Luteal phase length, endometrial edema, and behavior differentiate post-ovulatory events in a giant panda (Ailuropoda melanoleuca).

Despite decades of reproductive research on the giant panda (Ailuropoda melanoleuca), the post-ovulatory phase continues to confound zoologists in conservation and breeding centers around the world, often resulting in significant investments of time and resources without reproductive success. The purpose of this project was to document and compare post-ovulatory characteristics during a non-productive and productive breeding in the same individual in consecutive years. A multidisciplinary approach was used to monitor the visiting female giant panda at the Toronto Zoo through the luteal phase of her first two full reproductive cycles in 2014 and 2015. Monitoring occurred via urine-endocrine analysis, weekly ultrasound examinations, and continuous behavioral observations. The 2014 reproductive cycle consisted of a pseudopregnancy, characterized by an extended luteal phase (241 days), the identification of endometrial edema and folding during ultrasound examinations, and a lack of strong association between behavior patterns and urinary progestagen secretion. The 2015 reproductive cycle included increased feeding time through the primary progestagen rise compared to the previous year, followed by simultaneous decrease in appetite and increases in inactivity, resting, sitting upright, and pre-partum-associated behaviors. These changes began 25 days before the birth of twins on Day 153 post-ovulation. Both fetuses were detected via ultrasound 15 days pre-partum. These results suggest that an absence of pre-partum behaviors, ultrasound evidence of endometrial edema without a fetus, and an extended luteal phase may be indicative of pseudopregnancy in giant pandas. Simultaneous monitoring of morphology, behavior, and urinary-endocrine profiles showed clear differences between successful and unsuccessful reproductive years.

The Effects of Treatment with N-Acetyl Cysteine on Clinical Signs in Persistent Breeding-Induced Endometritis Susceptible Mares.

Persistent breeding-induced endometritis (PBIE) is a major cause of infertility in mares. Endometrial inflammation that persists until embryonic descent ultimately results in early embryonic death. A poor endometrial biopsy grade (IIb or III) has been identified as a risk factor for PBIE. Intrauterine fluid accumulation (>2 cm in depth), pathologic endometrial edema, and elevated intrauterine neutrophil levels are all clinical features of PBIE. Commonly applied treatment options include uterine lavage and oxytocin therapy. N-acetyl cysteine (NAC), a mucolytic used to treat bacterial endometritis in mares, has anti-inflammatory properties and was investigated as a potential treatment for PBIE. A randomized, blinded, cross-over design clinical trial used NAC before breeding in PBIE-susceptible mares (n = 9). Intrauterine infusion of 3.3% NAC was performed 12 hours before insemination, and endometrial cytology and endometrial biopsy samples were obtained at 12 and 60 hours after insemination. Endometrial biopsies were evaluated for the degree of inflammation present. Clinical signs of endometrial edema and intrauterine fluid volumes were assessed by transrectal ultrasound at 12 and then every 24 hours after breeding. Data were analyzed using repeated measures analysis of variance and a Mann Whitney Wilcoxon Test. Treatment with NAC did not improve clinical signs in PBIE-affected mares. However, endometrial biopsies from mares treated with NAC displayed more diffuse and severe neutrophil infiltration than control cycles. Further research using a larger population of mares is required to evaluate the effects of NAC treatment on the endometrium of PBIE-susceptible mares.

Single-Port Laparoscopic Treatment and Outcome of Dogs with Ovarian Remnant Syndrome: 13 Cases (2010-2018).

Ovarian remnant syndrome (ORS) is a condition resulting from incomplete removal of ovarian tissue during ovariectomy and/or ovariohysterectomy. Single-port laparoscopy (SPL) is an alternative to ventral midline laparotomy for treatment of ORS. Medical records of 13 client-owned female dogs who underwent SPL for the treatment of ORS were retrospectively reviewed to evaluate surgical technique and outcome. Dogs who had undergone a previous attempt at open ovariectomy or ovariohysterectomy were included. Major intraoperative complications did not occur and conversion to open laparotomy was not required. In 1 dog, an SPL + 1 technique was used, in which an additional port was placed cranial to the single-port device to aid in dissection and tissue manipulation. Median surgical time was 45 min (range, 30-90 min). Clinical signs related to estrus had resolved in 11 of 13 dogs with a median follow-up time of 18 mo. Two of 13 dogs were lost to follow-up at 3 mo postoperatively; however, signs of estrus had resolved at time of last follow-up. SPL treatment for ORS was feasible and successful in this cohort of dogs. Reduced surgical time was found in this study compared with previous reports investigating multiple-port laparoscopic treatment of ORS.

Canine cryptorchidism: An update.

This review article is aimed at providing an update on canine cryptorchidism based on the research studies published in this area over the last 26 years. Although a majority of these research studies were directed at investigating the genetic and molecular basis of cryptorchidism, the mechanisms underlying this problem in dogs are still not clear. Future studies using advanced genomic and molecular approaches and including a larger number of dogs of multiple breeds are required to further our understanding of canine cryptorchidism. Diagnosis and treatment of cryptorchidism in dogs have not received much attention. Research advancements include investigation of ultrasonography and anti-Müllerian hormone assay as potential diagnostic methods. Advancements in treatment include the use of laparoscopic-assisted cryptorchidectomy. Based on the available literature, this method appears to be the best for removal of abdominal cryptorchid testes from dogs. However, future studies that compare laparoscopic-assisted cryptorchidectomy with the traditional approaches are required for a more conclusive determination of the best method for cryptorchidectomy in dogs.

Single incision laparoscopic-assisted ovariohysterectomy for an ovarian tumor in a dog.

This report describes a single-incision, laparoscopic-assisted ovariohysterectomy in a 6-year-old, intact female Keeshond dog for the staging and treatment of a left-sided ovarian tumor. Abdominal access was obtained using a modified-Hasson technique allowing for placement of a multi-channel, single incision laparoscopic surgery port. Following carbon dioxide insufflation, superficial laparoscopic exploration of the abdominal cavity was performed and then both ovarian pedicles were sealed and divided using a vessel-sealing device. Laparoscopic-assisted ovariohysterectomy was performed with the aid of a wound retractor for exteriorization of the mass. There were no perioperative complications and the patient was discharged 1 day after surgery. Histopathology of the mass revealed an ovarian teratoma. Telephone follow-up 608 days after surgery revealed a good clinical outcome. Single-incision, laparoscopic-assisted ovariohysterectomy is technically feasible for the treatment of selected ovarian tumors in the dog.

Ovariohystérectomie à incision unique assistée par laparascopie pour une tumeur ovarienne chez une chienne. Ce rapport décrit une ovariohystérectomie à incision unique assistée par laparascopie chez une chienne Keeshond intacte âgée de 6 ans pour l'évaluation et le traitement d'une tumeur ovarienne du côté gauche. L'accès abdominal a été obtenu en utilisant une technique modifiée de Hasson pour le placement d'un accès chirurgical laparascopique à incision unique. Après l'insufflation de gaz carbonique, l'exploration laparascopique superficielle de la cavité abdominale a été réalisée et ensuite les deux pédicules ovariens ont été scellés et divisés à l'aide d'un dispositif de scellement des vaisseaux. L'ovariohystérectomie assistée par laparascopie a été réalisée à l'aide d'un rétracteur de plaie pour l'extériorisation de la masse. Il n'y a eu aucune complication péri-opératoire et la patiente a reçu son congé 1 jour après la chirurgie. L'histopathologie de la masse a révélé un tératome ovarien. Un suivi par téléphone 608 jours après la chirurgie a révélé un bon résultat clinique. L'ovariohystérectomie laparascopique à incision unique est techniquement réalisable pour le traitement de certaines tumeurs ovariennes chez les chiennes.(Traduit par Isabelle Vallières).

Vaginal impedometry for detection of optimal breeding time in bitches.

OBJECTIVE: To compare the efficacy of canine vaginal impedometry in identifying the preovulatory luteinizing hormone (LH) peak to that of currently used methods (serum progesterone concentration measurement, vaginal cytologic evaluation, and vaginoscopy). DESIGN: Prospective study. ANIMALS: 12 sexually intact female dogs. PROCEDURES: 12 mature postpubertal Beagle (n = 3), Beagle-cross (2), and hound-cross (7) bitches ranging from 7.5 to 27.5 kg (16.5 to 60.6 lb) were enrolled in the study. After the onset of spontaneous proestrus, determined on the basis of appearance of serosanguineous vaginal discharge, serum progesterone assays, vaginoscopy, vaginal cytologic evaluation, and vaginal impedometry were performed daily until approximately 4 days after peak LH concentration (day 0) as measured by radioimmunoassay. Vaginal impedometry was compared against serum progesterone concentration measurement, vaginal cytologic evaluation, and vaginoscopy as a method for accurately identifying the LH peak and therefore the optimal breeding time. Ten of 12 bitches were bred with subsequent assessment of embryos. RESULTS: Vaginal impedometry accurately predicted the preovulatory LH peak in 5 of 11 bitches. One bitch was removed from the study because data were not collected. Of the remaining 11 bitches, 6 had their LH peak on the day serum progesterone concentration first exceeded 2 ng/mL. Crenulation scores reached 1 (mean, 1.3; 95% confidence interval, 0.8 to 1.7) on day 0 as expected; however, these scores were not significantly different from those on days -1 or 1. Vaginal epithelial cell populations did not change noticeably on day 0. Nine of the 10 bitches that were bred produced viable embryos. CONCLUSIONS AND CLINICAL RELEVANCE: Results suggested that daily use of vaginal impedometry in bitches was unreliable as a method for monitoring periovulatory events. All techniques evaluated (ie vaginal impedometry, serum progesterone concentration assays, vaginoscopy and vaginal cytologic evaluation) frequently produced inaccurate results when used individually. Multiple methods should be used to identify optimal breeding time in dogs.

An evaluation of B-mode and color Doppler ultrasonography for detecting periovulatory events in the bitch.

When determining optimal breeding time in the bitch, specific periovulatory events must be identified. The main objectives were to relate ultrasonographic changes in ovarian blood flow, follicle/corpora lutea count and echotexture to periovulatory events, and to assess the efficacy of each for identifying these events. Twelve Beagle (N = 3), Beagle-cross (N = 2) and hound-cross (N = 7) bitches (body weight range, 7.5-27.5 kg) were examined daily from the onset of proestrus to approximately 4 days post-LH peak. Follicle and corpora lutea count and echotexture analyses were performed using B-mode ultrasound and ovarian blood flow analysis was performed using color Doppler ultrasound. Serum LH concentrations were analyzed by validated RIA. There was an increase (P < 0.05) in ovarian blood flow from the day of the preovulatory LH peak (605 pixels; confidence interval, 397-856), to 1 day after this peak (1092 pixels; confidence interval, 724-1535), enabling detection of the preovulatory LH peak. There were no significant changes in follicle/corpora lutea echotexture relative to days from the preovulatory LH peak. There were significant decreases in follicle/corpora lutea number between Days -1 and 3; Days -1 and 4; and Days 0 and 3, relative to the preovulatory LH peak. We concluded that color Doppler ultrasound performed once daily was more accurate in identifying the preovulatory LH peak than B-mode ultrasound and enabled prospective determination of ovulation.

Synaptically-competent neurons derived from canine embryonic stem cells by lineage selection with EGF and Noggin.

Pluripotent stem cell lines have been generated in several domestic animal species; however, these lines traditionally show poor self-renewal and differentiation. Using canine embryonic stem cell (cESC) lines previously shown to have sufficient self-renewal capacity and potency, we generated and compared canine neural stem cell (cNSC) lines derived by lineage selection with epidermal growth factor (EGF) or Noggin along the neural default differentiation pathway, or by directed differentiation with retinoic acid (RA)-induced floating sphere assay. Lineage selection produced large populations of SOX2+ neural stem/progenitor cell populations and neuronal derivatives while directed differentiation produced few and improper neuronal derivatives. Primary canine neural lines were generated from fetal tissue and used as a positive control for differentiation and electrophysiology. Differentiation of EGF- and Noggin-directed cNSC lines in N2B27 with low-dose growth factors (BDNF/NT-3 or PDGFαα) produced phenotypes equivalent to primary canine neural cells including 3CB2+ radial progenitors, MOSP+ glia restricted precursors, VIM+/GFAP+ astrocytes, and TUBB3+/MAP2+/NFH+/SYN+ neurons. Conversely, induction with RA and neuronal differentiation produced inadequate putative neurons for further study, even though appropriate neuronal gene expression profiles were observed by RT-PCR (including Nestin, TUBB3, PSD95, STX1A, SYNPR, MAP2). Co-culture of cESC-derived neurons with primary canine fetal cells on canine astrocytes was used to test functional maturity of putative neurons. Canine ESC-derived neurons received functional GABA(A)- and AMPA-receptor mediated synaptic input, but only when co-cultured with primary neurons. This study presents established neural stem/progenitor cell populations and functional neural derivatives in the dog, providing the proof-of-concept required to translate stem cell transplantation strategies into a clinically relevant animal model.

Migration of cells from the yolk sac to hematopoietic tissues after in utero transplantation of early and mid gestation canine fetuses.

BACKGROUND: In utero hematopoietic cell transplantation offers a means of early intervention for the treatment of diseases before birth. Delivery of cells to the yolk sac is a minimally invasive approach that results in low levels of chimerism. However, there is little information on the optimal doses, timing of delivery, and migration of transplanted cells from the yolk sac into the fetus. METHODS: Varying cell doses of mesenchymal stromal cells or bone marrow mononuclear cells labeled with fluorescent supraparamagnetic iron oxide nanoparticles and a fluorescent intracellular dye, 5- and 6-([(4-chloromethyl)benzoyl]-amino) tetramethylrhodamine, were transplanted under ultrasound guidance to the yolk sacs of day 25 or day 35 canine fetuses. Ex vivo whole body fluorescence imaging and microscopy of tissue sections were correlated with the presence of iron oxide in injected and control fetuses. RESULTS: Day 25 and day 35 recipients showed similar survival rates after injection of cells into yolk sacs, although increased fetal morality was associated with cell doses greater than 10 cells/kg to day 25 fetuses. The fluorescence and iron oxide signals were predominantly localized to the abdominal regions, with no fluorescence visible in yolk sacs. Microscopy of tissues revealed colocalization of fluorophore with iron oxide in donor cells detected in the fetal livers and bone marrow of recipients 7 and 17 days after receiving mesenchymal stromal cells or bone marrow mononuclear cells. CONCLUSIONS: These studies demonstrated that cells injected into the yolk sacs of early gestation canine fetuses migrate to recipient hematopoietic tissues. Thus, yolk sac injection offers a safe and effective approach for engraftment of cells to fetal hematopoietic tissues.

Assessing reproductive patterns and disorders in free-ranging dogs in Jodhpur, India to optimize a population control program.

The objectives were to test the hypothesis that estrus and pregnancy are seasonal in free-ranging female dogs (>3 mo old) in Jodhpur, India, and to determine litter size, and the prevalence of fetal resorption in this population. The prevalence of estrus and pregnancy was determined in 5400 free-ranging bitches (trapped and released) at the time of ovariohysterectomy. In a separate study, the uteri and ovaries of 246 free-ranging bitches were examined to determine litter size and fetal resorption. The bitches exhibited seasonal estrus and pregnancy (P < 0.00001), with a higher percentage of bitches in estrus or pregnant during the late monsoon season (September to November) compared to the other three seasons. The mean litter size based on embryo/fetal counts was 4.6 (95% CI = 4.0-5.3; n = 40) and based upon placental site counts was 4.4 (95% CI = 3.9-4.8; n = 105). Prevalence of fetal resorption was 32.6% (95% CI = 20.5-47.5; n = 43) with a mean of 2.8 resorptions per litter in those with at least one resorption (95% CI = 1.8-3.8; n = 14). This was the first study to estimate previous litter size of non-pregnant, free-ranging dogs based upon placental sites. Litter size data from this study will be used in a population demographic model to predict the long-term impact of animal birth control (ABC) on the free-ranging dog population in Jodhpur. Increasing the efforts to surgically sterilize bitches prior to the time of year of peak pregnancy or whelping will help maximize the impact of an ABC program on the Jodhpur free-ranging dog population.

Characterization of canine embryonic stem cell lines derived from different niche microenvironments.

Embryo-derived stem cells hold enormous potential for producing cell-based transplantation therapies, allowing high-throughput drug screening and delineating early embryonic development. However, potential clinical applications must first be tested for safety and efficacy in preclinical animal models. Due to physiological and genetic parity to humans, the domestic dog is widely used as a clinically relevant animal model for cardiovascular, neurodegenerative, orthopedic, and oncologic diseases. Therefore, we established numerous putative canine embryonic stem cell (cESC) lines by immunodissection of the inner cell mass (ICM), which we termed OVC.ID.1-23, and by explant outgrowths from whole canine blastocysts, named OVC.EX.1-16. All characterized lines were immunopositive for OCT4, SOX2, NANOG, SSEA-3, and SSEA-4; displayed high telomerase and alkaline phosphatase (ALP) activities; and were maintained in this state up to 37 passages ( approximately 160 days). Colonies from OVC.EX lines showed classic domed hESC-like morphology surrounded by a ring of fibroblast-like cells, whereas all OVC.ID lines exhibited a mixed cell colony of tightly packed cESCs surrounded by a GATA6+/CDX2- hypoblast-derived support layer. Spontaneous serum-only differentiation without feeder layers demonstrated a strong lineage selection associated with the colony niche type, and not the isolation method. Upon differentiation, cESC lines formed embryoid bodies (EB) comprised of cells representative of all germinal layers, and differentiated into cell types of each layer. Canine ESC lines such as these have the potential to identify differences between embryonic stem cell line derivations, and to develop or to test cell-based transplantation therapies in the dog before attempting human clinical trials.

Derivation and characterization of canine embryonic stem cell lines with in vitro and in vivo differentiation potential.

Embryonic stem cells (ESCs) represent permanent cell lines that can be maintained in an undifferentiated state. In an environment that induces differentiation, they form derivatives of the three embryonic germ layers: mesoderm, ectoderm, and endoderm. These characteristics give ESCs great potential for both basic research and clinical applications in the areas of regenerative medicine and tissue engineering. The establishment of ESCs from large animals that model human diseases is of significant importance. We describe the derivation of permanent canine cell lines from preimplantation-stage embryos. Similar to human ESCs, canine ESCs expressed OCT3/4, NANOG, SOX2, SSEA-3, SSEA-4, TRA-1-60, TRA-1-81, and alkaline phosphatase, whereas they expressed very low levels of SSEA-1. They maintained a normal karyotype and morphology typical of undifferentiated ESCs after multiple in vitro passages and rounds of cryopreservation. Plating cells in the absence of a feeder layer, either in attachment or suspension culture, resulted in the formation of embryoid bodies and their differentiation to multiple cell types. In vivo, canine ESCs gave rise to teratomas comprising cell types of all three embryonic germ layers. These cells represent the first pluripotent canine ESC lines with both in vitro and in vivo differentiation potential and offer the exciting possibility of testing the efficacy and safety of ESC-based therapies in large animal models of human disease.

A comparison of the cytobrush and uterine lavage techniques to evaluate endometrial cytology in clinically normal postpartum dairy cows.

This study compares cytobrush and lavage techniques for the assessment of endometrial cytology (EC) in clinically normal postpartum dairy cows. The EC samples were collected from Holstein cows (n = 35) during visit 1 (V1) at 20 to 33 d in milk (DIM) and 2 wk later during visit 2 (V2) at 34 to 47 DIM by using both techniques. A minimum of 100 cells were counted to determine the percentage of cells that were neutrophils (%PMN). The mean %PMN was significantly different between the techniques at V1 (P = 0.001), but not at V2 (P = 0.474). Overall, the %PM N decreased with time postpartum (r2 = 0.36; P = 0.001), but not within V1 (P > 0.05) or V2 (P > 0.1). Uterine diameter was negatively correlated with fluid recovery by the lavage technique (r2 = 0.41; P = 0.002). The mean %PMN was not influenced by the volume of fluid recovered in successful attempts, but 17% (12/70) of attempts yielded no fluid. In conclusion, the cytobrush technique is a consistent and reliable method for obtaining endometrial samples for cystologic examination from postpartum dairy cows.

Cyclosporine a slows the progressive renal disease of alport syndrome (X-linked hereditary nephritis): results from a canine model.

Alport syndrome refers to a hereditary disorder characterized by progressive renal disease and a multilaminar appearance to the glomerular basement membrane (GBM). In a small group of patients with Alport syndrome, cyclosporine A was reported to decrease proteinuria and maintain stable renal function over 7 to 10 yr of follow-up. The present study examined the effect of cyclosporine A on GBM structure and the progression to renal failure in a canine model of X-linked Alport syndrome. Affected male dogs and normal male dogs treated with cyclosporine A underwent serial renal biopsies. Body weight, serum concentrations of creatinine and albumin, and GFR were sequentially determined. Controls consisted of untreated dogs that developed end-stage renal failure by 8 mo of age. Renal biopsies were assessed for glomerulosclerosis and the percent of multilaminar GBM as measured by image analysis. Significant differences were found between treated and untreated affected dogs for weight, serum creatinine, and GFR. There was a significant delay in the progression of multilaminar change to the GBM, although treated affected dogs at termination had attained approximately 100% split GBM as did untreated affected dogs. A significant difference in the number of sclerotic glomeruli was also noted; treated dogs rarely developed obsolete glomeruli during the period studied. Interstitial fibrosis was not significantly affected by cyclosporine A treatment. These findings indicate that cyclosporine A is beneficial in slowing, but not stopping, the clinical and pathologic progression of Alport syndrome. At least part of this beneficial effect comes from a delayed deterioration of GBM structure, which in turn may be related to glomerular hemodynamics altered by cyclosporine A.

In utero injection of alpha-L-iduronidase-carrying retrovirus in canine mucopolysaccharidosis type I: infection of multiple tissues and neonatal gene expression.

Canine alpha-L-iduronidase (alpha-ID) deficiency is caused by a single base pair mutation in the alpha-ID gene, resulting in no enzyme activity in homozygous affected pups. The disease clinically resembles human mucopolysaccharidosis type I (MPSI). We used the canine MPSI model system to address the efficacy of a new retroviral vector, MND-MFG, containing the human alpha-ID cDNA (MND-MFG-alpha-ID) for direct in utero gene delivery to MPSI cells. In vitro, the MND-MFG-alpha-ID vector showed high-level, long-term expression of the transgene in both canine and human alpha-ID-deficient fibroblasts. The effectiveness of this vector for in utero gene transfer and expression in multiple tissues was assessed by injecting viral supernatants into MPSI fetuses and evaluating transduction efficiency and enzyme expression at various times after birth. Transduction of a spectrum of cell types and tissues was observed in all seven live-born pups and in one stillborn pup. Although enzyme activity was not detected in adult tissues from the seven surviving pups, significant alpha-ID enzyme activity was detected in both the liver and kidney of the deceased pup. Our combined gene delivery vector and in utero transfer approach, while encouraging in terms of overall gene transfer efficiency to multiple tissues and successful short-term gene expression, was unable to meet the important requirement of sustained in vivo gene expression.