Abstract
Effects of dietary biotin supplementation on serum biotin levels and physical properties of sole horn of 40 Holstein cows were evaluated. The mean serum biotin level in biotin-supplemented cows after 10 mo of biotin supplementation (1163.2 ± 76.2 pg/mL) was significantly higher (P = 0.007) than that in control cows (382.0 ± 76.2 pg/mL). The sole horn of biotin-supplemented cows was significantly harder (P = 0.026) and had a significantly lower moisture content (P = 0.021) than that of control cows. No morphologic differences in horn tubules or intertubular horn were found between the biotin-supplemented and control cows. The total lipid content of sole horn was significantly higher (P = 0.030) in the biotin-supplemented cows than in the control cows. These results suggest that dietary biotin supplementation causes increases in serum biotin levels and changes in physical properties and fat content of sole horn.
Résumé
Une évaluation des effets d’un apport alimentaire supplémentaire en biotine sur les niveaux sériques de biotine et les propriétés physiques de la corne de la sole a été faite sur 40 vaches de race Holstein. Après 10 mois de supplément de biotine le niveau sérique moyen de biotine chez les vaches avec apport supplémentaire (1163,2 ± 76,2 pg/mL) était significativement plus élevé (P = 0,007) que chez les vaches témoins (382,0 ± 76,2 pg/mL). La corne de la sole des vaches avec supplément de biotine était significativement plus dure (P = 0,026) et avait un contenu en humidité significativement plus faible (P=0,021) que les vaches témoins. Aucune différence morphologique dans les tubules de la corne ou dans la corne inter-tubulaire n’a été détectée entre les vaches avec apport supplémentaire et les vaches témoins. Le contenu en lipide total de la corne de la sole était significativement plus élevé (P = 0,030) chez les vaches avec apport en biotine comparativement aux vaches témoins. Les résultats suggèrent qu’un apport supplémentaire alimentaire en biotine entraîne une augmentation des niveaux sériques de biotine et des changements dans les propriétés physiques et le contenu en gras de la corne de la sole.
(Traduit par Docteur Serge Messier)
Introduction
Hoof disease is one of the most serious problems in the dairy industry. It leads to reductions in milk yield and fertility and to shortening of productive life (1). Hoof disease can be caused by various factors, including housing, behaviour, genetics, presence of infective agents, and nutrition (1).
We have reported that serum biotin levels were clearly different in normal cows versus those with laminitis and that the levels were closely related to claw horn quality (2). Biotin, like thiamine, is a sulfur-containing, water-soluble vitamin (3). Of its 8 isomers, only D-biotin is physiologically active, and it is the only form that occurs naturally (3). Biotin acts as a cofactor for enzymes and plays a key role in glycogenesis, fatty acid synthesis, and amino acid catabolism (4,5). Biotin is essential for normal differentiation of epidermal tissue, which is required for normal production of keratin and keratinization of claw horn tissue (6). The changes in claw horn structure during the course of laminitis resemble those observed in biotin-deficient calves (7). Biotin supplementation is effective for the prevention and healing of hoof disease in dairy cattle (8–12). However, its effect on the physical properties of claw horn has not been fully clarified.
The aim of this study was therefore to determine the effects of biotin supplementation on serum biotin levels and the physical properties of the sole horn in Holstein cows.
Materials and methods
Cows
The study was conducted on an experimental farm of Rakuno Gakuen University, Hokkaido, Japan. By random sampling methods, 40 Holstein–Friesian cows aged 2.8 to 7.2 y were divided into a biotin-supplemented group and a control group of 20 cows each. Cows from each group were then matched for stage of lactation. All of the cows were housed in tie stalls with concrete floors. In each stall, barley straw was used as bedding material (approximately 10 cm thick) on top of a rubber mat. The cows were given grass silage, alfalfa, and concentrate feed according to Japanese Feeding Standards (13), and they were milked twice a day. The mean 305-d mature equivalent milk production of milking cows in the herd was 8623 ± 2158 kg. Each day, each cow in the biotin–supplemented group was given 50 g of biotin-supplemented feed that included 20 mg of D-biotin.
Serum biotin level
Before the start of biotin supplementation and every month during the experimental period, a 10-mL blood sample was drawn from the coccygeal vein of each cow and allowed to clot. Serum biotin levels were determined by an enzyme-linked assay described by Bayer, Benhur, and Wilchek (14).
Sole horn condition and hoof trimming
Diagnoses of claw disorders were based on clinical signs, including pain, lameness, and abnormal stance, and lesions of the horn consisting of fairly extensive yellow and red discolouration of the horn of the sole and wall. We checked the condition of the claw horn at the start of the experiment and once a month during the experimental period, and no abnormalities in claw horns were observed in any of the cows. Hoof trimming was performed at the start of and during the 6th mo of the experimental period.
Physical properties of the sole horn
Physical properties of the sole horn were determined before and 10 mo after the start of biotin supplementation. The inner and outer claws of the front and rear hooves of each cow were brushed, and horn tissue was cut away to a depth of about 1 to 2 mm to produce a clean surface. A sample of horn tissue to a depth of about 2 to 3 mm was obtained; the sampling site is shown in Figure 1. The samples were dried at 180°C for 5 h to calculate their moisture content. Hardness was measured 3 times at the apex of the sole with a durometer, according to the method described by Baggot, Bunch, and Grill (15).
Figure 1.
Illustration of zones of the cow sole horn, including sampling site (•).
Morphology of horn tubules
Morphologic analysis was performed by the method of Kuwano (16) before and 10 mo after the start of biotin supplementation. Briefly, sole horn samples from the outer claws of the rear hooves (Figure 1) were fixed with 10% neutral formalin, dehydrated in a graded ethanol series, and embedded in resin. After polymerization, sections were cut to a width 0.5 to 1.0 mm with a diamond saw and then ground to a thickness of 30 to 40 μm. The sections were then stained with hematoxylin and eosin.
Total lipid content of sole horn
Total lipid content of the sole horn was determined, according to the method of Scaife, Meyer, and Grant (17), before and 10 mo after the start of biotin supplementation. Samples of sole horn from the outer claws of the rear hooves (Figure 1) were reduced to fine shavings with a rasp. Lipid extraction was performed at room temperature for successive intervals of 1 h in a 15-fold excess of chloroform–methanol (2:1). Total lipid was separated and the weight of the fat determined.
Statistical analysis
The data were analyzed by analysis of variance and Tukey’s t-test. Values of P < 0.05 were regarded as significant. Values are expressed as mean ± standard deviation (s).
Results
Serum biotin level
The changes in serum biotin level are shown in Figure 2. The mean serum biotin level in the biotin-supplemented cows after 10 mo of biotin supplementation was 1163.2 ± 76.2 pg/mL, significantly higher (P = 0.007) than that in the control cows (382.0 ± 76.2 pg/mL).
Figure 2.
Serum biotin levels (mean and standard deviation) in biotinsupplemented cows (•) and control cows (○). Asterisks indicate a significant difference (P < 0.001) from the mean for the control cows.
Hardness of sole horn
Before biotin supplementation, the hardness of sole horn of the 2 groups of cows was similar (Table I). After 10 mo of biotin supplementation, the inner and outer claws of the front hooves of the biotin-supplemented cows (52.0 ± 4.9 and 51.7 ± 3.4 units, respectively) were significantly harder (P = 0.026) than those of the control cows (45.2 ± 4.3 and 43.8 ± 5.2 units, respectively). After 10 mo of biotin supplementation, the inner and outer claws from the rear hooves of the biotin-supplemented cows (47.5 ± 2.7 and 46.9 ± 3.2 units, respectively) were also significantly harder (P = 0.018) than those of the control cows (42.8 ± 3.0 and 42.1 ± 3.5 units, respectively).
Table I.
Hardness of sole horn of biotin-supplemented and control cows before and after 10 mo of supplementation
| Hardness (unit)
|
|||
|---|---|---|---|
| Hoof and claw | Months of supplementation | Biotin-supplemented group | Control group |
| Front | |||
| Inner | 0 | 44.0 ± 4.2 | 44.8 ± 5.3 |
| 10 | 52.0 ± 4.9a | 45.2 ± 4.3 | |
| Outer | 0 | 42.8 ± 3.7 | 43.6 ± 5.4 |
| 10 | 51.7 ± 3.4a | 43.8 ± 5.2 | |
| Rear | |||
| Inner | 0 | 42.2 ± 2.6 | 41.9 ± 2.2 |
| 10 | 47.5 ± 2.7b | 42.8 ± 3.0 | |
| Outer | 0 | 43.2 ± 3.2 | 43.0 ± 5.1 |
| 10 | 46.9 ± 3.2b | 42.1 ± 3.5 | |
Significantly different (P = 0.026) from the value for the control cows
Significantly different (P = 0.018) from the value for the control cows
Moisture content of sole horn
Before biotin supplementation, the moisture content of the sole horn was almost the same in the 2 groups of cows (Table II). After 10 mo of biotin supplementation, the moisture content of the inner and outer claws of the front hooves of the biotin-supplemented cows (37.1% ± 3.3% and 36.1% ± 4.1%, respectively) was significantly lower (P = 0.021) than that of the control cows (44.0% ± 2.9% and 42.1% ± 2.1%, respectively). After 10 mo of biotin supplementation, the moisture content of the inner and outer claws of the rear hooves of the biotin-supplemented cows (44.2% ± 2.9% and 43.1% ± 3.0%, respectively) was also significantly lower (P = 0.016) than that of the control cows (49.3% ± 3.5% and 47.6% ± 3.2%, respectively).
Table II.
Moisture content of sole horn of biotin-supplemented and control cows
| Moisture content (%)
|
|||
|---|---|---|---|
| Hoof and claw | Months of supplementation | Biotin-supplemented group | Control group |
| Front | |||
| Inner | 0 | 42.5 ± 2.6 | 43.3 ± 2.2 |
| 10 | 37.1 ± 3.3a | 44.0 ± 2.9 | |
| Outer | 0 | 42.2 ± 2.3 | 41.0 ± 3.0 |
| 10 | 36.1 ± 4.1a | 42.1 ± 2.1 | |
| Rear | |||
| Inner | 0 | 48.5 ± 4.3 | 48.9 ± 2.9 |
| 10 | 44.2 ± 2.9b | 49.3 ± 3.5 | |
| Outer | 0 | 48.2 ± 5.6 | 48.5 ± 3.8 |
| 10 | 43.1 ± 3.0b | 47.6 ± 3.2 | |
Significantly different (P = 0.021) from the value for the control cows
Significantly different (P = 0.016) from the value for the control cows
Morphology of horn tubules
No morphologic differences were found between the sole horn of the biotin-supplemented cows and that of the control cows (Figure 3) or between the right and left hooves or the inner and outer claws of the 2 groups of cows.
Figure 3.
Horn tubules of the outer claws of the rear hooves of biotinsupplemented and control cows. Hematoxylin and eosin stain. Bar — 1 mm.
Total lipid content of sole horn
Before biotin supplementation, the total lipid content of the sole horn was similar in the 2 groups of cows (Table III). After 10 mo of biotin supplementation, the mean value for the biotin-supplemented cows (32.2 ± 3.2 mg/g) was significantly higher (P = 0.030) than that for the control cows (26.2 ± 3.0 mg/g). Significant differences in total lipid content of sole horn were not detected between the right and left hooves or between the inner and outer claws of the 2 groups of cows.
Table III.
Total lipid content of sole horn of biotin-supplemented and control cows
| Total lipid content (mg/g)
|
||
|---|---|---|
| Months of supplementation | Biotin-supplemented group | Control group |
| 0 | 23.8 ± 2.6 | 25.2 ± 2.8 |
| 10 | 32.2 ± 3.2a | 26.2 ± 3.0 |
Significantly different (P = 0.030) from the value for the control cows
Discussion
Biotin supplementation is effective for the prevention of hoof disease in dairy cattle (8–12). In this study, we examined the effects of biotin supplementation on serum biotin levels and the physical properties of sole horn in Holstein cows. After 1 mo of supplementation, the mean serum biotin level in biotin-supplemented cows was significantly higher than that in control cows. Claw horn takes approximately 3 mo to grow from the stratum basale (basal layer of keratin-producing cells) to the stratum corneum (horny layer) (18). The fact that the serum biotin concentrations in the biotin-supplemented cows remained high indicates that a 10-mo period of supplementation was sufficient for evaluating the effect on claw horn.
The front and rear sole horn of the biotin-supplemented cows was significantly harder than that of the control cows. Claw-horn hardness depends on the moisture content of the claw horn (19), and there is a negative correlation between hardness and moisture content. The moisture content of the inner and outer claws of the front and rear hooves of the biotin-supplemented cows was significantly lower than that of the control cows, indicating that the increase in horn hardness was due to a decrease in moisture content.
Moisture content is related to the microarchitecture (tubules and intertubular horn) and the biochemical composition of the horn (19). The tubules and the intertubular horn are composed of scleroproteins and fat and are crucial for the regulation of moisture content in the claw horn. High-quality horn has a large number of tubules per square millimetre (20), and the hardness of the horn is directly related to the number of tubules per unit area. We found no morphologic differences in horn tubules or intertubular horn between the biotin-supplemented and control cows. In a study of biotin-treated horses, Geyer and Schulze (21) found that the tubular structure had improved 16 mo after biotin supplementation and that the marrow was smaller than before, although they observed some decayed cortical cells microscopically. We speculate that biotin supplementation for 10 mo is sufficient to evaluate the physical properties of horn but not to evaluate histologic improvement.
The water content of horn is also related to the horn’s biochemical composition (22). Biotin acts as a cofactor with enzymes and plays a key role in glycogenesis, fatty acid synthesis, and amino acid catabolism (4,5). In this study, the total lipid content of sole horn of biotin-supplemented cows was significantly higher than that of control cows. Lipid is crucial to the regulation of water permeability of the claw (22). Our results suggest that an increase in the total lipid content of the sole horn induced by biotin supplementation is one of the factors causing changes in hardness and water content of sole horn in biotin-supplemented cows.
This study was performed in 1 herd of cows with 1 diet and 1 type of housing. It is therefore necessary to examine the effect of biotin supplementation in Holstein cows in other dairy farms, with the same experimental design.
Our results should stimulate further studies aimed at elucidating the mechanisms by which biotin supplementation prevents and improves claw horn disease.
Acknowledgments
The authors thank Dr. Eliott, Roche Products, Sydney, Australia, for his helpful comments and Dr. Tsuguaki Ohkuma, Roche Japan, Tokyo, for his help in the preparation of this article.
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