<i>Calotropis gigantea</i> flavonoid blends reduce wasteful ammoniagenesis and methanogenesis by inhibiting rumen protozoa

Askale Abrhaley; Tengfei Zhan; Lu Ma; Xiong Li; Dengpan Bu; Askale Abrhaley; Tengfei Zhan; Lu Ma; Xiong Li; Dengpan Bu

doi:10.48130/cas-0026-0016

Figures (2) Tables (2)

Figure 1.
(a) Proximate analysis of leaf powder showing dry matter (DM), ash, neutral detergent fiber (NDF), acid detergent fiber (ADF), ether extract (EE), and crude protein (CP) content. Descriptive statistical analysis of data presented in mean ± SD (% dry matter basis). (b) Flavonoid composition of Calotropis gigantea ethanolic extract, showing total flavonoid content (TFC) and individual flavonoid concentrations: hesperidin (HES), naringin (NAR), kaempferol (KAE), quercetin (QUE), and myricetin (MYR). Values presented as mean ± SD (µg/g extract).
Figure 2.
Sperman's correlation analysis between protozoal genera and rumen fermentation parameters. Color intensity represents correlation strength (red: positive; blue: negative) with significance denoted as * p < 0.05, ** p < 0.01, and *** p < 0.001.

Parameters	Treatments (4.5% DM)									SEM	p value
Parameters	CON	KAE	HES	NAR	KAE + HES	KAE + NAR	HES + NAR	KAE + HES + NAR	CGFE	SEM	p value
pH	6.47^a	6.46^ab	6.45^ab	6.37^cd	6.41^abc	6.43^abc	6.42^abc	6.40^bcd	6.34^d	0.01	< 0.001
TVFA (mM)	45.2^d	47.7^cd	48.9^cd	48.9^cd	51.0^cd	55.0^bcd	56.2^bc	62.4^b	80.8^a	2.17	< 0.001
Individual VFA (mol/100 mol)
Acetate	54.3^bc	55.7^ab	56.1^ab	54.8^bc	56.8^a	54.8^bc	53.1^c	53.3^c	47.3^d	0.38	< 0.001
Propionate	18.2^b	17.4^d	17.7^bcd	18.0^bc	17.5^cd	17.8^bcd	18.1^bc	18.3^b	20.0^a	0.12	< 0.001
Butyrate	24.0^bc	23.5^b	22.8^c	23.9^bc	22.4^c	23.9^bc	25.4^b	24.7^b	27.9^a	0.39	< 0.001
Isobutyrate	0.82^b	0.78^b	0.81^b	0.76^b	0.77^b	0.82^b	0.77^b	0.82^b	0.95^a	0.02	< 0.001
Valerate	1.38^c	1.35^c	1.41^bc	1.39^c	1.34^c	1.43^bc	1.43^bc	1.56^b	2.17^a	0.03	< 0.001
Isovalerate	1.25^b	1.22^b	1.21^b	1.19^b	1.18^b	1.26^b	1.24^b	1.29^b	1.58^a	0.03	< 0.001
A:P ratio	2.99^de	3.20^ab	3.16^abc	3.04^cde	3.24^a	3.08^bcd	2.94^de	2.91^e	2.37^f	0.03	< 0.001
NH₃-N, mg/dL	19.4^a	13.3^cd	17.6^ab	15.0^bcd	13.2^cd	13.7^cd	15.9^abc	12.9^cd	12.0^d	0.83	< 0.001
Methane*, mol/100mol	29.0^bc	29.7^a	29.5^ab	29.3^abc	29.7^a	29.3^ab	29.1^bc	28.8^c	27.0^d	0.09	< 0.001
MCP, mg/mL	1.94^bcd	2.01^bc	1.10^e	1.53^cde	1.80^bcd	1.77^bcd	1.43^de	2.13^b	3.14^a	0.11	< 0.001
* Estimated from standard stoichiometric equation: Methane = 0.45 × (Acetate proportion) − 0.275 × (Propionate proportion) + 0.40 × (Butyrate proportion)^[1,2]. TVFA total volatile fatty acids, VFA volatile fatty acids, NH₃-N ammonia nitrogen; MCP microbial crude protein. Fermentation parameters across treatments: control (CON), kaempferol (KAE), hesperidin (HES), naringin (NAR), their combinations, and Calotropis gigantea flavonoid extract (CGFE). Means with different superscripts differ (p < 0.05). SEM = standard error.

Table 1.

Effect of flavonoid treatments on rumen fermentation characteristics, ammonia-N, methane, and microbial protein.

Log₁₀ cells/mL	Treatments (4.5% DM)									SEM	p value
Log₁₀ cells/mL	CON	KAE	HES	NAR	KAE + HES	KAE + NAR	HES + NAR	KAE + HES + NAR	CGFE	SEM	p value
Total protozoa	5.59^a	5.46^bc	5.53^ab	5.51^abc	5.44^c	5.55^a	5.54^ab	5.35^d	5.33^d	0.02	< 0.001
Isotricha	3.27	2.64	1.75	1.36	1.75	2.04	1.39	1.00	1.80	0.48	0.079
Dasytricha	1.33	0.97	0.68	0.65	0.68	0.68	0.68	0.65	0.65	0.46	0.977
Entodinium	5.59^a	5.45^bc	5.52^abc	5.51^abc	5.44^c	5.54^a	5.53^ab	5.34^d	5.32^d	0.02	< 0.001
Epidinium	0.32	0.65	0.32	0.36	0.32	0.32	0	0.68	0.68	0.33	0.917
Diplodinium	0.97	1.72	1.00	1.82	0.71	0.71	0.32	0.68	1.00	0.48	0.433
Ophryoscolex	3.36	2.91	2.62	3.39	3.33	3.16	3.43	2.21	3.39	0.24	0.079
Prokaryotes (log₁₀ copies/mL)
Total bacteria*	11.9^ab	12.6^a	12.1^ab	12.0^ab	12.5^ab	12.0^ab	11.8^ab	12.4^ab	11.9^ab	0.17	0.006
Total Methanogens^⁑	9.23^d	10.1^abc	9.76^bcd	9.60^bcd	9.37^cd	10.3^ab	10.2^ab	10.6^a	10.2^ab	0.16	< 0.001
Control (CON), kaempferol (KAE), hesperidin (HES), naringin (NAR), Calotropis gigantea extract (CGFE). Means with different superscripts within rows differ (p < 0.05). SEM = standard error of the mean, * absolute abundance expressed as log₁₀ copies of 16S rRNA genes per mL culture, ^⁑absolute abundance expressed as log₁₀ copies of mcrA genes per mL culture.

Table 2.

Effect of different flavonoid treatments on microbial populations.