Experiment 1: Screening for immunomodulatory potency of the PGBE complex using MAC-T
The production of excessive reactive oxygen species, which causes oxidative stress and protein oxidation [
18], is involved in the pathogenesis of many types of inflammatory disease. Various natural resources from land plants and marine algae with antioxidant effects have been used to treat inflammatory disease. We tested the antioxidant potency of the PGBE complex at various combination rates by estimating its ABTS cation radical scavenging activity (
Table 2). All PGBE complexes at dilution rate of 0.16% showed strong antioxidant effects compared to ascorbic acid (1.0 mg/mL), but the radical scavenging activity of all PGBE complexes at dilution rates (0.08% to 0.01%) was weak and even disappeared. PGBE complex containing pinecone, garlic kernel, and brown seaweed midrib extracts (2:1:1; vol/vol) was comparable to the other combinations of PGBE (1:1:1 and 1:1:2; vol/vol), with the exception of PGBE combination 1:2:1; vol/vol (p = 0.0478). Consequently, 0.16% PGBE complex (1:1:1; vol/vol), as well as consideration of extract costs, was used to evaluate anti-inflammatory effects using MAC-T cells.
MAC-T cells, which are an immortalized epithelial cell line isolated from bovine mammary tissue, provide a useful
in vitro model for bovine lactation because they retain a number of biochemical and morphological characteristics typical of bovine primary MAC-T
in vivo [
14]. Therefore, we suggest that examination of the anti-inflammatory potency of the PGBE complex using MAC-T cells in response to LPS might be useful to understand the similar inflammatory responses of heat stressed dairy cows. COX-2 expression is an indicator of inflammatory responses in a variety of cells, including MAC-T [
19]. In order to identify the anti-inflammatory effects of PGBE complex (1:1:1, vol/vol), MAC-T cells were exposed to PGBE complex at concentrations of 0%, and 0.16% (vol/vol) for 6 h, followed by LPS treatment (0, 1 μg/mL) for 12 h. Western blot analysis showed that protein expression of COX-2 in MAC-T cells was elevated by LPS (1 μg/mL) as compared to non-LPS treatment (
Supplementary Figure S1). Interestingly, we found that pretreatment with 0.16% PGBE complex (1:1:1; vol/vol) attenuated LPS-induced COX-2 expression in MAC-T cells.
Taken together, these in vitro trials suggest that the combination of PGBE at 1:1:1 (vol/vol) had the strongest antioxidative potency and anti-inflammatory effects by suppressing COX-2 expression in MAC-T cells under LPS stimulation.
Experiment 2: Effects of a supplemental dietary mixture of PGBE on milk yield, milk composition, immune status, metabolic profiles, and behavior patterns in dairy cows experiencing heat stress
Based on the results of Experiment 1 and data previously described by Kim et al [
10], we further examined the effects of supplemental dietary 0.016% PGBE complex (1:1:1, vol/vol) on milk yield, milk composition, immune status, metabolic profiles, and behavior patterns in dairy cows during heat events in the summer period compared to those of the basal diet to evaluate whether PBGE complex has potential to improve milk production.
Although there was no significant difference in milk composition and feed intake between the control and 0.016% PGBE-treated groups during the whole experimental period, the PBGE mixture showed increased milk yield for 40 days as compared to the control group (p<0.05) (
Table 3). Comparing our observation, a study by Oh et al [
20] showed that milk composition and blood chemistry were not affected by treatment with 2 g per cow of garlic extract for 9 d. DMI tended to be lower for the garlic treatment, which increased feed efficiency but slightly decreased milk yield. A study by Yang et al [
21] investigated the effects of garlic (5 g per cow) and juniper berry (2 g per cow) essential oils on ruminal fermentation and on the site and extent of digestion in lactating cows, and reported that milk yield did not change. A prior study showed that incorporation of 0.016% pinecone oil into basal diet did not have a large influence on milk yield or metabolic and hematological parameters in lactating Holstein cows [
10]. A study by Hong et al [
5] found that feeding dietary brown seaweed byproducts at 2% and 4% for 12 months did not affect DMI, milk yield, or milk composition in Holstein cows. In addition, Benchaar et al [
22] observed no changes in milk yield or milk composition in cows supplemented with 2 g per day or 750 mg per day of a commercial mixture of essential oil compounds, reporting that milk protein and milk lactose content, as well as their yields, were not affected by the treatment. Similarly, milk efficiency, presented either as kg of milk or as fat-corrected milk per kg of DMI, did not differ among the groups. Although there was no observed improvement in milk composition following supplementation with the combined mixture of phytogenic extracts (garlic and pinecone) and marine extract (brown seaweed) in the current study, we suggest that supplementation with 0.016% PGBE at 1:1:1 (vol/vol) in basal diet was effective for improving the milk yield of lactating cows.
A study by Yun et al [
9] showed that a garlic bulb consists of the organosulfur compounds alliin, γ-glutamyl-S-allylcysteine, S-methyl cysteine sulfoxide, S-trans-1-propenylcysteine sulfoxide, S-2-carboxypropylglutathione, and S-allylcysteine. As aforementioned, the compounds in garlic, such as S-allylcysteine, show great antioxidant potential by increasing the activity of several antioxidant enzymes, such as GSH reductase, superoxide dismutase, and g-glutamate cysteine ligase [
8,
9]. A study by Kim et al [
10] showed that the major components of phytonic essential oil extracted from pinecone are γ-terpinene, dl-limonene, 2-β-pinene, and isolongifolene, and that the components of pinecone oil have multiple biological properties including antioxidant and suppression of cortisol. Brown seaweed is rich in a polysaccharide of alginic acid, and it been demonstrated that polysaccharide of fucoidan in brown seaweed shows anticoagulation, antitumor, anticancer, and antioxidation effects [
11,
12]. In the blood traits of cows, we did not observe a significant difference in antioxidant ability (TAC and GSH) (
Table 4). However, cows in the control group demonstrated significantly increased thiobarbituric acid reactive substances levels for 40 days (p<0.05), but cows supplemented with 0.016% PGBE in basal diet for 40 days showed tendency of an elevation of TAC (p = 0.0751) and GSH concentration (p = 0.0894) compared to those on day 0. Combined with the results in
Table 2, we suggest that 0.016% PGBE complex can scavenge free radicals, causing an antioxidant effect in lactating cows. There were no significant differences in metabolic (blood urea nitrogen, albumin, glucose, non-esterified fatty acid, triglyceride, gamma-glutamyl transpeptidase, and creatine) or hematological (white blood cell, lymphocyte, monocyte, and granulocyte) parameters between the control and 0.016% PGBE-treated groups.
The THI is a measure that accounts for the combined effects of environmental temperature and relative humidity on cattle to assess the risk of heat stress and prevent major effects. Heat stress causes changes in homeostasis and has been quantified by the measurement of milk production and physiological variables such as body temperature, respiratory rate, and behaviors of cows [
23]. According to a study by Gernand et al [
24], milk production is not affected by heat stress when mean THI values are under 68. However, milk production and feed intake begin to decline when beyond THI 68 [
24,
25]. During the experimental period of 40 days, animals were subjected to mild to moderate stress conditions (temperature, 25.4°C±2.58°C; humidity, 58.8%±14.15% [THI = 73.0±3.05]) in the current study (
Supplementary Figure S2). Considering the conditions using THI values, we further evaluated the data to determine the effects on milk yield and behavior patterns (rumination, eating, and ear surface temperature) of cows based on THI values such as stress threshold (temperature, 19.2°C±0.55°C; humidity, 78.8%± 4.53% [THI = 65.6±1.05]) and mild to moderate stress conditions (temperature, 26.1°C±0.33°C; humidity, 57.0%±2.29% [THI = 73.8±0.38]) (
Table 5). Milk yield between groups was not affected by THI, dietary supplementation, or THI interaction. For milk yield, however, cows experiencing mild to moderate stress (THI = 73.8) in the control group demonstrated tendency of a reduction of milk yield (p = 0.0781) compared with cows under the stress threshold (THI = 65.6). By contrast, when the THI value exceeded 72 (average THI = 73.8), the milk yield in 0.016% PGBE-treated group was greater as compared to that in the control group (p<0.05). Moreover, we observed a tendency of increase in milk yield of cows in the PGBE-treated group as compared to the control group under stress threshold conditions (p = 0.0897), suggesting that incorporation of 0.016% PGBE into the diet improved the milk yield as compared to non-treatment (control) under the stress threshold and even in mild to moderate stress conditions. Rumination is reduced in cows experiencing heat stress [
26]. A study by Moallem et al [
26] reported that the primary negative effect of THI is depression of rumination time, which subsequently leads to a reduction in DMI, followed by decreased milk yield. In the current study, the mild to moderate stress (THI = 73.8) led to a lower percentage of rumination time of cows in the control group compared to the initial period (control = −2.2%). There was no difference in the percentage of rumination time of cows in the PGBE-treated groups according to THI value. When THI was 73.8, the rumination time of cows in the 0.016% PGBE-treated group showed a tendency of an elevation compared to that in the control group (control = −2.2% vs 0.016% PGBE = 0.8%, p = 0.0914). In the increment, all cows showed a decline in eating and ear surface temperature as THI increased compared to the initial period. Cow behavior studies have shown that cows suffering heat stress eat more frequent, smaller meals and, to increase the surface area available for dissipating body temperature, stand for a longer time and pant as ambient temperature increases [
27]. Chewing and rumination might be impaired by panting in heat-stressed cows, particularly during the daytime. Despite a reduction in rumination in the control group, increased rumination of cows in the PGBE-treated group during the mild to moderate stress period was observed in the current study. We can hypothesize that cows supplemented with 0.016% PGBE might prevent deterioration of depression of rumination time, which subsequently leads to a reduction in DMI and milk yield, when THI was increased
In conclusion, the lactating dairy cows used in this study suffered mild to moderate heat stress throughout the experimental period (THI = 73.0). The milk production of cows experiencing heat stress in this trial showed an increase with supplementation of 0.016% PGBE complex in the diet. Besides showing antioxidant potency and anti-inflammatory ability, the PGBE complex (1:1:1 ratio, vol/vol), as shown in the aforementioned results in
Table 2 and
Supplementary Figure S1, prevented a decrease in the milk yield of cows during some periods of the trial (THI = 73.8). In particular, rumination time was negatively affected by hot conditions, but cows supplemented with PGBE complex exhibited increased rumination compared to the control. Our results suggest that incorporation of a combined mixture of PGBE to diet has the potential to improve milk yield and health status of cows under mild to moderate heat stress. Therefore, 0.016% PGBE (1:1:1 ratio, vol/vol) might be useful as an alternative anti-stressor in the diet of lactating dairy cows under hot conditions.