Use of microseaweeds (Chlorella pyrenoidosa) as a probiotic in dairy goats feeding

Abstract

Ten Majorera dairy goats were divided into two groups in order to observe the effects of the Chlorella pyrenoidosa oral administration on the colostrum and milk quality and on the animals' immune status. Treated animals received 5 g/day of seaweed from 40 days before partum to 40 days after partum, and blood, colostrum, and milk samples were obtained during the experimental period. No effects of the seaweed addition were observed on blood plasma IgG or chitotriosidase (CHT) activity, neither on colostrum/milk IgG, CHT activity nor on fatty acid profile.

Keywords:

• Chlorella pyrenoidosa
• IgG
• colostrum
• milk

1. Introduction

Recently, Argüello (2011) has reported the interest for new raw material in goat nutrition. That interest is shared with other livestock as hens (Celebi et al. 2011; Khan et al. 2011; Najafzadeh et al. 2011), broilers (Masoudi et al. 2011; Darabighane et al. 2012), rabbits (Djakalia et al. 2012), cattle (Jacob et al. 2012), or sheep (Llorente et al. 2011; Mota et al. 2011; Tirado-Estrada et al. 2011).

In particular, seaweed has been used for centuries. However, the cultivation of microseaweeds is only a few decades old (Spolaore et al. 2006). Nowadays, productive methods have been developed for the cultivation at high scale of many microseaweeds species. Microseaweeds can be incorporated into the feed for a wide variety of animals ranging from fish (aquaculture) to pets and farm animals as it was stated before. Many nutritional and toxicological evaluations have proved the suitability of algal biomass as feed supplement (Becker 2007).

With the incorporation of these microalgae to the livestock feeding, two goals are wanted: improving the quality of the products enhancing the nutritional quality and, on the other hand, reinforce the immune system of these animals. Changes in the composition of the milk and meat from supplemented animals have been reported (Moreno-Indias, Sanchez-Macias, Martinez-de la Puente et al. 2012), while, seaweeds positively affect the physiology and the external appearance of animals, for example.

Many genera have been used for animal feeding, being the most important: Arthrospira and Chlorella. Over 50% of the current world production of Arthrospira is used as feed supplement (Yamaguchi 1996), while Chlorella is produced by a wide number of companies (Spolaore et al. 2006). However, there is not a consensus about the concentration needed to produce the benefits. Most of the studies developed with microseaweeds used high amounts: Bichi et al. (2013) used 8 g/kg of dry matter; Toral et al. (2010) used among 8 and 24 g/kg of dry matter, both working with dairy ewes; and Papadopoulos et al. (2002) used until 94 g/ration of microseaweeds. These studies mainly found an enrichment of poly unsaturated fatty acid (PUFA) in milk due to the microseaweeds used are rich in these fatty acids. However, most of the studies are not focused on immunological functions. Studies in rats have demonstrated that Chlorella reinforces the immune system and protects rodents against bacteria and cancer and improves the production of immunoglobulin (Kanouchi et al. 2001). In humans there are also evidences of these benefits (Merchant & Andre 2001).

Commercial microseaweed products are usually expensive, and the amount needed to develop the benefits sought is high. The present study investigates whether the use of a few amount of microseaweeds administered as an oral dietary supplement is enough to develop mainly an immunomodulatory effect and some other benefits on the milk quality.

2. Material and methods

2.1. Animals

Experimental animal procedures were approved by the Ethical Committee of the Universidad de Las Palmas de Gran Canaria (ULPGC). Ten multiparous Majorera dairy goats from the ULPGC's farm (Arucas, Spain) were randomly assigned into two experimental groups according to the diet: control group (CG) received corn, soy 66, dehydrated Lucerne, and dehydrated beetroot, wheat straw and a vitamin–mineral corrector according to the guidelines of L'Institute National de la Reserche Agronomique (Jarrige 1990); the microseaweed group (SG) was fed with the same diet as CG plus 5 g/day of C. pyrenoidosa, and orally administered by a syringe. This procedure was used from 40 days before the expected parturition date to 40 days after partum. Dairy goats enrolled in the present study had been through the dry period during 2 months and did not show any health problems during the experimental period.

2.2. Samples

Goats were milked in a double stall parallel milking parlor (Alfa-Laval, Madrid, Spain) once a day (08:00). Colostrum and milk samples (100 ml from the whole available milk) of each goat were collected after milking, at partum and at day 1, 2, 3, 4, 5, 10, 20, 30, and 40 after parturition. All samples were collected into two aliquots: 50 ml of each was used to measure the chemical composition (fat and protein) immediately after sampling and 50 ml was frozen at −80 °C until fatty acid composition, IgG concentration and chitotriosidase activity (CHT) determination.

Blood samples were collected from the jugular vein immediately before the first treatment and onward, 7 days before the expected parturition date, at partum, 5, 10, 20, 30, and 40 days of lactation. Blood samples (5 ml) were collected in heparinized tubes. After collection, blood samples were centrifuged (2136 g, 5 min, 4 °C) and the obtained plasma was frozen at −80 °C in aliquots, until analysis.

2.3. Milk quality

Proximal composition (fat and protein) colostrum and milk contents were determined by routine laboratory procedures using the automated infrared method a DMA2001 Milk Analyzer (Miris Inc., Uppsala, Sweden).

Fatty acid profiles were determined as follows: milk fat was extracted according to the Rose-Gottlieb method, and fatty acid methyl esters were obtained as described by Moreno-Indias, Morales-delaNuez et al. (2012). Separation and quantification of the methyl esters were carried out on a gas chromatograph (Varian 3600; Varian, Harbor City, CA, USA) equipped with a split–splitless injector and a flame ionization detector. Methyl ester separation was carried out on a capillary column SP2560 (100 m × 0.25 mm i.d., 0.25 µm phase thickness; Supelco Inc., Bellefonte, PA, USA) with helium as the carrier gas (331 kPa). The injector and detector temperatures were set at 290 °C. The injection was done in split mode with a 1:100 split ratio. The temperature of the column was initially held at 75 °C for 1.5 min, increased to 190 °C at a rate of 8 °C/min, held at this temperature for 25 min, increased again to 230 °C at 15 °C/min, and held for an additional 4.5 min at 230 °C. Each fatty acid was identified with reference to the retention time of the standards (Sigma-Aldrich, St. Louis, MO, USA) and quantified with respect to the following internal standards: C13:0 (C10:0–C17:0), and C19:0 (C18:0–C18:3).

2.4. Immune parameters

Immune status was studied based on IgG concentration and CHT activity in plasma and colostrum and milk samples. IgG quantification was performed using a goat IgG enzyme-linked immunosorbent assay (ELISA) kit (Bethyl Laboratories, Montgomery, TX, USA) and fluorimetric assay. ChT activity was measured according to Arguello et al. (2008) by incubating 1 µL of undiluted colostrum or milk with 100 µL of a solution containing 22 mM of an artificial substrate (4-methylumbelliferyl-d-N,N′,N″triacetylchitotriose) in 0.5 M citrate phosphate buffer pH 5.2, for 15 min at 37 °C. The reaction was stopped with 5 ml of 0.5 M Na₂CO₃–NaHCO₃ buffer, pH 10.7. Fluorescence was measured with a fluorimeter (Perkin Elmer, Norwalk, CT, USA) at 365 nm excitation and 450 nm emission. ChT activity is expressed as nanomoles of substrate hydrolyzed per milliliter per hour (nmol ml⁻¹ hr⁻¹).

2.5. Statistical analyses

Statistical analyses were performed using SAS, Version 9.00 (SAS Institute Inc., Cary, NC, USA). The SAS PROC MIXED procedure for repeated measures was used to evaluate the effect of the supplementation of C. pyrenoidosa on the colostrum and milk quality and the immune parameters. Tukey's test was used to evaluate the differences between groups.

3. Results and discussion

Proximal composition of the colostrum/milk samples is shown in Table 1. There was no significant effect of seaweed addition on milk fat percentage (8.87 vs. 8.70% at partum and 5.20 vs. 5.81% at day 40, seaweed and control group, respectively). In addition, fat percentage decreased throughout the experiment in both groups, ranged from 8.87 to 5.20% and 8.70 to and 5.81%, seaweed and control group, respectively. This evolution follows a normal trend of a lactation curve, where fat values are higher in colostrum samples than in milk samples (Arguello et al. 2006a). Some authors such as Toral et al. (2010) have reported a reduction in fat content when marine algae was used as a supplementation, something that is not observed in the present study, probably due to the fact that the concentration used acts as a dietary supplement, nor as part of the diet itself as in other studies. Milk protein percentage decreased throughout the experiment in both groups, ranged from 20.28 to 3.10% and 21.53 to 3.88%, seaweed and control group, respectively, milk protein being significantly higher at first day of lactation in the seaweed group (13.20 vs. 8.30%, seaweed and control group, respectively), probably due to the high level of IgG at this stage (Moreno-Indias, Sanchez-Macias, Castro et al. 2012). Colostrum high protein content at birth is necessary for the offspring, as it has been reported by Arguello, Castro, Capote, Tyler et al. (2004).

Table 1. Colostrum and milk proximal compositions (fat and protein percentages) from partum to day 40.

		Days
		0	1	2	3	4	5	10	20	40	SEM
Fat (%)	SG	8.87^a	7.37^ab	7.54^ab	8.13^a	5.87^bc	6.11^bc	5.51^c	4.84^c	5.20^c	0.63
	CG	8.70^a	7.94^ab	7.25^abc	6.41^bc	5.97^cd	6.38^bcd	5.93^cd	5.62^cd	5.81^cd	0.70
Protein (%)	SG	20.28^a	13.20^bx	8.84^c	5.91^d	5.87^d	5.66^d	4.66^de	4.68^de	3.10^e	0.79
	CG	21.53^a	8.30^by	6.56^b	6.36^bc	5.64^cd	5.41^cd	4.56^cd	3.78^d	3.88^d	0.88

^a–e Means within a row with different superscript letters differ significantly (P < 0.05).

Regarding the fatty acid profiles, no differences were observed in individual fatty acids between groups, although these profiles were clearly influenced by the time, showing different evolutions (Table 2). Some fatty acids showed a tendency in their evolutions, such as C10:0 and C12:0 which were increased, or C16:0 which was decreased, this is according to findings described in other studies which established increases in the supply of long chain fatty acids which alter the synthesis of short- and medium-chain saturates (Chilliard et al. 2007).

Table 2. Evolution of colostrum and milk fatty acids profiles from day 0 to day 40.

		Days
		0	1	2	3	4	5	10	20	30	40	SEM
C4:0	SG	0.804^b	0.808^b	1.154^ab	1.188^ab	1.350^a	1.308^a	1.138^ab	1.088^ab	0.650^b	0.934^ab	0.074
	CG	0.790^b	1.015^b	1.308^ab	1.418^ab	1.148^b	1.103^b	1.203^ab	1.165^ab	1.673^a	0.787^b	0.085
C6:0	SG	0.742^c	0.920^bc	1.242^b	1.222^b	1.438^ab	1.422^ab	1.296^ab	1.530^ab	1.982^a	1.600^ab	0.094
	CG	0.707^c	0.945^c	1.202^bc	1.498^b	1.275^bc	1.310^bc	1.545^b	1.567^b	2.500^a	1.288^bc	0.150
C8:0	SG	0.766^d	0.998^d	1.248^cd	1.390^cd	1.730^bc	1.668^bc	1.580^c	2.108^bc	2.698^a	2.150^b	0.182
	CG	0.748^c	0.977^c	1.290^c	1.680^bc	1.540^bc	1.595^bc	2.057^b	2.128^b	3.348^a	2.045^b	0.230
C10:0	SG	2.586^d	3.154^d	3.744^cd	4.054^cd	4.730^cd	5.012^c	5.392^c	7.814^b	9.730^a	10.132^a	0.847
	CG	2.725^d	3.100^cd	3.947^cd	5.060^c	5.055^c	5.072^c	7.055^bc	7.338^b	11.748^a	8.412^b	0.867
C12:0	SG	1.494^c	1.578^c	1.674^bc	1.788^bc	2.092^bc	2.216^bc	2.404^b	3.562^a	4.182^a	4.150^a	0.333
	CG	1.628^c	1.563^c	1.740^c	2.110^c	2.225^bc	2.310^bc	2.888^bc	3.110^b	4.850^a	4.028^a	0.344
C14:0	SG	7.993^b	7.213^b	7.406^b	6.648^b	9.250^ab	6.468^b	9.855^a	6.586^b	9.793^ab	6.809^b	0.427
	CG	9.467^ab	7.006^bc	8.438^b	6.311^c	8.866^ab	7.177^bc	10.663^a	7.079^bc	10.601^a	6.931^bc	0.503
C16:0	SG	29.831^a	27.709^b	25.261^c	26.142^bc	25.858^bc	25.264^c	25.458^c	24.398^c	24.978^c	24.956^c	0.513
	CG	31.439^a	27.87^bc	25.382^c	25.849^bc	25.567^c	25.133^c	26.534^bc	25.722^c	28.11^b	25.128^c	0.628
C16:2	SG	1.479^a	1.437^ab	1.118^bc	1.386^ab	0.956^c	1.305^ab	0.772^c	1.254^b	0.866^c	1.199^b	0.077
	CG	1.436^ab	1.444^a	0.739^c	1.287^ab	0.936^c	1.269^ab	0.707^c	1.234^ab	0.826^c	1.204^b	0.089
C18:0	SG	12.168^b	12.185^b	14.691^a	12.720^ab	12.102^b	12.887^ab	11.675^b	13.377^ab	13.814^ab	13.787^ab	0.303
	CG	11.340^b	13.421^ab	14.454^a	13.212^ab	14.927^a	13.44^ab	11.450^b	14.043^ab	12.887^ab	15.272^a	0.417
C18:1n9c	SG	34.434^ab	36.204^ab	32.762^b	36.831^a	28.364^c	36.907^a	26.555^c	35.562^ab	25.007^c	34.294^ab	1.4
	CG	31.495^b	34.925^ab	29.714^b	36.567^a	28.032^bc	34.241^ab	20.850^c	33.670^ab	24.573^c	33.111^ab	1.573
C18:2n6c	SG	2.835^a	2.642^ab	2.445^ab	2.606^ab	2.420^b	2.785^a	2.477^ab	2.823^a	2.493^ab	2.702^ab	0.05
	CG	2.599^a	2.499^ab	2.471^ab	2.549^ab	2.276^ab	2.571^ab	0.782^c	2.691^a	2.134^b	2.694^a	0.18
SFA (%)	SG	57.79^bc	55.91^c	55.74^c	55.30^c	56.7^bc	58.16^bc	60.4^b	64.82^a	67.34^a	67.84^a	1.54
	CG	60.56^c	57.16^c	56.17^c	58.74^c	59.26^c	59.76^c	64.07^c	65.88^b	74.077^a	69.6^ab	1.83
MUFA (%)	SG	36.04^cd	37.92^a	38.48^a	38.77^a	37.54^c	36.25^c	34.60^cd	30.20^d	27.78^b	25.55^d	1.50
	CG	33.52^b	36.87^ab	38.32^a	35.74^ab	35.2^ab	34.81^ab	31.25^b	29.53^bc	22.32^c	26.21^c	1.60
PUFA (%)	SG	6.18^a	5.20^b	5.70^b	5.86^ab	5.74^ab	5.47^b	4.95^c	4.88^c	4.73^d	4.38^d	0.19
	CG	5.91^a	5.75^b	5.47^b	5.47^ab	5.49^ab	5.4^b	4.64^c	4.48^c	3.56^d	4.08^d	0.24
Atherogenic index	SG	1.50^b	1.33^b	1.24^b	1.20^b	1.23^b	1.33^b	1.46^b	2.00^a	2.15^a	2.09^a	0.11
	CG	1.85^bc	1.36^c	1.25^c	1.39^c	1.47^bc	1.42^bc	1.73^bc	1.92^b	2.995^a	2.48^a	0.17

Note: Atherogenic index (Ulbricht & Southgate 1991).

^a–d Means within a row with different superscript letters differ significantly (P < 0.05).

In the literature, some effects of the addition of marine algae have been reported as a reduction of the milk content of fatty acids with fewer than 16 C and increased those with more than 16 C (Toral et al. 2010), or a decrease in the ruminal outflow of C18:0 or an increase in the conjugated linoleic acid (CLA) content (Bichi et al. 2013), although the low amount of microseaweeds used in the present study did not affect these parameters. Attending to fatty acid groups, no significant effects of seaweed diet addition were observed in saturated fatty acid (SFA), monounsaturated fatty acid (MUFA) and PUFA percentages and atherogenic index (AI). Both groups displayed similar values until day 5 of lactation. However, there was a trend of raising the SFA percentage and AI and decreasing the MUFA and PUFA percentages in both groups from day 10 to day 40 of lactation.

No significant differences for blood plasma IgG concentration and ChT activity were observed between groups during the experiment (Table 3). Blood plasma IgG concentration peaked at day 20 in both groups (17.4 and 17.0 mg/ml, CG and SG, respectively); however, SG showed a slight increase earlier (5 day postpartum) than CG (10 days after partum), something that could suppose that at higher concentrations, the effect would be stronger. Similar evolution on blood plasma IgG concentration was described by Castro et al. (2006), who observed that the inclusion of CLA in the goats diet enhanced the blood IgG levels after partum. A time effect on colostrum IgG concentration was observed in both groups; the highest values were observed at partum (39.3 and 30.1 mg /ml in CG and SG, respectively) decreasing along the time, as it was expected.

Table 3. Blood plasma IgG concentrations and chitotriosidase activities before and postpartum.

		Days
		−40	−7	partum	5	10	20	30	40	SEM
IgG (mg/ml)	SG	6.39^a	9.68^a	9.28^a	13.36^b	14.89^bc	16.96^bc	15.44^bc	16.07^c	2.65
	CG	8.08^a	8.97^a	8.54^a	10.7^a	14.27^b	17.44^b	17.43^b	17.23^b	2.65
ChT (nmol/(ml/ h))	SG	5245.93^a	4882.9^ab	4821.56^ab	4362.61^ab	4688.21^ab	4942.13^ab	4871.84^ab	5456.43^b	599.16
	CG	5181.77	4896.06	4862.74	5354.40	5218.86	4974.52	5177.24	5673.50	669.88

^a–c Means within a row with different superscripts differ significantly (P < 0.05).

Blood plasma ChT activity ranged from 4896.1 to 5673.5 nmol/ml/hour in CG and from 4362.6 and 5456.4 nmol/ml/hour in SG group. At day 40 after microseaweed inclusion, ChT activity was significantly higher than before treatment.

Milk ChT activity (Table 4) peaked at partum in both groups (9253.2 and 10392.0 nmol/ml/hour for CG and SG, respectively). ChT is a secretory protein related to the immune system which is able to cleave chitin in the cell wall of fungi and nematodes (Aguilera et al. 2003). It has high activities in goat serum and colostrum (Arguello et al. 2008). These authors also reported higher values of ChT activity in goat colostrum than in milk. Colostrum/milk IgG displayed higher values at birth and lower at day 40, as it has been reported previously by Arguello et al. (2006b). Seaweed administration did not show any influence on colostrum/milk IgG concentration, and the IgG values are similar to those reported by Trujillo et al. (2007). It is important to remark that any influence that reduces the IgG concentration in colostrum may affect the immune passive transfer and growth of the goat kids (Arguello, Castro, Capote et al. 2004; Castro-Alonso et al. 2008).

Table 4. Colostrum/milk IgG concentrations and chitotriosidase activities.

		Days
		0	1	2	3	4	5	10	20	30	40	SEM
IgG (mg/ml)	SG	30.09^a^y	21.68^b^y	5.47^c	3.37^c	1.41^c	0.99^c	0.90^c	0.95^c	0.87^c	0.84^c	2.33
	CG	39.25^a^x	13.52^b^x	5.72^c	2.66^c	2.61^c	2.15^c	1.35^c	3.28^c	1.63^c	0.87^c	2.60
ChT (nM/(ml·h))	SG	10392.00^a	10162.00^b	6513.96^c	6145.27^cd	5825.00^cd	5613.79^cd	4661.87^de	3330.65^ef	2880.79^ef	2104.38^f	1167.72
	CG	9253.20^a	8391.45^a	7500.00^ab	6039.83^bc	5437.20^cd	4333.33^cd ^e	3666.67^de	3024.00^e	2786.20^e	2527.09^e	1348.37

^a–f Means within a row with different superscript letters differ significantly (P < 0.05).

^x–y Means within a column with different superscript letters differ significantly (P < 0.05).

4. Conclusions

In conclusion, the addition of 5 g/day of C. pyrenoidosa is not enough to show any differences on immunological or on milk quality parameters. These preliminary results suggest that the addition of 5 g of C. pyrenoidosa could have an effect on the goat immune status; however, the concentration of microseaweed added should be revised.