Tropical Plant Phytonutrient Improves the Use of Insect Protein for Ruminant Feed

Abstract

This work aimed to examine the effects of binding proteins from Gryllus bimaculatus with Sesbania grandiflora phytonutrient on gas dynamics, in vitro digestibility, and ruminal fermentation characteristics. For rumen fluid sources, two dairy bulls with permanent cannulas were used as donors. G. bimaculatus and S. grandiflora powder were combined in the following ratios: 100:0, 99:1, 98:2, 97:3, 96:4, 95:5, 94:6, and 93:7. As 7% of S. grandiflora and 3% of G. bimaculatus were combined, the rumen undegradable protein increased by 45.8% when compared to the control group (p < 0.05). All gas kinetics were not substantially different across treatments, except for cumulative gas output during 96 h of incubation (p < 0.05). Comparing the G. bimaculatus powder to S. grandiflora at a ratio of 95:5 to 93:7 revealed an increase in cumulative gas production (p < 0.05), compared to the other groups. Reduction of G. bimaculatus resulted in a linear increase of in vitro dry matter digestibility (IVDMD) and in vitro organic matter digestibility (IVOMD) at 24 h after incubation. The lowest level of G. bimaculatus—93% with 7% S. grandiflora—showed the greatest IVDMD and IVOMD (p < 0.05) when compared with the control group. Ruminal pH in all treatments remained constant after 4 and 8 h of in vitro incubation (p > 0.05). However, as the quantity of S. grandiflora in the sample increased, the concentration of ammonia-nitrogen (NH₃-N) linearly decreased (p < 0.05). Compared to the control group, the NH₃-N concentration at 4 h of incubation was decreased by 47% when 7% S. grandiflora and 93% G. bimaculatus were mixed. The alteration in the G. bimaculatus to S. grandiflora ratio did not affect the levels of acetic acid or butyric acid. However, when 93% of G. bimaculatus was combined with 7% of S. grandiflora at hour 4 of incubation, propionic acid concentration was moderately increased (p < 0.01) by 6.58 mmol/L. In conclusion, combining 93% G. bimaculatus with 7% S. grandiflora powder enhanced protein utilization, in vitro digestibility, propionate concentration, and cumulative gas production.

1. Introduction

The protein sources used in the animal feeding system will consistently be stocked with high-quality options at competitive prices [1]. Additionally, soybean meal is a major source of nutritional protein in the majority of animal production systems. Insect protein is a potential substitute for the usual plant proteins given to animals [2]. Insects are a protein source that is nutrient-dense and sustainably produced, according to the FAO [3]. In general, insects are more environmentally friendly sources of dietary protein for livestock than soybean meal, due to their low conversion ratio from the organic substrate to insect biomass over a short period and their low water requirement [4]. They have several advantages over proteins obtained from plants and animals. Insects also contain a high-quality fatty acid, a protein with a relative amino acid composition, and micronutrients, such as iron, magnesium, and selenium [5]. The utilization of insect products in ruminant diets, however, has only been the focus of a very small number of scientific studies [3,4]. As a result, there is still more research to be done on feeding ruminant insects.

Gryllus bimaculatus is the name of a species of cricket that belongs to the Gryllinae subfamily. G. bimaculatus is a promising insect species that contains high crude protein (CP) content, above 60–70% of dry matter (DM), with relatively good amino acid profiles, such as lysine, threonine, and tryptophan [2,5]. All of these advantages have increased interest in the concept of employing insects as a feed ingredient for animals, particularly ruminants. However, it has been well known that protein degradation in the rumen is rather “a wasteful process”, as more than 60% of the protein that enters the rumen is degraded rapidly to ammonia [6]. Even while some ammonia is absorbed through the rumen wall and some are used for microbial protein synthesis, the excess ammonia cannot be utilized by the animal and must be expelled by the urine. Protein bypass in the rumen is a strategy to protect the protein feed from rumen degradation and increase the protein amount that enters the abomasum [7], which can be obtained by physical treatment such as heat, chemical, formaldehyde, or tannin-saponin [8].

Tannins and saponins are widely distributed secondary compounds found in plants. By establishing a tannin–protein complex with protein through hydrogen bonding and controlling protein degradation in the rumen, tannin’s molecular structure allows it to control ruminal fermentation [8]. The tannin–protein complex is bound in the abomasum, where, although being stable at neutral pH levels and immune to rumen microbial oxidation, it dissociates at low pH values [7]. By reducing the quantity of proteins that are broken down in the rumen and increasing the flow of bypass proteins to the small intestine, tannin supplementation aims to improve overall protein digestion [9]. Sesbania grandiflora is a small leguminous plant grouped in the Fabaceae family and Sesbania genus [9]. It has edible flowers and leaves that are commonly eaten in Southeast Asia. S. grandiflora’s seed pods contain 35% CP, whereas tannins make up roughly 10% of the dry matter (DM) [10]. Previously, Unnawong et al. [9] found that 0.6% S. grandiflora supplementation prevented protein digestion by around 12% in cattle and may have enhanced rumen microorganisms and feed utilization. The effect of a high-protein bug like G. bimaculatus paired with a rumen bypass agent like S. grandiflora, on the other hand, has yet to be determined. It was hypothesized that a phytonutrient from S. grandiflora would enhance protein utilization and promote rumen fermentation when combined with an insect meal. Therefore, the purpose of this study was to investigate how using phytonutrients from S. grandiflora to bind proteins from G. bimaculatus affected the kinetics of gas, in vitro digestibility, and ruminal fermentation characteristics.

2. Materials and Methods

2.1. Substrate Preparation

G. bimaculatus was obtained from a local farmer in the Khon Kaen province of Thailand. G. bimaculatus was housed in big cages (120 cm W × 240 cm D × 80 cm H) in an evaporative house and kept in a climate-controlled chamber at 30 °C, with a 12-h day/night cycle and 30% humidity. The top cage was given ad libitum of chicken feed and vegetable waste. Each cage contained four egg cartons (30 × 30 × 30 cm) to increase crawl space. All cages received ad libitum water via plastic tubes (50 mL) with cotton balls at the end. The crickets were fed for approximately 30–35 days and brought to oven-drying at 60 °C for 48–72 h, then ground through a 1 mm sieve (Cyclotech Mill, Tecator, Höganäs, Sweden) and kept in a temperature-controlled room. Fresh S. grandiflora pods were harvested from Khon Kaen and Udon-Thani provinces (Thailand) from September to November 2020. The pods were sun-dried for a month before being ground to powder form through a 1 mm sieve (Cyclotech Mill, Tecator, Höganäs, Sweden).

2.2. Research Design

The research was designed according to a randomized complete block design, with incubation runs as a random effect (three replicates and three runs). The powders of G. bimaculatus and S. grandiflora were blended well to create a new product in the following proportions: 100:0, 99:1, 98:2, 97:3, 96:4, 95:5, 94:6, and 93:7, respectively, and then stored in bags until usage. S. grandiflora levels were used according to the recommendation by Unnawong et al. [9].

2.3. Chemical Composition Determination

The proximate composition of the samples was determined using the AOAC methodology, which included DM, OM, ash, CP, ether extract (EE), and crude fiber (CF). The saponin and tannin content of the S. grandiflora sample was calculated using data from Unnawong et al. [8,9], using Edeoga et al. [11] as a correction. In brief, 5 g of S. grandiflora were placed in an Erlenmeyer flask containing 80% methanol. The flask’s contents were then microwaved for 30 min before being transferred to a new flask with Whatman No. 41. The procedure was replicated four times in total. The sample was filtered before being evaporated in a revolving vacuum evaporator to a final volume of about 25 mL. A separatory funnel was used to separate the resulting solution from 99.9% ether. After that, the bottom-funnel residue was re-separated with butanol and washed twice with 5% NaCl. Following that, the residue was boiled in a water bath for 30 min at 80 °C. The crude saponin concentration of S. grandiflora was measured after oven drying the residue overnight at 60 °C. Using a modified Burns [12] approach, the tannin content was determined.

2.4. Rumen Degradable Protein Analysis

The substrate sample was determined for degradable protein percentage according to the protocol by Licitra et al. [13]. The amount of protease solution necessary for rumen-degradable protein (RDP) analysis was calculated using a tungstic acid technique to estimate the true protein (TP) of the feed sample. In a 125 mL Erlenmeyer flask, 0.5 g of dried material was weighed, followed by 50 mL of cold, distilled water and 8 mL of 10% sodium tungstate solution. The flask was maintained at room temperature overnight after being placed at 20 to 25 °C for 30 min to drop the pH to 2 with the addition of 10 mL of 5 M sulfuric acid. Whatman No. 541 was used to filter the mixture before it was transferred into a conical funnel. The residue was then washed twice with cold, distilled water before being transferred to a Kjeldahl flask to determine the amount of residual nitrogen. A total of 40 mL of borate-phosphate buffer were added, and the mixture was incubated for an hour at 39 °C. In the flask, the protease enzyme was introduced and stirred using a magnetic stirrer. After 18 h, the sample was filtered using Whatman No. 541 filter paper and then washed with 250 mL of cold, distilled water. The amount of N in the residue was estimated using the Kjeldahl technique, and the degradability as a proportion of total CP was computed: % RDP = (CP—rumen undegradable protein (RUP))/CP × 100.

2.5. Fermentation Characteristics Determination

Rumen liquor was donated by two fistulated dairy bulls, approximately 450 ± 30 kg of body weight. Individual pens were utilized to house the animals, and they were given access to TMR containing 14% and 75% of CP and TDN, respectively. Fresh feed was delivered twice daily (08:00 and 15:00), with 10% refusals permitted. Water and mineral blocks were available as options. Before morning feeding, ruminal fluid from cattle was obtained. The inoculated rumen fluid was then filtered through four layers of cheesecloth and stored in warmed thermos bottles before being transported to the laboratory. All procedures were anaerobic in condition and sterile between transfers of rumen liquid. To prepare the fermentation solution, rumen fluid and artificial saliva were created and combined in a 1:2 ratio. A total of 0.5 g of feed product samples and rice straw (70:30) were ground and weighed into an in vitro bottle (50 mL volume). Due to its excellent buffering capacity and modification by Cherdthong and Wanapat [14], artificial saliva generated from the composition of McDougall’s saliva was employed to adjust the pH in the rumen simulation system. A 60-mL syringe, with a 1.5-inch 20-gauge needle, was used to administer 40 mL of rumen solution combination to the bottles anaerobically, and the bottles were anaerobically cleaned using CO₂. In vitro, bottles with the individual substrate treatments were incubated in a thermal oven at 39 °C. By using several sets of bottles, the parameters were observed. First, gas release was monitored at 0, 2, 4, 6, 8, 12, 14, 16, 18, 24, 48, 72, and 96 h during the first batch of incubation to assess the gas’s kinetics. Another 24-h set incubation procedure was used to test digestibility. The last set’s pH, ammonia-nitrogen (NH₃-N), and volatile fatty acid (VFA) values were determined after 4 and 8 h of incubation. Three incubation runs were performed for each batch. Using a pressure transducer and a calibrated glass syringe, the gas release was measured at 0, 2, 4, 6, 8, 12, 14, 16, 18, 24, 48, 72, and 96 h during the incubation, as described by Cherdthong and Wanapat [14]. To fit cumulative gas production data, the Ørskov and McDonald [15] equation was used.

The samples were determined immediately at 4 and 8 h (incubate different sets of bottles) for ruminal pH and utilized for NH₃-N [16] and VFA analyses using gas chromatography (GC 8890; Agilent Technologies Ltd., Santa Clara County, CA, USA) equipped with a capillary column (molecular sieve 13×, 30/60 mesh, Alltech Associates Inc., Deerfield, IL, USA). After dissolving for 24 h after inoculation (incubate another set of bottles), the sample was filtered through pre-weighed Gooch crucibles (40 mm porosity), the DM residue was dried at 100 °C in a forced air oven for 24 h, and the DM residue was weighed for in vitro DM digestibility (IVDMD) determination according to a procedure previously used by Kaewpila et al. [17]. To determine in vitro OM digestibility (IVOMD), the dried residues were burned in a muffle furnace at 550 °C for 3 h.

2.6. Statistical Analysis

All data were analyzed as a randomized complete block design using the Proc ANOVA procedure of SAS^® OnDemand for Academics (SAS; Institute Inc., Cary, NC, USA). The model included the fixed effect of treatment (Gryllus bimaculatus and S. grandiflora powder ratios). The effect of the incubation run (block) was included as a random effect. The sampling time for pH, NH_3, VFA, and digestibility were included as a repeated measurement. Comparisons among treatment means were performed by orthogonal polynomials.

3. Result

3.1. Chemical Composition of Products

The proximate nutritional composition of treatments studied is represented in

Table 1. Chemical composition of G. bimaculatus, S. grandiflora, and rice straw.

Items	G. bimaculatus	S. grandiflora	Rice Straw
Dry matter, %	95.37	94.19	92.20
-----------------% of dry matter--------------------
Organic matter	92.02	93.86	90.61
Ash	7.96	6.14	9.39
Ether extract	27.79	4.42	1.46
Crude fiber	9.52	13.6	38.13
Crude protein	58.52	22.48	3.27

All express as % on a DM basis except for DM.

and

Table 2. Effect of manipulation of in vitro protein degradation from G. bimaculatus powder combined with phytonutrients from S. grandiflora on chemical composition, true protein (TP), and rumen undegradable protein (RUP).

Ratio of G. bimaculatus to S. grandiflora	DM, %	OM, %	Ash, %	EE, %	CF, %	CP, %	TP, %	RUP, %
T1 (100:0)	95.37 a	92.02	7.96	27.79	9.52 a	58.82 a	51.42 a	36.33 a
T2 (99:1)	95.75 a	92.77	7.21	27.35	9.29 ab	58.00 a	47.85 bc	37.95 a
T3 (98:2)	96.52 b	91.51	8.47	26.90	8.65 abc	56.31 b	46.62 bc	39.32 a
T4 (97:3)	97.34 c	91.77	8.22	29.32	8.41 bc	55.11 b	46.27 bc	46.19 b
T5 (96:4)	97.54 c	92.09	7.90	29.58	8.39 bcd	52.57 c	46.09 bc	45.58 b
T6 (95:5)	98.37 d	91.32	8.66	27.01	8.04 cd	52.65 c	45.19 bc	46.74 b
T7 (94:6)	98.43 d	91.92	8.07	26.14	7.48 de	52.14 cd	44.64 bc	47.01 b
T8 (93:7)	98.46 d	97.77	8.21	25.62	6.83 e	51.15 d	44.45 c	53.00 c

DM: dry matter; OM: organic matter; EE: ether extract; CF: crude fiber; CP: crude protein; TP: true protein; RUP: rumen undegradable protein; SEM: standard error of the mean; ^a,b,c: means with different superscripts within a colomn are significantly different at p ≤ 0.05.

. The average of OM, ash, and EE were similar among treatments (p > 0.05). DM, CF, CP, TP, and RUP were shown to differ significantly between treatments. As 7% of S. grandiflora and 3% of G. bimaculatus were combined, RUP increased by 45.8% when compared to the control group (p < 0.05). Most treatments contained high amounts of protein (51–58%) and fat (25–29%). The highest CP content was observed in 100% of G. bimaculatus (T1), but the ratio of G. bimaculatus to S. grandiflora at 93:7 (T8) contained the lowest (51.15%).

3.2. Kinetic, Cumulative Gas

The kinetic gas and cumulative gas production for all treatments were shown as total gas production, a (the gas production from the immediately soluble fraction), b (the gas production from the insoluble fraction), and c (the gas production rate constant for the insoluble fraction (b)); a + b is the potential extent of gas for the substrates studied and are shown in

Table 3. Effect of manipulation of in vitro protein degradation from G. bimaculatus powder combined with phytonutrients from S. grandiflora on kinetic of gas and gas production.

Ratio of G. bimaculatus to S. grandiflora	Kinetic of Gas, mL/0.5 g DM				Gas Production at 96 h, mL/0.5 gDM
	a	b	c	a + b
T1 (100:0)	0.47	57.38	0.0532	57.86	40.60 a
T2 (99:1)	0.49	56.50	0.0483	56.75	41.07 a
T3 (98:2)	0.53	54.40	0.0487	54.94	41.00 a
T4 (97:3)	0.85	55.81	0.0455	56.03	42.33 ab
T5 (96:4)	1.02	56.36	0.0539	57.39	40.87 a
T6 (95:5)	0.89	56.71	0.0575	57.61	43.00 b
T7 (94:6)	0.92	53.95	0.0594	54.87	43.53 b
T8 (93:7)	0.88	54.28	0.0565	55.17	46.23 b
SEM	0.14	1.07	0.003	1.11	1.53
Linear	0.08	0.17	0.14	0.16	0.02
Quadratic	0.33	0.30	0.79	0.33	0.85
Cubic	0.70	0.34	0.56	0.48	0.58

a: the gas production from the immediately soluble fraction; b: the gas production from the insoluble fraction; c: the gas production rate constant for the insoluble fraction (b); a + b: potential extent of gas; SEM: standard error of the mean; ^a,b: means with different superscripts within a column are significantly different at p ≤ 0.05.

. All gas kinetics were not substantially different across treatments, except cumulative gas output during 96 h of incubation (p < 0.05). Comparing the G. bimaculatus powder to S. grandiflora at a ratio of 95:5 (T6) to 93:7 (T8) revealed an increase in cumulative gas production (p < 0.05) compared to the other groups.

3.3. In Vitro Digestibility Characteristic

Table 4. Effect of manipulation of in vitro protein degradation from G. bimaculatus powder combined with phytonutrients from S. grandiflora on in vitro dry matter and organic matter digestibility.

Ratio of G. bimaculatus to S. grandiflora	In Vitro Dry Matter Digestibility, % DM	In Vitro Organic Matter Digestibility, % DM
	24 h after Incubation	24 h after Incubation
T1 (100:0)	47.28 a	67.66 a
T2 (99:1)	47.75 a	67.11 a
T3 (98:2)	47.71 a	67.23 a
T4 (97:3)	48.33 a	69.32 ab
T5 (96:4)	49.42 a	71.44 ab
T6 (95:5)	52.80 b	74.75 b
T7 (94:6)	57.29 b	78.18 b
T8 (93:7)	64.38 c	79.35 b
SEM	5.74	0.17
Linear	0.03	0.05
Quadratic	0.75	0.54
Cubic	0.95	0.22

_a,b,c means with different superscripts within a column are significantly different at p ≤ 0.05.

shows the effects of combining phytonutrients from S. grandiflora with G. bimaculatus powder on IVDMD and IVOMD. When G. bimaculatus was reduced, the IVDMD and IVOMD at 24 h after incubation linearly increased (p < 0.05). When compared to the control group, the lowest level of G. bimaculatus—93% with 7% S. grandiflora—showed the greatest IVDMD and IVOMD (p < 0.05).

3.4. In Vitro Fermentation Characteristics

Table 5. Effect of manipulation of in vitro protein degradation from G. bimaculatus powder combine with phytonutrients from S. grandiflora on pH and ammonia nitrogen.

Ratio of G. bimaculatus to S. grandiflora	pH		NH3-N, mg/100 mL
	4 h after Incubation	8 h after Incubation	4 h after Incubation	8 h after Incubation
T1 (100:0)	6.97	6.68	14.06 c	21.06 c
T2 (99:1)	6.96	6.67	13.69 c	20.69 c
T3 (98:2)	6.97	6.68	13.40 c	20.16 c
T4 (97:3)	6.98	6.68	12.17 c	20.55 c
T5 (96:4)	6.99	6.70	11.03 b	20.37 c
T6 (95:5)	6.98	6.70	10.75 b	18.77 b
T7 (94:6)	7.01	6.70	10.35 b	19.33 b
T8 (93:7)	7.01	6.72	7.44 a	15.45 a
SEM	0.009	0.01	0.89	1.06
Linear	0.40	0.49	0.05	0.02
Quadratic	0.40	0.10	0.63	0.90
Cubic	0.81	0.49	0.80	0.40

NH₃-N: ammonia nitrogen; DM: dry matter; OM: organic matter; SEM: standard error of the mean; ^a–c: means with different superscripts within a column are significantly different at p ≤ 0.05.

displays ruminal pH and NH₃-N. Ruminal pH in all treatments remained constant after 4 and 8 h of in vitro incubation (p > 0.05). However, as the quantity of S. grandiflora in the sample increased, the concentration of NH₃-N linearly decreased (p < 0.05). When 7% S. grandiflora and 93% G. bimaculatus were combined, the NH₃-N concentration at 4 h of incubation was reduced by 47% in comparison to the control group.

3.5. Total VFA Concentration

Table 6. Effect of manipulation of in vitro protein degradation from G. bimaculatus powder combined with phytonutrients from S. grandiflora on total volatile fatty acids, acetic acid, propionic acid, and butyric acid.

Ratio of G. bimaculatus to S. grandiflora	TVFAs, mmol/L		C2, mmol/L		C3, mmol/L		C4, mmol/L
	4 h after Incubation	8 h after Incubation	4 h after Incubation	8 h after Incubation	4 h after Incubation	8 h after Incubation	4 h after Incubation	8 h after Incubation
T1 (100:0)	75.05	107.00	53.41	70.78	5.61 c	18.23	7.03	9.66
T2 (99:1)	76.82	103.04	54.54	70.71	5.47 c	20.41	5.97	7.75
T3 (98:2)	52.52	104.76	39.24	74.17	6.42 bc	22.34	5.73	8.28
T4 (97:3)	49.51	101.31	40.60	72.00	6.94 bc	21.82	4.85	8.23
T5 (96:4)	56.60	100.23	39.60	69.70	7.46 b	23.59	4.63	8.69
T6 (95:5)	56.30	101.55	40.04	71.08	7.70 b	24.86	4.63	8.28
T7 (94:6)	54.31	100.93	40.44	73.66	11.10 a	24.56	3.60	6.85
T8 (93:7)	52.51	96.50	38.46	73.16	12.19 a	26.54	2.75	4.78
SEM	6.67	1.68	6.08	2.21	0.38	1.32	0.63	1.44
Linear	0.05	0.20	0.22	0.64	0.0002	0.36	0.09	0.89
Quadratic	0.80	0.91	0.98	0.73	0.44	0.85	0.91	0.88
Cubic	0.24	0.28	0.36	0.48	0.03	0.62	0.68	0.24

TVFAs: total volatile fatty acids profile; C2: acetic acid; C3: propionic acid; C4: butyric acid; SEM: standard error of the mean; ^a–c: means with different superscripts within a column are significantly different at p ≤ 0.05.

displays the proportions of the total VFA, acetic acid, propionic acid, and butyric acid. The average total VFA after 4 and 8 h of incubation, which varied from 52.51 to 75.05 and 96.50 to 107.00 mmol/L, respectively, did not alter between treatments (p > 0.05). The alteration in the G. bimaculatus to S. grandiflora ratio had no effect on the levels of acetic acid or butyric acid. However, when 93% of G. bimaculatus was combined with 7% of S. grandiflora at hour 4 of incubation, propionic acid concentration was moderately increased (p < 0.01) by 6.58 mmol/L.

4. Discussion

The increasing level of G. bimaculatus in products was linearly decreased in crude fiber, CP, TP, and RUP (p < 0.01). The high CP and fat content of G. bimaculatus matched that of Jayanegara et al. [2], who earlier reported a protein level of 58 percent in crickets. Furthermore, the current study verified previous reports that insects are high in protein and contain all of the essential amino acids [18]. Thus, insects are one of the potential protein sources for cattle feeding [19,20]. Based on the species, developmental stage, and nutritional quality, insects could have variable CP contents and amino acid compositions [21,22]. A substantial amount of fat was also found in G. bimaculatus, as was also shown in earlier investigations [2,23]. However, the TP was greater in the control and gradually decreased when the amount of cricket meal in the substrate dropped (p < 0.01). Furthermore, the RUP improved linearly by 45.8% as the amount of S. grandiflora in the product increased. This could be attributed to S. grandiflora’s high tannin concentration, which has been demonstrated to prevent protein breakdown in the rumen via amino bypass synthesis [24], implying that tannin is typically active in protein protection, either by protein complex synthesis or by suppressing microbial protein degradation. This reduction in RDP was supported by an increase in the RUP value [25].

At 96 h of incubation, gas production increased as G. bimaculatus levels decreased. This could be because G. bimaculatus has a high-fat content, which might limit the ability of microorganisms to break down the feed. A decrease in microbial activity, as well as a reduction in feed breakdown and fermentation, which both contribute to gas production, are ways that fat can be inhibited from producing gas [26]. Rumen microbial breakdown, particularly fiber degradation, has been demonstrated to be harmed by dietary EE, in accordance with the findings of Jayanegera et al. [2], who found that replacing soybean meal with cricket meal reduced the value of in vitro gas generation. Furthermore, consistent with our findings, gas reduction impacts due to dietary lipids under an in vitro fermentation system have repeatedly established that dietary fat content affects gas production [27,28,29].

The reduction of G. bimaculatus, which results in less fiber content, may be impacting the increase of IVDMD and IVOMD. Most of their exoskeleton (8–9%) is made up of chitin, a long-chained N-acetylglucosamine polymer. As a result of lower IVDMD and IVOMD when substantial insect inclusion occurs [30], this fiber component is known to be poorly assimilated by animals and may suppress rumen microbial activity [2,31]. Additionally, rumen microbial limitations and decreased digestibility may be caused by excessive fat content in insects [31,32]. Similar findings were made by Jayanegara et al. [33], who discovered that adding 14.5% oil-containing crickets to a diet reduced IVDMD by 15% when compared to the control group.

Ruminal pH is one of the indicators that help to regulate the rumen ecology and control rumen microbial activity [33,34]. The current finding showed that when insects were added together with plant phytonutrients, ruminal pH was discovered to be steady within a range of 6.76 to 7.01. This temperature range was suggested as appropriate for bacteria to break down the feed that animals ingest [35,36].

As the amount of insect protein was reduced from 100 to 93%, the concentration of NH₃-N linearly decreased. Because G. bimaculatus serves as a source of protein that bacteria can use to generate NH₃-N, low levels of CP result in low concentrations of NH₃-N [36,37]. The phytonutrient tannin, which is a component of S. grandiflora, may also affect the drop in NH₃-N concentration [38,39]. Tannins have a high affinity for proteins, despite having a lot of phenolic groups [40,41]. These offered several opportunities for peptides to bind with their carbonyl groups [7,11]. There were hydrogen bonds generated between the hydroxyl radicals of the phenolic groups and the oxygen of the amide groups, according to Kumar and Singh [39], who have described the involvement of protein protected by tannin in that complexity can originate from the peptide bonds of the protein. In particular, it was believed that hydrogen bonds were the main driver of tannin–protein complex formation, which reduced the rate of protein degradation in the rumen [8,40]. As a result, less NH₃-N was produced, allowing more amino acids to enter the small intestine and be utilized by animals [9,36]. This result was consistent with research by Unnawong et al. [9], which demonstrated that protein breakdown was reduced by 12% when cattle received 0.6% S. grandiflora.

The primary substrate for gluconeogenesis, as well as one of the principal fermentation products, of many rumen bacteria from carbohydrates is propionate [41]. The greatest level of S. grandiflora in the current study had the highest concentration of propionate when compared to the control group that did not receive any supplements. The reason for this might be that S. grandiflora contains sugar, which could serve as a source of fermentable carbohydrates for the synthesis of propionate [42]. As a result, increasing the quantity of the carbohydrate substrate available raises the concentration of propionate [43]. Nevertheless, the current study did not describe the sugar content of S. grandiflora pods. Similarly, Unnawong et al. [9] discovered that beef cattles’ propionate content increased considerably by 8.8% when S. grandiflora feeding was increased. Furthermore, propionate may be impacted by tannin as a result of an increase in S. grandiflora. Although there was no discernible influence on the total VFA by increasing the additional level of tannin, the rumen methane production may have been significantly inhibited as a result of the decreased acetate generation from pyruvate as the amount of tannin in the substrate increased [43]. Additionally, as seen in the current study, adding a tannin source along with insects might increase the concentration of propionate. Tannin’s biological function may involve preventing the growth of acetogenic bacteria while switching the route H₂ uses to produce propionate. Propionic bacterium agents performed better in free-H₂ [44]. Finally, a lower acetate to propionate ratio may be linked to increased propionate concentration [44].

5. Conclusions

In conclusion, the vitro digestibility, propionate concentration, and cumulative gas generation could all be improved by combining 7% S. grandiflora powder with 93% G. bimaculatus powder. The G. bimaculatus and S. grandiflora combination improved protein utilization by reducing NH₃-N levels in the rumen and promoting the rumen undegradable protein. Additional studies are required to determine the response of the proportion combining G. bimaculatus and S. grandiflora in in vivo testing.