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The skin and its microbiome function to protect the host from pathogen colonization and environmental stressors. In this study, using the Wisconsin Miniature Swine™ model, we characterize the porcine skin fungal and bacterial microbiomes, identify bacterial isolates displaying antifungal activity, and use whole-genome sequencing to identify biosynthetic gene clusters encoding for secondary metabolites that may be responsible for the antagonistic effects on fungi. Through this comprehensive approach of paired microbiome sequencing with culturomics, we report the discovery of novel species of Corynebacterium and Rothia. Further, this study represents the first comprehensive evaluation of the porcine skin mycobiome and the evaluation of bacterial-fungal interactions on this surface. Several diverse bacterial isolates exhibit potent antifungal properties against opportunistic fungal pathogens in vitro. Genomic analysis of inhibitory species revealed a diverse repertoire of uncharacterized biosynthetic gene clusters suggesting a reservoir of novel chemical and biological diversity. Collectively, the porcine skin microbiome represents a potential unique source of novel antifungals., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

Human papillomaviruses (HPVs) are the most common sexually transmitted infection in the United States and are a major etiological agent of cancers in the anogenital tract and oral cavity. Growing evidence suggests changes in the host microbiome are associated with the natural history and ultimate outcome of HPV infection. We sought to define changes in the host cervicovaginal microbiome during papillomavirus infection, persistence, and pathogenesis using the murine papillomavirus (MmuPV1) cervicovaginal infection model. Cervicovaginal lavages were performed over a time course of MmuPV1 infection in immunocompetent female FVB/N mice and extracted DNA was analyzed by qPCR to track MmuPV1 viral copy number. 16S ribosomal RNA (rRNA) gene sequencing was used to determine the composition and diversity of microbial communities throughout this time course. We also sought to determine whether specific microbial communities exist across the spectrum of MmuPV1-induced neoplastic disease. We, therefore, performed laser-capture microdissection to isolate regions of disease representing all stages of neoplastic disease progression (normal, low- and high-grade dysplasia, and cancer) from female reproductive tract tissue sections from MmuPV1-infected mice and performed 16S rRNA sequencing. Consistent with other studies, we found that the natural murine cervicovaginal microbiome is highly variable across different experiments. Despite these differences in initial microbiome composition between experiments, we observed that MmuPV1 persistence, viral load, and severity of disease influenced the composition of the cervicovaginal microbiome. These studies demonstrate that papillomavirus infection can alter the cervicovaginal microbiome.IMPORTANCEHuman papillomaviruses (HPVs) are the most common sexually transmitted infection in the United States. A subset of HPVs that infect the anogenital tract (cervix, vagina, anus) and oral cavity cause at least 5% of cancers worldwide. Recent evidence indicates that the community of microbial organisms present in the human cervix and vagina, known as the cervicovaginal microbiome, plays a role in HPV-induced cervical cancer. However, the mechanisms underlying this interplay are not well-defined. In this study, we infected the female reproductive tract of mice with a murine papillomavirus (MmuPV1) and found that key aspects of papillomavirus infection and disease influence the host cervicovaginal microbiome. This is the first study to define changes in the host microbiome associated with MmuPV1 infection in a preclinical animal model of HPV-induced cervical cancer. These results pave the way for using MmuPV1 infection models to further investigate the interactions between papillomaviruses and the host microbiome., Competing Interests: The authors declare no conflict of interest.

Porcine models are frequently used for burn healing studies; however, factors including anatomic location and lack of standardised wound methods can impact the interpretation of wound data. The objectives of this study are to examine the influence of anatomical locations on the uniformity of burn creation and healing in porcine burn models. To optimise burn parameters on dorsal and ventral surfaces, ex vivo and in situ euthanized animals were first used to examine the location-dependence of the burn depth and contact time relationship. The location-dependent healing in vivo was then examined using burn and excisional wounds at dorsal, ventral, caudal and cranial locations. Lactate dehydrogenase (LDH) and H&E were used to assess burn depth and wound re-epithelialization. We found that burn depth on the ventral skin was significantly deeper than that of the dorsal skin at identical thermal conditions. Compared with burns created ex vivo, burns created in situ immediately post-mortem were significantly deeper in the ventral location. In live animals, 2 out of 12 burn wounds were fully re-epithelialized after 14 days in contrast to complete re-epithelialization of all excisional wounds. Among the burn wounds, those at the cranial-dorsal site exhibited faster healing than at the caudal-dorsal site. This study showed that anatomical location is an important consideration for the consistency of burn depth creation and healing. These data support symmetric localization of treatment and control for comparative assessment of burn healing in porcine models to prevent misinterpretation of results and increase the translatability of findings to humans., (© 2024 The Author(s). Wound Repair and Regeneration published by Wiley Periodicals LLC on behalf of The Wound Healing Society.)

Slough is a well-known feature of non-healing wounds. This pilot study aims to determine the proteomic and microbiologic components of slough as well as interrogate the associations between wound slough components and wound healing. Ten subjects with slow-to-heal wounds and visible slough were enrolled. Aetiologies included venous stasis ulcers, post-surgical site infections and pressure ulcers. Patient co-morbidities and wound healing outcome at 3-months post-sample collection was recorded. Debrided slough was analysed microscopically, through untargeted proteomics, and high-throughput bacterial 16S-ribosomal gene sequencing. Microscopic imaging revealed wound slough to be amorphous in structure and highly variable. 16S-profiling found slough microbial communities to associate with wound aetiology and location on the body. Across all subjects, slough largely consisted of proteins involved in skin structure and formation, blood-clot formation and immune processes. To predict variables associated with wound healing, protein, microbial and clinical datasets were integrated into a supervised discriminant analysis. This analysis revealed that healing wounds were enriched for proteins involved in skin barrier development and negative regulation of immune responses. While wounds that deteriorated over time started off with a higher baseline Bates-Jensen Wound Assessment Score and were enriched for anaerobic bacterial taxa and chronic inflammatory proteins. To our knowledge, this is the first study to integrate clinical, microbiome, and proteomic data to systematically characterise wound slough and integrate it into a single assessment to predict wound healing outcome. Collectively, our findings underscore how slough components can help identify wounds at risk of continued impaired healing and serves as an underutilised biomarker., (© 2024 The Authors. Wound Repair and Regeneration published by Wiley Periodicals LLC on behalf of The Wound Healing Society.)

For decades research has centered on identifying the ideal balanced skin microbiome that prevents disease and on developing therapeutics to foster this balance. However, this single idealized balance may not exist. The skin microbiome changes across the lifespan. This is reflected in the dynamic shifts of the skin microbiome's diverse, inter-connected community of microorganisms with age. While there are core skin microbial taxa, the precise community composition for any individual person is determined by local skin physiology, genetics, microbe-host interactions, and microbe-microbe interactions. As a key interface with the environment, the skin surface and its appendages are also constantly exchanging microbes with close personal contacts and the environment. Hormone fluctuations and immune system maturation also drive age-dependent changes in skin physiology that support different microbial community structures over time. Here, we review recent insights into the factors that shape the skin microbiome throughout life. Collectively, the works summarized within this review highlight how, depending on where we are in lifespan, our skin supports robust microbial communities, while still maintaining microbial features unique to us. This review will also highlight how disruptions to this dynamic microbial balance can influence risk for dermatological diseases as well as impact lifelong health., (© 2023 The Author(s).)

The gut and liver are connected via the portal vein, and this relationship, which includes the gut microbiome, is described as the gut-liver axis. Hepatitis C virus (HCV) can infect the liver and cause fibrosis with chronic infection. HCV has been associated with an altered gut microbiome; however, how these changes impact metabolism across the gut-liver axis and how this varies with disease severity and time is unclear. Here we used multi-omics analysis of portal and peripheral blood, faeces and liver tissue to characterize the gut-liver axis of patients with HCV across a fibrosis severity gradient before (n = 29) and 6 months after (n = 23) sustained virologic response, that is, no detection of the virus. Fatty acids were the major metabolites perturbed across the liver, portal vein and gut microbiome in HCV, especially in patients with cirrhosis. Decreased fatty acid degradation by hepatic peroxisomes and mitochondria was coupled with increased free fatty acid (FFA) influx to the liver via the portal vein. Metatranscriptomics indicated that Anaerostipes hadrus-mediated fatty acid synthesis influences portal FFAs. Both microbial fatty acid synthesis and portal FFAs were associated with enhanced hepatic fibrosis. Bacteroides vulgatus-mediated intestinal glycan breakdown was linked to portal glycan products, which in turn correlated with enhanced portal inflammation in HCV. Paired comparison of patient samples at both timepoints showed that hepatic metabolism, especially in peroxisomes, is persistently dysregulated in cirrhosis independently of the virus. Sustained virologic response was associated with a potential beneficial role for Methanobrevibacter smithii, which correlated with liver disease severity markers. These results develop our understanding of the gut-liver axis in HCV and non-HCV liver disease aetiologies and provide a foundation for future therapies., (© 2022. This is a U.S. Government work and not under copyright protection in the US; foreign copyright protection may apply.)

Background: Increased microbial translocation (MT) into the systemic circulation is associated with liver disease progression. Microbial translocation has yet to be completely defined in chronic hepatitis B virus (HBV) and chronic hepatitis delta virus (HDV)., Methods: Our aim was to characterize MT and associated immune response in chronic HBV and HDV at various stages of disease. Serum from 53 HBV, 43 HDV, and 36 healthy control (HC) subjects was obtained. Subjects were categorized by aspartate aminotransferase-to-platelet ratio index into mild (<0.5), moderate, and severe (>1.0) disease. Cytokines, microbial products, and microbial deoxyribonucleic acid (DNA) levels were assessed in a single treatment-naive time point for each patient. Next-generation sequencing identified bacterial species present within patient sera., Results: The HBV and HDV subjects display higher serum concentrations of Gram-negative (G - ) bacterial lipopolysaccharide and fungal beta-glucan compared with HC (all P < .01). Gram-positive (G + ) bacterial peptidoglycan is higher in HBV compared to HDV and HC (both P < .0001). Within both disease cohorts, peptidoglycan correlates with interleukin (IL)-1b, IL-8, IL-12p70, and IL-13 (all Spearman's rho >0.45; P < .05). Next-generation sequencing from 7 subjects with detectable serum bacterial DNA revealed changes in abundance of bacterial taxa and a higher proportion of Gram-positive genera in severe disease. Greater G + /G - taxa ratio is associated with higher cytokine levels and disease markers., Conclusions: The HBV and HDV patients display increased translocation of bacterial and fungal products into serum. An increased proportion of Gram-positive genera is associated with disease progression. Correlations of peptidoglycan with antimicrobial cytokines suggest that particular microbial classes may contribute to systemic inflammation and possibly disease progression., (Published by Oxford University Press on behalf of Infectious Diseases Society of America 2020.)

From 2010 to 2015, 73 common marmosets ( Callithrix jacchus ) housed at the Wisconsin National Primate Research Center (WNPRC) were diagnosed postmortem with lymphocytic enterocolitis. We used unbiased deep-sequencing to screen the blood of deceased enterocolitis-positive marmosets for viruses. In five out of eight common marmosets with lymphocytic enterocolitis, we discovered a novel pegivirus not present in ten matched, clinically normal controls. The novel virus, which we named Southwest bike trail virus (SOBV), is most closely related (68% nucleotide identity) to a strain of simian pegivirus A isolated from a three-striped night monkey ( Aotus trivirgatus ). We screened 146 living WNPRC common marmosets for SOBV, finding an overall prevalence of 34% (50/146). Over four years, 85 of these 146 animals died or were euthanized. Histological examination revealed 27 SOBV-positive marmosets from this cohort had lymphocytic enterocolitis, compared to 42 SOBV-negative marmosets, indicating no association between SOBV and disease in this cohort ( p = 0.0798). We also detected SOBV in two of 33 (6%) clinically normal marmosets screened during transfer from the New England Primate Research Center, suggesting SOBV could be exerting confounding influences on comparisons of common marmoset studies from multiple colonies.

Background: Thrombocytopenia is a hallmark of advanced liver disease. Platelets, growth factors (GFs), and vascular integrity are closely linked factors in disease pathogenesis, and their relationship, particularly in early disease stages, is not entirely understood. The aim was to compare circulating platelets, growth factors, and vascular injury markers (VIMs) in hepatitis C-infected (HCV) patients with early fibrosis and cirrhosis., Methods: Retrospective evaluation of serum GFs and VIMs by ELISA were evaluated from twenty-six HCV patients. Analytes from an earlier time-point were correlated with MELD at a later time-point., Results: Platelets and GFs decreased, and VIMs increased with fibrosis. Platelets correlated positively with PDGF-AA, PDGF-BB, TGFB1, EGF, and P-selectin, and negatively with ICAM-3 and VCAM-1. P-selectin showed no correlations with VIMs but positively correlated with PDGF-AA, PDGF-BB, TGFB1, and EGF. Soluble VCAM-1 and ICAM-3 were linked to increasing fibrosis, liver enzymes, and synthetic dysfunction. Higher VCAM-1 and ICAM-3 and lower P-selectin at an earlier time-point were linked to higher MELD score at a later time-point., Conclusion: In chronic HCV, progressive decline in platelets and growth factors with fibrosis and their associations suggest that platelets are an important source of circulating GFs and influence GF decline with fibrosis. Enhanced markers of vascular injury in patients with early fibrosis suggest an earlier onset of endothelial dysfunction preceding cirrhosis. Associations of VIMs with platelets suggest a critical link between platelets and vascular homeostasis. Circulating markers of vascular injury may not only have prognostic importance but emphasize the role of vascular dysfunction in liver disease pathogenesis (NCT00001971).

Hepatitis C virus (HCV) infects 71 million individuals, and barriers to treatment remain. Bacterial translocation is a complication of chronic HCV infection, and this study evaluated circulating microbial components including lipopolysaccharide, peptidoglycan, and β-D-glucan in addition to their pattern recognition receptors and degree of hepatic macrophage uptake. The findings suggest that regulation of serum peptidoglycan and β-D-glucan differs from that of lipopolysaccharide. Additionally, macrophage activation in the liver may be better reflected by the degree of macrophage uptake than by circulating levels of microbial markers. These findings allow for a greater understanding of bacterial translocation and host immune activation during HCV infection., (Published by Oxford University Press on behalf of Infectious Diseases Society of America 2019.)

Background and Aim: Hepatitis delta virus (HDV) infection is the most rapidly progressive chronic viral hepatitis. Little is understood about the immune responses to HDV. This study aims to characterize the systemic immune environments of hepatitis B virus (HBV) and HDV patients at various disease stages., Methods: A total of 129 subjects were evaluated: 53 HBV, 43 HDV, and 33 healthy controls. HBV and HDV subjects were categorized by aspartate aminotransferase to platelet ratio index (APRI) into mild (APRI < 0.5), moderate, and severe (APRI > 1.0). Serum cytokines and immune markers were assessed at a single treatment-naïve time-point., Results: Type 1 cytokines are elevated in both HBV and HDV. Both groups show higher tumor necrosis factor-α (TNF-α), interleukin (IL)-12p40, and C-X-C motif chemokine ligand 9 when compared with controls (all P < 0.05). However, only HBV group displayed elevated γ-interferon compared with controls. Type 2 cytokines are elevated in HBV. HBV group shows higher IL-4, IL-13, and C-C motif chemokine ligand (CCL) 26 compared with healthy controls and HDV. Chemokines CCL2 and CCL13 are lower in HDV. When assessing ratios, HDV displays higher γ-interferon/IL-4, TNF-α/IL-4, and TNF-α/IL-13 ratios than HBV and controls., Conclusion: Hepatitis B virus and HDV subjects show similarly elevated type 1 cytokines. HDV subjects display relatively lower type 2 cytokines. These differences in the systemic immune environments, particularly the predominance of type 1 responses, may contribute to the comparatively rapid progression of HDV disease. Characterization of the imbalance in type 1 and type 2 immunity unique HDV has the potential to provide immunological insights for designing therapeutic targets in HDV-associated disease progression., (© 2019 Journal of Gastroenterology and Hepatology Foundation and John Wiley & Sons Australia, Ltd.)

Ubiquitination controls the stability of most cellular proteins, and its deregulation contributes to human diseases including cancer. Deubiquitinases remove ubiquitin from proteins, and their inhibition can induce the degradation of selected proteins, potentially including otherwise 'undruggable' targets. For example, the inhibition of ubiquitin-specific protease 7 (USP7) results in the degradation of the oncogenic E3 ligase MDM2, and leads to re-activation of the tumour suppressor p53 in various cancers. Here we report that two compounds, FT671 and FT827, inhibit USP7 with high affinity and specificity in vitro and within human cells. Co-crystal structures reveal that both compounds target a dynamic pocket near the catalytic centre of the auto-inhibited apo form of USP7, which differs from other USP deubiquitinases. Consistent with USP7 target engagement in cells, FT671 destabilizes USP7 substrates including MDM2, increases levels of p53, and results in the transcription of p53 target genes, induction of the tumour suppressor p21, and inhibition of tumour growth in mice.

We report herein the design, synthesis, and evaluation of macrocyclic peptidomimetics that bind to WD repeat domain 5 (WDR5) and block the WDR5-mixed lineage leukemia (MLL) protein-protein interaction. Compound 18 (MM-589) binds to WDR5 with an IC 50 value of 0.90 nM (K i value <1 nM) and inhibits the MLL H3K4 methyltransferase (HMT) activity with an IC 50 value of 12.7 nM. Compound 18 potently and selectively inhibits cell growth in human leukemia cell lines harboring MLL translocations and is >40 times better than the previously reported compound MM-401. Cocrystal structures of 16 and 18 complexed with WDR5 provide structural basis for their high affinity binding to WDR5. Additionally, we have developed and optimized a new AlphaLISA-based MLL HMT functional assay to facilitate the functional evaluation of these designed compounds. Compound 18 represents the most potent inhibitor of the WDR5-MLL interaction reported to date, and further optimization of 18 may yield a new therapy for acute leukemia.

Fms-like tyrosine kinase 3 internal tandem duplication (FLT3-ITD) is frequently detected in acute myeloid leukemia (AML) patients and is associated with a dismal long-term prognosis. FLT3 tyrosine kinase inhibitors provide short-term disease control, but relapse invariably occurs within months. Pim protein kinases are oncogenic FLT3-ITD targets expressed in AML cells. We show that increased Pim kinase expression is found in relapse samples from AML patients treated with FLT3 inhibitors. Ectopic Pim-2 expression induces resistance to FLT3 inhibition in both FLT3-ITD-induced myeloproliferative neoplasm and AML models in mice. Strikingly, we found that Pim kinases govern FLT3-ITD signaling and that their pharmacological or genetic inhibition restores cell sensitivity to FLT3 inhibitors. Finally, dual inhibition of FLT3 and Pim kinases eradicates FLT3-ITD(+) cells including primary AML cells. Concomitant Pim and FLT3 inhibition represents a promising new avenue for AML therapy.

AMPK is a master regulator of cellular metabolism that exerts either oncogenic or tumor suppressor activity depending on context. Here, we report that the specific AMPK agonist GSK621 selectively kills acute myeloid leukemia (AML) cells but spares normal hematopoietic progenitors. This differential sensitivity results from a unique synthetic lethal interaction involving concurrent activation of AMPK and mTORC1. Strikingly, the lethality of GSK621 in primary AML cells and AML cell lines is abrogated by chemical or genetic ablation of mTORC1 signaling. The same synthetic lethality between AMPK and mTORC1 activation is established in CD34-positive hematopoietic progenitors by constitutive activation of AKT or enhanced in AML cells by deletion of TSC2. Finally, cytotoxicity in AML cells from GSK621 involves the eIF2α/ATF4 signaling pathway that specifically results from mTORC1 activation. AMPK activation may represent a therapeutic opportunity in mTORC1-overactivated cancers., (Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.)

Down syndrome confers a 20-fold increased risk of B cell acute lymphoblastic leukemia (B-ALL), and polysomy 21 is the most frequent somatic aneuploidy among all B-ALLs. Yet the mechanistic links between chromosome 21 triplication and B-ALL remain undefined. Here we show that germline triplication of only 31 genes orthologous to human chromosome 21q22 confers mouse progenitor B cell self renewal in vitro, maturation defects in vivo and B-ALL with either the BCR-ABL fusion protein or CRLF2 with activated JAK2. Chromosome 21q22 triplication suppresses histone H3 Lys27 trimethylation (H3K27me3) in progenitor B cells and B-ALLs, and 'bivalent' genes with both H3K27me3 and H3K4me3 at their promoters in wild-type progenitor B cells are preferentially overexpressed in triplicated cells. Human B-ALLs with polysomy 21 are distinguished by their overexpression of genes marked with H3K27me3 in multiple cell types. Overexpression of HMGN1, a nucleosome remodeling protein encoded on chromosome 21q22 (refs. 3,4,5), suppresses H3K27me3 and promotes both B cell proliferation in vitro and B-ALL in vivo.

Here we report a comprehensive characterization of our recently developed inhibitor MM-401 that targets the MLL1 H3K4 methyltransferase activity. MM-401 is able to specifically inhibit MLL1 activity by blocking MLL1-WDR5 interaction and thus the complex assembly. This targeting strategy does not affect other mixed-lineage leukemia (MLL) family histone methyltransferases (HMTs), revealing a unique regulatory feature for the MLL1 complex. Using MM-401 and its enantiomer control MM-NC-401, we show that inhibiting MLL1 methyltransferase activity specifically blocks proliferation of MLL cells by inducing cell-cycle arrest, apoptosis, and myeloid differentiation without general toxicity to normal bone marrow cells or non-MLL cells. More importantly, transcriptome analyses show that MM-401 induces changes in gene expression similar to those of MLL1 deletion, supporting a predominant role of MLL1 activity in regulating MLL1-dependent leukemia transcription program. We envision broad applications for MM-401 in basic and translational research., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Mixed lineage leukemia 1 (MLL1) is a histone H3 lysine 4 (H3K4) methyltransferase, and targeting the MLL1 enzymatic activity has been proposed as a novel therapeutic strategy for the treatment of acute leukemia harboring MLL1 fusion proteins. The MLL1/WDR5 protein-protein interaction is essential for MLL1 enzymatic activity. In the present study, we designed a large number of peptidomimetics to target the MLL1/WDR5 interaction based upon -CO-ARA-NH-, the minimum binding motif derived from MLL1. Our study led to the design of high-affinity peptidomimetics, which bind to WDR5 with K(i) < 1 nM and function as potent antagonists of MLL1 activity in a fully reconstituted in vitro H3K4 methyltransferase assay. Determination of co-crystal structures of two potent peptidomimetics in complex with WDR5 establishes their structural basis for high-affinity binding to WDR5. Evaluation of one such peptidomimetic, MM-102, in bone marrow cells transduced with MLL1-AF9 fusion construct shows that the compound effectively decreases the expression of HoxA9 and Meis-1, two critical MLL1 target genes in MLL1 fusion protein mediated leukemogenesis. MM-102 also specifically inhibits cell growth and induces apoptosis in leukemia cells harboring MLL1 fusion proteins. Our study provides the first proof-of-concept for the design of small-molecule inhibitors of the WDR5/MLL1 protein-protein interaction as a novel therapeutic approach for acute leukemia harboring MLL1 fusion proteins., Competing Interests: Notes The authors declare the following competing financial interest(s): Asentage has licensed the technology related to this manuscript. Shaomeng Wang is a co-founder for Ascentage, owns stocks in Ascentage, and serves as a consultant for Ascentage.

MLL1 is a histone 3 lysine 4 (H3K4) methyltransferase and a promising new cancer therapeutic target. The catalytic activity of MLL1 is regulated by the formation of a core complex consisting of MLL1, WDR5, RbBP5, and Ash2L. The interaction between WDR5 and MLL1 plays an essential role in regulation of the H3K4 methyltransferase activity of MLL1 and targeting this interaction using small molecules may represent an attractive therapeutic strategy. In this study, we have defined the essential elements in MLL1 required for its high-affinity binding to WDR5. Our data showed that the minimal elements crucial for high-affinity binding of MLL1 to WDR5 are -CO-ARA-NH- motif and two intramolecular hydrogen bonds that stabilize the conformation of this motif. Two 3-mer peptides, Ac-ARA-NH(2) and Ac-ART-NH(2), were designed based upon MLL1 and H3 sequences and achieved K(i) values of 120 and 20 nM to WDR5, respectively. Our study provides a concrete basis for the design of potent peptidomimetics and nonpeptidic compounds to inhibit MLL1 activity by targeting the MLL1 and WDR5 interaction.

To establish the effect of sward height (SH) and concentrate supplementation on performance of grazing cattle, 24 crossbred Angus beef cows (535 kg BW) and calves (114 kg BW) were grouped by weight and calving date. They were randomly assigned to two SH treatments, either 4 to 8 cm or 8 to 11 cm, and fed three levels of supplement, high, low, or none, consisting of 6.24, 3.12, and 0 kg x animal(-1) x d(-1), respectively. The experiment was repeated over three 15-d periods in 1996: May (P1), June/July (P2), and August (P3). No SH x supplement level x period or SH x supplement level interactions (P > 0.10) were evident for responses tested. Cows on lower SH had greater (P < 0.08) DMI but spent an additional 1.3 h/d (P < 0.01) grazing compared with cows on higher SH. Sward height had no influence (P > 0.10) on forage DM digestibility (DMD). Forage DMI, DMD, and grazing time (GT) decreased (P < 0.05) as supplementation increased. Nonetheless, supplemented cows consumed more total DMI (P < 0.08) than unsupplemented cows. Cows consumed 2.4 kg/d more forage DM (P < 0.01) in P1 and P2 than in P3. Cows grazed 1.3 h/d (P < 0.01) less in P1 than in P2 and P3. Grazing efficiency (DMI/h GT) declined as supplementation increased and grazing season advanced to P3 (P < 0.01). Decreased forage DMI and grazing efficiency with increasing supplementation suggests that supplemented cattle should be able to maintain productivity while grazing at SH lower than unsupplemented cattle.

Cyclic Spanish Merino ewes were treated on Day 13 of the estrous cycle with 12 mg, i.m., FSH-P in saline (n = 9) or propylene glycol (n = 24), currently with 100 micrograms, i.m., Cloprostenol (Day 0). From Day-6 to Day 0, the ewes were observed daily by transrectal ultrasonography, after Day 0, ultrasonography was performed every 12 h for 72 h. Sizes and locations of > or = 2 mm follicles were recorded at each observation. The ovulation rate was determined by laparoscopy on Day 7 after estrus. The number of ovulations ranged from 0 to 6 in ewes treated with FSH-P in saline and from 0 to 16 in ewes receiving FSH-P in propylene glycol (P < 0.05). In the latter group, the response was bimodally distributed; about half of the females had 1 ovulation, whereas the remainder had > 4 with a mean of 7 ovulations. The ovulation rate was associated with 2 characteristics of the largest follicle present at treatment (Day 0). First, if the largest follicle on Day 0 had not changed in diameter from Day-1 to Day 0, then 7 of 9 ewes had > 3 ovulations; if the largest follicle had either increased or decreased, only 8 of 24 ewes had > 3 ovulations (P < 0.05). Second, there was a linear trend (P < 0.07) for ovulation rate to decrease as the persistence of the largest follicle at treatment increased; no ewe in which the largest follicle on Day 0 remained present for more than 36 h ovulated more than 6 follicles. As with the ovulation rate, the numbers of large follicles on Days 1.5, 2 and 2.5 varied with the interaction of change in diameter of the largest follicle on Day 0 from Day-1 to Day 0 and with vehicle. In summary, the superovulatory response was affected by the change in diameter from Day-1 to Day 0 of the largest follicle on Day 0 and the period required for that follicle to regress after treatment with FSH-P and cloprostenol.

Ovarian follicular waves were characterized before and after breeding in 52 lactating beef cows and 16 heifers. Effects of two (2 W) or three (3 W) waves of follicular development and associated patterns of concentrations of steroids in jugular serum on pregnancy rate were examined. Animals were observed for oestrus (= day 0) twice daily and inseminated artificially at second oestrus. Follicular development was monitored by ultrasonography and jugular blood samples were collected simultaneously on alternate days from day 6 after first oestrus until ovulation after the second oestrus and from day 6 after second oestrus (insemination) until next ovulation or day 24 of pregnancy. Pregnancy was determined by ultrasonography at 25 days after insemination. From individual patterns of growth or regression of the largest follicles, more of the 59 animals with oestrous cycles of 17 to 25 days had 2 W (51, 86%) than 3 W (8, 14%) during the oestrous cycle before breeding (P < 0.01). Cycles averaged 1.1 days longer (P < 0.10) and corpora lutea regressed later (P < 0.01) in animals with 3 W vs. those with 2 W, but mean oestradiol between 7 and 2 days before second oestrus (3.7 +/- 0.3 pg ml-1) did not differ between cycles with 2 W or 3 W. Ovulatory follicles in animals with 2 W differed from those with 3 W (P < 0.05) in day of detection (12.3 +/- 0.3 vs 16.5 +/- 0.5), growth rate (1.0 +/- 0.1 vs 1.5 +/- 0.1 mm day-1), interval from detection to ovulation (9.3 +/- 0.3 vs 6.3 +/- 0.7 days) and duration of dominance (4.0 +/- 0.2 vs 2.1 +/- 0.6 days). Pregnancy rates, 82% in cows with 2 W and 100% in cows with 3 W, did not differ (P > 0.05). During the period equivalent to an oestrous cycle after breeding, 29 (49%) of the 59 animals had 2 W and 30 (51%) had 3 W. Fewer animals with 2 W than 3 W after breeding became pregnant (16/23, 70% vs 26/27, 96%; P < 0.05), but patterns of concentrations of progesterone on days 6 through 14 or mean oestradiol on day 14 (2.6 +/- 0.2 pg ml-1) did not differ. In conclusion, fewer animals had 3 W than 2 W before breeding and fertility did not differ. During the equivalent of one oestrous cycle after breeding, approximately equal numbers of animals had 2 W or 3 W and fertility was greater for animals with 3 W.

Ovarian follicular dynamics were monitored in 12 surgically modified red deer hinds (ovaries adhered to vaginal wall) by transvaginal real-time ultrasonography during the luteal cycle, anoestrus and induction of superovulation. All 12 hinds showed evidence of regular luteal (plasma progesterone) cyclicity during the breeding season, although luteal tissue was not observed on the ultrasonograms. During the normal luteal cycle (14-22 days) total numbers of follicles > 3 mm did not vary significantly by day (range of means: 1.8-3.4; P > 0.05). A single large (> or = 6 mm) follicle was usually present on all days except immediately after ovulation (day 0). However, the appearance of new follicles (> or = 3 mm) was not random, and was greatest on day 1 and day 14 (P < 0.05). Tracking of individual follicles revealed irregular waves of emergence and disappearance of the largest follicle, with either one (n = 1), two (n = 3) or three (n = 5) waves observed across nine luteal cycles. New follicles (> or = 3 mm) emerged after regression or ovulation of a large follicle, suggesting a dominance effect. There were no significant differences in the overall mean numbers of follicles during early, mid- and late anoestrus (September, November and April, respectively) but follicle turnover was more rapid during mid-anoestrus as evidenced by a significantly greater number of new small (> 3 mm) follicles (P < 0.001). Administration of superovulatory doses of ovine FSH during the breeding season resulted in a marked increase in the appearance of new follicles within 48 h of initiation of the injection regimen. By termination at 96 h, the time of progesterone withdrawal, the mean number of follicles > 3 mm was significantly higher than for control hinds (9.8 versus 3.0; P < 0.0001). While most follicles ovulated progressively 2-7 days later, about 40% persisted beyond this period. The study demonstrated the presence of discrete patterns of antral follicle growth and regression during the breeding and non-breeding seasons, with the luteal cycle characterized by a variable number (1-3) of dominant follicle waves. Anoestrus represents a period of dynamic changes in follicular turnover.

Growth and regression of ovarian follicles with antral diameters > or = 2 mm were characterized during 15 estrous cycles by daily transrectal ultrasonography (7.5 MHz probe) in 9 ewes of Merino del Pais, a consistently monovular Spanish breed. Mean interovulatory interval was 17.5 +/- 0.5 days and ovulation rate was 1 in all ewes; of 60 to 116 follicles, > or = 2 mm observed during the entire estrous cycle, 13.0 +/- 1.2 reached a maximum diameter > or = 4 mm and 7.9 +/- 0.6 different follicles became the largest follicle in the animal at some point during the cycle. An average of 4.5 new follicles per ewe were detected each day, with no significant effect of day of cycle. Appearance of new follicles that grew to > or = 4 mm tended to differ during the first 8 days of the cycle, being highest on day 3 and lowest on day 6 (P < 0.10), but did not vary significantly during the last 6 days. Growth of new follicles from the day of detection to the next day differed between, but not within, periods, averaging 1.4 +/- 0.3 mm of the first 8 days of the cycle and 1.8 +/- 0.5 mm from day -6 through -1 (P < 0.05). Total number of follicles > or = 2 mm per ovary on days 1 through 8 varied with the interaction of ovary by day, being more variable in the non-CL ovary. During the last 7 days, a linear decline in total follicles was coupled with a linear increase in number of large follicles (P < 0.05). Differences in the size between the largest and second largest follicles were greater on days 5 through 8 than on days 1 through 4, did not differ with day of cycle on days -6 through -1, then increased on the last day from 1.5 mm to 2.9 mm (P < 0.001). In conclusion, the monovular Merino del Pais ewe showed a more rapid growth and turnover of ovarian follicles than other breeds studied, but identified 3-mm follicles did not emerge in other than a random distribution. There was little evidence of dominance until the ovulatory follicle had been identified.

Four crossbred beef steers (392 kg BW) fitted with ruminal cannulas were used in a 4 x 4 Latin square experiment with treatments arranged in a 2 x 2 factorial to evaluate effects of forage species (F) and reconstitution (R) on apparent digestibility, ruminal kinetics, and digesta functional specific gravity (FSG). Dietary treatments were alfalfa (Medicago sativa L.) and timothy (Phleum pratense L.) hays (90% DM) and the same hays reconstituted with water to 38% DM. Apparent DM digestibility was unaffected by R or F. Proportion of ruminal digesta with a FSG of .9 to 1.1 or 1.1 to 1.2 decreased linearly (P < .05) after feeding, whereas the proportion with FSG > 1.2 increased linearly (P < .01). Reconstitution decreased (P < .10) the proportion of ruminal digesta with FSG of 1.1 to 1.2 and increased (P < .10) the proportion with a FSG > 1.2 compared with the hay diets. Reconstitution decreased digesta passage rate for timothy forage; no influence on passage rate for alfalfa was detected (F x R, P < .10). Ruminal DM pool was increased by reconstitution (P < .10). Inert particles of 1-mm length passed from the rumen at a faster rate (P < .05) than 3-mm particles. Inert particles with SG of 1.32 passed from the rumen at a faster rate (P < .05) in the reconstituted alfalfa than in the dry alfalfa diet but at a slower rate in the reconstituted timothy than in the dry timothy diet. These results suggest that factors other than SG of the digesta can have a profound effect on passage of forage particles from the rumen.

Effects of day of estrous cycle and induced luteal regression on follicular dynamics in ewes were examined. In Exp. 1, ewes received PGF2 alpha (i.m.) on d 5, 8, or 11 after estrus (d 0) or saline on d 14 (n = 27, 27, 31, and 29, respectively). Ewes were laparotomized before treatment and again 48 h after induced estrus to record numbers, sizes, and locations of follicles > or = 2 mm and corpora lutea (CL). The two largest follicles (designated F1 and F2) and any of equal size (F1.5 and F2.5) were marked with India ink in stroma around the follicle. Ewes treated on d 8 ovulated fewer F1 and F1.5 (39%) than ewes treated on d 5 (69%; P < .05); d 11 (51%) and 14 (56%) were intermediate. In Exp. 2, ewes received PGF2 alpha on d 5 or 8 (n = 15/d). Ovaries were examined with transrectal ultrasonography daily from 4 d before PGF2 alpha until ovulation, and on d 5 and 8 after ovulation. Diameters, locations, and ranks (at time of PGF2 alpha) of follicles > or = 2 mm and CL were recorded. Ewes treated on d 8 ovulated fewer F1 and F1.5 (35%) than ewes treated on d 5 (86%; P < .05). Ovulatory follicles at time of PGF2 alpha were larger on d 5 (4.7 +/- .2 mm) than on d 8 (4.0 +/- .2 mm; P < .05).(ABSTRACT TRUNCATED AT 250 WORDS)

The development of ovarian follicular and luteal structures during the estrous cycle and early pregnancy in ewes was examined. Ewes were treated with prostaglandin F2 alpha to induce estrus; they were placed with either two fertile or two vasectomized rams and observed for estrus twice daily. Beginning at estrus (Day 0), ovaries were scanned for CL and for follicles > or = 2 mm by transrectal ultrasonography daily for the first 25 days of pregnancy (PREG, n = 17 ewes) or until Day 8 of the second estrous cycle (CYC, n = 15 ewes). Jugular blood samples were collected at ultrasonography and on Days 30, 35, and 40 of gestation for RIA of progesterone and estradiol-17 beta. Total follicles (17.6 +/- 1.3), number of follicles > or = 4 mm in diameter (11.3 +/- 0.6), and number of follicles that became the largest during the first 16 days of pregnancy or the first estrous cycle (4.9 +/- 0.3) did not differ between PREG and CYC ewes. Number of newly detected follicles each day and difference in mean diameter between the largest two follicles and all other follicles were similar between PREG and CYC ewes until luteal regression. At that time, difference in diameter between the largest two follicles and all other follicles increased in CYC ewes in parallel with estradiol-17 beta in serum. Mean concentrations of progesterone in serum were higher in PREG than in CYC ewes (2.4 +/- 0.1 vs. 2.0 +/- 0.1 ng/ml; p < 0.05) before onset of luteal regression, but mean areas of CL did not differ.(ABSTRACT TRUNCATED AT 250 WORDS)

In postpartum cows expected to have corpora lutea (CL) of normal (norgestomet-treated) compared to short (control) life spans, function of the largest follicle increases after an increase in concentrations of prostaglandin F2 alpha (PGF). To determine whether PGF alters follicular growth and subsequent life span of the CL, 43 crossbred beef cows (19 to 22 d postpartum) were assigned to one of four treatments: 1) control (C; n = 10), 2) control+PGF (CPGF; n = 10), 3) norgestomet (N; n = 13), 4) norgestomet+flunixin meglumine (NFM; n = 10). Flunixin meglumine inhibits prostaglandin endoperoxide synthase. On day 0, N and NFM cows received a 6 mg implant of norgestomet. From days 3 through 8, CPGF and NFM cows were injected every 8 hr with 10 mg PGF im or 1 g FM iv, respectively. Implants were removed on day 9. On day 11, each cow received 1000 IU of hCG im to induce formation of CL. Follicular growth was monitored by daily ultrasonography from days 6 through 11. In a majority of the cases (25/32), the largest follicle present on day 6 was still the largest on day 11; frequency of persistence did not differ with treatment. Rate of growth of the largest follicle was greater in CPGF than in N cows (.6 +/- .1 vs .3 +/- .1 mm/d, respectively; P less than .05) but did not differ between C and NFM cows (.4 +/- .1 and .5 +/- .1 mm/d, respectively). Concentrations of estradiol in NFM cows were higher (P less than .05) on day 3 and declined to concentrations similar to those of the other treatments on day 9.(ABSTRACT TRUNCATED AT 250 WORDS)

Data were obtained on 1039 recoveries of embryos from beef cows of four breeds at two locations, in clinic and on farm. General linear models procedures were utilized to determine the effects of breed, location, age of donor, dosage of follicle stimulating hormone (FSH) and the interaction of age and FSH on the following dependent variables: 1) the mean number of ova (unfertilized oocytes and embryos) recovered; 2) the mean number and percentage of embryos (fertilized; live and dead) recovered; and 3) the mean number and percentage of transferable embryos recovered. The interaction of age of donor and dosage of FSH with breed and location prevented the pooling of data over breed and location. The mean number of ova recovered was affected by age of the donor (Charolais-in clinic), or the interaction between age of donor and dosage of FSH (Polled Hereford-in clinic and -on farm and Simmental -on farm). The mean number of embryos was affected by age of donor (Polled Hereford-in clinic), dosage of FSH (Simmental-in clinic) or their interaction (Angus-on farm). The mean number of transferable embryos was affected by age of donor (Polled Hereford-in clinic and -on farm, Simmental-in clinic and Angus-on farm). General linear models procedures were utilized to determine the effects of the embryo (stage of development and quality) and the recipient (synchrony with the donor) on the rate of pregnancy. Rate of pregnancy varied with embryo quality score and synchrony of the recipient and the embryo. In conclusion, the superovulatory response was found to be highly breed-specific, and most of the variability in embryos produced was attributed to the number of ova recovered. However, the number of ova, embryos and transferable embryos recovered was further influenced by age of the donor, dosage of FSH or their interaction.

The quality of different classes of forage hay (C3, C4 grasses and legumes) was determined in intake and digestibility trials with mature cattle, sheep and goats. For all nine hays, DM and NDF digestibility by cattle and goats was higher (P less than .05) than by sheep, with no differences due to forage class. Cattle had a higher (P less than .01) DM intake than sheep or goats averaged across forage (92.6 vs 65.8 and 68.6 g/kg BW.75); hay intake was highest on legume, with no difference between C3 and C4 grasses. Mean NDF intake by cattle was greater than by sheep or goats (58.7 vs 39.6 and 42.6 g/kg BW.75); NDF intake for all animal species decreased in the order C4 grass greater than C3 grass greater than legume. Particle passage rates did not differ (P greater than .05) with forage class but were higher (P less than .02) for sheep and goats than for cattle. Prefeeding ruminal DM fill values, determined by emptying, were 10.6, 15.0 and 19.9 g/kg BW1.0 for alfalfa, orchardgrass and switchgrass hays fed to cattle, and 11.2, 11.3 and 16.5 g/kg BW1.0 for the same hays fed to sheep. Estimated turnover times for DM and NDF were shorter (P less than .05) for sheep than for cattle; DM turnover was longer for switchgrass than for alfalfa and orchardgrass, with no forage differences in NDF turnover between these two animal species. Results show that goats were superior to sheep in NDF digestion.

To examine effects of norgestomet pretreatment on development of follicles and their response to administration of gonadotropin releasing hormone (GnRH), 45 pluriparous suckled beef cows were assigned at random to receive a 6-mg implant of norgestomet for 9 d (inserted 24 d postpartum) or serve as untreated controls. Ovaries were obtained 48 h after removal of implants or 10 to 11 or 20 to 22 h after im administration of 150 micrograms GnRH at 48 h after removal of the implant. The largest follicle (F1) and all follicles within 3 mm in diameter of the F1 were dissected from the ovaries. Theca, granulosa and follicular fluid were separated and assayed for steroids and prostaglandins. Diameters and weights of F1 and weights of follicular components remained unchanged in control cows, but increased by 10 h and declined by 20 h in norgestomet-pretreated cows (treatment X time, quadratic, P less than .05). Ovarian volume and numbers of follicles at the surface of the ovary did not differ with treatment, but the diameter of the second-largest follicle (F2) was smaller (P less than .05) in norgestomet-pretreated cows than in controls (6.0 +/- .9 vs 8.2 +/- .7 mm). The F1 were embedded in the ovary in fewer norgestomet-pretreated than control cows (2/22 vs 8/23; P less than .05). Changes in steroids in F1 paralleled those in size (treatment X time, quadratic, P less than .05). Overall, F1 from norgestomet-pretreated cows had higher (P less than .05) contents of estradiol. Contents of prostaglandins in F1 follicles did not differ with treatment, but increased (P less than .05) following treatment with GnRH. The F2 had lower contents of estradiol than F1. It is suggested that norgestomet effected the maturation of a single follicle which produced more estradiol.

The objectives of this study were to determine 1) the ability of charcoal-extracted bovine follicular fluid (bFF) to suppress endogenous follicle stimulating hormone (FSH) at various stages of the estrous cycle and 2) the effects of suppression of FSH on luteal function and lengths of the current and subsequent estrous cycles. Twenty-six mature ewes were assigned randomly to receive 5 ml of either bFF or saline, subcutaneously, at 8-h intervals on d 1 through 5 (bFF n = 6; saline n = 3), d 6 through 10 (bFF n = 6; saline n = 3) or d 11 through 15 (bFF n = 6; saline n = 2) of the estrous cycle (d 0 = estrus). Blood was collected daily beginning at estrus and continued until the third estrus (two estrous cycles) or 40 d; more frequent samples were collected 2 h prior to initiation of treatment (0600), hourly for the first 8 h of treatment, then every 4 h until 0800 on the first day after treatment, and finally at 1600 and 2400 on that day. Plasma concentrations of FSH were lower (P less than .001) in bFF-treated than in saline-treated ewes. Treatment with bFF reduced (P less than .05) plasma concentrations of progesterone during the current but not during the subsequent estrous cycle. Treatment with bFF did not affect plasma concentrations of estradiol-17 beta. Administration of bFF on d 11 through 15 of the estrous cycle lengthened the interval from the decline in progesterone to estrus and the inter-estrous interval by approximately 3 and 4 d, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)