Health assessment of Holstein calves born after in vitro fertilization, biopsy-based genotyping at the blastocyst stage and subsequent embryo transfer.

Establishing methods for evaluating genomic estimated breeding values of bovine embryos can potentially increase the efficiency of breeding programs by transferring only embryos with a high genomic estimated breeding value. This may be achieved by analyzing DNA from trophectoderm biopsies. However, manipulation of bovine embryos is associated with a risk of impaired conceptus health. More knowledge on the health implications of embryonic handling procedures is required. In this study, we followed pregnancies after transfer of in vitro-produced (IVP) embryos and assessed the health of the offspring during the first 2 weeks of life. Three groups of calves were studied: i) freshly transferred non-biopsied embryos (39 transfers, 17 calves; Group B-/C-); ii) biopsied and freshly transferred IVP embryos (42 transfers, 21 calves; Group B+/C-); iii) biopsied and cryopreserved IVP embryos (17 transfers, 6 calves; Group B+/C+). Blood biochemical and hematologic values were compared between groups and to a control group of 13 calves produced by conventional artificial insemination. The pregnancy rate on day 50 and the calving rate did not differ among the groups, but the average gestation length of the B+/C+ group was significantly shorter and with wider variation than the two other groups. There was a tendency toward a higher average body weight at birth in group B+/C+ (45.1 kg) and the standard deviation in body weight was larger (11.7 kg) compared to the B-/C- (39.5 kg; 3.2 kg) and B+/C- (41.8 kg; 6 kg) groups. Body weight on day 14 was higher in the B+/C+ calves compared to the other groups. There was no difference in the biochemical and hematological values at birth between the groups and these were within the normal range. However, when compared to a group of calves produced by standard artificial insemination, significantly higher concentrations were found for the hepatic-related enzymes ALAT, ASAT, ALP, and GGT in group B-/C-and B+/C-, while only higher ALP concentrations were found in B+/C+ calves. The biochemical findings indicate higher heterogeneity in IVP calves compared to calves produced by artificial insemination. The more manipulated IVP embryos also showed increased heterogeneity in body weight at birth, with a shift toward heavier calves, which calls for closer attendance at parturition to handle dystocia in a timely manner and minimize fetal losses.

Assessing welfare risks in unowned unsocialised domestic cats in Denmark based on associations with low body condition score.

BACKGROUND: Populations of unowned unsocialised cats are present worldwide. Generally, there is concern about their welfare. Low body condition score (BCS) is a potentially relevant indicator that is relatively easy to assess: emaciated cats are likely to have welfare problems while thin cats may be at risk of becoming emaciated. The objective of this study was to assess the association of low BSC with a selection of factors relating to the host, disease, and infection in unowned unsocialised domestic cats. We necropsied 598 euthanised unowned unsocialised cats from Denmark. We recorded each cat's age-group, sex, and neuter status, together with its pregnancy status, the season and location of trapping, as well as gross lesions at necropsy. We also tested for feline immunodeficiency virus and feline leukaemia virus, recorded presence of ectoparasites, and a subsample of the cats were also tested for endoparasites. Cats with no or sparse adipose deposits were categorised as having low BCS, and logistic regression was used to determine the factors associated with low BCS. RESULTS: Of the cats, 11.4% had low BCS. Season, age-group and sex were associated with low BCS and confounded potential associations with other variables. Intact adult males and females in spring and early summer were at highest risk of low BCS. When these factors were taken into account, cats with biting lice had 2.8 (95% confidence interval (CI) 1.4-5.4) times higher odds of low BCS, and cats with gastro-intestinal findings (i.e., enlarged mesenteric lymph nodes, abdominal hernia, diarrhoea, obstructive foreign bodies, or diaphragmatic hernia) had 50 (95% CI 10-417) times higher odds of low BCS, than cats with no such findings. Cats with low BCS were primarily adult intact cats with tooth lesions and skin lesions, ear mite infection, and positive test result for feline immunodeficiency virus. CONCLUSIONS: The results highlight associations that can be used to define a risk profile: low BCS, notably in summer-autumn, in an unowned unsocialised cat was associated with underlying, less visible problems. Thus, low BCS can be more than a step towards being emaciated; it can also be an indicator of other underlying welfare problems.

How Serious Are Health-Related Welfare Problems in Unowned Unsocialised Domestic Cats? A Study from Denmark Based on 598 Necropsies.

Free ranging unsocialised domestic cats are widely believed to suffer from a high load of welfare problems. We assessed the validity of this belief by performing necropsies on the corpses of 598 unsocialised cats, originating from all parts of Denmark, that had been euthanised by two Danish cat welfare organisations. We selected a number of variables for health-related cat welfare that could be assessed through necropsy (e.g., gross lesions, ectoparasites and body condition) or by laboratory analysis (e.g., infection with feline immunodeficiency virus (FIV), and by feline leukaemia virus (FeLV)). Each finding was classified as having either a major or minor welfare impact on the cat. More than 83% of the cats had no major finding, and 54% had no finding indicating a welfare issue at all. More than 83% of the cats had a body condition within normal range. Only 0.3% were emaciated. The most common finding was infestation with ectoparasites, with 15.9% infected with lice, 12.3% with fleas, 4.7% with ticks, and 6.7% with ear mites. FIV and FeLV were detected in 9.2% and 1.2% of the cases, respectively. The most common lesion related to the cats' teeth. Overall, unsocialised cats in Denmark have a moderate level of health-related welfare problems.

Ethylene glycol toxicosis in milk-fed dairy calves.

BACKGROUND: Ethylene glycol (EG) (antifreeze) toxicosis has mostly been reported in dogs and cats, while reports on EG toxicosis in cattle are sparse. We report EG toxicosis in 25 milk-fed calves associated with a leak in the cooling pipes in a milk taxi. The milk taxi was connected to a geothermal heating system in which EG was used as antifreeze. CASE PRESENTATION: Although the assistant responsible for feeding milk to the calves observed a few blue-colored droplets of liquid on the surface of the milk in the milk taxi and suspected EG contamination, the milk was fed to the calves. Within hours, the calves became depressed and some died within the next 2 days. Necropsy and histopathology revealed widespread severe acute renal tubular necrosis with numerous birefringent crystals in the tubular lumen. Biochemical analysis of serum showed severe damage to the kidneys (marked azotemia) and hypochloremia, hyponatremia and hyperkalemia; findings consisting with metabolic acidosis. After feeding the calves, the assistant inspected the milk taxi and found a leaking cooling pipe. CONCLUSIONS: The suspected EG toxicosis was confirmed by the observation of renal tubular necrosis, numerous intratubular crystals, and metabolic acidosis. EG toxicosis due to leaking pipes connected to a geothermal heating system has not been reported previously. Alternative antifreeze products that are less toxic than EG are recommended for use if there is a risk of contamination of human and animal foodstuffs in case of a leak in the system.

Mild Microglial Responses in the Cortex and Perivascular Macrophage Infiltration in Subcortical White Matter in Dogs with Age-Related Dementia Modelling Prodromal Alzheimer's Disease.

BACKGROUND: Microglia contribute to Alzheimer's disease (AD) pathogenesis by clearing amyloid-ß (Aß) and driving neuroinflammation. Domestic dogs with age-related dementia (canine cognitive dysfunction (CCD)) develop cerebral amyloidosis like humans developing AD, and studying such dogs can provide novel information about microglial response in prodromal AD. OBJECTIVE: The aim was to investigate the microglial response in the cortical grey and the subcortical white matter in dogs with CCD versus age-matched cognitively normal dogs. METHODS: Brains from aged dogs with CCD and age-matched controls without dementia were studied. Cases were defined by dementia rating score. Brain sections were stained for Aß, thioflavin S, hyperphosphorylated tau, and the microglial-macrophage ionized calcium binding adaptor molecule 1 (Iba1). Results were correlated to dementia rating score and tissue levels of Aß. RESULTS: Microglial numbers were higher in the Aß plaque-loaded deep cortical layers in CCD versus control dogs, while the coverage by microglial processes were comparable. Aß plaques were of the diffuse type and without microglial aggregation. However, a correlation was found between the %Iba1 area and insoluble Aß 42 and N-terminal pyroglutamate modified Aß(N3pE)-42. The %Iba1 area was higher in white matter, showing phosphorylation of S396 tau, versus grey matter. Perivascular macrophage infiltrates were abundant in the white matter particularly in CDD dogs. CONCLUSION: The results from this study of the microglial-macrophage response in dogs with CCD are suggestive of relatively mild microglial responses in the Aß plaque-loaded deep cortical layers and perivascular macrophage infiltrates in the subcortical white matter, in prodromal AD.

A large deletion in the COL2A1 gene expands the spectrum of pathogenic variants causing bulldog calf syndrome in cattle.

BACKGROUND: Congenital bovine chondrodysplasia, also known as bulldog calf syndrome, is characterized by disproportionate growth of bones resulting in a shortened and compressed body, mainly due to reduced length of the spine and the long bones of the limbs. In addition, severe facial dysmorphisms including palatoschisis and shortening of the viscerocranium are present. Abnormalities in the gene collagen type II alpha 1 chain (COL2A1) have been associated with some cases of the bulldog calf syndrome. Until now, six pathogenic single-nucleotide variants have been found in COL2A1. Here we present a novel variant in COL2A1 of a Holstein calf and provide an overview of the phenotypic and allelic heterogeneity of the COL2A1-related bulldog calf syndrome in cattle. CASE PRESENTATION: The calf was aborted at gestation day 264 and showed generalized disproportionate dwarfism, with a shortened compressed body and limbs, and dysplasia of the viscerocranium; a phenotype resembling bulldog calf syndrome due to an abnormality in COL2A1. Whole-genome sequence (WGS) data was obtained and revealed a heterozygous 3513 base pair deletion encompassing 10 of the 54 coding exons of COL2A1. Polymerase chain reaction analysis and Sanger sequencing confirmed the breakpoints of the deletion and its absence in the genomes of both parents. CONCLUSIONS: The pathological and genetic findings were consistent with a case of "bulldog calf syndrome". The identified variant causing the syndrome was the result of a de novo mutation event that either occurred post-zygotically in the developing embryo or was inherited because of low-level mosaicism in one of the parents. The identified loss-of-function variant is pathogenic due to COL2A1 haploinsufficiency and represents the first structural variant causing bulldog calf syndrome in cattle. Furthermore, this case report highlights the utility of WGS-based precise diagnostics for understanding congenital disorders in cattle and the need for continued surveillance for genetic disorders in cattle.

Staphylococcus aureus infected embolic stroke upregulates Orm1 and Cxcl2 in a rat model of septic stroke pathology.

OBJECTIVE: Ischaemic brain lesions and brain abscesses are frequent in both human and animal cases of septic embolic stroke. However, existing models of brain infection do not reflect central aspects of septic embolic stroke. Our aim was to compare septic and non-septic embolic stroke in order to identify gene expressions, inflammatory mediators and brain damage in a rat model. METHODS: We created precisely located focal brain infarcts in a rat model of Staphylococcus aureus infected embolic stroke. To cause septic embolic stroke we used a fibrin-rich embolus with bacteria, while every rat in the control group received a non-infected embolus. 64 rats were randomized to receive sham-surgery, sterile embolic stroke or septic embolic stroke. All groups were compared for brain pathology, mortality, gene expressions and inflammatory mediators using histology and reverse transcription quantitative real-time PCR. RESULTS: Although infarct volumes did not differ, septic embolic stroke caused higher mortality than sterile embolic stroke (p=  0.002). Brain abscesses were observed only in the septic group. Approximately 400-500 fold increases were observed for Orm1 and Cxcl2 respectively (1.00E-08 < p < 1.92E-07) in the septic group compared to the sterile group, and these were the most dramatically regulated genes in septic embolic stroke compared to sterile embolic stroke. CONCLUSIONS: Septic embolic stroke caused brain abscesses, increased mortality and upregulated Orm1 and Cxcl2 gene expressions compared to non-infected embolic stroke. The dramatic Orm1 increase observed in the septic group is unprecedented and suggests a significant biological role of Orm1 during septic neuroinflammation.

Colonization of the bovine uterus by Candida kefyr.

BACKGROUND: While fungal infections of the bovine uterus are well-known diseases in pregnant cattle, very limited knowledge exists on the presence and significance of fungi in the uterus of non-pregnant cows. Presence of fungi in the uterine lumen of postpartum (pp) cows has been reported, but little attention has been paid to this as most studies of the bovine pp uterus have focused on bacteria. CASE PRESENTATION: Microscopy of uterine lavage cytology slides of three cows from one herd revealed the presence of numerous yeast-like organisms, which were located either free in the fluid or within macrophages. Two of the cows were around 30 days pp, while the third was 7 months pp. None of the cows had been treated with antibiotics. Culturing of the flush samples was unsuccessful, but Sanger sequencing of DNA extracted from an endometrial biopsy of one of the cows revealed the presence of Candida kefyr (Kluyveromyces marxianus). Fluorescence in situ hybridization examination of endometrial tissue sections of two cows using probes targeting 18S rRNA of the K. marxianus group was performed and revealed the presence of yeast cells on the endometrium. Histology was performed and demonstrated hyphal and non-hyphal yeast-like organisms on the surface of endometrium and in the crypts. Tissue invasion was restricted to the superficial part of the epithelium and although endometrial inflammation was present, this was mild and considered as not being caused by the fungi. One of the cows became pregnant and delivered a normal calf at term, while the two others were not bred. CONCLUSIONS: Candida kefyr is commonly isolated from milk of cows with mastitis, but has not been reported in association with other diseases of cattle. The infection was present as a monoculture in all three cows, but the fungi had only colonized the uterine lumen and the endometrial surface. Only a mild non-suppurative endometrial inflammation was present, but within the uterine luminal content, many macrophages having phagocytized yeast cells were present. Re-examination of the cows did not reveal a persistent infection, so the infection probably resolved spontaneously.

Presence of bacteria in the endometrium and placentomes of pregnant cows.

Bacterial invasion of the bovine uterus during the postpartum period occurs in most cows, but the general consensus is that these bacteria are eliminated before the next pregnancy. The pregnant uterus has therefore hitherto been considered a sterile environment, but this assumption has now been challenged by recent studies in humans, which indicate that bacteria can be present in the placenta of term pregnancies without causing abortion. The aim of the present study was therefore to investigate whether bacteria are present in the uterus of pregnant cows. Specimens were taken from the inter-caruncular endometrium and from placentomes of slaughtered pregnant cows (n = 43) and subjected to histology, fluorescence in situ hybridization and massive parallel sequencing. Bacteria were observed in the tissue from 90.7% (39/43) of the cows by fluorescence in situ hybridization. Fusobacterium necrophorum, Porphyromonas levii and Trueperella pyogenes were located within the endometrium, on the endometrial surface and in the caruncular stroma, but their presence was not associated with inflammation. Data from massive parallel sequencing of the 16S rRNA gene from a subset of 15 cows indicated that the most abundant bacteria were the families Porphyromonadaceae, followed by Ruminococcaceae and Lachnospiraceae. Our results indicate that the bovine uterus is not a sterile environment during pregnancy as previously assumed and that a cow can carry a pregnancy despite the presence of a few potentially pathogenic bacteria in the uterus.

An investigation of the microbiota in uterine flush samples and endometrial biopsies from dairy cows during the first 7 weeks postpartum.

Metritis and endometritis commonly occur in dairy cows after calving. Although numerous studies have been performed to identify the causative pathogens, a complete overview has not been done. Metagenomic studies have analyzed the bacterial populations of uterine flush samples from postpartum (pp) dairy cows, but the microbiota in the uterine luminal fluid may differ from the microbiota of the endometrium itself, and important putative pathogens may have been overlooked. In the present study, we compared the microbiota of the uterine lumen and the endometrium of healthy, metritic, and endometritic cows. Samples were collected from 68 Holstein dairy cows at 1, 4, and 7 weeks pp, and the data were analyzed by deep sequencing of the V1 and V2 hypervariable regions of the 16S ribosomal RNA gene. The results showed that Porphyromonadaceae, Fusobacteriaceae, Leptotrichiaceae, and Mycoplasmataceae may be associated with uterine disease. The microbiota of the uterine flush samples and the endometrial biopsies were correlated, but the microbiota of the biopsies was more diverse. Fusobacteriaceae and Leptotrichiaceae were not observed in the biopsies at week 7, whereas they accounted for 20% and 13%, respectively, of the bacterial populations in the flush samples. The Mycoplasmataceae family was observed in much higher quantity in the flush samples than in the biopsies of the endometritis groups at weeks 4 and 7. Our findings support the observations of previous metagenomic studies and illustrate the importance of including endometrial biopsies to obtain more detailed knowledge of the pp uterine microbiota.