Metabolic Abnormalities Detected in Phase II Evaluation of Doxycycline in Dogs with Multicentric B-Cell Lymphoma

Doxycycline has antiproliferative effects in human lymphoma cells and in murine xenografts. We hypothesized that doxycycline would decrease canine lymphoma cell viability and prospectively evaluated its clinical tolerability in client-owned dogs with spontaneous, nodal, multicentric, substage a, B-cell lymphoma, not previously treated with chemotherapy. Treatment duration ranged from 1 to 8 weeks (median and mean, 3 weeks). Dogs were treated with either 10 (n = 6) or 7.5 (n = 7) mg/kg by mouth twice daily. One dog had a stable disease for 6 weeks. No complete or partial tumor responses were observed. Five dogs developed grade 3 and/or 4 metabolic abnormalities suggestive of hepatopathy with elevations in bilirubin, ALT, ALP, and/or AST. To evaluate the absorption of oral doxycycline in our study population, serum concentrations in 10 treated dogs were determined using liquid chromatography tandem mass spectrometry. Serum levels were variable and ranged from 3.6 to 16.6 µg/ml (median, 7.6 µg/ml; mean, 8.8 µg/ml). To evaluate the effect of doxycycline on canine lymphoma cell viability in vitro, trypan blue exclusion assay was performed on canine B-cell lymphoma cell lines (17-71 and CLBL) and primary B-cell lymphoma cells from the nodal tissue of four dogs. A doxycycline concentration of 6 µg/ml decreased canine lymphoma cell viability by 80%, compared to matched, untreated, control cells (mixed model analysis, p < 0.0001; Wilcoxon signed rank test, p = 0.0313). Although the short-term administration of oral doxycycline is not associated with the remission of canine lymphoma, combination therapy may be worthwhile if future research determines that doxycycline can alter cell survival pathways in canine lymphoma cells. Due to the potential for metabolic abnormalities, close monitoring is recommended with the use of this drug in tumor-bearing dogs. Additional research is needed to assess the tolerability of chronic doxycycline therapy

Live Births from Domestic Dog (Canis familiaris) Embryos Produced by In Vitro Fertilization.

Development of assisted reproductive technologies (ART) in the dog has resisted progress for decades, due to their unique reproductive physiology. This lack of progress is remarkable given the critical role ART could play in conserving endangered canid species or eradicating heritable disease through gene-editing technologies-an approach that would also advance the dog as a biomedical model. Over 350 heritable disorders/traits in dogs are homologous with human conditions, almost twice the number of any other species. Here we report the first live births from in vitro fertilized embryos in the dog. Adding to the practical significance, these embryos had also been cryopreserved. Changes in handling of both gametes enabled this progress. The medium previously used to capacitate sperm excluded magnesium because it delayed spontaneous acrosome exocytosis. We found that magnesium significantly enhanced sperm hyperactivation and ability to undergo physiologically-induced acrosome exocytosis, two functions essential to fertilize an egg. Unlike other mammals, dogs ovulate a primary oocyte, which reaches metaphase II on Days 4-5 after the luteinizing hormone (LH) surge. We found that only on Day 6 are oocytes consistently able to be fertilized. In vitro fertilization of Day 6 oocytes with sperm capacitated in medium supplemented with magnesium resulted in high rates of embryo development (78.8%, n = 146). Intra-oviductal transfer of nineteen cryopreserved, in vitro fertilization (IVF)-derived embryos resulted in seven live, healthy puppies. Development of IVF enables modern genetic approaches to be applied more efficiently in dogs, and for gamete rescue to conserve endangered canid species

Development of embryos from Day 6 oocytes fertilized <i>in vitro</i> with sperm capacitated with Mg²⁺.

<p><b>Normal cleavage = black bars, delayed cleavage = grey bars</b>. (<b>A</b>) Proportion of oocytes collected on Day 5 versus Day 6 developing into embryos. Asterisks (*,**) indicate significant differences for day for total and delayed cleavage percentages, respectively, (<b>B</b>) Proportion of oocytes developing into embryos in unsupplemented versus P4-supplemented cNCSU, (<b>C</b>) An individual oocyte at collection though fertilization developing into an 8-cell embryo. A focused image of the presumptive zygote (14 hr mark) was not possible due to motility of attached sperm (<b>D</b>) Effect of male sperm donor on cleavage rates. Asterisks (*, **) indicate significant differences for that male in total and delayed cleavage, respectively.</p

Results of transfer of IVF embryos.

<p>(<b>A</b>) Embryo stage, status, transfer location and the results of IVF-derived embryo transfers. (<b>B</b>) Ultrasound image of a normally-developing embryo imaged Day 29 from Transfer #5. (<b>C</b>) 7 healthy puppies were born by planned Caesarian section. (<b>D</b>) Normally developing beagle puppy at 3 weeks of age.</p

Parentage Results.

<p>(<b>A</b>) Representative parentage results via Veterinary Genomics Laboratory with genetic parents (oocyte and sperm donor male), recipient bitch, and offspring. The recipient did not qualify as a parent of any offspring. (<b>B</b>) Summary table of parentage of the seven offspring.</p