Maternal pre- and postpartum supplementation of a Bacillus-based DFM enhanced cow and calf performance

Abstract

This study evaluated the effects of maternal supplementation of a Bacillus-based direct-fed microbial (DFM) on the physiology and growth performance of Bos indicus-influenced cow–calf pairs. On day 0 (~139 d before expected calving date), 72 fall-calving, Brangus crossbred beef heifers (20 to 22 mo of age) pregnant with first offspring were stratified by their initial body weight (BW; 431 ± 31 kg) and body condition score (BCS; 6.0 ± 0.36; scale 1 to 9), and randomly allocated into 1 of 12 bahiagrass pastures (1 ha and six heifers per pasture). Treatments were randomly assigned to pastures (six pastures per treatment) and consisted of heifers supplemented with 1 kg/d of soybean hulls (dry matter, DM) that was added (BAC) or not (CON) with DFM containing Bacillus subtilis and B. licheniformis (Bovacillus; Chr. Hansen A/S, Hørsholm, Denmark). Treatments were provided from days 0 to 242 (139 ± 4 d prepartum to 104 ± 4 d postpartum). Calves were weaned on day 242 (96 ± 30 d of age) and then allocated into 1 of 16 drylot pens and fed the same concentrate at 3.25% of BW (DM) until day 319. Maternal treatment effects were not detected (P ≥ 0.29) for herbage allowance and forage chemical composition. Heifer BCS on days 39 and 63 tended (P ≤ 0.09) to be greater for BAC vs. CON heifers, whereas heifer BCS on day 91 was greater (P = 0.01) for BAC vs. CON heifers. Heifer BCS did not differ (P ≥ 0.20) between treatments on days 179 and 242. Plasma glucose concentration did not differ from days 0 to 63 (P ≥ 0.14) but were greater (P < 0.01) on day 179 and tended (P = 0.09) to be greater on day 242 for BAC vs. CON heifers. Calf BW at birth, ADG from birth to weaning, and BW at weaning did not differ (P ≥ 0.19) between treatments, but calf BW at drylot exit (day 319) was greater (P = 0.05) for BAC vs. CON calves. Maternal treatment effects were not detected (P ≥ 0.42) for calf serum concentration of IgG at birth and postvaccination plasma concentrations of glucose, cortisol, and haptoglobin. Serum titers against bovine respiratory syncytial virus (BRSV) were greater (P = 0.04) for BAC vs. CON calves on day 287, whereas seroconversion against parainfluenza-3 virus (PI-3) was greater (P < 0.01) for BAC vs. CON calves on day 271. Thus, maternal supplementation of a Bacillus-based DFM increased prepartum BCS gain and postpartum plasma glucose concentration of heifers and led to positive carryover effects on postweaning BW gain and humoral immune response in their offspring.

Bacillus-based direct-fed microbials offered during late gestation and early lactation of Bos indicus-influenced beef heifers increased maternal body condition score at calving and led to positive effects on growth and humoral immune function in their offspring.

Introduction

In beef and dairy cattle nutrition, dietary inclusion of direct-fed microbials (DFM) has been used as an alternative to antibiotic feeding and to modulate ruminal fermentation, promote the establishment of beneficial microorganisms, and enhance fiber and overall nutrient digestibility (Krehbiel et al., 2003; Pan et al., 2022; Cappellozza et al., 2023). As a DFM, Bacillus spp. may inhibit potentially harmful pathogens (Copani et al., 2020), stimulate biofilm and mucin formation (Santano et al., 2020; Segura et al., 2020), enhance production of a wide variety of digestive enzymes (Elshaghabee et al., 2017; Luise et al., 2022), and anti-inflammatory molecules, improving intestinal barrier function and integrity (Rhayat et al., 2019). The gastrointestinal tract of mammalians is sterile in utero but undergoes rapid microbial colonization during and after birth (Fanaro et al., 2003) and can be regulated by maternal and offspring diet after birth (Fouhy et al., 2012; Rodriguez et al., 2015). Neonatal gut colonization is crucial for the developing gut and naïve immune system and has long-term health effects later in life (Hansen et al., 2012).

To our knowledge, studies evaluating the impacts of maternal supplementation of Bacillus-based DFM during critical periods of fetal and calf development (i.e., gestation and early postpartum periods) on overall cow and calf performance are lacking, particularly for Bos indicus-influenced beef cattle, which exhibit intrinsic differences in physiology and metabolism compared to Bos taurus beef cattle (Cooke et al., 2020). We hypothesized that maternal supplementation of a Bacillus-based DFM during gestation and early lactation would alter maternal physiology and enhance maternal body condition score (BCS) before calving, leading to positive carryover effects on the growth and immune function of their offspring. Therefore, the objective of this study was to evaluate the effects of maternal supplementation of a Bacillus-based DFM during prepartum and postpartum periods on physiology, immune function, and growth performance of their Bos indicus-influenced beef offspring.

Materials and Methods

The experiment was conducted at the University of Florida, Institute of Food and Agricultural Sciences, Range Cattle Research and Education Center, Ona, FL (27°23ʹN and 81°56ʹW) from May 2022 to November 2023. All animals used in this experiment were cared for by practices approved by the University of Florida—Institute of Animal Care and Use Committee (protocol #202111585).

Animals and diets

Maternal management

On day 0 (~139 d before expected calving date), 72 fall-calving, Brangus crossbred beef heifers (25.0% to 37.5% Bos indicus; 20 to 22 mo of age) pregnant with their first offspring were blocked according to their previous reproductive management into heifers bred by artificial insemination (n = 24; 4 Angus bulls) or natural 90-d breeding (n = 48; 4 Angus bulls). Within each block, heifers were stratified by their initial BW (431 ± 31 kg) and BCS (6.0 ± 0.36; scale 1 to 9), and randomly allocated into 1 of 12 bahiagrass pastures (1 ha and six heifers per pasture). Treatments were randomly assigned to pastures (six pastures per treatment) and consisted of heifers supplemented with 1 kg/d of loose soybean hulls dry matter (DM; Table 1) added (BAC) or not (CON) with a DFM supplement containing a combination of Bacillus subtilis and B. licheniformis (DSM5750 and DSM5749, respectively; Bovacillus; Chr. Hansen A/S, Hørsholm, Denmark) from days 0 to 242 (139 ± 4 d prepartum to 104 ± 4 d postpartum). The exact ingredient composition of the Bacillus-based DFM mixture is proprietary, but the commercial mixture was blended with soybean hulls (loose form) once weekly using a vertical mixer in amounts to provide on average 3 g of BAC per heifer daily and achieve an expected total colony forming unit (CFU) of 6.6 × 10⁹ per head daily. Each supplement (CON and BAC) was hand-delivered daily to each respective pasture at 0800 hours in plastic feed bunks located 1 m above ground to avoid calf consumption of maternal supplements. From days 0 to 242, heifers (and their first offspring) remained in their respective pasture assignment and each pasture was provided a complete salt-based trace mineral and vitamin supplement (17% Ca, 4% P, 21% NaCl, 1% Mg, 60 mg/kg Co, 1,750 mg/kg Cu, 350 mg/kg I, 60 mg/kg Se, 5,000 mg/kg Zn, 441 IU/g Vitamin A, 33 IU/g Vitamin D₃, and 0.44 IU/g of Vitamin E; University of Florida Cattle Research Winter Mineral; Vigortone, Brookville, OH) once every Monday at 0800 hours in amounts to provide on average 51 g of the trace mineral and vitamin supplement per heifer daily.

Table 1.

Average nutritional composition¹ of loose soybean hulls supplement (days 0 to 242) and limpograss hay offered to heifers (days 137 to 312), and limpograss hay (days 242 to 319) and concentrate offered to calves (days 258 to 319)

Item1	Soybean hulls, Heifers	Limpograss hay, Heifers	Limpograss hay, Calves	Concentrate, Calves
CP, %	14.0	3.1	3.3	21.0
ADF, %	38.4	40.4	38.0	32.6
NDF, %	48.0	80.2	80.0	45.5
TDN2, %	66.0	53.0	50.3	72.5
NEm3, Mcal/kg	1.44	0.95	0.95	1.71
NEg3, Mcal/kg	0.86	0.40	0.40	1.09
Ca, %	0.54	0.19	0.19	1.38
P, %	0.41	0.15	0.19	0.49
Mg, %	0.27	0.17	0.14	0.33
K, %	1.70	0.40	0.66	1.58
Na, %	<0.01	0.05	0.03	0.10
S, %	0.23	0.08	0.11	0.45
Fe, mg/kg	373	52	84	306
Zn, mg/kg	45	38	31	55
Cu, mg/kg	9.5	5.5	5.5	8.0
Mn, mg/kg	30	23	25	26
Mo, mg/kg	1.95	<0.01	0.33	0.65

¹Samples of limpograss hay offered to heifers were collected monthly from days 137 to 312, whereas samples of limpograss hay and concentrate offered to calves were collected weekly from days 258 to 319. All pooled samples were then dried at 60 °C in a forced-air oven for 72 h and then sent in duplicates to a commercial laboratory (Dairy One Forage Laboratory, Ithaca, NY) for wet chemistry analyses.

²Calculated as described by Weiss et al. (1992).

³Calculated using the equations proposed by the NASEM (2016).

All pastures contained one artificial shade structure consisting of ultraviolet-stabilized, high-density polyethylene fabric (1-mm thickness; black color; Agfabric, Corona, CA) located at the center of each pasture and set at 3 m height using a stainless steel tubing frame (3 × 6 m) to provide 4.5 m² of shade area per heifer (Turner, 2000; Van Laer et al., 2014). The shade fabric provided 70% protection against direct solar radiation and decreased ground surface temperature under the shade structure by 6 ± 0.4 °C (Izquierdo et al., 2023a). All pastures did not have access to natural shade. Heifers were monitored daily for calving and their first offspring was processed (weighed, tagged, and castrated if male) within 24 h after birth but after calf consumption of maternal colostrum. Calves were early weaned on day 242 (on average at 96 ± 30 d of age), except for six calves (n = 4 CON and 2 BAC), which were less than 52 d of age at the time of weaning and, therefore, remained with their mothers. Early weaning was implemented for calves older than 52 d of age (Moriel et al., 2014) to increase the reproductive success of primiparous Bos indicus-influenced beef cows (Arthington and Kalmbacher, 2003) and to avoid treatment-induced confounding effects on calf postweaning total DM intake (Izquierdo et al., 2023a). After weaning, heifers were combined into a single bahiagrass pasture (20 ha) and placed with three Brangus bulls (4 ± 2 years of age) from days 242 to 312. Bulls were confirmed fertile following breeding soundness exam conducted by a trained veterinarian 90 d before the start of the study. No visual signs of injury or difficulty in mounting were detected on all bulls. Pasture herbage mass on day 242 was limited (< 1,000 kg DM/ha), and hence, limpograss (Hemarthria altissima) hay was provided ad libitum for heifers and bulls from days 137 until 312 (Table 1). Weekly supplementation of 12.7 kg per heifer of sugarcane (Saccharum officinarum) molasses and urea (22% CP and 75% total digestible nutrients [TDN]; DM basis) was provided from days 242 until 312. The weekly molasses-based supplement amount was split in half and then offered every Monday and Thursday at 0800 hours in plastic tanks located 1 m above ground. Calving percentage of the first offspring (calves in utero when maternal treatments were provided) and second offspring (calves conceived from days 242 to 312) consisted of heifers that were diagnosed as pregnant and delivered a live calf at birth. The percentage of heifers pregnant with their second offspring was assessed on day 404 via rectal palpation by a trained veterinarian. Heifers that did not deliver a live calf at birth (n = 3 CON and 3 BAC) and heifer-calf pairs that were not early weaned (n = 6; as described above) were not used for blood collections and were removed from all statistical analyses of any heifer data.

First offspring management

Calves that were early weaned on day 242 (n = 60) remained in a single partially covered drylot pen (20 × 40 m; 1.5 m of bunk space per calf) for 16 d to overcome the stress of weaning. During this 16-d period, calves were provided limpograss hay ad libitum (Table 1) and 0.50 kg/d of a commercial supplement (guaranteed analysis, as fed: 14% CP, 1.0% fat, 18% fiber, 0.75% Ca, 0.40% P, and 0.40% NaCl, Purina, Gray Summit, MO). On day 258, calves were transferred to 1 of 16 pens (4 to 6 calves/pen; 15 × 5 m) in a drylot facility with covered concrete feed bunk using the same previous pasture distribution assigned to their dams. Calves were gradually adapted to concentrate (Table 1) by increasing the daily concentrate DM offered by 0.25% to 0.50% of BW/d from days 258 to 265, and then limit-fed the same concentrate at 3.25% of BW until day 319 (DM basis; Moriel et al., 2020). Calves were also offered limpograss hay at 0.50% of BW (DM basis) and a complete salt-based mineral supplement (same as described above; University of Florida Cattle Research Winter Mineral) from days 258 to 319. The mineral supplement was hand-mixed daily into the concentrate in equivalent amounts according to the number of calves in each respective pen to provide 51 g of mineral supplement per calf daily. On day 258, each calf received an oral drench of fenbendazole (5 mg/kg of BW; Safe-guard, Merck Animal Health, Summit, NJ) and vaccination against pathogens associated with bovine respiratory disease (2 mL subcutaneous; Bovi Shield Gold One Shot; Zoetis Inc., New York, NY) and Clostridium (2 mL subcutaneous; Ultrabac 7, Zoetis Inc., New York, NY). On day 272, each calf received booster vaccinations of Bovi Shield Gold 5 and Ultrabac 8 (2 mL subcutaneous; Zoetis Inc.). The vaccination protocol utilized herein was selected as our model to elicit an inflammatory response (Moriel et al., 2020; Izquierdo et al., 2023a). Calves were monitored daily by trained personnel, but no signs of health problems were detected.

Data and sample collection

Forage, supplement, and ambient conditions

Herbage mass (Gonzalez et al., 1990) and hand-plucked samples of pastures were obtained on days 0, 39, 63, 91, 119, and 137 to determine the concentrations of crude protein (CP) and in vitro digestible organic matter (IVDOM). Herbage allowance was calculated as the herbage mass divided by the respective total BW (heifer and calf) of each pasture on days 0, 39, 63, 91, 119, and 137 (Sollenberger et al., 2005). The evaluations of herbage mass and allowance were ceased on day 137 when ad libitum limpograss hay was provided. Samples of soyhulls (prior to mixing the commercial DFM mixture), limpograss hay, and sugarcane molasses offered to heifers were collected monthly from days 0 to 242, 137 to 312, and 242 to 312, respectively. Samples of concentrate-based diet and hay offered to calves were collected weekly from days 242 to 319. All forage and supplement samples were dried in a forced-air oven at 56 °C for 72 h following collection, and then ground to pass a 4-mm and 1-mm stainless steel screen, respectively, in a Wiley mill (Model 4, Thomas-Wiley Laboratory Mill, Thomas Scientific, Swedesboro, NJ). Samples of CON and BAC supplements were collected before the start of the study (day −14) and then weekly until day 242. Those samples were then sent to the laboratory (Chr. Hansen Inc., Milwaukee, WI) immediately after sampling to determine the concentrations of Bacillus spores (CFU/g of supplement), according to methodology reported by the European standard EN-15784 (2021), and to ensure that target Bacillus-based DFM mixture inclusion in BAC supplements and a nonsignificant presence (< 1.0 × 10⁵ CFU/g of supplement) of Bacillus spores in CON supplements were achieved. The commercial product was stored in the laboratory under controlled temperature and dry environment throughout the study.

Ambient environmental dry bulb temperature (T_db) and relative humidity (RH) were measured daily every 60 min from days 0 to 242 using a HOBO-U23 data logger (Onset Computer Corp., Bourne, MA) placed in a shaded area 3 m above ground (Moriel et al., 2022; Izquierdo et al., 2023a). The temperature humidity index (THI) was utilized as an indicator of heat stress (Dikmen and Hansen, 2009) and calculated according to NRC (1971): THI = (1.8 × T_db + 32) − [(0.55 − 0.0055 × RH) × (1.8 × T − 26)]. Average, minimum, and maximum THI were calculated daily but averaged and reported at 14-d intervals from days 0 to 242 (Figure 1). Heifer activity was evaluated by observing the percentage of heifers standing under shade, standing outside shade, laying under shade, laying outside shade, drinking water, and grazing at 1300 and 1700 hours (timeframe of highest THI levels within day) on days 0, 40, 64, 92, and 180 (24 h after heifers were worked in the chute and back to each respective pasture; Izquierdo et al., 2023a).

Figure 1.

Minimum, average, and maximum THI were measured daily but reported at 14-d intervals from days 0 to 242. Calculated according to NRC (1971) as: THI = (1.8 × T_db + 32) − ([0.55 − 0.0055 × RH] × [1.8 × T − 26]).

Heifers

Individual full BW and BCS of heifers were assessed once at 0800 hours (before morning supplementation) on days 0, 39, 63, 91 (near calving), 179 (after calving), 242 (start of the breeding season), and 312 (end of the breeding season). Heifer BCS was recorded by the same two trained technicians throughout the study (Wagner et al., 1988). Individual shrunk BW of pregnant heifers was not implemented because disturbed feeding behavior and prenatal physiological stress could interfere with fetal development and calf performance following birth (Littlejohn et al., 2016; Carroll et al., 2021). Blood samples were collected (10 mL) from all heifers 3 to 4 h after supplement consumption, via jugular venipuncture into sodium-heparin (158 USP) containing tubes (Vacutainer, Becton Dickinson, Franklin Lakes, NJ), on days 0, 39, 63, 179, and 242 to assess the plasma concentrations of cortisol and glucose.

First offspring

Individual calf shrunk BW, following 12 h of feed and water withdrawal, was recorded on days 242 (early weaning), 258 (drylot entry), and 319 (drylot exit) to calculate overall average daily gain (ADG) and gain:feed (G:F). Individual full BW of calves were obtained once at 0800 hours (before morning feeding) every 14 d from days 258 until 319 to adjust the concentrate DM amount offered to calves. Within 24 h after birth, blood samples (10 mL) were collected from 1 steer and 1 heifer per pasture via jugular venipuncture into tubes containing no additives (Vacutainer, Becton Dickinson) to determine serum concentration of immunoglobulin G (IgG). After weaning, blood samples (10 mL) were collected from all calves in drylot on days 258, 271, 272, 274, and 278, via jugular venipuncture into sodium-heparin (158 USP) containing tubes (Vacutainer, Becton Dickinson, Franklin Lakes, NJ) to determine the plasma concentrations of cortisol and haptoglobin. A second set of blood samples (10 mL) was collected from all calves in drylot via jugular venipuncture on days 271, 287, and 319 into tubes containing no additives (Vacutainer, Becton Dickinson) to determine serum titers against bovine respiratory syncytial virus (BRSV), bovine viral diarrhea virus type 1a (BVDV-1a), infectious bovine rhinotracheitis virus (IBR), and parainfluenza virus (PI-3). All blood samples were placed on ice immediately after collection, and then centrifuged at 1,200 × g for 25 min at 4 °C. Serum and plasma samples were harvested and then frozen at −20 °C until laboratory analysis.

Daily intake of hay, concentrate, and total were calculated daily from days 258 to 319 by subtracting the amount of hay and concentrate refused in each pen at 0800 hours (before morning feeding) from the respective amount of hay and concentrate offered in the previous day. Samples of hay and concentrate offered and refused were collected daily from each pen from days 258 to 319 and then dried at 60 °C in a forced-air oven for 72 h to determine the respective DM concentration of each sample and calculate daily DM intake (DMI) of hay, concentrate, and total (kg/day). Daily DMI of hay, concentrate, and total were also calculated as percentage of BW using the respective mean BW from days 258 to 319 of each pen.

Laboratory analyses

Samples of CON and BAC supplements and limpograss hay offered to heifers from days 0 to 242 and 137 to 312, respectively, and limpograss hay and concentrate offered to calves from days 258 to 319 were pooled across the entire respective sampling period, and then analyzed in duplicates by a commercial laboratory (Dairy One Forage Laboratory, Ithaca, NY) to determine the concentrations of CP (method 984.13; AOAC, 2006), TDN (Weiss et al., 1992), NEm, and NEg (NASEM, 2016; Table 1). Pasture sample analyses were performed in duplicates at the University of Florida Forage Evaluation Support Laboratory to assess the concentration of N (Gallaher et al., 1975) and IVDOM (Moore and Mott, 1974).

Serum concentration of IgG was evaluated using a bovine-specific ELISA kit (E11-118; Bethyl Laboratories, Inc., Montgomery, TX) with assay sensitivity at 0.69 ng/mL and an intra- and interassay CV of 4.22% and 6.80%, respectively. Plasma glucose concentration was determined using quantitative colorimetric kits (#G7521; Pointe Scientific Inc., Canton, MI). Plasma haptoglobin concentration was calculated by assessing the haptoglobin–hemoglobin and differences in peroxidase activity (Cooke and Arthington, 2013). Plasma cortisol concentration was determined using radioimmunoassay (#07221106, MP Biomedicals, Santa Ana, CA; Colombo et al., 2019). Intra- and interassay CV for glucose, haptoglobin, and cortisol were 4.12% and 4.99%, 4.17% and 9.20%, and 3.69% and 7.66%, respectively.

Serum antibody titers were determined by the Oklahoma Animal Disease and Diagnostic Laboratory using neutralization test panel developed for BSRV, BVDV-1a, PI-3, and IBR viruses (Rosenbaum et al., 1970). Serum titer of each virus was reported as the log₂ of the greatest dilution of serum that provided whole cell protection (lowest and greatest dilutions tested were 1:4 and 1:1,024, respectively). Samples with serum neutralization titers <4 were categorized as negative and assigned a value of 0, whereas samples with serum neutralization titer ≥4 were categorized as positive and assigned a value of 1. All assigned values (0 or 1) were then used to determine the percentage of calves with positive seroconversion (Moriel et al., 2020).

Statistical analyses

Pasture was the selected experimental unit for all statistical analyses. Pasture (maternal treatment) and heifer (pasture) or calf (pasture) were considered the random effects in all statistical analyses, excluding pasture data which considered pasture (maternal treatment) as the random effect. Nonbinary data were analyzed as a randomized complete block study using the MIXED procedure of SAS (SAS Institute Inc., Cary, NC, USA, version 9.4) with Satterthwaite approximation to determine the denominator degrees of freedom for the test of fixed effects. Herbage mass and allowance, heifer and calf BW, heifer BCS, and all blood data were analyzed as repeated measures and tested for fixed effects of maternal block, maternal treatment, day of the study, and all resulting interactions. Birth BW, ADG, G:F, and DMI (hay, concentrate, and total) of the first offspring were tested for fixed effects of maternal block, maternal treatment, and maternal treatment × block. Binary data (percentage of heifers calving, pregnant on day 404, male calves at birth, and calf-positive seroconversion) were analyzed as a randomized complete block design using the GLIMMIX procedure of SAS. Calf-positive seroconversion data were log₂-transformed before statistical analyses. All heifer reproductive data were tested for fixed effects of maternal block, maternal treatment, and maternal treatment × block. Calf-positive seroconversion data were log₂ transformed and then analyzed as repeated measures and tested for fixed effects of maternal block, maternal treatment, day of the study, and resulting interactions. Compound symmetry covariance structure was utilized in all repeated measures analyses because it generated the lowest Akaike information criterion, and therefore, heifer (pasture) were removed from the random effects in their respective statistical analyses and included as subjects. Heifer baseline plasma data obtained on day 0, calf age, and calf sex were initially included as covariates in all respective statistical analyses. Plasma data on day 0 remained in the model as covariate (P < 0.01) for all blood data, whereas calf ADG from birth to early weaning was covariate-adjusted for calf age (P = 0.07). Calf sex was removed from the model of all statistical analyses of calf growth performance (P ≥ 0.14). All results are reported as least-square means. Data were separated using PDIFF if a significant F-test was detected. Significance was set at P ≤ 0.05, and tendencies were noted if P > 0.05 and ≤ 0.10.

Results

Effects of reproductive management block × maternal treatment and reproductive management block were not detected for any variable analyzed in the study (P ≥ 0.11). Consequently, effects of reproductive management block and resulting interactions were removed from the model in all statistical analyses.

Pasture and maternal supplement

Effects of day of the study, but not maternal treatment × day of the study and maternal treatment (P ≥ 0.29), were detected (P < 0.01) for herbage mass, herbage allowance, forage CP, and IVDOM. Herbage mass did not differ between days 0 and 39 (P = 0.16), increased from days 39 to 63 (P < 0.01), decreased on day 91 (P = 0.05), peaked on day 119 (P < 0.01), and decreased on day 137 (P < 0.01; Table 2). Herbage allowance did not differ between days 0 and 39 (P = 0.11), gradually increased from days 63 to 119 (P ≤ 0.05) and decreased on days 137 (P < 0.01; Table 2). Forage IVDOM gradually decreased from days 0 to 137 (P ≤ 0.05; Table 2). Forage CP did not differ between days 0 and 39 (P = 0.83), decreased on day 63 (P ≤ 0.02), and did not differ among days 91, 119, and 137 (P ≥ 0.27; Table 2).

Table 2.

Herbage mass and allowance, CP, and IVDOM of bahiagrass pastures (six pastures per treatment; 1 ha and six heifers per pasture)¹.

Item	Maternal treatment2		SEM	P-value	Day of the study						SEM	P-value
	CON	BAC		Treatment	0	39	63	91	119	137		Day
Herbage mass, kg DM/ha	2,583	2,676	55.2	0.29	1,671a	1,387a	2,729b	2,348c	4,362e	3,279d	136	<0.01
Herbage allowance, kg DM/kg BW	0.97	0.99	0.038	0.80	0.65a	0.51a	0.99c	0.84b	1.56e	1.33d	0.059	<0.01
CP, % of DM	12.6	12.6	0.32	0.86	14.8c	14.7c	12.9b	11.5a	10.9a	10.5a	0.40	<0.01
IVDOM, %	44.1	43.9	0.87	0.88	55.0f	52.8e	48.6d	39.2c	36.0b	32.1a	1.02	<0.01

¹Herbage mass and hand-plucked samples of pastures were collected on days 0, 39, 63, 91, 119, and 137. Herbage allowance was calculated as the herbage mass divided by the total BW (heifer + calf) in each pasture. Effects of maternal treatment × day of the study were not detected (P ≥ 0.29) for herbage mass, herbage allowance, CP, and IVDOM.

²Treatments were randomly assigned to pastures (six pastures per treatment) and consisted of heifers assigned to receive soybean hulls supplementation (1 kg/) added or not with a DFM supplement (3 g/d) containing a combination of Bacillus subtilis and B. licheniformis (6.6 × 10⁹ CFU/d) from days 0 to 242.

^a-fWithin a row, means without a common superscript differ (P ≤ 0.05).

Maternal performance

Days on treatment during prepartum (142 ± 4 d vs. 135 ± 4 d for CON and BAC heifers, respectively) and postpartum periods (100 ± 4 d vs. 107 ± 4 d for CON and BAC heifers, respectively) did not differ (P = 0.29) between maternal treatments. Hence, maternal treatments were provided on average for 139 ± 4 d prepartum and 104 ± 4 d postpartum.

Effects of maternal treatment × day of the study were detected (P < 0.01) for heifer BCS but not (P = 0.65) for heifer BW (Table 3). Heifer BCS on days 0, 179, 242, and 312 did not differ (P ≥ 0.20) between maternal treatments. Heifer BCS on days 39 and 63 tended (P ≤ 0.09) to be greater, whereas heifer BCS on day 91 was greater (P = 0.01) for BAC compared to CON heifers (Table 3). Heifer BW change from days 0 to 91, 91 to 242, and 242 to 312 did not differ between maternal treatments (P ≥ 0.20). Heifer BCS change from days 0 to 91 was greater (P = 0.05) for BAC vs. CON heifers, whereas heifer BCS change from days 91 to 242 and 242 to 312 did not differ (P ≥ 0.28) between maternal treatments (Table 3).

Table 3.

Growth and reproductive performance of heifers allocated into 1 of 12 bahiagrass pastures (1 ha and six heifers per pasture) and then assigned to receive soybean hulls supplementation (1 kg/d) added (BAC) or not (CON) with a DFM supplement (3 g/d) containing a combination of Bacillus subtilis and B. licheniformis (6.6 × 10⁹ CFU/d) from days 0 to 242 (six pastures per treatment).

Item	Maternal treatment1		SEM	P-value2
	CON	BAC
Heifer BCS
Day 0	6.00	6.05	0.083	0.63
Day 39	6.25	6.46	0.083	0.09
Day 63	6.12	6.33	0.083	0.07
Day 91 (near calving)	6.09	6.37	0.083	0.01
Day 179	5.21	5.36	0.083	0.20
Day 242 (start of the breeding season)	5.04	5.04	0.083	0.95
Day 312 (end of the breeding season)	5.20	5.25	0.083	0.66
Heifer BCS change
Days 0 to 91	0.05	0.36	0.102	0.05
Days 91 to 242	−1.06	−1.33	0.167	0.28
Days 242 to 312	0.17	0.23	0.099	0.69
Heifer BW, kg
Day 0	431	431	4.8	0.96
Day 39	455	455	4.8	0.99
Day 63	464	462	4.8	0.75
Day 91 (near calving)	474	472	4.8	0.85
Day 179	430	421	4.8	0.17
Day 242 (start of the breeding season)	396	389	4.8	0.26
Day 312 (end of the breeding season)	400	397	4.8	0.62
Heifer BW chance, kg
Days 0 to 91	42	41	4.2	0.80
Days 91 to 242	−78	−83	7.6	0.63
Days 242 to 312	3	10	3.7	0.20
First offspring3
Calving4, % of total	95.8	91.3	4.22	0.45
Calving date, day of the study	142	135	4.1	0.22
Male calves, % of total	47.9	54.2	9.21	0.63
Calf birth BW, kg	28.1	29.5	0.99	0.34
Second offspring3
Pregnant on day 404, % of total	88.6	88.9	5.35	0.97
Calving4, % of total	84.1	87.5	7.83	0.76
Calving date, day of the study	554	556	4.6	0.61
Male calves, % of total	51.9	52.3	12.0	0.94

¹Maternal treatments were provided on average for 139 ± 4 d prepartum and 104 ± 4 d postpartum (days 0 to 242). Heifers were provided free-choice access to limpograss hay and 12.7 kg/wk of sugarcane molasses and urea from days 242 to 312. Calves were weaned on day 242 at 96 ± 30 d of age. After weaning, heifers were combined into a single group and placed with three Brangus bulls from days 242 to 312.

² P-value for the comparison of maternal treatments within each respective day of the study for heifer BCS and BW or P-value for the effects of maternal treatment for BCS and BW change.

³First offspring comprises calves that were in utero when maternal treatments were provided (on average from days 0 to 139). Second offspring comprises calves that were conceived during the breeding season (days 242 to 312).

⁴Calving percentage consists of heifers that were diagnosed as pregnant and delivered a live calf at birth. Heifers that did not deliver a live calf at birth were not used for blood collections and were removed from the study and all subsequent statistical analyses of any cow data.

Effects of maternal treatment × day of the study × hour of the day, maternal treatment × day of the study, maternal treatment × hour of the day, and maternal treatment were not detected (P ≥ 0.31) for any heifer activity variable (Table 4). Percentage of heifers calving their first offspring, calving date of the first offspring, percentage of male calves, and birth BW of the first offspring did not differ (P ≥ 0.22) between CON and BAC heifers (Table 4). Percentage of heifers pregnant with the second offspring on day 404 did not differ (P = 0.97) between CON and BAC heifers (Table 3). Percentage of heifers calving their second offspring, calving date of the second offspring, and percentage of male calves in the second offspring did not differ (P ≥ 0.61) between CON and BAC heifers (Table 4).

Table 4.

Plasma concentrations of cortisol and glucose of heifers allocated into 1 of 12 bahiagrass pastures (1 ha and six heifers per pasture) and then assigned to receive soybean hulls supplementation (1 kg/d) added (BAC) or not (CON) with a DFM supplement (3 g/d) containing a combination of Bacillus subtilis and B. licheniformis (6.6 × 10⁹ CFU/d) from days 0 to 242 (six pastures per treatment).

Item	Maternal treatment1		SEM	P-value2
	CON	BAC
Activity3, % of total heifers
Standing outside shade	34.4	42.0	7.24	0.48
Standing under shade	9.5	8.9	2.30	0.86
Laying outside shade	4.45	7.52	2.53	0.41
Laying under shade	20.5	14.1	5.69	0.45
Grazing	27.0	24.2	2.37	0.41
Drinking	3.1	3.4	1.62	0.91
Plasma cortisol, ng/mL
Day 0	12.65	12.02	1.41	0.75
Day 39	18.73	18.86	1.41	0.95
Day 63	14.30	16.39	1.41	0.29
Day 179	6.34	7.68	1.41	0.49
Day 242 (start of the breeding season)	12.33	14.55	1.41	0.26
Plasma glucose, mg/dL
Day 0	80.0	80.7	1.97	0.80
Day 39	80.0	82.4	1.97	0.37
Day 63	77.2	81.2	1.97	0.14
Day 179	76.5	84.3	1.97	0.005
Day 242 (start of the breeding season)	75.8	80.4	1.97	0.09

¹Maternal treatments were provided on average for 139 ± 4 d prepartum and 104 ± 4 d postpartum (days 0 to 242). Heifers were provided free-choice access to limpograss hay and 12.7 kg/wk of sugarcane molasses and urea from days 242 to 312. Calves were weaned on day 242 at 96 ± 30 d of age. After weaning, heifers were combined into a single group and placed with three Brangus bulls from days 242 to 312.

² P-value for the comparison of overall effects of maternal treatment for the respective heifer activity and comparison of maternal treatment within each respective day of the study for all remaining variables.

³Percentage of heifers standing under and outside the artificial shade, laying under and outside the artificial shade, drinking water, and grazing were recorded at 1300 and 1700 hours (periods of highest THI levels) on days 0, 40, 64, 92, and 180.

Effects of day of the study, but not maternal treatment × day of the study and maternal treatment (P ≥ 0.48), were detected (P < 0.01) for heifer plasma concentration of cortisol. Plasma concentrations of cortisol were greatest on day 39 (P < 0.01), greater on day 63 compared to day 0 (P < 0.01), did not differ between days 0 and 242 (P = 0.30), and were the least on day 179 (P < 0.01; Table 4). Effects of treatment × day of the study were detected (P = 0.02) for heifer plasma concentration of glucose. Plasma concentrations of glucose did not differ between CON and BAC heifers on days 0, 39, and 63 (P ≥ 0.14), but were greater (P = 0.005) for BAC vs. CON on day 179 and tended (P = 0.09) to be greater for BAC vs. CON on day 242 (Table 4).

Offspring performance

Effects of maternal treatment × day of the study were detected (P = 0.05) for the first offspring BW from days 242 to 319. First offspring BW did not differ (P ≥ 0.31) between treatments on days 242 and 258 (P ≥ 0.84) but was greater (P = 0.05) for BAC vs. CON calves on day 319 (Table 5). First offspring ADG from birth to early weaning and birth to drylot exit did not differ (P ≥ 0.19) between treatments, but first offspring ADG from drylot entry to exit was greater (P = 0.04) for BAC vs. CON calves (Table 5). Effects of day of the study, but not maternal treatment × day of the study and maternal treatment (P ≥ 0.57), were detected (P < 0.01) for hay, concentrate, and total DMI calculated as percentage of calf BW (Table 5). Effects of maternal treatment and maternal treatment × day of the study were not detected (P ≥ 0.32) for hay DMI calculated as kg/d (Table 5). Effects of maternal treatment tended (P ≤ 0.10) to be detected for concentrate and total DMI calculated as kg/d, which tended to be greater for BAC vs. CON calves (Table 5). Effects of maternal treatment were detected (P = 0.05) for G:F, which was greater for BAC vs. CON calves (Table 5).

Table 5.

Growth and DM intake of the first offspring born from heifers allocated into 1 of 12 bahiagrass pastures (1 ha and six heifers per pasture) and then assigned to receive soybean hulls supplementation (1 kg/d) added (BAC) or not (CON) with a DFM supplement (3 g/d) containing a combination of Bacillus subtilis and B. licheniformis (6.6 × 10⁹ CFU/d) from days 0 to 242 (six pastures per treatment).

Item2	Maternal treatment1		SEM	P-value3
	CON	BAC
BW, kg
Day 242 (early weaning)	81	81	3.1	0.98
Day 258 (drylot entry)	88	89	3.1	0.84
Day 319 (drylot exit)	149	158	3.1	0.05
ADG, kg/day
Birth to day 242	0.44	0.42	0.021	0.61
Days 258 to 319	1.01	1.11	0.035	0.04
Birth to day 319	0.68	0.72	0.020	0.19
Drylot performance (days 258 to 319)
Hay DMI, kg/day	0.46	0.50	0.028	0.32
Hay DMI, % of BW	0.37	0.38	0.021	0.65
Concentrate DMI, kg/day	3.48	3.70	0.079	0.06
Concentrate DMI, % of BW	2.87	2.89	0.025	0.78
Total DMI, kg/day	3.94	4.20	0.105	0.10
Total DMI, % of BW	3.24	3.27	0.038	0.73
G:F4	0.25	0.27	0.004	0.05

¹Maternal treatments were provided on average for 139 ± 4 d prepartum and 104 ± 4 d postpartum. Calves were early weaned on day 242 at 96 ± 30 d of age. From days 242 to 257, calves remained in a single drylot pen and were offered free-choice access to limpograss hay and 0.50 kg/d of a pelletized supplement. On day 258, calves were transferred to 1 of 12 drylot pens using the previous maternal pasture assignment distribution (4 to 6 calves/pen). Starting on day 258, calves were gradually adapted to concentrate by increasing diet DM offered by 0.25% to 0.50% of BW/d for 7 d. Then, calves were limit-fed the same concentrate at 3.25% of BW and limpograss hay at 0.5% of BW (DM basis) until day 319.

²Calf ADG from birth to day 242 were covariate-adjusted for calf age (P = 0.07). Calf age was removed from the model of all statistical analyses of calf variables (P ≥ 0.14).

³ P-value for the comparison of maternal treatments within each respective day of the study for calf BW or P-value for the effects of maternal treatment for all remaining variables.

⁴Calculated by dividing total BW gain by total DM intake (hay + concentrate) from days 258 to 319.

Effects of maternal treatment were not detected (P = 0.44) for serum concentration of IgG at birth (Table 6). Effects of day of the study, but not maternal treatment × day of the study and maternal treatment (P ≥ 0.42), were detected (P < 0.01) for plasma concentrations of glucose (Table 6), haptoglobin, and cortisol (Figure 2). Plasma concentration of glucose increased (P < 0.01) from days 258 to 271 (88.0 ± 1.49 vs. 101.1 ± 1.49 mg/dL, respectively), decreased (P < 0.01) on day 272, did not differ (P = 0.93) between day 272 and 274 (93.4 ± 1.49 vs. 93.5 ± 1.49 mg/dL), and then increased (P < 0.01) on day 278 (99.7 ± 1.49 mg/dL). Plasma concentration of cortisol peaked on day 272 (P ≤ 0.05), did not differ between days 258 vs. 278 and day 271 vs. 274 (P ≥ 0.12), and was greater on day 274 vs. 278 (P < 0.01; Figure 2). Plasma concentration of haptoglobin peaked on day 274 (P ≤ 0.05), and was greater on days 271 vs. 258, 272 vs. 271, and 278 vs. 258 and 271 (P ≤ 0.01; Figure 2). Effects of maternal treatment × day of the study and maternal treatment were not detected (P ≥ 0.18) for serum titers against BVDV-1a, IBR, and PI-3 (Table 6). Effects of maternal treatment × day of the study were detected (P = 0.05) for serum titers against BRSV, which was greater (P = 0.04) for BAC vs. CON calves on day 287 and did not differ (P ≥ 0.36) between treatments on days 271 and 319 (Table 6). Effects of maternal treatment × day of the study and maternal treatment were not detected (P ≥ 0.18) for positive seroconversion against BVDV-1a, BRSV, and IBR (Table 6). Effects of maternal treatment × day of the study were tended (P = 0.06) to be detected for positive seroconversion against PI-3, which was greater (P < 0.01) for BAC vs. CON calves on day 271 and did not differ (P ≥ 0.99) between treatments on days 287 and 319 (Table 6).

Table 6.

Plasma and serum measurements of the first offspring born from heifers allocated into 1 of 12 bahiagrass pastures (1 ha and six heifers per pasture) and then assigned to receive soybean hulls supplementation (1 kg/d) added (BAC) or not (CON) with a DFM supplement (3 g/d) containing a combination of B. subtilis and B. licheniformis (6.6 × 10⁹ CFU/d) from days 0 to 242 (six pastures per treatment).

Item1	Maternal treatment2		SEM	P 3
	CON	BAC
Serum IgG at birth, ng/mL	117	132	13.7	0.44
Plasma glucose, mg/dL	94.8	95.5	1.40	0.70
Plasma haptoglobin, mg/mL	0.49	0.49	0.023	0.86
Plasma cortisol, ng/mL	7.92	8.63	0.656	0.45
Serum titers, log 2
BVDV-1a	3.14	3.25	0.243	0.75
BRSV
Day 271	1.20	1.43	0.363	0.65
Day 287	1.63	2.50	0.363	0.04
Day 319	3.00	2.53	0.363	0.36
IBR	1.04	0.64	0.214	0.19
PI-3	7.77	7.64	0.182	0.59
Seroconversion, %
BVDV-1a	73.5	73.9	7.18	0.97
BRSV	50.7	59.3	4.78	0.21
IBR	23.3	25.6	7.65	0.84
PI-3
Day 271	80.0	93.3	3.57	0.009
Day 287	100.0	100.0	3.57	0.99
Day 319	100.0	100.0	3.57	0.99

¹Serum titers were reported as the log₂ of the greatest dilution of serum that provided complete protection of the cells. Positive seroconversion was determined as the percentage of calves with serum neutralization value ≥ 4.

²Maternal treatments were provided on average for 139 ± 4 d prepartum and 104 ± 4 d postpartum. First offspring were weaned on day 242 at 90 ± 30 d of age. On day 271, each calf received oral drench of fenbendazole (5 mg/kg of BW; Safe-guard, Merck Animal Health, Summit, NJ) and vaccination against pathogens associated with bovine respiratory disease (2 mL s.c.; Bovi Shield Gold One Shot; Zoetis Inc., New York, NY) and clostridium (2 mL s.c.; Ultrabac 7, Zoetis Inc.). On day 287, each calf received booster vaccinations of Bovi Shield Gold 5 (2 mL s.c.; Zoetis Inc.) and Ultrabac 8 (2 mL s.c.; Zoetis Inc.).

³ P-value for the comparison of maternal treatments within each respective day of the study for serum titers against BRSV and positive seroconversion against PI-3 or effects of maternal treatment for all remaining variables.

Figure 2.

Plasma concentrations of haptoglobin and cortisol of calves in drylot born from heifers allocated into 1 of 12 bahiagrass pastures (1 ha and six heifers per pasture) and then assigned to receive soybean hulls supplementation (1 kg/d) added (BAC) or not (CON) with a Bacillus-based DFM mixture (3 g/d) from days 0 to 242 (six pastures per treatment). Effects of day of the study, but not maternal treatment × day of the study and maternal treatment (P ≥ 0.42), were detected (P < 0.01) for plasma concentrations of haptoglobin and cortisol. ^a-eWithin the respective plasma measurement, means without common superscript differ (P ≤ 0.05).

Discussion

Maternal performance

To our knowledge, this is the first study to evaluate the effects of maternal supplementation of a Bacillus-based DFM and its short- and long-term effects on cow–calf performance; hence, direct comparisons with previous studies in beef cattle were not possible. Herbage mass and allowance and forage chemical composition did not differ among pastures offered CON and BAC supplementation, suggesting that BAC supplementation did not impact or was not sufficient to impact forage DMI of heifers and their calves. In addition, BAC supplementation did not impact heifer activity on pasture, calving date, and number of days on treatment during pre- and postpartum periods. Thus, forage quantity and chemical composition, heifer activity, and gestation length did not explain the maternal treatment-induced differences detected for heifer BCS throughout the study. We opted for collecting full BW rather than shrunk BW to avoid carryover effects of physiological stress during gestation, which could mask the potential effects of gestational BAC supplementation on offspring postnatal performance compared to CON supplementation. Nonetheless, heifer BW did not differ between CON and BAC heifers likely due to gut fill effects. Caution should be taken when translating in vitro data into in vivo performance results, but a previous in vitro study indicated that the inclusion of Bacillus-based DFM increased forage DM and NDF digestibility, regardless of forage nutritional value (Pan et al., 2022), which may explain the greater BCS of BAC vs. CON heifers from days 0 to 91. However, heifer BCS from days 91 to 142 did not differ between treatments. Perhaps the relatively high BCS loss from calving until early weaning in all heifers, due to the rapid decline in forage nutritive value combined with the increase in heifer nutrient requirements for lactation, may have alleviated any potential benefits of BAC supplementation during the postpartum period compared to CON supplementation.

Maternal nutrition during gestation often unleashes a cascade of modifications to animal metabolism (Moriel et al., 2021). Plasma concentration of cortisol is an important biomarker for physiological stress and often increases during stressful events, such as elevated environmental temperatures (Bernabucci et al., 2010; Abdelnour et al., 2019; Abduch et al., 2022). Plasma concentration of cortisol did not differ between CON and BAC heifers. However, in agreement with a previous study (Izquierdo et al., 2023a), plasma concentration of cortisol of both groups mimicked ambient THI, peaking on day 39 and decreasing until day 179, and then increasing on day 242 due to the early weaning-induced physiological stress (Moriel et al., 2014). Combined, these results indicate that CON and BAC heifers experienced similar levels of physiological stress from days 0 to 242, and that differences in BCS from days 39 to 91 were not related to different physiological stress responses. Plasma concentration of glucose remained relatively constant throughout the study, reflecting cattle’s ability to maintain glucose concentration despite the declining forage nutritional value and increased heifer nutrient requirements during late gestation and following parturition (NASEM, 2016). Nonetheless, plasma concentrations of glucose in BAC heifers were numerically greater on day 39, tended to be greater on day 179, and were greater on day 242 compared to CON heifers. Such effects on circulating glucose concentration perhaps can be explained by the numerically greater plasma concentration of cortisol in BAC vs. CON heifers from days 39 to 242, because cortisol elicits a plethora of biological effects on the body, including increased metabolism of carbohydrates and hepatic glucose production (Carroll and Fosberg, 2007). In addition, the combination of B. licheniformis and B. subtilis has been shown to increase the production of a wide variety and quantity of fibrolytic, amylolytic, proteolytic, and lipolytic enzymes (Elshaghabee et al., 2017; Luise et al., 2022), improving in vitro DM and NDF digestibility of multiple of different feedstuffs containing >32% NDF (Pan et al., 2022; Cappellozza et al., 2023). For instance, in vitro NDF digestibility increased by 13% after 24 h following incubation with a similar combination of B. licheniformis and B. subtilis as utilized in the present study (Cappellozza et al., 2023). Although not directly measured herein, it is plausible that BAC supplementation slightly increased total DM and NDF digestibility, enhancing total energy intake and plasma concentration of glucose, contributing with greater prepartum BCS following BAC supplementation.

Overall reproductive performance did not differ between CON and BAC heifers, which may have been masked by the optimal BCS before calving for both treatments (BCS > 6; Moriel et al., 2020) and the use of early weaning leading to increased pregnancy attainment (Arthington and Kalmbacher, 2003; Arthington and Minton, 2004). However, it is important to highlight that the present study was not designed to evaluate the reproductive success of beef heifers, and caution on interpreting reproductive data is needed due to the limited number of observations for statistical analysis of binary reproductive data.

Offspring performance

This is the first work evaluating the effects of maternal Bacillus-based supplementation during late gestation and early lactation on the performance of Bos indicus-influenced beef offspring. Hence, direct comparisons with other similar in utero Bacillus-based DFM supplementation in beef cattle were not possible. Maternal nutrition during late gestation impacts calf survivability (Corah et al., 1975) and postnatal growth and immune responses (Moriel et al., 2021). In the present study, serum concentration of IgG did not differ between calves born from CON and BAC heifers, which was not surprising as previous studies indicated that nutrient restriction (57% vs. 100% of nutrient requirements; Hough et al., 1990) and supplemental protein and energy (0 vs. 1 kg DM/cow daily; Palmer et al., 2022) during late gestation did not impact circulating concentration of IgG in the offspring.

Fetal growth is positively correlated with the nutrient supply available in utero (Bauer et al., 1998) and glucose and amino acids provide most substrates necessary for fetal growth (Bell, 1995). A 40% nutrient restriction in ewes decreased maternal plasma glucose concentration by 12%, fetal plasma glucose concentration by 16%, and reduced birth BW by 13% (Vonnahme, 2012). In the present study, first offspring birth BW did not differ between calves born from CON and BAC heifers, perhaps due to the lack of differences detected for maternal plasma concentration of glucose prior to calving. Although milk production of heifers was not measured herein, the lack of differences in first offspring BW at early weaning (day 242) and drylot entry (day 258) between CON and BAC calves suggests that BAC supplementation likely did not increase maternal milk output. However, maternal supplementation of BAC led to greater growth performance during the drylot period and increased calf BW at drylot exit by 9 kg compared to CON supplementation. Although daily supplementation of Bacillus-based DFM increased ADG of Holstein calves (Kowalski et al., 2009) and Nellore steers (Dias et al., 2022), the results detected herein likely reflect the maternal treatment-induced carryover effects on calf postweaning growth performance rather than calf intake of BAC supplements because feed troughs were set at a height to prevent calf consumption of maternal supplements. Hence, the improved growth performance during drylot can be partially explained by the greater prepartum BCS in BAC vs. CON heifers. In a previous study conducted with similar forage management and type, location, animal category, and age (Moriel et al., 2020), pregnant beef heifers were offered 0 or 1 kg/d of sugarcane molasses and urea for 57 ± 5 d prepartum and 17 ± 5 d postpartum. In that study, supplemented heifers gained BCS prior to calving, but their offspring BW at birth, early weaning, and drylot entry was not impacted by maternal treatment, whereas offspring BW at drylot exit increased by 9 kg in calves born from supplemented vs. nonsupplemented heifers. A plausible explanation for detecting maternal treatment-induced effects on offspring growth performance during drylot phase, but not from birth to early weaning, is the total energy and protein consumed by calves during each respective period. The relatively low nutritional value of forage (pasture + hay) consumed by both CON and BAC calves from birth to early weaning limited their growth performance, whereas the relatively high energy and protein consumption during drylot phase allowed BAC calves to express any maternal treatment-induced carryover effects on their growth potential (Moriel et al., 2020, 2021). Thus, Bacillus-based DFM supplementation might be an effective strategy to improve postweaning growth performance.

The mammalian gastrointestinal tract is sterile in utero but undergoes rapid colonization with a wide variety of microorganisms during and after birth. The neonatal colonization process is crucial for the developing gut and naïve immune system (Hansen et al., 2012) and modulated by maternal microbiota (Fanaro et al., 2003) and diet (Fouhy et al., 2012; Rodriguez et al., 2015). For instance, increased bacterial diversity during the first week of life has been shown to increase BW gain in older calves (Oikobomou et al., 2013). Analyses of fecal microbiome and plasma metabolome of heifers and their offspring provided additional explanations for the carryover effects detected for offspring postweaning growth and will be discussed in subsequent manuscripts. Therefore, the increased BAC offspring performance during drylot phase can be attributed due to greater maternal prepartum BCS gain and possibly the maternal/offspring interaction leading to neonatal gut colonization following birth as calves did not consume any maternal supplement due to the height of feed bunks. Additional potential mechanisms for the enhanced growth performance of BAC vs. CON calves during the drylot period might also include differences in offspring muscle transcriptome, body composition, gut barrier function, intestine integrity and inflammation, and postabsorptive metabolism following maternal supplementation of BAC (McAllister et al., 2011; Rhayat et al., 2019; Johnson et al., 2020; Moriel et al., 2021).

Prenatal exposure to stress and modifications to nutritional management can modulate the hypothalamic–pituitary–adrenal axis and alter circulating concentrations of cortisol and haptoglobin in the offspring (Littlejohn et al., 2016; Carroll et al., 2021; Moriel et al., 2021). Calves born from Bos taurus beef cows provided 70% of their energy requirements during the last 40 d of gestation had less plasma concentration of cortisol after vaccination compared to calves born to cows provided 100% of their energy requirements (Moriel et al., 2016). In addition, several studies reported a negative correlation between growth performance and circulating concentration of haptoglobin (Arthington et al., 2005; Qiu et al., 2007; Moriel and Arthington, 2013). Hence, we evaluated the innate immune response of calves following vaccination to potentially explain the detected differences in drylot growth performance between CON and BAC calves. Contrary to our expectations, plasma concentrations of cortisol and haptoglobin in the first offspring were not impacted by maternal treatments, which corroborates with previous studies using Bos indicus-influenced calves (Moriel et al., 2020; Palmer et al., 2020). It is possible that breed could partially explain the discrepancy in impacts of maternal gestational diet on offspring hypothalamic–pituitary–adrenal axis activity (Moriel et al., 2021).

Neutralizing serum antibody titers are often used as a marker of calf response to vaccination (Bolin, 1995), which differs among individuals and previous prenatal and postnatal management (Moriel et al., 2016, 2021; Izquierdo et al., 2023a, 2023b). In the current study, we evaluated calf vaccine response to four viruses associated with bovine respiratory disease. Maternal treatment effects were not observed for serum antibody titers and positive seroconversion against BVDV-1a and IBR. However, BAC supplementation led to a more responsive or earlier development of the humoral immune function in BAC calves, which had a greater percentage of positive seroconversion against PI-3 virus on the day of first vaccination and greater serum titers against BRSV virus 14 d after first vaccination compared to offspring born from CON heifers. This programmed, diverse virus-dependent humoral response to vaccination in offspring exposed to different in utero nutritional management was not surprising and has been reported in several previous studies conducted with similar cattle breed, forage management, and location (Moriel et al., 2020; Palmer et al., 2020, Palmer et al., 2021; Izquierdo et al., 2022, 2023a, 2023b; Vedovatto et al., 2022). Regardless of the mechanism, maternal supplementation of Bacillus-based DFM had positive carryover effects on early postvaccination humoral immune response in the offspring.

In summary, maternal supplementation of a Bacillus-based DFM from 139 ± 4 d prepartum and 104 ± 4 d postpartum increased maternal prepartum BCS gain, and postpartum plasma concentration of glucose compared to no Bacillus-based DFM supplementation. Despite the lack of calf consumption of maternal supplements, maternal supplementation of Bacillus-based DFM during gestation and early postpartum period led to positive carryover effects on calf postweaning BW gain and some indicators of humoral immune response to vaccination.

Glossary

Abbreviations

ADG

average daily gain

BAC

supplemental Bacillus

BCS

body condition score

BVDV-1a

bovine viral diarrhea virus type 1a

BRSV

bovine respiratory syncytial virus

BW

body weight

CFU

colony forming unit

CON

no supplemental Bacillus

CP

crude protein

DM

dry matter

DMI

dry matter intake

G:F

gain:feed

IBR

infectious bovine rhinotracheitis virus

IGF-1

insulin-like growth factor 1

IgG

immunoglobulin G

IVDOM

in vitro digestible organic matter

NDF

neutral detergent fiber

PI-3

parainfluenza-3 virus

TDN

total digestible nutrients

THI

temperature humidity index

Conflict of interest statement. The authors declare that there are no conflicts of interest in this study.