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We report the complete genome sequence of Mucilaginibacter strain 21P, which was isolated from estuarine soil contaminated with mine tailings from the Samarco disaster, which occurred in 2015 in Brazil. The genome sequence comprised 4,739,655 bp, with a G+C content of 43.2%, and harbors multiple antibiotic and metal resistance genes.

Benzophenones display several biological activities, including antioxidant, anticancer, and photoprotective. Furthermore, antioxidants can minimize both ultraviolet absorption and tumor development. In the present investigation, a series of twenty-six 1,2,3-triazole-benzophenone derivatives were synthesized and had their antioxidant, anticancer, and photoprotective effects evaluated. For the compounds synthesis, 4,4’-dihydroxybenzophenone (1a) and 2,4-dihydroxybenzophenone (1b) were propargylated, affording the alkynes bis(4-(prop-2-yn1-yloxy))benzophenone (2a) and (2-hydroxy-4-(prop-2-yn-1-yloxy))benzophenone (2b), respectively. The copper(I)-catalyzed azide-alkyne cycloaddition (CuAAC) reaction between the compounds 2a/2b and several benzyl azides gave the 1,2,3-triazole-benzophenone derivatives with yields ranging from 35 to 95%. The 1,2,3-triazole-benzophenone derivatives at the concentration of 0.2 μg mL−1 (a no-cytotoxic concentration) exhibited a solar protection factor (SPF) comparable to positive control benzophonen-3 (BP-3). Concerning their antioxidant and cytotoxic effects, the derivatives from 1b showed high in vitro antioxidant effects as well as cytotoxicity against A549 (lung carcinoma), MCF-7 (breast carcinoma), and HT-144 (metastatic melanoma) cell lines, without significant cytotoxicity to a non-cancerous cell line. Derivatives 19, 20, and 24 induced cell death and cell cycle arrest at G1/S in HT-144 melanoma cells.

Hereford heifers were assigned randomly to three superstimulation groups and given FSH for 4 days in the short FSH group (n = 5) and FSH starvation group, (n = 5) or for 7 days in the long FSH group (n = 4). In vivo oocyte maturation was induced with LH given 12 hours after the last FSH treatment in short and long FSH groups and 84 hours after the last FSH treatment in the FSH starvation group. The ovaries were removed by colpotomy 18 to 20 hours after LH treatment to aspirate cumulus-oocyte complexes (COCs) from follicles 8 mm or greater. Cumulus-oocyte complexes were graded morphologically, and oocytes were processed for either mitochondrial staining or for ATP assay. Collection efficiency was similar among treatment groups, but a greater proportion of COCs were expanded (P 0.01) and oocyte ATP content of the expanded COC tended to be greater (P 0.09) in the long FSH group than other two groups. Oocytes in the FSH starvation group had a greater proportion (P = 0.01) of mitochondrial clusters (i.e., fewer scattered individual mitochondria). Individual mitochondria and mitochondrial clusters in oocytes from the long FSH and FSH starvation groups had twice the relative staining intensity (P 0.01) compared to oocytes from the short FSH group. In summary, the long FSH superstimulation protocol yielded a greater proportion of expanded COCs that had oocytes with a scattered mitochondrial population and a greater ATP content than other two protocols. FSH starvation of 84 hours yielded a high proportion of grade 4 COCs characterized by a greater proportion of mitochondrial clusters within the oocyte.

Disentangling factors influencing suspended bacterial community structure across distribution system and building plumbing provides insight into microbial control strategies from source to tap. Water quality parameters (residence time, chlorine, and total cells) and bacterial community structure were investigated across a full-scale chlorinated drinking water distribution system. Sampling was conducted in treated water, in different areas of the distribution system and in hospital building plumbing. Bacterial community was evaluated using 16S rRNA gene sequencing. Bacterial community structure clearly differed between treated, distributed, and premise plumbing water samples. While Proteobacteria (60%), Planctomycetes (20%), and Bacteroidetes (10%) were the most abundant phyla in treated water, Proteobacteria largely dominated distribution system sites (98%) and taps (91%). Distributed and tap water differed in their Proteobacteria profile: Alphaproteobacteria was dominant in distributed water (92% vs. 65% in tap waters), whereas Betaproteobacteria was most abundant in tap water (18% vs. 2% in the distribution system). Finally, clustering of bacterial community profiles was largely explained by differences in chlorine residual concentration, total bacterial count, and water residence time. Residual disinfectant and hydraulic residence time were determinant factors of the community structure in main pipes and building plumbing, rather than treated water bacterial communities.

The present investigation describes the synthesis of a series of novel triazole derivatives from 4,4’-dihydroxybenzophenone along with their elastase inhibitory activity. The 1,2,3-triazoles were obtained via the copper(I)-catalyzed azide-alkyne cycloaddition reaction (CuAAC), also known as click reaction, between bis(4-(prop-2-yn-1-yloxy))benzophenone and several benzyl azides. It was found that five derivatives exhibited significant inhibitory effects, presenting half maximal inhibitory concentration (IC50) values in the range of 16.6 to 72.1 µM. The most active compound, namely bis(4-(1-(4-iodobenzyl)-1H-1,2,3-triazol-4-yl)methoxy)benzophenone (IC50 = 16.6 ± 1.9 µM), was found to bind to elastase with the inhibition constant (Ki ) of 11.12 µM, thereby illustrating competitive inhibitory behavior. Further, docking investigations provided insights on the possible binding mode of the most active compound with the elastase. A presente investigação descreve a síntese de uma série de novos derivados triazólicos de 4,4'-dihidroxibenzofenona juntamente com sua atividade inibidora da elastase. Os 1,2,3-triazóis foram obtido através da reação de cicloadição azida-alcino catalisada por cobre(I) (CuAAC), também conhecida como reação de clique, entre bis(4-(prop-2-in-1-iloxi))benzofenona e várias benzil azidas. Era descobriram que cinco derivados exibiram efeitos inibitórios significativos, apresentando metade inibitória máxima valores de concentração (IC50) na faixa de 16,6 a 72,1 µM. O composto mais ativo, ou seja, bis(4-(1-(4-iodobenzil)-1H-1,2,3-triazol-4-il)metoxi)benzofenona (IC50 = 16,6 ± 1,9 µM), foi encontrado para se ligar à elastase com a constante de inibição (Ki ) de 11,12 µM, ilustrando assim comportamento inibitório competitivo. Além disso, as investigações de encaixe forneceram informações sobre as possíveis modo de ligação do composto mais ativo com a elastase.

Antral follicle count (AFC) repeatability at the time of follicular wave emergence and duration of gonadotropin treatment in calves with small and large AFC affects the superstimulatory response of follicles. In Study I, the individual AFC was determined, calves were ranked as having a small, medium or large AFC, and a second count was performed prior to FSH treatments. There was a positive association between the number of follicles ≥1 mm after the first and second counts (r = 0.4; P = 0.003). In Study II, calves with small and large AFC were administered pFSH for 4 or 7 days, pLH 20 h after last pFSH administration and cumulus-oocyte-complexes (COC) were collected. In calves having large as compared with small AFC, number of follicles ≥6 mm were greater (P = 0.01) and COC collected were greater (P = 0.001). The proportion of large-sized follicles (>9 mm) was greater in the 7-day than in the 4-day gonadotropin treatment group (56.4 ± 8.3 and27.8 ± 7.5 %, respectively; P = 0.01). In Study III, there was a positive association between AFC and number of follicles ≥6 mm at the time of COC collection (r = 0.6; P = 0.003). In summary, the number of follicles at the time of follicular wave emergence was associated with the number of follicles recruited during subsequent waves of follicular development and ovarian response following gonadotropin superstimulation. Calves with a large AFC had more COC collected than calves with a small AFC.

The objective of the study was to determine relative effects of dose (200 or 350 mg) and duration (4 or 7 days) of superstimulatory treatment on the ovarian response in prepubertal calves. Calves with similar antral follicular counts at wave emergence (n = 24) were given eight doses of either 25 or 44 mg pFSH every 12 h for 4 days or 14 doses of either 14 or 25 mg pFSH for 7 days beginning at the time of follicular wave emergence and 12.5 mg of pLH im 12 h after the last FSH treatment. On Day 4 of pFSH treatment, calves given 14 mg had fewer follicles ≥3 mm than those given 25 mg (15.1 ± 1.9 and 27.9 ± 3.3, respectively; P = 0.04). At the end of treatment (24 h post-LH), number of follicles ≥9 mm was greater in calves of groups treated with 350 than 200 mg (13.5 ± 1.8 and 8.8 ± 1.3, respectively; P = 0.02) and calves of groups treated for 7 than 4 days (13.3 ± 1.8 and 9.0 ± 1.3, respectively; P = 0.03). The number of spontaneous ovulations was greater in calves of groups treated for 7 than 4 days as was the total number of ovulations (9.7 ± 0.9 and 6.9 ± 1.0, respectively; P ≤ 0.05). In summary, a dose of 25 mg of pFSH per treatment given twice daily for 7 days resulted in a greater ovarian response than other superstimulatory treatments in prepubertal calves.

The objective of the present study was to compare the effect of a single versus multiple doses of a 10-amino acid fragment of human (hKp) or murine (mKp) kisspeptin on LH secretion and the fate of the dominant follicle. In all experiments, a new wave was induced (Day 0) by ultrasound-guided ablation of >5 mm follicles, a progesterone device (CIDR) was placed in the vagina, animals given prostaglandin F2α analog im on Day 3.5 and 4, and hKp or mKp treatment given on Day 6. The experimental design maintained growth and ovulatory potential of the dominant follicle for 12 days and allowed hypothesis testing during the low-progesterone period (plasma progesterone ≤1.8 ng/ml on Day 6) wherein spontaneous wave emergence and ovulation did not occur between Day 6 and Day 12. In Experiment 1, heifers (n = 10/group) were given single iv dose of 45 mg hKp, 45 mg mKp, or 2 ml normal saline (control). Post-treatment plasma LH concentrations from 15 to 90 min were higher (P

Background Prolongation of superstimulatory treatment appears to be associated with a greater superovulatory response and with greater oocyte maturation in cattle. A genome-wide bovine oligo-microarray was used to compare the gene expression of granulosa cells collected from ovarian follicles after differing durations of the growing phase induced by exogenous FSH treatment. Cows were given a conventional (4-day) or long (7-day) superstimulatory treatment (25 mg FSH im at 12-h intervals; n = 6 per group), followed by prostaglandin treatment with last FSH and LH treatment 24 h later. Granulosa cells were harvested 24 h after LH treatment. Results The expression of 416 genes was down-regulated and 615 genes was up-regulated in the long FSH group compared to the conventional FSH group. Quantification by RT-PCR of 7 genes (NTS, PTGS2, PTX3, RGS2, INHBA, CCND2 and LRP8) supported the microarrays data. Multigene bioinformatic analysis indicates that markers of fertility and follicle maturity were up-regulated in the long FSH group. Conclusion Using the large gene expression dataset generated by the genomic analysis and our previous associated with the growth phase and gene expression changes post LH, we can conclude that a prolonged FSH-induced growing phase is associated with transcriptomic characteristics of greater follicular maturity and may therefore be more appropriate for optimizing the superovulatory response and developmental competence of oocytes in cattle.

Two experiments were carried out to determine the effect of Kisspeptin-10 on plasma LH concentrations and follicular dynamics during the luteal phase in cattle. We tested the hypothesis that a single treatment of Kisspeptin-10 will increase plasma LH concentration and the diameter of the dominant follicle, and induce ovulation during the luteal phase of the estrous cycle in cattle. In the Experiment 1, Hereford-cross heifers (n = 28, 14-16 mo) were given PGF2α im to induce luteolysis and ovulation. On Day 5 (Day 0 = ovulation), a new follicular wave was induced by ultrasound-guided follicular ablation. Heifers were treated on Day 10 (4 days after wave emergence) with 100 μg GnRH im (n = 9), 2 mL saline im (n = 7), 1 mg Kisspeptin-10 im (Kp im, n = 6) or 1 mg Kisspeptin-10 iv (Kp iv; n = 6). Blood samples were collected at -60, -15, 0, 5, 15min (0 min = time of injection) and every 15 min thereafter until 3 h. Transrectal ovarian ultrasonography was performed at 12 h intervals from Day 10-14. In Experiment 2, non-lactating beef cows on Day 5 were treated with 100 μg GnRH im (n = 9), saline im (n = 5), 10 mg of Kisspeptin-10 iv (Kp 10 mg; n = 5) or 15 mg of Kisspeptin-10 iv (Kp 15 mg; n = 5). Blood samples were collected at -15, 0, 15, 30, 60, 120, 180 min and twice daily ovarian ultrasonography was done from Day 5-10. In Expt 1, plasma LH concentrations increased for 1 h following Kp iv administration. The peak concentration occurred at 15 min and was higher in the Kp iv group than in the Kp im group (P = 0.01). The LH peak was 3.5-folds higher in the GnRH group than the Kp iv group (P 0.0001). In Expt 2, GnRH induced higher (P 0.001) plasma LH concentrations for all time-points than other groups. Kp 15 mg at peak (15min), 30 and 60 min induced higher (P 0.0001) plasma LH concentrations than Kp 10 mg and saline. Kisspeptin-treated animals did not ovulate in either experiment while GnRH induced ovulation (n = 5/9 in Expt 1; 9/9 in Expt 2). The diameter of the dominant follicle was greater (P = 0.02) at 12-48 h after kisspeptin treatment (Kp groups combined) than the Saline group in Expt 2. In conclusion, Kisspeptin-10 increased plasma LH concentrations and follicle size, and although plasma LH concentrations were higher after iv than im administration, but at the doses used, Kisspeptin-10 did not induce ovulation during the luteal phase in cattle.

Background\ud \ud The implementation of rapid drug susceptibility testing (DST) is a current global priority for TB control. However, data are scarce on patient-relevant outcomes for presumptive diagnosis of drug-resistant tuberculosis (pDR-TB) evaluated under field conditions in high burden countries.\ud \ud Methods\ud \ud Observational study of pDR-TB patients referred by primary and secondary health units. TB reference centers addressing DR-TB in five cities in Brazil. Patients age 18 years and older were eligible if pDR-TB, culture positive results for Mycobacterium tuberculosis and, if no prior DST results from another laboratory were used by a physician to start anti-TB treatment. The outcome measures were median time from triage to initiating appropriate anti-TB treatment, empirical treatment and, the treatment outcomes.\ud \ud Results\ud \ud Between February,16th, 2011 and February, 15th, 2012, among 175 pDR TB cases, 110 (63.0%) confirmed TB cases with DST results were enrolled. Among study participants, 72 (65.5%) were male and 62 (56.4%) aged 26 to 45 years. At triage, empirical treatment was given to 106 (96.0%) subjects. Among those, 85 were treated with first line drugs and 21 with second line. Median time for DST results was 69.5 [interquartile - IQR: 35.7–111.0] days and, for initiating appropriate anti-TB treatment, the median time was 1.0 (IQR: 0–41.2) days. Among 95 patients that were followed-up during the first 6 month period, 24 (25.3%; IC: 17.5%–34.9%) changed or initiated the treatment after DST results: 16/29 MDRTB, 5/21 DR-TB and 3/45 DS-TB cases. Comparing the treatment outcome to DS-TB cases, MDRTB had higher proportions changing or initiating treatment after DST results (p = 0.01) and favorable outcomes (p = 0.07).\ud \ud Conclusions\ud \ud This study shows a high rate of empirical treatment and long delay for DST results. Strategies to speed up the detection and early treatment of drug resistant TB should be prioritized.

Cryopreservation is known for its marked deleterious effects on embryonic health. Bovine compact morulae were vitrified or slow-frozen, and post-warm morulae were cultured to the expanded blastocyst stage. Blastocysts developed from vitrified and slow-frozen morulae were subjected to microarray analysis and compared with blastocysts developed from unfrozen control morulae for differential gene expression. Morula to blastocyst conversion rate was higher (P < 0.05) in control (72%) and vitrified (77%) than in slow-frozen (34%) morulae. Total 20 genes were upregulated and 44 genes were downregulated in blastocysts developed from vitrified morulae (fold change ≥ ± 2, P < 0.05) in comparison with blastocysts developed from control morulae. In blastocysts developed from slow-frozen morulae, 102 genes were upregulated and 63 genes were downregulated (fold change ≥ ± 1.5, P < 0.05). Blastocysts developed from vitrified morulae exhibited significant changes in gene expression mainly involving embryo implantation (PTGS2, CALB1), lipid peroxidation and reactive oxygen species generation (HSD3B1, AKR1B1, APOA1) and cell differentiation (KRT19, CLDN23). However, blastocysts developed from slow-frozen morulae showed changes in the expression of genes related to cell signaling (SPP1), cell structure and differentiation (DCLK2, JAM2 and VIM), and lipid metabolism (PLA2R1 and SMPD3). In silico comparison between blastocysts developed form vitrified and slow-frozen morulae revealed similar changes in gene expression as between blastocysts developed from vitrified and control morulae. In conclusion, blastocysts developed form vitrified morulae demonstrated better post-warming survival than blastocysts developed from slow-frozen morulae but their gene expression related to lipid metabolism, steroidogenesis, cell differentiation and placentation changed significantly (≥ 2 fold). Slow freezing method killed more morulae than vitrification but those which survived up to blastocyst stage did not express ≥ 2 fold change in their gene expression as compared with blastocysts from control morulae.

The objective was to determine the effects of the duration of progesterone exposure during the ovulatory wave on fertility (pregnancy rate) in beef cattle. We tested the hypothesis that short-progesterone exposure during the growing and early-static phase of the ovulatory follicle (analogous to the ovulatory wave of 3-wave cycles) is associated with higher fertility than a longer duration of exposure (analogous to the ovulatory wave of 2-wave cycles). Three to 5 days after ovulation, beef heifers (n = 172) and suckled beef cows (n = 193) were given an intravaginal progesterone-releasing device (CIDR) and 2.5 mg estradiol - 17β +50 mg progesterone im to induce a new follicular wave. Cattle were allocated to short- or long-progesterone exposure groups (for 3 and 6 d after wave emergence, respectively) after which prostaglandin F(2α) was administered and CIDR were removed. Forty-eight hours later, all cattle were given 12.5 mg pLH and artificially inseminated (AI) with frozen-thawed semen. The diameter of the two largest follicles and the corpus luteum were measured by transrectal ultrasonography at CIDR removal, insemination, and 36 h after insemination. Pregnancy diagnosis was done ultrasonically 38 and 65 d post-AI. There was no difference in pregnancy rates in short- vs long-progesterone exposure in heifers (53 vs 47%, P = 0.44) or cows (63 vs 58%, P = 0.51). However, the diameter of the ovulatory follicle at CIDR removal and AI was smaller in short- than in long-progesterone groups (P < 0.02), and larger in cows than in heifers (P < 0.006). In conclusion, short-progesterone exposure during the growing and early-static phase of the ovulatory follicle (similar to 3-wave cycles) was not associated with higher fertility than a longer progesterone exposure (similar to 2-wave cycles).

The objectives of this study were to determine the effect of LH on the blood flow to the ovaries of 4-month-old calves after 2 FSH stimulation protocols, and to examine the relationship between ovarian vascularity after superstimulation to the morphology of the cumulus–oocyte complexes (COC) and luteal function. We hypothesise that ovarian vascularity (detected by 3-dimensional (3D) analysis of Doppler ultrasound cineloops) will increase in response to LH, and the magnitude of change in vascularity would be predictive of (1) a greater proportion of expanded COC, (2) greater development of luteal tissue volume and vascularity at 3 and 7 days after follicle aspiration, and (3) higher levels of plasma progesterone. Ovarian superstimulation was initiated at the beginning of an induced follicular wave in 4-month-old beef calves (n = 16), and beef cattle >16 months of age (control group, adults; n = 8) using either a traditional 4-day or an extended 7-day FSH protocol (n = 8 calves and n = 4 controls per group). Power Doppler ultrasound cineloops were recorded immediately before (i.e. 12 h after the last FSH treatment) and 24 h after LH treatment (before ultrasound-guided follicular aspiration for oocyte collection) to assess ovarian vascularity, and 3 and 7 days after follicular aspiration to assess luteal tissue volume and vascularity. Video segments were analysed in Fiji and Imaris software to obtain the 3D ovarian vascularity index (ratio of blood flow volume to tissue volume). The ovarian vascularity index tended to increase >1.7-fold in response to exogenous LH in both prepubertal calves (pre-LH 1.5 ± 0.4% v. post-LH 2.6 ± 0.7%; P = 0.08) and adult cattle (pre-LH 2.2 ± 0.6% v. post-LH 4.7 ± 0.9%; P = 0.07). Calves with a recovery of >75% of expanded COC had a higher ovarian vascularity index (10.7 ± 2.6% v. 4.8 ± 1.6%; P = 0.06) and luteal vascularity index (15.7 ± 4.5% v. 5.7 ± 2.1%; P 10-fold higher concentration of plasma progesterone on Day 3 (12.7 ± 7.3 ng mL−1 v. 1.2 ± 0.4 ng mL−1; P 0.05) was found in control (adult) animals. In conclusion, there was an increase in ovarian vascularity resulting from LH treatment in prepubertal calves and adult cattle. A greater proportion of expansion of COC at 24 h after LH treatment (an indicator of follicular maturation) was related to higher ovarian and luteal vascularity on Day 7 after collection in prepubertal calves, but not in adults. Luteal vascularity on Day 3 was reflective of plasma progesterone concentration, and prepubertal calves in the 7-day FSH protocol had greater plasma progesterone than calves in the 4-day FSH protocol. The use of FSH in calves allows a greater number of follicles for oocyte collection as it does in adult cattle. Research was supported by an NSERC grant.

Gout manifests as recurrent episodes of acute joint inflammation and pain due to the deposition of monosodium urate (MSU) crystals within the affected tissue in a process dependent on NLRP3 inflammasome activation. The synthesis, activation, and release of IL-1β are crucial for MSU-induced inflammation. The current study evaluated the mechanism by which TNF-α contributed to MSU-induced inflammation. Male C57BL/6J or transgenic mice were used in this study and inflammation was induced by the injection of MSU crystals into the joint. TNF-α was markedly increased in the joint after the injection of MSU. There was inhibition in the infiltration of neutrophils, production of CXCL1 and IL-1β, and decreased hypernociception in mice deficient for TNF-α or its receptors. Pharmacological blockade of TNF-α with Etanercept or pentoxyfylline produced similar results. Mechanistically, TNF-α blockade resulted in lower amounts of IL-1β protein and pro-IL-1β mRNA transcripts in joints. Gene-modified mice that express only transmembrane TNF-α had an inflammatory response similar to that of WT mice and blockade of soluble TNF-α (XPro™1595) did not decrease MSU-induced inflammation. In conclusion, TNF-α drives expression of pro-IL-1β mRNA and IL-1β protein in experimental gout and that its transmembrane form is sufficient to trigger MSU-induced inflammation in mice.

The number of follicles recruited in successive waves are consistent in postpubertal cattle (Singh et al. 2004 Theriogenology 62, 227), but ovarian response to gonadotropin stimulation is highly variable among animals. We tested the hypotheses that the number of follicles present at wave emergence are repeatable and are predictive of superstimulatory response in prepubertal calves; therefore, we expected that calves with higher antral follicular counts (AFC; follicles = 1mm) at wave emergence will result in a greater number of follicles available for oocyte collection after a conventional 4-day gonadotropin treatment. Hereford crossbreed calves (n = 52; 5.1 to 6.8 months of age) were ranked according to the number of follicles = 1 mm detected in transrectal ultrasound video recordings of both ovaries at the time of wave emergence (First AFC; range: 12 to 53 follicles). Calves in the bottom (Low AFC; 32 follicles; n = 5) quartiles were selected for ovarian superstimulation. Emergence of a new follicular wave (Day 0) was induced by transvaginal follicle ablation (14 to 57 days after first AFC; 5.7 to 7.1 months of age), AFC were performed again (Second AFC), and calves were given eight 12-hourly IM injections of 25 mg of pFSH (Folltropin-V®, Bioniche Animal Health Inc., Belleville, Canada) beginning on Day 0.5. All calves were given 12.5 mg of pLH (Lutropin-V®, Bioniche Animal Health Inc.) IM 12 h after the last FSH and number of follicles equal to 3, 3 to 5, 6 to 8, and equal to 6 and 9 were counted 24 h after LH treatment (at the time of oocyte collection). A t-test was used to compare the number of follicles and ovarian response (Low v. High AFC). Values of Pearson (0.8; P

The review presents an overview of studies that examined the effects of follicular aging and maternal aging in the bovine model. The first of three main sections is a discussion of the developmental competence of oocytes from (1) the ovulatory follicle of 2-wave and 3-wave estrous cycles, (2) dominant follicles that develop under high or low LH pulse frequency, and (3) natural versus FSH-stimulated ovulatory follicles. The second section highlights the effects of maternal aging. Maternal aging in cattle is associated with (1) elevated circulating FSH concentrations, (2) reduced response to superstimulatory treatment, and (3) markedly decreased early embryonic development in cows >12 year of age. The third and final section on superstimulation protocols addresses the effects of the duration of FSH stimulation and withdrawal (i.e., FSH "starvation" or "coasting") on oocyte competence. Ovarian superstimulation for 4 days altered the expression of genes related to angiogenesis, and activated oxidative stress-response genes. Extending the duration of FSH stimulation from 4 to 7 days resulted in a greater and more synchronous ovulatory response and optimal oocyte maturation. The highest rates of blastocyst development in vitro were obtained when FSH support was discontinued for 44 to 68h and granulosa cell SMAD7 mRNA was predictive of this period. Longer periods of FSH starvation resulted in a loss of oocyte competence or ovulatory capability. By extending the bovine model to the transcriptome level, new approaches and treatments may be devised to resolve subfertility in women and animals.

The aim of the present study was to determine a set of reference genes in granulosa cells of dominant follicles that are suitable for relative gene expression analyses during maternal and follicular aging. Granulosa cells of growing and preovulatory dominant follicles were collected from aged and young cows (maternal aging study) and from FSH-stimulated follicles developing under different durations of FSH treatment (follicular aging study). The mRNA levels of the two commonly used reference genes (GAPDH, ACTB) and four novel genes (UBE2D2, EIF2B2, SF3A1, RNF20) were analysed using cycle threshold values. Results revealed that mRNA levels of GAPDH, ACTB, EIF2B2, RNF20, SF3A1 and UBE2D2 were similar (P > 0.05) between dominant follicle type, age and among follicles obtained after FSH-stimulation, but differed (P = 0.005) due to mRNA processing (i.e. with versus without amplification). The stability of reference genes was analysed using GeNorm, DeltaCT and NormFinder programs and comprehensive ranking order was determined using RefFinder. The mRNA levels of GAPDH and ACTB were less stable than those of UBE2D2 and EIF2B2. The geometric mean of multiple genes (UBE2D2, EIF2B2, GAPDH and SF3A1) is a more appropriate reference control than the use of a single reference gene to compare relative gene expression among dominant and FSH-stimulated follicles during maternal and/or follicular aging studies.

Our objective was to study the effect of superstimulation protocols on nuclear maturation of the oocyte and the distribution of lipid droplets in the ooplasm. Heifers (n = 4 each group) during the luteal phase were either treated with FSH for 4 days (Short FSH), FSH for 4 days followed by 84 h of gonadotropin free period (FSH Starvation) or for 7 days (Long FSH) starting from the day of wave emergence. In all groups, LH was given 24 h after induced luteolysis (penultimate day of FSH) and cumulus–oocyte complexes were collected 24 h later. Oocytes were stained for nuclear maturation (Lamin/chromatin) and lipid droplets (Nile red). The Long FSH group had a greater proportion of mature oocytes (metaphase II) compared with heifers in the Short FSH and FSH Starvation groups (59/100 vs 5/23 and 2/25, respectively; P

Microarray analysis was used to compare the gene expression of granulosa cells from dominant follicles with that of those after superstimulatory treatment. Cows were allocated randomly to two groups (superstimulation and control, n=6/group). A new follicular wave was induced by ablation of follicles ≥5 mm in diameter, and a progesterone-releasing device controlled internal drug release (CIDR) was placed in the vagina. The superstimulation group was given eight doses of 25 mg FSH at 12-h intervals starting from the day of wave emergence (day 0), whereas the control group was not given FSH treatment. Both groups were given prostaglandin F2α twice, 12 h apart, on day 3 and the CIDR was removed at the second injection; 25 mg porcine luteinizing hormone (pLH) was given 24 h after CIDR removal, and cows were ovariectomized 24 h later. Granulosa cells were collected for RNA extraction, amplification, and microarray hybridization. A total of 190 genes were downregulated and 280 genes were upregulated. To validate the microarray results, five genes were selected for real-time PCR (NTS, FOS, THBS1, FN1, and IGF2). Expression of four genes increased significantly in the three different animals tested (NTS, FOS, THBS1, and FN1). The upregulated genes are related to matrix remodeling (i.e. tissue proliferation), disturbance of angiogenesis, apoptosis, and oxidative stress response. We conclude that superstimulation treatment i) results in granulosa cells that lag behind in maturation and differentiation (most of the upregulated genes are markers of the follicular growth stage), ii) activates genes involved with the NFE2L2 oxidative stress response and endoplasmic reticulum stress response, and iii) disturbs angiogenesis.

Two and Three Wheeler industry in the Asian region is growing at a rapid pace, as commuting by these vehicles is more affordable and efficient, specially in the developing nations. However, tightening of emission legislations, aimed at creating a cleaner and healthier environment, has led to increasingly demanding efforts required in making more efficient engines / vehicles and also focus on effective aftertreatment systems. Catalytic converters, being the most preferred option for after-treatment solutions, play an important role in achieving the desired end results.

We examined whether progesterone (P4)-induced suppression of LH release in cattle can be overcome by an increased dose of exogenous gonadotropin-releasing hormone (GnRH) or pretreatment with estradiol (E2). In Experiment 1, postpubertal Angus-cross heifers (N = 32) had their 2 largest ovarian follicles ablated 5 d after ovulation. Concurrently, these heifers were all given a once-used, intravaginal P4-releasing insert (CIDR), and they were randomly assigned to be given either prostaglandin F(2alpha) (Low-P4) or no treatment (High-P4) at follicle ablation, and 12 h later. Six days after emergence of a new follicular wave, half of the heifers in each group (n = 8) were given either 100 or 200 microg of GnRH i.m. Plasma luteinizing hormone (LH) concentrations were higher in the Low- vs High-P4 groups, and in heifers given 200 vs 100 microg of GnRH (mean +/- SEM 15.4 +/- 2.2 vs 9.1 +/- 1.2, and 14.8 +/- 2.1 vs 9.8 +/- 1.4 ng/mL, respectively; P < or = 0.01). Ovulation rate was higher (P = 0.002) in the Low-P4 group (15/16) than in the High-P4 group (6/16), but it was not affected by GnRH dose (P = 0.4). In Experiment 2, heifers (n = 22) were treated similarly, except that 5.5 d after wave emergence, half of the heifers in each group were further allocated to be given either 0.25 mg estradiol benzoate i.m. or no treatment, and 8 h later, all heifers were given 100 microg GnRH i.m. Both groups treated with E2 (Low- and High-P4) and the Low-P4 group without E2 had higher peak plasma LH concentrations compared to the group with high P4 without E2 (12.6 +/- 1.8, 10.4 +/- 1.8, 8.7 +/- 1.3, and 3.9 +/- 1.2 ng/mL, respectively; (P < 0.04)). However, E2 pretreatment did not increase ovulation rates in response to GnRH (P = 0.6). In summary, the hypotheses that higher doses of GnRH will be more efficacious in inducing LH release and that exogenous E2 will increase LH release following treatment with GnRH were supported, but neither significantly increased ovulation rate.

The objective was to determine how maternal age influences the transcriptome of the dominant follicle during the preovulatory period. We tested the hypotheses that delayed ovulation in aged cows is associated with 1) altered gene expression of granulosa cells of the preovulatory follicle and 2) decreased synthesis of progesterone by granulosa cells of the preovulatory follicle. Granulosa cells of preovulatory follicles were obtained 24 h after LH treatment from aged Hereford cows (17.0 ± 2.5 years; n = 6) and their daughters (9.0 ± 0.6 years; n = 6), and compared using bovine specific microarrays (EMBV3, EmbryoGENE, Québec, QC, Canada). Results were confirmed by RT-qPCR. A total of 1340 genes or gene isoforms were expressed differentially (≥2-fold change; P ≤ 0.05) in aged cows v. their younger daughters. Differentially expressed up- and down-regulated genes were related to 1) LH response (↑RGS2, ↑SERPINE2, ↑PTGS2), 2) cellular differentiation and luteinization (↑TNFAIP6, ↑GADD45B, ↓VNN1), and 3) progesterone synthesis (↑STAR, ↑HSD3B2, ↑NR5A2, ↑NR4A1). Intra-follicular concentration of progesterone was lower (P

Our objective was to determine how follicular aging affects the distribution and content of mitochondrial population and ATP in in vivo-matured bovine oocytes. We hypothesised that in vivo-matured bovine oocytes obtained from aged follicles (84 h of gonadotropin starvation) have altered mitochondrial distribution and decreased cytoplasmic ATP content compared to those obtained immediately at the end of a superstimulatory protocol (no starvation). Follicular waves were synchronized by ablation 5 to 8 d after ovulation and a CIDR device was given. Starting on the day of wave emergence (Day 0), short FSH and FSH starvation groups (n = 5 heifers each) were given 8 doses of FSH im over 4 d and the long FSH group (n = 4) was given 14 doses over 7 d. Two doses of PGF were given on Day 4 (short FSH) or Day 7 (FSH starvation and long FSH groups), the CIDR was removed, and LH was given 24 h after second PGF treatment. The ovaries were removed 24 h later by colpotomy and cumulus-oocyte-complexes (COC) were collected from follicles ≥8 mm. Denuded oocytes were either stained with Mitotracker Deep Red FM and imaged by confocal microscopy or processed for ATP assay. Mitochondria numbers were assessed by segmentation of 3D datasets. Proportions of COC within each grade were compared using Fischer's exact test, and ATP and mitochondrial data were compared by analysis of variance. Short and long FSH groups had a greater proportion of Grade 1 expanded COC than the FSH starvation group (P = 0.02). The ATP content of oocytes (from expanded COC) tended to be higher in the long FSH group than short FSH (P = 0.09), and the FSH starvation group was intermediate. The ATP content of oocytes from compact COC did not differ among groups (P = 0.49). The proportion of mitochondrial clusters was highest (P = 0.01) and the proportion of individual mitochondria was lowest (P = 0.01) in the FSH starvation group compared to short and long FSH groups. Mitochondria from the long FSH and FSH starvation groups had twice the relative intensity compared to the short FSH group (P

The severe 'Capao Bonito' (CB) isolates of Citrus tristeza virus (CTV) have limited distribution in Brazil, but affect both sweet orange and rootstocks varieties, especially Pera sweet orange grafted on Rangpur lime. The CB complex has at least two different CTV types expressing different coat proteins, whose genes were cloned and expressed in Escherichia coli . These pro- teins, CB-104 and CB-22, were used as antigens, allowing us to obtain three groups of monoclonal antibodies (MAb). All CTV isolates from CB reacted strongly in DASI-ELISA with MAb 30 which seems to react with a conserved epitope. Whereas MAbs 30 and IC.04 react with conformational epitopes, MAbs 37 and 39 recognize linear epitopes on the viral coat protein. It is possible that MAbs 37, 39 and IC.04 can be used diagnostically, and may discriminate severe CB isolates.

We tested the hypotheses that extending the duration of follicular growth by superstimulation increases oocyte competence, and that FSH starvation at the end of superstimulatory treatment decreases oocyte competence. Heifers were randomly assigned to three groups: short FSH, FSH starvation, and long FSH (N = 8 per group). At 5 to 8 days after ovulation, follicle ablation was performed, and a progesterone-releasing device (CIDR) was placed intravaginally. Short FSH and FSH starvation groups were given eight doses of FSH im at 12-hour intervals, and the long FSH group was given 14 doses. PGF2α was administered twice (12 hours apart) and the CIDR was removed on Day 3 (Day 0 = wave emergence) in the short FSH group, and on Day 6 in the other two groups. Heifers were given LH 24 hours after CIDR removal and cumulus-oocyte complexes (COC) were collected 24 hours later. The COC were matured in vitro for 6 hours and fertilized in vitro; embryos were cultured for 10 days. A greater number of follicles ≥9 mm were detected in the long FSH group than in the FSH starvation and short FSH groups (25.4 ± 5.3, 11.0 ± 2.1, 10.6 ± 2.3, respectively; P < 0.03). A greater proportion of expanded COC were collected from the long FSH than from the FSH starvation group (P < 0.001), and the short FSH group was intermediate (93%, 54%, and 74%, respectively). The FSH starvation group had a greater proportion of poor quality oocytes than the short and long FSH groups (70%, 45%, and 33%, respectively; P < 0.001) and cleavage rate was lower (22%, 54%, and 56%, respectively; P = 0.003). The proportion of oocytes that developed into embryos (morulae and blastocysts on Day 9 after IVF) was also lower in the FSH starvation group than in the short and long FSH groups, (5% vs. 25% and 37%; P = 0.04); the latter two groups did not differ. The long FSH treatment resulted in 2.5 and 3.4 times more transferable embryos per animal (morulae and blastocysts) at Day 9 after IVF than the short FSH and FSH starvation groups (5.6, 2.5, and 1.7 embryos per heifer respectively; P = 0.04). In conclusion, extending the standard superstimulation protocol by 3 days enhanced the ovarian response to FSH treatment, and a period of FSH starvation after superstimulatory treatment compromised oocyte quality and the fertilization process.

We tested the hypotheses that extending the duration of follicular growth by superstimulation increases oocyte competence, and that FSH starvation at the end of superstimulatory treatment decreases oocyte competence. Heifers were allocated randomly to short FSH duration (n = 8), FSH starvation (n = 8), or long FSH duration (n = 8) groups. Five to 8 days after ovulation, transvaginal ultrasound-guided follicle ablation was done to synchronize follicle wave emergence, and a progesterone-releasing device (CIDR; Pfizer Animal Health, New York, NY, USA) was placed intravaginally. Short FSH and FSH starvation groups were given 8 doses of FSH (Folltropin-V; Bioniche Animal Health Inc., Belleville, ON, Canada) IM, whereas the long FSH group was given 14 doses of FSH at 12-h intervals, starting from the day of wave emergence (Day 0). Prostaglandin F2α (PGF) was administered twice, 12 h apart, on Day 3 in the short FSH group and on Day 6 in the other 2 groups. In all heifers, the CIDR was removed at the time of the second PGF treatment; pLH (Lutropin-V; Bioniche Animal Health Inc.) was given IM 24 h after CIDR removal, and cumulus–oocyte complexes (COC) were collected 24 h after pLH treatment. The COC were matured in vitro (6 h) and fertilized (IVF), and the embryos were cultured for 10 days. At 12 h after pLH, the long FSH group had a greater number of ≥9 mm follicles than the FSH starvation and short FSH groups (25.4 ± 5.3 v. 11.0 ± 2.1 and 10.6 ± 2.3, respectively; P

Growing dominant follicles at the time of selection from aged (n = 3; 15 ± 1.5 years) and young (n = 3; 6 ± 1.1 years) Hereford cows were compared using bovine-specific microarrays containing 40 000 targets. The objective of the study was to determine age-associated changes in transcriptome of granulosa cells at the time of dominant follicle selection. Cows were given prostaglandin F2α to cause ovulation (Day 0) and granulosa cells from dominant follicles were collected on Day 3 either by ultrasound-guided follicle aspiration or after ovariectomy. The mRNA was extracted, analyzed for quality, converted into antisense RNA, amplified, labelled with red and green florescent dyes, and hybridized with microarrays. Feature intensities were measured using Array-Pro software (Media Cybernetics Inc., Rockville, MD), and differentially expressed genes were obtained using FlexArray 1.6. A total of 169 transcripts were differentially expressed with a fold change of ≥2 (P ≤ 0.05) in aged cows v. young cows. Ingenuity System Pathway (IPA; Ingenuity Systems Inc., Redwood City, CA) analysis of these transcripts revealed that granulosa cells of aged cows exhibit (1) reduced capability to regulate gonadotropins [↓follistatin (FST), ↓inhibin beta A (INHBA), ↓inhibin beta B (INHBB)] and reduced responsiveness to gonadotropin-induced changes in cytoskeleton [↑tropomyosin 2 (TPM2), ↑actin gamma 2 (ACTG2), ↓tubulin beta] and extracellular matrix [↓tumor necrosis factor alpha-induced protein 6 (TNFAIP6), ↓versican (VCAN)], (2) inefficiency in processing lipids [↓low-density lipoprotein receptor (LDLR), ↓stearoyl-coenzyme A desaturase (SCD), ↑cluster of differentiation 36 (CD36), ↓sterol-C4-methyl oxidase-like (SC4MOL)] and synthesizing steroids [↓cytochrome P450, family 19, subfamily A, polypeptide 1 (CYP19A1), ↓cytochrome P450, family 51, subfamily A, polypeptide 1 (CYP51A1)], (3) decreased proliferation [↓proliferating cell nuclear antigen (PCNA)] and control of cell cycle check points [↓checkpoint kinase 1 (CHEK1), ↓centromere-associated protein E (CENPE)] and have poor intercellular communication [↓gap junction protein alpha 1 (GJA1)], and (4) higher expression of oxidative stress responsive genes [↑vanin-1 (VNN1), ↑vanin-2 (VNN2), and ↑glutathione peroxidase 3 (GPX3)]. A total of 6 transcripts: CYP19A1 (aromatase; P ≤ 0.1), VNN1 (P ≤ 0.05), INHBA (P ≥ 0.1), PCNA (P ≤ 0.001), TPM2 (P ≤ 0.1), and GJA1 (P ≤ 0.05) were selected to validate the microarray results via quantitative real-time PCR. In conclusion, granulosa cells of growing dominant follicles exhibit age-related changes in the transcriptome at the time of selection relative to young cows; changes that may explain follicle-associated loss of oocyte competence in aged cows. Research was supported by the Natural Sciences and Engineering Research Council of Canada (Ottawa, ON, Canada).

The objective was to determine the effect of the level of progesterone (P4) during the ovulatory wave on fertility. Low v. high progesterone concentrations which were expected to result in high v. low LH pulse-frequency were compared. Lactating, postpartum beef cows (n = 69) at random stages of the estrous cycle were given cloprostenol (PGF; Schering-Plough Animal Health, Pointe-Claire, QC, Canada) twice at an 11-day interval to synchronize ovulation (Day 0 = day of 2nd PGF). Cows were given 1.5 mg estradiol benzoate on Day 10 (7 days after expected ovulation). On Day 10, the Low-P4 group was given PGF and an intravaginal CueMate (Bioniche Animal Health, Belleville, ON, Canada) containing one P4 pod (expected to achieve a P4 plasma level of 1 ng mL–1). The High-P4 group also received a CueMate with one pod on Day 10, but no PGF was administered (i.e., cows were allowed to retain their CL for 4 days after expected emergence of the estradiol-induced wave). On Day 18, CueMates were removed and PGF was administered to both groups. Fifty-four to 56 h later, all animals received 12.5 mg of Lutropin-V (Bioniche) IM and were timed AI (TAI). Dominant follicle diameter was measured on Day 18 and at the time of AI. Pregnancy diagnosis was done by ultrasonography 27 days after AI to compare fertility between groups. Follicle diameter data were analyzed by Student's t-test and pregnancy rate data were compared by the chi-square test. The dominant follicle in the Low-P4 group was larger (P < 0.05) than in the high-P4 group on the day of the CueMate removal (Day 18) and on the day of the TAI (11.8 � 0.4 and 13.7 � 0.5 v. 13.3 � 0.6 and 15.3 � 0.6 in the High-P4 and Low-P4 groups, respectively). Pregnancy rate was similar (P = 0.16) between the groups (High-P4: 55.6% (20/36) and Low-P4: 44.4% (16/33)); however, a larger study is in progress to confirm these results. To conclude, Low-P4 resulted in a larger dominant follicle without any apparent effect on pregnancy rate. The project was funded by Saskatchewan ADF and NSERC. L. Pfeifer is funded through a CAPES scholarship. We acknowledge Schering-Plough and Bioniche for donation of pharmaceuticals.

In previous studies, giving eCG at CIDR removal significantly increased the pregnancy rate after timed-AI in beef cows. However, eCG is not universally available. Therefore, we tested the hypothesis that giving pFSH at CIDR removal might improve the pregnancy rate in a CIDR-based, Cosynch protocol in postpartum, suckled Bos taurus beef cows; a secondary objective was to compare pregnancy rates when GnRH v. pLH was used to synchronize wave emergence and ovulation. This work was conducted as two experiments (separate locations). All cows were given a CIDR (containing 1.9 g progesterone; Pfizer Animal Health, Montreal, QC, Canada) on Day 0 (without regard to stage of estrous cycle or cyclicity). On Day 7, CIDRs were removed, all cows were concurrently given 25 mg PGF (Lutalyse; Pfizer Animal Health, Groton, CT, USA), half were given 20 mg pFSH (Folltropin-V; Bioniche Animal Health, Belleville, ON, Canada), and all cows were timed-AI 54 h later (Day 9). In Experiment 1, 240 cows [94 � 10.8 days postpartum; body condition score (BCS: 1 = emaciated, 9 = obese; mean � SD): 5.8 � 0.4] were used; at CIDR insertion and AI, cows were allocated to receive either 100 µg GnRH (n = 160; Cystorelin, Merial Canada Inc., Victoriaville, QC, Canada) or 12.5 mg pLH (n = 80; Lutropin-V, Bioniche Animal Health). In Experiment 2, 109 cows (59.2 � 19.5 days postpartum; BCS: 5.6 � 1.1) were used; all received 100 µg GnRH (Cystorelin) at CIDR insertion and AI. In Experiment 1, three cows that lost their CIDR were excluded; pregnancy rates after timed-AI (logistic regression, backward selection: parity, pFSH, synchronizing treatment, and their interactions) were not different between cows given pFSH v. control cows (64.7 v. 65.2%; P > 0.80), nor between cows given GnRH v. pLH (62.7 v. 69.6%; P = 0.91). However, there was an interaction (P < 0.04) between parity and the synchronizing treatment; in primiparous cows, pregnancy rates were significantly lower in those given GnRH v. pLH (59.3 v. 83.3%). In Experiment 2, pregnancy rates after timed-AI (logistic regression, backward selection: parity, pFSH, and their interaction) were not different between cows given pFSH v. control cows (38.2 v. 42.6%; P > 0.6). In conclusion, our hypothesis was not supported; giving pFSH at CIDR removal did not significantly improve the pregnancy rate in a CIDR-based, Cosynch protocol in postpartum, suckled Bos taurus beef cows.

The objective was to investigate the use of Cue-Mate and porcine LH (pLH) or GnRH with or without presynchronization on pregnancy rate following timed AI (TAI) in Angus heifers (n = 462). Approximately half of the heifers (Control; n = 236) were treated at random stages of the estrous cycle, and the other half (Presynch; n = 226) received two injections of 500 �g cloprostenol (PGF: Estrumate�; Schering Plough Animal Health, Pointe-Claire, Quebec, Canada) 14 days apart; synchronization treatments were initiated 11 days after the second injection of PGF. On Day 0, heifers received an intravaginal progesterone device (Cue-Mate; Bioniche Animal Health, A/Asia Pty, Armidale, Australia) and were treated with 100 �g GnRH IM (Cystorelin�; Merial Canada Inc., Victoriaville, Quebec, Canada; n = 233) or 12.5 mg pLH (Lutropin-V�; Bioniche; n = 229). On Day 7, Cue-Mates were removed and heifers were given PGF IM. GnRH or pLH (same as the first treatment) was given concurrently with TAI on Day 9 (54–56 h after PGF) with frozen–thawed semen of one of 3 sires. Ultrasonographic examinations were performed in a subset of 182 heifers on Days 0 and 7 for CL and follicle development, and in all heifers on Days 41 to 49 for confirmation of pregnancy. Data were compared using CATMOD procedures in the Statistical Analysis System (SAS Institute, Inc., Cary, NC, USA). The proportion of cycling heifers on Day 0 was 90.7% (165/182). Pregnancy rate tended to differ among bulls (46.7, 56.9, and 61.4% for Bulls A, B, and C, respectively; P≤ 0.1). Heifers that ovulated in response to the first GnRH or pLH injection had a higher pregnancy rate than those that did not ovulate (66.3 vs. 51.9%; P ≤ 0.03). In addition, heifers treated with GnRH tended to have a higher ovulatory response to the first treatment and a higher pregnancy rate to TAI than those treated with pLH (60.9 and 60.5% vs. 50.0 and 52.4%, respectively; P ≤ 0.09). Although ovulatory response to the first GnRH or pLH treatment was 46.5 and 63.5% for Control and Presynch groups, respectively (P ≤ 0.02), pregnancy rates did not differ (59.8 vs. 53.1%; P ≥ 0.2). However, there was an interaction between presynchronization treatment and ovulatory response to the first injection of GnRH or pLH on pregnancy rates (P ≤ 0.02). The pregnancy rate was higher in Control heifers that ovulated (77.5%) to the first injection of GnRH or pLH than in Control heifers that did not ovulate (52.2%) or Presynch heifers that did (59.0%) or did not (51.4%) ovulate. In summary, Cue-Mate-treated heifers that ovulated in response to the first GnRH or pLH treatment had higher pregnancy rates to TAI. Although presynchronization with PGF increased the ovulation rate, it did not significantly affect the pregnancy rate.