Successful Treatment of a Chronic Recurrent Expanding Hematoma of the Thigh.

Chronic expanding hematoma (CEH) is a rare entity that poses diagnostic and therapeutic challenges due to persistent growth, risk of recurrence, and potential for blood loss anemia. The most common etiologies of CEH are trauma or surgery. It is thought to occur due to irritant effects of blood breakdown products, causing bleeding from capillaries in chronic granulation tissue. Although treatment of CEH is variable, complete surgical excision of the hematoma and its pseudocapsule is the gold standard. We present a case of a 15-year CEH that was initially treated with limited evacuation of the hematoma and cavity decortication, resulting in recurrence. Ultimately, the patient was managed with complete excision of the pseudocapsule, closure of the cavity with quilting sutures, application of an absorbable hemostatic agent, and placement of a large drain, resulting in a successful outcome. This case highlights the efficacy of a comprehensive surgical plan in addressing CEH, emphasizing the importance of pseudocapsule excision in its entirety to prevent recurrence.

Hexavalent Chromium Disrupts Oocyte Development in Rats by Elevating Oxidative Stress, DNA Double-Strand Breaks, Microtubule Disruption, and Aberrant Segregation of Chromosomes.

Environmental and occupational exposure to hexavalent chromium, Cr(VI), causes female reproductive failures and infertility. Cr(VI) is used in more than 50 industries and is a group A carcinogen, mutagenic and teratogenic, and a male and female reproductive toxicant. Our previous findings indicate that Cr(VI) causes follicular atresia, trophoblast cell apoptosis, and mitochondrial dysfunction in metaphase II (MII) oocytes. However, the integrated molecular mechanism of Cr(VI)-induced oocyte defects is not understood. The current study investigates the mechanism of Cr(VI) in causing meiotic disruption of MII oocytes, leading to oocyte incompetence in superovulated rats. Postnatal day (PND) 22 rats were treated with potassium dichromate (1 and 5 ppm) in drinking water from PND 22-29 and superovulated. MII oocytes were analyzed by immunofluorescence, and images were captured by confocal microscopy and quantified by Image-Pro Plus software, Version 10.0.5. Our data showed that Cr(VI) increased microtubule misalignment (~9 fold), led to missegregation of chromosomes and bulged and folded actin caps, increased oxidative DNA (~3 fold) and protein (~9-12 fold) damage, and increased DNA double-strand breaks (~5-10 fold) and DNA repair protein RAD51 (~3-6 fold). Cr(VI) also induced incomplete cytokinesis and delayed polar body extrusion. Our study indicates that exposure to environmentally relevant doses of Cr(VI) caused severe DNA damage, distorted oocyte cytoskeletal proteins, and caused oxidative DNA and protein damage, resulting in developmental arrest in MII oocytes.

Improved Resource Utilization Using WALANT Hand Surgery in Adolescents.

BACKGROUND: Cost, efficiency, patient preference, and safety have driven utilization of wide awake, local anesthesia, no tourniquet (WALANT) in hand surgery. This is not well documented in adolescents. We hypothesize that the use of WALANT with adolescents reduced time spent in the operating room (OR) and in the hospital when compared with patients who underwent surgery with traditional anesthesia (TA). METHODS: After institutional review board approval, we performed a retrospective review of patients aged 10 to 17 who underwent surgery at a regional hospital system including the level 1 pediatric trauma hospital. Operative notes were assessed for use of WALANT. We excluded those operations not traditionally amenable to WALANT. Using a propensity matched cohort, hospital time, OR time, and perioperative complications were recorded and compared to evaluate efficiency and perioperative safety. RESULTS: There were 28 cases in the WALANT group and 28 cases in the TA group after excluding cases not amenable to WALANT, and cases were propensity matched. Although the operative time (incision to closure) was similar, for WALANT patients, the in-room to procedure time (15 vs 22 minutes), procedure end to out-room time (5 vs 10 minutes), total room time (52.81 vs 63.68), and length of hospital stay (222 vs 342 minutes) were shorter than patients in the TA group. CONCLUSION: Our case series demonstrates time-savings both in the OR and in the hospital overall. Avoiding TA when WALANT is feasible may result in significant savings to hospital systems, patients, and payers while also freeing up anesthesia staff and perioperative nurses.

Maternal nutrient restriction in late pregnancy programs postnatal metabolism and pituitary development in beef heifers.

Maternal undernutrition during pregnancy followed by ad libitum access to nutrients during postnatal life induces postnatal metabolic disruptions in multiple species. Therefore, an experiment was conducted to evaluate postnatal growth, metabolism, and development of beef heifers exposed to late gestation maternal nutrient restriction. Pregnancies were generated via transfer of in vitro embryos produced using X-bearing sperm from a single Angus sire. Pregnant dams were randomly assigned to receive either 100% (control; n = 9) or 70% (restricted; n = 9) of their total energy requirements from gestational day 158 to parturition. From post-natal day (PND) 301 until slaughter (PND485), heifers were individually fed ad libitum in a Calan gate facility. Calves from restricted dams were lighter than controls at birth (P<0.05) through PND70 (P<0.05) with no difference in body weight from PND105 through PND485 (P>0.10). To assess pancreatic function, glucose tolerance tests were performed on PND315 and PND482 and a diet effect was seen with glucose area under the curve being greater (P<0.05) in calves born to restricted dams compared to controls. At slaughter, total internal fat was greater (P<0.05) in heifers born to restricted dams, while whole pituitary weight was lighter (P<0.05). Heifers from restricted dams had fewer growth hormone-positive cells (somatotrophs) compared to controls (P<0.05). Results demonstrate an impaired ability to clear peripheral glucose in heifers born to restricted dams leading to increased deposition of internal fat. A reduction in the number of somatotrophs may contribute to the adipogenic phenotype of heifers born to restricted dams due to growth hormone's known anabolic roles in growth, lipolysis, and pancreatic islet function.

Primary Extramedullary, Extradural Cervical Spine Seminoma.

While extragonadal seminomas resulting in spinal cord compression are rarely reported in the literature, most have been treated with surgical decompression followed by radiation therapy. In this report, we present the unique and interesting case of a 38-year-old man who initially presented as an outpatient with a chief complaint of axial neck pain and lateral thoracic wall pain. After an extensive malignancy workup, he was diagnosed with a primary cervical spine seminoma and was treated with a C6-T1 laminectomy with posterior spinal instrumentation from C5 to T2. He has since undergone chemotherapy with cisplatin, vinblastine, and bleomycin, and at 24-month follow-up, he remains asymptomatic with no signs of recurrent disease.

"Purse-String" Capsular Closure for Decreasing Dislocation Rates in Proximal Femur Replacements.

Hip joint dislocation is the most common complication after a proximal femur replacement. As the utilization of proximal femur replacements continues to increase, it becomes imperative for surgeons to find the optimal method to decrease postoperative dislocation and its sequelae. These cases often involve extensive soft-tissue deficits that require reconstruction to provide postoperative strength and stability. Patients report good functional outcomes; however, dislocation remains a concern. Although "described" previously in the literature, the authors illustrate the "purse-string" hip joint capsular closure technique to help other surgeons understand it and apply to their practice as deemed necessary. We also present the senior author's results with using a modified version of the "purse-string" hip joint capsular closure technique.

Bartonella henselae in a dog with ear tip vasculitis.

BACKGROUND: Bartonella henselae, a Gram-negative, zoonotic, alpha-proteobacteria has been previously implicated in association with cutaneous vasoproliferative lesions (bacillary angiomatosis), nodular panniculitis and multifocal erythema (erythema multiforme) in dogs. OBJECTIVE: Describe clinical, microbiological and histological lesions in a dog with ear margin vasculitis and B. henselae infection. ANIMALS: A 12-month-old, specific pathogen-free intact female beagle dog maintained in a vector-free laboratory animal resource facility. METHODS AND MATERIALS: Bartonella and Rickettsia serological evaluation, Bartonella and Rickettsia PCR, Bartonella alpha-proteobacteria growth medium (BAPGM) enrichment blood culture/PCR, histopathological investigation and confocal immunohistochemical evaluation. RESULTS: Serological investigation (seroreversion) and PCR testing of aural tissue biopsies failed to support Rickettsia rickettsii as a cause of the aural vasculitis; however, B. henselae, genotype San Antonio 2 DNA was amplified and sequenced from both ear tip margins and from normal-appearing abdominal skin. Seroconversion to B. henselae was documented retrospectively by IFA testing. Bartonella henselae organisms were visualized by confocal immunostaining within all three biopsies. Histopathology revealed small vessel necrotizing vasculitis and dermal necrosis. Bartonella henselae seroreversion and complete resolution of skin lesions occurred in conjunction with administration of oral doxycycline and enrofloxacin for six weeks. CONCLUSIONS AND CLINICAL IMPORTANCE: Bartonella henselae is an emerging zoonotic pathogen that has been associated with leucocytoclastic vasculitis in humans and may have had a contributing or causative role in the development of the cutaneous aural margin vasculitis in this beagle.

Metarrestin, a perinucleolar compartment inhibitor, effectively suppresses metastasis.

Metastasis remains a leading cause of cancer mortality due to the lack of specific inhibitors against this complex process. To identify compounds selectively targeting the metastatic state, we used the perinucleolar compartment (PNC), a complex nuclear structure associated with metastatic behaviors of cancer cells, as a phenotypic marker for a high-content screen of over 140,000 structurally diverse compounds. Metarrestin, obtained through optimization of a screening hit, disassembles PNCs in multiple cancer cell lines, inhibits invasion in vitro, suppresses metastatic development in three mouse models of human cancer, and extends survival of mice in a metastatic pancreatic cancer xenograft model with no organ toxicity or discernable adverse effects. Metarrestin disrupts the nucleolar structure and inhibits RNA polymerase (Pol) I transcription, at least in part by interacting with the translation elongation factor eEF1A2. Thus, metarrestin represents a potential therapeutic approach for the treatment of metastatic cancer.

Inhibition of EHMT2 Induces a Robust Antiviral Response Against Foot-and-Mouth Disease and Vesicular Stomatitis Virus Infections in Bovine Cells.

The genetic regulatory network controlling the innate immune system is well understood in many species. However, the role of the epigenetic mechanisms underlying the expression of immunoregulatory genes is less clear, especially in livestock species. Histone H3 lysine 9 dimethylation (H3K9me2) is an epigenetic modification associated with transcriptional silencing within the euchromatin regions. Euchromatic histone-lysine N-methyltransferase 2 (EHMT2; also known as G9a) is a crucial enzyme responsible for regulating the dynamics of this epigenetic modification. It has been shown that histone modifications play a role in regulating type I interferon (IFN) response. In the present study, we investigated the role of EHMT2 in the epigenetic regulation of bovine antiviral innate immunity and explored its therapeutic potential against viral infections. We evaluated the effects of pharmacological and RNAi-mediated inhibition of EHMT2 on the transcription of IFN-ß and other IFN-inducible antiviral genes, as well as its effect on foot-and-mouth disease virus (FMDV) and vesicular stomatitis virus (VSV) replication in bovine cells. We show that treatment of primary bovine cells with the synthetic EHMT2 inhibitor (UNC0638) either before or shortly after virus infection resulted in a significant increase in transcript levels of bovine IFN-ß (boIFN-ß; 300-fold) and other IFN-inducible genes, including IFN-stimulated gene 15 (ISG-15), myxovirus resistance 1 (Mx-1), Mx-2, RIG-I, 2',5'-oligoadenylate synthetase 1 (OAS-1), and protein kinase R (PKR). Expression of these factors correlated with a significant decrease in VSV and FMDV viral titers. Our data confirm the involvement of EHMT2 in the epigenetic regulation of boIFN-ß and demonstrate the activation of a general antiviral state after EHMT2 inhibition.

Molecular and preclinical basis to inhibit PGE2 receptors EP2 and EP4 as a novel nonsteroidal therapy for endometriosis.

Endometriosis is a debilitating, estrogen-dependent, progesterone-resistant, inflammatory gynecological disease of reproductive age women. Two major clinical symptoms of endometriosis are chronic intolerable pelvic pain and subfertility or infertility, which profoundly affect the quality of life in women. Current hormonal therapies to induce a hypoestrogenic state are unsuccessful because of undesirable side effects, reproductive health concerns, and failure to prevent recurrence of disease. There is a fundamental need to identify nonestrogen or nonsteroidal targets for the treatment of endometriosis. Peritoneal fluid concentrations of prostaglandin E2 (PGE2) are higher in women with endometriosis, and this increased PGE2 plays important role in survival and growth of endometriosis lesions. The objective of the present study was to determine the effects of pharmacological inhibition of PGE2 receptors, EP2 and EP4, on molecular and cellular aspects of the pathogenesis of endometriosis and associated clinical symptoms. Using human fluorescent endometriotic cell lines and chimeric mouse model as preclinical testing platform, our results, to our knowledge for the first time, indicate that selective inhibition of EP2/EP4: (i) decreases growth and survival of endometriosis lesions; (ii) decreases angiogenesis and innervation of endometriosis lesions; (iii) suppresses proinflammatory state of dorsal root ganglia neurons to decrease pelvic pain; (iv) decreases proinflammatory, estrogen-dominant, and progesterone-resistant molecular environment of the endometrium and endometriosis lesions; and (v) restores endometrial functional receptivity through multiple mechanisms. Our novel findings provide a molecular and preclinical basis to formulate long-term nonestrogen or nonsteroidal therapy for endometriosis.

Expression of porcine fusion protein IRF7/3(5D) efficiently controls foot-and-mouth disease virus replication.

UNLABELLED: Several studies have demonstrated that the delivery of type I, II, or III interferons (IFNs) by inoculation of a replication-defective human adenovirus 5 (Ad5) vector expressing IFNs can effectively control foot-and-mouth disease (FMD) in cattle and swine during experimental infections. However, relatively high doses are required to achieve protection. In this study, we identified the functional properties of a porcine fusion protein, poIRF7/3(5D), as a biotherapeutic and enhancer of IFN activity against FMD virus (FMDV). We showed that poIRF7/3(5D) is a potent inducer of type I IFNs, including alpha IFN (IFN-α), IFN-ß, and IFN-ω but not type III IFN (interleukin-28B), without inducing cytotoxicity. Expression of poIRF7/3(5D) significantly and steadily reduced FMDV titers by up to 6 log10 units in swine and bovine cell lines. Treatment with an IFN receptor inhibitor (B18R) combined with an anti-IFN-α antibody neutralized the antiviral activity in the supernatants of cells transduced with an Ad5 vector expressing poIRF7/3(5D) [Ad5-poIRF7/3(5D)]. However, several transcripts with known antiviral function, including type I IFNs, were still highly upregulated (range of increase, 8-fold to over 500-fold) by poIRF7/3(5D) in the presence of B18R. Furthermore, the sera of mice treated with Ad5-poIRF7/3(5D) showed antiviral activity that was associated with the induction of high levels of IFN-α and resulted in complete protection against FMDV challenge at 6, 24, or 48 h posttreatment. This study highlights for the first time the antiviral potential of Ad5-poIRF7/3(5D) in vitro and in vivo against FMDV. IMPORTANCE: FMD remains one of the most devastating diseases that affect livestock worldwide. Effective vaccine formulations are available but are serotype specific and require approximately 7 days before they are able to elicit protective immunity. We have shown that vector-delivered IFN is an option to protect animals against many FMDV serotypes as soon as 24 h and for about 4 days postadministration. Here we demonstrate that delivery of a constitutively active transcription factor that induces the production of endogenous IFNs and potentially other antiviral genes is a viable strategy to protect against FMD.

Sustained expression of insulin by a genetically engineered sertoli cell line after allotransplantation in diabetic BALB/c mice.

Immune-privileged Sertoli cells (SCs) exhibit long-term survival after allotransplantation or xenotransplantation, suggesting they can be used as a vehicle for cell-based gene therapy. Previously, we demonstrated that SCs engineered to secrete insulin by using an adenoviral vector normalized blood glucose levels in diabetic mice. However, the expression of insulin was transient, and the use of immunocompromised mice did not address the question of whether SCs can stably express insulin in immunocompetent animals. Thus, the objective of the current study was to use a lentiviral vector to achieve stable expression of insulin in SCs and test the ability of these cells to survive after allotransplantation. A mouse SC line transduced with a recombinant lentiviral vector containing furin-modified human proinsulin cDNA (MSC-EhI-Zs) maintained stable insulin expression in vitro. Allotransplantation of MSC-EhI-Zs cells into diabetic BALB/c mice demonstrated 88% and 75% graft survival rates at 20 and 50 days post-transplantation, respectively. Transplanted MSC-EhI-Zs cells continued to produce insulin mRNA throughout the study (i.e., 50 days); however, insulin protein was detected only in patches of cells within the grafts. Consistent with low insulin protein detection, there was no significant change in blood glucose levels in the transplant recipients. Nevertheless, MSC-EhI-Zs cells isolated from the grafts continued to express insulin protein in culture. Collectively, this demonstrates that MSC-EhI-Zs cells stably expressed insulin and survived allotransplantation without immunosuppression. This further strengthens the use of SCs as targets for cell-based gene therapy for the treatment of numerous chronic diseases, especially those that require basal protein expression.

Two methods for retrieving UV index for all cloud conditions from sky imager products or total SW radiation measurements.

Cloud effects on UV Index (UVI) and total solar radiation (TR) as a function of cloud cover and sunny conditions (from sky images) as well as of solar zenith angle (SZA) are assessed. These analyses are undertaken for a southern-hemisphere mid-latitude site where a 10-years dataset is available. It is confirmed that clouds reduce TR more than UV, in particular for obscured Sun conditions, low cloud fraction (<60%) and large SZA (>60°). Similarly, local short-time enhancement effects are stronger for TR than for UV, mainly for visible Sun conditions, large cloud fraction and large SZA. Two methods to estimate UVI are developed: (1) from sky imaging cloud cover and sunny conditions, and (2) from TR measurements. Both methods may be used in practical applications, although Method 2 shows overall the best performance, as TR allows considering cloud optical properties. The mean absolute (relative) differences of Method 2 estimations with respect to measured values are 0.17 UVI units (6.7%, for 1 min data) and 0.79 Standard Erythemal Dose (SED) units (3.9%, for daily integrations). Method 1 shows less accurate results but it is still suitable to estimate UVI: mean absolute differences are 0.37 UVI units (15%) and 1.6 SED (8.0%).

Preclinical derivation and imaging of autologously transplanted canine induced pluripotent stem cells.

Derivation of patient-specific induced pluripotent stem cells (iPSCs) opens a new avenue for future applications of regenerative medicine. However, before iPSCs can be used in a clinical setting, it is critical to validate their in vivo fate following autologous transplantation. Thus far, preclinical studies have been limited to small animals and have yet to be conducted in large animals that are physiologically more similar to humans. In this study, we report the first autologous transplantation of iPSCs in a large animal model through the generation of canine iPSCs (ciPSCs) from the canine adipose stromal cells and canine fibroblasts of adult mongrel dogs. We confirmed pluripotency of ciPSCs using the following techniques: (i) immunostaining and quantitative PCR for the presence of pluripotent and germ layer-specific markers in differentiated ciPSCs; (ii) microarray analysis that demonstrates similar gene expression profiles between ciPSCs and canine embryonic stem cells; (iii) teratoma formation assays; and (iv) karyotyping for genomic stability. Fate of ciPSCs autologously transplanted to the canine heart was tracked in vivo using clinical positron emission tomography, computed tomography, and magnetic resonance imaging. To demonstrate clinical potential of ciPSCs to treat models of injury, we generated endothelial cells (ciPSC-ECs) and used these cells to treat immunodeficient murine models of myocardial infarction and hindlimb ischemia.

Analgesic effects of tramadol, tramadol-gabapentin, and buprenorphine in an incisional model of pain in rats (Rattus norvegicus).

Postoperative pain management in laboratory animals relies heavily on a limited number of drug classes, such as opioids and nonsteroidal antiinflammatory drugs. Here we evaluated the effects of saline, tramadol, tramadol with gabapentin, and buprenorphine (n = 6 per group) in a rat model of incisional pain by examining thermal hyperalgesia and weight-bearing daily for 6 d after surgery. All drugs were administered preemptively and continued for 2 consecutive days after surgery. Rats treated with saline or with tramadol only showed thermal hyperalgesia on days 1 through 4 and 1 through 3 after surgery, respectively. In contrast, buprenorphine-treated rats showed no thermal hyperalgesia on days 1 and 2 after surgery, and rats given tramadol with gabapentin showed reduced thermal hyperalgesia on days 2 and 4. For tests of weight-bearing, rats treated with saline or with tramadol only showed significantly less ipsilateral weight-bearing on day 1 after surgery, whereas rats given either buprenorphine or tramadol with gabapentin showed no significant change in ipsilateral weight-bearing after surgery. These data suggest that tramadol alone provides insufficient analgesia in this model of incisional pain; buprenorphine and, to a lesser extent, tramadol with gabapentin provide relief of thermal hyperalgesia and normalize weight-bearing.

Expression of human FUS/TLS in yeast leads to protein aggregation and cytotoxicity, recapitulating key features of FUS proteinopathy.

Mutations in the fused in sarcoma/translocated in liposarcoma (FUS/TLS) gene have been associated with amyotrophic lateral sclerosis (ALS). FUS-positive neuropathology is reported in a range of neurodegenerative diseases, including ALS and fronto-temporal lobar degeneration with ubiquitin-positive pathology (FTLDU). To examine protein aggregation and cytotoxicity, we expressed human FUS protein in yeast. Expression of either wild type or ALS-associated R524S or P525L mutant FUS in yeast cells led to formation of aggregates and cytotoxicity, with the two ALS mutants showing increased cytotoxicity. Therefore, yeast cells expressing human FUS protein recapitulate key features of FUS-positive neurodegenerative diseases. Interestingly, a significant fraction of FUS expressing yeast cells stained by propidium iodide were without detectable protein aggregates, suggesting that membrane impairment and cellular damage caused by FUS expression may occur before protein aggregates become microscopically detectable and that aggregate formation might protect cells from FUS-mediated cytotoxicity. The N-terminus of FUS, containing the QGSY and G rich regions, is sufficient for the formation of aggregates but not cytotoxicity. The C-terminal domain, which contains a cluster of mutations, did not show aggregation or cytotoxicity. Similar to TDP-43 when expressed in yeast, FUS protein has the intrinsic property of forming aggregates in the absence of other human proteins. On the other hand, the aggregates formed by FUS are thioflavin T-positive and resistant to 0.5% sarkosyl, unlike TDP-43 when expressed in yeast cells. Furthermore, TDP-43 and FUS display distinct domain requirements in aggregate formation and cytotoxicity.

Viral particles of endogenous betaretroviruses are released in the sheep uterus and infect the conceptus trophectoderm in a transspecies embryo transfer model.

The sheep genome contains multiple copies of endogenous betaretroviruses highly related to the exogenous and oncogenic jaagsiekte sheep retrovirus (JSRV). The endogenous JSRVs (enJSRVs) are abundantly expressed in the uterine luminal and glandular epithelia as well as in the conceptus trophectoderm and are essential for conceptus elongation and trophectoderm growth and development. Of note, enJSRVs are present in sheep and goats but not cattle. At least 5 of the 27 enJSRV loci cloned to date possess an intact genomic organization and are able to produce viral particles in vitro. In this study, we found that enJSRVs form viral particles that are released into the uterine lumen of sheep. In order to test the infectious potential of enJSRV particles in the uterus, we transferred bovine blastocysts into synchronized ovine recipients and allowed them to develop for 13 days. Analysis of microdissected trophectoderm of the bovine conceptuses revealed the presence of enJSRV RNA and, in some cases, DNA. Interestingly, we found that RNAs belonging to only the most recently integrated enJSRV loci were packaged into viral particles and transmitted to the trophectoderm. Collectively, these results support the hypothesis that intact enJSRV loci expressed in the uterine endometrial epithelia are shed into the uterine lumen and could potentially transduce the conceptus trophectoderm. The essential role played by enJSRVs in sheep reproductive biology could also be played by endometrium-derived viral particles that influence development and differentiation of the trophectoderm.

Evaluation of culture systems for attachment and proliferation of epithelial cells cultured from ovine semen.

Different culture systems were evaluated for their ability to support attachment and proliferation of the somatic cells obtained from ovine semen. Ejaculates (n=14) were collected from eight rams representing three breeds, Dorper, Suffolk and Hampshire. All samples were processed immediately and somatic cells were obtained from 11 of the 14 ejaculates. These cells had classic epithelial morphology and expressed cytokeratin, indicating they were of epithelial origin. Cells from four rams with the greatest growth rates were used for subsequent studies. Cells were cultured in four different media for 5 days and total numbers of attached cells vs. total numbers of seeded cells were counted and compared each day. Four media were evaluated: (1) a supplemented medium composed of DMEM/F12, 10% fetal bovine serum (FBS), 10 ng/ml epidermal growth factor, 30 microg/ml bovine pituitary extract, 5 microg/ml insulin, 10 ng/ml cholera toxin, and 50 microg/ml gentamycin; (2) sheep fetal fibroblast (SFF)-conditioned medium; (3) swiss 3T3 fibroblast-conditioned medium; and (4) basic medium composed of DMEM/F12, 10% FBS, and 50 microg/ml gentamycin. Cell proliferation was greater in the supplemented medium, SFF-conditioned medium, and 3T3 fibroblast-conditioned medium compared to the basic medium by day 2 of culture (p<0.05, n=24), and greater in supplemented medium compared to the SFF-conditioned medium and 3T3 fibroblast-conditioned medium by day 4 of culture (p<0.05, n=24). Three different surfaces: (1) Matrigel basement membrane matrix-coated plastic; (2) collagen I-coated plastic; and (3) uncoated plastic were evaluated for their ability to support proliferation and attachment of the cells obtained from semen. Cell proliferation was greater when cells were cultured on the Matrigel-coated compared to the collagen I-coated and uncoated plastic by day 2 of culture (p<0.05, n=16). Cell attachment was greater when cells were plated on the Matrigel-coated and collagen I-coated plastic compared to the uncoated plastic (p<0.05, n=16). These studies describe an effective system for the culture and proliferation of epithelial cells obtained from ovine semen samples. The system may increase the likelihood of obtaining cells from frozen semen, which could be used for cloning to recover animals of genetic value in which semen is the only material that is available.

Synchronisation of canine germinal vesicle stage oocytes prior to in vitro maturation alters the kinetics of nuclear progression during subsequent resumption of meiosis.

Inhibition of meiosis before in vitro maturation (IVM) can improve meiotic competence in immature mammalian oocytes. Therefore, meiosis-inhibiting agents were evaluated singularly for the ability to arrest and synchronise germinal vesicle (GV) stage canine oocytes, and the most effective treatments were combined to improve meiotic resumption rates. Oocytes cultured in 2 ng mL(-1) oestradiol (E2), 10 IU mL(-1) eCG, or both (EG) for 72 h resulted in significantly fewer oocytes resuming meiosis in EG than the control, E2, or with eCG. Oocytes cultured in 50 or 100 micromol L(-1) of butyrolactone 1 or roscovitine (ROS) for up to 48 h did not resume meiosis nor increase subsequent meiotic resumption rates following IVM. A combination of 50 micromol L(-1) ROS and EG treatment for 48 h significantly increased the proportion of canine oocytes in meiotic arrest. More importantly, following 48 h of IVM, ROS+EG-treated oocytes demonstrated a dramatic increase in the ability to resume meiosis compared with the non-treated controls (51.3 +/- 8.2% and 10.8 +/- 4.5%, respectively; P < 0.05). These data indicate that chemical and biological meiotic inhibitors are effective at inducing GV arrest in canine oocytes. Furthermore, these inhibitors are reversible and beneficial to subsequent meiotic resumption in vitro.