Semen parameters and testicular dimensions in small breed dogs below ten-kilogram bodyweight.

Because of few available data on semen parameters in small breed dogs, the study aimed to analyse semen and measure testes of dogs ≤10.0 kg bodyweight. Semen was collected from 41 fertile stud dogs, which were divided based on bodyweight: group 1 ≤ 5.0 kg and group 2 between 5.1 and 10.0 kg. Median values for ejaculate volume (group 1: x ~ 1.2 ml; group 2: x ~ 2.2 ml), total sperm output (group 1: x ~ 110.7 × 106 ; group 2: x ~ 215.1 × 106 ) and testicular volume (group 1: left testicle x ~ 2.8 ml, right testicle x ~ 2.7 ml; group 2: left testicle x ~ 5.5 ml, right testicle x ~ 5.0 ml) were lower in group 1 compared to dogs of group 2 (p = .001; p = .001; both testes: p < .001). There was no difference in sperm concentration (p = .985). Based on these results, introduction of an additional weight group to the commonly used reference values is recommended, since values for ejaculate volume, total sperm output and testicular dimensions for dogs ≤5.0 kg bodyweight differed significantly from values of dogs with a bodyweight from 5.1 to 10.0 kg.

Toward evidence-based severity assessment in rat models with repeated seizures: I. Electrical kindling.

OBJECTIVE: Rodent epilepsy models can significantly contribute to our understanding of pathophysiological mechanisms and to validation of biomarker and target candidates. Evidence-based severity assessment is a presupposition for the ethical evaluation of animal experimentation allowances as well as for the development of efficacious refinement concepts. METHODS: Aiming to improve our understanding of the impact of experimental procedures and repeated seizures, we have completed a comprehensive behavioral and biochemical analysis assessing various parameters that can inform about the influence of an electrical kindling paradigm on well-being in rats. Thereby, we have focused on the immediate effects of phases with focal and generalized seizures with behavioral testing during kindling acquisition. RESULTS: Electrode implantation exerted mild effects on anxiety-associated behavior and reduced serum corticosterone at 3 weeks, but not 7 weeks, following surgery. Analysis in kindled rats excluded any relevant impact of focal seizures on behavioral and biochemical parameters. Assessment in rats with generalized seizures revealed an impact on nest complexity scores, nest soiling, and selected parameters in paradigms evaluating anxiety-associated behavior. Moreover, serum corticosterone levels, but neither hair corticosterone nor fecal corticosterone metabolite concentrations were lowered as a consequence of repeated generalized seizures. The assessment of various other behavioral and biochemical parameters did not reveal any other relevant effects of generalized seizures. Cross-correlation analysis suggested that assessment of nest building and maintenance can provide information comparable to that from more elaborate behavioral assays. This finding provides first evidence that nest scoring might serve as a simple and valid approach to evaluate rat well-being during routine assessment schemes. SIGNIFICANCE: The findings argue against a persistent level of pronounced distress and suggest a classification of the kindling paradigm as a model with moderate severity based on a longer-lasting mild impact on animal behavioral patterns. This suggestion provides a basis for a prospective and retrospective case-by-case severity assessment.

Anti-Müllerian hormone concentrations in queens throughout the estrous cycle.

Studies in cats and dogs have proven the usefulness of anti-Müllerian hormone (AMH) as a diagnostic tool to determine the castration status or to diagnose ovarian remnant syndrome. Yet the secretion pattern of AMH over the estrous cycle in queens has not been investigated so far. Seven healthy sexually intact female cats were examined daily for signs of estrous behavior over a trial period of 4 months. Five queens showed regular estrous behavior, 1 queen was mated in her first heat and 1 queen never showed any signs of heat. To distinguish between inter-estrus and metestrus progesterone levels were determined. Serum samples for AMH and progesterone measurement were collected from the regular cycling queens in late anestrus, at several times during heat, inter-estrus and metestrus, from the mated queen during her first heat and during pregnancy, and in the acycling queen at various times during the trial period. The measured AMH values in anestrus were significantly (P < 0.05) higher than in heat (P < 0.001), metestrus (P = 0.12) and inter-estrus (P = 0.449). In anestrus the median AMH levels were 10.26 ng/ml (range 4.96 to 22.90 ng/ml), in heat 5.97 ng/ml (range 3.32- 22.96 ng/ml), in inter-estrus 10.47 (range 3.35-22.96 ng/ml) and in metestrus 6.38 ng/ml (range 4.50-10.75 ng/ml. The pregnant cat showed median AMH concentrations of 6.47 ng/ml (range 5.60-9.80 ng/ml) during her pregnancy. The acycling queen had solely low AMH values with a median concentration of 0.39 ng/ml. In conclusion there were high variations of the AMH levels among and within the individual cats and between heat cycles in the single cat. Remarkable high AMH concentrations were measured in the younger queens of the study in their first estrous cycles and also in anestrus, when less ovarian activity is expected. Further studies are necessary to emphasize the reasons for these high AMH concentrations especially in young queens.

Vitrification and in-straw warming do not affect pregnancy rates of biopsied bovine embryos.

In the cattle industry, in vivo or in vitro embryo production combined with genotyping and cryopreservation technologies allows the selection and conservation of embryos carrying genes for desirable traits. This study aimed to assess the efficiency of a vitrification method suitable for in-straw warming of biopsied in vivo derived (IVD) bovine embryos. Three experiments were carried out using two methodologies: the Cryotop®, the gold standard vitrification and 3-step warming methodology, or the VitTrans, a vitrification/in-straw 1-step warming method that enables direct embryo transfer to the uterus. In experiment 1, intact and biopsied in vitro produced (IVP) day 7 expanded blastocysts were vitrified using the Cryotop® and warmed in 1- or 3-steps. No differences in survival rates were recorded at 24 h after warming for intact or biopsied IVP blastocysts irrespective of the warming procedure. In experiment 2, the effect of the time from trophectoderm (TE) biopsy to vitrification/in-straw warming on post-warming survival rate was assessed. No significant differences in survival were observed when blastocysts were vitrified/in-straw warmed immediately after biopsy or after 3 h in culture when compared to intact blastocysts. In experiment 3, IVD embryos were vitrified 3 h after biopsy using the Cryotop® or the VitTrans method and pregnancy rates were assessed at day 60 after transfer. Fresh, biopsied embryos served as control. Similar pregnancy rates were observed when IVD biopsied embryos were transferred fresh or vitrified/warmed by the Cryotop® or VitTrans method. No significant effect of the embryo quality or developmental stage was detected on the percentage of pregnant recipients when IVD biopsied embryos were transferred fresh or after vitrification. While fresh female IVD embryos produced significantly higher pregnancy rates than male embryos, there were no differences in pregnancy rates when male or female vitrified/warmed embryos were transferred. About 81% from the biopsies analyzed successfully determined the embryo sex, confirming that DNA was there, and it was efficiently amplified. To conclude, our findings indicate that both vitrification methodologies produced similar post-warming outcomes for both intact and biopsied IVP embryos. Besides, vitrification/in-straw warming of biopsied IVD bovine embryos did not affect the viability to originate pregnancy, being a useful option for their direct transfer in field conditions.

Equine Arteritis Virus (EAV) Outbreak in a Show Stallion Population.

(1) Background: Equine arteritis virus (EAV) infection causes reproductive losses and systemic vasculitis in susceptible equidae. The intact male becomes the virus' reservoir upon EAV infection, as it causes a chronic-persistent infection of the accessory sex glands. Infected semen is the main source of virus transmission. (2) Here, we describe acute EAV infection and spread in a stallion population after introduction of new members to the group. (3) Conclusions: acute clinical signs, acute phase detection of antigen via (PCR) nasal swabs or (EDTA) blood, and seroconversion support the idea of transmission via seminal fluids into the respiratory tract(s) of others. This outbreak highlights EAV's horizontal transmission via the respiratory tract. This route should be considered in a chronic-persistently infected herd, when seronegative animals are added to the group.

[Birth of a vital puppy following prolonged whelping in a Labrador Retriever]. / Geburt eines lebenden Labradorwelpen nach deutlich verlängerter Geburt.

An 8-year-old primiparous Labrador Retriever bitch was presented to the clinic 46 hours following the delivery of 2 vital and one stillborn puppy due to an increase of vaginal discharge. At the veterinary clinic, the dog spontaneously delivered an additional healthy puppy without any obstetric support. The findings of the general and gynaecological examinations as well as results of blood analyses were within normal limits. This case shows that extremely prolonged whelping process may be a rare but possible phenomenon in bitches. However, it is recommended to re-evaluate dam and fetuses regularly in case the inter-whelping interval exceeds the normal time span in order to recognize further signs of dystocia and induce obstetric support when clinically indicated.

Changes in anti-Müllerian hormone concentrations in bitches throughout the oestrous cycle.

Studies in female dogs proved the measurement of anti-Müllerian hormone (AMH) as a diagnostic tool to distinguish ovarioectomized from intact females or to diagnose ovarian remnant syndrome. Furthermore, its usability to diagnose a granulosa cell tumour and predict litter size was also investigated in female dogs. Although serum AMH was previously shown to increase during the transition from anoestrus to pro-oestrus in dogs, changes in AMH concentration over the entire oestrous cycle have not yet been determined. The aim of this study was to investigate the secretion pattern of AMH throughout the oestrous cycle in non-pregnant bitches. As a preliminary step, we tested our assay by measuring AMH concentrations in serum samples of 19 intact bitches (10 in anoestrus, 3 in pro-oestrus, 3 in oestrus, 3 in metoestrus) and 19 spayed females of different age and breed. For the main study, 20 healthy female dogs were examined (10 Beagles, and 10 Labrador crossbreeds, body weight 10-28 kg, age 1-6 years) during a normal oestrous cycle. Serum samples were collected in late anoestrus, at several times during pro-oestrus and oestrus, as well as 28 days and 4.5 months after ovulation. Blood collection was combined with a gynaecological examination including progesterone measurement. Serum concentration of AMH was determined using a chemiluminescence immunoassay validated for dog serum. In the preliminary test, intact bitches in various stages of the oestrous cycle had significantly higher serum AMH levels (0.19-1.45 ng/ml) than spayed females (0.01-0.06 ng/ml, P < 0.001). In cycling Beagle and Labrador crossbreed bitches participating in our main study, AMH concentrations were between 0.09 and 2.65 ng/ml with higher AMH concentrations in Beagles but overall high inter-individual and intra-individual variation. The highest AMH values were reached in the last three weeks before the onset of heat until six days before ovulation, and the lowest AMH concentrations were recorded during preovulatory oestrus in 6, metoestrus in 7 or anoestrus in 7 dogs. Serum AMH concentrations increased significantly from late anoestrus up to six days before ovulation and decreased significantly over the last three days before ovulation. A further significant decrease occurred from the last days of oestrus to metoestrus and mid anoestrus. This study shows that the used AMH assay can clearly distinguish between intact and spayed females and that the serum AMH pattern over the oestrous cycle is similar in all bitches, but with high variation among the breeds and among and within bitches. These changes in the AMH concentration are a challenge in timing serum sample collection and interpreting AMH values in bitches. Further studies are necessary to emphasize the factors influencing the AMH concentration.

The Munich MIDY Pig Biobank - A unique resource for studying organ crosstalk in diabetes.

OBJECTIVE: The prevalence of diabetes mellitus and associated complications is steadily increasing. As a resource for studying systemic consequences of chronic insulin insufficiency and hyperglycemia, we established a comprehensive biobank of long-term diabetic INSC94Y transgenic pigs, a model of mutant INS gene-induced diabetes of youth (MIDY), and of wild-type (WT) littermates. METHODS: Female MIDY pigs (n = 4) were maintained with suboptimal insulin treatment for 2 years, together with female WT littermates (n = 5). Plasma insulin, C-peptide and glucagon levels were regularly determined using specific immunoassays. In addition, clinical chemical, targeted metabolomics, and lipidomics analyses were performed. At age 2 years, all pigs were euthanized, necropsied, and a broad spectrum of tissues was taken by systematic uniform random sampling procedures. Total beta cell volume was determined by stereological methods. A pilot proteome analysis of pancreas, liver, and kidney cortex was performed by label free proteomics. RESULTS: MIDY pigs had elevated fasting plasma glucose and fructosamine concentrations, C-peptide levels that decreased with age and were undetectable at 2 years, and an 82% reduced total beta cell volume compared to WT. Plasma glucagon and beta hydroxybutyrate levels of MIDY pigs were chronically elevated, reflecting hallmarks of poorly controlled diabetes in humans. In total, ∼1900 samples of different body fluids (blood, serum, plasma, urine, cerebrospinal fluid, and synovial fluid) as well as ∼17,000 samples from ∼50 different tissues and organs were preserved to facilitate a plethora of morphological and molecular analyses. Principal component analyses of plasma targeted metabolomics and lipidomics data and of proteome profiles from pancreas, liver, and kidney cortex clearly separated MIDY and WT samples. CONCLUSIONS: The broad spectrum of well-defined biosamples in the Munich MIDY Pig Biobank that will be available to the scientific community provides a unique resource for systematic studies of organ crosstalk in diabetes in a multi-organ, multi-omics dimension.