no measurable effect on carcass composition traits was established.

Responses to n-3 fatty acid (LCPUFA) supplementation of gestating gilts, and lactating and weaned sows.

Feeding n-3 long-chain polyunsaturated fatty acids (LCPUFA) to gilts or sows has shown different responses to litter growth, pre-weaning mortality and subsequent reproductive performance of the sow. Two hypotheses were tested: (1) that feeding a marine oil-based supplement rich in protected n-3 LCPUFAs to gilts in established gestation would improve the growth performance of their litters; and (2) that continued feeding of the supplement during lactation and after weaning would offset the negative effects of lactational catabolism induced, using an established experimental model involving feed restriction of lactating primiparous sows. A total of 117 primiparous sows were pair-matched at day 60 of gestation by weight, and when possible, litter of origin, and were allocated to be either control sows (CON) fed standard gestation and lactation diets, or treated sows (LCPUFA) fed the standard diets supplemented with 84 g/day of a n-3 LCPUFA rich supplement, from day 60 of first gestation, through a 21-day lactation, and until euthanasia at day 30 of their second gestation. All sows were feed restricted during the last 7 days of lactation to induce catabolism, providing a background challenge against which to determine beneficial effects of n-3 LCPUFA supplementation on subsequent reproduction. In the absence of an effect on litter size or birth weight, n-3 LCPUFA tended to improve piglet BW gain from birth until 34 days after weaning (P = 0.06), while increasing pre-weaning mortality (P = 0.05). It did not affect energy utilization by the sow during lactation, thus not improving the catabolic state of the sows. Supplementation from weaning until day 30 of second gestation did not have an effect on embryonic weight, ovulation rate or early embryonic survival, but did increase corpora lutea (CL) weight (P = 0.001). Eicosapentaenoic acid and docosahexaenoic acid (DHA) levels were increased in sow serum and CL (P < 0.001), whereas only DHA levels increased in embryos (P < 0.01). In conclusion, feeding n-3 LCPUFA to gilts tended to improve litter growth, but did not have an effect on overall subsequent reproductive performance.

Temporal candidate gene expression in the sow placenta and embryo during early gestation and effect of maternal Progenos supplementation on embryonic and placental development.

The present study characterised gene expression associated with embryonic muscle development and placental vascularisation during early gestation in the pig and examined effects of Progenos supplementation in early pregnancy. Tissues were collected from commercial multiparous sows (n = 48) from Days 16 to 49 of gestation. In the placenta, qPCR revealed that vascular endothelial growth factor (VEGFA) expression did not change from Day 17 to 49 of gestation; however, KDR receptor and angiopoietin-1 and -2 expression were differentially regulated, with periods of high expression corresponding to two critical phases of angiogenesis in the pig. In the embryo, the pattern of myogenesis-related gene expression was consistent with available literature. A commercially available nutritional supplement Progenos (20 g day⁻¹ L-arginine) added to the diet of sows from either Day 15 to 29 (P15-29; n = 33), Day 30 to 44 (n = 29) or from Day 15 to 44 (n = 76) of gestation tended to increase (P = 0.058) embryonic growth rate compared with non-supplemented controls (n = 79) and angiogenin expression was higher (P = 0.028) at Day 30 of gestation in placentae from sows on the P15-29 Progenos treatment. These results are consistent with proposed beneficial effects of l-arginine on early embryonic development and placental vascularisation.

Restricted feed intake in lactating primiparous sows. I. Effects on sow metabolic state and subsequent reproductive performance.

The effects of feed restriction (60% of anticipated feed intake; Restrict; n=60) during the last week of a 21-day lactation in primiparous sows compared with feeding at 90% of anticipated feed intake (Control; n=60) on sow metabolic state, litter growth and sow reproductive performance after weaning were compared. Metabolisable energy (ME) derived from feed was lower, ME derived from body tissues was higher and litter growth rate was reduced (all P<0.05) in Restrict sows during the last week of lactation. Treatment did not affect weaning-to-oestrus interval, pregnancy rate, ovulation rate, embryonic survival or the number of live embryos (P>0.05) at Day 30 of gestation: However, embryo weight was greater (P<0.05) in Control than in Restrict sows (1.55±0.04vs 1.44±0.04g, respectively). These data suggest the biology of the commercial sow has changed and reproductive performance of contemporary primiparous sows is increasingly resistant to the negative effects of lactational catabolism. Overall, catabolism negatively affected litter weaning weight and embryonic development of the next litter, but the extent to which individual sows used tissue mobilisation to support these litter outcomes was highly variable.

Restricted feed intake in lactating primiparous sows. II. Effects on subsequent litter sex ratio and embryonic gene expression.

Expression of panels of candidate genes controlling myogenesis, angiogenesis and gender-specific imprinting of development were analysed in embryonic, placental and endometrial tissues recovered at Day 30 of gestation from a subset of primiparous sows that were either feed restricted (Restrict; n=17) or fed to appetite (Control; n=15) during the last week of the previous lactation. Embryos were also sex typed to investigate gender bias in response to treatments. Average embryonic weight was lower in the subset of Restrict compared with Control litters (1.38±0.07vs 1.59±0.08g, respectively) and the male:female sex ratio was higher (P<0.05) in embryos (litters) recovered from Restrict sows. Treatment affected (P≤0.05) the expression of embryonic and placental genes involved in insulin-like growth factor (IGF) 2 signalling, including IGF2, INSR and IGF2R. Embryonic expression of ESR1 was also affected by treatment (P<0.03) and sex×treatment interactions were observed for the expression of embryonic ESR1 (P<0.05) and placental ANGPT2 (P<0.03). At the molecular level, these results support the suggestion that changes in placental function are not the primary mechanism mediating detrimental effects of previous sow catabolism on early embryonic development in the feed-restricted lactational sow model. However, perturbations in the IGF2 system are implicated as mediators of these effects.

The effect of gilt age at first estrus and breeding on third estrus on sow body weight changes and long-term reproductive performance.

The objective of this trial was to determine the effect of age at first estrus on BW changes and long-term reproductive performance of sows. At approximately 100 d of age, prepubertal C22 gilts (n = 431) were allocated to trial. At a pen average of 140 d of age, gilts began daily direct contact with mature boars to stimulate onset of puberty. Gilts (n = 317, 73%) were recorded as cyclic by 180 d of age (select) and were classified on the basis of age at puberty into 3 puberty groups: 1) early puberty (EP; <153 d of age; n = 85); 2) intermediate puberty (IP; 154 to 167 d of age; n = 140); or 3) late puberty (LP; 168 to 180 d of age; n = 90). Gilts not exhibiting the standing reflex by 180 d of age were considered nonselect (NS; n = 91). Mean day to puberty and age at puberty attainment in each of the classifications were EP: 9.6 +/- 0.5 d and 147.4 +/- 0.5 d; IP: 19.3 +/- 0.5 d and 159.9 +/- 0.3 d; LP: 33.8 +/- 0.7 and 175.7 +/- 0.6 d, respectively. Fewer NS gilts (73.0%) were bred than were EP (97.7%), IP (93.2%), or LP (93.0%) gilts (P < 0.05). Total number of piglets born and born alive were not different between classifications and increased (P < 0.05) over successive parities in EP, IP, and NS gilts. For gilts initially served, there was no effect of puberty group classification on retention in the herd to farrow a third litter, but the rate of fallout per parity tended to be greatest for NS (17.2%) compared with EP (12.4%), IP (15.6%), and LP (14.2%) gilts (P < 0.08). Taken together, these data suggest that the response to a standardized protocol of boar stimulation can identify 50 to 75% of gilts that will have greatest lifetime productivity in the breeding herd. In the known cyclic (select) gilts, BW increased over the productive life of the sow, and EP gilts were lighter than LP gilts at every measured event (P < 0.05). Plasma IGF-1 only differed between puberty groups at d 100 of age (EP: 169.0 +/- 4.4; IP: 157.2 +/- 3.5; LP: 144.0 +/- 4.4 ng/mL), suggesting a mechanism linking IGF-1 status and age at puberty in the present study.

Synchronization of ovulation in cyclic gilts with porcine luteinizing hormone (pLH) and its effects on reproductive function.

The overall objective was to evaluate the use of porcine luteinizing hormone (pLH) for synchronization of ovulation in cyclic gilts and its effect on reproductive function. In an initial study, four littermate pairs of cyclic gilts were given altrenogest (15 mg/d for 14 d). Gilts received 500 microg cloprostenol (Day 15), 600 IU equine chorionic gonadotropin (eCG) (Day 16) and either 5mg pLH or saline (Control) 80 h after eCG. Blood samples were collected every 4h, from 8h before pLH/saline treatment to the end of estrus. Following estrus detection, transcutaneous real-time ultrasonography and AI, all gilts were slaughtered 6d after the estimated time of ovulation. Peak plasma pLH concentrations (during the LH surge), as well as the amplitude of the LH surge, were greater in pLH-treated gilts than in the control (P=0.01). However, there were no significant differences between treatments in the timing and duration of estrus, or the timing of ovulation within the estrous period. In a second study, 45 cyclic gilts received altrenogest for 14-18d, 600 IU eCG (24h after last altrenogest), and 5mg pLH, 750 IU human chorionic gonadotropin (hCG), or saline, 80 h after eCG. For gilts given pLH or hCG, the diameter of the largest follicle before the onset of ovulation (mean+/-S.E.M.; 8.1+/-0.2 and 8.1+/-0.2mm, respectively) was smaller than in control gilts (8.6+/-0.2mm, P=0.05). The pLH and hCG groups ovulated sooner after treatment compared to the saline-treated group (43.2+/-2.5, 47.6+/-2.5 and 59.5+/-2.5h, respectively; P<0.01), with the most synchronous ovulation (P<0.01) in pLH-treated gilts. Embryo quality (total cell counts and embryo diameter) was not significantly different among groups. In conclusion, pLH reliably synchronized ovulation in cyclic gilts without significantly affecting embryo quality.

Embryonic and fetal development in a commercial dam-line genotype.

During depopulation of a breeding unit within Swine Graphics Enterprises, extensive data were collected and used to examine relationships among ovulation rate, the pattern of prenatal loss, and placental and fetal development. Groups of Large White x Landrace females (n=447) were slaughtered between day 20-30, 50-55 or 85-90 of gestation, with approximately equal numbers of animals representing gilts and parity 1 (G/P1), parity 2-3 (P2/3), and parity >4 (P4+). Ovulation rate and embryo number were recorded for all animals. With the exception of the G/P1 animals, embryonic and placental weight were recorded for four conceptuses per sow on day 20-30; on day 85-90 two conceptuses per sow were dissected to determine placental and fetal development. Ovulation rate (22.7 +/- 0.2 overall) was higher (P <0.05) in P2/3 (23.6 +/- 0.4) and P4+ (24.7 +/- 0.4) than in G/P1 (20.2 +/- 0.5). Embryonic/fetal survival was 61.8 +/- 2.1% at day 20-30, 50.2 +/- 2.2% at day 50-55 and 48.7 +/- 1.9% at day 85-90 and the number of surviving conceptuses was higher (P <0.05) in the P2/3 sows than in other parity groups. There was no relationship between ovulation rate and number of live embryos at day 20-30 or 85-90. At day 20-30 and 85-90, embryo weight was positively correlated with placental weight, but neither placental weight nor embryonic/fetal weight was correlated with number of viable embryos. A parity by gestation day interaction existed; placental weight for P4+ (3.42 +/- 0.43 g) was less than for P2/3 (7.55 +/- 0.40 g) at day 20-30 (P <0.0001), whereas at day 85-90, placental weight of P2/3 (209.5 +/- 8.5 g) was less (P=0.05) than both G/P1 (235.7 +/- 7.3g) and P4+ (235.4 +/- 7.1 g). At day 85-90, fetal brain weight, relative to body weight (R2=0.61, P <0.0001), and fetal brain:liver weight ratio (R2=0.35; P <0.0001) were negatively related to mean fetal weight, and brain:liver weight ratio showed a trend towards a relationship with number of viable fetuses (P=0.08). Parity also affected brain:liver weight ratio (P=0.01). Clearly, high ovulation rates in the higher parity sows have the potential to cause excessive in utero crowding of conceptuses in the post-implantation period. Even with moderate crowding, increased brain:liver weight ratios in smaller fetuses in late gestation indicate that uterine capacity impacts fetal development as well as the number of surviving fetuses.

Impact of boar exposure on puberty attainment and breeding outcomes in gilts.

We examined the most effective method of boar exposure for the attainment of puberty in 89 gilts. At 160 days of age, we allocated gilts to daily direct contact with a vasectomized boar after movement of pen groups of gilts to a detection-mating area (DGB: n = 30); daily direct contact with boars in the gilt home pens (DBG: n = 31); or daily fenceline contact between boars and gilts housed in individual gilt stalls (FBG: n = 28). DGB gilts were younger (P < or = 0.05) than FBG gilts at puberty. Direct boar contact reduced the interval from initial boar contact to puberty in DGB and DBG gilts, compared to fenceline contact in FBG gilts (P < 0.05). There was no difference (P > or = 0.05) between treatment for pubertal weight, backfat, lifetime growth rate, or duration of first pubertal estrus. Backfat depth and leptin concentration at 160 days of age were positively correlated (P < or = 0.05). We detected no relationships between leptin or IGF-1 concentration at 160 days of age and the interval from initial exposure to a vasectomized boar to puberty (P > 0.05). Based on objective criteria, fenceline contact with a boar (BC) during artificial insemination improved the quality of artificial insemination compared to no boar contact (NC) (P < 0.05).

The effect of lean growth rate on puberty attainment in gilts.

Two hundred sixteen prepubertal Genex Manor hybrid F1 gilts were used to determine the impact of lean growth rate on sexual development of gilts. This study was composed of two experiments (Exp. 1 and Exp. 2). In Exp. 1, at approximately 96 d of age and 54 kg weight, gilts were allocated with respect to growth rate and litter origin to one of two dietary treatments: 1) a diet formulated to maximize lean growth potential (LP; n = 84) or 2) a diet formulated to produce a lower lean growth rate (LL; n = 84). In Exp. 2, at approximately 88 d of age and 50 kg weight, gilts were allocated with respect to growth rate and litter origin to one of two dietary treatments: 1) a diet formulated to maximize lean growth potential (LP; n = 24) or 2) a diet formulated to restrict lean growth further than was achieved in LL in Exp. 1 (RL; n = 24). All gilts were fed treatment diets for ad libitum consumption and housed in groups of six. Weight, backfat depth and loin depth, and feed intake were measured weekly. Starting at 135 d of age, gilts received 20 min of direct daily exposure to a boar as a pen group for pubertal stimulation. Puberty attainment was determined as the day gilts first exhibited the standing reflex in response to contact with a boar. At pubertal estrus, body weight, backfat depth, and loin depths were recorded. Diet affected (P < or = 0.05) estimated fat-free lean gain (LP, 424 vs LL, 347 g/d, Exp. 1; LP, 397 vs RL, 376 g/d, Exp. 2) during the growth period (start to stimulation). However, age at puberty was not affected by diet (LP, 157.3 vs LL, 157.6, Exp. 1; LP, 166.7 vs RL, 167.3, Exp. 2) or overall lean growth at stimulation (P > or = 0.05 in both experiments), confirming that innate variability in sexual development of commercial genotypes, rather than growth performance, determines onset of sexual maturity. A negative correlation between age at puberty and growth rate from 50 kg until puberty (P < or = 0.05) (LP, r = -0.40, LL, r = -0.36, Exp. 1; LP, r = -0.64, RL, r = -0.48, Exp. 2) was a consequence of reduced lean tissue growth during the stimulation period in later-maturing gilts.

Lack of an effect of prostaglandin injection at estrus onset on the time of ovulation and on reproductive performance in weaned sows.

We evaluated the effects of a PGF2alpha analogue on time of ovulation and reproductive performance in multiparous Camborough sows (n=47). At onset of first post-weaning estrus, sows received either an intravulval injection of 3.75 mg of prostaglandin analogue (PGF) or, served as a non-injected control (CON). Beginning 24 h after the onset of estrus, transcutaneous ultrasonography was carried out every six h to determine time of ovulation. At 36, 54, and 72 h after the onset of estrus, blood samples were taken for progesterone analysis. Weaning-to-estrus (WEI), duration of estrus, ovulation rate and number of live embryos at d 28 of gestation were recorded. Treatment had no effect (P > 0.05) on any parameters measured. Duration of estrus classified as less or greater than the overall mean also had no effect (P > 0.05) on any of the parameters measured. Results indicate that treatment did not advance ovulation nor did it improve reproductive performance in sows. Overall, a negative correlation of WEI with the ovulation rate (P = 0.0005, r = -0.54) was established.

Observed and reported expressive vocabulary and word combinations in bilingual toddlers.

The consistency of parental reports of expressive vocabulary and word combinations with observed expressive language among 21- to 27-month-old children exposed to English and Spanish on a regular basis was the focus of this investigation. Parental reports were obtained using the Spanish-English Vocabulary Checklist (Patterson, 1998), an adaptation of the Language Development Survey (Rescorla, 1989). The number of different words parents reported was correlated (r = .66) with the number of different words the children used during a 30-min videotaped interaction with the reporting parent. Parental reports of whether the child was combining words and estimates of proportion of the child's use of Spanish and English also were consistent with the children's language use during the 30-min language samples.

Identification of the minimal essential RNA sequences responsible for site-specific targeting of the Leishmania RNA virus 1-4 capsid endoribonuclease.

The Leishmania RNA virus 1-4 capsid protein possesses an endoribonuclease activity responsible for single-site-specific cleavage within the 450-nucleotide 5' untranslated region of its own viral RNA transcript. To characterize the minimal essential RNA determinants required for site-specific cleavage, mutated RNA transcripts were examined for susceptibility to cleavage by the virus capsid protein in an in vitro assay. Deletion analyses revealed that all determinants necessary for accurate cleavage are encoded in viral nucleotides 249 to 342. Nuclease mapping and site-specific mutagenesis of the minimal RNA sequence defined a stem-loop structure that is located 40 nucleotides upstream from the cleavage site (nucleotide 320) and that is essential for accurate RNA cleavage. Abrogation of cleavage by disruption of base pairing within the stem-loop was reversed through the introduction of complementary nucleotide substitutions that reestablished the structure. We also provide evidence that divalent cations, essential components of the cleavage reaction, stabilized the stem-loop structure in solution. That capsid-specific antiserum eliminated specific RNA cleavage provides further evidence that the virus capsid gene encodes the essential endoribonuclease activity.

Host cell proteins bind specifically to the capsid-cleaved 5' end of Leishmaniavirus RNA.

Leishmaniavirus (LRV) is a double-stranded RNA (dsRNA) virus that persistently infects some strains of the protozoan parasite, Leishmania. LRV generates a short transcript, corresponding to the 5' end of the positive-sense RNA (320 nt), via a cleavage event mediated by the viral capsid protein on the full-length positive sense RNA transcript. To address the possibility that the RNA cleavage represents a regulatory mechanism for maintaining persistent infection, the interactions between Leishmania cytoplasmic proteins and in vitro synthesized viral transcripts were studied. In gel mobility shift experiments, three specific RNA/protein complexes were formed between cellular proteins and the cleaved viral transcript, and three major proteins were labeled by UV cross-linking. No protein binding activity was observed for either the short (320 nt) or full-length RNA transcripts. However, the two cleavage reaction products were able to form stable RNA/RNA complexes. We present a model in which the virus is targeting its own transcript for cleavage to promote binding of host factors to cryptic domains inaccessible in the full-length transcript.

Overview of the Leishmaniavirus endoribonuclease and functions of other endoribonucleases affecting viral gene expression.

Leishmaniaviruses (LRV) are double-stranded RNA viruses that persistently infect some strains of the protozoan parasite Leishmania. The identification of a short viral RNA transcript led to our discovery of an endoribonuclease activity of the LRV capsid protein. Other known endoribonucleases serve a variety of diverse roles in the regulated balance of processing and degradation of both cellular and viral RNAs, thus determining the amount and functionality of specific RNA molecules in a cell at any given time. The consequence of LRV RNA cleavage on the LRV life cycle has not yet been determined. Here we review the LRV endoribonuclease and discuss potential roles for this endoribonuclease activity in the context of the involvement of other endoribonucleases in regulating viral gene expression and replicative capacity.