Effects of β-carotene-enriched dry carrots on β-carotene status and colostral immunoglobulin in β-carotene-deficient Japanese Black cows
Abstract
Data from 18 β-carotene-deficient Japanese Black cows were collected to clarify the effects of feeding β-carotene-enriched dry carrots on β-carotene status and colostral immunoglobulin (Ig) in cows. Cows were assigned to control or carrot groups from 3 weeks before the expected calving date to parturition, and supplemental β-carotene from dry carrots was 138 mg/day in the carrot group. Plasma β-carotene concentrations in the control and carrot groups at parturition were 95 and 120 μg/dL, and feeding dry carrots slightly improved plasma β-carotene at parturition. Feeding dry carrots increased colostral IgA concentrations in cows and tended to increase colostral IgG1, but colostral IgM, IgG2, β-carotene and vitamin A were not affected by the treatment. Feeding dry carrots had no effects on plasma IgG1, IgA and IgM concentrations in cows, but plasma IgG1 concentrations decreased rapidly from 3 weeks before the expected calving date to parturition. These results indicate that feeding β-carotene-enriched dry carrots is effective to enhance colostral IgA and IgG1 concentrations in β-carotene-deficient cows.
Introduction
Mortality and morbidity of neonates continue to be major problems in calves, and their most common disease is diarrhea, which can cause growth retardation and death of calves. Passive immunity is critical to the survival and health of neonates, and colostrum is a source of nutrients and immune components for neonatal calves (Blum 2006). Immunoglobulin (Ig) antibodies are main immune components in colostrum and the most abundant Ig in bovine colostrum is IgG rather than IgA and IgM (Stelwagen et al. 2009). IgG1 is the most frequent IgG isotype in bovine colostrum, and research consistently shows the importance of a sufficient amount of colostrum containing 50 g/L or higher levels of IgG1 to feed newborn calves immediately after birth (Cozler et al. 2016). IgA is the most abundant Ig isotype in mucosal secretions and provides protection against microbial antigens at mucosal surfaces in the guts (Fagarasan & Honjo 2003; Mora & von Andrian 2009), and feeding whey protein enhances mucosal IgA induction in the guts of Japanese Black calves (Yasumatsuya et al. 2012). However, the improvement of colostral IgG and IgA in Japanese Black cows is needed for the appropriate calf health and immune system, because the lower transfer of IgG and IgA from colostrum to neonatal calves was found in Japanese Black calves at 2 days of age (Nishiyama et al. 2011a; Yasumatsuya et al. 2013).
Supplemental vitamin A and β-carotene enhance the immune system in neonates (Chew & Park 2004; Rühl 2007). Feeding β-carotene to pregnant cows affected not only colostral β-carotene contents in cows but also β-carotene status in newborn calves (Kume & Toharmat 2001), and colostral β-carotene was positively correlated with colostral IgG or IgM in Japanese Black cows (Taniguchi et al. 2016). Supplemental β-carotene drastically increased serum β-carotene in Japanese Black calves (Nishiyama et al. 2011a), but β-carotene-deficient calves were found to have a higher incidence of diarrhea in the first week of life (Kume & Toharmat 2001). On the other hand, supplemental β-carotene or β-carotene-enriched dry carrots had no effect on colostral IgG, IgA and IgM in cows fed high β-carotene diets (Kaewlamun et al. 2011; Taniguchi et al. 2015), although supplemental β-carotene improved β-carotene status in cows. However, supplementation of β-carotene-enriched feeds is expected to improve colostral Ig in β-carotene-deficient cows, because of the higher incidence of diarrhea in β-carotene-deficient calves (Kume & Toharmat 2001).
The objective of this study was to clarify the effects of feeding β-carotene-enriched dry carrots on β-carotene status and colostral Ig in β-carotene-deficient Japanese Black cows.
Materials and Methods
Experimental design
This research was approved by the guide for the care and use of animals in Shiga Prefectural Livestock Technology Promotion Center (Hino, Japan). Eighteen Japanese Black cows were assigned to the control (n = 9) or carrot (n = 9) group from 3 weeks before the expected calving date to parturition by parity and age (Table 1). The gestation length was assumed to be 285 days, and cows were managed in paddocks during the dry period. Body weights (mean ± SD) of cows in the control and carrot groups before the experiment were 546 ± 26 kg and 509 ± 48 kg, respectively.
| Control | Carrot | SEM | P | |
|---|---|---|---|---|
| Number of cows | 9 | 9 | ||
| Parity | 4.6 | 5.1 | 0.9 | 0.666 |
| Age, months | 83.2 | 93.2 | 12.1 | 0.568 |
| Gestation length, days | 294 | 288 | 1 | 0.004 |
| Calf birth weight, kg | 34.8 | 31.7 | 1.6 | 0.182 |
| Colostrum | ||||
| IgG1, mg/mL | 60.0 | 78.5 | 6.8 | 0.074 |
| IgG2, mg/mL | 3.3 | 3.6 | 0.3 | 0.550 |
| IgA, mg/mL | 6.3 | 11.3 | 1.2 | 0.009 |
| IgM, mg/mL | 8.3 | 11.0 | 1.1 | 0.111 |
| β-carotene, μg/dL | 94.8 | 129.1 | 20.7 | 0.752 |
| Vitamin A, μg/dL | 963 | 1314 | 243 | 0.321 |
Cows in the control group were given 2 kg/day of concentrate and appropriate amounts of Bermudagrass hay and Italian ryegrass silages in the ratio of 1:1 on a dry matter basis to meet the total dry nutrient requirements of Japanese Black cows (Agriculture, Forestry, and Fisheries Research Council Secretariat 2008). Vitamins A, D and E were supplemented in the concentrate, but β-carotene was not supplemented in the concentrate. According to the analysis of β-carotene in feed (Taniguchi et al. 2015), β-carotene concentrations in Bermudagrass hay and Italian ryegrass silages on a dry matter basis were 0.3 and 15.7 mg/kg, respectively. Dry carrots were provided by Chubu Shiryo Co. Ltd (Ohbu, Japan), and 300 g/day of dry carrots were offered to the cows in the carrot group. Dry carrots contained 8.0% crude protein (CP), 11.2% neutral detergent fiber (NDF), 1.4% crude fat, 57.6% nitrogen free extract (NFE), 8.1% crude ash and 460 mg/kg β-carotene. As a result, supplemental β-carotene from dry carrots in the carrot group was 138 mg/day.
Sample collection and analyses of plasma and colostral components
Body weights of calves were measured at birth. Blood samples of cows were collected at 16.00 hours on 21 and 7 days before the expected calving date, and samples of blood and colostrum at birth were collected approximately within 2 h after parturition. Samples of colostrum were taken from each udder of the cows by hand and 50 mL colostrum was stored at −20°C for chemical analyses. Blood was sampled by a jugular vein puncture into ethylene diaminetetraacetic acid (EDTA)-2Na vacuum tubes and then centrifuged at 1670 × g for 10 min.
The immunoassay of colostral and plasma IgG1, IgA and IgM and colostral IgG2 in cows was determined as previously described (Nishiyama et al. 2011a; Yasumatsuya et al. 2013). Colostral and plasma IgG1, IgA and IgM and colostral IgG2 were measured using the Bovine IgG1, IgG2, IgA and IgM ELISA Quantitation Kit (Bethyl Laboratories, Montgomery, AL, USA) and ELISA Starter Accessory Package (Bethyl Laboratories) according to the manufacturer's instructions. Colostral and plasma β-carotene and colostral vitamin A were determined by high-performance liquid chromatography as previously described (Taniguchi et al. 2015; Wang et al. 2015). Plasma glucose, total protein, nonesterified fatty acid (NEFA) and urea N were determined by an Automatic Analyzer (8060 or 9130; Hitachi High-Technologies, Tokyo, Japan).
Statistics
Data of plasma components in cows were analyzed by least squares analysis of variance using the General Linear Model procedure of SAS (1997). The model was as follows;
Yijk=μ + Ti+C(i)j+Dk+TDik+eijk where μ is the overall mean, Ti is the effect of treatment, C(i)j is the random variable of cows nested in treatment, Dk is the effect of sampling day, TDik is the interactions and eijkl is the residuals. The differences between treatment and sampling day were tested by Tukey‑Kramer's multiple comparisons.
The General Linear Model procedure of SAS (1997) was used to analyze the effect of treatment on parity, age, calf birth weight, gestation length, colostral components or the ratio of β-carotene in cows. Relationships between colostral IgG1, IgG2, IgA or IgM and age, calf birth weights or gestation length in cows were examined by correlation analyses of SAS (1997). Significance was declared at P < 0.05 and trends were taken as P < 0.10.
Results
One calf in the carrot group died by dystocia, but no metabolic disorders were detected in the other cows and the health status of their calves was good at birth. Calf birth weights were not affected by the treatment, but the gestation length was significantly shorter (P < 0.01) in the carrot group (Table 1). Colostral IgA concentrations in the carrot group were significantly higher (P < 0.01) than those in the control group, and colostral IgG1 concentrations tended to be higher (P < 0.10) in the carrot group. However, colostral IgG2, IgM, β-carotene and vitamin A concentrations were not affected by the treatment. There were no relationships between colostral IgG1, IgG2, IgA or IgM and age, calf birth weights or gestation length in cows.
Plasma β-carotene concentrations were not affected by the treatment, but plasma β-carotene concentrations in the carrot group were at low levels at 21 days before the expected calving date (Fig. 1). Additionally, plasma β-carotene in the control group decreased (P < 0.05) from 21 days before the expected calving date to parturition, although plasma β-carotene in the carrot group remained almost constant. As a result, the ratios of plasma β-carotene at 21 days before the expected calving date on plasma β-carotene at parturition in the control and carrot groups were 0.53 and 0.94, respectively, and the ratio at 0.94 in the carrot group tended to be higher (P < 0.10) than that at 0.53 in the control group. Plasma IgG1, IgA and IgM concentrations were not affected by the treatment. Compared with plasma concentrations at 21 days before the expected calving date, plasma IgG1 decreased (P < 0.001) at parturition, but plasma IgM increased (P < 0.01) at parturition.
Plasma glucose, total protein, NEFA and urea N concentrations in Japanese Black cows were not affected by the treatment, but plasma NEFA concentrations in the carrot group were high at 21 days before the expected calving date (Fig. 2). Compared with plasma concentrations at 21 days before the expected calving date, plasma glucose (P < 0.001) and NEFA (P < 0.05) increased at parturition, but plasma urea N decreased (P < 0.01) at parturition.
Discussion
Vitamin A deficiency is associated with an increased risk of death from common childhood infections and supplementation of vitamin A decreases diarrhea and mortality in malnourished children (Mora & von Andrian 2009), and a higher incidence of diarrhea occurred in the β-carotene-deficient calves at 6 days of age (Kume & Toharmat 2001). High-quality silages contain large amounts of β-carotene, but the wide range of β-carotene in plasma and colostrum of cows may be due to the variable dietary levels of β-carotene and the uptake of β-carotene by the guts and mammary glands (Kume & Toharmat 2001; Johansson et al. 2014; Wang et al. 2015). Dietary β-carotene requirements were not determined in Japanese Black cows (Agriculture, Forestry, and Fisheries Research Council Secretariat 2008), but the optimal blood β-carotene concentration in dairy cows was determined above 200 μg/dL (Chew 1993). In the present study, plasma β-carotene concentrations in cows from 21 days before the expected calving date to parturition were below 200 μg/dL, and plasma β-carotene concentrations in the control and carrot groups at parturition were 95 and 120 μg/dL. Compared with the data in a similar experimental design of Japanese Black cows fed silages (Taniguchi et al. 2015), their plasma β-carotene concentrations in the control and carrot groups at parturition were 378 and 475 μg/dL, which were about four times higher than those in the present study. As a result, β-carotene status in Japanese Black cows was thought to be deficient in the present study.
Compared with the carrot group, energy status in the control group before the experiment may be slightly deficient due to high plasma NEFA at 21 days before the expected calving date, but low plasma β-carotene was found in the carrot group at 21 days before the expected calving date. However, because plasma glucose, total protein, NEFA and urea N concentrations in cows before parturition agreed with those in previous studies (Watanabe et al. 2014; Taniguchi et al. 2015), energy and protein status of Japanese Black cows was thought to be normal in the present study. Additionally, β-carotene-enriched dry carrots may be useful to improve β-carotene status in β-carotene-deficient cows, because the ratios of plasma β-carotene at 21 days before the expected calving date on plasma β-carotene at parturition in the carrot group tended to be higher than that in the control group.
The importance of adequate consumption of high-quality colostrum for acquisition of optimal nutrition and passive immunity is widely recognized in neonatal calves (Quigley & Drewry 1998). The bovine mammary gland plays an active role in regulating Ig concentrations in colostrum for the host defense of the mammary glands in cows and sufficient passive immunity in neonatal calves (Stelwagen et al. 2009). In the present study, the most abundant Ig in colostrum of Japanese Black cows was IgG1 rather than IgG2, IgA and IgM, which agreed with the colostral Ig in dairy cows (Stelwagen et al. 2009).
Feeding high-quality silages may be effective for enhancing colostral IgG in cows, because high colostral IgG in Japanese Black cows were related with high colostral β-carotene, vitamin A and α-tocopherol (Taniguchi et al. 2016). On the other hand, supplemental β-carotene at 1 g/day in late gestation increased β-carotene concentrations in blood and colostrum of dairy cows, but colostral IgG was not affected by the treatment (Kaewlamun et al. 2011). In the present study, feeding β-carotene-enriched dry carrots increased colostral IgA concentrations and tended to increase colostral IgG1 in β-carotene-deficient Japanese Black cows, but no effects were observed in colostral β-carotene, vitamin A, IgG2 and IgM. Although aging is a factor altering colostral IgG in cows (Kume & Tanabe 1993; Wang et al. 2015) and the gestation length was shorter in the carrot group, colostral IgG1 and IgA concentrations in Japanese Black cows were not affected by aging and gestation length. These results indicate that feeding β-carotene-enriched dry carrots is effective to improve colostral IgA and IgG1 in β-carotene deficient cows.
Colostral IgG and IgM originate from bovine plasma, while colostral IgA is synthesized within the mammary glands by plasma cells which have migrated from the gastrointestinal tract to the mammary glands (Wheeler et al. 2007; Taniguchi et al. 2016). Colostral IgG1 concentrations in the control group as well as the carrot group were above the sufficient level of 50 mg/mL in dairy cows (Cozler et al. 2016), but colostral IgG1 in beef cows were greater than those in dairy cows (Gui et al. 1994). Feeding β-carotene-enriched dry carrots had no effects on plasma IgG1 concentrations in the present study, but plasma IgG1 decreased rapidly from 3 weeks before the expected calving date to parturition. These results imply that β-carotene-enriched dry carrots may have predominant effects on the increased IgG1 transfer from plasma to colostrum in cows, and the higher level of colostral IgG1 is needed for the proper health and immunity in Japanese Black calves.
Several effects of carotenoids are thought to be mediated by their metabolism to vitamin A and subsequent mediation of all-trans retinoic acid (RA) receptor and retinoid X receptor response pathways (Rühl 2007; Nishida et al. 2014). Supplemental β-carotene at 30 or 50 mg/kg in the diet increased the numbers of IgA antibody-secreting cells in the mammary glands in lactating mice and enhanced IgA transfer from maternal milk to neonatal mice (Nishiyama et al. 2011a, 2011b). Supplemental β-carotene at 27 mg/day drastically increased serum β-carotene and slightly improved fecal IgA in calves at 14 days of age (Nishiyama et al. 2011a). Because feeding β-carotene-enriched dry carrots increased colostral IgA concentrations in β-carotene-deficient cows in the present study, the improvement of colostral IgA by β-carotene-enriched dry carrots may be partly due to the increased transfer of IgA antibody-secreting cells in the mammary glands.
Acknowledgments
The present study was supported in part by a grant from Ito Kinenzaidan (Tokyo, Japan).
