The supplementation of female dogs with live yeast Saccharomyces cerevisiae var. boulardii CNCM I-1079 acts as gut stabilizer at whelping and modulates immunometabolic phenotype of the puppies.

Time around parturition is a stressful period for both bitches and their puppies. The use of probiotics has been proposed, e.g., in pigs, to improve health status of sows, their reproductive performances and in turn, the health and performance of their progeny. The objective of the present study was to evaluate the impact, on both dams and puppies, of a supplementation of bitches with the live yeast Saccharomyces cerevisiae var. boulardii CNCM I-1079 (SB-1079) during the second part of the gestation and the lactation period. A total of 36 bitches of medium and large-sized breeds were enrolled. They were divided into two groups, one of which received 1.3 × 109 colony forming units of live yeast per day. At dam's level, SB-1079 yeast shaped a different microbiota structure between the two groups just after whelping, impacted alpha diversity and some plasma metabolites related to energy metabolism. Regarding reproductive performances, SB-1079 improved gross energy of the colostrum (1.4 vs. 1.2 kcal of ME/g) as well as the concentration of protein in milk at Day 7 after parturition (10.4 vs. 7.6%). SB-1079 also reduced the odds of having low birth weight in the litter. At puppy's level, a modulation of immunometabolic phenotype is suggested by the observation of increased growth rates during the early pediatric period (i.e., between 21 and 56 days of life, 225 vs. 190%) and a decrease of the IL-8:IL-10 ratio after vaccination against rabies (4.2 vs. 16.9). Our findings suggest that SB-1079 supplementation during gestation and lactation has the potential to enhance health of bitches and in turn health of puppies through maternal programming.

Composition and evolution of the gut microbiota of growing puppies is impacted by their birth weight.

Low birth weight puppies present an increased risk of neonatal mortality, morbidity, and some long-term health issues. Yet it has not been investigated if those alterations could be linked to the gut microbiota composition and evolution. 57 puppies were weighed at birth and rectal swabs were performed at 5 time points from birth to 28 days of age. Puppies were grouped into three groups based on their birth weight: low birth weight (LBW), normal birth weight (NBW) and high birth weight (HBW). 16S rRNA gene sequencing was used to highlight differences in the fecal microbiota. During the first three weeks, the relative abundance of facultative anaerobic bacteria such as E. coli, C. perfringens and Tyzzerella was higher in LBW feces, but they catch back with the other groups afterwards. HBW puppies showed higher abundances of Faecalibacterium and Bacteroides during the neonatal period, suggesting an earlier maturation of their microbiota. The results of this study suggest that birth weight impact the initial establishment of the gut microbiota in puppies. Innovative strategies would be desired to deal with altered gut microbiota in low birth weight puppies aiming to improve their survival and long term health.

Gut microbiota development in the growing dog: A dynamic process influenced by maternal, environmental and host factors.

Microorganisms of the gastrointestinal tract play a crucial role in the health, metabolism and development of their host by modulating vital functions such as digestion, production of key metabolites or stimulation of the immune system. This review aims to provide an overview on the current knowledge of factors shaping the gut microbiota of young dogs. The composition of the gut microbiota is modulated by many intrinsic (i.e., age, physiology, pathology) and extrinsic factors (i.e., nutrition, environment, medication) which can cause both beneficial and harmful effects depending on the nature of the changes. The composition of the gut microbiota is quickly evolving during the early development of the dog, and some crucial bacteria, mostly anaerobic, progressively colonize the gut before the puppy reaches adulthood. Those bacterial communities are of paramount importance for the host health, with disturbance in their composition potentially leading to altered metabolic states such as acute diarrhea or inflammatory bowel disease. While many studies focused on the microbiota of young children, there is still a lack of knowledge concerning the development of gut microbiota in puppies. Understanding this early evolution is becoming a key aspect to improve dogs' short and long-term health and wellbeing.

Management of Low Birth Weight in Canine and Feline Species: Breeder Profiling.

Low birth weight (LBW) has been identified as a major risk factor for neonatal mortality in many species. The aim of this survey was to determine the profiles of canine and feline breeders concerning their perceptions of, and management practices relating to, LBW individuals. An anonymous online survey was addressed to French cat and dog breeders in September 2019 via social networks. Multiple correspondence analysis and hierarchical clustering were used to explore breeders' profiles. Three clusters were identified among the 649 breeders included in this analysis. Cluster 1 (49%) included dog and cat breeders who weighed newborns (and thus identified LBW) and controlled nursing by the dam (controlled suckling) but did not warm them up. Cluster 2 breeders (21%) of both species did not weigh puppies or kittens to identify LBW or to monitor the evolution of their weight afterwards. Cluster 3 (30%) including mostly cat breeders who weighed neonates routinely as in Cluster 1, but they practiced artificial feeding rather than controlled suckling. This survey provides a basis for better understanding of perceptions and practices regarding LBW puppies and kittens. It will be useful to provide guidelines for neonatal management to increase their chances of survival.

Guidelines for postmortem examination of newborn dogs.

Neonatal mortality in puppies is a problem frequently encountered by dog breeders. Often, only postmortem examination allows diagnosis and implementation of measures to save the rest of the litter. This article presents the key steps of the postmortem examination, namely, autopsy, histopathology, bacteriology, molecular identification of pathogens, and coproscopy. Sampling, samples' conservation, and interpretation of the obtained results are presented as well as their relative importance for the final diagnosis. Finally, examples of the most frequent syndromes observed under postmortem examination in canine newborns, together with the results from a complementary analysis looking for infectious agents responsible for death, are discussed.

Low and very low birth weight in puppies: definitions, risk factors and survival in a large-scale population.

BACKGROUND: Neonatal mortality (over the first three weeks of life) is a major concern in canine breeding facilities as an economic and welfare issue. Since low birth weight (LBW) dramatically increases the risk of neonatal death, the risk factors of occurrence need to be identified together with the chances and determinants of survival of newborns at-risk. RESULTS: Data from 4971 puppies from 10 breeds were analysed. Two birth weight thresholds regarding the risk of neonatal mortality were identified by breed, using respectively Receiver Operating Characteristics and Classification and Regression Tree method. Puppies were qualified as LBW and very low birth weight (VLBW) when their birth weight value was respectively between the two thresholds and lower than the two thresholds. Mortality rates were 4.2, 8.8 and 55.3%, in the normal, LBW and VLBW groups, accounting for 48.7, 47.9 and 3.4% of the included puppies, respectively. A separate binary logistic regression approach allowed to identify breed, gender and litter size as determinants of LBW. The increase in litter size and being a female were associated with a higher risk for LBW. Survival for LBW puppies was reduced in litters with at least one stillborn, compared to litters with no stillborn, and was also reduced when the dam was more than 6 years old. Concerning VLBW puppies, occurrence and survival were influenced by litter size. Surprisingly, the decrease in litter size was a risk factor for VLBW and also reduced their survival. The results of this study suggest that VLBW and LBW puppies are two distinct populations. Moreover, it indicates that events and factors affecting intrauterine growth (leading to birth weight reduction) also affect their ability to adapt to extrauterine life. CONCLUSION: These findings could help veterinarians and breeders to improve the management of their facility and more specifically of LBW puppies. Possible recommendations would be to only select for reproduction dams of optimal age and to pay particular attention to LBW puppies born in small litters. Further studies are required to understand the origin of LBW in dogs.

Birth weight as a risk factor for neonatal mortality: Breed-specific approach to identify at-risk puppies.

In numerous species, low birth weight is a risk factor for neonatal mortality. In the canine species, definition of a low birth weight is complex due to the huge interbreed variability in size. To identify puppies at higher risk of neonatal death, data from 6,694 puppies were analysed. The data were collected from 75 French breeding kennels, examining 27 breeds and totaling 1,202 litters of puppies. Generalised linear mixed models allowed to identify birth weight, birth weight heterogeneity within the litter, and size of the breeding kennel as significant risk factors for neonatal mortality. Receiver Operating Characteristics (ROC) and classification and regression tree (CART) analyses were combined to define breed specific thresholds for birth weight allowing the identification of puppies at higher risk of neonatal mortality. Due to differences in birth weights between breeds, including when belonging to the same breed size, analyses were conducted at the breed level. First, ROC analysis thresholds were successfully established for 12 breeds (area under the ROC ≥ 0.70; sensitivity ≥ 75%; specificity: 45-68%) and they ranged from 162 g in the Maltese to 480 g in the Bernese Mountain dog. Secondly, CART analysis thresholds from 22 breeds ranged from 105 g in the Maltese and 436 g in the Boxer. Puppies were grouped into three categories according to birth weight: low, moderate and high risk of neonatal mortality (higher than the ROC threshold, between ROC and CART thresholds, and lower than the CART threshold respectively). In the current study, 44% of the puppies were classified as at moderate risk and 5.3% for a high risk of neonatal mortality. Thresholds defined by CART analysis (and not ROC analysis) were used to define low birth weight puppies and were sometimes quite different between breeds with similar birth weight distributions suggesting a variable relationship between birth weight reduction and neonatal death. These results allow the identification of puppies at an increased risk of neonatal death, thus requiring specific nursing to improve their chances of survival. With these high risk puppies identified, both animal welfare and kennel productivity is predicted to improve.

Passive immune transfer in puppies.

The puppy, born without immunoglobulins G (IgG), acquires a passive systemic immunity thanks to colostrum intake during the two first days of life. The quality of passive immune transfer (i.e. blood IgG concentration at two days of age), highly variable between litters and between puppies within litters, depends mainly on the time elapsed between birth and ingestion of colostrum, with limited influence of colostrum IgG concentration. Deficit in passive immune transfer, impacting puppy's health and neonatal mortality rate, can be indirectly diagnosed through blood gammaglutamyltransferases assay and evaluation of growth rate over the two first days of life. In the absence of maternal colostrum, few homo- and heterospecific immune sources are available and canine colostrum banking remains the optimal solution. Whereas passive immune transfer is crucial for survival during the neonatal period, it later interferes with response to vaccination. In addition to systemic passive immune transfer, maternal antibodies (mainly IgA) would provide local (digestive) immunity, ensuring mid-term protection of the puppies' gut together with probably long term training of the digestive immune system.

Oral Systemic Bioavailability of Bisphenol A and Bisphenol S in Pigs.

BACKGROUND: Given its hormonal activity, bisphenol S (BPS) as a substitute for bisphenol A (BPA) could actually increase the risk of endocrine disruption if its toxicokinetic (TK) properties, namely its oral availability and systemic persistency, were higher than those of BPA. OBJECTIVES: The TK behavior of BPA and BPS was investigated by administering the two compounds by intravenous and oral routes in piglet, a known valid model for investigating oral TK. METHODS: Experiments were conducted in piglets to evaluate the kinetics of BPA, BPS, and their glucuronoconjugated metabolites in plasma and urine after intravenous administration of BPA, BPS, and BPS glucuronide (BPSG) and gavage administration of BPA and BPS. A population semiphysiologically based TK model describing the disposition of BPA and BPS and their glucuronides was built from these data to estimate the key TK parameters that drive the internal exposure to active compounds. RESULTS: The data indicated that almost all the BPS oral dose was absorbed and transported into the liver where only 41% of BPS was glucuronidated, leading to a systemic bioavailability of 57.4%. In contrast, only 77% of the oral dose of BPA was absorbed and underwent an extensive first-pass glucuronidation either in the gut (44%) or in the liver (53%), thus accounting for the low systemic bioavailability of BPA (0.50%). Due to the higher systemic availability of BPS, in comparison with BPA, and its lower plasma clearance (3.5 times lower), the oral BPS systemic exposure was on average about 250 times higher than for BPA for an equal oral molar dose of the two compounds. CONCLUSION: Given the similar digestive tracts of pigs and humans, our results suggest that replacing BPA with BPS will likely lead to increased internal exposure to an endocrine-active compound that would be of concern for human health. https://doi.org/10.1289/EHP4599.

Is bisphenol S a safer alternative to bisphenol A in terms of potential fetal exposure ? Placental transfer across the perfused human placenta.

The aim of our study was to evaluate the bidirectional transfer of Bisphenol S (BPS) and its main metabolite, BPS Glucuronide (BPSG), using the model of perfused human placenta and to compare the obtained values with those of Bisphenol A (BPA) and BPA Glucuronide. Fourteen placentas at term were perfused in an open dual circuit with deuterated BPS (1 and 5 µM) and non-labelled BPSG (2.5 µM) and a freely diffusing marker antipyrine (800 ng/ml) in the presence of albumin (25 mg/ml). In a second experiment, the potential role of P-glycoprotein in the active efflux of BPS across the placental barrier was studied using the well-established P-glycoprotein inhibitor, PSC833 (2 and 4 µM). Placental transfer of BPS was much lower than that of BPA in both directions. The placental clearance index of BPS in the materno-fetal direction was three times lower than in the opposite direction, strongly suggesting some active efflux transport. However, our results show that P-glycoprotein is not involved in limiting the materno-fetal transfer of BPS. Placental transfer of BPSG in the fetal compartment was almost non-existent indicating that, in the fetal compartment, BPSG originates mainly from feto-placental metabolism. The feto-maternal clearance index for BPSG was 20-fold higher than the materno-fetal index. We conclude that the blood-placental barrier is much more efficient in limiting fetal exposure to BPS than to BPA, indicating that the placenta has a crucial role in protecting the human fetus from BPS exposure.

General and type 2 parvovirus-specific passive immune transfer in puppies - Evaluation by early growth.

Failure of passive immune transfer put puppies at a higher risk of neonatal and weaning mortality due to low immune protection against infectious agents. The aim of this study was to investigate the evolution of the general via serum IgG concentration (IgG) and the specific via serum maternally derived canine parvovirus type 2-specific antibody titer (CPV2 MDA) passive immune transfer within the first 4 weeks of age. Furthermore, the relationship between general and specific immune transfer and the possibility of non-invasive evaluation was assessed. Puppies (169) were weighed systematically between birth and Day 28. IgG and CPV2 MDA were assayed in serum samples at 2 and at 28 days of age. At Day 2, there was a positive correlation between IgG and CPV2 MDA (ρ = 0.71; p < 0.001). At Day 2, 17.9% (27/151) of puppies presented a deficit of passive immune transfer according to IgG result (defined as IgG < 2.3 g/L) and 25.8% (39/151) of puppies were under the minimal protective CPV2 MDA titer (defined as <1:160). No correlation between IgG and CPV2 MDA was observed at Day 28 (ρ = 0.14; p = 0.11). Growth rate within the first 48 hours <-2.7% allowed to distinguish puppies at high risk of the general and specific passive immune failure (Youden's index = 0.79 and 0.75, respectively). The threshold value of early growth rate, although applicable only in puppies non-supplemented with milk replacer, allows identifying via non-invasive way individuals requiring a special care. Further investigation of the mechanism of passive immune transfer in dogs is necessary to understand the relationship between the general and specific immunoglobulin levels.

Bisphenol S instead of Bisphenol A: Toxicokinetic investigations in the ovine materno-feto-placental unit.

Bisphenol S (BPS) is widely used as a substitute for Bisphenol A in consumer products. Despite its potential endocrine-disrupting effects and widespread exposure, toxicokinetic data, particularly during the critical period of pregnancy, are not available for BPS. The objectives of our study were to evaluate the mechanisms determining fetal exposure to BPS and to BPS glucuronide (BPSG) and to compare them with those prevailing for BPA. The disposition of BPS and BPSG was evaluated in the materno-fetal unit of the catheterized pregnant ewe model, following intravenous administrations of BPS and BPSG to mothers and their fetuses. In a second experiment, the rate of BPS accumulation in the fetal compartment was determined under steady-state conditions after repeated intravenous BPS administrations to the mother. In the maternal compartment, BPS was mainly metabolized into BPSG and totally eliminated in urine. Only 0.40% of the maternal dose was transferred to the fetus. However, once in the fetal compartment, 26% of the fetal dose was rapidly eliminated through placental transfer, while 46% of BPS was metabolized into BPSG which remained trapped in the fetal compartment. Thus, the elimination of BPSG from the fetal compartment required its back-conversion into bioactive BPS, leading to an 87% enhancement of the fetal BPS exposure. Our findings demonstrate that, despite the low materno-fetal placental transfer of BPS, this substitute for BPA is able to accumulate in the fetal compartment after repeated maternal exposure, leading to chronic fetal exposure to BPS in a range of concentrations similar to those of BPA.

Monitoring of the newborn dog and prediction of neonatal mortality.

Despite the high neonatal mortality rate in puppies, pertinent criteria for health evaluation of the newborns are not defined. This study was thus designed to measure and to characterize factors of variation of six health parameters in dog neonates, and to evaluate their value as predictors of neonatal mortality. A total of 347 purebred puppies under identical conditions of housing and management were examined within the first 8h after birth and then at Day 1. The first health evaluation included Apgar score, weight, blood glucose, lactate and ß-hydroxybutyrate concentration, rectal temperature and urine specific gravity (SG). The second evaluation at Day 1 included the same parameters, excluding Apgar score and weight. The mortality rate over the first 24h and over 21days of age was recorded. The early predictors of neonatal mortality in the dog were determined with generalized linear mixed models and receiver operating characteristic curves analyses. An Apgar score at or below 6 evaluated within the first 8h after birth was found associated with a higher risk of death during the first 24h. A reduced glucose concentration (≤92mg/dl) at Day 1 was found to be associated with higher mortality between 1 and 21days of age. Low-birth-weight puppies were characterized by both low viability (low Apgar score) and low blood glucose concentration, and thus were found indirectly at higher risk of neonatal mortality. This study promotes two low cost easy-to-use tests for health evaluation in puppies, i.e. Apgar scoring and blood glucose assay. Further investigation is necessary to establish if the strong relationship between blood glucose and neonatal survival reflects high energy requirements or other benefits from colostrum intake.

Characterization of the fecal microbiome during neonatal and early pediatric development in puppies.

Limited information is available describing the development of the neonatal fecal microbiome in dogs. Feces from puppies were collected at 2, 21, 42, and 56 days after birth. Feces were also collected from the puppies' mothers at a single time point within 24 hours after parturition. DNA was extracted from fecal samples and 454-pyrosequencing was used to profile 16S rRNA genes. Species richness continued to increase significantly from 2 days of age until 42 days of age in puppies. Furthermore, microbial communities clustered separately from each other at 2, 21, and 42 days of age. The microbial communities belonging to dams clustered separately from that of puppies at any given time point. Major phylogenetic changes were noted at all taxonomic levels with the most profound changes being a shift from primarily Firmicutes in puppies at 2 days of age to a co-dominance of Bacteroidetes, Fusobacteria, and Firmicutes by 21 days of age. Further studies are needed to elucidate the relationship between puppy microbiota development, physiological growth, neonatal survival, and morbidity.

Immunoglobulin G concentration in canine colostrum: Evaluation and variability.

Canine neonates are born hypogammaglobulinemic, and colostrum is their main source of immunoglobulins. The purpose of this study was to evaluate the immune quality of canine colostrum and its variability both among bitches and among mammary glands. The immune quality was estimated from immunoglobulin G (IgG) concentration (ELISA test). The correlation of IgG concentration with refractometry was evaluated. From a total of 44 bitches from 13 different breeds from a single breeding kennel, samples of colostrum and blood were collected one day after the parturition onset. Colostrum was collected separately from each pair of mammary glands (180 pairs). The mean colostrum IgG concentration in our population was 20.8 ± 8.1g/L (ranging from 8.0 to 41.7 g/L) with no influence of breed size, litter size, age of dam or serum IgG concentration. Colostrum IgG concentration varied widely among pairs of mammary glands within one bitch (variation coefficient: 42 ± 32.1%). Nevertheless, no single pair of mammary glands was found to produce regularly a secretion of higher quality. No difference in IgG concentration was recorded between anterior and posterior pairs either. The BRIX index and the refractive index were significantly, but moderately correlated with colostrum IgG concentration (r=0.53 and 0.42, respectively). This study demonstrates a great variability in immune quality of colostrum among bitches and among mammary glands within one bitch. Further studies on the suckling behavior of puppies and on determination of the minimal immune quality of colostrum are required to evaluate their impact of this high variability on neonatal mortality in dogs.

Effect of age, gestation and lactation on faecal IgA and calprotectin concentrations in dogs.

Faecal calprotectin and IgA have been suggested as non-invasive markers of gut health. Faecal calprotectin is a marker of intestinal inflammation in adults, whereas IgA has been suggested as a marker of intestinal immunity. The purpose of the present study was to evaluate the effect of gestation, lactation and age on faecal concentrations of these biomarkers. Thirty puppies, nineteen pregnant or lactating bitches and eighty-nine healthy control adult dogs were included in the study. Faeces were collected from the fourth week of gestation until the eighth week of lactation in pregnant and lactating bitches, and between 4 and 9 weeks of age in puppies. Faeces from the eighty-nine healthy control adult dogs were also collected. Faecal calprotectin and IgA concentrations were measured. Faecal calprotectin concentrations in control dogs were significantly lower than faecal calprotectin concentrations in puppies between 4 and 6 weeks of age (P < 0·001) or between 7 and 9 weeks of age (P = 0·004). Puppies between 4 and 6 weeks of age had significantly higher faecal IgA concentrations compared with puppies between 7 and 9 weeks of age (P = 0·001). Bitches during their second month of lactation had significantly lower faecal IgA concentrations compared with their first month of lactation (P = 0·049). Faecal calprotectin and IgA have been suggested as non-invasive and easily measured biomarkers of gut health in adults. However, the present study underlines that faecal IgA and calprotectin concentrations vary markedly depending of physiologic factors such as gestation, lactation and age. These factors need to be considered when these faecal biomarkers are used for evaluation of intestinal immunity or inflammation.

Protection against canine parvovirus type 2 infection in puppies by colostrum-derived antibodies.

During the first weeks of life puppies remain protected against canine parvovirus type 2 (CPV2) infection thanks to maternally derived antibodies (MDA) absorbed with colostrum after birth. The objective of the present study was to present the variability in CPV2-specific passive immune transfer and its consequences in puppies naturally exposed to the parvovirus. Seventy-nine puppies from one breeding kennel were included in the study at birth and followed until 56 d of age. Once per week the MDA titre for CPV2 specific antibodies was determined in blood. Viral excretion was also evaluated on a rectal swab by CPV2 PCR assay and puppies were weighed to determine growth rate. At 2 d of age, thirty-four out of seventy-nine puppies (43 %) had MDA ≤1:160 (designed group A) and forty-five puppies (57 %) had greater MDA titres (designed group B). The level of absorbed maternal antibodies was shown to be associated with breed size and growth rate during the first 48 h of life. The MDA level declined with age in all cases; however, the proportion of puppies with the antibody level considered as protective against CPV2 infection was significantly higher in group B compared with A from day 2 until 42. Among all puppies surviving until 56 d of age, sixty-seven out of seventy (95·7 %) underwent CPV2 infection. However, puppies from group A excreted CPV2 significantly earlier than puppies from group B. The present study demonstrates the link between passive immune transfer, in terms of level of specific MDA absorbed, and length of the protection period against parvovirus infection in weaning puppies.

The usefulness of Real Time Morphology software in semen assessment of teratozoospermic boars.

The aim of the present study was to compare two different techniques of sperm cell morphology evaluation in teratozoospermic boars: computer assisted semen morphology analysis and conventional assessment of stained semen smears. The semen samples were collected manually from 30 boars with reduced semen quality. In all samples the percentage of morphologically normal spermatozoa was below 70%. Computer assisted semen morphology assessment was performed using the Real Time Morphology (RTM) software (IVOS ver. 12.2, Hamilton Thorne Bioscience). The assessment was made by phase contrast optics, with the magnification of 20 x 3.8 and without staining. Conventional morphology assessment was performed by bright field microscopy with 1,000 x magnification after staining with Giemsa. At least 200 spermatozoa were evaluated per slide in both methods. The Bland-Altman plot indicated a general agreement between both methods of sperm morphology evaluation. The plots revealed the widest limits of agreement (mean ± 1.96 SD) for the percentage of midpiece anomalies (from -16 to 13.2), and the narrowest for the percentage of looped tail (from -1.49 to 1.09). The Bland Altman plot indicates general agreement between RTM and Giemsa staining in the percentage of major and minor defects. However, it was not possible to evaluate acrosomes using RTM. Otherwise, RTM proved to be a valuable tool in sperm morphology assessment, with accuracy equal to typical conventional methods.