The Impact of Feeding Non-defatted Silkworm Pupae Meal on Haemato-biochemical and Oxidative Stress Parameters of Crossbred Cattle Calves

Preweaned calves are raised under a variety of feeding programs with a wide variation in housing and environmental conditions. Numerous studies have been carried out on calf starter, but major emphasis is laid to explore the role of intake of calf starter in an early weaning programme (Kertz et al., 1979). Calves can be weaned early by promoting early intake of dry feed, by feeding of pre-starters along with milk or by feeding calf starter (Quigley et al., 1994). The process during which calves transition from their neonatal dependence on nutrients supplied from milk to nutrients supplied from solid feed (cereal grains and hay) is of substantial financial importance to the producer. Development of rumen is an important physiological challenge for young ruminants (Gupta et al., 2016) and this transition results in colossal metabolic ramifications to calf growth rate, for the reason that tissues must lean onto glucose supplied from milk to the metabolism of short-chain fatty acids as key energy substrates (Baldwin et al., 2004). In pre-weaned dairy calf, solid feed intake (especially high carbohydrate diet) invigorates rumen microbial proliferation and volatile fatty acid production, eventually initiating rumen development (Sua´rez​ et al., 2006; Gupta et al., 2016).
       
Calves upto 3-4 months of age cannot synthesize all the essential amino acids, thus calf starter must contain a good quality protein source. In India, many of the organized farms or farmers have adopted the practice of weaning calves at or above 3 months of age and include animal protein (especially fish meal) in the starter diet. The importance of fish protein is due to its high biological value protein (Tiwari et al., 2023), but the  quality of fish meal is very much variable and availability is uncertain (Islam, 1993; Nazneen, 1995). In addition, fish meal is often adulterated with other ingredients, e.g. fish bones, sand, sawdust (Ali, 1995). So, alternate feed ingredients should be utilised to replace the costly fish meal. In the present experiment as an alternate to costly fish meal, SWPM was used at graded levels in the starter ration, to assess its effect on haemato-biochemical and oxidative stress parameters of crossbred cattle calves.

Animal source
 
A total of fifteen healthy crossbred, female calves (30-40 days age) maintained at Mountain Livestock Research Institute (MLRI), Manasbal, SKUAST-K, Srinagar, were selected for the study. The trial animals were evenly divided in a random manner into three groups of five animals each viz., control (CON), treatment-1 (TRT-25) and treatment-2 (TRT-50), animals belonging to each treatment had average live weight of 35.21±2.72 kg. The experiment was conducted  from July to September, the average temperature during the experimental period was 20.8°C and the maximum temperature inside the shed was 24.7°C.
 
Experimental design and diets
 
The experimental design was CRD. The dietary treatments were control (CON), treatment-1 (TRT-25) and treatment-2 (TRT-50). In CON group, fish meal was added as an animal protein source in concentrate mixture at the level of 10%, while in treatment groups TRT-25 and TRT-50 groups fish meal was replaced by SWPM at the levels of 25% and 50%, respectively. The proximate composition of the experimental rations are presented in Table 1. The experiment was for a period of 90 days. The experimental animals were fed ration which constituted of chaffed green fodder (maize and sorghum), paddy straw and concentrate mixture individually in equal amount in the morning (08:00 am) and evening hours (04:00 pm) as per nutrient requirements of calves by ICAR (2013).


 
Haemato-biochemical studies
 
The estimation of haemoglobin (Hb g/dl) and packed cell volume (PCV %) was executed by using Drabkin’s solution and haematocrit tubes, while RBC and WBC counts was accomplished using haemocytometer. The blood glucose was evaluated after collection of blood by using Accu-Chek instant Glucometer. The estimation of ALT, AST, BUN, Creatinine, Total protein, Albumin and Total cholesterol was done by utilizing commercial diagnostic kits acquired from Span diagnostics limited, Surat, Gujarat, India. Globulin was estimated by subtracting albumin from total protein content and the A:G ratio was calculated by dividing albumin and the globulin concentrations.
 
Oxidative stress parameters
 
The activity of superoxide dismutase (SOD) was measured in hemolysate by an iodophenyl nitrophenol phenyl tetrazolium chloride modified method (RANSOD Kit, Randox, UK). The glutathione peroxidase (GPx) activity was evaluated by Paglia and Valentine’s method in whole blood, using RANSEL Kit (Randox, UK), while the catalase activity was subjected to hemolysate according to Aebi’s method. The quantification of serum malondialdehyde (MDA) was performed in serum by thiobarbituric acid method, for which reactive substances were quantified with thiobarbituric acid as peroxidation index.
 
Statistical analysis
The experimental data was evaluated by one-way ANOVA (SPSS Software, Base 23.0, for MacOS). The statistical equation used were as following:

Yij = μ + Ti + eij

where;
Yij = Parameter under analysis ij.
μ = Overall mean.
Ti = Effect due to treatments on the parameter under analysis.
eij = Experimental error for ij on the observation.
        The significance of mean difference was tested by Duncan’s New Multiple Range Test (DNMRT). The significance of data was considered at p≤0.05. The data is presented in the table as mean, SEM and p-values.

The dietary treatments showed no significant effect (p>0.05) on Hb (g/ dl) and PCV (%) levels (Table 2). The concentration of RBC’s also did not disclose any significance (p>0.05) up to 60 days of experiment, but at 90th day the results revealed significant difference (p≤0.05) where the values of TRT-50 were higher as compared to CON group (Table 2). The WBC count showed significant difference (p≤0.05) on 30^th day in which higher values were detected in groups incorporated with SWPM (TRT-50 and TRT-25) as opposed to CON group and from 60 days onwards non-significant difference (p>0.05) was observed between treatments (Table 2). The haematological framework is the most sensitive index to assess the health and pathological changes (Zhou et al., 2009). The assay of blood indices has endorsed to be a valuable approach for analysing the health status of farmed birds/animals as these indices provide consistent information on metabolic disorders and stress status in an experimental setting (Bahmani et al., 2001). From the present experiment it is evident that the hematological parameters remain unchanged; so, it specifies that replacement of fish meal with silkworm pupae meal had no antagonistic effects on the normal physiology of animals. The diet with different quantities of silkworm meal up to 100% level enhanced growth, hematological and physiological performance of cat fish was reported by Olaniyi and Babasanmi (2013). The values observed for all treatments suggest the nutritional sufficiency of all diets with no malnutrition status (Church et al., 1984). The RBC count showed non-significant differences (p<0.05) among treatments and the values were in normal range as reported by Bhuiyan (1998). These observations correlated with the health of the birds and no mortality was observed in the study as well. The values of RBC’s increases in association with superior quality protein in disease free animals was reported earlier (Ullah et al., 2017). These results also coincided with the results of Ijaiya and Eko (2009b) who reported blood sugar, packed cell volume, white blood cell, red blood cell, haemoglobin showed no significant differences (p<0.05) among the dietary treatment means when fish meal was replaced with silkworm caterpillar meal at 25%, 50%, 75% and 100% inclusion levels.


       
The existent study did not divulge any significant effect (p>0.05) on blood glucose (mg/ dl) levels of experimental animals fed SWPM, presented in Table 3. The results of total protein, albumin, globulin and total cholesterol differ significantly (p≤0.05) between the treatment groups, showing the higher values for the treatment groups fed 5.0% of SWPM (TRT-50) followed by the animals fed 2.5% of SWPM (TRT-25) and then the control (CON) group. The A:G ratio did not show any significant difference (p>0.05) between treatments. Data of liver function tests (ALT and AST) showed no significance (p>0.05) between treatments (Table 3). The kidney function tests (BUN and creatinine) also showed non-significant differences (p>0.05) between treatment groups (Table 3). The blood biochemistry is significant to access the animals’ health status (Lumeij, 2008) and are further implemented to estimate the safety of different feed ingredients. Recent investigations with laying hens have not con­firmed any effect from partial, or complete alternative of soybean meal in diets with either pupae meal, or black soldier fly larvae meal (Lalev et al., 2023; Ullah et al., 2017; Marono et al., 2017). The results of liver function tests (ALT and AST) were in harmony with Ullah (2016) who reported that on partial or complete replacement of soybean meal by silkworm meal (up to 0.05%) had no effect on liver enzymes. Numerically similar values of uric acid and creatinine con­firmed unchanged rate of protein catabolism and confirmed normal kidney functioning. Some researchers (Ullah, 2016; Marono et al., 2017; Bovera et al., 2018; Kim et al., 2020), also confirmed lack of any effect from the inclusion of black soldier fly larvae or pupae meals on protein metabolism. Gugolek et al. (2021) reported that the inclusion of SWPM in the diets of rabbits have shown an increase in the concentration of plasma albumin, while the total plasma protein levels remained unaffected. Same results were reported by Nandeesh et al., (1998) and Tona et al., (2006). Previous researches have demonstrated that the presence of total protein in blood is directly linked to the amount of available protein in the diet (Gasco et al., 2019). In the present study, concentration of albumin and globulin both increased in response to SWPM incorporation.


       
The present experiment revealed non-significant effect (p>0.05) on oxidative stress parameters viz., SOD, GPx, Catalase and MDA, of experimental animals fed SWPM, presented in Table 4. The values showed improved results by incorporating the SWPM in the ration of crossbred calves. Minimal work has been carried out on using silkworm pupae meal as a feed constituent in calf starter ration and especially on oxidative stress parameters of calves. The results of SOD and MDA showed disagreement with the findings of Ji et al., (2015), who revealed that high dietary silkworm pupae meal levels significantly decreased hepatic SOD activity and increased MDA content, showing that the diets containing higher silkworm pupae levels induced oxidative stress and lipid peroxidation and return to damage the hepatopancreas.

The verdict of the current experiment revealed that SWPM can be incorporated upto 50% replacing fish meal without manifesting any ill effect on haemato-biochemical and oxidative stress parameters of crossbred cattle calves. Thus, SWPM shows potential to replace costly fish meal as animal-based protein in the calf starter diets. Further studies are suggested on incorporation of SWPM as feed ingredient in calf starter ration for longer duration.

The experimental animals were handled as per approved trial protocol regulations of the Institutional Animal Ethics Committee of SKUAST-Kashmir.

The authors have no relevant financial or non-financial interests to disclose.