SUMMARY AND CONCLUSIONS
The Asian elephant (Elephas maximus) is listed as an endangered migratory species and protected under the Indian Wildlife Protection Act as well as protected from international trade by listing in Appendix-I. Elephants are a challenging species to keep in captivity, although they are considered a species of flagship conservation and thus a favourite species to exhibit. So, it is important to keep these animals in captivity, to understand their requirements better, and hopefully transition this knowledge to wild elephants. The elephants are mono-gastric, non-ruminant browsers as well as grazers, with the characteristics of hindgut fermentative metabolism. Due to the similarity of digestive behaviour, the horse is considered a model animal for calculating elephants’ nutrient requirements and designing diets.
The elephant’s hindgut harbors bacteria that degrade cellulose to VFA, CH4, and CO2 and get approximately 70% of its energy requirement. In addition, the Asian elephant deficit homologues of enzymes needed for cellulose degradation. Therefore, the degradation of cellulose in their feed depends on their intestinal microflora. Several strategies have been developed to prevent microbial disturbances in the animal intestine with the growing concerns about using antibiotics and other growth promoters like probiotics. Probiotics modulate the balance of hindgut bacterial communities and could be a possible strategy to control pathogen shedding and preventing disease resulted in improved health performance and nutritional status of the animal.
In the present study, the effect of probiotics supplementation was assessed in terms of nutrients, minerals, and energy utilization in captive Asian elephants. Besides this, other parameters like; faecal profile, faecal steroid hormone profile, the prevalence of GIT parasites, blood profile, and body measurements, etc. were also evaluated to ascertain elephants’ physical and physiological health.
Eighteen healthy, captive adult Asian elephants of 30-62 years age, similar sex, i.e., female, nearly similar body weight (3495 ± 133.34 Kg), and uniform conformation were selected at Elephant Village (Hathi Gaon), Jaipur. They were divided randomly into three similar groups of six each. All the elephants were housed in a hygienic and well ventilated individual enclosure, having a concrete floor with a feeding arrangement to collect each animal’s faeces separately. Animals had no access to the feed of other animals. A prophylactic dose of broad-spectrum anthelmintic bolus fenbendazole at 5mg/kg body weight was given to all the elephants before the experimental feeding of probiotics and acclimatized for twenty days. All the elephants were individually fed a measured quantity of experimental feed with ad libitum and access to clean drinking water daily.
The experimental elephants were distributed by complete randomized design into three groups, such as no probiotic in the CONT group (T1), Lactobacillus acidophilus in the LAB group (T2), and Saccharomyces cerevisiae in the SC group (T3). The elephants were stall-fed a consistent diet of green pearl millet forage (Pennisetum spp.) as a basal diet throughout the research period. The research was conducted for two months, including of 20 days preliminary period for adaptability with basal diet, 30 days feeding trial, and ten days observation period at the end of the experiment. The probiotics, i.e., Lactobacillus acidophilus and Saccharomyces cerevisiae, were supplemented during the feeding trial to all the elephants of T2 and T3 treatment groups, respectively. The probiotics were made available by Meteoric Biopharmaceuticals Pvt. Ltd, which contained 1 × 109 CFU/g concentrate of Lactobacillus acidophilus and 1 × 109 CFU/g concentrate of Saccharomyces cerevisiae, given at per 50 kg body weight orally.
During the last five days of the feeding trial, a digestibility trial was conducted to estimate the digestibility of various nutrients and feed intake. The feed and faecal samples were analyzed for proximate principles, fibre fractions, and other gross nutrients in different groups. The balance study of minerals and energy was also conducted. Blood samples were collected after the end of the feeding trial. In addition, the faecal samples were also collected for the estimation of faecal profile, faecal steroid hormone profile, and prevalence of GIT parasites. The body measurements and body condition score were also recorded during the experiment. The data were analyzed statistically as per Steel et al. (1997).
The chemical composition of proximate principles and other nutrients, fibre fractions, gross energy, and minerals contents of green pearl millet (bajra) forage were found to be nearly similar in the groups. The dry matter and crude protein contents were ranged between 17.02-17.70 and 5.55-5.84 per cent, respectively. The NDF, ADL, and GE contents were ranged between 69.15-71.0%, 7.08-7.95%, and 18.33-19.84 MJ/kg DM, respectively. The Ca and P contents were ranged between 0.41-0.43 and 0.38-0.40 per cent, respectively. However, the basal diet was low in CP content, but more or less; it met all the elephants’ nutritional requirements.
Statistically, non-significant effects were observed in terms of kg/day, per cent BW and g/kgBW0.75 for the intakes of all the proximate principles, fibre fractions, and other nutrients except for EE and total ash on probiotics supplementation. The overall average dry matter intakes in terms of kg/day, per cent BW and g/kgBW0.75 were found as 59.84 ± 3.22, 1.74 ± 0.10, and 132.78 ± 6.75, respectively. Significant effects were observed in terms of kg/day, per cent BW and g/kgBW0.75 for EE intake and in per cent BW and g/kgBW0.75 for total ash. The average intakes recorded for DM, OM, CF, NFE, total carbohydrates, and NDF were higher in SC (T3) group, followed by the LAB (T2) group, and then the CONT group, respectively. Higher intakes for all the nutrients except EE and total ash were found in probiotic groups as compared to CONT (T1) group. The outgo of proximate principles, fibre fractions, and other gross nutrients in faeces, in terms of kg/day and per cent BW was found nearly similar and could not reveal any significant effect of treatments. However, the hemicelluloses outgo showed a significant effect in probiotic groups.
Average per cent apparent digestibility coefficient of various nutrients in T1, T2 and T3 groups were 56.10 ± 2.99, 58.27 ± 2.73 and 57.85 ± 3.19 for dry matter, 54.36 ± 3.40, 58.25 ± 2.90 and 58.22 ± 3.45 for organic matter, 75.73 ± 2.59, 72.10 ± 3.76 and 76.39 ± 0.92 for crude protein, 64.41 ± 3.46, 55.43 ± 4.28 and 47.64 ± 9.77 for ether extract, 54.65 ±3.24, 55.92 ± 3.42 and 55.75 ± 3.44 for crude fibre, 50.89 ± 4.20, 58.54 ± 2.70 and 58.14 ± 3.93 for nitrogen free extract, 52.61 ± 3.70, 57.38 ± 2.96 and 57.09 ± 3.69 for total carbohydrates, 68.47 ± 2.54, 58.47 ± 2.37 and 53.93 ± 2.21 for total ash, and 57.51 ± 2.94, 61.02 ± 3.23 and 46.22 ± 2.38 for acid-insoluble ash, respectively. The digestibility of fibre fractions calculated as 49.70 ± 3.72, 53.37 ± 2.90 and 53.83 ± 3.95 for neutral detergent fibre, 48.07 ± 3.66, 45.62 ± 3.55 and 48.75 ± 4.46 for acid detergent fibre, 23.08 ± 6.87, 30.91 ± 4.89 and 21.40 ± 7.20 for acid detergent lignin, 70.62 ± 2.61, 69.25 ± 2.67 and 67.70 ± 2.02 for neutral detergent soluble, 52.99 ± 6.45, 67.78 ± 2.31 and 64.04 ± 3.19 for hemicelluloses, 54.58 ± 3.72, 49.97 ± 3.81 and 55.46 ± 4.35 for celluloses, 39.58 ± 4.87, 41.93 ± 13.73 and 32.36 ± 5.76 for NDF ash, and 28.34 ± 5.92, 35.30 ± 5.36 and 26.15 ± 1.96 for ADF ash, respectively. Statistical analysis of the data revealed a highly significant effect (P ≤ 0.01) of probiotics supplementation on the digestibility coefficient of total ash and acid-insoluble ash. The apparent digestibility coefficient of dry matter, organic matter, crude protein, ether extract, crude fibre, nitrogen free extract, total carbohydrates, neutral detergent fibre, acid detergent fibre, acid detergent lignin, neutral detergent solubles, hemicelluloses, cellulose, NDF ash, and ADF ash was remained unaffected due to probiotics supplementation. However, the apparent digestibility coefficient of almost nutrients except for EE, total ash, and NDS was comparatively higher in the supplemented groups. Further, the practical nutritional worth of the diet, determined in terms of %DCP, %TDN, and NR and the plane of nutrition determined as kg/day, %BW and g/kgBW0.75 for digestible dry matter intake, digestible organic matter intake, DCP intake, and TDN intake, could not reveal any significant effect of treatments. However, the practical nutritional worth and the plane of nutrition were almost higher in the supplemented groups.
The inclusion of probiotics on mineral consumption in terms of per day and per kg body weight and outgo per kg body weight basis and subsequently, their apparent absorption revealed non-significant effects. However, the intakes of Ca and Zn as g/d and Cu and Fe as mg/d were recorded higher, whereas the intakes of P, Mg, Fe, and Mn per kg BW were observed lower in probiotic groups. The outgo of P, Mg, Zn, Cu, Fe, and Mn (per kg BW) was recorded lower in probiotic fed groups. The average per cent apparent absorption in T1, T2 and T3 groups were 68.37 ± 3.25, 72.53 ± 2.71, and 70.70 ± 2.91 for calcium, 56.87 ± 3.33, 60.53 ± 2.86, and 58.93 ± 3.83 for phosphorus, 64.12 ± 1.27, 65.0 ± 3.40, and 66.96 ± 3.11 for magnesium, 18.17 ± 1.77, 18.95 ± 2.24, and 18.81 ± 4.37 for zinc, 21.31 ± 4.17, 21.64 ± 5.09, and 21.69 ± 6.07 for copper, 11.13 ± 2.54, 11.40 ± 1.63, and 11.21 ± 3.36 for iron and 9.33 ± 1.40, 9.20 ± 2.07, and 9.17 ± 1.76 for manganese, respectively. The apparent absorption of Ca, P, Zn, and Fe were recorded higher in the LAB group, whereas in the case of Mg and Cu, high in the SC group. Except for Mn, higher apparent absorption was observed in probiotics fed groups as compared to the control group.
The effects of probiotics supplementation on energy values; GE intake, DE intake, ME intake, and FE expressed as MJ/day, per cent BW and MJ/kgBW0.75, and per cent, apparent digestibility coefficient of energy were observed as non-significant in the treatment groups. The average per cent apparent digestibility coefficient in T1, T2, and T3 groups were 60.32 ± 2.74, 62.47 ± 3.08, and 63.28 ± 2.85, respectively. The intakes of GE, DE, ME, and apparent digestibility coefficient of energy were recorded higher in the SC group, followed by the LAB group and then the CONT group, respectively. The values of FE were not consistent with probiotics supplementation. The study revealed increased apparent digestibility and intake of energy values along with increased NDF digestibility and DM intake. Further, the energy balance profile; energy offered, DM intake, energy density, required MMR, and relative difference, calculated as ME MJ/Day, kg/day, ME MJ/kg DM, ME MJ/Day, and per cent, respectively, could not reveal any effect of treatments. However, the energy balance profile was recorded higher in the SC group, followed by the LAB group and then the CONT group.
The statistical analysis of the data of faecal profile; pH, lactic acid, coliform, and probiotic microbial counts like lactobacillus and Mould, determined on the 0 and 50th day of the study, did not exhibit any effect of probiotic supplementation in treatment groups. However, on comparing the data at different periods, the declined trend of average pH, coliforms, and mould counts was associated with increased lactic acid and lactobacillus count on the 50th day as compared to 0 day within all the respective groups. In contrast, pH, coliform, and mould counts were recorded higher, whereas lactic acid and lactobacillus counts were lower on day 50th in the LAB group, followed by the SC group then the CONT group, respectively. Thus, the study suggests a declining trend in lactobacillus count observed in probiotic groups as compared to CONT group, which could be implicative of positive health status.
The results of statistical analysis of the data of faecal steroid hormone profile; cortisol metabolite concentrations, and progesterone metabolite concentrations revealed non-significant effects in the probiotics fed groups. However, on comparing the data at different periods, an inconsistent rising trend in average cortisol and a gradually declined trend in progesterone were observed on the 50th day as compared to 0 day within all the respective groups. In addition, the average cortisol was found low in both the treatment groups as compared to the CONT group, whereas the average progesterone was observed lowest in the LAB group as compared to the CONT and the SC groups on the 50th day. Further, faecal samples, collected at different periods, were analyzed for the prevalence of gastrointestinal tract parasites, found to be negative for all the groups.
Regarding haematological study, the average red blood cell indices viz. RBC, Hb, haematocrit, MCV, MCH, and MCHC, did not exhibits, any statistical difference due to the effect of treatment. Highly significant (P < 0.01) effect of treatment was observed on per cent lymphocytes and neutrophils counts. Significant (P < 0.05) effect was recorded for mean platelet volume. However, it could not reveal any effect on other white blood cell indices viz.WBC, monocytes, eosinophils, and basophils as well as platelet cell indices viz., platelet counts, and plateletcrit. More or less, all haematological parameters were well within the reference range of the Asian elephant. The Difference in RBC, haematocrit, MCH, and MCHC was recorded in the present study. The biochemical parameters like serum metabolites viz. glucose, total protein, albumin, and cholesterol; renal function tests viz., creatinine, and blood urea nitrogen; serum enzymes viz., AST, ALT, and ALP; and serum minerals viz., calcium, and phosphorus, were remained unaffected due to probiotics supplementation and all these parameters were well within the normal reference range of the Asian elephant.
No statistical differences regarding the body measurements and body condition score were observed in different groups. The body weights recorded at different period exhibits more or less similar results. The average body condition score in T1, T2, and T3 groups were found to be 3.83 ± 0.17, 3.67 ± 0.21, and 3.83 ± 0.17, respectively.
On the basis of findings of the present study, it can be concluded that dietary supplementation of probiotics did not affect the intake and apparent digestibility coefficient of gross nutrients, minerals, and energy values as well as the practical nutritional worth of the diet, the plane of nutrition, and the energy balance profile in the Asian elephants. In addition, faecal profile, faecal steroid hormone profile, blood profile, and body measurements, and body condition score were also remained unaffected due to probiotics supplementation. However, the intake of EE and total ash, the outgo of hemicelluloses in faeces, apparent digestibility coefficient of total ash and acid-insoluble ash, lymphocytes, neutrophils counts, and mean platelet volume had its impact. Apparently, it seems that the intake and utilization of gross nutrients, minerals and energy values as well as the practical nutritional worth, plane of nutrition, and the energy balance profile were almost higher in the probiotic supplemented groups. Probiotics seem to induce some shift in faecal microflora, also. Probiotic was supplemented at the recommended dosage, but perhaps it was not enough to make a difference in a healthy elephant on a quality diet. The results of the present study suggest that probiotics L. acidophilus and S. cerevisiae can be safely incorporated up to 109 CFU/g concentrate for every 50 kg body weight per day into the diet, with no adverse effects on the general health of the elephants. However, further research needs to be conducted to solidify these results and examine the effect of larger doses on a mature, healthy elephant as well as to focus on the strains and viability of both the probiotics used, as the knowledge on their microbiome is limited. Overall, results provide a basis for future studies to investigate the effect of probiotics supplementation in land’s largest living animal.