Vicki N. Meyers-Wallen

Accuracy of canine parturition date prediction using fetal measurements obtained by ultrasonography.

The length of canine gestation is 65 days from the luteinizing hormone (LH) surge. Early and accurate determination of canine gestational age is useful for predicting and managing parturition. We performed a retrospective study on fetal measurements obtained by transabdominal ultrasonographic examination of 83 bitches (32 breeds) to estimate gestational age. Gestational age was estimated using two published tables correlating either (1). embryonic vesicle diameter (EVD), crown-rump length (CRL), body diameter (BD), and biparietal diameter (HD) to the LH surge in mid-gestational beagles or (2). BD and HD to parturition in late-gestation retrievers. Parturition date was predicted by obtaining the difference between the gestational age estimate and 65 days. Bitches were divided into four body weight (BW) groups based on nonpregnant body weight: small (<or=9 kg), medium (>9-20 kg), large (>20-40 kg), and giant (>40 kg). Mean+/-S.D. litter size (LS) was calculated for each BW group. The BW groups were then divided into small, average, or large LS groups. The accuracy of the prediction was not affected by LS but was affected by maternal body weight for small and giant BW groups only. When adjusted for weight, the accuracy of prediction within +/-1 day and +/-2 day intervals was 75 and 87%, respectively. Using stepwise logisitic regression, the most accurate prediction of parturition date was obtained when fetuses were measured at 30 days after the LH surge, regardless of body weight or LS. Parturition date predictions made after 39 days of gestation using only biparietal and BD fetal measurements were <50% accurate within +/-2 days.

Inherited ventricular arrhythmias and sudden death in German shepherd dogs

OBJECTIVES This report describes a unique group of German shepherd dogs with inherited ventricular arrhythmias and sudden death. Before death, these dogs have no evidence of cardiovascular failure. BACKGROUND There are few spontaneous animal models of sudden death that permit intensive investigation. METHODS To determine the temporal evolution of ventricular arrhythmias and to characterize the syndrome of sudden cardiac death in these dogs, 24-h ambulatory electrocardiographic (ECG) monitoring, echocardiograms, electrophysiologic testing and breeding studies were conducted. RESULTS The 24-h ambulatory ECGs from dogs that died showed frequent ventricular arrhythmias with rapid polymorphic ventricular tachycardia (rates > 480 beats/min). Affected dogs had a window of vulnerability for arrhythmias, with the highest incidence and severity of arrhythmias between 20 to 30 and 40 to 50 weeks of age. Affected dogs that died did not have prolongation of the QT interval over a spectrum of heart rates compared with unaffected dogs. The clinical arrhythmia was not induced in dogs during programmed electrical stimulation. Severely affected dogs monitored > 5 years did not develop any evidence of heart failure or cardiomyopathy, and no histopathologic abnormalities existed. Seventeen dogs died suddenly (age 4 to 30 months) and were either 1) found dead at first observation in the morning (n = 8), 2) observed to die during sleep (n = 4), 3) observed to die while resting after exercise (n = 3), or 4) observed to die during exercise (n = 2). All sudden deaths occurred between the end of September and April, with most (n = 11) during January and February. CONCLUSIONS The cause of the inherited severe ventricular arrhythmias and sudden death in these young German shepherd dogs is still undetermined. A purely arrhythmic disorder is supported by the lack of cardiac pathology. Moreover, the window of vulnerability to ventricular arrhythmias and the age and circumstances of death invite speculation about the role of the autonomic nervous system.

Accuracy of canine parturition date prediction from the initial rise in preovulatory progesterone concentration

Accurate prediction of parturition date is useful for clinical management of canine parturition. For nearly all normal canine pregnancies, parturition occurs 64-66 days from the LH peak, the timing of which cannot be differentiated from the initial sharp rise in serum progesterone (P4) concentrations. We sought to determine by retrospective analysis if prebreeding serum progesterone concentrations could accurately predict parturition date. Serum progesterone concentrations recorded as serial samples from 63 bitches (19 breeds) were analyzed. Progesterone concentrations were measured by radioimmunoassay (RIA) or chemiluminescent immunoassay (CLIA). The CLIA method was validated for use in determining P4 concentrations in canine serum and results were comparable to those obtained with RIA. Bitches were grouped by nonpregnant body weight (BW) and litter size (LS). Day 0 (D0), the day of preovulatory rise in serum P4, was defined as the day that P4 concentration rose to > or =l.5 ng/ml and was at least twice the baseline concentration. The predicted parturition date, 65 days following the day of preovulatory rise in serum P4 (D65), was compared to actual parturition date, the day the first pup was delivered. We determined that mean P4 concentration at D0 for all BW groups was 2.02+/-0.18 ng/ml and there was significant variation in P4 concentrations between BW groups after D1. In addition, we determined that the accuracy of parturition date prediction within a +/-1, +/-2, and +/-3 day interval using prebreeding serum progesterone concentrations was 67, 90, and 100%, respectively, and that the accuracy was not affected by body weight or litter size.

Sry-negative XX sex reversal in purebred dogs

The gene responsible for testis induction in normal male mammals is the Y‐linked Sry. However, there is increasing evidence that other genes may have testis‐determining properties. In XX sex reversal (XXSR), testis tissue develops in the absence of the Y chromosome. Previous polymerase chain reaction (PCR) assays indicated that autosomal recessive XXSR in the American cocker spaniel is Sry‐negative. In this study, genomic DNA from the breeding colony of American cocker spaniels and from privately owned purebred dogs were tested by PCR using canine primers for the Sry HMG box and by Southern blots probed with the complete canine Sry coding sequence. Sry was not detected by either method in genomic DNA of affected American cocker spaniels or in the majority (20/21) of affected privately owned purebred dogs. These results confirm that the autosomal recessive form of XXSR in the American cocker spaniel is Sry‐negative. In combination with previous studies, this indicates that Sry‐negative XXSR occurs in at least 15 dog breeds. The canine disorder may be genetically heterogeneous, potentially with a different mutation in each breed, and may provide several models for human Sry‐negative XXSR. A comparative approach to sex determination should be informative in defining the genetic and cellular mechanisms that are common to all mammals. Mol. Reprod. Dev. 53:266–273, 1999.

XX sex reversal in the American cocker spaniel dog: phenotypic expression and inheritance

SummaryThis study was conducted to define the range of phenotypic expression and mode of inheritance of XX sex reversal in the cocker spaniel dog. Breeding experiments produced F1, F1BC, and F2 generations in which 29 XX true hermaphrodites and 3 XX males were defined by chromosome constitution, serial histologic sections of the gonads, and examination of the internal and external genitalia. In XX true hermaphrodites, the most common combination of gonads was bilateral ovotestes, followed by ovotestis and ovary, then ovotestis and testis. The amount of testicular tissue in the two gonads was closely correlated within each true hermaphrodite. The distribution of testicular tissue within ovotestes of true hermaphrodites was consistent with the hypothesis that testicular differentiation is initiated in the center of the gonad and spreads outward. XX males had bilateral aspermatogenic testes and the internal ducts and external genitalia were more masculinized than in true hermaphrodites. Results of breeding experiments are consistent with autosomal recessive inheritance, the affected phenotype being expressed only in dogs with an XX chromosome constitution. The phenotypic expression and mode of inheritance of this disorder is compared to XX sex reversal in humans and other animals.

Gonadal and sex differentiation abnormalities of dogs and cats.

The molecular steps in normal sexual development were largely discovered by studying patients and animal models with disorders of sexual development (DSD). Although several types of DSD have been reported in the cat and dog, which are often strikingly similar to human DSD, these have been infrequently utilized to contribute to our knowledge of mammalian sexual development. Canine and feline cases of DSD with sufficient evidence to be considered as potential models are summarized in this report. The consensus DSD terminology, and reference to previous terminology, is used to foster adoption of a common nomenclature that will facilitate communication and collaboration between veterinarians, physicians, and researchers. To efficiently utilize these unique resources as molecular tools continue to improve, it will be helpful to deposit samples from valuable cases into repositories where they are available to contribute to our understanding of sexual development, and thus improve human and animal health.

A Single Base Pair Mutation Encoding a Premature Stop Codon in the MIS Type II Receptor Is Responsible for Canine Persistent Müllerian Duct Syndrome

Müllerian inhibiting substance (MIS), a secreted glycoprotein in the transforming growth factor-beta family of growth factors, mediates regression of the Müllerian ducts during embryonic sex differentiation in males. In persistent Müllerian duct syndrome (PMDS), rather than undergoing involution, the Müllerian ducts persist in males, giving rise to the uterus, fallopian tubes, and upper vagina. Genetic defects in MIS or its receptor (MISRII) have been identified in patients with PMDS. The phenotype in the canine model of PMDS derived from the miniature schnauzer breed is strikingly similar to that of human patients. In this model, PMDS is inherited as a sex-limited autosomal recessive trait. Previous studies indicated that a defect in the MIS receptor or its downstream signaling pathway was likely to be causative of the canine syndrome. In this study, the canine PMDS phenotype and clinical sequelae are described in detail. Affected and unaffected members of this pedigree are genotyped, identifying a single base pair substitution in MISRII that introduces a stop codon in exon 3. The homozygous mutation terminates translation at 80 amino acids, eliminating much of the extracellular domain and the entire transmembrane and intracellular signaling domains. Findings in this model could enable insights to be garnered from correlation of detailed clinical descriptions with molecular defects, which are not otherwise possible in the human syndrome.

Review and Update: Genomic and Molecular Advances in Sex Determination and Differentiation in Small Animals

Inherited disorders of sexual development are important to identify as a cause of inherited infertility or sterility in humans and animals. Investigation of these disorders in dogs and cats can identify new mutations, allowing us to eliminate inherited disorders from breeding populations, while contributing to the understanding of mammalian sexual development and differentiation. This review updates an overview of normal mammalian sexual development while discussing disorders of sexual development at three consecutive levels, as errors in sex chromosome constitution, gonadal sex determination or phenotypic sexual development. The molecular mechanisms controlling sexual development and current molecular methods to identify causative mutations are illustrated in three specific examples of abnormal sexual development reported in small animals: XX sex reversal, Persistent Mullerian Duct Syndrome and cryptorchidism. Identification of causative mutations and development of practical tests to identify carrier and affected animals will provide effective mechanisms to reduce the prevalence of these disorders in small animals.

Mutations in the RSPO1 coding region are not the main cause of canine SRY-negative XX sex reversal in several breeds.

This report details a case of SRY-negative XX sex reversal in a mixed breed dog and surveys affected dogs of several breeds for mutations in RSPO1 coding regions. Genomic DNA from the mixed breed case was evaluated for mutations in candidate genes. Sequencing identified a homozygous G to A transition in RSPO1 exon 4 that changes a highly conserved amino acid codon in the thrombospondin domain. The possibility that this was a single nucleotide polymorphism (SNP) could not be excluded by genotyping family members. Therefore, the coding region of RSPO1 was sequenced in a survey of affected dogs, which identified a T to C transition (exon 3) in some, the above G to A transition (exon 4) in others, and no change in the remaining affected dogs. Genotypes at these base pair positions were not uniquely associated with the affected phenotype in any breed, indicating the identified transitions are most likely SNPs, not causative mutations for this canine disorder. However, the possibility that polymorphisms play a modifier role, such as changing threshold or severity of phenotypic expression in a mixed breed dog, cannot be excluded. This study emphasizes the importance of canine pedigree, breed, and population studies in evaluating candidate mutations.

Development of New Stem Cell-based Technologies for Carnivore Reproduction Research

New reproductive technologies based on stem cells offer several potential benefits to carnivore species. For example, development of lines of embryonic stem cells in cats and dogs would allow for the generation of transgenic animal models, which could be used to advance both veterinary and human health. Techniques such as spermatogonial stem cell transplantation (SSCT) and testis xenografting offer new approaches to propagate genetically valuable individual males, even if they should die before producing sperm. These techniques might therefore have application to the conservation of endangered species of carnivores, as well as to biomedical research. Recently, our laboratory has successfully performed SSCT in the dog, with a recipient dog producing sperm of donor genetic origin. Testis xenografting has been used to produce sperm from pre-pubertal testis tissue from both cats and ferrets. These early steps reinforce the need not only for research on stem cell technologies, but also for additional research into complementary technologies of assisted reproduction in carnivores, so that the widest array of research and clinical benefits can be realized.