A social encounter drives gene expression changes linked to neuronal function, brain development, and related disorders in mice expressing the serotonin transporter Ala56 variant.

Multiple lines of evidence implicate the serotonin (5-HT) system in social function, including biomarker findings in autism spectrum disorder. In mice, knock-in of a rare Gly56Ala substitution in the serotonin transporter (SERT) causes elevated whole blood 5-HT levels, increased 5-HT clearance in the brain, and altered social and repetitive behavior. To further examine the molecular impact of this variant on social response, SERT Ala56 mutant mice and wildtype littermate controls were exposed to a social or non-social stimulus. We examined the differential activation of the prefrontal cortex, lateral amygdala, and medial amygdala, to social stimuli through RNA sequencing. Differentially expressed genes were enriched in axonal guidance signaling pathways, networks related to nervous system development and function, neurological and psychiatric disorders, and behavior. These identified pathways and networks may shed light on the molecular cascades underlying the impact of altered SERT function on social behavior.

Enhanced Social Dominance and Altered Neuronal Excitability in the Prefrontal Cortex of Male KCC2b Mutant Mice.

The K-Cl cotransporter KCC2 is essential in the development of the "GABA switch" that produces a change in neuronal responses to GABA signaling from excitatory to inhibitory early in brain development, and alterations in this progression have previously been hypothesized to play a causal role in autism spectrum disorder (ASD). We investigated the KCC2b (Slc12a5) heterozygous knockout mouse using a battery of rodent behavioral tests relevant to core and comorbid ASD symptoms. Compared to wild-type littermates, KCC2+/- mice were normal in standard measures of locomotor activity, grooming and digging behaviors, and social, vocalization, and anxiety-like behaviors. However, KCC2+/- mice exhibited increased social dominance behaviors and increased amplitude of spontaneous postsynaptic currents in the medial prefrontal cortex (PFC) that were previously implicated in governing social hierarchy and dominance behaviors. Treatment of wild-type mouse brain slices with the KCC2 inhibitor VU0240511 increased the amplitude and frequency of excitatory postsynaptic currents, partially recapitulating the phenotype of KCC2+/- mice. These findings indicate that the activity of KCC2 plays a role in social dominance, in parallel with effects on PFC signaling, further suggesting that KCC2 function has some relevance to social behavior but without the breadth of impact on autism-like behavior suggested by previous studies. Further testing could assess whether KCC2 alters other circuits and whether additional factors such as environmental insults may precipitate autism-related behavioral phenotypes. Autism Research 2019, 12: 732-743. © 2019 International Society for Autism Research, Wiley Periodicals, Inc. LAY SUMMARY: A mouse model of altered chloride transporter expression was used to look for a role in behaviors and brain function relevant to autism. There was an imbalance in signaling in the prefrontal cortex, and increased social dominance behavior, although other autism-related behaviors were not changed. These findings indicate that altered chloride transporter function affects prefrontal cortex function and social dominance without a broader impact on autism-like behaviors.

Effects of a social stimulus on gene expression in a mouse model of fragile X syndrome.

BACKGROUND: People with fragile X syndrome (FXS) often have deficits in social behavior, and a substantial portion meet criteria for autism spectrum disorder. Though the genetic cause of FXS is known to be due to the silencing of FMR1, and the Fmr1 null mouse model representing this lesion has been extensively studied, the contributions of this gene and its protein product, FMRP, to social behavior are not well understood. METHODS: Fmr1 null mice and wildtype littermates were exposed to a social or non-social stimulus. In one experiment, subjects were assessed for expression of the inducible transcription factor c-Fos in response to the stimulus, to detect brain regions with social-specific activity. In a separate experiment, tissue was taken from those brain regions showing differential activity, and RNA sequencing was performed. RESULTS: Immunohistochemistry revealed a significantly greater number of c-Fos-positive cells in the lateral amygdala and medial amygdala in the brains of mice exposed to a social stimulus, compared to a non-social stimulus. In the prelimbic cortex, there was no significant effect of social stimulus; although the number of c-Fos-positive cells was lower in the social condition compared to the non-social condition, and negatively correlated with c-Fos in the amygdala. RNA sequencing revealed differentially expressed genes enriched for molecules known to interact with FMRP and also for autism-related genes identified in the Simons Foundation Autism Research Initiative gene database. Ingenuity Pathway Analysis detected enrichment of differentially expressed genes in networks and pathways related to neuronal development, intracellular signaling, and inflammatory response. CONCLUSIONS: Using the Fmr1 null mouse model of fragile X syndrome, we have identified brain regions, gene networks, and molecular pathways responsive to a social stimulus. These findings, and future experiments following up on the role of specific gene networks, may shed light on the neural mechanisms underlying dysregulated social behaviors in fragile X syndrome and more broadly.

An autism-associated serotonin transporter variant disrupts multisensory processing.

Altered sensory processing is observed in many children with autism spectrum disorder (ASD), with growing evidence that these impairments extend to the integration of information across the different senses (that is, multisensory function). The serotonin system has an important role in sensory development and function, and alterations of serotonergic signaling have been suggested to have a role in ASD. A gain-of-function coding variant in the serotonin transporter (SERT) associates with sensory aversion in humans, and when expressed in mice produces traits associated with ASD, including disruptions in social and communicative function and repetitive behaviors. The current study set out to test whether these mice also exhibit changes in multisensory function when compared with wild-type (WT) animals on the same genetic background. Mice were trained to respond to auditory and visual stimuli independently before being tested under visual, auditory and paired audiovisual (multisensory) conditions. WT mice exhibited significant gains in response accuracy under audiovisual conditions. In contrast, although the SERT mutant animals learned the auditory and visual tasks comparably to WT littermates, they failed to show behavioral gains under multisensory conditions. We believe these results provide the first behavioral evidence of multisensory deficits in a genetic mouse model related to ASD and implicate the serotonin system in multisensory processing and in the multisensory changes seen in ASD.

Impact of Maternal Serotonin Transporter Genotype on Placental Serotonin, Fetal Forebrain Serotonin, and Neurodevelopment.

Biomarker, neuroimaging, and genetic findings implicate the serotonin transporter (SERT) in autism spectrum disorder (ASD). Previously, we found that adult male mice expressing the autism-associated SERT Ala56 variant have altered central serotonin (5-HT) system function, as well as elevated peripheral blood 5-HT levels. Early in gestation, before midbrain 5-HT projections have reached the cortex, peripheral sources supply 5-HT to the forebrain, suggesting that altered maternal or placenta 5-HT system function could impact the developing embryo. We therefore used different combinations of maternal and embryo SERT Ala56 genotypes to examine effects on blood, placenta and embryo serotonin levels and neurodevelopment at embryonic day E14.5, when peripheral sources of 5-HT predominate, and E18.5, when midbrain 5-HT projections have reached the forebrain. Maternal SERT Ala56 genotype was associated with decreased placenta and embryonic forebrain 5-HT levels at E14.5. Low 5-HT in the placenta persisted, but forebrain levels normalized by E18.5. Maternal SERT Ala56 genotype effects on forebrain 5-HT levels were accompanied by a broadening of 5-HT-sensitive thalamocortical axon projections. In contrast, no effect of embryo genotype was seen in concepti from heterozygous dams. Blood 5-HT levels were dynamic across pregnancy and were increased in SERT Ala56 dams at E14.5. Placenta RNA sequencing data at E14.5 indicated substantial impact of maternal SERT Ala56 genotype, with alterations in immune and metabolic-related pathways. Collectively, these findings indicate that maternal SERT function impacts offspring placental 5-HT levels, forebrain 5-HT levels, and neurodevelopment.

Incidence and risk factors of hepatocellular carcinoma after orthotopic liver transplantation.

BACKGROUND: The incidence of hepatocellular carcinoma has doubled over the past 2 decades, becoming the fifth most common cancer worldwide. Orthotopic liver transplant is the gold standard treatment for those with hepatocellular carcinoma meeting eligibility criteria, although recurrence rates of hepatocellular carcinoma after orthotopic liver transplant still remain an understudied obstacle. METHODS: We performed a single-center, retrospective, longitudinal study with the aim of determining the predominant baseline and follow-up variables associated with hepatocellular carcinoma recurrence. We gathered pre- and post-transplant data and conducted univariate and multivariate analysis to assess variables predicting hepatocellular carcinoma recurrence after orthotopic liver transplant. RESULTS: Between 2003 and 2015, 141 patients underwent orthotopic liver transplant for hepatocellular carcinoma. We identified 9 (6.4%) cases of documented hepatocellular carcinoma recurrence. Univariate analysis indicated that the difference in serum alpha-fetoprotein levels (most recent prior to transplant subtracted from maximum level) was lower in the hepatocellular carcinoma recurrence group (median 3 ng/mL vs 0 ng/mL, P = .052) as well as the pretransplant serum cholesterol level (median 158 mg/dL vs 113 mg/dL, P = .019) and days between hepatocellular carcinoma neoadjuvant treatment initiation and transplantation (median 122 vs 0, P = .045). Multivariate analysis revealed that a low pretransplant serum cholesterol level (<100 mg/dL) was independently associated with hepatocellular carcinoma recurrence (hazard ratio 11.0, P = .004). CONCLUSION: The risk of hepatocellular carcinoma recurrence after orthotopic liver transplant was low, at 6.4%, in this cohort. Low pretransplant serum cholesterol was the strongest predictor of recurrence and may help clinicians risk stratify patients for appropriate post-transplant monitoring and follow-up.

Depression symptom trends and health domains among lung cancer patients in the CanCORS study.

OBJECTIVES: Among lung cancer patients depression symptoms are common and impact outcomes. The aims of this study were to determine risk factors that contribute to persistent or new onset depression symptoms during lung cancer treatment, and examine interactions between depression symptoms and health domains that influence mortality. MATERIALS AND METHODS: Prospective observational study in five healthcare systems and 15 Veterans Affairs medical centers. Patients in the Cancer Care Outcomes Research and Surveillance (CanCORS) Consortium with lung cancer were eligible. The 8-item Center for Epidemiologic Studies Depression (CES-D) scale was administered at baseline and follow-up. Scores ≥4 indicated elevated depressive symptoms. Health domains were measured using validated instruments. We applied logistic regression and Cox proportional hazards modeling to explore the association between depression symptoms, health domains, and mortality. RESULTS: Of 1790 participants, 38% had depression symptoms at baseline and among those still alive, 31% at follow-up. Risk factors for depression symptoms at follow-up included younger age (OR=2.81), female sex (OR=1.59), low income (OR=1.45), not being married (OR=1.74) and current smoking status (OR=1.80); high school education was associated with reduced odds of depression symptoms at follow-up, compared with lesser educational attainment (OR=0.74) (all p values <0.05). Patients with depression symptoms had worse health-related quality of life, vitality, cancer-specific symptoms, and social support than patients without depression symptoms (all p<0.001). The association between depression symptoms and increased mortality is greater among patients with more lung cancer symptoms (p=0.008) or less social support (p=0.04). CONCLUSIONS: Patient risk factors for depression symptoms at follow-up were identified and these subgroups should be targeted for enhanced surveillance. Patients with depression symptoms suffer across all health domains; however, only more lung cancer symptoms or less social support are associated with worse mortality among these patients. These potentially modifiable health domains suggest targets for possible intervention in future studies.

Recent trends in testosterone testing, low testosterone levels, and testosterone treatment among Veterans.

Low serum testosterone (T) is common and increasingly prevalent with increased age. Recent studies report an 'epidemic' of T prescribing and concern about unnecessary T treatment. We investigated the number of men tested for T, the prevalence of low serum T levels, and initiation of T treatment among those with low T levels in men treated at Veterans Affairs (VA) facilities in the Northwest US (VISN 20). We identified male Veterans aged 40-89 years and examined yearly proportions of men tested for T, found to have low T levels (total T < 280 ng/dL, free T < 34 pg/mL, or bioavailable T < 84 ng/dL), and subsequently treated with T from 2002 to 2011. We excluded men who had T treatment in the year prior and men with diagnoses of prostate or breast cancer. Treatment initiation was defined as the first prescription for T within a year following a low T test. From 2002 to 2011, the yearly population of eligible men in VISN 20 increased from 129 247 to 163 572. The proportion of men who had serum T tests increased from 3.2% in 2002 to 5.8% in 2011. Among the tested men, the percentage of men with low T levels increased from 35.0 to 47.3%. However, the proportion of men with low T levels who were given T treatment within a year decreased from 31.0 to 28.0%. Despite large increases in T testing, and detection of men with low T levels, there was a slight decrease in the proportion of men with low T levels who were treated with T. The decrease in T treatment during this time period contrasts with other studies and may be related to higher comorbidity in Veterans and/or VA formulary restrictions on the use of transdermal T formulations.

Diagnostic performance of the PTSD checklist and the Vietnam Era Twin Registry PTSD scale.

AIMS: Self-report questionnaires are frequently used in clinical and epidemiologic studies to assess post-traumatic stress disorder (PTSD). A number of studies have evaluated these scales relative to clinician administered structured interviews; however, there has been no formal evaluation of their performance relative to non-clinician administered epidemiologic assessments such as the Composite International Diagnostic Interview (CIDI). We examined the diagnostic performance of two self-report PTSD scales, the PTSD checklist (PCL) and the Vietnam Era Twin Registry (VET-R) PTSD scale, compared to the CIDI. METHODS: Data were derived from a large epidemiologic follow-up study of PTSD in 5141 Vietnam Era Veterans. Measures included the PCL, VET-R PTSD scale and CIDI. For both the PCL and VET-R PTSD scale, ROC curves, areas under the curve (AUC), sensitivity, specificity, % correctly classified, likelihood ratios, predictive values and quality estimates were generated based on the CIDI PTSD diagnosis. RESULTS: For the PCL and VET-R PTSD scale the AUCs were 89.0 and 87.7%, respectively. Optimal PCL cutpoints varied from the 31-33 range (when considering sensitivity and specificity) to the 36-56 range (when considering quality estimates). Similar variations were found for the VET-R PTSD, ranging from 31 (when considering sensitivity and specificity) to the 37-42 range (when considering quality estimates). CONCLUSIONS: The PCL and VET-R PTSD scale performed similarly using a CIDI PTSD diagnosis as the criterion. There was a range of acceptable cutpoints, depending on the metric used, but most metrics suggested a lower PCL cutpoint than in previous studies in Veteran populations.

Comparative carcass and tissue nutrient composition of transgenic Yorkshire pigs expressing phytase in the saliva and conventional Yorkshire pigs.

A transgenic line of Yorkshire (YK) pigs named the Cassie (CA) line was produced with a low copy number phytase transgene inserted in the genome. The transgenic line efficiently digests P, Ca, and other major minerals of plant dietary origin. The objectives of this study were to 1) compare carcass and tissue nutrient composition and meat quality traits for third generation hemizygous CA line market BW finisher pigs (n = 24) with age-matched conventional YK finisher pigs (n = 24) and 2) examine effects of outbreeding with high-index conventional YK boars on modifying carcass leanness from the third to sixth generations in CA line finisher boars (n = 73) and gilts (n = 103). Cassie boars (n = 12) and CA gilts (n = 12) were fed diets without supplemental P and comparable numbers of age-matched YK boars and gilts fed diets containing supplement P were raised throughout the finisher phase. The pigs were slaughtered and then fabricated into commercial pork primals before meat composition and quality evaluation. Proximate and major micronutrient composition was determined on tissues including fat, kidney, lean, liver, and skin. The main difference observed was greater (P = 0.033) crude fat content in CA boar carcasses and increased (P < 0.04) leaf lard in both CA boars and gilts but no differences were observed (P = 0.895 and P = 0.223, respectively) in carcass backfat thickness as compared with YK pigs. There were no substantive differences in tissue composition, except for CA boar kidneys. Numerous changes in the mineral, fatty acid, and indispensable AA composition for CA boar kidneys were not apparent in CA gilts. These changes may point to adaptive physiological changes in the boar kidney necessary for homeostatic regulation of mineral retention related to phytase action rather than to insertion of the transgene. However, from a meat composition perspective, transgenic expression of phytase in the CA line of YK pigs had little overall effect on meat composition. Outbreeding of high-index CA gilts with high-index commercial YK boars linearly reduced (P = 0.002) back fat thickness with a corresponding linear increase (P = 0.001) in lean yield in finisher CA gilts, although no change in these parameters was observed in CA finisher boars. The increase in lean yield in CA gilts by selective breeding without affecting the level of salivary phytase activity documents the value of conventional genetic selection in conjunction with genetic modification.

Phytase properties and locations in tissues of transgenic pigs secreting phytase in the saliva.

A transgenic Cassie (CA) line of Yorkshire (YK) pigs was developed using a transgene composed of the mouse parotid secretory protein promoter linked to the Escherichia coli phytase gene integrated in chromosome 4. Previous studies documented that salivary secretion of phytase was sufficient to enable efficient digestion of plant feed phytate P. In the present study the catalytic properties and tissue distribution of the phytase in CA pigs were determined by a combination of enzymatic assays, immunohistochemistry, and immunoblots of tissue samples. The E. coli phytase had a mass of 44.82 kDa whereas the phytase secreted in CA saliva had a mass of 52.42 kDa as a result of glycosylation of the enzyme in the parotid gland. Despite the difference in size, the 2 enzymes exhibited similar substrate specificities, and substrate affinity ( K: m) and maximum hydrolytic activity ( V: max) catalytic properties. Phytase assays showed that the enzyme was present at high specific activity in the salivary glands with low activity in the soft palate and essentially none in the kidney, lean (muscle), liver, or skin of CA pigs and none in YK pigs. This conclusion was supported by immunoblot analysis using a polyclonal anti-phytase antibody. Immunohistochemical analysis of 83 different tissue locations of CA and YK pigs confirmed the ubiquitous presence of phytase in serous cells of the salivary glands and the localized presence of phytase in both serous and mixed cell types in the submucosal glands of the oropharynx; in the pharynx, tonsils, and esophagus; in some Bowman's glands in the nasal mucosa and eustachian tube; and in the prostate gland of CA boars. Furthermore, it showed the absence of phytase from the kidney, lean, liver, and skin of CA pigs. Phytase was not detected in any of the conventional YK tissues tested. The phytase was found to be glycosylated with the allergenic galactose-α-1,3-galactose (α-gal) epitope by immunoblotting using α-gal specific monoclonal antibodies. Galactose-α-1,3-galactose glycosylation of proteins is a common feature of pork and other red meats. The α-gal epitope was shown to be associated with a few proteins in muscle and skin but with the greatest number of proteins in kidney and parotid tissues of CA and YK pigs. The absence of phytase from the major food tissues and the displacement of other α-gal glycosylated proteins in the parotid glands by α-gal glycosylated phytase in conjunction with previously published data support the contention that expression of the novel phytase has minimal influence on pork quality and safety.

Mortality among veterans with type 2 diabetes initiating metformin, sulfonylurea or rosiglitazone monotherapy.

AIMS/HYPOTHESES: Despite oral hypoglycaemic medications being the most commonly used pharmacological treatments for type 2 diabetes, research is limited on their comparative safety, particularly their effects on overall mortality. We compared mortality risk with monotherapy initiation of four oral hypoglycaemic medications in a nationwide cohort of US veterans with type 2 diabetes. METHODS: We identified new users of oral hypoglycaemic medication monotherapy between 2004 and 2009 who received care for at least 1 year from the Veterans Health Administration.Patients were followed until initial monotherapy discontinuation,addition of another diabetes pharmacotherapy, death or end of follow-up. Mortality HRs were estimated using Cox regression adjusted for potential confounding factors. RESULTS: Among new users of metformin, sulfonylureas and rosiglitazone (185,360 men, 7,812 women), 4,256 (2.2%) died during follow-up. Average duration of medication use ranged from 1.4 to 1.7 years. Significantly higher mortality risk was seen for glibenclamide (known as glyburide in the USA and Canada) (HR 1.38, 95% CI 1.27, 1.50) or glipizide (HR 1.55,95% CI 1.43, 1.67) compared with metformin monotherapy,and for glipizide compared with rosiglitazone (HR 1.27, 95%CI 1.01, 1.59) or glibenclamide monotherapy (HR 1.12, 95%CI 1.02, 1.23). A significant sex­rosiglitazone interaction was seen (p=0.034) compared with metformin monotherapy, with women having a higher HR (HR 4.36, 95% CI 1.34, 14.20)than men (HR 1.19, 95% CI 0.95, 1.49). CONCLUSIONS/INTERPRETATIONS: Significantly higher mortality was associated with glibenclamide, glipizide and rosiglitazone use compared with metformin, and with glipizide use compared with rosiglitazone or glibenclamide. The potential for residual confounding by indication should be considered in interpreting these results.

Digestive utilization of phosphorus from plant-based diets in the Cassie line of transgenic Yorkshire pigs that secrete phytase in the saliva.

A line of transgenic Yorkshire pigs referred to as the Cassie (CA) line was generated, which possessed a stable, low copy number phytase transgene insertion that enabled phytase secretion in the saliva. This study was conducted to assess growth and efficacy for improving P, Ca, and other macromineral utilization in the CA pigs receiving diets typical of those used for commercial swine production. In Exp. 1, 12 CA boars and 12 CA gilts fed diets without supplemental P gained weight and exhibited feed efficiency similar to conventional age-matched 12 Yorkshire boars and 12 Yorkshire gilts raised on similar diets with supplemental P. Serum concentrations of P and Ca were similar for CA and Yorkshire pigs during the growing and finishing phases, indicating that the CA pigs were not P limited. In Exp. 2, 6 CA (13.1 kg BW) and 6 Yorkshire barrows (8.8 kg BW) were fed 3 diets (control; low in Ca and P; and low in Ca, P, and CP) over 3 phases. The CA barrows fed the diet without supplemental P retained 25 to 40% (P < 0.001), 77 to 91% (P < 0.001), and 27 to 56% (P < 0.001) more P during the weaning, growing, and finishing phases, respectively, than conventional Yorkshire barrows fed similar diets without supplemental P. In Exp. 3, CA and Yorkshire barrows of similar ages weighing 66.2 ± 1.7 kg (n = 10) and 50.0 ± 1.0 kg (n = 10), respectively, were used. The P retention of CA finisher barrows fed a diet without supplemental P was 34% greater (P < 0.001) than conventional Yorkshire barrows fed the same diet with 750 units of exogenous phytase/kg diet. Urinary Ca to P ratio in the CA pigs was 0.27, whereas that for the Yorkshire barrows was 30, thereby, indicating that the Yorkshire barrows suffered a P deficiency. Furthermore, digestive utilization of major electrolyte macrominerals, K and Na, was improved (P < 0.05) by 18 and 16%, respectively, in the CA finisher pigs compared with the conventional Yorkshire finisher pigs fed phytase; however, only K exhibited enhanced retention. In conclusion, the CA line pigs secrete sufficient phytase from the salivary glands to enable efficient digestion of plant P, Ca, and major electrolyte macrominerals.

Herpes zoster incidence in a multicenter cohort of solid organ transplant recipients.

BACKGROUND: Immunosuppressed patients are at increased risk for herpes zoster (HZ), but incidence in solid organ transplant (SOT) recipients has varied in multiple studies. To assess incidence of HZ, we examined patients who underwent SOT and received follow-up care within the large multicenter US Department of Veteran's Affairs healthcare system. METHODS: Incident cases of HZ were determined using ICD-9 coding from administrative databases. A multivariable Cox proportional hazards model, adjusted for a priori risk factors, was used to assess demographic factors associated with development of HZ. RESULTS: Among the 1077 eligible SOT recipients, the cohort-specific incidence rate of HZ was 22.2 per 1000 patient-years (95% confidence interval [CI], 18.1-27.4). African Americans (37.6 per 1000 [95% CI, 25.0-56.6]) and heart transplants recipients (40.0 per 1000 [95% CI, 23.2-68.9]) had the highest incidence of HZ. Patients transplanted between 2005 and 2007 had the lowest incidence (15.3 per 1000 [95% CI, 8.2-28.3]). In a multivariable model, African Americans (hazard ratio [HR] 1.88; 95% CI: 1.12, 3.17) and older transplant recipients (HR 1.13; 95% CI: 1.01, 1.27 [per 5-year increment]) had increased relative hazards of HZ. CONCLUSIONS: These data demonstrate that HZ is a common infectious complication following SOT. Future studies focused on HZ prevention are needed in this high-risk population.

Reproductive patterns in the domestic dog--a retrospective study of the Drever breed.

The aim of this study was to examine the differences, between seasons of the year, in the distribution of matings and whelpings, litter size, pup deaths, and sex ratio in domestic dogs. Furthermore, we wanted to examine the effects of age and parity of the bitch at the time of whelping on litter size, as well as the effect of litter size on gestational length. A final aim was to investigate the fertility and frequency of whelping problems in a private kennel of Drever dogs. Data from the Swedish Kennel Club (SKK) registry for the Drever breed during 1995-2006, comprising a total of 2717 litters, were analyzed together with more detailed data from a private, professional kennel of Drevers, with a total of 285 matings and 224 whelpings, during the same time period. The most matings took place during winter, and the fewest during summer; consequently, most whelpings occurred during the winter and spring seasons. Of the 285 mated bitches, 78.6% whelped, 6.25% experienced dystocia, and 5.36% underwent Cesarean section. The pup death rate was 7.6%. The largest litters were born during spring. Litter size was negatively correlated with duration of pregnancy (r=-0.18). Each pup more than average caused a shortening of the gestation by 0.25 days, and each pup less a corresponding lengthening. Bitches giving birth to their first litter after 4 years of age produced a smaller litter than younger bitches. Litter size decreased after 5 years in all bitches. The number of born pups at the private kennel increased from the first to the third parity, then decreased. The number of registered pups increased from the first to the second parity in the SKK data and from the second to the third parity in the data from the private kennel, then decreased. Mating a bitch only once resulted in a smaller litter size. None of the studied factors had any effect on the sex ratio of the pups. There were significant differences between males in whelping rate among the mated bitches, but no difference in mean litter size, which indicates a female problem rather than a male one. Available data suggest that the domestic dog is still under considerable seasonal influence, although modified by ambient and management factors.

Genomic differences between Fibrobacter succinogenes S85 and Fibrobacter intestinalis DR7, identified by suppression subtractive hybridization.

Fibrobacter is a highly cellulolytic genus commonly found in the rumen of ruminant animals and cecum of monogastric animals. In this study, suppression subtractive hybridization was used to identify the genes present in Fibrobacter succinogenes S85 but absent from F. intestinalis DR7. A total of 1,082 subtractive clones were picked, plasmids were purified, and inserts were sequenced, and the clones lacking homology to F. intestinalis were confirmed by Southern hybridization. By comparison of the sequences of the clones to one another and to those of the F. succinogenes genome, 802 sequences or 955 putative genes, comprising approximately 409 kb of F. succinogenes genomic DNA, were identified that lack similarity to those of F. intestinalis chromosomal DNA. The functional groups of genes, including those involved in cell envelope structure and function, energy metabolism, and transport and binding, had the largest number of genes specific to F. succinogenes. Low-stringency Southern hybridization showed that at least 37 glycoside hydrolases are shared by both species. A cluster of genes responsible for heme, porphyrin, and cobalamin biosynthesis in F. succinogenes S85 was either missing from or not functional in F. intestinalis DR7, which explains the requirement of vitamin B12 for the growth of the F. intestinalis species. Two gene clusters encoding NADH-ubiquinone oxidoreductase subunits probably shared by Fibrobacter genera appear to have an important role in energy metabolism.

Symptoms experienced by patients with peripheral vestibular disorders: evaluation of the Vertigo Symptom Scale for clinical application.

OBJECTIVES: To describe symptoms during an episode of dizziness in a sample of patients suffering from peripheral vestibular disorders and to compare them with the items in the Vertigo Symptom Scale. DESIGN: A descriptive study from a sample of patients with peripheral vestibular disorders. SETTING: Patients visiting a department of audiology at a university hospital. PARTICIPANTS: Twenty patients with peripheral vestibular disorders. The inclusion criteria were that the patient had had at least three spontaneous attacks of vertigo and/or was constantly unsteady during the last 3 months for at least 75% of the time when awake. MAIN OUTCOME MEASURES: Patients were instructed to complete a diary where they recorded symptoms that arose during an episode of dizziness. These symptoms were compared with the content of the Vertigo Symptom Scale. RESULTS: The most frequent symptoms as mentioned by the patients in their diaries were a feeling that things are spinning or moving around, nausea, feeling unsteady/about to lose one's balance, fatigue, headache, a feeling as if the ground you walk on is distant and ear-related such as tinnitus and a feeling of pressure in the ear. Pain in the heart or chest region, a heavy feeling in the arms or legs, pain in the lower part of the back and excessive sweating were not mentioned at all or by very few patients. Analysis showed that some of the symptoms included in the Vertigo Symptom Scale occurred less during an episode of dizziness than others in this sample of patients with peripheral vestibular disorders. CONCLUSION: It was found that the Vertigo Symptom Scale is an adequate base but may need to be developed for use in patients diagnosed with peripheral vestibular symptoms to be able to evaluate care and treatment.

Pregnancy in the domestic cat after vaginal or transcervical insemination with fresh and frozen semen.

The objective was to compare pregnancy rates in domestic cats using fresh semen for intravaginal artificial insemination (IVI), either at the time of hCG treatment for induction of ovulation, or 28 h later, and to compare pregnancy rates following IVI or transcervical intrauterine insemination (IUI) of frozen-thawed semen. Eighteen queens were inseminated during 39 estrus cycles. Fresh semen with 13.5+/-5.4 x 10(6) sperm (range, 6.8-22 x 10(6)) collected by electroejaculation from four male cats was used in Experiment 1, and cryopreserved semen (20 x 10(6) sperm, with 70+/-5% post-thaw motility) from one male cat was used in Experiment 2. Serum concentrations of estradiol-17beta and progesterone were determined in most queens on the day of AI and again 30-40 days later. Treatment with 100 IU of hCG 3 days after the onset of estrus induced ovulation in 95% of treated queens. Pregnancy rates to IVI with fresh semen at the time of hCG administration versus 28 h later were not different (P=0.58); overall 33% (5/15) of the queens became pregnant. For frozen-thawed semen, AI was consistently done 28h after hCG administration; IUI and IVI resulted in pregnancy rates of 41.7% (5/12), whereas no queen (0/12) became pregnant by IVI (P=0.0083). In conclusion, an acceptable pregnancy rate was obtained with frozen-thawed semen in the domestic cat by non-surgical transcervical IUI; this method might also be useful in other small felids.

Induced immotility during long-term storage at +5 degrees C does not prolong survival of dog spermatozoa.

This study investigated whether the immotility induced by the CLONE chilled semen extender prolongs the lifespan of dog spermatozoa stored at 5 degrees C, compared with a Tris-egg yolk-glucose (TG) extender, which maintains motility. Pooled semen was split in four aliquots, centrifuged, and the four sperm pellets mixed with TG extender; with the CLONE chilled semen (CL) extender; with TG extender mixed with an activator (TG+A(TG)); or with the CLONE extender mixed with the CLONE activator (CL+A(CL)). Samples were stored at 5 degrees C for 23 days and examined 12 times for sperm motility, plasma membrane and acrosome integrity, glucose consumption, and DNA fragmentation index (DFI). The experiment was performed in triplicate. Glucose consumption was not significantly different between extenders until the period 15-23 days, when it was higher in CL and CL+A(CL) than in TG (P=0.0055) and TG+A(TG) (P=0.0010). No breakdown of DNA chromatin (P>0.05) occurred until day 14. Spermatozoa preserved in TG or TG+A(TG) showed better values for all the different parameters throughout the experiment compared with sperm subjected to CL or CL+A(CL). In conclusion, the immotility induced by the CLONE chilled semen extender during long-term cold storage at 5 degrees C did not prolong the lifespan of spermatozoa compared with the lifespan following storage in Tris-egg yolk-glucose. In addition, our results indicate that good quality dog semen may possibly be stored for up to 14 days in TG extender at 5 degrees C, with retained fertilizing capacity. In vivo studies should, however, be performed to further support this conclusion.

Sperm morphology in the domestic cat, and its relation with fertility: a retrospective study.

Knowledge about normal ranges in semen quality and the association between sperm morphology and fertility in felids is limited. The aims of this retrospective study were to (1) define a normal spermiogram in cats; (2) evaluate possible effects of season, age and breed on sperm morphology; and (3) evaluate the relationship between sperm morphology and fertility. Semen samples collected by electroejaculation from 52 cats were evaluated for sperm morphology. The cats constituted two groups: a general population of cats (n = 48) and cats examined because of poor breeding records (n = 4). The general population was divided into household (n = 20), pedigree (n = 19) and colony cats (n = 9) and into three age classes, <12 months, 12-59 months and >or=60 months. The median percentage of normal spermatozoa in the general population was 44.0% (range 1.0-91.0%). Criteria were tentatively set for what was considered a normal spermiogram. The mean percentage of normal spermatozoa was higher during February to July than during August to January (p < 0.05). Pedigree cats had a lower mean percentage of normal spermatozoa than did household cats (p < 0.05). Age had no effect on the percentage of normal spermatozoa but was positively correlated with the percentage of proximal droplets. Of the cats with <40% normal spermatozoa (n = 19), all those with known breeding records (n = 11) had produced litters. The four cats examined because of poor breeding results had higher percentages of different sperm abnormalities than tentatively stipulated for the normal spermiogram. In two of these cats both sperm morphology and fertility changed over time.