Supplementation with sodium butyrate improves rumen fermentation, antioxidant capability, and immune function in dairy calves before weaning

doi:10.21203/rs.3.rs-25876/v1

Background

Short-chain fatty acids including butyrate have received increasing research interest as potential alternatives to antibiotic growth promoters (AGP) in animal production. This study was conducted to evaluate the effects of supplementation of sodium butyrate (SB) on the growth performance, rumen fermentation, antioxidant capacity, and immune response of calves before weaning. Healthy Holstein female calves (4-day-old; 40 ± 5 kg of body weight) were randomly allocated to 1 of 4 treatment groups (n = 10 per group). The control group was fed no SB (SB0), while the other groups were supplemented with 2% (SB2), 4% (SB4), or 6% (SB6) of SB/kg of dry matter. All calves were housed in individual hutches.

Results

The SB supplementation enhanced growth rate and improved feed conversion into body weight gain compared with the SB0 group. At 60 days of age, the rumen fluid pH increases quadratically with increased SB supplementation, and the ammonia nitrogen (NH₃-N) concentration of rumen fluid in the treatment groups were significantly lower than that of the SB0 group. There was a quadratic effect that indicated that the SB4 treatment was most effective in reducing the NH₃-N concentration. The concentration of volatile fatty acids and Acetic: Propionic in rumen fluid were not affected by SB in any groups. At 28 days of age, the serum level of maleic dialdehyde of the SB groups was significantly lower than that of the control group, and the glutathione peroxidase activity in the serum of group SB4 was significantly increased compared with the that of the control group. At 28 days of age, SB had a quadratic effect on serum immunoglobulin A concentration, with the greatest increase being observed in group SB4. At 60 days of age, the serum immunoglobulin G concentration increased linearly as SB levels increased.

Conclusions

Under the conditions of this study, there were positive effects of SB supplementation on growth performance, rumen fermentation, antioxidant ability, and immune function in calves before weaning. We recommended 4% as the optimal SB supplementation level to improve growth, antioxidant and immune function of calves before weaning.

Animal Science

Sodium butyrate

Calf

Antioxidant activity

Immune function

The digestive physiology of calves changes dramatically in the first months of life, and the transition from a monogastric to the functional ruminant digestive system is fraught with challenges [1]. The development of the gastrointestinal (GI) tract, especially the rumen, is one of the most important steps profoundly affecting the nutritional status and growth performance of every young calf and their adult lives. A successful development of the GI tract can decrease mortality and disease susceptibility and have important economic significance for producers [2]. The physiology of GI development is complex [3] and appears to be aided by some antibiotics for growth promotion [4]. However, extensive use of these antibiotics increases the development of antibiotic resistance in both humans and animals, posing a threat to public health [5–7]. Non-antibiotic alternatives are sought after as the use of antibiotics decreases to comply with government policy or voluntarily.

Butyric acid products (including acid and salt forms) have potential as feed additives to replace antibiotic growth promoters [8, 9]. Supplementation with butyric acid has been shown to have a positive impact on growth performance by enhancing proliferation, differentiation and function of gut tissues in both healthy and sick animals [10]. Sodium butyrate (SB) is a salt of butyric acid, a common short-chain fatty acid (SCFA) produced by anaerobic microbes fermenting the carbohydrates and fiber polysaccharides in the rumen and large intestines of ruminants [11]. Studies have shown that SB can promote the growth of calves, promote digestion and absorption in the small intestine [12], regulate inflammation, improve the antioxidant and immune properties of animals, increase feed intake and daily gain, and improve feed conversion efficiency in piglets and calves [12–16]. Several studies have evaluated SB for its ability to promote calf GI development and improve nutrient absorption [17–19].

However, the outcome of SB to promote calf growth and health has been discrepant. For example, supplementation with SB at 0.3 to 1% of dry matter (DM) increased feed intake in calves after weaning [15]. Rice et al. [20] found that as SB levels increased [from 0, to 0.25, 0.50, and 0.75 g of SB/kg of body weight (BW)], average daily gain (ADG), BW, and final BW of heifers also increased. However, Wanat et al. [21] reported conflicting results that even at 0.3%, 0.6%, and 0.9% of DM, microencapsulated SB added to starter mixture had a negative effect on the animal performance of calves, including linear decreases in ADG and BW in a dose-dependent manner. Slusarczyk et al. [22] shown that SB supplementation at 1–3% of DM was well tolerated in calves and resulted in improved growth performance, but it was found that SB supplementation at 3% of DM reduced feed intake although having a beneficial effect on calf growth and nutrient utilization. Moreover, most of the studies on SB has been focused on how SB could affect feed intake, rumen fermentation, and animal growth including rumen tissue growth. whereas the effects of supplementation SB in milk replace (MR) on antioxidant capacity and immune response of calves have yet to be determined. Therefore, the present study aimed to investigate the effects of different levels of SB on the growth performance, rumen fermentation, antioxidant capacity, and immune response of calves before weaning and ultimately to suggest the optimal dietary supplementation level of SB during the early period of growth and health of calves before weaning.

Animals, treatments, and management

The Institutional Animal Care and Use Committee at the Institute of Animal Sciences, the Chinese Academy of Agricultural Sciences approved all experimental procedures (protocol no. IAS 20180115). Forty healthy Holstein female calves (4-day-old; 40 ± 5 kg of BW) were randomly allocated to 1 of 4 treatment groups (n = 10 calves per group): The control group was fed no SB (SB0), while the other groups were supplemented with 2% (SB2), 4% (SB4), or 6% (SB6) of SB/kg of DM of milk. The doses of SB supplementation were adapted from the work of Gorka et al.[23]. Based on the estimated feed intake, the calculated intake of SB was 15 g, 30 g, and 45 g per day for the SB2, SB4, and SB6 groups, respectively, from 4 to 60 days of age. All calves were housed in individual hutches. Prior to the feeding experiment, all calves were fed 4 L of colostrum within 1 h after birth and were given two more feedings of colostrum at 6 h (2 L) and 18 h (1 L) after birth. All calves were fed milk from 2 to 20 days of age. From 21 to 23 days of age all calves were fed milk and MR mixed into water (1:5.6) (25% milk and 75% MR at 21 days of age, 50% milk and 50% MR at 22 days of age, 75% milk and 25% MR at 23 days of age). From 24 to 60 days of age, MR mixed into water (1:5.6) was supplied instead of milk. All calves were fed in individual buckets twice daily (0700 and 1500 h) from 2 to 60 days of age (2.5 L/time from 2 to 7 days of age; 3 L/time from 8 to 10 days of age; 3.5 L/time at 11 days of age; 4 L/time at 12 days of age; 4.5 L/time from 13 to 30 days of age; 6.5 L/time from 31 days to 50 days of age; 5.5 L/time at 51 days of age; 4.5 L/time at 52 days of age; and then 1 L was reduced per day (0.5 L/time separately) till weaning at 60 days of age). The chemical composition of the experimental feeds is presented in Table 1.

Sampling and analysis

The calves were weighed at 4, 14, 28, 42, and 60 days of age before morning feeding, and BW, body length, and heart girth were also recorded at those ages. Average daily gain (ADG) was calculated over each time interval. Intakes of starter feed were recorded daily at 0900 h, and intake of milk (and milk fortified with MR) was recorded twice daily at each feeding. Total dry matter intake (DMI) based on the consumption of milk and starter DMI for each calf. Feed-to-gain (F:G) ratio was calculated as the ratio of total DMI to ADG.

At 14, 28 and 60 days of age, a 25-mL sample of rumen content was collected from each calf using the oral tubing method two hours after the morning feeding. The first 5 mL was discarded to avoid contamination with saliva. The sample was squeezed through 4 layers of cheesecloth; the pH was measured immediately and then 6 mL of strained fluid was acidified with 3 mL of 0.5 M HCl and frozen at -20 ℃ for ammonia nitrogen (NH₃-N) analysis [24]. A 4-mL aliquot was prepared for volatile fatty acids (VFAs) analysis using gas chromatography as described by Erwin et al.[25].

Blood samples were taken from the external jugular vein at 2 hours after the morning feeding at 14, 28, and 60 days of age. At each collection, a duplicate 10 mL blood samples were placed into tubes without any additives. Serum was prepared by centrifugation at 3 000 g for 15 min at 4 ℃ and then stored at -20 ℃ until subsequent analysis. Concentrations of glutathione peroxidase (GSH-Px), superoxide dismutase (SOD), and maleic dialdehyde (MDA) were analyzed using respective commercial kits (Nanjing Jian Cheng Bioengineering Institute, Nanjing, China) as described previously [26]. The concentrations of immunoglobulin A (IgA), immunoglobulin G (IgG), and immunoglobulin M (IgM) in the serum were measured using IgG (F4042-A), IgA (F3995-A), and IgM (F6685-A) ELISA kits, respectively (Shanghai Panke Industrial Co., Shanghai, China).

Statistical analysis

All data were statistically analyzed using the Proc Mixed method of SAS, with linear and quadratic polynomial contrasts tested using the CONTRAST statement to determine the effects of SB on ADG, DMI, F:G ratio, rumen fermentation parameters, and blood parameters. The appropriate coefficients for the CONTRAST statement of this study were obtained using PROC IML's ORPOL function. The MIXED statistical model used for analysis was as follows:

Y_ijk = µ + T_i + D_j(i) + (TW)_ik + E_ijk,

where Y_ijk = dependent variable measured at week kth on the jth cow assigned to the ith treatment, µ = population mean, T_i = treatment effect, D_j(i) = random effect of the jth cow within the ith treatment, W_k = time effect, (TW) _ik = fixed interaction effect between treatment and time, E_ijk = random error associated with the jth cow assigned to the ith treatment at week kth. Fixed effects in the model included treatment, time, and treatment × time interaction. The variance for each calf was used as random effect. Significance was declared at P < 0.05 and trend was discussed at 0.05 < P < 0.10.

Growth performance

The effects of SB on ADG, milk DMI starter DMI, total DMI, and F:G ratio are presented in Table 2. There was no significant difference in milk DMI, starter DMI, or total DMI among the groups during the whole experimental period (P > 0.05). Between 4 to 60 days of age, the ADG was significantly higher in SB2, SB4, and SB6 than in SB0 (P < 0.05). Between 4 to 14 days of age and between 4 to 60 days of age, the F:G ratio of the SB2 group was significantly lower than that of SB0 (P < 0.05).

The BW and body size measurements are presented in Table 3. No significant difference (P > 0.05) was noted in heart girth among the groups during the whole experimental period. At 14 days of age, the BW in the treatment groups was significantly higher than that of the SB0 group. At 28 days of age, body height quadratically increased with increased SB supplementation (P < 0.05). At 60 days of age, the body length tended to increase quadratically (P = 0.075) as SB levels increased.

Rumen fermentation

At 14 days of age, the pH of rumen fluid tended to be increased quadratically (P = 0.092), while at 60 days of age, it was increased quadratically (P < 0.05) with the increased SB supplementation (Table 4), and the concentration of NH₃-N in rumen fluid was significantly lower (P < 0.05) in the treatment groups than in control group. There was a quadratic effect (P = 0.025) that indicated that the SB4 treatment was most effective in reducing the NH₃-N concentration. The concentration of VFAs and the ratio of Acetic: Propionic in rumen fluid were not affected by SB in any groups (P > 0.05).

Antioxidant capability in serum

The effects of the SB supplementation on the antioxidant capability in the calves before weaning are shown in Fig. 1. At 28 days of age, the plasma MDA concentrations linearly decreased as the SB supplementation amount increased and was significantly lower in the SB4 group than in the SB0 group (P < 0.05). The plasma GSH-Px activity was higher in the SB4 and the SB6 groups compared with the SB0 group (P < 0.05), while the serum SOD activity was not different among the groups (P = 0.066). At 60 days of age, the serum GSH-Px activity was quadratically increased (P < 0.05) with the increased SB supplementation and peaked at 4% SB.

Serum immunoglobulins

The SB supplementation numerically (P > 0.05) increased the titers of serum IgA, IgG, and IgM (Table 5). At 28 days of age, the serum IgA titer tended to be increased quadratically (P = 0.097) with the increased SB supplementation and peaked in the SB4 group. At 60 days of age, the serum IgG concentration increased linearly (P = 0.038) as SB levels increased. The supplementation with differing amounts of SB did not influence the serum IgM concentration of the calves (P > 0.05).

Sodium butyrate enhances feed utilization and ADG in calves before weaning

Several studies have shown that dietary supplementation with sodium butyrate has a positive effect on neonatal piglets and broiler chickens [12, 27, 28] and young calves [29]. The positive effects of sodium butyrate on the growth parameters observed in our study corroborate the previous studies and support the notion that butyrate supplementation is more effective when fed earlier rather than later after birth [14, 28]. In newborn calves, solid feed intake depends on the development of the rumen, the rumen tissue, rumen papillae, and the rumen microbiota [30, 31]. In the present study, we did not see any increase in feed intake by sodium butyrate supplementation. This is consistent with the reports by Similarly Hill et al. [15] and Vazquez-Mendoza et al. [32]. The supplementation with sodium butyrate did increase ADG, which concurs with the improved ADG previously observed in weaned calves supplemented with sodium butyrate [22, 33]. It should be noted that the ADG increase was substantial between days 4 and 14, by 42.9%, among the calves supplemented with 2% sodium butyrate. There was a linear trend in reducing the F:G ratio as sodium butyrate levels increased, and sodium butyrate supplementation at 2% decreased F:G ratio by nearly 14% throughout the feeding trial. The exact modes of action of sodium butyrate are not known. One study suggested that butyrate might enhance growth performance by improved feed digestibility [34], while another study proposed that butyrate might enhance the absorption capacity of nutrients by increasing the depth of the crypts and the length of small intestine villi, thus increasing the absorptive surface area [35]. Future wholistic studies are needed to elucidate the underlying mechanisms by integrating transcriptomic and proteomic approaches coupled with morphological and histochemical methodologies to investigate the growth and development of the host, especially the digestive system, and meta-omic approaches to investigate the rumen microbiome.

Sodium butyrate reduces the rumen NH₃-N concentration of calves before weaning

Rumen fermentation starts at a very young age, and VFAs can be found in the rumen of calves from the second week of their life [36]. In the rumen, butyrate confers multiple protective benefits, such as improving tight junctions, epithelial energy mobilization, and VFAs absorption capacity [2]. Studies have shown that butyric acid could lower the intestinal pH of calves [37], which can promote the GI colonization with beneficial bacteria [13]. However, our study showed that sodium butyrate significantly increased the rumen pH of calves, probably as the different pH values between butyric acid and sodium butyrate (6.0 vs. 8.0) [38], nevertheless, the pH in this study had no detrimental effects on rumen development. The ruminal VFAs concentration was not affected by the supplement with sodium butyrate, but improved the development of rumen in calves as indicated in previous findings [39, 40], which was due to the enhancement of the absorption of VFAs in rumen supplied with sodium butyrate in calves [2]. In our study, the growth and feed efficiency of calves were improved probably as the stimulating of rumen development with sodium butyrate [23, 39]. Interestingly, the NH₃-N concentration in the rumen fluid decreased linearly as sodium butyrate levels increased. Because the rumen concentration of NH₃-N reflects the balance of protein degradation NH₃-N uptake by rumen microbes synthesis [41], the decreased rumen NH₃-N concentration suggests improvement of nitrogen utilization in the calves. This premise is consistent with the decreased feed-to-gain ratio and increased ADG without any increase in feed intake we observed among the calves fed sodium butyrate. More research will need to be done to investigate the effect of sodium butyrate on nitrogen utilization in calves.

Sodium butyrate enhances the antioxidant capability of serum in calves before weaning

Calving leads to oxidative stress, which can increase free radical formation and damage the antioxidant systems of calves [42]. Oxidative stress can cause oxidative damages to tissue by reactive oxygen species (ROS) and reactive nitrogen and overwhelm the body's endogenous antioxidant protection capacity [43]. The antioxidative enzymes, such as SOD, GSH-Px, and CAT [44], are essential components of animal oxidative stress defense systems. In the present study, we evaluated how sodium butyrate might affect oxidative stress and the defense system of such stress. Compared to that of the control group, the GSH-Px activity increased with increasing sodium butyrate levels, while the serum MDA concentration decreased linearly. Although not significantly, sodium butyrate supplementation also numerically increased the activity of SOD in the serum compared with the control group at 60 days of age. In a study using chicken, dietary sodium butyrate increased the activity of SOD and decreased serum MDA concentration [45]. Ma et al. [46] showed that the alteration in antioxidant indices by sodium butyrate could suggest an improvement in the level of oxidative stress in the intestinal mucosa. Butyrate has also been shown to decrease the oxidative damages to human colorectal cells [47], reduce oxidative stress precipitated by colonic inflammation that is caused by cancer-induced destruction of the intestinal barrier [48], and alleviate oxidative stress induced by lipopolysaccharides in the intestinal epithelial Caco-2 cells and colonic mucosa [49] and in streptozotocin diabetic rats [50]. The discrepancies between our study and the above studies with respect to SOD may be attributable to differences in the levels of sodium butyrate and animals used. Nevertheless, the increased GSH-Px activity and decreased MDA concentration among the calves supplemented with sodium butyrate demonstrate the benefits of sodium butyrate supplementation to help the calves in coping with the oxidative stress from which they suffered in their young lives.

Sodium butyrate increases serum IgA and IgG concentration in calves before weaning

The three immunoglobulins (i.e., IgG, IgM, and IgA) are important indicators of the immune function of animals including calves as they can protect animals and humans against a variety of pathogens and viruses, activate the complement system, regulate the antibody-dependent cell-mediated cytotoxicity, and improve animal's immunity [51]. Butyrate has been found to have a profound impact on the immune system of humans and rodents [52]. Supplementation with sodium butyrate also increased the number of IgA⁺ cells, which later increased the production of secretory IgA in the jejunum of piglets [53] and increased serum IgG concentrations in pigs [54]. In the present study, supplementation of SB in MR has no effect on the immunoglobulin concentration, but had a tendency to increases the IgA and IgG concentration in serum of calf. It is worth noting that supplementation sodium butyrate in acidified milk did not affect the immunoglobulin concentration in the calf serum [55]. The differences in supplementation levels, methods, animals, and animal age might be among the factors that could be attributable to the discrepancies between our study and the other studies. Further research is warranted to further investigate if butyrate modulates immune system development and function in calves using other immunological analyses.

The addition of sodium butyrate to the diets of young calves increased growth performance and improve feed efficiency. The supplementation reduces the rumen NH₃-N concentration, improved immune functions as indicated by the numerically elevated concentration of IgA and IgM, and enhanced antioxidant capacity. Farm-level studies are needed to evaluate if sodium butyrate can improve calf growth and health. Mechanistic studies using physiological, immunological, transcriptomic, and proteomic methodologies and technologies are also needed to elucidate how butyrate enhance growth, antioxidant and immune functions in calves before weaning.

AGP: Antibiotic growth promoters; GI: Gastrointestinal; SB: Sodium butyrate; SCFA: Short-chain fatty acid; DM: Dry matter; ADG: Average daily gain; BW: body weight; MR: Milk replace; DMI: dry matter intake; F:G: feed-to-gain; NH₃-N: ammonia nitrogen; VFAs: Volatile fatty acids; GSH-Px: Glutathione peroxidase; SOD: Superoxide dismutase; MDA: Maleic dialdehyde; IgA: Immunoglobulin A; IgG: Immunoglobulin G; IgM: Immunoglobulin M.

Authors’ contribution

Experimental design was conducted by WL, LM, and DB. WL and ALTZL conducted the animal experiment. Data analysis was performed by WL, SG, and ALTZL. WL, LM, AE, and ZY wrote the manuscript. All authors reviewed the manuscript and read and approved the final manuscript.

Funding

This research was partially supported by the National Natural Science Foundation of China (award number: 31802092), the National Key Research and Development Program of China (award numbers: 2018YFE0101400 and 2017YFD0500502), the Agriculture Science and Technology Innovation Program (award number: ASTIP-IAS07), and Beijing Dairy Industry Innovation Team (award number: BAIC06-2020).

Availability of data and materials

The data analyzed during the current study are available from the corresponding author on reasonable request.

Ethics approval and consent to participate

The Institutional Animal Care and Use Committee at the Institute of Animal Sciences, the Chinese Academy of Agricultural Sciences approved all experimental procedures (protocol no. IAS 20180115).

Consent for publication

All the authors read and agree to the content of this paper and its publication.

Competing interests

The authors declare no competing interest.

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Hamer HM, Jonkers DM, Bast A, Vanhoutvin SA, Fischer MA, Kodde A, et al. Butyrate modulates oxidative stress in the colonic mucosa of healthy humans. Clin Nutr. 2009;28(1):88–93.
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Sharma B, Singh N. Attenuation of vascular dementia by sodium butyrate in streptozotocin diabetic rats. Psychopharmacology. 2011;215(4):677–87.
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Weber TE, Kerr BJ. Butyrate differentially regulates cytokines and proliferation in porcine peripheral blood mononuclear cells. Vet Immunol Immunopathol. 2006;113(1):139–47.
Huang C, Song P, Fan P, Hou C, Thacker PA, Ma X. Dietary sodium butyrate decreases postweaning diarrhea by modulating intestinal permeability and changing the bacterial communities in weaned piglets. J Nutr. 2015;145(12):2774–80.
Fang CL, Sun H, Wu J, Niu HH, Feng J. Effects of sodium butyrate on growth performance, haematological and immunological characteristics of weanling piglets. J Anim Physiol Anim Nutr. 2014;98(4):680–5.
Sun YY, Li J, Meng QS, Wu DL, Xu M. Effects of butyric acid supplementation of acidifed milk on digestive function and weaning stress of cattle calves. Livest Sci. 2019;225:78–84.

Table 1 Nutritional composition of the experimental feeds

Items	Milk	Items	Milk replacer^*	Starter Feed^*
Density (g/L)	1030.50	DM, %	96.06	97.23
Milk protein, %	3.50	CP, %	22.49	25.94
Milk fat, %	3.88	EE, %	9.35	3.01
Total solid, %	12.93	Ash, %	7.16	6.47
DM, %	12.30	NDF, %	0.78	16.03
Lactose, %	4.36	ADF, %	0.54	7.00
		Ca, %	1.15	0.94
		P, %	0.97	0.66

^* on DM basis.

Table 2 Effects of different levels of sodium butyrate on average daily gain (ADG), dry matter intake (DMI), and feed-to-gain (F:G) ratio in the calves before weaning

Items	Treatment¹				SEM	P-value
Items	SB0	SB2	SB4	SB6	SEM	Trt	Linear	Quadratic
4 to 14 days of age
ADG (kg/d)	0.35^ab	0.50^a	0.33^b	0.46^ab	0.04	0.041	0.490	0.902
Total DMI (g/d)	817.9	803.3	796.2	796.7	5.23	0.506	0.173	0.492
Milk DMI (g/d)	791.4	784.3	787.5	774.0	3.73	0.402	0.180	0.672
Starter DMI (g/d)	26.4	19.0	8.7	21.9	3.76	0.998	0.996	0.984
F:G ratio²	2.41^a	1.76^b	2.64^ab	1.88^ab	0.21	0.011	0.493	0.795
15 to 28 days of age
ADG (kg/d)	1.05	1.13	1.20	1.11	0.03	0.168	0.269	0.066
Total DMI (g/d)	1153.9	1162.9	1144.8	1153.3	3.33	0.758	0.384	0.715
Milk DMI (g/d)	1110.0	1121.2	1110.3	1110.8	2.72	0.541	0.784	0.408
Starter DMI (g/d)	43.9	41.7	34.5	42.4	2.11	0.820	0.753	0.507
F:G ratio	1.13^a	1.04^ab	0.97^b	1.06^ab	0.03	0.073	0.145	0.032
29 to 42 days of age
ADG (kg/d)	0.97	1.03	1.04	1.03	0.02	0.659	0.357	0.403
Total DMI (g/d)	1450.4	1468.4	1469.6	1462.9	1.33	0.996	0.876	0.852
Milk DMI (g/d)	1367.9	1383.6	1371.1	1367.2	3.82	0.335	0.666	0.172
Starter DMI (g/d)	82.5	84.8	98.6	95.7	3.60	0.935	0.586	0.925
F:G ratio	1.53	1.44	1.44	1.45	0.02	0.705	0.441	0.438
43 to 60 days of age
ADG (kg/d)	0.90	0.96	1.01	0.98	0.02	0.435	0.180	0.369
Total DMI (g/d)	1336.7	1382.2	1361.5	1319.0	8.26	0.932	0.687	0.634
Milk DMI (g/d)	1037.2	1036.7	1036.8	1036.1	0.23	0.165	0.055	0.778
Starter DMI (g/d)	299.5	345.6	324.7	282.9	13.87	0.843	0.782	0.409
F:G ratio	1.47	1.35	1.27	1.33	0.04	0.267	0.119	0.236
4 to 60 days of age
ADG (kg/d)	0.83^a	0.90^b	0.89^b	0.88^b	0.02	0.044	0.086	0.034
Total DMI (g/d)	1201.8	1202.8	1191.8	1186.9	3.90	0.932	0.545	0.886
Milk DMI (g/d)	1078.2	1080.7	1076.8	1073.1	1.59	0.370	0.165	0.317
Starter DMI (g/d)	123.6	122.2	114.9	113.8	1.59	0.978	0.696	0.866
F:G ratio	1.65^a	1.42^b	1.61^ac	1.46^bc	0.06	0.002	0.061	0.354

¹SB0, SB2, SB4, and SB6 = 0%, 2%, 4%, and 6% of sodium butyrate supplementation.

² F:G ratio was calculated by dividing average daily total DMI by ADG.

Means with different superscripts in a row differ significantly (P < 0.05).

Table 3 Effects of different levels of sodium butyrate on body weight (BW) and body size measurements of the calves before weaning

Items	Treatment¹				SEM	P-value
Items	SB0	SB2	SB4	SB6	SEM	Trt	Linear	Quadratic
4 days of age
BW (kg)	40.6	38.8	39.7	39.4	0.38	0.508	0.479	0.380
Body height (cm)	76.6	74.4	74.7	75.0	0.49	0.221	0.211	0.123
Body length (cm)	70.8	68.9	69.7	69.6	0.39	0.328	0.391	0.220
Heart girth (cm)	82.9	82.7	81.8	83.0	0.27	0.686	0.864	0.373
14 days of age
BW (kg)	44.20^ab	46.43^a	43.96^b	45.90^ab	0.61	0.041	0.460	0.842
Body height (cm)	76.75	78.02	77.52	77.02	0.28	0.271	0.884	0.072
Body length (cm)	73.03	73.75	72.88	72.45	0.27	0.600	0.407	0.416
Heart girth (cm)	87.26	87.02	86.63	86.89	0.13	0.905	0.578	0.686
28 days of age
BW (kg)	61.23	62.26	60.43	60.76	0.40	0.458	0.410	0.690
Body height (cm)	81.25	82.76	82.35	81.11	0.41	0.122	0.995	0.021
Body length (cm)	78.63	78.83	78.62	78.53	0.06	0.994	0.888	0.853
Heart girth (cm)	92.89	93.81	92.71	92.71	0.26	0.609	0.589	0.503
42 days of age
BW (kg)	75.23	75.42	75.14	76.9	0.41	0.637	0.340	0.483
Body height (cm)	86.08	87.22	87.54	87.17	0.31	0.639	0.318	0.389
Body length (cm)	84.35	86.17	85.50	84.83	0.39	0.495	0.906	0.156
Heart girth (cm)	97.13	100.16	97.85	98.92	0.66	0.196	0.486	0.299
60 days of age
BW (kg)	91.18	93.97	91.70	92.09	0.61	0.406	0.935	0.325
Body height (cm)	92.78	93.40	93.84	92.88	0.25	0.520	0.777	0.173
Body length (cm)	90.67	92.36	92.10	91.97	0.38	0.094	0.065	0.075
Heart girth(cm)	105.08	104.97	104.87	104.08	0.23	0.472	0.166	0.500

¹SB0, SB2, SB4, and SB6 = 0%, 2%, 4%, and 6% of sodium butyrate supplementation.

Means with different superscripts in a row differ significantly (P < 0.05).

Table 4 Effects of different levels of dietary sodium butyrate on rumen fermentation parameters of the calves before weaning

Items	Treatment¹				SEM	P-value
Items	SB0	SB2	SB4	SB6	SEM	Trt	Linear	Quadratic
14 days of age
pH	6.80	7.07	7.22	6.91	0.11	0.092	0.374	0.022
NH₃-N (mg/dL)	17.59	15.82	17.71	15.98	0.51	0.952	0.843	0.995
Acetic acid (mmol/mL)	17.35	15.98	22.72	19.79	1.48	0.606	0.193	0.948
Propionic acid (mmol/mL)	11.59	10.03	14.21	14.08	1.01	0.387	0.215	0.727
Isobutyric acid (mmol/mL)	0.48	0.52	0.74	0.55	0.06	0.108	0.223	0.132
Butyric acid (mmol/mL)	3.47	6.95	8.86	6.15	1.12	0.223	0.228	0.095
Isovaleric acid (mmol/mL)	0.59	0.60	0.88	0.69	0.07	0.184	0.220	0.310
Valeric acid (mmol/mL)	1.50	0.47	0.83	0.82	0.22	0.258	0.133	0.742
Total VFA (mmol/mL)	34.07	34.56	48.24	42.08	3.38	0.261	0.151	0.559
Acetic: Propionic	1.50	1.58	1.59	1.57	0.02	0.949	0.690	0.687
28 days of age
pH	6.38	6.59	6.34	6.40	0.09	0.609	0.755	0.601
NH₃-N (mg/dL)	21.93	19.79	18.25	22.65	1.00	0.488	0.511	0.496
Acetic acid (mmol/mL)	26.66	27.89	28.31	23.72	1.04	0.621	0.567	0.282
Propionic acid (mmol/mL)	22.00	19.89	18.74	29.91	2.52	0.188	0.207	0.077
Isobutyric acid (mmol/mL)	0.61	0.45	0.46	0.41	0.04	0.438	0.166	0.507
Butyric acid (mmol/mL)	6.67	5.33	5.30	5.53	0.32	0.819	0.603	0.596
Isovaleric acid (mmol/mL)	0.58	0.50	0.48	0.41	0.03	0.827	0.353	0.991
Valeric acid (mmol/mL)	1.39	1.20	1.49	0.81	0.15	0.743	0.501	0.586
Total VFA (mmol/mL)	57.91	56.52	53.45	62.36	1.85	0.452	0.147	0.245
Acetic: Propionic	1.39	1.39	1.54	1.13	0.09	0.277	0.358	0.164
60 days of age
pH	5.96^b	6.57^a	6.45^a	6.08^ab	0.15	0.020	0.732	0.003
NH₃-N (mg/dL)	23.60^a	14.67^b	13.75^b	15.29^b	2.28	0.023	0.014	0.025
Acetic acid (mmol/mL)	38.96	39.09	40.12	43.32	1.02	0.525	0.215	0.534
Propionic acid (mmol/mL)	53.09	54.40	48.75	58.58	2.02	0.287	0.518	0.249
Isobutyric acid (mmol/mL)	0.74	0.88	0.72	0.65	0.05	0.568	0.450	0.399
Butyric acid (mmol/mL)	11.75	16.02	15.24	15.31	0.96	0.366	0.211	0.226
Valeric acid (mmol/mL)	0.91	1.13	0.74	0.78	0.09	0.458	0.389	0.655
Isovaleric acid (mmol/mL)	4.13	3.83	4.25	5.09	0.27	0.476	0.254	0.364
Total VFA (mmol/mL)	109.59	115.36	109.67	123.83	3.35	0.339	0.219	0.520
Acetic: Propionic	0.74	0.72	0.84	0.76	0.03	0.135	0.342	0.431

¹SB0, SB2, SB4, and SB6 = 0%, 2%, 4%, and 6% of sodium butyrate supplementation.

Means with different superscripts in a row differ significantly (P < 0.05).

Table 5 Effects of different levels of sodium butyrate on serum Ig concentration in the calves before weaning

Items	Treatment¹				SEM	P-value
Items	SB0	SB2	SB4	SB6	SEM	Trt	Linear	Quadratic
28 days of age
IgA (µg/ml)	690.01	746.73	776.62	687.77	21.88	0.377	0.903	0.097
IgG (mg/ml)	5.63	5.72	5.85	5.50	0.08	0.941	0.884	0.584
IgM (µg/ml)	195.13	194.69	210.58	198.56	3.72	0.652	0.584	0.576
60 days of age
IgA (µg/ml)	741.43	735.83	755.76	748.21	4.31	0.992	0.850	0.984
IgG (mg/ml)	5.45	6.24	6.56	6.48	0.25	0.116	0.038	0.230
IgM (µg/ml)	205.40	212.32	200.50	217.81	3.81	0.682	0.580	0.630

¹SB0, SB2, SB4, and SB6 = 0%, 2%, 4%, and 6% of sodium butyrate supplementation.

Supplementation with sodium butyrate improves rumen fermentation, antioxidant capability, and immune function in dairy calves before weaning

Status:

Journal Publication

Version 1

Abstract

Background

Results

Conclusions

Figures

Introduction

Materials And Methods

Animals, treatments, and management

Sampling and analysis

Statistical analysis

Results

Growth performance

Rumen fermentation

Antioxidant capability in serum

Serum immunoglobulins

Discussion

Sodium butyrate enhances feed utilization and ADG in calves before weaning

Sodium butyrate reduces the rumen NH₃-N concentration of calves before weaning

Sodium butyrate enhances the antioxidant capability of serum in calves before weaning

Sodium butyrate increases serum IgA and IgG concentration in calves before weaning

Conclusions

Abbreviations

Declarations

References

Tables

Status:

Journal Publication

Version 1

Supplementation with sodium butyrate improves rumen fermentation, antioxidant capability, and immune function in dairy calves before weaning

Status:

Journal Publication

Version 1

Abstract

Background

Results

Conclusions

Figures

Introduction

Materials And Methods

Animals, treatments, and management

Sampling and analysis

Statistical analysis

Results

Growth performance

Rumen fermentation

Antioxidant capability in serum

Serum immunoglobulins

Discussion

Sodium butyrate enhances feed utilization and ADG in calves before weaning

Sodium butyrate reduces the rumen NH3-N concentration of calves before weaning

Sodium butyrate enhances the antioxidant capability of serum in calves before weaning

Sodium butyrate increases serum IgA and IgG concentration in calves before weaning

Conclusions

Abbreviations

Declarations

References

Tables

Status:

Journal Publication

Version 1

Sodium butyrate reduces the rumen NH₃-N concentration of calves before weaning