Technical note: Infusion,sampling, and vacuum-assisted collection devices for use in ruminally cannulated calves

Calves can be ruminally cannulated at young ages, but equipment size limitations preclude use of an infusion and sampling device in these small animals. Likewise, a procedure to easily evacuate rumen contents in young calves has not been described. Overcoming these technical complications related to assessment of ruminal passage kinetics, nutrient digestion, and volatile fatty acid absorption would aid in future studies advancing our knowledge of dairy calf nutrition. The first objective was to design and fabricate 2 devices (one device for infusion and sampling, and another for vacuum-assisted collection) suitable for use in young ruminally cannulated dairy calves. The second objective was to test the utility of these tools when performing procedures commonly used in ruminant nutrition research. A single weaned 62-d-old ruminally cannulated calf was used to evaluate the ability to infuse a solution of LiCoEDTA and sample rumen contents through the cannula cap over a period of 2 h to assess the rumen liquid passage rate (procedure 1). The device was capable of infusing the LiCoEDTA and sampling the rumen fluid, as evidenced by the presence of elevated Co concentrations in the sampled rumen fluid. Using the fluid samples obtained, liquid passage rate within the calf was estimated to be 40.2% of ruminal fluid/h. The second procedure tested the vacuum-assisted collection device and consisted of evacuating and weighing the rumen contents, which is considered a key preparatory step in washed reticulorumen technique experiments that aim to measure nutrient absorption. In agreement with existing literature, evacuated rumen contents represented approximately 4% of the calf's body weight. In conclusion, custom-built devices for infusion, sampling, and vacuum-assisted collection were efficacious when tested in a 62-d-old ruminally cannulated calf fed a diet of 100% texturized starter (18% crude protein, as-fed). Fellow scientists may employ and further modify these techniques to suit their needs when assessing passage kinetics, nutrient digestion, and volatile fatty acid absorption in calves.     

Technical note: The development of a methodology for ruminal and colon tissue biopsying of young Holstein dairy calves

The objectives of this study were to develop a methodology for biopsying the rumen and colon of young dairy calves and to collect suitable quality tissue samples for microscopic and gene expression analysis. Six Holstein dairy bull calves (45.0 ± 1.5 kg birth weight) were ruminally cannulated during the second week of life and weaned at the end of wk 6. Ruminal and colon tissue samples were collected at the end of wk 5, 6, 7, 8, and 12. Calves were not sedated but were restrained in a chute for sampling. The endoscope (100 cm length, 9.8 mm diameter) was introduced through the rumen cannula to harvest ruminal tissue. Endoscopic biopsies of the rumen with endoscopic biopsy forceps were unsuccessful 85% of the time because they were unable to shear the ruminal tissue. Thereafter, an Allis clamp was used to retrieve the blind sac through the rumen cannula to perform direct tissue biopsying with surgical scissors. To biopsy the colon, the lubricated distal tip of an endoscope was slowly inserted into the calf's anus. A total of 6 colon tissue samples (12.6 ± 0.74 mg) were collected per calf per time point from the distal colon 30 to 40 cm from the calf's anus using endoscopic biopsy forceps, which were inserted through the instrument channel. A new forcep was used between sites and calves. Between calves, the outside of the endoscope was washed with 4% chlorohexidine and rinsed with water and the instrument channel was washed with distilled water and 70% ethanol. Colon and ruminal samples were processed for histological measurements, and RNA was isolated and sequenced. High-quality RNA (RNA integrity number 8.8 ± 0.08) was collected from samples, and light and electron microscopy was performed on samples. In conclusion, endoscopic biopsying can be used for tissue harvest in the colon of young calves. However, it was found that collecting ruminal tissue by retracting the rumen from the cannula and taking samples with surgical scissors was more successful than an endoscopic biopsy. This method allows for tissue collection of the same animal throughout time, which can help the research community investigate the effect of weaning regimens, feed rations, and age on the structure and function of the gastrointestinal tract.     

Maternal rumen and milk microbiota shape the establishment of early-life rumen microbiota in grazing yak calves

Early-life gut microbial colonization and development exert a profound impact on the health and metabolism of the host throughout the life span. The transmission of microbes from the mother to the offspring affects the succession and establishment of the early-life rumen microbiome in newborns, but the contributions of different maternal sites to the rumen microbial establishment remain unclear. In the present study, samples from different dam sites (namely, oral, rumen fluid, milk, and teat skin) and rumen fluid of yak calves were collected at 6 time points between d 7 and 180 postpartum to determine the contributions of the different maternal sites to the establishment of the bacterial and archaeal communities in the rumen during early life. Our analysis demonstrated that the dam's microbial communities clustered according to the sites, and the calves' rumen microbiota resembled that of the dam consistently regardless of fluctuations at d 7 and 14. The dam's rumen microbiota was the major source of the calves' rumen bacteria (7.9%) and archaea (49.7%) compared with the other sites, whereas the potential sources of the calf rumen microbiota from other sites varied according to the age. The contribution of dam's rumen bacteria increased with age from 0.36% at d 7 to 14.8% at d 180, whereas the contribution of the milk microbiota showed the opposite trend, with its contribution reduced from 2.7% at d 7 to 0.2% at d 180. Maternal oral archaea were the main sources of the calves' rumen archaea at d 14 (50.4%), but maternal rumen archaea became the main source gradually and reached 66.2% at d 180. These findings demonstrated the potential microbial transfer from the dam to the offspring that could influence the rumen microbiota colonization and establishment in yak calves raised under grazing regimens, providing the basis for future microbiota manipulation strategies during their early life.     

Short communication: Effect of calf starter on rumen pH of Holstein dairy calves at weaning

The objective of this study was to evaluate the effect of feeding calf starter on rumen pH of dairy calves during weaning transition. Twenty Holstein bull calves were paired into 10 blocks by starting date of the study and body weight, and fed either milk replacer and hay (MR) or MR, hay, and a commercial texturized calf starter (MR+S) in a randomized complete block design. All calves were fed 750 g/d of milk replacer as the basal diet. Calves on MR+S treatment were also fed a calf starter ad libitum to maintain similar energy intake between calves within blocks, and MR calves were fed additional milk replacer that was equivalent to energy from calf starter intake. When MR+S calves consumed a calf starter at 680 g/d for 3 consecutive d, rumen pH of a MR+S calf and his MR counterpart was measured continuously for 3 d using a small ruminant rumen pH measurement system. Treatment did not affect minimum pH, mean pH, maximum pH, standard deviation of mean pH, and duration or area under pH 5.8, indicating that calf starter consumption did not appear to affect rumen pH. However, hay intake was negatively correlated to area under pH 5.8, with a breakpoint at 0.080 kg/d intake, suggesting hay intake might play an important role in mitigating ruminal acidosis in dairy calves during weaning transition.     

Effects of supplemental butyrate and weaning on rumen fermentation in Holstein calves

The objectives of this study were to determine the effects of the weaning transition and supplemental rumen-protected butyrate on subacute ruminal acidosis, feed intake, and growth parameters. Holstein bull calves (n = 36; age = 10.7 ± 4.1 d; ± standard deviation) were assigned to 1 of 4 treatment groups: 2 preweaning groups, animals fed milk replacer only (PRE-M) and those fed milk replacer, calf starter, and hay (PRE-S); and 2 postweaning groups, animals fed milk replacer, calf starter, and hay without supplemental rumen-protected butyrate (POST-S) or with supplemental rumen-protected butyrate at a rate of 1% wt/wt during the 2-wk weaning transition (POST-B). Milk replacer was provided at 1,200 g/d; starter, water, and hay were provided ad libitum. Weaning took place over 14 d by reducing milk replacer provision to 900 g/d in wk 7, 600 g/d in wk 8, and 0 g/d in wk 9. Rumen pH was measured continuously for 7 d during wk 6 for PRE-S and PRE-M and during wk 9 for POST-S and POST-B. After rumen pH was measured for 7 d, calves were euthanized, and rumen fluid was sampled and analyzed for volatile fatty acid (VFA) profile. Individual feed intake was recorded daily, whereas, weekly, body weights were recorded, and blood samples were collected. Compared with PRE-M, PRE-S calves tended to have a greater total VFA concentration (35.60 ± 11.4 vs. 11.90 ± 11.8 mM) but mean rumen pH was unaffected (6.25 ± 0.22 vs. 6.17 ± 0.21, respectively). Between PRE-S (wk 6) and POST-S (wk 9), calf starter intake increased (250 ± 219 vs. 2,239 ± 219 g/d), total VFA concentrations increased (35.6 ± 11.4 vs. 154.4 ± 11.8 mM), but mean rumen pH was unaffected (6.25 ± 0.22 vs. 6.40 ± 0.22, respectively). Compared with POST-S, POST-B calves had greater starter intake in wk 7, 8, and 9, but POST-B tended to have lower total VFA concentration (131.0 ± 11.8 vs. 154.4 ± 11.8 mM) and lower mean ruminal pH (5.83 ± 0.21 vs. 6.40 ± 0.22). In conclusion, the weaning transition does not appear to affect rumen pH and VFA profile, but supplementing rumen-protected butyrate during the weaning transition increased starter intake and average daily gain. Further, these data suggest that the ability of the rumen to manage rumen pH changes fundamentally postweaning. Why weaned calves with lower rumen pH can achieve higher calf starter intakes is unclear; these data suggest the effect of rumen pH on feed intake differs between calves and cows.     

Influence of weaning method on health status and rumen development in dairy calves

In the artificial rearing of dairy calves, the same feeding plan is applied to all animals during the milk-feeding period, with individual differences attributable to development or health status rarely considered. The aim of this study was 1) to analyze whether the parameters of feeding behavior automatically recorded by a feeding computer and weight gain are suitable for predicting the health status and rumen development of male dairy calves, and 2) to compare a conventional weaning method (end of milk provision at 12 wk of age, n = 23 calves) with a concentrate-dependent weaning method (with reduction in the milk amount depending on the consumption of concentrate, n = 24). The health status of each animal was evaluated daily by a scoring list (health score), and body temperature was measured automatically during each milk intake. In addition, the number of veterinary treatments per calf was recorded. Rumen development was assessed by measuring rumen papillae in 8 rumen areas after slaughter (n = 24, half of each treatment group). During the milk-feeding period, body temperature was elevated (≥39.5°C) on 40.8 and 43.2% of all days for calves on the concentrate-dependent weaning method and the conventional weaning method, respectively. Hay and concentrate intake (but not milk intake) and weight gain were clearly affected by health status. In addition, health score and the probability of being treated by a veterinarian were significantly related to decreases in concentrate consumption. During the milk-feeding period, increased body temperature, an increased number of veterinary treatments, and decreases in milk consumption were all associated with reduced weight gain. Calves on the concentrate-dependent weaning method were weaned at an average age of 76 d, which was significantly shorter than the age at the end of milk provision for conventionally fed calves (84 d). Weight gain and health status did not differ between treatment groups. Weight gain was positively associated with papillae length. A treatment effect on rumen development could not be found. We conclude that the concentrate-dependent weaning method allows a faster physiological development without any negative impact on rumen development, weight gain, or health status; we therefore recommend its use in practice.     

Invited review: Essential oils as modifiers of rumen microbial fermentation

Microorganisms in the rumen degrade nutrients to produce volatile fatty acids and synthesize microbial protein as an energy and protein supply for the ruminant, respectively. However, this fermentation process has energy (losses of methane) and protein (losses of ammonia N) inefficiencies that may limit production performance and contribute to the release of pollutants to the environment. Antibiotic ionophores have been very successful in reducing these energy and protein losses in the rumen, but the use of antibiotics in animal feeds is facing reduced social acceptance, and their use has been banned in the European Union since January 2006. For this reason, scientists have become interested in evaluating other alternatives to control specific microbial populations to modulate rumen fermentation. Essential oils can interact with microbial cell membranes and inhibit the growth of some gram-positive and gram-negative bacteria. As a result of such inhibition, the addition of some plant extracts to the rumen results in an inhibition of deamination and methanogenesis, resulting in lower ammonia N, methane, and acetate, and in higher propionate and butyrate concentrations. Results have indicated that garlic oil, cinnamaldehyde (the main active component of cinnamon oil), eugenol (the main active component of the clove bud), capsaicin (the active component of hot peppers), and anise oil, among others, may increase propionate production, reduce acetate or methane production, and modify proteolysis, peptidolysis, or deamination in the rumen. However, the effects of some of these essential oils are pH and diet dependent, and their use may be beneficial only under specific conditions and production systems. For example, capsaicin appears to have small effects in high-forage diets, whereas the changes observed in high-concentrate diets (increases in dry matter intake and total VFA, and reduction in the acetateto-propionate ratio and ammonia N concentration) may be beneficial. Because plant extracts may act at different levels in the carbohydrate and protein degradation pathways, their careful selection and combination may provide a useful tool to manipulate rumen microbial fermentation effectively. However, additional research is required to establish the optimal dose in vivo in units of the active component, to consider the potential adaptation of microbial populations to their activities, to examine the presence of residues in the products (milk or meat), and to demonstrate improvements in animal performance.     

From pre- to postweaning: Transformation of the young calf's gastrointestinal tract

The ruminant gastrointestinal tract (GIT) faces the challenge of protecting the host from luminal contents and pathogens, while supporting the absorption and metabolism of nutrients for growth and maintenance. The GIT of the calf in early life undergoes some of the most rapid microbial and structural changes documented in nature, and these adaptations in GIT function make the young calf susceptible to GIT diseases and disorders. Despite these challenges, the calf's GIT has a certain degree of plasticity and can sense nutrient supply and respond to bioactive ingredients. Calf GIT research has historically focused on the transition during weaning and characterizing ruminal papillae development using microscopy and digesta metabolite responses. Through the use of new molecular-based approaches, we have recently shown that delaying the age of weaning and providing a step-down weaning protocol is associated with a more gradual shift in ruminal microbiota to a postweaned state. In addition to ruminal adaptations during weaning, nutrient flow to the lower gut changes dramatically during weaning, coinciding with a wide array of structural and microbiological changes. Structural and gene expression changes suggest that the lower gut of the dairy calf undergoes alterations that may reduce barrier function when solid feeds are consumed. More recently, in vivo data revealed that the weaning transition increases total gut permeability of the calf. Interestingly, the lower gut may be able to communicate with the forestomach, meaning that a nutrient can be sensed in the lower gut and cause subsequent adaptations in the forestomach. An improved understanding of how diet, microbiota, and functional ingredients interact to affect growth and barrier function of the intestinal tract would greatly benefit the dairy calf industry. A mechanistic understanding of such adaptations would also aid in the formulation of specific management regimens and provision of functional ingredients required to characterize and enhance gut function in young calves.     

Effects of supplemental yeast (Saccharomyces cerevisiae) culture on rumen development, growth characteristics, and blood parameters in neonatal dairy calves

Yeast (Saccharomyces cerevisiae) culture was added to a texturized calf starter at 0 (control), 1, or 2% of dry matter to determine effects on intake, growth, blood parameters, and rumen development. Seventy-five Holstein calves (38 male; 37 female) were started on the experiment at 2 +/- 1 d of age and were studied for 42 d. Starter intake was measured, and fecal scoring was conducted daily. Growth and blood parameter measurements were recorded at weekly intervals. A subset of 6 male calves (2 per treatment) was euthanized at 5 wk of age, and rumen tissue was sampled for rumen epithelial growth measurements. An additional 6 male calves were euthanized at 6 wk of age for rumen epithelial growth measurements. Inclusion of yeast culture at 2% of the starter ration significantly increased starter and total dry matter intake, average daily gain, and daily hip width change when compared with the control treatment. Average daily gain was improved by 15.6% for the 2% yeast treatment. Daily change in hip height was also significantly greater for calves receiving 2% supplemental yeast culture than for calves receiving 1%. No significant treatment differences were observed for any other variables. These data suggest that the addition of yeast culture in a dairy calf starter at 2% enhances dry matter intake and growth and slightly improves rumen development in dairy calves.     

Symposium review: Re-evaluation of National Research Council energy estimates in calf starters

Provision of nutrients in appropriate amounts to meet nutrient requirements for growth, production, and reproduction is the basis for modern animal nutrition. Ration formulation systems predict nutrient requirements based on numerous inputs and then predict nutrient supply based on predicted intake and nutrient content of feeds. Energy systems are used to predict energy supply based on gross caloric content of feeds followed by adjustments for digestion and metabolism of ingested energy. Many models of energy supply use static coefficients of digestibility based on nutrient composition of feed. Other models partition digestion dynamically between ruminal and postruminal digestion but use static estimates of intestinal digestibility to predict energy supplied to the animal. In young calves, both ruminal fermentation and intestinal digestion are underdeveloped; therefore, existing models of energy supply might overestimate the energy available before complete gastrointestinal maturation. In a series of experiments, we reported that total-tract digestion of nutrients changes with advancing age and nutrient intake. Total-tract digestion was measured in calves from 3 to 16 wk of age when fed different amounts and types of milk replacers. Calves were also fed different types of calf starter for ad libitum consumption. Total-tract digestibility of protein, fat, neutral detergent fiber, and nonfiber carbohydrate (NFC) was used to calculate the metabolizable energy (ME) in starter. We used nonlinear regression to estimate the contribution of protein and fat from starter and milk replacer before weaning. Early in life, calculated ME of starter was low and increased with increasing intake of NFC. Cumulative intake of NFC was more highly correlated with changing ME values than other indices, including age, intake of milk replacer, or intake of other nutrients in starter. When calves consumed at least of 15 kg of NFC, ME calculated from digestibility measurements was similar to the ME calculated using National Research Council equations and indicated maturation of gastrointestinal digestion. Our data suggest that intake of NFC is critical to gastrointestinal maturation and the calf's ability to extract energy from calf starter.     

Effects of adding extra molasses to a texturized calf starter on rumen development, growth characteristics, and blood parameters in neonatal dairy calves

A texturized calf starter containing 5 (control) or 12% molasses [on a dry matter (DM) basis] was fed to dairy calves to determine effects on intake, growth, blood parameters, and rumen development. Forty-six Holstein calves (26 male and 20 female) were started at 2 +/- 1 d of age and studied for 42 d. Starter DM intake was measured and fecal scoring was conducted daily. Growth and blood parameter measurements were conducted weekly. A subset of 6 male calves (3 per treatment) was euthanized at 4 wk of age, and rumen tissue sampled for rumen epithelial growth measurements. Starter sugar content was significantly increased in the starter containing extra molasses. Postweaning and overall starter DM intake, overall total DM intake, daily heart girth change, and final heart girth were significantly decreased, whereas overall average daily gain tended to decrease when calves received starter containing 12% molasses. However, blood volatile fatty acid concentrations were significantly increased when calves received a starter containing 12% molasses. No significant differences were observed between calves receiving starters containing 5 or 12% molasses for all other variables. The data indicates that adding extra molasses to a texturized calf starter decreases intake and structural growth, possibly causing decreased weight gain, but increases blood volatile fatty acid concentrations and slightly increases ruminal development. However, feed handling and physical prehension problems in addition to the negative influences on calf growth and intake do not support increasing starter molasses content to 12% of the supplement.     

Effects of corn processing on growth characteristics, rumen development, and rumen parameters in neonatal dairy calves

Neonatal Holstein calves were fed texturized calf starters containing 33% whole (WC), dry-rolled (DRC), roasted-rolled (RC), or steam-flaked (SFC) corn to investigate how corn processing method affects intake, growth, rumen and blood metabolites, and rumen development. In the first experiment, 92 Holstein calves (52 male and 40 female) were started at 2 +/- 1 d of age and studied for 42 d. Starter dry matter (DM) intake was measured and fecal scoring conducted daily. Growth and blood parameter measurements were conducted weekly. A subset of 12 male calves (3/treatment) was euthanized at 4 wk of age and rumen tissue sampled for rumen epithelial development measurements. Experiment 2 consisted of 12 male Holstein calves ruminally cannulated at 7 +/- 1 d of age. Rumen fluid and blood samples were collected during wk 2 to 6. In the first experiment, postweaning and overall starter and total DM intake were significantly higher in calves fed starter with DRC than RC or SFC. Postweaning and overall starter and total DM intake were significantly higher in calves fed starter with WC than SFC. Postweaning average daily gain was significantly greater in calves fed starter with DRC than SFC. Blood volatile fatty acid concentrations were significantly higher in calves fed starter with SFC than in calves fed all other treatments. Papillae length and rumen wall thickness at 4 wk were significantly greater in calves fed starter with SFC than DRC and WC, respectively. In experiment 2, calves fed starter with WC had higher rumen pH and lower rumen volatile fatty acid concentrations than calves fed all other starters. Rumen propionate production was increased in calves receiving starter with SFC; however, rumen butyrate production was higher in calves fed starter with RC. Results indicate that the type of processed corn incorporated into calf starter can influence intake, growth, and rumen parameters in neonatal calves. Calves consuming starter containing RC had similar body weight, feed efficiency, and rumen development but increased structural growth and ruminal butyrate production when compared with the other corn processing treatments.     

Hay intake improves performance and rumen development of calves fed higher quantities of milk

Research to date has suggested that access to forage before weaning can limit rumen development in calves, but no research has yet addressed the role of forage for calves fed higher quantities of milk. This study compared performance and rumen development of calves provided high volumes (equivalent to approximately 20% of calf birth weight) of milk with and without access to hay. At d 3 of age, individually housed calves were randomly assigned to treatment (either ad libitum access to chopped grass hay or no forage; n = 15 calves per treatment, 10 heifers, and 5 bulls). All calves were provided ad libitum access to water and starter throughout the study. All calves were offered 8 L of milk/d from a nipple bottle from d 3 to 35, 4 L/d from d 36 to 53, and 2 L/d until weaning at d 56. Solid feed intake and growth parameters were monitored from d 3 to 70. At d 70, males from both treatments were slaughtered to measure rumen development parameters. Overall dry matter (DM) intake from solid feed did not differ between treatments before wk 5. However, during wk 6 to 10, calves fed forage consumed more total DM (starter plus hay) than did calves fed no forage. Hip and wither height, heart girth, and body barrel at d 3, 56, and 70 did not differ between treatments. Reticulorumen weight was heavier in calves fed hay versus those fed only starter (12.77 ± 1.29 vs. 7.99 ± 0.69 kg with digesta; 1.89 ± 0.05 vs.1.60 ± 0.09 kg without digesta). Body weight without digesta was similar in calves fed forage or no forage. Mean rumen pH was higher in calves fed hay compared with those fed no forage (5.49 ± 0.08 vs. 5.06 ± 0.04). In conclusion, provision of chopped hay to calves fed high volumes of milk can promote solid feed DM intake and rumen development without affecting BW gain.     

Interactions of alfalfa hay and sodium propionate on dairy calf performance and rumen development

The objective of this experiment was to investigate the effects of different levels of alfalfa hay (AH) and sodium propionate (Pro) added to starter diets of Holstein calves on growth performance, rumen fermentation characteristics, and rumen development. Forty-two male Holstein calves (40 ± 2 kg of birth weight) were used in a complete randomized design with a 3 × 2 factorial arrangement of treatments. Dietary treatments were as follows: (1) control = concentrate only; (2) Pro = concentrate with 5% sodium propionate [dry matter (DM) basis]; (3) 5% AH = concentrate + 5% alfalfa hay (DM basis); (4) 5% AH + Pro = concentrate + 5% alfalfa hay + 5% sodium propionate (DM basis); (5) 10% AH = concentrate + 10% alfalfa hay (DM basis); and (6) 10% AH + Pro = concentrate + 10% alfalfa hay + 5% sodium propionate (DM basis). All calves were housed in individual pens bedded with sawdust until 10 wk of age. They were given ad libitum access to water and starter throughout the experiment and were fed 2 L of milk twice daily. Dry matter intake was recorded daily and body weight weekly. Calves from the control, 10% AH, and 10% AH + Pro treatments were euthanized after wk 10, and rumen wall samples were collected. Feeding of forage was found to increase overall dry matter intake, average daily gain, and final weight; supplementing sodium propionate had no effect on these parameters. Calves consuming forage had lower feed efficiency than those on the Pro diet. Rumen fluid in calves consuming forage had higher pH and greater concentrations of total volatile fatty acids and molar acetate. Morphometric parameters of the rumen wall substantiated the effect of AH supplementation, as plaque formation decreased macroscopically. Overall, the interaction between forage and sodium propionate did not affect calf performance parameters measured at the end of the experiment. Furthermore, inclusion of AH in starter diets positively enhanced the growth performance of male Holstein calves and influenced both the macroscopic and microscopic appearances of the rumen wall. These benefits, however, were small when only sodium propionate was offered.     

The passage of lactic acid bacteria from silage into rumen fluid, in vitro studies

Inoculated silages sometimes improve cattle performance, possibly because of probiotic effects of lactic acid bacteria (LAB) silage inoculants. The cause of improved animal performance following feeding with inoculated silage is unclear. One issue in studying this phenomenon is to find out whether LAB pass from silage into the rumen fluid and survive in it. The purpose of the present study was to determine whether LAB from inoculated and uninoculated silages pass into the rumen fluid in vitro. Wheat and corn silages, uninoculated or inoculated with 1 of 10 commercial silage inoculant LAB, were prepared in glass jars. After ensiling, a 2.5-g silage sample was added to 25 mL of heat-sterilized or strained rumen fluid together with 5 g/L glucose, and incubated for 48 h at 39 degrees C. Analysis of the incubated rumen fluid included pH measurement, enumeration of LAB, and determination of lactic acid and volatile fatty acids (VFA). The pH of the rumen fluid decreased during incubation; both heat-sterilized and strained rumen fluid contained large numbers of LAB. The heat-sterilized rumen fluid contained lactic acid in addition to VFA, whereas the strained rumen fluid contained only VFA. The results indicate that LAB pass from silage samples into the rumen fluid in vitro and survive there. Their interactions with rumen microorganisms should be studied further to understand how some silage inoculant LAB exhibit probiotic effects in dairy cattle.     

Short communication: Does early-life administration of a Megasphaera elsdenii probiotic affect long-term establishment of the organism in the rumen and alter rumen metabolism in the dairy calf?

Megasphaera elsdenii is a bacterial species of the rumen that can utilize lactate to produce butyrate, a key volatile fatty acid often implicated in driving calf rumen development. Because lactate is abundant in the rumen of young calves, administration of M. elsdenii to increase butyrate production and thus promote calf rumen development is an appealing possibility. The main objective of this study was to determine whether M. elsdenii administration to calves via oral drench at 14 d of age affected its long-term establishment at 70 d postadministration. Ruminal volatile fatty acid and lactate profiles and blood glucose and β-hydroxybutyrate concentrations were also examined to determine potential influence on rumen metabolism. Six neonatal Holstein heifer calves were blocked on d 1 by body weight (41.3 ± 1.8 kg) and total serum protein (5.23 ± 0.16 g/dL) and assigned to either the M. elsdenii (n = 3) or control (n = 3) treatment groups. On d 14, calves in the M. elsdenii group orally received 25 mL of a commercially available M. elsdenii suspension, whereas calves in the control group received 25 mL of the same product that had been autoclaved. Rumen contents and blood samples were collected weekly from each animal until 84 d of age. The oral administration of M. elsdenii at 14 d did not increase the abundance of M. elsdenii 70 d postdosing, alter rumen fermentation, or change blood metabolites associated with butyrate. These results suggest that a single administration of the M. elsdenii probiotic may not affect the rumen establishment of the organism.     

Short communication: Comparison of pH,volatile fatty acids,and microbiome of rumen samples from preweaned calves obtained via cannula or stomach tube

The objective of this study was to compare rumen samples from young dairy calves obtained via a stomach tube (ST) or a ruminal cannula (RC). Five male Holstein calves (46 ± 4.0 kg of body weight and 11 ± 4.9 d of age) were ruminally cannulated at 15 d of age. Calves received 4 L/d of a commercial milk replacer (25% crude protein and 19.2% fat) at 12.5% dry matter, and were provided concentrate and chopped oats hay ad libitum throughout the study (56 d). In total, 29 paired rumen samples were obtained weekly throughout the study in most of the calves by each extraction method. These samples were used to determine pH and volatile fatty acids (VFA) concentration, and to quantify Prevotella ruminicola and Streptococcus bovis by quantitative PCR. Furthermore, a denaturing gradient gel electrophoresis was performed on rumen samples harvested during wk 8 of the study to determine the degree of similarity between rumen bacteria communities. Rumen pH was 0.30 units greater in ST compared with RC samples. Furthermore, total VFA concentrations were greater in RC than in ST samples. However, when analyzing the proportion of each VFA by ANOVA, no differences were found between the sampling methods. The quantification of S. bovis and P. ruminicola was similar in both extraction methods, and values obtained using different methods were highly correlated (R² = 0.89 and 0.98 for S. bovis and P. ruminicola, respectively). Fingerprinting analysis showed similar bacteria band profiles between samples obtained from the same calves using different extraction methods. In conclusion, when comparing rumen parameters obtained using different sampling techniques, it is recommended that VFA profiles be used rather than total VFA concentrations, as total VFA concentrations are more affected by the method of collection. Furthermore, although comparisons of pH across studies should be avoided when samples are not obtained using the same sampling method, the comparison of fingerprinting of a bacteria community or a specific rumen bacterium is valid.     

Changes in the rumen bacterial community in response to sunflower oil and fish oil supplements in the diet of dairy sheep

Rumen microbial biohydrogenation of dietary unsaturated fatty acids has a major effect on the process of developing healthier dairy products. This study aimed to investigate in vivo the effect of diet supplementation with sunflower (SO) and fish (FO) oils on the rumen bacterial community in dairy sheep. First, 32 lactating ewes, divided in 8 lots of 4 animals each (2 lots per treatment), were fed a high-concentrate total mixed ration supplemented with 0, 2% SO, 1% FO, or 2% SO plus 1% FO. After 21 d, rumen fluid samples were taken from each lot for DNA extraction and fluorescence in situ hybridization (FISH) analysis. In a second experiment, 5 cannulated ewes were first fed the same TMR, with the exception of a higher forage level, and then changed to the same diet supplemented with 2% SO plus 1% FO. After 0, 3, and 10 d, rumen content samples were taken for DNA extraction and FISH analysis (fluid). Total bacteria and the Butyrivibrio group were studied in microbial DNA by terminal RFLP analysis (T-RFLP), and real-time PCR was used to quantify Butyrivibrio bacteria that produce vaccenic acid or stearic acid. In rumen fluid samples, total bacteria and clostridial clusters IX and XIV were analyzed by FISH. Dietary supplementation with SO plus FO seemed to induce important changes in the total bacteria and Butyrivibrio populations, and a high interindividual variation was observed, and the speed of the effect of the lipid supplementation depended on the individual microbial composition. Analysis by T-RFLP and FISH showed increases in cluster IX bacteria with SO plus FO supplementation, presumably Quinella-like microorganisms. The abundances of vaccenic acid- and stearic acid-producing Butyrivibrio relative to total bacteria, estimated by real time PCR, were low (0.28 and 0.18%, respectively, in rumen fluid, and 0.86 and 0.81% in rumen contents) and only that of SA-producing bacteria seemed to be reduced by diets containing FO, although differences were only significant in lactating ewes. The T-RFLP analysis showed a variable effect of lipid supplementation on different bacteria of the family Lachnospiraceae, which includes the cultured bacteria known to be actively involved in rumen biohydrogenation. These results suggest that the latter bacteria do not play a dominant role in this process, and therefore other as-yet-uncultivated microorganisms might be more relevant.     

Technical note: technique for dissection and analysis of the rumen in young calves

This paper discusses a technique used to evaluate rumen development in young calves, including removal, dissection, and analysis of tissue. The method allowed for examination of the different sacs of the rumen (dorsal, ventral, cranial, and caudal) using scanning electron microscopy to measure papillae denseness and histology slides to measure papillae length and width. Computer software was used to produce accurate measurements of papillae. The rumens of young calves were dissected, and samples were taken from the cranial, caudal, ventral, and dorsal sections. Calves were part of a nutrition research study, and dietary treatments did have an effect on development measurements such as length, width, and papillae denseness.     

Analysis of rumen microbial populations in lactating dairy cattle fed diets varying in carbohydrate profiles and Saccharomyces cerevisiae fermentation product

The rumen microbial ecosystem is a critical factor that links diets to bovine physiology and productivity; however, information about dietary effects on microbial populations has generally been limited to small numbers of samples and qualitative assessment. To assess whether consistent shifts in microbial populations occur in response to common dietary manipulations in dairy cattle, samples of rumen contents were collected from 2 studies for analysis by quantitative real-time PCR (qPCR). In one study, lactating Holstein cows (n = 8) were fed diets in which a nonforage fiber source replaced an increasing proportion of forages and concentrates in a 4 × 4 Latin square design, and samples of ruminal digesta were collected at 9-h intervals over 3 d at the end of each period. In the second study, lactating Holstein cows (n = 15) were fed diets with or without the inclusion of a Saccharomyces cerevisiae fermentation product (SCFP) in a crossover design. In this study, rumen liquid and solid samples were collected during total rumen evacuations before and after feeding in a 42-h period. In total, 146 samples of ruminal digesta were used for microbial DNA isolation and analysis by qPCR. Validated primer sets were used to quantify total bacterial and anaerobic fungal populations as well as 12 well-studied bacterial taxa. The relative abundance of the target populations was similar to those previously reported. No significant treatment effects were observed for any target population. A significant interaction of treatment and dry matter intake was observed, however, for the abundance of Eubacterium ruminantium. Increasing dry matter intake was associated with a quadratic decrease in E. ruminantium populations in control animals but with a quadratic increase in E.ruminantium populations in cows fed SCFP. Analysis of sample time effects revealed that Fibrobacter succinogenes and fungal populations were more abundant postfeeding, whereas Ruminococcus albus tended to be more abundant prefeeding. Seven of the target taxa were more abundant in either the liquid or solid fractions of ruminal digesta. By accounting for the total mass of liquid and solid fractions in the rumen and the relative abundance of total bacteria in each fraction, it was estimated that 92% of total bacteria were found in the solid digesta fraction.