Effects of Supplementing Brown Seaweed By-products in the Diet of Holstein Cows during Transition on Ruminal Fermentation, Growth Performance and Endocrine Responses

Experimental design and diets

The study involved an examination of fermentation characteristics in the rumen using in vitro batch culture in triplicate with supplementation of BSB at various levels, namely 0% (control), 2% (2% BSB), and 4% (4% BSB) of dry matter, having replaced hay in the basal diets. Basal diets consisted of a mixture with commercial concentrates and hay approximately in the ratio of 35:65 as incubation substrates (Tables 1 and 2).

Table 1

Chemical composition of feeds used in Experiments 1 and 2 (% of dry matter unless otherwise stated)

Table 2

Ingredient and chemical composition of experimental diets used in Experiments 1 and 2 (% of dry matter unless otherwise stated)

Preparation of rumen inoculum and incubation procedure

Rumen inoculum used was obtained from a Holstein cow equipped with a permanent ruminal cannula. Once being taken, rumen contents were transferred to a laboratory using a thermos bottle kept at 39°C, immediately after which the rumen content was filtered with four layers cheesecloth to remove feed particles, and stored in a constant temperature bath maintained at 39°C. The filtered rumen fluid was mixed with McDougal’s buffer (McDougall, 1948) bubbled with CO₂ in 1:1 ratio, and used as a rumen inoculum. First, 0.25 g of substrate was placed in a 120 mL serum bottle, into which 25 mL of rumen inoculum was added under anaerobic conditions. The bottles were plugged with a butyl-rubber stopper and aluminum cap, and were incubated in a shaking incubator (SI-600R, Lab. Companion, Seoul, Korea) maintained at 39°C, 120 rpm, where the incubation times were 0, 3, 6, 9, 12, and 24 h. After completion of each incubation time, the pH was measured immediately using a pH meter (inoLab pH Level 1, Weilheim, Germany), and supernatant was sampled after centrifugation at 3,000 rpm for 15 min, and stored at −20°C for the analyses of ammonia nitrogen (NH₃-N) and volatile fatty acids (VFA). For VFA analysis, 4 mL of supernatant was added to 1 mL of 25% HPO₃ and stored at −72°C.

Sample analysis

Dry matter (DM), crude protein, ether extracts, organic matter, Ca and phosphorus of basal diets and BSB used were analyzed according to the method of AOAC (1995), and acid detergent fiber and neutral detergent fiber were analyzed according to the method of Van Soest et al. (1991). Analysis of ammonia-N was determined by using a spectrophotometer (Optizen 1142H, MECASYS Co., Ltd, Daejeon, Korea) after color fixation in the same quantities for phenol and alkali according to Chaney and Marbach (1962). An analysis of VFA was made with gas chromatography (VARIAN CP-3800, Varian Inc., Walnut Creek, CA, USA) according to the method by Erwin et al (1961) with a fused silica capillary column (30 m×0.32 mm, 0.5 μm film thickness, HP Innowax, Agilent Technologies Inc., Santa Clara, CA, USA) equipped with flame ionization detector.

Experimental design and animals

Initially, twenty-one Holstein heifers (average body weight 419 kg) were randomly divided into 3 groups, consisting of control, 2% (2% BSB) and 4% (4% BSB) supplementation of BSB by replacing a relatively similar portion of tall fescue based on nutritional requirements (Tables 1 and 2), and the experiment was conducted for 12 months at the research farm of Seoul National University, Korea. All experimental procedures were in accordance with the “Guidelines for Care and Use of Experimental Animals of Konkuk University”.

Sample collection and analysis

Ten-mL of blood was collected from the jugular veins in a heparin-treated vacuum tube (BD Vacutainer Systems Preanalytical Solutions, Becton, Dickinson and Company, Franklin Lakes, NJ, USA) at 9:00 AM before the start of the experiment, on 120th day and 240th day of pregnancy. After collection, the plasma was separated by centrifugation (4°C, 3,000 rpm, 15 min), and stored at −70°C until further analysis. Milk production was recorded daily by automatic recording with ALPRO SYSTEM (Alfa-Laval Agri., Peterborough, Ontario, Canada), and 30 days after parturition the milk was analyzed for milk protein, milk fat, lactose and solid-not-fat (SNF) using Milkoscan-133B (Foss, Hilleroed, Denmark), and the number of somatic cells was determined by using Fossomatic-300 (Foss).

Analysis of total estrogen, progesterone, triiodothyronine (T3) and thyroxine (T4) in blood was as follows; Total estrogen was measured by radioimmunoassay (RIA) method using DA Total Estrogen kit (ICN Biomedicals, Irvine, CA, USA), and progesterone measured using CoAT-A-OUNT Progesterone kit (Diagnostic Products Corporation, Los Angeles, CA, USA). T3 and T4 were determined by RIA method using RIA-mat-T3 and RIA-mat-T4 kits (Byk-Sangtec Diagnostica, Dietzenbach, Germany).

Animal performance

Animal performance in terms of daily gain and feed intake of Holstein cows is presented in Table 5. Daily gains with an average weight of 419 kg for a total of 275 days were approximately 0.7 kg/d for all three treatments as expected, indicating that addition of BSB did not influence daily gains. Alginic acids composing cell walls of seaweeds have been reported to have characteristics of forming viscous gel so that palatability preference is lowered, accompanied by a reduction in feed intakes (Beresford et al., 2000). Franklin et al. (1999) also reported that DM intake was significantly reduced as a result of daily supplementation of seaweeds for milking cows. On the other hand, the levels of supplement in the present study are not considered to have major impact on DM intake, as similar results were observed between treatments where total DM intakes were 10.58, 10.36, and 10.49 kg for the control, 2% BSB and 4% BSB, respectively. However, as this experiment was conducted with a group feeding regime and the results were presented with a mean value of all animals within a treatment, statistical analysis of the feed intake was not provided.

Table 5

Effects of dietary BSB supplementation on growth performance for 9 months in Holstein heifers in Experiment 2

Endocrine responses

Influences of adding BSB on various hormones in blood are given in Table 6. The pattern of estrogen before and after pregnancy indicated a normal pattern of estrogen. In terms of both estrogen and progesterone, no particular differences per stage were indicated among treatments. T3 and T4 are hormones synthesized in the thyroid glands, where I is an important precursor for the synthesis. Pattanaik et al. (2001) reported that contents of T3 and T4 in plasma had increased significantly compared with the control when I at the level of 0.05 mg and 0.075 mg per head a day was added in the basic feeds for goats. However, a pattern of increase in hormone synthesis in the thyroid glands was not exhibited per stage in the present study. In the case of T3, only a slight increase of 3.83% and 5.87% was observed when compared with the beginning of the study for a pregnancy of 120 days and 240 days, respectively.

Table 6

Effects of dietary BSB supplementation on endocrine response of Holstein dairy heifers in Experiment 21

Milk production and composition

Milk productions and milk composition 1 month after delivery of dairy cows are given in Table 7. DM intakes showed similar results among treatments with 21.05 kg, 20.88 kg, 20.52 kg for the control, 2% BSB, 4% BSB, respectively. Although the concentrations of estrogen and progesterone during pregnancy period, which tended to show higher values for 2% BSB compared with other treatments, were expected to influence duct development causing differences in milk production, no differences in milk production were brought about with resultant values of 27.14 kg, 28.77 kg, 27.65 kg corresponding to the control, 2% BSB and 4% BSB, respectively at 4th week of lactation. Contents of milk protein, milk fat, lactose, SNF were shown to be similar among treatments, indicating that addition of BSB during pregnancy period did not have an influence in the lactation period. Our results are somewhat different compared with previous reports. For example, Bendary et al. (2013) reported increased milk yield when dairy cows were supplemented with the mixture of seaweed and premix, and another study also has shown a similar result, an increased milk yield in high-yielding dairy cows (Hostens et al., 2011). The discrepancy in terms of milk yield between previous studies and our results remains to be determined. It is interesting to note, however, that in the report by Kellogg et al. (2006) where they concluded from a series of studies that supplementation of commercially available brown seaweed such as Tasco (Acadian Seaplants Ltd., Dartmouth, Nova Scotia, Canada) is beneficial for milk production in large dairy cows during moderate to severe heat stress, but not with cows at first-lactation where the body size is still relatively small compared with mature cows.

Table 7

Effects of dietary BSB supplementation on DMI, milk yield and composition of Holstein dairy cows in Experiment 2