Effect of Graded Levels of Spirulina (Arthropsira platensis) on Feed Intake and in vivo Digestibility of Trypsacum laxum in Guinea Pig (Cavia Porcellus L)

The study was conducted over a period of 15 days (from January 10 to 25, 2018) at the teaching and Research Farm of the University of Dschang, located in the Highlands of the West region of Cameroon at an altitude of 1410 m above sea level between latitude 5°26’ and longitude 10°26’. The annual rainfall is between 1500 and 2000 mm/year, with temperatures ranging from 10 ° C to 25 ° C. The climate is a Cameroonian equatorial climate temperate by the altitude, with the dry season from mid-november to mid-march and rainy season from mid-march to mid-november.

Twenty guinea pigs (Cavia porcellus), born at the Teaching and Research Farm (FAR) of the University of Dschang were used in this study. They were about 5 months old with an average weight of 450 ± 50 g. The animals were housed in single wired metabolic cages each measuring 80 cm x 50 cm x 30 cm and lined with mosquito nets, which were used to collect feces. Each cage was equipped with a feeder, a trough and a lighting device.

The basal diet was T. laxum harvested at the FAR and distributed to animals after preweaving for two hours on sun. Spirulina was used as a supplement and was purchased in markets around Lake Tchad. It was dried, crushed and incorporated at different levels to form the experimental rations. A 100 g sample of each feed, forage and spirulina was taken and dried at 60 ° C to constant weight in a Gallemkamp brand ventilated oven. Then, it was milled into powder of sieve size1 mm using a home-made trimarteau shredder of small size and kept in plastic bags for different bromatological analysis. The chemical composition as analyze in the laboratory of T. laxum, spirulina and the sample feed is presented in Table 1 and Table 2. Four rations were formulated (Table 2) according to the level of inclusion of spirulina. Apart from spirulina, all the ingredients used in the ration formulation were purchased from agricultural by-product dealers at the Dschang city market. The control diet did not contain spirulina while R1, R2 and R3 contained 2%, 4% and 6% Spirulina. All these rations were iso-nitrogenous (19% PB) and iso-caloric (2800 Kcal). To deal with a possible deficiency, vitamin C, (240 mg tablet in 1.5 liters of water) bought from a pharmacy was distributed to all animals daily in drinking water served ad libidum.

TS0: 40g/head/day of R0+Trypsacum laxum ad libitum (n = 5)

TS2: 40g / head /day of R1+Trypsacum laxum ad libitum (n = 5)

TS4: 40g / head /day of R2+Trypsacum laxum ad libitum (n = 5)

TS6: 40g / head /day of R3+Trypsacum laxum ad libitum (n = 5)

The guinea pigs were randomly distributed into labeled individual cages, and assigned to the 4 treatment diets in a completely randomized design with 5 replicates. The days trial lasted for 15 days (10 days for adaptation and 5 days for data collection). Each ration was served between 8 and 9 o’clock every day and left over feed, and the feces of each animal were collected and weighed daily before daily feeding and watering, for the assessment of feed intake. At the beginning of the test and at the end of each period, the animals were weighed to determine daily weight gain.

During the test, samples of 100 g of experimental feed and feces from each animal were taken daily and dried at 60 ° C to constant weight in a ventilated oven and milled to determine the digestibility of nutrients. At the end of the test, animals were fasted for 12 hours, and then sacrificed by cervical dislocation followed by bleeding. They were then dressed at the abdominal area and then eviscerated. The caecum was removed, sectioned, and their content was put into sterile tube to evaluate the caecal flora constitution according to the method described by Benson ¹⁵. Weighing was made using a digital scale balance with a capacity of 3 kg and a precision of 1 g. At the end of the study, mean values of feed intake, average daily gain, and apparent digestibility coefficients of nutrients were determined.

The dry matter (DM) and organic matterwas determined by drying 0.5 g of sample in an oven overnight at 100 ° C, ash was obtained by incineration at 500 ° C for 6 hours and organic matter (AOAC, 1990). Crude fiber (CF) was determined using the sheerer method and crude protein (CP) by the Kjeldahl method by AOAC ¹⁶. Apparent digestibility coefficients (ADC) were determined by the following formula:

 ADC = Ingested-excreted/ingestedx100

Feed intake, apparent digestibility coefficients and caecal flora constitution data were subjected to one-way analysis of variance. When significant differences existed between treatments, the separation of means was done by the Duncan test at 5% significance level ¹⁷. SPPS 20.0 software was used.

(Table 3) shows the dietary intake of guinea pigs during in vivo digestibility test This table shows that spirulina level significantly (p ˂ 0.05) influenced intake of T. laxum, and nutrients (DM, OM, CF and CP). The highest quantity of T. laxum DM ingested (63.22 g DM/head/day) was observed with animal fed on TS4 while the lowest (53.74 g DM /head/ day) was recorded with animals fed TS0 diet. However, ingestion of T. laxumDM by animal fed TS2 and TS6 groups was comparable (p ˃ 0.05). For the ingestion of T. laxumOM, the highest (56.75g DM /head/ day), was observed in animals fed with TS4 while the lowest (48.59 g DM /head/ day) was observed in animals fed TS0 but no significant difference (p ˃ 0.05). However, feed intake of T. laxum OM of the animals of TS2 and TS6 groups was comparable (p ˃ 0.05), but significantly (p ˂ 0.05) higher than that of TS0 group. The highest compound feed OM feed intake (19.03 g DM /head/day) was obtained with TS0 lot animals while the lowest (17.82 g DM /head/ day) was obtained with the animals of group TS2. The highest T. laxumCF intake (25.33 g DM /head/day) was obtained with TS4 animals fed on treatment TS4 while the lowest (21.69 g DM /head/ day) was observed with animals of group TS0. However, T. laxumCF intake was comparable (p ˃ 0.05) between the animals of groups TS0 and TS6 on one hand and those of treatment TS2 and TS4 on the other hand. The highest compound feed CF intake (1.99 g DM/head/day) was observed in TS0 animals while the lowest (1.70 g DM /head/day) was observed with animals of group TS6. The highest T. laxum CP intake (16.24 g DM/head/day) was observed with TS2 animals while the lowest (10.90 g DM/head/day) was observed with animals of group TS4. However, animals of TS0 and TS6 groups had similar CP intake (p ˃ 0.05). The highest compound feed CP feed intake (4.30 g DM/head/day) was observed with animals fed TS6 while the lowest (2.01 g DM/head/day) was observed in animals of group TS4. In addition, the CP intake of the compound feed of treatment TS0 and TS2 was comparable (p ˃ 0.05). Although, DM, OM and CP intake of the compound feed of animal of groups TS0, TS2, TS4 and TS6 were comparable (p ˃ 0.05).

On average, intake of DM, OM and CF significantly (p ˂ 0.05) increased with the level of spirulina in the diet. In fact, the total highest DM (83.89 g / animal/ day) was observed with animals of group TS4, while the lowest (74.39 g DM/head/day) was observed with animals of group TS0. However, the total DM intake of the animals of groups TS2 and TS6 was comparable (p ˃ 0.05), but significantly lower than that of group TS4. The highest total OM (75.63 g DM/head/day) was observed with animals of group TS4 while the lowest (67.62 g DM /head/ day) was observed with TS0 animals. However, OM intake of the animals from TS0, TS2 and TS4 was comparable (p ˃0.05). The total highest CF intake (27.18 g DM/head/day) was observed with TS4 animals while the lowest (23.67 g DM/head/day) was observed in animals of group TS0, but it was comparable (p ˃ 0.05) with the animals of TS0 and TS6 groups. The total CF intake of TS2 animal group was significantly higher than those of animal of group TS6. Total highest CP intake (16.24 g DM/head/day) was observed in TS2 animals, while the lowest (10.90 g DM/head/day) was observed with animals fed TS4 group. Intake of animals of TS2 group was significantly (p ˂ 0.05) higher than those of the other groups. Total CP intake of animals fed TS0 and TS6 diets were comparable.

The apparent digestibility coefficients (ADC) of dry matter (ADCDM), organic matter (ADCOM), crude protein (ADCCP) and crude fiber (ADCCF) with respect to the level of incorporation of spirulina in the diet was significantly (p < 0.05) improved. (Table 4) In fact, the highest apparent digestibility coefficients of dry matter (ADCDM) (86.20%), organic matter (ADCOM) (87.30%), crude cellulose (ADCCF) (87, 54%) and crude protein (ADCCP) (93.73%) were obtained in TS6 animals, while the lowest (75, 67%, 75, 62%, 88, 06% and 78, 05% respectively were observed in the control group (TS0). However, the ADCDM, ADCOM and ADCCF of TS0 and TS2 groups on one hand, and the TS4 and TS6 groups on the other hand were comparable. The ADCCP of the treated groups (TS2, TS4 and TS6) were comparable (p ˃ 0.05), but significantly (p ˂ 0.05) higher than that of TS0 group. Also, ADCCP of groups TS2 and TS4 were comparable (p ˃ 0.05).

Total DM intake was significantly higher in the supplemented animals. This is in agreement with the observations of Miégoué et al. ^{2, 5}, Kouakou et al. ¹¹, Ramirez-Riviera et al. ¹², Peiretti and Meineri ¹⁸, Noumbissi et al. ¹⁹ and Fotna ²⁰ who showed that the inclusion of protein source increased feed intake. This result shows the good palatability of spirulina in guinea pigs. Indeed, Marie-Christine ²¹ showed that supplementation with spirulina in the diet of pigs, poultry and broilers improve their acceptability, palatability and digestibility. Moreover, with a good protein (60 to 70%), mineral value ²² and a low fiber value (2%) ²¹, spirulina might allow sufficient proliferation of micro-organisms. In the case of cellulolytic microflora, its nutrients would be better digested, thereby accelerating the digestive transit ^{2, 23}. This has the effect of pushing the animal to consume more. Indeed, Quigley and Poppi ²⁴ showed that spirulina has the potential to increase the production of microorganisms and reduce its retention time in the rumen. Kana et al. ²⁵ reported a reduction in dietary intake with the increased level of spirulina associate to 50% of cassava root waste in poultry. Feed intake and digestibility of nutrients were generally higher in animals supplemented with spirulina (TS2, TS4 and TS6). The inclusion of Spirulina increased acceptability, palatability and even digestibility in our animals during the trial. The highest total DM feed intake (63.22 g DM/head/day) obtained was greater than 21.18, 56.8 and 50.61 g DM/head/day obtained respectively by Niba et al. ⁸, Miégoué et al. ² and Noumbissi et al. ¹⁹, but lower than 73.7 and 103.21; 115.80 and 170.9 g DM/head/day) obtained by Kouakou et al. ¹¹ and Egena et al. ²³ and Miégoué et al ². The observed difference could be attributed to the type of supplement used and to the quality of experimental feed.Supplementation increased total intake of dry matter but almost had opposite effect on ingestion of the compound feed. In fact, the ingestion of T. laxum was significantly higher in animals supplemented, than those of the control group. This observation contradicts those of Takele and Getachew ²⁷ on sheep, Kouakou et al. ¹¹in guinea pigs fed P. maximum and supplemented with Euphorbia heterophylla and Noumbissi et al. ¹⁹. This can be explained by the fact that, protein intake of spirulina would have stimulated palatability of guinea pigs. Moreover, several works by Razafindrajoana et al.²² and Marie-Christine ²¹ illustrated similar observation. The best feed intake of this grass obtained with the ration containing 4% spirulina (TS4) is explained by the fact that protein supplements would promote sufficient proliferation of intestinal microorganisms involved in in guinea pigs digestion. This could be related to the difference in chemical composition, notably the high CP content of T. laxum (1 3.86%) and fiber (37.77%) and its good palatability by guinea pigs ^{9, 28}.

Apparent digestibility of dry matter (ADCDM) and organic matter (ADCOM) increased with the inclusion level of spirulina in the diet. This result is consistent with that of Miégoué et al^{2, 5} who suggested that nitrogen (protein) supplementation improves DM digestibility by approximately 5 points, thereby improving the level of DM intake. Apparent digestibility of crude fiber (ADCCF) increased with increasing level of spirulina, which is corroborating with the observations of some authors ^{11, 12, 19} who used concentrate and Tithonia diversifolia as supplements. Also, low nitrogen intake in a low protein diet improves digestibility of cellulose ⁵. Protein supplementation of poor forages ensures favorable conditions in the digestive tract, for the proliferation of cellulolytic microflora, microbial proteins and reduces transit time in the rumen in dwarf guinea goats. Total weight gains (TWG) and daily weight gains (DWG) obtained from the highest digestibility test (6.60 g and 1.32 g / d) were observed with TS4 animals group, but low in TS6 group. TWG and DWG of TS4 are correlated with feed intake and digestibility of animals of this group. On the other hand, the decrease of these parameters in the animals of group TS6 was inversely correlated with their feed intake and digestibility. This remark could indicate the supplementation of spirulina at 6% would have an adverse effect on guinea pig and therefore the optimum level would be 4%.

The caecal flora (lactobacilli, enterobacilli and salmonella levels) was not influenced by the incorporation of spirulina in guinea pig diet. This shows a balance between the bacterial caecal populations due to the experimental feed. ^{2, 5} reported that stability of the caecal ecosystem is favored when the flow and nutrient composition entering the caecum is more stable. Moreover, the level of lactobacilli remained higher than that of enterobacilli irrespective of the ration. This corroborates with the observations made by Tsukahara ²⁹, Miégoué et al. ^{5, 30}. These authors reported that, guinea pig normal flora is predominantly anaerobic gram (+), grams (-) being present in lesser quantities. Spirulina can therefore be used as a source of protein in guinea pigs' diet without, however, running the risk of altering the composition of the caecal flora, which is generally unstable in order to ensure better digestibility of the nutrients. Losson ³¹ showed that a sudden change in the diet can lead to an imbalance of the flora that can lead to enteropathy. The lack of a significant difference in the caecal flora irrespective of the level of inclusion of spirulina can be explained by the chemical composition, especially their protein concentration, phycocianin and carotenoids which modulate the colonization of the digestive tract by microbial populations. Our results are contrary to those of Rasmussen et al. ³² who reported that Spirulina supplementation stimulates the development of certain strains of Clostridia and alters Bifidobacteria populations. Spirulina supplementation therefore tends to modulate the colonization of the digestive tract by microbial populations involved in the gut microbiota homeostasis.