Metabolomics during canine pregnancy and lactation.

During pregnancy and parturition, female dogs have to cope with various challenges such as providing nutrients for the growth of the fetuses, hormonal changes, whelping, nursing, milk production, and uterine involution. Metabolomic research has been used to characterize the influence of several factors on metabolism such as inter- and intra-individual factors, feeding, aging, inter-breed differences, drug action, behavior, exercise, genetic factors, neuter status, and pathologic processes. Aim of this study was to identify metabolites showing specific changes in blood serum at the different phases of pregnancy and lactation. In total, 27 privately owned female dogs of 21 different breeds were sampled at six time points: during heat, in early, mid and late pregnancy, at the suspected peak of lactation and after weaning. A validated and highly automated canine-specific NMR metabolomics technology was utilized to quantitate 123 measurands. It was evaluated which metabolite concentrations showed significant changes between the different time points. Metabolites were then grouped into five clusters based on concentration patterns and biochemical relationships between the metabolites: high in mid-pregnancy, low in mid-pregnancy, high in late pregnancy, high in lactation, and low in lactation. Several metabolites such as albumin, glycoprotein acetyls, fatty acids, lipoproteins, glucose, and some amino acids show similar patterns during pregnancy and lactation as shown in humans. The patterns of some other parameters such as branched-chain amino acids, alanine and histidine seem to differ between these species. For most metabolites, it is yet unstudied whether the observed changes arise from modified resorption from the intestines, modified production, or metabolism in the maternal or fetal tissues. Hence, further species-specific metabolomic research may support a broader understanding of the physiological changes caused by pregnancy that are likely to be key for the normal fetal growth and development. Our findings provide a baseline of normal metabolic changes during healthy canine pregnancy and parturition. Combined with future metabolomics findings, they may help monitor vital functions of pre-, intra-, and post-partum bitches and may allow early detection of illness.

Thyroid hormones in canine pregnancy and lactation.

It is believed that thyroid function has a significant effect on fertility and fetal development in mammals. So far, however, only few studies have been published about potential effects of the reproductive cycle stage on thyroid hormone concentrations in dogs. Therefore, over the course of 122 pregnant and non-pregnant cycles in healthy bitches, Thyroid stimulating hormone (TSH), free Thyroxine (fT4), total Thyroxine (tT4) and Progesterone (P4) were measured six times to assess the influence of the cycle stage and pregnancy on hormone concentrations. The aim was to evaluate established reference intervals for the thyroid hormones in a female study population. Of the 122 bitches, 98 became pregnant. Blood samples were collected during estrus, three times in pregnancy, during lactation and after weaning, or at equivalent times during and after estrus in non-pregnant dogs. No differences between pregnant and non-pregnant animals in any of the thyroid hormones were found. Hormone concentrations, however, differed significantly between the six samplings (p < .01). TSH initially declined during pregnancy, then rose again. The mean concentration of all dogs exceeded the overall upper reference limit of 0.70 ng/mL during lactation. Concentrations of tT4 and ft4 increased during the first third of pregnancy and then subsequently declined. The overall reference limits for tT4 were 0.47-3.20 µg/dL, and for fT4 4.86-29.60 pmol/L, but the reference intervals varied between the sampling dates. The observed patterns may reflect that maternal tT4 and fT4 seem to have important effects during early pregnancy, including a pronounced negative feedback effect on TSH. The initial increase and subsequent decline of tT4 and fT4 concentrations during the course of pregnancy is in accordance with findings in humans and may support the development of fetal thyroid function. The observed peak of TSH concentrations during lactation suggests that the demand for thyroid hormones in this phase is largest. Even if the underlying causes and mechanisms of thyroid regulation are not fully understood, the results of this study show relevant changes of hormone concentrations in the course of the sexual cycle and pregnancy. In that regard, cycle stage needs to be considered when assessing thyroid function in bitches.

The metabolic differences of anestrus, heat, pregnancy, pseudopregnancy, and lactation in 800 female dogs.

Introduction: Reproduction causes major hormonal and physiological changes to the female body. However, the metabolic changes occurring during canine reproduction are scarcely studied. Methods: In this cross-sectional study, we assessed the metabolic effects of canine reproductive status using a 1H NMR metabolomics platform optimized and validated for canine use. The study population consisted of a total of 837 healthy, intact female dogs in breeding age, of which 663 dogs were in anestrus, 78 in heat, 43 were pseudopregnant, 15 were pregnant, and 38 were lactating. The differences in metabolite profiles between these states were studied by the Kruskal-Wallis test with post-hoc tests performed using the Dunn's test, and visualized by box plots and a heatmap. The ability of the metabolite profile to differentiate pregnant dogs from non-pregnant ones was assessed by creating a multivariate Firth logistic regression model using forward stepwise selection. Results: Lactation, pregnancy and heat all were associated with distinct metabolic changes; pregnancy caused major changes in the concentrations of glycoprotein acetyls, albumin and creatinine, and smaller changes in several lipids, citrate, glutamine, and alanine. Pseudopregnancy, on the other hand, metabolically largely resembled anestrus. Lactation caused major changes in amino acid concentrations and smaller changes in several lipids, albumin, citrate, creatinine, and glycoprotein acetyls. Heat, referring to proestrus and estrus, affected cholesterol and LDL metabolism, and increased HDL particle size. Albumin and glycoprotein acetyls were the metabolites included in the final multivariate model for pregnancy detection, and could differentiate pregnant dogs from non-pregnant ones with excellent sensitivity and specificity. Discussion: These results increase our understanding of the metabolic consequences of canine reproduction, with the possibility of improving maternal health and ensuring reproductive success. The identified metabolites could be used for confirming canine pregnancy.

Diagnostic tests in canine andrology - What do they really tell us about fertility?

Dog breeders often require breeding soundness evaluations which include andrological examinations of the genital organs, hormone measurements, and semen analyses. During the past decades, a considerable number of research results have been published, allowing diagnoses of specific andrological conditions and fertility assessment. For specific examinations, however, no standard procedures have been defined and for some parameters different reference ranges have been published. Therefore, examination results from different facilities are difficult to compare and profound conclusions regarding health and fertility of a male dog are not always possible. Conventional semen examination, however, is still useful in identifying deviations or no deviations from normality, especially if confounding factors are taken into account and if the exam is repeated in case of inconclusive findings. A standardization of examination procedures and reference ranges would help to harmonize the exchange of examination results and interpretation of the findings.

Determination of reference intervals for nonesterified fatty acids in the blood serum of healthy dogs.

Background: Nonesterified fatty acids (NEFAs) are an important energy substrate in mammals. Measurement of the NEFA concentration in blood serum is common practice and enables reliable detection of a negative energy balance in several species. This parameter can be used to detect subclinical metabolic diseases or to optimise feeding to prevent severe negative energy balance. Since no reference values for dogs have been published, the aim of this study was to establish such values. Methods: Blood serum from 85 healthy dogs was examined with a multiparameter clinical chemistry analyser. Given that NEFA values are not usually normally distributed, reference intervals (RIs) were calculated nonparametrically using bootstrapping (5000 replicates) for the 90% confidence intervals. Results: The examined cohort had a median age of 62.16 months (2-180 months) and a median weight of 19.2 kg (3.0-55.0 kg) and comprised 27 (31.8%) males and 58 (68.2%) females, with 32 (37.6%) neutered or spayed. The fasting time was 5.9 h (range 0-23 h). The tested confounders age, sex, neuter status, bodyweight and body condition score did not significantly affect the NEFA concentrations. Conclusions: The NEFA RI for dogs in this study was 0.2-1.47 mmol/L. The results may be used to adjust food composition and amount in healthy dogs or to detect metabolic disorders. Further research on NEFA metabolism in dogs maintained in standardised conditions and in specific nutritional situations or with particular diseases is warranted.

What Kinds of Dogs Are Used in Clinical and Experimental Research?

BACKGROUND: Dogs are widely used in research to answer questions about canine or human conditions. For the latter, research dogs are often used as models, since they are physiologically more similar to humans than other species used in research and they share similar environmental conditions. From a veterinary perspective, research findings are widely based on academic research, and thus are generated under experimental conditions. In that regard, the question arises: do the dogs used for research adequately represent the dog population seen in veterinary practice? It may, for example, be assumed that Beagle dogs are often used as experimental animals. The objective of this study was to evaluate the signalment of dogs used in veterinary research. Furthermore, we aimed to assess other relevant criteria regarding the validity of clinical trials in the context of six different veterinary medicine specialties: cardiology, internal medicine, neurology, orthopaedics, reproduction, and surgery. METHODS: A literature search was conducted and 25 studies per specialty were randomly selected. The breed, sex, neuter status, median age, and median weight of the dogs used for clinical studies (n = 150) published between 2007 and 2019 were evaluated. RESULTS: In total, 596,542 dogs were used in the 150 trials. Breed information was given for 33,835 of these dogs (5.7%). Of the latter, 1.9% were Beagles. Nine clinical trials exclusively used Beagles. The most frequently used breeds were German Shepherds (7.3%), Labrador Retrievers (6.7%), and Golden Retrievers (4.7%). The major reporting deficits found were missing breed specification in 25.3% of the articles; missing information about the sex of the dogs in 16.2%; missing age and weight information in 22.7 and 32.7%, respectively; and missing neuter status in 38.7% of the clinical studies. The median sample size was 56 (Q1:29; Q3:365) dogs. CONCLUSIONS: The presented project revealed that Beagle dogs represent only a small proportion of dogs in veterinary research. Based on the evaluated publications, it seems that some relevant dog attributes differ between the specialties. The results, however, show deficits in the reporting of demographic data for the dogs. The need for an improvement in the documentation and/or reporting of animal signalment is obvious and should be addressed by authors, reviewers, and journal editors in the future.

Study design quality of research on dogs published in peer-reviewed journals.

BACKGROUND: In the past it has been criticised that only a low proportion of well-designed and well-reported studies in some medical specialities is available. The objective of this study was to systematically evaluate the quality of literature about canine medicine published in peer-reviewed journals in relation to six specific veterinary medicine specialities. METHODS: A literature search was conducted and 25 studies per speciality were selected. The quality of the articles (n = 150) published between 2007 and 2019 was evaluated with a validated checklist. RESULTS: In articles related to all specialities, deficits were found, such as not adequate number of animals in 60.0% of the studies. In 88.0%, information about housing and feeding of the dogs were not specified. In 69.4% of the prospective clinical studies, an ethical approval was reported, and written informed consent of the owners was obtained in 46.2%. CONCLUSIONS: The findings revealed extensive deficits in the design and reporting of studies in canine medicine. The demand for improvement is obvious and should be addressed by authors, reviewers and journal editors in the future. Our results underline that practitioners should critically appraise the quality of literature before implementing information into practice.

Ethics in canine reproduction - a survey among veterinarians who provide canine reproductive services.

OBJECTIVE: Several dog breeds suffer from health and welfare problems due to unfavorable anatomies and high prevalence of inherited diseases. Veterinarians should care for animal health and welfare but they may also be involved in breeding management including the use of reproductive techniques. MATERIAL AND METHODS: An online survey was conducted to find out how veterinarians who are involved in small animal reproduction regard ethical issues, which services they provide, how they discuss and handle ethical concerns and talk about them with breeders. RESULTS: In total, 83 participants mainly from Europe completed the online survey. Opinions on which interventions are ethical or not were heterogeneous. Most respondents (79.2 %) found it ethical to perform artificial insemination (AI) in dogs which did not reproduce naturally before. However, 62.7 % of participants stated that surgical insemination is not ethical. Elective cesarean sections are considered not ethical by approximately half of the participants. A closer look at responses to the questions of whether AI is ethical under specific conse quences suggests that most veterinarians are making ethical and value judgments in terms of what is justifiable or not. However, more than 80 % feel at least sometimes compromised by ethical conflicts. CLINICAL RELEVANCE: This research intended to foster discussion of this topic. International strategies should be developed to support solving conflicts and dilemmas related to ethical issues in dog breeding and assisted reproduction, as well as diminishing physical disabilities and heritable diseases.

Progesterone Concentrations during Canine Pregnancy.

Pregnancy and lactation are amongst the most challenging times of a bitch's life. Most studies focusing on the endocrinological aspect of pregnancy consider only a small number of animals. The aim of this study was to evaluate progesterone (P4) concentrations in a large number of bitches during early, mid and late pregnancy. In total, 126 bitches of various breeds were recruited following a thorough clinical and gynecological examination during estrus. Blood samples were collected three times (T1-T3) during pregnancy or from non-pregnant dogs in diestrus, and P4 was measured via chemiluminescence. At T1 (11-19 days post-ovulation (dpo)), serum P4 concentrations were 30.23 ± 6.65 ng/mL and 28.45 ± 6.26 ng/mL, at T2 (23-32 dpo) they were 22.73 ± 6.27 ng/mL and 22.59 ± 5.77 ng/mL and at T3 (52-60 dpo) they were 6.68 ± 2.18 ng/mL and 3.17 ± 2.26 ng/mL, in pregnant (n = 98) and non-pregnant (n = 23) dogs respectively. The P4 concentrations differed significantly between pregnant and non-pregnant animals at the last examination (p ≤ 0.001). In the context of hypoluteoidism, the gathered data yielded interesting results. Overall, 28 out of 98 pregnant bitches showed a greater decline (>15 ng/mL) in P4 concentrations from early to mid-pregnancy, and 56 bitches showed P4 concentrations lower than deemed adequate (>20 ng/mL at T1 and T2, >5 ng/mL at T3) according to existing recommendations. Despite not being supplemented with P4, none of those animals suffered from abortion or preterm delivery. Considering that supplementation of P4 can entail considerable risks for the bitch and the puppies, more research on P4 concentration patterns, diagnosis of hypoluteoidism and treatment indications and options is indicated.

[Taking body temperature in cattle - critical evaluation of an established diagnostic test]. / Messung der Körpertemperatur beim Rind ­ eine altbewährte Methode kritisch hinterfragt.

Measuring the body temperature is an essential component of the clinical examination of bovines. Next to its value for the diagnosis of diseases, body temperature also is an important value in clinical studies concerning estrus detection, calving time point prediction, or the evaluation of heat stress. This systematic review critically evaluates different methods of measuring bovine body temperature including rectal measurement, vaginal or ruminal temperature loggers, milk temperature, and infrared body surface thermography. Although body temperature measurement is a commonly employed and established diagnostic test, a close look at scientific studies displays multiple factors influencing body temperature data. The variability of results emphasizes the relevance of critically evaluating new measuring methods before introducing them into research or routine practice. Especially in terms of identifying specific cut-off values, i. e. for fever defined as body temperature > 39.5°C, the precision of the method is of importance, as the acquired values possess a high degree of influence on the veterinary decision taking process.

Critically Appraised Topics (CATs) in Veterinary Medicine: Applying Evidence in Clinical Practice.

Critically appraised topics (CATs) are evidence syntheses that provide veterinary professionals with information to rapidly address clinical questions and support the practice of evidence-based veterinary medicine (EBVM). They also have an important role to play in both undergraduate and post-registration education of veterinary professionals, in research and knowledge gap identification, literature scoping, preparing research grants and informing policy. CATs are not without limitations, the primary one relating to the rapid approach used which may lead to selection bias or restrict information identified or retrieved. Furthermore, the narrow focus of CATs may limit applicability of the evidence findings beyond a specific clinical scenario, and infrequently updated CATs may become redundant. Despite these limitations, CATs are fundamental to EBVM in the veterinary profession. Using the example of a dog with osteoarthritis, the five steps involved in creating and applying a CAT to clinical practice are outlined, with an emphasis on clinical relevance and practicalities. Finally, potential future developments for CATs and their role in EBVM, and the education of veterinary professionals are discussed. This review is focused on critically appraised topics (CATs) as a form of evidence synthesis in veterinary medicine. It aims to be a primary guide for veterinarians, from students to clinicians, and for veterinary nurses and technicians (hereafter collectively called veterinary professionals). Additionally, this review provides further information for those with some experience of CATs who would like to better understand the historic context and process, including further detail on more advanced concepts. This more detailed information will appear in pop-out boxes with a double-lined surround to distinguish it from the information core to producing and interpreting CATs, and from the boxes with a single line surround which contain additional resources relevant to the different parts of the review.

The bitch around parturition.

Around parturition, a bitch has to cope with various challenges such as hormonal changes, whelping, nursing, milk production, and uterine involution. Monitoring the health of bitches in this period is essential to detect potential illnesses and dystocia early. In that regard, it is elementary to know the normal progress and parameters during pregnancy, parturition and in the puerperium. Some research has been published in the past 50 years giving insights into hormonal and functional changes and findings including definitions of normal parturition and dystocia or puerperal conditions. However, taking a closer look into the literature reveals that for some issues heterogeneous data and varying conclusion were presented, indicating that further research is required. This paper gives an overview on endocrinology and methods to predict the time of parturition in the dog. Furthermore, the stages and mechanisms of parturition and signs of dystocia and puerperal health monitoring are discussed. Fields in which contradictory data have been published include for example the decline of hematocrit in the second half of pregnancy, the body temperature immediately before and after parturition, the interpretation of ultrasound findings, and the length of the canine puerperium. Even if thresholds are not clear for each parameter, examinations such as regular temperature measurement or ultrasound are important to monitor the bitches' health status. Nevertheless, for the practitioner it is important to know that research findings for some parameters used in daily practice are controversial.

Improving decision-making in complicated or rare cases-An approach towards Evidence-based Veterinary Medicine in small animal reproduction.

The concepts of Evidence-based Veterinary Medicine (EBVM) provide a methodological and systematic approach to include the best evidence from research into clinical decision-making. These concepts include steps as the search and assessment of relevant research findings and consideration of individual aspects. In addition, owners and other persons involved in animal health care should be included in shared decision-making. Some breeders have good basic knowledge concerning breeding management and characteristics of diseases and concerning advantages and disadvantages of different therapeutic approaches, while others are notable to understand complex medical interrelations or emergency situations. All these aspects need to be addressed when communicating and discussing different diagnostic, prophylactic and therapeutic options. In special fields, such as small animal reproduction, veterinarians often see animals with rare diseases or complex conditions so that an application of standard therapies and well-established textbook recommendations is not possible. To learn more about cases too rare for successful systematic research, the case collection tool REPROCASES (www.evssar.org/reprocases) is now available. The aim of this project is to gather information from specialists on small animal reproduction in a multicentre approach. If you see rare or not well-investigated cases such as cystic ovarian diseases, cryptorchidism or others, you are more than welcome to share your findings via the database. Even if this approach cannot completely replace standardized clinical trials, the idea is to gather more information on effects, prognosis, side effects and long-term fertility for specific conditions.

Teaching small animal reproduction via virtual patients.

Virtual patients have become an interesting alternative in medical education. Due to increasing demands regarding theoretical and clinical teaching and to improve an interdisciplinary approach, a new blended learning concept including virtual patients was developed and implemented in the veterinary curriculum of the Freie Universität Berlin. In the presented project, three virtual patients from the field of canine reproduction were developed. They focus on pregnancy diagnosis with suspected luteal insufficiency, pyometra and benign prostatic hyperplasia, respectively. The results of an evaluation by veterinary students of the 7th semester showed a high acceptance of virtual patients in a blended learning reproduction module in the interdisciplinary lectures. Students especially preferred videos, such as video lectures, hands-on videos and animations as well as a glossary for background information, to successfully and autonomously work on a virtual case. The content covered by the new modules that were developed in the context of this project is part of a spiral curriculum; they will be revised and enhanced during the clinical year.

Body temperature of bitches in the first week after parturition measured by ingestible loggers.

The objective of this study was to identify the physiological ranges of body temperature of bitches in the first 7 days after parturition by measurement with ingestible temperature loggers. Twenty bitches swallowed one ingestible temperature logger daily. Bitches were defined to be healthy by spontaneous parturition and leucocyte concentration. Mean core body temperatures of eight healthy bitches were (Mean ± SD) 38.8°C ± 0.40 on day 0 p.p., 38.9°C ± 0.47 on day 1 p.p., 38.9°C ± 0.35 on day 2 p.p. 38.7°C ± 0.31 on day 3 p.p., respectively. In the following days, the three remaining healthy bitches showed mean core body temperatures (Mean ± SD) of 38.8°C ± 0.30 on day 4 p.p., 38.6°C ± 0.35 on day 5 p.p., 38.5°C ± 0.27 on day 6 p.p. and 38.4°C ± 0.34 on day 7 p.p., respectively. Three out of the eight healthy bitches showed temperatures ≥39.5°C. Bitches with leucocytosis showed significant higher mean core body temperatures (39.0°C ± 0.49) than healthy bitches (38.8°C ± 0.39) during the first 3 days after parturition (p < 0.01). We conclude that the physiological ranges of body temperature of healthy bitches in the first days after parturition do not differ from those of healthy dogs in general, while the appearance of short episodes of febrile temperatures seems to be physiological. Puerperal bitches with leucocytosis show higher body temperatures increased by only 0.2°C.

Inter-observer agreement on a checklist to evaluate scientific publications in the field of animal reproduction.

This study's objective was to determine respondents' inter-observer agreement on a detailed checklist to evaluate three exemplars (one case report, one randomized controlled study without blinding, and one blinded, randomized controlled study) of the scientific literature in the field of bovine reproduction. Fourteen international scientists in the field of animal reproduction were provided with the three articles, three copies of the checklist, and a supplementary explanation. Overall, 13 responded to more than 90% of the items. Overall repeatability between respondents using Fleiss's κ was 0.35 (fair agreement). Combining the "strongly agree" and "agree" responses and the "strongly disagree" and "disagree" responses increased κ to 0.49 (moderate agreement). Evaluation of information given in the three articles on housing of the animals (35% identical answers) and preconditions or pretreatments (42%) varied widely. Even though the overall repeatability was fair, repeatability concerning the important categories was high (e.g., level of agreement=98%). Our data show that the checklist is a reasonable and practical supporting tool to assess the quality of publications. Therefore, it may be used in teaching and practicing evidence-based veterinary medicine. It can support training in systematic and critical appraisal of information and in clinical decision making.

Training evidence-based veterinary medicine by collaborative development of critically appraised topics.

In current veterinary education, skills such as retrieving, critically appraising, interpreting, and applying the results of published scientific studies are rarely taught. In this study, the authors tested the concept of team-based development of critically appraised topics (CATs) in training students in evidence-based veterinary medicine (EBVM). The 116 participants were in their fifth year and attending the clinical rotation at the Clinic for Animal Reproduction. Students developed 18 CATs of varying quality on topics of their choice. Preparing the CATs in teams stimulated discussion on the topic and the quality of the retrieved papers. Evaluation of the project revealed that more than 90% of the students endorsed training in critical appraisal of information in veterinary education. In addition, more than 90% considered the development of CATs an effective exercise for assessing the quality of scientific literature. A provided literature evaluation form was perceived as a useful tool for systematically summarizing a publication's quality. In conclusion, team-based development of CATs during clinical rotations is highly valuable for training in EBVM. Learning and intrinsic motivation seem to be enhanced by creating a situation similar to veterinary practice because the task is embedded into an authentic clinical problem. This approach to clinical training helps to prepare students to integrate evidence from literature into practice.

Training students to appraise the quality of scientific literature.

Implementing evidence-based veterinary medicine (EBVM) into clinical practice requires not only the ability to retrieve, interpret, and apply the results of published scientific studies, but also the ability to critically evaluate the quality of the literature. These skills, however, are not widely taught in the veterinary curriculum. The objective of this study was to test a literature evaluation form (LEF) designed to assist veterinary students in appraising the quality of literature on animal reproduction and to compare their ability to do so with that of students who were provided with a control form (CF). The 68 participants were in their fifth year of study and attended a clinical rotation at the Clinic for Animal Reproduction. Students in the LEF group determined the quality of two scientific papers, considering statements about study design, information content, and objectivity, and determined rating points to obtain an overall score. Participants using the CF ranked the quality of the article without the assistance of the quality assessment form. The LEF group was able to more correctly assess the quality of the literature and the variability of the chosen evidence levels was higher in the CF group. The questionnaire was found to be a useful tool for the systematic assessment of the quality of publications within a reasonable period of time. Seventy-eight per cent of the participants agreed that the LEF helps them evaluate the quality and validity of biomedical scientific information. We conclude that courses that introduce EBVM should be taught in the first semesters of the veterinary curriculum so that students can develop competence in defining a clinical problem, retrieving information from the literature, and developing independent critical thinking.