Artificial insemination with frozen semen in rabbits in Brazil: first litter reported

Brazilian rabbit breeding plays an important social role through family farming. However, one of the challenges faced is the need for new genetic material from genetically improved rabbits. The artificial insemination (AI) technique in Brazil is restricted to the use of fresh or chilled semen. Hence, this is the first report of the use of frozen semen and live kits in Brazil and probably in Latin America. The aims were to evaluate the commercial viability of frozen rabbit semen in Brazil, ranging from cryopreservation to insemination. Three bucks and three rabbit does of Flemish giant rabbits were used. Semen was collected, evaluated and diluted for freezing with CUNIFreeze. Semen was frozen in a 0.5 mL straw, thawed and then filled in a blue curve rabbit AI sheath with the doe in dorsal position with ovulation induced at the same time with buserelin acetate. The ejaculate volume was 0.8 ± 0.8 mL; the mass motility was 3.3 ± 0.6; and the total motility and vigor after dilution were 65% ± 5 points and 3.7 ± 0.6, respectively. Post-thawed, the motility and vigor were 20% ± 10 and 3,0 ± 0,0, respectively. The pregnancy rate was 66.6% with a litter sizes of 5 and 2 kits. Even with a lower result of litter size compared with refrigerated semen, this report brings a new scenario for rabbit breeding in Brazil, showing that frozen semen is a feasible technique for biobanking and mainly bringing the possibility of imported semen from genetically improved bucks solving inbreeding problems.

Abstract 61: A Novel Catalytic Antioxidant Carbon Nanoparticle Improves Outcome in Hyperglycemic Stroke in Rats at Clinically Relevant Recanalization Times

Introduction: While oxidative stress mediates many of the pathologies in stroke, antioxidant therapies have been unsuccessful. Current antioxidants are limited either by a narrow range of radicals quenched or by low capacity. Some can yield toxic intermediates. We developed a new class of antioxidant, highly modified carbon nanoparticles called PEGylated hydrophilic carbon clusters (PEG-HCCs). These 40 nM particles quench superoxide by a high capacity novel catalytic mechanism (PNAS 2015: 2343-8), are effective against - OH and recouple nitric oxide synthase. Here we report the effect of PEG-HCCs administered during reperfusion after transient MCA suture occlusion in the rat under varying glucose levels. Hyperglycemia occurs in 1/3 of stroke patients and worsens outcome in part by accelerating release of radicals during reperfusion. Methods: Variation in glucose was induced by streptozocin IP 2 days prior to MCAO. At 90 mins, 23 rats were injected IV with PBS or PEG-HCCs (4 mg/kg) and the suture removed. A second dose was given 2 hours later (dosing based on half-life and in-vitro efficacy). At 3 days, infarct size corrected for edema and neurological function (Bederson Score, BS) was obtained. An outcome curve was developed using a range of baseline glucose in controls and the effect of PEG-HCCs compared at rat’s baseline glucose values. Results: We found a strong sigmoidal relationship between infarct size and glucose level in control rats (Fig. R 2 =.93; p=.0001; blue curve: control arm ± 95% confidence intervals). All PEG-HCC treated rats showed infarct size below the confidence interval (red stars; p<.003 vs control). Preliminary results for 120 min occlusion showed improved BS compared to control (3.5±2.4 vs 5.4±1.8; p=.047). Conclusions: This novel, broadly active, high capacity antioxidant was able to improve outcome in a severe test of pre-clinical stroke at clinically relevant times. Studies are ongoing to assess the treatment window and longer term outcomes.

Long-Term Outcomes of Autologous Hematopoietic Cell Transplantation Using Melphalan and Total Body Irradiation Conditioning in Multiple Myeloma

Background: The prognosis of multiple myeloma (MM) has improved significantly over the last 20 years due to the introduction of autologous stem cell transplantation and novel drugs. The standard regimen for conditioning in MM is melphalan 200 mg/m2, since a study published in 2002 (Moreau et al.) showed a survival advantage of melphalan (MEL) only over melphalan- total body irradiation (MEL-TBI) conditioning. Ionizing radiation is an independent treatment modality and has activity in MM. We decided to reevaluate the outcome of MEL-TBI conditioning in the age of novel drugs. Patients and Methods: In a retrospective chart review, we identified 50 patients with MM who underwent autologous hematopoietic cell transplantation at Tulane University Medical Center and were conditioned with MEL-TBI between December 1995 and March 2012. Stem cells were collected following a stimulation by 10 µg/ kg filgrastim for 4 days. In case of insufficient collection, plerixafor was administered. Between 1995 and 2003 cyclophosphamide priming was used in most cases. Patients received melphalan 140 mg/m2 as a single dose given intravenously on day -5. TBI was administered in fractionated doses of 150 cGy between days -4 and -1 (total dose 12 Gy). Peripherally harvested stem cells were infused on day 0. Standard supportive measures were followed. Log-rank test and Kaplan-Meier curves were used to calculate overall survival (OS) and progression-free survival. Significance levels were standard (p<0.05). The expected survival for each patient was compared with data from the Louisiana Tumor Registry (matched with 5 year age interval and 4 year time of diagnosis interval). Results The OS from the date of diagnosis is shown in Fig. 1 (84.2 months). The mean survival from date of transplant to death or last follow-up is 70.1 months. No significant differences in OS were observed according to gender, race, age at transplant, or initial stage of disease. No significant differences were observed when patients transplanted in remission (CR, sCR or VGPR) were compared with patients with partial remission, stable or progressive disease. Patients treated more recently (2006-2013) had a trend to a longer survival compared with the earlier period (1995-2005). Seven out of 8 patients who were not in CR achieved CR post-transplant. The d100 survival in the earlier period was 83%, in the later period 97%. The 1 year survival increased from 72% in the earlier period to 91% in the later period. When the survival of patients transplanted at our center is compared with the observed survival of all MM patients in Louisiana, a dramatic difference becomes apparent (median survival 27 months versus 84 months, see Figure 1). Second malignancies were documented in 3 (6%) of our patients (T-cell lymphoma, renal cell carcinoma, possible myelodysplastic syndrome). Conclusion: Mel-TBI conditioning results in long-term survival, especially if the early toxicity associated with total body irradiation is ameliorated. The survival reported in this study is almost twice as reported by Moreau et al. The improvement is in large part due to salvage treatments and improved supportive care. However, MEL-TBI may be superior in certain subgroups of patients. We plan to perform a larger study comparing MEL-TBI to MEL only with risk stratification and to investigate the possible interaction between cyclophosphamide priming and early toxicity. According to our data, MEL-TBI does not result in increased second malignancies. The survival of patients who underwent transplant with MEL-TBI is superior to patients in a population-based registry (matched for age and time of treatment). Figure 1 Kaplan-Meier survival distribution of patients treated by autologous transplantation with MEL-TBI (green curve) compared with registry data (blue curve) from time of diagnosis to last follow-up or death Figure 1. Kaplan-Meier survival distribution of patients treated by autologous transplantation with MEL-TBI (green curve) compared with registry data (blue curve) from time of diagnosis to last follow-up or death Disclosures No relevant conflicts of interest to declare.