Contrast-enhanced ultrasonography of the thyroid gland in healthy dogs, hypothyroid dogs and dogs with non-thyroidal illness.

Diagnosis of canine hypothyroidism remains challenging, as non-thyroidal illness (NTI)-syndrome and medical treatment can influence thyroid hormone concentrations. Conventional ultrasound may give additional hints, however high interobserver variability has been described. Contrast-enhanced ultrasound (CEUS) allows detection of changes in tissue perfusion. The purpose of the present study was to assess the possible diagnostic value of CEUS regarding diagnosis of hypothyroidism. CEUS of the thyroid gland was performed in 52 healthy dogs, 16 hypothyroid dogs, and 20 NTI patients. The following perfusion parameters were calculated: Thyroid/carotid artery (TG/CA) ratios for peak enhancement (PE) and area under the curve (AUC), time to peak (TTP) and wash-in and wash-out rates (WiR, WoR) of the thyroid gland. Impact of sedation on perfusion parameters was investigated in 8 calm healthy dogs which were examined before and after sedation using midazolam and butorphanol. Significantly higher median TG/CA ratios for PE were detected for the left and right thyroid lobe in dogs with hypothyroidism (0.97/0.96) compared to healthy dogs (0.85/0.85) and dogs with NTI (0.84/0.84). AUCs were also significantly increased in hypothyroid dogs when compared to other groups. Dogs with NTI showed significantly lower WiR and WoR compared to other groups. Values for TTP were not significantly different between groups. Sedation had only impact on results of TTP which was significantly prolonged in sedated dogs. In conclusion, CEUS of the thyroid gland can provide an additional tool for diagnosis of hypothyroidism in dogs and support its differentiation from NTI. Sedation has limited impact on CEUS results.

Effect of metritis on in-vitro uterine contractility in cows during the puerperium.

Aim of this study was to determine the effect of metritis on in-vitro uterine contractility. Uteri obtained from 16 euthanized Holstein-Friesian cows were divided into two groups depending on whether metritis was absent (M-, n = 6) or present (M+, n = 10). Four longitudinal and four circular myometrial strips of all uteri were incubated in an organ bath. Spontaneous contractility was recorded in five consecutive 30-minute periods (T1-T5). This was followed by stimulation of one longitudinal and one circular strip with increasing concentrations of oxytocin, prostaglandinF2α (PGF2α), and calcium chloride (each during four 30-minute periods [T6-T9]). Strips in group M+ had higher minimum amplitude (minA) values at T1 and higher minA, mean amplitude (meanA), and area under the curve (AUC) values at T2 than strips in group M- (P ≤ 0.05). In the M+ group, the maximum amplitude (maxA), meanA, and AUC values of circular strips were higher than those of longitudinal strips during spontaneous contractility (T1, T4, and T5; P ≤ 0.05). The minA, meanA, and AUC values for strips in group M+ were higher than those in group M- when exposed to the highest concentration of PGF2α (P ≤ 0.05). During stimulation with PGF2α (T9), longitudinal strips had higher maxA values than the circular strips in group M+ (P ≤ 0.05). Spontaneous and stimulated contractility were temporarily increased in uteri with metritis compared to healthy uteri. Both myometrial layers, especially in uteri with metritis, reacted differently during spontaneous contractility and to stimulation with PGF2α.

Cows selected for divergent mastitis susceptibility display a differential liver transcriptome profile after experimental Staphylococcus aureus mammary gland inoculation.

Infection and inflammation of the mammary gland, and especially prevention of mastitis, are still major challenges for the dairy industry. Different approaches have been tried to reduce the incidence of mastitis. Genetic selection of cows with lower susceptibility to mastitis promises sustainable success in this regard. Bos taurus autosome (BTA) 18, particularly the region between 43 and 59 Mb, harbors quantitative trait loci (QTL) for somatic cell score, a surrogate trait for mastitis susceptibility. Scrutinizing the molecular bases hereof, we challenged udders from half-sib heifers having inherited either favorable paternal haplotypes for somatic cell score (Q) or unfavorable haplotypes (q) with the Staphylococcus aureus pathogen. RNA sequencing was used for an in-depth analysis of challenge-related alterations in the hepatic transcriptome. Liver exerts highly relevant immune functions aside from being the key metabolic organ. Hence, a holistic approach focusing on the liver enabled us to identify challenge-related and genotype-dependent differentially expressed genes and underlying regulatory networks. In response to the S. aureus challenge, we found that heifers with Q haplotypes displayed more activated immune genes and pathways after S. aureus challenge compared with their q half-sibs. Furthermore, we found a significant enrichment of differentially expressed loci in the genomic target region on BTA18, suggesting the existence of a regionally acting regulatory element with effects on a variety of genes in this region.

Genetic selection for bovine chromosome 18 haplotypes associated with divergent somatic cell score affects postpartum reproductive and metabolic performance.

The susceptibility of animals to periparturient diseases has a great effect on the economic efficiency of dairy industries, on the frequency of antibiotic treatment, and on animal welfare. The use of selection for breeding cows with reduced susceptibility to diseases offers a sustainable tool to improve dairy cattle farming. Several studies have focused on the association of distinct bovine chromosome 18 genotypes or haplotypes with performance traits. The aim of this study was to test whether selection of Holstein Friesian heifers via SNP genotyping for alternative paternal chromosome 18 haplotypes associated with favorable (Q) or unfavorable (q) somatic cell scores influences postpartum reproductive and metabolic diseases. Thirty-six heifers (18 Q and 18 q) were monitored from 3 wk before calving until necropsy on d 39 (± 4 d) after calving. Health status and rectal temperature were measured daily, and body condition score and body weight were assessed once per week. Blood samples were drawn twice weekly, and levels of insulin, nonesterified fatty acids, insulin-like growth factor-I, growth hormone, and ß-hydroxybutyrate were measured. Comparisons between the groups were performed using Fisher's exact test, chi-squared test, and the GLIMMIX procedure in SAS. Results showed that Q-heifers had reduced incidence of metritis compared with q-heifers and were less likely to develop fever. Serum concentrations of ß-hydroxybutyrate were lower and insulin-like growth factor-I plasma concentrations were higher in Q- compared with q-heifers. However, the body condition score and withers height were comparable between haplotypes, but weight loss tended to be lower in Q-heifers compared with q-heifers. No differences between the groups were detected concerning retained fetal membranes, uterine involution, or onset of cyclicity. In conclusion, selection of chromosome 18 haplotypes associated with a reduced somatic cell score resulted in a decreased incidence of postpartum reproductive and metabolic diseases in this study. The presented data add to the existing knowledge aimed at avoiding negative consequences of genetic selection strategies in dairy cattle farming. The underlying causal mechanisms modulated by haplotypes in the targeted genomic region and immune competence necessitate further investigation.

Correction to: Characterization of functional traits with focus on udder health in heifers with divergent paternally inherited haplotypes on BTA18.

The original article [1] contained an error whereby the captions to Figs 2 and 3 were mistakenly inverted; this has now been corrected.

Early Infection Dynamics of Dichelobacter nodosus During an Ovine Experimental Footrot In Contact Infection.

INTRODUCTION: Ovine footrot caused by Dichelobacter nodosus is a highly contagious and painful disease representing an economic as well as an animal welfare problem. In order to get more information on the infection dynamics, 26 lambs and 4 ewes enrolled in an in-contact infection trial were monitored over two weeks for the presence of D. nodosus-specific DNA. Two D. nodosus-positive ewes were housed together with 13 confirmed negative lambs. The control group consisted of another 13 lamb siblings and two confirmed D. nodosus-negative ewes. Every foot of all sheep was sampled seven times over the two weeks experiment period and subsequently analyzed for the presence of D. nodosus by quantitative real-time PCR. The control group was negative at the beginning and the end of the experiment and showed no clinical symptoms of footrot. The two positive ewes showed a high, but hundred fold differing level of virulent D. nodosus that remained constant over time with one of the ewes being also weakly positive for benign D. nodosus. All lambs of the infection group were positive for virulent D. nodosus at 14 days post infection (dpi). The first positive animals were observed on 3 dpi. The D. nodosus load remained at a low level and only increased in a few lambs at the end of the trial. Five of the contact lambs showed suspicious clincal signs (score 1-2) at 14 dpi corroborating the PCR results and indicating that the disease starts as early as two weeks after contact with positive sheep.

INTRODUCTION: Le piétin causé par Dichelobacter nodosus est une maladie hautement contagieuse et douloureuse qui représente à la fois un problème économique et de bien-être animal. Pour avoir plus informations sur la dynamique de l'infection, 26 agneaux et 4 brebis appartenant à un groupe d'essai d'infection par contact ont été contrôlés pendant deux semaines quant à la présence d'ADN spécifique de D. nodosus. Deux brebis positives pour D. nodosus ont été mises en contact avec 13 agneaux confirmés négatifs. Le groupe témoin était formé de 13 autres agneaux et deux brebis confirmées négatives. Sept échantillons sur écouvillon ont été prélevés sur chaque pied de chaque mouton au cours des deux semaines de la période expérimentale et analysés quant à la présence de D. nodosus par PCR quantitative en temps réel. Le groupe témoin était négatif au début et à la fin de l'expérience et n'a montré aucun symptôme clinique de piétin. Les deux brebis positives ont montré une forte présence de D. nodosus virulent, mais de cent niveaux différents, qui est restée constant dans le temps, l'une des brebis étant aussi faiblement positive pour D. nodosus bénin. Tous les agneaux du groupe infecté étaient positifs pour D. nodosus virulent 14 jours après l'infection (dpi). Les premiers animaux positifs ont été observés à 3 dpi. La charge de D. nodosus est restée faible et n'a augmenté que chez quelques agneaux à la fin de l'expérience. Cinq des agneaux en contact ont présenté des symptômes suspects (score 1-2) à 14 dpi, corroborant les résultats de la PCR et indiquant que l'infection commence dès deux semaines après le contact avec des moutons positifs.

Liver phosphorus content and liver function in states of phosphorus deficiency in transition dairy cows.

Phosphorus (P) deficiency in early lactating dairy cows is receiving increased attention because of incentives aiming at curtailing environmental pollution with P by reducing dietary P in ruminant diets. An in-vitro study using bovine hepatocytes incubated for 7 days with phosphate (Pi) concentrations of 0.9, 1.8 or 2.7 mmol/L, and an in-vivo study feeding late pregnant dairy cows diets with either adequate (0.28% and 0.44% in DM ante-partum and post-partum respectively) or low P content (0.15% and 0.20% in DM ante-partum and post-partum respectively) from 4 weeks before to 4 weeks after calving were conducted to explore effects of P deprivation on liver function. In vitro the relative abundance of mRNA of key enzymes of the carbohydrate metabolism in incubated hepatocytes and liver metabolites in culture medium were determined. In vivo health and productivity of experimental cows on low and adequate dietary P supply were monitored, and liver tissue and blood samples were obtained repeatedly. Liver tissue was assayed for its triacylglycerol-, mineral and water content as well as for the relative abundance of mRNA of enzymes of the carbohydrate-, fat- and protein metabolism. Reduced Pi-availability was not associated with altered enzyme transcription rates or metabolic activity in-vitro. The most prominent clinical finding associated with P deprivation in-vivo was feed intake depression developing after the first week of lactation. Accordingly cows on low P diets had lower milk yield and showed more pronounced increases in liver triacylglycerol after calving. Although the liver P content decreased in P deficient cows, neither negative effects on enzyme transcription rates nor on blood parameters indicative of impaired liver metabolic activity or liver injury were identified. These results indicate the P deprivation only indirectly affects the liver through exacerbation of the negative energy balance occurring as P deficient cows become anorectic.

Characterization of functional traits with focus on udder health in heifers with divergent paternally inherited haplotypes on BTA18.

BACKGROUND: A major challenge in modern medicine and animal husbandry is the issue of antimicrobial resistance. One approach to solving this potential medical hazard is the selection of farm animals with less susceptibility to infectious diseases. Recent advances in functional genome analysis and quantitative genetics have opened the horizon to apply genetic marker information for efficiently identifying animals with preferential predisposition regarding health traits. The current study characterizes functional traits with a focus on udder health in dairy heifers. The animals were selected for having inherited alternative paternal haplotypes for a genomic region on Bos taurus chromosome (BTA) 18 genetically associated with divergent susceptibility to longevity and animal health, particularly mastitis. RESULTS: In the first weeks of lactation, the q heifers which had inherited the unfavorable (q) paternal haplotype displayed a significantly higher number of udder quarters with very low somatic cell count (< 10,000 cells / ml) compared to their paternal half-sib sisters with the favorable (Q) paternal haplotype. This might result in impaired mammary gland sentinel function towards invading pathogens. Furthermore, across the course of the first lactation, there was indication that q half-sib heifers showed higher somatic cell counts, a surrogate trait for udder health, in whole milkings compared to their paternal half-sib sisters with the favorable (Q) paternal haplotype. Moreover, heifers with the haplotype Q had a higher feed intake and higher milk yield compared to those with the q haplotype. Results of this study indicate that differences in milk production and calculated energy balance per se are not the main drivers of the genetically determined differences between the BTA18 Q and q groups of heifers. CONCLUSIONS: The paternally inherited haplotype from a targeted BTA18 genomic region affect somatic cell count in udder quarters during the early postpartum period and might also contribute to further aspects of animal's health and performance traits due to indirect effects on feed intake and metabolism.

Establishing a model of primary bovine hepatocytes with responsive growth hormone receptor expression.

The liver becomes resistant to growth hormone before parturition in dairy cows (uncoupling of the somatotropic axis). However, the mechanism of growth hormone insensitivity has not been fully described. The aim of the present study was to improve a previous model of adult bovine hepatocytes in a sandwich culture system to ensure growth hormone receptor (GHR) expression. First, we modified the protocol for hepatocyte retrieval and tested the effect of short (18 min) and long (up to 30 min) warm ischemia on hepatocyte viability. Second, we used medium additives that affect GHR expression in vivo-insulin (INS), dexamethasone (DEX), both (INS+DEX), or no hormone additives (CTRL)-to ensure the functionality of hepatocytes, as measured by lactate dehydrogenase activity and urea concentration in the medium. We also used reverse transcriptase PCR of hepatocytes to evaluate expression of albumin (ALB), hepatocyte nuclear factor 4α (HNF4A), nuclear factor-κ-B-inhibitor α (NFKBIA), cytosolic phosphoenolpyruvate carboxykinase (PCK1), and vimentin (VIM) mRNA. Moreover, we analyzed the expression of GHRtot (GHR), GHR1A, insulin-like growth factor-1 (IGF1), and IGF binding protein-2 (IGFBP2) in response to exposure to media with the different compositions. Modification of the protocol (changes in rinsing and perfusion times, buffer composition, and the volume and standardization of collagenase) led to increased cell counts and cell viability. Short warm ischemia with the modified protocol significantly increased cell count (4.7 × 107 ± 1.9 × 107 vs. 3.5 × 106 ± 1.5 × 106 vital cells/g of liver) and viability (79.1 ± 8.4 vs. 37.1 ± 8.9%). Therefore, we gathered hepatocytes from the liver after short warm ischemia with the modified protocol. For these hepatocytes, lactate dehydrogenase activity was lower in media with INS and with DEX than in media with INS+DEX or CTRL; urea concentrations were highest at d 4 for INS+DEX. As well, HNF4A and ALB were more highly expressed in hepatocytes cultured with INS and INS+DEX than in those cultured with DEX or CTRL, and the substitution of DEX suppressed VIM and NFKBIA expression but increased PCK1 expression. The expression of GHR, GHR1A, and IGF1 was suppressed by dexamethasone (DEX and INS+DEX), whereas INS distinctly increased GHR, GHR1A, and IGF1 mRNA expression. Hepatocytes in a sandwich culture showed influenceable GHR expression; this study provides a model that can be used in studies examining factors that influence the expression and signal transduction of GHR in dairy cows.

Thyroid hormone profiles and TSH evaluation during early pregnancy and the transition period in dairy cows.

The thyroid hormone axis is one important endocrine system within a complex system of various factors and hormones necessary to establish and maintain pregnancy in cattle and in metabolic adaptations towards lactation. Pregnancy and lactation occur contemporaneously in dairy cows, and have major impacts on thyroid hormones as well as status of the hypothalamus-pituitary thyroid axis influences pregnancy and lactation. As especially the thyroid hormone axis with special focus to TSH is not well studies in the past, the aim of the study was to examine the thyroid hormone axis, especially in heifers during adaptation to the first lactation, to verify the association between lactation and thyroid hormones. Secondly, the early pregnancy period was the focus of the present investigation. To determine thyroid hormone patterns and especially thyroid-stimulating hormone (TSH) during the transition period, 12 primiparous Holstein Friesian cows and 41 pregnant and 34 non-pregnant pluriparous cows were sampled. During the transition period, thyroxin (T4) concentrations decreased after calving in primiparous cows, and the milk yield [kg] was negatively correlated with triiodothyronine (T3) and T4 concentrations. In pregnant and non-pregnant pluriparous cows, T3, T4, TSH and free T4 (fT4) decreased between the day of ovulation (day 0) and day 18. From day 18 of gestation to day 100 after AI, an increase in TSH and fT4 and a decrease in Reverse T3 (rT3) was observed in the pregnant cows. In conclusion, the results of the present study confirm that regulative processes within the thyroid hormone metabolism occur during early lactation. For the first time it could be shown in dairy cows that TSH and fT4 increased during early pregnancy whereas rT3 decreased indicating also in bovine species a TSH stimulatory effect by pregnancy.

Effects of intravenous infusion of E. coli lipopolysaccharide in early pregnant cows.

The objective was to characterize effects of Escherichia coli LPS (given i.v.) on corpus luteum (CL) and embryonic viability in early pregnant cattle. Eight non-lactating German Holstein cows were given 0.5 µg/kg LPS on 35 ± 3 day (mean ± s.e.m.) of pregnancy, whereas seven heifers, 41 ± 6 day pregnant, were given 10 mL saline (control group). Transrectal B-mode examinations of the CL were done at -1, 3, 6, 12, 24, 48, 72 and 96 h relative to treatment. Blood samples were collected at -1, 0.5, 1, 2, 3, 4, 6, 9, 12, 24, 48, 72 and 96 h. At 12 and 48 h, the CL was biopsied. None of the cows still in the experiment 10 day after LPS (n = 7) had embryonic loss. In LPS-treated cows, luteal area decreased (from 4.1 to 3.1 cm2; P ≤ 0.05) within 6 h and until 48 h. Luteal blood flow decreased by 39% (P ≤ 0.05) within the first 6 h after LPS, but returned to pre-treatment values by 48 h. Plasma P4 decreased by 62% (P ≤ 0.05), reached a nadir (2.7 ± 0.6 ng/mL) at 12 h after LPS and was not restored to pre-treatment (P ≤ 0.05). In luteal tissue, mRNAs for STAR and for FGF1 were lower (P ≤ 0.05) in LPS than in saline-treated cattle at 12 h, with no difference between groups at 48 h. Levels of mRNAs for CASP3 and FGF2 were not different between groups (P > 0.05) at 12 or 48 h after treatment. In conclusion, LPS transiently suppressed CL function, but did not induce embryonic mortality.

Dietary nitrogen and calcium modulate CYP27B1 expression in young goats.

In livestock, feeding a reduced nitrogen (N) diet is favored for economic and ecological reasons. Ruminants cope more easily with a reduced N diet than monogastric species. However, changes in mineral homeostasis such as a reduction in 1,25-dihydroxyvitamin D3 (1,25-(OH)2D3) concentrations, calcium (Ca), and IGF1 levels were observed in goats kept on a reduced N diet. The decrease in 1,25-(OH)2D3 occurred even during a simultaneous reduction in dietary N and Ca, whereas a solitary Ca reduction stimulated 1,25-(OH)2D3 synthesis. The aim of the present study was to examine the effects of N- and/or Ca-reduced diets on the expression of 24-hydroxylase (CYP24A1), 1-alpha-hydroxylase (CYP27B1), vitamin D receptor (VDR), retinoid X receptor alpha (RXRα), IGF1 receptor (IGF1R), Klotho, and fibroblast growth factor receptor 1c (FGFR1c) in kidneys of young goats. Four groups were kept on a control diet, an N-reduced diet, a Ca-reduced diet or an N- and a Ca-reduced diet. Renal expression of CYP24A1 was not affected, whereas CYP27B1 expression was significantly diminished in the N-reduced diet fed goats (P < 0.05) and significantly elevated with the Ca reduction (P < 0.001). The VDR expression was not modified, whereas RXRα (P < 0.05) and Klotho expression (P < 0.001) were stimulated during Ca reduction. The IGF1R (P < 0.05) and FGFR1c (P < 0.05) expression were enhanced with the N reduction. From these data, it can be concluded that the downregulation of renal CYP27B1 expression observed with dietary N reduction is probably mediated by a complex interaction between the somatotropic axis and the Klotho/FGF signaling pathway in young goats.

Proliferative and apoptotic changes in the healthy canine endometrium and in cystic endometrial hyperplasia.

Proliferation and apoptosis play an important role in cyclic changes in the healthy canine endometrium. Proteins of the Bcl-2-family are known to be involved in the regulation of apoptosis. However, only few reports mention their expression patterns during cystic endometrial hyperplasia (CEH). In order to correlate proliferative and apoptotic processes, expression of the apoptosis-regulatory proteins Bcl-2 (anti-apoptotic) and Bax (pro-apoptotic) in healthy and cystic hyperplastic endometrial tissue as well as in pyometra was investigated. Uteri from 33 bitches were assigned to three groups: group 1 - healthy endometrium (n = 12), group 2 - CEH (n = 17) and group 3 - pyometra (n = 4). Markers for proliferation (Ki-67) and apoptosis (activated caspase-3 and TUNEL-method) as well as expression of Bcl-2 and Bax were determined in all endometrial layers. For groups 1 and 2 this was done during endometrial gland secretion in mid and late luteal phase (mLP, lLP), endometrial reparation in early anestrus (eAE) and in the regenerated endometrium in late anestrus (lAE). For group 3 only the late luteal phase was investigated. In group 1, cyclic proliferation patterns were found predominantly in superficial glands (SG) and stroma, whereas progesterone-mediated low expression levels coincided with high apoptosis rates in the basal glands (BG). In eAE, higher apoptotic activity was detected compared to lLP and lAE. Bcl-2 and Bax expression levels showed an inverse cyclic relationship in all tissue layers. In the stroma, in eAE, a rise in proliferative activity and concomitant increase in anti-apoptotic Bcl-2 expression levels was found, indicating that this layer serves as a source for endometrial regeneration. In CEH, no or limited cyclic patterns of proliferation, apoptosis, Bcl-2 and Bax were found. Increased proliferation rates coincided with deregulated apoptosis. Besides the glandular compartments, the stroma played an important role in the pathogenesis of canine CEH. In case of pyometra, both proliferation and apoptosis increased, indicating irreversible damage of the inflamed canine endometrium. In conclusion Bcl-2 and Bax play a role both in the physiological regenerative processes of the cyclic canine endometrium and in deregulating proliferation and apoptosis in CEH and pyometra.

Effects of oxytocin and PGF2α on uterine contractility in cows with and without metritis-An in-vitro study.

The aim of this study was to investigate the effects of PGF2α and oxytocin in vitro on myometrial contractility in puerperal uteri. Thirteen puerperal uteri were removed and perfused after euthanasia of cows with (n=7) and without metritis (n=6). Measurement of uterine contractility was done using four piezoelectric crystals, which were implanted into the myometrium along the greater curvature of the uterine horn where fetal implantation occurred during the previous pregnancy. After 30min of equilibration, oxytocin (5 IU) or PGF2α (2.5mg Dinoprost) was administered randomly into both uterine arteries, and 30min later, the second administration of either oxytocin or PGF2α occurred. Treatment with oxytocin induced contractions in uteri with metritis and uteri without metritis (P<0.05). In uteri with metritis, greater uterine contractions occurred after stimulation with oxytocin than in uteri without metritis (P<0.05). Treatment with PGF2α did not (P>0.05) result in increased contractions in the uteri without metrtitis, however, induced an initial decrease in contractions followed by an increase (P<0.05) in contractions in uteri with metritis. Myometrial and endometrial gene expression of PGF2α (FPR) and oxytocin receptor (OTR) was greater (P<0.05) in uteri with metritis than in uteri without metritis. The results suggest that oxytocin, but not PGF2α, is an effective uterotonic drug in puerperal cows. Uteri in which metritis was diagnosed contracted more strongly after treatment with oxytocin than uteri in which metritis was not diagnosed. This effect was paralleled by greater gene expression of OTR as well as FPR in uteri with metritis compared with uteri in which metritis was not diagnosed.

Evaluation of intravenous regional anaesthesia and four-point nerve block efficacy in the distal hind limb of dairy cows.

BACKGROUND: Intravenous regional anaesthesia (IVRA) and hindfoot four-point nerve block anaesthesia (NBA) are recommended for local anaesthesia (LA) in the distal limb of dairy cows. Two studies were conducted to compare the efficacy, time until onset and stress responses to IVRA and NBA in dairy cows. In the first cross-over designed study, eight healthy unsedated German Holstein cows, restrained in lateral recumbency (LR) on a surgical tipping table, were treated with IVRA and NBA using procaine 2% as a local anaesthetic. Distal limb desensitization was tested by electrical (e-), mechanical (m-) and thermal (t-) nociceptive stimulation 10 min before and 15 and 30 min after LA. Hormonal-metabolic (blood concentrations of cortisol, lactate, non-esterified fatty acids, and glucose) and cardio-respiratory (heart and respiratory rate, mean arterial blood pressure) stress responses to treatment were assessed at predetermined intervals. In the second study, six healthy, unsedated German Holstein cows in LR were treated (crossover design) with IVRA and NBA. Short-interval e-stimulation was measured by the time until complete distal limb desensitization. RESULTS: In the first study, four of eight cows responded to e-stimulation 15 min after IVRA, while none of the cows treated with NBA responded until the safety cut-off level was reached. E-stimulation revealed complete desensitization of the distal limb 30 min after LA in all cows. Half of the cows did not respond to m- and t-stimulation before LA, so no further evaluation was performed. Stress reactions to IVRA and NBA treatment were similar, but differences may have been masked by stress response to LR restraint. In the second study, complete desensitization was achieved 12.5 min after NBA, while one of the six cows still responded to e-stimulation 20 min after IVRA. CONCLUSION: Hindfoot nerve block anaesthesia and intravenous regional anaesthesia induced complete desensitization of the distal hind limb in dairy cows. However, the anaesthesia onset after NBA was significantly faster than that of IVRA, which may be clinically relevant in the field, particularly when distal limb anaesthesia is required for major claw surgeries under time constraints.

Interferon-τ induced gene expression in bovine hepatocytes during early pregnancy.

Interferon-tau (IFNτ) is the conceptus derived specific early pregnancy signal in bovidae. Locally, IFNτ induces an IFNτ specific gene expression (ISG) in endometrial cells and by this it averts luteolysis of the corpus luteum (CL) by suppressing prostaglandin production. Moreover, it was shown that IFNτ also induces ISG in the liver in pregnant sheep and in liver biopsies from Holstein Friesian heifers on Day 18 of pregnancy. The objective of the present study was to confirm increased hepatic ISG in vivo on Day 18 of pregnancy and to prove if hepatocytes and not non-parenchymal cells react to IFNτ by using immunohistochemistry and primary bovine hepatocytes stimulated in vitro with recombinant bovine IFNτ. For the animal experiment, Angus heifers (n = 12) were cycle synchronized and the Day of ovulation (Day 0) was defined by ovarian ultrasonography and verified by progesterone < 0.1 ng/ml. Heifers were artificially inseminated either with sperm (n = 9) or with seminal plasma (mock control, n = 3). Early pregnancy was defined and detected by progesterone and pregnancy associated glycoprotein (PAG) concentration in blood before and after induced luteolysis by a PGF-injection on Day 21 in n = 3 inseminated heifers. A liver biopsy was taken on Day 18 for the analysis of gene (ISG 15, MX 1, MX 2 and OAS 1) and protein (OAS1) expression using qPCR and immunohistochemistry, respectively. Primary bovine hepatocytes were collected from bull liver using a two-step collagenase perfusion, cultured short-term in a monolayer and stimulated with IFNτ. Thereafter, gene expression was measured by qPCR. In liver biopsies obtained from pregnant heifers ISG was numerically higher expressed compared to biopsies from non-pregnant heifers. Furthermore, the OAS 1 protein expression was localized in hepatocytes on Day 18 of pregnancy. In vitro, primary bovine hepatocytes showed an increased mRNA expression of ISG after IFNτ stimulation. In conclusion, the findings confirm that IFNτ induces ISG in the parenchymal part of the liver in early pregnancy of cattle.

The myometrial contractility during late pregnancy in dairy cows, in vitro.

This study aimed to investigate the in vitro contractility of the myometrium and its relationship to the blood concentrations of estradiol-17ß (E2ß), progesterone (P4), 15-keto-13,14-dihydro-PGF2α (PGFM) and ionised calcium (Ca2+) prior to tissue harvest in 12 healthy Holstein-Friesian cows in late pregnancy. Three circular (CM) and 3 longitudinal myometrial (LM) strips were dissected during a caesarean section and mounted in an organ bath containing modified Krebs solution (KS). The spontaneous contractility was recorded during five 30-min time periods (T1 to T5), after which the strips were exposed to increasing concentrations of oxytocin (OT; 10-10-10-7M), a natural PGF2α-analogue (PGF; 10-7-10-4M) and KS (Cont) for four 30-min time periods (T6 to T9). The variables area under the curve (AUC), mean (MA) and maximal amplitude (maxA) were calculated for each T. The blood P4, E2ß, Ca2+ and PGFM values averaged 4.0±1.7ng/mL, 482.3±63.7 pg/mL, 0.8±0.3 mmol/L and 125.3±63.7pg/mL. The LM strips had greater AUC, MA, and maxA than CM, and OT caused greater AUC and MA in both muscle layers than PGF or control treatment (OT>PGF>Cont). Estradiol-17ß correlated with AUC and MA of LM at T1 to T5 (r=0.69; P≤0.05). In conclusion, LM and CM strips have different contractile performance but show enhanced activity when stimulated with OT and less activity after PGF stimulation if compared with Cont. Blood concentrations of E2ß may be useful as an indicator of uterine contractile performance in late pregnant cattle.

Different milk feeding intensities during the first 4 weeks of rearing dairy calves: Part 2: Effects on the metabolic and endocrine status during calfhood and around the first lactation.

Feeding dairy calves at high intensity has been demonstrated to increase milk yield in later life. We investigated the effect of 3 different feeding regimens in the preweaning period on the metabolic and endocrine status during calfhood and in heifers at the onset of the first lactation. In trial 1, 57 German Holstein calves were allocated to 3 different feeding groups: milk replacer restricted to 6.78 kg/calf per day, 11.5% solids (MR-res, n = 20), milk replacer 13.8% solids, ad libitum (MR-ad lib, n = 17), and whole milk ad libitum (WM-ad lib, n = 20). All calves received ad libitum colostrum for 3 d postnatal (p.n.). From d 4 to 27, all calves were fed according to their respective feeding regimen, resulting in average intakes of 6.38, 9.25, and 9.47 kg/d in MR-res, MR-ad lib, and WM-ad lib, respectively. Thereafter, all calves were fed according to the MR-res regimen until weaning at d 55 (gradually until d 69 p.n.). Blood samples were collected on d 0 before colostrum intake and on d 1, 3, 11, 22, 34, 43, 52, 70, 90, and 108 p.n. Liver biopsies were taken on d 19 and 100, and on d 22, 52, and 108 p.n. intravenous glucose tolerance tests were performed. The male calves (n = 8 to 10 per group) underwent also an insulin tolerance test on d 24, 54, and 110 p.n. The females (n = 28) from trial 1 were further reared and bred as common practice, and were enrolled in trial 2 when beginning the last trimester of pregnancy. Blood samples were collected monthly antepartum starting 91 d before calving and weekly (0-70 d) postpartum. Trial 1 was subdivided into 4 phases (P): P0 (d 0-1), P1 (d 2-27), P2 (d 28-69), and P3 (d 70-110 p.n.). In trial 1, the leptin and adiponectin concentrations increased with colostrum intake. Differences in fatty acids, insulin, adiponectin, revised quantitative insulin sensitivity check index (RQUICKI), and variables from the glucose tolerance tests were largely limited to P1. The MR-res group had greater RQUICKI and fatty acid values, and lower insulin and, as a trend, adiponectin concentrations than in 1 or both ad lib groups. These differences were partly sustained in P2 (fatty acids, adiponectin, and RQUICKI) and in P3 (adiponectin). The hepatic mRNA abundance of the gluconeogenic enzymes phosphoenolpyruvate carboxykinase and pyruvatcarboxylase increased from d 19 to 100. None of the blood variables were different between the groups when tested in pregnancy and lactation. Our results do not support a sustained deflection of metabolic regulation by rearing at different feeding intensities; nevertheless, the differences observed during rearing might influence nutrient utilization in later life or the cellular development of organs, such as the mammary gland, and thereby affect milk yield. Further studies involving greater animal numbers and, thus, improved power will help to sort out the mechanisms of programming body function in later life via nutrition in early life.

Monochorial diamniotic dizygotic twins in a German Shepherd Dog: A case report.

CASE REPORT: A 6.5-year-old clinically healthy German Shepherd Dog with regular oestrous cycles of 6 months was presented for pregnancy diagnosis on day 38 after ovulation (p.ov.). Ultrasonography revealed three individual placental sites in progressed resorption and two vital adequately developed foetuses sharing a joint placenta. On days 41 and 48 p.ov., sonographic signs indicated normal development of both foetuses, but on day 52 p.ov., both foetuses were found to be dead. A caesarean section was performed the same day. Examination of the removed uterus confirmed the diagnosis of a "twin" pregnancy with two foetuses sharing the same placental site but separate amniotic membranes. One foetus showed generalized oedema (anasarca). Bacterial culture of swabs taken from inside the placental cavity was negative. At histological examination of the uterus, no signs of inflammation were found. Serum relaxin concentrations (day 38, 41, 48 and 52. p.ov.) were consistent with those of bitches with normal pregnancies. Cytogenetic analysis of the two foetuses revealed dizygotic twins, one male and one female according to SRY-PCR. By genotyping 17 high-polymorphic canine microsatellites, it could be demonstrated that the two foetuses developed from two different oocytes.