Mechanism whereby nitric oxide (NO) infused chronically intrauterine in ewes is antiluteolytic rather than being luteolytic.

Nitric oxide (NO) has been reported to be luteolytic in vitro and in vivo in cows. However, an NO donor reversed PGF2alpha-induced inhibition of rat luteal progesterone secretion in vitro and an NO donor or endothelin-1 stimulated bovine luteal tissue secretion of prostaglandins E (PGE; PGE1, PGE2) in vitro without affecting progesterone or PGF2alpha secretion. In addition, chronic infusion of an NO donor into the interstitial tissue of the ovarian vascular pedicle adjacent the luteal-containing ovary prevented the decline in circulating progesterone, while a nitric oxide synthase (NOS) inhibitor did not affect luteolysis. The objective of this experiment was to determine whether an NO donor or NOS inhibitor infused chronically intrauterine adjacent to the luteal-containing ovary during the ovine estrous cycle was luteolytic or antiluteolytic. Ewes were treated either with vehicle (N=5), diethylenetriamine (DETA-control for DETANONOate; N=5), (Z)-1-[2-(2-aminoethyl)-N-(2-ammonioethyl)amino]diazen-1-ium-1,2-diolate (DETANONOate-long acting NO donor; N=6), l-arginine (N=5), l-nitro-arginine methyl ester (l-NAME-NOS inhibitor; N=6), or NG-monomethyl-l-arginine acetate (l-NMMA; NOS inhibitor; N=5) every 6h from 2400h (0h) on day 8 through 1800h on day 18 of the estrous cycle. Jugular venous blood and inferior vena cava plasma via a saphenous vein cathether 5cm anterior to the juncture of the ovarian vein and inferior vena cava were collected every 6h for analysis for progesterone and PGF2alpha and PGE, respectively, by RIA. Corpora lutea were collected at 1800h on day 18 and weighed. Weights of corpora lutea were heavier (P< or =0.05) in DETANONOate-treated ewes when compared to vehicle, DETA, l-arginine, l-NAME, or l-NMMA-treated ewes, l-arginine luteal weights were heavier than vehicle, DETA, l-arginine, l-NAME, or l-NMMA-treated ewes, and luteal weights of vehicle, DETA, l-NAME, or l-NMMA-treated ewes did not differ amongst each other (P> or =0.05). Profiles of progesterone in jugular venous blood on days 8-18 differed (P< or =0.05) in DETANONOate-treated ewes when compared to vehicle, DETA, l-arginine, l-NMMA or l-NAME-treated ewes, which did not differ (P> or =0.05) amongst each other. The PGE:PGF2alpha ratio profile in inferior vena cava plasma of DETANONOate-treated ewes was increased (P< or =0.05) when compared to all other treatment groups. In a second experiment, conversion of [3H PGE2] to [3H PGF2alpha] by day 15 ovine caruncular endometrium in vitro was determined in vehicle, DETA, or DETANONOate-treatment groups. Conversion of [3H PGE2] to [3H PGF2alpha] was decreased (P< or =0.05) only by DETANONOate. It is concluded that NO is not luteolytic during the ovine estrous cycle, but may instead be antiluteolytic and prevent luteolysis by altering the PGE:PGF2alpha ratio secreted by the uterus.

Is nitric oxide luteolytic or antiluteolytic?

Nitric oxide (NO) has been reported to be luteolytic based on treatment of cows in vivo with an inhibitor of nitric oxide synthase (NOS-produces NO), which delayed the decline in progesterone by two to three days [Jaroszewki J, Hansel, W. Intraluteal administration of a nitric oxide synthase blocker stimulates progesterone, oxytocin secretion and prolongs the life span of the bovine corpus luteum. Proc Soc Exptl Biol Med 2000;224:50-5; Skarzynski D, Jaroszewki J, Bah, M, et al. Administration of nitric oxide synthase inhibitor counteracts prostaglandin F(2alpha)-induced luteolysis in cattle. Biol Reprod 2003;68:1674-81]. The objective of this experiment was to determine the effect of a long acting NO donor or a NOS inhibitor infused chronically into the interstitial tissue of the ovarian vascular pedicle adjacent to the ovary with a corpus luteum on secretion of progesterone during the ovine estrous cycle. Ewes were treated either with Vehicle (N=5); Diethylenetriamine (DETA-control for DETA-NONOate; N=5); (Z)-1-[2-(2-aminoethyl)-N-(2-ammonioethyl) amino]diazen-1-ium-1,2-diolate (DETA-NONOate-long acting NO donor; N=6); or l-nitro-arginine methyl ester (l-NAME-NOS inhibitor; N=6) every 6 h from 24:00 h (0 h) on day 8 through 18:00 h on day 18 of the estrous cycle. Jugular venous blood was collected every 6h for analysis for progesterone and corpora lutea were collected at 18:00 h on day 18 and weighed. Weights of corpora lutea were heavier (P< or =0.05) in DETA-NONOate-treated ewes when compared to Vehicle, DETA, or l-NAME-treated ewes, which did not differ amongst each other (P> or =0.05). Profiles of progesterone in jugular venous blood on days 8-18 differed (P< or =0.05) in DETA-NONOate-treated ewes when compared to Vehicle, DETA, or l-NAME-treated ewes did not differ (P> or =0.05) amongst each other. It is concluded that NO is not luteolytic during the ovine estrous cycle, but may instead be antiluteolytic and prevent luteolysis.

Improvement of base-calling in multilane automated DNA sequencing by use of electrophoretic calibration standards, data linearization, and trace alignment.

We present a new method for the linearization and alignment of data traces generated by multilane automated DNA sequencing instruments. Application of this method to data generated with the Visible Genetics Open Gene DNA sequencing system (using MicroCel 700 gel cassettes, with a 25 cm separation distance) allows read lengths of > 1,000 nucleotides to be routinely obtained with high confidence and > 97% accuracy. This represents an increase of 10-15% in average read length, relative to data from this system that have not been processed in the fashion described herein. Most importantly, the linearization and alignment method allows usable sequence to be obtained from a fraction of 10-15% of data sets which, because of original trace misalignment problems, would otherwise have to be discarded. Our method involves adding electrophoretic calibration standards to the DNA sequencing fragments. The calibration standards are labeled with a dye that differs spectrally from the dye attached to the sequencing fragments. The calibration standards are identical in all the lanes. Analysis of the mobilities of the calibration standards allows correction for both systematic and random variation of electrophoretic properties between gel lanes. We have successfully used this method with two-dye and three-dye DNA sequencing instruments.

High performance DNA sequencing, and the detection of mutations and polymorphisms, on the Clipper sequencer.

The Visible Genetics Clipper sequencer is a new platform for automated DNA sequencing which employs disposable MicroCel cassettes and 50 microm thick polyacrylamide gels. Two DNA ladders can be analyzed simultaneously in each of 16 lanes on a gel, after labeling with far-red absorbing dyes such as Cy5 and Cy5.5. This allows a simultaneous bidirectional sequencing of four templates. We have evaluated the Clipper sequencer, by cycle-sequencing of an M13 single-stranded DNA standard, and by coupled amplification and sequencing (CLIP) of reverse-transcribed human immunodeficiency virus (HIV-1) RNA standards and clinical patient samples. (i) Limitations of instrument. We have examined basic instrument parameters such as detector stability, background, digital sampling rate, and gain. With proper usage, the optical and electronic subsystems of the Clipper sequencer do not limit the data collection or sequence-determination processes. (ii) Limitations of gel performance. We have also examined the physics of DNA band separation on 50 microm thick MicroCel gels. We routinely obtain well-resolved sequence which can be base-called with 98.5% accuracy to position approximately 450 on an 11 cm gel, and to position approximately 900 on a 25 cm gel. Resolution on 5 and 11 cm gels ultimately is limited by a sharp decrease in spacing between adjacent bands, in the biased reptation separation regime. Fick's (thermal) diffusion appears to be of minor importance on 6 cm or 11 cm gels, but becomes an additional resolution-limiting factor on 25 cm gels. (iii) Limitations of enzymology. Template quality, primer nesting, choice of DNA polymerase, and choice between dye primers and dye terminators are key determinants of the ability to detect mutations and polymorphisms on the Clipper sequencer, as on other DNA sequencers. When CLIP is used with dye-labeled primers and a DNA polymerase of the F667Y, delta(5'--> 3' exo) class, we can routinely detect single-nucleotide mutations and polymorphisms over the 0.35-0.65 heterozygosity range. We present an example of detecting therapeutically relevant mutations in a clinical HIV-1 RNA isolate.

Effects of relaxin on lactational performance in ovariectomized gilts.

In gilts, mammary lobulo-alveolar growth begins on about Day 80 of gestation and continues progressively until term. Relaxin in concert with estrogen plays a major role in promoting this mammary gland growth. The present study was conducted to determine the importance for lactational performance of prepartum relaxin-dependent growth of the mammary glands in ovariectomized gilts given progesterone to maintain pregnancy. Twenty-four gilts were either sham ovariectomized or bilaterally ovariectomized and assigned to four treatment groups: sham-ovariectomized control, ovariectomized progesterone-treated, ovariectomized progesterone- and (starting at Day 80) relaxin-treated, and ovariectomized progesterone- and (starting at Day 100) relaxin-treated. Piglets were delivered by cesarian section, and gilts were given uniform colostrum-replete foster litters (born of untreated mothers) to nurse from Day 1 to Day 28 of lactation. Prepartum mammary development appeared by visual examination to be greatly reduced in relaxin-deficient gilts. Stimulus of the mammary nipples by the nursing piglets, however, appeared to overcome relaxin-dependent differences in mammary development among treatments. There was no effect of treatment on the time piglets spent at the udder, piglet mortality, piglet weight at Day 21 of lactation, milk composition, mammary cross-sectional area, or sow weight change during lactation. We conclude that gilts devoid of circulating luteal relaxin can display normal lactational performance when given colostrum-replete foster litters.

Effects of relaxin administration in early gestation or prior to mating on uterine length and fetal survival in gilts.

The objective of this research was to determine the effect of administration of porcine relaxin to gilts during early gestation, or during the follicular phase of the estrous cycle immediately preceding mating, on the length of the uterus and consequently the number of surviving fetuses. Experiment 1 determined the individual and combined effects of 10 days of administration of relaxin (0.5 mg, 4 times daily), estradiol benzoate (1 mg, 2 times daily), and progesterone (50 mg, 2 times daily) on uterine wet weight and length in 58 ovariectomized 8-mo-old pubertal gilts. Relaxin alone had no effect on either uterine length or wet weight. Estrogen increased uterine wet weight, and this effect was augmented by relaxin. Progesterone increased uterine length, and this effect was augmented by estrogen. Combined treatment with progesterone, estrogen, and relaxin increased both uterine length and wet weight maximally. Experiment 2 determined the effects of relaxin administration in early gestation or prior to mating on uterine length and fetal survival in 75 unilaterally ovariectomized-hysterectomized gilts. Relaxin (0.5 mg, 4 times daily) was administered during three treatment periods, and uteri were collected on Day 40 of gestation. In this study, relaxin administration from Days 11 to 20 of gestation reduced the number of live fetuses (p = 0.01) and percentage survival (p = 0.01) and resulted in shorter uterine length (p = 0.01) and lower uterine wet weight (p = 0.03) than in controls, but did not affect length of uterus or uterine dry weight. Relaxin administration from Days 22 to 31 of gestation did not influence fetal survival, uterine length, uterine length per fetus, uterine wet weight, or uterine dry weight.(ABSTRACT TRUNCATED AT 250 WORDS)

Effect of oxygen and neostigmine on stillbirth and pig viability.

In this experiment, 98 multiparous Yorkshire sows blocked on farrowing date were randomly assigned to four treatments in a 2 x 2 factorial arrangement. Oxygen inhalation (6 L/min) by the sow was tested in an effort to reduce stillbirth rate and improve pig viability. This study also tested the effect of increasing uterine contractions using neostigmine (5 mg) to determine whether simultaneous administration of oxygen to the sow could improve the inconsistent response of stillbirth rate to neostigmine. Pig viability was assessed using viability scoring and blood gas analysis of samples obtained from the suborbital sinus immediately after birth. The farrowings averaged 12.2 pigs/litter, 7.8% stillbirths, and 3.5% low viability (viability score < or = 6) pigs. Treatment had no effect on stillbirth rate or pig viability. Oxygen treatment increased pO2 (P = .0002), increased pCO2 (P = .02), and decreased pH (P = .02) in sow venous blood sampled after the birth of the last pig, but it had no effect on pig venous blood gases. Neostigmine treatment had no effect on either sow or pig venous blood gases. Oxygen treatment doubled the length of the first birth interval after the start of treatment (P = .003) but had no effect on remaining birth intervals. Neostigmine had no effect on birth interval. It is suggested that the effect of oxygen on birth interval is the reason for the lack of improvement in stillbirth rate.

Variables related to the progress of parturition and probability of stillbirth in swine.

Sow and piglet variables related to probability of stillbirth and to viability score were analyzed in litters from 98 multiparous Yorkshire sows. Immediately after the birth of each piglet, viability was scored using Randall's method. Sow variables related to the probability of stillbirth were average birth weight of the litter (p = 0.0001), sow age (p = 0.001), sow condition score (p = 0.003), length of gestation (p = 0.005), and number of piglets in the litter (p = 0.01). Sow variables related to average viability score were average birth weight of the litter (p = 0.001), standard deviation in birth weight in the litter (p = 0.02), sow age (p = 0.03), sow condition score (p = 0.03), and length of gestation (p = 0.03). Piglet variables related to probability of stillbirth were piglet hemoglobin (p = 0.0001), position in the birth order (p = 0.0001), broken umbilical cord (p = 0.0004), and preceding birth interval (p = 0.0004). Piglet variables related to viability score were piglet hemoglobin (p = 0.0001), position in the birth order (p = 0.0001), broken umbilical cord (p = 0.0001), preceding birth interval (p = 0.0001), and birth weight (p = 0.004). Preceding birth interval was related to whether the piglet was live or stillborn (p = 0.0001), to position in the birth order (p = 0.003), and to the sex of the piglet (p = 0.03).The results demonstrated that sow and piglet variables were highly correlated to probability of stillbirth and to viability score. In addition it was also found that the probability of stillbirth was not associated with the duration of farrowing but with the number of piglets in the litter and piglet hemoglobin level. This study also found that lower weight piglets tend to have poor viability, but are not more prone to stillbirth as commonly suggested. These low viability piglets may survive if the necessary care is given during the farrowing process.

Massive splenomegaly in sarcoidosis.

We have presented a case of massive splenomegaly. Our patient was initially thought to have lymphoma, but at operation she was found to have sarcoidosis with splenic involvement. At 2250 g, the spleen was one of the largest recorded in the literature on sarcoidosis. Although the spleen is frequently involved in sarcoidosis, a review of 6074 cases showed that the incidence of actual splenomegaly is only 10%. In 628 of these cases the authors described various degrees of splenomegaly, but the incidence of massive splenomegaly was only 3%. We conclude that sarcoidosis must be considered in the differential diagnosis of splenomegaly.