Assessing the effect of ensiling moisture and sampling date on silage quality and subsequent yield of North American elderberry

doi:10.21203/rs.3.rs-7559441/v1

After harvesting the berries from the elderberry plant, the remaining plant residue can serve as a fodder for ruminants. A laboratory silo experiment was conducted to determine the effect of a) ensiling immediately after cutting [38–47% dry matter (DM), or after wilting (47–54% DM)] and b) ensiling dates: early post-harvest, mid post-harvest, and late post-harvest on the nutritive values and fermentation parameters of ensiled elderberry fodder. Early-harvest and unwilted fodder exhibited the lowest DM (P < 0.001) and the highest crude protein (CP: P < 0.024) in post-ensiled silage. Ensiling after later harvest dates significantly decreased total digestible nutrients and increased acid detergent fiber, neutral detergent fiber, and lignin (P < 0.01). Moisture by harvest date interactions were observed for calcium (P = 0.017), phosphorus (P = 0.005), magnesium (P < 0.01), potassium (P = 0.002), and sulfur (P = 0.03). Early harvested and unwilted fodder exhibited the greater forage mineral concentrations in post-ensiled silage compared with wilting levels and harvest dates. Iron, zinc, manganese, ash, non-fiber carbohydrate, net energy for lactose, net energy for maintenance, and relative feed value concentrations in post-ensiled silage from late harvest were lower than those from early harvest fodder (P < 0.05). Wilting and/or post-harvest ensiling dates impacted fermentation parameters such as butyric acid, pH, acetic acid, ammonia, lactic acid, formic acid, and succinic acid of post-ensiled elderberry. The timing of forage harvest, whether early or late, following the berry harvest did not affect berry yield in the following year. In conclusion, the post-ensiled nutritive values, including higher CP and most mineral concentrations and lower fiber levels, were observed in early harvest elderberry fodder compared with late harvest.

Elderberry

Silage Quality

Harvest Dates

Wilting Levels

Elderberry (Sambucus nigra subsp. canadensis) is a deciduous multi-stemmed woody shrub that typically grows to a height of about 1.5 to 3.1 meters, with a similar width. This plant is native to eastern and central North America (Charlebois, 2007) and can thrive in various soil types. The potential use of elderberry in the human food industry has been widely studied, as the anthocyanins and vitamins derived from elderberry are utilized as colorants and antioxidants in food preservatives (Domínguez et al., 2021). Elderberries are also processed into pies, jellies, ice creams, yogurts, and alcoholic beverages, and they are rich in essential nutrients, including carbohydrates, proteins, lipids, vitamins, and minerals (Thomas et al., 2020).

North American Elderberry ripens and becomes ready for harvest during mid-August to mid-September. After the berry harvest, leaves remain on the plant until seasonal leaf abscission in the late fall. The standard management practice for elderberry is to coppice the plant after berry harvest for the preparation of spring regrowth. Leafy biomass are generally disposed but research is needed to explore when biomass can be harvested and provide the greatest nutritive value, without affecting berry production in the following year.

Although some literature has reported that elderberry is highly palatable to grazing ruminants, utilizing the plant as a forage is not recommended due to the cyanogenic compounds in fresh elderberry plant (Panter, 2018). Additionally, the use of elderberry for grazing livestock has not been widely studied. Ensiling the late-season forage biomass could provide a solution to avoid potential toxicity and utilize elderberry plants as a livestock feed. Previous work has demonstrated that late-season harvest of elderberry biomass can be successfully ensiled that produce low-quality silage (crude protein, CP = 5.7%; acid detergent fiber, ADF = 68.1%; neutral detergent fiber, NDF = 77.8%; total digestible nutrient, TDN = 51%; Nieman and Conway-Anderson, 2022). Nutritive value of post-ensiled elderberry coppiced at green stage, immediately after the berry harvest and subsequent berry production is unknown.

The moisture at ensiling is a major factor that drives the production of fermentation acids and thereby, the reduction in pH. Lower silage pH (< 4.5) has greater stability (less spoilage) over time (Muck, 1988). Excessive moisture before ensiling results in poor fermentation and spoilage (Ridla et al., 2024). Early harvested elderberry have a lower dry matter (DM) percentage in comparison to late harvest (McKenzie, 2024). However, recommendations for DM percentage at ensiling and final pH differ based on forage species, post-harvest processing, and storage (Borreani et al., 2018). Wilting the forage after harvest to increase the DM percent reduces nutrient loss in the form of gases and effluents in freshly cut forages with low DM (Ribas et al., 2021). The optimum dry matter content required before ensiling falls in the range of 35 to 45% (McDonald et al., 1991).

Plants, including woody species, increase in fiber and lignin components, while CP and fermentable carbohydrates decrease, resulting in lower digestibility (Alstrup et al., 2016; Cabezas-Garcia et al., 2018). As a result of the lower concentrations of fermentable carbohydrates in mature forages, volatile fatty acid production is decreased. In addition, animal performance is limited when only low-quality forages/fodders are offered as feed. However, low-quality forages can be readily utilized in ruminant diets when supplementation is provided to fill nutritional deficiencies.

The objective of the study was to determine the a) effect of ensiling immediately after cutting (38–47% DM) and ensiling after wilting (47–54% DM) and b) ensiling dates early (15 Sept), mid (6 Oct), and late (30 Nov) after berry harvest on post-ensiled nutritional parameters. Nutritive parameters analyzed include CP, ADF, NDF, TDN, non-fiber carbohydrates (NFC), net energy for maintenance (NEM), net energy for lactation (NEL), net energy for gain (NEG), relative feed value (RFV), mineral concentrations, and fermentation parameters such as pH, propanediol, propanol, acetic acid, butyric acid, ammonia, lactic acid, succinic acid, and ethanol.

Experimental Site

The study was conducted at the Horticulture and Agroforestry Research Farm (HARF) near New Franklin, Missouri, USA (39°01'"N, 92°75'64"W). This area is comprised of silt loam, well-drained, loess soils that roll along the upland ridgetops, and low order streams converge into the Missouri river. Mean annual precipitation during the experimental period was 1060 mm (with approximately 64% of the precipitation occurred between April to September). Mean temperature during July was 25.6°C and mean temperature during January was − 2.06°C. About 2000 m² area was used for planting elderberry plants in four plots. Each plot had 4 rows of 25 m in length. Details regarding soil fertility, bed preparation, nutrient analysis before bed preparation, and planting details (row-to-row distance and plant-to-plant distance) are available in previous publication (McGowan et al., 2018).

Plant fodder harvest and ensiling

The study was conducted in 2 × 3 factorial design structure in a completely randomized design. Fodder was harvested at three time points after the harvest of berries in August (15 Sept, 6 Oct, and 30 Nov of 2023), and ensiled at immediately after cutting and chopping (38–47% DM) or after cutting, wilting, and chopping (47–54% DM). The target packing density was 240 kg DM/m³. Each treatment combination was replicated three times.

The elderberry canes (whole plant) were coppiced at 12.7 cm above the ground level. Harvested canes were passed through a commercial mulch chipper (Wallenstein BX42) to approximately 5 to 7.6-cm size. Chopped material was passed through a mesh to screen pieces greater than 7.6 cm in length. The screened material was divided into two, with one portion containing material that was ensiled immediately and other portion wilted on a tarp in open air and sunlight for approximately 3 hours before ensiling to target 55% DM. Target DM was estimated using quick method of microwave drying and measured with a balance scale (A&D Engineering, Model EK-30KL balance, Ann Arbor, MI, USA). Pre-ensiled subsamples were collected to determine DM and nutritional parameters prior to placing into the mini-silo for all treatment combinations, but not each replicate.

Eighteen laboratory polyvinyl chloride (PCV) silos were constructed (10.2 cm diameter × 29.2 cm height) with rubber end caps and metal brackets to secure the caps, which allowed for 2.4 L of elderberry material as described in (Nieman & Conway-Anderson, 2022). Silos were packed, sealed, and placed in dry indoor maintained at approximately 10°C during the fermentation period. After 60 days, the ensiled silos were opened, contents were removed, weighted, and samples were individually packed in Ziplock plastic bags, and frozen at -20°C until nutrient and fermentation analysis.

Determination of fruit yield

The field layout is described in detail in a previous publication (McGowan et al., 2018). Four plots of elderberry plants were established, with each plot further divided into four rows, each 25 m in length. In three plots, the elderberry plant was coppiced on three different dates (15 Sep, 6 Oct, and 30 Nov), while the fourth (control) plot was coppiced on the following year on March. The elderberry plants were spaced at 3 m apart between rows and 2.4 m apart within rows. To determine yield, berries were harvested on 6 August, 2024 when the fruit was fully set and ~ approximately 50% ripened. This should occur prior to any evidence of pest pressure that might artificially reduce yield data. Each block of silage treatment consists of 4 rows. From each row, the number of cymes were counted and snipped from each row as close as possible without leaving extra stem and weighed in bulk. Clippers were used to snip cymes, and they were weighed using a bucket and portable scale.

Nutritive Analysis

Pre-ensiled samples (150 g) were taken from each batch of wilted and unwilted elderberry fodder on each date for nutrient analysis, and another 150 g of pre-ensiled samples from each treatment combination were kept separately in a forced air oven (Blue M Electric, POM-1406F, Watertown, WI, USA) at 60°C for 48 hours to calculate DM content. The remaining 150 g of material was shipped to Dairy One Forage Laboratory (Ithaca, NY, USA) for nutrient analysis. Major nutrient analysis included CP, ADF, NDF, TDN, NEL, NEM, NFC, NEG, RFV, and minerals (Ca, P, Mg, K, Na, Fe, Zn, Cu, Mn, Mo, S, and Ash).

Crude protein was analyzed using the combustion technique with CN928 Carbon/Nitrogen determinator (AOAC, 2003), ADF and NDF were analyzed using the Ankom200 Fiber analyzer (ANKOM, 2020), and non-fiber carbohydrates were calculated with the formula: 100 - (CP + NDF + ether extract + ash). Similarly, TDN was calculated using the summative energy equation by Dairy One, while net energy for lactation, NEG, and NEM were also determined by Dairy One (NRC, 2001). The relative feed value was calculated by multiplying digestible dry matter by dry matter intake, and then dividing by 1.29 (Moore & Undersander, 2002). For mineral analysis, samples were digested in 50 mL MARS Xpress vessels (CEM Corporation, Matthews, NC, USA) using a MARS 6 Microwave Digestion System (CEM Corporation Matthews, NC USA), and mineral concentrations were measured using a Thermo iCAP Pro XP ICP (Thermo Fisher Scientific Inc. Waltham, MA, USA).

Fermentation Analysis

Post-ensiled fodder materials were sent to Rock River Laboratory (Watertown, WI, USA) to determine fermentation parameters 2,3-butanediol, pH, acetic acid, ammonia, butyric acid,1,2-Prop, ethanol, formic acid, lactic acid, and succinic acid. Ensiled samples were mixed with deionized water for pH determination. Volatile fatty acids (VFA) were extracted at a ratio of 1:10 (silage sample to deionized water), centrifuged, and the supernatant solution was combined with calcium hydroxide and copper sulfate. This mixture was centrifuged a second time, and the supernatant was analyzed using high-performance liquid chromatography equipped with a reverse-phase ion exclusion column and a refractive index detector (Waters Corporation, Milford, MA). The same supernatant collected during VFA production was used for ammonia-nitrogen analysis with the Skalar San +++ Segmented Flow SA 500 Analyzer (Skalar, Breda, The Netherlands) (Krom, 1980).

Statistical Analyses

Nutritional and fermentation parameters of silage were analyzed using the R statistical software program (R version 3.6.2) to examine the impact of unwilted or wilted, harvesting dates, and the interaction of wilting status and harvesting dates with a 2×3 factorial treatment in ANOVA. A Tukey test was performed to identify significant differences among treatments. Individual treatment means were summarized using Least-Squares Means (LSMEANS), and the level of significance was determined at P < 0.05, with tendencies toward significance between 0.10 and 0.05. Also, the measurement of elderberry fruit yield was compared among early, mid, and late sampling season plots, including control, using analysis of variance (ANOVA) in R statistical software.

Pre-Ensiled Nutrient Composition

Nutritional characteristics of wilted and un-wilted pre-ensiled and post-ensiled elderberry forage from the three harvest dates are provided in Table 1. Pre-ensiled samples were taken from each batch of wilted and unwilted elderberry fodder for each date, and do not represent individual lab silos, and therefore, averages were not analyzed statistically. Averages for wilted and un-wilted for pre-ensiled sample are largely similar. Post-ensiled samples for both mid-harvest and late harvest have slightly increased ADF and NDF proportions and decreased TDN and NFC proportions compared to pre-ensiled samples. Increases in the proportion of ADF and NDF are common in post-ensiled forages due to the loss or fermentation of readily fermentable carbohydrates (TDN and NFC) (Ridla et al., 2024), though not always present, and did not appear to occur for early harvest elderberry fodder in this study, though NFC was lower in post-ensiled samples.

Table 1

Nutritional parameters of pre- and post-ensiled elderberry fodder wilted or un-wilted and harvested at different dates
Dates		DM	CP	ADF	NDF	TDN	Ash	Lignin	NFC	NEL	NEM	NEG	RFV
Early harvest		%	%	%	%	%	%	%	%	Mcal/kg	Mcal/kg	Mcal/kg	%
Pre-ensiled	Un-wilted	48.45	7.5	44.2	54.4	52	4.88	15.8	29.1	1.12	1	0.45	93
	Wilted	51.02	7.3	42.2	52.3	54	5.41	16	29.8	1.18	1.07	0.52	100
Post-ensiled	Un-wilted	41.9	8.83	38.6	49.1	56.3	6.97	15.1	27.6	1.29	1.20	0.64	111
	Wilted	48.25	8	41.3	52.8	54	6.14	15.7	26.6	1.19	1.1	0.547	100
Mid harvest
Pre-ensiled	Un-wilted	47.80	5.5	53.1	62.9	45	3.3	20.2	23.4	0.89	0.74	0.2	70
	Wilted	53.01	5.9	51.4	62.9	47	3.09	19.3	22.4	0.94	0.83	0.29	72
Post-ensiled	Un-wilted	43.48	5.67	54.1	67.3	41	5.05	21.5	17.8	0.753	0.583	0.0567	64.7
	Wilted	45.92	6	54	66.4	41.7	5.21	21.5	17.7	0.783	0.617	0.103	66
Late harvest
Pre-ensiled	Un-wilted	49.58	4.7	60.4	71.8	38	4.42	24	17.9	0.62	0.43	0	54
	Wilted	57.16	4.8	60.4	73.6	37	4.1	24.2	16.3	0.57	0.39	0	53
Post-ensiled	Un-wilted	47.81	5.1	61.6	76	34.7	3.37	24.4	13.1	0.487	0.31	0	50
	wilted	54.6	4.8	62.3	77.1	34.7	3.15	23.8	12.9	0.46	0.297	0	48.7

Abbreviations: DM, Dry matter; CP, crude protein; ACP, Adjusted crude protein; ADF, acid detergent fiber; NDF, neutral detergent fiber; TDN, total digestible nutrients; Ash, Lignin, NFC, non-fiber carbohydrates; NEL, net energy for lactation; NEM, net energy for maintenance; NEG net energy for gain; RFV, relative feed value.

Post-ensiled nutrient composition.

Nutritional parameters of post-ensiled elderberry fodder wilted or unwilted and three harvest dates are presented in Table 2. Significant interaction was observed between wilting × harvest dates for DM (P < 0.001) and CP (P = 0.024). The lowest DM% (P < 0.05) was observed in early harvest and unwilted elderberry, while the greatest DM% was observed in late harvest and wilted. All unwilted harvesting differed from one another, and all wilted versus unwilted silage within the same harvesting time differed from one another except the mid date harvest. Generally, CP was greater in early harvest fodders than in late harvest fodders, with mid-harvest intermediate, except mid-harvest unwilted fodder was not different from late-harvest unwilted fodder. Wilting by harvest date interactions were observed for Ca (P = 0.017), P (P = 0.005), Mg (P < 0.01), K (P = 0.002), and S (P = 0.03), in which early-harvest × unwilted had the greatest forage mineral concentrations compared with late harvest irrespective of wilting. Mid-harvest mineral concentrations of P, Mg, K, and S were intermediate between early-harvest and late-harvest, irrespective of wilting. Harvesting date affected NDF and ADF, which values were greatest in late harvest (NDF, P < 0.01; ADF, P < 0.01), followed by mid harvest, and lowest during early harvest. Such trend was reversed for TDN (P < 0.01). By harvesting date, Fe concentrations were greatest from mid harvest, lowest from late harvest, and similar to both from early harvest (P = 0.008). Ensiled Mn (P < 0.001), ash% (P < 0.001), lignin (P < 0.001), NFC (P < 0.001), NEL (P < 0.001), NEM (P < 0.001), and RFV (P < 0.001) were greatest for early harvest elderberry forage followed by mid and late harvest. Ensiled Mo (P < 0.01) and NEG (P < 0.001) were greater in early harvested ensiled silage compared with mid and late harvest, whereas mid and late harvest were not different with each other. Ensiled Zn was similar from early and mid-harvest and lower from late harvest (P < 0.001). Only Mn had a significant wilting effect without an interaction, the concentration of Mn (P = 0.04) was greater for unwilted elderberry fodder silage than wilted.

Table 2

Nutritional values of elderberry silage by main effect and interaction for wilted or unwilted fodder and three different harvesting dates (early, mid, and late growth season).
Treatment	DM	CP	NDF	ADF	TDN	Ca	P	Mg	K	Na	Fe
Wilted	%	%	%	%	%	%	%	%	%	%	ppm
Wilted	49.0^a	6.27^a	65.5^a	52.5^a	43.4^a	0.949^a	0.182^a	0.234^b	0.971^a	0.0041^a	114^a
Unwilted	42.9^b	6.53^a	64.1^a	51.4^a	44^a	0.994^a	0.190^a	0.249^a	1.009^a	0.0032^a	111^a
SEM	0.305	0.10	0.581	0.59	0.643	0.016	0.003	0.003	0.012	0.0004	14.3
P-value	< 0.001	0.098	0.13	0.14	0.55	0.074	0.105	0.01	0.053	0.163	0.88
Date
Early	43.1^b	8.42^a	51.0^c	40^c	55.2^a	1.255^a	0.267^a	0.333^a	1.372^a	0.0045^a	123.3^ab
Mid	44.0^b	5.83^b	66.9^b	54^b	41.3^b	1.045^b	0.180^b	0.240^b	0.875^b	0.0036^a	154.7^a
Late	50.6^a	4.95^c	76.5^a	62^a	34.7^c	0.615^c	0.112^c	0.152^c	0.723^c	0.0028^a	60.8^b
SEM	0.37	0.13	0.1	0.59	0.78	0.02	0.003	0.004	0.015	0.0005	17.6
P-value	< 0.001	< 0.001	< 0.001	< 0.001	< 0.001	< 0.001	< 0.001	< 0.001	< 0.001	0.116	0.008
Wilting × Date
Early × Unwilted	38.6^e	8.83^a	49.1^c	38.6^c	56.3^a	1.333^a	0.283^a	0.36^a	1.447^a	0.0033^a	122.3^a
Early × Wilted	47.7^b	8.0^a	52.8^c	41.3^c	54^a	1.177^b	0.250^b	0.307^b	1.297^b	0.0056^a	124.3^a
Mid × Unwilted	43.0^d	5.67^bc	67.3^b	54.1^b	41^b	1.033^c	0.177^c	0.237^c	0.857^c	0.003^a	146.3^a
Mid × Wilted	45.1^cd	6.0^b	66.4^b	54^b	41.7^b	1.057^bc	0.183^c	0.243^c	0.893^c	0.0043^a	163^a
Late × Unwilted	47.0^bc	5.10^cd	76^a	61.6^a	34.7^c	0.617^d	0.11^d	0.15^d	0.723^d	0.0033^a	65.7^a
Late × Wilted	54.1^a	4.8^d	77.1^a	62.3^a	34.7^c	0.613^d	0.113^d	0.153^d	0.723^d	0.0023^a	56^a
SEM	0.528	0.182	1.01	0.84	1.1	0.028	0.005	0.005	0.021	0.0007	24.9
P-value	< 0.001	0.024	0.11	0.28	0.39	0.017	0.005	< 0.01	0.002	0.105	0.87
Abbreviations: DM, Dry matter; CP, crude protein; ACP, Adjusted crude protein; ADF, acid detergent fiber; NDF, neutral detergent fiber; TDN, total digestible nutrients; Ca, calcium; P, Phosphorus; Mg, Magnesium; K, Potassium; Na, Sodium; Fe, Iron.
SEM, standard error of the mean
Lowercased letters, means without a common letter differ (P < 0.05).

Table 2

continue: Nutritional values of elderberry silage by main effect and interaction for two wilted or unwilted fodders and three different harvesting dates (early, mid and late growth season).
Treatment	Zn	Cu	Mn	Mo	S	Ash	Lignin	NFC	NEL	NEM	NEG	RFV
Wilted	ppm	ppm	ppm	ppm	%	%	%	%	McalL/kg	McalL/kg	McalL/kg	%
Wilted	27.9^a	4.56^a	91.8^b	1.04^a	0.0822^a	4.83^a	20.4^a	19.1^a	0.812^a	0.671^a	0.217^a	71.6^a
Unwilted	29.3^a	4.89^a	96.8^a	1.07^a	0.0844^a	5.13^a	20.3^a	19.5^a	0.842^a	0.699^a	0.232^a	75.3^a
SEM	0.5	0.13	2.27	0.035	0.001	0.10	0.29	0.47	0.019	0.023	0.019	1.48
P-value	0.07	0.10	0.04	0.51	0.27	0.08	0.89	0.55	0.28	0.42	0.57	0.095
Date
Early	30.8^a	4.67^a	121.7^a	1.18^a	0.1117^a	6.55^a	15.4^c	27.1^a	1.24^a	1.152^a	0.593^a	105.7^a
Mid	29.5^a	5.00^a	87.2^b	0.99^b	0.0783^b	5.13^b	21.5^b	17.7^b	0.768^b	0.60^b	0.080^b	65.3^b
Late	25.5^b	4.50^a	74.0^c	0.99^b	0.06^c	3.26^c	24.1^a	13^c	0.473^c	0.303^c	0.00^b	49.3^c
SEM	0.63	0.16	2.7	0.04	0.001	0.13	0.36	0.57	0.023	0.029	0.023	1.81
P-value	< 0.001	0.13	< 0.001	0.01	< 0.001	< 0.001	< 0.001	< 0.001	< 0.001	< 0.001	< 0.001	< 0.001
Wilting × Date
Early × Unwilted	32.3^a	5.00	127.7^a	1.23^a	0.1167^a	6.97^a	15.1^c	27.6^a	1.287^a	1.203^a	0.64^a	111.3^a
Early × Wilted	29.^3ab	4.33	115.7^a	1.13^a	0.1067^a	6.14^a	15.7^c	26.6^a	1.193^a	1.1^a	0.546^a	100^b
Mid × Unwilted	30^a	5.00	87^b	0.99^a	0.0767^b	5.05^b	21.5^b	17.8^b	0.753^b	0.583^b	0.0567^b	64.7^b
Mid × Wilted	29^ab	5.00	87.3^b	0.99^a	0.08^b	5.21^b	21.5^b	17.7^b	0.783^b	0.617^b	0.103^b	66^b
Late × Unwilted	25.7^b	4.67	75.7^bc	0.99^a	0.06^c	3.37^c	24.4^a	13.1^c	0.487^c	0.31^c	0.0^b	50^c
Late × Wilted	25.3^b	4.33	72.3^c	0.99^a	0.06^c	3.15^c	23.8^ab	12.9^c	0.460^c	0.297^c	0.0^b	48.7^c
SEM	0.89	0.236	2.79	0.06	0.002	0.18	0.51	0.81	0.03	0.058	0.03	2.56
P-value	0.33	0.39	0.11	0.64	0.03	0.062	0.53	0.84	0.21	0.27	0.13	0.067
Abbreviations: Zn, zinc; Cu, copper; Mn, Manganese; Mo, Molybdenum; S, Sulphur; Ash, Lignin, NFC, non-fiber carbohydrates; NEL, net energy for lactation; NEM, net energy for maintenance; NEG net energy for gain; RFV, relative feed value.
SEM, standard error of the mean.
Lowercased letters, means without a common letter differ (P < 0.05).

Fermentation parameters

Fermentation parameters of wilted and unwilted ensiled elderberry fodder harvested at three different dates are presented in Table 3. An interaction between wilting and harvest date for ensiled elderberry was observed for pH in which early harvest fodders had lower pH than mid and late harvest fodders, except unwilted late-harvest fodders which did not differ, while both mid-harvest fodders did not differ from wilted and late harvest fodders. An interaction was observed for butyric acid (P < 0.001). Butyric acid was greater in unwilted early harvest fodder compared to wilted early harvest fodder, and wilted early harvesting fodders did not differ from unwilted mid-harvest fodders. Mid-harvest and late-harvest fodders did not differ based on wilting, and only unwilted mid-harvest was greater than both late-harvest fodders, while mid-harvest wilted did not differ from either late harvest fodder. Butyric acid was < 0.04% for wilted mid-harvest fodders and < 0.007% both late-harvest fodders.

Table 3

Fermentation values for elderberry silage by main effect and interaction for wilted or unwilted fodder and three different harvesting dates (early, mid and late growth season).
Treatment	pH	Butyric	Acetic acid	NH3	Lactic acid	2,3-Butanediol	Formic acid	Succinic acid	1,2-Prop	Ethanol
Wilted	%	%	%	%	%	%	%	%	%	%
Wilted	5.23^a	0.0509^b	0.196^b	0.0287^b	1.22^b	0.0986^a	0.0104^a	0.158^a	0.0006^a	0.563^a
Unwilted	5.00^b	0.1331^a	0.268^a	0.034^a	1.53^a	0.1072^a	0.009^a	0.199^a	0.00^a	0.557^a
SEM	0.032	0.009	0.015	0.001	0.09	0.021	0.0008	0.022	0.0004	0.057
P-value	0.0002	< 0.001	0.007	0.02	0.048	0.7830	0.59	0.22	0.33	0.94
Date
Early	4.76^c	0.20^a	0.3^a	0.055^a	2.752^a	0.00^b	0.0153^a	0.17^b	0.00^a	0.532^a
Mid	5.42^a	0.0723^b	0.278^a	0.0228^b	0.814^b	0.255^a	0.0101^b	0.329^a	0.001^a	0.667^a
Late	5.17^b	0.0035^c	0.119^b	0.0162^b	0.57^b	0.054^b	0.0048^c	0.038^c	0.00^a	0.482^a
SEM	0.039	0.011	0.02	0.001	0.122	0.026	0.001	0.027	0.0005	0.070
P-value	< 0.001	< 0.001	< 0.001	< 0.001	< 0.001	< 0.001	< 0.001	< 0.001	0.39	0.203
Wilting × Date
Early × Unwilted	4.74^b	0.285^a	0.328^a	0.0593^a	2.985^a	0.00^c	0.016^a	0.1933^abc	0.00^a	0.545^a
Early × Wilted	4.78^b	0.1153^b	0.272^a	0.0507^a	2.52^a	0.00^c	0.014^ab	0.146^bc	0.00^a	0.519^a
Mid × Unwilted	5.32^a	0.1073^bc	0.338^a	0.025^b	0.912^b	0.273^a	0.0093^bcd	0.367^a	0.00^a	0.714^a
Mid × Wilted	5.53^a	0.0373^cd	0.217^ab	0.0207^b	0.715^b	0.237^ab	0.011^abc	0.2903^ab	0.002^a	0.619^a
Late × Unwilted	4.94^b	0.007^d	0.138^b	0.0177^b	0.702^b	0.049^c	0.0033^d	0.0383^c	0.00^a	0.413^a
Late × Wilted	5.39^a	0.00^d	0.1^b	0.0147^b	0.439^b	0.059^bc	0.0063^cd	0.0377^c	0.00^a	0.551^a
SEM	0.055	0.015	0.027	0.0025	0.172	0.037	0.0014	0.039	0.0008	0.1
P-value	0.0093	< 0.001	0.31	0.53	0.725	0.816	0.17	0.63	0.39	0.51
1-Prop, 2-Butanol, and propionic acid were not detected on samples.
Lowercased letters, means without a common letter differ (P < 0.05)
SEM, standard error of the mean

Unwilted ensiled elderberry fodder had greater concentrations of acetic acid (P = 0.007), ammonia (P = 0.02), and lactic acid (P = 0.048) compared with wilted fodder silage. Early harvest ensiled forage had greater ammonia (P < 0.001) and lactic acid concentrations (P < 0.01) compared with mid and late harvest, which did not differ. Early and late harvest ensiled fodders had lower 2,3-butanediol compared with mid-harvest (P < 0.001). Acetic acid concentrations were greater from early and mid-harvest fodders compared with late harvest (P < 0.01). Formic acid concentrations were greatest in early harvest followed by mid, which differed from late harvest (P < 0.001). Succinic acid concentrations were greatest from mid harvest fodder, followed by early harvest, which was greater than late harvest (P < 0.001).

Yield

Elderberry plants were coppiced on different dates: early harvest (Sept 15), mid harvest (Oct 6), late harvest (Nov 30) in 2023, along with a control in March 2024 to correspond with typical commercial management practices. The control was included only in the berry yield, not in the silage study. Elderberry fruit was harvested when 50% of the berries had ripened (in August 2024). In the current study, whole plant coppicing for fodder during early, mid, or late season had no effect on successive berry yield in the next season (P = 0.072; Table 4).

Table 4

Berry yield in elderberry plants for early, mid, and late season coppiced plants
Treatment	Yield (Kilograms)
Control	5.00
Early	2.51
Mid	4.29
Late	5.11
SEM	1.52
P-value	0.072
Standard error of mean, SEM

Nutrient composition of ensiled elderberry

American elderberry is rapidly expanding in the U.S. for its use in nutraceutical products as well as food products such as jelly, jam, and liqueur (Domínguez et al., 2021; Thomas et al., 2020). After berry harvest in mid-August, the leaves remain on the plant until natural senescence in late fall. The standard management practice involve coppicing the plant to prepare for spring regrowth (Byers et al., 2012). Leafy biomass is generally composted of or disposed. There is an opportunity for the whole plant to be used as a forage source for ruminant livestock. Several species of elderberry plant contain cyanogenic compounds, so grazing or browsing the plant is not recommended (Panter, 2018). American elderberry leaf biomass likely presents a low risk of toxicity when utilized as a ruminant feed supplement and thus can be detrimental to ruminant health (McKenzie, 2024). The threat of acute toxicity in forages can be reduced by wilting and ensiling, as cyanogenic compounds volatilize during wilting or degraded during ensiling (Gruss et al., 2022). Coppiced and ensiled elderberry from the first harvest date in September, regardless of wilting, had comparable nutritive value (8.42% CP; 51% NDF; 40% ADF; 55% TDN) to silage prepared from the most common grass forages in the US. Coblentz et al. (2020) reported 7.1, 10.1, and 7.5% CP of ensiled meadow fescue, orchardgrass, and tall fescue forages respectively. However, NDF was high in those forages (62–65%), and ADF was low (36–39%) compared to first harvest ensiled elderberry in the current study. The nutritive value of the second and third harvest ensiled elderberry was significantly lower in CP, with high NDF and ADF. This was expected because the elderberry plant still had pliable stems and leafy biomass at the early harvest, while in later harvests, leaves senesced and stems lignified.

The effects of wilting prior to ensiling on nutritional parameters was of interest in this study as wilting could be used as a management strategy to reduce cyanogenic compounds, but may reduce fodder nutritive value or the ability to ensile due to low DM. Notable parameters such as CP, pH, and TDN were not affected by wilting prior to ensiling. Similarly, a meta-analysis evaluating wilting effects on different forages found no effects on CP, pH, and in vitro dry matter digestibility (Ridla et al., 2024), though, wilting increased DM, NDF, and ADF of the ensiled biomass (Ridla et al., 2024). In this study, wilting increased the DM percentage of ensiled elderberry silage but did not influence ADF and NDF. Generally, NDF and ADF proportions increase as sugars lost through respiration which continues during wilting, in this study, the wilting time may have been too short for significant loss of carbohydrates or sugar content of fodders was initial low.

Cow at maintenance requires 0.18% Ca, 0.18% P, 0.6% K, 0.04 to 0.1% Mg, 0.08% Na, 0.15% S, 10 ppm Cu, 50 ppm Fe, 40 ppm Mg, 30 ppm Zn, and 0.5–2.5 ppm Mo (Eric Bailey, 2017). Higher mineral requirements are needed during heat stress, parasite infection, and reproduction (Masters et al., 2019). Most of the forages in the pasture contain less macro- and micro-minerals than are required for livestock growth (Julian et al., 2020; Masters et al., 2019). Therefore, supplementation of salt and minerals is always recommended for livestock production. Shrubs, unlike common forages, contain higher levels of minerals than livestock need (Julian et al., 2020; Masters et al., 2019). However, elderberry, which is considered a shrub, has lower mineral levels than most livestock require. Several factors influence mineral concentration in plants, such as forage species, plant parts, growth stage, soil fertility, nitrogen fertilization, liming, season, and plant parts (McDowell & Valle, 2000). As plants mature, there is a decrease in several micro-minerals, including Cu, Se, and Co (McDowell & Valle, 2000). Additionally, wilting results in the loss of minerals such as Zn, Fe, and Mg (Zhao, 2014). In this study, most minerals from early-season harvests were higher compared to mid and late harvests. However, elderberry silage, regardless of harvest date or wilting level, cannot fulfill several macro and micro-nutrients, such as Na, S, and Cu, for cows during their dry period, when nutritional requirements are lowest. Earlier reports of mineral analysis from Sudan × sorghum silage from the Midwest revealed deficiencies in several essential minerals for the animals (Corah & Dargatz, 1996). Similarly, corn silage samples submitted to Dairy One lab and Dairyland (WI, MN) showed deficiencies in key macro-elements (Linn et al., 2004). Slight mineral deficiencies might not be a critical factor for animal feed use, as most producers supplement additional minerals for their cattle, so this may not be a significant concern.

Fermentation parameters

Ensiling may be the preferred method for storing elderberry biomass for feed, as producers may not be able to adequately dry fodders or temperatures in late fall may not allow for drying. Forages suitable for ensiling must contain (40–60 DM%) and fermentable carbohydrates to create adequate conditions for the microbes to carry out fermentation. Adequate fermentation is required to reduce the pH at least below 5.5 for long-term storage stability. Across treatments, elderberry silage pH ranged from 4.7 to 5.5. Most legumes and grass silage fall within this reference range (Kung Jr et al., 2018). A negative linear relationship exists between forage moisture at ensiling and pH, greater forage moisture at ensiling allows for better fermentation conditions producing more volatile fatty acids that reduce the post-ensiled pH. Lactic acid is a primary acid responsible for the drop in silage pH. In the current study, the lactic acid concentration ensiled silage ranged from 2.8% for early harvest to 0.6% in late harvest fodder. Unwilted fodders (38–47% DM) resulted in lower pH compared to wilted (47–54% DM) due to available moisture for fermentation, and early harvested fodder also had lower pH, due to combination of greater fodder moisture concentrations and available nutrients for fermentation compared to later harvested fodders that had lower TDN, CP, and greater NDF, ADF, and lignin.

Lactic acid concentration in ensiled forages shows a positive linear relationship with dry matter (DM) content during storage. Coblentz et al. (2020) measured the lactic acid concentration in round-bale alfalfa-orchardgrass silage ensiled at various DM percentages. The regression curve of lactic acid concentration versus forage DM percentage displayed a cubic response. Lactic acid levels were lower (< 0.6% of DM) when forage was ensiled at 50% DM, but increased to 3.5% of DM when forage (alfalfa-orchardgrass mix) was ensiled at 35% forage DM, reaching a plateau before declining (Coblentz et al., 2020). In the current study, lactic acid concentrations in unwilted (38–47% DM) ensiled elderberry were higher compared to wilted (47–54% DM). The values found align with the range reported by (Coblentz et al., 2020).

Similar to lactic acid, the concentration of acetic acid in the silage is negatively correlated with the DM % of the forage before ensiling (Kung Jr et al., 2018). Legume silage ensiled at 45–55% DM results in 0.5-2.0% acetic acid concentration, whereas corn ensiled at high moisture levels (70–75%) and low DM percentages (25–35%) results in less than 0.5% acetic acid concentration. In line with other studies, we found that elderberry ensiled at lower dry matter percentage (DM%, 38–47%) had a lower acetic acid concentration compared to that stored at high DM% (47–54%). Propionic acid and butyric acid concentrations can also be indicator of silage quality. No propionic acid was detected in any of the post-ensiled samples. Ideally, propionic acid should be less than 0.1% of DM in legume, grass, and corn silage. Well-fermented forages will have low levels of propionic acid. Similarly, silages high in butyric acid (> 1%) have low nutritive value and considered unpalatable. Acceptable silage contains < 3% acetic acid, < 1% butyric acid, and < 0.5% propionic acid. Butyric acid in our study ranged from 0.003 to 0.2%, which is within the lower range reported for most legume, grass, and corn silages (Kung Jr et al., 2018). The normal level of ammonia in corn silage is 0.11% (Kung Jr et al., 2018). High ammonia concentration in the silo results in excessive protein breakdown and are also associated with high moisture silage. Ammonia values ranged from 0.01 to 0.05%, indicating the low CP content of fodder at ensiling and fermentation conditions that did not result in excessive protein degradation. High ethanol levels are generally indicative of poor fermentation, but levels in this study were in the acceptable range and did not differ. Formic acid and succinic acid concentrations were low and did not were only affected by harvest date likely as a result of greater fodder quality. Digestible energy in silage can be expressed as net energy for lactation (NEL), net energy for maintenance (NEM), and net energy for gain (NEG). Most values found in other silages are within the reference range between 0.5 to 1 (https://www.agproud.com/articles/55071-the-potential-of-small-grain-silage). Most early harvest ensiled elderberry samples were within this above reference range; however, showed much lower values.

Berry yield

In the current study, there were no effects of whole plant coppicing for fodder during the early, mid, or late season on successive berry yield in the following season.

Fall coppiced elderberry material may serve as a viable fodder source for livestock. Coppiced and ensiled elderberry from the first harvest date in September, regardless of moisture at ensiling, had comparable nutritive value to silage prepared from the most common grass forages in the U.S. With CP levels of 8.4% and NDF values near 51%, unwilted early harvested fodder may meet the nutritional requirements of beef cows, but the greater levels of ADF% (40%) and lignin near 15% resulting in low digestibility, cattle would still likely require an energy supplement for maintenance, gestation, and lactation. Post-ensiled fodder had an acceptable pH (< 5.5) for long-term storage and concentrations and proportions of volatile fatty acid were within acceptable ranges for silages. Early harvested elderberry (40% DM) successfully ensiled (< 4.7 pH) and is the best option for livestock feeding as CP reduced greatly from early to mid-harvest.

Acknowledgements

We would like to sincerely thank USDA ARS for their funding to complete this study and the University of Missouri, Horticulture and Agroforestry Research Farm (HARF), for providing space, support, and their active involvement in the project.

Author contribution

Conceptualization - Roshani Sharma Acharya; Christine C. Nieman; Ashley C. Conway-Anderson. Methodology – Roshani Sharma Acharya. Data curation- Roshani Sharma Acharya. Formal analysis and investigation - Roshani Sharma Acharya; Christine C. Nieman. Funding acquisition - Christine C. Nieman; Ashley C. Conway-Anderson. Investigation – Roshani Sharma Acharya. Writing - Original draft preparation – Roshani Sharma Acharya, Christine C. Nieman; Ashley C. Conway-Anderson. Writing-review & editing - Roshani Sharma Acharya; Christine C. Nieman; Ashley C. Conway-Anderson.

Funding

This material was based on work supported by USDA-ARS Dale Bumpers Small Farms Research Center CRIS number 6020-21310-011-000D. Any opinions, findings, conclusions, or recommendations expressed in this publication are those of the authors and do not necessarily reflect the view of the USDA.

Data availability

Data will be available upon request.

Competing interest:

The authors declare no competing interests.

Conflict of interest:

The authors have no competing interests to declare that are relevant to the content of this article.

Alstrup L, Søegaard K, Weisbjerg MR (2016) Effects of maturity and harvest season of grass-clover silage and of forage-to-concentrate ratio on milk production of dairy cows. J Dairy Sci 99 (1): 328–340
ANKOM (2020) Acid Detergent Fiber in Feeds - Filter Bag Technique; ANKOM Technology: Macedon, NY, USA
AOCC (2003) The Association of Official Analytical Chemists. In: Official Methods of Analysis. Washington, DC, USA
Bailey E (2017) Mineral Supplements for Beef Cattle. https://extension.missouri.edu/publications/g2081
Borreani G, Tabacco E, Schmidt RJ, Holmes BJ et al (2018) Silage review: Factors affecting dry matter and quality losses in silages. J of Dairy Sci 101(5): 3952–3979
Byers PL, Thomas AL, Cernusca MM et al (2012) Growing and Marketing Elderberries in Missouri (2012) https://mospace.umsystem.edu/xmlui/handle/10355/50222
Cabezas-Garcia EH, Krizsan SJ, Shingfield KJ et al (2018) Effects of replacement of late-harvested grass silage and barley with early-harvested silage on ruminal digestion efficiency in lactating dairy cows. J of Dairy Sci 101(2): 1177–1189.
Charlebois D (2007) Elderberry as a Medicinal Plant. Issues in new crops and new uses. ASHS Press, Alexandria, VA pp. 284-292.
Coblentz WK, Akins MS, Cavadini JS (2020) Fermentation characteristics and nutritive value of baled grass silages made from meadow fescue, tall fescue, or an orchardgrass cultivar exhibiting a unique nonflowering growth response. J of Dairy Sci 103(4): 3219–3233.
Corah LR, Dargatz D (1996) Forage analyses from cow/calf herds in 18 states. Beef Cow/Calf Health and Productivity Audit. Washington (DC): USDA, Animal and Plant Health Inspection Service, Veterinary Service, National Health Monitoring System. https://www.aphis.usda.gov/sites/default/files/chapa_dr_forageanal.pdf
NRC (2001) Nutrient requirements of dairy cattle: National Academies Press. Nutrient Requirements of Dairy Cattle, 7^th ed: Washington
Domínguez R, Pateiro M, Munekata PE et al (2021). Potential use of elderberry (Sambucus nigra L.) as natural colorant and antioxidant in the food industry. A review. Foods 10(11): 2713
Gruss SM, Johnson KD, Johnson SK et al (2022) Managing Prussic Acid Hazard in Sorghum. https://www.extension.purdue.edu/extmedia/AY/AY-378-W.pdf
McKenzie EL (2024) American Elderberry (Sambucus nigra Subsp. candensis) Biomass Nutritive Value Assessment as a Potential Feed Source for Ruminant Livestock. Master's thesis University of Missouri-Columbia.
Julian AA, Scasta JD, Stam BR et al (2020) Mineral element concentrations of common grass and shrub species on sheep winter range in Wyoming: Insights for mineral supplementation strategies. Transl Anim Sci 4 (Supplement_1): S11–S16.
Krom MD (1980) Spectrophotometric determination of ammonia: A study of a modified Berthelot reaction using salicylate and dichloroisocyanurate. Analyst, 105(1249): 305–316.
Kung Jr L, Shaver RD, Grant RJ et al (2018) Silage review: Interpretation of chemical, microbial, and organoleptic components of silages. J of Dairy Sci 101(5): 4020–4033
Linn J, Salfer J, Martens D et al (2004) Guide to evaluating corn silage quality. https://www.midwestforage.org/pdf/109.pdf
Masters DG, Norman HC, Thomas DT (2019) Minerals in pastures—Are we meeting the needs of livestock? Crop Pasture Sci 70(12): 1184–1195
McDonald P, Henderson AR, Heron SJE (1991) The biochemistry of silage. Chalcombe Publications. Marlow Bucks, UK, 340
McDowell LR, Valle G (2000) Major minerals in forages. In Forage evaluation in ruminant nutrition. pp. 373–397, https://doi.org/10.1079/9780851993447.0373
McGowan KG, Byers PL, Jose S et al (2018) Flower production and effect of flower harvest on berry yields within six American elderberry genotypes. XXX International Horticultural Congress IHC2018: III International Berry Fruit Symposium 1265, 99–106. https://www.actahort.org/books/1265/1265_14.htm
Moore JE, Undersander DJ (2002) Relative forage quality: An alternative to relative feed value and quality index. Proceedings 13th Annual Florida Ruminant Nutrition Symposium, 32, 16–29 https://www.academia.edu/download/68175673/Relative_Forage_Quality_An_Alternative_t20210718-29286-7pupcz.pdf
Muck RE (1988) Factors influencing silage quality and their implications for management. J of Dairy Sci, 71(11): 2992–3002
National Reserach Council (2001) Nutrient Requirement of Dairy Cattle, 7th ed,: The National Academic Press: Washington, DC, USA
Nieman CC, Conway-Anderson AC (2022) Effects of Packing Density and Inoculation with Lactic Acid-Producing Bacteria to Evaluate the Potential for North American Elderberry (Sambucus canadensis L.) Fodder as Silage. Agronomy 12(12): 3212
Panter KE (2018) Cyanogenic glycoside–containing plants. In Vet. Toxico. pp. 935–940. https://www.sciencedirect.com/science/article/pii/B9780128114100000647
Ribas WFG, Monção FP, Rocha VR et al (2021) Effect of wilting time and enzymatic-bacterial inoculant on the fermentative profile, aerobic stability, and nutritional value of BRS capiaçu grass silage. Rev. Bras. Zootecn 50:e20200207
Ridla M, Albarki HR, Risyahadi ST et al (2024) Effects of wilting on silage quality: A meta-analysis. Ani. Biosci 37(7): 1185–1195.
Technology A (2006) Acid detergent fiber in feeds filter bag technique. ANKOM Technology Method 5, Ankom Technology Macedon, NY.
Thomas AL, Byers PL, Vincent PL et al (2020) Medicinal Attributes of American Elderberry. In Á. Máthé (Ed.), MAPs of North America 6:119–139. https://doi.org/10.1007/978-3-030-44930-8_5
Zhao X (2014) Content of macro-and microminerals in wrapped forages for horses. SLU, Dept. of Animal Nutrition and Management (until 231231). http://stud.epsilon.slu.se/7332/

No competing interests reported.

Assessing the effect of ensiling moisture and sampling date on silage quality and subsequent yield of North American elderberry

Status:

Version 1

Abstract

Introduction

Materials and methods

Experimental Site

Plant fodder harvest and ensiling

Determination of fruit yield

Nutritive Analysis

Fermentation Analysis

Statistical Analyses

Results

Pre-Ensiled Nutrient Composition

Fermentation parameters

Yield

Discussion

Nutrient composition of ensiled elderberry

Fermentation parameters

Berry yield

Conclusion

Declarations

References

Additional Declarations

Status:

Version 1