An in-depth investigation of the microbiota and its virulence factors associated with severe udder cleft dermatitis lesions.

Udder cleft dermatitis (UCD) is a skin condition affecting the anterior parts of the udder in dairy cattle. In the present study, we aimed to shed light on the microbiota in severe UCD lesions versus healthy udder skin by putting forward a taxonomic and functional profile based on a virulence factor analysis. Through shotgun metagenomic sequencing, we found a high proportion of bacteria in addition to a low abundance of archaea. A distinct clustering of healthy udder skin versus UCD lesion samples was shown by applying principal component analysis and (sparse) partial least squares analysis on the metagenomic data. Proteobacteria, Bacillota, and Actinomycetota were among the most abundant phyla in healthy udder skin samples. In UCD samples, Bacteroidota was the most abundant phylum. At genus level, Bifidobacterium spp. had the highest relative abundance in healthy skin samples, whereas Porphyromonas spp. and Corynebacterium spp. had the highest relative abundance in UCD samples. In the differential abundance analysis, Porphyromonas spp. and Bacteroides spp. were significantly differentially abundant in UCD samples, whereas Bifidobacterium spp., Staphylococcus sp. AntiMn-1, and Staphylococcus equorum were more commonly found in healthy samples. Moreover, the abundance of several treponeme phylotypes was significantly higher in lesion samples. The streptococcal cysteine protease speB was among the most abundant virulence factors present in severe UCD lesions, while a plethora of virulence factors such as the antitoxin relB were downregulated, possibly contributing to creating the ideal wound climate for the dysbiotic community. Network analysis showed healthy lesion samples had a large network ofpositive, correlations between the abundances of beneficial species such as Aerococcus urinaeequi and Bifidobacterium angulatum, indicating that the healthy skin microbiome forms an active protective bacterial network, which is disrupted in case of UCD. In UCD samples, a smaller microbial network mainly consisting of positive correlations between the abundances of Bacteroides fragilis and anaerobic Bacteroidota was exposed. Moreover, a high correlation between the taxonomic data and virulence factors was revealed, concurrently with 2 separate networks of microbes and virulence factors. One network, matching with the taxonomic findings in the healthy udder skin samples, showcased a community of harmless or beneficial bacteria, such as Bifidobacterium spp. and Butyrivibrio proteoclasticus, associated with hcnB, hcnC, relB, glyoxalase, and cupin 2. The other network, corresponding to UCD samples, consisted of pathogenic or facultative pathogenic and mainly anaerobic bacteria such as Treponema spp., Mycoplasmopsis spp., and bovine gammaherpesvirus 4, that correlated with virulence factors SpvB, fhaB, and haemagglutination activity domain-associated factor. Our results point toward a dysbiotic community with a notable decrease in diversity and evenness, with a loss of normal skin inhabitants and innocuous or useful species making way for predominantly anaerobic, facultative pathogens. The shift in the abundance of virulence factors such as fhaB and SpvB could play a role in the manifestation of a local micro-environment favorable to the microbiome associated with udder skin lesions. Lastly, the presence of specific networks between microbial species, and between microbes and virulence factors was shown.

Effect of overconditioning on the hepatic global gene expression pattern of dairy cows at the end of pregnancy.

Overconditioning is a risk factor for upregulated pre- and postpartum fat mobilization. Therefore, we hypothesized that overconditioning at the end of pregnancy leads to the accumulation of lipids in the liver and modifications of the hepatic gene expression pattern. The aim of this study was to evaluate the effect of normal- versus overconditioning on the hepatic transcriptomic profile of dairy cows at the end of pregnancy. Ten dry multiparous Holstein cows were killed 2 wk before expected calving. Body condition score (BCS) and backfat thickness (BFT) were evaluated, and blood samples for nonesterified fatty acids (NEFA) were taken before cows were killed. After cows were killed, liver biopsy samples were collected for further assessment of total lipids and RNA sequencing. Five cows were classified as normal-conditioned (median BCS = 3, range 2.75-3.5) and 5 as overconditioned (median BCS = 4, range 4-5). Regression models confirmed that normal-conditioned cows had lower BFT (1.29 ± 0.29 cm; least squares means ± standard error) and serum NEFA (0.16 ± 0.04 mmol/L) in comparison to overconditioned cows (3.14 ± 0.43 cm and 0.38 ± 0.07 mmol/L for BFT and NEFA, respectively). Total liver lipid percentage tended to be lower in normal- versus overconditioned cows (4.63 ± 0.40% and 6.06 ± 0.44%, respectively). In comparison to the mean liver lipid percentage of the normal- and overconditioned cows, 1 overconditioned cow had a relatively low (5.21%) and 1 normal-conditioned cow had a relatively high (6.07%) liver lipid percentage. Differentially expressed genes analysis (edgeR quasi-likelihood method) showed that normal-conditioned cows presented 11 upregulated and 12 downregulated genes in comparison to overconditioned cows. Linear discriminant analysis effects size revealed 133 differentially expressed genes between normal- versus overconditioned cows. Notably, the liver of normal-conditioned cows had upregulated genes associated with liver functionality (ALB, SELENOP, IGF1, and IGF2). On the other hand, overconditioned cows had upregulated genes associated with the acute-phase response (C3, HPX, and, LBP). High basal lipolysis in overconditioned cows at the end of pregnancy increased liver lipid content, and this may alter the hepatic gene expression pattern to a pro-inflammatory state.

Influence of adipocyte size and adipose depot on the number of adipose tissue macrophages and the expression of adipokines in dairy cows at the end of pregnancy.

The aim of this study was to determine the number of adipose tissue macrophages (ATM) and the mRNA expression of adipokines [adiponectin (ADIPOQ), leptin (LEP), interleukin 6 (IL6), tumor necrosis factor (TNF), and interleukin 10 (IL10)] in different adipose depots from cows with a variable body condition score (BCS) at the end of the dry period. We hypothesized that the number of ATM and the expression of these adipokines depend on adipocyte size and the anatomical location of the adipose depot. Subcutaneous, omental, mesenteric, perirenal, and intrapelvic adipose tissue samples were taken immediately after euthanasia of 10 Holstein Friesian dairy cows (upcoming parity 2 to 5, age 3.9 ± 1.4 yr; mean ± standard deviation) at the end of pregnancy (actual days of pregnancy at the moment of euthanasia: 269 ± 5 d). During the dry period, all animals received similar diets to meet but not exceed requirements. Five animals were considered to have a normal BCS (2.5-3.5) and 5 animals were considered to be over-conditioned (BCS = 3.75-5). Body weight of the animals at the moment of euthanasia was 717 ± 77 kg. Expression of the different genes was determined by reverse transcription quantitative real-time PCR. Adipocyte size was determined by measuring the area of 100 adipocytes on histological sections. Average adipocyte area was 10,475 ± 1,019, 8,500 ± 780, 10,383 ± 1,227, 11,466 ± 1,039, and 11,087 ± 1,632 µm2 for the subcutaneous, mesenteric, omental, intrapelvic, and perirenal adipose depot, respectively. Immunohistochemistry using anti-bovine CD172a antibodies was performed to determine the proportion of ATM (the number of CD172a-positive cells per 100 adipocytes, given as a percentage). Expression of LEP, IL6, and TNF was positively associated with adipocyte size, whereas no association could be detected between ADIPOQ and IL10 with the size of the adipocytes. The omental adipose depot was especially infiltrated with ATM (1.92 ± 0.55, 1.10 ± 0.33, and 8.28 ± 2.24% for the subcutaneous, mesenteric, and omental adipose depot, respectively). The proportion of ATM was positively associated with the size of the adipocytes in the omental and mesenteric adipose depot. Expression of ADIPOQ, LEP, IL6, TNF, and IL10 differed among depots, which suggests differences in inflammatory characteristics depending on the anatomical location of depots. In conclusion, the results of the present study confirm the adipose tissue as a potential source of inflammatory mediators and demonstrate ATM infiltration, especially in the omental adipose depot.

Distribution of inflammation and association between active and chronic alterations within the endometrium of dairy cows.

Objectives of this study were twofold: (i) to assess the association between polymorphonuclear (PMN) counts and chronic alterations within the bovine endometrium and (ii) to determine the distribution of inflammation throughout the endometrium of clinically healthy dairy cows. Holstein-Friesian cows (n = 32) from a single dairy farm were selected for this experiment. Before slaughtering, a complete reproductive examination was performed to discard any type of clinical disease. After slaughtering, reproductive tracts were collected, and the endometrium was sampled at 8 pre-defined locations. At each location, endometrial biopsies (EBs) and cytology (CY) samples were harvested. Histopathology samples were stained with haematoxylin-eosin (EB-HE) and naphthol-AS-D-chloroacetate-esterase (EB-naphthol), while CY samples were stained with Wright-Giemsa. In the EB-HE samples, parameters assessed were epithelium height, mononuclear cells infiltration, lymphocytic aggregates, periglandular fibrosis, angiosclerosis and haemorrhage. In EB-naphthol and CY slides, PMNs counts were evaluated. Binomial logistic regression was used to assess the association between the number of PMNs present in both the EB-naphthol and CY samples and alterations identified in the EB-HE samples and to analyse the distribution of the histopathological alterations (EB-HE). A Poisson mixed-effect model was used to analyse the distribution of PMNs within the endometrium. A significant positive association was found between the PMN counts and the mononuclear cells infiltration. The presence of erythrocytes was associated with higher odds to detect PMNs in the stratum compactum. Significantly, higher infiltration of PMNs and mononuclear cells were detected in the uterine body and the right horn region. Concluding, CY is a technique that allows the evaluation of PMN counts and therefore only evaluates active inflammation. A complete assessment of endometrial health can only be obtained using EB. To optimize the sensitivity to diagnose endometrial inflammation in cows, adjacencies of the corpus uteri should be considered as the preferred region to harvest samples.

A novel cytologic sampling technique to diagnose subclinical endometritis and comparison of staining methods for endometrial cytology samples in dairy cows.

The present article describes a study of the diagnosis of subclinical endometritis in dairy cows having two principal aims: first, to validate a novel technique for taking endometrial cytology samples to diagnose subclinical endometritis in dairy cows. Second, to compare the percentage of polymorphonuclear cells (PMNs) in cytology samples stained with Diff-Quik versus a specific staining method for PMNs, naphthol-AS-D-chloroacetate-esterase (CIAE). In the first experiment, Holstein-Friesian cows (n = 204) were used to take two cytology samples at the same time using the conventional cytobrush (CB) and the new cytotape (CT). Both devices were assembled within the same catheter allowing sampling at the same time, and approximately at the same location. Cytotape consisted of a 1.5-cm piece of paper tape rolled on the top of an insemination catheter covered with a double guard sheet. Parameters used to evaluate both methods were: PMNs percentage, total cellularity, quality of the smears, and red blood cell contamination. The concordance correlation coefficient analysis was used to assess agreement between continuous and Pearson chi-square tests for categorical variables. Agreement between the percentage of PMNs in both methods was good ρ = 0.84 (0.79, 0.87) with a minor standard error of 2%. Both methods yielded similar total cellularity (P = 0.62). Cytotape yielded better quality smears with more intact cells (P < 0.01) while samples that were taken by CB were more likely to be bloody (P < 0.01). Hence, CT and CB methods yielded smears with a similar PMNs percentage and a total number of cells, but CT provided smears with higher quality and significantly less blood contamination. For the second experiment, 114 duplicate cytology slides were stained using both Diff-Quik and CIAE. Agreement between PMNs percentage in both staining techniques was good ρc = 0.84 (0.78, 0.89) with a standard error of only 2%. Hence, Diff-Quik was confirmed as an easy, fast, and high-quality staining technique, which can be routinely used to stain endometrial cytology samples satisfactorily.

Is knee function better with contemporary modular bicompartmental arthroplasty compared to total knee arthroplasty? Short-term outcomes of a prospective matched study including 68 cases.

BACKGROUND: Bicompartmental knee arthroplasty (BKA) was developed to treat medial tibiofemoral and patellofemoral osteoarthritis while preserving the anterior cruciate ligament to optimise knee kinematics. Our objective here was to compare the probability of achieving forgotten knee status and the functional outcomes at least two years after BKA versus total knee arthroplasty (TKA). We hypothesised that contemporary modular BKA produced better functional outcomes than TKA after at least two years, for patients with similar pre-operative osteoarthritic lesions. MATERIAL AND METHODS: We conducted a two-centre prospective controlled study of 34consecutive patients who underwent BKA between January 2008 and January 2011. Each patient was matched on age, gender, body mass index, preoperative range of knee flexion, centre, and surgeon to a patient treated with TKA. An independent observer evaluated all 68 patients after six and 12months then once a year. Forgotten knee status was defined as a 100/100 value of the Forgotten Joint Score (FJS-12) and each of the five KOOS subscales. We also compared the two groups for knee range of motion, Knee Society Scores (KSSs), Timed Up-and-Go test (TUG), and UCLA Activity Score. RESULTS: At a mean follow-up of 3.8±1.7 years, the probability of forgotten knee status was significantly higher in the BKA group (odds ratio, 4.64; 95% confidence interval, 1.63-13.21; P=0.007, Chi(2) test). Mean post-operative extension was not significantly different between the groups, whereas mean range of knee flexion was significantly greater in the BKA group (130°±6° vs. 125°±8° after TKA; P=0.03). The BKA group had significantly higher mean values for the knee and function KSSs, TUG test, and UCLA score (P<0.04 for all four comparisons). CONCLUSION: After at least two years, contemporary unlinked BKA was associated with greater comfort during everyday activities (forgotten knee) and better functional outcomes, compared to TKA. These short-term results require validation in randomised trials with longer follow-ups. LEVEL OF EVIDENCE: III, case-control study.

Pathways of the dominant follicle after exposure to sub-luteal circulating progesterone concentrations are different in lactating dairy cows versus non-lactating heifers.

With the increased use of different synchronization programs in cattle, attention is given to the progesterone concentration during development of the ovulatory follicle. It has been shown that low peripheral progesterone concentrations during follicular development may lead to decreased fertility. To investigate the effect of low progesterone concentrations on the fate of the dominant follicle, a study was conducted where cycles of dairy cows and heifers were manipulated to induce the development of the first dominant follicle without progesterone (PLACEBO) or under sub-luteal progesterone concentrations from a progesterone releasing intravaginal device (PRID Delta(®)). After insertion of the devices, daily follow up was performed by transrectal ultrasonography to identify and measure follicular development and blood samples were taken to determine the circulating progesterone concentration. Follow up was continued until the ovulation of a follicle occurred. After ovulation, the fate of the first dominant follicle was identified as arrested, atretic or ovulatory. Arrest was defined as persistence of the dominant follicle followed by ovulation whereas atresia was defined as regression of the dominant follicle and subsequent growth and ovulation of a new follicle. During PLACEBO treatment, heifers ovulated earlier and smaller follicles in comparison to cows. During PRID Delta(®) treatment, heifers had greater progesterone concentrations compared to cows and arrest of the dominant follicle occurred more in cows in comparison to heifers. In cycles where the dominant follicle was arrested, the ovulatory follicle was larger in comparison to cycles where the dominant follicle was atretic.

A field study to unravel factors that are significantly associated with the secretory activity of the corpus luteum during the first three postpartum cycles in high yielding dairy cows, based on the amount of steroidogenic and endothelial cells present in the luteal tissue.

Fourteen multi- and eight primiparous high-yielding dairy cows were followed from the first till the fourth ovulation postpartum. Cows were randomly divided into two groups and supplemented with soybean (group I; n = 11) or rapeseed meal (group II; n = 11). Both groups were subjected to a biopsy sampling of the corpus luteum (CL) at cycle day 9. The luteal capillary network (visualized by Bandeiraea simplicifolia) was denser in cycles 2 and 3 (p = 0.0005). The same was seen for the surface occupied by steroidogenic cells (visualized by 3ß-hydroxysteroiddehydrogenase) (p = 0.0001). The peripheral blood progesterone concentration showed an increasing trend with increasing cycle number and was higher in primiparous cows (p = 0.013), which had also larger glands on cycle day 9. The area occupied by endothelial cells was positively correlated with the area occupied by steroidogenic cells (r = 0.59; p < 0.0001). Both the areas occupied by endothelial and by steroidogenic cells were negatively correlated with the blood concentration of nonesterified fatty acids (NEFAs) (respectively, r = -0.377; p = 0.004 and r = -0.355; p = 0.007). We can conclude that primiparous cows generally have higher peripheral progesterone levels during the first three cycles after calving which is associated with a larger CL. In comparison with those of the first post-partum cycle, corpora lutea of cycles 2 and 3 have a denser capillary network and a larger area of steroidogenic cells, while these are only associated with a trend of higher peripheral progesterone concentrations.

Feeding soybean meal increases the blood level of isoflavones and reduces the steroidogenic capacity in bovine corpora lutea, without affecting peripheral progesterone concentrations.

Thirty-three Holstein-Friesian cows were followed from 14 days pre partum until the fourth ovulation post partum. Housing conditions and basic ration were identical for all animals. Concentrates were individually supplemented according to the daily milk production level, using two different types of protein rich concentrates: soybean meal and rapeseed meal. Soybean and rapeseed meal are known to be respectively high and low in isoflavones. Cows were randomly divided into three groups and blocked for parity. Group I (n=11) was supplemented with soybean meal and acted as control group. Groups II (n=11) and III (n=11) were respectively supplemented with soybean and rapeseed meal and were subjected to a biopsy sampling of the corpus luteum at day 9 of the first three postpartal estrous cycles. Soybean meal supplementation to lactating dairy cows (1.72 kg on average) induced an increase in the blood concentration of equol, dihydrodaidzein, o-desmethylangolensin in both soy groups and resulted in a reduced area occupied by steroidogenic (P=0.012) and endothelial cells (P=0.0007) in the luteal biopsies. Blood concentrations of equol and glycitein were negatively correlated with the areas occupied by steroidogenic (r=-0.410 with P=0.0002, respectively r=-0.351 with P=0.008) and endothelial cells (r=-0.337 with P=0.01, respectively r=-0.233 with P=0.085) in the 3 first estrous cycles. The latter however did not affect the diestrous peripheral blood progesterone concentration.

Topographic distribution of the different cell types, connective tissue and vascular tissue/lumina within a functional bovine corpus luteum and its association with breed, type of fixation protocol and stage during the cycle.

In the present study, we analysed the effect of fixative, breed, luteal stage and location on the nuclear density, volume density of connective tissue and vascular tissue/lumina within a bovine luteal gland in view of the development of an in vivo sampling technique to longitudinally monitor luteal histophysiology. The inner zone defined as the zone geometrically closest to the centre of the gland shows a significantly lower nuclear density (for all cell types) and a higher volume density of collagen fibres and vessels when compared with the outer zone (p < 0.001). The nuclear density in luteal glands from Holstein-Friesian cows is not significantly different from that in Belgian Blue cows, nor is it in stage II vs stage III glands. The collagen fibre content was significantly lower in glands of Belgian Blue cows (p = 0.01) and in younger glands (p = 0.003). Hence, it seems that the lower nuclear density in the inner zone was compensated by a higher amount of collagen fibres. As the type of fixative applied has a significant effect on the nuclear density of the different cell types, the present study warrants future research to further optimize the fixation protocol. As a conclusion, we can state that the topographic difference in nuclear distribution for the different cell types in a bovine luteal gland is only significant when comparing the inner vs the outer zone. This implies that if a sample representative for the whole gland has to be taken, for example, when taking an in vivo sample, it is necessary that the biopsy goes through the inner zone and contains the total diameter of the gland.

The fatty acid profile of subcutaneous and abdominal fat in dairy cows with left displacement of the abomasum.

The objective of this study was to determine the fatty acid (FA) profile and assess desaturase indices of nonesterified fatty acids (NEFA) in the blood, as well as in the abdominal (ABD) and subcutaneous (SUBC) fat stores, in dairy cows with left displacement of the abomasum (LDA). Blood, ABD, and SUBC samples were taken from 50 Holstein cows offered for surgery to correct LDA. The FA profile of the 3 compartments was determined by gas chromatography after lipid extraction, methylation, and, in the case of blood plasma, separation of lipid classes. The most abundant FA in all 3 compartments were 16:0, 18:0, and 18:1 cis-9, with a total proportion of 82.5, 68.0, and 74.1g/100 g of FA in ABD, NEFA, and SUBC, respectively. A principal component analysis was performed on the entire FA profile as well as on the Δ(9)-desaturase indices (14:1 cis-9/14:0, 16:1 cis-9/16:0, 18:1 cis-9/18:0). The principal component analysis extracted 2 principal components (PC), representing 51.6% (PC1) and 21.1% (PC2) of the total variance in FA composition of the 3 compartments. The loading plot for the regression factors revealed a strong positive correlation between PC1 with the Δ(9)-desaturase indices and the proportions of 14:1 cis-9 and 16:1 cis-9, and revealed a negative correlation with the proportion of 18:0 and saturated FA. The correlation with PC2 was positive for the proportion of unsaturated FA, 18:2n-6, and 18:3n-3, and negative for the proportion of 14:0, 16:0, and saturated FA. The SUBC could be distinguished from the NEFA and ABD by a positive score for PC1, whereas differentiation among the latter 2 compartments could be made by a positive (NEFA) or negative (ABD) score for PC2. The Δ(9)-desaturase indices for C14 and C16 differed between all compartments but were numerically closer for NEFA and ABD versus NEFA and SUBC. The desaturase indices of the main FA (18:1 cis-9 and 18:0) did not differ between NEFA and ABD. These results support the existence of a different FA composition in ABD compared with SUBC. The greater similarity between the FA profiles of ABD and NEFA compared with SUBC and NEFA and the closer desaturase indices of ABD and NEFA support the hypothesis of a preferential mobilization of ABD fat in dairy cows with LDA.

Two atypical cases of granulosa cell tumours in Belgian Blue heifers.

This paper describes two atypical cases of granulosa cell tumours (GCTs) in Belgian Blue heifers. In the first case, an 18-month-old heifer died after ovariectomy, and a granulosa-theca cell tumour was diagnosed post-mortem. The second heifer (31 months of age) was near term and was successfully treated surgically for a malignant granulosa cell tumour. Unfortunately, no further reproductive data are available, as the heifer was sold for slaughter. This is the first report describing GCTs in Belgian Blue beef cattle.

Immunohistochemical visualization of insulin receptors in formalin-fixed bovine ovaries post mortem and in granulosa cells collected in vivo.

Insulin is crucial for granulosa cell (GC) function, follicle growth and ovulation in cows; low insulin levels increase the risk for anoestrus. Apart from insulin concentration, alterations in the insulin receptor (IR) density on GC may affect follicular growth and steroidogenesis. Data about the IR protein distribution in the bovine follicle are scarce. Therefore, we aimed to develop a quantifiable staining method for IR protein on histological sections of bovine follicles in different developmental stages, and to apply this technique on GC obtained in living cows. In a first experiment, bovine ovaries were collected post mortem, formalin fixed, routinely processed, and stained with monoclonal murine IR-antibodies, peroxidase-labeled goat anti-mouse antibodies, and substrate chromogen. Based on their diameter, follicles were morphologically classified as small antral (SAF; n = 141), dominant (DF; n=28) or subordinate (SF; n=8); DF and SF were further classified as healthy or atretic based on the ratio of estrogen and progesterone concentrations in their follicular fluid. Using specialized software, the proportion of pixels displaying a positive staining signal was computed as a measure for IR density in three selected follicular regions: GC, theca (T) and stroma (STR). Results were analyzed in an ANOVA model with follicle type, region and health status as fixed factors. In SAF, DF, and SF, IR density was notably higher in GC than T or STR; the latter two displayed very low or no IR presence. The IR density in SAF was stronger than in DF and tended to be stronger than in SF. Staining intensity was not altered in atretic compared to healthy follicles. In corpus luteum, cumulus-oocyte complexes and pre-antral follicles, no IR could be detected. In a second experiment, GC samples were collected from 20 live cows on 30 and 70 d post partum by transvaginal follicular fluid aspiration, projected on glass slides, and stained using the protocol described above. Most samples yielded sufficient GC and IR was clearly visualized. However, objective quantification of the staining signal was impeded by extensive variation in the arrangement and density of GC and the amount of cellular debris on the slides. Altogether, strong IR presence in GC, most notably in SAF, suggests acquisition of IR as a key event in early follicle growth. Furthermore, we have developed a quantifiable staining technique for bovine follicles that may be applicable for GC obtained in live cows, although this method requires further standardization.

Pre-operative and operative difficulties during bovine caesarean section in Belgium and associated risk factors.

The aim of this study was to describe the difficulties encountered during caesarean section (CS) in practice on mainly Belgian Blue (BB) cows and to identify the associated risk factors. Data were collected using a questionnaire completed by veterinarians of the Ambulatory Clinic of the Faculty of Veterinary Medicine, Ghent University (Belgium), immediately after performing a CS. Results revealed that the BB breed had fewer severe pre-operative problems than animals of other breeds, most probably due to the fact that CSs in this breed are performed in a very early stage of parturition. Mild as well as severe difficulties were more likely to happen during surgery in cows that had undergone a CS before. Cows at their second or third surgery had a significantly higher proportion of larger calves and more skin scar tissue was present, both increasing the risk of abdominal wall muscular and uterine haemorrhage. These factors can all lead to problems with exteriorization and suturing of the uterus; hence special attention is needed when performing a CS on a cow which has experienced the procedure before. The more experienced veterinarians were less likely to report difficulties during the operation than less experienced surgeons.

Protocol of the Caesarean section as performed in daily bovine practice in Belgium.

In the early part of the 20th century, veterinarians began to perform Caesarean section (CS) in cows. At first, it was a final remedy to save the calf and/or the cow in more complicated obstetrical cases. As in Belgium, the Belgian Blue (BB) breed has become the predominant beef breed, CS are currently performed on a routine basis in daily practice. The overwhelming success of the breed is based on its hypermuscularity and exceptional carcass characteristics with high killing out (>70%). These and other characteristics made the breed popular and newborn animals valuable. Consequently, farmers do not want to take any risk of losing the calf during birth. This, together with the fact that bovine practitioners perform CS successfully on a routine basis, has made CS an elective operation. It is, however, important to remember that CS is still a major abdominal operation and that complications are not uncommon both during and after the operation. To end up with a good success rate, it is very important to follow a high-quality and sterile surgical procedure. The current paper describes the standard protocol by which CS are performed by the veterinarians of the Ambulatory Clinic of the Department of Reproduction, Obstetrics, and Herd Health of the Faculty of Veterinary Medicine in Ghent (Belgium). The aim of the article is to provide a successful protocol as regards CS. We especially focus on those factors which are known to be limiting for the success of the surgery and highlight the most recent recommendations to optimize the outcome of the operation.

Effects of beta-OH butyrate on bovine granulosa and theca cell function in vitro.

In this study, the effects of beta-OH butyrate (BHB) levels, associated with a negative energy balance, on bovine granulosa and theca cell function were investigated in vitro. Granulosa and theca cells of healthy large follicles (>8 mm), obtained from slaughterhouse ovaries, were cultured in serum free medium containing 0, 0.5, 1 or 1.5 mm BHB and 3 mm glucose, to mimic the situation in the early postpartum dairy cow. Hormone concentrations (progesterone, oestradiol-17beta and/or androstenedione) in spent medium and cell numbers were measured after 48 h of culture. No effects of BHB on theca cell numbers or on steroid production were observed. In granulosa cells, all BHB treatments evenly increased cell numbers (p < 0.05), while they reduced progesterone and oestradiol-17beta production per cell (p < 0.05). These effects may be attributed to the use of BHB as energy source which is however differently metabolized than glucose. Conclusively, in the presence of physiological glucose concentrations BHB can modulate granulosa but not theca cell function in vitro.

Effect of non-esterified fatty acids on bovine theca cell steroidogenesis and proliferation in vitro.

Elevated serum non-esterified fatty acid (NEFA) levels associated with a negative energy balance (NEB) may affect ovarian function and hence reproductive performance in high-yielding dairy cows. We have investigated the individual and combined effects of the three major NEFAs on bovine theca cell proliferation and steroidogenesis in vitro. Theca cells from healthy large follicles (>8 mm) obtained from slaughterhouse ovaries were cultured in serum free medium in the presence of 0, 50, 150 and 200 microM of palmitic acid (PA; C16:0); 0, 50, 150 and 250 microM of stearic acid (SA; C18:0); and/or 0, 50, 150 and 250 microM of oleic acid (OA; C18:1). Progesterone and androstenedione concentrations were measured in spent medium after 48 h of culture and cell numbers were determined spectrophotometrically per culture well. Cell viability was assessed by annexin-V FITC/propidium iodide staining. Only the treatment with 200 microM of PA inhibited cell proliferation (P<0.001) when tested individually, both of the mixtures tested (M1=100 microM of PA, 130 microM of SA and 140 microM of OA; M2=200 microM PA, 260 microM of SA and 280 microM of OA) reduced cell numbers (P<0.001). Progesterone and androstenedione production, both per well and per 10(4) cells, were not affected by any of the treatments, with the exception of M2. This mixture reduced progesterone production per well and per 10(4) cells (P<0.05). The effects observed were most likely caused by the cytotoxic action of the NEFAs, as demonstrated by the increased percentage of early apoptotic (M1) and late apoptotic/necrotic cells (M1 and M2) in the combination treatments (P<0.05). When combined, elevated physiological concentrations of PA, SA and OA can modulate theca cell proliferation and steroidogenesis in vitro by reducing theca cell viability. These NEFAs may be one of the mediators through which NEB compromises ovarian functioning and thus fertility in high-yielding dairy cows.

Hormonal and metabolic profiles of high-yielding dairy cows prior to ovarian cyst formation or first ovulation post partum.

The present study aimed to investigate the pathogenesis of cystic ovarian disease (COD) in high-yielding dairy cows postpartum (pp). Hormonal and metabolic profiles during the first 3 weeks pp as well as during the final week prior to ovulation/cyst formation, were compared between dairy cows that developed either an ovulatory follicle (OV) or a cyst (CYST) < day 60 pp. Thirty-four lactations of 28 high-yielding (9500 kg/305 days) Holstein-Friesian dairy cows were studied. Ovaries of cows were scanned twice a week from day 10 pp on, until ovulation/cyst formation. Milk yield data, body condition scores and blood samples, for determination of oestradiol-17beta, insulin, beta-OH-butyrate and non-esterified fatty acids, were collected simultaneously. Milk samples for progesterone analysis were collected daily. Four lactations were excluded from further analysis because of irregular pp ovarian cyclicity, excluding COD. Eight lactations (26.7%) developed a cyst, while 22 lactations ovulated < days 60 pp. Ovulation and cyst formation occurred at similar times pp. Metabolic and hormonal profiles did not differ between CYST and OV lactations during the first 3 weeks pp. In the final week prior to cyst formation/ovulation, insulin concentrations were lower in CYST than in OV lactations while no differences were observed for any of the other parameters tested. In two lactations, cyst formation was preceded by suprabasal progesterone and increased oestradiol-17beta concentrations. These results suggest that cyst formation in high-yielding dairy cows pp is associated with lower insulin levels but not with other distinct hormonal and metabolic alterations. However from this study, we cannot exclude the involvement of subtle hormonal and metabolic changes in the pathogenesis of ovarian cysts. Suprabasal progesterone, and altered oestradiol-17beta concentrations, seem to play a minor role in cyst formation.

Effect of non-esterified fatty acids on bovine granulosa cell steroidogenesis and proliferation in vitro.

In high-yielding dairy cows, the negative energy balance (NEB) during the first weeks post partum may influence dominant follicle growth and steroidogenesis. Since non-esterified fatty acid (NEFA) concentrations are elevated during NEB and are shown to be toxic for several cell types, we investigated the individual and combined effects of the three main NEFA's on granulosa cell proliferation and steroidogenesis in vitro. Granulosa cells from large follicles were cultured for two days in serum free medium in the presence of palmitic (C16:0) (PA), stearic (C18:0) (SA) and/or oleic acid (C18:1) (OA). Addition of 150, 300 or 500 microM of PA and SA inhibited cell proliferation (P<0.05) while OA only elicited such an effect at 500 microM (P<0.01). In the combination treatment (150 microM of each fatty acid), cell numbers were also reduced (P<0.01). These inhibitory effects on cell number are partly due to the induction of apoptosis by these NEFA's, as was demonstrated by annexin V-FITC/propidium iodide staining of the granulosa cells. Oestradiol-17beta production was stimulated by all doses of PA, by 300 and 500 microM of SA and by 500 microM of OA (P<0.05). Combined treatment with 150 microM of each fatty acid also stimulated oestradiol-17beta production per 10(4) cells (P<0.05). We can conclude that PA, SA and to a lesser degree OA modulate granulosa cell proliferation and steroidogenesis in vitro. These effects may be involved in the occurrence of ovarian dysfunction during the postpartum period in high-yielding dairy cows.

Fatal meningitis in a calf caused by Mannheimia varigena.

Mannheimia varigena was identified as the etiologic agent of meningitis in a young Belgian White Blue heifer calf. Species identification of the bacterium was done by phenotyping and molecularly confirmed by tDNA-PCR. Standard bacteriological examination might fail to differentiate species belonging to the genus Mannheimia.