Evaluating benefits of neonatal calf gut-originated probiotics, as direct-fed microbials (DFMs), during the weaning transition to improve calf health

Objective II: Evaluate benefits of neonatal calf gut-originated DFM during the weaning transition period to modulate health of male calves, local inflammatory response, and performance.

Animals and Treatments: Thirty-eight, one-day-old male Holstein dairy calves will be obtained and housed at the University of Idaho Dairy Center. Calves received povidone iodine navel dip and were fed 150mg Igg commercial colostrum within 8 h of birth. Within 48-72 hours of birth, serum samples were collected from each calf and total proteins measured to assess failure of passive transfer (FPT). Calves were blocked by BW at birth and randomly assigned to one of four treatments.  Two animals were pulled from the original study due to clinical illness and the need for antibiotic therapy during the study.

This study examined the effects of probiotic supplementation using a calf gut-originated direct fed Lactobacillus agili, L. delbrueckii, L. mucosae, and L. rueteri 1x10⁹ CFU/d (control vs supplemented) and weaning pace (abrupt vs gradual) on feed intake, growth, vitals, perineal area, and hematological values in dairy calves in a 2 x 2 factorial arrangement of treatments. Holstein bull calves (n=38) were blocked by birth weight and randomly assigned to one of four treatments: Abrupt-Control (AC; n=10), Abrupt-Supplemented (AP; n=10), Gradual-Control (GC; n=9), and Gradual-Supplemented (GP; n=9). 3.4L milk replacer (22% CP, 22% fat) was fed twice daily. Water, calf starter (18% CP) and chopped alfalfa were provided ad libitum, with intake of milk replacer, calf starter, and alfalfa recorded daily. The probiotic was supplemented in milk replacer once daily, for 7 days, beginning 4 days prior to weaning. Abrupt weaning occurred over days 54-57 in 3 incremental step-downs of 1.13L. Gradual weaning occurred over days 49-63 in 7 incremental step-downs of 0.49L.  Health parameters including fecal scores, hydration status, respiration rate, heart rate and temperature as well as body weights were measured weekly. Blood hematology was measured at days 3 and 7 of age, onset of treatment and the day after completion of weaning.  Fresh fecal samples from the abrupt-weaned calves will be collected on d 3, d 7, onset of treatment, and day after completion of weaning.  Fresh fecal samples and sterile fecal swabs were collected and placed into sterile vials and immediately stored at -20°C until further processing to determine total bacteria, total coliform, and bacterial shedding (Laegreid et al., 1999; Rosa et al., 2021).

One day after weaning, the animals were humanely euthanized and samples of feces, rumen, duodenum, jejunum, ileum, large intestine, liver, mesenteric lymph nodes, abdominal and perirenal adipose tissues were obtained.  All samples were placed in 2cc cyro tubes, immersed in liquid nitrogen and stored in -80 to determine microbiome (feces) and gene expression of inflammatory markers (tissues). Tissues will also be preserved (Mavangira et al., 2015) to determine oxylipids profile.

Analyses: A complete blood count will be determined in a hematology analyzer (VETSCAN HM5, Hematology Analyzer, Zoetis, Parsippany, NJ). Serum NEFA and albumin concentration will be determined via enzymatic-colorimetric assay, according to manufacturer instructions (Wako Chemical, Dallas, TX; Sigma Aldrich, St. Louis, MO). Serum β-hydroxybutyrate, haptoglobin, and glucose concentration will be determined through colorimetric methods established by our team, as described by Tsai (2021). Serum amyloid A and cortisol will be determined with a commercial ELISA assay (Sigma Aldrich, St. Louis, MO; Arbor Assays, Ann Arbor, MI). Serum total protein will be determined using a refractometer, as described by Hernandez et al. (2016). On d 49, d 57 (the abrupt-wean), and d 63 (the gradual-wean). PMN and PBMC will be isolated by gradient centrifuging using Histopaque 1077 and 1119 (Sigma Aldrich, St. Louis, MO) and washed with Hank's Balanced Salt Solution (Watts et al., 2013; Tsai et al., 2017). PMN and PBMC will be used to determine the gene expression of inflammatory markers: total RNA from PMN and PBMC will be extracted using NucleoSpin® RNA kit (Macherey Nagel, Düren, Germany) according to the manufacturer’s protocol. The quality of samples and cDNA synthesis will be carried out as previously described by our team (Tsai, 2021). Real-time reversetranscribed PCR will be carried out in a ViiA 7 Real-Time PCR system (Applied Biosystems, Foster City, CA) using custom-designed TaqMan MGB probes (Rezamand and McGuire, 2011; Rezamand et al., 2012), on targeted genes (IL-1, IL-6, IL-8, TNF-α, INF-γ, ICAM, and NF-κB). 

Analysis: Total RNA extraction and cDNA synthesis will be carried out on PMN and tissue samples following the procedures described in the previous section. Real-time reverse-transcribed PCR will be carried out in a ViiA 7 Real-Time PCR system (Applied Biosystems, Foster City, CA) as detailed in Objective I. Statistical analysis: The effect of weaning pace and bDFM will be compared as main fixed effects. In repeated measures, the treatment and time effect will be compared using best variance/covariance structures with the lowest AIC. Treatment effects will be analyzed in ANOVA using the mixed-model procedure of SAS (9.4) with treatment as fixed effect, calf and BW at birth as random effects, and day as a repeated measure, when appropriate. Significant differences will be declared when P < 0.05 and trends at P ≤ 0.10.

Objective I: Evaluate benefits of neonatal calf gut-originated bDFM during the weaning transition to improve heifer calves’ health and performance Location: Commercial Dairy Farm, Dunrenton and University of Idaho commercial Dairy

Animals and Treatments: Thirty-six, one-day-old female Holstein dairy calves will be selected from a collaborating commercial dairy farm. Newborn calves will be cared for according to the farm’s management protocol; separation from dams after calving; navel dipping using povidone iodine; and feeding of 6 L of maternal colostrum within 8 h of birth. Between 48 -72 h of birth serum will be collected from each calf and total proteins measured to assess failure of passive transfer (FPT). Calves will be blocked by body weight (BW) at birth and randomly assigned to one of four treatments (n=10 per treatment) in a 2 × 2 factorial arrangement of treatments in a randomized complete block design. One factor will be weaning pace (abrupt vs. gradual), and the other factor will be DFM supplementation (0 vs. 1 x 109 CFU). Weaning will either be abrupt (over 2 d) from 55 – 57 d of age in 3 incremental step downs, or gradual (over 14 d) from d 49 – 63 in 7 incremental step downs. With each weaning group, half the calves will be fed bDFM and the other half will be fed a placebo (ground corn). For abrupt-weaned calves, bDFM will be supplemented beginning at d 48, continued through the weaning process and discontinued at d 57. For the gradual-weaned calves, bDFM will begin on d 42, continue through the gradual weaning process and be discontinued at d 63. The treatment groups are as follows:

1) Gradual-Control: gradual weaning without bDFM supplementation

2) Abrupt-Control: abrupt weaning without bDFM supplementation

3) Gradual-bDFM: gradual weaning with bDFM supplementation (1 × 109 CFU/d)4)

Control-bDFM: abrupt weaning with bDFM supplementation (1 × 109 CFU/d) Feeding and Management: Calves will be housed in individual-covered hutches, bedded on sand, and with free-access to water. From d 2 to d 7, animals will be fed by bottle twice daily up to 8 L of transition milk replacer (consisting of 50% milk replacer at 150 g/L and 50% colostrum replacer at 170 g/L). After d 7, calves will be fed twice daily, up to 1,200 g/d of milk replacer (26% CP, 16% fat). Total milk consumption will be controlled across all treatments. During this period, calves will have free access to calf starter and chopped alfalfa hay. Daily milk replacer and solid feed intake will be recorded in addition to weekly BW to assess average daily gain and feed efficiency. Sampling: Once a week fecal scores, hydration status, respiration rate, heart rate, and rectal temperatures will be recorded to assess health status as previously described (Wolfe et al., 2022). Any calf requiring antibiotic therapy during the experiment will be removed from the study. Blood samples will be collected at approximately 0900 h on d 3, d 7, d 48 (onset of bDFM treatment), d 57 (conclusion of abrupt weaning), d 42 (onset of bDFM treatment) and d 63 (conclusion of gradual weaning). Samples will be collected in EDTA blood tubes for plasma separation and other tubes without additive for serum separation. Blood samples will be centrifuged at 800 × g for 10 min. Serum will be stored at -20°C to determine blood metabolites and plasma will be stored at -80°C to determine oxylipids.  Fresh fecal samples from the abrupt-weaned calves will be collected on d 3, d 7, d 48, and d 57 and collected from the gradual-weaned calves on d 3, d 7, d 42, and d 63. One additional sample will be collected from both groups on d 120. Fresh fecal samples will be collected via rectal stimulation into sterile vials and immediately stored at -20°C until further processing to determine total bacteria, total coliform, and bacterial shedding (Laegreid et al., 1999; Rosa et al., 2021).