The effect of maternal supply of rumen-protected protein to Holstein Friesian cows during the dry period on the transfer of passive immunity and colostral microbial composition.

The objective of this study was to analyze if maternal supply of rumen-protected protein during the dry period can affect the IgG concentration and microbial composition of colostrum and the IgG absorption and fecal microbial composition in the calf. Seventy-four multiparous Holstein Friesian (HF) dairy cows were stratified per parity and randomly assigned to one of 2 different dry period diets, a diet with a low crude protein (CP) level (LP) and a diet with a high CP level (HP) by addition of rumen-undegraded protein (RUP; formaldehyde-treated soybean meal, Mervobest, Nuscience, Drongen, Belgium). Colostrum was collected within 1 h after calving and IgG concentration was quantified by radial immunodiffusion analysis. Forty-nine calves (23 female and 26 male) were enrolled in the trial with a 2 × 2 factorial design, with prenatal and postnatal treatment as the 2 independent variables. This led to 4 experimental groups: LPLP, LPHP, HPLP, and HPHP, in which the first 2 letters refer to the prenatal treatment (diet of the dam) and the last 2 refer to the postnatal treatment (diet of the colostrum-producing cow). Calves received 3× 2 L of colostrum within 2, 6, and 24 h after birth. Meconium and feces were collected solely from female calves (n = 18) by digital palpation of the rectum, immediately after birth and before colostrum administration and at d 3 of age. Microbial DNA was extracted from meconium (n = 9), feces (n = 15), and colostrum (n = 49). Amplicon sequencing of the bacterial V3-V4 region of the 16S rRNA gene was performed for characterization of the bacterial communities. Colostrum IgG concentration was higher in cows that were supplemented with RUP, especially in cows entering their second lactation (LSM ± SEM 61.3 ± 2.3 vs. 55.2 ± 2.8 g of IgG/L). Calves born out of LP cows that received colostrum from HP cows (LPHP) had a lower serum IgG level compared with HPHP and LPLP calves (LSM ± SEM 14.2 ± 1.3 vs. 18.8 ± 1.2 and 20.9 ± 1.3 g of IgG/L in HPHP and LPLP, respectively). The most abundant phyla in colostrum were Proteobacteria (48.2%), Firmicutes (24.8%), Bacteroidetes (9.5%), and Actinobacteria (5.0%). The most abundant phyla in calf meconium and feces were Firmicutes (42.5 and 47.5%), Proteobacteria (21.7% and 33.7%), Bacteroidetes (16.8% and 15.7%), and Actinobacteria (2.9% and 3.1%). There was no difference in the overall microbial communities between colostrum from HP and LP cows. However, 2 genera (both members of the family Lachnospiraceae) were more abundant in colostrum from HP cows compared with LP cows. The microbial composition of meconium, feces and colostrum differed from each other. Fecal samples were more similar to each other and are characterized by a lower intersample diversity compared with colostrum and meconium samples. To conclude, increasing the CP level by addition of RUP in the dry period diet affected the colostrum IgG concentration and the transfer of passive immunity, but did not change the overall microbial composition of colostrum nor of meconium and feces in the calf.

Prediction of first test day milk yield using historical records in dairy cows.

The transition between two lactations remains one of the most critical periods during the productive life of dairy cows. In this study, we aimed to develop a model that predicts the milk yield of dairy cows from test day milk yield data collected in the previous lactation. In the past, data routinely collected in the context of herd improvement programmes on dairy farms have been used to provide insights in the health status of animals or for genetic evaluations. Typically, only data from the current lactation is used, comparing expected (i.e., unperturbed) with realised milk yields. This approach cannot be used to monitor the transition period due to the lack of unperturbed milk yields at the start of a lactation. For multiparous cows, an opportunity lies in the use of data from the previous lactation to predict the expected production of the next one. We developed a methodology to predict the first test day milk yield after calving using information from the previous lactation. To this end, three random forest models (nextMILKFULL, nextMILKPH, and nextMILKP) were trained with three different feature sets to forecast the milk yield on the first test day of the next lactation. To evaluate the added value of using a machine-learning approach against simple models based on contemporary animals or production in the previous lactation, we compared the nextMILK models with four benchmark models. The nextMILK models had an RMSE ranging from 6.08 to 6.24 kg of milk. In conclusion, the nextMILK models had a better prediction performance compared to the benchmark models. Application-wise, the proposed methodology could be part of a monitoring tool tailored towards the transition period. Future research should focus on validation of the developed methodology within such tool.

Exploring the microbial composition of Holstein Friesian and Belgian Blue colostrum in relation to the transfer of passive immunity.

For centuries, multicellular organisms have lived in symbiosis with microorganisms. The interaction with microorganisms has been shown to be very beneficial for humans and animals. During a natural birth, the initial inoculation with bacteria occurs when the neonate passes through the birth canal. Colostrum and milk intake are associated with the acquisition of a healthy gut flora. However, little is known about the microbial composition of bovine colostrum and the possible beneficial effects for the neonatal calf. In this prospective cohort study, the microbial composition of first-milking colostrum was analyzed in 62 Holstein Friesian (HF) and 46 Belgian Blue (BB) cows by performing amplicon sequencing of the bacterial V3-V4 region of the 16S rRNA gene. Calves received, 3 times, 2 L of their dam's colostrum within 24 h after birth. Associations between colostral microbial composition and its IgG concentration, as well as each calf's serum IgG levels, were analyzed. Colostrum samples were dominated by the phyla Proteobacteria, Firmicutes, Bacteroidetes, and Actinobacteria. The 10 most abundant genera in the complete data set were Acinetobacter (16.2%), Pseudomonas (15.1%), a genus belonging to the Enterobacteriaceae family (4.9%), Lactococcus (4.0%), Chryseobacterium (3.9%), Staphylococcus (3.6%), Proteus (1.9%), Streptococcus (1.8%), Enterococcus (1.7%), and Enhydrobacter (1.5%). The remaining genera (other than these top 10) accounted for 36.5% of the counts, and another 8.7% were unidentified. Bacterial diversity differed significantly between HF and BB samples. Within each breed, several genera were found to be differentially abundant between colostrum of different quality. Moreover, in HF, the bacterial composition of colostrum leading to low serum IgG levels in the calf differed from that of colostrum leading to high serum IgG levels. Results of the present study indicate that the microbes present in colostrum are associated with transfer of passive immunity in neonatal calves.

Effect of lycopene supplementation to bovine oocytes exposed to heat shock during in vitro maturation.

We investigated the effect of the antioxidant lycopene supplemented into the in vitro maturation medium (TCM-199 with 20 ng/mL epidermal growth factor and 50 mg/mL gentamycin) in a heat shock (HS) model to mimic in vivo heat stress conditions. Bovine cumulus-oocyte complexes were supplemented with 0.2 µM lycopene (or not supplemented; control) under HS (40.5 °C) and non-HS (NHS; 38.5 °C) during maturation. After 22 h of maturation, we evaluated the nuclear status of the oocytes, the level of reactive oxygen species (ROS) production, and the respective blastocyst development and quality (via differential staining). Data were fitted in logistic and linear regression models, and the replicates were set as a random effect. The nuclear maturation was higher in NHS (84.0 ± 3.2%; least square mean ± standard error) than HS control (60.4 ± 4.3%; P < 0.001). Remarkably, the nuclear maturation in HS lycopene (71.7 ± 4.1%) was similar to NHS control (P = 0.7). Under HS conditions lycopene reduced ROS production (27.4 ± 4.8; relative fluorescence units (RFU)) in comparison to HS control (33.8 ± 1.8 RFU; P = 0.009). However, the ROS production in NHS lycopene (18.9 ± 2.0 RFU) was similar to NHS control (18.7 ± 1.8 RFU; P = 0.9). The cleavage rate in HS lycopene (76.1 ± 3.3%) was not lower than NHS lycopene (83.3 ± 2.5%; P > 0.1). On the day 8 of embryo development, the blastocyst rate was higher for NHS lycopene (55.2 ± 4.7%) versus NHS control (44.5 ± 4.7%; P = 0.04), but under HS the day 8 blastocyst rate was similar between control (29.9 ± 4.2%) and lycopene (32.3 ± 4.2%; P = 0.9). Lycopene supplementation increased the cell number of the embryos (total cell, trophectoderm, and inner cell mass numbers) under NHS conditions (P > 0.03). The apoptotic cell ratio was lower in lycopene (NHS and HS) versus control (NHS and HS) (P > 0.04). Lycopene has the ability to scavenge oocyte ROS and improved the cleavage rate of embryos under HS conditions. However, this could not be translated to a higher blastocyst development, which remained lower under HS. Results of our study indicate that antioxidant supplementation like lycopene during the maturation of bovine cumulus-oocyte complexes may be routinely used to improve blastocyst rate and quality under standard maturation conditions.

Invited review: MicroRNAs in bovine colostrum-Focus on their origin and potential health benefits for the calf.

Colostrum is the first milk produced by a cow after she gives birth. Compared with mature milk, it has a high concentration of immunoglobulin G. Calves are born without circulating antibodies, thus ingestion of colostrum is necessary to protect the calf against pathogens in the first challenging weeks of life. In addition to the life-saving supply of antibodies, colostrum contains minerals, vitamins, growth factors, and immune cells. Recently, microRNAs (miRNAs) were added to that list. MicroRNAs are short, non-coding RNA molecules that can regulate gene expression at the post-transcriptional level. They are thought to act as key regulators of diverse biological and developmental processes. Colostrum contains higher amounts of miRNAs than mature milk; immune- and development-related miRNAs are prominent. Their expression pattern in milk is likely to be influenced by maternal nutrition and environment. The fat content of the maternal diet appears to have a major effect on expression of miRNAs in milk and in the neonate. The immunological state of the mammary gland seems to affect miRNA expression as well. In cows diagnosed with subclinical mastitis, alterations in the expression of miRNAs in milk have been observed. It is believed that miRNAs in colostrum and milk are signaling molecules passed from mother to newborn. They are packaged in extracellular vesicles, which makes them resistant to the harsh conditions in the gastrointestinal tract. Therefore, they can reach the small intestine, where they are absorbed and transferred into the bloodstream. MicroRNAs are important for the development of the intestines. For example, miRNAs stimulate cell viability, proliferation, and stem cell activity of the intestinal epithelium. Furthermore, miRNAs seem to act as key players in the development of the complete immune system. They can, among other things, regulate B- and T-cell differentiation and affect interleukin production of macrophages. The abundance of miRNAs in colostrum and milk and the possibility for their absorption in the intestines of the neonate supports the hypothesis that these tiny molecules are important for the development of the newborn. The probable relation of diet to the expression of miRNAs by the mother creates a possible avenue to optimize expression of miRNAs and improve neonatal maturation.

Season of birth is associated with first-lactation milk yield in Holstein Friesian cattle.

The aim of the present research was to assess factors associated with first-lactation milk yield in dairy heifers, including maternal and environmental factors, factors related to the development of the heifer and factors related to its offspring such as gender of the calf. In addition, the potential underlying mechanism, in particular metabolic adaptations, was further explored. Data on body growth, reproduction and milk yield of 74 Holstein Friesian heifers on three herds in Flanders (Belgium) were collected. At birth, body measurements of the heifers were recorded and blood samples were taken (in order) to determine basal glucose and insulin concentrations. Body measurements were assessed every 3 months until first calving, and gender and weight of their first calf were recorded. Information on fertility and milk yield of the heifer and its dam were collected from the herd databases. Daily temperature and photoperiod were recorded from the database of the Belgian Royal Meteorological Institute. Linear mixed models were run with herd as a random factor, to account for differences in herd management. Heifers grew 867±80.7 g/day during their first year of life and were inseminated at 14.8±1.34 months. First calving took place at 24.5±1.93 months, at a weight of 642±61.5 kg and heifers produced 8506±1064 kg energy corrected milk during their first 305-day lactation. Regression models revealed that none of the maternal factors such as milk yield and parity, nor the growth of the heifer during the 1st year of life were associated with milk yield during first lactation. Age, and to a lesser extent BW at first parturition were positively associated with first-lactation milk yield. In addition, the season of birth, but not calving, had a significant influence on milk yield, with winter-born heifers producing less than heifers born in any other season. The lower yielding winter-born heifers had higher insulin concentrations at birth, whereas glucose concentrations were similar, the latter being suggestive for lower insulin sensitivity of the peripheral tissues. Furthermore, environmental temperature at the end of gestation was negatively correlated with neonatal insulin concentrations. In conclusion, results of the present study suggest heifers born during the hotter months are born with a higher peripheral insulin sensitivity, finally leading to a higher first-lactation milk yield.

Effect of ß-hydroxybutyric acid, parity, and body condition score on phenotype and proliferative capacity of colostral mononuclear leukocytes of high-yielding dairy cows.

In neonatal calves, the ingestion of colostrum is imperative for preventing infectious diseases. Investigations into the transfer of passive immunity of cattle have primarily focused on the importance of colostral immunoglobulins, with a recent increase in focus on understanding the role of colostral leukocytes. The main objective of the present study was to measure the influence of parity, body condition score, serum nonesterified fatty acids, and serum ß-hydroxybutyrate concentrations of periparturient cows on phenotype and mitogen- and antigen-induced proliferative capacity of bovine colostral leukocytes. Holstein-Friesian cows (n=141) were intramuscularly vaccinated at 60 and 30 d before the expected parturition date with a tetanus toxoid vaccine. Of these 141 animals, 28 primiparous and 72 multiparous cows were sampled immediately after parturition. Colostrum mononuclear cell populations were identified by flow cytometry using bovine cluster of differentiation markers, and the proliferative capacity of these cells was determined using a (3)H-thymidine proliferation assay. Under-conditioned cows had a significantly higher percentage of colostral macrophages than normal-conditioned animals, whereas over-conditioned cows had significantly more colostral B-lymphocytes. Serum ß-hydroxybutyrate was significantly associated with higher numbers of colostral T-lymphocytes and macrophages. Heifers had significantly higher mitogen- and antigen-induced proliferation of their colostral leukocytes than third parity or older cows. In conclusion, body condition score, parity, and serum ß-hydroxybutyrate concentration of periparturient high-yielding dairy cows were shown to influence the number of colostral macrophages or the mitogen- and antigen-induced proliferation of colostral leukocytes, possibly influencing the cellular immunity of the newborn calf.

Evaluation of a protocol to reduce the incidence of neonatal calf diarrhoea on dairy herds.

Calf diarrhoea causes substantial economic losses in cattle herds worldwide. Neonatal calves are particularly sensitive to infections with enteropathogens. The present study focused on prevention against the main infectious causes of neonatal calf diarrhoea i.e. Escherichia coli, rota- and coronavirus, and Cryptosporidium parvum. Dairy herds (n=24) with a high percentage of neonatal calves scouring (>10%) were included and calves were sampled for the presence of these four enteropathogens. To decrease diarrhoea problems among neonatal calves, a standard protocol was tested on 13 herds (treatment group) where both C. parvum and either E. coli or rota- or coronavirus were identified as being involved, the other 11 herds served as control group. The protocol consisted of 2 points of action: preventive vaccination of dams against E. coli, rota- and coronavirus, and preventive administration of halofuginone lactate to newborn calves. The average percentage of calves suffering from neonatal diarrhoea (39.7% versus 14.3%, P<0.01) and the average percentage of faecal samples positive for C. parvum (34% versus 11%, P<0.05) differed significantly between control herds and treatment herds after implementation of the protocol. No significant differences between control and treatment group were observed in the percentage of calves excreting E. coli, rotavirus and coronavirus, both before and at the end of the trial. Furthermore, risk factors potentially associated with the development of neonatal calf scours were determined. Non-significant results were obtained for the effect of the protocol on duration of diarrhoea and the effect of the colostral IgG quantity on the risk of diarrhoea. Passive immunity transfer status of the calves, measured both before the onset and at the end of the study, were non-significant between groups.

Influence of a marine algae supplementation on the oxidative status of plasma in dairy cows during the periparturient period.

This study was part of a larger study that addressed the effects of marine algae (ALG) supplementation in the ration of high yielding periparturient dairy cows. The objectives were to induce milk fat depression (MFD) in early lactation by feeding docosahexaenoic acid (DHA) from ALG and to determine the effects on milk production, milk components and metabolic status early post partum. This study focuses on the oxidative status in the plasma during the ALG supplementation. Plasma samples were collected from 16 Holstein Friesian cows at the day of parturition and at -1, 2, 4 and 6 weeks relative to calving with half of the cows receiving the ALG supplement (44gDHA/d) from 3 weeks pre partum on. The following parameters were measured in plasma: ferric reducing ability of plasma (FRAP), α-tocopherol level, glutathione peroxidase activity (GSH-Px) and thiobarbituric acid reactive substances (TBARS) concentration. There was a significant effect of time for FRAP and α-tocopherol indicating changes in the plasma oxidative status around parturition. The ALG supplementation was successful in creating a milk fat depression (MFD) but could not improve the energy balance. Feeding of ALG significantly increased lipid peroxidation as measured by TBARS, probably through their high content of n-3 polyunsaturated fatty acids.

The effect of marine algae in the ration of high-yielding dairy cows during transition on metabolic parameters in serum and follicular fluid around parturition.

Sixteen Holstein cows were assigned to 2 groups to evaluate the caloric and metabolic effect of feeding marine algae (ALG) from 3 wk prepartum until 12 wk postpartum. Milk production characteristics and the profiles of hormones and metabolites in the serum were monitored from -7 to 46 d in milk (DIM) and in follicular fluid (FF) from 14 to 46 DIM. All cows received a corn- and grass silage-based partially mixed ration supplemented with concentrate and protein supplement. In the diet of the ALG group, 2 kg of the concentrate was replaced by a concentrate containing ALG (44 g/d of docosahexaenoic acid). Diets were isocaloric (net energy basis) and equal in intestinal digestible protein. The ALG diet increased milk yield (41.2 vs. 38.2 kg/d) and decreased milk fat yield (1.181 vs. 1.493 kg/d) and milk fat content (31.6 vs. 40.7 g/kg). Protein yield (1.336 vs. 1.301 kg/d) was not affected but a tendency toward decreased milk protein content (32.8 vs. 34.7 g/kg) was observed. Marine algae supplementation increased the ß-hydroxybutyric acid (BHBA) concentration in FF of the ALG cows compared with that in the controls (0.992 vs. 0.718 mmol/L). The total protein concentration in FF was decreased in ALG (62.9 vs. 67.6 g/L). Plasma and serum metabolites did not significantly differ between treatments except for a tendency toward a lower concentration of urea in the serum of the control compared with ALG (4.69 vs. 5.13 mmol/L). Based on metabolizable energy calculations, a daily energy-sparing effect of 3.48 Mcal was obtained due to milk fat depression (MFD). The concomitant increase in milk yield suggests that at least part of this spared energy is used to stimulate milk production. Theoretically, 3.48 Mcal of ME could lead to an increase in milk yield of 7.43 kg/d, which is higher than the observed 3 kg/d. However, when evaluating nutrient requirements during MFD in early lactation, we calculated that increased milk production is caused by a propionate-saving effect of 2.71 mol in the udder when milk fat is depressed. Concurrent increased BHBA concentrations in FF in the ALG group cannot be attributed to a worsened energy status of the animals because all other indicators contradict any change in energy balance, indicating that BHBA might not be an appropriate metabolic parameter to estimate the energy balance in early lactating dairy cows during MFD.

Pathogen group specific risk factors at herd, heifer and quarter levels for intramammary infections in early lactating dairy heifers.

Risk factors for intramammary infections caused by coagulase-negative staphylococci, contagious major pathogens and environmental major pathogens in early lactating heifers were evaluated at the herd, heifer and quarter levels. In total, 764 quarters of 191 dairy heifers in 20 randomly selected farms in Flanders (Belgium) were sampled. Quarter milk samples were collected between 1 and 4 days in milk and between 5 and 8 days in milk for bacteriological culture. Data were analyzed using multivariable, multilevel logistic regression analysis. Higher average herd milk somatic cell count (>200,000 cells/mL), not having an effective fly control strategy, contact with lactating cows prior to calving and moderate to severe udder edema prior to calving increased the odds of intramammary infections caused by contagious major pathogens. Poor heifer hygiene and lack of mineral/vitamin supplementation prior to calving were risk factors for intramammary infection caused by environmental major pathogens. Teat apex colonization with coagulase-negative staphylococci prior to calving seemed to protect quarters against intramammary infections caused by major pathogens. Poor heifer hygiene before calving, a non-clipped udder and not practicing of teat dipping prior to calving increased the odds of intramammary infection with coagulase-negative staphylococci. Although management is important in the prevention and control of intramammary infections in early lactating heifers, most variation in the prevalence of intramammary infections resided at the heifer and quarter levels, indicating that the susceptibility for intramammary infections around calving is mainly determined by heifer and quarter characteristics.

Heifer and quarter characteristics associated with periparturient blood and milk neutrophil apoptosis in healthy heifers and in heifers with subclinical mastitis.

Polymorphonuclear neutrophilic leukocytes (PMNL) play an important role in the first line cell-mediated immune defense of the body in general and of the mammary gland against mastitis pathogens in particular. Reduced viability of PMNL close to parturition may explain the high incidence of infectious diseases and the high prevalence of intramammary infections (IMI) in periparturient dairy heifers. Apoptosis of blood PMNL 1 wk before the expected calving date and of blood and milk PMNL at 1 to 4 d in milk was determined using flow cytometry. Information on heifer and gland characteristics was collected before calving and in early lactation. Data were analyzed using multivariable, multilevel regression analysis. Supplementation of a commercial mineral/vitamin mix before calving was associated with less blood (14.4 +/- 2.9 vs. 22.4 +/- 2.1%) and milk PMNL apoptosis (19.0 +/- 1.1 vs. 26.4 +/- 0.9%) near calving, presumably related to higher blood selenium concentrations. Both blood and milk PMNL apoptosis showed seasonal variation with the highest proportion of apoptotic cells between January and March (32.0 +/- 6.1 and 34.6 +/- 2.7%, respectively) and April and June (31.3 +/- 5.7 and 37.8 +/- 2.3%, respectively). Heifers losing 0.25 points or more of their body condition in the periparturient period had higher proportions of apoptotic blood PMNL in early lactation compared with heifers losing less than 0.25 points (24.0 +/- 2.8 vs. 16.6 +/- 1.7%). Milk PMNL apoptosis was less pronounced in quarters having teat orifices colonized with non-aureus staphylococci before calving (18.9 +/- 1.0 vs. 29.4 +/- 1.0%). The variation in blood PMNL apoptosis before and after calving mainly resided at the heifer level (71.4 and 98.4% of the total variation, respectively), whereas the variation in milk PMNL apoptosis mainly resided at the heifer (45.7% of the total variation) and quarter levels (45.5% of the total variation). These data imply that the impaired blood and milk PMNL viability in periparturient heifers can be reduced by optimization of certain heifer management practices such as supplementation of minerals/vitamins, and pasture and feeding strategies.

Tetracycline-resistance in lactose-positive enteric coliforms originating from Belgian fattening pigs: degree of resistance, multiple resistance and risk factors.

Between March and October 2003 a field study was conducted in 50 randomly selected pig herds to assess the degree of tetracycline-resistance in lactose-positive enteric coliforms (LPEC) originating from fattening pigs and to evaluate the combined effects of various husbandry conditions on the development and persistence of antimicrobial-resistant bacteria. Data on housing, management and antimicrobial-drug consumption were collected, as well as faecal samples at three production stages: end of the nursery period (mean age: 72 days), end of the grower period (mean age: 125 days) and end of the finisher period (mean age: 186 days). The degree of tetracycline-resistant LPEC was determined by means of an agar dilution method. Tetracycline-resistant LPEC were found in every herd. The overall degree of tetracycline-resistance in LPEC was 56.8% (S.D. 22.4%). Only a very weak relation was found between the degrees of TETR in the different production stages within the same herd, indicating that the degree of TETR is mainly associated with the production stage rather than with the farm as a whole. The risk factor analysis showed that besides the antimicrobial-drug use, other factors like inside pen hygiene can influence the development and maintenance of antimicrobial-resistant bacteria in the gastrointestinal tracts of pigs. It was also observed that tetracycline-resistance in commensal Escherichia coli is often linked with resistance to other antimicrobial drugs like ampicillin and trimethoprim-sulphonamides. These results illustrate that the epidemiology of antimicrobial resistance is influenced by antimicrobial-drug use, cross-resistance development and non-antimicrobial risk factors.