Dietary supplementation of rumen native microbes improves lactation performance and feed efficiency in dairy cows.

The objectives of this study were to determine the effects of 2 dietary microbial additives on productive performance and feed efficiency when supplemented to diets of Holstein cows. One hundred seventeen Holstein cows were enrolled at 61 d (31-87 d) postpartum in a randomized complete block design experiment. Cows were blocked by parity group, as nulliparous or multiparous cows and, within parity, by pretreatment ECM yield. Within block, cows were randomly assigned to one of 3 treatments administered as top-dress for 140 d. Treatments consisted of either 100 g of corn meal containing no microbial additive (CON; 15 primiparous and 25 multiparous), 100 g of corn meal containing 5 g of a mixture of Clostridium beijerinckii and Pichia kudriavzevii (G1; 4 × 107 cfu of C. beijerinckii and 1 × 109 cfu of P. kudriavzevii; 14 primiparous and 24 multiparous), or 100 g of corn meal containing 5 g of a mixture of C. beijerinckii, P. kudriavzevii, Butyrivibrio fibrisolvens, and Ruminococcus bovis (G2; 4 × 107 cfu of C. beijerinckii, 1 × 109 cfu of P. kudriavzevii, 1 × 108 cfu of B. fibrisolvens, and 1 × 108 cfu of R. bovis; 15 primiparous and 24 multiparous). Intake of DM, milk yield, and BW were measured daily, whereas milk composition was analyzed at each milking 2 d a week, and body condition was scored twice weekly. Milk samples were collected on d 60 and 62 in the experiment and analyzed for individual fatty acids. The data were analyzed with mixed-effects models with orthogonal contrast to determine the effect of microbial additive (MA; CON vs. 1/2 G1 + 1/2 G2) and type of microbial additive (TMA; G1 vs. G2). Results are described in sequence as CON, G1, and G2. Intake of DM (22.2 vs. 22.4 vs. 22.4 kg/d), BW (685 vs. 685 vs. 685 kg) and the daily BW change (0.40 vs. 0.39 vs. 0.39 kg/d) did not differ among treatments; however, feeding MA tended to increase BCS (3.28 vs. 3.33 vs. 3.36). Supplementing MA increased yields of milk (39.9 vs. 41.3 vs. 41.5 kg/d), ECM (37.9 vs. 39.3 vs. 39.9 kg/d), fat (1.31 vs. 1.37 vs. 1.40 kg/d), TS (4.59 vs. 4.75 vs. 4.79 kg/d), and ECM per kg of DMI (1.72 vs. 1.76 vs. 1.80 kg/kg). Furthermore, cows fed MA increased yields of preformed fatty acids in milk fat (>16C; 435 vs. 463 vs. 488 g/d), particularly UFA (367 vs. 387 vs. 410 g/d), such as linoleic (C18:2 cis-9,cis-12; 30.9 vs. 33.5 vs. 35.4 g/d) and α-linolenic acids (C18:3 cis-9,cis-12,cis-15; 2.46 vs. 2.68 vs. 2.82 g/d) on d 60 and 62 in the experiment. Collectively, supplementing G1 and G2 improved productive performance of cows with no differences between the 2 MA.

Evaluating differences in milk production, reproductive performance, and survival associated with vaginal discharge characteristics and fever in postpartum dairy cows.

The objective was to assess differences in productive and reproductive performance, and survival associated with vaginal discharge characteristics and fever in postpartum dairy cows located in western and southern states of the United States. This retrospective cohort study included data from 3 experiments conducted in 9 dairies. Vaginal discharge was evaluated twice within 12 DIM and scored on a 5-point scale. The highest vaginal discharge score observed for each cow was used to allocate them into 1 of 5 possible groups (VD group) as follows: VD 1 and 2 (VD 1/2; n = 1,174) = clear mucus or lochia with or without flecks of pus; VD 3 (n = 1,802) = mucopurulent with <50% pus; VD 4 (n = 1,643) = mucopurulent with ≥50% of pus or nonfetid reddish-brownish mucus, n = 1,643; VD 5 = fetid, watery, and reddish-brownish, n = 1,800. All VD 5 cows received treatment according to each herd's protocol. Rectal temperature was assessed in a subset of VD 5 cows, and subsequently divided into fever (rectal temperature ≥39.5°C; n = 334) and no fever (n = 558) groups. A smaller proportion of cows with VD 5 (67.6%) resumed ovarian cyclicity compared with VD 1/2 (76.2%) and VD 4 (72.9%) cows; however, a similar proportion of VD 5 and VD 3 (72.6%) cows resumed ovarian cyclicity. A smaller proportion of VD 5 (85.8%) cows received at least one AI compared with VD 1/2 (91.5%), VD 3 (91.0%), or VD 4 (91.6%) cows. Although we did not detect differences in pregnancy at first AI according to VD, fewer cows with VD 5 (64.4%) were pregnant at 300 DIM than cows with VD 1/2 (76.5%), VD 3 (76.2%), or VD 4 (74.7%). Hazard of pregnancy by 300 DIM was smaller for VD 5 compared with VD 1/2, VD 3, or VD 4 cows. A greater proportion of VD 5 cows were removed from the herd within 300 DIM compared with other VD groups. Milk production was 760 kg lower within 300 DIM for VD 5 compared with VD 2, VD 3, and VD 4, whereas VD 2, VD 3, and VD 4 had similar milk production. We did not detect an association between fever at diagnosis of VD 5 and reproductive performance or milk production. A greater proportion of VD 5 cows without fever were removed from the herd by 300 DIM compared with VD 5 cows with fever. Differences in productive and reproductive performance, and removal of the herd were restricted to fetid, watery, and reddish-brownish vaginal discharge, which was independent of fever.

Differences in uterine and serum metabolome associated with metritis in dairy cows.

Objectives were to evaluate differences in the uterine and serum metabolomes associated with metritis in dairy cows. Vaginal discharge was evaluated using a Metricheck device (Simcro) at 5, 7, and 11 d in milk (DIM; herd 1) or 4, 6, 8, 10, and 12 DIM (herd 2). Cows with reddish or brownish, watery, and fetid discharge were diagnosed with metritis (n = 24). Cows with metritis were paired with herdmates without metritis (i.e., clear mucous vaginal discharge or clear lochia with ≤50% of pus) based on DIM and parity (n = 24). Day of metritis diagnosis was considered study d 0. All cows diagnosed with metritis received antimicrobial therapy. The metabolome of uterine lavage collected on d 0 and 5, and serum samples collected on d 0 were evaluated using untargeted gas chromatography time-of-flight mass spectrometry. Normalized data were subjected to multivariate canonical analysis of population using the MultBiplotR and MixOmics packages in R Studio. Univariate analyses including t-test, principal component analyses, partial least squares discriminant analyses, and pathway analyses were conducted using Metaboanalyst. The uterine metabolome differed between cows with and without metritis on d 0. Differences in the uterine metabolome associated with metritis on d 0 were related to the metabolism of butanoate, amino acids (i.e., glycine, serine, threonine, alanine, aspartate, and glutamate), glycolysis and gluconeogenesis, and the tricarboxylic acid cycle. No differences in the serum metabolome were observed between cows diagnosed with metritis and counterparts without metritis on d 0. Similarly, no differences in uterine metabolome were observed between cows with metritis and counterparts not diagnosed with metritis on d 5. These results indicate that the establishment of metritis in dairy cows is associated with local disturbances in amino acid, lipid, and carbohydrate metabolism in the uterus. The lack of differences in the uterine metabolome on d 5 indicates that processes implicated with the disease are reestablished by d 5 after diagnosis and treatment.

Blood metabolomics and impacted cellular mechanisms during transition into lactation in dairy cows that develop metritis.

The objective of this study was to identify metabolites associated with metritis and use them for identification of cellular mechanisms affected during transition into lactation. Holstein cows (n = 104) had blood collected in the prepartum period (d -14 ± 6 relative to calving), at calving (d 0), and at the day of metritis diagnosis (d 7 ± 2 after calving). Cows with reddish or brownish, watery, and fetid discharge were diagnosed with metritis (n = 52). Cows with metritis were paired with herdmates without metritis (n = 52) based on days in milk. The metabolome of plasma samples was evaluated using untargeted gas chromatography time-of-flight mass spectrometry. Univariate analyses included t-tests and fold change analyses. Metabolites with false discovery rate adjusted P ≤ 0.10 on t-tests were used for partial least squares discriminant analysis coupled with permutational analysis using 2,000 permutations. Metabolites with false discovery rate adjusted P ≤ 0.10 on t-tests were also used for enriched pathway analyses and identification of cellular processes. Cows that developed metritis had affected cellular processes associated with lower amino acid metabolism in the prepartum period, greater lipolysis, cell death, and oxidative stress at calving and at metritis diagnosis, and greater leukocyte activation at calving, but lower immune cell activation at metritis diagnosis. In summary, cows that developed metritis had plasma metabolomic changes associated with greater lipolysis, oxidative stress, and a dysregulated immune response which may predispose cows to metritis development.

Evaluation of luteolysis, follicle size, and time to ovulation in Holstein heifers treated with two different analogs and doses of prostaglandin-F2α.

Objectives were to evaluate the effect of 2 analogs of PGF2α (cloprostenol vs. dinoprost) and 2 doses (1 injection vs. 2 injections) on luteolysis, follicle diameter, hormonal concentrations, and time to ovulation in dairy heifers. Holstein heifers were fitted with automated estrus detection devices and had their estrous cycle synchronized using PGF2α and an intravaginal insert containing progesterone. Heifers detected in estrus were blocked by weight and randomly assigned to 1 of 4 treatments in a 2 × 2 factorial arrangement: cloprostenol on d 7 after estrus (CLOx1; n = 45), cloprostenol on d 7 and 8 after estrus (CLOx2; n = 41), dinoprost on d 7 after estrus (DINx1; n = 43), or dinoprost on d 7 and 8 after estrus (DINx2; n = 44). Treatment with the first injection of PGF2α was defined as experiment d 0. Area and blood flow of corpus luteum (CL) and diameter of follicles >5 mm were recorded every 12 h from d 0 to estrus and every 6 h thereafter until ovulation. Blood was sampled every 6 h from d 0 until ovulation. Heifers treated with cloprostenol had shorter interval to luteolysis (± SEM; CLOx1 = 23.5 ± 2.2, CLOx2 = 22.9 ± 2.2, DINx1 = 32.6 ± 2.7, DINx2 = 26.4 ± 2.1 h); however, time to ovulation was not affected by treatment. A smaller proportion of heifers treated with a single injection of PGF2α underwent luteolysis compared with heifers treated with 2 injections (CLOx1 = 84.6 ± 6.2, CLOx2 = 100.0 ± 0.0, DINx1 = 59.7 ± 9.8, DINx2 = 96.3 ± 2.7%). Proportion of heifers that ovulated was smaller for DINx1 compared with other treatments (CLOx1 = 88.8 ± 5.1, CLOx2 = 100.0 ± 0.0, DINx1 = 55.2 ± 9.7, DINx2 = 94.4 ± 3.4%). Ovulatory follicle diameter was larger for DINx1 (18.2 ± 2.7 mm) compared with DINx2 (17.4 ± 2.7 mm), whereas dose did not affect the diameter of the ovulatory follicle in heifers treated with cloprostenol (CLOx1 = 17.6 ± 2.7 vs. CLOx2 = 17.8 ± 2.8 mm). Among heifers that underwent luteolysis, progesterone concentrations from 18 to 36 h after treatment were lesser in heifers treated with cloprostenol compared with those treated with dinoprost. Type of PGF2α did not affect progesterone concentrations past 36 h from treatment; however, heifers treated with 2 PGF2α injections had lesser progesterone concentrations and CL blood flow from 36 to 72 h after treatment compared with heifers that received a single PGF2α injection.

Effects of metritis treatment strategies on health, behavior, reproductive, and productive responses of Holstein cows.

Our objectives were to compare the effects of ceftiofur crystalline free acid (CCFA) and ampicillin trihydrate (AMP) treatments of cows diagnosed with metritis on uterine health, behavior, reproductive, and productive responses. A controlled randomized clinical trial was designed. Metritis was defined as vaginal discharge (VD) = 5 (fetid, watery, red/brown) within 21 d in milk (DIM) and rectal temperature (RT) <39.5°C, whereas VD = 5 and RT ≥39.5°C was defined as puerperal metritis. On the day of diagnosis (d 0), cows were paired by parity and severity of metritis (metritis vs. puerperal metritis) and assigned randomly to the AMP and CCFA treatments. Cows enrolled in the AMP (n = 308) treatment were moved to a nonsalable-milk pen, where they were treated once daily for 5 d, and were moved back to their original pen 72 h after the last treatment (d 7). Cows enrolled in the CCFA (n = 310) treatment remained in their original pen and received 2 treatments of CCFA, 72 h apart. Rectal temperature was measured daily from d 0 to 6 and on d 11. Vaginal discharge was evaluated on d 4, 6, and 11 to assess cure. Cure was defined as the absence of treatment with additional antimicrobial before experiment d 11, VD <5, and RT <39.5°C. Cows were examined at 28 ± 3 DIM for purulent VD (PVD) and at 35 ± 3 DIM for cytological endometritis. Pregnancy was diagnosed at 40 ± 3 and 60 ± 7 d after first and second artificial inseminations. Cure of metritis did not differ between treatments on d 11 (AMP = 64.6 ± 3.1, CCFA = 63.5 ± 3.1%). Cows treated with AMP had greater RT from experiment d 1 to 6 compared with cows treated with CCFA (AMP = 39.1 ± 0.02, CCFA = 39.0 ± 0.02°C). Cows in the AMP treatment had greater prevalence of PVD at 28 ± 3 DIM (AMP = 82.6 ± 2.3, CCFA = 74.4 ± 2.7%) and tended to have greater prevalence of cytological endometritis at 35 ± 3 DIM (AMP = 77.8 ± 6.2 vs. CCFA = 61.7 ± 7.5%) than CCFA-treated cows. Treatment did not affect the hazard of pregnancy among multiparous cows; however, among primiparous cows, CCFA treatment reduced the hazard of pregnancy and increased the median days to pregnancy (AMP = 145 vs. CCFA = 169 d). Finally, average daily milk yield up to 14 wk postpartum was not affected by treatment (AMP = 38.0 ± 0.4, CCFA = 37.5 ± 0.4 kg). We conclude from the current experiment that CCFA was more effective in reducing RT and improving uterine health of metritic cows; however, the improved hazard of pregnancy of primiparous cows treated with AMP is important and warrants further investigation.

Failure of clinical cure in dairy cows treated for metritis is associated with reduced productive and reproductive performance.

Objectives were to assess reproductive and productive outcomes associated with failure of clinical cure in dairy cows diagnosed with metritis following antimicrobial therapy. This retrospective cohort study included data from 3 experiments performed in 5 dairies. Metritis was characterized by the presence of watery, fetid, reddish-brownish vaginal discharge within 21 DIM (study d 0). Cows not diagnosed with metritis (i.e., cows may have had other diseases postpartum; NoMT; n = 1,194) were paired based on lactation number and calving date. All cows with metritis received antimicrobial therapy (ampicillin or ceftiofur). Clinical cure was evaluated on d 10 based on vaginal discharge score, and cows were categorized as cured (MTC; n = 1,111) or not cured (MTnoC; n = 299). Purulent vaginal discharge (28 ± 3 or 32 ± 3 DIM), cytological endometritis (35 ± 3 or 39 ± 3 DIM), and estrous cyclicity (50 ± 3 and 64 ± 3, 36 ± 3 and 50 ± 3, or 37 ± 5 and 51 ± 5 DIM) were evaluated in subgroups of cows. Proportions of cows with purulent vaginal discharge and cytological endometritis were greatest for MTnoC (91.7 and 91.4%), intermediate for MTC (74.0 and 73.3%), and smallest for NoMT (38.1 and 36.4%). Proportion of cyclic cows was smaller for MTnoC compared with MTC and NoMT (62.0, 71.0, and 71.0%). Pregnancy per artificial insemination following first service was smaller for cows with metritis compared with their counterparts with no metritis (NoMT = 28.1, MTC = 26.1, MTnoC = 22.0%). Pregnancy loss tended to be greater for MTnoC compared with MTC (NoMT = 11.5, MTC = 11.1, MTnoC = 18.4%). Hazard of pregnancy by 300 DIM was smallest for MTnoC, intermediate for MTC, and greatest for NoMT. Death by 60 DIM (3.9, 1.1, and 0.6%) and removal from herd by 300 DIM (26.3, 17.4, and 15.4%) were greatest for MTnoC compared with MTC and NoMT, respectively. Milk production among multiparous cows was smaller for MTnoC compared with MTC and NoMT in the first 10 mo postpartum, whereas MTC produced less milk compared with NoMT only during the first 2 mo postpartum (NoMT = 42.0 ± 0.22, MTC = 40.6 ± 0.28, MTnoC = 37.7 ± 0.54 kg/d). Failure of clinical cure was not associated with milk yield in primiparous cows (NoMT = 35.2 ± 0.31, MTC = 33.9 ± 0.31, MTnoC = 35.0 ± 0.52 kg/d). Cows diagnosed with metritis that do not undergo clinical cure by 10 d of onset of antimicrobial therapy have impaired reproductive performance, reduced milk production, and increased risk of leaving the herd.

Using chitosan microparticles to treat metritis in lactating dairy cows.

The main objective of this study was to evaluate the efficacy of intrauterine administration of chitosan microparticles (CM) in curing metritis in dairy cows. A secondary objective was to evaluate the effects of metritis treatments on milk yield, survival, and reproductive performance. Cows with a fetid, watery, red-brownish vaginal discharge were diagnosed with metritis. Holstein cows (n = 826) with metritis from 3 dairies located in northern Florida were blocked by parity (primiparous or multiparous) and, within each block, randomly assigned to one of 3 treatments: CM (n = 276) = intrauterine infusion of 24 g of CM dissolved in 40 mL of sterile distilled water at the time of metritis diagnosis (d 0), 2 (d 2), and 4 (d 4) d later; ceftiofur (CEF; n = 275) = subcutaneous injection of 6.6 mg/kg ceftiofur crystalline-free acid in the base of the ear at d 0 and d 3; Control (CON; n = 275) = no treatment applied at metritis diagnosis. All groups could receive escape therapy if condition worsened. Cure was considered when vaginal discharge became mucoid and not fetid. A group of nonmetritic (NMET; n = 2,436) cows was used for comparison. Data were analyzed by generalized linear mixed and Cox's proportional hazard models. Cows in CM and CON had lesser risk of metritis cure on d 12 than cows in CEF (58.6 ± 5.0 vs. 61.9 ± 4.9% vs. 77.9 ± 3.9, respectively). The proportion of cows culled within 60 days in milk (DIM) was greater for cows in CM than for cows in CEF and CON (21.5 ± 2.7 vs. 9.7 ± 1.9 vs. 11.3 ± 2.0%, respectively). Treatment did not affect rectal temperature or plasma nonesterified fatty acids, ß-hydroxybutyrate, and haptoglobin concentrations. Milk yield in the first 60 DIM differed for all treatments, and it was lowest for CM (35.8 ± 0.3 kg/d), followed by CON (36.8 ± 0.3 kg/d) and CEF (37.9 ± 0.3 kg/d). The hazard of pregnancy up to 300 DIM was lesser for CM than CEF (hazard ratio = 0.62; 95% CI: 0.50-0.76), for CM than CON (hazard ratio = 0.77; 95% CI: 0.62-0.95) and for CON than CEF (hazard ratio = 0.80; 95% CI: 0.65-0.99). Culling was greater, and milk yield and fertility were lesser for CEF than NMET. In summary, CM did not improve the cure of metritis, and was detrimental to milk yield, survival, and fertility compared with CON. In contrast, CEF increased the cure of metritis, milk yield, and fertility compared with CM and CON. Finally, the negative effects of metritis on milk yield culling and fertility could not be completely reversed by CEF.

Effects of timing of artificial insemination and treatment of semen with a Slo3 potassium channel blocker on fertility of dairy heifers subjected to the 5-day CIDR-Synch protocol.

Two experiments were conducted to evaluate the effects of the timing of artificial insemination (AI) and incorporation of the Slo3 K+ channel blocker 4-(4-chlorophenyl)butyl-diethyl-heptylammonium to semen extender (CSE) on pregnancy per AI (P/AI) and pregnancy loss in dairy heifers. In experiment 1, Holstein heifers were subjected to the 5-d CIDR-Synch protocol: d -8 GnRH and controlled internal drug-release device (CIDR); d -3 PGF2α and CIDR removal; d -2 PGF2α; d 0 GnRH) and assigned randomly to receive timed AI with control semen on d 0 (72-CON; n = 104), control semen on d -1 (48-CON; n = 100), or CSE-treated semen on d -1 (48-CSE; n = 98). Heifers were fitted with collar-mounted automated estrus detection devices to monitor physical activity and rumination. In experiment 2, Holstein heifers were subjected to the 5-d CIDR-Synch protocol and received a mount detection patch at the first PGF2α injection. Heifers detected in estrus before d 0 were inseminated on the same day, whereas those not detected in estrus received the second GnRH injection and timed AI on d 0. Heifers were assigned randomly to receive AI with control (AI-CON; n = 148) or CSE-treated semen (AI-CSE; n = 110). Four bulls with proven fertility were used in both experiments, and ejaculates from each sire were divided and processed as CON or CSE. Pregnancy was diagnosed by transrectal ultrasonography at 29 and 54 d after AI. Data were analyzed by logistic regression, and statistical models included the fixed effects of treatment and enrollment week. In experiment 1, orthogonal contrasts were built to assess the effects of day of AI (72-CON vs. 48-CON + 48-CSE) and treatment of semen with CSE (48-CON vs. 48-CSE). Pregnancy per AI on d 29 (72-CON = 60.8, 48-CON = 35.2, 48-CSE = 39.8%) and d 54 (72-CON = 58.2, 48-CON = 31.6, 48-CSE = 36.2%) was greater for heifers inseminated on d 0 compared with d -1. However, no effect of semen extender on P/AI was observed in heifers inseminated on d -1. In experiment 2, P/AI tended to be greater for AI-CSE than for AI-CON on d 29 (58.6 vs. 47.3%) and d 54 after AI (55.6 vs. 43.7%). Advancing AI by 24 h decreased the likelihood of pregnancy, and use of CSE was unable to overcome the expected asynchrony between insemination and ovulation. Nevertheless, incorporation of CSE in semen processing tended to improve P/AI when heifers received AI upon detected estrus or timed AI concurrently with the final GnRH of the 5-d CIDR-Synch protocol.

Impact of assisted reproduction techniques on subsequent reproductive performance of dairy heifers and lactating cows.

Use of assisted reproduction techniques (ART) such as ovum pick-up for in vitro embryo production (OPU-IVP) and multiple ovulation for embryo transfer (MOET) in livestock has increased in recent years. Objectives of this retrospective cohort study were to evaluate the association between ART and reproductive performance of donor dairy heifers and cows until third lactation. Holsteins donors subjected to MOET (n = 1087) or OPU-IVP (n = 551) were matched with non-donor females (n = 4112) based on date of birth allowing for a maximal difference of 10 days. Binary variables were analyzed using multivariable logistic regression whereas time to first breeding and time to pregnancy were analyzed using multivariable Cox's proportional hazard models and Kaplan-Meier survival curves. Contrasts were built to assess the association between donor status (non-donor vs. MOET + OPU-IVP), type of ART (MOET vs. OPU-IVP) and number of OPU-IVP procedures. Non-donor heifers had greater hazards of first breeding, pregnancy, and first calving compared with donor counterparts. Proportion of pregnant heifers after first breeding was reduced for donors compared with non-donors, which was primarily explained by a reduced proportion of OPU-IVP donors that became pregnant. Although proportion of pregnant cows after first breeding was not associated with ART group during first lactation, hazards of first breeding and pregnancy relative to the end of voluntary wait period were reduced for OPU-IVP donors. During second and third lactations, hazard of first breeding was greater for non-donors compared with donor counterparts. However, proportion of pregnant cows after first breeding and pregnancy hazard were not associated with ART groups. Culling was greater for non-donors compared with donors across all lactations. However, proportion cows culled during first lactation that was attributed to reproductive causes tended to be greater for donor compared with non-donor cows. Assisted reproduction techniques were associated with reduced reproductive performance in dairy heifers and cows but did not increase overall culling.

Size of the exon 1-CAG repeats of the androgen receptor gene employed as a molecular marker in the diagnosis of Turner syndrome in girls with short stature.

Turner syndrome (TS) is one of the most common chromosomal abnormalities among girls. Complete monosomy of X chromosome is responsible for almost 50% of all cases of TS, and mosaicism and X anomaly are detected in the other half. It has already been demonstrated that early diagnosis of these children allows appropriate growth hormone treatment with better final height prognosis and introduction of estrogen at an ideal chronological age. Sixty-four short-stature girls were selected and the clinical data obtained were birth weight and height, weight and height at the first medical visit and target height. Other clinical data including cardiac and renal abnormalities, otitis, Hashimoto thyroiditis, cubitus valgus, short neck, widely separated nipples, and pigmented nevi were obtained from the patients' medical records. The aim of the present study was to evaluate the screening of a group of short-stature girls for TS based on the number of CAG repeats of the androgen receptor gene analyzed by GeneScan software. Patient samples with two alleles (heterozygous) were 49/64 (76.5%) and with one allele (homozygous) were 15/64 (23.5%). A karyotype was determined in 30 patients, 9 homozygous and 21 heterozygous. In the homozygous group, 6/9 were 45,X and 3/9 were 46,XX. In the heterozygous group, 17/21 were 46,XX, and 4/21 were TS patients with mosaicism (45,X/46,XX; 45,X/46XiXq; 46XdelXp). The pattern obtained by GeneScan in two patients with mosaicism in the karyotype was an imbalance between the peak heights of the two alleles, suggesting that this imbalance could be present when there is a mosaicism. The frequency of TS abnormalities (18.7%) did not differ between TS and 46,XX girls. Thus, it is important to accurately assess the incidence of TS in growth-retarded girls, even in the absence of other dysmorphisms. In this study, we diagnosed 6 cases of TS 45,X (9.4%) by molecular analysis, with a 100% sensitivity and 85% specificity. This molecular analysis was able to detect all cases of TS 45,X and the majority of mosaicisms, without the need for more X chromosome markers. In conclusion, determining the number of CAG repeats of the androgen receptor gene analyzed by GeneScan was a fast method with high sensitivity for the detection of TS 45,X, suggesting that it could be interesting as a method for screening a population of growth-retarded girls.

Alternagin-C, a disintegrin-like protein from the venom of Bothrops alternatus, modulates alpha2beta1 integrin-mediated cell adhesion, migration and proliferation.

The alpha2beta1 integrin is a major collagen receptor that plays an essential role in the adhesion of normal and tumor cells to the extracellular matrix. Alternagin-C (ALT-C), a disintegrin-like protein purified from the venom of the Brazilian snake Bothrops alternatus, competitively interacts with the alpha2beta1 integrin, thereby inhibiting collagen binding. When immobilized in plate wells, ALT-C supports the adhesion of fibroblasts as well as of human vein endothelial cells (HUVEC) and does not detach cells previously bound to collagen I. ALT-C is a strong inducer of HUVEC proliferation in vitro. Gene expression analysis was done using an Affimetrix HU-95A probe array with probe sets of approximately 10,000 human genes. In human fibroblasts growing on collagen-coated plates, ALT-C up-regulates the expression of several growth factors including vascular endothelial growth factor, as well as some cell cycle control genes. Up-regulation of the vascular endothelial growth factor gene and other growth factors could explain the positive effect on HUVEC proliferation. ALT-C also strongly activates protein kinase B phosphorylation, a signaling event involved in endothelial cell survival and angiogenesis. In human neutrophils, ALT-C has a potent chemotactic effect modulated by the intracellular signaling cascade characteristic of integrin-activated pathways. Thus, ALT-C acts as a survival factor, promoting adhesion, migration and endothelial cell proliferation after binding to alpha2beta1 integrin on the cell surface. The biological activities of ALT-C may be helpful as a therapeutic strategy in tissue regeneration as well as in the design of new therapeutic agents targeting alpha2beta1 integrin.

Adhesion of the human pathogen Sporothrix schenckii to several extracellular matrix proteins.

The pathogenic fungus Sporothrix schenckii is the causative agent of sporotrichosis. This subcutaneous mycosis may disseminate in immunocompromised individuals and also affect several internal organs and tissues, most commonly the bone, joints and lung. Since adhesion is the first step involved with the dissemination of pathogens in the host, we have studied the interaction between S. schenckii and several extracellular matrix (ECM) proteins. The binding of two morphological phases of S. schenckii, yeast cells and conidia, to immobilized type II collagen, laminin, fibronectin, fibrinogen and thrombospondin was investigated. Poly (2-hydroxyethyl methacrylate) (poly-HEMA) was used as the negative control. Cell adhesion was assessed by ELISA with a rabbit anti-S. schenckii antiserum. The results indicate that both morphological phases of this fungus can bind significantly to type II collagen, fibronectin and laminin in comparison to the binding observed with BSA (used as blocking agent). The adhesion rate observed with the ECM proteins (type II collagen, fibronectin and laminin) was statistically significant (P < 0.05) when compared to the adhesion obtained with BSA. No significant binding of conidia was observed to either fibrinogen or thrombospondin, but yeast cells did bind to the fibrinogen. Our results indicate that S. schenckii can bind to fibronectin, laminin and type II collagen and also show differences in binding capacity according to the morphological form of the fungus.

ATL-1, a synthetic analog of lipoxin, modulates endothelial permeability and interaction with tumor cells through a VEGF-dependent mechanism.

Lipoxins (LX) and 15-epi-LX are lipids with a potent inhibitory effect on angiogenesis, in different models in vivo and in vitro. ATL-1, a synthetic analog of 15-epi-LXA4, inhibits various actions stimulated by vascular endothelial growth factor (VEGF). However, LX actions on endothelial cells (EC) in tumor-related contexts are still unknown. Here, we investigated the modulation of EC by ATL-1, in a model that mimics tumor extravasation. We observed that the analog inhibited endothelial permeability induced by VEGF, through the stabilization of VE-cadherin/ß-catenin-dependent adherens junctions. We tested the ability of MV3 cells, a highly metastatic melanoma cell line, to transmigrate across unchallenged EC monolayers for 18 h, as compared to NGM normal melanocytes. ATL-1 was able to inhibit only melanoma extravasation. MV3 cells secrete large amounts of VEGF and we observed that ATL-1 per se did not alter this ability. Melanoma cells skills to crossing endothelial monolayers were due to the steady accumulation of tumor-derived VEGF. When endothelial cells were challenged with exogenous VEGF, added at levels comparable to those secreted by MV3 cells over 18 h, and a short-term (4h) transendothelial migration assay was performed, both melanoma and melanocyte cells were able to extravasate, and ATL-1 was able to block the passage of both cells. These results indicate that ATL-1 has a potent inhibitory effect on the permeability induced by VEGF, and that this pharmacological effect could be used to block tumor extravasation across endothelial barriers, with a possible prospect of reducing the haematogenic spread of cancer cells.

[Dispersion of Biomphalaria straminea in the State of Sergipe: a comparative study within an interval of 19 years]. / Dispersão de Biomphalaria straminea no Estado de Sergipe: um estudo comparativo com dezenove anos de intervalo.

The aim of the current paper was to study in the state of Sergipe the population distribution and interactions between two species of Biomphalaria, the snail vectors of Schistosoma mansoni in the northeastern Brazil. Data collected in 1969 showed that B. straminea, with only one exception, was limited to the semi-dry region, while B. glabrata was found to live exclusively in the forest region, both in the state of Sergipe. This spatial distribution seemed to suggest that the above Biomphalaria species used to dominate specific territories. Snail collections made in 1988 in the same 37 places searched in 1969, showed that B. straminea has invaded territories previously occupied by B. glabrata, suggesting that a process of competitive displacement is taking place between these two closely related species. Natural snail infection rates were determined and some ecological aspects of the snail breeding places were registered.

Involvement of fungal cell wall components in adhesion of Sporothrix schenckii to human fibronectin.

Systemic sporotrichosis is an emerging infection potentially fatal for immunocompromised patients. Adhesion to extracellular matrix proteins is thought to play a crucial role in invasive fungal diseases. Here we report studies of the adhesion of Sporothrix schenckii to the extracellular protein fibronectin (Fn). Both yeast cells and conidia of S. schenckii were able to adhere to Fn as detected by enzyme-linked immunosorbent binding assays. Adhesion of yeast cells to Fn is dose dependent and saturable. S. schenckii adheres equally well to 40-kDa and 120-kDa Fn proteolytic fragments. While adhesion to Fn was increased by Ca(2+), inhibition assays demonstrated that it was not RGD dependent. A carbohydrate-containing cell wall neutral fraction blocked up to 30% of the observed adherence for the yeast cells. The biochemical nature of this fraction suggests the participation of cell surface glycoconjugates in binding by their carbohydrate or peptide moieties. These results provide new data concerning S. schenckii adhesion mechanisms, which could be important in host-fungus interactions and the establishment of sporotrichosis.

Size of the exon 1-CAG repeats of the androgen receptor gene employed as a molecular marker in the diagnosis of Turner syndrome in girls with short stature

Turner syndrome (TS) is one of the most common chromosomal abnormalities among girls. Complete monosomy of X chromosome is responsible for almost 50% of all cases of TS, and mosaicism and X anomaly are detected in the other half. It has already been demonstrated that early diagnosis of these children allows appropriate growth hormone treatment with better final height prognosis and introduction of estrogen at an ideal chronological age. Sixty-four short-stature girls were selected and the clinical data obtained were birth weight and height, weight and height at the first medical visit and target height. Other clinical data including cardiac and renal abnormalities, otitis, Hashimoto thyroiditis, cubitus valgus, short neck, widely separated nipples, and pigmented nevi were obtained from the patients’ medical records. The aim of the present study was to evaluate the screening of a group of short-stature girls for TS based on the number of CAG repeats of the androgen receptor gene analyzed by GeneScan software. Patient samples with two alleles (heterozygous) were 49/64 (76.5%) and with one allele (homozygous) were 15/64 (23.5%). A karyotype was determined in 30 patients, 9 homozygous and 21 heterozygous. In the homozygous group, 6/9 were 45,X and 3/9 were 46,XX. In the heterozygous group, 17/21 were 46,XX, and 4/21 were TS patients with mosaicism (45,X/46,XX; 45,X/46XiXq; 46XdelXp). The pattern obtained by GeneScan in two patients with mosaicism in the karyotype was an imbalance between the peak heights of the two alleles, suggesting that this imbalance could be present when there is a mosaicism. The frequency of TS abnormalities (18.7%) did not differ between TS and 46,XX girls. Thus, it is important to accurately assess the incidence of TS in growth-retarded girls, even in the absence of other dysmorphisms. In this study, we diagnosed 6 cases of TS 45,X (9.4%) by molecular analysis, with a 100% sensitivity and 85% specificity. This molecular analysis was...

Alternagin-C, a disintegrin-like protein from the venom of Bothrops alternatus, modulates alpha2ß1 integrin-mediated cell adhesion, migration and proliferation

The alpha2ß1 integrin is a major collagen receptor that plays an essential role in the adhesion of normal and tumor cells to the extracellular matrix. Alternagin-C (ALT-C), a disintegrin-like protein purified from the venom of the Brazilian snake Bothrops alternatus, competitively interacts with the alpha2ß1 integrin, thereby inhibiting collagen binding. When immobilized in plate wells, ALT-C supports the adhesion of fibroblasts as well as of human vein endothelial cells (HUVEC) and does not detach cells previously bound to collagen I. ALT-C is a strong inducer of HUVEC proliferation in vitro. Gene expression analysis was done using an Affimetrix HU-95A probe array with probe sets of 10,000 human genes. In human fibroblasts growing on collagen-coated plates, ALT-C up-regulates the expression of several growth factors including vascular endothelial growth factor, as well as some cell cycle control genes. Up-regulation of the vascular endothelial growth factor gene and other growth factors could explain the positive effect on HUVEC proliferation. ALT-C also strongly activates protein kinase B phosphorylation, a signaling event involved in endothelial cell survival and angiogenesis. In human neutrophils, ALT-C has a potent chemotactic effect modulated by the intracellular signaling cascade characteristic of integrin-activated pathways. Thus, ALT-C acts as a survival factor, promoting adhesion, migration and endothelial cell proliferation after binding to alpha2ß1 integrin on the cell surface. The biological activities of ALT-C may be helpful as a therapeutic strategy in tissue regeneration as well as in the design of new therapeutic agents targeting alpha2ß1 integrin.

Adhesion of the human pathogen Sporothrix schenckii to several extracellular matrix proteins

The pathogenic fungus Sporothrix schenckii is the causative agent of sporotrichosis. This subcutaneous mycosis may disseminate in immunocompromised individuals and also affect several internal organs and tissues, most commonly the bone, joints and lung. Since adhesion is the first step involved with the dissemination of pathogens in the host, we have studied the interaction between S. schenckii and several extracellular matrix (ECM) proteins. The binding of two morphological phases of S. schenckii, yeast cells and conidia, to immobilized type II collagen, laminin, fibronectin, fibrinogen and thrombospondin was investigated. Poly (2-hydroxyethyl methacrylate) (poly-HEMA) was used as the negative control. Cell adhesion was assessed by ELISA with a rabbit anti-S. schenckii antiserum. The results indicate that both morphological phases of this fungus can bind significantly to type II collagen, fibronectin and laminin in comparison to the binding observed with BSA (used as blocking agent). The adhesion rate observed with the ECM proteins (type II collagen, fibronectin and laminin) was statistically significant (P<0.05) when compared to the adhesion obtained with BSA. No significant binding of conidia was observed to either fibrinogen or thrombospondin, but yeast cells did bind to the fibrinogen. Our results indicate that S. schenckii can bind to fibronectin, laminin and type II collagen and also show differences in binding capacity according to the morphological form of the fungus